Liposome-entrapped plasmid DNA: characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 mu g) encoding the S (small) region o f hepatitis B surface antigen was incorporated by the dehydration-rehydrati on method into liposomes composed of 16 mu mol egg phosphatidylcholine (PC) , 8 mu mol dioleoylphosphatidylcholine (DOPE) and 1,2-diodeoyl-3-(trimethyl ammonium)propane (DOTAP)(cationic liposomes) or phosphatidylglycerol (anion ic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA u sed, respectively. Mixing of preformed cationic liposomes with 100 mu g pla smid DNA also led to high complexation values of 73-97%. As expected, the a ssociation of DNA with preformed anionic liposomes was low (9%). Further wo rk with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential , fluorescent microscopy and gel electrophoresis in the presence of the ani on sodium dodecyl sulphate (SDS) indicate that, under the conditions employ ed, interaction of DNA with preformed cationic SUV as above, or with cation ic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent ), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing an ionic SDS molecules. However, dehydration of the DNA-SUV complexes and subs equent rehydration, generates submicron size liposomes incorporating most o f the DNA in a fashion that prevents DNA displacement through anion competi tion. It is suggested that, in this case, DNA is entrapped within the aqueo us compartments, in between bilayers, presumably bound to the cationic char ges. (C) 2000 Elsevier Science B.V. All rights reserved.