利用穿膜胜肽改善帶正電高分子之轉染效率;The Use of Cell Penetrating Peptides and for Improving the Transfection Efficiency of Polyethylenimine

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题名:	利用穿膜胜肽改善帶正電高分子之轉染效率;The Use of Cell Penetrating Peptides and for Improving the Transfection Efficiency of Polyethylenimine
作者:	林則緯;Lin,Tse-wei
贡献者:	化學工程與材料工程研究所
关键词:	基因傳送;穿膜胜肽;聚乙烯亞胺;gene transfer;cell penetrating peptides;polyethylenimine
日期:	2012-08-23
上传时间:	2012-09-11 18:13:05 (UTC+8)
出版者:	國立中央大學
摘要:	本研究利用PEI(750 kDa & 25 kDa)結合穿膜胜肽作為基因載體，並以正負電荷吸引之方式，將之與質體DNA以不同胺基/磷酸根莫耳比(N/P ratio)組裝成奈米粒子。首先，由BCA assay測定細胞總蛋白量，我們發現R9與SAP10具有良好的生物適合性，然而IL在高濃度時具有明顯的細胞毒性。將PEI結合穿膜胜肽(Cell-penetrating peptides, CPP)作為基因載體能有效改善純PEI的轉染效率，以PEI/R9效率最佳。其中我們還發現在CPPs輔助下可更有效提升高分子量PEI(750 kDa)的轉染效率。我們將幾組轉染較佳的PEI與CPP結合的載體進行物理性質量測，由動態雷射散射粒徑分析儀與掃描式電子顯微鏡測得粒子之粒徑大小分佈於200 nm-400 nm之間；而表面電位介於10~20 mV，均為適合基因傳送之條件。以電泳方式簡單測試其包覆率，可發現粒子表層的PEI能將質體DNA完整包覆形成穩定的粒子。藉由改變合成粒子的順序，我們證實轉染的增進主要是基於複合於粒子上的CPPs的效果。最後，我們探討CPP輔佐轉染的機制，發現此系統CPPs不但可以促進複合體進入細胞的效率(尤其是R9)；還能夠輔助複合體以質子海綿效應以外的路徑脫離內胞(endosome)；另外藉由競爭實驗，我們證實IL及SAP10可以增加質體DNA與載體分離的機會。因此，透過本研究我們了解CPP可以幫助陽離子高分子進行基因傳送，這些結果將有助於發展安全且高效率的非病毒載體以應用於基因治療。關鍵字：聚乙烯亞胺、穿膜胜肽、基因傳送In this study, we would like to investigate if the combination cell penetrating peptides (CPPs) and polyethylenimine (PEI) may facilitate gene delivery. Polyethylenimine (PEI, 750 kDa & 25 kDa) were combined with cell-penet rating peptides(CPPs) as gene vectors. By electrostatic interaction, self- assemble nanoparticles using PEI, CPPs, and plasmid DNA were prepared in different amine/phosphate (N/P) ratios. The BCA assay was applied to evaluate the cytotoxicity of CPPs. Both R9 and SAP-10 were biocompatible compared to IL which may induced serious toxicity at high concentration. By combing PEI and CPPs as gene vehicles, transfection efficiencies can be highly improved over the pure PEI delivery, which was especially efficient to promote gene transfer of PEI with molecular weight of 750 kDa, a poor vector when it was solely administrated. Dynamics light scattering (DLS) assay and scanning electron microscopy (SEM) were performed to characterize PEI/CPPs/DNA complex. Complexed particle demonstrated sizes between the 200 nm to 400 nm and zeta potentials between +10 to +20 mV, suggesting that these nanoparticles should be suitable for gene delivery. The electrophoresis data indicated that the PEI and CPPs could complex DNA completely to form a stable particle. The experiment of changing complexation order revealed that only complexed CPPs may help DNA delivery. Finally, the delivery mechanism of CPPs was studied. It suggested that CPPs should not only increase cellular uptake, but also promote complexes escaping from endosome by route other than proton sponge effect. In addition, the competition experiment demonstrated that IL and SAP10 should interfere the interaction between PEI and DNA to increase the chance of dissociation when they were internalized. Through this study, the adjuvant effects of CPPs on facilitating gene delivery using cationic polymer were studied, which should be beneficial to development a safe and highly efficiency nonviral vector for gene therapy.Key words：polyethylenimine, cell-penetrating peptides, gene delivery
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