抗原遞送方式對於誘導有效之免疫反應至關重要。文獻指出，將抗原以奈米粒子的形式包覆並傳遞，可增加其被抗原呈現細胞胞吞之效率，提高疫苗之免疫效力。本研究以帶正電之幾丁聚醣(Chitosan)與帶負電之玻尿酸(Hyaluronic acid)包覆抗原(Ovalbumin; OVA)，形成聚電解質奈米粒子，並以微針傳輸至真皮層以增加奈米粒子被抗原呈現細胞胞吞的機會，期望提高抗原的免疫效果。所製備出之奈米粒子大小約210 ± 17.9 nm(n=4)，表面電性為+37.5 ± 2.4 mV(n=4)，由動態光散射儀測得奈米粒子溶液為單分散(monodisperse)，顯示溶液分散良好且大小均一。為評估奈米粒子對於免疫細胞之胞吞影響，以高(40 μg OVA/mL)及低(10 μg OVA/mL)濃度之含抗原奈米粒子與巨噬細胞(Raw264.7)進行共培養，發現奈米粒子之包覆有助於提升細胞吞噬抗原的能力，亦促進了Raw264.7細胞的活化，且於細胞毒性測試確認細胞存活率皆達85%以上，證實材料具良好之細胞相容性。將含OVA之奈米粒子包覆進聚麩胺酸鹽(Poly-Gamma glutamate, γ-PGA)微針，由豬皮穿刺之組織切片結果得穿刺深度達約705.0 ± 40.7 μm(n=4)，成功將奈米粒子傳輸至表皮及真皮層。將微針回溶於水並以TEM觀察，發現奈米粒子之型態未受微針的製程影響而有聚集或變形。將OVA或含OVA之奈米粒子，分別以皮下注射或γ-PGA微針之方式施打於Sprague Dawley大鼠中，進行免疫試驗。由血中抗體濃度可知，皮下注射奈米粒子所引起之抗體值持續上升至第8週達到高峰，可產生較皮下注射OVA組高4倍之抗體量。將奈米粒子與微針結合，則可在第8週時產生較皮下注射OVA組增加8倍之抗體量，證實了以微針經皮傳輸包覆抗原的奈米粒子可作為一新穎、準確而有效率之疫苗傳遞系統。

Antigen delivery is vital for inducing potent immune responses. Several researches have shown that using nanoparticles to deliver antigens may enhance the immune responses to antigens by promoting endocytosis into immune cells. In this study, we encapsulated the model antigen ovalbumin (OVA) in polyelectrolyte nanoparticles based on chitosan (CS) as polycations and hyaluronic acid (HA) as polyanions, following by further encapsulation to Poly-gamma-glutamate (γ-PGA) microneedles for enhanced uptake from antigen presenting cells (APC) and better immune responses through transcutaneous immunization. Spherical polyelectrolyte nanoparticles with size 210.0 ± 17.9 nm (n=4) and zeta potential +37.5 ± 2.4 mV were characterized and DLS results showed monodispersity, indicating that the nanoparticle solution was well dispersed with homogeneous sizes of nanoparticles. The in vitro cell experiments showed that CS/HA nanoparticles had a strong ability to deliver antigen to macrophages (Raw264.7) and could further promote macrophages to maturation while maintaining low cytotoxicity. OVA-loaded CS/HA NPs were encapsulated into γ-PGA MNs through centrifugation, and the skin cryotome image confirmed the effective transmission of the nanoparticles to the dermis with an average depth of 705.0 ± 40.7 μm(n=4). The TEM results demonstrated that the morphology of NPs was stable after MN dissolution. In vivo study was assessed with Sprague Dawley (SD) rats assigned to the following groups: Subcutaneous (S.C) or γ-PGA MN injection of free form OVA or OVA-loaded NPs (100ug of OVA). Blood analysis demonstrated that S.C NPs induced 4 times of OVA-specific antibody levels than that of soluble OVA, and the synergistic effect of both OVA-loaded NPs and MNs could trigger 8 times of OVA-specific immune responses than that of soluble OVA while maintaining high induction till week 8th, which indicated that the combination of NP and MN system provided a novel design approach for accurate and efficient drug delivery system.

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