Sphingolipid-Containing Cationic Liposomes for Topical Delivery of Bioactive Material Including Genetic Material
This patent application claims the priority of U.S. provisional patent application No. 60/167,867, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to methods for preparing cationic liposomes, containing sphingolipids, for topical delivery of bioactive material. More specifically, it relates to a new lipid vehicle for topical delivery, offering, for example, an improved transfection and penetration efficiency of genetic material. The topical delivery system is capable of transporting a multitude of active ingredients, including drugs, genetic material or cosmaceuticals to hair follicles or deep into the skin.
BACKGROUND OF THE INVENTION
Gene therapy is a new modality generally aimed at providing intracellular introduction and expression of therapeutic genes in human somatic cells for treatment or prevention of both inherited and acquired diseases. (Erning et al, British Journal ofPlastic Surgery, 1997; 50: 491-
500) Problems encountered in therapy using recombinant proteins, such as the necessity of frequent administrations and protein denaturation, may be circumvented in gene therapy. Presently, one of the most important in the field of gene therapy is the development of effective delivery vehicles. Experimental gene delivery vehicles such as adenoviral and retroviral vectors have proven to be highly effective for gene transfer. Philip et al. (U.S. Patent No. 5,861,314) describes adeno-associated viral (AAV) liposomes and methods related to the delivery of therapeutic genes in certain cell lines. Problems associated with such vectors include
immunogenicity, transient expression and mutagenicity. Recent developments in the use of biodegradable synthetic polymer poly-lactide-co-glycoside microspheres and nanospheres as experimental gene delivery vehicles have partially addressed some of these problems, although this polymer has been observed to induce an inflammatory response (van der Giessen, et al, Circulation 1996; 94: 1690- 1697). The induction of inflammatory responses when viral vectors are used in vivo has not yet been adequately resolved.
Since the 1960's, liposomes, ball-like structures having a diameter of about 100 nm or more, have been used as model membranes to study the transport of molecules across bilayers, lipid protein interactions, and physiochemical properties of amphipathic molecules. Liposomes have also been used in dermo-pharmacotherapy. Liposomes can be loaded with water-soluble or oil soluble biologically active agents for controlled release, for instance, by solublizing the agent in the lipid or aqueous phase used to prepare the liposomes. Limiting factors in the use of liposomes as carriers for topical delivery of pharmacological agents include storage instability, low manufacturing reproducibility, low entrapment efficiency, and drug leakage. In addition, large molecules normally cannot penetrate mammalian skin, and dermally applied liposomes thus have limited ability to penetrate the skin.
Cationic liposomes are the most extensively studied liposomes in drug delivery. Alipid- DNA complex is easily achieved due to the electrostatic interaction between the cationic liposomes and negatively charged polynucleotides. Such a system can be used to influence hair growth, color, and appearance. A non-invasive technique such as topical gene delivery would be very useful in treating many cutaneous disorders.
Lipofection is a highly efficient, lipid-mediated DNA-transfer procedure. Under appropriate conditions, a eukaryotic cell is able to take up exogenous DNA, a portion of the DNA becoming localized in the nucleus. Techniques utilizing cationic liposomes have been developed to facilitate the delivery of functional DNA into the cell. (Feigner et al, Proc Natl
Acad Sci 1987; 84:7413-7417).
The delivery of active ingredients through liposomes to the hair follicle or skin is well established. Li et al. (In vitro Cell Dev. Biol. 1992; 28 A: 679-681) reported an in vivo topical delivery of calcien and melanin to the hair follicle through liposome based systems. Yu et al, (J. Invest. Dermatol. 1999; 112: 370-375) reported that the gene transfer efficiency of DNA topically applied alone was equal to or greater than that achieved in the
topical application of a DNA/liposome complex In an earlier publication, Alexander and Akhurst (Hum Mol Genet 1995, 4 2279-285) reported that topical application of the β- galactosidase gene with liposomes failed more often than succeeded in terms of its transaction efficiency A common feature in the experiments performed by these separate groups was their use of commercially available cationic lipids called DOTAP (dioleoyl trimethyl ammonium propane), DOTMA (dioleoyl trimethylamrnoniumchloride) or "lipofectin"
Simply replacing the DOTMA with other structurally related cationic lipids such as sterarylamine and dioctadecyldimethylammonium bromide, showed little DNA transfection efficacy (Feigner et al, Proc Natl Acad Sci U S A 1987, 84 7413-7) Although the use of a cationic lipid is an essential part of a vector system for gene delivery, the in vivo transfection efficiency also depends on other factors
In PCT US98/07645, a method for topical delivery of nucleic acids to amammalian cell in vivo is described The delivery system was a liposomal composition comprising both nonionic lipids and a cationic lipid U S Patent No 5,851,818 describes improvements in a method for preparing a plasmid-liposome complex for in vivo transfection The improvements include the selection of a condensing agent to condense the plasmid prior to contact with the liposomes, an appropriate working medium, and the ratio of liposome lipid to plasmid Also disclosed are DNA plasmid-liposome complexes formed by the method Takeuchi et al , (FEBS Letters 1996, 397 207-209) describe the effect of zeta potential of cationic liposomes as one of the important factors which control gene transfection Crommelin (J Pharm Sci 1984, 73 1559-63) describes the influence of lipid composition and ionic strength on the physical stability of liposomes
The use of conventional liposomes as carriers for the topical delivery of pharmacological agents is, however, associated with many drawbacks, including their instability to storage, low reproducibility of manufacture, low entrapment efficiency, and the leakage of drugs
There is thus a need in the art for stable delivery vehicles, by which drugs and genetic material can be transported more effectively across the stratum corneum and into deeper skin layers
SUMMARY OF THE INVENTION
This invention provides cationic liposomes containing drugs or genetic material for topical gene delivery The lipid content of the liposomes includes at least one sphingolipid and at least one cationic lipid The liposomes can also include at least one neutral lipid The invention further relates to methods of prepaπng said cationic liposomes
In another aspect, the invention provides cationic liposomes in which a therapeutic agent is incorporation, and the preparation thereof
In yet another aspect the invention provides a therapeutic formulation including said cationic lipid In still another aspect, the invention provides a method of administering cationic liposomes to a human or other animal including the topical administration of an effective amount of a therapeutic agent such as e.g.. a gene, drug, or cosmetic agent In a preferred embodiment, the active agent is a nucleic acid, or a plasmid comprising a nucleic acid or gene
BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Images showing transfection of the β-galactosidase gene into keratinocyte cells Dark areas indicate transfected cells A and C transfection achieved using a vector system developed according to the invention B and D transfection achieved using a commercially available liposomal transfecting dispersion known as "Lipofectin" Keratinocytes were treated with 2 μg of DNA (A and B), and with 5 μg of DNA (C and D)
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a method for prepaπng cationic liposomes, containing certain sphingolipids, for topical drug delivery in the field of, e.g., gene therapy One example of such a preparation showed high efficiency in comparison to a commercial liposomal preparation,
Lipofectin (Life Technologies, Rockville, MD), exclusively marketed for liposomal gene transfection research purposes Lipofectin may, in certain instances, be toxic
The present invention also provides a cationic posome composition for gene delivery containing neutral and cationic lipids having a desired zeta potential to aid in the transfection of cells These cationic liposomes are typically between 100 and 250 nm in diameter and can be applied topically for the delivery of an active agent In a preferred embodiment, the
topically applied cationic liposomes can be used for the transfection of cells with a nucleic acid, e.g.. a plasmid.
General Definitions
The term "delivery vehicle" or simply "vehicle" as used herein herein refers to carrier molecules used to deliver or deposit pharmacological or cosmetic agents into the skin. These therapeutic agents include drugs, genetic material or cosmaceuticals.
The term " topical carriers" as used herein refers to vehicles suitable for topical application of drugs or cosmetics and includes any such liquid or non-liquid solvent, diluent or like material known in the cosmetic and medicinal arts, for forming any liquid or semisolid gel, cream, ointment, emulsion, aerosol, foam, lotion, or the like, and that does not adversely affect living animal tissue or interact with other components of the composition in a deleterious manner. Topical carriers are used to provide the compositions of the invention in their preferred liquid form. Non-limiting examples of suitable topical carriers for use herein include water, liquid alcohols, liquid glycols, liquid polyalkated protein hydroyslates, liquid lanolin and lanolin derivatives, and like materials, and mixtures thereof.
The terms" pharmacologically active agent", "cosmetic agent", "bioactive agent" or simply "active agent" as used herein refer to any chemical material or compound suitable for topical administration which produces any desired local effect. Non-limiting examples of such substances include antifungal agents, chemotherapeutic agents, antibiotics, anti-microbial agents, antiviral agents, hormones, cutaneous growth enhancers, including those for the hair and nails, hair care agents, antipsoriatics, retinoids, anti-acne medicaments, antineoplastic agents, topical anesthesetics, phototherapeutic agents, sunscreen, cutaneous protection agents, alpha-hydroxy acids (including lactic acid and glycolic acid), insect repellants and the like.
The term "effective amount" of a pharmacologically or therapeutically active agent or cosmetic agent refers to a nontoxic but sufficient amount of a compound to provide the desired local effect and performance at a reasonable benefit/risk ratio attending any medical treatment.
The term "zeta potential" is the measurement of the charge on the electric double layer of a dispersed particle. This charge may be affected by DNA contained in the liposomes. The charge on the liposome may, in turn, affect the transfection efficiency, which depends on a
successful fusion of the liposome with the cell membrane. Zeta potential is measured by a Zetasizer model Zetasizer 3000 (Malvern Instruments, Inc., Southborough, MA).
The term "cationic liposome" is a liposome made from cationic lipids that carry a net positive charge.
Molecular Biology Definitions
In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques may be employed within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; and F.M. Ausubel et al. (eds.), Current
Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
Proteins and enzymes can be made in a cell using instructions in DNA and RNA, according to the genetic code. "Transcription" is the process by which a DNA sequence or gene having instructions for a particular protein or enzyme is "transcribed" into a corresponding sequence of RNA. "Translation" is the process by which the RNA sequence is
"translated" into the sequence of amino acids which form the protein or enzyme.
The terms "polynucleotide" or "nucleic acid molecule", used interchangeably herein, refer to polymers of nucleotide residues or nucleotide bases. Such residues include, e.g., inosine, adenosine, guanosine, cytosine, uracil and thymidine, as well as modified bases, such as, e.g., thiouracil, thioguanine and fluorouracil. Nucleic acids also include "double stranded" and "single stranded" DNA and RNA, as well as backbone modifications thereof (for example, methylphosphonate linkages) and "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone.
The polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3 '-non-coding regions and the like. The nucleic acids may also be modified by many means known in the art. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin and the like.
A "gene" is a polynucleotide which codes for a functional product, including mRNA tRNA rRNA, a polypeptide, and/or a protein. Typically, a gene product is a functional protein. A gene may also comprise regulatory (i.e., non-coding) sequences as well as coding sequences. Exemplary regulatory sequences include promoter sequences, which determine, for example, the conditions under which the gene is expressed. The transcribed region of the gene may also include untranslated regions including introns, a 5'-untranslated region (5'-UTR) and a 3 '-untranslated region (3'-UTR). For purposes of the present invention, the terms "gene" and "nucleic acid" can be used interchangeably.
A "polypeptide" or "protein" is a polymer of amino acids that are linked together by chemical bonds called "peptide bonds".
Liposomes
Lipids self-assemble into structures having one or more lipid bilayers, each of which include two opposing monolayers of amphipatic lipids surrounding an aqueous compartment. The amphipatic lipid molecules have a polar (hydrophilic) headgroup region covalently linked to one or two non-polar (hydrophobic) acyl chains. Energetically unfavorable interactions between the hydrophobic acyl chains and the aqueous medium are generally believed to induce lipid molecules to rearrange such that their polar headgroups are oriented towards the aqueous medium, while the acyl chains reorient towards the interior of the bilayer. An energetically stable structure is formed in which the acyl chains are effectively shielded from contact with the aqueous medium.
A variety of amphipathic lipids may form bilayers (liposomes), including those having saturated or unsaturated acyl chains of 10-24 carbons. Suitable polar groups include phosphorylcholine, phosphorylethanolamine, phosphorylserine, phosphorylglycerol and phosphorylinositol. Suitable acyl chains include laurate, myristate, palmitate, stearate and oleate chains. Liposomal bilayers can further contain sterol, such as cholesterol. Liposomes may also contain nonphospholipids such as sphingolipids, ceramide. cerebroside, glycosphingolipid, free fatty acids, eicoanoids and lipid vitamins.
Stability, rigidity, and permeability of liposomes are altered by changes in lipid composition. Membrane fluidity generally depends on the composition of the fatty acyl chains of the lipids. The fatty acyl chain may be in an ordered, rigid state, or in a relatively disordered
-8- fluid state Factors affecting rigidity include chain length, the degree of saturation of the fatty acyl chains, and temperature Longer acyl chains interact more strongly with each other, thus increasing fluidity with shorter chains, and unsaturated fatty acyl chains are more flexible than saturated fatty acyl chains Transition of the membrane from a rigid to a fluid state occurs as the temperature is raised above the melting temperature The melting temperature is dependent upon the length and degree of saturation of the fatty acyl chain
Liposomes may be multilamellar or uniiamellar Multilamellar vesicles contain concentric membranes with numerous enclosed aqueous compartments Large and small uniiamellar vesicles contain one single bilayer and one enclosed aqueous compartment
Cationic Liposomes
The liposomes of the present invention consists of lipid bilayers containing an active drug, e.g., a DNA plasmid inside or outside of the structure The lipids of the cationic liposomes include at least one sphingolipid and at least one cationic lipid such as, e.g., a charged phospholipid The particle diameter of the liposomes of the present invention preferably ranges from about 100 to about 250 nm The appearance of the liposomes are generally slightly turbid, and the pH is between about 5 and about 6, preferably about 5 5 These liposomes typically have a zeta potential of between 25 mV and 35 mV. preferably about 30 mV
In a preferred embodiment, the cationic lipid is a phospholipid Preferred phospholipids include, but are not limited to, soy lecithin, phospholipon 80, 90, and 90H, Varisoft preparations (Witco Corp , Dublin, OH), and combinations thereof
In another embodiment of the invention, the sphingolipid is Ceramide Preferred sphmgolipids include, but are not limited to, Ceramide I- VI, galactosyl ceramides, glycosyl ceramides, lactosyl ceramides, and combinations thereof A particularly preferred sphingolipid is Ceramide III
In another embodiment, the sphingolipid content of the cationic liposomes is within the range from about 0 01% to about 1% by weight (w/w) of the lipid bilayer More preferably, the sphingolipid content is from about 0 02 to about 0 2%, by weight (w/w) of the lipid bilayer
In still another embodiment, the sphingolipid content of the cationic liposomes is within the range from about 0 01%) to about 1% in weight by volume More preferably, the
-9- sphingolipid content is from about 0 02 to about 0 2% in weight by volume (w/v) of the lipid bilayer
In yet another embodiment of the invention, the ceramide is Ceramide III, and the phospholipids are Phosphilipon 90 G (American Lecithin Co , Danbury, CT) and Varisoft 432PPG Varisoft 423 PPG consists of 67% quarternary compound which is critical to the transfection efficiency
In a preferred embodiment, the amount of Ceramide III is from about 0 75 to about 1 5 % (w/v), the amount of Phospholipon 90-G is from about 0 1 to about 0 2 % (w/v), and the amount of Varisoft 432 PPG is from about 0 02 to about 0 08% (w/v)
Plasmids for Gene Therapy
Plasmids suitable for use in this context are preferably circularized or closed double- stranded nucleic acid molecules having sizes preferably in the 5-40 Kbp (kilo basepair) range. The plasmids are constructed according to methods known in the art and include a therapeutic nucleic acid or gene, i.e., a nucleic acid gene to be translated and/or transcribed in gene therapy, under the control of suitable promoter and terminator control elements, and other elements necessary for replication within the host cell and/or integration into the host-cell genome Methods for preparing plasmids useful for gene therapy in genes or other mammals are widely known and referenced
The nucleic acids or genes to be introduced for gene therapy by the complex of the invention generally fall into one of three categories
The first category includes those genes which are intended to overcome a gene deficiency or defect in the subject, i e , where the subject fails to produce active, endogenous protein at all or within normal levels, and the gene introduced in the plasmid is intended to make up this deficiency The second includes polypeptides encoded by the nucleic acids designed to treat any existing pathology, such as cancer, or a pathogenic condition such as viral infection
The third class includes genes intended to produce a mRNA transcript that can act as an antisense molecule to inhibit an undesirable protein expression, such as overexpression of proteins specific for tumor growth, or expression of viral proteins
EXAMPLES
The invention is further illustrated by the following non-limiting examples.
EXAMPLE 1: Preparation of a Cationic Liposome Phosphlipon 90G 0.12g (American Lecithin Company, Danbury, CT) and Ceramide
III 0.04g (Centerchem Inc., Stamford, CT) were dissolved in 2 ml ethanol (Aaper, Shelby, KY) by heating in a water bath. Sodium chloride (0.5 g) ( Sigma Chem Co., St. Louis, MO), and lg Varisoft 432PPG (Witco Corporation, Dublin, OH) were dissolved in 96.79 ml deionized water and warmed to approximately 60-70°C. The water phase was added to the ethanol phase at the approximate temperature of 70-80°C. The suspension was microfluidized
(Microfluidics, Newton, MA) using high pressure/ high shear 5 times without a cooling loop.
EXAMPLE 2: Transfection
The cationic liposomal formulation was mixed with plasmid EDFP (enhanced green fluorescent protein) and β-galactosidase ( Oral Biology and Pathology, SUNY, Stony Brook,
NY ) 0.5-5 mg. The human keratinocyte and 293 HEK cell lines (Oral Biology and Pathology, SUNY, Stony Brook, NY) were transfected with both the cationic liposomal formulation and the known compound lipofectin and transfection was compared. Cells were plated one or two days before lipofection at 400,000-500,000 cells per p35 well (corresponding to an area of about 9 cm2). Cells were between 40-60% confluent of the day of lipofection.
Liposomes prepared as described above were mixed gently into serum-free, antibiotic free media (SFM), at a volume ratio of about 1 : 100 liposome: SFM, and gently mixed for about 45 minutes prior to transfection. Two milligrams of DNA encoding for β-galactosidase were diluted into 100 ml serum-free antibiotic free media, and thirty minutes later combined with
100 ml liposome preparation. The cell culture media (1 ml) was replaced with 200 μ\ liposome/DNA solution and 800 μl SFM. The cells were incubated at 37° C for about 5 hours. After 5 hours the liposome/DNA solution was replaced with regular cell culture media. The next day, cells were identified for DNA product expression by excitation with UN light to
visualize green fluorescent protein (GPF). Efficiency of gene transfection was compared to that of the commercially available product Lipofectamine. The cationic liposome vector system showed 50%ι transfection efficiency with keratinocytes compared to 4% by lipofectin. The high efficiency was attributed to the use of the a combination of lipids which are similar to the stratum corneum and balancing the cationic charge as determined by zeta potential.
Images depicting the transfection efficiency of β-galactosidase gene in keratinocytes are shown in FIGURE 1, in which the dark areas indicate transfected cells. FIGURE 1 shows that significantly more number of cells are transfected by the vector system of the present invention as compared to lipofectin.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Patents, patent applications, and publications are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.