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# Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties

Slavica Stankic, Sneha Suman, Francia Haque, Jasmina Vidic

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Slavica Stankic, Sneha Suman, Francia Haque, Jasmina Vidic. Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties. Journal of Nanobiotechnology, 2016, 14 (1), pp.73. 10.1186/s12951-016-0225-6. [hal-01391064](https://hal.sorbonne-universite.fr/hal-01391064)

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# Pure and multi metal oxide nanoparticles: synthesis, antibacterial and cytotoxic properties

Slavica Stankic<fnref n="1" />,²*, Sneha Suman³, Francia Haque<fnref n="1" />,² and Jasmina Vidic<fnref n="4" />,⁵,⁶*

**Abstract**

The antibacterial activity of metal oxide nanoparticles has received marked global attention as they can be specifically synthesized to exhibit significant toxicity to bacteria. The importance of their application as antibacterial agents is evident keeping in mind the limited range and effectiveness of antibiotics, on one hand, and the plethora of metal oxides, on the other, along with the propensity of nanoparticles to induce resistance being much lower than that of antibiotics. Effective inhibition against a wide range of bacteria is well known for several nano oxides consisting of one metal (Fe₃O₄, TiO₂, CuO, ZnO), whereas, research in the field of multi-metal oxides still demands extensive exploration. This is understandable given that the relationship between physicochemical properties and biological activity seems to be complex and difficult to generalize even for metal oxide nanoparticles consisting of only one metal component. Also, despite the broad scope that metal oxide nanoparticles have as antibacterial agents, there arise problems in practical applications taking into account the cytotoxic effects. In this respect, the consideration of polymetallic oxides for biological applications becomes even greater since these can provide synergetic effects and unify the best physicochemical properties of their components. For instance, strong antibacterial efficiency specific of one metal oxide can be complemented by non-cytotoxicity of another. This review presents the main methods and technological advances in fabrication of nanostructured metal oxides with a particular emphasis to multi-metal oxide nanoparticles, their antibacterial effects and cytotoxicity.

**Keywords:** Multi-metal oxide nanoparticles, Nanoparticles synthesis, Antibacterial activity, Cytotoxicity

**Review**

**Background**

Nanomaterials have numerous applications in areas ranging from catalysis, photonics, molecular computing, energy storage, fuel cells, tunable resonant devices, sensing to nanomedicine. This is due to an increase in reactivity when compared to their micro-sized counterparts since nanoscaled materials exhibit larger surface-to-volume ratio which provides unsaturated and, thus, more reactive surface atoms. To consider nanoparticles for biological applications, such as drug delivery,

biosensing, imaging and antibacterial therapeutics, several key requirements have to be fulfilled. The first is to deal with the engineered nanoparticles of well characterized composition, size, crystallinity and morphology. The second implies manipulation of stabilized, non-agglomerated nanomaterials in order to control dosing. Finally, the most crucial requirement is their biocompatibility. Despite very fast expansion of the bionanotechnology in the last 30 years, there are many challenges facing these three requirements. Relevant works that aimed at correlated synthesis, stabilization and surface modification of nanoparticles with their biological effects and decreased toxicity have shown that there is no general rule.

Presently, microbial resistance to antibiotics has been reaching a critical level. In exploring various options to address this problem, inorganic nanomaterials, like metal oxide nanoparticles, have emerged as

<footnote>*Correspondence: slavica.stankic@insp.upmc.fr; jasmina.vidic@inra.fr</footnote>

<footnote n="1">¹ CNRS, Institut des Nanosciences de Paris (INSP), UMR 7588, 4 Place Jussieu, 75252 Paris Cedex 05, France</footnote>

<footnote n="4">⁴ Virologie et Immunologie Moléculaires, UR892, INRA, Paris Saclay University, Jouy en Josas, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
promising candidates since they possess greater durability, lower toxicity and higher stability and selectivity when compared to organic ones. Nanostructured metal oxides have already been extensively studied for their promising use in technology. This has resulted in development of numerous reproducible procedures for the synthesis of nanoparticles with desired characteristics—like size, shape, morphology, defects in the crystal structure, monodispersity—providing a rich background for research relevant to antibacterial applications. Characterization of these nanoparticles can be helpful in modifying and tuning their antibacterial and cytotoxic effects. For instance, it has been established that the antibacterial activity increases with decreasing the particles size [1]. In contrast, the crystallographic orientation appears to have no effect on antibacterial activity [2], whereas increasing the lattice constants enhances the antibacterial activity [3]. It has also been proposed that different morphologies and crystal growth habits can affect the antibacterial activity [4]. Hence, the synthesis technique employed is functional in determining the biological characteristics of a given nanoparticle. As potential novel anti-bacterial agents, metal oxide nanoparticles like $Fe_3O_4$, $TiO_2$, CuO and ZnO are being thoroughly investigated. Their relatively low toxicity against human cells [5], low cost [6], size-dependent effective inhibition against a wide range of bacteria, ability to prevent biofilm formation [7] and even eliminate spores [8] make them suitable for application as anti-bacterial agents in the fabric [7], skincare products [9], biomedical [10] and food-additive industries [11]. However, research to understand cytotoxic effects and the corresponding mechanisms is necessary to adapt this class of nanomaterials for safe applications.

Recent achievements in nanotechnology of metal oxides include elaboration of nanostructured oxides consisting of two or more metallic components. Their potential applications are immense due to their unique electronic, optical, magnetic and other physicochemical properties [12]. Multi-metal oxide nanoparticles, like $Zn_xMg_{1-x}O$, Ta-doped ZnO, Ag/$Fe_3O_4$ nanocomposites, are being studied extensively as potential antimicrobial agents owing to the beneficial synergistic effects of their components. These nanoparticles have shown promising solutions to problems seen in pure metal oxide nanoparticles, like high cytotoxicity or agglomeration. In this paper, we have discussed the existing synthesis routes and the antibacterial activity of metal oxide nanoparticles with a particular focus on polymetallic oxides. Additionally, a strong emphasis has been given to their cytotoxic nature.

## Synthesis methods of metal oxide nanoparticles

Before exploring the antibacterial properties of metal oxide nanoparticles, a review of the various synthesis methods has been described. We make broadly a division of synthesis methods into three categories: solution based, vapor state and biological methods. Such division is based on the type of the medium in which the oxidation reaction takes place. The choice of synthesis method determines the physicochemical characteristics of the metal oxide nanoparticle, such as the size, dispersity, type of intrinsic and/or extrinsic defects, morphology and crystal structure. An example is given in Fig. 1 for nano-ZnMgO fabricated via three different synthesis methods. Corresponding TEM images show that this polymetallic oxide can be found in form of regular cubes of similar size (chemical vapor synthesis, CVS ZnMgO), a mixture of cubes and tetrapods (metal combustion, Smoke ZnMgO) and irregular nanorods (sol–gel ZnMgO). It was, furthermore, shown that despite cubic and hexagonal phase, that are thermodynamically most stable for pure MgO and ZnO, respectively, CVS technique allows for stabilization of one crystal structure in ZnMgO. Diffractions specific of only cubic crystal phase were observed in the corresponding XRD pattern while other measurements demonstrated that Zn-atoms replace Mg atoms on the surface of nanocubes [13]. The surface segregation of Zn-atoms is highlighted by green color surrounding cubes in the illustration of Fig. 1. In contrast, phase separation is most probably the reason for the presence of two types of shapes in ZnMgO powder obtained via metal combustion.

All these physicochemical properties, that are evidently in a strong correlation with the synthesis route, determine nanoparticles surface energies and, thus, their interaction with biological entities.

## Solution based synthesis

**Sonochemical method** In sonochemical methods, solution of the starting material (for e.g. metallic salts) is subjected to a stream of intensified ultrasonic vibrations which breaks the chemical bonds of the compounds. The ultrasound waves pass through the solution causing alternate compression and relaxation. This leads to acoustic cavitation i.e. formation, growth and implosive collapse of bubbles in the liquid. In addition, the change in pressure creates microscopic bubbles that implose violently leading to emergence of shock waves within the gas phase of the collapsing bubbles. Cumulatively, the effect of millions of bubbles collapsing produces an excessive amount of energy that is released in the solution. Transient temperatures of ~5000 K, pressure of ~1800 atm and cooling rates above $10^{10}$ K/s have been recorded at the localized
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cavitational implosion hotspots [14]. The excessively high rate of cooling process is found to affect the formation and crystallization of the obtained products [15]. This method has been used to synthesize a wide range of nano-materials as metals, alloys, metal oxides, metal sulfides, metal nitrides, metal-polymer composites, metal chalcogenides, metal carbides etc. [16]. Examples of reported metal oxides synthesized by this method include TiO₂ [17], ZnO [18], CeO₂ [19], MoO₃ [20], V₂O₅ [21], In₂O₃ and Eu/Dy-doped In₂O₃ [22], ZnFe₂O₄ [23], PbWO₄ [24], BiPO₄ [25], ZnAl₂O₄ and ZnGa₂O₄—pure and doped with varying combinations of Dy⁺³, Tb⁺³, Eu⁺³ and Mn⁺² [26], Fe₃O₄ [27], BaFe₁₂O₁₉ [28] and Mn-doped γ-Fe₂O₃ [29]. Using this method, enhanced photocatalytic properties in the case of TiO₂ [17] or varying magnetism of iron-oxide nanoparticles [30–32] have been reported. The advantages associated with sonochemical methods include uniform size distribution, a higher surface area, faster reaction time and improved phase purity of the metal oxide nanoparticles as observed by various research groups mentioned in references listed above.

**Co-precipitation method**

**Co-precipitation method** involves precipitating the oxo-hydroxide form from a solution of a salt precursor (metal salts like nitrates or chlorides) in a solvent (like water) by using a precipitating medium. Once a critical concentration of species in solution is reached, a short burst of nucleation occurs followed by growth phase. This method has been employed in synthesizing metal oxides like ZnO [33], MnO₂ [34], BiVO₄ [35], MgO [36], Ni₁-xZnxFe₂O₄ [37], SnO₂ [38], Cu-doped ZnO [39], MgFe₂O₄ [40], Ni-CeZrO₂ [41] and Y₂O₃:Eu⁺³ [42]. Co-precipitation is commonly used for preparing magnetic nanoparticles such as magnetite by using a base, usually NaOH and NH₄OH, for alkaline co-precipitation of ferrous and ferric salts dissolved in water in stoichiometric amounts [34, 43, 44]. The use of NaOH, KOH and (C₂H₅)₄NOH as a precipitating medium has established that pH, the nature of alkali, the slow or fast addition of alkaline solution and the drying modality of synthesized powders affect the size, paramagnetic properties and degree of agglomeration of the synthesized magnetite nanoparticles [44]. In addition, the use of sur-
factants has been seen to be useful in optimizing further the surface characteristics [42]. The advantages of this method are low cost, mild reaction conditions like low synthesis temperature, the possibility to perform direct synthesis in water, simplicity of processing, the ease of scale-up, flexibility in modulation of core and surface properties [39, 44].

**Solvothermal method** These methods are employed to prepare a variety of nanomaterials by dispersing the starting material in a suitable solvent and subjecting it to moderately high temperature and pressure conditions which lead to product formation. An organometallic complex of titanium, orthobutoxide, was for instance used for the synthesis of TiO₂ nanoparticles [45]. When the reaction is performed using water as the solvent, the method is called hydrothermal synthesis. Chemical parameters (type, composition and concentration of the reactants, ratio-solvent/reducing agent) and thermodynamic parameters (temperature, pressure and reaction time) affect the final particle formation. It was also observed that basicity and hydrolysis ratio of the reacting medium together with the steric or electrostatic stabilization of the reactive molecules affect the nucleation and growth steps, which in turn control the particle size, shape, composition and crystal structure of particles. For instance, varying the hydrolysis ratio allows to synthesize either metal or (oxy)hydroxide or oxide nanoparticles [46]. Nanoparticles of Nb₂O₅, MgO, TiO₂, MnFe₂O₄, CoFe₂O₄ and Fe₃O₄ have been synthesized using polyol as the solvent [46–50]. Solvothermal methods have successfully been employed to prepare various nanocomposites displaying a combination of the properties of their parent nanoparticles. Zhai et al. [51] have synthesized novel water-soluble nanohybrids composed of shape-tuned Ag cores and a Fe₃O₄ shell. Graphene-TiO₂ nanocomposites [52], CoFe₂O₄@BaTiO₃ nanocomposites [53], a series of multifunctional magnetic core–shell hetero-nanostructures (Fe₃O₄@NiO and Fe₃O₄@Co₃O₄) [54] are some other examples. This method, moreover, allows for the preparation of ultra-small nanoparticles (<5 nm) such as 2.5 × 4.3 nm TiO₂ nanoparticles [55] and 1.6 ± 0.3 nm WOₓ nanoparticles [56]. In the latter case, it was shown that, the use of reducing/oxidizing agents may strongly affect both, the size (use of an oxidizing agent led to particles with diameters smaller then 1 nm) and the shape (use of a reducing agent led to rod-shaped nanoparticles). Tian et al. have shown that adjusting the ratio of reducing agent and solvent can tune the particle size of magnetite nanoparticles from ~6 to 1 nm [57] while iron oxide nanostructures could be produced in different morphologies—such as, nanocubes [58] and hollow spheres [59]—by this synthesis route. Another advantage of this

technique is the use of suitable surfactants that can tune the particle characteristics and limit their agglomeration. For example, using a zwitterionic surfactant, smaller ZnO particle sizes were obtained as compared with those obtained from surfactant-free hydrothermal reaction [60]. Du et al. have reported surfactant assisted solvothermal technique to prepare mixed metal oxide nanoparticles like barium ferrite (BaFe₁₂O₁₉) and Co-Ti-doped barium-ferrite nanoparticles (Ba(CoTi) × Fe₁₂ – 2 × O₁₉) with high-purity crystalline phase, small particle size and good magnetic properties [61].

**Sol–gel method** Main steps of sol–gel method include the hydrolysis of metalorganic compound precursors, like alcoxysilane [62] to produce corresponding oxohydroxide, followed by condensation to form a network of the metal hydroxide. After hydroxide polymerizes it forms a dense porous gel the subsequent drying and heating of which leads to the production of ultrafine porous oxides in the desired crystal phase. The method has been used to synthesize a variety of metal oxide nanoparticles, like TiO₂ [63], ZnO [64], MgO [65], CuO [66], ZrO₂ and Nb₂O₅ [67] and nanocomposites, like LiCoO₂ thin film [68], Cu doped ZnO nanoparticles [69], CuO/Cu₂O nanocomposites [70], Ce-doped ZrO₂ [71], oxides of Hf, Ta and Nb [72]. Moreover, sol gel method is promising in doping of Group 5 oxides, which is generally a challenge. It is seen as a clean, surfactant free technique to synthesize high quality nanocrystals of doped metal oxide nanoparticles with magnetic properties like cobalt doped Hf-oxide nanoparticles [72]. To eliminate/reduce limitations associated with this method, researchers have incorporated certain modifications. For instance, Corr et al. have reported a modified one-step sol–gel aqueous approach for the synthesis of iron oxide-silica nanocomposite [62]. The modification consisted of employing ultrasonic conditions to overcome the effects of high temperature conditions (up to 600 °C) which could lead to oxidation of the products. Under the effect of ultrasound vibrations, high temperatures and pressures could instantaneously be generated and then dissipated in the local environment of the particles avoiding oxidation [73]. This technique has also been used to prepare novel nanocomposites like InNbO₄, a photocatalytically active ternary metal oxides semiconductor [74]. Sol–gel method, moreover, allows for a formation of multi-metal oxides instead of a mixture of the individual binary oxides—as shown for SnO₂-doped In₂O₃ [75]. Also it provides the particle size to be tuned by simply varying the gelation time [76]. In addition, it has been reported that supercritical fluids can be used to synthesize nanoparticles like TiO₂, ZrO₂, Al₂O₃, TiO₂-SiO₂, SiO₂-Al₂O₃ and ZrO₂/TiO₂ hybrid oxide nanotubes [77].
**Microwave-assisted method** This method has been of increasing interest as it is relatively low energy and time consuming [78]. The reaction times are reduced from a few hours to several minutes without compromising the particle purity or size. Faster reaction rates can be achieved by employing high heating rates which favor rapid nucleation and formation of small, highly monodisperse particles. Microwave-assisted methods involve quick and uniform heating of the reaction medium with no temperature gradients through two mechanisms: dipolar polarization and ionic conduction. Highly crystalline nanoparticles of MnO, Fe₃O₄, CeO₂, CaO, BaTiO₃, ZnO, Cr₂O₃, CoO, Mn₂O₃ and MgO have been successfully synthesized using microwave-assisted routes [79–82]. Automation allows control over the reaction conditions and hence facilitates manipulation of particle size, morphology and crystallinity [83]. The choice of starting metal oxides precursors (as acetates, chlorides, isopropyls) and solvents (as ethylene glycol, benzene) can govern reaction success, particle size and crystal structure [80].

**Microemulsion method** This method comprises two immiscible phases (oil and water) which are separated by a monolayer of surfactant molecules forming two binary systems—water/surfactant and oil/surfactant—such that the hydrophobic tails of the surfactant molecules are dissolved in the oil phase and the hydrophilic head groups in the aqueous phase. Broadly the method comprises of mixing appropriate amounts of the surfactant, oil, water and the metallic precursor (for instance, organometallic precursor can be added as a solution in the oily phase) by stirring at room temperature to prepare a homogenized phase [84]. Reducing/oxidizing/precipitating agents are then added, under vigorous stirring, to enable sedimentation of the nanoparticles. The microemulsions act as nanoreactors for synthesis of the nanoparticles. This is then followed by centrifugation, wash cycles and drying/calcination. Shape and size can be manipulated in these methods by affecting the various self-assembled structures formed in the binary systems [85]. This method was used to synthesize iron oxide nanoparticles [86], NiO [85], CeO₂ [84], TiO₂ [87], ZnO [88], CuO [89], and nanocomposites like BaAlO₂ [90], iron-oxide doped alumina nanoparticles [91]. The ability to control the formation of different kinds of core–shell structures with sub-nanometric resolution is seen as a major benefit of this technique [92]. Additionally, the method also provides the possibility to manipulate size and morphology of nanoparticles by adjusting parameters such as concentration and type of surfactant, the type of continuous phase, the concentration of precursors and molar ratio of water to surfactant. The disadvantage associated with this method involves the necessity of several washing processes and further sta-

bilization treatment due to aggregation of the produced nanoparticles [86]. Modifications have been incorporated to overcome these disadvantages. For instance, reverse microemulsion technique has been used to produce monodisperse spherical ZnO nanoparticles. The modification was that ZnO nanoparticles were not directly produced in the microemulsion but by the thermal decomposition of zinc glycerolate microemulsion product during subsequent calcination process [93]. The modified technique prevented agglomeration whereas the calcination temperature and concentration of surfactant could be varied in order to tune the particle size and morphology of the ZnO nanoparticles, respectively.

## Vapor state synthesis

**Laser ablation method** This method is used to generate nanoparticles by laser irradiation of immersed targets of colloidal solutions generated from bulk materials immersed in aqueous or non-aqueous solvents [94]. The method has been used to synthesize ZnO [95], NiO [96], SnO₂ [97], ZrO₂ [98], iron-oxide [99], Al₂O₃ [100] but also ternary metal oxides like Au-SnO₂ [101], Cu/Cu₂O [102]. The size of the nanoparticles can be controlled by manipulating two parameters: laser fluence and the nature of the liquid media [103, 104]. Indeed, the size of the nanoparticles increases with increase in thickness of the molten layer, which in turn increases with increase in laser fluence. The nature of the liquid plays an important role as the vapor pressure of the liquid and provides the recoil pressure under which the molten layer transforms into nanoparticles. Liu et al. [105] have established laser ablation of metal targets in aqueous environments to generate nanoparticles of oxides of Ti and Ni with well-controlled phase, size and size distribution, along with high production rate. Some of the drawbacks associated with laser ablation are related to propensity for nanoparticle agglomeration, lack of long term stabilization in solution and the need for capping [106].

**Chemical vapor based methods** In chemical vapor deposition (CVD), substrates are heated to high temperatures and exposed to precursor materials in the gaseous state. The precursors react or decompose on the substrate surface to form nanomaterial. In chemical vapor synthesis (CVS) approach, within a flow reactor pure metal or metal–organic salts are by heating transformed into the vapor phase and introduced into a hot-wall reactor where they react with the oxidizing agent under conditions that favor the chemical [107, 108]. Usually an inert gas, such as Ar, is used to carry the gaseous reactants to the reaction zone where nucleation and crystal growth occur. Finally, the product that is also in the gas phase is carried to a much cooler zone where it due to such temperature gra-
dient transforms into a solid state and can get collected. These techniques are extensively employed to produce uniform and contamination-free metal oxide nanoparticles and films; such as ZnO nanowires and films [109] and defect-free ZnO nanoparticles [110], nanocubes and nanospheres of magnetite [111], Cu₂O [112], MgO and CaO [113], SnO₂ [114], SrO [115], CoO and Co₃O₄ [116]. When multi-metal oxides are considered, this technique allows for the production of B-doped ZnO [117], euro-pium doped yttria (YO: Eu) [118], Li-doped MgO [119], Ca-doped [92, 120]. Moreover, via CVS technique Zn²⁺ cations may selectively replace Mg²⁺ surface cations preferentially at the edges and corners of MgO nanocubes that resulted in unique optical and chemical surface properties of ternary ZnₓMg₁₋ₓO nanoparticles [13]. Reproducibility is another advantage associated with this method [121]. Careful choice of experimental parameters such for instance the nature and/or concentration of the oxidizing agent used has a major effect on the nucleation process and consequently affects the average size of the particles. This has been reported for MgO nanoparticles which could be produced via CVS technique in the average size ranging from 3, 5 or 11 nm—depending whether N₂O or O₂ or dry air were used as the oxidizing agent [122]. Control over particle size can be also realized by varying the reaction temperature [110] since the nucleation and growth kinetics can be controlled by manipulation of temperature and reactant concentration [123]. Reactant delivery, reaction energy input and product separation may also affect the characteristics and quality of the product. These techniques can be modified to obtain desirable attributes in the nanoparticles and eliminate limitations associated with volatility of the reactants and degree of agglomeration. Some examples are laser assisted [124], electrospray assisted [125], thermally activated/pyrolytic, metalorganic, plasma-assisted and photo CVD methodologies [126]. For instance, electrospray assisted chemical vapor deposition (ES-CVD) was employed to synthesize non-agglomerated spherical titanium and zirconium oxide nanoparticles [125]. Djenadic and Winterer [124] have used laser assisted technique to synthesize TiO₂ and Co-doped ZnO magnetic semiconducting nanoparticles.

**Combustion method** In this synthesis method, pure metallic precursor is heated by different techniques to evaporate it into a background gas in which the second reactant i.e. oxidizing agent is admixed. The synthesis starts with an initialization in which the metal is only partially heated for the oxidation reaction to start. Thereafter, the heat required for the following metal evaporation is produced in situ by the combustion reactions itself. Even though this process is very successful commercially, the

coupling of the particle production to the flame chemistry makes this a complex process that is rather difficult to control. However, the control over partial pressure of oxidizing agent that determines the nucleation and growth can affect the particle size to some extent, as it has been shown for MgO nanosmoke [127]. Nanoparticles of ZnO [128], FeO [129], CuO, Mn₂O₃, MgO [127], CdO and Co₃O₄ [130] or Ag supported on MgO surface [131], Co₃O₄ on CuO nanowire arrays (Co₃O₄@CuO) [132], La₀.₈₂Sr₀.₁₈MnO₃ [133]. Another example of using this synthesis route for the production of polynuclear oxides was shown in the work by Vidic et al. [134]. In this paper a phase separation—an existence of both, the hexagonal ZnO and cubic MgO crystal phases—has been demonstrated. Despite this disadvantage relatively good antibacterial efficiency and biocompatibility of ZnMgO nanoparticles were shown. Modifications in combustion technique, such as reported by Lee and Choi who have used a CO₂ laser to re-heat flame-synthesis technique, affects nanoparticle morphology and degree of agglomeration of TiO₂ nanoparticles [135]. Wegner et al. [136] have employed a modification by using a critical flow nozzle to extract synthesized titania nanoparticles from the flame to quench particle growth and agglomeration.

**Template/surface-mediated synthesis** The major strategies employed in this type of fabrication are electrochemical [137], electroless and sol-gel [138], chemical polymerization [139], and chemical vapor deposition [140]. Consequently, as reaction between metal and oxidizing agent may take place in different medium this method can be attributed to both of the previously listed classes of synthesis. The method is based on fabrication of the desired nanomaterial within the pores or channels of a nanoporous template. Depending on the properties of the template, various morphologies of nanomaterial such as rods, fibrils, and tubules, can be prepared. This method can be used to synthesize self-assembly systems with tubular and fibrillary like nanostructures with small diameters [141]. Highly monodisperse nanostructures with enhanced activities, uniform morphology and a high specific surface area can be obtained using this synthesis method [142]. Examples are mesoporous MoO₃ nanoparticles with improved electrochemical properties [143], α-Fe₃O₄ and Co₃O₄ [144], Fe₂O₃ [145] and mesoporous NiMn₂Oₓ [144]. The templates used for such synthesis methods mainly are track-etch membranes, porous alumina and other nanoporous structures, like mesoporous zeolites [146, 147]. Carbon nanotubes have been used for the fabrication of a variety of metal oxide nanoparticles like PbO, Bi₂O₃, V₂O₅, SiO₂, Al₂O₃, MoO₃, MnO₂, Co₃O₄, ZnO, and WO₃ [148–150]. The choice of precursor, fixa-
are considered. Biological method occurs as a good alternative but the studies on biogenic synthesis methods are scanty and much work is necessary to improve their efficiency in a first place. Chemical and physical methods are definitely superior in producing larger quantities of nanoparticles but their main advantage over biological is the ability to control the size and shape. “Biocompatible production” needs, therefore, more active research to widen commercialization prospects.

## Metal oxide nanoparticle in aqueous solution

Physico-chemical properties of metal oxide nanoparticles are surface specific and directly dependent on their surface-to-bulk ratio. Therefore, nanoparticles manipulation and storage may modify their fundamental properties. The classical approach of surface science studies employs experimental techniques which preserve pristine properties of nanoparticles. Such techniques imply ultra-high or at least high vacuum conditions i.e. conditions in which the residual pressure of air components is minimized and the surface modifications negligible. However, biological applications typically expose nanoparticles to aqueous environment in which their surfaces may undergo a series of physico-chemical modifications. Accordingly, nanoparticles characteristics, as well as their dispersion and stability have also to be examined in water and biological media or fluids. Indeed, particle dissolution, aggregation/agglomeration and protein corona formation on the particle surfaces may take place in aqueous solutions leading to properties that strongly differ to the ones characteristic for as-synthesized forms.

## Stabilization and biocompatibility of metal oxide nanoparticles

Notably, metal oxide nanoparticles dissolve partially in water solutions which leads to the modification of their morphology in which formation of new crystallographic phases may take place [158]. The propensity to dissolve in water depends on the composition and structure of the nanoparticles, as was demonstrated for nano-ZnO [159]. The dissolution rate was also shown to strongly depend on nanoparticles size [160]. The significantly higher dissolution rate was observed for CVS-MgO cubes (~5 nm average size) than for smoke-MgO cubes (~80 nm average size) produced by magnesium combustion in air. Small CVS-MgO particles were shown to be completely transformed into Mg(OH)₂ in a water solution. In contrast, on larger smoke-MgO nanoparticles the formed surface hydroxide led to a self-inhibition resulting in only partial dissolution and surface faceting [160]. In addition, the aggregation of metal oxide nanoparticles in water solutions is a common phenomenon. Among

others, a typical example which undergoes strong aggregation in water is TiO₂ nanopowder [161, 162].

Two strategies for nanoparticle dispersion in water are mainly employed: electrostatic stabilization and steric repulsion. In the course of electrostatic stabilization, particles do not aggregate due to their equal charges i.e. electrostatic repulsion. This method is simple to realize but demands well defined pH and ionic strength of the solution and the control of the presence of reactive species such as OH- or H₃O⁺ ions that can modify the surface charge of metal oxide particles. Considering steric repulsion, the surface of nanoparticle is modified by an appropriate hydrocarbon polymer or a bio-macromolecule. Such stabilizing molecules can be adsorbed or grafted onto the nanoparticles surface to prevent direct contact between them and, thus, their aggregation. Consequently, the nanoparticles remain dispersed in water solution even upon pH changes or salt concentration [163].

Bovine serum albumin is a commonly used stabilization agent as it spontaneously forms a protein corona around metal oxides particles [162]. The advantage of using albumin lies in its biological role to nonspecifically bind various molecules and its natural and abundant presence in biological fluids, such as blood. As albumin is a charged biomacromolecule, its adsorption on the metal oxides allows both nanoparticle steric and electrostatic stabilization. However, albumin adsorption on nanoparticles is not always stable and may, thus, be inefficient for some applications. For instance, when nano-ZnMgO was added to cell culture medium containing albumin as the most abundant protein, the protein corona consisted of many other proteins from the medium [164]. This indicated that over time the most abundant protein in initially formed corona may be replaced by proteins which are less abundant but have higher affinities to interact with nanoparticles' surface. In such cases, superior surface-active agents have to be used for effective nanoparticle stabilization in a given medium. For instance, the prevention of nanoparticle aggregation and the achievement of their stable dispersion in an aqueous solution might be obtained by adding a mild detergent, as Tween-20 or P-20. Also, a recent study has shown that nontoxic polycarbonate ethers may efficiently substitute albumin to stabilize TiO₂ giving a suspension of non-aggregated nano-TiO₂ in various cell culture media tested [165].

## Antibacterial activity of metal oxide nanoparticles

Several metal oxides in form of nanoparticles have been reported to exhibit marked antibacterial activity allowing efficient eradication of various bacterial strains. This fact has attracted significant interest of environmental, agricultural and health care industries that are searching for
newer and better agents to control or prevent bacterial
infections. Many studies have been undertaken to explain
the efficacy and mechanisms of antibacterial action of
metal oxide nanoparticles but the existent literature is
still controversial and incomplete. It was demonstrated,
however, that when applied at well-defined sizes, crys-
tal structure and concentrations, these nanoparticles are
highly effective inhibitors against a wide range of bacte-
ria. Although their exact antibacterial mechanism is still
under debate, some distinctive mechanisms have been
proposed, which include reactive oxygen species (ROS)
formation, metal-ion release, particle internalization into
bacteria and direct mechanical destruction of bacterial
cell wall and/or membrane (Fig. 2). Metal oxide nano-
particles may show bacteriostatic or bactericidal effect.
In case of bacteriostatic effect, treated bacteria do not die
but stop to reproduce or grow. If treated bacterial cells
are removed from the solution containing nanoparticles,
they re-start to grow. This can be easily tested by plating
these bacterial cells onto new nanoparticle-free agar. In
case of bactericidal effect, no bacterial colonies can be
observed upon re-plating treated bacteria onto nano-
particle-free agar. Depending on the experimental con-
ditions, nanoparticle concentration and bacterial strain,

a particular type of metal oxides nanoparticle may have
bacteriostatic or bactericidal effect as shown for ZnO
[166] or TiO₂ [167].

Different ions, small molecules (such as H₂O₂), free
radicals (like, OH, ¹O₂) or superoxide ions (such as O₂⁻)
are examples of highly reactive ROS species which can be
produced on the surface of metal oxide nanoparticles and
can induce bacterial cell death. ROS-induced damages
and bacterial death comprise oxidative stress, oxidative
lesions and membrane lipid peroxidation. In addition,
ROS can harm bacterial components such as proteins
and nucleic acids. For instance, oxidative stress induced
by Ag₂O nanoparticles was shown to damage the DNA
of *E. coli* which led to the interruption of the bacterial
cell cycle and induction of bacterial death [168]. Also,
CuO nanoparticles were shown to generate ROS, namely
superoxide anions, when adsorbed onto the bacterial cell
surfaces or internalized into bacterial cells. Formed ROS
induced bactericidal effect in both Gram-positive (S.
*aureus*) and Gram-negative (*E. coli*) bacteria [169].

The physicochemical characteristics of metal oxide
nanoparticles, such as size, crystal structure defects,
composition and surface charge, are directly associated
with enhanced antibacterial effects. The synthesis and

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Metal oxide nanoparticles interracting with bacteria. Molecular mechanisms of antibacterial activities of metal oxide nanoparticles</figcaption></figure>
treatment procedures employed can tune these charac-
teristics, as discussed in the previous sections, and hence
produce the desired antibacterial efficacy. For instance,
nanoparticles of smaller sizes (<20 nm) can easily pen-
etrate into bacterial cells and may release toxic metal ions
upon dissolution [170]. Thus, smaller particles are usu-
ally the most efficient antibacterial agents. However, this
is not the case when decrease in size leads to enhanced
aggregation. Also, defects present at the nanoparticles'
surface influence strongly antibacterial efficiency. Point
defects, such as atoms at edges and in corners give rise
to an abrasive surface that may cause the injury of the
bacterial cell wall or membrane. For instance, it was
proposed that partial dissolution of nano-ZnO in water
medium results in formation of surface defects giving an
uneven surface texture due to rough edges and corners.
This surface roughness was shown to be responsible for
mechanical damage of the cell membrane of *E. coli*. Wang
et al. [171] have also suggested that the crystallographic
orientation and type of surface plane can influence anti-
bacterial efficiency of ZnO nanowires. They showed that
randomly oriented ZnO nanowires were more efficient in
killing *E. coli* than regularly oriented ones. This is prob-
ably due to different spatial arrangements of ZnO.

Surface charge was also shown to play an important
role in membrane damage and particle internaliza-
tion. Bacterial membranes and cell walls are typically of
negative total charge. Electrostatic attractions can occur
between bacterial surfaces and metal oxide nanoparti-
cles of positive zeta-potential, like observed for positively
charged nano-ZnO and negatively charged *C. jejuni* cells.
Xie et al. [172] proposed that upon binding to bacte-
rial surface, ZnO nanoparticles disrupted the cell mem-
brane causing morphological changes and measurable
membrane leakage in *C. jejuni*. Moreover, even particles
of negative zeta potential may damage cell membranes
since interactions cannot only be electrostatic, but Van
der Waals and hydrophobic as well. Metal oxide nano-
particles may specifically bind some moieties within
membrane barrier surface such as phosphate, amine or
carboxyl groups in lipids and proteins and subsequently
induce bacterial death. It is worth noting that metal oxide
nanoparticles remain tightly bound to the surface of
damaged or dead bacteria which may modify their effec-
tive concentration in the given solution over time.

Since metal oxide nanoparticles with varying phys-
icochemical characteristics exhibit different antibacte-
rial mechanisms and effects, oxide nanoparticles with a
combination of two or more metals can be developed for
efficient elimination of various bacterial strains includ-
ing those highly resistant to existing treatments. Table 1
summarizes some examples of multi-metal oxide nano-
particles tested for their applications in eradication of

different bacterial strains. Interestingly, some multi-metal
oxide nanoparticles show higher antimicrobial activity
when compared to their pure components of similar size.

For instance, nanostructured ZnMgO produced by
combustion technique exhibit advantageous properties
from both of its pure components: high antibacterial
activity of nano-ZnO and low cytotoxicity of nano-MgO
[134]. This mixed metal oxide inhibited Gram-positive
bacteria (*B. subtilis*) completely and Gram-negative bacte-
ria (*E. coli*) partially upon 24 h treatment [134]. ZnMgO
nanoparticles were shown to damage bacterial cells by
causing extensive injury to membranes that resulted
in a leakage of the cell content as illustrated in Fig. 3.
Comparatively, pure ZnO nanorods and nanotetrapods
exhibited the highest but nonselective activity as they
completely eradicated both bacterial strains and mam-
malian HeLa cells, under the same treatment protocol
[134]. In contrast, pure MgO nanocubes only partially
inhibited bacterial growth being at the same time harm-
less to mammalian cells.

In case of Zn/Fe oxide nanocomposites, antibacte-
rial effectiveness similar to that of ZnO nanoparticles,
was observed [173]. However, no particle agglomeration,
typical for nano-ZnO in water solutions was detected.
Compared to nano-ZnO, the pure Fe₃O₄ lacks significant
antibacterial efficiency, but exhibits good colloidal stabil-
ity [173]. It was observed that both the antibacterial effect
and stability of Zn/Fe oxide nanocomposite in an aqueous
medium can be optimized by changing the ratio of Zn/Fe.
The study suggested that hydroxyl radicals were formed at
the surface of zinc oxide. These active oxygen derivatives
were proposed to damage bacterial cells of *E. coli* and *S.
aureus*. Since similar mechanisms were not observed for
zinc ferrite, it appears that iron oxide contributes only
towards good colloidal stability of the composite. In
another study, Fe³⁺-ions were used to dope nano-ZnO
in order to induce the formation of lattice defects in
ZnO nanocrystals and thus to enhance its antibacterial
efficiency. It was shown that Fe-doped ZnO nanoparti-
cles efficiently inhibited *E. coli* bacterial growth without
being toxic to mammalian cells [193]. Fe³⁺-ions acted as
an impurity in the ZnO nanostructure that enhanced the
overall antimicrobial activity. Similarly, it was observed
that sea urchin-like ZnO doped with 5 % iron had a strong
antimicrobial activity, as it killed up to 95 % *C. albicans*
and *A. flavus* [194]. Inserting Fe³⁺-ions into ZnO lattice
increased antibacterial efficiency by decreasing the size
of ZnO nanoparticles and favoring the formation of sea
urchin-like structure. Moreover, Fe³⁺-ions have the capac-
ity to kill bacteria by destroying both cell walls and mem-
branes due to their strong reduction ability. Also, binding
Fe³⁺-ions to biomolecules may cause protein denatura-
tion, DNA damaging and enzyme function alternating.
<table><caption>Table 1 Some examples of mixed and doped metal oxide nanoparticles that were tested for their antibacterial activity</caption><thead><tr><td>Metal oxides nanoparticle</td><td>Synthesis/doping method</td><td>Bacterial strain tested</td><td>References</td></tr></thead><tbody><tr><td>Zn/Fe oxide</td><td>Sol gel</td><td>S. aureus; E. coli</td><td>[173]</td></tr><tr><td>Zn/Mg oxide</td><td>Combustion</td><td>E. coli; B. subtilis</td><td>[134]</td></tr><tr><td>ZnO/Au</td><td>Photo-reduction</td><td>E. coli; S. aureus</td><td>[174]</td></tr><tr><td>TiO<sub>2</sub>/Ag</td><td>Reactive magnetron sputtering</td><td>S. aureus</td><td>[175]</td></tr><tr><td>Fe<sub>3</sub>O<sub>4</sub>/Ag</td><td>Template based</td><td>S. aureus</td><td>[176]</td></tr><tr><td>Ta-doped ZnO</td><td>Sol gel</td><td>B. subtilis; S. aureus; E. coli; P. aeruginosa</td><td>[177]</td></tr><tr><td>Fe-doped ZnO</td><td>Sol gel</td><td>E. coli</td><td>[178]</td></tr><tr><td>Ce-doped ZnO</td><td>Sonochemical</td><td>E. coli</td><td>[179]</td></tr><tr><td>Nd-doped ZnO</td><td>Co-precipitation</td><td>E. coli; K. pneumoniae</td><td>[180]</td></tr><tr><td>Zn-doped CuO</td><td>Sonochemical</td><td>E. coli; S. aureus</td><td>[181]</td></tr><tr><td>Zn-doped TiO<sub>2</sub></td><td>Electrospinning</td><td>E. coli; S. aureus</td><td>[182]</td></tr><tr><td>Ag-doped TiO<sub>2</sub></td><td>TiO<sub>2</sub>-Sol gel<br>Ag-doped TiO<sub>2</sub>-Solvothermal</td><td>E. coli; S. aureus</td><td>[183]</td></tr><tr><td>Cu-doped TiO<sub>2</sub></td><td>Flame Synthesis</td><td>M. smegmatis; S. oneidensis</td><td>[184]</td></tr><tr><td>Cu-doped TiO<sub>2</sub></td><td>Co-precipitation</td><td>E. coli</td><td>[185]</td></tr><tr><td>Li-doped MgO</td><td>Sol gel</td><td>E. coli</td><td>[186]</td></tr><tr><td>Cu-doped MgO</td><td>Co-precipitation</td><td>E. coli</td><td>[187]</td></tr><tr><td>Ag-doped SiO<sub>2</sub></td><td>SiO<sub>2</sub>-Sol gel<br>Doped SiO<sub>2</sub>- Co-precipitation</td><td>P. aeruginosa; S. aureus; E. coli</td><td>[188]</td></tr><tr><td>Mn- and Fe-doped ZnO</td><td>Co-precipitation</td><td>S. aureus; E. coli; K. pneumoniae; S. typhi; P. aeruginosa; B. subtilis</td><td>[189]</td></tr><tr><td>Zn- and/or Y-doped TiO<sub>2</sub></td><td>Sol gel</td><td>C. albicans; S. aureus</td><td>[190]</td></tr><tr><td>Zn/Ce/SO<sub>4</sub><sup>2-</sup>-doped TiO<sub>2</sub></td><td>Sol gel</td><td>E. coli; S. aureus</td><td>[191]</td></tr><tr><td>Ag-TiO<sub>2</sub>-doped SiO<sub>2</sub></td><td>Sol gel</td><td>E. coli</td><td>[192]</td></tr></tbody></table>

Guo et al. [177] have reported that when ZnO nanoparticles were doped with Ta, the bactericidal activity was revealed to be higher than that of pure nano-ZnO. The introduction of Ta<sup>5+</sup>-ions into ZnO crystal structure induced changes in structure, morphology and surface defect concentration giving larger lattice parameter, smaller grain size and more active defect sites and hydroxyl groups—formed upon particles dissolution in water. In consequence, the surface reactivity of nano-ZnO could be dramatically increased by Ta-doping. The antimicrobial activity of Ta-doped ZnO nanoparticles was tested on *B. subtilis*, *S. aureus* (Gram-positive bacteria) and *E. coli* and *P. aeruginosa* (Gram-negative bacteria) under dark ambient and visible light irradiation. The incorporation of Ta<sup>5+</sup>-ions into ZnO significantly improved the bacteriostatic effect of ZnO nanoparticles on *E. coli*, *S. aureus* and *B. subtilis* in the absence of light, while both Ta-doped ZnO and pure ZnO nanoparticles showed increased bactericidal efficacy on *P. aeruginosa*, *E. coli* and *S. aureus* under visible light illumination. It was proposed that the high valence of Ta<sup>5+</sup> might generate Zn vacancy or oxygen interstitial to keep the electric neutral equilibrium in the crystal structure. This, in return, increased the productions of ROS. In addition, the high valence of Ta<sup>5+</sup> increased electrostatic

attractions between metal oxide nanoparticles and the bacterial surface which also facilitated the bactericidal action. The differences in bactericidal efficiencies observed with various strains may originate from different structure and composition of the bacteria tested. For instance, Gram-negative bacteria possess a double membrane bilayer while Gram-positive bacteria are limited only by one lipid bilayer.

In another study, He et al. [174] have observed that deposition of small Au particles of 3 nm diameter onto the surface of ZnO nanoparticles—at a very low ZnO/Au molar ratio (0.2 %)—significantly enhanced the photocatalytic and antibacterial activity of ZnO. Indeed, deposition of Au onto ZnO nanoparticles resulted in production of holes and electrons at the particle surface which dramatically increased light-induced generation of hydroxyl radical, superoxide and singlet oxygen. When incubated with *E. coli*, the ZnO/Au hybrid nanostructures showed about three times higher antibacterial efficiency than pure ZnO nanoparticles. Also, ZnO nanoparticles doped with both Mn and Fe ions (10 % molar ratio) exhibited higher antibacterial activities as compared to 1 % loading or pure ZnO when incubated with *S. aureus*, *E. coli*, *K. pneumoniae*, *S. typhi*, *P. aeruginosa* and *B. subtilis* [189]. The enhancement in antimicrobial
Nano-ZnMgO-*E. coli*

<figure><img src="image_6.png" /></figure>

Nano-ZnMgO-*B. subtilis*

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 3** Treating *E. coli* and *B. subtilis* with ZnMgO nanoparticles. TEM images of Gram-negative bacteria *E. coli* (blue) and Gram-positive bacteria *B. subtilis* (rose) treated with mixed ZnMgO nanoparticles. Note nanoparticles association with bacterial cells, the leakage of cell content and particles aggregations in the bacterial grown medium. Images were obtained at MIMA2 MET platform in INRA Jouy en Josas. Bar 1000 nm</figcaption></figure>

effectiveness was attributed to the increased generation of ROS due to the synergistic effects of Mn and Fe loading. When bound to the bacterial surface Fe- and Mn-doped ZnO nanoparticles induced an apparition of holes on the membrane surfaces, which subsequently led to cell death. Interestingly, these doped nanoparticles shown higher efficiency against Gram-negative than against Gram-positive bacteria.

Similarly, TiO₂ nanoparticles coated with Ag nanoparticles showed increased antibacterial effectiveness against S. aureus compared to pure TiO₂ [175]. The proposed mechanism involved a direct mechanical destruction of bacterial cells upon binding of nanoparticles to their surfaces. The final effect was enhanced by bactericidal activity of released silver from the particle surface. In addition to enhanced antibacterial activity, Ag/TiO₂ hybrid structures showed higher durability compared to pure TiO₂. In another study, TiO₂ nanoparticles were doped with zinc and/or yttrium in order to increase their antibacterial activity [190]. It was shown that bactericidal efficiency of the obtained Zn-Y/TiO₂ nanomaterials strongly depended on the synthesis procedure but also

on composition and irradiation with visible light. Zinc-doped TiO₂ nanoparticles were much more efficient than yttrium-doped ones when C. albicans or S. aureus were treated for 30 min upon visible light irradiation. However, the double-doped Zn-Y/TiO₂ nanoparticles revealed the highest antibacterial activities compared to pure TiO₂, Zn-doped TiO₂ or Y-doped TiO₂ when exposed to visible light. Since antibacterial activity of Zn-Y/TiO₂ nanoparticles was weaker in dark than that in visible irradiation the mechanism seemed to be related to the generation of toxic hydroxyl radical upon illumination. Moreover, co-doped nanoparticles were shown to release Zn and Y ions, both highly toxic for bacteria since they easily penetrate cell membrane barrier.

Another approach consisted in applying a mild solvothermal method to synthesize Ag-doped TiO₂ nanosheet films. When film attachment method was used to estimate Ag-TiO₂ activities against *E. coli* and *S. aureus* growth, the excellent performance in killing bacteria under UV light and in the dark was observed [183]. Ag-doped TiO₂ combined the advantages of highly efficient antibacterial effects of Ag with low cost production of
TiO₂ nanoparticles. In addition to acting as an antimicrobial auxiliary agent in this complex material, silver also acted as a sink for electrons and redox catalyst which enhanced the photo-oxidation ability of TiO₂. Indeed, Ti⁴⁺-ions in TiO₂ substituted by monovalent Ag⁺-ions increased the density of defects and generation of oxygen vacancies, which improved antibacterial performance of the nanosheets. However, possible applications of this nanomaterial are limited by the cytotoxicity of Ag–TiO₂ nanocomposites, especially those that contain more than 4 % of silver. Likewise, Cu-doped TiO₂ have been observed to exhibit higher antibacterial activity than pure nano-TiO₂. Moreover, Cu-doped TiO₂ nanoparticles of 20 nm diameter synthesized by a flame aerosol method significantly reduced the growth rate of *M. smegmatis*, but did not affect the growth of *S. oneidensis* at 20 mg/L. In contrast, pure TiO₂ had no effect on growth of the two strains even at 100 mg/L [184]. Cu-doped TiO₂ nanoparticles, similarly to non-doped TiO₂, agglomerated in the bacterial medium and, thus, probably did not directly damage bacterial cellular structures. The overall inhibitory effect on *M. smegmatis* growth suggested that Cu²⁺ and TiO₂ might have synergistic effects and that TiO₂ nanoparticles served as a carrier and concentrator of highly efficient copper ions which resulted in an enhancement of antibacterial efficiency compared to pure CuO. The toxicity of Cu-doped TiO₂ was probably driven by the release of Cu²⁺-ions since the corresponding antibacterial effectiveness increased with an increase of copper content. Interestingly, *S. oneidensis MR-1* was able to tolerate high concentrations of Cu²⁺-ions as capable to enzymatically reduced ionic copper in a culture medium.

Pure nano-MgO exhibits only mild antimicrobial activity against both Gram-positive and Gram-negative bacteria but has an advantage in being synthesized from available and economical precursors. Metal-ion doping has been shown to be an effective method to improve its antibacterial efficiency. However, Rao et al. [195] have shown that doping MgO with different metal ions may give opposite effects on nanoparticles' antibacterial properties. Li-doped MgO was more efficient than pure MgO, while Zn- and Ti-doped nano-MgO displayed poorer antibacterial activity than MgO. The authors concluded that doping with Li⁺ promoted the generation of oxygen vacancies and increased the basicity of the oxide, which favorited generation and stabilization of superoxide anion, O₂⁻. In contrast, Ti²⁺ and Zn²⁺, having higher valence than Li⁺, less efficiently favored these two phenomena although Ti-doped MgO was somehow more efficient than Zn-doped MgO in eliminating *E. coli*— which was ascribed to smaller sizes of Ti-doped MgO compared to those of Zn-doped MgO nanoparticles.

Although CuO nanoparticles have been shown to possess marked antibacterial activities, they usually have to be applied in higher doses and are not efficient against all bacterial strains. However, when used in a nanocomposite form, CuO has been shown to be highly efficient. For instance, Zn-doped CuO nanocomposite in a colloidal suspension form or deposited on the fabric shown a 10,000 times enhancement in the antibacterial activity against *E. coli* and *S. aureus* bacteria compared to pure ZnO or CuO [181]. Physicochemical characterization of the nanocomposite suggested that Zn-ions were incorporated into the crystalline lattice of CuO. Such nanocomposite produced larger amount of ROS in an aqueous solution and subsequently more toxic OH radical, superoxide anions and singlet oxygen than its pure metal oxides components. Although it is difficult to compare antibacterial efficiency of one nanocomposite towards different bacterial strains, it appears that bacteria rich in amine and carboxyl groups at the surface, like *B. subtilis*, bind more strongly CuO and thus is more sensitive to its bactericidal effects.

## Toxicity of mixed metal oxide nanoparticles

Understanding the mechanisms involved in interaction of metal oxide nanoparticles with mammalian cells is required for any safe practical application. Presently, we lack knowledge about the general mechanism by which these nanoparticles bind and interact with eukaryotic cells. Similar to antibacterial activity, the cytotoxicity of metal oxide nanoparticles is also dependent on their physicochemical characteristics. Metal oxide nanoparticles of similar size but of different compositions usually show varying cytotoxic effects. For instance, when toxicities of CuO, TiO₂, ZnO, CuZnFe₂O₄, Fe₃O₄ and Fe₂O₃ were compared in vitro using human alveolar epithelial cells A549, CuO was shown to induce a high percentage of cell death together with DNA damage, while TiO₂ could only trigger DNA damage [196]. Interestingly, pure Fe₃O₄ and Fe₂O₃ were almost harmless while mixed CuZnFe₂O₄ nanoparticles strongly damaged DNA. Such results strongly suggest that metal oxide nanoparticles of different compositions interact with living cells through different mechanisms—some of which are schematically presented in Fig. 4.

Lai et al. [197] have shown that ZnO nanoparticles are the most, TiO₂ nanoparticles the second most and MgO nanoparticles the least effective in induction of human cell death. A study of 19 different metal oxide nanoparticles suggested that the most important factor that determines their toxicity is the inherent toxicity of the metal-ions released [198]. Another recent study of 11 types of metal oxide nanoparticles of similar sizes (<20 nm diameter) suggested that differences in their
<figure><img src="image_10.png" /><figcaption>**Fig. 4** Metal oxide nanoparticles interreacting with mammalian cell. Various mechanisms of metal oxide nanoparticles toxicity towards mammalian cells</figcaption></figure>

toxicities might be explained by two principal aspects: release of metal-ions, which is observed as the main mechanism for ZnO and CuO, and induction of ROS generation, observed for $Mn_3O_4$ and $Co_3O_4$ [199]. Interestingly, all nanoparticles tested in this study were shown to be internalized by A549 cells. This suggests that ROS formation and metal-ion release may be triggered from internalized nanoparticles within cells.

Presently, surface coating of metal oxide nanoparticles is employed to modify their toxicity. Nevertheless, the same coating may enhance or reduce the toxic effects depending on their initial surface properties. This was shown for different crystals of nano-ZnO stabilized with trichlorododecylsilane [200]. Probably, the final toxicity reflects physicochemical modifications obtained upon coating such as changes in particle aggregation state, dissolution, zeta potential, and ion and free radical releasing to a solution. Thus, the primary determinant of particle toxicity seems to be its starting surface property and not the coating. Furthermore, the same nanomaterial may show different reactivity, and consequently, toxicity in different media. For instance, the toxicity of nano-ZnO comes partially from the released $Zn^{2+}$-ions into aqueous biological media. It was shown that its toxicity can be lowered using a medium containing phosphate ions [201]. Indeed,

the formation of Zn-phosphate inactivates the hazardous $Zn^{2+}$-ions. Similarly a metal-ion chelator diethylene triamine pentaacetic acid can be used to decrease toxicity of metal oxide nanoparticles [202]. Although mild determinants used preventing nanoparticle aggregation are supposed to be interactive, they may additionally alter the toxicity of a given nanoparticle [203].

Finally, the toxicity can also be reduced by doping metal oxide nanoparticles with other metal ions. For instance, nano-ZnO released toxic $Zn^{2+}$-ions and generated ROS causing mitochondria perturbations, cell inflammation and induced cytotoxicity in treated lungs and embryos [204]. All these pro-oxidative and pro-inflammatory effects were reduced by iron doping of nano-ZnO [204]. A uniform distribution of Fe atoms throughout the ZnO crystal structure enhanced the crystal stability in an aqueous solution and reduced dissolution of doped nanoparticles in biological media [205]. Nano-ZnO was more effective in inducing cellular death than nano-MgO [134] but, surprisingly, mixed nano-ZnMgO nanoparticles, containing less than 5 % of zinc were inoffensive to mammalian cells, thus, behaving safely as a pure nano-MgO [134].

Penetration of metal oxide nanoparticles in eukaryotic cells may be prevented by particles binding to specific
biomolecules, such as membrane proteins [5]. In other cases, internalized nanoparticles are degraded in cellular endosomes or liposomes and then metabolized. Consequently, such metal oxide nanoparticles are considered safe as they neither affect cell viability nor induce apoptosis. The specific features of nanoparticle interaction with cells depend on the surface energy of the particles, which may be modulated by synthesis procedure or functionalization of their surfaces. The practical application of metal oxide nanoparticles as bactericidal agents is, thus, possible at certain conditions and particle concentrations at which there is low or no toxicity against mammalian cells, as demonstrated for ZnO [5], Fe₂O₃ [206] or Ag₂O₃ [207].

The application of metal oxide nanoparticles as new antibacterial agents strikingly depends on their cytotoxic nature. Meanwhile, it is important to mark that many studies dealing with cytotoxicity of metal oxide nanoparticles are being done with nanoparticles of not well characterized physicochemical properties. Also, the standardized testing procedure for toxicity assessment of nanoparticles is lacking. This implies that our understanding of cytotoxic mechanisms is incomplete and non-uniform. Taking into account that metal oxide nanoparticles are a class of nanomaterials with the highest global annual production, we expect that the progress in addressing their cytotoxicity will be made rapidly.

## Conclusions

Multi-metal oxide nanoparticles are promising candidates for antibacterial applications if the synergic effects of their constituents are effectively harnessed. Numerous already existing synthesis methods provide a rich base that may fuel research devoted to such applications. This is particularly important keeping in mind that the type of the synthesis affects properties like size, shape, morphology, dispersity, presence and type of stress and defects in the crystal which in turn determines their interaction with bacterial and mammalian cells. The reactivity may also be determined by their solubility and degree of agglomeration. Even though no general conclusion has been established regarding the mechanism of metal oxide nanomaterials interacting with living cells and microorganisms, the commonly proposed once are: ROS formation, interaction with cell membrane, particle internalization and binding with specific targets such as proteins or DNA. In polymetallic oxides, physicochemical parameters of the corresponding components are altered which may lead to novel reactivity towards living organisms. Some multi-metal oxide nanoparticles have shown lesser tendency to aggregate in biological solutions and fluids resulting in an increased antibacterial activity while being highly biocompatible when compared to their components. Taking into account the numerous pure metal/

metal oxide components that can be combined to obtain desired complementary effects a plethora of polymetallic oxide nanoparticles can be created with properties specific in terms of their antibacterial activity, biocompatibility and monodispersity in biological media.

## Abbreviations

ROS: reactive oxygen species; CVD: chemical vapour deposition; CVS: chemical vapour synthesis; ES-CVD: electro-spray assisted chemical vapor deposition.

## Authors' contributions

SSt, SSu, and JV prepared the body of this paper and were all equally contributing to this work. FH performed synthesis of ZnMgO nanoparticles shown in Fig. 1. All authors read and approved the final manuscript.

## Author details

¹ CNRS, Institut des Nanosciences de Paris (INSP), UMR 7588, 4 Place Jussieu, 75252 Paris Cedex 05, France. ² UPMC-Université Paris 06, INSP, UMR 7588, Paris, France. ³ Birla Institute of Technology & Science, Pilani Campus, Vidya Vihar, Pilani, Rajasthan, India. ⁴ Virologie et Immunologie Moléculaires, UR892, INRA, Paris Saclay University, Jouy en Josas, France. ⁵ School of Material Science and Engineering, Nanyang Technological University, 50 Nanyang Ave, Singapore 639798, Singapore. ⁶ NTU-HJU-BGU CREATE Programme, 1 Create Way, Research Wing # 02-06 to 08, Singapore 138602, Singapore.

## Acknowledgements

We acknowledge BioAsia Grant 35976PH to JV and SSt, and the grant of the Agence Nationale de la Recherche SENSAIV (ANR-15-MRSE-0028-01) to JV. We thank the MIMA2 MET platform for its expertise and access to electron microscopy equipment. We also thank Jean Michel Guigner from IMPMC, Paris for recording TEM images shown in Fig. 1.

## Competing interests

The authors declare that they have no competing interests.

Received: 15 September 2016 Accepted: 12 October 2016

Published online: 24 October 2016

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