奈米金被細胞攝入後會變得難以掌控，攝入一段時間後奈米金會被細胞吐出，而單純的奈米金塗佈表面又有穩定性的疑慮。為了要解決以上問題，本研究將葉酸 (Folic acid, FA) 奈米金粒子鑲嵌進PDLLA (Poly(D,L-lactic acid)) 奈米纖維中，製備具有長時間拉曼增強效果SERS (Surface enhanced Raman scattering) 的複合型材料，當細胞培養於複合材料表面，細胞表面和奈米金接觸時並不會造成胞吞及胞吐作用，且奈米金的露出程度可藉由纖維的表面崩解而進行控制，利於長時間的骨細胞特性追蹤。
本研究利用葉酸當作封端劑及還原劑，以一步加熱法合成出葉酸-奈米金粒子，並透過調整不同的葉酸濃度控制葉酸-奈米金粒子形狀，合成的葉酸奈米金粒子分為球型以及非球型，球型的葉酸奈米金粒子及非球型葉酸奈米金粒子粒徑分別為52.7±2.1 nm與63.3±1.7 nm， FTIR表面官能基檢測結果證實奈米金表面有葉酸的存在。在4℃下存放5天後，介達電位分別為-23 mV與-38 mV，處於中高度穩定狀態，經儲存30天後性質均未改變，證明具有良好的穩定性。
利用靜電紡絲技術製備PDLLA纖維，並將葉酸奈米金包埋進奈米纖維中，接著用SEM觀測不同添加量對於纖維型態的影響。奈米金添加量提升時，纖維仍具有良好的分散性及大小均一性，TGA及XRD結果亦證實纖維中具有奈米金粒子，TEM結果則顯示纖維內部奈米粒子的分佈頗為均勻。透過長時間浸泡奈米纖維觀察其膨潤現象，在第五天時纖維表面逐漸瓦解，此時葉酸-奈米金粒子出現明顯裸露。在體外細胞實驗中觀察到，鑲嵌式奈米纖維具有良好的生物相容性，當奈米金粒子在纖維中的濃度提高至0.53 wt%時，並不會干擾細胞活性、鹼性磷酸酶分泌及礦化的表現。
在骨分化前期時，無論是添加奈米金粒子或將細胞培養於鑲嵌式奈米纖維上，兩者皆能有效提升拉曼訊號。在骨分化中期與後期，鑲嵌式奈米纖維裸露出來的奈米金有效提升骨分化指標的拉曼訊號，至第二十天仍能看到明顯的骨分化蛋白與礦化表現。相對地，單純使用奈米金粒子的組別已無法觀察到顯著的骨分化訊號，以上結果證明利用包埋奈米金之奈米纖維可以進行骨細胞分化的長時間追蹤。
未來可以調整拉曼激發光波長以減少螢光訊號，或將纖維包埋不同種類奈米粒子進行SERS檢測，藉此開發出增強效果更顯著的複合型材料。也可選用不同細胞進行SERS分析，比較不同細胞間的拉曼訊號增強效果。

In previous studies, GNPs (Gold nanoparticles) were unmanageable after being ingested by cells and then would leave cells with invalid surface-enhanced Raman spectra (SERS) effects. It would be difficult to identify all the differentiation markers throughout the cell culture process. The direct coating of GNPs would lead to low stability, too. In order to solve these problems, the folic acid-gold nanoparticles (FA-GNPs) were embedded into PDLLA (Poly(D,L-lactic acid)) nanofibers. The composite materials developed in this research presented SERS effects with good duration and stability, allowing the long-term tracking of osteogenic differentiation.
In this study, FA was used as a capping and reducing agent to synthesize FA-GNPs by a one-step heating process. The shape of FA-GNPs was controlled to be spherical or non-spherical by adjusting FA concentrations. The particle sizes of spherical and non-spherical FA-GNPs were 52.7±2.1 nm and 63.3±1.7 nm, respectively. The surface functional groups were detected by FTIR, and the existence of FA on particle surfaces was confirmed. After being stored at 4℃ for 5 days, the particle size was unchanged. The zeta potential was -23 mV for spherical GNPs and -38 mV for non-spherical GNPs, indicating the high stability of suspended FA-GNPs.
The FA-GNPs were embedded into PDLLA nanofibers prepared by electrospinning, and SEM results indicated that the nanofibers were uniform and continuous with FA-GNPs addition. The existence and of FA-GNPs was confirmed by XRD and TGA. The uniform distribution of GNPs in nanofibers were testified according to TEM images. The swelling tests showed that the embedded GNPs were partially exposed after a 5-day incubation. From in vitro experiments, the composite nanofibers presented good biocompatibility. When FA-GNPs concentration was lower than 0.53 wt%, the cell activity, alkaline phosphatase (ALP) and biomineralization were not interfered.
In the early stage of osteogenic differentiation, either adding FA-GNPs or culturing cells on composite nanofibers effectively enhanced the SERS signal intensity of differentiation indicators. However, the Raman signals of late osteogenic differentiation on nanofibers with FA-GNPs were still significant, but nanoparticles without embedding were almost ineffective in signal enhancement. The results supported that the duration of FA-GNPs in SERS was promoted successfully by embedding in nanofibers, allowing the long-tern monitoring of cellular fate by using Raman spectroscopy.

In the future, the optimized wavelength of Raman excitation and different nanoparticles can be applied, which would reduce fluorescence noises and enhance reinforcement in SERS detection. Different cells can also be examined for the prevailing uses of the technique developed in this study.

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