利用酸鹼敏感型高分子材料做為控制病毒或基因傳遞之載體於生物醫學上之應用

Switchable gene or virus delivery system using pH-sensitive polymeric carrier for biomedical application

曾意芳(Yi-Fang Zeng)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.10376

摘要

無論是使用病毒或非病毒的載體去進行基因傳遞，如何有效的調控一直是個重要的課題，本篇論文中，以高分子材料做為基質或載體，搭配不同的酸鹼敏感性特質去控制基因傳遞的效率，達到更有效的轉導作用。在本論文的第二章節中，介紹我們以生物可相容及對酸鹼敏感的水膠做為基質，將帶有綠色螢光表現基因的重組腺相關病毒包埋於水膠中，利用水膠的特性在局部將病毒釋放而達到基因物質傳遞的效果，並藉由聚組胺酸添加的多寡來控制水膠吸收水分的含量，而直接影響水膠的膨脹及水解程度，藉此可控制包埋於水膠內的病毒釋放量及速率。由實驗結果得知，添加聚組胺酸成分的水膠可更有效率的將病毒釋放，並在人類的纖維瘤細胞株 HT-1080中可得到明顯的轉導效果，隨著聚組胺酸添加濃度的增加，局部基因轉導的效果也更加明顯，但過量添加聚組胺酸將會引起細胞的發炎反應。
接下來於第三個章節中，我們利用支鏈聚乙烯亞胺取代病毒載體，將小分子核醣核酸與聚乙烯亞胺所形成的聚合體送入腫瘤細胞進行基因沉默機制，並藉由添加聚組胺酸及聚麩胺酸來達到聚合體對酸鹼敏感的感受性，利用細胞內環境酸鹼值的差異將聚合體送入細胞內釋放出小分子核糖核酸，最後於細胞核內成功進行表現。此研究中顯示，添加兩種胺基酸後的聚乙烯亞胺高分子材料適合在腫瘤細胞特有的酸性環境中使用，並且明顯的降低了原有的細胞毒性。
由上述研究可知，利用細胞環境酸鹼值的不同來開發具有酸鹼敏感特性的高分子材料做為基因傳遞的載體可增加基因轉導的標靶性，無論是使用病毒或小分子核糖核酸做為基因傳遞的來源，皆可獲得印證。說明了添加具有酸鹼敏感性特質的反應劑於非病毒型載體上可增進基因傳遞系統的轉導效率，並且可更準確的達到在特定部位表現的效果。

關鍵字

基因傳遞 ； 酸鹼敏感型 ； 水膠 ； 病毒 ； 聚乙烯亞胺 ； 小分子核醣核酸

並列摘要

Controlled gene delivery is a highly activated approach using viral or nonviral carriers. We used different strategies of gene transfer based on polymeric matrix or carrier for improved their efficiency. In the section of Chapter 2, we designed a biodegradable and pH sensitive polymeric matrix for localized delivery of recombinant adeno-associated virus serotype 2 (rAAV2). The rAAV2 containing the green fluorescent protein gene (rAAV2-GFP) were loaded into poly(ethylene glycol) (PEG) hydrogels, with and without incorporation of poly-L-histidine (polyHis). The fraction of polyHis used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of rAAV2-GFP. As a result, release and transduction efficiency of the rAAV2-GFP from PEG-polyHis hydrogel in human HT-1080 fibrosarcoma cells increased significantly compared to a PEG hydrogel. Transduction rate can be controlled by the hydrogels’ polyHis concentration and is sensitive to localized decreases in pH consistent with inflammation. In the section of Chapter 3, we successfully constructed a high-efficient dual functional gene vector based on polyethylenimine (PEI)-based nonviral vectors. A small interfering RNA (siRNA)-loaded polyelectrolyte constructed with branched polyethylenimine (bPEI) and copolymer, consisting of PEG, histidine (His), and glutamic acid (Glu) was developed in order to provide a tumor acidosis-triggered delivery system with low cytotoxicity.
In summary, we provide some insight into strategies developed for nonviral vectors to overcome intracellular barriers, using the strategy of pH-controllable to nuclear targeting, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization expression.

並列關鍵字

gene delivery ； pH-sensitive ； hydrogel ； virus ； polyethylenimine ； siRNA

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