螢光奈米鑽石的特性和生物應用：專一性、光毒性和能量轉移研究

Abstract

近來奈米鑽石在生物醫學上的應用極具潛力，因為螢光奈米鑽石具有良好的光學特性和生物相容性。本研究主要分為三部份：(1)將140 nm紅光奈米鑽石製作成專一性的探針，利用螢光影像研究運鐵蛋白和海拉細胞膜表面受體的作用力；(2)探討不同表面官能基40 nm和140 nm奈米鑽石對胞吞作用的影響，以及觀察奈米鑽石大量進入細胞後的毒性，並利用光熱效應選擇性殺死癌細胞；(3)首次探討綠光和紅光奈米鑽石間是否有螢光共振能量轉移，以拓展生物醫學上的應用。實驗結果顯示，透過運鐵蛋白和受體的作用力，螢光奈米鑽石成功變成專一性的探針並進入細胞，其螢光光譜和螢光生命期特性亦不受外在環境影響。利用流式細胞儀的分析，發現螢光奈米鑽石-運鐵蛋白複合物的胞吞作用效率，約是胺化奈米鑽石的2倍。奈米鑽石有效率的進入細胞後，我們觀察到細胞的生長速度變慢。此外，在532 nm的雷射照射下，專一性進入細胞的奈米鑽石可產生光熱效應，選擇性將被標靶的癌細胞殺死，使用的照射能量是低於殺死未被標靶細胞所需能量的二分之一。綠光奈米鑽石的螢光範圍，和紅光奈米鑽石的吸收範圍有良好的重疊，可形成很好的螢光共振能量轉移配對。我們分別將抗體和抗原固定在綠光和紅光奈米鑽石表面，利用抗原-抗體作用力相結合，其螢光共振能量轉移的效率約是18 %。本研究結果顯示螢光奈米鑽石在生醫應用的可能性，有潛力變成專一性探針和扮演成光熱治療試劑去殺死癌細胞。

Recently, fluorescent nanodiamonds (FNDs) have attracted considerable attention as optical probes in biological and medicinal applications because of their special optical properties and great biocompatibility. In this study, we investigated FNDs as specifically targeted probes to image the interactions of transferrins (Tf) with transferrin receptors (TfR) on HeLa cells, the mechanisms involved in the cellular uptake of surface-modified FNDs, the effects of particle size on the specificity of cellular uptake, and evaluated their cytotoxicity and phototoxicity following particle internalization, and the efficiency of fluorescence resonance energy transfer (FERT) between green and red fluorescent nanodiamonds (gFND and rFND). With Tf immobilized on the surface of rFNDs, we demonstrated that rFND-Tf bioconjugates successfully targeted HeLa cells through Tf-TfR interactions and the surface effects of chemical interactions on the emission of rFND are negligible. Through an analysis of the efficiency of internalized rFND-Tf and rFND-NH2 by flow cytometry, the endocytosis of rFND-Tf particles was highly effective and nearly twice as efficient as that of rFND-NH2 particles. The cytotoxicity of internalized particles resulted in decreasing the rate of proliferation of rFND-treated cells. An examination of the photothermal effect of internalized rFND-Tf particles under irradiation using a 532 nm laser revealed that the rFND-treated cells could be killed selectively using less than half the energy required for untreated cells. gFND and rFND could be a proper FRET pair because the emission of gFND overlaps well with the absorption of rFND. With antibody and antigen immobilized on the surface of gFND and rFND, we observed that the efficiency of FRET was 18 % after the combination of gFND and rFND through antigen-antibody interactions. Our results demonstrated that bioconjugated FNDs could be highly effective in the targeting of cancer cells for fluorescence imaging and photokilling with a minimum of collateral cell damage.