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 400n 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.

中文翻译：

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用于传统 VIT-Venom 免疫疗法的现代脂质体和蜂毒制剂的设计

传统的毒液免疫疗法使用在缓冲液中或吸附在 Al (OH)3 中的完整蜂毒注射，这种方式既昂贵又耗时。因此，通过减少注射来提高这种治疗的安全性和有效性的新策略将受到普遍关注。它将提高患者的依从性并提供社会经济效益。脂质体在药物递送方面有着悠久的传统，因为它们增加了治疗指数并避免了药物降解和继发效应。然而，蜂毒蜂毒肽 (Mel) 和磷脂酶 (PLA2) 会破坏磷脂膜。我们的中心思想是通过组氨酸烷基化和/或色氨酸氧化（分别使用 pbb、对溴苯甲酰溴和/或 NBS-N-溴代琥珀酰亚胺）抑制 PLA2 和 Mel 活性，使它们在稳定的脂质体中封装成为可能。我们坚信该配方无毒但具有免疫原性。在本文中，我们通过紫外线、圆二色性和荧光光谱法展示了化学修饰期间和之后以及与脂质体相互作用之后的整个蜂毒构象表征。PLA2 和 Mel 活性通过 400nm 处的浊度变化、罗丹明渗漏和溶血间接测量。天然全蜂毒 (BV) 呈现 78.06% 的 α-螺旋含量。BV 的烷基化 (A-BV) 和琥珀酰化 (S-BV) 增加了其 α-螺旋含量的 0.44% 和 0.20%。双修饰毒液（SA-BV）的α-螺旋含量增加了0.74%。BV 化学修饰诱导了 Trp 观察到的蛋白质构象的另一个变化，该变化相对于天然完整 BV 被掩埋。证明脂质体膜必须含有 pbb (SPC:Cho:pbb, 26:7:1) 作为成分以保护它们免于聚集和/或融合。与改性毒液孵育后，含有 pbb 的膜保持相同的浊度 (100%)，而不含 pbb 的膜则显示出 15% 的尺寸减小。这种尺寸减小被解释为膜降解，并被 50% 罗丹明泄漏证实。证实我们的解释的另一个事实是观察到在用 pbb 和 NBS (SA-BV) 进行毒液修饰后对溶血活性的 100% 抑制。当 SA-BV 与脂质体相互作用时，观察到其他蛋白质构象变化，其特征是 SA-BV α-螺旋含量增加了 1.93%，并且在更疏水的环境中存在色氨酸残基。换句话说，SA-BV 与脂质体膜相互作用，但这种相互作用不能有效地引起聚集、漏出或融合。设计了一种由 SA-BV 组成的稳定制剂，封装在由 SPC:Cho:pbb 组成的脂质体中，比例为 26:7:1。具有负表面电荷 (–24.29 mV) 的 202.5 nm 大单层囊泡封装了 95% 的 SA-BV。现在，可以在 VIT 上分析该配方。