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WO2000062813A2 - Lipides peg cationiques et méthodes d'utilisation - Google Patents

Lipides peg cationiques et méthodes d'utilisation Download PDF

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Publication number
WO2000062813A2
WO2000062813A2 PCT/CA2000/000451 CA0000451W WO0062813A2 WO 2000062813 A2 WO2000062813 A2 WO 2000062813A2 CA 0000451 W CA0000451 W CA 0000451W WO 0062813 A2 WO0062813 A2 WO 0062813A2
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WO
WIPO (PCT)
Prior art keywords
lipid
cpl
peg
splp
group
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PCT/CA2000/000451
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English (en)
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WO2000062813A3 (fr
Inventor
Pieter R. Cullis
Tao Chen
David B. Fenske
Lorne R. Palmer
Kim Wong
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The University Of British Columbia
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Application filed by The University Of British Columbia filed Critical The University Of British Columbia
Priority to CA002370690A priority Critical patent/CA2370690A1/fr
Priority to AU40962/00A priority patent/AU783647B2/en
Priority to EP00920309A priority patent/EP1173600A2/fr
Priority to JP2000611949A priority patent/JP5117648B2/ja
Publication of WO2000062813A2 publication Critical patent/WO2000062813A2/fr
Priority to JP2001577996A priority patent/JP2004508012A/ja
Priority to US09/839,707 priority patent/US7189705B2/en
Priority to AU5454801A priority patent/AU5454801A/xx
Priority to AU2001254548A priority patent/AU2001254548B2/en
Priority to EP01927519A priority patent/EP1355670A2/fr
Priority to CA002406654A priority patent/CA2406654A1/fr
Priority to PCT/CA2001/000555 priority patent/WO2001080900A2/fr
Publication of WO2000062813A3 publication Critical patent/WO2000062813A3/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
    • 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
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • This invention relates to cationic lipid conjugates, and more particularly, to cationic polymer lipid conjugates and lipid-based drug formulations thereof, containing one or more bioactive agents.
  • Lipid-based non-viral systems include cationic lipid plasmid DNA complexes. Limitations of these systems include large sizes, toxicity and instability of the complexes in the serum. Unfortunately, the foregoing drawbacks limit the applications for these complexes.
  • researchers have devoted tremendous effort to the design of long circulation stealth liposomes that can be used for systemic delivery (see, Papahadjopoulos, D. et al., Proc. Natl. Acad. Sci. 88:11460-1 1464 (1991); Klibanov, A.L. et al., J.
  • stealth liposomes are often comparatively inefficient at facilitating cellular uptake and therefore the therapeutic efficacy is reduced.
  • the molecular mechanism of liposomal longevity in vivo can be attributed to steric hindrance resulting from hydrophilic polymer surface barriers.
  • the hydrophilic polymer barriers prevent or reduce the rate of the adsorption of macromolecules from the blood and sterically inhibit both electrostatic and hydrophobic interactions between liposomes and blood components.
  • the longevity of stealth liposomes has been increased by the insertion of hydrophilic polymers, the cellular uptake of the stealth liposomes often is inefficient.
  • SPLPs stabilized plasmid-lipid particles
  • PEG poly(ethyleneglycol)
  • Enhancing intracellular delivery of liposomes and/or their contents represents one of the major remaining problems in the development of the next generation of drug delivery systems.
  • general methods for increasing the interactions of liposomes with cells need to be developed.
  • attempts include the use of specific targeting information on the liposome surface, such as an antibody (see, Meyer, O. et al., Journal of Biological Chemistry 273:15621-15627 (1998); Kao, G.Y. et ai, Cancer Gene Therapy 3:250-256 (1996); Hansen, C.B.
  • the present invention relates to new conjugates that can be incorporated or inserted into stabilized plasmid lipid particles to enhance transfection efficiencies.
  • the conjugates of the present invention possess a lipid anchor for anchoring the conjugate into the bilayer lipid particle, wherein the lipid anchor is attached to a non- immunogenic polymer, such as a PEG moiety, and wherein the non-immunogenic polymer is, in turn, attached to a polycationic moiety, such as a positively charged moiety.
  • a non- immunogenic polymer such as a PEG moiety
  • the non-immunogenic polymer is, in turn, attached to a polycationic moiety, such as a positively charged moiety.
  • the present invention provides a compound of Formula I:
  • A is a lipid moiety attached to a non-immunogenic polymer.
  • W in Formula I, is a non-immunogenic polymer, and
  • Y in Formula I, is a polycationic moiety.
  • the compounds of Formula I contain groups that give rise to compounds having the general structure of Formula II:
  • "A” is a lipid, such as a hydrophobic lipid.
  • "X” is a single bond or a functional group that covalently attaches the lipid to at least one ethylene oxide unit, i.e., (-CH - CH 2 -O-).
  • "Z” is a single bond or a functional group that covalently attaches the at least one ethylene oxide unit to a cationic group.
  • "Y” is a polycationic moiety.
  • the index "n” is an integer ranging in value from about 6 to about 160.
  • the present invention relates to a lipid-based drug formulation comprising:
  • a W Y i wherein: A, W and Y have been defined;
  • the lipid-based drug formulation is in the form of a liposome, a micelle, a virosome, a lipid-nucleic acid particle, a nucleic acid aggregate and mixtures thereof.
  • the bioactive agent is a therapeutic nucleic acid or other drugs.
  • the present invention relates to a method for increasing intracellular delivery of a lipid-based drug delivery system, comprising: incorporating into the lipid-based drug delivery system a compound of Formulae I or II, thereby increasing the intracellular delivery of the lipid-based drug delivery system.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; (3) “derived lipids” such as steroids.
  • vesicle-forming lipid is intended to include any amphipathic lipid having a hydrophobic moiety and a polar head group, and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by most phospholipids.
  • Vesicle-adopting lipid is intended to include any amphipathic lipid which is stably incorporated into lipid bilayers in combination with other amphipathic lipids, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.
  • Vesicle-adopting lipids include lipids that on their own tend to adopt a non-lamellar phase, yet which are capable of assuming a bilayer structure in the presence of a bilayer-stabilizing component.
  • DOPE dioleoylphosphatidylethanolamine
  • Bilayer stabilizing components include, but are not limited to, polyamide oligomers, peptides, proteins, detergents, lipid-derivatives, PEG- lipid derivatives such as PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides (see, U.S. Application Serial No. 08/485,608, now U.S. Patent No. 5,885,613, which is incorporated herein by reference).
  • amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
  • Amphipathic lipids are usually the major component of a lipid vesicle. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocychc group(s).
  • apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocychc group(s).
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
  • amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and ⁇ -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipid described above can be mixed with other lipids including triglycerides and sterols.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
  • hydrophopic lipid refers to compounds having apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic or heterocychc group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane and 1 ,2-dialkyl-3-aminopropane.
  • diacylglycerolyl denotes 2-fatty acyl chains, R 1 and R 2 having independently between 2 and 30 carbons bonded to the 1- and 2 -position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation.
  • Diacylglycerol groups have the following general formula:
  • dialkylglycerolyl denotes two C ⁇ -C o alkyl chains bonded to the 1- and 2-position of glycerol by ether linkages.
  • Dialkylglycerol groups have the following general formula:
  • N-N-dialkylamino denotes
  • l,2-diacyloxy-3-aminopropane denotes 2-fatty acyl chains Ci-
  • acyl groups can be saturated or have varying degrees of unsaturation.
  • the 3-position of the propane molecule has a -NH- group attached.
  • l,2-dialkyl-3-aminopropane denotes 2-alkyl chains (C 1 -C 30 ) bonded to the 1- and 2-position of propane by an ether linkage.
  • the 3-position of the propane molecule has a -NH- group attached.
  • l,2-dialkyl-3-aminopropanes have the following general formula:
  • non-cationic lipid refers to any neutral lipid as described above as well as anionic lipids.
  • anionic lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoyl phosphatidylethanolammes, N-succinyl phosphatidylethanolammes, N- glutarylphosphatidylethanolamines, lysophosphatidylglycerols, and other anionic modifying groups joined to neutral lipids.
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.
  • lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 3 -(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Choi”) and N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl am
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and l,2-dioleoyl-s «-3- phosphoethanolamine (“DOPE”), from GIBCO/BRL, Grand Island, New York, USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA”) and("DOPE”), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine ("DOGS”) in ethanol from Promega Corp., Madison, Wisconsin, USA).
  • LIPOFECTIN® commercially available cationic
  • lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA and the like.
  • fusogenic refers to the ability of a liposome or other drug delivery system to fuse with membranes of a cell.
  • the membranes can be either the plasma membrane or membranes surrounding organelles. e.g., endosome, nucleus, etc. Fusogenesis is the fusion of a liposome to such a membrane.
  • dendrimer includes reference to branched polymers that possess multiple generations. In dendrimers, each generation creates multiple branch points.
  • ligand includes any molecule, compound or device with a reactive functional group and includes lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, targeting agents, or toxins.
  • the foregoing list is illustrative and not intended to be exhaustive.
  • the term “ATTA” or "polyamide” refers to, but is not limited to, compounds disclosed in U.S. Patent Application Serial No. 09/218,988, filed December 22, 1998. These compounds include a compound having the formula
  • R is a member selected from the group consisting of hydrogen, alkyl and acyl
  • R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are bound form an azido moiety
  • R 2 is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid
  • R 3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR 4 R ⁇ wherein R 4 and R 3 are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4; and q is 0 or 1. It will be apparent to those of skill in the art that other polyamides can be used in the compounds of the present invention.
  • alkyl denotes branched or unbranched hydrocarbon chains, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso- butyl, tertbutyl, octa-decyl and 2-methylpentyl.
  • These groups can be optionally substituted with one or more functional groups which are attached commonly to such chains, such as, hydroxyl, bro o, fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio.
  • heterocyclyl aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groups such as trifluoromethyl, 3- hydroxyhexyl, 2- carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl and the like.
  • alkylene refers to a divalent alkyl as defined above, such as methylene (-CH 2 -), propylene (-CH 2 CH 2 CH 2 -), chloroethylene (-CHC1CH 2 -), 2- thiobutene (-CH 2 CH(SH)CH 2 CH 2 -), l-bromo-3-hydroxyl-4-methylpentene (- CHBrCH 2 CH(OH)CH(CH 3 )CH 2 -), and the like.
  • alkenyl denotes branched or unbranched hydrocarbon chains containing one or more carbon-carbon double bonds.
  • alkynyl refers to branched or unbranched hydrocarbon chains containing one or more carbon-carbon triple bonds.
  • aryl denotes a chain of carbon atoms which form at least one aromatic ring having preferably between about 6-14 carbon atoms, such as phenyl, naphthyl, indenyl, and the like, and which may be substituted with one or more functional groups which are attached commonly to such chains, such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form aryl groups such as biphenyl, iodobiphenyl, methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chlorophenyl, hydroxyphenyl, methoxyphenyl, formylphenyl, acet
  • alkoxy denotes -OR-, wherein R is alkyl.
  • amido denotes an amide linkage: -C(O)NR- (wherein R is hydrogen or alkyl).
  • amino denotes an amine linkage: -NR-, wherein R is hydrogen or alkyl or a terminal NH 2 .
  • carboxyl denotes the group -C(O)O-
  • carbonyl denotes the group -C(O)-
  • carbonate indicates the group -OC(O)O-.
  • basic amino acid refers to naturally-occurring amino acids as well as synthetic amino acids and/or or amino acid mimetics having a net positive charge at a selected pH, such as physiological pH. This group includes, but is not limited to, lysine, arginine, asparagine, glutamine, histidine and the like.
  • phosphorylethanolamino denotes the group -OP(O)(OH)OCH 2 CH 2 NH-.
  • phqsphorylethanolamido denotes the group -
  • phospho denotes a pentavalent phosphorous moiety - P(O)(OH)O-.
  • phosphoethanolamino denotes the group - P(O)(OH)OCH 2 CH 2 NH-.
  • phosphoethanolamido denotes the group- P(O)(OH)OCH 2 CH 2 NHC(O)-.
  • ethylene oxide unit denotes the group -OCH 2 CH 2 -.
  • CPL refers to a cationic-polymer-lipid e.g., cationic-PEG- lipid.
  • Preferred CPLs are compounds of Formulae I and II.
  • d-DSPE-CPL-M is encompassed by the term “CPL1” which refers to a DSPE-CPL having one positive charge.
  • the "d-" in d-DSPE-CPL-M indicates that the CPL contains a fluorescent dansyl group. It will be apparent to those of skill in the art that a CPL can be synthesized without the dansyl moiety, and thus the term “DSPE-CPL-M" is encompassed by in the term "CPLl” as defined above.
  • d-DSPE-CPL-D is encompassed by the term “CPL2” which refers to DSPE-CPL having two positive charges.
  • d-DSPE-CPL-Tl is encompassed by the term “CPL3” which refers to DSPE-CPL having three positive charges.
  • d-DSPE-CPL-Ql is encompassed by the term “CPL4a” which refers to DSPE-CPL having four positive charges.
  • d-DSPE-CPL-Q5 or, alternatively, DSPE-PEGQuad5, or, alternatively, DSPE-CPL-4, are all encompassed by the term “CPL4 (or CPL4b)" which refer to a DSPE-CPL having four positive charges.
  • CPL4 or CPL4b
  • CPLs were synthesized which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T), and 4 (quad, or Q) positive charges. Narious Quad CPLs were synthesized, hence these are numbered Ql through Q5.
  • HBS Hepes-buffered saline
  • Rho-PE rhodamine-phosphatidylethanolamine
  • LUNs refers to "large unilamellar vesicles.”
  • Figure 1 illustrates a structural design of a cationic-polymer lipid (CPL) conjugate.
  • Figure 2 illustrates a synthetic scheme for the preparation of cationic- PEG-lipid conjugates having varying amount of charged head groups (a.) Et 3 N/CHCl 3 ; (b.) TFA /CHC1 3 ; c. Et 3 N / CHCI 3 No, N ⁇ -di-t-Boc-L-Lysine N-hydroxysuccinide ester.
  • Figure 3 illustrates a CPL incorporated liposome.
  • the large unilamellar vesicles (LUV) have incorporated different examples of CPLs (CPLl, CPL2, CPL4, and CPL 8, respectively).
  • FIG. 4 illustrates a distribution of DSPE-CPL-4 between the inner/outer leaflets of a liposomal membrane.
  • CPL-4-LUNs (DSPC/Chol/DSPE-CPL-4, 55:40:5 mole %) were prepared by extrusion method as described herein.
  • the distribution of outer leaflet CPLs was quantified by a fluorescamine assay.
  • the following assay was used for the outer leaflet CPLs.
  • An appropriate amount of CPL-4-LUNs was diluted with 1M Borate buffer (pH 8.5) and cooled in ice-water.
  • FIG. 5 illustrates a cellular uptake study of CPL-4 LUNs in BHK cells in PBS-CMG.
  • the controls were LUNs (DSPC/Chol, 60:40) and CPL-4-LUNs (DSPC/Ch/DSPE-CPL-4, 55:40:5) were prepared by extrusion as described herein.
  • Figure 6 illustrates cellular uptake of CPL-4-LUNs in BHK cells in DMEM (with 10% FBS). The control used LUNs (DSPC/Chol, 60:40).
  • CPL-4-LUNs DSPC/Ch/DSPE-CPL-4, 55:40:5), which were prepared by extrusion as described herein.
  • Figure 7 illustrates a cellular uptake of CPL-liposomes in BHK cells in
  • LUNs DSPC/Chol, 60:40
  • CPL-LUNs DSPC/Chol/DSPE-CPL, 55:40:5
  • FIG 8 illustrates the preparation of CPL-LUNs by detergent dialysis.
  • Lipids were codissoived in chloroform at the indicated ratios, following which the solvent was removed by nitrogen gas and high vacuum.
  • the lipid mixture was dissolved in detergent/buffer (OGP in HBS) and dialysed against HBS for 2-3 days.
  • the LUNs, which formed during dialysis, were then fractionated as shown on Sepharose CL-4B.
  • Panel A Fractionation of DOPE/DODAC/CPL4[3.4K] /PEGCerC20/Rho-PE (79.5/6/4/10/0.5);
  • Panel B Fractionation of DOPE/DODAC/CPL4[lK]/ PEGCerC20/Rho-PE (79.5/6/4/10/0.5);
  • Panel C Fractionation of DOPE/DODAC/CPL4[3.4K] /PEGCerC20/Rho-PE (71.5/6/12/10/0.5).
  • Figure 9 Panel A illustrates the insertion of DSPE-CPL-Q5 into DOPC LUNs (100 nm).
  • DOPC LUNs 2.5 ⁇ mol lipid
  • 0.214 ⁇ mol DSPE- CPL-Q5 total volume 300 ⁇ L
  • HEPES-buffered saline 1 mL fractions were collected and assayed for dansyl-labelled CPL and rhodamine-PE as described herein.
  • Panel B illustrates the insertion of DSPE-CPL-Q5 into LUNs (100 nm) composed of DOPE/DOD AC/PEG-Cer-C20 (84/6/10).
  • LUNs (5 ⁇ mol lipid) were incubated with 0.43 ⁇ mol DSPE-CPL-Q5 (total volume 519 ⁇ l) at 60°C for 3 hours, following which the sample was applied to a column of Sepharose CL-4B equilibrated in HEPES-buffered saline. The elution of free CPL is also shown, demonstrating a straightforward method for isolation of the CPL-LUN.
  • Panel C illustrates retention of DSPE-CPL-Q5 in LUNs (100 nm) composed of DOPE DODAC/PEG-Cer-C20 (84/6/10).
  • the main LUN fraction from Fig. 9 Panel B was re-applied to a column of Sepharose CL-4B equilibrated in HEPES-buffered saline. 1 mL fractions were collected and assayed for dansyl-labelled CPL and rhodamine-PE as described.
  • Figure 10 illustrates the effect of time and temperature on the insertion of d-DSPE-CPL-Ql into DOPE/DODAC/PEG-Cer-C20 LUNs.
  • 3 ⁇ mol lipid was combined with 0.17 ⁇ mol CPL (total volume 240 ⁇ l).
  • 1 ⁇ mol of lipid was withdrawn and cooled in ice to halt insertion of CPL.
  • the samples were passed down a column of Sepharose CL-4B to remove excess CPL, and assayed for CPL insertion.
  • Figure 11 illustrates the effect of initial CPL/lipid ratio on final CPL insertion levels.
  • Initial CPL/lipid molar ratios were 0.01 1, 0.024, 0.047, 0.071, 0.095, and 0.14.
  • Final mol % inserted were 0.8, 1.8, 3.4, 5.0, 6.5, and 7.0.
  • Right-hand axis is %-insertion.
  • Figure 12 illustrates the insertion of DSPE-CPL-Q1 and DSPE-CPL-Q5 into neutral vesicles.
  • the initial CPL/lipid molar ratio was 0.065 for Ql (2.5 ⁇ mol lipid and 0.21 ⁇ mol CPL) and 0.034 for Q5. Samples were incubated at 60°C for 3 hours.
  • DOPC and DOPC/Chol LUNs were prepared by extrusion, while the others were prepared by detergent dialysis. As described herein, the presence of 4% methanol in the Q5 samples appear to account for the higher insertion observed for this sample. Sample compositions were as follows: DOPC/Chol (55/45), DOPC/PEG-Cer-C20 (90/10), DOPC/Chol/PEG-Cer-C20 (45/45/10).
  • Figure 13 illustrates the effect of chain length of PEG-Cer on mol-% CPL inserted.
  • LUNs composed of DOPE/DODAC/PEG-Cer-C20 (84/6/10), DOPE/DODAC/PEG-Cer-C14 (84/6/10), and DOPE/DODAC/PEG-Cer-C8 (79/6/15) were incubated in the presence of between 2-8.6 mol % d-DSPE-CPL-Ql at 60°C for 3 hrs.
  • Figure 14 illustrates the effect of PEG-Cer-C20 content on insertion of d- DSPE-CPL-Q5.
  • Vesicles composed of DOPC/DODAC/PEGCerC20, with the latter lipid ranging from 4-10 mol %, were incubated in the presence of CPL-Q5 (initial CPL/lipid molar ratio 0.071).
  • Figure 15 illustrates the uptake of CPL-LUVs incubated in PBS/CMG on BHK cells. Approximately 10 5 BHK cells were incubated with 20 nmol of DOPE DODAC/PEGCerC20 (84/6/10) LUVs containing (1) no CPL, (2) 8% DSPE-
  • Panel A illustrates a structure of the CPL .
  • Panel B illustrates a protocol for the insertion of CPL 4 into the SPLP system.
  • FIG 17 Panel A illustrates a model for DOPE/DODAC/PEG-Cer-C20 LUNs, i.e., a standard liposome containing a PEG-lipid (or "stealth" lipid).
  • Panel B illustrates the same LUNs with CPL (i.e. long chain) inserted.
  • “Long chain” refers to the polymer W being the same length or greater length than the polymer component of the PEG-lipid. Thus, the charged group of the CPLl is immediately exposed to the outside environment.
  • Panel C illustrates the same LUNs with CPL with a short chain inserted.
  • a "short chain” CPL wherein polymer W is shorter than the corresponding polymer of the PEG-lipid.
  • Figure 18 Panel A illustrates a time-course for the uptake of SPLP system
  • Panel B illustrates transfection efficiencies of 1.5 ⁇ g/mL pLuc obtained using the SPLP system compared to those obtained using complexes after 4 hour ( ⁇ ) or 8 hour ( ⁇ ) incubations.
  • Figure 19 illustrates a column profile, following insertion of 3.5 mol
  • Figure 21 illustrates a time course for the uptake of 20 ⁇ M of SPLP possessing 0% ( ⁇ ), 3% (0), or 4% (•) CPL 4 in BHK cells.
  • Figure 22 illustrates transfection of BHK cells by SPLP (2.5 ⁇ g/mL pLuc) following insertion of various mol % of the CPL 4 compared to SPLP alone (0% CPL). Transfections were carried out by incubating the samples on top of the cells for 4 or 9 hours and replacing with complete media for a complete 24 hours incubation (see also Figure 33).
  • Figure 23 tabulates CPL insertion results.
  • Figure 24 also tabulates CPL insertion results.
  • Figure 25 illustrates the post-insertion method for preparation of CPL- containing liposomes.
  • the preformed liposomes were made of DSPC/Chol (55:45, mo mol).
  • the CPL was incubated with the preformed liposomes at 60°C for 2 hour.
  • Panel A illustrates separation of free CPL and CPL-LUNs by gel filtration after post- insertion.
  • Panel B illustrates elution of fraction 10 (Panel A) on a Sepharose CL-4B column.
  • Figure 26 illustrates cellular uptake of the stealth liposomes containing DSPE-CPLs in BHK cells in DMEM (10% FBS).
  • Control LUNs DSPC/Chol/PEG-PE, 56:40:4
  • CPL-LUNs DSPC/Chol/PEG-PE/CPL, 55.5:40:2:2 were prepared by extrusion as described herein.
  • Figure 27 illustrates cellular uptake of stealth liposomes containing DSPE-CPLs in BHK cells in PBS-CMG.
  • Control LUVs DSPC/Chol/PEG-PE, 56:40:4
  • CPL-LUVs DSPC/Chol/PEG-PE/CPL, 55.5:40:2:2 were prepared by extrusion as described herein.
  • FIG. 28 Panel A: Chemical structures of various CPLs; Panel B: Chemical structures of various CPLs. Note that CPL (Panel A) is identical to CPL 4b (Panel B); and Panel C: Chemical structures of various CPLs.
  • Figure 29 illustrates a synthetic embodiment to generate compounds of the present invention.
  • FIG 30 illustrates a structure of dansylated CPL .
  • CPL possesses four positive charges at the end of a PEG 34 oo molecule which is attached to a DSPE molecule.
  • the CPL is dansylated by incorporation of a dansylated lysine.
  • Figure 31 illustrates an effect of cation concentration on the deaggregation of SPLP-CPL .
  • QELS quasi-elastic light scattering
  • Measurement of the mean diameter ⁇ standard deviation of the particles in the presence of differing amounts of the cation were made using a Nicomp Model 270 Submicron Particle Sizer.
  • the diameters of the particles do not dramatically change, however, the Gaussian distributions do get broader.
  • the standard deviations were used as a measure of deaggregation with smaller deviations indicating less aggregation.
  • Figure 32 illustrates uptake of SPLP containing various percentages of CPL 4 .
  • Panel A Time course for the uptake of 20 ⁇ M SPLP possessing 0 mol % (•), 2 mol % ( ⁇ ), 3 mol % (A), or 4 mol % ( ⁇ ) CPL 4 and DOPE:DODAC complexes (T) by BHK cells. The insertion of the CPL into SPLP and the preparation of complexes was performed as described herein The mol % of CPL in the SPLP-CPL was also determined, as described herein. BHK cells were plated in 12-well plates at lxlO 3 cells/well.
  • Figure 33 illustrates tansfection of BHK cells by SPLP (5.0 ⁇ g/mL pLuc) following insertion of various mole percentages of CPL (2, 3, and 4 mol %).
  • the CPL was inserted into SPLPs using the procedure described herein.
  • SPLP (0 mol % CPL) and DOPE:DODAC (1 :1) complex transfections were also performed.
  • BHK cells were plated at lxlO 4 in 96-well plates. Transfections were carried out by incubating the samples [20 ⁇ L (SPLP-CPL + CaCl 2 ) + 80 ⁇ L of complete media] on the cells for 4 hours followed by a 24 hour complete incubation.
  • the CaCl 2 concentration again is diluted to 20% of the original concentration. Following the 24 hour incubation, the cells were lysed with lysing buffer and the luciferase and BCA assays were performed (see Figure 22).
  • Figure 34 illustrates the effect of [Cation], Ca 2+ (•) and Mg 2 " ( ⁇ ), on the transfection of SPLP-CLP 4 (5.0 ⁇ g/mL pLuc) on BHK cells.
  • MgCl 2 was mixed with DMEM +10% FBS and the mixtures were applied to lxlO 4 BHK cells plated in a 96-well plate. Following a complete 48 hour incubation, the transfection media was removed and the cells were lysed with lysing buffer and the luciferase activity and protein content were measured as described earlier.
  • Figure 35 illustrates the effect of [Cation], Ca 2+ (•) and Mg 2- ( ⁇ ), on the lipid binding and uptake of 80 ⁇ M SPLP-CPL on BHK cells.
  • FIG. 36 illustrates transfection of SPLP-CPL , SPLP and complexes (each containing 5.0 ⁇ g/mL pCMVLuc) at longer time points.
  • Transfection of SPLP- CPL 4 (4 mol % CPL 4 ) + 40 mM initia ⁇ CaCl 2 (•), SPLP (T), DOPE:DODAC complexes ( ⁇ ), and Lipofectin complexes ( ⁇ ) was performed on 1x10 BHK cells.
  • FIG. 37 illustrates transfection potency and toxicity of SPLP-CPL compared to Lipofectin complexes.
  • Figure 38 illustrates the transfection of BHK cells using both long and short chained CPLs.
  • the presence of the short chained PEG in the CPL results in a decrease by a factor of about 4 compared to the transfection by the long chained CPL.
  • Figure 39 illustrates the transfection of Neuro-2a cells.
  • SPLP + 4 mol % CPL4-lk produces 4 orders of magnitude of gene expression more than SPLP alone in Neuro-2a cells.
  • Figure 40 illustrates in vivo pharmacokinetics of SPLP containing a short chain CPL 4 .
  • the present invention provides cationic-polymer-lipid conjugates (CPLs), such as distal cationic-poly(ethylene glycol)-lipid conjugates that can be incorporated into conventional and stealth liposomes for enhancing, inter alia, cellular uptake.
  • CPLs cationic-polymer-lipid conjugates
  • the CPLs of the present invention have the following architectural features: (1) a lipid anchor, such as a hydrophobic lipid, for incorporating the CPLs into the lipid bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol, for linking the lipid anchor to a cationic head group; and (3) a polycationic moiety, such as a naturally occurring amino acid, to produce a protonizable cationic head group.
  • the present invention provides a compound of Formula I:
  • A is a lipid moiety such as an amphipathic lipid, a neutral lipid or a hydrophobic lipid that acts as a lipid anchor.
  • Suitable lipid examples include vesicle-forming lipids or vesicle adopting lipids and include, but are not limited to, diacylglycerolyls, dialkylglycerolyls. N-N-dialkylaminos, l,2-diacyloxy-3- aminopropanes and l,2-dialkyl-3-aminopropanes.
  • W is a polymer or an oligomer, such as a hydrophilic polymer or oligomer.
  • the hydrophilic polymer is a biocompatable polymer that is non- immunogenic or possesses low inherent immunogenicity.
  • the hydrophilic polymer can be weakly antigenic if used with appropriate adjuvants.
  • Suitable non- immunogenic polymers include, but are not limited to, PEG, polyamides, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers and combinations thereof.
  • the polymer has a molecular weight of about 250 to about 7000 daltons.
  • Y is a polycationic moiety.
  • polycationic moiety refers to a compound, derivative, or functional group having a positive charge, preferably at least 2 positive charges at a selected pH, preferably physiological pH.
  • Suitable polycationic moieties include basic amino acids and their derivatives such as arginine, asparagine, glutamine, lysine and histidine; spermine; spermidine; cationic dendrimers; polyamines; polyamine sugars; and amino polysaccharides.
  • the polycationic moieties can be linear, such as linear tetralysine, branched or dendrimeric in structure.
  • Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2 to about 12 positive charges, and more preferably between about 2 to about 8 positive charges at selected pH values.
  • the selection of which polycationic moiety to employ may be determined by the type of liposome application which is desired.
  • the charges on the polycationic moieties can be either distributed around the entire liposome moiety, or alternatively, they can be a discrete concentration of charge density in one particular area of the liposome moiety e.g., a charge spike. If the charge density is distributed on the liposome, the charge density can be equally distributed or unequally distributed. All variations of charge distribution of the polycationic moiety are encompassed by the present invention.
  • the lipid "A”, and the non-immunogenic polymer “W”, can be attached by various methods and preferably, by covalent attachment. Methods known to those of skill in the art can be used for the covalent attachment of "A” and “W”. Suitable linkages include, but are not limited to, amide, amine, carboxyl, carbonate, carbamate, ester and hydrazone linkages. It will be apparent to those skilled in the art that "A” and “W” must have complementary functional groups to effectuate the linkage. The reaction of these two groups, one on the lipid and the other on the polymer, will provide the desired linkage.
  • the lipid is a diacylglycerol and the terminal hydroxyl is activated, for instance with NHS and DCC, to form an active ester, and is then reacted with a polymer which contains an amino group, such as with a polyamide (see, U.S. patent Application No. 09/218,988, filed December 22, 1998), an amide bond will form between the two groups.
  • a polymer which contains an amino group such as with a polyamide
  • "W” is bound, preferably covalently bound, to "Y".
  • a covalent attachment of "W” to "Y” can be generated by complementary reactivity of functional groups, one on the polymer and the other on the polycationic moiety.
  • an amine functional group on "W” can be reacted with an activated carboxyl group, such as an acyl chloride or NHS ester, to form an amide.
  • an activated carboxyl group such as an acyl chloride or NHS ester
  • the desired coupling can be obtained.
  • Other activated carboxyl groups include, but are not limited to, a carboxylic acid, a carboxylate ester, a carboxylic acid halide and other activated forms of carboxylic acids, such as a reactive anhydride.
  • Reactive acid halides include for example, acid chlorides, acid bromides, and acid fluorides.
  • the polycationic moiety can have a ligand attached, such as a targeting ligand.
  • a ligand attached such as a targeting ligand.
  • the cationic moiety maintains a positive charge.
  • the ligand that is attached has a positive charge.
  • Suitable ligands include, but are not limited to, a compound or device with a reactive functional group and includes lipids, amphipathic lipids, carrier compounds, bioaffinity compounds, biomaterials, biopolymers, biomedical devices, analytically detectable compounds, therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional groups, other targeting moieties, or toxins.
  • A is a lipid moiety such as, an amphipathic lipid, a neutral lipid or a hydrophobic lipid moiety.
  • Suitable lipid examples include, but are not limited to, diacylglycerolyl, dialkylglycerolyl, N-N-dialkylamino, l,2-diacyloxy-3-aminopropane and l,2-dialkyl-3-aminopropane.
  • X is a single bond or a functional group that covalently attaches the lipid to at least one ethylene oxide unit.
  • Suitable functional groups include, but are not limited to, phosphatidylethanolamino, phosphatidylethanolamido, phosphoro, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, carbonate, amido, thioamido, oxygen, NR wherein R is a hydrogen or alkyl group and sulfur.
  • the lipid "A” is directly attached to the ethylene oxide unit by a single bond.
  • the number of ethylene oxide units can range from about 1 to about 160 and preferably from about 6 to about 50.
  • Z is a single bound or a functional group that covalently attaches the ethylene oxide unit to the polycationic moiety.
  • Suitable functional groups include, but are not limited to, phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate, carboxyl, amido, thioamido, NR wherein R is a member selected from the group consisting of hydrogen atom or alkyl group.
  • the terminal ethylene oxide unit is directly attached to the polycationic moiety.
  • Y is a polycationic moiety as described above in connection with Formula I.
  • index "n” is an integer ranging in value from about 6 to about 160.
  • the residue is then purified by repeated precipitation of the chloroform mixture solution with diethyl ether until disappearance of the lipid using chromatography.
  • the purified CPL conjugate is dissolved in a solvent, followed by addition of TFA, and the solution is stirred at room temperature. The solution can again be concentrated under a nitrogen stream.
  • the residue is then purified by repeated precipitation of the mixture with diethyl ether to offer a lipid-PEG-NH 2 , such as a DSPE-PEG-NH 2 or, alternatively, DSPE-CPL- 1 with one protonizable cationic head group.
  • the ratio of the phosphoryl-lipid anchor and the distal primary amine can then be measured by phosphate and flourescamine assays as described herein.
  • the number of protonizable amino groups can be increased to create a polycationic moiety.
  • the polycationic moiety can be increase from about 2 to about 16 positive charges.
  • the positive charges can be incorporated using any number of suitable polycationic moieties such as lysine, arginine, asparagine, glutamine, histidine, polyamines and derivatives or combinations thereof.
  • the number of cationic groups, such as amino groups can be readily controlled during the CPL synthesis.
  • the present invention provides a lipid-based drug formulation comprising:
  • the lipid-based drug formulation of the present invention comprises the second lipid, such as a PEG-lipid derivative.
  • the lipid-based drug formulations of the present invention comprise (a) a compound of Formula II:
  • the lipid-based drug formulation of the present invention comprises the second lipid, such as a PEG-lipid derivative.
  • the CPLs can be utilized in a variety of ways including, for example, in lipid-based drug formulations.
  • the lipid-based formulations can be in the form of a liposome, a micelle, a virosome, a lipid- nucleic acid particle, a nucleic acid aggregate and other forms which can incorporate or entrap one or more bioactive agents.
  • the lipid-based drug formulations of the present invention comprise a second lipid.
  • the compounds of Formulae I and II can be used in lipid-based formulations such as those described in for example, the following copending U.S. Patent Applications Serial Numbers 08/454,641, 08/485,458, 08/660,025, 08/484,282, 60/055,094, 08/856,374, 60/053,813 and 60/063,473, entitled "Methods for
  • lipid components and CPLs used in forming the various lipid-based drug formulations will depend, in part, on the type of delivery system employed.
  • the lipids used in the CPL will generally be selected from a variety of vesicle-forming or vesicle-adopting lipids, typically including phospholipids and sterols, such as phosphatidylenthanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), which have been suitably functionahzed, and the like.
  • PE phosphatidylenthanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • PA phosphatidic acid
  • the lipids used in the CPL will generally be selected from sterylamines, alkylamines, C3-C22 alkanoic acids, lysophospholipids, detergents and the like.
  • the acyl chains can be varied in length and can be saturated or possess varying degrees of unsaturation. The more saturated the acyl chains the more rigid the membrane. Higher degrees of unsaturation impart more fluidity into the vesicle's membrane.
  • the other lipid components e.g., lipids, cationic lipids, neutral lipids, non-cationic lipids, etc.
  • lipids, cationic lipids, neutral lipids, non-cationic lipids, etc. will vary depending on the drug delivery system employed. Suitable lipids for the various drug delivery systems will be readily apparent to those of skill in the art.
  • cationic lipids can be included in the formulation, e.g., liposome, micelle, lipid-nucleic acid particle, etc.
  • Nucleic acid is negatively charged and can be combined with a positively charged entity to form a lipid complex suitable for formulation and cellular delivery.
  • cationic lipid generally refers to a lipid with a cationic head group situated at or near the liposome membrane (when incorporated in a liposome).
  • CPLs are distinguished from cationic lipids by the polymer "W" which in certain instances, has the effect of placing the cationic charge at a significant distance from the membrane.
  • suitable cationic lipids include, but are not limited to, the following: DC-Choi, (see, Gao, et ai, Biochem. Biophys. Res. Comm., 179:280-285 (1991); DDAB; DMRIE; DODAC (see, United States Patent Application Serial Number 08/316,399, filed September 30, 1994, which is incorporated herein by reference); DOGS; DOSPA; DOTAP; and DOTMA.
  • N,N- dioleoyl-N,N-dimethylammonium chloride is used in combination with a phosphatidylethanolamine.
  • the CPL-liposomes of the present invention are optimized for systemic delivery applications.
  • the polymer length in the CPL is shorter than the normal neutral PEG chains (M.W. 2000-5000 Daltons) used for stealth liposomes.
  • the shorter polymer in the CPL is about 250 to about 3000 Daltons and more preferably, about 1000 to about 2000 Daltons.
  • the second lipid is for example, a PEG 3 oo-lipid and the compound of Formula I is, for example, A- PEGiooo-Y- (see, Figure 17C).
  • the shorter polymer when used, it is believed that the distal charge(s) of the CPL is hidden within the normal PEG exclusion barrier, thus allowing retention of long circulation lifetimes while at the same time, extending the positive charges away from the liposomal surface.
  • This embodiment enhances interactions between liposomes and a target cell.
  • the use of different sized polymers, such as PEG chains, in the CPLs and the neutral PEG-lipids used to modulate vesicle circulation and cellular uptake, allows for a new generation of stealth liposomes as drug carriers. It is believed that the optimized polymer length can vary with the specific conditions such as in vitro or in vivo applications, local or systemic administration, and different lipid formulations.
  • the polymer length in the CPL has a larger MW than the normal neutral PEG chains used for stealth liposomes.
  • the second lipid is for example, a PEGiooo-lipid and the compound of Formula I has a formula of, for example, A- PEG 3 oo-Y- (see, Figure 17B).
  • the type of CPL i.e. the length of the polymer chain, and the amount of cationic charge per molecule, and the amount of such CPL in a formulation e.g., SPLP, can be optimized to obtain the best balancing of clearance properties.
  • long chain CPLs and higher levels of such CPLs are to be preferred to increase transfection.
  • a fusogenic liposome or virosome is provided. It will be readily apparent to those of skill in the art that the CPLs of the present invention can advantageously be incorporated into various types of fusogenic liposomes and virosomes. Such fusogenic liposomes and virosomes can be designed to become fusogenic at the disease or target site. Those of skill in the art will readily appreciate that a number of variables can be used to control when the liposome or virosome becomes fusogenic.
  • the fusogenic liposome comprises: a lipid capable of adopting a non-lamellar phase, yet capable of assuming a bilayer structure in the presence of a bilayer-stabilizing component (such as a PEG-lipid derivative); and a bilayer- stabilizing component reversibly associated with the lipid to stabilize the lipid in a bilayer structure.
  • a bilayer-stabilizing component such as a PEG-lipid derivative
  • a bilayer- stabilizing component reversibly associated with the lipid to stabilize the lipid in a bilayer structure.
  • the composition and concentration of the BSC by controlling the composition and concentration of the BSC, one can control the rate at which the BSC is degraded, i.e., broken down, by endogenous systems, e.g., endogenous enzymes in the serum, and, in turn, the rate at which the liposome becomes fusogenic.
  • endogenous systems e.g., endogenous enzymes in the serum
  • lipids When the effective cross-sectional areas of the polar head group and the hydrophobic region buried within the membrane are similar then the lipids have a cylindrical shape and tend to adopt a bilayer conformation.
  • Lipids with head groups that are large relative to their hydrophobic domain, such as lysophospholipids have an inverted cone shape and tend to form micelles in aqueous solution.
  • phase preference of a mixed lipid system depends, therefore, on the contributions of all the components to the net dynamic molecular shape.
  • a combination of cone-shaped and inverted cone-shaped lipids can adopt a bilayer conformation under conditions where either lipid in isolation cannot (see, Madden and Cullis, Biochim. Biophys. Acta, 684: 149-153 (1982)).
  • the lipids which can be used to form the fusogenic liposomes of the present invention are those which adopt a non-lamellar phase under physiological conditions or under specific physiological conditions, e.g., in the presence of calcium ions, but which are capable of assuming a bilayer structure in the presence of a BSC.
  • Such lipids include, but are not limited to, phosphatidylenthanolamines, ceramides, glycolipids. or mixtures thereof.
  • Other lipids known to those of skill in the art to adopt a non-lamellar phase under physiological conditions can also be used.
  • the fusogenic liposome is prepared from a phosphatidylethanolamine.
  • the phosphatidylethanolamine can be saturated or unsaturated.
  • the phosphatidylyethanolamme is unsaturated.
  • the fusogenic liposome is prepared from a mixture of a phosphatidylethanolamine (saturated or unsaturated) and a phosphatidylserine. In another equally preferred embodiment, the fusogenic liposome is prepared from a mixture of a phosphatidylethanolamine (saturated or unsaturated) and a cationic lipid.
  • the lipid-based drug formulations of the present invention comprise a bilayer stabilizing component (BSC).
  • BSCs include, but are not limited to, polyamide oligomers, peptides, proteins, detergents, lipid-derivatives, PEG-lipids such as PEG coupled to phosphatidylethanolamine, and PEG conjugated to ceramides (see, U.S. Patent No. 5,885,613, which is incorporated herein by reference).
  • the bilayer stabilizing component is a PEG-lipid, or an ATTA-lipid.
  • the PEG or the ATTA of the BSC has a greater molecular weight compared to the polymer "W" of the CPL. In other instances, the BSC has a smaller molecular weight compared to the "W" of the polymer.
  • the present invention encompasses all such variations.
  • lipids adopting a non-lamellar phase under physiological conditions can be stabilized in a bilayer structure by BSCs which are either bilayer forming themselves, or which are of a complementary dynamic shape.
  • the non-bilayer forming lipid is stabilized in the bilayer structure only when it is associated with, i.e., in the presence of, the BSC.
  • the BSC be capable of transferring out of the liposome, or of being chemically modified by endogenous systems such that, with time, it loses its ability to stabilize the lipid in a bilayer structure. Only when liposomal stability is lost or decreased can fusion of the liposome with the plasma membrane of the target cell occur.
  • the BSC-lipid therefore, is "reversibly associated" with the lipid and only when it is associated with the lipid is the lipid constrained to adopt the bilayer structure under conditions where it would otherwise adopt a non-lamellar phase.
  • the BSC-lipids of the present invention are capable of stabilizing the lipid in a bilayer structure, yet they are capable of exchanging out of the liposome, or of being chemically modified by endogenous systems so that, with time, they lose their ability to stabilize the lipid in a bilayer structure, thereby allowing the liposome to become fusogenic.
  • the CPL is present in the lipid-based formulation of the present invention at a concentration ranging from about 0.05 mole percent to about 50 mole percent. In a presently preferred embodiment, the CPL is present at a concentration ranging from 0.05 mole percent to about 25 mole percent. In an even more preferred embodiment, the CPL is present at a concentration ranging from 0.05 mole percent to about 15 mole percent.
  • concentration of the CPL can be varied depending on the CPL employed and the rate at which the liposome is to become fusogenic.
  • the liposomes contain cholesterol. It has been determined that when cholesterol-free liposomes are used in vivo. they have a tendency to absorb cholesterol from the plasma lipoproteins and cell membranes. Cholesterol, if included, is generally present at a concentration ranging from 0.2 mole percent to about 50 mole percent and, more preferably, at a concentration ranging from about 35 mole percent to about 45 mole percent.
  • CPL-liposomes are discussed herein.
  • Two general techniques include "post-insertion,” that is, insertion of a CPL into for example, a pre-formed liposome vesicle, and "standard” techniques, wherein the CPL is included in the lipid mixture during for example, the liposome formation steps.
  • the post- insertion technique results in liposomes having CPLs mainly in the external face of the liposome bilayer membrane, whereas standard techniques provide liposomes having CPLs on both internal and external faces.
  • post-insertion involves forming vesicles (by any method), and incubating the pre-formed vesicles in the presence of CPL under appropriate conditions (usually 2-3 hours at 60°C). Between 60-80% of the CPL can be inserted into the external leaflet of the recipient vesicle, giving final concentrations up to 7 mol % (relative to total lipid).
  • the method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-Ceramide).
  • the CPL-LUNs of the present invention can be formed by extrusion.
  • CPL-LUNs can be accomplished using a detergent dialysis or ethanol dialysis method, for example, as discussed in U.S. Patent ⁇ os. 5,976,567 and 5,981,501, both of which are incorporated herein by reference. The extrusion method and the detergent dialysis method are explained in detail in the Example section.
  • Suitable methods include, but are not limited to, sonication, extrusion, high pressure/homogenization, micro fluidizati on, detergent dialysis, calcium-induced fusion of small liposome vesicles, and ether-infusion methods, all of which are well known in the art.
  • One method produces multilamellar vesicles of heterogeneous sizes.
  • the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • the film may be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form.
  • a suitable solvent such as tertiary butanol
  • This film is covered with an aqueous buffered solution and allowed to hydrate, typically over a 15-60 minute period with agitation.
  • the size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents, such as deoxycholate.
  • Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is an effective method for reducing liposome sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller-pore membranes, to achieve gradual reduction in liposome size.
  • liposomes having a size ranging from about 0.05 microns to about 0.40 microns are preferred.
  • lipid-based drug formulations and compositions of the present invention are useful for the systemic or local delivery of bioactive agents such as therapeutic agents, prophylactic agents and diagnostic agents.
  • bioactive agents such as therapeutic agents, prophylactic agents and diagnostic agents.
  • Such delivery systems are described in greater detail in, for example, the following copending U.S. Patent Applications Serial Numbers 08/454,641, 08/485,458, 08/660,025, 08/484,282, 60/055,094, 08/856,374, 60/053,813 and 60/063,473, the teachings of all of which are incorporated herein by reference.
  • liposomes refers generally to liposomes; however, it will be readily apparent to those of skill in the art that this same discussion is fully applicable to- the other drug delivery systems of the present invention (e.g., micelles, virosomes, lipid- nucleic acid particles, etc.).
  • the compositions can be loaded with a therapeutic agent and administered to the subject requiring treatment.
  • the therapeutic agents which are administered using the present invention can be any of a variety of drugs which are selected to be an appropriate treatment for the disease to be treated or prevented.
  • the drug will be an antineoplastic agent, such as vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.
  • Especially preferred antitumor agents include, for example, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylative agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs. It may also be desirable to deliver anti- infective agents to specific tissues by the present methods.
  • compositions of the present invention can also be used for the selective delivery of other drugs including, but not limited to, local anesthetics, e.g., dibucaine and chlorpromazine; beta-adrenergic blockers, e.g., propranolol, timolol and labetolol; antihypertensive agents, e.g., clonidine and hydralazine; anti-depressants, e.g., imipramine, amitriptyline and doxepim; anti- conversants, e.g., phenytoin; antihistamines, e.g., diphenhydramine, chlorphenirimine and promethazine; antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin, and cefoxitin; antifungal agents, e.g., miconazole, terconazole, econazole, isoconazole, butaconazole
  • cationic lipids can be used in the delivery of therapeutic genes or oligonucleotides intended to induce or to block production of some protein within the cell.
  • Nucleic acid is negatively charged and may be combined with a positively charged entity to form a lipid complex or a fully encapsulated stable plasmid- lipid particle.
  • antisense oligonucleotides are directed to targets such as c-myc, bcr-abl, c-myb, ICAM-1, C-erb B-2 and BCL-2.
  • the CPLs of the present invention are also useful in the delivery of peptides, nucleic acids, plasmid DNA, minichromosomes and ribozymes.
  • Another clinical application of CPLs of this invention is as an adjuvant for immunization of both animals and humans.
  • Protein antigens such as diphtheria toxoid, cholera toxin, parasitic antigens, viral antigens, immunoglobulins, enzymes and histocompatibility antigens, can be incorporated into or attached onto the liposomes containing the CPLs of the present invention for immunization purposes.
  • Liposomes containing the CPLs of the present invention are also particularly useful as carriers for vaccines that will be targeted to the appropriate lymphoid organs to stimulate an immune response.
  • Liposomes containing the CPLs of the present invention can also be used as a vector to deliver immunosuppressive or immunostimulatory agents selectively to macrophages.
  • glucocorticoids useful to suppress macrophage activity and lymphokines that activate macrophages can be delivered using the liposomes of the present invention.
  • Liposomes containing the CPLs of the present invention and containing targeting molecules can be used to stimulate or suppress a cell.
  • liposomes incorporating a particular antigen can be employed to stimulate the B cell population displaying surface antibody that specifically binds that antigen.
  • Liposomes incorporating growth factors or lymphokines on the liposome surface can be directed to stimulate cells expressing the appropriate receptors for these factors.
  • bone marrow cells can be stimulated to proliferate as part of the treatment of cancer patients.
  • Liposome-encapsulated antibodies can be used to treat drug overdoses.
  • liposomes having encapsulated antibodies to be delivered to the liver has a therapeutic advantage in clearing substances, such as toxic agents, from the blood circulation. It has been demonstrated that whereas unencapsulated antibodies to digoxin caused intravascular retention of the drug, encapsulated antibodies caused increased splenic and hepatic uptake and an increased excretion rate of digoxin.
  • Liposomes containing the CPLs of this invention also find utility as carriers for introducing lipid or protein antigens into the plasma membrane of cells that lack the antigens.
  • histocompatibility antigens or viral antigens can be introduced into the surface of viral infected or tumor cells to promote recognition and killing of these cells by the immune system.
  • liposomes containing the CPLs of the present invention can be used to deliver any product (e.g., therapeutic agents, diagnostic agents, labels or other compounds) including those currently formulated in PEG-derivatized liposomes.
  • product e.g., therapeutic agents, diagnostic agents, labels or other compounds
  • targeting moieties that are specific to a cell type or tissue.
  • targeting moieties such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and monoclonal antibodies, has been previously described (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044, the teachings of which are incorporated herein by reference).
  • the targeting moieties can comprise the entire protein or fragments thereof.
  • the diagnostic targeting of the liposome can subsequently be used to treat the targeted cell or tissue.
  • the toxin when a toxin is coupled to a targeted liposome, the toxin can then be effective in destroying the targeted cell, such as a neoplasmic cell.
  • the present invention provides a method for increasing intracellular delivery of a lipid-based drug formulation, comprising: incorporating into the lipid-based drug formulation, a compound of Formulae I or II, thereby increasing the intracellular delivery of the lipid based drug formulation compared to a formulation without a compound of Formulae I or II.
  • the compounds of Formulae I or II increase intracellular delivery about 10 fold to about 1000 fold and preferably, about 10 fold to about 100000 fold.
  • the present invention provides a method of increasing the blood-circulation time of a parenterally administered lipid-based drug formulation, the method comprising: incorporating into the lipid-based drug formulation about 0.1 to 20 mole percent of a compound of Formulae I or II.
  • the present invention provides a method for transfection of a cell with a lipid-based drug formulation, comprising: contacting the cell with a lipid-
  • SUBST ⁇ UTE SHEET RULE 26 based drug formulation having about 0.1 to 20 mole percent of a compound of Formulae I or II.
  • a method for increasing the transfection of a cell with a lipid-based drug formulation comprising: contacting the cell with a lipid-based drug formulation having about 0.1 to 20 mole percent of a compound of Formulae I or II, whereby the transfection efficiency of the lipid-based drug formulation is increased compared to the transfection efficiency of a lipid-based drug formulation without the compound of Formulae I or II.
  • the lipid-based drug formulations or compositions, e.g., liposomes, prepared using the CPLs of this invention can be labeled with markers that will facilitate diagnostic imaging of various disease states including tumors, inflamed joints, lesions, etc.
  • these labels will be radioactive markers, although fluorescent labels can also be used.
  • gamma-emitting radioisotopes is particularly advantageous as they can easily be counted in a scintillation well counter, do not require tissue homogenization prior to counting and can be imaged with gamma cameras.
  • Gamma- or positron-emitting radioisotopes are typically used, such as
  • the liposomes can also be labelled with a paramagnetic isotope for purposes of in vivo diagnosis, as through the use of magnetic resonance imaging (MRI) or electron spin resonance (ESR).
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • Methods of loading conventional drugs into liposomes include, for example, an encapsulation technique, loading into the bilayer and a transmembrane potential loading method.
  • an encapsulation technique the drug and liposome components are dissolved in an organic solvent in which all species are miscible and concentrated to a dry film.
  • a buffer is then added to the dried film and liposomes are formed having the drug incorporated into the vesicle walls.
  • the drug can be placed into a buffer and added to a dried film of only lipid components. In this manner, the drug will become encapsulated in the aqueous interior of the liposome.
  • the buffer which is used in the formation of the liposomes can be any biologically compatible buffer solution of, for example, isotonic saline, phosphate buffered saline, or other low ionic strength buffers. Generally, the drug will be present in an amount of from about 0.01 ng/mL to about 50 mg/mL.
  • the resulting liposomes with the drug incorporated in the aqueous interior or in the membrane are then optionally sized as described above.
  • Transmembrane potential loading has been described in detail in U.S. Patent No. 4,885,172, U.S. Patent No. 5,059,421, and U.S. Patent No. 5,171,578, the contents of which are incorporated herein by reference.
  • the transmembrane potential loading method can be used with essentially any conventional drug which can exist in a charged state when dissolved in an appropriate aqueous medium.
  • the drug will be relatively lipophilic so that it will partition into the liposome membranes.
  • a transmembrane potential is created across the bilayers of the liposomes or protein-liposome complexes and the drug is loaded into the liposome by means of the transmembrane potential.
  • the transmembrane potential is generated by creating a concentration gradient for one or more charged species (e.g., Na + , K + and/or H + ) across the membranes.
  • This concentration gradient is generated by producing liposomes having different internal and external media and has an associated proton gradient. Drug accumulation can than occur in a manner predicted by the Henderson-Hasselbach equation.
  • the liposome compositions of the present invention can by administered to a subject according to standard techniques.
  • pharmaceutical compositions of the liposome compositions are administered parenterally, i.e., intraperitoneally, intravenously, subcutaneously or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously by steady infusion.
  • suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
  • the pharmaceutical compositions can be used, for example, to diagnose a variety of conditions, or treat a diseased state.
  • the diseases include, but are not limited to, inflammation associated with rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), septic shock, and acute and chronic inflammation, including atopic dermatitis and psoriasis.
  • various neoplasms and tumor metastases can be treated.
  • compositions for intravenous administration which comprise a solution of the liposomes suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.9% isotonic saline, and the like.
  • These compositions can be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the concentration of active ingredient in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • the amount of composition administered will depend upon the particular label used (i.e., radiolabel, fluorescence label, and the like), the disease state being diagnosed and the judgment of the clinician.
  • Distal cationic-poly(ethylene glycol)-lipid conjugates were designed, synthesized and incorporated into conventional and stealth liposomes for enhancing cellular uptake.
  • the present approach uses either inert, nontoxic or naturally occurred compounds as components for the CPL synthesis.
  • CPLs were synthesized with the following architectural features: 1) a hydrophobic lipid anchor of DSPE for incorporating CPLs into liposomal bilayer; 2) a hydrophilic spacer of polyethylene glycol for linking the lipid anchor to the cationic head group; and 3) a naturally occurring amino acid (L-lysine) was used to produce a protonizable cationic head group. The number of charged amino groups can be controlled during the CPL synthesis.
  • DSPE-CPLs were almost quantitatively inco ⁇ orated into liposomal bilayer by a hydration-extrusion method. Quite su ⁇ risingly, in an in vitro model, it was confirmed for the first time that liposomes possessing distal positively charged polymer conjugates with preferably four or more charges efficiently bind to host cell surfaces and enhance cellular uptake in mammalian cells.
  • DSPE Distearoyl-sn-glycero-3- phosphoethanolamine
  • DSPC l,2-distearoyl-sn-glycero-3-phosphocholine
  • DSPE- PEG2000 l,2-distearoyl-3-phosphatidylethanolamine-PEG2ooo
  • TFA trifluoroacetic acid
  • CPL cationic-poly (ethylene glycol)-lipid conjugate
  • DSPE-CPL (cationic-polyethylene glycol)-DSPE conjugate
  • DSPE- CPL-2 DSPE-CPL with two positive charges
  • DSPE-CPL-4 DSPE-CPL with four positive charges
  • DSPE-CPL-8 DSPE-CPL with eight positive charges
  • Rh-PE (or Rho- PE), l,2-dioleoyl-sn
  • Synthesis of DSPE-CPL- 1 To a solution of DSPE ( 121 mg, 161 mmol) and Et 3 N (200 ⁇ L) in CHC1 3 (2 mL) at 45 °C was added t-Boc-NH-PEG 3400 - CO2NHS (500 mg, 147 ⁇ mol in 2 mL dry CHCI 3 ), and the solution was stirred for 3 hr at ambient temperature. The solution was concentrated under a nitrogen stream to dryness. The residue was purified by repeat precipitation of the chloroform mixture solution with diethyl ether until disappearance of DSPE spot on TLC.
  • the purified DSPE-PEG conjugate was dissolved in 2 mL CHC13 followed by addition of 2 mL TFA, and the reaction solution was stirred at room temperature for 4 hr. The solution was again concentrated under a nitrogen stream to dryness. The residue was purified by repeat precipitation of the chloroform mixture solution with diethyl ether to offer DSPE-PEG- NH 2 as DSPE-CPL-1 with one protonable cationic head group: yield 500 mg (120 ⁇ mol, 80%); R f 0.4 (CHC /MeOH, 9/1, v/v); The ratio of phosphoryl-lipid anchor and the distal primary amine was measured by phosphate and flourescamine assays and 1H NMR.
  • the residue was purified by repeat precipitation of the chloroform mixture solution with diethyl ether until disappearance of t-Boc-Lysine spot on TLC.
  • the purified DSPE-PEG-conjugates were dissolved in 2 mL CHC1 followed by addition of 2 mL TFA, and the reaction solution was stirred at room temperature for 4 hr. The solution was again concentrated under a nitrogen stream to dryness.
  • DSPE-CPL2 The residue was purified by repeat precipitation of the chloroform mixture solution with diethyl ether to offer DSPE-CPL2: yield 250 mg (57 ⁇ mol, 95%); R f 0.4 (CHCl 3 /MeOH, 9/1, v/v); The ratio of phosphoryl-lipid anchor and the distal primary amine was 1 measured by phosphate and flourescamine assays.
  • DSPE-CPL4 and DSPE- CPL8 were synthesized in a similar manner.
  • LUN Large unilamellar vesicles
  • lipid mixtures DSPC/Chol, 60:40 mol/mol
  • DSPE-CPLs as set out in Table 3
  • Rh-PE in chloroform a lipid mixture containing trace amounts of Rh-PE in chloroform.
  • the trace amount of solvent was then removed under a vacuum overnight.
  • the lipid film was hydrated in HBS buffer (pH 7.5) with or without HPTS (50 mM) by vortex mixing.
  • MSNs multilamellar vesicles
  • Nuclepore polycarbonate filters
  • Extrusion device Lipex Biomembranes, Inc., Vancouver, BC, Canada
  • Uninco ⁇ orated DSPE-CPLs and in some cases untrapped free HPTS were removed by chromatography using a 1.1 x 20 cm Sepharose CL-6B column (Sigma Chemical Co., St. Louis, MO, USA) equilibrated with HBS buffer.
  • Liposome size was determined by quasi-elastic light scattering (QELS) using a Nicomp 370 submicron particle sizer (Santa Barbara, CA).
  • CPL distal positively charged cationic polymer lipid conjugates
  • the present approach uses inert, nontoxic and naturally occurring compounds, e.g., amino acids, as components for the CPL synthesis.
  • Several CPLs were designed with the following architectural features: 1 ) a hydrophobic lipid anchor for inco ⁇ orating the CPLs into the liposomal bilayer; 2) a hydrophilic spacer for linking the lipid anchor to the cationic head group; and 3) a cationic head group.
  • the amount and nature of the cationic group can be changed according to the final application.
  • a naturally occurring amino acid, L-lysine was used to produce a protonizable amino group. The number of amino group can be controlled during the CPL synthesis.
  • Lipid composition Size (nm) CPL ⁇ nco ⁇ .(%) 1, DSPC/Ch(60:40) no -
  • LUVs containing CPL can be formed by a detergent dialysis method.
  • the LUVs contain DOPE, DODAC, PEG-Cer-C20, and CPL 4 [3.4K] (or CPL [1K]). Two preparations were made with the CPL comprising 4 mol % of the original lipids: TABLE 4
  • the lipids indicated above were co-dissolved in chloroform, which was then removed under nitrogen followed by 2 hours under high vacuum.
  • the dry lipid mixture (10 ⁇ mol total) was then hydrated in 83 ⁇ L of 1 M OGP and 1 mL Hepes- buffered saline (20 mM Hepes 150 mM NaCI pH 7.5) at 60°C with vortexing until all the lipid was dissolved in the detergent solution.
  • the lipid-detergent mixture was transferred to Slide-A-Lyzer dialysis cassettes, and dialysed against at least 2 L HBS for 48 hours, with a least two changes of buffer in that time. Removal of detergent by dialysis results in formation of LUVs.
  • the lipid samples were fractionated on a column of Sepharose CL-4B (see Figures 8A and 8B). The fractionation profiles show LUVs formed with either CPL 4 [3.4K] or CPL 4 [1K].
  • the final concentration of CPL in the LUV fraction (fractions 7 - 10) was estimated from initial and final dansyl/rhodamine ratios, and from estimating the proportion of total dansyl and rhodamine fluorescence present in the LUV peak. Essentially identical results were obtained.
  • LUVs containing CPL 4 can be formed by detergent dialysis. Not all of the CPL 4 is inco ⁇ orated into the vesicle, and the proportion that is inco ⁇ orated falls as the initial CPL/lipid molar ratio is increased. In the present case, beginning with 4 mol % CPL, about 3 mol % was inco ⁇ orated into the LUV. For an initial CPL content of 12 mol %, a final content of 6 mol % was achieved. It is also worth noting that the behavior of the CPL 4 [1K] is very similar to that of the CPL 4 [3.4K]. This is also true in post-insertion studies. In certain instances, the ideal length of the hydrophilic spacer will allow the cationic groups to extend out from the liposomal surface at a distance shorter than the normal neutral PEG that is typically being used to provide stealth properties for increased liposomal circulation lifetimes.
  • a non-specific targeting approach is described that involves increasing the electrostatic attraction between liposomes and cells by inco ⁇ oration of positively-charged lipid molecules into preformed vesicles.
  • This approach leads to dramatic increases in cell binding uptake in vitro in BHK cells.
  • the methodology is demonstrated to work for neutral vesicles and for vesicles composed of lipids used in the construct of lipid-based gene carriers.
  • the approach outlined herein thus has numerous applications ranging from delivery of conventional drugs to gene therapy.
  • CPLs Two types were synthesized which differed in the lipid anchor portion of the molecule.
  • the anchor was distearoylglycerol (DSG), while the other contained distearoylphosphatidylethanolamine (DSPE).
  • the molecule consists of the anchor portion, to which is attached a PEG3400 chain. At the end of the PEG chain, a charged "headgroup” is attached, often made up of lysine residues linked together.
  • CPLs were synthesized which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T), and 4 (quad, or Q) positive charges.
  • Quad CPLs were synthesized, hence these are numbered Ql through Q5.
  • the nomenclature chosen to describe these compounds specifies the type of lipid anchor and the identity of the headgroup (e.g., d-DSPE-CPL-Q5). The lower case "d” indicates a dansylated derivative.
  • vesicles were formed using a detergent dialysis method (see, Wheeler, J.J., et al. (1999) Stabilized plasmid-lipid particles: construction and characterization. Gene Therapy 6, 271-281, the teachings of which are inco ⁇ orated herein by reference).
  • the lipids, as described in Example II, were co-dissolved in chloroform in the appropriate ratios, following which the chloroform was removed under a stream of nitrogen and placed under high vacuum for 2 hours.
  • Vesicles of DOPC and DOPC/Chol were prepared by extrusion as previously described (Hope, M.J., et al, (1985) Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812, 55-65).
  • CPLs cationic PEG-lipids
  • the insertion levels of CPL were measured by fluorescence.
  • the vesicles contained either 0.25 mol % or 0.5 mol % rhodamine-PE, and the CPL contained a dansyl group.
  • a 15 ⁇ L aliquot (initial fraction) was set aside for analysis.
  • the amount of CPL inserted into the vesicles could then be quantified by measuring the initial dansyl/rhodamine (D/R) fluorescence ratio, and the D/R ratio of the isolated CPL-LUVs.
  • D/R dansyl/rhodamine Fluorescence parameters: for the rhodamine assay, the excitation wavelength was 560 nm, and the emission wavelength was 590 nm.
  • the excitation wavelength was 340 nm, and the emission wavelength was 510 nm. In general, the excitation and emission slit widths were 10 and 20 nm, respectively.
  • the assay was performed as follows: to an aliquot of the initial sample (2-3 ⁇ L) or the CPL-LUV (20-40 ⁇ L) was added 30 ⁇ L of 10% Triton X-100 followed by 2 mL of HBS. The fluorescence levels of both the dansyl and rhodamine labels were read consecutively using a wavelength program as per the above parameters with an emission filter of 410 nm. The %-insertion was calculated as follows:
  • the free CPL elutes in a broad peak centered at 16 mLs, which is separate from the vesicle peak, allowing for easy isolation of the CPL-LUV.
  • the DSPE- CPL-Q5 is retained and does not exchange out of the vesicles.
  • the CPL-LUN fraction from Figure 9(B) was re-eluted on the column of Sepharose CL-4B. As shown in Figure 9(C), all of the CPL remains with the LUVs. The effects of incubation temperature and time on the insertion process are shown in Figure 10.
  • DSPE-CPL-Q1 was incubated in the presence of DOPE/DODAC/PEGCerC20 (84/6/10) at room temperature, 40°C, and 60°C, with aliquots withdrawn at 1, 3, and 6 hours.
  • the highest insertion levels were achieved at 60 °C, which was therefore used in subsequent insertions. Although slightly higher insertion was obtained at 6 hr, we chose 3 hr to minimize sample degradation.
  • CPL-LUVs were examined by fluorescence microscopy, using a rhodamine filter. While control LUVs exhibited no signs of aggregation, significant levels were observed for CPL-LUVs. However, it was found that addition of 40 mM CaCl 2 completely prevented this effect.
  • oligopeptides see, Zalipsky et al, Bioconjugate Chemistry 6, 705-708 (1995); Zalipsky et al, Bioconjugate Chemistry 8, 111-118 (1997)) oligosaccharides (see, Zalipsky et al, Bioconjugate Chemistry 8, 111-118 (1997)), folate (see, Gabizon et al, Bioconjugate Chemistry 10, 289-298 (1999); Lee et al, Journal of Biological Chemistiy 269, 3198-3204 (1994); Reddy et al.
  • the approach involves the insertion of novel cationic-PEG-lipids into pre-formed liposomes, leading to a cationic vesicle in which the positive charge involved in cell interaction is located some distance away from the vesicle surface.
  • the process is illustrated in Figure 16 for the insertion of a CPL into sterically-stabilized LUVs composed of DOPE, the cationic lipid DODAC, and PEG-Cer-C20.
  • This lipid composition was chosen for study for two reasons: first, it allows for efficient entrapment of plasmid DNA within small vesicular structures by virtue of the presence of positively charged DODAC (see, Wheeler et al. Gene Therapy 6, 271-281 (1999)), and thus has potential as a gene delivery system (see below).
  • this composition is representative of the many sterically-stabilized drug delivery systems which contain PEG-lipids. Insertion of CPLs leads to localization of positive charge above the surface PEG layer, thereby allowing electrostatic interactions between the CPLs and cell surfaces. This should lead to increased cellular interactions for both conventional- and PEG-containing liposomes.
  • the CPLs are conjugates of DSPE, a dansyl-lysine moiety, the hydrophilic polymer PEG 34 oo, and a mono- or multivalent cationic headgroup.
  • the PEG functions as a spacer, separating the charged headgroup from the lipid anchor and vesicle surface.
  • Incubation of a wide variety of neutral and cationic LUVs with micellar CPLs resulted in the inco ⁇ oration of up to 6-7 mol % (relative to total vesicle lipid) of CPL in the outer vesicle monolayer (see tables in Figures 23 and 24).
  • the insertion efficiency was quite high, with approximately 70 - 80% of added CPL inco ⁇ orating into the LUVs (see tables in Figures 23 and 24).
  • the most important factors influencing the CPL insertion levels were the incubation temperature ( Figure 10) and initial CPL/lipid ratio ( Figure 11).
  • the composition of the liposome was found to affect the final CPL levels to a lesser degree (see tables in Figures 23 and 24).
  • the CPL-LUV could be efficiently separated from free CPL by gel exclusion chromatography. Similar insertion levels were obtained for all CPLs, with headgroup charges ranging from one to four charges per molecule . With this knowledge, vesicles could be prepared containing a desired level of CPL with reasonable accuracy.
  • the CPL 3 sample has a greater positive charge than the CPL 4b sample, and yet exhibits only 1/3 the uptake. It would appear that localization of a sufficient positive charge density at the distal end of the CPL molecule is an important parameter in ensuring interaction with cells. At least four charges seem to be required for efficient cell binding to occur.
  • the protocol described for insertion of CPL into conventional and sterically-stabilized CPL is ideal for demonstrating the methodology using in vitro applications.
  • the added positive charge is physically distant from the surface, and is available for interactions with cells. This is particularly important for polymer-coated vesicles that are designed for minimal interactions with serum proteins and cells such as macrophages.
  • this system may not be ideal for in vivo applications, where it may be desirable to initially hide or screen the CPL charge to reduce clearance and allow accumulation of the vesicles at the tissue of choice.
  • alternative embodiments employ shorter PEG spacer chains in the CPL, or longer PEG chains in the PEG-Cer molecules.
  • the PEG-Cer molecules are known to exchange out of the particle during circulation see, Webb et al, Biochimica et Biophysica Acta 1372, 272- 282 (1998)], which would leave the CPL exposed for cellular interactions.
  • the cationic liposomes employed in the present study are composed of a fusogenic lipid (DOPE), a cationic lipid (DODAC), and a stabilizing lipid (PEG-Cer-C20), the latter of which imparts long-circulating properties to the vesicles.
  • DOPE fusogenic lipid
  • DODAC cationic lipid
  • PEG-Cer-C20 stabilizing lipid
  • This lipid composition was modeled after a new class of lipid-based DNA carrier systems known as stabilized plasmid-lipid particles (SPLPs) see, Wheeler et al. Gene Therapy 6, 271-281 (1999)). SPLPs are small (70 nm) particles that encapsulate a single plasmid molecule.
  • SPLPs thus represent the first carrier systems with real potential for systemic in vivo gene therapy applications.
  • the approach described here greatly enhances the tranfection potency of these particles by increasing cellular binding and uptake, which leads to increased intracellular delivery of plasmids.
  • CPL in conventional formulations (e.g., anticancer drugs) also leads to increased efficacy.
  • SPLPs SPLPs
  • the 4 mol % CPL shows the greatest increase in transfection: approximately 4500 times higher, followed by the 3% and then the 2% CPL samples. Therefore, the presence of the CPL, DSPE-Quad5 in the SPLP increased in both uptake and transfection to levels comparable to or above those achieved with the complexes.
  • the final samples were prepared to contain 2, 3, or 4 mol % of the CPL.
  • the dansyl assay involved preparing a standard curve of 0.5 to 2.5 mol % of dansylated CPL in BBS and determining the concentration of the CPL in the sample.
  • the phospholipid was extracted from the SPLP by extracting the lipid using the Bligh-Dyer extraction technique (Bligh & Dyer, 1952) and then performing a Fiske- Subarrow assay on the organic phase of the extraction.
  • the PicoGreen assay was performed by comparing the sample in the presence of PicoGreen and Triton X-100 using a DNA standard curve. The final % insertion of the CPL was determined by dividing the CPL concentration by the lipid concentration.
  • the optimal time for insertion of the CPL into the SPLP was determined using SPLP prepared with 0.5 mol % Rh-DSPE. 15 nmol of the dansylated CPL (DSPE- Quad5) was mixed with 200 nmol of the labeled SPLP and the sample was incubated at 60°C for various time points (0.5, 1, 2, 3, and 4 hours). At these time points the sample was removed from the water bath and was passed down a Sepharose CL-4B column. The major fraction was collected from the column and the dansyl to rhodamine fluorescence ratios were measured.
  • the parameters used for the rhodamine fluorescence were a ⁇ ex of 560 nm and a ⁇ em of 600 nm and for the dansyl fluorescence were a ⁇ ex of 340 m and a ⁇ em of 510 nm.
  • the excitation and emission slit widths for both of these were 10 nm and 20 nm, respectively.
  • Freeze-Fracture EM Freeze-fracture EM was performed on the 2%, 3%, and 4% CPL samples by methods which will be described by K. Wong 5. Serum Stability of Particles: The stability of the DNA within these
  • CPL-SPLP was determined by incubating the samples (25 ⁇ L), containing 6 ⁇ g of plasmid DNA (pLuc) for various time periods (0, 1, 2, and 4 hours) in 50% mouse serum (25 ⁇ L) at 37°C. At each time point, other than the zero time point, 1 l ⁇ L of the mixture was removed, the volume was made up to 45 ⁇ L using water and the samples were placed on ice. The DNA was then extracted from the lipid using one volume of phenohchloroform (1:1). Following a 20 min centrifugation in a microfuge, the top aqueous phase was removed. The zero time point was obtained by removing 5.5 ⁇ L of the sample prior to serum addition and performing the extraction.
  • pLuc plasmid DNA
  • the rhodamine fluorescence of the lysate was then measured on a fluorometer using a ⁇ ex of 560 nm and a ⁇ em of 600 nm using slit widths of 10 and 20 nm, respectively. An emission filter of 430 nm was also used. A 1.0 L microcuvette was used. The lipid uptake was determined by comparison of the fluorescence to that of a lipid standard (5 nmol). This value was then normalized to the amount of cells present by measuring the protein in 50 ⁇ L of the lysate using the BCA assay.
  • BHK cells were plated in 24-well plates in complete media. These were incubated overnight at 37°C in 5% CO 2 .
  • SPLP, SPLP + 75mM CaCl 2 , DOPE:DODAC (1 :1)/DNA complexes, and CPL-SPLP systems (2, 3, and 4 mol % CPL) containing 2.5 ⁇ g of DNA were made up to lOO ⁇ L using HBS or HBS + 75mM CaCl 2 and were placed on the cells. Then 400 ⁇ L of complete media was added to this. At 4 and 9 hours, the transfection media was removed and replaced with complete media containing penicillin and streptomycin for a complete 24 hour transfection.
  • the cells were lysed with lysis buffer containing Triton X-100. Following this lysis, 10- 20 ⁇ L of the lysated was transferred to a 96-well luminescence plate. The luminescence of the samples on the plate were measured using a Luciferase reaction kit and a plate luminometer. The luciferase activity was determined by using a luciferase standard curve and was normalized for the number of cells by measuring the protein with the BCA assay on 10-20 ⁇ L of the lysated.
  • Figures 18A and B show that the uptake and transfection of the SPLP system is on the order of 10 times lower than complexes.
  • the CPL, DSPE-Quad5 will be used in the following studies. Its structure is shown in Figure 16A. This molecule possesses four positive charges at the end of a PEG oo molecule, which has been covalently attached to the lipid DSPE. The inco ⁇ oration of this CPL into empty liposomes of the same composition as the SPLP has been described previously in the above examples.
  • the DSPE-Quad5 was inco ⁇ orated into SPLPs containing DOPE:PEG- CerC20:DODAC (84: 10:6) at various concentrations of the CPL (from 2-4 mol %).
  • the inco ⁇ oration efficiencies for the various CPL percentages were between 70 and 80% of the initial.
  • gel filtration chromatography was employed in order to separate the SPLPs possessing the CPL from the uninco ⁇ orated CPL.
  • a typical column profile for the 3% DSPE-Quad5 is shown in Figure 19A.
  • the CPL, lipid, and DNA all eluted from the column at the same time in a single peak. There was however a small amount of uninco ⁇ orated CPL that eluted at a later stage.
  • the diameter of these particles containing the CPL was determined by QELS to be 125 mn compared to the SPLP, which had a diameter of 109 nm. To observe the structure of these particles compared to the SPLP in the absence of the CPL, freeze- fracture EM was performed
  • the serum stability of the SPLP in the presence and absence of various amounts of the CPL was assayed (data not shown). Incubating free DNA with 50% mouse serum for only 1 hour results in its complete degradation. The serum stability of the CPL-SPLPs was similar to that for the SPLP system. This indicates that the DNA in the CPL-SPLP is as protected as that in the SPLP system without CPL.
  • Figure 21 shows the time course for the uptake of rhodamine labeled SPLP in the presence (2, 3, or 4 mol %) and absence of the DSPE-Quad5 (0%).
  • the uptake of the 4% system is higher than the 3% system, which is higher than the 2% system, and all three are much higher than the system without CPL.
  • Fig. 22 shows 4 h and 9 h time points of the same formulations.
  • EXAMPLE V This example illustrates the inco ⁇ oration of a CPL into a Stabilized
  • SALP Antisense-Lipid Particle
  • Distearoylphosphatidylcholine was purchased from Northern Lipids (Vancouver, Canada).
  • DODAP or AL-1 l,2-dioleoyloxy-3-dimethylammoniumpropane
  • Cholesterol was purchased from Sigma Chemical Company (St. Louis, Missouri, USA).
  • PEG-ceramides were synthesized by Dr. Zhao Wang at Inex Pharmaceuticals Co ⁇ . using procedures described in PCT WO 96/40964, inco ⁇ orated herein by reference. [ 3 H] or [ 14 C]-CHE was purchased from NEN (Boston, Massachusetts, USA).
  • lipids were > 99% pure. Ethanol (95%), methanol, chloroform, citric acid, HEPES and NaCI were all purchased from commercial suppliers. Lipid stock solutions were prepared in 95% ethanol at 20 mg/mL (PEG- Ceramides were prepared at 50 mg/mL).
  • SALPs are first prepared according to the methods set out in PCT Patent Application No. WO 98/51278, published 19 November 1998, and inco ⁇ orated herein by reference. See also, J.J. Wheeler et al, (1999), Gene Therapy, 6, 271-281. Briefly, a l ⁇ mer of [3H]-phosphorofhioate oligodeoxynucleotide Inx-6295 (human c-myc) having sequence 5' T AAC GTT GAG GGG CAT 3' (SEQ ID.
  • the antisense-lipid mixture was subjected to 5 cycles of freezing (liquid nitrogen) and thawing (65°C), and subsequently was passed 10X through three stacked 100 nm filters (Poretics) using a pressurized extruder apparatus with a thermobarrel attachment (Lipex Biomembranes). The temperature and pressure during extrusion were 65°C and 300-400 psi (nitrogen), respectively.
  • the extruded preparation was diluted with 1.0 mL of 300 mM citric acid, pH 3.8, reducing the ethanol content to 20%.
  • the extruded sample was dialyzed (12 000- 14 000 MW cutoff; SpectraPor) against several liters of 300 mM citrate buffer, pH 3.8 for 3-4 hours to remove the excess ethanol.
  • the sample was subsequently dialyzed against HEPES-buffered saline (HBS), pH 7.5, for 12-18 hours to neutralize the DODAP and release any antisense that was associated with the surface of the vesicles.
  • Encapsulation was assessed either by analyzing the pre-column and post-column ratios of [ ⁇ ]-antisense and [ 14 C]-lipid or by determining the total pre-column and post-column [ ⁇ ]-antisense and [ l4 C]-lipid radioactivity.
  • CPL is inco ⁇ orated after the SALPs are prepared. Approximately 5 ⁇ mol SALP were mixed with 3-10 mol % CPL (i.e., 0.15-0.5 ⁇ mol CPL). CPL were stored as micellar solutions in HBS, or in methanol. When CPL was added in methanol, the final methanol concentration of 3-4%. The mixtures were incubated overnight at room temperature or at 40°C. Uninco ⁇ orated CPL was removed from the SALP preparation by column separation (Sepharose CL-4B equilibrated with HBS, 75mM CaCL 2 at pH 7.5). Inco ⁇ oration efficiency was between 34 and 60%. It is anticipated that other organic solvents may improve inco ⁇ oration efficiency.
  • distal positively charged cationic poly(ethylene glycol) lipid conjugates were synthesized and assessed for their efficacy at enhancing the cellular uptake of CPL-inco ⁇ orated liposomes. It was confirmed that distal charged polymer conjugates bound to a liposome surface enhanced liposome uptake in mammalian cells in vitro.
  • CMC critical micelle concentration
  • CPLi Compared to a control, reduced cell uptake was observed for CPLi, a moderate increase for CPL 2 (2 fold), and a large increase for both CPL 4 and CPL 8 .
  • the similar degree of increase resulting from CPL and CPL 8 indicates a charge density of four in the CPLs satisfies the requirement for maximum enhanced cellular uptake.
  • DSPC l,2-Distearoyl-sn-glycero-3- phosphocholine
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • the phosphate concentration of the CPL was determined using the Fiske- Subarrow phosphorus assay (see, Fiske, C.H., and Subbarow, Y. (1995) The colorimetric determination of phosphorous. J. Biol. Chem. 66, 375-400.).
  • the primary amine concentration in the CPL was determined using the fluorophore, fluorescamine.
  • a fluorescamine solution (0.6mg/mL) in acetone was prepared.
  • An aliquot of CPL solution in FIBS (2-4 ⁇ L) was made up to 250 ⁇ L with 200 mM sodium borate, pH 8.0. To this mixture, 50 L of the fluorescamine solution was added dropwise with vortexing, followed by 1700 ⁇ L of water.
  • tBoc-NH-PEG 340 o-CO 2 -(N ⁇ - dansyl)iysine-NHS (2) was prepared as follows. A solution of tBoc-NH-PEG 34 oo-CO 2 - (N ⁇ -dansyl)lysine (1) (500 mg, 132 ⁇ mol) and NHS (31.5 mg, 274 ⁇ mol) in 2 mL of dry chloroform was added to DCC (42.8 mg, 207 ⁇ mol) dissolved in 1 mL of dry chloroform. The reaction mixture was stirred for 2 h at room temperature.
  • DSPE 120.6 mg, 161 ⁇ mol
  • Dansylated CPLi (4) Trifluoroacetic acid (TFA) (2 mL) was added to a solution of dansylated CPLi-tBoc (3) (550 mg, 121 ⁇ mol) in 2 mL of chloroform and stirred for 4 h at room temperature. The solution was concentrated to a thick paste and chloroform/ether washed three times. After the removal of ether, the solid was dissolved in 6 mL of chloroform/methanol (2:1) and washed with 1.2 mL of 5% sodium bicarbonate. The chloroform phase was extracted, dried and redissolved in 6 mL chloroform/methanol (2:1) and washed with 1.2 mL distilled water.
  • TFA Trifluoroacetic acid
  • Dansylated CPL 2 -tBoc (5) A solution of N ⁇ ,N ⁇ -di-tBoc-L-lysine-N- hydroxysuccinimide ester (105 mg, 236 ⁇ mol) in 2 mL dry chloroform was gradually added to a solution of dansylated CPLi (4) (510 mg, 112 ⁇ mol) in 2 mL chloroform containing 200 ⁇ L triethylamine and stirred at room temperature for 3 h. The completion of the reaction was indicated by the disappearance of primary amine as visualized by ninhydrin assay on TLC.
  • the reaction mixture was concentrated to a thick paste and chloroform/ether washed ( ⁇ 3 times) until the disappearance of excess N ⁇ ,N ⁇ -di-tBoc-L- lysine-N-hydroxysuccinimide ester as checked by TLC.
  • the product was dissolved in 6 mL chloroform/methanol (2:1) and washed with 1.2 mL 0.1 M HCl.
  • the chloroform phase was extracted, dried, redissolved in 6 mL chloroform/methanol (2: 1) and washed with 1.2 mL distilled water.
  • the chloroform phase was concentrated to a thick paste and the purified compound was obtained through a chloroform/ether wash and vacuum dried. Yield: 510 mg (96%).
  • CPL -tBoc (7) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (170 mg, 383 ⁇ mol) with dansylated CPL 2 (6) (455 mg, 95 ⁇ mol). Yield: 475 mg (96%).
  • TLC sica gel
  • chloroform methanol 85: 15
  • CPL 8 -tBoc (9) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (70 mg, 158 ⁇ mol) with dansylated CPL 4 (8) (100 mg, 19 ⁇ mol). Yield: 112 mg (96%).
  • TLC sica gel chloro form/methanol (85:15) R/0.58. ⁇ NMR (CDCI 3 ).
  • the CPL were synthesized by repeated coupling reaction steps involving amines and NHS-activated carbonate groups as outlined in Figure 29. This consists of (a) inco ⁇ orating the dansyl fluorescent label to the hydrophilic PEG spacer, (b) coupling of the DSPE anchor, and (c) attachment of the cationic headgroup to the lipid.
  • the heterobifunctional PEG polymer tBoc-NH-PEG 340 o-CO 2 -NHS (MW 3400) was chosen for two reasons. Firstly, it was commercially available. Secondly, it is insoluble in ether that provided a very convenient means of purifying its derivatives, 1 - 10. Other reagents were used in excess to ensure the complete conversion of the PEG polymer to its derivatives. The excess reagents were soluble in ether and therefore could be removed by washing in ether during purification.
  • Inco ⁇ oration of the fluorescent label, N ⁇ -dansyl lysine, to the PEG polymer by coupling the ⁇ -amino group of dansyl lysine with the NHS activated carbonate of PEG gave the lysine derivative 1.
  • the DSPE anchor was coupled via intermediate 2 that was formed by the esterification of 1 using NHS and DCC.
  • the resulting PEG lipid, 3, was deprotected by removing the tBoc to form CPLi, 4, with one positive charge.
  • the positive charges in the other CPL are carried by the amino groups of lysine.
  • the 1H NMR spectra showed well-resolved resonances for the PEG, tBoc and acyl chains of DSPE at approximately 3.61, 1.41 and 1.21 ppm, respectively, and for the resonances of the dansyl moiety (aromatic protons at 7.1-8.5 ppm; methyl protons at 2.8-3.0 ppm). From the integrated signal intensities of the former three peaks, it was found that the ratio of tBoc/PEG or tBoc/DSPE was 1.0, 2.1, 4.0, and 8.1 for CPLi-tBoc, CPL 2 -tBoc, CPL 4 -tBoc, and CPL 8 - tBoc, respectively.
  • the CPL described here possess several attributes which may increase their usefulness relative to other cationic lipids. Firstly, the phospholipid anchor will readily allow efficient inco ⁇ oration of CPL into liposomal systems. Secondly, the dansyl label will permit accurate and convenient quantification of the CPL in the bilayer using fluorescence techniques. Finally, the valency of the cationic headgroup in the CPL can easily be modified using lysine residues.
  • tBoc tert-butyloxycarbonyl
  • tBoc-NH-PEG ⁇ ooo-CO 2 -NHS tBoc protected and NHS activated PEG 1 000
  • CPL cationic poly(ethylene glycol) lipid conjugate
  • CPLi CPL with one positive charge
  • CPL 2 CPL with two positive charges
  • CPL 4 CPL with four positive charges
  • DCC N,N'-dicyclohexyl-carbodiimide
  • DCU dicyclohexyl urea
  • NHS N-hydroxysuccinimide
  • DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • PEGiooo poly( ethylene glycol) with an average
  • Fluorescamine was obtained from Molecular Probes (Eugene,OR). Trifluoroacetic acid, diethyl ether, methanol, triethylamine, and chloroform were obtained from Fisher Scientific (Vancouver, BC). All other reagents were used without further purification.
  • the filtrate containing tBoc-NH-PEG ⁇ ooo-CO 2 -(N ⁇ -dansyl)lysine-NHS (2), was slowly added to a solution of DSPE (365 mg, 488 ⁇ mol) in 3 mL of dry chloroform and 300 ⁇ L of triethylamine.
  • DSPE 365 mg, 488 ⁇ mol
  • the dissolution of DSPE in dry chloro form, and triethylamine required warming to 65 °C.
  • the reaction mixture was stirred overnight at room temperature, it was filtered to remove some precipitate (unreacted DSPE) and dried to a viscous paste.
  • the paste was dissolved in chloroform methanol (2: 1), washed with dilute HCl and water as before.
  • Dansylated CPL 2 -tBoc (5) A solution of N ⁇ ,N ⁇ -di-tBoc-L-lysine-N- hydroxysuccinimide ester (350 mg, 789 ⁇ mol) in 3 mL dry chloroform was gradually added to a solution of dansylated CPLi (4) (750 mg, 400 ⁇ mol) in 3 mL chloroform containing 300 ⁇ L triethylamine and stirred at room temperature for 3 h. The completion of the reaction was indicated by the disappearance of primary amine as visualized by ninhydrin assay on TLC.
  • CPL -tBoc (7) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (500 mg, 1127 ⁇ mol) with dansylated CPL 2 (6) (650 mg, 292 ⁇ mol). Besides washing with dilute HCL and water no further attempts were made to purify CPL -tBoc before deblocking to generate CPL 4 . Yield: 800 mg (Crude). TLC (silica gel) chloroform /methanol (85:15) ⁇ .58 (dansyl peak only).
  • CPL (8) Dansylated CPL (8).
  • the synthesis of CPL (8) was the same as that of CPLi (4) by deprotecting dansylated CPL -tBoc (7) (800 mg).
  • the final product was purified by column chromatography using silica gel 60, 70 - 230 mesh, and chloroform methanol ammonia solution (65:25:4 v/v). Yield: 300 mg (38%).
  • TLC sica
  • SPLP SPLP composed of DOPE:DODAC:PEG-CerC 20 (84:6:10) and containing the plasmid pLuc, a modified marker gene expressing luciferase, was supplied by INEX Pharmaceuticals Inc.
  • SPLP-CPL 4 Dansylated CPL was prepared in our laboratory and inco ⁇ orated into SPLP as follows: SPLP at a dose of 500 nmol lipid was incubated with different amounts of CPL (12.5, 19, and 30 nmol) at 60°C for 2 to 3 hours in Hepes Buffered Saline, pH 7.5 (HBS) to achieve a final inco ⁇ oration of 2, 3, and 4 mol %, respectively. SPLP-CPL was separated from uninco ⁇ orated CPL by gel filtration chromatography on a Sepharose CL-4B column equilibrated in HBS. Fractions (lmL) were collected and assayed for CPL, phospholipid and DNA contents.
  • Phospholipid Assay Phospholipid was determined by first extracting the lipids from SPLP using the Bligh-Dyer technique, and then measuring phosphate in the organic phase according to the Fiske-Subbarow method (see, Bligh EG, Dyer WJ A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37: 91 1-917; and Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925; 66: 375-400.).
  • DNA Assay DNA content was measured using the PicoGreen Assay kit
  • the lipids DOPE, PEG- CerC 20 , DODAC, and rhodamine-DOPE (Rh-PE), all stocks in CHC1 3 were mixed together in a molar ratio of (83.5:10:6:0.5) and the CHCI 3 was completely evaporated.
  • the resulting lipid film was dissolved in 20 mM octyl glucopyranoside (OGP) and 200 ⁇ g/mL of plasmid DNA was added to a total volume of 1 mL.
  • OGP octyl glucopyranoside
  • the resulting sample was passed down a DEAE Sepharose column and the effluent was run on a discontinuous sucrose gradient as described previously, (see, Gabizon A, Papahadjopoulos D. Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc Natl Acad Sci USA 1988; 85: 6949-6953.).
  • the resulting rhodamine-labeled SPLP possessed a DNA Lipid ratio of -60 ⁇ g/ ⁇ mol. 3. Determination of Optimal Incubation Time for Insertion ofCPL4 Into SPLP
  • Submicron Particle Sizer Then the salt (CaCl 2 or MgCl 2 ) was added to concentrations from 20 mM to 70 mM. At each interval the mean diameter ⁇ standard deviation was determined by QELS. The mean diameter of the particles hardly changes with increasing [Cation], however, the QELS Gaussian distribution gets broader. Therefore, the standard deviations were used as a measure of deaggregation.
  • SPLP-CPL4 and SPLP Freeze-fracture EM were performed on the SPLP-CPL 4 (no CaCl ), SPLP- CPL; + 40 mM CaCl 2 , and SPLP, according to Wheeler et al. (see, Wheeler JJ et al. Stabilized plasmid-lipid particles: construction and characterization. Gene Therapy 1999; 6: 271-281.).
  • the SPLP-CPL 4 contained 4 mol % CPL 4 .
  • the micrographs of SPLP-CPL 4 in the presence and absence of CaCl 2 , were compared to show the visual effect of Ca 2+ on the aggregation. Vesicle diameters of the SPLP-CPL + 40 mM CaCl 2 and SPLP were analyzed by QELS using a Nicomp Model 270 Submicron Particle Sizer. 6. Serum Stability of SPLP -CPU Particles
  • the serum stability of the SPLP-CPL containing various % of CPL were determined by mixing the particles with mouse serum to a final serum concentration of 50% v . These mixtures were then incubated for 0, 1, 2, or 4 hours at 37°C. At these time points, a volume of the mixture containing about 1 ⁇ g of plasmid DNA was removed and the DNA was extracted from the lipid and protein using a phenohchloroform extraction. The resulting DNA solutions were then run on a 1% agarose gel following which the DNA was transferred to nitrocellulose and a Southern blot was performed.
  • the cells used were a transformed BHK cell line (tk-).
  • tk- transformed BHK cell line
  • lxlO 3 BHK cells were grown on 12-well plates overnight in 2 mL of complete media (DMEM + 10% FBS) at 37°C in 5% CO 2 .
  • SPLP, SPLP-CPL 4 + 40 mM CaCl 2 , or DOPE:DODAC complexes (200 ⁇ L), each containing 0.5 mol % Rh-PE as lipid marker were mixed with 800 ⁇ L of complete media and this mixture was added to the top of the cells at a lipid dose of 20 ⁇ M.
  • Spectrophotometer using a ⁇ ex of 560 nm and a ⁇ em of 600 nm with slit widths of 10 and 20 nm, respectively. An emission filter of 430 nm was also used. Lipid uptake was determined by comparison of the fluorescence in the lysate to that of a lipid standard and normalized to the amount of cells as determined by the BCA protein assay (Pierce, Rockford, IL).
  • This uptake experiment was performed with the same SPLP-CPL 4 (containing 0.5 mol % Rh-PE) as above. 5xl0 4 BHK cells were plated overnight in 1 mL of complete media in 24-well plates.
  • the SPLP-CPL 4 (40 nmol) was mixed with CaCl 2 or MgCl at various initial concentrations of 20 mM to 70 mM in a total volume of 100 ⁇ L. To this was added 400 ⁇ L of complete media resulting in final [Cation] of 4 mM to 14 mM. This mixture was then added to the top of the cells and the cells incubated for 4 hours.
  • SPLP and SPLP-CPL containing between 2 and 4 mol % CPL, were prepared to deliver 0.5 ⁇ g of DNA in a total volume of 20 ⁇ L using HBS (SPLP), or HBS + 40 mM CaCl 2 (SPLP-CPL 4 ) and were added to 90 ⁇ L of complete media. Samples were incubated with the cells for 4 hours. The transfection media was then replaced with complete media for a complete 24 hour incubation. Cells were then lysed with 100 ⁇ L of lysis buffer, and 40 ⁇ L of the lysate was transferred to a 96-well luminescence plate.
  • SPLP HBS
  • SPLP-CPL 4 HBS + 40 mM CaCl 2
  • Luciferase activity was determined using a Luciferase reaction kit (Promega, Madison, WI), a luciferase standard (Boehringer-Manheim), and a ML3200 microtiter plate, luminometer from Molecular Dynamics (Chantilly, VA).
  • Lipofectin complexes for 24 or 48 hours. After the incubation period the cells were immediately lysed and the luciferase activity was measured and was normalized to the amount of protein present, as above.
  • the protein concentration after cell lysis at 24 and 48 hours was measured and compared for the SPLP-CPL 4 + 40 mM CaCl 2 and the Lipofectin complexes.
  • SPLP-CPL 4 (5.0 ⁇ g/mL) with either CaCh or MgC ⁇ at concentrations of 20 mM to 70 mM were combined in a volume of 20 ⁇ L and mixed with complete media, resulting in final [Cation] of 4 mM to 14 mM. Following incubation on the cells for 48 hours, the cells were washed and lysed, and the luciferase activity and protein content were measured as above.
  • SPLP-CPL containing encapsulated pEGFP (kindly supplied by Inex), that expresses GFP (green fluorescence protein), using the detergent dialysis procedure, (see, Wheeler et al. supra). 400 ⁇ g/mL of pEGFP was encapsulated within 10 mM DOPE:PEG- CerC20:DODAC (84:10:6), followed by the insertion of 4 mol % of CPL 4 . DOPE:DODAC complexes and Lipofectin complexes containing pEGFP were also prepared at a charge ratio of 1.5: 1. The transfections were performed as described earlier at a DNA dose of 5.0 ⁇ g/mL.
  • SPLP-CPL4 aggregate following insertion ofCPL4 and de- aggregate following addition of divalent cations.
  • the molar ratio of DOPE:PEG-CerC 20 was 11.0( ⁇ 0.1):1. This corresponds to a system of DOPE:PEG-CerC 20 :DODAC of (81.6:10.9:7.4). From the results, 79.7 ⁇ 0.9 % of the PEG-CerC 20 remains following CPL 4 insertion. This corresponds to a final mol % of PEG-CerC 2 o of 5.9 ⁇ 0.1 mol %. This means that about 1.5 ⁇ 0.1 mol % of PEG-CerC 2 o was replaced during the insertion of CPL .
  • the outer leaflet will possess 4.4 ⁇ 0.1 mol % of PEG-CerC 20 after insertion. Since we inserted -4.5 mol % CPL 4 into SPLP (9.0 mol % in the outer leaflet), resulting in a total of 13.4 ⁇ 0.1 mol % of total PEG in the outer leaflet.
  • SPLP-CPL4 exhibit enhanced uptake into BHK cells and dramatically enhanced transfection potency.
  • the next set of experiments was aimed at determining the influence of inco ⁇ orated CPL on the uptake of SPLP into BHK cells and the resulting transfection potency of the SPLP-CPL system.
  • SPLP containing up to 4 mol % CPL were prepared in the presence of 40 mM CaCl 2 and were added to BHK cells (final CaCl 2 concentration 8 mM) and incubated for varying times. The cells were then assayed for associated SPLP-CPL as indicated in Methods.
  • uptake of SPLP that contain no CPL 4 is minimal even after 8 h of incubation, however uptake is dramatically improved for SPLP containing 3 mol % or higher levels of CPL 4 .
  • SPLP containing 4 mol % CPL 4 exhibit accumulation levels at 8 h that are approximately 50- fold higher than achieved for SPLP.
  • This enhanced uptake can be visually detected using fluorescence micrographs of BHK cells following incubation with rhodamine-labeled SPLP and SPLP-CPL 4 for 4 h.
  • the presence of 4 mol % CPL 4 clearly results in improved levels of cell-associated SPLP.
  • the transfection properties of SPLP, SPLP-CPL 4 and plasmid DNA- cationic lipid complexes were examined using the incubation protocol usually employed for complexes. This consisted of incubation of 10 4 BHK cells with SPLP, SPLP-CPL 4 and complexes containing 0.5 ⁇ g pCMVLuc for 4 h, followed by removal of SPLP, SPLP-CPL 4 or complexes that are not associated with the cells, replacement of the media, incubation for a further 20 h and then assaying for luciferase activity.
  • the SPLP-CPL 4 preparations contained 7 mM CaCl 2 in the incubation medium. As shown in Figure 33, the presence of the CPL 4 resulted in dramatic increases in the transfection potencies of the SPLP system. SPLP-CPL 4 containing 4 mol % CPL 4 exhibited luciferase expression levels some 3xl0 3 higher than achieved with SPLP. (.see.Mok et al, Biochim Biophys Acta, 1419:137-150 (1999)).
  • Ca2+ is required for transfection activity of SPLP-CPL 4 . It was of interest to determine the influence of Ca 2+ on the transfection activity of SPLP-CPL 4 .
  • SPLP containing 4 mol % CPL 4 were incubated with BHK cells for 48 h in the presence of 0-14 mM MgCl 2 and CaCl 2 and the luciferase activities then determined. As shown in Figure 34, the transfection activity was influenced by the presence of Ca 2+ in the transfection medium. At the optimum CaCl concentration of 10 mM, SPLP-CPL 4 exhibited transfection potencies that were more than 10 4 times higher than if MgCl 2 was present.
  • SPLP-CPL4 Uptake of SPLP-CPL into BHK cells was monitored following a 4 h incubation in the presence of 0-14 mM MgCl 2 and CaCl 2 . As shown in Figure 35 the amount of SPLP-CPL 4 taken up by BHK cells is the same for both Mg 2+ and Ca 2+ - containing media. The uptake of the SPLP-CPL 4 decreases as the concentration of divalent cations increases, which likely arises due to shielding of the negatively charged binding sites for the CPL 4 on the surface of the BHK cells. 5. SPLP-CPL4 exhibit transfection potencies in vitro that are comparable to or greater than achieved using complexes.
  • DOPE:DODAC (1:1) complexes 1.5:1, c.r.
  • DOPE/DOTMA [1 : 1] complexes 1.5:1 c.r.
  • the transfection potency of the SPLP-CPL4 increases markedly with increased incubation times, suggesting that a limiting factor for transfection achieved at a 4 h incubation time was the rate of uptake of the SPLP-CPL system.
  • transfection levels are achieved that are comparable to those achieved by Lipofectin or DOPE/DODAC complexes.
  • SPLP-CPL4 are non-toxic and efficient transfection agents.
  • SPLP-CPL contains low levels of cationic lipid and are potentially less toxic than complexes.
  • the toxicity of SPLP-CPL4 and complexes was assayed by determining cell viability following a 48 h exposure to levels of SPLP-CPL and complexes corresponding to 0.5 ⁇ g plasmid and -30 nmol total lipid. As shown in Figure 37B, SPLP-CPL 4 exhibit little if any toxicity. Cell survival was only 30% after a 48 h incubation with Lipofectin complexes whereas -95% of the cells were viable following a 48 hour incubation with SPLP-CPL 4 .
  • the efficiency of transfection is also an important parameter.
  • the proportion of cells transfected were estimated using plasmid carrying the green fluorescent protein (GFP) gene. Transfection was detected by expression of the fluorescent protein inside a cell employing fluorescence microscopy. As shown in Figure 37A and 38B, approximately 35% of the cells at 24 h and 50% at 48 h were transfected by SPLP-CPL 4 , with no apparent cell death. In contrast, Lipofectin complexes exhibit maximum transfection efficiencies of less than 35% and only -50% cell survival after the 24 h transfection period. Similar low transfection efficiencies and high toxicities were also seen with DOPE:DODAC complexes.
  • CPL 4 increases the transfection potency of the SPLP system.
  • the presence of the CPL increases the uptake of SPLP into the BHK cells, however the increase in transfection potency is almost entirely dependent on the additional presence of Ca 2+ . It may be noted that, following an 8 h incubation, the presence of 4 mol % CPL increases the uptake of SPLP into BHK cells by approximately 50-fold, whereas the transfection potency (in the presence of Ca 2+ ) is increased by a factor of -10 4 .
  • the final area of discussion concerns the advantages of the SPLP-CPL 4 system over other non-viral vectors, which include the well-defined modular nature of the SPLP-CPL system as well as toxicity and potency issues.
  • the well-characterized nature of the SPLP-CPL 4 as small, homogeneous, stable systems containing one plasmid per particle contrast with non-viral systems such as plasmid DNA-cationic lipid complexes which are large, inhomogeneous, unstable systems containing ill-defined numbers of plasmids per complex.
  • SPLP are basic components of more sophisticated systems, such as SPLP-CPL 4 , which can be constructed in a modular fashion.
  • SPLP-CPL 4 are markedly less toxic to BHK cells in tissue culture. This is presumably related to the low proportions of cationic lipid contained in SPLP as compared to complexes.
  • the transfection potency and efficiency of SPLP-CPL 4 is clearly comparable to the levels that can be achieved with complexes. It should be noted that this finding suggests that models of transfection by complexes that involve.
  • the superiority of SPLP-CPL 4 compared to commercially available complex systems e.g. Lipofectin
  • the first point revolves around the placement of the charge. Whereas on complexes the charges are located on the surface of the lipid bilayer, the SPLP-CPL possess charges on the vesicle surface which are localized a good distance from the liposomal surface, above the protective PEG coating which surrounds the liposome. In the case of the complexes, proteins binding to the liposome surface can lead to recognition and clearance by macrophages of the RES.
  • the charge on the surface of the bilayer is protected by the PEG coating, such that this should not occur.
  • the charge on the SPLP-CPL will allow the association of the liposomes with cells resulting in eventual uptake and transfection.
  • the size and serum stability of the SPLP-CPL 4 compared to complexes are important parameters for effective gene delivery systems, especially if one wishes to approach the capabilities of viral systems.
  • the SPLP-CPL 4 have been shown here to be of relative small size (-100 nm) compared to complexes, which are frequently on the order of microns in diameter. The small size should allow for accumulation at sites with larger fenestration (e.g. tumors, and inflammation sites), (see, Kohn et al. Lab Invest; 67:596-607 (1992)).
  • DNA in the SPLP-CPL 4 was shown to be protected from the external environment (t.e. inaccessible to degradation by DNase within serum), whereas DNA in complexes is susceptible to DNase. (see, Wheeler et al, Gene Therapy; 6:271-281 (1999)).
  • This example shows transfection rates of BHK cells by long- versus short- chained CPLs.
  • CPL (PEG 3.4k) and CPL(PEG Ik) were generated and each inserted into a separate SPLP system containing PEG- 2 ooo-Cer C20 as described above.
  • Figure 38 illustrates transfection rates of the CPLs having a PEG 3.4k versus a CPL having a PEG Ik.
  • the short-chained PEG in the CPL results in a decrease by a factor of about 4 compared to the transfection by the long chained CPL.
  • the long chain CPL (PEG 4 oo) sticks out above the surface, whereas the short chain CPL (PEGiooo) is buried (masked) in the surface of the SPLP.
  • the reduced in vitro transfection of the short chain CPL clearly suggests that it has improved in vivo circulation.
  • media is removed from cells and they are washed 2x with PBS then frozen at -70°C.
  • Cells are lysed with 150-200 ⁇ L lx CCLR; then shaken 5 minutes on plate shaker. 20 ⁇ L lysate is transferred to a 96-well luminescence plate. Plates are read to determine luciferase activity.
  • This in vivo example discloses pharmacokinetics and biodistribution of CPL 4 -l-k LUNs (SPLPs containing short chain CPLs) in C57/bl6 mice.
  • SPLPs containing short chain CPLs Different SPLP formulations containing increasing amounts of CPL-4-lk are assayed in vivo to determine optimal clearance characteristics.
  • CPL 4 -lk SPLPs are prepared according to previous protocols. Before use, all samples are characterized to determine actual composition prior to administration. All samples are filter sterilized prior to dilution to working concentration. All samples are to provided in sterile crimp top vials. All vials are labeled with the formulation date, lipid composition, and specific activity. 3 [H]CHE is inco ⁇ orated at 1 ⁇ Ci/mg Lipid. The following formulations are made and analyzed:
  • mice were treated with 3 [H]CHE-LUV administered by tail vein IN. in a total volume of 200 ⁇ l . Mice receive one treatment only. At the indicated time-points mice are weighed, sacrificed, and blood will be collected by cardiac puncture then evaluated for 3 [H]CHE. Formulations are expected to be well tolerated. Mice are treated according to certified animal care protocols. Any mice exhibiting signs of distress associated with the treatment are terminated at the discretion of vivarium staff. All mice are terminated by CO 2 inhalation followed by cervical dislocation. Measurement of 3 [H]CHE from blood is determined according to standard protocols. In vivo pharmacokinetics of SPLP containing short chain CPL 4 are illustrated in Figure 40.

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Abstract

Cette invention a trait à des conjugats polymère-lipide-cationique (CPL) tel que des conjugats poly(éthylène glycol)-lipide-cationique distaux pouvant être incorporés à des liposomes classiques ou à des liposomes furtifs ou à toute autre formulation à base de lipide pour renforcer l'absorption cellulaire. Les CPL selon l'invention comportent une fraction lipidique, un polymère hydrophile et une fraction polycationique. L'invention concerne également une méthode permettant de renforcer la libération intracellulaire d'acides nucléiques.
PCT/CA2000/000451 1999-04-20 2000-04-20 Lipides peg cationiques et méthodes d'utilisation WO2000062813A2 (fr)

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CA002370690A CA2370690A1 (fr) 1999-04-20 2000-04-20 Lipides peg cationiques et methodes d'utilisation
AU40962/00A AU783647B2 (en) 1999-04-20 2000-04-20 Cationic peg-lipids and methods of use
EP00920309A EP1173600A2 (fr) 1999-04-20 2000-04-20 Lipides peg cationiques et m thodes d'utilisation
JP2000611949A JP5117648B2 (ja) 1999-04-20 2000-04-20 カチオン性ｐｅｇ脂質および使用方法。
PCT/CA2001/000555 WO2001080900A2 (fr) 2000-04-20 2001-04-20 Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
JP2001577996A JP2004508012A (ja) 2000-04-20 2001-04-20 エンドソーム膜不安定剤を用いたｓｐｌｐ媒介性トランスフェクションの強化方法
US09/839,707 US7189705B2 (en) 2000-04-20 2001-04-20 Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers
AU5454801A AU5454801A (en) 2000-04-20 2001-04-20 Methods of enhancing splp-mediated transfection using endosomal membrane destabilizers
AU2001254548A AU2001254548B2 (en) 2000-04-20 2001-04-20 Enhanced stabilised plasmid-lipid particle-mediated transfection using endosomal membrane
EP01927519A EP1355670A2 (fr) 2000-04-20 2001-04-20 Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale
CA002406654A CA2406654A1 (fr) 2000-04-20 2001-04-20 Procedes permettant d'ameliorer la transfection a mediation splp (particule plasmide-lipide stabilisee) au moyen de destabilisateurs de la membrane endosomale

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AU783647B2 (en) 2005-11-17
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JP2002542341A (ja) 2002-12-10
WO2000062813A3 (fr) 2001-08-09
JP2012122075A (ja) 2012-06-28
EP1173600A2 (fr) 2002-01-23
CA2370690A1 (fr) 2000-10-26

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