Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • 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
  • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome

Definitions

• lipid complexes are currently used as delivery vehicles for a number of molecules where sustained release or target release to specific biological sites is desired.
• nucleic acids charged nucleic acid-lipid complexes are utilized to enhance transfection efficiencies in somatic gene transfer by facilitating the attachment of nucleic acids to the targeted cells.
• Success in somatic gene therapy depends on the efficient transfer and expression of extracellular DNA to the nucleus of eucaryotic cells, with the aim of replacing a defective or adding a missing gene (1).
• Viral-based carriers of DNA are presently the most common method of gene delivery, but there has been a tremendous activity in developing synthetic nonviral vectors.
• cationic liposomes in which the overall positive charge of the cationic liposome-DNA (CL-DNA) complex enhances transfection by attaching to anionic animal cells, have shown gene expression in vivo in targeted organs, and human clinical protocols are ongoing (2-4).
• Cationic liposome transfer vectors exhibit low toxicity, nonimmunogenicity, and ease of production, but their mechanism of action remains largely unknown with transfection efficiencies varying by up to a factor of 100 in different cell lines (2-6).
• Feigner et al. (3) originally proposed a "bead-on-string" structure of the CL-DNA complexes picturing the DNA strand decorated with distinctly attached liposomes.
• Electron microscopy (EM) studies have reported on a variety of structures including string-like structures and indications of fusion of liposomes in metal-shadowing EM (13), oligolamellar structures in cryo-TEM (14), and tube-like images possibly depicting lipid bilayer-covered DNA observed in freeze-fracture EM (15).
• the invention provides novel compositions involving macromolecule-lipid complexes and methods for making them. These compositions and methods of the invention are significant improvements in the field of macromolecule-lipid complex synthesis, macromolecule targeting and delivery to various biological systems.
• the present invention provides methods for making macromolecule-lipid complexes and methods for controlling components of the macromolecule-lipid complexes such as the membrane thickness and intermolecular spacing of the complex constituents.
• the method comprises mixing a lipid combination (e.g., a neutral lipid and a charged lipid) in a sufficient amount with a macromolecule so as to form a complex with specific geometric and charge qualities.
• a lipid combination e.g., a neutral lipid and a charged lipid
• the relative amounts of (1) the charged and neutral lipids, (2) the weight amount and/or the macromolecule and (3) the assembly solution conditions distinct complexes can be generated having desired isoelectric point or charged states.
• an extremely versatile molecular targeting and delivery system can be developed for a variety of applications.
• the invention has applications in the numerous methods which utilize lipids and various macromolecules such as gene therapy, nucleic acid based vaccine development and peptide and protein delivery.
• Figure 1(A) is a series of high resolution differential interference contrast microscopy images of cationic liposome-DNA complexes showing the formation of distinct condensed globules in mixtures of different lipid to DNA weight ratios.
• the scale bar is lO ⁇ m.
• Figure 1(B) is a plot of the average size of the lipid-DNA complexes measured by dynamic light scattering.
• Figure 2(A) is a series of small-angle x-ray scattering scans in water as a function of different lipid to DNA weight ratio (L/D). (Inset is under extreme dilute conditions).
• Figure 2(B) is plot of the spacing d and doNA as a function of L D.
• Figure 2(C) is a series of small-angle x-ray scattering scans of the lamellar L ⁇ phase of DOPC/DOTAP water mixtures done at lower resolution (rotating anode x-ray generator).
• Figure 3(A) is a schematic picture of the local arrangement in the interior of lipid-DNA complexes.
• Figure 3(B) is a micrograph of the DNA-lipid condensates under bright light.
• Figure 3(C) is a micrograph of DNA-lipid condensates under crossed polarizers.
• Figure 4(A) is a series of small-angle x-ray scattering scans of CL-DNA complexes at approximately the isoelectric point.
• Figure 4(B) is d D A and d from figure 4(A) plotted as a function of L/D.
• Figure 4(C) the average domain size of the ID lattice of DNA chains derived from the width of the DNA peaks shown in 4(B).
• Figure 5a is a schematic representation showing the macromolecule-lipid complex formation from the negatively charged DNA and positively charged liposomes. Schematics of lamellar and inverted hexagonal complex
• Figure 5b is the powder X-ray diffraction patterns of two distinct ( and ) liquid- crystalline phases of CL-DNA complexes.
• Figures 6a-d are video-microscopy images of CL-DNA complexes in H ⁇ 7 and t a .
• Figure 7 are two SAXS scans obtained following the transformation from £ a to H ⁇ phase in the case when the macromolecule is DNA (Left) or a polynucleotide T (right).
• Figure 8 shows the variation of structural parameters in l a and H ; complexes with the three different types of polyelectrolytes and correlative schematic diagrams showing the structure of a unit cell in the three H restrictive complexes (with DNA, Poly-T, or PGA as the macromolecule).
• Figure 9 is a schematic of DNA-lipid complex oriented in microchannels with applications in nanolithograph and separations (or in oriented multilayers).
• lipid means any surfactant both biologically and non- biologically derived.
• lipid combination means any mixture of two or more lipids.
• sufficient amount means a concentration of a given component that is determined to be adequate to produce the desired effect or characteristic.
• complex means a substance composed of two or more molecules, components, or parts.
• isoelectric point state means the set of conditions under which the electric charge of the complex is approximately zero.
• negative state means the set of conditions under which the electric charge of the complex has a net negative charge.
• positive state means the set of conditions under which the electric charge of the complex has a net positive charge.
• charged state means the set of conditions under which the electric charge of the complex has some net charge or zero charge.
• the term "the macromolecule interaxial distance (di i)" means the perpendicular distance between the cylinder axis of neighboring macromolecules or the average distance between macromolecules.
• membrane thickness of the lipid combination means the thickness of a bilayer of lipid molecule made up of a particular lipid combination.
• macromolecule area (A M ) means the cross section area of the macromolecule.
• micromolecule density (P M ) means the density of the macromolecule.
• lipid density (P L ) means the density of the lipid combination.
• inverted hexagonal complex phase means the phase wherein the lipid combination forms a monolayer around the macromolecule (i.e., with lipid tails pointing outward); thereby creating a lipid monolayer macromolecule tube which then assembles into a hexagonal lattice.
• regular hexagonal complex phase means the phase wherein the lipid combination assembles into a cylindrical rod (i.e. with lipid tails pointing inward) and macromolecule attached to the outer surface of the rod; thereby creating cylindrical rods with attached macromolecules which then assemble in a hexagonal lattice.
• the invention provides methods for regulating the structure of a charged macromolecule- lipid complex having a selected characteristic or multiple characteristics. These characteristics include interaxial distance (d M ), membrane thickness of the lipid combination ( ⁇ m ), macromolecule area (A M ), macromolecule density (P M ), lipid density (P L ). and the ratio (L/D) between the weight of the lipid combination (L) and the weight of the macromolecule (D).
• d M interaxial distance
• ⁇ m membrane thickness of the lipid combination
• a M macromolecule area
• P M macromolecule density
• P L lipid density
• the benefits of being able to precisely control the micromolecular structure of macromolecule-lipid complexes is that it will be possible to tailor make specific structures which have defined chemical and biological activities. For example specific structural attributes of cationic lipid-DNA structures are known to impact transfection efficiencies in different biological systems. By being able to manipulate these structural attributes, the chance of success in somatic gene therapy, which depends on the efficient transfer and expression of extracellular DNA to the nucle
• the complex comprises a macromolecule and lipid combination.
• both the macromolecule and lipid combination are charged.
• the charge of the lipid combination is typically opposite of the charge of the macromolecule.
• distinct complexes can be generated having selected isoelectric point or charged states.
• the lipid combination and the macromolecule can be associated so as to form a complex in an isoelectric point state,
• the lipid combination and the macromolecule can be associated so as to form a complex in a positively charged state.
• the lipid combination and the macromolecule can be associated so as to form a complex in a negatively charged state.
• a lipid combination comprises a neutral lipid and a charged lipid.
• the ratio of the neutral lipid component relative to the charged lipid component can be 70/30, 50/50, 0/100, or 10/90. It clear that in the embodiment, wherein the ratio of the neutral lipid component relative to the charged lipid component is 0/100, a lipid combination is not used but only a single lipid component is used.
• suitable macromolecules include nucleic acid molecules, peptides, proteins, polysaccharides, combinations of a protein and carbohydrate moiety and a synthetic macromolecule of non-biological origin, e.g., doped polyacetylene macromolecules (J.G.S. Cowie "Polymers Chemistry and Physics of Modern Materials", Chapter 7, (Blackie Academic & Professional Press) (1993)).
• neutral lipids include but are not limited to: dioleoyl phosphatidyl cholin, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dicaproyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dioctanoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dicapryl- sn-glycero-3-phosphoethanolamine, l,2-dilauroyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dipentadecanoyl-sn-glycero-3- phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine, l,2-dipalmit
• Suitable charged lipids include, but are not limited to, l,2-diacyl-3- trimethylammonium-propane, 1 ,2-dimyristoyl-3-trimethylammonium-propane, 1 ,2- dipalmitoyl-3-trimethylammonium-propane, l,2-distearoyl-3-trimethylammonium- propane, l,2-diacyl-3-dimethylammonium-propane, l,2-dimyristoyl-3- dimethylammonium-propane, l,2-dipalmitoyl-3-dimethylammonium-propane, 1,2- distearoyl-3-dimethylammonium-propane, and 1 ,2-dioleoyl-3-dimethylammonium- propane.
• the nucleic acid molecule can be single stranded, double stranded, triple stranded or quadruple stranded.
• the nucleic acid molecule can be DNA or RNA.
• the DNA or RNA can be naturally occurring or recombinantly-made. Alternatively, it can be a synthetic polynucleotide.
• the polynucleotides include nucleic acid molecules having non-phosphate backbones which improve binding.
• the macromolecule may be linear, circular, nicked circular, or supercoiled.
• the method comprises selecting a selected characteristic or characteristics described above and modulating one or more of the non- selected characteristics from the group so as to regulate the structure of the macromolecule-lipid complex having the selected characteristic.
• PM density of macromolecule (g/cc)
• PL densities of membrane, d m the membrane thickness, and A the macromolecule area.
• the method comprises modulating any of the characteristics associated with the charged macromolecule-lipid complex as described above so as to regulate the structure of the macromolecule-lipid complex having the selected characteristic.
• the method further comprises determining amounts of the macromolecule and the lipid combination so selected which would be sufficient to achieve the selected characteristic or characteristics thereby regulating the structure of the complex. In one embodiment this can be accomplished by selecting a selected characteristic or multiple characteristics to be achieved. These characteristics are macromolecule interaxial distance (d M ), membrane thickness of the lipid combination ( ⁇ m ), macromolecule area (A M ), macromolecule density (PM), lipid density (P L ), and the ratio (L/D) between the weight of t O uie n iu iuiii ⁇ iii ⁇ u ⁇ n ⁇ L,) ana tne weignt ot the macromolecule ). ⁇ ucn mc characteristics not selected can be modulated so as to achieve the selected characteristic. After determining the proper amounts, the method provides mixing the macromolecule with the lipid combination in the amount so determined.
• the method provides selecting a charged macromolecule and lipid combination, wherein the charge of the lipid combination is opposite of the charge of the macromolecule.
• the interaxial distance is in a range between 24.5 and 60 angstroms. In another example, the interaxial distance is about 60 angstroms.
• d M (L/D) (AMPM)/(5 Rand,PL)-
• the macromolecule-lipid complex can be a multilamellar structure wherein the lipid combination forms alternating lipid bilayers and macromolecule monolayers.
• the macromolecule-lipid complex can form either an inverted hexagonal complex phase or a regular hexagonal complex phase.
• the complex whether part of a multilamellar or hexagonal structure, comprises macromolecules associated with the lipid in an arrangement that can be regulated and controlled in accordance with the method of the invention.
• the lipid combination and the macromolecule are associated so as to form a complex in an isoelectric point state and the complex has macromolecules exhibiting interaxial spacing of greater than 24.5 angstroms.
• the resulting complex can have a charge of about zero.
• the lipid and the macromolecule is associated so as to form a complex in an isoelectric point state, wherein the amount of the neutral lipid component relative to the charged lipid component ranges from 2 to 95 percent.
• the resulting complex can have a charge of about zero.
• the lipid and the macromolecule can associate so as to form a complex in a charged state, wherein the amount of the neutral lipid component relative to the charged lipid component ranges from 55 to 95 percent.
• the resulting complex can have a net charge.
• the lipid combination can form a bilayer membrane to which charged macromolecules are associated, and wherein the relative amounts of the lipid components generate the lipid bilayer membrane having a thickness of between 25 and 70 angstroms.
• the lipid combination can form a bilayer membrane to which charged macromolecules are associated and wherein the relative amounts of the lipid components generate the lipid bilayer membrane having a thickness of between 41 and 60 angstroms.
• the lipid combination can form a bilayer membrane to which charged macromolecules are associated, and wherein the relative amounts of the lipid components generate the lipid bilayer membrane having a thickness of between 32 and 48 angstroms.
• the lipid combination can form a monolayer membrane to which charged macromolecules are associated, and wherein the relative amounts of the lipid components generates the lipid monolayer membrane having a thickness of between 12 and 40 angstroms.
• the resulting complex can form a monolayer (also referred to herein as being in a hexagonal phase, e.g. inverted hexagonal or regular hexagonal).
• the lipid combination can form a monolayer membrane to which charged macromolecules are associated and wherein the relative amounts of the lipid components generate the lipid monolayer membrane having a thickness of between 15 and 35 angstroms.
• the lipid combination can form a monolayer membrane to which charged macromolecules are associated, wherein the relative amounts of the lipid components generate the lipid monolayer membrane having a thickness of between 16 and 30 angstroms.
• the invention further provides a macromolecule-lipid complex produced by the methods of the invention described above.
• the resulting macromolecule-lipid complex comprises a lipid combination having a charged lipid component and a neutral lipid component; and a charged macromolecule.
• the charge of the lipid combination being opposite of the charge of the macromolecule.
• the lipid combination macromolecule associate thereby forming a complex in an isoelectric point state. In this state the lipid combination forms a bilayer membrane to which the charged macromolecule is associated and the relative amounts of the neutral lipid component relative to the charged lipid component generates the lipid bilayer membrane having a thickness of between 25 and 75 angstcpms.
• the lipids form a bilayer membrane to which the macromolecule is associated, wherein the relative amounts of the lipid components generate the lipid bilayer membrane having a thickness of between 25 and 75 angstroms; and the conformation of the complex has macromolecule exhibiting interaxial spacing of a range between 50 and 75 angstroms.
• the invention further provides a process for generating formulations which form the basis for the processing of templates and for producing molecular sieves with precise control over pore size.
• the invention provides a process for creating a pattern on a surface using complexes having regulated structures made using the methods described above.
• the invention provides a process for creating a material having selected properties such as optical, mechanical, electronic, optoelectronic, or catalytic characteristics not previously realized from components of the material.
• This process comprises applying a macromolecule-lipid complex to a surface.
• the complex must have a regulated structure created by the methods of the invention.
• the process further provides applying molecules which make up the material onto the complex, wherein the molecules self-assemble based on its interactions with the complex.
• the complex is then removed from the surface thereby creating the material having a selected property.
• the complex can be in a multilamellar, regular hexagonal, or inverted hexagonal phase.
• the resulting material can function as a molecular sieve having precise pore size.
• the invention further provides a molecular sieve produced by the process above.
• the present invention provides nucleic acid-lipid complexes comprising a charged lipid combination and a charged nucleic acid molecule.
• the charge of the lipid combination is opposite of the charge of the nucleic acid molecule.
• the resulting complex has a desired isoelectric point state and nucleic acids exhibiting interaxial spacing of greater than 24.5 angstroms.
• the interaxial spacing range is about between 24.5 and 60 angstroms.
• the interaxial spacing is about 60 angstroms.
• the conformation of the resulting complex can be a multilamellar structure with alternating lipid bilayers and nucleic acid monolayers.
• nucleic acid molecules include, but are not limited to, deoxyribonucleic acid (DNA), ribonucleic acid (RNA).
• the macromolecules may be linear, circular, nicked circular or supercoiled.
• the nucleic acid molecules can have phosphate backbones but not necessarily so. Alternatively, nucleic acid molecules having non-phosphate backbones which improve binding are also encompassed within this invention.
• the complex comprises a charged lipid combination; and a charged nucleic acid molecule.
• the charge of the lipid combination can be opposite of the charse of the nucleic acid molecule.
• the lipid and the nucleic acid molecule are associated so as to form a complex in an isoelectric point state. In this state, the relative amounts of the lipid components generates the lipid bilayer membrane having a thickness of between 25 and 75 angstroms. Additionally, the conformation of the complex has nucleic acids exhibiting interaxial spacing of a range between 50 and 75 angstroms.
• the present invention further provides macromolecule-lipid complexes comprising a charged lipid combination; and a charged macromolecule.
• suitable macromolecules include, but are not limited to, nucleic acid molecules such as single or double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or hybrids thereof, or modified analogs thereof of varying lengths.
• the macromolecule can be a peptide, a protein (or modified analogs thereof).
• the macromolecule may be a drug such as a chemotherapeutic agent or a modified analog thereof.
• the charge of the lipid combination is opposite of the charge of the nucleic acid molecule. Also, the lipid and the nucleic acid molecule are associated so as to form a complex in an isoelectric point state.
• the lipid combination can have a charge lipid component and a neutral lipid component.
• the amount of the neutral lipid component relative to the charged lipid component can range from 2 to 95 percent.
• the amount of the neutral lipid component relative to the charged lipid component ranges from 55 to 95 percent.
• the ratio of the neutral lipid component relative to the charged lipid component can be 70/30.
• Suitable lipids include, but are not limited to, dioleoyl phophatidyl choline or dioleoyl phophatidyl ethanolamine and dioleoyl triethylammonium propane combination.
• the lipid combination can be a charged lipid combination and the macromolecule can be a charged macromolecule.
• the lipids form a bilayer membrane in the complex to which the charged macromolecule i ⁇ - can be associated.
• the charge of the lipid combination can be opposite of the charge of the nucleic acid molecule.
• the lipid and the nucleic acid molecule are associated so as to form a complex in an isoelectric point state. Additionally, the relative amounts of the lipid components generates the lipid bilayer membrane having a thickness of between 25 and 75 angstroms.
• the lipid and the nucleic acid molecule are associated so as to form a complex in a positively charged state, wherein the lipids form a bilayer membrane to which charged macromolecule is associated, and the relative amounts of the lipid components generates the lipid bilayer membrane having a thickness of between 41 and 75 angstroms.
• the lipid and the nucleic acid molecule are associated so as to form a complex in a negatively charged state, wherein the lipids form a bilayer membrane to which charged macromolecule is associated, and the relative amounts of the lipid components generates the lipid bilayer membrane having a thickness of between 32 and 75 angstroms.
• the lipid can be dioleoyl phophatidyl cholin or dioleoyl phophatidyl ethanolamine and dioleoyl triethylammonium propane.
• the charge of the lipid combination in the complex can be opposite of the charge of the nucleic acid molecule.
• the dioleoyl phophatidyl cholin or dioleoyl phophatidyl ethanolamine and dioleoyl triethylammonium propane form a bilayer membrane to which the charged macromolecule is associated in an isoelectric point state, wherein the relative amounts of dioleoyl phophatidyl cholin or dioleoyl phophatidyl ethanolamine lipids relative to the dioleoyl triethylammonium propane generates the lipid bilayer membrane having a thickness of between 25 and 75 angstroms.
• the amount of the neutral lipid component relative to the charged lipid component ranges from 0 to 95 percent and whose charge is approximately zero.
• the amount of the neutral lipid component relative to the charged lipid component ranges from 55 to
• complexes may be relatively simple or may consist of a highly ordered structure.
• conformation of such a complex can include a multilamellar structure with alternating lipid bilayers and nucleic acid monolayers.
• the invention further provides formulations which form the basis for the processing of templates and for producing molecular sieves with precise control over pore size.
• Cationic liposomes complexed with DNA are promising synthetically based nonviral carriers of DNA vectors for gene therapy.
• the solution structure of CL-DNA complexes was probed on length scales from subnanometer to micrometer by synchrotron x-ray diffraction and optical microscopy.
• the addition of either linear ⁇ -phage or plasmid DNA to CLs resulted in an unexpected topological transition from liposomes to optically birefringent liquid crystalline condensed globules.
• X-ray diffraction of the globules reveals a novel multilamellar structure with alternating lipid bilayer and DNA monolayers.
• ⁇ -DNA chains form a one-dimensional lattice with distinct interhelical packing states.
• the ⁇ -DNA interaxial spacing expands between 24.5 and 60 angstroms upon lipid dilution and is indicative of a long-range electrostatic-induced repulsion possibly enhanced by chain undulations.
• the CLs consisted of binary mixtures of lipids which contained either DOPC (dioleoyl phosphatidyl cholin) or DOPE (dioleoyl phosphatidyl ethanolamine) as the neutral co-lipid and DOTAP (dioleoyl trimethylammonium propane) as the cationic lipid.
• DOPC dioleoyl phosphatidyl cholin
• DOPE dioleoyl phosphatidyl ethanolamine
• DOTAP dioleoyl trimethylammonium propane
• a mixture of DOPE/DOT AP (1:1, wt:wt) was prepared in a 20 mg/ml chloroform stock solution. 500 ml was dried under nitrogen in a narrow glass beaker and desiccated under vacuum for 6 hours. After addition of 2.5 ml Millipore water and 2 hx incubation at 40°C the vesicle suspension was sonicated by clarity for 10 minutes. The resulting solution of liposomes, 25 mg/ml was filtered through 0.2 ⁇ m Nucleopore filters. For optical measurements the concentration of SUV used was between 0.1 mg/ml and 0.5 mg/ml. All lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama).
• the DOTAP/DOPC and DOTAP/DOPE CLs had a size distribution ranging between 0.02 to 0.1 ⁇ m in diameter, with a peak around 0.07 ⁇ m (18).
• Escherichia coli DNA and pBR322 plasmid DNA (4361 bp); the latter, consisted of a mixture of nicked circular and supercoiled DNA. Purified ⁇ -phage DNA and pBR322
• I * plasmid were purchased from Biolabs, New England. Optical and x-ray data were taken with linear ⁇ prepared in 2 ways: (1) used as delivered, and (2) by heating to 65°C and reacting with a surplus of a 12-base oligo complementary to the 3' COS end. Subsequently the DNA was ligated (T4 DNA ligase, Fischer). The methods gave the same result. For the optical experiments the DNA concentration used was between 0.01 mg/ml and 0.1 mg/ml. Condensation of CLs with ⁇ -DNA was directly observed using differential interference microscopy (DIC) and fluorescence microscopy. A Nikon Diaphot 300 equipped for epifluorescence and high resolution DIC was used.
• DIC differential interference microscopy
• Figure 1A shows high-resolution DIC images of CL-DNA complexes forming distinct condensed globules in mixtures of different lipid to DNA weight ratio (L/D); scale bar is 10 ⁇ m.
• the size of the globules appears to be only weakly dependent on the length of the DNA in similar experiments carried out with Escherichia coli DNA or pBR322 plasmid (4361 bp).
• Figure 2A shows a series of SAXS scans of CL-DNA complexes in excess water as a function of different lipid to DNA weight ration (L/D).
• the Bragg reflections at g 0 o ⁇ is a series of SAXS scans of CL-DNA complexes in excess water as a function of different lipid to DNA weight ration (L/D).
• the Bragg reflections at g 0 o ⁇ is a series of SAXS scans of CL-DNA complexes in excess water as a function of different lipid to DNA weight ration (L/D).
• FIG. 2B shows the spacings d and D NA as a function of L/D show that (i) d is nearly constant and (ii) two distinct states of DNA packing, one where the complexes are positive (L/D > 5, t o NA approximately 46 A) and the other state where the complexes are negative (L/D > 5, C? DNA approximately 35 A)
• the DNA-lipid condensates were prepared from a 25 mg/ml liposome suspension and a 5 mg/ml DNA solution.
• the solutions were filled in 2 mm diameter quartz capillaries with different ratios L/D respectively and mixed after flame sealing by gentle centrifugation up and down the capillary.
• Figure 3 A shows a schematic picture of the local arrangement in the interior of lipid-DNA complexes (shown at two different concentrations in Figure 1A and in Figure 3B below.
• the semiflexible DNA molecules are represented by rods on this molecular scale.
• the neutral and cationic lipids comprising the membrane are expected to locally demix with the cationic lipids (red) more concentrated near the DNA.
• the membrane thickness and water gap are denoted by ⁇ m and ⁇ w , respectively (Fig. 3A).
• the middle broad peak q DNA arises from DNA-DNA correlations and gives dpNA - ⁇ /qoNA (Fig. 2B, solid circles).
• the multilamellar structure with intercalated DNA is also observed in CL-DNA complexes containing supercoiled DNA both in water, and also in Dulbecco's Modified Eagle Medium used in transfection experiments in gene therapy applications.
• This novel multilamellar structure of the CL- DNA complexes is observed to protect DNA from being cut by restriction enzymes.
• the intercalation of ⁇ -DNA between membranes in CL-DNA complexes was found to protect it against a HincflH restriction enzyme which cuts naked ⁇ -DNA at 7 sites (22).
• lamellar condensates coexist with excess giant liposomes in the positive state, and with excess DNA in the negative state.
• the multilamellar structure of the complex (with ⁇ -DNA) and the distinct DNA interhelical packing states was also found in SAXS data in binary mixtures of cationic lipids which contained DOPE [which has a high transfection efficiency (2)] as the neutral co-lipid.
• DOPE which has a high transfection efficiency (2)
• the driving force for higher order self-assembly is the release of counterions.
• the cationic lipid tends to fully neutralize the phosphate groups on the DNA in effect replacing and releasing the originally condensed counterions (both those bound to the ID DNA and to the 2D cationic membranes) in solution.
• the DNA-lipid condensates were prepared from a 25 mg/ml liposome suspension
• FIG. 1A A typical SAXS scan in mixtures at the optical microscopy concentrations (Fig. 1A) is shown in Fig. 2A (inset) which exhibits the same features and confirms that the local multilayer and DNA structure (Fig. 3A) is unchanged between the two concentrations.
• the x-ray samples consisted of connected yet distinct globules (Fig. 3B). What is remarkable is the retention of the globule morphology consistent with what was observed at lower concentrations in DIC (Fig. 1 A).
• Fig. 3C Under crossed polarizers (Fig. 3C) LC defects, both focal conies and spherulites (32), resulting from the smectic-A-like layered structure of the DNA-lipid globules are evident.
• the globules at the lower concentrations show similar LC defects.
• Figure 4B shows d O ⁇ A and d from (A) plotted as a function of L/D (see Fig. 2A for notation). Circles are synchrotron data, and triangles are rotating anode. The solid line is the prediction of a packing calculation (with no adjustable parameters) where the DNA chains form a space-filling ID lattice.
• Figure 4C shows the average domain size of the ID lattice of DNA chains derived from the width of the DNA peaks shown in (B) [corrected for resolution and powder averaging broadening effects].
• the DNA interaxial spacing can be calculated rigorously from simple geometric considerations. If we assume that all of the DNA is adsorbed between the bilayers and that the orientationally ordered DNA chains separate to fill the increasing lipid area as L/D increases, while maintaining a ID lattice (Fig. 3A), then:
• the solid line in Fig. 4B is then obtained from Eq. 1 with no adjustable parameters and clearly shows a remarkable agreement with the data over the measured interaxial distance from 24.5 to 73.5 A.
• This example provides the hexagonal phase of a cationic lipid-polyelectrolyte complex (an embodiment of a macromolecule-lipid complex).
• This embodiment is a LC structure of the complex achieved by varying the lipid composition. It is a novel LC phase with DNA double-strands surrounded by lipid monolayers arranged on a regular hexagonal lattice. This embodiment interacts differently with giant negatively charged liposomes, compared to the lamellar phase, and represents the simplest model of outer cellular membranes.
• Example 1 shows that mixing linear DNA with liposomes of DOPC/DOTAP mixtures leads to a topological transition into CL-DNA complexes of lamellar structure L c a , where DNA monolayers are sandwiched between lipid bilayers (43).
• L c a lamellar structure
• L c a lipid bilayers
• cationic lipids can be extended to deliver other negatively charged biopolymers into cells, in particular polypetide-based drugs and single-stranded oligonucleotides for antisense therapy (23, 24).
• polypetide-based drugs and single-stranded oligonucleotides for antisense therapy 23, 24.
• these polyelectrolytes also form complexes with cationic lipids of lamellar and hexagonal structure, similar to the CL-DNA complexes. Comparison of the three types of complexes allows to gain an insight on how the polyelectrolyte charge density and diameter tune the interactions between lipids and polymer, shifting the phase boundaries between L c a and HJ, complexes.
• Figure 5a shows the formation pathway of a complex from the free DNA and liposomes.
• 1-DNA in solution has a random-coil configuration of ⁇ 1 ⁇ m diameter.
• the Cls consisting of binary DOPE/DOTAP mixture have an average size of 0.06 ⁇ m.
• both DNA and lipid charges are partially neutralized by their respective counterions.
• cationic lipids replace DNA counterions, releasing the [Na + ] and [Cf] ions into solution with a very large entropic free energy gain (of order k B T per released counterion). The result is a close association between DNA and lipid in a compact complex of ⁇ 0.2 ⁇ m size.
• the overall charge of the complex is determined by the weight ratio r of cationic lipid and DNA.
• the complexes are positive for r>2.2 and negative for r ⁇ 2.2, indicating that charge reversal occurs when complexes are stoichiometrically neutral with one positive lipid per each negatively charged nucleotide base.
• the internal structure of the complex changes completely with DOPE/DOTAP ratio.
• the complex is lamellar L c a for ⁇ PE ⁇ 0.41 and has inverted hexagonal H° u structure for ⁇ P E >0.7.
• SAXS Small-angle x-ray scattering
• micellar void in the H j phase is ⁇ 28A, again sufficient for a DNA molecule with approximately two hydration shells.
• the complexes appear as highly dynamic birefringent aggregates when viewed with video-enhanced optical microscopy (Figure 6a,b).
• Each complex consists of several connected blobs close to charge neutrality, with the aggregates becoming smaller and eventually dissociating into individual blobs with the increasing complex charge.
• the shape of aggregates is different in the two complex phases: the L c a phase forms linear structures, while in the H), phase the aggregates are predominantly branched.
• Microscopy of DNA and lipids with appropriate fluorescent labels allows us to image their respective distributions in the complex.
• the membrane of giant anionic liposome is a good model of the outer cell membrane - the first barrier to the complex on its way to DNA delivery.
• H 7 and L complexes interact with model anionic lipid membranes.
• the L° a complexes attached to anionic membrane remain stable for many hours.
• the compact complex morphology can be seen in DIC as well as in DNA and lipid fluorescence.
• HCA complexes A strikingly different behavior is observed with H Compute complexes.
• DOPE forms stable H ⁇ phases
• DOTAP has stable lamellar structures.
• the internal structure of the complex will be affected by several comparable free energy contributions. Since DOPE monolayers have negative spontaneous curvature and bending energy of only a few k B T *", increasing ⁇ rm will allow the lipid layers to curve around the polyelectrolites, forming the Hj j structure. Additionally, the lipid head-group area and correspondingly chain length will adjust itself so as to further minimize the free energy of the system, since the stretching energy of the lipid chain is only slightly greater then the bending energy of the monolayers.
• phase a reasonable phase boundary may be only achieved if the head-group area is substantially smaller, resulting in stretching of the lipid chains and increase in lipid layer spacing.
• stronger electrostatic interaction and small polymer diameter result in crowding of lipid heads.
• the additional free energy of stretching the chains may be the cause of the very narrow region of stability of pure Hj phase in CL-PGA system.
• Figure 5a shows the schematic of the complex formation from the negatively charged
• Figure 5b provides the powder X-ray diffraction patterns of the two distinct liquid- crystalline phases of CL-DNA complexes.
• Figures 6a-b provides video-microscopy images of CL-DNA complexes in (a) HJ, and (b) L c a phases.
• complexes were viewed in DIC (left), lipid fluorescence (middle) and DNA fluorescence (right).
• cationic lipids were labeled with 0.2 mol% of D ⁇ PE-TexasRed and DNA was labeled with Yo Yo- 1 iodide at 1 dye molecule/15bP ratio.
• the complex morphology is different in the two phases: branched in the H tI and linear in the L c a phase.
• Figures 6c-d provides video microscopy of positively charged Hj j (c) and L c a (d) complexes that interact differently with the negatively charged giant liposomes.
• the lamellar complexes simply stick to the liposomes and remain stable for many hours, retaining their blob-like morphology.
• the blobs are localized in DIC as well as lipid and DNA fluorescence modes.
• the hexagonal complexes break-up and spread immediately after attaching to giant liposomes, indicating a fusion process between the complex and the liposome lipid bilayer. Spreading of the complex is evident in both lipid and DNA fluorescence modes.
• Giant unilamellar liposomes were prepared from the mixtures of 90%) DOPC (neutral) and 10% DOPG (negatively charged) lipids.
• Scale bar is lO ⁇ m in both DIC and fluorescence images.
• Figure 7 provides SAXS scans following the transformation from L c to Hj j phase with increasing amount of DOPE for complexes with DNA (i) and poly-Thymine (ii).
• the dashed line indicates L c a phase peaks.
• the H u complexes coexist with the excess H ⁇ phase of pure DOPE (peaks marked with arrows).
• Figure 8 shows variation of structural parameters in L c a and Hj j complexes with the three different types of polyelectrolites (i) 1-DNA, (ii) poly-Thymine (polyT), (iii) polyglutamic acid (PGA).
• a « -J3/2 d were a is the repeat distance of pure Hj, and d is the membrane repeat distance in pure L c a complex.
• PGA polyglutamic acid
• Microstructures with submicron linewidths as substrates for confining and orienting this multilamellar CL-DNA structure is shown schematically in Fig. 9.
• the oriented multilamellar structure would have many important technological applications. For example, in developing nano-scale masks in lithography and molecular sieves with nanometer scale cylindrical pores (Fig. 9).

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