Applications of Positively-charged Lipid Nanoparticles Complexed with CpG Oligodeoxynucleotide as Immunostimulators

疫苗的發展在醫學上是一個非常重要的貢獻，不僅是傳染性疾病如麻疹、小兒麻痺、流行性感冒，皆因為有相對應疫苗的開發而逐漸控制了感染的暴發。近年來，許多研究者也相繼將目標對準了癌症，如乳癌、卵巢癌等，希望可以發展出疫苗來減輕癌症對病患及社會的負擔。其中，微脂粒奈米粒子在佐劑中也佔有一個重要的角色。在小於1000nm的尺寸下，這種顆粒性的遞送系統有一些優點：抗原可以有緩慢釋放的效果以延長抗原和免疫細胞的作用時間、奈米尺寸的大小和很多病源體的大小相近所以免疫系統可以容易的辨識並處理，此外，奈米顆粒可以同時促進細胞免疫以及體液免疫反應的發生。另外，CpG這種沒有甲基化的大約20 mer的短鍊核苷酸，可以模擬細菌和病毒中的DNA，藉著類鐸受體9 (Toll-like receptor 9, TLR9)的訊息傳遞，產生細胞激素更加刺激抗原專一性T細胞反應的發生。 所以，本實驗應用正價的脂質奈米粒子作為佐劑載體，以正負電荷交互作用在粒子上吸附帶負電的蛋白質作為抗原，以避免共價鍵修飾蛋白質造成的結構破壞而影響免疫反應產生。同時也因為本微脂粒帶有正電可以直接吸附TLR9致效劑CpG短鍊核苷酸，而誘導免疫反應朝向抗病毒以及抗癌的方向。以綠色螢光蛋白 (EGFP)作為和微脂粒吸附的蛋白質，以不同的比例混合反應後處理人類子宮頸癌HeLa細胞和小鼠巨噬細胞，觀察進入細胞的效率，可以發現約在正負電比例為6:1時，也就是約7.5 nmol的GEC-Chol加上5 μg的綠色螢光蛋白，正價微脂粒處理HeLa細胞可以達到最大的遞送效率。實驗加上CpG短鍊核苷酸處理小鼠巨噬細胞，可以同時將蛋白質與CpG短鍊核苷酸送入巨噬細胞中。以即時定量聚合酶連鎖反應決定各實驗組的細胞激素mRNA表達量後發現，GEC-Chol/Chol正價微脂粒可以順利引起免疫反應，顯示出GEC-Chol/Chol正價微脂粒或許可以做為免疫刺激佐劑之用途。此外，其遞送蛋白質與寡核苷酸進入細胞的效率在未來可能有可以應用之處，如siRNA的遞送與定量送入活性蛋白質研究細胞中訊息傳遞路徑等。The investigation of vaccines plays an important role in the progression of public health affairs. The discovery of vaccines effectively reduced the severe outbreak of diseases. In recent years, some researchers have shifted the aim of vaccine development against cancers such as breast cancer, ovarian cancer and cervical cancer. It is somewhat more difficult to develop an efficient anticancer vaccine because of the immunotolerance characteristics of cancer cells. In order to solve the problem, many adjuvants were discovered and tested. The lipid-based nanoparticles were used in the development of efficient anticancer adjuvants. There are some ideal properties of lipid-based nanoparticles in the application of adjuvant application. The particulate effect is favorable for antigen presenting cells to uptake and process. It behaves a depot effect that prolongs the duration of antigen exposure time. Therefore, we can combine different adjuvants in a particulate form to further enhance the immune stimulatory ability. Previous studies have shown that when lipid nanoparticles combined with oligonucleotides containing unmethylated cytosine-guanidine residues (CpG ODN), for elicit strong immune response. Thus, many researchers tried to combine cationic surfactants or cationic lipids with CpG ODN for enhancing cellular or humoral immune responses. In our laboratory, a cationic cholesterol-based amphipathic material named 3β-[N-(2-guanidinoethyl) carbamoyl] cholesterol (GEC-Chol), which is originally designed to be applied in gene delivery and other nucleic acid delivery in vitro and in vivo, is applied to deliver CpG ODN and a model protein antigen, EGFP, in this study. In these experiments, I chose GEC-Chol/Chol to coat ultrasmall superparamagnetic iron oxide (USPIO) in a micelle-like formulation and hope this particle can not only possess adjuvant behavior but with MRI contrast characteristics. As a result, the GEC-Chol/Chol-coated USPIO can transduce EGFP and CpG ODN into primary-cultured murine macrophages in as fast as 2 h incubation in vitro. And the most efficient lipid-protein formulation is 10 μg/ml EGFP mixed with 15 μM GEC-Chol. Furthermore, using DiI-labeled GEC-Chol/Chol-coated USPIO, non-adherent cells, such as splenocytes, U937 and HL-60 cells could be effectively labelled. Besides, EGFP and CpG ODN could also be transduced into those non-adherent cells. Finally, the real-time PCR reveals that GEC-Chol/Chol-coated USPIO complexed with CpG ODN, and could enhance proinflammatory cytokine production in comparable to previously published formulations. According to these results, we suggested that, GEC-Chol/Chol-coated USPIO particle could deliver EGFP protein and CpG ODN into macrophages simultaneously and also could be used to label non-adherent cells although in a lesser degree compared with macrophages. From the preliminary in vitro experiment, it shown the elevated cytokine mRNA production. The in vivo responses need to be further investigated.口試委員會審定書 i誌 ii文摘要 iiibstract iv寫表 vi表目錄 ix論 1.1 正價微脂粒 1.1.1 正價微脂粒之應用 1.1.2 以正價微脂粒遞送蛋白質 1.1.3 以正價微脂粒作為佐劑 2.2 類鐸受體 3.2.1 類鐸受體分類 3.2.2 類鐸受體訊息傳遞路徑 4.3 CpG寡核苷酸 4.4 氧化鐵奈米粒子 4.4.1 分離物質 5.4.2 磁振造影的顯影劑 5.4.3 藥物與基因遞送 5.4.4 磁致熱效應 6.5 研究動機與目的 6驗材料與方法 8.1 實驗材料 8.1.1 藥品 8.1.2 實驗動物 8.1.3 細胞株 8.1.4 CpG免疫刺激性寡核苷酸 9.1.5 氧化鐵奈米粒子 9.1.6 脂質 9.1 7 質體 9.1.8 儀器 10.2 實驗方法 11.2.1 GEC-Chol製作 11.2.2 小鼠骨髓誘導巨噬細胞 12.2.3 小鼠脾臟細胞分離 13.2.4 細胞繼代培養 13.2.5 微脂粒和氧化鐵複合體製備 14.2.6 綠色螢光蛋白表達與純化 14.2.7 CT-26細胞膜蛋白萃取 16.2.8 以FITC螢光分子標定CT-26細胞膜蛋白 16.2.9 蛋白質定量 17.2.10 以綠色螢光蛋白和CpG寡核苷酸與微脂粒氧化鐵複合粒子處理細胞 17.2.11 以雷射共軛焦顯微鏡觀察粒子在細胞中的分布 19.2.12 細胞總RNA抽取 20.2.13.1 巨噬細胞mRNA反轉錄作用 21.2.13.2 即時定量聚合酶連鎖反應 21驗結果 23.1 正價微脂粒氧化鐵複合粒子含蛋白質和CpG短鍊核苷酸之製備與分析 23.1.1 綠色螢光蛋白之純化與CT-26細胞膜蛋白之萃取 23.1.2 抗原蛋白質和CpG寡核苷酸與GEC-Chol/Chol正價微脂粒氧化鐵複合粒子之大小分析 23.2正價微脂粒氧化鐵複合粒子遞送不同免疫細胞之效率分析 24.2.1 正價微脂粒複合粒子遞送rEGFP進入細胞之研究 24.2.2正價微脂粒氧化鐵複合粒子遞送抗原蛋白與寡核苷酸進入骨髓巨噬細胞 25.2.3 正價微脂粒氧化鐵複合粒子遞送蛋白與寡核苷酸進入懸浮型細胞之效率分析 25.3 正價微脂粒氧化鐵複合粒子含有抗原蛋白質和CpG對細胞免疫反應刺激之分析 26論 28.1 短鍊核苷酸與蛋白質和正價微脂粒氧化鐵複合體之粒徑大小分析 28.2 GEC-Chol/Chol正價微脂粒氧化鐵複合體遞送CpG寡核苷酸與蛋白質進入骨髓誘導巨噬細胞之分析 28.3 GEC-Chol/Chol正價微脂粒氧化鐵複合體遞送CpG寡核苷酸與蛋白質進入懸浮性細胞之分析 29.4 遞送癌細胞膜蛋白與免疫刺激物質CpG到免疫細胞中 30.5 GEC-Chol/Chol正價微脂粒氧化鐵複合體遞送CpG寡核苷酸具有效引發免疫刺激細胞激素之基因表達 30.6 GEC-Chol/Chol正價微脂粒氧化鐵複合粒子之應用範圍 31表和說明 33.1 論文圖表 33.2 圖表說明 40考文獻 4