Targeted Delivery Of MiR-148b By Reduction Sensitive Nano-carrier To Inhibit Metastasis And Invasion Of HuH-7 Cells

Hepatocellular carcinoma(HCC)is a type of primary liver cancer with high mortality,which has the characteristic of intrahepatic metastasis.Hepatocellular carcinoma can invade into the portal vein and other parts of the liver,leading to postoperative recurrence,making it difficult to completely cure hepatocellular carcinoma.RNA interference is a good method to solve the problem caused by metastasis and invasion of hepatocellular carcinoma.Among them,miR-148 b is a related gene of hepatocellular carcinoma metastasis and invasion,which is low expression in metastatic liver cancer cells and can regulate the metastasis and invasion of hepatocellular carcinoma.However,naked miR-148 b can be easily degraded by serum nuclease in the blood after intravenous injection,and miR-148 b has poor targeting ability that can not be effectively accumulated in the tumor site.In order to solve the above problems,this paper takes the gene nanocarrier as a breakthrough to construct a reduction sensitive gene nano-system for targeted delivery of miR-148 b from the perspective of active targeting and efficient delivery,the focus is to investige the reduction responsiveness,tumor targeting,biocompatibility,and the inhibition of migration and invasiveness of nanosystems.The aim is to provide a highly efficient and low-toxic delivery system for miR-148 b that can actively target tumors,and solve the problem of hepatocellular carcinoma metastasis and invasion.In this paper,a low molecular weight branched polyethyleneimine(BPEI)with low toxic was used as a raw material to prepare a cationic carrier SSBPEI with high molecular weight and high transfection rate by introducing a disulfide bond(-SS-).In order to improve the biocompatibility and tumor targeting of SSBPEI,polyethylene glycol(PEG)and CC9 peptide were introduced to prepare CC9-PEG-SSBPEI nanocarriers.The CC9-PEG-SSBPEI can effectively carry miR-148 b through charge interaction to form CC9-PEG-SSBPEI/miR-148 b nanoparticles.Both zeta potential analysis and gel retardation assay confirmed that CC9-PEG-SSBPEI can stably bind to miR-148 b and protect miR-148 b from being degraded by nuclease.The DLS method tested the particle size of the nanoparticles and found that the N/P ratio had a great influence on the particle size and dispersion of the nanoparticles.When N/P=10:1,the particle size was about 150 nm,and the nanoparticles reached the optimum state.The AFM and TEM images showed that the nanoparticles were uniformly dispersed in a spherical shape.In addition,CC9-PEG-SSBPEI/miR-148 b nanoparticles had reduction responsiveness,which can be degraded and released miR-148 b in GSH/DTT environment.FACS analysis and confocal microscopy showed that CC9-PEG-SSBPEI could promote the uptake of miR-148 b by Hu H-7cells,and confirmed that the nanosystem was targeted to Hu H-7 cells.It was found that CC9-PEG-SSBPEI can increase the expression of miR-148 b in Hu H-7 cells and effectively inhibit the expression of NRP-1 gene in Hu H-7 cells by q RT-PCR analysis.Wound healing assay and transwell migration assay showed that CC9-PEG-SSBPEI/miR-148 b can effectively inhibit the metastasis,invasion and growth of Hu H-7 cells.More importantly,CC9-PEG-SSBPEI/miR-148 b has a lower hemolysis rate(<5 %),and MTS method found that CC9-PEG-SSBPEI has good biocompatibility.