Huc-MSC-derived exosomes modified with the targeting peptide of aHSCs for liver fibrosis therapy | Journal of Nanobiotechnology

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Cell culture

Rat hepatic stellate cells (HSC-T6) (Procell Life Science & technology Co., Ltd, Wuhan, China), rat liver cells (BRL-3A), rat kidney epithelial-like cells (NRK-52E) and rat cardiomyoblast cells (H9C2) (Chinese Academy of Science, Shanghai, China) were maintained in DMEM (Biological Industries, Israel) containing 10% fetal bovine serum (FBS) (HyClone, USA) in an incubator with 5% CO2 at 37 °C.

Screening the targeting peptide of HSC-T6 cells

Phage display biopanning was performed according to a previous description, with certain modifications [51]. HSC-T6 cells were selected as the positive target cells, and BRL-3A cells were selected as the negative bound cells for whole-cell subtractive screening from the Ph.D. C7C™ Phage Display Library (New England Biolabs, USA). A total of 4 rounds of biopanning were performed, 2 × 1011 plaque-forming units (pfu) of collected phage were used for each round, and Tween 20 was increased in a stepwise manner to 0.2%. Twenty phage clones were randomly selected, and amplified. Sanger sequencing was performed by GENEWIZ, Inc., (Suzhou, China). The data were analyzed using Chromas 2.6.5 (South Brisbane, Australia).

Enzyme linked immunosorbent assay (ELISA)

Cell-based ELISA, HSC-T6, BRL-3A, NRK-52E and H9C2 cells were plated in 96-well plates (5 × 104 cells/well) until they adhered and covered the bottom of the wells. The cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 15 min. After three washes, the cells were blocked with 5% BSA for 1 h at 37 °C. Next, 2 × 108 pfu of picked phages were incubated separately with each cell type for 1 h at room temperature (RT). The wells were washed 5 times, and 100 µl of horseradish peroxidase HRP-conjugated anti-M13 antibody (Abcam, UK) (1:500) was introduced into to each well for 1 h at 37 °C. After PBS washes, TMB substrate solution (Boster, China) was added to the wells and incubated for 10 min at RT. The reaction was terminated by the TMB stop solution (0.5 M H2SO4) (Boster, China). The plates were read on a full wavelength microplate reader (BioTek, Germany) at 450 nm. According to the sequencing and ELISA results, a specific peptide was identified in the HSC-T6 cell affinity clones, and designated HSTP1. A peptide with the same amino acids as HSTP1 in a randomized order as the negative control was designed as rcHSTP1. The cationic penetrating peptide RKKRRQRRR (peptide Tat) was used as the positive control. All peptides were attached to fluorescein-5-isothiocyanate (FITC) labeling and synthesized by Chinese Pepide (Hangzhou, China).

ELISA for CCL2 secreted by HSC-T6 cells, HSC-T6 cells were treated with TGF-β1 and different exosomes for 24, 48, and 72 h; then, the supernatants were centrifuged at 10,000g for 10 min and collected for analysis. In addition, the media from untreated were collected as the control. Commercial CCL2 ELISA kits (Elabscience, China) were used to detect the content of CCL2 in each group.

Peptide-affinity assay

For immunocytofluorescence analysis, HSC-T6 cells, and control BRL-3A cells, and NRK-52E cells were plated in 35 mm glass bottom petri dishes (Thermo, USA) for 12 h (1 × 104 cells/well). HSC-T6 cells were incubated with increasing concentrations (5, 15, 25, 50 µM) of FITC-HSTP1 for 1 h. For the examination of HSTP1 specificity, HSC-T6, BRL-3A, NRK-52E, and H9C2 cells were incubated with 15 µM FITC-HSTP1 for 1 h at RT. The cells were also incubated with 150 µM rhodamine phalloidin (Solarbio, China) for 30 min, and counterstained with DAPI (2 µg/ml) for 5 min. The cells were observed by laser scanning confocal fluorescence microscopy (Olympus, Japan).

For flow cytometry analysis, HSC-T6 cells (1 × 106) were harvested and resuspended to a single cell suspension, and then divided into quarters followed by incubation with 15 µM FITC-labeled peptides for 30 min in the dark. HSC-T6 cell affinity properties of peptides were measured using flow cytometry (Beckman Coulter, USA). BRL-3A cells were treated in the same manner.

For immunofluorescencehistochemistry analysis, paraformaldehyde-fixed rat livers were stained with FITC-HSTP1, FITC-rcHSTP1 and anti-α-SMA antibodies (SAB, USA), followed by CY3-conjugated secondary antibodies. Tissue slides were imaged by fluorescence microscopy (Olympus, Japan).

Isolation and culture of huc-MSCs

Huc-MSCs were obtained from fresh umbilical cord tissue of healthy donors using the explant method. All procedures were approved by the ethics committee. The two arteries and one vein in the umbilical cord were removed using a scalpel. Wharton’s jelly was minced into 2 mm3 blocks. After culture for 3 h at 37 °C, DMEM/F12 (Biological Industries, Israel) containing 15% FBS was added to T75 flasks. Approximately 7 days later, the cells gradually migrated out. Huc-MSCs were incubated with FITC-labeled primary antibodies raised against CD45 and CD73, and APC-labeled primary antibodies raised against CD105 and HLA-DR (Biolegend, USA) for 30 min at RT. After washing and resuspension in PBS, the samples were analyzed by a flow cytometer (BD, USA).

Plasmid construct and virus package

The fusion gene sequence of Lamp2b and HSTP1 (Lamp2b + HSTP1) was synthesized and purified by Shanghai GeneChem Co., Ltd. (Shanghai, China). After being digested by Agel restriction enzymes, the lentivirus-based vectors Ubi-MCS-SV40-EGFP-IRES-puromycin and Lamp2b + HSTP1 were expanded by polymerase chain reaction (PCR), and products were run on a gel for purification. Then, the cDNA of Lamp2b + HSTP1 was added to the vector via Exnase ™ II (Clontech, USA) for 30 min at 37 ℃, followed by driving the recombinant plasmid vector into competent cells. An individual colony was picked out from agarose plates. Sequencing was conducted after bacterial explanation. Lentivirus was generated by transfection of HEK 293 T cells with the gene-transfer-plasmids, the packaging plasmid helper 1.0 and the envelope-plasmid helper 2.0. After forty-eight hours, the lentivirus suspension was harvested, and concentrated by ultracentrifugation at 25,000 r for 2 h at 4 °C.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)

The supernatants were collected to isolate exosomes via ultracentrifugation according to a previous study [52]. The supernatants were centrifuged at 800 g for 15 min followed by 10,000 rpm for 30 min at 4 °C to remove cells and debris and then ultrafilter and concentrated at 2000 rpm for 20 min using ultrafiltration centrifugal tube (Millipore, USA). Concentrated supernatants were centrifuged at 140,000g for 90 min at 4 °C in a Type Ti100 rotor using an XL-100K ultracentrifuge (Beckman). After resuspension in PBS, the exosome pellet was ultracentrifuged again for 90 min at 140,000g. Finally, the exosomes were resuspended in PBS, filtered using a 0.22-μm filter (Millipore, USA), and analyzed with a Enhance BCA Protein Assay kit (Beyotime, China). Approximately 6 mg of exosomes are obtained per 500 ml of supernatant. The purified exosomes were resuspended in PBS (200 µl) and further diluted by 1- to 10- hundred folds for analysis. The samples were fixed with 2.5% glutaraldehyde overnight at 4 °C. Ten microliters of the mixture were applied to copper grids and stained with 1% phosphotungstic acid for 1 min. The dried grid was observed by a Tecnai G2 TEM (FEI, USA). NTA was conducted using a Zeta View system (NanoSight, UK) to automatically track the Brownian motion and size distribution data of exosomes in real time.

Exosome uptake analysis

For investigation of the uptake of HSTP1-Exos by HSC-T6 cells, Blank-Exos, Lamp2b-Exos, and HSTP1-Exos were labeled with Dil (Thermo Fisher Scientific, USA) and cocultured with HSC-T6 cells. After 1 h and 3 h, the percentage of uptake of exosomes by HSC-T6 cells was examined by flow cytometry (Beckman Coulter, USA). Then, we labeled the HSC-T6 cells with DiO (Thermo Fisher Scientific, USA). The labeled exosomes were cocultured with HSC-T6 cells for 1 and 3 h. Images of exosomes and HSC-T6 cells were observed under confocal fluorescence microscopy (Nikon, Japan).

LDs staining

HSC-T6 cells were divided into five groups: the normal control group, TGF-β group (cells were treated with 10 ng/ml TGF-β1), TGF-β + Blank-Exos group (cells were treated with 10 ng/ml TGF-β1 plus 50 µg/ml blank-exosomes), TGF-β + Lamp2b-Exos group (cells were treated with 10 ng/ml TGF-β1 plus 50 µg/ml lamp2b-exosomes), and TGF-β + HSTP1-Exos group (cells were treated with 10 ng/ml TGF-β1 plus 50 µg/ml HSTP1-exosomes). Cells were plated in 6-well plates for 12 h, followed by administration of the corresponding drugs for 24, 48, and 72 h. HSC-T6 cells were fixed for 15 min with 4% paraformaldehyde on the indicated culture day. Then, the cells were incubated with ORO (Sigma, Germany) working stain for 30 min, followed by 15 s of rinsing in 60% isopropanol. The nuclei were counterstained with hematoxylin for less than 1 min before phase contrast microscope observation (Olympus, Japan).

Transwell assay and live-cell imaging

HSC-T6 cells were also divided into the control group, TGF-β group, TGF-β + Blank-Exos group, TGF-β + Lamp2b-Exos group, and TGF-β + HSTP1-Exos group, and treated with the corresponding drugs for 48 h. Each group of cells was harvested, and 2 × 104 cells in 200 µl of nonserum medium were placed in the upper chamber of an insert. The lower chamber was filled with 700 µl of DMEM with 20% FBS. Migrated cells on the underside of the filter, were fixed and stained with 1% crystal violet for 10 min. Three randomly selected fields were photographed and counted under a phase contrast microscope (Olympus, Japan). HSC-T6 cells (3 × 103 cells/well) were seeded in 96-well culture plates. After attachment, the cells were treated with TGF-β1 and exosomes. Cell count was detected using Cytation C5 (BioTek, USA).

Immunofluorescence staining

HSC-T6 cells, divided into the control group, TGF-β group, TGF-β + Blank-Exos group, TGF-β + Lamp2b-Exos group, and TGF-β + HSTP1-Exos group, were seeded on slides in 6-well plates, followed by administration of the corresponding drugs for 48 h, and 72 h. The cells were permeabilized with 0.5% Triton X-100 for 20 min and blocked with 3% bovine serum albumin (BSA) for 1 h. Then, the cells were incubated with anti-α-SMA antibody (SAB, USA) for 16 h at 4 °C, followed by incubation with FITC-conjugated secondary antibody for 1 h at RT. The nuclei were counterstained with DAPI, and images were taken using a fluorescence microscope.

Western blot and real-time PCR

The BCA protein assay kit was used to determine the protein concentration. Proteins were fractionated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was blocked with 3% BSA in Tris-buffered saline containing 0.05% Tween 20. Antibodies against CD9, CD63, TSG101, GAPDH (Abcam, USA), Lamp2 (Santa Cruz, USA), CoL1α1, α-SMA, and β-actin (SAB, USA) were used to quantify protein expression by Western blotting. Total RNA was extracted with TRIzol (TaKaRa, Japan). Real-time PCR was performed using an Mx3000 real-time PCR detection system (Agent, USA). The 2Ct method was used to calculate the relative expression.

Animal treatment

Four-week-old male SD rats (n = 120) weighing 120–150 g were purchased from the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences (Lanzhou, China). After a week-long acclimation period, rats were randomly divided into normal control (NC) groups and experimental groups. The experimental group was subjected to intraperitoneal injections of 2 ml CCl4/olive oil (1:1, v/v)/kg body weight 2 times per week for up to 6 weeks to induce the liver fibrosis model. Then, the rats in the experimental group were divided into 4 groups: PBS, Blank-Exos, Lamp2b-Exos, and HSTP1-Exos groups. Rats in the PBS group were given 500 µl of PBS twice a week for 6 weeks, and rats in the Blank-Exos, Lamp2b-Exos, and HSTP1-Exos groups were given corresponding exosomes 2.5 mg/kg in 500 µl PBS twice a week for 6 weeks. Blank-Exos, Lamp2b-Exos, and HSTP1-Exos, labeled with DiR, were intravenously injected via the tail vein to determine tissue distribution via a VISQUE in vivo smart imaging system (Vieworks, Korea). Blank-Exos, Lamp2b-Exos, and HSTP1-Exos, labeled with Dil, were intravenously injected via the tail vein to determine the cell distribution via cryosections. The tissues were embedded with OCT and sliced at − 20 °C; then, liver tissue slices were immunostained with anti-α-SMA (SAB, USA), or anti-CD68 (Abcam, UK) antibodies followed by FITC-conjugated secondary antibodies (Abcam, UK). Tissue slides were imaged by fluorescence microscopy (Olympus, Japan).

At the end of the experiments, liver tissues were removed and fixed in 4% neutral formaldehyde, embedded in paraffin and sectioned. HE staining, Masson’s trichrome staining, and Sirius red staining were used for analysis of histological structure and fibrotic area, respectively. The positive area was assessed with ImageJ 1.8.0 software (National Institutes of Health, USA). CD68+CD163+ double positive macrophages were analyzed by immunofluorescence staining with anti-CD68 and anti-CD163 antibodies (Abcam, UK) followed by CY3- and FITC-labeled goat anti-rabbit IgG antibodies (Abcam, UK) respectively. For immunohistochemistry analysis, the slices were probed with primary targeted against rat α-SMA (SAB, USA) and CCL2 (Proteintech, China), and stained with 3,3’-diaminobenzidine. The degree of liver fibrosis in different rats in PBS, Blank-Exos, Lamp2b-Exos, and HSTP1-Exos groups were assessed according to the Metavir score system. Under 400-times microscope, 5 pathological areas were randomly selected. The CCL2 and α-SMA protein expression was quantified based on the semi-quantitative Histoscore, which was calculated by an assessment of both the percentage of positive cells and the intensity of staining (0, non-staining; 1, weak; 2, median; or 3, strong). This analysis was performed by two independent reviewers.

Statistical analysis

All data are expressed as the mean ± SD. Independent sample t test was applied for comparing data between two groups, and one-way analysis of variance (ANOVA) was utilized for data comparison among multiple groups. P-values less than 0.05 were considered statistically significant. Statistical analyses were performed using Prism 8 software (GraphPad, USA).

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