Preparation and Characterization of Magnetic Fe3O4/CdWO4 and Fe3O4/CdWO4/PrVO4 Nanoparticles and Investigation of Their Photocatalytic and Anticancer Properties on PANC1 Cells

Fe3O4/CdWO4 and Fe3O4/CdWO4/PrVO4 magnetic nanoparticles were prepared at different molar ratios of PrVO4 to previous layers (Fe3O4/CdWO4) via the co-precipitation method assisted by a sonochemical procedure, in order to investigate the photocatalytic performance of these systems and their cytotoxicity properties. The physico-chemical properties of these magnetic nanoparticles were determined via several experimental methods: X-ray diffraction, energy dispersive X-ray spectroscopy, Fourier transformation infrared spectroscopy and ultraviolet-visible diffuse reflection spectroscopy, using a vibrating sample magnetometer and a scanning electron microscope. The average sizes of these nanoparticles were found to be in the range of 60–100 nm. The photocatalytic efficiency of the prepared nanostructures was measured by methylene blue degradation under visible light (assisted by H2O2). The magnetic nanosystem with a 1:2:1 ratio of three oxide components showed the best performance by the degradation of ca. 70% after 120 min of exposure to visible light irradiation. Afterwards, this sample was used for the photodegradation of methyl orange, methyl violet, fenitrothion, and rhodamine-B pollutants. Finally, the mechanism of the photocatalytic reaction was examined by releasing •OH under UV light in a system including terephthalic acid, as well as O2−, OH, and hole scavengers. Additionally, the cytotoxicity of each synthesized sample was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against the human cell line PANC1 (cancer), and its IC50 was approximately 125 mg/L.


Preparation of Fe 3 O 4 Nanoparticles
First, 0.01 moles of FeCl 3 ·6H 2 O and 0.005 moles of FeCl 2 ·4H 2 O were dissolved in 200 mL of distilled water and transferred to a three-neck flask [49]. A mechanical stirrer was used for stirring the solution for 60 min. Then, by adding 2 M NaOH at 30 • C in the presence of gaseous N 2 , the precipitation process was carried out. The reaction system was kept at 70 • C for 5 h, and the pH of the solution was ± 12. Afterward, the system was cooled to room temperature, and the precipitation was separated via a permanent magnet and washed with distilled water until the pH was 7. Finally, the Fe 3 O 4 nanoparticles were washed with acetone and dried at 70 • C. In situ co-precipitation (assisted by the ultrasonic approach) was used for the preparation of these nanoparticles. First, 0.544 g of noncalcified as-prepared Fe 3 O 4 /CdWO 4 (sample S1) was dispersed in 70 mL of distilled water under ultrasonic irradiation for 15 min. Then, in two separate containers, 50 mL of 0.01 M NH 4 VO 3 and Pr(NO 3 ) 3 ·6H 2 O were prepared to obtain sediments with a molar ratio of 1:2:0.5 (sample S2), and the NH 4 VO 3 solution was added to the dispersed nanoparticles. The ultrasonic probe was placed in the mixture for the generation of ultrasonic waves with a frequency and power of 20 KHz and 400 W, respectively. The contents of the Pr(NO 3 ) 3 ·6H 2 O solution were added dropwise to the reaction vessel for 5 min and then exposed to ultrasound irradiation for 15 min. To fabricate sediments with various molar ratios, quantities of 0.087 g NH 4 VO 3 and 0.325 g Pr(NO 3 ) 3 ·6H 2 O (with molar ratios of 1:2:0.75, sample S3), 0.117 g NH 4 VO 3 and 0.44 g Pr(NO 3 ) 3 ·6H 2 O (1:2:1, sample S4), as well as 0.175 g NH 4 VO 3 and 0.651 g Pr(NO 3 ) 3 ·6H 2 O (1:2:1.5, sample S5) were used in the same approach. The sediments were separated with a magnet, washed with distilled water and ethanol, and then dried in the oven at 75 • C. Finally, the synthesized nanoparticles were placed at 550 • C for 3 h.
To achieve a more accurate understanding of the synthesis of nanocomposites, the proposed mechanism is expressed as follow:

Assessment of Photocatalytic Performance
To identify the optimal sample, the photocatalytic performance of all the synthesized samples was assessed by MB photodegradation under visible light. In each experiment, 60 mg of the dispersed photocatalyst was added to 300 mL of 25 ppm MB solution. Then, 1 mL of 25% H 2 O 2 was added to the photoreactor. Previous to exposure to the visible spectrum (250 W xenon lamp), the solution was stirred in darkness for 20 min to reach an adsorption/desorption equilibrium between the catalyst and the MB solution. Then, 4 mL of the solution was kept in darkness for 10 min and then for 20 min under the light. Next, the solutions were centrifuged at 5000 rpm for 5 min to separate the catalysts. A UV-Vis spectrophotometer was used to determine the outcome of the photodegradation of MB. Finally, the photocatalytic activity of the S4 sample was determined via the photodegradation of 10 mg/L of MO and 10 mg/L of MV.
The photodegradation of MB under UV light was evaluated. In each experiment, 30 mg of the photocatalyst was added to 300 mL of 20 mg/L MB solution. Before irradiation under UV light (50 W Hg lamp), the solution was stirred in the dark for 20 min to get an adsorption/desorption equilibrium between the catalyst and the MB solution. Then, 4 mL of the solution was pipetted every 10 min and centrifuged at 5000 rpm for 5 min to separate the catalyst. The concentration of MB solution was calculated via a UV-Vis spectrophotometer to identify the result of the photodegradation. Afterward, the optimized sample was used for the photodegradation of 15 mg/L MO, 15 mg/L FNT, and 20 mg/L RhB via the same method.

Photodegradation Mechanism
Hydroxyl radicals ( • OH) produced at the photocatalyst/water interface were analysed via terephthalic acid (TA) as a probe via a photoluminescence technique. A high fluorescence intensity of 2-hydroxyterephthalic acid is achieved by TA with • OH. Therefore, the intensity of fluorescence is directly proportional to the concentration of • OH. The experiments were similar to the photocatalytic testing under ultraviolet irradiation. The reaction was performed as follows: 0.03 g of photocatalyst (0.1 g/L) was added into the 300 mL aqueous solution of terephthalic acid with a concentration of 0.0005 M (0.451 g in 0.5 L distilled water) along with 0.002 M NaOH (0.04 g in 0.5 L distilled water). The principal oxidative species in the photocatalytic procedure were obtained, respectively, via the super oxide radical ( • O 2− ), • OH, and holes, using benzoquinone, tert-butanol, and citric acid. First, 300 mL of 25 mg/L MB and 3 mmol from one of the scavengers was added to the solution. Next, 0.03 g of a dispersed photocatalyst was subjected to the ultraviolet irradiation. Finally, 4 mL of each solution was centrifuged, and the process was monitored through a UV-Vis spectrophotometer.

Cell Culture
PANC1 cell lines were prepared from the National Cell Bank of Iran (NCBI, Tehran). The cell line was grown in RPMI 1640 medium (Gibco) and supplemented with 10% (v/v) FBS and penicillin/streptomycin (100 IU/mL and 100 µg/mL, respectively). The cells were incubated and preserved at 37 • C with 5% CO 2 . As soon as confluence reached ca. 85%, the cells were rinsed with pure RPMI and gathered using a 0.25% trypsin/EDTA solution. Each test was performed 3 times.

MTT Assay
An MTT assay was used to assess the cytotoxicity of the extract on the PANC1 cells. The potential of viable cells was determined via the production of a blue formazon catalyst from yellow tetrazolium salt through mitochondrial dehydrogenase. The cells were collected and plated in a 96-well plate (Nunc, Denmark) at a density of 104 cells/well and were treated with varying concentrations of nanoparticles (2, 1, 0.5, 0.25, 0.125, 0.063, 0.0315, and 0.0157 mg/mL). For 1 and 2 days, the microplates were incubated at 37 • C and 5% CO 2 . Then, the supernatants were discarded, and 100 µL of DMSO was added to each well and further incubated for 20 min. The ELISA plate reader was used at λ = 570 nm. The percentage of cell cytotoxicity and viability was achieved using the following relation [29]:

Characterization of Synthesized Nanostructures
The X-ray diffraction (XRD) patterns of the powder materials of Fe 3 O 4 , Fe 3 O 4 /CdWO 4 and the Fe 3 O 4 /CdWO 4 /PrVO 4 sample (S4) are shown in Figure S1, and the main reflections are summarized in Table S1. As seen in Figure S1a As illustrated in Figure S2, the EDX spectrum of the Fe 3 O 4 /CdWO 4 /PrVO 4 (sample S4) nanoparticles is made up of six varied elements, Fe, O, Cd, W, Pr, and V. Furthermore, no impurity peaks were found, indicating that the Fe 3 O 4 /CdWO 4 /PrVO 4 nanoparticles have a high purity.
The surface morphologies of the Fe 3 O 4 /CdWO 4 and Fe 3 O 4 /CdWO 4 /PrVO 4 nanoparticles (samples S1 and S4) were studied using scanning electron microscopy (SEM). The structures of S1 and S4 exhibit porous morphologies, with numerous channels and outcroppings (Figure 1a,b). All of the samples are composed of aggregates of the nanoparticles with sizes under 100 nm. The Fe 3 O 4 /CdWO 4 nanoparticles formed aggregates with an average size of 60-70 nm (Figure 1c). The S4 sample shows a uniform morphology with larger crystallites in the range of 90-100 nm (Figure 1d). In addition, the outcomes proved that the particle size obtained by SEM is larger than that obtained by XRD. This refers to the point where the SEM images show the aggregates of many inorganic crystallites.
The particle sizes of the synthesized nanoparticles were also estimated from the high-resolution transmission electron microscopy (HRTEM) (Figure 1e (Figure 1f).
In Figure 2, the magnetic behaviour of the nanosized structures was verified via the hysteric curve at 300 K and the nearly saturated nature. The results showed the magnetic contribution of the as-fabricated Fe 3 O 4 /CdWO 4 /PrVO 4 nanoparticles at room temperature. Furthermore, the VSM data validated that the as-fabricated products could be classified as paramagnetic nanomaterials, and their magnetization values were approximately 51 and 0.13 emu/g for the Fe 3 O 4 and Fe 3 O 4 /CdWO 4 /PrVO 4 nanoparticles at room temperature, respectively.  The band gap energies of the prepared nanoparticles were examined via UV-Vis absorption spectroscopy, as shown in Figure 3. From the UV-Vis spectra (Figure 3a), the band gap was found by extrapolating the steepest portion of the (αhν) 1/2 vs. hν plot by using Tauc's formula: Tauc's plots were made for the Fe 3 O 4 /CdWO 4 (S1) and Fe 3 O 4 /CdWO 4 /PrVO 4 (S4) samples gaps of the material (Figure 3b). Using Equation (14), the energy gaps were calculated for the S1 and S4 samples and were determined to be 3.1 and 2.8 eV, respectively.
To identify the functional groups and oxide metal bonding in Fe 3 O 4 , Fe 3 O 4 /CdWO 4 (sample S1) and Fe 3 O 4 /CdWO 4 /PrVO 4 (sample S4) before and after the calcination process, and after photocatalysis in the presence of the MB dye, Fourier transformation infrared (FT-IR) spectroscopy was used; the results are presented in Figure 4. The FT-IR spectra were recorded between the 450 cm −1 and 3500 cm −1 wavelengths at room temperature.   (Figure 4a) [51]. Some additional absorption bands at 455, 716, 819, and 893 cm −1 were observed for the CdWO 4 nanostructures, and they are attributed to the vibration modes present in these nanoparticles after the absorption of the infrared wavelength (Figure 4b). The peak at 455 cm −1 may be attributed to the Cd-O stretching vibration mode, whereas the peaks at 716 and 819 cm −1 were due to O-W-O, and the peak at 820 cm −1 was due to Cd-O-W [36,37,52,53]. The FT-IR spectra confirm the presence of stretching and bending vibrations of metal cations, such as the Cd-O, O-W-O and Cd-O-W bands in the CdWO 4 structure. Figure 4c,d shows the FT-IR spectra of the S4 sample before and after calcination, respectively. The peak at 816 cm −1 (Figure 4c), which was replaced by 827 cm −1 (Figure 4d), is related to the calcination process. The peak became more intense and may be related to the vibrational modes of the V-O bond. The small absorption peak at 451 cm −1 belongs to the Pr-O vibration frequency [54]. Figure 6e shows the FT-IR spectra of the S4 sample after the photodegradation of MB by UV light irradiation. No change in the absorption spectra of the inorganic nanoparticles was observed, which indicates that these nanoparticles are stable and were not destroyed during the photocatalytic reactions.

Photocatalytic Performance
The photocatalytic properties of the synthesized samples, assisted by 100 mL MB in 1 mL H 2 O 2 , under visible light irradiation, were tested to find the nanoparticles with the highest photocatalytic performance. The efficiency of H 2 O 2 , a non-catalytic pollutant photodegradation, was also studied with and without light to evaluate the photocatalytic properties of the synthesized nanoparticles. The results are shown in Figure 5a. Additionally, the kinetics of the photocatalytic processes in terms of the irradiation time were determined (−ln(C/C 0 )). The mathematical analysis is presented in Figure 5b. The slope of the linear regression was utilized as the first-order reaction rate constant. A comparison of all samples clearly showed ( Figure 5) that the higher photocatalytic activity was performed by S4 (Fe 3 O 4 /CdWO 4 /PrVO 4 ). S4 has a good potential to eliminate all organic contaminants.
Similar experiments on the photodegradation of MV and MO were performed with the same sample (S4), and the results are displayed in Figure 6. Some additional photocatalytic degradation tests were carried out using MB, MO, FNT, and RhB pollutants to gain a better understanding of the S4 properties under an ultraviolet wavelength, and the results are shown in Figure 7. Therefore, the reduced size of sample S4 led to an increase in the surface of the nanoparticles as well as more absorption under ultraviolet rays, improving the production of the radical species and resulting in the enhancement of the degradation of dyes.
The photocatalytic degradation of pollutants occurs via the reactive sample, after the light absorption and the electron-hole formation by the photocatalyst [55]. The terephthalic acid (TA) photoluminescence technique was used to study the generation of active • OH radicals for all samples, as summarized in Table S2 [38]. By means of • OH by TA, 2-hydroxyl-TA could be formed. This has a high fluorescence radiation, and as a result the • OH could be monitored incidentally once we monitored the changes in the fluorescence intensity of the TA solution. The change in the fluorescence intensity of 2-hydroxyl-TA is shown in Figure S3. Therefore, any increase in • OH can lead to an increase in 2-hydroxyl-TA, which has a fluorescence property. Hence, the production of • OH radicals will be improved, and as a result, the degradation of the dyes will be enhanced.    As shown in Figure S3, the UV irradiation time is directly proportional to the fluorescence intensity. However, the intensity of • OH reaches its minimum value during the first 10 min in the absence of irradiation. This indicates the formation of • OH all over the photocatalyst with ultraviolet waves. Due to the factors described in the absence of UV light, we obtained a minimal amount of • OH. Conversely, in the presence of UV light, the fluorescence was intensified; this was interpreted as a larger production of • OH, which led to an increase in the degradation of the dyes.
Trapping tests of holes (h+) were used to establish the principal oxidative samples via superoxide radical (O 2 •− ) and • OH, citric acid, benzoquinone, as well as tert-butanol [10]. As shown in the picture, citric acid can remove holes (h+) in the solution, which results in the degradation of the dyes by up to 92%. Therefore, holes (h+) have no significant effect on the solution. However, without O 2 •− or • OH, the degradation of the dyes was observed at levels of 35% and 76%, respectively. These findings The outcomes of applying these scavengers for the photodegradation of MB (25 ppm) are represented in Figure 8. The addition of a superoxide scavenger to the studied solution could lead to a reduction of the photocatalytic performance of inorganic nanoparticles by up to two thirds. Interestingly, the addition of a hydroxyl scavenger resulted in a noticeable decrease in the photocatalytic performance (less than half). However, the addition of a holes scavenger had an insubstantial influence on the photocatalytic activity of the inorganic nanoparticles.

Cytotoxicity Effect on PANC1 Cells
The MTT assay shows that the S4 nanocomposite had a toxic effect on a PANC1 cell line in a dose-depended manner, and its IC50 was approximately 125 mg/L ( Figure 9). Additionally, Figure  S4 presents the microscopic photos of PANC1 cells with S4 at the three different concentrations. The presented studies confirmed that the toxicity of the S4 sample was reduced by reducing its concentration. The in vitro studies indicated that the Fe 3 O 4 /CdWO 4 /PrVO 4 nanoparticles were able to inhibit the growth of the PANC1 cancer cells. Therefore, these inorganic nanoparticles have some potential to be developed as new and novel anticancer agents for the treatment of pancreatic cancer based on the outcome provided as primary evidence [56].

Conclusions
In summary, magnetic Fe3O4/CdWO4 and Fe3O4/CdWO4/PrVO4 nanostructures were prepared at various molar ratios of inorganic salts via the co-precipitation method assisted by the ultrasonic technique. XRD, EDS, SEM, and FTIR methods established the presence of the desired nanoparticles with different transition metals. The DRS data showed an important reduction in the band gap when adding PrVO4 to the "core" phases (Fe3O4 and CdWO4). The DRS test showed that the OBGEs of Fe3O4 and the S4 sample were 3.1 and 2.8 eV, respectively. The VSM test determined the MS values of Fe3O4 and S4, which were 50.9 and 0.13 emu/g, respectively. The highest photocatalytic activity was shown by Fe3O4/CdWO4/PrVO4, with a ratio of 1:2:1 (S4). The degradation of MB with a 70% yield under visible light was observed. This sample was also used for the photodegradation of MO, MV, FNT, and RhB under visible and UV light. The IC50 of the S4 sample on a PANC1 cell line was approximately 125 mg/L, as determined by the MTT assay.

Conclusions
In summary, magnetic Fe 3 O 4 /CdWO 4 and Fe 3 O 4 /CdWO 4 /PrVO 4 nanostructures were prepared at various molar ratios of inorganic salts via the co-precipitation method assisted by the ultrasonic technique. XRD, EDS, SEM, and FTIR methods established the presence of the desired nanoparticles with different transition metals. The DRS data showed an important reduction in the band gap when adding PrVO 4 to the "core" phases (Fe 3 O 4 and CdWO 4 ). The DRS test showed that the OBGEs of Fe 3 O 4 and the S4 sample were 3.1 and 2.8 eV, respectively. The VSM test determined the MS values of Fe 3 O 4 and S4, which were 50.9 and 0.13 emu/g, respectively. The highest photocatalytic activity was shown by Fe 3 O 4 /CdWO 4 /PrVO 4 , with a ratio of 1:2:1 (S4). The degradation of MB with a 70% yield under visible light was observed. This sample was also used for the photodegradation of MO, MV, FNT, and RhB under visible and UV light. The IC50 of the S4 sample on a PANC1 cell line was approximately 125 mg/L, as determined by the MTT assay.