Materials
Zn (NO3)2-6H2O was purchased from Innochem (Peking, China), 2-Methylimidazole, MnCO were purchased from Aladdin’s Reagent (Shanghai, China). DOX was provided by Yuan Ye (Shanghai, China). Calcein AM, DNA damage detection kit, ROS assay kit, and crystal violet reagent were purchased from Beyotime Biotechnology (Shanghai, China). CCK-8 cell proliferation and cytotoxicity assay kit was provided by Solarbio (Peking, China). Apoptosis detection kit was purchased from Vazyme (Nanjing, China). Matrigel and transwell plates were supplied by Corning, USA. 4% paraformaldehyde was provided by Biosharp (Peking, China). FITC fluorescent dye was purchased from MedChemExpress (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (USA). Rabbit polyclonal antibody to STING (DF12090), rabbit polyclonal antibody to phospho-STING (AF7416), rabbit polyclonal antibody to phospho-IRF3 (AF2436) and rabbit polyclonal antibody to phospho-TBK1 (AF8190) were purchased from Affinit (Jiangsu, China). β-Actin mouse antibody, anti-rabbit or anti-mouse horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Abmart (Shanghai, China). Anti-CD11c antibody (E-AB-F0991C), anti-CD86 antibody (E-AB-F0994D), anti-CD80 antibody (E-CK-A107), anti-CD8 antibody (E-AB-F1104D), anti-CD4 antibody (E-AB-F1097C) were purchased from Elabscience (Shanghai, China). Anti-CD45 antibody (557659), anti-F4/80 antibody (565411), anti-CD11b antibody (552850), and anti-CD206 (568808) antibody were purchased from BD Biosciences (USA). Primers for qPCR were purchased from Tsingke (Peking, China). Primer sequences are shown in Supplementary Table S1.
Preparation of ZMD NPs
Synthesis of ZIF-8
92 mg of dimethylimidazole (2-MIM) was dissolved in 10 ml of methanol, and subjected to sonication for 2 min to achieve complete dissolution. 43.2 mg of Zn (NO3)2-6H2O was dissolved in 10 ml of methanol, and sonicated for 2 min until fully dissolved. The two solutions were mixed and stirred at room temperature at a speed of 1,000 rpm for 1 h. A white suspension was obtained after the reaction. The synthesized NPs were collected by centrifugation at 12,000 rpm for 10 min, washed three times with methanol, and freeze-dried to obtain ZIF-8 NPs (Scheme 1).
Synthesis of ZIF-8@MnCO
ZIF-8@MnCO was prepared by dissolving 10 mg of MnCO in 10 ml of methanol, 92 mg of 2-MIM in 10 ml of methanol, 43.2 mg of Zn (NO3)2-6H2O in 10 ml of methanol, and each solution is sonicated for 2 min to ensure complete dissolution. The MnCO solution was then added to the Zn (NO3)2-6H2O solution and gently mixed. Following this, the combined solution was added dropwise to the 2-MIM solution at a rate of one drop every 5 s, while continuously stirring at a speed of 1000 rpm. The reaction was allowed to proceed at room temperature for 1 h. The resulting products were collected by centrifugation (12,000 rpm,10 min), washed three times with methanol, and stored after freeze-drying.
Synthesis of ZIF-8@MnCO@DOX
The synthesis of ZIF-8@MnCO@DOX was executed using an in situ ‘one-pot’ approach. Initially, 10 mg of MnCO was dissolved in 10 ml of methanol, 92 mg of 2-MIM was dissolved in 10 ml of methanol, and 10 mg of DOX was dissolved in 10 ml of distilled water, with each solution sonicated for 2 min to ensure complete dissolution. Next, 43.2 mg of Zn (NO3)2-6H2O was dissolved in the DOX solution. The MnCO solution was then incorporated into the mixture of DOX and Zn (NO3)2-6H2O, followed by gentle shaking to ensure uniform mixing. The mixed solution was added dropwise into the 2-MIM solution utilizing a dropping funnel, at a rate of one drop every 5 s, while continuously stirring with a speed of 1,000 rpm. Once the dripping was completed, the reaction was covered with plastic wrap and stirred at room temperature for an additional hour. The product was collected via centrifugation (12,000 rpm,10 min), washed three times with distilled water and freeze-dried to prepare ZMD.
Characterization of ZIF-8@MnCO@DOX
The size and zeta potential of the NPs were measured by dynamic light scattering (DLS). The morphology of the NPs was observed by transmission electron microscopy (TEM). Elemental mapping was used to confirm the elemental composition of the NPs.
The DOX absorption peak was detected by UV spectrophotometry and the standard curve of DOX was plotted as a basis for the calculation of DOX concentration. The release of DOX from ZMD was studied by dialysis assay. A certain amount of ZMD was filled into a dialysis bag and incubated in solvents of different pH at room temperature with stirring. 1 ml of solvent was removed and replenished with fresh solvent at different time points, followed by measurement of DOX in the solvent using a fluorescence spectrophotometer. The formula for calculating the amount of DOX released is as follows:
$$\:Cumulative\:release\:amount=\frac{{M}_{t}}{{M}_{total}}\times\:100\%$$
Mt is the amount of DOX released into the solvent at the time point, Mtotal is the total amount of DOX in the dialysis bag.
To evaluate the release of manganese ions from ZMD, a quantity of ZMD was placed in phosphate buffer solution (PBS). The filtrate after ultrafiltration was collected at predetermined time points, the release of manganese ions was detected by Inductively Coupled Plasma (ICP), and the cumulative release was calculated.
Stability testing of ZMD
An amount of ZMD was dissolved in DMEM medium containing 10% FBS and PBS buffer with pH 7.4 (n = 3), respectively. The particle size change of ZMD in the sample solution was measured at intervals.
Cell culture
Human HCC cell huh7 and mouse HCC cell hepa1-6 were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin liquid. All cells were cultured in a humidified incubator at 37 °C in the presence of 5% CO2.
Cytotoxicity assay in vitro
HCC cells were seeded in 96-well plates at a density of 3000 cells per well. Each well was treated by adding different concentrations of different agents for 24 h. After the cells were attached to the wall, the old medium was aspirated and 100 µl of medium containing 10 µL of CCK-8 was added, and the plates were incubated at 37 °C for 2 h in a light- protected environment. After that, the absorbance of each well was measured at 450 nm wavelength and the half maximal inhibitory concentration (IC50) value was calculated.
Cellular uptake assay
FITC dye was incubated with ZMD to synthesize fluorescently labelled ZMD. FITC-ZMD was incubated with HCC cells for 4 h. After that, the medium was aspirated and the cells were washed three times with PBS to remove the drug that did not enter the cells. The cells were stained with 2-(4-amidinophenyl)-6-indolylamidine dihydrochloride (DAPI) and imaged using a laser confocal microscope. The experiment was repeated and cells were collected to detect FITC fluorescence intensity in the cells using a flow cytometer (CytoFlex, Beckman Coulter).
Live-dead staining assay
HCC cells in logarithmic growth phase were inoculated into six-well plates at a density of 2 × 105 cells per well and cultured for 8 h in fresh medium containing 30 ug/ml of different NPs before being subjected to 6 Gy radiation treatment. After 24 h of incubation, 1 mL of live-dead staining solution was added, and the cells were incubated in an incubator protected from light for 15 min. The cells were observed and photographed using a fluorescence inverted microscope at 490 nm and 533 nm excitation wavelengths.
Wound healing assay
Cells were seeded in 6-well plates and scraped vertically with a 200 µl pipette when the cells reached approximately 90% confluency. After washing three times with PBS, images of 0 h HCC cells were taken. Next, the cells were treated with different NPs and underwent radiotherapy, and continued to be cultured with medium containing 2% FBS for 24 h. The images were captured with the microscope.
Transwell invasion assay
Cells from different treatment groups were pre-incubated with serum-free DMEM medium for 12 h, and then inoculated into the upper chamber of transwell plates (pre-coated with 70 µL of Matrigel in the upper chamber of the transwell plates and incubated at 37℃ for 4 h), and the lower chamber was added with DMEM containing 20% of FBS. After 24 h of incubation, the infiltrating cells were fixed with formalin, and stained with crystal violet for 15 min. and then observed and counted by inverted microscope (Olympus Corporation).
Cell colony formation assay
HCC cells were inoculated in 6-well plates at a density of 5 × 103 cells/well and cultured for 12 h. Then the cells were treated with medium containing different agents (at a concentration of 30 µg/ml) for 8 h and the radiotherapy group was irradiated with 6 Gy. The cells were continued to be cultured for 1 week. Cells were fixed with paraformaldehyde and stained with crystal violet reagent. The number of infiltrating cells was observed and counted using an inverted microscope (Olympus).
Detection of intracellular ROS
HCC cells were inoculated into six-well plates at a concentration of 2 × 105 cells/well. After the cells were wall-adhered, they were treated with different NPs for 8 h. After fluid exchange, the radiotherapy group was irradiated with 6 Gy, and the culture medium was removed after 24 h of further incubation. The cells were washed three times with PBS, then treated with 5 µM DCFH-DA solution, and incubated at 37 °C for 30 min in light-avoidance incubation. The amount of ROS production was detected by flow cytometry.
γH2Ax fluorescence detection
2.0 × 105 cells per well were inoculated into six-well plates and cultured for 24 h. Then the medium was replaced with new medium containing different NPs (concentration of 30 µg/mL) for 8 h. Subsequently, the cells in the radiotherapy group were irradiated with 6 Gy and continued to be cultured for 24 h. After cell fixation, cells were incubated with γH2Ax primary antibody for 1 h at room temperature, followed by goat anti-rabbit IgG labeled primary antibody. Imaging analysis was performed under a microscope.
Western blot
Cell samples were lysed on ice in radio immunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors and phosphatase inhibitors. After centrifugation at 12,000 rpm for 20 min, the supernatant of each sample was collected and the protein concentration in the samples was determined by the bicinchoninic acid assay (BCA). All protein samples were stored at -80 or -20 °C. Proteins in each sample were separated using a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel at 80 V and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, NJ, USA) at 200 mA. The membranes were blocked with 5% skimmed milk for 1 h at room temperature and then incubated overnight at 4 °C with the corresponding primary antibody. Anti-rabbit or anti-mouse HRP-labeled secondary antibodies were incubated for 1 h at room temperature, and the bands were visualized with a chemiluminescent kit.
Apoptosis detection
Cells were collected by trypsinization (without ethylenediamine tetra acetic acid) and washed twice with PBS, then stained with Annexin V and propidium iodide (PI) at a concentration of 50 µg/mL for 15 min at room temperature in a dark room. The apoptosis level was detected by flow cytometry and analyzed by FlowJo.
Maturation and activation of bone marrow-derived dendritic cells (BMDCs)
Mouse femoral and tibial bone marrow cells were cultured with RIPM 1640 medium containing GM-CSF (1000 U/ml) and IL-4 (1000 U/ml). The medium was changed every other day and the cells were collected on day 7 for subsequent studies. Dendritic cell (DC) cells treated with different drugs were collected, washed with PBS and stained with anti-CD80, anti-CD86 and anti-CD11c antibodies. The maturation of BMDC cells was then detected by flow cytometry.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RNA was extracted from cells using Trizol reagent and RNA purification kit, and the quantity and quality of RNA was confirmed using a NanoDrop spectrophotometer. Next, we synthesized cDNA using a reverse transcription kit with gDNA remover (Vazyme). 2 × Color SYBR Green qPCR Master Mix (Vazyme) was used for RT-qPCR experiments on the light cycler 480 instrument (Roche).
RNA sequencing (RNA-seq)
Absolute quantitative transcriptome sequencing was entrusted to the Institute of Hydrobiology, Chinese Academy of Sciences. 2.0 × 105 cells per well were seeded and cultured in six-well plates for 24 h, then replaced with a new medium containing PBS or ZMD (concentration of 30 µg/mL) for 24 h. Cells were collected, mRNA was enriched from total RNA using oligo (dT) magnetic beads, and mRNA fragments were sheared into short fragments of approximately 300 bp using lysis buffer. The mRNA was used as a template to synthesize cDNA, and PCR amplification was performed after purification and modification to ensure that the effective concentration of the library was greater than 2 nM. The sequencing was performed on the Illumina platform. RNA sequencing results were analyzed using R version 4.2.0 (https://www.r-project.org/). Differential gene expression analysis was performed using the R package “DESeq2” with the thresholds of Fold Change ≥ 2 and FDR
Immunofluorescence
After the cells or tumor tissues were fixed and punched, they were incubated with the corresponding primary antibody for 1 h at room temperature, then incubated with the corresponding anti-rabbit or anti-mouse fluorescently labelled secondary antibody for 50 min at room temperature before being imaged and analyzed under the microscope.
Tumor model establishment, in vivo fluorescence imaging and anti-cancer therapy
C57BL/6 mice (male, 4–6 weeks) were purchased from Wuhan sblbio Co. Ltd. and housed in a specific pathogen-free (SPF) facility. All animal experiments were conducted under the guidelines approved by the Experimental Ethics Committee of the First Clinical College of Wuhan University (Institutional Animal Care and Use Committee issue number: WDRM 20240906 C). 1 ml of PBS solution containing 7 × 106 hepa1-6 cells was injected subcutaneously into the right lumbar rib side of C57 mice. When the tumor volume increased to 500 mm3, 100 µL of saline containing FITC-labelled NPs (1 mg/ml) was injected into the tail vein of the mice. The fluorescence signals on the tumors at different time points were measured using the IVIS imaging system while maintaining inhalation anesthesia. Twenty-four hours after drug injection, mice were euthanized, and tumor tissues and major organs were collected for fluorescence imaging to observe the distribution of the drug in the body.
After modelling as described above, tumor volume and mouse body weight were measured every other day. After three times of injections of NPs into the tail vein and three times of radiotherapy treatments (n = 5), the mice were euthanized, and orbital blood, tumor tissue, and major organs were collected for subsequent analysis.
In the lung metastasis model, mice were injected with 100 µl of saline containing 1 × 106 hepa1-6 cells via tail vein to generate lung metastases. Subsequently, the mice were randomly divided into 8 groups (n = 3) to receive drug and radiation treatment. 30 days later, the mice were euthanized to obtain lung tissue sections for immunohistochemical experiments.
Flow cytometry analysis on tumor tissues
Tumor tissues were split into small pieces and incubated with medium containing 0.8 mg/mL collagenase D, 0.2 mg/mL DNA zyme, and 0.1 mg/mL hyaluronic acid (Meilunbio, China) at 37 °C for 30 min, and then single-cell suspensions were obtained by passing through 70 μm nylon mesh. After Percoll (Meilunbio, China) was added to the cell suspension, the cells in the lower layer were collected by gradient centrifugation and closed, labeled with immunocyte markers using appropriate antibodies, and incubated for 30 min at room temperature before being analyzed on a flow cytometer. Figure S9 shows the exact process.
Blood biochemical index
Blood collected from the orbits of each treatment group (n = 3) was centrifuged at 3000 rpm for 15 min at 4 °C to collect serum, which was analyzed on an Impact 400 clinical biochemical automatic analyzer (USA, Gilford) for ALT, AST, UA, CREA, and other biochemical parameters.
H&E staining
After all mice were sacrificed, lungs, hearts, spleens, livers and kidneys were collected and stained with hematoxylin-eosin (H&E) staining, and the pathology of each tissue was observed under a microscope.
Statistical analyses
The measurement data were expressed as the mean ± standard deviation (SD). All statistical analyses were performed using Prism software (GraphPad Prism version 10.1.2; www.graphpad.com). Statistical analysis was performed by two-tailed Student’s t-test for comparison between two groups and one-way analysis of variance (ANOVA) followed by Turkey’s post-test for comparison of three or more groups. Survival rates of mice were compared using the Kaplan-Meier method. p values p p p p
