Nir Responsive Nanoenzymes Via Photothermal Ablation And Hypoxia Reversal To Potentiate The STING-dependent Innate Antitumor Immunity

Objective:This research aims to design a near infrared light(NIR)-responsive nanoenzyme(PBMn/OVA NE),which is constructed by doping manganese into the ovalbumin(OVA)-templated Prussian blue(PB)nanoparticles.The nanoenzyme can be used in combination with photothermal ablation and hypoxia reversal to enhance STING-dependent innate antitumor immunity.Then,the photothermal conversion performance and catalase activity,immune regulation as well as antitumor effect of PB-Mn/OVA NEs are investigated in vitro and in vivo.Methods:1.PB-MN/OVA NEs were prepared by one-step method.During the synthesis process,manganese was doped into prussian blue nanoparticles by ion exchange using OVA as a template,and PB-MN/OVA NEs was centrifugally collected.2.The physical properties of PB-Mn/OVA NEs were characterized by Malvern particle size analyzer,high resolution transmission electron microscopy(TEM)and X-ray diffractometer(XRD),and the chemical properties were characterized by Fourier infrared spectroscopy(FTIR)and X-ray photoelectron spectroscopy(XPS).3.The PB-Mn/OVA NEs solution was irradiated by 808 nm NIR to explore the optimal power density and concentration,and then the photothermal conversion efficiency was calculated according to the relevant formula.4.The degradation of the characteristic peaks of PB-Mn/OVA at different p H was measured by ultraviolet visible absorption(UV-Vis)spectrometer.The oxygen content of hydrogen peroxide catalyzed by PB-Mn/OVA NEs was determined by an oxygen solubilizer.5.PB-Mn/OVA NEs was co-incubated with mouse erythrocytes,mouse embryonic fibroblasts(MEFs)and mouse breast cancer cells(4T1)to explore its compatibility with blood and normal/tumor cells.After co-incubating PB-Mn/OVA NEs with 4T1 cells,CCK-8 kit was used to detect 4T1 cell viability after 808 laser irradiation.The photothermal killing effect of PBMn/OVA NEs on 4T1 cells was investigated by staining live and dead cells after laser irradiation.ICD markers were detected by ELISA kit.6.Western blot was used to investigate the effect of PB-Mn/OVA NEs on cGAS-STING pathway in DC2.4 cells.The changes of maturity and antigen presentation ability of DC2.4treated with PB-Mn /OVA NEs were analyzed by flow cytometry.7.Thermal imager and magnetic resonance imaging system were used to verify the thermal and nuclear magnetic imaging capabilities of the PB-Mn/OVA NEs.8.The mouse subcutaneous breast cancer models were constructed,and the expressions of DC cells,cytotoxic T cells and memory T cells in lymph nodes and spleen of mice were analyzed by flow cytometry after different treatments.9.The mouse subcutaneous breast cancer models were constructed,and the tumor size and survival of mice were detected after different treatments,so as to explore the anti-tumor effect of PB-Mn/OVA NEs on mouse breast cancer.Result:1.PB-Mn/OVA NEs with dark blue granules were prepared successfully.2.PB-Mn/OVA NEs showed good dispersibility in de-ionized water with uniform particle size of about 160 nm.The structure was square under transmission electron microscope.The mapping results were consistent with the XPS results,showing that the PB-Mn/OVA NEs were rich in C,N,Fe,and Mn elements.FTIR meant that PB-Mn/OVA NEs and PB had similar characteristic peak C≡N,2070 cm-1,XRD results showed that PB-Mn/OVA NEs and PB had similar crystal structure.The above results proved that manganese was successfully doped into Pb-Mn /OVA NEs,and the physical and chemical characteristics were similar to the original PB nanoparticles.3.According to a series of experiments on photothermal conversion performance,the photothermal conversion efficiency of PB-Mn/OVA NEs was about 30% and the photothermal stability was excellent.4.PB-Mn/OVA NEs slowly degraded at p H6.5 and 4.5,and the lower the p H,the faster the degradation.PB-Mn/OVA NEs showed good catalase activity and could decompose H2O2 to produce O2.5.When the maximum solution concentration was 400 μg/m L,PB-Mn/OVA NEs showed excellent blood and cell compatibility,hemolysis rate was lower than 6%,and cell viability was higher than 85%.After 5 minutes of laser irradiation,PB-Mn/OVA NEs showed significant tumor killing ability and reduced 4T1 cell viability to less than 10%.6.PB-Mn/OVA NEs activated the cGAS-STING pathway in DC2.4 cells with a concentration-dependent manner,and increased the level of type I interferon.At the same time,it could promote the maturation and antigen presentation ability of DC2.4 cells.7.The temperature of PB-Mn/OVA NEs could rise to about 60℃ within 5 minutes in vivo,showing good thermal imaging ability.in addition,the PB-Mn/OVA NEs demonstrated excellent T1-weighted imaging capabilities both in vivo and in vitro,and had the potential to be used as a nuclear magnetic contrast agent.8.The expression of DC cells,cytotoxic T cells and memory T cells in lymph nodes and spleen of mice was analyzed.The results showed that PB-Mn/OVA NEs could increase the content of the above cells and had the ability to activate anti-tumor immune response in vivo.9.Within 20 days after treatment in mice,the tumor volume of PB-Mn/OVA NEs group decreased and did not relapse,and the life span of 60% mice was extended more than 60 days.HE staining illustrated no obvious lesions in organs.The results explained that PB-Mn/OVA NEs had excellent anti-tumor ability and no obvious side effects.Conclusion:1.In this paper,a PB-Mn/OVA NEs was synthesized by oxidative polymerization and ion exchange.2.The resultant PB-Mn/OVA NEs exhibited favorable catalase activity to produce oxygen,which was conducive to alleviate the tumor hypoxic microenvironment.Under 808 nm NIR irradiation,the PB-Mn/OVA NEs with outstanding photothermal conversion effificiency of 30%signififi-cantly destroyed tumor cells by inducing immunogenic cell death(ICD).3.The PB-Mn/OVA NEs could activate the cGAS-STING pathway to promote the maturation and the antigen cross-presentation ability of DCs,which further activated cytotoxic T lymphocytes and memory T lymphocytes.