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