Materials
Mitoxantrone hydrochloride and phytic acid were purchased from Shanghai Yi En Chemical Technology Co., LTD. (Shanghai, China). Phosphate buffer solution (PBS) was purchased from Beijing Dingguo Changsheng Biological Products Co., LTD. (Beijing, China). Rpmi-1640 medium, penicillin-streptomycin mixture, trypsin cell digestion solution and fetal bovine serum were purchased from GIBCO (USA). Anhydrous FeCl3 was from Sigma Aldrich Co., LTD. (Shanghai, China). BCA kit and RIRP lysate were purchased from Beijing Solaibao Technology Co., LTD. (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sinopharm. (Shanghai, China). MTT was purchased from Sigma (USA). Anti-phospho-smad2, Rabbit Anti-Smad2, Rabbit Anti-CRT-FITC and Rabbit Anti-GAPDH were purchased from Beijing Boosen Biotechnology Co., LTD. (Beijing, China). Goat Anti-rabbit IgG-HRP was purchased from Hunan Aijia Biotechnology Co., LTD. (Hunan, China). 4% paraformaldehyde fixation solution was purchased from Suzhou Zeke Biotechnology Co., LTD. (Jiangsu, China). InVivoMab Anti-Mouse CTLA-4 was purchased from BioXcell Co. (USA). Ultrapure water was made in the laboratory.
Cells
Mouse dendritic cells (DC cells), mouse prostate cancer cells (RM-1 cells), and human peripheral leukemia T cells (Jurkat T cells) used in this study were purchased from Shanghai Yuchi Biotechnology Co., LTD. (Shanghai, China). The specific experimental operations were carried out in a sterile ultra-clean workbench. All cells were cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin-streptomycin (50U/ml) in a 5% CO2 atmosphere at 37°C.
Animals
Healthy 6-week-old male C57B16j mice were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Changsha, China), and fostered in the SPF Animal experimental center of Hunan Provincial People's Hospital. The daily diet and water activities of mice were not restricted, and the light requirements in line with physiological activities were given. All animal experiments in this study were approved by the Laboratory Animal Ethics Committee of Hunan Provincial People's Hospital and met the requirements of the National Law on the Use of Laboratory Animals of the People's Republic of China.
Preparation And Characterization Of Mto@pa/fe Mof
At room temperature, 375 µl PA solution (2 mg/mL), 200 µl MTO aqueous solution (0.125 mg/mL) and 750 µl FeCl3 aqueous solution (0.5 mg/mL) were mixed under ultrasonic water bath for 5 min, and then centrifuged (10000 rpm, 10 min) to remove the supernatant and collect the MTO@PA/Fe3+ MOF. The nanoparticles were washed twice by ultrapure water, and resuspended in water at 4°C for further uses.
The characteristic UV-Vis absorption peak of MTO at 609 nm was used to establish the standard curve for MTO quantification. The MTO content in the supernatant after centrifugation was quantified to calculate the encapsulation efficiency. The nanoparticles were freeze-dried to obtain the total mass of nanoparticles to allow the quantification of drug loading.
Encapsulation efficiency (EE%) = (1- MTO content in supernatant /feeding MTO) × 100%
Drug loading (DL%) = (MTO mass in nanoparticles/total mass of nanoparticles) × 100%.
The particle size, dispersion coefficient (PDI) and ζ potential of the nanoparticles were measured using a Nano-ZS90 particle size analyzer. The absorption spectrum was detected by uv-2600 uv-visible spectrophotometer. The microstructure of the nanoparticles was studied by Tecnai G2 F20 field emission transmission electron microscope (TEM). The stability of nanoparticles was assessed by redissolving nanoparticles in different buffer conditions (water, PBS, pH 7.4, and 1640 medium containing 10%FBS), and the dynamic size was measured at various timepoints under a 37 ℃ water bath.
In Vitro Cytotoxicity Study
Methyl thiazolyl tetrazolium (MTT) assay was used to test the toxic effect of nanoparticles on cells. The cells were seeded at a density of 5000 cells/well in 96-well plates (100 µl/well), and the periphery of the well plate was blocked with sterile PBS and cultured for 24 h in a cell incubator. The culture substrates were discarded by centrifugation after cell adherence. Then, each formulation with various concentrations was added with 5 multiple Wells in each group. In addition, a cell-free zeroing group and a drug-free control group were set up. The cells were incubated for 24 h, followed by washing twice with PBS. Then, MTT solution (0.5 mg/mL) was added, and the cells were incubated for another 4 h in a cell incubator. After discarding the supernatant, 150 µL DMSO solution was added to each well for 10–15 min incubation at 37°C in a constant temperature shock chamber. The absorbance value (OD) of each well at 490 nm was measured, and the cell survival rate was calculated.
Induction Of Immunogenic Cell Death In Vitro
RM-1 cells were seeded in 24-well plates (4×104 cells/well) for 24 h incubation, and the culture substrates were discarded. The cells were washed twice with PBS, and free MTO or MTO@PA/Fe3+ MOF with equivalent drug concentration of 200 nM was added. A control group without drug was set up. After 4 h incubation, the culturing media and the cells were collected, respectively. The ATP release was calculated by an ATP Assay Kit. For the cell samples, 0.5 µg Anti-CRT-FITC or Anti-HMGB1-FITC antibody was added. The samples were incubated at 4 ℃ for 1 h in the dark, screened, and analyzed by flow cytometry (FCM). Enzyme-linked immunosorbent assay (ELISA) kit was used to detect the expression level of CRT and HMGB1 on RM-1.
Activation And Blockade Of Tgf-β Signaling Pathway
The expression of P-SMAD protein was detected by Western blot (WB) to detect the expression of TGF-β signaling pathway. DC cells and Jurkat T cells were seeded in 6-well plates (2×105 cells/well). Each cell was divided into 4 groups, each with one of the following treatments for 24 h: control group, TGF-β1 group, MTO group, and MTO@PA/Fe3+ MOF group. For free MTO and MTO@PA/Fe3+ MOF groups, the equivalent drug concentration was 200 nM. Except for the control group, the other three groups were incubated with TGF-β1 complete medium solution (20 ng/mL) for 30 min. At the end of cell incubation, cells were lysed using RIPA lysate. Then proteins were collected, and the protein concentration was quantified according to the BCA protein quantification method to unify the protein concentration in each group. The resulting protein samples were refrigerated in a -20°C refrigerator until use. The extracted proteins were detected by gel electrophoresis using Western blot (WB), and finally placed in the visualizer for imaging analysis.
Hemolysis Test
The blood was collected and washed with physiological PBS until the supernatant was clarified. The red blood cell suspension was prepared and stored at 4°C for later use. The blood cell suspension (200 µL) was mixed with nanoparticles (800 µL). The nanoparticles were replaced by pure water for positive control, while the nanoparticles were replaced by normal saline for negative control. After 4 h incubation at 37 ℃, the mixture was centrifuged at 1000 rpm for 5 min. The absorbance value of the supernatant at 577 nm was measured and the hemolysis rate was calculated. Hemolysis rate (%) = (Ab sample -AB negative) / (Ab positive -Ab negative) × 100%.
Pharmacokinetics Study
The healthy SD mice were intravenously injected with free MTO or MTO@PA/Fe3+ MOF (n = 4 for each group, with drug dose of 1 mg/kg). The, the blood samples were collected from the posterior orbital nerve plexus of the mice at each timepoint. The plasma was collected via centrifugation, followed by adding icy methanol to allow protein precipitation. After 90 s vortex, the sample was centrifugated at 12000 rpm for 15 min. Afterwards, the plasma drug concentration was measured by a HPLC system.
Construction Of Tumor-bearing Tumor Model
The right leg of mice was shaved and sterilized. The femoral condyle of mice was drilled with a 1 mL sterile syringe, and then 10 µL of RM-1 cell suspension (1×108 cells/mL) was absorbed with a 25 µL micro syringe for cell injection. Four days after tumor cell inoculation, all experimental mice were subcutaneously injected with 100 µl DEGARELIX (50 mg/kg) to establish a castration-resistant prostate cancer bone metastasis model. The tumor formation in the femur of mice was observed regularly. When the tumor grew, the length and width of the tumor were measured with vernier calipers, and the tumor volume was calculated.
Biodistribution Study
The tumor-bearing mice were randomly grouped, and administrated with free MTO or MTO@PA/Fe3+ MOF with equivalent drug dose of 1 mg/kg. At 12 h after treatment, the mice were sacrificed, and major organs and tumor tissues were collected, weighted and homogenized. After centrifugation, the supernatant was collected for HPLC quantification.
Anti-tumor Efficacy Evaluation
The tumor-bearing mice were randomly divided into four groups, each receiving one of the following treatments: control; αCTLA-4; MTO@PA/Fe3+ MOF; MTO@PA/Fe3+ MOF plus αCTLA-4. The drugs were applied at days 0 (when tumors were palpable on the body surface), 3, 6, and 9. The nanoparticles (1 mg/kg MTO) were injected via the tail vein, while CTLA-4 antibody was injected intraperitoneally (the first dose was 8 mg/kg, and the next three doses were 4mg/kg). Tumor volume was measured every 2 days until the completion of 4 doses, and tumor growth and appearance were recorded. On the 12th day after administration, the whole-body bone CT imaging was performed to observe the bone destruction of the mice in each group. At the same time, the mice were sacrificed to collect the tumors for for weighting and pathological analyses.
Safety Evaluation
The body weight of mice was measured every other day from the first administration. After treatments, the serum samples were collected to measure the biochemical indexes, and the major organs of the mice were subjected to H&E staining pathological examination.
Statistical analysis
All experimental data were processed by GraphPad-Prism 8.0 software, and statistical analysis was performed by two-sample t-test and multi-sample ANOVA comparison. Significance was defined as: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.