壁虎抗肿瘤活性单体透明质酸纳米粒肝靶向抗肿瘤研究

Object: .To prepare Glycyrrhetinic acid modified hyaluronic acid (GA-HA) nanoparticles as the Gekko swinhonis anti-neoplasm active component(GSAAC) carrier for liver targeting therapy, while analyzing Pharmacokinetics and antitumor efficacy of livertargeting GA-HA nanoparticles loaded with the GSAAC..Method: .(1).Conjugation of glycyrrhetinic acid modified hyaluronic acid (GA-HA) are produced by cross-linking method, its structural properties are investigated with 1H NMR. GA-HA nanoparticles are prepared by ultrsonic method, their physicochemical properties are performed by dynamic light scattering (DLS). Zeta potential critical aggregation concentration (CAC), biocompatibility and acute toxicity of GA-HA nanoparticles are also tested. .(2).Prepare GA-HA nanoparticles loaded with GSAAC monomer, evaluate the encapsulation efficiency(EE) and loading capacity(LE), test cytotoxicity and cellular uptake with MTS assay, confocal microscopy and flow cytometry respectively. .(3).The USPIO-labeled GA-HA nanoparticles are produced by chemical cross-linking method and injected into SD rats intravenously, its distribution are investigated at 5, 10,20 and 30mins with 3.0T MR molecular imaging method respectively. Drug-loaded nanoparticles are injected intravenously to beagle dogs and mice, drug concentration in blood and various organs are performed by triple quadrupole mass spectrometry. .(4).In order to study the anti-tumor efficancy of drug-loaded nanoparticles, we establish human hepatocarcinoma model in nude mice, and monitor the tumor size using NMR after intravenous injection of drug-loaded nanoparticles. Sacrifice mice and prepare specimen for pathological analysis (tumor volume, weight, HE staining, etc.) after various time point (7d, 14d and 21d). We also study the cell proliferation (PCNA), microvessle density and apoptosis (TUNEL / of caspase3) in human hepatocarcinoma tisse. So as to provide theoretic basis for reasonable and effective use of the GSAAC.

目的：制备壁虎抗肿瘤活性单体肝靶向透明质酸纳米粒，分析其药物代谢规律和靶向抗肿瘤作用。方法：①化学偶联法合成甘草次酸修饰透明质酸耦合物，核磁共振分析其结构。超声法制备纳米粒，分析其理化性质和生物相容性。②制备载壁虎活性单体透明质酸纳米粒，确定其包封率、载药量，MTS法检测载药纳米粒细胞毒性，共聚焦显微镜和流式细胞仪分析其细胞摄入特征。③化学偶联法合成USPIO标记的纳米粒，静脉注射SD大鼠后于5min、10min、20min和30min，3.0TMR分子影像实时监测纳米粒在大鼠体内的分布。静脉注射载药纳米粒，三重四极杆质谱测量动物(犬)血液、各脏器(小鼠)药物浓度。④构建人肝癌裸鼠模型，静脉注射载药纳米粒，应用MR定期检测癌块的变化，并于不同时间点（7d、14d、21d）处死裸鼠，进行病理学分析，肝癌组织细胞增殖、微血管密度及凋亡分析。研究结果将为中药壁虎合理有效用药提供可靠的理论依据。

本研究制备了甘草次酸修饰透明质酸肝靶向纳米粒（GA-HA）和pH响应肝靶向的混合纳米胶束（GA-HA/His-HA），分别包载壁虎抗肿瘤单体成分腺嘌呤(Adenine, A)和临床常用抗肿瘤药物阿霉素( adriamycin, ADM)，研究载药纳米粒的肝组织靶向性及其肝靶向抗肿瘤作用。. 成功制备了取代度为3.8GA-HA纳米粒，采用Dir/GA-HA纳米粒尾静脉注射昆明小鼠，注射1-3小时，小动物活体成像显示Dir/GA-HA在小鼠肝组织富集，呈明显的肝组织靶向性。. GA-HA纳米粒包载壁虎抗肿瘤活性单体腺嘌呤（adenine, A），成功制备了载药量为12.65%、包封率为87.27%的载腺嘌呤肝靶向性纳米粒A/GA-HA。按载药量2mg/kg体重尾静脉注射SD大鼠，测量动物血浆药物浓度，结果显示载药纳米粒在生物体内的存留时间延长。按载药量5mg/kg体重尾静脉注射昆明小鼠，不同时间点处死动物，检测A在不同组织中的分布。结果显示，肝组织相对摄取率re=2.62，re大于1表示药物制剂在该器官或组织有靶向性，肝组织相对于心、肺、肾的靶向效率te均大于1，表示药物制剂对靶器官比某非靶器官有选择性；肝组织药物峰浓度比te=2.02，ce值愈大，表明改变药物分布的效果愈明显。可见腺嘌呤经包载后在生物体内具有很好的肝靶向性。. MTT法细胞学实验及裸鼠荷瘤动物检测纳米粒对药物的抗肿瘤活性影响。10mg/kg载药纳米粒（A/GA-HA）腹腔注射，15d后处死荷瘤裸鼠，剥离瘤块并称重，SP法检测肿瘤细胞增殖核抗原（PCNA）和CD31表达。结果显示A/GA-HA的抑瘤作用明显增强，肿瘤组织细胞增殖指数PI显著降低，肿瘤组织微血管密度显著降低。显示A/GA-HA具有肝组织靶向性及更好的抗肿瘤作用。. 本研究还制备了兼具肝靶向性和pH响应性的透明质酸纳米混合胶束（GA-HA/His-HA），核磁共振氢谱表征化合物的结构后包载AMD，成功制备载药量为8.14%、包封率为91.37%的载药混合胶束。体外细胞学实验显示AMD/GA-HA/His-HA混合胶束与单纯药物相比有更好的肿瘤细胞增殖抑制作用，且表现良好的pH响应性。小鼠荷瘤动物实验显示，AMD/GA-HA/His-HA与单纯AMD相比，抗肿瘤作用明显增强。