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开发安全有效的疫苗是预防传染病爆发的有效手段。由于新型抗原的免疫原性弱，因此佐剂的开发成为了疫苗研发的重点之一。复合佐剂能够同时改善免疫强度和改变免疫类型，已成为疫苗佐剂研发的一大趋势。临床应用的乳液佐剂由于能够诱导良好的体液免疫获得了广泛关注，但不能胞内递送抗原，也无法诱导细胞免疫；而颗粒佐剂能有效被细胞内吞，能诱导良好的细胞免疫，但无法模拟天然病原体的柔性。因此，本论文提出将颗粒引入乳液佐剂中组成复合佐剂，经过合理设计可实现其细胞摄取和细胞内行为研究，从而实现颗粒-乳液复合佐剂的协同增效，并考察颗粒在乳液中的不同分布对免疫机制的影响。 论文具体开展的研究工作如下： 1.优化多种壳聚糖基颗粒的制备工艺，为颗粒-乳液复合佐剂研究提供多样化的颗粒材料。改进实验室的快速膜乳化-热固化技术、以及开发新型的单相凝胶成球技术，使用壳聚糖温敏水凝胶制备得到了微米级和亚微米级的壳聚糖基颗粒。开发均质-热固化技术、优化壳聚糖-TPP离子胶凝法、优化纳米沉淀法，利用分子间的离子相互作用，制备得到了纳米级的壳聚糖颗粒。对壳聚糖颗粒的粒径大小、均一性、表面电荷以及亲疏水性进行表征，为颗粒引入乳液中构建稳定的颗粒-乳液复合佐剂提供了多样化的颗粒材料。 2.将颗粒引入乳液内部，构建颗粒-乳液复合佐剂水包油包颗粒乳液（CSSE），其能模拟病原体的动态内吞过程，实现高效细胞摄取。CSSE乳液能够高效包埋颗粒和抗原。其与细胞膜接触时，外部应力和内部颗粒作用下发生明显形变，呈扁平状，从而增大与细胞膜的接触区域；同时颗粒在CSSE内吞过程中持续暴露以提供足够的摄取信号，从而显著增强抗原提呈细胞（antigen-presenting cells，APCs）的摄取及活化。作为口蹄疫（foot-and-mouth disease virus，FMDV）疫苗佐剂，与商品化疫苗ISA206相比，CSSE乳液能够显著增强体液免疫反应及细胞毒性T细胞（cytotoxic T lymphocyte，CTL）免疫反应，具有明显的佐剂节省效应，是一种有潜力的、安全有效的兽用疫苗佐剂。 3. 将颗粒引入乳液界面，构建颗粒-乳液复合佐剂Pickering乳液（CSPE），其具有胞内向高分子稳定乳液转变的特征，实现高效可控的溶酶体逃逸，具有优异的细胞免疫。由于Pickering乳液的柔性，CSPE乳液在细胞膜表面发生应力形变，模拟天然病原体的变形性，促进了细胞对疫苗的高效摄取及细胞活化。在溶酶体的酸性条件下，CSPE乳液界面的壳聚糖颗粒向壳聚糖分子链转变，壳聚糖分子上的氨基质子化，利用乳液界面对颗粒的空间限域效应，实现了CSPE乳液向分子链稳定的乳液转变，同时也实现了CSPE乳液的质子累积效应。通过调节乳液上质子数目，实现了对溶酶体逃逸效率的调控。最终在高效的溶酶体逃逸和分子链稳定的乳液作用下，显著提高了抗原在细胞质内的交叉递呈。CSPE乳液能够显著促进细胞免疫应答尤其是CTL免疫应答，诱导Th1型免疫偏向，在EG7-OVA和B16-MUC1的预防性及治疗性肿瘤模型中均能显著抑制肿瘤的生长和延长小鼠生存期。 4. 研究颗粒分布在乳液外-界面-内部对颗粒-乳液复合佐剂免疫机制及佐剂效应的影响。构建了由壳聚糖颗粒和角鲨烯乳液组成的颗粒-乳液复合佐剂，颗粒分别分布在乳液外部、乳液界面以及乳液内部。结果表明，颗粒分布在乳液内部和界面产生的抗原储库效应明显，APCs募集能力强，淋巴结协同递送能力强；注射部位的组织中，颗粒分布在乳液内部和界面分别分泌更多的Th2和Th1型细胞因子。结果表明，颗粒分布在乳液界面具有最佳的Th1型免疫偏向，颗粒分布在乳液内部具有最佳的Th2型免疫偏向，颗粒分布在乳液外部诱导的体液及细胞免疫效果均不及前两者。;Developing efficient and safe vaccines is an effective means of preventing outbreaks of infectious diseases. Due to the weak immunogenicity of the new antigens, the development of adjuvants has become one of the focuses of vaccine research and development. Compound adjuvants are promising adjuvants because they can simultaneously improve immune strength and change the type of immunity. Emulsion adjuvants for clinical applications have received widespread attention because they are able to induce considerable humoral immunity, but they cannot deliver antigens intracellularly or induce cellular immunity. Meanwhile, particle adjuvants can be effectively endocytosed by cells and induce favorable cellular immunity, but they don’t have the ability to simulate the deformability of natural pathogens. Therefore, this dissertation proposes to introduce particles into emulsions to form compound adjuvants. After reasonable design, they can realize the study of cell uptake and intracellular behavior, so as to achieve synergistic immune effects. After that, the influence of different distributions of particles in emulsion on the mechanism of action also can be investigated.Detailed work done in this dissertation are listed below:1. Multiple preparation processes of chitosan particles were optimized, providing diversified chitosan particles for investigating particle-emulsion compound adjuvants. We prepared micron and submicron particles employing the novel premix membrane emulsification technology and chitosan thermosensitive hydrogel. And nanoparticles were prepared by self-solidification, ionic-crosslinking, or nanoprecipitated method utilizing ionic interaction between molecules. Size, homogeneity, surface charge and hydrophilicity of chitosan particles were characterized. These particle candidates were then introduced into emulsion to construct stable particle-emulsion compound adjuvants.2. Constructing a novel particles-in-oil-in-water emulsion (CSSE), which was capable of simulating the dynamic endocytosis process of pathogens to achieve high uptake efficiency and eventually enhanced the immune response. Particles and antigens were embedded in the interior of emulsion. With the assistance of particles, CSSE exhibited amplified deformability and the vaccine-cell contact zone was increased. Additionally, its configurational transitions, which offered sustained exposure of sheltered uptake signals including antigens and stimulator chitosan during endocytosis, resulted in efficient antigen delivery to APCs and immunocyte activation. As a result, CSSE formulation robustly induced both humoral and cellular immunity against foot-and-mouth disease virus (FMDV), with an adjuvant-sparing effect, compared to the commercial adjuvant ISA206. These advantages prove CSSE to be a promising veterinary vaccine adjuvant with superior safety and effectiveness.3. Constructing special Pickering emulsion (CSPE) with the characteristic of changing from particles-stabilized emulsion to polymer-stabilized emulsion, which facilitated efficient and controllable lysosomal escape, thus inducing excellent cellular immunity. CSPE underwent mechanical deformation on the cell membrane, mimicking the deformability of natural pathogens, which was favorable for efficient uptake of vaccines and cells activation. Chitosan nanoparticles could convert to molecular chains and amino groups on them were protonated. CSPE imposed the spatial restriction effect of interface on the particles, which facilitated its conversion to polymer-stabilized emulsion and achieves proton accumulation effect simultaneously. By adjusting the number of protons, the regulation of lysosomal escape efficiency was realized. Eventually, by utilizing the efficient lysosomal escape and polymer-stabilized emulsion, the antigen cross-presentation was conspicuously ameliorated. In comparison with commercial adjuvants, the Pickering emulsion significantly promoted cellular immunity, especially the CTL immune response, with Th1-bias. As a result, Pickering emulsion showed excellent performance in EG7-OVA lymphoma and B16-MUC1 melanoma, resulting in a remarkable reduction in tumor growth and extension in survival time.4. Studying the effects of particle distributions in emulsion (outside / surface / inside) on the mechanism of action, and adjuvanticity. Three compound adjuvant formulations composed of chitosan particles and squalene emulsion were successfully constructed. The particles were distributed inside the emulsion droplets, on the surface, and over the water phase, respectively. It was concluded that distributing particles into the emulsion or on its surface induced significant antigen-depot effect, strong cells recruitment, and simultaneous delivery to lymph node. Robust Th2 or Th1 cytokines were secreted at the injection site induced by emulsions with particles distributed inside or on the surface, respectively. The results showed that particles in the emulsion droplets induced Th2 immune bias, and particles on the surface of emulsion resulted in Th1 bias. Meanwhile, emulsions with particles distributed outside induced weaker humoral and cellular immunity than the above two formulations.
|邹勇娟. 颗粒引入对传统乳液佐剂效应的影响[D]. 中国科学院大学,2020.|
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