Optical Materials 80 (2018) 77–86

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Optical Materials
journal homepage: www.elsevier.com/locate/optmat

Tuning the luminescence of ZnO:Eu nanoparticles for applications in biology T

and medicine
Jarosław Kaszewskia,b,c,∗, Paula Kiełbika,b, Ewelina Wolskac, Bartłomiej Witkowskic,
Łukasz Wachnickic, Zdzisław Gajewskia, Marek Godlewskic, Michał M. Godlewskia,b
a
Veterinary Research Centre, Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW,
Nowoursynowska 100, 02-797, Warsaw, Poland
b
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Nowoursynowska 159, 02-776, Warsaw, Poland
c
Institute of Physics, Polish Academy of Sciences, al. Lotników 32/46, 02-668, Warsaw, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: Zinc oxide nanoparticles were synthesized with microwave hydrothermal technique and tested as luminescent
Zinc oxide contrast for biological imaging. Luminescence was activated by Eu3+ ions embedded in the nanoparticle matrix
Hydrothermal in the increasing concentrations of 1, 5 and 10 %mol. It was found that europium did not create a separate
Europium crystalline phase up to the concentration as high as 5 %mol. However, Eu3+ ions did not substitute Zn2+ in the
Luminescence
host lattice, but allocated in the low symmetry environment. It was proposed that europium was locating in the
Imaging
Nanoparticles
inter-grain space or on the surface of nanoparticles. The luminescence intensity in ZnO:Eu, as well as the size of
particles, increased with the Eu ion concentration. Moreover, in 10 %mol Eu sample, the separate phase of Eu-
hydroxide was identified with crystals of micrometre length. Interestingly, in vivo study revealed, that contrary
to the in silico experiments, following gastric gavage, the brightest nanoparticle-related luminescence signal was
observed at 1 %mol. concentration of Eu. Since the alimentary uptake of nanoparticles was related to their size,
we concluded that the increase in luminescence at 5 and 10 %mol. Eu concentrations was associated with the
largest ZnO:Eu and Eu-hydroxide particles that did not cross the gastrointestinal barrier.

1. Introduction general expectations for drug-carrying nanoparticles. The final con-

There are many potential applications of nanoparticles in the
biology and medicine. Those include imaging or/and drug delivery. For
the nanoparticle to be considered a carrier of the drug, there are certain
criteria to be fulfilled [1]: ability to contain drug, stability in the serum,
targeting to disease spot, ability to release drug, finally, the carrier must
either undergo bio-degradation or be biologically inert. Promising
properties of nanostructures, predisposing these materials for future use
in biomedical fields, were always based on their size and, as a result,
specific distribution in the living organism. These include the ability to
overcome natural barriers of the body (i.e. intestinal or blood-brain
barrier), barriers often tight or impermeable for either ionic drug forms
or macromolecules [2]. Our recent studies proved preferential alloca-
tion of studied nanoparticles to the sites of primary and metastatic tu-
mours, most likely associated with the enhanced permeabilization and
retention effect (EPR) [3]. Furthermore, oxide nanoparticles were
proved to transfer and deposit biologically active macromolecules from
the lumen of the GI tract to the brain [4]. These findings conclude the
sideration was also fulfilled with ZnO-based nanocrystals being biode-
gradable [5] or ZrO2 and Y2O3-matrices exhibiting no negative effects
during their passage throughout the body [2,6]. Nowadays, most of
available studies regarding biomedical application of nanoparticles
within the lining organisms, were based on their intravenous applica-
tion [7–9]. This approach involved implementation of further, specific
requirements like stability in suspension, no aggregation, and preven-
tion of acute changes in the bloodstream. On the other hand, oral route
of application of nanostructures, based on natural mechanisms of
macromolecule uptake from the gut was confirmed safe and effective
for oxide-based nanoparticles [2,10,11]. Any development of the
medical use of nanoparticles has to be preceded by detailed examina-
tion of the uptake mechanisms, the distribution and the fate of these
materials in the living body. It has been demonstrated in various stu-
dies, that optical methods of tracking were viable and powerful tools to
evaluate uptake and distribution of administrated nanoparticles
[2,12–14].
Zinc oxide, ZnO, is wide bandgap semiconductor (3.37 eV) with

∗
Corresponding author. Veterinary Research Centre, Department of Large Animal Diseases with Clinic, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW,
Nowoursynowska 100, 02-797, Warsaw, Poland.
E-mail address: kaszewski@ifpan.edu.pl (J. Kaszewski).

https://doi.org/10.1016/j.optmat.2018.04.028
Received 11 January 2018; Received in revised form 13 April 2018; Accepted 15 April 2018
Available online 25 April 2018
0925-3467/ © 2018 Elsevier B.V. All rights reserved.
J. Kaszewski et al. Optical Materials 80 (2018) 77–86

high exciton binding energy (60 meV). It crystallized in the zinc blende
(sphalerite, cubic), wurtzite (hexagonal) or rocksalt (cubic) structures
[15,16]. These properties rendered ZnO suitable as host lattice for
lanthanides. However, luminescence activated by Eu3+ substituting
Zn2+ in wurtzite type ZnO was not commonly described in literature
[17]. Europium ions could be introduced into the hexagonal zinc oxide
matrix by numerous methods including solid state [18], microemulsion
[17] and implantation methods [19]. Usually, it was reported, that
Eu3+ ions were active in the low symmetry sites. Additionally, surface
to volume ratio in the material had the strong influence on the intensity
of europium luminescence, which suggested that Eu3+ ions were pre-
dominantly grouped on the surface of the ZnO crystals [20]. Also a
number of reports associated the europium luminescence with their
placement at C3v symmetry sites in zinc oxide lattice [21,22]. Finally,
interstitial oxygen atoms were found to play an important role in the
balance charge difference and energy transfer of Eu [23,24].
Trivalent europium ions exhibited red luminescence when asso-
ciated with inorganic crystalline matrices and glasses, in other com-
plexes, solutions, polymers, liquid crystals, zeolites and metal organic
frameworks [25]. Radiative emission was related to the 5D0→7FJ Fig. 1. X-ray diffraction patterns of ZnO:Eu nanoparticles doped with various
(J = 0-6) electronic transitions within 4f manifold of Eu3+ ions. Lu- europium content.
minescence of europium ions was sensitive to the molecular sur-
roundings was even suggested as a probe of local environment sur-
Table 1
rounding the ion [26]. Sizes of nanoparticles measured using diffraction reflexes (100), (002) and
Hydrothermal technique was employed as the methods of ZnO:Eu (101); calculated using Scherrer's method (MCS) and FW1/4/5 M method
nanoparticle preparation. Since reactions took place in the aqueous (< R >).
environment, the products were not contaminated with supporting
Mean crystallite size MCS [nm]
chemicals used in the syntheses. Application of microwave radiation as
a heating agent additionally increased purity of the reaction environ- Eu content [%mol.] (100) (002) (101)
ment, because the resistance heating elements did not contact directly
the reaction mixture [27]. Also, microwave hydrothermal route per- 1 35 37 33
5 35 42 35
mitted to bypass the calcination step, as the material was already 10 37 46 31
crystalline [28]. Furthermore, kinetics of reactions were enhanced, re-
sulting in the significantly higher quality of product. Finally, our ap- Average grain size < R > [nm]
proach assumed reduction in the number of chemicals used in the
Eu content [%mol.] (100) (002) (101)
synthesis and to minimize the use of compounds containing impurities.
Metals were introduced into the reaction mixture as nitrate(V) and 1 51 51 46
precipitation agent was aqueous ammonia solution, therefore only ni- 5 49 55 49
trogen was present in the process as a subsidiary element. This way we 10 49 55 44
have successfully prepared zirconium dioxide [29], yttrium oxide [30],
europium oxide [31], different forms of zinc oxide [32,33] and zinc
aluminate [34]. 2.2. Nanoparticles characterization

Structural characterization of obtained samples was performed with

2. Experiment
2.1. Nanoparticles preparation
Nanoparticles of ZnO doped with europium were prepared as fol-
lows. In the 200 ml of distilled water Zn(NO3)2·6H2O (98%, Sigma-
Aldrich) and Eu(NO3)3·5H2O (99.9%, Sigma-Aldrich) salts were dis-
solved to form clear solution. Then 1 M solution of NaOH was prepared
to act as precipitation agent. The main solution's pH was changed to the
value of 8 and white precipitate appeared. The residue was washed
with distilled water and suction filtered. Then it was placed in the teflon
reaction vessel of 100 ml volume to take 40 ml and it was filled in with
distilled water to the final volume of 80 ml and stirred. The microwave
hydrothermal process was performed in Ertec Magnum II reactor with
700 W microwave heating. Process was conducted under 4 MPa by
20 min, then heating was turned off and reaction mixture was left to
cool down. Then product was collected to the glass evaporation dish
and dried overnight at 40 °C. Afterwards, resulting lumps were ground
in agate mortar to form uniform powder. Nanoparticles were prepared
to achieve 1, 5 and 10 %mol. concentrations of Eu.
Panalytical X'Pert Powder Diffractometer operating with CuKα radia-
tion and Hitachi SU-70 scanning electron microscope equipped with
GATAN Mono CL3 system of cathodoluminescence measurements and
EDX characteristic radiation detector. Photoluminescence (PL) and
photoluminescence excitation (PLE) spectra were measured at the room
temperature with the Horiba/Jobin-Yvon Fluorolog-3 spectro-
fluorimeter equipped with 450 W xenon lamp, detection iHR550
monochromator and Hamamatsu R928P photomultiplier. Zeta potential
and other suspension properties were measured using Beckman Coulter
DelsaMax Pro light scattering analyser equipped with 50 mW diode
pumped solid state (DPSS) single longitudinal mode 532 nm laser and
measurement cell pressurization system.
2.3. Nanoparticle uptake and biodistribution in mice
To determine and compare usefulness of ZnO nanoparticles doped
with various concentrations of Eu, a water suspensions of ZnO nano-
particles doped with either 1 %mol., 5 %mol. or 10 %mol. Eu (10 mg/
ml in reverse osmosis (RO) water; 0.3 ml/mouse) were alimentary ad-
ministrated (IG) to adult (3–6 month) Balb-c mice (n = 4 per each ex-
perimental group). Control group (n = 4) received 0.3 ml of RO water
IG. All procedures were approved by the Local Ethical Committee (#

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J. Kaszewski et al. Optical Materials 80 (2018) 77–86

Fig. 3. SEM images of ZnO nanoparticles: A - 1 %mol. Eu, B - 5 %mol. Eu, C - 10

%mol. Eu.

Table 2
EDX elemental analysis results for all the samples.
Contribution [at. %] ZnO 1 %mol. Eu ZnO 5 %mol. Eu ZnO 10 %mol. Eu

Fig. 2. Grain size distribution plots in directions (100), (002) and (101) for the O 50.49 ± 0.34 52.81 ± 0.31 54.48 ± 0.31
ZnO nanoparticles consisting of: A - 1 %mol. Eu, B - 5 %mol. Eu and C - 10 % Zn 48.95 ± 0.21 44.94 ± 0.18 41.72 ± 0.40
mol. Eu. Eu 0.55 ± 0.11 2.24 ± 0.08 3.80 ± 0.09

44/2012). No pathological, nor behavioural changes were observed in

Collected tissues and organs were dehydrated in the tissue processor
mice following application of nanoparticles. For cytometry, mice were
(Leica TP 1020, KAWA.SKA), embedded in paraffin blocks, cut into
sacrificed and internal organs collected 3h after IG (for all experimental
6 μm sections and mounted on the silane-coated microscope slides.
groups) and also for 24 h and 7 d for the 1 %mol. and control groups.

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J. Kaszewski et al. Optical Materials 80 (2018) 77–86

Fig. 4. Room temperature photoluminescence (PL) and photoluminescence excitation (PLE) spectra for all examined samples. A - PL λexc = 278 nm, B - PL
λexc = 395 nm, C - PL λexc = 465 nm, D - PLE λem = 616 nm.

Table 3
Decay times of Eu3+ ions luminescence excited at 525 nm (5D1 level).
Sample τ1 [μs] I1 [%] τ2 [μs] I2 [%]

1% Eu 173 32 407 68
5% Eu 76 11 338 89
10% Eu 79 9 199 91

Deparaffined samples were then counterstained with Hoechst 33342

Fig. 5. Decay curves of emission from 5D1 level of Eu3+ ions in ZnO:Eu samples
containing various europium concentration: A - 1 %mol., B - 5 %mol., C - 10 %
mol.
(Sigma-Aldrich) to visualize cell nuclei.
For quantitative evaluation, an index of cells positive for ZnO:Eu
luminescence in analysed tissues was evaluated by the SCANˆR scan-
ning cytometry (Olympus Polska). Specimens of collected organs were
also visualized under Leica SP8-WLL (KAWA.SKA) confocal microscope
for qualitative study – with x20 magnification in the sequence scan
mode, 405 nm excitation and 420-430 emission filter for Hoechst 33342
(cell nuclei) and 619 vs. 625–650 nm spectra for ZnO:Eu.
2.4. Statistical evaluation

Acquired data were expressed as the mean values ± SEM. For sta-
tistical evaluation of SCANˆR experiments, one-way ANOVA with

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J. Kaszewski et al. Optical Materials 80 (2018) 77–86

Fig. 6. Cathodoluminescence spectra of the ZnO nanoparticles containing 1, 5

and 10 % mol. Eu.

Table 4
Zeta potential and hydrodynamic radius values for the aqueous suspension of
ZnO:Eu nanoparticles.
Eu content [%mol.] Zeta potential [mV] Hydrodynamic radius [nm]

1 20.9 96.2
5 31.5 88.8
10 33.7 333.7

Tukey-Kramer multiple comparison test were applied using GraphPad

InStat 3.10. For all tests, significance was set at 95% or 99% confidence
level with p ≤ 0.05 treated as significant and p ≤ 0.01 and p ≤ 0.001,
as highly significant.

3. Results and discussion

3.1. Structural properties

X-ray diffraction (XRD) patterns were shown in Fig. 1 along with

reference patterns. All the samples contained mainly wurtzite type zinc
oxide phase (PDF 75-0576), however in the sample with 10 %mol. Eu
also a hexagonal europium hydroxide phase (PDF 18-0504) was ob-
served. Reflexes responsible for Eu(OH)3 phase were marked by arrows
on diffraction pattern. Reflexes were size-broadened and sizes of crys-
tallites were calculated using Scherrer's [35] and FW1/4/5 M [36,37]
methods and were shown in Table 1. The calculations were made in
three crystallographic directions (100), (002) and (101), with reflexes
located at 2θ = 31.5, 34.2 and 36.1° respectively. Mean crystallite sizes
(MCS) were in range 31–46 nm and average grain sizes (< R >) were in
range 44–55 nm for all the samples. Values calculated using FW1/4/
5 M method were higher than those obtained with Scherrer's method,
however trends in the crystallite sizes were preserved: in (100) direc-
tion calculated sizes did not change significantly with europium con- Fig. 7. Size distributions of the ZnO:Eu nanoparticles in water suspension
tent, in (002) they increased and in (101) decreased. Grain size dis- measured using DLS technique. Europium concentration: A – 1 %mol., B – 5 %
mol., C – 10 %mol.
tributions were also calculated for three different crystallographic
directions in the ZnO crystals and were shown in Fig. 2. These direc-
tions were responsible for: (100) lateral walls of the elementary hex- elongated in c-axis.
agonal prism in wurtzite structure, (002) plane perpendicular to the Scanning electron microscopy (SEM) images were shown in Fig. 3.
first one (c axis) and (101) a crosswise in relation to the base of the There two populations of crystals were observed: the first sized below
prism. Grain size distributions (GSD) in various directions were not 100 nm with uniform round shapes and the other with shapes strongly
changing significantly, sizes were ranging from 30 to 60 nm in all of the depending on the Eu content and sizes reaching μm range. The smaller
samples. Crystallites growth was the largest in (002) direction for all grains were present in considerable quantities in 1 and 5 %mol. Eu
the europium concentrations, meaning the primary nanoparticles were samples. The amount of larger nanocrystals increased depending on the

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J. Kaszewski et al.
Fig. 8. Mean index ( ± SEM) of cells positive for the ZnO nanoparticles doped
with 1 %mol., 5 %mol. or 10 %mol. Eu in duodenum, liver, kidney and brain 3h
following oral administration. Significant difference of *P ≤ 0.05, **P ≤ 0.001
vs. control were marked. No of animals within each group: n = 4.
Eu concentration: from flower-like multi crystals in 1 %mol. sample to
the pitted double cone shaped structure numerous at 5 %mol. and
dominant at 10 %mol. A third type of crystals in the sample with 10 %
mol. Eu was also observed (Fig. 3C). There were long needle-like
structures up to few μm long and usually few hundred nm wide. This
phase was attributed to europium hydroxide and was observed before
as a result of microwave hydrothermal process [31]. Only the smallest
fraction of nanoparticles followed the grain size distribution calcula-
tions based on the XRD. Sizes of the rest of the objects seen on the SEM
images were much larger comparing to the crystallite sizes calculations.
It suggested that the large grains were intrinsically not coherent and
contained smaller crystalline domains detected in diffraction mea-
surements.
Energy dispersive X-ray spectroscopy (EDX) results were shown in
Table 2. Only the oxygen, zinc and europium were detected in the
samples. The main contribution to the samples' composition had O and
Zn atoms, as they formed ZnO matrix. The ratio O:Zn increased with the
europium content. However, the ratio changes did not lead to final
relation O to Me 1:1. This ratio was larger indicating oxygen was pre-
sent in excess in relation to the stoichiometry of zinc oxide. This be-
haviour suggested that substitution of the Zn ions by Eu did not apply to
all of the europium atoms. The main origin of this phenomenon was
charge difference between Zn2+ and Eu3+ ions, which had to be
compensated by introduction of structural defects into the ZnO crystal
lattice. However, all the samples were similarly defected, in-
dependently from the Eu content, measured by means of deep level
emission (DLE) (see Fig. 5). Other reason for such behaviour might have
been increasing formation of non-crystalline phase assisted by Eu ad-
dition. However, all the samples were crystalline with well resolved X-
ray diffraction reflexes, not excluding non-ordered phases present in the
samples. Thus, we concluded that the true origin of phenomenon was
composition of the porous phase with Eu allocating on the nanoparticle
surface or intergrain space, in the non-crystalline layer. Decrease of the
size of ZnO nanoparticles in the sample containing 10 %mol. Eu could
also be attributed to this phenomenon. The ratios of O:Eu was as follows
in particular samples: for 1 %mol. Eu, 2.8; for 5 %mol. Eu, 3.5 and for
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Optical Materials 80 (2018) 77–86
10 %mol. Eu, 3.4. The rates oxygen to europium were counted after full
Zn:O 1:1 reaction and it is approximation. The excess oxygen atoms are
associated with the intergrain/surface Eu3+ ions, beyond ZnO nano-
particles. Again this indicated, that europium ions tended to locate in
the low symmetry environment of the nanoparticles not in the crys-
tallites. Segregation of europium ions is related to the difference in
Eu3+ and Zn2+ charges, its ionic radii and may have origin in the
difference of chemical properties. Zinc solution chemistry varies from
europium [38], which might led to non-uniform precipitation.
3.2. Luminescence properties
Photoluminescene (PL) and photoluminescence excitation (PLE)
spectra were shown in Fig. 4 for various excitation wavelengths
(279 nm, 395 nm, 465 nm) and 616 nm emission. Excitation wave-
lengths (Fig. 4D) were chosen to directly excite oxide matrix (Fig. 4A),
5
L6 (Fig.4B) and 5D2 (Fig. 4C) levels of europium ions. Spectra with
excitation at 278 nm were shown in Fig. 4A. There was a weak near
band edge emission (NBE) and much more intense luminescence of the
deep levels (DLE) caused by defects in the ZnO crystalline structure.
Much more intense DLE indicates strongly defected structure of crys-
tals. Characteristic Eu3+ emission lines did not appear suggesting ab-
sence of the Eu3+ energetic coupling with ZnO host lattice. Samples
excited at 395 nm (Fig. 4B) shown both defect related and Eu3+-related
luminescence. Excitation at 465 nm (5D2 level) resulted in less intense
band emission in relation to the 4f intrashell luminescence, allowing the
estimation of Eu3+ site symmetry [25]. Samples consisting of 1 and 5 %
mol. Eu, presented a very similar Eu3+ site symmetry, with 5D0→7F2
transition dominating the spectrum and suggesting non-centrosym-
metric character of the sites. In the sample of 10 %mol. Eu, spectrum
changes and narrow lines responsible for 5D0→7FJ transitions appeared
(Fig. 4C). Generally, the spectrum was fusion of broad lines present in
lower concentration samples and narrow lines seen at 10 %mol. Narrow
lines were predominant and were identified as C3h site symmetry,
which matches with cationic positions in hexagonal europium hydro-
xide. High concentration of Eu lead to crystallization of separate
europium compound, hence higher intensity of luminescence. The sites
symmetries in samples with 1 and 5 %mol. of europium are low, and
suggest that dopant is located in the amorphous fraction of the nano-
particle. In ZnO doped with europium, broadening of the Eu3+ emission
lines is caused by segregation of dopant ions on the surface of nano-
particles [20]. Luminescence decay curves were shown in Fig. 5 and the
values of lifetime components and intensities in Table 3. Excitation
wavelength was 525 nm, therefore directly into the 5D1 level of Eu3+.
All the curves were determined biexponential and values of τ1 and τ2
were calculated using formula:
−t −t
I (t ) = I0 + I1⋅e ( τ1 ) + I2⋅e ( τ2 ) (1)
where I0 - background, I1,2 - intensities of the components, τ1,2 - lifetime
components, t - time.
Values of lifetime components decreased with growth of Eu con-
centration. Shorter component dropped from 173 to 79 μs and longer
one from 407 to 199 μs. Times were overall shorter, as the content of
europium increased and therefore probability of non-radiative pro-
cesses in all the samples also increased. Shorter decay times were re-
lated to the emission from the surface layer or intergrain or europium
hydroxide either crystalline or amorphous, region enriched with Eu.
Concentration of europium enriched phase depended on the overall Eu
concentration. Longer time component was responsible for emission
from inside the ZnO crystallites and was also shortening, accordingly
with the increase of Eu concentration in the lattice.
Cathodoluminescence spectra of ZnO:Eu nanoparticles were shown
in Fig. 6. All of the nanoparticles were severely defected within their
crystallographic structure. It was indicated by high DLE to NBE lumi-
nescence intensity ratio in all of the samples. Eu3+ related
J. Kaszewski et al. Optical Materials 80 (2018) 77–86

Fig. 9. Differences in the fluorescence intensity of ZnO:Eu-related red luminescence reflecting different mol.% of Eu dopant in ZnO matrix observed by confocal
microscopy. Cell nuclei counterstained by HOECHST 33342 (blue fluorescence). Representative microphotographs from duodenum, liver, kidney and brain in adult
mice collected 3h after administration of nanoparticles. Lens magn. 20×. (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)

luminescence was also seen as low intensity broadened lines peaking at
595, 619 and 698 nm. Near band edge luminescence was the most in-
tense and therefore testifying least defected structure of ZnO matrix in
the sample with 1 %mol. of Eu.
Aqueous suspensions of ZnO:Eu nanoparticles doped with various
concentration of europium were prepared and were analysed by dy-
namic light scattering (DLS) technique. Results were presented in
Table 4 and Fig. 7. Zeta potential, as well as hydrodynamic radii of the
ZnO:Eu nanoparticles changed in the function of europium content.
Nanoparticles were positively charged and the suspension stability
increased with the Eu content. For the 1 %mol. Eu sample it was located
in the unstable region, however 5 and 10 %mol. Eu nanoparticles
formed charge stabilized suspensions. The objects suspended in the
water were sized approximately below one hundred nm, except for 10
%mol. Eu sample, where average size was also dependent on the
europium hydroxide crystallization. Distribution of hydrodynamic radii
were shown in Fig. 7. In samples 1 and 5 %mol. Eu size distributions
were peaking ca. 100 nm and in the sample 10 %mol. Eu sizes were
much more scattered and peaking around 100 nm as well as
500–600 nm.

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J. Kaszewski et al.
Fig. 10. Changes in the intensity of the ZnO:Eu-related red fluorescence (1 %mol. of Eu) observed by confocal microscopy in selected tissues, cell nuclei counter-
stained by HOECHST 33342 (blue fluorescence). Lens magn. 20×. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
3.3. Luminescence of europium doped biodegradeble nanoparticles in the
organism of a mouse
The index of cells positive for ZnO:Eu nanoparticles doped with
varying molar concentrations of Eu (red luminescence) was quantita-
tively evaluated with the scanning cytometry for the duodenum, liver,
kidneys and brain (Fig. 8). In all tested organs 3 h after IG the index of
nanoparticle-positive cells was significantly higher when compared to
the control group. Administration of nanoparticles doped with 1 %mol.
of Eu reflected the highest index of cells positive for Eu-luminescence in
all organs except for the brain. In duodenum, liver and kidney the index
of positive cells exceeded 40%. However, with subsequent increase of %
mol. of Eu in ZnO nanoparticles, a gradual decrease in the positive cells
index was observed.
Microphotographs from selected organs were obtained using con-
focal the microscope. Qualitative comparison of the nanoparticle-re-
lated luminescence yield within collected tissues 3 h after IG revealed
confirmed the highest-nanoparticle related signal for 1 %mol. Eu na-
noparticles (Fig. 9). Hence, nanoparticles of this dopant concentration
were used to evaluate dynamics of tissue distribution of ZnO:Eu
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Optical Materials 80 (2018) 77–86
(Fig. 9).
For biodegradable nanoparticles (in our case ZnO-based), the ima-
ging methods applied in this study (scanning cytometry and confocal
microscopy) seemed to be crucial for understanding of the uptake and
distribution patterns of nanoparticles within the living organism
(Figs. 8–10). These analyses could not be comparatively accurate in the
case of implementation the methodology only based on mineral ana-
lyses, as we proved previously [14]. Based on the luminescence ana-
lyses, one can conclude a rapid biodistribution of nanoparticles, after
their efficient uptake from the gastrointestinal track. It was clearly
visible at results for duodenum and liver 3h following IG (Figs. 8 and
10). Kidneys were previously hypothesized as an organ responsible for
the elimination of nanoparticles from the body [5,39–41]. In our study
a significantly higher level of nanoparticle-positive cells was observed
3h after IG as well as at confocal microphotographs in further time
points (Figs. 8 and 10). In the liver, a second key organ involved in the
elimination of nanoparticles, an interesting phenomenon was observed
(Fig. 8). We speculate that persistence of nanoparticle-related red lu-
minescence 7d after IG may indicate that nanoparticles could undergo
recirculation between internal organs, as well as reabsorption process
J. Kaszewski et al.
when nanoparticles eliminated with bile were picked up by the duo-
denal enterocytes.
Interesting results were obtained from the brain, where both scan-
ning cytometry (Fig. 8) and confocal microscopy (Fig. 9) showed a
rapid distribution of nanoparticles through the tightly-controlled blood
brain barrier into the brain tissue. This provided an insight into the
possible use of ZnO-based NPs as carriers of pharmaceuticals into the
nerve tissue. Other studies also indicated an efficient process of nano-
particle elimination from brain tissue [6].
The luminescence intensity of Eu-doped ZnO nanoparticles varied
depending on the molar concentrations of Eu in the tissues. We con-
cluded that 1 %mol. of Eu-doped ZnO nanoparticles revealed the most
efficient imaging ability in the tissues (Fig. 9). Thus this concentration
was selected for further analyses of the distribution patterns of nano-
particles in selected organs. We speculated, that the contradictory re-
sults of the in silico and in vivo studies, especially regarding the lumi-
nescence intensity of tested nanoparticles, hinge on their ability to cross
the barriers in the living organism. It was noted, that with subsequent
increase of %mol. of Eu, the number of nanoparticles either combined
into or crystallised into the large composites (Figs. 3 and 7). This
phenomenon may have resulted in the improved luminescence, how-
ever the intestinal uptake of such crystallites would be null [2].
Nevertheless, Eu-doped ZnO nanoparticles revealed both excep-
tional imaging properties in the body and ability to overcome extremely
difficult for exogenous material physiological barrier – blood brain
barrier. Presented results were obtained for oral (intra-gastric) admin-
istration of nanostructures, which promote the examined nanos-
tructures for promising, future use in biomedical fields od study. As
mentioned in the introduction, an oral way of administration is con-
siderably easier and no requirements for nanoparticle-contained sus-
pension are necessary, in contrast to intravenous administration.
4. Summary
Results presented in this study illustrate an innovative approach in
the research of nanoparticle distribution within the living organism. Up
to now, similar studies were largely focused on quantitative analyses of
ion concentrations in reference for nanoparticles. That approach, al-
though common, was known to introduce a significant error in the
measurements when biodegradable nanoparticles were used (for ref.
see Ref. [5]). In the current study, visual investigation of ZnO nano-
particles in the organs and tissues was possible because based on the
doping of nanoparticles with europium (red luminescence). Varying
concentrations of Eu dopant were used to evaluate the usefulness of this
material for medical and biological imaging. ZnO:Eu nanoparticles
were used to investigate the dynamics of alimentary uptake and fol-
lowing nanoparticle distribution to the organs of adult mice. Material
contained either 1, 5 or 10 %mol. of europium as luminescence acti-
vator. Nanoparticles were prepared using microwave hydrothermal
technique and exhibited complex structure with primary nanoparticles
sized in range 30–50 nm and hundreds of nanometers to single micro-
meters large agglomerates of the same. 10 %mol. Eu sample also con-
tained few micrometers long rods of Eu(OH)3. Excess of oxygen and
luminescence only from low symmetry sites, in 1 and 5 %mol. Eu
samples suggested that europium was not substituting zinc in the ZnO
host lattice, but located in amorphous environment. Also, oxide matrix
was not coupled with Eu3+ ions by means of luminescence excitation.
Initial in silico evaluation prompted the use of 10 %mol. Eu nano-
particles for biological experiments, as obtained luminescence intensity
was the highest. However, in the biological experiment, the brightest
signal and the most efficient uptake and tissue distribution was ob-
served in for the 1 %mol. Eu concentration. The most probable ex-
planation for this phenomenon was that the brightest crystalline
structures within 5 and 10 %mol. formulations were also the largest
with limited gastrointestinal uptake (for ref. see Ref. [12]).
Furthermore, these findings stress the importance of the actual
85
Optical Materials 80 (2018) 77–86
animal studies in the development of nanomaterials for the medical
applications.
Acknowledgements
This work was supported by National Science Centre, grant num-
bers: 2012/05/E/NZ4/02994 and 2012/06/A/ST7/00398.
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