Syntheses, characterization, and suppression efficiency of silver & silver iodide nanoparticle for proliferation, migration, and invasion in follicular thyroid carcinoma cells

In this study, a chemical co-precipitation method has been employed, silver iodide (AgI NPs) and silver nanoparticles (AgNPs) have been synthesized. UV–vis, FTIR, x-ray diffraction, FESEM, TEM, and other techniques have been used to examine the optical and structural properties of AgNPs and AgI NPs. The UV–vis absorption spectra gave the highest peak at 400 nm for AgNPs and AgI NPs at 434 nm. The x-ray data showed that the prepared AgNPs and AgI NPs were nanocrystalline cubic structures with crystallite sizes of 18 nm and 51 nm, respectively. The FESEM results show that synthesized AgNPs and AgI NPs agglomerate and aggregate. TEM data revealed that AgNPs have a quasi-spherical shape and Gaussian size distribution type. TEM analysis of AgI NPs with different magnifications revealed primarily spherical and well dispersed AgI NPs. TEM histogram shows that the particles were highly monodispersed AgNPs and AgI NPs with an average diameter of 11.5, 24.28 nm, respectively. According to the MTT assay results of FTC133cells, the cytotoxic action IC50 of AgNPs was (52.74 μg ml−1) and for AgI nanoparticles was (95.22 μg ml−1). It has been found that FTC133 cellular uptake was concentration, size- and time-dependent for both AgNPs and AgI NPs. The migrated FTC133 cell rates were reduced following AgNPs treatment to 75.7% and for AgI NPs treatment to 60% compared with the control group. Furthermore, Invasive FTC133 cell rates were reduced by 60% in the AgNPs treatment group and by 55.71 percent in the AgI NPs treatment group compared to the control group.


Introduction
Thyroid cancer is a common type of cancer in the head and neck area. Most thyroid cancer cases are differentiated thyroid carcinoma, including papillary thyroid carcinoma and follicular thyroid carcinoma [1]. Follicular thyroid cancer (FTC) is the second furthermost common differentiated thyroid cancer and credits for about 10%-15% of all thyroid cancers. The relative rate of FTC is higher in iodine-deficient areas, accounting for up to 40% of all cases of thyroid cancer disease [2]. The initial treatment of thyroid cancer was surgical and depended, for the most part, on the extent of the local condition. The nanomaterials have wide biomedical applications such as photothermal therapy [3], photodynamic therapy [4], a drug targeted delivery [5], genetic therapy [6], immunotherapy [7], etc.
Due to their availability, material characteristics, and capacity to improve drug specificity against cancer cells, metallic nanoparticles have been established as diagnosing markers or drug delivery systems in cancer therapy [8]. In addition, they can easily infiltrate the cellular environment due to their small size. Furthermore, NPs can target specific cancer cells both actively and passively [9]. Silver nanoparticles (AgNPs) are among the most promising metal nanoparticles due to their exceptional antibacterial and confined surface plasmon resonance capabilities. These qualities include broad-spectrum antimicrobials, ground Raman spectroscopy, chemical/biological sensors and biomedical materials, biomarkers, and so on [10]. Moreover, the toxicity of with 0.1 M (0.425 gm AgNo3 in 25 distilled water) in the presence of 0.2 g sodium dodecyl sulfate NaC1 2 H 25 SO 4 (Didactic Co. Barcelona Spain) as a surfactant with vigorous stirring. Then the solution turned yellowish-white, then the precipitate was separated by centrifugation at (6500 r.p.m) and washed with double-distilled water and ethanol to remove impurities several times, and then dried.

Characterization of synthesized NPs 2.2.1. Optical properties
The AgNPs and AgI NPs absorption spectra were calculated and examined in solution samples using a UV-vis spectrophotometer (Double Beam Li-2800) and (T60, LONDON) equipped with a Deuterium and Tungsten lamp. The used wavelength of (200-900) nm in the Materials Laboratory for Postgraduate Studies at the Faculty of Science/Mustansiriyah University.

Fourier transforms infrared spectrometer (FT-IR)
The absorption of electromagnetic energy in the frequency range generates Fourier transform infrared spectra (400 to 4000 cm −1 ). In the molecule, different functional groups and structural characteristics are absorbed in different frequencies. As a result, the frequency and intensity of absorption reveal the molecule's band structures and structural geometry, as measured by Thermo Fisher Scientific Corporation's FT-IR at Daypetronic Co. in Tehran, Iran. Additionally, the infrared spectra of the emission and absorption were obtained for the synthesized AgNPs and AgI NPs powder samples.

X-ray diffraction (XRD)
The orientations of Ag and AgI (NPs) grown samples have been investigated by XRD measurements in Daypetronic Co. Tehran-Iran. The XRD measurements were performed at room temperature using a '(Instrument: Panalytical X 'Pert Pro, USA)', which was outfitted with a Cu tube for producing monochromatic Cu kα x-ray radiation. The incident beam was in the 2θ mode over the range of (5.0131°−79.9711°), with a step size (2θ = 0.0260) and operated at a voltage of 40 kV and a filament current of 40 mA. The phase identification for all the samples reported in this work has been performed by matching the peak positions and intensities in XRD patterns to those patterns in the JCPDS (Joint Committee on Powder Diffraction Standards) database. The crystallite size was estimated using the Debye-Scherrer's formula (D=Kλ/β cosθ) [24] where D is the crystalline size; λ is the wavelength of x-ray (λ=0.154056 nm for (CuKα); β is the full width at half maximum (FWHM) of the Braggs peak (in radians); θ is the diffraction angle of the reflection. The dislocation density (δ) of the synthesized NPs was estimated from equations (δ=1/D 2 ) and the micro-strain (ε) from (ε=β cos/4) [25].

Field emission scanning electron microscope (FESEM)
The surface morphological characterization and elemental analysis were carried out using Field Emission Scanning Electron Microscopy (FE-SEM) integrated with Energy Dispersive x-ray (EDX) analyzer. The synthesized AgNPs and AgI NPs have been examined by '(EBSD Instrument: ZEISS SIGMA VP, Germany)' in Daypetronic Co. Tehran-Iran. Additionally, EDX analysis was used to estimate the elemental structure, purity, and the percentage of each metal in the structured powder of synthesized AgNPs and AgI NPs.

Transmission electron microscope (TEM)
The size and shape of AgNPs and AgI NPs were determined by transmission electron microscopy (TEM) type (TEM Instrument: ZEISS LEO 912 AB-, Germany) by accelerating the voltage (100 kV) in Daypetronic Co./ Tehran -IRAN. The measurement was done on particle diameter and the histogram of the size distribution. Thus, it is possible to calculate sample dispersion information.

Biomedical tests 2.3.1. FTC-133 cell line
The Pharmacology Centre for Natural Product Research and Drug Discovery, University of Malaya, Malaysia, provided the follicular thyroid carcinoma (FTC-133) (Thyroid: lymph node metastasis, Human). Cell Line Description: FTC-133 was obtained from a lymph node metastasis of follicular thyroid carcinoma in a 42-yearold male. FTC-133 got from a lymph node metastasis of follicular thyroid carcinoma, not the primary tumor. In this paper, we investigated FTC-133 cultured for 7 and 14 days. All cell lines were sophisticated as monolayers in a humidified atmosphere (5% CO 2 ) at 37°C. During experiments, the cell lines were cultured in a serum-free DMEM/HAM-F12 medium. In the medium, cells were grown various times, as shown in the experiments. Aluminum foil was used to hide the cells from light. One day after seeding the cells, RA and DMSO were added to prevent any interactions with cell attachment to the culture dishes. Tumor cells were grown until they were roughly 90% confluent and then dissociated into single-cell suspensions from tissue culture flasks using 0•05 percent EDTA in PBS for 3 min for all experiments.

WRL-68 cell line
The human liver cell line WRL-68 which used as a normal cell because its morphology is similar to that of hepatocytes and liver primary cultures. Albumin and alpha-fetoprotein are secreted by cells, and liver-specific enzymes like alanine aminotransferase are expressed to produce the solutions and medium for cell culture according to [26,27].

MTT cytotoxicity assay
The tests were carried out in triplicate, and the IC50 values for the nanoparticles synthesized AgNPs and AgI NPs were determined using a log dosage inhibition curve. For compliance with [28], follow the manufacturer's recommendations. In 96-well plates, the cells (1×10 4 to 1×10 6 cells ml −1 ) were grown to a final volume of 200 L of complete culture media per well. The plates were topped with sterile parafilm, gently stirred, and incubated at 37°C with 5% CO2 for 24 h. The medium was withdrawn after incubation, and 200 μl of a 2-fold serial dilution of AgNPs and AgI NPs solutions (25, 50, 100, 200, 400 μg ml −1 ) was added to the wells. At each concentration and control, triplicate tests were carried out. Each concentration and control were tested in triplicate. The plates were incubated at 37°C for 24 h with 5% CO 2 . Following the extract exposure, 10 ml of MTT solution was applied to each well. The plates were then incubated for another 4 h at 37°C with 5% CO 2 . After carefully removing the medium, each well was filled with 100 ml of DMSO solubilization solution and incubated for 5 min. At a wavelength of 575 nm, the absorbance was measured using an ELISA reader (Bio-rad, Germany). The untreated cells control (100% cell viability) was used to express the percentage of the cell viability. The IC50 was calculated using statistical analysis of the optical density values. The following equation can be used to solve this problem [29]: ( ) =Viability optical densty of sample optical density of control % 100%

Cellular uptake and western blot analyses of AgNPs & AgI NPs
FTC133 cells were obtained from American Type Culture Collection (ATCC) with the help of cooperation between Cardiff University, Cardiff and the collaboration of a researcher from Brighton University, UK. The stock AgNPs & AgI NPs suspensions were sonicated and freshly diluted to appropriate concentrations in the cell medium according to [30]. Each analysis included control cells that had not been treated. The toxicity endpoints were evaluated in control and exposed cells using mean fluorescence intensity to examine the cellular absorption of fluorescent nanoparticles after the exposure period.
• Western blot analysis For FTC133 cell movement and invasion rate measurements, total protein was extracted according to the manufacturer's protocol and used for protein expression analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to load and separate the protein samples (30 g of lysates from FTC-133 cells). The protein was electrotransferred on a polyvinylidene difluoride membrane after electrophoresis (Immobilon-P; Millipore).

Statistical analysis
Analysis of Variance (One-Way) In this section, ANOVA was utilized. SPSS version 23 was used to assess the significance of the findings and their connection. Statistical significance was defined as a P value of less than 0.05. At least three separate studies' data were provided, each with a different interpretation of the meaning.

UV-vis absorption analyses
To study the optical measurements of synthesized AgNPs and AgI NPs UV-vis absorption spectrophotometer was applied, and the scanned wavelength range was from 200 nm to 800 nm. The optical properties of spherical AgNPs are highly reliant on the diameter of the nanoparticle. Figure 1(A) depicts the electronic absorbance spectra of synthesis AgNPs, which mainly absorbed light and had a peak near 450 nm, with a surface plasmon resonance band observed between (400-475) nm. The maximum absorption band of AgNPs was monitored at 424 nm by UV-vis spectra, which was qualified to surface plasmon excitation. These results are in a match with previous study results by (Katarzyna Ranoszek et al 2017) [31].
The absorbance spectra for synthesized AgI NPs shown in figure 1(B) revealed the highest intensity peak observed in the visible area near 425 nm, indicating surface plasmon excitation. There is an excellent match with previous data for AgI NPs reported by (Raid A Ismail et al 2018) [14].

Fourier transform infrared spectroscopy (FTIR) analysis
Fourier transforms infrared (FTIR) spectroscopy is an essential instrument for functional groups observation. It was employed in this work to characterize the diverse functional groups involved in the reduction of the  [32], G Narasimha [33], and M Z H Khan et al [34]. Figure 2(B) shows the FTIR spectrum of synthesized AgI NPs by chemical method. The observed bands identify the functional groups in AgI NPs; the FTIR spectrum shows intense absorption bands at 3439.29 and 1619.72 cm −1 can be assigned to O-H (alcohol) stretching and bending vibrations, respectively. In addition to weak intensity bands at 3932.8 and 3786.75 cm −1 ascribed to the OH groups [35]. The peak found at 2925.38 cm −1 is corresponding to the CH 2 stretching vibration of SDS (sodium dodecyl sulfonate). The appearance of this peak suggests that a trace amount of SDS has been coated on the surface of AgI nanoparticles. So, the presence of purified water on the nanoparticles is indicated by these findings. The band at 1385.19 cm −1 can be exemplified the N=O symmetry stretching. The band at 615.45 cm −1 belongs to the stretching of the Iodo-compound (C-I) [36]. The band at 1024.39 cm −1 corresponds to the C-N stretching of amines. Previous reports provided support for the current data by J (Safei and M A Ghasemzadeh) [24,37].    The determined crystallite sizes values for AgNPs were in the range of (12-23) nm. The mean crystallite size was (18)

Field emission scanning electron microscope (FE-SEM)
The morphological structure of the nanoparticle's products of Ag and AgI is determined by FE-SEM analysis. Figures 5(A)-(D) reveals FE-SEM images of Ag NPs prepared by a chemical method deposited on a glass substrate with different magnification. The morphology of AgNPs displays their uniform distribution and is relatively homogeneous. Also, these NPs are assembled into a cluster of spherical-like shapes with different diameters due to the stability of cluster distribution and colloid formation. The particle sizes estimated from FE-SEM images range from (108.7-to 236.8 nm), which are larger than the sizes obtained from XRD analysis and TEM images using Scherrer's formula. This can be attributed to larger clusters deposited on the film's surface due to an aggregate structure composed of tiny grains on the glass substrate during drying. In other words, XRD is size-dependent and defect-free, whereas FESEM directly visualizes the particles without regard for the degree of crystal defects. Several researchers have reported such particle size differences between XRD and FE-SEM [33,38]. FE-SEM images in figures 6(A)-(D) show the produced AgI NPs powder specimen prepared by a chemical method with different magnifications. FE-SEM pics explain the morphology of AgI with relatively a homogenous size, and these NPs assembled in semi-spherical-like shape with some ledge and multi-branch objects with diameters ranging between (22-73) nm due to agglomeration effect. Notice that there is a good agreement with the derived values from the XRD pattern of the AgI NPs analysis.

Energy dispersive x-ray (EDX) analysis:
To determine the ratio of the elements in any compounds, an Energy Dispersive x-ray (EDX) analysis was performed. In this study, it was used to show the presence of silver (Ag) and Iodine (I) in the mixture with a specific ratio. The analyses were mentioned in different patterns, as shown in the figure 7 for AgNPs and figure 8 for AgI NPs. The EDX-spectrum in figure 7 clearly demonstrates the high quality of silver (Ag) in AgNPs, with a weight percentage of the presence of silver Ag being 95%. Besides, other peaks for oxygen 1.2% and carbon 3.8% percentage were also observed. On the other hand, the presence of the silver (Ag) peak in figure 8 for the AgI NPs specimen is 51.5%, and Iodine (I) is 48.5% with no contamination in the AgI NPs powder specimen. A peak related to the Au element was observed in the EDX spectrum of AgNPs and AgI NPs, which probably originated from the SEM chamber contaminations.
3.1.6. TEM micrograph TEM micrograph of AgNPs nanoparticles is shown in figures 9(A)-(D) with different magnifications, which demonstrates spherical shape and a narrow particle size distribution. Dispersible particles were observed under TEM analyses even at high magnification. The TEM histogram in figure 9(E), obtained using Image J software of at least 100 particles, shows the spherical particles have an average diameter of 11.5 nm and a standard deviation of 1.3 nm. The minimum diameter of AgNPs particles was 5.5 nm, and the maximum was 39.5 nm. The TEM   analysis of AgI NPs with different magnifications shown in figures 10(A)-(D) revealed primarily spherical and well-dispersed silver iodide nanoparticles. With a low level of aggregation, some nanoparticles took on an uneven shape. The monodispersed nanoparticles are the most common, as seen in the TEM histogram data in figure10(E). The particle's size with homogenous distribution ranged between 9.9 nm and 50 nm in diameter as the minimum and maximum values, with an average diameter of 24.28 nm and a standard deviation of 1.47 nm.

Cell viability (MTT Assay)
To examine the cytotoxicity of synthesized AgNPs and AgI NPs on follicular thyroid cancer cells FTC133 cell line and the normal cell WRL68 cell line were used, with (1×10 5 ml −1 ) cells per well in their exponential growth phase. They were incubated with increasing the AgNPs and AgI NPs concentrations for 24 h. The cell viability was expressed as a percentage of the untreated control (100% cell viability), which was investigated by MTT assay. Cytotoxicity results of FTC-133 cell viability after 24 h of treatment with various concentrations of each AgNPs and AgI NPs (25 to 400 μg ml −1 ) are shown in figures 11 and 12, respectively. All results indicated that a decrease in cell viability in a dose-dependent manner and both AgNPs and AgI NPs solutions resulted in a significant decrease in the survival rate of FTC-133 cells in dose dependence (P<0.0001). For AgNPs figure 11 at a concentration of (25 μg ml −1 ), there is no significant difference between FTC133 carcinoma and normal cell WRL68, but there are significant differences at higher concentrations of (50,100,200 μg ml −1 ). At concentrations (50,100 g ml −1 ) in the table 3, we found that the viability rate of the normal cell WRL68 remained nearly unchanged at 94.64, 92.13%, whereas the viability rate of the FTC133 carcinoma decreased to 78.63% and 64.51%, respectively. And a higher cell death rate of FTC133 was 55.13% at the highest concentration (400 μg ml −1 )     death rate of 55.58% for FTC133 was at the highest concentration of (400 μg ml −1 ) and 31.4% for the normal cell WRL68. The determined IC50 value of AgI NPs for FTC133 was (95.22 μg ml −1 ) and (208.5 μg ml −1 ) for normal cell WRL68.
The cytotoxicity results showed the dose-dependent cytotoxicity of AgNPs and AgI NPs on follicular thyroid cancer cell line FTC133 more than standard cell line WRL68 at particular concentrations. Therefore, it can conclude that the cytotoxic effect of AgNPs and AgI NPs depends on cell type. In comparison between the cytotoxic effect of AgNPs and AgI NPs on FTC133 and WRL68, significant toxicity was observed in AgNPs, and   Because bigger AgNPs release silver ions slowly [39], and smaller particles penetrate cells more quickly than larger particles [40], smaller AgNPs are more hazardous than larger nanoparticles. Size-dependent cellular interactions have also been studied [41]. Also, could the cytotoxicity of AgNPs and AgI NPs be due to the type of capping agent used in nanoparticle synthesis, revealing the effect of capping agent type [42]. The type of coating depends on the capping agent properties such as organic capping agents and inorganic capping agents [19] for AgI NPs the FTIR results displayed the existence of organic groups in the coated layer of it which reduced the cytotoxicity effect at a particular dose [43].

FTC133 cellular uptake for AgNPs and AgI NPs
A flow cytometer was used to examine the cellular uptake of AgNPs and AgI NPs in FTC133 to understand better the factors that contribute to toxicity. The variation of the incubation time was 0−120 min, the AgNPs and AgI NPs suspended in media with two concentrations of 10 mM and 20 mM in addition to control and dosed cells. Figures 14(A) and (B) shows the fluorescence intensity rate as a function of incubation time for FTC133 of AgNPs and AgI NPs, respectively, to detect differences in cellular AgNPs and AgI NPs uptake. Figure 15  demonstrates that AgNPs and AgI NPs uptake is strongly time-dependent, and nanoparticle uptake is relatively rapid. Notably, there is a significant uptake within an hour with the AgNPs concentration of 3.05 mg/10 8 cells and 2 mg/10 8 cells for AgI NPs. After that, the cellular uptake continues to increase at approximately the same rate until the four-hour limit. Thus, after six hours, the rate of FTC133 cells ingesting AgNPs and AgI NPs begins to grow, albeit at a slower rate. After a long (6 h) incubation period, the FTC133 cellular uptake reached stable values for AgI NPs, while the increase continued at the same low rate at (8 h) for AgNPs to get regular discounts. That behavior could be because the cell is saturated. The cellular uptake results in this study indicate that FTC133 is concentration-and time-dependent for both AgNPs and AgI NPs. Previous work demonstrates that their uptake increases by increasing the time and engagements in which cells are exposed to NPs [44,45]. Also, it can be observed that, throughout the exposure time, the rate of FTC133 ingested AgNPs was higher than that of AgI NPs taken up, confirming with that the cytotoxicity results which proved the AgNPs are more effective on FTC133 cells than the AgI NPs. That could be due to particle size differences between AgNPs and AgI NPs, as mentioned above in the TEM results, with AgNPs having an average diameter of 12.3 nm and AgI NPs having an average diameter of 26.25 nm, implying that FTC133 cellular uptake was size-dependent in the test. The results observed in this current study agree with a previous study that found the smaller AgNPs go into cells more quickly than, the bigger size particles [44,45]. Other reasons could be taken into account in the shape of NPs. From FESEM and TEM tests, the morphological structure is highly monodispersed spherical shape of AgNPs specific surface area than that semi-spherical like with some of the ledges in the morphological form of the AgI  NPs. As a result, the FTC133 cellular uptake in this study could be shape-dependent. Previous studies have found that the shape-dependence of cellular internalization causes significant differences in the uptake of different nanoparticles [46,47].

FTC133 migration and invasion for AgNPs and AgI NPs treatment
Western blot technique was performed here to access the effects of AgNPs, and AgI NPs on the migration and invasion ability of FTC133 cells by analyzing the migration and invasion-related protein as shown in figure 16(a), the protein levels were decreased after AgNPs and AgI NPs treatments. The evaluated values of migrated and invasive FTC133 cell rates were reduced after AgNPs and AgI NPs therapy, as shown in figures 16(b) and (c). In figure 16(b), the migrated cell rate decreased when the FTC133 cells were treated with AgNPs to 76% and 60% with AgI NPs treatment compared to the control group. Also, it can be observed that the invasive cell rate decreased to 60% of the control group for AgNPs treatment and decreased to 55.71% in the cells treated with AgI NPs as shown in figure 16(c).

Conclusions
The novelty of this work relies on the study the cytotoxic effects of AgNPs on follicular thyroid cancer cells (FTC133 cell line). Also, up to our knowledge the study of the anticancer efficiency for AgI NPs at a first time in our article. The synthesis of AgNPs and AgI NPs via a chemical co-precipitation approach was characterized in this study, and their potential for targeting follicular thyroid cancer was demonstrated. According to the MTT assay, dose-dependent cytotoxicity of manufactured AgNPs and AgI NPs with cytotoxic effect varies by cell type, with AgNPs having a cytotoxic impact higher than AgI NPs on follicular thyroid cancer cells and normal cells. Furthermore, in a concentration, size, and time-dependent way, follicular thyroid carcinoma takes up AgNPs more than AgI NPs, according to the uptake data. Furthermore, AgI NPs inhibited FTC133 cell migration and invasion more effectively than AgNPs.