Preparation Of Mesoporous Silica Nanoparticles Containing Trifluoperazine And Study Of Anti-Tumor Effects

Breast cancer（BC）is a complex disease that seriously endangers women’s health and life,and its incidence is increasing year by year.The current treatment for breast cancer is based on traditional chemotherapy drugs,such as Adriamycin,Paclitaxel and Cisplatin,but these drugs have disadvantages such as cardiotoxicity,suppression of the bone marrow hematopoietic system and destruction of the human immune system.What’s more,tumor cells will develop drug resistance after repeated contact with a certain drug,thus leading to a gradual deterioration of the subsequent anti-cancer effect.These shortcomings of traditional drugs have increased the demand for innovative anti-cancer drugs,but the progress of new drug development is slow due to long cycle time,high cost and high risk.In this context,"new use of old drugs"has become a very important strategy.Trifluoperazine（TFP）,a phenothiazine derivative,was approved in the United States in 1959 for the treatment of anxiety and psychiatric disorders.In recent years,studies have reported that TFP can inhibit the growth of a variety of tumors,such as breast cancer,glioblastoma,and lung cancer.However,TFP can easily cross the blood-brain barrier（BBB）and reach high concentrations in the brain thus affecting the central nervous system（CNS）,leading to side effects such as drowsiness,dizziness and extrapyramidal reactions in patients.In this regard,our group proposes to encapsulate TFP in mesoporous silica nanocarriers that cannot cross the blood-brain barrier,thus reducing its central pharmacological effects while improving the efficacy of tumor treatment.The research carried out in this subject is as follows:MSN-NH₂was firstly prepared,and then polyethylene glycol（PEG）and anisamide（AA）were modified on its surface to obtain nanoparticles named MSN@PEG-AA.The structures of the nanoparticles were characterized by transmission electron microscopy,laser particle size and zeta potential analyzer,infrared spectrophotometer,specific surface and porosity analyzer,and differential thermal thermogravimetric simultaneous analyzer.The results showed that the prepared MSN@PEG-AA nanoparticles and drug-loaded nanoparticles（TFP@MSN@PEG-AA,TMPA）were uniformly dispersed with homogeneous particle size.The surface area（SBET）of MSN-NH₂was calculated from the nitrogen adsorption/desorption curves as～471.316 m²/g,and the pore volume（Vp）and pore diameter（DBJH）were～0.968 cm³/g and～4.358 nm,respectively.Infrared spectroscopy and DTG mapping both proved the successful preparation of MSN@PEG-AA.A method for the determination of TFP content was established by high-performance liquid chromatography（HPLC）and a methodological study of the method was carried out.The loading and release of TFP were also investigated.The results showed that the established method for the determination of the content of TFP had good specificity,good precision and reproducibility,and good linearity in the concentration range of 2.5μg/mL to 80μg/mL.The drug loading and encapsulation rates of the prepared drug-loaded nanoparticles（TFP@MSN@PEG-AA,TMPA）were 13.1%and 65.2%,respectively.The release experiments showed that the TMPA nanoparticles had pH-responsive drug release behavior,and the release rate of TFP in PBS buffer at pH 5.5 was the fastest and the cumulative release amount was the highest,reaching 70.9%;the pH-responsive drug release of TMPA nanoparticles could reduce the release of TFP in normal tissue sites and increase the drug concentration in tumor tissues,which could exert anti-tumor effects more efficiently.The safety of MSN@PEG-AA nanoparticles was evaluated using hemolytic assay,and the cytotoxicity of TMPA and its intracellular uptake were examined by MTT method and fluorescence inverted microscopy.The results of hemolysis assay showed that MSN@PEG-AA nanoparticles had good hemocompatibility and good safety.The results of MTT assay showed that the inhibitory effects of free TFP and TMPA nanoparticles on three types of breast cancer cells（MDA-MB-231,MCF-7 and 4T1）were concentration-dependent and time-dependent;with the extension of the action time,TFP in TMPA nanoparticles was gradually released completely,and TMPA nanoparticles could achieve an inhibitory effect comparable to that of free TFP.The results of cell uptake experiments showed that the green fluorescence of 4T1 cells was significantly reduced after pre-incubation with the sigma receptor inhibitor haloperidol（HP）,which confirmed that the nanoparticles entered the tumor cells through sigma receptor-mediated endocytosis and had the ability to specifically target the tumor cells.The intracellular drug release assay showed that the drug-loaded nanoparticles could release the drug normally in 4T1 tumor cells,and the amount of release increased with time.The in vivo anti-tumor effects of TMPA nanoparticles were investigated by constructing a 4T1 tumor-bearing Balb/c mouse model.The results showed that the tumor volume growth of mice in saline group was rapid,and the tumor volume growth of mice in free TFP group,TMP nanoparticles group and TMPA nanoparticles group was inhibited to some extent,among which the TMPA nanoparticles group had stronger inhibitory ability on the tumor volume growth and stronger anti-tumor activity;the change curve of mouse body weight indicated that the TMPA nanoparticles group had lower toxic side effects.H&E staining results showed that each group of drugs had almost no effect on the organ tissue morphology of mice,and there was almost no apoptosis of tumor cells in the saline group,and there were different degrees of apoptosis of tumor cells in the free drug group and the drug-loaded nanoparticle group,among which,the TMPA nanoparticle group showed a higher apoptotic effect.A method for the determination of TFP in various tissues and tumors of mice by HPLC was established,and a methodological study was also performed.The results showed that the analytical method had good specificity,good precision and reproducibility,and good linearity in the concentration range of0.025μg/mL～2μg/mL.The results of in vivo drug distribution experiments showed that there was no statistical difference between the distribution of TFP group and TMPA nanoparticles group in heart,liver,spleen,lung,kidney and tumor;while in brain tissue,the concentration of TFP group was significantly higher than that of TMPA nanoparticles group,indicating that wrapping TFP in the nanocarrier MSN@PEG-AA could significantly reduce the amount of TFP into brain tissue and decrease the central nervous side effects.In summary,the TMPA nanoparticles prepared in this project have great potential in tumor therapy by reducing the effect on the central nervous system while exerting the anti-tumor effect of TFP.