Encapsulation of Plasmid DNA in Stabilized Plasmid – Lipid Particles Composed of Different Cationic Lipid Concentration for Optimal Transfection Activity

Abstract

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.

Very Long Chain N 4 ,N 9 -Diacyl Spermines: Non-Viral Lipopolyamine Vectors for Efficient Plasmid DNA and siRNA Delivery

“…Lindner and coworkers reported the use of very long chain lipids e.g. erucoyl and compared them with other shorter chains in their designed cationic lipids (67), while Cullis and co-workers have studied the variation of the length of the acyl chains contained in the hydrophobic anchor from octanoyl to myristoyl to arachidoyl (68)(69)(70). Using confocal laser scanning microscopy with one labelling solution (Invitrogen) containing both Alexa Fluor 594 wheat germ agglutinin (5 μg/ml) for cell membrane labelling, and Hoechst 33342 (2 μΜ) for nuclei labelling, we have shown (Fig.…”

Targeting of stabilized plasmid lipid particles to hepatocytesin vivoby means of coupled lactoferrin

“…Although targeting to the hepatocytes was very efficient, no significant transfection was observed, neither in hepatocytes nor in any other cell type in the liver or in any organ for that matter. Transfection in vivo with untargeted SPLPs of B16 melanoma cells in mice has been reported by Saravolac et al (2000). The B16 cells are tumor cells, which are rapidly proliferating cells, and, therefore, more prone to transfection.…”

Cell-Specific Targeting of Lipid-Based Carriers for ODN and DNA

“…Moreover, in vivo gene expression was observed upon intraperitoneal injection of SPLPs in mice inoculated intraperitoneally with B16 tumor cells (Saravolac et al, 2000;Zhang et al, 1999).…”