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

20 Pages·2016·2.73 MB·English

by  Slavica Stankic

Stankic et al. J Nanobiotechnol (2016) 14:73 Journal of Nanobiotechnology
DOI 10.1186/s12951-016-0225-6
REVIEW Open Access
Pure and multi metal oxide
nanoparticles: synthesis, antibacterial
and cytotoxic properties
Slavica Stankic1,2*, Sneha Suman3, Francia Haque1,2 and Jasmina Vidic4,5,6*
Abstract
Th 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 (Fe O , TiO , CuO, ZnO), whereas, research in the field of multi-metal oxides still demands extensive
3 4 2
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 biosensing, imaging and antibacterial therapeutics, sev-
Background eral key requirements have to be fulfilled. The first is to
Nanomaterials have numerous applications in areas deal with the engineered nanoparticles of well character-
ranging from catalysis, photonics, molecular comput- ized composition, size, crystallinity and morphology. The
ing, energy storage, fuel cells, tunable resonant devices, second implies manipulation of stabilized, non-agglom-
sensing to nanomedicine. This is due to an increase in erated nanomaterials in order to control dosing. Finally,
reactivity when compared to their micro-sized counter- the most crucial requirement is their biocompatibility.
parts since nanoscaled materials exhibit larger surface- Despite very fast expansion of the bionanotechnology in
to-volume ratio which provides unsaturated and, thus, the last 30 years, there are many challenges facing these
more reactive surface atoms. To consider nanoparti- three requirements. Relevant works that aimed at corre-
cles for biological applications, such as drug delivery, lating synthesis, stabilization and surface modification of
nanoparticles with their biological effects and decreased
toxicity have shown that there is no general rule.
*Correspondence: [email protected]; [email protected]
1 CNRS, Institut des Nanosciences de Paris (INSP), UMR 7588, 4 Place Presently, microbial resistance to antibiotics has
Jussieu, 75252 Paris Cedex 05, France been reaching a critical level. In exploring various
4 Virologie et Immunologie Moléculaires, UR892, INRA, Paris Saclay
options to address this problem, inorganic nanomate-
University, Jouy en Josas, France
Full list of author information is available at the end of the article rials, like metal oxide nanoparticles, have emerged as
© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 2 of 20
promising candidates since they possess greater dura- Synthesis methods of metal oxide nanoparticles
bility, lower toxicity and higher stability and selectiv- Before exploring the antibacterial properties of metal
ity when compared to organic ones. Nanostructured oxide nanoparticles, a review of the various synthesis
metal oxides have already been extensively studied for methods has been described. We make broadly a divi-
their promising use in technology. This has resulted sion of synthesis methods into three categories: solution
in development of numerous reproducible proce- based, vapor state and biological methods. Such division
dures for the synthesis of nanoparticles with desired is based on the type of the medium in which the oxida-
characteristics—like size, shape, morphology, defects tion reaction takes place. The choice of synthesis method
in the crystal structure, monodispersity—providing a determines the physicochemical characteristics of the
rich background for research relevant to antibacterial metal oxide nanoparticle, such as the size, dispersity, type
applications. Characterization of these nanoparticles of intrinsic and/or extrinsic defects, morphology and
can be helpful in modifying and tuning their antibac- crystal structure. An example is given in Fig. 1 for nano-
terial and cytotoxic effects. For instance, it has been ZnMgO fabricated via three different synthesis methods.
established that the antibacterial activity increases Corresponding TEM images show that this polymetallic
with decreasing the particles size [1]. In contrast, the oxide can be found in form of regular cubes of similar size
crystallographic orientation appears to have no effect (chemical vapor synthesis, CVS ZnMgO), a mixture of
on antibacterial activity [2], whereas increasing the lat- cubes and tetrapods (metal combustion, Smoke ZnMgO)
tice constants enhances the antibacterial activity [3]. and irregular nanorods (sol–gel ZnMgO). It was, further-
It has also been proposed that different morphologies more, shown that despite cubic and hexagonal phase,
and crystal growth habits can affect the antibacterial that are thermodynamically most stable for pure MgO
activity [4]. Hence, the synthesis technique employed and ZnO, respectively, CVS technique allows for stabi-
is functional in determining the biological character- lization of one crystal structure in ZnMgO. Diffractions
istics of a given nanoparticle. As potential novel anti- specific of only cubic crystal phase were observed in the
bacterial agents, metal oxide nanoparticles like Fe O , corresponding XRD pattern while other measurements
3 4
TiO , CuO and ZnO are being thoroughly investi- demonstrated that Zn-atoms replace Mg atoms on the
2
gated. Their relatively low toxicity against human cells surface of nanocubes [13]. The surface segregation of Zn-
[5], low cost [6], size-dependent effective inhibition atoms is highlighted by green color surrounding cubes
against a wide range of bacteria, ability to prevent bio- in the illustration of Fig. 1. In contrast, phase separa-
film formation [7] and even eliminate spores [8] make tion is most probably the reason for the presence of two
them suitable for application as anti-bacterial agents types of shapes in ZnMgO powder obtained via metal
in the fabric [7], skincare products [9], biomedical [10] combustion.
and food-additive industries [11]. However, research All these physicochemical properties, that are evi-
to understand cytotoxic effects and the corresponding dently in a strong correlation with the synthesis route,
mechanisms is necessary to adapt this class of nano- determine nanoparticles surface energies and, thus, their
materials for safe applications. interaction with biological entities.
Recent achievements in nanotechnology of metal
oxides include elaboration of nanostructured oxides Solution based synthesis
consisting of two or more metallic components. Their Sonochemical method In sonochemical methods, solu-
potential applications are immense due to their unique tion of the starting material (for e.g. metallic salts) is
electronic, optical, magnetic and other physicochemi- subjected to a stream of intensified ultrasonic vibrations
cal properties [12]. Multi-metal oxide nanoparticles, like which breaks the chemical bonds of the compounds. The
Zn Mg O, Ta-doped ZnO, Ag/Fe O nanocomposites, ultrasound waves pass through the solution causing alter-
x 1−x 3 4
are being studied extensively as potential antimicrobial nate compression and relaxation. This leads to acoustic
agents owing to the beneficial synergistic effects of their cavitation i.e. formation, growth and implosive collapse
components. These nanoparticles have shown promis- of bubbles in the liquid. In addition, the change in pres-
ing solutions to problems seen in pure metal oxide nano- sure creates microscopic bubbles that implode violently
particles, like high cytotoxicity or agglomeration. In this leading to emergence of shock waves within the gas phase
paper, we have discussed the existing synthesis routes of the collapsing bubbles. Cumulatively, the effect of mil-
and the antibacterial activity of metal oxide nanoparticles lions of bubbles collapsing produces an excessive amount
with a particular focus on polymetallic oxides. Addition- of energy that is released in the solution. Transient tem-
ally, a strong emphasis has been given to their cytotoxic peratures of ~5000 K, pressure of ~1800 atm and cooling
nature. rates above 1010 K/s have been recorded at the localized
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 3 of 20
Fig. 1 Various shapes of ZnMgO nanoparticles produced by dufferent synthesis routes. TEM images of ZnMgO nanoparticles obtained via three
different synthesis methods at the Paris Institute of Nanosciences and the illustrations of the corresponding crystal forms. All powders were kept at
P < 10−5 mbar after the synthesis while the microscopic measurements were performed on bare powders in order to analyze the initial morphology
resulting from the corresponding fabrication route. Surface segregation of Zn-atoms is highlighted by green color surrounding MgO cubes in the
illustration representing CVS method
cavitational implosion hotspots [14]. The excessively high Co‑precipitation method Co-precipitation method
rate of cooling process is found to affect the formation involves precipitating the oxo-hydroxide form from a
and crystallization of the obtained products [15]. This solution of a salt precursor (metal salts like nitrates or
method has been used to synthesize a wide range of nano- chlorides) in a solvent (like water) by using a precipitat-
materials as metals, alloys, metal oxides, metal sulfides, ing medium. Once a critical concentration of species in
metal nitrides, metalpolymer composites, metal chalco- solution is reached, a short burst of nucleation occurs fol-
genides, metal carbides etc. [16]. Examples of reported lowed by growth phase. This method has been employed
metal oxides synthesized by this method include TiO in synthesizing metal oxides like ZnO [33], MnO [34],
2 2
[17], ZnO [18], CeO [19], MoO [20], V O [21], In O BiVO [35], MgO [36], Ni Zn Fe  O   [37], SnO [38],
2 3 2 5 2 3 4 1-x x 2 4 2
and Eu/Dy-doped In O [22], ZnFe O [23], PbWO [24], Cu-doped ZnO [39], MgFe O [40], Ni–CeZrO [41]
2 3 2 4 4 2 4 2
BiPO [25], ZnAl O and ZnGa O —pure and doped with and Y O :Eu+3 [42]. Co-precipitation is commonly used
4 2 4 2 4 2 3
varying combinations of Dy+3, Tb+3, Eu+3 and Mn+2 [26], for preparing magnetic nanoparticles such as magnetite
Fe O [27], BaFe O [28] and Mn-doped γ-Fe O [29]. by using a base, usually NaOH and NH OH, for alkaline
3 4 12 19 2 3 4
Using this method, enhanced photocatalytic properties in co-precipitation of ferrous and ferric salts dissolved in
the case of TiO [17] or varying magnetism of iron-oxide water in stoichiometric amounts [34, 43, 44]. The use of
2
nanoparticles [30–32] have been reported. The advan- NaOH, KOH and (C H ) NOH as a precipitating medium
2 5 4
tages associated with sonochemical methods include uni- has established that pH, the nature of alkali, the slow or
form size distribution, a higher surface area, faster reac- fast addition of alkaline solution and the drying modal-
tion time and improved phase purity of the metal oxide ity of synthesized powders affect the size, paramagnetic
nanoparticles as observed by various research groups properties and degree of agglomeration of the synthesized
mentioned in references listed above. magnetite nanoparticles [44]. In addition, the use of sur-
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 4 of 20
factants has been seen to be useful in optimizing further technique is the use of suitable surfactants that can tune
the surface characteristics [42]. The advantages of this the particle characteristics and limit their agglomera-
method are low cost, mild reaction conditions like low tion. For example, using a zwitterionic surfactant, smaller
synthesis temperature, the possibility to perform direct ZnO particle sizes were obtained as compared with those
synthesis in water, simplicity of processing, the ease of obtained from surfactant-free hydrothermal reaction [60].
scale-up, flexibility in modulation of core and surface Du et al. have reported surfactant assisted solvothermal
properties [39, 44]. technique to prepare mixed metal oxide nanoparticles
like barium ferrite (BaFe O ) and Co-Ti-doped barium-
12 19
Solvothermal method These methods are employed to ferrite nanoparticles (Ba(CoTi)   Fe  – 2   O ) with
× 12 × 19
prepare a variety of nanomaterials by dispersing the start- high-purity crystalline phase, small particle size and good
ing material in a suitable solvent and subjecting it to mod- magnetic properties [61].
erately high temperature and pressure conditions which
lead to product formation. An organometallic complex Sol–gel method Main steps of sol–gel method include
of titanium, orthobutoxide, was for instance used for the the hydrolysis of metalorganic compound precursors,
synthesis of TiO nanoparticles [45]. When the reaction is like alcoxysilane [62] to produce corresponding oxo-
2
performed using water as the solvent, the method is called hydroxide, followed by condensation to form a network
hydrothermal synthesis. Chemical parameters (type, com- of the metal hydroxide. After hydroxide polymerizes it
position and concentration of the reactants, ratio-solvent/ forms a dense porous gel the subsequent drying and heat-
reducing agent) and thermodynamic parameters (temper- ing of which leads to the production of ultrafine porous
ature, pressure and reaction time) affect the final particle oxides in the desired crystal phase. The method has been
formation. It was also observed that basicity and hydroly- used to synthesize a variety of metal oxide nanoparticles,
sis ratio of the reacting medium together with the steric or like TiO [63], ZnO [64], MgO [65], CuO [66], ZrO and
2 2
electrostatic stabilization of the reactive molecules affect Nb O [67] and nanocomposites, like LiCoO thin film
2 5 2
the nucleation and growth steps, which in turn control the [68], Cu doped ZnO nanoparticles [69], CuO/Cu O nano-
2
particle size, shape, composition and crystal structure of composites [70], Ce-doped ZrO [71], oxides of Hf, Ta and
2
particles. For instance, varying the hydrolysis ratio allows Nb [72]. Moreover, sol gel method is promising in dop-
to synthesize either metal or (oxy)hydroxide or oxide ing of Group 5 oxides, which is generally a challenge. It
nanoparticles [46]. Nanoparticles of Nb O , MgO, TiO , is seen as a clean, surfactant free technique to synthesize
2 5 2
MnFe O , CoFe O and Fe O have been synthesized high quality nanocrystals of doped metal oxide nano-
2 4 2 4 3 4
using polyol as the solvent [46–50]. Solvothermal meth- particles with magnetic properties like cobalt doped Hf-
ods have successfully been employed to prepare various oxide nanoparticles [72]. To eliminate/reduce limitations
nanocomposites displaying a combination of the proper- associated with this method, researchers have incorpo-
ties of their parent nanoparticles. Zhai et al. [51] have syn- rated certain modifications. For instance, Corr et al. have
thesized novel water-soluble nanohybrids composed of reported a modified one-step sol–gel aqueous approach
shape-tuned Ag cores and a Fe O shell. Graphene-TiO for the synthesis of iron oxide-silica nanocomposite [62].
3 4 2
nanocomposites [52], CoFe O @BaTiO nanocompos- The modification consisted of employing ultrasonic con-
2 4 3
ites [53], a series of multifunctional magnetic core–shell ditions to overcome the effects of high temperature con-
hetero-nanostructures (Fe O @NiO and Fe O @Co O ) ditions (up to 600 °C) which could lead to oxidation of
3 4 3 4 3 4
[54] are some other examples. This method, moreover, the products. Under the effect of ultrasound vibrations,
allows for the preparation of ultra-small nanoparticles high temperatures and pressures could instantaneously be
(<5 nm) such as 2.5   4.3 nm TiO nanoparticles [55] and generated and then dissipated in the local environment
× 2
1.6   0.3 nm WO nanoparticles [56]. In the latter case, it of the particles avoiding oxidation [73]. This technique
± x
was shown that, the use of reducing/oxidizing agents may has also been used to prepare novel nanocomposites like
strongly affect both, the size (use of an oxidizing agent led InNbO , a photocatalytically active ternary metal oxides
4
to particles with diameters smaller then 1 nm) and the semiconductor [74]. Sol–gel method, moreover, allows for
shape (use of a reducing agent led to rod-shaped nano- a formation of multi-metal oxides instead of a mixture of
particles). Tian et al. have shown that adjusting the ratio the individual binary oxides—as shown for SnO -doped
2
of reducing agent and solvent can tune the particle size of In O [75]. Also it provides the particle size to be tuned by
2 3
magnetite nanoparticles from ~6 to 1 nm [57] while iron simply varying the gelation time [76]. In addition, it has
oxide nanostructures could be produced in different mor- been reported that supercritical fluids can be used to syn-
phologies—such as, nanocubes [58] and hollow spheres thesize nanoparticles like TiO , ZrO , Al O , TiO -SiO ,
2 2 2 3 2 2
[59]—by this synthesis route. Another advantage of this SiO -Al O and ZrO /TiO hybrid oxide nanotubes [77].
2 2 3 2 2
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 5 of 20
Microwave‑assisted method This method has been of bilization treatment due to aggregation of the produced
increasing interest as it is relatively low energy and time nanoparticles [86]. Modifications have been incorporated
consuming [78]. The reaction times are reduced from a to overcome these disadvantages. For instance, reverse
few hours to several minutes without compromising microemulsion technique has been used to produce
the particle purity or size. Faster reaction rates can be monodisperse spherical ZnO nanoparticles. The modifi-
achieved by employing high heating rates which favor cation was that ZnO nanoparticles were not directly pro-
rapid nucleation and formation of small, highly mono- duced in the microemulsion but by the thermal decompo-
disperse particles. Microwave-assisted methods involve sition of zinc glycerolate microemulsion product during
quick and uniform heating of the reaction medium with subsequent calcination process [93]. The modified tech-
no temperature gradients through two mechanisms: dipo- nique prevented agglomeration whereas the calcination
lar polarization and ionic conduction. Highly crystalline temperature and concentration of surfactant could be
nanoparticles of MnO, Fe O , CeO , CaO, BaTiO , ZnO, varied in order to tune the particle size and morphology
3 4 2 3
Cr O , CoO, Mn O and MgO have been successfully syn- of the ZnO nanoparticles, respectively.
2 3 2 3
thesized using microwave-assisted routes [79–82]. Auto-
mation allows control over the reaction conditions and Vapor state synthesis
hence facilitates manipulation of particle size, morphol- Laser ablation method This method is used to gener-
ogy and crystallinity [83]. The choice of starting metal ate nanoparticles by laser irradiation of immersed tar-
oxides precursors (as acetates, chlorides, isopropyls) and gets of colloidal solutions generated from bulk materials
solvents (as ethylene glycol, benzene) can govern reaction immersed in aqueous or non-aqueous solvents [94]. The
success, particle size and crystal structure [80]. method has been used to synthesize ZnO [95], NiO [96],
SnO [97], ZrO [98], iron-oxide [99], Al O [100] but
2 2 2 3
Microemulsion method This method comprises two also ternary metal oxides like Au-SnO [101], Cu/Cu O
2 2
immiscible phases (oil and water) which are separated by [102]. The size of the nanoparticles can be controlled
a monolayer of surfactant molecules forming two binary by manipulating two parameters: laser fluence and the
systems—water/surfactant and oil/surfactant—such that nature of the liquid media [103, 104]. Indeed, the size of
the hydrophobic tails of the surfactant molecules are dis- the nanoparticles increases with increase in thickness of
solved in the oil phase and the hydrophilic head groups the molten layer, which in turn increases with increase in
in the aqueous phase. Broadly the method comprises of laser fluence. The nature of the liquid plays an important
mixing appropriate amounts of the surfactant, oil, water role as the vapor pressure of the liquid and provides the
and the metallic precursor (for instance, organometallic recoil pressure under which the molten layer transforms
precursor can be added as a solution in the oily phase) by into nanoparticles. Liu et al. [105] have established laser
stirring at room temperature to prepare a homogenized ablation of metal targets in aqueous environments to gen-
phase [84]. Reducing/oxidizing/precipitating agents are erate nanoparticles of oxides of Ti and Ni with well-con-
then added, under vigorous stirring, to enable sedimen- trolled phase, size and size distribution, along with high
tation of the nanoparticles. The microemulsions act as production rate. Some of the drawbacks associated with
nanoreactors for synthesis of the nanoparticles. This is laser ablation are related to propensity for nanoparticle
then followed by centrifugation, wash cycles and drying/ agglomeration, lack of long term stabilization in solution
calcination. Shape and size can be manipulated in these and the need for capping [106].
methods by affecting the various self-assembled struc-
tures formed in the binary systems [85]. This method was Chemical vapor based methods In chemical vapor depo‑
used to synthesize iron oxide nanoparticles [86], NiO [85], sition (CVD), substrates are heated to high temperatures
CeO [84], TiO [87], ZnO [88], CuO [89], and nanocom- and exposed to precursor materials in the gaseous state.
2 2
posites like BaAlO [90], iron-oxide doped alumina nano- The precursors react or decompose on the substrate
2
particles [91]. The ability to control the formation of dif- surface to form nanomaterial. In chemical vapor synthe‑
ferent kinds of core–shell structures with sub-nanometric sis (CVS) approach, within a flow reactor pure metal or
resolution is seen as a major benefit of this technique [92]. metal–organic salts are by heating transformed into the
Additionally, the method also provides the possibility vapor phase and introduced into a hot-wall reactor where
to manipulate size and morphology of nanoparticles by they react with the oxidizing agent under conditions that
adjusting parameters such as concentration and type of favor the chemical [107, 108]. Usually an inert gas, such as
surfactant, the type of continuous phase, the concentra- Ar, is used to carry the gaseous reactants to the reaction
tion of precursors and molar ratio of water to surfactant. zone where nucleation and crystal growth occur. Finally,
The disadvantage associated with this method involves the product that is also in the gas phase is carried to a
the necessity of several washing processes and further sta- much cooler zone where it due to such temperature gra-
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 6 of 20
dient transforms into a solid state and can get collected. coupling of the particle production to the flame chemis-
These techniques are extensively employed to produce try makes this a complex process that is rather difficult
uniform and contamination-free metal oxide nanopar- to control. However, the control over partial pressure
ticles and films; such as ZnO nanowires and films [109] of oxidizing agent that determines the nucleation and
and defect-free ZnO nanoparticles [110], nanocubes and growth can affect the particle size to some extent, as it
nanospheres of magnetite [111], Cu O [112], MgO and has been shown for MgO nanosmoke [127]. Nanoparti-
2
CaO [113], SnO [114], SrO [115], CoO and Co O [116]. cles of ZnO [128], FeO [129], CuO, Mn O , MgO [127],
2 3 4 2 3
When multi-metal oxides are considered, this technique CdO and Co O [130] or Ag supported on MgO surface
3 4
allows for the production of B-doped ZnO [117], euro- [131], Co O on CuO nanowire arrays (Co O @CuO)
3 4 3 4
pium doped yttria (YO: Eu) [118], Li-doped MgO [119], [132], La Sr MnO [133]. Another example of using
0.82 0.18 3
Ca-doped [92, 120]. Moreover, via CVS technique Zn2+ this synthesis route for the production of polymetal-
cations may selectively replace Mg2+ surface cations pref- lic oxides was shown in the work by Vidic et al. [134]. In
erentially at the edges and corners of MgO nanocubes that this paper a phase separation—an existence of both, the
resulted in unique optical and chemical surface proper- hexagonal ZnO and cubic MgO crystal phases—has been
ties of ternary Zn Mg O nanoparticles [13]. Reproduc- demonstrated. Despite this disadvantage relatively good
x 1−x
ibility is another advantage associated with this method antibacterial efficiency and biocompatibility of ZnMgO
[121]. Careful choice of experimental parameters such for nanoparticles were shown. Modifications in combustion
instance the nature and/or concentration of the oxidizing technique, such as reported by Lee and Choi who have
agent used has a major effect on the nucleation process used a CO laser to re-heat flame-synthesis technique,
2
and consequently affects the average size of the particles. affects nanoparticle morphology and degree of agglomer-
This has been reported for MgO nanoparticles which ation of TiO nanoparticles [135]. Wegner et al. [136] have
2
could be produced via CVS technique in the average size employed a modification by using a critical flow nozzle to
ranging from 3, 5 or 11 nm—depending whether N O extract synthesized titania nanoparticles from the flame
2
or O or dry air were used as the oxidizing agent [122]. to quench particle growth and agglomeration.
2
Control over particle size can be also realized by vary-
ing the reaction temperature [110] since the nucleation Template/surface‑mediated synthesis The major strat-
and growth kinetics can be controlled by manipulation of egies employed in this type of fabrication are electro-
temperature and reactant concentration [123]. Reactant chemical [137], electroless and sol–gel [138], chemical
delivery, reaction energy input and product separation polymerization [139], and chemical vapor deposition
may also affect the characteristics and quality of the prod- [140]. Consequently, as reaction between metal and oxi-
uct. These techniques can be modified to obtain desirable dizing agent may take place in different medium this
attributes in the nanoparticles and eliminate limitations method can be attributed to both of the previously listed
associated with volatility of the reactants and degree of classes of synthesis. The method is based on fabrication
agglomeration. Some examples are laser assisted [124], of the desired nanomaterial within the pores or channels
electrospray assisted [125], thermally activated/pyrolytic, of a nanoporous template. Depending on the properties
metalorganic, plasma-assisted and photo CVD method- of the template, various morphologies of nanomaterial
ologies [126]. For instance, electrospray assisted chemi- such as rods, fibrils, and tubules, can be prepared. This
cal vapor deposition (ES-CVD) was employed to synthe- method can be used to synthesize self-assembly systems
size non-agglomerated spherical titanium and zirconium with tubular and fibrillary like nanostructures with small
oxide nanoparticles [125]. Djenadic and Winterer [124] diameters [141]. Highly monodisperse nanostructures
have used laser assisted technique to synthesize TiO and with enhanced activities, uniform morphology and a high
2
Co-doped ZnO magnetic semiconducting nanoparticles. specific surface area can be obtained using this synthesis
method [142]. Examples are mesoporous MoO nanopar-
2
Combustion method In this synthesis method, pure ticles with improved electrochemical properties [143],
metallic precursor is heated by different techniques to α-Fe O and Co O [144], Fe O [145] and mesoporous
3 4 3 4 2 3
evaporate it into a background gas in which the second NiMn O [144]. The templates used for such synthesis
2 x
reactant i.e. oxidizing agent is admixed. The synthesis methods mainly are track-etch membranes, porous alu-
starts with an initialization in which the metal is only par- mina and other nanoporous structures, like mesoporous
tially heated for the oxidation reaction to start. Thereafter, zeolites [146, 147]. Carbon nanotubes have been used for
the heat required for the following metal evaporation is the fabrication of a variety of metal oxide nanoparticles
produced in situ by the combustion reactions itself. Even like PbO, Bi O , V O , SiO , Al O , MoO , MnO , Co O ,
2 3 2 5 2 2 3 3 2 3 4
though this process is very successful commercially, the ZnO, and WO [148–150]. The choice of precursor, fixa-
3
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 7 of 20
tion method and loading allow for the control of nano- Choice of synthesis method
particles size and shape. Sun et al. have established that As presented above, a broad variety of techniques for
the size and shape of reaction container along with simple fabrication of nanostructured metal oxides exists. The
modifications in the container opening accessibility can reason for it stems mostly from their vast technological
have significant impact on the crystal growth and thereby applications. Except biological, all described methods can
the properties such as particle size, mesostructure order- provide metal oxide nanocrystals of high quality, with
ing and crystallinity [145]. In addition, the choice of con- precisely defined particles size or shape—the properties
tainer has been associated with reproducibility of crystal- which play a major role when antibacterial efficiency is
lite size or shape for the same nanomaterials. under question. However, for most of the above men-
tioned techniques, it is not possible to establish control
Biological synthesis over all the involved characteristics simultaneously, more
Nature is able to synthesize a variety of metal oxides so when synthesizing polymetallic oxide nanoparticles.
nanomaterials under ambient conditions [151]. As bio- In this perspective, the most efficient is chemical vapor
compatibility is one of the most important requirements synthesis that provides in addition a very high crystal
for any nanomaterial used in the field of nanomedicine, purity—similar to other vapor based techniques. Another
extensive research for synthesis techniques using micro- exceptional advantage of chemical vapor synthesis is,
organisms is currently undertaken. For instance, magnet- however, the stabilization of otherwise unstable crystal
ite nanocrystals have been synthesized in magnetotactic phase. For instance, ZnO in cubic crystal structure can
bacteria as a part of their magnetic navigation device only be obtained under very high pressures. However,
[152]. ZnO nanoparticles were synthesized from leaf CVS allows for c-ZnO to be dispersed within MgO sur-
extracts [153]. Raliya and Tarafdar [154] have synthe- face [13]. This is very important given that the type of the
sized ZnO, MgO and TiO nanoparticles by using fun- crystal phase may also affect the antibacterial efficiency
2
gus. In these syntheses, an enzymatic reaction replaces of the considered oxide which may exist in more than one
the chemicals process which eliminates the production of structure. However, the relation between crystal phase
toxic wastes and is more environment-friendly. In addi- and antibacterial efficiency is not clearly provided in the
tion, a biological synthesis is lesser energy intensive than literature. For instance, despite a complete phase sepa-
its physicochemical counterparts. The particles generated ration in smoke ZnMgO that occurred in the course of
by these processes have higher catalytic reactivity, greater metal combustion synthesis, its surprisingly good anti-
specific surface area if not coated with a lipid layer [155, bacterial activity was evidenced [134].
156]. In some cases, nanoparticles produced in microor- Nanoparticles’ agglomeration, that plays a signifi-
ganisms are purified coated with protein corona which cant role in determining the antibacterial efficiency, is
confers their physiological solubility and stability. These another issue at hand. The tendency for the agglomera-
may be critical for biomedical applications and is the tion is favored by electrostatic forces between particles
bottleneck of some purification methods. The biologi- itself, i.e. even when they are not dissolved (Fig. 1). Some
cal synthesis is supported by the fact that the majority of of solution-based fabrication techniques use surfactants
the bacteria inhabit ambient conditions of varying tem- [42] which, in addition to affecting particles size, tend
perature, pH, and pressure. By varying parameters like to decrease the agglomeration degree between particles.
microorganism type and strain, its growth phase, culture In such cases, however, the presence of foreign, mostly
growth medium, pH, substrate concentrations, tempera- organic, groups attached to the surface of primary metal
ture, reaction time, addition of non-target ions and a oxide nanoparticles must be considered—the situation
source compound of the wanted nanoparticle it is pos- where we switch actually to composites and deal no
sible to control size of particle and their monodispersity more with pure mono or multi oxides. Moreover, solu-
[157]. Compared to chemical and physical methods, the tion based techniques struggle with the problem of con-
main drawback associated with biological synthesis is the taminations present in a resulting metal oxide product.
inability to obtain desired size and/or shape of nanopar- Indeed, nanoparticles remain frequently contaminated
ticles along with a low yield. Slow in general, this process with anions present in the precursor salts despite multi-
may take several hours and even a few days. Moreover, ple and obligatory washing cycles.
the decomposition of formed nanoparticles may take Another issue that needs simultaneous in-depth study
place after a certain period of time. Due to its biocom- is the cytotoxic nature of these metal oxide nanopar-
patibility, however, this process remains very attractive ticle. Research on determining the characteristics that
when it comes to the production of potential antibacte- can produce concomitant low harmful cytotoxic effects
rial agents. is still in its infancy, especially when polymetallic oxides
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 8 of 20
are considered. Biological method occurs as a good alter- others, a typical example which undergoes strong aggre-
native but the studies on biogenic synthesis methods are gation in water is TiO nanopowder [161, 162].
2
scanty and much work is necessary to improve their effi- Two strategies for nanoparticle dispersion in water are
ciency in a first place. Chemical and physical methods are mainly employed: electrostatic stabilization and steric
definitely superior in producing larger quantities of nan- repulsion. In the course of electrostatic stabilization, par-
oparticles but their main advantage over biological is the ticles do not aggregate due to their equal charges i.e. elec-
ability to control the size and shape. “Biocompatible pro- trostatic repulsion. This method is simple to realize but
duction” needs, therefore, more active research to widen demands well defined pH and ionic strength of the solu-
commercialization prospects. tion and the control of the presence of reactive species
such as OH- or H O+ ions that can modify the surface
3
Metal oxide nanoparticle in aqueous solution charge of metal oxide particles. Considering steric repul-
Physico-chemical properties of metal oxide nanoparticles sion, the surface of nanoparticle is modified by an appro-
are surface specific and directly dependent on their sur- priate hydrocarbon polymer or a bio-macromolecule.
face-to-bulk ratio. Therefore, nanoparticles manipulation Such stabilizing molecules can be adsorbed or grafted
and storage may modify their fundamental properties. onto the nanoparticles surface to prevent direct contact
The classical approach of surface science studies employs between them and, thus, their aggregation. Consequently,
experimental techniques which preserve pristine proper- the nanoparticles remain dispersed in water solution
ties of nanoparticles. Such techniques imply ultra-high or even upon pH changes or salt concentration [163].
at least high vacuum conditions i.e. conditions in which Bovine serum albumin is a commonly used stabiliza-
the residual pressure of air components is minimized and tion agent as it spontaneously forms a protein corona
the surface modifications negligible. However, biologi- around metal oxides particles [162]. The advantage of
cal applications typically expose nanoparticles to aque- using albumin lies in its biological role to nonspecifi-
ous environment in which their surfaces may undergo a cally bind various molecules and its natural and abundant
series of physico-chemical modifications. Accordingly, presence in biological fluids, such as blood. As albumin is
nanoparticles characteristics, as well as their disper- a charged biomacromolecule, its adsorption on the metal
sion and stability have also to be examined in water and oxides allows both nanoparticle steric and electrostatic
biological media or fluids. Indeed, particle dissolution, stabilization. However, albumin adsorption on nanopar-
aggregation/agglomeration and protein corona formation ticles is not always stable and may, thus, be inefficient for
on the particle surfaces may take place in aqueous solu- some applications. For instance, when nano-ZnMgO was
tions leading to properties that strongly differ to the ones added to cell culture medium containing albumin as the
characteristic for as-synthesized forms. most abundant protein, the protein corona consisted of
many other proteins from the medium [164]. This indi-
Stabilization and biocompatibility of metal oxide cated that over time the most abundant protein in ini-
nanoparticles tially formed corona may be replaced by proteins which
Notably, metal oxide nanoparticles dissolute partially in are less abundant but have higher affinities to interact
water solutions which leads to the modification of their with nanoparticles’ surface. In such cases, superior sur-
morphology in which formation of new crystallographic face-active agents have to be used for effective nanopar-
phases may take place [158]. The propensity to dissolute ticle stabilization in a given medium. For instance, the
in water depends on the composition and structure of the prevention of nanoparticle aggregation and the achieve-
nanoparticles, as was demonstrated for nano-ZnO [159]. ment of their stable dispersion in an aqueous solution
The dissolution rate was also shown to strongly depend might be obtained by adding a mild detergent, as Tween-
on nanoparticles size [160]. The significantly higher 20 or P-20. Also, a recent study has shown that nontoxic
dissolution rate was observed for CVS-MgO nano- polycarbonate ethers may efficiently substitute albumin
cubes (~5 nm average size) than for smoke-MgO cubes to stabilize TiO giving a suspension of non-aggregated
2
(~80 nm average size) produced by magnesium combus- nano-TiO in various cell culture media tested [165].
2
tion in air. Small CVS-MgO particles were shown to be
completely transformed into Mg(OH) in a water solu- Antibacterial activity of metal oxide nanoparticles
2
tion. In contrast, on larger smoke-MgO nanoparticles the Several metal oxides in form of nanoparticles have been
formed surface hydroxide led to a self-inhibition result- reported to exhibit marked antibacterial activity allowing
ing in only partial dissolution and surface faceting [160]. efficient eradication of various bacterial strains. This fact
In addition, the aggregation of metal oxide nanoparticles has attracted significant interest of environmental, agri-
in water solutions is a common phenomenon. Among cultural and health care industries that are searching for
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 9 of 20
newer and better agents to control or prevent bacterial a particular type of metal oxides nanoparticle may have
infections. Many studies have been undertaken to explain bacteriostatic or bactericidal effect as shown for ZnO
the efficacy and mechanisms of antibacterial action of [166] or TiO [167].
2
metal oxide nanoparticles but the existent literature is Different ions, small molecules (such as H O ), free
still controversial and incomplete. It was demonstrated, radicals (like, OH, 1O ) or superoxide ions (suc2h 2as O−2)
2
however, that when applied at well-defined sizes, crys- are examples of highly reactive ROS species which can be
tal structure and concentrations, these nanoparticles are produced on the surface of metal oxide nanoparticles and
highly effective inhibitors against a wide range of bacte- can induce bacterial cell death. ROS-induced damages
ria. Although their exact antibacterial mechanism is still and bacterial death comprise oxidative stress, oxidative
under debate, some distinctive mechanisms have been lesions and membrane lipid peroxidation. In addition,
proposed, which include reactive oxygen species (ROS) ROS can harm bacterial components such as proteins
formation, metal-ion release, particle internalization into and nucleic acids. For instance, oxidative stress induced
bacteria and direct mechanical destruction of bacterial by Ag O nanoparticles was shown to damage the DNA
2
cell wall and/or membrane (Fig. 2). Metal oxide nano- of E. coli which led to the interruption of the bacterial
particles may show bacteriostatic or bactericidal effect. cell cycle and induction of bacterial death [168]. Also,
In case of bacteriostatic effect, treated bacteria do not die CuO nanoparticles were shown to generate ROS, namely
but stop to reproduce or grow. If treated bacterial cells superoxide anions, when adsorbed onto the bacterial cell
are removed from the solution containing nanoparticles, surfaces or internalized into bacterial cells. Formed ROS
they re-start to grow. This can be easily tested by plating induced bactericidal effect in both Gram-positive (S.
these bacterial cells onto new nanoparticle-free agar. In aureus) and Gram-negative (E. coli) bacteria [169].
case of bactericidal effect, no bacterial colonies can be The physicochemical characteristics of metal oxide
observed upon re-plating treated bacteria onto nano- nanoparticles, such as size, crystal structure defects,
particle-free agar. Depending on the experimental con- composition and surface charge, are directly associated
ditions, nanoparticle concentration and bacterial strain, with enhanced antibacterial effects. The synthesis and
Fig. 2 Metal oxide nanoparticles interracting with bacteria. Molecular mechanisms of antibacterial activities of metal oxide nanoparticles
Stankic et al. J Nanobiotechnol (2016) 14:73 Page 10 of 20
treatment procedures employed can tune these charac- different bacterial strains. Interestingly, some multi-metal
teristics, as discussed in the previous sections, and hence oxide nanoparticles show higher antimicrobial activity
produce the desired antibacterial efficacy. For instance, when compared to their pure components of similar size.
nanoparticles of smaller sizes (<20 nm) can easily pen- For instance, nanostructured ZnMgO produced by
etrate into bacterial cells and may release toxic metal ions combustion technique exhibit advantageous properties
upon dissolution [170]. Thus, smaller particles are usu- from both of its pure components: high antibacterial
ally the most efficient antibacterial agents. However, this activity of nano-ZnO and low cytotoxicity of nano-MgO
is not the case when decrease in size leads to enhanced [134]. This mixed metal oxide inhibited Gram-positive
aggregation. Also, defects present at the nanoparticles’ bacteria (B. subtils) completely and Gram-negative bacte-
surface influence strongly antibacterial efficiency. Point ria (E. coli) partially upon 24 h treatment [134]. ZnMgO
defects, such as atoms at edges and in corners give rise nanoparticles were shown to damage bacterial cells by
to an abrasive surface that may cause the injury of the causing extensive injury to membranes that resulted
bacterial cell wall or membrane. For instance, it was in a leakage of the cell content as illustrated in Fig. 3.
proposed that partial dissolution of nano-ZnO in water Comparatively, pure ZnO nanorods and nanotetrapods
medium results in formation of surface defects giving an exhibited the highest but nonselective activity as they
uneven surface texture due to rough edges and corners. completely eradicated both bacterial strains and mam-
This surface roughness was shown to be responsible for malian HeLa cells, under the same treatment protocol
mechanical damage of the cell membrane of E. coli. Wang [134]. In contrast, pure MgO nanocubes only partially
et al. [171] have also suggested that the crystallographic inhibited bacterial growth being at the same time harm-
orientation and type of surface plane can influence anti- less to mammalian cells.
bacterial efficiency of ZnO nanowires. They showed that In case of Zn/Fe oxide nanocomposites, antibacte-
randomly oriented ZnO nanowires were more efficient in rial effectiveness similar to that of ZnO nanoparticles,
killing E. coli than regularly oriented ones. This is prob- was observed [173]. However, no particle agglomeration,
ably due to different spatial arrangements of ZnO. typical for nano-ZnO in water solutions was detected.
Surface charge was also shown to play an important Compared to nano-ZnO, the pure Fe O lacks significant
3 4
role in membrane damage and particle internaliza- antibacterial efficiency, but exhibits good colloidal stabil-
tion. Bacterial membranes and cell walls are typically of ity [173]. It was observed that both the antibacterial effect
negative total charge. Electrostatic attractions can occur and stability of Zn/Fe oxide nanocomposite in an aqueous
between bacterial surfaces and metal oxide nanoparti- medium can be optimized by changing the ratio of Zn/Fe.
cles of positive zeta-potential, like observed for positively The study suggested that hydroxyl radicals were formed at
charged nano-ZnO and negatively charged C. jejuni cells. the surface of zinc oxide. These active oxygen derivatives
Xie et al. [172] proposed that upon binding to bacte- were proposed to damage bacterial cells of E. coli and S.
rial surface, ZnO nanoparticles disrupted the cell mem- aureus. Since similar mechanisms were not observed for
brane causing morphological changes and measurable zinc ferrite, it appears that iron oxide contributes only
membrane leakage in C. jejuni. Moreover, even particles towards good colloidal stability of the composite. In
of negative zeta potential may damage cell membranes another study, Fe3+-ions were used to dope nano-ZnO
since interactions cannot only be electrostatic, but Van in order to induce the formation of lattice defects in
der Waals and hydrophobic as well. Metal oxide nano- ZnO nanocrystals and thus to enhance its antibacterial
particles may specifically bind some moieties within efficiency. It was shown that Fe-doped ZnO nanoparti-
membrane barrier surface such as phosphate, amine or cles efficiently inhibited E. coli bacterial growth without
carboxyl groups in lipids and proteins and subsequently being toxic to mammalian cells [193]. Fe3+-ions acted as
induce bacterial death. It is worth noting that metal oxide an impurity in the ZnO nanostructure that enhanced the
nanoparticles remain tightly bound to the surface of overall antimicrobial activity. Similarly, it was observed
damaged or dead bacteria which may modify their effec- that sea urchin-like ZnO doped with 5 % iron had a strong
tive concentration in the given solution over time. antimicrobial activity, as it killed up to 95 % C. albicans
Since metal oxide nanoparticles with varying phys- and A. flavus [194]. Inserting Fe3+-ions into ZnO lattice
icochemical characteristics exhibit different antibacte- increased antibacterial efficiency by decreasing the size
rial mechanisms and effects, oxide nanoparticles with a of ZnO nanoparticles and favoring the formation of sea
combination of two or more metals can be developed for urchin-like structure. Moreover, Fe3+-ions have the capac-
efficient elimination of various bacterial strains includ- ity to kill bacteria by destroying both cell walls and mem-
ing those highly resistant to existing treatments. Table 1 branes due to their strong reduction ability. Also, binding
summarizes some examples of multi-metal oxide nano- Fe3+-ions to biomolecules may cause protein denatura-
particles tested for their applications in eradication of tion, DNA damaging and enzyme function alternating.