- 學位論文
雙功能中孔洞奈米藥物載體於治療神經退化性疾病之設計
Design of Nanodrug for Neurodegenerative Disease by using Dual-functionalized Mesoporous Silica Nanoparticle
摘要
阿茲海默症為一種神經退化性疾病且隨著年齡增長而增加，根據統計2014年全球超過4千萬人因病所苦。但目前為止，尚未找到有效的方法來治療或者延緩發病，因此治療阿茲海默症的方法，其研究及開發上相當的急迫性與重要性。 RhoG(質體DNA)屬於GTPases家族成員之一，根據過去文獻顯示RhoG藉由活化下游蛋白Rac與cdc42以調節細胞骨架誘導神經細胞分化。此外，薑黃素(Curcumin, CUR)萃取自薑黃，具有抗腫瘤以及抗發炎反應等藥物活性，過去在神經退化性疾病研究上發現其與發炎反應息息相關，因此薑黃素在治療阿茲海默症上非常具有潛力，然而薑黃素本身不溶於水，因此在生物應用上受到限制。 在本次研究上，我們使用PEG修飾的奈米材料(mesoporous silica nanoparticle，簡稱MSN)作為藥物載體，因為MSN具有中孔洞結構、易修飾和好的生物相容性等優點，能夠同時載入Curcumin (於中孔洞結構中)與攜帶RhoG(藉由材料表面修飾正電荷吸)。另外，為了增加細胞吞噬與基因轉染效率，我們引入TAT peptide於實驗設計上，我們稱此奈米藥物為CUR@FMSN(+)/RhoG-TAT。當CUR@FMSN(+)/RhoG-TAT被neuro-2a (N2a)細胞吞噬後，我們藉由流式細胞儀鑑定吞噬效率以及定量活性氧化物質(ROS)，西方點墨法定量發炎反應相關蛋白表現，以及螢光顯微鏡觀察轉染結果。 在我們系統中，除了證明材料無生物毒性和好的細胞吞噬效率（接近99%吞噬）外並解決了薑黃素難溶於水所造成生物應用上的限制，此外我們也觀察到CUR@FMSN(+)/RhoG-TAT具有抗氧化、抗發炎及幫助神經生長等多重功能，因此我們認為MSN在神經退化性疾病的應用上具有相當大的潛力。
並列摘要
Alzheimer's disease (AD), a progressive neurodegenerative disorder (ND) of the elderly, affects more than 44 million people worldwide. So far, there is no effective method that treats or delays the progression of AD. Therefore, development of strategies for AD therapy is very important goal. RhoG, a member of small Rho family guanine nucleotide triphosphatases (GTPases), is able to induce neurite outgrowth through downregulating activation of Rac and cdc42, resulting in reorganizing the actin cytoskeleton and subsequent morphological changes. In addition, a well-known polyphenol compound, Curcumin, which was found from the rhizome of turmeric with antioxidant and anti- inflammatory properties, has already been noted for its therapy potential in neurodegenerative disease especially AD. However, due to limitations of poor bioavailability and aqueous solubility, curcumin is not suitable for biomedical applications. In our work, taking advantage of mesoporous silica nanoparticle (MSN), including its structure, easy modification and good biocompatibility, we proposed a concept of PEGylated MSN-based dual therapy and designed the nanodrug that contains curcumin(CUR) by MSN inner channel loading; MSN surface positive charge modification for RhoG gene adsorption; and TAT peptide enhanced the RhoG gene nuclear delivery and nonendocytosis mechanisms, named CUR@FMSN(+)/RhoG-TAT. MSN as a drug carrier improved the stability and bioavailability of curcumin. On the basis of the results shown in this study, Flow cytometry and confocal microscopy demonstrated the good cell uptake efficiency (approaching 99%) and localization of CUR@FMSN(+)/RhoG-TAT in neuro-2a (N2a) cell line. The expression levels of inflammatory related proteins inhibited by CUR@FMSN(+)/RhoG-TAT were carried out by using Western blotting. The levels of paraquat-induced ROS reduced by nanodrug were stained by DHE assays and quantified by flow cytometry. Our results evaluated that CUR@FMSN(+)/RhoG-TAT not only stimulated neurite outgrowth but also allowed neuron cells to against ROS-induced damage in N2a cells. Hence, the application of MSN might be a promising candidate method for treating NDs.
參考文獻
延伸閱讀
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