Sulfur Dioxide Nanomedicine Enhances The Therapeutic Effects Of Doxorubicin And PD-L1 Monoclonal Antibody In Melanoma And Correlated Mechanism Analysis

Research backgroundMelanoma is a malignant tumor derived from melanocytes.Chemotherapy and immunotherapy are important treatment methods for advanced melanoma.However,melanoma has relatively poor sensitivity to chemotherapy drugs.Recently,immunotherapy has made unprecedented progress in the treatment of advanced melanoma.Unfortunately,nearly half of the patients have no response or transient response to immunotherapy.Therefore,it's essential to explore effective targets to enhance the therapeutic effects of chemotherapy and immunotherapy in melanoma.In recent years,it has been found that the changes of internal environment of tumor cells,such as intracellular redox balance,are closely related to the therapeutic effects of chemotherapy and immunotherapy.Reactive oxygen species(ROS)is an intracellular oxidative active substance.Researches have shown that the increase of ROS in tumor cells will reduce the expression of P-glycoprotein(P-gp,a protein that promotes drug efflux)and increase chemotherapy sensitivity.In addition,studies have verified that excessive ROS can cause strong oxidative stress in tumor cells,induce immunogenic cell death(ICD)and improve immunity.Therefore,increasing the level of ROS in tumor cells for destroying the redox balance in tumor cells is expected to enhance the therapeutic effects of chemotherapy and immunotherapy in melanoma.There are many ways to increase the content of ROS in tumor cells,including application of sulfur dioxide(SO₂)which is an oxidizing gas molecule to induce strong oxidative stress in tumor cells.Previous studies have shown that SO₂is closely related to the body pathophysiological activities.The sulfur-containing amino acids in the human body will produce endogenous SO₂ during the body metabolism.Small doses of SO₂ play an important role in the regulation of cardiovascular system.However,excessive SO₂ will increase intracellular ROS level and induce oxidative stress.Therefore,increasing the content of SO₂ in the tumor site is expected to increase the level of ROS and enhance the chemotherapy and immunotherapy effects.In view of this,we designed tumor microenvironment-responsive SO₂nanomedicine and further combined SO₂ gas therapy with melanoma chemotherapy drug doxorubicin(DOX)or immunotherapy drug PD-L1 monoclonal antibody.SO₂is expected to disrupt the redox balance in tumor cells,increase the level of intracellular ROS,and enhance the therapeutic effects of DOX and PD-L1 monoclonal antibody.Based on the above research background,we conducted the research as follows.Part One Reduction-responsive SO2 nanomedicine enhances the therapeutic effect f doxorubicin in melanoma and correlated mechanism analysisPurposeConstruct a novel reduction-responsive SO₂nanomedicine(PDDN)and explore the inhibitory effect of SO₂ on melanoma and correlated mechanisms.In addition,DOX was encapsulated into the core of PDDN to form DOX-loaded nanomedicine(PDDN-DOX),which combines gas therapy with chemotherapy and aims to enhance the chemotherapy effect of melanoma.Methods1.Preparation of nanomedicne.In brief,the target polymer m PEG-g-Dex(DIBA)are synthesized through chemical conjugation.And the synthesized m PEG-g-Dex(DIBA)can self-assemble into PDDN in water.Furthermore,DOX was encapsulated into the core of PDDN to form DOX-loaded nanomedicine(PDDN-DOX).2.In vitro experiments.Flow cytometry and laser confocal microscopy were used to detect the internalization of nanoparticles by melanoma cells.DEACA and DCFH-DA fluorescence probes were utilized to detect the level of SO₂ and ROS in the tumor cells.MTT was used to detect the inhibitory effects of the tested drugs.Western blot was used to detect whether PDDN can reduce the expression of P-gp.3.In vivo experiment.Evaluate the distribution and anti-tumor effect of PDDN-DOX by constructing murine melanoma in situ and lung metastasis models.Detect the expression of SO₂ and ROS in the tumor by staining the tumor sections with fluorescence probes.Immunofluorescence and immunohistochemical methods were utilized to test the apoptosis level and P-gp expression of the tumor tissues.Results1.PDDN and PDDN-DOX were successfully synthesized.The result of transmission electron microscopy(TEM)indicated that PDDN and PDDN-DOX had spherical structures.The hydrodynamic radiuses measured by dynamic light scattering were44.4±3.2 nm and 51.2±6.1 nm,respectively.In addition,the UV absorption spectrum indicated that DOX was successfully loaded in the PDDN carrier.2.In vitro experiments:PDDN could be endocytosed by melanoma cells and released SO₂,which exhausted intracellular GSH,increased the level of ROS and reduced the expression of P-gp protein on the cell surface.In addition,PDDN-DOX could be internalized and released SO₂ and DOX simultaneously in the tumor cell.The results of MTT showed that the synergy index of SO₂ and DOX is less than 1,indicating that they have a synergistic anti-tumor effect.3.In vivo experiments:PDDN-DOX can significantly inhibit the growth of subcutaneous and lung metastatic melanoma.The results of fluorescence staining and immunohistochemical of tumor tissues suggested that PDDN could increase the level of SO₂ as well as ROS in the tumor and reduce the expression of P-gp protein on the cell surface,which contributed to reducing the efflux of chemotherapy drugs and increasing the sensitivity of chemotherapy..ConclusionThe reduction-responsive SO₂nanomedicine PDDN was successfully constructed.The obtained PDDN could responsively release SO₂ in melanoma cells,increase intracellular ROS level and decrease the expression of tumor cell surface P-gp protein.In addition,DOX was encapsulated into the core of PDDN to form DOX-loaded nanomedicine(PDDN-DOX).Furthermore,SO₂ and DOX showed significant synergistic effects both in vivo and in vitro.Part Two Mitochondrial targeted SO2 nanomedicine enhances the therapeutic effect of PD-L1 monoclonal antibody in melanoma and correlated mechanism nalysisPurposeBased on the results of Part One,in order to improve the utilization of SO₂ and induce stronger oxidative stress,we designed a mitochondrial targeted SO₂ nanomedicine(RhoB-DNs)in this part and explore the inhibitory effect of RhoB-DNs on melanoma and correlated mechanisms of regulating tumor immune microenvironment.In addition,the combination of RhoB-DNs and PD-L1 monoclonal antibody may provide a new idea for improving tumor immunotherapy.Methods1.Preparation of nanomedicine:In brief,the mitochondrial targeted SO₂nanomedicine(RhoB-DNs)was prepared through chemical conjugation-induced self-assembly method.Laser confocal microscopy was performed to detect the co-localization of RhoB-DNs and mitochondria.Fluorescence probes were used to detect intracellular SO₂ and ROS levels,respectively.Besides,MTT analysis was utilized to test the inhibitory effects of RhoB-DNs on melanoma cells.2.In order to verify whether RhoB-DNs can induce ICD of melanoma cells,we incubated B16-F10 cells with RhoB-DNs.And then we evaluated the level of ICD by testing the expression of calreticulin(CRT)as well as the release of high mobility protein(HMGB1)and adenosine triphosphate(ATP).Vaccination and re-challenge experiment was used to verify whether RhoB-DNs could induce ICD in vivo.3.We constructed murine skin melanoma model and tested the inhibitory effect of RhoB-DNs.At the end of treatment,the tumors and spleens were obtained for immune analysis.In addition,we further combined RhoB-DNs and PD-L1 monoclonal antibody in this study to combat subcutaneous and metastasis melanoma.It is planned to test the treatment effect of this combination therapy on melanoma.Results1.The RhoB-DNs was successfully synthesized.TEM result indicated that it had a spherical structure and its diameter is 111.9±2.27 nm according to DLS.Laser confocal microscopy results showed that RhoB-DNs could target mitochondria and release SO₂ in the cells and increase intracellular ROS level.2.In vitro experiments confirmed that RhoB-DNs could induce the apoptosis of melanoma cell,at the same time enhance the expression of CRT on the tumor cell surface,and release HMGB1 and ATP into the supernatant,which indicated that RhoB-DNs can induce ICD.3.The results of the vaccination and re-challenge experiment(the gold standard of ICD)showed that on the 20th day of the re-challenge,all mice in the PBS group developed tumors(10/10),while 5/10 mice in the RhoB-DNs group did not develop tumors,which indicated that RhoB-DNs could induce ICD in vivo..4.In vivo experimental results showed that RhoB-DNs could increase the infiltration of activated dendritic cells and CD8⁺T lymphocytes in the tumor.RhoB-DNs and PD-L1 onoclonal antibody could significantly inhibit tumor growth,which played a synergistic anti-tumor effect.ConclusionThe mitochondrial-targeted SO₂ nanomedicine(RhoB-DNs)was successfully constructed.The obtained RhoB-DNs could increase the intracellular level of ROS,induce ICD of melanoma,effectively activate anti-tumor immunity.In addition,the combination of RhoB-DNs and PD-L1 monoclonal antibody can significantly combat in situ and metastatic melanoma.Based on the above two parts,we confirmed that SO₂ can break the redox balance in tumor cells,improve the level of intracellular ROS and induce the oxidative stress of tumor cells.On one hand,it can reduce the cell expression of P-gp,reduce the efflux of DOX,increase the accumulation of DOX,and enhance the therapeutic effect of elanoma.On the other hand,it can induce the ICD of tumor cells and promote the recognition of immune cells,therefore activate anti-tumor immunity and improve the therapeutic effect of PD-L1 monoclonal antibody.In summary,this study combines SO₂ gas therapy with melanoma chemotherapy or immunotherapy.It provides new ideas for improving the therapeutic effect of melanoma and provides a theoretical basis for the transformation of basic research into clinical practice.