EP2396365A2 - Polymères réductibles pour l'administration de gènes non viraux - Google Patents

Polymères réductibles pour l'administration de gènes non viraux

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Publication number
EP2396365A2
EP2396365A2 EP10741516A EP10741516A EP2396365A2 EP 2396365 A2 EP2396365 A2 EP 2396365A2 EP 10741516 A EP10741516 A EP 10741516A EP 10741516 A EP10741516 A EP 10741516A EP 2396365 A2 EP2396365 A2 EP 2396365A2
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EP
European Patent Office
Prior art keywords
copolymer
lysine
biodegradable
nanoplex
pdna
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Withdrawn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
EP10741516A
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German (de)
English (en)
Other versions
EP2396365A4 (fr
Inventor
Malavosklish Bikram
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University of Houston
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University of Houston
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Publication date
Application filed by University of Houston filed Critical University of Houston
Publication of EP2396365A2 publication Critical patent/EP2396365A2/fr
Publication of EP2396365A4 publication Critical patent/EP2396365A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates generally to the fields of copolymer chemistry and gene delivery systems. More specifically, the present invention relates to the design and synthesis of a novel biodegradable polymer, for example, reducible linear L-lysine-modified copolymers (LLC).
  • LLC reducible linear L-lysine-modified copolymers
  • Nonviral gene delivery systems based on cationic polymers for the treatment of both inherited and acquired diseases. This is due to the dissemination of many disease pathways in which the modulation of expressed proteins or genes via gene therapy has the potential of significantly improving the treatment options of patients.
  • Natural cationic polymers such as chitosan (1) and atelocollagen (2) or synthetic cationic polymers including poly(L-lysine) (PLL) (3), poly(ethylenimine) (PEI) (4-6), and dendrimers (4,7) have been widely explored as a means of delivering therapeutic nucleic acids to target cells.
  • biodegradable carriers have been designed to overcome these hurdles as well as to increase transfection efficiencies by facilitating the unpacking of the polymer/pDNA polyplexes after cellular uptake (8).
  • biodegradable polymers such as polyurethanes (PUs) that contain tertiary amines on the backbone and primary, secondary, and tertiary amines on the side chains have been synthesized as nonviral gene delivery vectors (9).
  • glycidol was conjugated into the structure.
  • backbone modifications resulted in higher transfection efficiencies comparable to the well-known non- degradable gene carrier poly(2-(dimethylamino)ethyl methacrylate (PDMAEMA) and lower cytotoxicities.
  • PDMAEMA non- degradable gene carrier poly(2-(dimethylamino)ethyl methacrylate
  • hydrolytically degradable poly(_-amino esters) were developed as cationic polymers for gene transfer, which produced about four times higher gene expression in human embryonic stem cells with minimal toxicity (10).
  • hydrolysable polymers such as poly(ester amines) (11-13), poly(esters) (14), ketalized PEI (15-17), chitosans (18), dendrimers (4,7), and polyphosphazenes (19-21) have been developed as alternatives to non-degradable polymers for gene delivery.
  • Poly(L-lysine) and its derivatives have been shown to be very effective gene delivery carriers with much less cytotoxicity compared with PEI (30-36). However, these carriers also pose a significant problem of prolonged cytotoxicity in clinical applications due to their high molecular weight.
  • the present invention is directed to a biodegradable copolymer or a pharmaceutical composition thereof.
  • the copolymer comprises a reducible polymer linearly modified with lysine.
  • the present invention is directed to a related biodegradable copolymer further comprising a moiety effective to sequester a reactive group conjugated along the copolymer backbone.
  • the present invention is directed to another related biodegradable copolymer further comprising a targeting moiety.
  • the present invention is directed to yet another related biodegradable copolymer further comprising a targeting moiety one or more moieties effective to facilitate endosomal escape.
  • the present invention is directed to a related biodegradable, linear lysine- modified copolymer having the chemical structure:
  • the present invention also is directed to a nanoplex delivery system.
  • the delivery system comprises the biodegradable copolymer described herein; and a cargo molecule complexed thereto.
  • the present invention is directed to a related nanoplex delivery system comprising the biodegradable, linear lysine-modified copolymer having the chemical structure described supra and a nucleic acid or polynucleotide complexed thereto.
  • the present invention is directed further to a method for treating a pathophysiological condition in a subject.
  • the method comprises delivering the cargo molecule comprising the nanoplex delivery system described herein one or more times to a cell associated with the pathophysiological condition in the subject, where transfection of the cell with the cargo molecule elicits a therapeutic response, thereby treating the pathophysiological condition.
  • the present invention is directed to a related method further comprising administering one or more times concurrently or consecutively one or more other therapeutic compounds or pharmaceutical compounds to the subject.
  • the present invention is directed further still to a method for increasing biocompatibility of a polymeric delivery system upon delivery to a subject.
  • the method comprises synthesizing a linear copolymer from a polymer comprising a reducible bond along the polymer backbone and lysine and complexing the synthesized linear lysine-modified copolymer with a biomolecule thereby forming a nanoplex.
  • the nanoplex is delivered to the subject whereupon reduction of the polymer backbone, the biomolecule is released from the nanoplex and the nanoplex degrades into biodegradable lysine subunits, thereby improving biocompatibility of the polymeric delivery system with the subject.
  • the present invention is directed to a related method further comprising conjugating diethylamine along the copolymer backbone to sequester lysine hydroxyl groups.
  • the present invention is directed to another related method further comprising conjugating one or both of a targeting moiety or one or more moieties effective to facilitate endosomal escape to the linear lysine-modified copolymer.
  • Figure 2 is the 1 H NMR spectra of final bioreducible lysine copolymer (LLC).
  • Figure 3 shows an agarose gel electrophoresis of LLCs and PLL with plasmid DNA polyplexes as a function of N/P ratio (Lane 1, naked pDNA; lanes 2-11, LLC/pDNA and PLL/pDNA at N/P ratios of 1: 1, 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 40: 1. 50: 1 and 100: 1).
  • Figures 5A-5B show the Zeta potential ( ⁇ ) of LLC/pCMV-Luc polyplexes as a function of N/P ratio ( Figure 5A).
  • Figure 5B A representative zeta potential profile of the polyplexes prepared from the LLCs with pCMV-luc plasmids at N/P ratio of 25/1 is shown ( Figure 5B).
  • Data represented as mean ⁇ SD, N 3.
  • Figures 6A-6C show the In-vitro transfection efficiency of the polyplexes of pCMV-Luc with LLC polymer in HDFs (Figure 6A), MCF-7s (Figure 6B) and 4TIs (Figure 6C) cells in comparison with same N/P ratio PLL. Data are averages of three parallel experiments.
  • Figures 8A-8D are fluorescence microscopy images of transfected HDF cells with pCMV-EGFP complexed with either branched PEI (Figure 8A), lipofectamine ® ( Figure 8B) and reducible LLCs (Figure 8C) at N/P ratio of 25/1 after 48 hr of transfection.
  • Figures lOA-l ⁇ C show confocal microscopy images of transfected HDF cells with EMA-labeled pCMV-Luc using reducible LLCs at N/P ratio of 25/1 after four (Figure 10A), eight ( Figure 10B) and twelve hours (Figure 10C) post transfection.
  • Figures HA- HB show the agarose gel electrophoresis of final LLC polymer with plasmid DNA polyplexes at different nitrogen/phosphate (N/P) ratios at the conditions of without DTT (Lane 1, naked pDNA; lanes 2-11, LLC/pDNA at N/P ratios of 1: 1, 5: 1, 10: 1, 15: 1,
  • N/P nitrogen/phosphate
  • Figures 12A-12B shows the effect of DTT on the release of EMA-labeled pCMV-Luc from LLC/pDNA polyplexes using fluorescence spectroscopy and gel electrophoresis.
  • Lanes 1-6 in Figure 12A represented free DNA, DNA with 4 mM of DTT, LLCs with DNA, PLL with DNA, LLCs with DNA and 4 mM of DTT and PLL with DNA and 4 mM
  • Figure 13 shows the DNase I protection assay for LLC/pDNA polyplexes at 25/1
  • Lanes 1-5 and 6-10 represent dissociated and reisolated plasmids from naked pDNA and polymer/pDNA polyplexes respectively after exposure to DNase 1 (1) without DTT and (2) with 4 mM DTT for 120 min followed by electrophoresis on a 1 % agarose gel at 80 V for 60 min
  • linear copolymer refers to a polymer derived from two (or more) monomelic species comprising a single main chain.
  • the linear lysine-modified copolymers described herein may comprise, but are not limited to, a plurality of units with lysine-modified N.N'cystamine bisacrylamide (CBA).
  • the linear copolymer may be further modified along the copolymer backbone with moieties suitable to enhance complexation with a nucleic acid, such as DNA, plasmid DNA (pDNA) or a gene and/or with targeting moieties or moieties effective to facilitate endosomal escape.
  • a nucleic acid such as DNA, plasmid DNA (pDNA) or a gene and/or with targeting moieties or moieties effective to facilitate endosomal escape.
  • pDNA plasmid DNA
  • N/P ratio refers to the number of nitrogen residues of the copolymer per DNA phosphates in the complex.
  • the term "subject” refers to any recipient of the reducible linear lysine-modified copolymers and/or the nanoplexes comprising the same.
  • a biodegradable copolymer comprising a reducible polymer linearly modified with lysine.
  • the biodegradable copolymer may comprise a moiety effective to sequester a reactive group conjugated along the copolymer backbone.
  • the reactive group is the lysine hydroxyl group and the sequestering moiety is diethylamine.
  • the biodegradable copolymer may comprise a targeting moiety.
  • the biodegradable copolymer may comprise one or more moieties effective to facilitate endosomal escape.
  • the reducible polymer may comprise a disulfide bond.
  • the reducible polymer may be N,N'cystamine bisacrylamide.
  • the biodegradable copolymer may comprise a linear lysine modified N,N'cystamine bisacrylamide and a diethyleneamine conjugated to the lysine hydroxy group.
  • the biodegradable copolymer may have the following chemical structure where n is 1 to about 10 repeating units. Preferably, n is about 6 to about 8 repeating units
  • the present invention provides a biodegradable, linear lysine-modified copolymer having the chemical structure described supra where n is 1 to about 10 repeating units.
  • biodegradable copolymers may comprise a pharmaceutical composition and a pharmaceutically acceptable carrier.
  • a nanoplex delivery system comprising the biodegradable copolymer, as described supra and a cargo molecule complexed thereto.
  • the cargo molecule may comprise a nucleic acid, a polynucleotide or other biomolecule.
  • Representative examples of the cargo molecule are DNA, plasmid DNA, siRNA, introgens, or antisense nucleotides.
  • the nanoplex may have a nitrogen: phosphate ratio of about 1: 1 to about 50: 1.
  • a nanoplex delivery system comprising the biodegradable, linear lysine-modified copolymer having the chemical structure as described supra and a nucleic acid or polynucleotide complexed thereto.
  • a method for treating a pathophysiological condition in an subject comprising delivering the cargo molecule comprising the nanoplex delivery system, as described supra, one or more times to a cell associated with the pathophysiological condition in the subject, where transfection of the cell with the cargo molecule elicits a therapeutic response, thereby treating the pathophysiological condition. Further to this embodiment the method comprises administering one or more times concurrently or consecutively one or more other therapeutic compounds or pharmaceutical compounds to the subject.
  • a method for increasing biocompatibility of a polymeric delivery system upon delivery to a subject comprising synthesizing a linear copolymer from a polymer comprising a reducible bond along the polymer backbone and lysine; complexing the synthesized linear lysine-modified copolymer with a biomolecule thereby forming a nanoplex; and delivering the nanoplex to the subject, whereupon reduction of the polymer backbone, the biomolecule is released from the nanoplex and the nanoplex degrades into biodegradable lysine subunits, thereby improving biocompatibility of the polymeric delivery system with the subject.
  • the method comprises conjugating diethylamine along the copolymer backbone to sequester lysine hydroxyl groups. Further still the method comprises conjugating one or both of a targeting moiety or one or more moieties effective to facilitate endosomal escape to the linear lysine-modified copolymer.
  • the reducible bond is a disulfide bond.
  • the polymer may be N.N'cystamine bisacrylamide.
  • the biomolecule may be a nucleic acid or a polynucleotide. Representative examples are a DNA, a plasmid DNA, an siRNA, an nitrogen, or an antisense nucleotide.
  • the linear lysine-modified copolymer may have the chemical structure and n repeating units as described supra.
  • the copolymers may comprise a reducible linear L-lysine-modified copolymer (LLC) which comprises a reducible disulfide bond in the polymer backbone.
  • LLC reducible linear L-lysine-modified copolymer
  • a representative unit of the copolymer is shown in Example 1.
  • the LLCs of the present invention may be synthesized using standard and well- known chemical synthetic procedures. A Michael addition reaction is utilized to form a covalent bond between the lysine sidechain amino group and the beta alkenyl carbon of an acrylamide moiety of N.N'cystamine bisacrylamide (CBA) to form the linear copolymer.
  • CBA N.N'cystamine bisacrylamide
  • the copolymer backbone comprises disulfide bonds in main chains, which are totally stable in an extracellular oxidizing environment and are degraded rapidly in an intracellular reducing environment.
  • the lysine-modified copolymer may comprise about 1-10 polymeric repeating units, preferably 6-8 repeating units.
  • the LLCs may be further conjugated to a moiety, such as, but not limited to, diethyleneamine, along the copolymer backbone suitable to incease or improve complexation with a nucleic acid or polynucleotide.
  • diethyleneamine is suitable to sequester the free lysine hydroxyl group.
  • This backbone modification allows a cargo, such as plasmid DNA, other polynucleotides, e.g., siRNA, introgens or antisense nucleotides, or other biomolecules to be complexed efficiently with the cationic copolymer to form stable and small particle sized nanoplexes that enhance transfection efficiencies.
  • the linear lysine-modified copolymers further may comprise a targeting moiety to facilitate delivery of the LLC complexed to a nucleic acid to a cell of interest.
  • Effective targeting moieties are standard and well-known in the art and may include, inter alia, antibodies, receptor ligands and other peptides.
  • the LLCs further may comprise known and standard molecules and/or moieties suitable to facilitate endosomal escape, for example, but not limited to an ionophore.
  • the linear lysine-modified copolymers comprise a delivery vehicle and may form nanoplexes with a suitable nucleic acid or polynucleotide,.
  • the polyplexes may have an N/P ratio from about 1/1 to about 50/1.
  • the polyplexes may be delivered to the cell cytosol using standard and well-known transfection techniques. It is contemplated that the LLCs are effective to deliver a nucleic acid to the cell nucleus.
  • the DNA load is released into the cytoplasm allowing efficient gene or other nucleic acid or polynucleotide delivery.
  • biodegradability upon cleavage of the disulfide bond within the polymer backbone, the broken down subunits, which are lysine monomer units, are completely biodegradable.
  • conjugation of the ethylenediamine component within the polymer backbone with an 80% conjugation efficiency enhances polymer water solubility and increases polymer backbone cationic charge which subsequently increases nucleic acid binding affinity.
  • the biodegradable, reducible LLC DNA nanoplexes may be used to treat a pathophysiological condition in a subject, for example, a cancer or other disease or disorder for which the DNA or polynucleotide complexed with the LLCs would provide a therapeutic benefit upon transfection and expression. It also is contemplated that the LLCs can form nanoplexes with other biomolecules effective to elicit a therapeutic effect upon delivery to one or more cells associated with the pathophysiological condition. As is known in the art the biodegradable LLCs or the nanoplexes may comprise a pharmaceutical composition having a pharmaceutically acceptable carrier. The nanoplexes may be administered one or more times to the subject.
  • the nanoplexes or pharmaceutical compositions may be administered with one or more other therapeutic molecules or pharmaceuticals either concurrently or consecutively. It is well-known in the art to determine an effective dose and dosage schedule depending on the pathophysiological condition and its progression or remission and, inter alia, the age, sex, and health of the subject.
  • N,N_-cystaminebisacrylamide (CBA) is purchased from PolySciences, Inc. (Warrington, PA).
  • Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), trypsin-like enzyme (TrypLE Express), Maxiprep kit, and Dulbecco's phosphate buffered saline (PBS) are purchased from Invitrogen- Gibco (Carlsbad, CA).
  • Luciferase assay system with reporter lysis buffer and RQl RNase-free DNase I enzymes are purchased from Promega (Madison, WI).
  • SlowFade ® Gold antifade reagent, ethidium monoazide and DAPI are purchased from Invitrogen (Carlsbad, CA).
  • PD-IO column is purchased from Pharmacia Biotech (Uppsala, Sweden).
  • Bicinchoninic acid protein assay reagent (BCA) kit is purchased from Pierce (Rockford, IL). All materials and solvents are used as received without further purification.
  • Formazan concentration is determined with a BioTek instruments' ELx800TM system equipped with BioTek's Gen5TM Reader Control and Data Analysis Software (Winooski, VT). Chemluminescence generated from the luciferin protein is assayed using BioTek Instruments Synergy 2 luminometer system equipped with BioTek's Gen5TM Reader Control and Data Analysis Software (Winooski, VT). 1 H NMR spectra are obtained using a General Electric (GE) QE-300 300 MHz, (Boston, MA) and chemical shifts ( ⁇ ) are reported in parts per million (ppm).
  • GE General Electric
  • ⁇ chemical shifts
  • Matrix assisted laser desorption ionization time-of -flight (MALDI-TOF) data are obtained using a Voyager-DE STR Biospectrometry Workstation from Applied Biosystems (Foster City, CA).
  • PDA photodiode array
  • ELSD-LTII Low temperature evaporative light scattering detector
  • pH measurements are determined with a Corning pH meter 340 purchased from Corning Incorporated (Corning, NY).
  • Particle size and zeta potential ( ⁇ ) of the nanoplexes are determined with BI-200SM dynamic light scattering instrument (DLS, Brookhaven Instrument Corporation, Holtsville, NY). Fluorescence microscopy data is obtained using a fluorescence microscope
  • LLC/pCMV-Luc complexes are prepared at a 25/1 N/P ratio with a final pDNA concentration of 0.01 mg/mL. 6 units (6U) RQl RNase- free DNase I is then added to either naked pCMV-Luc (12 ⁇ g) or complexes (12 ⁇ g pDNA) and the samples are incubated at 37 "C.
  • the viscosity (0.8905 mPa s) and refractive index (1.333) of pure water at 25 0 C are used and measurements for particle size are reported as the effective mean diameters.
  • Smoluchowski's equation is used to calculate the zeta potential values from the electrophoretic mobility. Measurements for each sample are repeated three times and reported as mean values ⁇ standard deviations.
  • a gel retardation assay is performed with a plasmid coding for the luciferase gene (pCMV-Luc).
  • the polymer/pDNA complexes are prepared by mixing 1 ⁇ g of the pCMV-Luc plasmid with increasing amounts of synthesized reducible LLC in 5% glucose to form complexes at different N/P ratios.
  • the polymer/pDNA complexes at predetermined N/P ratios of 1/1, 5/1, 10/1, 15/1, 20/1, 25/1, 30/1, 40/1 and 50/1 are then subjected to electrophoresis on a 1 % (w/v) agarose gel containing 0.5 ⁇ g/mL ethidium bromide for 30 min at 120 V in IX tris-acetate-EDTA (TAE) buffer.
  • TAE IX tris-acetate-EDTA
  • the gel is run at 120 V for 30 min and the location of the DNA bands is visualized with a UV transilluminator (254 nm) using Alpha lnnotech FluoChemTM 8900 MultilmageTM Light cabinet operated through AlphaEaseFCTM gel documentation software.
  • DTT reduction of LLC Gel retardation assay using DTT reagent
  • a solution of optimized LLC/pDNA polyplexes at an N/P ratio of 25/1 are prepared as described above and the DTT reagent is added at different concentrations from 0.1 to 4 mM.
  • an N/P ratio of 25/1 is used as it showed maximum DNA condensation.
  • Polyplexes are incubated in Eppendorf tubes at room temperature for 30 min. After incubation, the polyplexes are electrophoresed on a 1% agarose gel containing ethidium bromide with tris- acetate-EDTA (TAE) running buffer at 120 V for 30 min. The location of the DNA bands is visualized with a UV illuminator (254 nm) using a gel documentation system.
  • TAE tris- acetate-EDTA
  • DTT reduction of LLC Fluorescence spectroscopy using DTT reagent To determine whether all of the pDNA was released from the LLC/pDNA polyplexes, the reduction of the disulfide bonds with DTT was monitored with fluorescence microscopy. Briefly, LLCs were complexed with EMA-labeled pCMV-Luc for 60 min at an optimized N/P ratio of 25/1. Afterwards, 4 mM of DTT was added and the fluorescence was monitored for 120 min. The excitation and emission wavelengths were 480 ⁇ 20 nm and 600 ⁇ 35 nm respectively. All of the fluorescence spectroscopy data were normalized against the free pDNA and pDNA with 4 mM DTT fluorescence. Each sample was prepared in triplicates and the data was reported as mean ⁇ standard deviations.
  • HDFs, 4T Is, and MCF-7s cells In vitro transfection efficiencies of LLC/pDNA polyplexes LLC mediated transfection is evaluated in HDFs, 4T Is, and MCF-7s cells by using the reporter plasmid pCMV-Luc.
  • HDFs and MCF-7s are maintained in DMEM containing 10 % FBS, streptomycin (100 ⁇ g/mL) and penicillin (100 units/mL) at 37 °C in a humidified atmosphere with 5 % CO 2 and RPMI media is used for the 4Tl cell line.
  • Cells are seeded in 6- well plates at a density of 5 x 10 5 cells/well for at least 24 hrs prior to transfection.
  • DNA is complexed with the LLC and PLL at predetermined N/P (1, 5, 10, 15, 20, 25, 30, 40 and 50) ratios in phosphate buffer and incubated for 30 min before use.
  • the medium in each well is replaced with fresh serum-free medium.
  • Polyplexes (2 ⁇ g DNA/well) are incubated with the cells for 4 hrs at 37 "C.
  • the media is then replaced with 2 ml of fresh complete medium and the cells are incubated for an additional 44 hrs.
  • the cells are then washed with PBS, treated with 200 ⁇ L cell lysis buffer, and incubated for 15 min. Cellular debris is removed by centrifugation at 8,000 rpm for 2-3 min.
  • the luciferase activity in the cell lysate (25 ⁇ L) is measured using a luciferase assay kit ( 100 ⁇ L luciferase assay buffer) on a BioTek Instruments Synergy 2 chemiluminometer.
  • the relative luminescent unit (RLU) of luciferase expression is normalized against the protein concentration in the cell extracts as measured with a BCA protein assay kit.
  • the LLC/pDNA polyplexes that produced the highest transfection efficiency with the least cytotoxicity in the cell lines tested was designated as the optimized N/P ratio and utilized in subsequent studies. Each sample was prepared in triplicates and the data was reported as mean ⁇ standard deviations.
  • LLC -mediated transfections were evaluated in HDFs cells as described in the previous section with the exception of using complete DMEM media containing 10% fetal bovine serium (FBS) instead of serum-free media. DNA was complexed with the LLCs or PLL control at the N/P ratio that gave the highest transfection efficiency in this cell line for both polymers based on the serum free transfection study (N/P ratio of 50/1 for PLL and N/P ratio of
  • HDF cells were transfected with bPEI and Lipofectamine ® complexed with the pCMV-EGFP plasmids at an optimized N/P ratio of 25/1 (least cytotoxicity as described above) and 10/1 for LLCs and bPEI respectively.
  • the optimized N/P of 10/1 for bPEI was chosen as it yielded the highest transfection efficiency with the least cytotoxicity in HDF cells (data not shown).
  • Lipofectamine ® /pDNA conjugates were prepared as per the manufacturer's instructions. Briefly, HDF cells were cultured on 2 X 2 glass slides placed into the wells of a 6 well plate.
  • polyplexes prepared with pCMV-EGFP were added to the cells, which were incubated for an additional 4 hr.
  • the cells were washed in IX PBS, pH 7.4 and fixed in 2 % freshly prepared formaldehyde for 15 min.
  • the HDF cells expressing EGFP were imaged and counted using an Olympus BX 51 Series fluorescence microscope per total amount of cells.
  • the maximum excitation and emission wavelengths for EGFP detection were 488 ⁇ 20 nm and 509 ⁇ 20 nm respectively.
  • HDF, MCF-7s, and 4TIs cells are seeded in a 6-well plate at a density of 5.0 x 10 5 cells/well and incubated for at least 24 hrs.
  • pCMV-Luc is complexed with the LLC and PLL at predetermined N/P ratios (1, 5, 10, 15, 20, 25, 30, 40 and 50) in 5 % glucose and incubated for 30 min.
  • Polyplexes (2 ⁇ g DNA/well) are incubated with the cells for 4 hrs in serum-free media followed by 20 hrs in complete media.
  • MTT solution 120 ⁇ L, 2 mg/mL in PBS
  • Sample OD 570 Absorbance of the transfected cells.
  • Control OD 570 Absorbance of the untransfected cells.
  • the cells were washed and fixed with 2 % freshly prepared formaldehyde in IX PBS buffer for 15 min at predetermined time intervals of 0, 4, 8, and 12 hr. The cells were then counterstained with a 10 ⁇ g/ml 4,6-diamidino- 2-phenylindole (DAPI) solution to visualize the cells' nuclei.
  • DAPI 4,6-diamidino- 2-phenylindole
  • the labeled cells were then imaged using an Olympus BX 51 confocal laser scanning microscope on an inverted IX 81 frame.
  • the EMA-labeled pDNA was excited using 488 - 541 nm illumination (TRITC range), while the DAPI stain was imaged at 358 nm.
  • HDFs Human dermal fibroblasts
  • MCF-7s human breast adenocarcinoma cells
  • 4TIs metastatic mouse breast cancer cells
  • ATCC American Type Culture Collection
  • MA Mouse adipose stromal cells
  • MA were a generous gift from Professor Ke-He Ruan (University of Houston) and cultured in DMEM-F12 medium supplemented with 10% Newborn Calf Serum (NCF) and maintained as above.
  • a firefly luciferase reporter gene is inserted into a pCI plasmid vector driven by the (CMV) cytomegalovirus immediate-early promoter (Promega, Madison, WI) to yield the pCMV- Luc plasmid, which is amplified in E. coli DH5 ⁇ and isolated by standard Maxiprep kit.
  • the pCMV-Luc and pCMV-EGFP (enhanced green fluorescence protein construct under the control of the CMV promoter) vectors are a generous gift from Professor Sung Wan Kim (University of Utah).
  • the plasmids are amplified via transformation into DH5 ⁇ competent cells and purified using a QIAGEN Endofree Maxi Plasmid Purification kit.
  • pCMV-Luc pDNA is fluorescently labeled with the fluorophore ethidium monoazide (EMA).
  • EMA fluorophore ethidium monoazide
  • To 200 ⁇ g of pCMV-Luc in 2 ml of H 2 O is added 5 ⁇ g of ethidium monoazide.
  • the solution is exposed to UV light of principal wavelength 312 nm for 2 min after incubation for 10 minutes.
  • PD-10 columns are then used to purify the plasmid.
  • CsCl is added to a concentration of 1.1 g/ml to remove intercalated but not covalently bound ethidium and is gently mixed until it dissolved.
  • the data are presented as means of at least three replicates and standard deviations; differences are analyzed using the two-tailed student's t-test and a probability of less than or equal 0.001 is taken as very highly significant (***), between 0.01 and 0.001 is considered highly significant (**) and between 0.05 and 0.01 is taken as significant (*).
  • GraphPad Prism ® version 5 is used to conduct the statistical analysis.
  • the lysine monomers are activated by neutralization of the acid form of the amino acid with sodium hydroxide (NaOH).
  • NaOH sodium hydroxide
  • the activated lysine is then reacted with CBA in a Michael addition reaction.
  • 730.6 mg (4 moles) L-lysine HCl and 1060 mg (4 moles) CBA are weighed and transferred to a 50 mL round bottom flask.
  • 160 mg (4 moles) of NaOH is used to neutralize the HCl present in the L-lysine starting material.
  • 10 mL of methanol/water (MeOH/H 2 O) mixture of 80/20 volume ratio are added to the flask, which is then stirred in an oil bath at 45 "C to dissolve the reagents.
  • the reaction is then stirred in the dark under a nitrogen atmosphere for 2 days (Fig. 1, Intermediate 1).
  • the resulting solution is then dissolved in ultrapure water and the reaction solution is purified via dialysis (MWCO 2000) against water for 2 days to remove low molecular weight polymeric biproducts and remaining traces of the starting materials.
  • the dialyzed solution is then transferred to a sterile conical tube and lyophilized for 2 days.
  • the resulting polymers are characterized with proton nuclear magnetic resonance ( 1 H NMR), gel permeation chromatography (GPC), and matrix assisted laser desorption ionization-time of flight (MALDI-TOF).
  • the molecular weights of the various polymers are determined with size exclusion chromatography (SEC) and mass spectrometry with MALDI ionization.
  • SEC size exclusion chromatography
  • ELSD evaporative light scattering detector
  • a polyethylene glycol standard kit is used to construct the calibration curves.
  • the matrix for MALDI-TOF is saturated alpha-cyano-4- hydroxycinnamic acid dissolved in 50/50 acetonitrile/water with 0.1% TFA with a polymer concentration of 1 mg/ml. An equal volume (8 ⁇ L) of the polymer and the matrix prepared are spotted and air-dried on the plate.
  • N-boc ethylenediamine is selectively conjugated along the polymer backbone.
  • 5 different copolymers are prepared by varying the amounts of EDC and N-boc. Briefly, 100 mg each of reducible LLC (0.033 mmoles) are weighed and transferred to five different round bottom flasks to which 6 ml of deionized water is added to each flask to fully dissolve the polymers with stirring.
  • the acid-labile N-boc amine protection group present on the terminal end of the conjugated ethylenediamine is removed with TFA/H 2 O mixture (75/25 v/v). Briefly, 100 mg (0.033 mmoles) of reducible LLC is added to a round bottom flask to which 10 mL of deionized H 2 O is added. Afterwards, 30 ml of TFA is added. The dissolved copolymers are then stirred for 30 min at room temperature (Fig. 1, final product). The final polymers are then purified by dialysis (MWCO 2000) against deionized water for 2 days to remove the free N-boc groups and TFA.
  • MWCO 2000 dialysis
  • the dialyzed polymers are then transferred to a sterile conical tube and dried on a lyophilizer for 2 days.
  • the resulting polymers are characterized with 1 H NMR, GPC, and MALDI-TOF.
  • the amount of primary amines in intermediate 1, intermediate 2, and the final product is quantified with a ninhydrin assay at 570 nm.
  • the assay is conducted at 100 0 C for 15 min. Glycine is used to construct the standard curves.
  • the number of conjugated ethylenediamine per polymer chain for the final polymer using 5, 10, 15, 20 and 25 excess molar ratio of EDC and N-boc-ethylenediamine is 1.95, 3.33, 3.84, 7.51 and 2.3 respectively (Table 1).
  • the reducible LLC copolymers with 20 excess molar ratio of EDC and N-boc-ethylenediamine showed the maximum amount of ethylenediamine conjugation as determined with 1 H NMR and it is then designated the optimum reducible LLC and selected for use in subsequent assays.
  • the molecular weight of the synthesized LLC copolymers is determined using SEC and MALDI-TOF.
  • the number average molecular weight (Mn) of intermediate 1, intermediate 2 and final product as determined by SEC are 3489, 3118, and 3601 Da with a polydispersity index (PDI) of 1.08, 1.10 and 1.06 respectively (Table 2).
  • the number average molecular weight (Mn) of the LLC copolymers as determined by MALDI-TOF supported the calculated SEC data with molecular weights of 3538, 31 18 and 3468 Da lor intermediate 1, intermediate 2 and the final product respectively.
  • the PDI of each is found to be 1.097, 1.10 and 1.09 respectively (Table 2).
  • the reducible LLCs with 94% conjugated EDA was then used in subsequent assays as the final LLCs. PLL was able to effectively condense pDNA from an N/P ratio of 1/1 as evident by the absence of free pDNA from wells of the gel (Fig.
  • LLC/pDNA polyplexes are prepared through diluting 1.74 mg/mL aqueous solution of plasmid DNA
  • LLC coplymer with pCMV-Luc is determined by dynamic light scattering.
  • the average particle size of LLC/pDNA polyplexes of N/P ratios 1/1, 5/1, 10/1, 15/1, 20/1 and 25/1 are found to be 231.9 ⁇ 22.6, 180.6 ⁇ 13.3, 158.3 ⁇ 12.9, 150.6 ⁇ 21.3, 151.6 ⁇ 12.5, 141.7 ⁇ 11.5, 143.9 ⁇ 10.1, 130.3 ⁇ 1 1.6 and 100.3 ⁇ 24.4 nm respectively (Fig. 4).
  • the PLL/pDNA polyplexes of N/P ratios 1/1, 5/1, 10/1, 15/1, 20/1 and 25/1 had average particle sizes of 258.7 ⁇ 41.0, 182.2 ⁇ 15.5, 176.6 ⁇ 23.2, 148.2 ⁇ 19.3, 121.3 ⁇ 14.6, 125.9 ⁇ 11.5, 112.9 ⁇ 8.1, 1 17.6 ⁇ 7.3 and 112.7 ⁇ 38.1 nm respectively (Fig. 4).
  • the particle size distribution of the polyplexes prepared from the LLC with pCMV-luc is found to be homogenous and unimodal as represented in the LLC polyplexes of N/P ratio of 25/1 (Fig. 4B).
  • the zeta potential of LLC/pDNA polyplexes of N/P ratios 1/1, 5/1, 10/1, 15/1, 20/1 and 25/1 are -4.1 ⁇ 4.4, -1.0 ⁇ 2.8, 0.9 ⁇ 2.0, 5.8 ⁇ 2.5, 5.0 ⁇ 1.2, 8.4 ⁇ 2.1, 8.3 ⁇ 2.5, 15.0 ⁇ 2.7, 16.6 ⁇ 2.8 mV respectively (Figs. 5A-5B).
  • the zeta potential of the control PLL/pDNA polyplexes of N/P ratios 1/1, 5/1, 10/1, 15/1, 20/1 and 25/1 are -3.0 ⁇ 2.2, 2.1 ⁇ 2.6, 0.5 ⁇ 4.7, 4.9 ⁇ 2.0, 5.9 ⁇ 2.3, 6.9 ⁇ 1.0, 13.0 ⁇ 2.0, 10.2 ⁇ 3.4, 14.0 ⁇ 4.4 mV respectively with excellent fit to the theoretical polynomial values (Fig. 4B).
  • the particle size distribution of the LLC/pDNA polyplexes showed that N/P ratios greater than 5/1 can efficiently condense pDNA into nanoparticles with effective diameters of less than 150 nm (Figs. 4A-4B).
  • the LLC copolymers produced slightly larger polyplexes than PLL, which could be attributed to the lower molecular weight chains as compared to the high molecular weight PLL chains.
  • the majority of the polyplexes remained constant at ⁇ 150 nm at various N/P ratios in comparison to PLL, which showed an average particle size of ⁇ 125 nm from an N/P ratio of 20/1.
  • the particle size distribution of the LLC/pDNA polyplexes was found to be homogenous (Fig.
  • the zeta potential of the LLC/pDNA polyplexes ranged from ⁇ -4 ⁇ 4.4 mV for N/P ratio of 1/1 to a maximum surface charge of ⁇ 17 ⁇ 2.85 mV for N/P ratio of 50/1 (Figs. 5A-5B) and the zeta potential of the PLL/pDNA polyplexes ranged from ⁇ -3 ⁇ 2.21 for N/P ratio of 1/1 to a maximum surface charge of ⁇ 14 ⁇ 4.3 mV for N/P ratio of 50/1 (Figs. 5A-5B).
  • the luciferase assay showed that LLC/pDNA polyplexes of N/P ratio 40/1 resulted in a 5.5-fold higher transfection efficiency in comparison to the optimal PLL control at an N/P ratio of 50/1 in HDF cells (p ⁇ 0.05) (Fig. 6A).
  • the LLC/pDNA polyplexes of N/P ratio 20/1 showed a 3-fold higher gene transfection efficiency than the optimal PLL control at an N/P ratio of 20/1 in MCF-7 cells (p ⁇ 0.05) (Fig. 6B).
  • the LLC/pDNA polyplexes at an N/P ratio of 50/1 resulted in a 4.4-fold higher gene transfection efficiency than the optimal PLL control at an N/P ratio of 50/1 (p ⁇ 0.05) (Fig. 6C).
  • These higher transfection efficiencies of the LLCs as compared to the PLL control could be attributed to the more efficient release of the pDNA from the LLC polyplexes into the cytosol of the cells as a result of reduction of the disulfide bonds along the polymer backbone.
  • the higher transfection efficiencies of the LLCs could also be due to the higher cell viabilities of the cells treated with the LLC polyplexes as compared with the PLL control (Fig. 8A-8C, discussed below).
  • the LLC/pDNA polyplexes at an N/P ratio of 25/1 was selected as the optimum complexes for use in subsequent studies since these produced high transfection efficiencies in both HDF and MCF-7 cells with the least cytotoxicity in all cell lines.
  • the MTT assay showed that LLC polyplexes produced lower cytotoxicity compared to PLL at N/P ratios up to 50/1. LLC polyplexes at 20/1 N/P ratio maintained the highest cell viability in all three cell lines in comparison to PLL at the same N/P ratio. PLL at 20/1 N/P ratio showed a decrease in cell viability to 82 %, 77 % and 81 % for HDF, MCF-7 and MA cells respectively with ⁇ 100 % cell viability for LLC/pDNA polyplexes in the three cell lines (Fig. 9A-9C).
  • LLC polyplexes showed ⁇ 100 % cell viability for the three cell lines as compared to PLL at the same N/P ratio, which produced an 81 %, 75 % and 60 % reduction in cell viability in for HDF, MCF-7 and MA cells respectively. Moreover, a cell viability of -100 % was maintained for LLC/pDNA polyplexes as compared to a reduced 75 % cell viability for PLL from an N/P ratio of 1/1 to 30/1 for MCF- 7 cells (p ⁇ 0.05). Finally, LLC/pDNA polyplexes showed negligible cytotoxicity in MA cells at N/P ratios from 1/1 to 25/1 compared to PLL (p ⁇ 0.05).
  • the confocal microscopy data showed red punctate staining indicative of the presence of EMA-labeled pDNA in the cytosol, aggregated around the nucleus, and possibly within the nucleus of DAPI-stained cells transfected with LLC/pDNA polyplexes after 4, 8, and 12 hrs (Figs. lOA-lOC).
  • the delivery of the pDNA in and around the nucleus could reflect the efficient reduction of the disulfide bonds of the copolymer chains and hence efficient release of pDNA that was trafficked to the nucleus.
  • the mechanism of pDNA release from the LLC/pDNA polyplexes was investigated in two reduction assays with the optimized 25/1 N/P ratio polyplexes.
  • a gel retardation assay was used to verify pDNA release from the polyplexes as a function of DTT concentration. This data showed that the reduction of the disulfide bonds occurred from a low concentration of ⁇ 0.5 mM, which was apparent from the increase in fluorescence in the wells but that higher concentrations of ⁇ 3 mM DTT were required to completely reduce the disulfide bonds so as to release the pDNA from the polyplexes (Fig. 1 IA, lanes 10 - 12).
  • the DNase I protection assay showed that the LLCs were able to form tight polyplexes, which completely protected the pDNA from degradation by the endonucleases for up to 2 hr (Fig. 13, lane 10).
  • the naked pDNA was fully degraded by DNase I in 20 min, as shown in Fig. 11, lane 2, and in 40 min, all the naked pDNA was completely degraded. This is opposed to the pDNA that was released from the polyplexes only after the addition of DTT for 60 min. LLC polyplexes protected the pDNA at all time points from endonuclease digestion as shown by the intact pDNA bands on the stained agarose gel (Fig. 13, lanes 6-10).

Abstract

La présente invention concerne des copolymères biodégradables et des systèmes d'administration par nanoplexes comportant de tels copolymères et une molécule cargo, tel qu'un acide nucléique, un polynucléotide ou autre molécule. Les copolymères biodégradables peuvent comporter un polymère réductible modifié linéairement par la lysine, par exemple un N,N'-cystamine bisacrylamide linéaire modifié par la lysine. Les copolymères biodégradables peuvent également être conjugués à une fraction séquestrante, telle que la diéthylamine. Les copolymères biodégradables peuvent aussi comporter un ou deux parmi une fraction de ciblage et une ou des fractions pour faciliter la fuite endosomale. L'invention concerne également des procédés permettant le traitement d'une condition pathophysiologique et l'amélioration de la biocompatibilité d'un système d'administration polymérique lors de l'administration à un sujet au moyen des copolymères biodégradables et des nanoplexes.
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AHN C-H ET AL: "Synthesis of biodegradable multi-block copolymers of poly(l-lysine) and poly(ethylene glycol) as a non-viral gene carrier", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 97, no. 3, 7 July 2004 (2004-07-07), pages 567-574, XP004519519, ISSN: 0168-3659, DOI: 10.1016/J.JCONREL.2004.04.002 *
MALAVOSKLISH BIKRAM ET AL: "-lysine)- g -histidine Multiblock Copolymers for Nonviral Gene Delivery", MACROMOLECULES, vol. 37, no. 5, 1 March 2004 (2004-03-01), pages 1903-1916, XP055079715, ISSN: 0024-9297, DOI: 10.1021/ma035650c *
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