次單位疫苗因其良好的生物安全性以及較低的大規模生產成本一直是疫苗應用開發的熱門候選，而微針做為一種藥物傳輸裝置其經皮給藥的特性使其能與疫苗接種途徑中理論效果最好的皮內給藥完美契合，本研究在此之上加入了金奈米疫苗作為中介點，結合其奈米載體以及免疫佐劑的特性進一步最佳化疫苗的傳遞。本研究分為兩大部分:第一部分著重金奈米—卵清蛋白奈米載體疫苗之開發、基礎性質鑑定以及免疫功能之評估，第二部分則整合金奈米載體疫苗與幾丁聚醣(具免疫佐劑特性的天然高分子)微針實際驗證該雙佐劑免疫增強策略。
第一部份:次單位疫苗常受限於較弱的免疫原性以及在製劑加工過程(如乾燥、濃縮)中的不穩定性。本研究使用金奈米粒子(GNP)開發一種兼具結構穩定性與自佐劑特性(Self-adjuvanticity）的奈米載體平台，以作為後續微針遞送系統的核心元件。利用檸檬酸還原法合成，並透過硫醇-聚乙二醇-羧酸(HS-PEG-COOH)進行表面修飾，隨後，利用化學接枝方式將模型抗原卵清蛋白(Ovalbumin, OVA)共價接枝於GNP表面，形成GNP-OVA奈米載體疫苗。經過表面修飾與抗原接枝後，奈米粒子的水合粒徑由的13.8 nm增加至約28.6 nm，且表面電位由強負電轉趨中性，證實了表面修飾的成功。實驗證實 PEG層所提供的立體障礙賦予了該奈米載體疫苗極佳的膠體穩定性，使其在未添加額外冷凍保護劑的情況下，仍能抵抗凍乾製程中的脫水應力與離子強度變化等物理影響，回溶後不發生聚集，此特性確立了其適用於微針製程的可行性。細胞實驗結果顯示GNP-OVA具有良好的生物相容性，且相較於物理混合劑型，化學接枝策略能顯著提升抗原呈現細胞的攝取效率(6.4倍的平均螢光強度)，並有效促進免疫呈現分子MHC-II的上調。活體動物實驗進一步證實，於小鼠皮下注射GNP-OVA能誘導出顯著優於純抗原及物理混合組的抗原特異性IgG效價，且具備良好的免疫記憶效應。
第二部份:本章節整合奈米載體疫苗的免疫增強功能與微針的經皮傳輸特性進行雙重佐劑免疫增強策略的實際開發，將GNP-OVA奈米疫苗負載於具備免疫刺激特性的幾丁聚醣微針中，透過材料功能的互補，達成從細胞層級的精準遞送到組織層級的免疫環境重塑。研究首先探討奈米載體的抗原接枝比例(O/G ratio)對細胞之影響，發現O/G=1的配方能最大程度地促進樹突細胞(DC2.4)表面共刺激分子CD86與MHC-II的表現。活體影像系統(IVIS)追蹤顯示，GNP-OVA 能有效促進抗原向淋巴結的引流與累積。本研究利用壓模成型法製備出具有聚乳酸(PLA)支撐基座的GNP-OVA/幾丁聚醣複合微針，機械強度測試顯示其單根微針之耐受力達1.1 N，且在活體大鼠皮膚上的平均穿刺深度為897.9 ± 96.6 μm (n = 5)，確保疫苗能精確遞送至真皮層。活體釋放追蹤顯示，幾丁聚醣微針能作為抗原儲存庫在皮膚內提供長達 28 天以上持續性的抗原釋放。組織切片染色進一步揭示，微針的植入能主動招募CD86+免疫細胞聚集於注射部位，形成有利於抗原捕獲的發炎微環境。動物免疫實驗證實，此雙重佐劑微針平台所誘導的IgG 抗體效價顯著優於傳統皮下注射，並能同時促進Th1與Th2的免疫應答。
本研究成功建構了一套整合「微觀奈米載體」與「巨觀微針基質」的複合式經皮疫苗平台，透過幾丁聚醣微針的抗原儲庫、內源性免疫刺激等特性與金奈米載體的尺寸效應、仿病毒結構的結合，本研究確立了雙重佐劑機制自巨觀至微觀的結合，不僅實現了抗原的穩定封裝與長效緩釋，更證實能有效調控免疫細胞活化，誘導出強效的免疫應答。此模組化載體平台具備高度的可擴展性，除了現有的模型抗原外，未來可進一步應用於癌症免疫治療或流感等需誘導強烈免疫反應之疫苗開發。

Subunit vaccines have emerged as promising candidates for vaccine development due to their superior biosafety and cost-effective scalability. Meanwhile, microneedles serve as an ideal drug delivery system, enabling transdermal administration that aligns perfectly with intradermal vaccination, which is theoretically the most efficient inoculation route. Based on this foundation, this study incorporates gold nanovaccines as a pivotal intermediary, leveraging their properties as both nanocarriers and immune adjuvants to further optimize vaccine delivery. The research is divided into two main parts: Part I focuses on the development, physicochemical characterization, and immunological function evaluation of gold nanoparticle-ovalbumin nanovaccines; Part II integrates these nanocarriers with chitosan microneedles (a natural polymer with immunostimulatory properties) to practically validate this dual-adjuvant immune enhancement strategy.
Part I: Subunit vaccines are often limited by their weak immunogenicity and instability during processing steps such as drying and concentration. In this study, gold nanoparticles (GNPs) were utilized to develop a nanocarrier platform featuring both structural stability and self-adjuvanticity, serving as the core component for the subsequent microneedle system. The GNPs were synthesized via citrate reduction, surface-modified with Thiol-PEG-Carboxyl (HS-PEG-COOH), and covalently conjugated with the model antigen ovalbumin (OVA) to form the GNP-OVA nanovaccine. Following modification and grafting, the hydrodynamic diameter increased from 13.8 nm to approximately 28.6 nm, while the surface potential shifted from strongly negative to near-neutral, confirming successful functionalization. Experiments demonstrated that the steric stabilization provided by the PEG layer endowed the nanovaccine with exceptional colloidal stability. This allowed it to withstand physical stresses—such as dehydration and ionic strength fluctuations—during freeze-drying without the need for additional cryoprotectants, ensuring no aggregation occurred upon reconstitution. This characteristic established its suitability for microneedle fabrication. Cellular assays indicated that GNP-OVA exhibited high biocompatibility; moreover, the chemical grafting strategy significantly enhanced uptake efficiency by antigen-presenting cells (6.4-fold higher mean fluorescence intensity compared to physical mixtures) and effectively promoted the upregulation of MHC-II molecules. In vivo animal studies further confirmed that subcutaneous injection of GNP-OVA in mice induced antigen-specific IgG titers significantly superior to those of pure antigen or physical mixtures, while establishing a robust immune memory effect.
Part II: This section focuses on the practical application of a dual-adjuvant immune enhancement strategy by integrating the immunogenic nanocarriers with the transdermal capabilities of microneedles. The GNP-OVA nanovaccine was loaded into chitosan microneedles, which possess intrinsic immunostimulatory properties. This combination leverages material complementarity to achieve precise delivery at the cellular level and immune environment remodeling at the tissue level. Investigation of the antigen grafting ratio (O/G ratio) revealed that an O/G ratio of 1 maximally promoted the expression of co-stimulatory molecules CD86 and MHC-II on dendritic cells (DC2.4). In vivo imaging system (IVIS) tracking showed that GNP-OVA effectively facilitated antigen drainage and accumulation in lymph nodes. Using a compression molding method, GNP-OVA/chitosan composite microneedles were prepared on a polylactic acid (PLA) supporting base. Mechanical testing indicated a withstand force of 1.1 N per needle, with an average in vivo penetration depth of 897.9 ± 96.6 μm (n = 5) in rat skin, ensuring precise dermal delivery. Release profiles demonstrated that the chitosan microneedles acted as an antigen depot, providing sustained release within the skin for over 28 days. Histological analysis further revealed that microneedle implantation actively recruited CD86+ immune cells to the injection site, creating an inflammatory microenvironment conducive to antigen capture. Immunization studies confirmed that this dual-adjuvant platform induced IgG antibody titers significantly higher than traditional subcutaneous injection and simultaneously promoted Th1 and Th2 immune response.
In conclusion, this study successfully constructs a composite transdermal vaccine platform that integrates nanocarriers with a microneedle matrix to achieve macroscopic-microscopic adjuvanticity transition. By synergizing the antigen depot and endogenous immunostimulatory properties of chitosan microneedles with the size effects and virus-mimetic structure of gold nanocarriers, this research establishes a seamless dual-adjuvant mechanism spanning macroscopic to microscopic scales. This approach not only accomplishes stable antigen encapsulation and long-term sustained release but also effectively regulates immune cell activation to induce potent immune responses. The modular nature of this platform offers high scalability; beyond the current model antigen, it holds significant potential for future applications in cancer immunotherapy or the development of vaccines requiring robust immune induction, such as those for influenza.

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