Physicochemical properties and in vitro toxicity of cationic liposome cDNA complexes.
The purpose of this study was to elucidate the interaction of cationic liposomes and plasmid cDNA by examining their ultrastructure, zeta potential, stability in aqueous media and protection from DNaseI digestion; their potential for hemolysis and platelet aggregation was evaluated as it may serve as an in vitro toxicity screen. Liposomes consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and dioleylphosphatidylethanolamine (DOPE) were complexed with plasmid constructs of ovine prostaglandin G/H synthase (pCMV4-PGH) or human alpha 1-antitrypsin (pCMV4-AAT) at lipid:plasmid (L/P) ratios of 3:1-8:1 (w/w). The electron micrographs showed bead-like attachment of liposomes to cDNA and coating of plasmid strands. The zeta potential showed isoelectric points at L/P ratios of 3.5-4 (DOTMA/DOPE) and 5.5-6.5, corresponding to a pKa of 6.45 (DC-Chol/DOPE). Liposome cDNA complexes were stable in water, saline and 5% dextrose for 48 h, but precipitated instantaneously in PBS. An increase in the L/P ratio corresponded with increased protection from DNaseI digestion. DOTMA/DOPE liposomes alone were highly hemolytic and DC-Chol/DOPE liposomes moderately hemolytic; hemolysis was abolished by cDNA complexation, with the exception of very high (> or = 7:1) L/P ratios. Both liposomes alone and cDNA complexes caused transient serum turbidity, while none caused platelet aggregation. It was concluded that current cationic lipid cDNA formulations are metastable and appear to have very little if any toxicity with respect to hemolytic potential and untoward interaction with other blood components.
Schreier
H
H
Advanced Therapies Inc., Novato, CA 94949, USA. mlsb86a@prodigy.com
Gagné
L
L
Bock
T
T
Erdos
G W
GW
Druzgala
P
P
Conary
J T
JT
Müller
B W
BW
D018076:DNA, Complementary / Q000008:administration & dosage* / Q000737:chemistry / Q000633:toxicity; D003851:Deoxyribonucleases / Q000494:pharmacology; D004337:Drug Carriers; D004355:Drug Stability; D015316:Genetic Therapy; D006801:Humans; D008081:Liposomes; D008854:Microscopy, Electron; D010957:Plasmids; D010974:Platelet Aggregation / Q000187:drug effects
D018076:DNA, Complementary; D004337:Drug Carriers; D008081:Liposomes; D003851:Deoxyribonucleases