Materials. Soya bean lecithin (Lip) was obtained from Sinopharm group chemical reagent Co., Ltd. 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG) and Iron (III) chloride hexahydrate (FeCl3‧6H2O) was supplied by Xiamen Sinobang Biological Technology Co., Ltd. Polyacrylic acid (PAA, 50 wt% in H2O, Mw ~ 50,000), DCFH-DA, Glu, cystine (Cys) were bought from Sigma-Aldrich. DFO, GSH and VE was purchased from Mitsubishi Chemical Corporation. Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium and fetal calf serum (FBS) were acquired from Life Technologies Co., Ltd. All solvents and reagents were obtained commercially.
Synthesis and exfoliation of FeOCl plates. FeOCl plates were obtained by a solid-phase method. The polished FeCl3‧6H2O was tiled into the bottom of 5 mL crucible and covered for reaction for 1 h in a high-temperature oven (240°C, the heating rate was 10°C per minute). After cooling to room temperature, FeOCl plates were fully ground until there was no agglomeration, and cleaned with a large amount of anhydrous acetone to remove the unreacted FeCl3‧6H2O, then dried overnight in a vacuum oven (90°C).
Exfoliation of the FeOCl plates was performed by dispersing the FeOCl plates (30 mg) in acetonitrile (30 mL). The suspensions were broken by sonication in an ice water bath for 3 h (500 W, 5 s/5 s), and in a water bath for 48 h (37°C, 480 W). The supernatant was collection after 10 min of centrifugation at 8000 rpm, and then nanosheets were gathered by centrifugation at 13000 rpm for 20 min.
Modification of nanosheets. 3 mL PAA (10 mg/mL) were added dropwise into 30 mL FeOCl nanosheets solution (1 mg/mL) under magnetic stirring. After 5 h, [email protected] nanosheets were gathered by centrifugation (13000 rpm, 20 min) and then washed for 3 times by water. [email protected], Lip and DSPE-PEG were mixed with the mass ratio of 1:1:1 and stirred 24 h. Finally, the obtained product ([email protected]) was stored in water at 4°C after centrifugation (13000 rpm, 20 min) and washing by water for 3 times.
Characterization. X-ray diffraction (XRD) analysis was carried out on a 2.2 kW X-ray diffractometer using Cu (60 kV, 55 mA) radiation. SEM and TEM images were obtained by a Hitachi S4800 and a JEOL JEM-1400, respectively. A SHIMADZU UV-1750 spectrophotometer and a Nicolet iS10 FTIR spectrometer were used for UV-vis-NIR spectra and Fourier transform infrared (FTIR) spectra, respectively. The size and zeta potential of products were measured by Malvern Zeta sizer Nano ZS (model: ZEN 3600). The concentration of iron ion was determined by -an inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500ce) Confocal fluorescence images were recorded by Olympus FV1000 laser scanning confocal microscopy. Atomic force microscope (AFM, Multimode 8) was used to determine the thickness of the nanosheets.
Release of Fe. 1 mL 50 µg/mL FeOCl before and after exfoliation was added into dialysis membrane (6000 Da), respectively, and incubated in 20 mL PBS solution (pH 7.4 or pH 5.0) under shaking at 37°C (100 rpm). At pre-determined time intervals, 1 mL dialysate was extracted for analysis by ICP, and 1mL corresponding fresh medium was replenished into remaining dialysate.
Detection of ·OH and LPO. To compare the ·OH generation ability of [email protected] and [email protected] under different pH, colorimetric method was employed based on the oxidation of TA. More precisely, 300 µL sample solution (200 µg/mL), 300 µL H2O2 aqueous solution (10 mM) and 300 µL TA (5 mM) was transferred into 2.1 mL PBS (pH = 7.4, 6.5, 5.0). The mixture was tested under 435 nm excitation light after shaking 12 h at 37°C in the dark. In addition, MB was exploited to detect the ROS production of [email protected] and [email protected] under various H2O2 concentrations. 2 mL sample solution (250 µg/mL) and 1 mL MB (30 mg/L) were respectively added into 2 mL H2O2 aqueous solution (0, 1, 5, 10, 20 mM) and then the mixture was kept at 37°C for 12 h. After centrifugation (10000 rpm, 10 min), the absorbance value of MB at 665 nm was detected.
Xylenol orange method was used to test the production of LPO. 100 µg/mL [email protected] (with/without 10 mM H2O2), 100 µg/mL [email protected] (with/without 10 mM H2O2) and xylenol orange were placed in a constant temperature shaker at 37°C for 24 h, followed by dialysis (100 Da) to remove excess H2O2 and iron ions for another 48 h with changing water frequently. 1 M H2SO4 was used to adjust the pH of sample to less than pH 6.5 after dialysis. 5 mL sample was added into 100 µL detection solution (0.3 mM FeSO4, 20 mM H2SO4 and 150 µM xylenol orange), followed by reacting in a 30°C water bath for 30 min. The supernatant was collection for further test after centrifugation.
Cell culture. 4T1 murine breast cancer cells, U87 human glioma cells and L929 mouse fibroblast cells were incubated in RMPI 1640 medium containing 10% FBS at 37°C in a humidified atmosphere containing 5% CO2.
In vitro cell toxicity test. MTT assay was used to evaluated the in vitro cytotoxicity. L929, 4T1 and U87 cells were seeded into 96-well plates (1 × 104 per well) and incubated 24 h. Then, 200 µL fresh medium (with or without H2O2) containing different concentrations of [email protected] or [email protected] nanosheets were co-incubated with cells for 24 h or 48 h. After co-incubation, the cells were washed with PBS and treated by 100 µL of 10% MTT solution in each well for 4 h. After that, the MTT solution was replaced by 200 µL DMSO for 30 min. Finally, the absorbance at 570 nm was measured by a microplate reader (TECAN, infinite M200 PRO).
For flow cytometric analysis, 1 mL cell suspension (1 × 105 cells) were cultured with [email protected], [email protected], [email protected] + H2O2 and [email protected] + H2O2 for 24 h, respectively. The cells were collected by centrifugation and washing by PBS. Diluted Annexin V-FITC and PI solutions were added successively to stain the cells, and then these cells were analyzed using a flow cytometry () (MoFlo, XDP, USA).
For calcien AM/PI staining observation, 500 µL 4T1 cells (1 × 104/mL) were plated for 12 h in culture dishes with a diameter of 6 cm, then the medium was replaced by 500 µL medium (with or without H2O2) containing different concentrations of [email protected] or [email protected] for 24 h. 5 µL PI (16 mM) and 5 µL AM (4 mM) were added into 10 mL PBS. After washing by PBS, 1 mL above calcien AM/PI solution was used to stain cells for 30 min at 37°C, followed by fluorescent microscope observation.
In vitro ROS detection. Confocal laser scanning microscopy (CLSM) was used to observe the levels of ROS in 4T1 cells. 5 × 104 4T1 cells were seeded onto a round coverslip. After attachment the cells were cultured for 12 h in a 24-well plate, followed by treating with the medium containing 300 µg/mL [email protected] or [email protected] nanosheets and 0.3 mM H2O2 for 2, 4 or 6 h. After being washed by PBS, 200 µL of 10 µM DCFH-DA was added, and the cells were further cultured for 20 min to measure the generation of ROS. Hoechest 33258 dye was utilized for nuclear staining. The fluorescent signal of DCF and Hoechest 33258 was tested by CLSM.
A MDA assay kit was utilized to measure the intracellular LPO content. 1 × 106 cells per well were seeded into 6-well plates and then incubated with 500 µg/mL [email protected] and [email protected] for 12 h respectively. Cell suspension was obtained by repeated blowing, and then supernatant was discarded after centrifugation to collect cell precipitation. 1 mL MDA extract was added to the cell precipitation, and an ultrasonic crusher was used (200 W, 3 s on/10 s off, 7 min) for crushing. The supernatant was obtained after centrifugation (8500 rpm, 4°C, 10 min), followed by adding working fluid to the supernatant according to the MDA test kit and keeping at 100°C for 1 h. The supernatant was collected by centrifugation, followed by measuring the absorbance of above supernatant at 450 nm, 532 nm and 600 nm via microplate reader. Intracellular MDA content was calculated according to the specification of the kit instructions.
For ferroptosis mechanism analysis, 4T1 cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured for 12 h. [email protected] or [email protected] was added into each well, and then immediately added media with 10 mM DFO, 1 µM Fer-1, 5 mM GSH, 2 mM Glu, 100 µM VC and 100 µM VE, respectively. The cells were then co-incubated for 24 h, followed detecting cell viability using MTT assay.
Animal model building. Female BALB/c mice (4 − 6 weeks old) were purchased from Xiamen University Laboratory Animal Center and used in accordance with the guidelines of the Chinese National Science and Technology Committee. BALB/c tumor-bearing mice models were established by subcutaneous injecting of 100 µL 4T1 tumor cells (1 × 108 cells) into the flank of mice. Further experiments were performed when the tumor volume increased to 50 mm3.
In vivo imaging. To observe the distribution of the nanosheets in vivo, fluorochrome Cy 5.5 was loaded onto [email protected] nanosheets. Two female Balb/c mice with tumor volume of about 200 mm3 were injected with 200 µL of Cy 5.5-labeled [email protected] and free Cy 5.5 into the tail vein with a dose of 20 mg/kg, respectively. The whole-body fluorescence imaging pictures were taken at 1, 3, 5, 7, 9, 11, 24, 36, and 48 h after injection by the mouse optical imaging system (parameter: Ex 630 nm, Em 645–680 nm).
In vivo therapy. Eighteen female Balb/c mice with tumor volume of about 50 mm3 were divided into three groups at random: control group (PBS injection), the [email protected] group and the [email protected] group. The tumor-bearing mice were treated with different medicament (200 µL) by injecting via the tail vein with a dose of 20 mg/kg on the 0, 1, 3, 5, 7, 9, 12th day. The tumor volume and weight of mice were continuously measured and photos were taken once a week. After treatment for 14 days, tumor of each mouse was stripped and taken pictures. At the same time, the major organs, including heart, liver, spleen, lung, and kidney, were dissected and fixed with 4% glutaraldehyde solution, dehydrated with alcohol, embedded in paraffin and stained with H&E and observed.
Determination of iron content in living animal organs. After injecting PBS, [email protected] and the [email protected] for 24 hours, 3 mice were taken from each group and each mouse was dissected to get the heart, liver, spleen, lung, kidney and tumor for weighing. Then, nitric acid was used to nitrolysis all organizations. After the tissue is completely oxidized and nitrified, digestion solution was diluted (concentration of nitric acid < 5%), followed by filtration with a 0.22 µm microporous filter membrane for ICP-MS testing. The iron content of each organ is calculated based on the wet weight and the ICP-MS data.
Hemolysis experiment. Blood was taken by extracting the eyeballs from three healthy female Balb/c mice, and centrifuged at 4°C at 2000 rpm for 5 min to separate red blood cells. 200 µL erythrocyte suspension were mixed with 800 µL of deionized water (positive control), PBS pH 7.4 (negative control) and [email protected] PBS solutions with various concentrations (25, 50, 100, 200, 300, and 500 µg/mL), respectively. After culturing at 37°C for 24 h, centrifugation was implemented (2000 rpm, 5 min, 4°C), followed by testing the absorbance with a multifunctional microplate reader at 541 nm. The hemolysis percentage is calculated as follows:
Among them, AS, AC(-) and AC(+) represent the absorbance of the experimental group containing [email protected], the negative control group containing PBS and the positive control group containing deionized water, respectively.
Analysis of blood biochemical indexes. Blood was taken from the eyeball of twelve female Balb/c mice received different treatment (PBS group, [email protected] group and [email protected] group) after therapy for one week, then centrifuged at 2000 rpm for 5 min at 4°C. The supernatant plasma was carefully aspirated and tested the content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urinary nitrogen (BUN) and creatinine (CRE) content.