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Chitosan coating of copper nanoparticles reduces in vitro toxicity and increases inflammation
in the lung
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2013 Nanotechnology 24 395101
(http://iopscience.iop.org/0957-4484/24/39/395101)
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IOP PUBLISHING
NANOTECHNOLOGY
Nanotechnology 24 (2013) 395101 (10pp)
doi:10.1088/0957-4484/24/39/395101
Chitosan coating of copper nanoparticles
reduces in vitro toxicity and increases
inflammation in the lung
Kristan L S Worthington1,2 , Andrea Adamcakova-Dodd3 ,
Amaraporn Wongrakpanich2 , Imali A Mudunkotuwa4 ,
Kranti A Mapuskar5 , Vijaya B Joshi2 , C Allan Guymon1 ,
Douglas R Spitz5 , Vicki H Grassian4 , Peter S Thorne3 and
Aliasger K Salem1,2,6
1
Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa,
Iowa City, IA 52242, USA
2
Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy,
University of Iowa, Iowa City, IA 52242, USA
3
Department of Occupational and Environmental Health, College of Public Health, University of Iowa,
Iowa City, IA 52242, USA
4
Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City,
IA 52242, USA
5
Free Radical and Radiation Biology and Toxicology Programs, Department of Radiation Oncology,
Holden Comprehensive Cancer Center, Carver College of Medicine, University of Iowa, Iowa City, IA
52242, USA
E-mail: aliasger-salem@uiowa.edu
Received 26 February 2013, in final form 11 July 2013
Published 5 September 2013
Online at stacks.iop.org/Nano/24/395101
Abstract
Despite their potential for a variety of applications, copper nanoparticles induce very strong inflammatory
responses and cellular toxicity following aerosolized delivery. Coating metallic nanoparticles with
polysaccharides, such as biocompatible and antimicrobial chitosan, has the potential to reduce this toxicity. In
this study, copper nanoparticles were coated with chitosan using a newly developed and facile method. The
presence of coating was confirmed using x-ray photoelectron spectroscopy, rhodamine tagging of chitosan
followed by confocal fluorescence imaging of coated particles and observed increases in particle size and zeta
potential. Further physical and chemical characteristics were evaluated using dissolution and x-ray diffraction
studies. The chitosan coating was shown to significantly reduce the toxicity of copper nanoparticles after 24 and
52 h and the generation of reactive oxygen species as assayed by DHE oxidation after 24 h in vitro. Conversely,
inflammatory response, measured using the number of white blood cells, total protein, and
cytokines/chemokines in the bronchoalveolar fluid of mice exposed to chitosan coated versus uncoated copper
nanoparticles, was shown to increase, as was the concentration of copper ions. These results suggest that coating
metal nanoparticles with mucoadhesive polysaccharides (e.g. chitosan) could increase their potential for use in
controlled release of copper ions to cells, but will result in a higher inflammatory response if administered via
the lung.
(Some figures may appear in colour only in the online journal)
Nanotechnology plays an increasingly central role in
technological innovation in a broad range of fields ranging
from cosmetics to sustainable energy. Nanoparticles (NPs)
often combine useful properties of bulk materials such as
magnetism, conductivity and stability with a very high
surface to volume ratio, increasing reactivity. Thus, many
important applications for metal NPs are receiving research
6 Address for correspondence: Division of Pharmaceutics and Translational
Therapeutics, College of Pharmacy, The University of Iowa, S228 PHAR,
115 South Grand Avenue, Iowa City, IA 52242, USA.
0957-4484/13/395101+10$33.00
1
c 2013 IOP Publishing Ltd Printed in the UK & the USA
Nanotechnology 24 (2013) 395101
K L S Worthington et al
chitosan properties lend themselves well to wound-healing
applications, including its biocompatibility in mammals
[20, 21] and antimicrobial [22, 23] properties. These
properties have led to a diverse number of medical
applications, including hemostatic wound dressings [24–26],
direct wound filling [27], drug [28–31] and gene [32–35]
delivery, and tissue engineering [36–39]. Although some have
investigated various chitosan coating methods for metal oxide
NPs [40] and even studied their biocompatibility [41], no
study to date has shown the impact of chitosan coating of Cu
NPs on in vitro toxicity and in vivo inflammatory responses.
Such information is critical to allow application involving
polysaccharide coatings of metallic NPs.
In this study, the effect of chitosan coatings on Cu NP
physical properties and toxicity were evaluated. The coated
particles were optimized and thoroughly characterized using
dynamic light scattering, fluorescent tagging, microscopy,
and surface analysis. The impact of the chitosan coating on
the level of toxicity induced in vitro was assessed using
human adenocarcinomic alveolar (A549) cells, as well as
the in vivo inflammatory response of mice following nasal
instillation of coated particles, characterized by differential
cell counts and cytokine/chemokine concentration in lung
lavage analysis. The information gained through this work
provides a basis for understanding the effect of chitosan on
NP toxicity and how polysaccharide coatings on NP surfaces
alter their characteristics and behavior in biological settings.
attention–attention that could impact society in a variety
of important ways. For example, the magnetic properties
of iron oxide NPs lend themselves well to biomedical
applications such as targeting, imaging, and hyperthermia
treatments [1]. Due to their high conductivity, copper
(Cu) NPs are most traditionally used for applications such
as nanofluids [2, 3] to facilitate increased heat transfer
and for high throughput catalysis applications [4, 5]
due to increased reactivity with higher surface area.
More recently, however, studies have demonstrated the
antimicrobial activity of Cu NPs against methicillin-resistant
Staphylococcus aureus (MRSA) and Escherichia coli in
suspension [6] and Saccharomyces cerevisiae, Escherichia
coli, and several other microbes when released in a controlled
manner from polymer films [7]. Other potential biomedical
applications have been demonstrated with other types of metal
nanoparticles, including photo-thermal ablation of tumor
cells [8] and medical imaging [9, 10]. Furthermore, the
release of copper ions from NPs could prove useful in
diseases associated with abnormally low accumulation of
copper ions in certain regions of the body, such as Menkes
disease [11, 12].
Despite their potential use, copper and other metal oxide
NPs are limited by their widely demonstrated toxic properties.
Indeed, metal oxide NPs have been shown to negatively
affect the reproduction and embryonic development of white
worms [13] and zebra fish embryos [14]. Likewise, the
effects of metal oxide NPs on mammalian cells and on
whole organisms can be severe, affecting the central nervous
system [15], and especially the lungs upon inhalation [16].
In a murine model, host defense against bacterial infections
was shown to be significantly lowered by exposure to copper
oxide NPs in a dose-dependent manner [17]. Another study
conducted comparing the toxicity of iron (Fe) and copper
nanoparticles (Cu NPs) using a murine model by Pettibone
et al clearly shows greater inflammatory responses triggered
by Cu NPs than Fe NPs [16]. Furthermore, a study by Yang
et al has shown that Cu NPs with greater surface oxidation
have higher ROS generating capacity [18]. These researchers
also observed a correlation between the surface ligand chain
length and the extent of oxidation, which consequently affects
the ROS generation and toxicity of the Cu NPs. Therefore,
research to understand how to overcome this inherent toxicity
is critical, since addressing the issue is necessary for future
applications of metal oxide NPs.
Coating metallic NPs with polysaccharides can overcome
the drawbacks mentioned above by increasing stability,
improving size distributions, increasing biocompatibility
and introducing chemical groups that allow for further
functionalization of the NPs [19]. Furthermore, these long
chain carbohydrate molecules can potentially inhibit surface
oxidation of the nanoparticles that may lead to increased
toxicity [18]. Chitosan, an attractive polysaccharide for
coating Cu NPs, is a naturally occurring polysaccharide
that is directly obtained by deacetylation of chitin, the
main component of crustacean shells and fungal cell walls.
Additionally, chitosan is cationic in solutions of dilute acid
and one of the most abundant biopolymers on earth. Many
1. Results and discussion
A chitosan coating was applied to Cu NPs to study the
effect of the polysaccharide on NP physical properties,
toxicity in vitro, and inflammatory response in vivo upon
nasal instillation. The physical properties were first examined
visually using transmission electron microscopy (TEM,
figure 1). The copper particles alone (Cu NPs, figure 1(a))
appear to have a smooth, round morphology. The particles
aggregate in water to some degree, making applications using
copper particles in water difficult. A direct coating method
was first attempted by mixing Cu NPs with a solution of
chitosan in acetic acid and buffer. This method, however,
caused even more significant aggregation and dissolution of
copper ions from the particles. Additionally, the solution
appeared to gel, trapping the Cu NPs in a semi-solid matrix.
To stabilize the Cu NPs in the aqueous environment and
protect them from dissolution, a pre-coating of surfactant
(Tween R 80) was applied prior to coating the particles
with chitosan. The hydrophobic chains of the Tween R 80
molecule adsorb via Van der Waals forces (physisorption)
to the hydrophobic Cu NPs [42], leaving a hydrophilic
external layer to the coating. It has been proposed that the
π orbital associated with the carbon–carbon double bond
in the surfactant’s alkyl chain also contributes to its strong
interaction with metal NP surfaces and increases its ability to
stabilize them [43]. Thus, this initial coating process should
help to solubilize Cu NPs, as has been shown for other
NPs [44]. The copper particles coated only with Tween R 80
(Cu NPs + Tw, figure 1(b)), however, exhibited a rough
2
Nanotechnology 24 (2013) 395101
K L S Worthington et al
Figure 1. TEM images of Cu NPs with (a) no coating (Cu NPs), (b) a Tween R 80 coating (Cu NPs + Tw), and (c) a Tween R 80 coating
followed by chitosan coating (Cu NPs + Tw + Ch).
Figure 2. Physical characterization of coated and uncoated Cu NPs. Shown are (a) confocal microscopy images of Cu NPs with no coating
(inset), and coated with Tween R 80 and rhodamine conjugated chitosan; (b) XPS data in the N 1s binding energy region for Cu NPs, Cu
NPs + Tw, and Cu NPs + Tw + Ch where only the Cu NPs + Tw + Ch show the presence of nitrogen on the surface of nanoparticles; and
(c) XRD characterization of Cu NPs, Cu NPs + Tw, Cu NPs + Tw + Ch, and unprocessed Cu NPs.
Table 1. Size, zeta potential, and dissolution of Cu NPs with and
without coating.
morphology and were heavily aggregated, potentially because
the concentration of Tween R 80 used was above the critical
micelle concentration of only 10 µM [45].
We hypothesize that charged, hydrophilic chitosan
molecules can adsorb to the externally presented hydrophilic
groups of the Tween R molecules by physisorption, or Van der
Waals interactions. Once the chitosan coating was applied (Cu
NPs + Tw + Ch, figure 1(c)), these particles demonstrated
a smooth, spherical morphology and aggregated to a lesser
extent than the copper particles alone or the Tween R -coated
particles, likely due to increased repulsive forces between the
positively charged nanoparticles. Further study of chitosan’s
interaction with polysorbate surfactants that are coated on NPs
would certainly be beneficial for understanding the processes
involved in the coating process described herein.
To provide a visual confirmation of chitosan coated
on the NP surface, Rhodamine B conjugated chitosan
derivatives were prepared and used to coat Cu NPs in the
same manner as regular chitosan. Because Rhodamine B
is fluorescent and Cu NPs are not, the chitosan on the
particle surface could now be detected using fluorescence
microscopy (figure 2(a)). To further characterize the coated
particles, the hydrodynamic diameter, charge, and surface
Cu NPs
a
Radius (nm)
Zeta
Potential
(mV)a
Dissolved Cu
(ppm)
Cu NPs + Tw
Cu NPs + Tw + Ch
b
187 ± 10 495 ± 66
15.4 ± 3.2 13.6 ± 0.3b
260 ± 25
43.4 ± 1.7
15.7 ± 0.1 12.4 ± 0.6
27.2 ± 0.4
a
n = 3.
Data quality report indicated poor data quality and high likelihood
of aggregation.
b
composition were determined using dynamic light scattering
(DLS) zeta potential measurements and x-ray photoelectron
spectroscopy (XPS), respectively. For ease of comparison,
the size and charge information is summarized in table 1.
The measured hydrodynamic diameter of the Cu NPs before
coating, after pre-coating with Tween R 80, and after chitosan
coating were 187 ± 10 nm, 495 ± 66 nm, and 260 ± 25 nm,
respectively. Although these measured sizes are much larger
than those observed using TEM, the overall size trends
were consistent; chitosan-coated particles were slightly larger
3
Nanotechnology 24 (2013) 395101
K L S Worthington et al
than Cu NPs alone and the Tween R 80 coated NPs were
highly aggregated and difficult to measure. The charge
measurements showed a significant increase in positive charge
(from 15.4 ± 3.2 to 43.4 ± 1.7 mV) when Cu NPs were
coated with chitosan. Since chitosan is a cationic molecule,
these results give evidence that the Cu NPs were, in fact,
coated with chitosan. As a final confirmation of the chitosan
coating, the surface composition of the NPs was analyzed
using XPS. All three NP formulations (Cu NPs, Cu NPs + Tw,
Cu NPs + Tw + Ch) exhibited peaks at three corresponding
bonding energies standard for copper, confirming the presence
of Cu. XPS analysis of the N1s region (figure 2(b)) showed
no representative signals for the Cu NPs or Cu NPs + Tw
samples. On the other hand, a very distinct peak at a binding
energy of 390 mV was observed for the Cu NPs + Tw + Ch,
indicating the presence of nitrogen, the source of which being
the chitosan free amine groups. The relative ratios of various
atoms on the NP surface were also estimated by XPS. The
number of carbon and oxygen atoms relative to the number of
copper atoms on the surface increased dramatically (∼550%
and ∼680%, respectively) between the Cu NPs and Cu
NPs + Tw + Ch groups, likely due to the addition of the high
molecular weight polysaccharide. Coated and uncoated NPs
were also characterized by XRD for their bulk composition
to identify any changes taking place during the process of
coating. The Cu NPs oxidize during the treatment process
into CuO NPs (see figure 2(c)). The diffraction patterns
indicate that the unprocessed Cu NPs also contain CuO,
likely due to surface oxidation of Cu NPs. As noted by the
manufacturer, these NPs are in fact ‘partially’ passivated with
oxygen. This process can leave behind a surface coating that
contains CuO and Cu2 O phases as discussed previously [16].
Additionally, over time this oxide layer can become even
thicker under ambient storage conditions [46]. However, here
it is noted that upon treatment, a complete oxidation of
the CuO is observed. It is possible that when the NPs are
sonicated, the increase in temperature and breaking of the
aggregates increases the exposed surface area, facilitating the
oxidation process. Dissolution studies indicated that uncoated
and Tween R 80 coated NPs showed similar extents of copper
dissolution (table 1). Coating with chitosan, however, resulted
in a two-fold increase in copper dissolution. Furthermore,
solutions containing Cu NPs + Ch were darker blue in
color than those containing Cu NPs, Cu NPs + Tw, or Cu
NPs + Tw + Ch, which is due to dissolved Cu2+ ions. These
characteristics suggest that chitosan enhances dissolution
when in direct contact with the Cu NP surface. Upon coating
with the Tween R 80, chitosan assisted dissolution is inhibited.
Therefore, Tween R acts as a protective coating that inhibits
nearly complete dissolution of Cu NPs. It should be noted that
because all of the particles examined have a significant oxide
component, as demonstrated in figure 2(c), the deliberate
coatings applied (i.e. Tween R 80 and chitosan) can be
considered independently with regard to the resulting toxicity
and inflammatory response.
After fully characterizing the physical properties of the
coated Cu NPs, the in vitro toxicity was analyzed in human
alveolar epithelial (A549) cells using a standard MTS cell
Figure 3. Toxicity of coated and uncoated Cu NPs to A549 cells.
Toxicity was measured relative to untreated cells of coated and
uncoated Cu NPs on A549 cells exposed for (a) 24 and (b) 52 h.
Statistical analysis was performed by a two-way analysis of
variance followed by Bonferroni post-tests. DHE oxidation levels
(c) of A549 cells after 24 h of exposure to coated and uncoated Cu
NPs. Statistical analysis was performed by a Kruskal–Wallis test
followed by Dunn’s post-tests. For all panels, the statistical
significance shown is relative to Cu NPs. Error bars represent the
standard error mean, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
viability assay. To confirm that the differences in toxicity
observed in A549 cells could be extrapolated to other cell
lines, the treatments were also tested in HEK-293 (human
embryonic kidney) cells. The cells were very sensitive to Cu
NPs and Cu NPs + Tw and less-so to their chitosan-coated
counterparts (Cu NPs + Tw + Ch) after 24 and 52 h
of exposure (figure 3(a)). Cells treated with 5 µg Cu
NPs + Tw + Ch were four times more viable after 24 h
and five times more viable after 52 h than those treated
4
Nanotechnology 24 (2013) 395101
K L S Worthington et al
Table 2. Characterization of lung tissue and fluid after nasal instillation of coated and uncoated Cu NPs. Except where noted, data were
obtained from the post-necropsy BAL fluid. Data are expressed as mean ± standard error.
Naı̈ve
Cu NPs
Cu NPs + Tw
Cu NPs + Tw + Ch
Cu in lung tissuea
(µg g−1 dry weight)
Cua (µg l−1 )
Total cells/mouse
(×10−3 )
Total protein
(µg ml−1 )
LDH activity
(U l−1 )
—
30.4 ± 5.3
27.9 ± 5.8
28.8 ± 6.6
—
36.4 ± 13.4
46.2 ± 13.7
88.5 ± 26.5
168 ± 15
765 ± 102
756 ± 89
1920 ± 777
63.6 ± 1.8
142 ± 19
172 ± 24
378 ± 111b
21 ± 4
31 ± 5
27 ± 7
73 ± 14c
a
Data were corrected for concentration of Cu in the naı̈ve group.
p = 0.063.
c p < 0.01, activity of LDH in BAL fluid was significantly higher in Cu NPs + Tw + Ch (one-way ANOVA followed
by Tukey test) compared to Cu NPs.
b
with 5 µg Cu NPs. The median lethal dose (LD50 ) of
Cu NPs + Tw + Ch was the highest of all treatments
for both exposure times, while the chitosan-coated Cu NPs
had the lowest LD50 values in each case. Similar results
were observed in the HEK-293 cells (data not shown). This
decrease in toxicity is also correlated with a two-fold decrease
in the cells’ generation of reactive oxygen species (figure 3(b))
when treated with Cu NPs + Tw + Ch compared to Cu
NPs. The presence of chitosan coating on Cu NPs apparently
lowers the environmental stress for cells, increasing their
viability relative to other Cu NP exposures. These dramatic
differences in dose-related toxicity in vitro demonstrate that
coating Cu NPs with chitosan protects exposed cells from Cu
NPs and significantly reduces their toxicity, thus increasing
their biocompatibility and perhaps suitability for various
biomedical applications.
The effect of chitosan coating of Cu NPs on the
inflammatory response upon nasal instillation was also
investigated. Twenty-four hours after nasal instillation of Cu
NPs, Cu NPs + Tw, or Cu NPs + Tw + Ch, exposed
mice had lost an average of 12% of their starting body
weight (average weight loss of 2.81 ± 0.41, 2.56 ± 0.32
and 2.59 ± 0.43 g, respectively). After necropsy, analyses of
cellularity and concentration of cytokines/chemokines in the
bronchoalveolar lavage (BAL) fluid and copper in the excised
lung tissue were performed.
The concentration of Cu (NPs and ions) deposited in the
lung tissue of all exposed animals (adjusted for the Cu in
control mice) was about 30 µg g−1 lung dry weight (table 2).
The concentration of Cu ions in the BAL fluid supernatants
was also measured. This analysis revealed a higher copper
ion concentration in the BAL fluid of lungs exposed to
chitosan-coated Cu NPs than the other treatments (table 2).
This result corresponds well with the increased dissolution of
copper from chitosan-coated Cu NPs ex vivo (table 1).
The total number of cells recovered in BAL fluid
(table 2) was the highest in the group of animals exposed to
chitosan-coated copper NPs (1920 ± 777 × 103 cells/mouse),
and while the number of cells from the animals exposed
to uncoated Cu NPs was much lower (765 ± 102 ×
103 cells/mouse), this difference was not shown to be
statistically significant. Similarly, the concentration of total
protein and activity of LDH in the supernatants of BAL
fluid were higher in the chitosan-coated Cu NP group
compared to the Cu NPs group (table 2, p < 0.01 and
Figure 4. Inflammatory response in the post-necropsy lung BAL
fluid 24 h after nasal instillation of coated and uncoated Cu NPs.
Differential cell counts (a) show the number of macrophages,
neutrophils and lymphocytes, while chemokine/cytokine analysis.
(b) shows the levels of selected inflammatory markers. Statistical
analyses were performed by Kruskal–Wallis tests for each cell type
or chemokine/cytokine followed by Dunn’s post-tests. Statistical
comparisons shown are relative to the naive group for each cell type
or inflammatory marker. The differences between all other groups
are not statistically significant. Error bars represent the standard
error mean, n = 5–7, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
p = 0.063, respectively). The number of neutrophils (key
inflammatory cells in the lung) in the BAL fluid (figure 4(a))
was 3.5 times higher in the chitosan-coated Cu NPs group
(1580 ± 799 × 103 cells/mouse) than the Cu NPs alone
group (456 ± 108 × 103 cells/mouse). Correspondingly, the
percentage of neutrophils in the BAL fluid was highest in the
chitosan-coated Cu NPs group followed by Cu NPs alone and
Cu NPs + Tw groups (71%, 56% and 51%, respectively).
Out of six selected inflammatory cytokines/chemokines
that were analyzed in the supernatants of BAL fluid
(figure 4(b)), IL-6 and KC had higher levels in chitosan-coated
5
Nanotechnology 24 (2013) 395101
K L S Worthington et al
in toxicity in vitro and in vivo in the lung, chitosan-coated
Cu NPs for the controlled release of copper ions in the
treatment of copper deficiency diseases such as Menkes
disease could still be beneficial, if a more appropriate route of
administration could be identified. Although chitosan coating
was, in fact, shown to increase the inflammatory response
of Cu NPs administered via nasal instillation, it was also
shown to dramatically decrease toxicity in vitro. Thus, this
study demonstrates the importance of not only a thorough
analysis of NP physical properties, but also a careful analysis
of toxicity, especially regarding making inferences about in
vivo exposures and effects based on in vitro studies.
Cu NPs than uncoated Cu NPs, corresponding well with the
higher neutrophil counts in figure 4(a). The concentration
of IFN-γ and TNF-α were below the lower limit of
detection (LLOD, 1.03 and 2.02 pg ml−1 , respectively) in
all experimental groups. These in vivo results show that
chitosan-coated Cu NPs elicit higher pulmonary inflammatory
responses than their uncoated counterparts. The higher
response is demonstrated by the increased total cell and
neutrophil numbers in the BAL fluid as well as increased
concentrations of inflammatory cytokine/chemokines (IL-6
and KC) in mice exposed to Cu NPs + Tw + Ch as
opposed to Cu NPs and Cu NPs + Tw. Likewise, pulmonary
cytotoxicity represented by increased activity of LDH in the
BAL fluid was higher in groups exposed to chitosan-coated
Cu NPs. Chitosan-coated Cu NPs are more likely to have
a high residence time in the lungs due to the well-known
mucoadhesive properties of chitosan [47, 48]. Chitosan has
been shown, in fact to enhance pulmonary delivery of
calcitonin from PLGA nanospheres by this phenomenon
coupled with the opening of tight junctions [48]. The inability
to clear these coated NPs causes a higher concentration of
copper (from NPs and ions) to remain stagnant during the
first 24 h after exposure, eliciting a higher inflammatory
response than non-mucoadhesive Cu NPs that can be cleared
relatively quickly from the lungs. Furthermore, chitosan has
been shown to have a powerful effect on the tight junctions
and permeability of mucus-rich epithelial cells [49]; an effect
that is enhanced when the chitosan is in NP form [50, 51].
3. Materials and methods
3.1. Reagents
Cu NPs (25 nm, partially passivated with 10% oxygen,
Nanostructured and Amorphous Materials, Inc., Houston,
TX), Tween R 80 (Fisher Scientific, Hampton, NH), sodium
bicarbonate (Sigma-Aldrich, St Louis, MO), ethylenediaminetetraacetic disodium salt (EDTA, Fischer Scientific),
and sodium dodecyl sulfate (SDS, Research Products
International Corporation, Mount Prospect, IL) were used as
received.
Chitosan (2 g, low molecular weight, 96.1% deacetylation, 1% w/v in 1% v/v acetic acid 35 cps, Sigma-Aldrich)
was dissolved in dilute acetic acid (200 ml, 1% v/v), vacuum
filtered, and precipitated using 1 N sodium hydroxide. The
precipitate was then mixed with 500 ml of purification
buffer (0.1 M sodium bicarbonate, 20 mM EDTA, 0.5% w/v
SDS) for 30 min, filtered, rinsed, and dialyzed using a
Snakeskin R dialysis tube (MWCO 10 000) against nanopure
water for two days (water was replaced approximately every
12 h). After dialysis, the chitosan was vacuum filtered again,
resuspended in a small amount of nanopure water, frozen
overnight at −20 ◦ C, and lyophilized.
2. Conclusions
The addition of a chitosan coating to Cu NPs changed physical
properties and toxicity in vitro and in vivo. The size of
the NPs increased by roughly 50%, but the morphology
of the chitosan-coated particles was smooth and round, in
contrast to aggregated and rough Tween R 80 coated Cu
NPs. The presence of chitosan coating was confirmed using
fluorescent imaging of Cu NPs coated with Rhodamine B
conjugated chitosan. The presence of cationic chitosan on the
particle surface also caused the particle charge to increase
significantly. XPS analysis demonstrated the presence of
chitosan on the surface of the coated NPs via the clearly
detectable presence of nitrogen. The in vitro toxicity of
chitosan-coated Cu NPs was significantly lower than uncoated
Cu NPs for two different cell types, two time points, and a
range of doses. LD50 values were highest for chitosan-coated
NPs and lowest for Cu NPs + Tw, indicating that coating
with chitosan protects cells in culture from the toxic effects
of Cu NPs. Conversely, an increase in inflammatory response
was observed for mice exposed to chitosan-coated Cu NPs
versus uncoated Cu NPs. These results suggest that coating
metal NPs with mucoadhesive polysaccharides (e.g. chitosan)
decreases their ability to be cleared from the lungs, prolonging
the exposure of cells and tissue to toxic metal oxides and
producing a dramatic acute inflammatory response. In future
studies, coating of Cu NPs with chitosan and its effect on
toxicity should be evaluated by intramuscular, intravenous and
subcutaneous routes of administration. Despite the differences
3.2. Cu NP coating
Cu NPs (50 mg) were placed in a 20 ml glass vial and
suspended in 10 ml of a dilute solution of Tween R 80
(5 mg ml−1 nanopure water) by mixing overnight with a
magnetic stir bar. The resulting solution was then dialyzed
(Snakeskin R dialysis tube, MWCO 3500) against nanopure
water for 6 h (water was replaced approximately every
90 min). Meanwhile, a solution of chitosan was prepared by
mixing chitosan (50 mg) with 1% v/v acetic acid (10 ml)
to homogeneity, adjusting the pH to ∼5.8 with 1 N sodium
hydroxide, and then adding acetate buffer (50 mM, pH 5.5)
to a final volume of 20 ml. To accomplish the final chitosan
coating, 1 ml of dialyzed particles was mixed with 9 ml of
the chitosan solution overnight. The finished particles were
then centrifuged at 4000 rpm for 15 min, the supernatant was
decanted, and the particles were resuspended to the desired
concentration with nanopure water.
6
Nanotechnology 24 (2013) 395101
K L S Worthington et al
in 0.1 M 2-(N-morpholino)ethanesulfonic (MES) buffer
(1 ml) with the pH adjusted to 6. After mixing for two
hours, this solution (1 ml) was added to a 5 mg ml−1
chitosan solution (9 ml), prepared as described previously,
and allowed to react for six hours. The resulting product
was dialyzed (Snakeskin R dialysis tube, MWCO 10 000)
against nanopure water for two days and lyophilized. The
rhodamine conjugated chitosan was used to coat Cu NPs
using the same method as described above. For imaging,
two coverslips were placed on a glass slide spaced about
3 cm apart to form a thin chamber. A drop of copper
particles coated with fluorescent-labeled chitosan was added
between coverslips. Another coverslip was placed on top of
this chamber to facilitate uniform thickness and to spread
the drop. The arrangement was sealed using clear fingernail
polish. Images were acquired using a Bio-Rad Radiance 2100
multi-photon microscope (Bio-Rad Laboratories, Hercules,
CA). The images were processed using ImageJ (Image
Processing and Analysis in Java, Version 1.46b).
3.3. Particle size analysis
The size of coated and uncoated Cu NPs was measured by
JEOL JEM-1230 transmission electron microscope equipped
with a Gatan UltraScan 1000 2k × 2k CCD acquisition
system. A small drop (10 µl) of sample solution was
left on a 400-mesh TEM copper grid that was pre-coated
with a Formvar R 0.5% solution in ethylene dichloride
film (Electron Microscopy Sciences, Hatfield, PA) for
2 min. Whatman R filter paper was then used to remove
any excess liquid and the grid was air dried. The TEM
images were processed using ImageJ (Image Processing and
Analysis in Java, Version 1.46b). The hydrodynamic diameter
(number-weighted) and surface charge of particles in solution
were measured in distilled water at 25 ◦ C using dynamic
light scattering (Zeta Sizer Nano ZS, Malvern Instruments,
Southborough, MA).
3.4. Composition analysis
X-ray diffraction (XRD) patterns were collected using a
Rigaku Miniflex II Diffractometer with a Co source. XRD
analysis was conducted according to the following protocol.
The solutions containing Cu NPs, Cu NPs + Tw and Cu
NPs + Tw + Ch were centrifuged at 22 000 rpm for 1 h in air
tight centrifuge vials and the supernatant carefully removed,
leaving ∼200 µl in the vial. Then using a micropipette,
the solid was mixed well with the remaining solution and
placed on the XRD slides, dried overnight in a dessicator, and
diffraction patterns were collected. The unprocessed sample
of Cu NPs was also analyzed to investigate the effects of
oxidation during the treatment process.
3.6. Dissolution of the coated and uncoated particles
The dissolution studies were conducted using a Varian inductively coupled plasma optical emission spectrophotometer
(ICP-OES). The experiments were conducted according to
the following protocol. Cu NPs, Cu NPs + Tw and Cu
NPs + Tw + Ch solutions were sonicated for 5 min and
a 1.5 ml aliquot was filtered using 0.2 µm filters (Xpertec)
into centrifuge vials. The filtered samples were centrifuged
at 22 000 rpm for 45 min to ensure maximum removal of
any remaining Cu NPs in the filtered solutions. From the
supernatant 0.5 ml was carefully transferred into 4.5 ml of 2%
HNO3 followed by the analysis by ICP-OES with a calibration
curve of 5, 10, 25 and 50 ppm. A total of five replicates were
conducted for each sample.
3.5. Surface analysis
The surface functionality of the coated Cu NPs was analyzed
using x-ray photoelectron spectroscopy (XPS, Axis Ultra
XPS, Kratos, Chestnut Ridge, NY) and confocal microscopy.
For XPS analysis, sample preparation followed a standard
procedure. Briefly, 20 µl droplets of each solution were
carefully pipetted onto a small square of clean, heavy
duty aluminum foil. The droplets were allowed to dry,
and then the resulting spot was analyzed using XPS. The
resulting data were calibrated and analyzed using XPS
software (CasaXPS 2.3.15, Casa Software Ltd, Estepona,
Spain). The area under the curve for each atomic signal
was calculated using an ‘area under the curve’ function
of another data analysis software (GraphPad Prism version
5.02 for Windows, GraphPad Software, San Diego, CA)
and used to estimate ratios of the number of atoms on
the surface of the particles. The confocal microscopic
analysis was conducted using rhodamine conjugated chitosan
prepared according to the following protocol. A 5 mg ml−1
chitosan solution was prepared as previously described.
100 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
HCl (EDC, Thermo Scientific, Rockford, IL), 50 mg
N-hydroxysulfosuccinimide (sulfo-NHS, Thermo Scientific),
and 5 mg Rhodamine B (Sigma-Aldrich) were dissolved
3.7. Cytotoxicity analysis in vitro
The cytotoxicity of chitosan-coated Cu NPs was determined
using an MTS assay. A549 (adenocarcinomic human alveolar
basal epithelial) cells were seeded at a density of 1 × 104
cells/well in RPMI-1640 medium (Gibco R , Life Technologies
Corporations, NY) at 37 ◦ C and 5% CO2 in a 96-well plate.
The media were supplemented with 10% fetal bovine serum
(FBS, Atlanta Biologicals, GA), 10 mM HEPES (Gibco R ),
50 µg ml−1 gentamicin sulfate (Cellgro, VA), 1 mM sodium
pyruvate (Gibco R ), and 1 mM GlutamaxTM (Gibco R ).
After 24 h, the medium was discarded and replaced with
100 µl fresh media in each well. Various concentrations
(0.01–0.09 µg ml−1 ) of each treatment in phosphate buffered
saline (PBS, Invitrogen) were then added to each well at a
volume of 100 µl. The cells were incubated for either 24
or 52 h, the treatment removed, and fresh media (100 µl)
added. 20 µl of reagent (CellTiter 96 R AQueous One Solution
Cell Proliferation Assay, Promega Corporation, Madison,
WI), which changes from MTS tetrazolium to formazan
in proportion to the number of live cells present, was
added to each well. The cells were incubated between 1
7
Nanotechnology 24 (2013) 395101
K L S Worthington et al
and 4 h, then the formazan product was quantified by
spectrophotometric analysis (SpectraMax Plus384, Molecular
Devices, Sunnyvale, CA) using the absorbance at 490 nm,
according to the company protocol.
Reactive oxygen species (ROS) production was estimated
by measuring the oxidation of dihydroethidium (DHE, Molecular Probes, Eugene, Oregon), which becomes fluorescent
upon oxidation with superoxide. A549 cells (2–4 × 105
cells per 60 mm Petri dish) were cultured 24 h, exposed
to one of the three NP treatments (2 ml at 50 µg Cu NPs
per ml) for 24 h, and then tested for DHE oxidation as
described previously [52]. Briefly, the cells were washed
once with PBS and labeled at 37 ◦ C for 40 min in PBS
containing 5 mM pyruvate with DHE (10 µM; in 1% DMSO).
Culture plates were placed on ice to stop the labeling,
trypsinized, and resuspended in ice-cold PBS. Samples
were analyzed using a FACScan flow cytometer (Becton
Dickinson Immunocytometry Systems, Inc., Mountain View,
CA; excitation 488 nm, emission 585 nm band-pass filters).
The mean fluorescence intensity (MFI) of 10 000 cells was
analyzed in each sample and corrected for auto-fluorescence
from unlabeled cells. The MFI data were normalized to levels
from A549 cells treated with PBS only.
with no exposure) served as a control group. All mice
were euthanized 24 h postexposure by overdose inhalation
of isoflurane, cervical dislocation and exsanguinations, after
which bronchoalveolar lavage (BAL) fluid and lung tissues
were collected.
3.10. Bronchoalveolar lavage (BAL) fluid
The lungs from six mice in each group were lavaged in situ
three times with approximately 1 ml of sterile saline (0.9%
sodium chloride solution, Baxter, Deerfield, IL, USA). The
lavage fluid was centrifuged (800g for 5 min at 4 ◦ C), the total
white blood cells were counted using a hemocytometer and
the supernatants were stored at −80 ◦ C for later analyses. For
differential cell counts, resuspended cells in Hank’s balanced
salt solution and fetal calf serum, were spun (800g, 3 min,
Cytospin 4, Thermo Shandon, Thermo Scientific, Waltham,
MA, USA) onto microscope slides and air dried. The cells
were then stained using HEMA 3 R stain set (PROTOCOL R ,
Fisher Scientific Company LLC, Kalamazoo, MI) and the
number of macrophages, neutrophils and lymphocytes (total
of 400 total cells per each animal) were counted.
A Bradford protein assay (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) was used to measure total protein
levels in BAL fluid supernatants and lactate dehydrogenase
(LDH) activity was determined by a Cytotoxicity Detection
Kit (Roche Diagnostics, Penzberg, Germany). Concentrations
of selected inflammatory cytokines/chemokines (tumor
necrosis factor [TNF]-α, interferon [IFN]-γ , interleukin
[IL]-6, IL-12(p40), keratinocyte-derived cytokine [KC], and
macrophage inflammatory protein [MIP]-1α) were measured
in the BAL fluid using a multiplexed fluorescent bead-based
immunoassay (Bio-Rad Laboratories, Inc., Hercules, CA).
The lowest limit of detection (LLOD) for cytokine assays
was calculated by dividing the lowest detected point on the
√
standard curve by 2. (IL-6 = 0.52, IL-12(p40) = 0.72,
IFN-γ = 1.03, KC = 1.42, MIP-1α = 1.47 and TNF-α =
2.02 pg ml−1 .)
3.8. Animal models
An inflammation mouse model was used in these studies.
Mice (C57Bl/6, males, six weeks old) were purchased from
The Jackson Laboratory (Bar Harbor, ME). The instillation
protocol was approved by the Institutional Animal Care and
Use Committee and complied with the NIH Guidelines.
Animals were housed in a vivarium in polypropylene,
fiber-covered cages in HEPA-filtered Thoren caging units
(Hazelton, PA, USA) in the Pulmonary Toxicology Facility
at the University of Iowa. They were acclimatized for
ten days prior to the exposures. Food (sterile Teklad 5%
stock diet, Harlan, Madison, WI, USA) and water (via an
automated watering system) was provided ad libitum and a
12-h light–dark cycle was maintained in the animal room.
3.9. Nasal instillation exposure
3.11. Copper analysis in exposure solutions, lung tissue, and
BAL fluid
Animals in each experimental group (n = 6): Cu NPs, Cu
NPs + Tw, and Cu NPs + Tw + Ch, were exposed to
tested material by nasal instillation. Each mouse was exposed
twice with 50 µl (Cu NPs and Cu NPs + Tw groups)
and with 100 µl (Cu NPs + Tw + Ch) of exposure
solution with a 1 h interval between each dosing. The total
dose of Cu NPs in each experimental group, as measured
by inductively coupled plasma-mass spectrometry (ICP-MS,
X Series, Thermo Scientific, Waltham, MA, USA) was
approximately 30 µg/mouse (33.5, 34.3, and 29.0 µg/mouse
in Cu NPs, Cu NPs + Tw, and Cu NPs + Tw + Ch group,
respectively). Suspensions of tested materials were prepared,
as described above and vortexed immediately before the
instillation exposure. Nasal instillation was conducted under
anesthesia by inhalation of isoflurane (3%) using a precision
Fortec vaporizer (Cyprane, Keighley, UK). Naı̈ve mice (mice
The lungs from mice in each group were harvested at
the time of necropsy and stored at −80 ◦ C. Tissues were
dried using a freeze dryer for approximately 16 h at 1.3 ×
10−1 mBar and −50 ◦ C (Labconco Corp., Kansas City,
MO, USA) and subsequently weighed. Dried lungs were
placed in 50 ml digestion tubes and high purity nitric
acid and hydrochloric acid were added (Optima grade,
Fisher Scientific, Pittsburgh, PA, USA). Tissues were then
digested in 36-well HotBlockTM (Environmental Express, Mt.
Pleasant, SC, USA) at 95 ◦ C for 6 h. The concentration
of Cu ions was determined by inductively coupled plasmamass spectrometry (ICP-MS, X Series, Thermo Scientific,
Waltham, MA, USA). Each sample was spiked with an
internal standard at 20 µg l−1 .
8
Nanotechnology 24 (2013) 395101
K L S Worthington et al
3.12. Statistical analysis
[8]
The results were analyzed using the statistical analysis
package included in the analytical software (GraphPad Prism
5.02). For the in vitro results, a two-way analysis of variance
(ANOVA) was performed, followed by Bonferroni post-tests
between each group. When the data could not be assumed
to be normal in distribution (ROS and in vivo results), a
Kruskal–Wallis one-way analysis of variance on ranks test
was performed, followed by Dunn’s multiple comparison
tests. Data are expressed as a mean ± standard error. P-values
less than 0.05 were considered significant.
[9]
[10]
[11]
[12]
Acknowledgments
[13]
The authors gratefully acknowledge support from the
Environmental Health Sciences Research Center (NIH
P30 ES005605). Other sources of support include the
American Cancer Society (RSG-09-015-01-CDD), the National Cancer Institute at the National Institutes of Health
(1R21CA13345-01/1R21CA128414-01A2/UI Mayo Clinic
Lymphoma SPORE), and the National Science Foundation
(CBET-0933450). K Worthington thanks the University of
Iowa Graduate College for additional support. The authors
would also like to gratefully acknowledge Dr David Peate
for his generosity with ICP-MS, the Central Microscopy
Research Facility at the University of Iowa for the use
of TEM, confocal microscopy, and XPS equipment, Jonas
Baltrusaitis for technical support in performing the XPS
analyses, and the Radiation and Free Radical Research Core
Lab and P30-CA086862 for technical support in measuring
DHE oxidation.
[14]
[15]
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[17]
[18]
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