Oncolytic Virion-like Stapled Peptide Nanoprodrug For Enhanced Immunometabolic Cancer Therapy

Compared with traditional chemotherapeutics,anti-tumor peptides have shown high affinity,great biocompatibility,modular design and synthetic convenience,and have unique advantages in the treatment of cancer.In addition,peptides have been reported to trigger immunogenic cell death after lysis of tumor cells,indicating their potential for cancer immunotherapy.However,the therapeutic effect of the conventional linear peptides has been hindered by their poor stability,short half-life,and weak cell permeability.Stapled peptides with aromatic cross-links provide improved stability and potent cell permeability.Although traditional treatments such as chemotherapy can induce immunogenic cell death,the existence of multiple immunosuppression in tumor region leads to the insufficient efficacy of immune responces.In particular,the IDO enzyme-mediated immune metabolism pathway can significantly affect the activity of T cells,thereby affecting the activation of the immune system.We herein report a virion-like nanoprodrug sKLA-NLG NPs which was engineered by conjugation of the stapled KLA peptide with an IDO inhibitor NLG919.The main contents and conclusions of this research are as follows:1.Synthesis of the stapled peptide-drug conjugates sKLA-NLG:The KLA peptide（H-KLCKLAKKLCKLAK-NH₂）was synthesized based on solid-phase peptide synthesis（SPSS）.The NLG-SA was conjugated with the peptide through amid condensation.The successful synthesis of the conjugates were analized and confirmed by HPLC and LC-MS.Theα-helical conformation of the stapled peptides was proved by circular dichroism spectroscopy.The diameter and zeta potential of sKLA-NLG NPs were 145.4±1.91 nm and+42.2±0.17 m V,the morphology of nanoparticles were observed by transmission electron microscopy.The release rate of NLG919 from nanoprodrug can reach 85.2%within 48 hours.2.Evaluation of the in vitro therapeutic properties of the nanoprodrugs:The cellular uptake efficiency of sKLA-NLG NPs was evaluated by fluorescence imaging and flow cytometry.It was shown that the cellular uptake efficiency of stapled nanoprodrug was 2.5times higher than that of the KLA-NLG NPs.The mitochondria-induced apoptosis was evaluated by JC-1,TEM and Weatern blot.The cytotoxicity was evaluated by Alarm blue assay.The immunogenic cell death induction were evaluated by detecting the expression of CRT,HMGB1 and ATP.The inhibition effect on IDO enzyme was evaluated by detecting the metabolite kynurenine after co-incubation.3.Evaluation of the in vivo immunotherapeutic effect of nanoprodrugs:In vivo prolonged biodistribution of sKLA-NLG NPs was evaluated by fluorescence imaging.Bilateral 4T1 tumor model on Balb/c mice were established,and the immunotherapy efficiency was evaluated by tracking the tumor growth curves.The TGI of primary and distant tumors in the combined treatment group reahed 88.0%and 97.3%,respectively.The activation of the immune response was evaluated by flow cytometry,immunohistochemistry,cytokine detection,etc.The great biosafety of the treatments was evaluated by routine blood tests,etc.