奈米金粒子-doxorubicin結合物之細胞毒性研究:粒徑相關性和細胞內分佈情形

Abstract

本次研究是利用奈米金粒子(GNP)作為抗癌藥物doxorubicin(DOX)的載體,使DOX對細胞的毒性降低,並探討分子間的作用機制,了解毒性降低的原因。將雙官能基之橋接分子16-mercaptohexadecanoic acid (HS(CH 2 15 COOH, 16-MHA)置換且吸附於奈米金粒子表面上,置換後的奈米金粒子簡稱為GNP-MHA 。再將抗癌藥物DOX與GNP-MHA反應,讓DOX與GNP結合在一起，簡稱為GNP -DOX。我們選用的奈米金粒子的粒徑大小介於5奈米到50奈米之間。合成的GNP -DOX經由長時間的透析做純化。此外,我們用UV-visible鑑定奈米金粒子與藥物結合的效率。以未經任何化學反應處理的Free DOX作為positive control。人類卵巢癌細胞(PA-1)於培養箱培養12小時,達穩定生長狀態後給予藥物(free DOX, 5-nm GNP-DOX, 8-nm GNP- DOX, 12-nm GNP- DOX, 17-nm GNP- DOX, 37-nm GNP- DOX, 和50-nm GNP -DOX)24和48小時,實驗結果顯示, GNP和DOX結合之後, 藥物對癌細胞的毒性均較 Free DOX 低,且藥物大部份分佈在細胞核周圍。GNP-DOX conjugates於細胞內的分佈情形,指出藥物對細胞毒性的降低,可能是因為GNP-DOX conjugates 進入細胞核的效率較差。 並且,從p53和caspase-3的免疫螢光染色結果顯示, GNP-DOX造成細胞死亡的原因與free DOX相同, 是經由藥物誘發細胞凋亡。

The aim of this study is to explore possible toxicity reducing effect of gold nanoparticle (GNP) as drug carrier bringing doxorubicin (DOX) into cells and study the molecular mechanism of reducing effects. 16-mercaptohexadecanoic acid (MHA) was first bound to GNP and served as cross-linker. The GNP-MHA conjugate reacted with DOX to generate GNP-DOX conjugate. GNPs ranged from 5-nm to 50-nm. The efficiency of chemical reaction was monitored by UV-visible. The conjugates were purified through serial dialysis. Free DOX was applied as positive control. PA-1 cells was cultured for 48 hours and incubated with free DOX, 5-nm GNP-DOX, 8-nm GNP- DOX, 12-nm GNP- DOX, 17-nm GNP- DOX, 37-nm GNP- DOX, and 50-nm GNP -DOX. In addition, GNPs of various diameters were incorporated into free DOX to serve as control. All of GNP-DOX conjugates were reduced toxicity. Cellular locations of GNP-DOX conjugates indicated that the reducing effect might be due to GNPs-DOX conjugates translocate into nucleus difficultly. Increased nuclear localization of p53 and caspase-3 was observed in cells exposed to free DOX and GNP-DOX. The result indicate that free DOX and GNP-DOX induce cell death through apoptosis.