Precise engineering of ultrasound-mediated cerium oxide nanoparticles: Investigations on antioxidant, antibacterial activity and human osteomyelitis

In this study, the antibacterial activity of cerium oxide nanoparticles on two Gram-negative and three Gram-positive foodborne pathogens was investigated. CeO 2 nanoparticles (CeO 2 NPs) were synthesized by a Wet Chemical Synthesis route, using the precipitation method and the Simultaneous Addition of reactants (WCS–SimAdd). The as-obtained precursor powders were investigated by thermal analysis (TG–DTA), to study their decomposition process and to understand the CeO 2 NPs formation. The composition, structure, and morphology of the thermally treated sample were investigated by FTIR, Raman spectroscopy, X-ray diffraction, TEM, and DLS. The cubic structure and average particle size ranging between 5 and 15 nm were evidenced. Optical absorption measurements (UV–Vis) reveal that the band gap of CeO 2 NPs is 2.61 eV, which is smaller than the band gap of bulk ceria. The antioxidant effect of CeO 2 NPs was determined, and the antibacterial test was carried out both in liquid and on solid growth media against ve pathogenic microorganisms, namely Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. Cerium oxide nanoparticles showed growth inhibition toward all ve pathogens tested with notable results. This paper highlights the CeO 2 NPs showed antibacterial activity with signicant variations due to the differences in the membrane structure and cell wall composition among the two groups tested. Consequently, synthesized CeO 2 NPs can be potential candidates for the treatment of osteomyelitis.


Introduction
Osteomyelitis is an in ammation of the bone caused by infective micro-organisms. The usual treatment of osteomyelitis mainly involves fundamental surgical debridement of the infected bone, lling the bone defect, adequate soft tissue coverage and antibiotic therapy [1][2][3][4]. A prolonged course of antibiotic therapy is required, most often weeks and sometimes longer. The oral delivery of antibiotics can result in systemic toxicity associated with renal and liver complications and poor penetration into the targeted site. Hence, an alternative strategy of antibiotic delivery should be explored [5][6][7][8]. Recently, development of a local drug delivery system received much attention since antibiotics are delivered locally, the side effects and the risk of overdose of oral administration can be avoided and high concentration of drugs can effectively reach the targeted site. Bioactive materials in combination with antibiotics are very useful for the development of local drug delivery systems because they play a vital role in subsequent bone regeneration at the infected site [9][10][11][12].
Antibiotics or antimicrobials have been widely utilized in various elds, such as medicine, food industry, agriculture, livestock, water treatment for diseases prevention, and pathogens eradication [13][14][15]. Nevertheless, there is an increased concern related to multidrug-resistant microorganisms. Worldwide, numerous scientists direct their efforts toward nding new alternatives for combating pathogens [16][17][18].
Among the many alternatives, such as plant extracts, antimicrobial peptides, and bee products, metal or metal oxide nanoparticles are recently receiving increased attention [19][20][21][22]. The properties found in the nanoparticle form of these materials can signi cantly differ from those of their bulk tantamount. Speci c properties, such as considerably high contact surface and selectivity in the mediation of chemical transformations, facilitate their use in various areas. Nanoparticles with various morphologies are utilized in multiple areas, including medicine, for the treatment or diagnosis of pathogen-ghting systems or improvement of the fuel quality [23][24][25].
Metal oxide nanoparticles, such as CeO 2 , have signi cant action on the antimicrobial activity and offer the plausibility of e cient pathogen removal from different environments. Furthermore, the stability and the slow release of metal ions from the nanoparticles are key features which make their usage advantageous [26][27][28][29]. In comparison to other metal oxide nanoparticles, CeO 2 has the quality of being an antioxidant due to the reversibility of its transfer from the reduced state into the oxidized state and resumes the process. Its uses as an abrasive in semiconductor fabrication, or as a component in catalytic converters for auto machines exhaust systems, as a fuel additive to boost combustion, as an electrolyte for fuel, and as a UV-light absorber are also well-known [30][31][32][33][34]. Recently, nanoceria has been used as a colorimetric indicator for antioxidants activity due to the color changes induced by the modi cations of cerium oxidation states at nanoparticle surface. It was established that CeO 2 NPs have the capacity to initiate their mechanism by the modulation of the oxygen environment. The properties of these CeO 2 NPs prove important possible biomedical applications. Scattered literature regarding the antibacterial activity of CeO 2 NPs is available [18,35,36]. The effect against E. coli and B. subtilis a comparison of the antibactericidal activity of bulk and nano CeO 2 NPs against the same Gram-negative bacteria and cytotoxicity on nitrogen-xing bacteria are reported. Many studies have reported the synthesis of ceria nanoparticles (CeO 2 NPs), using different cerium salts (acetate or nitrate) and different reactants (citric acid, ammonia, or hydrogen peroxide), and organic solvents/stabilizers (oleic acid, oleylamine, or diphenyl ether) to prevent particle agglomeration. The addition of these organic species to the aqueous solution directly affects the stabilization level, the nucleation, and the growth processes of the CeO 2 NPs in the homogeneous solutions [37][38][39][40][41].
The aim of our work was the investigation of antibacterial properties of CeO 2 NPs against a wider range of pathogens, namely Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. A simple, reproducible, controllable, and low-cost wet-chemical synthesis method was used for the preparation of CeO 2 NPs. This precipitation-type method consists of the simultaneous addition of reactants (SimAdd) in the presence of an anti-agglomeration agent, under pH control, with the formation of cerium oxalate type salt as a precursor. Based on the precursor characterization-thermal analysis and FTIR-the particle dimensionality and aggregation can be controlled.
The CeO 2 NPs were characterized by using spectroscopic and microscopic methods, and their antioxidant and antibacterial activity was tested on two Gram-negative and three Gram-positive pathogens, respectively. hydroxide solution was added to prevent the agglomeration of the particles. The post-precipitation stage consisted of 24 h aging, separation by ltering, and drying. The precursor nano-powders were intimately mixed with NH4Cl ( ux), and the thermal treatment was performed at 400 °C/1 h, in air, at a heating rate of 300 °C/h. The thermally treated powders were washed several times with distilled water, until the ltrate was Cl− free. The solution was heated at 80 °C in an oven till half of its volume. The precursor was then kept in the microwave oven, operated at a power of 800 W with the frequency 2450 MHz for 10 min. A yellowish brown precipitate started to appear which suggested the formation of cerium oxide nanoparticles. A yellow aqueous solution (2 mM) was obtained by dispersing a speci c mass of ceria nps in distilled water.

Characterization of cerium oxide nanoparticles (CeO 2 NPs)
The thermal decomposition process of cerium oxalate-type precursor was monitored by thermal analysis, using a Mettler Toledo TGA/SDTA851 (Greifensee, Switzerland) system, a platinum crucible, and a heating rate of 10 K/min, in a static air atmosphere up to 500 °C. The chemical nature of the precursor and nal oxide was analyzed by Fourier Transform Infrared Spectroscopy (FTIR), using a Tensor 27 Bruker FTIR spectrophotometer (Bruker Optik GmbH, Germany). The crystalline structure of the CeO 2 NPs-powders was determined by X-ray diffraction (XRD) investigations, which were performed at room temperature, by means of a Bruker AXS D8 Discover diffractometer (Bruker, Karlsruhe, Germany) operating at 40 kV, 40 mA, CuKα radiation λ = 1.54056 Å. The hydrodynamic diameter was determined by dynamic light scattering (DLS) measurements, using a Brookhaven Instruments Corporation (Holtsville, NY, USA), a goniometer, and a laser light-scattering system. The acquisition time was set to 90 s, a laser radiation wavelength of 632.8 nm was used, and the angle at which data acquisition was performed was 90°. During long-term storage, the solution gradually lost its stability due to the formation of nanoparticle aggregates. TEM was utilized to characterize the morphology of CeO 2 NPs. A JEOL JEM1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA, USA) was used, operating at an accelerating voltage of 100 kV and equipped with a MegaViewIII CCD camera. The optical characterization was performed by a Jasco V 530 spectrophotometer (Jasco Corporation, Tokyo, Japan), in the range of 200-800 nm. The Raman spectra of the CeO 2 NPs were recorded at room temperature, using a Renishaw in Via Re ex Raman Microscope (New Mills, UK) equipped with a RenCam CCD detector. The Raman system was operated with 532 nm laser line, and the spectra were recorded by adding 4 accumulations, each with a 40 s exposure time, acquired by using 13.5 mW laser power with a 4 cm −1 resolution.

Antioxidant properties of CeO 2 NPs scavenging effect on ABTS radicals
The scavenging properties of CeO 2 NPs against radical cations ABTS + (2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)) were determined according to the procedure described in other studies [19,42,43], adapted to a microplate of 96 wells. The ABTS assay is based on the capacity of a sample to scavenge the ABTS radical cation (ABTS. ABTS + ), as compared to a standard antioxidant (Trolox). The blue-green ABTS + solution was produced by the reaction between a 7 mM aqueous solution of ABTS and a 2.45 mM potassium persulfate, in a dark medium, and at room temperature, for 12-16 h before use. ABTS+ working solution was obtained by diluting the stock solution with ethanol, resulting in an absorbance of 0.70 ± 0.02 AU at 734 nm. Then, 20 µL of Trolox or CeO 2 NPs solution at different concentrations were added to 170 µL ABTS + solutions, and the absorbance was measured after 6 min of incubation, in the dark, and at room temperature, using a microplate reader. The results were expressed as µmol Trolox per Gram sample.

Disk diffusion method
About 15 mL of sterile nutrient agar (Bioaqua, Targu-Mures) was poured into the sterile Petri dish.
Triplicate plates were inoculated with 200 µL of the overnight culture (approximately 108 CFU/mL) of the targeted pathogenic bacteria, which were spread on the plate, using sterile Drigalski spatulas. On the solid medium were gently placed up to 6 paper disks that were soaked with a xed concentration of CeO 2 NPs solution. The plates were incubated for 24 h at 37 ± 1 °C, prior to the determination of results. The zone of inhibition around each of the paper disks was measured and expressed as millimeters in diameter. Gentamicin was used as the control [47][48][49].

Minimum bactericidal concentration (MBC)
To prevent the possibility of misinterpretation due to the turbidity of insoluble compounds, the MBC was established by sub-culturing the above (MIC) serial dilutions after the incubation time in nutrient agar plates, using a 0.01 mL loop, and further incubating the dilutions at 37 °C for 24 h. The MBC was considered to be the lowest concentration that prevents the growth of the bacterial colony on this solid medium [53][54][55].

Bacterial viability using confocal microscopy
The confocal laser scanning microscope -Zeiss LSM 710 (Oberkochen, Germany) -is a method that allowed us to view live and dead bacteria. Bacteria cells culture (~108 CFU) were treated with 50 µg/mL of CeO 2 NPs for 24 h. A control with no treatment was made for each pathogen. Prior to the confocal microscopy assay, the bacteria cells were mixed, for coloration, with a LIVE/DEAD® BacLight™ bacterial viability Kit, L-7007, for 15 min, in the dark. Aliquots of 50 µL of the colored bacteria were placed on a microscope slide, xed using a ame, rinsed, and dried.

Time kill assay
The potential CeO 2 NPs was subject to the time killing assay. An inoculum of the tested pathogens (10 µL), at a concentration of 108 CFU/mL, was mixed with 100 µL of solution containing growth medium and nanoparticles, to a nal concentration of 50 µg/mL. The negative controls were the samples in which no nanoparticles were utilized. The growth of bacterial species was assessed at every 1 h interval, by measuring the optical density at 550 nm with the microplate reader [56][57][58].

Results And Discussion
It is well-known that the critical step in the synthesis of metal oxide by wet-chemical methods is the thermal decomposition of the obtained precursor [59][60][61]. The thermal analysis method (DTA-TG) allowed us to investigate the chemical transformation of the oxalate-type precursor into oxide powders during heating (Figure 1a and b). The thermal behavior of the synthetized precursor through simultaneous DTA-TG measurements reveals two weight-loss processes. In the low temperature range, the DTA curve illustrates an endothermic process with the maximum temperature centered around 150 °C, accompanied by a mass loss of 23.8% on the TG curve. This rst decomposition step is attributed to the dehydration of the precursor with the loss of around 10 water molecules and the formation of anhydrous cerium oxalate. The second weight loss (~28.5%) is observed at temperatures ranging between 250 and 350 °C and corresponds to the oxidative decomposition of the oxalate group, as indicated by the exothermic effect, whose maxima is located at 298 °C. Almost no weight loss is observed at temperatures higher than 350 °C in the TG curve, indicating that the oxalate precursor was completely decomposed with the formation of CeO 2 NPs. This decomposition behavior con rms that the chemical composition of the oxalate precursor is close to the molecular formula of Ce 2 (C 2 O 4 ) 3 •10H 2 O, which is in concordance with the literature data. Based on these observations, one can assume that the precursor decomposition takes place according to the following chemical reactions: To identify the crystalline phases and to estimate the average particle sizes, an XRD investigation was performed. The XRD pattern of the oxide powders obtained after annealing at 400 °C for 1 h is presented in Figure 1c. All the re ections were indexed with those of the pure cubic uorite structure of CeO2 (JCDD PDF 034-0394). The average crystallite sizes of the sample were calculated by using the Debye-Scherrer formula: Dp = 0:9.λ/ β cosθ, where Dp is the average crystallite size, λ is the wavelength of the CuKα line, θ is the Bragg angle, and β is the Full Width at Half-Maximum (FWHM) of the diffraction peak in radians.  Raman spectroscopy is one of the major tools utilized in the study of crystallinity, purity, and any defect levels connected to the nanomaterials. A typical Raman spectrum for bulk CeO 2 NPs with cubic uorite structure presents a single strong peak at 464 cm −1 , assigned to the F 2g symmetry modes of CeO 2 NPs.
The Raman spectrum of the synthetized CeO 2 NPs (Figure 3b) exhibits a strong Raman absorption peak at 461 cm −1 and two weak absorption peaks at 610 and 740 cm −1 . The Raman peak at 461 cm −1 corresponds to the triply degenerate F 2g mode of symmetric stretching vibration of oxygen ions around Ce 4+ cations and therefore con rms the cubic uorite structure. The slight broadness of the peak is attributed to the reduction of the particle size to nanometric ranges. The shift in the peak position toward the lower energy value and the broad peaks at 610 and 740 cm −1 are due to the presence of point defects generated by oxygen vacancies.
The UV-Vis spectrum is shown in Figure 4. To evaluate the antioxidant potential of CeO 2 NPs, the ABTS (2,2'-azino-bis(3-ethylbenzthiazoline)-6sulphonic acid) assay was tested. The ABTS method is indicated for a suitable and continuous spectrophotometric measurement of any potential radical formations. ABTS creates a characteristic cation radical (ABTS + ) that can be easily followed and can allow the measurement of the initial rate of radical formation. The obtained results ( Figure 5) have clearly demonstrated that the CeO 2 NPs is a radical scavenger and can inhibit the ABTS+ radical formation in a dose-dependent manner. The results were expressed as Trolox equivalents. Trolox is a water-soluble analogue of vitamin E. Trolox is an antioxidant that has been shown to prevent lipid peroxidation in vivo. Previous publications have shown that vitamin E is effective in preventing oxidative harm from iron and other redox-active metals in vivo and in vitro, likely by reacting with hydroxyl radicals or downstream radical intermediates. It can be easily concluded that the antioxidant activity increases in direct proportionality with the nanoparticle concentration.
Compared with bulk, CeO 2 NPs exhibit superior properties due to their small size, which allows a rapid adsorption of pathogens. Many studies have shown that the CeO 2 NPs exhibit excellent antibacterial activity against Gram-positive and Gram-negative bacteria due to the generation of reactive oxygen species (ROS). In healthy cells, CeO 2 NPs act as antioxidants by scavenging ROS at a physiological pH, while, in pathogens-under low-pH environment-CeO 2 NPs act as a pro-oxidant by generating ROS and producing cell damage.
The antibacterial activity of CeO 2 NPs was evaluated by using the microdilution method, and the corresponding MIC and MBC values were evaluated against two Gram-negative and three Gram-positive pathogens. The correspondent values are given in Table 1 and Table 2, respectively. The cerium oxide nanoparticles demonstrated antibacterial properties against all the tested pathogens in relative low concentrations. The antibacterial activity of CeO 2 NPs discloses that the same concentration of nanoparticles against Escherichia coli and Salmonella typhimurium resulted in different diameters of inhibition, with these being 9 and 10 mm, respectively. Regarding the Gram-positive pathogens, the highest inhibition was registered in the case of Listeria monocytogenes, followed by Bacillus cereus and Staphylococcus aureus. The MBC results reveal that the highest sensitivity of solely 1.07 g/L CeO 2 NPs can be observed in Salmonella typhimurium and Listeria monocytogenes. The difference in the action of CeO 2 NPs against the Gram-positive and Gram-negative pathogens is primarily related to the different structure and compactness of their cell walls. Usually, Gram-positive bacteria have a thicker, waxy cell wall, making them more resistant to the antimicrobial activity of CeO 2 NPs in comparison with Gram-negative bacteria.
For example, the Gram-positive Bacillus cereus has a cell wall of 55.4 nm, while the Gram-negative S. typhimurium has a cell wall of only 2.4 nm. Even if important functional differences can be seen between the Gram-positive and Gram-negative bacteria cell wall, in the DNA-based molecular taxonomy, some pathogens have a similar response to the same antibacterial agents.
The antibacterial effect of CeO 2 NPs is con rmed by the confocal microscopy images from Figure 6 and 7. The results clearly evidence that the inhibitory effect of CeO 2 NPs is present at a lower concentration, with respect to gentamicin (the standard drug). This fact guarantees the presence of the antibacterial action of CeO 2 NPs against the tested pathogens.
The time kill assay shows an inhibitory effect in a time-dependent manner, in Figure 8 and 9, indicating that CeO 2 NPs interaction with the tested pathogens results in cell damage. It can be observed that the bacterial growth was inhibited from the rst hour.
A feasible mechanism of action can be explained by the fact that the CeO 2 NPs are carrying positive charges, and the bacteria are charged negatively, inducing electromagnetic attraction. This attraction ensures direct contact between nanoparticles and bacteria, leading to oxidation and bacterial cellular death.

Conclusions
Cerium oxide nanoparticles have been designed through a wet chemical method by using the simultaneous addition of reactants (WCS-SimAdd). A better understanding of the decomposition mechanism for as-obtained oxalate precipitate was achieved by DTA-TG and FTIR analyses. The cubic uorite structure of the cerium oxide nanoparticles was determined by XRD and Raman spectroscopy. TEM and DLS revealed the morphology, showing that the sizes are in the range of 5-20 nm, with pseudospherical shapes and a lower tendency to form aggregates. The oxygen vacancies and formation of Ce 3+ evidenced by Raman and UV-Vis spectroscopies are correlated to the particle low-dimensionality.
Experiments prove that the synthetized CeO 2 NPs exhibit excellent antibacterial activity against the ve tested bacterial species: E. coli, S. typhimurium, L. monocytogenes, S. aureus, and B. cereus. Low MIC values were registered, a result that suggests the suitable antibacterial characteristics of CeO 2 NPs.
Antibacterial action is attributable to the direct interaction between the cerium oxide nanoparticles and the bacteria which induces the cellular death of these pathogens. In overview, this study evidenced the potential antibacterial property of cerium oxide nanoparticles, a fact that leads to new approaches in the development of biomedical and food applications. Further studies, such as long-term treatment, are important for a better understanding and the application of CeO 2 NPs in various antibacterial applications. Consequently, the present study offers a bone substitute material with the combined properties of CeO 2 NPs for the treatment of osteomyelitis.