Laboratório de Microesferas e Lipossomas, Centro de Biotecnologia, I. Butantan, São Paulo, Brasil.
J Liposome Res. 2011 Jun;21(2):116-23. doi: 10.3109/08982104.2010.491072. Epub 2010 Jun 4.
Chitosan (α-(1-4)-amino-2-deoxy-β-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.
壳聚糖（α-（1-4）-氨基-2-脱氧-β-D-葡聚糖）是甲壳素的脱乙酰形式，甲壳素是一种来自甲壳类动物外壳的多糖。其独特的特性，如正电荷、可生物降解性、生物相容性、无毒性和刚性结构，使其成为口服疫苗传递系统的理想选择。我们制备了壳聚糖（Chi）和聚乙烯醇（PVA）夹心的反相蒸发囊泡（REV）。然而，在这种方法中，仍然存在一些与蛋白质稳定化相关的问题需要解决。在蛋白质纳米封装的反胶束相期间，疏水面扩展，导致界面吸附，随后是蛋白质展开和聚集。在这里，使用光谱和免疫学技术来确定霍夫曼系列离子对二联类毒素（Dtxd）在反胶束相期间稳定性的影响。在水溶液中使用的盐与 Dtxd 溶解度和构象的变化之间建立了相关性。Dtxd 的α-螺旋含量相当稳定，这使我们得出结论，封装发生时没有蛋白质聚集或没有疏水性残基暴露。亲盐性 PO(2-)(4) 避免了 98%的 Dtxd 聚集。该离子用于在存在 50 mM PO(2-)(4)的情况下制备稳定的 Dtxd 和免疫识别的 REV-Chi-PVA 制剂。在这些条件下，Dtxd 保留了其免疫学特性。因此，在接触 CH(3)CO(2)C(2)H(5)开始其纳米封装后，我们可以在理想条件下获得最大的 Dtxd 溶解度和稳定性。这是一项技术突破，因为简单的盐添加溶液避免了异源蛋白质的使用。
