Materials and animals

Iron (III) chloride (FeCl3) and 2-methylimidazole (2-MI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc nitrate hexahydrate [Zn (NO3)2·6H2O] were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Gossypol (Gp) was obtained from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Thiazolyl blue tetrazolium bromide, Annexin V-FITC, JC-1 and propidium iodide (PI) were provided by the Beyotime Institute of Biotechnology (Shanghai, China). Cytochrome C, caspase-9, caspase-3, Bcl-2, Bax and GAPDH were all obtained from Abcam (Cambridge, UK). PKM2, LDHA, SDHA, HSP70 and HSP90 were provided by Cell Signaling Technology (Massachusetts, USA). Dithiothreitol, glycine and Tris-base were from Biosharp (Anhui, China). PMSF, TEMED, bromophenol blue and acrylamid were purchased from Amresco (WA, USA). DMEM, FBS, PBS buffer and trypsin–EDTA solution (0.25%) were bought from Gibco (Carlsbad, CA). All other chemical reagents were of analytical or HPLC grade.

HepG2 cells were provided by Cell Resource Center, Peking Union Medical College (PCRC) (Beijing, China). Male BALB/c nude mice (6–8 weeks, 19–23 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All animal experiment procedures were approved by the Institutional Animal Care and Use Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, and followed the Guide for the Care and Use of Laboratory Animals.

Preparation and characterization of ZIAMH nanocompositesSynthesis of ZIF-8@ArBu&ICG

40 mg Zn (NO3)2·6H2O, 0.77 g 2-methylimidazole, 1 mg ArBu and 1 mg ICG were dissolved in 5 mL methanol, stirring at room temperature for 5 min. After centrifugation, desired precipitate was washed with water and dried for further use.

Synthesis of ZIF-8@ArBu&ICG@MPN (ZIAM)

The material ZIF-8@ArBu &ICG, Gp and FeCl3·6H2O were mixed in defined ratio (1:0.75:0.36 w/w). After being stirred overnight at room temperature, the mixture was dialyzed in deionized water, and the dialysis melium was interplaced by fresh deionized water every 2 h. Nanoparticles were collected by centrifugation and washed three times with deionized water. ZIF-8@MPN of control group was synthesized in similar way.

Synthesis of ZIAMH

The HA solution (1.0 mg/mL) was stirred with ZIAM at a ratio of 1:50 in deionized water for 2 h, and ZIAMH was obtained by centrifugation, resuspended in PBS, and kept at 4 °C for further experiments.

Characterization of ZIAMH

The morphology of the nanoparticles was observed by transmission electron microscope (HITACHI, Tokyo, Japan). The particle size, dispersion index (PDI) and Zeta potential of the prepared ZIAMH nanoparticles were measured by a Malvern NanoZS90 (UK). The phase composition and microstructure of the nanoparticles were further analyzed using Powder X-ray diffraction (PXRD), The element distribution was analyzed by Energy Dispersive Spectrometer (EDS). The surface area of the nanoparticles was measured by Brunauer − Emmett − Teller (BET) method. The loading content (LC) and encapsulation efficacy (EE) of the ArBu and ICG in the nanoparticles was measured by UV − vis spectra, and was determined according to the following equations.

Performance investigation of ZIAMH nanomaterialsIn vitro drug release

The in vitro ArBu and ICG release from nanoparticles was determined at 37 °C in PBS containing 0.5% Tween-80 (pH 4.0, pH 5.6 or pH 6.5). The quantities of released ArBu and ICG drug were determined by UV–vis spectrophotometer, respectively. Each sample was measured for three times.

Detection of ·OH

Methylene blue (MB) was introduced to detect the hydroxyl radical (·OH) generated by ZIAMH. Using methylene blue (MB) as an indicator, the generation of ·OH was detected by UV–Vis spectrophotometer. 100 μg and 200 μg of ZIAMH was mixed with MB (10 μg/mL) and H2O2 (150 μM), respectively, to investigate the effect of sample size on the chemical kinetic catalytic ability.

The photothermal properties of ZIAMH

The temperature elevation curve and photothermal stability of ZIAMH were measured under 808 nm NIR laser irradiation in vitro. Firstly, the thermal stability of ZIAMH was established. The temperature variation curves of ZIAMH solution at different concentrations (125, 250, 500, 1000 μg/mL) were measured under the irradiation of 808 nm NIR with a power density of 1.5 W/cm2 according to the previous study [44]. FLIR Thermal CAM (E50) was used to record the real-time thermal image of the sample, and FLIR Examiner software was used to quantify and draw the temperature elevation curve. Secondly, the thermal stability of ZIAMH was investigated. 1 mL of ZIAMH solution was irradiated with 808 nm NIR laser for 600 s and cooled down to room temperature, followed by three cycles of laser irradiation. The temperature change of the solution was recorded in real time curve.

Hemolysis Assay

The hemolysis of nanoparticles was determined by co-culture with human red blood cells according to previously reported method. Red blood cells were isolated by centrifugation at 5000 rpm, then were rinsed with tris buffer and finally diluted to 5% blood cell stock solution. The concentration of the material was started from 1000 μg/mL, which was diluted using a double dilution method. The material was then incubated with an equal amount of red blood cell samples in a 37% CO2 incubator for 1 h. The PBS group was used as negative control. 1% Triton X-100 solution was defined as positive control (which induced 100% hemolysis). After centrifugation, the supernatant was collected and its absorbance was measured at 540 nm. Triplicates were made for each test, the data were recorded as mean ± standard deviation (SD, n = 3). The hemolytic percentage was estimated by the following equation:

$$}\left( \% \right) \, = \, \left( }_}} - }_}} } \right) \, / \, \left( }_}} - }_}} } \right) \, \times 00\%$$

where Am is the absorbance of cells treated by ZIAMH, An is the absorbance of the negative control, and Ap is the absorbance of the positive control.

In vitro cellular uptake

HepG2 cells were inoculated in confocal laser scanning microscope (CLSM) plates at a density of 1 × 105 cell/ well for 24 h incubation. Free ICG (0.5 μg/mL), ZIF-8@ArBu&ICG@MPN (ZIAM, 0.625 mg/mL) and ZIAMH (0.625 mg/mL) were added to the cells before incubation for 0, 0.5, 1, 2 or 4 h. HepG2 cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Pre-cooled TritonX-100 was added to destroy the cell membrane. After washing with PBS, HepG2 cells were stained with DAPI for 10 min. The fluorescence intensity was imaged and analyzed by laser confocal microscopy at an excitation wavelength of 750 ± 10 nm and an emission wavelength of 810 ± 20 nm.

Cytotoxicity evaluation

HepG2 were seeded in 96-well plates with 5 × 103 cell/ well for 24 h, and then different gradient concentrations of ZIAMH were added to investigate the cytotoxicity. Next, ZIF-8@MPN@HA (ZMH), ArBu, ArBu&ICG + NIR and ZIAMH + NIR were added and incubated for 24 h. The cell viability value was detected using MTT colorimetry and calculated as follows:

$$}\left( \% \right) \, = }_})}} /}_})}} \times 00\%$$

Flow cytometry analysis of apoptosis

The cell apoptosis mechanism was explored by flow cytometry using Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). HepG2 cells were inoculated into 6-well plate for 24 h, follow by 24 h incubation with PBS、ZMH (0.625 mg/mL), ArBu (90 μM), ArBu (90 μM)&ICG (50 μg) and ZIAMH (0.625 mg/mL), during which ArBu&ICG group and ZIAMH group were irradiated by 1.5 w/ 5 min/ well of 808 nm laser post 4-h incubation. Collected cell pellets were washed with cool PBS and resuspended up to 1 × 106 cells/mL with binding buffer. 100 μL of the cell suspensions and 5 μL of AnnexinV-FITC were then gently mixed in falcon tube, followed by 30 min dark incubation at room temperature, with 5 μL of PI added in the middle of incubation. The suspension was then replenished with 400 μL of binding buffer and analyzed immediately.

Live/Dead assay

HepG2 cells were planted in 6-well plates at a density of 3 × 105 cells/ well, and then treated with PBS, ZMH (0.625 mg/mL), ArBu (90 μM), ArBu (90 μM)&ICG (5 0 μg), ZIAMH (0.625 mg/mL) for 24 h, during which ArBu&ICG group and ZIAMH group were irradiated with 1.5 W/ well 808 nm laser for 5 min after 4 h of treatment. The harvested cells were stained with 10 nM of calcein AM and PI dye for 10 min at 37℃. Cells were washed with PBS before analysis by confocal microscopy.

Measurement of mitochondrial membrane potential

HepG2 cells were treated with PBS, ZMH (0.625 mg/mL), ArBu (90 μM), ArBu&ICG + NIR (90 μM ArBu & 50 μg ICG), ZIAMH + NIR (0.625 mg/mL) for 24 h, during which ArBu&ICG group and ZIAMH group were irradiated with 1.5 W/ well 808 nm laser for 5 min after 4 h of treatment. The harvested cells were then incubated with 1 mL JC-1 working solution for 20 min. After washing, the cells were resuspended with 500 μL of JC-1 working solution for analysis by BD FACSCalibur flow cytometer.

Western blot analysis

Total protein was extracted from HepG2 cells or nude mouse tumors using RIPA lysis buffer (Beyotime, China) with 1% protease inhibitor PMSF (Amresco, China). The protein content was determined using a BCA protein assay kit (Beyotime, China). 30 μg of total protein were taken for SDS-PAGE electrophoresis, and wet transferred to polyvinylidene fluoride (PVDF) membrane (Merck, Germany), based on the protein quantification results. After blocking with 5% skimmed milk/TBST solution at room temperature for 1 h, the PVDF membrane was incubated with the indicated primary antibodies: caspase-9 (1:500), caspase-3 (1:1000), cytochrome C (1:1000), Bcl-2 (1:1000), Bax (1:1000), PKM2 (1:500), LDHA (1:1000), SDHA (1:500), HSP70 (1:1000) and HSP90 (1:1000), and was shaken overnight at 4 °C. After washing with TBST, the PVDF membrane was incubated with the corresponding secondary antibody (1:10,000) for 45 min at room temperature. The protein band was acquired by Tanon 5200 Chemiluminescence Imaging System (Shanghai, China) and GAPDH was used as internal control.

Seahorse cell energy metabolic analysis

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HepG2 cells were detected by Seahorse XF-96 system (Agilent, Santa Clara, USA) according to the instructions of Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit. The HepG2 cells were seeded into Seahorse XF cell culture plates and were treated with PBS, ZMH, ArBu, ArBu&ICG + NIR and ZIAMH. The two indicators OCR and ECAR were measured to evaluate the energy metabolism status and mitochondrial function of cells, and the experimental data was analyzed using wave software and report generator.

Cellular ROS analysis

Cellular reactive oxygen species (ROS) levels were determined using a 2,7-dichloro-fluorescein diacetate (DCFH-DA) assay kit (Beyotime, #S0033M) following the manufacturer's protocol. HepG2 cells were seeded into confocal small dish cultured for 24 h at 37 °C and incubated with ZMH, ArBu, ArBu&ICG + NIR and ZIAMH + NIR for 4 h, respectively. Subsequently, the cells were washed with PBS three times and incubated with 10 μM DCFH-DA for 30 min. Then, the cells were observed by CLSM.

In vivo NIR fluorescence imaging of ZIAMH

For NIR fluorescence imaging and biodistribution analysis, HepG2 tumor-bearing mice were intravenously injected with ICG or ZIAMH. The tumor-bearing mice were imaged using an animal imaging system (Xtreme, Bruke) at 0.5, 4, 8, 24, and 48 h post-injection. Mice were sacrificed 48 h after administration, and tumor tissues and main organs (including heart, liver, spleen, lung and kidney) were isolated for fluorescence imaging and semi-quantitative analysis. The excitation wavelength of ICG was 750 ± 10 nm and the emission wavelength was 810 ± 20 nm.

In vivo photothermal imaging of ZIAMH

To evaluate the in vivo photothermal effect of ICG-loaded ZIAMH nanoparticles, PBS and ZIAMH were intravenously injected to tumor-bearing mice, the thermal change of tumor irradiated at 808 nm NIR laser was monitored. Moreover, 24 h after intravenous injection was selected as the irradiation time and ICG accumulation reached its peak at this time. The mice were anesthetized with isoflurane, at predetermined time, and then 808 nm NIR laser (1.5 W/cm2) was injected to the tumor site. The mice were thermally imaged with an infrared thermal imager (FLIR E50) every 0.5 min for a total of 5 min. Quantitative analysis on imaging results and thermal variation data was performed by FLIR tools.

In vivo tumor inhibition evaluation

To establish a xenograft tumor nude mouse model, 6 × 106 HepG2 cells/well were subcutaneously injected into male BALB/c nude mice based on a previously described study with some minor changes [16]. After the tumor volume reached approximately 100 mm3, HepG2 tumor-bearing mice were randomly divided into five groups (n = 3): PBS group, ZMH group, ArBu group, ArBu + ICG + NIR group, PD-L1 group, ZIAMH + NIR group and ZIAMH + PD-L1 + NIR group, afterward the mice were intravenously injected with the above drug formulations. For photothermal therapy, 24 h after injection, the tumor site was irradiated with 808 nm NIR laser (1.5 W/cm2) for 5 min. The tumor volume and mouse body weight (bw) were measured every two days for a total of 10 days, and the tumor volume was calculated according to the following formula: V (mm3) = length × (width)2/2. The tumor inhibition ratio percentage = (bw control group -bw treatment group)/ bw control group × 100%. The tumor-bearing mice were sacrificed 21 days after intravenous administration, and the heart, liver, lung, spleen and kidney of the mice were obtained for hematoxylin and eosin (H&E) staining to evaluate the morphology of cancer cells.

Histochemical staining

For hematoxylin and eosin (H&E) staining, tumor tissues and main organs (heart, liver, spleen, lung and kidney) of HepG2 tumor-bearing mice were fixed in 4% formaldehyde solution and then placed in different concentrations of ethanol for dehydration. Subsequently, the tissues were cleared with xylene, immersed and embedded in molten paraffin. The wax blocks were cut into sections and dewaxed with xylene, then rehydrated and rinsed with water, and stained with hematoxylin and eosin. The coverslips were covered with neutral balsam mounting medium for sealing. The tumor and major tissue sections were observed and photographed with a microscope. For CD4 immunohistochemical staining, the tissue fixation and section steps are similar to H&E staining, expected that a CD4 monoclonal antibody was added to specifically identify immune cell subpopulations that express CD4 molecules.

RNA-sequencing analysis

The tumor tissues were ground into powder using liquid nitrogen and total RNA was extracted by Trizol (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The transcriptome library was constructed using a TruSeq Stranded mRNA LTSample Prep Kit (Illumina, USA), and RNA-sequencing analysis was conducted by Shanghai OE Biotech Co., Ltd (Shanghai, China).

Fastq software was used to perform quality control analysis on the preprocessed data. The clean data were mapped to Homo sapiens (GRCh38.p13) by HISAT2. The Fragments Per Kilobase Million (FPKM) value of each gene was calculated by Cufflinks, and the sequence counts for per gene were acquired by HTSeq-count. Differentially expressed genes (DEGs) analysis was carried out using an R package, DESeq2. Data with P < 0.05 and log2 |(fold change)|> 1 was considered as significant DEGs. Principal Component Analysis (PCA), heatmap analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using Omicshare cloud tools (https://www.omicshare.com/tools/).

Targeted metabolomics of glucose metabolic pathways

The tumor tissues in each group were homogenized with threefold volume of saline solution. 100 μL of the homogenate were added with 0.2% formic acid-acetonitrile, then were vortexed for 60 s and sonicated for 10 min in an ice-cold sonication bath. The mixture was centrifuge at 12,000 r/min for 15 min at 4 °C. The supernatant was taken for UPLC-MS/MS targeted metabolomic analysis.

The UPLC-6500 + triple quadrupole MS/MS system (SCIEX, CA, USA) was applied for quantitative analysis of TCA cycle metabolites in the above tumor tissues. A Waters UPLC® HSS PFP column (2.1 mm × 100 mm, 1.8 μm) was used for sample separation at 35 ℃. The mobile phase consisted of (A) water containing 0.05% formic acid and (B) acetonitrile containing 0.05% formic acid with a flow rate of 0.3 mL/min. The following gradient program of chromatographic condition was performed: 0–4 min, 2% B; 4–6 min, 2%–98% B; 6–10 min, 98% B; 10–10.1 min, 98%–2% B; 10.1–14 min, 2% B. The injection volume was 3 µL. The MS spectrum conditions were optimized as follows: ESI source negative ion mode; multiple reaction monitoring (MRM) detection mode; spray voltage, ± 4500 V; atomization temperature, 550 ℃; curtain gas, 35 psi; collision air pressure, 9 psi; atomization gas pressure (N2) and gas pressure (N2) were both 55 psi. The parameters of metabolites including Q1 Mass, Q3 Mass, collision energy (CE) and decluttering potential (DP) were listed in Table S1.

Statistical analysis

All data were analyzed by GraphPad Prism software (Version 9.0, SanDiego, USA) and expressed as means ± standard deviation (SD). One-way ANOVA and t-test statistical analysis were used for all experimental data. The significant differences were defined as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.