Stabilized plasmid-lipid particles: factors influencing plasmid entrapmentand transfection properties

Previous work has shown that plasmid DNA can be encapsulated in small 'stab ilized plasmid-lipid particles' (SPLP) composed of 1,2-dioleyl-3-phosphatid ylethanolamine (DOPE), the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) (PEG) conjugated ceramides (PEG- Cer), employing a detergent dialysis procedure. These SPLP have potential a s vectors for in vivo gene therapy. This study is aimed at characterizing t he influence of the cationic lipid and PEG-Cer species on SPLP formation an d in vitro transfection properties. It is shown that the transfection poten cy of SPLP is sensitive to the cationic lipid species employed, the size of the PEG polymer incorporated in the PEG-ceramide and the length of the acy l chain contained in the ceramide anchor. With regard to the influence of c ationic lipid, the transfection levels achieved were highest for SPLP conta ining N-[2,3-(dioleyloxy)propyl]-N,N-dimethyl-N-cyanomethylammonium chlorid e (DODMA-AN) and lowest for SPLP containing 3-beta-[N-(N',N'-dimethylaminoe thyl)carbamoyl]-cholesterol (DC-CHOL), according to the series DODMA-AN > N -[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)>DODAC >N ,N-distearyl-N,N-dimethylammonium chloride (DSDAC)>DC-CHOL. Incorporation o f short (PEG(750)) PEC polymers in the PEG-ceramide components resulted in modest improvements in transfection levels over PEG(2000) and PEG(5000) pol ymers, however variation of the length of the acyl chain contained in the h ydrophobic ceramide anchor from octanoyl (PEG-CerC(8)) to myristoyl (PEG-Ce rC(14)) to arachidoyl (PEG-CerC(20)) had the most dramatic effects. Transfe ction levels achieved for SPLP containing PEG-CerC(8) were substantially la rger than observed for SPLP containing PEG-CerC(14) or PEG-CerC(20), consis tent with a requirement for the PEG-ceramide to dissociate from the SPLP su rface for maximum transfection potency. It is also shown that the ability o f SPLP to be accumulated into cells is a dominant factor influencing transf ection potency, and that the transfection potency of SPLP that are accumula ted is at least equivalent to that of cationic lipid-plasmid DNA complexes. (C) 1999 Elsevier Science B.V. All rights reserved.