The Anti-Tumor Evaluation Of Vascular Normalization Based Photodynamic-Immunotherapy Combined Therapeutic Nanoplatform

Photodynamic therapy(PDT),as a promising therapeutic modality,has the advantages of less invasiveness and less systemic toxicity.It kills tumor cells in two ways.On the one hand,photosensitizers(PSs)-based PDT directly kill tumor cells by generating cytotoxic reactive oxygen species(ROS).On the other hand,PDT can cause immunogenic cell death(ICD),thereby activating immune responses for immunotherapy.Therefore,PDT itself can be called photodynamic immunotherapy.However,due to the intricate tumor microenvironment(TME),its therapeutic effect is severely hampered.Firstly,oxygen is essential for PDT to produce ROS,but hypoxic TME greatly weakens the efficiency of ROS production during PDT.In order to obtain sufficient nutrients to meet the needs of rapid growth and proliferation,tumor cells will continuously secrete excessive vascular endothelial growth factor(VEGF),and result in an inefficient blood supply and aggravating hypoxia TME.Therefore,abnormal vasculature is the primary obstacle to PDT.In addition,the transport and infiltration of immune effector cells into tumor sites in the immune cycle is an indispensable step,in which the functioning vasculature plays a vital role.However,the dysfunctional vasculature prevents immune cells entering into tumors.Even the immune cells that have reached the tumor site still need to overcome other immunosuppressive mechanisms in TME,such as indoleamine-2,3-dioxygenase(IDO).Thus,immunosuppressive TME caused by abnormal tumor vasculature and excessive IDO is an important obstacle to immunotherapy.Based on the above,hypoxia and immunosuppressive TME caused by overexpression of VEGF and IDO severely limit the effect of PSs-based photodynamic immunotherapy.Therefore,it is imperative to build PSs-based nanoplatforms to solve the above obstacles and realize effective photodynamic immunotherapy for anti-tumor therapy.Until now,there have been few reports on therapeutic modality that can simultaneously enhance PDT and immunotherapy to achieve effective photodynamic immunotherapy.Herein,we rationally designed a PSs nanoplatform,which can simultaneously enhance photodynamic immunotherapy by improving hypoxia and immunosuppressive TME.This nanoplatform(named as CAM NPs)was constructed through self-assembly of chlorin e6(Ce6,PSs),axitinib(AXT,tyrosine kinase inhibitor)and 1-methyl-D-tryptophan(1MT,IDO inhibitor)with the assistance of human serum albumin(HSA),which could effectively inhibit tumor growth and metastasis by enhancing photodynamic immunotherapy.In our nanoplatform,hydrophobic pockets of biocompatible HSA facilitated the binding of hydrophobic molecules for effective delivery.The CAM NPs were constructed by the self-assembly of Ce6,AXT and 1MT with the help of HSA.The results of FT-IR spectroscopy and UV-vis spectroscopy showd that the successful preparation of CAM NPs.TEM and DLS results demonstrated the spherical CAM NPs with size of 170 nm and with the zeta potential of～10.5 mV.The results of singlet oxygen production in vitro showed the CAM NPs could stably generate ROS for PDT.Besides,the hemolysis ratio of CAM NPs was extremely low,indicating that CAM NPs was biocompatible.Melanoma cells(B16F10)were selected to evaluate the anti-tumor effects and immune effects of CAM NPs in vitro.The significant higher fluorescence intensity was observed in CAM NPs groups and the highest internalization efficiency was found after 6 h of incubation,which was select as the optimal laser irradiation time for the in vitro laser-based studies.The cytotoxicity experiments results showed that the CAM NPs had higher cytotoxicity than that of free drugs.IDO assay revealed the CAM NPs was an efficient platform to inhibit the IDO pathway.Eversion of calreticulin(CRT)to the cell membrane,the secretion of adenosine triphosphate(ATP)and the release of high mobility group box 1(HMGB1)from the nucleus had proved the CAM NPs with laser irradiation could result in excellent ICD for immunotherapy.Apoptosis experiments illustrated that the CAM NPs could cause stronger cell apoptosis by activating the anti-tumor immune response.C57BL/6 female mice bearing melanoma(B16F10 cells)were selected to study the anti-tumor effects and mechanisms of CAM NPs in vivo.The in vivo biodistribution experiment showed CAM NPs could accumulate preferentially in the tumor site and the strongest fluorescent signals was found after 6 h of incubation which was select as the optimal laser irradiation time.In bilateral tumor models,CAM NPs could not only significantly inhibit primary tumor growth,but also inhibit the growth of the abscopal tumor.Furthermore,the CAM NPs could improve the hypoxic TME through the normalization of blood vessels.The increasion of effector T cells,the decreasion of Tregs and the up-regulation of various immune-promoting cytokines were found in post-treatment mice,indicated that CAM NPs could induce an effective anti-tumor immune response.Besides,lung metastasis experimental was established to further explore the anti-metastatic effects of CAM NPs.The result showed that CAM NPs could effectively suppress the metastasis tumor.In summary,this topic systematically studied the application of PSs-based nanoplatform for PDT combined with immunotherapy.The prepared CAM NPs could simultaneously improve hypoxia and immunosuppressive TME via combined PDT with vascular normalization and immune regulation.The CAM NPs thus demonstrated an amplified photodynamic immunotherapy,exhibiting excellent therapeutic efficacy against tumor growth and metastasis,thereby providing a powerful therapeutic nanoplatform.