以短鏈胜?接枝聚乙烯亞胺來進行基因輸送應用之研究; The use of short peptides conjugated PEI for gene delivery application

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Title:	以短鏈胜?接枝聚乙烯亞胺來進行基因輸送應用之研究;The use of short peptides conjugated PEI for gene delivery application
Authors:	葉喬淳;Yeh,Chiao-chun
Contributors:	化學工程與材料工程學系
Keywords:	聚乙烯亞胺;胜?;基因傳送;polyethylenimine;peptides;gene delivery;bioconjugation;endosomal escape;indolicidin
Date:	2013-07-26
Issue Date:	2013-08-22 11:39:26 (UTC+8)
Publisher:	國立中央大學
Abstract:	本研究將短鏈胜?接枝於聚乙烯亞胺當作基因載體，並以正負電荷吸引方式，將此載體與質體DNA以不同胺基/磷酸根莫耳比(N/P ratio)組裝成奈米粒子。我們可以藉由動態雷射散射粒徑分析儀測得粒子粒徑大小約分佈在200nm-600nm間；大部份表面電位則為正電，皆為可接受之基因傳送條件。此外，改質過後的PEI，依然能提供質體DNA良好的包覆率。而由MTT分析顯示，改質過後的PEI毒性比未改質前毒性較少，甚至沒有毒性。PEI接枝R9、SAP10兩組載體幾乎沒有轉染效率，IL接枝組不僅有轉染還且優於純PEI組別。我們利用螢光標定以追蹤質體DNA，發現改質過的PEI只有IL接枝組能有效將DNA送入到細胞中。利用Bafilomycin A1 及Chloroquine藥物來探討PEI-IL轉染機制，可發現以PEI-IL所送入的DNA，其內胞逃脫非完全藉由質子海綿效應。最後我們以分子動態模擬來探討這些接枝的胜?與脂雙層間之交互作用，發現胜?的疏水端須能進入脂雙層方可穩定其與細胞膜的作用力，進而促進粒子的被吞噬，其中IL的tryptophan 扮演疏水端進入脂雙層的關鍵角色。 In this study, short peptides were conjugated with polyethyleneimine for the application of gene delivery. By electrostatic interaction, self- assembled nanoparticles using PEI-CPPs, and plasmid DNA were prepared in different amine/phosphate (N/P) ratios. The dynamic laser scattering experiment demonstrated that most of particles formed by peptide modified PEI were between the 200 to 600 nm with positive surfaces, suggesting that these nanoparticles were appropriate for gene transfer. In addition, the modified PEI effectively bound DNA. The MTT assays suggested that modification PEI reduced the cytotoxicity. PEI conjugated either by R9 or SAP10 were unable to transfect cells. In contrast, IL conjugated PEI demonstrated better transfection efficiency than that using sole PEI. We used fluorescein labeling to track plasma DNA delivery. Only PEI-IL can carry DNA to cells. To determine the endosomal escape mechanism of PEI-IL, bafilomycin A1 & chloroquine were applied during transfection. Their results suggested that internalized DNA should be released mainly by proton sponge effect however, the grafted IL may also perturb endosome membrane and eventually causes membrane disruption. Finally, we used molecular dynamic simulation to elucidate the mechanism of grafted CPP interact with lipid bilyaer. The results indicated that only peptides with hydrophobic domain entering lipid bilayer can stabilize their interactions to cell membrane. In addition, tryptophan in indolicidin plays an important role to the insertion of IL to lipid bilayer.
Appears in Collections:	[National Central University Department of Chemical & Materials Engineering] Electronic Thesis & Dissertation

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