Artemisinin (99%), 2-methylimidazole (98%), zinc nitrate (ZnNO3; 98%), Ta (98%), ferrous sulfate (FeSO4; 98%), and anhydrous methanol were provided by Aladdin-Reagent Co. Ltd. (Shanghai, China).
Fabrication of TA-Fe/[email protected] nanoparticles
For the preparation of Art nanoparticles, 200 mg of Art was dissolved in 1 mL of anhydrous methanol, and 2 g of 2-methylimidazole (the solvent was 8 mL of absolute methanol) was slowly added to the obtained Art solution. Under magnetic stirring, 0.2 g of zinc nitrate (the solvent was 1 mL of absolute methanol) was slowly added. Finally, the volume of the solution was adjusted to 15 mL and stirred for 10 min to obtain a light white solution. After centrifugation at 10,000 rpm, the sample was washed thrice with methanol. The supernatant from the first centrifugation was maintained to measure the content of Art, while the precipitant was freeze dried to obtain solid state for further use.
For the preparation of TA-Fe/[email protected] nanoparticles, 40 mg of solid Art nanoparticles was dissolved in water (1 mL). Ta (80 mg) (Ta solvent was 2 mL of deionized water) was slowly added to the nanoparticle solution and the volume of the solution was adjusted to 76 mL. After stirring for 20 min, 20 mg of FeSO4 (the solvent was 4 mL of deionized water) was slowly added to the solution. After repeated stirring for 30 min, a dark purple solution was obtained. The solution was centrifuged at 10,000 rpm, washed thrice with water, and the precipitant was freeze dried to obtain solid statefor further use TA-Fe/[email protected]
Transmission electron microscopy (TEM) and dynamic light scattering analysis of TA-Fe/[email protected] nanoparticles
TEM (JEM-1230; JEOL,Tokyo, Japan) was used to determine the morphological and elemental composition of each part of the nanoparticle. Dynamic light scattering and zeta potential (DLS; Zetasizer Nano system Malvern Instruments, Malvern, United Kingdom) were used to evaluate the particle size and electrical stability of the nanoparticles.
Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis of TA-Fe/[email protected] nanoparticles
Fourier transform infrared spectroscopy (VERTEX 70; Bruker, Bremen, Germany) and thermogravimetric analysis were used to analyze the composition of the constituents of the nanoparticles.
Measurement of encapsulation efficiency and loading capacity
High-performance liquid chromatography (HPLC) (Agilent 1200; Agilent Technologies, Santa Clara, CA.) was used to measure the amount of Art in the supernatant. The drug loading and encapsulation rates of ART can be calculated as follows:
In vitro release and pH-response of TA-Fe/[email protected] nanoparticles
The treated dialysis membrane wrapped with 2 mg of nanoparticles was placed in 50 mL of phosphate-buffered saline (PBS) with pH of 7.4 and 5.0, respectively, and shaken continuously at 37°C. The solution outside the dialysis membrane was sampled at 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h after the initiation of the experiment. The contents of Art in the buffer solution were measured using HPLC.
MDA‐MB‐231 and L929 cell lines were acquired from the American Type Culture Collection (American TypeCulture Collection, Manassas, VA, USA). Cells were cultured at 37°C and 5% CO2 humidity in RPMI-1640 medium (Solarbio, Beijing, China), which was supplemented with 10% fetal bovine serum (Cyclone,Utah, USA), 100 µg/mL of sodium pyruvate, penicillin, and streptomycin (Solarbio Beijing, China).
Cellular toxicity test in vitro
Cell viability was determined using 3-(4, 5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (Beyotime Biotechnology, Nanjing, China). MDA-MB-231 cells and L929 cells were cultured in standard cell media in 96-well plates (5,000 cells per well) and incubated in 5% CO2 at 37°C for 24 h. The fluid in the well was discarded, and 100 μL per well of the serum-free medium with PBS and different concentrations of ART, TA-Fe/ZIF, TA-Fe/[email protected], deferoxamine (MedChemExpress, Shanghai, China), N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK; MedChemExpress), and ferrostatin-1 (MedChemExpress) were added to the 96-well plates. After 48 h, 10 μL of MTT (5 mg/mL) was added and incubated for 4 h. Finally, an automatic enzyme marker (BioTek Instruments Inc., USA) was used to measure the absorbance in each well. Results were expressed as the percentage of cell viability.
Calcein-acetoxymethyl (Calcein-AM) staining assay
MDA-MB-231 cells were cultured in 24-well plates (2×104 cells per well) and incubated for 24 h. Subsequently, the cells were treated with different concentrations of TA-Fe/[email protected] nanoparticles for 24 h. After discarding the medium, the cells were stained with Calcein-AM (Beyotime Biotechnology, Shanghai, China) in the dark at 4°C for 20 min and observed under a fluorescence inverted microscope (Olympus, Tokyo, Japan).
Flow cytometry for apoptosis
MDA-MB-231 cells were placed in a six-well plate at a density of 2.5×105 cells per well under the same conditions. After treatment with PBS, ART, TA-Fe/ZIF, and TA-Fe/[email protected] were applied for 24 h. Subsequently, the cells were centrifuged and collected from the six-well plate. After propidium iodide and Annexin-V double staining (Annexin V‐FITC Kit; Beckman Coulter, Marseille, France), flow cytometry was used to detect.
In vitro reactive oxygen species (ROS) determination assay
The determination of ROS content in cells was performed using a ROS fluorescence probe (dichloro-dihydro-fluorescein diacetate [DCFH-DA]; Beyotime Biotechnology, Shanghai, China). MDA-MB-231cells were cultured into six-well plates (2.5×105 cells per well) and incubated in 5% CO2 at 37°C for 24 h. The fluid from the wells was discarded, and cells were treated as follows: blank control group (serum-free medium), positive control group and experimental group (different concentrations of nanoparticles). Following incubation for 8 h at 37°C, 0.1% DCFH-DA was added to each well and the cells were incubated for 30 min. Cells unresponsive to DCFH-DA were removed with PBS and observed under a fluorescence inverted microscope (Olympus, Tokyo, Japan).
Malondialdehyde (MDA) and GSH content determination
The MDA assay kit (TBA method; Jiancheng Bioengineering, Nanjing, China) and GSH assay kit (Beyotime Biotechnology, Shanghai, China) were used to measure the intracellular levels of MDA and GSH. After treatment with PBS, ART, TA-Fe/ZIF and TA-Fe/[email protected], MDA-MB-231 cells were collected and counted. The intracellular content of MDA and GSH was determined according to the instructions provided in the kits.
Western blot analysis
The MDA-MB-231 cells which treated with different nanoparticles were lysed with RIPA lysis buffer. After the protein concentration was determined, the proteins of different samples were separated using 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The nitrocellulose membrane which loaded with sample proteins was blocked by 0.5% BSA protein solution for 1 h, and the nitrocellulose membrane and the primary antibodies were incubated for 24 h at 4°C. We rinsed the primary antibodies from the nitrocellulose membrane with TBST and continued to place it with the corresponding secondary antibodies at ordinary temperature for 2 hours. After washing off the secondary antibodies on the in cellulose membrane, the nitrocellulose membrane was used a chemiluminescent solution and observed under the gel imaging system.
All experiments were repeated at least thrice. All data were statistically analyzed using the SPSS version 22.0 software (IBM Corp., Armonk, USA). The results were expressed as the mean ± standard deviation. P-values <0.05 denoted statistical significance.