Hepatocellular carcinoma is a major health problem, the second highest incidence of cancer in China, and the most common gastrointestinal cancer. No effective drug exists for patients with advanced hepatocellular carcinoma. In recent years, Chinese medicine has achieved considerable success against hepatocellular carcinoma. Hepatocellular carcinoma belongs to diagnosis of blood stasis in Chinese medicine which is treated by promoting blood circulation. Salvia miltiorrhiza is one of traditional Chinese medicine, and its main active ingredient is Tanshinone IIA. Tanshinone IIA has showed obvious anti-cancer effects on a variety of cancer cells, especially liver cancer cells, it can inhibit proliferation and induce apoptosis, and its anti-tumor effect which has attracted scientists’ common concern. However, due to the poor water solubility and poor uptake in tissue and cells of Tanshinone IIA, its clinical application is limitted. It is important to develop safe and effective drug delivery systems for anti-cancer drugs in rational use of traditional Chinese medicine in anti-cancer. Purpose: Glycyrrhetinic acid, a mediator of liver targeting was used to funtional system with liver targeting and redox sensitive of polymeric micelles system (TAN IIA /GAPEG-SS-PLGA), which is a preparation of safe, efficient, targeted, less toxic drug delivery system in anti-hepatoma drug delivery of Tanshinone IIA. Method: GA-PEG-SS-PLGA were synthesized by chemical grafting method and characterized by 1H NMR and FTIR. TAN IIA/GA-PEG-SS–PLGA micelles were prepared by solvent evaporation method and the drug loading, encapsulation efficiency, size distribution, the zeta potential, redox sensitivity, in vitro release properties were evaluated. The cytotoxicity of blank micelles and drug loading micelles were evaluated by MTT assay in HepG2 and A549 cells. In vitro anti-tumor activity of TAN IIA/GAPEG-SS–PLGA in HepG2 was evaluated by the expression of intracellular ROS, cell cycle arrest, and cell apoptosis, and in vivo antitumor study. Fluorescence microscopy and cell flow cytometry were used for qualitative and quantitative cellular uptake. Results: TAN IIA/GA-PEG-SS–PLGA micelles have been prepared. The mean particle size (157.6±1.3 nm, PDI=0.18±0.01) and Zeta potential (-40.3±1.3 mV) were measured by dynamic laser light scattering (DLS), which conformed to the TEM image. The drug loading and encapsulation efficiency of TAN IIA/GA-PEG-SS–PLGA micelles were 6.7±0.1% and 74.1±0.6%, respectively. The GA-PEG-SS–PLGA micelles showed redox-sensitivity in PBS with 10 mM DTT and size of GA-PEG-SS-PLGA micelle increased from 185 nm to 1000 nm after 8 h, while the mean particle size of GA-PEG- PLGA micelle size was stable at 145 nm over 24 h in 10 mM (DTT). In vitro release revealed that approximately 90% and 20% of TAN IIA were released from GA-PEGSS-PLGA micelles and GA-PEG-PLGA micelles, respectively, in 10 mM DTT in 24 h. The MTT assay in HepG2 and A549 cells revealed TAN IIA/loaded GA-PEG-SSPLGA micelles exhibited blatant hepatoma-targeting and extremely enhanced antitumor effects in HepG2 cells with the half maximal inhibitory concentration (IC50) of 1.50 μmol/L, which approximately 1.8-fold lower than that of PEG-SS-PLGA and 3.3-fold lower than that of free TAN IIA under the same conditions. The anticancer effect of the TAN IIA/GA-PEG-SS-PLGA micelles was investigated in HepG2 cells. The intracellular ROS level of TAN IIA/loaded GA-PEG-SS-PLGA micelles was obviously up-regulated and 3.4 times higher than free TAN IIA. TAN IIA/GA-PEGSS–PLGA micelles significantly enhanced S phase arrest and increased rates of apoptosis in HepG2 cells. It is to be noted, the cellular uptake was evaluated by nile red-labeled GA-PEG-SS-PLGA micelles by confocal laser scanning microscopy and the results showed that payload was efficiently released into cytoplasm and entered into the nuclei in HepG2 cells but little nile red found in the nuclei in A549 cells. Flow cytometry showed that intercellular payload level of nile red/GA-PEG-SS-PLGA micelles was much higher than that of free Tanshinone IIA in HepG2 cells, which has significantly enhanced the ability of nile red/GA-PEG-SS-PLGA micelles to enter into cancer cells, thus contributing to improve the anti-tumor effect. Conclusions: In this study, glycyrrhetinic acid-mediated liver targeting and redoxsensitive micelles, a nano carrier of Tanshinone IIA, were evaluated by nanosystems, analysisi of in vitro anti-tumor activity and mechanisms, cellular uptake research, liver targeting and redox-sensitive dual mechanisms. TAN IIA/GA-PEG-SS–PLGA can significantly improve the anti-hepatocellular carcinoma effects. These hepatomatargeted and redox-responsive GA-PEG-SS-PLGA micelles can be a potential drug carrier for poor water soluble active ingredients and improveing their clinical application against HCC.

肝癌在我國癌症發病率中高居第二位，是最常見的消化道腫瘤，目前臨床對 中晚期肝癌尚無特效藥物。近年來中醫藥抗肝癌研究取得一定成績，肝癌的臨床 表現在中醫診斷學屬於血瘀證，中醫多採用活血化瘀法治療。丹參是一味經典和 有效的活血化瘀中藥，其主要成分和有效成分 丹參酮 IIA 表現出較明顯抗腫瘤 作用，對多種實體腫瘤尤其是肝癌具有較好的抑制腫瘤增殖及誘導細胞凋亡的作 用，其抗腫瘤作用受到科學家的普遍關注。但由於其存在水溶性差，組織及細胞 攝取差，抗腫瘤作用不如化學藥物強等不足，很大程度上限制了其臨床運用，開 發安全有效的新型遞藥系統已成為合理利用中藥抗腫瘤的重要手段之一。 目的：以甘草次酸為肝靶向介導物，同時基於腫瘤細胞特異微環境，構建肝 靶向和還原敏感雙重機制介導的丹參酮 IIA 聚合物膠束系統（TAN IIA/GA-PEGSS-PLGA），為製備安全、高效、靶向、低毒的丹參酮 IIA 抗肝癌遞藥系統奠定 基礎。 方法：首先採用基團活化化學接枝法合成 GA-PEG-SS-PLGA 聚合物材料， 並採用核磁共振和紅外分光光度法對其進行表徵鑒定；隨後，採用溶劑揮發法製 備載藥聚合物膠束，並對其載藥量、包封率、納米膠束粒徑分佈、電位、還原敏 感性、體外釋藥性能等性質進行了考察；其次，採用 MTT 法，以人肺癌 A549 細 胞為對照組，考察丹參酮 IIA 聚合物膠束對人肝癌 HepG2 細胞的體內和體外的 抗腫瘤作用，並進行了細胞內 ROS 表達、細胞週期阻滯、細胞凋亡分析檢測表 達等一系列抗肝癌機制的研究，以及體內抑制腫瘤的考察，同時利用共聚焦顯微 鏡和流式細胞儀對聚合物膠束在細胞內的攝取情況進行定性和定量分析。 結果：本研究成功合成並製備出 GA-PEG-SS-PLGA 聚合物膠束，其粒徑為 157.6±1.3nm（PDI=0.18±0.01）；Zeta 電位為-40.3±1.3mV。透射電子顯微鏡下觀 察發現，GA-PEG-SS-PLGA 聚合物膠束基本呈類球形，且分散性良好，無聚合粘 連。聚合物膠束包載丹參酮 IIA 的包封率為 74.1±0.6%，載藥量為 6.7±0.1%。該 聚合物膠束表現出較強的還原敏感性，在 10 mM DTT 溶液中，聚合物膠束粒徑 在 24 小時內從 185 nm 增長到 1000 nm，同時有 90%的丹參酮 IIA 從 GA-PEGSS-PLGA 聚合物膠中釋放出來，而無還原敏感的膠束粒徑在 24 小時內均保持在 145 nm左右，相同時間內只有 20%的丹參酮 IIA 從中釋放出來。MTT 實驗結果表明丹參酮 IIA 聚合物膠束能顯著抑制 HepG2 的增殖。藥物 作用 48 小時後，丹參酮 IIA 聚合物膠束的 IC50 為 1.50 μmol/L，比無介導機制的 載藥膠束低 1.8 倍，比原藥低 3.3 倍。採用丹參酮 IIA 聚合物膠束作用 HepG2 細 胞 2 小時後，細胞內 ROS 水準明顯升高，細胞核皺縮變小，提示可能與其誘導 細胞凋亡有關；定量分析結果表明，丹參酮 IIA 聚合物膠束作用細胞後，細胞內 ROS 水準是原藥作用的 3.4 倍，並且顯著增強對 HepG2 細胞 S 期阻滯率，增加 HepG2 細胞凋亡率。通過尼儸紅標記 GA-PEG-SS-PLGA 聚合物膠束，我們用共 聚焦顯微鏡和細胞流式儀觀察到包載尼儸紅的 GA-PEG-SS-PLGA 聚合物膠束在 HepG2 細胞的攝取顯著高於無介導機制的聚合物膠束以及遊離尼儸紅，主要分 佈於細胞質和細胞核周圍，由此提示 GA-PEG-SS-PLGA 載藥膠束較原藥相比， 其進入腫瘤細胞的能力顯著增強，從而有利於提高藥物抗腫瘤活性。 結論：本研究採用甘草次酸介導肝靶向，同時結合還原敏感釋藥機制的聚合 物膠束作為丹參酮 IIA 的載體 ，通過載體材料的評價、納米體系理化評價、體 外抗腫瘤活性及機制分析、細胞攝取的研究揭示，丹參酮 IIA 聚合物膠束具有肝 靶向及還原敏感雙重作用機制，能顯著提高抗肝癌作用，為難溶性中藥抗腫瘤活 性成分臨床應用提供了新的研究基礎和研究方向。 關鍵字：膠束；還原敏感；甘草次酸；肝靶向；肝癌治療； 丹參酮 IIA