中文摘要：
乳腺癌是世界上第二大常见癌症,也是女性中最常见的癌症,威胁着女性生命健康。就目前而言,化疗依旧是常用治疗手段。然而,受一些因素的阻碍,化疗的疗效不佳,如大部分化疗药物水溶性差,体内消除快,毒性大,缺乏肿瘤部位靶向性,以及肿瘤细胞易对化疗药物表现多药耐药性(Multidrug resistance,MDR)。本课题选用乳腺癌化疗一线药物紫杉醇(Paclitaxel,PTX)用于抗肿瘤研究,考虑到化疗的种种限制因素,尤其是乳腺癌的MDR,选择与P-糖蛋白(P-glycoprotein,P-gp)抑制剂槲皮素(Quercetin,QC)联合应用克服MDR,增强化疗效果。此外,基于肿瘤细胞内的还原条件及细胞表面过表达的CD44受体,设计了硫酸软骨素(Chondroitin sulfate,ChS)涂覆、PTX和QC共载的介孔二氧化硅纳米粒(MSNs-ChS@PQ)给药系统,此系统改善了 PTX和QC的水溶性,具备了还原响应型的药物控释行为、良好的生物相容性、肿瘤靶向性以及肿瘤部位滞留时间延长的特性。在体内外各项研究中,此系统均表现逆转乳腺癌细胞MDR,增强化疗的效果,为克服乳腺癌的MDR提供了一种可行的方法。本课题研究内容包括六部分:1.PTX和QC含量测定方法建立采用UV-Vis分光光度计对紫外吸收明显的QC建立方法学,用高灵敏度的高效液相色谱(High Performance Liquid Chromatography,HPLC)建立 PTX 的含量测定方法。结果显示,二者检测方法的专属性和线性关系良好,精密度和回收率均符合方法学要求。2.MSNs-ChS@PQ的制备与表征采用溶胶-凝胶法制备了介孔二氧化硅纳米粒(Mesoporous silica nanoparticles,MSNs),并进一步对MSNs表面依次氨基化和羧酸化,之后通过酰胺反应引入还原响应型二硫键,最后接枝ChS,得到MSNs-ChS。借助动态光散射法、扫描电镜、高分辨透射电镜、氮气吸附和热重分析对得到的纳米粒进行表征,结果显示MSNs-ChS 的水合粒径为 224.6±0.7 nm,Zeta 电位为-10.90±0.64 mV,PDI 为0.14±0.01,分散性良好,透射电镜下可以观察到MSNs-ChS呈球形,表面有厚度约为25 nm的ChS薄膜,热重分析结果显示ChS接枝率为19.2%。对在乙醇中分散性较好的MSNs-COOH实现载药,然后进一步合成得到MSNs-ChS@PQ。考察了 PTX/QC不同投药比对载药量的影响,结合细胞毒性实验,选择PTX/QC=1(w/w)为最佳投药比,PTX和QC的载药量分别为5.29±0.38%和 5.13±0.19%。提取家兔血红细胞研究MSNs-ChS的血液相容性,结果显示在0.1～1 mg/mL浓度范围下,MSNs-ChS的溶血率均在5%以下,安全性较好,可用以静脉给药。3.MSNs-ChS@PQ的体外释放研究采用透析袋法研究MSNs-ChS@PQ的药物释放行为,在释放介质中加入谷胱甘肽(Glutathione,GSH)模拟肿瘤细胞内的还原环境,考察了 MSNs-ChS@PQ在不同条件下的药物释放情况。结果发现在不含GSH的释放介质中,PTX和QC在36h内释放量均在20%以下,而在20mMGSH释放介质中,二者的释放量大幅度提高,分别为66%和53%,表明MSNs-ChS@PQ具有还原响应型的释放特性。4.MSNs-ChS@PQ的体外抗肿瘤研究考察了耐阿霉素人乳腺癌细胞(MCF-7/ADR细胞)对纳米粒的摄取行为,结果显示其摄取行为呈现时间依赖性,MSNs-ChS可以经CD44受体介导的内吞作用进入细胞,具有主动靶向性。通过体外细胞毒性、活/死细胞染色、免疫荧光微管染色、细胞凋亡、细胞周期实验和P-gp检测实验证实了 MSNs-ChS@PQ可以逆转MCF-7/ADR细胞的MDR,增强化疗疗效以及诱导P-gp水平下降。5.MSNs-ChS的体内生物分布研究将鼠源的乳腺癌细胞(4T1细胞)接种在雌性BALB/c小鼠右腋下建立荷瘤动物模型,建模成功后,尾静脉注射载碘化1,1'-双十八烷基-3,3,3',3'-四甲基吲哚三羰花青(1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide,DiR)的纳米粒(MSNs-ChS@DiR),通过小动物活体/离体成像技术考察了纳米粒在小鼠体内不同时间段的生物分布及24 h后在各组织器官的含量,证实了 MSNs-ChS@DiR纳米粒具有肿瘤靶向性,相比于游离DiR,可以在肿瘤部位长时间滞留。6.MSNs-ChS@PQ的体内抗肿瘤研究将MCF-7/ADR细胞接种于雌性BALB/c裸鼠右腋,建立肿瘤模型,尾静脉注射方式给药,通过肿瘤体积变化曲线、瘤重、肿瘤切片H&E染色和TUNEL分析等实验评价MSNs-ChS@PQ的体内药效,证实了单载PTX纳米粒对耐药肿瘤的低效现象,也凸显了 MSNs-ChS@PQ体内逆转肿瘤MDR的效果。通过对生理盐水组和MSNs-ChS@PQ组小鼠的主要组织器官进行H&E染色分析,初步证实了 MSNs-ChS@PQ的体内安全性。
英文摘要：
As the second most common cancer in the world,breast cancer is most prevalent of all female cancer cases,which threatens women's life and health.At present,chemotherapy is still a common treatment.However,chemotherapy has been limited by mountainous factors,such as poor water solubility of most chemotherapeutic drugs,rapid drug elimination in vivo,high toxicity,insufficient tumor targeting,and the multidrug resistance(MDR)of tumor cells.In this project,paclitaxel(PTX),a first-line drug for breast cancer chemotherapy,was selected for anti-tumor research.Considering the various limiting factors of chemotherapy,especially MDR for breast cancer,P-glycoprotein(P-gp)inhibitor quercetin(QC)was used in combination with PTX to overcome MDR and enhance chemotherapy effect.Furthermore,based on the high redox environment of tumor cells and the overexpressed CD44 receptor on the cell surface,a chondroitin sulfate(ChS)coated PTX/QC-loaded mesoporous silica nanoparticles(MSNs-ChS@PQ)drug delivery system was developed.In addition to solving the problem of poor water solubility of PTX and QC,this nanosystem possessed redox-responsive drug release property at tumor,good biocompatibility,tumor targeting,and prolonged tumor retention.In various studies in vitro and in vivo,this system reversed the MDR of breast cancer cells and enhanced the effect of chemotherapy,providing a feasible approach to overcome MDR in breast cancer.This project was consisted of the following six parts.1.Method establishment of PTX and QC content determinationA UV-Vis spectrophotometer was used for QC content measurement.And the determination method of PTX content was built by high performance liquid chromatography(HPLC)with high-sensitivity.The results suggested that the specificity and linearity of the two methods were good.The precision and recovery were in line with the methodological requirements.2.Preparation and characterization of MSNs-ChS@PQMSNs were prepared by sol-gel method.The surface of the MSNs was aminated and carboxylated in turn,and then a redox-responsive disulfide bond was introduced via an amide reaction.Finally,ChS was grafted to obtain MSNs-ChS.The obtained nanoparticles were characterized by dynamic light scattering method,scanning electron microscope,high-resolution transmission electron microscope,nitrogen adsorption and thermogravimetric analysis.The results showed that the hydrated size of MSNs-ChS was 224.6±0.7 nm,and the zeta potential was-10.90±0.64 mV.The PDI was 0.14±0.01,exhibiting the good dispersibility of MSNs-ChS.According to transmission electron microscopy,MSNs-ChS was observed spherical and a clear ChS film with a thickness of about 25 nm on the surface.Thermogravimetric analysis showed that the grafting rate of ChS was 19.2%.Considering the good dispersibility in ethanol,MSNs-COOH was selected for drug loading and further modification to obtain MSNs-ChS@PQ.The effects of different PTX/QC feeding ratios on drug loading were investigated.Combined with the results of cytotoxicity experiments,PTX/QC=1(w/w)was selected as the optimal feeding ratio.And the drug loading contents of PTX and QC were 5.29±0.38%and 5.13±0.19%,respectively.The red blood cells of rabbit were extracted to study the blood compatibility of MSNs-ChS.The results showed that the hemolytic rate of MSNs-ChS was below 5%at a concentration range of 0.1-1 mg/mL,illustrating its safety for intravenous administration.3.In vitro release of MSNs-ChS@PQThe dialysis bag method was used to study the drug release behavior of MSNs-ChS@PQ.Glutathione(GSH)was added to the release medium to simulate the redox environment of tumor cells and the drug release of MSNs-ChS@PQ in different release media was investigated.It was found that the release percentages of PTX and QC were both less than 20%within 36 h in the release medium without GSH.When the release medium contained 20 mM GSH,the release percentages of PTX and QC increased significantly,which were 66%and 53%,respectively.The phenomenon indicated that MSNs-ChS@PQ had redox-responsive release characteristic.4.Anti-tumor research of MSNs-ChS@PQ in vitroThe uptake behavior of doxorubicin-resistant human breast cancer cells(MCF-7/ADR cells)to nanoparticles was investigated.The results showed that its uptake behavior was time-dependent and the cellular uptake of MSNs-ChS was based on CD44 receptor-mediated endocytosis with active targeting.In vitro cytotoxicity,live/dead cell staining,immunofluorescent microtubule staining,apoptosis,cell cycle analysis and P-gp detection experiments verified the effects of MSNs-ChS@PQ in reversing MDR of drug-resistant cells,enhancing chemotherapy efficacy,and decreasing P-gp level.5.Biodistribution of MSNs-ChS@PQ in vivoMouse breast cancer cells(4T1 cells)were injected into the right armpit of female BALB/c mice to establish a tumor-bearing animal model.After successful modeling,DiR-loaded nanoparticles were injected into the tail vein,and the biodistribution of nanoparticles in mice at different time periods as well as the fluorescence intensity in main organs after 24 h were confirmed by in vivo/ex vivo imaging technology.Nanoparticles exhibited tumor-targeting property and prolonged tumor retention time.6.Anti-tumor research of MSNs-ChS@PQ in vivoMCF-7/ADR cells were injected into the right armpit of female BALB/c nude mice to establish a tumor-bearing animal model.Then mice were administrated by tail vein injection.The efficacy of MSNs-ChS@PQ in vivo was evaluated according to tumor volume growth curves,tumor weight,H&E staining and TUNEL analysis of tumor section,confirming the ineffectiveness of PTX-loaded nanoparticles on drug-resistant tumors and highlighting the effect of MSNs-ChS@PQ to reverse tumor MDR in vivo.The main organs of mice in the saline group and the MSNs-ChS@PQ group were subjected to H&E staining,reflecting the safety of the nanoparticles in vivo.
