PH/hyaluronidase Dual Response Nano Drug Delivery System For Combination Therapy In Non-small-cell Lung Cancer

Objective:Non-small cell lung cancer(NSCLC)accounts for 80% of all lung cancers with high morbidity and mortality.Chemotherapy is still the main therapeutic method for non-small cell lung cancer at different stages.However,chemotherapy drugs have defects such as low water solubility,poor targeting and large side effects.These defects will reduce the efficacy of chemotherapy.The application of stimulus-responsive nano drug delivery system is an effective solution to improve the efficacy of chemotherapy.Although the application of stimuli-responsive nano drug delivery systems has improved the tumor treatment efficiency of traditional chemotherapy,single chemotherapy still has the disadvantages of low bioavailability and large side effects.One of the effective ways to solve these problems is to combine chemotherapy with other treatments such as immunotherapy,phototherapy,chemodynamic therapy,gene therapy,etc.The combination therapy could improve the bioavailability of drugs,reduce side effects and overcome drug resistance of tumor cells,achieve a more pronounced therapeutic effect.Methods:In this work,RCDs-HA@AZD and PCN-CuO-HA@AZD nano drug delivery system were prepared.The morphology,elemental composition,surface functional groups and optical properties of the materials were characterized by TEM,XRD,FT-IR,XPS,UV-vis and FL.The drug release rates and hemolysis rates of RCDs-HA@AZD and PCN-CuOHA@AZD were studied.The photothermal properties of RCDs-HA@AZD were evaluated under 660 nm laser irradiation.The fluorescence imaging ability of RCDs-HA@AZD was studied using A549 cells and HLF cells.The therapeutic effects of RCDs-HA@AZD were evaluated by MTT and AM-PI co-staining experiments.The ·OH generation ability of PCNCuO-HA@AZD was investigated using reactive oxygen probe.The uptake of the PCNCuO-HA@AZD by cells was explored using A549 cells and HLF cells.Flow cytometry was used to study the apoptosis rate of PCN-CuO-HA@AZD.The mouse models of non-small cell lung cancer were established to determine the in vivo distribution,therapeutic efficacy and biosafety of PCN-CuO-HA@AZD.Results:The characterization results of RCDs-HA@AZD and PCN-CuO-HA@AZD demonstrated that the above nano drug delivery system synthesized successful.The drug release rates of RCDs-HA@AZD and PCN-CuO-HA@AZD were 51.4% and 50%,respectively.Hemolysis experiments showed indicated that the hemolysis rates of the nano drug delivery system were 0.63% and 1.83%,respectively.The temperature of RCDsHA@AZD can reach 46.7°C under 660 nm laser irradiation for 10 min.After incubating A549 cells and HLF cells with RCDs-HA@AZD,A549 cells showed bright red fluorescence,while HLF cells only showed appeared weak fluorescence.MTT results displayed that the survival rate of A549 cells was in the range of 90.29%-18.74% when the cells incubated with RCDs-HA@AZD(10-200 μg/m L)and irradiated with 660 nm laser.AM-PI staining indicates that green fluorescence and red fluorescence coexisted in the monotherapy group,while red fluorescence existed throughout the field of view in chemotherapy/PTT group.PCN-CuO-HA@AZD could generate ·OH in the acidic environment and then oxidize TA to fluorescent TAOH.After incubating with PCN-CuO-HA@AZD,the A549 cells showed bright fluorescence,while HLF cells showed only weak fluorescence.The survival rate of A549 cells incubated with 10-150 μg/m L concentrations of PCN-CuO-HA@AZD was 86%-23.75%.AM-PI staining shows exhibited that green fluorescence and red fluorescence coexisted in the monotherapy group,while red fluorescence existed throughout field of vision in the chemotherapy/PDT combined treatment group.The apoptosis rate of A549 cells was 80% after incubating with PCN-CuO-HA@AZD(150 μg/m L).The fluorescence intensity in the tumor tissue gradually increased and reached the highest level after injection for 9h.The tumor volume was decreased significantly after 14 days of combined treatment.During the treatment period,the body weight of mice in each group did not change significantly.H&E staining results indicated that the cells morphology of the main organs was normal.The blood biochemistry and hematology analysis displayed that there was no significant difference between experimental groups and control groups.Conclusion:The drug release of RCDs-HA@AZD and PCN-CuO-HA@AZD all exhibited p H and hyaluronidase dual stimulus responsiveness.Under 660 nm laser irradiation,RCDsHA@AZD has a good photothermal effect.RCDs-HA@AZD can emit red fluorescence in cells and has good cell imaging ability.MTT assay and AM-PI double staining assay revealed that RCDs-HA@AZD had favorable therapeutic efficacy on non-small cell lung cancer.PCN-CuO-HA@AZD has a high ability to generate ·OH.Cell uptake experiments indicated that PCN-CuO-HA@AZD had excellent targeting ability to tumor cells.MTT assay and AM-PI double staining assay demonstrated that PCN-CuO-HA@AZD had excellent combination therapy effect on non-small cell lung cancer.Apoptosis experiments indicated that PCN-CuO-HA@AZD could effectively induce cancer cell apoptosis.In vivo experiments proved that PCN-CuO-HA@AZD could aggregate to the tumor site with obvious tumor therapeutic effect and high biosafety.