A calcium phosphate-based gene delivery system

• Published in: Journal of biomedical materials research 2005-07, Vol.74A (1), p.40-48
• Main Authors: Fu, Huihua, Hu, Yunhua, McNelis, Tim, Hollinger, Jeffrey O.
• Format: Article
• Language: English
• Subjects: Animals; beta-Galactosidase - metabolism; calcium phosphate; Calcium Phosphates; Cell Line; Cell Survival - drug effects; Cultispheres; DNA - administration & dosage; DNA - biosynthesis; Drug Storage; Excipients; gene delivery; Gene Transfer Techniques; Histocytochemistry; L-Lactate Dehydrogenase - metabolism; Lac Operon - genetics; Mice; Microscopy, Electron, Scanning; Particle Size; plasmid DNA; Transfection; transfection efficiency
• ISSN: 1549-3296; 0021-9304; 1552-4965; 1097-4636
• DOI: 10.1002/jbm.a.30267

Although nonviral vectors have lower transfection efficiency than viral vectors, the excellent safety profile of nonviral vectors is appealing for gene therapy. An efficient, simple nonviral vector gene delivery system has been designed that includes plasmid DNA–calcium phosphate precipitates (pDNA‐CaP) and porous collagen spheres (Cultispheres™). The hypothesis for this study was the pDNA‐CaP would achieve efficient plasmid DNA transfection and the porous collagen spheres would provide a suitable delivery carrier system for three‐dimensional (3D) administration. To test the hypothesis, plasmid DNA including the LacZ reporter gene encoding β‐galactosidase was precipitated with CaP to form particles of compacted LacZ‐CaP and delivered directly or by Cultispheres™ to cells in vitro. The transfection efficiency was determined by β‐galactosidase gene expression. Results indicated that pLacZ‐CaP promoted 25–84% of transfection efficiency in a broad cell line spectrum and in flexible experimental conditions. Maximum transfection efficiency was achieved by having mostly nano‐sized partles (50–200 nm in diameter) of pDNA‐CaP precipitates. Seeding density of 0.7–4 × 104 cells/cm2 provided sufficient transfection efficiency, and storage of pDNA‐CaP at 4°C was most efficient to preserve transfection efficacy for up to 3 days. The pDNA‐CaP worked well in the presence of serum and serum‐free conditions and was less cytotoxic than the liposomes. Cultispheres™ carrying plasmid LacZ‐CaP was an effective 3D system for gene delivery. The technique described here is a simple and safe procedure to deliver genes, and may have application to regenerate bone and other tissues. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res, 2005