| In this project,carboxymethyl chitosan(CMCS)was used as the carrier,the photosensitizer Rose Bengal and the active ingredient of traditional Chinese medicine oxymatrine were simultaneously loaded to prepare a nano-drug loading system with good redox responsiveness.In order to overcome the problems of short half-life,low uptake efficiency and poor stability of the two drugs,and play a synergistic effect,at the same time,the intelligent release of drugs in tumor cells can be realized.The main research methods and results of this topic are as follows:Under the action of the catalyst,the linking arm cystamine(CYS)containing disulfide bonds reacted through the amide bond,and RB was grafted on the carrier CMCS to synthesize the CMCS-CYS-RB polymer prodrug.The structure of the CMCS-CYS-RB polymer prodrug was confirmed by ~1H-NMR and IR spectroscopy.The content of RB in the CMCS-CYS-RB polymer prodrug was determined by UV spectrophotometry to be 10.67%.The release of RB in CMCS-CYS-RB polymer prodrug in phosphate buffered saline solution(PBS,p H 7.4)with different concentrations of glutathione(GSH)was determined by dynamic dialysis method.The results proved that the CMCS-CYS-RB prodrug had good redox responsiveness,when the concentration of GSH was 2μM,the drug release amount was only10.37%;when the concentration of GSH was 10 m M,the disulfide bond was broken,the CMCS-CYS-RB prodrug released RB intelligently in a time-dependent manner and the drug release amount reached 76.44%.Single drug nanoparticles(CMCS-CYS-RB NPs)and oxymatrine-loaded combined drug delivery nanoparticles(OMT/CMCS-CYS-RB NPs)were prepared by ion crosslinking,and the best mass ratio of CMCS-CYS-RB prodrugs to OMT was studied with the drug loading and encapsulation efficiency as indexes.The results showed that when the mass ratio of CMCS-CYS-RB prodrugs to OMT was5:1,the drug loading amount was 10.01%,and the encapsulation efficiency was59.37%.The average particle sizes of CMCS-CYS-RB NPs and OMT/CMCS-CYS-RB NPs were 149.6 nm and 179 nm,respectively,with negative Zeta potential.The morphology of the nanoparticles was observed to be spherical shape under transmission electron microscope,and the two nanoparticles could maintain good stability within 72 hours.The in vitro release of RB and OMT in OMT/CMCS-CYS-RB nanoparticles was studied by dynamic dialysis.The analysis of drug cumulative release curves showed that the nanoparticles had good redox responsiveness.Through the in vitro singlet oxygen study,it was found that the combined drug nanoparticles can generate more reactive oxygen species and have a good photodynamic effect.Cal-27 cells(oral cancer cells)and L929 cells(fibroblasts)were used as model cells,and the CCK8 method was used to investigate the toxicity of the carrier,free drugs and nanoparticles.The OMT/CMCS-CYS-RB nanoparticles could play a synergistic effect to improve the anti-tumor effect and had lower toxic and side effects.OMT/CMCS-CYS-RB nanoparticles could be efficiently taken up by Cal-27cells.Compared with free drugs,OMT/CMCS-CYS-RB nanoparticles could induce Cal-27 cells to produce more reactive oxygen species.At the same time,the mitochondrial membrane potential of Cal-27 cells decreased significantly,thereby inducing more tumor cell apoptosis and necrosis.The proteins related to PI3K/AKT pathway and apoptosis pathway were detected by western blotting,and it was found that the anti-tumor mechanism of nanoparticles was to inhibit the activation of PI3K/AKT signaling pathway and trigger the apoptosis of tumor cells through ROS-mediated oxidative stress and mitochondrial apoptosis pathway.The OMT/CMCS-CYS-RB nanoparticles prepared in this study have good redox responsiveness and sustained release,and effectively overcome the problems of Rose Bengal and oxymatrine in vivo application.OMT/CMCS-CYS-RB nanoparticles not only plays a synergistic role in enhancing the curative effect,but also reduces the toxic and side effects on the body,which has certain potential for the treatment of oral cancer. |
PDF Full Text Request