發展以螢光奈米鑽石攜帶短干擾核醣核酸之生物醫學應用

發展安全且高效能的生物載體在癌症以及許多疾病的治療上面扮演至關重要的角色。以基因標靶治療為例，也就是利用設計特定基因序列的遺傳序列送入病人體內，藉由調整該基因的表現來治療相關的遺傳或後天疾病，這種治療方式已廣泛應用在許多癌症的臨床治療上面。其中短片段雙股核醣核酸干擾小體(siRNA)係藉由設計與特定基因相關的短片段雙股核醣核酸序列送入體內，抑制該基因的表現量來治療與該基因相關的疾病的方式，已被視為十分具有潛力也廣泛應用在臨床治療的重要研究領域。目前雖然有許多備受矚目的載體，但其本身的結構以及組成均可能造成對生物體的毒性，不僅如此，這些多功能的載體在生物體內長期累積可能造成免疫以及其方面的傷害，因此能夠發展新一代結合影像診斷及標靶治療於一身，最重要的是具有活體相容性的多功能奈米載體，更是生醫材料領域研究的當務之急！螢光奈米鑽石是經由高速粒子束撞擊，再以熱能結合創造出晶格缺陷，這個真空的晶格空缺使螢光奈米鑽石在約560 nm波長的光激發之下，可以有效且穩定地放出約700 nm波長的大紅色永恆的螢光。而且，螢光奈米鑽石具有高光穩定性、完全無光漂白、且沒有blinking的獨特性質，使螢光觀察得以長期且穩定的執行。螢光奈米鑽石不具生物毒性，而且其表面特性很容易處理成含氧的官能基，再進一步修飾得以與核酸分子或是蛋白質作結合。本篇研究將以口腔癌細胞為基礎，系統性的研究螢光奈米鑽石與標的細胞及活體之間的生物交互作用，並找出適合的生物分子裝載條件，期望能達到最終發展口腔癌治療的依據。我們以短片段雙股核醣核酸干擾小體與螢光奈米鑽石及帶電荷聚合體結合，並且施加輕微短時間的離心作用，找到生物分子裝載最佳化之條件。並且利用奈米鑽石的螢光做為細胞追蹤工具，研究螢光奈米鑽石進入細胞並於期內所引發之交互作用。我們也同時使用冷光胜肽標定異體移植口腔癌之小鼠，了解螢光奈米鑽石結合特定雙股短片段核醣核酸在生物系統之作用效果與情形。我們的結果顯示，螢光奈米鑽石可作為極佳的生物分子載體，並證明我們可利用螢光奈米鑽石在無毒性反應下，達到更佳的基因抑制的效果。此外我們也發現螢光奈米鑽石除了可藉由一般的細胞胞吞作用進入細胞以外，更能夠引發細胞的大胞飲作用。而離心的作用增加粒子與細胞表面接觸的頻率，因而增加活化細胞的胞吞作用，以達到更佳的基因抑制效果。我們藉由深入的探討螢光奈米鑽石與生物系統的交互作用，可望發展出可以追蹤目標細胞的核酸裝載系統，成為多功能奈米載體在癌症治療的應用上極具潛力的明日之星!

關鍵字

基因治療；螢光奈米鑽石；短干擾核醣核酸

並列摘要

Delivery technique holds the key to successful treatment of cancer and many diseases. For example, gene therapy is a promising method which manipulates the defective genes by delivery nucleotides into cells to treat inherited and acquired diseases that are currently considered incurable. It has been exercising in many clinical trials. RNA interference has been considered one of the most promising therapeutic platforms by introducing siRNA into the cell and switch off certain disease-relating genes. There are many vectors reported to be useful for genetic delivery, however, most of them have the concerns of cytotoxicities due to the component molecule and the induction of long-term damage. For these reasons, an efficient delivery system for siRNA remains to be developed.Type Ib fluorescent nanodiamonds (FNDs) can emit no photobleaching and no photoblinking fluorescence from their nitrogen-vacancy point defects, at a spectral range well suited for long term observation in living cells. They have good biocompatibility and can be easily functionalized for specific or nonspecific binding with biomolecules. In this study, based on the experiences of oral cancer, we propose to use cell and animal based oral cancer models to address the biological responses elicited by FNDs, and to simultaneously assess the best combinations of functionalization and cargo loading for future applications. We combined the FND with siRNA and common transfection reagents, applied with mild centrifugation to explore the gene-inhibition efficiency. In the same time, we studied the uptake mechanism and cellular response to the internalization of bare and cargo loaded FNDs. Besides, we tested the genetic delivery efficiency of FND-optimized siRNA complexes into xenografe bioluminescence-marked oral cancer animal models. From our results, FND showed dramatic improvement in silencing effect with good biocompatibility. And FND may be internalized into cells by macropinocytosis besides normal endocytosis pathways. We found that centrifugation facilitated the entrance of FND by increasing the contact between the particles and cellular surface, thus triggered more macropinocytosis responses to include more siRNA-combined FNDs. We provide the first and fundamental knowledge for a well-grounded development in biomedical applications of the FNDs. This is a powerful and promising achievement for subsequent improvements towards the final achievement of revolutionary new capabilities in the diagnosis, tracing and curing of cancer, as well as the treatment of other difficult diseases.

並列關鍵字

gene therapy ； fluorescent nano-diamond ； siRNA

參考文獻

1. Ofek, P., Fischer, W., Calderon, M., Haag, R. & Satchi-Fainaro, R. In vivo delivery of small interfering RNA to tumors and their vasculature by novel dendritic nanocarriers. FASEB J (2010).

2. Kim, S.H., Jeong, J.H., Kim, T.I., Kim, S.W. & Bull, D.A. VEGF siRNA delivery system using arginine-grafted bioreducible poly(disulfide amine). Mol Pharm 6, 718-726 (2009).

3. Shen, Y. Advances in the development of siRNA-based therapeutics for cancer. IDrugs 11, 572-578 (2008).

4. Devi, G.R. siRNA-based approaches in cancer therapy. Cancer Gene Ther 13, 819-829 (2006).

5. Aagaard, L. & Rossi, J.J. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59, 75-86 (2007).

國際替代計量

發展以螢光奈米鑽石攜帶短干擾核醣核酸之生物醫學應用

全文下載

主題瀏覽