Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

Tuning ceria catalysts in aqueous media at the nanoscale: how do surface charge and surface defects determine peroxidase- and haloperoxidase-like reactivity.

Designing the shape and size of catalyst particles, and their interfacial charge, at the nanometer scale can radically change their performance. We demonstrate this with ceria nanoparticles. In aqueous media, nanoceria is a functional mimic of haloperoxidases, a group of enzymes that oxidize organic substrates, or of peroxidases that can degrade reactive oxygen species (ROS) such as H2O2 by oxidizing an organic substrate. We show that the chemical activity of CeO2-x nanoparticles in haloperoxidase- and peroxidase-like reactions scales with their active surface area, their surface charge, given by the ζ-potential, and their surface defects (via the Ce3+/Ce4+ ratio). Haloperoxidase-like reactions are controlled through the ζ-potential as they involve the adsorption of charged halide anions to the CeO2 surface, whereas peroxidase-like reactions without charged substrates are controlled through the specific surface area SBET. Mesoporous CeO2-x particles, with large surface areas, were prepared via template-free hydrothermal reactions and characterized by small-angle X-ray scattering. Surface area, ζ-potential and the Ce3+/Ce4+ ratio are controlled in a simple and predictable manner by the synthesis time of the hydrothermal reaction as demonstrated by X-ray photoelectron spectroscopy, sorption and ζ-potential measurements. The surface area increased with synthesis time, whilst the Ce3+/Ce4+ ratio scales inversely with decreasing ζ-potential. In this way the catalytic activity of mesoporous CeO2-x particles could be tailored selectively for haloperoxidase- and peroxidase-like reactions. The ease of tuning the surface properties of mesoporous CeO2x particles by varying the synthesis time makes the synthesis a powerful general tool for the preparation of nanocatalysts according to individual needs.

Generalized synthesis of NaCrO2 particles for high-rate sodium ion batteries prepared by microfluidic synthesis in segmented flow.

NaCrO2 particles for high-rate sodium ion batteries were prepared on a multigram scale in segmented flow from chromium nitrate and sodium nitrate using a segregated flow water-in-oil emulsion drying process. Microfluidic processing is an environmentally friendly and rapid synthetic method, which can produce large-scale industrial implementation for the production of materials with superior properties. The reaction time for NaCrO2 particles was reduced by almost one order of magnitude compared to a normal flask synthesis and by several orders of magntitude compared to a conventional solid-state reaction. In addition, it allows for an easy upscaling and was generalized for the synthesis of other layered oxides NaMO2 (M = Cr, Fe, Co, Al). The automated water-in-oil emulsion approach circumvents the diffusion limits of solid-state reactions by allowing a rapid intermixing of the components at a molecular level in submicrometer-sized micelles. A combination of Raman and nuclear magnetic resonance spectroscopy (1H, 23Na), thermal analysis, X-ray diffraction and high resolution transmission electron microscopy provided insight into the formation mechanism of NaCrO2 particles. The new synthesis method allows cathode materials of different types to be produced in a large scale, constant quality and in short reaction times in an automated manner.

High-throughput synthesis of CeO2 nanoparticles for transparent nanocomposites repelling Pseudomonas aeruginosa biofilms.

Preventing bacteria from adhering to material surfaces is an important technical problem and a major cause of infection. One of nature's defense strategies against bacterial colonization is based on the biohalogenation of signal substances that interfere with bacterial communication. Biohalogenation is catalyzed by haloperoxidases, a class of metal-dependent enzymes whose activity can be mimicked by ceria nanoparticles. Transparent CeO2/polycarbonate surfaces that prevent adhesion, proliferation, and spread of Pseudomonas aeruginosa PA14 were manufactured. Large amounts of monodisperse CeO2 nanoparticles were synthesized in segmented flow using a high-throughput microfluidic benchtop system using water/benzyl alcohol mixtures and oleylamine as capping agent. This reduced the reaction time for nanoceria by more than one order of magnitude compared to conventional batch methods. Ceria nanoparticles prepared by segmented flow showed high catalytic activity in halogenation reactions, which makes them highly efficient functional mimics of haloperoxidase enzymes. Haloperoxidases are used in nature by macroalgae to prevent formation of biofilms via halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). CeO2/polycarbonate nanocomposites were prepared by dip-coating plasma-treated polycarbonate panels in CeO2 dispersions. These showed a reduction in bacterial biofilm formation of up to 85% using P. aeruginosa PA14 as model organism. Besides biofilm formation, also the production of the virulence factor pyocyanin in is under control of the entire quorum sensing systems P. aeruginosa. CeO2/PC showed a decrease of up to 55% in pyocyanin production, whereas no effect on bacterial growth in liquid culture was observed. This indicates that CeO2 nanoparticles affect quorum sensing and inhibit biofilm formation in a non-biocidal manner.

Defect-controlled halogenating properties of lanthanide-doped ceria nanozymes.

Marine organisms combat bacterial colonization by biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidase enzymes, whose activity can be emulated by nanoceria using milli- and micromolar concentrations of Br- and H2O2. We show that the haloperoxidase-like activity of nanoceria can greatly be enhanced by Ln substitution in Ce1-xLnxO2-x/2. Non-agglomerated nanosized Ce1-xLnxO2-x/2 (Ln = Pr, Tb, particle size < 10 nm) was prepared mechanochemically from CeCl3 and Na2CO3 followed by short calcination. Lanthanide metals could be incorporated into the CeO2 host without solubility limit, as shown for Tb. The distribution of the Ln3+ defect sites in the CeO2 host structure was analyzed by electron spin resonance spectroscopy. Ce3+ and superoxide O2- species are present at surface sites. Their formation is promoted by increasing dopant concentration. Ce1-xLnxO2-x/2 was prepared in copious amounts by ball-milling. This energy-saving and residue-free method can be upscaled to industrial scale. The surface defect chemistry of Ce1-xLnxO2-x/2 was unravelled by vibrational spectroscopy. It is associated with the mechanochemical preparation and leads to enhanced catalytic activity. Although Ce0.9Pr0.1O1.95 had a lower BET surface area than pure CeO2, its catalytic activity, calibrated by oxidative bromination of phenol red, was much higher because the ζ-potential increased from 15 mV (for CeO2) to 30 mV (for Ce0.9Pr0.1O1.95). This facilitates adsorption of Br- in aqueous conditions and explains the high catalytic activity of the Ln-substituted CeO2. Ce1-xLnxO2-x/2 is an effective and "green" nanoparticle haloperoxidase mimic for antifouling applications, as no chemicals other than the ubiquitous Br- and H2O2 (generated in daylight) are required, and only natural metabolites are released into the environment.

Transparent polycarbonate coated with CeO2 nanozymes repel Pseudomonas aeruginosa PA14 biofilms.

Highly transparent CeO2/polycarbonate surfaces were fabricated that prevent adhesion, proliferation, and the spread of bacteria. CeO2 nanoparticles with diameters of 10-15 nm and lengths of 100-200 nm for this application were prepared by oxidizing aqueous dispersions of Ce(OH)3 with H2O2 in the presence of nitrilotriacetic acid (NTA) as the capping agent. The surface-functionalized water-dispersible CeO2 nanorods showed high catalytic activity in the halogenation reactions, which makes them highly efficient functional mimics of haloperoxidases. These enzymes are used in nature to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). Bacteria-repellent CeO2/polycarbonate plates were prepared by dip-coating plasma-treated polycarbonate plates in aqueous CeO2 particle dispersions. The quasi-enzymatic activity of the CeO2 coating was demonstrated using phenol red enzyme assays. The monolayer coating of CeO2 nanorods (1.6 µg cm-2) and the bacteria repellent properties were demonstrated by atomic force microscopy, biofilm assays, and fluorescence measurements. The engineered polymer surfaces have the ability to repel biofilms as green antimicrobials on plastics, where H2O2 is present in humid environments such as automotive parts, greenhouses, or plastic containers for rainwater.

Nanocomposite antimicrobials prevent bacterial growth through the enzyme-like activity of Bi-doped cerium dioxide (Ce1-xBixO2-δ).

Preventing bacterial adhesion on materials surfaces is an important problem in marine, industrial, medical and environmental fields and a topic of major medical and societal importance. A defense strategy of marine organisms against bacterial colonization relies on the biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidases, a class of metal-dependent enzymes, whose activity can be emulated by ceria nanoparticles. The enzyme-like activity of ceria was enhanced by a factor of 3 through bismuth substitution (Ce1-xBixO2-δ). The solubility of Bi3+ in CeO2 is confined to the range 0 < x < 0.25 under quasi-hydrothermal conditions. The Bi3+ cations are located close to the nanoparticle surface because their ionic radii are larger than those of the tetravalent Ce4+ ions. The synthesis of Ce1-xBixO2-δ (0 < x < 0.25) nanoparticles was upscaled to yields of ∼50 g. The halogenation activity of Ce1-xBixO2-δ was demonstrated with phenol red assays. The maximum activity for x ≈ 0.2 is related to the interplay of the ζ-potential of surface-engineered Ce1-xBixO2-δ nanoparticles and their BET surface area. Ce0.80Bi0.20O1.9 nanoparticles with optimized activity were incorporated in polyethersulfone beads, which are typical constituents of water filter membrane supports. Although Ce1-xBixO2-δ nanoparticles are not bactericidal on their own, naked Ce1-xBixO2-δ nanoparticles and polyethersulfone/Ce1-xBixO2-δ nanocomposites showed a strongly reduced bacterial coverage. We attribute the decreased adhesion of the Gram-negative soil bacterium Pseudomonas aeruginosa and of Phaeobacter gallaeciensis, a primary bacterial colonizer in marine biofilms, to the formation of halogenated signaling compounds. No biocides are needed, H2O2 (formed in daylight) and halide are the only substrates required. The haloperoxidase-like activity of Ce1-xBixO2-δ may be a promising starting point for the development of environmentally friendly, "green" nanocomposites, when the use of conventional biocides is prohibited.