Influence of formulation of ZnO nanoblokes containing metallic ions dopants on their cytotoxicity and protective factors: An in vitro study on human skin cells exposed to UVA radiation

Graphical abstract

Highlights

• •The [Zn(O)/M] (M = Mg, Al, Ca, Ti) synthesize by thermal method and characterize.
• •The metal dopants percolate into crystal lattice of ZnO and stable it.
• •The [Zn(O)/M] shows very low amount of Zn⁺² release into culture medium.
• •Intracellular reactive oxygen species generation decrease under UVA radiation.
• •The [Zn(O)/M] protects of human skin cells against UVA radiation.

Abstract

Application of ZnO nanoparticles in sunscreens exposes human skin with their adverse effects, which correlates to dissolution/translocation of free Zn⁺² ions. The possibility of decreasing solubility and therefore, reducing toxicity, by structural modifications have been discussed as a solution. The present investigation has developed new metallic lattices of ZnO to reduce cytotoxicity of ZnO nanoparticles. Novel metal-promoted Zn-based nanocomposites ([Zn(O)/M], M = Mg, Al, Ca, Ti) were synthesized and their physicochemical properties and their cytotoxicity were evaluated. Solubility and release studies showed that modification of ZnO structure decreases release of Zn⁺² into culture medium. XRD and UV absorbance analyses showed that metallic-dopants percolate into crystalline lattice of ZnO. This phenomenon is basic reason for stability of Zn-based network. Cell culture studies and MTT assay on human skin cells (HFF-1) exposed to UVA radiation showed that the level of protection of [Zn(O)/M] compounds were more than of [ZnO]. Dichlorofluoroscein diacetate-ROS assay and Zn⁺² release experiments indicated that [Zn(O)/M] nanocomposites decreased the level of ROS generation and Zn⁺² release in compared to ZnO, indicating higher safety of nanocomposites. This study shows that the synthesized Zn-based nanocomposites have potential to be used as safer and more effective sunscreens than ZnO.

1. Introduction

Exposure of human skin with sunlight and therefore UVA, UVB, and UVC radiations, is the major environmental factor that affects the structure of skin and develops skin cancers. Ozone layer efficiently absorbs UVC and 95% of UVB [1]. The UV radiation generates reactive oxygen species-ROS by photochemical interactions and lead to DNA, proteins, and lipids damages [[2], [3], [4]]. Public health authorities recommend the use of sunscreens, including ZnO, to limit sun light (UVA and UVB radiation) damages [5].

ZnO compounds are interesting class of semiconductor compounds which have been used in sunscreens because of their ability to filter ultraviolet UVA and UVB light. In recent years, manufacturers have started using nano-sized forms of these metal oxide in sunscreens [6]. These new formulation resolved the problem of the unsightly white film of traditional sunscreens and created a vehicle that is more transparent, less viscous, and blends into the skin more easily [7]. With the rapidly increasing application of ZnO particles, several growing community and scientific concern warned to increased health and environmental risks of metallic nanoparticles due to interactions and release of them in the environment and microorganism [8].

Previous reports have shown that nano-ZnO exerts toxicity in mammalian cells as a result of particle dissolution in the tissue culture medium [9,10]. Zn⁺² ions release in intracellular space induce generation of ROS and cell death [11,12]. Oppositely, some metal nanoparticles may show more toxicity than either their ionic forms or their parent compounds [13]. More soluble compounds like ZnO and FeO show greater acute toxicity than nanoparticles with extremely low solubility such as CeO₂ and TiO₂ [14]. An increasing number of research articles have focused on the cytotoxicity of different types of engineered nanomaterials via the generation of ROS [15,16].

Modifying structure of ZnO nanoparticles is a suitable way for promoting their properties. Over the past decade, it has been shown that Zn-based nanomaterials with multi-metallic structures (Zn(O)-M, M = secondary metal) show new and effective interesting properties that were not seen in mono-metallic structures (ZnO) [[17], [18], [19], [20], [21], [22]]. Previous reports have shown that altering the ZnO matrix through iron doping decreases particle dissolution in tissue culture media and leading to a reduction its toxicity [9]. Also, the synergetic effects of the Cu/Zn bimetallic nanoparticles distributed in the ACFs/CNFs (activated carbon fibres/carbon nanofibres) resulted from the relatively slower release of the Cu nanoparticles located at the tip of the CNFs and faster release of the Zn nanoparticles dispersed in the ACF at the in-vitro hemolytic test [23].

The doping metal on ZnO particles, helps stabilizing both structure and surface morphology of ZnO particles by preventing the release of Zn⁺² ions in solution [17,24,25]. Incorporation of doping element into ZnO crystal can change lattice parameters and defects (intrinsic/extrinsic) concentration [[26], [27], [28]]. For example, incorporation of metal ions in the semiconductor networks can lead to the formation of electron trapping sites and promote charge transfer from ZnO to metal ions, thus resulting in the enhanced photoreaction of surface adsorbed species [29,30]. With doping of nonmetal ions (e.g., N, S, C, F, etc.) into ZnO lattice, dopants create intra-band-gap states close to the conduction or valence band edges that induce visible light absorption. Unlike metal ions, nonmetal ions are less likely to form recombination centers, and thus are more effective in enhancing visible light activity [31].

Therefore, the multi-metallic nanoparticle not only retains most of the properties of the individual nano-metals but also possess several superior physicochemical properties which could prove beneficial for human health [[32], [33], [34]]. This strategy is employed here to synthesis safer ZnO nanoparticles. In spite of the considerable research into the potential hazardous effects of ZnO bulk- and nano-particles under environmental and biological conditions, attempts have not been made to design a potentially safer ZnO compound. Therefore, it was decided here to develop bimetallic novel heterometallic Zn-based nanocomposites ([Zn(O)/M], M = Mg, Al, Ca, Ti) via thermal method. The effects of metal-doping Zn-based nanocomposites on the solubility, release, UV-blocking behavior, cytotoxicity, and protective effects of them on cultured human skin (HFF-1) cells exposed with UVA radiation were then investigated.

To date, two approaches have been developed to synthesis metal doping ZnO: direct doping method of partial replacement of zinc by either Group I elements such as Na [35], K [36], Li [37] or oxygen by Group V elements such as: N [38], P [39], As [40], and Sb [41]. To the best of our knowledge, there is no report on the fabrication of [Zn(O)/M] (M = Mg, Al, Ca, Ti) nanocomposites and investigation of cytotoxicity and protective effects of them on human skin cells.

2. Materials and methods

2.1. Materials

CaCl₂·2H₂O (CAS number (CN): 10035-04-8), MgCl₂·6H₂O (CN: 7791-18-6), AlCl₃ (CN: 7446-70-0), TiCl₄ (CN: 7550-45-0), polyethylene glycol (PEG, M.W. = 35000 g/mol, CN: 25322-68-3), 1-Hexadecyltrimethylammonium bromide (CTAB, ≥98%, CN: 57-09-0), absolute ethanol (CN: 64-17-5), and dimethyl sulfoxide (DMSO, CN: 67-68-5) were purchased from Merck, Germany. Zn(OAc)₂·2H₂O (CN: 5970-45-6, ≥99.99%), commercial ZnO (nanoparticle morphology, CN: 1314-13-2), hydrazinium hydroxide (65 wt%, CN: 7803-57-8), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, ≥97%, CN: 4091-99-0), phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, CN: 298-93-1), and cellulose dialysis tube were purchased from Sigma-Aldrich, USA. Normal saline (NS) and lansulin were purchased from Exir Pharmaceutical Company, Iran. Penicillin was purchased from Jabiribn Hayyan Pharmaceutical Company, Iran. All compounds were purchased with analytical grades and these were used without further purification.

2.2. Preparation of the metal-doping Zn-based nanocomposites

In a typical synthesis, 0.02 g paraffin was dissolved in 2 mL ultrapure water at 50 °C (solution 1). After 5 min, 0.05 mmol CTAB was added to solution 1. In a separate beaker, 4.50 mmol Zn(OAc)₂·2H₂O was dissolved in 4 mL ultrapure water (solution 2). 0.50 mmol chloride salt (CaCl₂, MgCl₂·6H₂O, AlCl₃, TiCl₄) was dissolved separately in 2 mL of ultrapure water (solution 3). The solution 2 was then added drop-wise to solution 1 under stirring followed by solution 3. Then, 2 mL hydrazinium hydroxide (7.49 × 10⁻⁴ mmol, 65 wt%) was added drop-wise to the above solution. Finally, 0.05 mmol CTAB and 5.70 × 10⁻⁴ mmol PEG were added to the reaction solution under stirring for 5 min until the solution became homogeneous. The resulting homogeneous solution was sealed at 50 °C for 1 h (thermal method). Then the resulting precipitate was collected, filtered, and washed with ultrapure water and then with absolute ethanol for several times to remove any possible impurities and finally dried in vacuum before characterization.

The concentration of Zn was 43.70 Wt.% in the all of the Zn-based compounds which was determined using atomic absorption spectrometry (AAS). Preparation conditions for all compounds are summarized in Table 1.

Table 1.

Experimental parameters for the preparation various formulations of the metal-doping Zn-based nanocomposites. See text for details.

No.	Compound code	[Zn(O)/M] formula	Ma	[M] weight percent
1	F1T	[ZnO]	–	–
2	F2T	[Zn(O)/Mg]	Mg	4.9
3	F3T	[Zn(O)/Al]	Al	12.1
4	F4T	[Zn(O)/Ca]	Ca	15.0
5	F5T	[Zn(O)/Ti]	Ti	5.5

^aM = Co-metal additive in zinc oxide compounds.

2.3. Methods

2.3.1. Atomic absorption spectrometer (AAS)

The Zn⁺² and co-metals (Mg⁺², Ca⁺², Al⁺³, Ti⁺⁴) contents were determined by Perkin Elmer Atomic Absorption Spectrometer 1100 model, United Kingdom, with a deuterium background corrector and zinc, magnesium, calcium, aluminum, and titanium hollow cathode lamps.

2.3.2. X-ray diffraction (XRD)

Crystalline nature of compounds were recorded by X’Pert PRO X-ray diffractometer, Philips Company, Netherlands, using CuK_α radiation (1.5405 Å) in the 2θ range of 20–80.

2.3.3. Scanning electron microscopy (SEM)

The external morphology of compounds was characterized by AIS 2100 microscope model with resolution 3.0 nm at 30 KeV, Seron Technologies INC, Korea. Samples were prepared by coating of solid particles into a conductive layer (gold).

2.3.4. Transmission electron microscopy (TEM)

The internal morphology of compounds was characterized by Philips-FEI 208S model, Philips Company, USA. Samples were prepared by dispersing solid product in ethanol by sonication. Few drops of the suspension were deposited on copper grids, which were then put into the desiccators for drying, followed by imaging.

2.3.5. Particle size analysis

Particle size has pronounced effects on the spectral properties of semiconductor materials [42]. The size distribution of compounds in culture medium was obtained using a Dynamic Light Scattering (DLS), Malvern, HPPS-5001 model equipped with a green laser at 532 nm, United Kingdom.

2.3.6. Fourier transformation infra-red (FT-IR) spectra

The surface properties of compounds were investigated by analysis of an ALPHA FT-IR spectrophotometer model of Bruker Company, Germany, at a spectral resolution of 4 cm⁻¹ using KBr pellets in the range of 400–4000 cm⁻¹ with a delicate beam condenser and a liquid nitrogen cooled MCT (mercury cadmium telluride)-Detector.

2.3.7. UV absorption properties

2.3.7.1. Diffuse reflectance spectroscopy (DRS)

In order to calculate the precise value of optical band gaps (E_g, eV), as a measure of UV protection properties of semiconductors, UV–vis DRS analysis on compounds (powder) was employed here by Kubelka-Munk theory [43]. This analysis was recorded on Varian’s Cary 100 UV–vis model spectrophotometer, USA.

2.3.7.2. UV–vis spectra

UV–vis spectra of 20 mg/L ZnO loading of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium after 3 min sonication was recorded by CECIL Aurius Series CE 2021 UV/Vis Spectrophotometer, United Kingdom, using quartz cells of 1 cm optical path.

2.3.7.3. UV spectra for UV-blocking measurement

“Ultraviolet protection factor” (UPF) can be directly investigated using the ability of materials to block the UV radiation in the range of 280–400 nm [44]. The UV-blocking activity of 20 mg/L ZnO loading of F1T, F2T, F3T, F4T, F5T, and commercial ZnO was measured by a UV transmittance analyzer (CECIL Aurius Series CE 2021 UV/Vis Spectrophotometer) in culture medium, after 3 min sonication, using quartz cells of 1 cm optical path at room temperature.

2.4. Solubility measurement

The solubility of compounds in culture medium at 25 °C was studied in a beaker using samples of F1T, F2T, F3T, F4T, F5T, and commercial ZnO with 30 mg/L ZnO solid loading. Solutions (300 mL) were stirred for 24 h, while covered with aluminum foil to inhibit any photo-induced changes. Quantitative measurements were done at time intervals of 0.5, 6, 12, 18, and 24 h.

To separate solid particles from solution, solutions were passed through 0.2 μm filter. This was then followed by centrifugation at 12,000 rpm (3.15 × 10⁴ rcf) for 60 min. Concentration of Zn⁺² ions in supernatant was measured by AAS. The supernatants were also investigated under UV–vis light in the range of 200–700 nm to make sure that particulates are absent in the supernatant. No distinct peaks were observed, indicating that there is no particulate material in the supernatants after filtering and centrifugation. The pHs of the supernatants were also measured by a Metrohm 744 pH Meter.

2.5. Release of Zn⁺² ions from Zn-based compounds

Release of Zn⁺² ions of F1T, F2T, F3T, F4T, F5T, and commercial ZnO with a ZnO solid loading of 20 mg/L in culture medium were carried out by dialysis method. Dialysis experiments were carried out in cellulose acetate dialysis tubing (12 KDa cutoff) filled with 3 mL nanoparticles dispersion and immersed in 100 fold of its volume of culture medium (300 mL) at 37 °C and covered with aluminum foil to inhibit any photo-induced dissolution. The dialysis was carried out under slow stirring (200 rpm) with a magnetic stirrer and the samples were drawn of receptor medium at time intervals of 0.5, 3, 6, 9, 12, 15, 18, 21, 24, and 48 h. The concentration of released Zn⁺² was detected by AAS and the cumulative amount of the released Zn⁺² was calculated and plotted against time.

2.6. Stability of nanoparticles in culture medium

2.6.1. Aggregation/particle size study

Understanding the stability of nanoparticles in liquid solution in terms of aggregation is very important. Particle size analysis is used as an indicator of nanoparticles stability. To evaluate this, particle sizes of F1T, F2T, F3T, F4T, and F5T suspensions in culture medium at room temperature were studied over time (1, 6, 12, 24, and 48 h).

2.6.2. Sedimentation and turbidity measurements

Based on colloidal theories on particle aggregation [45] and sedimentation [46], the dispersion stability of metallic nanoparticles can greatly influence on their bioavailability. The precipitation stability of the 20 mg/L ZnO loading of F1T, F2T, F3T, F4T, and F5T solutions in culture medium was measured using UV–vis spectrophotometer. The λ_max was measured at different times (during 48 h). The normalized absorbance A_i/A₀ (A_i = absorbance as a function of time, A₀ = initial absorbance) was calculated as a measure of sedimentation.

Turbidimetric test of 20 mg/L of ZnO loading of F1T, F2T, F3T, F4T, and F5T solutions in culture medium was also studied by 2100P-HACH Turbidimeter-USA (NTU-Normal Turbidity Unit) at room temperature over 48 h.

2.7. Cell culture studies

Cytotoxicity and protection effects of M-promoted ZnO nanocomposites (M = Mg, Ca, Al, Ti) were investigated using human skin (HFF-1) cells. Cells were seeded at a density of 4 × 10⁴ cells per well of a 24-well plates [47] in complete culture medium (DMEM, 20% (v/v) heat-inactivated FBS, 10% (v/v) penicillin, and 100 IU ml⁻¹ insulin). Cells were incubated for 24 h at 95/5% air/CO₂ incubator (ASTEC, BENCH-TOP Incubator EC-9, Japan) and 37 °C. Cells were exposed to Zn compounds and UV radiation for cytotoxicity and ROS measurement studies, as discussed below.

2.7.1. Exposure to Zn-based compounds

Cells were treated with 20 mg/L of F1T, F2T, F3T, F4T, and F5T for 24 h. Intracellular ROS production was then measured by ROS assay, as discussed later. The culture medium was also collected and centrifuged at 14,000 rpm (4.29 × 10⁴ rcf). The concentration of released Zn²⁺ in all cellular supernatant was measured by AAS. The Zn²⁺ concentration in cells not exposed to nanocompounds suspension was served as a control.

2.7.2. UVA exposure

Exposing the skin cells to sunlight, especially to UVA radiation, leads to the photochemical generation of ROS [47,48]. It seems that Zn-based compounds can scavenge radiation-induced reactive oxygen (oxygen, hydroxyl, and superoxide radicals) intermediates in vitro [49,50]. To determine the protective effect of the Zn-based compounds (F1T, F2T, F3T, F4T, and F5T) in this research, Zn-treated cells were exposed to UVA radiation for 30 min at 2 W/cm² using UV lamp (mercury vapor lamp, with spectrum from 315 to 400 nm and maximum intensity at 365 nm). The distance between cells and UV source was kept at 50 cm to prevent over heating during irradiation [50]. A compensated Kipp-Zonen thermopile coupled to a digital voltmeter was used to measure the UVA actual energy received by cells.

2.7.3. ROS measurement

High intracellular mitochondria ROS levels and oxidative assault have been cited as important factors for cytotoxicity-carcinogenesis of skin cells induced by UVA radiation and nanoscale metal oxides [[51], [52], [53]]. The generation of ROS by UVA radiation and Zn-based compounds (F1T, F2T, F3T, F4T, and F5T) in the HFF-1 cells was determined by a fluorometric assay using the intracellular oxidation of the molecular probe DCFH-DA [54].

Firstly, cells were treated with 20 mg/L F1T, F2T, F3T, F4T, and F5T as described above. Cells were then washed with NS and PBS, and then incubated with 40 μM DCFH-DA for 30 min. At the end of incubation, supernatant containing the fluorescence of DCF, which is the oxidized product of DCFH, was measured with a fluorescence spectrophotometer (Infinite^® 200 PRO, Tecan, Switzerland) using an excitation of 485 nm and an emission of 530 nm. The DCF concentration in cells not exposed to Zn-based compounds was used as control.

Secondary, Zn (F1T, F2T, F3T, F4T, and F5T)-treated cells were exposed to UVA radiation as described above. At the end of incubation, as described above, supernatant containing the fluorescence of DCF was measured with a fluorescence spectrophotometer. The DCF concentration in cells not exposed to Zn-based compounds and UVA radiation was used as control.

2.7.4. MTT assay

The cell viability after nanoparticles (F1T, F2T, F3T, F4T, and F5T with 20 mg/L) and UVA treatment was measured by MTT assay in the form of percentage relative death to an untreated control, without the Zn-based nanocompounds and UVA radiation. Measurements done by using ELISA microplate reader at 570 nm (BioTek, ELx808 Absorbance Microplate Reader, USA), as described by Alarifi and Ali [55].

2.8. Statistical analysis

Results were expressed as mean values and standard deviation (±SD) and the significance of the difference observed was analyzed by the Student’s t-test. In all tests, a probability value of P < 0.05 was considered as statistically significant.

Also all the experiments were performed in triplicate and repeated three times to ensure reproducibility.

3. Results and discussion

3.1. Characterization the Zn-based compounds

3.1.1. Co-metal-content by AAS analyses

Atomic absorption studies showed that F2T, F3T, F4T, and F5T contained about 4.9, 12.1, 15.0, and 5.5 wt.% Ca⁺², Mg⁺², Al⁺³, and Ti⁺⁴ ions respectively when 0.5 mmol chloride salt (CaCl₂, MgCl₂·6H₂O, AlCl₃, TiCl₄) was used for synthesis (Table 1). Higher amount of chloride salt (0.75 mmol) did not change the M (co-metal)-content of the particles in considerable amount. The loading percent of Ca⁺², Mg⁺², Al⁺³, and Ti⁺⁴ ions at this concentration (0.75 mmol) was found to be 5.1, 12.0, 15.9, and 5.0 wt.% in F2T, F3T, F4T,and F5T, respectively.

3.1.2. Crystalline and phase structure studies by XRD

Fig. 1 shows XRD patterns of F1T, F2T, F3T, F4T, and F5T. Zinc oxide showed a hexagonal structure with JCPDS card 36-1451. Bimetallic compounds also showed hexagonal structures, except for F3T that shows an amorphous structure. For all the bimetallic nanocomposites, shifts in the peaks angle of F1T XRD pattern (Fig. 1a) relative to ZnO were observed (Fig. 1b–e). The peak shifting may indicate the substitution of Zn⁺² by M⁺ⁿ (Mg⁺², Al⁺³, Ca⁺², and Ti⁺⁴) in hexagonal-ZnO crystal due to smaller ionic size of M⁺ⁿ (Mg⁺², Al⁺³, Ca⁺², and Ti⁺⁴, 0.072, 0.053, 0.01, and 0.074 nm, respectively) compared to Zn⁺² (0.074 nm). No diffraction peaks from co-metals species of Mg and Ti such as magnesium oxides and titanium oxides were detectable for F2T and F5T (Fig. 1b, e). For F3T and F4T, aluminum oxide and calcium oxide species were distinguishable (Fig. 1c, d).

Fig. 1.

XRD patterns of the (a) F1T ([ZnO]), (b) F2T ([Zn(O)/Mg]), (c) F3T ([Zn(O)/Al]), (d) F4T ([Zn(O)/Ca]), and (e) F5T ([Zn(O)/Ti]). The circles in (c) and (d) show aluminum oxide and calcium oxide, respectively.

3.1.3. Morphology, surface structure, and particle size analyses

Fig. 2 shows the typical SEM and TEM images of F2T, F3T, F4T, and F5T. As shown in Fig. 2, all nanocomposites (F2T, F3T, F4T, F5T) showed nano-metric size in the one dimensional (1-D) of their configuration (nanoflake morphology). Apparent nanoflakes of F2T and F3T had thinner diameters (about 25–50 nm, Fig. 2a, b) compared to F4T and F5T (about 50–100 nm, Fig. 2c, d). Also F4T and F5T exhibited monodisperse and homogeneous hexagonal configuration as angular forms (Fig. 2c, d).

Fig. 2.

SEM and TEM images of the (a) F2T ([Zn(O)/Mg]), (b) F3T ([Zn(O)/Al]), (c) F4T ([Zn(O)/Ca]), and (d) F5T ([Zn(O)/Ti]).

Results of particle size analysis of F2T, F3T, F4T, and F5T are shown in Fig. 3 and Table 2. Particle size of ZnO (F1T without co-metal) is about 336 nm (Fig. 3a, Table 2). When Mg was added as co-metal, particle size was reduced by-about 2–4 times to about 140 nm (F2T, [Zn(O)/Mg], Fig. 3b, Table 2). F4T and F5T also provided smaller particle sizes (Fig. 3d, e, Table 2). But, Al-containing system (F3T) showed higher particle size than F1T (576 nm) (Fig. 3c, Table 2). The reason behind this phenomenon may be due to inhibition/acceleration of the growth rate of nucleations in the presence of co-metals during the synthesis process [19]. Polydispersity Index (PDI) of the synthesized Zn-based compounds shows uniformity in size (Fig. 3, Table 2).

Fig. 3.

Sample particle size distribution of the (a) F1T ([ZnO]), (b) F2T ([Zn(O)/Mg]), (c) F3T ([Zn(O)/Al]), (d) F4T ([Zn(O)/Ca]), (e) and F5T ([Zn(O)/Ti]) in culture medium.

Table 2.

Particle size (nm) and PDI of various formulations of the Zn-based compounds in culture medium (mean ± SD, n = 3).

Sample	Formula	Particle size (nm)	PDI
F1T	[ZnO]	336 ± 2.7	0.5 ± 0.1
F2T	[Zn(O)/Mg]	138 ± 2.4	0.1 ± 0.0
F3T	[Zn(O)/Al]	575 ± 5.0	0.7 ± 0.2
F4T	[Zn(O)/Ca]	246 ± 8.0	0.3 ± 0.2
F5T	[Zn(O)/Ti]	185 ± 5.5	1.0 ± 0.0

Fig. 4 shows FT-IR spectra of F2T, F3T, F4T, and F5T. Peaks at 1330–1350, 1550–1570, and 2800–2900 cm⁻¹ are attributed to PEG. Peaks at 1400–1500, 2800, and 2900 cm⁻¹ are attributed to CTAB. These data show that PEG and CTAB are present on composites surface.

Fig. 4.

FT-IR spectra of the (a) F2T ([Zn(O)/Mg]), (b) F3T ([Zn(O)/Al]), (c) F4T ([Zn(O)/Ca]), and (d) F5T ([Zn(O)/Ti]).

3.1.4. UV absorption properties

Fig. 5, Fig. 6 show the absorbance and transmittance properties of F1T, F2T, F3T, F4T, F5T, and commercial ZnO solutions. All compounds exhibited high transparency in the visible region (400–700 nm, Fig. 5a–e). The UV absorbance properties in the bimetallic nanocomposites were improved significantly (except the F3T, [Zn(O)/Al], Fig. 5, Fig. 6). It seems that the trapped UV light increased in 1-D structures which improved the optical path for the UV light absorption (morphology effect of compounds) [56,57]. According to anisotropic properties, crystalline materials with different morphologies and surfaces (different geometric and electronic structures) have different defect intensities [58] and active centers. These sites capture photo-induced electrons, recombination of photo-induced electrons and holes [59], and consequently different optical properties.

Fig. 5.

Absorption spectra of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium.

Fig. 6.

Transmittance spectra of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium.

In order to determine the precise value of energy band gap (E_g) of F1T, F2T, F3T, F4T, F5T, and commercial ZnO, we used optical absorption method on UV–vis diffuse reflectance by Kubelka-Munk theory [43] (Table 3). Band gap of ZnO in F1T is selected as control value (3.07 eV). It is clear that the absorption index metal-doped ZnO samples are showed the red-shifted (F3T) and blue-shifted (F2T, F4T, and F5T) in the band gap energy compared to undoped ZnO sample. These shifts in metal-doped ZnO samples are attributed to exchange interaction between the band electrons (ZnO) and the electrons of valance orbitals of the metal ion (metals doping) substituting cation [60]. This interaction result is moving of valance band and conductive band of metal-doped ZnO samples (Table 3). Also according to results of absorption and transmittance spectra, the unprecedented magnitude of both spectra (Fig. 5, Fig. 6) for metal-doped ZnO samples may be due to creation of new, accessible, and localize electronic states in the band gap of them that are known to induce UV–vis-light absorption and transmittance. This phenomenon is expected to increase UV-carrier concentrations which lead to the Burstein-Moss effect [61]. Also special particle size (Fig. 3, Table 3) and morphology (Fig. 2) of metal-doped ZnO samples might affect band gap and electronic sites due to quantum confinement effects of metals particles in these properties.

Table 3.

Optical band gap (E_g, eV) values of F1T, F2T, F3T, F4T, F5T and commercial ZnO.

Sample	Formula	Eg (eV)	Changes in comparison to ZnO
F1T (Control)	[ZnO]	3.07	0
F2T	[Zn(O)/Mg]	3.37	+0.30
F3T	[Zn(O)/Al]	2.75	−0.32
F4T	[Zn(O)/Ca]	3.37	+0.30
F5T	[Zn(O)/Ti]	3.6	+0.53
Commercial ZnO	ZnO	3.4	+0.33

3.2. Time-dependent stability tests of Zn-based compounds

3.2.1. Aggregation measurement

Particle size of F1T, F2T, F3T, F4T, and F5T suspensions in culture medium and room temperature was showed in Table 4. It can be said that particle sizes of F2T, F4T, and F5T suspensions are stable over 48 h. However particle sizes of F1T and F3T increased about 65 and 67 nm during 48 h possibly due to agglomeration in culture medium.

Table 4.

Particle size (nm) of F1T, F2T, F3T, F4T, and F5T during 48 h in culture medium (mean ± SD, n = 3).

Sample	Zero time	1 h	6 h	12 h	24 h	48 h
F1T [ZnO]	475 ± 1.3	475 ± 4.4	475 ± 0.0	530 ± 1.5	535 ± 2.8	540 ± 2.3
F2T [Zn(O)/Mg]	178 ± 3.0	178 ± 1.5	179 ± 4.9	185 ± 3.0	181 ± 4.1	181 ± 5.0
F3T [Zn(O)/Al]	333 ± 4.1	345 ± 1.0	350 ± 6.2	400 ± 3.8	400 ± 4.5	400 ± 2.9
F4T [Zn(O)/Ca]	154 ± 3.2	179 ± 6.0	180 ± 3.0	178 ± 3.3	181 ± 1.5	180 ± 4.3
F5T [Zn(O)/Ti]	210 ± 1.5	223 ± 1.0	220 ± 2.4	225 ± 1.7	225 ± 1.5	225 ± 2.9

3.2.2. Sedimentation and turbidity measurements

Fig. 7A, B shows the time-dependent sedimentation and turbidity rates of F1T, F2T, F3T, F4T, and F5T suspensions in culture medium during 48 h. The initial sedimentation rate of all Zn-based compounds is very fast nearly till 2 h (Fig. 7A). The F2T and F5T suspensions (Fig. 7Ab, e) are more stable compared to F1T, F3T, and F4T suspensions (Fig. 7Aa, c, d) over 48 h.

Fig. 7.

(A) Sedimentation and (B) turbidimetric measurements of F1T, F2T, F3T, F4T, and F5T in culture medium and room temperature (mean ± SD, n = 3).

The initial turbidity of F3T and F5T suspensions (Fig. 7Bc, e) is more compared to F1T, F2T, and F4T suspensions (Fig. 7Ba,b,d). Turbidity value of F1T and F3T suspensions was zero after 48 h (Fig. 7Ba, c).

3.3. Solubility of Zn-based compounds in culture medium

Table 5 shows the solubility and pH of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium at room temperature after equilibrium.

Table 5.

[Zn⁺²] ions solubility and pH of saturated solutions of F1T, F2T, F3T, F4T, F5T and commercial ZnO in culture medium (mean ± SD, n = 3).

Sample	Formula	[Zn+2] (mg/L)	pH
Commercial ZnO	ZnO	11.2 ± 0.8	8.5
F1T	[ZnO]	8.9 ± 0.2	7.7
F2T	[Zn(O)/Mg]	2.7 ± 0.1	7.5
F3T	[Zn(O)/Al]	5.9 ± 0.4	7.6
F4T	[Zn(O)/Ca]	3.6 ± 0.2	7.7
F5T	[Zn(O)/Ti]	0.1 ± 0.0	7.3

Solubility of prepared ZnO (F1T, 8.9 mg/L) is close to commercial ZnO (11.2 mg/L, Table 5). There is a decrease in solubility for all metal-doping ZnO nanocomposites (F2T, F3T, F4T, F5T) compared to undoped ZnO (F1T). This phenomenon may be due to change in crystalline lattice and structural strength of the metal–oxide bond (vacancies, interstitial elements, degree of ordering, and etc., Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Table 2, Table 3) of metal-doping Zn-based nanocomposites [9,62]. Also among metal-doping Zn-based nanocomposites, F5T shows lower solubility (0.1 mg/L) compared to F2T, F3T, and F4T (2.7–5.9 mg/L). The pH of suspensions was in the range of 7.3–7.7 (Table 5).

3.4. Release of Zn⁺² ions from the Zn-based nanocompounds in culture medium

The observed cytotoxicity of ZnO nanoparticles has been attributed to dissolve Zn⁺² of their network in environment [63]. In vitro toxic concentrations of Zn⁺² typically exceed 10 mg/L [64]. Fig. 8 shows cumulative released Zn⁺² profiles of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium at 37 °C during 48 h. For commercial ZnO, Zn⁺² ions released completely (Fig. 8f). For undoped ZnO compound, Zn⁺² ions released about 80% and reached to steady state after about 5 h (F1T, Fig. 8a).

Fig. 8.

Cumulative released Zn⁺² profiles of F1T, F2T, F3T, F4T, F5T, and commercial ZnO in culture medium and 37 °C for 48 h (mean ± SD, n = 3).

Metal-doping ZnO nanocomposites showed Zn⁺² ions release of lower than 30% (F2T, F3T, F4T, F5T, Fig. 8b–e). F3T and F4T showed a burst release followed by a steady state after about 8 h and 30 and 22% of Zn⁺² ions released after 48 h, respectively (Fig. 8c, d). F2T and F5T were found stable with lowest release. F5T showed very lower release of about 6% and F2T only about 15%, (Fig. 8b, e) after 6 h.

Also the release medium of F2T, F3T, and F4T was seen to contain about 3, 76, and 44% of Mg⁺², Al⁺³, and Ca⁺² ions released, respectively, but for F5T, no Ti⁺⁴ was detected in the release medium.

3.5. Release of Zn⁺² ions from nanocompounds in the presence of HFF-1 cells

Amount of released Zn⁺² ions in the presence of cells was 12, 33, 21, and 0% after exposure to 20 mg/L of F2T, F3T, F4T, and F5T solutions, respectively (Fig. 9). Results showed that the nature of ZnO structure affects the amount of released Zn⁺² ions in the presence of HFF-1 cells.

Fig. 9.

Zn⁺² release from F1T, F2T, F3T, F4T, and F5T in the presence of HFF-1 cell over 24 h (a) pure amount and (b) percent (mean ± SD, n = 3).

Also 67% Zn⁺² ions released after exposure to 20 mg/L of F1T (undoped ZnO compound, Fig. 9). According to above results, stability of metal-doped ZnO nanocomposites (F2T, F3T, F4T, F5T) has increased compared to undoped ZnO (F1T). The metal dopant (Mg⁺², Al⁺³, Ca⁺², Ti⁺⁴), similar to useful metallic chelator at crystalline structure of ZnO, avoid released Zn⁺² ions of ZnO network into culture medium.

No co-metal ions (Mg⁺², Al⁺³, Ca⁺², Ti⁺⁴) were detected for all metal-doping Zn-based nanocomposites (F2T, F3T, F4T, F5T) in the cellular medium in the presence of HFF-1 cells.

For F2T, F3T, F4T, the percent of released Zn⁺² ions in the presence of HFF-1 cells (Fig. 9) was similar to cell-free conditions (Fig. 8b–d). However, F5T did not show any released Zn⁺² ions in the presence of HFF-1 cells (Fig. 9), opposite to plain culture medium that showed a 6% release (Fig. 8e).

3.6. Intracellular ROS production

The ROS production percentages in Zn-treated cells relative to untreated ones were 31, 3, 9, 4, 0% for F1T, F2T, F3T, F4T, F5T, respectively (Fig. 10a). These results showed that doped compounds have lower effect on formation of intracellular ROS compared to F1T. This phenomenon may be due to high stability of ZnO particles in the presence of co-metal.

Fig. 10.

Nature-dependent measurements of intracellular ROS production (pure amount and percent) in HFF-1 cells (a) for cells exposed to Zn-based compounds (in compared to untreated control) and (b) for cells exposed to Zn-based compounds and UVA radiation (in compared to untreated control) (mean ± SD, n = 3).

When the cells were treated with UVA radiation in the presence of Zn-based compounds, 52, 20, 44, 37, 8% ROS were formed for F1T, F2T, F3T, F4T, F5T, respectively (Fig. 10b). It seems that photo-catalytic protection of ZnO after incorporation of co-metal in its network was increased due to change in the local crystal lattice environment (strength bond, vacancies, degree of ordering of elements, and etc.) of metal ions guest in ZnO host. However, among these compounds, F5T acts as a strong preservative and F2T, F3T, F4T act as a relatively lower preservative against UVA radiation.

Fig. 10a, b also shows that the effects of UVA radiation on intracellular ROS generation are more than that of Zn-based compounds.

3.7. Cell viability study

Nature-dependent protective effect of Zn-based compounds in HFF-1 cells after exposure to UVA radiation was demonstrated by MTT assay (Fig. 11). Cells showed a viability of about 9% when was treated with F1T (ZnO). For F2T and F5T, cell viability was increased sharply to 81 and 109%, respectively. Others Zn-based compounds also showed higher viabilities than ZnO, 44 and 32% for F3T, and F4T respectively. Among metal-doped ZnO nanocomposites, the lower protective effects of F3T and F4T compared to F2T and F5T were attributed to interaction aluminum oxide and calcium oxide on ZnO surface in F3T and F4T, respectively (Fig. 1c, d).

Fig. 11.

Nature-dependent cell viability of HFF-1 cells exposed to Zn-based compounds and UVA radiation (mean ± SD, n = 3).

This phenomenon might be discussed by band-gap energy as discussed below. As according to DRS results (Table 3), due to interaction between the band electrons of ZnO and the electrons of valance orbitals of co-metal (Ca⁺², Mg⁺², Al⁺³, Ti⁺⁴), new defects were appeared in band gap of ZnO. Therefore band gap position of composites was changed and new storage energy source was formed. It seems that the new electronic states (E_g) in the metal-doped ZnO composites are greatly responsible for special absorbance/transmittance property (photo-catalyst activity) of these composites. Therefore these composites act as a protective agents for cells against UVA radiation.

4. Conclusion

In summary, different ZnO nanocomposites containing Mg⁺², Al⁺³, Ca⁺², and Ti⁺⁴ (as co-metals) were synthesized and characterized here. Dopant ions were uniformly incorporated and replaced zinc sites in the hexagonal crystal lattice. These newly developed nanocomposites were able to protect of human skin cells (HFF-1) against UVA radiation photodamage. The protection was more than plain ZnO and the cytotoxicity of the doped nanocompounds was lower than non-doped ZnO.

This study is an initial attempt toward synthesis of the bimetallic zinc nanostructures. Modification of synthetic approach, purity of chemical of nanocomposites, and application of these compounds in a wide range of human skin protection, including preparation of a safer nano-zinc sunscreen are in progress in our laboratories.