title: A calcium phosphate-based gene delivery system.
abstract: 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 (Cultispherestrade mark). 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 beta-galactosidase was precipitated with CaP to form particles of compacted LacZ-CaP and delivered directly or by Cultispherestrade mark to cells in vitro. The transfection efficiency was determined by beta-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 x 10(4) cells/cm2 provided sufficient transfection efficiency, and storage of pDNA-CaP at 4 degrees 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. Cultispherestrade mark 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.
authors.affiliation: Bone Tissue Engineering Center, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Huihua
authors.initials: H
authors.lastname: Fu
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Hu
authors.forename: Tim
authors.initials: T
authors.lastname: McNelis
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Hollinger
mesh_terms: D000818:Animals; D002130:Calcium Phosphates; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000008:administration & dosage / Q000096:biosynthesis; D004356:Drug Storage; D005079:Excipients; D018014:Gene Transfer Techniques; D006651:Histocytochemistry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008855:Microscopy, Electron, Scanning; D010316:Particle Size; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D002130:Calcium Phosphates; D005079:Excipients; D004247:DNA; D007770:L-Lactate Dehydrogenase; D001616:beta-Galactosidase