Human specimens

Medical history from 26 non-fibrotic HCC patients and 38 fibrotic HCC patients were collected for survival analysis. Immunohistochemistry were performed in a subset of patients with fibrotic HCC or non-fibrotic HCC. All 64 patients with HCC were obtained from Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine (SUTCM) and approved by SHUTCM Clinical Research Ethics Committee.

Cell culture

Mouse hepatoma cell line Hepa1-6, human hepatocellular carcinoma cell line Huh7, LSEC cell line SK-Hep1 and HSC cell line LX2 were maintained in high glucose Dulbecco’s modified Eagle Medium (DMEM, Gibco) supplemented with 10% bovine calf serum (BCS) (Hyclone), 1% penicillin and streptomycin (Hyclone). Two μg/mL puromycin was used additionally for Hepa1-6-luciferase (luc) cells. All cells were grown at 37 °C in a humidified incubator with 5% CO2.


Simvastatin with over 99% purity (Sigma-Aldrich MO, USA) were used for the study. Mannan and L-NAME were purchased from Sigma-Aldrich. DiD was purchased from ThermoFisher Scientific. Poly(lactic-co-glycolic acid (PLGA)-Polyethylene glycol (PEG)-NH2 was purchased from NANOSOFT POLYMERS. Other synthetic materials as previously reported were obtained from Sigma-Aldrich MO, USA [20].


In vivo anti-mouse IgG, CD1d, CD8 and CD4 mAb were purchased from BioXcell (West Lebanon, NH). Primary and secondary antibodies used for immunoblotting, immunostaining and flow cytometry are listed in Additional file 1: Table S1.

Murine HCC models establishment

Four-week-old male C57BL/6 mice were purchased from Jackson Laboratory. All animal regulations and procedures were accepted by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill. For fibrotic HCC model, 4-week-old male immune-competent C57BL/6 mice received intraperitoneal injection of CCl4 (1 μL/g, diluted in 100 μL olive oil) twice a week for 4 weeks. Age-matched mice administered with olive oil served as control. Intrahepatic injection of 5 × 106 Hepa1-6-luc cells in 50 μL PBS was performed at the end of the fourth week. Mice were sacrificed at 3 weeks post-tumor cell inoculation or at humane endpoint. The tumors were isolated from the liver and measured with caliper. The volume was calculated with the formula of length × width × width/2, and the average value of each group was equal to the sum of total volumes divided by the number of mice. The tumor growth indicated by luciferase intensity was monitored using IVIS (Perkin Elmer, CA) imaging every 2 days. Tumor and matched non-tumor liver tissues were collected for section analysis. For advanced-stage HCC model, hemi-splenic injection of Hepa1-6 cells was performed after 4-week CCl4 exposure. To be specific, an incision located below the left rib cage was made to exteriorize the spleen. The spleen was tied and cut into two parts, each containing an intact vascular pedicle for each half of the spleen. The distal section of the spleen was inoculated with 1 × 106 Hepa1-6 cells in 150 μL PBS. The half spleen containing inoculated cells were resected 5 min after inoculation allowing the cancer cells to enter the portal vein. The other half of the spleen was returned to the cavity to keep the immune system competent.

Synthesis and characterization of the drug-loaded mannan modified NPs

The PLGA copolymers were obtained by a previously reported method [20, 21]. The PLGA-PEG and PLGA-PEG-MAN were first synthesized. Mannan attachment to the particle surface was achieved through covalent attachment by conjugation chemistry of the COOH-terminus of PLGA and mannan [22].

Two mg simvastatin was dissolved in 200 µL DMSO, and then mixed with 2 mg PLGA-PEG-MAN and 6 mg PLGA-PEG in the drug solution. The drug-polymer mixture was added dropwise to 10 mL deionized water while stirring, and stirred for 4 h. Nanoparticles were purified by ultrafiltration using 10 k cutoff columns at 4000×g.

The particle size and zeta potential of nanoparticle were measured by Malvern Nano-ZS (Malvern Instruments, UK). The morphology was analyzed by transmission electron microscopy (TEM). Five μL particles were added on 400-mesh carbon-filmed copper grids (Agar Scientific) for 2 min and stained with 2% (w/w) uranyl acetate before imaging using TEM (JEOL JEM1230).

The drug encapsulation efficiency (EE) was calculated by (Entrapped Drug/Drug Added)*100%. Loading capacity (LC) was calculated by (Entrapped Drug/Nanoparticle Weight)*100%.

In vivo treatment studies

For intra-hepatic inoculation HCC model, the mice with liver tumors were treated 3 days after the tumor cell inoculation. Five groups of mice were separately given PBS, blank PLGA NPs, simvastatin PLGA NPs (20 mg/kg), simvastatin PLGA-MAN NPs (20 mg/kg) by tail vein injection and simvastatin free drug oral gavage (40 mg/kg) every other day for totally 5 times. The anti-tumor efficacy was regularly assessed using IVIS (Perkin Elmer, CA) imaging every 2 days and survival was recorded. Some mice were sacrificed 3 days after treatment, the tumors and matched non-tumor liver tissues were collected and prepared for section study and immune effect evaluation. For immune cell depletion study, after Hepa1-6-Luc intrahepatic inoculation, PBS, IgG, anti-CD1d, anti-CD4 or anti-CD8 antibody (100 µg) was intraperitoneally injected to mice every other day for totally 5 injections 1 day before NPs treatment. For hemi-splenic inoculation HCC model, intervention was applied 5 days after tumor cell inoculation. Four groups of mice were given separately by PBS, simvastatin PLGA-MAN NPs (20 mg/kg), anti-PD-L1 antibody (100 µg) or combination. NPs tail vein injection and anti-PD-L1 antibody intraperitoneal injection were performed every other day for a total 5 times.

Mouse liver sinusoidal endothelial cell isolation

Mouse LSECs were isolated as previously described [23]. In brief, livers were perfused through the portal vein and digested with a collagenase solution. After mincing the liver, cells were filtered and centrifuged at 50×g to remove hepatocytes. Non-parenchymal cells were then separated by differential centrifugation using a Percoll gradient. Kupffer cells were eliminated by plastic pre-culture for 30 min. LSEC were collected for RNA extraction.

Gene expression profiling

SK-Hep1 cells in triplicate were treated with simvastatin (20 μM) and control for 24 h. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and checked using RNase free agarose gel electrophoresis. mRNAs were isolated and fragmented to about 200 base pairs length and reverse transcribed into cDNA using the QuantiTect Reverse Transcription kit (Qiagen). The cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, Netherland), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The cDNA library products were size selected by agarose gel electrophoresis, PCR amplified and sequenced using Illumina HiSeqTM4000 platform (Illumina, San Diego, CA). Transcript-level expression analysis of sequencing data was performed using HISAT and StringTie software (,shtml) [24]. Differential transcripts of chemokines with stringent cutoff coefficient of less than 0.05 were obtained to align to GO database ( for protein functional annotation corresponding to immune population.

MTT assay

Cell viabilities were assessed by MTT assay. 10^4 cells were seeded in 96-well plates per well overnight and subjected to different treatments. Five mg/mL MTT (Alfa Aesar) reagent was added for 4 h at 37 °C, and then the supernatant was discarded. The formazan was resuspended in 100 μL of DMSO and absorbance was examined by a spectrometer (Hidex Chameleon).


Liver tissues with tumor or major organs including hearts, livers, spleens, lungs, and kidneys were collected and were fixed in 4% paraformaldehyde (PFA). Fixed samples were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome at UNC histology facility. PFA-fixed samples were embedded with optimum cutting temperature compound and sectioned at 8 μm thickness. For immunohistochemistry (IHC), sections were incubated with primary antibodies at 4 °C overnight, washed, and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Digital images were taken using brightfield light microscope (Olympus BX61). Immunofluorescence (IF) was performed using fluorescent antibodies and counterstained with Prolong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific). Antibodies are listed in Additional file 1: Table S1. Apoptotic cells were stained with a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) kit (Promega, Madison, WI). Images were taken using laser-scanning confocal fluorescence microscope (Zeiss LSM 710). Liver samples fixed with 4% PFA by perfusion through portal vein were sectioned using vibratome at UNC microscopy services laboratory and prepared for scanning electron microscopy (Zeiss Supra 25 FESEM). Five random microscopic fields were selected and quantified by ImageJ software. Porosity was measured as percentage of LSEC surface occupied by fenestrae in SEM in liver tissue. Fenestration frequency was calculated with total number of fenestrations divided by total area of LSEC surface.

Flow cytometric analysis

Single-cell suspensions from tumor tissue were harvested in MACs buffer (1 × PBS + 2 mM EDTA + 0.5% BSA, filter sterile), then subjected to conjugated staining with fluorescence. At least 10,000 live cells were subjected to flow cytometric analysis on a flow cytometer (Becton Dickinson LSR II). Experimental data were analyzed using FlowJo software. The antibodies used are listed in Additional file 1: Table S1.


Cells were lysed in RIPA lysis buffer with protease inhibitors. Total lysates were quantified by a BCA Protein Assay Kit (Biorad, CA). Thirty μg protein samples were used for immunoblotting analysis. After incubating with appropriate primary and secondary antibodies, the immunoreactions were visualized with Western HRP substrate (Thermo, Rockford, IL). The antibodies used are listed in Additional file 1: Table S1.

Quantitative real-time polymerase chain reaction (RT-PCR) assay

Total RNA was extracted from cells or the whole tumor using an RNeasy microarray mini kit (Qiagen, Hilden, Germany) and was reverse-transcribed to cDNA with an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed in a 7500 RT-PCR system. The PCR primers are listed in Additional file 1: Tables S2 and S3.

Nitric oxide assay

Nitric oxide (NO) amount was assessed using Nitric Oxide Colorimetric Assay Kit (BioVision, Milpitas, CA) in accordance to the manufacturer’s manual. Briefly, SK-Hep1 cells were treated with different concentrations of simvastatin (0, 5, 20 μM) for 24 h and the supernatants were collected. Equal aliquot of samples in every group were mixed with Nitrate Reductase Buffer and incubated at room temperature for 1 h. The mixture was then added Griess Reagent and color was developed in 10 min. The absorbance was measured at 540 nm and NO concentration was calculated referring to a standard curve. The experiments were done in triplicate.


0.05% (wt) of lipophilic carbocyanine DiD (ThermoFisher) was used to formulate the DiD-labeled PLGA-PEG or PLGA-PEG-MAN NP. In mice bearing fibrotic tumor, 24 h following tail vein injection of DiD-labeled NP, major organs and liver with tumors were collected and analyzed using IVIS (Perkin Elmer, CA) with the excitation wavelength at 640 nm and the emission wavelength at 670 nm.

Blood chemistry analysis

Seven days after 5 doses of nanoparticle injections, whole blood was obtained from the normal mice. Blood routine including RBC, WBC, PLT, HGB, and HCT was tested using the whole blood. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CRE) in serum were analyzed as indicators of hepatic and renal function using the serum.

Statistical analysis

GraphPad Prism 8 (GraphPad Software, La Jolla, California, USA) was used for statistical analysis. The independent Student t-test was used to compare data between the two groups. A one-way analysis of variance test with Bonferroni correction was used to compare data in multiple groups of mice. Pearson correlation coefficient was calculated to show the correlation of two parameters. Two-way ANOVA with a Bonferroni post-hoc test was used to estimate the difference in multiple groups of mice when considering two factors of time and treatment. Kaplan–Meier survival analysis was used to determine the overall survival rates and tumor incidence-free rates, the differences were compared by the log-rank Mantel-Cox test. Data averages from each group are presented as mean ± SD; *p < 0.05, **p < 0. 01, ***p < 0. 001.

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