Construction of nanoparticles encapsulating doxorubicin (DOX) and PD-L1 siRNA (NPDOX/siPD-L1) and its characterization
NPDOX/siPD-L1 was constructed according to a previously reported method. In brief, block copolymer PEG-PLA (Sigma-Aldrich, St. Louis, MO, USA), cationic lipid DOTAP (Avanti Polar Lipids, Alabaster, AL, USA), and DOX (Sigma-Aldrich) were combined at a ratio of 10:1:1 according to a single emulsion method (Sun et al. 2015). The formed NPDOX was further loaded with PD-L1 siRNA (siPD-L1, Suzhou Ribo Life Science Co., Ltd., Suzhou, China). After that, the gel retardation assay was used to determine the weight ratios of DOTAP to siPD-L1. Dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90) was used to determine particle size and distribution. Transmission electron microscopy (TEM, JEOL JEM2010 200 kV) was used to observe the size and morphology of NPDOX/siPD-L1.
Cell lines and animals
HCC cell lines including murine H22 and human HepG2 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in the complete medium supplied with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 IU/ml penicillin at 37 °C in the presence of 5% CO2.
Mouse bone marrow-derived dendritic cells (BMDCs) and human peripheral blood dendritic cells (PBDCs) were isolated and cultured according to previously reported methods (Madaan et al. 2014; Grievink et al. 2016). For BMDCs isolation and culture, bone marrow progenitors were washed out from shin bone and thigh-bone of the mice, then cultured in the medium containing IL-4 (1 ng/ml) and granulocyte-macrophage colony-stimulating factor (10 ng/ml) (PeproTech, Rocky Hill, NJ). After culturing for 48 h, non-adherent cells were gently washed out. The remaining cell clusters were cultured. Medium was changed every other day. On day 7, the cells were collected for further experiments. For PBDCs isolation and culture, PBDCs were purified from non-adherent cells by an iso-osmotic Percoll density gradient, to yield low-density cells. Then, the low-density cells were plated to remove the monocytes. This treatment was repeated. The cells were cultured in the complete culture medium.
In the present study, H22 cells (1 × 106 cells) were co-cultured with BMDCs (1 × 106 cells) at a ratio of 1:1 in the 12-well plates. Similarly, human HepG2 cells (1 × 106 cells) were co-cultured with PBDCs (1 × 106 cells) at a ratio of 1:1 in the 12-well plates. After incubation for 24 h or 48 h, the cells and supernatant were collected for further assays.
H22-OVA cells were constructed by transfecting plasmid encoding OVA (VectorBuilder, USA) into H22 cells. OT-1 mice were purchased from the Cyagen Biosciences Inc (Suzhou, China).
Measurement of ATP
After the cells were treated with DOX or nanoparticles for 24 h, the release of ATP was determined using a chemiluminescent ATP determination kit (Life Technologies, Pleasanton, CA, USA), according to the document of the manufacturers.
Flow cytometry
The surface exposure of calreticulin (CRT) was determined using flow cytometry. H22 and HepG2 cells were treated with Doxorubicin (DOX, 0.25 μM) for 24 h and then labeled with anti-CRT-PerCP-Cy5.5 (antibody online. com. Catalog No. ABIN2486728). The percentage of CRT positive cells was quantified based on Propidium iodide (PI) negative cell populations.
To analyze cellular uptake of DOX and PD-L1 siRNA, H22 cells were incubated with indicated antibodies that were labeled with fluorescence Dye. siRNA was labeled with FITC. FITC signal and DOX signal were detected for determining cellular uptake of PD-L1 siRNA and DOX, respectively.
The populations of matured dendritic cells (DCs) and cytotoxic T cells in the tumor tissues were also detected using flow cytometry (BD FACSCalibur, San Jose, CA, USA) according to a previously reported method (Shang et al. 2018). APC-labeled CD80 (Clone: 16-10A1, BioLegend) and PE-labeled CD86 antibodies were applied to determine the population of DCs. APC-labeled CD4 (Clone: GK1.5, BioLegend) and FITC-labeled CD8 (Clone:53-6.7, BioLegend) were applied to determine the population of cytotoxic T cells. The results were analyzed using software FlowJo (LLC, Ashland, Oregon, USA).
Detection of cell apoptosis
In the present study, cell apoptosis was determined using flow cytometry. Annexin V/PI double staining was applied. H22-OVA cells were treated with nanoparticles for 24 h. Next, the treated H22-OVA cells were co-cultured with CD8 + T cells that were isolated from OT-1 mice in 9 mm petri-dish. Cells were then harvested and suspended in the binding buffer containing FITC-labeled Annexin-V and PI followed by incubation in the dark for 15 min. Then the population of apoptotic cells (Annexin-V+PI+) was measured by flow cytometry and data were analyzed using software FlowJo.
Quantitative Real-Time reverse transcription(qRT)-PCR
RNA extraction kit was used to isolate RNA from the cells, according to the manufacturer’s document. Reverse transcriptase was used in the RT reaction. The Melt curves were used to analyze the accuracy. The expressions of each gene were calculated using 2-△△Ct values. The mRNA expression values of PD-L1 were normalized to that of GAPDH.
ELISAs
The supernatant was collected from different treatment groups. The productions of the cytokines including high mobility group box 1 protein (HMGB1), transforming growth factor (TGF)-β, IL12p70, and interferon (IFN)-γ were determined using specifics ELISAs according to the manufacturers’ instruction (DAKEWE, Beijing, China).
Western blot
The protein was extracted according to the previous methods (Yang et al. 2017). In brief, a cold RIPA buffer containing protease inhibitor was used to lyse the cells or tumor tissues. After that, the extraction buffer was centrifuged at 13,000 g for 10 min to remove the cell debris and other insoluble materials. The BCA protein assay kits were applied to qualify the concentrations of extracted proteins.
An equal amount of proteins was loaded and separated using the 10% SDS gel. After that, the gel was transfer to a PVDF membrane, which was blocked with 5% non-fat milk at room temperature for 2 h. Next, a primary antibody against S-HMGB1 (ab18256, Abcam) or PD-L1 (ab205921, Abcam) was used to incubate with the membrane at 4 °C overnight. Appropriated secondary antibodies conjugated with HRP were used and the imaging system was applied to qualify the expressions of each target proteins. In the present study, bovine serum albumin (BSA) was used as an internal control for soluble HMGB1(S-HMGB1). β-actin was used as an internal control for PD-L1.
H22 tumor-bearing animal model
Murine H22 cells (1 × 107 cells/per mouse) were subcutaneous injected into the C57BL/6 mice. When tumor volumes reached between 50 and 100 mm3, the mice were intravenously injected (i.v.) with PBS, NPDOX, NPsiPD-L1, or NPDOX/siPD-L1 at every 3 days. The nanoparticles contained DOX at a dose of 2.5 mg/kg and siPD-L1 at a dose of 2 mg/kg. Tumor volumes were recorded every 3 days. Volumes were calculated using an equation [tumor volume (mm3) = (tumor length) × (tumor width)2 × 0.5]. At the end of the experimental period, the mice were sacrificed and tumor tissues were collected. The mRNA and protein levels of PD-L1 were measured using qRT-PCR and western blot, respectively. The experimental protocol was supported by the Ethic Commitment of Hangzhou First People’s Hospital.
Histopathological analysis
At the end of the experimental period, the mice were sacrificed and the tumor tissues were collected. After the tumor tissues were fixed in 10% formalin solution, the tissues were embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed and the slides were observed under a microscope.
PCNA immunohistochemistry
To analyze the cell proliferation in the tumor tissues, PCNA immunohistochemistry was applied. Paraffin sections were incubated with mouse primary antibody against PCNA followed by incubation with secondary antibody. Avidin–biotin complex procedure was applied. Five randomly chosen sections PCNA immunostained for each group were viewed at a magnification of × 1000 using an image analyzer system.
Statistical analysis
SPSS 13.0 and GraphPad prism were used in this study. Data were shown as mean ± S.D. One- or two-way analysis of variance with multiple comparisons and Student–Newman–Keuls (SNK) test were performed. A p value that less than 0.05 was thought as a statistical significance between the two groups.