INTERACTION OF ZINC OXIDE NANOPARTICLES WITH HUMAN RED BLOOD CELLS
P P Shirsekar, N Kanhe, V L Mathe, 1, Y K Lahir and P M Dongre*
Department of Biophysics, University of Mumbai, Kalina Campus, Santa Cruz (E),Mumbai, India-400 098
1
Department of Physics, University of Pune, Pune, India-411007
*corresponding author Email:[email protected]
ABSTARCT: Wereport on the interaction of ZnO nanoparticles (ZnO NPS) with human red blood cells in relation to the osmotic
fragility, hemolytic activity, cell integrity and morphology. Various microscopic techniques like fluorescence microscope, bright
field and differential image contrast techniques have been used. Highest degree of osmotic fragility (in terms of hemolysis) was
noticed at lower concentration of ZnOnano-particles and lowest/sensitive at higher concentrations. Changes in the morphological
features of RBCs were noticed due to the interaction of ZnO NPs. Stability of RBCs was observed at lower concentration of ZnO
NPs. Fluorescence study indicated concentration dependent interaction of ZnO NPs with RBCs. The overall results suggest that
ZnO NPs are compatible to red blood cell at lower concentration. The possible mechanisms of interaction between ZnO NPs and
RBCs have been described.
Keywords: ZnO NPs, osmotic fragility, stability, fluorescence activity
inducers of histological and apoptotic changes in the hepatic
cells of rat (Alarifi et al, 2013). Pardeshi et al (2014) reported
that ZnO NPs are toxic to hepatocytes of chicken embryo. In
present work, we have studied the interaction between ZnO
nanoparticles and Red blood cells with reference to
morphological features, fragilityand fluorescence activity.
INTRODUCTION:
The current interest is to understand the interaction of nano
scaled matter with living systems for the development of
devices that can be applicable in biology and medical
investigations such as biosensor, targeted drug delivery, bioimaging, bio-mimicking devices etc. Nanoscale materials
possess a unique physico-chemical property due to their size
and shape; they have ability to move across most of the
biological barriers. When nano materials are disbursed within
biological fluid, these get rapidly covered by biological
macromolecules specifically proteins(Mariam et al 2011).It is
considered to be an important phenomenon associated with
biological materials. Uptake of nano particles in cells/tissues is
due to the surface interaction. The interaction of nano particles
with macromolecules or cell may be favorable or unfavorable.
However, silver nano particles are able to cause derogative
effects on the histological and hematological parameters in
albino rats (Brunner et al, 2006). ZnO NPs have been studied
as a useful oxide in cosmetic industry. ZnO nano particles
because of their possible biocompatibility are quite suitable for
cellular and animal models (Zheng et al 2009). (Hussein et al
2009) have reported that ZnO nano particles are suppose to be
non-toxic, biosafe and possibly biocompatible, hence, being
used in biosensor; the surface of nano particles is
photosensitive and this is the one of the causes of ROS
generation and results in oxidative stress leading to cellular
and/or tissue damage. Mesosilica NPs can reduce the
hemolytic activity of human red blood cells, (Slowing et al
2009); iron oxide nano particles have the capacity to change
the physiological parameters like pH of red blood cells
(Moersdorf et al 2010); silver NPs increase the osmotic
fragility of RBCs (Ozturk and Ozdemir 2013, Osama and
Hussein 2014). The interaction of nano particles with RBCs
can either change or deform their morphology. The ZnO NPs
induce a differential cytotoxic response between human
immune cell subset; lymphocytes are the most resistant while
monocytes are the most susceptible (Henley et al 2009).
Different sizes, doses and exposure of gold nano particles
caused histological alterations in rat (Mohamed and Bashir
2012). ZnO NPs induce selective killing of cancerous cells
(Mohamed et al 2012). TiO2 nanoparticles are potential
MATERIALS AND METHODS
SYNTHESIS AND CHARACTERIZATION OF ZnONPs
: Zinc oxide nanoparticles were synthesized by homogeneous
gas phase condensation method using DC-Transferred Arc
Thermal Plasma Reactor (DC-TATPR). Initially the reaction
chamber was evacuated to a base pressure of 10-3 Torr. Then
oxygen was filled in the chamber at 500Torr pressure. After
igniting the Ar plasma oxygen was purged through the side
port and was adjusted so as to maintain a 500 Torr constant
operating pressure in the reactor using throttling valve. The
typical operating conditions for the synthesis of
nanocrystallineZnO summarized in this process, argon
plasma plume was allowed to strike on graphite anode on
which zinc metal plate is placed. Interaction of high enthalpy
of plasma with material results in melting and evaporation of
zinc. These flying metallic vapors react with ambient oxygen
in reactor inside/near the periphery of plasma plume, results in
homogeneous nucleation of ZnO (g) species. During its flight
further growth takes place and restricted just outside the
periphery of plasma plume where they collide with the cooler
ambient oxygen. Thus, steep temperature gradient arrested the
growth results in nanostructured form formation and gets
deposited over the inner wall of water cooled hemispherical
dome and chamber. The synthesized powder was used for
structural, morphological and optical characterization using
BRUKER AXE D8 ADVANCE X-RAY Diffractometer,
TECNAT 200G (200kV), Transmission Electron Microscopy
(TEM) and JASCO UV- visible spectrometer respectively.
Hemolysis/Fragility Study: Whole blood samples were
collected in EDTA voiles and treated with RBC diluting fluid
to lyse the WBCs. In order to lyse the RBCs the resultant
mixture was treated with varied concentrations between 0.1to
0.6% NaCl. The process of lysis was noted by hemocytometer.
The Fouroescence Labeling :Fluorescence labeling for red
blood cells was done with few modifications; the mixture of
99
0.8% NaCl, Dimethyl Sulfoxide (DMSO), Fluorescein (10M), Zinc Oxide (20μg/ml, 30μg/ml, 40μg/ml) were used for
RBCs. Tris-Buffered Ringer's solution was prepared by
dissolving Sodium Chloride 7.708g, Potassium Chloride
0.350g, Calcium Chloride 0.294g, Magnesium Sulfate
(MgSO4,7H2 O) 0.288g and Tris 2.544g in 1liter. (The pH is
10.25. Titrate with 1 M HCL till the pH of the reaction mixture
is 7.4).Tris-buffered Ringer's albumin solution (TRA) was
prepared by heating the reaction mixture (500ml) to 370C; 2.5
gm of Bovine serum albumin were added and dissolve by
constant stirring. The pH was adjusted to 7.4 using 1M NaOH.
The EDTA blood samples were washed thrice with Trisbuffered Ringer's solution and centrifuged; the RBCs were
collected in the form of a pallet at the bottom. The WBCs
(upper layer) was discarded. The remaining part was treated
with fluorescein dye (10-6 M), 0.1 ml DMSO and 10ml of TRA
were added. The resultant mixture was incubated for 2 hours at
room temperature. The resultant mixture was washed thrice
using TRA. This step was to remove unbound dye. The
reaction mixture was divided into two parts- first part was
subjected to fluorescence microscopy immediately while the
second part was incubated at 40 C overnight thereafter
subjected to fluorescence microscopy. The resultant mixture
was incubated at room temperature for 2 hours and then kept at
40C overnight and was treated with TRA to remove unbound
dye followed by centrifugation at 18000 rpm to ensure
complete removal of unbound dye. Thereafter, the mixture was
subjected to fluorescence microscopy. Treatment of RBCs
with ZnO nanoparticles: normal RBCs were treated with 20
µg/ml, 30µg/ml and 40µg/ml of ZnO NPs. The respective
reaction mixtures were incubated for half an hour at room
temperature. These reaction mixtures were centrifuged at 2000
rpm for 10 minutes. Supernatants were removed from the
respective mixtures and were subjected to fluorescence
microscopy to observe fluorescence intensity; these were
confirmed by fluorimetry.
Stability Test For Red Blood Cells:Stability test of RBCs was
carried out to evaluate the interaction of the normal RBCs in
relation to ZnO NPs using 0.2% trypan blue. This was
followed by incubation at 370C for 10 minutes. The final
mixtures were subjected for observation for viability staining.
6
RESULTS AND DISCUSSIONS
To date, most of the research seems to be focused on
applications of nano particles in biology and medicine due
their unique physico-chemical properties. Nano particles have
shown strong action against, pathogens, viruses and fungus. It
is important to understand their mechanisms of interaction at
cellular and molecular level. However, interaction of NPs with
active biological molecules, cells, tissues and its consequences
at physiological level are yet to be understood. Introduction of
NPs in blood and their interaction with plasma proteins and
blood cells is essential in biomedical applications including
bio safety concern.
In the present study, the effect of ZnO NPs on RBCs have been
investigated in context with the osmotic fragility, hemolysis
and morphological features of RBCs. Fragility of RBCs
represents stress on the membrane due to osmotic and
mechanical stress; osmotic stress is due to the pressure caused
by the flow of hypotonic solution; osmotic fragility is also
related to the composition, integrity, size of the cell and/or
surface area to volume ratio. The fragility of red blood cells is
related to some of the diseased conditions like hereditary
spherocytosis, hypernatremia, anemia, thalassemia, and
sickle cell anemia, (Rodak et al 2007, Fischback et al 2008 and
Greer et al 2008). Mechanical fragility of RBCs refers to the
stress caused due to some kind of shear stress during
diagnostic testing of blood, handling devices, manipulation of
blood during dialysis or intra-operative auto transfusion,
storage of red blood cells (storage lesions) and/or applications
during blood transfusion and blood bank, (Asaio 2002, Asaio
2005, Yazer et al 2008 and Vox Sang 2010).
This investigation indicatesleast chances of mechanical
fragility on RBCs because these cells exhibited 97% stability,
even at 100µg/ml (Table 3), while no significant osmotic
fragility in terms of hemolysis observed with the increase of
concentration of ZnO NPs under isotonic conditions (Table 1,
3 and Fig 3). The interaction of ZnO nano wires with L929 cell
line caused stimulation in reproduction at the 100 µg/ml (Li et
al 2008). Mesoporous silica nanoparticles (Slowing et al
2009) and TiO2 (Ghosh et al 2013) caused hemolytic activity
towards mammalian RBCs. Ozturk and Ozdemir (2013) have
found that Al+3 can increase the osmotic fragility of RBC.
When RBCs were treated with the various concentrations of
NaCl and ZnO NPs, hemolysis/osmotic fragility was noticed
i.e. 0.1% NaCl and 20µg/ml ZnO NPs) not when treated with
higher concentrations of ZnO NPs and this was confirmed
using light microscopy (Table 3). Bright field microscopy was
adapted to confirm the structural and morphological changes
(Figs 1b, 1c). Osama and Hussein (2014) have observed
fluctuations in the hematological parameters such as WBC
count, Hb contents, mean corpuscular volume and decline in
RBC count when silver nanoparticles were introduced
intraperitoneally in rats (less than 100 nm and 2000 mg/kg
body weight).
In current study, all of the RBCs did not hemolyzed at the same
time when treated with ZnO NPs, some RBCs took more time,
and some became crenated while some remained turgid for
relatively longer duration (Fig 1a, 1b,1c, and 2).Zhao et al
(2011) have observed that silica NPs have the capacity to
change the size and surface properties of red blood cells
without affecting the morphology of membrane while others
can deform. Similarly in this study ZnO NPs were found
toinfluence the membrane of red blood cells without breaking
iti.e. degree of hemolysis increased slightly with increase in
the concentration of ZnO NPs under isotonic condition (Table
3). Hemolytic activity was observed at different hypotonic
conditions involving different concentration of ZnO NPs;
percentage hemolysis were found at 0.1%, 0.2%, 0.3% and
0.4% of NaCl, as reference points. These concentrations of
NaCl were involved while studying the effects of ZnO NPs on
hemolysis of red blood cells, the degree of hemolysis declined
with increased concentration of ZnO NPs (Table 1). This
observation indicates that ZnO NPs do not facilitate
hemolysis of Red blood cells at least at higher concentrations.
The stability is another important aspect of monitoring
biocompatibility of nano particle. Li et al (2008) observed that
ZnO nanowires are biocompatible and biologically safe in
lower concentration to biosystem. The current observations
on the stability of RBCs with reference to ZnO nanoparticle
100
are in agreement to their findings. Heng et al (2010a) observed
>99% stability in cells which were unexposed to H2O2 in
negative control up to ZnO NPs 10 µg/ml and stability declined
above this concentration of ZnO NPs. The current study
exhibited around 97% stability of red blood cells with
reference to 100 µg/ml ZnO NPs (Table 3). Engineered ZnO
NPs (431 nm) showed aggregation formation on RBCs when
suspended in buffer, (Simundic et al 2013).
Fluorescence is one of the important biophysical tools to study
images in life science and it is more useful in almost all
situations specifically in those conditions where one needs to
distinguish a particular component from the inconspicuous
and complex region (Bob Carr 2013). The study reveals that
there is no interference between ZnO NPs and normal RBCs.
Effect of NPs on fluorescein binding was studied using
fluorimetric and fluorescence microscopy (Fig 2). Both of
observations showed correlations between changes in
fluorescence intensity with respect to the change in ZnO NPs
concentrations; decline in fluorescence intensity with increase
in ZnO NPs concentration was observed (Figs 4a, 4b, 4c, 4d
and 5a to5c, Tables2 and 3). This change might be due to
modifications of surface property by ZnO NPs. Hence, to
confirm these observations, we used bright field microscopy
and cell stability test. The morphological changes were noticed
under bright field microscopy. The RBCs did not showed any
effect after treatment with ZnO NPs, 97% stability of red blood
cells was obtained after treating cells with 100 µg/ml of ZnO
NPs. Percentage of non-stable RBCs was found to be
negligible. While bright field microscopic images showed
slight changes on RBC structure which might be responsible
for fluorescence binding to RBC membrane (Figs 1b,1c and 2);
fluorescence intensity was obtained from RBCs by specifying
excitation (495 nm) and emission wave length (520 nm) in
presence of fluorescein dye. The fluorescence intensity was
obtained only from bound probes to RBCs and not from
unbound probes. Fluorescence activity declined even when
buffer solution and the increased concentrations of ZnO NPs
were used (Table 2 and 3).
Stoker (2012) has described crenation as a process in which a
cell undergoes contraction when subjected to hypertonic
solution or conditions; this contraction is due to the loss of
water from the cell. This loss of water causes shrinkage of the
cytoplasm as a result of this cell membrane shows some sharp
notches or fine projections. Kanshan sky et al (2010) suggested
that crenated stage of red blood cells is likely to be the result of
either development of some abnormalities in the lipids and
proteins of the cell membrane and/or some clinical
condition/infection. This affects the normal functioning of red
blood cells. In the present study, red blood cells exhibited
increased degree of crenation when treated with 1%, 2% and
3% of NaCl only, but when subjected to exposure to ZnO NPs
varied degree of crenation was observed; lower concentrations
caused no crenation and higher concentrations caused intense
crenation in more number of red blood cells, (Figs6a to 9d).
The overall results indicate that ZnO NPs induce physical
modifications in red blood cell membrane when treated with
higher concentration of ZnO NPs. This mechanism is yet to be
studied at molecular level. Kristine (2009) explained that
white patch/dot in the center of the red blood cell is pellor zone
or zone of central pellor and this zone is around ⅓ of the
diameter of the normal red blood cell; this state of red blood
cell is called normochromic. If the pellor zone is more in a
given red blood cell such cells are considered to be
hypochromic – and it is indicative of anemic condition. Pellor
zone and peripheral zone exhibited variations when exposed
to varied concentrations of ZnO NPs; pellor zone was found to
be indistinct when treated with lower concentrations while
higher concentrations did not affect the pellor zone and the
peripheral zone, (Figs 7a to 9d). Qualitative variations in size
of red blood cells were noticed resulted due to hypertonic
NaCl and lower concentrations of ZnO NPs; reduction in size
specifically in crenated red blood cells was noticed when
treated with 20µg/ml of ZnO NPs in 1%, 2% and 3% NaCl.
Higher concentrations of ZnO NPs were found to be relatively
ineffective in this parameter (Figs 7a to 9d). In this study the
pattern of distribution of red blood cells was affected when
exposed to 30µg/ml and 40µg/ml of ZnO NPs in 1% NaCl and
20 µg/ml, 40 µg/ml, 50 µg/ml in 2% NaCl. This behavior of
red blood cells may indicate some physical interaction with
ZnO NP. Turgidity is another aspect which was found to be
affected at least in case of 40µg/ml in 2% NaCl; it may be
possible that at this concentration inward flow might have
been elevated. The red blood cells were seen to exhibit some
degree of aggregation in response to interaction with higher
concentration of ZnO NPs like 30 µg/ml, 40 µg/ml and
50µg/ml in 1% NaCl and also at 50µg/ml in 2% NaCl (Figs 7d
to 9d).
CONCLUSION
In this study the influence of ZnO NPs of 25 nm was carried
out. These NPs caused certain changes in osmotic fragility.
Non significant hemolysis of human RBCs was found to be in
gradual manner, red blood cells lysed at different intervals
under isotonic conditions but when treated with ZnO NPs the
degree of hemolysis increased with the increase of
concentration of ZnO NPs. Under varying hypotonic
conditions the percentage hemolysis declined with the
increase of ZnO NPs concentrations. A correlation has been
observed between changes in fluorescence intensity and
change in the concentration of ZnO NPs; the fluorescence
intensity declined with rise in the concentration. RBCs
exhibited scanty fluorescence activities under the influence of
ZnO NPs. Human RBCs were found to be very stable even
after treatment with 100 µg/ml ZnO NPs. These nanoparticles
were found to affect the distribution or dispersal of RBCs
under observation. The distribution of RBCs was observed to
be either somewhat linear or as small aggregates; uniform
dispersal was not observed. Further, the pellor zone and
peripheral zone of RBCs were found to be affected, peripheral
zone was found to be reduced with the increase of
concentration of ZnO NPs and even crenation exhibited
variations with rise in concentrations of these NPs. Present
investigation indicates that ZnO NPs (25 nm) appear to be
hemocompatible within 20 µg/ml to 100 µg/ml range but may
cause some changes in the fragility, dispersal in the medium
and morphological fluctuations in human red blood cells. The
details of the mechanism involved in the interaction between
ZnO NPs at cellular and the membrane of RBCs need further
elaboration.
101
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102
1a
1b
1c
Figure 1a – Unlabeled RBCs immediately observed under
hypotonic condition showing swollen, crenation and lysed
cells. Figure 1b – Unlabeled RBCs in isotonic solution – under
Bright field. Figure 1c – RBCs labeled with fluorescein
without ZnO NPs – under Bright field
2
Figure (2) –Fluorescein labeled RBCs treated with zinc oxide
nanoparticles. Number of crenated cells increased when
treated with ZnO NPs (100 μg/ml) Indicate crenated cells.
Table 2 Fluorescence intensity with different
ZnO concentrations under hypotonic conditions
Figure (3) – Isotonic and hypotonic concentrations (0.10.4%
NaCl) used with varying (0.0-50.0μg/ml) ZnO NPs
concentrations (as indicated) are used for determining
percentage hemolysis;
4a
NaCl
ZnO NPs
Fluorescence
concentration
concentration
intensity
(%)
(µg/ml)
0.7
20
16.668
0.7
30
15.63
0.7
40
Not detected
Fluorescence Intensity was not able to detected
below 0.7% NaCl
Table 3 Hemolysis and stability under isotonic conditions
4b
4c
4d
5aFigure (4a) RBC labeled with Fluorescein; Figure (4b)
RBCs + 20μg/ml ZnO; Figure (4c) RBCs + 30μg/ml ZnO;
Figure (4d) RBCs + 40 μg/ml ZnO;
5a
5b
5c
Figure (5a to 5c). Fluorescently tagged RBCs tagged with
fluorescence under hypotonic condition with varying ZnO
NPs concentration; (5a) RBCs + Fluorescent dye + NaCl
(0.7%); (5b) RBCs + Fluorescent dye + NaCl(0.7%) +
20μg/ml ZnO NPs; (5c) RBCs + Fluorescent dye +
NaCl(0.7%) + 30μg/ml ZnO NPs.
6a
6b
8a
8b
6c
6d
8c
8d
Figure (8a) RBCs + NaCl (2%) + ZnO NPs (20μg/ml); Figure
(8b) RBCs + NaCl (2%) + ZnO NPs (30 μg/ml); Figure (8c)
RBCs + NaCl (2%) + ZnO NPs (40 μg/ml);Figure (8d) RBCs
+ NaCl (2%) + ZnO NPs (50 μg/ml).
Figure (6a) RBCs (0.85% NaCl); Figure (6b) RBCs + NaCl
(1%); Figure (6c) RBCs + NaCl (2%); Figure (6d) RBCs +
NaCl (3%).
7a
7b
7c
7d
9a
9b
9c
9d
Figure (9a) RBCs + NaCl (3%) + ZnO NPs (20 μg/ml) Figure
(9b) RBCs + NaCl (3%) + ZnO NPs (30 μg/ml); Figure (9c)
RBCs + NaCl (3%) + ZnO NPs (40 μg/ml); Figure (9d) RBCs
+ NaCl (3%) + ZnO NPs (50 μg/ml)
8aFigure (7a) RBCs + NaCl (1%) + ZnO NPs (20μg/ml);
Figure (7b) RBCs + NaCl (1%) + ZnO NPs (30 μg/ml); Figure
(7c) RBCs + NaCl (1%) + ZnO NPs (40 μg/ml); Figure (7d)
RBCs + NaCl (1%) + ZnO NPs (50 μg/ml).
104