CATIONIC LIPIDS FOR INTRACELLULAR DELIVERY OF BIOACTIVE
SUBSTANCES
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a method of delivering bioactive substances, particularly nucleic acids, into cells. This invention describes a series of novel biodegradable cationic lipids as transport agents. Preferred cationic lipids have two hydroxyl groups and combined with a neutral lipid form a cationic liposome. Negatively charged molecules, e.g. plasmid DNA, for complex with these cationic liposomes. Drugs or molecules that can be delivered using these cationic liposomes range from DNA plasmids, RNAs, proteins to small molecular weight drugs. Biodegradable cationic liposomes ofthe invention can also be used to aid the virus particle mediated gene transfer.
2. Background
Effective delivery of nucleic acid to cells or tissue with high levels of expression are continued goals of gene transfer technology. As a consequence ofthe general inability to achieve those goals to date, however, clinical use of gene transfer methods has been limited.
Ideal gene delivery vehicles should be bioabsorable, non-toxic, non- immunogenic, stable during storage and after administration, able to access target cells, and suitable for efficient gene expression. As many studies demonstrate, the limitations of viral vectors make synthetic vectors an attractive alternative.
Cationic liposome (Lipoplex) and cationic polymers are among the two major types of non-viral gene delivery vectors. Toxicity data on cationic polymers suggests that many polymers used for transfections are most effective at concentrations that are just
subtoxic. Illustrative polymers include polyamino acids (e.g. poly-L-lysine, poly-L- ornithine), polyamidoamine dendrimers, chitosan, polyethylenimine, poly((2- dimethylamino)ethyl methacrylate). The entry ofthe complexes may be mediated by the membrane destabilizing effects of cationic polymers. Several observations have suggested that liposomal systems are relatively unstable after the administration.
Significant toxicity has been shown to be associated to liposomal vectors, especially the fusogenic phospholipid (neutral lipid), include the down regulation of PKC dependent immunomodulator synthesis, macrophage toxicity, neurotoxicity, acute pulmonary inflammation, etc. Moreover, both vectors are far less efficient comparing with the viral counter-part. Searching for a safe and efficient gene carrier will still remain a major challenge in the field of non- viral gene delivery.
Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant device applications. Sometimes, it is also desirable for such polymers to be, not only biocompatible, but also biodegradable to obviate the need for removing the polymer once its therapeutic value has been exhausted.
Conventional methods of drug delivery, such as frequent periodic dosing, are not ideal in many cases. For example, with highly toxic drugs, frequent conventional dosing can result in high initial drug levels at the time of dosing, often at near-toxic levels, followed by low drug levels between doses that can be below the level of their therapeutic value. However, with controlled drug delivery, drug levels can be more nearly maintained at therapeutic, but non-toxic, levels by controlled release in a predictable manner over a longer term.
If a biodegradable medical device is intended for use as a drug delivery or other controlled-release system, using a polymeric carrier is one effective means to deliver the therapeutic agent locally and in a controlled fashion, see Langer et al., Rev. Macro. Chem. Phys., C23(l), 61 (1983). As a result, less total drug is required, and toxic side effects can be minimized. Polymers have been used as carriers of therapeutic agents to effect a localized and sustained release. See Chien et al., Novel Drug Delivery Systems
(1982). Such delivery systems offer the potential of enhanced therapeutic efficacy and reduced overall toxicity.
SUMMARY OF THE INVENTION The present invention provides a new class of cationic lipids, cationic liposomes comprising a cationic lipid ofthe invention and a neutral lipid, liposome compositions comprising at least one biologically active substance dispersed in a cationic liposome of the invention and methods of preparing and using such cationic liposomes and compositions to deliver biologically active substances to specified tissues or cells. A preferred application includes the localized, controlled release of at least a portion of one or more bioactive substances from a cationic liposome ofthe invention into a specified tissue or cell. In another preferred application, cationic liposomes ofthe invention are effective gene delivery agents for localized delivery of DNA to specified tissues or cells in gene therapy.
The present invention provides cationic lipids according to Formula I:
Formula I wherein: Ri is chosen from the group consisting of C .
6cycloalkyl having between about 1 and 6 alicyclic rings, and (CR
6R
7)
b-Y-Z;
R2 is selected from the group consisting of hydrogen, optionally substituted C_. 6alkyl, optionally substituted C3-8cycloalkyl, and optionally substituted C3_ cycloalkyl C_. ealkyl; R3 and R4 are independently selected at each occurrence from the group consisting of optionally substituted Cι_3alkylene, optionally substituted Cs-scycloalkylene; X is a pharmaceutically acceptable anion; Y is -OCO2-, -NR6CO2- or -NR6CONR7-;
Z is an optionally substituted steroid derivative, optionally substituted C_.36alkyl. optionally substituted C2-36alkenyl, optionally substituted C2-36alkynyl, or optionally substituted C3.36cycloalkyl having between about 1 and 6 alicyclic rings which may optionally comprise 0-3 heteroatoms in the alicyclic rings; R6 and R7 are each independently selected at each occurrence from the group consisting of hydrogen and C.-6alkyl; and b is a positive integer.
Preferred cationic lipids according to Formula I are biocompatible or biodegradable. Typically a biodegradable cationic lipid ofthe invention are biocompatible before, during and after biodegredation.
The present invention also features cationic liposomes comprising at least on neutral lipid and at least one cationic lipid of Formula I. Preferred cation liposomes are biocompatible and degrade in vivo or in vitro to the component lipids. Preferably both the cationic lipid and neutral lipid components ofthe liposome are biocompatible.
The present invention further comprises biocompatible cationic liposome compositions comprising at least one biologically active substance; a neutral lipid; and a cationic lipid according to Formula I.
The invention further includes methods of making biocompatible cationic liposomes comprising a cationic liposome according to Formula I and a neutral lipid, the method comprising the steps of: providing a neutral lipid and a cationic lipid according to Formula I; dissolving the neutral lipid and the cationic lipid in an organic solvent to form a lipid mixture; hydrating the lipid mixture in water; and incubating the aqueous lipid mixture under conditions conducive to formation of cationic liposomes comprising a neutral lipid and a cationic lipid according to Formula I..
In another aspect ofthe invention, a method of preparing a biocompatible cationic liposome composition is provided, the composition comprising a cationic lipid, a neutral lipid and a biologically active substance, the method comprising the steps of: providing at least one biocompatible cationic liposome by the method of preparing a cationic liposome ofthe invention and at least one biologically active substance; and contacting the cationic liposome with the biologically active substance under conditions conducive to the inclusion of at least a portion ofthe biologically active substances into the cationic liposome resulting in a biocompatible cationic liposome composition comprising a biologically active substance.
In yet another aspect ofthe invention, a method for the controlled release of a biologically active substance is provided, the method comprises the steps of: providing a biocompatible cationic liposome composition comprising: (a) at least one biologically active substance; and
(b) A cationic lipid according to Formula I; and contacting the cationic liposome composition in vivo or in vitro with a biological fluid, cell or tissue under conditions conducive to the delivery of at least a portion ofthe biologically active substance to the biological fluid, cell or tissue so that the biologically active substance is released in a controlled manner.
The invention also provides method for gene therapy, wherein a gene or gene fragment, e.g., a DNA sequence is transfected in a controlled fashion into a specified tissue or cell. The method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
(a) at least a portion of at least one gene; and
(b) A cationic lipid according to Formula I; and contacting the micelle composition in vivo or in vitro with a biological fluid, cell or tissue under conditions conducive to the delivery of at least a portion ofthe gene to the biological fluid, cell or tissue such that gene therapy occurs.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plot ofthe cytotoxicity of BHEM-Chol/DOPE, BHEM-Chol and Transfast™; and
FIG. 2 is a graph comparing transfection efficiency of BHEM-Chol/DOPE and Transfast™.
DETAILED DESCRIPTION OF THE INVENTION The present invention features biocompatible cationic lipids, cationic liposomes comprising a biocompatible cationic lipid and a biocompatible neutral lipid and cationic liposome compositions comprising a biologically active substance dispersed within a cationic liposome ofthe invention. The invention further provides methods of manufacturing cationic liposomes and cationic liposome compositions and method s of using biocompatible cationic liposome compositions ofthe invention for localized controlled release of biologically active substances at on in a specified tissue or cell and for use in gene therapy applications.
Prefened cationic lipids of Formula I include those cationic lipids according to
Formula II:
wherein:
R1 and X are as defined in Formula I;
R2 is hydrogen or Cι_6alkyl; and m and n are independently selected integers from about 1 to about 6;
Other prefened cationic lipids of Formula I include those lipids according to Formula III:
wherein
R1 is chosen from the group consisting of C3.36cycloalkyl having between about 3 and about 5 alicyclic rings, and -(CH2)b-Y-Z; R2 is methyl or ethyl; X is a pharmaceutically acceptable anion. Y is -OCO2-, -NR6CO2- or -NR6CONR7-;
Z is an optionally substituted steroid derivative, optionally substituted Cι-36alkyl, optionally substituted C2-36alkenyl, optionally substituted C2.36alkynyl, or optionally substituted C3_36cycloalkyl having between about 1 and 6 alicyclic rings which may optionally comprise 0-3 heteroatoms in the alicyclic rings; R6 and R7 are each independently selected at each occurrence from the group consisting of hydrogen and Cι-6alkyl; and b is a positive integer.
Particularly preferred cationic lipids of Formula I include those cationic lipids according to Formula IN
HO-(CH2)
wherein m and n are independently selected integers from about 1 to about 6; b is a positive integer from about 1 to about 6; and the steroid ring structure
is optionally be substituted at one or more steroid ring atoms with one or more substitutents chosen from the group consisting of Cι-ι
2alkyl, C
2-ι
2alkenyl, C
2.ι
2alkynyl, and C
3.
8cycloalkyl and two or more substitutents can combine to form additional carbocyclic or heteroalicyclic rings which can be fused or spiro to the steroid ring structure.
A particularly preferred R1 group for cationic lipids according to any of Formula I, II or III include R1 groups according to Formula N:
wherein the steroid ring structure can optionally be substituted at one or more steroid ring atoms with one or more substitutents chosen from the group consisting of Ci- ι2alkyl, C2-.2alkenyl, C .ι2alkynyl, and C3.8cycloalkyl and two or more substitutents can combine to form additional carbocyclic or heteroalicyclic rings which can be fused or spiro to the steroid ring structure.
The invention also provides cationic liposomes comprising a neutral lipid and a cationic lipid according to any one of Formulae I, II, III, or IV. Particularly preferred cationic lipids suitable for use in cationic liposomes ofthe invention include an R1 according to Fonnula V.
Prefened cationic liposomes comprising a catiomc lipid according to Formula I, II, m, or IV are biocompatible before, during and after dissolution ofthe liposome.
Other prefened cationic liposomes ofthe invention comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, II, III or IN wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1:10 by weight.
Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited. Prefened neutral lipids include those selected from steroids, di(C6-C36)alkyl phosphatidyl (hydroxyalkyl) amines and di(C6-C36)alkenyl phosphatidyl (hydroxyalkyl) amines and the like. Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
In another embodiment, the invention provides cationic liposome compositions comprising cationic liposome ofthe invention and a biologically active substance disposed therein.
Prefened cationic liposome compositions ofthe invention comprise at least one biologically active substance, a neutral lipid and a cationic lipid according to any one of Formulae I, II, III, or IN. Particularly prefened cationic lipids suitable for use in cationic liposome compositions include those having the R1 group according to Formula V.
In prefened embodiments, at least one ofthe biologically active substances dispersed in the cationic liposome is selected from the group consisting of DΝA, RΝA, proteins, and small molecule therapeutics.
Additionally prefened are cationic liposome compositions comprising at least one biocompatible cationic lipid according to Formula I, II, III, or IN, a neutral lipid and at
least one negatively charged or neutral biologically active substance. Prefened biologically active substances include DNA (inclusive of cDNA), RNA (inclusive of mRNA, tRNA and the like), proteins, and small molecule therapeutics.
Prefened cationic liposome compositions comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, π, III or IV wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1 : 10 by weight.. The cationic liposome compositions further comprise about 1 to about 65 parts by weight ofthe biologically active substance relative to the combined weight ofthe neutral lipid and cationic lipid of the cationic liposome.
Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited. Prefened neutral lipids include those selected from steroids, di(C6-C36)alkyl phosphatidyl (hydroxyalkyl) amines and di(C6-C36)alkenyl phosphatidyl (hydroxyalkyl) amines, and the like. Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
In another embodiment, the present invention provides methods for preparing cationic liposomes ofthe invention. The methods comprise the steps of: providing a neutral lipid and a cationic lipid according to any one of Formulae I, π, III or IV; dissolving the neutral lipid and the cationic lipid in an organic solvent to form a lipid mixture; hydrating the lipid mixture in water; and incubating the aqueous lipid mixture under conditions conducive to formation of cationic liposomes comprising a neutral lipid and a cationic lipid according to Formula I..
Preferably cationic liposomes prepared by the methods ofthe invention comprise a mixture of neutral lipid and cationic lipid according to any one of Formulae I, II, HI or IN wherein the ratio of neutral lipid to cationic lipid in the mixture is between about 10:1 and about 1:10 by weight.
Neutral lipids suitable for use in cationic liposomes ofthe invention are biocompatible, but are otherwise not particularly limited. Prefened neutral lipids include those selected from steroids, di(C6-C36)alkyl phosphatidyl (hydroxyalkyl) amines and di(C6-C36)alkenyl phosphatidyl (hydroxyalkyl) amines, and the like. Non limiting examples of particularly prefened neutral lipids include DOPE and cholesterol.
The present invention further provides a method of preparing a biocompatible cationic liposome composition comprising a cationic lipid, a neutral lipid and a biologically active substance, the method comprising the steps of: providing at least at least one biologically active substance and one biocompatible cationic liposome ofthe invention comprising a neutral lipid and at least one cationic lipid according to any one of Formulae I, II, III or IN; and contacting the cationic liposome with the biologically active substance under conditions conducive to the inclusion of at least a portion ofthe biologically active substances into the cationic liposome resulting in a biocompatible cationic liposome composition comprising a biologically active substance.
In yet another embodiment, the present invention provides methods for the controlled release of a biologically active substance from a cationic liposome composition ofthe present invention. The method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
(a) at least one biologically active substance; and
(b) at least one neutral lipid and at least one cationic lipid according to any one of Formulae I, II, III, or IN; and contacting the cationic liposome composition in vivo or in vitro with a biological fluid, cell or tissue under conditions conducive to the delivery of at least a portion ofthe biologically active substance to the biological fluid, cell or tissue so that the biologically active substance is released in a controlled manner.
h prefened embodiments, the biologically active substance is released in a controlled fashion in vivo or alternatively in vitro. In other prefened embodiments, the biologically active substance is released extracellularly or intracellularly.
Prefened biologically active substances that are suitable for controlled release from cationic liposome compositions ofthe invention include substances selected from the group consisting of DNA, RNA, proteins, and small molecule therapeutics.
The present invention further provides methods for gene therapy. The method comprising the steps of: providing a biocompatible cationic liposome composition comprising:
(a) at least a portion of at least one gene; and
(b) a cationic liposome comprising at least one neutral lipid and at least one cationic lipid according to any one of Formulae I. II, III, or IN; and contacting the micelle composition in vivo or in vitro with a biological fluid, cell or tissue under conditions conducive to the delivery of at least a portion ofthe gene to the biological fluid, cell or tissue such that gene therapy occurs.
In prefened gene therapy methods ofthe invention, the cationic liposome composition comprising a gene or gene fragment is contacted with a specified cell or tissue in vivo. Alternatively, the cationic liposome composition comprising the gene or gene fragment is contacted with the specified tissue or cell in vitro. The invention also provides methods wherein the DΝA sequence or gene is released extracellularly or intracellularly.
In prefened therapeutic and gene therapy methods ofthe invention, the method of administration ofthe cationic liposome composition comprising a biologically active substance is not particularly limited. However in certain prefened embodiments, cationic liposome compositions are administered orally or by injection into a tissue such as a muscle, an internal organ, a region ofthe spinal cord or the like.
Suitable subjects for in vivo gene therapy using the compositions and methods of the invention are typically mammals. Particularly prefened mammals include rodents, including mice and rats, livestock such as sheep, pig, cow and the like and primates, particularly humans, however other subjects are also contemplated as within the scope of the present invention. Further, the compositions and methods ofthe present invention are also suitable for in vitro gene therapy applications.
The gene delivery systems ofthe present invention can achieve gene transfer efficiencies in vitro that are superior to commercially available cationic liposome preparations such as Transfast™. Furthermore, the cationic liposomal delivery systems ofthe invention offer numerous technical advantages including improved biocompatibility (FIG. 1) and biodegradability ofthe gene delivery system, e.g., the liposome degrades in vitro or in vivo to molecular cationic lipids and neutral lipids; the versatility of cationic lipids of Formula I can be tailored by variation of one or more groups or substituents to control cationic liposome properties such as charge density, charge ratio, liposome stability, rate of degredation, hydrophilicity/hydrophobicity and other physical properties; and specific cell or tissues can be targeted by introduction of one or more specific ligands into the charged lipid according to Formula I or in the neutral lipid component ofthe liposome thereby enhancing delivery ofthe biologically active substance to the specified location.
Nucleic acid administered in accordance with the invention may be any nucleic acid (DNA or RNA) including genomic DNA, cDNA, mRNA and tRNA. These constructs may encode a gene product of interest, e.g. a therapeutic or diagnostic agent. A wide variety of known polypeptides are known that may be suitably administered to a patient in accordance with the invention.
For instance, for administration to cardiac myocytes, nucleic acids that encode vasoactive factors may be employed to treat vasoconstriction or vasospasm. Nucleic acids that encode angiogenic growth factors may be employed to promote revascularization. Suitable angiogenic growth factors include e.g. the fibroblast growth
factor (FGF) family, endothelial cell growth factor (ECGF) and vascular endothelial growth factor (VEGF; see U.S. Patents 5,332,671 and 5,219,739). See Yanagisawa- Miwa et al., Science 1992, 257:1401-1403; Pu et al., JSurgRes 1993, 54:575-83; and Takeshita et al., Circulation 199 A, 90:228-234. Additional agents that maybe administered to ischemic heart conditions, or other ischemic organs include e.g. nucleic acids encoding transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor α and tumor necrosis factor β. Suitable vasoactive factors that can be administered in accordance with the invention include e.g. atrial natriuretic factor, platelet-derived growth factor, endothelin and the like.
For treatment of malignancies, particularly solid tumors, nucleic acids encoding various anticancer agents can be employed, such as nucleic acids that code for diphtheria toxin, thymidinekinase, pertussis toxin, cholera toxin and the like. Nucleic acids encoding antiangiogenic agents such as matrix metalloproteases and the like also can be employed. See J.M. Ray et al. Eur Respir J 1994, 7:2062-2072.
For other therapeutic applications, polypeptides transcribed by the administered nucleic acid can include growth factors or other regulatory proteins, a membrane receptor, a structural protein, an enzyme, a hormone and the like.
Also, as mentioned above, the invention provides for inhibiting expression or function of an endogenous gene of a subject. This can be accomplished by several alternative approaches. For example, antisense nucleic acid may be administered to a subject in accordance with the invention. Typically, such antisense nucleic acids will be complementary to the mRNA ofthe targeted endogenous gene to be suppressed, or to the nucleic acid that codes for the reverse complement ofthe endogenous gene. See J.H. Izant et al., Science 1985, 229:345-352; and L.J. Maher II et al., Arch Biochem Biophys 1987, 253:214-220. Antisense modulation of expression of a targeted endogenous gene can include antisense nucleic acid operably linked to gene regulatory sequences.
Alternatively, nucleic acid may be administered which antagonizes the expression of selected endogenous genes (e.g. ribozymes), or otherwise interferes with function of the endogenous gene or gene product.
The nucleic acid to be administered can be obtained by known methods, e.g. by isolating the nucleic acids from natural sources or by known synthetic methods such as the phosphate triester method. See, for example, Oligonucleotide Synthesis, IRL Press (MJ. Gait, ed. 1984). Synthetic oligonucleotides also may be prepared using commercially available automated oligonucleotide synthesizers. Also, as is known, if the nucleic acid to be administered is mRNA, it can be readily prepared from the conesponding DNA, e.g. utilizing phage RNA polymerases T3, T7 or SP6 to prepare mRNA from the DNA in the presence of ribonucleoside triphosphates. The nucleotide sequence of numerous therapeutic and diagnostic peptides including those discussed above are disclosed in the literature and computer databases (e.g., GenBank, EMBL and Swiss-Prot). Based on such information, a DNA segment may be chemically synthesized or may be obtained by other known routine procedures such as PCR.
To facilitate manipulation and handling ofthe nucleic acid to be administered, the nucleic acid is preferably inserted into a cassette where it is operably linked to a promoter. The promoter should be capable of driving expression in the desired cells. The selection of appropriate promoters can be readily accomplished. For some applications, a high expression promoter is prefened such as the 763 -base pair cytomegalovirus (CMV) promoter. The Rous sarcoma (RSV) (Davis et al., Hum Gene Ther, 1993, 4:151) and MMT promoters also maybe suitable. Additionally, certain proteins can be expressed using their native promoter. Promoters that are specific for selected cells also may be employed to limit transcription in desired cells. Other elements that can enhance expression also can be included such as an enhancer or a system that results in high expression levels such as a tat gene or a tar element. A cloning vehicle also may be designed with selective receptor binding and using the promoter to provide temporal or situational control of expression.
Typical subjects to which nucleic acid will be administered for therapeutic application include mammals, particularly primates, especially humans, and subjects for xenotransplant applications such as a primate or swine, especially pigs. For veterinary applications, a wide variety of subjects will be suitable, e.g. livestock such as cattle, sheep, goats, cows, swine and the like; poultry such as chickens, ducks, geese, turkeys and the like; and pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects including rodents (e.g. mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.
The effective dose of nucleic acid will be a function ofthe particular expressed protein, the target tissue, the subject (including species, weight, sex, general health, etc.) and the subject's clinical condition. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art using conventional dosage determination tests. Additionally, frequency of administration for a given therapy can vary, particularly with the time cells containing the exogenous nucleic acid continue to produce the desired polypeptide as will be appreciated by those skilled in the art. Also, in certain therapies, it may be desirable to employ two or more different proteins to optimize therapeutic results.
The concentration of nucleic acid within a polymer nanoparticle or micelle can vary, but relatively high concentrations are prefened to provide increased efficiency of nucleic acid uptake. More specifically, prefened nanoparticles and micelles comprise a polyphosphate-nucleic acid complex and includes between about 1% to 70% by weight of the nucleic acid. More preferably, the micelle or nanoparticle comprises about 10 to about 60 % nucleic acid by weight or 10%, 20%, 30%, 40%, 50% or 60% by weight of the nucleic acid.
As indicated above, various substituents ofthe various Formulae are "optionally substituted", including R1, R2, R3, R4, R5, R6, and R7 of Formula I-TV. When substituted, those substituents may be substituted by other than hydrogen at one or more available positions, typically 1 to about 6 positions or more typically 1 to about 3 or 4 positions, by
one or more suitable groups such as those disclosed herein. Suitable groups that may be present on a "substituted" R , R , R , R , R , R , and R group or other substituent include e.g. halogen such as fluoro, chloro, bromo and iodo; cyano; hydroxyl; nitro; azido; alkanoyl such as a Cι_6 alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups having those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groups including those moieties having one or more sulfinyl linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkylsulfonyl groups including those moieties having one or more sulfonyl linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; aminoalkyl groups such as groups having one or more N atoms and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; carbocyclic aryl having 6 or more carbons, particularly phenyl (e.g. an Ar group being a substituted or unsubstituted biphenyl moiety); aralkyl having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with benzyl being a prefened group; aralkoxy having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, with O-benzyl being a prefened group; or a heteroaromatic or heteroalicyclic group having 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pynolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pynolidinyl.
As used herein, the term "amphiphilic" is intended to include polymers which comprises two or more domains or groups which are linked together wherein at least one domain or group or domain is a polar, hydrophilic group and at least one domain or group is a non-polar hydrophobic group. Amphiphilic polymers ofthe invention typically
comprise a polar, hydrophilic main chain having non-polar hydrophobic groups or domains pendant therefrom.
As used herein, the term "a positively charged or positively chargeable group" is intended to include both positively charged functional groups such as phophonium groups, quaternary ammonium groups and other charged groups and also chargeable functional groups that can reversibly protonated to yield a positively charged group, e.g., typical chargeable groups include primary, secondary and tertiary amines, amides and other functional groups which comprise a proton acceptor and can be protonated in aqueous media at or around neutral pH.
As used herein, "alkyl" is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkylene, having the specified number of carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, i-propyl, n-butyl, s-bufyl, t-butyl, n-pentyl, and s-pentyl. Alkyl groups typically have 1 to about 36 carbon atoms. Typically lower alkyl groups have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms. Prefened lower alkyl groups are C1-C20 alkyl groups, more prefened are Cι-ι2-alkyl and Cι-6-alkyl groups. Especially prefened lower alkyl groups are methyl, ethyl, and propyl. Typically higher alkyl groups have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms. Prefened higher alkyl groups are C4-C36 alkyl groups, more prefened are C8_24-alkyl and Cπ-.s-alkyl groups.
As used herein, "heteroalkyl" is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkyl groups include the following groups. Prefened alkylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alylthio groups having 1, 2, 3, or 4 carbon
atoms are particularly prefened. Prefered alkylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened alkylsulfonyl groups include those groups having one or more sulfonyl (SO2) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened aminoalkyl groups include those groups having one or more primary, secondary and or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
As used herein, "heteroalkenyl" is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkenylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkenyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkenyl groups include the following groups. Prefened alkylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkenylthio groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefered alkenylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkenylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened alkenylsulfonyl groups include those groups having one or more sulfonyl (SO2) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkenylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened aminoalkenyl groups include those groups having one or more primary,
secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkenyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
As used herein, "heteroalkynyl" is intended to include branched, straight-chain and cyclic saturated aliphatic hydrocarbon groups including alkynylene, having the specified number of carbon atoms and at least one heteroatom, e.g., N, O or S. Heteroalkynyl groups will typically have between about 1 and about 20 carbon atoms and about 1 to about 8 heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 to about 4 heteroatoms. Prefened heteroalkynyl groups include the following groups. Prefened alkynylthio groups include those groups having one or more thioether linkages and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkynylthio groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefered alkynylsulfinyl groups include those groups having one or more sulfoxide (SO) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkynylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened alkynylsulfonyl groups include those groups having one or more sulfonyl (SO2) groups and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Alkynylsulfonyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened. Prefened aminoalkynyl groups include those groups having one or more primary, secondary and/or tertiary amine groups, and from 1 to about 12 carbon atoms, more preferably from 1 to about 8 carbon atoms, and still more preferably from 1 to about 6 carbon atoms. Aminoalkynyl groups having 1, 2, 3, or 4 carbon atoms are particularly prefened.
As used herein, "cycloalkyl" is intended to include saturated and partially unsaturated ring groups, having the specified number of carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Also included are carbocyclic ring
groups with ine or more olefinic linkages between two or more ring carbon atoms such as cyclopentenyl, cyclohexenyl and the like. Cycloalkyl groups typically will have 3 to about 8 ring members.
In the term "(C3_6 cycloalkyl)Cι-4 alkyl", as defined above, the point of attachment is on the alkyl group. This term encompasses, but is not limited to, cyclopropylmethyl, cyclohexylmethyl, cyclohexylethyl.
As used here, "alkenyl" is intended to include hydrocarbon chains of straight, cyclic or branched configuration, including alkenylene having one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl and propenyl. Alkenyl groups typically have 1 to about 36 carbon atoms. Typically lower alkenyl groups have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms. Prefened lower alkenyl groups are Cι-C2o alkenyl groups, more prefened are Cι_ι2-alkenyl and Cι-6-alkenyl groups. Especially prefened lower alkenyl groups are vinyl, and propenyl. Typically higher alkenyl groups have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms. Prefened higher alkenyl groups are C4-C36 alkenyl groups, more prefened are C8.24-alkenyl and Cι2-ι_-alkenyl groups.
As used herein, "alkynyl" is intended to include hydrocarbon chains of straight, cyclic or branched configuration, including alkynylene, and one or more triple carbon-carbon bonds which may occur in any stable point along the chain. Alkynyl groups typically have 1 to about 36 carbon atoms. Typically lower alkynyl groups have about 1 to about 20, 1 to about 12 or 1 to about 6 carbon atoms. Prefened lower alkynyl groups are Cι-C
2o alkynyl groups, more prefened are C_-_
2- alkynyl and Cι-
6- alkynyl groups. Especially prefened lower alkyl groups are ethynyl, and propynyl. Typically higher alkynyl groups have about 4 to about 36, 8 to about 24 or 12 to about 18 carbon atoms. Prefened higher alkynyl groups are C
4-C
36 alkynyl groups, more prefened are C
8_
24- alkynyl and Cι
2_ι
8- alkynyl groups.
As used herein, "haloalkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 or more halogen (for example
where v = 1 to 3 and w =1 to (2v+l). Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl. Typical haloalkyl groups will have 1 to about 16 carbon atoms, more typically 1 to about 12 or 1 to about 6 carbon atoms.
As used herein, "a steroid derivative" is defined as an optionally substituted steroid group. A steroid is defined as a group of lipids that contain a hydrogenated cyclopentanoperhydrophenanthrene ring system. Some ofthe substances included in this group are progesterone, adrenocortical hormones, the gonadal hormones, cardiac aglycones, bile acids, sterols (such as cholesterol), toad poisons, saponins and some of the carcinogenic hydrocarbons. Prefened steroid derivatives include the sterol family of steroids, particularly cholesterol. Particularly prefened steroid derivatives include alkylene carboxamic acid steryl esters, e.g., -alkylene-NH-CO-O-steryl.
As used herein, "alkoxy" represents an alkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy. Alkoxy groups typically have 1 to about 16 carbon atoms, more typically 1 to about 12 or 1 to about 6 carbon atoms.
Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. A stable compound or stable structure is meant to imply a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an effective therapeutic agent.
As used herein, the term "aliphatic" refers to a linear, branched, cyclic alkane, alkene, or alkyne. Prefened aliphatic groups in the biodegradable amphiphilic
polyphosphate ofthe invention are linear or branched and have from 1 to 36 carbon atoms. Prefened lower aliphatic groups have 1 to about 12 carbon atoms and prefened higher aliphatic groups have about 10 to about 24 carbon atoms,
As used herein, the term "aryl" refers to an unsaturated cyclic carbon compound with 4n+2 electrons where n is a non-negative integer, about 5-18 aromatic ring atoms and about 1 to about 3 aromatic rings.
As used herein, the terms "heterocyclic" and "heteroalicyclic" refer to a saturated or unsaturated ring compound having one or more atoms other than carbon in the ring, for example, nitrogen, oxygen or sulfur. Typical heterocyclic groups include heteroaromatic and heteroalicyclic groups that have about a total of 3 to 8 ring atoms and 1 to about 3 fused or separate rings and 1 to about 3 ring heteroatoms such as N, O or S atoms. Illustrative heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lΗ-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl;- l,2,5oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pynolidinyl, pynolinyl, 2H-pynolyl, pynolyl, quinazolinyl, quinolinyl, 4Η-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, l,3.4thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,
thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl. 1,3,4-triazolyl, and xanthenyl.
A typical in vitro toxicity assay would be performed with live cells, such as HeLa cells, according to the procedure outlined in Example 2 wherein polymers ofthe invention are evaluated using the stardard WST-1 dye reduction assay. HeLa cells seeded onto a multi well plate and incubated with 100 μ L of DMEM medium complemented with 10 % fetal bovine serum (FBS) containing a polymer ofthe invention. After culturing for 24-72 hours, ten microliters of WST-1 reagent (Boehringer Mannheim) was added to each well. After several hours, the absorbance ofthe supernatant at 450 m was measured against a 655 nm reference. Cytotoxicity of polymers was measured based on cell viability.
Preferably, however, the biocompatible cationic liposome composition comprises both:
(a) at least one biologically active substance and
(b) a biocompatible cationic liposome comprising a neutral lipid and a cationic lipid according to any one of Formula I, II, III or IN.
Biologically active substances ofthe invention can vary widely with the purpose for the composition. The active substance(s) may be described as a single entity or a combination of entities. The delivery system is designed to be used with biologically active substances having high water-solubility as well as with those having low water- solubility to produce a delivery system that has controlled release rates. The term "biologically active substance" includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function ofthe body; or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment. Prefened biologically active substances include negatively charged and neutral substances. Particularly prefened
biologically active substances are DNA, RNA, proteins and negatively charged or neutral therapeutic small molecules.
Non-limiting examples of useful biologically active substances include the following expanded therapeutic categories: anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-coagulants, anti-convulsants, anti- dianheals, anti-emetics, anti-infective agents, anti-inflammatory agents, anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents, anti-thrombotic agents, anti-uricemic agents, anti-anginal agents, antihistamines, anti-tussives, appetite suppressants, biologicals, cerebral dilators, coronary dilators, decongestants, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxatives, mineral supplements, mucolytic agents, neuromuscular drugs, peripheral vasodilators, psychotropics, sedatives, stimulants, thyroid and anti-thyroid agents, uterine relaxants, vitamins, antigenic materials, and prodrugs.
Specific examples of useful biologically active substances from the above categories include: (a) anti-neoplasties such as androgen inhibitors, antimetabolites, cytotoxic agents, immunomodulators; (b) anti-tussives such as dextromethorphan, dextrometho han hydrobromide, noscapine, carbetapentane citrate, and chlophedianol hydrochloride; (c) antihistamines such as chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; (d) decongestants such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloids such as codeine phosphate, codeine sulfate and morphine; (f) mineral supplements such as potassium chloride, zinc chloride, calcium carbonates, magnesium oxide, and other alkali metal and alkaline earth metal salts; (g) ion exchange resins such as cholestryramine; (h) anti- anhythmics such as N-acetylprocainamide; (i) antipyretics and analgesics such as acetaminophen, aspirin and ibuprofen; (j) appetite suppressants such as phenylpropanolamine hydrochloride or caffeine; (k) expectorants such as guaifenesin; (1)
antacids such as aluminum hydroxide and magnesium hydroxide; (m) biologicals such as peptides, polypeptides, proteins and amino acids, hormones, interferons or cytokines and other bioactive peptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO and insulin; (n) anti-infective agents such as anti-fungals, anti-virals, antiseptics and antibiotics; and (o) antigenic materials, partricularly those useful in vaccine applications.
Preferably, the biologically active substance is selected from the group consisting of polysaccharides, growth factors, hormones, anti-angio genesis factors, interferons or cytokines, DNA, RNA, proteins and pro-drugs. In a particularly prefened embodiment, the biologically active substance is a therapeutic drug or pro-drug, more preferably a drug selected from the group consisting of chemotherapeutic agents and other anti-neoplasties, antibiotics, anti-virals, anti-fungals, anti-inflammatories, anticoagulants, an antigenic materials. Particularly prefened biologically active substances are DNA and RNA sequences that are suitable for gene therapy.
The biologically active substances are used in amounts that are therapeutically effective. While the effective amount of a biologically active substance will depend on the particular material being used, amounts ofthe biologically active substance from about 1% to about 65% have been easily incorporated into the present delivery systems while achieving controlled release. Lesser amounts may be used to achieve efficacious levels of treatment for certain biologically active substances.
In addition, the biocompatible cationic liposome compositions ofthe invention can also comprise additional neutral or cationic lipids, so long as they do not interfere undesirably with the biodegradation characteristics ofthe composition. Mixtures of two or more cationic lipids according to any one of Formulae I-IN may offer even greater flexibility in designing the precise release profile desired for targeted drug delivery.
Pharmaceutically acceptable carriers may be prepared from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disintegrants, colorants, bulking agents, flavorings, sweeteners and
miscellaneous materials such as buffers and adsorbents in order to prepare a particular medicated composition.
In its simplest form, a biocompatible therapeutic agent delivery system consists of a dispersion ofthe therapeutic agent in a liposome comprising a cationic lipid and a neutral lipid. The therapeutic agent is typically released as the liposome disperses in vivo into soluble products that can be excreted from the body.
As a drug delivery device, the cationic liposome compositions ofthe invention provide a matrix capable of sequestering a biologically active substance and provide predictable, controlled delivery ofthe substance. The liposome then disperses to individiual lipid molecules or smaller aggregates of lipid molecules which are readily excreted from the patient.
The following examples are offered by way of illustration and are not intended to limit the invention in any manner.
Example 1. Synthesis and characterization of BHEM- Choi.
Scheme 1.
N-(2-bromoethyl)carbarmoyl cholesterol: To a mixture of 2-bromoethylamine hydrobromide (17.42 g, 85.0 mmol) and cholesteryl chloroformate (34.7 g, 77.3 mmol) in 300 ml of chloroform cooled to -30 °C, was added triethylamine (24 ml, 172 mmol). This mixture was stined overnight at room temperature, and washed with 1 N HCI in saturated NaCl (150 ml x3) and washed once with saturated NaCl solution (150 ml). The solution obtained was dried over anhydrous magesium sulfate. The crude product afetr removing the solvent was recrystallized from ethanol once and then acetone once to give white powder (yield 35. Og, 84%). TLC analysis gave Rf=0.68 in mixture of toluene, hexane and methanol (8:8:1, v/v/v). m.p. 125-128 °C.
Step B:
N,N-bis-(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonylamino-ethyl) ammonium bromide (BHEM-Chol): N-(2-bromoethyl)carbarmoyl cholesterol (4.77 g, 7.77 mmol) and N-methyldiethanolamine (1.15 g, 9.65 mmol) were combined in 50 ml of dried toluene, and refluexed overnight. TLC revealed that all of N-(2- bromoethyl)carbarmoyl cholesterol was consumed. The mixture was poured to large volumes of ether and the precipitate was collected. The crude product was further purified by recrystallization from ethanol twice then once from the mixture of acetonitrile and ethanol (2:1, v/v) to yield white powder (3.86 g, 73%).
Example 2. Preparation of cationic liposome with BHEM-Chol.
BHEM-Chol was dissolved in chloroform at a concentration of 10 mg.ml with a helper lipid DOPE (dioleoyl phosphatidylethanolamine) at 1:1 molar ratio (1:1.12, w/w). Alternatively, cholesterol was used as the helper lipid at 5:4 molar ratio (2.1:1, w/w). The mixture was evaporated to dryness in a round-bottom flask using a rotary evaporator at room temperature. The resulted lipid film was dried under vacuum over night. The lipid film was then re-hydrated in sterile water to a concentration of 2 mg/ml based on the weight of cationic lipid. The suspension was incubated at 50 °C for 30 min. with
shaking, and then sonicated using a bath-sonicator for 2 min. at room temperature to form homogenized liposomes.
Example 3. Assay for the cytotoxicity of BHEM-Chol and cationic liposomes containing BHEM-Chol.
A kidney fibroblast cell line, COS-7 (African green monkey) was maintained in
Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL), lOOU/ml penicillin (Sigma- Aldrich, St. Louis, MO), 100 μg/ml streptomycin (Sigma- Aldrich) and 2 mM L- glutamine (Gibco-BRL) at 37°C (5% CO2). Cells were seeded at a density of lxlO4 per well in 96-well plate. Twenty-four hours later, culture medium was replaced with 100 μl of serum-free DMEM medium containing different concentrations of BHEM-Chol or BHEM-Chol/DOPE liposomes. Cells were incubated with the reagents for 4 hours at 37 °C. TransFAST™ was used as a control, and was incubated with cells for one hour at the conesponding concentration. After the above incubation period, lOOμl of DMEM medium containing 20% serum was added to each well. Cells were cultured for another 20 hours. Ten micro liters of WST-1 reagent (Roche Molecular Biochemicals, Basel, Switzerland) was added to each well and allowed to react for 4 hours at 37°C. The absorbance ofthe supernatant was measured at 450 nm using a microplate reader (Bio- Rad Labs, Hercules, CA) with 655 nm as the reference wavelength. The assay results (Figure 19) indicated that BHEM-Chol exhibited much lower cytotoxicity in culture than the commercial liposome Transfast™, one ofthe best standard transfection reagents in vitro. In the COS-7 cell culture model described above, LD50 of BHEM-Chol and Transfast™ was 38.6 μg/ml and 17.2 μg/ml, respectively. When combined with a neutral lipid DOPE at 1:1 molar ratio, BHEM-Chol showed significantly reduced cytotoxicity. The LD50 ofthe liposome (BHEM-Chol/DOPE) increased at least 14 times. Over 80% ofthe cells remained viable when the total lipid concentration was 500 μg/ml. It is worth noting that TransFast™ Reagent is comprised ofthe synthetic cationic lipid (+)-N,N-[bis (2-hydroxyethyl)-N-methyl-N-[2,3-
di(tetradedecanoyloxy)propyl] ammonium iodide and the neutral lipid, DOPE at a 1:1 molar ratio. BHEM-Chol/DOPE and Transfast™ have similar compositions.
Example 4. Complexation of cationic liposomes (BHEM-Chol/DOPE) with DNA.
A suspension of BHEM-Chol/DOPE liposomes or DC-Chol/DOPE liposomes in OPTI-MEM (Gibcol-BRL) was mixed with various amount of plasmid DNA solution at room temperature by vortexing for about 10 sec. The complexes were incubated at room temperature for 30 min and used directly. To evaluate the binding efficiency of DNA to the cationic liposomes (BHEM-Chol/DOPE), samples after incubation were analyzed by gel electrophoresis analysis (0.8% agrose).
Gel mobility analysis showed that complete binding of DNA occurred at a charge ratio of 1 : 1 and above.
Example 5. Transfection efficiency of liposome-DNA complexes in cell lines and primary hepatocytes.
Human embryonic kidney epithelial cell line (HEK 293) and African green monkey kidney fibroblast cell line (COS-7) were maintained in DMEM supplemented with 10% FBS, lOOU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Primary hepatocytes were gift from Chia SM and Yu H (National University of Singapore), they were harvested from male Wistar rat by a 2-step in situ collagenase perfusion as reported (Chia SM, et al. Hepatocyte encapsulation for enhanced cellular functions. Tissue Engineering 6(5):481-95 (2000)). The isolated hepatocytes were cultured in Hepatozym Serum free medium (SFM, Gibco-BRL) in a humidified atmosphere with 5% CO2. The culture medium was supplemented with 0.1 μM dexamethasone and 1% Penicillin and Streptomycin.
Cells were seeded in 24-well microtiter plates (Nalgen NUNC hit. Naperville, IL) 24 hours before transfection at a density of 8xl04 for HEK 293, 2xl04 for COS-7 cell.
Hepatocytes were seeded on collagen coated wells at a density of 5xl05. At the time of transfection, the medium in each well was replaced with 0.5 ml of Opti-MEM.
Liposome-DNA complexes were incubated with the cells for 2 hours at 37°C. The medium was then replaced with 1 ml of fresh complete medium and cells were further incubated for 72 hours. All the transfection tests were performed in triplicate. After the incubation, cells were washed with PBS and permeabilized with 100 μl of cell lysis buffer (Promega Co., Madison, WI). The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega Co., Madison, WI) on a luminometer (Lumat 9507, EG&G Berthold, Bad Wildbad, Germany). The light units (LU) were normalized against protein concentration in the cell extracts, which was measured using a protein assay kit (Bio-Rad Labs, Hercules, CA).
Transfection conditions using BHEM-Chol/DOPE liposomes was optimized with regards to charge ratio of BHEM-Chol and DNA, and dose of plasmid DNA. Maximal transfection occuned at a charge ratio of 1:1 to 1:2, and at a DNA dose of 2 μg/ml. Under these conditions, transfection efficiency was compared with Transfast™ in 293 cells, COS-7 cells and primary hepatocytes. As shown in Figure 20, BHEM-Chol/DOPE liposome transfected HEK293 cells at the similar level as Transfast™. It performed particularly well in COS-7 cells (about 40 times higher than Transfast™). Even in primary hepatocytes, which is hard to transfect in vitro, BHEM-Chol/DOPE liposomes induced a luciferase expression level at least 100 times higher than background, which was about 7 to 10 times higher than Transfast™ transfection.
The invention has been described in detail with reference to prefened embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope ofthe invention.