Mechanism Of MIM Protein Regulating Macrophages Endocytosis Of Iron Oxide Nanomaterials

Iron oxide nanoparticles?IONPs?are widely used in the biomedical applications based on their unique physical,chemical,thermal and mechanical properties and good biocompatibility.IONPs have a series of applications ranging from MRI contrast enhancement,heating mediators for hyperthermia,cell labelling and tracking,cell sensing,drug/gene delivery carriers and theragnostics.When the IONPs applicated in vivo,can be recognized and cleared rapidly by macrophages via opsonization,and gradually enriched in the mononuclear phagocytic system?MPS?organs such as liver and spleen.Therefore,monocytes and macrophages are a major challenge for nanomaterial applications in vivo.This is why the process of nanoparticle endocytosis by macrophages is an important focus area for researchers.Missing-in-metastasis?MIM?is also known as metastasis suppressor 1?MTSS1?,a scaffolding protein interacting with multiple partners to regulate both actin dynamics and membrane dynamics,which is highly expressed in macrophages and regulates receptor-mediated endocytosis.This is important for IONP applications in vivo,i.e.,the long circulating time or retention time,accumulation in target site,etc.The studies of this dissertation are as following:?1?The biological effects of iron oxide nanoparticles on monocytes and macrophages were systematically investigated.Biosafety evaluation of IONPs was performed including MTT,Annexin V-FITC/PI apoptosis,ROS,etc.The effects of IONPs on macrophage adhesion and cell morphology were observed microscopically.The effect of IONPs on the differentiation of monocytes into macrophages was evaluated by CD11b.The effect of IONPs on iron metabolism was detected by fluorescence quantitative PCR,evaluated the changes in mRNA levels of transferrin,transferrin receptor 1,ferroportin 1,divalent metal ion transporter?DMT1?,and ferritin.?2?MIM protein participates in the regulation of cell membrane function through the reaction with PI?4,5?P₂,such as depolarization of cell membrane.The regulation of cell membrane shape via MIM is beneficial to the hydrolysis of PIP2 by PLC to generate IP₃.Increased IP₃ awakens IP₃R to mediate Ca²⁺releasing from ER calcium store.The combination of MIM and PIP2 was increased after treated with IONPs,which activated the hydrolysis of PIP2 by PLC,regulate IP₃/Ca²⁺signal,and participate in endocytosis of macrophages.?3?We investigated the effect of MIM expression?downregulation and overexpression?on IONPs endocytosis,and found that MIM can promoted the endocytosis.MIM was evidently co-localized with CLC and CHC after 4 h treated with IONPs,and involved in clathrin-mediated endocytosis.Co-immunoprecipitation and computational results suggested that MIM bind CLC via its PRD domain.?4?By analyzing the influence of nanomaterials such as IONPs on the intracellular distribution of MIM,it was found that MIM protein can promote the intracellular transport of nanoparticles from endocytosis to lysosomes.Fluorescence co-localization and co-immunoprecipitation experiments confirmed that MIM can bind to Rab5 and Rab7 in sequence,promote the transport of IONPs from early endosomes to late endosomes,and finally locate in lysosomes.