| Texto completo | |
| Autor(es): |
Silva, Tatiana C.
[1]
;
Moura, Sergio De Paula
;
Ramos, Henrique R.
[2, 3]
;
De Araujo, Pedro S.
[2]
;
Bueno Da Costa, Maria H.
[4]
Número total de Autores: 5
|
| Afiliação do(s) autor(es): | [1] Univ Fed Sao Paulo, Disciplina Clin Med, Dept Med, Sao Paulo - Brazil
[2] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-09500900 Sao Paulo - Brazil
[3] Inst Butantan, Ctr Biotechnol, Mol Biotechnol Lab, Sao Paulo - Brazil
[4] Inst Butantan, Lab Microesferas & Lipossomas, BR-05503900 Sao Paulo - Brazil
Número total de Afiliações: 4
|
| Tipo de documento: | Artigo Científico |
| Fonte: | Journal of Liposome Research; v. 18, n. 4, p. 353-368, 2008. |
| Citações Web of Science: | 1 |
| Resumo | |
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 (PLA(2)) destroy the phospholipid membranes. Our central idea was to inhibit the PLA(2) and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBSN-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 PLA(2) and Mel activities were, measured indirectly by changes in turbidity at 400(nm), rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06% of alpha-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20% of its alpha-helical content. The double-modified venom (S-A-BV) had a 0.74% increase of alpha-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 alpha-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. (AU) | |
| Processo FAPESP: | 06/04088-6 - Desafio farmacotecnológico para microencapsulação de melitina em lipossomas |
| Beneficiário: | Sérgio de Paula Moura |
| Modalidade de apoio: | Bolsas no Brasil - Pós-Doutorado |
| Processo FAPESP: | 07/05466-7 - Design of a modern liposome and bee venom formulation for the traditional VIT - venom immunotherapy |
| Beneficiário: | Maria Helena Bueno da Costa |
| Modalidade de apoio: | Auxílio à Pesquisa - Reunião - Exterior |
| Processo FAPESP: | 00/14228-3 - Lipossomos e outros complexos lipídicos no estudo de vetorização de vacinas |
| Beneficiário: | Maria Helena Bueno da Costa |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |
| Processo FAPESP: | 02/07293-9 - Microencapsulação de venenos de animais peçonhentos em lipossomos estabilizados de fosfolipídeos naturais |
| Beneficiário: | Pedro Soares de Araujo |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa de Apoio à Propriedade Intelectual (PAPI/Nuplitec) |
| Processo FAPESP: | 05/04514-2 - Mecanismo de interação de melitina com fosfolipídeos no contexto de microencapsulações em lipossomas ou interação melitina/lipídeos |
| Beneficiário: | Maria Helena Bueno da Costa |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |