Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity

Citation
Eg. Saravolac et al., Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity, J DRUG TAR, 7(6), 2000, pp. 423-437
Citations number
38
Categorie Soggetti
Pharmacology & Toxicology
Journal title
JOURNAL OF DRUG TARGETING
ISSN journal
1061186X → ACNP
Volume
7
Issue
6
Year of publication
2000
Pages
423 - 437
Database
ISI
SICI code
1061-186X(2000)7:6<423:EOPDIS>2.0.ZU;2-1
Abstract
In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have sh own that plasmid DNA can be entrapped in "stabilized plasmid-lipid particle s" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lip id dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anch or containing an arachidoyl acyl group (PEG-CerC(20)). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lip id at a 1 : 1 lipid ratio, The objective of this study was to construct SPL Ps with increased cationic lipid contents that result in maximum transfecti on levels. A phosphate buffer detergent dialysis technique is described res ulting 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 a nchor for the PEG moiety (PEG-CerC(8)) permitting a quick exchange out of t he SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either lineariz ed 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 d etermined by cryo-electron microscopy. The high plasmid-to-lipid ratio obse rved corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microsco py. SPLP containing a range of DODAC concentrations were tested for in vitr o and in vivo transfection. In vitro. in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC c oncentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasi ng the cationic lipid concentration improved transfection in part due to de creased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection acti vity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor c ells was increased by approximately one magnitude as compared to cationic l ipid: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-ye ar period. Thus this phosphate buffer detergent dialysis technique can be u sed to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.