University of South Carolina Scholar Commons

Theses and Dissertations

Summer 2019

The Health Implication and Application of Silver and Iron Oxide Nanoparticles

Sahar Pourhoseini

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Recommended Citation Pourhoseini, S.(2019). The Health Implication and Application of Silver and Iron Oxide Nanoparticles. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/5412

This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. 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 © Copyright by Sahar Pourhoseini 2019 All Rights Reserved.

ii 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.

iii 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).

iv 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

v 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

vi increasing demand for obesity treatments, nanomedicine approaches, coupled with our results, may provide substantial contributions toward progress in this area.

vii 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

viii 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 5 2.5×10 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

ix 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

x 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

xi 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

xii 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

xiii 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

xiv CHAPTER 1

INTRODUCTION

1 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

2 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].

3 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

4 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

5 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].

6 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.

7 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).

8 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

9 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

10 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.

11

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).

12 CHAPTER 2

UPTAKE AND TOXICITY OF SILVER NANOPARTICLES AND

SILVER IONS TO HUMAN PERIPHERAL BLOOD MONONUCLEAR

CELLS

13 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

-1 5 treatment. In 1000 µg L AgNO3 exposure, up to 11 ng Ag was detected in 2.5× 10 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

14 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

15 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

16 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;

17 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

18 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

19 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

20 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,

21 153]. 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

22 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

5 -1 culture plates at a density of 2.5×10 cells. PVP-AgNP and AgNO3 stock (25 mg L ) 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

23 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

-1 (as positive control), AgNO3 -PVP and PVP (10, 100, 500, and 1000 µg L ) 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

24 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.

25 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

26 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

-1 AgNO3 in UHPW and media at two concentrations (500 and 1000 µg L ). 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.

27 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

-1 AgNO3 treatments (Figure 2.7). At concentrations of 500 and 1000 µg L 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%

-1 in AgNO3 of total Ag in the treatments. Furthermore, at the 1000 µg L 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).

28 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

-1 concentrations of 0 to 1000 µg L . 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

-1 (LDH test) became significant from 10 µg L for AgNO3-PVP and continually increased

-1 up to 500 µg L . 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

-1 µg L 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

29 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

30 [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

31 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

32 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

-1 that was detected in the cells in AgNO3 treatment compared to NP at 500 and 1000 µg L 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

33 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

-1 Ag uptake in AgNP and AgNO3 treatment at 10 and 100 µg L Ag concentration.

-1 However, at 500 and 1000 µg L 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

-1 assay) in 500 µg L 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

34 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].

-1 Cytotoxicity of both PVP-AgNPs and AgNO3 was initiated from 100 µg L 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

-1 100 µg L [139]. LDH and MTS results are in agreement in PVP-AgNP, AgNO3, and

-1 AgNO3-PVP exposure at 500 µg L , 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

-1 results did not support MTS results at 1000 µg L 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

-1 of 1000 µg L 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

35 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

36 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.

37 Table 2.1 List of chemicals used in NP synthesis. Chemicals used in core-shell (Ag@Au@Ag) and PVP-coated AgNPs synthesis.

Chemical Chemical MW No. Supplier Name Structure (gramMol-1) 1 Ag 107 Ag 106.9 Isoflex 2 Ag 109 Ag 108.9 Isoflex 3 Ag Ag 107.9 Fisher Scientific Sodium 4 NaBH 37.83 Sigma-Aldrich borohydride 4 5 Sodium citrate C6H5O7.2H2O.3Na 294.10 Sigma-Aldrich 6 Nitric acid HNO3 63.01 Fisher Scientific Gold (III) 7 AuCl 303.33 Sigma- Aldrich chloride 3 8 PVP (C6H9O) n 10,000 Sigma-Aldrich

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.

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

39 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

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.

Mass of Ag in exposure (ng) 10 100 500 1000 Cell 0.07 0.39 2.40 4.97 Supernatant 5.17 42.46 212.77 419.88 PVP-AgNP Total loss 4.76 57.15 285.62 576.15 Cell/Sup. (%) 1.35 0.91 1.12 1.18 Cell 0.06 0.36 4.56 10.85 Supernatant 5.49 59.77 120.88 166.46 AgNO 3 Total loss 4.45 39.87 374.56 822.69 Cell/Sup. (%) 1.09 0.60 3.79 6.52

41 Table 2.5 Mass balance of Ag in the exposure for each person. Ag mass balance (ng) in 5 2.5×10 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 Cell 0.06 0.31 1.76 5.41 PVP-AgNP Supernatant 6.23 38.02 153.61 312.95 Total loss 3.71 61.67 344.62 681.63 Cell 0.05 0.32 4.54 8.14 AgNO3 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 Cell 0.06 0.34 2.13 4.57 PVP-AgNP Supernatant 3.59 33.17 163.68 425.14 Total loss 6.53 66.49 334.19 570.30 Cell 0.05 0.30 4.98 14.44 AgNO3 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 Cell 0.07 0.51 2.75 5.96 PVP-AgNP Supernatant 4.53 39.64 301.33 457.06 Total loss 5.40 59.85 195.93 536.97 Cell 0.06 0.42 4.97 6.95 AgNO3 Supernatant 4.78 43.79 83.65 198.16 Total loss 5.16 55.79 411.38 794.88

42 Table 2.5 Mass balance of Ag.

Person 4 Mass of Ag in exposure (ng) 10 100 500 1000 Cell 0.07 0.46 3.58 6.78 PVP-AgNP Supernatant 4.55 48.19 206.93 404.74 Total loss 5.38 51.35 289.49 588.48 Cell 0.08 0.51 5.50 11.86 AgNO3 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 Cell 0.08 0.38 2.63 4.31 PVP-AgNP Supernatant 6.15 45.34 213.65 409.12 Total loss 3.76 54.28 283.73 586.57 Cell 0.08 0.39 4.47 13.54 AgNO3 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 Cell 0.07 0.32 1.56 2.79 PVP-AgNP Supernatant 5.99 50.41 237.40 510.26 Total loss 3.94 49.27 261.04 486.95 Cell 0.07 0.23 2.91 8.84 AgNO3 Supernatant 5.57 76.22 120.41 199.83 Total loss 4.36 23.55 376.68 791.32

43

a

b

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.

44 a b

c d

e

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 a b

c d

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 a

b

c

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 PVP-AgNP treatment a b

AgNO3 treatment c d

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

a b

c d

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).

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

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 a

b

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 a

b

c

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 CHAPTER 3

CAN PVP-COATED IRON OXIDE NANOPARTICLES BE USED TO

PREVENT WEIGHT GAIN IN A HIGH-FAT DIET MOUSE MODEL

56 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

57 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

58 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

59 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.

60 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

61 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-

62 012) 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-

63 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

64 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

65 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.

66 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.

67 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.

68 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,

69 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).

70 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

71 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

72 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

73 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,

74 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

75 [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

76 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.

77

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.

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. 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: 84% and olive oil: 89%.

81

a

b

c d

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).

82 a b

c

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 a

b

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

a b

c

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 a b c

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 CHAPTER 4

OVERALL CONCLUSION

87 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.

88 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

89 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.

90

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