利用正電明膠奈米粒子做為具免疫刺激性抗原載體應用於黏膜抗原傳遞之研究

疫苗自過去的研究以來，一直都是利用活化人體內的免疫系統來治療其疾病以及傳染病…等等，但是從過去到現在，疫苗的給予途徑一直以來都是以打針注射的方式最為常見，但也因為針劑所造成的疼痛以及恐懼，導致疫苗的給予經常會有些阻礙，因此想藉由黏膜的途徑來進行疫苗的給予來有效的改善其缺點。從過去疫苗的製作上也經常會以其活化的效率以及對於接種的本體安全性之間來進行考量，從活毒疫苗、減毒疫苗以及死毒疫苗上，可以確定隨著疫苗上所帶有的病原體本上總量越多以及活性越強，產生的效率理所當然會更好，但也造成了安全上的風險，為了減低其安全上的風險也因此研發出了次單位疫苗以及DNA疫苗，將病原體上帶有的片段或是DNA藉由疫苗的方式帶入體內活化，但同樣隨著劑量越多，也有可能造成注入疫苗的本體基因被過量的劑量產生改變甚至造成基因突變的風險，為了改善其劑量的使用並能達到良好的活化效率，在我的研究中是想利用正電改質後的明膠奈米載體作為傳輸系統，並將其載體攜帶著疫苗上所需要的抗原-卵白蛋白(OVA)以及免疫刺激物-聚肌胞苷酸(Poly(I:C))，最後將製備好的抗原傳輸系統藉由黏膜點鼻的途徑進行給予疫苗並活化其動物體內的免疫系統，並在最後以淋巴癌細胞(表面帶有抗原OVA)為治療目標並觀察其預防腫瘤成長效果! 在本研究中利用明膠奈米載體經過改質後已成功的讓明膠奈米載體在中性的環境下仍然能維持帶有大量的正電，並且找出了適當的參數能有效的將抗原OVA以及免疫刺激物poly(I:C)吸附於載體上，並將其特性(大小、PDI以及電性)維持在我們所需要的範圍內，明膠奈米載體有成功的修飾正電並且成功的吸附抗原及Poly(I:C)並且吸附完後仍帶有正電，並提高特性進行傳遞，將各種吸附完抗原及免疫刺激物的奈米載體，藉由細胞實驗以及動物實驗中得到各種數據，從細胞實驗中，得到了在帶有抗原及免疫刺激物的載體仍能維持電性以及有利於細胞的吞噬，並藉由流式細胞儀得到了經過功能性載體的刺激後，樹突狀細胞表面的CD80&86得到了足夠的活化，並藉由細胞激素的分析看到了隨著poly(I:C)的增加，在經過刺激後的細胞分泌出了IL-6、IL-12以及TNF-α，也隨著吸附量的增加，分泌量也隨之增加。後續的動物實驗則藉由脾臟細胞的外刺激再次確定其經過活化後的免疫系統經過外來相同抗原是否能達到同樣的分泌反應，並藉由分泌出來各種細胞激素(TH1:IFN-γ；TH2:IL-4、IL-5、IL-6)的量來推斷其免疫系統內活化的方向，並藉由抽取血清以及鼻肺部表面的黏膜液，分析其體內隨著疫苗的刺激後的抗體變化，自以上可以知道隨著抗原搭配奈米載體的增加能有效促進適應性免疫分化往TH2的方向活化；然而在加入免疫刺激物poly(I:C)後能有效的提升其適應性免疫並誘導其分化往TH1的方向成長，並藉由分泌出的細胞激素，已經由文獻證實可以有效促使毒殺型T細胞的分化並藉由此細胞達到毒殺特定細胞的功能，因此藉由腫瘤植入實驗來證明其免疫活化的效果，自結果來看能確定本研究中所使用的帶有抗原及免疫刺激物的奈米載體，一方面能有效的降低其用量並達到良好的抑制腫瘤生長的效果，從此證據可以證明研究中所製備的載體能誘發專一性的抗體免疫反應以及細胞激素的提升以及腫瘤抑制效果且達到提升其專一性免疫的活化!

關鍵字

免疫刺激物；正電明膠奈米載體；抗原傳遞；黏膜免疫；疫苗

並列摘要

In the past, the vaccine has been research to attack the disease. This disease is including about the infectious disease. However, up to now the vaccine administrations are used to inject the vaccine in body. The injection owing to cause the pain, many people don’t want to accept the vaccine injection. Because of this reason, we want to use the mucosal delivery to change the disadvantage of the injection. The history of making vaccine has been used to achieve the goals which are better effective result and the safe. However, Live/attenuated vaccines have the good result but they are not safe for the patient. And the inactivated vaccine has decreased the dosage for the patient safe but it was not effective for curing the disease. We need to find the method which can decrease the dosage and have the better treatment effect. So we can use the delivery system to overcome this aim. The successful vaccine needs three important things including with antigen, delivery system and immune-stimulator. In this study, the Ovalbumin (OVA) was used to be the antigen model. The positive gelatin nanoparticles were used to be the material of delivery system. And the Poly (I: C) was used to be the immune-stimulator. Poly (I: C) and OVA were exposed on the outer surface of positive gelatin nanoparticles. The particle sizes of each positive gelatin nanoparticle with OVA and Poly (I: C) were controlled to smaller than 500nm. And the absorption of OVA at this particle has up to 95%. We demonstrated the Poly (I:C) and OVA incorporated positive gelatin nanoparticles effectively facilitated antigen uptake by mouse bone-marrow derived dendritic cells (BMDCs) and macrophage in vitro, led to higher expression of maturation markers, including CD80&86, and induced higher production of pro-inflammatory cytokine. In vivo use the C57BL/6 mice and the immunization procedure was repeated 2times at 2 weeks interval. C57BL/6 mice immunized by intranasal with the Poly (I: C) and OVA incorporated positive gelatin nanoparticles produced high levels of OVA-specific IgG antibodies in their serum and secretory-IgA (s-IgA) in nasal wash fluid. Spleen cells from mice receiving the Poly (I: C) and OVA incorporated positive gelatin nanoparticles were re-stimulated with OVA and showed significantly augmented levels of IFN-γ. In addition, intranasal administration of the Poly (I: C) and OVA incorporated positive gelatin nanoparticles resulted in complete protection against EG7 tumor challenge in C57BL/6 mice. Taken together, these results indicate that nasal administration of the Poly (I: C) and OVA incorporated positive gelatin nanoparticles mediates the development of an effective immunity against tumors and might be useful for further clinical anti-tumor application.

並列關鍵字

Immuno-stimulator ； Positive gelatin nanoparticle ； Antigen delivery ； Mucosal immunity ； Vaccine

參考文獻

1. Kambayashi, T., Laufer, T. M., Atypical MHC class II-expressing antigen-presenting cells: can anything replace a dendritic cell? Nat Rev Immunol, 2014. 14(11): p. 719-730.

2. Mathew, E.c., Antigen presenting cell. Uptodate, 2015.

3. Hafner, A.M., Corthesy, B., Merkle, H. P., Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant. Adv Drug Deliv Rev, 2013. 65(10): p. 1386-1399.

4. Ardavin, C., Origin, precursors and differentiation of mouse dendritic cells. Nat Rev Immunol, 2003. 3(7): p. 582-590.

5. Young-Jun, L., Dendritic Cell Subsets and Lineages, and Their Functions in Innate and Adaptive Immunity. Cell press, 2001. 106(3): p. 259- 262.

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利用正電明膠奈米粒子做為具免疫刺激性抗原載體應用於黏膜抗原傳遞之研究

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