| 摘要: | COVID-19 mRNA 疫苗的成功使得其輸送載體逐漸被重視，目前市
面上mRNA載體多為脂質奈米顆粒與脂質體，然而胜肽具有多樣性且可客
製化的優勢，因此本研究探討先前設計的ILs胜肽在mRNA輸送的可行
性。DLS及TEM分析中發現ILs為直徑約50nm的柱狀結構而ILs/DOPE
則為50-100 nm球形結構。在加入mRNA後ILs會被纏繞吸附成700nm的
聚集體，而ILs/DOPE除了會因mRNA而聚集，也有出現150nm較大的囊
泡，證實ILs/DOPE能以纏繞或包覆的方式與mRNA結合。透過EtBr包覆
性實驗以及電泳實驗得知ILs/DOPE在較低N/P ratio下就能達到完整且穩
定的包覆。推測ILs會因其柱狀結構的空間效應，導致對mRNA的包覆效
果不佳，而ILs/DOPE囊泡能均勻分布在mRNA周圍。接著透過共軛交顯
微鏡與流式細胞儀，發現兩種載體均透過胞吞作用進入細胞，但
ILs/DOPE攝取率更好且所攝入的mRNA能自內體逃脫。透過MTT細胞毒
性實驗得知兩種載體只要N/P ratio 不超過30，細胞存活率能達80%以
上。最後透過GFP mRNA轉染實驗證實兩種載體都能有效輸送mRNA
且，且ILs:DOPE為1:0.3具最佳效果。利用冷光酶mRNA轉染實驗量化
轉染效率，發現ILs/DOPE明顯較ILs具更佳的轉染效果，其最佳結果能
媲美Lipofectamine轉染試劑，證實我們開發的自組裝胜肽具有輸送
mRNA轉染的潛力，未來能用於如mRNA疫苗開發及基因治療等領域。;Due to the success of COVID-19 mRNA vaccine, its delivery carrier has
gradually been valued. Currently, most mRNA carriers on the market are lipid
nanoparticles and liposomes. However, peptides have the advantages of diversity
and customizability. Therefore, this study will explore the feasibility of previously
designed ILs peptides in mRNA delivery. DLS and TEM analysis found that ILs
is a columnar structure with a diameter of about 25nm, while ILs/DOPE is a
spherical structure of 50-100 nm. After adding mRNA, ILs will be adsorbed into
700nm aggregates, and ILs/DOPE will not only be aggregated by mRNA, but also
appear 150nm larger vesicles, confirming that ILs/DOPE can bind to mRNA in a
entangled or coated manner. Through EtBr coating experiments and
electrophoresis experiments, it is known that ILs/DOPE can achieve complete and
stable coating at a lower N/P ratio. It is speculated that ILs will have a poor coating
effect on mRNA due to the spatial effect of its columnar structure, while
ILs/DOPE vesicles can be evenly distributed around mRNA. Then, through
conjugate microscopy and flow cytometry, it was found that both vectors entered
the cells through endocytosis, but ILs/DOPE had a better uptake rate and the
mRNA taken in could escape from the endosome. Through the MTT cytotoxicity
experiment, it was known that as long as the N/P ratio of the two vectors did not
exceed 30, the cell survival rate could reach more than 80%. Finally, through the
transfection experiment of GFP mRNA, it was confirmed that both vectors could
effectively deliver mRNA, and ILs:DOPE had the best effect when it was 1:0.3.
Using cold light experiments to quantify the transfection efficiency, it was found
that ILs/DOPE had a significantly better transfection effect than ILs, and its best
result was comparable to Lipofectamine transfection reagent, proving that the
self-assembling peptide we developed has the potential to deliver mRNA
transfection, and can be applied in the future to fields such as mRNA vaccine
development and gene therapy. |
