Ferric trichloride, ferrous sulfate, ammonium hydroxide, n-hexane, isopropyl alcohol, tetraethyl orthosilicate (TEOS), cetyl trimethyl ammonium bromide (CTAB), anhydrous sodium carbonate, N, 'N-dimethyl formamide, butanedioic anhydride and octane were purchased from Sinopharm Chemical Reagent Corporation, Shanghai, China. Oleic acid (OA), oleylamine (OAm) and 3-aminopropyltrimethoxysilane (ATPES) were purchased from Aladdin Industrial Corporation, Shanghai, China. Silver nitrate, propidium iodide (PI) was purchased from Shanghai yuanye Bio-Technology Co., Ltd., Calcein-AM was purchased from Yeasen Biotech Co., Ltd., Shanghai, China. Methylthiazolydiphenyl-tetrazolium bromide (MTT) was purchased from Dalian Meilun Biotechnology Co., Ltd., China. All of the above reagents were used as received without further purification, and deionized water was used throughout the experiment.
Preparation of OA-modified Fe3O4 NPs.
OA-coated Fe3O4 NPs were prepared by a chemical co-precipitation method . In brief, 12 mmol FeCl3 and 6 mmol FeSO4 were dissolved in 80 mL of deionized water, and the mixture was heated to 40 ℃ in a water bath and 16 mL of ammonia water was added. After heating to 80 ℃, 5 mL of OA was added and reacted for 2 h. The product was collected by magnetic separation, then dissolved in n-octane, and the precipitate was removed by suction filtration. The supernatant was precipitated with excess absolute ethanol, and the precipitate was washed with anhydrous ethanol and deionized water alternately, and dried in vacuum.
Preparation of Fe3O4-Ag NPs.
Silver nitrate (34 mg) and oleylamine (800 μL) were dispersed in 15 mL of n-hexane, under 70 ℃ and mixing, 1 mL of OA-coated Fe3O4 dispersion (in n-hexane, 5 mg/mL) was added, and the temperature was set to 80 ℃, reacted for 2 h. After reaction, the mixture was cooled down to room temperature, excess ethanol was added for precipitation, and the precipitate was washed with ethanol and dried in vacuum.
Preparation of Fe3O4[email protected]2 NPs.
50 mg of Fe3O4-Ag particles was dispersed in 20 mL of isopropanol and sonicated for 15 min. Under 40 °C and 300 rpm stirring, 1.8 mL of deionized water and 1 mL of ammonia water were added, and after 10 min of reaction, 50 μL of TEOS was injected to the mixture. After 2 h reaction, the particles were washed with ethanol and water. Then the particles were dispersed in 10 mL water and sonicated for 15 min, then transferred to a mixed solution of 15 mL water, 15 mL ethanol and 0.275 mL ammonia water with 75 mg CTAB, and stirred at room temperature for 30 min at 300 rpm. Afterwards, 125 μL TEOS was added to the dispersion and stirred for 6 h, and the particles were washed alternately with ethanol and water. The particles were ultrasonically dispersed with 424 mg of sodium carbonate in 20 mL of water, reacted at 50 °C for 10 h, and washed alternately with deionized water and ethanol. The particles were then dispersed in 15 mL of water, transferred to a reaction vessel and reacted at 100 °C for 2 h. Last the particles were washed with deionized water and dried in vacuum.
Particle surface modification.
For amino group modification, 10 mg of the particles were dispersed in 30 mL of ethanol, 1 mL of ATPES was dispersed in 10 mL of ethanol, these dispersions were mixed and stirred at room temperature for 8 h, then the mixture was heated to 105 °C and refluxed for 2 h. For carboxyl group modification, 10 mg particles were dispersed in 50 mL N, N'-dimethylformamide containing 2 wt% succinic anhydride, and the mixture was stirred at room temperature for 24 h, and the particles were collected and washed with absolute ethanol, last dried in vacuum before use.
The overall preparation process of Fe3O4[email protected]2 NPs is presented in Scheme 1.
Drug loading and in vitro release
1 mg of nanoparticles were dispersed in 1 mL of phosphate buffer saline (PBS, with DOX, at a concentration of 0.5 mg/mL) and stirred for 8 h. After the particles were magnetically separated, the UV absorbance of the solution was measured at 480 nm to deduce the encapsulation efficiency and drug loading, and they can be calculated by using the following formulas:
Encapsulation efficiency (%) =Weight loaded DOX / Weight total DOX×100%
Drug loading (%) =Weight loaded DOX / Weight Fe3O4[email protected]2/DOX×100%
For plotting drug release profiles, 1 mg of drug-loaded particles were dispersed in 3 mL of neutral PBS (pH 7.4) or acidic PBS (pH 5.4), and the dispersions were shaken on an orbital shaker at 37 °C, 100 rpm. An aliquot of the supernatant was taken at intervals and the corresponding release medium was replenished in time. The UV absorbance of the removed supernatant was measured at a wavelength of 480 nm, and the cumulative release amount of the drug at each time point was calculated, and each sample was measured three times in parallel.
MCF-7 cells were chosen for cytotoxicity test and apoptosis study. Toxicity of blank particles was tested by MTT method, briefly, cells were first transplanted to a 96-well plate with a density of 104 cells per well, and cells in each well were incubated with 100 μL of culture medium (Dulbecco’s modified Eagle’s medium with fetal bovine serum and antibiotics) at 37 °C for 24 h. Then the medium in each well was replaced by 100 μL of fresh medium (free of fetal bovine serum) or medium in which the particles were dispersed to a concentration of 0.1, 0.5, 1, 5, 10, 50, 100 μg/mL. After a subsequent incubation for 48 h, the particle suspensions were discarded, and the wells were washed twice with PBS. 80 μL of the medium and 20 μL of MTT solution (5 mg/mL) were added into each well followed by 4 h incubation. Afterwards, the solution in each well was discarded, 100 μL of dimethyl sulfoxide was added and shaken for 10 min on an orbital shaker. Lastly, the absorbance at 490 nm of the solution in each well was measured for cell viability estimation using a microplate reader (SynergyH1, BioTec, USA). Cells cultured with particle-free media were used as references. For cell apoptosis study, DOX loaded particles (0.1, 0.5, 1, 5, 10, 50, 100 μg/mL, with a drug loading of 28.4%) were used instead of blank particles.
Calcein-Am and propidium iodide (PI) were used as dyes to indicate the living cells and the middle and late apoptotic cells respectively. The MCF-7 cell suspension was planted in a 24-well plate at a concentration of about 300 μL per well and incubated for 24 h. Then blank particles and DOX loaded particles were added into the culture medium and co-incubated for another 24 h. Afterwards, the culture medium was discarded, and the cells were washed with PBS, then 50 μL of 10 μg/mL Calcein-Am was added to each well and cultured for 15 min. Subsequently the cells were washed twice with PBS, followed by adding 50 μL of 20 μg/mL PI to each well and culturing for 10 min. Last the cells were washed twice with PBS and were placed under an inverted fluorescence microscope (Ti-U, Nikon, Japan) for observation.
NIR laser assisted cell apoptosis.
The MCF-7 cell suspension was planted in a 24-well plate at a concentration of about 300 μL per well and incubated for 24 h, and the medium was discarded, 300 μL of culture medium having blank particles or DOX loaded particles were added. The cells were irradiated with 808 nm laser at an intensity of 0.84 w/cm2 for 5 min, and then cultured for 24 h. After discarding the medium and washing with PBS, 50 μL of 10 μg/mL Calcein-Am was added to each well and cultured for 15 min, then each well was washed twice with PBS, and added with 50 μL 20 μg/mL PI followed by another culturing for 10 min. Lastly, the wells were washed twice with PBS, and the cells were observed under an inverted fluorescence microscope.