Materials

2-Methylimidazole (2-MI), tetrahydrofuran (THF), trichloroacetic acid, p-dimethylaminobenzaldehyde, L-tryptophan, and acetic acid were obtained from Rhawn Reagent Co., Ltd (Shanghai, China). Zinc nitrate hexahydrate (Zn [NO3]2·6H2O) was obtained from Beijing Bailingwei Technology Co., Ltd (Beijing, China). D-1-MT, Gem and thiazolyl blue tetrazolium bromide (MTT) were obtained from Aladdin Chemistry Co. Ltd (Shanghai, China). Chlorin e6 (Ce6) was obtained from Glpbio Technology Inc. (Montclair, California). HOS and MG63 were obtained from ATCC, K7M2 and MC3T3-E1 were obtained from ProCell (Wuhan, China). DMEM was obtained from Gibco (Gibco, USA). Fetal bovine serum albumin was obtained from Biological Industries (Beit-Haemek, Israel). Crystal violet dye and cell cycle and apoptosis detection kit were obtained from Biyuntian Biotechnology (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was obtained from Yeasen Biotech (Shanghai, China). IFN-γ was obtained from NovoProtein (Shanghai, China). FITC anti-mouse CD3 Antibody, APC anti-mouse CD4 Antibody, PE anti-mouse CD8a Antibody, FITC anti-mouse CD11c Antibody, APC anti-mouse CD80 Antibody, PE anti-mouse CD86 Antibody, PE/Cy5.5 anti-mouse CD11b Antibody, PE anti-mouse Ly6G Antibody, Alexa Fluor® 488 anti-mouse Ly6C Antibody were obtained from Biolegend (California, USA). Red blood cell lysis solution was obtained from Solarbio (Beijing, China).

Synthesis of HA/ZIF-8@Gem/D-1-MT NPs

HA/ZIF-8@Gem/D-1-MT NPs were synthesized using the one-pot synthetic method described previously [15] with minor modifications. Briefly, 40 mg of Gem and 40 mg of D-1-MT were dissolved in 4 mL THF (50%) as solution A, 100 mg of Zn (NO3)2·6H2O was dissolved in 0.4 mL double-distilled water (ddH2O) as solution B and 1 g of 2-MI was dissolved in 4 mL ddH2O as solution C. Solution B was stirred (600 rpm, 5 min) and solution A was added dropwise with stirring for 10 min. Then, the mixture was added dropwise with stirring (800 rpm) to solution C and stirred for a further 15 min. After centrifugation (13,000 rpm, 30 min) and washing with ddH2O (3 times), the ZIF-8@Gem/D-1-MT NPs product was obtained. 100 mg of ZIF-8@Gem/D-1-MT NPs was resuspended in 30 mL HA solution (3%, w/v), then the suspension was further sonicated using probe sonication for 30 min and magnetically stirred (1000 rpm) for 24 h. After centrifugation and washing with ddH2O for 3 times, the HA/ZIF-8@Gem/D-1-MT NPs product was obtained. For bioluminescent imaging, 40 mg of Ce6, 40 mg of Gem and 40 mg of D-1-MT was dissolved in 4 mL THF (50%) as solution D, which was sequentially stirred with solution B and solution C for 15 min. After centrifugation (13,000 rpm, 30 min) and washing with ddH2O for 3 times, the HA/ZIF-8@Gem/D-1-MT/Ce6 NPs product was obtained.

Characterization of HA/ZIF-8@Gem/D-1-MT NPs

The Faraday-Tyndall effect was measured using a 532 nm laser in a dark chamber. The hydrodynamic diameter was measured using a ZetaView PMX110 instrument. The zeta potential of HA/ZIF-8@Gem/D-1-MT NPs were measured with a Malvern Zetasizer Nano-ZS90. The morphology was examined using a Hitachi HT-7800 transmission electron microscope (TEM). The x-ray diffraction (XRD) spectra of HA/ZIF-8@Gem/D-1-MT NPs were obtained with a PANalytical X Pert PRO X-ray diffractometer. NPs were disrupted in HCl solution (pH 1), and the fluorescence intensity measured (Excitation wavelength = 280 nm, Emission wavelength = 344 nm) for D-1-MT and UV absorbance (Wavelength = 271 nm) for Gem to construct appropriate standard curves for detecting drug loading capacity. The release of Gem from HA/ZIF-8@Gem/D-1-MT NPs was carried out using a dialysis method. Briefly, HA/ZIF-8@Gem/D-1-MT NPs were placed into dialysis bags (MWCO = 3500 Da), each dialysis bag containing 1 mL solution. After sealing, all samples were immerged in 49 mL PBS (pH 5.0, 6.8 and 7.4) at 37 °C with rotation at 80 rpm. 1 mL of release medium was then withdrawn and replaced with 1 mL fresh medium at predetermined times. Release of Gem and D-1-MT was calculated from the related standard curves. All assays were conducted in parallel in triplicate.

Cell culture

The HOS, MG63, K7M2 and MC3T3-E1 cells were incubated in DMEM culture medium with 10% fetal bovine serum albumin and 1% streptomycin-penicillin. The cells were kept at 37 °C and 5% CO2 in a moist environment.

In vitro cell uptake

MG63 and K7M2 were seeded at 2 × 105 cells/well in 6-well plates. At 80% confluence, these cells were incubated with fresh medium containing 10 μM Ce6, ZIF-8@Gem/D-1-MT/Ce6 NPs, or HA/ZIF-8@Gem/D-1-MT/Ce6 NPs at different time points (0 h, 1 h, 2 h, 4 h, 6 h). The same volume of PBS at 0 h was used as a control. After incubation, trypsin was used to digested the adherent cells into single-cell suspensions, and cell pellets were collected by centrifugation (800 rpm, 3 min). Afterwards, cells were washed with pre-cooled PBS to remove medium, and resuspended in PBS (100 μL) for flow cytometric analysis (BD FACSCelesta, USA). 1 × 104 cells were recorded for each sample. Additionally, cells were washed three times for 5 min each after being treated with different formulations, then fixed with 4% paraformaldehyde for 15 min, and the cell images captured using an inverted fluorescence microscope (Nikon, Tokyo, Japan).

Metabolic activity

A MTT assay was used to evaluate the toxicity and anti-OS effects of various nanosystems. HOS, MG63, and K7M2 cells were chosen as tumor models to evaluate the ability of anti-OS, while MC3T3-E1 cells were selected as normal osteoblasts to study toxicity. Briefly, these cells were plated at 5 × 103 cells per well in 96-well plates. After reaching 80% confluence, cells were treated with D-1-MT or Gem, either freely in solution or in the nanoplatform. After 4 h, the supernatant was discarded, and cells were washed with PBS before the addition of fresh cell medium. After 24 h, a standard MTT assay and data analysis were carried out as reported previously [7].

Colony formation assay

MG63 and K7M2 were seeded at 2 × 105 cells/well in 6-well plates. At 80% confluence, cells were incubated with fresh medium containing 5 μM D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs. After 4 h of intervention, the supernatant was discarded, MG63 and K7M2 cells were washed, digested and plated at 600 cells/well in 12-well plates. After 7 days of culture, the adherent cells were fixed with pre-cooled 4% paraformaldehyde for 30 min and then stained with crystal violet dye for 10 min. Cell colonies were counted using ImageJ. Each set of experiments was replicated 3 times.

Apoptosis

MG63 and K7M2 cells were plated at 2 × 105 cells/well in 6-cm dishes. At 80% confluence, cells were treated with 5 μM D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs. After 4 h, the medium was renewed, and the cells were cultured for a further 24 h. The suspension and adherent cells were collected and incubated in 100 μL of binding buffer containing 5 μL of Annexin V-FITC and 10 μL PI (Yeasen) for 30 min in the dark. Fluorescence data were acquired on a BD FACSCelesta flow cytometer, and 1 × 104 cells were recorded for each sample.

Cell cycle assay

MG63 and K7M2 cells were plated at 1 × 105 cells/well in 6-well plates. To synchronize the cell cycle, the medium was replaced with serum-free medium for 24 h when 70%-80% confluent. Following serum starvation, cells were treated with complete medium containing 1 μM D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs. After 4 h of intervention, the supernatant was replaced with fresh medium after washing three times with PBS. After 24 h, the adherent cells were trypsinized into single-cell suspensions, washed with pre-cooled PBS, fixed with 70% ethanol at 4 ℃ for 2 h, and washed again with pre-cooled PBS. Cells were stained in 500 μL cycle dye solution (v: v: v; dyeing buffer: propidium iodide dye: RNase A; 100: 5: 1) at 37 °C for 30 min, shielded from light. Fluorescence data were acquired on a BD FACSCelesta flow cytometer, and 1 × 104 cells were recorded for each sample. The cell cycle was analyzed using Modfit LT V5.0.

Migration assay

MG63 and K7M2 cells were seeded at 5 × 105 cells/well in 6-well plates. To reduce cytotoxicity effects in scratch assays, we chose 0.4 μM D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs as the intervention concentration based on previous MTT results. After the cell confluence reached 90–100%, three parallel linear scratches per well were created using a 200 µL pipette tip, and the nonadherent cells at the scratch edges were washed out with PBS before the intervention started. After 4 h, the medium was replaced with low serum media (1%) and timing commenced. Scratches were photographed at 0, 24, and 48 h on an inverted fluorescence microscope (Nikon, Tokyo, Japan). The cell migration rate was calculated using the gap area method using ImageJ software.

Invasion assay

25 μL of 10% matrigel (v: v; Matrigel: DMEM; 1: 9) was evenly spread on the bottom of the upper chamber and left at 37 ℃ for setting. After serum starvation for 24 h, MG63 or K7M2 cells were treated with 0.4 μM D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs for 4 h, washed by PBS, and seeded at 2 × 105 cells/well into the upper chamber with serum-free media. The upper chamber was then put into a 24-well plate containing 600 μL of complete media. Following incubation for 24 h, the upper chamber was put into 24-well plate containing 600 μL of 4% paraformaldehyde for 30 min. After the non-invading cells were scraped off, the upper chamber was put into a 24-well plate containing 600 μL of crystal violet dye for 10 min. The stained cells were observed and photographed with an Olympus SZX16 Stereomicroscope. Cells were counted using ImageJ software.

IDO activity assay

IDO enzyme activity in cells was evaluated by measuring the concentration of Kyn [16]. To make the inhibitory effect more visible, 100 ng/mL IFN-γ and 100 μM L-tryptophan were used to enhance IDO enzyme activity [17]. Briefly, K7M2 cells were cultured in transwell chamber with conditioned medium (90% DMEM, 10% fetal bovine serum, 100 ng/mL IFN-γ, 100 μM L-tryptophan). After 80% confluence, cells were treated with D-1-MT (5 μM) or Gem (5 μM), either freely in solution or in NPs form for 72 h. After centrifugation (1000 rpm, 10 min), 1.5 mL supernatant was collected and mixed with 100 μL of 30% trichloroacetic acid at 50℃ for 30 min. Samples were again centrifuged for 1000 min at 2000 × g then 1 mL supernatant was collected and mixed with 1 mL fresh Ehrlich reagent (v/v; 4-dimethylaminobenzaldehyde: glacial acetic acid; 1:50) at room temperature for 30 min. The absorbance at 492 nm was read on a Synergy H1 plate reader (BioTek).

Cell sorting

After the mice were killed, the spleens were dissociated to make single-cell suspension. pan T-cells (CD3+), Th cells (CD3+CD4+) and CTLs (CD3+CD8+) were purified by flow sorting after incubated with CD3-FITC, CD4-APC, and CD8-PE. B cells and T cells in the single-cell suspension were removed by immunomagnetic enrichment with B220 and CD90.2 microbeads for obtaining M-MDSCs and G-MDSCs. Unabsorbed lymphocytes were incubated with CD11b-PE/Cy5.5, Ly6C-Alexa Fluor® 488 and Ly6G-PE. After that, G-MDSCs and M-MDSCs were purified by flow sorting.

Co-culture experiment

K7M2 cells were treated with 5 μM of D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs, or HA/ZIF-8@Gem/D-1-MT NPs for 4 h or 24 h. After that, the cells were cocultured with purified immune cells in medium (90% RPMI1640, 10% fetal bovine serum, 100 μM L-tryptophan, and 100 ng/mL IFN-γ) at a ratio of 1:10 for 72 h.

ELISA

The levels of TNF-α and IL-2 in coculture supernatant using the ELISA kits (Shanghai Enzyme-linked, China) following the manufacturer’s recommendations.

Orthotopic OS model

Male BALB/c mice (6 weeks old) were bought from Chengdu YaoKang Biological Technology Co., Ltd. After 1 week acclimatization, mice were anesthetized with 4% chloral hydrate (300 mg/kg). Wells were punched in the marrow cavities of the tibia using a 10 mL syringe needle and filled with 10 µL of K7M2 cell suspension (1 × 107 cells/mL). After the procedure, mice were warmed under a heating lamp until they revived.

Living imaging

To examine the targeting properties of NPs, 10 mg HA/ZIF-8@Gem/D-1-MT/Ce6 NPs were intravenously injected in mice with the equal mass of ZIF-8@Gem/D-1-MT/Ce6 NPs (12 mg) and Ce6 (1.2 mg) used as controls. Mice (n = 3 per group) were anaesthetized using 3% isoflurane for induction and 1.5% isoflurane for maintenance and photographed at 1, 2, 4, 8, 24, 72 h post-injection using the IVIS Lumina III system.

In vivo antitumor efficacy

Mice were randomly divided into six cohorts and treated at 14 d after inoculation (n = 4). Briefly, tumor-bearing mice were treated with 200 μL saline solution, D-1-MT, Gem, Gem/D-1-MT, ZIF-8@Gem/D-1-MT NPs or HA/ZIF-8@Gem/D-1-MT NPs at equivalent concentration (20 mg/kg of D-1-MT/Gem) via tail vein injection. Tumor volumes (V) were measured as calculated by the following formula: V (mm3) = length × width2 × 0.5. The tumor volumes and body weights of mice were recorded every 3 days until day 21 post the start of treatment. Tumors were then removed, weighed and photographed.

Tumor immune infiltration

The staining procedures of Th cells (CD3+CD4+), CTLs (CD3+CD8+), G-MDSCs (CD11b+Ly6CLy6G+) and M-MDSCs (CD11b+Ly6C+Ly6G) were following the manufacturer’s instructions. Stained cells were analyzed by flow cytometry using FACScan (BD Biosciences), data were analysed by FlowJo software, 1 × 104 cells were recorded for each sample.

Safety profile

For histopathological observation, the heart, lungs, liver, spleen, and kidney were removed and repeatedly washed in pre-cooled PBS to clear red blood cells. They were then fixed with 4% paraformaldehyde for 48 h, and embedded in paraffin for H&E staining. For hematological analysis, orbital venous blood was collected and left at room temperature overnight to clot. 150 μL of serum was analyzed for AST, ALT, BUN, and creatinine (Cre) levels.

Statistical analysis

Statistical analysis was performed using GraphPad prism software. All data were analyzed by one way analysis of variance (ANOVA). Asterisks (*) and/or pound sign (#) denote the statistical significance (*/#P < 0.05, **/## P < 0.01, and ***/### P < 0.001).

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