醇溶玉米蛋白/酪蛋白鈉奈米粒子系統於小干擾RNA基因傳遞之製備與特性之應用研究

基因治療已經成為研究的趨勢之一，小干擾RNA更是成為一個有潛力的治療方式。在本研究中利用在酸性環境下帶正電的醇溶玉米蛋白和帶負電的小干擾RNA以及酪蛋白鈉形成的奈米粒子將小干擾RNA轉染致癌細胞中，研究中使用的小干擾RNA是抑制 Integrin-linked kinase(ILK)基因，抑制此基因將抑制癌細胞的增生以及轉移和促使癌細胞的凋亡。單純的醇溶玉米蛋白奈米粒子在離子強度較高的環境下容易形成較大的粒子或是沉澱，因此加入酪蛋白鈉使得粒子能夠穩定的存在上述的環境進而達到轉染的效果。在從不同廠牌中的醇溶玉米蛋白中，利用形成的奈米粒子大小以及利用蛋白質電泳判斷甲型醇溶玉米蛋白含量來篩選使用的醇溶玉米蛋白來源。在本研究中，醇溶玉米蛋白奈米粒子搭載小干擾RNA的粒徑約為100至250奈米，為帶負電的粒子，經由螢光顯微鏡觀察由醇溶玉米蛋白轉染有螢光標記的小干擾RNA，可以發現螢光在細胞中的存在，證明此系統在細胞實驗中能夠將小干擾RNA攜帶進入細胞。研究中轉染效果利用即時聚合酶鏈式反應和商業轉染試劑TransIT TKO®比較並沒有明顯的差異，且以ILK圍標的的小干擾RNA在細胞存活率中也有明顯的抑制生長效果。

關鍵字

醇溶玉米蛋白；小干擾RNA ；奈米載體；基因遞送

並列摘要

Gene therapy has become a promising way of the treatment of cancer and other disease. The aim of this study is to develop a system to deliver small interfering RNA (siRNA) to inhibit the proliferation and migration of cancer cell. In this system, the nanoparticle is design to overcome the obstacles of gene deliver in vitro or even in vivo, protecting and deliver siRNA to the target cancer cells. Zein, a prolamin from corn is the main material to form a complex with negative charge siRNA and sodium caseinate in low pH environment. The empty zein nanoparticle without sodium caseinate is about (113.7±1.43 nm) in water. But the zein particles become larger (≥1000 nm) or precipitation after existing in higher ionic strength environment. The purpose of adding sodium caseinate to the process of forming zein nanoparticles is to maintain the particle size and avoid precipitation in higher ionic strength environment. The zein-sodium caseinate nanoparticles is stable in Hank’s balanced salt solution (HBSS) after 24 hours (174.0±1.93 nm). Gel retardation assay showed the complexation of the zein nanoparticle with siRNA. The silencing effect in vitro is compared with commercial product, TransIT TKO®. Real-time PCR showed barely difference between zein nanoparticles and positive control. About 80% silencing rate was shown in real-time PCR. The target of the siRNA is integrin-linked kinase (ILK), silencing of the ILK mRNA inhibited cells migration and proliferation. The silencing effect was measured by real-time PCR and compared with negative control and positive control. The MTT assay showed good inhibition of cell proliferation after transfection of zein-siRNA nanoparticle.

並列關鍵字

Zein ； Sodium caseinate ； Small interfering RNA ； Gene delivery ； Integrin-linked kinase

參考文獻

1. Jeong, J.H., T.G. Park and S.H. Kim, Self-assembled and nanostructured siRNA delivery systems. Pharmaceutical Research, 2011. 28(9): p. 2072-85.

2. Kim, W.J. and S.W. Kim, Efficient siRNA delivery with non-viral polymeric vehicles. Pharm Res, 2009. 26(3): p. 657-66.

3. Resnier, P., T. Montier, V. Mathieu, J.P. Benoit and C. Passirani, A review of the current status of siRNA nanomedicines in the treatment of cancer. Biomaterials, 2013. 34(27): p. 6429-43.

4. Grimm, D., Small silencing RNAs: State-of-the-art. Advanced Drug Delivery Reviews, 2009. 61(9): p. 672-703.

5. Geusens, B., N. Sanders, T. Prow, M. Van Gele and J. Lambert, Cutaneous short-interfering RNA. Expert Opinion on Drug Delivery, 2009. 6(12): p. 1333-1349.

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醇溶玉米蛋白/酪蛋白鈉奈米粒子系統於小干擾RNA基因傳遞之製備與特性之應用研究

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