【作者】 杨文智；

【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， 2009， 博士

【摘要】 天然多糖具有亲水、安全、稳定、无毒、生物可降解性、自然界存在广泛、易制备和价格便宜等优点;同时,多糖分子链有较多不同种类的基团,经化学和生化方法修饰,可获得相应的功能化多糖衍生物。因此,应用多糖及其衍生物作为药物载体的研究报告日益增多。普鲁兰多糖具有水溶性、无毒、多羟基易化学修饰和缺少免疫原性等优点,可做药物载体。内源性胆固醇分子具有环状结构,导致分子有一定的疏水性。而选择人体内源性小分子琥珀酸为连接臂代替文献报道的1,6-己二异氰酸酯,将胆固醇接枝到普鲁兰多糖分子链上,获得新型安全的两亲性胆固醇基普鲁兰(CHSP),此方法也为胆固醇基普鲁兰的合成提供了新策略。本文还报道合成胆固醇基羧甲基普鲁兰(CHS-CMP)。课题研究的具体内容如下:(1)合成新型两亲性胆固醇基普鲁兰(CHSP)、胆固醇基羧甲基普鲁兰(CHS-CMP)和FITC标记的CHSP(FITC-labeled CHSP)的普鲁兰糖衍生物。用红外(FT-IR)、核磁共振(NMR)、粉末晶体衍射(XRD)、差示扫描量热仪(DSC)、紫外-可见分光光度计和荧光分光光度计对合成普鲁兰衍生物进行表征。通过FT-IR、NMR、XRD和DSC实验证明,合成了两亲性胆固醇基普鲁兰。用NMR法和硫酸铵比色法计算系列CHSP的胆固醇取代度,每100糖单元取代3.87～5.70个胆固醇。采用琥珀酸连接臂,合成胆固醇基羧甲基普鲁兰(CHS-CMP),并对其中间体和CHS-CMP材料进行FT-IR和~1H NMR表征。以CHSP和FITC为原料,在二月桂酸二丁基锡(DBTDL)催化下,合成FITC-labeled CHSP并经FT-IR和~1H NMR表征,通过NMR和荧光分光光度法计算衍生物的FITC取代度,每100糖单元取代4.3个FITC。(2)通过溶胀超声法或透析法制备CHSP和FITC-labeled CHSP自聚集水凝胶纳米粒。用~1H NMR、动态激光散射(DLS)、透射电镜(TEM)和稳态荧光探针法对CHSP纳米粒物化性质进行表征。由于CHSP材料的胆固醇取代度不同,获得粒径在51.8～73 nm自聚集水凝胶纳米粒。CHSP分子结构中无极性取代基,其纳米粒在蒸馏水中的zeta电位绝对值近似为零。而CHSP材料的临界聚集浓度(cac)的大小直接与胆固醇取代度有关,当胆固醇取代度增大,cac浓度减小。采用透析法可制备粒径在50 nm左右的FITC-labeled CHSP自聚集纳米粒。(3)载药CHSP和载药FITC-labeled CHSP自聚集纳米粒的制备。用动态激光散射(DLS)、透射电镜(TEM)、紫外-可见分光光度计(UV-Vis)、XRD粉末晶体衍射和差示扫描量热(DSC)对载药纳米粒进行表征。以米托葸醌(MTO)作为抗瘤模型药物,用透析法制备CHSP载药纳米粒。动态光散射测得载药纳米粒的粒径为153.1～174.2 nm。UV-Vis法检测载药纳米粒的包封率为83.3～91.7%,载药量为4.35%～14.29%。透射电镜下观察,载药CHSP纳米粒为规则的球形。随着CHSP材料胆固醇取代度增大,纳米粒的载药量和包封率均增大。对照MTO药物晶体衍射峰,载药CHSP纳米粒的MTO药物的晶体峰消失,XRD结果表明,负载的MTO包入纳米粒内部。DSC实验结果同样证明这一结论。载药CHSP纳米粒的体外释放显示,MTO释放与释放介质的pH有关,pH低的释放介质药物释放快。但在pH6.8或7.4释放介质中,能缓慢释放MTO。药物释放与载体CHSP的胆固醇取代度有关,取代度越大,载药纳米粒缓释效果越明显。采用离子梯度法,用CHSP纳米粒包载强荧光性的表阿霉素(EPI),与包载MTO的CHSP纳米粒相比,包载EPI纳米粒的载药量和包封率都偏低。用透析法制备了负载EPI的FITC-labeled CHSP纳米粒。(4)CHSP自聚集纳米粒、载药CHSP自聚集纳米粒和载药FITC-labeled CHSP纳米粒的细胞实验评价。采用MTT、激光共聚焦显微镜、流式细胞仪对细胞实验进行评价。向HeLa肿瘤细胞中加入高达200μg/mL的CHSP自聚集水凝胶纳米粒的培养基,此浓度的CHSP纳米粒对共孵育的HeLa细胞的生长无抑制作用;无论是MTO还是EPI载药纳米粒,当载药纳米粒中药物浓度在1～10μg/mL,均显示出对HeLa细胞的毒性。将相同药物浓度的载药CHSP纳米粒和药物溶液的培养基加入HeLa细胞中,共孵育相同时间,载药纳米粒对肿瘤细胞生长抑制作用强于游离药物。流式细胞仪检测结果表明,肿瘤细胞对于载药纳米粒吞噬作用强于游离药物。激光共聚焦显微镜下观测的结果同样证明这一结论。为了观测CHSP材料自聚集水凝胶纳米粒进入HeLa细胞的情况,选择50 nm左右的FITC-labeled CHSP自聚集水凝胶纳米粒与HeLa细胞共孵育,随孵育时间延长,在激光共聚焦显微镜下可观测到纳米粒由粘附于肿瘤细胞膜,逐渐进入细胞浆,直到最后分布包括细胞核在内的整个细胞。而观测负载表阿霉素的FITC-labeled CHSP纳米粒与HeLa细胞共孵育2 h的照片,发现药物和CHSP纳米粒均可进入肿瘤细胞核。(5)负载表阿霉素CHSP自聚集水凝胶纳米粒的wistar大鼠药代实验初步研究。采用高效液相法检测不同时间游离EPI和载药的CHSP纳米粒在大鼠血液中的药物含量结果显示,载药纳米粒在血液中药物含量较单独EPI药物明显增加,载药纳米粒在大鼠体内可达到长循环和缓释效果。本文合成未见文献报道的两亲性胆固醇基普鲁兰、胆固醇基羧甲基普鲁兰和FITC标记的CHSP的普鲁兰糖衍生物。安全无毒的两亲性CHSP材料可在水中形成自聚集凝胶纳米粒并可用作包载疏水性抗瘤药物的纳米载体;wistar大鼠药代实验表明,包载EPI的CHSP纳米粒在大鼠体内能达到长循环和缓释作用;而FITC-labeled CHSP纳米粒可用作材料导入细胞的标志物,用其纳米粒与细胞共孵育,可有效观测纳米粒入胞的过程。更多还原

【Abstract】 Polysaccharides are highly stable,safe,non-toxic,hydrophilic,biodegradable, abundant resources in nature and low cost in their processing.Moreover,there are various derivable groups on molecular chains,so that polysaccharides can be easily modified and result in many kinds of derivatives by chemically and biochemically.Due to above outstanding merits,polysaccharides and their derivatives have received more and more attention in the area of drug delivery systems.Pullulan has many advantages as a macromolecular drug carrier,e.g.highly water-soluble,non-toxic,multiple hydroxyl groups that can readily be modified chemically,lacks immunogenicity,and usefulness as a plasma expander.Cholesterol has the hydrophobic property due to the presence of hydrophobic moieties such as cyclopentenophenanthrene nucleus in its molecule. Succinic acid is a natural compound existing in the human body which can be used as a linker between hydrophobic groups and hydrophilic polysaccharides skeleton.Therefore, using succinic acid,small endogenous molecule of the body’s metabolic process,replace the exogenous 1,6-hexyl diisocyanate.This provides a new strategy for the effective and safe synthesis cholesterol-modified pullulan.It also is of great significance to further expand its application.Based on above information,we developed a new method to synthesize CHSP or CHS-CMP by directly grafting proper cholesterol residues onto pullulan or O-carboxymethyl pullulan.The main content of this research are shown as follows.(1) Cholesterol-modified pullulan(CHSP) conjugate with succinyl linkage was synthesized and characterized by fourier transform infrared(FT-IR),proton nuclear magnetic resonance(~1H NMR),and X-ray diffraction(XRD).The degree of substitution (DS)of cholesterol moiety determined by ~1H NMR ranged from 3.87 to 5.70 cholesterol groups per hundred glucose units.The CHS-CMP was also synthesized by using cholesterol succinate reacted with O-carboxylmethyl pullulan,and the intermediates and pullulan derivatives were characterized by FT-IR and ~1H NMR.The synthesis of FITC-labeled CHSP was reacted CHSP and FITC catalyzed by DBTDL.The DS of FITC grafted onto CHSP was characterized by NMR and fluorescence spectrophotometry.(2) CHSP self-aggregated nanoparticles were prepared by probe sonication in aqueous media and analyzed by dynamic laser light-scattering(DLS),zeta potential, transmission electron microscopy(TEM) and the fluorescence probe technologies.These novel nanoparticles were almost spherical in shape,and their size,ranging from 51.8 to 73.0 nm,could be controlled by DS of cholesterol moiety.The zeta potentials of CHSP self-aggregated nanoparticles were near zero in aqueous media.The value of critical aggregation concentration(cac) was dependent on the DS of cholesterol moiety. FITC-labeled CHSP self-aggregated nanoparticles with D_h about 50 nm can be obtained by a dialysis method.(3) Mitoxantrone(MTO) was loaded into the CHSP nanoparticles by dialysis method.MTO-loaded CHSP self-aggregated nanoparticles were almost spherical in shape and their size increased from 153.1 to 174.2 nm with the MTO-loading capacity increasing from 4.35%to 14.29%.The encapsulation efficiency(EE) of the process and loading capacity(LC) of the nanoparticles increased with increasing cholesterol DS. XRD powder patterns showed that crystal peaks of MTO disappeared when MTO was entrapped into CHSP nanoparticles.DSC experimental results also proved the same conclusions.The release behavior of MTO from CHSP self-aggregated nanoparticles was studied in vitro.The results showed that the release behavior of MTO from CHSP nanoparticles exhibited a sustained release,and MTO release rate decreased with the pH increase of the release media.Epirubicin(EPI) was also physically entrapped inside the CHCS self-aggregated nanoparticles by remote loading method,and the EE and LC of EPI-loaded CHSP nanoparticles appeared rather lower than those of MTO-loaded CHSP self-aggregated nanoparticles.(4) The study of cell cytotoxicity in vitro showed CHSP self-aggregated nanoparticles had no antitumor activity even the CHSP concentration at 200ug/mL. However,whether MTO loaded CHSP or EPI loaded CHSP nanoparticles showed antirumor activity when adjusted the drug concentration at 1～10μg/mL.Moreover,the cytotoxicity of the MTO or EPI loaded nanoparticles was higher than free drug with the same concentration.The result of flow cytometry and confocal image ananlysis revealed that EPI loaded CHSP self-aggregated nanoparticles appeared much easier cellular endocytic than free EPI.The analysis of intracellular distribution of FITC-labeled CHSP self-aggregated nanoparticles in HeLa cell was carried out,and the result showed that the FITC labeled nanoparticles accumulated in cell membrane firstly,and then the nanoparticles passed through cell membrane and arrived at cellular nuclear with incubation time prolonged.EPI loaded FITC-CHSP nanoparticles cell entry also confirmed by the same way.(5) The EPI loaded CHSP nanoparticles and free drugs were injected intravenously to wistar mice respectively,and the preliminary study about Pharmacokinetics was carried out.The concentration of EPI at different times in blood was detected by high performance liquid.The result showed that the EPI-loaded CHSP nanoparticles achieved long-circulating and sustained-release effect.更多还原