Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403

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Journal of Photochemistry & Photobiology, B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Photo-induced toxicity of tungsten oxide photochromic nanoparticles T

a b b b b a
A.L. Popov , N.M. Zholobak , O.I. Balko , O.B. Balko , A.B. Shcherbakov , N.R. Popova ,
⁎
O.S. Ivanovac, A.E. Baranchikovc, V.K. Ivanovc,d,
a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
b
Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv D0368, Ukraine
c
Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia
d
National Research Tomsk State University, Tomsk 634050, Russia

A R T I C L E I N F O A B S T R A C T

Keywords: We synthesised a new type of photochromic tungsten oxide nanoparticles, analysed their photocatalytic activity
Tungsten oxide and carried out a thorough analysis of their effect on prokaryotic and eukaryotic organisms. Ultrasmall hydrated
Nanoparticles tungsten oxide nanoparticles were prepared by means of hydrothermal treatment of tungstic acid in the presence
Phototoxicity of polyvinylpyrrolidone as a template, stabiliser and growth regulator. Tungstic acid was synthesised through an
Apoptosis
ion-exchange method using sodium tungstate solution and a strongly acidic cation exchange resin.
UV irradiation
Upon illumination, photochromic nanoparticles of WO3 were shown to increase greatly their toxicity against
both bacterial (both gram-positive and gram-negative – P. aeruginosa, E. coli and S. aureus) and mammalian cells
(primary mouse embryonic fibroblasts); under the same conditions, fungi (C. albicans) were less sensitive to the
action of tungsten oxide nanoparticles. UV irradiation of primary mouse fibroblasts in the presence of WO3
nanoparticles demonstrated a time- and dose-dependent toxic effect, the latter leading to a significant decrease
in dehydrogenase activity and an increase in the number of dead cells. WO3 nanoparticles were photo-
catalytically active under both UV light and even diffused daylight filtered through a window glass, leading to
indigo carmine organic dye discolouration.
The obtained experimental data not only show good prospects for biomedical applications of tungsten tri-
oxide, but also demonstrate the need for clear control of biosafety when it is used in various household materials
and appliances.

1. Introduction
The modern technological paradigm requires research and devel-
opment into new materials with specific physical and chemical prop-
erties. Recently, much attention has been paid to the development of
various types of stimuli-responsive materials, which are able to change
their characteristics in response to external factors. For instance, in the
context of energy-saving, smart photo- or electrochromic materials
have been developed which are able to control the throughput of visible
light and solar radiation into buildings by having different transmit-
tance levels depending on changing needs [1]. One of the most pro-
mising materials for photochromic films or coatings is nanocrystalline
tungsten oxide, an n-type wide-bandgap semiconductor with a chemical
type of chromism [2,3]. The electro-, photo- and chemochromic prop-
erties of tungsten oxide (WO3) are widely used in electronic displays,
optical modulators, windows with adjustable light transmission, rear-
view mirrors in cars, etc. [4–7]. Under light irradiation, exposure to
chemical reagents or the application of an electric field, stoichiometric
WO3 (faintly yellowish) undergoes a number of serial transformations,
reversibly forming brightly coloured products [8]:

⁎
Corresponding author.
E-mail address: van@igic.ras.ru (V.K. Ivanov).

https://doi.org/10.1016/j.jphotobiol.2017.11.021
Received 15 August 2017; Received in revised form 14 November 2017; Accepted 15 November 2017
Available online 20 November 2017
1011-1344/ © 2017 Elsevier B.V. All rights reserved.
A.L. Popov et al.
Fig. 1. Schematic diagram of the interaction mechanisms between light and WO3 nanoparticles in semiconductor (A) and plasmonic (B) states. Photocatalysis, autophotoreduction and
plasmonic heating are demonstrated.
In addition to the above-mentioned applications, tungsten oxide is
one of the most thoroughly studied photocatalysts [9–15] for organic
dye degradation. WO3 nanoparticles of different morphologies, ob-
tained by the hydrothermal-microwave method, have been shown to
possess photocatalytic properties with respect to the discolouration of
rhodamine B, indigo carmine and tetracycline hydrochloride under
UV–vis irradiation [11]. WO3 nanoparticles obtained by surfactant-as-
sisted sonication have provided a high rate of rhodamine B and indigo
carmine degradation under xenon lamp irradiation [12]. Multi-phase
WO3 samples possess improved photocatalytic activity, (as demon-
strated by the bleaching of rhodamine B), that can be attributed to a
decrease in the electron-hole recombination rate, owing to the forma-
tion of interphase junctions [13]. The efficient degradation of methy-
lene blue dye by tungsten oxide nanoplates, synthesised using a hy-
drothermal or microwave-hydrothermal method, has been shown under
UV light irradiation [14]. The photocatalytic activity of WO3 nano-
particles (obtained by annealing (NH4)xWO3 − y at 500 °C in air) and
nanorods (prepared using a hydrothermal method using Na2WO4, HCl,
(COOH)2 and NaHSO4 precursors at 200 °C) has been confirmed by the
decomposition of methyl orange in an aqueous solution under UV light
irradiation [15].
The common mechanism for organic dye degradation by photo-
active semiconductor (photocatalyst) is represented in the left part of
Fig. 1, A:
When a photocatalyst absorbs light that has an energy that exceeds
the semiconductor's bandgap, an electron of the valence band is pro-
moted to the conduction band, thus creating an electron (e−)/hole (h+)
pair. Due to the semiconductor's photoexcitation (WO3 → WO3⁎) and
the generation of electron/hole pairs, oxidation-reduction reactions
take place at the surface: electrons reduce and holes oxidise the mole-
cules of the surrounding substrate (Sub). Upon contact between the dye
and the photoexcited nanoparticle, direct redox decomposition of the
dye molecule occurs (Sub = Dye). More often, the first stage of pho-
tocatalysis is the redox transformation of water and/or oxygen
(Sub = H2O, O2), forming the reactive oxygen species (ROS), which
then destroy other substances, (the indirect redox decomposition of the
dye molecule). Obviously, the photocatalytic activity of the material
can affect not only the decomposition of organic dyes, but also the
biological components of living cells.
The mechanism of tungsten oxide photo-induced chromism (au-
tophotoreduction) is represented in the right part of Fig. 1, A. The ex-
cited electrons cause the reduction of tungsten (6 +) to (5 +) ions,
leading to the formation of coloured, non-stoichiometric, hydrated
tungsten oxide (HxWO3, 0 < x < 1); the holes oxidise substrate (for
example, water [16]), forming ROS, such as hydroxyl radicals. Upon
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Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403
contact with living cells, these ROS can cause oxidative stress, resulting
in cell death. Another specific tungsten oxide feature is that it possesses
photoactivity at a wide range of wavelengths. The most widely explored
TiO2-photocatalysts are effective under UV light only, while WO3-based
ones can change their bandgap upon irradiation, and thus tungsten
trioxide could be an effective visible light photocatalyst, wherein the
absorbed efficiency of sunlight can be enhanced enormously.
The autophotoreduction of tungsten oxide is accompanied by for-
mation of free charge carriers, so photochromic colouration of tungsten
oxide occurs due to the local surface plasmon resonance (LSPR) arising
from appreciable free carrier concentrations [17,18]. It is well known
that the interaction of plasmonic particles and light (HxWO3 → HxWO3⁎
photoexcitation) leads not only to redox processes on their surface (left
part of Fig. 1, B), but also to the partial conversion of electromagnetic
energy into heat (right part of Fig. 1, B). The volumetric generation of
heat within the plasmonic nanoparticle Q(r, t) is affected by the in-
tensity of light, the particle's internal electromagnetic field distribution,
and the thermal and electrical conductivity of the particle's material
[18]:
1 4πnk
σE (̃ r , t ) ∙E ̃ (r, t ); σ =
∗
Q (r , t ) =
2 λi μc
where Ẽ(r, t) and Ẽ∗(r, t) are the generated electric field and its complex
conjugate within the nanoparticles; σ – electrical conductivity at optical
frequencies; λi – incident light wavelength; μ – relative magnetic per-
meability of the nanoparticle, and n and k – the real and imaginary
parts of the refraction index of the nanoparticle, respectively. Plas-
monic nanoparticles of non-stoichiometric tungsten oxide have been
used for photothermal cancer treatment upon IR irradiation [18–23].
Thus, volumetric heating of WO3 nanoparticles upon irradiation can
bring an additional contribution to cytotoxicity, and thus should be
taken into account.
For this paper, we synthesised a new type of photochromic tungsten
oxide nanoparticles, analysed their photocatalytic activity and carried
out a thorough analysis of their effect on prokaryotic and eukaryotic
organisms. WO3 nanoparticles possess both dark and light cytotoxicity,
which significantly distinguishes them from common photocatalysts,
and opens up new possibilities for their practical use.
2. Materials and Methods
2.1. Tungsten Oxide Nanoparticles
Ultrasmall tungsten oxide nanoparticles were synthesised by means
of hydrothermal treatment of tungstic acid in the presence of
A.L. Popov et al.
polyvinylpyrrolidone (PVP K-30, average mol. wt. 40,000) as a tem-
plate, stabiliser and growth regulator. Tungstic acid was prepared
through an ion-exchange method using sodium tungstate (Na2WO4)
solution and a strongly acidic cation exchange resin (Amberlite®
IR120). Briefly, ion exchange resin (in a hydrogen form) was swollen in
water and loaded into a glass column of 200 ml volume. Then, 100 ml
of 0.05 M sodium tungstate solution was passed through the column,
dropwise, 4 g of PVP was added to the obtained eluent, and the solution
was transferred to a flask and stirred for 4 h under reflux. During
heating, the clear sol of tungsten trioxide was formed, as evidenced by
the appearance of a UV absorption band at 325 nm and a Tyndall cone.
Transmission electron microscopy (TEM) and selected area electron
diffraction (SAED) measurements were performed using a Leo912 AB
Omega analytical transmission electron microscope operating at an
accelerating voltage of 100 kV. Energy dispersive X-ray (EDX) spectra
were recorded using a Carl Zeiss NVision 40 scanning electron micro-
scope equipped with an Oxford Instruments X-Max detector (80 mm2)
at an accelerating voltage of 20 kV.
For the toxicological experiments, the obtained sol was diluted with
water to produce 0.1–25.0 mg/ml colloid solutions.
2.2. Photocatalytic Activity Measurements
2.2.1. Dye
Indigo carmine was purchased from Sigma-Aldrich (Aldrich,
#57000) and used as received, without further purification.
2.2.2. Photocatalytic Activity Test
The photocatalytic activity of the obtained nanoparticles was eval-
uated in the reaction of photodecomposition of indigo carmine in an
aqueous solution under UV irradiation, without a detailed study of the
reaction intermediates. The source of ultraviolet radiation was a ver-
tically installed DELUX T8 36 W G13 UV lamp (analogue of Philips TUV
G15 T8) with a maximum emission at 253.7 nm. The reaction con-
tainers were 1 × 1 cm cuvettes (standard quartz cells for spectro-
photometer), located around an UV lamp at a distance of 10 cm.
Cuvettes were filled with 0.25 mM of indigo carmine aqueous solution
and colourless tungsten oxide sols with various concentrations in the
range of 0–0.18 mM. The temperature of the environment was held at
25 ± 0.5 °C. The concentration of the dye in the cuvettes was de-
termined every 4–16 min, and then the cuvettes were shaken and im-
mediately returned back to their location near the lamp. The con-
centration change of the dye was determined using an Agilent
Technologies Cary 5000 UV–Vis spectrophotometer at the adsorption
maximum of the dye λ = 600–620 nm. The removal of indigo carmine
(%) was calculated according to the equation:
C0 − Ct ∗
Dye removal (%) = 100
C0
where C0 and Ct are the initial concentration of indigo carmine and the
concentration of indigo carmine at a given irradiation duration (t), re-
spectively. A similar series of experiments was performed using natural
scattered illumination. For this, the cuvettes were exposed on a win-
dowsill and illuminated by diffused summer sunlight at noon (average
European latitude), which passed through two conventional 3 mm si-
licate glasses.
2.3. Bacterial Toxicity Study
2.3.1. Cell Cultures
Four reference strains from the Ukrainian collection of micro-
organisms (UCM, Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine) were tested: Staphylococcus
aureus UCM B-904 (ATCC 25923), Escherichia coli UCM B-906 (ATCC
25922), Pseudomonas aeruginosa UCM B-907 (ATCC 27853) and
Candida albicans UCM Y-2681 (ATCC 10231).
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Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403
Microorganisms were cultured in a slant meat-peptone agar medium
at 37°C for 24 h. Then they were suspended in a physiological isotonic
solution (0.85% NaCl) and adjusted to the McFarland turbidity stan-
dard no.4 (1.2 × 109 CFU/ml). Obtained suspensions were diluted, by
saline, to the required concentrations and further used to determine the
antimicrobial properties of WO3 nanoparticles.
2.3.2. Bacterial Viability Assays
Two antimicrobial susceptibility tests were used: disc diffusion and
dilution assay. The agar overlay assay was carried out as described
elsewhere [24,25]; it was shown that this technique is not suitable for
nanoparticle toxicity study, due to their low diffusion capacity in agar.
The suspension dilution assay was carried out as described elsewhere
[26,27]; this technique proved to be adequate for the evaluation of
bactericidal activity of the WO3 nanoparticles.
Briefly, the experiments were carried out in 1.5 ml Eppendorf tubes;
0.1 ml of WO3 nanoparticle sols of different concentrations (test group),
or 0.1 ml of distilled water (control), were added to 0.9 ml of a sus-
pension of microorganisms having a total titer of about 105 CFU/ml. All
the experiments were carried out in two parallel sets, under light and
dark conditions.
The number of viable microorganisms was measured both in the
control samples, and in the test tubes after incubation with nano-
particles in the dark and under UV irradiation for various intervals of
time. Bacterial suspensions incubated under the same conditions, but
without nanoparticles, were used as a control. To determine the titer of
microorganisms, 0.1 ml of bacterial suspension was sampled, then im-
mediately diluted using the method of serial tenfold dilutions, and then
10 μl aliquots were plated on nutrient agar. Cells were cultivated at
37°С for 24 h and the number of cell colonies on agar plates was
counted. The number of grown colonies was extrapolated to 1 ml of
suspension and measured in CFU/ml.
2.3.3. UV Irradiation
Test tubes were exposed to ultraviolet irradiation through UV-A
(λmax = 340 nm) Wood's lamp (Philips, 15 W). Samples were placed at
a distance of 30 cm from the lamp for 30 or 60 min; the control samples
were kept under similar conditions, at room temperature and in the
dark. Control tubes not containing nanoparticles were kept under both
light and dark conditions.
2.4. Mammalian Cells Toxicity Study
2.4.1. Cell Culture
The culture of primary mouse embryonic fibroblasts was obtained
from embryos of SHK white mice on day 13 of pregnancy, according to
the previously published protocol [28]. The cells were cultured in
DMEM/F12 (1:1) medium, with the addition of 10% fetal calf serum
and 100 U/ml penicillin/streptomycin, under 5% СО2 at 37°С. All ex-
periments were conducted using cultures taken from passages 1–2.
2.4.2. UV Irradiation
Primary mouse embryonic fibroblasts of the experimental group
were irradiated for 10, 15 or 20 mins with the UV light (pulsed UV lamp
with a continuous spectrum of radiation in the range of 200–700 nm
Melitta Alpha 05, 50 W cm− 2); the cells in the control group were not
irradiated. In the next step, cells were grown for an additional 24 h in
the CO2-incubator at 37 °C, under a humidified atmosphere of 5% CO2
(v/v), and were subsequently sampled for morphology determination,
live/dead viability or MTT assay.
2.4.3. MTT Assay
Determination of activity of mitochondrial and cytoplasmic dehy-
drogenases in living cells was performed using the MTT assay, which is
based on the reduction of a colourless tetrazolium salt (3-[4,5-di-
methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT). After an
A.L. Popov et al.
interval of 24 h after UV irradiation, a standard MTT assay was per-
formed.
2.4.4. Live/Dead Viability Assay
To evaluate the cytotoxic effects of WO3 nanoparticles in combi-
nation with UV light irradiation, a Live/Dead Viability Kit (Invitrogen,
Life Technologies) was used. Cells attached to the 12-well plates were
processed according to the manufacturer's protocol and visualised and
photographed 25 min after adding the kit, using an Axiovert 200
fluorescence-light microscope (Carl Zeiss, Germany), and recorded
using a Canon A620 digital camera (Canon, USA). A green signal (SYTO
9, λ = 485/498 nm) characterised the live cells and a red signal (pro-
pidium iodide, λ = 535/617 nm) characterised the dead cells. For each
cell group, four fields in each well were examined.
To evaluate the cytotoxic effect of the WO3 nanoparticles in com-
bination with UV irradiation, primary mouse embryonic fibroblasts
were incubated for 24 h in the cell growth medium, supplemented with
different concentrations of WO3 nanoparticles. Next, the medium was
changed for a fresh one without the WO3 nanoparticles, and the cell
cultures were irradiated for 10, 15 and 20 min. After 24 h, the cultures
were sampled for the morphological study, the live/dead viability and
MTT assay.
3. Results and Discussion
3.1. Tungsten Oxide Nanoparticles
Highly photochromic, ultra-small, WO3 nanoparticles were synthe-
sised using the protocol proposed, based on the hydrothermal treatment
of tungstic acid in the presence of polyvinylpyrrolidone (Fig. 2). Ultra-
Fig. 2. A – TEM image and SAED pattern; B – EDX spectrum of WO3 nanoparticles. C, D – absorbance of WO3 sols upon exposure to direct sunlight; the spectra were taken in the dark
every 2 min. C – UV–Vis-NIR spectra, D – dynamics of the relative change of the optical density of the UV–Vis-NIR bands of WO3 sol when exposed in the dark.
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Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403
small WO3 nanoparticles demonstrated a very high rate of photo-
chromic colouration, which takes place especially easily in an aqueous
medium. Our preliminary data indicate that the bandgap of thus syn-
thesised tungsten oxide was size-dependent. The optical bandgap cal-
culated using the absorbance spectra (Fig. 1, C) was Eg ≈ 3.2 eV, while
for the bulk material Eg0 = 2.6 eV [29].
3.2. Photocatalytic Activity
Indigo carmine (C16H8N2Na2O8S2) dye is prone to photodegradation
under illumination, and is often used in connection with the photo-
catalytic activity of nanoparticles tests [30–33], including tungsten
oxide nanoparticles [11,12,32,33]. Our data indicate that the synthe-
sised WO3 sols did possess photocatalytic activity in the processes of
indigo carmine discolouration under illumination (Fig. 3). According to
UV–vis measurements, no absorption bands intrinsic to WO3 nano-
particles in the range of 600–800 nm were observed until complete
indigo carmine discolouration (Fig. 1S).
Indigo carmine is light-sensitive dye and can be easily decomposed
under intense UV irradiation (Fig. 3, A, curve “0”). Photocatalytically
active tungsten oxide nanoparticles drastically accelerated the rate of
dye decomposition in a concentration-dependent manner (Fig. 3, A,
curves “1”–“5”). Moreover, the photocatalytic activity of WO3 nano-
particles remains at nearly the same high level (Fig. 3, A, curves “1”-
“5”) even upon illumination with diffused daylight filtered through a
common window, which contained only a small fraction of UV com-
ponent and could not directly destroy the dye molecules (Fig. 3, B,
curve “0”). This fact should be taken into account by the users of “smart
windows” containing tungsten oxide nanoparticles. In turn, according
to our observations, WO3 sols illuminated by UV/yellow-filtered light
A.L. Popov et al.
Fig. 3. Relative dynamics of indigo carmine dye decomposition by WO3 nanoparticles
upon UV (A) and diffused daylight (B) illumination. Indigo carmine concentration –
0.25 mM, tungsten oxide concentrations: “0” – 0 mM (control); “1” – 0.015 mM; “2” –
0.03 mM; “3” – 0.06 mM; “4” – 0.09 mM; “5” – 0.18 mM. (For interpretation of the re-
ferences to colour in this figure legend, the reader is referred to the web version of this
article.)
showed negligible photocatalytic activity (Fig. S2).
3.3. Toxicity of WO3 to Bacterial Cells
3.3.1. Antibacterial Activity of Tungsten Oxide Nanoparticles without UV
Irradiation
Tungsten oxide nanoparticles were found to be toxic for most of the
bacteria strains studied, even without UV irradiation. Thus, at a con-
centration of 25 mg/ml, WO3 nanoparticles drastically decreased E. coli
viability after 5 min of contact; longer exposures (30 and 60 min)
completely sterilised the cultural media. Diluted solutions of WO3 sol
decreased bacterial viability in a concentration-dependent manner
(Fig. 4). The same effect was registered for S. aureus and P. aeruginosa.
The latter strain was the most sensitive to tungsten oxide nanoparticles:
an abrupt decline (by two to three orders of magnitude) in bacterial
viability was observed during the first 5 min of contact with nano-
particles in the concentration range of 10.0–25.0 mg/ml, whereas, after
30 min, there were no viable cells in the medium (Fig. 4). C. albicans
yeasts were found to be non-sensitive to WO3 nanoparticles, and no
changes in viability were found in the whole range of concentrations
studied (Fig. 4). Thus, the sensitivity of the studied microorganisms to
the bactericidal action of WO3 nanoparticles decreased in the following
sequence: P. aeruginosa > S. aureus > E. coli > C. albicans.
Existing data on the antibacterial activity of tungsten oxide nano-
particles are somewhat contradictory. Some studies have shown low
dark cytotoxicity of nanoparticles to microorganisms. For instance, the
toxicity of 12 metal-based nanoparticles (including WO3) to algae,
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Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403
Fig. 4. Antibacterial activity of WO3 nanoparticles to various bacterial strains and yeasts
after 30 min of exposure without UV irradiation.
protozoa and bacteria (including S. aureus and E. coli) was recently
tested [34]: for most of the test species, tungsten oxide nanoparticles
were found to be non-toxic at concentrations below 0,1 mg/ml. Simi-
larly, antibacterial activity of some metal and metal oxide nanoparticles
against peri-implantitis pathogens Prevotella intermedia, Porphyromonas
gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomyce-
temcomitans was studied [35]. Tungsten oxide had the lowest cytoxo-
city, and the antibacterial activity of the nanoparticles decreased in
the following sequence: Ag > Ag + CuO > Cu2O > CuO > Ag
+ ZnO > ZnO > TiO2 > WO3. On the other hand, a number of re-
searchers reported the high antibacterial activity of tungsten oxide.
Moreover, tungsten species have been shown to damage bacterial
genomic material [36]. For example, the genotoxic effects of nano-WO3
were assessed in bacteria using a reverse mutation assay in S. typhi-
murium [37], or by analysing the direct interaction of WO3 nanoplates
with naked DNA plasmid, which was further transferred into E. coli cells
for a mutation study [38]. WO3 prepared from peroxotungstic acid
showed high antibacterial activity against Staphylococcus aureus and
Klebsiella pneumonia, even in the dark [39]. Ghasempour et al. [40]
studied the antibacterial activity of tungsten oxide nanorods/microrods
against E. coli under visible light irradiation and in the dark. The na-
norods (with diameters of 50–90 nm) of WO3 could inactivate ~58% of
the bacteria in the dark and > 92% after 24 h of visible light irradia-
tion. The stronger antibacterial activity of WO3 samples synthesised at
the lowest temperature was assigned to the higher density of the pho-
togenerated electron-hole pairs on the surface and high bound water
content, as well as O–H groups on the surface of the tungsten oxide
films. Disinfection of E. coli was also investigated [41] using an im-
mobilised thin film tungsten trioxide photocatalyst in a visible light-
driven photoelectrocatalytic batch cell. Optimal disinfection efficiency
(> 99% within 15 min) was registered when the WO3 catalyst was il-
luminated under closed circuit conditions. Thus, the irradiation dras-
tically enhanced the microbiocidal ability of WO3 nanoparticles.
3.3.2. Antibacterial Activity of WO3 Nanoparticles under UV-Irradiation
Since WO3 nanoparticles have a significant bactericidal effect in the
concentration range of 5.0–25.0 mg/ml, a study of UV-enhanced anti-
bacterial activity was carried out at a minimal concentration of 0.5 mg/
ml.
Preliminary studies on the bactericidal activity of ultraviolet irra-
diation under the experimental conditions chosen were performed; for
the most sensitive strain, P. aeruginosa, the decrease in viability after
60 min of UV exposure was about 10–15% (4.6 × 108 CFU/ml before,
and 4.0 × 108 CFU/ml after irradiation). The same UV dose slightly
reduced the viability of S. aureus (by 5%), and virtually did not affect E.
coli (Table 1).
A.L. Popov et al. Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403

Table 1 least two major mechanisms that can be involved in the antimicrobial
WO3 nanoparticles' effect on the viability of microorganisms. effect of nanoparticles, namely direct action and/or indirect interaction
[43]. In different microorganisms, nanoparticles are piled up differently
Initial concentration The time of incubation – 60 min The fold of
inhibition in the vicinity of a bacterial cell. In most cases, the nanoparticles are
Without NP 0.5 mg/ml WO3 located directly on the membrane; under stress conditions, C. albicans
NP forms exopolysaccharide capsules, which prevent contact between the
particles and the cell. In this case, the direct damaging action of the
P. aeruginosa 9.0 × 105 1.8 × 106 9.5 × 105 1.89
P. aeruginosa + 60 min UV 1.2 × 106 3.6 × 105 3.33 particles on the cell membrane is absent. In turn, an indirect me-
(340 nm) chanism, involving the nanoparticle-mediated formation of ROS (Fig. 1)
The fold of inhibition 1.5 2.64 in the microenvironment, with subsequent damaging of the cell mem-
E. coli 2.0 × 105 1.6 × 105 1.2 × 105 1.33 brane, makes probably only a small contribution to the bactericidal
E. coli + 60 min UV 1.6 × 105 3.5 × 104 4.57
action of photoactive WO3 nanoparticles.
(340 nm)
The fold of inhibition 1.0 3.43 The observed differences in the number of viable microorganisms
S. aureus 5.7 × 104 6.6 × 104 6.2 × 104 1.06 may depend on their structure and physiology: for example, the sensi-
S. aureus + 60 min UV 6.3 × 104 1.7 × 102 370.59 tivity of microorganisms to the phototoxic effect of WO3 nanoparticles
(340 nm)
(gram-positive > gram-negative > eukaryotes) correlates with the
The fold of inhibition 1.04 364.71
cell wall structure of the investigated microorganisms.

3.4. Mammalian Cells

Tungsten oxide nanoparticles are typically adjudged to be of low

Fig. 5. The influence of WO3 nanoparticles (0,5 mg/ml), UV irradiation (340 nm, 60 min)
and their combination on the viability of various bacterial and yeast strains.
The sensitivity of the studied microorganisms to the bactericidal
action of WO3 nanoparticles under UV irradiation was found to de-
crease in the following sequence: S. aureus > E. coli > P.
aeruginosa > C. albicans (Fig. 5).
Thus, concentrations in excess of 5.0 mg/ml of tungsten oxide na-
noparticles demonstrated a significant bactericidal effect on P. aerugi-
nosa, E. coli and S. aureus strains (Fig. 4). UV irradiation of bacteria in
the presence of the minimally studied concentration of WO3 nano-
particles (0.5 mg/ml) provided an increase in their bactericidal activity
by 3–5 times for P. aeruginosa and E. coli, whereas S. aureus was the
most sensitive to the phototoxic effect of WO3 nanoparticles: the
number of viable organisms decreased > 350 times after 60 min of UV
irradiation in the presence of 0.5 mg/ml WO3 nanoparticles. Possibly,
this very specific action of tungsten oxide nanoparticles on S. aureus is
due to the presence of teichoic acids located within the cell walls of
these bacteria. These phosphate-containing bacterial copolymers can
inhibit the penetration of WO3 nanoparticles through a membrane,
decreasing the dark toxicity of nanoparticles. On the other hand, in-
teraction of WO3 with teichoic acid can result in the formation of
tungsten phosphates on the surface of nanoparticles, which increases
their catalytic activity. The irradiation of the particles leads to the re-
duction of W6 + to W5 + and W4 +; phosphate species stabilise tungsten
ions in a low valence state, preserving their stronger catalytic activity
[42], and so the phototoxicity of WO3 nanoparticles becomes higher for
this strain.
WO3 nanoparticles had virtually no toxic effect on C. albicans, in the
dark or under UV irradiation, which may have been due to its dense,
chitin-containing cellular envelope. It is well known that there are at
toxicity to mammalian cells. Recently, the toxicity of 11 metal oxide
nanoparticles (including WO3) in the concentration range of 3–100 μg/
ml to three mammalian cell types in vitro was tested [44]; tungsten
oxide nanoparticles were found to be non-toxic in concentrations below
100 μg/ml. Chitosan-capped WO3 nanoparticles demonstrated no sig-
nificant dark cytotoxicity to U266 cell line at concentrations up to
5 mg/ml after 24 h of incubation [45]. Chitosan- [45] and poly-ε-ca-
prolactone-coated [46] tungsten trioxide nanoparticles are low-toxic
and can be used as a contrast agent for X-ray computer tomography. In
mammalian systems, the genotoxic potential of WO3 has been eval-
uated using a transformation assay in BALB/c cells [47]. The interac-
tion of human genomic DNA and WO3 nanoparticles was registered and
characterised by Fourier transform infrared spectroscopy, photo-
luminescence spectroscopy, agarose gel-electrophoresis and polymerase
chain reaction [48].
According to the MTT test, pretreatment of primary mouse fibro-
blast cells with WO3 nanoparticles at concentrations of 5, 10 and
25 mg/ml led to a significant decrease in their viability (up to 50% of
the control) after 24 h of incubation (Fig. 6). Lower WO3 nanoparticle
concentrations (1, 0.5 and 0.1 mg/l) did not cause a significant de-
crease in dehydrogenase activity, and cell viability was maintained at
the level of control values. UV irradiation of primary mouse fibroblasts
enhanced the toxic effect of WO3 nanoparticles, depending on exposure
time, whereas incandescent light illumination (colour temperature of
2700 K) showed no significant effect (Fig. S3). The strongest decrease in
viability (23% of the control) was observed with 20 min UV irradiation
Fig. 6. Influence of WO3 nanoparticles under UV irradiation (200–700 nm, 10–20 min)
on the dehydrogenase activity of primary mouse embryonic fibroblasts after 24 h of in-
cubation (MTT assay).

400
A.L. Popov et al.
Fig. 7. Morphology and viability analysis of primary mouse embryonic fibroblasts upon UV irradiation in the presence of different concentrations of WO3 nanoparticles (DIC-Syto 9/PI).
Scalebar: 20 μm.
and the highest WO3 nanoparticle concentration (25 mg/ml). At a
concentration of 10 and 5 mg/ml, WO3 nanoparticles, upon 10 and
15 min' UV irradiation, also significantly reduced the viability of the
cells, increasing the number of dead cells (Fig. 7). It is also worth noting
that UV irradiation of the cells in the presence of WO3 nanoparticles led
to the detachment of cells from the substrate and their scalding, which
was most likely due to structural disturbances in the focal contacts of
cells and the cytoskeleton, as a result of active ROS generation by WO3
401
Journal of Photochemistry & Photobiology, B: Biology 178 (2018) 395–403
nanoparticles. Additionally, it should be noted that WO3 nanoparticles
inhibited the growth and proliferation of primary mouse fibroblasts in a
dose-dependent manner, even without exposure to UV radiation
(Fig. 8). The mechanism of the WO3 nanoparticles cytostatic effect can
be explained by the direct interaction of nanoparticles with phosphate
groups in the DNA molecule, which subsequently led to the disruption
of its structure and the replication process as a whole. There was also
indirect evidence of the ability of WO3 nanoparticles to inhibit enzyme
A.L. Popov et al.
Fig. 8. The growth rate of primary mouse fibroblasts in the presence of WO3 nano-
particles. The cells were plated in a 96-well plate at a density of 30,000/cm2; 6 h later,
WO3 nanoparticles were added and, each day, the living cells were counted using a he-
mocytometer (Goryaev chamber).
activity (e.g. Taq polymerase activity) via surface adsorption, sug-
gesting that the toxicity of WO3 nanoparticles has a complex nature.
The obtained experimental data not only show good prospects for
biomedical applications of tungsten trioxide, but also demonstrate the
need for clear control of biosafety when it is used in various household
materials and appliances.
4. Conclusions
A method for tungsten oxide nanoparticles synthesis is proposed.
Upon UV irradiation, WO3 nanoparticles possess high photocatalytic
activity in an indigo carmine dye photodecomposition reaction.
Tungsten oxide nanoparticles showed both light and dark cytotoxic
effects against prokaryotic microorganisms and eukaryotic cells. We
have identified their different sensitivity to the effects of WO3 nano-
particles, which, apparently, is associated with the morphological fea-
tures of their cell membrane structure and different metabolic activ-
ities. Under UV illumination, the toxicity of tungsten oxide
nanoparticles has been shown to increase notably to both bacterial and
mammalian cells. The toxic effect of tungsten oxide nanoparticles is
probably due to their high photocatalytic activity, which leads to cel-
lular membrane damage, disruption of metabolism and cell death. This
fact is to be taken into account during the development of new pho-
tosensitive materials, coatings and end products with photosensitive
properties. Possibly, it will be useful for auto-cleaning and disinfecting
the outer surfaces of smart windows.
Acknowledgment
The procedure for WO3 nanoparticles synthesis was elaborated with
support from the Russian Science Foundation (project 16-13-10399).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jphotobiol.2017.11.021.
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