Construction Of An Enzymatic Peptide Self-assembly System And Its Application For Cancer Selective Therapy

The abnormal metabolism of tumor cells creates a specific tumor microenvironment,typically characterized by acidic p H,high glutathione（GSH）concentration,overexpression of certain enzymes and lack of oxygen.These biological features provide directions for the selective treatment of cancer.In recent years,supramolecular nanodrugs formed by in situ enzymatic self-assembly（EISA）have been widely used in tumor therapy due to their advantages of controlled quality,deep penetration,long-term retention and reduced toxicity.Lactate,a marker of tumor metabolic reprogramming,relies on the transport output of monocarboxylate transporter protein 4（MCT 4）and is involved in multiple signaling pathways in tumor tissues,while maintaining the acidic microenvironment of tumors,thus contributing to tumor progression.Therefore,it is a new anti-tumor strategy to induce cellular acidosis by blocking the normal lactate transport output of tumor cells and allowing intracellular lactate accumulation.Based on this,we rationally designed an in situ EISA system for selective cancer therapy by inducing tumor acidification and apoptosis.First,the peptide derivative target molecule（LND-G^DF^DF_P^DY）as well as the control molecules（LND-GGG_P^DY and Nap-G^DF^DF_P^DY）were synthesized by standard Fmoc solid-phase synthesis method,and the successful synthesis was verified by NMR hydrogen spectroscopy and high-resolution mass spectrometry.The results of in vitro self-assembly experiments showed that LND-G^DF^DF_P^DY was able to form hydrogels by alkaline phosphatase（ALP）-catalyzed self-assembly.Transmission electron microscopy revealed the microstructure of the hydrogel as intertwined nanofibers.The results of circular dichroism（CD）experiments also verified the enzymatic self-assembly property of the target molecule,which showed that the secondary structure of LND-G^DF^DF_P^DY changed from an irregular structure to aβ-sheet structure after the addition of ALP to the solution.The dynamic light scattering（DLS）assay tested the critical in vitro assembly concentration of the peptide derivative at 136μM,indicating its good in vitro EISA ability.Next,the in situ EISA properties of the peptide derivatives were verified by cell surface self-assembly experiments,and the sequence of action of the compounds with sequential dual effects was verified using the fluorescent molecule NBD-G^DF^DF_P^DY.The results of the subsequent CCK-8 assay showed that the constructed in situ EISA system had a selective killing effect on tumor cells,as demonstrated by the typical concentration-dependent toxic effect of LND-G^DF^DF_P^DY on both 4T1 and He La tumor cells with high ALP expression;while no significant cytotoxicity was observed for 3T3 normal cells with low ALP expression.The results of apoptosis and scratch assays indicated that LND-G^DF^DF_P^DY could exert significant pro-apoptotic and anti-migratory effects on 4T1 tumor cells.western blot and immunofluorescence assays showed that the in situ EISA system could enhance the interaction between LND and MCT 4 through assembly-induced retention effects,and effectively inhibit the expression of lactate transporter protein MCT4 JC-1 mitochondrial membrane potential probe and cellular oxygen consumption rate（OCR）assays demonstrated that the in situ EISA system caused damage to mitochondrial function after internalization into tumor cells,thus blocking the tricarboxylic acid cycle and pyruvate consumption and disrupting the balance of pyruvate and lactate conversion.The fluorescence of intracellular lactate level and intracellular p H were further examined,and the results showed that the intracellular lactate level increased after in situ EISA system treatment,effectively inducing acidosis in tumor cells.Finally,the potential of the in situ EISA system as a sensitizer for radiotherapy was verified by clone formation assay,comet assay,γ-H₂AX immunofluorescence assay and cell cycle assay.The results demonstrated that it could significantly improve the sensitivity of 4T1 tumor cells toγ-rays and possess good radiotherapy sensitizing effect.Subsequently,in vivo radiotherapy synergistic anti-tumor experiments were conducted using 4T1 tumor-bearing mice,and the results showed that the in situ EISA system had outstanding synergistic anti-tumor effects with radiotherapy during treatment,and had significant inhibitory effects on tumor proliferation,and the tumor inhibition rate could reach about90%.Post-treatment analysis of tumor tissues using H&E staining,immunohistochemistry and immunofluorescence assays showed that the tumor tissues showed inhibition of MCT4 protein,damage to mitochondria,alleviation of the hypoxic environment and the appearance of extensive apoptosis.In addition,body weight trends were monitored during treatment,blood biochemistry and routine blood tests were performed after treatment,and pathological sections of the major organs were analyzed by H&E staining.The results of all tests and analyses showed no abnormalities,indicating that the in situ EISA system has good biocompatibility.