Biocompatible Ag/Fe-Enhanced TiO2 Nanoparticles as an Effective Compound in Sunscreens

In this work, valuable biocompatible Ag/Fe-enhanced TiO2 nanoparticles are comparatively prepared by a conventional wet chemistry method (sol-gel) and a rapid, efficient, hybrid unconventional method (microwave-assisted hydrothermal synthesis). In order to establish their application as effective compounds in sunscreens, the obtained powders were first structurally and morphologically characterized, analyses from which their nanodimensional character, crystalline structure and thermal behavior were highlighted. The evaluation of sunscreen effectiveness is based on the determination of the sun protection factor (SPF). It was observed that silver enhancing increases the SPF significantly, especially when compared to the pristine samples. The obtained Ag/Fe-enhanced TiO2 powders were also evaluated from the point of view of their biocompatibility on amniotic fluid stem cells, and the results indicated an enhance of cell proliferation when exposed to the synthesized nanostructures.


Introduction
Throughout life, there are many factors that can lead to skin degradation, but the most common one is the exposure to ultraviolet radiation (UV). While UVC radiation (200-290 nm wavelength) is absorbed by the ozone layer, both UVB (290-320 nm wavelength) and UVA (320-400 nm wavelength) radiation are well identified as contributors to various molecular events that facilitate skin carcinogenesis and eventually lead to skin cell injury, melanoma and non-melanoma skin cancer [1][2][3]. The detrimental effects of UV radiation on epidermal cells consist in DNA alteration and reactive oxygen species (ROS) formation, which later interact with other molecules, triggering oxidative harm. The UV exposure also amplifies nitric oxide (NO) concentrations, that in high levels becomes detrimental to the human body and interacts as a free radical with ROS to produce additional NO end-products [4,5].
To prevent these adverse effects of UV radiation, scientists have introduced sunscreen creams. The effectiveness of a sunscreen cream is given by the SPF (sunscreen protection factor), determined by

Sol-Gel Synthesis
For the sol-gel method, the TTIP:H 2 O:C 3 H 8 O = 1:155:1.5 ratio was used and 0.4% molar silver or iron precursors were added to obtain titanium dioxide particles enhanced with silver or iron ions. In order to obtain the anatase phase, the pH was adjusted to 2.5 by adding HNO 3 . The resulting solutions were stirred on a magnetic hob at 82.5 • C for 24 h at reflux (corresponding to the boiling temperature of the isopropanol), then centrifuged and washed with distilled water several times until obtaining a neutral pH, and dried in the oven at 45 • C for 72 h.

Microwave-Assisted Hydrothermal Synthesis
For the microwave-assisted hydrothermal method the ratio TTIP:H 2 O = 1:100 was used. The general steps implied in this type of synthesis were previously described by Paduraru et al. [28]: Particularly, the precursors for Ti and Ag/Fe, along with distilled water and nitric acid, were weighed and transferred into Teflon vials. Afterwards, the vials (placed in a rack) were automatically pulled down into the reaction chamber, sealed and pre-pressurized with nitrogen. The obtained reaction mixtures were then subjected to a microwave-assisted hydrothermal treatment of 10 min, at 200 • C and the pressure of 20 bars, then cooled to room temperature. After the autoclaving process, the samples were centrifuged and washed several times with distilled water until obtaining a neutral pH and dried in the oven at 45 • C for 72 h.

Structural and Morphological Characterization
In order to investigate the crystalline phases, the samples were analyzed using the PANalytical Empyrean equipment (from Malvern PANalytical, Bruno, Nederland) in Bragg-Brentano geometry equipped with a Cu anode (λ CuKα = 1.541874 Å) X-ray tube. The X-ray diffractogram (XRD) was acquired in the range of 10 • -80 • 2θ angle, with an acquisition step of 0.02 • and an acquisition time of 100 s per step. The scanning electron microscopy (SEM) images were performed with an Inspect F50 microscope coupled with an energy dispersive spectrometer (EDS) (Thermo Fisher-former FEI, Eindhoven, Nederland), while transmission electron microscopy (TEM) images were obtained using a Tecnai G2 F30 S-Twin microscope (Thermo Fisher-former FEI, Eindhoven, Nederland) equipped with scanning transmission electron microscopy (STEM)/ high-angle annular dark-field (HAADF) detector, EDS and energy filtered transmission electron microscopy (EFTEM)-electron energy loss spectroscopy (EELS) spectrometer. The Raman spectra were realized with a LabRAM HR Evolution instrument (from Horiba, Longjumeau, France), using a 633 nm LASER and 50× lens, while thermogravimetric (TG) analysis and differential thermal analysis (DTA) were performed using Shimadzu DTG-TA-50H equipment (from Shimadsu, Sanjo, Japan) at 20-900 • C.

Sun Protection Factor (SPF) Evaluation
For the determination of the SPF, the UV-VIS Evolution 300 Spectroscope (Thermo Fisher, Eindhoven, Nederland) was used, by performing a scan with a step of 5 nm on dispersions that contained 5% (wt) Ag/Fe-TiO 2 /TiO 2 powder in distilled water. In order to determine the SPF of the obtained samples, Mansur's relation (1) was used for wavelengths between 290 and 320 nm [29]: where EE is the erythemal effect spectrum; I represents the solar intensity; Abs is the absorbance of sunscreen product and CF represents the correction factor (= 10). The values of EE × I are constants and predetermined by Sayre et al. [30].
2.6. Biocompatiblity In-Vitro 2.6.1. Quantitative In-Vitro Evaluation of Biocompatibility-MTT Assay The obtained Ag/Fe-enhanced TiO 2 powders were first evaluated from the point of view of their biocompatibility by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis, performed on mesenchymal stem cells isolated from amniotic fluid (AFSC). The cells were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Missouri, MO, USA) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) and changed twice a week. A dedicated MTT assay kit (Vybrant®MTT Cell Proliferation Assay Kit, Thermo Fischer Scientific, Massachusetts, MA, USA) was used. The analysis involves the cultivation of AFSC cells in 96-well plates, with a seeding density of 3000 cells/well, in the presence of the obtained Ag/Fe-enhanced TiO 2 powders, for 72 h. Then 10 µL (12 mM) MTT was added and the cells were incubated at 37 • C for 4 h. Thereafter, 100 µL SDS-HCl solution (1 mg Sodium Dodecyl Sulphate + 10 mL HCl, 0.01 M) was added, followed by vigorous pipetting to solubilize the formed formazan crystals. The optical density (OD) of solubilized formazan reading was performed after 1 h using a TECAN Infinite M200 spectrophotometer (Männedorf, Switzerland) at 570 nm.

Oxidative Stress Assessment-GSH-Glo Glutathione Assay
For this analysis, AFSC were seeded at a density of 3000 cells/well in 300 µL DMEM, supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin/neomycin) in plates with 96 wells. After 24 h of seeding, the cells were treated with the synthesized Ag/Fe-enhanced TiO 2 powders and incubated for 72 h. The GSH-Glo Glutathione Assay kit (Promega, WI, USA) was used for oxidative stress assessment. The kit measures the amount of glutathione (GSH) that is produced by cells and converted to oxidized glutathione. The amount of glutathione transformed is directly proportional with the amount of glutathione S-transferase (GST) enzyme that catalyzes the oxidation reaction, which is also linked to the formation of a luminescent luciferin. If the light emission is poor, glutathione production is inhibited and thus oxidative stress is increased, while in the case of an intense light, more glutathione has been synthesized and thus the cell is less stressed.
Initially, 100 µL 1X GSH-Glo Reagent was added, followed by incubation at 37 • C for 30 min. Subsequently, 100 µL Luciferin Detection Reagent was added and incubated at 37 • C for another 15 min. After 15 min, the medium was well homogenized and the plate was read on the luminometer (Microplate Luminometer Centro LB 960, Berthold, Germany) [31].

Evaluation of Cell Morphology and Viability by Fluorescence Microscopy
The biocompatibility of the obtained Ag/Fe-enhanced TiO 2 powders with AFSC was also evaluated based on fluorescence microscopy using RED CMTPX fluorophore (Thermo Fischer Scientific, Massachusetts, MA, USA), which is a cell tracker for long-term tracing of living cells. The CMTPX tracker was added in cell culture treated with the nanoparticles and the viability and morphology of the AFSC was evaluated after 5 days. The CMTPX fluorophore was added in the culture medium at a final concentration of 5 µM, incubated for 30 min in order to allow the dye penetration into the cells. Next, the AFSC were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken with Olympus CKX 41 digital camera driven by CellSense Entry software (Olympus, Tokyo, Japan) [28,31].

Results and Discussions
According to Figure 1, on the ATD curve, around the temperature of 100 • C, an endothermic effect determined by the evaporation of the water used for synthesis was observed and also the evaporation of isopropanol from the sample which is accompanied by a mass loss effect. The exothermic effect correlated with an observed mass loss between 200 and 300 • C is due to the degradation of the excess titanium precursor (TTIP). According to Figure 1, on the ATD curve, around the temperature of 100 °C, an endothermic effect determined by the evaporation of the water used for synthesis was observed and also the evaporation of isopropanol from the sample which is accompanied by a mass loss effect. The exothermic effect correlated with an observed mass loss between 200 and 300 °C is due to the degradation of the excess titanium precursor (TTIP). In the case of samples obtained by hydrothermal method (Figure 2), on the TG curve, a mass loss (approx. 8%) representative of several thermal effects is determined. Around the temperature of 694 °C for the pure TiO2 sample and 718 °C for the enhanced samples, an exothermic effect is observed, without mass loss, which may be due to the anatase-rutile polymorphic transformation.  1 5). At the same time, the diffraction peaks specific for the plane (2 1 1), characteristic for brookite was identified (according to the ICDD PDF4+ file 00-002-0514). Table 1 presents the full width at half maximum (FWHM) values corresponding to the peaks associated to most intense plane signal, (1 0 1), along with the calculated crystallite size. In the case of samples obtained by hydrothermal method (Figure 2), on the TG curve, a mass loss (approx. 8%) representative of several thermal effects is determined. Around the temperature of 694 • C for the pure TiO 2 sample and 718 • C for the enhanced samples, an exothermic effect is observed, without mass loss, which may be due to the anatase-rutile polymorphic transformation. According to Figure 1, on the ATD curve, around the temperature of 100 °C, an endothermic effect determined by the evaporation of the water used for synthesis was observed and also the evaporation of isopropanol from the sample which is accompanied by a mass loss effect. The exothermic effect correlated with an observed mass loss between 200 and 300 °C is due to the degradation of the excess titanium precursor (TTIP). In the case of samples obtained by hydrothermal method (Figure 2), on the TG curve, a mass loss (approx. 8%) representative of several thermal effects is determined. Around the temperature of 694 °C for the pure TiO2 sample and 718 °C for the enhanced samples, an exothermic effect is observed, without mass loss, which may be due to the anatase-rutile polymorphic transformation.  1 5). At the same time, the diffraction peaks specific for the plane (2 1 1), characteristic for brookite was identified (according to the ICDD PDF4+ file 00-002-0514). Table 1 presents the full width at half maximum (FWHM) values corresponding to the peaks associated to most intense plane signal, (1 0 1), along with the calculated crystallite size.  1 5). At the same time, the diffraction peaks specific for the plane (2 1 1), characteristic for brookite was identified (according to the ICDD PDF4+ file 00-002-0514). Table 1 presents the full width at half maximum (FWHM) values corresponding to the peaks associated to most intense plane signal, (1 0 1), along with the calculated crystallite size.   The FWHM change due to the enhancing effect, this variation is related to the crystallite size being modified by the incorporation of Ag or Fe species in the TiO2 crystalline structure.
The Raman analysis for the samples obtained by sol-gel method and by microwave-assisted hydrothermal method are presented in Figures 5 and 6.    The FWHM change due to the enhancing effect, this variation is related to the crystallite size being modified by the incorporation of Ag or Fe species in the TiO2 crystalline structure.
The Raman analysis for the samples obtained by sol-gel method and by microwave-assisted hydrothermal method are presented in Figures 5 and 6.  The FWHM change due to the enhancing effect, this variation is related to the crystallite size being modified by the incorporation of Ag or Fe species in the TiO 2 crystalline structure.
The Raman analysis for the samples obtained by sol-gel method and by microwave-assisted hydrothermal method are presented in Figures 5 and 6.  The Raman bands are found at 153, 407, 521, and 642 cm −1 for the samples obtained by the solgel method and at 151, 404, 521, and 644 cm −1 for the samples obtained by the hydrothermal method. The above mentioned vibrational modes are specific to the anatase phase of titania. At the same time, in the case of enhanced samples obtained by both methods of synthesis, a decrease in intensity is observed. This data suggests the presence of Ag/Fe in the structure of the obtained samples. Also, no other bands specific for silver oxide or iron oxide have been identified through the Raman technique [32].
For the pristine and enhanced TiO2 samples, SEM analyses (Figure 7) show that the obtained particles have a nanostructured nature. The particles obtained by the hydrothermal method are uniform from a morphological point of view with spheroidal shapes. They are organized in agglomerates with existing dimensions in the micrometric domain and have polyhedral shape.  The Raman bands are found at 153, 407, 521, and 642 cm −1 for the samples obtained by the solgel method and at 151, 404, 521, and 644 cm −1 for the samples obtained by the hydrothermal method. The above mentioned vibrational modes are specific to the anatase phase of titania. At the same time, in the case of enhanced samples obtained by both methods of synthesis, a decrease in intensity is observed. This data suggests the presence of Ag/Fe in the structure of the obtained samples. Also, no other bands specific for silver oxide or iron oxide have been identified through the Raman technique [32].
For the pristine and enhanced TiO2 samples, SEM analyses (Figure 7) show that the obtained particles have a nanostructured nature. The particles obtained by the hydrothermal method are uniform from a morphological point of view with spheroidal shapes. They are organized in agglomerates with existing dimensions in the micrometric domain and have polyhedral shape. The Raman bands are found at 153, 407, 521, and 642 cm −1 for the samples obtained by the sol-gel method and at 151, 404, 521, and 644 cm −1 for the samples obtained by the hydrothermal method. The above mentioned vibrational modes are specific to the anatase phase of titania. At the same time, in the case of enhanced samples obtained by both methods of synthesis, a decrease in intensity is observed. This data suggests the presence of Ag/Fe in the structure of the obtained samples. Also, no other bands specific for silver oxide or iron oxide have been identified through the Raman technique [32].
For the pristine and enhanced TiO 2 samples, SEM analyses (Figure 7) show that the obtained particles have a nanostructured nature. The particles obtained by the hydrothermal method are uniform from a morphological point of view with spheroidal shapes. They are organized in agglomerates with existing dimensions in the micrometric domain and have polyhedral shape.  Following the average particle distribution, for the titanium dioxide powder, the average particle size is 7.24 nm, the largest statistics being in the 5-8 nm range, but their size is between 1-13 nm.
Transmission electron microscopy images for the Fe-enhanced sample ( Figure 9) reveal a spheroidal morphology, and electron diffraction on selected area highlights the diffraction rings specific to interplanar distances for the (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 0 4), (1 1 6), (2 1 5) planes for titanium dioxide in the form of anatase and diffraction ring specific to interplanar distances for the (2 1 1) plane for titanium dioxide in the form of brookite. According to the histogram the average particle size is 7 nm, the largest statistics being in the diameter range of 6-8 nm. Following the average particle distribution, for the titanium dioxide powder, the average particle size is 7.24 nm, the largest statistics being in the 5-8 nm range, but their size is between 1-13 nm.
Transmission electron microscopy images for the Fe-enhanced sample ( Figure 9) reveal a spheroidal morphology, and electron diffraction on selected area highlights the diffraction rings specific to interplanar distances for the (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 0 4), (1 1 6), (2 1 5) planes for titanium dioxide in the form of anatase and diffraction ring specific to interplanar distances for the (2 1 1) plane for titanium dioxide in the form of brookite. According to the histogram the average particle size is 7 nm, the largest statistics being in the diameter range of 6-8 nm. Figure 8. The TEM and HRTEM images (a), SAED patterns (b) and particle size distribution (c) corresponding to TiO2 SG sample.
Following the average particle distribution, for the titanium dioxide powder, the average particle size is 7.24 nm, the largest statistics being in the 5-8 nm range, but their size is between 1-13 nm.
Transmission electron microscopy images for the Fe-enhanced sample ( Figure 9) reveal a spheroidal morphology, and electron diffraction on selected area highlights the diffraction rings specific to interplanar distances for the (1 0 1), (0 0 4), (2 0 0), (1 0 5), (2 0 4), (1 1 6), (2 1 5) planes for titanium dioxide in the form of anatase and diffraction ring specific to interplanar distances for the (2 1 1) plane for titanium dioxide in the form of brookite. According to the histogram the average particle size is 7 nm, the largest statistics being in the diameter range of 6-8 nm. The results of the MTT analysis show that the samples do not produce cytotoxicity in the tested conditions, all registered absorbance levels being higher than those associated to the control values (the AFSC cells without being in contact with the us obtained powders) at 24, 48 and also 72 h. These results suggest in fact a favoring of AFSC proliferation when in contact with Ag/Fe-enhanced TiO2 The results of the MTT analysis show that the samples do not produce cytotoxicity in the tested conditions, all registered absorbance levels being higher than those associated to the control values (the AFSC cells without being in contact with the us obtained powders) at 24, 48 and also 72 h. These results suggest in fact a favoring of AFSC proliferation when in contact with Ag/Fe-enhanced TiO 2 powders, continuously sustained through a larger period of time-72 h ( Figure 10). All results are represented as mean ± standard error, n = 3, * p < 0.05. Figure 9. The TEM and HRTEM images (a), SAED patterns (b) and particle size distribution (c) corresponding to TiO2@Fe SG sample.
The results of the MTT analysis show that the samples do not produce cytotoxicity in the tested conditions, all registered absorbance levels being higher than those associated to the control values (the AFSC cells without being in contact with the us obtained powders) at 24, 48 and also 72 h. These results suggest in fact a favoring of AFSC proliferation when in contact with Ag/Fe-enhanced TiO2 powders, continuously sustained through a larger period of time-72 h ( Figure 10). All results are represented as mean ± standard error, n = 3, * p < 0.05. It is observed that after the first 24 h, the best answer is given by the silver-enhanced samples. This result is most likely due to the antimicrobial activity of silver. After 48 h, all values are increasing compared to control, but after 72 h most values approach the value of the control. The results obtained after 72 h may be a consequence of the release of nanoparticles from cells. It is observed that after the first 24 h, the best answer is given by the silver-enhanced samples. This result is most likely due to the antimicrobial activity of silver. After 48 h, all values are increasing compared to control, but after 72 h most values approach the value of the control. The results obtained after 72 h may be a consequence of the release of nanoparticles from cells.
In the oxidative stress assessment, the amount of glutathione, a natural antioxidant produced by cells during a normal functioning process, was measured. A high glutathione concentration is associated with the cells being less stressed by the presence of complex nano-systems (unaffected biochemical activity). Therefore, after 24 h it is observed that the level of GSH is slightly lower than in the case of the control sample, but the differences registered are not of significant biological value. Hence we can consider that the samples do not produce significant oxidative stress at the cell level ( Figure 11), in accordance with the previously MTT results. In the oxidative stress assessment, the amount of glutathione, a natural antioxidant produced by cells during a normal functioning process, was measured. A high glutathione concentration is associated with the cells being less stressed by the presence of complex nano-systems (unaffected biochemical activity). Therefore, after 24 h it is observed that the level of GSH is slightly lower than in the case of the control sample, but the differences registered are not of significant biological value. Hence we can consider that the samples do not produce significant oxidative stress at the cell level ( Figure 11), in accordance with the previously MTT results. By the AFSC cells interaction with the sol-gel obtained anatase nanoparticles, it is observed that the morphology of the cytoskeleton changes ( Figure 12). The cells become elongated and few retain their spherical shape. In the case of the AFSC cells interaction with the hydrothermal obtained anatase nanoparticles the same change in the morphology of the cytoskeleton is observed, with higher deformation than in the case of the control (especially in the case of non-enhanced samples) and very few cells maintain their initial form. The observed changes are specific to a normal fibroblast-like phenotype, with abundant filopodia, which means that the cells are viable, with an active metabolism and are well attached to the substrate. The occurrence of these pseudopodia is generated by the actin cytoskeleton, a vital protein with high activity in cell interactions and migration [28]. In the oxidative stress assessment, the amount of glutathione, a natural antioxidant produced by cells during a normal functioning process, was measured. A high glutathione concentration is associated with the cells being less stressed by the presence of complex nano-systems (unaffected biochemical activity). Therefore, after 24 h it is observed that the level of GSH is slightly lower than in the case of the control sample, but the differences registered are not of significant biological value. Hence we can consider that the samples do not produce significant oxidative stress at the cell level ( Figure 11), in accordance with the previously MTT results. By the AFSC cells interaction with the sol-gel obtained anatase nanoparticles, it is observed that the morphology of the cytoskeleton changes ( Figure 12). The cells become elongated and few retain their spherical shape. In the case of the AFSC cells interaction with the hydrothermal obtained anatase nanoparticles the same change in the morphology of the cytoskeleton is observed, with higher deformation than in the case of the control (especially in the case of non-enhanced samples) and very few cells maintain their initial form. The observed changes are specific to a normal fibroblast-like The SPF assessment is a quantitative measurement of the effectiveness of a sunscreen formulation. The in vitro SPF is useful for a screening test during product development, as a prerequisite of the in vivo SPS measure. For the determination of the sun protection factor (SPF) the absorbance values for each sample were taken at wavelengths between 290 to 320 nm (Table 2).  Table 2 highlights that the absorbance values for enhanced samples are much higher than for non-enhanced samples. The SPF of the samples is highly related to the light absorption and their absorption wavelength range. Using Mansur's formula, it was possible to determine the value of the sun protection factor for each sample. The results are presented in Table 3. According to Table 3, it can be observed that, in most cases, silver enhancing increases the SPF significantly due to the much higher absorbance value than for non-enhanced samples. The SPF with the highest value is given by a suspension containing sol-gel-derived silver-enhanced anatase nanoparticles. The SPF values obtained for the sol-gel-derived iron-enhanced nanoparticles, as well as hydrothermal-derived silver-enhanced nanoparticles, are also close to the best obtained value. Given that, in regular sunscreen creams, the proportion of nanoparticles is usually 10 wt.%, a reduction of the nanoparticle fraction to half of that value still holds adequate SPF values, which make the above mentioned samples suitable for use in sun protection creams even in 5 wt.%.

Conclusions
When synthesizing TiO 2 nanoparticles by wet chemistry (sol-gel versus microwave-assisted hydrothermal synthesis methods) and adding metallic materials, such as Ag or Fe, a series of improvements can be observed, especially from the point of view of their applications. Anatase polymorphic TiO 2 phase was obtained as a principal crystalline compound in all synthesized samples, with a crystallite size varying from 4 to 8 nm, and a monomodal particles size distribution, with medium diameter range of 6-8 nm. As a prospective compound in sunscreens, the main studied properties are the ability to absorb, scatter and reflect UV rays, quantified by SPF value. In the present study it is shown that the highest SPF value is given by a suspension containing sol-gel-derived silver-enhanced anatase nanoparticles. Corroborating these results with the high biocompatibility proved by the obtained samples for all applied biological characterization methods, we can propose the usage of Ag/Fe-enhanced TiO 2 nanoparticles as an effective compound in sunscreens.