| 摘要: | 這幾年因為病毒的多變，例如肆虐將近三年的新冠病毒，或最近正在流行的猴痘病毒，導致人類的健康產生越來越多的危害。所以快速開發新又有效的疫苗，並提高現有的疫苗針對特定疾病的功效，變成了是一種與時間賽跑的挑戰。
褐藻醣膠（Fucoidan）近年來已被研究證實，具有抑制腫瘤功效，也能活化巨噬細胞、B細胞、T細胞及自然殺手細胞（NK Cell）等免疫細胞的活性，提升人體的免疫能力，也被研究出具有減輕癌症治療帶來的副作用的潛力。聚乙烯亞胺（polyetherimide，PEI）是一種陽離子聚合物，其本身帶正電荷的特性，能當作一種佐劑，可與帶負電的蛋白質或生醫材料形成奈米粒子。其本身的電位，可提高與帶負電荷的細胞膜結合的機率，提升奈米粒子被細胞吞噬之機會。卵清白蛋白（ovalbumin，OVA），是一種模式蛋白，目前廣泛運用在免疫學研究上，可用來研究蛋白質所引發的各種免疫反應。
本篇實驗的設計，是利用帶正電荷的聚乙烯亞胺（polyetherimide，PEI）與帶負電荷的褐藻醣膠以靜電交互作用力，組成奈米顆粒，作為包覆蛋白質之載體。本研究製備了兩種奈米粒子OVA@PEI_NPs與OVA@Fucoidan-PEI_NPs (OVA@Fu-PEI_NPs)，經實驗結果證明，兩者皆可有效的包覆OVA，且加入褐藻醣膠的奈米顆粒，更有效的提高包覆效率，達到了80.42%。所製備出來的奈米粒子大小平均約250nm，可有效的被樹突細胞所吞噬。而體外細胞實驗證實，此奈米粒子對於細胞來說是相當安全的。另外，將奈米粒子與樹突細胞株培養，可測出細胞激素TNF-α 和 IL-6 的濃度上升。動物實驗證明，所製備的奈米粒子在小鼠體內不會造成重要器官組織細胞的異常。在血清抗體反應的實驗中，小鼠體內的anti-OVA IgG與IgG1的濃度，在施打奈米粒子之後，其濃度比鋁鹽做為佐劑的表現更好。將奈米粒子免疫過的小鼠脾臟細胞，與OVA培養刺激後，可以使OVA特異性淋巴細胞增殖。實驗證實新開發的OVA@Fu-PEI_NPs的奈米顆粒可有效的被樹突細胞吞噬，活化樹突細胞在動物體內引發抗原特異性抗體反應。本論文所開發的奈米粒子可應用在更多不同類型的疫苗開發，是相當具有研究與發展價值的。
本篇實驗的設計，是利用帶正電荷的聚乙烯亞胺（polyetherimide，PEI）與帶負電荷的褐藻醣膠以靜電交互作用力，組成奈米顆粒，作為包覆蛋白質之載體。本研究製備了兩種奈米粒子OVA@PEI_NPs與OVA@Fucoidan-PEI_NPs (OVA@Fu-PEI_NPs)，經實驗結果證明，兩者皆可有效的包覆OVA，且加入褐藻醣膠的奈米顆粒，更有效的提高包覆效率，達到了80.42%。所製備出來的奈米粒子大小平均約250nm，可有效的被樹突細胞所吞噬。而體外細胞實驗證實，此奈米粒子對於細胞來說是相當安全的。另外，將奈米粒子與樹突細胞株培養，可測出細胞激素TNF-α 和 IL-6 的濃度上升。動物實驗證明，所製備的奈米粒子在小鼠體內不會造成重要器官組織細胞的異常。在血清抗體反應的實驗中，小鼠體內的anti-OVA IgG與IgG1的濃度，在施打奈米粒子之後，其濃度比鋁鹽做為佐劑的表現更好。將奈米粒子免疫過的小鼠脾臟細胞，與OVA培養刺激後，可以使OVA特異性淋巴細胞增殖。實驗證實新開發的OVA@Fu-PEI_NPs的奈米顆粒可有效的被樹突細胞吞噬，活化樹突細胞在動物體內引發抗原特異性抗體反應。本論文所開發的奈米粒子可應用在更多不同類型的疫苗開發，是相當具有研究與發展價值的。
In recent years, due to the variability of viruses, such as the widespread COVID-19 caused by the novel coronavirus for nearly three years and the recent outbreak of monkeypox, human health has been increasingly threatened. Therefore, the rapid development of new and effective vaccines and the improvement of existing vaccines' effectiveness against specific diseases have become a race against time.
For the past few years, fucoidan has been studied and proven to have tumor-inhibiting effects. It can activate immune cells such as macrophages, B cells, T cells, and natural killer cells (NK cells), enhancing the body's immune capabilities. Moreover, it has been researched for its potential in reducing the side effects of cancer treatments. Polyetherimide (PEI) is a cationic polymer known for its positive charge characteristics. It can be used as an adjuvant to form nanoparticles with negatively charged proteins or biomaterials. The positive charge of PEI can increase the probability of binding with negatively charged cell membranes, thereby enhancing the chances of nanoparticle uptake by cells. Ovalbumin (OVA) is a model protein widely used in immunological research to study various immune responses triggered by proteins.
The design of this experiment involved the use of positively charged polyetherimide (PEI) and negatively charged fucoidan to form nanoparticles through electrostatic interactions. These nanoparticles were used as carriers for protein encapsulation. Two types of nanoparticles, OVA@PEI_NPs and OVA@Fucoidan-PEI_NPs, were prepared in this study. The experimental results demonstrated that both OVA@PEI_NPs and OVA@Fucoidan-PEI_NPs effectively encapsulated OVA. Importantly, the addition of fucoidan in the nanoparticles significantly improved the encapsulation efficiency, reaching 80.42 %. The average size of the prepared nanoparticles was approximately 250 nm, making them suitable for uptake by dendritic cells. The in vitro study confirmed the safety of these nanoparticles.
Furthermore, co-culturing nanoparticles with dendritic cell lines resulted in an increase in the concentrations of cytokines, TNF-α and IL-6, released by the cells. Animal experiments demonstrated that the prepared nanoparticles did not cause abnormalities in the tissue sections collected from major organs. In serum antibody response experiments, the concentrations of anti-OVA IgG and IgG1 in serum collected from mice that received the administration of the nanoparticles showed better performance compared to those that received OVA mixed with aluminum salts (OVA@Alum), a widely used adjuvant. Finally, the splenocytes from mice immunized with the OVA@Fucoidan-PEI_NPs, when restimulated with OVA in vitro, exhibited a higher proliferation index compared to those collected from mice that received OVA@Alum.
All in all, various experiments have demonstrated that the newly developed OVA@Fucoidan-PEI_NPs can be efficiently phagocytosed by dendritic cells, effectively activating dendritic cells, and subsequently inducing antigen-specific antibody responses in the animal body. The nanoparticles developed in this study have the potential to be applied in the development of vaccines for different types, making them highly valuable for research and development. |
