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
A photoactivated liposome release system that is generally applicable for triggered release of encapsulated hydrophilic materials is described. This approach to phototriggered release, derived from the known effects of plasmalogen photooxidation on membrane permeability in whole cells and model membrane systems, relies on producing a lamellar phase change or increase in permeability upon cleaving its constitutive lipids to single-chain surfactants using 630-820 nm light to sensitize the photooxidation of the plasmalogen vinyl ether linkage. Semi-synthetic plasmenylcholine liposomes containing encapsulated calcein and a membrane-bound sensitizer, such as zinc phthalocyanine, tin octabutoxyphthalocyanine, or bacteriochlorophyll a, were prepared by extrusion. Irradiation of air-saturated liposome solutions enhanced membrane permeability toward calcein and Mn2+, and promoted membrane fusion processes compared to non-irradiated or anaerobic controls. Bacteriochlorophyll a sensitization produced the fastest observed photoinitiated release rate from these liposomes (100% calcein release in less than 20 min; 800 nm irradiation at 300 mW); the observed release rate was two orders of magnitude slower for egg lecithin liposomes prepared and irradiated under identical experimental conditions. Liposome aggregation, interlipidic particle formation, and membrane fusion between adjoining liposomes was observed by 31P-NMR, freeze-fracture/freeze-etch TEM, and cryo-TEM as a function of irradiation time. The use of near-infrared sensitizers and the capacity of photolyzed plasmenylcholine liposomes to undergo membrane fusion processes make photodynamic therapy with these liposome-borne sensitizers an attractive adjunct to biochemical targeting methods.
Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-be generated. The SPLP produced could be isolated from 281) has shown that plasmid DNA can be entrapped in empty vesicles by sucrose density gradient centrifugation, 'stabilized plasmid-lipid particles' (SPLP) containing the and exhibited a narrow size distribution (62 ± 8 nm, as fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), determined by freeze-fracture electron microscopy) and a low levels (5-10 mol%) of cationic lipid, and stabilized by high plasmid-to-lipid ratio of 65 g/ mol (corresponding to a polyethyleneglycol (PEG) coating. The PEG moieties are one plasmid per particle) regardless of the DODAC conattached to a ceramide anchor containing an arachidoyl tent. It was found that isolated SPLP containing 20-acyl group (PEG-CerC 20 ). These SPLP exhibit low trans-24 mol% DODAC resulted in optimum transfection of COSfection potencies in vitro, due in part to the long residence 7 and HepG2 cells in vitro, with luciferase expression levels time of the PEG-CerC 20 on the SPLP surface. In this work comparable to those achieved for plasmid DNA-cationic we employed SPLP stabilized by PEG attached to ceralipid complexes. In vivo studies employing an intraperimide containing an octanoyl acyl group (PEG-CerC 8 ), toneal B16 tumor model and intraperitoneal administration which is able to quickly exchange out of the SPLP, to of SPLP also demonstrated maximum luciferase develop systems that give rise to optimized in vitro and in expression for DODAC contents of 20-24 mol% and sigvivo (regional) transfection. A particular objective was to nificantly improved gene expression in tumor tissue as achieve cationic lipid contents that give rise to maximum compared with complexes. We conclude that SPLP stabiltransfection levels. It is shown that by performing the dialyized by PEG-CerC 8 and containing 20-24 mol% cationic sis procedure in the presence of increasing concentrations lipid are attractive alternatives to plasmid DNA-cationic of citrate, SPLP containing up to 30 mol% of the cationic lipid complexes for regional gene therapy applications. lipid dioleoydimethylammonium chloride (DODAC) could
A previous study has shown that plasmid DNA can be encapsulated in lipid particles (SPLP, "stabilized plasmid lipid particles") of approximately 70 nm diameter composed of 1,2-dioleoyl-3-phosphatidyl-ethanolamine (DOPE), the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) conjugated to ceramide (PEG-Cer) using a detergent dialysis process (Wheeler et al. (1999) Gene Therapy 6, 271-281). In this work we evaluated the potential of these SPLPs as systemic gene therapy vectors, determining their pharmacokinetics and the biodistribution of the plasmid and lipid components. It is shown that the blood clearance and the biodistribution of the SPLPs can be modulated by varying the acyl chain length of the ceramide group used as lipid anchor for the PEG polymer. Circulation lifetimes observed for SPLPs with PEG-CerC14 and PEG-CerC20 were t(1/2) = approximately 1 and approximately 10 h, respectively. The SPLPs are stable while circulating in the blood and the encapsulated DNA is fully protected from degradation by serum nucleases. The accelerated clearance of SPLPs with PEG-CerC14 is accompanied by increased accumulation in liver and spleen as compared to PEG-CerC20 SPLPs. Delivery of intact plasmid to liver and spleen was detected. Significant accumulation (approximately 10% of injected dose) of the long circulating SPLPs with PEG-CerC20 in a distal tumor (Lewis lung tumor in the mouse flank) was observed following i.v. application and delivery of intact plasmid to tumor tissue at approximately 6% injected dose/g tissue is demonstrated.
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