Liver-targeted delivery of TSG-6 by calcium phosphate nanoparticles for the management of liver fibrosis

Mesenchymal stem cells (MSCs) transplantation is a promising antifibrotic strategy but facing clinical controversies. Inspired by advances in nanomedicine, we aimed to bypass these clinical barriers of MSCs by identifying the key antifibrotic molecule of MSCs and developing a specific liver-targeting nanocarrier. Methods: Cytokines secreted by MSCs were examined with serum stimulation of cirrhotic patients. Immunohistochemistry, microarray, immunoblotting, and quantitative real-time PCR (qRT-PCR) were applied to identify the critical antifibrotic cytokine and to discover its role in modulating antifibrotic effects. Biomineralization method was used to prepare calcium phosphate nanoparticles (NPs). The targeting and therapeutic efficiency of NPs were evaluated by in vivo imaging and biochemical studies on fibrotic mice induced by CCl4. Results: The stimulated MSCs exhibited high-level expression of Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6). On animal study, exogenous administration of TSG-6 alone can ameliorate liver fibrosis while TSG-6 knocked MSCs (Lv-TSG-6 MSCs) lost antifibrotic effects. Further studies verified the importance of TSG-6 and identified its antifibrotic mechanism by modulating M2 macrophages and increasing matrix metalloproteinase 12 (MMP12) expression. Additionally, we found a feedback loop between TSG-6, MMP12 and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β), which may improve our understanding of the aggravating process of cirrhosis and antifibrotic mechanisms of TSG-6 and MSCs. Based on these findings, we developed calcium phosphate nanoparticles (CaP@BSA NPs) by biomineralization method using bovine serum albumin (BSA) as the biotemplate. Imaging tracking and drug loading studies showed specific liver targeting and high TSG-6 loading efficacy of as-prepared CaP@BSA NPs. In vivo therapeutic study further demonstrated the improved therapeutic effects of TSG-6 loaded CaP@BSA. Conclusions: TSG-6 was a major antifibrotic cytokine of MSCs, TSG-6 loaded CaP@BSA NPs showed specific liver accumulation and improved therapeutic effects, which indicated translational potentials of CaP@BSA as a promising drug carrier for the liver disease management.


Primary liver macrophages isolation and supernatant collection
Primary liver macrophages were isolated using a modified two-step collagenase perfusion method [3]. Briefly, mice were anesthetized, and the peritoneal cavity was exposed and a cannula was inserted into inferior vena cava. Then the mice liver was flushed with Ca 2+ -free perfusion buffer at 37 °C to remove the blood at a flow rate of 20 mL/min for approximately 10 min. The entire liver was excised and the connective tissues were carefully removed. Then the liver was digested with 30 mL of collagenase IV buffer (17104019, Gibco, NY) at 37 °C with a flow rate of 10 mL/min for 15 min. The catheter was then removed and the liver was transferred to a dish containing 20-30 mL DMEM. The Glisson's capsule of the liver was peeled back and the cells were shacked out in the liquid. The cell suspension was filtered through a 100 μm gauze mesh filter. Hepatocytes were separated from nonparenchymal cells by three rounds of low-speed centrifugation at 50 g. The supernatant containing the macrophages cells was collected. Slowly layer the supernatant on the Percoll gradient with a 15 mL 4 / 41 pipette and centrifuge the mixture at 2,300 rpm for 30 min at 4 °C. Carefully collected the middle interphase and transfer the cells to a new 50 mL tube. Then the cell precipitates were collected after centrifugation at 1,500 rpm for 10 min at 4 °C. To determine the effects of different treatment (PBS, MSCs, Lv-shTSG-6 MSCs, TSG-6) on cytokines releasing, the primary liver macrophages were isolated at 24 h post cell transplantation. Cultured in vitro for another 24 h, the supernatant of culture medium was collected for ELISA and macrophages were harvested for PCR analysis.

Histological evaluation
The tissues were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. The paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin (HE), for routine histological examination and Sirius Red for fibrosis evaluation. Quantification of collagen fiber was assessed using Image-Pro Plus software (v6.0, Media Cybernetics Inc.).

Measurement of blood biochemistry
The mice serum obtained from each time point, and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (ALB) were determined using an automatic biochemistry analyzer (Hitachi 7600-120, Hitachi, Japan) [4].

Immunofluorescence (IF) staining
Tissue sections were prepared and incubation with anti-α-SMA antibody (A2547, 1  Ex Taq II (DRR820A, Takara, Tokyo) and CFX96 Touch™ real-time PCR System (Bio-Rad, CA) were used for amplification. Specific primer for each transcript was list in Supplemental Table S6. All reactions were performed in triplicated. GAPDH mRNA was used as an internal control to normalize mRNA expression.

Immunoblotting
The proteins from cells or tissue samples were prepared by the RIPA lysis buffer (Beyotime Biotechnology, China) with proteinase inhibitors and phosphatase inhibitors (Roche, Basel). Extracted protein lysates were quantified by Bradford method using Bio-Rad protein assay (Bio-Rad Laboratories, CA) and 12 μg of protein was loaded to SDS-PAGE, followed by transfer to nitrocellulose membranes (Bio-Rad Laboratories). Membranes were blocked with TBS-T containing 5% skim milk. After blocking, the membrane was incubated with primary antibodies overnight at 4 °C. Information of used primary antibodies was listed in Supplementary Table S7. After 30 min incubation with peroxidaseconjugated secondary antibodies (Jackson immune research laboratories, PA), the blots were developed by an enhanced chemiluminescence kit (Thermo Fisher Scientific, IL). Image Lab software was used to quantify the western blot bands.

ELISA
The human TSG-6 and MMP12 levels were measured using ELISA kit (ELH-TSG6, ELH-MMP12, RayBiotech, GA). The levels of MMP12 in patients with hepatitis B virus-related cirrhosis and primary biliary cirrhosis were detected respectively. The clinical information of patients in these two groups was shown in supplemental Table 4 and Table 5

Microarray assays of hepatic mRNA
Total RNA isolated from livers of fibrosis mice with or without TSG-6 treatment was studied by mouse gene expression microarray (n = 3, SurePrint G3 Gene Expression Microarrays, Agilent Technologies, Inc, CA). The experimental procedure was performed in accordance with manufacturer's instructions, and data interpretation was done by CapitalBio (CapitalBio Technology Co. Ltd, China).
The GEO ID of the expression profile data is GSE121669.

IHC
Human tissue array containing 28 cases of liver fibrosis and 16 cases of normal liver were brought from Alenabio (LV20812a, Xi'an, China) and standard procedures of immunohistochemistry staining (IHC) were strictly followed. Briefly, sections were dewaxed and rehydrated by xylol and alcohol. After antigen retrieval, 1% H2O2 was introduced to block endogenous peroxidase activity. After 30 min goat serum for blocking, the slices were incubated either with anti-MMP12 antibody (Abcam, ab52897) overnight at 4 °C. Information of used antibodies was listed in Supplemental Table S7. After washing with PBS, the sections were incubated 1 h at room temperature with secondary antibody, then visualized by diaminobenzidine (DAB).

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Firstly, BSA was labeled by IRdye 800CW NHS ester (IR800) according to the manufactory instructions [5]. Briefly, 50 mg BSA was dissolved in 5 mL saline, the pH value was adjusted to 8.5-8.7 by using Na2CO3 aqueous solution (50 mM). Then, a certain volume of IR800 (10 mM) in DMSO was added in the above solution under vigorous stirring, the molar ratio of dye to BSA was set as 1.8, and the DMSO concentration was kept below 2% (v/v) in the reaction system. After stirring for 2 h under room temperature, the reaction mixture was purified by centrifugal ultrafiltration (10k MWCO, 8,500 rpm, 5 min), and concentrated by DMEM to 1 mL. The UV−Vis absorption and fluorescence spectra of the IR800 labeled BSA (IR800-BSA) were detected on a Carry 60 UV-Vis spectrophotometer (Agilent Technologies, CA and a fluorescence spectrophotometer (Thermo Scientific Lumina, Germany), respectively. For the preparation of CaP@BSA-IR800, 200 μL of BSA-IR800 was added in 0.8 mL DMEM medium, sealed, and incubated at 37 °C for 24 h to reach equilibrium. Then, 10 μL CaCl2 (1 M) was added into the reaction system for another 24 h. After incubation, the product was separated by centrifugation (13,500 rpm, 15 min), washed by deionized water for several times.

In vitro TSG-6 loading and releasing
The typical in vitro drug loading and releasing experiments were carried out as following: CaP@BSA NPs (240 μg) were dispersed in 240 μL PBS 7.4 containing 30 μg TSG-6. The suspension was sealed and then shaken at a constant rate (120 rpm) at 37 °C for 4 h. The drug-loaded product was separated by centrifugation, and the remained TSG-6 in the supernatant was determined by the TSG-6 ELISA kit (R&D Systems Inc.) according to the standard protocol. The drug loading efficiency was calculated by the formula: drug loading efficiency = ((original TSG-6 -remained TSG-6)/original TSG-6) × 100%. For the release experiments, the obtained TSG-6 loaded CaP@BSA NPs was re-dispersed in 720 μL PBS 7.4, and then sub-packed in three tubes (240 μL per tube) at 37 °C with constant shaking (120 rpm). The drug release medium (120 μL) was withdrawn for analysis at given time intervals and replaced with the same volume of fresh PBS. At each time point, the reaction system was centrifugated to separate released drug and solid particles. The released drug was determined by the TSG-6 ELISA kit (R&D Systems Inc.) and normalized to the total released drug amount, which was determined by the complete dissolution of particles at pH 5.

The pH-responsive degradation experiments
The degradation experiments of CaP@BSA NPs was carried out in phosphate buffer solution (PBS) with different pH values (pH 7.4, 6.8, 6.5, 6.2, and 5.0) at 37 °C under constant shaking (120 rpm).
Briefly, 1 mL CaP@BSA suspension (5 mg/mL) was placed in dialysis bags (MWCO = 3500), and then the dialysis bag was sealed to dialysis against another 19 mL of PBS at pH 7.4, 6.8, 6.5, 6.2, and 5.0 respectively to simulate different biological environments. The supernatant solution (5 mL) was withdrawn for ICP analysis to measure the concentrations of Ca 2+ ions at given time intervals and replaced with the fresh PBS with the same volume and the same pH value.

Biocompatibility of BSA-CaP NPs.
The biocompatibility of CaP@BSA NPs was evaluated by 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazollium bromide (MTT) assay. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C for 48 h. Then, the cells were seeded in 96-well flat-bottom microassay plates at a concentration of 5 × 10 3 cells per milliliter and incubated for 24 h. After that, the media were replaced with fresh media containing CaP@BSA NPs (10−400 μg/mL) and co-cultured for 48 h. The sample-free tissue culture plate was used 9 / 41 as the control. Cell viability was quantified by MTT assay and calculated based on the formula: cell viability = (OD490 nm of the sample/OD490 nm of the control) × 100%, the cell viability of the control group was set as 100%, and the data represents the mean value of four parallel measurements.

Hemolysis assay
The red blood cells (RBCs) were isolated from serum by centrifugation of the mixture containing 0.5 mL blood sample and 1 mL PBS 7.4 at 4,500 rpm for 3.5 min. After washed by PBS for five times, the purified RBCs were diluted to 5 mL. Then, 0.