本實驗室於去年時開發出第一代可鑲嵌式的幾丁聚醣(chitosan, CS)微針貼片，證明在相同劑量的抗原(500 μg OVA)下，CS微針可引起較肌肉注射更高且更持久的免疫反應，但因接種劑量較高，導致在接種初期各組的抗體差異不明顯，而本年度的目標為研發一款同時結合快速及緩慢釋放特性的微針貼片，此款微針利用聚麩胺酸(gamma-polyglutamic acid, γ-PGA)的快速水溶特性及幾丁聚醣的緩慢降解性質製成，將低劑量的抗原(100 μg OVA)以1:3的比例包覆於γ-PGA及CS中，達到類似於傳統疫苗接種上的Prime-Boost兩階段免疫接種效果，並希望能在接種初期即快速提高體內抗體的濃度，達到快速產生免疫保護力的效果。後端支撐軸的部分改用生物相容性好且水溶性更佳的聚麩胺酸來製備，取代第一代的高分子聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)和聚乙烯醇(polyvinyl alcohol, PVA)所製備的支撐軸，由體外豬皮及體內大鼠穿刺測試證實，此微針貼片具有足夠之機械強度可刺破角質層，讓微針針體部分鑲嵌於富含抗原呈現細胞之表皮層及真皮層中(深度約800~900 μm)，並且穿刺後所產生之些微紅腫及傷口都可在三天內完全癒合。而將微針包覆螢光抗原後刺入老鼠背部皮膚，並利用非侵入式活體分子影像系統(In vivo imaging spectrum, IVIS)及共軛焦顯微成像技術，證實聚麩胺酸部分的抗原可在7天內被完全釋放，而幾丁聚醣微針部分能在體內緩慢降解並持續釋放抗原長達至少28天。在大鼠免疫試驗結果證實，結合快速釋放與緩慢釋放抗原的CS/γ-PGA微針可在接種2週後引起明顯較高的專一性抗體反應，且持續至少12週。證實結合快速及緩釋的CS/γ-PGA微針，能比第一代的CS微針更快速刺激抗體的產生，且明顯降低抗原的使用量，只需一半的抗原就能刺激出更強烈的免疫反應，這種具有Prime-Boost免疫接種策略的微針，有機會取代傳統多次針劑接種模式，成為新一代的免疫接種方式。

Currently, polymeric microneedles (MNs) are composed of water-soluble polymers and rapidly release encapsulated vaccines. However, these dissolving MNs can not be capable to offer an antigen-depot effect for better vaccination. They usually require repeated administration to induce a protective immunity. In this study, we develop a two-layered MN system, composed of chitosan (CS) MNs and poly--glutamic acid (-PGA) supporting, to provide a two-stage vaccine release strategy. When inserted into the skin, highly hydrophilic -PGA supporting can be quickly dissolved in skin interstitial fluid to release encapsulated payload to “prime” the immune system. The biodegradable CS MN can be embedded in the skin for extended delivery of antigens to spur an immune response. Skin insertion results showed MNs were inserted and then embedded between epidermis and dermis, which are rich in antigen-presenting cells. The MN-created microchannels recovered completely within 3 days. When the OVA-loaded MNs were applied to the rat skin in vivo, confocal microscopic and IVIS images showed that OVA can be rapidly released from the γ-PGA supporting and then gradually released from the CS MNs in a sustained manner. Compared to the first generation CS MNs and the traditional intramuscular immunization, the CS/-PGA MNs induced a significantly higher OVA specific antibody response from the second week to the twelfth weeks. The proposed MN system with two-stage release behavior achieves a prime-boost vaccination from a single administration. We expect that the two-layered -PGA/chitosan MN technology may serve as an alternative to repeated immunizations and become an effective single-dose formulation for vaccine.

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