【作者】 李磊；

【导师】 张其清；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2010， 博士

【摘要】 本研究利用内源性小分子胆固醇疏水性修饰可德兰衍生物——羧甲基可德兰合成新型两亲性高分子聚合物并用以制备自聚集纳米粒子,用抗肿瘤药物表阿霉素为模型药考察载体对小分子药物的包埋及释放行为；利用体外细胞培养技术考察载药纳米粒子的抗瘤效应及细胞毒性,利用动物实验考察载药纳米粒子在动物体内的药代动力学和不同组织的分布情况。主要研究内容及结果如下：采用化学的方法合成了一系列不同取代度的胆固醇基羧甲基可德兰(CCMC),并用电位滴定、红外光谱(FT-IR)、氢核磁光谱(1H NMR)、X射线衍射(XRD)、差示扫描量热仪(DSC)、高效液相仪(HPLC)对原料、中间产物以及产物的化学结构及物理特征进行了表征；用硫酸铁氨比色法计算出系列CCMC的胆固醇取代度,取代度分别为2.3,3.5和6.4(%,每一百个羧甲基可德兰糖单元)。采用透析加探头超声法在水中制备了CCMC自聚集纳米粒子；并用透射电镜(TEM)、动态光散射仪(DLLS)、Zeta-电位仪和荧光光谱对CCMC自聚集纳米粒的形态、大小以及在水溶液中的性质进行了表征,结果表明,CCMC在水相中能形成规则球形的纳米粒子,粒径(144～233 nm)随着胆固醇取代度的增加而减小；CCMC自聚集纳米粒子的Zeta-电位在蒸馏水中为负值,说明带负电荷的羧甲基位于纳米粒子的表面；CCMC材料的临界聚集浓度(2.6×10-2～9.2×10-2mg／mL)与胆固醇的取代度有关,当胆固醇取代度增大时其临界聚集浓度减小以抗肿瘤药物表阿霉素为模型药物,采用硫酸铵梯度法制备了负载表阿霉素的CCMC自聚集纳米粒子,结果表明,载药量随着药物和载体材料比的增加而增加,包封率为78.0%～65.6%。透射电镜(TEM)观察显示,载药纳米粒子为均匀的球形,表面较空白纳米粒子粗糙；这可能是由于有一部分药物吸附在纳米粒子表面。动态光散射(DLLS)结果表明载药纳米粒子的粒径为294.4～510.4 nm,且随着载药量的增加而增加,表阿霉素在CCMC自聚集纳米粒子中的释放速率与释放介质的pH值和载药量相关,释放速率随释放介质pH值的升高而减慢,随载药量的增加而减慢。CCMC自聚集空白纳米粒子、载药CCMC纳米粒子对HeLa细胞的体外细胞实验表明,当空白纳米粒子浓度高达100μg/mL时对HeLa细胞的生长无抑制作用。当载药纳米粒子或游离药物中药物浓度在0.01～10μg／mL范围内时有抑瘤作用；当药物浓度<1μg/mL时载药纳米粒子对HeLa细胞的抑制作用比游离药物稍强；当药物浓度在1～10μg/mL时载药纳米粒子对HeLa细胞的抑制作用比游离药要明显增强；同时,随着药物与细胞孵育时间的增加其对细胞的抑制作用也随之增加。流式细胞仪及激光共聚焦显微镜研究结果表明,游离药物主要分布于细胞核而载药纳米粒子分布在细胞核及细胞浆,同时HeLa细胞对载药纳米粒子的摄取明显要高于对游离药物的摄取。载药CCMC纳米粒子、游离药物的Wister大鼠体内药代动力学实验和组织分布实验结果表明,载药CCMC纳米粒子在大鼠体内可达到长循环和缓慢释放的作用,既能减少药物的毒副作用,也能更好地延长药物作用时间；载药CCMC纳米粒子能显著改变药物在大鼠各脏器的分布情况,大大降低药物对心脏的毒副作用,同时也避免了肾小球的过滤作用。载药CCMC自聚集纳米粒子、游离药物的抗肿瘤药效学研究结果表明,载药CCMC纳米粒子能降低药物的毒性,给药第14天,载药CCMC自聚集纳米粒子与游离药物均表现出显著的肿瘤抑制作用,尤其是载药CCMC纳米粒子组对肿瘤的抑制作用随着时间的延长而增强,在给药14天,肿瘤抑制作用显著强于游离药物组。这可能是由于载药CCMC纳米粒子在体内具有长循环作用,这种长循环的作用赋予了载药CCMC纳米粒子在被动靶向过程中具有EPR效应,增加药物在肿瘤部位的蓄积,从而提高抗肿瘤效率。总之,胆固醇基羧甲基可德兰衍生物可以通过自聚集的方法制备成纳米粒子,制备方法简单可行。CCMC自聚集纳米粒子可以作为抗肿瘤药物载体包载双亲性或疏水性药物,延缓药物的释放从而降低药物的毒副作用,可望将其作为一种新型的抗肿瘤药物载体用于抗肿瘤药物制剂的开发。更多还原

【Abstract】 In this study, we synthesized novel polymeric amphiphiles by cholesterol conjugated carboxymethyl curdlan (CCMC) and prepared its self-assembled nanoparticles. Epirubicin (EPB) was chosen as a model drug to assess the potential of CCMC self-assembled nanoparticles as a carrier for anti-cancer drugs, the loading capability and release behavior were studied. The in vitro anti-tumor effects and cell toxicity of EPB-loaded CCMC self-assembled nanoparticles were studied by cell culture. The in vivo pharmacokinetics and biodistribution studies were investigated by animal experiments. The main contents and results are as follows:A series of CCMC conjugates with different degrees of substitution (DS) of the cholesterol moieties were synthesized and their phychemical properties were characterized by couductometric titration, fourier transform infrared (FT-IR), proton nuclear magnetic resonance (1H NMR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and high performance liquid chromatography (HPLC). The degree of substitution (DS) of cholesterol moiety was 2.3,3.5 or 6.4 cholesterol groups per hundred sugar residues of CM-curdlan determined by high performance liquid chromatography (HPLC) method. CCMC self-assembled nanoparticles were prepared by probe sonication and dialysis in distilled water and their characteristics were analyzed by transmission electron microscopy (TEM), dynamic laser light scattering (DLLS), Zeta-potential and fluorescence spectroscopy. TEM observation demonstrated that CCMC self-assembled nanoparticles were roughly spherical in shape, the mean diameter of CCMC self-assembled nanoparticles were in the range of 144-233 nm, which depending on the DS value. The mean diameter of self-assembled nanoparticles decreased as the DS increasing. The zeta potentials of CCMC self-assembled nanoparticles were very negative in distilled water, which indicated the negatively charged carboxyl groups of CMC covered nanoparticles. The critical aggregation concentration (CAC) values of the CCMC conjugates defined as the threshold concentration of self-assemble formation of polymeric amphiphiles by intra-and/or intermolecular association depends on the DS value of cholesterol, and the CAC values decrease with an increase in the DS of cholesterol.EPB was chosen as a model drug, and we used the remote loading method to physically entrap it into CCMC self-assembled nanoparticles in this study. The drug loading contents increased with the weight ratio of EPB to CCMC self-assembled nanoparticles increasing, while the drug entrapment efficiency decreased from 78.0% to 65.6% at the same time. Transmission electron microscopy (TEM) showed that EPB-loaded CCMC self-assembled nanoparticles were almost spherical in shape and had a coarser surface compared with the blank CCMC nanoparticles. We believed this is because some EPB was adsorbed to the surface of nanoparticles. Dynamic laser light scattering (DLLS) showed that the mean diameter of EPB-loaded CCMC self-assembled nanoparticles increased from 294.4 to 510.4 nm with the increase of EPB contents. In vitro release studies showed that EPB release from EPB-loaded CCMC self-assembled nanoparticles was sensitive to the pH of the release media as well as the drug loading contents, and the release rate decrease with the pH of the release media and the drug loading contents increase.The cellular cytotoxicity and cellular uptake were accessed by using Human Cervix Carcinoma (HeLa) cells. The blank CCMC self-assembled nanoparticles did not show any cytotoxic effect on HeLa cells even at the concentration of 100μg/mL. However, the EPB-loaded CCMC self-assembled nanoparticles and the free EPB showed anti-tumor activity at the drug concentration of 0.1 to 10μg/mL in vitro. EPB-loaded CCMC self-assembled nanoparticles exhibited slightly more cytotoxic to HeLa cells at low EPB concentrations (<1μg/mL), while the inhibitory effects of EPB-loaded CCMC self-assembled nanoparticles increased more rapidly with drug concentration increased from 1 to 10μg/mL compared with the free EPB. In addition, the cellular cytotoxicity increased significantly with the cell incubation time increases. Flow cytometry and confocal image anlysis revealed that the free EPB was mainly distributed within the nucleus, while the EPB-loaded CCMC self-assembled nanoparticles showed a broader distribution within the cells. The cellular uptake of EPB-loaded CCMC self-assembled nanoparticles was better than that of free EPB.The in vivo pharmacokinetics and biodistribution were investigated in Wister rats and revealed that the EPB-loaded CCMC self-assembled nanoparticles showed a much longer circulation time and sustained release effect in rats, which may implied reduced side effects and thus have clinical significance. The EPB-loaded CCMC self-assembled nanoparticles significantly changed the tissue biodistribution of the original drug which greatly reduced the systemic toxicity of EPB while helped to bypass glomerular filtration and was finally eliminated after the formulation yielded to metabolism in the liver. The results of in vivo anti-tumor efficacy of free EPB and EPB-loaded CCMC self-assembled nanoparticles indicated that both free EPB and EPB-loaded CCMC self-assembled nanoparticles could inhibit tumor growth obviously compared with the control group on day 14 after administration, in particular, the EPB-loaded CCMC self-assembled nanoparticles showed better anti-tumor activity as the time going compared with free EPB, with a significant difference on day 14 after administration. This maybe attributed that the EPB-loaded CCMC self-assembled nanoparticles could reach tumor site through EPR effect and maintain the effective therapeutic concentration for a long period of time.In short, cholesterol conjugated carboxymethyl curdlan derivatives could from nanoparticles by self-assembled manner, the method of prepaing nanoparticles was simple and feasible. CCMC self-assembled nanoparticles can be used as anti-tumor drug carrier of both amphipathic and hydrophobic drugs that sustain the release effect and reduce the systemic toxicity and is expected as a new carrier for anti-tumor drugs delivery.更多还原