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Title: | 開發脂質複合奈米粒子之mRNA載體平台 Developing an mRNA Carrier Platform of Lipid Hybrid Nanoparticles |
Authors: | 許煜婕 Yu-Chieh Hsu |
Advisor: | 黃義侑 Yi-You Huang |
Keyword: | 信使核糖核酸,載體平台,脂質複合奈米粒子,疫苗,基因治療, mRNA,carrier platform,lipid hybrid nanoparticle,vaccine,gene therapy, |
Publication Year : | 2023 |
Degree: | 碩士 |
Abstract: | 在嚴重特殊傳染性肺炎疫情爆發後,大眾對於mRNA疫苗的關注度大幅上升,mRNA的各項應用及遞送技術也受到各界重視。除了感染性疾病疫苗外,mRNA也被應用於癌症治療及預防,以及蛋白質替代療法等新興領域,對於許多疾病皆具有應用潛力。
本研究使用編碼增強型綠色螢光蛋白(enhanced green fluorescent protein, EGFP)之mRNA,探討脂質/高分子複合奈米粒子及脂質複合奈米粒子二種系統,期望透過不同遞送材料及組成的測試,開發出合成簡易、成本較低的mRNA載體平台。在脂質/高分子複合奈米粒子部分,選用PCL-PEI作為高分子材料,結合PCL的生物降解性及PEI帶正電可與帶負電之核酸分子結合的特性,再經過脂質包覆提升親脂性,增進細胞攝取。然而目前在小鼠胚胎成纖維細胞株NIH-3T3及人類胚胎腎細胞株HEK-293皆未觀察到綠色螢光表現,推測可能原因包含溶劑DMSO對RNA結構的破壞性及形成粒子粒徑過大。而在脂質複合奈米粒子方面,進行不同脂質組成的測試,透過奈米粒子之mRNA有效包覆率及對HEK-293細胞的轉染效率等分析,找出最佳化製備條件為所含脂質總濃度次高、正電荷脂質比例最高的LNP6組別(各成分莫耳比例為37.5 % DOTAP、43.125% DOPE、3.75% Stearylamine、2.1875 % SM-102、12.5 % Cholesterol、0.9375 % DSPE-PEG2000),接著測試以魚精蛋白先行與mRNA縮合,再透過該條件之脂質組成包覆,發現與僅以脂質包覆的組別相比,粒徑較為均一,但細胞轉染效率較低,推測為魚精蛋白與mRNA之間的結合過於緊密所致。綜合而言,本研究探討之不同系統的mRNA載體平台中,以脂質複合奈米粒子的LNP6組別效果最佳,可以此為基礎,開發出合成簡易、成本較低的mRNA載體平台。 After the COVID-19 outbreak, there has been a significant increase in public interest in mRNA vaccines. The attention has extended to various applications and delivery technologies of mRNA. Apart from vaccines for infectious diseases, mRNA has also found utility in emerging fields such as cancer treatment and prevention, as well as protein replacement therapy. It holds immense potential for application in numerous diseases. This study aimed to investigate two systems of lipid/polymer composite nanoparticles and lipid composite nanoparticles using mRNA encoding enhanced green fluorescent protein (EGFP). The goal was to develop an mRNA carrier platform that is easy to synthesize and cost-effective by testing different delivery materials and compositions. In the case of lipid/polymer hybrid nanoparticles, PCL-PEI was chosen as the polymer material due to the biodegradability of PCL and the positive charge of PEI, which allows it to bind to negatively charged nucleic acid molecules. Lipids were then coated onto the particles to enhance lipophilicity and improve cellular uptake. However, no green fluorescence was observed in the mouse embryonic fibroblast cell line NIH-3T3 or the human embryonic kidney cell line HEK-293. Possible reasons include the destructive effect of the solvent DMSO on the RNA structure and the excessive size of the particles formed. As for lipid hybrid nanoparticles, different lipid compositions were tested. The encapsulation efficiency of nanoparticles and the transfection efficiency of HEK-293 cells were analyzed to identify the optimal lipid composition, and we found that with second highest total lipid concentration and highest ratio of positively charged lipids, LNP6, composed of 37.5 % DOTAP, 43.125% DOPE, 3.75% Stearylamine, 2.1875 % SM-102, 12.5 % Cholesterol and 0.9375 % DSPE-PEG2000, exhibited the best performance. Subsequently, the composition of LNP6 was selected to test the influence of protamine. We first condensed protamine with mRNA and then coated it with the specified lipid composition. The results showed that compared to the group only composed of lipids, the particle size was relatively uniform. However, the cell transfection efficiency was low, possibly due to the excessively strong binding between protamine and mRNA. In conclusion, among the different mRNA carrier platforms discussed in this study, the LNP6 group of lipid hybrid nanoparticles demonstrated the most promising results. This finding paves the way for the development of an mRNA carrier platform that is simple to synthesize and cost-effective. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90705 |
DOI: | 10.6342/NTU202301548 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 醫學工程學研究所 |
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