Studies On Functional Mesoporous Silica Nanoparticles Loading Paclitaxel And Quercetin For Reversing Multidrug Resistance In Breast Cancer

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.