| Research Article |
Open Access |
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| Rheological Parameters Assessment in Serum, Plasma and Whole Blood of
Rats after Administration of Gold Nanoparticles of Different Sizes: In vivo |
| Mohamed Anwar K Abdelhalim1* and Mohsen Mahmoud Mady1,2
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| 1Department of Physics and Astronomy, College of Science, King Saud University, Saudi Arabia |
| 2Department of Biophysics, Cairo University, 12613 Giza, Egypt |
| *Corresponding author: |
Mohamed Anwar K. Abdelhalim
Associate Professor,
Department of Physics and Astronomy
College of Science, King Saud University
P.O.
2455, Riyadh 11451, Saudi Arabia
E-mail: abdelhalimmak@yahoo.com |
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| Received May 25, 2012; Accepted July 16, 2012; Published July 18, 2012 |
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| Citation: Abdelhalim MAK (2012) Rheological Parameters Assessment in Serum,
Plasma and Whole Blood of Rats after Administration of Gold Nanoparticles
of Different Sizes: In vivo. J Nanomed Nanotechol 3:145. doi:10.4172/2157-7439.1000145 |
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| Copyright: © 2012 Abdelhalim MAK. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
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| Abstract |
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| Background: The evaluation of blood rheology has been underutilised in clinical practice. We performed an array
of rheological parameters measurements to quantify the responses of rat plasma, serum and whole blood to gold
nanoparticles (GNPs) of different sizes. |
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| Methods: GNPs of various sizes were used in this study. Doses of 0.05 ml of the GNPs were administered to the
animals via intraperitoneal injection for a period of 3 days. Blood samples with volumes of nearly 2 ml were obtained
from each rat. Various rheological parameters, such as %torque, shear stress (SS), shear rate (SR), viscosity, plastic
velocity, yield stress, consistency index and flow index, were measured in rat plasma, serum and whole blood. |
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| Results: The relationship between SS and SR for rat serum, plasma and whole blood showed linear behaviour
with the 10, 20 and 50 nm GNPs. The viscosities of rat serum, plasma and whole blood with GNPs were decreased
with increasing the SR and showed non-linear behaviour. The viscosity of blood serum and plasma was measured at a
range of shear rates from 200 to 1375 s-1, while the viscosity of whole blood was measured at 75 to 600 s-1. |
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| Conclusions: The GNP size has a considerable influence on the various rheological parameters for rat blood
at a fixed temperature of 37°C. The decrease in viscosity of 50 nm GNPs compared to 10 and 20 nm GNPs may be
attributed to decrease in number of NPs and GNP surface area. It can be concluded that the GNPs probably cause
erythrocyte deformability, and their interactions with blood proteins may cause a decrease in serum, plasma and whole
blood viscosities under a given level of applied SS and SR compared to the control. This study suggests that further
experimental work taking nanoparticle surface properties into consideration should be performed. |
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| Keywords |
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| Gold nanoparticle sizes; Serum; Plasma; Whole blood;
Rheological parameters; Rats |
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| Introduction |
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| Hemorheology is the study of the flow properties of blood and its
elements (plasma and formed elements, including erythrocytes, white
blood cells and platelets). There is increasing evidence that the flow
properties of blood are among the main determinants of proper tissue
perfusion, and alterations in these properties play significant roles in
disease processes; therefore, knowledge of these properties is vital to
our understanding of Hemorheology [1]. |
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| Blood viscosity is determined by plasma viscosity, haematocrit
(volume fraction of erythrocytes, which constitutes 99.9% of the
cellular elements) and the mechanical behaviour of erythrocytes.
Therefore, erythrocyte mechanics represent the major determinant
of the flow properties of blood. Erythrocytes have unique mechanical
behaviours that can be discussed in terms of erythrocyte deformability
and aggregation [2]. |
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| Whole-blood viscosity is a predictor of stroke, carotid intima-media
thickening, and carotid atherosclerosis. However, in most studies,
whole blood viscosity was measured at a few non-specific shear rates,
and these data do not reflect the complete rheological characteristics
found in these studies [3]. |
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| Blood is a unique fluid. It exhibits non-Newtonian characteristics,
and its viscosity is dependent on the shear rate. The major determinants
of whole-blood viscosity are hematocrit, plasma viscosity, and red cell
aggregation and deformation under conditions of low and high shear
rates [4-6]. |
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| In hyperviscosity syndromes, plasma viscosity is a better indicator
in follow-up examinations. In rheumatoid arthritis, sensitivity and
specificity of plasma viscosity are better than that of C-reactive protein. The plasma fibrinogen concentration and plasma viscosity
are elevated in unstable angina pectoris and stroke, and their higher
values are associated with higher rates of major adverse clinical events.
The elevation of plasma viscosity is correlated with the progression of
coronary and peripheral artery diseases. Thus, plasma viscosity should
be measured routinely in medical practice [7]. |
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| Nanoparticle studies are becoming much more common owing to
their novel physical and chemical attributes in biological and medical
applications [8-11]. |
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| The Surface Plasmon Resonance (SPR) property of NPs allows the
use of GNPs in many biological and medical applications. GNPs are
used as immunostaining marker particles for electron microscopy and
as chromophores for immunoreactions and nucleic acid hybridisation
[12,13]. Their application for gene delivery into cells has also been
reported [14-17]. In addition, GNPs have attracted attention as photothermal
agents in hyperthermia treatment [18]. |
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| The sizes of the NPs are similar to the sizes of most biological
molecules. For this reason, the GNPs can be used for both in vivo and in vitro biomedical applications. Therefore, increased attention has been
placed on the applications of NPs in biology and medicine. |
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| Viscoelastic properties can be applied to the quality control of raw
materials, final products, and manufacturing processes. Furthermore,
the release of a drug from a semi-solid carrier is influenced by the
carrier’s rheological behaviour. The effect of certain parameters,
such as the storage time and temperature, on the quality of the
GNPs as pharmaceutical products can be investigated via rheological
measurements. Rheological analysis can be used as a sensitive tool in
predicting the physical properties of the GNPs of different sizes. |
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| The objective of this study was to examine the effects of the daily
intraperitoneal administration of 0.05 ml of the GNPs of different sizes
(10, 20 and 50 nm) for 3 days on various rheological parameters in rat
serum, plasma and whole blood over a wide range of shear rates. |
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| Materials and Methods |
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| GNP sizes |
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| GNPs (in sizes of the 10, 20 and 50 nm) were purchased (Product
MKN-Au) and used in this study. The GNPs were in aqueous solution
(10 mm GNPs: Product MKN-Au-010, concentration 0.01% Au; 20 nm
GNPs: Product MKN-Au-020, concentration 0.01% Au; 50 nm GNPs:
Product MKN-Au-050, concentration 0.01% Au). |
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| Animals |
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| Healthy male Wistar-Kyoto rats (8-12 weeks old; approximately
250 g body weight) that were obtained from the Laboratory Animal
Centre (LAC) (College of Pharmacy, King Saud University) were
housed in pairs in humidity- and temperature-controlled ventilated
cages on a 12 h day/night cycle. A rodent diet and water were provided.
Fifty rats were individually caged and divided into the control group
(NG: n=8), group 1 (A: infusion of the 20 nm GNPs for 3 days; n=6),
group 2 (A: infusion of the 10 nm GNPs for 3 days; n=6) and group 3
(A: infusion of the 50 nm GNPs for 3 days; n=6). All experiments were
conducted in accordance with the guidelines approved by King Saud
University Local Animal Care and Use Committee. |
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| GNPs administration and preparation of serum, plasma and
whole blood |
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| Doses of 0.05 ml of the GNPs (10, 20 and 50 nm) in aqueous
solutions were administered to the animals via intraperitoneal
administration for a period of 3 days. The rats were anesthetised by
inhalation of 5% isoflurane until muscular tonus relaxed. Blood and
several organs (liver, heart, lung and kidney) were collected from each
rat. |
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| Blood samples of nearly 2 ml were collected into three polypropylene
tubes: the 1st tube for serum, the 2nd tube for plasma and the 3rd tube for
whole blood. The serum was prepared by allowing the blood to clot at
37°C. The blood was then centrifuged at 3,000 rpm for ten minutes. The
blood for plasma was collected in EDTA. Whole blood was prepared by
adding 0.8 ml of heparin to 0.8 ml of collected blood. |
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| Experimental setup and rheological parameters |
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| The following experimental setup was used to measure several
rheological parameters in rat serum, plasma and whole blood after
the administration of the 10, 20 and 50 nm GNPs. The rheological
parameters tested were viscosity, %torque, Shear Stress (SS), Shear
Rate (SR), plastic viscosity, yield stress, and consistency index. These
parameters were measured using a Brookfield LVDV-III Programmable rheometer (cone-plate viscometer; Brookfield Engineering Laboratory,
Incorporation, Middleboro, USA) that was supplied with a temperature
bath controlled by a computer. The rheometer was guaranteed to
be accurate within ±1% of the full scale range of the spindle/speed
combination, and in-use reproducibility is within ± 0.2%. |
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| The rheological parameters were measured at 37°C. The
temperature inside the sample chamber was carefully monitored using
a temperature sensor during the viscosity measurements. |
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| A cone and plate sensor with a diameter of 2.4 cm and an angle of
0.8 was used. The rheometer was calibrated using standard fluids. This
viscometer has a viscosity measurement range of 1.5-30,000 mPa s. |
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| The spindle type (SC-40) and its speed combinations produce
results with high accuracy when the applied torque is within the range
of 10-100%; the appropriate spindle was chosen accordingly. |
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| An aqueous solution of 0.5 ml of each size of GNP was poured into
the sample chamber of the rheometer. The spindle was immersed and
rotated in these gold nanofluids in a speed range of 20 to 180 RPM in
20 minutes. The viscous drag of the GNP aqueous solution against the
spindle was measured by the deflection of the calibrated spring. |
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| Statistical analysis |
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| The results of this study were expressed as mean ± standard
deviation (Mean ± SD). To assess the significance of the differences
between the control group and the 3 experimental groups (G1A, G2A
and G3A), a statistical analysis was performed using one-way analysis
of variance (ANOVA) for repeated measurements, with significance
assessed at the 5% confidence level. |
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| Results and Discussions |
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| Rheological parameters measurements |
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| This study is unique in examining the relationships between various
rheological parameters in rat plasma, serum and whole blood after
the administration of 0.05 ml of the GNPs of various sizes at a fixed
temperature of 37°C and a wide range of shear rates using a Brookfield
LVDV-III Programmable rheometer. |
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| The data in the present study suggest that at these shear rates,
serum, plasma and whole blood viscosities are influenced by the size
and shape of the administered GNPs, the number of NPs and the GNP
surface area. |
|
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| The viscosities of blood serum and plasma were measured at a
shear rate range of 200 to 1375 s-1, while the viscosity of whole blood
was measured at a shear rate range of 75 to 600 s-1. The shear stress (SS)
and shear rate (SR) of G1A (20 nm), G2A (10 nm) and G3A (50 nm)
for plasma, serum and whole blood were linearly related as shown in
Figures 1, 3 and 5, and the SS and SR relationships of serum, plasma
and whole blood can be written as shown in Table 1. |
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Figure 1: The relationship between shear stress and shear rate for blood
plasma of rat at shear rate range of 200-1375 s-1. |
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Figure 2: The relationship between viscosity and shear rate for blood plasma
of rat at shear rate range of 200-1375 s-1. |
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Figure 3: The relationship between shear stress and shear rate for blood
serum of rat at shear rate range of 200-1375 s-1. |
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Table 1: The relationship between shear stress and shear rate for plasma, serum and whole blood of rat at wide range of shear rate. |
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| The viscosities of rat serum, plasma and whole blood with the 50
nm GNPs were significantly lower than those with the 10 nm GNPs.
The decrease in viscosity of the 50 nm GNP solutions may be attributed
to the decrease in the number of GNPs and decreased GNP surface
area. For all of the GNPs, the highest viscosity values were observed
for whole blood compared to plasma and serum as shown in Figures
2, 4 and 6. |
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Figure 4: The relationship between shear stress and shear rate for blood
serum of rat at shear rate range of 200-1375 s-1. |
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Figure 5: The relationship between shear stress and shear rate for whole
blood of rat at shear rate range of 75-600 s-1. |
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Figure 6: The relationship between viscosity and shear rate for whole blood of
rat at shear rate range of 75-600 s-1. |
|
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| The viscosities of rat serum, plasma and whole blood with the10, 20
and 50 nm GNPs were decreased with increasing the SR. The viscosity and SR relationships of rat serum, plasma and whole blood with the10,
20 and 50 nm GNPs were non-linearly related as shown in Figures 2,
4 and 6. |
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| This study suggests that the decrease in serum, plasma and whole
blood viscosities with the 10, 20 and 50 nm GNPs may be attributed
to the decrease in haematocrit and cytoplasmic haemoglobin
concentration of erythrocytes, erythrocyte deformability (degree of shape change under a given level of applied shear stress and shear rate),
and the changes induced in the viscoelastic properties of erythrocyte
membranes. |
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| Plasma viscosity is determined by the water content and
macromolecular components of plasma, so the factors that affect blood
viscosity are the plasma protein concentration and the types of proteins
present in the plasma. However, these effects are minor compared to the
effect of hematocrit, so they are effectively insignificant. An elevation
of plasma viscosity correlates with the progression of coronary and
peripheral vascular diseases. Anaemia can lead to decreased blood
viscosity, which may lead to heart failure [19]. |
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| The shape change of erythrocytes under applied forces is reversible,
and the biconcave-discoid shape, which is normal for most mammals,
is maintained after the removal of the deforming forces. In other words,
erythrocytes behave like elastic bodies, but they also resist shape change
under the influence of deforming forces. This viscoelastic behaviour of
erythrocytes is determined by the following three properties: 1) their
biconcave-discoid geometric shape provides extra surface area for the
cell, enabling shape changes without increasing the surface area; this
type of shape change requires significantly smaller forces than those
required for shape changes with surface area expansion; 2) cytoplasmic
viscosity, which reflects the cytoplasmic haemoglobin concentration
of erythrocytes; and 3) the viscoelastic properties of the erythrocyte
membrane, which are mainly determined by their special membrane
skeletal network [20]. |
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| The effect of a protein on plasma viscosity depends on its molecular
weight and structure. A less spheroid shape, higher molecular weight,
higher aggregating capacity, and higher temperature or pH sensitivity of
a protein will lead to higher plasma viscosity [7]. Plasma is a Newtonian
fluid, and its viscosity does not depend on flow characteristics. Its
normal value is 1.10-1.30 mPa s at 37°C, which is independent of age
and gender [7]. The measurement has high stability and accuracy, so
the detection of small alterations may be pathologically important.
Inflammation and tissue injuries resulting in changes in plasma
proteins can increase its value with high sensitivity but low specificity
[19-21]. |
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| GNPs strongly associate with essential blood proteins, where
the binding constant and degree of cooperatively of particle−protein
binding depends on the particle size and the native protein structure.
Moreover, the thickness of the adsorbed protein layer (bare NP
diameter <50 nm) progressively increases with the NP size, which
causes effects that have potential importance for understanding the
general NP aggregation in biological media and the interactions of NP
with biological materials [21]. |
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| In this manuscript the author used GNPs with three different
sizes that are available commercially as models for rheological study.
However, it is not clear that the surface properties of the GNPs are
critically important influencing the interaction with the proteins in the
blood. Illustration on the surface properties would help to gain deeper
insight into interactions between GNPs and blood, thus the rheological
behaviour of blood and GNPs is relevant. Therefore, this study suggests
that further experimental work taking nanoparticle surface properties
into consideration should be performed. |
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| Conclusions |
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| This study indicates that GNP size has a considerable influence on
the various rheological parameters measured in rat serum, plasma and
whole blood at 37°C. |
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| The decrease in viscosity of blood fluids in the presence of the 50
nm GNPs compared to the 10 and 20 nm GNPs may be attributed to
the decrease in the number of GNPs and the decreased GNP surface
area. |
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| The viscosities of rat serum, plasma and whole blood with the10, 20
and 50 nm GNPs were decreased with increasing the SR. The viscosity
and SR relationships of rat serum, plasma and whole blood with the10,
20 and 50 nm GNPs showed non-linear behaviour. For all of the GNPs,
the highest viscosity values were observed in whole blood compared to
plasma and serum. |
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| Based on the present results of erythrocyte rheological properties,
it can be concluded that the GNPs would probably cause erythrocyte
deformability. |
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| Authors’ Contributions |
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| AMAK analysed data and interpreted and wrote the final draft of this
manuscript. The animal model used in this study was obtained from the Laboratory
Animal Centre (College of Pharmacy, King Saud University). AMAK conceived the
plan and design and obtained research grants for this study. The authors have read
and approved the final manuscript. |
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| Acknowledgements |
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| The authors are very grateful to NPST. This research was financially supported
by the National Science and Technology Innovation Plan (NSTIP), Research No.
08-ADV206-02 and Research No. 09-NAN670-02, College of Science, King Saud
University, Saudi Arabia. |
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