Development of Highly Stable and Low Toxic Cationic Liposomes for Gene Therapy

Abstract

Cationic liposomes with high stability and low cytotoxicity for gene therapy have been developed. Luciferase plasmid DNA (pLuc) was used as a model gene. The empty liposomes and niosomes were prepared by freeze dried empty liposomes (FDEL) method. The entrapment of pLuc in the liposomes was by reconstitution of the lyophilized dried vesicles with the plasmid solution. The morphology of the vesicles showing multilamellar structure was characterized by transmission electron microscope (TEM) and cryo-TEM. Cytotoxicity of the vesicular formulations was investigated on mouse melanoma cell lines (B₁₆F₁₀) by MTT assay. Cationic liposomes and niosomes containing the cationic lipid DDAB were less cytotoxic than other bilayer vesicular formulations. The pLuc entrapped in the cationic DPPC/Chol/DDAB liposomes (at 1:1:1 molar ratio) exhibited higher stability than other vesicular formulations and the pLuc in solution when stored at 4, 30 and 50°C for 8 weeks. The entrapment efficiency determined by gel electrophoresis and gel documentation of the pLuc in this liposomal formulation was 100%. Luciferase gene expression of pLuc-loaded in cationic liposomes (lipoplexes) in HeLa cell lines was evaluated from luciferase activity determined by a luminometer at 24 and 48 h incubation. Percentages of cell proliferation of the lipoplexes on HeLa cell line at 24 and 48 h incubation were evaluated by the WST-1 assay. When the amount of DPPC or cholesterol was increased in the lipoplexes, the higher amount of DDAB was needed to protect pLuc from enzymatic degradation. However, DPPC and cholesterol exceeded 33 and 50% mol, respectively gave no gene expression. The DPPC/Chol/DDAB (at 1:1:1 molar ratio) lipoplexhas demonstrated moderately lower luciferase gene expression and low cytoxicity. This lipoplex with the DDAB/pLuc weight ratio of 14:1 was the most desirable formulation for gene therapy because of its high stability, high luciferase gene expression and low cytotoxicity.

Key words

Cationic liposomes, cytotoxicity, stability - Gene therapy - Liposomes, gene therapy - Luciferase plasmid

• References

• 1 Almofti MR, Harashima H, Shinohara Y, Almofti A, Baba Y, Ki-wada H. Cationic liposome-mediated gene delivery: Biophysical study and mechanism of internalization. Arch Biochem Bio-phys. 2003; 410: 246-253
• 2 Anderson M, Omri A. The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations. Drug Deliv. 2004; 11: 33-39
• 3 Anwer K, Kao G, Rolland A, Driessen WHR, Sullivan SM. Pep-tide-mediated gene transfer of cationic lipid/plasmid DNA complexes to endothelial cells. Drug Target. 2004; 12 (4) 215-221
• 4 Bally MB, Harvie P, Wong FM, Kong S, Wasan EK, Reimer DL. Biological barriers to cellular delivery of lipid-based DNA Carriers. Adv Drug Deliv Rev. 1999; 38: 291-315
• 5 Banerjee R. Liposomes: Applications in Medicine. Biomater Appl. 2001; 16 (3) 1-21
• 6 Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol. 1965; 13: 238-252
• 7 Campbell RB, Balasubramanian SV, Straubinger R. Phospholip-id-cationic lipid interactions: influences on membrane and vesicle properties. Biochim Biophys Acta. 2001; 1512: 27-39
• 8 Choosakoonkriang S, Wiethoff CM, Smith IG, Russell MC. Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA. J Biol Chem. 2001; 276 (ll) 8037-8043
• 9 Congiu A, Pozzi D, Esposito C, Castellano C, Mossa G. Correlation between structure and transfection efficiency: a study of DC-Chol-DOPE/DNA complexes. Colloid Surface B. 2004; 36: 43-48
• 10 Domashenko A, Gupta S, Cotsarelis G. transgenes to human hair follicle progenitor cells using topical lipoplex. Nature Biotechnol. 2000; 18: 420-423
• 11 Esposito C, Generosi J, Mossa G, Masotti A, Castellano AC. The analysis of serum effects on structure, size and toxicity of DDAB - DOPE and DC Chol - DOPE lipoplexes contributes to explain their different transfection efficiency. Colloid Surface B. 2006; 53: 187-192
• 12 Hao Y, Zhao F, Li N, Yang Y, Li K. Studies on a high encapsulation of colchicine by a niosome system. IntJ. Pharm. 2002; 244: 73-80
• 13 Hong K, Zheng W, Papahadjopoulos D. Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery. FEES Letters. 1997; 400: 233-237
• 14 Huang Z, Li W, MacKay JA, Francis C, Szoka Jr. Thiocholesterol-based lipids for ordered assembly of bioresponsive gene carriers. Mol Ther. 2005; 11: 409-417
• 15 Inagaki A, Tsuchiya K, Sakai H, Imura T, Ohkubo T, Tsubaki N et al Effectes of dialkyldimethylammonium bromides on the membrane properties of cationic liposomes. J Oleo Sci. 200 54 (7) 383-388
• 16 Ishiwata H, Suzukia N, Andoa S, Kikuchi H, Kitagawa T. Characteristics and biodistribution of cationic liposomes and their DNA complexes. J Control Release. 2000; 69: 139-148
• 17 Jensen TG. Gene transfer into human epidermis as an experimental model for somatic gene therapy. Dan Med Bull. 2004; 51: 155-66
• 18 Kikuchi H, Suzuki N, Ebihara K, Morita H, Ishii Y, Kikuchi A et al Gene delivery using liposome technology. J Control Release. 1999; 62: 269-277
• 19 Koltover I, Salditt T, Rädler JO, Saflnya CR. An inverted hexagonal phase of cationiclliposome-DNA complexes related to DNA release and delivery. Science. 1998; 281: 78-81
• 20 Lai MZ, Duzgunes N, Szoka FC. Effects of replacement of the hydroxyl group of cholesterol and tocopherol on the thermotropic behavior of hospholipid-membranes. Biochemistry. 1985; 24: 1646-1653
• 21 Li XS, Gao X, Son SF, Hofland H, Huang L. DC-Choi lipid system in gene transfer. J Control Release. 1996; 39: 373-381
• 22 Liang X, Mao G, Ng KYS. Mechanical properties and stability measurement of cholesterol-containing liposome on mica by atomic force microscopy. J Colloid Interf Sci. 2004; 278: 53-62
• 23 Manosroi J, Dhumtanoma P, Manosroi A. Anti-proliferative activity of essential oil extracted from Thai medicinal plants on KB and P388 cell lines. Cancer Lett. 2005; 235: 111-120
• 24 Manosroi A, Panyosak A, Rojanasakul Y, Manosroi J. Characteristics and anti-proliferative activity of azelaic acid and its derivatives entrapped in bilayer vesicles in cancer cell lines. J Drug Target. 2007; 15 (5) 334-341
• 25 Manosroi A, Wongtrakul P, Manosroi J, Sakai H, Sugawara F, Yuasa M et al Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids Surf B Biointerfaces. 2003; 30: 129-138
• 26 Meilander NJ, Pasumarthy MK, Kowalczyk TH, Cooperb MJ, Bellamkonda RV. Sustained release of plasmid DNA using lipid microtubules and agarose hydrogel. J Control Release. 2003; 88: 321-331
• 27 Nguyen LT, Atobe K, Barichello JM, Ishida T, Kiwada H. Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes. Biol Pharm Bull. 2007; 30 (4) 751-757
• 28 Nichols WW, Ledwith BJ, Manam SV, Troilo PJ. Potential DNA vaccine integration into host cell genome. Ann NY Acad. Sci. 1995; 27 (772) 30-39
• 29 Nishikage S, Koyama H, Miyata T, Ishii S, Hamada H, Shigematsu H. In vivo electroporation enhances plasmid-based gene transfer of basic fibroblast growth factor for the treatment of ischemic limb. J Surg Res. 2004; 120: 37-46
• 30 Parker AL, Newman C, Briggs S, Seymour L, Sheridan PJ. Nonviral gene delivery: techniques and implications for molecular medicine. Expert Rev Mol Med. 2003; 5: 1-15
• 31 Patil SD, Rhodes DG, Burgess DJ. Anionic liposomal delivery system for DNA transfection. AAPSJ. 2004; 6: 1-10
• 32 Prazeres DM, Ferreira GNM, Monteiro GA, Cooney CL, Cabrai JMS. Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks. Trends Biotechnol. 1999; 17: 169-174
• 33 Presti FT, Pace RJ, Chen SI. Cholesterol-phospholipid interaction in membranes. Stoichiometry and molecular packing of cholesterol-rich domains. Biochemistry. 1982; 21: 3831-3835
• 34 Shahiwala A, Misra A. Studies in topical application of niosomally entrapped Nimesulide. J Pharm Pharm Sci. 2002; 5: 220-225
• 35 Tang FX, Hughes JA. use of dithiodiglycolic acid as a tether for cationic lipids decreases the cytotoxicity and increases transgene expression of plasmid DNA in vitro. Bioconjugate Chem. 1999; 10: 791-796
• 36 Tokunaga M, Hazemoto N, Yotsuyanagi T. Effect of oligopeptides on gene expression: comparison of DNA/peptide and DNA/peptide/liposome complexes. Int J Pharm. 2004; 269: 71-80
• 37 Torchilin VP, Levchenko TS, Rammohan R, Volodina N, Sternberg BP, D’Souza GM. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome - DNA complexes. PNAS. 2003; 100: 1972-1977
• 38 Wang D, Jing N, Lin Q. Stearylamine Liposome as a New Efficient Reagent for DNA Transfection of Eukaryotic Cells. Biochem Biophys Res Commun. 1996; 226: 450-455
• 39 Weintraub H, Cheng PF, Conrad K. Expression of transfected DNA depends on DNA topology. Cell. 1986; 46: 115-122
• 40 Wiethoff CM, Gill ML, Koe GS, Koe JG, Russell MC. The structural organization of cationic lipid-DNA complexes. J Biol Chem. 2002; 277 (47) 44980-44987
• 41 Wolff JA, Vladimir B. The mechanism of naked DNA uptake and expression. Adv Genetics. 2005; 54: 1-20
• 42 You J, Kamihira M, Iijima S. Surfactant-mediated gene transfer for animal cells. Cytotechnology. 1997; 25: 45-52
• 43 You J, Kamihira M, Iijima S. Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer. J Biochem. 1999; 125: 1160-1167
• 44 Zhang L, Nolan E, Kreitschitz S, Rabussay DP. Enhanced delivery of naked DNA to the skin by non-invasive in vivo electroporation. Biochim Biophys Acta. 2002; 1572: 1-9