Enzyme Sensitive Hydrogel For Timing Sequential Release Of MiRNAs And The Regulation Of Macrophage Phenotype By MiR-155

The early inflammatory stage of bone repair is the key to the whole bone repair.Macrophages sequential polarization to pro-inflammatory M1 and anti-inflammatory M2 phenotype had been proved to be the core physiological events involved in the inflammatory phase of bone repair.Sequential regulation of M1 and M2 phenotypes of macrophages in inflammatory stage by miRNAs is a new strategy and research hotspot for promoting bone repair.Therefore,it is necessary to design a delivery system that can follow the natural bone repair process to sequentially release miRNAs.In this paper,bovine serum albumin(BSA)was used as a model.The timing sequential release system loaded with two kinds of BSA nanoparticles was designed.First,hydrogels were synthesized by Michael addition reaction between 8 arm maleimides modified polyethylene glycol(8-arm-PEG-Mal)and matrix metalloproteinase-7(MMP-7)sensitive peptides.MMP-7 is a natural physiological signal that simulates the transition from M1 phenotype to M2 phenotype of macrophages in fracture microenvironment.Second,two kinds of BSA nanoparticles were added into hydrogel by physical embedding and chemical bonding,respectively.Finally,the physically embedded nanoparticles were released in large quantities by physical diffusion in the early stage.With the occurrence of MMP-7,the peptides in the hydrogel system were cleaved by MMP-7,then chemically linked nanoparticles were rapidly released due to the degradation of hydrogel.The characterization and timing release ability of hydrogels were studied by Fourier transform infrared spectrometry,scanning electron microscopy and in vitro release experiments.Based on this,we further replaced BSA with miRNA-155 to verify the timing sequential release capability of the hydrogel system loaded with miRNAs nanoparticles.At the same time,flow assistant cytometry sorting technique,laser confocal fluorescence microscopy and enzyme linked immunosorbent assay(ELISA)were used to investigate the effect of miRNA-155 nanoparticles on M1 phenotype polarization of macrophages before and after the release of hydrogel system.The results showed that the prepared hydrogels were characterized as a porous network with an average pore size of about 60 μm.The energy storage moduli were between 1000-4800 Pa,and they had low cytotoxicity and good biocompatibility.More importantly,Pep-PEG-4% hydrogel system could achieve the sequential release of nanoparticles,with the release of about 80% of the physically loaded nanoparticles in the first three days.When MMP-7 appeared 3 days later,chemically linked nanoparticles were rapidly released with degradation of hydrogel,and the cumulative release amount was up to 100%.The prepared miRNA-155 nanoparticles had uniform particle size,low cytotoxicity and weak electropositive properties.The uptake rate of miRNA-155 nanoparticles remained about 80% before and after released.It was proved that miRNA-155 nanoparticles could upregulate the expression level of proinflammatory cytokines,such as i NOS,TNF-α and IL-6,and promote M1 polarization of macrophages.This sequential release hydrogel system,which can follow the natural bone repair process,provides a novel material design strategy for promoting bone repair by sequential release of miRNA-155 and the other miRNA that regulates macrophage M2 phenotype in the future.