利用磷脂質促進硬脂基化胜?之基因輸送;The Use of Phospholipid to Promote Gene Delivery of Stearylated Peptide Vehicles

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Title:	利用磷脂質促進硬脂基化胜?之基因輸送;The Use of Phospholipid to Promote Gene Delivery of Stearylated Peptide Vehicles
Authors:	謝勗元;Sie, Syu-Yuan
Contributors:	化學工程與材料工程學系
Keywords:	細胞穿膜胜?;基因輸送;非病毒載體;CPP;Gene delivery;non-viral vector;Indolicidin
Date:	2021-10-28
Issue Date:	2021-12-07 11:37:38 (UTC+8)
Publisher:	國立中央大學
Abstract:	細胞穿膜胜?(CPP)能與細胞膜相互作用，可作為藥物載體並具備基因輸送的潛力。Indolicidin(IL) 是一種帶正電的CPP，先前研究中將IL之C端硬脂基化，稱為ILs，結果發現ILs擁有自組裝效果，可作為基因載體遞送質體DNA進行轉染。為提升ILs與DNA結構穩定性，本研究嘗試加入輔助性脂質DOPE，以不同莫耳比與ILs混合來製備胜?/脂質複合物。臨界微胞濃度測試(CMC)及DLS分析證實ILs能與DOPE帶正電的微結構，而SAXS結果顯示ILs呈現六角相結構而ILs/DOPE會自組裝成單層囊泡，其形態也透過TEM進行確認。電泳膠體實驗證實，將DOPE加入ILs可提高與DNA複合效果，但肝素競爭實驗結果顯示DNA和載體的結合主要還是倚賴帶正電的ILs，因為ILs/DOPE表面上的ILs較少，少量添加DOPE(P0.3L)會降低載體與DNA的親和力。流式細胞儀及ONPG轉染分析結果顯示，P0.3L擁有最佳的細胞攝取效果及轉染效率。雷射共軛焦顯微鏡結果顯示ILs/DOPE所帶入的DNA可自內體逃脫，為了瞭解其轉染機制，分別進行了Chloroquine、Bafilomycin A1以及酸性環境下鈣黃綠素滲漏實驗，其結果表明ILs/DOPE在酸化環境下可瓦解內體膜，促進內體逃脫。這些結果顯示DOPE的添加的確可此促進ILs的基因輸送效果。;Cell-penetrating peptides (CPPs) can interact with cell membranes and thus are potential carriers for gene delivery. Indolicidin (IL) is a cationic CPP. Our previous study has stearylated C-terminal of IL and denoted it as ILs. ILs can self-assemble as gene carriers to deliver plasmid DNA for transfection. In order to improve the stability of microstructure of gene carriers, we included dioleoylphosphatidylethanolamine (DOPE), a helper lipid, with ILs. Critical micelle concentration (CMC) and DLS analyses confirmed that ILs and DOPE can form cationic microstructures. The SAXS results indicated that ILs presented a hexagonal phase structure and ILs/DOPE self-assembled a monolayer vesicle, and their morphologies were confirmed by TEM. Electrophoresis experiments showed that the addition of DOPE to ILs can improve their complexation efficiency to DNA, but the results of heparin competition revealed that DNA complexation was mainly related to cationic ILs molecules. Because there were fewer ILs on the surface of ILs/DOPE, the affinity of ILs/DOPE to DNA was lower than that of the ILs group. Flow cytometry and transfection experiments indicated that P0.3L exhibited the best cell uptake and transfection efficiency. Confocal microscopy showed that DNA delivered by ILs/DOPE could escape from endosomes. The results of chloroquine, Bafilomycin A1, and calcein leakage experiments in acidic environment all indicated that ILs/DOPE disrupted endosomal membranes and promoted DNA to escape from the acidic endosome. These results all suggested that the addition of DOPE can facilitated ILs-mediated gene delivery.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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