A New Sterilization Strategy Using TiO 2 Nanotubes for Production of Free Radicals that Eliminate Viruses and Application of a Treatment Strategy to Combat Infections Caused by Emerging SARS-CoV-2 during the COVID-19 Pandemic

: Synthesized titanium oxide nanoparticles (TiO 2 -NPs) nanotubes were used for the disinfection of new emerging corona virus-19 (SARS-CoV-2) in this study. The newly synthesized TiO 2 -NPs (nanotubes) were characterized by chemical spectroscopic analysis Fourier-transform infrared spectroscopy and ultraviolet FT-IR and UV. The chemical purity and Zeta potential distribution of the TiO 2 -NPs (nanotubes) were evaluated to conﬁrm their nano-range, and their surface morphology was determined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR-TEM), and energy dispersive X-ray analysis (EDX). The antiviral activity of the TiO 2 -NPs (nanotubes) against SARS-CoV-2 was evaluated using 10% (Dimethyl sulfoxide) DMSO and dist.H 2 O using a cytotoxicity assay and inhibitory concentration assay (to determine the cytotoxic half concentration CC 50 and half maximal inhibitory concentration IC 50 ). The current results conﬁrmed that TiO 2 -NPs exhibit strong anti-SARS-CoV-2 activity at very low cytotoxic concentrations in vitro with a non-signiﬁcant selectivity index (CC 50 /IC 50 ≤ 1). The obtained results indicate that TiO 2 -NPs and nanotubes have potent antiviral activity at a very low concentrations (IC 50 = 568.6 ng/mL), with a weak cytotoxic effect on the cellular host (CC 50 = 399.1 ng/mL). Thus, we highly recommend the use of TiO 2 -NPs (nanotubes) in vitro and in wall coatings as a potent disinfectant to combat SARS-CoV-2 with little irritation of the cellular hosts. Furthermore, we also recommend more and excessive prospective studies on the complexation of natural active or natural compounds with TiO 2 -NPs (nanotubes) to minimize their cytotoxicity, enhance their antiviral activity, and increase their inhibition of SARS-CoV-2. sheet of cells treated with TiO 2 -NPs showing high enlarged patches around SRAS-CoV-2 cells, thus demonstrating the ability of TiO 2 -NPs (nanostructures) to disinfect SARS-CoV-2 and offer a high level of inhibition to its growth. Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (hCoV-19/Egypt/NRC-03/2020).


Introduction
SARS-CoV-2 is considered a Ribonucleic acid "RNA" virus. RNA viruses cause numerous diseases, particularly in mammals, and are considered major pathogens in many diseases including Hepatitis C Virus (HCV), Ebola disease, SARS, influenza, measles and retrovirus, and human immunodeficiency virus (HIV) [1]. CoV infections first affect the upper respiratory tract of mammals. Coughing and fever are considered major symptoms of CoV infection in humans, and breathing difficulty is also evident as a result of SARS-CoV-2. COVID-19 s clinical symptoms include severe respiratory disorders caused by SARS-CoV-2, such as excessive inflammation and oxidation [2]. Scheme 1. Graphical abstract for the experimental protocol and summarization of the study.

Synthesis of Titanium Oxide Nanostructure
TiO2-NPs were synthesized according to [16] in two steps. In the first, 0.2 M TiO2 was synthesized by adding 0.80 g of TiO2 to 100 mL of H2O dist. and stirring for 2 h. Another solution with 10.32 g of citrate of tri-sodium (50 mL H2O) was synthesized and added dropwise to the first solution. This rate was maintained for about 2 h. Then, a precipitate was produced in the solution; the precipitate was washed 3 times before centrifuging for 15 min. The precipitate was then dried in an oven at 1100 °C for about 24 h, yielding TiO2-NP powder. A quantity of 20 mL HNO3 solution was then mixed with the remaining samples and sonicated for 1 h, before the addition of 0.1 M of sodium tri citrate followed by 2 h sonication. The synthesized samples were then washed 4 times. TNTs were prepared using a hydrothermal process described in [17]. Then, 5 g of TiO2-NPs prepared by the sol-gel method were mixed with 500 mL of a 10 M NaOH aqueous solution, followed by hydrothermal treatment at 150 °C (TNTs) in a Teflon-lined autoclave for 12 h. After the hydrothermal reaction, the treated powders were washed thoroughly with distilled water and 0.1 M HCl, and subsequently filtered and dried at 80 °C for 1 day. To achieve the desired TNT size and crystallinity, the powders were calcined in air at 500 °C for 1 h [16,17]. The resulting TiO2-NPs were used to testing their activity against SARS-CoV-2.

Chemical Characterization of TiO2-Nanostructures
By using tools of analysis, the shape and size of TiO2-Nps were characterized using images of scanning electron microscopy (Tokyo, Japan), transmition electron microscopy (Tokyo, Japan) and high resolution transmition electron microscopy (Nakagyo-ku, Kyoto, Japan). Ti and O analysis measured using EDX. Electronic spectrum of (Nakagyo-ku, Kyoto, Japan) TiO2-nanostructure was analyzed using UV-Vis spectroscopy (Nakagyo-ku, Kyoto, Japan). The IR analysis performed mixing TiO2-Nanostrucure powder with KBr. The X-ray diffraction patterns were recorded on X'Pert PRO PAN-analytical X-ray powder diffraction (Nakagyo-ku, Kyoto, Japan), target copper with secondary monochromate.

Synthesis of Titanium Oxide Nanostructure
TiO 2 -NPs were synthesized according to [16] in two steps. In the first, 0.2 M TiO 2 was synthesized by adding 0.80 g of TiO 2 to 100 mL of H 2 O dist. and stirring for 2 h. Another solution with 10.32 g of citrate of tri-sodium (50 mL H 2 O) was synthesized and added dropwise to the first solution. This rate was maintained for about 2 h. Then, a precipitate was produced in the solution; the precipitate was washed 3 times before centrifuging for 15 min. The precipitate was then dried in an oven at 1100 • C for about 24 h, yielding TiO 2 -NP powder. A quantity of 20 mL HNO 3 solution was then mixed with the remaining samples and sonicated for 1 h, before the addition of 0.1 M of sodium tri citrate followed by 2 h sonication. The synthesized samples were then washed 4 times. TNTs were prepared using a hydrothermal process described in [17]. Then, 5 g of TiO 2 -NPs prepared by the sol-gel method were mixed with 500 mL of a 10 M NaOH aqueous solution, followed by hydrothermal treatment at 150 • C (TNTs) in a Teflon-lined autoclave for 12 h. After the hydrothermal reaction, the treated powders were washed thoroughly with distilled water and 0.1 M HCl, and subsequently filtered and dried at 80 • C for 1 day. To achieve the desired TNT size and crystallinity, the powders were calcined in air at 500 • C for 1 h [16,17]. The resulting TiO 2 -NPs were used to testing their activity against SARS-CoV-2.

Chemical Characterization of TiO 2 -Nanostructures
By using tools of analysis, the shape and size of TiO 2 -Nps were characterized using images of scanning electron microscopy (Tokyo, Japan), transmition electron microscopy (Tokyo, Japan) and high resolution transmition electron microscopy (Nakagyo-ku, Kyoto, Japan). Ti and O analysis measured using EDX. Electronic spectrum of (Nakagyo-ku, Kyoto, Japan) TiO 2 -nanostructure was analyzed using UV-Vis spectroscopy (Nakagyo-ku, Kyoto, Japan). The IR analysis performed mixing TiO 2 -Nanostrucure powder with KBr. The X-ray diffraction patterns were recorded on X'Pert PRO PAN-analytical X-ray powder diffraction (Nakagyo-ku, Kyoto, Japan), target copper with secondary monochromate.

Zeta Potential Measurements
It is a technique for assessment of the particle surface charge in the solution, which is directly related to the stability of the nanoparticle suspension. Zeta potential values revealed the stability of the prepared nanoparticles and nanotubes. The pH effect on the size of particles and the zeta potential distributions of TiO 2 -Nps (nanostructures) were evaluated by using pH from 5 to 12 by drop wise addition of solutions of 0.1 M NaOH or HCl.

Cytotoxicity Assay
To evaluate the cytotoxic half concentration (CC 50 ) (maximal) of TiO 2 -NPs, stock solutions of TiO 2 -NPs in 10% concentration of DMSO were prepared in "dist.H 2 O" and diluted to the solutions of Dulbecco's Modified Eagle Medium DMEM. Cytotoxic activity of TiO 2 -NPs was evaluated and tested in VERO-E6 cells by using MTT assay. VERO-E6 cells were completely seeded in (3 × 10 5 cells/mL, about 100 µL/each well) concentration, then incubated for 24 h in CO 2 (5%) at 37 • C for (24 h) later, VERO-E6 cells, the host for SARS-CoV-2 were treated with different concentrations of TiO 2 -NPs in triplicates. Then, the upper supernatants were completely discarded, and the monolayers were excessively washed with sterile (PBS) buffer phosphate saline for (3 times) and addition of (20 µL of TiO 2 -NPs) of MTT solution to 96 well-plates and incubated for about 4 h at 37 • C. The formed crystals of formazan were dissolved with isopropanol treated with HCl. Their absorbance was measured at λ max 540 nm with 620 nm as a reference λ by using a multiwell plate reader (Nakagyo-ku, Kyoto, Japan). The % of cytotoxicity as compared to the untreated cells was determined by the following Equation (1).
The % cytotoxicity against sample conc. was used to calculate the conc. which exhibited 50% of the cellular cytotoxicity (CC 50 ).

Electronic Spectrum of TiO2-NPs
The UV-Vis absorption spectra of the synthesized TiO2-NPs (nanostructures) yielded a broad absorption spectrum that appeared at a wavelength of 350 nm. This result is consistent with those in the literature [19]. Figure 2 shows the electronic spectrum of TiO2-NPs. The electronic spectrum of TiO2 is shown in Figure 3, after heating at 150 °C. The peak refers to titanium oxide produced, strong peak resulted at wavelength of 350 nm This was due to the band intrinsic absorption gap for titanium oxide produced due to the electronic transition from the valence band to the conduction band [20]. The band gap energy (Eg) of titanium oxide is estimated according to Eg = hc/λ, where c is the light speed, equal to 3 × 10 8 m/s; h is the constant plank, equal to 6.626 × 10 −34 Js; and λ is the wavelength (m) [21,22]. The energy of band gap is equal to 3.26 eV.

Electronic Spectrum of TiO 2 -NPs
The UV-Vis absorption spectra of the synthesized TiO 2 -NPs (nanostructures) yielded a broad absorption spectrum that appeared at a wavelength of 350 nm. This result is consistent with those in the literature [19]. Figure 2 shows the electronic spectrum of TiO 2 -NPs. The electronic spectrum of TiO 2 is shown in Figure 3, after heating at 150 • C. The peak refers to titanium oxide produced, strong peak resulted at wavelength of 350 nm. This was due to the band intrinsic absorption gap for titanium oxide produced due to the electronic transition from the valence band to the conduction band [20]. The band gap energy (Eg) of titanium oxide is estimated according to Eg = hc/λ, where c is the light speed, equal to 3 × 10 8 m/s; h is the constant plank, equal to 6.626 × 10 −34 Js; and λ is the wavelength (m) [21,22]. The energy of band gap is equal to 3.26 eV.

Electronic Spectrum of TiO2-NPs
The UV-Vis absorption spectra of the synthesized TiO2-NPs (nanostructures) yielded a broad absorption spectrum that appeared at a wavelength of 350 nm. This result is con sistent with those in the literature [19]. Figure 2 shows the electronic spectrum of TiO2 NPs. The electronic spectrum of TiO2 is shown in Figure 3, after heating at 150 °C. Th peak refers to titanium oxide produced, strong peak resulted at wavelength of 350 nm This was due to the band intrinsic absorption gap for titanium oxide produced due to th electronic transition from the valence band to the conduction band [20]. The band gap energy (Eg) of titanium oxide is estimated according to Eg = hc/λ, where c is the ligh speed, equal to 3 × 10 8 m/s; h is the constant plank, equal to 6.626 × 10 −34 Js; and λ is th wavelength (m) [21,22]. The energy of band gap is equal to 3.26 eV.

Zeta Potential and Size Distribution Intensity
The diameter of TiO2-NPs (nanostructures) and Zeta potential in dist.H2O at pH value of ~7.36 determined by ultrasonication using Dynamic light scattering DLS of prepared dispersions using 0.2 mg of TiO2-NPs in 10 mL of H2O deionized. TiO2-NPs (nanostructures) particles' diameter average of TiO2-NPs was measured to be 470.6 nm with zeta potential of −5.92 mV and Polydispersity index pdI = 0.364. Zeta potential and size of particles TiO2-NPs were determined (Figure 3a,b) using pH 6−12 and the zeta potential decreases while the size of particles increases by increasing the pH. Measurements of TiO2-NPs (nanostructures) surface area attributed to at various TiO2-NPs (nanostructures) burning temperatures occurred by N2-adsorption of N2 at 77 K. As temperature of calcination elevated from (29 m 2 ·g −1 ) for 82 ± 10 nm TiO2-NPs (nanostructures) at 100 °C to (7 m 2 ·g −1 ) for 265 ± 8 nm at 600 °C surface area of TiO2-NPs (nanostructures) decreased which is convenient with a previous study [20]. Table 1 shows the mean particle size or the average dynamic sizes of the TiO2-NPs (nanostructures) were found 953.6 d·nm with pdI equal to 0.526.

Zeta Potential and Size Distribution Intensity
The diameter of TiO 2 -NPs (nanostructures) and Zeta potential in dist.H 2 O at pH value of~7.36 determined by ultrasonication using Dynamic light scattering DLS of prepared dispersions using 0.2 mg of TiO 2 -NPs in 10 mL of H 2 O deionized. TiO 2 -NPs (nanostructures) particles' diameter average of TiO 2 -NPs was measured to be 470.6 nm with zeta potential of −5.92 mV and Polydispersity index pdI = 0.364. Zeta potential and size of particles TiO 2 -NPs were determined (Figure 3a,b) using pH 6−12 and the zeta potential decreases while the size of particles increases by increasing the pH. Measurements of TiO 2 -NPs (nanostructures) surface area attributed to at various TiO 2 -NPs (nanostructures) burning temperatures occurred by N 2 -adsorption of N 2 at 77 K. As temperature of calcination elevated from (29 m 2 ·g −1 ) for 82 ± 10 nm TiO 2 -NPs (nanostructures) at 100 • C to (7 m 2 ·g −1 ) for 265 ± 8 nm at 600 • C surface area of TiO 2 -NPs (nanostructures) decreased which is convenient with a previous study [20]. Table 1 shows the mean particle size or the average dynamic sizes of the TiO 2 -NPs (nanostructures) were found 953.6 d·nm with pdI equal to 0.526.  Figure 4a,b represents a scanning electron microscopy (SEM) image of TiO 2 -NPs (nanostructures). The morphology of the TiO 2 -NPs (nanotubes) was examined, and the size of the grains was nearly uniform. The morphology surfaces of the prepared TiO 2 -NPs (nanostructures) powders were measured using SEM analytical techniques, such as wide plates and nodes with homogenously sized distributions for TiO 2 -NPs. Figure 4b shows an FE-SEM image of the sample anatase TiO 2 -NPs nanotubes, which were grown at 150 • C for 12 h, and exhibit a pure tube-like structure. The length of the TiO 2 -NPs nanotubes is several micrometers, their diameter is approximately 50 to 100 nm, and they are very uniform, relatively clean, and smooth-surfaced. Figure 3 shows the surface morphology of the electrode film on the FTO glass. Chemical composition analysis by EDX confirmed the Ti and O peaks in the TiO 2 -NPs (nanostructures). The sample elemental analysis resulted in values of 78% for Ti and 20% for O, which confirms the high purity of TiO 2 -NPs (nanotubes) [21,22].  Figure 4a,b represents a scanning electron microscopy (SEM) image of TiO2-NPs (nanostructures). The morphology of the TiO2-NPs (nanotubes) was examined, and the size of the grains was nearly uniform. The morphology surfaces of the prepared TiO2-NPs (nanostructures) powders were measured using SEM analytical techniques, such as wide plates and nodes with homogenously sized distributions for TiO2-NPs. Figure 4b shows an FE-SEM image of the sample anatase TiO2-NPs nanotubes, which were grown at 150 °C for 12 h, and exhibit a pure tube-like structure. The length of the TiO2-NPs nanotubes is several micrometers, their diameter is approximately 50 to 100 nm, and they are very uniform, relatively clean, and smooth-surfaced. Figure 3 shows the surface morphology of the electrode film on the FTO glass. Chemical composition analysis by EDX confirmed the Ti and O peaks in the TiO2-NPs (nanostructures). The sample elemental analysis resulted in values of 78% for Ti and 20% for O, which confirms the high purity of TiO2-NPs (nanotubes) [21,22].

Transmission Electron Microscope (TEM) and High Resolution Transmittance Electron Micrographs (HRTEM) of TiO2-NPs
The morphology of the TiO2-NPs (nanostructures) sample was determined by using TEM. Figure 5a,b shows a typical TEM image of the TiO2-NPs. The size and morphology of the TiO2-NPs (nanostructures) were estimated using HRTEM micrographs. TiO2-NP nanoparticles were found in the 40-60 nm range with a spherical to polygonal medium. HRTEM Figure 6a,b showed a lattice space of 0.22 for TiO2 planes.

Transmission Electron Microscope (TEM) and High Resolution Transmittance Electron Micrographs (HRTEM) of TiO 2 -NPs
The morphology of the TiO 2 -NPs (nanostructures) sample was determined by using TEM. Figure 5a

XRD of TiO2-Nanotubes
XRD for TiO2-NPs (nanostructures) is shown in Figure 7a which provides the sol-gel pattern at 450 °C, confirming the presence of anatase and rutile phases' mixture. Prominent anatase peaks and prominent rutile peaks of TiO2-NPs (nanostructures) clearly presented using XRD pattern confirming the nanoparticle structure. The patterns of the TiO2

XRD of TiO2-Nanotubes
XRD for TiO2-NPs (nanostructures) is shown in Figure 7a which provides the sol-gel pattern at 450 °C, confirming the presence of anatase and rutile phases' mixture. Prominent anatase peaks and prominent rutile peaks of TiO2-NPs (nanostructures) clearly presented using XRD pattern confirming the nanoparticle structure. The patterns of the TiO2

XRD of TiO 2 -Nanotubes
XRD for TiO 2 -NPs (nanostructures) is shown in Figure 7a which provides the sol-gel pattern at 450 • C, confirming the presence of anatase and rutile phases' mixture. Prominent anatase peaks and prominent rutile peaks of TiO 2 -NPs (nanostructures) clearly presented using XRD pattern confirming the nanoparticle structure. The patterns of the TiO 2 nanotube films prepared at a hydrothermal temperature of 180 • C for 14 h are shown in Figure 8. By the hydrothermal method, TiO 2 nanoparticles were observed to be transformed into the anatase phase. From XRD patterns (Figure 7b), the TiO 2 -NPs (nanostructures) possess crystallized highly anatase structure with high degree of purity. The rutile peaks for TiO 2 -NPs nanotubes, refers to complete transformation to anatase.
Coatings 2021, 11, x FOR PEER REVIEW 9 of 1 nanotube films prepared at a hydrothermal temperature of 180 °C for 14 h are shown i Figure 8. By the hydrothermal method, TiO2 nanoparticles were observed to be trans formed into the anatase phase. From XRD patterns (Figure 7b), the TiO2-NPs (nanostruc tures) possess crystallized highly anatase structure with high degree of purity. The rutil peaks for TiO2-NPs nanotubes, refers to complete transformation to anatase.

Cytotoxicity of TiO2-NPs and Inhibition Activity against SARS-CoV-2
TiO2-NPs showed potent antiviral activity against SARS-CoV-2 in very low concen tration of 568.6 ng/mL. The MTT assay was used to evaluate potential cell cytotoxicity o TiO2-NPs. The results showed that 399.1 ng/mL of TiO2-NPs significantly reduced the vi ability of VERO-E6 cells (Figure 8).

Anti-Viral Activity and Cytotoxicity of Titanium Oxide Nanoparticles and Inhibition Activity Against SARS-CoV-2
TiO2-NPs (nanotubes) showed potent antiviral activity against SARS-CoV-2 in ver low concentration of 568.6 ng/mL, by using MTT assay to elucidate the potential cell cy totoxicity of TiO2-NPs. The results showed that 399.1 ng/mL of TiO2-NPs (nanostructures nanotube films prepared at a hydrothermal temperature of 180 °C for 14 h are shown in Figure 8. By the hydrothermal method, TiO2 nanoparticles were observed to be trans formed into the anatase phase. From XRD patterns (Figure 7b), the TiO2-NPs (nanostruc tures) possess crystallized highly anatase structure with high degree of purity. The rutil peaks for TiO2-NPs nanotubes, refers to complete transformation to anatase.

Cytotoxicity of TiO2-NPs and Inhibition Activity against SARS-CoV-2
TiO2-NPs showed potent antiviral activity against SARS-CoV-2 in very low concen tration of 568.6 ng/mL. The MTT assay was used to evaluate potential cell cytotoxicity o TiO2-NPs. The results showed that 399.1 ng/mL of TiO2-NPs significantly reduced the vi ability of VERO-E6 cells (Figure 8).

Anti-Viral Activity and Cytotoxicity of Titanium Oxide Nanoparticles and Inhibition Activity Against SARS-CoV-2
TiO2-NPs (nanotubes) showed potent antiviral activity against SARS-CoV-2 in very low concentration of 568.6 ng/mL, by using MTT assay to elucidate the potential cell cy totoxicity of TiO2-NPs. The results showed that 399.1 ng/mL of TiO2-NPs (nanostructures 2 θ/°

Cytotoxicity of TiO 2 -NPs and Inhibition Activity against SARS-CoV-2
TiO 2 -NPs showed potent antiviral activity against SARS-CoV-2 in very low concentration of 568.6 ng/mL. The MTT assay was used to evaluate potential cell cytotoxicity of TiO 2 -NPs. The results showed that 399.1 ng/mL of TiO 2 -NPs significantly reduced the viability of VERO-E6 cells (Figure 8).

Anti-Viral Activity and Cytotoxicity of Titanium Oxide Nanoparticles and Inhibition Activity Against SARS-CoV-2
TiO 2 -NPs (nanotubes) showed potent antiviral activity against SARS-CoV-2 in very low concentration of 568.6 ng/mL, by using MTT assay to elucidate the potential cell cytotoxicity of TiO 2 -NPs. The results showed that 399.1 ng/mL of TiO 2 -NPs (nanostructures) significantly reduced the viability of VERO-E6 cells as shown in (Figure 8) and images of the host cells "Vero-E6" cell after the incubation with SARS-CoV-2 (after the stage of infection) which showed a high potent anti-viral activity of TiO 2 -NPs against SARS-Cov-2, the cells' sheet that was treated with TiO 2 -NPs showing enlarged holes and patches around SRAS-CoV-2 as shown in (Figure 9A-C).  Differences between zinc oxide nanoparticles (ZnO-NPs) inhibition activity [23][24][25][26][27] on SARS-CoV-2 cells as cited in our previous work [28] and inhibition activity of titanium oxide nanoparticles and its nanotubes (TiO2-NPs) as shown in Table 2 clarified that TiO2-NPs has more potent antiviral activity against SARS-CoV-2 than ZnO-NPs, and showed a higher CC50/IC50 ratio in TiO2-NPs (0.7) than ZnO-NPS. Table 2. Comparison between inhibition activity for SARS-CoV-2 (differences between antiviral activity of both nanoparticles against SARS-CoV-2).

Discussion
Viral prevention remains a major objective for the maintenance of human health, and the essential concept is the inhibition of viral replication. Current antivirals have been weakened due to the elevation of drug resistance, particularly during the COVID-19 pandemic [23].
Thus, the release of new effective anti-COVID-19 treatments is urgently needed.
Nanoparticles provide significant benefits because they are highly effective as antiviral agents, even in low concentrations. In addition, nanoparticles have vital characteristics that ensure their suitability for different types of coating [24][25][26][27].
The present study reported that TiO 2 -NPs (nanostructures) have potent anti-SARS-CoV-2 activity with very low cytotoxicity, as indicated by their SI value (IC 50 /CC 50 ≤ 1). This finding is consistent with [16], who revealed that TiO 2 -NPs (nanostructures) have stronger antiviral activity and low cytotoxicity when coated with Polyethylene Glycated -NPs, compared to TiO 2 -NPs alone. Results of the current study thus highlight the use of TiO 2 -NPs as a disinfecting agent against SARS-CoV-2 with low cytotoxicity. Similarly, we suggest using TiO 2 -NPs (nanostructures) coated with Polyethylene Glycated (polyethylene Glycated), which plays a major role in enhancing and elevating the antiviral activity against SARS-CoV-2 virus, and may reduce the cellular cytotoxicity of TiO 2 -NPs (nanostructures) for the host cells.
Additional confirmation of our findings and suggestion to coat TiO 2 -NPs (nanotubes) was provided in our previous research [28]. A recent explanation is that TiO 2 -NPs produce Ti +2 ions and thus produce a large quantity of reactive oxygen species (ROS); these free radicals can damage proteins, lipids, carbohydrates, and DNA, and eventually lead to cellular apoptosis [29,30]. Therefore, we suggest coating TiO 2 -NPs (nanostructures) with polyethylene glycol or natural active compounds that can provide strong anti-SARS-CoV-2 activity to reduce cytotoxicity and prevent the release of reactive oxygen by the masking of TiO 2 -NPs (nanostructures).
Nanoscience has recently identified an essential vital role of nanoparticle agents due to their direct inhibition of a wide range of microbes. Nanoparticles such as TiO 2 -NPs (nanotubes) are considered to be effective agents for carrying a large number of drugs and vaccines. Additionally, the size of nanomaterials accelerates their drug delivery involvement due to their surface functions. As a result, nanoparticles have the capability to co-transport drugs and numerous agents [31].
The relationship between SARS-CoV-2A and this vital aspect of nanomaterials is evidence of the competitive ability of the host with a viral binding surface cellular receptor because receptors of ACE2 are essential in the action of SARS-CoV-2 entering host cells [32]. Hence, blocking of ACE2 receptors could help in fighting SARS-CoV-2 [33]. Therefore, preventing SARS-CoV-2 infection in host cells plays an important role in combatting viral infection. To prevent viruses entering the human body, it is important to prepare nano-formulas using recently developed nanotechnology to prevent viral entry via ACE2 receptors [34]. Additionally, the catalytic activity and stability of ACE can be enhanced using nanoparticles to inhibit entry of SARS-CoV-2 [34,35]. Therefore, if TiO 2 -NPs (nanotubes) are used in coatings for hospitals walls, the spread of SARS-CoV-2 may be significantly reduced. Thus, we proposed a hypothetical antiviral mechanism for TiO 2 -NPs [36][37][38][39], including ACE2, which is able to block the SARS-CoV-2 entry into the cellular host by inhibiting its attachment to the ACE2 receptors [40].
The current finding broadly supports our previous work [28], which revealed that metal nanoparticles, ZnO-NPs, demonstrated significant activity against different types of microorganisms, including most viruses. The reported mechanism was the same as that shown in the current study; however, TiO 2 -NPs revealed more antiviral activity with less cytotoxicity than ZnO-NPs.
As indicated in previous studies [17], TiO 2 -NPs (nanostructures) have attracted increasing attention as an antimicrobial agent. TiO 2 -NPs (nanostructures) have the ability to bind to the bacterial cellular wall and can enter the cellular membrane via a significant number of modifications, resulting in bacterial cellular death [41]. Another mechanism is the development of "pits" at the cellular surface, resulting in the accumulation of TiO 2 -NPs (nanostructures) [42], in turn leading to the accumulation of free radicals that cause cellular death [43,44]. Due to the global COVID-19 pandemic, the transmission of the SARS-CoV-2 virus largely occurs by direct viral attachment to cellular hosts. TiO 2 will be of significant benefit by inducing the triggering of free radicals on the viral cell surface and thus causing the death of the virus. This concept confirms our findings relating to the anti-SARS-CoV-2 and inhibition activities in very low concentrations, and is considered to be a promising result.
The results of the current study are consistent with the findings of [45], who demonstrated that TiO 2 -NPs (nanostructures) have a large number of potential applications due to their high chemical stability, wide photoactivity, high pH range, and the presence of numerous active adsorption sites on their surface, which may help in absorbing pollutants. Due to their high photoactivity, TiO 2 -NPs (nanostructures) are widely applied to the removal of water pollutants in water treatment.
TiO 2 has a wide range of antimicrobial activity, as shown in several studies using TiO 2 against different types of microorganisms, such as viruses [46,47]. The efficiency of TiO 2 -NPs (nanotubes) is related to their surface reactions, surface morphology, and surface size ratio [48]. Researchers have focused on TiO 2 synthesis with new features that make it suitable for a large number of green environmental applications, such as the effective control of viral infections. Negatively charged TiO 2 -NPs (nanostructures) can block viruses and inhibit their attachment to host cells.
It was somewhat unexpected to obtain data that confirm the anti-COVID-19 activity of TiO 2 -NPs (nanotubes) with low cytotoxicity. These findings verify the anti-SARS-CoV-2 activity of TiO 2 -NPs at a low concentration (568.6 ng/mL) with low cytotoxicity, which can be alleviated by coating TiO 2 -NPs (nanotubes) with natural active compounds or polymeric compounds, such as PEGylated. These coatings may reduce the diameter of the particles and moderate the triggering of cytotoxic effects of ROS on the cellular host, thus achieving effective anti-SARS-CoV-2 action in low concentrations. These findings reveal the potential for the use of TiO 2 -NPs (nanostructures) to combat COVID-19.
The use of metal oxides in nanotechnology represents a new strategy for combatting microorganisms, owing to direct antiviral activity or enhancing an organism's resistance to the pathogen. In this study, the ability of TiO 2 -NPs (nanostructures) to inhibit SARS-CoV-2 and, thus, their antiviral activity were tested. The results showed a significant decline in virus activity. In addition, our results also showed the high efficacy of TiO 2 -NPs nanotubes.
Previous studies showed that hepatic enzymes were elevated in experiments on male rats, indicating that damage had occurred in the plasma membrane structure due to the presence of reactive oxygen species (ROS) produced during the oxidative stress. These results are in agreement with El-Shenawy et al. [49] who found that the elevation of hepatic enzymes could be due to oxidative stress induced by TiO 2 nanostructures, which caused an imbalance in the permeability of the plasma membrane. Elevated levels of serum hepatic enzymes are indicative of cellular leakage and the loss of the functional integrity of cell membranes in tissues, which, in turn, signify tissue injury [49].
Different metal oxide nanotubes have been developed using different methodologies, precursors, and fibers. TiO 2 -NPs nanotubes have been obtained using different techniques [23][24][25]. However, no development has been carried out with antiviral purposes, particularly against the SARS-CoV-2 virus.
TiO 2 is one of the most widely applied nanomaterials and is extensively used in food applications [50]. TiO 2 -NPs nanostructures have been previously developed with antimicrobial goals. TiO 2 nanotubes have been shown to reduce E. coli and S. aureus. Recently, Jian et al. developed TiO 2 and silver loaded-TiO 2 antibacterial agents using the sol-assay method with minimal inhibition concentration [51,52].
TiO 2 -NPs (nanostructures) have been reported to induce the excessive production of free radicals and thus cause oxidative stress and a series of inflammatory processes and cytotoxicity [53]. These findings are in accordance with the current findings, which showed that prepared TiO 2 -NPs nanotubes induced excessive free radicals, resulting in oxidative stress that may induce cytotoxicity in the context of SARS-CoV-2.
Despite the promising properties of TiO 2 -NPs (nanostructures), research is underway to change the NPs' surfaces' characteristics in order to elevate the efficiency of reactive oxygen species generation and to improve its chemical properties. Surface-modified TiO 2 -NPs can make it to be doped with various metal ions [54] or combined with various dyes [55,56] and this will very helpful for coatings of walls with this new synthesized nanotubes of TiO 2 -NPs (nanostructures) that can give sterilization option for coatings. Surface complexes that enhancing TiO 2 as photosensitizers include transition metals. Inorganic ligands, such as CN − , F − , PO 4 3− can also link surface titanium. If the molecule absorbs a photon with energy higher than its value, it passes to an excited state and can trigger more electrons that are negatively charged in the conduction band, leaving positively charged holes in the valence band. Freeē may attack surrounding O 2 and H 2 O molecules to form reactive oxygen species, including superoxide (O 2− ), (H 2 O 2 ), and hydroxyl radical (•OH) [57]. These forms of superoxides are highly unstable in the biological systems and react with the cellular components causing necrotic cell death or apoptotic. It has also been proven that TiO 2 -NPs (nanostructures) inhibit bacterial multidrug resistance [36]. Human health threats from SARS viruses have been present over time. Uncontrolled bat, avian mouse and human SARS-CoV-dispersal can impact both global public health and economic stability [58]. All these finding confirmed the obtained results and recommend using synthesized (TiO 2 -NPS) (nanostructures) as a sterilization agent against SARS-CoV-2.

Conclusions
TiO 2 -NPs (nanostructures) were fully chemically characterized using different spectroscopic tools. The synthesized titanium nanotubes have a potent antiviral activity against SARS-CoV-2 at low concentration and can trigger many free radicals that may cause oxidative stress to SARS-CoV-2, which is a very promising result in combating SARS-CoV-2. Thus, we recommend more prospective studies applied to TiO 2 -NPs immobilization with other active compounds that may reduce this cytotoxicity and enhance its anti-SARS-CoV-2 activity.