US20140065204A1 - Compositions and methods for delivering nucleic acid to a cell - Google Patents

Compositions and methods for delivering nucleic acid to a cell Download PDF

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US20140065204A1
US20140065204A1 US13/967,664 US201313967664A US2014065204A1 US 20140065204 A1 US20140065204 A1 US 20140065204A1 US 201313967664 A US201313967664 A US 201313967664A US 2014065204 A1 US2014065204 A1 US 2014065204A1
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lipid
nucleic acid
canceled
liposome
dna
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Mark E. Hayes
Dimitri B. Kirpotin
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Merrimack Pharmaceuticals Inc
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Priority to US15/004,481 priority patent/US10087464B2/en
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Priority to US16/025,503 priority patent/US20190017073A1/en
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    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A61K47/48823—
    • 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
    • A61K47/6913—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 the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • 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
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • nucleic acids into living cells is an important process in modern biological research, industry, and medicine. Efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy.
  • gene therapy involves the transfer of normal, functional genetic material into specific cells to correct an abnormality due to a deficient or defective gene product.
  • a variety of methods have been developed to facilitate both in vivo, in vitro, or ex vivo gene transfer.
  • Nucleic acid therapies involve the transfer of natural or synthetic oligonucleotides and polynucleotides into normal and/or pathological cells with the purpose of correcting or eliminating the diseased cells.
  • antisense oligonucleotides and interfering RNAs such as siRNAs and shRNAs are used to block undesirable pathways of protein expression in the cells.
  • Plasmids e.g., plasmids that comprise one or more protein-encoding sequences, may be introduced into cells to correct a cellular defect associated with a defective or absent gene or gene product, or to induce tumor cell death.
  • Polynucleotide inductors of immunity such as poly(I, C) or oligo- and polynucleotides having methylated GC pairs are used to increase the patients' defense against pathogens such as viruses or cancer cells.
  • Ribozymes are ribonucleic acids that catalyze selective degradation of other polynucleotides in the diseased cells, for example, in cancer or virus-infected cells. Because oligo- and polynucleotides generally have low permeability through cell membranes, and are quickly eliminated from the body, there is the need for oligo/polynucleotide delivery vehicles that would allow enhanced intracellular delivery and protection from degradation and/or elimination from the body.
  • One useful method for providing nucleic acid therapies is to encapsulate therapeutic nucleic acids into liposomes suitable for administration to a patient.
  • Liposome technology has been developed and commercialized for the delivery of conventional pharmaceutical agents, but to date therapeutic nucleic acid containing liposomes have not been commercialized.
  • many publications demonstrate that liposome-plasmid DNA complexes can mediate efficient transient expression of a gene in cultured cells but poor in vivo transfection efficiencies. Unlike viral vector preparations, liposome-nucleic acid complexes can have insufficient stability, and thus can be unsuitable for systemic injection.
  • a liposome to deliver a nucleic acid to a cell can be enhanced by adding adequate levels of cationic lipids to the lipid bilayer of the liposome, but cationic lipids are not cell specific. Because many diseases, such as cancers, are limited to specific organs, tissues, or cell types, it is desirable to transfer nucleic acids in an organ, tissue, or cell selective fashion.
  • Immunoliposomes liposomes comprising exterior antibody functionalities, are capable of achieving such cell selective transfer of nucleic acids to cells.
  • the presence of large amounts of cationic lipids on liposomes a useful for achieving introduction of a therapeutic entity within the liposome into a cell.
  • immunoliposomes however, amounts of cationic lipids that are effective to achieve such an effect can result in undesirable non-specific binding to cells, which decreases the ability to specifically direct a liposome-nucleic acid complex to a target cell or cell type.
  • the present invention relates to liposome-nucleic acid complexes and to methods of making and using such complexes.
  • a method for preparing a liposome comprising a nucleic acid and a lipid component comprising: combining a lipid component and a nucleic acid component in a mixture comprising: water; a water-miscible organic solvent; and a polyamine; under conditions such that a liposome comprising the nucleic acid and the lipid component is formed.
  • the polyamine is an oligoethyleneimine, a polyethyleneimine, or a polyaminoC 2 -C 10 alkane.
  • the polyamine is selected from the group consisting of spermine, spermidine, and putrescine.
  • the polyamine is spermine.
  • the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA. In certain embodiments, the RNA is siRNA. In certain embodiments, the RNA is shRNA. In other embodiments the RNA is a double-stranded dicer substrate RNA.
  • the lipid component comprises a non-cationic lipid.
  • the non-cationic lipid is a neutral lipid.
  • the neutral lipid comprises DOPC, DOPE, cholesterol, or PEG-DSG.
  • the liposome further comprises a cationic lipid.
  • the cationic lipid comprises DOTAP or DOSPA.
  • the lipid component contains no cationic lipid.
  • the water-miscible organic solvent comprises methanol, ethanol, 1-propanol, or 2-propanol. In certain embodiments, the water-miscible organic solvent comprises ethanol. In certain embodiments, the ratio of water to water-miscible organic solvent is between about 2:1 and 1:2. In certain embodiments, the ratio of water to water-miscible organic solvent is about 1:1.
  • the ratio of polyamine nitrogen to nucleic acid phosphate groups is at least about 0.5. In certain embodiments, N/P is between about 0.8 and about 1.5.
  • the step of combining is performed at a temperature not greater than about 60° C. In certain embodiments, the step of combining is performed at a temperature between about 40° C. and about 50° C.
  • the lipid component comprises a cationic lipid and the ratio of cationic lipid nitrogen to nucleic acid phosphate groups (N/P) is about 0.5 or less.
  • the lipid component comprises a neutral phospholipid and no cationic lipid, and wherein the nucleic acid and the lipid component are present at a ratio of from 5 nmol lipid per microgram of the nucleic acid to 20, 30, 40, 50, 60, 70, 80, 90, or 100 (optionally 5-20 or 30, preferably 10) nmol lipid per microgram of the nucleic acid, and wherein the liposome is from 30 to 500 nanometers in diameter.
  • the step of combining is performed at a pH not less than about 6.5. In certain embodiments, the step of combining is performed at a pH between about 7.0 and about 8.0. In certain embodiments where the liposomes will comprise RNA, the step of combining is performed at a pH between about 5.5 and about 6.5.
  • the invention provides a composition comprising a liposome in an aqueous medium, the liposome having an interior and an exterior, wherein the liposome comprises: a nucleic acid; a polyamine; and a lipid component;
  • the lipid component comprises a neutral phospholipid and essentially no cationic lipid
  • the nucleic acid and the lipid component are present at a ratio of from 5 nmol lipid per microgram of the nucleic acid to 20, 30, 40, 50, 60, 70, 80, 90, or 100 (optionally 5-20 or 30, preferably 10) nmol lipid per microgram of the nucleic acid, and wherein the liposome is from 30 to 500 nanometers in diameter.
  • the polyamine is oligoethyleneimine, polyethyleneimine, or a polyaminoC 2 -C 10 alkane.
  • the polyamine is selected from the group consisting of spermine, spermidine, and putrescine.
  • the polyamine is spermine
  • the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA. In certain embodiments, RNA is siRNA. In certain embodiments, the RNA is shRNA.
  • the non-cationic lipid comprises DOPC, DOPE, cholesterol, PEG-DSG.
  • the lipid component is essentially free of cationic lipid (e.g., has less than 0.1% cationic lipid by weight).
  • the lipid component comprises a cationic lipid and a non-cationic lipid.
  • the cationic lipid comprises DOTAP or DOSPA.
  • the liposome is from 70 to 300 nanometers in diameter.
  • a method of using of a liposome comprising: attaching an internalizing antibody or a fragment thereof, which antibody or fragment binds to a specific cell surface antigen, to the exterior of the liposome, wherein the liposome with the antibody attached is internalized by a cell expressing at least 100,000 or at least 1,000,000 molecules of the antigen when contacted and incubated with the cell under internalizing conditions.
  • internalization of the liposome into the cell results in alteration of a property of the cell.
  • the liposome further comprises an internalizing antibody or a fragment thereof attached to the exterior of the liposome, wherein the antibody or fragment binds to a specific cell surface antigen.
  • a method of delivering a nucleic acid to a cell comprising: contacting the cell with a composition comprising a liposome comprising an internalizing antibody or a fragment thereof attached to the exterior of the liposome, wherein the antibody or fragment binds to a specific cell surface antigen, and wherein the liposome with the internalizing antibody or a fragment thereof attached is internalized by a cell expressing at least 100,000 or at least 1,000,000 molecules of the antigen when contacted and incubated with the cell under internalizing conditions.
  • a method of treating a patient in need thereof with a nucleic acid comprising administering to the patient an effective amount of a composition comprising a liposome comprising an internalizing antibody or a fragment thereof attached to the exterior of the liposome, wherein the antibody or fragment binds to a specific cell surface antigen, under conditions such that the patient is treated for a condition responsive to nucleic acid therapy.
  • a composition prepared by any of the methods described herein comprising a liposome in an aqueous medium, wherein the liposome comprises: a nucleic acid; a polyamine; and a lipid component.
  • the lipid component comprises a neutral phospholipid and no cationic lipid and wherein the nucleic acid and the lipid component are present at a ratio of from 5 nmol lipid per microgram of the nucleic acid to 20, 30, 40, 50, 60, 70, 80, 90, or 100 (optionally 5-20 or 30, preferably 10) nmol lipid per microgram of the nucleic acid, and wherein the liposome is from 30 to 500 nanometers in diameter.
  • the composition comprises no more than about 5 mol % or 1 mol % DOPE. In certain embodiments, the composition is essentially free of DOPE (e.g., has less than 0.1% DOPE by weight). In certain embodiments, transfection of the nucleic acid into the cell is at least about 10% more efficient (or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more efficient) when DOPE is absent or, if present, is present at a concentration of no more than 5 mol. % of total lipid, compared to transfection of the nucleic acid into the cell with a composition identical except for the presence of cationic lipid at a concentration of more than 5 mol.
  • the transfection efficiency is transfection efficiency of a composition identical except for the presence of DOPE at a concentration of 10 mol. % of total lipid.
  • the % dye accessibility of the composition is at least about 10% greater (or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater) when the DOPE is absent or, if present, is present at a concentration of no more than 5 mol. % of total lipid, compared to the % dye accessibility of a composition identical except for the presence of cationic lipid at a concentration of more than 5 mol. % of total lipid; in certain embodiments, the % dye accessibility is dye accessibility of a composition identical except for the presence of DOPE at a concentration of 10 mol. % of total lipid.
  • the liposome further comprises an internalizing antibody or a fragment thereof attached to the exterior of the liposome, wherein the antibody or fragment binds to a specific cell surface antigen.
  • FIGS. 1A and 1B are graphs showing the effect of pH on GFP expression and luciferase expression, respectively.
  • FIG. 2 is a graph showing the time course of temperature equilibration of 50 vol. % ethanol solution, after immersion in 60° C. oil bath.
  • FIG. 3 shows the results of fluorescent microscopy of cells treated with liposomes (Her2 targeted and plain non-targeted) for 24 h. (1 sec exposure using Rh filter set). The values on the right indicate the theoretical charge, determined by taking the ratio of cationic lipid to DNA, and the % dye accessibility which indicates the % DNA entrapped within the liposome.
  • FIG. 4 is a graph showing the % dye accessibility of liposomes prepared by varying the amount of the cationic lipid component, keeping other components constant.
  • the formulation was DOSPA/DOPC/Chol/DOPE/PEG-DSG/DiI(3)-DS where the composition was X/15/10/4/0.3/0.03 nmol/ ⁇ g DNA.
  • FIG. 5 shows the chemical structures of spermine, a typical phospholipid, dioleoyl-sn-glycero-phosphatidylcholine (DOPC) and a 3D depiction of spermine interaction with DNA double helix.
  • DOPC dioleoyl-sn-glycero-phosphatidylcholine
  • FIGS. 6A and 6B are (A) a chart showing entrapment analysis of liposomes containing a fixed amount of cationic lipid in the formulation to bind 10% of the available anionic phosphates charges in addition to varying amounts of spermine, and (B) a table displaying liposome size before and after sterile filtering through a 0.2 um PES filter and filtering efficiency.
  • FIGS. 7A and 7B are (A) a chart showing entrapment analysis of liposomes containing a fixed amount of cationic lipid in the formulation to bind 25% of the available anionic phosphates charges in addition to varying amounts of spermine, and (B) a table displaying liposome size before and after sterile filtering through a 0.2 um PES filter and filtering efficiency.
  • FIGS. 8A and 8B are (A) a chart showing entrapment analysis of liposomes containing a fixed amount of cationic lipid in the formulation to bind 50% of the available anionic phosphates charges in addition to varying amounts of spermine, and (B) a table displaying liposome size before and after sterile filtering through a 0.2 um PES filter and filtering efficiency.
  • FIG. 9 shows microscopy of MCF7/clone18 cells after 24 h exposure to DiI(3)-DS labeled liposomes-DOSPA (0.1) Formulation (1 ⁇ 4 sec fluorescence; 8 sec phase contrast).
  • NT designates plain, “non-targeted” formulations, T “targeted” anti-Her2 receptor formulations.
  • N/P indicates the nitrogen/phosphate ratio of DOSPA/DNA respectively.
  • FIG. 10 shows microscopy of MCF7/clone18 cells after 24 h exposure to DiI(3)-DS labeled liposomes-DOSPA (0.25) Formulation (1 ⁇ 4 sec fluorescence; 8 sec phase contrast).
  • NT designates plain, “non-targeted” formulations, T “targeted” anti-Her2 receptor formulations.
  • N/P indicates the nitrogen/phosphate ratio of DOSPA/DNA respectively.
  • FIG. 11 shows microscopy of MCF7/clone18 cells after 24 h exposure to DiI(3)-DS labeled liposomes-DOSPA (0.25) Formulation (1 ⁇ 4 sec fluorescence; 8 sec phase contrast).
  • NT designates plain, “non-targeted” formulations, T “targeted” anti-Her2 receptor formulations.
  • N/P indicates the nitrogen/phosphate ratio of DOSPA/DNA respectively.
  • FIG. 12 shows an SDS-PAGE gel showing purified liposomes samples and standards containing varying amounts of F5-PEG-DSPE.
  • FIG. 13 shows the absorbance at 260 nm of various siRNA solutions at temperatures.
  • FIG. 14 shows an SDS-PAGE gel showing F5-PEG-DSPE content of purified liposomes that were heated at various temperatures to test insertion efficiency of targeting conjugate.
  • liposomal compositions for the delivery of nucleic acids can be prepared using organic-aqueous monophase assembly procedures, and/or incorporating polyamine or polymeric amine additives.
  • the amount of cationic lipid used in the liposomal compositions reduced (compared to conventional cationic liposomes) or eliminated.
  • the nucleic acid is predissolved in an organic-aqueous monophase at neutral pH or higher and having relatively low buffering capacity
  • the lipid and/or other liposome-forming component is dissolved in an organic-aqueous monophase at pH lower than neutral and having relatively higher buffering capacity
  • the two solutions are combined at a temperature elevated above ambient, and quickly cooled down to temperature ambient or below, to avoid inactivation of the nucleic acid.
  • pH below neutral e.g., less than pH 6.5
  • an aqueous-organic monophase e.g., of 50 vol. % ethanol
  • shorter exposure times (1-2 min) and/or maintaining of the solution at higher pH (pH 6.5 or higher) preserves the transfection capacity of the DNA.
  • lower pHs may be preferable.
  • compositions for introducing a nucleic acid into a cell comprising the nucleic acid in a complex with a polyamine or a polymeric amine, in a liposome also comprising lipids, wherein the lipids include a relatively low amount of a cationic lipid, or do not include a cationic lipid at all, and where the amount of the nucleic acid entrapped in the liposome is high, for example, 5-30 nmol lipid per microgram of the nucleic acid.
  • compositions have no or very little transfection activity in the absence of ligand-directed targeting to cells, where, for example, the ligand is internalized by the cells, but have surprisingly high transfection activity when there is a cell-targeting ligand appended to the liposome surface, the transfection activity even exceeding that of the similar liposomes containing high levels (e.g., greater than 25% of total lipids) cationic lipid.
  • nucleic-acid carrying liposomes comprising non-cationic lipids, such as, phosphatidylcholines (PC) and cholesterol, with or without hydrophilic polymer-modified lipids, such as, PEG-phosphatidylethanolamine (PEG-PE), PEG-distearoylglycerol (PEG-DSG), or PEG-di(C 12 -C 18 )alkylamine, (PEG being optionally methylated at the non-lipid-derivatized end) resulted in poor encapsulation efficiencies.
  • PC phosphatidylcholines
  • hydrophilic polymer-modified lipids such as, PEG-phosphatidylethanolamine (PEG-PE), PEG-distearoylglycerol (PEG-DSG), or PEG-di(C 12 -C 18 )alkylamine, (PEG being optionally methylated at the non-lipid-derivatized end) resulted in poor
  • the present inventors unexpectedly found that combining of a non-cationic lipid, such as dioleoyl-PC (DOPC) and cholesterol, optionally with PEG-DSG (PEG m.w. 2,000), and a nucleic acid in an organic-aqueous monophase, such as, for example, 50 vol.
  • a non-cationic lipid such as dioleoyl-PC (DOPC) and cholesterol
  • PEG-DSG PEG m.w. 2,000
  • nucleic acid in an organic-aqueous monophase, such as, for example, 50 vol.
  • % aqueous ethanol preferably at a temperature above ambient, for example, at 50-60° C., with subsequent cooling to the ambient temperature and removal of the ethanol by dialysis, results in a very efficient entrapment of the nucleic acid into small ( ⁇ 300 nm in size) liposomal particles, evidenced by decreased accessibility of the nucleic acid to a nucleic-acid-binding hydrophilic dye (20-30% accessibility, or at least 70%-80% DNA incorporated and protected from the dye).
  • liposomes did not transfect the nucleic acid into the cells, but when an internalizable, cell-specific ligand (e.g., an scFv antibody) was appended to the liposomes, they transfected the nucleic acid very effectively, as evidenced by the expression of the GFP transgene (see Examples 1-9, infra).
  • an internalizable, cell-specific ligand e.g., an scFv antibody
  • the greater encapsulation efficiency in the absence of a cationic lipid is contrary to the conventional teaching of the art that states the need for a cationic lipid to impart affinity of the nucleic acid molecule for hydrophobic lipid phase and to ensure the solubility of the nucleic acid in the presence of an organic solvent, where the organic solvents are used to combine the nucleic acid and the lipid components of the liposomes.
  • hydrophilic polymer-conjugated lipid e.g., PEG-DSPE
  • PEG-DSPE hydrophilic polymer-conjugated lipid
  • helper lipid such as DOPE
  • lipid composition of the liposomes did not increase the effectiveness of transfection.
  • the range of non-precipitating ratios of the polyamine or polymeric amine to the nucleic acid in the organic-aqueous monophase can be determined by turbidimetric titration.
  • a suitable ratio of positive charge (polyamine or polymeric amine) to negative charge (nucleic acid phosphate groups) is about 0.9:1 or about 1:1.
  • the nucleic acid was quite poorly encapsulated in the liposomes after mixing in an organo-aqueous monophase and removal of the organic solvent (dye accessibility about 80-90%).
  • the DNA was combined with a polymeric amine or a polyamine, as indicated above, and when the amount of a cationic lipid was reduced, or when a cationic lipid was a single hydrocarbon-chain lipid, or when the cationic lipid was completely omitted from the composition, the DNA was incorporated into the liposomes of DOPC and cholesterol very effectively (see Examples 1-9, infra).
  • the particles had small size, near the volume-weighed average of 200 nm, and when delivered via appended cell-internalizing ligand to the cancer cells in vitro, the distribution of the liposome material, according to the fluorescent microscopy data, was more diffuse and uniform than the distribution of similarly prepared liposomes containing cationic lipids, which was more punctate and appeared to concentrate in discrete subcellular compartments. This observation was in agreement with more effective transgene (GFP) expression of the delivered plasmid DNA in these cells by the inventive liposomes, compared to those containing cationic lipids in usual amounts (i.e., about 0.5 to 2.0 positive-to-negative (DNA) charge ratio).
  • GFP transgene
  • the liposomes of non-cationic lipids and polyamine/polymeric amine-nucleic acid formed effectively when the components were combined in 50 vol. % ethanol-low ionic aqueous buffer at pH 7.0-7.5, at 50-60° C., by mixing the solutions of the lipid component and the DNA-polyamine/polymeric amine component, followed by cooling and removal of ethanol, e.g. by dialysis against a neutral aqueous buffer at physiological concentration of sodium chloride. It was unexpectedly found that this process, albeit not including a “high-transition temperature” lipid component, gave better results if the components were combined at the elevated (50-60° C.), rather than ambient, temperature.
  • cationic lipid is art-recognized and refers to lipid moieties having a net positive charge.
  • Examples of cationic lipids include dioleyl-N,N-dimethylammonium chloride (“DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 3 ⁇ -(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”); N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”);
  • DODAC di
  • neutral lipid is art-recognized and refers to lipid moieties having no net charge.
  • a neutral lipid may have no charge, or may be zwitterionic.
  • neutral lipids include 1,2-dioleoyl-sn-3-phosphoethanolamine (“DOPE”); 1,2-dioleoyl-sn-glycero-3-phosphocholine (“DOPC”); polyethylene glycol-distearoylglycerol (“PEG-DSG”); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (“EPC”); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (“POPC”); and sterols such as cholesterol.
  • DOPE 1,2-dioleoyl-sn-3-phosphoethanolamine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • PEG-DSG polyethylene glycol-
  • liposome refers to conventional liposomes (having an aqueous phase in the interior) as well as to nanoparticles having a lower internal water content.
  • a nucleic acid useful in embodiments of the present invention is selected according to the biological or physiological effect desired to be produced, e.g. by its delivery into living cells. Such selection is well known to the skilled artisans in the fields of molecular biology and medicine.
  • a nucleic acid is a polymeric material that is a nucleic acid or resembles in its structure and function a nucleic acid in that it exhibits a backbone of covalently linked repetitive molecular units (also referred to as monomers) and has a biological or physiological effect.
  • a nucleic acid may include natural, modified or synthetic bases and backbone elements.
  • a nucleic acid may be of natural or synthetic origin and may include a nucleic acid (i.e., a polymer that comprises a plurality of nucleic acid bases attached to a backbone of covalently linked repetitive molecular units), DNA, RNA, natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA)), double-stranded RNAs that are about 30 base pairs in length and can act as dicer enzyme substrates, nucleoprotein, peptide, nucleic acid, ribozyme, DNA-containing nucleoprotein, such as an intact or partially deproteinated viral particles (virions), oligomeric and polymeric anionic compounds other than DNA (for example, acid polysaccharides and glycoproteins), and the like.
  • a nucleic acid i.e., a polymer that comprises a plurality of nucleic acid bases attached to a back
  • RNA it is preferably DNA or RNA, and is more preferably DNA carrying a sequence of an expressible gene or siRNA.
  • Antisense oligonucleotides are another preferred type of nucleic acids.
  • transfection without limitation to any particular kind of nucleic acid or to any particular function that may be performed in the cell by a nucleic acid so transferred.
  • the transfection may be performed on cells in the body of a subject to be treated (in vivo) or on cells maintained outside a subject (in vitro or ex vivo).
  • the terms “transfection” and “delivery” will be used interchangeably in this description.
  • liposomes may contain more than one kind of nucleic acid in respect to structure, function, or nucleotide sequences.
  • a “polyamine” or “polymeric amine”, as used herein, is a non-lipid compound having multiple (at least two) basic nitrogen moieties capable of having a positive charge.
  • Polyamines include linear, branched, or cyclic compounds having from 2 to 20 amino groups.
  • a polyamine has a molecular weight of not more than about 2000 daltons, or not more than about 1800 daltons, or not more than about 600 daltons, or not more than about 400, 300, or 250 daltons.
  • the polyamine has no more than twenty carbon atoms in total.
  • polyamines examples include spermine, spermidine, putrescine, and polyaminoC 2 -C 10 alkanes (such as a ⁇ , ⁇ -aminoC 2 -C 10 alkane), in which the aliphatic chain can be interrupted by one or two nitrogen atoms.
  • the polyamine is spermine
  • Polymeric amines include compounds such as oligoethyleneimine, polyethyleneimine, and the like.
  • compositions provided herein are prepared by providing a first solution containing the nucleic acid and a second solution containing the lipid component, mixing the two solutions together under suitable conditions so that a liposome is prepared.
  • one or both of the first and second solutions comprises a water-miscible organic solvent.
  • the water-miscible organic solvent generally maintains complete miscibility with water (single liquid phase or monophase) under the conditions chosen for the lipid component-nucleic acid combining and organic solvent amount reducing steps described below, i.e., over the entire range from about 0.01 vol. % up to about 60 vol. %.
  • the water-miscible organic solvent of this step is preferably an alcohol, or an aprotic solvent, and is preferably one suitable for use in biological preparation.
  • the alcohol solvent include methanol, ethanol, 1-propanol, 2-propanol, 2-butanol, tert-butanol, ethylene glycol, diethylene glycol, propylene glycol, glycerol, methylcellosolve (ethylene glycol monomethyl ether), methylcarbitol (diethylene glycol monomethyl ether) and the like.
  • Methanol, ethanol or tert-butanol are preferred, particularly ethanol.
  • Aprotic solvents include an ether, an ester, a ketone, a nitrile, an amide, or a sulfoxide.
  • the aprotic solvent is preferably ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethyleneglycol dimethyl ether, dioxane, tetrahydrofurane, acetone, methylethylketone, acetonitrile, dimethylformamide, or dimethylsulfoxide.
  • a lipid component solution can be combined with a nucleic acid solution under conditions that are sufficient to form the desired microparticulate complex.
  • the selected nucleic acid is combined with the lipid component in a solution having a single liquid phase (i.e., monophasic) comprising water and water-miscible organic solvent selected as described above.
  • the monophasic composition is a mixture characterized by the absence of liquid-liquid interfaces, without regard to its optical clarity, as discussed below.
  • the lipid component and nucleic acid can be combined using any method known in the art.
  • the percentage of the organic solvent by volume present in the resulting aqueous/organic solvent mixture will vary according to the type of nucleic acid and lipid component used in the process. This percentage may range from about 10% vol.
  • the temperature range at which the process takes place is above the freezing point of the aqueous/organic solvent mixture, but below the boiling point of the organic solvent; it will typically vary from about 0° C., to no more than 100° C. under ambient conditions of pressure. Temperatures above ambient, such as in the range of 30° C. to 70° C., are preferred, especially about 40° to about 65° C. In general, the temperature can be higher for longer, more stable nucleic acids; conversely, the temperature can be lower for shorter, less stable oligonucleotides such as siRNA or shRNA, to reduce denaturation of the oligonucleotide duplex.
  • One preferred method is to prepare a solution of the nucleic acid in an essentially aqueous medium, prepare the lipid component as a solution in the organic solvent, and combine the two solutions, for example by mechanical mixing, in the volume ratio providing in the mixture the necessary content of the organic solvent.
  • the content of the organic solvent in the resulting mixture preferably provides for partial dehydration and/or condensation of the nucleic acid, while keeping the nucleic acid in a dissolved state; and at the same time, the organic content solubilizes the lipid component into a non-vesicular form, such as, for example, micellar form.
  • Another preferred method is to prepare the nucleic acid solution in a single fluid phase containing water and a first volume percentage of the water-miscible organic solvent, prepare the lipid component solution in a single fluid phase containing water and a second volume percentage of the water-miscible organic solvent, and combine these two solutions, for example by mechanical mixing, in the volume ratio providing in the mixture the necessary content of the organic solvent as specified below.
  • the first and second volume percentages of the organic solvent in these two solutions are preferably the same.
  • the volume percentage of the organic solvent in the first (nucleic acid) solution is preferably chosen to facilitate the transition e.g., of the nucleic acid molecule into condensed and/or less hydrated, form, while the volume percentage of the organic solvent in the second (lipid component) solution solubilizes, e.g., a lipid into a non-vesicular, such as micellar, form.
  • the content of the organic solvent in the nucleic acid and lipid solutions, as well as in the resulting mixture to satisfy both the need to facilitate nucleic acid dehydration and/or condensation, and the need for lipid solubilization.
  • a lipid component is provided in the neat form, preferably in the form having high surface area, such as a film deposited on an insoluble substrate, and then contacted with the nucleic acid solution in a single fluid phase containing water and water-miscible organic solvent in the volume percentage to satisfy the need for nucleic acid dehydration/condensation and/or lipid component solubilization, which percentage is more particularly defined below.
  • Contacting of the neat lipid component with the nucleic acid solution is preferably accompanied by mechanical agitation, such as slow rotation or reciprocation of the vessel in which the contacting is conducted, so that the lipid component is solubilized and contacts the nucleic acid, preferably in a condensed state, to ensure formation of the microparticulate complex.
  • the agitation typically continues until essentially all of the neat lipid component is solubilized.
  • the organic solvent in the resulting nucleic acid/lipid component solution is preferably present at the volume concentration at which both the nucleic acid, such as nucleic acid, and the lipid component are independently molecularly or micellarly soluble. That is, the organic-aqueous monophase produced after combining the nucleic acid with the lipid component would be able to dissolve either the nucleic acid or the lipid component in the form of a molecular or micellar solution without the need of both nucleic acid and lipid component to be present during the dissolution.
  • the lipid component when the content of the organic solvent, and/or the temperature at which nucleic acid and lipid component are combined, is decreased, the lipid component forms a self-assembled, non-micellar, condensed phase, such as bilayer, inverted hexagonal, cubic, liquid crystalline, or amorphous phase.
  • Such lipid components are known in the art.
  • Bilayer-forming lipid components in aqueous environment typically form enclosed structures, such as vesicles.
  • the ability of the lipid component to form a self-assembled, condensed phase upon reduction of the organic solvent concentration in the monophase is independent of whether or not a nucleic acid is present.
  • Exemplary classes of lipid component that form self-assembled, non-micellar, condensed phases in aqueous environment are known in the art and can be selected by one of ordinary skill in the art using no more than routine experimentation.
  • exemplary lipid components are designated as Class I insoluble, non-swelling amphiphiles (spread on interface to form stable monolayer: water-insoluble or having very low solubility) and Class II—insoluble, swelling amphiphiles (spread to form stable monolayer at interface and are insoluble but swell in water to form lyotropic liquid crystals).
  • Class I insoluble, non-swelling amphiphiles swipe on interface to form stable monolayer: water-insoluble or having very low solubility
  • Class II—insoluble, swelling amphiphiles stamp to form stable monolayer at interface and are insoluble but swell in water to form lyotropic liquid crystals.
  • the particular concentration of the organic solvent selected for any given mixture would depend on the nature of the organic solvent, the lipid component, and the nucleic acid; the temperature at which the components are combined; the ionic strength of the aqueous component; and the concentration of lipid component and/or nucleic acid in the mixture.
  • the organic solvent, the nucleic acid, the aqueous component, and the lipid component are selected according to the needs of a particular application, a skilled artisan being guided by this specification, would easily establish the required concentration of the organic solvent by performing simple solubility tests known in the art.
  • the molecular or micellar nature of the dissolved nucleic acid and/or lipid can be determined by dynamic light scattering.
  • micellar or especially molecular (true) solutions have substantially lower light scattering than those containing particles, vesicles, filaments, or other elements comprising aggregated nucleic acid or lipid component phases.
  • Other methods know in the art, such as NMR, ESR probe, and fluorescent probe methods can be used to detect the presence of nucleic acid or lipid component in the state other than micellar or molecular solution.
  • the amount of an organic solvent in the mixture is so elected as to provide for nucleic acid and lipid component to be independently micellarly or molecularly soluble in the resulting aqueous-organic solvent monophase. Typically this amount is from about 10 vol. % to about 60 vol. %, preferably from 30 vol. % to 55 vol. %, and most preferably from about 45 vol. % to about 55 vol. %.
  • the lipid component can comprise neutral or cationic lipids.
  • the amount of cationic lipid is present at a concentration of no more than 5 mol. % of total lipid (optionally no more than 4 mol. %, 3 mol. %, 2 mol. %, 1 mol. %, or 0.5 mol. % of total lipid, or essentially free of cationic lipid).
  • Any particular amount of non-cationic lipids will depend on the nature of this lipid, the chosen cationic lipid, the nucleic acid, and the organic solvent.
  • Sterols may be present in the amount of up to 100% of the non-cationic lipid.
  • sterols such as for example, cholesterol
  • the nucleic acid solution and the lipid are preferably combined at the temperature above ambient and above the highest of the phase transition temperatures of the lipids present in the solution, but below the boiling point of the organic solvent, more preferably between about 30° C. and about 80° C., yet more preferably between about 40° C. and about 70° C., and optimally between about 50° C. and 65° C.
  • the precise temperature at which the nucleic acid and lipid component are combined also provides for molecular or micellar dissolution of both components in the chosen monophase. This temperature can be determined, for example, by the solubility tests described above.
  • Aqueous component of the fluid phase is preferably of low ionic strength, i.e., at or below the physiological value (that of 144 mM NaCl), more preferably below that of 50 mM NaCl, and most preferably less than that of 10 mM NaCl.
  • Ionic strength is defined as one-half the sum of concentrations of all ions in a solution multiplied by the square of their ionic charges. Without being limited by a particular theory, it is believed that low ionic strength at the lipid component/nucleic acid combining step reduces the risk of liposome aggregation and precipitation and eliminates the requirement of sterically stabilizing lipid components to be present during this step.
  • the aqueous component may also contain buffer substances to maintain the desired pH, typically in the range from about 3.0 to about 10.0, more preferably in the physiological pH from about 4.0 to about 9.0. In certain embodiments, the pH is between about 5.5 and about 6.5.
  • the amount of the buffer substance is chosen to keep the ionic strength low, within the above range of ionic strength.
  • the polyamine or polymeric amine is added to the solution of the nucleic acid prior to mixing of the nucleic acid solution with the lipid component solution.
  • the amount of the organic solvent in the mixture is reduced to effect formation of liposomes. It is believed that reduction of the organic solvent contents promotes lipid bilayer formation around the condensed nucleic acid/lipid core, this effecting the formation and stabilization of liposomes. Thus, the amount of organic solvent is preferentially reduced to the point of self-assembly of the nucleic acid/lipid complex into particles. If, as evidenced, for example, by particle size measurements, the formation of nucleic acid/lipid particles occurs at the monophase step, removal of organic solvent is optional, and may serve the purpose of, for example, improving biocompatibility of the transfecting formulation.
  • the amount of organic solvent is preferably reduced to, or below, the point where bilayer formation is achieved. Generally, this amount is less than about 20 vol. %. Most preferably, essentially all of the organic solvent is removed, e.g. down to about 0.01 vol. %; however in some topical applications, such as the nucleic acid delivery to the cells of skin, it is advantageous to a pharmaceutically acceptable organic solvent and retain a percentage of the solvent (e.g. ethanol) in the composition. Reduction of the organic solvent is achieved by any means available in the art, such as, for example, by dialysis, gel-chromatography, absorption, evaporation under reduced pressure, ultrafiltration, size-exclusion chromatography, lyophilization, or a combination thereof.
  • the liposomes also enables the liposomes to be transferred into appropriate medium for storage or final use.
  • the ionic strength of the medium can be brought up to physiological value (that of 144 mM NaCl), for example, by addition of the concentrated salt solution, followed by mixing. It was unexpectedly found that the liposomes remain stable against aggregation in physiological salt solutions even in the absence of aggregation-preventing polymer-lipid conjugates.
  • the temperature at which the organic solvent is removed is preferably the one at which the nucleic acid was combined with the lipid component.
  • the temperature can be first brought to ambient or below up to refrigeration temperature of 4-8° C. The latter is more suitable when low phase transition temperature lipids, such as the ones containing unsaturated fatty acid chains (phase transition temperature ⁇ 4° C.), are used.
  • liposomes provided herein have reduced water content compared to conventional liposomes. See, e.g., U.S. Patent Application Publication No. 2007/0171077, the contents of which are incorporated herein by reference, for discussion of certain compositions having reduced water content and methods of making such compositions.
  • the lipid shell surrounds the nucleic-acid-containing core closely so that between the core and the shell there is little space holding extraneous small molecules (solutes).
  • the aqueous content of the inner space enclosed by the shell is less than 50%, and more preferably, 20% or less of that calculated from the particle size. The latter value corresponds to the expected amount of water immobilized in the hydration layer of the nucleic acid contained within the particle.
  • liposomes of these embodiments in an aqueous medium may contain encapsulated water in the amount approximating the hydration water immobilized by the encapsulated nucleic acid.
  • the liposome contains encapsulated water in the amount approximating the hydration water immobilized by the encapsulated nucleic acid.
  • the liposome has an interior having an enclosed interior volume containing the nucleic acid in a condensed state and the enclosed interior has an aqueous content of less than about 50% of the volume, as calculated from liposome particle size, or alternately, the aqueous content is 20% of the volume or less.
  • a liposome as provided herein also optionally contains associated therewith a ligand that facilitates the liposome's entry into a cell, i.e., a cell-specific ligand.
  • the ligand is a chemical moiety, such as a molecule, a functional group, or fragment thereof, which is specifically reactive with the cell of choice while being less reactive with other cells thus giving the liposome an advantage of transferring nucleic acids, selectively into the cells of choice.
  • binding affinity By being “reactive” it is meant having binding affinity to a cell or tissue, or being capable of internalizing into a cell wherein binding affinity is detectable by any means known in the art, for example, by any standard in vitro assay such as ELISA, flow cytometry, immunocytochemistry, surface plasmon resonance, etc.
  • a ligand binds to a particular molecular moiety—an epitope, such as a molecule, a functional group, or a molecular complex associated with a cell or tissue, forming a binding pair of two members. It is recognized that in a binding pair, any member may be a ligand, while the other being an epitope.
  • binding pairs are known in the art.
  • Exemplary binding pairs are antibody-antigen, hormone-receptor, enzyme-substrate, nutrient (e.g. vitamin)-transport protein, growth factor-growth factor receptor, carbohydrate-lectin, and two polynucleotides having complementary sequences.
  • Fragments of the ligands are to be considered a ligand and may be used so long as the fragment retains the ability to bind to the appropriate cell surface epitope.
  • the ligands are proteins and peptides comprising antigen-binding sequences of an immunoglobulin. More preferably, the ligands are antigen-binding antibody fragments lacking Fc sequences.
  • Such preferred ligands are Fab fragments of an immunoglobulin, F(ab) 2 fragments of immunoglobulin, Fv antibody fragments, or single-chain Fv antibody fragments. These fragments can be enzymatically derived or produced recombinantly.
  • the ligands are preferably internalizable ligands, i.e., the ligands that are internalized by the cell of choice for example, by the process of endocytosis.
  • ligands with substitutions or other alterations, but which retain the epitope binding ability may be used.
  • the ligands are advantageously selected to recognize pathological cells, for example, malignant cells or infectious agents.
  • Ligands that bind to cell surface epitopes are preferred.
  • One especially preferred group of ligands are those that form a binding pair with the tyrosine kinase growth factor receptors which are overexpressed on the cell surfaces in many tumors.
  • Exemplary tyrosine kinase growth factors are the VEGF receptor, FGF receptor, PDGF receptor, IGF receptor, EGF receptor, TGF-alpha receptor, TGF-beta receptor, HB-EGF receptor, ErbB2 receptor, ErbB3 receptor, and ErbB4 receptor.
  • EGF receptor vIII and ErbB2 (HER2) receptors are especially preferred in the context of cancer treatment using liposomes as these receptors are more specific to malignant cells, while scarce on normal ones.
  • the ligands are selected to recognize the cells in need of genetic correction, or genetic alteration by introduction of a beneficial gene, such as: liver cells, epithelial cells, endocrine cells in genetically deficient organisms, in vitro embryonic cells, germ cells, stem cells, reproductive cells, hybrid cells, plant cells, or any cells used in an industrial process.
  • a beneficial gene such as: liver cells, epithelial cells, endocrine cells in genetically deficient organisms, in vitro embryonic cells, germ cells, stem cells, reproductive cells, hybrid cells, plant cells, or any cells used in an industrial process.
  • the ligand may be attached to the liposome by any suitable method available in the art.
  • the attachment may be covalent or non-covalent, such as by adsorption or complex formation.
  • the attachment preferably involves a lipophilic molecular moiety capable of conjugating to the ligand by forming a covalent or non-covalent bond, and referred to as an “anchor”.
  • An anchor has affinity to lipophilic environments such as lipid micelles, bilayers, and other condensed phases, and thereby attaches the ligand to a lipid-nucleic acid microparticle. Methods of the ligand attachment via a lipophilic anchor are known in the art.
  • an amount of a lipophilic anchor effective to provide ligand conjugation is included into the lipid component, e.g. lipid, prior to, or during, the liposome formation.
  • the conjugate of an anchor and a ligand can be first formed, and then incorporated into liposomes by addition to the lipid prior to the liposome formation, or by addition of the conjugate to the aqueous suspension of liposomes after their formation.
  • a particularly suitable mode of ligand attachment to liposomes is by using a ligand conjugated to a lipophilic anchor through an intermediate hydrophilic polymer linker.
  • the ligand moves freely above the microparticle surface and can react even with hard-to-reach epitopes on the cell surface.
  • Ligands conjugated to lipophilic anchors via a hydrophilic polymer intermediate linker advantageously become stably associated with preformed nucleic acid-lipid liposomes during co-incubation of the conjugated ligands and the liposomes in an aqueous medium.
  • U.S. Pat. No. 6,210,707 the contents of which are incorporated herein by reference.
  • targeting moieties and their incorporation into liposomes see, e.g., U.S. Pat. No. 7,244,826; U.S. Pat. No. 7,507,407; and U.S. Pat. No. 6,794,128; the contents of each of these patents are incorporated herein by reference.
  • the liposomes can further comprise other components beneficial for its function of transfecting cells. These can be viewed as transfection-enhancing components, i.e., an entity associated with the liposome that improves the delivery of an exogenous nucleic acid to a living cell.
  • transfection-enhancing components may include, without limitation, endosome-escape agents, nuclear localization factors, triggerable means for enhanced transfer into cytosol, pH-sensitive compounds, heat and radiation-triggerable release, and membrane fusion promoters such as membrane fusion-enhancing or membrane fusion-inducing compounds, intracellular nucleic acid release-enhancing or inducing components, transcription factors, and promoter-modulating compounds.
  • the liposomes provided herein are capable of entrapping high percentages of the nucleic acid in the nucleic acid solution. For example, in preferred embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75% 80%, 85% or 90% of the nucleic acid present in the nucleic acid solution (prior to mixing with the lipid component solution) is entrapped within the liposomes. The extent of trapping of the nucleic acid can be measured using assays such as the assays described in the Examples herein.
  • the ratio of lipid to nucleic acid is of from 1 nanomole (nmol) lipid per microgram of the nucleic acid to 20, 30, 40, 50, 60, 70, 80, 90, or 100 (optionally 2.5-20 or 30, preferably 10, or 5-20, or 5-10) nmol lipid per microgram of the nucleic acid. It has now been found that entrapment of the nucleic acid is more efficient when the ratio of lipid to nucleic acid is at least 2.5 nmol lipid per microgram of the nucleic acid.
  • the liposome is from 30 to 500 nanometers (nm) in diameter. In other embodiments, the liposome is from about 40 to about 250 nm, or from about 40 to about 200 nm in diameter.
  • compositions comprising the liposomes provided herein are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., normal saline will be employed as the pharmaceutically acceptable carrier.
  • suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • the concentration of liposomes in pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension.
  • the amount of liposome administered will depend upon the particular lipids and targeting ligands used, the disease state being treated, the therapeutic agent being delivered, and the judgment of the clinician. Generally the amount of liposomes administered will be sufficient to deliver a therapeutically effective dose of the particular nucleic acid.
  • immunoliposome dosages may be between about 0.01 and about 50 mg per kilogram of body weight, or between about 0.1 and about 10 mg/kg of body weight.
  • the pharmaceutical compositions are administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, by direct injection into the brain (intrathecally or via convection enhanced delivery) or intramuscularly.
  • the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection.
  • Particular formulations which are suitable for this use are well known in the art.
  • the formulations will comprise a solution of the liposomes suspended in an acceptable carrier, preferably an aqueous carrier.
  • an aqueous carriers may be used, e.g., water, buffered water, 0.9% isotonic saline, and the like.
  • compositions may be sterilized, e.g., by filtration.
  • the resulting aqueous solutions may be packaged for use as is. In certain instances the solutions can be frozen.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the nanoparticles (liposomes) provided herein can be used to treat any of the cancers disclosed in U.S. Pat. No. 7,846,440, the contents of which are incorporated herein by reference.
  • the nanoparticles (liposomes) can used for cancer treatment in conjunction with one or more anti-cancer agents disclosed in U.S. Pat. No. 7,846,440 (e.g., as disclosed in the Table of anti-cancer agents spanning Col. 19-Col. 24 of U.S. Pat. No. 7,846,440).
  • the polyamine was mixed with GFP plasmid DNA in 50% ethanol/50% 20 mM MES, pH 5.1.
  • the lipid mixtures were dissolved in 50% ethanol/50% 2 mM TE buffer, pH 8.0.
  • the polyamine mixture and the lipid mixture were heated at 60° C. for 2 minutes, mixed, and allowed to cool to room temperature with stirring.
  • the mixture was transferred to a dialysis cassette (MWCO 10,000) and dialysed against saline ( ⁇ 2) and then HBS, pH 7.25. After 4 h total dialysis time, samples were removed and tested for DNA concentration, dye accessibility and size. Picogreen® dye accessibility assay was performed as described in Example 12, infra.
  • PEI MW 600, PEI MW 1800 and Spermine were used as the polyamine molecules.
  • N/P 1.33 DNA undergoes aggregation in 50% ethanol mixtures, observed by the appearance of turbidity.
  • Samples were immunotargeted by the addition of an antiHer2-lipid conjugate, and the GFP transfection measured.
  • Samples were prepared as above and targeted (T) by addition of 15 ⁇ g/ ⁇ mol PC of F5-PEG-DSPE antibody-lipid conjugate by an insertion methodology described, e.g., in U.S. Pat. No. 6,210,707.
  • Non-targeted (NT) particles are those particles to which no antibody conjugate has been added.
  • SKBR3 cells (ATCC® #HTB-30TM, grown in McCoy's 5A media with 10% FBS) were plated at a density of 100,000 cells/well in a 24 well plate the day previously. DNA was mixed with LipofectamineTM 2000 as described by the manufacturer's instructions, and 1 ⁇ g DNA per well was added to the cells. 1 ⁇ g of encapsulated DNA, and free DNA was also added to each well (in duplicate). After 24 h, the cells were washed and 1 mL media added. After a further 24 h, the cells were assayed for the green fluorescent protein (GFP). This entailed being washed twice with Hank's' BSS solution and viewed under a fluorescence microscope for GFP positive cells. GFP expression is calculated as the number of fluorescent cells/total number of cells ⁇ 100 over many fields of view (n>3).
  • GFP expression is calculated as the number of fluorescent cells/total number of cells ⁇ 100 over many fields of view (n>3).
  • Lipid amounts are shown as DOTAP/DOPC/Chol/DOPE/CHEMS/CHIM/DiI(3)-DS.
  • Formulation 1 as above in the ratios 6/20/7/5/6/3.67/0.47/0.05 nmol per 1 ⁇ g DNA
  • Formulation 6 as above in the ratios 3/16/4/15/5/0.225/0.045 nmol per 1 ⁇ g DNA with PEI 1800 added to DNA at N/P
  • Polyamines can be successfully incorporated into liposomes without causing extensive aggregation of the particles. However, it is noticeable that the liposomes were more susceptible to dye accessibility and the liposomes were a little larger. It is unknown at this point, whether the increase in dye accessibility is due to incomplete DNA entrapment or the liposomes give reduced protection to the encapsulated DNA.
  • formulation 8 gave DNA protection at all; this formulation gave the highest GFP signal of any of the samples in the transfection assay.
  • Cationic liposomes using the formulation DOTAP/POPC/Chol do not form properly (they aggregate during dialysis) at +/ ⁇ 1.67.
  • liposomal particles can be made without using any cationic lipid, that entrap >75% of the DNA.
  • the liposomes were prepared as in Example 3, Formulation 1, except that Hexadecyltrimethylammonium bromide (HTAB) and tetradecyltrimethylammonium bromide (TTAB) were used instead of DOTAP.
  • HTAB Hexadecyltrimethylammonium bromide
  • TTAB tetradecyltrimethylammonium bromide
  • Single-chain cationic amphiphiles can be used to make low-cationic-lipid liposomes with polyamine/polymeric amine pre-condensed DNA.
  • DiI(3)-DS is included for purposes of facilitating the experiments, but would not typically be included in liposomes for therapeutic use.
  • nucleic acid-condensing agent e.g., spermine
  • Formulations A, B, and E had dye accessibility of about 20%.
  • Sample C had somewhat higher dye accessibility.
  • the particle with the highest dye accessibility and largest size was sample D. Therefore, lipid amounts of 500/333/8.3 mmol of the components per mg DNA may be too low, and it is preferable to use ratios greater than this. It is also possible that too high ratios may lead to liposomes that do not contain any DNA, so the minimum amount of lipid that stably entraps DNA would be preferable. Therefore sample B or C is optimal.
  • Formulation 5 DOPC/Chol/Peg-DSG/DiI(3)-DS 0% PEG-DSG 1000/667/0/1.67
  • Formulation 6 DOPC/Chol/Peg-DSG/DiI(3)-DS 0.5% PEG-DSG 1000/667/8.33/1.67
  • Formulation 7 DOPC/Chol/Peg-DSG/DiI(3)-DS 1.0% PEG-DSG 1000/667/16.7/1.67
  • Formulation 8 DOPC/Chol/Peg-DSG/DiI(3)-DS 2.0% PEG-DSG 1000/667/33.3/1.67
  • Formulation 9 DOPC/Chol/Peg-DSG/DiI(3)-DS 5.0% PEG-DSG 1000/667/83.3/1.67
  • Non-targeted (NT) liposomes are those particles to which no antibody conjugate has been added.
  • SKBR3 cells (grown in McCoy's 5A media with 10% FBS) were plated at a density of 100,000 cells/well in a 24 well plate the day previously. DNA was mixed with LipofectamineTM 2000 as described by the manufacturer's instructions, and 1 ⁇ g DNA per well was added to the cells. 1 ⁇ g of liposome-encapsulated DNA, and free DNA was also added to each well (in duplicate). After 24 h, the cells were washed and 1 mL media added. After a further 24 h, the cells were assayed for the green fluorescent protein (GFP). This entailed being washed twice with Hank's BSS solution and viewed under a fluorescence microscope for GFP positive cells. GFP expression is calculated as the number of fluorescent cells/total number of cells ⁇ 100 over many fields of view (n>3).
  • GFP expression is calculated as the number of fluorescent cells/total number of cells ⁇ 100 over many fields of view (n>3).
  • PEG-DSG is beneficial for optimal liposome formation. Greater than 0.5 mol. % allows for reasonably efficient DNA encapsulation and gives rise to small particles. Less than 0.5 mol. % causes larger particles to form. Increased PEGylation does not inhibit GFP expression.
  • Samples were prepared as above and targeted by addition of 15 mg/mmol PC of F5-PEG-DSPE antibody-lipid conjugate. The insertion procedure is initiated by heating at 60° C. for 30 min. The resulting solutions were rapidly cooled in iced water, samples sterilized by passing through 0.45 ⁇ m PES filter. Samples were also tested for gene expression by addition to SKBR3 cells, and counting GFP-positive cells 48 h later.
  • DOPE as a “helper” lipid for transfection by lipid-based carriers
  • inclusion of DOPE did not increase the transfection efficiency of these liposomes.
  • targeted samples containing DOPE have substantially higher dye accessibilities than corresponding samples having less than 10% DOPE.
  • spermine on its own does not cause DNA to be expressed intracellularly, indicating that it is the combination of the targeted-liposome entrapping precondensed DNA that causes gene expression.
  • GFP and luciferase reporter plasmids were prepared in buffered solutions of pH ranging from approximately 4 to 7.2 at 50 ⁇ g/ml DNA concentration from stock DNA solutions in TE, pH 8.0 buffer.
  • the two chosen buffers were Hepes and MES, both at a concentration of 5 mM.
  • Hepes and MES Hepes and MES, both at a concentration of 5 mM.
  • ethanol a 1 mL aliquot of the above solutions, equal volumes of ethanol were added, mixed, and the resultant solutions incubated at 60° C. for 10 min. After cooling, the samples were dialysed against Hepes buffered saline, (HBS), pH 7.25 overnight.
  • the samples were sterile filtered under aseptic conditions and the DNA concentrations were determined by UV absorbance at 260 nm.
  • SKBR3 cells grown in McCoy's 5A media with 10% FBS were plated at a density of 100,000 cells/well in a 24 well plate the day previously. DNA was mixed with LipofectamineTM 2000 as described by the manufacturer's instructions, and 1 ⁇ g DNA per well was added to the cells. After 6 h, the cells were washed and 1 mL media added. After a further 18 h, the cells were assayed for the respective reporter gene.
  • GFP plasmid this entailed being washed twice with Hank's BSS solution and viewed under a fluorescence microscope for GFP-positive cells, or for luciferase expression by lysing in a 0.1M sodium phosphate solution and reading luciferase using a luminometer after injection with luciferin.
  • a standard curve was prepared by using purified luciferase, and protein measured by using the MicroBCATM assay (Pierce).
  • GFP expression is calculated as the number of fluorescent cells/total number of cells ⁇ 100 over many fields of view (n>3). Luciferase quantity was expressed as ng luciferase per ⁇ g total protein. Results are shown below.
  • Results are shown in FIG. 2 . It was found that about two minutes is sufficient for solution to reach desired temperature.
  • DNA 50 ⁇ g in an ethanolic solution (50% v/v) of 2 mM TE buffer was mixed with an equal volume of an ethanolic solution (50% v/v) of 20 mM MES, pH 5.1 after incubation at 60° C. for 2 min, and the resultant solution dialysed, sterilized by microfiltration, and its transfection activity was studied using LipofectamineTM 2000 as described above.
  • the results were as follows: untreated DNA (positive control) 100 ⁇ 1.4%; treated DNA, 72.7 ⁇ 9.7%.
  • Treating DNA in the above manner minimizes the damage done to the plasmid, and retains greater than 70% of the reporter gene activity.
  • Liposomes were made so that the net electrical charge on the liposomes was varied from negative to positive, accomplished by altering the ratio of cationic lipid to DNA in the formulation.
  • Liposomes were prepared using a cationic lipid, neutral zwitterionic phospholipids DOPC and DOPE, cholesterol, a PEGylated lipid and a fluorescent lipid (to aid visualization of particles) in such a manner that only the amount of cationic lipid was varied, using the formulation DOSPA/DOPC/Chol/DOPE/PEG-DSG/DiI(3)-DS where the composition was X/15/10/4/0.3/0.03 nmol/ ⁇ g DNA.
  • the liposomes were made, they were tested for DNA entrapment and ability to interact with cultured human cancer cells that over-express the HER2 receptor (SKBR3), by fluorescence microscopy ( FIG. 3 ).
  • DNA (or siRNA/oligonucleotide) entrapment is measured by a simple, quick fluorescence based assay called the Picogreen® Dye Accessibility Assay. It is based around an idea that a molecule such as Picogreen® (Invitrogen) is highly fluorescent once bound to DNA. In its unbound state it has low fluorescence. Once Picogreen® is added to a solution of liposomes it quickly binds to any “accessible” DNA, thus exhibiting fluorescence. This accessible dye may be bound to totally unencapsulated DNA (free DNA) or DNA that is partially or poorly encapsulated. The dye does not cross lipid bilayers; therefore any DNA that is properly encapsulated will be shielded from the dye.
  • Picogreen® Dye Accessibility Assay a simple, quick fluorescence based assay. It is based around an idea that a molecule such as Picogreen® (Invitrogen) is highly fluorescent once bound to DNA. In its unbound state it has low fluorescence.
  • Picogreen® has the ability to bind the total amount of DNA present. By extrapolation to standard curves, the concentration of accessible and total DNA can be measured. The ratio of each gives us the “% Dye Accessibility” and is calculated as follows:
  • nucleic acids can confer entrapment into liposomes without the use of any cationic lipid. It has also been found that lower molecular weight nucleic acids, such as 18-mer oligonucleotides and siRNA, can also be effectively entrapped without the use of any cationic lipid.
  • Liposomes were prepared according to the formulation DOSPA/DOPC/Chol/DOPE/Peg-DSG/DiI(3)-DS varying the amount of DOSPA and spermine while keeping all other lipids constant.
  • the entrapment efficiency, size, filterability (efficiency of passing through a sterilizing 0.2 ⁇ m filter) and cellular uptake of liposomes were measured.
  • DOSPA 0.25 a formulation that has enough DOSPA to bind to 25% of the charges i.e., DOSPA/DOPC/Chol/DOPE/Peg—DSG/DiI(3)—DS with a molar lipid ratio of 0.1875/30/20/8/1.74/0.058 nmol per 1 ⁇ g oligo is referred to as DOSPA 0.25.
  • This small amount of cationic lipid was supplemented with spermine to aid entrapment of the oligo, and is indicated below as N/P (referred to as total N from spermine to DNA phosphate).
  • FIGS. 9-11 show that the liposomes prepared with low amounts of cationic lipid, supplemented with spermine, exhibit highly specific targeted uptake, with no observable non-specific cellular interactions.
  • Liposomes can be targeted by co-incubation with F5-PEG-DSPE micelles and heating at 37° C. overnight.
  • the initial amount of protein conjugate that was added was 15 ⁇ g/umol PL.
  • the amounts of conjugate associated with the liposomes was 13.64 ⁇ 0.64 and 10.59 ⁇ 0.39 for the two formulations tested. This represents a great than 70% incorporation efficiency. This indicates that liposomes that contain siRNA and low amounts of cationic lipids can readily be targeted by this method.
  • siRNA/shRNA Nucleic acids in general, but more specifically siRNA/shRNA are extremely sensitive to degradation during storage, and are best stored frozen. Special handling precautions are recommended for working with siRNA/shRNA. RNases are abundant are present on most surfaces that come into human contact and should be thoroughly cleaned with special RNase removing solutions. In short, it is very easy to cause siRNA/shRNA degradation through even the most careful handling procedures. As the knockdown effect of RNAi is so sequence-specific, partial degradation often adversely effects the ability of a given siRNA/shRNA to have the desired knockdown effect. Therefore, storing the liposomes in a medium that prevents either chemical degradation and/or biological degradation from nucleases would be of great benefit.
  • liposomes could be stored frozen by combining with a cryoprotectant solution of 10% sucrose before freezing was tested.
  • Sucrose is known to aid in the freezing of liposomes by keeping the phospholipid headgroup hydrated and preventing lipid realignment and bilayer deformation.
  • the present process allows for assembly of DNA—lipid liposomes in an environment where particle-forming lipids would not form a condensed phase beyond a micelle, and the DNA would still be soluble by itself prior to combination. It was found that such conditions could be satisfied in a number of different organic solvent/water mixtures including 50% (v/v) ethanol. However, this is usually achieved by heating the lipid and DNA solution prior to combination. For encapsulation of closed circular plasmid DNA, the optimal temperature was found to be 60° C. However, it is well known that shorter pieces of DNA (and RNA) can “melt” or denature at lower temperatures. DNA melting is the process by which double-stranded (deoxy)ribonucleic acid unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases.
  • RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm. Therefore the delivery of double-stranded siRNA is very important. Denaturation of siRNA or shRNA before encapsulation would likely result in a non-active particle; therefore it is prudent to measure the melting temperature of each siRNA or shRNA sequence prior to encapsulation.
  • RISC RNA-induced silencing complex
  • Tm melting temperature
  • siRNA melting transition temperature defined as the temperature at which 50% of the siRNA is in the single-stranded form and 50% in the double-stranded form
  • siRNA Buffer Tm ° C. Ssb 5 mM Hepes, pH 7.25 41.5 Ssb Ethanol/5 mM Hepes, pH 7.25 53.0 Ctrl 5 mM Hepes, pH 7.25 57.0 Ctrl Ethanol/5 mM Hepes, pH 7.25 68.0
  • Ssb 5′-ACAACAGACUUUAAUGUAA-3′ control: 5′-UCUUUUAACUCUCUUCAGG-3′
  • FIG. 13 shows that the control siRNA is stable at 45° C. to unwinding. Therefore, we used this temperature as a stable temperature to compare to liposomes containing the less stable SSB siRNA at 45° C. and at a lower temperature, 40° C.
  • the insertion of the targeting ligand for plasmid liposomes usually takes place at 60° C.
  • the insertion efficiency of the same liposomes containing SSB siRNA at lower temperatures was tested in order to evaluate if liposomes can be targeted by this methodology without elevated temperatures (see FIG. 14 ).
  • targeting ligand F5-PEG-DSPE
  • siRNA or shRNA
  • the liposome formulation mostly consists of DOPC and cholesterol, and the low lipid transition temperature likely aids incorporation of the scFv-lipid conjugate into the bilayer.
  • Short sequences of RNA and short oligonucleotides can be entrapped in liposomes that contain either no cationic lipid, or very small amounts of cationic lipid, and will help the liposome formation process and provide more reproducible formulations.
  • the amount of cationic lipid in a formulation is typically less than 0.5% total lipid, which is far less than used in many alternative formulations, e.g.
  • typical non-targeted SNALP formulations contain 30% cationic lipid, rendering them useful for targeting such organs as liver, but not ideal for reaching distal tumor sites for targeted delivery (Morrissey D V et al., Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs, Nature Biotechnology (2005) August; 23(8):1002-7).

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