Abstract
The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17∶83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofect Amine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.
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References
Patil SD, Burgess DJ. DNA-based biopharmaceuticals: therapeutics for the 21st century.AAPS NewsMagazine. 2003;6:27.
Patil SD, Rhodes DG, Burgess DJ. DNA-based therapeutics and DNA delivery systems: a comprehensive review.AAPS PharmSci. 2004; In press.
Romano G, Micheli P, Pacilio C, Giordano A. Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications.Stem Cells. 2000;18(1):19–39.
Vorburger SA, Hunt KK. Adenoviral gene therapy.Oncologist. 2002;7:46–59.
Eastman SJ, Scheule RK. Cationic lipid:pDNA complexes for the treatment of cystic fibrosis.Curr Opin Mol Ther. 1999;1:186–196.
Luo D, Saltzman WM. Synthetic DNA delivery systems.Nat Biotechnol. 2000;18:33–37.
Liu F, Huang L. Development of non-viral vectors for systemic gene delivery.J Control Release. 2002;78(I-3):259–266.
Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.Nat Med. 2001;7:33–40.
Zhdanov RI, Podobed OV, Vlassov VV. Cationic lipid-DNA complexes—lipoplexes—for gene transfer and therapy.Bioelectrochemistry. 2002;58:53–64.
Brown MD, Schatzlein AG, Uchegbu IF. Gene delivery with synthetic (non viral) carriers.Int J Pharm. 2001;229(I-2):1–21.
Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.Proc Natl Acad Sci USA. 1987;84:7413–7417.
Audouy SA, de Leij LF, Hoekstra D, Molema G. In vivo characteristics of cationic liposomes as delivery vectors for gene therapy.Pharm Res. 2002;19:1599–1605.
Marshall J, Yew NS, Eastman SJ, Jiang C, Scheule RK, Cheng SH. Cationic lipid-mediated gene delivery to the airways. In: Huang L, Hung M-C, Wagner E, eds.Nonviral Vectors for Gene Therapy. San Diego, CA: Academic Press, 1999:39–68.
Lappalainen K, Jaaskelainen I, Syrjanen K, Urtti A, Syrjanen S. Comparison of cell proliferation and toxicity assays using two cationic liposomes.Pharm Res. 1994;11:1127–1131.
Dokka S, Toledo D, Shi X, Castranova V, Rojanasakul Y. Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes.Pharm Res. 2000;17:521–525.
Filion MC, Phillips NC. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells.Biochim Biophys Acta 1997;1329:345–356.
Armeanu S, Pelisek J, Krausz E, et al. Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo.Mol Ther. 2000;1:366–375.
Fillion P, Desjardins A, Sayasith K, Lagace J. Encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with fluid liposome-encapsulated antisense oligonucleotides.Biochim Biophys Acta. 2001;1515:44–54.
Lakkaraju A, Dubinsky JM, Low WC, Rahman Y-E. Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.J Biol Chem. 2001;276:32000–32007.
Akhtar S, Basu S, Wickstrom E, Juliano RL. Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).Nucleic Acids Res. 1991;19:5551–5559.
Patil SD, Rhodes DG. Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study.Nucleic Acids Res. 2000;28:2439–2445.
Perrie Y, Gregoriadis G. Liposome-entrapped plasmid DNA: characterization studies.Biochim Biophys Acta. 2000;1475:125–132.
Safinya CR. Structures of lipid-DNA complexes: supramolecular assembly and gene delivery.Curr Opin Struct Biol. 2001;11:440–448.
Koltover I, Salditt T, Radler JO, Safinya CR. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery.Science. 1998;281:78–81.
Hope MJ, Mui B, Ansell S, Ahkong QF. Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs.Mol Membr Biol. 1998;15:1–14.
Tilcock CPS, Cullis PR, Gruner SM. Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures.Biochemistry. 1988;27:1415–1420.
Xu Y, Szoka FC Jr. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection.Biochemistry. 1996;35:5616–5623.
Szoka FC Jr, Xu Y, Zelphati O. How are nucleic acids released in cells from cationic lipid-nucleic acid complexes?.J Liposome Res. 1996;6:567–587.
Sulpice J-C, Moreau C, Devaux PF, Zachowski A, Girand F. Antagonist effects of Ca2+ and spermine on phosphatidylinositol 4,5-bis-phosphate-mediated transmembrane redistribution of phospholipids in large unilamellar vesicles and in erythrocytes.Biochemistry. 1996;35:13345–13352.
Williamson P, Kulick A, Zachowski A, Schlegel RA, Devaux PF. Calcium induces transbilayer redistribution of all major phospholipids in human erythrocytes.Biochemistry. 1992;31:6355–6360.
Bucki R, Bachelot-Loza C, Zachowski A, Giraud F, Sulpice J-C. Calcium induces phospholipid redistribution and microvesicle release in human erythrocyte membranes by independent pathways.Biochemistry 1998;37:15383–15391.
Patil SD, Rhodes DG. Conformation of oligodeoxynucleotides associated with anionic liposomes.Nucleic Acids Res. 2000;28:4125–4129.
Merrick P, Nieminen AL, Harper RA, Herman B, Lemasters JJ. Cytotoxicity screening of surfactant-based shampoos using a multiwell fluorescence scanner: correlation with Draize eye scores.Toxicol In Vitro. 1992;6:543–547.
Tari AM, Fuller N, Boni LT, Collins D, Rand P, Huang L. Interactions of liposome bilayers composed of 1,2-diacyl-3-succinylglycerol with protons and divalent cations.Biochim Biophys Acta. 1994;1192:253–262.
Almofti MR, Harashima H, Shinohara Y, Almofti A, Li W, Kiwada H. Lipoplex size determines lipofection efficiency with or without serum.Mol Membr Biol. 2003;20:35–43.
Ross PC, Hui SW. Lipoplex size is a major determinant of in vitro lipofection efficiency.Gene Ther. 1999;6:651–659.
Turek J, Dubertret C, Jaslin G, Antonakis K, Scherman D, Pitard B. Formulations which increase the size of lipoplexes prevent serum-associated inhibition of transfection.J Gene Med. 2000;2:32–40.
Audouy S, Molema G, De Leij L, Hoekstra D. Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability.J Gene Med. 2000;2:465–476.
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Published: October 18, 2004.
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Patil, S.D., Rhodes, D.G. & Burgess, D.J. Anionic liposomal delivery system for DNA transfection. AAPS J 6, 29 (2004). https://doi.org/10.1208/aapsj060429
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DOI: https://doi.org/10.1208/aapsj060429