Study On Exocytosis Mechanism Of Superparamagnetic Fe₃O₄ Nanoparticles With Different Sizes

In recent years,superparamagnetic iron oxide nanoparticles(SPIONs)have developed rapidly in the field of biomedicine,such as cell tracing,magnetic resonance imaging,drug carrier and gene therapy.The effective internalization of nanoparticles is the key to the realization of the effect of cancer diagnosis and treatment.Therefore,it is urgent to study the interaction mechanism between nanoparticles and cells in order to realize the effective internalization of nanoparticles.The endocytosis and exocytosis of nanoparticles affect the effective internalization,subcellular distribution,aggregation,metabolic pathways and metabolites in the nanoparticle,and then determine the efficacy and biosafety of nanoparticles.The study of the mechanism of endocytosis and exocytosis of functionalized nanoparticles has become a hot spot in the study of nanoscale.Therefore,we choose superparamagnetic iron oxide nanoparticles(SPIONs)as model nanomaterials and macrophages as model cells,the exocytosis pathway and metabolic regulation mechanism of different sizes of Fe3O4 nanoparticles were investigated through the q PCR detection of cellular transmission electron microscopy,exocytosis pathway and specific interference experiments of the associated proteins regulated by the exocytosis pathway.Three different sizes of oil dispersible Fe3O4 nanoparticles were prepared by the organic phase high temperature thermal decomposition method(corresponding to the average core size of 2nm,7N m and 14 nm).Fe3O4 nanoparticles with different sizes of polyethylene glycol(PEG)were obtained by the ligand exchange method.The corresponding average hydration diameters were 7.8nm,15.3 nm and 30.7 nm,respectively.Macrophage(RAW264.7)was used as a model cell to analyze the process of exocytosis by plasma inductively coupled emission spectrometry(ICP-AES).Transmission electron microscopy(TEM),real-time fluorescence quantitative detection and protein disturbance analysis were used to investigate the exocytosis pathway and the regulation mechanism of exocytosis.The results of the study show that:(1)Fe3O4 nanoparticles intracellular endocytosis and exocytosis behavior cultured in the presence and size of the nanoparticles time-dependent.The exocytosis of SPION1,SPION2 and SPION3 increased with the prolongation of the incubation time,and the rate of increasing increased from fast to slow.The relationship between the amount of exocytosis and time conforms to the single exponential growth equation.(2)By studying the transmission electron microscope results at different time points during the exocytosis process of Fe3O4 nanoparticles,we can know that : After endocytosis,SPIONs is located in the organelles such as Golgi body,mitochondria and lysosome.The nanoparticles will also cause autophagy by the cells to form autophagic bodies containing Fe3O4 nanoparticles.(3)Q uantitative analysis of Rab protein showed that SPIONs' s exocytosis pathway mainly includes: recycling endosomes pathway,Golgi pathway,lysosomal pathway,melanosome pathway and autophagosome pathway.(4)The results of protein interference analysis further verify the correctness of the pathway of the Golgi pathway and the recirculating internal body,and calculate the transport and circulation rate of the nanoparticles carried by the branching pathway through the inhibition of the perce ntage.