Huc-MSC-derived Exosomes Modified With HSTP1 For Liver Fibrosis Therapy

Objective:Liver fibrosis is caused by chronic recurrent liver injury from different causes such as viral infection,metabolic disorders,cytotoxic drugs and immune attack,and has become a global health problem.Regardless of the underlying cause,the activation of hepatic stellate cells（HSCs）and the excessive deposition of extracellular matrix（ECM）secreted by activated HSCs（aHSCs）are key steps in the progression of liver fibrosis.Therefore,aHSCs become the target cells of liver fibrosis therapy.Mesenchymal stem cells（MSCs）transplantation has become a hot research topic in regenerative medicine in recent years due to its anti-inflammatory,anti-fibrotic,and antioxidative stress effects,which inhibit the activation and proliferation of HSCs.However,the clinical application of MSCs is limited by their differentiation into inappropriate cell types,potential tumorigenicity,and the effects of cell infusion on microvessels.The regulatory effect of stem cells on surrounding tissues and host cells is mainly attributed to paracrine,while the exosomes are the main substance of stem cells paracrine secretion.Exosomes possess similar functions to their parent cells and have a significant regulatory effect on aHSCs.Therefore,they are considered a safe and effective cell-free reagent.However,unmodified exosomes injected into the bloodstream,only a small amount would be ingested by aHSCs.This leads to off-target effects of exosomes,causing not only the waste of biological agents but also the inability of aHSCs to uptake to an effective therapeutic dose.In this study,we aim to utilize biopanning of a phage-display peptide library to obtain the specificity-bonding targeting peptide HSTP1 for aHSCs.Using genetic engineering technology to display the targeting peptide on the surface of exosomes,enabling them to target and delivery to aHSCs,and improving their anti-fibrotic effect,providing new treatment strategies for clinical reversal of liver fibrosis.Method1.Screening of the HSCs-T6 cell-targeting peptide.Using BRL-3A cells as negative adherent cells,HSC-T6 cells as positive target cells,Ph.D.^TM-C7C phage display peptide library kit containing 2×10¹¹pfu phages was subjected to four rounds of subtractive screening by"negative adsorption,positive adsorption,washing,elution,recovery amplification,purification".Twenty phage plaques were randomly selected for sequencing and the affinity of phages with HSCs-T6 cells was determined by enzyme-linked immunosorbent assay（ELISA）.The peptide sequence with the highest repeat rate was used as the target peptide and was named HSTP1,and a randomly mismatched sequence was named rcHSTP1 as a negative control peptide sequence.The targeting ability of the purpose peptide HSTP1 to aHSCs was detected by flow cytometry,fluorescence cell chemistry and fluorescence tissue chemistry.2.Construction of exosomes targeting aHSCs.Human umbilical cord mesenchymal stem cells（huc-MSCs）were isolated by tissue block adhesion,and the cell phenotype was identified by flow cytometry.Fusion protein of HSTP1 and lysosome-associated membrane glycoprotein 2b（Lamp2b）was engineered genetically,and huc-MSCs were divided into blank control group,negative control group,Lamp2b overexpression group（Lamp2b+）,and fusion protein overexpression group（Lamp2b+HSTP1）.The reliability of the fusion protein construction was tested by PCR,agarose gel electrophoresis,and Western Blot.Exosomes were extracted from the cell culture supernatant using ultracentrifugation.Exosomes morphology characterization and particle size analysis were performed by transmission electron microscopy（TEM）and nanoparticle tracking analysis（NTA）technology.The surface marker proteins of exosomes such as CD9,CD63,and TSG101 were detected by Western Blot.3.In vitro cell experiment to verify the anti-fibrotic effects of exosomes modified with HSTP1.Blank control exosomes（Blank-Exos）,Lamp2b overexpressing exosomes（Lamp2b-Exos）,and fusion protein overexpressing exosomes（HSTP1-Exos）were labeled with Di L fluorescent dye,and co-cultured with HSC-T6cells.Flow cytometry and fluorescence cell chemistry were used to detect the targeting ability of HSTP1-Exos to bind with HSC-T6 cells.The inhibition of Bank-Exos,Lamp2b-Exos,and HSTP 1-Exos on HSCs-T6 cells was analyzed by Oil red O（ORO）staining,transwell assay,live-cell imaging,α-SMA cytoskeleton immunofluorescence staining,and Western Blot.4.In vivo animal experiments to verified the anti-fibrotic ability of exosomes modified with HSTP1.Rats were divided into the control and experimental groups.The rats in the experimental group were induced by carbon tetrachloride to establish a liver fibrosis model,which after successful modeling was used for in vivo tracing of exosomes,in vivo targeted detection and intervention therapy.Blank-Exos,Lamp2b-Exos,and HSTP1-Exos were labeled with Di R fluorescent dye,and injected into rats through the tail vein.The distribution of exosomes in the body was observed using the vivo imaging system.Blank-Exos,Lamp2b-Exos and HSTP1-Exos were labeled with Di L fluorescent dye,and injected into rats through the tail vein.24 hours later,rat liver,spleen and kidney were taken to make frozen sections to detect the targeting of HSTP1-Exos binding to aHSCs in liver by fluorescence histochemistry.Twenty-four rats from the remaining experimental group were randomly selected and divided into PBS,Blank-Exos,Lamp2b-Exos,and HSTP1-Exos group,and gave the corresponding intervention,respectively.6 weeks later,rat liver tissues were collected,analyzed and compared with the pathological changes.The effect of exosomes interventions on macrophage polarization was measured by fluorescence histochemistry,immunohistochemistry and ELISA.The role of HSTP1 modification in exosomes regulation of macrophage polarization was preliminarily explored in combination with the literature.Results:1.After four rounds of subtraction selection,the phage was significantly enriched,with a recovery rate in the fourth round that was 46 times higher than that in the first round.Sequencing results showed that phages P3,P5,P8,P9,and P15 had the same exogenous peptide sequence.Using ELISA,it was found that the OD_450nm values of the aforementioned phages binding to HSC-T6 cells were significantly higher than those binding to BRL-3A,NRK-52E,and H9C2 cells（P<0.001）.HSTP1 and its sequence were not homologous to known genes and proteins,and were characterised as exogenous peptides.The fluorescence cytochemistry results showed that the fluorescence intensity of FITC-HSTP1 binding to HSC-T6 cells was significantly higher than that of FITC-HSTP1 binding to BRL-3A,NRK-52E and H9C2 cells（P<0.001）.Flow cytometry results showed that the average fluorescence intensity of FITC-HSTP1 binding to HSC-T6 cells was significantly higher than that of FITC-rcHSTP1（P<0.001）.The fluorescence histochemical results showed that the Manders’colocalization coefficients M1 and M2 of FITC-HSTP1 andα-SMA were significantly higher than those of FITC-rcHSTP1 andα-SMA（P<0.01）.2.Microscopic observation showed that the isolated huc-MSCs were visualized as fibroblast-like cells,with spindle,compact,vortex growth and regular arrangement.Flow cytometry showed positive expression of specific antigens CD73 and CD105and negative expression of antigens CD45 and HLA-DR in the isolated huc-MSCs.The targeted fusion protein was constructed,and PCR with Lamp2b universal primers confirmed that the target genes of huc-MSCs in Lamp2b+group and Lamp2b+HSTP 1 group were overexpressed.PCR with a specific primer for Lamp2b+HSTP1fusion genes resulted in effective amplification in only the huc-MSCs of Lamp2b+HSTP1 group.Western blot detection of Lamp2 protein expression showed that Lamp2 protein was successfully overexpressed in the Lamp2b+and Lamp2b+HSTP1 groups compared to the Blank control and Negative control groups（P<0.01）.TEM revealed the exosomes with unequal sizes and saucer-or concave hemispherical-like structures,with low-density bright areas visible in the vesicles.NTA confirmed that the average diameter of the extracellular vesicles was within the range of exosomes particle sizes.Western blot detection of extracted exosomes showed high expression of the signature proteins CD9,CD63,and TSG101.3.Flow cytometry results showed that the binding efficiency of HSTP1-Exos with HSC-T6 cells was significantly higher than that of Lamp2b-Exos and Blank-Exos at both 1 h and 3 h（P<0.05）.Fluorescent cytochemistry results demonstrated that after co-incubation for 1 h and 3 h,HSC-T6 cells in the HSTP1-Exos group exhibited more red fluorescence compared to the Blank-Exos and Lamp2b-Exos groups.ORO staining results indicated that at 48 h and 96 h,HSC-T6 cells in the HSTP1-Exos group exhibited more prominent cytoplasmic lipid droplets（LDs）accumulation than those in the Blank-Exos and Lamp2b-Exos groups.Transwell cell migration assay results showed that the number of HSC-T6 cells that penetrated the membrane in the HSTP1-Exos group was significantly lower than that in the Blank-Exos and Lamp2b-Exos groups（P<0.001）.Live-cell imaging results demonstrated that the cell counts in the HSTP 1-Exos group was significantly less than that in the Blank-Exos and Lamp2b-Exos groups after 72 h（P<0.05）.Immunofluorescence staining forα-SMA revealed that HSC-T6 cells in the HSTP1-Exos group had longer cell synapses and smaller cell bodies.Western blot results showed that the expression levels ofα-SMA and collagen I（COL-1）in HSC-T6 cells in the HSTP1-Exos group were significantly lower than those in the Blank-Exos and Lamp2b-Exos groups（P<0.05）.4.Animal in vivo imaging observations showed that compared with Blank-Exos and Lamp2b-Exos,the fluorescent of HSTP1-Exos could be more rapidly enriched in the liver area of rats,and the fluorescent was relatively stronger.The fluorescent of the three groups gradually increased before the 24-hour time point,and the fluorescent intensity reached its peak at 24 hours,and then gradually weakened.Although there was no statistical difference in the fluorescent intensity of the three groups of liver tissues in vitro,the average fluorescence intensity of the HSTP1-Exos group was higher than that of the Blank-Exos and Lamp2b-Exos groups.The fluorescent histochemical results showed that HSTP1-Exos had a significant co-localization withα-SMA,and the Manders’colocalization coefficients M1 and M2 were significantly higher than those of the Blank-Exos and Lamp2b-Exos groups（P<0.001）.In frozen sections of spleen and kidney,Di L-labeled HSTP1-Exos accumulated less（P<0.05）.The histopathological results of the liver tissue in the liver fibrosis rat model after intervention treatment showed that compared with Blank-Exos and Lamp2b-Exos,the HSTP1-Exos group had lessα-SMA-positive areas and collagen deposition in rat liver tissue（P<0.05）.The fluorescent histochemical results showed that compared with Blank-Exos and Lamp2b-Exos,HSTP1-Exos were more easily avoided from being phagocytosed by macrophages.The percentage of CD68⁺CD163⁺（M2）macrophages in the HSTP1-Exos group was significantly lower than that in the Blank-Exos and Lamp2b-Exos groups（P<0.001）.ELISA detected the concentration of CC chemokine ligand 2（CC chemokine ligand 2,CCL2）in the supernatant of HSC-T6 cells,and found that compared with Blank-Exos and Lamp2b-Exos,HSTP1-Exos could more effectively inhibit the secretion of CCL2 by HSC-T6 cells.Scatter plot analysis showed a significant positive correlation betweenα-SMA and CCL2 expression at different fibrosis levels,with an immunohistochemical score correlation coefficient of r=0.962 and P<0.001.HSTP1-Exos could more effectively inhibit the secretion of CCL2 by HSC-T6 cells,thereby further effectively suppressing M2 macrophages polarization induced by the CCL2/CC chemokine receptor 2（CCR2）axis.Conclusion1.HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs.It also provides a potential ligand for targeted delivery of drug peptides,oligonucleotides,liposomes,inorganic nanoparticles,and exosomes.2.Modification of HSTP1 improves the targeting of huc-MSCs-derived exosomes to aHSCs,promotes the activation inhibition of aHSCs,and improved the therapeutic efficacy of exosomes to reverse liver fibrosis.3.The modification of HSTP1 promoted the inhibition of huc-MSCs-derived exosomes to aHSCs induced M2 macrophages polarization through the CCL2/CCR2 axis.