Materials
Carboxylated polyethylene glycol was purchased from AMINBIC. Ethylene glycol, FeCl3–6H2O, FeCl2–4H2O, Sodium thiosulfate pentahydrate (Na2S2O3·5H2O), and copper sulfate (CuSO4) were obtained from Merck Company (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma Company (St. Louis, MO, USA). Penicillin–streptomycin, fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI) 1640, and Trypsin–EDTA (0.25%) were provided from Gibco (Invitrogen, USA). The nitric oxide assay kit was obtained from Biocore Diagnostik Company (ZellBio GmbH, Germany). The fluorimetric hydrogen peroxide assay kit was prepared from Sigma-Aldrich Company (St. Louis, MO, USA). cDNA synthesis kit and SYBR Green qPCR Master Mix were purchased from Takara company (Takara Bio Inc., Japan). Furthermore, HT-29 cell lines were obtained from the Pasteur Institute of Iran.
Synthesis of nanoparticles
For preparation of SPIONs (Fe3O4), a solution of FeCl3 and FeCl2 was prepared in a molar ratio of 2:1 (FeCl3 = 3.2442 g and FeCl2 = 1.2675 g in 25 mL deionized water) and stirred under an N2 atmosphere at 90 °C. In the next step, an adequate amount of 28% ammonia was added to the solution to the pH of 10. After 30 min stirring, the resultant product was washed three times with deionized water to remove impurities and then dried.
0.15 g of magnetite nanoparticles were dispersed in 20 mL of pure ethylene glycol and stirred at 120 °C. Thereafter, 0.8 g of CuSO4 and 1.9 g of Na2S2O3. 5H2O were added to the solution, respectively, and refluxed for 90 min at 140 °C. Finally, the formed product was washed three times with deionized water and then dried.
Carboxylated polyethylene glycol was used to functionalize the surface of nanoparticles. For this purpose, 0.1 g of nanoparticles obtained from the previous step were dispersed in 20 mL of deionized water by ultrasound for 60 min. Subsequently, 10 mL of a solution containing 50 mg of polyethylene glycol carboxyl was added to it and sonicated for 20 min. Thereafter, the solution was stirred at room temperature for 24 h. Finally, the resultant product was washed three times with deionized water and dried. The obtained nanoparticles were stored in dried form.
Characterization of nanoparticles
The morphology of Fe3O4 and Fe3O4@Cus–PEG NPs were characterized by transmission electron microscope (TEM) (Zeiss LEO906, Jena, Germany) at 100 kV. The mean hydrodynamic diameter and size distribution of synthesized nanoparticles were determined using a dynamic light scattering (DLS) system (Nanoflex, Particle Metrix, Germany).
The X-ray diffraction (XRD) spectra of Fe3O4@Cus NPs were determined using an D8-advance (Bruker, Germany) powder diffractometer equipped with a Cu Kα radiation source (λ = 1.54187 A°) and scanned in a range from 20 to 80˚.
Biological experiments
Cell culture
The experiments were performed on human colorectal adenocarcinoma (HT-29) cell line. The cell lines were cultured in RPMI-1640 culture medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and incubated at 37 °C in 5% CO2. The experiments were carried out in the logarithmic phase of cell growth.
Analysis of cytotoxicity
3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity of Fe3O4@Cus–PEG NPs on the HT-29 cell lines. The cells at a density of 8 × 103 cells/well were seeded in a 96 well-plate and incubated in RPMI-1640 medium at 37 °C in 5% CO2 for 24 h. Then, the culture medium was replaced with one containing different concentrations of Fe3O4@Cus–PEG NPs (0, 0.5, 5, 10, 20, 25, 30, and 40 mg/mL) and incubated for an additional 24 h. After that, the medium was discarded and 100 μL of MTT solution (5 mg of MTT powder was dissolved in 1 mL of PBS) was added to each well and incubated for 4 h. The viable cells can reduce the tetrazolium salt to formazan crystals, which have a purple color. After incubation for 4 h, the MTT solution was removed and the formazan crystals were dissolved with 100 μL DMSO. The relative viability was determined by measuring the optical density (OD) of samples at 570 nm. This experiment was carried out in triplicate and repeated three times. The percentage of cell viability was calculated according to Eq. 1 and plotted as a function of nanoparticle concentrations:
$$\mathrm{Cell \, viability} \left(\%\right)=\frac{\mathrm{average \,OD\, of\, treated \,samples}}{\mathrm{average \,OD \,of\, untreated\, samples}} \times 100$$
(1)
Moreover, the inhibitory concentration values (IC50 and IC10) of nanoparticles were calculated based on the dose–response curve using the Compusyn software.
Radiation treatment and radio-sensitivity evaluation of cancer cells
To evaluating the in vitro radio-sensitization effects of the synthesized nanoparticles, HT-29 cell lines were treated in eight groups: (1) control, (2) Fe3O4@Cus–PEG NPs (5 mg/mL), (3) 2 Gy X-rays, (4) 4 Gy X-rays, (5) 6 Gy X-rays (6) NPs + 2 Gy X-rays, (7) NPs + 4 Gy X-rays, and (8) NPs + 6 Gy X-rays.
After culturing HT-29 cell lines for 24 h (at a density of 104 cells/cm2), the cells were treated with Fe3O4@Cus–PEG nanoparticles at the concentration of 5 mg/mL. After 24 h treatment, HT-26 cells were washed three times to remove traces of NPs. Subsequently, the cells were irradiated with 6-MV X-ray photons (200 cGy/min) from a medical linear accelerator (Siemens, Germany) at doses of 0, 2, 4, and 6 Gy X-rays and incubated for an additional 24 h.
To evaluate the cytotoxicity effects of various treatments and radio-sensitization effects of NPs, MTT assay, reactive oxygen species analysis, Nitric oxide (NO) assay, glutathione peroxidase (GPX) enzyme activity measurement, colony formation analysis, and quantitative real-time PCR (q-RT-PCR) assay for Bax, Bcl-2, and caspase-3 genes were performed.
Metabolic assay
The effects of various treatments on metabolic activity and cell viability were assessed by MTT assay. After treating the cells with ionizing radiation or nanoparticles, the cells were incubated for 24 h. Afterward, the culture medium was replaced with MTT solution and the assay was performed as described in section "Analysis of cytotoxicity". The activity of NADH-dependent cellular oxidoreductase enzymes reveals the number of viable cells present.
The long-term cytotoxicity of treatments
Cell survival and long-term cytotoxicity of treatments were quantified using the clonogenic assay. After treatment as described in section "Radiation treatment and radio-sensitivity evaluation of cancer cells", the HT-29 cells were re-cultured in 60 mm Petri dishes and incubated in the presence of RPMI-1640 culture medium supplemented with 10% FBS for 8 days at 37 °C and 5% CO2 humidified atmosphere.
The number of seeded cells per dish should be appropriate with the type of treatment to obtain the countable number of colonies. Therefore, the number of cultured cells for control, Fe3O4@Cus–PEG NPs, 2 Gy X-rays, 4 Gy X-rays, 6 Gy X-rays, NPs + 2 Gy X-rays, NPs + 4 Gy X-rays, and NPs + 6 Gy X-ray groups were 200, 200, 500, 1000, 2000, 1000, 2000, and 4000, respectively.
After 8 days, the cells were fixed with a 2% formaldehyde solution for 15 min and stained with Crystal Violet for 20 min. The photographic images of Petri dishes containing colonies were prepared and the number of cell colonies (a group of more than 50 cells) was counted. The plating efficiency (PE) was calculated according to Eq. 2. This was used to determine the surviving fraction (SF) for each treatment by Eq. 3:
$$\mathrm{PE}\, \left(\%\right)=\frac{\mathrm{The \, number \,of \,colonies \,countd}}{\mathrm{The \,number \,of \,cell \,seeded}}\times 100$$
(2)
$$\mathrm{SF}=\frac{\mathrm{PE\, treated}}{\mathrm{PE \,control}}$$
(3)
Moreover, survival curves were plotted as survival fractions against radiation doses (alone or combined with NPs) and fitted to the Linear Quadratic Model by OriginPro software according to the following equation:
$$\mathrm{SF}={\mathrm{exp}}^{{-\alpha D- \beta D}^{2}}$$
(4)
where SF is the cell survival fraction, D is the radiation dose (Gy), α is a single hit that induces double-strand break (DSB) of two chromosomes (linear part of the curve), and β is double hits that induce DSB of two chromosomes (quadratic part of the curve). The parameters of ɑ, β, D10, D37, D50, and SF2 were obtained from the curves. D10, D37, and D50 are doses necessary to reduce the SF of cells to 10%, 37%, and 50%, respectively. SF2 is the survival fraction of cells at 2 Gy X-rays. The sensitivity enhancement ratio (SER) is a principal factor to determine the efficacy of radiosensitizer agents. The SER of Fe3O4@Cus–PEG nanoparticles was calculated using the following equation:
$$\mathrm{SER}=\frac{{D}_{50} (\mathrm{without \,sensitizer})}{{D}_{50} (\mathrm{with \,sensitizer})}$$
(5)
Cellular ROS measurement
To determine the amount of intracellular hydrogen peroxides as reactive oxygen species that were generated by X-ray radiation or nanoparticles, the Fluorescent Hydrogen Peroxide assay Kit (Sigma-Aldrich Company) was used. Master mix solution was prepared according to the kit manual. After 24 h of cell treatments, 50 µL of the Master mix solution was added into each sample and the cells were incubated for 30 min at room temperature. Peroxidase substrate generates a red fluorescent product after reacting with the intracellular hydrogen peroxides. The fluorescence intensity was measured by a fluorescent microplate reader at 540 nm excitation and 590 nm emission. The concentration of intracellular hydrogen peroxides produced by various treatments is proportional to the fluorescence intensity.
Nitric oxide (NO) assay
Nitric oxide has a very short half-life, but its content can be calculated indirectly by measuring concentrations of nitrates and nitrites in biological fluids by the nitric oxide assay. According to the kit manual (ZellBio GmbH, Germany), the supernatants of samples were carefully collected and 300 μL of the samples were added to the related name test tubes. 10 μL R1 reagent was added to each tube and the tubes were centrifuged. Subsequently, 100 μL supernatants of the tubes and 100 μL standards were transferred into related microwells. 100 μL ready R2, 50 μL ready R3, and 50 μL ready R4 were added into all wells and incubated for 30 min at 37 °C. Nitrates and nitrites in the solutions can react with the chromogenic agent and produce a pink compound. The color intensity was measured by the microplate reader at 540 nm and is proportional to the nitric oxide concentration.
Glutathione peroxidase (GPX) enzyme activity measurement
Glutathione Peroxidase is an anti-oxidant enzyme and catalyzes the reduction of hydrogen peroxide to water by reducing glutathione. GPX has a key role to protect the cells from oxidative damage. After 24 h of cell treatments, the supernatants of samples were collected and the GPX enzyme activity was quantified using the ZellBio GmbH assay kit according to the manual (ZellBio GmbH, Germany). Finally, the yellow color intensity was measured by the microplate reader at 412 nm and indirectly related to the GPX enzyme activity.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
To investigate apoptosis as one of the mechanisms involved in the death of cancer cells, the expression of apoptotic-related genes (Bax, Bcl-2, and caspase-3) was evaluated by q-RT-PCR analysis. Following the treatments, RNA was extracted from cells using a Trisol solution (Gene all, South Korea) according to the kit manual. Total RNA was reverse transcribed into the single-strand complementary DNA (cDNA) using the Prime Script cDNA synthesis kit (Takara Bio Inc., Japan). The primer sequences were as follows:
Bcl-2.
F 5′-CTGTGGATGACTGAGTACCTG-3′
R 5′-GAGACAGCCAGGAGAAATCA-3′
Bax
F 5′-GACTCCCCCCGAGAGGTCTT-3′
R 5′-ACAGGGCCTTGAGCACCAGTT‐3′
Caspase-3
F 5′-TGTCATCTCGCTCTGGTACG-3′
R 5′-AAATGACCCCTTCATCACCA-3′
Housekeeping gene (GAPDH)
F 5′-CAAGATCATCAGCAATGCCT-3′
R 5′-GCCATCACGCCACAGTTTCC-3′.
Real-time PCR was performed on an ABI Plus one system using SYBR® Premix Ex Taq™ (Takara Bio Inc., Japan). The reaction conditions were pre-denaturation at 94 °C for 3 min; followed by 40 cycles of denaturation at 94 °C for 10 s, annealing at 59 °C for 30 s, and extension at 72 °C for 20 s. The expression of Bax, Bcl-2, and Caspase-3 genes was normalized to the housekeeping gene and quantified using the 2−ΔΔCt method.
Combined effect of nanoparticles and ionizing radiation
The combined effects of Fe3O4@Cus–PEG NPs and ionizing radiation (at different doses) were evaluated using equations established by Ito et al. (Ito et al., 2007). [NP], [IR], and [NP + IR] representative the percentage of cell viability after treatments with Fe3O4@Cus–PEG NPs, ionizing radiation, and a combination of nanoparticles with ionizing radiation, respectively. The combined effects were calculated as follows:
[NP + IR] < [NP] × [IR]/100, synergistic effect.
[NP + IR] = [NP] × [IR]/100, additive effect.
[IR] < [NP + IR], if [NP] < [IR], antagonistic effect.
Statistical analysis
All assays were carried out in triplicate and repeated three times. Data were presented as mean ± standard deviation (SD). To statistically analyze the data, one-way analysis of variance (ANOVA) analysis was performed by GraphPad Prism software (version 6). P < 0.05 was considered to be statistically significant.