Abstract
The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.
Key Words: PLGA, Microspheres, Plasmid DNA, Controlled Release
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References
- 1.Luo D, Woodrow-Mumford K, Belcheva N, Saltzaman WM. Controlled DNA delivery systems. Pharm. Res. 1999;16:1300–1307. doi: 10.1023/A:1014870102295. [DOI] [PubMed] [Google Scholar]
- 2.Crystal RG. Transfer of genes to humans early lessons and obstacles to success. Science. 1995;270:404–410. doi: 10.1126/science.270.5235.404. [DOI] [PubMed] [Google Scholar]
- 3.Thieny AR, Rabinovich P, Peng B, Mahan LC, Bryant JL, Gallo RC. Characterization of liposome-mediated gene delivery expression, stability and phamacokinetics of plasmid DNA. Gene Ther. 1997;4:226–237. doi: 10.1038/sj.gt.3300350. [DOI] [PubMed] [Google Scholar]
- 4.felgner PL, Ringold GM. Cationic liposome-mediated transfection. Nature. 1989;337:387–388. doi: 10.1038/337387a0. [DOI] [PubMed] [Google Scholar]
- 5.Wasan EK, Fairchild A, Bally MB. Cationie liposome-plasmid DNA complexes used of gene transfer retain a significant trapped volume. J of Pharm Sci. 1998;87:5–14. doi: 10.1021/js970265k. [DOI] [PubMed] [Google Scholar]
- 6.Felgner PL, Tsai YJ, Sukhu L, Wheeler CJ, Manthorpe M, Marshall J, Cheng SH. Improved cationic lipid formulations for in vivo gene therapy. Ann NY Acad Sci. 1995;772:126–139. doi: 10.1111/j.1749-6632.1995.tb44738.x. [DOI] [PubMed] [Google Scholar]
- 7.Liu Y, Liggitt D, Zhong W, Tu G, Gaensler K, Debs R. Cationic liposome-mediated intravenous gene delivery. J Biol Chem. 1995;270:24864–24870. doi: 10.1074/jbc.270.42.24864. [DOI] [PubMed] [Google Scholar]
- 8.Xu Y, Szoka FC. Mechanism of DNA release from cationieliposome/DNA complexes used in cell transfection. Biochem. 1996;35:5616–5626. doi: 10.1021/bi9602019. [DOI] [PubMed] [Google Scholar]
- 9.Niven R, Pearlman R, Wedeking T, et al. Biodistribution of racholabeled lipid-DNA complexes and DNA in mice. J Pharm Sci. 1998;87:1292–1299. doi: 10.1021/js980087a. [DOI] [PubMed] [Google Scholar]
- 10.Pouton CW, Lucas P, Thomas BJ, Uduehi AN, Milroy DA, Moss SH. Polycation-DNA complexes for gene delivery: a comparison of the biopharmacentical properties of cationic polypeptides and cationic lipids. J Contr Rel. 1998;53:289–299. doi: 10.1016/S0168-3659(98)00015-7. [DOI] [PubMed] [Google Scholar]
- 11.Mumper RJ, Duguid JG, Anwer K, Barron MK, Nitta H, Rolland AP. Polyvinyl derivatives as novel interactive polymers for controlled gene delivery tomuscle. Pharm Res. 1996;13:701–709. doi: 10.1023/A:1016039330870. [DOI] [PubMed] [Google Scholar]
- 12.MacDonald RC, Rakhmanova VA, Choi KL, Rosenzweig HS, Lahiri MK. O-ethylphosphatidylcholine: a metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J Phann Sci. 1999;88:896–904. doi: 10.1021/js990006q. [DOI] [PubMed] [Google Scholar]
- 13.DeLuca PP, Mehta RC, Hausberger AG, Thanoo BC. Biodegradable polyesters for drug and polypeptide delivery. In: El-Nokaly MA, Piatt DM, Charpentier BA, editors. Polymeric drug delivery systems. Washington, DC: American Chemical Society; 1993. pp. 53–79. [Google Scholar]
- 14.Mehta RC, Jeyanthi R, Calis S, Thanoo BC, Burton KW, DeLuca PP. Biodegradable microspheres as depot system for parenteral delivery of peptide drugs. J. Cont. Rel. 1994;29:375–384. doi: 10.1016/0168-3659(94)90082-5. [DOI] [Google Scholar]
- 15.DeLuca PP. Formulation Strategies for Proteins and Peptides. J. KorPharm Sci. 1993;23:S51–S60. [Google Scholar]
- 16.Capan Y, Woo BH, Gebrekidan S, Ahmed S, DeLuca PP. Influence of formulation parameters on the characteristics of poly(D, L-lactide-co-glycolide) microspheres containing poly(L-lysine) complexed plasmid DNA. J Cont Rel. 1999;60:279–286. doi: 10.1016/S0168-3659(99)00076-0. [DOI] [PubMed] [Google Scholar]
- 17.Capan Y, Woo BH, Gebrekidan S, Ahmed S, DeLuca PP. Stability of poly(L-lysine)-complexed plasmid DNA during mechanical stress and DNase I treatment. Pharm Dev Technol. 1999;4:491–498. doi: 10.1081/PDT-100101386. [DOI] [PubMed] [Google Scholar]
- 18.Yasuhiko T, Yoshito I. Effect of the size and surface charge of polymer microspheres on their phagocytosis by macrophages. Biomaterials. 1988;9:356–362. doi: 10.1016/0142-9612(88)90033-6. [DOI] [PubMed] [Google Scholar]
- 19.Kanke M, Morlier E, Geissler R, Powell R, Kaplan A, DeLuca PP. Interaction of microspheres with Blood constituents II: Uptake of biodegradable particles by macrophages. J. Parenteral Sci and Tech. 1986;40:114–118. [PubMed] [Google Scholar]
- 20.Middaugh CR, Evans RK, Montgomery DL, Casmiro DR. Analysis of Plasmid DNA from a pharmaceutical Perspective. J. Pharm. Sci. 1998;87:130–146. doi: 10.1021/js970367a. [DOI] [PubMed] [Google Scholar]