Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Abstract

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)₃ in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA₂) destroy the phospholipid membranes. Our central idea was to inhibit the PLA₂ and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS- N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA₂ and Mel activities were measured indirectly by changes in turbidity at 400_{n m}, rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of α-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its α-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of α-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15% size decrease. This size decrease was interpreted as membrane degradation and was corroborated by a 50% rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100% inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93% on S-A-BV α-helical content and the presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (–24.29 mV) encapsulated 95% of S-A-BV. This formulation can, now, be assayed on VIT.

Keywords:

• liposome
• stabilization of liposomes
• drug delivery
• bee venom immunotherapy

Acknowledgments

FAPESP (2007/05466-7, 2005/04514-2, 2002/07293-9, 2000/14228-3), CNPq (300648/2005-7) and Fundação Butantan. S.P.M. receives a postdoctoral fellowship from FAPESP (06/04088-6), H.R.R. receives a PhD fellowship from FAPESP (03/06011-2), and T.C.S. has a master fellowship from CAPES.