Materials
Hydroxycamptothecin (HCPT), Ce6, GOX, cholesterol, 1,3-diphenylisobenzofuran (DPBF), tetramethylbenzidine (TMB), methylene blue (MB), glucose and egg yolk lecithin were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). DSPE was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd. (Chongqing, China). Iron (III) chloride hexahydrate (FeCl3⋅6H2O) and dithiodiglycolic acid were acquired from Aladdin-Reagent Co. Ltd. (Shanghai, China). β-actin, SLC7A11 and GPX4 antibody were obtained from Cell Signaling Technology (Boston, MA, USA). HIF-1α, VEGF, PKM2 and HK2 antibody were obtained from Proteintech Group Inc. (Chicago, IL, USA). P-gp antibody were brought from Santa Cruz Biotechnology (Dallas, TX, USA). Hoechst 33,342 and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Aladdin (Shanghai, China). ROS assay kit, annexin VFITC apoptosis detection kit and Calcein/PI Cell Viability/Cytotoxicity Assay Kit were obtained from Beyotime Biotechnology Co. Ltd (Shanghai, China). ATPlite was obtained from PerkinElmer Life Sciences (Boston, MA, USA). GSH kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ROS-ID®Hypoxia/Oxidative stress detection kit was obtained from Enzo Life Sciences (Farmingdale, NY, USA). C11-BODIPY was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The H2O2 and malondialdehyde (MDA) assay kit were purchased from Solarbio (Beijing, China). All other organic solvents used in this study were analytical grade.
Instruments
Transmission electron microscopy (TEM, FEI Tecnai G2 F20) and scanning electron microscopy (SEM, Hitachi Rcgulus8100) were conducted to analyze the size and morphology of Ce6@HGMOF. Zeta potential was measured on Malvern Zetasizernano. Absorbance was recorded on a UV − vis spectrophotometer (Thermo Fisher Scientific). Surface area and pore distributions were obtained on N2 adsorption with a physical adsorption apparatus (Micromeritics ASAP 2020). Fourier Transform Infrared Spectrometer (BRUKER) was used to determine the chemical structures. The dissolved oxygen analyzer (JPB-607 A) was purchased from Shanghai INESA Scientific Instrument Co., Ltd (Shanghai, China). Anaerobic culture tanks and anaerobic bags were purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). 660 nm near infrared laser was brought from Xi’an Lei Ze Electronic Technology Co. Ltd (Xi’an, China). Flow cytometry was analyzed on BD Beckman Coulter flow cytometer (Brea, CA). CLSM images were recorded on a Zeiss LSM 780 microscope.
Preparation of NPs
Synthesis of MOF. Referring to the previous method, use a peristaltic pump to slowly add FeCl3 · 6H2O solution (100 mg dissolved in 4 mL of deionized water) to the dithiodiethylene glycol solution (100 mg dissolved in 16 mL of water) [12]. Magnetic stirring for about 30 min. When the liquid turns creamy yellow, centrifuge at 8000–10,000 rpm for 10 min to remove the supernatant.
Synthesis of Ce6@HGMOF. Ce6@HGMOF was prepared by thin lipid film hydration and sequential extrusion method. Dissolve GOX in water, mix with the above synthetic MOF, and mix with the magnetic force for 24 h. Centrifuge at 10,000 rpm and wash three times for collection. Meanwhile, liposomes loaded with Ce6-HCPT were prepared. 200 mg of egg yolk lecithin, 50 mg of cholesterol, 4 mg of DSPE, 5 mg of Ce6 and 5 mg of HCPT (ethanol/dichloromethane, 1:1) were thoroughly mixed in 20 mL of dichloromethane solution, and the solvent was removed by rotary evaporation at 40 ℃. Then the membrane liposomes were hydrated with deionized water and completely dispersed by contact ultrasound treatment for 20 min. Mix drug-loaded liposome solution with drug-loaded MOF and press Ce6@HGMOF nanoparticles repeatedly. Finally, centrifuge the above nanoparticles (3000 rpm) for 10 min, remove the supernatant, and wash the deionized water 3 times to collect.
Drug loading content and encapsulation efficiency
To determine the amount of drug loaded, Ce6@HGMOF was placed in a constant temperature ice box at 4 °C for contact sonication for 2 h followed by centrifugation (13,000 rpm, 20 min). The payloads of HCPT and Ce6 were obtained using a UV-vis spectrophotometer. The loading of GOX was determined by BCA protein assay.
Drug/Fe ions release of the Ce6@HGMOF
To assess drug release from HCPT, free HCPT and Ce6@HGMOF were sealed in dialysis bags with a MWCO of 1000 Da, after which the bags were placed in different beakers. An appropriate amount of PBS with pH 5.4 was added to each beaker with or without 10 mM GSH, and all dialysis bags were shaken at 300 rpm and a portion of the supernatant buffer was collected at predetermined time intervals. Drug release was observed by measuring the HCPT content with a UV-Vis spectrophotometer. A similar method was used for the Fe ions release experiments, in which Ce6@HGMOF was sealed in dialysis bags with MWCO of 1000 Da, after which the bags were placed in different beakers. An appropriate amount of PBS at pH 7.4 or 5.4 was added to each beaker with or without 10 mM GSH, all dialysis bags were shaken at 300 rpm and a portion of the supernatant buffer was collected at predetermined intervals. Iron ions were measured by using ICP-MS.
Drug stability
For drug stability analysis, Ce6@HGMOF solutions were placed in centrifuge tubes and sealed in the dark at 4℃. At intervals, nanoparticles were extracted from the corresponding centrifuge tubes and measured for particle size and PDI. To detect the decomposition of nanoparticles, Ce6@HGMOF was added to PBS solution at pH 6.5. Ce6@HGMOF solution was irradiated (660 nm, 100 mW/cm2, 5 min) and GSH (10 mM) was added at corresponding time, and the decomposition of nanoparticles after treatment was observed by measuring particle size.
Assessment of extracellular and intracellular GSH
The GSH depletion capacity of MOF was first examined outside the cells. 10 mM of GSH solution was mixed with MOF (10 mg/mL) and stirred, and the GSH content in the solution was measured at different time points (0, 2, 4, 12 and 24 h), and the GSH solution without MOF was used as the control group. Immediately afterward, the GSH depletion capacity of MOF was assessed intracellularly. EC109 cells were seeded in 10 cm cell culture dishes and cultured for 24 h before use. After various treatments, the cells were harvested, washed twice with PBS, then contact sonication at 150 W, the 30 s and repeated 10 times, then centrifuged at 8000 rpm for 10 min. The supernatant was collected and placed in a low-temperature ice bath box at 4 ℃, then the change of intracellular GSH content was calculated according to the absorbance value of the enzyme marker according to the instruction s book of the GSH kit.
Measure of⋅OH levels in the solution
The production of ·OH was detected with MB. MB solution (64 µg/mL) was prepared with10 mM H2O2 and 5 mM of glucose mixed or unmixed into different groups, respectively. MOF was added to the resulting mixed solution and incubated, and finally the samples were centrifuged and the absorbance change of the supernatant at 664 nm was recorded by UV-Vis spectroscopy.
Catalase-like activity of MOF
The catalase-like activity of as-prepared MOF toward the oxidation of TMB was tested as follows: 10 mg/mL MOF aqueous dispersion was added into 0.1 M acetate buffer (pH 4.0), then incubated with 0.1 mL of 10 mM TMB solution for 20 min under room temperature. The resulting solution was measured by the UV-vis spectrograph at the wavelength 652 nm.
Oxygen generation
Evaluate the production of O2 in vitro by measuring the amount of dissolved oxygen. In short, add 10 mg/mL of MOF to a 10% H2O2 solution, with ddH2O as the control. Then, the production of O2 was detected using a portable dissolved oxygen meter.
Singlet oxygen detection
DPBF probe was applied to measure the 1O2 generation. DPBF solution 200 µL (10 mM in DMSO) was added to the 2 mL Ce6@HGMOF solution (0.4 mg/mL calculated by Ce6) under laser irradiation (660 nm, 100 mW/cm2, 20 min). The absorbance of DPBF at 420 nm was recorded every 2 min.
Glucose consumption studies and pH effect of GOX
Co-incubate GOX with glucose to detect changes in pH value. Compare the pH changes of GOX in the presence or absence of glucose, and compare the pH changes at different concentrations. Incubate 5 mg of GOX with 0, 5, 10, 15 and 20 mM glucose, incubate the solution at ambient temperature for 2 h, gently shake, and monitor pH changes using pH reagent paper.
Cell culture
The esophageal squamous cell carcinoma line EC109 was obtained from the American Type Culture Bank (VA, USA). These cells were cultured in DMEM (BASF, Shanghai, China) containing 10% fetal bovine serum and 1% antibiotics (penicillin-streptomycin).
ATPlite assay
For cell proliferation assay, EC109 cells (3 × 103 cells per well) were seeded overnight in complete medium in 96-well plates and then cultured with different concentrations of (1) MOF, (2) Ce6 + Laser, (3) Ce6@MOF + Laser, (4) Ce6@GMOF + Laser and (5) Ce6@HGMOF + Laser (660 nm, 100 mW/cm2, 5 min). After 48 h, the medium was removed, then ATPlite solution was added and shaken for 8 min protected from light, and the luminescence value of each well was detected using an enzyme marker to calculate the proliferation inhibition effect.
Cell apoptosis analyses by FACS
For apoptosis analysis, EC109 cells were seeded on 6 well plates with different treatments, (1) control, (2) MOF, (3) Ce6 + Laser, (4) Ce6@MOF + Laser, (5) Ce6@GMOF + Laser and (6) Ce6@HGMOF + Laser (Calculated by MOF, 50 µg/mL; Calculated by Ce6 5 µg/mL) (660 nm, 100 mW/cm2, 5 min). After 24 h of culture, cells were collected and stained with Annexin VFITC/PI Apoptosis Detection Kit. Analysis was performed using a flow cytometer.
Live/Dead staining assays
EC109 cells were treated with (1) Control, (2) MOF, (3) Ce6 + Laser, (4) Ce6@MOF + Laser, (5) Ce6@GMOF + Laser and (6) Ce6@HGMOF + Laser (Calculated by MOF, 50 µg/mL; Calculated by Ce6 5 µg/mL) (660 nm, 100 mW/cm2, 5 min) for 24 h. The cell death was also analyzed by live/dead staining assay.
Western blot
The expression of HIF-1α, VEGF, Pgp, GPX4, SLC7A11, PKM2 and HK2 in EC109 esophageal cancer cells or tumor tissues were determined by western blot. After treating the cells in different ways for 24 h, cell lysates were prepared with a lysis solution containing protease inhibitors. A similar approach was used for tumor tissues by adding 500 µL of strong lysate per 50 mg of tumor tissue at 10%. After BCA quantification, sampling and electrophoresis were performed, chemiluminescence was performed after incubation with primary and secondary antibodies, and analysis was performed by “Image J” software.
Detection of hypoxia and ROS in cancer cells
The ROS-ID Hypoxia/Oxidative Stress assay kit was purchased from Enzo Life Sciences and EC109 cells were treated differently according to grouping and cultured for 24 h. The assay kit mixture was added sequentially to the cancer cells according to the manufacturer’s instructions. After incubation for 30 min, the cells were thoroughly washed and the ROS and hypoxia signals were monitored by fluorescence microscopy.
Detection of LPO in cancer cells
EC109 cells were seeded in 12-well plates and cultured overnight. The cells were treated with (1) Control, (2) MOF, (3) Ce6@MOF + Laser, (4) Ce6@GMOF + Laser, (5) Ce6@HGMOF + Laser (Calculated by MOF, 50 µg/mL) (660 nm, 100 mW/cm2, 5 min) for 24 h. Cells were stained with C11-BODIPY (LPO probe) and fluorescence images of the cells were obtained using fluorescence microscopy.
In vitro cell uptake assay
EC109 cells were seeded in 20 mm dishes at a density of 1.5 × 103 cells per well and incubated with Ce6@HGMOF NPs (Calculated by MOF, 50 µg/mL) for 12 h. The culture medium was removed and cells were rinsed three times with PBS. The cells were stained with Hoechst 33,342 for nuclei and LysoTracker Red for lysosomes and then fixed with 5% glutaraldehyde solution and observed under confocal laser scanning microscopy.
Biodistribution
Ce6 or Ce6@HGMOF NPs (Calculated by MOF, 4 mg/kg; Calculated by Ce6, 0.4 mg/kg) were administered via tail vein to tumor-bearing mice at different time points (2, 6, 12 and 24 h) and measured by in vivo fluorescence imaging. Then, after the euthanasia of the mice, tumors and major organs, including the heart, liver, spleen, lungs and kidneys, were collected. The Biodistribution of Ce6@HGMOF NPs in vivo was assessed using the VISQUE InVivo Elite imaging system.
In vivo anticancer therapy
All the experimental procedures involving animals were performed according to the guidelines approved by Shanghai Research Center of the Southern model Institutional Animal Care and Use Committee (IACUC) (IACUC No. 2023-0015). BALB/c male nude mice (6 weeks, 15–20 g) were obtained from Beijing Viton Lever Laboratory Animal Technology Co. Ltd (Beijing, China) and were housed in an SPF environment. 50 µL of 2 × 106 EC109 cell suspension in well-cultured culture condition and in logarithmic growth was co-mixed with 50 µL of Matrigel, and inoculated in the right buttock of the mice to establish a mouse model of esophageal cancer tumor-bearing mice. Additionally, we utilized a 660 nm near-infrared light laser to treat PDT co-treated mice with 100 mW/cm2, 5 min of phototherapy. The EC109 tumor-bearing BALB/c nude mice were randomly assigned to 6 groups (n = 6 per group): (1) Control, (2) MOF, (3) Ce6 + Laser, (4) Ce6@MOF + Laser, (5) Ce6@GMOF + Laser, (6) Ce6@HGMOF + Laser (Calculated by MOF, 4 mg/kg; Calculated by Ce6, 0.4 mg/kg) (660 nm, 100 mW/cm2, 5 min). The tumor sizes and body weights were measured every other day for 18 days after treatment (tumor volume = W2 × L/2, W = width, L = length). The relative tumor volumes were calculated for each mouse as V/V0 (V0 was the tumor volume when the treatment was initiated).
MDA assays
According to the manufacturer’s instructions, 0.1 mg of tumor tissue was added to 1 mL of the extract solution, and the supernatant was centrifuged after sufficient crushing and lysis for detection, and finally the proteins of different samples were normalized to calculate the MDA content in the tissue.
Immunofluorescence staining
To confirm that the strategy we designed can effectively alleviate hypoxia at tumor sites, reduce the differentiation of tumor cell vascular endothelium and promote lipid peroxidation in tumor cells. We observed the fluorescence intensity changes of Tunel, HIF-1 α, CD31 and LPO in tumor tissues by immunofluorescence staining.
Safety evaluation
After the mice were sacrificed, serum was collected and ALT, AST, ALB, CREA, UN, UA were measured by the Medical Testing Department of Longhua Hospital, Shanghai University of Traditional Chinese Medicine to assess the health changes of the mice.
Statistical analysis
Data could be expressed as mean ± standard deviation. The student’s t-test was used for statistical comparisons. The differences were set to be significant at *p < 0.05 and very significant at **p < 0.01 and ***p < 0.001.
