Introduction
============

Among the rare earth elements (the 15 lanthanides, along with scandium and yttrium), cerium is the most abundant element, at 66.5 ppm in the Earth's crust compared with copper (60 ppm) or tin (2.3 ppm).[@b1-ijn-8-3395],[@b2-ijn-8-3395] As a highly abundant material, ceria (or cerium oxide \[CeO~2~\]) is technologically important due to its wide range of applications, for example, in catalysts for the elimination of toxic auto exhaust gases,[@b3-ijn-8-3395],[@b4-ijn-8-3395] oxygen sensors,[@b5-ijn-8-3395],[@b6-ijn-8-3395] fuel cells,[@b7-ijn-8-3395]--[@b9-ijn-8-3395] electrochromic thin films,[@b10-ijn-8-3395],[@b11-ijn-8-3395] and so on. Upon the transition from a macrosized particle to a nanocrystalline particle, CeO~2~ significantly changes its physicochemical properties to, usually, possess a high density of nanocrystalline CeO~2~ interfaces.[@b12-ijn-8-3395] Thus, nanostructured CeO~2~ has attracted extensive attention in the field of nanotechnology due to improvements in redox properties, transport properties, and high surface to volume ratios, which may be of value for various biological applications.

However, although CeO~2~ nanoparticles have been studied for numerous traditional scientific and engineering applications, there have been almost no studies regarding the potential biomedical applications of CeO~2~ until recently, when researchers demonstrated that CeO~2~ nanoparticles possess antioxidant properties at physiological pH values. The ability of these nanoparticles to act as an antioxidant lies in their ability to reversibly switch from Ce^3+^ to Ce^4+^.[@b13-ijn-8-3395] In recent studies, this antioxidative ability enabled the application of CeO~2~ nanoparticles to protect against radiation damage, oxidative stress, and inflammation.[@b14-ijn-8-3395]--[@b16-ijn-8-3395] For example, Tsai et al reported that free radical scavenging could be achieved in murine insulinoma βTC-tet cells by the introduction of crystalline CeO~2~ nanoparticles.[@b17-ijn-8-3395]

Moreover, in previous studies, due to safety concerns for biomedical applications, the toxicity of zinc oxide (ZnO), CeO~2~, and titanium oxide (TiO~2~) was compared based on dissolution and oxidative stress properties.[@b18-ijn-8-3395],[@b19-ijn-8-3395] The results showed that the CeO~2~ nanoparticles could suppress the generation of reactive oxygen species (ROS) and induce cellular resistance to an exogenous source of oxidative stress, to protect cells from oxidation injury. However, concerns over CeO~2~ toxicity, biocompatibility, and avoidance of immune-system clearance remained. Thus, to further improve the clinical use of CeO~2~ nanoparticles, previous efforts have coated CeO~2~ with biocompatible dextran and PAA; enhanced stability in aqueous solutions has also been reported for these coated CeO~2~ nanoparticles.[@b15-ijn-8-3395],[@b31-ijn-8-3395]

To build on these exciting medical applications of CeO~2~ nanoparticles, the present study investigated, for the first time, the influence of dextran- and PAA-coated CeO~2~ nanoparticles on bacteria growth. *Pseudomonas aeruginosa* is an aerobic gram-negative bacterium that is an important cause of various infections, especially in hospitals. Hospitalized patients may be colonized with *P. aeruginosa* on admission or during their hospital stay, and *P. aeruginosa* can be isolated from nearly any conceivable source within hospitals.[@b20-ijn-8-3395],[@b21-ijn-8-3395] It accounts for 11%--13.8% of all nosocomial infections when a microbiological isolate is identifiable.[@b22-ijn-8-3395] Infections caused by *P. aeruginosa* are not only common,[@b23-ijn-8-3395],[@b24-ijn-8-3395] but are also associated with high morbidity and mortality when compared with other bacterial pathogens.[@b25-ijn-8-3395],[@b26-ijn-8-3395] An additional concern involves the increased antimicrobial resistance of nosocomial *P. aeruginosa* isolates.[@b27-ijn-8-3395] Even more troublesome, *P. aeruginosa* attaches to both biotic and abiotic surfaces, forming a biofilm. Once formed, the *P. aeruginosa* infections are much more difficult to treat, due to their formation of exopolysaccharides. Biofilms of *P. aeruginosa* have been found on numerous medical devices, including central venous catheters, contact lenses, mechanical heart valves, intrauterine devices, and indwelling urinary catheters.[@b28-ijn-8-3395],[@b29-ijn-8-3395] Thus, it is significant to develop active and biocompatible molecules that do not rely on antibiotics, to which bacteria may develop a resistance, and that kill bacteria at early stages of infections, before biofilm formation.

Therefore in this study, for the first time, due to concerns over the biocompatibility, toxicity, and immune system clearance of pristine CeO~2~ nanoparticles, we investigated the effectiveness of dextran- and PAA-coated CeO~2~ nanoparticles in inhibiting bacterial growth, specifically, *P. aeruginosa*. The results showed that such coated CeO~2~ nanoparticles significantly inhibited the growth of *P. aeruginosa*, revealing a promising application of CeO~2~ nanoparticles to prevent bacterial infections that should be further explored.

Materials and methods
=====================

Synthesis, coating, and characterization of the cerium oxide nanoparticles
--------------------------------------------------------------------------

The dextran- or PAA-coated CeO~2~ nanoparticles were synthesized according to previously described procedures.[@b15-ijn-8-3395],[@b30-ijn-8-3395],[@b31-ijn-8-3395] Briefly, a solution of cerium (III) nitrate (1.0 M, 99%) (Sigma-Aldrich Corp, St Louis, MO, USA) in water (5.0 mL) was mixed with an aqueous solution of either PAA (0.5 M) (Sigma-Aldrich Corp) or dextran (1.0 M) (Sigma-Aldrich Corp). The resulting mixtures were then added to an ammonium hydroxide solution (30.0 mL, 30%) (Sigma-Aldrich Corp) under continuous stirring for 24 hours, at room temperature. The preparation was then centrifuged at 4000 rpm for two 30-minute cycles to settle down any debris and large agglomerates. The supernatant solution was then purified from free polymers and other reagents and then concentrated using a 30K Amicon® Ultra-4 Centrifugal Filter Units (EMD Millipore Corp, Billerica, MA, USA). The final dextran- and PAA-coated CeO~2~ nanoparticle preparations were stored at a 1.5 mg CeO~2~/mL concentration in deionized water until further used; such concentrations have previously been shown to be nontoxic to mammalian cells.[@b15-ijn-8-3395],[@b30-ijn-8-3395],[@b31-ijn-8-3395]

The various polymer-coated CeO~2~ nanoparticle preparations were characterized by dynamic light scattering (DLS) to determine the size, zeta potential, and surface charge of the nanoparticles (Zetasizer; Malvern Instruments Ltd, Malvern, UK). Fourier transform infrared (FTIR) spectrometry analysis was performed on vacuum dried samples to verify the surface functionalities of the nanoparticles (Spectrum™ 100 Optica; PerkinElmer Inc, Waltham, MA, USA).

Bacterial assays
----------------

A bacteria cell line of *P. aeruginosa* was obtained in freeze-dried form from the American Type Culture Collection (catalog number 27853; Manassas, VA, USA). The bacterial cells were propagated in 30 mg/mL tryptic soy broth (TSB) (catalog number 22092; Sigma-Aldrich Corp) for 24 hours in an incubator (37°C, humidified, 5% carbon dioxide). When the second passage of bacteria reached its stationary phase, the second passage was frozen in one part TSB and one part 40% sterile glycerol (catalog number G5516; Sigma-Aldrich Corp). Before bacterial seeding, a sterile 10 μL loop was used to withdraw bacteria from the prepared frozen stock, the bacteria were streaked onto a TSB Agar plate (catalog number A1296; Sigma-Aldrich Corp), and the TSB agar plate was incubated for 20 hours, to form single bacterial colonies. Then, a single colony was collected using a sterile loop and was incubated in a 15 mL sterile test tube containing 3 mL of TSB, for 20 hours. The bacterial solution was diluted to a concentration of 10^7^ bacteria/mL, which was assessed by measuring the optical density of the bacterial solution, using a standard curve correlating optical densities and bacterial concentrations. The optical densities were measured at 562 nm, using a SpectraMax M5 Microplate Reader (Molecular Devices LLC, Sunnyvale, CA, USA).

Two concentrations of both dextran- or PAA-coated CeO~2~ nanoparticles were separately tested against bacterial growth. The solutions of CeO~2~ nanoparticles were mixed with bacterial solutions (10^7^ bacteria/mL) at a 1:10 or 1:5 volume ratio and cultured for either 1, 6, or 24 hours in the incubator. Solutions of sterile TSB were mixed with bacteria solutions as blank controls. After the treatment, the solutions with bacteria were diluted with a phosphate-buffered saline (PBS) (catalog number P4417; Sigma-Aldrich Corp) solution and were spread on agar plates, and the bacteria colonies were counted after 18 hours of incubation.

Bacterial tests were conducted in triplicate and repeated three times. Data were collected, and the differences were assessed with the probability associated with a one-tailed Student's *t*-test. Statistical analyses were performed using Microsoft® Excel® 2010 (Microsoft Corporation, Redmond, WA, USA).

Results and discussion
======================

Cerium oxide nanoparticle characterization
------------------------------------------

The CeO~2~ nanoparticle preparations used in this study were aqueous suspensions of nanoparticles coated with either PAA or dextran. The synthetic procedure used to make these nanoparticles is highly reproducible and yielded nanoparticles with a CeO~2~ core of 3--4 nm, by transmission electron microscopy (TEM).[@b15-ijn-8-3395],[@b30-ijn-8-3395],[@b31-ijn-8-3395] Specifically, the hydrodynamic diameter of the PAA-coated CeO~2~ nanoparticles was 5 nm, while the size for the dextran-coated CeO~2~ nanoparticles was 14 nm.[@b15-ijn-8-3395],[@b30-ijn-8-3395],[@b31-ijn-8-3395] Confirmation of the polymer coating was accessed by FTIR, as previously described[@b15-ijn-8-3395],[@b30-ijn-8-3395],[@b31-ijn-8-3395] and by zeta potential, confirming the presence of a highly negative charged polymer coating for the PAA-coated CeO~2~ nanoparticles (ζ = −45 mV) and a lower negative charge (close to neutral) for the dextran-coated CeO~2~ nanoparticles (ζ = −2 mV).

Bacterial assays
----------------

As shown in [Figures 1](#f1-ijn-8-3395){ref-type="fig"} and [2](#f2-ijn-8-3395){ref-type="fig"}, the CeO~2~ nanoparticles did not show significant inhibition of *P. aeruginosa* growth compared with the TSB controls after 1 hour of treatment. However, after 6 hours of treatment, both concentrations of the dextran-coated CeO~2~ nanoparticles were found to have significantly inhibited the growth of *P. aeruginosa*, by about 20% compared with the TSB controls (*P* \< 0.05). The PAA-coated CeO~2~ nanoparticles showed significant inhibition of *P. aeruginosa* growth only for the higher concentration (bacterial solution:CeO~2~ nanoparticles = 5:1).

Most importantly, the growth of *P. aeruginosa* was strongly inhibited in the presence of either the dextran- or PAA-coated CeO~2~ nanoparticles after 24 hours compared with the TSB controls (*P* \< 0.01). When the volume ratio of bacterial solution to CeO~2~ nanoparticles was 10:1, *P. aeruginosa* growth was inhibited by 40.12% and 26.88% in the presence of dextran- and PAA-coated CeO~2~ nanoparticles, respectively, compared with the TSB controls. When the concentration of CeO~2~ nanoparticles doubled, the inhibition of *P. aeruginosa* growth reached 55.41% and 36.44% for the dextran- and PAA-coated CeO~2~ nanoparticles, respectively. Critically, all of this inhibition occurred without the use of antibiotics, with only the use of the CeO~2~ nanoparticles.

Based on the results of bacterial assays, the inhibition to *P. aeruginosa* growth by dextran- and PAA-coated CeO~2~ nanoparticles was more significant as the treatment time increased to 24 hours. In addition, the dextran-coated CeO~2~ nanoparticles showed a stronger inhibition of bacterial growth than did the PAA-coated CeO~2~ nanoparticles, especially after treatment for 6 and 24 hours. One possible reason the dextran coating and PAA coating could affect the bacterial growth differently is that the size of the dextran-coated CeO~2~ nanoparticles was 14 nm and the size of PAA-coated CeO~2~ nanoparticles was 5 nm, which translates to an altered ability to interact with bacteria membranes. In addition, as mentioned, the surface energy of the dextran- and PAA-coated CeO~2~ nanoparticles was different, which could also have strongly influenced their interactions with bacteria cell membranes. Moreover, as expected, from 6 hours to 24 hours, the bacteria propagated quickly in the TSB control, but the bacteria numbers decreased even more during this time period with the treatment with both kinds of coated CeO~2~ nanoparticles.

Importantly, the mechanism of how dextran- and PAA-coated CeO~2~ nanoparticles prevented bacterial growth remains unexplored. Since CeO~2~ nanoparticles have been shown to suppress the generation of ROS,[@b19-ijn-8-3395] their antibacterial properties demonstrated here, for the first time, could be different from those of many other nanoparticles (such as ZnO nanoparticles, silver \[Ag\] nanoparticles, and so on) that are believed to kill bacteria mainly through the generation of ROS. Although requiring additional investigation, the possible mechanism for the antibacterial action of the coated CeO~2~ nanoparticles formulated here could be that the dextran and PAA coatings interacted with specific proteins on the bacterial cell membranes to alter the permeability of the cell membranes, thus, killing the bacterial cells. Moreover, it is possible that the dextran or PAA coated CeO~2~ nanoparticles studied here may have promoted the adsorption, from the TSB, of proteins that are known to inhibit bacteria growth (such as fibronectin, mucin, vitronectin, etc).[@b32-ijn-8-3395] Future studies should also investigate how universal such bacterial inhibition is, by exposing such coated CeO~2~ nanoparticles to other bacteria (such as *Staphylococcus aureus* and *Escherichia coli*).

Conclusion
==========

In conclusion, the growth of *P. aeruginosa* was significantly inhibited in the presence of either dextran- or PAA-coated CeO~2~ nanoparticles after 24 hours. The effectiveness of bacterial-growth inhibition reached 55.41% for the dextran-coated CeO~2~ nanoparticles compared with the TSB control. This study revealed, for the first time, that the characterized coated CeO~2~ nanoparticles could potentially serve as a novel antibacterial agent in biomedical applications. Further studies, such as investigation of longer-term treatment and elucidating the mechanism of action, are important to achieve a better understanding and the application of such coated CeO~2~ nanoparticles in numerous antibacterial applications.

The authors thank Northeastern University and the University of Central Florida for funding.

**Disclosure**

The authors report no conflicts of interest in this work.

![Inhibited *Pseudomonas aeruginosa* growth in the presence of ceria nanoparticles at three time points tested (1, 6, and 24 hours).\
**Notes:** The volume ratio of bacterial solution (10^7^ bacteria/mL) and ceria nanoparticles was 10:1. Data is represented as mean ± standard deviation, n = 3; \**P* \< 0.01 compared with the TSB control group, after 24 hours of treatment. \*\**P* \< 0.05 compared with the TSB control group, after 6 hours of treatment.\
**Abbreviations:** Ce, ceria; Dex, dextran; PAA, polyacrylic acid; TSB, tryptic soy broth.](ijn-8-3395Fig1){#f1-ijn-8-3395}

![*Pseudomonas aeruginosa* growth in the presence of ceria nanoparticles at three time points tested (1, 6, and 24 hours).\
**Notes:** The volume ratio of bacterial solution (10^7^ bacteria/mL) and ceria nanoparticles was 5:1. Data is represented as mean ± standard deviation, n = 3; \**P* \< 0.01 compared with the TSB control group, after 24 hours of treatment. \*\**P* \< 0.05 compared with the TSB control group after, 6 hours of treatment.\
**Abbreviations:** Ce, ceria; Dex, dextran; PAA, polyacrylic acid; TSB, tryptic soy broth.](ijn-8-3395Fig2){#f2-ijn-8-3395}
