Integrin α6-targeting Self-assembled Nanopeptide In Diagnosis And Treatment Of Central Nervous System Leukemia

Central nervous system leukemia(CNS-L)refers to the disease state in which leukemia cells infiltrate into the meninges or brain parenchyma.Most CNS-L is developed from acute lymphoblastic leukemia(ALL).About 3-5%of newly diagnosed leukemia patients and about 30-40%of relapsed patients have central nervous system leukemia;In the absence of central nervous system-oriented preventive treatment,5%of patients will develop into central nervous system leukemia in the course of disease progression.At present,it is recommended to use methotrexate or cytarabine high-dose systemic chemotherapy combined with intrathecal injection.However,systemic high-dose chemotherapy is highly toxic to organs and easy to cause adverse reactions.New therapies such as antibody immunotherapy and CAR-T cell therapy are easy to cause adverse reactions such as cytokine release syndrome.Therefore,exploring and optimizing the treatment of central nervous system leukemia has become an urgent clinical problem.Integrin a6 is highly expressed in many kinds of tumors and participates in many processes such as tumor genesis,development,distant metastasis and invasion.Integrin is highly expressed in acute lymphoblastic leukemia(ALL)cells.It interacts with laminin to promote the migration of ALL cells to cerebrospinal fluid and induce central nervous system leukemia.Therefore,integrin a6 is an ideal target for the diagnosis and targeted treatment of central nervous system leukemia.In the previous study,we screened and obtained a6 targeting peptide RWY through phage display technology and second-generation sequencing technology.Using molecular imaging technology,the tumor targeting effect of RWY was verified in liver cancer,breast cancer and central nervous system leukemia.On the basis of the previous research,this study designed a series of targeted peptide mutants using systematic peptide optimization strategy.The targeted peptide RD with stronger affinity had screened by microscale thermophoresis.Targeted site of RD and integrin α6 were found by molecular docking.The tumor targeting effect of RD on central nervous system leukemia was verified at the cellular and mouse levels by flow cytometry binding assay and molecular imaging experiments.Based on the targeted peptide RD,anti-tumor peptide KLA and self-assembled peptide Gffy,self-assembled nanopeptide RD-AP-Gffy was designed and synthesized.The minimum polymerization concentration,particle size and Zata potential were measured.The results showed that RD-AP-Gffy was consistent with the basic characteristics of nanoparticles.RD-AP-Gffy had strong affinity to integrin α6.In many tumor cells and leukemia cells with high expression of integrin α6,we used CCK8 activity detection and SYTOX dead cell nucleic acid staining to compare the cytotoxicity of RD-AP-Gffy and a series of control peptides.The result proved that RD-AP-Gffy had a powerful killing effect on tumor cells.The proapoptotic effect of RD-AP-Gffy on leukemia cells was verified by flow cytometry,WB detection and transcriptome sequencing.Using molecular imaging technology,the tumor targeting of RD-AP-Gffy was verified in the subcutaneous tumor and meningeal metastasis model mice of leukemia.The targeted anti-tumor effect of nanopeptide RD-AP-Gffy was verified in the meningeal metastatic tumor model mice of leukemia.In the combined drug test,the therapeutic effect of the combination of nanopeptide RD-AP-Gffy and methotrexate on central nervous system leukemia was verified.This study provides a new method for improving the treatment of central nervous system leukemia,and has important clinical transformation application value.