title: Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.
abstract: Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We conclude that SPLP stabilized by PEG-CerC8 and containing 20-24 mol% cationic lipid are attractive alternatives to plasmid DNA-cationic lipid complexes for regional gene therapy applications.
authors.affiliation: INEX Pharmaceuticals Corporation, Burnaby, BC, Canada.
authors.forename: Y P
authors.initials: YP
authors.lastname: Zhang
authors.forename: L
authors.initials: L
authors.lastname: Sekirov
authors.forename: E G
authors.initials: EG
authors.lastname: Saravolac
authors.forename: J J
authors.initials: JJ
authors.lastname: Wheeler
authors.forename: P
authors.initials: P
authors.lastname: Tardi
authors.forename: K
authors.initials: K
authors.lastname: Clow
authors.forename: E
authors.initials: E
authors.lastname: Leng
authors.forename: R
authors.initials: R
authors.lastname: Sun
authors.forename: P R
authors.initials: PR
authors.lastname: Cullis
authors.forename: P
authors.initials: P
authors.lastname: Scherrer
mesh_terms: D000818:Animals; D019556:COS Cells; D003956:Dialysis; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000628:therapy; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection / Q000379:methods*
chemical_list: D008055:Lipids; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C107332:dioleoylphosphatidylethanolamine-polyethyleneoxide; C015831:dimethyldioctadecylammonium; D011092:Polyethylene Glycols