Construction Of PD-L1 Blocking Peptide-based Tumor Microenvironment Responsive Nanoplatform And Its Antitumor Research

In recent years,immune checkpoint blocking therapy(ICB)has attracted extensive attention in cancer treatment,especially in the PD-1/PD-L1 pathway.In PD-1/PD-L1 pathway immunoblocking therapy,PD-L1 is mainly exposed to the surface of tumor cells,such as melanoma cells,non-small cell lung cancer cells,lung cancer and colorectal cancer cells.PD-L1 can bind to the receptor PD-1 on the surface of T lymphocytes,which makes T cells lose their function and cannot kill tumor cells,thus making tumor cells produce immune escape and promoting tumor development.Studies have shown that PD-1/PD-L1 inhibitors can effectively promote the proliferation of helper T cells and cytotoxic T lymphocytes,and enhance the function of T cells and enhance immune response.Although PD-1/PD-L1 checkpoint inhibitors have unique pharmacological effects in the treatment process,their ICB response rate in many tumors is low due to immune resistance,which has some limitations.Photodynamic therapy(PDT)kills tumor cells by transforming oxygen into reactive oxygen species(ROS),such as singlet oxygen species(O2),hydrogen peroxide(H2O2)and hydroxyl radical(OH),by irradiating photosensitizer(PSs)with laser.Studies have found that reactive oxygen species produced by PDT can cause the immunogenic cell death(ICD)of tumor cells,thus activating the organism’s anti-tumor immune response.However,free photosensitizers have the problems of poor water solubility,low targeting,insufficient accumulation in tumor tissue during PDT,and poor therapeutic effect Therefore,although photodynamic can induce a certain degree of anti-tumor immune response,it is usually not enough to inhibit the growth and metastasis of residual tumor cells after photodynamic alone.In view of the problems and advantages of the above two therapies,the combined application of photodynamic therapy and immune checkpoint blocking therapy,using tumor antigens generated by photodynamic therapy to induce the host to develop an anti-tumor immune response,which is expected to improve the treatment efficiency of immune checkpoint blocking therapy and inhibit tumor metastasis.In the current study of combination therapy,intratumoral injection of immune checkpoint blocking antibody is usually used.The cost of antibody drugs is high,and immune related adverse events(irAEs)put forward strict requirements for the accurate loading of anti-PD-L1 antibody in nanopreparations.However,it is a difficult task to accurately control the loading content of PD-L1 antibody,because physical encapsulation will inevitably lead to batch differences,while chemical modification is limited by the low stability of the antibody.In order to solve the above problems,we proposed a molecular engineering strategy,using anti-PD-L1 peptide(APP)instead of anti-PD-L1 antibody to develop an efficient,low toxic and economic nano platform that can accurately control the content of PD-L1 inhibitor.Due to the overexpression of matrix metalloproteinase-2(MMP-2)in tumor microenvironment,the PD-L1 inhibitory peptide and photosensitizer are linked by the MMP-2 responsive peptide sequence as the linker to form an amphiphilic molecule(IR780-M-APP).APP and photosensitizer IR780 were linked by MMP-2 responsive peptide fragment(PLGLAG)to obtain IR780-M-APP,which is amphiphilic and can self assemble into nanoparticles(NPs).From the molecular structure,it has a precise APP loading(48.4 wt%).Specifically,part of IR780 in IR780-M-APP NPs endows the nanoparticles with photodynamic therapeutic effect and the ability to trigger ICD,while part of app can block PD-1/PD-L1 pathway to promote immune response.The IR780-M-APP can spontaneously assemble into nanoparticles in solution,which can release app precisely under the action of high concentration of MMP-2 in tumor tissue.APP binds with PD-L1 on the cell membrane of tumor,blocking PD-1/PD-L1 pathway;the remaining fragments become smaller nanoparticles,enhancing the penetration of photosensitizer in tumor tissue and uptake of tumor cells with laser irradiation can induce ICD,stimulate T cell activation and proliferation,and enhance the immunotherapeutic effect of PD-L1 immunosuppressive peptide.Therefore,the photodynamic immunotherapy nano platform constructed,not only directly kills primary tumors,but also effectively eradicates metastatic and invasive tumors.Current molecular engineering strategies may provide more opportunities for the design of advanced nano platforms for cancer treatment.The main research contents are as follows:1.Construction and characterization of IR780-M-APP NPsWe covalently linked the photosensitizer IR780 with the anti-PD-L1 peptide(APP)through the MMP-2 responsive peptide sequence to synthesize the MMP-2 responsive amphiphilic molecule,namely IR780-M-APP.IR780-M-APP NPs were successfully prepared by nanoparticles precipitation method.The physicochemical properties,in vitro optical activity,MMP-2 responsiveness and hemolysis rate of IR780-M-APP NPs were studied.The results showed that the nanoparticle preparation was a green solution.The synthesis of IR780-M-APP was confirmed by 1H-NMR,MALDI-TOF-MS and UV-Vis.The results of TEM showed that IR780-M-APP was successfully synthesized Dynamic light scattering(DLS)showed that the size of the nanoparticles was about 151.1 nm.In vitro reactive oxygen species experiments showed that IR780-M-APP NPs had good photodynamic activity in vitro.MMP-2 pretreatment experiment showed that IR780-M-APP NPs had good MMP-2 response,MALDI-TOF-MS experiment confirmed that the molecular weight of the two fragments after the response was in line with the theoretical value;TEM results showed that the segment containing photosensitizer after the response would become smaller nanoparticles(34.12 nm).2.In vitro cellular anti-tumor evaluation of IR780-M-APP NPsWe used B16F10 cells to establish in vitro anti-tumor cell model to evaluate the anti-tumor effect of IR780-M-APP NPs combined with photodynamic immunotherapy at the cellular level in vitro,and conducted a preliminary study on its immune effect:in vitro cell experiment,under the condition of MMP-2 pretreatment,the cellular uptake of IR780-M-APP NPs showed that IR780-M-APP NPs could inhibit the growth of tumor cells.The results showed th at the uptake effect of NPs was better than that of free IR780;the cytotoxicity test showed that IR780-M-APP NPs had a strong killing effect on cells under laser irradiation;the intracellular ROS level test and apoptosis test showed that IR780-M-APP NPs had a strong photodynamic effect and induced more efficient apoptosis under laser irradiation;the eversion test of CRT showed that IR780-M-APP NPs had a strong killing effect on cells under laser irradiation.The binding ability of PD-L1 on B16F10 cells showed that M-APP(MMP-2 sensitive bond linked anti-PD-L1 peptide)pretreated with matrix metalloproteinase-2 and IR780-M-APPNPs blocked the binding of PD-1/PD-L1 to PD-L1,indicating that the chemical modification of IR780 had little effect on the anti-PD-L1 peptide3.In vivo photodynamic and immunotherapeutic anti-tumor evaluation of IR780-M-APPNPsThe B16F10 melanoma bilateral model and lung metastasis model were established in female C57BL/6 mice.The antitumor effect and immune effect of IR780-M-APP NPs in vivo were studied by in vivo drug distribution experiment,bilateral tumor inhibition test and anti-lung metastasis test.In vivo drug distribution experiment verified that the accumulation ability of nanoparticles in tumor site was higher than free IR780;bilateral tumor inhibition experiment results showed that IR780-M-APP NPs+Laser can effectively inhibit the growth of tumor in situ,and has a good inhibitory effect on distant tumor through immune effect.IR780-M-APP NPs+Laser can promote the infiltration of lymphocytes in tumor sites and can effectively reduce regulatory T cells(Tregs).At the same time,we detected the content of helper T cells(Ths),cytotoxic T lymphocytes(CTLs),Tregs and serum cytokines in the spleen of mice after treatment,which proved that the nanoparticles can effectively enhance the anti-tumor immunity in mice IR780-M-APP NPs can effectively inhibit the lung metastasis of tumor cells.In summary,we have constructed IR780-M-APP amphiphilic molecular assembly with tumor microenvironment response by molecular engineering strategy,and realized photosensitizer and PD-L1 The simultaneous delivery of inhibitory peptides and the responsive release of tumor microenvironment can enhance the immunogenicity of the system,improve the immune response efficiency of PD-L1 inhibitory peptides,inhibit tumor recurrence and metastasis,and provide a new treatment idea for effectively solving tumor recurrence and metastasis.