Preparation And Evaluation Of Doxorubicin-loaded Chondroitin Sulfate A-ss-vitamin E Succinate Micelles

Objective:In this study,reduction-sensitive chondroitin sulfate A（CSA）-vitamin E succinate（TOS）polymer（CST）with disulfide bond was synthesized,and adipic dihydrazide-linked（ADH）CSA-TOS polymer（CAT）was synthesized as a non-reduction-sensitive control.The physicochemical properties,cytotoxicity and antitumor effect of DOX-loaded micelles on tumor bearing nude mice were studied to evaluate their reductive response.Methods:CST and CAT were characterized by infrared spectrometer,nuclear magnetic resonance and pyrene fluorescence.The particle size and morphology of DOX-loaded micelles were determined by dynamic light scattering and transmission electron microscopy.The antitumor drug DOX was used as the model drug,and the DOX-loaded CST（D-CST）micelles and DOX-loaded CAT（D-CAT）micelles were prepared by a dialysis method.The drug loading and encapsulation efficiency were determined by UV spectrophotometry.The drug release characteristics of DOX-loaded micelles in phosphate buffer solution（PBS,p H 7.4）containing 20m M reduced glutathione（GSH）at 37℃were investigated.A549 cells were pretreated with 20m M GSH,and the uptake of A549 cells was observed by laser confocal microscopy（CLSM）.The cytotoxicity of DOX-loaded micelles and DOX·HCl against A549,AGS and C6 cells was evaluated by MTT colorimetry.The antitumor effects of DOX-loaded micelles on A549 tumor bearing nude mouse model were studied.Results:The critical micelle concentrations（CMC）of CST and CAT were 0.033 and0.041 mg/m L,respectively.The drug loading of D-CST and D-CAT were 13.0±0.9%and 11.4±1.1%,and their encapsulation efficiencys were 74.9±5.8%and 64.7±6.7%,and the particle sizes were 268±11.2 and 232±12.4 nm,respectively.The morphology of DOX-loaded micelles was nearly spherical.In 20m M GSH containing PBS,24%of DOX was released from D-CAT micelles,and more than 54.1%of DOX was released from D-CST micelles within 120 h.In the cell uptake experiment,for D-CST micelles,the fluorescence intensity in GSH-pretreated A549 cells was significantly higher than that in the unpretreatment A549 cells group.In the 24 h cytotoxicity test,the order of toxicity was DOX·HCl>D-CST>D-CAT.The IC₅₀ values of DOX·HCl,D-CST and D-CAT were 1.22,5.16 and 10.88μg/m L for A549 cells,1.26,3.61 and6.63μg/m L for AGS cells,and 0.791,3.909 and 6.638μg/m L for C6 cells,respectively.Compared with 5%glucose group,DOX·HCl,D-CST and D-CAT groups significantly inhibited tumor growth（P<0.001）,D-CST and D-CAT had stronger inhibitory effect on tumor growth than DOX·HCl（P<0.01 and P<0.05,respectively）.The tumor volume in D-CST group was 1.4 times smaller than that in D-CAT group（P<0.05）.Compared with 5%glucose group,the body weight of nude mice in DOX·HCl group decreased after treatment（P<0.01）and weight gain in drug-loaded micelles groups（P>0.05）;The pathological results showed that large nuclei and spherical or spindle-shaped tumor cells with wide distribution were observed in the tumor tissues of the 5%glucose,CAT and CST groups.Compared with the DOX·HCl and D-CAT groups,the apoptosis of tumor cells was more obvious in the D-CST micelle group.Conclusions:CST micelles have good drug loading capacity and encapsulation efficiency.In vitro release experiment simulates the internal environment of human tumor reductivity,which verifies that D-CST has reductive sensitivity of intracellular drug release.Animal experiments showed that D-CST micelle reduces the toxic and side effects of DOX on normal cells,and it has the strongest effect in vivo and improves the anti-tumor effect of DOX.In conclusion,reduction-sensitive CST micelles can be used as delivery carriers for cancer chemotherapy,providing a theoretical basis for the construction of intelligent release nanomaterials.