the health implication and application of silver and iron
TRANSCRIPT
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Theses and Dissertations
Summer 2019
The Health Implication and Application of Silver and Iron Oxide The Health Implication and Application of Silver and Iron Oxide
Nanoparticles Nanoparticles
Sahar Pourhoseini
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The Health Implication and Application of Silver and Iron Oxide Nanoparticles
by
Sahar Pourhoseini
Bachelor of Science
Azad University of Mashhad, 2005
Master of Science
Islamic Azad University of Tehran, Science and Research Branch, 2012
Submitted in Partial Fulfillment of the Requirements
For the Degree of Doctor of Philosophy in
Environmental Health Sciences
The Norman J. Arnold School of Public Health
University of South Carolina
2019
Accepted by:
Jamie R. Lead, Major Professor
Alan W. Decho, Committee Member
E. Angela Murphy, Committee Member
Eric P. Vejerano, Committee Member
Cheryl L. Addy, Vice Provost and Dean of the Graduate School
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© Copyright by Sahar Pourhoseini 2019
All Rights Reserved.
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DEDICATION
This dissertation research and doctoral degree is dedicated to my beloved parents
and family for endless support and encouragement, to my husband for his love and constant
support, to my friends and to everyone who has helped me achieve this goal.
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ACKNOWLEDGEMENTS
I would like to express my deepest appreciation to my advisor, Dr. Jamie Lead for
the continuous support, guidance and patience. As my teacher and mentor, you have taught
me more than I could give credit for here. I would like to acknowledge my supportive
dissertation committee members, Dr. Angela Murphy, Dr. Alan Decho and Dr. Eric
Vejerano, whose advice and guidance were invaluable. Your time and efforts are really
appreciated. I also thank Dr. Geoff Scott and Dr. Dwayne Porter for their support during
my PhD. Special thanks to Dr. Reilly Enos and Dr. Kandy Velázquez for their continuous
support, guidance and encouragement. I am grateful to Elizabeth Caulder for endless
support and providing me personal and professional guidance, and to my colleagues for
encouragement and collaboration. The author thanks the SmartState Center for
Environmental Nanoscience and Risk (CENR) for financial support. This work was
partially supported by a SPARC Graduate Research Grant from the office of the Vice
President for Research at the University of South Carolina and by the National Science
Foundation (NSF).
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ABSTRACT
Nanoscience and nanotechnology have successfully created different
nanomaterials, which are defined as materials with at least one dimension between 1-100
nm. This small size gives nanomaterials unique and novel properties compared with bulk
material, such as a high surface area, specific mechanical, optical and magnetic properties.
These novel properties have stimulated the interest of using nanoparticles in a wide range
of consumer products and applications including biomedical, pharmaceutical and drug
delivery. Silver and iron oxide nanoparticles attracted more attention commercially due to
their antibacterial and magnetic properties, respectively. So, the overall objective of this
dissertation was: 1) to examine the implication of well-characterized polyvinylpyrrolidone-
coated silver nanoparticles (PVP-AgNPs) on human peripheral blood mononuclear cells
(PBMCs) at environmentally relevant concentrations, and 2) to investigate the application
of iron oxide nanoparticles in fat removal using high-fat diet-fed mice.
AgNPs biological behavior, such as bio-uptake and accumulation, may be affected
by their novel properties and they might show different behavior than bulk material and
free Ag ions. There is discrepancy in the results as to whether Ag toxicity is caused by
nanoparticulate or ions from NP dissolution. In most nanotoxicology studies lack of NP
characterization in the relevant exposure media biased the NP dose and toxicity outcome.
This study aimed to use in house synthesized and well- characterized PVP-coated AgNPs
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for investigation of their bio-uptake and toxicity in PBMCs. Synthesis protocol was
successful in generating spherical and monodisperse PVP-coated AgNPs. PVP-AgNPs
showed more stability in the cell exposure media and no aggregation was observed at any
exposed concentrations. AgNPs and silver nitrate (which was used as Ag ion control)
indicated the same overall toxicity. Although, after normalizing toxicity to the amount of
Ag that was taken up by cells, AgNPs seemed to be more toxic to cells than Ag ions. Our
results emphasize the importance of sufficient characterization of nanoparticles in proper
exposure media over the exposure time to have a better understanding of nanoparticle
behavior and interaction in biological systems for evaluating risk assessment in human
exposure.
Iron oxide NPs which are frequently used in nanomedicine, have recently been
investigated for oil removal purposes [1]. However, in vitro and in vivo studies on iron
oxide NPs toxicity have revealed little consistency of result due to the usage of different
NP characterizations and concentrations. In this study we used PVP-coated iron oxide NPs
(that were used successfully before for oil removal from aqueous solutions) to remove
dietary fat from mice fed with a high-fat diet. Our animal data demonstrated that oral
exposure of PVP-coated iron oxide nanoparticles at concentrations up to 20 mg/kg mouse
body weight did not cause any significant decrease in the body weight and fat percentage
of high-fat diet-fed mice. Although, iron uptake by liver cells and some effects on glucose
metabolism in HNP treatment was detected compared to controls. No increase in
inflammatory markers were observed using high doses of nanoparticle treated mice
compared with high-fat diet. Further studies using different concentrations of NPs with
modified surface coatings should be performed to validate our findings. Given the
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increasing demand for obesity treatments, nanomedicine approaches, coupled with our
results, may provide substantial contributions toward progress in this area.
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TABLE OF CONTENTS
Dedication .......................................................................................................................... iii
Acknowledgements ............................................................................................................ iv
Abstract ................................................................................................................................v
List of Tables ..................................................................................................................... ix
List of Figures ......................................................................................................................x
List of Abbreviations ....................................................................................................... xiii
Chapter 1: Introduction ........................................................................................................1
Chapter 2: Uptake and toxicity of silver nanoparticles and silver ions to human peripheral
blood mononuclear cells ....................................................................................................13
Chapter 3: Can PVP-coated iron oxide nanoparticles be used to prevent weight gain in a
high-fat diet mouse model .................................................................................................56
Chapter 4: Overall conclusion ...........................................................................................87
References ..........................................................................................................................91
ix
LIST OF TABLES
Table 2.1 List of chemicals used in NP synthesis. Chemicals used in core-shell
(Ag@Au@Ag) and PVP-coated AgNPs synthesis ............................................................38
Table 2.2 Summary of Characterization of core-shell NPs (Ag@Au@Ag).
Characterization of core-shell NP synthesis in each step using DLS and UV-Vis ............39
Table 2.3 AgNP characterization results. Characterization of PVP-AgNPs in UHPW. DLS
and TEM size are reported as mean ± standard deviation. ................................................40
Table 2.4 Mass balance of Ag in the exposure. Ag mass balance in the supernatant and cell
in AgNP and AgNO3 treatment measured by ICP-MS. Cell data were collected after three
washes with PBS and shows internalized and strongly bound Ag. Total loss indicates loss
of Ag during washes or experimental process. Ratio of cell to supernatant in percent for
Ag uptake ...........................................................................................................................41
Table 2.5 Mass balance of Ag in the exposure for each person. Ag mass balance (ng) in
2.5×105 cells (absorbed or cell surface-attached) in AgNP and AgNO3 treatments for each
of 6-individuals measured by ICP-MS. Data are presented as mean± standard error (n=3).
............................................................................................................................................42
x
LIST OF FIGURES
Figure 1.1 Routes of human exposure to Ag. The extensive use of nanosilver in consumer
products introduces multiple ways for human exposure................................................... 12
Figure 2.1 Progressive formation of Ag@Au and Ag@Au@Ag NPs using UV-Vis. a)
Addition of Au in solution to AgNPs to form Ag@Au NPs. As Au is added to the AgNP,
the Ag peak shift to higher wavelength that shows formation of Au layer. b) Formation of
Ag@Au@Ag by adding Ag to Ag@Au NPs. Progression of wavelength to lower numbers
as being coated by Ag. .......................................................................................................44
Figure 2.2 AgNP characterization using DLS, UV-Vis, TEM and EDX. Characterization
of stock PVP-AgNPs using DLS (a), UV-Vis spectra (b), TEM images with different
magnifications (c and d), and EDX imaging (e). ...............................................................45
Figure 2.3 STEM images of 1000 µg L-1 PVP-AgNPs and particle size distribution obtained
by ImageJ. PVP-AgNP in RPMI media (without cells) at (a and b) T= 0, size distribution=
29 ± 0.49 nm and (c and d) T= 24 hours, size distribution= 17 ± 11 nm in the same condition
as cell exposure (5% CO2 and 37°C incubator). ...............................................................46
Figure 2.4 Behavior of PVP-AgNPs in media during 24-hour exposure. UV-Vis spectra of
PVP-AgNPs in RPMI media in (a) 1000, (b) 500 and (c) 100 µg L-1 PVP-AgNP
concentration as a function of time. The lowest concentration of exposure (10 µg L-1 PVP-
AgNP) did not show any signal due to detection limit of UV-Vis ....................................47
Figure 2.5 Dissolved and total Ag concentrations in PVP-AgNPs vs. AgNO3 during
exposure time. Dissolved Ag concentration measurements using centrifugal ultrafiltration
units (a and c), and total Ag concentration (b and d) in RPMI media at T=0 and T=24 hour
measured by ICP-MS. All experiments were performed in triplicate and are reported as
mean ± SE (n=3) ................................................................................................................48
Figure 2.6 Dissolved and total concentrations of Ag in AgNO3 in UHPW and media.
Dissolved Ag concentrations were measured using centrifugal ultrafiltration units (a and c)
and total Ag concentration (b and d) in UHPW and RPMI media at T=0 and T=24 hours
were measured by ICP-MS. Data are reported as mean ± SE (n=3) ..................................49
Figure 2.7 Ag uptake (ng) in 2.5×105 cells. Results of absorbed or cell surface-attached
Ag in PVP-AgNP and AgNO3 treatments measured by ICP-MS after 24-hour exposure.
Data are presented as mean ± SE (n=6). * statistically different from 10 µg L-1, ^
statistically different from 100 µg L-1, # statistically different from 500 µg L-1, and ¥
statistically different when compared with AgNP treatment ................................................. 50
xi
Figure 2.8 Ag uptake (ng) in 2.5×105 cells for each person. Results for absorbed or cell
surface-attached Ag after treatment of PBMCs in 6 individuals. The amount of Ag in cells
after 3 washes with buffer that was measured by ICP-MS ................................................51
Figure 2.9 Effects of PVP-AgNP, AgNO3, AgNO3-PVP, and PVP on PBMCs. PBMCs
were exposed for 24 hours to different concentrations of PVP-AgNP, AgNO3, AgNO3-
PVP, and PVP (0-1000 µg L-1) in RPMI. The effect on cell viability was measured using
LDH leakage (a). Cell metabolic activity was assessed by MTS assay (b) and results is
expressed as percentage of reduction values of untreated cells. Data are presented as mean
± SE as the average of six independent experiments (n=6). * shows statistically significant
difference from the control (0), and # shows statistically significant difference when
compared with PVP-AgNP (p < 0.05) ...............................................................................52
Figure 2.10 Cell viability results for each person. Cell viability of PBMCs assessed by
LDH leakage after 24 hours treatment with representative concentration of PVP-AgNP,
AgNO3, AgNO3-PVP, and PVP. Data represents the means of six replicates ± standard
error ....................................................................................................................................53
Figure 2.11 Cell metabolic activity results for each person. Metabolic activity of PBMCs
measured by MTS assay after 24 hours exposure at the representative concentrations for 6
individuals. Data represents the means of six replicates ± standard error .........................54
Figure 2.12 Pearson correlation between uptake and toxicity of Ag. Correlation between
uptake and toxicity of Ag in AgNP (a) and AgNO3 (b and c) treatment. There was a
moderate positive correlation between the two variables (uptake and toxicity) for AgNPs:
r = 0.56, p = 0.01 (a) and AgNO3 excluding person 5: r = 0.52, p = 0.04 (b). No correlation
was detected in AgNO3 including person 5: r = 0.314, p = 0.2 (c) ....................................55
Figure 3.1 Schematic diagram of flow-through synthesis of PVP-coated iron oxide NPs
(figure not to scale). Adopted from Kadel et al. 2018 ......................................................78
Figure 3.2 Size characterization of iron oxide NP. DLS size distribution plot indicated a
mean size of 116 ± 4.9 nm .................................................................................................79
Figure 3.3 Fluorescence spectra for the seawater before and after oil removal using iron
oxide NPs. Oil removal efficiency was calculated as 94% from synthetic seawater ........80
Figure 3.4 Fluorescence spectra of oil removal from stomach fluid using iron oxide NPs.
Oil was removed from stomach solution after 1-hour separation time using a magnet.
Percent oil removal was calculated as: canola oil: 85%, vegetable oil: 85% and olive oil:
89%. ...................................................................................................................................81
Figure 3.5 Body morphology and food intake after 16 weeks of treatment. Body weight,
food intake and fat pad weights after 16 weeks of treatment (low-fat diet (LFD), high-fat
diet (HFD), low nanoparticle (LNP), medium nanoparticle (MNP) and high nanoparticle.
Mice were fed for 16 weeks. a) body weight; b) food intake; c) fat pad weight; and d) body
composition measured by dual-energy X-ray absorptiometry (DEXA). Data are presented
xii
as means ± SE; n =10 mice per group. Bar graphs not sharing a common letter are
significantly different (p < 0.05) ........................................................................................82
Figure 3.6 Metabolic data after 16 weeks of treatment. Mice were administered with (low-
fat diet (LFD), high-fat diet (HFD), low nanoparticle (LNP), medium nanoparticle (MNP)
and high nanoparticle for 16 weeks. a) fasting blood glucose levels (5h); b) glucose
tolerance test (GTT); and c) corresponding area under the curve (AUC). Values are
presented as mean ± SE; n =10 mice per group. Bar graphs not sharing a common letter
differ significantly from each other (p < 0.05 ....................................................................83
Figure 3.7 Iron measurement in feces and tissues using ICP-MS after 16 weeks of dietary
treatment. Iron measurement after 16 weeks of treatment ((low-fat diet (LFD), high-fat diet
(HFD), low nanoparticle (LNP), medium nanoparticle (MNP) and high nanoparticle
(HNP), in a) feces and b) brain, heart, kidney and liver. Values are presented as mean ±
SE; n= 4 mice per group. Bar graphs not sharing a common letter differ significantly (p <
0.05) ...................................................................................................................................84
Figure 3.8 Hepatic lipid accumulation and inflammatory mediators. a) liver weight after
euthanasia, low-fat diet (LFD), high-fat diet (HFD), low nanoparticle (LNP), medium
nanoparticle (MNP) and high nanoparticle (HNP); b) hepatic lipid accumulation; c) hepatic
gene expression of EMR1; gene expression of hepatic inflammatory mediators: MCP-1
(monocyte chemoattractant protein-1, TNF-α (tumor necrosis factor-α) and IL-6
(Interleukin-6) normalized to the HMBS and H2AFV. Data are presented as mean ± SE; n
= 10 mice per group. Bar graphs not sharing a common letter are significantly different (p
< 0.05) ................................................................................................................................85
Figure 3.9 Mice diet. Mice diet modified as: Fat (40% kcal); SF (12% kcal), USF (28%
kcal), a) high nanoparticle; b) medium nanoparticles; c) low nanoparticles. ....................86
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LIST OF ABBREVIATIONS
Ag ................................................................................................................................. Silver
Ag@Au ......................................................................... Silver core-gold shell nanoparticles
AgNO3............................................................................................................... Silver nitrate
AgNPs ................................................................................................... Silver Nanoparticles
Au .................................................................................................... Aurum (Latin), for gold
DLS ................................................................................................ Dynamic light scattering
EDX ................................................................................................ Energy dispersive x-ray
EMR1 ............................ EGF-like module-containing mucin-like hormone receptor-like 1
ENP .................................................................................................Engineered nanoparticle
FBS ......................................................................................................... Fetal bovine serum
HFD................................................................................................................... High-fat diet
HNO3.....................................................................................................................Nitric acid
HNP........................................................................................................... High nanoparticle
Human PBMCs ................................................ Human peripheral blood mononuclear cells
ICP-MS ...................................................... Inductively coupled plasma mass spectrometry
ICP-OES .................................. Inductively coupled plasma optical emission spectrometry
IL-6 ................................................................................................................... Interleukin-6
LFD .................................................................................................................... Low-fat diet
LNP ........................................................................................................... Low nanoparticle
MCP-1 ......................................................................... Monocyte chemoattractant protein-1
MNP .................................................................................................... Medium nanoparticle
xiv
NaBH4 ...................................................................................................Sodium borohydride
NPs .................................................................................................................. Nanoparticles
PBS .............................................................................................. Phosphate buffered saline
PDI ........................................................................................................ Polydispersity index
pH .................................................................... Potential of hydrogen, a measure for acidity
PVP ..................................................................................................... Polyvinylpyrrolidone
rpm .........................................................................................................Rotation per minute
STEM ............................................................... Scanning transmission electron microscopy
TEM ............................................................................... Transmission electron microscopy
TNF-α.............................................................................................. Tumor necrosis factor-α
UHPW ................................................................................................. Ultra-high pure water
UV-Vis ................................................................................ Ultraviolet visible spectrometry
1
CHAPTER 1
INTRODUCTION
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Nanoscience, nanotechnology and nanoparticles
Nanoscience, which refers to the study and manipulation of structures with at least
one dimension between 1-100 nm (according to definition of the European Union [2], the
American Society for Testing Materials [3] and the Scientific Committee on Emerging and
Newly-Identified Health Risks [4]), and nanotechnology which is a multidisciplinary field
that refers to the application of nanoscale material in the products, have been created
different types of nanomaterials (NMs). The properties of these material, such as electrical,
thermal, mechanical, magnetic and optical, change as their size shifts to the nanoscale and
this stimulates the increasing interest in the NM [5]. For example gold will show higher
reactive properties in the nanosized scale than bulk material [6] and the yellow color of
bulk gold turns into red when it approaches nano size [6, 7]. The difference between bulk
and nanosized properties is due to their high surface area over volume ratio that increases
the number of atoms on the surface and increase in interacting faces [8, 9] which lead to
surface reactivity [10, 11]. Changes in surface reactivity and crystalline structure may
influence NM behavior such as increase in toxicity as it is shown that titanium dioxide and
aluminum oxide NPs have more toxic effects than the bulk material [12].
1.1 NM synthesis methods
Different methods are used for synthesizing nanomaterials and fabricating
nanostructures. These methods can be grouped into two categories: 1) top-down approach
(physical method) and the 2) bottom-up approach (chemical methods) [13-15]. Top-down
method begins from bulk material and then reduces to nanoscale using high-pressure
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homogenization or milling [16]. In contrast, the bottom-up approach starts NMs from
atoms and molecules and builds up to nanostructures through nucleation.
1.2 NP classification
Nanoparticles (NPs) can be classified into different classes based on their origin,
shape or size. Based on origin of NPs, they can be grouped as 1) naturally occurring NPs,
2) incidental NPs, and 3) manufactured or engineered NPs [17-19]. Natural NPs are
generated by natural processes and offers a wide range of small particles from inorganic
ash and mineral particles found in the air, to selenium and sulfur NPs that are produced by
yeast and bacteria [20]. Some natural NPs such as iron oxide NPs produced from wind-
blow mineral dust, serves as a micronutrient for phytoplankton which controls the CO2
levels in the aquatic environment [21]. Incidental NPs are produced unintentionally as a
byproduct of some other activities such as combustion processes. For example, platinum
and rhodium NPs released from automotive catalytic converters (which driving speed and
traffic could control the releasing level of these NPs) [22]. Manufactured or Engineered
NPs (ENPs) are produced for specific purposes by humans to serve as an ingredient in
some products. ENPs may be produced in different and particular shapes and compositions
for different purposes such as in science, technology, medicine and industry. ENPs are
produced with different capping agents to make them more persistent in the environment
(media) and they are made in definitive sizes and shapes to achieve their unique
physicochemical properties. These factors can affect the risk associated with ENPs and
they can be more toxic than natural occurring NPs. Therefore, it is reasonable that ENPs
become the main focus of current research [17, 23, 24].
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1.3 NM exposure
The novel properties of NMs have accelerated the production and utilization of NPs
in many consumer products. Human may be exposed to engineered NMs through
occupational, environmental and consumer exposure based on The Royal Society/ Royal
Academy of Engineering report (2004). Being exposed to occupational exposure is
probable at all phases of a NMs’ life cycle. NM exposure could occur while experimental
procedures are happening to develop new materials (under laboratory conditions) which
could cause incidental spills of materials. In the final product, exposure may take place
during packaging, transport or storage and finally, exposure may happen while NM product
is being disposed of (at the end of product lifetime). In many of these products, exposure
can happen deliberate (usually by ingestion or dermal application) or incidental (including
inhalation). So, for every NM there is different routes of exposure depending on the
production, use, and clearance.
1.4 AgNPs application
In the Nanodatabase (http://nanodb.dk/) as of 2018, more than 1100 consumer
products containing NPs are listed which there are 379 reported products including AgNPs
(35%) and making AgNPs the most commercialized NP in the market. Because of high
electrical and thermal conductivity [25-27], as well as their chemical stability and catalytic
activity of Ag, they are valuable in microelectronics and medical imaging [28]. In addition,
AgNPs show a broad spectrum of antibacterial and antiviral activity [29-32].
Antimicrobial properties of AgNPs make them extremely popular in different consumer
products such as soaps, plastics, food packaging, cleaning products, antibacterial wound
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dressings, cosmetics, personal care products and textiles [25, 29, 33-39]. Ag containing
textiles are popular for their antibacterial effect and their ability to reduce sweating odor
[40]. AgNPs are also used as coating for medical products [41, 42] such as urinary
catheters [43], scalpels and needles [44].
1.5.1 Human exposure to AgNP
This rapid increase in AgNP usage in consumer products raises concern about its
consequent adverse effect on environment and human health [17]. Ingestion, injection,
inhalation, dermal and ocular contacts are possible routs of human exposure to AgNPs
(Figure 1.1). A high dose of Ag may lead to argyria which is associated with the irreversible
deposition of Ag in the skin [45]. Stammberger reported that application of Ag-containing
nose spray for a year led to argyria [46] also in a clinical case it was reported that argyria
happened after taking Ag-containing food supplements for a few months [47]. The World
Health Organization (WHO) offers a 10 g Ag over the lifetime of human as a no observed
adverse effect level (NOAEL), which matches to 0.1 mg L-1 Ag concentration in tap water
(Guidelines for drinking water qualities, 1996). However, the allowed tap water Ag
concentration in Germany is 0.01 mg Ag L-1 (Trinkwasserverordnung).
1.5.2 AgNP transformations
AgNPs are very reactive (due to their high surface area and surface energy) and are
susceptible to dynamically transform in biological media. These transformations could
change the behavior of NPs such as dissolution, aggregation and sulfidation and complicate
the evaluation of risk associated to NP exposure [22]. To have a better evaluation of NP
bioavailability, bio-uptake and toxicity, we should have an understanding about the type
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and magnitude of NP transformations [22]. Surface interactions of NPs with biological
components (such as proteins) will affect their physicochemical properties (size, shape,
concentration and surface charge). In case of proteins, formation of a protein corona on
the surface of NPs decreases the reactive oxygen production and reduces the toxic effect
[48]. Aggregation and dissolution are two key NP transformation processes which
determine NP toxicity and bioavailability [17, 49]. Considering all this factors,
comprehensive characterization of NPs in nanotoxicology studies is essential for pristine
NPs, during exposure and after exposure in the proper exposure media.
1.5.3 AgNP vs. Ag ion toxicity
Toxicity of both NPs and ions in metal-based NPs has been investigated and relative
role of these two has not been fully explained. Depending on dissolved (ion) or particulate
uptake, bioaccumulation and toxicity will be different as Baek et al., (2011) reported higher
toxicity of copper and nickel oxide NPs than ions in bacteria [50]. In some NPs such as
zinc oxide, dissolution alone could explain toxicity effects [51]. However, in NPs such as
ceria and titanium that are determined as poorly soluble NPs, dissolution is playing the
minimal role in toxicity [52, 53]. AgNP is considered as partially soluble NP for
nanotoxicology purposes and many studies have been performed to investigate NP vs ion
bio-uptake and toxicity [54, 55]. However, current methods for investigating the
contribution of particles and ions in toxicity (uptake and accumulation) are relatively
unreliable and require more development. Some studies suggest that toxicity can be mostly
explained by dissolved Ag from NP dissolution [56], whereas other studies indicate that
the residual effect of NPs is contributing to toxicity along with ions [57, 58].
7
1.5.4 Core-shell NPs
To overcome the uncertainty in identifying NP and ion role, bimetallic and
isotopically labeled NPs have been recently used to measure bio-uptake and accumulation
of metals in organisms [59-61]. Bimetallic NPs become very popular because of their
fascinating sensing, electronic and optical properties [62] and higher functionality by
combining multiple NMs which each holding unique advantage. The properties can be
modified by changing either the creating materials or the core to shell ratio. The coating
of the core NPs has different reasons such as surface modification, stability, dispersibility
and controlled release of the core material [63]. Bimetallic NPs can have different forms
such as alloy, nanowire and core-shell [64]. Between these types, bimetallic gold (Au) and
Ag core-shell NPs have attracted more attention [65]. Outer Ag shell protects Au core
from interacting with the media, therefore Au will be protected from dissolution and can
be used as an internal standard, and Ag and Au ratios can be used to explain the Ag
transformations (such as dissolution) in the media [66]. Physiochemical properties of
bimetallic core-shell NPs uniquely depend on the composition of NP and thickness of core
and shell that is determined by their end use and application [67]. Isotopically labeled NPs
provided enhanced sensitivity to evaluate the rate of particles delivery to organism [68-72].
Isotopically labeled NPs enhance the signal to noise ratio of the enriched isotope against
the background (non-enriched isotope). This property of NPs significantly decrease the
detection limits within organisms by at least two fold [61]. In bio-uptake and
bioaccumulation studies, isotopically labeled core-shell NPs allow the direct calculation of
ion and NP uptake, since the core always remains as a particle, while the shell partially
dissolves.
8
1.5.5 AgNP toxicity on human
Cell toxicity induced by AgNPs has been studied in cells in vitro and in a wide
range of organisms including bacteria, algae, nematodes, fish and mice [23, 25, 73-80]. It
has been shown previously that injected AgNPs are translocated to the circulatory system
and accumulate in the main organs (especially kidney, liver and spleen) [81]. Takenaka
et al., reported that AgNPs can move into blood circulation system through blood
pulmonary barrier and indicate a systemic distribution [82]. After entering the blood, NPs
come in direct contact with peripheral blood mononuclear cells (PBMCs) that is consists
of mostly monocytes and lymphocytes (B and T cells) which are involved in cellular host
defense system [83]. A few studies have been done to assess Ag uptake and toxicity on
human blood cells [84-86], but they did not consider NP characterization and
transformations in the exposure media that could alter NP toxicity.
1.5 Engineered iron oxide NPs
Engineered magnetic iron oxide NPs have been intensively established due to their
biomedical and in vivo applications such as contrast agents in magnetic resonance imaging
(MRI), targeted delivery of drugs and genes, cell separation, tissue repair and detoxification
[87-95], (because of their inherent low toxicity and magnetism property).
The most common used techniques for iron oxide NPs synthesis include: thermal
decomposition, hydrothermal reactions [96], micro-emulsion [97] and co-precipitation
method [98]. Among these synthesis techniques, co-precipitation in aqueous solution is
most widely used to synthesize superparamagnetic iron oxide NPs [99, 100] since it is more
environmentally sustainable (needs low-toxicity organic solvent or does not need that).
9
Because of the colloidal nature of iron oxide NPs, their synthesis is a complex process.
Almost all biomedical applications of iron oxide NPs require high magnetization value, a
size smaller than 100 nm and a narrow size distribution [101]. Therefore, defining
experimental conditions leading to a monodisperse NP and a reproducible process are the
challenges in the synthesis process.
1.6.1 Iron oxide NP toxicity
Although the potential advantage of iron oxide NPs for therapeutic purposes is
considerable, still there are some concerns about their potential toxicity and cellular
damage on human associated with these particles. The same nanoscale properties such as
large surface area, increased surface reactivity, increase tendency to diffuse across
biological membranes (because of nano-size) can potentially enhance their cytotoxicity
[10, 102, 103]. Different studies demonstrated that iron oxide NPs are safe and non-toxic
at concentrations below 100 µg L-1 [104, 105]. Alabresm et al. measured acute toxicity of
PVP-coated iron oxide NPs on copepod and they did not observe any toxicity up to 25 mg
L-1 NP concentration [106]. In most cytotoxicity tests on human and murine cell lines, iron
oxide NPs did not show toxic effects up to 100 µg L-1 exposure concentration and toxicity
was dependent on cell type and duration of exposure [107-110].
1.6.2 Obesity
Obesity is a widespread disease/syndrome in the modern world. It can noticeably
reduce the quality of life and competes with smoking as the most common cause of
premature death [111]. The rate of obesity including childhood and adolescent obesity has
increased during the past decade and nearly 40% of U.S. adults have obesity [112]. Diet
10
composition, excess calorie intake, genetics, decrease in exercise and the built environment
are risk factors that cause obesity and associated co-morbidity [113, 114]. Obesity is
associated with more than 40 diseases and some health complications such as coronary
heart disease, hypertension, stroke, type 2 diabetes and at least 13 different types of cancer
[112, 115, 116]. The health care costs for obesity now is a major concern across developed
countries. According to CDC, the medical costs of obesity in the U.S. are estimated as
$147 billion annually [112] and research suggests that individuals with obesity have 42%
higher health care costs than normal weight people [112]. There are different weight-loss
strategies including dietary modification, behavior modification, exercise, medication and
surgery. Some of the weight-loss drugs that are approved by U.S. Food and Drug
Administrative (FDA) for treating obesity include: Orlistat (Xenical), Phentermine and
Sibutramine (Meridia). Medications are used as part of morbid obesity treatment, but they
also can have serious side effects and have failed to provide an effective solution. Currently
there is no non-surgical method to achieve major weight loss and maintain that [111].
Bariatric surgery includes the procedure of sleeve gastrectomy, biliopancreatic bypass,
gastric banding, and gastric bypass. Surgical options have their strengths and weaknesses
as well such as perioperative mortality, early adverse event and late adverse event.
1.6.3 Iron oxide NPs application
With nanotechnology development, novel solutions for environmental remediation
are emerging. For example, particles are used for cleaning of contaminated sites [117, 118].
In this way manufactured magnetic iron oxide NPs are remarkable for oil remediation
because of their magnetic property, low toxicity and low cost [119, 120]. Oil removal
efficiency of iron oxide NPs has been studied before, and results showed 100% oil removal
11
in ultra-pure water, synthetic freshwaters and seawater [1]. With this introduction, we
aimed to investigate the potential ability of iron oxide NPs to remove dietary fat in vivo
using a high-fat-diet mouse model.
1.6 Dissertation overview
In short, the production and consumption of NMs in many consumer products have
been accelerated due to the novel properties and advanced synthesis method of NMs. This
rapid increase in the commercialization of NPs raises concern about their safety and their
potential toxic effect on human health. Therefore, the overall aim of this dissertation is to
investigate the implication and applications of silver and Iron Oxide NPs on Mammals.
This dissertation is organized as follows:
Chapter 2 investigates the uptake and toxicity of PVP-AgNPs and Ag ions (as control)
to human PBMCs in physiologically relevant concentrations. It provides characterization
data of synthesized AgNP in exposure media and during exposure time, as well as the
uptake and toxicity of AgNPs and Ag ions in PBMCs.
Chapter 3 describes the efficiency of a facile and cost-effective synthesized PVP-coated
iron oxide NPs to remove dietary fat and decrease body weight in a high-fat diet-fed mice
compared with controls (LFD and HFD). It provides results of changes in body weight,
food intake and body composition of mice after 16 weeks of treatment, as well as iron
accumulation in feces and tissues and hepatic inflammatory markers (TNF-α and MCP-1).
Chapter 4 discusses and summarizes findings of this dissertation.
12
Figure 1.1 Routes of human exposure to Ag. The extensive use of nanosilver in consumer
products introduces multiple ways for human exposure. (Adapted from Wen et al., 2016).
13
CHAPTER 2
UPTAKE AND TOXICITY OF SILVER NANOPARTICLES AND
SILVER IONS TO HUMAN PERIPHERAL BLOOD MONONUCLEAR
CELLS
14
2.1 ABSTRACT
Silver nanoparticles (AgNPs) are widely used in medical applications due to their
antibacterial and antiviral properties. Despite the increasing study of AgNPs, their toxicity
and their effect on human health is poorly understood. This is largely a result of such issues
as poor control of NP properties as well as a lack of proper characterization and inadequate
quantified bioaccumulation. The aim of this study is to investigate the uptake and toxicity
of well-characterized PVP-coated AgNPs on primary human cells (peripheral blood
mononuclear cells) at physiologically relevant concentrations (10 - 1000 µg L-1). AgNPs
were synthesized in-house and characterized using a multi-method approach. Results
indicated relatively stable NPs in RPMI media with a decrease in size and greater
polydispersity after 24 hours of exposure. No aggregation of NPs was observed in the
STEM images in the RPMI media during exposure time (24 hours). A dose-dependent
relationship between uptake and Ag concentration was detected for both AgNP and AgNO3
treatment. In 1000 µg L-1 AgNO3 exposure, up to 11 ng Ag was detected in 2.5× 105 cells,
which is about 2-fold higher than 500 µg L-1 exposure. There was almost 2-fold increase
in the Ag uptake into the cells in the AgNO3 treatment than NPs. However, LDH assay
did not show any significant difference in cytotoxicity of AgNPs and Ag ions.
2.2 INTRODUCTION
Nanotechnology is a rapidly growing industry that creates nanoscale products with
novel physio-chemical properties [121]. Nanoparticles (NPs) are particles with at least one
dimension between 1-100nm, which gives them unique properties and innovative
15
applications [122]. These particles are used widely in industry (oil remediation) [123],
numerous commercial products such as cosmetics, electronics, and textiles, and in different
biomedical and drug-delivery applications including the treatment of diseases [17, 124,
125]. Silver nanoparticles (AgNPs) are one of the most commercialized NPs on the market
and are widely used in medical products such as ointments, bandages and wound dressings
[126-128] due to their antibacterial and antiviral properties [29, 30]. In fact, the use of
AgNP in commercially-available products is anticipated to double in the next five years
[129]. AgNPs have the potential to prevent bacterial colonization on different surfaces,
such as catheters and prostheses [130, 131], which raises concern about Ag release in the
body and their possible negative effects on human health. For instance, the silver
concentration in the blood of people without any occupational or medicinal exposure is
<1µg L-1 [132]. However, in burn patients who are treated with silver containing
antibacterial creams, Ag concentration in the blood can be found up to a concentration of
310 µg L-1 [132]. Additionally, AgNPs that are used in food-related products might alter
gut microbiota and modulate the systemic homeostasis of the intestinal tract [133, 134].
A human body might be exposed to AgNPs through injection, ingestion, inhalation,
and dermal and ocular contact [36]. As soon as AgNPs enter the body, they are translocated
to the circulatory system and can come in direct contact with human peripheral blood
mononuclear cells (PBMCs) before ultimately being distributed to the main organs where
they accumulate [81]. PBMCs consist of monocytes and lymphocytes (B and T cells) and
are responsible for the cellular host defense against foreign substances [83]. These cells
secrete different inflammatory mediators after activation, including pro- and anti-
inflammatory cytokines. Despite the increased usage and likely increased exposure of
16
AgNPs, there is a lack of quantitative analysis of bio-uptake and potential cytotoxicity of
PBMCs after exposure to well-characterized AgNPs. Different studies have investigated
the bioavailability and cytotoxicity of AgNPs using different cell lines and rodent models
[126, 135-137]. However, these immortalized cell lines are more resistant to toxicity
effects that may be induced by AgNPs [138]. Furthermore, there are only a few studies on
the bio-uptake and exposure of AgNPs on human PBMCs [84-86, 139-143]. Few, if any,
of these studies investigate procurement, characterization and bio-uptake of NPs,
transformations in exposure media, or use proper controls at relevant exposure
concentrations.
In the present study, we initially decided to synthesize and use isotopically labeled
core-shell NPs to be able to directly calculate ion and particulate uptake and toxicity in the
PBMCs. Considering that, in the core-shell NPs core remains as particulate, while the
outer shell dissolves partially. We have synthesized unlabeled core-shell
silver@gold@silver (Ag@Au@Ag) NPs following the method described by Merrifield et
al. [144]. As synthesis of core-shell NPs is a more complex process than single NPs and it
needs more alloted time and cost than single material NPs, we decided to optimize
experiments using PVP-coated AgNPs, before moving to core-shell NPs.
The overall aim of this study was to investigate the bio-uptake and toxicity of well-
characterized PVP-coated AgNPs on PBMCs at physiologically relevant concentrations.
The specific objectives include;
1) Investigate if cell culture media (RPMI) and exposure condition are capable of
transforming PVP-coated AgNPs. We hypothesized that PVP-AgNPs shows
17
more stability in the media. We investigated this through experiments
measuring dissolution and aggregation of AgNPs during exposure time in the
cell culture media.
2) Quantify the uptake of Ag by human PBMCs in RPMI. We hypothesized that
uptake is faciliated by ionic Ag resulting from NP dissolution. To test this, we
exposed PBMCs to PVP-AgNPs and AgNO3 and measured Ag concentrations
within cells over the exposure time.
3) Investigate and compare toxicity of Ag in the form of NP and ion exposed to
human PBMCs. We hypothesized that Ag in the form of ion is more toxic to
cells than AgNP. To test this hypothesis, we assessed cell viability and cell
metabolic activity in PBMCs.
Comprehensive characterization of NPs plays a critical role in order to have a better
eastimation of hazards and risks associated to the nanomaterials. The goal of this study is
to emphasize the importance of NP characterization in nanotoxicology studies to avoid
over- or under- estimation of NP hazard assessment.
2.3 MATERIALS AND METHODS
2.3.1 Core-shell Ag@Au@Ag nanoparticle synthesis
The tri-layered core@shell@shell NPs (Ag@Au@Ag) with both unlabeled Ag as
the core and outer layer and isotopically labeled Ag107 as core and Ag109 as outer shell
(Ag107@Au@Ag109) were synthesized following Merrifield and Lead 2016 protocol
[144]. This synthesis includes four methodological steps as follows: 1) AgNO3 synthesis;
18
2) AgNP seed production and wash; 3) Adding Au layer (gold capping); and 4) outer Ag
layer (silver capping). The Ag metal powder (Ag107, Ag109, or unlabeled Ag) was
converted to AgNO3 by boiling powder in HNO3. A 50 mg of Ag powder was weight
graph metrically into a plastic boat and then washed out completely into the mortar using
10 mL of concentrated HNO3 (trace metal grade). The suspension was pipette up and down
and mixed until Ag is fully dissolved. The mortar was placed on the hot plate with a watch
glass as a cap inside the fume hood and temperature was adjusted to 120°C. The mortar
was checked every 20-30 minutes and was swirled to avoid AgNO3 crystals from drying
and burning. Heat was removed once all acid was evaporated and only white crystals of
AgNO3 was remained in the mortar. All the AgNO3 powder was scrapped from the mortar
and was collected into a dark, acid washed, scintillation vial for later AgNP production.
Ag seed NPs (for both labeled and unlabeled Ag) were synthesized using a standard
sodium borohydride reduction process following Romer et al., 2013 [145]. UHPW (400
mL) was gravimetrically dispensed into a conical flask. The flask was placed on the 200°C
set hot plate while vigorously stirred to boil. Proper amounts of AgNO3, silver citrate and
NaBH4 solutions were prepared and NaBH4 stock was diluted 10-fold. Once UHPW was
boiling AgNO3 and silver citrate were added simultaneously and allowed to stir for a few
seconds. Then 0.5 mL of diluted NaBH4 was added dropwise (~ drop per 10 seconds). As
soon as NaBH4 was added, solution turned yellow. The solution was heated for a further
30 minutes, and then heat was turned off and solution was allowed to cool to room
temperature. Synthesized batch was placed in dark place overnight. After synthesis
AgNPs were washed by ultrafiltration using a diafiltration technique to avoid drying and
NP aggregation as well as to remove any excess and unreacted AgNO3 or NaBH4 from
19
solution. Millipore ultracel 3 kDa ultrafiltration disc was placed in the base of a plastic
petri dish and was thoroughly rinsed three times with UPW and then was completely soak
in UPW for 30 minutes. Ultrafiltration device was set up and 200 mL of AgNP stock was
transferred to the pressure filtration apparatus. AgNPs were washed using nitrogen gas
(100 psi). When half of the total volume of AgNP batch was filtered, 100 mL of citrate
solution was added to make the original volume. The citrate solution was used to reduce
NP growth or aggregation due to loss of surface citrate [146]. Washing process was
repeated at least three times. AgNPs are light sensitive and in all the procedures they were
protected from light.
A gold (III) chloride solution was prepared and was protected from light by
wrapping in aluminum foil. A 50 mL of citrate washed AgNP seed solution was placed on
hot plate and heated to 60°C while moderately stirring using a magnet. One drop of gold
chloride solution was added to the heated AgNP seed in a 30 seconds time interval. After
each 5 minutes of adding Au, 1 mL aliquot of sample was taken and measured by the UV-
Vis until spectra peak showed a peak around 470 nm wavelength. At this point heat was
stopped and Ag@Au solution cooled to room temperature while stirring. After adding Au
cap color of solution changed from yellow to peach-red. Washing process was repeated as
explained in the AgNP seed section.
Ag@Au NPs were heated to 100°C while stirring and solutions of AgNO3 and
citrate were prepared. One drop of each solution mentioned above was added to NP batch
simultaneously and dropwise. After adding each drop and 5 minutes of stirring 1 mL
aliquot of sample was taken and measured by UV-Vis to monitor the growth of Ag outer
layer. After the surface plasmon resonance reached 410 nm heat was removed and
20
Ag@Au@Ag solution was stirred to cool to room temperature and was washed the same
way as explained above.
2.3.1 Synthesis and characteriozation of PVP-AgNPs
Citrate-capped AgNPs (cit-AgNPs) were synthesized by the standard reduction of
silver nitrate (AgNO3) in trisodium citrate as described in previous publications [145, 147,
148]. Briefly, separate solutions of AgNO3, trisodium citrate, and the reducing agent
sodium borohydride (NaBH4) were prepared. AgNO3 and trisodium citrate were mixed
together while stirring vigorously and NaBH4 was added in a dropwise fashion. The
solution was heated slowly to boiling, heated for a further 90 minutes, and then left
overnight to cool in the dark. After synthesis, the NPs were washed by ultrafiltration (3
kDa cellulose membrane EMD Millipore) using a diafiltration technique to avoid drying
and NP aggregation as well as to remove any excess and unreacted AgNO3 ions or NaBH4.
The washing process was repeated at least three times and after each wash the trisodium
citrate solution was added to make the original volume. A citrate solution was used to
reduce NP growth/aggregation due to re-equilibration and loss of surface citrate [146]. The
final reactant concentrations were < 1% of the original. Cit-AgNPs were then recapped
with polyvinylpyrrolidone (PVP) using the ligand exchange approach [147, 149]. A
solution of PVP10 (Mw 10000, sigma Aldrich) was added to the NP batch while stirring
vigorously for one hour. PVP is a non-toxic polymer [150, 151] used to sterically stabilize
particles by strongly binding to the AgNP core [147, 148] and protect them from
dissolution and aggregation in complex media [149, 152].
Z-average hydrodynamic diameter and polydispersity index (PDI) of PVP coated
AgNPs (PVP-AgNPs) were measured using dynamic light scattering (DLS) with a Malvern
21
Zetasizer NanoZS (Malvern Instruments, MA, USA). The colloidal stability (zeta
potential) of PVP-AgNPs was measured by laser Doppler electrophoresis (Malvern
Zetasizer NanoZS, MA, USA). The mean of at least three consecutive measurements was
reported. Absorption spectra of PVP-AgNPs was recorded in triplicate over the
wavelength range of 200-800 nm using a UV-Vis spectrometer (UV-2600, Shimadzu Co.,
Kyoto, Japan). Transmission electron microscope (TEM) and scanning transmission
electron microscope (STEM) samples were prepared using an ultracentrifugation-based
method in UHPW and RPMI media, respectively. Briefly, a carbon coated copper grid
(300 mesh; Agar scientific) was placed into a clear plastic centrifuge tube and 4 mL of
sample was added into the tube very slowly. The tube was covered by parafilm and was
centrifuged using vacuum centrifugation for 1 hour at 50,000 rpm. The grid was rinsed
thoroughly with high-purity water and left overnight to fully dry. The grids were then
imaged by a Hitachi HT8700 and HD2000. The PVP-AgNP batch was checked for purity
using Energy-dispersive X-ray spectrometry. The final concentration of the PVP-AgNP
batch was measured using inductively coupled plasma optical emission spectrometry (ICP-
OES; Varian 710-ES). PVP-AgNP characterization in media without cells was conducted
using UV-Vis spectrometer and STEM. Dissolution rate was measured at relevant
exposure concentrations of PVP-AgNPs and at 0 and 24-hour time points using ICP-MS
(inductively coupled plasma mass spectrometry- NexION 350D, Perkin Elmer Inc.,
Massachusetts, USA).
2.3.2 Measurement of the concentration of total, dissolved, and NP fraction
When exposed to oxygen, AgNPs oxidize through oxidative dissolution
mechanisms and Ag ions will be released from the surface of AgNPs to the solution [84,
22
153]. To determine the extent of ionic silver release from PVP-AgNPs in media (without
cells), we used a centrifugal ultrafiltration method. Aliquots of samples were taken at 0
and 24-hour time points and were subsequently added to the top of centrifugal
ultrafiltration tubes (Pall Corporation, Microsep Advance with 3kDa omega; maximum
volume 4 mL) and centrifuged at 4000 rpm for 20 minutes at 20°C (Eppendorf 5810R
centrifuge) [154, 155]. The original (total Ag) and ultrafiltered (dissolved Ag) samples
were then acidified with concentrated nitric acid (70% HNO3) and diluted to 1% acid prior
to the measurement of Ag concentration with ICP-MS. Each sample was measured in
triplicate and Indium (In115) was used as an internal standard. The detection limit was
calculated as 0.024 µg Ag L-1. The validity of analytical method was checked every 12
measurements with 2 quality controls (Blank and 5 µg Ag L-1). A serial dilution of Ag
standard solution (1000 µg mL-1, VWR analytical, USA) was used for calibration curve.
To measure and compare the loss of Ag (dissolved or PVP-AgNPs) on the ultrafilter
membrane or sorption to the exposure tube, 500 and 1000 µg L-1 concentrations of Ag (as
AgNO3) was prepared in UHPW and RPMI media. Samples were collected at 0 and 24-
hour time points and were ultrafiltered and measured by ICP-MS in triplicate as previously
described and were used to correct samples results.
2.3.3 Isolation and culture of peripheral blood mononuclear cells (PBMCs)
Human blood for 6 individual people was purchased from the New York Blood
Bank (New York City, NY) and PBMCs were isolated by gradient centrifugation using
Ficoll-paque Plus (GE Healthcare Bio-Sciences AB, Sweden). In brief, peripheral blood
from healthy volunteers was diluted with an equal volume of sterile phosphate buffer saline
(PBS; Gibco by life Technologies) and slowly layered onto the Ficoll-paque media
23
solution. Tubes were centrifuged at 500 g for 30 to 40 minutes at 18°C. After
centrifugation a thin layer of mononuclear cells could be detected between plasma and
Ficoll-paque media. The layer of mononuclear cells was transferred to a sterile tube and
was washed with sterile PBS three times. After the last wash, cells were resuspended in
RPMI1640 media (Sigma- Aldrich, Taufkirchen, Germany), supplemented with 10% fetal
bovine serum (FBS; Gibco by Life Technologies) and 100 IU/mL pen/strep (Gibco by Life
Technologies) and counted using trypan blue. The isolated cells were adjusted to 2.5×105
in 24 well-plate (Costar, ME, USA) for cell culture and bio-uptake experiments, and
2.5×104 cells in 96 well-plate (Corning Incorporated, NY, USA) for cell viability and
metabolic activity test.
2.3.4 Uptake of Ag into PBMCs and mass balance
ICP-MS was used to quantify the uptake of Ag in PBMCs after exposure to PVP-
AgNPs and AgNO3 (as control). Freshly isolated PBMCs were seeded in 24-well cell
culture plates at a density of 2.5×105 cells. PVP-AgNP and AgNO3 stock (25 mg L-1) were
diluted with cell-culture water (Sigma-Aldrich, Taufkirchen, Germany) and added to attain
final concentrations of 10, 100, 500, and 1000 µg L-1 prior to exposure. The AgNP solution
was sonicated for 20 minutes to reduce NP agglomeration immediately before exposure.
The plates were then incubated for 24 hours at 37°C and 5% CO2. Each exposure study
was run in triplicate. After 24 hours each well was collected, and the cells and supernatant
were separated using a centrifuge (10 min x 300g). The supernatant was used for Ag
analysis to determine the amount of Ag that was not taken up by PBMCs. Cells were
washed three times with PBS using a centrifuge (15 min x 300g) and then lysed and
24
digested with concentrated HNO3. The Ag ion or AgNP uptake by cells was measured by
quantifying the total Ag concentration in the digested samples using ICP-MS.
2.3.5 Measurements of viability and metabolic activity of cells
To investigate cytotoxicity of Ag on PBMCs we used two different assays that
measure cell damage (LDH) assay and metabolic activity (MTS) assay. LDH is a
colorimetric assay that measures the membrane integrity and quantitatively quantify lactate
as a substrate that is released from lysed cells into the media as a biomarker. LDH
cytotoxicity assay (Dojindo laboratories, Kyoto, Japan) was performed according to
manufacturer instructions. In brief, immediately after isolation of PBMCs, cells were
treated in the absence (control) and presence of increasing doses of PVP- AgNPs, AgNO3
(as positive control), AgNO3 -PVP and PVP (10, 100, 500, and 1000 µg L-1) in 96 well
plates for 24 hours in 6 replicates. Subsequently, 100µl of the assay buffer was added to
each well and incubated at room temperature in the dark. After 30 minutes, 50µl of the
stop solution was added to each well and the LDH activity of the samples was assessed by
measuring NADH absorbance at 490nm.
Cell metabolic activity was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium (MTS; Promega, Madison, WI)
assay. MTS assay measures the insoluble formazan that is accumulated in the
metabolically active cells. The MTS assay was performed according to the manufacturer’s
protocol. Briefly, isolated PBMCs were exposed to 0, 10, 100, 500, and 1000 µg L-1 of
PVP- AgNPs, AgNO3, AgNO3 -PVP and PVP in 96 well plates in 6 replicates for 24 hours.
Subsequently, MTS dye was added to each well, and incubated at 37°C in a 5% CO2
25
humidified incubator for 4 additional hours. The optical density of reduced MTS was
measured at 490 nm using a 96-well plate reader spectrophotometer.
The potential interference of particles with LDH and MTS assay was also examined
in the cell free conditions and subtraction of background absorbance was applied where
necessary.
2.3.6 Statistical analysis
Data for uptake, cell viability and metabolic activity are expressed as means ±
standard errors (SEs) for six separate donors. Statistical analyses were evaluated using the
ANOVA with random effects to detect the significant differences between the treatment
and control groups. P-values with less than 0.05 were considered statistically significant.
All analyzes were performed using SAS software (SAS 9.4).
2.4 RESULTS
2.4.1 Core-shell NP characterization
Characterization of core-shell NPs in different synthesis steps are shown in Table
2.2. Characterization of core-shell NPs (Ag@Au@Ag) in water resulted in an absorption
spectrum (measured by UV-Vis) that was indicative of Ag@Au@Ag NPs (407 nm), an
average hydrodynamic size of 28.23 ± 9.3 nm and a polydispersity index (PDI) of 0.12 ±
0.04. Figure 2.1a and b shows the progression of the Ag wavelength to form Ag@Au and
Ag@Au@Ag, respectively.
26
2.4.1 PVP-AgNP characterization
In this study we used a multi-method approach to characterize pristine AgNPs
including DLS, UV-Vis, TEM and ICP-MS. DLS showed a Z-average hydrodynamic size
of 33.7 ± 0.7 nm (mean ± standard deviation) and PDI as 0.18, indicating a reasonably
monodisperse particle suspension (Table 2.3). A representative curve is shown in Figure
2.2a. The zeta potential was measured as -10.3 ± 2.1 mV for PVP-AgNPs. UV-Vis spectra
exhibited a characteristic absorption peak at 390 nm for Ag (Figure 2.2b). A mean core
size of 4.9 ± 0.3 nm was indicated by TEM imaging and confirmed a spherical shape of
NPs (Figure 2.2c and d), which was proven to be pure silver by performing EDX analysis
(Figure 2.2e). The peaks of other elements (C, Cu, and O) in EDX analysis are due to grid
material that was used for sample preparation. DLS estimates the hydrodynamic size of
NPs, which includes the hydration shell around NPs and should be the same as, or larger
than the core size of NPs measured by TEM. Characterization values for pristine PVP-
AgNP can be found in Table 2.3.
STEM images of 1000 µg L-1 PVP-AgNPs in RPMI media at the time points of 0-
and 24-hour exposure are shown in Figure 2.3. STEM images showed a mean size of 29
± 4.9 nm at the T=0 hour in RPMI (Figure 2.3a), which is larger than the size of pristine
NPs measured by TEM in the UHPW (4.9 ± 0.3 nm, p < 0.05). STEM images did not show
any evidence of aggregation of NPs. After 24 hours in exposure media, PVP-AgNPs
remained spherical, with a mean size of 17 ± 11 nm (Figure 2.3c). Figure 2.3b and d show
a skewed distribution and greater polydispersity of PVP-AgNPs in the RPMI media after
24 hours of exposure. Extinction spectra of PVP-AgNPs at 100, 500 and 1000 µg L-1
concentrations in exposure media during 24-hour time point are presented in Figure 2.4. A
27
decrease in the UV-Vis absorbance was observed for all concentrations. A small red shift
and broadening in the Ag peak, especially in 1000 µg L-1 PVP-AgNP concentration was
observed during 24-hour exposure. Full width half maximum (FWHM) for both 500 and
1000 µg L-1 exposure concentration increased (44 nm to 81 nm and 49 nm to 140 nm, for
500 and 1000 µg L-1, respectively) after 24-hour exposure.
2.4.2 Dissolved Ag measurement using ICP-MS
Ag dissolution in the culture media was measured after addition of PVP-AgNPs at
T=0 hour (sample collection was performed immediately after addition of AgNP) and T=24
hour, which matches the total exposure time in the experiment using centrifugal
ultrafiltration method. For PVP-coated AgNPs, no dissolved Ag was measured in the
filtrate during the exposure time (T=0 and 24 hours, Figure 2.5a). The original Ag
concentrations were found nearly unchanged under the experimental conditions (Figure
2.5b). The dissolution experiment was repeated at the same condition in the presence of
AgNO3 as a control for Ag ions. Figure 2.5c and d show that Ag concentration dropped
from 60 µg L-1 to less than 0.5 µg L-1 for 500 µg Ag L-1 exposure, and from 600 µg Ag L-
1 to less than 4 µg L-1 for 1000 µg L-1 at T=0 hour. After 24 hours of exposure, the Ag
concentration dropped from 190 µg L-1 to 0.1 µg L-1 for the 500 µg L-1 concentration, and
from 200 µg L-1 to 0.3 µg L-1 for the 1000 µg L-1. To confirm the results in the media, and
to compare the dissolution in media and UHPW, the dissolution test was repeated using
AgNO3 in UHPW and media at two concentrations (500 and 1000 µg L-1). Results
indicated whole recovery of total Ag in UHPW at both concentrations and Ag loss in media
and in ultrafiltered samples. A representative image for dissolved and total Ag
measurement for AgNO3 in UHPW and media is shown in Figure 2.6.
28
2.4.3 Ag uptake to the cells and mass balance
After 24 hours of exposure, the total Ag content that was considered internalized or
strongly bound to the cells was measured using ICP-MS in the digested samples after three
washes. An increase in cellular binding and uptake of Ag in PBMCs was observed with
increase in exposure concentration from 10 µg L-1 to 1000 µg L-1 for both PVP-AgNPs and
AgNO3 treatments (Figure 2.7). At concentrations of 500 and 1000 µg L-1 more Ag was
detected in the cells in the Ag ion treatment than AgNP (p < 0.05). No significant
difference in the uptake of Ag in PBMCs was detected in AgNP and AgNO3 treatments in
the lower concentrations (10 and 100 µg L-1).
Ag content was measured in the supernatant using ICP-MS and mass balance was
calculated. Mass balance results (Table 2.4) indicate that after 24-hour exposure at the
1000 µg L-1 concentration the measured internalized Ag was 0.5% in PVP-AgNP and 1%
in AgNO3 of total Ag in the treatments. Furthermore, at the 1000 µg L-1 concentration,
42% and 16.6% of total Ag was detected in the supernatant as NP and ion, respectively.
Figure 2.8 shows Ag content that is internalized or attached to cell surface of PBMCs in
AgNPs and AgNO3 treatment in each of 6 individuals. Mass balance data for each person
for AgNP and AgNO3 treatments are presented in Table 2.5.
The ratio of cell to supernatant uptake did not show any significant increase in PVP-
AgNP treatment as a result of the increase in dose. However, in AgNO3 treatment this ratio
increased in a dose-dependent response (Table 2.4).
29
2.4.4 Impact of PVP-AgNP and Ag ion on PBMC viability and metabolic
activity
Cell membrane integrity as a marker for cell viability was measured by LDH
release. PBMCs were exposed to PVP-AgNP, AgNO3, AgNO3-PVP and PVP at
concentrations of 0 to 1000 µg L-1. AgNO3 was used as a positive control and AgNO3-
PVP was used to examine possible interactions between Ag ions and PVP in cell toxicity.
In PBMCs exposed to PVP, no significant increase in LDH release was detected at any
concentration. Both PVP-AgNPs and AgNO3 increased LDH release in a dose dependent
manner compared to the control (without Ag), starting from 100 µg L-1 concentration
(Figure 2.9a). No significant effect was observed in 10 µg L-1 concentration. Cell toxicity
(LDH test) became significant from 10 µg L-1 for AgNO3-PVP and continually increased
up to 500 µg L-1. No significant difference was observed between PVP-AgNP and AgNO3
(or AgNO3-PVP) treatments at any concentration.
The effect of Ag on metabolic activity of PBMCs was measured by the MTS assay.
Similar to the cell viability test, cells were exposed to different concentrations of PVP-
AgNP, AgNO3, AgNO3-PVP and PVP for 24 hours. Metabolic activity of cells was
elevated for all treatments at 10 and 100 µg L-1 compared to the control (0) (Figure 2.9b).
PVP treatment did not cause any decrease in metabolic activity at any concentration. A
significant decrease in cell metabolic activity for PVP-AgNP, AgNO3, AgNO3-PVP,
compared to the control group, started at 500 µg L-1 and continued at 1000 µg L-1. At 1000
µg L-1 Ag concentration, a significant difference was observed between AgNO3 and
AgNO3-PVP with PVP-AgNP treatment. The highest reduction in metabolic activity was
found with AgNO3-PVP treatment, which elicited a 20% decrease in metabolic activity
30
compared to the control group. Results for cell viability and metabolic activity of PBMCs
for each person are presented in Figures 2.10 and 2.11, respectively.
A Pearson correlation coefficient was calculated to assess the relationship between
uptake and cytotoxicity (LDH assay) of PVP-AgNPs and AgNO3 (Figure 2.12). A
moderate but significant positive correlation was observed between uptake and toxicity of
AgNPs (r = 0.56, p = 0.01). With AgNO3 treatment, Person 5 displayed a different trend
in cell viability and Pearson correlation; considering Person 5 in the correlation, uptake
and cytotoxicity did not show significant relationship (r = 0.314, p = 0.2). However,
excluding Person 5 from analysis changed the correlation to a moderate but significant
correlation between uptake and toxicity (r = 0.52, p = 0.04).
2.5 DISCUSSION
Because of antibacterial properties, AgNPs have been used in several consumer
products and therapeutic purposes which increases the human exposure. Most of the
previous studies investigating uptake and toxicity of AgNPs on human PBMCs have failed
to examine transformation of NPs during the exposure time in the exposure media and to
use physiologically relevant Ag concentrations. Therefore, the purpose of this study was
to investigate the uptake and toxicity of well-characterized AgNPs on PBMCs at low
concentrations.
Physicochemical properties (such as small size, high surface area per mass and
surface reactivity) of nanoparticles determine their interaction with biological systems
31
[156]. Inadequate characterization of nanoparticles may lead to over- or under- estimation
of nanoparticle hazard assessment. PVP-coated AgNPs were synthesized and
characterized in UHPW and cell culture media (RPMI) containing 10% FBS before and
after exposure time using a multi-method approach. DLS results confirmed a
monodisperse AgNP solution with a relatively narrow distribution. STEM images
indicated an increase in size when transitioning from water to media and a subsequent
reduction after 24 hours in the RPMI. In addition, the polydispersity showed an increase
in the media. The increase in size and polydispersity are likely related to (partial)
dissolution of existing NPs and re-precipitation of existing NPs, AgNP ripening or forming
new small NPs through nucleation. Subsequent reduction in size is explained by dissolution
of AgNPs, through oxidation [153]. This interpretation is supported by UV-Vis spectra
that shows a decrease in the absorbance of the Ag peak, a red shift and greater
polydispersity. Although not directly studied, dissolved silver chloride complexes and
other particles were also likely formed [157]. There is no evidence that aggregation played
a large role in NP transformations due to protection by the PVP, or possible protein
interactions in the RPMI media [158, 159]. This result is in agreement with a previous
study that found that aggregation of PVP-coated AgNPs was not observed in the hMSC
cell culture media using focus ion beam (FIB) [160]. Furthermore, Kittler et. al. reported
that media containing 10% FCS (fetal calf serum) will prevent PVP-AgNPs from
agglomeration by either coating NPs or inducing ligand exchanging of PVP with proteins
[161].
Dissolved oxygen in the solutions tend to oxidize AgNPs resulting in Ag ion release
from the NP surface. The Ag ion release kinetics depend on temperature, media
32
composition, size and surface functionalization of NPs [162]. It has been suggested that
toxicity of Ag has been linked to Ag ion release and availability [159, 163, 164], whereas
others have suggested a direct effect related to the AgNP [57]. As the exposure media
(RPMI) is a complex culture media that contains different proteins and factors, it can alter
the surface reactivity of NPs and change the possible mechanisms for uptake and toxicity.
We measured the amount of Ag release during the exposure time in UHPW and RPMI
media. We could not detect any Ag in the dissolved fraction of PVP-coated AgNPs.
However, in the AgNO3 dissolution test in RPMI, 100% recovery of Ag in the dissolved
fraction and total exposure was not obtained. The dissolved Ag measurement for AgNO3
in RPMI suggests a very quick loss of Ag with the 500 µg L-1 Ag concentration. After 24
hours of exposure Ag come back into suspension to some extent (3 fold). However, in the
1000 µg L-1 Ag concentration, there is a continuous loss over exposure time. These results
suggest that some Ag ion is lost either by attaching to the container wall or during the
filtration process due to forming larger complexes with biomolecules in the media or
reprecipitation (or a mixture of both events) resulting in the formation of an Ag complex
with a molecular weight above the nominal value of the 3kDa filter membrane. Since
RPMI media contains chloride (in the form of potassium chloride and sodium chloride)
that can lead to reprecipitation of Ag ions as silver chloride or precipitate within a protein
and form an agglomeration of proteins [157]. Furthermore, cysteine and proteins
containing a thiol group are found in FBS that have high affinity to Ag ions and NPs.
Cysteine could form a complex with Ag ions in the RPMI and be removed from exposure
whish is supported by previous works that cysteine was used to decrease Ag ion availability
in the exposure media (acts like a strong Ag ion ligand) for determining AgNP and Ag ion
33
toxicity contribution [74, 165]. Dissolution results indicate that PVP-coated AgNPs are
more consistent and stable in the exposure media, whereas dissolved Ag is much more
prone to transformation.
Though the exact mechanism of NP uptake into the cells is not fully understood,
endocytosis has been mentioned as a possible mechanism [151, 166]. Previous studies
have shown NP uptake into PBMCs using flow cytometry [84], electron microscopy [139]
and combined FIB (focused ion beam milling)/SEM [160]. We used ICP-MS to quantify
the total amount of Ag (internalized or strongly bound) in digested PBMCs after 24 hours
of exposure to PVP-AgNPs and dissolved Ag. For both AgNP and Ag-ion exposure, the
amount of Ag that was taken up by cells increased as Ag concentration increased, which is
consistent with previous literature [167]. There was a significant difference between Ag
that was detected in the cells in AgNO3 treatment compared to NP at 500 and 1000 µg L-1
concentrations (Figure 4). As it was shown in previous studies that a higher amount of Ag
ion was measured in cells in comparison to those which were exposed to AgNPs at the
same doses [168]. The higher uptake of Ag ions in the cells could be due to higher uptake
rate for dissolved Ag than AgNP [169]. A limitation of our investigation is that we did not
measure the amount of dissolved Ag in digested cells exposed to AgNPs which could be
released due to dissolution of AgNPs. This transformation of AgNPs to Ag ions may cause
toxicity that is referred to as the Trojan-horse type mechanism (that AgNPs acts as a carrier
for Ag ions) in the case that toxicity is attributed to Ag ions [170, 171].
It is important to point out that the type of cell and the kinetics of cellular uptake
may also impact NP uptake [160]. Human PBMCs primarily consist of monocytes and
lymphocytes (mostly T cells). Monocytes are known for their phagocytic properties and
34
phagocytosis is suggested as one of possible mechanisms for Ag uptake by cells [126].
The number of monocytes in the blood stream of different individuals could be elevated
due to chronic inflammatory diseases or viral infection. By using flow cytometry and
FIB/SEM it has been shown that in PBMCs exposed to AgNPs and AgNO3, monocytes
accumulate more Ag in the form of NP in the cytoplasm than T lymphocytes [84, 160].
Therefore, the higher ratio of monocytes to lymphocytes could affect uptake of Ag in
PBMCs. As Figure 2.8 shows different Ag uptake levels for each individual.
Mass balance calculation results did not show any significant differences between
Ag uptake in AgNP and AgNO3 treatment at 10 and 100 µg L-1 Ag concentration.
However, at 500 and 1000 µg L-1 the amount of Ag uptake in AgNO3 treatment was almost
2 times higher than AgNP treatment [169]. For all treatments and concentrations, we could
not get 100% recovery, and some Ag was lost during exposure and sample preparation (e.g.
attached to exposure plate, pipet tips, centrifuge tubes and during cell washes).
Cell to supernatant uptake percentages showed an increase in AgNO3 treatment
with an increase in the exposure concentration. Interestingly, in cell viability test (LDH
assay) in 500 µg L-1 AgNP and AgNO3 treatment same cytotoxicity was detected.
However, cell to supernatant ratio showed more than a 3-fold increase in AgNO3 treatment
compare to AgNP.
Toxicity may be affected by different parameters including particle size, surface
area, incubation time, cell type, NP and cell concentration, growth condition and culture
media. In previous studies, different viability assays were used to measure cytotoxicity of
AgNPs in human PBMCs [84-86, 139-143]. In this study we used LDH leakage assay for
35
measuring cell viability and MTS assay for cell metabolic activity measurement.
Metabolic activity of cells showed an increase for all treatments at 10 and 100 µg L-1
compared to the control, which is consistent with the results of Greulich et. al. who found
an increase in cell activity at sub-lethal concentrations of AgNPs in hMSCs [159].
Cytotoxicity of both PVP-AgNPs and AgNO3 was initiated from 100 µg L-1 Ag
concentration in LDH assay, as was reported by Orta-Garcia et. al. They measured PBMCs
viability using trypan blue exclusion assay and reported toxicity of AgNPs starting from
100 µg L-1 [139]. LDH and MTS results are in agreement in PVP-AgNP, AgNO3, and
AgNO3-PVP exposure at 500 µg L-1, which means a decrease in the number of live cells
leads to a decrease in metabolic activity. Interestingly, in the100 µg L-1 exposure, more
dead cells were detected in LDH while metabolic activity was increased. This could be
explained by the higher mitochondrial activity of live cells because of the treatments. LDH
results did not support MTS results at 1000 µg L-1 for AgNO3 and AgNO3-PVP treatments
(results for LDH not presented). This variation between LDH and MTS results possibly
came from some artifacts that lead to a false negative outcome. In the background test that
was done for both LDH and MTS assay (without cells), an increase in the LDH absorbance
of 1000 µg L-1 AgNO3 and AgNO3-PVP compared to the control (0) was detected, which
indicates inactivation of LDH molecules by ions in the exposure media [172, 173].
In this study, we reported AgNP cytotoxicity started at low concentrations (100 µg
L-1). However, in some of the previous studies that MTT assay was used for measuring
cell viability, any decrease in cell viability (metabolic activity) was not detected even at
high concentrations of AgNPs. There is evidence that Ag toxicity to cells occurs through
oxidative stress by the generation of reactive oxygen species (ROS) which leads to the
36
release of pro-inflammatory cytokines and ultimately cause cell death [174, 175]. MTT
assay measures mitochondrial activity and in the situation that elevated ROS is expected,
mitochondrial activity will increase. There could be different reasons that a decrease in
MTT activity may not be seen even at high concentrations of AgNPs; 1) Using such a high
NP concentration (1- 15 mg L-1) increases the possibility and rate of NP aggregation.
Toxicity, reactivity, fate, transport and risk of NPs is related to aggregation. Large
aggregated NPs tend to be deposited in the exposure media and therefore become less
available to the cells. 2) Cytotoxicity of Ag could increase mitochondrial activity in live
cells but still lead to some cell death. In this case, there might be a smaller number of live
cells that is not reflected in the mitochondrial activity. Therefore, prudent interpretation
and validation of LDH and MTS (MTT) results should be considered in nanotoxicology
studies.
Cell uptake was normalized against Ag concentration that was available to the cells
in the exposure media for both AgNP and AgNO3. Cell to supernatant ratio suggested that
for the same added Ag mass, lower uptake was detected in the AgNPs treatment than
AgNO3. It suggests that as per unit mass of Ag that was taken up by cells, NPs showed
more toxicity than Ag ions, or some other toxicity mechanisms that are involved in Ag
cytotoxicity. However, cell viability results show the same cytotoxicity for both AgNP
and AgNO3. The role of the epidermal growth factor receptor (EGFR) in toxicity of PM2.5
and diesel exhaust particles (DEP) was studied and an upregulation of EGFR ligand was
confirmed in human airway epithelial cells [176, 177]. Unfried et al. suggested that EGF
receptor activation by NPs may lead to different cellular outcomes such as apoptosis and
37
proliferation through the activation of the EGF receptor [178]. However, for engineered
NPs, EGFR signaling needs to be investigated more.
A moderate correlation between uptake and toxicity of AgNPs and AgNO3 was
displayed using the Pearson correlation coefficient (excluding person 5 from calculations).
Person 5 in the toxicity experiment showed a different trend compared to other samples. It
is not clear if this discrepancy was a result of variability between individuals or a result of
experimental error.
In summary, our study suggested that our complex exposure media (RPMI) could
alter NP characterization and properties that is a precursor for assessing their toxicity and
uptake. Comprehensive characterization of NPs in toxicology studies is an important factor
as there may or may not be a positive relationship between NPs and their potential toxic
effect.
2.6 LIMITATIONS
There are some methodological limitations for this study including, but not limited
to, a small sample size due to cost of sample collection and lack of time. In addition, no
information was provided by the blood bank regarding sex, race, specific sicknesses (that
could affect the ratio of white blood cells), age, body mass index (BMI), diet or different
ratio of cells in the blood (monocytes and lymphocytes) of donors.
38
Table 2.1 List of chemicals used in NP synthesis. Chemicals used in core-shell
(Ag@Au@Ag) and PVP-coated AgNPs synthesis.
No. Chemical
Name
Chemical
Structure
MW
(gramMol-1) Supplier
1 Ag 107 Ag 106.9 Isoflex
2 Ag 109 Ag 108.9 Isoflex
3 Ag Ag 107.9 Fisher Scientific
4 Sodium
borohydride NaBH4 37.83 Sigma-Aldrich
5 Sodium citrate C6H5O7.2H2O.3Na 294.10 Sigma-Aldrich
6 Nitric acid HNO3 63.01 Fisher Scientific
7 Gold (III)
chloride AuCl3 303.33 Sigma- Aldrich
8 PVP (C6H9O) n 10,000 Sigma-Aldrich
39
Table 2.2 Summary of Characterization of core-shell NPs (Ag@Au@Ag).
Characterization of core-shell NP synthesis in each step using DLS and UV-Vis.
DLS UV-Vis
Sample Size (nm) PDI Wavelength (nm)
AgNP seed 25.46 0.18 391
Ag@Au 26.31 0.23 440
Ag@Au@Ag 28.23 0.12 407
40
Table 2.3 AgNP characterization results. Characterization of PVP-AgNPs in UHPW.
DLS and TEM size are reported as mean ± standard deviation.
Parameter Measurement
DLS size (nm) 33.7 ± 0.7
Polydispersity index (PDI) 0.18
Position of maximum UV-Vis absorbance (nm) 390
TEM size (nm) 4.9 ± 0.3
41
Table 2.4 Mass balance of Ag in the exposure. Ag mass balance in the supernatant and
cell in AgNP and AgNO3 treatment measured by ICP-MS. Cell data were collected after
three washes with PBS and shows internalized and strongly bound Ag. Total loss indicates
loss of Ag during washes or experimental process. Ratio of cell to supernatant in percent
for Ag uptake.
Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.07 0.39 2.40 4.97
Supernatant 5.17 42.46 212.77 419.88
Total loss 4.76 57.15 285.62 576.15
Cell/Sup. (%) 1.35 0.91 1.12 1.18
AgNO3
Cell 0.06 0.36 4.56 10.85
Supernatant 5.49 59.77 120.88 166.46
Total loss 4.45 39.87 374.56 822.69
Cell/Sup. (%) 1.09 0.60 3.79 6.52
42
Table 2.5 Mass balance of Ag in the exposure for each person. Ag mass balance (ng) in
2.5×105 cells (absorbed or cell surface-attached) in AgNP and AgNO3 treatments for each
of 6-individuals measured by ICP-MS. Data are presented as mean± standard error (n=3).
Person 1 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.06 0.31 1.76 5.41
Supernatant 6.23 38.02 153.61 312.95
Total loss 3.71 61.67 344.62 681.63
AgNO3
Cell 0.05 0.32 4.54 8.14
Supernatant 5.85 57.03 70.21 119.42
Total loss 4.10 42.65 425.25 872.43
Person 2 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.06 0.34 2.13 4.57
Supernatant 3.59 33.17 163.68 425.14
Total loss 6.53 66.49 334.19 570.30
AgNO3
Cell 0.05 0.30 4.98 14.44
Supernatant 6.21 41.29 79.46 142.15
Total loss 3.74 58.40 415.56 843.42
Person 3 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.07 0.51 2.75 5.96
Supernatant 4.53 39.64 301.33 457.06
Total loss 5.40 59.85 195.93 536.97
AgNO3
Cell 0.06 0.42 4.97 6.95
Supernatant 4.78 43.79 83.65 198.16
Total loss 5.16 55.79 411.38 794.88
43
Table 2.5 Mass balance of Ag.
Person 4 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.07 0.46 3.58 6.78
Supernatant 4.55 48.19 206.93 404.74
Total loss 5.38 51.35 289.49 588.48
AgNO3
Cell 0.08 0.51 5.50 11.86
Supernatant 5.35 70.86 96.16 163.53
Total loss 4.57 28.64 398.34 824.61
Person 5 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.08 0.38 2.63 4.31
Supernatant 6.15 45.34 213.65 409.12
Total loss 3.76 54.28 283.73 586.57
AgNO3
Cell 0.08 0.39 4.47 13.54
Supernatant 5.19 69.46 114.16 158.99
Total loss 4.73 30.16 381.36 827.47
Person 6 Mass of Ag in exposure (ng)
10 100 500 1000
PVP-AgNP
Cell 0.07 0.32 1.56 2.79
Supernatant 5.99 50.41 237.40 510.26
Total loss 3.94 49.27 261.04 486.95
AgNO3
Cell 0.07 0.23 2.91 8.84
Supernatant 5.57 76.22 120.41 199.83
Total loss 4.36 23.55 376.68 791.32
44
Figure 2.1 Progressive formation of Ag@Au and Ag@Au@Ag NPs using UV-Vis.
a) Addition of Au solution to AgNPs to form Ag@Au NPs. As Au is added to the AgNP,
the Ag peak shift to higher wavelength that shows formation of Au layer. b) Formation
of Ag@Au@Ag by adding Ag to Ag@Au NPs. Progression of wavelength to lower
numbers as being coated by Ag.
b
a
45
Figure 2.2 AgNP characterization using DLS, UV-Vis, TEM and EDX.
Characterization of stock PVP-AgNPs using DLS (a), UV-Vis spectra (b), TEM images
with different magnifications (c and d), and EDX imaging (e).
a
c d
e
b
46
Figure 2.3 STEM images of 1000 µg L-1 PVP-AgNPs and particle size distribution
obtained by ImageJ. PVP-AgNP in RPMI media (without cells) at (a and b) T= 0, size
distribution= 29 ± 0.49 nm and (c and d) T= 24 hours, size distribution= 17 ± 11 nm in
the same condition as cell exposure (5% CO2 and 37°C incubator).
a
c
b
d
47
Figure 2.4 Behavior of PVP-AgNPs in media during 24-hour exposure. UV-Vis
spectra of PVP-AgNPs in RPMI media in (a) 1000, (b) 500 and (c) 100 µg L-1 PVP-
AgNP concentration as a function of time. The lowest concentration of exposure (10 µg
L-1 PVP-AgNP) did not show any signal due to detection limit of UV-Vis.
a
b
c
48
PVP-AgNP treatment
AgNO3 treatment
Figure 2.5 Dissolved and total Ag concentrations in PVP-AgNPs vs. AgNO3
during exposure time. Dissolved Ag concentration measurements using centrifugal
ultrafiltration units (a and c), and total Ag concentration (b and d) in RPMI media at
T=0 and T=24 hour measured by ICP-MS. All experiments were performed in
triplicate and are reported as mean ± SE (n=3).
a b
c d
49
Figure 2.6 Dissolved and total concentrations of Ag in AgNO3 in UHPW and media.
Dissolved Ag concentration were measured using centrifugal ultrafiltration units (a and
c) and total Ag concentration (b and d) in UHPW and RPMI media at T=0 and T=24
hours were measured by ICP-MS. Data are reported as mean ± SE (n=3).
a b
c d
50
Figure 2.7 Ag uptake (ng) in 2.5×105 cells. Results of absorbed or cell surface-attached
Ag in PVP-AgNP and AgNO3 treatments measured by ICP-MS after 24-hour exposure.
Data are presented as mean± SE (n=6). * statistically different from 10 µg L-1, ̂ statistically
different from 100 µg L-1, # statistically different from 500 µg L-1, and ¥ statistically
different when compared with AgNP treatment.
51
Figure 2.8 Ag uptake (ng) in 2.5×105 cells for each person. Results for absorbed or
cell surface-attached Ag after treatment of PBMCs in 6 individuals. The amount of Ag
in cells after 3 washes with buffer that was measured by ICP-MS.
52
Figure 2.9 Effects of PVP-AgNP, AgNO3, AgNO3-PVP, and PVP on PBMCs. PBMCs
were exposed for 24 hours to different concentrations of PVP-AgNP, AgNO3, AgNO3-
PVP, and PVP (0-1000 µg L-1) in RPMI. The effect on cell viability was measured using
LDH leakage (a). Cell metabolic activity was assessed by MTS assay (b) and results is
expressed as percentage of reduction values of untreated cells. Data are presented as mean
± SE as the average of six independent experiments (n=6). * shows statistically significant
difference from the control (0), and # shows statistically significant difference when
compared with PVP-AgNP (p < 0.05).
a
b
53
Figure 2.10 Cell viability results for each person. Cell viability of PBMCs assessed
by LDH leakage after 24 hours treatment with representative concentration of PVP-
AgNP, AgNO3, AgNO3-PVP, and PVP. Data represents the means of six replicates ±
standard error.
54
Figure 2.11 Cell metabolic activity results for each person. Metabolic activity of
PBMCs measured by MTS assay after 24 hours exposure at the representative
concentrations for 6 individuals. Data represents the means of six replicates ± standard
error.
55
Figure 2.12 Pearson correlation between uptake and toxicity of Ag. Correlation
between uptake and toxicity of Ag in AgNP (a) and AgNO3 (b and c) treatment. There was
a moderate positive correlation between the two variables (uptake and toxicity) for AgNPs:
r =0.56, p = 0.01 (a) and AgNO3 excluding person 5: r =0.52, p = 0.04 (b). No correlation
was detected in AgNO3 including person 5: r = 0.314, p = 0.2 (c).
a
b
c
56
CHAPTER 3
CAN PVP-COATED IRON OXIDE NANOPARTICLES BE USED TO
PREVENT WEIGHT GAIN IN A HIGH-FAT DIET MOUSE MODEL
57
3.1 ABSTRACT
Obesity has become a public health concern for developed countries. More than
one-third of the adult U.S. population is considered as obese. Current obesity treatments
are inefficient and may have harmful side effects and consequences. However,
nanotechnology offers new applications in medicine and drug delivery. Iron oxide
nanoparticles (NPs) have the ability to be used as nanocarriers and as contrast agents in
nanomedicine and magnetic resonance imaging, respectively. In addition, they have been
recently used for oil remediation from oil-water mixture. This study was performed to
investigate if iron oxide NPs have the potential to remove dietary fat and promote weight
loss in mice fed a high-fat diet. Polyvinylpyrrolidone (PVP)-coated iron oxide NPs were
synthesized and administered orally to mice through food ingestion. Three different doses
of PVP-coated iron oxide NPs were administered in the mice diet (≈ 1 mg/kg body weight
(low-nanoparticle, LNP; n=10), ≈ 5 mg/kg body weight (medium-nanoparticle, MNP;
n=10) and ≈ 20 mg/kg body weight (high-nanoparticle, HNP; n=10); n=10/group). Control
groups consisted of mice consuming a low-fat diet or high-fat diet (n=10) without the
addition of NPs. Mouse body weight and food intake was recorded weekly for 16 weeks.
After 16 weeks of diet, body composition and glucose tolerance test were assessed. Iron
accumulation in the liver, kidney, heart, brain, as well as feces was measured using ICP-
MS. Liver was investigated for lipid accumulation and the presence of inflammatory
mediators via qRT-PCR. There was a significant effect of HFD to increase mice body
weight relative to LFD control. However, no significant differences among the HFDs in
mice body weights was detected. LNP treatment resulted in an increase in fat mass and
body fat percentage when compared to all other groups. There was a high fat diet effect to
58
increase fasting blood glucose levels and lipid accumulation in the liver compared to LFD
control group. Although, no differences in fasting blood glucose levels and lipid
accumulation among the HFD treated groups was observed. A dose-dependent increase in
iron excretion was detected in the feces of the mice treated with nanoparticles.
Additionally, iron concentration was significantly elevated in the liver of the high
nanoparticle group relative to the high-fat diet control group. A significant decrease in
macrophage marker (EGF-like module-containing mucin-like hormone receptor-like
1(EMR1)) and macrophage recruitment marker (Monocyte chemoattractant protein 1
(MCP-1) was found in high nanoparticle treatment when compared with high-fat diet
group. This study suggests that consumption of iron oxide nanoparticles in high-fat diet
food (in the dose up to 20 mg/kg) does not positively effect body weight. However, it
might paradoxically modulate macrophages and glucose metabolism.
3.2 INTRODUTION
Obesity is a universal epidemic. Due to the consumption of high-fat diets and
increasing inactive lifestyles, the number of obese individuals has increased greatly across
the world. It has been reported that in the U.S. alone more than one third of the adult
population are obese [179]. Consequently, there has been a concern about health problems
related to obesity such as diabetes, cancer, metabolic syndrome, and cardiovascular
diseases and an increase in health care costs [180]. As a result, multiple weight-loss
strategies have been developed including medication and surgery. Medications such as
Orlistat (Xenical) and Qsymia are used as treatment for morbid obesity, but they also can
59
have serious side effects such as increase heart rate , oily rectal leakage [181], liver injury
[182], insomnia and dizziness [183]. Currently, there is no non-surgical method that can
both achieve and sustain major weight loss. Surgical options, such as bariatric surgery and
liposuction, promote rapid weight loss. However, this invasive treatment can lead to post-
operative complications including acid reflux, infection, ulcers, bowel obstruction, and
weight gain [184, 185].
Nanoparticles (NPs) have been broadly used in different fields such as
nanomedicine and drug delivery. They have a unique characteristic that is related to their
small size which increases their surface to volume ratio providing them novel physical and
chemical properties. NPs are used widely in personal care, as well as in food and medical
products. Among the variety of NPs used in biomedical applications, iron oxide NPs have
attracted significant attention due to their small size and stable magnetic characterization.
Iron oxide NPs have been used in many applications such as, in vivo NMR technology
[186], nanocarriers in drug delivery, contrasting agents for magnetic resonance imaging
(MRI) in clinical diagnosis [187-189] and gene delivery [190]. Recently, PVP-coated Iron
oxide NPs have been used successfully in oil remediation from oil-water mixtures [1].
Iron oxide NP toxicity has been examined in vitro [108, 110, 191, 192] (using
different cell lines) and in vivo [193-195]. An increase in lipid peroxidation in RAW
macrophages after 2 hours incubation with iron oxide NPs was reported by Stroh et al.,
[186] which was abolished after 24 hours. They also demonstrated that iron oxide NPs
does not cause any long term cytotoxicity, even though they are uptaken by RAW cells
[186]. Moreover, exposure of iron oxide NPs (10-20 µg mL-1) on murine (IL-4-activated
bone marrow derived) and human (promonocytic THP-1 cells) macrophage models did not
60
cause cell toxicity after 24 hour (up to 100 µg mL-1 exposure) despite the internalization of
NPs, triggeration of cell activation and alteration in iron metabolism [196]. On the other
hand, intraperitoneally administration of iron oxide NPs was shown to cause cardiotoxicity
through oxidatative stress in albino mice [193]. The discrepancy observed in iron oxide
NP toxicity in the literature could be explained by many factors including different coating
of NP, size of NPs, routes of exposure, frequency and duration of exposure.
Our study was designed to investigate the efficiency of iron oxide NPs as an
effective treatment to reduce fat from high-fat diet mice model. The specific objectives
include;
1) Investigate if our FT synthesized PVP-coated iron oxide NPs are capable to
remove oil in vitro. We hypothesized that adding PVP-coated iron oxide NPs
to synthetic seawater and stomach solution will remove oil efficiently. To test
this, we measured and calculated oil removal percentage using a
spectrofluorometer.
2) Investigate if there is any difference in food consumption between different
diets (with and without NPs). We hypothesized that there is no difference
between food intake with different diets. We studied this by measuring food
intake weekly.
3) Investigate if iron oxide NPs are capable to induce weight loss in a pre-clinical
model of obesity. We hypothesized that mice that consume a high-fat diet rich
in NPs will loss body weight due to the removal of fat by NPs. To test this
hypothesis, we assessed body weight (weekly), body composition (DEXA),
and weight fat depots at sacrifice.
61
4) Investigate if the iron that is consumed in the food will be excreted in the feces
or accumulated in tissues (liver, kidney, heart, and brain). We hypothesized
that part of the excess iron will be excreted through the feces. To test this
hypothesis, we measured iron content in tissues and feces at the end of the
study.
5) Investigate if iron oxide NPs are capable to reduce lipid accumulation and
inflammation in the liver and improve glucose metabolism in a pre-clinical
model of obesity. We hypothesized that mice that consume a high-fat diet rich
in NPs will reduce hepatic lipid and improve glucose metabolism compared
with high-fat diet. To test this hypothesis, we assessed hepatic lipid
accumulation, measured macrophage and inflammatory markers, fasting blood
glucose, and glucose tolerance in mice fed high-fat diet mixed with NPs.
To our knowledge, this is the first study which used engineered iron oxide NPs to
examine fat removal and body weight loss in a mouse model of obesity.
3.3 METHODS
3.3.1 Synthesis and characterization of iron oxide NPs
Different synthesis methods have been established to produce iron oxide NPs for
environmental remediation. In this study, we used a hydrothermal flow-through synthesis
(FT) method developed by Kadel et al. [197], to produce cost-effective large-scale
synthesis of iron oxide NPs. In brief, 1.5 mmol ferric chloride (FeCl2.4H2O, 98%, Alfa
Aesar), 4 mmol ferrous chloride (FeCl3.6H2O, >98%, BDH) and 0.3 mmol
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polyvinylpyrrolidone (PVP) (Mw 10 kDa, Sigma-Aldrich) were dissolved in 6.25 mL
UHPW at 90°C. Iron salts and PVP solution was pumped continuously into the reaction
chamber along with 6.25 mL ammonium hydroxide (NH4OH, 28-30%, BDH) at the same
flow rate. The reaction chamber was kept at 90°C inside a water bath. A black precipitate
of iron oxide NPs that came out of the chamber was collected. NPs were washed with
UHPW using magnetic decantation and re-dispersed in UHPW with sonication. Iron salts
were used as precursors and NH4OH was used as a precipitation agent. A schematic of a
typical FT synthesis is shown in Figure 3.1.
Z-average hydrodynamic diameter and polydispersity index (PDI) of iron oxide
NPs were measured using dynamic light scattering (DLS) with a Malvern Zetasizer
NanoZS (Malvern Instruments, MA, USA). The mean of at least eight consecutive
measurements at 25°C after 2 minutes of temperature equilibrium was reported. PDI is a
measurement of the quality of NP size distribution. Zeta potential (colloidal stability) of
NPS was measured by a Laser Doppler Electrophoresis (Malvern Zetasizer NanoZS, MA,
USA).
Total iron concentration was measured using inductively coupled plasma optical emission
spectrometer (ICP-OES; Varian 710-ES) after washing and removing iron dissolved
residual.
3.3.2 Oil removal from water
Oil separation in seawater was performed using 0.15 g L-1 oil-water mixture. Oil
concentration was chosen based on previous literature data [198]. Synthetic seawater (30
ppt) was prepared following the U.S. Environmental Protection Agency (EPA-821-R-02-
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012) protocol [199]. Crude oil, which is representative of the Deepwater Horizon spill
(reference MC252 surrogate oil; sample ID: A0068H, Aecom Environment), was used for
oil-water mixture removal experiment. Oil was mixed with seawater and sonicated for 30
minutes. Iron oxide nanoparticles were then added to the oil mixture to obtain suspension
of 14 mg L-1 of the NPs and sonicated again for 5 minutes. NPs were separated using a 1
½ inches cubic neodymium magnet (magnets of Grade N 52, K&J Magnetics INC.) for 1-
hour separation time. After separation, solution was collected for fluorescence
spectroscopy measurements. Emission spectra of samples were measured at excitation
wavelength of 337 nm and emission range from 350 to 650 nm using a Horiba Jobin Yvon
Fluorolog-3 spectrofluorometer. These wavelengths were used for detecting polycyclic
aromatic hydrocarbons (PAHs) and other aromatics [200, 201].
3.3.3 Oil separation from stomach solution (in vitro)
The physiologically extraction test was done using human gastric solution and three
different oil samples: vegetable oil (Crisco), canola oil (Carnili) and olive oil (Pompian)
purchased from grocery store. The gastric solution was prepared following Turner and IP
protocol [202]. Briefly, 0.5 g of sodium malate, 0.5 g of sodium citrate, 1.25 g of pepsin
A, 500 µL of acetic acid and 420 µL lactic acid was dissolved in 1 L of UHPW. The pH
was adjusted as 2.5 by adding HCL and labeled as gastric solution. Five mL of gastric
solution was centrifuged at 3500 rpm for 10 minutes. Supernatant was collected and stored
in a 25 mL volumetric flask. UHPW (5 mL) was added to the pellet, centrifuged again
(3500 rpm for 10 minutes) and supernatant was transferred to a 25 mL flask. The flask
solution was then diluted to mark with UHPW. The following mix was used as stomach
solution. A 23 mg from each oil was then added to 10 mL of stomach solution. PVP-
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coated iron oxide NPs were added for a final suspension of 20 mg L-1 of NPs. This mixture
was placed in an incubator/shaker at 37°C for 2 hours. Then NPs were separated from the
suspension using a magnet for 30 minutes. Emission spectra of samples was recorded
using fluorescence spectroscopy at the same wavelength as the seawater oil removal
experiment. The stomach solution and the sample before NP treatment were used as
control.
3.3.4 Animal
Pathogen-free, adult C57BL/6J (wild-type) male mice (n=50) were obtained from
Jackson Laboratory (Bar Harbor, Maine). Mice (n=10/group) were housed five per cage
in a temperature-controlled room at 23-24°C maintained on a 12h light/dark cycle and they
had ad libitum access to food and water for the duration of the experiment. All animals
were treated according to the National Institutes of Health (NIH) Guide for the Humane
Care and Use of Laboratory Animals and local Institutional Animal Care and Used
Committee (IACUC) standards. All experiments were approved by the IACUC of the
University of South Carolina.
3.3.5 Diets
Mice at 4 weeks of age were randomly divided into 5 groups with 10 mice in each
group. Mice were assigned to one of five treatment diets as follows: low-fat diet of 10%
kcal fat (LFD, AIN-76A), high-fat diet of 40% kcal (HFD, SF 12% kcal, USF 28% kcal)
and three HFD supplemented with: low concentration of PVP-coated iron oxide NP (LNP,
1 mg kg-1 NPs), medium NP concentration (MNP, 5 mg kg-1 NPs) and high NP
concentration ( 20mg kg-1 NPs) (BioServ, Frenchtown, NJ). Diet ingredients was chose
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based on a typical content of fat in american diet. The doses of PVP-coated iron oxide NPs
were chosen based on previous literature [107, 108, 193, 203] in in vivo and in vitro animal
and human studies [204]). The doses selected to use in our study were of physiological
relevance in humans, making them lower than the dose administered in other studies in
rodents [193, 194, 203].
3.3.6 Body weights, food intake and body composition
Body weight and food intake were recorded on a weekly basis after starting the
treatments for 16 weeks. Body composition was assessed at the end of treatments (16
weeks). For this procedure, mice were briefly anesthesized (using isoflurane) and lean
mass, fat mass and percent body fat were measured using a Dual- Energy X-ray
Absorptiometry (DEXA, LUNAR, Madison, WI).
3.3.7 Glucose Tolerance Test
A glucose tolerance test (GTT) was performed at week 16 after 5 hours of fasting
the animals. After the determination of fasting blood glucose levels, each animal received
an intraperitoneal glucose average 1.5 g/kg lean body mass of glucose (D-glucose, Sigma,
St.Louis, MO). Blood glucose levels were measured after 15, 30, 60 and 120 minutes using
glucometer (Bayer Contour, Michawaka, IN).
3.3.8 Tissue collection
After 16 weeks of dietary treatment, mice were sacrificed under anesthesia (using
isoflurane) for tissue collection. Liver, kidney, brain, heart, epididymal, mesentery as well
as kidney fat pads were collected, weighed and immediately frozen in liquid nitrogen.
Whole blood was collected from the inferior vena cava using heparinized syringes, and
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plasma was separated from blood samples using a centrifuge at 4°C at 4000 rpm for 10
minutes. Tissue and plasma samples were stored at -80°C for further experiments.
3.3.9 Iron content measurement in tissues and feces
Iron content in the selected tissues (kidney, heart, liver, brain) and feces was
quantified using Finnigan ELEMENT XR double focusing magnetic sector field
inductively coupled plasma mass spectrometer (SF-ICP-MS). Tissues were weighed, cut
and digested using concentrated nitric acid (70% HNO3) followed by heating on a heat
block using sealed Teflon tubes. Samples were then diluted to 1% acid and filtered (if
needed) prior to the measurement of iron concentration with SF-ICP-MS. Rh was used as
internal standard and the detection limit was calculated as 0.018 µg/L iron. The validity of
the analytical method was checked every five measurements with two quality controls
(blank and standard). A serial dilution of iron standard solution (High Purity Standard,
1000 µg mL-1) was used for calibration curve.
3.3.10 Hepatic lipid extraction
Lipids in the liver were isolated using modified Folch extraction method [205] as
previously described [206, 207]. Liver tissues were weighed and homogenized using 2:1
chloroform-methanol (v/v) in 5 mL tubes. After adding NaCl solution and centrifugation,
a biphasic system was obtained and the clear solution at the bottom of the tube that
contained most of all the tissue lipids was collected in the glass bottles. Samples on the
chloroform layer were dried using nitrogen gas in glass bottles and lipids were assessed
gravimetrically.
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3.3.11 Liver gene expression
Quantification of liver gene expression for selected macrophage markers (EMR1)
and inflammatory mediators (tumor necrosis factor (TNF-α), monocyte chemotactic
protein (MCP-1), and interleukin (IL)-6) were performed. Liver samples were
homogenized and total RNA was extracted using Trizol reagent (Invitrogen, Calsbad, CA).
The extracted RNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water and
quantified spectrophotometrically at 260 nm wavelength. RNA was used to synthesize
cDNA and quantified RT-PCR analysis was done using Taqman Gene Expression assays.
Potential reference genes hydroxymethylbilane synthase (HMBS) and H2A histone family
member V (H2AFV) were used as internal control for liver tissue.
3.3.12 Measurement of vitamin A and E in plasma
Plasma samples were used to quantify lipid soluble vitamins (A and E) using LC
MS/MS. * Still waiting for results.
3.3.13 Statistical analysis
The data were analyzed using Prism 7 (GraphPad Software, USA). Comparisons
were tested with one-way analysis of variance (ANOVA) followed by a Student-Newman-
Keuls post hoc analysis. Differences were considered statistically significant when p <
0.05 in all experiments. Results are expressed as the mean ± standard error.
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3.4 RESULTS
3.4.1 NP synthesis and characterization
PVP-coated iron oxide NPs were synthesized using a continuous hydrothermal FT
following Kadel et al.’s protocol. The mean hydrodynamic diameter of NPs was measured
using DLS in water and was recorded as 116 ± 4.9 nm with an average PDI of 0.26 (Figure
3.2). Zeta-potential was measured as 10.6 ± 0.8 mV from 10 repeated measurements. Other
characterization data are reported in the previous study [197].
3.4.2 Oil separation from seawater using PVP-coated iron oxide NPs
To check the efficiency of NPs and compare that with previous studies, oil removal
property of PVP-coated iron oxide NPs was analyzed using synthetic seawater. Figure 3.3
shows the fluorescence spectra for the seawater with and without oil, before and after NP
treatment. The concentration of NP that was used for oil separation was 14 mg L-1. A
significant decrease in fluorescence spectra for seawater after NP treatment was observed.
Results showed the percentage of oil removal as 94% from seawater.
3.4.3 Oil separation from stomach fluid using iron oxide NPs
The oil removal in the stomach solution was examined using canola, olive and
vegetable oil. Figure 3.4 shows a significant reduction in fluorescence spectra for all three
oils that were used. According to these results, the percentage of oil removal was
calculated as 85%, 84% and 89% in canola, vegetable and olive oil, respectively.
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3.4.4 Body weight and fat pad weights
Body weight and fat pad weights are presented in Figure 3.5. Mice were fed LFD,
HFD, or HFD supplemented with different concentrations of PVP-coated iron oxide NPs
for 16 weeks. The mice consuming HFD and LNP had significantly elevated body weights
compared with LFD (control diet) starting at week 2 (p < 0.05). This effect began later for
HNP and MNP mice (week 4 and 5 compared with LFD, respectively) (p < 0.05). No
significant difference in body weight was observed between HFD and HFD supplemented
with different concentrations of PVP-coated iron oxide NPs througout the study.
Measuring individual food intake for each mouse was not possible, because they
were housed five mice per cage. However, no significant differences was observed
between all diets in weekly food intake during the study.
For all fat-pad depots, the HFD-fed mice showed an increase in fat mass compared
with LFD-fed mice (control) (p < 0.05) (Figure 3.5c). Although, there was no significant
difference between HFD and HFD supplemented with NPs in epididymal fat weight, the
LNP treated group showed a greater mesentery and peri-renal fat weight than HFD, MNP
and HNP (p < 0.05).
3.4.5 Body composition
Body composition analysis was performed after 16 weeks of diet (Figure 3.5d). In
the HFD and HFD supplemented with NPs-fed mice, fat mass, and fat percentage had
increased significantly when compared to control group (LFD) (p < 0.05). LNP group had
enhanced fat mass and fat percentage compare to HFD, MNP and HNP mice (p < 0.05).
There were no significant differences in body fat and fat percentages between HFD, MNP,
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and HNP-fed mice. No differences in lean mass was observed between groups at the end
of the study.
3.4.6 Effect of PVP-coated iron oxide NP diet on glucose metabolism
Glucose tolerance test (GTT) was performed on mice after 16 weeks of diet. HFD
and HFDs supplemented with iron oxide NPs-fed mice showed elevated fasting blood
glucose (Figure 3.6a) and GTT AUC (area under the curve) (Figure 3.6c) when compared
to control group (LFD) (p < 0.05). No significant difference was observed in the fasting
blood glucose between HFD and HFD with NPs. LNP and MNP treatments also displayed
elevated GTT AUC compared with HFD, demonstrating a dysfunctional glucose
metabolism (p < 0.05). There was no significant difference in GTT AUC between HFD
and HNP-fed diet treatment.
3.4.7 Iron content in feces and tissues
As expected, we observed similar iron content in the feces of LFD and HFD treated
groups. A significant dose dependent increase in iron content was observed in the feces
(Figure 3.7a) of PVP-coated iron oxide NP treated animals when compare to LFD and HFD
groups (p < 0.05). Then, we investigated if PVP-coated iron oxide NPs accumulated in the
brain, heart, kidney, and liver. For this we selected only the HNP group as they should
have the highest amount of iron. HFD group was used as a control to compare iron
accumulation in tissues. No changes in iron content was observed in the brain, heart, and
kidney between HNP and HFD treated mice. However, we observed a significant increase
in the iron content in the liver of HNP treated mice when compared to HFD group (Figure
3.7b) (p < 0.05).
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3.4.8 Hepatic lipid accumulation and inflammatory markers
To determine if PVP-coated iron NPs were able to remove lipid content and
decrease inflammation in the liver, we measured liver mass, hepatic lipid accumulation,
and the gene expression of macrophages (EMR1), a chemokine (MCP-1), and cytokines
(TNF-α and IL-6) markers. HFD and HFDs supplemented with NPs treatments displayed
a significant increase in the liver weight (hepatomegaly) (Figure 3.8a) (p < 0.05) and
accumulation of lipids in the liver (hepatic steatosis) when compared to LFD treatment (p
< 0.05). Mice treated with different concentrations of NPs did not show any significant
difference between liver weight and hepatic lipid accumulation when compared to HFD
treatment (Figure 3.8b).
Inflammatory markers was assessed via RT-PCR in LFD, HFD, and HNP treated
mice. HFD treated mice displayed an increase in EMR1, MCP-1, and TNF-α expression
when compared to LFD treatment (Figure 3.8c and d, p < 0.05). HNP treatment
significantly decreased gene expression of EMR1and MCP-1 when compared to HFD
group (Figure 3.8c and d, p < 0.05). Despite a reduction in EMR1 in the liver of HNP
treated mice, no effect was detected in the gene expression of TNF-α and IL-6 compared
with HFD group (Figure 3.8e). No significant difference was observed in IL-6 gene
expression across examined groups (Figure 3.8f).
3.5 DISCUSSION
Obesity is one of the current health challenges in the America, mainly because it is
associated with inflammatory metabolic disorders (such as insulin resistance, steatosis
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hepatis and type 2 diabetes) [208]. Current treatments (including non-surgical and surgical
therapies) have their own limitations and side effects [182-185]. Nanomaterials have been
considered as possible medicines for gene/drug delivery and imaging [209]. Among
nanomaterials, iron oxide NPs have attracted a great deal of attention, especially in imaging
and nanomedicine because of their small size and magnetic properties [210]. Recently,
PVP-coated iron oxide NPs have been used for oil removal [1] and results indicated
excellent removal efficiencies under laboratory conditions from aqueous solutions [1].
However, it is not known if in complex scenarios such as, in vivo systems, whether PVP-
coated iron oxide NPs are capable to remove excess oil/fat from animals. In this study, we
tested for the first time the ability of PVP-coated iron oxide NPs (synthesized in house) to
remove dietary fat in in vivo model using mice fed with HFD.
PVP-coated iron oxide NPs were synthesized using a hydrothermal FT [197].
According to DLS data and distribution profile, it is possible to conclude that NPs
presented a monodisperse NPs (mean size of 116 ± 4.9 nm, PDI: 0.2). The narrow DLS
peak suggests a homogenous system with size close to the mean. Zeta-potential, which
was measured as 10.6 ± 0.8 mV, confirmed steric stabilization of NPs by PVP and an
absence of a significant charge on the NPs [149]. The synthesis procedure of iron oxide
NPs is influenced by many factors such as time, solution pH and temperature; therefore,
synthesis should be controlled carefully to obtain consistent and reproducible NPs [211].
The magnetic properties of iron oxide NPs allow separation of NPs from water and
oil mixture using a magnet. Hydrophobic PVP, which is a non-toxic polymer [150] and
coating agent, is capable of absorbing oil through interaction between hydrophobic fraction
of PVP and hydrocarbons [197]. It is suggested that hydrophobic moieties of PVP coating
73
absorbs oil from the solution onto the NP surface and then the magnetic property of iron
oxide NP easily separates NPs associated with oil from the solution [197]. Previously it
was shown that PVP-coated iron oxide NPs are capable of removing almost all aromatic
and a majority of the aliphatic oil components [212]. In this study, we tested the ability of
our synthesized PVP-coated iron oxide NPs to remove oil from synthetic seawater; oil
removal percentage was calculated as 94%, which is acceptably close to the number which
Kadel et al., and Mirshahghassemi et al., achieved (got more than 98% of oil removal in
the same media) [1, 197]. In vitro experiments that were performed using stomach solution
and three different oils resulted in 85%, 84% and 89% oil removal for canola, vegetable
and olive oil, respectively, which shows an acceptable percentage of oil removal. The
reduction in the efficiency for the oil removal may be because of the synthesis method (FT
synthesis) that was used to increase the efficiency and decrease the cost of synthesis. This
problem could be solved by adding larger masses of iron oxide NPs and/or increasing the
separation time.
To obtain comprehensive information on the efficiency of NPs on fat removal in
vivo, we administered different concentrations of PVP-coated iron oxide NPs (1, 5 and 20
mg/kg mouse body weight) in the food. Additionally, we used a LFD and HFD without
NPs as control groups. After 16 weeks of diet-treatments we observed no significant
differences in body weight of mice treated with NPs when compared between themselves
and to the HFD control group. LNP treatment showed higher total fat pad, fat mass and
body fat percentage compared with other diets. However, no changes were observed in
their body weight, lean mass and liver weight. This leads us to suggest that perhaps the
low concentration of PVP-coated iron oxide NPs is accumulating in the fat tissue and might
74
be contributing to the increase of fat mass in these mice. We believe we did not see increase
in fat mass in MNP and HNP mice because NPs at these concentrations might have some
aggregation. We could visibly observe NPs aggregation in the food of medium and high
NP concentration (Figure 3.9). If NPs are aggregated even before mice consumption, this
could affect the ability of NPs to bind to the oil/fat and decrease NP efficiency. A more
specific oil binding phase on the particles, rather than only PVP might help with
aggregation and the oil removal property of iron oxide NPs.
Obesity is associated with an increase in the prevalence of type 2 diabetes,
hypertension and impaired glucose metabolism. To assess if a diet supplemented with
PVP-coated iron oxide NPs influence glucose metabolism, we measured fasting blood
glucose at the end of 16 weeks. It has previously been shown that HFD feeding elevates
blood glucose levels and cause insulin resistance in rodents [213]. Our results showed that
NP treatment did not mitigate impaired glucose metabolism. Surprisingly, LNP and MNP
treatment showed a greater degree of impaired in glucose metabolism (GTT AUC) than
HFD treated mice. Once again, this effect on HNP mice might have been hindered due to
increases in aggregation in the diet.
Iron was measured in the feces and selected tissues (brain, heart, kidney and liver)
to investigate the fate of iron in the body. In the feces, a dose dependent increase in the
iron excretion was found as the concentration of iron in the diet increased. Considering
the iron background in the LFD and HFD (that was added to the food as a supplement by
the food company), we can conclude that most of the iron that is excreted through feces is
from NP consumption (48%, 69% and 81% for LNP, MNP and HNP, respectively). In the
iron measurements from selected tissues, no significant difference was observed in brain,
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heart, kidney. However, liver from HNP treated mice showed higher accumulation of iron
than the liver of HFD mice (LNP and MNP were not measured) which suggests iron is
taken up by the liver cells. Our result is consistent with previous studies who found more
NPs in the liver after treatment with iron oxide NPs [214-216]. A biodistribution study of
DMSA-iron oxide NPs in vivo showed that NPs are transformed to organs that are
responsible for iron regulation (liver and spleen) [217]. The ability of liver to uptake more
NPs is referred to as an active mononuclear phagocyte system (specially Kupffer cells)
which works as an immune network to remove alien material [218]. In addition, due to the
large surface area of NPs, the interaction between macrophages and NPs could be
facilitated, leading to fast recognition and clearance by macrophages. This was shown by
Stroh et al., who reported a massive uptake of iron NPs by RAW macrophages without any
effect on cell viability [186]. However, more studies are needed to better understand the
interaction between PVP-coated iron oxide NP treatment and glucose metabolism. A
limitation of the study is that we were not able to calculate iron mass balance due to the
study design.
Obesity is considered as a low-grade inflammatory disease that is characterized by
infiltration of macrophages in the tissues, which leads to inflammatory mediators secretion
(such as TNF-α and IL-6) [219, 220]. Interestingly, we found a significant decrease in
gene expression of macrophage (EMR1) and macrophage chemokine (MCP-1) markers in
the liver tissue of HNP treatment relative to HFD (almost as low as LFD group). Others
have reported an increase in monocyte recruitment in bronchoalveolar macrophages
exposed to DMSA-coated iron oxide NPs [221]. IL-6 is known as a factor that mediates
inflammatory responses in obesity and its elevated levels in plasma was reported before
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[222]. However, we did not find any increase in IL-6 in the liver which is consistent with
previous studies [220, 223]. TNF-α as an inflammatory factor exhibited an increase in the
HFD-fed mice, as was shown previously [220]. However, we could not detect any
significant difference in TNF-α between HFD and HNP groups. The liver is recognized as
the main organ of iron deposition and cell injury [224]. TNF-α has potent proinflammatory
and cytotoxic effects and is the major mediator secreted form Kupffer cells that are hepatic
resident macrophages. Continued induction of TNF-α expression is an important feature
of chronic inflammatory liver disease [225] and the results of She et al., showed iron up-
regulate expression of TNF-α in Kupffer cells [226]. The inability to detect a decrease in
TNF-α gene expression in the liver (despite the decrease in the macrophage gene
expression) may be due to the ability of other cells (such as hepatocytes) to compensate for
the macrophages and secrete more TNF-α. However, a more comprehensive analysis is
needed to determine the reason for the significant decrease in hepatic macrophages and
stable gene expression of IL-6 resulting from PVP-coated iron oxide NP treatment. One
limitation of this study was the gene expression measurement, which was performed only
by qRT-PCR.
Although our in-vitro tests showed promising fat removal results using PVP-coated
iron oxide NPs from stomach solution, our data in a more complex model in-vivo was not
able to mimic this effect. Oral exposure of PVP-coated iron oxide NPs mixed in a high-
fat diet at concentrations up to 20 mg/kg body weight did not show any effect on body
weight loss and/or fat accumulation in the liver. In fact, low and medium concentration of
NPs might aggravate glucose metabolism. The discrepancy between in-vivo and in-vitro
model could be due to NP aggregation in HFD, ingestion and excretion process, and
77
interaction of PVP-coated iron NPs with other cells. Therefore, further studies, such as,
optimizing routs, concentration and surface modifications of NPs are necessary to
corroborate if iron oxide NPs are capable to remove oil/fat in in-vivo systems.
78
Figure 3.1 Schematic diagram of flow-through synthesis of PVP-coated iron oxide
NPs (figure not on scale). Adopted from Kadel et al. 2018.
79
Figure 3.2 Size characterization of iron oxide NP. DLS size distribution plot indicated a
mean size of 116 ± 4.9 nm.
80
Figure 3.3 Fluorescence spectra for the seawater before and after oil removal. Oil
removal efficiency was calculated as 94% from synthetic seawater.
81
Figure 3.4 Fluorescence spectra of oil removal from stomach fluid using iron oxide
NPs. Oil was removed from stomach solution after 1-hour separation time using a magnet.
Percent oil removal was calculated as: canola oil: 85%, vegetable oil: 84% and olive oil:
89%.
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Figure 3.5 Body morphology and food intake after 16 weeks of treatment. Body
weight, food intake and fat pad weights after 16 weeks of treatment (low-fat diet (LFD),
high-fat diet (HFD), low nanoparticle (LNP), medium nanoparticle (MNP) and high
nanoparticle. Mice were fed for 16 weeks. a) body weight; b) food intake; c) fat pad weight;
and d) body composition measured by dual-energy X-ray absorptiometry (DEXA). Data
are presented as means ± SE; n =10 mice per group. Bar graphs not sharing a common
letter are significantly different (p < 0.05).
a
b
c d
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Figure 3.6 Metabolic data after 16 weeks of treatment. Mice were administered with
(low-fat diet (LFD), high-fat diet (HFD), low nanoparticle (LNP), medium nanoparticle
(MNP) and high nanoparticle for 16 weeks. a) fasting blood glucose levels (5h); b) glucose
tolerance test (GTT); and c) corresponding area under the curve (AUC). Values are
presented as mean ± SE; n =10 mice per group. Bar graphs not sharing a common letter
differ significantly from each other (p < 0.05).
a b
c
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Figure 3.7 Iron measurement in feces and tissues using ICP-MS after 16 weeks of
dietary treatment. Iron measurement after 16 weeks of treatment ((low-fat diet (LFD),
high-fat diet (HFD), low nanoparticle (LNP), medium nanoparticle (MNP) and high
nanoparticle (HNP), in a) feces and b) brain, heart, kidney and liver. Values are presented
as mean ± SE; n= 4 mice per group. Bar graphs not sharing a common letter differ
significantly (p < 0.05).
a
b
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Figure 3.8 Hepatic lipid accumulation and inflammatory mediators. a) liver weight
after euthanasia, low-fat diet (LFD), high-fat diet (HFD), low nanoparticle (LNP), medium
nanoparticle (MNP) and high nanoparticle (HNP); b) hepatic lipid accumulation; c) hepatic
gene expression of EMR1; gene expression of hepatic inflammatory mediators: MCP-1
(monocyte chemoattractant protein-1, TNF-α (tumor necrosis factor-α) and IL-6
(Interleukin-6) normalized to the HMBS and H2AFV. Data are presented as mean ± SE; n
= 10 mice per group. Bar graphs not sharing a common letter are significantly different (p
< 0.05).
a b
c
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Figure 3.9 Mice diet. Mice diet modified as: Fat (40% kcal); SF (12% kcal), USF (28%
kcal), a) high nanoparticle; b) medium nanoparticles; c) low nanoparticles.
a
b
c
87
CHAPTER 4
OVERALL CONCLUSION
88
The purpose of this dissertation was to investigate the effect and application of Ag
and iron oxide NPs in mammals (using human blood cells and mice model, respectively).
This aim was achieved by:
A) Investigating the uptake and cytotoxicity of AgNP and AgNO3 (as control) to human
PBMCs.
The overall conclusions are summarized as below:
1) Uptake and toxicity of AgNPs and AgNO3 were investigated using well-
characterized PVP-coated AgNPs on human PBMCs. In summary, our study
showed that PVP-AgNPs are more stable in the RPMI media than Ag ions that
could be associated with a decrease in the bioavailability of Ag ions to the cells.
2) We determined that a combination of AgNP transformations is happening in the
complex cell culture media that influence NP uptake and cytotoxicity.
3) In terms of overall observed toxicity, AgNPs and AgNO3 showed the same
cytotoxicity. Although, in terms of toxicity normalized to the amount of Ag that is
taken up by the cells, particles seem to be more toxic to PBMCs than ions.
4) This data underscores the importance of sufficient characterization of NPs in proper
exposure media for duration of exposure, to have a better understanding of NP
behavior and interaction in biological systems for evaluating risk assessment in
human exposure.
B) Investigating the efficiency of iron oxide NPs to remove fat from a HFD-fed-mouse
model.
89
The overall conclusions are summarized as below:
5) Using PVP-coated iron oxide NPs we could remove canola, vegetable, and olive
oil from stomach solution with high removal efficiency percentages (85%, 84% and
89%, respectively).
6) Despite the promising results in vitro, orally administration of PVP-coated iron
oxide NPs through food at concentrations up to 20 mg/kg did not show any effect
on body weight loss and lipid content in the liver of high fat diet-fed mice.
7) Some degree of glucose metabolism impairment was detected in NP treatment
groups. However, inflammatory markers in the liver (TNF-α and IL-6) did not
show any elevation. Further studies are needed to better understand the role of
PVP-coated iron oxide NPs in metabolic processes.
8) As our in-vitro data showed reassuring results, we are optimistic that with some
modifications in the experimental design, such as doses of NPs, surface
modification of NPs, and a more reliable and applicable route of exposure, iron
oxide NPs can be used to investigate if iron oxide NPs can be used as an anti-obesity
treatment.
In this study, we investigated the impact of nanotechnology and nanomaterials in
mammals. Our research results can be used for further advancement research and to add
information for potential rules and regulations for the utilization and application of
nanoparticles in commercial products.
While the number of publications in the nanotechnology and nanotoxicology field
is increasing each year, no standardize protocol is published for systematic analyzing of
90
toxicology of NMs (Nat Nano, 2012). Factors like cell viability assays, NPs
characterization, culture media conditions and toxicology assays that are not standardized
could make differences in the results and in the results interpretation.
91
REFERENCES
1. Mirshahghassemi, S. and J.R. Lead, Oil recovery from water under
environmentally relevant conditions using magnetic nanoparticles. Environmental
science & technology, 2015. 49(19): p. 11729-11736.
2. BSI-PAS71. Vocabulary-Nanoparticles. 2005.
3. American Society for Testing and Materials. Standard terminology relating to
nanotechnology.2006;Availablefrom: http://www.oalib.com/references/11954774.
4. Risks, s.C.o.E.a.N.I.H. The appropriateness of the risk assessment methodology in
accordance with the Technical Guidance Documents for new and existing
substances for assessing the risks of nanomaterials. 2007.
5. Klaine, S.J., et al., Nanomaterials in the environment: behavior, fate,
bioavailability, and effects. Environmental toxicology and chemistry, 2008. 27(9):
p. 1825-1851.
6. Daniel, M.-C. and D. Astruc, Gold nanoparticles: assembly, supramolecular
chemistry, quantum-size-related properties, and applications toward biology,
catalysis, and nanotechnology. Chemical reviews, 2004. 104(1): p. 293-346.
7. Kelly, K.L., et al., The optical properties of metal nanoparticles: the influence of
size, shape, and dielectric environment. 2003, ACS Publications.
8. Roduner, E., Size matters: why nanomaterials are different. Chemical Society
Reviews, 2006. 35(7): p. 583-592.
9. Sun, C.Q., Size dependence of nanostructures: Impact of bond order deficiency.
Progress in solid state chemistry, 2007. 35(1): p. 1-159.
10. Oberdörster, G., V. Stone, and K. Donaldson, Toxicology of nanoparticles: a
historical perspective. Nanotoxicology, 2007. 1(1): p. 2-25.
11. Madden, A.S., M.F. Hochella Jr, and T.P. Luxton, Insights for size-dependent
reactivity of hematite nanomineral surfaces through Cu2+ sorption. Geochimica et
cosmochimica acta, 2006. 70(16): p. 4095-4104.
12. Wang, H., R.L. Wick, and B. Xing, Toxicity of nanoparticulate and bulk ZnO,
Al2O3 and TiO2 to the nematode Caenorhabditis elegans. Environmental
Pollution, 2009. 157(4): p. 1171-1177.
13. Tolaymat, T.M., et al., An evidence-based environmental perspective of
manufactured silver nanoparticle in syntheses and applications: a systematic
review and critical appraisal of peer-reviewed scientific papers. Science of the
Total Environment, 2010. 408(5): p. 999-1006.
14. Ju-Nam, Y. and J.R. Lead, Manufactured nanoparticles: an overview of their
chemistry, interactions and potential environmental implications. Science of the
total environment, 2008. 400(1-3): p. 396-414.
92
15. Rane, A.V., et al., Methods for Synthesis of Nanoparticles and Fabrication of
Nanocomposites, in Synthesis of Inorganic Nanomaterials. 2018, Elsevier. p. 121-
139.
16. Petschacher, C., et al., Thinking continuously: a microreactor for the production
and scale-up of biodegradable, self-assembled nanoparticles. Polymer chemistry,
2013. 4(7): p. 2342-2352.
17. Nowack, B. and T.D. Bucheli, Occurrence, behavior and effects of nanoparticles
in the environment. Environmental pollution, 2007. 150(1): p. 5-22.
18. Kendall, M. and S. Holgate, Health impact and toxicological effects of
nanomaterials in the lung. Respirology, 2012. 17(5): p. 743-758.
19. Baalousha, M. and J.R. Lead, Overview of Nanoscience in the Environment.
Environmental and human health impacts of nanotechnology. Wiley-Blackwell
Publishing Ltd, Hoboken, NJ, 2009: p. 1-25.
20. Griffin, S., et al., Natural nanoparticles: A particular matter inspired by nature.
Antioxidants, 2018. 7(1): p. 3.
21. Keller, A.A., et al., Stability and aggregation of metal oxide nanoparticles in
natural aqueous matrices. Environmental science & technology, 2010. 44(6): p.
1962-1967.
22. Lowry, G.V., et al., Transformations of nanomaterials in the environment. 2012,
American Chemical Society.
23. Handy, R.D., et al., Manufactured nanoparticles: their uptake and effects on fish—
a mechanistic analysis. Ecotoxicology, 2008. 17(5): p. 396-409.
24. Handy, R.D., R. Owen, and E. Valsami-Jones, The ecotoxicology of nanoparticles
and nanomaterials: current status, knowledge gaps, challenges, and future needs.
Ecotoxicology, 2008. 17(5): p. 315-325.
25. Fabrega, J., et al., Silver nanoparticles: behaviour and effects in the aquatic
environment. Environment international, 2011. 37(2): p. 517-531.
26. Prabhu, S. and E.K. Poulose, Silver nanoparticles: mechanism of antimicrobial
action, synthesis, medical applications, and toxicity effects. International nano
letters, 2012. 2(1): p. 32.
27. Seiler, H.G., H. Sigel, and A. Sigel, Handbook on toxicity of inorganic compounds.
1988.
28. Monteiro, D.R., et al., The growing importance of materials that prevent microbial
adhesion: antimicrobial effect of medical devices containing silver. International
journal of antimicrobial agents, 2009. 34(2): p. 103-110.
29. Morones, J.R., et al., The bactericidal effect of silver nanoparticles.
Nanotechnology, 2005. 16(10): p. 2346.
30. Navarro, E., et al., Toxicity of silver nanoparticles to Chlamydomonas reinhardtii.
Environmental science & technology, 2008. 42(23): p. 8959-8964.
31. Ahamed, M., M.S. AlSalhi, and M. Siddiqui, Silver nanoparticle applications and
human health. Clinica chimica acta, 2010. 411(23-24): p. 1841-1848.
32. Franci, G., et al., Silver nanoparticles as potential antibacterial agents. Molecules,
2015. 20(5): p. 8856-8874.
93
33. García-Barrasa, J., J.M. López-de-Luzuriaga, and M. Monge, Silver nanoparticles:
synthesis through chemical methods in solution and biomedical applications.
Central European journal of chemistry, 2011. 9(1): p. 7-19.
34. Dallas, P., V.K. Sharma, and R. Zboril, Silver polymeric nanocomposites as
advanced antimicrobial agents: classification, synthetic paths, applications, and
perspectives. Advances in colloid and interface science, 2011. 166(1-2): p. 119-
135.
35. Benn, T., et al., The release of nanosilver from consumer products used in the home.
Journal of environmental quality, 2010. 39(6): p. 1875-1882.
36. Wijnhoven, S.W.P., et al., Nano-silver–a review of available data and knowledge
gaps in human and environmental risk assessment. Nanotoxicology, 2009. 3(2): p.
109-138.
37. Weir, E., et al., The use of nanoparticles in anti-microbial materials and their
characterization. Analyst, 2008. 133(7): p. 835-845.
38. Tulve, N.S., et al., Characterization of silver nanoparticles in selected consumer
products and its relevance for predicting children's potential exposures.
International journal of hygiene and environmental health, 2015. 218(3): p. 345-
357.
39. Yoon, S.-D., M.-H. Park, and H.-S. Byun, Mechanical and water barrier properties
of starch/PVA composite films by adding nano-sized poly (methyl methacrylate-co-
acrylamide) particles. Carbohydrate Polymers, 2012. 87(1): p. 676-686.
40. Gorjanc, M., et al., The influence of water vapor plasma treatment on specific
properties of bleached and mercerized cotton fabric. Textile research journal, 2010.
80(6): p. 557-567.
41. Furno, F., et al., Silver nanoparticles and polymeric medical devices: a new
approach to prevention of infection? Journal of Antimicrobial Chemotherapy,
2004. 54(6): p. 1019-1024.
42. Knetsch, M.L. and L.H. Koole, New strategies in the development of antimicrobial
coatings: the example of increasing usage of silver and silver nanoparticles.
Polymers, 2011. 3(1): p. 340-366.
43. Beattie, M. and J. Taylor, Silver alloy vs. uncoated urinary catheters: a systematic
review of the literature. Journal of clinical nursing, 2011. 20(15‐16): p. 2098-2108.
44. Eby, D.M., H.R. Luckarift, and G.R. Johnson, Hybrid antimicrobial enzyme and
silver nanoparticle coatings for medical instruments. ACS applied materials &
interfaces, 2009. 1(7): p. 1553-1560.
45. Alexander, J.W., History of the medical use of silver. Surgical infections, 2009.
10(3): p. 289-292.
46. Stammberger, H., Argyrosis of the nasal mucosa (author's transl). Laryngologie,
Rhinologie, Otologie, 1982. 61(5): p. 234-237.
47. Bowden, L.P., et al., Rapid onset of argyria induced by a silver‐containing dietary
supplement. Journal of cutaneous pathology, 2011. 38(10): p. 832-835.
48. Casals, E., et al., Hardening of the nanoparticle–protein corona in metal (Au, Ag)
and oxide (Fe3O4, CoO, and CeO2) nanoparticles. Small, 2011. 7(24): p. 3479-
3486.
94
49. Dale, A.L., G.V. Lowry, and E.A. Casman, Much ado about α: reframing the
debate over appropriate fate descriptors in nanoparticle environmental risk
modeling. Environmental Science: Nano, 2015. 2(1): p. 27-32.
50. Baek, Y.-W. and Y.-J. An, Microbial toxicity of metal oxide nanoparticles (CuO,
NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus
aureus. Science of the total environment, 2011. 409(8): p. 1603-1608.
51. Franklin, N.M., et al., Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and
ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the
importance of particle solubility. Environmental science & technology, 2007.
41(24): p. 8484-8490.
52. Collin, B., et al., Environmental release, fate and ecotoxicological effects of
manufactured ceria nanomaterials. Environmental Science: Nano, 2014. 1(6): p.
533-548.
53. Merrifield, R.C., et al., Synthesis and characterization of polyvinylpyrrolidone
coated cerium oxide nanoparticles. Environmental science & technology, 2013.
47(21): p. 12426-12433.
54. Bondarenko, O., et al., Particle-cell contact enhances antibacterial activity of silver
nanoparticles. PLoS One, 2013. 8(5): p. e64060.
55. Krystek, P., et al., Exploring influences on the cellular uptake of medium-sized
silver nanoparticles into THP-1 cells. Microchemical Journal, 2015. 120: p. 45-50.
56. Leclerc, S. and K.J. Wilkinson, Bioaccumulation of Nanosilver by Chlamydomonas
reinhardtii Nanoparticle or the Free Ion? Environmental science & technology,
2013. 48(1): p. 358-364.
57. Kroll, A., et al., Extracellular polymeric substances (EPS) of freshwater biofilms
stabilize and modify CeO2 and Ag nanoparticles. PLoS One, 2014. 9(10): p.
e110709.
58. Piccapietra, F., et al., Intracellular silver accumulation in Chlamydomonas
reinhardtii upon exposure to carbonate coated silver nanoparticles and silver
nitrate. Environmental science & technology, 2012. 46(13): p. 7390-7397.
59. Khan, F.R., et al., Stable isotope tracer to determine uptake and efflux dynamics of
ZnO nano-and bulk particles and dissolved Zn to an estuarine snail. Environmental
science & technology, 2013. 47(15): p. 8532-8539.
60. Croteau, M.-N., et al., Isotopically modified silver nanoparticles to assess
nanosilver bioavailability and toxicity at environmentally relevant exposures.
Environmental Chemistry, 2014. 11(3): p. 247-256.
61. Croteau, M.-N.l., et al., Bioaccumulation and toxicity of CuO nanoparticles by a
freshwater invertebrate after waterborne and dietborne exposures. Environmental
science & technology, 2014. 48(18): p. 10929-10937.
62. Huang, J., et al., Ag dendrite-based Au/Ag bimetallic nanostructures with strongly
enhanced catalytic activity. Langmuir, 2009. 25(19): p. 11890-11896.
63. Ghosh Chaudhuri, R. and S. Paria, Core/shell nanoparticles: classes, properties,
synthesis mechanisms, characterization, and applications. Chemical reviews,
2011. 112(4): p. 2373-2433.
64. Mizukoshi, Y., et al., Characterization and catalytic activity of core− shell
structured gold/palladium bimetallic nanoparticles synthesized by the
95
sonochemical method. The Journal of Physical Chemistry B, 2000. 104(25): p.
6028-6032.
65. Nishida, N., et al., Fabrication of Liquid Crystal Sol Containing Capped Ag− Pd
Bimetallic Nanoparticles and Their Electro-Optic Properties. The Journal of
Physical Chemistry C, 2008. 112(51): p. 20284-20290.
66. Merrifield, R.C., C. Stephan, and J.R. Lead, Single-particle inductively coupled
plasma mass spectroscopy analysis of size and number concentration in mixtures
of monometallic and bimetallic (core-shell) nanoparticles. Talanta, 2017. 162: p.
130-134.
67. Shon, Y.-S., et al., Monolayer-protected bimetal cluster synthesis by core metal
galvanic exchange reaction. Langmuir, 2002. 18(10): p. 3880-3885.
68. Gulson, B., et al., Small amounts of zinc from zinc oxide particles in sunscreens
applied outdoors are absorbed through human skin. Toxicological Sciences, 2010.
118(1): p. 140-149.
69. Dybowska, A.D., et al., Synthesis of isotopically modified ZnO nanoparticles and
their potential as nanotoxicity tracers. Environmental pollution, 2011. 159(1): p.
266-273.
70. Misra, S.K., et al., Isotopically modified nanoparticles for enhanced detection in
bioaccumulation studies. Environmental science & technology, 2011. 46(2): p.
1216-1222.
71. Croteau, M.-N., et al., A novel approach reveals that zinc oxide nanoparticles are
bioavailable and toxic after dietary exposures. Nanotoxicology, 2011. 5(1): p. 79-
90.
72. Larner, F., et al., Tracing Bioavailability of ZnO Nanoparticles Using Stable
Isotope Labeling. Environmental Science & Technology, 2012. 46(21): p. 12137-
12145.
73. Klaine, S.J., et al., Nanomaterials in the environment: behavior, fate,
bioavailability, and effects. Environmental Toxicology and Chemistry, 2008. 27(9):
p. 1825-1851.
74. Navarro, E., et al., Environmental behavior and ecotoxicity of engineered
nanoparticles to algae, plants, and fungi. Ecotoxicology, 2008. 17(5): p. 372-386.
75. Rico, C.M., et al., Interaction of nanoparticles with edible plants and their possible
implications in the food chain. Journal of agricultural and food chemistry, 2011.
59(8): p. 3485-3498.
76. Handy, R.D., et al., Effects of manufactured nanomaterials on fishes: a target organ
and body systems physiology approach. Journal of Fish Biology, 2011. 79(4): p.
821-853.
77. Baun, A., et al., Ecotoxicity of engineered nanoparticles to aquatic invertebrates:
a brief review and recommendations for future toxicity testing. Ecotoxicology,
2008. 17(5): p. 387-395.
78. Kim, T.H., et al., Size‐dependent cellular toxicity of silver nanoparticles. Journal
of Biomedical Materials Research Part A, 2012. 100(4): p. 1033-1043.
79. Desai, S.R., X.N. Verlecar, and U. Goswami, Genotoxicity of cadmium in marine
diatom Chaetoceros tenuissimus using the alkaline Comet assay. Ecotoxicology,
2006. 15(4): p. 359-363.
96
80. Johnson, A., E. Carew, and K.A. Sloman, The effects of copper on the
morphological and functional development of zebrafish embryos. Aquatic
Toxicology, 2007. 84(4): p. 431-438.
81. Tang, J., et al., Distribution, translocation and accumulation of silver nanoparticles
in rats. Journal of nanoscience and nanotechnology, 2009. 9(8): p. 4924-4932.
82. Takenaka, S., et al., Pulmonary and systemic distribution of inhaled ultrafine silver
particles in rats. Environmental Health Perspectives, 2001. 109: p. 547-551.
83. Beisel, W.R., Nutrition and immune function: overview. The Journal of nutrition,
1996. 126(suppl_10): p. 2611S-2615S.
84. Greulich, C., et al., Cell type-specific responses of peripheral blood mononuclear
cells to silver nanoparticles. Acta biomaterialia, 2011. 7(9): p. 3505-3514.
85. Ghosh, M., et al., In vitro and in vivo genotoxicity of silver nanoparticles. Mutation
Research/Genetic Toxicology and Environmental Mutagenesis, 2012. 749(1): p.
60-69.
86. Shin, S.-H., et al., The effects of nano-silver on the proliferation and cytokine
expression by peripheral blood mononuclear cells. International
immunopharmacology, 2007. 7(13): p. 1813-1818.
87. Jain, T.K., et al., Iron oxide nanoparticles for sustained delivery of anticancer
agents. Molecular Pharmaceutics, 2005. 2(3): p. 194-205.
88. Chourpa, I., et al., Molecular composition of iron oxide nanoparticles, precursors
for magnetic drug targeting, as characterized by confocal Raman
microspectroscopy. Analyst, 2005. 130(10): p. 1395-1403.
89. Bulte, J.W., Intracellular endosomal magnetic labeling of cells. Methods Mol Med,
2006. 124: p. 419-39.
90. Burtea, C., et al., Contrast agents: magnetic resonance, in Molecular imaging I.
2008, Springer. p. 135-165.
91. Boutry, S., et al., Specific E‐selectin targeting with a superparamagnetic MRI
contrast agent. Contrast media & molecular imaging, 2006. 1(1): p. 15-22.
92. Babes, L., et al., Synthesis of iron oxide nanoparticles used as MRI contrast agents:
a parametric study. Journal of colloid and interface science, 1999. 212(2): p. 474-
482.
93. Sonvico, F., et al., Metallic colloid nanotechnology, applications in diagnosis and
therapeutics. Current pharmaceutical design, 2005. 11(16): p. 2091-2105.
94. Corot, C., et al., Recent advances in iron oxide nanocrystal technology for medical
imaging. Advanced drug delivery reviews, 2006. 58(14): p. 1471-1504.
95. Modo, M.M. and J.W. Bulte, 22 An Outlook on Molecular and Cellular
MRImaging. Molecular and Cellular MR Imaging, 2007: p. 405.
96. Wan, J., et al., A soft-template-assisted hydrothermal approach to single-crystal
Fe3O4 nanorods. Journal of Crystal Growth, 2005. 276(3-4): p. 571-576.
97. Chin, A.B. and I.I. Yaacob, Synthesis and characterization of magnetic iron oxide
nanoparticles via w/o microemulsion and Massart's procedure. Journal of materials
processing technology, 2007. 191(1-3): p. 235-237.
98. Wu, W., Q. He, and C. Jiang, Magnetic iron oxide nanoparticles: synthesis and
surface functionalization strategies. Nanoscale research letters, 2008. 3(11): p. 397.
97
99. Reddy, L.H., et al., Magnetic nanoparticles: design and characterization, toxicity
and biocompatibility, pharmaceutical and biomedical applications. Chemical
reviews, 2012. 112(11): p. 5818-5878.
100. Hasany, S., et al., Systematic review of the preparation techniques of iron oxide
magnetic nanoparticles. Nanosci. Nanotechnol, 2012. 2(6): p. 148-158.
101. Laurent, S., et al., Magnetic iron oxide nanoparticles: synthesis, stabilization,
vectorization, physicochemical characterizations, and biological applications.
Chemical reviews, 2008. 108(6): p. 2064-2110.
102. Brunner, T.J., et al., In vitro cytotoxicity of oxide nanoparticles: comparison to
asbestos, silica, and the effect of particle solubility. Environmental science &
technology, 2006. 40(14): p. 4374-4381.
103. Nel, A., et al., Toxic potential of materials at the nanolevel. Science, 2006.
311(5761): p. 622-627.
104. Karlsson, H.L., et al., Copper oxide nanoparticles are highly toxic: a comparison
between metal oxide nanoparticles and carbon nanotubes. Chemical research in
toxicology, 2008. 21(9): p. 1726-1732.
105. Ankamwar, B., et al., Biocompatibility of Fe3O4 nanoparticles evaluated by in
vitro cytotoxicity assays using normal, glia and breast cancer cells.
Nanotechnology, 2010. 21(7): p. 075102.
106. Alabresm, A., et al., Use of PVP-coated magnetite nanoparticles to ameliorate oil
toxicity to an estuarine meiobenthic copepod and stimulate the growth of oil-
degrading bacteria. Environmental Science: Nano, 2017. 4(9): p. 1859-1865.
107. Kanagesan, S., et al., Synthesis, characterization, and cytotoxicity of iron oxide
nanoparticles. Advances in Materials Science and Engineering, 2013. 2013.
108. Jeng, H.A. and J. Swanson, Toxicity of metal oxide nanoparticles in mammalian
cells. Journal of Environmental Science and Health Part A, 2006. 41(12): p. 2699-
2711.
109. Hussain, S.M., et al., In vitro toxicity of nanoparticles in BRL 3A rat liver cells.
Toxicology in vitro, 2005. 19(7): p. 975-983.
110. Hilger, I., et al., Cytotoxicity of selected magnetic fluids on human adenocarcinoma
cells. Journal of magnetism and magnetic materials, 2003. 261(1-2): p. 7-12.
111. O'Brien, P.E., Bariatric surgery: mechanisms, indications and outcomes. Journal
of gastroenterology and hepatology, 2010. 25(8): p. 1358-1365.
112. ASMBS, A.S.f.M.a.B.S., Obesity in America. 2018.
113. Farooqi, I.S., Genetic, molecular and physiological insights into human obesity.
European journal of clinical investigation, 2011. 41(4): p. 451-455.
114. Rius, B., et al., Resolution of inflammation in obesity-induced liver disease.
Frontiers in immunology, 2012. 3: p. 257.
115. Bhaskaran, K., et al., Body-mass index and risk of 22 specific cancers: a
population-based cohort study of 5· 24 million UK adults. The Lancet, 2014.
384(9945): p. 755-765.
116. Ogden, C.L., et al., Prevalence of obesity among adults and youth: United States,
2011–2014. 2015.
117. Karn, B., T. Kuiken, and M. Otto, Nanotechnology and in situ remediation: a
review of the benefits and potential risks. Environmental health perspectives, 2009.
117(12): p. 1813-1831.
98
118. Yan, W., et al., Iron nanoparticles for environmental clean-up: recent
developments and future outlook. Environmental Science: Processes & Impacts,
2013. 15(1): p. 63-77.
119. Chen, X., Molecular imaging probes for cancer research. 2012: World Scientific.
120. Xue, Z., et al., Special wettable materials for oil/water separation. Journal of
Materials Chemistry A, 2014. 2(8): p. 2445-2460.
121. Zhang, X.-F., W. Shen, and S. Gurunathan, Silver nanoparticle-mediated cellular
responses in various cell lines: an in vitro model. International journal of molecular
sciences, 2016. 17(10): p. 1603.
122. Kawasaki, E.S. and A. Player, Nanotechnology, nanomedicine, and the
development of new, effective therapies for cancer. Nanomedicine:
Nanotechnology, Biology and Medicine, 2005. 1(2): p. 101-109.
123. Alabresm, A., et al., A novel method for the synergistic remediation of oil-water
mixtures using nanoparticles and oil-degrading bacteria. Science of the Total
Environment, 2018. 630: p. 1292-1297.
124. Piccinno, F., et al., Industrial production quantities and uses of ten engineered
nanomaterials in Europe and the world. Journal of Nanoparticle Research, 2012.
14(9): p. 1109.
125. Wagner, V., et al., The emerging nanomedicine landscape. Nature biotechnology,
2006. 24(10): p. 1211-1217.
126. AshaRani, P.V., et al., Cytotoxicity and genotoxicity of silver nanoparticles in
human cells. ACS nano, 2008. 3(2): p. 279-290.
127. Ip, M., et al., Antimicrobial activities of silver dressings: an in vitro comparison.
Journal of medical microbiology, 2006. 55(1): p. 59-63.
128. Melaiye, A., et al., Silver (I)-imidazole cyclophane gem-diol complexes
encapsulated by electrospun tecophilic nanofibers: formation of nanosilver
particles and antimicrobial activity. Journal of the American Chemical Society,
2005. 127(7): p. 2285-2291.
129. Silver Nanoparticles Market By Application (Electronics & Electrical, Healthcare,
Food & Beverages, Textiles) And Segment Forecasts To 2022. 2015.
130. Samuel, U. and J.P. Guggenbichler, Prevention of catheter-related infections: the
potential of a new nano-silver impregnated catheter. International Journal of
Antimicrobial Agents, 2004. 23: p. 75-78.
131. Gosheger, G., et al., Silver-coated megaendoprostheses in a rabbit model—an
analysis of the infection rate and toxicological side effects. Biomaterials, 2004.
25(24): p. 5547-5556.
132. Wan, A.T., et al., Determination of silver in blood, urine, and tissues of volunteers
and burn patients. Clinical Chemistry, 1991. 37(10): p. 1683-1687.
133. Williams, K., et al., Effects of subchronic exposure of silver nanoparticles on
intestinal microbiota and gut-associated immune responses in the ileum of
Sprague-Dawley rats. Nanotoxicology, 2015. 9(3): p. 279-289.
134. Van Den Brûle, S., et al., Dietary silver nanoparticles can disturb the gut
microbiota in mice. Particle and fibre toxicology, 2015. 13(1): p. 38.
135. Park, J., et al., Size dependent macrophage responses and toxicological effects of
Ag nanoparticles. Chemical communications, 2011. 47(15): p. 4382-4384.
99
136. Carrola, J., et al., Metabolomics of silver nanoparticles toxicity in HaCaT cells:
structure–activity relationships and role of ionic silver and oxidative stress.
Nanotoxicology, 2016. 10(8): p. 1105-1117.
137. Kaiser, J.-P., et al., Cytotoxic effects of nanosilver are highly dependent on the
chloride concentration and the presence of organic compounds in the cell culture
media. Journal of nanobiotechnology, 2017. 15(1): p. 5.
138. Stępkowski, T.M., K. Brzóska, and M. Kruszewski, Silver nanoparticles induced
changes in the expression of NF-κB related genes are cell type specific and related
to the basal activity of NF-κB. Toxicology in Vitro, 2014. 28(4): p. 473-478.
139. Orta‐García, S.T., et al., Analysis of cytotoxic effects of silver nanoclusters on
human peripheral blood mononuclear cells ‘in vitro’. Journal of Applied
Toxicology, 2015. 35(10): p. 1189-1199.
140. Paino, I.M.M. and V. Zucolotto, Poly (vinyl alcohol)-coated silver nanoparticles:
Activation of neutrophils and nanotoxicology effects in human hepatocarcinoma
and mononuclear cells. Environmental toxicology and pharmacology, 2015. 39(2):
p. 614-621.
141. Haase, H., A. Fahmi, and B. Mahltig, Impact of silver nanoparticles and silver ions
on innate immune cells. Journal of biomedical nanotechnology, 2014. 10(6): p.
1146-1156.
142. Yang, E.-J., et al., Inflammasome formation and IL-1β release by human blood
monocytes in response to silver nanoparticles. Biomaterials, 2012. 33(28): p. 6858-
6867.
143. Flower, N.A., et al., Characterization of synthesized silver nanoparticles and
assessment of its genotoxicity potentials using the alkaline comet assay. Mutation
Research/Genetic Toxicology and Environmental Mutagenesis, 2012. 742(1-2): p.
61-65.
144. Merrifield, R.C. and J.R. Lead, Preparation and characterization of three-layer,
isotopically labelled core-shell nanoparticles; a tool for understanding
mechanisms of bioavailability. NanoImpact, 2016.
145. Römer, I., et al., The critical importance of defined media conditions in Daphnia
magna nanotoxicity studies. Toxicology letters, 2013. 223(1): p. 103-108.
146. Cumberland, S.A. and J.R. Lead, Particle size distributions of silver nanoparticles
at environmentally relevant conditions. Journal of chromatography A, 2009.
1216(52): p. 9099-9105.
147. Römer, I., et al., Aggregation and dispersion of silver nanoparticles in exposure
media for aquatic toxicity tests. Journal of Chromatography A, 2011. 1218(27): p.
4226-4233.
148. Cumberland, S.A. and J.R. Lead, Synthesis of NOM-capped silver nanoparticles:
size, morphology, stability, and NOM binding characteristics. ACS Sustainable
Chemistry & Engineering, 2013. 1(7): p. 817-825.
149. Tejamaya, M., et al., Stability of citrate, PVP, and PEG coated silver nanoparticles
in ecotoxicology media. Environmental science & technology, 2012. 46(13): p.
7011-7017.
150. Blinova, I., et al., Toxicity of two types of silver nanoparticles to aquatic
crustaceans Daphnia magna and Thamnocephalus platyurus. Environmental
Science and Pollution Research, 2013. 20(5): p. 3456-3463.
100
151. Greulich, C., et al., Uptake and intracellular distribution of silver nanoparticles in
human mesenchymal stem cells. Acta biomaterialia, 2011. 7(1): p. 347-354.
152. Hitchman, A., et al., The effect of environmentally relevant conditions on PVP
stabilised gold nanoparticles. Chemosphere, 2013. 90(2): p. 410-416.
153. Cai, W., H. Zhong, and L. Zhang, Optical measurements of oxidation behavior of
silver nanometer particle within pores of silica host. Journal of applied physics,
1998. 83(3): p. 1705-1710.
154. Sikder, M., et al., A rapid approach for measuring silver nanoparticle
concentration and dissolution in seawater by UV–Vis. Science of The Total
Environment.
155. Georgantzopoulou, A., et al., Effects of silver nanoparticles and ions on a co-
culture model for the gastrointestinal epithelium. Particle and fibre toxicology,
2015. 13(1): p. 9.
156. Wei, L., et al., Silver nanoparticles: synthesis, properties, and therapeutic
applications. Drug discovery today, 2015. 20(5): p. 595-601.
157. Loza, K., et al., The dissolution and biological effects of silver nanoparticles in
biological media. Journal of Materials Chemistry B, 2014. 2(12): p. 1634-1643.
158. Murdock, R.C., et al., Characterization of nanomaterial dispersion in solution
prior to in vitro exposure using dynamic light scattering technique. Toxicological
sciences, 2008. 101(2): p. 239-253.
159. Greulich, C., et al., Studies on the biocompatibility and the interaction of silver
nanoparticles with human mesenchymal stem cells (hMSCs). Langenbeck's
Archives of Surgery, 2009. 394(3): p. 495-502.
160. Ahlberg, S., et al., PVP-coated, negatively charged silver nanoparticles: A multi-
center study of their physicochemical characteristics, cell culture and in vivo
experiments. Beilstein journal of nanotechnology, 2014. 5(1): p. 1944-1965.
161. Kittler, S., et al., The influence of proteins on the dispersability and cell-biological
activity of silver nanoparticles. Journal of Materials Chemistry, 2010. 20(3): p.
512-518.
162. Chernousova, S. and M. Epple, Silver as antibacterial agent: ion, nanoparticle, and
metal. Angewandte Chemie International Edition, 2013. 52(6): p. 1636-1653.
163. Boyle, D. and G.G. Goss, Effects of silver nanoparticles in early life-stage zebrafish
are associated with particle dissolution and the toxicity of soluble silver.
NanoImpact, 2018. 12: p. 1-8.
164. Kittler, S., et al., Synthesis of PVP‐coated silver nanoparticles and their biological
activity towards human mesenchymal stem cells. Materialwissenschaft und
Werkstofftechnik: Entwicklung, Fertigung, Prüfung, Eigenschaften und
Anwendungen technischer Werkstoffe, 2009. 40(4): p. 258-264.
165. Kawata, K., M. Osawa, and S. Okabe, In vitro toxicity of silver nanoparticles at
noncytotoxic doses to HepG2 human hepatoma cells. Environmental science &
technology, 2009. 43(15): p. 6046-6051.
166. Luther, E.M., et al., Accumulation of silver nanoparticles by cultured primary brain
astrocytes. Nanotechnology, 2011. 22(37): p. 375101.
167. Chakraborty, N., et al., Biorecovery of gold using cyanobacteria and an eukaryotic
alga with special reference to nanogold formation–a novel phenomenon. Journal
of Applied Phycology, 2009. 21(1): p. 145.
101
168. Liu, W., et al., Impact of silver nanoparticles on human cells: effect of particle size.
Nanotoxicology, 2010. 4(3): p. 319-330.
169. Yu, S.-j., et al., Quantification of the uptake of silver nanoparticles and ions to
HepG2 cells. Environmental science & technology, 2013. 47(7): p. 3268-3274.
170. Park, E.-J., et al., Silver nanoparticles induce cytotoxicity by a Trojan-horse type
mechanism. Toxicology in Vitro, 2010. 24(3): p. 872-878.
171. Gliga, A.R., et al., Size-dependent cytotoxicity of silver nanoparticles in human
lung cells: the role of cellular uptake, agglomeration and Ag release. Particle and
fibre toxicology, 2014. 11(1): p. 11.
172. Vrček, I.V., et al., Comparison of in vitro toxicity of silver ions and silver
nanoparticles on human hepatoma cells. Environmental toxicology, 2016. 31(6):
p. 679-692.
173. Han, X., et al., Validation of an LDH assay for assessing nanoparticle toxicity.
Toxicology, 2011. 287(1-3): p. 99-104.
174. Akter, M., et al., A systematic review on silver nanoparticles-induced cytotoxicity:
Physicochemical properties and perspectives. Journal of advanced research, 2018.
9: p. 1-16.
175. Gloire, G., S. Legrand-Poels, and J. Piette, NF-κB activation by reactive oxygen
species: fifteen years later. Biochemical pharmacology, 2006. 72(11): p. 1493-
1505.
176. Blanchet, S., et al., Fine particulate matter induces amphiregulin secretion by
bronchial epithelial cells. American journal of respiratory cell and molecular
biology, 2004. 30(4): p. 421-427.
177. Auger, F., et al., Responses of well-differentiated nasal epithelial cells exposed to
particles: role of the epithelium in airway inflammation. Toxicology and applied
pharmacology, 2006. 215(3): p. 285-294.
178. Unfried, K., et al., Cellular responses to nanoparticles: target structures and
mechanisms. Nanotoxicology, 2007. 1(1): p. 52-71.
179. Méndez‐Sánchez, N., et al., Current concepts in the pathogenesis of nonalcoholic
fatty liver disease. Liver International, 2007. 27(4): p. 423-433.
180. Nguyen, D.M. and H.B. El-Serag, The epidemiology of obesity. Gastroenterology
Clinics, 2010. 39(1): p. 1-7.
181. Cheah, J., Orlistat (Xenical) in the management of obesity. ANNALS-ACADEMY
OF MEDICINE SINGAPORE, 2000. 29(4): p. 419-420.
182. Douglas, I.J., et al., Orlistat and the risk of acute liver injury: self controlled case
series study in UK Clinical Practice Research Datalink. Bmj, 2013. 346: p. f1936.
183. Daneschvar, H.L., M.D. Aronson, and G.W. Smetana, FDA-approved anti-obesity
drugs in the United States. The American journal of medicine, 2016. 129(8): p. 879.
e1-879. e6.
184. Albaugh, V.L. and N.N. Abumrad, Surgical treatment of obesity. F1000Research,
2018. 7.
185. Sugerman, H.J., et al., Bariatric surgery for severely obese adolescents. Journal of
Gastrointestinal Surgery, 2003. 7(1): p. 102-108.
186. Stroh, A., et al., Iron oxide particles for molecular magnetic resonance imaging
cause transient oxidative stress in rat macrophages. Free Radical Biology and
Medicine, 2004. 36(8): p. 976-984.
102
187. Mulens, V., M.d.P. Morales, and D.F. Barber, Development of magnetic
nanoparticles for cancer gene therapy: a comprehensive review. ISRN
Nanomaterials, 2013. 2013.
188. Mejías, R., et al., Liver and brain imaging through dimercaptosuccinic acid-coated
iron oxide nanoparticles. Nanomedicine, 2010. 5(3): p. 397-408.
189. Mejías, R., et al., Dimercaptosuccinic acid-coated magnetite nanoparticles for
magnetically guided in vivo delivery of interferon gamma for cancer
immunotherapy. Biomaterials, 2011. 32(11): p. 2938-2952.
190. Chatterjee, J., Y. Haik, and C.-J. Chen, Size dependent magnetic properties of iron
oxide nanoparticles. Journal of Magnetism and Magnetic Materials, 2003. 257(1):
p. 113-118.
191. Hussain, S., et al., In vitro toxicity of nanoparticles in BRL 3A rat liver cells.
Toxicology in vitro, 2005. 19(7): p. 975-983.
192. Mahmoudi, M., et al., Cell toxicity of superparamagnetic iron oxide nanoparticles.
Journal of colloid and interface science, 2009. 336(2): p. 510-518.
193. Manickam, V., et al., Recurrent exposure to ferric oxide nanoparticles alters
myocardial oxidative stress, apoptosis and necrotic markers in male mice.
Chemico-biological interactions, 2017. 278: p. 54-64.
194. Sundarraj, K., et al., Repeated exposure to iron oxide nanoparticles causes
testicular toxicity in mice. Environmental toxicology, 2017. 32(2): p. 594-608.
195. Dhakshinamoorthy, V., V. Manickam, and E. Perumal, Neurobehavioural toxicity
of iron oxide nanoparticles in mice. Neurotoxicity research, 2017. 32(2): p. 187-
203.
196. Rojas, J.M., et al., Superparamagnetic iron oxide nanoparticle uptake alters M2
macrophage phenotype, iron metabolism, migration and invasion. Nanomedicine:
Nanotechnology, Biology and Medicine, 2016. 12(4): p. 1127-1138.
197. Kadel, K., S. Mirshahghassemi, and J.R. Lead, Facile flow-through synthesis
method for production of large quantities of polyvinylpyrrolidone-coated magnetic
iron oxide nanoparticles for oil remediation. Environmental Engineering Science,
2018. 35(2): p. 67-75.
198. Labson, V.F., et al., Estimated minimum discharge rates of the deepwater horizon
spill-interim report to the flow rate technical group from the mass balance team.
2010.
199. Weber, C.I., Methods for measuring the acute toxicity of effluents and receiving
waters to freshwater and marine organisms. 1991: Environmental Monitoring
Systems Laboratory, Office of Research and ….
200. Pantoja, P.A., et al., Prediction of crude oil properties and chemical composition
by means of steady-state and time-resolved fluorescence. Energy & Fuels, 2011.
25(8): p. 3598-3604.
201. Ryder, A.G., Analysis of crude petroleum oils using fluorescence spectroscopy, in
Reviews in Fluorescence 2005. 2005, Springer. p. 169-198.
202. Turner, A. and K.-H. Ip, Bioaccessibility of metals in dust from the indoor
environment: application of a physiologically based extraction test. Environmental
science & technology, 2007. 41(22): p. 7851-7856.
103
203. Sundarraj, K., et al., Iron oxide nanoparticles modulate heat shock proteins and
organ specific markers expression in mice male accessory organs. Toxicology and
applied pharmacology, 2017. 317: p. 12-24.
204. Winer, J.L., C.Y. Liu, and M.L. Apuzzo, The use of nanoparticles as contrast media
in neuroimaging: a statement on toxicity. World neurosurgery, 2012. 78(6): p. 709-
711.
205. Folch, J., M. Lees, and G. Sloane Stanley, A simple method for the isolation and
purification of total lipides from animal tissues. J biol Chem, 1957. 226(1): p. 497-
509.
206. Enos, R.T., et al., A low dose of dietary quercetin fails to protect against the
development of an obese phenotype in mice. PloS one, 2016. 11(12): p. e0167979.
207. Velázquez, K.T., et al., miR155 deficiency aggravates high‐fat diet‐induced
adipose tissue fibrosis in male mice. Physiological reports, 2017. 5(18).
208. Buettner, R., J. Schölmerich, and L.C. Bollheimer, High‐fat diets: modeling the
metabolic disorders of human obesity in rodents. Obesity, 2007. 15(4): p. 798-808.
209. McBain, S.C., H.H. Yiu, and J. Dobson, Magnetic nanoparticles for gene and drug
delivery. International journal of nanomedicine, 2008. 3(2): p. 169.
210. Lee, N. and T. Hyeon, Designed synthesis of uniformly sized iron oxide
nanoparticles for efficient magnetic resonance imaging contrast agents. Chemical
Society Reviews, 2012. 41(7): p. 2575-2589.
211. Mamani, J.B., L.F. Gamarra, and G.E.d.S. Brito, Synthesis and characterization of
Fe3O4 nanoparticles with perspectives in biomedical applications. Materials
Research, 2014. 17(3): p. 542-549.
212. Song, J.E., et al., Hydrophobic interactions increase attachment of gum arabic-and
PVP-coated Ag nanoparticles to hydrophobic surfaces. Environmental science &
technology, 2011. 45(14): p. 5988-5995.
213. Jornayvaz, F.R., et al., A high-fat, ketogenic diet causes hepatic insulin resistance
in mice, despite increasing energy expenditure and preventing weight gain.
American Journal of Physiology-Endocrinology and Metabolism, 2010. 299(5): p.
E808-E815.
214. de Jesus Felismino, C., et al., Effect of obesity on biodistribution of nanoparticles.
Journal of controlled release, 2018. 281: p. 11-18.
215. Salimi, M., et al., Biodistribution, pharmacokinetics, and toxicity of dendrimer-
coated iron oxide nanoparticles in BALB/c mice. International journal of
nanomedicine, 2018. 13: p. 1483.
216. Sharma, A., et al., Physical characterization and in vivo organ distribution of
coated iron oxide nanoparticles. Scientific reports, 2018. 8(1): p. 4916.
217. Mejías, R., et al., Long term biotransformation and toxicity of dimercaptosuccinic
acid-coated magnetic nanoparticles support their use in biomedical applications.
Journal of Controlled Release, 2013. 171(2): p. 225-233.
218. MacParland, S.A., et al., Phenotype determines nanoparticle uptake by human
macrophages from liver and blood. ACS nano, 2017. 11(3): p. 2428-2443.
219. Castoldi, A., et al., The macrophage switch in obesity development. Frontiers in
immunology, 2016. 6: p. 637.
104
220. Enos, R.T., K.T. Velázquez, and E.A. Murphy, Insight into the impact of dietary
saturated fat on tissue-specific cellular processes underlying obesity-related
diseases. The Journal of nutritional biochemistry, 2014. 25(6): p. 600-612.
221. Valois, C.R., et al., The effect of DMSA-functionalized magnetic nanoparticles on
transendothelial migration of monocytes in the murine lung via a β2 integrin-
dependent pathway. Biomaterials, 2010. 31(2): p. 366-374.
222. Straub, R., et al., Hormone replacement therapy and interrelation between serum
interleukin-6 and body mass index in postmenopausal women: a population-based
study. The Journal of Clinical Endocrinology & Metabolism, 2000. 85(3): p. 1340-
1344.
223. Bader, J.E., et al., Macrophage depletion using clodronate liposomes decreases
tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon
cancer. American Journal of Physiology-Gastrointestinal and Liver Physiology,
2017. 314(1): p. G22-G31.
224. Powell, L.W., et al. Hemochromatosis: genetics and pathogenesis. in Seminars in
liver disease. 1996. © 1996 by Thieme Medical Publishers, Inc.
225. McClain, C.J. and D.A. Cohen, Increased tumor necrosis factor production by
monocytes in alcoholic hepatitis. Hepatology, 1989. 9(3): p. 349-351.
226. She, H., et al., Iron activates NF-κB in Kupffer cells. American Journal of
Physiology-Gastrointestinal and Liver Physiology, 2002. 283(3): p. G719-G726.