建立一種新型微脂體與聚合物之複合體來做為核酸疫苗與次單位疫苗的共同載體

Abstract

近年來多盛行以非病毒性載體來當作基因治療及疫苗開發等載體系統。 因此在本篇研究當中，主要是建立一種新型微脂體與聚合物之複合體，進而使其當作核酸或是蛋白質的載體系統。新型的複合體則是由兩種聚合物聚乙烯亞胺(PEI)及聚乙二醇(PEG)與大豆沙拉油經過快速的超音波震盪製作而成。 進一步分析其基本物理性質包括利用穿透式電子顯微鏡觀察複合體之外觀以及使用動態粒徑散射儀分析其顆粒大小分布約在212.2 奈米至312.1奈米範圍之中，並且偵測此複合體之表面電位來確立其具有正電性質。此外，透過洋菜膠電泳方式證明此複合體具有緊密吸附核酸的能力並且可以保護核酸免於核酸酶的破壞。同時，經由細胞轉染實驗發現此複合體可以有效的在纖維母細胞中提升核酸的轉染效率並且增強基因表現。 再者，經由實驗證明此複合體確實可有效的提升巨噬細胞對抗原的吞噬能力與誘發老鼠免疫細胞較高量的腫瘤壞死因子, 這都說明了此複合體具有擔任疫苗佐劑功能的潛力。 然而在動物實驗上, 我們使用胃幽門螺旋桿菌相關的熱休克蛋白與尿素酶蛋白當作抗原, 進ㄧ步觀察核酸抗原及蛋白質抗原與複合體共同進行免疫老鼠的實驗, 則證實可誘發較高量的免疫反應產生。 因此，在本篇研究中已建立了ㄧ個核酸與蛋白質傳遞系統, 此系統則是以微脂體與聚合物之複合體來當作載體，並且評估此載體在未來疫苗開發的濳力上，可由於製作的方便性及較低的材料價格, 使其可當作動物農場所使用的核酸疫苗與次單位疫苗之共同傳輸系統。

In this study, a novel liposome-polymer transfection complex (LPTC) was developed with inexpensive cost and used as DNA or protein delivery system. LPTC was fabricated via the sonication and composed of soybean oil, polyethylenimine (PEI) and polyethylene glycol (PEG). After preparation of LPTC, the morphology of particles and their physical properties were conducted with TEM, DLS and Zeta-Sizer. The particles of LPTC showed round shape and fuzzy edge around the surface of particle captured by TEM. In addition, the particle sizes of LPTC were in the major range of 212.2 nm to 312.1 nm and the zeta-potential (surface charge) was strongly positive (+38.7 mV). Moreover, we examined the ability of DNA condensation and the protection from DNase I digestion in agarose gel electrophoresis. In the in vitro tests, to clarify the gene delivery efficacy, we evaluated the transfection efficiency of LPTC/DNA complexes in Balb/3T3 cells and the transfection efficiency increased as the charge ratios of LPTC to DNA increasing. LPTC enhanced the cellular uptake of antigen in mouse macrophage cells and also stimulated TNF-alpha release in naïve mice splenocyte that both showed the potential to be adjuvant in vaccine development. In vivo studies, using H. pyrori relative Hsp60 and Urease B as antigen model, we observed that vaccination of BALB/c mice with LPTC complexed DNA and protein enhanced humoral immune response. Therefore, these results showed that we have developed a DNA and protein delivery system by liposome-polymer transfection complex with inexpensive cost and successfully applied in the development of DNA and subunit vaccine. The success of this design may provide an economical vaccination alternative for farm animal use.