WO2003057190A1 - Encapsulation efficace d'acides nucleiques dans des liposomes de taille moyenne - Google Patents
Encapsulation efficace d'acides nucleiques dans des liposomes de taille moyenne Download PDFInfo
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- WO2003057190A1 WO2003057190A1 PCT/US2002/041814 US0241814W WO03057190A1 WO 2003057190 A1 WO2003057190 A1 WO 2003057190A1 US 0241814 W US0241814 W US 0241814W WO 03057190 A1 WO03057190 A1 WO 03057190A1
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- 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—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- 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
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- This invention concerns a method of preparing liposomes containing a nucleic acid encapsulated therein, liposomes containing a nucleic acid encapsulated therein prepared by said method, and methods of using the liposomes containing the nucleic acid.
- the method of preparing the liposomes of the present invention has the advantages of being simple and able to generate primarily small liposomes of relatively homogeneous particle size with a high entrapment efficiency.
- the liposomes containing a plasmid DNA encapsulated therein are useful in transfection of cells with high transfection efficiencies.
- Gene therapy involves the delivery of a gene of interest to inside the cells of a subject in need of the therapy.
- gene delivery systems There are two major groups of gene delivery systems used in gene therapy: viral and nonviral delivery systems.
- Viral delivery systems e.g. , using adenoviruses or herpes simplex II viruses, are quite efficient, but the systems suffer disadvantages of toxicity, immunogenicity of the viral components, potential risk of reversion of the virus to a replication-competent state, potential introduction of tumor igenic mutations, lack of targeting mechanism, limitations in DNA capacity and difficulty in large-scale production.
- Non-viral delivery systems are cationic liposome-DNA complexes, i.e., lipoplexes, liposome containing a DNA encapsulated therein along with a DNA condensing agent, or polymer complexes, i.e., polyplexes (see Shangguan et al, Gene Therapy 7:769-783, 2000). These non-viral delivery systems protect the DNA from extracellular DNases by condensation (in lipoplexes and polyplexes) or physical separation of the DNA from the extracellular environment via a lipid bilayer (in true liposomes carrying the DNA). The true liposomes of the prior art carrying the DNA require the inclusion of a DNA condensing agent, e.g.
- the method of the present invention prepares liposomes containing a nucleic acid encapsulated therein without any requirement of the DNA condensing agent.
- the present invention is related to the use of liposomes as carrier of the nucleic acid.
- the liposomes prepared by the method of the present invention are useful in gene therapy if the nucleic acid encapsulated is a DNA.
- Liposomes are lipid vesicles having at least one aqueous phase completely enclosed by at least one lipid bilayer membrane. Liposomes can be unilamellar or multilamellar. Unilamellar liposomes are liposomes having a single lipid bilayer membrane. Multilamellar liposomes have more than one lipid bilayer membrane with each lipid bilayer membrane separated from the adjacent lipid bilayer membrane by an aqueous layer. The cross sectional view of multilamellar vesicles is often characterized by an onion-like structure. Liposomes are known to be useful in drug delivery, so many studies have been conducted on the methods of liposome preparation. Descriptions of these methods can be found in numerous reviews (e.g.
- a method of preparing multilamellar liposome was first reported by Bangham et al. (J. Mol. Biol. 13:238-252, 1965).
- phospholipids were mixed with an organic solvent to form a solution.
- the solution was then evaporated to dryness leaving behind a film of phospholipids on the internal surface of a container.
- An aqueous medium is added to the container to form multilamellar vesicles (hereinafter referred to as MLVs).
- MLVs multilamellar vesicles
- Small unilamellar vesicles hereinafter referred to as SUVs were prepared using sonication (Huang, Biochemistry 8:346-352, 1969).
- a phospholipid was dissolved in an organic solvent to form a solution, which was dried under nitrogen to remove the solvent.
- An aqueous phase was added to produce a suspension of vesicles.
- the suspension was sonicated until a clear liquid was obtained, which contained a dispersion of SUVs.
- LUVs Large unilamellar vesicles
- aqueous phase was added directly into the lipid solution in a ratio of the aqueous phase to the organic solvent of 1:3 to 1:6.
- the mixture of the lipid/organic solvent/aqueous phase was briefly sonicated to form a homogenous emulsion of inverted micelles.
- the organic solvent was then removed from the mixture in a two-step procedure, in which the mixture was evaporated at 200-400 mm Hg until the emulsion became a gel, which was then evaporated at 700 mm Hg to remove all the solvent allowing the micelles to coalesce to form a homogeneous dispersion of mainly unilamellar vesicles known as reverse-phase evaporation vesicles (hereinafter referred to as REVs) (e.g., see Papahaduopoulos, U.S. Patent No. 4,235,871).
- REVs reverse-phase evaporation vesicles
- a phospholipid was dispersed with a detergent, such as cholate, deoxycholate or Triton X-100, in an aqueous phase to produce a turbid suspension.
- the suspension was sonicated to become clear as a result of the formation of mixed micelles.
- the detergent was removed by dialysis or gel filtration to obtain the liposomes in the form of mostly large unilamellar vesicles (e.g., see Enoch et al. , Proc. Natl. Acad. Sci. USA, 76: 145-149, 1979).
- the liposomes prepared by the detergent removal method suffer a major disadvantage in the inability to completely remove the detergent, with the residual detergent changing the properties of the lipid bilayer and affecting retention of the aqueous phase.
- MPVs monophasic lipid vesicles
- SUVs lipid vesicles having a plurality of lipid bilayers.
- REVs Relatively lipid vesicles
- a lipid or lipid mixture and an aqueous phase were added to a water-miscible organic solvent in amounts sufficient to form a monophase. The solvent was then evaporated to form a film. An appropriate amount of the aqueous phase was added to suspend the film, and the suspension was agitated to form the MPVs.
- the gelatinous material was briefly heated at 51°C until the material liquified.
- the resulting liquid was centrifuged to form lipid vesicles containing the biologically active agent.
- the aqueous supernatant was removed and the pellet of lipid vesicles was washed several times.
- the modification of Minchey et al. was that the biologically active agent and the lipid were maintained as hydrated at all times to avoid the formation of a film of the biologically active agent and lipid upon the complete removal of all the aqueous phase. During evaporation of the organic solvent, the presence of a gel indicated that the monophase was hydrated.
- the present invention solves the problems by presenting a new relatively simple method of making liposomes containing a nucleic acid encapsulated therein having a high entrapment efficiency and of relatively homogeneous size.
- the method of the present invention is especially useful in encapsulating a plasmid DNA in liposomes.
- the liposomes so prepared using the gel hydration method of the present invention are useful in the transfection of eukaryotic cells due to their high transfection efficiency.
- the liposomes prepared by the method of the present invention are useful in gene therapy.
- the present invention involves the formation of liposomes via the hydration of a gel or a liquid containing gel particles, wherein the gel or the liquid containing gel particles comprise at least one liposome-forming lipid in a water- miscible organic solvent, preferably at a high concentration, and an aqueous medium, preferably in a small amount.
- One of the aspects of the present invention concerns a method of preparing liposomes containing at least one nucleic acid encapsulated therein, said method comprising the following steps:
- the amount of the at least one fusogenic lipid is at least about 20 % , at least about 30% , at least about 40%, at least about 50% , at least about 60%, at least about 70% , at least about 75 % or at least about 80% by weight of the lipid content of the gel or the liquid containing gel particles in step (A) or (B).
- the gel or the liquid containing gel particles of step (A) or (B) is prepared by a method comprising the following steps:
- step (II) (a) mixing the mixture of step (I)(a) with aqueous medium Y and optionally the nucleic acid to form the gel or liquid containing gel particles; or (b) mixing the mixture of step (I)(b) with the nucleic acid and aqueous medium Y to form the gel or liquid containing gel particles, wherein aqueous media X and Y are the same or different.
- the gel or the liquid containing gel particles of step (A) or (B) is prepared by a method comprising the following steps:
- step (ii) mixing the liposomes of step (I)(a)(i) with the at least one nucleic acid;
- step (ii) mixing the liposomes of step (I)(b)(i) with the at least one nucleic acid;
- step (ii) mixing the liposomes of step (I)(c)(i) with aqueous medium U and the at least one nucleic acid;
- step (ii) mixing the liposomes of step (I)(d)(i) with aqueous medium U and the at least one nucleic acid;
- step (e) forming liposomes comprising the at least one liposome-forming lipid and the at least one fusogenic lipid in the presence of the at least one nucleic acid by a method other than the instant method; (II) (a) mixing the product of step (I)(b), (I)(c) or (I)(d) with the water-miscible organic solvent to form the gel or the liquid containing gel particles; or
- step (b) mixing the product of step (I)(a) or (I)(e) with aqueous medium V and the water-miscible organic solvent to form the gel or the liquid containing gel particles, wherein aqueous media U and V are the same or different.
- aqueous media U and V are the same or different.
- liposomes containing the nucleic acid encapsulated therein as prepared by any of the above preparation methods.
- the present invention is also directed toward methods of using the liposomes containing the nucleic acid encapsulated therein as prepared by any of the above preparation methods in cell transfection, gene therapy, vaccination or diagnosis.
- the nucleic acid encapsulated is a DNA, especially a plasmid DNA
- the liposomes containing the nucleic acid encapsulated therein are useful for transfection of cells.
- Figure 1 shows, under a light microscope (magnification 400X), NAPE/DOPC (in a 70/30 molar ratio, with a volume ratio of aqueous phase :ethanol of 2: 1) liposomes prepared according to the method of the present invention before (top panel) and after (bottom panel) extrusion through a membrane filter having a 0.4 ⁇ m pore size.
- Figure 2 depicts the appearance of NAPE/DOPC (70/30) liposomes prepared according to the method of the present invention under freeze-fracture electron microscopy.
- Figure 3 depicts the appearance of NAPE/DOPC (70/30) liposomes prepared according to the method of the present invention under cryo electron microscopy.
- Figure 4 shows the encapsulation efficiencies and particle sizes of NAPE/DOPC (70/30) liposomes containing DNA prepared according to the method of the present inventions
- sample # column
- “extr” standed for extruded through a membrane filter of 400 nm pore size and "wash” represented samples washed with an aqueous medium.
- Three particle sizes were given for the samples in the order of: mean particle diameter weighted by number, mean particle diameter weighted by light reflection intensity and mean particle diameter weighted by volume. The particle sizes were below 400 nm. Also shown were the final DNA concentration, lipid concentration and ratio of DNA to lipid in the liposomes.
- Figure 5 shows the results of fractionation of NAPE/DOPC liposomes prepared according to the method of the present invention in a 5-20% sucrose gradient.
- the lipids were homogeneously distributed with no phase separation.
- the liposomes in the peak fractions had entrapment of 2.1 +/- 0.2 ⁇ l/ ⁇ mol of lipids.
- the open squares, labeled "p/pc" represented the phosphate to choline weight ratios, as determined by the respective assays, of the fractions separated by the sucrose gradient.
- Figure 6 is the phase diagram of a lipids-ethanol-aqueous buffer system, wherein the lipids were NAPE/DOPC (70/30, molar ratio).
- the three axes of the ternary phase diagram show the individual weight fractions of the three components (lipids, ethanol or aqueous buffer) based on the sum of the weight of the three components.
- the mixture was a clear liquid.
- the mixture existed as a cloudly liquid.
- the mixture was in a clear gel state.
- the mixture existed as a cloudy gel.
- Figure 7 shows the light scattering of 100 ⁇ g/ml enhanced green fluorescence protein (hereinafter referred to as EGFP) plasmid DNA in ethanol- LSB solution with or without 200 mM sodium chloride, wherein “LSB” represented “low salt buffer. "
- EGFP enhanced green fluorescence protein
- Figure 8 shows the transfection of OVCAR-3 cells with N-C12- DOPE/DOPC (70/30) liposomes (washed to remove unencapsulated DNA) prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent, wherein the liposomes (washed to remove unencapsulated DNA) contained EGFP plasmid DNA encapsulated therein.
- the transfection activity was determined based on the expression of the EGFP plasmid DNA in the OVCAR-3 cells. The transfection activity did not require any plasmid DNA condensing agent or any extrusion, which was a liposome size reduction process.
- FIG 9 shows the transfection of OVCAR-3 cells with N-C12- DOPE/DOPC (70/30) liposomes (washed to remove unencapsulated DNA) prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent, wherein the liposomes (washed to remove unencapsulated DNA) contained luciferase plasmid DNA encapsulated therein.
- the transfection activity was determined based on the expression of the luciferase gene in the plasmid DNA in the OVCAR-3 cells.
- the liposomes could transfect the OVCAR-3 cells in the presence of 10% serum (FBS stands for fetal bovine serum) with or without targeting via transferrin.
- FBS fetal bovine serum
- FIG. 10 shows the transfection of OVCAR-3 cells with N-C12- DOPE/DOPC (70/30) liposomes prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent, wherein the liposomes contained luciferase plasmid DNA encapsulated therein.
- the transfection activity was determined based on the expression of the luciferase gene in the plasmid DNA in the OVCAR-3 cells.
- the liposomes could transfect the OVCAR-3 cells at physiological Ca 2+ and Mg 2+ concentrations, i.e. , about 1.2 mM Ca 2+ and 0.8 mM Mg 2+ .
- Figure 11 shows the transferrin mediated binding of N-C12-DOPE/DOPC (70/30) liposomes prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent (see Example 13). The binding experiment was conducted in the presence of 10% FBS.
- Figure 12 shows the transferrin mediated transfection of N-C12- DOPE/DOPC (70/30) liposomes prepared by the gel-hydration method of the present invention using ethanol as the water-miscible organic solvent, wherein the liposomes contained PGL-3 plasmid DNA encapsulated therein.
- the experiment was conducted in the presence of 10% FBS.
- Figure 13 shows the transfection activity of liposomes prepared with pure DOPC, DOPC/N-C12-DOPE (8:2 molar ratio), DOPC/N-C12-DOPE (6:4 molar ratio), DOPC/N-C12-DOPE (4:6 molar ratio), DOPC/N-C12-DOPE (2:8 molar ratio) or pure N-C12-DOPE using the gel hydration method of the present invention in OVCAR-3 cells in culture. After incubation of the cells with the liposomes, the expression of the EGFP gene in the cells was determined by measuring the intensity of green fluorescence.
- Figure 14 shows the encapsulation efficiencies, for dextran fluorophores, of NAPE/DOPC (70/30) liposomes prepared using the gel hydration method of the present invention or using a process for making stable plurilamellar vesicles (SPLV).
- the NAPE/DOPC liposomes prepared according to the gel-hydration method of the present invention had a much higher encapsulation efficiency than the NAPE/DOPC liposomes prepared using the SPLV process.
- the method of preparing liposomes containing a nucleic acid encapsulated therein of the present invention involves hydration of a mixture of at least one nucleic acid at least one liposome-forming lipid, at least one fusogenic lipid and a water-miscible organic solvent in the form of a gel or a liquid containing gel particles.
- the liposome-forming lipid and the fusogenic lipid are typically dissolved in the water-miscible organic solvent, preferably at high concentrations.
- the mixture is typically mixed with a small amount of an aqueous medium to form the gel or the liquid containing gel particles. Hydration of the gel or the liquid containing gel particles leads to direct formation of liposomes without any additional manipulation, such as evaporation or sonication, normally required in prior art methods.
- the gel or the liquid containing gel particles may go through a wax stage before forming liposomes, but no additional manipulation, such as evaporation or sonication, is required other than an optional hydration of a waxy substance if the intermediate waxy substance is formed upon hydration of the gel or the liquid containing gel particles. For instance, when certain saturated liposome-forming lipids are used in the methods, the gel or gel particles go through the wax stage upon hydration before liposome formation.
- step (A) or (B) does not involve the use of any hydrating agent for the formation of the gel or the liquid containing gel particles.
- the hydrating agent is a compound having at least two ionizable groups, one of which ionizable groups is capable of forming an easily dissociative ionic salt, which salt can complex with the ionic functionality of the liposome-forming lipid.
- the hydrating agent inherently does not form liposomes in and of itself and the hydrating agent must also be physiologically acceptable.
- the at least two ionizable groups of the hydrating agent are of opposite charge.
- a phospholipid content of the gel or the liquid containing gel particles in step (A) or (B) is not 15 to 30% by weight of the gel or the liquid containing gel particles.
- a phospholipid content of the gel or the liquid containing gel particles in step (A) or (B) is not 15 to 30% by weight of the gel or the liquid containing gel particles and the content of the water-miscible organic solvent is not 14 to 20% by weight of the gel or the liquid containing gel particles.
- nucleic acid condensing agent e.g., a polycation of charge +3 or higher such as poly ly sine, poly amine and hexammine cobalt (III), is used.
- to directly form the liposomes means that the liposomes are formed without requiring any additional procedure or manipulation, such as evaporation or sonication, other than going through an optional intermediate stage of formation of a waxy substance if certain liposome- forming lipids are used.
- step (III) (A) leads directly to the formation of the liposomes having the nucleic acid entrapped without the requirement of any additional procedure or manipulation, such as evaporation or sonication, other than the hydration of a waxy intermediate if certain saturated liposome-forming lipids are used.
- the aqueous medium X, aqueous medium Y, aqueous medium Zl and/or aqueous medium Z2 is preferably an aqueous buffer.
- the aqueous buffer include citrate buffer, Tris buffer, phosphate buffer and a buffer containing sucrose or dextrose.
- the gel or the liquid containing gel particles and aqueous medium Zl are mixed by either adding aqueous medium Zl to the gel or the liquid containing gel particles, or adding or infusing the gel or the liquid containing gel particles into aqueous medium Zl.
- the at least one "liposome-forming lipid” is any lipid that is capable of forming liposomes.
- the at least one "liposome-forming lipid” is a lipid that can form lipid bilayers. Examples of the liposome-forming lipid include phospholipids, glycolipids and sphingolipids.
- the phospholipids that are liposome-forming include phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and N-acyl phospatidylethanolamine.
- the liposome-forming phospholipid examples include phospholipids selected from the group consisting of dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-oleoyl- 2-palmitoyl-sn-glycero-3-phosphocholine, l,2-dioleoyl-sn-glycero-3-[phospho-rac- ( 1 -gly cerol)] , 1 , 2-dipalmitoy 1-sn-gly cer o-3 - [phospho-rac-( 1 -gly cerol)] , 1,2- distearoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] , 1 ,2-dimyristo
- the at least one liposome-forming lipid is phosphatidylcholine, e.g., dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, l-palmitoyl-2-oleoy 1-sn- gly cero-3-phosphocholine and 2-palmitoyl-l-oleoyl-sn-glycero-3-phosphocholine, or N-acyl phosphatidylethanolamine, e.g.
- the at least one "fusogenic lipid” is a lipid that, upon incorporation into a liposome, increases the fusogenicity of the liposome and examples of the "fusogenic lipid” include N-acyl phosphatidylethanolamine (see Meers et al, U.S. Patent No. 6,120,797, the disclosure of which is herein incorporated by reference).
- the at least one liposome-forming lipid and the at least one fusogenic lipid are the same or different.
- the at least one liposome-forming lipid is also a fusogenic lipid.
- the N-acyl phosphatidylethanolamine is liposome- forming and also increases the fusogenicity of the liposomes (see U.S. Patent No. 6,120,797).
- N-acyl phosphatidylethanolamine that can be used include N-decanoyl phosphatidylethanolamine, N-undecanoyl phosphatidylethanolamine, N-dodecanoyl phosphatidylethanolamine, N-tridecanoyl phosphatidylethanolamine, and N- tetradecanoyl phosphatidylethanolamine, e.g.
- the fusogenicity-increasing N-acyl phosphatidylethanolamine is preferably N-dodecanoyl phosphatidylethanolamine and more preferably l,2-dioleoyl-sn-glycero-N-dodecanoyl-3- phosphoethanolamine .
- the liposome prepared by the method of preparing liposomes containing the nucleic acid encapsulated therein of the present invention can further comprise a sterol.
- the sterol is cholesterol.
- the sterol can be added in step (A) or (B) of the method.
- the liposomes prepared by the preparatory methods of the present invention can comprise one or a combination (at any ratio) of the following lipids (if a lipid is both liposome-forming and fusogenic, only one lipid is required but optionally at least one of the other lipids can be included in a combination; if a lipid is liposome-forming and not fusogenic, another lipid which is fusogenic is required but optionally at least one of the other lipids can be included in a combination; and if a lipid is fusogenic and not liposome-forming, another lipid which is liposome-forming is required but optionally at least one of the other lipids can be included in a combination): phosphatidylcholines, phosphatidylglycerols, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, headgroup modified phospholipids, headgroup modified phosphatidylethanolamines,
- the lipids can be added in step (A) or (B) when the gel or the liquid containing gel particles are mixed (the lipids can be a part of the gel or the liquid containing gel particles, or the lipids can be mixed with the aqueous medium and the gel or the liquid containing gel particles) or added before the gel or the liquid containing gel particles is formed (the lipids the lipids can be mixed with the water-miscible organic solvent, or the lipids can be a part of the liposome formed by a method other than the method of the present invention).
- At least one charged lipid is added in preparing the liposomes having the nucleic acid encapsulated therein.
- the at least one charged lipid can be added in step (I) and/or step (II) to form the gel or the liquid containing gel particles.
- the gel or the liquid containing gel particles comprising at least one charged lipid, at least one liposome-forming lipid, at least one fusogenic lipid, the water-miscible organic solvent and the nucleic acid is used, wherein the at least one charged lipid, the at least one liposome-forming lipid and the at least one fusogenic lipid are the same or different.
- the "charged lipid” is a lipid having a net negative or positive charge in the molecule.
- Examples of the charged lipid include N-acyl phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (i.e., cardiolipin) and phosphatidic acid.
- the water-miscible organic solvent is an organic solvent that, when mixed with water, forms a homogeneous liquid, i.e., with one phase.
- the water-miscible organic solvent can be selected from the group consisting of acetaldehyde, acetone, acetonitrile, allyl alcohol, allylamine, 2-amino-l-butanol, 1-aminoethanol, 2-aminoethanol, 2-amino-2-ethyl-l,3- propanediol, 2-amino-2-methyl-l-propanol, 3-aminopentane, N-(3- aminopropyl)morpholine, benzylamine, bis(2-ethoxyethyl) ether, bis(2- hydroxyethyl) ether, bis(2-hydropropyl) ether, bis(2-methoxyethyl) ether, 2- bromoethanol, meso-2,3-butanediol
- Acetonitrile, C C 3 alcohols and acetone are preferred examples of the water- miscible organic solvent.
- the C,-C 3 alcohols are preferably methanol, ethanol, 1- propanol, 2-propanol, ethylene glycol and propylene glycol, and more preferably ethanol, 1-propanol or 2-propanol, with ethanol being the most preferred.
- an organic solvent such as ethanol or acetone, of relatively low toxicity can be used.
- the liposomes prepared according to the method of the present invention would not be expected to pose any significant toxicity threat even when the liposomes contain a residual amount of the water-miscible organic solvent.
- the total amount of the at least one liposome-forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) can range from about 1 % by weight of the gel or the liquid containing gel particles to the sum of the solubility limit of the at least one liposome-forming lipid and the solubility limit of the at least one fusogenic lipid in the water-miscible organic solvent.
- the total amount of the at least one liposome- forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) can have a lower limit of about 5%, about 10%, about 15%, about 20%, about 30%, about 40%), about 50%, about 60% or about 70% by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B), and an upper limit of about 95% by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B).
- the total amount of the at least one liposome-forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) can have a lower limit of about 5%, about 10%, about 15%, about 20%, about 30%), about 40%, about 50%, about 60% or about 70% by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B), and an upper limit of about 90%> by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B).
- the total amount of the at least one liposome-forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) can have a lower limit of about 5%, about 10%, about 15%, about 20%, about 30%o, about 40%, about 50%, about 60% or about 70% by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B), and an upper limit of about 85% by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B).
- the total amount of the at least one liposome-forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) can also be from about 5% to about 80%, about 10%) to about 80%, about 15% to about 80%), about 20% to about 80%, about 30% to about 80%, about 40% to about 80%), about 50% to about 80%, about 60% to about 80%, about 70% to about 80%), about 10% to about 70%, about 20% to about 60%, or about 30% to about 50%) by weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with the aqueous medium Zl in step (A) or (B).
- the total amount of the at least one liposome-forming lipid and the at least one fusogenic lipid in the gel or the liquid containing gel particles before the mixing with the aqueous medium Zl in step (A) or (B) ranges from about 60% to about 90%), or is about 45%>, by weight of gel or the liquid containing gel particles.
- aqueous medium Zl is preferably mixed with the gel or the liquid containing gel particles in increments.
- Mixing in increments has the advantage of yielding a higher entrapment efficiency compared with mixing the entire amount of aqeuous medium Zl with the gel or the liquid containing gel particles in one step.
- the size of the increment can be up to about 100%, up to about 90% , up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20% , up to about 10% , up to about 5%, up to about 2%, up to about 1 % , up to about 0. % , up to about 0.1 % , up to about 0.05 % or up to about 0.01 % of the weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with any aqueous medium Zl.
- the size of the increment can also be from about 0.001 % to about 10% , from about 0.001 % to about 5%, from about 0.001 % to about 1 % or from about 0.001 % to about 0.1 % of the weight of the gel or the liquid containing gel particles before the gel or the liquid is mixed with any aqueous medium Zl.
- Figure 6 shows the phase diagram of a lipids/water-miscible organic solvent/aqueous medium system that can be used in the liposome preparatory method of the present invention, wherein the lipids are N-C12-DOPE/DOPC (70/30, molar ratio). Ethanol was the water-miscible organic solvent and Tris buffer was the aqueous medium.
- the three axes of the ternary phase diagrams show the individual weight fractions of the three components (lipids, ethanol or aqueous buffer). In the ternary phase diagram, the liquid or solution zone, the gel zone and the liposome zone are depicted.
- ternary phase diagrams can be generated by a person skilled in the art without undue experimentation for other lipid(s)/water-miscible organic solvent/aqueous medium systems.
- the method of the present invention can, however, be practiced without the ternary phase diagrams.
- the ternary phase diagrams are used to show the general relationship between the fluid zone, gel zone and liposome zone for the particular lipid(s)/water-miscible organic solvent/aqueous medium system used in the method.
- the liposomes are washed with an aqueous medium by centrifugation, gel filtration or dialysis.
- Liposomes are useful as delivery vehicles of encapsulated substances.
- the method of the present invention can be used to encapsulate at least one nucleic acid in liposomes.
- the liposomes containing the at least one nucleic acid encapsulated therein prepared by the method of the present invention have the advantages of a high entrapment efficiency and a relatively homogeneous particle size.
- the method of preparing the liposomes of the present invention allows relatively rapid production of the liposomes at a low cost.
- the method of the present invention has the additional advantage of being easily controlled and modified, e.g., by selecting a batch or continuous operation, to fit the special requirements of different formulations.
- the at least one nucleic acid encapsulated in the liposomes of the present invention is an oligonucleotide, RNA or DNA.
- the oligonucleotide that can be encapsulated can be of about 5 to about 500 bases in size.
- Examples of RNA that can be encapsulated in the liposomes prepared according to the present invention are anti-sense RNA and RNA interference, i.e. , RNA,.
- the DNA that can be encapsulated in the liposomes prepared according to the present invention includes a plasmid DNA.
- the plasmid DNA can be of up to 20 kb, up to 15 kb, up to 10 kb, from about 0.5 kb to about 20 kb, from about 1 kb to about 15 kb, from about 2 kb to about 10 kb or from about 3 kb to about 7 kb in size.
- Liposomes of the present invention containing the plasmid DNA are useful in gene therapy, transfection of eukaryotic cells and transformation of prokaryotic cells.
- the liposomes prepared by the method of the present invention containing a plasmid DNA encapsulated therein have a high transfection efficiency.
- the liposomes of the present invention having at least one nucleic acid encapsulated therein can be administered to a subject in need of the nucleic acid via an oral or parenteral route (e.g., intravenous, intramuscular, intraperitoneal and intrathecal routes) for therapeutic or diagnostic purposes.
- the dose of the liposomes to be administered is dependent on the nucleic acid involved, and can be adjusted by a person skilled in the art based on the health of the subject and the medical condition to be treated or diagnosed. For diagnostic purposes, some the liposomes of the present invention can be used in vitro.
- the present invention comprises a method of preventing or treating a health disorder in a subject in need of the treatment or prevention, said method comprises administering the liposomes containing a nucleic acid encapsulated therein as prepared by one of the above methods in the subject.
- the nucleic acid can be an RNA, such as anti-sense RNA or RNA ; , or plasmid DNA.
- the present invention encompasses a method of transfecting cells with a DNA, said method comprises using the liposomes containing a DNA encapsulated therein by mixing the liposomes prepared according to the liposome preparatory method of the present invention with the cells with optional incubation.
- the DNA preferably is a plasmid DNA.
- the plasmid DNA preferably contains a gene of interest for the transfection.
- a concentration of the nucleic acid is up to about 40 mg/ml, up to about 30 mg/ml, up to about 20 mg/ml, up to about 10 mg/ml or up to about 5 mg/ml.
- the liposomes containing the nucleic acid encapsulated therein prepared by the method of the present invention can further comprise a targeting agent to facilitate the delivery of the nucleic acid to a proper target in a biological system.
- the targeting agent examples include antibodies, a molecule containing biotin, a molecule containing streptavidin, or a molecule containing a folate or transferrin molecule.
- N-C 12-DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dodecanoyl
- DOPC 1,2-dioleoy 1-sn-gly cer o-3 -phosphocholine
- POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
- DSPC 2-distear oy 1-sn-gly cero-3 -phosphocholine
- DSPG l,2-distearoyl-sn-glycero-3-[phospho-rac-(l-glycerol)]
- EGFP plasmid DNA enhanced green fluorescence protein plasmid DNA
- N-C12-DOPE and 14.2 mg of DOPC were co-dissolved in 100 ⁇ l ethanol.
- a volume of 100-200 ⁇ l of an aqueous solution containing a biological active substance was injected into the lipid ethanol solution under intense mixing.
- 1.8 ml of a hydration buffer 300 mM sucrose, 10 mM Tris, 1 mM NaCl, pH 7.0 was slowly added to the sample to form a suspension of liposomes.
- any unencapsulated material was removed by washing (one wash consisted of (1) sedimenting the liposomes in an aqueous phase, (2) replacing the supernatant with fresh aqueous phase, and (3) resuspending the pellet) the liposomes three times via 10,000 g centrifugation.
- the nucleic acid to be encapsulated was a EGFP plasmid DNA or PGL-3 plasmid
- the liposome-forming lipid to be used was a mixture of N-C12- DOPE/DOPC (in a molar ratio of 70/30), generally the following procedure could be used to prepare the liposomes with gel hydration.
- the lipid mixture N-C12- DOPE/DOPC (in a molar ratio of 70/30), was dissolved in ethanol at a concentration of about 600 mM.
- the plasmid DNA was added in an aqueous solution at a concentration of about 1 to 4 mg/ml to the lipid ethanol solution to form a clear gel.
- the gel was hydrated by adding an aqueous buffer (10 mM Tris, 1 mM sodium chloride, 300 mM sucrose, pH 7.0) under intense mixing. The gel turned cloudy and finally collapsed after additional aqueous solution was added.
- the so formed liposome suspension was washed (a wash was (1) sedimenting the liposomes in an aqueous phase, (2) replacing the supernatant with a fresh aqueous phase, and (3) resuspending the pellet) by centrifugation to remove any free plasmid DNA.
- NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of DOPC and 400 ⁇ g of EGFP plasmid DNA.
- Light micrographs (Olympus BH-2, New York/New Jersey Scientific) of these liposomes before and after five passes of extrusion through a membrane filter with 400 nm pore size were taken at a magnification of 400X (see Figure 1, top and bottom panels).
- NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of DOPC and 400 ⁇ g PGL-3 plasmid DNA (a commercially available plasmid DNA containing luciferase as a reporter gene). Freeze fracture electron replicas were made and observed at magnifications of about 43,000X (see Figure 2).
- NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of
- DOPC and 400 ⁇ g of EGFP plasmid DNA Liposomes samples were placed on Quantifoil 2/2 grids, blotted with a filtering paper to form a uniform thin film of liquid 1-2 mm in thickness, and flush-frozen by plunging into liquid ethane.
- NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of DOPC and 400 ⁇ g PGL-3 or EGFP plasmid DNA. Their particle sizes were measure by a Submicron Particle Sizer (model 370), from NICOMP Particle Sizing Systems, Inc. Mean particle diameters (nm), as measured by number, intensity or volume weighting, were smaller than 400nm ( Figure 4).
- NAPE/DOPC liposomes (70:30, molar ratio) were prepared by the gel hydration process (as set forth in Example 1) using 36.7 mg of NAPE, 14.2 mg of DOPC and 400 ⁇ g PGL-3 or EGFP plasmid DNA.
- the liposomes had DNA: lipid ratios of about 1-2 ⁇ g/ ⁇ mole ( Figure 4), as determined by a phosphate assay and Picogreen assay (Shangguan et al., Gene Therapy, 769-783, 2000), respectively.
- the plasmid DNA was protected against DNase I digestion as described in Shangguan et al.
- a 5-20% continuous sucrose gradient was obtained by mixing a 10 mM Tris buffer, pH 7, containing 140 mM NaCl, and the same buffer, containing 20% sucrose instead of NaCl.
- the liposomes were loaded on top of the gradient and centrifuged for 17 hours at 35,000 rpm. The centrifugation yielded a single band of liposomes centered at approximately 10% sucrose.
- the contents of the centrifuge tubes were fractionated starting from the bottom.
- the concentrations of the total phosholipids and DOPC were determined using phosphate and choline assays. In all fractions examined, the phosphate to choline ratios were nearly the same: 3 +0.2 (see Figure 5), which indicates compositional homogeneity of mixed lipid liposomes.
- NAPE/DOPC - Ethanol - Aqueous Phase Diagram Different amounts of 5-60 mg of NAPE/DOPC lipid mixtures (70:30, molar ratio) were dissolved in 38-190 mg ethanol to reach lipid concentrations of 3%, 14%, 18% . 25%, 31 %, 40% , and 60% (wt/wt).
- a 5 mM HEPES buffer pH 7.5 was added incrementally to the lipid solutions at increments of 20-25 mg under intense mixing. The total weight of added buffer was recorded each time when the mixtures underwent a phase change.
- NAPE/DOPC lipid mixtures (70:30, molar ratio) were suspended in 34-77 mg of a 5 mM HEPES buffer (pH 7.5) to reach lipid concentrations of 25% , 33% , 43% , and 60% (wt/wt). Ethanol was added incrementally to the lipid suspensions at increments of 15-30 mg under intense mixing. The total weight of added ethanol was recorded each time when the mixtures underwent a phase change. A ternary lipids - ethanol - aqueous phase diagram was constructed by connecting the critical points at which the mixture underwent any phase change (Figure 6).
- a volume of 85.7 ⁇ l of a EGFP plasmid DNA stock solution (3.5mg EGFP plasmid DNA/ml) was added to each of 0-97% (wt/wt) ethanol solutions.
- the ethanol solution contained 200 mM NaCl. 90° light scattering of the EGFP plasmid DNA at 875 nm in different ethanol solutions was presented in Figure 7. This experiment was conducted to determine the effect of ethanol on the plasmid DNA.
- the 200 mM NaCl solution was used to mimic the ionic strength in the gel containing N-C12-DOPE.
- the N-C12-DOPE/DOPC (70:30) liposomes containing the EGFP plasmid DNA were made by the gel hydration method as described above. Half of the sample was extruded through a 400 nm filter five times before removal of unencapsulated DNA.
- OVCAR3 cells were plated in 96 well plates at 2 x 10 5 cells/ml in 0.1 ml/well of RPMI 1640 with 10% heat inactivated fetal bovine serum (FBS). The cells were allowed to grow for approximately 40-48 hours before transfections were performed. At this point the cells were at confluency.
- Transfection solutions (0.1 ml/well for 96 well plates) were prepared by dilution of appropriate liposome samples to approximately 2 mM total lipid (for equal lipid transfection) into medium with 0.5% FBS.
- the plates were aspirated to remove medium and washed once with Dulbecco's phosphate buffered saline (PBS) followed by aspiration.
- PBS Dulbecco's phosphate buffered saline
- the transfection solution was then added to the wells and incubated at 37 °C for 3 hours. After incubation, the wells were aspirated and a medium containing 10% heat inactivated FBS was added to each well.
- the N-C12-DOPE/DOPC (70:30) liposomes containing PGL-3 plasmid were made by the gel hydration method as described above. Transfections without transferrin were performed as described in example 10, except that in one of the transfection assays, 10% FBS instead of 0.5% FBS was used.
- the liposome samples were first mixed with equal volumes of a 2 mg/ml poly-lysin transferrin conjugate at a concentration of 20 mM for 10 minutes, and then this mixture was diluted 10 times with Hank's balanced salt solution (HBSS) without Ca 2+ /Mg 2+ containing 10% FBS before being applied to the cells.
- HBSS Hank's balanced salt solution
- the level of luciferase expression was determined by the Bright-glow luciferase assay (Clontech).
- the N-C12-DOPE/DOPC (70:30) liposomes containing PGL-3 plasmid were made by the gel hydration method as described above.
- the transfections were performed as described in example 10, in the presence of 0.5% FBS and without targeting, except that various volumes of CaCl 2 and MgCl 2 solution were added to 500 ⁇ l of the transfection solution before their addition to the cells at 100 ⁇ l per well to test the Ca 2+ /Mg 2+ dependence of the transfection activity.
- the level of luciferase expression was determined by the Bright-glow luciferase assay (Clontech).
- the N-C12-DOPE/DOPC (70:30) liposomes had transfection activity at physiological concentrations of Ca 2+ -Mg 2+ , i.e. , about 1.2 mM Ca 2+ and 0.8 mM Mg 2+ ( Figure 10).
- Example 13 Transferrin Mediated Binding of N-C12-DOPE/DOPC (70:30) Liposomes in 10% FBS ( Figure 11)
- the N-C12-DOPE/DOPC (70:30) liposomes containing fluorescent lipid probe Dil at a 0.1 % (wt%) concentration were prepared by the ethanol gel hydration method as described above.
- the liposomes were incubated with OVCAR-3 cells in the presence of 10% FBS and various concentrations of transferrin as described in Example 11. After a 3 hour incubation at 37 °C, the cells were washed three times with PBS and dissolved in 1 % C12E8. Cell associated Dil fluorescence was measured at an emission wavelength of 620 nm, with an excitation wavelength of 560 nm. Binding of the liposome sample showed a small increase with increasing transferrin concentration (Figure 11).
- the N-C12-DOPE/DOPC (70:30) liposomes containing PGL-3 plasmid were made by the gel hydration method as described above.
- the transfections were performed as described in Example 11, in the presence of 10% FBS and with various concentrations of transferrin for targeting.
- the level of luciferase expression was determined by the Bright-glow luciferase assay (Clontech).
- the liposome sample showed a transferrin dependent increase of transfection activity
- the liposomes containing a EGFP plasmid DNA and the following lipids or lipid mixtures including 100% DOPC, DOPC/N-C12-DOPE (8:2 molar ratio), DOPC/N-C12-DOPE (6:4 molar ratio), DOPC/N-C12-DOPE (4:6 molar ratio),
- DOPC/N-C12-DOPE (2:8 molar ratio), and 100% N-C12-DOPE, were made by the ethanol gel hydration method as described above.
- the transfection assay was performed as described in Example 10.
- Encapsulation of Dextran N-C12-DOPE/DOPC liposomes were prepared by the gel hydration process (as set forth in Example 1) using 36.7mg of N-C12-DOPE, 14.2 mg of DOPC and 100 ⁇ l of one of the following dextran stock solutions (5 mg/ml): tetramethyl rhodamine (MW 70,000), tetramethyl rhodamine (MW 2,000,000) or fluorescein (MW 70,000, lysine fixable).
- NAPE/DOPC liposomes 70:30, molar ratio
- SPLV method 1.13 ml of N-C12-DOPE/DOPC lipid mixtures(60 mM total lipid, 70:30 molar ratio) in chloroform were mixed with 100 ⁇ l of one of the following dextran stock solutions (5 mg/ml): tetramethyl rhodamine (MW 70,000), tetramethyl rhodamine (MW 2,000,000) or fluorescein (MW 70,000, lysine fixable). The mixture was sonicated briefly to form an emulsion.
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AU2002359892A AU2002359892A1 (en) | 2001-12-31 | 2002-12-31 | Efficient nucleic acid encapsulation into medium sized liposomes |
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Cited By (20)
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WO2009082817A1 (fr) | 2007-12-27 | 2009-07-09 | Protiva Biotherapeutics, Inc. | Silençage de l'expression de la polo-like kinase à l'aide d'un arn interférent |
WO2009129319A2 (fr) | 2008-04-15 | 2009-10-22 | Protiva Biotherapeutics, Inc. | Réduction au silence de l'expression du gène csn5 au moyen d'arn interférant |
WO2010020935A2 (fr) | 2008-08-18 | 2010-02-25 | Universidade Do Minho | Utilisation de monooléine comme nouveau lipide adjuvant dans la lipofection |
EP2395012A2 (fr) | 2005-11-02 | 2011-12-14 | Protiva Biotherapeutics Inc. | Molécules d'ARNsi modifiées et leurs utilisations |
WO2011160062A2 (fr) | 2010-06-17 | 2011-12-22 | The Usa As Represented By The Secretary, National Institutes Of Health | Compositions et procédés pour traiter des affections inflammatoires |
WO2012106490A1 (fr) | 2011-02-03 | 2012-08-09 | The Government Of The U.S.A., As Represented By The Secretary, Department Of Health & Human Services | Vaccins multivalents pour le virus de la rage et des filovirus |
WO2013075132A1 (fr) | 2011-11-17 | 2013-05-23 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Compositions de commutateurs arn thérapeutiques et procédés d'utilisation |
WO2013124327A1 (fr) | 2012-02-21 | 2013-08-29 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Récepteurs tim comme cofacteurs d'entrée de virus |
WO2013124324A1 (fr) | 2012-02-21 | 2013-08-29 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Récepteurs tam comme cofacteurs d'entrée de virus |
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WO2016054421A1 (fr) | 2014-10-02 | 2016-04-07 | Protiva Biotherapeutics, Inc | Compositions et méthodes d'extinction de l'expression du gène du virus de l'hépatite b |
WO2016197132A1 (fr) | 2015-06-04 | 2016-12-08 | Protiva Biotherapeutics Inc. | Traitement d'une infection à virus de l'hépatite b à de l'aide de crispr |
WO2017019891A2 (fr) | 2015-07-29 | 2017-02-02 | Protiva Biotherapeutics, Inc. | Compositions et méthodes de silençage de l'expression du gène du virus de l'hépatite b |
WO2017147540A1 (fr) | 2016-02-25 | 2017-08-31 | Applied Biological Laboratories, Inc. | Compositions et procédés de protection contre des agents pathogènes et des substances irritantes aériens |
WO2017176596A1 (fr) | 2016-04-04 | 2017-10-12 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Vaccins multivalents contre le virus de la rage et des coronavirus |
DE212016000074U1 (de) | 2015-04-09 | 2018-01-02 | Bikanta Corp. | Bildgebende Systeme mit fluoreszenten Nanodiamanten |
WO2019046426A1 (fr) | 2017-08-30 | 2019-03-07 | Applied Biological Laboratories, Inc. | Compositions et méthodes de protection contre des agents pathogènes et irritants en suspension dans l'air |
WO2019101882A1 (fr) | 2017-11-23 | 2019-05-31 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nouveau procédé de traitement d'une infection par le virus de la dengue |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5741513A (en) * | 1990-02-08 | 1998-04-21 | A. Natterman & Cie. Gmbh | Alcoholic aqueous gel-like phospholipid composition, its use and topical preparations containing it |
US5980935A (en) * | 1996-05-15 | 1999-11-09 | Kirpotin; Dmitri | Cationic lipids and methods of use therefor |
-
2002
- 2002-12-31 AU AU2002359892A patent/AU2002359892A1/en not_active Abandoned
- 2002-12-31 WO PCT/US2002/041814 patent/WO2003057190A1/fr not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5741513A (en) * | 1990-02-08 | 1998-04-21 | A. Natterman & Cie. Gmbh | Alcoholic aqueous gel-like phospholipid composition, its use and topical preparations containing it |
US5980935A (en) * | 1996-05-15 | 1999-11-09 | Kirpotin; Dmitri | Cationic lipids and methods of use therefor |
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EP2395012A2 (fr) | 2005-11-02 | 2011-12-14 | Protiva Biotherapeutics Inc. | Molécules d'ARNsi modifiées et leurs utilisations |
WO2009082817A1 (fr) | 2007-12-27 | 2009-07-09 | Protiva Biotherapeutics, Inc. | Silençage de l'expression de la polo-like kinase à l'aide d'un arn interférent |
WO2009129319A2 (fr) | 2008-04-15 | 2009-10-22 | Protiva Biotherapeutics, Inc. | Réduction au silence de l'expression du gène csn5 au moyen d'arn interférant |
EP2770057A1 (fr) | 2008-04-15 | 2014-08-27 | Protiva Biotherapeutics Inc. | Silençage de l'expression du gène CSN5 au moyen d'ARN interférant |
WO2010020935A2 (fr) | 2008-08-18 | 2010-02-25 | Universidade Do Minho | Utilisation de monooléine comme nouveau lipide adjuvant dans la lipofection |
WO2011160062A2 (fr) | 2010-06-17 | 2011-12-22 | The Usa As Represented By The Secretary, National Institutes Of Health | Compositions et procédés pour traiter des affections inflammatoires |
WO2012106490A1 (fr) | 2011-02-03 | 2012-08-09 | The Government Of The U.S.A., As Represented By The Secretary, Department Of Health & Human Services | Vaccins multivalents pour le virus de la rage et des filovirus |
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WO2013075132A1 (fr) | 2011-11-17 | 2013-05-23 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Compositions de commutateurs arn thérapeutiques et procédés d'utilisation |
US9035039B2 (en) | 2011-12-22 | 2015-05-19 | Protiva Biotherapeutics, Inc. | Compositions and methods for silencing SMAD4 |
WO2013124324A1 (fr) | 2012-02-21 | 2013-08-29 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Récepteurs tam comme cofacteurs d'entrée de virus |
WO2013124327A1 (fr) | 2012-02-21 | 2013-08-29 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Récepteurs tim comme cofacteurs d'entrée de virus |
WO2014016152A1 (fr) | 2012-07-27 | 2014-01-30 | Institut National De La Sante Et De La Recherche Medicale | Utilisation du cd147 comme récepteur pour l'adhésion impliquant les pili des méningocoques à l'endothélium vasculaire |
WO2014046983A1 (fr) | 2012-09-21 | 2014-03-27 | Intensity Therapeutic | Procédé de traitement du cancer |
WO2016054421A1 (fr) | 2014-10-02 | 2016-04-07 | Protiva Biotherapeutics, Inc | Compositions et méthodes d'extinction de l'expression du gène du virus de l'hépatite b |
DE212016000074U1 (de) | 2015-04-09 | 2018-01-02 | Bikanta Corp. | Bildgebende Systeme mit fluoreszenten Nanodiamanten |
WO2016197132A1 (fr) | 2015-06-04 | 2016-12-08 | Protiva Biotherapeutics Inc. | Traitement d'une infection à virus de l'hépatite b à de l'aide de crispr |
WO2017019891A2 (fr) | 2015-07-29 | 2017-02-02 | Protiva Biotherapeutics, Inc. | Compositions et méthodes de silençage de l'expression du gène du virus de l'hépatite b |
WO2017147540A1 (fr) | 2016-02-25 | 2017-08-31 | Applied Biological Laboratories, Inc. | Compositions et procédés de protection contre des agents pathogènes et des substances irritantes aériens |
WO2017176596A1 (fr) | 2016-04-04 | 2017-10-12 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Vaccins multivalents contre le virus de la rage et des coronavirus |
EP4104854A2 (fr) | 2016-04-04 | 2022-12-21 | The United States of America as represented by the Secretary of the Department of Health and Human Services | Vaccins multivalents pour le virus de la rage et les coronavirus |
WO2019046426A1 (fr) | 2017-08-30 | 2019-03-07 | Applied Biological Laboratories, Inc. | Compositions et méthodes de protection contre des agents pathogènes et irritants en suspension dans l'air |
WO2019101882A1 (fr) | 2017-11-23 | 2019-05-31 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nouveau procédé de traitement d'une infection par le virus de la dengue |
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