【作者】 李雪；

【导师】 刘伟；

【作者基本信息】 郑州大学 ， 药剂学， 2019， 硕士

【摘要】 癌症是当今世界严重危害人类生命与健康的疾病之一,它分为多种类型,它的发病率和致死率均较高,目前,临床上治疗癌症的主要治疗手段有化疗、放疗、手术治疗、热疗（Hyperthermia,HT）、超声疗法等及将两种或者两种以上的治疗方法联合起来治疗癌症。因此,本课题拟构建半乳糖化壳聚糖（galactosylated chitosan,GC）修饰的磁性介孔二氧化硅（Magnetic mesoporous silica nanoparticles,MMSNs）负载奈达铂（Nedaplatin,NDP）的双重靶向给药系统（NDP@MMSN-COOH-GC NPs）,将该系统联合光热治疗（photothermal therapy,PTT）,具有以下优势:（1）半乳糖化壳聚糖修饰的磁性介孔二氧化硅载体的主动靶向作用使药物可以有效蓄积在肿瘤部位,减小全身毒性,提高生物安全性;（2）纳米载体凭借自身良好的光热效应,使得光热治疗大大增强了制剂的体内和体外抗肿瘤效果。相关研究如下:1、NDP@MMSN-COOH-GC NPs的制备与表征。采用共沉淀法制备水溶性Fe₃O₄。接着以Fe₃O₄为磁核,以正硅酸四乙酯为硅源,CTAB为模板剂,采用溶胶-凝胶法制备磁性介孔二氧化硅,并对MMSNs表面进行羧基化修饰,再以戊二醛作为交联剂,利用羧基可以和半乳糖化壳聚糖上的氨基作用,制备GC修饰的磁性介孔二氧化硅纳米粒子（MMSN-COOH-GC NPs）,通过红外,粒径电位,扫描电镜,透射电镜和氮气吸附-脱附等进行表征,并考察了纳米载体在808激光照射不同时间下的升温情况,最后以载药量和包封率为指标进行处方工艺的筛选,通过优化制剂制备的条件,最终得到具有双重靶向作用的NDP@MMSN-COOH-GC NPs制剂。红外光谱图显示Fe₃O₄磁核已被成功制备,MMSNs表面已经成功修饰上氨基和羧基,半乳糖化壳聚糖也已被成功包覆在MMSNs-COOH上;透射电子显微镜图显示,Fe₃O₄粒径为15 nm左右,MMSN–COOH NPs和MMSN–COOH–GC NPs粒径增至100 nm左右;Zeta电位图显示经柠檬酸钠修饰之后的Fe₃O₄的Zeta电位是-12.6 mV,此外,MMSNs表面成功修饰氨基后,电位为正,增加到+16.7 mV,而再次修饰羧基后,电位变为-21.8 mV,最后,MMSN-COOH NPs成功包覆GC后,电位变为+18.0 mV。磁性测试结果显示Fe₃O₄纳米粒子的饱和磁化值（Ms）为65 emu/g,具有超顺磁性,MMSN-COOH NPs和MMSN-COOH-GC NPs饱和磁化值分别为38 emu/g和28 emu/g;N₂吸附-脱附曲线表明MMSN-COOH NPs的等温线符合IUPAC分类的IV型等温线,这说明MMSN-COOH NPs属于介孔材料。此外,通过BET法计算MMSN-COOH NPs的比表面积为568.80 m2/g,通过BJH计算其孔体积为1.15 cm3/g,孔径为6.3 nm。与超纯水对比,纳米载体在808激光的照射下均能够快速升温,在3分钟内,温度均可升至43℃以上,能达到光热治疗的目的。最后,经过各种条件筛选,最优处方所制备的NDP@MMSN-COOH-GC NPs,载药量为24.6%±1.34%,包封率为33.09%±1.22%。制剂的体外释放结果表明,NDP@MMSN–COOH–GC NPs组相比NDP@MMSN–COOH NPs一组,具有一定的缓释作用。此外,体外释药实验还表明在NIR激光的照射下由磁性纳米粒子产生的热量可以加速药物从制剂中释放。2、NDP@MMSN-COOH-GC NPs联合光热治疗的体外抗肿瘤活性研究。以人肺腺癌A549细胞为模型,进行一系列体外实验以研究NDP@MMSN-COOH-GC NPs制剂联合光热治疗的体外抗肿瘤活性及机制。细胞毒结果表明空白载体对A549细胞无明显毒性,此外,NDP@MMSNs-COOH-GC NPs联合光热治疗后可以更有效抑制肿瘤细胞的增殖;细胞摄取结果表明我们所构建的NDP@MMSNs-COOH-GC NPs载药系统可以更多的被A549细胞摄取;细胞周期实验结果表明NDP及其制剂（不）联合光热治疗和单独的光热治疗均能将A549细胞阻滞在S期,诱导细胞凋亡;细胞凋亡实验结果表明最终治疗组NDP@MMSNs-COOH-GC NPs+NIR laser的总凋亡率最高,远高于其他任何实验组,诱导了更多A549细胞的凋亡。上述结果说明NDP@MMSNs-COOH-GC NPs联合光热治疗可以有效的抑制肿瘤细胞的增殖,具有显著的体外抗肿瘤活性。3、NDP@MMSN-COOH-GC NPs联合光热治疗的体内抗肿瘤活性研究。以荷S180瘤的KM雌性小鼠为动物模型,进行一系列体内实验以研究NDP@MMSN-COOH-GC NPs制剂联合光热治疗的体内抗肿瘤效果。小动物活体成像结果显示,IR783@MMSN-COOH-GC NPs（肿瘤部位绑有磁铁）可以将药物有效靶向至肿瘤部位,24 h后肿瘤组织仍然有较强的荧光信号,证明了载体的靶向性。体内药效学结果显示NDP@MMSN-COOH-GC NPs+MT+NIR laser组有最好的治疗效果,抑瘤效果最佳。此外,有结果显示,相比奈达铂原料药组,其他各实验组小鼠的体重均未出现明显的下降趋势,说明制剂和载体均有较高的生物安全性。HE染色结果表明各组的心肝脾肺肾并未有明显的病理学改变,再次说明了制剂的安全性。但是肿瘤组织HE染色结果表明NDP@MMSN-COOH-GC NPs+MT+NIR laser组肿瘤细胞出现大部分凋亡坏死的现象,比其余几组更加明显。综合以上实验结果,证明本课题所制备的NDP@MMSN-COOH-GC NPs联合光热治疗能够有效抑制肿瘤增殖,且生物安全性良好。更多还原

【Abstract】 Cancer is one of the diseases that seriously endanger human life and health in the world today,it is divided into many types,it has a high incidence and fatality rate,at present,the main treatment methods for clinical treatment of cancer are chemotherapy,radiotherapy,surgical treatment,hyperthermia（HT）,ultrasound therapy,etc.,and two or more treatments are combined to treat cancer.Therefore,the subject is to be constructed based on Nedaplatin（NDP）as a basic drug,galactosylated chitosan（GC）modified magnetic mesoporous silica nanoparticles（MMSNs）as the carrier of the dual targeting drug delivery system NDP@MMSN-COOH-GC NPs,the system combined with photothermal therapy（PTT）has the following advantages:（1）The active targeting of GC modified MMSNs allows the drug to accumulate effectively in the tumor site,avoid systemic toxicity and improve biosecurity;（2）Nanocarriers by virtue of its own good photothermal effect,so that photothermal therapy greatly increased the preparation of in vivo and in vitro anti-tumor effect.The relevant experiments of this study are as follows:1.The synthesis and characterization of NDP@MMSN-COOH-GC NPs.Water-soluble Fe₃O₄ was prepared by co-precipitation method.Then with the Fe₃O₄as the magnetic nucleus,the TEOS as the silicon source,CTAB as the template agent,the magnetic mesoporous silica nanoparticles were prepared by Sol-gel method,and its surface was carboxyl modified,with Glutaraldehyde as crosslinking agent,using the role of carboxyl and galactosylated chitosan amino acid,the MMSN-COOH-GC NPs coated with GC were characterized by infrared,DLS,SEM,TEM and nitrogen adsorption-desorption,and the warming of the carriers at different times of 808 laser irradiation was investigated.Finally,the prescription process was screened with the EE%and DL%as the index,and the NDP@MMSN-COOH-GC NPs preparation with double targeting effect was finally obtained by optimizing the preparation conditions of the preparation.Infrared spectroscopy showed that the Fe₃O₄ has been successfully prepared,the MMSNs surface has been successfully modified on the amino and carboxyl,GC has been successfully coated on the MMSNs-COOH,TEM diagram showed that the Fe₃O₄ particle size is about 15nm,MMSN-COOH NPs and MMSN-COOH-GC NPs particle size increased to about 100nm;Zeta potential graph showed that the zeta potential of sodium citrate modified Fe₃O₄ was-12.6 mV,in addition,after the MMSNs surface successfully modified the amino,the potential was positive,increased to+16.7 mV,and once again modified the carboxyl,the potential became-21.8 mV,after the MMSN-COOH NPs was coated with GC,the potential became+18.0 mV.The magnetic test results showed that the saturation magnetization value（Ms）of Fe₃O₄ nanoparticles was 65 emu/g,which has ultra-paramagnetic magnetism,and the saturation magnetization values of MMSN-COOH Nps and MMSN-COOH-GC NPs were 38 emu/g and 28 emu/g respectively.The N₂adsorption-desorption curve indicated that the isotherm of MMSN-COOH NPs conformed to the IV isotherm of IUPAC classification,which showed that MMSN-COOH NPs belong to mesoporous materials.In addition,the specific surface area was calculated by BET method was 568.80 m2/g,and the pore volume calculated by BJH was 1.15 cm3/g and the aperture is 6.3 nm.Compared with deionized water,nanocarriers can quickly heat up to 43℃under 808 laser irradiation within 3 minutes,so that they can achieve the purpose of photothermal therapy.Finally,after a variety of conditions screening,the drug loading and the encapsulation efficiency of NDP@MMSN-COOH-GC NPs were 24.6%±1.34%and 33.09%±1.22%.The resultsofinvitroreleaseof thepreparationshowed that the NDP@MMSN-COOH-GC NPs group had a sustained release effect,compared with the NDP@MMSN-COOH NPs group.In addition,it also shows that the heat generated by magnetic nanoparticles under the exposure of NIR lasers can accelerate drug release from preparations.2.The antitumor efficiency of NDP@MMSN-COOH-GC NPs combined with PTT in vitro.Based on the A549 cells of human lung adenocarcinoma,the anti-tumor activity and mechanism of NDP@MMSN-COOH-GC NPs in vitro were studied by a series of in vitro experiments.The cytotoxicity results showed that the blank nanocarriers had no obvious toxicity to the A549 cell,in addition,NDP@MMSNS-COOH-GC NPs combined with PTT could more effectively inhibit the proliferation of tumor cells,and the cell uptake results showed that the nanocarriers could effectively enter to the A549 cell.The results of cell cycle experiments showed that NDP and its preparation combined with or without PTT and PTT all can block cell blocking in S period and induce apoptosis.The results of apoptosisexperimentshowedthatthetotalapoptosisrateof NDP@MMSNS-COOH-GC NPs+NIR laser was the highest,and more induced apoptosis of A549 cells.The above results showed that NDP@MMSNS-COOH-GC NPs combined with PTT can obviously inhibit the proliferation of tumor cells and has good anti-tumor activity in vitro.3.The antitumor efficiency of NDP@MMSN-COOH-GC NPs combined with PTT in vivo.A series of in vivo experiments were carried out to study the anti-tumor activity of NDP@MMSN-COOH-GC NPs combined with photothermal therapy based on the S180 tumor-bearing mice model The results of Near infrared（NIR）fluorescence in vivo imaging showed that IR783@MMSN-COOH-GC NPs（the tumor site was bound with magnets）could effectively target the drug to the tumor site,and the tumor tissue still had strong fluorescence signal after 24h,which proved the targeting ability of the nanocarriers.The results of changes of relative tumor volume showed that the NDP@MMSN-COOH-GC NPs+MT+NIR laser group had the best therapeutic effect and the best tumor suppressor effect.In addition,the results showed that there was no significant decrease in body weight of other groups compared with the NDP group,indicating that the preparation and nanocarriers had high biosecurity.The results of HE staining showed that there was no obvious pathological change in the heart,liver,spleen,lung and kidney of each group,which explained the safety of the preparation.However,the results of HE staining showed that most of the apoptosis necrosis occurred in tumor cells in NDP@MMSN-COOH-GC NPs+MT+NIR laser group.In summary,it is proved that the NDP@MMSN-COOH-GC NPs combined with PTT can effectively inhibit the proliferation of tumor,and it has the good biosecurity.更多还原