CD147单抗介导的α-常春藤皂苷壳聚糖纳米粒的制备及其肝肿瘤靶向性研究

发布时间：2018-06-10 04:30

本文选题：CD147单抗 + α-常春藤皂苷　；参考：《苏州大学》2014年硕士论文

【摘要】：目的：以壳聚糖（Chitosan，CS）为载体，以水难溶性药物-常春藤皂苷（-Hederin，-Hed）为模型药物，制备-常春藤皂苷壳聚糖纳米粒（-Hed-CS-NP， HCS），再以肝肿瘤细胞表面高度表达的CD147单抗为靶头，将其偶联修饰于纳米粒表面制备CD147介导的-常春藤皂苷壳聚糖纳米粒（-Hed-CS-CD147-NP， αHCD），从而通过纳米粒的缓释效果和单抗介导的主动靶向性达到减毒增效的作用。方法：（1）以平均粒径、多分散指数（Polydisperse Index，P.I.）和包封率（EntrapmentEfficiency，EE）为综合指标，采用乳化-溶剂扩散法制备HCS，并采用单因素试验和正交试验设计，考察纳米粒处方和制备工艺。红外（FT-IR）、X射线衍射（XRD）、差示扫描量热分析（DSC）等对αHCS纳米粒进行表征，同时系统考察纳米粒的体外释放行为及其相关药剂学性质。（2）采用EDC/NHS酰胺化反应制备αHCD，并对纳米粒进行粒径测定、FT-IR和XRD表征；采用BCA试剂盒和ELISA法分别建立CD147单抗的含量和活性测定方法；依据HepG2细胞对不同单抗修饰量纳米粒的摄取结果，选择合适的单抗修饰量。（3）以未载药的CD147单抗介导的壳聚糖纳米粒（VoidCS-CD147-NP，VCD）为对照，MTT法考察CD147单抗对肝肿瘤细胞活性的抑制作用、-Hed及其纳米粒对HepG2、SMMC-7721、HSC的细胞毒作用；采用流式细胞术检测原料药及其纳米粒对HepG2细胞的凋亡效果和对其细胞周期的影响。(4)流式细胞术方式考察HepG2在2h和24h内αHCS和αHCD的摄取率，HSC细胞作为对照细胞考察两种纳米粒的摄取差异性，并通过加入过饱和CD147进行纳米粒的竞争性抑制试验；多种细胞内吞抑制剂对两种纳米粒的摄取机制进行探讨，并考察两种纳米粒在体内的摄取分布过程。（5）分别以生理盐水组为阴性对照，环磷酰胺为阳性对照，HepG2细胞裸鼠肿瘤模型腹腔注射给予-Hed及其纳米粒后，考察其体内药效学；采用小动物活体成像技术，动态考察纳米粒在荷HepG2细胞裸鼠体内的组织分布及其肿瘤靶向性。结果：（1）乳化溶剂扩散法制备的αHCS纳米粒粒径分布均匀，平均粒径（92.7±2.77）nm，P.I.（0.134±0.036），包封率（75.63±4.06）%；透射电镜观察纳米粒外观形态圆整；FT-IR、XRD、DSC表征结果表明药物已分散于壳聚糖纳米粒中；体外释放显示壳聚糖纳米粒具有一定的缓释特性。（2）CD147能通过酰胺反应偶联修饰到αHCS表面；与αHCS的粒径相比，αHCD的粒径变化较小；偶联所得αHCS中CD147单抗饱和修饰量为（6.55±0.234）μg/mgNP，结合不同修饰量对纳米粒摄取结果影响、单抗的活性和过多修饰量可能产生的空间位阻效应等因素，本实验最终确定修饰量为1.5μg/mgNP，且单抗活性保留率为（57.07±0.60）%。（3）MTT实验结果表明未载药单抗修饰的壳聚糖纳米粒VCD及本实验所用CD147单抗修饰量均对肝肿瘤细胞无细胞毒作用，原料药及其纳米粒对HepG2、 SMMC-7721的IC50值为αHCDHCS-Hed，以HSC细胞为对照，提示与单抗介导的主动靶向性有关；通过细胞凋亡实验结果与HepG2细胞MTT实验相一致；在细胞周期实验中发现细胞在S期的百分比有所增加。（4）HepG2细胞对纳米粒摄取具有时间依赖性，且αHCD的摄取明显高于HCS，另外HSC对两种纳米粒摄取无差异，结合过饱和竞争抑制实验中αHCD摄取率明显下降，说明αHCD纳米粒对肝肿瘤细胞具有更好的亲和性；从摄取抑制剂实验结果可发现载药纳米粒的内吞摄取过程属于能量依赖型，需通过形成有被小窝内陷和网格蛋白介导的内吞进入细胞，且与细胞膜结构稳定性和流动性息息相关。倒置荧光显微镜结果显示纳米粒1h时大多聚集在细胞膜表面，3h时细胞摄入纳米粒明显增多。（5）体内药效学实验中， HCS（79.02%）和αHCD（88.43%）纳米粒的抑瘤率远高于原料药中剂量组（53.83%）；纳米粒组抑瘤率均在70%以上，且存在剂量依赖性。近红外荧光成像实验结果发现，尾静脉给药1h后，两种纳米粒在肿瘤部位和肝脏可见较为集中的荧光分布，在6h时，肿瘤部位荧光强度有所减弱，12h后纳米粒荧光开始减弱。结论：乳化溶剂扩散法制备的αHCS大小分布均匀、外观形态圆整、包封率高，有缓释特性，体外体内药效学实验表明空白纳米粒与细胞具有生物相容性，， αHCD对肝肿瘤细胞的毒副作用的增强和肝肿瘤细胞对该纳米粒摄取的增多与CD147单抗介导的主动靶向性有关。综上所述， αHCD具有应用潜能并有望成为一种新型的难溶性药物的载体。
[Abstract]:Objective: using Chitosan (CS) as the carrier and using the water-insoluble drug -Hederin (-Hed) as the model drug, the -Hed-CS-NP (HCS) of ivy saponins (-Hed-CS-NP, HCS) was prepared, and then the CD147 mAb expressed in the surface of the liver tumor cells was used as the target, and the coupling modified to the nanoparticles surface to prepare the CD147 mediated ivy. Saponins chitosan nanoparticles (-Hed-CS-CD147-NP, alpha HCD) can achieve the effect of attenuation and synergism through the sustained release effect of nanoparticles and the active targeting mediated by monoclonal antibody.
Methods: (1) using the average particle size, Polydisperse Index, P.I. and EntrapmentEfficiency (EE) as a comprehensive index, HCS was prepared by emulsion solvent diffusion method, and the formulation and preparation process of nanoparticles were investigated by single factor test and orthogonal design. Infrared (FT-IR), X ray diffraction (XRD), differential scanning calorimetry analysis (DSC) to characterize alpha HCS nanoparticles and investigate the release behavior of nanoparticles in vitro and their related pharmaceutical properties. (2) the preparation of alpha HCD by EDC/NHS amidation, particle size determination, FT-IR and XRD characterization, and BCA reagent box and ELISA method, respectively, to determine the content and activity of CD147 McAbs; According to the results of HepG2 cells' uptake of different mAb modifier nanoparticles, the appropriate mAb modifier was selected. (3) the CD147 mAb mediated chitosan nanoparticles (VoidCS-CD147-NP, VCD) was used as the control, and the MTT method was used to investigate the inhibitory effect of CD147 mAb on the activity of liver tumor cells, and the cytotoxicity of -Hed and its nanoparticles on HepG2, SMMC-7721, HSC. The effect of flow cytometry was used to detect the effect of API and its nanoparticles on the apoptosis of HepG2 cells and its effect on its cell cycle. (4) flow cytometry was used to investigate the uptake of HepG2 in 2H and 24h, the uptake of alpha and alpha in 2H and 24h. HSC cells were used as control cells to investigate the difference in the uptake of the nanoparticles, and by adding a supersaturated CD147. The competitive inhibition test of nanoparticles, the uptake mechanism of two kinds of nanoparticles by multiple cell endocytosis inhibitors and the uptake of the two nanoparticles in the body were investigated. (5) the negative control of the saline group, cyclophosphamide as the positive control, and the intraperitoneal injection of the HepG2 nude mouse tumor model to -Hed and its nanoscale The pharmacodynamics of the nanoparticles in vivo were investigated and the tissue distribution and tumor targeting of nanoparticles in nude mice bearing HepG2 cells were dynamically investigated by using small animal living imaging techniques.
Results: (1) the particle size distribution of alpha HCS nanoparticles was evenly distributed, the average particle size (92.7 + 2.77) nm, P.I. (0.134 + 0.036), and encapsulation rate (75.63 + 4.06)%; transmission electron microscopy was used to observe the appearance of nanoparticles. The characterization results of FT-IR, XRD, and DSC showed that the drug was dispersed in chitosan nanoparticles, and the release of chitosan in vitro (2) CD147 can be modified to the surface of alpha HCS through amide reaction. Compared with the particle size of alpha HCS, the particle size of alpha HCD is smaller, and the saturation modification of CD147 mAb in alpha HCS is (6.55 + 0.234) mu g/mgNP, and the effects of different modifiers on the results of nanofilm uptake, the activity of McAbs and the excess modification In this experiment, the amount of space hindrance was 1.5 g/mgNP, and the retention rate of the monoclonal antibody was (57.07 + 0.60). (3) the results of MTT experiment showed that the chitosan nanoparticles VCD modified by unloaded McAb and the CD147 McAb modification used in this experiment had no cytotoxic effect on liver tumor cells. The IC50 value of rice grain to HepG2, SMMC-7721 is alpha HCDHCS-Hed and HSC cells as the control, which is related to the active targeting mediated by McAb; the results of apoptosis experiments are consistent with the MTT experiment of HepG2 cells, and the percentage of cells in S phase is increased in cell cycle experiments. (4) HepG2 cells have time for nanoparticles uptake. The uptake of alpha HCD was significantly higher than that of HCS, and there was no difference in the uptake of two nanoparticles in HSC. The uptake of alpha HCD decreased significantly in the combination of supersaturated competition inhibition experiments, indicating that the alpha HCD nanoparticles had better affinity to the liver tumor cells. The quantity dependent type should be entered into cells by the formation of endocytic endocytosis mediated by trift and gridin, which is closely related to the stability and fluidity of the cell membrane structure. The inverted fluorescence microscope results showed that most of the nanoparticles 1H aggregated on the surface of the cell membrane, and the cell intake nanoparticles increased significantly at 3H. (5) in the pharmacodynamic experiment, HCS (79.02) The tumor inhibition rate of%) and alpha HCD (88.43%) nanoparticles was much higher than that in the medium dose group (53.83%), and the tumor suppressor rate of the nanoparticles was more than 70%, and there was a dose dependence. The results of the near infrared fluorescence imaging test found that after the tail vein was given 1H, the two nanoparticles were more concentrated in the tumor site and the liver, and the tumor site was at 6h. The fluorescence intensity decreased, and the fluorescence of nanoparticles decreased after 12h.
Conclusion: the size distribution of alpha HCS in the emulsion solvent diffusion method is uniform, the appearance is round, the encapsulation efficiency is high, and it has the sustained release characteristics. In vitro, the pharmacodynamic experiment in vitro shows that the blank nanoparticles have biocompatibility with the cells, the enhancement of the toxic side effects of alpha HCD on the liver tumor cells and the increase of the liver tumor cells to the nanoparticles and CD147 single In conclusion, alpha HCD has potential for application and is expected to become a new carrier of insoluble drugs.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R735.7

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