EP2197897A1 - Amino acid pairing-based self assembling peptides and methods - Google Patents

Amino acid pairing-based self assembling peptides and methods

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Publication number
EP2197897A1
EP2197897A1 EP08800379A EP08800379A EP2197897A1 EP 2197897 A1 EP2197897 A1 EP 2197897A1 EP 08800379 A EP08800379 A EP 08800379A EP 08800379 A EP08800379 A EP 08800379A EP 2197897 A1 EP2197897 A1 EP 2197897A1
Authority
EP
European Patent Office
Prior art keywords
peptide
ellipticine
amino acid
asn
self
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08800379A
Other languages
German (de)
French (fr)
Other versions
EP2197897A4 (en
Inventor
Pu Chen
Hong Yang
Shane Fung
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Waterloo
Original Assignee
University of Waterloo
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Filing date
Publication date
Application filed by University of Waterloo filed Critical University of Waterloo
Publication of EP2197897A1 publication Critical patent/EP2197897A1/en
Publication of EP2197897A4 publication Critical patent/EP2197897A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/8605—Porous electrodes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present invention relates to self-assembling peptides that form nanostructures, methods of preparing the peptides, and uses of the same.
  • the spontaneous organization of molecules into structurally well-defined arrangements due to non-covalent interactions is referred to as "molecular self- assembly".
  • the resulting supramolecular structure typically provides nanoarchitectures with very defined macroscopic properties (Whitesides et al., (1991) Science 254: 1312-1319 "Whitesides et al. (1991)").
  • the self-assembly of peptides not only relates to many naturally occurring states of proteins, such as amyloid fibrillogenesis (Aggeli et al. (1999)), but also provides useful biomolecular building blocks for a wide variety of supramolecular fabrications (Zhang et al. (1993); Chen (2005); Zhang (2002)).
  • the new self-assembling peptides is a class of ionic-complementary, amphiphilic peptides, e.g., EAK made of glutamic acid (E), alanine (A), and lysine (K) residues (Zhang et al. (1993) and U.S. Patent No. 5,670,483 to Zhang et al.).
  • EAK glutamic acid
  • A alanine
  • K lysine residues
  • This new class of peptides originates from zuotin, a yeast protein that preferentially binds to left-handed Z-DNA (Zhang et al. (1993)).
  • the molecular structure of these peptides contains alternating positive and negative charges, enabling ionic-complementarity.
  • EAKl 6-11 is an example of an ionic-complementary amino acid-based self assembling peptide. EAKl 6-11 peptides demonstrate self-assembly into a variety of configurations in a concentration-dependent manner when plated on a mica surface (Jun et al. (2004) Biophys. J. 87:1249-1259).
  • the critical aggregation concentration (CAC) of EAK16-II was determined to be 0.06 mM (Fung et al. (2003)). Furthermore, it was shown that the sequence of the EAK peptide is a critical determinant of the nanostructure that results upon peptide aggregation (Jun et al. (2004) Biophys. J. 87:1249-1259). pH was also shown to be critical for peptide aggregation (Hong et al. (2003)).
  • Gazit et al. have studied peptide self-assembly in the context of short peptide fragments that form well-organized amyloid fibrils, responsible for a number of protein aggregation diseases (Azriel and Gazit (2001) J. Biol. Chem. 276:34156- 34161; Gazit (2002) FASEB J. 16:77-83; Gazit (2002) Bioinformatics 18:880-883). It has been also demonstrated that peptides from 3 to 6 amino acids in length and containing aromatic residues can form amyloid fibrils that further assemble to form ⁇ - pleated sheets (Maji et al. (2004) Tetrahedron 60:3251-3259). Due to their ability to form ⁇ -sheet-rich fibrils, amyloid peptides have a suggested use as building blocks for nano-electronics (reviewed in Reches and Gazit (2006) Current Nanoscience 2: 105-111).
  • Dipeptides of phenylalanine have been shown to be sufficient to self-assemble into peptide nanotubes (Reches and Gazit (2003) Science 300:625-627). It was then demonstrated that the self-assembled phenylalanine dipeptide can be used to form peptide-nanotube platinum-nanoparticle composites (Song et al. (2004) Chem. Commun. 9:1044-1045). Additional studies have indicated the use of such peptide nanotubes in electrochemical biosensing applications (Yemini et al. (2005) Nano Letters 5: 183-186).
  • U.S. Patent No. 7,179,784 describes surfactant-like self-assembling peptides that form nanotubes.
  • the disclosed peptides specifically contain amino acids having nonpolar, noncharged side chains in combination with amino acids having cationic
  • peptides are amphiphilic in nature and tend to aggregate in order to isolate the hydrocarbon chain from contact with water.
  • the basic principle behind the self-assembly of these peptides is the formation of a polar interface that separates the hydrocarbon and water regions.
  • U.S. Patent Application Publication No. 2005/0164361 discloses that manipulation of the environment of a self-assembling peptide can assist in the control of the nucleation and propagation of the peptides.
  • U.S. Patent Application Publication No. 2005/0181973 discloses self- assembling peptides having two amino acid domains.
  • the first domain has alternating hydrophobic and hydrophilic amino acids and mediates self-assembly into macroscopic structures when the amino acids are present in unmodified form.
  • the second amino acid domain is unable to self-assemble on its own.
  • Typical second amino acids domains of the peptides disclosed in this application mimic a biologically active peptide motif found in a naturally occurring protein, such as a component of the basement membrane.
  • U.S. Patent Application Publication No. 2005/0209145 discloses self assembling amphiphilic peptides capable of binding to growth factors through specific non-covalent interactions.
  • Typical peptides disclosed in this application include an alkyl tail, a ⁇ -sheet forming peptide sequence, and a bio-active peptide sequence.
  • U.S. Patent Application Publication No. 2005/0272662 describes peptide- amphiphile compositions which include a first peptide-amphiphile having a hydrophilic region and an ionic charge, and a second peptide-amphiphile having a hydrophilic region and an opposite ionic charge. Each hydrophilic region has an associated biological signal. Examples of peptides disclosed are YIGSR and IKVAV.
  • U.S. Patent Application Publication No. 2006/0079454 discloses tubular, planar and spherical nanostructures that consist of aggregated self-assembling peptides.
  • the peptides of the application include aromatic amino acids and can consist entirely of aromatic amino acids, and are no more than 4 amino acids in length.
  • U.S. Patent Application Publication No. 2006/0084607 discloses amphiphilic peptide chains having alternating hydrophilic and hydrophobic amino acids.
  • the peptides are at least 8 amino acids in length and compatible structurally and are complementary such that they self-assemble into ⁇ -pleated sheets, forming a macroscopic scaffold.
  • the present invention provides ⁇ -stranded peptides that are self-complementary and assemble into nanostructures. These peptides are designed on the basis of amino acid pairing properties of amino acids. It is demonstrated that these peptides are useful in enhancing solubility of hydrophobic drugs. Various uses of such ⁇ -strand peptide-based nanostructures include drug delivery, biomolecule sensory, and biofuel cell applications.
  • the invention provides a self-complementary ⁇ -strand peptide having alternating hydrogen bonding proton donor amino acid segments and hydrogen bonding proton acceptor amino acid segments, that self assembles into a nanostructure.
  • the peptide has a length from two to forty amino acids.
  • the peptide has at least one proton donor and one proton acceptor segment, each of which consists of at least one amino acid.
  • Such peptides are not comprised of alternating hydrophobic and hydrophilic amino acid segments.
  • the invention provides a self-complementary ⁇ -strand peptide having one of the following structures: a) (A x B y C z ) w A z (I), and b) (A x B y C z ) w (C' x B' y A' z ) w (II)
  • A, A', B, B', C and C are each a hydrogen bonding amino acid, and are either a proton donor or a proton acceptor amino acid;
  • x and y are each independently an integer from 1 to 10;
  • z is an integer from 0 to 10; and
  • w is an integer from 1 to 20.
  • A is complementary to A'
  • B is complementary to B'
  • C is complementary to C.
  • the invention provides a self-complementary ⁇ -strand peptide having one of the following structures: a) A x B y C z ... ; and (III), and b) A x B y C z ... C z 'B y 'A x ' (IV).
  • A, A', B, B', C and C are each independently a donor amino acid or an acceptor amino acid, and are each self-complementary. These amino acids are further selected from the group consisting of a hydrogen bond donor amino acid, a hydrogen bond acceptor amino acid, a positively charged amino acid, a negatively charged amino acid, and a van der Waals' interacting amino acid.
  • A is complementary to A'
  • B is complementary to B'
  • C is complementary to C.
  • the invention provides a self-complementary ⁇ -strand peptide having at least one hydrogen bonding amino acid pair, at least one ionic- complementary amino acid pair, and at least one hydrophobic amino acid pair, for fo ⁇ ning a nanostructure.
  • the peptide has a length from four to forty amino acids.
  • the invention provides a self-assembled nanostructure consisting of aggregated units of a peptide having one of the following structures: a) (A x ByC z ) w I; and b) (A x B y C z ) w A x II.
  • A, B and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20.
  • the nanostracture formed from the self assembled peptides is one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
  • the invention provides a self assembled nanostructure consisting of aggregated units of a peptide having the general formula (V):
  • A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10.
  • the nanostructure formed from the self assembled peptides is one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
  • the invention provides pharmaceutical compositions comprising the ⁇ -strand peptides described in combination with a therapeutic agent.
  • the invention provides a kit for delivering a material to a patient, including a pharmaceutical composition comprising a self assembled ⁇ - strand peptide and a therapeutic agent; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; instructions for preparing the pharmaceutical composition for use; instructions for mixing the composition with other agents; and instructions for introducing the composition into a subject.
  • the invention provides a method of preparing a self- assembling peptide having amino acid pairing properties for manufacture of a nanostructure.
  • the method includes the steps of: designing a ⁇ -strand peptide consisting of amino acids that are capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction with a complementary amino acid; and generating a peptide from two to forty amino acids in length consisting of at least one amino acid pair capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction, and having complementary amino acid pairing and stereochemistry with a second peptide.
  • the invention provides a method for detecting a biomolecule of interest.
  • the method includes the steps of: forming a nanostructure from a ⁇ -strand peptide upon self assembly of the peptide; adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; coupling a reporter molecule capable of providing a measurable signal to the peptide- coated surface of the nanostructure; and providing the biomolecule of interest.
  • the invention provides a use of a ⁇ -strand peptide, for identification of inhibitors of protein aggregation disease.
  • Figure 1 is a flow chart of amino acid pairing (AAP)-based self assembling peptide design.
  • Figure 2A is a photograph of a glass vial containing pyrene in the absence of peptide
  • Figure 2B is a photograph of a glass vial containing 0.1 mg/mL ellipticine in the presence of decreasing concentrations of EAKl 6-11 peptide (0.5, 0.1 and 0 mg/mL).
  • FIG. 3A is a graph depicting the release rate of pyrene from the EAKl 6-11 peptide as a function of pyrene concentration (measured in ⁇ mol/L) against time (measured in hours) (from Keyes-Baig et al. (2004) J. Am. Chem. Soc. 126: 7522-7532 "Keyes-
  • Figure 3B is a scanning electron (SE) micrograph of pyrene: EAK at a ratio of 78: 1 in solution. Dotted lines extend from the micrograph to the graph of Figure 3A to corresponding pyrene release curves (from Keyes-Baig et al. (2004)).
  • SE scanning electron
  • Figure 3C is a SE micrograph of pyrene:EAK at a ratio of 16:1 in solution. Dotted lines extend from the micrograph to the graph of Figure 3A to corresponding pyrene release curves (from Keyes-Baig et al. (2004)).
  • Figure 4 is a series of AF micrographs of ⁇ -amyloid peptide (1-42 amino acids) on a hydrophobic HOPG surface (A-C) and on a mica surface (D-H). Images were captured at 512 s intervals. A schematic of the ⁇ -amyloid peptide amino acid sequence is depicted in (I). Arrows indicate that the A ⁇ assembly on hydrophobic graphite (HOPG) and mica may dictate A ⁇ aggregation at the hydrophobic interior of the lipid membrane and the hydrophilic (negatively charged) head portion, respectively.
  • HOPG hydrophobic graphite
  • mica may dictate A ⁇ aggregation at the hydrophobic interior of the lipid membrane and the hydrophil
  • Figure 5 is a schematic of nanofiber nucleation and growth of EAK 16-11 peptides on a mica surface. Peptide monomers are indicated by rectangular boxes. Charges within the peptide are indicated (- and +).
  • Figure 6 is a schematic of peptide monomer assembly on a mica surface where positive charges within the peptide are attracted to the negative charge of mica. Two peptides are shown, oriented horizontally.
  • Figure 7 is a series of schematics of EAKl 6-11 peptide dimers assembling on a mica surface in solutions of varying pH: A) water (neutral); B) 1 mM HCl; C) 10 mM HCl; D) 1 mM NaOH; and E) 10 mM NaOH. In 10 mM NaOH, the peptide dimers do not adsorb on mica, resulting no fiber formation. This illustrates how solution pH controls the surface-assisted peptide assembly on mica.
  • Figure 8 indicates the chemical structures of peptides of the present invention consisting of amino acids capable of hydrogen bonding.
  • the arrangement of hydrogen bonding pairs in a synthesized peptide was varied in terms of the number of repeating pair units (peptide length), for example QN and NS versus QNQN and NSNS or NSNSNSNS, and the design of the repeating unit itself (e.g. QN versus QQNN).
  • Figure 9A is a graph of absorbance plotted against wavenumber (cm "1 ) determined using Fourier Transform Infrared (FTIR) spectroscopy for peptides QN, QNQN, QQNN and NS4 (NSNSNSNS).
  • FTIR Fourier Transform Infrared
  • Figure 9B is a graph of Circular Dichroism (CD) (mdeg) plotted against wavelength (nm) for peptides QN, QNQN, QQNN and NS4. CD spectra were used to determine the secondary structures of the peptides in the bulk solution.
  • CD Circular Dichroism
  • Figure 10 is a graph of Thiofiavine T (ThT) assay fluorescence spectra data for peptides NS4, QNQN, QN, QQNN and ThT alone. Normalized intensity is plotted against wavelength (nm). The increase in ThT fluorescence responds to the abundance of ordered ⁇ -sheets in solution.
  • Thiofiavine T (ThT) assay fluorescence spectra data for peptides NS4, QNQN, QN, QQNN and ThT alone. Normalized intensity is plotted against wavelength (nm). The increase in ThT fluorescence responds to the abundance of ordered ⁇ -sheets in solution.
  • Figure HA is a scanning electron microscopy (SEM) image of a QN peptide (peptide concentration of 0.1 mg/mL).
  • Figure HB is a SEM image of a QNQN peptide (peptide concentration of 0.1 mg/niL).
  • Figure HC is a SEM image of a QQNN peptide (peptide concentration of 0.1 mg/niL).
  • Figure HD is a SEM image of a NS peptide (peptide concentration of 0.1 mg/mL).
  • Figure HE is a SEM image of a NSNS peptide (peptide concentration of 0.1 mg/mL).
  • Figure HF is a SEM image of a NSNSN peptide (peptide concentration of 0.1 mg/mL).
  • Figure HG is a SEM image of a NSNSNSNS peptide (peptide concentration of 0.1 mg/mL).
  • Figure 12A is a graph of ThT assay fluorescence spectra data for varying concentrations (mM) of the NS4 peptide.
  • the arrow points to a peak in the graph indicative of the increase of ThT fluorescence according to peptide concentrations.
  • FIG. 12B is a graph of the data in Figure 12A plotting normalized intensity values against the log concentration ( ⁇ M). The arrow points to a peak in the graph indicative of the critical aggregation concentration (CAC).
  • CAC critical aggregation concentration
  • Figure 13 A is a schematic of the chemical structure of the ACS peptide (FEFQFNFK) of the present invention.
  • Figure 13B is a schematic of the molecular structure of the AC8 peptide. An ionic bonding pair, a hydrogen bonding (HB) pair and a hydrophobic residue are indicated at the arrows.
  • Figure 14A is a graph of surface tension (measured in mJ/m 2 ) as a function of time for the AC8 peptide at varying concentrations indicated.
  • Figure 14B is a graph of the equilibrium surface tension obtained from Figure 14A as a function of peptide concentration ( ⁇ M). The intersection of the two straight lines indicates the CAC.
  • Figure 15A is a graph of ThT fluorescence spectra data plotting fluorescence intensity (a.u.) against wavelength (nm) for the AC8 peptide.
  • Figure 15B is a graph of the ThT fluorescence spectra data of Figure 15A plotted against peptide concentration ( ⁇ M). The intersection of the two straight lines indicates the CAC of ACS of approximately 20 ⁇ M.
  • Figure 16A is a graph of 8-anilino-l-naphthalenesulfonic acid (ANS) fluorescence spectra data (a.u.) for the AC8 peptide plotted against wavelength (run). The arrow indicates peak fluorescence at 475 nm.
  • ANS 8-anilino-l-naphthalenesulfonic acid
  • Figure 16B is a graph of the ANS fluorescence spectra data of Figure 16A plotted against peptide concentration ( ⁇ M). The intersection of the two straight lines indicates the CAC of AC8 of approximately 20 ⁇ M.
  • Figure 17A is a graph of static light intensity plotting normalized light intensity data for the AC8 peptide against peptide concentration ( ⁇ M). The intersection of the two straight lines indicates the CAC of AC 8 of approximately 20 ⁇ M.
  • Figure 17B is a graph of the number-based size distribution (%) (hydrodynamic diameter in nm) for varying concentrations of AC8 peptide.
  • Figure 18A is an Atomic Force (AF) micrograph of self-assembled AC8 peptides at a concentration of 2.2 ⁇ M.
  • AF Atomic Force
  • Figure 18B is an AF micrograph of self-assembled AC8 peptides at a concentration of 5 ⁇ M.
  • Figure 18C is an AF micrograph of self-assembled AC8 peptides at a concentration of lO ⁇ M.
  • Figure 18D is an AF micrograph of self-assembled AC8 peptides at a concentration of 40 ⁇ M.
  • Figure 18E is an AF micrograph of self-assembled AC8 peptides at a concentration of 87 ⁇ M.
  • Figure 18F is a graph of absorbance plotted against wavenumber (cm "1 ) determined using FTIR spectroscopy for the AC8 peptide, indicating ⁇ -sheet rich secondary structure.
  • Figure 19 is a schematic of AC8 peptide monomer assembly at concentrations below the CAC (left side) and above the CAC (right side), where concentrations of AC8 above the CAC result in a mixture of mature fibers, protofibrils and peptide monomers.
  • Figure 2OA is a graph of MCF-7 cell viability (measured as % viability) in the presence of various concentrations of EAK peptide ( ⁇ g/mL) in combination with ellipticine (red bars) compared to EAK peptide alone (blue bars). Black and green bars represent the medium and water solvent control, respectively.
  • Figure 2OB is a photograph of a series of glass vials containing various concentrations of EAK peptide (mg/mL) or water alone with 0.1 mg/mL ellipticine.
  • Figure 2OC is a SE micrograph of 0.1 mg/mL ellipticine in solution with 0.5 mg/mL
  • FIG. 2OD is a SE micrograph of 0.1 mg/mL ellipticine in solution with 0.1 mg/mL
  • Figure 2OE is a SE micrograph of 0.1 mg/mL ellipticine alone in solution.
  • Figure 21A is a graph of A549 cell viability in the presence of various solutions (cell culture medium, H 2 O, 3.3% DMSO); ellipticine (EPT) alone (0.1 mg/mL); or various peptides (EAK-p, EAKIC, EFK, NS4, FEQNK, QN, QNQN or QQNN) at a concentration of 0.1 mg/mL with (red bars) or without ellipticine (blue bars). All experiments looking at cell viability in the presence of peptide were conducted on cells cultured in the presence of serum.
  • Figure 21B is a graph of A549 cell viability in the presence of various solutions (cell culture medium, H 2 O, 3.3% DMSO); ellipticine (EPT) alone: or various peptides
  • FIG. 22 is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various peptides in the presence of ellipticine (EPT). Arrows point to crystal EPT, neutral EPT and protonated EPT, respectively.
  • Figure 23A is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various concentrations of AC8 peptide ( ⁇ M) in combination with 0.04 mg/mL ellipticine (in 1.33% DMSO), indicating ability of AC8 peptide to solubilize EPT.
  • Figure 23B is a graph of the data of Figure 23A plotting fluorescence intensity against peptide concentration, showing peptide concentration-dependent solubilization of EPT.
  • Figure 24A is a graph of A549 cell viability in various control media (1.3% DMSO,
  • Figure 24B is a graph of A549 cell viability in the presence of EAK peptide at varying concentrations in 1.3% DMSO in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • Figure 24C is a graph of MCF-7 cell viability in various control media (1.3% DMSO, PBS, H 2 O, medium alone).
  • Figure 24D is a graph of MCF-7 cell viability in the presence of EAK peptide at varying concentrations in 1.3% DMSO in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • Figure 24E is a graph of fluorescence emission spectra of EPT with different AC8 concentrations of 5, 9 and 100 ⁇ g/mL combined with 0.04 mg/mL EPT.
  • the solutioin contains 1.3% DMSO.
  • the solutions were tested on both A549 and MCF-7 cells as shown in Figure 24B and D.
  • Figure 25A is a series of bar graphs depicting A549 cell viability in the presence of medium alone; H 2 O; varying concentrations of EAK peptide (125 or 25 mg/mL); 25 mg/mL AC8 peptide; ellipticine in water (EPT-H; 25 ⁇ g/mL); or ellipticine in DMSO
  • Figure 26A is a bar graph depicting A549 cell viability upon culture in medium (M), water (H 2 O), or varying concentrations ( ⁇ g/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • M medium
  • H 2 O water
  • EPT ellipticine
  • Figure 26C is a bar graph depicting MCF-7 cell viability upon culture in medium (M), water (H 2 O), or varying concentrations ( ⁇ g/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • Figure 27A is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various concentrations of AC8 peptide (mg/mL) in combination with 0.04 mg/mL ellipticine.
  • Figure 27B is a zoom-in graph of fluorescence emission (normalized intensity) plotted against wavelength (nm) from Figure 27A.
  • Figure 27C is a bar graph depicting A549 cell viability upon culture in medium (M), water (H 2 O), or varying concentrations ( ⁇ g/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • M medium
  • H 2 O water
  • EPT ellipticine
  • Figure 27E is a bar graph depicting MCF-7 cell viability upon culture in medium (M), water (H 2 O), or varying concentrations ( ⁇ g/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
  • Figure 28 is a series of plots of AOD,- as a function of total EAK peptide concentration at pH 4 (A-C) and pH 7(D-F) at varying concentrations of dGi 6 (A, D), dCie (B, E) and dGCi 6 (C, F) oligonucleotides.
  • Figure 29 is a pair of plots of v/P/ versus v for the binding of EAK peptide to a guanine hexadecamer (dGi 6 ), a cytosine hexadecamer (dCi 6 ), and their duplex (dGC ⁇ ) at pH 4 (A) and pH 7 (B).
  • Figure 31 is graph of anisotropy (o) and the calculated percentage ( ⁇ ) of the FAM- dCi 6 in aggregates upon the addition of EAK peptide.
  • Figure 32 is a pair of plots of UV absorption spectra of dCi 6 -Rh in the presence and absence of EAK at pH 7.
  • A 3.9 ⁇ M dC, 6 -Rh (•), 60 ⁇ M EAK(+), 3.9 ⁇ M dC, 6 -Rh and 60 ⁇ M EAK before centrifugation (-) and after centrifugation (x);
  • B dCi6-Rh at concentration of 3.9 ⁇ M (•), 2.0 ⁇ M (-), and 1.3 ⁇ M (x).
  • Figure 33 is a photoimage of 20 % PAGE of 3.6 ⁇ M of dGQe mixed with EAK at (A) pH 4 and (B) pH 7 (the EAK concentrations are 0, 6, 60, 120 ⁇ M in lanes 1, 2, 3, and 4, respectively).
  • Figure 34 is a pair of plots of fluorescence anisotropy of a 0.1 mg/mL EAK solution containing 1 mol% of FAM-EAK in the presence (•) and in the absence (o) of (A)
  • Figure 35 is a pair of plots of data obtained from Steady States Light Scattering (SLS) experiments performed on (o) buffer and solutions containing (A) 3.6 ⁇ M dGi6 and
  • Figure 36 is a series of Atomic Force Microscopy (AFM) images of the EAK- oligonucleotide (ODN) complexes formed in a solution containing 3.6 ⁇ M dGj ⁇ and
  • EAK 0.1 mg/mL EAK, imaged in solution at pH 4 at: (A) 8 mins.; (B) 60 mins.; (C) 70 mins.; and (D) 75 mins. Images in C and D are zoomed in views of the marked areas in B and C, respectively.
  • Figure 37 is a population histogram of the EAK-ODN complexes as a function of particle diameter determined by dynamic light scattering for EAK-ODN solutions containing 7.2 ⁇ M of dGi ⁇ with increasing EAK concentration at pH 4. The sample of
  • EAK alone was measured 40 minutes after preparation, whose concentration was 0.1 mg/mL (60 ⁇ M); the concentration of dGi 6 alone was 7.2 ⁇ M.
  • Figure 38 is pair of Stern- Volmer plots for a solution of 3.6 ⁇ M of fluorescently labeled dCi 6 free and bound to 0.2 mg/mL of EAK at pH 4 quenched by KI. Solid lines represent the fits to the Stern- Volmer equation with parameters listed in Table
  • Figure 39 is a graph depicting scattered light intensity (measured in absorbance units by Dynamic Light Scattering (DLS)) plotted against time (hours) for 0.5 mg/mL
  • FIG. 40 is a time course series of AF micrographs showing EAKl 6-11 peptide nanofiber growth on a mica surface in pure water from adsorbed nanofiber "seeds"(top panels) and fiber clusters (bottom panels). The green arrows indicate the nanofiber growth from both active ends of the "seed” and the red arrows points to a reference spot (top panels).
  • Figure 41 is a series of AF micrographs showing nanostructure formation of 2 ⁇ M EAK16-II peptides on a mica surface under conditions of increasing pH (10 mM HCl, 1 mM HCl, H 2 O, ImM NaOH, and 1OmM NaOH, respectively). Lines are drawn from the various micrographs to a graph plotting zeta potential (mV) against solution pH at the pH value corresponding to the solution.
  • pH 10 mM HCl, 1 mM HCl, H 2 O, ImM NaOH, and 1OmM NaOH, respectively.
  • Figure 42A is a graph of nanofiber frequency (measured in %) on a mica surface plotted against fiber growth rate (measured in nm/s) for 2 ⁇ M EAKl 6-11 peptide in solutions of varying pH (1 mM HCl, water, and 1 mM NaOH).
  • Figure 42B is a table correlating solution conditions with pH and average nanofiber growth rate for 2 ⁇ M EAKl 6-11 peptide on mica.
  • Figure 43A is an AF micrograph of EAK16-II (0.05 mg/mL) on a mica surface, imaged in water.
  • Figure 43B is an AF micrograph of EAKl 6-11 (0.05 mg/mL) on a HOPG surface, imaged in water.
  • Figure 44A is a schematic of peptide monomer self-assembly on a HOPG surface. Arrows indicate direction of nanofiber growth. In order to maximize the hydrophobic interactions between the peptide and the HOPG surface, peptide molecules tend to arrange themselves following the HOPG lattice to cover the most carbon atoms. This leads to the orientation of the peptide nano fibers at 60 or 120 degree angles with each other.
  • Figure 44B is an AF micrograph of 10 ⁇ M EAKl 6-11 peptide assemblies on a HOPG surface, showing fiber alignment at 60° and 120° angles, resembling the HOPG lattice shown in Figure 44A.
  • Figure 44C is a schematic of an EAK16-II peptide monomer with an indication of coverage of HOPG lattices based on the dimension of the peptide and the HOPG lattice.
  • Figure 45 is a series of photomicrographs indicating change in amphiphilicity of the surfaces upon the modification with peptides: A) water droplet on mica (no peptide); B) water droplet on EAK-modified mica surface; C) water droplet on HOPG (no peptide); and D) water droplet on EAK-modified HOPG surface.
  • Figure 46A is an AF micrograph and corresponding cross section analysis on a bare HOPG surface.
  • Figure 46B is an AF micrograph showing EAK peptide-modified HOPG surface.
  • Figure 46C is an AF micrograph and corresponding cross section analysis of glucose oxidase (GOx) molecules on a bare HOPG surface. The rough background indicates that the GOx may be denatured on the HOPG surface.
  • Figure 46D is an AF micrograph showing GOx on an EAK peptide-modified HOPG surface. More ball-shape GOx appeared on the modified HOPG surface as compared to Figure 46C, showing that the modified surface provides a more biocompatible environment for GOx adsorption.
  • FIG 47 is a schematic of the chemical reactions that occurs between glutamic acids (E) from EAK peptides and the amine groups of enzymes such as GOx on a modified surface.
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • NHS n- hydroxysuccinimide.
  • Figure 48 is a series of graphs cyclic voltammograms of 1 mM K 3 Fe(CN) 6 depicting the electrochemical characterization of an EFK peptide/HOPG and a bare HOPG electrode for varying scan rates from 2 mV/s to 100 mV/s. At low scan rates, the presence of EAKl 6-11 nanofiber coatings does not block the electron transfer significantly.
  • Figure 49 is a graph of the cyclic voltammetry of a GOx-immobilized EAK peptide/HOPG electrode, plotting current ( ⁇ A) against potential (V) compared to Ag/AgCl at a scan speed of 2 mV/s.
  • Figure 50 is a graph depicting current ( ⁇ A) as a function of potential (V) compared to Ag/AgCl for a GOx/EAK peptide/HOPG electrode (20 mM glucose, 1 cm 2 HOPG).
  • Figure 51A is a graph of GOx-immobilized EAK peptide/HOPG electrode current ( ⁇ A) plotted against time (seconds) with different glucose concentrations (0-20 mM).
  • FIG. 5 IB is a graph of GOx-immobilized EAK peptide/HOPG electrode current ( ⁇ A) plotted against glucose concentration (mM). The data can be fitted using nonlinear regression with the equation below to obtain the I max and the K M ra n.- I A - 1 n nBIaX - maximum current; K m : Michaelis constant for enzyme-substrate complex.
  • Figure 52 is a graph indicating storage stability of a GOx-immobilized EAK peptide/HOPG electrode up to one month.
  • Figure 53 is a graph showing operational stability of a GOx-immobilized EAK peptide/HOPG electrode.
  • Figure 54A is a schematic of a biofuel cell using a GOx-immobilized EAK peptide/HOPG electrode.
  • Figure 54B is a schematic indicating the mechanism of electron (e-) and proton (H + ) transfer of an O 2 /H 2 biofuel cell using the laccase and hydro genase enzymes.
  • Figure 55 is a schematic of a metallic nanowire fabrication based on self-assembly of a modified EAK peptide coupled with electrochemical reduction and enhancement steps.
  • Figure 56 illustrates absorption spectra of siRNA in pH 7.3 HEPES buffer with increasing peptide concentration (from 0 to 40 ⁇ M).
  • Figure 57 illustrates hypochromicity of siRNA at 260 nm as a function of R9 concentration for siRNA concentrations of 1.5 ⁇ M (o), 3.0 ⁇ M ( ⁇ ), and 4.5 ⁇ M ( ⁇ ). Solid lines are the line of best fit generated by Prism. Error bars represent the largest standard deviation from 3 replicates at each siRNA concentration.
  • Figure 58 illustrates hypochromicity of siRNA at 260 nm as a function of +/- charge ratio for siRNA concentrations of 1.5 ⁇ M (o), 3.0 ⁇ M ( ⁇ ), and 4.5 ⁇ M (*). Solid lines are the line of best fit generated by Prism.
  • Figure 59 illustrates CD spectra of 3.0 ⁇ M siRNA (top) and 100 ⁇ M R9 (bottom) in HEPES buffer (6 mM HEPES-NaOH, 20 mM NaCl, 0.2 mM MgC12, pH 7.3). The spectra showed a typical A-DNA structure for the siRNA and random coil structure for R9.
  • Figure 60 illustrates circular dichroic spectra of siRNA in pH 7.3 HEPES buffer with increasing peptide concentration (from 0 to 40 ⁇ M). (from top to bottom)
  • Figure 61 illustrates the relative change in ellipticity of siRNA at 260 nm as a function of R9 concentration for siRNA concentrations of 1.5 ⁇ M (o) and 3.0 ⁇ M ( ⁇ ).
  • Solid lines are the line of best fit generated by Prism.
  • Figure 62 illustrates the relative change in ellipticity of siRNA at 260 nm as a function of +/- charge ratio for siRNA concentrations of 1.5 ⁇ M (o) and 3.0 ⁇ M ( ⁇ ). Solid lines are the line of best fit generated by Prism.
  • Figure 63 illustrates hydrodynamic diameter and Zeta potential of CTGF siRNA-R9 complexes at 1.5 ⁇ M siRNA. Zeta potential of siRNA and siRNA-R9 complexes is expressed in solid bars; Zeta potential of R9 is represented by diagonal bar; and size is represented by a solid line. Error bars represent the standard deviation from 3 replicates.
  • Figure 64 illustrates absorbance at 260 nm of siRNA (1.5 ⁇ M) and siRNA-R9 (1.5 ⁇ M / 150 ⁇ M) complex solution upon 2 M salt addition.
  • the absorbance of siRNA only and siRNA-R9 complex solutions are monitored prior to salt addition (white), 2 hours after salt addition (diagonal), and one day after salt addition (black). Error bars represent the standard deviation from 3 replicates.
  • Figure 65 illustrates calculated binding isotherm for CTGF siRNA-R9 complexes. Following the analysis developed by Bujalowski and Lohman, the calculated free peptide concentrations were infeasible.
  • Figure 66 illustrates the molecular structure of EAKl 6-II: AEAEAKAKAEAEAKAK; A is alanine, E glutamic acid and K lysine.
  • Figure 67 illustrates the ellipticine fluorescence from the peptide-ellipticine suspension over time.
  • A Fluorescence spectra of ellipticine as a function of time;
  • B the normalized fluorescence intensities at 468 nm (diamonds) and 520 nm (squares) as a function of time.
  • the ellipticine concentration is 1.0 mg/mL and the peptide concentration is 0.2 mg/mL.
  • Figure 68 illustrates static light scattering of 0.2 mg/mL EAKl 6-11 solution at 400 nm before (diamonds) and after mechanical stirring for 30 h (squares).
  • Figure 69 illustrates the effect of peptide concentration on the complex formation.
  • the ellipticine concentration was fixed at 1.0 mg/mL with various EAKl 6-11 concentrations ranging from 0 to 0.5 mg/mL.
  • Figure 70 illustrates the effect of ellipticine concentration on the complex formation.
  • the 0.2 and 0.5 mg/mL EAKl 6-11 were used with different ellipticine concentrations from 0.1 to 1.0 mg/mL.
  • Figure 71 illustrates the fluorescence spectra of the peptide-ellipticine suspensions after 24 h stirring with 0.1 mg/mL ellipticine and various peptide concentrations of 0- 0.5 mg/mL. Inset indicates the crystalline ellipticine fluorescence.
  • Figure 72 illustrates the effect of ellipticine concentration on the complex formation.
  • (A) The time-dependent ellipticine fluorescence showing the release of ellipticine from the complex made of 0.05 mg/mL EAK 16-11 and 0.1 mg/mL ellipticine into the EPC vesicles.
  • (B) The transfer profiles of ellipticine from different peptide-ellipticine complexes to the EPC vesicles.
  • the complexes were made of 0.1 mg/mL ellipticine with various EAKl 6-11 concentrations: 0.05 (triangles), 0.1 (crosses), 0.2 (squares) and 0.5 mg/mL (circles).
  • the solid lines represent the fitting curves to the data points using either Equation 2 or Equation 3.
  • the excitation and emission wavelengths are 295 and 436 nm, respectively.
  • Figure 73 SEM images of the peptide-ellipticine complexes with 0.1 mg/mL ellipticine and different EAKl 6-11 concentrations: (a) 0.5 mg/mL, (b) 0.2 mg/mL and (c) 0.05 mg/mL.
  • Figure 74 Viability of MCF-7 and A549 cells treated with the complexes for 24 h at different peptide-to-ellipticine ratios (a) and upon serial dilution (b). The complex at 5: 1 ratio was used for the serial dilution.
  • the complexes were prepared with a fixed ellipticine concentration of 0.1 mg/mL with various EAKl 6-11 concentrations of 0.02- 1.0 mg/mL.
  • Figure 75 SEM images of the peptide-ellipticine complexes with 0.1 mg/mL ellipticine and different EAKl 6-11 concentrations: (a) 0.5 mg/mL, (b) 0.2 mg/mL and (c) 0.05 mg
  • Figure 75.2 Fluorescence images showing cellular uptake of ellipticine at 37 0 C and 4 0 C in A549 and MCF-7 cells with different treatments. Green color represents ellipticine fluorescence. First column shows phase contrast images, and the insets are the corresponding fluorescence images. Figure 76. Intensity-based size distribution of the EPC vesicles.
  • FIG 77 Photographs of the peptide-ellipticine suspensions after 24 h stirring with 0.1 mg/mL ellipticine and various peptide concentrations of 0-0.5 mg/mL.
  • Figure 78 (a) Calibration curve of various ellipticine concentrations in the EPC vesicles, (b) Corresponding UV absorption of ellipticine in (a).
  • Figure 79 illustrates relative UV-vis absorbance change ( ⁇ OD r ) over time of the dilute-5 (solid) and dilute-10 (crosshatch) solutions generated by mixing 8.6 ⁇ M dGi ⁇ with: (A) 10.5 ⁇ M EAK16IV at pH 4; (B) 48.2 ⁇ M EAKl 6IV at pH 7; (C) 24.1 ⁇ M EAK16II at pH 4; and (D) 60 ⁇ M EAK 1611 at pH 7.
  • Figure 80 illustrates (A) the absorption spectra of solution made of 5.0 ⁇ M dCj ⁇ and 120 ⁇ M EAK16II at pH 4. The supernatant of the solution after centrifugation (x); The solution was dried, resuspended in pH 11 buffer, and vortexed. Before (D) and after (-) centrifuging the resulting solution. (B) Relative UV-vis absorbance change ( ⁇ OD r ) as a function of time for the sample obtained by resuspending aggregates made of 8.6 ⁇ M dCi 6 and 0.1 mg/mL EAK16-IV at pH 4 (o) and pH 7 (•) in pH 9.5 buffer.
  • Figure 81 illustrates the fluorescence spectra of an 8.6 ⁇ M Fl-dCi 6 -Rh solution with orwithout 60 ⁇ M EAKl 6IV at pH 4.
  • the solutions at pH 4 were diluted 10 times with pH 4 buffer and kept at 25 0 C for 1 day.
  • the EAK-ODN aggregates were then incubated with 0.7 U/ ⁇ L exonuclease I: (A) without EAKl 6IV: 20 min ( ⁇ ), 30 min (+), and control, i.e., without the nuclease treatment (o); (B) with EAKl 6IV: 20 min (+), 60 min ( ⁇ ), 90 min (-), and non-treated control (x).
  • the solution was centrifuged 1 day after preparation.
  • Figure 82 is a calibration curve correlating the normalized /D//A ratio to the percentage of degraded ODNs.
  • the /D//A ratio was normalized to that of the intact Fl- dCi 6 -Rh.
  • Figure 83 illustrates (A) the effect of the EAK peptide sequence on ODN protection against nuclease degradation.
  • Figure 84 illustrates (A) the fluorescence spectra of the EAK-ODN aggregates prepared with 8.6 ⁇ M of FWCi 6 -Rh and lO ⁇ M of EAK16IV at pH 4. control (x), nuclease-treated 30 min and the solution was centrifuged one day after sample preparation ( ⁇ ). (B) the percentage of degraded ODN in the EAK-ODN aggregates as a function of EAK16IV concentration.
  • Figure 85 illustrates the effect of centrifugation on the nuclease resistance of the EAK-ODN aggregates made of 8 ⁇ M FWCi 6 -Rh and 60 ⁇ M EAKl 6IV at pH 4: (A) 30 min after sample preparation, the solution was (D) or was not (•) centrifuged and then diluted 10 times. 24 h later, the resulting solution was incubated with exonuclease I. The percentage of degraded ODN was recorded over time; (B) 30 min after preparation, the solution was centrifuged before it was diluted 10 times.
  • Figure 87 illustrates AFM images of the peptide nanostructures: (a) EAKl 6-II; (b) EAKl 6-IV; (c) EFKl 6-II.
  • the peptide concentration is 0.5 mg/mL.
  • the scale bar is 200 urn.
  • Figure 88. The hydrophobicity of the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 and their assemblies by dynamic surface tension (a) and ANS fluorescence (b).
  • the inset is the ANS fluorescence control with the absenceof peptides.
  • the peptide concentration is 0.5 mg/mL, and the ANS concentration is 10 ⁇ M.
  • Figure 89 illustrates the formation of peptide-ellipticine complexes
  • (a) are photographs of the complexes with the three peptides at different peptide concentrations and the ellipticine in pure water as a control.
  • the insets show the spectra of the complexes with low peptide concentrations.
  • Figure 90 illustrates the maximum suspension (%) of ellipticine in aqueous solution stabilized by the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 and with the absence of peptides.
  • Figure 91 illustrates the size distribution of the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at 0.5 mg/mL in pure water (a) and the complexes with 0.5 mg/m
  • Figure 92 is SEM images of the complexes with the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at different peptide concentrations and ellipticine crystals in pure water as the control.
  • Figure 93 illustrates the cellular toxicity of the peptides EAK16-II, EAK16-IV and
  • the viability of non-treated cells is 1 (M: cells were treated with culture medium).
  • M cells were treated with pure water (dark green bar);
  • drug control cells were treated with ellipticine in pure water with the absence of peptides
  • Figure 94 illustrates cellular toxicity of the complexes formulated with the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at a peptide concentration of 0.5 mg/mL and their serial dilutions in water for A549 cells (a) and MCF-7 cells (b).
  • EPT ellipticine.
  • Figure 95 illustrates the mass spectrum of EAK16-II.
  • Figure 96 illustrates the mass spectrum of EAKl 6-IV.
  • Figure 97 illustrates the mass spectrum of EFK16-II.
  • Figure 98 illustrates HPLC data of EAKl 6-II. The purity of the peptide is around
  • Figure 99 illustrates HPLC data of EAKl 6-IV. The purity of the peptide is around
  • the present invention concerns peptides that self-assemble into various nanostructures. These peptides are designed based on the capacity of the individual amino acids to participate in hydrogen bonding, electrostatic, hydrophobic and van der Waals' interactions.
  • the resulting self assembled nanostructures can be used in a variety of technological or biomedical functions, including drug delivery and solubility, biosensors and biofuel cells.
  • amino acids are defined as any of the 20 essential amino acids. These include those that are naturally occurring as well as non-natural amino acids such as D-forms, ⁇ and ⁇ derivatives.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as follow: alanine (Ala, A); arginine (Arg, R); asparigine (Asp, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamine (GIn, Q); glutamic acid (GIu, E); glycine (GIy, G); histidine (His, H); isoleucine (He, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (VaI, V).
  • peptide refers to a chain of amino acids.
  • the peptide is from 2 to 40 amino acids in length.
  • self-assembly refers to the process of atoms, molecules or peptides forming regular shaped structures in response to general conditions in the environment.
  • self-assembly refers to the aggregation of peptides into an ordered structure.
  • self-assembling peptide refers to a peptide that can interact noncovalently with another peptide, of the same or different amino acid sequence to form an organized structure, under near thermodynamic equilibrium conditions.
  • hydrogen bonding refers to chemical bonding in which a hydrogen atom of one molecule is attracted to an electronegative atom, especially a nitrogen or an oxygen, usually of another molecule. Hydrogen bonding occurs between amino acids of peptides and, more particularly, hydrogen bonding occurs between atoms of the amino acids that form the peptide backbone. However, hydrogen bonding between atoms in the amino acid side chains contributes to the stabilization and induces assembly of the peptide. Side chain interactions are more important in shorter peptides.
  • ⁇ -strand refers to a single continuous stretch of amino acids adopting an extended conformation and involved in hydrogen bonding.
  • ⁇ sheet refers to an assembly of ⁇ -strands that are hydrogen- bonded to each other, ⁇ -strands arrange to form ⁇ -sheets in parallel or anti-parallel arrangement.
  • a determination of parallel or anti-parallel conformation is based on the arrangement of the peptide direction from N to C terminus. In parallel arrangement, all peptides are aligned in the same direction from N to C terminus. In anti-parallel conformation, alternating peptides are aligned in opposite direction (i.e.
  • the first peptide is aligned N to C terminus relative to a second peptide which is aligned C to N terminus).
  • Parallel arrangement can involve peptide shifting resulting in peptide termini that are staggered relative to one another. At least half the length of the peptide is involved in interpeptide interactions.
  • peptides typically align to provide flush ends. This is typically indicative of end-to-end complementary peptides.
  • ionic-complementarity refers to the characteristic of an alternating arrangement of negatively and positively charged residues in a specific pattern. Typical charge distributions are: type I (-+); type II (—++); and type IV ( — ++++), and any repetitions of these charge distributions or combinations thereof.
  • hydrophobic refers to the tendency of a substance to repel water or to be incapable of completely dissolving in water.
  • hydrophilic refers to the property of being able to readily absorb moisture and having strongly polar groups that readily interact with water.
  • amphiphilic refers to the tendency of a substance to have both hydrophobic and hydrophilic properties.
  • Amphiphilic peptides consist of polar, water-soluble amino acids (hydrophilic) and nonpolar, water-insoluble hydrocarbon amino acids (hydrophobic) arranged in a specific sequence so that the peptide molecule has distinguishable hydrophilic and hydrophobic regions.
  • the te ⁇ n "functional moiety" as used herein refers to an additional, short peptide sequence that confers some particular biological activity to the peptide, and is capable of associating with other molecules/materials.
  • functional moieties include molecular and cell recognition sequences, cell membrane penetration sequences, and metal ion binding motif. Functional moieties confer specific function to peptides, however, they do not necessarily contribute to peptide self-assembly.
  • the term "cell targeting moiety” as used herein refers to a short peptide sequence that can selectively associate with specific cells. The targeting moiety is capable of binding to a cell surface and preferably comprises a member of a binding pair, for example an antibody or parts thereof (e.g.
  • the targeting moiety comprises a low molecular weight protein such as, but not limited to, lysozyme.
  • Cell targeting moieties are not involved in peptide self-assembly.
  • Nanostructure refers to a structure with arrangement of its parts in the nanometre scale. Nanostructures can form any shape of any one of one (ID), two (2D), or three dimensions (3D), including nanosurfaces, nanofibrils, nanorods, nanowires, nanofiber networks, nanospheres, nanospirals, and combinations thereof. Nano textured surfaces have one dimension on the nanoscale, i.e., only the thickness of the surface of an object is between 0.1 and 100 nm. Nanotubes have two dimensions on the nanoscale, i.e., the diameter of the tube is between 0.1 and 100 nm; its length could be much greater. Finally, spherical nanoparticles have three dimensions on the nanoscale, i.e., the particle is between 0.1 and 100 nm in each spatial dimension.
  • oligonucleotide refers to a molecule composed of 30 or fewer nucleotides.
  • the term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), modified or unmodified.
  • RNA may be in the form of small nuclear RNA (snRNA), microRNA (miRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), antisense RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), and ribozymes.
  • the oligonucleotides may be single stranded or double stranded.
  • biosensor refers to a device that detects, records, and transmits information regarding a physiological change or process.
  • a biosensor uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds, usually by electrical, thermal or optical signals.
  • biofuel cell refers to a device that transforms raw materials into electrical power in the presence of biocatalysts, enzymes, or whole cell organisms.
  • biomaterials such as self-assembling peptides, participate in biofuel cell activity by providing a biocompatible environments for enzyme immobilization and potentially improving the electron transfer between the fuel substrates and oxidizers and the electrodes.
  • therapeutic agent refers to any compound or composition for treating a disease or condition and includes, without being limited thereto, drugs, small protein molecules and oligonucleotides.
  • therapeutic agents include, but are not limited to, anticancer agents including paclitaxel, ellipticine, camptothecin, doxorubicin and adriamycin, and oligonucleotide-based agents. Hydrophobic and hydrophilic agents are included in the definition.
  • stabilizing agent refers to the materials that are introduced to a peptide-drug complex to enhance the complex stability in vivo. It will be appreciated by one skilled in the art that the stabilizing agent will be chosen in order to avoid detrimental interaction or reaction with other components in a composition, or reduce the solubility of pharmaceutically active agent.
  • stabilizing agents for use with the present invention include fatty ester of glycerol, fatty ester of polyethylene glycol (PEG), fatty ester of propylene glycol, fatty acid,
  • pharmaceutical excipient refers to an inactive substance used as a carrier for the active ingredients of a medication.
  • pharmaceutical excipients may suitably be selected from one or any combination of dextrose, sorbitol, mannitol, starch, dextrin, maltodextrin, lactose, magnesium stearate, calcium stearate, talc, microcrystalline cellulose, hydroxypropylmethylcellulose and hydroxyethylcellulose. Other appropriate excipients may be used.
  • biocatalyst refers to an enzyme that catalyzes the oxidation/reduction reaction.
  • electrode refers to an electrically conductive material that transfers electrons to or from the reduction/oxidation reaction center.
  • the term “mediator of electron transfer” as used herein refers to a molecule that helps transfer electrons from the electrochemical reaction center to the electrode
  • fuel source refers to the materials that can undergo oxidation/reduction reaction to generate electronic current.
  • Self-assembling peptides designed based on amino acid pairing properties may be used to form a wide variety of nanostructures.
  • An Amino Acid Pairing (AAP) strategy of the present invention for designing self-assembling peptides is based on the ability of amino acids to interact with one another through at least one of ionic complementarity, hydrogen bonding, hydrophobic and van der Waals' interactions ( Figure 1).
  • the AAP-based peptide design provides complementary interactions that achieve certain stereochemical and physicochemical stability, resulting in pair affinity and minimum pairing free energy.
  • asparagine-asparagine (asn-asn or N- N) pairs are possible and can be included in the peptide sequence due to the ability of Asn to participate in hydrogen bonding interactions both as a proton donor and as a proton acceptor.
  • Nanostructures form when self-assembling peptides approach each other and undergo pairing interactions between complementary amino acids, typically forming antiparallel or parallel ⁇ -sheet secondary structures.
  • amino acids acting as proton acceptors include arginine, tiytophan, tyrosine, lysine, histidine, aspartic acid, threonine, cysteine, serine, asparigine, glutamic acid, methionine and glutamine. All of these amino acids, except for methionine, also act as proton donors. Bonding pairs were identified based on the positions of the proton acceptor and proton donor within the amino acid. Bonding pairs were further classified as being more soluble or less soluble. More soluble hydrogen bonding amino acids that function as proton acceptors at the 3 position are aspartic acid and histidine.
  • Aspartic acid is capable of hydrogen bonding with 3 -position proton donor amino acid serine while histidine is capable of hydrogen bonding with threonine.
  • 4-position proton acceptors tryptophan, histidine and glutamine hydrogen bond with proton donor aspartic acid, while arginine and glutamic acid hydrogen bond with 4-position proton donor asparigine.
  • Less soluble hydrogen bonding amino acid pairs include: asparigine (3- position proton acceptor) and threonine, serine or cysteine; methionine (3 -position proton acceptor) with cysteine or serine; 4-position proton acceptor glutamine with asparagine; and 5-position proton acceptor tyrosine with glutamine or tryptophan as indicated in Table 4.
  • Peptides containing less soluble hydrogen bonding amino acid pairs from 2-8 amino acids in length (from 1 to 4 amino acid pairs), can be synthesized.
  • Exemplary peptides consist of glutamine (Q)-asparagine (N) or asparagine (N)-serine (S) amino acid pairs.
  • Peptides may be subjected to end protection consisting of acetylation at the amino terminus and amidation at the carboxy terminus.
  • Peptides may have a varying number of amino acids, as well as varying numbers of contiguous hydrogen bonding pairs.
  • single pair peptides can be synthesized (e.g. QN, NS) as well as double (e.g. QNQN and NSNS or QQNN) and quadruple pairs (e.g. NSNSNSNS).
  • peptides having amino acids that participate in all of ionic pairing, hydrophobic pairing, and hydrogen bonding can be synthesized. Alternating hydrophobic and hydrophilic amino acid residues contribute to the amphiphilicity of the peptide.
  • the invention provides a method of preparing a self- assembling peptide having amino acid pairing properties for manufacture of a nanostructure.
  • the method includes the steps of: designing a ⁇ -strand peptide consisting of amino acids that are capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction with a complementary amino acid; and generating a peptide from two to forty amino acids in length consisting of at least one amino acid pair capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction, and having complementary amino acid pairing and stereochemistry with a second peptide.
  • FTIR Fourier Transform Infrared
  • CD Circular Dichroism
  • ThT fluorescence spectra may be used to analyze the secondary structure of the peptides.
  • FTIR spectra can be useful in indicating the ability of the peptides to form ⁇ -sheet structures while ThT fluorescence can be used to indicate ⁇ -sheet rich peptide assemblies.
  • the invention provides a self-complementary ⁇ -strand peptide having alternating hydrogen bonding proton donor amino acid segments and hydrogen bonding proton acceptor amino acid segments, that self assembles into a nanostructure.
  • the peptide has a length from two to forty amino acids.
  • the peptide has at least one proton donor and one proton acceptor segment, each of which consists of at least one amino acid.
  • Such peptides are not comprised of alternating hydrophobic and hydrophilic amino acid segments.
  • a /3-strand peptide of the present invention further includes at least one functional moiety at at least one of an N-terminus and C- terminus of the peptide.
  • the functional moiety is selected from a cell targeting moiety, a metal ion binding motif and a cell membrane penetration moiety.
  • the hydrogen bonding occurs between the side chains of complementary amino acids of the peptides of the present invention.
  • the complementary peptides assemble into a parallel confo ⁇ nation.
  • the complementary peptides assemble into an anti-parallel conformation.
  • the peptides can assemble in either an end-to-end or staggered peptide arrangement.
  • peptides of the present invention can assemble into a staggered arrangement, wherein between one and 20 amino acids in the peptide form hydrogen bonds with complementary amino acids in a second peptide.
  • the hydrogen bonding proton donor amino acid for a ⁇ - strand peptide of the present invention is selected from the group consisting of Arg, Trp, Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu and GIn.
  • the hydrogen bonding proton acceptor amino acid for a /3-strand peptide of the present invention is selected from the group consisting of Arg, Trp, Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu, Met and GIn.
  • the present invention provides a self-complementary ⁇ - strand peptide comprising at least one hydrogen bonding amino acid pair, at least one ionic-complementary amino acid pair, and at least one hydrophobic amino acid pair, and having a length from four to forty amino acids, for forming a nanostructure.
  • this peptide has the formula: (A w B x A y C z ) n A a B b (V) where A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10.
  • the hydrophobic amino acid is selected from the group consisting of VaI, He, Leu, Met, Phe, Trp, Cys, Ala, Tyr, His, Thr, Ser, Pro, GIy, Arg and Lys.
  • the charged amino acid is selected from the group consisting of His, Arg, Lys, Asp and GIu.
  • the present invention includes an amino acid sequence Phe-Glu-Phe-Gln-Phe-Asn-Phe-Lys (AC8) (SEQ ID NO: 6). The invention further includes self-assembled nanostructures of this peptide.
  • the invention provides a self-complementary ⁇ -strand peptide having one of the following structures: a) (A x B y C z ) w A z (I), and b) (A x B y C z ) w (C' x B' y A' z ) w (II)
  • A, A', B, B', C and C are each a hydrogen bonding amino acid, and are either a proton donor or a proton acceptor amino acid; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20.
  • A is complementary to A', B is complementary to B', and C is complementary to C.
  • the invention provides a self-complementary ⁇ -strand peptide having one of the following structures: a) A x B y C z ... ; and (III), and b) A x B y C z ...C z 'B y 'A x ' (IV).
  • A, A', B, B', C and C are each independently a donor amino acid or an acceptor amino acid, and are each self-complementary. These amino acids are further selected from the group consisting of a hydrogen bond donor amino acid, a hydrogen bond acceptor amino acid, a positively charged amino acid, a negatively charged amino acid, and a van der Waals' interacting amino acid.
  • A is complementary to A'
  • B is complementary to B'
  • C is complementary to C.
  • the invention provides a self-complementary ⁇ -strand peptide having at least one hydrogen bonding amino acid pair, at least one ionic- complementary amino acid pair, and at least one hydrophobic amino acid pair, for forming a nanostructure.
  • the peptide has a length from four to forty amino acids.
  • 3-strand peptides of the present invention include GIn- Asn, Gln-Asn-Gln-Asn (SEQ ID NO: 1), Gln-Gln-Asn-Asn (SEQ ID NO: 2), Asn-Ser, Asn-Ser-Asn-Ser (SEQ ID NO: 3), Asn-Ser-Asn-Ser-Asn (SEQ ID NO: 4), and Asn- Ser-Asn-Ser-Asn-Ser-Asn-Ser (SEQ ID NO: 5).
  • the invention further includes nanostructures formed of these peptides.
  • the peptides of the present invention may be used to form various nanostructures including nanofibers and nanofiber networks, nanotubes, nanowires, nanospheres and nanospirals, as well as combinations of these structures.
  • Peptides present at a Critical Aggregation Concentration (CAC) may assemble into various structures in a concentration-dependent manner. At concentrations below the CAC, peptides may form a mixture of single layer protofibrils and monomers while above the CAC, peptides may form mature fibers and fiber bundles, or other nanostructures.
  • the nanostructure formed by the peptide aggregation is dependent on the concentration of the peptide in solution as well as the pH of the solution (reviewed in Chen (2005) Colloids and Surfaces A: Physiochem. Eng.
  • the CAC of a peptide may be determined using ThT fluorescence as well as surface tension, ANS fluorescence, and steady state light scattering assays.
  • the invention provides a self-assembled nanostructure consisting of aggregated units of a peptide having one of the following structures: a) (A ⁇ ByC z ) w I; and b) (A x B y C z ) w A x II.
  • A, B and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20.
  • the nanostructure formed from the self assembled peptides can be one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
  • the invention provides a self assembled nanostructure consisting of aggregated units of a peptide having the general formula (V): (A w B x A y C z ) n A a B b (V).
  • A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10.
  • the nanostructure fo ⁇ ned from the self assembled peptides can be one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
  • the peptides of the present invention can be used in controlled release drug delivery applications.
  • EAKl 6 peptides could be used to encapsulate pyrene, a highly hydrophobic compound ( Figure 2; Keyes-Bag et al. (2004) J. Am. Chem. Soc. 126: 7522-7532). It further demonstrated that the ratio of pyrene to peptide resulted in different surface coatings ( Figure 3).
  • the presently disclosed peptides may be used to solubilize hydrophobic therapeutic agents, such as ellipticine, an anti-cancer drug in its protonated or neutral form. Furthermore, the peptides are useful as drug delivery vehicles.
  • the solubilized neutral ellipticine by the newly designed peptides was more protected and stable inside the peptide vehicles upon 16-fold dilution in water.
  • the peptides may also be used in gene and oligonucleotide delivery applications.
  • the present inventors have also discovered that EAK- 16-1, EFKl 6-11 and EAKl 6-11 can be used to solubilize hydrophobic therapeutic agents and for gene and oligonucleotide delivery applications.
  • compositions of the present invention can include the disclosed peptides and any suitable excipient or stabilizer as is known to a person skilled in the art. Further the mode of administration of the therapeutic agents and pharmaceutical compositions of the present invention are not particularly restricted and suitable modes of administration are within the purview of persons of skill in the art and include without limitation oral administration and intravenous administration.
  • the invention provides a kit for delivering a therapeutic agent to a patient, including a pha ⁇ naceutical composition comprising a self assembled ⁇ -strand peptide and a therapeutic agent; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; instructions for preparing the pharmaceutical composition for use; instructions for mixing the composition with other agents; and instructions for introducing the composition into a subject.
  • a kit for delivering a therapeutic agent to a patient including a pha ⁇ naceutical composition comprising a self assembled ⁇ -strand peptide and a therapeutic agent; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; instructions for preparing the pharmaceutical composition for use; instructions for mixing the composition with other agents; and instructions for introducing the composition into a subject.
  • the peptides of the present invention may assemble on different surfaces, either hydrophobic or hydrophilic in nature. It has been demonstrated that the ⁇ - amyloid peptide (amino acids 1-42) is capable of assembling on different templates including mica (hydrophilic) and HOPG (hydrophobic graphite) (Figure 4; Kowalewski and Holtzman (1999) 96:3688-3693). The size and shape of the ⁇ - amyloid aggregates, as well as the kinetics of their formation, were shown to be dependent on the physicochemical nature of the surface.
  • Amphiphilic peptides may be seeded onto charged, hydrophilic surfaces, such as mica, and elongate into nanofibers in a concentration-dependent manner ( Figures 5, 6).
  • the driving force for the amphiphilic peptide deposited on a charged surface is primarily electrostatic interaction ("peptide-surface interaction").
  • Peptides readily align at both ends of the peptide and "seed” to achieve nanofiber growth.
  • the growth of the nanofiber can be subsequently controlled by adjusting the pH of the solution, shown in the schematic of Figure 7.
  • the same peptides seeded onto a hydrophobic surface such as HOPG can assemble into different nanostructures. For example, EAKl 6 peptides align at 60° and 120° angles to one another.
  • peptides of the present invention can be shaped into specific nanostructures depending on the nature of the surface on which they are seeded. Peptide-modification of a surface can result in changes to the surface wettability. Peptide-modified surfaces may be used for biomolecule sensing applications. Peptides may bind to biomolecules such as enzymes for use in sensing enzymatic substrates, and immobilize the biomolecules onto a surface.
  • the present invention provides a fuel cell.
  • a biofuel cell is a device that generates electricity from a fuel source by an electrochemical process using biocatalysts.
  • Peptides of the present invention can be immobilized onto electrodes where they are bound to biosensory molecules such as enzymes. Enzymatic substrates can be added to drive the catalytic reaction.
  • biofuel cells have strong storage and operational stabilities.
  • the invention provides a use of a ⁇ -strand peptide, for identification of inhibitors of protein aggregation disease.
  • Peptides of the present invention may be used to generate models to study protein conformation diseases, such as Alzheimer's and prion disorders. Similar to the ⁇ -amyloid peptide (1-42 amino acids), peptides of the present invention can be seeded onto various surfaces and studied for assembly as well as for inhibition of assembly.
  • Peptides of the present invention may be used in proteomics and bioinformatics applications. Molecular dynamics simulations can be applied to screen all potential pairs of amino acids that can complementarily interact with each other.
  • Peptides can be used to modify array surfaces where the functional residues of the peptide can immobilize DNA and protein molecules onto the array.
  • the invention provides a method for detecting a biomolecule of interest.
  • the method includes the steps of: forming a nanostructure from a ⁇ -strand peptide upon self assembly of the peptide; adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; coupling a reporter molecule capable of providing a measurable signal to the peptide- coated surface of the nanostructure; and providing the biomolecule of interest.
  • the biomolecule is selected from proteins, nucleic acids, carbohydrates and viruses.
  • the biomolecule of interest is glucose.
  • Peptides of the present invention may be used for various coating applications including: coating for the prevention of biofouling; biocompatible surface coating; and coating for functionalized chromatographic columns. Peptides can be used to modify surface coatings and to provide a biocompatible environment for protein/enzyme immobilization.
  • the sequence of the peptide determines the secondary structure and nanostructure of the self-assembled peptide aggregation, and influences their applicability in drug delivery, chemical sensing, biofuel cells, and models for the study of protein aggregation diseases.
  • Amino acids that participated in hydrogen bonding, and the position of the atoms serving as proton donors and proton acceptors, are given in Table 1.
  • Amino acids capable of pairing are given in Table 2.
  • Soluble and less soluble hydrogen bonding amino acid pairs are listed in Tables 3 and 4, respectively.
  • the existence of repulsion forces results in the resistance of amino acids hydrogen pairing in cases of amino acids that contains a charge (see Table 3). For this reason, non-charged amino acid pairs capable of hydrogen bonding were of particular interest.
  • Q-N and N-S amino acid pairs represented the most hydrophilic amino acid pairs except the pairs involving charged amino acids (E, D, R and K).
  • Peptides were designed and synthesized such that they contained varying combinations of either Q- N or N-S amino acid pairs.
  • the arrangement of hydrogen bonding pairs in a synthesized peptide was varied in terms of the number of repeating pair units (peptide length) and the design of the repeating unit itself.
  • Various peptides designed and evaluated for self-assembly are listed in Tables 5 and 6.
  • NSNSNSNS contained the maximum ⁇ -sheet secondary structure (-50%), as shown in Figures 9 A and 9B.
  • This result was consistent with results of the ThT assay, where the increase in the fluorescence signals of ThT corresponds to an increase in the amount of ⁇ -sheets in the peptide assemblies.
  • the NS4 peptide provided the maximum fluorescence intensity of ThT, indicating the peptide contained maximum content of ⁇ -sheets.
  • the hydrodynamic size of a peptide aggregation in pure water was characterized using a dynamic light scattering approach. The results of the assay are given in Table 7. The average hydrodynamic size of a peptide aggregation was found to be ⁇ 100 nm. The apparent size of the aggregation in pure water indicated that the larger aggregates observed with scanning electron microscopy (SEM) were formed in aqueous solutions rather than on the mica surface.
  • SEM scanning electron microscopy
  • a peptide was designed such that it contained each of hydrogen bonding, electrostatic, and hydrophobic bonding amino acid pairs.
  • the resulting peptide was referred to as AC 8.
  • AC 8 contains 8 amino acids in sequence with one exemplary hydrogen bonding pair (QN), one exemplary ionic-complementary pair (EK), and two hydrophobic residue pairs (FF) ( Figure 13A).
  • Hydrophobic amino acids were incorporated to create a hydrophobic interior for encapsulation and stabilization of hydrophobic compounds ( Figure 13B). These hydrophobic residues also enhanced the peptide-peptide association; the charged residues enhanced the solubility of the peptide and resulting peptide assemblies; and the hydrogen bonding amino acid pairs stabilized the peptide assemblies.
  • the release rates can be controlled by adjusting the peptide-to-pyrene ratio during the formulation (Figure 3) Increased concentration of EAK peptide was shown to increase the solubility of the more protonated ellipticine in pure water ( Figure 20B).
  • the effects of EAK-ellipticine complexes on cancer cells was evaluated in the MCF-7 cell line. Cell viability was not affected by any control treatment ( Figure 20A), however, cell viability was shown to be reduced upon treatment with ellipticine in complex with increasing concentrations of EAK ( Figure 20A).
  • SE micrographs indicated the size of ellipticine-peptide complexes in the presence ( Figures 2OC and D) and absence of EAK peptide ( Figure 20E).
  • Peptides tested for their ability to deliver the hydrophobic anticancer agent ellipticine were: 1) the ionic-complementary peptides EAKl 6-11 (crude -70% and pure > 95% having the amino acid sequence AEAEAKAKAEAEAKAK), EAKKl 6 (crude, having the amino acid sequence AEAEAKAKAKAKAK), EFKl 6-II (crude, having the amino acid sequence FEFEFKFKFEFEFKFK); T) hydrogen bonding-based peptides NS4 (90% pure, amino acid sequence NSNSNSNS), QN (95% pure), QNQN (92% pure), QNQN, and QQNN (92% pure); and 3) peptides containing each of hydrogen bonding, ionic and hydrophobic bonding amino acid pairs including FEQNK (or AC8, 95% pure, having the amino acid sequence FEFQFNFK).
  • the peptide concentrations were fixed at 0.1 mg/mL for complexing with ellipticine.
  • the formulation consisted of 3.3 % (v/v) DMSO in aqueous solution and the steps of: a) dissolving a high concentration of ellipticine in DMSO; b) preparing a 0.1 mg/niL peptide solution in pure water and sonicating for 10 minutes; and c) adding aliquots of ellipticine-DMSO into peptide solutions to achieve a final ellipticine concentration of 0.1 mg/mL (3Ox dilution).
  • Peptide-mediated delivery of ellipticine was tested in two cancer cell lines: the non-small cell lung cancer cell line A549 (1x10 4 cells/well seeded in 96 well plate) and the breast cancer cell line MCF-7 (2x10 4 cells/well seeded in 96 well plate). Cells were treated with the peptide-drag complexes at a final ellipticine concentration of 25 ⁇ g/mL in medium, for 24 hours. Cell viability was determined using the MTT assay.
  • AC 8 peptides were further evaluated for their ability to complex with ellipticine and enhance solubilization of the therapeutic agent. It was shown that increasing concentrations of AC8 resulted in the increase of ellipticine fluorescence at -430 nm as shown in Figure 23A. The results indicate that the AC8 peptides can help stabilize neutral ellipticine in a peptide concentration-dependent manner. When plotting the fluorescence intensities against AC8 concentrations in Figure 23B, it is clearly seen that the dramatic increase in fluorescence occurs at the AC 8 concentration of -20 ⁇ M, which is veiy close to the CAC of the AC8 as described in 0059.
  • EXAMPLE 6 Cytotoxicity of AC8 and EAK peptides in complex with ellipticine Cell viability assays were performed to determine cytotoxicity of peptide- ellipticine complexes over a period of 48 hours. Peptides used in these experiments were EAKl 6-11 and AC8. The results of the assays demonstrated that all of the peptides were effective in killing A549 cells at 48 hours post-treatment ( Figure 25A), and also MCF-7 cells ( Figure 25B). In these experiments, the concentration of ellipticine in the complex was maintained at 0.04 mg/mL and prepared in the absence of DMSO to eliminate the possibility of cytotoxic effects due to DMSO.
  • ellipticine crystals were added to the peptide solutions to give an ellipticine concentration of 0.04 mg/mL. 0.5 mg/mL and 0.1 mg/mL peptides were used to prepare the complex. The final ellipticine concentration in the medium was 25 ⁇ g/mL.
  • a treatment time of 48 hours was shown to be required for efficacy of the ellipticine- AC 8 complexes in killing cancer cells.
  • the peptide-drug complex was still effective in killing cancer cells with equal or slightly higher efficacy compared with the control group.
  • the protocol and the formulation method were then modified accordingly to re-evaluate the peptide concentration effect.
  • the new formulation method was as follows: a) 0.4 mg/mL ellipticine was dissolved in THF; b) aliquots of the ellipticine-THF solution was added to a glass vial, and air applied to evaporate the THF completely; c) 1 or 2 mL of a peptide solution
  • the fluorescence spectra data indicated utility of AC8 peptides in solubilizing neutral ellipticine in aqueous solution. Varying concentrations of AC 8 were evaluated for the ability to complex with ellipticine. Cell viability assays were performed on cells treated with the AC8-drug complexes in order to determine a possible relationship between complex formation and the CAC of AC8. The AC8 concentrations tested were: 0.5, 0.2, 0.1, 0.04, 0.01 and 0.005 mg/mL, the CAC of AC8 being around 0.0115 mg/mL. The toxicity of the complexes was tested in each of the A549 (Figure 27C) and MCF-7 ( Figure 27E) cell lines.
  • the EAKl 6-11 peptide was evaluated for its ability to bind to both single- and double-stranded oligodeoxynucleotides (ODNs), namely a guanine hexadecamer (dGi ⁇ ), a cytosine hexadecamer (dCi 6 ), and their duplex (dGCi ⁇ ).
  • ODNs oligodeoxynucleotides
  • dGi ⁇ guanine hexadecamer
  • dCi 6 cytosine hexadecamer
  • dGCi ⁇ duplex
  • dGi 6 and dCi 6 hexadecamers were chosen to assess how a purine or a pyrimidine affects the binding of a self-assembling peptide to an oligonucleotide.
  • Most therapeutic antisense oligonucleotides and siRNAs are short nucleic acids of less than 22 nucleotides in length, thus, the choice of 16mer ODNs was within the usual therapeutic oligonucleotide length range.
  • Peptide-oligonucleotide complexes were formed using the EAKl 6-11 peptide, in combination with both single- and double-stranded oligoucleotides (ODNs), namely a guanine hexadecamer (dGi ⁇ ), a cytosine hexadecamer (dC]g) and a duplex of the two (dGC )6 ).
  • ODNs single- and double-stranded oligoucleotides
  • the pH 4 buffer was made with 0.171 M acetic acid and 0.029 M sodium acetate adjusted with acetic acid (35);
  • the pH 7 buffer was made with 0.01 M tris(hydroxymethyl)methylamine and 0.005 M sodium sulfate adjusted with sulfuric acid (Akinrimisi et al. (1963) Biochemistry 2: 340-344);
  • the pH 11 buffer was made with 0.1 M glycine and 0.1 M sodium chloride adjusted with sodium hydroxide (Bolumar et al. (2003) Appl. Environ. Microbio.
  • EAKl 6-11 peptide was purchased from CanPeptide Inc. (Quebec, Canada) and the C-terminus carboxyfiuorescein labeled EAK peptide (FAM-EAK) was purchased from Research Genetics (Alabama, USA) and used without further purification.
  • FAM-EAK carboxyfiuorescein labeled EAK peptide
  • FAM-dCi ⁇ (dCi 6 labeled with carboxyfiuorescein at the 5'-end), and dCi6-Rh (dCi6 labeled with carboxytetramethylrhodamine at the 3 '-end) were obtained with 95 % purity from Eurogentec North America (San Diego, USA) with HPLC purification.
  • the ODN sequences are listed in Table 8.
  • dsODNs double-stranded ODNs
  • Table 8 Type, name, and sequence of oligonucleotides (ODNs) and self- assembling peptide.
  • UV- Vis absorption spectra were obtained on a Hewlett-Packard 8452A diode array spectrophotometer (California, USA) using a 50 ⁇ L quartz cuvette from Hellma (M ⁇ llheim, Germany).
  • Samples for the construction of binding isotherms were prepared at pH 4, 7, and 11. For each pH, two ODN concentrations of about 3 ⁇ M and 7 ⁇ M were used. At each ODN concentration, six to seven samples were prepared with EAK concentrations ranging from 0 to 0.2 mg/mL (equivalent to 0-120 ⁇ M) by mixing the ODN solution with different amounts of EAK powder. The resulting solutions were stirred vigorously for a few seconds with a vortex mixer and incubated at 25 0 C for 30 mins. The EAK-ODN aggregates formed in the solution were removed by centrifugation at 14,000 rpm for 2 minutes with a Centrifuge 5410 from Eppendorf (Hamburg, Germany).
  • the supernatant was collected and its absorbance was measured on the spectrophotometer at wavelengths between 190 and 800 nm. Beer's Law was used to determine the total ODN concentration and the concentration of the ODN left in the supernatants from the absorbance of the ODN at 260 nm of the initial solution (OD 0 ) and the supernatant (OD S ), respectively.
  • the term (OD 0 -OD S )/OD 0 was defined as the relative UV- Vis absorbance change AOD 1 -.
  • the obtained AOD 1 - were then analyzed using the ligand binding density function (23) to generate binding isotherms.
  • Fluorescence spectra of dCi ⁇ labeled with carboxyfluorescein at the 5 '-end (FAM-dCi ⁇ ), dCi6 labelled with carboxytetramethylrhodamine at the 3 '-end (dCi 6 - Rh), and EAK labeled with carboxyfluorescein at the C-terminus (FAM-EAK) were acquired on a Photon Technology International steady-state fluorometer (New Jersey, USA) equipped with a Ushio UXL-75Xe Xenon arc lamp and PTI 814 photomultiplier detection system.
  • the peak absorption wavelength of the solution was chosen as the excitation wavelength ( ⁇ ex ).
  • Fluorescence decays were acquired by the time-correlated single photon counting technique on a time-resolved fluorometer (IBH system 2000, Glasgow, UK). Samples containing 3.6 ⁇ M of the chromophore-labeled dCi 6 were prepared in the presence and the absence of 0.2 mg/mL (120 ⁇ M) EAK at pH 4. The excitation wavelength (l ex ) and emission wavelength ( ⁇ em ) were set to the wavelength corresponding to the absorption and emission maxima of the chromophores.
  • Xe x and ⁇ em were 452 nm and 514 nm, respectively; for dCig-Rh, ⁇ ex and ⁇ em were 560 nm and 580 nm, respectively.
  • a right angle configuration was used between the excitation and emission monochromators.
  • AU decay curves were collected over 512 channels and with a total of 20,000 counts in the channel of maximum intensity. The analysis of the decay curves started by acquiring the instrument response function obtained with a scattering solution, which was then convoluted with a sum of exponentials shown in Equation 2 (Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy. Plenum Publisher, New York):
  • SLS Steady-State Light Scattering
  • DLS Dynamic Light Scattering
  • a Picoscan atomic force microscope (Molecular Imaging, Arizona, USA) was used to study the morphology of the EAK-ODN complexes/aggregates in solution. It was operated in magnetic AC (MAC) tapping mode in solution using magnetically coated cantilevers, Type II MAClevers (Molecular Imaging, Arizona, USA), with a spring constant of 0.5 N/m and a resonance frequency of ⁇ 27 kHz at room temperature. A volume of 400 ⁇ L of each solution was deposited on a freshly cleaved mica surface, inside a Teflon liquid chamber, where the AFM images were acquired.
  • MAC magnetic AC
  • the MvH model described the initial complexation process only, and did not take into account the second aggregation step. However, the MvH model was determined to be useful for the following reasons: first, a goal of the study was to find differences or similarities in the process leading to the formation of EAK-ODN aggregates under different experimental conditions, e.g., pHs and nucleotide types. Second, the binding constant K calculated from the MvH model showed a trend for the binding strength of EAK to different ODNs and under various solution conditions, which are also comparable to those reported for the binding of other DNA/peptide pairs. With these considerations, the binding isotherms (Figure 29) were fitted with EQN 5. Effect of pH on Binding The binding of EAK to dCi ⁇ , dGig, and dGCi 6 was monitored at pH 4, 7, and
  • AOD r increased with increasing EAK concentration at pH 7 for both the ssODNs and the dsODN; however, it required higher EAK concentration to reach the plateau. Furthermore, at a given ODN concentration, the AOD r values at pH 7 were significantly lower than those at pH 4 suggesting that increasing the pH from 4 to 7 resulted in a much weaker binding between EAK and the ODNs. At pH 11, no binding of EAK to the ODNs was detected as the EAK concentration was varied from O to 120 ⁇ M and AOD 1 - equaled zero.
  • the AOD r values for dGi6 were higher than those for dCi 6 at a given EAK concentration, suggestive of the fact that more EAK binds to dGj ⁇ than to dQg.
  • the A0D r values for the ssODNs were consistently higher than those obtained for the dGCi 6 duplex at the same EAK concentration, suggesting that EAK molecules bind more strongly to the ssODNs than to the dsODN.
  • Nature of the ODNs Remaining in the Supernatant after Centrifugation The fraction of the ODN in the EAK-ODN aggregates was determined from the relative UV absorbance change of the solution, A0D r .
  • Fluorescence anisotropy reflected changes in the rotational correlation time of the chromophore, which was related to the hydrodynamic volume of the species to which the chromophore was attached.
  • Solutions containing 3.6 ⁇ M of FAM-dGCi6 or of FAM-dCi 6 were mixed at pH 4 and pH 7 with EAK concentrations ranging from O to 0.2 mg/mL (0-120 ⁇ M). The solutions were centrifuged and the fluorescence anisotropy of the supernatants was measured. The anisotropy of the supernatants was plotted in Figure 31 as a function of EAK concentration.
  • the fluorescence anisotropy of FAM-labeled ODNs equaled 0.11 ⁇ 0.01 and 0.04 ⁇ 0.01 at pH 4 and pH 7, respectively.
  • the anisotropy of the FAM-labeled ODN species remaining in the supernatants ranged from 0.08 to 0.12 and 0.03 to 0.04 at pH 4 and pH 7, respectively, close to that of the FAM-labeled ODN, demonstrating that unimers of ODN existed in the centrifuged solutions; no EAK-ODN aggregates were detected.
  • UV- Vis absorption experiments were performed to demonstrate the existence of EAK-ODN complexes in the supernatant that were not detected in the anisotropy experiments.
  • the absorption spectra of the samples containing 3.9 ⁇ M of dCi 6 -Rh in the presence and absence of 60 ⁇ M EAK were acquired before and after centrifugation.
  • the absorption spectrum of a mixture of dCi 6 - Rh and EAK was different from that of free dC I6 -Rh.
  • Mixing dC I6 -Rh with EAK induced a decrease in the absorbance at the 563 nm band characteristic of dCi 6 -Rh, and a new prominent absorption band at 524 nm.
  • the ratio of absorbance at 524 and 563 nm (OD524/OD563) for dCi ⁇ -Rh was 0.50 ⁇ 0.01 and was concentration- independent as shown in Figure 32B. This ratio changed to 0.89 and 0.78 after mixing with EAK, before and after centrifugation, respectively.
  • EAK-ODN aggregates To identify the pathway leading to the formation of EAK-ODN aggregates, the time scale over which EAK self-assembled in solution was estimated. To this end, the fluorescence anisotropy of EAK and EAK-dG] 6 mixtures was measured at pH 4 and 7 over a one hour period immediately after sample preparation using carboxyfluorescein-labeled EAK (FAM-EAK). These experiments were performed with 0.1 mg/mL (60 ⁇ M) EAK solutions where 1 in 100 EAK molecules was fluorescently labeled. To these solutions, 5 ⁇ M of dGi 6 was added.
  • the hydrodynamic diameter of the EAK-ODN aggregates in solution was obtained by DLS.
  • the hydrodynamic diameter of the EAK-dGi 6 aggregates at pH 4 with varying concentrations of EAK was measured 30 min after sample preparation ( Figure 37).
  • the dGi 6 solutions at 7.2 ⁇ M exhibited a species with a hydrodynamic diameter of ⁇ 7.5 nm which was attributed to isolated ODNs in solution.
  • the diameter of the species present in the dG] 6 solution increased to around 150 nm upon addition of EAK and remained constant as the EAK concentration was increased from 0.01 to 0.04 mg/mL (6-24 ⁇ M) suggesting that EAK bound first to ODN molecules, followed by aggregate formation.
  • Equation 5 was used to obtain the binding constant, K, and the binding site size, n.
  • K and n values obtained for the binding of EAK to the ODNs at various pH values are listed in Table 10.
  • the "equilibrium constants" obtained for the binding of EAK onto the ODNs ranged from 7.0 x 10 3 to 7.6 x 10 4 M "1 .
  • ODNs located inside the aggregates would be less accessible to the solvent than those located on the surface, and hence be protected from the outside environment.
  • the accessibility of the ODN to the solvent was measured by performing fluorescence dynamic quenching experiments.
  • the fluorescence emission of a solution containing 3.6 ⁇ M of the labeled dCi 6 was monitored as the quencher KI was added to the solution in the presence or absence of 0.2 mg/mL EAK.
  • the potassium ion concentration was maintained constant and equal to 0.3 M by addition of K 2 SO 4 to the KI solution, ensuring constant ionic strength.
  • the I 0 II ratio was plotted as a function of iodide concentration in Figures 38A and B for fluorescein and rhodamine, respectively.
  • the quantities I 0 and / represent the fluorescence intensity of the chromophore without and with quencher, respectively.
  • the I 0 II ratio was found to increase linearly with iodide concentration for both cliromophores in the absence or presence of EAK. However the increase was stronger in the absence of EAK.
  • the fluorescence decays of the FAM- dC] 6 -EAK mixture were acquired in the absence and presence of 0.2 M KI.
  • the IJI ratio obtained from the steady-state fluorescence measurements equaled 1.9 for a concentration of 0.2 M KI, a value comparable to the ⁇ j ⁇ ratio. Fitting the I 0 II vs.
  • k q is determined from the ratio Ksv/ ⁇ o where T 0 is the lifetime of the chromophore in the absence of quencher (Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy. Plenum Publisher, New York)
  • the average lifetime ⁇ 0 was used to calculate the bimolecular quenching rate constant k q from the slope Ksv of the plots in Figure 38.
  • the values of Ksv, T 0 , and k q are listed in Table 12.
  • k q for FAM-dCi6 and dCi 6 -Rh equaled 2.5x lO 9 JVT 1 S "1 and 3.5 ⁇ lO 9 MV 1 , respectively.
  • the bimolecular quenching rate constant was related to the quenching efficiency, size, and diffusion coefficient of the chromophore and quencher.
  • k q decreased from 2.5x lO 9 M ⁇ V to 1.5x lO 9 M -1 S "1 .
  • k q decreased from 3.5X lO 9 MT 1 S “1 to 2.Ox IO 9 M- 1 S "1 .
  • Y K 1 K (EQN 7) where k q b and k/ are the bimolecular quenching constants for the chromophore- labeled ODNs in the presence and absence of EAK, respectively.
  • the values of ⁇ for the two different labels are listed in Table 12.
  • the relative accessibility changes of dCi 6 for both types of labels were similar and smaller than unity, being 0.60 and 0.57 for the 5 '-labeled FAM-dCi ⁇ and the 3 '-labeled dCi 6 -Rh, respectively. Since ⁇ is smaller than 1.0, the accessibility of the 5'- and 3 '-ends of dCj 6 to the solvent was reduced after the binding of EAK to dCj 6 . Since the ⁇ values corresponding to the two labels are similar, the accessibility of both ends of dCi 6 to the solvent was reduced by the same extent, about 40 %.
  • EXAMPLE 8 USE OF PEPTIDES FOR FORMING NANOWIRES
  • nanofiber growth occurs when the nanofiber "seeds" ( Figure 40 top panels) and fiber clusters ( Figure 40 bottom panels) that are formed in the solution adsorb on the surface.
  • the surface serves the purpose of adsorbing the peptides and permitting elongation from the ends of a peptide nucleus as exemplified in the schematic of Figure 5.
  • BIOMOLECULAR SENSING Peptides of the present invention can be used in biomolecular sensing applications.
  • EAKl 6-11 peptides self-assembled into different nanostructures depending on the surface on which the peptides were seeded ( Figure 43).
  • Peptides formed nanofibers when seeded onto a mica (hydrophilic) surface at a concentration of 0.05 mg/mL.
  • the same peptide tended to self-assemble into patterned nanofibers at 60° and 120° angles to one another when seeded onto HOPG (hydrophobic) ( Figure 43B, Figure 44).
  • EAKl 6-11 peptides were shown to alter the hydrophobicity of HOPG and induce it to be hydrophilic.
  • the substrate for a biomolecular sensing application should be confuctive and chemically inert (e.g. HOPG). Since the EAK 16-11 peptides have a unique amphiphilic structure with hydrophilic resides on one side and hydrophobic residues on the other side, it was proposed that the hydrophobic side of the molecules were capable of interacting with the hydrophobic HOPG surface.
  • the exposed hydrophilic resides of glutamic acid (E) and lysine (K) contain functional amine and carboxylic acid groups, which can be used to immobilize many proteins and enzymes for a molecular sensing application.
  • the wettability of a surface may affect the adsorption of biomolecules and cells as well as the enzyme immobilization.
  • the amphiphilic properties of the EAK peptides are capable of altering the surface wettability ( Figure 45).
  • Glucose oxidase was used as a model enzyme to test if the amino acid pairing peptides of the present invention could be used to modify a surface for a biomolecular sensing application.
  • Two exemplary ionic pairing peptides were used for this study: EAK16-II; and EFK16-II.
  • the morphologies of GOx on bare HOPG and EAK/HOPG were characterized using atomic force microscopy (AFM). GOx tended to denature upon coating on HOPG, but maintained a native ball shape on EAK/HOPG ( Figure 46).
  • FIG. 49 The activity of immobilized GOx on EAK/HOPG electrode was examined using cyclic voltammetry (CV).
  • Figure 49 showed the CV of a GOx-immobilized EAK/HOPG electrode in 100 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM ferrocenecarboxylic acid (FCA, mediator) in the absence and presence of 20 mM glucose.
  • FCA ferrocenecarboxylic acid
  • Figure 49 upper profile
  • an electrocatalytic anodic current was observed ( Figure 49, lower profile), indicating that GOx molecules were immobilized on the surface and maintained good activity.
  • Glucose biosensors using peptides of the present invention were examined for the ability to act as an anode for a biofuel cell (Figure 54A).
  • the peptide-modified electrodes could also be used to generate a cathode, such as an oxygen electrode, in a biofuel cell. Laccase could be immobilized to an EAK or EFK/HOPG electrode.
  • Peptide modified electrodes can, thus, be used to generate biofuel cells, including glucose-oxygen ( Figure 54A) and hydrogen-oxygen (Figure 54B) biofuel cells.
  • EXAMPLE 11 PHYSICOCHEMICAL CHARACTERIZATION OF siRNA- PEPTIDE COMPLEXES
  • RNA interference Short interfering RNAs (siRNAs) trigger RNA interference (RNAi), where the complementary mRNA is degraded, resulting in silencing of the encoded protein.
  • a delivery carrier is desired to increase the solution stability of siRNA and improve its cellular uptake to overcome its rapid enzymatic degradation and low transfection efficiency.
  • the physicochemical properties of the carrier-drug complexes including size, surface charge and surface chemistry are essential factors for the development of a suitable formulation of siRNA therapeutics.
  • RNA interference (RNAi) is an evolutionary conserved mechanism that performs a sequence specific, post transcriptional gene silencing through the use of short RNAs.
  • RNAi can be triggered by several sub-types of short RNAs, which include short interfering RNA (siRNA), micro RNA (miRNA), tiny non-coding RNA (tncRNA), small modulatory RNA (smRNA), and short hairpin RNA (shRNA) Double stranded RNA (dsRNA) can act as a precursor of RNAi in invertebrates to obtain siRNAs upon its cleavage by the Dicer. Once the siRNA is located in the cytosol, Ago2 cleaves the sense strand of the siRNA (Matranga, C.et al. Cell 2005, 123, 607-20.; Rand, T. A. et al. Cell 2005, 123, 621-29.).
  • the anti-sense strand will be thermodynamically favored to incorporate to the RNA induced silencing complex (RISC).
  • RISC RNA induced silencing complex
  • the anti-sense sequence of the siRNA that is incorporated into the RISC would pair with its complementary mRNA sequence.
  • the mRNA is then cleaved enzymatically by Ago2. Since the cleaved RNA fragments lack either the cap structure m7G or the polyA tail, which are essential to RNA stability, this leads to further degradation of the mRNA molecule. Since mRNA is the precursor to protein translation, the protein encoded by such mRNA thus cannot be synthesized.
  • RNAi takes place in the cytosol, the inability of hydrophilic drugs to effectively travel across the hydrophobic core of the plasma membrane is also a major obstacle for their therapeutic application.
  • NAs nucleic acids
  • the carriers associated or covalently conjugated with the NAs, are designed to prolong drug circulation time, to improve membrane permeation, and to increase cell targeting capabilities, while being biocompatible and biodegradable.
  • a safe drug delivery system should exert minimal side effects, that is, minimal cytotoxicity and inflammatory response, especially to non- targeted cells.
  • Hydrophobic or highly charged particles can interact with opsonins, where the resulting complexes are removed from circulation by phagocytes and the reticuloendothelial system (Vonarbourg, A. et al. Biomater. 2006, 27, 4356-73. "Vonarbourg et al.
  • CGF TAT derived cell penetrating peptide Arginine-9
  • R9 TAT derived cell penetrating peptide
  • the CTGF siRNA was chosen as the model siRNA for this study. It had a sense sequence of 5 'CGGUGUACCGAGCCCAGAUdTdT 3' and an antisense sequence of 5'AUCUCCGCUCGGUACACCGdTdT 3'. It was purchased from Dharmacon (processing option A4; Lafayette, CO). The molar concentrations of siRNA were determined by absorption spectroscopy, using an extinction coefficient of 355,021 L/mol'cm. Crude R9 peptide with N-terminal acetylation and C-terminal amidation (AcN-RRRRRRRRR-CNH2) was purchased from the Sheldon Biotechnology Center at McGiIl University (Montreal, QC).
  • siRNA-R9 complexes Prior to use, siRNA and R9 peptides were first dissolved in Milli-Q water separately (Millipore, USA), divided in aliquots in microcentrifuge tubes, and stored in -2O 0 C after drying in Eppendorf Vacufuge Concentrator 5301.
  • SiRNA at concentrations 1.5 ⁇ M, 3.0 ⁇ M, and 4.5 ⁇ M was first suspended in HEPES buffer (6 mM HEPES-NaOH, 20 niM NaCl, 0.2 mM MgC12, pH 7.3), then added to the dried peptide vials to achieve a final peptide concentration ranging from 0-60 ⁇ M
  • HEPES buffer (6 mM HEPES-NaOH, 20 niM NaCl, 0.2 mM MgC12, pH 7.3
  • the resulting complex solutions were stirred vigorously for 10 s with a vortex mixer and incubated for 3 hours at room temperature.
  • UV- Vis Absorbance UV- Vis absorption spectra of each sample were obtained on a Hewlett-
  • Spectropolarimeter (Jasco, USA). Spectra were acquired from samples in a 55 ⁇ L, 3mm path length quartz cuvette at 25 0 C. Spectra were scanned from 400 to 200 nm at 200 nm/min, with a response time of 2 s and pitch of 1 nm. Spectra shown are the average of 3 replicates. Hydrodynamic Diameter and Zeta Potential Measurement
  • the hydrodynamic diameter of siRNA-R9 complexes was measured by dynamic light scattering (DLS) and Zeta potential by laser doppler velocimetry (LDV) at 25 0 C using a Zetasizer Nano ZS (Malvern, UK) equipped with a 4 mW He-Ne laser operating at 633 nm. All measurements were performed at 25 0 C at a measurement angle of 173°.
  • SiRNA and R9 stock solutions were separately filtered through 0.2 ⁇ m non-protein binding syringe filters (Pall, USA) prior to complexation.
  • the size and Zeta potential are presented as the mean value ⁇ standard deviation from three measurements of at least 10 runs per measurement.
  • Salt effect on siRNA-R9 binding High concentration of salt can destabilize non-covalent interactions, including electrostatic interactions and hydrogen bonds.
  • Two solutions were first prepared, one with 1.5 ⁇ M siRNA only and the other with an addition of 150 ⁇ M of R9. One hour after peptide addition, 2 M sodium chloride was separately added to the two solutions.
  • UV- Vis absorbance spectra of the two solutions were monitored before salt addition, two hours after salt addition and one day after salt addition. UV- Vis Absorbance The interaction between CTGF siRNA and a cell penetrating peptide R9 has been investigated with various spectroscopic methods.
  • the UV-Vis absorbance spectra of 3.0 ⁇ iM siRNA in the absence and presence of R9 at concentrations ranging from 0 - 40 ⁇ M are shown in Figure 56.
  • the characteristic peaks of siRNA at 210 nm and 260 nm are due to the presence of phosphate groups and nucleotide base pairs, respectively.
  • the addition of R9 induced a decrease in the absorbance of the complex solution.
  • the hypochromic effect on siRNA absorbance due to peptide addition is more pronounced with increasing peptide concentration, until reaching saturation at peptide concentrations above 32 ⁇ M. However, above this concentration significant sedimentation and turbidity can decrease the adsorption and minimize the suitability of UV-spectroscopy method.
  • the hypochromic effect at 260 nm upon peptide addition, expressed in terms of peptide concentration, is quantified for siRNA concentrations of 1.5 ⁇ M, 3.0 ⁇ M, and 4.5 ⁇ M.
  • a plot of hypochromicity can also be presented with respect to charge ratio, which is a normalization of R9 concentration with respect to siRNA concentration, expressed in terms of the charge ratio of positively charged R9 to negatively charged siRNA.
  • Figure 58 A plot of hypochromicity can also be presented with respect to charge ratio, which is a normalization of R9 concentration with respect to siRNA concentration, expressed in terms of the charge ratio of positively charged
  • the relative change in absorbance initially increases (UV absorbance at 260 nm initially decreases) with increasing peptide concentration and eventually reaches a plateau after reaching saturation at 32 ⁇ M of R9, where only 7.8% of initial absorbance remained upon saturation.
  • hypochromicity is plotted against +/- charge ratio for siRNA concentrations of 1.5 ⁇ iM, 3.0 ⁇ M, and 4.5 ⁇ M, a significant portion of the curves is overlapped.
  • the relative change in absorbance reaches its maximum at a charge ratio (+/-) of 2.2, which corresponds to a molecular binding ratio of 10.3 peptides per siRNA.
  • ⁇ oA electric dipole transition moment
  • the CD spectra of siRNA-R9 complexes prepared at various R9 concentrations at 3.0 ⁇ M siRNA are shown in Figure 60.
  • the siRNA has characteristic peaks around 210 nm and 265 nm, which, when compared to the CD spectrum of established nucleic structures (Bloomfield et al. (2000)), confirms that the siRNA possesses a right handed structure, similar to that of A-DNA.
  • concentration of R9 increases, the ellipticity of complex solutions decreases progressively, until reaches a plateau at R9 concentrations above 35 ⁇ M.
  • ⁇ r ( ⁇ 0 - ⁇ )/ ⁇ 0
  • G 0 is the initial ellipticity of the free siRNA
  • ⁇ is the observed ellipticity of the sample containing siRNA-peptide complexes.
  • CD measures the difference in absorption spectrum between left-handed and right handed polarized light. Therefore, CD provides sensitive and unique spectra for chiral molecules, and it has been widely used in structural determination of proteins and nucleic acids.
  • R9 concentrations With increasing R9 concentrations, the ellipticity of complex solution decreased, while the maximum and minimum peaks experience negligible shifting, which suggested that the structure of the siRNA experienced minimal structural changes upon interacting with R9.
  • the decrease in ellipticity of siRNA due to increasing peptide concentration can be attributed to the decrease in absorbance of the nucleosides in siRNA-R9 complexes, similar to the spectra obtained by UV- Vis spectroscopy. Hydrodvnamic Diameter and Zeta Potential Measurements
  • CTGF siRNA adopts the structure of the right-handed A form of DNA in solution, with a measured hydrodynamic diameter of 5.21 nm, which is very close to the theoretical value of 5.46 nm for a 21 base pair siRNA (Lodish, H. et al. Molecular Cell Biology, W. H.
  • R9 peptide adopts a random coil structure (also confirmed by CD), and its hydrodynamic diameter is found to be 6.81 nm. With increasing peptide concentration, both the hydrodynamic diameter and Zeta potential of the complex solution increased. The size of the complexes increases until its value reaches 1055.8 nm.
  • the Zeta potential of CTGF siRNA in HEPES is -36.2 mV, which reflects the contribution from the 42 negative charges on the phosphate group at neutral pH; whereas the Zeta potential of R9 in HEPES is 28.1 mV, due to the positively charged guanidino group.
  • the increase in Zeta potential is the most pronounced when the charge ratio is between 1.43 and 8.57 mV, while its value increased consistently upon additional peptide addition.
  • the rate of increase in the hydrodynamic diameter is at maximum when the charge ratio is also between 1.43 and 8.57, where the value of the surface charge is low.
  • the increase in hydrodynamic diameter strongly demonstrated that the two species interact with each other. Further, since the hydrodynamic diameter increased by almost 200 fold, the results also indicate that siRNA and R9 forms large aggregates upon complexation, confirming the earlier observation in the UV- Vis absorbance measurements.
  • UV- Vis spectroscopy and CD can detect the complexation of siRNA and R9 only at a charge ratio below 2.2:1; further complexation and aggregation phenomena cannot be detected by these two methods.
  • UV-Vis absorbance and CD signal has reached saturation at a charge ratio of ⁇ 2.2: 1, which is very close to the charge ratio corresponding to the isoelectric point of the siRNA-R9 complexes (around 3). Since size and surface charge are the essential parameters contributing to the activation of the complement system.
  • peptide concentration can be used to control the size and surface charge of the siRNA-R9 complexes so that the interaction between the siRNA-peptide complexes and the immune system can be minimized during delivery.
  • siRNA-R9 complexes are unstable in solutions with high ionic strength, in which the complexes dissociated and resulted in restoration of siRNA absorbance.
  • the dissociation of complexes was demonstrated in a salt solution with a very high ionic strength (2 M); the stability of siRNA-R9 complexation in various salt concentrations, including physiological conditions, requires further investigations. Nevertheless, ionic strength can be used as another parameter that controls the complexation reaction in subsequent formulation considerations.
  • the highest binding ratio of R9 to siRNA determined from DLS is 39.1 : 1 (corresponding to charge ratio of 8.4:1).
  • the difference in binding ratios is possibly due to the difference in signal contributions between absorption and light scattering. Since the signal from absorption measurements is solely contributed by the nucleoside bases, it cannot represent the extent of the overall reaction when there are preferences in any of the binding sites.
  • the salt dissociation experiment has shown that siRNA and R9 react through non-covalent interaction.
  • the physicochemical characterization of CTGF siRNA-R9 complexes presented here have shown that various methods can be used to control the properties of the siRNA-peptide complexes, which provides necessary information for the formulation of siRNA therapeutics with peptide carriers.
  • SiRNA has 42 negative charges per molecule and R9 has 9 positive charges per molecule. Therefore, it is expected that siRNA molecules can interact with multiple R9 molecules through columbic forces. Furthermore, two hydrogen atoms from the guanidino group of each arginine can hydrogen bond with the oxygen and nitrogen at the purine base of guanine within the major groove of the GC base pair. It is anticipated that electrostatic interaction and hydrogen bonding are the major driving forces for the interaction between siRNA and R9. In other words, siRNA is present as a macromolecule that can interact with multiple R9 ligands in a non-sequence-specific and non-covalent manner. In order to characterize the complexation reaction quantitatively, it is essential to obtain an accurate equilibrium binding isotherm.
  • hypochromic effect of siRNA absorbance at 260 nm is observed upon its interaction with R9.
  • siRNA concentrations at a fixed siRNA concentration three titration curves expressed in terms of hypochromicity were obtained at siRNA concentrations of 1.5 ⁇ M, 3.0 ⁇ M, and 4.5 ⁇ M.
  • siRNA and R9 would establish equilibrium between the free siRNA sites, free peptide and bound siRNA sites.
  • a siRNA molecule consists of 21 base pairs and it forms a double helical structure with two 3' overhangs. It can be viewed as a linear lattice with N repeating units. It is assumed that each R9 covers the same number of phosphate groups ( ⁇ ) on a siRNA. Since hypochromicity is observed upon R9-siRNA complexation, it follows that the extinction coefficients of the complexes is lower than that of the free siRNA. If there are r binding state and each binding state i has a distinct extinction coefficient, then according to the Beer's Law, the optical density of a R9-siRNA complex solution, OD o bs, can be expressed as
  • the binding density of the siRNA at state i, v h is defined as the number of peptide molecules bound per siRNA phosphate group
  • Equation 1 L ⁇ is the bound peptide concentration for complexes in stage i. Substitute Equations 2 and 3 into Equation 1,
  • Equation 2 the equilibrium binding constant, which is related to the equilibrium quantities through where M b is the sum siRNA phosphate concentrations for all binding states, and it is equal to ⁇ Mu- Substitute reaction site conservation equation (Equation 2) into Equation 6 and express in terms of binding density (Equation 3), one obtains
  • Equation 7 From Equation 7, it is shown that the overall binding density is only a function of free peptide concentration. Equation 5 illustrated that the experimental observed hypochromicity is a unique function of the binding density, whereas Equation 7 demonstrated that the binding density is a unique function of the free peptide concentration. In other words, hypochromicity is also a unique function to the free peptide concentration, related through the binding density. As a result, the free peptide concentration and binding density will be constant for a number of different combinations of total peptide and total siRNA concentrations (L 1 ,M 1 ) taken at a constant hypochromicity value, given that it follows the peptide mass conservation equation
  • Fitted values of total peptide concentration are calculated at chosen hypochromicity values for siRNA concentrations of 1.5 ⁇ M, 3.0 ⁇ M, and 4.5 ⁇ M. Linear regression is performed according to Equation 8 to obtain binding densities and free peptide concentrations at each hypochromicity value. However, the calculated free peptide concentration is negative in the experimental relevant range, which means that this analysis is not applicable to this experimental system.
  • Figure 65 is a plot of binding densities versus free peptide concentrations. Since the signal contributed by the siRNA is solely from the nucleoside bases and it is possible that the decrease in absorbance cannot reflect the interactions that undergo other modes of interaction, such as electrostatic interaction with the phosphate backbone and charge dipole interaction with the sugar ring. Furthermore, aggregation of complexes also affects the applicability of this method.
  • EXAMPLE 12 INVESTIGATION OF ABILITY OF EAKl 6-11 TO STABILIZE HYDROPHOBIC ANTICANCER AGENT
  • the ability of the self-assembling peptide EAKl 6-11 in stabilizing the hydrophobic anticancer agent ellipticine was investigated. The formation of peptide-ellipticine suspensions was monitored with time until equilibrium was reached. The equilibration time was found to be dependent on the peptide concentration. When the peptide concentration was close to its critical aggregation concentration (CAC, ⁇ 0.1 mg/mL), the equilibration time was minimal at 5 h.
  • CAC critical aggregation concentration
  • EAK16-II and ellipticine concentrations Two molecular states (protonated or crystalline) of ellipticine could be stabilized. These different states of ellipticine significantly affected the release kinetics of ellipticine from the peptide-ellipticine complex into the egg phosphatidylcholine (EPC) vesicles, which were used to mimic cell membranes. The transfer rate of protonated ellipticine from the complex to the vesicles was much faster than that of crystalline ellipticine. This observation may also be related to the size of the resulting complexes as revealed from the scanning electron (SE) micrographs. In addition, the complexes with protonated ellipticine were found to have a better anticancer activity against two cancer cell lines, A549 and MCF-7.
  • SE scanning electron
  • the ideal drug delivery vehicle should have the following properties: biocompatible, biodegradable, suitable size, high loading capacity, extended circulation time, and capable of accumulating at required pathological sites in the body.
  • Peptides have shown much potential for drug delivery. The most attractive aspect of peptide-mediated drug delivery is the natural propensities of many peptides for cell penetration and targeting. As a result, many novel delivery systems involve peptides to achieve targeted delivery for anticancer therapeutics and to cross the cell membrane barrier for gene/siRNA delivery. Peptide-based delivery systems have also shown the potential to deliver therapeutic proteins, bioactive peptides, small molecules and nucleic acids.
  • a special class of self-assembling, ionic-complementary peptides taught in the present application represents a new and promising biomaterial for constructing drug delivery nanocarriers.
  • the unique amphiphilic structure and the ability of self- assembly of these peptides allow them to encapsulate both hydrophobic chemotherapeutics and hydrophilic protein and oligonucleotides.
  • no detectable immune response was observed when these peptides were introduced into animals.
  • EAKl 6-11 A representative self-assembling, ionic-complementary peptide, EAKl 6-11 ( Figure 66), has been shown to encapsulate hydrophobic compounds readily and stabilize them in aqueous solution. Work using pyrene as a model hydrophobic compound demonstrated the potential of this peptide in the delivery of hydrophobic anticancer drugs. EAK16-II was shown to stabilize pyrene microcrystals in aqueous solution at a concentration ten-thousand fold beyond its solubility in water, indicating a very high loading efficiency. The encapsulated pyrene can be released from the peptide coatings into liposomes and the release rate can be controlled by changing the peptide-to-pyrene ratio during the encapsulation.
  • This peptide has been used to stabilize microcrystals of the anticancer agent ellipticine in aqueous solution (see Example 3).
  • the stabilized ellipticine microcrystals can have a concentration several hundred times more than its solubility.
  • Ellipticine was selected as the model hydrophobic anticancer drug in our studies for the following reasons: first, the fluorescence property of ellipticine enables us to monitor the interaction of ellipticine with the peptide and locate it in different micro-environments. Second, ellipticine is extremely hydrophobic with a low water solubility of ⁇ 0.62 ⁇ M at neutral pH, (Liu, J. et al.
  • the fluorescence technique is the primary tool to characterize the complex formation and the release kinetics, where the change of ellipticine fluorescence is monitored over time.
  • Scanning electron microscopy (SEM) is applied to characterize the dimensions of the peptide-ellipticine complexes.
  • the complexes with various peptide-to- ellipticine ratios (by mass) are further tested on their cellular toxicity against two cancer cell lines, A549 and MCF-7. The cells are treated for different time periods (4- 48 h) to reveal the time-dependent toxicity of the complexes.
  • Figure 67a shows the fluorescence spectra of the complex suspension with 1.0 mg/mL ellipticine and 0.2 mg/mL EAKl 6-11 at different times. Initially, the fluorescence spectrum exhibits a characteristic of protonated ellipticine with a peak located around 520 nm after 0.5 h stirring. This peak rises with time and reaches a maximum after 6 h; it then decreases with time.
  • the band at 468 nm is characterized as the ciystal form of ellipticine.
  • ellipticine crystals There was no trace of fluorescence from ellipticine crystals (at 468 nm) initially; although ellipticine was in crystalline form when just mixed with the peptide solution, the ellipticine crystals were large, unable to suspend in solution, and thus precipitated at the bottom of the sample vial, not contributing to the fluorescence signal detected.
  • EAKl 6-11 assemblies may consume the negatively charged glutamic acid residues as they are complementary to the lysine residues in the assemblies. This in turn reduces the amount of free glutamic acid residues that are able to stabilize the protonated ellipticine.
  • the pH of the EAKl 6-11 solution was found to increase from -4.6 (fresh) to 6.4 (30 h after preparation), which is slightly above the pKa of ellipticine. The combination of these two effects can induce deprotonation of ellipticine, thereby explaining the disappearance of protonated ellipticine over time.
  • the peptide assembly does not likely inhibit the formation of stable ellipticine microcrystals.
  • these peptide assemblies are mainly made of ⁇ -sheets, which are amphiphilic with hydrophobic and hydrophilic regions on the opposite sides. The hydrophobic region can still interact with hydrophobic ellipticine microcrystals to form stable peptide-ellipticine suspensions. It has been shown that EAK 16-11 can adsorb on hydrophobic surfaces and assemble into stable ⁇ -sheet rich nanostmctures.
  • Figure 69 shows the peptide concentration effect on the formation of peptide- ellipticine complexes at a fixed ellipticine concentration of 1.0 mg/mL.
  • the peptide concentration ranges from 0.05 to 0.5 mg/mL.
  • the fluorescence intensity at 468 nm (crystalline ellipticine) increases with time and reaches a plateau for all peptide concentrations used, but the time required to reach equilibrium is dependent on the peptide concentration ( Figure 69a).
  • the equilibration time is at minimum ( ⁇ 5 h) when the peptide concentration is around 0.1 mg/mL, which is the CAC of the peptide. When the peptide concentration is away from the CAC, the equilibration time increases (>10 h).
  • the change in the fluorescence intensity at 520 nm is also strongly dependent on the peptide concentration as shown in Figure 69b.
  • the protonated ellipticine can be seen to form and over time to disappear.
  • the protonated ellipticine stays for a longer time (40 h) with a higher peptide concentration (0.5 mg/mL).
  • the fluorescence of potonated ellipticine only appears in the first 2 h and quickly disappears afterwards. Below the CAC, no significant protonation of ellipticine is observed.
  • the peptide solution has a relatively high pH, which prohibits ellipticine protonation. But under such a condition, microcrystals of ellipticine can form over time. Such formation of ellipticine microcrystals becomes faster with increasing peptide concentration up to its CAC.
  • a low pH below the pKa of ellipticine, is observed. Such a low pH allows protonated ellipticine to form ( Figure 69b).
  • the solution pH starts to increase and the number of available glutamic acid residues reduces. (The glutamic acid residues can stabilize the protonated ellipticine.)
  • deprotonation of ellipticine occurs shortly after the initial protonation.
  • the peptide EAKl 6-11 is capable of stabilizing ellipticine microcrystals and protonated ellipticine in aqueous solution in a time-dependent and peptide concentration-dependent manner.
  • the state of ellipticine could be critically important in its function as a therapeutic agent. It has been reported that the neutral form of ellipticine is active against various tumors (Garbett et al. (2004); Sainsbury, M. Ellipticine; In The Chemistry of Antitumour Agents, Wilman, D. E. V., Ed.
  • the slight initial decrease in intensity of the protonated ellipticine is likely the results of inner-filter effect as the solution solubilizes more ellipticine with time.
  • the protonated ellipticine is stable for at least 50 h, the duration of the experiment, under continuous mechanical stirring. During this time period, the solution remains clear with a yellow-orange color.
  • This particular combination of ellipticine and peptide concentrations suggests that a prolonged state of protonated ellipticine can be established. This will affect ellipticine release kinetics, and probably its therapeutic efficiency. Release of Ellipticine from the Complex into EPC Vesicles
  • the suspension with 0.5 mg/mL EAKl 6-11 has a pronounced peak located ⁇ 525 nm (protonated state) while those with peptide concentrations ranging from 0.05 to 0.2 mg/mL exhibit a peak of ⁇ 468 nm (crystalline state) ( Figure 71, inset).
  • the very different properties of the complex suspensions according to the peptide concentration could also have significant effects on the ellipticine release (see below).
  • FIG. 72a A typical transfer curve of ellipticine from the complex to the EPC vesicles, or liposomes, is shown in Figure 72a.
  • the complex was made with 0.05 mg/mL EAKl 6-11 and 0.1 mg/mL ellipticine in pure water with 24 h stirring.
  • the fluorescence intensity at 436 nm increases with time and approaches to a plateau after 20,000 s.
  • the fitting parameters A and B are 17.5 ⁇ 0.36 and 230000 ⁇ 13400 (1/M), respectively.
  • [EP J] represents the concentration of ellipticine within the range of 0-20 ⁇ M.
  • Figure 72b shows four transfer profiles of ellipticine concentration in the vesicles ([EPT V ]) with time (h). Each curve corresponds to the transfer of ellipticine into the vesicles from different peptide-ellipticine complexes made with various
  • EAKl 6-11 concentrations All profiles have a similar trend with a fast increase initially and gradually approaching a plateau.
  • the very high initial values of the transfer profile from the complexes with 0.5 mg/mL EAK16-II indicate a burst release of ellipticine from the complex into the vesicles within 30 s. This is reasonable since the 0.5 mg/mL EAKl 6-11 solution can stabilize protonated ellipticine ( Figures 70 and 5 71). These protonated ellipticine molecules may easily migrate into the lipid bilayers, causing a sudden increase in the ellipticine concentration in the vesicles.
  • [EPT V ] ⁇ t) [EPT V ] eq (l - a x e- « - a 2 e ⁇ ' ) (3)
  • [EPT v ](t), [EPT v ] eq and [EPT v ]o are the ellipticine concentration in the vesicles at time t, at equilibrium and at time zero, respectively
  • k, ki and f ⁇ are the rate0 constants
  • aj and a ⁇ are the pre-exponential factors and ai + ⁇ X 2 - 1.
  • Equation 2 The particular transfer profile with 0.5 mg/mL EAKl 6-11 was fitted with Equation 2 where [EPT v ]o ⁇ O due to an initial burst transfer of ellipticine into the vesicles; the other three profiles were fitted well with Equation 3 as the initial transfer in these cases was very small and can be negligible.
  • the rate constants are summarized in Table 16.5 Comparing the average rate constants for each transfer profile, it can be seen that the rate of transfer of ellipticine from the complexes into the vesicles increases with the peptide concentration during the preparation of peptide-ellipticine complexes. Table 16.
  • EAKl 6-11 can stabilize ellipticine in protonated or crystalline form in aqueous solution, depending on the peptide and ellipticine concentrations.
  • Protonated ellipticine can be stabilized in the complexes formulated with a combination of 0.5 mg/mL EAKl 6-11 and 0.1 mg/mL ellipticine.
  • the ratio of peptide-to-ellipticine is smaller than 5:1 (by mass)
  • the stabilized ellipticine is predominantly in crystalline form in the complexes (Figure 71). This may indicate that the 5:1 ratio is important in determining the molecular states of ellipticine in the complexes. It is expected that the different molecular states will have different therapeutic effects against cancer cells.
  • protonated ellipticine tends to interact with negatively charged cell membranes, and accumulate at the membrane surface; the hydrophobic moiety of ellipticine further helps it cross the cell membrane. This also implies that the internalization of ellipticine may not be through energy-dependent endocytosis.
  • Figure 74b shows the toxicity of the complex prepared at a 5: 1 ratio and its serial dilution in water (2, 4, 8 and 16 times). The 2 times dilution does not affect the toxicity of the complex significantly for both cells. Further dilution greatly reduces the complex toxicity against MCF-7 cells; it also decreases the toxicity against A549 cells, but to a lesser degree. Normally, the decrease in cell viability should be gradual and smooth due to the decrease in drug concentration upon dilution.
  • the observed trend is not gradual, but rather a sharp change at more than 2x dilution. This may be related to the instability of the complexes upon dilution in water.
  • the complex containing protonated ellipticine is pH sensitive; extensive dilution is expected to increase the solution pH, leading to the deprotonation of ellipticine to form ellipticine microcrystals.
  • the toxicity of the diluted complexes reduces, similar to that of the complexes prepared at a lower peptide-to-ellipticine ratio. Note that a drastic decrease in complex toxicity upon dilution for MCF-7 cells provides additional evidence that MCF-7 cells are more sensitive to protonated ellipticine.
  • FIG. 75.1 The uptake of ellipticine by the two cell lines is shown in Figure 75.1.
  • the cells are treated with complexes at two ratios of 5: 1 and 1: 1, the ellipticine control and the peptide control for 5, 15 and 30 min.
  • the treatment with peptide alone does not exhibit any fluorescence (1 st column, insets), which is reasonable since there is no ellipticine in the system and EAKl 6-11 is not fluorescent.
  • the fluorescence signals are too dim to be seen initially, but increase with time. A stronger fluorescence signal can be observed for the treatments of complexes at 5 min and become more pronounced at later times.
  • the ellipticine fluorescence seems to accumulate in the cell nuclei. Similar phenomena are observed for MCF-7 cells ( Figure 75.1b).
  • unspecific uptake may be solved by introducing cell targeting moieties onto the peptide sequence to achieve active targeting.
  • An example is the cyclic peptide motif c-NGRGEQ-c, which has been found to strongly bind to several non-small cell lung cancer cell lines including A549, CaIu-I and H178.
  • a vasoactive intestinal peptide (VIP) can selectively bind to many breast cancer cell lines such as MCF-7 (Moody, T. W., et al. The development of VIP-ellipticine conjugates. Regul. Pept. 123, 187-192 (2004); Moody, T. W. et al. VIP-ellipticine derivatives inhibit the growth of breast cancer cells. Life Sci. 71, 1005-1014 (2002)).
  • VIP vasoactive intestinal peptide
  • EAKl 6-II a self-assembling, ionic-complementary peptide
  • EAKl 6-II hydrophobic anticancer agent ellipticine in aqueous solution.
  • Different combinations of peptide and ellipticine concentrations can stabilize either protonated or crystalline ellipticine for an extended time.
  • the ellipticine can be released from the complexes into a cell membrane mimic. The release rate is related to the peptide concentration used in the complexation. By optimizing the process of complex formation, one could obtain desired complex dimensions and drug release property. These factors and molecular states of ellipticine can have significant impacts on the cellular toxicity of the peptide-ellipticine complexes.
  • the size of the peptide-ellipticine complexes can be controlled from micrometers to hundreds of nanometers.
  • the particle size will significantly affect the circulation in the blood stream, binding to the cells and uptake by the cells.
  • the size of the complexes ranging from 100 nm to 500 nm would be ideal for passive targeting to solid tumors via the enhanced permeability and retention (EPR) effect (Gu et al. (2007); Brigger, I. etl al. Nanoparticles in Cancer Therapy and Diagnosis. Adv. Drug Del. Rev. 2002, 54, 631-651; Greish, K.
  • the ionic-complementary self-assembling peptide EAKl 6-11 was found to be able to stabilize the hydrophobic anticancer agent ellipticine in aqueous solution. Both microcrystal and protonated forms of ellipticine can be obtained in the complexes.
  • the complex formation in water is peptide concentration-dependent. When the peptide concentration was close to its CAC ( ⁇ 0.1 mg/mL), the equilibration time for complex formation could be as short as 5 h. At higher and lower peptide concentrations, the time required to reach equilibrium became much longer. High peptide concentrations facilitated the formation of protonated ellipticine during the complexation while low peptide concentrations favored crystalline ellipticine formation.
  • EAKl 6-11 is capable of stabilizing protonated or crystalline ellipticine in aqueous solution depending on the peptide-to- ellipticine ratio during the fo ⁇ nulation. Above the ratio of 5:1 (by weight), the stabilized ellipticine is protonated; below this ratio ellipticine is in crystalline form in the complexes.
  • the two molecular states of stabilized ellipticine in the complexes exhibit different toxicity against two cancer cell lines, A549 and MCF-7, with the protonated being more toxic. Such an effect is more pronounced for MCF-7 cells than for A549 cells. This is probably due to the fact that MCF-7 cells are more sensitive to protonated ellipticine.
  • the complexes with protonated ellipticine are not stable upon dilution in water.
  • the uptake of ellipticine in both cell lines appears to follow a diffusion mechanism. It is found that ellipticine can still be taken up by both cancer cells at 4 0 C, where the energy-dependent endocytosis pathway is blocked.
  • the complexation of ellipticine with EAKl 6-11 seems not to alter the internalization pathway of ellipticine. However, it significantly enhances the uptake over a shorter time period ( ⁇ 5 min).
  • the anticancer agent ellipticine (99.8% pure) was purchased from Sigma- Aldrich (Oakville, Canada) and used as received.
  • Egg Phosphatidylcholine (EPC, powder, > 99% pure) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Ethylenediaminetetraacetic acid (EDTA) was from Bio-Rad Laboratories (Mississauga, Canada).
  • Tris(hydroxymethyl)methylamine (Tris) and glacial acetic acid were bought from BDH Inc. (Toronto, Canada). Tetrahydrofuran (THF, reagent grade 99%) was obtained from Calendon Laboratories Ltd. (Georgetown, Canada).
  • Cell culture reagents including Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-ETDA, were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada).
  • Phosphate buffer saline (PBS) and penicillin-streptomycin (p/s, 10000 U) were obtained from MP Biomedicals Inc. (Solon, OH, USA).
  • the mixture was then extruded using a LiposoFast-Basic extruder (Avestin Inc., Ottawa, Canada) with a polycarbon
  • the supernatant was collected and stored at 4 0 C before use.
  • the EPC concentration was determined to be 7.1 x 10 "4 M using the method described in our previous publication.
  • the size of the EPC liposomes was characterized by a Dynamic Light Scattering (DLS) technique; their hydrodynamic diameter (intensity-based) was found to be around 200 nm. Formation of Peptide-Ellipticine Complexes
  • peptide-ellipticine complexes To make peptide-ellipticine complexes, certain amounts of ellipticine crystals were added into fresh EAKl 6-11 solutions (0.02-1.0 mg/mL) to have ellipticine concentrations of 0.1-1.0 mg/mL.
  • the fresh peptide solutions were prepared by dissolving peptide powder in pure water (18.2 M ⁇ , MiUi-Q AlO synthesis), followed by the sonication for 10 min.
  • the peptide-ellipticine mixtures were stirred at 900 rpm on a magnetic stir plate throughout the complexation experiment. At specified times, the mixtures were transferred to a quartz cuvette to acquire fluorescence spectra of ellipticine on a steady-state spectrofluorometer (Photon Technology International, London, Canada). The test was performed once every hour for the first 20 h and less frequently for the remaining period until an equilibrium state was reached. 1 mg/mL of ellipticine in pure water was prepared as a control
  • the peptide assembly without ellipticine was investigated. For this purpose, 0.2 mg/mL of fresh EAKl 6-11 solution was prepared and stirred for 30 h at 900 rpm. The peptide assembly was characterized by static light scattering (at 400 nm) acquired on the steady-state spectrofluorometer and compared with the fresh peptide solution (0 h). The light scattering intensity of air was obtained as the standard to correct for the lamp fluctuations.
  • the complexes were newly prepared with a fixed ellipticine concentration of 0.1 mg/mL and various peptide concentrations ranging from 0.05 to 0.5 mg/mL. The samples were continuously stirred for 24 h to ensure that equilibrium was reached in the mixture. The steady-state fluorescence spectra of the complexes were acquired just before the release experiments (to show the states of ellipticine in complexes different from that in EPC vesicles).
  • the complexes were prepared with 0.1 mg/mL ellipticine and fresh EAK16-II solutions at concentrations of 0.02, 0.1, 0.2, 0.5 and 1.0 mg/ml, generating five peptide-to-ellipticine ratios of 10: 1, 5: 1, 2: 1, 1 :1 and 1:5 (by mass), respectively.
  • the EAK16-II-ellipticine mixtures were under mechanical stirring at 900 rpm for 24 h.
  • An ellipticine control in pure water (with the absence of EAKl 6-II) at the same ellipticine concentration was prepared for comparison, following the same procedure.
  • the complexes at a 5: 1 ratio were diluted serially (2x, 4x, 8x and 16x) in pure water to study the complex stability. All vials and solvents were sterilized and the samples were prepared in a biological safety cabinet to avoid possible contamination. The appearance of the peptide-ellipticine suspensions was recorded in conjunction with the ellipticine fluorescence spectra, to determine the different molecular states of ellipticine in the complexes. Ellipticine Release into Liposome Vesicles
  • the release of ellipticine from the complex into the EPC vesicles was continuously monitored on the spectrofluorometer over time.
  • the experiments were conducted with the following procedure: 100 ⁇ L of the peptide-ellipticine dispersion were transferred into a quartz cuvette and mixed with 2.9 mL of EPC vesicles. The 30 times dilution of the complex upon mixing with the vesicles was to ensure that the final ellipticine concentration was in the range where the calibration curve was applicable.
  • the cuvette was then put in the spectrofluorometer with gentle magnetic stirring, covered with a parafilm on top (to eliminate the water evaporation during the course of measurement) before starting to collect the fluorescence over time. The time required to prepare the sample before starting a time-dependent fluorescence measurement was less than 30 s.
  • Steady-State Fluorescence Measurements The ellipticine fluorescence was acquired on the Photon Technology
  • the fluorescence intensity at 468 nm and 520 nm were obtained by taking the average from 458 to 478 nm and 510 to 530 nm, respectively.
  • a standard (2 ⁇ M ellipticine in ethanol, sealed and degassed) was used in each run to correct the lamp intensity variations.
  • the standard fluorescence intensity I s was obtained by taking the average of the fluorescence from 424 to 432 nm (peak at ⁇ 428 nm).
  • the kinetics of the ellipticine release from the complex into the EPC vesicles was monitored by acquiring the time-dependent ellipticine fluorescence at 436 nm over a 7 h time span at 5 s intervals. All solutions reached equilibrium within 7 h as the fluorescence intensities reached a plateau during the experimental time span. The same standard sample as described above was used to obtain /, (at 428 nm over 10 min) to correct for the day-to-day fluctuations. For each release experiment, the fluorescence was recorded while the solution was gently stirred in the spectrafluorometer. Scanning Electron Microscopy (SEM)
  • a LEO model 1530 field emission SEM (GmbH, Oberkochen, Germany) was employed to study the morphology and dimensions of the peptide-ellipticine complex.
  • the SEM samples were prepared by depositing 20 ⁇ iL of the complex suspensions on a freshly cleaved mica surface. The mica was affixed on an SEM stub using a conductive carbon tape. The sample was placed under a Petridish-cover for 10 min to allow the complexes adhering to the mica surface. It was then washed once with a total of 100 ⁇ L pure water and air-dried in a desicator overnight. All samples were coated with a 20 nm thick gold layer prior to imaging; the images were acquired using the secondary electron (SE2) mode at 5 kV.
  • SE2 secondary electron
  • the cells were cultured in DMEM containing 10% FBS and 1% p/s at 37°C and with 5% CO 2 . When the cells grew to reach -95% confluence, they were detached from the cell culture dishes with trypsin-EDTA, centrifuged at 500 rpm for 5 minutes, and resuspended in fresh cell culture media at concentrations of 5 x 10 4 and 1 x 10 5 cells/mL for A549 and MCF-7 cells, respectively.
  • A549 and MCF-7 Two cancer cell lines, A549 and MCF-7 (from ATCC), were used to investigate the uptake of EAK16-II-ellipitcine complexes in vitro. They were cultured in DMEM with 10% FBS at 37 0 C with 5% CO 2 . The cells were then seeded on a 12-well plate with cell densities of 5 x 10 4 and 1 x 10 5 cells/well for A549 and MCF-7, respectively, followed by 48 h incubation prior to the treatments. The prolonged incubation time was to enhance the cell adhesion and avoid significant cell loss under intensive rinsing during the subsequent cell fixing procedure. The treatments were added into each well and incubated for 5, 15 and 30 min.
  • the complexes at two peptide- to-ellipticine ratios 5:1 (125 ⁇ g/mL:25 ⁇ g/mL) and 1:1 (25 ⁇ g/mL:25 ⁇ g/mL), the ellipticine control (25 ⁇ g/mL), and the peptide control (125 ⁇ g/mL).
  • the treated cells were washed with PBS 3 times, and fixed with 4% PFA in PBS, followed by another 3 times washing with PBS.
  • the cells were examined with a fluorescence microscope (Nikon Eclipse 8Oi); a green fluorescence filter was used to collect the fluorescence signals of ellipticine, and phase contrast images were acquired to observe the cell morphology.
  • the temperature-dependent cellular uptake of ellipticine was conducted at 37 0 C and 4°C to examine whether the internalization of ellipticine occurs through an endocytosis pathway (Derossi, D. et al. Cell internalization of the third helix of the antennapedia homeodomain is receptor-independant. J. Biol. Chem. 271, 18188- 18193 (1996); Vives, E. et al. TAT peptide internalization: seeking the mechanism of entry. Curr. Protein Peptide Sci. 4, 125-132 (2003)). The same cell density (as above) was used for cell seeding with 48 h incubation.
  • the plates Prior to treating the cells, the plates were incubated at 37 0 C or 4 0 C for 30 min, allowing the culture media to reach the equilibrium temperature. The same treatments (two complexes, ellipticine control and peptide control) were applied with an incubation time of 30 min. The cells were then fixed following the same procedure as before, and examined with the fluorescence microscope. Supporting Information
  • the hydrodynamic diameter of the EPC vesicles was obtained via dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The experiments were conducted with appropriate viscosity and refractive index settings, and the temperature was maintained at 25 0 C during the measurement. A small-volume (45 ⁇ L) black quartz cuvette with a 3 mm light path was used. The scattered light intensities of the samples at the angle of 173 degree were collected. The intensity-based size distribution of the EPC vesicles was obtained with the multimodal algorithm CONTIN (Provencher, S. W. A Constrained Regulation Method for Inverting Data Represented by Linear Algebraic or Integral Equations. Comput. Phys.
  • a calibration curve of ellipticine fluorescence from the ellipticine-EPC suspensions is needed.
  • concentration range of ellipticine was selected to be 10 "6 -10 "4 M based on our previous experience on pyrene release experiments.
  • the ellipticine-EPC suspensions were prepared using the following procedure: ellipticine crystals were dissolved in THF to make the ellipticine-THF stock solution with ellipticine concentration of 1 niM.
  • the UV- Vis absorption of the ellipticine-EPC suspensions was acquired and plotted as a function of ellipticine concentration to show whether ellipticine was completely dissolved in the EPC dispersions.
  • the fluorescence of a control sample made of the buffer saturated with ellipticine was acquired. The fluorescence of the control was found to be ⁇ 280 folds smaller than that of 1 ⁇ M ellipticine in the EPC dispersion. Such a small fluorescence signal can be negligible.
  • the observed fluorescence of the ellipticine-EPC dispersions should represent the fluorescence of ellipticine in the vesicles.
  • the emission fluorescence of ellipticine in EPC vesicles were collected at 436 nm over 1 min and averaged to yield the intensity for each ellipticine concentration.
  • the excitation and emission slit widths were set at 0.5 mm and 0.25 mm, respectively.
  • the intensities were corrected with an ellipticine standard (2 ⁇ M in ethanol, sealed and degassed) to account for lamp fluctuations.
  • the I s was obtained by taking the average of fluorescence at 428 nm over 1 min after each run.
  • Figure 78 shows the calibration curve of the ellipticine fluorescence in EPC vesicles.
  • the ellipticine fluorescence increases to a maximum with the increase of ellipticine concentration from 1 to ⁇ 20 ⁇ M, and then slightly decreases with a further increase in ellipticine concentration (up to 100 ⁇ M).
  • the decrease of ellipticine fluorescence at high ellipticine concentrations can be related to the inner- filter effect. This usually occurs when the concentration of a fluorophore is high enough with right angle detection. However, the decrease of the fluorescence due to the inner-filter effect is usually more dramatic (-50%), which does not seem to match with the present case.
  • Figure 78b shows the concentration-dependent UV absorption of the ellipticine in EPC vesicles used for generating the calibration curve. It can be seen that the ellipticine absorbance at 295 nm increases initially and reaches to a plateau when the ellipticine concentration is greater than 20 ⁇ M. This trend correlates well with that of the calibration curve. A plateau observed in Figure 78b indicates that ellipticine is saturated in the vesicles (formed at this particular lipid concentration of 7.1 x 10 "4 M). This confirms that the inner-filter effect is not the cause of the slight decrease in the calibration curve at ellipticine concentrations above 20 ⁇ M.
  • the molar ratio of ellipticine to the EPC lipid can be roughly estimated to be 0.028. Such a small number reflects the fact that such liposomes may not be an effective carrier for ellipticine when loading capacity is concerned; this is because the drug is inserted into the membrane, rather than inside the aqueous interior.
  • the exact reason for the slight decrease in the ellipticine fluorescence at high ellipticine concentrations (> 20 ⁇ M) in Figure 78a is still under study.
  • the rising region in the calibration curve can be used to convert the fluorescence signals from the transfer profile to the ellipticine concentration in vesicles. This can be done by using a simple exponential equation to fit the rising region in Figure 78a (1-20 ⁇ M):
  • the fitting parameters A and B are 17.5 ⁇ 0.36 and 230000 ⁇ 13400 (VM), respectively.
  • [EPT] represents the concentration of ellipticine within the range of 0-20 ⁇ M. For a given fluorescence intensity of ellipticine (///, ⁇ 17.5), one can obtain the corresponding ellipticine concentration using Equation S 1.
  • Antisense oligodeoxynucleotides have great potential for down regulating certain genes (Stein C. et al. Antisense strategies for oncogene inactivation. Semin Oncol 2005;32: 563-72; Crooke ST. Therapeutic applications of oligonucleotides. Biotechnology 1992;10:882-6.)
  • ODNs Antisense oligodeoxynucleotides
  • the production of faulty proteins is inhibited when antisense ODNs reach the cytoplasm or nucleus of cells where they bind specifically to the mRNA or DNA target (Wang H et al. Antisense anticancer oligonucleotide therapeutics. Curr Cancer Drug Targets 2001; 1 : 177-96 "Wang et al.
  • a delivery system should be designed to protect the ODNs during all the different steps involved in the trafficking of ODNs from outside the cell to its ultimate target inside the cell.
  • the current ODN delivery systems are often composed of cationic lipids (Parekh H. S. et al. Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition. Bioorg Med Chem 2006; 14:4775-80) polymers (Park T.G. et al. Current status of polymeric gene delivery systems.
  • An interesting delivery system consists of a class of self-assembling peptides of the EAK family made of glutamic acid (E), alanine (A), and lysine (K) residues.
  • E glutamic acid
  • A alanine
  • K lysine residues.
  • These self-assembling peptides have an amphiphilic structure consisting of alternating negatively and positively charged amino acids on one side of the backbone, and hydrophobic amino acids on the other side.
  • the ionic complementarity together with hydrogen bonding, hydrophobicity, and van der Waals interactions, promotes the self- assembly of the peptides into aggregates, which are highly stable against extreme pH, various proteases, and denaturing agents (Zhang S. et al. Unusually stable /3-sheet formation of an ionic self complementary oligopeptide.
  • EAK-ODN aggregates generated with EAKl 6IV and EAKl 611 have been characterized.
  • EAK16IV and EAK16II have the same amino acid composition but different charge distributions, respectively given by the charge sequences — + + + + and -- + + — + at neutral pH.
  • Both EAK peptides were found to bind to ODNs more strongly at pH 4 than at pH 7, due to favorable electrostatic attraction at pH 4 (Wang et al. (2001)).
  • EAKl 6IV binds to ODNs more strongly than EAKl 611 does at the same pH due to the larger density of positively charged amino acids at one end of the EAKl 6IV molecule.
  • the present example provides conclusive evidence that the EAK peptides are suitable for use as gene carriers by investigating how the EAK-ODN aggregates resist nuclease degradation and how their stability is affected by dilution, pH, and centrifugation.
  • the ODN sequences are listed in Table 17.
  • Escherichia coli exonuclease (20 U/ ⁇ L) and its 1O x degradation buffer (670 mM glycine-KOH (pH 9.5 at 25 0 C), 67 mM MgCl 2 , 1OmM DTT) were purchased from Fermentas Canada Inc. (Burlington, Canada). Table 17. Type name, and sequence of ODNs and self-assembling EAK peptides
  • UV-vis absorption spectra were obtained on a Hewlett-Packard 8452A diode array spectrophotometer (California, USA) using a 50 ⁇ L quartz cuvette from Hellma (M ⁇ llheim, Germany). Samples containing 8.6 ⁇ m ODNs and different amounts of the EAK peptides were prepared at pH 4 and pH 7 at 25°C. Half an hour after mixing, the EAK-ODN mixtures were diluted 5- and 10-fold with the buffer used to prepare the mixtures. The resulting samples are referred to as "dilute-5" and "dilute-10" samples, respectively.
  • UV-vis spectroscopy together with centrifugation was utilized to measure over time the percentage of ODN in the EAK-ODN aggregates after dilution.
  • the EAK-ODN aggregates formed in solution can be collected by centrifuging the solution at 10,000 rpm for 2 min. The supernatant was collected and its absorbance was measured on the spectrophotometer at wavelengths between 190 and 800 nm. Beer's Law was used to determine the total ODN concentration and the concentration of the ODN left in the supernatant, from the absorbance of the ODN at 260nm of the initial solution (OD 0 ) and the supernatant (OD 5 ), respectively.
  • sample preparation for the evaluation of the nuclease resistance of the oligonucleotides in the EAK-ODN aggregates was conducted as follows. A certain amount of stock EAK and Fl-dCi 6 -Rh solutions were mixed and the mixture solution was vortexed for a few seconds. The resulting EAK- ODN solutions were kept at 25 0 C. Half an hour later, the mixtures were diluted 10 times with the buffer used to prepare the solutions and kept at 25 0 C. Incubation of the EAK-ODN aggregates with nuclease
  • EAK-ODN solutions were centrifuged at 10,000 rpm for 2min.
  • the pellets containing the EAK-ODN aggregates were collected and incubated with 0.7U/ ⁇ L exonuclease I in degradation buffer (pH 9.5) at 25 0 C.
  • Samples without nuclease treatment were used as controls. They were prepared by incubating the EAK-ODN aggregates or free ODN solutions with 1 x degradation buffer under the same conditions as those treated with exonuclease I.
  • 55 ⁇ L of solution was removed at different points in time and inactivated by the addition of 2 ⁇ L of 40% sodium dodecyl sulphate.
  • the ⁇ OD r value of the solution prepared at pH 4 with EAKl 61 V and dGi 6 equals 0.46 ⁇ 0.01.
  • the ⁇ OD r value did not change much within the experimental period after the solution had been diluted 5 or 10 times.
  • the fact that no decrease in ⁇ OD r is observed indicates that the aggregates prepared at pH 4 with EAK16IV and dGi 6 do not dissociate after a 10-fold dilution.
  • the ⁇ OD, value for the solution prepared at pH 7 with EAKl 6IV and dGi 6 equals 0.72 ⁇ 0.01. Note that this higher ⁇ OD r value is a result of the higher EAK16IV concentration used to prepare the solution at pH 7.
  • Figs. 79C and 79D present the - ⁇ OD r vs. time profile for the "dilute-5" and "dilute-10" solutions prepared by mixing 8.6 ⁇ M dGie with either 24.1 ⁇ M EAKl 611 at pH 4 (Fig. 79C) or 60 ⁇ M EAK16II at pH 7 (Fig. 79D). No decrease in the ⁇ OD r value over time was observed regardless of the pH at which the solutions were prepared.
  • FRET was used to monitor the nuclease degradation of an ODN located inside the EAK-ODN aggregates.
  • the ODN was labelled at one end with a fluorescence donor (fluorescein) and at the other end with a fluorescence acceptor (rhodamine).
  • fluorescein fluorescein
  • rhodamine fluorescence acceptor
  • EAK contains four glutamic acids (GIu, E) and four lysines (Lys, K) with pKa values of 4.25 and 10.53, respectively. According to these pKa values, the EAK molecules are expected to be negatively charged at pH 11, and not to interact with the negatively charged ODNs due to electrostatic repulsion. Thus, adjusting the solution pH to 11 may provide a means to dissociate or break down the EAK-ODN aggregates.
  • EAK-ODN aggregates containing 5.0/XM of dCi 6 and 120/XM of EAK16II at pH 4 were prepared and divided into two aliquots. The first aliquot was used to make sure that all ODN molecules were incorporated in the EAK-ODN aggregates. The UV-vis absorbance of the solution at the peak wavelength (276 nm) equaled 0.75. It dropped to about zero after centrifugation (Fig. 80A). This result indicates that no free ODN was left in the supernatant and that all ODNs were incorporated into aggregates, which could be removed by centrifugation. The second aliquot was dried, resuspended in pH 11 buffer, before being vortexed for a few seconds.
  • EAK-ODN aggregates prepared at pH 4 would fully disaggregate when the solution was brought to pH 11, but would undergo only partial dissociation when the solution pH would increase from 4 to 9.5 suggests that at pH 9.5, some lysines in EAKl 6IV remain positively charged and ensure that strong electrostatic attraction is retained between EAKl 6IV and the ODN.
  • Nuclease resistance of the ODN in the EAK-ODN aggregates The ODN protection against the nuclease provided by the EAK peptides was monitored by performing FRET experiments with FWC 16 -Rh. The effect of several factors, i.e. peptide sequence, pH, peptide concentration, and centrifugation, on the protection of the ODN encapsulated in the EAK-ODN aggregates was investigated. Control solutions of the Fl-dCi 6 -Rh prepared in the presence or absence of
  • EAKl 6IV at pH 4 were incubated in the pH 9.5 buffer solution without exonuclease I for 30 min. FRET took place as seen from the two emission peaks at 517 and 583 ran that correspond to the emission of the fluorescein donor and the rhodamine acceptor, respectively (Fig. 81).
  • the ratio of the donor to the acceptor fluorescence intensity at the peak wavelengths (/ D //A) for the ODN is about 1.4 (Fig. 81A) and 1.2 (Fig. 81B) without and with EAK, respectively.
  • the emission spectra were also taken at different points in time after incubating the ODN or EAK-ODN solutions prepared at pH 4 with exonuclease I (Fig. 81).
  • the fluorescence of the donor immediately increased 2.6-fold as that of rhodamine decreased, indicating a lower extent of FRET and significant degradation of the ODN.
  • Fig. 81A indicates that a substantial percentage of the ODN is degraded within 20 min, a result consistent with the expectation that the unprotected ODNs are rapidly degraded by nucleases.
  • Fig. 83A shows the percentage of dC) 6 degraded by exonuclease I when Fl- dCi 6 -Rh was prepared in the presence or absence of EAKl 6IV or EAKl 611, at pH 4 or pH 7.
  • Fig. 83A indicates that about 85 ⁇ 10% of free Fl-dC ]6 -Rh prepared at pH 4 are degraded within 30 min of nuclease incubation.
  • Fl-dCi6-Rh complexed with EAKl 6IV at pH 4 shows significant nuclease resistance against exonuclease I.
  • the aggregates prepared with EAKl 61 V and Fl-dC I6 -Rh at pH 4 protect Fl-dCi 6 -Rh against nuclease degradation even after being incubated at pH 9.5 for 2h. It suggests that Fl-dCi 6 -Rh is located inside the EAK-ODN aggregates where it remains inaccessible to the nuclease.
  • the protection from degradation afforded by the EAK-ODN aggregates to the ODN represents a desired property for using EAKl 6IV as a carrier for ODN delivery.
  • the aggregates made of EAK16IV and Fl-dCi 6 -Rh were prepared at a pH which is much more acidic than the pH of 9.5 of the degradation buffer.
  • EAK-ODN aggregates prepared with Fl-dCi 6 -Rh and EAKl 6IV at pH 7 offer some protection from nuclease degradation (Fig. 83A).
  • EAKl 6IV binds more strongly to dC i6 than EAK16II does.
  • the aggregates prepared with EAKl 6IV and dCi 6 at pH 7 dissociate when the pH is adjusted to 9.5 (Fig. 80B), small EAK-ODN complexes are still present in the solution that may protect the ODN.
  • EAKl 6IV bears four positively charged amino acids at its C-terminal whereas pairs of positively charged amino acids are distributed throughout the back-bone of EAKl 611. The distribution of lysines in EAKl 6IV might facilitate the formation of more cohesive aggregates that prevent nuclease degradation. AU these observations clearly indicate that the aggregates prepared with ODN and EAK 16IV at pH 4 provide the ODNs with better protection against nuclease degradation. Effect of peptide concentration on nuclease resistant
  • the aggregates resulting from the mixture at pH 4 of 8.6 ⁇ M Fl-dCi 6 -Rh and 60 ⁇ M EAKl 6IV provide 100% protection for Fl-dCi 6 -Rh against nuclease degradation (Figs. 81B and 83A).
  • the protection afforded by the EAK-ODN aggregates was also examined at lower EAK concentrations.
  • 8.6 ⁇ M Fl- dCi6-Rh and 10 ⁇ M EAK16IV was used to prepare the EAK-ODN aggregates at pH 4.
  • the resulting aggregates fail to protect Fl-dCi 6 -Rh against nuclease degradation (Fig. 84A).
  • the aggregates with 8 ⁇ M of Fl-dCi6-Rh and 60 ⁇ M of EAK 16IV were prepared at pH 4 and centrifuged for the first time 24 h after sample preparation. They were kept at 25°C and were centrifuged once a day for the next 3 days. The aggregates showed significant stability against nuclease degradation up to 3 days after sample preparation (Fig. 85C).
  • EAKl 6-IV is a promising carrier for ODN delivery.
  • the EAK-ODN aggregates generated with EAKl 6IV at pH 4 are stable at acidic pH and confer nuclease resistance to the ODNs even after having been diluted or centrifuged.
  • the stability of the EAK-ODN aggregates after dilution of the solution was determined by UV-vis absorption.
  • the aggregates were found to be stable, undergoing no detectable dissociation over ⁇ 20h after the solutions were diluted 5- and 10-fold with the buffer used for their preparation.
  • the protection of the oligonucleotides against nuclease afforded by the EAK-ODN aggregates was investigated with FRET when the aggregates were generated with different EAK peptides at pH 4 and pH 7.
  • EAK-ODN aggregates were prepared with similar concentration of EAKIV at pH 7 or of EAKl 611 at either pH 4 or pH 7.
  • the ability of the EAK-ODN aggregates to protect dCi 6 correlates well with the binding strength of the EAK peptides to dCi ⁇ .
  • the effect that centrifugation has on the ability of the EAK-ODN aggregates to protect the ODN from nuclease degradation was also investigated. If the first centrifugation was applied 24 h after sample preparation, the EAK-ODN aggregates confer nuclease resistance to the ODN even after being centrifuged once per day for 4 days.
  • EAK-ODN aggregates lost their nuclease protection ability. This observation led to the suggestion that the EAK-ODN aggregates need to age for more than 30min in order to generate the morphology that can protect the ODNs from degradation.
  • EXAMPLE 14 SEQUENCE EFFECT OF SELF-ASSEMBLING PEPTIDES ON THE COMPLEXATION AND IN VITRO DELIVERY OF THE HYDROPHOBIC ANTICANCER DRUG ELLIPTICINE
  • peptide sequence effects on the drug formulation and in vitro delivery were investigated.
  • Three self-assembling peptides, EAKl 6-II, EAKl 6-IV and EFKl 6-11 were chosen to investigate the effects of charge distribution (type II vs. type IV) and hydrophobicity (alanine A vs. phenylalanine F).
  • a hydrophobic anticancer agent, ellipticine was selected as a model drug.
  • the self-assembled nanostructures of these peptides were first characterized by AFM; the hydrophobicity of the peptides dissolved in aqueous solution was studied via surface tension measurements and fluorescence spectroscopy using a hydrophobic fluorescent probe.
  • EAKl 6-IV has a different charge distribution of type IV ( — ++++) from EAKl 6-11 as type II (-++--++), while the difference between EFKl 6-11 and EAKl 6-11 is a more hydrophobic residue F replacing A in EAKl 6-II.
  • the peptide self-assembled nanostructures are different among the three peptides.
  • the distribution of negative and positive charges towards the two ends of an EAKl 6-IV molecule at neutral pH is reported to cause the folding of the peptide molecule to form a /3-rurn structure, resulting in the formation of globular nanostructures (Hong Y. et al. (2003) Effect of amino acid sequence and pH on nano fiber formation with self-assembling peptides EAKl 6-11 and EAKl 6-IV. Biomacromolecules 4: 1433-1442 "Hong et al. (2003)"; Jun S. et al. (2004) Self- assembly of the ionic peptide EAK16: the effect of charge distribution on self- assembly.
  • EAKl 6-II has a preferable stretched molecular structure and likely self assembles into ⁇ - sheet rich nano fibers (Jun et al. (2004)).
  • the nanostructures of the two peptides are shown in Figure 87A and B at a peptide concentration of 0.5 mg/mL.
  • EAK16-II forms straight nanofibers, connecting to networks ( Figure 87A), whereas EAK 16-IV self-assembles into many more globular aggregates and some short nanofibers ( Figure 87B).
  • short nanofibers of EAKl 6-IV may be due to a relatively low pH ( ⁇ 5) at such a high peptide concentration: when the pH is low enough, some of the negatively charged residues can be neutralized so that the intramolecular ionic interaction is weakened. Thus, some peptides remain in a stretched form, facilitating the formation of nanofibers (Hong et al. (2003)).
  • the nanostructures of EFKl 6-11 are also different from those of EAKl 6-11 as shown in Figure 87.
  • EFKl 6-11 forms predominant nanofibers and these fibers tend to aggregate into fiber clusters.
  • TMs aggregation of nanofibers is probably due to a stronger hydrophobic interaction between them.
  • Such a stronger hydrophobic interaction is expected to come from the more hydrophobic phenylalanine (F) residues in the EFKl 6-11 sequence, compared with the alanine (A) residues in EAKl 6-II. This is probably why the nanofibers of EFK 16-11 tend to form fiber clusters, but those of EAKl 6-11 are dispersed and form fiber networks.
  • FIG 88a shows the surface tension as a function of time for the three peptides at a peptide concentration of 0.5 mg/mL. For each profile, the surface tension decreases fast initially and slowly approaches equilibrium. This change with time corresponds to the dynamic process of the adsorption of peptide molecules/ assemblies at the air-liquid interface, leading to the decrease in surface tension (Eastoe .T, Dalton JS (2000) Dynamic surface tension and adsorption mechanisms of surfactants at the air-water interface.
  • EAKI6-IV has a lower equilibrium surface tension than EAKl 6- II is probably due to the formation of /3-turn structure through intramolecular ionic interaction in EAKl 6-IV. This conformational change may cause the exposure of hydrophobic alanine residues toward the aqueous phase, resulting in a slight increase in hydrophobicity of the molecule and lowering the surface tension (Hong et al.
  • Figure 88b shows the fluorescence spectra of the ANS probe in the three peptide solutions comparing to that in pure water (black line and the inset).
  • the ANS fluorescence spectrum has a peak of ⁇ 470 nm in the EFKl 6-11 solution. The changes in ANS fluorescence intensity and peak position indicate that the ANS probe is in different environments.
  • ANS is a widely used probe to study protein aggregation as well as cell membrane composition and function due to its extreme sensitivity to the changes in the polarity of the probed environment (Torrent J. et al. (2004) High pressure induces scrapie-like prion protein misfolding and amyloikd fibril formation. Biochemistry 43: 7162-7170; Lindgren M. et al. (2005) Detection and characterization of aggregates,. Prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. Biophys J 88: 4200- 4212; Slavik J (1982) Anilinonaphthalene sulfonate as a probe of membrane composition and function.
  • the hydrophobicity determined by the two methods may refer to two different situations.
  • Surface tension is a solution property and based on the molecular adsorption at the interface, affecting the surface free energy.
  • the adsorption process involves three steps: i) diffusion of the molecules from the bulk to the sub-interface; ii) transfer of the molecules from the sub-interface to the interface; iii) rearrangement of the molecules at the interface (Biswas M.E. et al. (2005) Modeling of adsorption dynamics at air-liquid interfaces using statistical rate theory (SRT). J. Colloid Interface Sci 286: 14-27.).
  • SRT statistical rate theory
  • the surface tension may reflect predominantly the properties of peptide monomers and small peptide assemblies, rather than those of the large peptide aggregates.
  • ANS fluorescence depends pronouncedly on the local probe environment. The binding of ANS to peptide monomers may not significantly affect its fluorescence properties as it still "feels" surrounding solvent molecules (i.e., water in this case). Only when the ANS probe is enclosed in a different environment from the solvent does its fluorescence greatly change. Therefore, the observed changes in ANS fluorescence in Figure 88b should result from the properties of peptide assemblies/aggregates. This is probably why the difference between EAKI6-II and EAKI6-IV from surface tension is not observed by the ANS fluorescence.
  • protonated ellipticine can be stabilized by ionic interaction with the negatively charged residues (glutamic acid E in this case) of the peptide.
  • the ellipticine microcrystals are stabilized by peptide assemblies coating on the surface. When ellipticine is protonated, it can dissolve in aqueous solution and cause the solution to have a yellow, transparent appearance. On the other hand, the suspended ellipticine microcrystals make the solution turbid and cloudy.
  • EAKl 6-11 and EAKl 6-IV can stabilize protonated or crystalline ellipticine while ellipticine stabilized by EFKl 6-11 may be predominantly in microcrystal form.
  • the molecular state of ellipticine can be further elucidated by the ellipticine fluorescence spectra. It has been found that protonated ellipticine molecules have a fluorescence peak at ⁇ 520 nm while the fluorescence peak at ⁇ 430 nm is attributed to neutral ellipticine molecules; crystalline ellipticine exhibits a fluorescence peak at ⁇ 470 nm with an extremely low intensity.
  • the fluorescence spectra of the complexes with the three peptides, EAKl 6-II, EAKl 6-IV and EFKl 6-II are shown in Figure 89 b, c and d, respectively.
  • the complexes with 0.5 mg/mL peptide have a fluorescence peak located ⁇ 520 nm, indicating that ellipticine is protonated.
  • the spectra have a peak close to 470 run with an extremely low intensity (insets in Figure 89b and c), representing crystalline ellipticine.
  • the complexes with EFKl 6-11 exhibit a fluorescence spectrum with a major peak located at ⁇ 435nm and a small shoulder covering the wavelengths from 470 to 570 nm ( Figure 89d), very different from those of protonated and crystalline ellipticine.
  • the peak located at -435 nm represents neutral (non-charged) ellipticine, present as individual molecules in a much less polar environment.
  • the peak intensity is proportional to the EFKl 6-11 concentration.
  • crystalline and protonated ellipticine can coexist in the suspensions as indicated by the turbid appearance of the suspensions and a shoulder from the fluorescence spectra.
  • the fluorescence signals from crystalline ellipticine are too small to be seen compared to those of neutral ellipticine.
  • the different quantum yields and overlapping of the fluorescence signals from the three molecular states of ellipticine make it difficult to determine the percentage of each state among the three in the complexes. However, the total amount of stabilized ellipticine can be obtained.
  • ellipticine can be uptaken and stabilized in the solution as protonated, neutral or crystalline ellipticine. Not all given ellipticine can be stabilized and suspended in solution; the deposition of ellipticine thin film can be observed at the bottom of most sample vials.
  • the amount of stabilized ellipticine varies with the types of peptides and peptide concentrations. The highest maximum suspension is found to be -71% (by Wt.) by 0.5 mg/mL EAKl 6-II. At the same peptide concentration, such a value decreases to -56% for EAKl 6-IV and to -46 for EFK 16-11.
  • EAKl 6-11 appears to be the most effective peptide among the three at stabilizing protonated ellipticine (at a high peptide concentration of 0.5 mg/mL.); EFKl 6-11, on the other hand, can stabilize neutral ellipticine (in addition to crystalline and protonated ellipticine), and it has less variation in the maximum suspension with different peptide concentrations. Size of the Complexes
  • the size distribution of the peptide assemblies and complexes at a peptide concentration of 0.5 mg/mL is shown in Figure 91.
  • the peptide assemblies have a broad size distribution from 10 to several hundred nanometers (Figure 91a). They all have a major size population around 30 nm and a second one corresponding to a shoulder located at -300 nm, 100 nm and 200 nm for EAK16-II, EAKl 6-IV and EFKl 6-II, respectively.
  • the size distribution of EAKl 6-11 obtained here correlates well with our earlier findings, and the two populations represent short peptide nanofibers and fiber clusters.
  • the dimensions of the complexes range from hundreds of nanometers to several micrometers regardless of the peptide concentrations. However, the morphology of these complexes looks different according to the peptide concentration. At 0.04 mg/mL, the majority of the complexes are also rod-like although they seem to be shorter and more dispersed than those with EAKl 6-11 and EAKl 6-IV; at higher peptide concentrations, the complexes appear to have irregular shapes. In addition, more membrane-like structures are observed in the background with the increase in EFKl 6-11 concentration. These membrane-like EFKl 6-11 assemblies could play an important role in stabilizing neutral ellipticine molecules.
  • EAKl 6-11 and EAKl 6-IV can solubilize protonated ellipticine or encapsulate ellipticine microcrystals, depending on the peptide concentration.
  • EAKl 6-II can stabilize neutral ellipticine molecules in addition to the other two states in aqueous solution; the amount of neutral ellipticine that can be carried by EFKl 6-11 assemblies is peptide concentration dependent.
  • the size and structure of the complexes also depend on the type of peptide and peptide concentration.
  • Figure 93 shows the viability of both A549 and MCF-7 cancer cells upon being treated with peptide-ellipticine complexes for 48 h.
  • A549 cells Figure 93a
  • all peptide-ellipticine complexes reduce the cell viability to less than 0.3 compared with the viability of non-treated cells (viability is 1).
  • the toxicity of complexes is 2-folds higher than that of the ellipticine control with the absence of peptides (light green bar).
  • the peptide controls have some toxicity to the cells, causing the decrease of viability to the values between 0.6 and 0.8.
  • the protonated ellipticine has a positive which can interact with a negatively charged cell membrane surface, leading to accumulation of ellipticine at the cell membrane surface.
  • a small molecule with a hydrophobic characteristic is expected to cross the cell membrane easily into the cytoplasm.
  • the protonated ellipticine molecules release much faster from the complexes compared with that from ellipticine microcrystals, due to the differences in complex size and a relatively weak interaction between protonated ellipticine and the peptide in the complexes.
  • the viability increases from -0.25 to -0.5 for A549 cells; for MCF-7 cells, it remains unchanged at -0.57 up to 4 times dilution and then slightly increases to -0.65 for 16 times dilution.
  • Such a good stability may result from a stronger interaction between EFKl 6-11 and ellipticine in the complexes due to a higher hydrophobicity of the peptide.
  • a possible increase in solution pH after dilution should not affect the state of the stabilized neutral ellipticine molecules or ellipticine microcrystals. It is worth noting that although these complexes are not as effective as protonated ellipticine at killing cancer cells, their stability is much better, which is especially important for practical applications in clinics where drug dilution always occurs after administration into the bloodstream.
  • EAKl 6-11 can solubilize protonated ellipticine in nano scale complexes with high anticancer activity against both A549 and MCF-7 cells, but these complexes are pH sensitive and not very stable after dilution.
  • the complexes formulated with 0.5 mg/mL EFK 16-11 are more stable upon dilution, but most of their sizes are in the micrometer range and their anticancer activity is relatively low. Nevertheless, these results provide essential information to design an appropriate peptide sequence that would optimize the delivery of hydrophobic anticancer drugs.
  • EFKl 6-11 was able to stabilize both neutral and crystalline ellipticine within the range of tested peptide concentrations; the amount of neutral ellipticine that can be stabilized was proportional to the peptide concentration.
  • the different molecular states of stabilized ellipticine in the complexes greatly affected the anticancer activity of the complexes and their stability upon dilution in water.
  • the complexes with protonated ellipticine were found to be very effective at killing both A549 and MCF-7 cells with a cell viability close to zero; however, these complexes were not very stable and their anticancer activity reduced significantly after serial dilution in water.
  • the mass spectra and HPLC data are presented in Figures 95, 96, 97, 98, 99.
  • the N-terminus and C-terminus of the peptide were protected by acetyl and amino groups, respectively. At pH ⁇ 7, A and F are neutral, while E and K are negatively and positively charged, respectively.
  • the anticancer agent ellipticine (99.8% pure) and l-anilinonaphthalene-8-sulfonic acid (ANS) were purchased from Sigma-Aldrich (Oakville, ON, Canada) and used as received. Tetrahydrofuran (THF, reagent grade 99%) and dimethyl sulfoxide (DMSO, spectral grade >99%) were from Calendon Laboratories Ltd. (Georgetown, ON, Canada) and Sigma-Aldrich (Oakville, ON, Canada), respectively.
  • Cell culture reagents including Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-E I'DA were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Phosphate buffer saline (PBS) and penicillin-streptomycin (p/s, 10000 U) were obtained from MP Biomedicals Inc. (Solon, OH, USA). Sample preparation
  • peptide powder Appropriate amounts of the peptide powder were first dissolved in pure water (18 ⁇ ; Millipore MiUi-Q system) to obtain fresh peptide solutions at concentrations of 0.5, 0.2 and 0.04 mg/ml ("crude" peptide concentration). The solution was then sonicated in a bath sonicator (Branson, model 2510) for 10 min. The peptide solution at a concentration of 0.5 mg/mL was used to study the differences among the three peptides in self-assembled nanostructures, hydrophobicity and surface activity.
  • the peptide-ellipticine complexes were prepared by adding 1 mL of the fresh peptide solution into a glass vial containing a thin film of 0.04 mg ellipticine at the bottom, followed by mechanical stirring at 900 rpm for 24 h. 1 mL of pure water, instead of peptide solution, was also added to another vial to make a control sample.
  • the purpose of using a relatively low ellipticine concentration of 0.04 mg/mL in this study was to obtain distinguishable cellular toxicity of the complexes and the control sample.
  • the amount of suspended ellipticine in solution was determined by the ellipticine UV-absorption.
  • the peptide-ellipticine suspension was diluted 20 times in DMSO (resulting in a solvent mixture of 95% DMSO and 5% water by volume) to dissolve ellipticine from the complexes. 80 ⁇ L of the solution were then transferred to a quartz microcell (70 ⁇ L) with a 1 cm light path and tested on a UV- Vis spectrophotometer (Biochrom Ultraspec 4300 Pro, Cambridge, England).
  • absorbance (Abs) ecd
  • e the molar extinction coefficient
  • c the molar concentration of ellipticine
  • d the optical path length (cm)
  • the extinction coefficient was obtained as 59000 ⁇ 1100 (R 2 >0.995) from the linear fitting of ellipticine absorption as a function of ellipticine concentration (2-20 ⁇ M) prepared in a mixture of 95% DMSO and 5% water.
  • the suspension concentration of ellipticine was averaged from 3 measurements, and compared with the given ellipticine concentration of 0.04 mg/mL. Since not all ellipticine in the thin film at the bottom of the vials could be stabilized and suspended in solution, the comparison of the suspension concentration with the given ellipticine concentration (0.04 mg/mL) would thus provide the maximum percentage of the ellipticine suspension at each formulation condition.
  • Atomic Force Microscopy AFM
  • the peptide self-assembled nanostmcrures were imaged on a PicoScanTM (Molecular Imaging, Phoenix, AZ) in pure water.
  • the samples were prepared with the following procedure: 10 ⁇ L of 0.5 mg/mL peptide solution ( ⁇ 15 min after solution preparation) were put on a freshly cleaved mica substrate, which was fixed on an AFM sample plate; a custom made AFM liquid cell was fastened on top of the mica substrate. The solution was incubated for 10 s to allow the peptide assemblies to adhere to the mica surface. The surface was then washed with pure water 15 times, and 500 ⁇ L of pure water were added into the cell prior to AFM imaging.
  • a scanner with a maximum scan area of 6x6 ⁇ m 2 was used to acquire the AFM images. It was operated with a tapping mode using silicon nitride cantilevers with a nominal spring constant of 0.58 N/m (DNP-S, Digital Instruments, Santa Barbara, CA) and a typical tip radius of 10 nm. For the best imaging quality, the tapping frequency was typically set between 16 kHz and 18 IcHz and the scan rates controlled between 0.8 and 1 line/s. The experiments were conducted in an environmentally-controlled chamber at room temperature to avoid evaporation of the solution. AU AFM images were obtained at a resolution of 256 x 256 pixels.
  • ADSA-P Axisymmtratic Drop Shape Analysis-Profile
  • 60 ⁇ L of the mixed solution were transferred to a quartz microcell and tested on a spectrafluorometer (Photon Technology International, Type QM4-SE, London, Canada) with a continuous xenon lamp as the light source.
  • the sample was excited at 360 nm and the emission spectra were collected from 420 to 670 nm.
  • the excitation and emission slit widths were set at 0.5 mm and 1.25 mm, respectively (0.5 and 1.25 mm corresponds to 2 and 5 nm band path).
  • the spectra were normalized with light scattering of air at 360 nm, to correct the lamp fluctuations.
  • DLS Dynamic Light Scattering
  • SEM Scanning Electron Microscopy
  • AU samples were coated with a 20 nm thick gold layer prior to SEM imaging; the images were acquired using the secondary electron (SE2) mode at 5 kV.
  • SE2 secondary electron
  • In vitro cell viability studies Two types of cancer cells, non-small cell lung cancer cell A549 and breast cancer cell MCF-7 were used for in vitro cellular toxicity studies on the peptide- ellipticine complexes. The cells were cultured in DMEM containing 10% FBS and 1 % p/s at 37°C and with 5% CO 2 .
  • MTT assay was used to determine the cell viability after different treatments.
  • MTT solution was added to each well of the treated plates.
  • the plates were incubated for 4 h prior to the addition of 100 ⁇ L of the solubilization solution (anhydrous isopropanol with 0.1 N HCl and 10% Triton X-100). After overnight incubation, the absorbance at 570 nm was recorded on a microplate reader (BMG FLUOstar OPTIMA) and subtracted by the background signals at 690 nm. The absorption intensities were averaged from 4 replicates for each treatment and normalized to that obtained from the untreated cells (negative control) to generate the cell viability.
  • solubilization solution anhydrous isopropanol with 0.1 N HCl and 10% Triton X-100

Abstract

The invention relates to self assembling β-strand peptides for forming nanostructures, compositions containing the peptides, and methods of forming the peptides. The invention further relates to uses of these peptides in drug delivery and enhancement of drug solubility, biomolecule detection, and biocatalysis applications. The peptides of this invention are further useful in models of protein aggregation disease.

Description

AMINO ACID PAIRING-BASED SELF ASSEMBLING PEPTIDES
AND METHODS FIELD OF THE INVENTION
The present invention relates to self-assembling peptides that form nanostructures, methods of preparing the peptides, and uses of the same. BACKGROUND OF THE INVENTION
The spontaneous organization of molecules into structurally well-defined arrangements due to non-covalent interactions is referred to as "molecular self- assembly". The resulting supramolecular structure typically provides nanoarchitectures with very defined macroscopic properties (Whitesides et al., (1991) Science 254: 1312-1319 "Whitesides et al. (1991)").
The ability of these entities to self-assemble has been a key factor in developing these entities for novel materials. For example, the last decade showed that molecular self-assembly of a biopolymer plays a key role in the discovery and design of biomaterials, with applications in the field of medical technology and, more specifically, regenerative medicine and drug delivery systems (Langer and Vacanti (1993) Science 260:920-926; Hubbell (1996) MRS Bulletin 21(11):33-35).
An important issue in nanotechnology is the spontaneous formation of highly ordered structures at the nanoscale level (Whitesides et al. (1991); Zhang (2003) Biotechnology 21: 1171-1178; Lazzari et al. (2006) J. Nanosci. Nanotechnol. 6:892- 905). The way in which simple building blocks, such as peptides, recognize each other and assemble guides the formation of ordered nanostructures. It is a combination of many different covalent interactions including electrostatic interactions, hydrogen bonds, hydrophobic interactions and aromatic stacking interactions that guide the organization.
Self-assembling peptides have recently emerged as one of the most promising biomolecular materials in bio-nanotechnology research (Zhang et al. (1993) Proc. Natl. Acad. Sci. USA 90:3334-3338 "Zhang et al. (1993)"; Chen (2005) Colloids Surf. A 261 :3-24 "Chen (2005)"; Zhang (2002) Biotechnol. Adv. 20:321-339 'Zhang (2002)'; Aggeli et al. (1999) MoI. Med. Today 5:512-513 "Aggeli et al. (1999)"; and Zhang et al. (1994) Biopoly. 34:663-672). The self-assembly of peptides not only relates to many naturally occurring states of proteins, such as amyloid fibrillogenesis (Aggeli et al. (1999)), but also provides useful biomolecular building blocks for a wide variety of supramolecular fabrications (Zhang et al. (1993); Chen (2005); Zhang (2002)).
Among the new self-assembling peptides is a class of ionic-complementary, amphiphilic peptides, e.g., EAK made of glutamic acid (E), alanine (A), and lysine (K) residues (Zhang et al. (1993) and U.S. Patent No. 5,670,483 to Zhang et al.). This new class of peptides originates from zuotin, a yeast protein that preferentially binds to left-handed Z-DNA (Zhang et al. (1993)). The molecular structure of these peptides contains alternating positive and negative charges, enabling ionic-complementarity. This ionic complementarity, together with hydrogen bonding, hydrophobic, and van der Waals interactions promotes self-assembly of the peptide molecules into highly stable aggregates (Chen (2005); Hong et al. (2003) Biomacromol. 4: 1433-1442 "Hong et al. (2003)"; and Fung et al. (2003) Biophys. J. 85:537-548 "Fung et al. (2003)"). The nano/microstructures constructed from the peptide self-assembly have found many biomedical applications, including scaffolding for tissue engineering (Zhang et al. (1995) Biomater. 16:1385-1393 and Holmes et al. (2000) Proc. Natl. Acad. Sci. USA 97:6728-6733) and biological surface patterning (Zhang (2002)). It has been shown recently that these peptides can encapsulate a hydrophobic organic compound and unload it into a model cell membrane in a controlled manner (Keyes-Bag et al. (2004) J. Am. Chem. Soc. 126:7522-7532). EAKl 6-11 is an example of an ionic-complementary amino acid-based self assembling peptide. EAKl 6-11 peptides demonstrate self-assembly into a variety of configurations in a concentration-dependent manner when plated on a mica surface (Jun et al. (2004) Biophys. J. 87:1249-1259). The critical aggregation concentration (CAC) of EAK16-II was determined to be 0.06 mM (Fung et al. (2003)). Furthermore, it was shown that the sequence of the EAK peptide is a critical determinant of the nanostructure that results upon peptide aggregation (Jun et al. (2004) Biophys. J. 87:1249-1259). pH was also shown to be critical for peptide aggregation (Hong et al. (2003)).
Gazit et al. have studied peptide self-assembly in the context of short peptide fragments that form well-organized amyloid fibrils, responsible for a number of protein aggregation diseases (Azriel and Gazit (2001) J. Biol. Chem. 276:34156- 34161; Gazit (2002) FASEB J. 16:77-83; Gazit (2002) Bioinformatics 18:880-883). It has been also demonstrated that peptides from 3 to 6 amino acids in length and containing aromatic residues can form amyloid fibrils that further assemble to form β- pleated sheets (Maji et al. (2004) Tetrahedron 60:3251-3259). Due to their ability to form β-sheet-rich fibrils, amyloid peptides have a suggested use as building blocks for nano-electronics (reviewed in Reches and Gazit (2006) Current Nanoscience 2: 105-111).
Dipeptides of phenylalanine have been shown to be sufficient to self-assemble into peptide nanotubes (Reches and Gazit (2003) Science 300:625-627). It was then demonstrated that the self-assembled phenylalanine dipeptide can be used to form peptide-nanotube platinum-nanoparticle composites (Song et al. (2004) Chem. Commun. 9:1044-1045). Additional studies have indicated the use of such peptide nanotubes in electrochemical biosensing applications (Yemini et al. (2005) Nano Letters 5: 183-186).
U.S. Patent No. 7,179,784 describes surfactant-like self-assembling peptides that form nanotubes. The disclosed peptides specifically contain amino acids having nonpolar, noncharged side chains in combination with amino acids having cationic
(positively charged) and/or anionic side chains (negatively charged). These peptides are amphiphilic in nature and tend to aggregate in order to isolate the hydrocarbon chain from contact with water. The basic principle behind the self-assembly of these peptides is the formation of a polar interface that separates the hydrocarbon and water regions.
U.S. Patent Application Publication No. 2002/0160471 describes peptide scaffolds useful in the repair and replacement of various tissues. Once again, the application concerns the use of amphiphilic self-assembling peptides having alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible, for this purpose.
U.S. Patent Application Publication No. 2005/0164361 discloses that manipulation of the environment of a self-assembling peptide can assist in the control of the nucleation and propagation of the peptides.
U.S. Patent Application Publication No. 2005/0181973 discloses self- assembling peptides having two amino acid domains. The first domain has alternating hydrophobic and hydrophilic amino acids and mediates self-assembly into macroscopic structures when the amino acids are present in unmodified form. The second amino acid domain is unable to self-assemble on its own. Typical second amino acids domains of the peptides disclosed in this application mimic a biologically active peptide motif found in a naturally occurring protein, such as a component of the basement membrane.
U.S. Patent Application Publication No. 2005/0209145 discloses self assembling amphiphilic peptides capable of binding to growth factors through specific non-covalent interactions. Typical peptides disclosed in this application include an alkyl tail, a β-sheet forming peptide sequence, and a bio-active peptide sequence.
U.S. Patent Application Publication No. 2005/0272662 describes peptide- amphiphile compositions which include a first peptide-amphiphile having a hydrophilic region and an ionic charge, and a second peptide-amphiphile having a hydrophilic region and an opposite ionic charge. Each hydrophilic region has an associated biological signal. Examples of peptides disclosed are YIGSR and IKVAV.
U.S. Patent Application Publication No. 2006/0079454 discloses tubular, planar and spherical nanostructures that consist of aggregated self-assembling peptides. The peptides of the application include aromatic amino acids and can consist entirely of aromatic amino acids, and are no more than 4 amino acids in length.
U.S. Patent Application Publication No. 2006/0084607 discloses amphiphilic peptide chains having alternating hydrophilic and hydrophobic amino acids. The peptides are at least 8 amino acids in length and compatible structurally and are complementary such that they self-assemble into β-pleated sheets, forming a macroscopic scaffold.
Most of the self-assembling peptides known in the art consist of alternating hydrophobic and hydrophilic amino acids or segments of amino acids. There remains a need to design peptides based on properties of amino acids other than charge. SUMMARY OF THE INVENTION
In one embodiment, the present invention provides β-stranded peptides that are self-complementary and assemble into nanostructures. These peptides are designed on the basis of amino acid pairing properties of amino acids. It is demonstrated that these peptides are useful in enhancing solubility of hydrophobic drugs. Various uses of such β-strand peptide-based nanostructures include drug delivery, biomolecule sensory, and biofuel cell applications.
In one embodiment, the invention provides a self-complementary β-strand peptide having alternating hydrogen bonding proton donor amino acid segments and hydrogen bonding proton acceptor amino acid segments, that self assembles into a nanostructure. The peptide has a length from two to forty amino acids. The peptide has at least one proton donor and one proton acceptor segment, each of which consists of at least one amino acid. Such peptides are not comprised of alternating hydrophobic and hydrophilic amino acid segments.
In another embodiment, the invention provides a self-complementary β-strand peptide having one of the following structures: a) (AxByCz)wAz (I), and b) (AxByCz)w(C'xB'yA'z)w (II) A, A', B, B', C and C are each a hydrogen bonding amino acid, and are either a proton donor or a proton acceptor amino acid; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20. A is complementary to A', B is complementary to B', and C is complementary to C. In another embodiment, the invention provides a self-complementary β-strand peptide having one of the following structures: a) AxByCz ... ; and (III), and b) AxByCz... Cz'By'Ax' (IV).
A, A', B, B', C and C are each independently a donor amino acid or an acceptor amino acid, and are each self-complementary. These amino acids are further selected from the group consisting of a hydrogen bond donor amino acid, a hydrogen bond acceptor amino acid, a positively charged amino acid, a negatively charged amino acid, and a van der Waals' interacting amino acid. A is complementary to A', B is complementary to B', and C is complementary to C.
In another embodiment, the invention provides a self-complementary β-strand peptide having at least one hydrogen bonding amino acid pair, at least one ionic- complementary amino acid pair, and at least one hydrophobic amino acid pair, for foπning a nanostructure. The peptide has a length from four to forty amino acids.
In another embodiment, the invention provides a self-assembled nanostructure consisting of aggregated units of a peptide having one of the following structures: a) (AxByCz)w I; and b) (AxByCz)wAx II.
A, B and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20. The nanostracture formed from the self assembled peptides is one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
In another embodiment, the invention provides a self assembled nanostructure consisting of aggregated units of a peptide having the general formula (V):
(AwBxAyCz)nAaBb (V).
A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10. The nanostructure formed from the self assembled peptides is one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
In another embodiment, the invention provides pharmaceutical compositions comprising the β-strand peptides described in combination with a therapeutic agent. In another embodiment, the invention provides a kit for delivering a material to a patient, including a pharmaceutical composition comprising a self assembled β- strand peptide and a therapeutic agent; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; instructions for preparing the pharmaceutical composition for use; instructions for mixing the composition with other agents; and instructions for introducing the composition into a subject.
In another embodiment, the invention provides a method of preparing a self- assembling peptide having amino acid pairing properties for manufacture of a nanostructure. The method includes the steps of: designing a β-strand peptide consisting of amino acids that are capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction with a complementary amino acid; and generating a peptide from two to forty amino acids in length consisting of at least one amino acid pair capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction, and having complementary amino acid pairing and stereochemistry with a second peptide.
In another embodiment, the invention provides a method for detecting a biomolecule of interest. The method includes the steps of: forming a nanostructure from a β-strand peptide upon self assembly of the peptide; adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; coupling a reporter molecule capable of providing a measurable signal to the peptide- coated surface of the nanostructure; and providing the biomolecule of interest. In another embodiment, the invention provides a use of a β-strand peptide, for identification of inhibitors of protein aggregation disease.
BRIEF DESCRIPTION OF THE FIGURES
These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:
Figure 1 is a flow chart of amino acid pairing (AAP)-based self assembling peptide design.
Figure 2A is a photograph of a glass vial containing pyrene in the absence of peptide
(0 rng/mL) or in the presence of EAKl 6-11 peptide (AEAEAKAKAEAEAKAK) (0.5 mg/mL). The chemical structure of pyrene is shown below.
Figure 2B is a photograph of a glass vial containing 0.1 mg/mL ellipticine in the presence of decreasing concentrations of EAKl 6-11 peptide (0.5, 0.1 and 0 mg/mL).
The last vial (far right) is a control without EAKl 6-11 peptide. The chemical structure of ellipticine is shown below. Figure 3A is a graph depicting the release rate of pyrene from the EAKl 6-11 peptide as a function of pyrene concentration (measured in μmol/L) against time (measured in hours) (from Keyes-Baig et al. (2004) J. Am. Chem. Soc. 126: 7522-7532 "Keyes-
Baig et al. (2004)").
Figure 3B is a scanning electron (SE) micrograph of pyrene: EAK at a ratio of 78: 1 in solution. Dotted lines extend from the micrograph to the graph of Figure 3A to corresponding pyrene release curves (from Keyes-Baig et al. (2004)).
Figure 3C is a SE micrograph of pyrene:EAK at a ratio of 16:1 in solution. Dotted lines extend from the micrograph to the graph of Figure 3A to corresponding pyrene release curves (from Keyes-Baig et al. (2004)). Figure 4 is a series of AF micrographs of β-amyloid peptide (1-42 amino acids) on a hydrophobic HOPG surface (A-C) and on a mica surface (D-H). Images were captured at 512 s intervals. A schematic of the β-amyloid peptide amino acid sequence is depicted in (I). Arrows indicate that the Aβ assembly on hydrophobic graphite (HOPG) and mica may dictate Aβ aggregation at the hydrophobic interior of the lipid membrane and the hydrophilic (negatively charged) head portion, respectively.
Figure 5 is a schematic of nanofiber nucleation and growth of EAK 16-11 peptides on a mica surface. Peptide monomers are indicated by rectangular boxes. Charges within the peptide are indicated (- and +).
Figure 6 is a schematic of peptide monomer assembly on a mica surface where positive charges within the peptide are attracted to the negative charge of mica. Two peptides are shown, oriented horizontally. Figure 7 is a series of schematics of EAKl 6-11 peptide dimers assembling on a mica surface in solutions of varying pH: A) water (neutral); B) 1 mM HCl; C) 10 mM HCl; D) 1 mM NaOH; and E) 10 mM NaOH. In 10 mM NaOH, the peptide dimers do not adsorb on mica, resulting no fiber formation. This illustrates how solution pH controls the surface-assisted peptide assembly on mica. Figure 8 indicates the chemical structures of peptides of the present invention consisting of amino acids capable of hydrogen bonding. The arrangement of hydrogen bonding pairs in a synthesized peptide was varied in terms of the number of repeating pair units (peptide length), for example QN and NS versus QNQN and NSNS or NSNSNSNS, and the design of the repeating unit itself (e.g. QN versus QQNN). Figure 9A is a graph of absorbance plotted against wavenumber (cm"1) determined using Fourier Transform Infrared (FTIR) spectroscopy for peptides QN, QNQN, QQNN and NS4 (NSNSNSNS). FTIR spectra were used to determine the secondary structures of the peptides on the surface (dried). Figure 9B is a graph of Circular Dichroism (CD) (mdeg) plotted against wavelength (nm) for peptides QN, QNQN, QQNN and NS4. CD spectra were used to determine the secondary structures of the peptides in the bulk solution.
Figure 10 is a graph of Thiofiavine T (ThT) assay fluorescence spectra data for peptides NS4, QNQN, QN, QQNN and ThT alone. Normalized intensity is plotted against wavelength (nm). The increase in ThT fluorescence responds to the abundance of ordered β-sheets in solution.
Figure HA is a scanning electron microscopy (SEM) image of a QN peptide (peptide concentration of 0.1 mg/mL). Figure HB is a SEM image of a QNQN peptide (peptide concentration of 0.1 mg/niL).
Figure HC is a SEM image of a QQNN peptide (peptide concentration of 0.1 mg/niL). Figure HD is a SEM image of a NS peptide (peptide concentration of 0.1 mg/mL).
Figure HE is a SEM image of a NSNS peptide (peptide concentration of 0.1 mg/mL).
Figure HF is a SEM image of a NSNSN peptide (peptide concentration of 0.1 mg/mL).
Figure HG is a SEM image of a NSNSNSNS peptide (peptide concentration of 0.1 mg/mL).
Figure 12A is a graph of ThT assay fluorescence spectra data for varying concentrations (mM) of the NS4 peptide. The arrow points to a peak in the graph indicative of the increase of ThT fluorescence according to peptide concentrations.
Normalized intensity is plotted against wavelength (nm). Figure 12B is a graph of the data in Figure 12A plotting normalized intensity values against the log concentration (μM). The arrow points to a peak in the graph indicative of the critical aggregation concentration (CAC).
Figure 13 A is a schematic of the chemical structure of the ACS peptide (FEFQFNFK) of the present invention. Figure 13B is a schematic of the molecular structure of the AC8 peptide. An ionic bonding pair, a hydrogen bonding (HB) pair and a hydrophobic residue are indicated at the arrows.
Figure 14A is a graph of surface tension (measured in mJ/m2) as a function of time for the AC8 peptide at varying concentrations indicated. Figure 14B is a graph of the equilibrium surface tension obtained from Figure 14A as a function of peptide concentration (μM). The intersection of the two straight lines indicates the CAC.
Figure 15A is a graph of ThT fluorescence spectra data plotting fluorescence intensity (a.u.) against wavelength (nm) for the AC8 peptide. Figure 15B is a graph of the ThT fluorescence spectra data of Figure 15A plotted against peptide concentration (μM). The intersection of the two straight lines indicates the CAC of ACS of approximately 20 μM. Figure 16A is a graph of 8-anilino-l-naphthalenesulfonic acid (ANS) fluorescence spectra data (a.u.) for the AC8 peptide plotted against wavelength (run). The arrow indicates peak fluorescence at 475 nm.
Figure 16B is a graph of the ANS fluorescence spectra data of Figure 16A plotted against peptide concentration (μM). The intersection of the two straight lines indicates the CAC of AC8 of approximately 20 μM.
Figure 17A is a graph of static light intensity plotting normalized light intensity data for the AC8 peptide against peptide concentration (μM). The intersection of the two straight lines indicates the CAC of AC 8 of approximately 20 μM. Figure 17B is a graph of the number-based size distribution (%) (hydrodynamic diameter in nm) for varying concentrations of AC8 peptide.
Figure 18A is an Atomic Force (AF) micrograph of self-assembled AC8 peptides at a concentration of 2.2 μM.
Figure 18B is an AF micrograph of self-assembled AC8 peptides at a concentration of 5 μM.
Figure 18C is an AF micrograph of self-assembled AC8 peptides at a concentration of lO μM.
Figure 18D is an AF micrograph of self-assembled AC8 peptides at a concentration of 40 μM. Figure 18E is an AF micrograph of self-assembled AC8 peptides at a concentration of 87 μM.
Figure 18F is a graph of absorbance plotted against wavenumber (cm"1) determined using FTIR spectroscopy for the AC8 peptide, indicating β-sheet rich secondary structure. Figure 19 is a schematic of AC8 peptide monomer assembly at concentrations below the CAC (left side) and above the CAC (right side), where concentrations of AC8 above the CAC result in a mixture of mature fibers, protofibrils and peptide monomers.
Figure 2OA is a graph of MCF-7 cell viability (measured as % viability) in the presence of various concentrations of EAK peptide (μg/mL) in combination with ellipticine (red bars) compared to EAK peptide alone (blue bars). Black and green bars represent the medium and water solvent control, respectively. Figure 2OB is a photograph of a series of glass vials containing various concentrations of EAK peptide (mg/mL) or water alone with 0.1 mg/mL ellipticine.
Figure 2OC is a SE micrograph of 0.1 mg/mL ellipticine in solution with 0.5 mg/mL
EAK peptide. Figure 2OD is a SE micrograph of 0.1 mg/mL ellipticine in solution with 0.1 mg/mL
EAK peptide.
Figure 2OE is a SE micrograph of 0.1 mg/mL ellipticine alone in solution.
Figure 21A is a graph of A549 cell viability in the presence of various solutions (cell culture medium, H2O, 3.3% DMSO); ellipticine (EPT) alone (0.1 mg/mL); or various peptides (EAK-p, EAKIC, EFK, NS4, FEQNK, QN, QNQN or QQNN) at a concentration of 0.1 mg/mL with (red bars) or without ellipticine (blue bars). All experiments looking at cell viability in the presence of peptide were conducted on cells cultured in the presence of serum.
Figure 21B is a graph of A549 cell viability in the presence of various solutions (cell culture medium, H2O, 3.3% DMSO); ellipticine (EPT) alone: or various peptides
(EAK-p, EAKIC, EFK, NS4, FEQNIC, QN, QNQN or QQNN) with (darker bars) or without ellipticine (lighter bars). All experiments looking at cell viability in the presence of peptide were conducted on cells cultured in the absence of serum for the first 4 hour incubation. Figure 22 is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various peptides in the presence of ellipticine (EPT). Arrows point to crystal EPT, neutral EPT and protonated EPT, respectively.
Figure 23A is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various concentrations of AC8 peptide (μM) in combination with 0.04 mg/mL ellipticine (in 1.33% DMSO), indicating ability of AC8 peptide to solubilize EPT.
Figure 23B is a graph of the data of Figure 23A plotting fluorescence intensity against peptide concentration, showing peptide concentration-dependent solubilization of EPT. Figure 24A is a graph of A549 cell viability in various control media (1.3% DMSO,
PBS, H2O, medium alone). Figure 24B is a graph of A549 cell viability in the presence of EAK peptide at varying concentrations in 1.3% DMSO in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
Figure 24C is a graph of MCF-7 cell viability in various control media (1.3% DMSO, PBS, H2O, medium alone).
Figure 24D is a graph of MCF-7 cell viability in the presence of EAK peptide at varying concentrations in 1.3% DMSO in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
Figure 24E is a graph of fluorescence emission spectra of EPT with different AC8 concentrations of 5, 9 and 100 μg/mL combined with 0.04 mg/mL EPT. The solutioin contains 1.3% DMSO. The solutions were tested on both A549 and MCF-7 cells as shown in Figure 24B and D.
Figure 25A is a series of bar graphs depicting A549 cell viability in the presence of medium alone; H2O; varying concentrations of EAK peptide (125 or 25 mg/mL); 25 mg/mL AC8 peptide; ellipticine in water (EPT-H; 25 μg/mL); or ellipticine in DMSO
(EPT-D; 25 μg/mL) for various time points over the course of 48 hours of treatment
(intervals are indicated at the tops of the graphs). All peptide-EPT combinations were prepared in the absence of DMSQ. Red/dark purple bars indicate the presence of EPT with the peptide while blue/light purple bars indicate peptide alone. Figure 25B indicates similar data as in Figure 25A for MCF-7 cells.
Figure 26A is a bar graph depicting A549 cell viability upon culture in medium (M), water (H2O), or varying concentrations (μg/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
Figure 26B is a bar graph depicting the effects of ACS peptide dilution on A549 cell viability in the presence of ellipticine (EPT) (Dl=2-fold dilution; D2=4-fold dilution;
D3=8-fold dilution; and D4=l 6-fold dilution).
Figure 26C is a bar graph depicting MCF-7 cell viability upon culture in medium (M), water (H2O), or varying concentrations (μg/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT). Figure 26D is a bar graph depicting the effects of AC8 peptide dilution on MCF-7 cell viability in the presence of ellipticine (EPT) (Dl=2-fold dilution; D2=4-fold dilution; D3=8-fold dilution; and D4=16-fold dilution). Figure 27A is a graph of emission fluorescence (normalized intensity) plotted against wavelength (nm) for various concentrations of AC8 peptide (mg/mL) in combination with 0.04 mg/mL ellipticine.
Figure 27B is a zoom-in graph of fluorescence emission (normalized intensity) plotted against wavelength (nm) from Figure 27A.
Figure 27C is a bar graph depicting A549 cell viability upon culture in medium (M), water (H2O), or varying concentrations (μg/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT).
Figure 27D is a bar graph depicting the effects of AC8 peptide dilution on A549 cell viability in the presence of ellipticine (EPT) (Dl=2-fold dilution; D2=4-fold dilution;
D3=8-fold dilution; and D4=l 6-fold dilution).
Figure 27E is a bar graph depicting MCF-7 cell viability upon culture in medium (M), water (H2O), or varying concentrations (μg/mL) of AC8 peptide in the presence (red bars) or absence (blue bars) of ellipticine (EPT). Figure 27F is a bar graph depicting the effects of AC8 peptide dilution on MCF-7 cell viability in the presence of ellipticine (EPT) (Dl=2-fold dilution; D2=4-fold dilution; D3=8-fold dilution; and D4= 16-fold dilution).
Figure 28 is a series of plots of AOD,- as a function of total EAK peptide concentration at pH 4 (A-C) and pH 7(D-F) at varying concentrations of dGi6 (A, D), dCie (B, E) and dGCi6 (C, F) oligonucleotides.
Figure 29 is a pair of plots of v/P/ versus v for the binding of EAK peptide to a guanine hexadecamer (dGi6), a cytosine hexadecamer (dCi6), and their duplex (dGC^) at pH 4 (A) and pH 7 (B).
Figure 30 is a series of plots of fluorescence anisotropy of the supernatant of an EAK peptide solution mixed with 3.6 μM of: (A) FAM-dGCi6 at pH 4 (A6x =452 nm, λem
=514 nm); (B) FAM-dGCi6 at pH 7 (λex =494 nm, λem =514 nm); (C) FAM-dQe at pH
4 (Λ« =452 nm, Aem =514 nm); and (D) FAM-dC]6 at pH 7 (/W =494 nm, Aem =514 nm).
Figure 31 is graph of anisotropy (o) and the calculated percentage (■) of the FAM- dCi6 in aggregates upon the addition of EAK peptide.
Figure 32 is a pair of plots of UV absorption spectra of dCi6-Rh in the presence and absence of EAK at pH 7. (A) 3.9 μM dC,6-Rh (•), 60 μM EAK(+), 3.9 μM dC,6-Rh and 60 μM EAK before centrifugation (-) and after centrifugation (x); (B) dCi6-Rh at concentration of 3.9 μM (•), 2.0 μM (-), and 1.3 μM (x).
Figure 33 is a photoimage of 20 % PAGE of 3.6 μM of dGQe mixed with EAK at (A) pH 4 and (B) pH 7 (the EAK concentrations are 0, 6, 60, 120 μM in lanes 1, 2, 3, and 4, respectively).
Figure 34 is a pair of plots of fluorescence anisotropy of a 0.1 mg/mL EAK solution containing 1 mol% of FAM-EAK in the presence (•) and in the absence (o) of (A)
3.6 μM of dGie at pH 4 (/U =452 nm and λem=5H nm) and (B) 4.3 μM of dGi6 at pH
7 (Xex =494 nm and λem =514 nm). Figure 35 is a pair of plots of data obtained from Steady States Light Scattering (SLS) experiments performed on (o) buffer and solutions containing (A) 3.6 μM dGi6 and
0.1 mg/mL EAK, (□) 0.1 mg/mL EAK, (x) 3.6 μM dG]6 at: (A) pH 4 and (B) pH 7. nm.
Figure 36 is a series of Atomic Force Microscopy (AFM) images of the EAK- oligonucleotide (ODN) complexes formed in a solution containing 3.6 μM dGjβ and
0.1 mg/mL EAK, imaged in solution at pH 4 at: (A) 8 mins.; (B) 60 mins.; (C) 70 mins.; and (D) 75 mins. Images in C and D are zoomed in views of the marked areas in B and C, respectively.
Figure 37 is a population histogram of the EAK-ODN complexes as a function of particle diameter determined by dynamic light scattering for EAK-ODN solutions containing 7.2 μM of dGiό with increasing EAK concentration at pH 4. The sample of
EAK alone was measured 40 minutes after preparation, whose concentration was 0.1 mg/mL (60 μM); the concentration of dGi6 alone was 7.2 μM.
Figure 38 is pair of Stern- Volmer plots for a solution of 3.6 μM of fluorescently labeled dCi6 free and bound to 0.2 mg/mL of EAK at pH 4 quenched by KI. Solid lines represent the fits to the Stern- Volmer equation with parameters listed in Table
12. (A) FAM-dC]6 (A), FAM-dCi6-EAK (D), and (B) dCi6-Rh (A), dCiβ-Rh-EAK
(□).
Figure 39 is a graph depicting scattered light intensity (measured in absorbance units by Dynamic Light Scattering (DLS)) plotted against time (hours) for 0.5 mg/mL
EAKl 6-II peptide (filtered with a 0.22 μm) filter) in water, 1 mM HCl, 10 mM HCl or 1O mM NaOH. Figure 40 is a time course series of AF micrographs showing EAKl 6-11 peptide nanofiber growth on a mica surface in pure water from adsorbed nanofiber "seeds"(top panels) and fiber clusters (bottom panels). The green arrows indicate the nanofiber growth from both active ends of the "seed" and the red arrows points to a reference spot (top panels).
Figure 41 is a series of AF micrographs showing nanostructure formation of 2 μM EAK16-II peptides on a mica surface under conditions of increasing pH (10 mM HCl, 1 mM HCl, H2O, ImM NaOH, and 1OmM NaOH, respectively). Lines are drawn from the various micrographs to a graph plotting zeta potential (mV) against solution pH at the pH value corresponding to the solution.
Figure 42A is a graph of nanofiber frequency (measured in %) on a mica surface plotted against fiber growth rate (measured in nm/s) for 2 μM EAKl 6-11 peptide in solutions of varying pH (1 mM HCl, water, and 1 mM NaOH). Figure 42B is a table correlating solution conditions with pH and average nanofiber growth rate for 2 μM EAKl 6-11 peptide on mica.
Figure 43A is an AF micrograph of EAK16-II (0.05 mg/mL) on a mica surface, imaged in water.
Figure 43B is an AF micrograph of EAKl 6-11 (0.05 mg/mL) on a HOPG surface, imaged in water. Figure 44A is a schematic of peptide monomer self-assembly on a HOPG surface. Arrows indicate direction of nanofiber growth. In order to maximize the hydrophobic interactions between the peptide and the HOPG surface, peptide molecules tend to arrange themselves following the HOPG lattice to cover the most carbon atoms. This leads to the orientation of the peptide nano fibers at 60 or 120 degree angles with each other.
Figure 44B is an AF micrograph of 10 μM EAKl 6-11 peptide assemblies on a HOPG surface, showing fiber alignment at 60° and 120° angles, resembling the HOPG lattice shown in Figure 44A. Figure 44C is a schematic of an EAK16-II peptide monomer with an indication of coverage of HOPG lattices based on the dimension of the peptide and the HOPG lattice. Figure 45 is a series of photomicrographs indicating change in amphiphilicity of the surfaces upon the modification with peptides: A) water droplet on mica (no peptide); B) water droplet on EAK-modified mica surface; C) water droplet on HOPG (no peptide); and D) water droplet on EAK-modified HOPG surface. Figure 46A is an AF micrograph and corresponding cross section analysis on a bare HOPG surface. Figure 46B is an AF micrograph showing EAK peptide-modified HOPG surface.
Figure 46C is an AF micrograph and corresponding cross section analysis of glucose oxidase (GOx) molecules on a bare HOPG surface. The rough background indicates that the GOx may be denatured on the HOPG surface. Figure 46D is an AF micrograph showing GOx on an EAK peptide-modified HOPG surface. More ball-shape GOx appeared on the modified HOPG surface as compared to Figure 46C, showing that the modified surface provides a more biocompatible environment for GOx adsorption.
Figure 47 is a schematic of the chemical reactions that occurs between glutamic acids (E) from EAK peptides and the amine groups of enzymes such as GOx on a modified surface. EDC: l-ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS: n- hydroxysuccinimide.
Figure 48 is a series of graphs cyclic voltammograms of 1 mM K3Fe(CN)6 depicting the electrochemical characterization of an EFK peptide/HOPG and a bare HOPG electrode for varying scan rates from 2 mV/s to 100 mV/s. At low scan rates, the presence of EAKl 6-11 nanofiber coatings does not block the electron transfer significantly.
Figure 49 is a graph of the cyclic voltammetry of a GOx-immobilized EAK peptide/HOPG electrode, plotting current (μA) against potential (V) compared to Ag/AgCl at a scan speed of 2 mV/s. Figure 50 is a graph depicting current (μA) as a function of potential (V) compared to Ag/AgCl for a GOx/EAK peptide/HOPG electrode (20 mM glucose, 1 cm2 HOPG). Figure 51A is a graph of GOx-immobilized EAK peptide/HOPG electrode current (μA) plotted against time (seconds) with different glucose concentrations (0-20 mM). Figure 5 IB is a graph of GOx-immobilized EAK peptide/HOPG electrode current (μA) plotted against glucose concentration (mM). The data can be fitted using nonlinear regression with the equation below to obtain the Imax and the K Mran.- I A -1 nnBIaX - maximum current; Km: Michaelis constant for enzyme-substrate complex. Figure 52 is a graph indicating storage stability of a GOx-immobilized EAK peptide/HOPG electrode up to one month.
Figure 53 is a graph showing operational stability of a GOx-immobilized EAK peptide/HOPG electrode. Figure 54A is a schematic of a biofuel cell using a GOx-immobilized EAK peptide/HOPG electrode.
Figure 54B is a schematic indicating the mechanism of electron (e-) and proton (H+) transfer of an O2/H2 biofuel cell using the laccase and hydro genase enzymes. Figure 55 is a schematic of a metallic nanowire fabrication based on self-assembly of a modified EAK peptide coupled with electrochemical reduction and enhancement steps.
Figure 56 illustrates absorption spectra of siRNA in pH 7.3 HEPES buffer with increasing peptide concentration (from 0 to 40 μM). (from top to bottom) Figure 57 illustrates hypochromicity of siRNA at 260 nm as a function of R9 concentration for siRNA concentrations of 1.5 μM (o), 3.0 μM (■), and 4.5 μM (^). Solid lines are the line of best fit generated by Prism. Error bars represent the largest standard deviation from 3 replicates at each siRNA concentration. Figure 58 illustrates hypochromicity of siRNA at 260 nm as a function of +/- charge ratio for siRNA concentrations of 1.5 μM (o), 3.0 μM (■), and 4.5 μM (*). Solid lines are the line of best fit generated by Prism.
Figure 59 illustrates CD spectra of 3.0 μM siRNA (top) and 100 μM R9 (bottom) in HEPES buffer (6 mM HEPES-NaOH, 20 mM NaCl, 0.2 mM MgC12, pH 7.3). The spectra showed a typical A-DNA structure for the siRNA and random coil structure for R9. Figure 60 illustrates circular dichroic spectra of siRNA in pH 7.3 HEPES buffer with increasing peptide concentration (from 0 to 40 μM). (from top to bottom) Figure 61 illustrates the relative change in ellipticity of siRNA at 260 nm as a function of R9 concentration for siRNA concentrations of 1.5 μM (o) and 3.0 μM (■). Solid lines are the line of best fit generated by Prism. Figure 62 illustrates the relative change in ellipticity of siRNA at 260 nm as a function of +/- charge ratio for siRNA concentrations of 1.5 μM (o) and 3.0 μM (■). Solid lines are the line of best fit generated by Prism. Figure 63 illustrates hydrodynamic diameter and Zeta potential of CTGF siRNA-R9 complexes at 1.5 μM siRNA. Zeta potential of siRNA and siRNA-R9 complexes is expressed in solid bars; Zeta potential of R9 is represented by diagonal bar; and size is represented by a solid line. Error bars represent the standard deviation from 3 replicates.
Figure 64 illustrates absorbance at 260 nm of siRNA (1.5 μM) and siRNA-R9 (1.5 μM / 150 μM) complex solution upon 2 M salt addition. The absorbance of siRNA only and siRNA-R9 complex solutions are monitored prior to salt addition (white), 2 hours after salt addition (diagonal), and one day after salt addition (black). Error bars represent the standard deviation from 3 replicates.
Figure 65 illustrates calculated binding isotherm for CTGF siRNA-R9 complexes. Following the analysis developed by Bujalowski and Lohman, the calculated free peptide concentrations were infeasible. Figure 66 illustrates the molecular structure of EAKl 6-II: AEAEAKAKAEAEAKAK; A is alanine, E glutamic acid and K lysine.
Figure 67 illustrates the ellipticine fluorescence from the peptide-ellipticine suspension over time. (A) Fluorescence spectra of ellipticine as a function of time; (B) the normalized fluorescence intensities at 468 nm (diamonds) and 520 nm (squares) as a function of time. The ellipticine concentration is 1.0 mg/mL and the peptide concentration is 0.2 mg/mL.
Figure 68 illustrates static light scattering of 0.2 mg/mL EAKl 6-11 solution at 400 nm before (diamonds) and after mechanical stirring for 30 h (squares). Figure 69 illustrates the effect of peptide concentration on the complex formation. The normalized fluorescence intensities of peptide-ellipticine suspensions as a function of time at 468 nm (A) and 520 nm (B). The ellipticine concentration was fixed at 1.0 mg/mL with various EAKl 6-11 concentrations ranging from 0 to 0.5 mg/mL.
Figure 70 illustrates the effect of ellipticine concentration on the complex formation. The normalized fluorescence intensities of peptide-ellipticine suspensions as a function of time at 468 nm (A) and 520 nm (B). The 0.2 and 0.5 mg/mL EAKl 6-11 were used with different ellipticine concentrations from 0.1 to 1.0 mg/mL. Figure 71 illustrates the fluorescence spectra of the peptide-ellipticine suspensions after 24 h stirring with 0.1 mg/mL ellipticine and various peptide concentrations of 0- 0.5 mg/mL. Inset indicates the crystalline ellipticine fluorescence. Figure 72. (A) The time-dependent ellipticine fluorescence showing the release of ellipticine from the complex made of 0.05 mg/mL EAK 16-11 and 0.1 mg/mL ellipticine into the EPC vesicles. (B). The transfer profiles of ellipticine from different peptide-ellipticine complexes to the EPC vesicles. The complexes were made of 0.1 mg/mL ellipticine with various EAKl 6-11 concentrations: 0.05 (triangles), 0.1 (crosses), 0.2 (squares) and 0.5 mg/mL (circles). The solid lines represent the fitting curves to the data points using either Equation 2 or Equation 3. The excitation and emission wavelengths are 295 and 436 nm, respectively.
Figure 73. SEM images of the peptide-ellipticine complexes with 0.1 mg/mL ellipticine and different EAKl 6-11 concentrations: (a) 0.5 mg/mL, (b) 0.2 mg/mL and (c) 0.05 mg/mL. Figure 74. Viability of MCF-7 and A549 cells treated with the complexes for 24 h at different peptide-to-ellipticine ratios (a) and upon serial dilution (b). The complex at 5: 1 ratio was used for the serial dilution. The complexes were prepared with a fixed ellipticine concentration of 0.1 mg/mL with various EAKl 6-11 concentrations of 0.02- 1.0 mg/mL. Figure 75. Time-dependent toxicity of the EAK16-II-ellipticine complexes against MCF-7 (a) and A549 (b) cells. EPT-H2O: ellipticine control (in pure water). Figure 75.1 Fluorescence images showing cellular uptake of ellipticine in A549 (a) and MCF-7 (b) cells. Green color is from ellipticine fluorescence. The first column shows the phase contrast images with corresponding fluorescence images as the insets. * denotes half exposure time.
Figure 75.2 Fluorescence images showing cellular uptake of ellipticine at 37 0C and 4 0C in A549 and MCF-7 cells with different treatments. Green color represents ellipticine fluorescence. First column shows phase contrast images, and the insets are the corresponding fluorescence images. Figure 76. Intensity-based size distribution of the EPC vesicles.
Figure 77. Photographs of the peptide-ellipticine suspensions after 24 h stirring with 0.1 mg/mL ellipticine and various peptide concentrations of 0-0.5 mg/mL. Figure 78. (a) Calibration curve of various ellipticine concentrations in the EPC vesicles, (b) Corresponding UV absorption of ellipticine in (a).
Figure 79 illustrates relative UV-vis absorbance change (ΔODr) over time of the dilute-5 (solid) and dilute-10 (crosshatch) solutions generated by mixing 8.6μM dGiβ with: (A) 10.5 μM EAK16IV at pH 4; (B) 48.2 μM EAKl 6IV at pH 7; (C) 24.1 μM EAK16II at pH 4; and (D) 60 μM EAK 1611 at pH 7.
Figure 80 illustrates (A) the absorption spectra of solution made of 5.0 μM dCjβ and 120 μM EAK16II at pH 4. The supernatant of the solution after centrifugation (x); The solution was dried, resuspended in pH 11 buffer, and vortexed. Before (D) and after (-) centrifuging the resulting solution. (B) Relative UV-vis absorbance change (ΔODr) as a function of time for the sample obtained by resuspending aggregates made of 8.6 μM dCi6 and 0.1 mg/mL EAK16-IV at pH 4 (o) and pH 7 (•) in pH 9.5 buffer. Figure 81 illustrates the fluorescence spectra of an 8.6μM Fl-dCi6-Rh solution with orwithout 60μM EAKl 6IV at pH 4. Half an hour after preparation, the solutions at pH 4 were diluted 10 times with pH 4 buffer and kept at 250C for 1 day. The EAK-ODN aggregates were then incubated with 0.7 U/μL exonuclease I: (A) without EAKl 6IV: 20 min (Δ), 30 min (+), and control, i.e., without the nuclease treatment (o); (B) with EAKl 6IV: 20 min (+), 60 min (Δ), 90 min (-), and non-treated control (x). The solution was centrifuged 1 day after preparation.
Figure 82 is a calibration curve correlating the normalized /D//A ratio to the percentage of degraded ODNs. The /D//A ratio was normalized to that of the intact Fl- dCi6-Rh. The solid line represents the best fit to the equation: normalized /D//A = A X % degraded ODN+1 with A = 0.0410 ± 0.002 and R2 = 0.999. Figure 83 illustrates (A) the effect of the EAK peptide sequence on ODN protection against nuclease degradation. About 8.6 μM Fl-dCi6-Rli was mixed with 60 μM EAK16IV or EAK16II for 30 min at pH 4 (crosshatch) and pH 7 (solid) before it was diluted 10 times. The solution was centrifuged 1 day after preparation. (B) shows the percentage of Fl-dCi6-Rh in the EAK-ODN aggregates one day after 10-fold dilution at pH 4 (crosshatch) and pH 7 (solid).
Figure 84 illustrates (A) the fluorescence spectra of the EAK-ODN aggregates prepared with 8.6μM of FWCi6-Rh and lOμM of EAK16IV at pH 4. control (x), nuclease-treated 30 min and the solution was centrifuged one day after sample preparation (■). (B) the percentage of degraded ODN in the EAK-ODN aggregates as a function of EAK16IV concentration.
Figure 85 illustrates the effect of centrifugation on the nuclease resistance of the EAK-ODN aggregates made of 8μM FWCi6-Rh and 60μM EAKl 6IV at pH 4: (A) 30 min after sample preparation, the solution was (D) or was not (•) centrifuged and then diluted 10 times. 24 h later, the resulting solution was incubated with exonuclease I. The percentage of degraded ODN was recorded over time; (B) 30 min after preparation, the solution was centrifuged before it was diluted 10 times. About 24 h later, the tJV-vis spectra of the resulting solution (x) and its supernatant after centrifugation (») were recorded; (C) percentage of degraded ODN. The EAK-dCi6 mixture solution was centrifuged for the first time 24 h after sample preparation, and accumulatively centrifuged once a day for the next 3 days. The resulting solution was incubated with exonuclease I for 60 min. Figure 86 illustrates the molecular structures and sequences of EAK16-II, EAKl 6-IV and EFKl 6-II. N and C termini are protected by acetylation and amidation, respectively.
Figure 87 illustrates AFM images of the peptide nanostructures: (a) EAKl 6-II; (b) EAKl 6-IV; (c) EFKl 6-II. The peptide concentration is 0.5 mg/mL.The scale bar is 200 urn. Figure 88. The hydrophobicity of the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 and their assemblies by dynamic surface tension (a) and ANS fluorescence (b). The inset is the ANS fluorescence control with the absenceof peptides. The peptide concentration is 0.5 mg/mL, and the ANS concentration is 10 μM. Figure 89 illustrates the formation of peptide-ellipticine complexes, (a) are photographs of the complexes with the three peptides at different peptide concentrations and the ellipticine in pure water as a control. The normalized fluorescence spectra of ellipticine in the complexes with EAK16-II (b), EAK16-IV (c) and EFKl 6-11 (d). The insets show the spectra of the complexes with low peptide concentrations. Figure 90 illustrates the maximum suspension (%) of ellipticine in aqueous solution stabilized by the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 and with the absence of peptides. Figure 91 illustrates the size distribution of the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at 0.5 mg/mL in pure water (a) and the complexes with 0.5 mg/mL
EAKl 6-π and EAKl 6-IV (b) by DLS.
Figure 92 is SEM images of the complexes with the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at different peptide concentrations and ellipticine crystals in pure water as the control.
Figure 93 illustrates the cellular toxicity of the peptides EAK16-II, EAK16-IV and
EFK16-II and their complexes with ellipticine for A549 cells (a) and MCF-7 cells (b).
The viability of non-treated cells is 1 (M: cells were treated with culture medium). For the solvent control, cells were treated with pure water (dark green bar); for the drug control, cells were treated with ellipticine in pure water with the absence of peptides
(light green bar). Blue bars represent the peptide controls where no ellipticine was added.
Figure 94 illustrates cellular toxicity of the complexes formulated with the three peptides EAKl 6-II, EAKl 6-IV and EFKl 6-11 at a peptide concentration of 0.5 mg/mL and their serial dilutions in water for A549 cells (a) and MCF-7 cells (b). EPT: ellipticine.
Figure 95 illustrates the mass spectrum of EAK16-II.
Figure 96 illustrates the mass spectrum of EAKl 6-IV. Figure 97 illustrates the mass spectrum of EFK16-II.
Figure 98 illustrates HPLC data of EAKl 6-II. The purity of the peptide is around
73%.
Figure 99 illustrates HPLC data of EAKl 6-IV. The purity of the peptide is around
83%. DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns peptides that self-assemble into various nanostructures. These peptides are designed based on the capacity of the individual amino acids to participate in hydrogen bonding, electrostatic, hydrophobic and van der Waals' interactions. The resulting self assembled nanostructures can be used in a variety of technological or biomedical functions, including drug delivery and solubility, biosensors and biofuel cells.
In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, a person skilled in the art will understand, given the context, that circumstances exist in which the invention may be practiced without specific preferred features.
In the following description, reference is made to certain terms of the art, some of which are defined below. "Amino acids" are defined as any of the 20 essential amino acids. These include those that are naturally occurring as well as non-natural amino acids such as D-forms, β and γ derivatives. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as follow: alanine (Ala, A); arginine (Arg, R); asparigine (Asp, N); aspartic acid (Asp, D); cysteine (Cys, C); glutamine (GIn, Q); glutamic acid (GIu, E); glycine (GIy, G); histidine (His, H); isoleucine (He, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (VaI, V).
The term "peptide" as used herein refers to a chain of amino acids. In particular, the peptide is from 2 to 40 amino acids in length.
The term "self-assembly" as used herein refers to the process of atoms, molecules or peptides forming regular shaped structures in response to general conditions in the environment. In particular, self-assembly refers to the aggregation of peptides into an ordered structure. The term "self-assembling peptide" as used herein refers to a peptide that can interact noncovalently with another peptide, of the same or different amino acid sequence to form an organized structure, under near thermodynamic equilibrium conditions.
The term "hydrogen bonding" as used herein refers to chemical bonding in which a hydrogen atom of one molecule is attracted to an electronegative atom, especially a nitrogen or an oxygen, usually of another molecule. Hydrogen bonding occurs between amino acids of peptides and, more particularly, hydrogen bonding occurs between atoms of the amino acids that form the peptide backbone. However, hydrogen bonding between atoms in the amino acid side chains contributes to the stabilization and induces assembly of the peptide. Side chain interactions are more important in shorter peptides.
The term "β-strand" as used herein refers to a single continuous stretch of amino acids adopting an extended conformation and involved in hydrogen bonding. The teπn "β sheet" refers to an assembly of β-strands that are hydrogen- bonded to each other, β-strands arrange to form β-sheets in parallel or anti-parallel arrangement. A determination of parallel or anti-parallel conformation is based on the arrangement of the peptide direction from N to C terminus. In parallel arrangement, all peptides are aligned in the same direction from N to C terminus. In anti-parallel conformation, alternating peptides are aligned in opposite direction (i.e. the first peptide is aligned N to C terminus relative to a second peptide which is aligned C to N terminus). Parallel arrangement can involve peptide shifting resulting in peptide termini that are staggered relative to one another. At least half the length of the peptide is involved in interpeptide interactions. In anti-parallel arrangement, peptides typically align to provide flush ends. This is typically indicative of end-to-end complementary peptides.
The term "ionic-complementarity" as used herein refers to the characteristic of an alternating arrangement of negatively and positively charged residues in a specific pattern. Typical charge distributions are: type I (-+); type II (—++); and type IV ( — ++++), and any repetitions of these charge distributions or combinations thereof.
The term "hydrophobic" as used herein refers to the tendency of a substance to repel water or to be incapable of completely dissolving in water.
The term "hydrophilic" as used herein refers to the property of being able to readily absorb moisture and having strongly polar groups that readily interact with water.
The teπn "amphiphilic" as used herein refers to the tendency of a substance to have both hydrophobic and hydrophilic properties. Amphiphilic peptides consist of polar, water-soluble amino acids (hydrophilic) and nonpolar, water-insoluble hydrocarbon amino acids (hydrophobic) arranged in a specific sequence so that the peptide molecule has distinguishable hydrophilic and hydrophobic regions.
The teπn "functional moiety" as used herein refers to an additional, short peptide sequence that confers some particular biological activity to the peptide, and is capable of associating with other molecules/materials. Examples of functional moieties include molecular and cell recognition sequences, cell membrane penetration sequences, and metal ion binding motif. Functional moieties confer specific function to peptides, however, they do not necessarily contribute to peptide self-assembly. The term "cell targeting moiety" as used herein refers to a short peptide sequence that can selectively associate with specific cells. The targeting moiety is capable of binding to a cell surface and preferably comprises a member of a binding pair, for example an antibody or parts thereof (e.g. single chain, Fab-fragments), or a receptor ligand. In another preferred embodiment, the targeting moiety comprises a low molecular weight protein such as, but not limited to, lysozyme. Cell targeting moieties are not involved in peptide self-assembly.
The term "nanostructure" as used herein refers to a structure with arrangement of its parts in the nanometre scale. Nanostructures can form any shape of any one of one (ID), two (2D), or three dimensions (3D), including nanosurfaces, nanofibrils, nanorods, nanowires, nanofiber networks, nanospheres, nanospirals, and combinations thereof. Nano textured surfaces have one dimension on the nanoscale, i.e., only the thickness of the surface of an object is between 0.1 and 100 nm. Nanotubes have two dimensions on the nanoscale, i.e., the diameter of the tube is between 0.1 and 100 nm; its length could be much greater. Finally, spherical nanoparticles have three dimensions on the nanoscale, i.e., the particle is between 0.1 and 100 nm in each spatial dimension.
The term "oligonucleotide" as used herein refers to a molecule composed of 30 or fewer nucleotides. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), modified or unmodified. RNA may be in the form of small nuclear RNA (snRNA), microRNA (miRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), antisense RNA, short hairpin RNA (shRNA), small interfering RNA (siRNA), and ribozymes. The oligonucleotides may be single stranded or double stranded. The term "biosensor" as used herein refers to a device that detects, records, and transmits information regarding a physiological change or process. In particular, a biosensor uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds, usually by electrical, thermal or optical signals. The term "biofuel cell" as used herein refers to a device that transforms raw materials into electrical power in the presence of biocatalysts, enzymes, or whole cell organisms. In particular, biomaterials, such as self-assembling peptides, participate in biofuel cell activity by providing a biocompatible environments for enzyme immobilization and potentially improving the electron transfer between the fuel substrates and oxidizers and the electrodes.
The term "therapeutic agent" as used herein refers to any compound or composition for treating a disease or condition and includes, without being limited thereto, drugs, small protein molecules and oligonucleotides. Suitably, therapeutic agents include, but are not limited to, anticancer agents including paclitaxel, ellipticine, camptothecin, doxorubicin and adriamycin, and oligonucleotide-based agents. Hydrophobic and hydrophilic agents are included in the definition.
The term "stabilizing agent" as used herein refers to the materials that are introduced to a peptide-drug complex to enhance the complex stability in vivo. It will be appreciated by one skilled in the art that the stabilizing agent will be chosen in order to avoid detrimental interaction or reaction with other components in a composition, or reduce the solubility of pharmaceutically active agent. Examples of stabilizing agents for use with the present invention include fatty ester of glycerol, fatty ester of polyethylene glycol (PEG), fatty ester of propylene glycol, fatty acid,
Labrasol(R), Capmul MCM(R), Captex 200 (R), Captex 300 (R) or mixtures thereof.
The term "pharmaceutical excipient" as used herein refers to an inactive substance used as a carrier for the active ingredients of a medication. Without being limited thereto, pharmaceutical excipients may suitably be selected from one or any combination of dextrose, sorbitol, mannitol, starch, dextrin, maltodextrin, lactose, magnesium stearate, calcium stearate, talc, microcrystalline cellulose, hydroxypropylmethylcellulose and hydroxyethylcellulose. Other appropriate excipients may be used.
The term "biocatalyst" as used herein refers to an enzyme that catalyzes the oxidation/reduction reaction.
The term "electrode" as used herein refers to an electrically conductive material that transfers electrons to or from the reduction/oxidation reaction center.
The term "mediator of electron transfer" as used herein refers to a molecule that helps transfer electrons from the electrochemical reaction center to the electrode The term "fuel source" as used herein refers to the materials that can undergo oxidation/reduction reaction to generate electronic current.
Self-assembling peptides designed based on amino acid pairing properties may be used to form a wide variety of nanostructures. An Amino Acid Pairing (AAP) strategy of the present invention for designing self-assembling peptides is based on the ability of amino acids to interact with one another through at least one of ionic complementarity, hydrogen bonding, hydrophobic and van der Waals' interactions (Figure 1). The AAP-based peptide design provides complementary interactions that achieve certain stereochemical and physicochemical stability, resulting in pair affinity and minimum pairing free energy. For example, asparagine-asparagine (asn-asn or N- N) pairs are possible and can be included in the peptide sequence due to the ability of Asn to participate in hydrogen bonding interactions both as a proton donor and as a proton acceptor. Nanostructures form when self-assembling peptides approach each other and undergo pairing interactions between complementary amino acids, typically forming antiparallel or parallel β-sheet secondary structures.
Of the 20 essential amino acids, 13 have been identified as capable of hydrogen bonding via corresponding side chains. Those amino acids acting as proton acceptors include arginine, tiytophan, tyrosine, lysine, histidine, aspartic acid, threonine, cysteine, serine, asparigine, glutamic acid, methionine and glutamine. All of these amino acids, except for methionine, also act as proton donors. Bonding pairs were identified based on the positions of the proton acceptor and proton donor within the amino acid. Bonding pairs were further classified as being more soluble or less soluble. More soluble hydrogen bonding amino acids that function as proton acceptors at the 3 position are aspartic acid and histidine. Aspartic acid is capable of hydrogen bonding with 3 -position proton donor amino acid serine while histidine is capable of hydrogen bonding with threonine. Similarly, 4-position proton acceptors tryptophan, histidine and glutamine hydrogen bond with proton donor aspartic acid, while arginine and glutamic acid hydrogen bond with 4-position proton donor asparigine. 5- position proton acceptor tyrosine hydrogen bonds to the corresponding proton donors glutamic acid and arginine, while lysine hydrogen bonds with glutamine, tryptophan and histidine. Less soluble hydrogen bonding amino acid pairs include: asparigine (3- position proton acceptor) and threonine, serine or cysteine; methionine (3 -position proton acceptor) with cysteine or serine; 4-position proton acceptor glutamine with asparagine; and 5-position proton acceptor tyrosine with glutamine or tryptophan as indicated in Table 4.
Peptides containing less soluble hydrogen bonding amino acid pairs, from 2-8 amino acids in length (from 1 to 4 amino acid pairs), can be synthesized. Exemplary peptides consist of glutamine (Q)-asparagine (N) or asparagine (N)-serine (S) amino acid pairs. Peptides may be subjected to end protection consisting of acetylation at the amino terminus and amidation at the carboxy terminus. Peptides may have a varying number of amino acids, as well as varying numbers of contiguous hydrogen bonding pairs. For example, single pair peptides can be synthesized (e.g. QN, NS) as well as double (e.g. QNQN and NSNS or QQNN) and quadruple pairs (e.g. NSNSNSNS).
In addition to peptides only of amino acids forming hydrogen bonding pairs, peptides having amino acids that participate in all of ionic pairing, hydrophobic pairing, and hydrogen bonding can be synthesized. Alternating hydrophobic and hydrophilic amino acid residues contribute to the amphiphilicity of the peptide.
In one embodiment, the invention provides a method of preparing a self- assembling peptide having amino acid pairing properties for manufacture of a nanostructure. The method includes the steps of: designing a β-strand peptide consisting of amino acids that are capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction with a complementary amino acid; and generating a peptide from two to forty amino acids in length consisting of at least one amino acid pair capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction, and having complementary amino acid pairing and stereochemistry with a second peptide.
Fourier Transform Infrared (FTIR), Circular Dichroism (CD) and Thioflavine T (ThT) fluorescence spectra may be used to analyze the secondary structure of the peptides. FTIR spectra can be useful in indicating the ability of the peptides to form β-sheet structures while ThT fluorescence can be used to indicate β-sheet rich peptide assemblies.
In one embodiment, the invention provides a self-complementary β-strand peptide having alternating hydrogen bonding proton donor amino acid segments and hydrogen bonding proton acceptor amino acid segments, that self assembles into a nanostructure. The peptide has a length from two to forty amino acids. The peptide has at least one proton donor and one proton acceptor segment, each of which consists of at least one amino acid. Such peptides are not comprised of alternating hydrophobic and hydrophilic amino acid segments. In one embodiment, a /3-strand peptide of the present invention further includes at least one functional moiety at at least one of an N-terminus and C- terminus of the peptide. In one embodiment, the functional moiety is selected from a cell targeting moiety, a metal ion binding motif and a cell membrane penetration moiety.
In one embodiment, the hydrogen bonding occurs between the side chains of complementary amino acids of the peptides of the present invention. In one embodiment, the complementary peptides assemble into a parallel confoπnation. In another embodiment, the complementary peptides assemble into an anti-parallel conformation. The peptides can assemble in either an end-to-end or staggered peptide arrangement. In one embodiment, peptides of the present invention can assemble into a staggered arrangement, wherein between one and 20 amino acids in the peptide form hydrogen bonds with complementary amino acids in a second peptide.
In one embodiment, the hydrogen bonding proton donor amino acid for a β- strand peptide of the present invention is selected from the group consisting of Arg, Trp, Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu and GIn. In one embodiment, the hydrogen bonding proton acceptor amino acid for a /3-strand peptide of the present invention is selected from the group consisting of Arg, Trp, Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu, Met and GIn. In one embodiment, the present invention provides a self-complementary β- strand peptide comprising at least one hydrogen bonding amino acid pair, at least one ionic-complementary amino acid pair, and at least one hydrophobic amino acid pair, and having a length from four to forty amino acids, for forming a nanostructure. In one embodiment, this peptide has the formula: (AwBxAyCz)nAaBb (V) where A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10. Suitably, the the hydrophobic amino acid is selected from the group consisting of VaI, He, Leu, Met, Phe, Trp, Cys, Ala, Tyr, His, Thr, Ser, Pro, GIy, Arg and Lys. Suitably, the charged amino acid is selected from the group consisting of His, Arg, Lys, Asp and GIu. In one embodiment, the present invention includes an amino acid sequence Phe-Glu-Phe-Gln-Phe-Asn-Phe-Lys (AC8) (SEQ ID NO: 6). The invention further includes self-assembled nanostructures of this peptide.
In another embodiment, the invention provides a self-complementary β-strand peptide having one of the following structures: a) (AxByCz)wAz (I), and b) (AxByCz)w(C'xB'yA'z)w (II)
A, A', B, B', C and C are each a hydrogen bonding amino acid, and are either a proton donor or a proton acceptor amino acid; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20. A is complementary to A', B is complementary to B', and C is complementary to C. In another embodiment, the invention provides a self-complementary β-strand peptide having one of the following structures: a) AxByCz... ; and (III), and b) AxByCz...Cz'By'Ax' (IV).
A, A', B, B', C and C are each independently a donor amino acid or an acceptor amino acid, and are each self-complementary. These amino acids are further selected from the group consisting of a hydrogen bond donor amino acid, a hydrogen bond acceptor amino acid, a positively charged amino acid, a negatively charged amino acid, and a van der Waals' interacting amino acid. A is complementary to A', B is complementary to B', and C is complementary to C.
In another embodiment, the invention provides a self-complementary β-strand peptide having at least one hydrogen bonding amino acid pair, at least one ionic- complementary amino acid pair, and at least one hydrophobic amino acid pair, for forming a nanostructure. The peptide has a length from four to forty amino acids.
Self-complementary |3-strand peptides of the present invention include GIn- Asn, Gln-Asn-Gln-Asn (SEQ ID NO: 1), Gln-Gln-Asn-Asn (SEQ ID NO: 2), Asn-Ser, Asn-Ser-Asn-Ser (SEQ ID NO: 3), Asn-Ser-Asn-Ser-Asn (SEQ ID NO: 4), and Asn- Ser-Asn-Ser-Asn-Ser-Asn-Ser (SEQ ID NO: 5). The invention further includes nanostructures formed of these peptides.
The peptides of the present invention may be used to form various nanostructures including nanofibers and nanofiber networks, nanotubes, nanowires, nanospheres and nanospirals, as well as combinations of these structures. Peptides present at a Critical Aggregation Concentration (CAC) may assemble into various structures in a concentration-dependent manner. At concentrations below the CAC, peptides may form a mixture of single layer protofibrils and monomers while above the CAC, peptides may form mature fibers and fiber bundles, or other nanostructures. The nanostructure formed by the peptide aggregation is dependent on the concentration of the peptide in solution as well as the pH of the solution (reviewed in Chen (2005) Colloids and Surfaces A: Physiochem. Eng. Aspects 26:3-24.) The CAC of a peptide may be determined using ThT fluorescence as well as surface tension, ANS fluorescence, and steady state light scattering assays. In another embodiment, the invention provides a self-assembled nanostructure consisting of aggregated units of a peptide having one of the following structures: a) (AχByCz)w I; and b) (AxByCz)wAx II.
A, B and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20.
The nanostructure formed from the self assembled peptides can be one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
In another embodiment, the invention provides a self assembled nanostructure consisting of aggregated units of a peptide having the general formula (V): (AwBxAyCz)nAaBb (V).
A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10. The nanostructure foπned from the self assembled peptides can be one of a nanofibril, a nanowire, a nanosurface and a nanosphere.
The peptides of the present invention can be used in controlled release drug delivery applications. Previously, it was demonstrated that EAKl 6 peptides could be used to encapsulate pyrene, a highly hydrophobic compound (Figure 2; Keyes-Bag et al. (2004) J. Am. Chem. Soc. 126: 7522-7532). It further demonstrated that the ratio of pyrene to peptide resulted in different surface coatings (Figure 3). The presently disclosed peptides may be used to solubilize hydrophobic therapeutic agents, such as ellipticine, an anti-cancer drug in its protonated or neutral form. Furthermore, the peptides are useful as drug delivery vehicles. The solubilized neutral ellipticine by the newly designed peptides was more protected and stable inside the peptide vehicles upon 16-fold dilution in water. The peptides may also be used in gene and oligonucleotide delivery applications. The present inventors have also discovered that EAK- 16-1, EFKl 6-11 and EAKl 6-11 can be used to solubilize hydrophobic therapeutic agents and for gene and oligonucleotide delivery applications.
Pharmaceutical compositions of the present invention can include the disclosed peptides and any suitable excipient or stabilizer as is known to a person skilled in the art. Further the mode of administration of the therapeutic agents and pharmaceutical compositions of the present invention are not particularly restricted and suitable modes of administration are within the purview of persons of skill in the art and include without limitation oral administration and intravenous administration.
In one embodiment, the invention provides a kit for delivering a therapeutic agent to a patient, including a phaπnaceutical composition comprising a self assembled β-strand peptide and a therapeutic agent; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; instructions for preparing the pharmaceutical composition for use; instructions for mixing the composition with other agents; and instructions for introducing the composition into a subject.
The peptides of the present invention may assemble on different surfaces, either hydrophobic or hydrophilic in nature. It has been demonstrated that the β- amyloid peptide (amino acids 1-42) is capable of assembling on different templates including mica (hydrophilic) and HOPG (hydrophobic graphite) (Figure 4; Kowalewski and Holtzman (1999) 96:3688-3693). The size and shape of the β- amyloid aggregates, as well as the kinetics of their formation, were shown to be dependent on the physicochemical nature of the surface. Amphiphilic peptides may be seeded onto charged, hydrophilic surfaces, such as mica, and elongate into nanofibers in a concentration-dependent manner (Figures 5, 6). The driving force for the amphiphilic peptide deposited on a charged surface is primarily electrostatic interaction ("peptide-surface interaction"). Peptides readily align at both ends of the peptide and "seed" to achieve nanofiber growth. The growth of the nanofiber can be subsequently controlled by adjusting the pH of the solution, shown in the schematic of Figure 7. The same peptides seeded onto a hydrophobic surface such as HOPG can assemble into different nanostructures. For example, EAKl 6 peptides align at 60° and 120° angles to one another. Thus, peptides of the present invention can be shaped into specific nanostructures depending on the nature of the surface on which they are seeded. Peptide-modification of a surface can result in changes to the surface wettability. Peptide-modified surfaces may be used for biomolecule sensing applications. Peptides may bind to biomolecules such as enzymes for use in sensing enzymatic substrates, and immobilize the biomolecules onto a surface.
In one embodiment, the present invention provides a fuel cell. The ability of the peptides of the present invention to assemble on different surfaces to fabricate bioactive electrodes makes them useful for biofuel cell applications. A biofuel cell is a device that generates electricity from a fuel source by an electrochemical process using biocatalysts. Peptides of the present invention can be immobilized onto electrodes where they are bound to biosensory molecules such as enzymes. Enzymatic substrates can be added to drive the catalytic reaction. Such biofuel cells have strong storage and operational stabilities.
In one embodiment, the invention provides a use of a β-strand peptide, for identification of inhibitors of protein aggregation disease.
Peptides of the present invention may be used to generate models to study protein conformation diseases, such as Alzheimer's and prion disorders. Similar to the β-amyloid peptide (1-42 amino acids), peptides of the present invention can be seeded onto various surfaces and studied for assembly as well as for inhibition of assembly.
Peptides of the present invention may be used in proteomics and bioinformatics applications. Molecular dynamics simulations can be applied to screen all potential pairs of amino acids that can complementarily interact with each other.
This information is useful in peptide library development. Specific applications include use in manufacturing protein/peptide and DNA arrays for high throughput screening and validation studies. Peptides can be used to modify array surfaces where the functional residues of the peptide can immobilize DNA and protein molecules onto the array.
In one embodiment, the invention provides a method for detecting a biomolecule of interest. The method includes the steps of: forming a nanostructure from a β-strand peptide upon self assembly of the peptide; adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; coupling a reporter molecule capable of providing a measurable signal to the peptide- coated surface of the nanostructure; and providing the biomolecule of interest. In one embodiment, and without being limited thereto, the biomolecule is selected from proteins, nucleic acids, carbohydrates and viruses. In one embodiment, the biomolecule of interest is glucose.
Peptides of the present invention may be used for various coating applications including: coating for the prevention of biofouling; biocompatible surface coating; and coating for functionalized chromatographic columns. Peptides can be used to modify surface coatings and to provide a biocompatible environment for protein/enzyme immobilization.
The sequence of the peptide determines the secondary structure and nanostructure of the self-assembled peptide aggregation, and influences their applicability in drug delivery, chemical sensing, biofuel cells, and models for the study of protein aggregation diseases.
The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention. EXAMPLE 1: HYDROGEN BONDING AMINO ACID PAIRING TAAP)
Amino acids that participated in hydrogen bonding, and the position of the atoms serving as proton donors and proton acceptors, are given in Table 1. Amino acids capable of pairing are given in Table 2. Soluble and less soluble hydrogen bonding amino acid pairs are listed in Tables 3 and 4, respectively. The existence of repulsion forces results in the resistance of amino acids hydrogen pairing in cases of amino acids that contains a charge (see Table 3). For this reason, non-charged amino acid pairs capable of hydrogen bonding were of particular interest.
Table 1. List of amino acids that can participate in hydrogen bonding and the position of the atoms in the amino acids that serve as acceptor and donor
Table 2. Classifications of amino acids that can participate in hydrogen bonding in terms of their function (donor or acceptor) and the position of the atoms in the amino acids that serves as acceptor and donor
Table 3. Classifications of soluble amino acid hydrogen bonding pairs in terms of the position of the atoms in the amino acids that serves as hydrogen bonding acceptor and donor
* denotes the requirement of the neutral amino acid in order to participate in hydrogen bonding with others. This can be done by adjusting solution pH to protonate or deprotonate the amino acids. Table 4. Classifications of less soluble amino acid hydrogen bonding pairs in terms of the position of the atoms in the amino acids that serves as hydrogen bonding acceptor and donor
Q-N and N-S amino acid pairs represented the most hydrophilic amino acid pairs except the pairs involving charged amino acids (E, D, R and K). Peptides were designed and synthesized such that they contained varying combinations of either Q- N or N-S amino acid pairs. The arrangement of hydrogen bonding pairs in a synthesized peptide was varied in terms of the number of repeating pair units (peptide length) and the design of the repeating unit itself. Various peptides designed and evaluated for self-assembly are listed in Tables 5 and 6.
Table 5. Peptide sequences containing single Q-N-type 1 pair, repetitive Q-N- type 1 pair and Q-N-type 2 pair
Table 6. Peptide sequences containing single N-S-typel pair and repetitive N-S- typel pairs
The secondary structure components of the QN and NS peptides listed in Tables 5 and 6 (also shown in Figure 9) were characterized with Fourier Transform Infra Red
(FTIR) and circular dichroism (CD) spectroscopy. Both techniques showed that all the peptides adopted a mixture of secondaiy structures including β-sheets, α-helices and random coils. In addition, the β-sheet content was found to increase with an increase in the number of repetitive amino acid pair units (compare the absorbance at 1620 cm" x (β-sheet) for QN, having a single pair unit, with that of QNQN, having two pairing units). It was found that the β-sheet content in type 1 amino acid arrangement (single amino acid pairing unit, e.g. QN) was higher than that in type 2 (repeated amino acid pairing unit, e.g. QQNN).
Among all the hydrogen bonding amino acid pairing peptides tested, NSNSNSNS (NS4) contained the maximum β-sheet secondary structure (-50%), as shown in Figures 9 A and 9B. This result was consistent with results of the ThT assay, where the increase in the fluorescence signals of ThT corresponds to an increase in the amount of β-sheets in the peptide assemblies. As shown in Figure 10, the NS4 peptide provided the maximum fluorescence intensity of ThT, indicating the peptide contained maximum content of β-sheets.
The self-assembled nanostructures of various QN and NS hydrogen bonding pair peptides provided at a concentration of 0.1 mg/ml were determined (Figures 1 IA-G). Most of peptides tested were shown to be capable of self-assembly into various nanofibril structures. Peptide QQNN showed less fibrillar, but more amorphous aggregation (Figure 11 C). Even peptides containing a single amino acid pair, such as QN and NS were shown to form nano fibrils (Figures HA and HD, respectively).
The hydrodynamic size of a peptide aggregation in pure water was characterized using a dynamic light scattering approach. The results of the assay are given in Table 7. The average hydrodynamic size of a peptide aggregation was found to be ~ 100 nm. The apparent size of the aggregation in pure water indicated that the larger aggregates observed with scanning electron microscopy (SEM) were formed in aqueous solutions rather than on the mica surface.
Table 7. The hydrodynamic diameter of 300 μM hydrogen bonding based amino acid pairing peptides
It has been shown previously that peptide self-assembly is concentration-dependent (Fung et al. (2003) Biophys. J. 85:537-548). The concentration dependence of the hydrogen bonding pair NS4 peptide was studied by ThT fluorescence assays as this peptide had previously demonstrated maximum fluorescence intensity in the assay. As shown in Figure 12A, the fluorescence intensity of ThT at 480 nm was maintained upon increase in starting peptide concentration. Once the peptide concentration reached ~90 mM, the fluorescence intensity was shown to increase, indicative of a critical aggregation concentration (CAC) of -90 mM for the NS4 peptide (Figure 12B). EXAMPLE 2: All-complementary based amino acid pairing peptide
A peptide was designed such that it contained each of hydrogen bonding, electrostatic, and hydrophobic bonding amino acid pairs. The resulting peptide was referred to as AC 8. AC 8 contains 8 amino acids in sequence with one exemplary hydrogen bonding pair (QN), one exemplary ionic-complementary pair (EK), and two hydrophobic residue pairs (FF) (Figure 13A). Hydrophobic amino acids were incorporated to create a hydrophobic interior for encapsulation and stabilization of hydrophobic compounds (Figure 13B). These hydrophobic residues also enhanced the peptide-peptide association; the charged residues enhanced the solubility of the peptide and resulting peptide assemblies; and the hydrogen bonding amino acid pairs stabilized the peptide assemblies.
Concentration dependent assembly of the AC8 peptide was studied by surface tension measurements (Figures 14A and 14B), fluorescence spectroscopy using ThT (Figures 15A and 15B) and ANS assays (Figures 16A and 16B), static light scattering technique (Figure 17A), and dynamic light scattering (Figure 17B). The results of each of these assays indicated that the critical aggregation concentration (CAC) of AC8 was around 10-15 μM as shown in Figures 14B, 15B, 16B and 17A. The size and the morphology of AC8 peptide assemblies in pure water were determined (Figures 18A-E). The hydrodynamic size of the AC8 peptide assemblies was found to increase with increasing peptide concentration from the DLS measurements (Figure 17B). AFM images showed that the AC8 peptide formed nanofiber bundles at peptide concentrations above the CAC; however, below the CAC, the AC8 peptide formed a single fiber layer of 1 nm in height. The secondary structure of the peptide was characterized with FTIR technique as shown in Figure 17F and shown to be predominantly β-sheet structure. Based on the results obtained, a model of AC8 assembly was proposed (Figure 19). Below the CAC, the peptide formed a single layer of β-sheet fibers; above the CAC, the β-sheet fibers were shown to further interact with each other via hydrophobic residue pairs and hydrogen bonding pairs. EXAMPLE 3: EAK peptides for use in drug encapsulation
The utility of the EAK peptides for delivering a hydrophobic compound was tested for the hydrophobic anticancer agent ellipticine. Previously, it was shown that pyrene solubility was increased in the presence of EAKl 6-11 peptides (Figure 2) (Keyes-Baig et al. (2004) J. Am. Chem. Soc. 126: 7522-7532). The solubility of pyrene was shown to be further dependent on the concentration of EAKl 6-11 peptide in solution (Figure 2). The encapsulated pyrene can be released into liposome cell mimics. The release rates can be controlled by adjusting the peptide-to-pyrene ratio during the formulation (Figure 3) Increased concentration of EAK peptide was shown to increase the solubility of the more protonated ellipticine in pure water (Figure 20B). The effects of EAK-ellipticine complexes on cancer cells was evaluated in the MCF-7 cell line. Cell viability was not affected by any control treatment (Figure 20A), however, cell viability was shown to be reduced upon treatment with ellipticine in complex with increasing concentrations of EAK (Figure 20A). SE micrographs indicated the size of ellipticine-peptide complexes in the presence (Figures 2OC and D) and absence of EAK peptide (Figure 20E).
EXAMPLE 4: Use of AAP peptides in hydrophobic drug solubilization and delivery
Peptides tested for their ability to deliver the hydrophobic anticancer agent ellipticine (0.1 mg/mL) were: 1) the ionic-complementary peptides EAKl 6-11 (crude -70% and pure > 95% having the amino acid sequence AEAEAKAKAEAEAKAK), EAKKl 6 (crude, having the amino acid sequence AEAEAKAKAKAKAKAK), EFKl 6-II (crude, having the amino acid sequence FEFEFKFKFEFEFKFK); T) hydrogen bonding-based peptides NS4 (90% pure, amino acid sequence NSNSNSNS), QN (95% pure), QNQN (92% pure), QNQN, and QQNN (92% pure); and 3) peptides containing each of hydrogen bonding, ionic and hydrophobic bonding amino acid pairs including FEQNK (or AC8, 95% pure, having the amino acid sequence FEFQFNFK). The peptide concentrations were fixed at 0.1 mg/mL for complexing with ellipticine. The formulation consisted of 3.3 % (v/v) DMSO in aqueous solution and the steps of: a) dissolving a high concentration of ellipticine in DMSO; b) preparing a 0.1 mg/niL peptide solution in pure water and sonicating for 10 minutes; and c) adding aliquots of ellipticine-DMSO into peptide solutions to achieve a final ellipticine concentration of 0.1 mg/mL (3Ox dilution). Peptide-mediated delivery of ellipticine was tested in two cancer cell lines: the non-small cell lung cancer cell line A549 (1x104 cells/well seeded in 96 well plate) and the breast cancer cell line MCF-7 (2x104 cells/well seeded in 96 well plate). Cells were treated with the peptide-drag complexes at a final ellipticine concentration of 25 μg/mL in medium, for 24 hours. Cell viability was determined using the MTT assay.
Initial experiments were conducted on A549 cells cultured in medium with (Figure 21A) or without serum (Figure 21B), in order to investigate the effects of serum on peptide-drug complexes. Cells were first cultured in serum-free medium for 4 hours. It was demonstrated that complexes of ellipticine (0.1 mg/mL in 3.3% DMSO) with any of EAKl 6 -II, EFKl 6-11 or AC8 peptides were effective in killing A549 cells when compared to cells treated with the uncomplexed drug alone after 24 hour treatments (indicated by the arrows in Figures 21A and B). There was no toxicity associated with the peptides alone as determined by cell viability assays. Serum was not found to have a significant effect on the treatment, indicating that the complex remained stable in serum. The fluorescence spectra data for each peptide complex with ellipticine is given in Figure 22. The complexes with the AC8 and EFK 16-11 peptides exhibited a peak at ~430nm, indicating the presence of a neutral form of ellipticine (Fung et al. (2006) J. Phys. Chem. 110: 11446-11454), which must be stabilized in the relatively hydrophobic environment provided by the peptides. The corresponding fluorescence spectra of the peptide-drug complexes are shown in Figure 22. It was shown that the complexes with EFKl 6-11 and AC8 exhibited a strong fluorescence signal at ~430 nm while other peptide-drug complexes did not. The complexes with EAKl 6-11 (pure) had fluorescence signals at ~ 470 nm and -520 nm; the former represents crystalline form of ellipticine while the later indicates the ellipticine is protonated. Complexes with the other peptides had similar fluorescence signals to those of ellipticine alone in water, indicating these peptides did not help stabilize ellipticine in aqueous solution. The protonation of ellipticine with EAK 16-11 peptide is probably due to the presence of counter ions that provide the protons and lower the solution pH.
EXAMPLE 5: AC8 peptides for use in drug encapsulation
AC 8 peptides were further evaluated for their ability to complex with ellipticine and enhance solubilization of the therapeutic agent. It was shown that increasing concentrations of AC8 resulted in the increase of ellipticine fluorescence at -430 nm as shown in Figure 23A. The results indicate that the AC8 peptides can help stabilize neutral ellipticine in a peptide concentration-dependent manner. When plotting the fluorescence intensities against AC8 concentrations in Figure 23B, it is clearly seen that the dramatic increase in fluorescence occurs at the AC 8 concentration of -20 μM, which is veiy close to the CAC of the AC8 as described in 0059. It may be concluded that the mature fibers/fiber bundles of AC8 may be more efficient to stabilize the neutral ellipticine. These data demonstrate that AC8 can solubilize neutral ellipticine in aqueous solution in a peptide concentration-dependent manner.
In a second series of experiments, the effects of AC8 peptide-ellipticine complexes on cell viability were tested in both A549 and MCF-7 cells cultured in the presence of serum in the medium. 0.04 mg/mL ellipticine was prepared in the presence of 1.3 % DMSO and cells were treated for 24 hours. AC8-ellipticine complexes were found to be cytotoxic to A549 cells and to MCF-7 cells to a lesser extent (Figures 24A-D). Figure 24E shows the fluorescence spectra profile of various concentrations of AC8 in complex with ellipticine. The peak at ~430nm is indicative of neutral ellipticine that was stabilized by the AC8 peptides. EXAMPLE 6: Cytotoxicity of AC8 and EAK peptides in complex with ellipticine Cell viability assays were performed to determine cytotoxicity of peptide- ellipticine complexes over a period of 48 hours. Peptides used in these experiments were EAKl 6-11 and AC8. The results of the assays demonstrated that all of the peptides were effective in killing A549 cells at 48 hours post-treatment (Figure 25A), and also MCF-7 cells (Figure 25B). In these experiments, the concentration of ellipticine in the complex was maintained at 0.04 mg/mL and prepared in the absence of DMSO to eliminate the possibility of cytotoxic effects due to DMSO. In order to prepare the drug, amounts of ellipticine crystals were added to the peptide solutions to give an ellipticine concentration of 0.04 mg/mL. 0.5 mg/mL and 0.1 mg/mL peptides were used to prepare the complex. The final ellipticine concentration in the medium was 25 μg/mL.
A treatment time of 48 hours was shown to be required for efficacy of the ellipticine- AC 8 complexes in killing cancer cells. In the absence of DMSO, the peptide-drug complex was still effective in killing cancer cells with equal or slightly higher efficacy compared with the control group. The protocol and the formulation method were then modified accordingly to re-evaluate the peptide concentration effect.
The new formulation method was as follows: a) 0.4 mg/mL ellipticine was dissolved in THF; b) aliquots of the ellipticine-THF solution was added to a glass vial, and air applied to evaporate the THF completely; c) 1 or 2 mL of a peptide solution
(varying concentrations of peptide) was added to the vial to obtain a final ellipticine concentration of 0.04 mg/mL; and d) the solution was stirred at 900 rpm for 20 hours prior to testing.
Four AC8 concentrations were tested in complex with ellipticine: 125μM, 50μM, lOμM and 2.5μM. Peptides of varying concentrations were complexed with ellipticine and assayed for cytotoxicity in the A549 and MCF-7 cell lines after 48 hour treatments. AC8 was shown to be effective at killing A549 cells when complexed with ellipticine (Figure 26A). The cytotoxic effects of AC8-ellipticine appeared to be peptide concentration dependent. While AC8-ellipticine complexes were effective at killing A549 cells, the complexes were less effective at killing cells of the MCF-7 cell line and there was not a notable concentration-dependent effect (Figure 26C). Series dilutions of the peptide-drug complex with 0.2 mg/mL AC8 in pure water were performed in order to evaluate the stability of the complex. Figures 26B and D shows that the dilution of the complex up to 8 fold still maintains the toxicity compare to non-diluted control sample. This indicates that the ellipticine-AC8 complex is stable and effective upon dilution.
The fluorescence spectra data indicated utility of AC8 peptides in solubilizing neutral ellipticine in aqueous solution. Varying concentrations of AC 8 were evaluated for the ability to complex with ellipticine. Cell viability assays were performed on cells treated with the AC8-drug complexes in order to determine a possible relationship between complex formation and the CAC of AC8. The AC8 concentrations tested were: 0.5, 0.2, 0.1, 0.04, 0.01 and 0.005 mg/mL, the CAC of AC8 being around 0.0115 mg/mL. The toxicity of the complexes was tested in each of the A549 (Figure 27C) and MCF-7 (Figure 27E) cell lines. The ellipticine concentration was fixed at 0.04 mg/mL for all complexes and prepared in the absence of DMSO. No significant difference in the toxicity of the peptide-drug complex with varying AC8 concentrations was observed (Figures 27C and 27E). Series dilutions of the peptide-drug complex with 0.2 mg/mL AC8 in pure water were performed in order to evaluate the stability of the complex. These complexes were stable after serial dilution in pure water as shown in Figures 27D and 27F. EXAMPLE 7: AAP PEPTIDES IN OLIGONUCLEOTIDE DELIVERY
Self-assembling peptides were evaluated for their utility in nucleic acid delivery applications. The EAKl 6-11 peptide was evaluated for its ability to bind to both single- and double-stranded oligodeoxynucleotides (ODNs), namely a guanine hexadecamer (dGiδ), a cytosine hexadecamer (dCi6), and their duplex (dGCiδ). Given that many peptide-nucleic acid interactions are not sequence-specific and result from non-covalent molecular interactions (Schwarz and Watanabe (1983) J. MoI .Biol. 163: 467-484; McGhee and von Hippel (1974) J. MoI. Biol. 86: 469-489; Muller et al. (1991) Biochemistry 30: 3709- 3715; Scatchard (1949) Ann. N. Y. Acad. Sci. 51: 660-672; Latt, and Sober (1967) Biochemistry 6: 3293-3306; Crothers (1968) Biopoly. 6: 575-584; Schwarz (1970) Eur. J. Biochem. 12: 442-453; Epstein (1978) Biophy. J. 8: 327-339; Tsodikov et al. (2001) Biophy. J. 81: 1960-1969; and Bujalowski and Lohman (1987) Biochemistry 26: 3099-3106), the dGi6 and dCi6 hexadecamers were chosen to assess how a purine or a pyrimidine affects the binding of a self-assembling peptide to an oligonucleotide. Most therapeutic antisense oligonucleotides and siRNAs are short nucleic acids of less than 22 nucleotides in length, thus, the choice of 16mer ODNs was within the usual therapeutic oligonucleotide length range. Peptide-oligonucleotide complexes were formed using the EAKl 6-11 peptide, in combination with both single- and double-stranded oligoucleotides (ODNs), namely a guanine hexadecamer (dGiδ), a cytosine hexadecamer (dC]g) and a duplex of the two (dGC)6). Proof of the binding of EAK to the ODNs was obtained by conducting absorption measurements on the supernatant of centrifuged solutions of the EAK-ODN mixtures; PAGE, fluorescence anisotropy, and UV- Vis absorption measurements were conducted to confirm that the quantity AOD1- was an appropriate representation of the fraction of ODNs incorporated in the EAK-ODN aggregates; fluorescence anisotropy and steady-state light scattering experiments were used to establish the time scale over which the EAK-ODN aggregates formed and to confirm it was much shorter than that over which the self-assembly of EAK occurred; the size of the EAK-ODN aggregates was determined using AFM and dynamic light scattering; the modified MvH model was used to compare the binding of EAK to the ODNs under different pHs and nucleotide types; and the accessibility of the ODNs to the solvent once encapsulated inside the EAK-ODN aggregates was investigated by performing fluorescence quenching experiments. Materials
All reagents were of analytical grade and obtained from BDH (Poole, UK). The pH dependence of the binding of EAK to the ODNs was investigated by performing experiments at three different pHs. The pH 4 buffer was made with 0.171 M acetic acid and 0.029 M sodium acetate adjusted with acetic acid (35); the pH 7 buffer was made with 0.01 M tris(hydroxymethyl)methylamine and 0.005 M sodium sulfate adjusted with sulfuric acid (Akinrimisi et al. (1963) Biochemistry 2: 340-344); the pH 11 buffer was made with 0.1 M glycine and 0.1 M sodium chloride adjusted with sodium hydroxide (Bolumar et al. (2003) Appl. Environ. Microbio. 69: 227-232). The EAKl 6-11 peptide was purchased from CanPeptide Inc. (Quebec, Canada) and the C-terminus carboxyfiuorescein labeled EAK peptide (FAM-EAK) was purchased from Research Genetics (Alabama, USA) and used without further purification. Four single-stranded oligodeoxynucleotides (ssODNs), namely dGiβ, dCi6,
FAM-dCiδ (dCi6 labeled with carboxyfiuorescein at the 5'-end), and dCi6-Rh (dCi6 labeled with carboxytetramethylrhodamine at the 3 '-end) were obtained with 95 % purity from Eurogentec North America (San Diego, USA) with HPLC purification. The ODN sequences are listed in Table 8. Solutions of the double-stranded ODNs (dsODNs) at different pHs were prepared one day before use by mixing two equimolar amounts of complementary ssODNs in the corresponding buffer in Eppendorf tubes, placing the tubes in a 95 0C water bath for 5 minutes, turning off the water bath, and letting the solution slowly cool to room temperature.
Table 8. Type, name, and sequence of oligonucleotides (ODNs) and self- assembling peptide.
UV- Vis Absorbance
UV- Vis absorption spectra were obtained on a Hewlett-Packard 8452A diode array spectrophotometer (California, USA) using a 50 μL quartz cuvette from Hellma (Mϋllheim, Germany).
Samples for the construction of binding isotherms were prepared at pH 4, 7, and 11. For each pH, two ODN concentrations of about 3 μM and 7 μM were used. At each ODN concentration, six to seven samples were prepared with EAK concentrations ranging from 0 to 0.2 mg/mL (equivalent to 0-120 μM) by mixing the ODN solution with different amounts of EAK powder. The resulting solutions were stirred vigorously for a few seconds with a vortex mixer and incubated at 25 0C for 30 mins. The EAK-ODN aggregates formed in the solution were removed by centrifugation at 14,000 rpm for 2 minutes with a Centrifuge 5410 from Eppendorf (Hamburg, Germany). The supernatant was collected and its absorbance was measured on the spectrophotometer at wavelengths between 190 and 800 nm. Beer's Law was used to determine the total ODN concentration and the concentration of the ODN left in the supernatants from the absorbance of the ODN at 260 nm of the initial solution (OD0) and the supernatant (ODS), respectively. The term (OD0-ODS)/OD0 was defined as the relative UV- Vis absorbance change AOD1-. The obtained AOD1- were then analyzed using the ligand binding density function (23) to generate binding isotherms. Polyacrylamide Gel Electrophoresis (PAGE)
PAGE was applied to detect whether EAK-dsODN complexes/aggregates were present in the supernatant after centrifugation. Samples containing 3.6 μM of dGCie with 0, 0.01, 0.1, or 0.2 mg/mL (0, 6, 60, or 120 μM) of EAIC were prepared at pH 4 and 7. The supernatants obtained by centrifuging the samples were analyzed with non-denaturing 20 % PAGE. The polyacrylamide gels were 14 cm long x 15 cm wide x 0.5 mm thick. PAGE was performed in the corresponding buffer at 10 V/cm for 2 hrs. The gels were stained with a solution of 0.5 mg/mL ethidium bromide for 30 mins. before being visualized with a Bio-Rad UV Transilluminator gel document system (California, USA). Steady-State Fluorescence
Fluorescence spectra of dCiβ labeled with carboxyfluorescein at the 5 '-end (FAM-dCiό), dCi6 labelled with carboxytetramethylrhodamine at the 3 '-end (dCi6- Rh), and EAK labeled with carboxyfluorescein at the C-terminus (FAM-EAK) were acquired on a Photon Technology International steady-state fluorometer (New Jersey, USA) equipped with a Ushio UXL-75Xe Xenon arc lamp and PTI 814 photomultiplier detection system. The peak absorption wavelength of the solution was chosen as the excitation wavelength (λex). For the FAM-dCi6 and FAM-EAK solutions, λex was 452 nm at pH 4 and 494 nm at pH 7, respectively; for the dCi6-Rh solutions, Xex was 560 nm at both pH 4 and pH 7. Steady-State Fluorescence Anisotropy
Anisotropy of the supematants obtained after centrifuging the solutions was measured using the steady-state fluorometer with polarizers fitted on the excitation and emission monochromators. Each sample was excited with vertically polarized light, and the fluorescence intensity was separately detected with the emission polarizer set in the vertical {lyγ) and horizontal (Jγu) orientations. In a fluorescence anisotropy experiment, the polarization dependence of the emission monochromator was corrected by the G factor (37). To obtain the G factor, the excitation polarizer was set to the horizontal direction, and the fluorescence intensities were measured individually with vertical (IHV) and horizontal (IHH) emission polarization. The G factor is given by the IHv I IHH ratio. The fluorescence anisotropy (r) was calculated using Equation 1(37):
r = Ivv ~ GI™ (EQN l)
1 YY T" ZiKJl Yff The wavelength corresponding to the maximum absorption of FAM was chosen as the excitation wavelength (λex). The fluorescence intensity was monitored at 514 run, the peak wavelength of the emission spectra. Time-Resolved Fluorescence Decays
Fluorescence decays were acquired by the time-correlated single photon counting technique on a time-resolved fluorometer (IBH system 2000, Glasgow, UK). Samples containing 3.6 μM of the chromophore-labeled dCi6 were prepared in the presence and the absence of 0.2 mg/mL (120 μM) EAK at pH 4. The excitation wavelength (lex) and emission wavelength (λem) were set to the wavelength corresponding to the absorption and emission maxima of the chromophores. For FAM-dCi6, Xex and λem were 452 nm and 514 nm, respectively; for dCig-Rh, λex and λem were 560 nm and 580 nm, respectively. A right angle configuration was used between the excitation and emission monochromators. AU decay curves were collected over 512 channels and with a total of 20,000 counts in the channel of maximum intensity. The analysis of the decay curves started by acquiring the instrument response function obtained with a scattering solution, which was then convoluted with a sum of exponentials shown in Equation 2 (Lakowicz, J. R. 1999. Principles of Fluorescence Spectroscopy. Plenum Publisher, New York):
/(t) = f>/β →/τ' (2)
(=1 where i=l, 2,... N; N is the minimum number of exponentials required to achieve a good fit. The fitting parameters were optimized using the Marquardt-Levenberg algorithm. The fits of the fluorescence decays were deemed satisfactory when the rf value was smaller than 1.3 and the residuals and autocorrelation function of the residuals were randomly distributed around zero. Steady-State Light Scattering (SLS) SLS intensity of solutions of EAK, ODNs, and their mixtures was monitored at right angle with the steady-state fluorometer by irradiating the solution at 350 nm where the ODNs and EAK do not absorb or emit. Dynamic Light Scattering (DLS) The hydrodynamic diameter of the EAK-ODN complexes/aggregates in the pH 4 buffer was obtained with a Zetasizer Nano ZS instrument equipped with a 4 mW He-Ne laser operating at 633 nm (Malvem, UK). AU measurements were performed at 25 0C at a measurement angle of 173°. Dust particles were found to be absent in the buffer solutions used to prepare the samples, as confirmed by dynamic light scattering. DLS experiments were performed 30 mins. after sample preparation. Atomic Force Microscopy (AFM)
A Picoscan atomic force microscope (Molecular Imaging, Arizona, USA) was used to study the morphology of the EAK-ODN complexes/aggregates in solution. It was operated in magnetic AC (MAC) tapping mode in solution using magnetically coated cantilevers, Type II MAClevers (Molecular Imaging, Arizona, USA), with a spring constant of 0.5 N/m and a resonance frequency of ~27 kHz at room temperature. A volume of 400 μL of each solution was deposited on a freshly cleaved mica surface, inside a Teflon liquid chamber, where the AFM images were acquired.
Interaction of EAK with ODN resulted in aggregates composed of EAK and ODN. The fraction of ODNs in the aggregates could be obtained from the relative change in absorbance, AODn described above. The procedure for building binding isotherms consisted of using three or four ODN concentrations and plotting the fraction of ODN in the aggregates, i.e., AODr, as a function of the total peptide concentration. Each plot was fitted reasonably well with the equation A0Dr = A *Pt /(I +B XP1), where A and B are regression parameters listed in Table 9:
Table 9. Parameters A and B retrieved from the fits of the AODr versus [EAK] plots shown in Figure 28 with the empirical equation: AOD \ = A x [EAK] / (7+5x[EAK]) pH 4 dG 16
Concentration 2.0 μM 4.0 μM 5.9 μM 9.5 μM
A (IO4) 18.454 ± 3.465 4.513 ± 0.507 3.574 ± 0.613 2.846 ± 0.248
B (IQ4) 18.088 ± 3.853 3.645 ± 0.599 2.750 ± 0.751 2.053 ± 0.306
pH 4 dC16 Concentration 1.5 μM 3.0 μM 5.0 μM 7.3 μM
A (104) 19.262 ±2.549 9.747 ± 0.981 6.954 ±1.074 5.871 ±0.583
B (104) 22.309 ±3.481 10.073 ± 6.547 ±1.328 5.996 ±0.823
1.249
pH4 dGC16
Concentration 2.0 μM 4.0 μM 7.5 μM 9.0 μM
A(IO4) 4.871 ±0.671 2.436 ± 1.599 ±0.153 1.247 ±0.140
0.414
B (104) 5.539 ±1.048 2.440 ± 1.367 ±0.254 0.878 ±0.236
0.670
pH7 dG16
Concentration 1.0 μM 2.0 μM 4.0 μM 6.0 μM
A(IO4) 3.309 ±0.387 2.612 ± 1.834±0.163 1.237 ±0.095
0.176
B (104) 3.318 ±0.587 2.425 ± 1.636 ±0.267 0.844 ±0.160
0.269
pH7 dC16
Concentration 1.0 μM 2.0 μM 4.0 μM 6.0 μM
A(IO4) 1.341 ±0.082 1.001 ± 0.752 ±0.036 0.621 ±0.034
0.027
B (104) 1.686 ±0.175 1.035 0.585 ±0.085 0.339 ±0.081
±0.063
pH7 dGC16
Concentration 1.0 μM 2.0 μM 4.0 μM
A(IO4) 0.748 ±0.019 0.631 ±0.035 0, ,503 ±0.011
B (104) 0.442 ±0.038 0.306 ±0.077 0. ,082 ±0.023
Sets of v and Pf were obtained from the plot of Pt vs. Dt for a given AOD1-. Seven-to- ten AOD r values were chosen. For each AOD1- value, the value of Pt was read from the fits of the curves shown in Figure 28 and plotted as a function of Dt. Then Pf and v were obtained from the intercept and slope of the plot of Pt vs. Dt. Plotting v/Pf versus v generated the binding isotherms (Figure 51). The interaction/binding between EAK and ODN molecules most likely starts with the formation of EAK-ODN complexes, which is followed by further association of the complexes into aggregates. This process is described in Scheme I:
Scheme I ODN + EAK Z=^ [ODN, EAK] complexes Z=Z [ODN, EAK] aggregates where Kc and Ka are the equilibrium constants for the formation of EAK-ODN complexes from unimolecular ODN and EAK and for the formation of EAK-ODN aggregates from the association of EAK-ODN complexes, respectively. To facilitate the analysis of the binding isotherms (i.e., the v/Pf versus v trends), the modified non-cooperative McGhee and von Hippel (MvH) model (McGhee and von Hippel (1974) J. MoI. Biol. 86: 469-489 and Tsodikov et al. (2001) Biophy. J. 81: 1960-1969) was used: The MvH model described the initial complexation process only, and did not take into account the second aggregation step. However, the MvH model was determined to be useful for the following reasons: first, a goal of the study was to find differences or similarities in the process leading to the formation of EAK-ODN aggregates under different experimental conditions, e.g., pHs and nucleotide types. Second, the binding constant K calculated from the MvH model showed a trend for the binding strength of EAK to different ODNs and under various solution conditions, which are also comparable to those reported for the binding of other DNA/peptide pairs. With these considerations, the binding isotherms (Figure 29) were fitted with EQN 5. Effect of pH on Binding The binding of EAK to dCiβ, dGig, and dGCi6 was monitored at pH 4, 7, and
11. Solutions were prepared with different EAK concentrations at two ODN concentrations. The absorbance of the solution supernatant was measured after the EAK-ODN aggregates had been centrifuged out. The relative change in absorbance, AOD1-, was calculated and plots of AOD1- as a function of the total EAK concentration were generated for each ODN concentration at pH 4 and 7 (Figure 30). AODr was found to increase with increasing total EAK concentration, eventually leveling off for the single-stranded dGig and dCiδ at pH 4, indicative of more EAK bound to the ODNs with increasing EAK concentration, until the ssODNs were saturated. AODr increased with increasing EAK concentration at pH 7 for both the ssODNs and the dsODN; however, it required higher EAK concentration to reach the plateau. Furthermore, at a given ODN concentration, the AODr values at pH 7 were significantly lower than those at pH 4 suggesting that increasing the pH from 4 to 7 resulted in a much weaker binding between EAK and the ODNs. At pH 11, no binding of EAK to the ODNs was detected as the EAK concentration was varied from O to 120 μM and AOD1- equaled zero. The AODr values for dGi6 were higher than those for dCi6 at a given EAK concentration, suggestive of the fact that more EAK binds to dGjβ than to dQg. The A0Dr values for the ssODNs were consistently higher than those obtained for the dGCi6 duplex at the same EAK concentration, suggesting that EAK molecules bind more strongly to the ssODNs than to the dsODN. Nature of the ODNs Remaining in the Supernatant after Centrifugation The fraction of the ODN in the EAK-ODN aggregates was determined from the relative UV absorbance change of the solution, A0Dr. Fluorescence anisotropy reflected changes in the rotational correlation time of the chromophore, which was related to the hydrodynamic volume of the species to which the chromophore was attached. Solutions containing 3.6 μM of FAM-dGCi6 or of FAM-dCi6 were mixed at pH 4 and pH 7 with EAK concentrations ranging from O to 0.2 mg/mL (0-120 μM). The solutions were centrifuged and the fluorescence anisotropy of the supernatants was measured. The anisotropy of the supernatants was plotted in Figure 31 as a function of EAK concentration. The fluorescence anisotropy of FAM-labeled ODNs equaled 0.11 ± 0.01 and 0.04 ± 0.01 at pH 4 and pH 7, respectively. When the FAM- labeled ODN solution was mixed with increasing EAK concentrations, the anisotropy of the FAM-labeled ODN species remaining in the supernatants ranged from 0.08 to 0.12 and 0.03 to 0.04 at pH 4 and pH 7, respectively, close to that of the FAM-labeled ODN, demonstrating that unimers of ODN existed in the centrifuged solutions; no EAK-ODN aggregates were detected. To estimate the resolution limit of the fluorescence anisotropy experiments, the anisotropy of a 1 μM solution of FAM-dCi6 was measured as a function of EAK concentration at pH 4 and was plotted (data not shown). With the K and n values listed in Table 10, retrieved from the analysis of the binding isotherms using Equation 5, the fraction of ODNs incorporated in the EAK-ODN aggregates was calculated and plotted (data not shown):
Table 10. Binding constant K and binding site size n retrieved from the fits of the data with Equation 5 pH 4 pH 7 pH l l
ODNs n K (IO4M"1) n K (IO4 M'1) N K (M"1)
dGi6 1.66 ±0.05 7.6 ±1.2 1.16 ± 0.1 3.4 ± 0.5 No Interaction
dC!6 1.96 ± 0.02 4.7 ± 0.6 ^ ± 2.0 ± 0.2 No Interaction
dGCi6 3.16 ± 0.2 4.2 ± 0.8 2.6 ± 0.3 0.7 ± 0.02 No Interaction
* Each fit has R > 0.94 with at least 5 data points.
It was determined that -10 mol% of ODNs was required in the EAK-ODN aggregates to induce a detectable change in fluorescence anisotropy.
UV- Vis absorption experiments were performed to demonstrate the existence of EAK-ODN complexes in the supernatant that were not detected in the anisotropy experiments. The absorption spectra of the samples containing 3.9 μM of dCi6-Rh in the presence and absence of 60 μM EAK were acquired before and after centrifugation. As shown in Figure 32, the absorption spectrum of a mixture of dCi6- Rh and EAK was different from that of free dC I6-Rh. Mixing dC I6-Rh with EAK induced a decrease in the absorbance at the 563 nm band characteristic of dCi6-Rh, and a new prominent absorption band at 524 nm. The ratio of absorbance at 524 and 563 nm (OD524/OD563) for dCiβ-Rh was 0.50 ± 0.01 and was concentration- independent as shown in Figure 32B. This ratio changed to 0.89 and 0.78 after mixing with EAK, before and after centrifugation, respectively.
Using the extinction coefficients of the rhodamine dimer reported in the literature (Hamman et al. (1996) J. Biol. Chem. 271: 7568-7573), the data given in Figure 3OA were analyzed to estimate the concentrations of ODNs present in the solution as unimers (1.39 μM), complexes (1.63 μM), and aggregates (0.88 μM). In this situation, ΔODr equaled (0.88 μM)/(3.9 μM) = 0.23, close to the value of 0.28 obtained from the K and n values listed in Table 10. Despite the substantial amount of complexes remaining in the supernatant after centrifugation, no increase in anisotropy was observed in the supernatant (Figure 30C), whereas the presence of aggregates in the solution resulted in an observable increase in the anisotropy of the solution. Non-denaturing PAGE experiments were conducted to investigate the nature of dsODN in the supernatant after centrifugation. Samples containing 3.6 μM of dGCj6 mixed with 0, 6, 60, or 120 μM of EAK were centrifuged and their supernatants were run on a 20 % native gel at pH 4 and pH 7. As shown in Figure 33A, for the gel obtained at pH 4 and stained with ethidium bromide, the bands corresponding to the supernatants of EAK-dGCi6 mixtures (Lanes 2-4) appeared at the same position as the band corresponding to dGCi6 (Lane 1). A similar observation was obtained for the gel run at pH 7 with the EAK-dGCi6 mixtures (Figure 33B). These data demonstrate that the centrifugation separated the EAK-ODN aggregates from the original EAK-ODN solution, and indicate the existence of free EAK and ODN molecules, as well as EAK-ODN complexes. Pathway of the EAK-ODN Binding
To identify the pathway leading to the formation of EAK-ODN aggregates, the time scale over which EAK self-assembled in solution was estimated. To this end, the fluorescence anisotropy of EAK and EAK-dG]6 mixtures was measured at pH 4 and 7 over a one hour period immediately after sample preparation using carboxyfluorescein-labeled EAK (FAM-EAK). These experiments were performed with 0.1 mg/mL (60 μM) EAK solutions where 1 in 100 EAK molecules was fluorescently labeled. To these solutions, 5 μM of dGi6 was added. The steady-state fluorescence anisotropy of EAK and the EAK-dGi6 mixtures was measured at pH 4 and 7 as a function of time (Figures 34A and B). Whether at pH 4 or 7, the fluorescence anisotropy of FAM-EAK alone remained constant as a function of time and equaled 0.10 and 0.05, respectively. On the other hand, addition of dG]6 to the EAK solution resulted in a large increase in anisotropy, reflective of a large increase in the hydrodynamic volume of the species present in solution. These species were the EAK-ODN aggregates which could be centrifuged out.
The interaction between individual EAK and ODN molecules was further confirmed by SLS experiments on EAK and EAK-dGi6 mixtures. The light scattered by the EAK solution remained constant within the first 20 mins. at pH 4 and pH 7 as shown in Figures 35A and 35B, respectively. As a control, the light scattered by dGiβ in the buffer solution and by the buffer solution alone was also monitored for 20 mins. The SLS intensity of both solutions remained constant and close to the SLS intensity of the solution containing EAK only. These results indicated that the size of EAK remained constant during the first 20 mins. after sample preparation. In comparison, the SLS intensity of the EAK-ODN mixtures increased initially and then leveled off within 30 mins. at both pHs.
Size Characterization of EAK-ODN Aggregates by AFM and DLS The size of the EAK-ODN aggregates was characterized using AFM and DLS.
Images of the dGiβ-EAK solution were captured 8 and 60 mins. after sample preparation (Figure 36). Both small fibers and large aggregates were found after 8 and 60 mins. and the sizes of these objects did not change during the 1 hour time period. The zoomed in view of the image captured at 60 mins. showed that the smaller fibers were 10 nm wide, 5.2 nm tall and over 100 run long. The large aggregates appeared to be giant clusters of small fibers with lateral dimensions of 650 nm and a height of 35 nm. Two control experiments were performed: no nanostructure could be detected when a 2 μM dGiβ solution was imaged by AFM. Sparsely distributed globular structures with a diameter of 34 nm and a height of 1 nm were observed for the 0.1 mg/niL (60 μM) EAK solution 45 mins. after solution preparation.
In addition, the hydrodynamic diameter of the EAK-ODN aggregates in solution was obtained by DLS. The hydrodynamic diameter of the EAK-dGi6 aggregates at pH 4 with varying concentrations of EAK was measured 30 min after sample preparation (Figure 37). The dGi6 solutions at 7.2 μM exhibited a species with a hydrodynamic diameter of ~ 7.5 nm which was attributed to isolated ODNs in solution. The diameter of the species present in the dG] 6 solution increased to around 150 nm upon addition of EAK and remained constant as the EAK concentration was increased from 0.01 to 0.04 mg/mL (6-24 μM) suggesting that EAK bound first to ODN molecules, followed by aggregate formation. The hydrodynamic diameter increased to 1000 and 2000 nm when the EAK concentration was increased to 60 and 90 μM, respectively. Under the conditions indicated in Figure 37, dGiβ was fully incorporated in the EAK-ODN aggregates for an EAK concentration of 60 μM. Since 0.1 mg/mL (60 μM) of EAK exhibited a size of ~ 4 nm 40 minutes after preparation (Figure 37), it indicated that EAK did not self-assemble during that time and was present as single molecules. The observed large diameters in the EAK-ODN mixture solution were due to the aggregates of ODN and EAK in solution. Binding parameters The curves shown in Figure 28 were used to generate plots of v/P/ as a function of v. Equation 5 was used to obtain the binding constant, K, and the binding site size, n. The K and n values obtained for the binding of EAK to the ODNs at various pH values are listed in Table 10. The "equilibrium constants" obtained for the binding of EAK onto the ODNs ranged from 7.0 x 103 to 7.6 x 104 M"1. In comparison, oligolysine and lysine-rich peptides such as KWKGK, KWKO, K4N4, K4N6, and Kn (n=3-8) have been reported to bind to oligo- and polynucleotides with binding constants in the range of l χ l03~lx l05 M-' (Latt and Sober (1967) Biochemistry 6: 3293-3306; Baffin et al. (2004) Nucl. Acids Res. 32: 3271-3281; Matsuno et al. (2001) Biochemistry 40: 3615-3622; and Roy et al. (1992) Biochemistry 31: 6241-6245).
The base composition of the ODN sequences, guanine in dGiβ and cytosine in dCi6, appeared to affect the binding of EAK to the ODNs since EAK bound more strongly to dG]6 than to dCie at both pH 4 and 7 (Table 10). The data listed in Table 10 indicate that EAK binds preferentially to ssODNs rather than to their duplex. This phenomenon has been observed for other peptides (Simeoni et al. (2003) Nucl. Acids Res. 31: 2717-2724). This favorable interaction disappeared in the case of dsODNs, since each nucleotide base hydrogen-bonded with the opposite base in the complementary strand of the duplex. Solvent Accessibility of ODNs in the EAK-ODN Aggregates
One important aspect of the structure of the EAK-ODN aggregates was whether the ODNs were located inside or outside the aggregates. ODNs located inside the aggregates would be less accessible to the solvent than those located on the surface, and hence be protected from the outside environment. The accessibility of the ODN to the solvent was measured by performing fluorescence dynamic quenching experiments. dCi6 was labeled either at the 5'-end with fluorescein (FAM-dCi6, λex = 452 nm, λem = 514 nm) or at the 3 '-end with rhodamine (dCi6-Rh, λex = 560 nm, λem = 580 nm) to investigate whether both ends of dCiβ are protected from the solvent. The fluorescence emission of a solution containing 3.6 μM of the labeled dCi6 was monitored as the quencher KI was added to the solution in the presence or absence of 0.2 mg/mL EAK. Throughout these experiments, the potassium ion concentration was maintained constant and equal to 0.3 M by addition of K2SO4 to the KI solution, ensuring constant ionic strength. As the KI concentration increased, the fluorescence intensity of fluorescein or rhodamine decreased. The I0II ratio was plotted as a function of iodide concentration in Figures 38A and B for fluorescein and rhodamine, respectively. The quantities I0 and / represent the fluorescence intensity of the chromophore without and with quencher, respectively. The I0II ratio was found to increase linearly with iodide concentration for both cliromophores in the absence or presence of EAK. However the increase was stronger in the absence of EAK.
The fluorescence decays of FAM-dCi6 and dCi6-Rh were acquired in the presence and absence of EAK. They were fitted with two exponentials according to Equation 2. The pre-exponential factors and decay times obtained from the fits are listed in Table 11:
Table 11. Pre-exponential factors and decay times obtained from the analysis of the fluorescence decays of the fluorescent ODNs in the absence and presence of EAK in pH 4 buffer, respectively
2 τi (ns) (X1 τ2 (ns) OC2 τ (ns)* t
FAM-dC16
3. 9 0.67 2.0 0.33 3.3 1. .12 (without KI)
EAK-FAM-dC16
3. 7 0.63 1.7 0.37 3.0 1. 00 (without KI)
EAK-FAM-dC16
?. 8 0.43 1.0 0.57 1.7 1 15
(with KI)
3. 9 0.60 1.5 0.40 2.9 1. .13 (without KI)
EAK-dCie-Rh
3. 8 0.58 1.3 0.42 2.8 1. 37 (without KI)
■fWimP1
Although fluorescein itself displays a single lifetime of 4.1 ns (Sjoback et al. (1995) Spectrochim. Acta Part A, 51: L7-L21), the decay became more complex when fluorescein was covalently attached to an ODN where it showed two decay times. This phenomenon has been reported (Sjoback et al. (1998) Biopoly. 46: 445-453) to be due to a change in the conformation of fluorescein when it is covalently linked to an ODN. The binding of EAK onto the labeled ODN did not change the lifetime of the chromophore significantly as shown in Table 11.
To ensure that the quenching is dynamic, the fluorescence decays of the FAM- dC]6-EAK mixture were acquired in the absence and presence of 0.2 M KI. The average lifetime of the decays was determined and the ratio of the average lifetime of the solution without KI (r0) over that of the solution with 0.2 M KI (τ) was found to be 1.8, i.e. T0ZT =I .8. The IJI ratio obtained from the steady-state fluorescence measurements equaled 1.9 for a concentration of 0.2 M KI, a value comparable to the τjτ ratio. Fitting the I0II vs. [F] trends shown in Figure 38 with the Stern- Volmer equation given in Equation 6 yields the bimolecular quenching rate constant, kq, which reflects the accessibility of the chromophore to the solvent (Lakowicz, J. R.
(1999) Principles of Fluorescence Spectroscopy. Plenum Publisher, New York).
I0 / I = \ + Ksv [Q] = l + kgτ0 [Q] (EQN 6)
The slopes of the straight lines referred to as the Stern- Volmer constants, Ksv, are listed in Table 12:
Table 12. Stern- Volmer quenching constants IQy, bimolecular quenching rate constant kq, fluorescence lifetime τ0, and relative accessibility change γ when dCj6 is in the absence or presence of 0.2 mg/mL of EAK in pH 4 buffer.
5' end fluorescein labeling 3' end rhodamine labeling
ODNs
Ksv To K Ksv K (M-1) (ns) (1O9 M-1S"1) r (M-1) (ns) (109M-1S-1) r 2.8 3.5 ± 0.1 (free)
Fluorescently labeled EAK- 4.6 ± 3.0 1.5 ± 0.1 0.60 » * 2.7 2.0 ± 0.1 0.57 dC16 0.1 (bound)
As indicated in Equation 6, kq is determined from the ratio Ksv/τo where T0 is the lifetime of the chromophore in the absence of quencher (Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy. Plenum Publisher, New York)
Since the fluorescence decay of the choromophores without quencher is biexponential, the average lifetime τ0 was used to calculate the bimolecular quenching rate constant kq from the slope Ksv of the plots in Figure 38. The values of Ksv, T0 , and kq are listed in Table 12. In the absence of EAK, kq for FAM-dCi6 and dCi6-Rh equaled 2.5x lO9 JVT1S"1 and 3.5χlO9 MV1, respectively. The bimolecular quenching rate constant was related to the quenching efficiency, size, and diffusion coefficient of the chromophore and quencher. Since the iodide ions have quenching efficiencies near unity (Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy. Plenum Publisher, New York) and the overall sizes of the two labeled ODNs were comparable, the differences in kq found for FAM-dCi6 and dC I6-Rh (Table 12) were taken to be indicative of differences in the efficiency of iodide at quenching the excited chromophores. Indeed, FAM and Rh were neutral and positively charged at pH 4, respectively. In the presence of EAK, kq decreased for both FAM-dCi6 and dC I6-Rh. For FAM-dC]6, kq decreased from 2.5x lO9 M~V to 1.5x lO9 M-1S"1. Similarly, for dCi6-Rh, kq decreased from 3.5X lO9 MT1S"1 to 2.Ox IO9 M-1S"1.
The change in accessibility of the ODNs upon EAK binding was quantified with the relative accessibility change, γ, defined by:
Y = K 1K (EQN 7) where kq b and k/ are the bimolecular quenching constants for the chromophore- labeled ODNs in the presence and absence of EAK, respectively. The values of γ for the two different labels are listed in Table 12. The relative accessibility changes of dCi6 for both types of labels were similar and smaller than unity, being 0.60 and 0.57 for the 5 '-labeled FAM-dCiβ and the 3 '-labeled dCi6-Rh, respectively. Since γ is smaller than 1.0, the accessibility of the 5'- and 3 '-ends of dCj6 to the solvent was reduced after the binding of EAK to dCj6. Since the ^values corresponding to the two labels are similar, the accessibility of both ends of dCi6 to the solvent was reduced by the same extent, about 40 %.
EXAMPLE 8: USE OF PEPTIDES FOR FORMING NANOWIRES
The ability of EAKl 6-11 to self-assemble in a pH-dependent manner is shown in Figure 39, which indicates the results of dynamic light scattering experiments. The results show that EAKl 6-11 self-assembles in water but not in acid and basic solutions. The pH will affect the peptide assembly on surfaces (see below) as well. Thus, the pH control could provide a means to regulate the peptide assembly into nanofibers, which can serve as a template for nanowire fabrication (Figure 55). The growth of EAK16-II peptides on a mica surface was shown (Figure 40). EAK peptides were shown to self-assemble into nanofibrils when plated on a mica surface as the peptide assembly reached equilibrium in water as shown in Figure 39. The nanofiber growth occurs when the nanofiber "seeds" (Figure 40 top panels) and fiber clusters (Figure 40 bottom panels) that are formed in the solution adsorb on the surface. The surface serves the purpose of adsorbing the peptides and permitting elongation from the ends of a peptide nucleus as exemplified in the schematic of Figure 5.
The ability of EAK peptides to assemble on a mica surface has been demonstrated (Hong et al. (2007) J. Am. Chem. Soc. Accepted). Adjustment of the pH of the peptide solution resulted in changes to the nanostructure formed (Figure 41). The affinity of EAK peptides to mica was shown to decrease with increasing pH. Growth rates were also shown to differ depending on the environmental pH (Figures 42A and B). Schematics of the effects of solution pH on nanofiber growth on a mica surface are shown in Figure 7. It is noted that nanofiber assembly is limited in extreme acidic and basic environments. By adjusting the solution pH, the length of the EAK nanofibers can be controlled. In addition, the density of the nanofiber "seeds" can be altered depending on the pH. In combination, one may be able to fabricate the nanofibers with desired dimensions and amounts on the surface. This is an important step towarding nanowire fabrication via controllable peptide assembly (see schematic of Figure 55). In addition, studying the assembly of the disclosed peptides on surfaces could serve as a model system to investigate the complicated amyloid fibrillogenesis. EXAMPLE 9: BIOMOLECULAR SENSING Peptides of the present invention can be used in biomolecular sensing applications. EAKl 6-11 peptides self-assembled into different nanostructures depending on the surface on which the peptides were seeded (Figure 43). Peptides formed nanofibers when seeded onto a mica (hydrophilic) surface at a concentration of 0.05 mg/mL. However, the same peptide tended to self-assemble into patterned nanofibers at 60° and 120° angles to one another when seeded onto HOPG (hydrophobic) (Figure 43B, Figure 44).
Furthermore, EAKl 6-11 peptides were shown to alter the hydrophobicity of HOPG and induce it to be hydrophilic. In general, the substrate for a biomolecular sensing application should be confuctive and chemically inert (e.g. HOPG). Since the EAK 16-11 peptides have a unique amphiphilic structure with hydrophilic resides on one side and hydrophobic residues on the other side, it was proposed that the hydrophobic side of the molecules were capable of interacting with the hydrophobic HOPG surface. The exposed hydrophilic resides of glutamic acid (E) and lysine (K) contain functional amine and carboxylic acid groups, which can be used to immobilize many proteins and enzymes for a molecular sensing application. The wettability of a surface may affect the adsorption of biomolecules and cells as well as the enzyme immobilization. The amphiphilic properties of the EAK peptides are capable of altering the surface wettability (Figure 45).
Glucose oxidase (GOx) was used as a model enzyme to test if the amino acid pairing peptides of the present invention could be used to modify a surface for a biomolecular sensing application. Two exemplary ionic pairing peptides were used for this study: EAK16-II; and EFK16-II. The morphologies of GOx on bare HOPG and EAK/HOPG were characterized using atomic force microscopy (AFM). GOx tended to denature upon coating on HOPG, but maintained a native ball shape on EAK/HOPG (Figure 46). These results indicated that the presence of EAKl 6-11 nanofibers on the HOPG surface changed the hydrophobicity of HOPG, improved the biocompatibility of the surface for enzyme immobilization. The procedure for immobilization of GOx on EAK/HOPG electrode is shown in Figure 47. The glutamic acid provided the functional group. The electrochemical property of EAKl 6-11 on HOPG surface is characterized with a redox probe of K3Fe(CN)6. As shown in Figure 48, the presence of EFKl 6-11 nanofibers on HOPG electrode did not block electron transfer significantly at both slower and faster scan speeds (indicated in mV/s), indicating that the peptide-modified surface provided a much better electrode surface than thiol, bunte salts and polymer surface modifiers, which have been shown to dramatically block electron transfer.
The activity of immobilized GOx on EAK/HOPG electrode was examined using cyclic voltammetry (CV). Figure 49 showed the CV of a GOx-immobilized EAK/HOPG electrode in 100 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM ferrocenecarboxylic acid (FCA, mediator) in the absence and presence of 20 mM glucose. In the absence of glucose, the characteristic redox response of FCA was observed (Figure 49, upper profile). In the presence of glucose, an electrocatalytic anodic current was observed (Figure 49, lower profile), indicating that GOx molecules were immobilized on the surface and maintained good activity.
The effect of applied potentials on the steady state currents was examined
(Figure 50). The steady state current initially increased with the applied potential and reached a plateau at 0.45V. Since a higher applied potential would cause additional side reactions, a potential of 0.45 V was used to obtain steady state currents to plot the calibration curve.
The i-t curves of the fabricated enzyme sensor toward various concentrations of glucose were generated (Figure 51A). Steady state currents were obtained at 120 s to plot the calibration curve shown in Figure 51B. The current increased with increased glucose concentration linearly up to 10 mM, a concentration which overlaps with the clinical detection range of glucose (4-7 mM). These results indicated EAK/HOPG electrode can be used to fabricate enzyme sensors.
In addition, the GOx immobilized EAK/HOPG electrode showed lower Km compared to an unmodified electrode, indicating the enzyme electrode has higher affinity for glucose (Table 13):
Table 13. Comparison of unmodified electrode with EAK and EFK/HOPG electrodes
GOx in solution GOx immobilized on electrode
A comparison of self-assembling peptide-modified electrodes for molecular sensing with other surface modifiers was performed (Table 14). The self-assembling peptides EAK and EFK were found to be less hindering to electron transfer, and exhibited higher affinities for glucose (lower Km), higher maximum currents, and relatively higher sensitivities than thiol and bunte salts.
Table 14. Comparison of EFK peptide/HOPG electrode with other surface modifiers
*From Dong et al. (1995) Bioelectrochem. Bioenerg. 36:73-76; and Sun (2007) University of Waterloo PhD Thesis, entitled "Biosensing at an individually addressable electrochemical array".
The storage and measurement stabilities of GOx immobilized EFK/HOPG electrodes were determined (Figure 52). As shown in Figure 52, after one month of storage, the enzyme electrode retained 87% of its original current, indicative of long term storage stability. Operational stability of the electrode was also measured (Figure 53). The EFK peptide electrode was subjected to cyclic scanning 50 times. The data demonstrated that the electrode retained 87% of its original current, indicative of relative stability of the electrode with continuous use. EXAMPLE 10: BIOFUEL CELL
Glucose biosensors using peptides of the present invention were examined for the ability to act as an anode for a biofuel cell (Figure 54A). The peptide-modified electrodes could also be used to generate a cathode, such as an oxygen electrode, in a biofuel cell. Laccase could be immobilized to an EAK or EFK/HOPG electrode. Peptide modified electrodes can, thus, be used to generate biofuel cells, including glucose-oxygen (Figure 54A) and hydrogen-oxygen (Figure 54B) biofuel cells. EXAMPLE 11: PHYSICOCHEMICAL CHARACTERIZATION OF siRNA- PEPTIDE COMPLEXES
Short interfering RNAs (siRNAs) trigger RNA interference (RNAi), where the complementary mRNA is degraded, resulting in silencing of the encoded protein. A delivery carrier is desired to increase the solution stability of siRNA and improve its cellular uptake to overcome its rapid enzymatic degradation and low transfection efficiency. The physicochemical properties of the carrier-drug complexes including size, surface charge and surface chemistry are essential factors for the development of a suitable formulation of siRNA therapeutics. RNA interference (RNAi) is an evolutionary conserved mechanism that performs a sequence specific, post transcriptional gene silencing through the use of short RNAs. RNAi can be triggered by several sub-types of short RNAs, which include short interfering RNA (siRNA), micro RNA (miRNA), tiny non-coding RNA (tncRNA), small modulatory RNA (smRNA), and short hairpin RNA (shRNA) Double stranded RNA (dsRNA) can act as a precursor of RNAi in invertebrates to obtain siRNAs upon its cleavage by the Dicer. Once the siRNA is located in the cytosol, Ago2 cleaves the sense strand of the siRNA (Matranga, C.et al. Cell 2005, 123, 607-20.; Rand, T. A. et al. Cell 2005, 123, 621-29.). Further, since the 5' of the anti-sense strand is less thermodynamically stable than the 5' of the sense strand (Khvorova, A. et al. Cell 2003, 115, 209-16), the anti-sense strand will be thermodynamically favored to incorporate to the RNA induced silencing complex (RISC). The anti-sense sequence of the siRNA that is incorporated into the RISC would pair with its complementary mRNA sequence. The mRNA is then cleaved enzymatically by Ago2. Since the cleaved RNA fragments lack either the cap structure m7G or the polyA tail, which are essential to RNA stability, this leads to further degradation of the mRNA molecule. Since mRNA is the precursor to protein translation, the protein encoded by such mRNA thus cannot be synthesized.
When administered, a significant portion of the siRNA is excreted through the reticuloendothelial system possibly due to their small size and hydrophilicity. Furthermore, they are subjected to enzymatic degradation during circulation and within the cell. As a result, the potency of the siRNA drugs is decreased, and in some cases an increase of drug dosage is required to compensate these effects. Since RNAi takes place in the cytosol, the inability of hydrophilic drugs to effectively travel across the hydrophobic core of the plasma membrane is also a major obstacle for their therapeutic application.
Recently, the carrier-mediated delivery system has become a prevalent approach for improving the cellular delivery of nucleic acids (NAs). The carriers, associated or covalently conjugated with the NAs, are designed to prolong drug circulation time, to improve membrane permeation, and to increase cell targeting capabilities, while being biocompatible and biodegradable. A safe drug delivery system should exert minimal side effects, that is, minimal cytotoxicity and inflammatory response, especially to non- targeted cells. Hydrophobic or highly charged particles can interact with opsonins, where the resulting complexes are removed from circulation by phagocytes and the reticuloendothelial system (Vonarbourg, A. et al. Biomater. 2006, 27, 4356-73. "Vonarbourg et al. (2006)"); Passirani, C; Benoϊt, J. P. Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids, Mahato, R. L, Ed.; CRC Press: 2005; Chapter 6. "Passirani & Benoϊt. (2005)"), which essentially decreases the effective drug concentration. Neutrally charged particles are found with a lower opsonization rate than charged particles due to the decrease in electrostatic interactions with opsonins (Vonarbourg et al. (2006); Owens, D. E., Ill; Peppas, N.A. Int. J. Pharm 2006, 307, 93-102 "Owens & Peppas (2006)".) Particle size also has an effect on the rate of opsonization. Smaller carrier- drug complexes, which have a smaller surface area, display slow opsonization and thus a longer bloodstream circulation time (Vonarbourg et al. (2006); Passirani & Benoϊt. (2005).) Since many carrier-drag complexes are mainly internalized through the temperature and energy dependent endocytosis pathways, small complexes, with size 100-200 nm, may also facilitate cellular internalization (Vonarbourg et al. (2006); Owens et al. (2006).) Upon cellular internalization, the concentration of various enzymes within the vesicle increases and the pH is about 5.0 during the transition from early to late endosomes. The increase in enzymatic degradation and acute pH changes would decrease the drug potency if the carrier cannot escape from or withstand the harsh environment within the late endosome, or later in the lysosome. In order to achieve cellular targeting, specific molecules, such as antibodies, that can interact with surfaces of the targeted cells, can be grafted onto the surface of the carrier-drug complexes (Liang, H. F. et al. Biomater. 2006, 27, 2051-59; Hashida, M. et al. J.Controlled Release 1999, 62, 253-62.). For passive cellular targeting, the carrier-drug complex should exhibit physiochemical properties similar to the targeting site, such that it can easily diffuse to the targeted cells (Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46, 6387-92; D'Souza, A. J. M.; Topp, E. M. J Pharm.Sci. 2004, 93, 1962-79.). Release kinetics of the drug from the carrier-drag complexes is another essential aspect in the carrier design. It is desirable to have high drag packing density within the complex, where the drug can be released in a controllable manner that is below the toxic concentration but above the minimum therapeutic concentration. Thus, the determination of size, surface charge and surface chemistry is essential when characterizing the physico chemical properties of carrier-drug complexes. A siRNA sequence that corresponds to the connective tissue growth factor
(CTGF) was chosen as the model siRNA for its potential of breast cancer treatment. The TAT derived cell penetrating peptide Arginine-9 (R9) was chosen as the model peptide. Two separate statistical studies of nucleic acid-protein complexes have shown that arginine has a high frequency to interact with nucleic acids (Lejeune, D. et al. Proteins 2005, 61, 258-71; Jones, S. D. D. T. A. et al. Nucleic Acids Res. 2001, 29, 943-54). It was anticipated that electrostatic interaction and hydrogen bonding are the major driving forces for the interaction between siRNA and R9. In most cases, the long ranged columbic forces first bring the molecules in close proximity, followed by energy minimization through other forms of non-covalent interactions such as charge- dipole interaction, hydrogen bonds and Van der Waals interactions.
UV-Vis spectroscopy, circular dichroism, dynamic light scattering, Zeta potential measurements were used to investigate the physicochemical properties of CTGF siRNA-R9 complexes, including their equilibrium binding ratio, complex structure, size, and surface charge. The driving force for the complexation reaction was also verified by a salt addition experiment. siRNA and R9 Peptide
The CTGF siRNA was chosen as the model siRNA for this study. It had a sense sequence of 5 'CGGUGUACCGAGCCCAGAUdTdT 3' and an antisense sequence of 5'AUCUCCGCUCGGUACACCGdTdT 3'. It was purchased from Dharmacon (processing option A4; Lafayette, CO). The molar concentrations of siRNA were determined by absorption spectroscopy, using an extinction coefficient of 355,021 L/mol'cm. Crude R9 peptide with N-terminal acetylation and C-terminal amidation (AcN-RRRRRRRRR-CNH2) was purchased from the Sheldon Biotechnology Center at McGiIl University (Montreal, QC). Peptide identity was confirmed by mass spectroscopy and HPLC. Other reagents were all commercially available and were of analytical grade. Preparation of siRNA-R9 complexes Prior to use, siRNA and R9 peptides were first dissolved in Milli-Q water separately (Millipore, USA), divided in aliquots in microcentrifuge tubes, and stored in -2O0C after drying in Eppendorf Vacufuge Concentrator 5301. SiRNA at concentrations 1.5 μM, 3.0 μM, and 4.5 μM was first suspended in HEPES buffer (6 mM HEPES-NaOH, 20 niM NaCl, 0.2 mM MgC12, pH 7.3), then added to the dried peptide vials to achieve a final peptide concentration ranging from 0-60 μM The resulting complex solutions were stirred vigorously for 10 s with a vortex mixer and incubated for 3 hours at room temperature. UV- Vis Absorbance UV- Vis absorption spectra of each sample were obtained on a Hewlett-
Packard 8452A diode array spectrophotometer (California, USA) at wavelengths between 190 nm and 364 run, using a 75 μL quartz cuvette. Background absorbances were subtracted from the acquired signal. Circular Dichroism Circular dichroism (CD) measurements were preformed with a J-810
Spectropolarimeter (Jasco, USA). Spectra were acquired from samples in a 55 μL, 3mm path length quartz cuvette at 250C. Spectra were scanned from 400 to 200 nm at 200 nm/min, with a response time of 2 s and pitch of 1 nm. Spectra shown are the average of 3 replicates. Hydrodynamic Diameter and Zeta Potential Measurement
The hydrodynamic diameter of siRNA-R9 complexes was measured by dynamic light scattering (DLS) and Zeta potential by laser doppler velocimetry (LDV) at 25 0C using a Zetasizer Nano ZS (Malvern, UK) equipped with a 4 mW He-Ne laser operating at 633 nm. All measurements were performed at 250C at a measurement angle of 173°. SiRNA and R9 stock solutions were separately filtered through 0.2 μm non-protein binding syringe filters (Pall, USA) prior to complexation. The size and Zeta potential are presented as the mean value ± standard deviation from three measurements of at least 10 runs per measurement. Salt effect on siRNA-R9 binding High concentration of salt can destabilize non-covalent interactions, including electrostatic interactions and hydrogen bonds. Two solutions were first prepared, one with 1.5 μM siRNA only and the other with an addition of 150 μM of R9. One hour after peptide addition, 2 M sodium chloride was separately added to the two solutions. UV- Vis absorbance spectra of the two solutions were monitored before salt addition, two hours after salt addition and one day after salt addition. UV- Vis Absorbance The interaction between CTGF siRNA and a cell penetrating peptide R9 has been investigated with various spectroscopic methods. The UV-Vis absorbance spectra of 3.0 μiM siRNA in the absence and presence of R9 at concentrations ranging from 0 - 40 μM are shown in Figure 56. The characteristic peaks of siRNA at 210 nm and 260 nm are due to the presence of phosphate groups and nucleotide base pairs, respectively. The addition of R9 induced a decrease in the absorbance of the complex solution. The hypochromic effect on siRNA absorbance due to peptide addition is more pronounced with increasing peptide concentration, until reaching saturation at peptide concentrations above 32 μM. However, above this concentration significant sedimentation and turbidity can decrease the adsorption and minimize the suitability of UV-spectroscopy method. Hypochromicity of siRNA spectra was also observed upon peptide addition at siRNA concentrations of 1.5 μM and 4.5 μM. It is also noted that the red/blue shift at absorption maxima (210 nm and 260 nm) is negligible.
The hypochromic effect on siRNA due to the addition of R9 at a given wavelength can be quantified by the relative change in absorbance, ΔODr, defined as ΔODr=(ODo-OD)/ODo where OD0 is the initial absorbance of the free siRNA and OD is the observed absorbance of the sample containing siRNA-peptide complexes. The hypochromic effect at 260 nm upon peptide addition, expressed in terms of peptide concentration, is quantified for siRNA concentrations of 1.5 μM, 3.0 μM, and 4.5 μM. (Figure 57) A plot of hypochromicity can also be presented with respect to charge ratio, which is a normalization of R9 concentration with respect to siRNA concentration, expressed in terms of the charge ratio of positively charged R9 to negatively charged siRNA. (Figure 58)
At 3.0 μM siRNA, the relative change in absorbance initially increases (UV absorbance at 260 nm initially decreases) with increasing peptide concentration and eventually reaches a plateau after reaching saturation at 32 μM of R9, where only 7.8% of initial absorbance remained upon saturation. When hypochromicity is plotted against +/- charge ratio for siRNA concentrations of 1.5 μiM, 3.0 μM, and 4.5 μM, a significant portion of the curves is overlapped. The relative change in absorbance reaches its maximum at a charge ratio (+/-) of 2.2, which corresponds to a molecular binding ratio of 10.3 peptides per siRNA.
An important parameter for the determination of the optical properties of nucleic acids is the electric dipole transition moment (μoA), which represents the movement of charge density during the transition from the ground state to the excited state (Bloomfield, V. A. et al. Nucleic acids: structures, properties, and functions, University Science Books: Sausalito, Calif., 2000 "Bloomfield et al. (2000)".). A larger dipole moment would result in a higher absorption band. In addition to contribution of absorbance from individual nucleic acid bases, interaction between nucleic acid bases or other species in solution also affect their absorption intensities. When the resultant transition dipole moment is decreased, a decrease in absorbance occurs, and it is described as hypochromic. On the other hand, when the resultant transition dipole moment is increased, an increase in absorbance occurs and it is described as hyperchromia When the guanidino groups from R9 interact with nucleoside bases via hydrogen bonding, electron density contributed by the nucleic acid bases will be delocalized which resulted in a decrease in the magnitude of the electric dipole transition moment, and thus hypochromicity in the UV- Vis spectra.
The analysis method developed by Bujalowski and Lohman (Bujalowski, W.; Lohman, T. M. Biochem. 1987, 26, 3099-106 "Bujalowski & Lohman (1987)".) was applied to the absorbance data at 260 nm in attempt to obtain the equilibrium binding parameters between CTGF siRNA and R9 (see Figure 59). However, the analysis method was found to be not applicable to this experimental system. The signal contributed by the siRNA at 260 nm is solely from the nucleoside bases and it is possible that the decrease in absorbance cannot reflect the interactions that underwent other modes of interaction, such as electrostatic interaction with the phosphate backbone. More likely, aggregation of complexes, which was not included in the derivation developed by Bujalowski and Lohman, also affects the applicability of this method. Circular Dichroism
The CD spectra of siRNA-R9 complexes prepared at various R9 concentrations at 3.0 μM siRNA are shown in Figure 60. The siRNA has characteristic peaks around 210 nm and 265 nm, which, when compared to the CD spectrum of established nucleic structures (Bloomfield et al. (2000)), confirms that the siRNA possesses a right handed structure, similar to that of A-DNA. As the concentration of R9 increases, the ellipticity of complex solutions decreases progressively, until reaches a plateau at R9 concentrations above 35 μM. Similarly, the relative change in ellipticity, Δθr, is defined as Δθr=(θ0-Θ)/Θ0, where G0 is the initial ellipticity of the free siRNA and θ is the observed ellipticity of the sample containing siRNA-peptide complexes. A plot of the relative change in ellipticity over increasing R9 concentration, expressed separately in terms of R9 concentration and +/- charge ratio, is given in Figure 61 and Figure 62, respectively. By monitoring the changes in the CD spectra upon titration of peptide, the stoichiometry was found to be 10.7 R9 peptides per siRNA at saturation (+/- charge ratio of 2.2), which agrees with the results given by UV absorbance. Previous study on the binding between poly-L-arg and nucleotides (Wagner, K. G. Eur.J.Biochem. 1969, 10, 261.) has obtained a binding ratio of 2 arginine to one nucleotide, which corresponds to binding ratios between siRNA and R9 obtained by both UV and CD in this study.
CD measures the difference in absorption spectrum between left-handed and right handed polarized light. Therefore, CD provides sensitive and unique spectra for chiral molecules, and it has been widely used in structural determination of proteins and nucleic acids. With increasing R9 concentrations, the ellipticity of complex solution decreased, while the maximum and minimum peaks experience negligible shifting, which suggested that the structure of the siRNA experienced minimal structural changes upon interacting with R9. The decrease in ellipticity of siRNA due to increasing peptide concentration can be attributed to the decrease in absorbance of the nucleosides in siRNA-R9 complexes, similar to the spectra obtained by UV- Vis spectroscopy. Hydrodvnamic Diameter and Zeta Potential Measurements
The size and surface charge of the siRNA-R9 complexes are characterized by measuring the hydrodynamic diameter and Zeta potential (Figure 63). CTGF siRNA adopts the structure of the right-handed A form of DNA in solution, with a measured hydrodynamic diameter of 5.21 nm, which is very close to the theoretical value of 5.46 nm for a 21 base pair siRNA (Lodish, H. et al. Molecular Cell Biology, W. H.
Freeman & Co.: New York, 1999.). R9 peptide adopts a random coil structure (also confirmed by CD), and its hydrodynamic diameter is found to be 6.81 nm. With increasing peptide concentration, both the hydrodynamic diameter and Zeta potential of the complex solution increased. The size of the complexes increases until its value reaches 1055.8 nm. The Zeta potential of CTGF siRNA in HEPES is -36.2 mV, which reflects the contribution from the 42 negative charges on the phosphate group at neutral pH; whereas the Zeta potential of R9 in HEPES is 28.1 mV, due to the positively charged guanidino group. The increase in Zeta potential is the most pronounced when the charge ratio is between 1.43 and 8.57 mV, while its value increased consistently upon additional peptide addition. The rate of increase in the hydrodynamic diameter is at maximum when the charge ratio is also between 1.43 and 8.57, where the value of the surface charge is low. The increase in hydrodynamic diameter strongly demonstrated that the two species interact with each other. Further, since the hydrodynamic diameter increased by almost 200 fold, the results also indicate that siRNA and R9 forms large aggregates upon complexation, confirming the earlier observation in the UV- Vis absorbance measurements.
According to the DVLO theory for colloidal systems (Hunter, R. J. Zeta potential in colloid science: principles and applications, Academic Press: London, 1981), the energy barrier resulting from the repulsive force prevents two particles approaching one another and adhering together. However, when sufficient energy is given to overcome the barrier, the attractive forces will pull them into contact where they adhere strongly and/or irreversibly together. When the complexes have low Zeta potential, electrostatic repulsion is low thus the particles in solution can adhere to each other and result in large aggregates. The results from DLS and Zeta potential suggest that the complexation reaction is saturated when the charge ratio is above 8.57, which is higher than the values obtained by UV- Vis spectroscopy and CD. Thus, it is suggested that the hydrogen bonding sites between nucleosides and R9 is first depleted among the three. It can be seen that UV- Vis spectroscopy and CD can detect the complexation of siRNA and R9 only at a charge ratio below 2.2:1; further complexation and aggregation phenomena cannot be detected by these two methods. UV-Vis absorbance and CD signal has reached saturation at a charge ratio of ~2.2: 1, which is very close to the charge ratio corresponding to the isoelectric point of the siRNA-R9 complexes (around 3). Since size and surface charge are the essential parameters contributing to the activation of the complement system. As seen in Figure 63, peptide concentration can be used to control the size and surface charge of the siRNA-R9 complexes so that the interaction between the siRNA-peptide complexes and the immune system can be minimized during delivery.
Salt effect on siRNA-R9 binding
Ionic interaction contributed by salts can destabilize non-covalent interactions. To this end, a high concentration of sodium chloride (2 M) was added to the siRNA- R9 complex solution to investigate the driving force of the complexation reaction. Similar to the UV-Vis absorbance results above, hypochromicity is seen when excess peptide is added to siRNA, where the absorbance dropped by 83.0% in one hour following peptide addition (Figure 64). Two hours after the addition of 2 M sodium chloride, the absorbance of the complex solution increased and its absorbance is 13.1% lower than the absorbance of siRNA under the same treatment. The absorbance of the complex solution was monitored after one day and the absorbance remained the same.
The addition of salt increased the ionic strength of the solution, which weaken the non-covalent interactions in solution. Therefore, the restoration of absorbance upon salt addition to siRNA-R9 complex solution indicated that the interaction between siRNA and R9 are based on non-covalent interactions, which mainly includes electrostatic interaction, hydrogen bonding, and Van der Waals interaction. This result also indicates that the siRNA-R9 complexes are unstable in solutions with high ionic strength, in which the complexes dissociated and resulted in restoration of siRNA absorbance. The dissociation of complexes was demonstrated in a salt solution with a very high ionic strength (2 M); the stability of siRNA-R9 complexation in various salt concentrations, including physiological conditions, requires further investigations. Nevertheless, ionic strength can be used as another parameter that controls the complexation reaction in subsequent formulation considerations.
The fundamental aspects of using peptide earners for siRNA delivery have been explored using CTGF siRNA and R9 as a model at neutral pH (pH 7.3). Hypochromicity of siRNA absorbance was observed in UV and CD spectra upon addition of R9. The highest binding ratio for siRNA-R9 complexes was determined to be 10.3 R9 to one siRNA (corresponding to 2.2 in +/- charge ratio). Since peak shifts are negligible in UV and CD spectra, siRNA may not undergo significant structural changes. Using DLS, it is demonstrated that siRNA and R9 readily formed aggregates through molecular association, with a maximum hydrodynamic diameter of ~lμm at siRNA saturation. Aggregation is due to the decrease in surface charge at increasing peptide concentration demonstrated by Zeta potential measurements. The highest binding ratio of R9 to siRNA determined from DLS is 39.1 : 1 (corresponding to charge ratio of 8.4:1). The difference in binding ratios is possibly due to the difference in signal contributions between absorption and light scattering. Since the signal from absorption measurements is solely contributed by the nucleoside bases, it cannot represent the extent of the overall reaction when there are preferences in any of the binding sites. Furthermore, the salt dissociation experiment has shown that siRNA and R9 react through non-covalent interaction. The physicochemical characterization of CTGF siRNA-R9 complexes presented here have shown that various methods can be used to control the properties of the siRNA-peptide complexes, which provides necessary information for the formulation of siRNA therapeutics with peptide carriers. Equilibrium Binding Parameter Analysis - Supplementary Information for EX. 11
SiRNA has 42 negative charges per molecule and R9 has 9 positive charges per molecule. Therefore, it is expected that siRNA molecules can interact with multiple R9 molecules through columbic forces. Furthermore, two hydrogen atoms from the guanidino group of each arginine can hydrogen bond with the oxygen and nitrogen at the purine base of guanine within the major groove of the GC base pair. It is anticipated that electrostatic interaction and hydrogen bonding are the major driving forces for the interaction between siRNA and R9. In other words, siRNA is present as a macromolecule that can interact with multiple R9 ligands in a non-sequence-specific and non-covalent manner. In order to characterize the complexation reaction quantitatively, it is essential to obtain an accurate equilibrium binding isotherm.
Hypochromic effect of siRNA absorbance at 260 nm is observed upon its interaction with R9. By increasing peptide concentrations at a fixed siRNA concentration, three titration curves expressed in terms of hypochromicity were obtained at siRNA concentrations of 1.5 μM, 3.0 μM, and 4.5 μM.
According to thermodynamics, the interaction between siRNA and R9 would establish equilibrium between the free siRNA sites, free peptide and bound siRNA sites. A siRNA molecule consists of 21 base pairs and it forms a double helical structure with two 3' overhangs. It can be viewed as a linear lattice with N repeating units. It is assumed that each R9 covers the same number of phosphate groups (ή) on a siRNA. Since hypochromicity is observed upon R9-siRNA complexation, it follows that the extinction coefficients of the complexes is lower than that of the free siRNA. If there are r binding state and each binding state i has a distinct extinction coefficient, then according to the Beer's Law, the optical density of a R9-siRNA complex solution, ODobs, can be expressed as
ODobs = εfMf + ∑ εbi\Mbi (1)
where respectively <£/-and Mf are the extinction coefficient and molar concentrations of phosphate groups of unbound siRNA, _%,• and Mu are extinction coefficient and molar concentrations of phosphate groups of bound siRNA in state i, I is the light path length of cuvette. On the other hand, the mass conservation equation between Mf, Mb and Mt, which represents the free, bound and total siRNA phosphate concentrations, is
M, = Mf + ∑Mbt (2)
Furthermore, the binding density of the siRNA at state i, vh is defined as the number of peptide molecules bound per siRNA phosphate group
where, L^ is the bound peptide concentration for complexes in stage i. Substitute Equations 2 and 3 into Equation 1,
ODobs = ε,mt + nlMt '∑vl {εbι - εt) (4)
(=1
Given that the absorbance of the free siRNA, ODi is simply εβdh the relative change . in absorbance expressed in terms of hypochromicity (H), can be related to the extent of binding by
OD1 /-. i n K )
where Aεri is the relative change in extinction coefficient at state /. Therefore, the experimentally observed hyprochromicity is only a function of binding density vj. Further, to show that the fraction of siRNA bound is only a function of the free peptide concentration Lf, consider the equilibrium between the free siRNA sites, free peptide and bound siRNA sites:
1 Tf siRNA free sites + — Peptide « *-.iiRNA occupied sites n
where K represents the equilibrium binding constant, which is related to the equilibrium quantities through where Mb is the sum siRNA phosphate concentrations for all binding states, and it is equal to ΣMu- Substitute reaction site conservation equation (Equation 2) into Equation 6 and express in terms of binding density (Equation 3), one obtains
From Equation 7, it is shown that the overall binding density is only a function of free peptide concentration. Equation 5 illustrated that the experimental observed hypochromicity is a unique function of the binding density, whereas Equation 7 demonstrated that the binding density is a unique function of the free peptide concentration. In other words, hypochromicity is also a unique function to the free peptide concentration, related through the binding density. As a result, the free peptide concentration and binding density will be constant for a number of different combinations of total peptide and total siRNA concentrations (L1 ,M1 ) taken at a constant hypochromicity value, given that it follows the peptide mass conservation equation
L1 = L1 + M^ v1 (8) i
When two siRNA titration curves were obtained, the binding density and free peptide concentration at each hypochromicity can be obtained with a set of two simultaneous equations of Equation 8. When three or more siRNA titration curves were obtained, the above quantities can be obtained through linear regression of Equation 8. Through the use of the above analysis technique by Lohman and Bujalowski (Bujalowski & Lohman (1987)), a model independent binding isotherm can be obtained without applying any assumptions.
Fitted values of total peptide concentration are calculated at chosen hypochromicity values for siRNA concentrations of 1.5 μM, 3.0 μM, and 4.5 μM. Linear regression is performed according to Equation 8 to obtain binding densities and free peptide concentrations at each hypochromicity value. However, the calculated free peptide concentration is negative in the experimental relevant range, which means that this analysis is not applicable to this experimental system. Figure 65 is a plot of binding densities versus free peptide concentrations. Since the signal contributed by the siRNA is solely from the nucleoside bases and it is possible that the decrease in absorbance cannot reflect the interactions that undergo other modes of interaction, such as electrostatic interaction with the phosphate backbone and charge dipole interaction with the sugar ring. Furthermore, aggregation of complexes also affects the applicability of this method.
EXAMPLE 12: INVESTIGATION OF ABILITY OF EAKl 6-11 TO STABILIZE HYDROPHOBIC ANTICANCER AGENT In this Example, the ability of the self-assembling peptide EAKl 6-11 in stabilizing the hydrophobic anticancer agent ellipticine was investigated. The formation of peptide-ellipticine suspensions was monitored with time until equilibrium was reached. The equilibration time was found to be dependent on the peptide concentration. When the peptide concentration was close to its critical aggregation concentration (CAC, ~0.1 mg/mL), the equilibration time was minimal at 5 h. With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be stabilized. These different states of ellipticine significantly affected the release kinetics of ellipticine from the peptide-ellipticine complex into the egg phosphatidylcholine (EPC) vesicles, which were used to mimic cell membranes. The transfer rate of protonated ellipticine from the complex to the vesicles was much faster than that of crystalline ellipticine. This observation may also be related to the size of the resulting complexes as revealed from the scanning electron (SE) micrographs. In addition, the complexes with protonated ellipticine were found to have a better anticancer activity against two cancer cell lines, A549 and MCF-7.
The ideal drug delivery vehicle should have the following properties: biocompatible, biodegradable, suitable size, high loading capacity, extended circulation time, and capable of accumulating at required pathological sites in the body. Peptides have shown much potential for drug delivery. The most attractive aspect of peptide-mediated drug delivery is the natural propensities of many peptides for cell penetration and targeting. As a result, many novel delivery systems involve peptides to achieve targeted delivery for anticancer therapeutics and to cross the cell membrane barrier for gene/siRNA delivery. Peptide-based delivery systems have also shown the potential to deliver therapeutic proteins, bioactive peptides, small molecules and nucleic acids.
A special class of self-assembling, ionic-complementary peptides taught in the present application represents a new and promising biomaterial for constructing drug delivery nanocarriers. The unique amphiphilic structure and the ability of self- assembly of these peptides allow them to encapsulate both hydrophobic chemotherapeutics and hydrophilic protein and oligonucleotides. Moreover, no detectable immune response was observed when these peptides were introduced into animals. (Holmes, T. C. et al. Extensive Neurite Outgrowth and Active Synapse Formation on S elf- Assembling Peptide Scaffolds. Proc. Natl. Acad. Sci. USA 2000, 97, 6728-6733; Zhang, S. et al. Spontaneous Assembly of a Self-Complementary Oligopeptide to Form a Stable Macroscopic Membrane. Proc. Natl. Acad. Sci. USA 1993, 90, 3334-3338; Zhang, S. et al. Self-Complementary Oligopeptide Matrices Support Mammalian Cell Attachment. Biomaterials 1995, 16, 1385-1393.) These peptides can spontaneously organize themselves into nano/micro structures that may provide a protected and stable environment for the therapeutic molecules, and facilitate passive targeting. (Gu, F. X. et al. Targeted Nanoparticles for Cancer Therapy. Nanotoday 2007, 2, 14-21 "Gu et al. (2007)".) An additional advantage of using such peptide-based carriers is the ease of sequence modification and design to incorporate peptide cell penetration and active targeting capabilities.
A representative self-assembling, ionic-complementary peptide, EAKl 6-11 (Figure 66), has been shown to encapsulate hydrophobic compounds readily and stabilize them in aqueous solution. Work using pyrene as a model hydrophobic compound demonstrated the potential of this peptide in the delivery of hydrophobic anticancer drugs. EAK16-II was shown to stabilize pyrene microcrystals in aqueous solution at a concentration ten-thousand fold beyond its solubility in water, indicating a very high loading efficiency. The encapsulated pyrene can be released from the peptide coatings into liposomes and the release rate can be controlled by changing the peptide-to-pyrene ratio during the encapsulation. This peptide has been used to stabilize microcrystals of the anticancer agent ellipticine in aqueous solution (see Example 3). The stabilized ellipticine microcrystals can have a concentration several hundred times more than its solubility. Ellipticine was selected as the model hydrophobic anticancer drug in our studies for the following reasons: first, the fluorescence property of ellipticine enables us to monitor the interaction of ellipticine with the peptide and locate it in different micro-environments. Second, ellipticine is extremely hydrophobic with a low water solubility of ~0.62 μM at neutral pH, (Liu, J. et al. Polymer-Drug Compatibility: a Guide to the Development of Delivery Systems for the Anticancer Agent, Ellipticine. J. Pharm. Sci. 2004, 93, 132-144) which is comparable with that of the model hydrophobic compound pyrene. Third, its great anticancer activity makes ellipticine one of the promising candidates in cancer chemotherapy. (Garbett, N. C; Graves, D. E. Extending Nature's Leads: the Anticancer Agent Ellipticine. Curr. Med. Chem. 2004, 4, 149-172 "Garbett & Graves (2004)".) Fourth, the discovery of severe side effects of ellipticine derivatives during clinical trials suggests that a novel delivery system is required. (Garbett & Graves (2004); Clarysse, A. et al. Phase II Study of 9- Hydroxy-2NMethylellipticinium Acetate. Eur. J. Cancer Clin. Oncol. 1984, 20, 243- 247.) In this study, we investigated the effect of peptide and ellipticine concentration on the formation of peptide-ellipticine complexes in aqueous solution over time. This will elucidate the kinetics of the complex formation in relation to peptide self- assembly. A new methodology is applied to study the release kinetics of ellipticine from the peptide-ellipticine complexes to EPC vesicles as cell membrane mimics. The fluorescence technique is the primary tool to characterize the complex formation and the release kinetics, where the change of ellipticine fluorescence is monitored over time. Scanning electron microscopy (SEM) is applied to characterize the dimensions of the peptide-ellipticine complexes. The complexes with various peptide-to- ellipticine ratios (by mass) are further tested on their cellular toxicity against two cancer cell lines, A549 and MCF-7. The cells are treated for different time periods (4- 48 h) to reveal the time-dependent toxicity of the complexes. Time Dependence of the Formation of Peptide-Ellipticine Complexes To investigate the details of the complexation kinetics, the change in the ellipticine fluorescence of the peptide-ellipticine dispersions was monitored over time. Figure 67a shows the fluorescence spectra of the complex suspension with 1.0 mg/mL ellipticine and 0.2 mg/mL EAKl 6-11 at different times. Initially, the fluorescence spectrum exhibits a characteristic of protonated ellipticine with a peak located around 520 nm after 0.5 h stirring. This peak rises with time and reaches a maximum after 6 h; it then decreases with time. Meanwhile, a shoulder located at ~ 468 nm becomes pronounced with time and eventually forms a peak after 9 h. The band at 468 nm is characterized as the ciystal form of ellipticine. There was no trace of fluorescence from ellipticine crystals (at 468 nm) initially; although ellipticine was in crystalline form when just mixed with the peptide solution, the ellipticine crystals were large, unable to suspend in solution, and thus precipitated at the bottom of the sample vial, not contributing to the fluorescence signal detected.
The intensity changes of the two peaks at 468 and 520 nm were plotted with time in Figure 67b. It can be clearly seen that the intensity at 520 nm increases for the first 6 h to a maximum and then decreases to the initial level after 1O h. On the other hand, the intensity at 468 nm increases with time and reaches a plateau after -15 h. The latter indicates that ellipticine crystals or microcrystals are gradually stabilized in aqueous solution, to form a peptide-ellipticine suspension with time. The final state of stabilized ellipticine is in crystalline form and equilibrium is reached after -15 h (considering both I468 and I52o reaching equilibrium).
The change in the fluorescence of protonated ellipticine with such a trend may indicate a special mechanism of the complex formation. It is speculated that the fresh peptide solution could facilitate the formation of protonated ellipticine. Since ellipticine has a pKa of ~6 (pyridine-like nitrogen), it can be protonated in a weak acidic environment ((Garbett & Graves (2004); Sbai, M. et al. Use of Micellar Media for the Fluorimetric Deteπnination of Ellipticine in Aqueous Solutions. J. Pharm. Biomed. Anal. 1996, 14, 959-965 "Sbai et al. (1996)"; El Hage Chahine, J. M. et al. Kinetics and Thermodynamics of the Formation of Inclusion Complexes Between Cyclodextrins and DNA-Inercalating Agents. Inclusion of Ellipticine in G- Cyclodextrin. J. Chem. Soc. Perkin Trans. II 1989, 629-633.) A fresh 0.2 mg/mL EAK16-II in pure water has a pH value of -4.6, which can cause the protonation of ellipticine. In addition, the peptide molecules consisting of negatively charged glutamic acid residues may help stabilize the protonated ellipticine upon interaction. A similar phenomenon has been reported that highly negatively charged SDS micelles can stabilize protonated ellipticine in pure water (Fung, S. Y. et al. Solvent Effect on the Photophysical Properties of the Anticancer Agent Ellipticine. J. Phys. Chem. A 2006, 110, 11446-11454; Sb.ai et al. (1996)) The amount of protonated ellipticine increases during the first several hours to a maximum and then disappears when the equilibrium is established. The diminishing of the protonated ellipticine prior to equilibrium may be related to the peptide self-assembly over time and its associated events. As shown in Figure 68, a 0.2 mg/mL EAKl 6-11 solution can significantly increase the scattered light intensity 30 h after preparation, which is 6 fold higher than that of the fresh peptide solution. This is the evidence of peptide assembly over time under constant mechanical stirring. The formation of EAKl 6-11 assemblies may consume the negatively charged glutamic acid residues as they are complementary to the lysine residues in the assemblies. This in turn reduces the amount of free glutamic acid residues that are able to stabilize the protonated ellipticine. Meanwhile, the pH of the EAKl 6-11 solution was found to increase from -4.6 (fresh) to 6.4 (30 h after preparation), which is slightly above the pKa of ellipticine. The combination of these two effects can induce deprotonation of ellipticine, thereby explaining the disappearance of protonated ellipticine over time.
On the other hand, the peptide assembly does not likely inhibit the formation of stable ellipticine microcrystals. In fact, these peptide assemblies are mainly made of β-sheets, which are amphiphilic with hydrophobic and hydrophilic regions on the opposite sides. The hydrophobic region can still interact with hydrophobic ellipticine microcrystals to form stable peptide-ellipticine suspensions. It has been shown that EAK 16-11 can adsorb on hydrophobic surfaces and assemble into stable β-sheet rich nanostmctures.
Concentration Effect on the Complex Formation
Figure 69 shows the peptide concentration effect on the formation of peptide- ellipticine complexes at a fixed ellipticine concentration of 1.0 mg/mL. The peptide concentration ranges from 0.05 to 0.5 mg/mL. The fluorescence intensity at 468 nm (crystalline ellipticine) increases with time and reaches a plateau for all peptide concentrations used, but the time required to reach equilibrium is dependent on the peptide concentration (Figure 69a). The equilibration time is at minimum (~5 h) when the peptide concentration is around 0.1 mg/mL, which is the CAC of the peptide. When the peptide concentration is away from the CAC, the equilibration time increases (>10 h). The change in the fluorescence intensity at 520 nm (protonated ellipticine) is also strongly dependent on the peptide concentration as shown in Figure 69b. Above the CAC, the protonated ellipticine can be seen to form and over time to disappear. The protonated ellipticine stays for a longer time (40 h) with a higher peptide concentration (0.5 mg/mL). At the CAC, the fluorescence of potonated ellipticine only appears in the first 2 h and quickly disappears afterwards. Below the CAC, no significant protonation of ellipticine is observed.
The overall equilibration time of the peptide-ellipticine complexation at different EAKl 6-11 concentrations is listed in Table 15. The reported values were estimated from Figure 69, considering that both processes of ellipticine protonation and formation of stable ellipticine microcrystals have reached steady-state or equilibrium. Note that when the equilibrium of the both processes is reached, the final state of the stabilized ellipticine is mainly in crystalline form. It can be clearly seen that the overall equilibration time is strongly dependent on the peptide concentration and approaches a minimum when the peptide concentration is close to the CAC. This phenomenon may be related to the solution pH at various peptide concentrations and the peptide self-assembly. Table 15: Peptide concentration-dependent equilibration time and solution pH
[EAK] (mg/mL) Estimated equilibration time (h) pH of fresh peptide solution
__
0.50 40
0.20 15 4.6
0.10 7 5.2
0.08 4 5.5
0.05 12 6.0 Notes:
1. The equilibration time was estimated from Figure 69, considering that both I468 and I520 reach equilibrium. 2. The pH was measured from freshly prepared peptide solutions.
At peptide concentrations below the CAC, the peptide solution has a relatively high pH, which prohibits ellipticine protonation. But under such a condition, microcrystals of ellipticine can form over time. Such formation of ellipticine microcrystals becomes faster with increasing peptide concentration up to its CAC. At the CAC, a low pH, below the pKa of ellipticine, is observed. Such a low pH allows protonated ellipticine to form (Figure 69b). However, over time peptide assemblies start to appear at this concentration; meanwhile the solution pH starts to increase and the number of available glutamic acid residues reduces. (The glutamic acid residues can stabilize the protonated ellipticine.) As a result, deprotonation of ellipticine occurs shortly after the initial protonation.
At peptide concentrations above the CAC, a longer equilibration time is observed. This is probably due to the combined effects of the ellipticine protonation and microcrystal formation. The pH of a fresh peptide solution decreases with an increase in peptide concentration as shown in Table 15. At a concentration above the CAC, the solution has a pH below 5, which can induce the protonation of ellipticine. The lower the solution pH is, the more ellipticine can be protonated. The protonated ellipticine will gradually disappear with time while more stable ellipticine microcyrstals form. It takes a longer time for protonated ellipticine to disappear at a higher peptide concentration, leading to a longer equilibration time.
The peptide EAKl 6-11 is capable of stabilizing ellipticine microcrystals and protonated ellipticine in aqueous solution in a time-dependent and peptide concentration-dependent manner. The state of ellipticine could be critically important in its function as a therapeutic agent. It has been reported that the neutral form of ellipticine is active against various tumors (Garbett et al. (2004); Sainsbury, M. Ellipticine; In The Chemistry of Antitumour Agents, Wilman, D. E. V., Ed. Blackie and Son Ltd.: Glasgow, UK, 1990; pp 411-435.) Further in vitro and in vivo studies will elucidate the effects of the different states of ellipticine on its activity against cancer cells. The ellipticine concentration effect on the complex formation was investigated with 0.2 mg/mL EAKl 6-11 and three ellipticine concentrations: 1.0, 0.5 and 0.1 mg/mL. The change in fluorescence of crystal and protonated ellipticine is shown in Figure 70a and b, respectively. At this peptide concentration, the ellipticine concentration does not seem to affect the overall equilibration time significantly. The time for the peptide-ellipticine suspension formation (both ellipticine protonation and formation of ellipticine microcrystals should reach equilibrium) is all ~10 h.
Interestingly, a particular combination of 0.1 mg/mL ellipticine with 0.5 mg/mL EAKl 6-II, increased from 0.2 mg/mL, can stabilize protonated ellipticine for a prolonged time (Figure 70, crosses). The intensity at 520 nm decreases slightly at the beginning and reaches a plateau after 1O h, while that at 468 nm remains constant at a low value over time. The very low intensity at 468 nm is within the background noise, indicating that no ellipticine microcrystals can be detected. This result shows that most ellipticine is protonated and solubilized in the solution. The slight initial decrease in intensity of the protonated ellipticine is likely the results of inner-filter effect as the solution solubilizes more ellipticine with time. The protonated ellipticine is stable for at least 50 h, the duration of the experiment, under continuous mechanical stirring. During this time period, the solution remains clear with a yellow-orange color. This particular combination of ellipticine and peptide concentrations suggests that a prolonged state of protonated ellipticine can be established. This will affect ellipticine release kinetics, and probably its therapeutic efficiency. Release of Ellipticine from the Complex into EPC Vesicles
So far, we have shown the formation of peptide-ellipticine dispersions in water, either in microcrystal or protonated form. It is important to investigate how ellipticine releases from the peptide complex, and the release kinetics. This was done by mixing the complex with liposome vesicles, which mimic the cell membrane.
Four peptide concentrations (0.05, 0.1, 0.2 and 0.5 mg/mL) were used to form stable peptide-ellipticine dispersions with 0.1 mg/mL of ellipticine, to study the release kinetics. The samples were stirred for 24 h to ensure that equilibrium was reached. At 0.5 mg/mL of EAK16-II, the dispersion looks more yellow and less turbid comparing to others at lower peptide concentrations, providing the additional evidence to the results of Figure 70 that the 0.5 mg/mL EAKl 6-11 solution can stabilize protonated ellipticine for a prolonged time. The different states of ellipticine at equilibrium can be clearly seen from their characteristic fluorescence spectra as shown in Figure 71. The suspension with 0.5 mg/mL EAKl 6-11 has a pronounced peak located ~525 nm (protonated state) while those with peptide concentrations ranging from 0.05 to 0.2 mg/mL exhibit a peak of ~468 nm (crystalline state) (Figure 71, inset). The very different properties of the complex suspensions according to the peptide concentration could also have significant effects on the ellipticine release (see below).
It is noted that the fluorescence of ellipticine, either protonated or in crystalline form upon interaction with peptides, is very different from that of ellipticine in EPC vesicles. Ellipticine in the vesicles exhibits a strong fluorescence signal at -436 nm, which is characterized as neutral, monomeric ellipticine. Such a signal can be well distinguished from those of the protonated ellipticine (~520 nm) and ellipticine crystals (weak fluorescence at ~468 nm). The transfer of ellipticine from the complex into the vesicles can be monitored by the change in ellipticine fluorescence at 436 nm over time.
A typical transfer curve of ellipticine from the complex to the EPC vesicles, or liposomes, is shown in Figure 72a. The complex was made with 0.05 mg/mL EAKl 6-11 and 0.1 mg/mL ellipticine in pure water with 24 h stirring. The fluorescence intensity at 436 nm increases with time and approaches to a plateau after 20,000 s. The reason that the initial point starts slightly above zero (t = 0) is probably due to the burst release of ellipticine into the vesicles during the initial sample mixing time (<30 s).
The transfer curve based on the ellipticine fluorescence can be related to its concentration accumulation in vesicles using a calibration curve. It was found that the vesicles were saturated with ellipticine when ellipticine concentration was above ~20 μM. Thus, the rising region in the calibration curve can be used to convert the fluorescence signals from the transfer profile to the ellipticine concentration in vesicles with a simple exponential equation: I = A(l - e-B[Epr]) (1)
The fitting parameters A and B are 17.5 ± 0.36 and 230000 ± 13400 (1/M), respectively. [EP J] represents the concentration of ellipticine within the range of 0-20 μM. For a given fluorescence intensity of ellipticine (I/Is<\1.5), one can obtain the corresponding ellipticine concentration using Equation 1. Figure 72b shows four transfer profiles of ellipticine concentration in the vesicles ([EPTV]) with time (h). Each curve corresponds to the transfer of ellipticine into the vesicles from different peptide-ellipticine complexes made with various
EAKl 6-11 concentrations. All profiles have a similar trend with a fast increase initially and gradually approaching a plateau. The very high initial values of the transfer profile from the complexes with 0.5 mg/mL EAK16-II indicate a burst release of ellipticine from the complex into the vesicles within 30 s. This is reasonable since the 0.5 mg/mL EAKl 6-11 solution can stabilize protonated ellipticine (Figures 70 and 5 71). These protonated ellipticine molecules may easily migrate into the lipid bilayers, causing a sudden increase in the ellipticine concentration in the vesicles. On the other hand, other peptide concentrations (0.05-0.2 mg/mL) do not stabilize protonated ellipticine but ellipticine microcrystals. The migration of ellipticine molecules from the microcrystals to the vesicles involves the molecularly dissolving of ellipticine,0 which is time consuming. Therefore, their transfer profiles do not have a large sudden increase, and start at values close to zero ellipticine concentration.
To better compare the transfer kinetics for the four different EAKl 6-11 concentrations, the profiles were fitted to one of the following exponential equations. The second equation was used if the first could not satisfactorily describe the5 dynamics:
[EPTvit) = [EPTv\q - ([EPTv]eq -[EPTv]o ykt (2)
[EPTV ]{t) = [EPTV ]eq (l - axe-« - a2e^' ) (3) where [EPTv](t), [EPTv]eq and [EPTv]o are the ellipticine concentration in the vesicles at time t, at equilibrium and at time zero, respectively; k, ki and fø are the rate0 constants; aj and a^ are the pre-exponential factors and ai + <X2 - 1. The particular transfer profile with 0.5 mg/mL EAKl 6-11 was fitted with Equation 2 where [EPTv]o ≠O due to an initial burst transfer of ellipticine into the vesicles; the other three profiles were fitted well with Equation 3 as the initial transfer in these cases was very small and can be negligible. The rate constants are summarized in Table 16.5 Comparing the average rate constants for each transfer profile, it can be seen that the rate of transfer of ellipticine from the complexes into the vesicles increases with the peptide concentration during the preparation of peptide-ellipticine complexes. Table 16. Transfer rates of ellipticine from peptide-ellipticine complexes to EPC liposomes [EAK] 0.5 mg/mL* 0.2 mg/mL 0.1 mg/mL 0.05 mg/mL Ic1 (1/h) 3.13 ± 0.14 5.20 ± 0.04 3.52 ± 0.03 2.53 ± 0.02
0.363 ± 0.002 0.345 ± 0.002 0.348 ± 0.003
k2 (1/h) n/a 0.75 ± 0 .002 0.62 ± 0 .002 0.30 ± 0.003
Cl2 0.637 ± 0.002 0.655 ± 0.003 0.652 ± 0.002
K(XVg 3.13 ± 0. 14 2.36 ± 0 .02 1.62 ± 0 .01 1.08 ± 0.01
R2 0.976 0.999 0.999 0.999
^Denotes the fitting with Equation 2; all others with Equation 3. All the fitting parameters are significantly different from the statistical analysis. kavg = aiki + a2k2; α/ + a2 = 1
This trend is opposite to that of our previous studies on the pyrene release from 5 the EAK16-II coatings into the EPC vesicles. The higher EAK16-II concentration used to form peptide-pyrene complexes results in a thicker coating on the pyrene microcrystals, which in turn causes a slower release rate. In the present case of ellipticine, the higher peptide concentration induces the protonation of ellipticine and formation of smaller complexes as visualized by the SEM images in Figure 73. The size of the complexes with 0.5 mg/mL EAK16-II (Figure 73a) is much smaller than that with 0.2 mg/mL EAKl 6-11 (Figure 73b). A further decrease in the peptide concentration (0.05 mg/mL) will result in a bigger size of the complexes as shown in Figure 73c. Since ellipticine in the vesicles is molecularly solubilized, the transfer process must involve the release of individual ellipticine molecules from the complexes; the bigger the complexes are, the longer the release of ellipticine from the complexes will be. Therefore, a slower transfer rate of ellipticine was observed at low peptide concentrations. Cellular Toxicity of EAK16-II-Ellipticine Complexes
So far, we have shown that EAKl 6-11 can stabilize ellipticine in protonated or crystalline form in aqueous solution, depending on the peptide and ellipticine concentrations. Protonated ellipticine can be stabilized in the complexes formulated with a combination of 0.5 mg/mL EAKl 6-11 and 0.1 mg/mL ellipticine. When the ratio of peptide-to-ellipticine is smaller than 5:1 (by mass), the stabilized ellipticine is predominantly in crystalline form in the complexes (Figure 71). This may indicate that the 5:1 ratio is important in determining the molecular states of ellipticine in the complexes. It is expected that the different molecular states will have different therapeutic effects against cancer cells.
Here a series of peptide-to-ellipticine ratios (at a fixed ellipticine concentration of 0.1 mg/mL) was used to form peptide-ellipticine complexes, and their cellular toxicity was investigated on two cancer cell lines, A549 and MCF-7. It has been found that the solutions turn yellow and transparent at ratios of 5: 1 and 10: 1, indicating the formation of protonated ellipticine in the complexes. At ratios below 5: 1, the solutions appear to be turbid as the stabilized ellipticine is predominantly in crystalline form. However, the ellipticine control sample remains transparent with chunks of ellipticine crystals floating on top or at the bottom of the vial, owing to its extreme hydrophobicity with a veiy low solubility in water (~0.6 μM). These results confirm our previous observations and provide evidence that whether protonated or crystalline ellipticine can be stabilized is related to the peptide-to-ellipticine ratio.
As shown in Figure 74a. The complexes at the ratios of 5:1 and 10:1 are effective at killing both cancer cells, leading to low cell viability (less than 0.25). Below the 5: 1 ratio, the anticancer activity of the complexes decreases significantly, and is similar to the ellipticine control (no peptides). The dramatic change in the complex toxicity is probably related to the molecular state of ellipticine in the complexes. The protonated ellipticine appears to be more effective at killing cancer cells than crystalline ellipticine. This may be due to fast release kinetics of protonated ellipticine from the complexes (Figure 72b). In addition, protonated ellipticine tends to interact with negatively charged cell membranes, and accumulate at the membrane surface; the hydrophobic moiety of ellipticine further helps it cross the cell membrane. This also implies that the internalization of ellipticine may not be through energy-dependent endocytosis.
Note that the protonated ellipticine seems to be more active at killing MCF-7 cells than A549 cells, causing an almost zero MCF-7 cell viability. Such an effect may be due to the fact that MCF-7 cells are more sensitive to protonated ellipticine. Thus, these results may lead to a notion of selecting appropriate formulations to treat different cancer cells. By adjusting the mass ratio of EAKl 6-11 versus ellipticine, one can obtain different molecular states of ellipticine in the complexes as well as the complex dimensions, for specific cancer cells. The stability of a given formulation upon dilution is an important factor in determining its applicability in clinical usage. Since the complexes at 5:1 ratio show a good anticancer activity against both cancer cell lines, we further carried out serial dilution of such complexes in pure water and test their stability in relation to the cellular toxicity. Figure 74b shows the toxicity of the complex prepared at a 5: 1 ratio and its serial dilution in water (2, 4, 8 and 16 times). The 2 times dilution does not affect the toxicity of the complex significantly for both cells. Further dilution greatly reduces the complex toxicity against MCF-7 cells; it also decreases the toxicity against A549 cells, but to a lesser degree. Normally, the decrease in cell viability should be gradual and smooth due to the decrease in drug concentration upon dilution. However, the observed trend is not gradual, but rather a sharp change at more than 2x dilution. This may be related to the instability of the complexes upon dilution in water. One possible reason is that the complex containing protonated ellipticine is pH sensitive; extensive dilution is expected to increase the solution pH, leading to the deprotonation of ellipticine to form ellipticine microcrystals. As a result, the toxicity of the diluted complexes reduces, similar to that of the complexes prepared at a lower peptide-to-ellipticine ratio. Note that a drastic decrease in complex toxicity upon dilution for MCF-7 cells provides additional evidence that MCF-7 cells are more sensitive to protonated ellipticine. The time-dependent toxicity of the complexes was further investigated and the results are shown in Figure 75. Two ratios, 1: 1 (squares) and 5:1 (circles), were used to examine the difference in toxicity between crystalline and protonated ellipticine, respectively. They were compared with the ellipticine control (crosses) in the absence of EAKl 6-II. It can be seen that the two different cell lines exhibit different patterns in the time-dependent viability in response to the treatments. For MCF-7 cells (Figure 75a), the complexes at the 5: 1 ratio are so effective that the cell viability decreases to less than 0.5 after 4 h treatment; it decreases further to almost zero after 24 h treatment. Meanwhile, the complexes at the 1: 1 ratio and ellipticine control have almost no effect at 4 h, before gradually decreasing the viability to about 0.5 by 48 h. On the other hand, no distinguishable difference in cell viability can be observed among the three treatments at 4 h for A549 cells (Figure 75b); with time, all treatments cause significant cell death, although the effect is more pronounced for the complexes at 5: 1 ratio. Almost zero viability is achieved after 48 h treatment with protonated ellipticine (5:1 ratio).
The above results indicate that again, MCF-7 cells are very sensitive to protonated ellipticine while both protonated and crystalline ellipticine are effective at killing A549 cells. However, the reason behind this is still unclear. One may speculate that such a phenomenon probably results from the differences in uptake of ellipticine in the complexes by A549 and MCF-7. Or it may be due to the different nature of the two cancer cell lines in response with ellipticine. Further experiments are required to study this phenomenon. Cellular Uptake of Ellipticine in A549 and MCF-7 Cells
The uptake of ellipticine by the two cell lines is shown in Figure 75.1. The cells are treated with complexes at two ratios of 5: 1 and 1: 1, the ellipticine control and the peptide control for 5, 15 and 30 min. For A549 cells (Figure 75.1a), the treatment with peptide alone does not exhibit any fluorescence (1st column, insets), which is reasonable since there is no ellipticine in the system and EAKl 6-11 is not fluorescent. When the cells are treated with ellipticine control, the fluorescence signals are too dim to be seen initially, but increase with time. A stronger fluorescence signal can be observed for the treatments of complexes at 5 min and become more pronounced at later times. After 30 min treatment of the complexes at the 5: 1 ratio, the ellipticine fluorescence seems to accumulate in the cell nuclei. Similar phenomena are observed for MCF-7 cells (Figure 75.1b).
These results show that the uptake of ellipticine is fast. This is likely because ellipticine is a small hydrophobic molecule, and can easily cross the cell membrane barrier once reaching its outer surface. However, the presence of peptides appears to enhance the rate of uptake. One reason is that peptides can stabilize large amounts of protonated or crystalline ellipticine in aqueous solution. The high concentration of ellipticine in solution facilitates its diffusion into the cells. In addition, protonated ellipticine seems to have a much stronger tendency to be taken up by both cell lines, especially MCF-7 cells. This may due to a stronger interaction between positively charged ellipticine and negatively charged cell membranes. Besides, protonated ellipticine is released much faster from the complexes than crystalline ellipticine. It is worth noting that the subtle difference in ellipticine uptake from the complexes at two different ratios cannot explain the much higher toxicity of the complexes at the 5:1 ratio over the other. Other factors may determine the toxicity of protonated and crystalline ellipticine. The toxicity difference may be related to variations in the amount of ellipticine uptaken at the two ratios, although it is not directly reflected from these fluorescence images. To confirm whether the internalization of ellipticine is through direct permeation across the cell membranes, the uptake of ellipticine is conducted at two different temperatures, 370C and 4°C. Most cellular uptake through the endocytosis pathway is energy dependent, which can be blocked at a temperature as low as 4 0C (Rejman, J. et al. Size-dependent internalization of particles via the pathways of clathrinand caveolae-mediated endocytosis. Biochem. J. 377, 159-169 (2004)). As shown in Figure 75.2, the uptake of ellipticine in both cancer cells can be observed after 30 min treatments with the complexes as well as the ellipticine control. Evidently, there are no significant differences in ellipticine uptake between 370C and 4°C, although the ellipticine fluorescence is weak at 40C for the treatment of ellipticine control. The temperature-independent uptake of ellipticine indicates that the passive diffusion of ellipticine into the cells is probably the primary cell internalization mechanism.
From these results, the complexation of ellipticine with EAKl 6-11 seems not to alter the internalization pathway of ellipticine; however, it enhances the rate of cellular uptake by increasing the amount of ellipticine suspended in solution. Note that such a fast and unspecific cellular uptake may not be ideal for anticancer drug delivery as it would cause harmful side effects to healthy tissue during delivery. To overcome this problem, one could increase the interaction between ellipticine and peptides to slow the release process. For example, one could increase the hydrophobicity of the peptide through sequence design, which may increase the interaction between the hydrophobic ellipticine and peptides. On the other hand, unspecific uptake may be solved by introducing cell targeting moieties onto the peptide sequence to achieve active targeting. An example is the cyclic peptide motif c-NGRGEQ-c, which has been found to strongly bind to several non-small cell lung cancer cell lines including A549, CaIu-I and H178. A vasoactive intestinal peptide (VIP) can selectively bind to many breast cancer cell lines such as MCF-7 (Moody, T. W., et al. The development of VIP-ellipticine conjugates. Regul. Pept. 123, 187-192 (2004); Moody, T. W. et al. VIP-ellipticine derivatives inhibit the growth of breast cancer cells. Life Sci. 71, 1005-1014 (2002)). Thus, a proper design of peptide sequence becomes very important in the development of self-assembling peptide- mediated delivery for hydrophobic anticancer drugs.
The study presented demonstrates that a self-assembling, ionic-complementary peptide, EAKl 6-II, can stabilize the hydrophobic anticancer agent ellipticine in aqueous solution. Different combinations of peptide and ellipticine concentrations can stabilize either protonated or crystalline ellipticine for an extended time. The ellipticine can be released from the complexes into a cell membrane mimic. The release rate is related to the peptide concentration used in the complexation. By optimizing the process of complex formation, one could obtain desired complex dimensions and drug release property. These factors and molecular states of ellipticine can have significant impacts on the cellular toxicity of the peptide-ellipticine complexes.
The results obtained here will be important in the next phase studies on the peptide-based delivery of ellipticine in vivo. First, the size of the peptide-ellipticine complexes can be controlled from micrometers to hundreds of nanometers. The particle size will significantly affect the circulation in the blood stream, binding to the cells and uptake by the cells. The size of the complexes ranging from 100 nm to 500 nm would be ideal for passive targeting to solid tumors via the enhanced permeability and retention (EPR) effect (Gu et al. (2007); Brigger, I. etl al. Nanoparticles in Cancer Therapy and Diagnosis. Adv. Drug Del. Rev. 2002, 54, 631-651; Greish, K. Enhanced Permeability and Retention of Macromolecular Drugs in Solid Tumors: a Royal Gate for Targeted Anticancer Nanomedicines. J. Drag Targeting 2007, 15, 457-464; Maeda, H. et al. Mechanism of Tumor-Targeted Delivery of Macromolecular Drugs, Including the EPR Effect in Solid Tumor and Clinical Overview of the Prototype Polymeric Drag SMANCS. J. Control. Release 2001, 74, 47-61.) Second, different molecular states of stabilized ellipticine in solution can be obtained depending on peptide and ellipticine concentrations used in the formulation. This will have a varying impact on the anticancer activity and therapeutic efficacy (Li, X. et al. Doxorubicin Physical State in Solution and Inside Liposomes Loaded Via a PH Gradient. Biochim. Biophys. Acta 1998, 1415, 23-40; Garcia-Carbonero, R.; Supko, J. G. Current Perspectives on the Clinical Experience, Pharmacology, and Continued Development of the Camptothecins. Clin. Cancer Res. 2002, 8, 641-661; Hatefi, A.; Amsden, B. Camptothecin Delivery Methods. Pharm. Res. 2002, 19, 1389-1399.) Current results demonstrate that the complexes with protonated ellipticine are more effective at killing MCF-7 cells in vitro. Third, the release rate of ellipticine from the complex can be tuned, in relation to the size of the complex and the molecular state of stabilized ellipticine. The in vivo animal studies are currently undergoing.
The ionic-complementary self-assembling peptide EAKl 6-11 was found to be able to stabilize the hydrophobic anticancer agent ellipticine in aqueous solution. Both microcrystal and protonated forms of ellipticine can be obtained in the complexes. The complex formation in water is peptide concentration-dependent. When the peptide concentration was close to its CAC (~0.1 mg/mL), the equilibration time for complex formation could be as short as 5 h. At higher and lower peptide concentrations, the time required to reach equilibrium became much longer. High peptide concentrations facilitated the formation of protonated ellipticine during the complexation while low peptide concentrations favored crystalline ellipticine formation. With a combination of 0.1 mg/mL ellipticine and 0.5 mg/mL EAKl 6-II, protonated ellipticine can be stabilized. The transfer rate of ellipticine from its peptide complexes into EPC vesicles was dependent on the peptide concentration used during the peptide-ellipticine formulation. A higher peptide concentration resulted in a faster release rate, relating to the fact that higher peptide concentrations favor protonation of ellipticine and formation of smaller complexes. In addition, the size of the EAK16-II- ellipticine complex could be tuned by adjusting the peptide-to-ellipticine ratio (by mass) during formulation. The cellular toxicity results indicated that the complexes ( > 5: 1 ratio) with protonated ellipticine were effective at killing both MCF-7 and A549 cells, although their stability upon dilution in water was not very good. This study demonstrates the capability of ionic-complementary, self-assembling peptides as carriers for hydrophobic anticancer drug delivery.
From this study, it can be concluded that EAKl 6-11 is capable of stabilizing protonated or crystalline ellipticine in aqueous solution depending on the peptide-to- ellipticine ratio during the foπnulation. Above the ratio of 5:1 (by weight), the stabilized ellipticine is protonated; below this ratio ellipticine is in crystalline form in the complexes. The two molecular states of stabilized ellipticine in the complexes exhibit different toxicity against two cancer cell lines, A549 and MCF-7, with the protonated being more toxic. Such an effect is more pronounced for MCF-7 cells than for A549 cells. This is probably due to the fact that MCF-7 cells are more sensitive to protonated ellipticine. The complexes with protonated ellipticine are not stable upon dilution in water. The uptake of ellipticine in both cell lines appears to follow a diffusion mechanism. It is found that ellipticine can still be taken up by both cancer cells at 40C, where the energy-dependent endocytosis pathway is blocked. The complexation of ellipticine with EAKl 6-11 seems not to alter the internalization pathway of ellipticine. However, it significantly enhances the uptake over a shorter time period (~5 min). These results demonstrate that the EAK16-II-ellipticine complexes with protonated ellipticine are effective at killing cancer cells in vitro; the existence of some unfavorable properties of the complexes, such as unspecific cellular uptake, suggests that appropriate design of the peptide sequence is essential for future development of self-assembling peptide carriers. Materials
The peptide EAK16-II (Mw = 1657 g/mol, crude) was purchased from CanPeptide Inc. (Montreal, Canada) and used without further purification. It has a sequence of AEAEAKAKAEAEAKAK (Chart 1), where A corresponds to alanine, E to glutamic acid and K to lysine. The N-terminus and C-terminus of the peptide were protected by acetyl and amino groups, respectively. The anticancer agent ellipticine (99.8% pure) was purchased from Sigma- Aldrich (Oakville, Canada) and used as received. Egg Phosphatidylcholine (EPC, powder, > 99% pure) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Ethylenediaminetetraacetic acid (EDTA) was from Bio-Rad Laboratories (Mississauga, Canada).
Tris(hydroxymethyl)methylamine (Tris) and glacial acetic acid were bought from BDH Inc. (Toronto, Canada). Tetrahydrofuran (THF, reagent grade 99%) was obtained from Calendon Laboratories Ltd. (Georgetown, Canada). Cell culture reagents, including Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-ETDA, were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Phosphate buffer saline (PBS) and penicillin-streptomycin (p/s, 10000 U) were obtained from MP Biomedicals Inc. (Solon, OH, USA). Liposome Preparation
EPC powders (~5 g) were weighed and dissolved in 125 mL of a buffer solution containing 25 mM Tris/acetic acid (pH = 7.0) and 0.2 niM EDTA. The mixture was then extruded using a LiposoFast-Basic extruder (Avestin Inc., Ottawa, Canada) with a polycarbonate membrane (100 nm pore size) to obtain uniform liposome dispersions at room temperature. The dispersions were further diluted (4x) with the same Tris/acetic acid buffer. This was followed by centrifugation at 4000 rpm for 1 h to eliminate the larger vesicles and possible contaminates. The supernatant was collected and stored at 4 0C before use. The EPC concentration was determined to be 7.1 x 10"4 M using the method described in our previous publication. The size of the EPC liposomes was characterized by a Dynamic Light Scattering (DLS) technique; their hydrodynamic diameter (intensity-based) was found to be around 200 nm. Formation of Peptide-Ellipticine Complexes
To make peptide-ellipticine complexes, certain amounts of ellipticine crystals were added into fresh EAKl 6-11 solutions (0.02-1.0 mg/mL) to have ellipticine concentrations of 0.1-1.0 mg/mL. The fresh peptide solutions were prepared by dissolving peptide powder in pure water (18.2 MΩ, MiUi-Q AlO synthesis), followed by the sonication for 10 min. The peptide-ellipticine mixtures were stirred at 900 rpm on a magnetic stir plate throughout the complexation experiment. At specified times, the mixtures were transferred to a quartz cuvette to acquire fluorescence spectra of ellipticine on a steady-state spectrofluorometer (Photon Technology International, London, Canada). The test was performed once every hour for the first 20 h and less frequently for the remaining period until an equilibrium state was reached. 1 mg/mL of ellipticine in pure water was prepared as a control.
Since the peptide can self-assemble over time, it is expected that a competition may exist between the peptide-peptide association and the peptide-ellipticine complexation during the drug formulation. To better understand such a complicated process, the peptide assembly without ellipticine was investigated. For this purpose, 0.2 mg/mL of fresh EAKl 6-11 solution was prepared and stirred for 30 h at 900 rpm. The peptide assembly was characterized by static light scattering (at 400 nm) acquired on the steady-state spectrofluorometer and compared with the fresh peptide solution (0 h). The light scattering intensity of air was obtained as the standard to correct for the lamp fluctuations.
For the ellipticine release experiments, the complexes were newly prepared with a fixed ellipticine concentration of 0.1 mg/mL and various peptide concentrations ranging from 0.05 to 0.5 mg/mL. The samples were continuously stirred for 24 h to ensure that equilibrium was reached in the mixture. The steady-state fluorescence spectra of the complexes were acquired just before the release experiments (to show the states of ellipticine in complexes different from that in EPC vesicles). For cellular toxicity tests, the complexes were prepared with 0.1 mg/mL ellipticine and fresh EAK16-II solutions at concentrations of 0.02, 0.1, 0.2, 0.5 and 1.0 mg/ml, generating five peptide-to-ellipticine ratios of 10: 1, 5: 1, 2: 1, 1 :1 and 1:5 (by mass), respectively. The EAK16-II-ellipticine mixtures were under mechanical stirring at 900 rpm for 24 h. An ellipticine control in pure water (with the absence of EAKl 6-II) at the same ellipticine concentration was prepared for comparison, following the same procedure. The complexes at a 5: 1 ratio were diluted serially (2x, 4x, 8x and 16x) in pure water to study the complex stability. All vials and solvents were sterilized and the samples were prepared in a biological safety cabinet to avoid possible contamination. The appearance of the peptide-ellipticine suspensions was recorded in conjunction with the ellipticine fluorescence spectra, to determine the different molecular states of ellipticine in the complexes. Ellipticine Release into Liposome Vesicles
The release of ellipticine from the complex into the EPC vesicles was continuously monitored on the spectrofluorometer over time. The experiments were conducted with the following procedure: 100 μL of the peptide-ellipticine dispersion were transferred into a quartz cuvette and mixed with 2.9 mL of EPC vesicles. The 30 times dilution of the complex upon mixing with the vesicles was to ensure that the final ellipticine concentration was in the range where the calibration curve was applicable. The cuvette was then put in the spectrofluorometer with gentle magnetic stirring, covered with a parafilm on top (to eliminate the water evaporation during the course of measurement) before starting to collect the fluorescence over time. The time required to prepare the sample before starting a time-dependent fluorescence measurement was less than 30 s. Steady-State Fluorescence Measurements The ellipticine fluorescence was acquired on the Photon Technology
International spectrafluorometer (Type QM4-SE, London, Canada) with a continuous xenon lamp as the light source. For each sample, approximately 3mL of solution were transferred from a vial into a square quartz cell (1 cm x 1 cm) through a pasteur glass pipette. AU samples containing ellipticine were excited at 294 nm and the emission spectra were collected from 320 to 650 nm. The excitation and emission slit widths were set at 0.25 mm and 0.5 mm, respectively (0.25 mm corresponds to 1 nm band path). The fluorescence intensity at 468 nm and 520 nm were obtained by taking the average from 458 to 478 nm and 510 to 530 nm, respectively. A standard (2 μM ellipticine in ethanol, sealed and degassed) was used in each run to correct the lamp intensity variations. The standard fluorescence intensity Is was obtained by taking the average of the fluorescence from 424 to 432 nm (peak at ~428 nm).
The kinetics of the ellipticine release from the complex into the EPC vesicles was monitored by acquiring the time-dependent ellipticine fluorescence at 436 nm over a 7 h time span at 5 s intervals. All solutions reached equilibrium within 7 h as the fluorescence intensities reached a plateau during the experimental time span. The same standard sample as described above was used to obtain /, (at 428 nm over 10 min) to correct for the day-to-day fluctuations. For each release experiment, the fluorescence was recorded while the solution was gently stirred in the spectrafluorometer. Scanning Electron Microscopy (SEM)
A LEO model 1530 field emission SEM (GmbH, Oberkochen, Germany) was employed to study the morphology and dimensions of the peptide-ellipticine complex. The SEM samples were prepared by depositing 20 μiL of the complex suspensions on a freshly cleaved mica surface. The mica was affixed on an SEM stub using a conductive carbon tape. The sample was placed under a Petridish-cover for 10 min to allow the complexes adhering to the mica surface. It was then washed once with a total of 100 μL pure water and air-dried in a desicator overnight. All samples were coated with a 20 nm thick gold layer prior to imaging; the images were acquired using the secondary electron (SE2) mode at 5 kV. Cellular Toxicity Tests
Two cancer cell lines, non-small cell lung cancer cell A549 and breast cancer cell MCF-7, were used for in vitro cellular toxicity studies on the EAK16-II- ellipticine complexes. The cells were cultured in DMEM containing 10% FBS and 1% p/s at 37°C and with 5% CO2. When the cells grew to reach -95% confluence, they were detached from the cell culture dishes with trypsin-EDTA, centrifuged at 500 rpm for 5 minutes, and resuspended in fresh cell culture media at concentrations of 5 x 104 and 1 x 105 cells/mL for A549 and MCF-7 cells, respectively. For each type of cell, 200 μL of the cell suspensions were added into each well of a clear, flat bottom, 96-well plate (Costar) and incubated for -24 h. The old media were taken out and replaced with 150 μL fresh culture media, followed by an addition of 50 μL treatments (including the complexes and control samples) into each well, resulting in a 4-fold dilution of the treatments. The plates were incubated for 4, 8, 12, 24 and 48 h prior to performing the cell viability assay. The experimental setup contained several control groups for each plate, including negative control (medium), solvent control, peptide control and drug control. MTT assay
(TOXl from Sigma-Aldrich, Oakville, ON, Canada) was used to determine the cell viability after each treatment. 5 mg of solid MTT was first dissolved in 3 mL PBS solution, followed by a 10-time dilution in the culture medium. All the treatments were taken out, and 100 μL of the MTT solution was then added to each well of the treated plates. The plates were incubated for 4 h prior to the addition of 100 μL solubilization solution (anhydrous isopropanol with 0.1 N HCl and 10% Triton X- 100). After overnight incubation, the absorbance at 570 nm was collected on a microplate reader (BMG FLUOstar OPTIMA) and subtracted by the background signals at 690 nm. The absorption intensities were averaged from 4 replicates for each treatment and normalized to that obtained from the untreated cells (negative control) to generate the cell viability (i.e., the cell viability of the negative control is 1). Cellular Uptake Studies
Two cancer cell lines, A549 and MCF-7 (from ATCC), were used to investigate the uptake of EAK16-II-ellipitcine complexes in vitro. They were cultured in DMEM with 10% FBS at 37 0C with 5% CO2. The cells were then seeded on a 12-well plate with cell densities of 5 x 104 and 1 x 105 cells/well for A549 and MCF-7, respectively, followed by 48 h incubation prior to the treatments. The prolonged incubation time was to enhance the cell adhesion and avoid significant cell loss under intensive rinsing during the subsequent cell fixing procedure. The treatments were added into each well and incubated for 5, 15 and 30 min. Four treatments were tested: the complexes at two peptide- to-ellipticine ratios, 5:1 (125 μg/mL:25 μg/mL) and 1:1 (25 μg/mL:25 μg/mL), the ellipticine control (25 μg/mL), and the peptide control (125 μg/mL). The treated cells were washed with PBS 3 times, and fixed with 4% PFA in PBS, followed by another 3 times washing with PBS. The cells were examined with a fluorescence microscope (Nikon Eclipse 8Oi); a green fluorescence filter was used to collect the fluorescence signals of ellipticine, and phase contrast images were acquired to observe the cell morphology.
The temperature-dependent cellular uptake of ellipticine was conducted at 37 0C and 4°C to examine whether the internalization of ellipticine occurs through an endocytosis pathway (Derossi, D. et al. Cell internalization of the third helix of the antennapedia homeodomain is receptor-independant. J. Biol. Chem. 271, 18188- 18193 (1996); Vives, E. et al. TAT peptide internalization: seeking the mechanism of entry. Curr. Protein Peptide Sci. 4, 125-132 (2003)). The same cell density (as above) was used for cell seeding with 48 h incubation. Prior to treating the cells, the plates were incubated at 37 0C or 4 0C for 30 min, allowing the culture media to reach the equilibrium temperature. The same treatments (two complexes, ellipticine control and peptide control) were applied with an incubation time of 30 min. The cells were then fixed following the same procedure as before, and examined with the fluorescence microscope. Supporting Information
Size characterization of the EPC vesicles via the DLS measurements, photographs of peptide-ellipticine complexes, and calibration curves of ellipticine fluorescence and absorption in the EPC vesicles is provided below. See also http://pubs.acs.org. Size Characterization of the EPC Vesicles
The hydrodynamic diameter of the EPC vesicles was obtained via dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The experiments were conducted with appropriate viscosity and refractive index settings, and the temperature was maintained at 25 0C during the measurement. A small-volume (45 μL) black quartz cuvette with a 3 mm light path was used. The scattered light intensities of the samples at the angle of 173 degree were collected. The intensity-based size distribution of the EPC vesicles was obtained with the multimodal algorithm CONTIN (Provencher, S. W. A Constrained Regulation Method for Inverting Data Represented by Linear Algebraic or Integral Equations. Comput. Phys. Commun. 1982, 27, 213-227), provided in the software package Dispersion Technology Software 5.0 (Malvern Instruments, Worcestershire, U.K.). Six measurements were performed to generate the intensity-based size distribution plot as shown in Figure 76. The sizes of the vesicles were found to be ~200 nm in diameter. The liposomes with such a size using the extrusion method are classified as large unilaminar vesicles (LUVs) (Hope, M. J. et al. Production of Large Unilamellar Vesicles by a Rapid Extrusion Procedure. Characterization of Size Distribution, Trapped Volume and Ability to Maintain a Membrane Potential. Biochim. Biophys. Acta 1985, 512, 55-65; Sabin, J. et al. Characterization of Phospholipid + Semmifluorinated Alkane Vesicle System. Colloid. Surfaces B. Biointerfaces 2006, 47, 64-70; Tirosh, O. et al. Hydration of Polyethylene Glycol- Grafted Liposomes. Biophys. J. 1998, 74, 1371-1379.) Photographs of the Peptide-Eϋipticine Complexes
Four peptide concentrations (0.05, 0.1, 0.2 and 0.5 mg/mL) were used to form peptide-ellipticine dispersions with 0.1 mg/mL of ellipticine. The samples were stirred for 24 h to ensure that equilibrium was reached. Figure 77 shows that the complexes are formed with the presence of EAKl 6-11 compared to ellipticine only in water (far left vial). At 0.5 mg/mL of EAKl 6-II, the dispersion looks more yellow and less turbid comparing to others at lower peptide concentrations. This exceptional appearance is the additional evidence to the results of Figure 70 that the 0.5 mg/mL EAKl 6-11 solution can stabilize protonated ellipticine for a prolonged time. Calibration Curves of Ellipticine Fluorescence and Absorption
In order to relate the fluorescence signals of ellipticine in the EPC vesicles to the ellipticine concentration, a calibration curve of ellipticine fluorescence from the ellipticine-EPC suspensions is needed. The concentration range of ellipticine was selected to be 10"6-10"4 M based on our previous experience on pyrene release experiments. The ellipticine-EPC suspensions were prepared using the following procedure: ellipticine crystals were dissolved in THF to make the ellipticine-THF stock solution with ellipticine concentration of 1 niM. Aliquots of ellipticine-THF solution were put into a 4 mL vial and THF was evaporated under a stream of filtered air (0.45 μm) to have a film of ellipticine at the bottom of the vial. 3 mL of the EPC dispersions were added and the samples were stirred for -24 h to allow the saturation of ellipticine in the EPC vesicles. The ellipticine fluorescence was then acquired. It should be noted that ellipticine at high concentrations may not 100% dissolved in the vesicles made of 7.1 x 10"4 M EPC. Therefore, the UV- Vis absorption of the ellipticine-EPC suspensions was acquired and plotted as a function of ellipticine concentration to show whether ellipticine was completely dissolved in the EPC dispersions. To ensure that the fluorescence signal of ellipticine-EPC dispersions was from the ellipticine in the vesicles, the fluorescence of a control sample made of the buffer saturated with ellipticine was acquired. The fluorescence of the control was found to be ~280 folds smaller than that of 1 μM ellipticine in the EPC dispersion. Such a small fluorescence signal can be negligible. Thus, the observed fluorescence of the ellipticine-EPC dispersions should represent the fluorescence of ellipticine in the vesicles.
In order to generate an accurate calibration curve, the emission fluorescence of ellipticine in EPC vesicles were collected at 436 nm over 1 min and averaged to yield the intensity for each ellipticine concentration. The excitation and emission slit widths were set at 0.5 mm and 0.25 mm, respectively. The intensities were corrected with an ellipticine standard (2 μM in ethanol, sealed and degassed) to account for lamp fluctuations. The Is was obtained by taking the average of fluorescence at 428 nm over 1 min after each run.
Figure 78 shows the calibration curve of the ellipticine fluorescence in EPC vesicles. The ellipticine fluorescence increases to a maximum with the increase of ellipticine concentration from 1 to ~20 μM, and then slightly decreases with a further increase in ellipticine concentration (up to 100 μM). Normally, the decrease of ellipticine fluorescence at high ellipticine concentrations can be related to the inner- filter effect. This usually occurs when the concentration of a fluorophore is high enough with right angle detection. However, the decrease of the fluorescence due to the inner-filter effect is usually more dramatic (-50%), which does not seem to match with the present case.
Figure 78b shows the concentration-dependent UV absorption of the ellipticine in EPC vesicles used for generating the calibration curve. It can be seen that the ellipticine absorbance at 295 nm increases initially and reaches to a plateau when the ellipticine concentration is greater than 20 μM. This trend correlates well with that of the calibration curve. A plateau observed in Figure 78b indicates that ellipticine is saturated in the vesicles (formed at this particular lipid concentration of 7.1 x 10"4 M). This confirms that the inner-filter effect is not the cause of the slight decrease in the calibration curve at ellipticine concentrations above 20 μM. In addition, the molar ratio of ellipticine to the EPC lipid can be roughly estimated to be 0.028. Such a small number reflects the fact that such liposomes may not be an effective carrier for ellipticine when loading capacity is concerned; this is because the drug is inserted into the membrane, rather than inside the aqueous interior. However, the exact reason for the slight decrease in the ellipticine fluorescence at high ellipticine concentrations (> 20 μM) in Figure 78a is still under study.
The rising region in the calibration curve can be used to convert the fluorescence signals from the transfer profile to the ellipticine concentration in vesicles. This can be done by using a simple exponential equation to fit the rising region in Figure 78a (1-20 μM):
I = A(l - e-B[EPT] ) (Sl)
The fitting parameters A and B are 17.5 ± 0.36 and 230000 ± 13400 (VM), respectively. [EPT] represents the concentration of ellipticine within the range of 0-20 μM. For a given fluorescence intensity of ellipticine (///, < 17.5), one can obtain the corresponding ellipticine concentration using Equation S 1.
EXAMPLE 13 PROTECTION OF OLIGODEOXYNUCLEOTIDES AGAINST NUCLEASE DEGRADATION THROUGH ASSOCIATION WITH SELF- ASSEMBLING PEPTIDES
Antisense oligodeoxynucleotides (ODNs) have great potential for down regulating certain genes (Stein C. et al. Antisense strategies for oncogene inactivation. Semin Oncol 2005;32: 563-72; Crooke ST. Therapeutic applications of oligonucleotides. Biotechnology 1992;10:882-6.) The production of faulty proteins is inhibited when antisense ODNs reach the cytoplasm or nucleus of cells where they bind specifically to the mRNA or DNA target (Wang H et al. Antisense anticancer oligonucleotide therapeutics. Curr Cancer Drug Targets 2001; 1 : 177-96 "Wang et al. (2001)".) However, before the ODNs can reach their target site, they have to cross the cellular membrane, escape from the endosomal compartment, leave their carriers behind and hybridize with the target sequence (Zhao H. et al. Delivery of G3139 using releasable PEG-linkers: impact on pharma cokinetic profile and anti-tumor efficacy. J Control ReI 2007; 119: 143-52; Garcia-Chaumont C. et al. Delivery systems for antisense oligonucleotides. Pharm Therap 2000; 87:255-77.) During these trafficking steps, rapid degradation of the ODNs can happen and result in shorter ODN fragments, which are more likely to complement non-target mRNA sequences rather than the target sequence (Crooke ST. Progress in antisense technology: the end of the beginning. Methods Enzymol 2000;313:3-45.) Thus, the sequence specificity and efficacy of the ODNs are compromised and their success in therapeutic applications becomes limited.
One approach that circumvents the stability problems of antisense ODNs aims at developing suitable delivery systems. Ideally, a delivery system should be designed to protect the ODNs during all the different steps involved in the trafficking of ODNs from outside the cell to its ultimate target inside the cell. The current ODN delivery systems are often composed of cationic lipids (Parekh H. S. et al. Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition. Bioorg Med Chem 2006; 14:4775-80) polymers (Park T.G. et al. Current status of polymeric gene delivery systems. Adv Drug Del Rev 2006;58:467-86) or peptides (Thierry A.R. et al. Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides. Biochim Biophys Acta 2006;1758:364-74.)
An interesting delivery system consists of a class of self-assembling peptides of the EAK family made of glutamic acid (E), alanine (A), and lysine (K) residues. These self-assembling peptides have an amphiphilic structure consisting of alternating negatively and positively charged amino acids on one side of the backbone, and hydrophobic amino acids on the other side. The ionic complementarity, together with hydrogen bonding, hydrophobicity, and van der Waals interactions, promotes the self- assembly of the peptides into aggregates, which are highly stable against extreme pH, various proteases, and denaturing agents (Zhang S. et al. Unusually stable /3-sheet formation of an ionic self complementary oligopeptide. Biopolymers 1994;34:663-72 "Zhang et al. (1994").) These peptides do not cause significant cytotoxicity, arouse minimal immune response and inflammation in the host, and are more biocompatible than many other carrier materials (Zhang et al. (1994)). The positively charged amino acids of the EAK peptides facilitate the complexation of the peptides with ODNs through electrostatic attraction. The inherent self-assembling capability of the EAK peptides further enables the association of the resulting complexes into aggregates (Wang et al. (2001)). The ODNs inside the EAK-ODN aggregates exhibit reduced accessibility to the surrounding solvent, implying that their accessibility to nucleases might also be reduced with an associated enhancement of the ODN stability.
EAK-ODN aggregates generated with EAKl 6IV and EAKl 611 have been characterized. EAK16IV and EAK16II have the same amino acid composition but different charge distributions, respectively given by the charge sequences — + + + + and -- + + — + + at neutral pH. Both EAK peptides were found to bind to ODNs more strongly at pH 4 than at pH 7, due to favorable electrostatic attraction at pH 4 (Wang et al. (2001)). EAKl 6IV binds to ODNs more strongly than EAKl 611 does at the same pH due to the larger density of positively charged amino acids at one end of the EAKl 6IV molecule. The present example provides conclusive evidence that the EAK peptides are suitable for use as gene carriers by investigating how the EAK-ODN aggregates resist nuclease degradation and how their stability is affected by dilution, pH, and centrifugation.
To assess the resistance of a given ODN against nuclease degradation after the EAK-ODN aggregates had been subject to different treatments, an assay was established whereby the EAK-ODN aggregates were generated with an ODN labeled at each end with a fluorescent donor or acceptor. In the EAK-ODN aggregates, fluorescence resonance energy transfer (FRET) took place. Upon addition of a nuclease, a reduction in FRET indicated the degradation of the doubly labeled ODN (Uchiyama H. et al. Detection of undegraded oligonucleotides in vivo by fluorescence resonance energy transfer. J Biol Chem 1996; 271 :380-4.) The extent of ODN degradation obtained after subjecting the EAK-ODN aggregates to various treatments was determined quantitatively from the reduction in FRET efficiency. This information was used to generate EAK-ODN aggregates that would have optimal stability and efficiency at protecting ODNs from nuclease degradation. Materials
All reagents were of analytical grade and obtained from BDH (Poole, UK). The pH 4 buffer was made with 0.171 M acetic acid and 0.029 M sodium acetate, adjusted with acetic acid (Alcinrimisi E.O. et al. Properties of helical polycytidylic acid. Biochemistry 1963;2:340-4 "Akinrimisi et al. (1963)"); the pH 7 buffer was made with 0.01 M tris(hydroxymethyl)methylamine and 0.005 M sodium sulfate, adjusted with sulfuric acid (Akinrimisi et al. (1963)); the pH 11 buffer was made with 0.1 M glycine and 0.1 M sodium chloride, adjusted with sodium hydroxide (Bolumar T. et al. Purification and characterization of a prolyl aminopeptidase from debaryomyces hansenii. Appl Environ Microbiol 2003;69:227-32.) The EAKl 611 and EAKl 6IV peptides, whose sequences are, respectively n-AEAEAKAKAEAEAKAK- c and n-AEAEAEAEAKAKAKAK-c (Table 17) were purchased from Research Genetics (Alabama, USA) and used without further purification. A cytosine hexadecamer doubly labeled with fluorescein at the 5 '-end and rhodamine at the 3'- end, namely Fl-dCi6-Rh, and two unlabeled guanine and cytosine hexadecamers, namely dGi6 and dCiβ, were obtained with 95% purity after HPLC purification from Eurogentec North America (San Diego, USA). The ODN sequences are listed in Table 17. Escherichia coli exonuclease (20 U/μL) and its 1O x degradation buffer (670 mM glycine-KOH (pH 9.5 at 250C), 67 mM MgCl2, 1OmM DTT) were purchased from Fermentas Canada Inc. (Burlington, Canada). Table 17. Type name, and sequence of ODNs and self-assembling EAK peptides
Dissociation of the EAK-ODN aggregates upon dilution probed by UV-vis absorbance
UV-vis absorption spectra were obtained on a Hewlett-Packard 8452A diode array spectrophotometer (California, USA) using a 50 μL quartz cuvette from Hellma (Mϋllheim, Germany). Samples containing 8.6μm ODNs and different amounts of the EAK peptides were prepared at pH 4 and pH 7 at 25°C. Half an hour after mixing, the EAK-ODN mixtures were diluted 5- and 10-fold with the buffer used to prepare the mixtures. The resulting samples are referred to as "dilute-5" and "dilute-10" samples, respectively. UV-vis spectroscopy together with centrifugation was utilized to measure over time the percentage of ODN in the EAK-ODN aggregates after dilution. As outlined in an earlier study (Wang et al. (2001)), the EAK-ODN aggregates formed in solution can be collected by centrifuging the solution at 10,000 rpm for 2 min. The supernatant was collected and its absorbance was measured on the spectrophotometer at wavelengths between 190 and 800 nm. Beer's Law was used to determine the total ODN concentration and the concentration of the ODN left in the supernatant, from the absorbance of the ODN at 260nm of the initial solution (OD0) and the supernatant (OD5), respectively. The term (ODo-ODs)/ODo was defined as the relative UV-vis absorbance change, ΔODr, which yields the fraction of ODN trapped in the EAK-ODN aggregates and which can he centrifuged out. Preparation of the EAK-ODN aggregates for nuclease resistance studies
Unless mentioned otherwise, sample preparation for the evaluation of the nuclease resistance of the oligonucleotides in the EAK-ODN aggregates was conducted as follows. A certain amount of stock EAK and Fl-dCi6-Rh solutions were mixed and the mixture solution was vortexed for a few seconds. The resulting EAK- ODN solutions were kept at 250C. Half an hour later, the mixtures were diluted 10 times with the buffer used to prepare the solutions and kept at 250C. Incubation of the EAK-ODN aggregates with nuclease
Twenty-four hours after dilution, the EAK-ODN solutions were centrifuged at 10,000 rpm for 2min. The pellets containing the EAK-ODN aggregates were collected and incubated with 0.7U/μL exonuclease I in degradation buffer (pH 9.5) at 250C. Samples without nuclease treatment were used as controls. They were prepared by incubating the EAK-ODN aggregates or free ODN solutions with 1 x degradation buffer under the same conditions as those treated with exonuclease I. For both nuclease-treated samples and non-treated controls, 55 μL of solution was removed at different points in time and inactivated by the addition of 2μL of 40% sodium dodecyl sulphate. The obtained samples were dried with a Savant Model ISSI lO DNA SpeedVac system (Minnesota, USA) and redissolved in 55 μL of pH 11 buffer to completely release the ODN from the EAK-ODN aggregates. In the end, each sample was centrifuged at 10,000 rpm for 2 min and the supernatant was collected for the FRET measurements. FRET measurements The fluorescence spectra of the supernatant were acquired on a Photon
Technology International steady-state fluorometer (New Jersey, USA) equipped with a LPS 220-B xenon arc lamp and a PTI 814 photomultiplier detection system. The emission spectra were acquired from 500 to 700 nm while exciting the solution at the absorbance peak wavelength of the fluorescein donor (λeX = 494nm). The fluorescence intensity ratio of the donor to the acceptor (-D//A) was determined by monitoring the fluorescein and rhodamine fluorescence at 517 and 583nm, respectively. Dissociation of the EAK-ODN aggregates upon dilution The solutions of the EAK-ODN aggregates prepared at pH 4 and pH 7 were diluted to investigate whether the aggregates would dissociate upon dilution. Since the binding of EAKl 6IV to ODNs at pH 4 is stronger than at pH 7 (see below), 8.6 μM dGie was mixed with 10.5 μM EAK16IV at pH 4, but with 48.2 μM EAK16IV at pH 7. Stable aggregates are expected to form in solution 30 min after preparation, and the resulting solution was diluted 5 or 10 times with the buffer which was used to prepare the mixture solution. The relative UV-vis absorbance change, ΔOD,-, which reflects the percentage of ODNs in the EAK-ODN aggregates, was determined before and after dilution.
The ΔODr value of the solution prepared at pH 4 with EAKl 61 V and dGi6 equals 0.46±0.01. As evident from Fig. 79A, the ΔODr value did not change much within the experimental period after the solution had been diluted 5 or 10 times. The fact that no decrease in ΔODr is observed indicates that the aggregates prepared at pH 4 with EAK16IV and dGi6 do not dissociate after a 10-fold dilution. Similarly, the ΔOD,, value for the solution prepared at pH 7 with EAKl 6IV and dGi6 equals 0.72±0.01. Note that this higher ΔODr value is a result of the higher EAK16IV concentration used to prepare the solution at pH 7. This value remained unchanged after the solution was diluted 5 or 10 times with the pH 7 buffer (Fig. 79 B). The data shown in Figs. 79A and B suggest that, although the binding of EAK16IV to dGi6 at pH 4 is stronger than at pH 7, aggregates made with EAK 16IV and dGi6 at pH 4 and pH 7 do not undergo any detectable dissociation upon an up to 10-fold dilution.
The absence of dissociation exhibited by the EAK-ODN aggregates was found not only with EAKl 6IV, but also with EAKl 611. Figs. 79C and 79D present the - ΔODr vs. time profile for the "dilute-5" and "dilute-10" solutions prepared by mixing 8.6μM dGie with either 24.1μM EAKl 611 at pH 4 (Fig. 79C) or 60μM EAK16II at pH 7 (Fig. 79D). No decrease in the ΔODr value over time was observed regardless of the pH at which the solutions were prepared. This result demonstrates that the aggregates of EAKl 611 and dGi6 prepared at pH 4 and pH 7 do not dissociate upon a 10-fold dilution of the solutions. As shown in Fig. 79, neither a 5- nor a 10-fold dilution can induce a detectable break-up of the EAK-ODN aggregates formed at pH 4 and pH 7 for up to 1 day. Following this promising observation, the nuclease resistance of the ODN inside the EAK-ODN aggregates was investigated. Breaking down the EAK-ODN aggregates
FRET was used to monitor the nuclease degradation of an ODN located inside the EAK-ODN aggregates. The ODN was labelled at one end with a fluorescence donor (fluorescein) and at the other end with a fluorescence acceptor (rhodamine). The distance between the two labels increases if the ODN is degraded and the donor and acceptor diffuse away from each other. Under such conditions, a decrease in FRET efficiency occurs. However, if the ODN is initially encapsulated inside the EAK-ODN aggregate, the degraded ODN might not be able to diffuse out of the EAK-ODN aggregate, leaving the distance between donor and acceptor unaffected. In order for FRET to correctly reflect the degradation of the ODN, it is necessary to ensure that the EAK-ODN aggregate can be dissociated or broken down to smaller complexes during these experiments.
EAK contains four glutamic acids (GIu, E) and four lysines (Lys, K) with pKa values of 4.25 and 10.53, respectively. According to these pKa values, the EAK molecules are expected to be negatively charged at pH 11, and not to interact with the negatively charged ODNs due to electrostatic repulsion. Thus, adjusting the solution pH to 11 may provide a means to dissociate or break down the EAK-ODN aggregates.
UV-vis experiments were carried out to confirm this expectation. First, the
EAK-ODN aggregates containing 5.0/XM of dCi6 and 120/XM of EAK16II at pH 4 were prepared and divided into two aliquots. The first aliquot was used to make sure that all ODN molecules were incorporated in the EAK-ODN aggregates. The UV-vis absorbance of the solution at the peak wavelength (276 nm) equaled 0.75. It dropped to about zero after centrifugation (Fig. 80A). This result indicates that no free ODN was left in the supernatant and that all ODNs were incorporated into aggregates, which could be removed by centrifugation. The second aliquot was dried, resuspended in pH 11 buffer, before being vortexed for a few seconds. The absorption of the resulting solution was measured. If the ODN molecules were not released from the EAK-ODN aggregates, the difference in UV absorbance obtained before and after centrifugation of this solution should be similar to the difference obtained for the first aliquot, namely 0.00-0.75 = -0.75. The fact that the UV absorbance of this solution at 276 nm before and after centrifugation was, respectively 0.73 and 0.71 (Fig. 80A) and yielded a difference in UV absorbance equal to 0.71-0.73 =0.00 demonstrates that most aggregates were successfully dissociated or broken down to smaller complexes, which could not be centrifuged out of the supernatant, when the solution pH was brought from 4 to 11. This extraction method was applied in the next series of experiments to determine the nuclease resistance of the ODNs encapsulated in the EAK-ODN aggregates.
Since bringing the solution pH from 4 to 11 breaks up the EAK-ODN aggregates, the effect that a pH value of 9.5, that of the E. coli exonuclease I degradation buffer, would have on the stability of the EAK-ODN aggregates needed to be determined. To this end, 8.6 μM of dCi6 was mixed with 0.1 mg/mL EAK16-IV at pH 4 and 7. The resulting solutions were centrifuged 30 min after sample preparation. The aggregates made of EAK16IV and dCi6 were collected, resuspended in the pH 9.5 buffer, and vortexed. The ΔODr value was monitored over time (Fig. 80B). It was found that the fraction of ODNs incorporated inside the EAK-ODN aggregates prepared at pH 4 decreased from 0.85 to 0.60 3 h after the pH was increased to 9.5. In comparison, this fraction decreased from 0.85 to about 0.05 when the pH of the solution was brought from 7 to 9.5. In other words, increasing the pH to 9.5 broke up less than 30% of the EAK-ODN aggregates prepared at pH 4 but almost 100% of the aggregates prepared at pH 7. These results suggest that the aggregates initially prepared at pH 4 are more stable than those prepared at pH 7 when the pH is increased to 9.5. This might be due to the fact that binding of the EAK peptides to ODNs at pH 4 is much stronger than at pH 7. The 30% reduction in aggregation observed for the EAK-ODN aggregates prepared at pH 4 might be the result of aggregates being broken down to either small complexes or free ODN molecules.
That the EAK-ODN aggregates prepared at pH 4 would fully disaggregate when the solution was brought to pH 11, but would undergo only partial dissociation when the solution pH would increase from 4 to 9.5 suggests that at pH 9.5, some lysines in EAKl 6IV remain positively charged and ensure that strong electrostatic attraction is retained between EAKl 6IV and the ODN. Nuclease resistance of the ODN in the EAK-ODN aggregates The ODN protection against the nuclease provided by the EAK peptides was monitored by performing FRET experiments with FWC16-Rh. The effect of several factors, i.e. peptide sequence, pH, peptide concentration, and centrifugation, on the protection of the ODN encapsulated in the EAK-ODN aggregates was investigated. Control solutions of the Fl-dCi6-Rh prepared in the presence or absence of
EAKl 6IV at pH 4 were incubated in the pH 9.5 buffer solution without exonuclease I for 30 min. FRET took place as seen from the two emission peaks at 517 and 583 ran that correspond to the emission of the fluorescein donor and the rhodamine acceptor, respectively (Fig. 81). The ratio of the donor to the acceptor fluorescence intensity at the peak wavelengths (/D//A) for the ODN is about 1.4 (Fig. 81A) and 1.2 (Fig. 81B) without and with EAK, respectively.
The emission spectra were also taken at different points in time after incubating the ODN or EAK-ODN solutions prepared at pH 4 with exonuclease I (Fig. 81). For the ODN alone (Fig. 81A), the fluorescence of the donor (fluorescein) immediately increased 2.6-fold as that of rhodamine decreased, indicating a lower extent of FRET and significant degradation of the ODN. Fig. 81A indicates that a substantial percentage of the ODN is degraded within 20 min, a result consistent with the expectation that the unprotected ODNs are rapidly degraded by nucleases. In contrast, the EAK-ODN aggregates generated with EAKl 6IV at pH 4 are not degraded by exonuclease I even after 90 min, as indicated by the similar emission spectra acquired before and after nuclease treatment (Fig. 81B). This result is consistent with the fluorescence quenching experiments, which showed that an ODN incorporated in an EAK matrix is less accessible to the solvent than a free ODN is (Wang et al. (2001)). To monitor the degradation of the double labeled ODN in a quantitative manner, a 0.46/XM solution of the FWCi6-Rh was used to represent the non-degraded ODN sample, whereas a solution of 0.46 μu Fl-dCiβ and 0.46 μu dCi6-Rh was used to mimic a situation where 100% of all ODN would be degraded. The solution of Fl- dCi6-Rh was then mixed with the solution containing Fl-dCi6 and dCi6-Rh in varying proportions. The /D//A ratios of the mixtures were determined and normalized to the /D//A ratio of the intact ODN solution (Fig. 82). The normalized /D//A ratio was found to increase linearly with the percentage of singly labeled ODN present in solution. The plot in Fig. 82 was then used as a calibration curve to determine quantitatively the percentage of ODNs, which were degraded by the nuclease Effect of EAK sequence and solution pH on nuclease resistance
Fig. 83A shows the percentage of dC)6 degraded by exonuclease I when Fl- dCi6-Rh was prepared in the presence or absence of EAKl 6IV or EAKl 611, at pH 4 or pH 7. Fig. 83A indicates that about 85±10% of free Fl-dC]6-Rh prepared at pH 4 are degraded within 30 min of nuclease incubation. In contrast, Fl-dCi6-Rh complexed with EAKl 6IV at pH 4 shows significant nuclease resistance against exonuclease I. The aggregates prepared with EAKl 61 V and Fl-dC I6-Rh at pH 4 protect Fl-dCi6-Rh against nuclease degradation even after being incubated at pH 9.5 for 2h. It suggests that Fl-dCi6-Rh is located inside the EAK-ODN aggregates where it remains inaccessible to the nuclease. The protection from degradation afforded by the EAK-ODN aggregates to the ODN represents a desired property for using EAKl 6IV as a carrier for ODN delivery. In these experiments, the aggregates made of EAK16IV and Fl-dCi6-Rh were prepared at a pH which is much more acidic than the pH of 9.5 of the degradation buffer. Although Fig. 8OB shows that about 30% of the aggregates prepared with EAKl 6IV and Fl-dCi6-Rh at pH 4 dissociate when the pH is increased to pH 9.5, the dissociation products, whatever their nature, still provide significant protection against nuclease (Fig. 83A). This might be due to the formation of some small EAK-ODN complexes in the solution as a result of the dissociation process following the increase of the solution pH. The existence of these small complexes has been demonstrated in an earlier study. They have been shown to contain a few ODN strands and to be too small to be centrifuged out (Wang et al. (2001)). In comparison with EAKl 6IV, EAKl 611 did not provide significant protection to the ODN. About 75% of all Fl-dCi6-Rh are degraded when aggregates are generated between Fl-dC I6-Rh and EAKl 611 at pH 4. This might be due to the binding of EAK16II to being much weaker than that of EAK16IV to dCi6.
When Fl-dC i6-Rh is dissolved in the pH 7 buffer without EAK peptide and is then incubated with the nuclease solution, it is strongly degraded within a few minutes, as was observed for Fl-dCi6-Rh when it was dissolved in the pH 4 buffer with no EAK peptide. The fraction of Fl-dCi6-Rh which is allowed to form aggregates with EAKl 611 at pH 7 is degraded to the same extent as Fl-dC I6-Rh when no EAK is added to the solution. This indicates that the EAK-ODN aggregates prepared with EAKl 611 at pH 7 do not offer any protection to the ODNs against nuclease. On the other hand, EAK-ODN aggregates prepared with Fl-dCi6-Rh and EAKl 6IV at pH 7 offer some protection from nuclease degradation (Fig. 83A). One possible explanation for this result might be that EAKl 6IV binds more strongly to dCi6 than EAK16II does. Although the aggregates prepared with EAKl 6IV and dCi6 at pH 7 dissociate when the pH is adjusted to 9.5 (Fig. 80B), small EAK-ODN complexes are still present in the solution that may protect the ODN.
The much weaker protection provided by both EAK peptides at pH 7 as well as by EAKl 611 at pH 4 does correlate well with the relative binding strength of the peptides to the ODN. As shown in Fig. 83B, all dCi6 strands are incorporated into EAK-ODN aggregates when EAKl 6IV and dCi6 are mixed at pH 4. EAK-ODN aggregates prepared by mixing dCi6 with EAKl 6IV at pH 7, EAKl 611 at pH 4, and EAKl 611 at pH 7 result in a similar ΔODr value reflecting similar binding strength. Interestingly, these three conditions for preparing EAK-ODN aggregates result also in similar levels of ODN protection against exonuclease I in Fig. 83A. The correlation observed between the weaker protection afforded by those EAK-ODN aggregates that are generated via a weaker binding of the EAK peptide to an ODN suggests that the sequence of the peptide which strongly affects the binding strength plays a critical role in protecting the ODN in the aggregates from nuclease degradation. EAKl 6IV bears four positively charged amino acids at its C-terminal whereas pairs of positively charged amino acids are distributed throughout the back-bone of EAKl 611. The distribution of lysines in EAKl 6IV might facilitate the formation of more cohesive aggregates that prevent nuclease degradation. AU these observations clearly indicate that the aggregates prepared with ODN and EAK 16IV at pH 4 provide the ODNs with better protection against nuclease degradation. Effect of peptide concentration on nuclease resistant
The aggregates resulting from the mixture at pH 4 of 8.6μM Fl-dCi6-Rh and 60μM EAKl 6IV provide 100% protection for Fl-dCi6-Rh against nuclease degradation (Figs. 81B and 83A). The protection afforded by the EAK-ODN aggregates was also examined at lower EAK concentrations. To this end, 8.6μM Fl- dCi6-Rh and 10μM EAK16IV was used to prepare the EAK-ODN aggregates at pH 4. The resulting aggregates fail to protect Fl-dCi6-Rh against nuclease degradation (Fig. 84A). The I^IIA ratio of the sample after nuclease treatment equaled 6.3, significantly higher than the 1.6 value of the non-treated control. According to the calibration curve shown in Fig. 82, these /D//A ratios imply that 73% of the ODN were degraded. It appears that to achieve 100% protection, a minimum EAKl 6IV concentration is necessary, which was found in Fig. 84B to equal 60 μu for an Fl-dCi6-Rh concentration of 8.6μM (Fig. 84B).
Effect of centrifugation on nuclease resistance
30min after preparing a solution of EAK16IV and Fl-dCi6-Rh at pH 4, the EAK-ODN solution was centrifuged at 10,000 rpm for 2 min. The precipitates were resuspended in the supernatant before being vortexed for a few seconds. The resulting solution was diluted 10-fold with the same buffer used to prepare the original solution and treated with the nuclease as described in the experimental section. The above procedure resulted in around 50% of the ODN being degraded within 20 min and 75% within 60 min of nuclease incubation (empty symbol, Fig. 85A). By comparison, almost no ODN degradation occurred for the aggregates, which were centrifuged 24 h after sample preparation (solid symbol, Fig. 85A). One reason for such a significant effect of centrifugation on the aggregate stability might be that the process of aggregate formation is disrupted when centrifugation is applied just 30min after sample preparation. However, the UV-vis spectra recorded for the aggregates centrifuged 30 min after sample preparation show that ΔODr = 0.95 or that 95% of all ODNs are encapsulated in the EAK-ODN aggregates (Fig. 85B), a ΔODr value similar to that obtained for the aggregates which were centrifuged 24 h after sample preparation. The fact that little nuclease protection was detected for the aggregates centrifuged 30min after sample preparation suggests that the aggregate morphology might change over a time scale longer than 30 min and that centrifuging the EAK- ODN solution within 30 min results in the loss of ODN protection against nuclease.
The interruption of the "maturing" process necessary to achieve stable aggregates has been shown to strongly affect the morphology of the aggregates made of protein and polyelectrolyte (Woon-Soo K. et al. Aging characteristics of protein precipitates produced by polyelectrolyte precipitation in turbulently agitated reactor. Chem Eng Sci 2002;57:4077-85). When put in the context of our experiments, this suggests that the aggregate might still be maturing 30 min after sample preparation and that by applying centrifugation within that period, the aggregation process might be interrupted, resulting in a change of aggregate morphology that may expose the ODN to the nuclease. As observed, the aggregates which were centrifuged 24 h after sample preparation showed significant nuclease protection even after incubation with the nuclease for up to 2 h (solid symbol in Fig. 85A). These results indicate that the EAK16IV-0DN aggregates might require a minimum period of 24h after sample preparation to achieve ODN protection against nuclease degradation.
Since centrifugation is a standard process used routinely in biochemistry laboratories, the effect that centrifugation has on the EAK-ODN aggregates needed to be investigated over time. To this end, the aggregates with 8 μM of Fl-dCi6-Rh and 60 μM of EAK 16IV were prepared at pH 4 and centrifuged for the first time 24 h after sample preparation. They were kept at 25°C and were centrifuged once a day for the next 3 days. The aggregates showed significant stability against nuclease degradation up to 3 days after sample preparation (Fig. 85C). While the nuclease treatment did not degrade all ODNs 4 days after the sample had been prepared and centrifuged four times, the EAK-ODN aggregates started to lose their ability to protect the ODN against nuclease degradation. These results suggest that if EAK16IV were to be used to deliver ODNs, the EAK-ODN aggregates would need to be given enough time (24 h in this study) before they can be employed and be used within 4 days, which is acceptable in drug therapy.
The results obtained in this study confirm that EAKl 6-IV is a promising carrier for ODN delivery. The EAK-ODN aggregates generated with EAKl 6IV at pH 4 are stable at acidic pH and confer nuclease resistance to the ODNs even after having been diluted or centrifuged.
The stability of the EAK-ODN aggregates after dilution of the solution was determined by UV-vis absorption. The aggregates were found to be stable, undergoing no detectable dissociation over ~20h after the solutions were diluted 5- and 10-fold with the buffer used for their preparation. The protection of the oligonucleotides against nuclease afforded by the EAK-ODN aggregates was investigated with FRET when the aggregates were generated with different EAK peptides at pH 4 and pH 7. The results demonstrated remarkable nuclease protection of the ODN when the EAK-ODN aggregates were prepared with relatively high concentration of EAK16IV at pH 4. Much less efficient protection was obtained when the EAK-ODN aggregates were prepared with similar concentration of EAKIV at pH 7 or of EAKl 611 at either pH 4 or pH 7. The ability of the EAK-ODN aggregates to protect dCi6 correlates well with the binding strength of the EAK peptides to dCiβ. The effect that centrifugation has on the ability of the EAK-ODN aggregates to protect the ODN from nuclease degradation was also investigated. If the first centrifugation was applied 24 h after sample preparation, the EAK-ODN aggregates confer nuclease resistance to the ODN even after being centrifuged once per day for 4 days. However, if the first centrifugation was applied 30 min after sample preparation, the EAK-ODN aggregates lost their nuclease protection ability. This observation led to the suggestion that the EAK-ODN aggregates need to age for more than 30min in order to generate the morphology that can protect the ODNs from degradation. EXAMPLE 14 SEQUENCE EFFECT OF SELF-ASSEMBLING PEPTIDES ON THE COMPLEXATION AND IN VITRO DELIVERY OF THE HYDROPHOBIC ANTICANCER DRUG ELLIPTICINE
In this example, peptide sequence effects on the drug formulation and in vitro delivery were investigated. Three self-assembling peptides, EAKl 6-II, EAKl 6-IV and EFKl 6-11 were chosen to investigate the effects of charge distribution (type II vs. type IV) and hydrophobicity (alanine A vs. phenylalanine F). A hydrophobic anticancer agent, ellipticine was selected as a model drug. The self-assembled nanostructures of these peptides were first characterized by AFM; the hydrophobicity of the peptides dissolved in aqueous solution was studied via surface tension measurements and fluorescence spectroscopy using a hydrophobic fluorescent probe. These characteristics of the three peptides were expected to impact their complexation with ellipticine, in terms of ellipticine molecular states and the size of the resulting complexes. The anticancer activity of the foπnulation was tested in vitro against two cancer cell lines: non-small cell lung cancer cell A549 and breast cancer cell MCF-7. The stability of the complexes after serial dilutions in aqueous solution was further investigated.
Subtle differences in the peptide sequence affect the properties of the peptide assemblies, the formation of the peptide-ellipticine complexes, and the cellular toxicity of the complexes. Three self-assembling, ionic-complementary peptides, EAK16-II, EAK16-IV and EFKl 6-11 were used in this study. The latter two peptides are derived from the first one EAKl 6-11 All peptides have 16 amino acids in sequence with 3 amino acid components: E, K and A or F, as shown in Figure 86. EAKl 6-IV has a different charge distribution of type IV ( — ++++) from EAKl 6-11 as type II (-++--++), while the difference between EFKl 6-11 and EAKl 6-11 is a more hydrophobic residue F replacing A in EAKl 6-II.
The peptide self-assembled nanostructures are different among the three peptides. The distribution of negative and positive charges towards the two ends of an EAKl 6-IV molecule at neutral pH is reported to cause the folding of the peptide molecule to form a /3-rurn structure, resulting in the formation of globular nanostructures (Hong Y. et al. (2003) Effect of amino acid sequence and pH on nano fiber formation with self-assembling peptides EAKl 6-11 and EAKl 6-IV. Biomacromolecules 4: 1433-1442 "Hong et al. (2003)"; Jun S. et al. (2004) Self- assembly of the ionic peptide EAK16: the effect of charge distribution on self- assembly. Biophys J 87: 1249-1259 ("Jun et al. (2004)"). EAKl 6-II, on the other hand, has a preferable stretched molecular structure and likely self assembles into β- sheet rich nano fibers (Jun et al. (2004)). The nanostructures of the two peptides are shown in Figure 87A and B at a peptide concentration of 0.5 mg/mL. EAK16-II forms straight nanofibers, connecting to networks (Figure 87A), whereas EAK 16-IV self-assembles into many more globular aggregates and some short nanofibers (Figure 87B). The formation of short nanofibers of EAKl 6-IV may be due to a relatively low pH (<5) at such a high peptide concentration: when the pH is low enough, some of the negatively charged residues can be neutralized so that the intramolecular ionic interaction is weakened. Thus, some peptides remain in a stretched form, facilitating the formation of nanofibers (Hong et al. (2003)).
The nanostructures of EFKl 6-11 are also different from those of EAKl 6-11 as shown in Figure 87. EFKl 6-11 forms predominant nanofibers and these fibers tend to aggregate into fiber clusters. TMs aggregation of nanofibers is probably due to a stronger hydrophobic interaction between them. Such a stronger hydrophobic interaction is expected to come from the more hydrophobic phenylalanine (F) residues in the EFKl 6-11 sequence, compared with the alanine (A) residues in EAKl 6-II. This is probably why the nanofibers of EFK 16-11 tend to form fiber clusters, but those of EAKl 6-11 are dispersed and form fiber networks.
The hydrophobicity of the three peptides and their assemblies is further characterized by surface activity and fluorescence measurements, and the results are shown in Figure 88. Figure 88a shows the surface tension as a function of time for the three peptides at a peptide concentration of 0.5 mg/mL. For each profile, the surface tension decreases fast initially and slowly approaches equilibrium. This change with time corresponds to the dynamic process of the adsorption of peptide molecules/ assemblies at the air-liquid interface, leading to the decrease in surface tension (Eastoe .T, Dalton JS (2000) Dynamic surface tension and adsorption mechanisms of surfactants at the air-water interface. Adv Colloid Interface Sci 85: 103-144.) Comparing the surface tensions of the three profiles at 2 h (near equilibrium), they follow a trend: EAK16-II>EAK16-IV>EFK16-II. In general, the lower the surface tension is, the more hydrophobic the molecule is. Thus, the hydrophobicity of the three peptides and their assemblies (coexisting in solution) has a reversed trend: EFK16-II>EAK16-IV>EAKI6-II. This is reasonable that EFKl 6-11 is the most hydrophobic peptide among the three as it consists of phenylalanine residues, which is more hydrophobic than alanine residues in EAKl 6-11 and EAKl 6- IV. The reason why EAKI6-IV has a lower equilibrium surface tension than EAKl 6- II is probably due to the formation of /3-turn structure through intramolecular ionic interaction in EAKl 6-IV. This conformational change may cause the exposure of hydrophobic alanine residues toward the aqueous phase, resulting in a slight increase in hydrophobicity of the molecule and lowering the surface tension (Hong et al. (2003).) Figure 88b shows the fluorescence spectra of the ANS probe in the three peptide solutions comparing to that in pure water (black line and the inset). The normalized fluorescence intensities of ANS in different solutions follow a trend: EFKl 6-II»EAKl 6-11 =EAK16-IV>H20. Meanwhile, the peak positions of the spectra are different; it locates at ~520 nm in pure water (inset), but shifts to -485 run in EAKl 6-11 and EAKl 6-IV solutions. The ANS fluorescence spectrum has a peak of ~470 nm in the EFKl 6-11 solution. The changes in ANS fluorescence intensity and peak position indicate that the ANS probe is in different environments. ANS is a widely used probe to study protein aggregation as well as cell membrane composition and function due to its extreme sensitivity to the changes in the polarity of the probed environment (Torrent J. et al. (2004) High pressure induces scrapie-like prion protein misfolding and amyloikd fibril formation. Biochemistry 43: 7162-7170; Lindgren M. et al. (2005) Detection and characterization of aggregates,. Prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. Biophys J 88: 4200- 4212; Slavik J (1982) Anilinonaphthalene sulfonate as a probe of membrane composition and function. Biochim Biophys Acta 694: 1-25 "Slavik (1982)). A less polar environment will cause a shift of the fluorescence spectrum of ANS toward lower wavelengths (blue shift) and a significant increase in the fluorescence quantum yield (Slavik (1982)). Thus, the changes in ANS fluorescence in different peptide solutions (Figure 88b) can be related to the hydrophobicity of the local environment where ANS resides. This leads to a conclusion that EFKl 6-11 provides a more hydrophobic environment for ANS than the other two peptides.
The hydrophobicity determined by the two methods may refer to two different situations. Surface tension is a solution property and based on the molecular adsorption at the interface, affecting the surface free energy. The adsorption process involves three steps: i) diffusion of the molecules from the bulk to the sub-interface; ii) transfer of the molecules from the sub-interface to the interface; iii) rearrangement of the molecules at the interface (Biswas M.E. et al. (2005) Modeling of adsorption dynamics at air-liquid interfaces using statistical rate theory (SRT). J. Colloid Interface Sci 286: 14-27.). Considering diffusion to be the rate limiting step, small molecules are expected to rapidly accumulate at the interface due to their faster diffusion rate than large ones. Thus, in the self-assembling peptide systems, the surface tension may reflect predominantly the properties of peptide monomers and small peptide assemblies, rather than those of the large peptide aggregates. On the other hand, ANS fluorescence depends pronouncedly on the local probe environment. The binding of ANS to peptide monomers may not significantly affect its fluorescence properties as it still "feels" surrounding solvent molecules (i.e., water in this case). Only when the ANS probe is enclosed in a different environment from the solvent does its fluorescence greatly change. Therefore, the observed changes in ANS fluorescence in Figure 88b should result from the properties of peptide assemblies/aggregates. This is probably why the difference between EAKI6-II and EAKI6-IV from surface tension is not observed by the ANS fluorescence.
It is found above that different peptide sequences affect the peptide assemblies and their properties. Such effects further influence the formation of peptide-ellipticine complexes. The results are shown in Figure 89. The differences among the complexes made of the three peptides can be directly visualized from the appearance of the suspensions (Figure 89a). For EAK16-II, the peptide-ellipticine solutions appear to be slightly turbid at peptide concentrations of 0.2 and 0.04 mg/mL, indicating the formation of large colloidal suspensions. However, at a concentration of 0.5 mg/mL, the solution becomes clearer with a light yellow color (far left vial). Similar appearances of the peptide-ellipticine solutions are found for EAKl 6-IV (central three vials) except that the solution looks less yellow at a peptide concentration of 0.5 mg/mL. For EFKl 6-II, all solutions look cloudy. Compared with the control sample (with the absence of peptides, far right vial) that remains colorless and transparent, the changes in the solution appearance of the peptide-ellipticine samples reveal that ellipticine has been uptaken by the peptides and stabilized in solution. The different appearances of the solutions may indicate different molecular states of ellipticine in the complexes. Studies on the complexation of EAK16-II with ellipticine have demonstrated that two molecular states of ellipticine, either protonated or crystalline, can be obtained in the complexes depending on the peptide and ellipticine concentrations. The protonation of ellipticine usually occurs at a higher peptide concentration, related to a relatively low solution pH (<5, pKa of ellipticine is ~6) (Garbett NC, Graves DE (2004) Extending nature's leads: the anticancer agent ellipticine. Curr Med Chem 4: 149-172 "Garbett & Graves (2004)"); protonated ellipticine can be stabilized by ionic interaction with the negatively charged residues (glutamic acid E in this case) of the peptide. The ellipticine microcrystals are stabilized by peptide assemblies coating on the surface. When ellipticine is protonated, it can dissolve in aqueous solution and cause the solution to have a yellow, transparent appearance. On the other hand, the suspended ellipticine microcrystals make the solution turbid and cloudy. Thus, by looking at the appearance of the samples, one can possibly predict that EAKl 6-11 and EAKl 6-IV can stabilize protonated or crystalline ellipticine while ellipticine stabilized by EFKl 6-11 may be predominantly in microcrystal form.
The molecular state of ellipticine can be further elucidated by the ellipticine fluorescence spectra. It has been found that protonated ellipticine molecules have a fluorescence peak at ~520 nm while the fluorescence peak at ~430 nm is attributed to neutral ellipticine molecules; crystalline ellipticine exhibits a fluorescence peak at ~470 nm with an extremely low intensity. The fluorescence spectra of the complexes with the three peptides, EAKl 6-II, EAKl 6-IV and EFKl 6-II, are shown in Figure 89 b, c and d, respectively. For EAK16-II and EAK16-IV, the complexes with 0.5 mg/mL peptide have a fluorescence peak located ~520 nm, indicating that ellipticine is protonated. At peptide concentrations below 0.5 mg/mL, the spectra have a peak close to 470 run with an extremely low intensity (insets in Figure 89b and c), representing crystalline ellipticine. Interestingly, the complexes with EFKl 6-11 exhibit a fluorescence spectrum with a major peak located at ~435nm and a small shoulder covering the wavelengths from 470 to 570 nm (Figure 89d), very different from those of protonated and crystalline ellipticine. The peak located at -435 nm represents neutral (non-charged) ellipticine, present as individual molecules in a much less polar environment. The peak intensity is proportional to the EFKl 6-11 concentration. These results indicate that EFKl 6-11 can stabilize neutral, molecular ellipticine in aqueous solution; in contrast, the other two molecular states of ellipticine, protonated and crystalline, can be formed in the complexes with EAKl 6-11 and EAKl 6-IV. EFKl 6-11 assemblies provide a more hydrophobic environment than those of EAKl 6-11 and EAKl 6-IV as shown in Figure 88b, possibly facilitating the stabilization of neutral ellipticine molecules. In addition to neutral ellipticine, crystalline and protonated ellipticine can coexist in the suspensions as indicated by the turbid appearance of the suspensions and a shoulder from the fluorescence spectra. The fluorescence signals from crystalline ellipticine, however, are too small to be seen compared to those of neutral ellipticine. The different quantum yields and overlapping of the fluorescence signals from the three molecular states of ellipticine make it difficult to determine the percentage of each state among the three in the complexes. However, the total amount of stabilized ellipticine can be obtained.
To determine how much ellipticine that can be stabilized in solution by the peptides, aliquots of the peptide-ellipticine suspensions were diluted into DMSO, and the UV absorption of ellipticine was collected. The ellipticine absorbance was then converted to corresponding ellipticine concentration in the suspensions. This concentration was compared with the given ellipticine concentration (0.04 mg/mL) to obtain the maximum suspension (%) as shown in Figure 90. Initially in the preparation, ellipticine is in solid form as a thin film at the bottom of the vial. With the help of the peptides and mechanical stirring over time, ellipticine can be uptaken and stabilized in the solution as protonated, neutral or crystalline ellipticine. Not all given ellipticine can be stabilized and suspended in solution; the deposition of ellipticine thin film can be observed at the bottom of most sample vials. The amount of stabilized ellipticine varies with the types of peptides and peptide concentrations. The highest maximum suspension is found to be -71% (by Wt.) by 0.5 mg/mL EAKl 6-II. At the same peptide concentration, such a value decreases to -56% for EAKl 6-IV and to -46 for EFK 16-11. The lowest maximum suspension appears to be -13% by 0.04 mg/mL EAKl 6-IV, which is 3 folds higher than the control (-4.5%) with the absence of peptides. The amount of ellipticine suspended by the peptide the peptide in water is found to be much higher than the reported solubility in water (-0.6 μM) (Liu J. et al. (2004) Polymer-drug compatibility: a guide to the developments of delivery systems for the anticancer agent, ellipticine. J Pharm Sci 93: 132-144.). With the peptide concentration, the maximum suspension varies largely for EAKl 6-11 and EAK16-1V but not for EFKl 6-11. Overall, EAKl 6-11 appears to be the most effective peptide among the three at stabilizing protonated ellipticine (at a high peptide concentration of 0.5 mg/mL.); EFKl 6-11, on the other hand, can stabilize neutral ellipticine (in addition to crystalline and protonated ellipticine), and it has less variation in the maximum suspension with different peptide concentrations. Size of the Complexes
The size distribution of the peptide assemblies and complexes at a peptide concentration of 0.5 mg/mL is shown in Figure 91. For all three peptides, the peptide assemblies have a broad size distribution from 10 to several hundred nanometers (Figure 91a). They all have a major size population around 30 nm and a second one corresponding to a shoulder located at -300 nm, 100 nm and 200 nm for EAK16-II, EAKl 6-IV and EFKl 6-II, respectively. The size distribution of EAKl 6-11 obtained here correlates well with our earlier findings, and the two populations represent short peptide nanofibers and fiber clusters. When the peptides interact with ellipticine to form complexes, the size distributions change significantly as shown in Figure 91b. Only the size distributions of the complexes with EAKl 6-11 and EAKl 6-IV are shown in the plot because the size of the complexes with EFKl 6-11 is very polydispersed and over the detection limit of the instrument. The EAK16-II-ellipticine complexes have a relatively wider size distribution than EAK16-IV-ellipticine complexes; two size populations with one around 90 nm and the other around 500 nm can be found in both distributions.
SEM imaging was applied as a complementary method to examine the size and morphology of the complexes for the three peptides at different peptide concentrations. The representative images are shown in Figure 92. It is clearly seen that the dimensions of the complexes with 0.5 mg/mL EAK16-II and EAKl 6-IV are in the range of 100-200 nm. For these two peptides, at peptide concentrations below 0.5 mg/mL, the size of the complexes can be as large as several micrometers.These complexes tend to have a rod-like or fiber-like structure, aggregating into bundles or entanglements. Such structures are very different from ellipticine crystals suspended in water (control).
For EFKl 6-II, the dimensions of the complexes range from hundreds of nanometers to several micrometers regardless of the peptide concentrations. However, the morphology of these complexes looks different according to the peptide concentration. At 0.04 mg/mL, the majority of the complexes are also rod-like although they seem to be shorter and more dispersed than those with EAKl 6-11 and EAKl 6-IV; at higher peptide concentrations, the complexes appear to have irregular shapes. In addition, more membrane-like structures are observed in the background with the increase in EFKl 6-11 concentration. These membrane-like EFKl 6-11 assemblies could play an important role in stabilizing neutral ellipticine molecules. This may explain the increase in the fluorescence intensity of neutral ellipticine as a function of EFKl 6-11 concentration shown in Figure 89d. Meanwhile, the ellipticine microcrystals could be stabilized by the amphophilic EFKl 6-11 monomers and small assemblies via forming peptide coatings on the surface of the crystals, leading to the formation of cloudy suspensions at all peptide concentrations. Cellular Toxicity of the Complexes and Their Dilutions
From the characterization of the complexes above, it can be summarized that peptide sequence will affect the molecular state of ellipticine in the peptide-ellipticine complexes/assemblies. EAKl 6-11 and EAKl 6-IV can solubilize protonated ellipticine or encapsulate ellipticine microcrystals, depending on the peptide concentration. EFKl 6-II, on the other hand, can stabilize neutral ellipticine molecules in addition to the other two states in aqueous solution; the amount of neutral ellipticine that can be carried by EFKl 6-11 assemblies is peptide concentration dependent. The size and structure of the complexes also depend on the type of peptide and peptide concentration. To gain more insight concerning these differences in the molecular state of ellipticine as well as the size and structure of the complexes, we investigated their cellular toxicity against two cancer cell lines and the stability of the complexes upon dilution in water. The information regarding the complex stability after dilution would be useful for later animal studies and preclinical experiments.
Figure 93 shows the viability of both A549 and MCF-7 cancer cells upon being treated with peptide-ellipticine complexes for 48 h. For A549 cells (Figure 93a), all peptide-ellipticine complexes reduce the cell viability to less than 0.3 compared with the viability of non-treated cells (viability is 1). The toxicity of complexes is 2-folds higher than that of the ellipticine control with the absence of peptides (light green bar). The peptide controls have some toxicity to the cells, causing the decrease of viability to the values between 0.6 and 0.8. The much lower cell viability resulted from the peptide-ellipticine complexes compared with that from the ellipticine control is probably due to the fact that the peptides can stabilize large amounts of ellipticine in aqueous solution as shown in Figure 90. Interestingly, the cells treated with the complexes formulated with 0.5 mg/mL EAKl 6-11 and EAKl 6- IV have almost zero viability. This may indicate that protonated ellipticine is more effective at killing A549 cells than other forms of ellipticine in the complexes. Such a result seems to contradict to the already known fact that neutral ellipticine is the active form to suppress the cancer cell growth (Garbett & Graves (2004).)
The high efficacy of protonated ellipticine against cancer cells may be explained in the following. First, the protonated ellipticine has a positive which can interact with a negatively charged cell membrane surface, leading to accumulation of ellipticine at the cell membrane surface. In addition, such a small molecule with a hydrophobic characteristic is expected to cross the cell membrane easily into the cytoplasm. Second, the protonated ellipticine molecules release much faster from the complexes compared with that from ellipticine microcrystals, due to the differences in complex size and a relatively weak interaction between protonated ellipticine and the peptide in the complexes. This accelerates the diffusion speed of ellipticine from the complexes to the cells, facilitating a fast cellular uptake of ellipticine. Third, although EFKl 6-11 is capable of stabilizing neutral ellipticine molecules, the amount of stabilized molecules are probably low; the release rate can be slow due to a possibly stronger hydrophobic interaction between neutral ellipticine and EFKl 6-11 in the complexes. This is probably why the complex prepared with 0.5 mg/mL EFKl 6-11 has much less effect on the cellular toxicity than protonated ellipticine stabilized by EAKl 6-11 and EAKl 6-IV at the same peptide concentration. For MCF-7 cells, the efficacy of protonated ellipticine on anti-proliferation of the cells becomes more significant when compared with the other forms of ellipticine (Figure 93b). The lowest cell viability for the complexes with neutral ellipticine and/or ellipticine crystals is around 0.5, which is about 70% of the viability for the ellipticine control (-0.7). This percentage can be as low as -25% in the case of A549 cells. Such a difference may imply that the peptide-ellipticine suspensions are less effective to MCF-7 cells than to A549 cells. However, the complexes with protonated ellipticine have similar efficacy at killing both cells, although the reason behind is still unclear. It could be related to the different sensitivity, internalization pathway and/or cell defense mechanism of the two cells in response to ellipticine. Nevertheless, these results provide evidence that the molecular state of ellipticine in the complexes significantly affects their cellular toxicity. Accordingly, one should be aware that selection of an appropriate formulation method is important in treating different cancer cells. Figure 94 shows the toxicity of the complexes with 0.5 mg/mL EAK16-II,
EAKl 6-IV and EFKl 6-11 upon serial dilution in water against both cell lines. The ellipticine control is diluted the same way for comparison. It is clearly seen that dilution has significant effect on the toxicity of the complexes with EAKl 6-11 and EAKl 6-IV, where ellipticine is stabilized in protonated form. For A549 cells (Figure 94a), the cell viability is very low and less than 0.05 with these complexes before dilution; it increases largely to above 0.6 for 16 times dilution of the complexes. A similar trend is found for MCF-7 cells as the viability increases from less than 0.05 to above 0.7 (Figure 94b). Such changes imply that the complexes may not be stable, altering the protonated form of ellipticine after dilution in water. This instability of complexes is probably due to the rising of solution pH, leading to the deprotonation of ellipticine and the formation of ellipticine microcrystals after dilution. This may explain why a sudden increase in cell viability occurs upon 2 times dilution for MCF- 7 cells as they seem to be more sensitive to protonated ellipticine than ellipticine microcrystals. The EFK16-II-ellipticine complexes, on the other hand, exhibit good stability upon dilution in water. The viability increases from -0.25 to -0.5 for A549 cells; for MCF-7 cells, it remains unchanged at -0.57 up to 4 times dilution and then slightly increases to -0.65 for 16 times dilution. Such a good stability may result from a stronger interaction between EFKl 6-11 and ellipticine in the complexes due to a higher hydrophobicity of the peptide. In addition, a possible increase in solution pH after dilution should not affect the state of the stabilized neutral ellipticine molecules or ellipticine microcrystals. It is worth noting that although these complexes are not as effective as protonated ellipticine at killing cancer cells, their stability is much better, which is especially important for practical applications in clinics where drug dilution always occurs after administration into the bloodstream.
Overall, this study demonstrated the effect of peptide sequence on its ability at stabilizing hydrophobic ellipticine in protonated, neutral as well as crystalline forms in aqueous solution. The difference in charge distribution (type II vs. type IV) on the peptide sequence seems not to have much effect on the complex formation and the molecular state of ellipticine in the complexes. The size, anticancer activity and stability of the complexes are very similar, although the charge distribution does affect, to some degree, the peptide assemblies: nanofibers vs. globular aggregates. It may be because the complexation of ellipticine with EAKl 6-11 and EAKl 6-IV is mainly based on the peptide monomers but not on the peptide assembles. The increase in hydrophobicity of the peptide by replacing alanine (A) with phenylalanine (F), however, significantly alters the molecular state of ellipticine in the complexes, the complex stability and its therapeutic effect due to the following reasons: (i) the EFKl 6-11 assemblies provide a more hydrophobic, enclosed environment where neutral ellipticine molecules can be solubilized; (ii) a stronger hydrophobic interaction between ellipticine and EFKl 6-11 may further enhance the stability of the complexes upon dilution.
Different peptide sequences have different advantages in formulating the ellipticine drug. For example, 0.5 mg/mL EAKl 6-11 (or EAKl 6-IV) can solubilize protonated ellipticine in nano scale complexes with high anticancer activity against both A549 and MCF-7 cells, but these complexes are pH sensitive and not very stable after dilution. In contrast, the complexes formulated with 0.5 mg/mL EFK 16-11 are more stable upon dilution, but most of their sizes are in the micrometer range and their anticancer activity is relatively low. Nevertheless, these results provide essential information to design an appropriate peptide sequence that would optimize the delivery of hydrophobic anticancer drugs. One could utilize the advantages of different molecular states of ellipticine to improve the delivery efficacy, through a proper peptide design to form a stable, peptide nanocarriers, which can encapsulate neutral or crystalline ellipticine; if such a carrier enters cells through endocytosis, the encapsulated ellipticine becomes protonated at low pH in the lysosomes, and the protonated ellipticine can be released and cross the lysosome membrane into cytoplasm.
In conclusion, three ionic-complementary self-assembling peptides, EAKl 6- II, EAKl 6-IV and EFKl 6-II, with different charge distributions and hydrophobicities were found to be able to stabilize the hydrophobic anticancer agent ellipticine in aqueous solution. Ellipticine was stabilized in the form of microcrystals, protonated and neutral molecules depending on the peptide sequence and the peptide concentration. 0.5 mg/mL EAK16-II and EAK16-IV stabilized protonated ellipticine to form nano-complexes while crystalline ellipticine was obtained in the complexes with these peptides at lower peptide concentrations. On the other hand, EFKl 6-11 was able to stabilize both neutral and crystalline ellipticine within the range of tested peptide concentrations; the amount of neutral ellipticine that can be stabilized was proportional to the peptide concentration. The different molecular states of stabilized ellipticine in the complexes greatly affected the anticancer activity of the complexes and their stability upon dilution in water. The complexes with protonated ellipticine were found to be very effective at killing both A549 and MCF-7 cells with a cell viability close to zero; however, these complexes were not very stable and their anticancer activity reduced significantly after serial dilution in water. The complexes formulated with EFKl 6-11 (containing neutral ellipticine and ellipticine micro- crystals), on the contrary, appeared to be stable after serial dilution, although their original anticancer activity was relatively low. These results showed that the differences in charge distribution of the peptides did not have much effect on the complex formation and their cellular toxicity, whereas the increase in peptide hydrophobicity could strengthen the interaction between the peptide and ellipticine, which gives the stability of their complexes upon dilution. This study provides necessary information on peptide sequence design to construct functional peptide carriers for hydrophobic anticancer drug delivery. Materials
Three self assembling, ionic-complementary peptides EAKl 6-11 (Mw = 1657 g/mol, crude), EAKI6-IV (Mw = 1657 g/mol, crude) and EFKl 6-11 (Mw = 2265 g/mol, crude) were obtained from CanPeptide Inc. (Pointe-Claire, Quebec, Canada) and used without further purification. The mass spectra and HPLC data are presented in Figures 95, 96, 97, 98, 99. The N-terminus and C-terminus of the peptide were protected by acetyl and amino groups, respectively. At pH~7, A and F are neutral, while E and K are negatively and positively charged, respectively. The anticancer agent ellipticine (99.8% pure) and l-anilinonaphthalene-8-sulfonic acid (ANS) were purchased from Sigma-Aldrich (Oakville, ON, Canada) and used as received. Tetrahydrofuran (THF, reagent grade 99%) and dimethyl sulfoxide (DMSO, spectral grade >99%) were from Calendon Laboratories Ltd. (Georgetown, ON, Canada) and Sigma-Aldrich (Oakville, ON, Canada), respectively. Cell culture reagents including Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and trypsin-E I'DA were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Phosphate buffer saline (PBS) and penicillin-streptomycin (p/s, 10000 U) were obtained from MP Biomedicals Inc. (Solon, OH, USA). Sample preparation
Appropriate amounts of the peptide powder were first dissolved in pure water (18 Ω; Millipore MiUi-Q system) to obtain fresh peptide solutions at concentrations of 0.5, 0.2 and 0.04 mg/ml ("crude" peptide concentration). The solution was then sonicated in a bath sonicator (Branson, model 2510) for 10 min. The peptide solution at a concentration of 0.5 mg/mL was used to study the differences among the three peptides in self-assembled nanostructures, hydrophobicity and surface activity.
The peptide-ellipticine complexes were prepared by adding 1 mL of the fresh peptide solution into a glass vial containing a thin film of 0.04 mg ellipticine at the bottom, followed by mechanical stirring at 900 rpm for 24 h. 1 mL of pure water, instead of peptide solution, was also added to another vial to make a control sample. The purpose of using a relatively low ellipticine concentration of 0.04 mg/mL in this study was to obtain distinguishable cellular toxicity of the complexes and the control sample. To make a thin film of ellipticine at the bottom of the vials, 100 μL of 0.4 mg/mL ellipticine stock solution in THF was transferred to the vials, and dried with gently blowing of filtered air (0.22 μm pore size filter) for ~5 min. All the vials and solvents were sterilized and the samples were prepared in a biological safety cabinet to avoid possible contamination, for especially cell culture experiments. For dynamic light scattering (DLS) measurements, the solvents were filtered, and the samples were made in the biosafety cabinet to eliminate potential dust contamination. The complexes were photographed with a digital camera (Cannon PowerShot A95) and characterized with several techniques to obtain complex dimensions and molecular states of the ellipticine in the complexes. Determining the maximum suspension concentration of ellipticine
The amount of suspended ellipticine in solution was determined by the ellipticine UV-absorption. The peptide-ellipticine suspension was diluted 20 times in DMSO (resulting in a solvent mixture of 95% DMSO and 5% water by volume) to dissolve ellipticine from the complexes. 80 μL of the solution were then transferred to a quartz microcell (70 μL) with a 1 cm light path and tested on a UV- Vis spectrophotometer (Biochrom Ultraspec 4300 Pro, Cambridge, England). The absorbance at 295 nm was converted to the ellipticine concentration using Beer- Lamberts law: absorbance (Abs) = ecd, where e is the molar extinction coefficient, c is the molar concentration of ellipticine, and d is the optical path length (cm) (Lakowicz JR (1999) Principles of Fluorescence Spectroscopy, New York: Kluwer Academic/Plenum Publishers.). The extinction coefficient was obtained as 59000±1100 (R2>0.995) from the linear fitting of ellipticine absorption as a function of ellipticine concentration (2-20 μM) prepared in a mixture of 95% DMSO and 5% water. The suspension concentration of ellipticine was averaged from 3 measurements, and compared with the given ellipticine concentration of 0.04 mg/mL. Since not all ellipticine in the thin film at the bottom of the vials could be stabilized and suspended in solution, the comparison of the suspension concentration with the given ellipticine concentration (0.04 mg/mL) would thus provide the maximum percentage of the ellipticine suspension at each formulation condition. Atomic Force Microscopy (AFM)
The peptide self-assembled nanostmcrures were imaged on a PicoScan™ (Molecular Imaging, Phoenix, AZ) in pure water. The samples were prepared with the following procedure: 10 μL of 0.5 mg/mL peptide solution (~15 min after solution preparation) were put on a freshly cleaved mica substrate, which was fixed on an AFM sample plate; a custom made AFM liquid cell was fastened on top of the mica substrate. The solution was incubated for 10 s to allow the peptide assemblies to adhere to the mica surface. The surface was then washed with pure water 15 times, and 500 μL of pure water were added into the cell prior to AFM imaging. A scanner with a maximum scan area of 6x6 μm2 was used to acquire the AFM images. It was operated with a tapping mode using silicon nitride cantilevers with a nominal spring constant of 0.58 N/m (DNP-S, Digital Instruments, Santa Barbara, CA) and a typical tip radius of 10 nm. For the best imaging quality, the tapping frequency was typically set between 16 kHz and 18 IcHz and the scan rates controlled between 0.8 and 1 line/s. The experiments were conducted in an environmentally-controlled chamber at room temperature to avoid evaporation of the solution. AU AFM images were obtained at a resolution of 256 x 256 pixels. Surface tension measurements The dynamic surface tension of fresh peptide solutions was measured over a period of 2 h using the Axisymmtratic Drop Shape Analysis-Profile (ADSA-P) technique. The experimental setup and operation of ADSA-P were described in Yang H. et al. (2006) Anion effect on the nanostructure of a metal ion binding self- assembling peptide. Langmuir 22: 8553-8562 and the references therein. Fluorescence spectroscopy
The hydrophobicity of the three peptides and their assemblies was investigated via ANS fluorescence (Slavik J. (1982)) Anilinonaphthalene sulfonate as a probe of membrane composition and function. Biochim Biophys Acta 694: 1-25; Cardamone M, Puri NK (1992) Spectrofiuorimetric assessment of the surface hydrophobicity of proteins. Biochem J 282: 589-593.) 10 μM ANS solution was prepared in a 10 mM phosphate buffer at pH 6. The fresh peptide solutions were mixed with the same volume of the ANS solution on a vortex mixer for 10 s. The ANS solution was also mixed with the same volume of pure water as a control sample. 60 μL of the mixed solution were transferred to a quartz microcell and tested on a spectrafluorometer (Photon Technology International, Type QM4-SE, London, Canada) with a continuous xenon lamp as the light source. The sample was excited at 360 nm and the emission spectra were collected from 420 to 670 nm. The excitation and emission slit widths were set at 0.5 mm and 1.25 mm, respectively (0.5 and 1.25 mm corresponds to 2 and 5 nm band path). The spectra were normalized with light scattering of air at 360 nm, to correct the lamp fluctuations.
To study the molecular states of ellipticine in the complexes, 60 μL of the peptide-ellipticine suspensions were transferred to a microcell and tested on the spectrafluorometer. The excitation was set to be 294 nm and the emission was collected from 320 to 650 run, The excitation and emission slit widths were set at 0.5 mm and 0.25 mm, respectively. The intensities were corrected with an ellipticine standard (2 μM in ethanol, scaled and degassed), to account for lamp fluctuations. Dynamic Light Scattering (DLS) The dimension of the peptide assemblies (0.5 mg/mL) and the complexes from the peptide-ellipticine suspensions was investigated on a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) with appropriate viscosity and reflactive index settings, and the temperature was maintained at 25°C during the measurement. A quartz microcell (45μL) with a 3 mm light path was used. The scattered light intensities of the samples at the angle of 173° were collected. The intensity-based size distribution was obtained with the multimodal algorithm CONTIN (Provencher SW (1982) A constrained regulation method for inverting data represented by linear algebraic or integral equations. Comput Phys Commun 27: 213-227.) Scanning Electron Microscopy (SEM) A LEO model 1530 field emission SEM (GmbH, Oberkochen, Germany) was employed to study the morphology and dimensions of the peptide-ellipticine complexes. The SEM sample was prepared by depositing 10 μL of the peptide- ellipticine suspensions on a freshly cleaved mica surface. The mica was affixed on an SEM stub using a conductive carbon tape. The sample was placed under a Petridish- cover for 10 min to allow the complexes to adhere onto the mica surface. It was then washed once with a total of 100 μL pure water and air-dried in a desicator overnight. AU samples were coated with a 20 nm thick gold layer prior to SEM imaging; the images were acquired using the secondary electron (SE2) mode at 5 kV. In vitro cell viability studies Two types of cancer cells, non-small cell lung cancer cell A549 and breast cancer cell MCF-7 were used for in vitro cellular toxicity studies on the peptide- ellipticine complexes. The cells were cultured in DMEM containing 10% FBS and 1 % p/s at 37°C and with 5% CO2. When cells grew to reach -95% confluence, they were detached from cell culture flasks with trypsin-EDTA and resuspended in the cell culture media at concentrations of 5 x 104 and 1 x 105 cells/mL for A549 and MCF-7 cells, respectively. For each type of cell, 200 μL of the cell suspensions were added into each well of a clear, flat bottom 96-well plate (Costar) and incubated overnight. 50 μL of the treatments (including the complexes and control samples) were then added to the wells each containing 150 μL of fresh culture media. The plates were incubated for 48 h prior to perform the cell viability assay.
MTT assay was used to determine the cell viability after different treatments.
5 mg of solid MTT was dissolved in 3 mL PBS solution, followed by 10 times dilution in the culture medium. All the treatments were taken out before 100 μL of the
MTT solution was added to each well of the treated plates. The plates were incubated for 4 h prior to the addition of 100 μL of the solubilization solution (anhydrous isopropanol with 0.1 N HCl and 10% Triton X-100). After overnight incubation, the absorbance at 570 nm was recorded on a microplate reader (BMG FLUOstar OPTIMA) and subtracted by the background signals at 690 nm. The absorption intensities were averaged from 4 replicates for each treatment and normalized to that obtained from the untreated cells (negative control) to generate the cell viability.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
All documents cited herein are incorporated by reference as if they were specifically incorporated in their entirety. However, the citing of any document herein does not constitute an acknowledgement of it as prior art.

Claims

Claims
1. A self-complementary β-strand peptide comprising alternating hydrogen bonding proton donor amino acid segments and hydrogen bonding proton acceptor amino acid segments, and having a length from at least two to forty hydrogen bonding amino acids, for forming a nanostructure, wherein: a) the hydrogen bonding proton donor amino acid segment consists of at least one amino acid; b) the hydrogen bonding proton acceptor amino acid segment consists of at least one amino acid; c) the β-strand peptide comprises at least one proton donor segment and at least one proton acceptor segment; and d) the peptide is not comprised of alternating hydrophobic and hydrophilic amino acid segments, wherein the self-complementary β-strand peptides assemble into the nanostructure.
2. The self-complementary β-strand peptide of claim 1 wherein hydrogen bonding occurs between side chains of complementary amino acids,
3. The self-complementary β-strand peptide of claim 1 wherein the self- complementary peptides assemble in one of parallel or anti-parallel conformation.
4. The self-complementary β-strand peptide of claim 3 wherein the peptides further assemble as one of end-to-end or staggered peptide arrangement.
5. The self-complementary β-strand peptide of claim 4 wherein the peptides are assembled in staggered arrangement and at least one to at least 20 amino acids in the peptide form hydrogen bonds with complementary amino acids in a second peptide.
6. The self-complementary β-strand peptide of claim 1, wherein the hydrogen bonding proton donor amino acid is selected from the group consisting of Arg, Trp,
Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu and GIn.
7. The self-complementary β-strand peptide of claim 1, wherein the hydrogen bonding proton acceptor amino acid is selected from the group consisting of Arg, Trp, Tyr, Lys, His, Asp, Thr, Cys, Ser, Asn, GIu, Met and GIn.
8. The self-complementary β-strand peptide of claim 1, wherein the nanostructure is selected from the group consisting of nanofibers, nanotubes, nanoplanes and nanospheres.
9. The self-complementary β-strand peptide of claim 1 wherein at least one hydrogen bonding amino acid is non-charged.
10. The self-complementary β-strand peptide of claim 1 having a structure selected from the group consisting of: a) (AxByCz)wAz (I); and b) (AxByCz)w(C'xB'yA'z)w (II) where A, A', B, B', C and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20, wherein A is complementary to A', B is complementary to B', and C is complementary to C.
11. The self-complementary β-strand peptide of claim 1 having a structure selected from the group consisting of: a) AxByCz... ; and (III), and b) AxByC2... Cz'By'Ax' (IV), where A, A', B, B', C and C are each independently a donor amino acid or an acceptor amino acid, are each self-complementary, and are selected from the group consisting of a hydrogen bond donor amino acid, a hydrogen bond acceptor amino acid, a positively charged amino acid, a negatively charged amino acid, and a van der Waals' interacting amino acid, and wherein A is complementary to A', B is complementary to B', and C is complementary to C.
12. The self-complementary β-strand peptide of claim 1 selected from the group consisting of: Gln-Asn, Gln-Asn-Gln-Asn (SEQ ID NO: 1), Gln-Gln-Asn-Asn (SEQ ID NO: 2), Asn-Ser, Asn-Ser-Asn-Ser (SEQ ID NO: 3), Asn-Ser-Asn-Ser-Asn (SEQ ID NO: 4), and Asn-Ser-Asn-Ser-Asn-Ser-Asn-Ser (SEQ ID NO: 5).
13. The self-complementary β-strand peptide of claim 1 further comprising a functional moiety at at least one of an N- terminus and C-terminus of the peptide, wherein the functional moiety is selected from the group comprising a cell targeting moiety, a metal ion binding motif, and a cell membrane penetration moiety.
14. The peptide of claim 1 wherein the amino acids are selected from the group comprising naturally occurring amino acids, D-amino acids, β amino acid derivatives, and γ amino acid derivatives.
15. A self-complementary β-strand peptide comprising at least one hydrogen bonding amino acid pair, at least one ionic-complementary amino acid pair, and at least one hydrophobic amino acid pair, and having a length from four to forty amino acids, for forming a nanostructure.
16. The self-complementary β-strand peptide of claim 15 wherein the self- complementary peptides assemble in one of parallel or anti-parallel conformation.
17. The peptide of claim 15 having the general formula:
(AwBxAyCz)nAaBb (V) where A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10.
18. The peptide of claim 15 wherein the hydrophobic amino acid is selected from the group consisting of VaI, He, Leu, Met, Phe, Trp, Cys, Ala, Tyr, His, Thr, Ser, Pro, GIy, Arg and Lys.
19. The peptide of claim 15 wherein the charged amino acid is selected from the group consisting of His, Arg, Lys, Asp and GIu.
20. The peptide of claim 15 wherein the hydrogen bonding amino acid is selected from the group consisting of a proton donor and a proton acceptor.
21. The peptide of claim 15 further comprising at least one pi pairing amino acid selected from the group consisting of Tyr, Phe and Trp.
22. The peptide of claim 15 consisting of the amino acid sequence Phe-Glu-Phe- Gln-Phe-Asn-Phe-Lys (AC8) (SEQ ID NO: 6).
23. The peptide of claim 15 wherein amino acids A, B and C are one of independently self-complementary and independently complementary to a non-self amino acid, and the peptide self-assembles into parallel β-sheets.
24. The peptide of claim 15 wherein amino acid A, B and C, respectively, is each complementary to an amino acid in a second peptide other than A, B and C, respectively, and the peptide self-assembles into anti-parallel β-sheets.
25. A self-assembled nanostructure consisting of aggregated units of a peptide having a structure selected from the group consisting of : a) (AxByCz)w I; and b) (AxByCz)wAx II where A, B and C are each a hydrogen bonding amino acid selected from the group consisting of proton donors and proton acceptors; x and y are each independently an integer from 1 to 10; z is an integer from 0 to 10; and w is an integer from 1 to 20, wherein the nanostructure is selected from the group consisting of a nanofibril, a nanowire, a nanosurface and a nanosphere.
26. The self-assembled nanostructure of claim 25, wherein the peptide is selected from the group consisting of Gln-Asn, Gln-Asn-Gln-Asn (SEQ ID NO: 1), Gln-Gln-
Asn-Asn (SEQ ID NO: 2), Asn-Ser, Asn-Ser-Asn-Ser (SEQ ID NO: 3), Asn-Ser-Asn-
Ser-Asn (SEQ ID NO: 4), and Asn-Ser-Asn-Ser-Asn-Ser-Asn-Ser (SEQ ID NO: 5).
27. A self assembled nanostructure consisting of aggregated units of a peptide having the general formula (V):
(AwBxAyCz)nAaBb (V) where A, B and C are each an amino acid selected from the group consisting of a hydrophobic amino acid, a charged amino acid, and a hydrogen bonding amino acid, and A, B and C are each different; w, x, y and z are each independently an integer from 1 to 5; a and b are each independently an integer from 0 to 2; and n is an integer from 1 to 10, wherein the nanostructure is selected from the group consisting of a nanofibril, a nanowire, a nanosurface and a nanosphere.
28. The self-assembled nanostructure of claim 27, wherein the peptide is Phe-Glu-
Phe-Gln-Phe-Asn-Phe-Lys (SEQ ID NO: 6). •
29. A pharmaceutical composition comprising the peptide of claim 15 and a therapeutic agent.
30. The pharmaceutical composition of claim 29 further comprising a pharmaceutically acceptable excipient.
31. The pharmaceutical composition of claim 29 further comprising a stabilizing agent.
32. The pharmaceutical composition of claim 29, wherein the peptide is Phe-Glu-
Phe-Gln-Phe-Asn-Phe-Lys (SEQ ID NO: 6).
33. The pharmaceutical composition of claim 29, wherein the therapeutic agent is selected from the group comprising drugs, small protein molecules, oligonucleotides, siRNA, miRNA, and shRNA.
34. The phaπnaceutical composition of claim 33, wherein the therapeutic agent is a hydrophobic drug.
35. The pharmaceutical composition of claim 34, wherein the therapeutic agent is an anti-cancer agent selected from the group comprising paclitaxel, ellipticine, camptothecin, doxorubicin, and adriamycin.
36. The pharmaceutical composition of claim 33, wherein the therapeutic agent is hydrophilic.
37. The pharmaceutical composition of claim 36, wherein the therapeutic agent is an oligonucleotide.
38. A pharmaceutical composition comprising the peptide of claim 1 and a therapeutic agent.
39. The pharmaceutical composition of claim 38 further comprising a pharmaceutically acceptable excipient.
40. The pharmaceutical composition of claim 38 further comprising a stabilizing agent.
41. The pharmaceutical composition of claim 38, wherein the peptide is selected from the group consisting of Gln-Asn, Gln-Asn-Gln-Asn (SEQ ID NO: 1), Gln-Gln- Asn-Asn (SEQ ID NO: 2), Asn-Ser, Asn-Ser-Asn-Ser (SEQ ID NO: 3), Asn-Ser-Asn- Ser-Asn (SEQ ID NO: 4), and Asn-Ser-Asn-Ser-Asn-Ser-Asn-Ser (SEQ ID NO: 5).
42. A kit for delivering a material to a patient, comprising:
the pharmaceutical composition of any one of claims 27 and 36; and one or more of an electrolyte, a buffer, a delivery device, a vessel suitable for mixing the composition with one or more other agents; and instructions for preparing the pharmaceutical composition for use, instructions for mixing the composition with other agents, and instructions for introducing the composition into a subject.
43. A method of preparing a self-assembling peptide having amino acid pairing properties for manufacture of a nanostructure, comprising: a) designing a peptide consisting of amino acids that are capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction with a complementary amino acid; and b) generating a peptide from two to forty amino acids in length consisting of at least one amino acid pair capable of at least one of hydrogen bonding, electrostatic interaction, hydrophobic interaction, and van der Waals' interaction, and having complementary amino acid pairing and stereochemistry with a second peptide.
44. Use of the peptide of claim 15 for delivery of a therapeutic agent to a cell.
45. The use of claim 44, wherein the therapeutic agent is hydrophobic.
46. The use of claim 44, wherein the therapeutic agent is hydrophilic.
47. The use of claim 44, wherein the therapeutic agent is an oligonucleotide.
48. A method for detecting a biomolecule of interest comprising: a) forming a nanostructure from the peptide of claim 1 upon self assembly of the peptide; b) adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; c) coupling a reporter molecule capable of providing a measurable signal to the peptide-coated surface of the nanostructure; and d) providing the biomolecule of interest.
49. The method of claim 48, wherein the peptide is selected from the group consisting of EAKl 6-1, EFKl 6-11 and EAKl 6-II.
50. The method of claim 49 wherein the biomolecule of interest is selected from the group consisting of proteins, nucleic acids, carbohydrates, and viruses.
51. The method of claim 50, wherein the biomolecule of interest is glucose.
52. A method for detecting a biomolecule of interest comprising: a) forming a nanostructure from the peptide of claim 15 upon self assembly of the peptide; b) adsorbing the peptide to an electrode surface, allowing electron transfer and immobilization of biocatalysts; c) coupling a reporter molecule capable of providing a measurable signal to the peptide-coated surface of the nanostructure; and d) providing the biomolecule of interest.
53. The method of claim 52 wherein the peptide is Phe-Glu-Phe-Gln-Phe-Arg- Phe-Lys.
54. The method of claim 52 wherein the biomolecule of interest is selected from the group consisting of proteins, nucleic acids, carbohydrates, and viruses.
55. The method of claim 52, wherein the biomolecule of interest is glucose.
56. A use of the peptide of claim 1 for forming a biofuel cell.
57. The use of claim 56 wherein the biofuel cell further comprises an electrode; a biocatalyst; a fuel source; and a membrane.
58. The use of claim 56, wherein the peptide is selected from the group comprising EAKl 6-11 and EFKl 6-II.
59. A use of the peptide of claim 15 for forming a biofuel cell.
60. The use of claim 59 wherein the biofuel cell further comprises an electrode; a biocatalyst; a fuel source; and a membrane.
61. The use of claim 59, wherein the peptide is Phe-Glu-Phe-Gln-Phe-Arg-Phe- Lys.
62. Use of the peptide of claim 1, for identification of inhibitors of protein aggregation disease.
63. Use of the peptide of claim 1 for altering the wettability of a surface.
64. Use of the peptide of claim 1 for altering the biocompatibility property of a surface.
EP08800379A 2007-08-30 2008-08-29 Amino acid pairing-based self assembling peptides and methods Withdrawn EP2197897A4 (en)

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Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2757108B1 (en) * 2009-03-09 2016-08-31 Menicon Co., Ltd. Self-assembling peptide and peptide gel with high strength
CN102199195B (en) * 2011-03-07 2014-04-02 华南理工大学 Half-path charge matching amphiphilic self assembling short peptide, and application thereof as nanometer hemostatic material and hydrophobic drug carrier
EP3375457B1 (en) 2011-11-24 2020-06-24 Positec Power Tools (Suzhou) Co., Ltd Peptide sequence design and use thereof for peptide-mediated sirna delivery
CN102911258B (en) * 2012-07-06 2014-07-30 江苏大学 Method for forming two-dimensional polypeptide nanosheets by regulating assembly of polypeptides with organic micromolecules
EP2698162A1 (en) 2012-08-15 2014-02-19 Credentis AG Method for producing a composition for treating a tooth lesion
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RU2673820C2 (en) 2013-09-25 2018-11-30 Кредентис Аг Dental care product for tooth whitening
CN104725478B (en) 2013-12-18 2018-12-25 纳肽得有限公司 Polypeptide compound, the assembly of polypeptide compound and siRNA and its application
EP3696536A1 (en) 2015-04-14 2020-08-19 Illumina, Inc. A method of manufacturing a substrate and a method of analyzing biomolecules capable of generating light emissions
EP3248591A1 (en) 2016-05-24 2017-11-29 Credentis AG Method and product for caries treatment
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CN108659103A (en) * 2018-08-30 2018-10-16 安徽工程大学 A kind of ion complementary type self-assembling peptides and preparation method thereof
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EP3831360A1 (en) 2019-12-04 2021-06-09 Credentis AG Personal dental care product for preventing demineralisation
CN111040026B (en) * 2019-12-10 2021-03-05 中山大学附属口腔医院 Amyloid hexapeptide and application thereof in broad-spectrum inhibition of bacterial and fungal biofilms
CN111840547B (en) * 2020-06-15 2023-04-28 山西振东泰盛制药有限公司 Preparation method of pemetrexed magnetic self-assembled nano composite particles for injection
CN111748016B (en) * 2020-07-03 2023-05-02 四川大学华西医院 Atypical hydrophobic amino acid-based self-assembled short peptide and application thereof
US11154470B1 (en) 2020-07-31 2021-10-26 vVardis AG Anti-inflammatory and senolytic dental care product with tooth whitening characteristics
EP3988509A1 (en) * 2020-10-21 2022-04-27 Hellma GmbH & Co. KG Production of quartz glass mouldings of different grey to black colouration for optical applications
CN114660160A (en) * 2020-12-22 2022-06-24 陈璞 Method for determining minimum loading binding ratio in polypeptide and siRNA (small interfering ribonucleic acid) co-assembly body
CN112760349A (en) * 2021-02-04 2021-05-07 海南华研胶原科技股份有限公司 Method for extracting soybean peptide by enzymolysis method
CN113477194A (en) * 2021-06-21 2021-10-08 江苏大学 Method for regulating and controlling length distribution of one-dimensional self-assembly nano material based on polymer coating

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0457314A1 (en) * 1990-05-18 1991-11-21 Clintec Nutrition Company Nutrient compositions containing peptides
WO2002068601A2 (en) * 2001-02-28 2002-09-06 Skubitz Keith M Small peptides capable of modulating the function of cd66 (ceacam) family members
WO2004007532A2 (en) * 2002-07-15 2004-01-22 University Of Leeds Beta-sheet forming peptides and material made thereof
WO2006014570A2 (en) * 2004-07-06 2006-02-09 3D Matrix, Inc. Purified amphilic peptide compositions and uses thereof
WO2006081249A2 (en) * 2005-01-25 2006-08-03 Cell Therapeutics, Inc. Conjugates of biologically active proteins having a modified in vivo half-life

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7179784B2 (en) * 2001-07-10 2007-02-20 Massachusetts Institute Of Technology Surfactant peptide nanostructures, and uses thereof
US20050118494A1 (en) * 2003-12-01 2005-06-02 Choi Sung H. Implantable biofuel cell system based on nanostructures
WO2006047315A2 (en) * 2004-10-25 2006-05-04 The Brigham And Women's Hospital, Inc. Targeted delivery of biological factors using self-assembling peptide nanofibers
JP2008525822A (en) * 2004-12-28 2008-07-17 ナノミックス・インコーポレーテッド Nanoelectronic device for DNA detection / recognition of polynucleotide sequences

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0457314A1 (en) * 1990-05-18 1991-11-21 Clintec Nutrition Company Nutrient compositions containing peptides
WO2002068601A2 (en) * 2001-02-28 2002-09-06 Skubitz Keith M Small peptides capable of modulating the function of cd66 (ceacam) family members
WO2004007532A2 (en) * 2002-07-15 2004-01-22 University Of Leeds Beta-sheet forming peptides and material made thereof
WO2006014570A2 (en) * 2004-07-06 2006-02-09 3D Matrix, Inc. Purified amphilic peptide compositions and uses thereof
WO2006081249A2 (en) * 2005-01-25 2006-08-03 Cell Therapeutics, Inc. Conjugates of biologically active proteins having a modified in vivo half-life

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CARRICK ET AL: "Effect of ionic strength on the self-assembly, morphology and gelation of pH responsive beta-sheet tape-forming peptides", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 63, no. 31, 21 June 2007 (2007-06-21) , pages 7457-7467, XP022124611, ISSN: 0040-4020, DOI: 10.1016/J.TET.2007.05.036 *
CHEN ET AL: "Self-assembly of ionic-complementary peptides: a physicochemical viewpoint", COLLOIDS AND SURFACES. A, PHYSICACHEMICAL AND ENGINEERING ASPECTS, ELSEVIER, AMSTERDAM, NL, vol. 261, no. 1-3, 1 July 2005 (2005-07-01), pages 3-24, XP027802761, ISSN: 0927-7757 [retrieved on 2005-07-01] *
JÖRG GSPONER, URS HABERTHÜR, AMEDEO CAFLISCH: "The role of side-chain interactions in the early steps of aggregation: Molecular dynamics simulation of an amyloid-forming peptide from the yeast prion Sup35", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE OF THE USA, vol. 100, no. 9, 29 April 2003 (2003-04-29), pages 5154-5149, XP002678609, *
See also references of WO2009026729A1 *

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