| Liver cancer is the third most common malignant tumor and the second leading cause of death in China.The early stage of liver cancer is not prominent,and it is often found in the middle and late stages,so the cure rate is low and the mortality rate is high.At present,chemotherapy is still the most common clinical method for cancer treatment.Studies have found that As2O3(ATO)has a certain clinical therapeutic effect on liver cancer.However,due to the rapid immune clearance of ATO by the human body,the uptake dose at the tumor site is insufficient,and the clinical effect is poor.High doses of ATO have greater toxic and side effects.The rapid development of nanotechnology has promoted the research of arsenic nano-drug delivery systems.However,the previous arsenic nano-drug delivery systems have problems such as arsenic leakage,low encapsulation efficiency,and complicated preparation process,and most of them are loaded with arsenic by physical action,and the arsenic content and release behavior are difficult to control.Therefore,designing an arsenic nano-drug delivery system that can effectively deliver ATO,control arsenic release behavior,and have high anti-liver cancer biological activity is of great significance for the clinical application of ATO in the treatment of liver cancer.In this study,the coupling grafting method was used to modify chitosan with deoxycholic acid(DCA),polyethylene glycol(PEG),folic acid(FA)and N-acetyl-L-cysteine(NAC)respectively,especially chitosan derivatives with different thiol content were prepared,and the multifunctional chitosan derivatives were characterized by infrared spectroscopy,elemental analysis and ultraviolet spectroscopy,etc.By adjusting the ratio of reactants,chitosan derivatives with DCA substitution degree of 5.2%-10.7%and PEG substitution degree of 0.8%-13.2%were synthesized,and their self-assembly ability was studied by pyrene fluorescent probe and TEM.In order to obtain chitosan self-assembled nanoparticles with better self-assembly ability and better self-assembly morphology,the chitosan derivative DCA 10.7-CS-PEG3.1 with DCA substitution degree of 10.7%and PEG substitution degree of 3.1%was selected for further discussion.In view of the difficulty in regulating the release behavior of ATO loaded by physical action,and there were problems such as arsenic leakage,in view of the reversible characteristics of the As?-S bond in the GSH reduction environment,the thiol-containing multifunctional chitosan derivatives prepared above were prepared by a simple self-assembly method.Arsenic-loaded chitosan nanoparticles based on chemical interaction(As?-S)loaded ATO were prepared by assembly method.TEM and DLS studies showed that the arsenic-loaded chitosan nanoparticles have good self-assembled morphology and good dispersibility,indicating that the nanoparticles can be more compact based on the loading of ATO based on the As?-S bond.The surfaces of arsenic-loaded chitosan nanoparticles were positively charged,which was more favorable to act on negatively charged cancer cells.The drug loading capacity of arsenic-loaded chitosan nanoparticles was tested by AFS,and the results showed that the arsenic-loaded chitosan nanoparticles loaded with ATO based on physical action can only load 5.6 ATO per CS molecule,while the arsenic-loaded chitosan nanoparticles with a thiol substitution degree of 20.2%can load 20 ATO per CS molecule.Compared with physical loading of ATO,As?-S can effectively increase the arsenic loading,and the ATO loading can be controlled by regulating the thiol content.In vitro drug controlled release studies showed that DCA10.7-CS-PEG3.1-FA-NAC-ATO has a GSH response in drug release.At 24 h,the cumulative release rate of ATO in PBS of arsenic-loaded chitosan nanoparticles with a thiol substitution degree of 20.2%was 23.3%,when the GSH concentration in the system was increased to 10 mM,the cumulative release rate of ATO was as high as 95.8%,which was consistent with the reversibility of the As?-S bond in the GSH reduction environment,which could effectively control the release behavior of ATO.Arsenic-loaded chitosan nanoparticles had good suspension stability in vitro and can be stored stably at 4? for 2 weeks.which was of great significance for in vitro storage and in vivo circulation.In order to study the anti-cancer activity of arsenic-loaded chitosan nanoparticles,the biological evaluation of rsenic-loaded chitosan nanoparticles was carried out in HepG2 cells.The cytotoxicity,cellular uptake,cellular distribution and apoptosis of arsenic-loaded chitosan nanoparticles were investigated by HepG2 cells.The results showed that the unloaded chitosan nanoparticles had good biocompatibility.Arsenic-loaded chitosan nanoparticles had strong cytotoxicity to HepG2 human hepatoma cells,and the cytotoxicity of ATO loaded by As?-S was higher than that of arsenic-loaded chitosan nanoparticles loading ATO based on physical action and free ATO,but for 293T human renal epithelial cells,the cytotoxicity had the opposite result.The IC50 was consistent with the cytotoxicity results,for HepG2 cells,the IC50 of arsenic-loaded chitosan nanoparticles with a degree of thiol substitution of 20.2%was 0.88 ± 0.28 ?g/mL,which was lower than that of arsenic-loaded chitosan nanoparticles(1.10 ± 0.31 ?g/mL)based on physical action and free ATO(1.59 ± 0.29 pg/mL),while for 293T cells,the IC50 results were opposite.The analysis was mainly because the As?-S bond can stabilize ATO and reduce the release of ATO in the extracellular base fluid,and the modification of FA can effectively improved its targeting effect on HepG2 cells,thereby increasing the accumulation of ATO in HepG2 cells.The cellular uptake situation further indicated and confirmed that FA modification could endow the arsenic-loaded chitosan nanoparticles with the ability to target liver cancer cells.Quantitative analysis further showed that As?-S could better stabilize ATO and increase the cellular uptake of ATO.Arsenic-loaded chitosan nanoparticles with a degree of thiol substitution of 14.1%induced a total apoptosis rate of 90.4%in HepG2 cells,while the total apoptotic rates of cells with physical loading and free ATO were 79.6%and 75.1%,respectively,further indicating that As?-S Loading ATO could increase the availability of ATO,thereby inducing more apoptosis of hepatoma cells.The pharmacokinetics of arsenic-loaded chitosan nanoparticles were investigated in healthy SD rats,and the pharmacokinetics,liver tumor targeting properties and in vivo anti-hepatocellular carcinoma activity of arsenic-loaded chitosan nanoparticles were investigated in tumor-bearing mice.The results showed that DCA10.7-CS-PEG3.1-FA-NAC20.2-ATO had better pharmacokinetic properties,and its half-life in blood circulation was about 10 times that of free ATO.In addition,the drug-loading system had good liver tumor targeting,which could effectively promote the accumulation of ATO in liver tumor sites.The in vivo anti-tumor results showed that the tumor inhibition rate of arsenic-loaded chitosan nanoparticles was as high as 86.4%after 21 days of treatment of tumor-bearing mice,which greatly improved the anti-tumor effect of ATO and significantly reduced the toxic and side effects on normal organs and tissues. |
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