Encapsulation of native crotoxin in liposomes: A safe approach for the production of antivenom and vaccination against Crotalus durissus terrificus venom

Abstract

Crotoxin, the neurotoxic component of Crotalus durissus terrificus (Cdt) venom that displays phospholipase A 2 activity, was successfully encapsulated into dehydration-rehydration vesicles (DRV/crotoxin) and reverse-phase evaporation vesicles (REV/crotoxin) made from sphingomyelin and cholesterol. The encapsulation efficiency of native crotoxin was higher in DRV/crotoxin than in REV/crotoxin. DRV/crotoxin was not toxic when i.v. inoculated in mice at a dose of crotoxin as high as 91 times its ld 50 or when s.c. inoculated at 42 times its ld 50. On the other hand, crotoxin released from DRV/crotoxin retained its original toxicity. REV/crotoxin was found to be at least 1.9 times more toxic than DRV/crotoxin. The fact that DRV/crotoxin retained crotoxin more efficiently than REV/crotoxin may account for the difference in acute toxicity between the two preparations. DRV/crotoxin, when s.c. inoculated in mice, induced anti-crotoxin antibodies that protected animals against the lethal effect of Cdt venom. Following immunization with three doses of DRV/crotoxin (3 × 20 μg of crotoxin/mouse) and challenge with 8 × ld 50 of Cdt venom, 75% of mice were protected. The DRV/crotoxin preparation was compared to crotoxin emulsified in Freund's adjuvant (FCA/crotoxin). DRV/crotoxin was found to be less toxic than FCA/crotoxin, and to induce lower levels of anti-crotoxin antibodies but similar levels of protection when inoculated at high doses (20 or 70 μg crotoxin/mouse). When DRV/crotoxin was adsorbed to alum at the time of immunization, it induced antibody and protection levels comparable to those produced by FCA/crotoxin.