Study Of PD-L1 Tumor-Targeted Nanomedicine For Synergistic Therapy Of Anti-Tumor Immune Activation By Photodynamic Therapy

Cancer is one of the leading causes of death in the world.Due to population aging,it is increasing year by year in China.At present,there are more and more ways to treat cancer.However,traditional antineoplastic drugs have low drug utilization,poor biocompatibility,and large side effects,so antineoplastic therapy faces great challenges.The rise of nanomedicine has optimized the structure of anti-tumor drugs,improved the retention rate of drugs in tumors,and promoted anti-tumor therapy.CVRARTR,a PD-L1 tumor-targeting peptide,is a biocompatible nanocarrier that preferentially targets tumor cells with high levels of PD-L1 expression and competitively binds to PD-L1,such as colorectal cancer tumor cells CT26,blocking the binding of PD1 on the surface of T cells to PD-L1 on the membrane of tumor cells.It can restore T cell activity and enhance anti-tumor immunity to avoid tumor cell immune escape.Photodynamic Therapy(PDT)is a local tumor treatment method.After irradiation with specific wavelength infrared light,reactive oxygen species are generated,which cause endoplasmic reticulum stress and Immunogenic cell death(ICD)of tumor cells.It is one of the methods of tumor immunotherapy.Macitentan(MAC)is a specific drug for the treatment of pulmonary arterial hypertension.At the same time,it can improve the tumor immune microenvironment,activate the proliferation of T cells,down-regulate Tregs cells,and promote anti-tumor immunotherapy.PD-L1 tumor-targeting peptides can form new polypeptide chains by solid-phase synthesis bonded to photosensitizer protoporphyrin,which can self-assemble to form nano-micelles and encapsulate macittetan to form new nano-drug Fmoc-K(Pp IX)CVRARTR@Macitentan(PM).Nanodrug PM specifically targets the tumor site through EPR effect and competitively blocks the binding of PD1and PD-L1.After photodynamic therapy,PM induces the production of ICD,stimulates the proliferation of T cells,and inhibits the proliferation and metastasis of tumors.ObjectiveIn this paper,we aimed to construct PD-L1 tumor-targeted nanomedicine for Photodynamic Therapy(PDT)and photodynamic activated immune synergistic therapy,aiming at the low bioavailability,poor biological safety and high side effects of traditional anti-tumor drugs.Pd-L1-targeted photosensitizer bond encapsulated T cell agonist Macitentan(MAC)to form a novel nanomedicine FMOC-K(Pp IX)CVRARTR@Macitentan(PM).The tumor localization of nanodrugs was achieved by PD-L1-targeting peptides,blocking the specific binding of PD-L1 and PD-L,and exposing the tumor surface antigens.Combined with T cell activator Macitentan(MAC)to restore the activity of T cells and promote the proliferation of T cells.At the same time,Protoporphyrin IX(Protoporphyrin IX)-induced photodynamic therapy(PDT)can promote tumor cell Immunogenic death(ICD),and the combination of the two can further enhance anti-tumor immunotherapy.Therefore,the study of PD-L1 tumor-targeted nanomedicine through photodynamic activation of anti-tumor immune synergistic therapy needs to be further elucidated.MethodsIn order to solve the problems of low bioavailability of anti-tumor drugs,short tumor retention time,poor biological safety,and high side effects.We constructed a PD-L1-targeting peptide FMOC-K(Pp IX)CVRARTR based on amino acid peptide chain as bonding photosensitizer,and encapsulated T cell agonist Macitentan(MAC)to form FMOC-K(Pp IX)CVRart R@macitentan nanomedicine PM.The nanodrug was formed by self-assembly of a PD-L1-targeted peptide photosensitizer bond to form a nano-micelle encapsulated with the free T cell agonist drug macittetan.1.PD-L1 tumor-targeting peptide photosensitizer bond was synthesized by amino acid solid phase,and the correctness of peptide series was detected by mass spectrometer.2.Cell uptake assay was used to compare the targeting of PM in cell lines with different PD-L1 expression levels.3.Particle size,Zeta potential and morphology of PM were characterized by scanning electron microscopy and Malvern particle size analyzer.4.DCFH-DA and SOSG were used as indicators to evaluate the photodynamic reactive oxygen species(ROS)production effect of PM by CLSM.5.Immunofluorescence assay was used to detect the expression of ICD-related immune markers in the cell membrane and nucleus of CT26 cells induced by PM under CLSM.6.Western-blot was used to detect the effect of PM on the induction of ICD-related proteins CRT and HMGB-1 in CT26 cells.7.The anti-tumor effect of PM was studied by MTT assay,live/dead cell double staining and flow cytometry.8.CT26 mouse tumor model was established to evaluate the anti-tumor activity and biosafety of PM in vivo.A mouse model of pulmonary metastases was established to study the therapeutic effect of PM-induced immune response on distant metastases.9.To investigate the mechanism of PM treatment on CT26 orthotopic and metastatic tumors by intratumoral immune cell analysis.Results1.The results of ES detection by mass spectrometry showed that the molecular weight and the number of amino acids of the PD-L1 tumor-targeted too photosensitizer bond were correct,and the solid phase synthesis of PD-L1tumor-targeted too photosensitizer bond was successful.2.After the cell line with no PD-L1 expression level endocytosed PM under CLSM,the attachment of PM on the cell membrane showed that the PD-L1 high expression CT26 cell membrane attachment was obvious3.The PM was oval and uniformly distributed under the TEM with the same size.The diameter of PM was about 255nm and the zeta potential was about 22.8 m V under a Darwin particle size meter,which was stable in aqueous solution.4.Compared with the dark treatment group,SOSG produced the highest level of reactive oxygen species at the wavelength of 630nm.When DCFH-DA was used as ROS indicator,the level of ROS production in CT26 cells was significantly increased under the same infrared light irradiation,which was observed as fluorescent green under CLSM.5.The level of ICD marker protein calreticulin(CRT)was significantly increased and the level of high mobility group box 1(HMGB-1)was significantly decreased after endocytosis of nanomedicine PM by Western-Blot.6.After the nanodrug PM was endocytosed into CT26 cells,the CRT immunofluorescence on the membrane of CT26 cells was significantly increased under CLSM under the incubation of the corresponding CRT and HMGB-1immunofluorescence primary antibody and secondary antibody,indicating that the membrane transfer of calreticulin after photodynamic therapy.However,HMGB-1showed obvious immunofluorescence in the nucleus after photodynamic therapy,indicating that the nuclear transfer of high mobility group protein occurred.7.MTT assay showed that PM had phototoxicity and low dark toxicity on CT26 in a concentration-dependent manner.The results of live/dead cell double staining and apoptosis assay showed that the cell death of PM exposed to light for 3 minutes was significantly higher than that of the dark treatment group and the blank group.8.PM was highly accreted into CT26 tumors 8 hours after tail vein injection,and the tumor growth was significantly inhibited after IR irradiation.No obvious abnormalities were found in the HE stained sections of the heart,liver and kidney of the mice.In the lung metastasis model,there was no obvious metastasis in the lung of the PM group,and the ratio of red and white pulp in the spleen of the PM group was normal.9.The results of immunocyte analysis showed that the proportion of CD3CD8~+T cells in PM group was significantly higher than that in control group,and CD3~+CD4~+FOXP3~+T cells was significantly lower than that in control group.Conclusions1.The PD-L1 tumor-targeting peptide self-assembled to form a beam encapsulated T cell agonist MAC to form a stable nanomedicine PM,which effectively targeted colorectal cancer cells CT26.2.Nanomedicine PM significantly enhanced the intracellular ROS generation ability of photosensitizer.3.PM significantly inhibited tumor growth and metastasis by inducing the production of ICD and inhibiting the growth of Treg cells,while activating the proliferation of T cells.