Construction Of Two-Way "Homing" PH/Enzyme Response Nano-Controlled Release System And Study Of The Tumor Chemo-Immunotherapy

Objective: In order to accurately deliver chemotherapeutic drugs and immunotherapeutic agents to different target cells,respectively,and avoid the killing of macrophages by chemotherapeutic drugs,we constructed a two-way "homing" pH/ enzyme dual-responsive nano drug delivery system.Then,the response characteristics,M2 tumor-associated macrophage(TAM)phenotype remodeling,antitumor activity and anti-metastatic ability in vitro and in vivo were systematically studied.Methods:(1)Construction and characterization of nanosystems Firstly,the chemotherapeutic drug doxorubicin(DOX)was loaded into the protein cage to form DOX@HFn nanoparticles by utilizing its reversibly dissociation-reassemble characters upon pH changes.The galactoside-functionalized zwitterionic liposome Res@PGZL loaded with resveratrol(Res)was then synthesized by membrane dispersion method.Finally,DOX@HFn nanoparticles were attached to the surface of Res@PGZL via amide bond to obtain DOX@HFn-PGZL@Res system.The nano-systems were characterized by UV-vis spectroscopy and nuclear magnetic resonance spectroscopy.The size and morphology of the system were investigated by dynamic light scattering(DLS)and transmission electron microscopy(TEM).(2)The study of dual-responsive characteristics in vitro The enzyme responsiveness of the system to MMP was investigated by HPLC and TEM.And the acid-response controlled release characteristics of the system were investigated by studying the release of DOX and Res at different pH in vitro.(3)TAM phenotypic remodeling,antitumor activity and anti-metastasis in vitro Fluorescence microscopy and flow cytometry were used to investigate the specific targeting of DOX@HFn and Res@GZL.The phenotypic remodeling of M2-TAMs by Res@GZL was analyzed by ELISA kit and Western blot.In addition,the cytotoxicity of the blank vector and the inhibitory effect of the delivery system on breast cancer cells were examined by MTT assay.The anti-metastatic ability of the system was studied by scratch and Transwell experiments.(4)The studies of targeting and anti-tumor activity in vivo The system was labeled with IR783,and the tissue distribution of free IR783 and IR783@HFn-PGZL in mice was studied by in vivo fluorescence imaging system.The BALB/c female mice bearing 4T1 breast cancer were used as animal models,the anti-tumor effect and side effects of the delivery system were analyzed by the changes of tumor volume,HE staining pathological section and Tunel staining.(5)In vivo anti-metastasis study Similarly,BALB/c female mice were used to construct metastasis prevention model and metastasis model of 4T1 breast cancer,and the anti-metastasis effect of the nano-system was studied by metastatic nodules and lung pathological sections of lung tissue.Results: DOX@HFn-PGZL@Res was analyzed by DLC and TEM,the results showed that the system was spherical,with a particle size of about 130 nm and a potential of about-15 mV.HPLC and TEM results showed that the dual drug-loaded nanosystem can be separated into DOX@HFn and Res@GZL in response to MMP2.The results of in vitro release showed that the release rate of the system under intracellular acidic conditions was significantly higher than that of normal physiological conditions.In addition,in vitro cell experiments indicated that DOX@HFn and Res@GZL had good targeting to their respective target cells,which can avoid the damage of macrophage by chemotherapeutic drugs.The blank vector of this system was safe and non-toxic,the DOX@HFn-PGZL@Res group had a significant inhibitory effect on the proliferation of cancer cells.In vivo imaging results showed that the accumulation of the system in tumor tissue was significantly higher than other major organ tissues,it still had excellent tumor targeting even in mice.In vivo pharmacodynamics and anti-metastatic results indicated that DOX@HFn-PGZL@Res combined chemotherapy and immunotherapy significantly inhibited tumor growth and metastasis and was almost non-toxic to normal tissues.On the other hand,the results of phenotypic remodeling of M2-TAMs in vitro and in vivo also showed that Res@GZL nanosystem can successfully repolarize M2-TAMs into M1-TAMs,improve the immunosuppressive microenvironment,and exert a positive immune response.Conclusion: The DOX@HFn-PGZL@Res nanosystem prepared in the study can simultaneously load chemotherapeutic drugs and immunotherapeutics,isolate in situ in tumor tissue by responding to MMP2.Then it can two-way recognize cancer cells and M2 macrophages,improve immunosuppression,and effectively inhibit tumor growth and metastasis through chemotherapy and immunotherapy.