Reactive Oxygen Species Responsive Micelles Based Two-photon Photodynamic Therapy In Combination With Immune Checkpoint Therapy Against Breast Cancer

Breast cancer is the most frequent cancer among Chinese women and the incidence of breast cancer occupies the first position in malignant cancers,which is significantly higher than the world's average morbidity and appears an increasing trend.The mostly used treatment of breast cancer is surgery,with the combinational treatment of chemotherapy,radiotherapy and endocrine therapy,the clinical efficacy has been significantly improved.However,surgery remains high recurrence rate,chemotherapy and endocrine therapy always accompanied with strong side-effects,thereby exploring new treatment strategy for breast cancer is necessary.Photodynamic therapy?PDT?is a high efficient anti-cancer therapy,which utilize laser with specific wavelength to excite the photosensitizers accumulated in tumor site,then generate a large amount of ROS through the reaction of photosensitizer and oxygen in tumor cells,ultimately induce cancer cell apoptosis and necrosis through oxidative damage.PDT is a non-invasive therapy,with tumor targeting,clear effect and can be applied repeatedly with no drug resistance.However,most photosensitizers are water insoluble and easy to form aggregates in the body,which results in low ¹O₂ yields.Besides,common photosensitizers are lack of active tumor targetability,and mostly using poor penetration visible light as light source,which limits their applications.Besides killing the tumor cells directly,PDT can also induce acute inflammation,enhance tumor immunogenicity and activate CTL to stimulate anti-tumor immune reaction preliminary and induce ICD.CTL is an important immune cell to kill tumor,but the high expression of CTLA-4 and PD-1 in tumor microenvironment inhibited its anti-tumor activity.It will result tumor cells escaping from the immune system and metastasis.Fortunately,in recent years,using monoclonal antibody as an inhibitor of immune checkpoint,have effectively blocked the corresponding inhibitory signal which could promote CTL's activation.Based on this,this research focuses on applying 2PA-PDT based on ROS responsive micelles combined immune checkpoint therapy,to activate and enhance antitumor immune effect.Firstly,we designed the mPEG decorated dendritic polymer,which can co-encapsulate photosensitizer and two photo compound to enhance their water-solubility.Moreover,this nano drug delivery system is sensitive to ROS which can release the drug after accumulating in tumor tissues;secondly,the drug delivery system carries novel strong2PA absorption compound,which can be excited by NIR laser and indirectly excite the photosensitizer through FRET and extend the penetration depth of PDT;finally,checkpoint inhibitor was followed up to activate the inhibited CTL,enhancing the“distant effect”which was aimed for inhibiting tumor growth and metastasis,and reducing the damage to the body during therapeutic process.In this subject,we firstly synthesized ROS sensitive dendritic polymer with mPEG decorated?pHPMA-CA-mPEG?,and characterized the polymer by SEC.Then pHPMA-CA-mPEG was used in the preparation of PPa/Tb co-loaded micelles?CLM?,and optimized for drug loading efficiency,encapsulation efficiency and FRET efficiency,determined CLM's size,morphology,stability,CMC,2PA section,in vitro release profile and singlet oxygen yield.The in vitro cell line study was carried on mouse 4T1 breast cancer cells,the dark cytotoxicity and phototoxicity were evaluated by CCK-8 test,cellular uptake efficiency and intracellular ROS productivity were further investigated.Flow cytometry and immunofluorescence were used to test the expression of CRT on cell's surface after2PA-PDT.The in vivo study was carried on mouse 4T1 breast cancer model,the targetability of CLM was evaluated by in vivo animal imaging system,and the anti-cancer efficacy was evaluated according to TGI and metastatic nodules in lungs,side effects were studied through mice body weights and organic observation.The results showed that the ROS-responsive polymer can co-encapsulate the two-photo compound Tb and photosensitizer PPa,and formed Tb/PPa co-loaded micelle?CLM?.CLM was exhibited unniform spheres with an average size of around 110 nm.The encapsulation efficiency of Tb and PPa were both above 75%after optimization,and these micelles can maintain stable under a refrigeration condition in one week;the 2PA section of CLM was about 300?37?/GM at 808 nm,and got good singlet oxygen yield under irradiation,these micelles exhibited specific ROS-responsive ability and can release the cargoes under 808nm laser.The 4T1 cells can uptake CLM quickly and it was proven no obvious dark toxicity under a concentration of 20?M CLM,cell viability of 4T1 cells decreased to 25%after 3min irradiation;the IF and CFM results showed that the expression of CRT was enhanced along with the laser's strength.The accumulation of CLM in mice orthotopic breast cancer model reached maximum at 24 h after injection.The results of anti-cancer efficacy study showed that the combined therapy of targeted 2PA-PDT and immune therapy can inhibit the orthotopic tumor growth and lung metastasis obviously,and the TGI achieved approximately 66%,the body weights and anatomy analyses of therapeutic mice exhibited no obvious abnormality.In conclusion,the ROS-responsive nanoscale drug delivery system can realize targeted2PA-PDT as well as activating immune reaction in the tumor site.The anti-cancer efficacy can be further enhanced after combing immune checkpoint therapy,and this combined therapy is expected to provide new ideas for targeting treatment of metastatic breast cancer.