RESUMO
Chitosan (α-(1-4)-amino-2-deoxy-ß-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, PO(2-)(4). This ion was used to prepare a stable Dtxd and immunologically recognized REV-Chi-PVA formulation in the presence of 50 mM of PO(2-)(4). Under these conditions, the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH(3)CO(2)C(2)H(5) to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough, because a simple solution, such as salt, addition avoided heterologous protein use.
Assuntos
Quitosana/química , Toxoide Diftérico/química , Composição de Medicamentos/métodos , Lipossomos/química , Conformação Molecular , Acetatos/química , Administração Oral , Adsorção , Animais , Toxoide Diftérico/imunologia , Toxoide Diftérico/metabolismo , Ensaio de Imunoadsorção Enzimática , Cavalos , Íons , Lipossomos/imunologia , Lipossomos/metabolismo , Micelas , Tamanho da Partícula , Fosfatos/química , Álcool de Polivinil/química , Estabilidade Proteica , Desdobramento de Proteína , Sais , Vacinas/química , Vacinas/imunologiaRESUMO
Microspheres of polymers like poly(lactic-co-glycolic acid) (PLGA) have been studied as a vehicle for controlled release vaccines. They require materials and processes that might change the protein antigenicity. Lactic acid is produced during microsphere degradation that occurs in tandem with protein liberation. In addition, most of the proteins that have been used in microencapsulation studies contain Thimerosal((R))(TM) and this can introduce another undesirable effect for their stability. We demonstrated in vitro that the thiosalycilic acid (TSA), produced after the reduction of TM by lactic acid, reduces the S-S bridge of the previously incubated diphtheric toxoid (Dtxd). This reduction is immediately followed by blocking the two -SH formed by the same TSA molecules. In the light of these conclusions it is necessary now, to reinterpret the in vitro protein degradation-stabilization data in the presence of PLGA microspheres, mainly for those proteins which contain S-S. We propose that all the PLGA microspheres microencapsulation studies and protein structural considerations should be done in the absence of TM as preservative.
Assuntos
Toxoide Diftérico/química , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Conservantes Farmacêuticos/química , Timerosal/química , Portadores de Fármacos/química , Interações Medicamentosas , Estabilidade de Medicamentos , Microesferas , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, by adding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of Q-Sepharose previously equilibrated with 500 mM Tris, pH 5.0-9.0 (increments of 0.5 pH units). The best condition for the Dtxd monomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300 mM NaCl. After studying the gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500 mM Tris, pH 9.0, was prepared. The purification factor for Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a very realistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with "toxoid highly purified toxin" is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts of European Pharmacopoeia to recommend its use both for children and adult vaccination.
Assuntos
Toxoide Diftérico/química , Íons , Adsorção , Cromatografia Líquida de Alta Pressão/métodos , Toxoide Diftérico/isolamento & purificação , Concentração de Íons de Hidrogênio , Sefarose/química , Cloreto de Sódio/metabolismoRESUMO
Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.