以蛋白質所包覆合成的金奈米團簇(gold nanoclusters,NCs)具有好的螢光特性及生物相容性，蛋白質上多種胺基酸提供了可修飾不同分子的優點，使之成為具有功能性與專一性的探針。研究中在牛血清蛋白質金奈米團(BSA-AuNCs)表面修飾上雌激素(17α-ethinyl estradiol, EE2)，利用一段聚乙二醇(polyethylene glycol, PEG)具有疊氮基(azide)和胺基(amine)，雌激素上的炔基(alkyne)能與疊氮基經由銅金屬催化行環化加成反應，形成1,4-雙取代-1,2,3-三氮唑的五員雜環。而PEG另一端胺基則可利用交聯試劑與蛋白質上的羧酸基經由醯胺化反應鍵結。將此合成出來的E2-BSA-AuNCs應用在乳癌細胞顯影上，在具有雌激素受體的MCF-7乳癌細胞中，E2-BSA-AuNCs會隨著時間由細胞膜慢慢進入細胞核，在時間2小時時已完全進入細胞核，推測E2-BSA-AuNCs是經由細胞膜的受體辨認而進入到細胞中並進入到細胞核中，而對照組BSA-AuNCs則無法進入細胞內；另以市售螢光染料Cyanine 3上修飾上雌激素(E2-Cy3)作為比較，反應時間在6小時才進入到細胞核，表示E2-BSA-AuNCs具有更好的生物相容性。而在不具雌激素受體MD-MBA-231乳癌細胞實驗結果顯示，E2-BSA-AuNCs不具有螢光訊號表現，但是E2-Cy3在細胞質卻有螢光表現，推測E2-Cy3是藉由被動傳輸進入到細胞中。另外以MTT試驗測試比較細胞增殖速率，實驗結果顯示在相同的藥品濃度下E2-BSA-AuNCs比E2-PEG具有較高的增殖率，顯示所合成的E2-BSA-AuNCs更容易進入到細胞核中作用。實驗中所合成的BSA-AuNCs及E2-BSA-AuNCs以質譜儀作為鑑定，金奈米團簇為15個金原子所組成，而有5個E2-PEG鍵結在蛋白質表面；而在原子力顯微鏡下，有修飾E2-PEG的粒子尺寸比未修飾的直徑變大約2nm；而E2-PEG與E2-Cy3的合成鑑定，經由質譜儀與核磁共振光譜作為鑑定。

Protein-protected fluorescent gold nanoclusters (AuNCs) have attracted many attentions due to their superior properties such as low toxicity, high biocompatibility and feasible functionalization. In this study, bovine serum albumin-protected AuNCs (BSA-AuNCs) was synthesized and functionalized with 17β-estradiol-azide-poly(ethylene glycol) conjugates (E2-PEG) via carbodiimide crosslinker chemistry (EDC/NHS) and used to selectively target estrogen receptor positive breast cancer cell. E2-PEG derivative was synthesized from Ethinyl Estradiol (EE2) by CuI-assisted azide-alkyne cycloaddition. The products, BSA-AuNCs and E2-BSA-AuNCs were purified by gel filtration chromatography or sucrose density gradient and characterized by fluorescence spectroscopy. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Fourier Transform Infrared spectroscopy (FT-IR), Matrix assisted laser desorption mass spectroscopy (MALDI-MS). The data showed each BSA-AuNCs contains eighteen Au atoms and five E2-PEG molecules with an emission maximum at around 640 nm (380 nm excitation) which was not much altered by E2-PEG conjugation. Furthermore, the volume of BSA-AuNCs (or E2-BSA-AuNCs) was found to expand by four times as compared to that of the native BSA. This resulted in a 4-fold decreased in density as observed by sucrose density gradient and yielded a loosely packed E2-BSA-AuNCs assembly. Based on a time course study using confocal fluorescence microscopy, E2-BSA-AuNCs were shown to selectively target estrogen receptor positive MCF-7(ER+) breast cancer cell by membrane ER assisted endocytosis rather than by passive diffusion of the free drug. In contrast, E2-Cy3 could not differentiate MCF-7(ER+) versus MDA-MB-231(ER-) cell due to the passive transport of the free estrogen. MTT assay reveals that AuNCs conjugation increases the estrogen potency by 1.6 folds due to increased rates of nuclei transport.

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