Fluorescent Nanodiamonds for Biomedical Applications: Cytotoxicity, In vitro and In vivo studies

螢光奈米鑽石於生醫上之應用: 毒性、活體外與活體內之研究

蜜瑪拉(VAIJAYANTHIMALA.V)

指導教授 ： 張煥正 ； 倪其焜

國立清華大學/理學院/化學系/博士(2011年)

https://doi.org/10.6843/NTHU.2011.00247

摘要

Fluorescent nanodiamond is a novel carbon based nanoparticle with a variety of important and unique properties such as photostability, biocompatibility, facile surface functionalizability making it as a novel nanoparticle for both in vitro and in vivo applications. Nanodiamond upon high energy ion beam irradiation and subsequent annealing at high temperature, results in the formation of (N-V)0 and (N-V)- defect centers which emit far red fluorescence under visible light excitation. Aforementioned properties make FND as a robust nanoparticle for cell labeling, cell tracking, in vitro and in vivo imaging applications.

In brief, this research begins with the cytotoxicity studies of FNDs in various cell lines. Our studies suggested that FNDs are biocompatible to different cell lines including HeLa, 3T3-L1 and 489-2.1 cells. Further studies showed that the FND cellular uptake is by energy dependent, clathrin mediated endocytosis. Based on the cell doubling time measurements, we found ~10% of FNDs are exocytosed both in short term as well as in long term. In addition to the fluorescence, light scattering also serve as an important parameter that could be utilized during cell sorting. Furthermore, FNDs can serve as an alternative probe to the commonly used dye like CFSE. We have demonstrated the feasibility of using FNDs for in vivo imaging. Injected FNDs accumulate in axillary lymph node and we also developed a new powerful methodology for the quantitation of FNDs in tissues and organs. Finally, we have also demonstrated that FND can be used as a fluorescent marker for in vivo stem cell tracking. In conclusion, FND can serve as an ideal nanoparticle for different biological applications.

關鍵字

螢光穩定性 ； 螢光奈米鑽石 ； 細胞倍增時間 ； 活體幹細胞 ； 活體影像

並列摘要

螢光奈米鑽石為碳原子組成的新穎材料，許多重要且獨特的性質，如：螢光穩定性、生物相容性、表面修飾多元性等，使其成為生物體外及活體影像應用的重要材料。經過高能離子束靶擊及高溫淬火的奈米鑽石內，產生(N-V) 0、(N-V)- 缺陷中心，以可見光激發此類缺陷中心 ， 可放出近紅外波長的螢光。上述性質使螢光奈米鑽石具有細胞標記、細胞追蹤及生物體外及活體影像應用的潛力。 此篇論文首先研究螢光奈米鑽石對不同細胞株的細胞毒性，結果顯示螢光奈米鑽石在3T3-L1, 489-2.1 細胞株內生物相容性好; 進一步研究顯示, 細胞藉由網格蛋白輔助 (clathrin mediated） 吞噬螢光奈米鑽石； 細胞倍增時間 （cell doubling time）實驗中， 約百分之十的螢光奈米鑽石在短期及長時間內被細胞排出 ，除了螢光之外 ， 散射光亦是細胞分選的重要參數，本實驗證明螢光奈米鑽石亦可做細胞分選的探針， 與常用的有機染料CFSE具有相似功能 ； 螢光奈米鑽石亦展現活體影像的應用潛力 ， 注射入老鼠的螢光奈米鑽石累積於輔助淋巴結中 ， 並由本實驗發展的方法進行組織及器官內螢光奈米鑽石的定量。最後，螢光奈米鑽石亦被證實可做為活體幹細胞追蹤的螢光標籤。總結，螢光奈米鑽石為生物應用的理想材料。

並列關鍵字

Fluorescent Nanodiamonds ； Flowcytometry ； Endocytosis ； Exocytosis ； Invivo imaging

參考文獻

9. Fu, C.-C.; Lee, H.-Y.; Chen, K.; Lim, T.-S.; Wu, H.-Y.; Lin, P.-K.; Wei, P.-K.; Tsao, P.-H.; Chang, H.-C.; Fann, W., Characterization and application of single fluorescent nanodiamonds as cellular biomarkers. Proceedings of the National Academy of Sciences 2007, 104 (3), 727-732.

24. Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C., Nanoparticles in Medicine: Therapeutic Applications and Developments. Clinical Pharmacology and Therapeutics 2007, 83 (5), 761-769.

28. Schrand, A. M.; Huang, H.; Carlson, C.; Schlager, J. J.; Ōsawa, E.; Hussain, S. M.; Dai, L., Are Diamond Nanoparticles Cytotoxic? The Journal of Physical Chemistry B 2006, 111 (1), 2-7.

3. Moustakas TD: Synthetic Diamond: Emerging CVD Science and Technology. Spear KE, Dismukes JP (Eds). John Wiley, New York, USA 145–192 (1994).

4. Dion I, Baquey C, Monties JR: Diamond – the biomaterial of the 21st century? Int. J. Artif. Organs 16, 623–627 (1993).