WO1997043363A1 - Cationic lipids and methods of use therefor - Google Patents
Cationic lipids and methods of use therefor Download PDFInfo
- Publication number
- WO1997043363A1 WO1997043363A1 PCT/US1997/008120 US9708120W WO9743363A1 WO 1997043363 A1 WO1997043363 A1 WO 1997043363A1 US 9708120 W US9708120 W US 9708120W WO 9743363 A1 WO9743363 A1 WO 9743363A1
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- Prior art keywords
- compound
- substance
- cells
- arg
- cationic
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Classifications
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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
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
- A61K47/544—Phospholipids
-
- 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
- A61K47/6907—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 microemulsion, nanoemulsion or micelle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
Definitions
- the present invention relates generally to a non-toxic lipid conjugated with a cationic amino acid containing a guanidino group.
- a non-toxic lipid conjugated with a cationic amino acid containing a guanidino group Specifically, the naturally-occuring lipid dioleoylphosphatidylethanolamine (DOPE) is combined with the naturally-occurring amino acid Arginine.
- DOPE naturally-occuring lipid dioleoylphosphatidylethanolamine
- Arginine lipid dioleoylphosphatidylethanolamine
- These compounds are useful for encapsulating and delivering pharmaceuticals and poly and oligonucleotides. These compounds improve over current compounds, because they are composed of non-toxic and, in the case of Arginine conjugated with DOPE (Arg-PE), natural components, and therefore result in minimal unwanted side effects.
- Methods of use of the cationic lipids are also claimed.
- Cationic lipids have been described in the past. Most of the cationic lipids previously described involve synthetic (non-naturally-occurring) components. The three patents provided in this background to the invention also describe many non-naturally- occurring components.
- Fig 1. Effect of Arg-PE and commercially available cationic lipids on the growth of cultured KB31 cells using lipids alone.
- Fig. 2. Effect of Arg-PE and commercially available cationic lipids on the growth of cultured KB31 cells using lipid -DNA complexes. DNA/lipid ratios are 1:18 for LFA, TP, LF; 1:9 for TA, Arg-PE; 1:27 for LCE.
- Fig. 3 Expression of Iuciferase reporter gene in C26 cells transfected with complexes of pCMVLUC with various cationic lipids.
- Fig. 4 Expression of Iuciferase reporter gene in H1048 cells transfected with complexes of pCMVLUC with various cationic lipids.
- Fig. 5 Expression of Iuciferase reporter gene in KB 31 cells transfected with complexes of pCMVLUC with various cationic lipids.
- This invention relates, inter alia, to materials used in facilitating the delivery of nucleic acids and oligonucleotides into living cells.
- the utility of such delivery is recognized in the practice of biomedical research and industry, biotechnology, and medicine.
- cationic lipids for facilitating the entry of functional nucleic acids and oligo nucleotides into living cells has been described in the scientific and patent literature.
- the array of molecular structures of such lipids as reviewed, for example, in Remy, cited above, and Behr, cited above, demonstrates that cationic properties of such lipids have been provided by introduction of a positively-charged group, or groups, based on the ammonium function.
- ammonium group (pK 9.2) is a weaker base than guanidine group (pK 12.7) present in the natural protein amino acid arginine, while the use of strong bases such as quaternary ammonium groups renders a molecule of cationic lipid more toxic and less biodegradable by the cell. It is also noteworthy that protamines, natural polypeptides with the highest DNA-compacting ability, have about 60% arginine content.
- An underlying concept of the present invention is to employ a guanidine-bearing group with an arginine residue in an amphipatic construct as a candidate for a nucleic acid cellular delivery vehicle which would be readily degraded by cellular enzymes, and fragments resulting from such degradation would be natural ubiquitous metabolites of a cell, ln one aspect of the invention, the hydrophilic arm has charged groups, (either negative or zwitterionic in nature), which are useful for forming bilayers in the physiological pH and ionic environment. Liposomal delivery is therefore more advantagous using the present invention. Most specifically, a compound which bears a phosphatidyl group is disclosed in the present invention. For example, N-L-Arginyl- phosphatidyl-ethanolamine is provided by the present invention.
- Arg-PE N -arginyl-PE
- This compound possesses a net cationic charge due to the presence of one acidic and two basic groups, one of the latter being a guanidine group of the arginyl residue.
- This molecule would be easily split by cellular peptidases into its original components, arginine and PE, both of which are natural cellular constituents.
- Arg-PE for its ability to deliver functional plasmid DNA and phosphorothioate oligonucleotide (PS ON) into living cells.
- PS ON functional plasmid DNA and phosphorothioate oligonucleotide
- DOGS polycationic lipid
- the inventive lipid was highly active for the DNA delivery into the cells even without the use of a"helper lipid. Helper lipids are required for the activity of previously known monocationic lipids.
- Toxicity of most currently available cationic lipids is a limiting factor in their practical uses.
- the present invention is not limited to the above-described embodiment, Arg-PE, which is merely an example of possible embodiments. More broadly, the invention covers a group of materials whose molecules are capable of bearing a net cationic charge in an aqueous solution and are capable of being degraded in the living cells into non-toxic, metabolizable fragments comprising (1) a guanidino domain as a bearer of the cationic charge; (2) a hydrophobic domain capable of causing the molecule to form micellular structures in aqueous medium and (3) a hydrophilic arm Unking together the above two domains.
- the invented lipids may be formulated alone, or in the mixture with other (non-cationic) lipids, or even combined with other cationic lipids, in the form of micellular solution, or bilayer vesicles (liposomes), in an aqueous medium, and brought into contact with a polynucleotide (DNA or RNA), or ohgonucleotide, prior to administration to the cells.
- the lipid may be formulated as a solution in a water-miscible organic solvent, such as ethanol, and combined with the poly or oligonucleotide in an aqueous medium prior to administration to the cells.
- the invented materials alone are capable of forming bilayer vesicles (liposomes) in an aqueous buffer. Since cationic liposomes are known to be the instruments for intracellular dehvery of substances other than nucleic acids (Debs etal., 265 J. Biol. Chem. 10189 (1990)), the liposomes formed by the invented lipids have utility for the cellular delivery of substances other than poly- or oligonucleotides, such as, for example, proteins and various pharmaceuticals.
- the present invention therefore provides methods for treating various disease states, so long as the treatment involves transfer of material into cells. In particular, treating the following disease states using the present invention is mcluded within the scope of this invention: cancer, infectious diseases, inflammatory diseases and genetic hereditary diseases.
- Example 1 and the final product was purified by chromatography. Yield of Arg-PE: 33.5 mg (24% of theory). Molar ratio of phosphate to primary amino group: theory, 1.0; found 0.88 ⁇ 0.08.
- DOTAP- DOPE 1:1 was prepared from the mixture (1:1 by weight) of 1,2 dioleoyloxy-3- trimethylammoniopropane (DOTAP, Avanti PolarLipids, USA) and DOPE by dissolving in distilled water at the concentration 2 mg/ml.
- DDAB-DOPE 1:2.5 (also known as LipofectACE®, Gibco BRL, USA) was prepared from the mixture of dioctadecyl- di methylammonium bromide (DDAB, Sigma, USA) and DOPE in the weight ratio 1 :2.5 dissolved in distilled water at concentration of 3 mg/ml with brief sonication. All lipid solutions were sterilized by filtration though 0.2 micrometer cellulase acetate filter, unless supplied sterile by manufacturers.
- Human epidermoid carcinoma (KB31) cells were grown at 37°C and 5% CO 2 in
- RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum.
- the cells were plated into a 12-well cell culture plate at 1.5xl() 5 cells/well in 1 ml of the growth medium.
- 1 microgram of bacterial plasmid (pUC- CM VLUC) with Iuciferase reporter gene under control of the CMV promoter was added to the cell media, either alone, or in the complex with cationic lipids as indicated below, prepared at a variety of lipid-to-DNA ratios.
- the complexes were prepared by mixing the lipid, as supplied by the manufacturer, and 1 microgram of the plasmid in 40 microliters of HEPES-NS.
- HEPES-BSS HEPES at pH 7.4
- HEPES-BSS HEPES at pH 7.4
- EDTA 3 mM EDTA
- HEPES-BSS 0.1 M potassium phosphate
- the extracts were assayed for Iuciferase by luminometry and for total protein using protein dye method (Bio-Rad, USA). The results are displayed in Figure 5 and Table 1.
- Table 1 Comparative expression of LUC gene in KB cells transfected with pUC- CMVLUC plasmid alone or in a complex with Arg-PE or commercially available cationic lipids.
- Cells were grown as described in Example 4, plated at 5xl03/well in 96-well cell culture plates, and acclimated for 48 hours. Lipids alone, or in the complex with pUC- CMVLUC plasmid at the indicated ratio were added to the cell medium at concentrations of 3.2-100 microgram of lipid per 1 ml of growth medium. After 24 hours exposure to the hpids, the hpid-containing media were removed, and the cells were further incubated in fresh growth medium for 65 hours. At the end of incubations, the cell viability was determined by MTT assay as described in T. Mossman 65 J. Immunol. Methods 55 (1983). The results are shown in Figures 1 and 2.
- Arg-PE displayed the least toxicity when given either as such or in the form of DNA-lipid complexes. 7. Delivery of a phosphorothioate oligonucleotide into the cells via complexes with Arg-PE and commercial cationic lipids.
- NCI-H1048 Human small cell lung carcinoma cells
- RPMI-1640 medium with 10% heat-inactivated fetal calf serum.
- the cells were exposed for 7 hours in a serum-free medium with a fluorescein-labeled 24-mer phosphorothioate ohgonucleotide (0.5 micro-M final concentration), alone or in a complex with lipids obtained as described in Examples 2 and 3.
- the equal volume of serum-supplemented medium was added, and the cells were incubated for another 18 hours.
- cellular accumulation of the ohgonucleotide was assayed by flow cytometry using fluorescein label fluorescence.
- Table 2 The results indicate that Arg-PE enhanced the uptake of the oligonucleotide by the cells at least as effectively as the tested commercial cationic hpids.
- NCI-A549 Human lung adenocarcinoma cells (NCI-A549, American Type Culture Collection) were grown on Permanox® chamber slides in RPMI 1470 medium supplemented with
- the medium was aspirated, the cells washed 4 times with HEPES-buffered balanced salt solution, and immediately examined through a fluorescence microscope. The microscopic examination showed bright green nuclear fluorescence and substantial cytoplasmic deposits of granular fluorescent material in 100% of the cells. Accumulation of the oligonucleotide in NCI-A549 cells, incubated with F-ON under the same conditions, but in the absence of Arg-PE, was undetectable.
- Mouse colon carcinoma cells (C26) were grown, incubated with plasmid pCMVLUC in the presence of Arg-PE or commercially available cationic lipids, and assayed for Iuciferase expression as described in Example 5 above. The following results were obtained, indicating that Arg-PE was more effective for the plasmid delivery into C26 cells than other monocationic lipids and as effective as the polycationic lipid Transfectam®.
- NCI-H1048 Human extrapulmonary small cell carcinoma cells (NCI-H1048), American Type Culture Collection) were grown, incubated with plasmid pCMVLUC in the presence of Arg-PE or commercially available cationic hpids, and assayed for Iuciferase expression as described in the Example 5 above. The foUowing results were obtained, indicating that Arg-PE was as effective for the plasmid delivery into NCI-H1048 cells as a polycationic Transfectam®. Results are shown in Figure 4 and Table 4.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002268900A CA2268900A1 (en) | 1996-05-15 | 1997-05-14 | Cationic lipids and methods of use therefor |
AU31248/97A AU3124897A (en) | 1996-05-15 | 1997-05-14 | Cationic lipids and methods of use therefor |
EP97926489A EP0923630A4 (en) | 1996-05-15 | 1997-05-14 | Cationic lipids and methods of use therefor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/648,558 | 1996-05-15 | ||
US08/648,558 US5980935A (en) | 1996-05-15 | 1996-05-15 | Cationic lipids and methods of use therefor |
Publications (1)
Publication Number | Publication Date |
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WO1997043363A1 true WO1997043363A1 (en) | 1997-11-20 |
Family
ID=24601289
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/008120 WO1997043363A1 (en) | 1996-05-15 | 1997-05-14 | Cationic lipids and methods of use therefor |
Country Status (5)
Country | Link |
---|---|
US (1) | US5980935A (en) |
EP (1) | EP0923630A4 (en) |
AU (1) | AU3124897A (en) |
CA (1) | CA2268900A1 (en) |
WO (1) | WO1997043363A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AT413707B (en) * | 2004-07-19 | 2006-05-15 | Voest Alpine Ind Anlagen | METHOD AND DEVICE FOR METALING |
WO2008137758A3 (en) * | 2007-05-04 | 2009-04-09 | Nastech Pharm Co | Amino acid lipids and uses thereof |
WO2011003834A1 (en) | 2009-07-09 | 2011-01-13 | Marina Biotech, Inc. | Amphoteric liposomes comprising imino lipids |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1433478A (en) * | 1999-12-30 | 2003-07-30 | 诺瓦提斯公司 | Novel colloid synthetic vectors for gene therapy |
KR100381512B1 (en) * | 2000-09-21 | 2003-04-26 | 굿젠 주식회사 | Cationic lipids and use thereof |
KR100373844B1 (en) * | 2000-09-21 | 2003-02-26 | 굿젠 주식회사 | Cationic lipids and method for preparing the same |
KR100373845B1 (en) * | 2000-09-21 | 2003-02-26 | 굿젠 주식회사 | Cationic lipids and method for preparing the same |
US20030138407A1 (en) * | 2001-11-02 | 2003-07-24 | Patrick Lu | Therapeutic methods for nucleic acid delivery vehicles |
WO2003057190A1 (en) * | 2001-12-31 | 2003-07-17 | Elan Pharmaceuticals, Inc. | Efficient nucleic acid encapsulation into medium sized liposomes |
AU2002357396A1 (en) * | 2001-12-31 | 2003-07-24 | Elan Pharmaceuticals, Inc. | Efficient liposomal encapsulation under mild conditions |
EP1474107A4 (en) * | 2002-01-09 | 2010-01-20 | Transave Inc | Efficient liposomal encapsulation under mild conditions |
US20090191259A1 (en) * | 2002-01-09 | 2009-07-30 | Transave, Inc. | Efficient liposomal encapsulation |
US8496961B2 (en) | 2002-05-15 | 2013-07-30 | Sutter West Bay Hospital | Delivery of nucleic acid-like compounds |
EP1619951B1 (en) | 2003-04-21 | 2011-06-22 | Epeius Biotechnologies Corporation | Methods and compositions for treating disorders |
US20070178066A1 (en) * | 2003-04-21 | 2007-08-02 | Hall Frederick L | Pathotropic targeted gene delivery system for cancer and other disorders |
US9925276B2 (en) | 2013-03-14 | 2018-03-27 | Epeius Biotechnologies Corporation | Thymidine kinase gene |
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US4559324A (en) * | 1982-07-28 | 1985-12-17 | Takeda Chemical Industries, Ltd. | Polypeptide-diesters, their production and use |
US4804539A (en) * | 1986-07-28 | 1989-02-14 | Liposome Technology, Inc. | Ophthalmic liposomes |
US5286634A (en) * | 1989-09-28 | 1994-02-15 | Stadler Joan K | Synergistic method for host cell transformation |
Family Cites Families (4)
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CN1003524B (en) * | 1985-10-14 | 1989-03-08 | 株式会社日立制作所 | Electroless gold plating solution |
US5264618A (en) * | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
US5364884A (en) * | 1993-01-21 | 1994-11-15 | Baylor College Of Medicine | Arginine compounds as ocular hypotensive agents |
US5614503A (en) * | 1993-11-12 | 1997-03-25 | Aronex Pharmaceuticals, Inc. | Amphipathic nucleic acid transporter |
-
1996
- 1996-05-15 US US08/648,558 patent/US5980935A/en not_active Expired - Fee Related
-
1997
- 1997-05-14 AU AU31248/97A patent/AU3124897A/en not_active Abandoned
- 1997-05-14 WO PCT/US1997/008120 patent/WO1997043363A1/en active Application Filing
- 1997-05-14 CA CA002268900A patent/CA2268900A1/en not_active Abandoned
- 1997-05-14 EP EP97926489A patent/EP0923630A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4559324A (en) * | 1982-07-28 | 1985-12-17 | Takeda Chemical Industries, Ltd. | Polypeptide-diesters, their production and use |
US4804539A (en) * | 1986-07-28 | 1989-02-14 | Liposome Technology, Inc. | Ophthalmic liposomes |
US5286634A (en) * | 1989-09-28 | 1994-02-15 | Stadler Joan K | Synergistic method for host cell transformation |
Non-Patent Citations (1)
Title |
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See also references of EP0923630A4 * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AT413707B (en) * | 2004-07-19 | 2006-05-15 | Voest Alpine Ind Anlagen | METHOD AND DEVICE FOR METALING |
WO2008137758A3 (en) * | 2007-05-04 | 2009-04-09 | Nastech Pharm Co | Amino acid lipids and uses thereof |
US7939505B2 (en) | 2007-05-04 | 2011-05-10 | Marina Biotech, Inc. | Amino acid lipids and uses thereof |
US8501824B2 (en) | 2007-05-04 | 2013-08-06 | Marina Biotech, Inc. | Amino acid lipids and uses thereof |
AU2008247488B2 (en) * | 2007-05-04 | 2014-02-27 | Marina Biotech, Inc. | Amino acid lipids and uses thereof |
US8877729B2 (en) | 2007-05-04 | 2014-11-04 | Marina Biotech, Inc. | Amino acid lipids and uses thereof |
US9339461B2 (en) | 2007-05-04 | 2016-05-17 | Marina Biotech, Inc. | Arginine-based lipids for delivery of therapeutics |
US9731016B2 (en) | 2007-05-04 | 2017-08-15 | Marina Biotech, Inc. | Tyrosine-based lipids for delivery of therapeutics |
EP3434259A1 (en) * | 2007-05-04 | 2019-01-30 | Marina Biotech, Inc. | Amino acid lipids and uses thereof |
WO2011003834A1 (en) | 2009-07-09 | 2011-01-13 | Marina Biotech, Inc. | Amphoteric liposomes comprising imino lipids |
EP2823810A1 (en) | 2009-07-09 | 2015-01-14 | Marina Biotech, Inc. | Amphoteric liposomes comprising imino lipids |
US11541010B2 (en) | 2009-07-09 | 2023-01-03 | Biontech Delivery Technologies Gmbh | Amphoteric liposomes comprising imino lipids |
Also Published As
Publication number | Publication date |
---|---|
EP0923630A1 (en) | 1999-06-23 |
US5980935A (en) | 1999-11-09 |
AU3124897A (en) | 1997-12-05 |
CA2268900A1 (en) | 1997-11-20 |
EP0923630A4 (en) | 2003-05-21 |
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