WO2003094971A1 - Lipides cationiques pour la liberation intracellulaire de substances bioactives - Google Patents

Lipides cationiques pour la liberation intracellulaire de substances bioactives Download PDF

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Publication number
WO2003094971A1
WO2003094971A1 PCT/SG2003/000109 SG0300109W WO03094971A1 WO 2003094971 A1 WO2003094971 A1 WO 2003094971A1 SG 0300109 W SG0300109 W SG 0300109W WO 03094971 A1 WO03094971 A1 WO 03094971A1
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optionally substituted
group
alkyl
cationic
cycloalkyl
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PCT/SG2003/000109
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English (en)
Inventor
Kam Leong
Wen Jie
Hai Quan Mao
Jun Wang
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Johns Hopkins Singapore Pte Ltd
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Priority to AU2003237763A priority Critical patent/AU2003237763A1/en
Publication of WO2003094971A1 publication Critical patent/WO2003094971A1/fr

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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
    • 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
    • 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
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • 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

Definitions

  • the present invention generally relates to a method of delivering bioactive substances, particularly nucleic acids, into cells.
  • This invention describes a series of novel biodegradable cationic lipids as transport agents.
  • Preferred cationic lipids have two hydroxyl groups and combined with a neutral lipid form a cationic liposome.
  • Drugs or molecules that can be delivered using these cationic liposomes range from DNA plasmids, RNAs, proteins to small molecular weight drugs.
  • Biodegradable cationic liposomes ofthe invention can also be used to aid the virus particle mediated gene transfer.
  • Ideal gene delivery vehicles should be bioabsorable, non-toxic, non- immunogenic, stable during storage and after administration, able to access target cells, and suitable for efficient gene expression. As many studies demonstrate, the limitations of viral vectors make synthetic vectors an attractive alternative.
  • Cationic liposome (Lipoplex) and cationic polymers are among the two major types of non-viral gene delivery vectors. Toxicity data on cationic polymers suggests that many polymers used for transfections are most effective at concentrations that are just subtoxic. Illustrative polymers include polyamino acids (e.g. poly-L-lysine, poly-L- ornithine), polyamidoamine dendrimers, chitosan, polyethylenimine, poly((2- dimethylamino)ethyl methacrylate). The entry ofthe complexes may be mediated by the membrane destabilizing effects of cationic polymers. Several observations have suggested that liposomal systems are relatively unstable after the administration.
  • Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant device applications. Sometimes, it is also desirable for such polymers to be, not only biocompatible, but also biodegradable to obviate the need for removing the polymer once its therapeutic value has been exhausted.
  • a biodegradable medical device is intended for use as a drug delivery or other controlled-release system
  • using a polymeric carrier is one effective means to deliver the therapeutic agent locally and in a controlled fashion, see Langer et al., Rev. Macro. Chem. Phys., C23(l), 61 (1983). As a result, less total drug is required, and toxic side effects can be minimized.
  • Polymers have been used as carriers of therapeutic agents to effect a localized and sustained release. See Chien et al., Novel Drug Delivery Systems (1982). Such delivery systems offer the potential of enhanced therapeutic efficacy and reduced overall toxicity.
  • the present invention provides a new class of cationic lipids, cationic liposomes comprising a cationic lipid ofthe invention and a neutral lipid, liposome compositions comprising at least one biologically active substance dispersed in a cationic liposome of the invention and methods of preparing and using such cationic liposomes and compositions to deliver biologically active substances to specified tissues or cells.
  • a preferred application includes the localized, controlled release of at least a portion of one or more bioactive substances from a cationic liposome ofthe invention into a specified tissue or cell.
  • cationic liposomes ofthe invention are effective gene delivery agents for localized delivery of DNA to specified tissues or cells in gene therapy.
  • the present invention provides cationic lipids according to Formula I:
  • Ri is chosen from the group consisting of C . 6 cycloalkyl having between about 1 and 6 alicyclic rings, and (CR 6 R 7 ) b -Y-Z;
  • R 2 is selected from the group consisting of hydrogen, optionally substituted C_. 6 alkyl, optionally substituted C 3 - 8 cycloalkyl, and optionally substituted C 3 _ cycloalkyl C_. ealkyl; R 3 and R 4 are independently selected at each occurrence from the group consisting of optionally substituted C ⁇ _ 3 alkylene, optionally substituted Cs-scycloalkylene; X is a pharmaceutically acceptable anion; Y is -OCO2-, -NR 6 CO 2 - or -NR 6 CONR 7 -; Z is an optionally substituted steroid derivative, optionally substituted C_. 36 alkyl.
  • 36 cycloalkyl having between about 1 and 6 alicyclic rings which may optionally comprise 0-3 heteroatoms in the alicyclic rings; R 6 and R 7 are each independently selected at each occurrence from the group consisting of hydrogen and C.- 6 alkyl; and b is a positive integer.
  • Preferred cationic lipids according to Formula I are biocompatible or biodegradable.
  • a biodegradable cationic lipid ofthe invention are biocompatible before, during and after biodegredation.
  • the present invention also features cationic liposomes comprising at least on neutral lipid and at least one cationic lipid of Formula I.
  • Preferred cation liposomes are biocompatible and degrade in vivo or in vitro to the component lipids.
  • Preferably both the cationic lipid and neutral lipid components ofthe liposome are biocompatible.
  • the present invention further comprises biocompatible cationic liposome compositions comprising at least one biologically active substance; a neutral lipid; and a cationic lipid according to Formula I.
  • the invention further includes methods of making biocompatible cationic liposomes comprising a cationic liposome according to Formula I and a neutral lipid, the method comprising the steps of: providing a neutral lipid and a cationic lipid according to Formula I; dissolving the neutral lipid and the cationic lipid in an organic solvent to form a lipid mixture; hydrating the lipid mixture in water; and incubating the aqueous lipid mixture under conditions conducive to formation of cationic liposomes comprising a neutral lipid and a cationic lipid according to Formula I..
  • a method of preparing a biocompatible cationic liposome composition comprising a cationic lipid, a neutral lipid and a biologically active substance, the method comprising the steps of: providing at least one biocompatible cationic liposome by the method of preparing a cationic liposome ofthe invention and at least one biologically active substance; and contacting the cationic liposome with the biologically active substance under conditions conducive to the inclusion of at least a portion ofthe biologically active substances into the cationic liposome resulting in a biocompatible cationic liposome composition comprising a biologically active substance.
  • a method for the controlled release of a biologically active substance comprises the steps of: providing a biocompatible cationic liposome composition comprising: (a) at least one biologically active substance; and
  • the invention also provides method for gene therapy, wherein a gene or gene fragment, e.g., a DNA sequence is transfected in a controlled fashion into a specified tissue or cell.
  • the method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
  • FIG. 1 is a plot ofthe cytotoxicity of BHEM-Chol/DOPE, BHEM-Chol and TransfastTM;
  • FIG. 2 is a graph comparing transfection efficiency of BHEM-Chol/DOPE and TransfastTM.
  • the present invention features biocompatible cationic lipids, cationic liposomes comprising a biocompatible cationic lipid and a biocompatible neutral lipid and cationic liposome compositions comprising a biologically active substance dispersed within a cationic liposome ofthe invention.
  • the invention further provides methods of manufacturing cationic liposomes and cationic liposome compositions and method s of using biocompatible cationic liposome compositions ofthe invention for localized controlled release of biologically active substances at on in a specified tissue or cell and for use in gene therapy applications.
  • Prefened cationic lipids of Formula I include those cationic lipids according to
  • R 1 and X are as defined in Formula I;
  • R 2 is hydrogen or C ⁇ _ 6 alkyl; and m and n are independently selected integers from about 1 to about 6;
  • Other prefened cationic lipids of Formula I include those lipids according to Formula III:
  • R 1 is chosen from the group consisting of C 3 . 36 cycloalkyl having between about 3 and about 5 alicyclic rings, and -(CH 2 ) b -Y-Z; R 2 is methyl or ethyl; X is a pharmaceutically acceptable anion.
  • Y is -OCO 2 -, -NR 6 CO 2 - or -NR 6 CONR 7 -;
  • Z is an optionally substituted steroid derivative, optionally substituted C ⁇ - 36 alkyl, optionally substituted C 2 - 36 alkenyl, optionally substituted C 2 . 36 alkynyl, or optionally substituted C 3 _ 36 cycloalkyl having between about 1 and 6 alicyclic rings which may optionally comprise 0-3 heteroatoms in the alicyclic rings; R 6 and R 7 are each independently selected at each occurrence from the group consisting of hydrogen and C ⁇ - 6 alkyl; and b is a positive integer.
  • Particularly preferred cationic lipids of Formula I include those cationic lipids according to Formula IN
  • n and n are independently selected integers from about 1 to about 6; b is a positive integer from about 1 to about 6; and the steroid ring structure
  • steroid ring atoms is optionally be substituted at one or more steroid ring atoms with one or more substitutents chosen from the group consisting of C ⁇ - ⁇ 2 alkyl, C 2 - ⁇ 2 alkenyl, C 2 . ⁇ 2 alkynyl, and C 3 . 8 cycloalkyl and two or more substitutents can combine to form additional carbocyclic or heteroalicyclic rings which can be fused or spiro to the steroid ring structure.
  • R 1 group for cationic lipids include R 1 groups according to Formula N:
  • the steroid ring structure can optionally be substituted at one or more steroid ring atoms with one or more substitutents chosen from the group consisting of Ci- ⁇ 2 alkyl, C 2 - .2 alkenyl, C . ⁇ 2 alkynyl, and C 3 . 8 cycloalkyl and two or more substitutents can combine to form additional carbocyclic or heteroalicyclic rings which can be fused or spiro to the steroid ring structure.
  • the invention also provides cationic liposomes comprising a neutral lipid and a cationic lipid according to any one of Formulae I, II, III, or IV.
  • Particularly preferred cationic lipids suitable for use in cationic liposomes ofthe invention include an R 1 according to Fonnula V.
  • Prefened cationic liposomes comprising a catiomc lipid according to Formula I, II, m, or IV are biocompatible before, during and after dissolution ofthe liposome.
  • prefened cationic liposomes ofthe invention comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, II, III or IN wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1:10 by weight.
  • Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited.
  • Prefened neutral lipids include those selected from steroids, di(C 6 -C 36 )alkyl phosphatidyl (hydroxyalkyl) amines and di(C 6 -C 36 )alkenyl phosphatidyl (hydroxyalkyl) amines and the like.
  • Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
  • the invention provides cationic liposome compositions comprising cationic liposome ofthe invention and a biologically active substance disposed therein.
  • Prefened cationic liposome compositions ofthe invention comprise at least one biologically active substance, a neutral lipid and a cationic lipid according to any one of Formulae I, II, III, or IN.
  • Particularly prefened cationic lipids suitable for use in cationic liposome compositions include those having the R 1 group according to Formula V.
  • At least one ofthe biologically active substances dispersed in the cationic liposome is selected from the group consisting of D ⁇ A, R ⁇ A, proteins, and small molecule therapeutics.
  • cationic liposome compositions comprising at least one biocompatible cationic lipid according to Formula I, II, III, or IN, a neutral lipid and at least one negatively charged or neutral biologically active substance.
  • biologically active substances include DNA (inclusive of cDNA), RNA (inclusive of mRNA, tRNA and the like), proteins, and small molecule therapeutics.
  • Prefened cationic liposome compositions comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, ⁇ , III or IV wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1 : 10 by weight.
  • the cationic liposome compositions further comprise about 1 to about 65 parts by weight ofthe biologically active substance relative to the combined weight ofthe neutral lipid and cationic lipid of the cationic liposome.
  • Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited.
  • Prefened neutral lipids include those selected from steroids, di(C 6 -C 36 )alkyl phosphatidyl (hydroxyalkyl) amines and di(C 6 -C 36 )alkenyl phosphatidyl (hydroxyalkyl) amines, and the like.
  • Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
  • the present invention provides methods for preparing cationic liposomes ofthe invention.
  • the methods comprise the steps of: providing a neutral lipid and a cationic lipid according to any one of Formulae I, ⁇ , III or IV; dissolving the neutral lipid and the cationic lipid in an organic solvent to form a lipid mixture; hydrating the lipid mixture in water; and incubating the aqueous lipid mixture under conditions conducive to formation of cationic liposomes comprising a neutral lipid and a cationic lipid according to Formula I..
  • cationic liposomes prepared by the methods ofthe invention comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, II, HI or IN wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1:10 by weight.
  • Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited.
  • Prefened neutral lipids include those selected from steroids, di(C 6 -C 36 )alkyl phosphatidyl (hydroxyalkyl) amines and di(C 6 -C 36 )alkenyl phosphatidyl (hydroxyalkyl) amines, and the like.
  • Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
  • the present invention further provides a method of preparing a biocompatible cationic liposome composition comprising a cationic lipid, a neutral lipid and a biologically active substance, the method comprising the steps of: providing at least at least one biologically active substance and one biocompatible cationic liposome ofthe invention comprising a neutral lipid and at least one cationic lipid according to any one of Formulae I, II, III or IN; and contacting the cationic liposome with the biologically active substance under conditions conducive to the inclusion of at least a portion ofthe biologically active substances into the cationic liposome resulting in a biocompatible cationic liposome composition comprising a biologically active substance.
  • the present invention provides methods for the controlled release of a biologically active substance from a cationic liposome composition ofthe present invention.
  • the method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
  • Prefened biologically active substances that are suitable for controlled release from cationic liposome compositions ofthe invention include substances selected from the group consisting of DNA, RNA, proteins, and small molecule therapeutics.
  • the present invention further provides methods for gene therapy.
  • the method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
  • a cationic liposome comprising at least one neutral lipid and at least one cationic lipid according to any one of Formulae I. II, III, or IN; and contacting the micelle composition in vivo or in vitro with a biological fluid, cell or tissue under conditions conducive to the delivery of at least a portion ofthe gene to the biological fluid, cell or tissue such that gene therapy occurs.
  • the cationic liposome composition comprising a gene or gene fragment is contacted with a specified cell or tissue in vivo.
  • the cationic liposome composition comprising the gene or gene fragment is contacted with the specified tissue or cell in vitro.
  • the invention also provides methods wherein the D ⁇ A sequence or gene is released extracellularly or intracellularly.
  • the method of administration ofthe cationic liposome composition comprising a biologically active substance is not particularly limited.
  • cationic liposome compositions are administered orally or by injection into a tissue such as a muscle, an internal organ, a region ofthe spinal cord or the like.
  • Suitable subjects for in vivo gene therapy using the compositions and methods of the invention are typically mammals.
  • Particularly prefened mammals include rodents, including mice and rats, livestock such as sheep, pig, cow and the like and primates, particularly humans, however other subjects are also contemplated as within the scope of the present invention.
  • the compositions and methods ofthe present invention are also suitable for in vitro gene therapy applications.
  • the gene delivery systems ofthe present invention can achieve gene transfer efficiencies in vitro that are superior to commercially available cationic liposome preparations such as TransfastTM. Furthermore, the cationic liposomal delivery systems ofthe invention offer numerous technical advantages including improved biocompatibility (FIG.
  • the liposome degrades in vitro or in vivo to molecular cationic lipids and neutral lipids
  • the versatility of cationic lipids of Formula I can be tailored by variation of one or more groups or substituents to control cationic liposome properties such as charge density, charge ratio, liposome stability, rate of degredation, hydrophilicity/hydrophobicity and other physical properties
  • specific cell or tissues can be targeted by introduction of one or more specific ligands into the charged lipid according to Formula I or in the neutral lipid component ofthe liposome thereby enhancing delivery ofthe biologically active substance to the specified location.
  • Nucleic acid administered in accordance with the invention may be any nucleic acid (DNA or RNA) including genomic DNA, cDNA, mRNA and tRNA. These constructs may encode a gene product of interest, e.g. a therapeutic or diagnostic agent.
  • a wide variety of known polypeptides are known that may be suitably administered to a patient in accordance with the invention.
  • nucleic acids that encode vasoactive factors may be employed to treat vasoconstriction or vasospasm.
  • Nucleic acids that encode angiogenic growth factors may be employed to promote revascularization. Suitable angiogenic growth factors include e.g. the fibroblast growth factor (FGF) family, endothelial cell growth factor (ECGF) and vascular endothelial growth factor (VEGF; see U.S. Patents 5,332,671 and 5,219,739).
  • FGF fibroblast growth factor
  • ECGF endothelial cell growth factor
  • VEGF vascular endothelial growth factor
  • Additional agents that maybe administered to ischemic heart conditions, or other ischemic organs include e.g. nucleic acids encoding transforming growth factor ⁇ (TGF- ⁇ ), transforming growth factor ⁇ (TGF- ⁇ ), tumor necrosis factor ⁇ and tumor necrosis factor ⁇ .
  • Suitable vasoactive factors that can be administered in accordance with the invention include e.g. atrial natriuretic factor, platelet-derived growth factor, endothelin and the like.
  • nucleic acids encoding various anticancer agents can be employed, such as nucleic acids that code for diphtheria toxin, thymidinekinase, pertussis toxin, cholera toxin and the like. Nucleic acids encoding antiangiogenic agents such as matrix metalloproteases and the like also can be employed. See J.M. Ray et al. Eur Respir J 1994, 7:2062-2072.
  • polypeptides transcribed by the administered nucleic acid can include growth factors or other regulatory proteins, a membrane receptor, a structural protein, an enzyme, a hormone and the like.
  • the invention provides for inhibiting expression or function of an endogenous gene of a subject.
  • antisense nucleic acid may be administered to a subject in accordance with the invention.
  • antisense nucleic acids will be complementary to the mRNA ofthe targeted endogenous gene to be suppressed, or to the nucleic acid that codes for the reverse complement ofthe endogenous gene. See J.H. Izant et al., Science 1985, 229:345-352; and L.J. Maher II et al., Arch Biochem Biophys 1987, 253:214-220.
  • Antisense modulation of expression of a targeted endogenous gene can include antisense nucleic acid operably linked to gene regulatory sequences.
  • nucleic acid may be administered which antagonizes the expression of selected endogenous genes (e.g. ribozymes), or otherwise interferes with function of the endogenous gene or gene product.
  • the nucleic acid to be administered can be obtained by known methods, e.g. by isolating the nucleic acids from natural sources or by known synthetic methods such as the phosphate triester method. See, for example, Oligonucleotide Synthesis, IRL Press (MJ. Gait, ed. 1984). Synthetic oligonucleotides also may be prepared using commercially available automated oligonucleotide synthesizers. Also, as is known, if the nucleic acid to be administered is mRNA, it can be readily prepared from the conesponding DNA, e.g.
  • RNA segment may be chemically synthesized or may be obtained by other known routine procedures such as PCR.
  • the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter.
  • the promoter should be capable of driving expression in the desired cells. The selection of appropriate promoters can be readily accomplished. For some applications, a high expression promoter is prefened such as the 763 -base pair cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • RSV Rous sarcoma
  • MMT also maybe suitable. Additionally, certain proteins can be expressed using their native promoter. Promoters that are specific for selected cells also may be employed to limit transcription in desired cells.
  • a wide variety of subjects will be suitable, e.g. livestock such as cattle, sheep, goats, cows, swine and the like; poultry such as chickens, ducks, geese, turkeys and the like; and pets such as dogs and cats.
  • rodents e.g. mice, rats, hamsters
  • rabbits, primates, and swine such as inbred pigs and the like.
  • the effective dose of nucleic acid will be a function ofthe particular expressed protein, the target tissue, the subject (including species, weight, sex, general health, etc.) and the subject's clinical condition.
  • Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests.
  • frequency of administration for a given therapy can vary, particularly with the time cells containing the exogenous nucleic acid continue to produce the desired polypeptide as will be appreciated by those skilled in the art.
  • the concentration of nucleic acid within a polymer nanoparticle or micelle can vary, but relatively high concentrations are prefened to provide increased efficiency of nucleic acid uptake. More specifically, prefened nanoparticles and micelles comprise a polyphosphate-nucleic acid complex and includes between about 1% to 70% by weight of the nucleic acid. More preferably, the micelle or nanoparticle comprises about 10 to about 60 % nucleic acid by weight or 10%, 20%, 30%, 40%, 50% or 60% by weight of the nucleic acid.
  • substituents ofthe various Formulae are "optionally substituted", including R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 of Formula I-TV.
  • those substituents may be substituted by other than hydrogen at one or more available positions, typically 1 to about 6 positions or more typically 1 to about 3 or 4 positions, by one or more suitable groups such as those disclosed herein.
  • suitable groups that may be present on a "substituted" R , R , R , R , R , R , and R group or other substituent include e.g.
  • alkanoyl such as a C ⁇ _ 6 alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups having those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groups including those moieties having one or more
  • an Ar group being a substituted or unsubstituted biphenyl moiety
  • aralkyl having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with benzyl being a prefened group
  • aralkoxy having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with O-benzyl being a prefened group
  • a heteroaromatic or heteroalicyclic group having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g.
  • amphiphilic is intended to include polymers which comprises two or more domains or groups which are linked together wherein at least one domain or group or domain is a polar, hydrophilic group and at least one domain or group is a non-polar hydrophobic group.
  • Amphiphilic polymers ofthe invention typically comprise a polar, hydrophilic main chain having non-polar hydrophobic groups or domains pendant therefrom.
  • a positively charged or positively chargeable group is intended to include both positively charged functional groups such as phophonium groups, quaternary ammonium groups and other charged groups and also chargeable functional groups that can reversibly protonated to yield a positively charged group, e.g., typical chargeable groups include primary, secondary and tertiary amines, amides and other functional groups which comprise a proton acceptor and can be protonated in aqueous media at or around neutral pH.
  • alkyl is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkylene, having the specified number of carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n- propyl, i-propyl, n-butyl, s-bufyl, t-butyl, n-pentyl, and s-pentyl.
  • Alkyl groups typically have 1 to about 36 carbon atoms. Typically lower alkyl groups have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms.
  • Prefened lower alkyl groups are C 1 -C 20 alkyl groups, more prefened are C ⁇ - ⁇ 2 -alkyl and C ⁇ - 6 -alkyl groups. Especially prefened lower alkyl groups are methyl, ethyl, and propyl. Typically higher alkyl groups have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms. Prefened higher alkyl groups are C 4 -C 36 alkyl groups, more prefened are C 8 _ 24 -alkyl and C ⁇ - . s-alkyl groups.
  • heteroalkyl is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkyl groups include the following groups.
  • Prefened alkylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alylthio groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefered alkylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms.
  • Alkylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefened alkylsulfonyl groups include those groups having one or more sulfonyl (SO 2 ) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefened aminoalkyl groups include those groups having one or more primary, secondary and or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • heteroalkenyl is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkenylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkenyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkenyl groups include the following groups.
  • Prefened alkylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms.
  • Alkenylthio groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefered alkenylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms.
  • Alkenylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefened alkenylsulfonyl groups include those groups having one or more sulfonyl (SO 2 ) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkenylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefened aminoalkenyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkenyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • heteroalkynyl is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkynylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkynyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkynyl groups include the following groups.
  • Prefened alkynylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkynylthio groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefered alkynylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkynylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • SO sulfoxide
  • Prefened alkynylsulfonyl groups include those groups having one or more sulfonyl (SO 2 ) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms.
  • Alkynylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • Prefened aminoalkynyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkynyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
  • cycloalkyl is intended to include saturated and partially unsaturated ring groups, having the specified number of carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Also included are carbocyclic ring groups with ine or more olefinic linkages between two or more ring carbon atoms such as cyclopentenyl, cyclohexenyl and the like. Cycloalkyl groups typically will have 3 to about 8 ring members.
  • (C 3 _ 6 cycloalkyl)C ⁇ - 4 alkyl as defined above, the point of attachment is on the alkyl group. This term encompasses, but is not limited to, cyclopropylmethyl, cyclohexylmethyl, cyclohexylethyl.
  • alkenyl is intended to include hydrocarbon chains of straight, cyclic or branched configuration, including alkenylene having one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl and propenyl.
  • Alkenyl groups typically have 1 to about 36 carbon atoms.
  • lower alkenyl groups typically have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms.
  • Prefened lower alkenyl groups are C ⁇ -C 2 o alkenyl groups, more prefened are C ⁇ _ ⁇ 2 -alkenyl and C ⁇ - 6 -alkenyl groups.
  • Prefened lower alkenyl groups are vinyl, and propenyl.
  • higher alkenyl groups typically have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms.
  • Prefened higher alkenyl groups are C 4 -C 36 alkenyl groups, more prefened are C 8 . 24 -alkenyl and C ⁇ 2 - ⁇ _-alkenyl groups.
  • alkynyl is intended to include hydrocarbon chains of straight, cyclic or branched configuration, including alkynylene, and one or more triple carbon-carbon bonds which may occur in any stable point along the chain.
  • Alkynyl groups typically have 1 to about 36 carbon atoms.
  • lower alkynyl groups typically have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms.
  • Prefened lower alkynyl groups are C ⁇ -C 2 o alkynyl groups, more prefened are C_-_ 2 - alkynyl and C ⁇ - 6 - alkynyl groups.
  • Especially prefened lower alkyl groups are ethynyl, and propynyl.
  • higher alkynyl groups typically have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms.
  • Prefened higher alkynyl groups are C 4 -C 36 alkynyl groups, more prefened are C 8 _ 24 - alkynyl and C ⁇ 2 _ ⁇ 8 - alkynyl groups.
  • haloalkyl examples include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.
  • Typical haloalkyl groups will have 1 to about 16 carbon atoms, more typically 1 to about 12 or 1 to about 6 carbon atoms.
  • a steroid derivative is defined as an optionally substituted steroid group.
  • a steroid is defined as a group of lipids that contain a hydrogenated cyclopentanoperhydrophenanthrene ring system. Some ofthe substances included in this group are progesterone, adrenocortical hormones, the gonadal hormones, cardiac aglycones, bile acids, sterols (such as cholesterol), toad poisons, saponins and some of the carcinogenic hydrocarbons.
  • Prefened steroid derivatives include the sterol family of steroids, particularly cholesterol.
  • Particularly prefened steroid derivatives include alkylene carboxamic acid steryl esters, e.g., -alkylene-NH-CO-O-steryl.
  • alkoxy represents an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge.
  • alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
  • Alkoxy groups typically have 1 to about 16 carbon atoms, more typically 1 to about 12 or 1 to about 6 carbon atoms.
  • a stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an effective therapeutic agent.
  • aliphatic refers to a linear, branched, cyclic alkane, alkene, or alkyne.
  • Prefened aliphatic groups in the biodegradable amphiphilic polyphosphate ofthe invention are linear or branched and have from 1 to 36 carbon atoms.
  • Prefened lower aliphatic groups have 1 to about 12 carbon atoms and prefened higher aliphatic groups have about 10 to about 24 carbon atoms,
  • aryl refers to an unsaturated cyclic carbon compound with 4n+2 electrons where n is a non-negative integer, about 5-18 aromatic ring atoms and about 1 to about 3 aromatic rings.
  • heterocyclic and “heteroalicyclic” refer to a saturated or unsaturated ring compound having one or more atoms other than carbon in the ring, for example, nitrogen, oxygen or sulfur.
  • Typical heterocyclic groups include heteroaromatic and heteroalicyclic groups that have about a total of 3 to 8 ring atoms and 1 to about 3 fused or separate rings and 1 to about 3 ring heteroatoms such as N, O or S atoms.
  • heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, l ⁇ -indazolyl, indolenyl, ind
  • a typical in vitro toxicity assay would be performed with live cells, such as HeLa cells, according to the procedure outlined in Example 2 wherein polymers ofthe invention are evaluated using the stardard WST-1 dye reduction assay.
  • HeLa cells seeded onto a multi well plate and incubated with 100 ⁇ L of DMEM medium complemented with 10 % fetal bovine serum (FBS) containing a polymer ofthe invention.
  • FBS fetal bovine serum
  • the biocompatible cationic liposome composition comprises both:
  • a biocompatible cationic liposome comprising a neutral lipid and a cationic lipid according to any one of Formula I, II, III or IN.
  • Biologically active substances ofthe invention can vary widely with the purpose for the composition.
  • the active substance(s) may be described as a single entity or a combination of entities.
  • the delivery system is designed to be used with biologically active substances having high water-solubility as well as with those having low water- solubility to produce a delivery system that has controlled release rates.
  • biologically active substance includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function ofthe body; or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • Prefened biologically active substances include negatively charged and neutral substances. Particularly prefened biologically active substances are DNA, RNA, proteins and negatively charged or neutral therapeutic small molecules.
  • Non-limiting examples of useful biologically active substances include the following expanded therapeutic categories: anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-coagulants, anti-convulsants, anti- dianheals, anti-emetics, anti-infective agents, anti-inflammatory agents, anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents, anti-thrombotic agents, anti-uricemic agents, anti-anginal agents, antihistamines, anti-tussives, appetite suppressants, biologicals, cerebral dilators, coronary dilators, decongestants, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxatives, mineral supplements
  • useful biologically active substances from the above categories include: (a) anti-neoplasties such as androgen inhibitors, antimetabolites, cytotoxic agents, immunomodulators; (b) anti-tussives such as dextromethorphan, dextrometho han hydrobromide, noscapine, carbetapentane citrate, and chlophedianol hydrochloride; (c) antihistamines such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; (d) decongestants such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloids such as codeine phosphate, codeine sulfate and morphine; (f) mineral supplements such as potassium chloride, zinc chloride, calcium carbonates
  • the biologically active substance is selected from the group consisting of polysaccharides, growth factors, hormones, anti-angio genesis factors, interferons or cytokines, DNA, RNA, proteins and pro-drugs.
  • the biologically active substance is a therapeutic drug or pro-drug, more preferably a drug selected from the group consisting of chemotherapeutic agents and other anti-neoplasties, antibiotics, anti-virals, anti-fungals, anti-inflammatories, anticoagulants, an antigenic materials.
  • Particularly prefened biologically active substances are DNA and RNA sequences that are suitable for gene therapy.
  • the biologically active substances are used in amounts that are therapeutically effective. While the effective amount of a biologically active substance will depend on the particular material being used, amounts ofthe biologically active substance from about 1% to about 65% have been easily incorporated into the present delivery systems while achieving controlled release. Lesser amounts may be used to achieve efficacious levels of treatment for certain biologically active substances.
  • biocompatible cationic liposome compositions ofthe invention can also comprise additional neutral or cationic lipids, so long as they do not interfere undesirably with the biodegradation characteristics ofthe composition.
  • Mixtures of two or more cationic lipids according to any one of Formulae I-IN may offer even greater flexibility in designing the precise release profile desired for targeted drug delivery.
  • Pharmaceutically acceptable carriers may be prepared from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disintegrants, colorants, bulking agents, flavorings, sweeteners and miscellaneous materials such as buffers and adsorbents in order to prepare a particular medicated composition.
  • a biocompatible therapeutic agent delivery system consists of a dispersion ofthe therapeutic agent in a liposome comprising a cationic lipid and a neutral lipid.
  • the therapeutic agent is typically released as the liposome disperses in vivo into soluble products that can be excreted from the body.
  • the cationic liposome compositions ofthe invention provide a matrix capable of sequestering a biologically active substance and provide predictable, controlled delivery ofthe substance.
  • the liposome then disperses to individiual lipid molecules or smaller aggregates of lipid molecules which are readily excreted from the patient.
  • N-(2-bromoethyl)carbarmoyl cholesterol To a mixture of 2-bromoethylamine hydrobromide (17.42 g, 85.0 mmol) and cholesteryl chloroformate (34.7 g, 77.3 mmol) in 300 ml of chloroform cooled to -30 °C, was added triethylamine (24 ml, 172 mmol). This mixture was stined overnight at room temperature, and washed with 1 N HCI in saturated NaCl (150 ml x3) and washed once with saturated NaCl solution (150 ml). The solution obtained was dried over anhydrous magesium sulfate.
  • N,N-bis-(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonylamino-ethyl) ammonium bromide (BHEM-Chol): N-(2-bromoethyl)carbarmoyl cholesterol (4.77 g, 7.77 mmol) and N-methyldiethanolamine (1.15 g, 9.65 mmol) were combined in 50 ml of dried toluene, and refluexed overnight. TLC revealed that all of N-(2- bromoethyl)carbarmoyl cholesterol was consumed. The mixture was poured to large volumes of ether and the precipitate was collected. The crude product was further purified by recrystallization from ethanol twice then once from the mixture of acetonitrile and ethanol (2:1, v/v) to yield white powder (3.86 g, 73%).
  • BHEM-Chol was dissolved in chloroform at a concentration of 10 mg.ml with a helper lipid DOPE (dioleoyl phosphatidylethanolamine) at 1:1 molar ratio (1:1.12, w/w).
  • DOPE dioleoyl phosphatidylethanolamine
  • cholesterol was used as the helper lipid at 5:4 molar ratio (2.1:1, w/w).
  • the mixture was evaporated to dryness in a round-bottom flask using a rotary evaporator at room temperature.
  • the resulted lipid film was dried under vacuum over night.
  • the lipid film was then re-hydrated in sterile water to a concentration of 2 mg/ml based on the weight of cationic lipid.
  • the suspension was incubated at 50 °C for 30 min. with shaking, and then sonicated using a bath-sonicator for 2 min. at room temperature to form homogenized liposomes.
  • Example 3 Assay for the cytotoxicity of BHEM-Chol and cationic liposomes containing BHEM-Chol.
  • a kidney fibroblast cell line, COS-7 African green monkey was maintained in
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • lOOU/ml penicillin Sigma- Aldrich, St. Louis, MO
  • 100 ⁇ g/ml streptomycin Sigma- Aldrich
  • 2 mM L- glutamine (Gibco-BRL) at 37°C (5% CO 2 ).
  • Cells were seeded at a density of lxlO 4 per well in 96-well plate.
  • culture medium was replaced with 100 ⁇ l of serum-free DMEM medium containing different concentrations of BHEM-Chol or BHEM-Chol/DOPE liposomes.
  • Cells were incubated with the reagents for 4 hours at 37 °C.
  • TransFASTTM was used as a control, and was incubated with cells for one hour at the conesponding concentration.
  • lOO ⁇ l of DMEM medium containing 20% serum was added to each well.
  • Cells were cultured for another 20 hours.
  • Ten micro liters of WST-1 reagent (Roche Molecular Biochemicals, Basel, Switzerland) was added to each well and allowed to react for 4 hours at 37°C.
  • the LD 50 ofthe liposome (BHEM-Chol/DOPE) increased at least 14 times. Over 80% ofthe cells remained viable when the total lipid concentration was 500 ⁇ g/ml.
  • TransFastTM Reagent is comprised ofthe synthetic cationic lipid (+)-N,N-[bis (2-hydroxyethyl)-N-methyl-N-[2,3- di(tetradedecanoyloxy)propyl] ammonium iodide and the neutral lipid, DOPE at a 1:1 molar ratio.
  • BHEM-Chol/DOPE and TransfastTM have similar compositions.
  • BHEM-Chol/DOPE liposomes or DC-Chol/DOPE liposomes in OPTI-MEM (Gibcol-BRL) was mixed with various amount of plasmid DNA solution at room temperature by vortexing for about 10 sec. The complexes were incubated at room temperature for 30 min and used directly. To evaluate the binding efficiency of DNA to the cationic liposomes (BHEM-Chol/DOPE), samples after incubation were analyzed by gel electrophoresis analysis (0.8% agrose).
  • Example 5 Transfection efficiency of liposome-DNA complexes in cell lines and primary hepatocytes.
  • HEK 293 Human embryonic kidney epithelial cell line
  • COS-7 African green monkey kidney fibroblast cell line
  • Primary hepatocytes were gift from Chia SM and Yu H (National University of Singapore), they were harvested from male Wistar rat by a 2-step in situ collagenase perfusion as reported (Chia SM, et al. Hepatocyte encapsulation for enhanced cellular functions. Tissue Engineering 6(5):481-95 (2000)).
  • the isolated hepatocytes were cultured in Hepatozym Serum free medium (SFM, Gibco-BRL) in a humidified atmosphere with 5% CO 2 .
  • the culture medium was supplemented with 0.1 ⁇ M dexamethasone and 1% Penicillin and Streptomycin.
  • Hepatocytes were seeded on collagen coated wells at a density of 5xl0 5 .
  • the medium in each well was replaced with 0.5 ml of Opti-MEM. Liposome-DNA complexes were incubated with the cells for 2 hours at 37°C. The medium was then replaced with 1 ml of fresh complete medium and cells were further incubated for 72 hours. All the transfection tests were performed in triplicate. After the incubation, cells were washed with PBS and permeabilized with 100 ⁇ l of cell lysis buffer (Promega Co., Madison, WI).
  • the luciferase activity in cell extracts was measured using a luciferase assay kit (Promega Co., Madison, WI) on a luminometer (Lumat 9507, EG&G Berthold, Bad Wildbad, Germany).
  • the light units (LU) were normalized against protein concentration in the cell extracts, which was measured using a protein assay kit (Bio-Rad Labs, Hercules, CA).
  • Transfection conditions using BHEM-Chol/DOPE liposomes was optimized with regards to charge ratio of BHEM-Chol and DNA, and dose of plasmid DNA. Maximal transfection occuned at a charge ratio of 1:1 to 1:2, and at a DNA dose of 2 ⁇ g/ml. Under these conditions, transfection efficiency was compared with TransfastTM in 293 cells, COS-7 cells and primary hepatocytes. As shown in Figure 20, BHEM-Chol/DOPE liposome transfected HEK293 cells at the similar level as TransfastTM. It performed particularly well in COS-7 cells (about 40 times higher than TransfastTM).
  • BHEM-Chol/DOPE liposomes induced a luciferase expression level at least 100 times higher than background, which was about 7 to 10 times higher than TransfastTM transfection.

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Abstract

L'invention concerne des liposomes cationiques biodégradables constitués d'un mélange d'un lipide cationique et d'un lipide neutre, des compositions de liposomes cationiques et des méthodes d'utilisation de ces composés pour la libération contrôlée d'une substance bioactive dans un tissu ou des cellules spécifiques. Parmi les lipides cationiques utilisés de préférence dans les liposomes cationiques, on trouve des liposomes cationiques comprenant un groupe chargé positivement et deux groupes hydroxyle qui sont capables de complexer une ou plusieurs substances bioactives. Parmi les méthodes préférées figurent la libération contrôlée de substances bioactives et la thérapie génique faisant appel aux liposomes cationiques et aux compositions constituées de ces liposomes.
PCT/SG2003/000109 2002-05-10 2003-05-10 Lipides cationiques pour la liberation intracellulaire de substances bioactives WO2003094971A1 (fr)

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US20050260276A1 (en) * 2004-05-19 2005-11-24 Agency For Science, Technology And Research Methods and articles for the delivery of therapeutic agents
WO2013116126A1 (fr) * 2012-02-01 2013-08-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques biodégradables de faible masse moléculaire pour la délivrance d'oligonucléotides

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050260276A1 (en) * 2004-05-19 2005-11-24 Agency For Science, Technology And Research Methods and articles for the delivery of therapeutic agents
US8821859B2 (en) * 2004-05-19 2014-09-02 Agency For Science, Technology And Research Methods and articles for the delivery of therapeutic agents
WO2013116126A1 (fr) * 2012-02-01 2013-08-08 Merck Sharp & Dohme Corp. Nouveaux lipides cationiques biodégradables de faible masse moléculaire pour la délivrance d'oligonucléotides

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