Stabilized plasmid-lipid particles: construction and characterization

Abstract

A detergent dialysis procedure is described which allows of up to 70% and permits inclusion of 'fusigenic' lipids such encapsulation of plasmid DNA within a lipid envelope, as dioleoylphosphatidylethanolamine (DOPE). The in vitro where the resulting particle is stabilized in aqueous media transfection capabilities of SPLP are demonstrated to be by the presence of a poly(ethyleneglycol) (PEG) coating. strongly dependent on the length of the acyl chain conThese 'stabilized plasmid-lipid particles' (SPLP) exhibit an tained in the ceramide group used to anchor the PEG polyaverage size of 70 nm in diameter, contain one plasmid mer to the surface of the SPLP. Shorter acyl chain lengths per particle and fully protect the encapsulated plasmid from result in a PEG coating which can dissociate from the digestion by serum nucleases and E. coli DNase I. Encap-SPLP surface, transforming the SPLP from a stable parsulation is a sensitive function of cationic lipid content, with ticle to a transfection-competent entity. It is suggested that maximum entrapment observed at dioleoyldimethylam-SPLP may have utility as systemic gene delivery systems monium chloride (DODAC) contents of 5 to 10 mol%. The for gene therapy protocols. formulation process results in plasmid-trapping efficiencies

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

“…Preparation of stable plasmid-lipid particles (SPLP) Plasmid pCMVluc18 was encapsulated as SPLP using the detergent dialysis method 19 (Saravolac et al, in preparation). The SPLP contained 42.5:42.5:15 mol% of DOPE:DODAC:PEG-CerC 8 , at a lipid:DNA ratio of 15:1 (w/w).…”

“…19 The lipoplexes used in this study were prepared using the cationic lipid dioleoyldimethylammonium chloride (DODAC) in a 1:1 mixture with the fusogenic lipid 1,2-sn-dioleoylphosphatidylethanolamine (DOPE). The other cationic lipid transfection system examined here, SPLP, are fully encapsulated DNA particles, assembled using a detergent dialysis method.…”

“…The other cationic lipid transfection system examined here, SPLP, are fully encapsulated DNA particles, assembled using a detergent dialysis method. 19 The encapsulated system differs from lipoplexes in terms of its method of assembly, uniform morphology, smaller size (70-150 versus 500-700 nm) greater serum stability and ability to protect the plasmid from serum nucleases. 19 SPLPs are of potential interest in nonviral gene transfer due to their improved pharmacokinetic properties and the potential to add targeting ligands to the exterior surface.…”

“…19 The encapsulated system differs from lipoplexes in terms of its method of assembly, uniform morphology, smaller size (70-150 versus 500-700 nm) greater serum stability and ability to protect the plasmid from serum nucleases. 19 SPLPs are of potential interest in nonviral gene transfer due to their improved pharmacokinetic properties and the potential to add targeting ligands to the exterior surface. 19 Understanding the relationship between cell cycle and transfection should provide insight into the mechanism of cationic lipid-mediated transfection which will enable the rational engineering of these systems for gene therapy applications.…”

“…19 SPLPs are of potential interest in nonviral gene transfer due to their improved pharmacokinetic properties and the potential to add targeting ligands to the exterior surface. 19 Understanding the relationship between cell cycle and transfection should provide insight into the mechanism of cationic lipid-mediated transfection which will enable the rational engineering of these systems for gene therapy applications.…”