標靶藥物載體之劑型開發用於肝組織再生之研究

Abstract

慢性肝炎及肝癌一直是國人十大死因之一，為了克服上述問題，肝臟的治療研究也普遍受到重視，對肝癌病患而言，目前常見的有肝切除手術、電燒、栓塞和標靶治療。肝細胞具備極強的再生能力，在接受物理刺激或化學刺激後，肝細胞會進行有絲分裂增生，然而目前治療肝臟碰到的主要問題為治療後肝機能低落造成肝臟復原率不佳。因此本研究首次嘗試將 Targeted drug delivery system (TDDS) 與肝組織再生的概念整合，以自製的標靶藥物載體來包埋能提升肝機能的小分子藥物來加速肝臟切除或電燒手術後的恢復速度，以期減少治療後肝細胞無法順利增生產生機能所導致的風險。 Mesoporous silica nanoparticles (MSNs) 是近年來研究上極為重視的奈米藥物載體，已被用於癌症的標靶治療，然而利用 TDDS 來標靶正常細胞，以其讓正常細胞能提升其自身機能的研究卻很少。本研究利用 MSNs 來包埋能促使細胞有絲分裂的 Lysine，再利用具有高生物相容性的 Chitosan 來修飾 MSNs-Lysine 的外表 (Chitosan-MSNs-Lysine; CS-ML)，Chitosan 不但能使 MSNs-lysine 在透過胞吞作用進入細胞時不會破壞細胞膜，並且還具有 pH-control 功能。最後再利用能與肝細胞表面受器結合的 Glycyrrhizin (GL) 將其 conjugated 在 CS-ML 上 (Glycyrrhizin-Chitosan-MSNs-lysine; GL-CS-ML)，預期這樣設計的標靶藥物載體 GL-CS-ML 能確實達到標靶肝細胞的功能。本研究結果證明：(1) 低濃度的 MSNs 對於肝細胞生長無毒害 (< 0.1mg/mL)，(2) 濃度為 0.1ml/mL 的 Lysine 能提升 30% 的肝蛋白分泌量，並且具備跟 HGF 有相似的機能可刺激肝細胞由休止期重新回到細胞周期的效果，(3) CS-ML 和 GL-CS-ML 的 Lysine 包埋率最大可達 70%，(4) MSNs 表面電位和粒徑大小分別為 -31.8mV 和 148nm，而 CS-ML 的表面電位和粒徑大小則為 48.9mV 和 222nm，且經過 TEM 的觀察能證實 Chitosan 能成功修飾於 MSNs 上，(5) 由流式細胞儀檢測，肝細胞胞吞 GL-CS-ML 的量是胞吞 CS-ML 的 1.8 倍，並且肝細胞在與 NIH-3T3 共培養的情況下，肝細胞胞吞 GL-CS-ML 的比例較高，(6) GL-CS-ML 對肝蛋白和尿素分泌量分別提升約 40% 和 48%。本研究成功地將設計的標靶藥物載體 GL-CS-ML 用於肝細胞的體外培養，並進一步證實有促使肝細胞再生與增進肝機能的潛能，相信這樣的研究對未來奈米材料應用於肝細胞培養的相關議題上具有很大的發展空間。

Mesoporous silica nanoparticles (MSNs) are solid materials possessing a honeycomb-like porous structure and hundreds of empty channels, it has been developed recently and regarded as an efficient drug carrier in the targeted drug delivery system (TDDS). However, most of the TDDS are designed to kill the tumor cells, and there are fewer related-reports about the cell-targeted strategy for enhancing the functionality of targeted cells. In this study, we applied MSNs to carry lysine for hepatocyte culture since lysine has been proved to enhance the mitosis of cell. Moreover, MSNs were encapsulated with chitosan to enhance the biocompatibility (Chitosan-MSNs-Lysine; CS-ML), and glycyrrhizin (GL) were conjugated to CS-ML for hepatocyte targeting (Glycyrrhizin-Chitosan-MSNs-Lysine; GL-CS-ML). Results have shown that: (1) MSNs showed no cytotoxicity to the hepatocytes in low concentration (<0.1mg/mL). (2) The albumin concentration in hepatocyte cultures with 0.1 mg/mL of lysine was increased 30% higher (this results are consistent with HGF) than that in control condition. (3) The maximum loading capacity of lysine on CS-ML and GL-CS-ML are both 70%. (4) MSNs particles showed a negative zeta potential of -31.8mV compared to the CS-ML, which showed highly positive zeta potentials of 48.9mV. The average particle size of CS-ML (222nm) was found to be relatively larger than MSNs (148nm). (5) The cell uptake amount of GL-CS-ML is 1.8 times higher than that in CS-ML in hepatocyte cultures. (6) The albumin and urea concentration in hepatocyte cultures with GL-CS-ML was separately increased 40% and 48% higher than that in control condition. In conclusion, GL-CS-ML has a high potential in hepatocyte-target study. Its significant impacts are far-reaching at scientific and industrial aspects and will reinforce our existing research strengths in liver tissue engineering.