Effect of membrane composition and of co-encapsulation of immunostimulants in a liposome-entrapped crotoxin.

Crotoxin isolated from the venom of Crotalus durissus terrificus (South American rattlesnake) was incorporated into liposomes by the dehydration-rehydration vesicle method using different membrane compositions and the co-encapsulation of immunostimulants. Crotoxin was also encapsulated into liposomes formed from a non-phospholipid amphiphile, a mixture of polyoxyethylene 2-cetyl ether, dicetyl phosphate and cholesterol. The preparations were characterized in relation to stability, toxicity and the protection of mice against whole venom after immunization. All liposome preparations were quite stable, retaining more than 75% of the originally encapsulated crotoxin after 1 week of incubation at physiological temperature. Co-encapsulation with lipopolysaccharide increased the leakage of crotoxin. In contrast, co-encapsulation of the lipid moiety of lipopolysaccharide did not influence the stability of liposomes. Toxicity of liposomes was dependent on membrane composition. Liposomes made with phospholipids that were resistant to phospholipase A(2) activity were less toxic. Mice immunized with three doses of the 1 x LD50 of crotoxin encapsulated into liposomes, and with associated immunostimulants, were protected against challenge with 8 x subcutaneous LD50 of C. durissus terrificus venom. Using the same immunization schedule, liposomes made from a non-phospholipid mixture and without immunostimulants achieved 100% protection.