Will the Bacteria Survive in the CeO2 Nanozyme-H2O2 System?

As one of the nanostructures with enzyme-like activity, nanozymes have recently attracted extensive attention for their biomedical applications, especially for bacterial disinfection treatment. Nanozymes with high peroxidase activity are considered to be excellent candidates for building bacterial disinfection systems (nanozyme-H2O2), in which the nanozyme will promote the generation of ROS to kill bacteria based on the decomposition of H2O2. According to this criterion, a cerium oxide nanoparticle (Nanoceria, CeO2, a classical nanozyme with high peroxidase activity)-based nanozyme-H2O2 system would be very efficient for bacterial disinfection. However, CeO2 is a nanozyme with multiple enzyme-like activities. In addition to high peroxidase activity, CeO2 nanozymes also possess high superoxide dismutase activity and antioxidant activity, which can act as a ROS scavenger. Considering the fact that CeO2 nanozymes have both the activity to promote ROS production and the opposite activity for ROS scavenging, it is worth exploring which activity will play the dominating role in the CeO2-H2O2 system, as well as whether it will protect bacteria or produce an antibacterial effect. In this work, we focused on this discussion to unveil the role of CeO2 in the CeO2-H2O2 system, so that it can provide valuable knowledge for the design of a nanozyme-H2O2-based antibacterial system.


Introduction
Nanozymes, as a type of nanomaterial with enzyme-like activities, have recently garnered considerable researchers' attention toward their biological and biomedical applications [1][2][3]. Thus far, various types of nanomaterials have been reported to have catalytic activities similar to those of peroxidase, catalase, superoxide dismutase, oxidase, and other natural enzymes, including different metal and metal oxide nanoparticles, carbon-based nanomaterials, as well as some organic nanostructures [4][5][6][7]. Compared with natural enzymes, nanozymes exhibit several advantages such as tunable size and activity, facile preparation, low cost, and high stability against denaturing [8,9]. These superior properties sufficiently meet the demands for diverse biological and biomedical applications. Indeed, nanozymes have recently been frequently used for disease treatment, such as cancer therapy, bacterial disinfection, as well as Alzheimer's disease treatment [10][11][12].
Bacterial infection has long been a major threat for human health. Antibiotics treatment is currently a golden standard for bacterial disinfection. However, the overuse of antibiotics greatly promotes antibiotic resistance development in bacterial pathogens. Therefore, the exploration of new antimicrobial strategies is urgently demanded [13][14][15][16]. Nanozymebased antibacterial strategies have been recently demonstrated to be a promising new approach for bacterial infection treatment [17][18][19][20][21][22][23][24]. For example, Qu et al. developed graphene quantum dots (GQDs) that can act as a peroxidase mimic to enhance the antibacterial capacity of H 2 O 2 and can be used for wound disinfection [25]. Wang and coworkers reported Cu 2 WS 4 nanocrystals (CWS NCs) with excellent antibacterial activity owing to their high intrinsic peroxidase activity that can catalyze the decomposition of H 2 O 2 to form ·OH, so that it inhabits wound bacterial infection [26]. Besides, Qu and his team also proposed that MOF/Ce-based nanozymes with dual enzymatic activities can not only disperse biofilms ascribed to the Ce complex, but also kill bacteria on-site, thereby avoiding the proliferation of bacteria and the recurrence of biofilms [27]. Notably, most of these works were based on the peroxidase activity of nanozymes to promote the reactive oxygen species (ROS) generation of H 2 O 2 to kill bacteria [28][29][30][31][32][33][34]. Thus, the nanozyme with high peroxidase activity can be regarded as an excellent candidate for building a bacterial disinfection nanozyme system.
The cerium oxide nanoparticle (Nanoceria, CeO 2 ) is a classical nanozyme with high peroxidase activity [35,36]. According to the criterion that high peroxidase activity is more conducive to promoting the production of ROS, the CeO 2 -H 2 O 2 nanozyme system would be very efficient for bacterial disinfection. However, CeO 2 could show multiple enzymelike activities owing to different shapes and sizes [8,37]. In addition to high peroxidase activity, CeO 2 also possesses high superoxide dismutase activity and antioxidant activity, which can be used as a ROS scavenger. In view of the fact that CeO 2 has both the activity of promoting ROS production and the opposite activity of ROS scavenging, it is meaningful to discuss which activity will play the dominating role in the CeO 2 -H 2 O 2 system, and whether it will protect bacteria or produce an antibacterial effect (Scheme 1). Herein, we focus on this discussion to unveil the role of CeO 2 in the CeO 2 -H 2 O 2 system, which will offer valuable knowledge for the design of a nanozyme-H 2 O 2 based antibacterial system. Nanozyme-based antibacterial strategies have been recently demonstrated to be a promising new approach for bacterial infection treatment [17][18][19][20][21][22][23][24]. For example, Qu et al. developed graphene quantum dots (GQDs) that can act as a peroxidase mimic to enhance the antibacterial capacity of H2O2 and can be used for wound disinfection [25]. Wang and coworkers reported Cu2WS4 nanocrystals (CWS NCs) with excellent antibacterial activity owing to their high intrinsic peroxidase activity that can catalyze the decomposition of H2O2 to form ·OH, so that it inhabits wound bacterial infection [26]. Besides, Qu and his team also proposed that MOF/Ce-based nanozymes with dual enzymatic activities can not only disperse biofilms ascribed to the Ce complex, but also kill bacteria on-site, thereby avoiding the proliferation of bacteria and the recurrence of biofilms [27]. Notably, most of these works were based on the peroxidase activity of nanozymes to promote the reactive oxygen species (ROS) generation of H2O2 to kill bacteria [28][29][30][31][32][33][34]. Thus, the nanozyme with high peroxidase activity can be regarded as an excellent candidate for building a bacterial disinfection nanozyme system. The cerium oxide nanoparticle (Nanoceria, CeO2) is a classical nanozyme with high peroxidase activity [35,36]. According to the criterion that high peroxidase activity is more conducive to promoting the production of ROS, the CeO2-H2O2 nanozyme system would be very efficient for bacterial disinfection. However, CeO2 could show multiple enzymelike activities owing to different shapes and sizes [8,37]. In addition to high peroxidase activity, CeO2 also possesses high superoxide dismutase activity and antioxidant activity, which can be used as a ROS scavenger. In view of the fact that CeO2 has both the activity of promoting ROS production and the opposite activity of ROS scavenging, it is meaningful to discuss which activity will play the dominating role in the CeO2-H2O2 system, and whether it will protect bacteria or produce an antibacterial effect (Scheme 1). Herein, we focus on this discussion to unveil the role of CeO2 in the CeO2-H2O2 system, which will offer valuable knowledge for the design of a nanozyme-H2O2 based antibacterial system. Scheme 1. A schematic representation of the question to be answered in this work.

Preparation and Characterization of CeO2 Nanozyme
The CeO2 nanozyme was first synthesized according to a facile solvothermal method, of which the inorganic salt Ce (NO3)3·6H2O and organic acid C2H5COOH were selected as the initial materials without any surfactants [27]. As shown in Figure 1a, the transmission electron microscopy (TEM) images demonstrated the formation of CeO2 nanospheres with uniform dispersion. Dynamic light scattering (DLS) analysis was performed to determine the distribution of the hydration particle size of CeO2, which is placed at around 150 nm from Figure 1b. Furthermore, the X-ray photoelectron energy spectrum was employed to analyze the element and chemical value state of CeO2 nanospheres. We can observe from Figure 1c that there were main peaks such as Ce 4+ 3d3/2 and Ce 4+ 3d5/2 at around 914.2 and 896.0 eV in the Ce 3d core spectrum, and the Ce 3+ 3d3/2 and Ce 3+ 3d5/2 peaks are subscribed at 898.4 and 880.0 eV, respectively. In addition, the crystalline and phase information were implied by powder X-ray diffraction (XRD) patterns. As shown in Figure 1d, there were four clearly specific peaks, and the peaks at 2θ = 28.5°, 32.8°, 47.4°, and 56.4° can be subscribed to the characteristic (111), (200), (220), and (311) reflections of the face-centered Scheme 1. A schematic representation of the question to be answered in this work.

Preparation and Characterization of CeO 2 Nanozyme
The CeO 2 nanozyme was first synthesized according to a facile solvothermal method, of which the inorganic salt Ce (NO 3 ) 3 ·6H 2 O and organic acid C 2 H 5 COOH were selected as the initial materials without any surfactants [27]. As shown in Figure 1a, the transmission electron microscopy (TEM) images demonstrated the formation of CeO 2 nanospheres with uniform dispersion. Dynamic light scattering (DLS) analysis was performed to determine the distribution of the hydration particle size of CeO 2 , which is placed at around 150 nm from Figure 1b. Furthermore, the X-ray photoelectron energy spectrum was employed to analyze the element and chemical value state of CeO 2 nanospheres. We can observe from Figure 1c that there were main peaks such as Ce 4+ 3d 3/2 and Ce 4+ 3d 5/2 at around 914.2 and 896.0 eV in the Ce 3d core spectrum, and the Ce 3+ 3d 3/2 and Ce 3+ 3d 5/2 peaks are subscribed at 898.4 and 880.0 eV, respectively. In addition, the crystalline and phase information were implied by powder X-ray diffraction (XRD) patterns. As shown in Figure 1d, there were four clearly specific peaks, and the peaks at 2θ = 28.  Figure 1d). All the above results demonstrate the successful synthesis of the CeO 2 nanoparticles.
cubic structure of CeO2 nanocrystals (JCPDS No. 43-1002, the red line in Figure 1d). All the above results demonstrate the successful synthesis of the CeO2 nanoparticles.

The Peroxidase Activity of CeO2 Nanozyme
In this work, we utilized a classical colorimetric assay to estimate the peroxidase activity of CeO2 nanospheres. TMB (3,3',5,5'-tetramethylbenzidine) was used as a substrate.
In the presence of peroxidase mimics and H2O2, colorless TMB can be oxidized to blue oxTMB, which exhibits two specific absorbance peaks at 370 and 652 nm. As shown in Figure 2a, neither the nanozyme nor H2O2 alone could produce any color changes, but there appeared two sharp and strong peaks at 370 and 652 nm with the presence of both CeO2 and H2O2, which indicated that CeO2 nanospheres had intrinsic peroxidase enzymatic activity. Likewise, we investigated the effect of pH on its peroxidase activity, and the nanozyme was demonstrated to possess optimistic catalytic activity at a pH range from 4.0 to 6.0 (Figure 2b).

The Peroxidase Activity of CeO 2 Nanozyme
In this work, we utilized a classical colorimetric assay to estimate the peroxidase activity of CeO 2 nanospheres. TMB (3,3',5,5'-tetramethylbenzidine) was used as a substrate.
In the presence of peroxidase mimics and H 2 O 2 , colorless TMB can be oxidized to blue oxTMB, which exhibits two specific absorbance peaks at 370 and 652 nm. As shown in Figure 2a, neither the nanozyme nor H 2 O 2 alone could produce any color changes, but there appeared two sharp and strong peaks at 370 and 652 nm with the presence of both CeO 2 and H 2 O 2 , which indicated that CeO 2 nanospheres had intrinsic peroxidase enzymatic activity. Likewise, we investigated the effect of pH on its peroxidase activity, and the nanozyme was demonstrated to possess optimistic catalytic activity at a pH range from 4.0 to 6.0 (Figure 2b).

ROS Generation
Considering that the catalytic capacity of most of the nanozymes with peroxidase activity may originate from the decomposition of H2O2 to generate ROS hydroxyl radicals (·OH), we further investigated the generation of ·OH in the CeO2-H2O2 system to verify the peroxidase activity of the CeO2 nanozyme. Terephthalic acid (TA), which could capture ·OH to generate fluorescent product 2-hydroxy terephthalic acid (TAOH) (Figure 3a), was used as a fluorescence probe for the tracing of ·OH. As shown in Figure 3b, compared with the fluorescent intensity of TA alone, there was a remarkable increase with the addition of H2O2. However, when we added the CeO2 nanozyme to the H2O2-TA system, the fluorescent intensity did not show any increase but sharply decreased to near the background intensity, which was contrary to what we predicted. This may be because CeO2 possesses certain antioxidant properties as obstacles to the decomposition of H2O2 to generate ·OH [37]. In other words, CeO2 shows considerable ROS scavenging capacity in the CeO2 nanozyme-H2O2 system.

The Evaluation of Antibacterial Activity of CeO2-H2O2 System
Finally, the antibacterial activities were investigated by using a Gram-negative bacterium (Escherichia coli, E. coli) as a model strain. In view of the effect of the pH value on the nanozyme activity, all the bacterial experiments were performed at pH 6.0 with PBS (phosphate-buffered saline) as the buffer. As shown in Figure 4b, CeO2 (0.25 mg/mL) alone caused a weak antibacterial effect. H2O2 showed concentration-dependent antibacterial activity. At a low concentration of 0.5 mM, H2O2 showed a weak antibacterial activity

ROS Generation
Considering that the catalytic capacity of most of the nanozymes with peroxidase activity may originate from the decomposition of H 2 O 2 to generate ROS hydroxyl radicals (·OH), we further investigated the generation of ·OH in the CeO 2 -H 2 O 2 system to verify the peroxidase activity of the CeO 2 nanozyme. Terephthalic acid (TA), which could capture ·OH to generate fluorescent product 2-hydroxy terephthalic acid (TAOH) (Figure 3a), was used as a fluorescence probe for the tracing of ·OH. As shown in Figure 3b, compared with the fluorescent intensity of TA alone, there was a remarkable increase with the addition of H 2 O 2 . However, when we added the CeO 2 nanozyme to the H 2 O 2 -TA system, the fluorescent intensity did not show any increase but sharply decreased to near the background intensity, which was contrary to what we predicted. This may be because CeO 2 possesses certain antioxidant properties as obstacles to the decomposition of H 2 O 2 to generate ·OH [37]. In other words, CeO 2 shows considerable ROS scavenging capacity in the CeO 2 nanozyme-H 2 O 2 system.

ROS Generation
Considering that the catalytic capacity of most of the nanozymes with peroxidase activity may originate from the decomposition of H2O2 to generate ROS hydroxyl radicals (·OH), we further investigated the generation of ·OH in the CeO2-H2O2 system to verify the peroxidase activity of the CeO2 nanozyme. Terephthalic acid (TA), which could capture ·OH to generate fluorescent product 2-hydroxy terephthalic acid (TAOH) (Figure 3a), was used as a fluorescence probe for the tracing of ·OH. As shown in Figure 3b, compared with the fluorescent intensity of TA alone, there was a remarkable increase with the addition of H2O2. However, when we added the CeO2 nanozyme to the H2O2-TA system, the fluorescent intensity did not show any increase but sharply decreased to near the background intensity, which was contrary to what we predicted. This may be because CeO2 possesses certain antioxidant properties as obstacles to the decomposition of H2O2 to generate ·OH [37]. In other words, CeO2 shows considerable ROS scavenging capacity in the CeO2 nanozyme-H2O2 system.

The Evaluation of Antibacterial Activity of CeO2-H2O2 System
Finally, the antibacterial activities were investigated by using a Gram-negative bacterium (Escherichia coli, E. coli) as a model strain. In view of the effect of the pH value on the nanozyme activity, all the bacterial experiments were performed at pH 6.0 with PBS (phosphate-buffered saline) as the buffer. As shown in Figure 4b, CeO2 (0.25 mg/mL) alone caused a weak antibacterial effect. H2O2 showed concentration-dependent antibacterial activity. At a low concentration of 0.5 mM, H2O2 showed a weak antibacterial activity

The Evaluation of Antibacterial Activity of CeO 2 -H 2 O 2 System
Finally, the antibacterial activities were investigated by using a Gram-negative bacterium (Escherichia coli, E. coli) as a model strain. In view of the effect of the pH value on the nanozyme activity, all the bacterial experiments were performed at pH 6.0 with PBS (phosphate-buffered saline) as the buffer. As shown in Figure 4b, CeO 2 (0.25 mg/mL) alone caused a weak antibacterial effect. H 2 O 2 showed concentration-dependent antibacterial activity. At a low concentration of 0.5 mM, H 2 O 2 showed a weak antibacterial activity (Figure 4c). When the concentration of H 2 O 2 was increased to 1.5 mM, almost all the bacteria were killed, indicating a significant high antibacterial activity (Figure 4d). Thereafter, the bacterial effect of the CeO 2 nanozyme-H 2 O 2 system was investigated. As shown in Figure 4e, compared with H 2 O 2 (0.5 mM) alone, the presence of CeO 2 (0.25 mg/mL) did not cause an obvious change in the number of bacteria, which demonstrated that the CeO 2 nanozyme did not promote the killing effect of H 2 O 2 against bacteria in this nanozyme-H 2 O 2 system. Interestingly, compared with H 2 O 2 (1.5 mM) alone, the presence of CeO 2 (0.25 mg/mL) caused an obvious increase in the number of bacteria, which indicated that the CeO 2 greatly inhibited the killing effect of H 2 O 2 against bacteria in this nanozyme-H 2 O 2 system (Figure 4g). This role was further found to be in a concentration-dependent manner. When we reduced the CeO 2 concentration to 0.1 mg/mL, the effect of CeO 2 on the bacterial killing activity of H 2 O 2 decreased accordingly (Figure 4f). On the contrary, the effect of CeO 2 on the bacterial killing activity of H 2 O 2 was dramatically increased with the increase in the concentration of CeO 2 to 0.4 mg/mL. The further study demonstrated that CeO 2 played a similar protection role in the nanozyme-H 2 O 2 system toward the Gram-positive bacterium (Bacillus subtilis, B. subtilis) (Figure 4f-i). All the above results manifested that the peroxidase activity of CeO 2 did not contribute to the antibacterial effect of the nanozyme-H 2 O 2 system. Instead, the ROS scavenging capacity of CeO 2 protected the bacteria from being killed by H 2 O 2 . Although the phenomenon we observed may be different from other enzymes with multiple activities, our work would provide valuable new knowledge for the design of the nanozyme-H 2 O 2 -based antibacterial system.  Figure 4c). When the concentration of H2O2 was increased to 1.5 mM, almost all the bacteria were killed, indicating a significant high antibacterial activity (Figure 4d). Thereafter, the bacterial effect of the CeO2 nanozyme-H2O2 system was investigated. As shown in Figure 4e, compared with H2O2 (0.5 mM) alone, the presence of CeO2 (0.25 mg/mL) did not cause an obvious change in the number of bacteria, which demonstrated that the CeO2 nanozyme did not promote the killing effect of H2O2 against bacteria in this nanozyme-H2O2 system. Interestingly, compared with H2O2 (1.5 mM) alone, the presence of CeO2 (0.25 mg/mL) caused an obvious increase in the number of bacteria, which indicated that the CeO2 greatly inhibited the killing effect of H2O2 against bacteria in this nanozyme-H2O2 system (Figure 4g). This role was further found to be in a concentration-dependent manner. When we reduced the CeO2 concentration to 0.1 mg/mL, the effect of CeO2 on the bacterial killing activity of H2O2 decreased accordingly (Figure 4f). On the contrary, the effect of CeO2 on the bacterial killing activity of H2O2 was dramatically increased with the increase in the concentration of CeO2 to 0.4 mg/mL. The further study demonstrated that CeO2 played a similar protection role in the nanozyme-H2O2 system toward the Grampositive bacterium (Bacillus subtilis, B. subtilis) (Figure 4f-i). All the above results manifested that the peroxidase activity of CeO2 did not contribute to the antibacterial effect of the nanozyme-H2O2 system. Instead, the ROS scavenging capacity of CeO2 protected the bacteria from being killed by H2O2. Although the phenomenon we observed may be different from other enzymes with multiple activities, our work would provide valuable new knowledge for the design of the nanozyme-H2O2-based antibacterial system.

Chemicals and Instrument
All reagents and solvents were purchased from commercial sources. Hydrogen-peroxide is an AR reagent with a concentration of 30% in water. The degree of purity of other compounds was at least 98%, and they were used without any further treatment.

Chemicals and Instrument
All reagents and solvents were purchased from commercial sources. Hydrogenperoxide is an AR reagent with a concentration of 30% in water. The degree of purity of other compounds was at least 98%, and they were used without any further treatment. PL spectra were collected by a FL-970 Fluorescence Spectrometer (slit width of 2.5 nm and PMT voltage of 600 V). UV-vis adsorption spectra were measured by a UV-2600 spectrometer (SHIMADZU, Japan). The size of nanoparticles was monitored with a SZ-100V2 Nano Particle Analyzer. The X-ray diffraction (XRD) patterns of the products were determined on a DX-2700 X-ray diffractometer with Cu Kα radiation (λ = 1.5416 Å), with an operation voltage and current maintained at 35 kV and 25 mA, respectively. Xray photoelectron spectroscopy (XPS) measurements were conducted on a Kratos AXIS Ultra DLD photoelectron spectrometer with Al Kα X-ray radiation as the X-ray source for excitation. The nanostructure of products was analyzed with a 120 kV JEM-1400Flash transmission electron microscope (TEM) with a Gatan Rio16 digital camera. Samples for TEM were prepared by dropping dilute solutions of nanoparticles onto carbon-coated copper grids and then maintained at 40 • C to wait for the solvent to evaporate.

Synthesis of CeO 2 Nanozyme
The CeO 2 nanozyme was synthesized by one simple solvothermal method right in this work. Briefly, 1 g of Ce(NO 3 ) 3 ·6H 2 O was dissolved in 1 mL of ultrapure water. Then, 1 mL of C 2 H 5 COOH and 30 mL of glycol were added in the above solution with stirring until the formation of a uniform solution. Following that, the mixture solution was transferred to an autoclave and heated to 180 • C for 200 min to obtain the initial product. The product was first centrifuged at 5000 rpm for 10 min to remove large particles; then, the supernatant was centrifuged at 10,000 rpm for 10 min, and the precipitation was washed with ethanol and ultrapure water twice. Ultimately, the product was redispersed into ultrapure water for later use.

Detection of Peroxidase Activity of CeO 2
The peroxidase activity was investigated by a catalytic reaction of the TMB with the assistance of H 2 O 2 , and the catalytic performance was characterized by a specific peak at 652 nm. Typically, the reaction solution (200 µL) containing PBS buffer solution (0.2 M, 20 µL), TMB (80 mM, 2 µL), H 2 O 2 (10 mM, 40 µL), and CeO 2 nanozyme (40 µL) was incubated at room temperature for 10 min, and the blue product (oxTMB) was then monitored by a UV-spectrum at 652 nm.

Detection of OH
The detection of ·OH was dependent on the reaction of TA and ·OH. TA has weak fluorescence in the absence of ·OH, but it has unique fluorescence around 425 nm in the presence of ·OH ascribed to the generation of 2-hydroxy terephthalic acid.
The reaction solutions containing TA, TA + H 2 O 2 , and TA + H 2 O 2 + nanozymes were reacted for 2 h and then centrifuged to separate the supernatant. The fluorescent intensity of the supernatant at around 430 nm was determined by a FL-970 fluorescence spectrometer. The concentrations of TA, H 2 O 2 , and CeO 2 nanozyme were 0.5 mM, 1 mM, 0.25 mg/mL, respectively.

Antibacterial Experiment
The spread plate method was utilized to measure the bacterial number under different treatments. E. coli was coped with five different groups: (a) without any addition, (b) CeO 2 , (c) H 2 O 2 , and (d) CeO 2 + H 2 O 2 groups. In brief, the mono-colony of E. coli on a solid Luria-Bertani (LB) agar plate was diverted to 2 mL of liquid LB culture medium and grown at 37 • C for 5 h under 180 rpm rotation. After finishing this process, we chose 0.3 as the initial optical density of bacteria at OD 600 nm . Then, 20 µL of as-prepared bacteria solution was mixed up with different groups we mentioned above with a 200 µL total volume and incubated in 24-cell culture plates at a reaction system (pH = 5, PBS buffer solution 20 mM) for 2 h. Thereafter, the bacteria solution was transferred from the 24-well plate to the solid medium by the spread plate method and was cultured at 37 • C for 12 h. A similar method was used in the B. subtilis experiment.

Conclusions
In summary, we successfully synthesized a CeO 2 nanozyme with both the peroxidase and ROS scavenging activity. Based on this, we investigated the function between the CeO 2 -H 2 O 2 system and bacteria. The results of the bacterial experiments demonstrated that the peroxidase activity of CeO 2 did not contribute to the antibacterial effect in the CeO 2 -H 2 O 2 system; instead, the ROS scavenging capacity of CeO 2 could protect the bacteria from being killed by H 2 O 2 . Our present work not only unveils the role of CeO 2 in the CeO 2 -H 2 O 2 system toward bacteria but also provides valuable new knowledge for the design of nanozyme-H 2 O 2 -based antibacterial systems.