Construction Of Atezolizumab-modified Nanoparticles Loaded With Dihydrotanshinone I And Study Of Its Anti-tumor Effec

At present,chemotherapy,surgical treatment and radiation therapy are common tumor treatment methods in clinical practice.Single therapy has its drawbacks,and the combination of different treatment methods has become the main strategy for tumor treatment.Research has shown that chemotherapy drugs can directly act on tumors,leading to tumor cell death,deregulating the immunosuppressive effect of the tumor tissue,enhancing the anti-tumor effect of immunotherapy.At the same time,immunotherapy can also make tumor cells sensitive to chemotherapy and inhibit the DNA damage repair mechanism after chemotherapy.Based on this,the combination of chemotherapeutic agents and immune checkpoint inhibitors(Immune checkpoint inhibitors,ICIs)has a high application prospect.Studies have shown that dihydrotanshinone I(Dihydrotanshinone I,DHT),as the active ingredient of Chinese miltiorrhiza,can be used to treat a variety of malignant tumors,induce tumor cell apoptosis and inhibit tumor growth.It is a promising anti-tumor active ingredient of Chinese herbal medicine.Programmed death molecule-1(PD-1)is mainly expressed on the surface of activated B lymphocytes,T lymphocytes and macrophages,while programmed death 1 ligand 1(PD-L1)is mainly expressed on the surface of various tumor cells.The combination of the two can inhibit the function of CD4+T lymphocytes and CD8+T lymphocytes,induce lymphocyte apoptosis,and promote tumor resistance to lymphocyte killing,leading to immune escape of tumor cells.ICIs atezolizumab(ATEZO)can specifically bind to PD-L1,block the PD-1/PD-L1 signaling pathway,enhance the killing effect of T lymphocytes on tumors,and effectively inhibit tumor growth.In this study,ATEZO was modified on the surface of DHT loaded polymer nanoparticle to construct tumor cell PD-L1 targeted nanoparticles(ATEZO DHT NP),achieved the synergistic anti-tumor effect of immune checkpoint blockade and immunogenic cell death(ICD),solved the problem of low response rate of chemotherapy and related adverse reactions caused by immunotherapy,put forward effective targeted co-administration strategy,improved the tumor treatment effect of drugs,and explored new methods of tumor treatment.1 Preparation and Characterization of ATEZO DHT NPDihydrotanshinone I nanoparticle(DHT NP)were prepared by emulsion solvent diffusion method.The optimal preparation scheme of nanoparticle was obtained by single factor experiment and orthogonal experiment optimization.The concentration of organic phase was 1.5%,the aqueous phase was 2%sodium cholate solution,the volume ratio of organic phase to aqueous phase was 1:1.8,and the mass ratio of drug to carrier was 1:2.The drug loading and encapsulation efficiency of DHT NP determined by ultra-efficient liquid chromatography(UPLC)were 11.73%and 54.49%,respectively.The optimal experimental condition for the thiolation of ATEZO were obtained through the Ellman’s reagent method.The molar ratio of ATEZO to Traut’s reagent was 1:20,and the reaction time was 30 minutes.The sulfhydrated atezolizumab reacted with the maleimide group on the surface of DHT NP,and modified ATEZO on the surface of DHT NP.The ATEZO coupling rate of ATEZO DHT NP was 56.93%,and the drug loading was 14.43%,measured by BCA kit.ATEZO modified DHT NP nanoparticles(ATEZO DHT NP)were successfully prepared.The particle size of DHT NP was determined by the Marvin particle size analyzer to be 184.9 nm,with a Zeta potential of-18.55 mV.The particle size of ATEZO DHT NP was 246.9 nm,with a Zeta potential of-19.57 mV.The morphology of the nanoparticles observed by transmission electron microscope shows that the nanoparticle are nearly spherical and evenly distributed.Continuous time measurement using a particle size analyzer showed that the particle size of DHT NP and ATEZO DHT NP did not change significantly within a week,indicating their good stability.The hypoxia of poly(lactic-co-glycolic acid)-p-aminoazobenzene-poly(ethylene glycol)-maleimide(PLGA-Azo-mPEG-Mal)polymer materials was investigated by UV-vis spectroscopy,confirming that the Azo bond between PLGA and PEG molecules can be degraded in anaerobic environment.The cumulative DHT release of ATEZO DHT NP in the first 10 h was measured by UPLC,and then slow DHT release was observed within the next 62 h,indicating sustainable drug release properties of ATEZO DHT NP.2 Evaluation of the in vitro anti-tumor effect of ATEZO DHT NPIn this study,a gastric cancer HGC-27 cell model was used to investigate the ATEZO IR808 NP uptake and PD-L1 targeting ability of cells under normoxic and hypoxic conditions.Results in both environments showed that the ATEZO IR808 NP group exhibited better cellular uptake effects compared to the free IR 808 group,and after 30 minutes of ATEZO pretreatment,a decrease in the fluorescence intensity of ATEZO IR808 NP was observed due to the blocking effect of ATEZO.The results of the MTT pharmacodynamics experiment showed that after 48 hours of administration,under hypoxic conditions,the IC50 value of ATEZO DHT NP was 0.125μmol/L,significantly lower than the IC50 values of other groups(P<0.05).At a DHT concentration of 8 μmol/L,the apoptosis of tumor cells was determined by flow cytometry.The apoptosis rate of the ATEZO DHT NP group was 44.2%,and compared with other groups,ATEZO DHT NP induced cell apoptosis more effectively(P<0.05).The results of cell apoptosis experiment,cytotoxicity experiment,and cell uptake experiment match,indicating that the uptake effect of DHT NP cells modified with ATEZO was enhanced,thereby enhancing the anti-tumor effect.3 Evaluation of the anti-tumor effect of ATEZO DHT NP in nude mice in vivoOn the HGC-27 tumor transplantation BALB/c nude mouse model,observation was conducted using an in vivo imaging system.It was found that the fluorescence intensity of ATEZO IR808 NP at the tumor site gradually increased within 8 h,and the fluorescence remained significant within 24 h,indicating that ATEZO IR808 NP has strong tumor targeting ability in vivo.After 24 h,important tissues and tumor tissues of the nude mice were immediately removed.In vitro imaging showed that ATEZO IR808 NP mainly accumulated in the tumor tissue,rather than accumulating in the normal tissues,the tumor fluorescence intensity in the ATEZO IR808 NP group was approximately 1.5 times that of the IR808 NP group.Record the changes in tumor volume of the HGC-27 tumor transplantation BALB/c nude mouse model throughout the entire administration period.The tumor volume of the ATEZO DHT NP group was significantly reduced compared to the DHT NP group.The results of HE staining and TUNEL staining of tumor tissue sections showed that the ATEZO DHT NP group had better anti-tumor efficacy than the DHT NP group.Monitoring body weight showed no significant decrease,and there was no significant difference in plasma levels of ALT,AST,CRE and BUN compared to the normal group(P>0.05),The HE staining results of heart,liver,spleen,lung and kidney sections also showed that the drug had no significant toxic or side effects.4 Evaluation of the anti-tumor effect of ATEZO DHT NP in mice in vivoThe tumor volume changes of the MFC tumor transplantation BALB/c mouse model were recorded throughout the entire administration period.The tumor volume of the ATEZO DHT NP group was significantly reduced compared to other groups.The HE and TUNEL staining results of tumor tissue sections showed that the ATEZO DHT NP group had the best anti-tumor effect.During the entire administration period,body weight was monitored and there was no significant decrease in body weight,and ALT,AST,the levels of CRE and BUN were not significantly different from the normal group(P>0.05),the HE staining results of heart,liver,spleen,lung and kidney sections also showed that the drug had no significant toxic or side effects.Flow cytometry analysis of lymphocyte infiltration in the tumor and spleen of MFC tumor transplantation BALB/c mouse model showed that the proportion of CD4+T cells and CD8+T cells in the tumor after ATEZO DHT NP treatment was 1.837 and 1.256 times higher than that in the DHT NP group,respectively,and the proportion of CD4+CD25+T cells was decreased.Detection of immune related cytokines in tumor supernatant and serum using ELISA kits,including tumor necrosis factor-α(TNF-α),Interferon-γ(IFN-γ)and transforming growth factors-β(TGF-β)compared to the DHT NP group,the ATEZO DHT NP group secretes more TNF-α and IFN-γ in the tumor supernatant and serum,and TGF-β less secretion(P<0.01).The above results indicate that ATEZO DHT NP can improve the tumor immune suppression microenvironment and activate the tumor immune system.Detect the expression of immune checkpoint blocker related protein PD-L1 and ICD related protein CRT and HMGB1 through immunoblotting.The expression of PD-L1 increased after DHT,DHT NP and Lps-P treatment,while the expression of PD-L1 decreased after DHT+ATEZO and ATEZO DHT NP treatment,indicating that DHT+ATEZO and ATEZO DHT NP could specifically bind to PD-L1 and block the PD-1/PD-L1 signaling pathway.The expression levels of CRT and HMGB1 in each treatment group were upregulated,indicating that the drug could induce ICD and relieve tumor immune suppression.In conclusion,this study developed targeted therapeutic tumor nanoscale drugs(ATEZO DHT NP)combining PD-L1 blockade with immunogenic cell death.ATEZO DHT NP selectively releases DHT in cancer cells overexpressing PD-L1,inducing a strong anti-tumor immune response through ICD effect.At the same time,the surface modification of nanoparticles with atezolizumab can not only promote the accumulation of nanoparticles at the tumor site,but also serve as an inhibitor of the PD-1/PD-L1 pathway.In vitro cell experiments have shown that ATEZO DHT NP can significantly inhibit the growth of tumor cells in hypoxic environments.In vivo studies in two animal models showed that ATEZO DHT NP has tumor targeting,obvious anti-tumor effect,high drug safety,and can improve the tumor suppression rate by increasing the proportion of CD3+T cells,CD4+T cells,and CD8+T cells,reducing CD4+CD25+T cells,and promoting the expression of immune related cytokines.Therefore,this study provides a safe and effective combination of ICD therapy and immune checkpoint blockade therapy(ICB),providing hope for future clinical conversion.