In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid
DNA can be entrapped in "stabilized plasmid
lipid particles" (SPLP) using low levels (5-10 mol%) of cationic
lipid, the fusogenic
lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a
ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic
lipid complexes formed with
liposomes composed of cationic and neutral
lipid at a 1:1
lipid ratio. The objective of this study was to construct SPLPs with increased cationic
lipid contents that result in maximum transfection levels. A
phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol%
DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized
DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by
sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-
lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a
lipid bilayer surrounding the plasmid
DNA as visualized by cryo-electron microscopy. SPLP containing a range of
DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the
DODAC concentration in the SPLP was decreased from 42.5 to 24 mol%
DODAC. Decreasing the cationic
lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16
tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol%
DODAC. Gene expression observed in
tumor cells was increased by approximately one magnitude as compared to cationic
lipid/
DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this
phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic
lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.