INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR

AND MONOCYTIC CELLULAR RESPONSE

Thesis

Submitted to

The School of Engineering of the

UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for

The Degree of

Master of Science in Bioengineering

by

Katherine Eileen Burns

UNIVERSITY OF DAYTON

Dayton, Ohio

August 2018

INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR

AND MONOCYTIC CELLULAR RESPONSE

Name: Burns, Katherine Eileen

APPROVED BY:

______Kristen K. Comfort, Ph.D. Robert J. Wilkens, Ph.D., P.E. Advisory Committee Chair Committee Member Assistant Professor Professor Chemical and Materials Engineering Chemical and Materials Engineering

______Matthew E. Lopper, Ph.D. Committee Member Associate Professor Chemistry

______Robert J. Wilkens, Ph.D., P.E. Eddy M. Rojas, Ph.D., M.A., P.E. Associate Dean for Research and Dean Innovation School of Engineering Professor School of Engineering

ii

ABSTRACT

INFLUENCE OF FLUID DYNAMICS ON SILVER NANOPARTICLE BEHAVIOR

AND MONOCYTIC CELLULAR RESPONSE

Name: Burns, Katherine Eileen University of Dayton

Advisor: Dr. Kristen K. Comfort

Nanoparticles (NPs) are being increasingly used in many industries and consumer products. As they become more prevalent in consumer goods and applications, a new area of study, nanotoxicology, which explores the safety of these novel materials, has emerged. The toxicity of a particular NP can be due to many tunable physicochemical properties, such as size, core composition, morphology, and surface charge. That toxicity can also be affected by the environment surrounding the NP, including whether the system is static or dynamic, if the cells are grown within a 2-dimensional or 3- dimensional space, and the composition of the surrounding fluid.

Currently, most nanotoxicity testing occurs in a standard cell-based in vitro model. These models do not take the true physiological environment in which NP exposure occurs, such as pH or the dynamic nature of the human body, into account. This investigation sought to understand some of the effects, toxicological or otherwise, of silver nanoparticles (AgNPs) on the U937 monocytic cell within both a static and dynamic exposure condition. Dynamic flow was created using a peristaltic pump,

iii operating at a flow rate to produce an average tube-side linear velocity of 0.2 cm/s; the known velocity within capillaries. As the U937 cell line grew in suspension, the cells themselves were moving with the AgNPs throughout the duration of the exposure under dynamic conditions.

The addition of the fluid dynamics had minimal effect on the physicochemical properties of the AgNPs themselves. However, the interactions of the AgNPs with the cells were greatly increased with the addition of the dynamic fluid movement. This increase in nano-cellular interactions also augmented AgNP-dependent bioresponses, including reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) leakage, heat shock protein 27 (HSP27) activation, and activation of an inflammatory response.

These observed alterations to cellular viability, stress, and inflammatory markers between static and dynamic exposure conditions suggest that the incorporation of physiologically relevant conditions in an in vitro model enhance the cellular model and could provide a mechanism to bridge the gap between in vitro and in vivo models.

iv

DEDICATION

Dedicated to my wonderful parents, Dave and Diane, who have supported me and encouraged me to always continue learning, and to Andrew, my greatest cheerleader.

v

ACKNOWLEDGMENTS

First, I would like to thank Dr. Kristen Comfort for her incredible guidance, support, and patience. I have learned so much about this massively broad field from her, and I am eternally grateful for the opportunity to learn from a woman as passionate about her chosen profession as she is. A special thanks to my committee as well, for taking the time out of their busy schedules to be committee members.

I would also like to thank the rest of the faculty and staff of the Chemical and

Materials Engineering Department at the University of Dayton. I have been supported by each and every person in the department for the last seven years, and simply would not be where I am without them.

Funding for this research came from the Dayton Area Graduate Studies Institute at Wright-Patterson Air Force Base, and the National Science Foundation, each of which

I am very grateful to.

Finally, I owe so many thanks to my friends and family. They have pushed me to grow both academically and personally and helped to shape me into the person I am today. They are my sounding boards, confidants, and greatest cheerleaders. I am so lucky to know each and every one of them.

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TABLE OF CONTENTS

ABSTRACT ...... iii

DEDICATION ...... v

ACKNOWLEDGMENTS ...... vi

LIST OF FIGURES ...... x

LIST OF TABLES ...... xii

LIST OF ABBREVIATIONS AND NOTATIONS ...... xiii

CHAPTER 1 BACKGROUND ...... 1

1.1– Nanoparticles ...... 1

1.1.1 – Unique Properties of Nanoparticles ...... 1

1.1.2 – Applications of Nanoparticles ...... 6

1.2 – Silver Nanoparticles ...... 8

1.2.1 – Applications of Silver Nanoparticles ...... 8

1.3 – Nanotoxicity ...... 9

1.3.1 – Tunable Properties and Toxicity ...... 10

1.3.2 – Observable Signs of Nanotoxicity ...... 13

1.4 – Physiologically Relevant Models ...... 15

vii

1.5 – References ...... 20

CHAPTER 2 EXPERIMENTAL PROCEDURES ...... 29

2.1 – Introduction ...... 29

2.2 – In Vitro Cell Growth and Monitoring ...... 29

2.2.1 – Cell Culture...... 30

2.2.2 – Cell Counting ...... 31

2.2.3 – Cell Spinning ...... 32

2.2.4 – Cell Lysing ...... 33

2.3 – NP Characterization ...... 34

2.3.1 – Transmission Electron Microscopy (TEM) ...... 35

2.3.2 – Dynamic Light Scattering (DLS) ...... 36

2.3.3 – Zeta Potential ...... 37

2.3.4 – Ultraviolet-Visible Spectroscopy (UV-Vis) ...... 38

2.4 – NP Exposure and Response Characterization ...... 39

2.4.1 – AgNP Exposures...... 39

2.4.2 – AgNP Internalization by U937 Cells ...... 44

2.5 – Cellular Responses to NP Exposure ...... 46

2.5.1 – Lactate Dehydrogenase (LDH) ...... 47

2.5.2 – Reactive Oxygen Species (ROS) ...... 48

2.5.3 – Cytokine ELISAs ...... 53

viii

2.6 – Signaling Responses to NP Exposure ...... 55

2.6.1 – BCA Protein Assay (BioRad) ...... 55

2.6.2 – Stress ELISAs ...... 57

2.7 – Statistical Analysis ...... 59

2.8 – References ...... 60

CHAPTER 3 RESULTS, DISCUSSION, AND CONCLUSIONS ...... 62

3.1 – Introduction ...... 62

3.2 – Results ...... 62

3.2.1 – NP Characterization Results ...... 62

3.2.2 – Cellular Association Results ...... 67

3.2.3 – Cellular Response Results ...... 68

3.3 – Discussions ...... 76

3.3.1 – Discussion of NP Characterization Results ...... 76

3.3.2 – Discussion of Cellular Association Results ...... 78

3.3.3 – Discussion of Cellular Response Results ...... 79

3.4 – Conclusions ...... 84

3.5 – References ...... 85

ix LIST OF FIGURES

Figure 1.1: Examples of physicochemical characteristics of NPs which may affect how they interact with surroundings. These examples include 1) individual NP size, 2) surface charge, 3) composition of the NP, and 4) morphology or shape of the NP...... 2

Figure 1.2 [11]: Size-induced color change of spherical gold nanoparticle solution. As the size of the NP increases, the color changes from a rusty orange color (left) to purple (right) due to the plasmonic light scattering...... 3

Figure 1.3 [38]: Some common uses for NPs and the exposure pathways they may encounter...... 10

Figure 1.4: A549 cells photographed under fluorescent microscopy after a) a static growth, or b) a dynamic growth of 24 hours [52]. The cells grown in an in vitro environment which incorporated fluid dynamics have a shape more similar to lung cells seen in vivo...... 18

Figure 2.1: Representative TEM images of a) spherical 5 nm AgNPs and b) spherical 50 nm AgNPs ...... 35 Figure 2.2: An example of a UV-Vis spectral profile for both 5 nm and 50 nm AgNPs between 300-700 nm ...... 38 Figure 2.3: The peristaltic pump, inside the incubator, used to create physiologically relevant flow rates during experimentation. The lower right image shows how the tubing fits into a 24-well plate...... 40

Figure 3.1: TEM Images of a) 5 nm AgNPs and b) 50 nm AgNPs confirming spherical morphology and uniformity [1]...... 64 Figure 3.2: Plot showing the absorbance of both the 5 nm and 50 nm AgNPs using UV-Vis. The two peaks are sharp, and the 50 nm peak is shifted about 50 nm to the right, as expected. [1] ...... 65 Figure 3.3: The percentage of NPs taken up by the U937 cells after a 24-hour exposure in either a static or dynamic environment. The dynamic exposures of both the 5 and 50 nm particles show a significant increase in internalization when compared with their static counterpart. *Denotes statistical significance between static and dynamic conditions...... 68

x

Figure 3.4: A comparison of ROS produced by the monocytes after 24 hours of exposure to 2.5 µg/mL of each NP size and environmental condition compared to a static, untreated control. *Denotes statistical significance from untreated control. †Denotes statistical significance between static and dynamic conditions...... 69 Figure 3.5: The activation of NF-κB of each experimental exposure compared to a static, untreated control. The static exposures showed a higher activation of this transcription factor than the dynamic exposures did. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions ...... 71 Figure 3.6: A comparison of the activation of HSP 27 by AgNP treated U937s to a static, untreated control. The extent of activation appears to correspond to both the NP size and the extent to which the AgNPs were taken up by the U937s. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions...... 72 Figure 3.7: A comparison of 5 nm AgNP exposures to an untreated, static control. A small concentration did not seem to influence the LDH leakage of U937s, but a larger concentration seems to have influenced a large increase in the LDH levels, which correspond to the internalization of the AgNPs within the U937s. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions...... 73 Figure 3.8: A chart showing the comparison of the secretion of IL-6 across all four experimental conditions with a static, untreated control. The 5 nm particles appear not to have influenced the secretion of the inflammatory response, only the larger particles were able to do so. *Denotes statistical significance from untreated control ...... 75 Figure 3.9: IL-8 secretion induced by the four experimental AgNP exposures compared to a static, untreated control. This plot shows little to no difference occurred between the static and dynamic exposures to the same NP. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions...... 76

xi

LIST OF TABLES

Table 1.1: Summary of advantages and disadvantages of nanotoxicity testing through in vitro and in vivo models [52]...... 16

Table 2.1: Blood circulation properties, adapted from Introduction to Bioengineering [2] ...... 40

Table 3.1: A Summary of all size-related data obtained from AgNP Characterization analysis. This includes primary and agglomerate sizes [2]...... 66 Table 3.2: A Summary of all Zeta Potential Data for both 5 nm and 50 nm AgNPs [2]… ...... 66

xii

LIST OF ABBREVIATIONS AND NOTATIONS

AgNP Silver Nanoparticle

Au Gold

BCA Bicinchoninic Acid

CO2 Carbon Dioxide

DCFH-DA 2',7'-dichlorodihydrofluorescein diacetate

DI Deionized

DMSO Dimethyl Sulfoxide

EGF Epidermal Growth Factor

ELISA Enzyme-Linked Immunosorbent Assay

FBS Fetal Bovine Serum

HaCaT Human Keratinocyte Cells

HSP Heat Shock Protein

HRP Horseradish Peroxidase

ID Inner Diameter

xiii

IGF Insulin-like Growth Factor

IL Interleukin

LDH Lactate Dehydrogenase

NF Nuclear Factor

NP Nanoparticle

PEG Polyethylene Glycol

PSA Penicillin-Streptomycin-Amphotericin

PVP Poly(vinylpyrrolidone)

RCF Relative Centrifugal Force

ROS Reactive Oxygen Species

RPM Rotations Per Minute

RPMI Roswell Park Memorial Institute

TiO2 Titanium Dioxide

TNF Tumor Necrosis Factor

UV-Vis Ultraviolet-Visible Spectroscopy

U937 Human Myeloid Cell

ZnO Zinc Oxide

xiv

CHAPTER 1

BACKGROUND

1.1– Nanoparticles

1.1.1 – Unique Properties of Nanoparticles

In recent years nanoparticles (NPs) have been utilized increasingly due to their unique physicochemical properties, which significantly differ from those of their bulk material

[1]–[4]. A NP is any material in which one of its dimensions measures less than or equal to 100 nm [5], [6]. These different properties, including primary particle size, agglomeration, core composition, porosity, surface chemistry, and reactivity (Figure 1.1), make NPs advantageous in many industries [2], [4], [6]–[9]. In fact, it was forecasted that the market for NP-enabled products would reach over 3.5 trillion dollars in 2018 [10].

NPs can also be of many different morphologies, such as spheres, rods, fibers, or disks, which can impact how the NP interacts with its biological surroundings [2], [4].

1

Figure 1.1: Examples of physicochemical characteristics of NPs which may affect how they interact with surroundings. These examples include 1) individual NP size, 2) surface charge, 3) composition of the NP, and 4) morphology or shape of the NP.

For example, the primary particle size of an NP, which is tunable during synthesis, impacts the wavelength of absorbance of a fluid in solution. Gold NPs, in particular, visibly exhibit this property, also known as plasmonic behavior. As gold nanospheres increase in size, the solution color changes from a rusty orange color to a more purple color, as can be seen in Figure 1.2 [11]. This is due to the fact that the surface plasmons, or the movement of the electrons on the gold NP surface, cause significant scattering of visible light [12]. As the particle size increases, less light is being absorbed and more light is scattered, shifting the color of the gold NP solution. This phenomenon can also be seen in nanorods, but as a function of changing aspect ratio (length:radius), demonstrating that this phenomenon is not unique to spherical particles. A gold nanorod with a smaller aspect ratio will produce a turquoise color in suspension, whereas nanorods with a longer aspect ratio will exhibit an olive-green color [11].

2

Figure 1.2 [11]: Size-induced color change of spherical gold nanoparticle solution. As the size of the NP increases, the color changes from a rusty orange color (left) to purple (right) due to the plasmonic light scattering.

Individual particle size has been shown to affect the way that a NP interacts with its environment, particularly the biological system. Generally speaking, the smaller the NP the higher the rates of cellular association, interaction, final cellular distribution, and cellular elimination [4], [13]. This size-dependence is what makes NPs so promising for the next-generation of drug-delivery technologies, as the alteration of size has a direct correlation to the degree of NP delivery. However, active cellular endocytosis, the internalization of material into the cell, can also lead to a cytotoxic response, meaning that there exists a direct link between primary particle size, NP delivery, and NP-induced cellular death [4].

3 Apart from individual particle size, the agglomerate size of NPs has been found to affect the nano-cellular interface and biological response. All NPs agglomerate to some degree when suspended in solution. However, the composition of the solution causes NPs to aggregate differently, primarily based on the ionic content of the fluid [14]. Excessive agglomeration can cause the NPs to lose stability and fall out of solution, thereby reducing the efficacy of nano-based applications. As such, the medium in which the NPs are suspended during application must be taken into account in order to accurately characterize the NPs and ensure that the desired properties are maintained.

Surface chemistry is another tunable property of NPs. The coating of a NP can alter the surface charge, thereby modifying the extent of cellular interactions and the degree of NP internalization or association with cells [4], [15], [16]. Common coatings and functionalization for NPs include many different polymers and proteins, as they can aid in biocompatibility through variance of surface charge [17]. The surface charge is also related to NP agglomeration, with subsequent consequences including cellular uptake and biocompatibility [4]. A recent study showed that cellular uptake of silver nanoparticles

(AgNPs) in Caco-2 intestinal epithelial cells could be varied by changing the coating of the NP, which modified the particle surface charge [15]. Their results found that lowering the surface charge, as measured via zeta potential, a measure of electrokinetic potential in suspensions containing insoluble microscopic particles, resulted in less cellular uptake by the Caco-2 cells [15]. Another study showed that polymeric and PEGylated coatings on iron oxide NPs could vary the cellular uptake in both human umbilical vein endothelial cells (HUVEC) and murine macrophages [16]. Particularly in the macrophages, a

4 decrease in cellular uptake was observed when polyethylene glycol (PEG) was added to the amphiphilic polymer coating [16].

Each of these tunable properties can work together or against one another to create NPs which will be delivered to a specific area in the body. Even if size, surface charge, and surface chemistry are the same, a NP with a different core or morphology will react differently when exposed to the same conditions [4], [18]. AgNPs and iron oxide NPs of the same size and morphology were coated with the same material and studied for internalization in a Caco-2 cellular model [18]. The internalization of the AgNPs was significantly greater than the iron oxide NPs, even when compared to a 4X concentration of the iron oxide NPs [18]. This significant difference despite so many similar tunable properties helps deepen the understanding of how these properties could work together for many medical applications of NPs.

Not only can these physicochemical properties determine how the NP will react within its surrounding environment, but recent studies have begun elucidating how the environment itself may also influence NP behavior [4]. This has been proven to be true in some fluids with lower pH, such as physiological fluids, which have been shown to dissolve metal

NPs with core compositions such as silver (Ag) and zinc oxide (ZnO) [19]. A low pH has also been shown to influence the agglomeration of AgNPs, with NPs displaying greater agglomeration within an acidic environment [19]. This aligns with the emerging knowledge that the physicochemical properties of NPs can be modified by their

5 environmental surroundings [2], [20], [21]. However, there is a lack of published knowledge pertaining to how these environmental factors, such as pH, or presence of fluid dynamic conditions, may affect NP behavior.

1.1.2 – Applications of Nanoparticles

The dramatic increase in the usage of NPs has affected a number of markets, including consumer, medical, energy, industrial, and military sectors [6], [22]. These applications include many products that the average consumer would encounter, such as sunscreens, cosmetics, food packaging, pharmaceuticals, baby products, and personal hygiene products [13], [23]. Some commonly used NPs include Ag, titanium dioxide (TiO2),

ZnO, and gold (Au) [21], [24]–[29]. Due to their prevalence in consumer goods and applications, AgNPs were selected as the experimental NP in this work, with a further discussion of these applications in the following section.

TiO2 NPs are one of the more frequently utilized metal oxide NPs in many industries due to their anti-corrosive properties and high stability [30], [31]. These NPs can also be used as a pigment, often white pigment, making it highly utilized across many sectors [25],

[31]. Most of the TiO2 produced for pigment is used in paint, but it is also used in cosmetics, pharmaceuticals, foods, and many other markets [25], [30]. In the cosmetics field, TiO2 NPs can be used to increase the UV protectivity in sunscreens and other products which serve to guard against harmful UV rays, like facial cosmetics [30]. In the food industry, chewing gum and candies have been shown to contain the highest

6 concentration of TiO2 NPs [25]. In the medical field, TiO2 NPs are being investigated for use in bioimaging or treatment for various skin conditions [31].

ZnO NPs are most often used as food additives to help improve the antimicrobial packing of food [26], [32]. In addition, ZnO NPs are also being incorporated into sunscreens, and other coatings, to mitigate UV exposure, as well as other personal care products and cosmetics [27], [32]. Furthermore, these NPs have applications in the medical industry as well. ZnO NPs have been shown to have antibacterial properties for both gram-negative and gram-positive bacteria [32]. However, as more studies have been performed on ZnO

NPs, it has been shown to be quite toxic in certain environmental conditions [33]. This is where more physiologically relevant in vitro modeling will help to evaluate safety in humans.

Gold nanoparticles (AuNPs) are used across many sectors but are being explored for numerous biomedical applications including bioimaging, drug delivery, and cancer ablation [21], [24], [28], [34]. These particles, in the form of nanorods, give off a response in both the near-infrared and visible light regions of the spectrum [29]. Gold nanorods will absorb light at the same frequency at which they scatter it [29]. This lends itself to better predictability to those who seek to use AuNPs, which is why it is among one of the most investigated and utilized NPs [21]. However, it is documented that

AuNPs can agglomerate a great deal in a biological system, which can lead to alterations in particle behavior from what is predicted [24]. Despite this, AuNPs are being

7 investigated quite heavily for next-generation drug delivery applications, due to their general biocompatibility and their recognized plasmonic capabilities [28].

1.2 – Silver Nanoparticles

AgNPs are one of the more commonly used NPs across many different industries, due to their numerous beneficial properties, including catalytic, plasmonic, and antimicrobial potential [23]. Because of this, the properties of AgNPs are well researched and documented [32]. The combination of high utilization and known biological responses following exposure were the rationale for incorporating AgNPs into the design of this study.

1.2.1 – Applications of Silver Nanoparticles

AgNPs are being used in hundreds of applications due to their distinctive antimicrobial, antibacterial, and plasmonic properties [7], [8], [23], [35], [36]. The robust antimicrobial properties of AgNPs lend themselves to use in the medical field in product coatings on medical devices, dental resin, surgical meshes, and different bandages and wound dressings, as well as air sanitizer sprays, detergents, and wet wipes [7], [37]. AgNPs have also been shown to increase the lifespan of inserts for joint replacements, as they can decrease the wear on the polymer [7].

AgNPs can also be found in consumer products such as detergents, air sanitizers, baby bottles, washing machines, cosmetics, and numerous other products where antimicrobial

8 agents are desired [1], [7], [8], [23], [35]. Personal care products, in particular, such as shampoo, soap, and toothpaste often utilize AgNPs [7]. They can even be found in various fabrics, as textile engineering has begun to exploit the favorable properties

AgNPs have to offer [7], [8], [23]. Furthermore, a recent study found AgNPs in multiple parts of plush children’s toys and sippy cups [23].

The plasmonic properties of AgNPs can be exploited in sensors, circuits, and imaging [7],

[36]. Biosensors incorporating AgNPs show promise for detecting proteins which other biosensors have difficulty detecting [7]. Bioimaging is another promising use for AgNPs, as an alternative to fluorescent dyes, which can only be used once per dose [7].

1.3 – Nanotoxicity

Along with all the tunable physicochemical characteristics of NPs that differ from their bulk materials, their toxicity can differ from their bulk material as well [18]. As such, much of the NP research currently being pursued is dedicated to understanding the toxicity of many different NPs being used in numerous ways. This specific branch of this body of work, called nanotoxicology, is an incredibly important piece of the NP research being conducted worldwide. The field of nanotoxicology overall seeks to provide a better understanding of any health effects which may be caused by NPs, and what the appropriate protocols are to study those effects and attain ethical, reliable results [9].

9 Nanotoxicity studies are imperative, as there has been a rising concern regarding the safety of direct exposure to NPs. This exposure can occur through the use of many of the consumer products discussed previously in this chapter, which is visually represented in

Figure 1.3 [38]. In particular, people may encounter these NPs through inadvertent inhalation, absorption through the skin, ingestion, or use in future drug-delivery systems

[1], [3], [9], [37], [39]. Therefore, it is necessary to evaluate the safety of multiple types of exposure to these particles, particularly in lung, skin, and intestinal models, prior to utilization.

Figure 1.3 [38]: Some common uses for NPs and the exposure pathways they may encounter.

1.3.1 – Tunable Properties and Toxicity

The toxicity of NPs can vary as the tunable properties of the NPs themselves change [4],

[13]. For example, the surface charge of the NP can play a large part in the cytotoxicity

10 of the particle. As surface charge plays a vital role in attracting and binding proteins in biological media, this property directly impacts both the protein corona surrounding the

NP and the extent of NP internalization [13]. The surface charge of a NP has also been shown to be able to play a role in the ability for NPs to translocate through the blood- brain barrier [38]. Positively charged NPs have been shown to elicit an immediate toxic response at the blood-brain barrier, while studies suggest that lower concentrations of neutral or negatively charged NPs could potentially be used for drug delivery across the blood-brain barrier without being toxic [38]. In general, negatively charged NPs are found to be less cytotoxic than positively charged particles, likely due to the fact that positively charged particles are taken up into the cell at a higher rate [2], [4].

The size and morphology of the NP can also greatly impact how the NP interacts with the surrounding cellular system. For example, a NP with a rod-like shape can have an increased level of interaction with the cell, if it is situated longitudinally when compared to spherical NPs of the same core, due to the increased surface area [4]. Furthermore, a disc shape is less likely to be taken up by a phagocytic cell, than a spherical particle [4].

While shape plays a large part in the cellular interactions, a more critical property in terms of cytotoxicity is the size of the particle [2]. Generally, the smaller the NP, the more likely it is to be taken up into the cell through endocytosis [4]. This, in turn, has been shown to cause an increase in the visibility of stress markers, as well as a higher degree of cell death [2].

11 The local environment has been shown to affect cellular uptake as well. A study performed with AuNPs in a HaCaT cell model supplemented with physiological fluids showed that the addition of physiological fluids can not only change the protein corona but also the toxicity of the NP [28]. Another study showed that copper oxide (CuO) NPs elicited a significantly lower cellular viability and higher oxidative stress in HaCaT cells when artificial interstitial fluid was included in the culture environment rather than traditional culture media [3]. However, this is not true of all NPs with all cell types and biological surroundings. The same study found little difference in cellular viability or stress in HaCaTs when TiO2 NPs were studied in both culture media and artificial interstitial fluid [3].

Of course, while all of the previously mentioned properties have an effect on cytotoxicity, the actual substances themselves determine much of the toxicity, in terms of both the core composition and the coating of the NP. The core composition has been more directly linked to observable signs of cytotoxicity than any other property, and certain materials tend to be more cytotoxic than others, such as silver and copper [2], [4].

A study using both iron oxide and silver NPs of the same size, coating, and surface charge found that the two particles behaved quite differently in a Caco-2 cellular model

[18]. While some of the toxicity could potentially be lessened by certain coatings, which can slow down or speed up endocytosis, as well as keep certain NPs from dissolving and releasing toxins, often the core composition will still be a major factor [2], [4], [18].

12 1.3.2 – Observable Signs of Nanotoxicity

There are many ways to obtain nanotoxicity data, using both in vivo and in vitro exposures to NPs, and each of these observable signs discussed below can in some way, be linked to cellular death. One of the most common ways to begin an investigation into toxicity is by looking for the production of reactive oxygen species (ROS). Studies have demonstrated that the presence of oxidative stress, including ROS, is a precursor of apoptosis and can be utilized as a means to predict the degree of cellular death [8], [9],

[40]. Another common way to study cytotoxicity is to understand the cellular viability following NP exposure. This can be done by using a viability assay, like lactate dehydrogenase (LDH) or cellular reduction viability, like 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) [9], [40]–[44].

Along with the elevation of ROS levels and cellular viability, there are other pieces of the puzzle which must be added in order to fully understand the extent of nanotoxicity of any given particle. These include the activation of the immune system and other measures of cell stress or inflammation. There are multiple studies which show that cellular exposure to NPs can influence the production of certain cytokines, such as TNF-α and various interleukins, generally, those which are considered proinflammatory [1], [40], [44], [45].

These can be observed through the use of enzyme-linked immunosorbent assays

(ELISAs) or western blot, however, an ELISA is significantly more common now, as it is more time efficient and repeatable.

13 In addition to the activation of an inflammatory response and the production of ROS,

AgNP exposure can activate alternative stress markers, which are less commonly studied, such as HSP27, and NF-κB. HSP27 is a heat shock protein which is generally present in cells at low levels under normal conditions [46]. However, HSP27 can have a rapidly increased expression in cells undergoing stress from a myriad of factors [46]. HSP27 expression has been shown to link to the expression of other measurable cellular responses such as TNF-α, NF-κB, and IL-1β, as well as the cellular process of apoptosis

[47]. NF-κB controls the production of some of the markers of cellular stress, as it responds to multiple inflammatory activators [48]. It has been known to promote the expression of IL-6, a known proinflammatory cytokine, as well as lessen ROS [48].

Observed bioresponses have been correlated directly to a number of the physicochemical properties of AgNPs. Primary size of particles has been shown to affect cytotoxicity and

ROS generation, with smaller AgNPs showing a higher deposition in lung cells after inhalation, and generating more ROS and causing a greater loss in cell viability in macrophages [35], [40], [49]. In longer-term exposure studies, it has been shown that

AgNPs can generate silver ions through dissolution, which can cause a shift in cell functionality [50]. The coating of AgNPs has also been shown to help mitigate toxicity, with one study showing that a hydrocarbon coating could minimize toxicity even in environments containing physiological fluids, whereas a polysaccharide coating could not

[1].

14 Observed bioresponses following NP exposure have been shown to correlate to a number of physiochemical properties in other classes of NPs beyond silver. For example, studies have been published showing that NPs which have agglomerated can show toxicity similar to a larger particle, rather than its smaller, individual particle size [44], [45].

Additionally, the core composition of NPs can have a huge part to play in toxicity. A study performed on three metal oxide NPs (CuO, ZnO, and TiO2) showed that different core compositions result in different cell viabilities when human colorectal adenocarcinoma cells (HT29) were identically exposed to each respective NP [51]. The three different metal oxides were introduced to the HT29 cells in the same concentration and under the same conditions, yet the concentration of internalized NPs and dependent cellular responses differed vastly for each core composition [51].

1.4 – Physiologically Relevant Models

Because of the large number of NPs being developed, as well as the numerous exposure pathways, nanotoxicology safety assessments are carried out on various cell models, a summary of which can be seen in Table 1.1. Currently, most assessments being conducted occur in an in vitro model. The in vitro model utilizes cell lines which can be grown on a plate or in suspension in a flask. These models are advantageous due to their flexibility, low cost, and rapid performance [4], [9], [37], [52]. In vitro models also offer the advantage of less variability among trials [9]. In vivo models, or animal models, can be used as well, but they are more expensive and have many regulations that go along with them, so generally in vitro models are favored for high throughput screening [9],

[52].

15 Table 1.1: Summary of advantages and disadvantages of nanotoxicity testing through in vitro and in vivo models [52].

Both of these types of testing are incredibly important to the nanotoxicology field and have been extensively utilized. However, currently, there are very few physiological similarities between in vitro and in vivo models, producing a poor correlation between NP exposure systems [37], [41], [53], [54]. For example, Demokritou et al bring attention to the fact that CeO2 NPs showed minimal toxicity in in vitro model testing, but in vivo model testing of the same NPs showed signs of cytotoxicity and inflammation [53]. A study performed with TiO2 NPs in both in vivo rat and in vitro rat lung epithelial cell models showed that the results from the two models were quite different [41]. Multiple toxicity parameters were examined, all related to oxidative stress, however, since the tests utilized could not be performed on both model types, it is difficult to relate them precisely [41].

These model-based discrepancies seen in the study performed by Han et al and discussed in depth by Demokritou et al have called significant attention to the fact that traditional in vitro systems are not physiologically relevant for safety and efficacy screenings [41],

16 [53]. Standard in vitro testing cannot replicate many of the toxicological effects of NPs observed in in vivo models, as standard in vitro modeling does not account for coordinated tissue responses, physiological fluids the NP may encounter, or the influence of the fluid dynamics of the body [1], [9]. This is where enhanced in vitro models can play a large role in bridging the gap between standard in vitro models and in vivo systems.

Enhanced in vitro models are ones that retain the advantages of traditional cell-based models but incorporate physiologically relevant transformations. One example of an enhanced in vitro model would be a cellular model incorporating a relevant physiological fluid, such as interstitial, alveolar, and lysosomal fluids, aligning with the utilized cell line. These can be samples of the biological fluid obtained from in vivo systems or an artificially synthesized fluid. The use of artificial physiological fluids allows for a more reliable, reproducible, and accurate in vitro model aiding in the discovery of the full breadth of nanotoxicological responses a particular NP may cause [1], [3], [21]. Early studies of NPs exposed to physiological fluids show that toxicity of numerous NPs may increase after exposure to biological fluids [1], [3], [28]. These studies have also shown that physiological fluids change the way that NPs agglomerate, as well as the way they are internalized by cells [1], [3], [9], [21], [24].

Another way to enhance an in vitro model is to incorporate dynamic flow. The human body, due to the cardiovascular system, is never static. Therefore, including a dynamic

17 element in an in vitro model allows scientists to better predict how a NP may affect certain cell types, particularly when it comes to toxicity and cellular uptake [54]. When studied using fluorescent microscopy, A549 alveolar epithelial cells grown in vitro were shown to elongate under physiologically relevant flow conditions, as they would in vivo, an example of which can be seen in Figure 1.4 [21], [52].

a) b)

Figure 1.4: A549 cells photographed under fluorescent microscopy after a) a static growth, or b) a dynamic growth of 24 hours [52]. The cells grown in an in vitro environment which incorporated fluid dynamics have a shape more similar to lung cells seen in vivo.

Early studies using a microfluidic device to induce dynamic flow have identified differential nanotoxicity results versus a traditional static model [54]. A study using citrate-capped spherical AuNPs and a lab-built microfluidic device showed that the viability of HUVECs after a 24-hour exposure was significantly higher than the viability of the same cells exposed to the AuNPs in a static environment for 24 hours [54].

Another study utilizing a peristaltic pump showed that spherical AuNPs in a range of sizes (5nm – 30nm) agglomerated less in environments incorporating dynamic flow, thereby modifying the physicochemical properties [55]. The same study also showed that

18 cellular interaction and deposition of the AuNPs was lower in a dynamic co-culture of astrocytes and endothelial cells (representative cell types of the blood-brain barrier) than the static culture, determined to be due to the lack of sedimentation forces in the dynamic exposure [55].

Some studies have even combined both fluid dynamics and biological fluids, to create an enhanced in vitro environment. One study used the A549 cellular model along with artificial alveolar fluid and dynamic flow in order to create a more biologically accurate microenvironment [21]. The study found that some cellular responses varied with the inclusion of alveolar fluid, yet were not influenced by dynamic flow, while other responses were altered with the incorporation of dynamic flow but not the alveolar fluid

[21]. The investigators showed that the incorporation of both in their in vitro modifications resulted in data which can better reflect the complex environment of the lung.

Overall, utilizing enhanced in vitro models has many benefits and increases the physiological relevance of the model [56]. In addition to having a model which more directly relates to an in vivo model while avoiding what some see as ethically ambiguous, it is significantly easier to recover NPs from an in vitro exposure than it is to do so from in vivo exposures [37]. However, to date, all of the studies which explored the impact of fluid dynamics on NP internalization and subsequent bioeffects focused on adherent cells.

The goal of this study was to expand these experimental efforts to cells which grow in

19 suspension and would be susceptible to the influences of dynamic flow, such as the U937 monocytic cell line. This research sought to understand how fluid dynamics, specifically physiologically relevant fluid motion, would alter the nano-cellular interface and resultant cellular responses when U937 cells and AgNPs were transported together in a dynamic exposure environment.

1.5 – References

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28 CHAPTER 2

EXPERIMENTAL PROCEDURES

2.1 – Introduction

Throughout the course of this research, numerous experiments were conducted for the purpose of both characterization of particles, as well as to aid in the novel discoveries associated with the exposure of suspended cells to NPs in dynamic environments. These experiments contained numerous protocols and procedures, all of which are explicitly discussed in this section. Along with procedures, the purpose and goal of each experiment are stated as it pertains to the goal of the overall research initiative.

2.2 – In Vitro Cell Growth and Monitoring

In vitro tests are faster and more cost-efficient than in vivo testing, which can take months. These in vitro tests are also considered to be more ethically acceptable as they do not deal with an animal model. As such, in vitro experiments were utilized throughout the duration of this research. U937 monocytes, purchased from American Type Culture

Collection (CRL-1593.2), were cultured in suspension within tissue flasks throughout the duration of the research.

29

2.2.1 – Cell Culture

Purpose: In vitro cell growth allows the investigator to perform experiments with the live cell line in a completely controlled environment. The culturing technique discussed below is the process that was utilized for the U937 cells to grow in suspension.

Procedure:

1. The U937 cell line was purchased from American Type Culture Collection to

culture in the lab.

2. Cells were suspended in a treated flask with 20 – 30 mL of culture media, a

solution of 1640 RPMI (Gibco) media supplemented with 2% penicillin-

streptomycin-amphotericin (PSA) (Gibco) and 10% fetal bovine serum (FBS)

(Gibco). The flask was kept in a 37°C incubator (Thermo Fischer Scientific) with

5% CO2.

3. Every two to three days, the culture was split by aspirating approximately half of

the suspended cells by using a vacuum aspirator.

4. Once aspirated, fresh culture media was pipetted into the flask to return the

suspension to its original volume.

5. The flask was then returned to the incubator until ready to be split again or

utilized in an experiment.

30

2.2.2 – Cell Counting

Purpose: When running in vitro tests, it is necessary to ensure that the cell number in each well is the same, in order to directly compare the collected data between wells. To do this in an accurate and timely manner, a cell counting procedure was run, which resulted in cell densities given in cells/mL.

Procedure:

1. Flasks were gently swirled to ensure an evenly mixed suspension.

2. 10 µL of cells were pipetted from the flask into a clean well of a 96 well plate.

3. Using a new tip, 10 µL of Trypan Blue (Invitrogen) was pipetted into the same

well.

4. The combined 20 µL was pipetted up and down in the well to mix the two liquids

completely.

5. 10 µL of the mixed solution was pipetted onto one side of a Countess Automated

Cell Counter slide.

6. The cartridge was then inserted into a Countess Automated Cell Counter and the

counting program was run.

a. Digital readout gives total cell count, live count, dead count, and percent

viability, based on Trypan Blue analysis.

b. To be considered acceptable for experimentation, cells should have at least

a 90% viability.

31 2.2.3 – Cell Spinning

Purpose: The U937 cell line, in its monocytic form, grows in suspension, rather than adhering to a culture plate. Therefore, during cell splits, not all of the culture media was removed. To ensure that any contaminants were removed from the culture, cells were spun in a centrifuge so that the entirety of the culture media was removed every week.

Procedure:

1. Suspended cells and culture media were pipetted from their flasks into an even

number of either 2 mL microcentrifuge tubes (Thermo Scientific) or 15 mL

centrifuge tubes (Thermo Scientific).

2. Tubes were placed in the centrifuge with the carriers angled at 45°.

3. The centrifuge was run for 10 minutes between 800 and 900 RCF.

4. Tubes were then gently removed from the centrifuge, ensuring that the cell pellet

at the bottom of the tube remained intact and did not re-enter suspension.

5. The culture media supernatant was carefully aspirated from all tubes, being sure

not to disturb the cell pellet.

6. Cell pellets were then reconstituted with a small volume of fresh culture media in

each tube, generally about 1 mL between two microcentrifuge tubes or 1 mL per

15 mL tube.

7. The culture media was pipetted up and down in each tube to ensure that all cells

re-entered suspension in the fresh culture media.

32 8. Finally, the resuspended cells were pipetted into a fresh flask, diluted to an

appropriate volume with more culture media, and returned to the 37°C, 5% CO2

incubator or combined into one tube to be counted and used for experimentation.

2.2.4 – Cell Lysing

Purpose: For experimental analysis, U937s cells were broken open, or lysed, in order to collect intracellular contents. Once the contents of the cells were collected, it was possible to characterize their contents using numerous experimental protocols.

Procedure:

1. Cells, treated or untreated, were pipetted into 2 mL microcentrifuge tubes and

spun in a microcentrifuge angled at 45° at 850 RCF for 10 minutes.

2. The supernatant was then aspirated from the tubes, leaving the cell pellet behind

in the bottom of the tube.

3. The cells were then treated with ice-cold lysis buffer (Cell Signaling

Technologies) in each tube (volume varied for different experimental procedures).

a. Lysis buffer contained protease inhibitors to prevent proteolysis following

collection.

4. Each tube was vortexed for 30 seconds immediately after treatment and placed on

ice for 10 minutes.

5. After 10 minutes, tubes were vortexed again for 30 seconds and placed back on

ice for another 10 minutes.

33 6. Tubes were then spun at 14,000 RPM in the microcentrifuge for 10 minutes at

4°C.

7. After the 10-minute spin, the supernatant from each tube was carefully pipetted

into a fresh microcentrifuge tube, leaving the cell membranes behind.

8. The lysis supernatant was then used for further experimentation. For

experimentation later that day, the supernatant was placed in the refrigerator until

needed. For experimentation taking place at least 12 hours later, the supernatant

was placed in the freezer to maintain the integrity of the sample.

2.3 – NP Characterization

As discussed in Chapter 1, there are many tunable characteristics of NPs which influence specific NP behavior. These characteristics, such as size, surface chemistry, and charge, can alter how the NPs interact with their surroundings and subsequent biological responses. To ensure that the AgNPs used for experimentation were uniform in primary size, surface charge, agglomerate size, and morphology, a number of experiments were run on the AgNPs in an acellular environment. These experiments were run on NPs exposed within both static and dynamic environments to ensure that the introduction of dynamic flow did not alter these characteristics.

The experimental particles utilized in this study were 5 nm and 50 nm poly(vinylpyrrolidone) (PVP) coated AgNPs, purchased from nanoComposix. These particles were the same ones simultaneously used in a second project. Therefore, all

34 AgNP characterization assessments were previously performed by an undergraduate student in Dr. Comfort’s lab.

2.3.1 – Transmission Electron Microscopy (TEM)

Purpose: Transmission Electron Microscopy was used to visualize the experimental

AgNPs. For the purpose of this experiment, TEM was used to confirm the primary size, spherical morphology, and uniformity of both the 5 nm and 50 nm AgNPs. An example of a TEM image for both AgNPs can be seen in Figure 2.1. The procedure that was used is summarized below.

Figure 2.1: Representative TEM images of a) spherical 5 nm AgNPs and b) spherical 50 nm AgNPs.

Procedure [1]:

1. The AgNP stock solutions were removed from storage at 4°C and vortexed to

ensure a homogeneous particle solution.

35 2. Using a micropipette, one drop of AgNP solution (about 20 μL) was placed onto a

TEM grid (Electron Microscopy Sciences) and dried.

3. Once dry, the TEM grid with the AgNPs was imaged using a Hitachi H-7600

microscope.

4. The spherical morphology of the AgNPs was verified via inspection using the

obtained TEM images.

5. The average primary particle size was determined using Image J software. Image

J compares pixel numbers to a known size standard, included in the TEM image,

to produce particles sizes. For each set of AgNPs, approximately 25 particles were

sized to determine average primary size.

2.3.2 – Dynamic Light Scattering (DLS)

Purpose: DLS was used to examine the agglomeration of the AgNPs both in water and in

1640 RPMI media. This analysis was run on both particle sizes in both liquids to assess the extent to which the proteins within media altered AgNP agglomeration. The procedure that was used is summarized below.

Procedure [1]:

1. The AgNP stock solutions were removed from dark, refrigerated storage and

vortexed to ensure a homogeneous solution.

2. The 5000 µg/mL stock solutions were diluted to 25 μg/mL in either water or

culture media.

36 3. The AgNP samples were translocated into an assay cuvette and placed into a

Malvern Zetasizer Nano ZS.

4. DLS analysis was performed on each sample in triplicate, with pre-programmed

settings for silver. The hydrodynamic diameter, which is equivalent to the

agglomerate size, was recorded in nm.

2.3.3 – Zeta Potential

Purpose: Zeta Potential analysis was employed to determine the surface charge of the 5 nm and 50 nm AgNPs. This analysis was run in solutions of both water and 1640 RPMI culture media so as to characterize the degree through which the proteins in the culture media altered the surface charge of the AgNPs. The procedure that was used is summarized below.

Procedure [1]:

1. The 5 nm and 50 nm AgNP stock solutions were removed from refrigerated, dark

storage and vortexed to ensure a homogeneous solution.

2. The 5000 μg/mL stock solutions were diluted to 25 μg/mL in either water or

culture media.

3. The AgNP samples were translocated into an assay cuvette and placed into a

Malvern Zetasizer Nano ZS.

4. Zeta potential analysis was performed on each sample in triplicate, with pre-

programmed settings for silver. The surface charge for each independent trial was

recorded in mV.

37 2.3.4 – Ultraviolet-Visible Spectroscopy (UV-Vis)

Purpose: UV-Vis was used to analyze the size uniformity of the AgNP solutions.

Different sized AgNPs will absorb light at a slightly different wavelength, with larger particles demonstrating a red shift in the spectrum. To confirm uniformity, a single, sharp plasmonic peak is the desired result from this experiment. An example of what these peaks should look like can be seen in Figure 2.2. The procedure that was used is summarized below.

Figure 2.2: An example of a UV-Vis spectral profile for both 5 nm and 50 nm AgNPs between 300-700 nm.

Procedure [1]:

1. The AgNP stock solutions were removed from storage and vortexed to ensure a

homogeneous solution.

2. The 5000 μg/mL stock solutions were diluted to 25 μg/mL in either water or

culture media.

3. 100 μL samples of the diluted AgNP stocks were placed into individual wells of a

96-well plate.

38 4. The well plate was inserted into a SpectraMAX Plus 190 microplate reader and

the absorbance of the samples was analyzed at wavelengths between 300 and 700

nm, in 10 nm increments.

5. The spectral profiles were observed by plotting the collected absorbance data.

2.4 – NP Exposure and Response Characterization

In order to determine how exactly the AgNPs affected cellular functionality after exposure, numerous tests were run to determine how much the AgNPs interacted with and influenced the monocytes. This data could then be correlated with cellular viability, stress, inflammation, and signaling. This section discusses all of those protocols.

2.4.1 – AgNP Exposures

Purpose: The overall goal of this research was to determine the effects of AgNPs on

U937 monocytes. As such, the monocytes had to be exposed to AgNPs in a controlled environment. These procedures outline the different ways in which the monocytes were exposed to the NPs. The dynamic exposure employed a peristaltic pump to create a tube- side velocity of 0.2 cm/s, corresponding to the average velocity seen in capillaries and venules, creating a velocity of 0.003 cm/s across the bottom of the well (Table 2.1) [2].

This pump, as well as the setup of the tubing and cell culture plate, can be seen in Figure

2.3 below.

39 Table 2.1: Blood circulation properties, adapted from Introduction to Bioengineering [2]

Figure 2.3: The peristaltic pump, inside the incubator, used to create physiologically relevant flow rates during experimentation. The lower right image shows how the tubing (ID 0.0625 in) fits into a 24-well plate.

40 Static Procedure:

1. U937 cells were pipetted into 2 mL microcentrifuge tubes.

2. Cells were spun for 10 minutes between 850 – 1000 RCF, in order to remove

them from suspension.

3. The supernatant was aspirated from each tube, leaving the cell pellet at the bottom

of each tube.

4. Cells were reconstituted with a small volume of fresh media, about 1 mL per two

tubes.

5. Reconstituted cells were then combined into one 15 mL tube, gently mixed, and

counted according to the protocol in Section 2.2.2.

a. The desired concentration of all experimentation done was 1.5×105

cells ⁄mL.

6. The U937s were diluted to the appropriate cell volume with fresh culture media to

attain the desired cell concentration at the desired volume, found by using the

following equation.

cells 5 cells (cell count ⁄mL)(volume of solution mL) = (1.5×10 ⁄mL)(x mL)

x = diluted volume mL

7. The diluted solution was then evenly split into five 15 mL tubes, one tube for each

treatment of the cell suspension, as follows:

a. Untreated control, 5 nm particles - 2.5 μg/mL, 5 nm particles – 25 μg/mL,

50 nm particles – 2.5 μg/mL, 50 nm particles – 25 μg/mL

8. The appropriate amount of AgNPs was calculated, based on the stock

concentration of 5000 µg/mL using the following equations:

41 1 μg μg a. For 2.5 μg/mL: (diluted cell volume μL)(2.5 ⁄ )=(x)(5000 ⁄ ) 5 mL mL

1 μg μg b. For 25 μg/mL: (diluted cell volume μL)(25 ⁄ )=(x)(5000 ⁄ ) 5 mL mL

9. Each tube was treated according to the above calculations and was mixed by

inverting each tube a few times.

10. 4 independent samples per treatment condition were plated in 24-well plates

(Thermo Fisher Scientific) with 750 µL of sample per well

11. Plates were incubated for 24 hours in a 37 °C incubator with 5% CO2, in order to

allow the cells time to equilibrate.

Dynamic Procedure:

Note: Due to the number of cassettes available on the peristaltic pump this experiment was run separately for 5 nm NPs and 50 nm AgNPs

1. U937 cells were pipetted into 2 mL microcentrifuge tubes.

2. The cells were spun in a microcentrifuge with tubes angled at 45° at 850 RCF for

10 minutes.

3. Culture media was aspirated, leaving the cell pellets behind in the tubes.

4. Cells were reconstituted with 1 mL of fresh culture media for every two 2 tubes.

The contents of the tubes were then gently mixed and pipetted into a clean 15 mL

tube. A 10 µL sample was taken to count cells according to the protocol in

Section 2.2.2.

5 cells a. The desired cell concentration for experimentation was 1.5×10 ⁄mL.

42 5. Concentrated cell/media solution was combined with fresh media in 50 mL tube

(Thermo Scientific) to reach the desired cell concentration using the equation

below.

cells 5 cells (cell count ⁄mL)(volume of solution mL) = (1.5×10 ⁄mL)(x mL)

x = diluted volume mL

6. A total of three 15 mL tubes were prepared from the counted cells, representing

the three experimental conditions of 1) untreated control, 2) AgNPs at a

concentration of 2.5 μg/mL, and 3) AgNPs at a concentration of 25 μg/mL, based

on the equations below.

1 μg μg (2.5 ⁄ )(diluted cell volume - 2250 μL)=(5000 ⁄ )(x μL) 3 mL mL

1 μg μg (25 ⁄ )(diluted cell volume - 2250 μL)=(5000 ⁄ )(x μL) 3 mL mL

x = volume of AgNP stock μL

7. The appropriate amount of AgNPs were pipetted into both tubes which were then

inverted to mix.

8. 750 μL of untreated cell solution was pipetted into three wells of a 24-well plate

for a static comparison.

9. 2.25 mL of untreated control cells or cells treated with either 2.5 μg/mL or 25

μg/mL of AgNPs were pipetted into three wells each of the same 24-well plate.

a. The volume, 2.25 mL, comes from the addition of 1.5 mL (required to

prime the tubing) to the 0.750 mL working volume of a 24-well plate

43 10. Inlet and outlet tubes from the pump were fit into each dynamic well through

holes drilled in the 24-well plate lid.

11. The pump was then turned on and the tubing was primed.

12. The speed was then decreased to 0.900 RPM to mimic capillary flow. This

operation rate resulted in a tube-side linear velocity of 0.2 cm/s.

13. The plate and pump were placed in an incubator at 37°C with 5% CO2 for 24

hours.

14. After 24 hours, the liquid was allowed to exit from the tubes back into the wells,

and the cells were collected for analysis.

2.4.2 – AgNP Internalization by U937 Cells

Purpose: This experiment was used to determine the amount of AgNPs that was internalized by the cells, which is known to directly correlate to observed cellular responses.

Static Procedure:

1. To begin, the static protocol for AgNP exposure discussed in Section 2.4.1 was

followed. A total of 40 mL of cell/media solution was prepared for this

experiment using that protocol.

2. After a 24-hour exposure, each well was transferred to a 2 mL microcentrifuge

tube and labeled. Tubes were then spun in a microcentrifuge for 10 minutes at

1000 RCF.

44 3. The supernatant from each tube was transferred into a fresh 2 mL microcentrifuge

tube, leaving the cell pellet behind to be discarded.

4. For each experimental run, 100 µL samples were pulled from the wells, in

triplicate, and transferred to a clean 96-well plate.

5. Using UV-Vis an absorbance spectrum was run on the plate from 300 – 700 nm,

increasing by 10 nm each pass, using a Synergy 4 BioTek microplate reader. The

quantity of AgNPs remaining in solution was determined by performing an area

under the curve analysis for each AgNP, utilizing previously generated calibration

curves. The quantity of internalized AgNPs was determined by comparing the

initial dosage and the final solution concentrations.

6. After transferring these aliquots, all remaining supernatant was placed in a -20°C

freezer for storage for later analysis.

Dynamic Procedure:

1. The dynamic AgNP exposure protocol was followed to prepare 23 mL of

cell/media solution for experimentation according to the protocol spelled out in

Section 2.4.1.

2. Following the 24-hour exposure, the solution from each well was pipetted into 2

mL microcentrifuge tubes, one tube for every static well and two tubes for every

dynamic well.

3. All microcentrifuge tubes were spun at 850 RCF for 10 minutes.

45 4. After spinning in the centrifuge, the supernatant from each trial was pipetted from

into fresh tubes.

5. Three 100 μL samples were pulled off the top of each tube containing the 50 nm -

25 µg/mL samples using a pipette and placed into a well of a 96-well plate.

6. After transferring these aliquots, all remaining supernatant was placed in a -20°C

freezer for storage for later analysis.

7. Prepared standards (5 nm and 50 nm AgNPs) were plated in 3 wells of the same

96-well plate (Thermo Fischer Scientific) that contained the experimental

samples. Using UV-Vis, on a Synergy 4 BioTek microplate reader, an absorbance

spectrum was run on the plate from 300 – 700 nm, increasing by 10 nm each pass.

The quantity of AgNPs remaining in solution was determined by performing an

area under the curve analysis for each AgNP, utilizing previously generated

calibration curves. The quantity of internalized AgNPs was determined by

comparing the initial dosage and the final solution concentrations.

2.5 – Cellular Responses to NP Exposure

It is imperative that, immediately after exposure to AgNPs, cellular responses are measured, or the samples are preserved so that the sample maintains its integrity. The tests performed on the supernatant included tests to measure cell stress and inflammation, as well as cytokine responses. These cytokine responses are measures of stress specifically linked to immune and inflammatory responses. The results of these tests were compiled to determine overall cytotoxicity after NP exposure.

46 2.5.1 – Lactate Dehydrogenase (LDH)

Purpose: LDH levels released by cells are an indicator of cellular apoptosis, as the LDH is exocytosed into the surroundings following cellular death. Released LDH levels are known to be directly proportional to the degree of cellular death, which will indicate the overall cytotoxicity of the AgNPs.

Procedure:

1. Previously frozen supernatants, collected during static and dynamic

internalization analyses, were thawed.

2. The frozen Assay Buffer (Promega) was thawed in a drawer, keeping it away

from light. Once thawed, 12 mL of buffer was pipetted 12 into a bottle of

Substrate Mix (Promega).

3. The substrate solution was gently shaken to mix and placed in a drawer to keep it

away from light until needed.

4. 50 µL of each thawed experimental supernatant was pipetted into a well of a 96-

well plate.

5. 50 µL of reconstituted Substrate Solution was then pipetted into each well

containing sample.

6. The 96-well plate was then covered in aluminum foil and placed in a drawer to

incubate at room temperature for 30 minutes without light.

7. After 30 minutes, 50 µL of Stop Solution (Promega) was added to each well

containing an experimental sample.

47 8. The absorbance of the plate was read at 490 nm, with the absorbance being

directly correlated to the degree of LDH release.

2.5.2 – Reactive Oxygen Species (ROS)

Purpose: Reactive oxygen species are produced intracellularly when a cell is experiencing stress. As the amount of ROS increases, it indicates that the cells are experiencing higher levels of intracellular stress, which is a precursor for apoptosis.

Therefore, quantifying the amount of ROS present is a good way to determine the cytotoxic potential of the AgNPs. Due to the light-sensitive nature of this procedure, all experimentation was carried out with minimal ambient lighting.

Static Procedure:

1. U937 cells were pipetted into 2 mL microcentrifuge tubes and spun at 850 RCF

for 10 minutes.

2. After the 10 minutes, the supernatant in each tube was aspirated, being sure not to

disturb the cell pellet.

3. Cells were reconstituted with 1 mL of fresh culture media for every two tubes.

4. Reconstituted cells were pipetted into one 15 mL tube and gently mixed. Cells

were then counted according to the protocol in Section 2.2.2.

5 cells a. Desired count for experimentation 1.5×10 ⁄mL

48 5. Based on the cell count, concentrated cell/media solution was pipetted into 2

microcentrifuge tubes and spun again for 10 minutes at 850 RCF.

6. While the two tubes were in the microcentrifuge, the fluorescent probe solution

was prepared to quantify the amount of ROS, as follows.

a. 8.4 mg of the powdered fluorescent probe for U937 cells, DCFH-DA

(Invitrogen), was weighed out in a tared 2 mL tube.

b. The DCFH-DA was reconstituted with 1.74 mL of DMSO to create a 10

mM stock of the probe solution.

c. A 100 µM stock of DCFH-DA was created by mixing 12 mL of culture

media with 120 µL of the 10 mM probe stock in a 15 mL tube. The 100

µM stock was vortexed to ensure a uniform concentration throughout the

tube.

d. The remainder of the 10 mM stock was covered in aluminum foil and

placed in the refrigerator for storage.

7. When the microcentrifuge was finished, the supernatant was aspirated from the

two tubes, leaving the cell pellet behind.

8. The cells were resuspended in the prepared 100 μM probe stock and pipetted into

one 15 mL tube.

9. The 15 mL tube containing the probe stock/cell mixture was incubated for 30

minutes at 37°C with 5% CO2.

49 10. After the 30-minute incubation period, the contents of the 15 mL tube were

pipetted into microcentrifuge tubes, which were spun for 10 minutes at 850 RCF.

11. After spinning, the supernatant was aspirated from the tubes and cells were

reconstituted with fresh culture media to a total volume of 20 mL.

12. The 20 mL of cell/media mixture was divided into tubes with 4 mL of cell/media

solution in each, one for each of the five experimental cell treatments, which are

as follows:

a. Untreated control, 5 nm AgNPs - 2.5μg/mL and 25μg/mL, 50 nm AgNPs -

2.5μg/mL and 25μg/mL

13. Appropriate aliquots of AgNPs were pipetted into the four tubes to be treated and

gently mixed. μg μg (2.5 ⁄mL)(4 mL) = (5000 ⁄mL)(x mL)

x = 0.002 mL = 2 μL

a. 2 μL of AgNPs to 2.5 μg/mL tubes

b. 20 μL of AgNPs to 25 μg/mL tubes

14. Each sample was plated in four wells of a 24-well plate, with 750 μL of

cell/media/NP solution per well.

15. The plate was incubated at 37°C with 5% CO2 for 24 hours.

16. The next day, four 100 μL samples from each well were transferred to a 96-well

plate, resulting in 16 wells for each exposure condition.

50 17. The plate was then placed in a Synergy 4 BioTek microplate reader where a

fluorescence reading was taken, with excitation and emission wavelengths of 495

and 520, respectively.

Dynamic Procedure:

Note: Due to the amount of tubing available, this experiment was run separately for 5 nm

NPs and 50 nm NPs

1. One flask of U937 cells spun for 10 minutes at 850 RCF, after which the

supernatant was aspirated, leaving the cell pellet behind.

2. Cells were reconstituted with fresh culture media and pipetted into one 15 mL

tube. The reconstituted cells were counted according to the protocol discussed in

Section 2.2.2.

5 cells a. Desired concentration for experimentation was 1.5×10 ⁄mL.

3. The total required volume was determined based on the cell count. This volume of

cells was then pipetted into three microcentrifuge tubes and spun at 850 RCF for

10 minutes.

4. While the cells were spinning, the DCFH-DA probe was made as previously

described in the Static Procedure of this section.

5. After spinning, the supernatant from the cell/media solution was aspirated,

leaving the cell pellet behind. The cells were then resuspended in the prepared

100 µM DCFH-DA probe in one 15 mL tube.

51 6. The 15 mL tube was incubated at 37°C and 5% CO2 for 30 minutes.

7. The contents of the 15 mL tube were pipetted into microcentrifuge tubes and spun

at 850 RCF for 10 minutes.

8. The supernatant was aspirated from the microcentrifuge tubes and the remaining

solid contents of the tube were reconstituted with the necessary amount of fresh

culture media to maintain the desired cell concentration, and gently mixed.

9. 6.8 mL of cell/media solution was then pipetted into two different 15 mL tubes,

one for a 2.5 µg/mL concentration and one for 25 µg/mL.

10. A calculation was performed to determine the amount of AgNPs necessary for the

two concentrations. 휇푔 휇푔 (2.5 ⁄푚퐿)(6800 휇퐿) = (5000 ⁄푚퐿)(푥 휇퐿)

푥 = 3.4 휇퐿

11. AgNPs were pipetted into the tubes based on the correct concentration based on

the above calculation.

a. 3.4 µL for 2.5 µg/mL

b. 34 µL for 25 µg/mL

12. All cells were then plated in a 24-well plate, with 750 µL of cell/media solution

for the static wells containing the 0 NP control and 2.25 mL for the dynamic

wells.

13. Once all cells were plated, the pump tubes were placed in all dynamic wells, the

tubing was primed, and the pump speed was set to run at 0.900 RPM. The pump

52 was housed in the incubator to ensure the proper cellular conditions of 37°C and

5% CO2 for 24 hours.

14. After 24 hours, the cells were recollected from the tubing into the 24-well plate.

For each experimental well, 100 µL of cells were transferred into four wells of a

96-well plate.

15. The 96-well plate was placed into a Synergy 4 BioTek microplate reader where

ROS levels were analyzed via fluorescence analysis.

2.5.3 – Cytokine ELISAs

Purpose: The cytokines IL-1β, IL-6, IL-8, IL-10, and TNF-α have all been known to be markers of immune responses in human monocytes [3] - [7]. IL-1β enhances the production of IL-2, which regulates T-cell growth and differentiation [3], [8]. IL-6 is not generally produced by normal cells, but its production is induced by a variety of viral infections, making it an excellent marker of cell health [4]. IL-8 has been known to increase the ability for specific types of white blood cells to permeate inflamed or injured tissues in the body [5]. IL-10 has been shown to be an excellent anti-inflammatory agent in humans [6]. TNF-α has been known to respond to numerous immune stimuli, such as a tissue injury or infection, and can induce local blood clotting [7]. These tests were run on the supernatant of monocytic cultures in order to determine the immunological response to a 24-hour exposure to AgNPs.

53 Concentrations of secreted cytokines were determined via ELISA kits from Invitrogen.

The full manufacturer protocols were followed and summarized below.

Procedure for Cytokine ELISAs [3]-[7]:

1. The human cytokine standard was diluted with the Standard Diluent Buffer

included in the ELISA kit to its designated concentration, then gently mixed and

used immediately

2. The cytokine standard was then further diluted seven more times to create various

standards for the samples to be compared to

a. IL-1β standards ranged from 250 pg/mL to 0 pg/mL

b. IL-6 standards ranged from 500 pg/mL to 0 pg/mL

c. IL-8 standards ranged from 1000 pg/mL to 0 pg/mL

d. IL-10 standards ranged from 500 pg/mL to 0 pg/mL

e. TNF-α standards ranged from 1500 pg/mL to 0 pg/mL

3. After the standards were prepared, Streptavidin-HRP was prepared via dilution

with HRP Diluent.

4. For each experimental sample and premade standard, 100 µL (50 µL for IL-1β)

were pipetted into a well of one of the pre-coated ELISA strips.

5. After samples and standards were plated, 50 µL (100 µL for IL-1β) of cytokine

biotin conjugate was added to each well.

6. The plate was then covered and incubated for two hours at room temperature.

7. After the incubation period, the wells were washed four times with reconstituted

1x wash buffer.

54 8. 100 µL of 1x Streptavidin-HRP was pipetted into each well. The plate was

covered and incubated at room temperature for 30 minutes.

9. After the incubation, the wells were washed four times.

10. After the second wash, 100 µL of the included stabilized chromogen was pipetted

into each well. The plate incubated in the dark, at room temperature, for 30

minutes.

11. Following the incubation, 100 µL of the included Stop Solution was pipetted into

each well, causing a color change from blue to yellow.

12. The absorbance at 450 nm was read on a Synergy 4 BioTek microplate reader.

2.6 – Signaling Responses to NP Exposure

Frequently, a signaling response accompanies a cellular loss of viability, which helps explain the mechanism of cytotoxicity. Similarly, when a cell becomes stressed or inflamed, there is a signal which releases the stress or inflammatory indicators, similar to the secretion of cytokines. In an attempt to determine which specific signaling pathway the U937s used, a number of phosphorylation sandwich ELISAs were run, which helps to identify which signaling pathways were “turned on”.

2.6.1 – BCA Protein Assay (BioRad)

Purpose: The BCA protein assay is a way to determine the protein concentration of a cellular lysate. This process occurs after lysis when the cell walls have been opened and the intracellular contents are collected. As ELISAs have a target protein range, it is

55 important to not exceed the maximum amount of protein loaded. Following BCA analysis, if it was found that the protein concentration within the lysate superseded recommended levels, the samples were diluted prior to experimentation.

Procedure for U937 Number Optimization:

1. U937s were transferred to microcentrifuge tubes and spun at 850 RCF for 10

minutes.

2. After spinning, the supernatant of the tubes was aspirated and reconstituted with

fresh culture media in a 15 mL tube.

3. The cells were then counted, according to Section 2.2.2 and based on the cell

density, multiple experimental concentrations of U937s were generated, as

follows:

a. Target concentrations were 0.5×105, 1.5×105, 3.0×105, 5.0×105, 1.0×106,

2.0×106, 2.5×106 and 3.0×106 cells/mL

4. Following the generation of the experimental U937 concentrations, the samples

were spun for 10 minutes at 850 RCF in a microcentrifuge.

5. The cells were then lysed according to the cell lysis protocol discussed in Section

2.2.4.

6. Protein standards were then diluted according to the BCA assay protocol.

Standards were diluted using the same lysis buffer that was employed to lyse the

U937s.

a. Standard concentrations ranged from 0 - 2000 µg/mL.

56 7. 25 µL of either protein standard or experimental lysate was pipetted into two

wells of a 96-well plate.

8. The Working Reagent for the BCA protein assay was then prepared as per the

manufacturer’s instructions.

a. 17.65 mL Reagent A and 353 µL Reagent B, both included in the BCA

protein assay kit, were pipetted into a 50 mL tube.

9. 200 µL of Working Reagent was then pipetted into each well containing standard

or sample. The plate was gently tapped to mix the contents of the wells.

10. The plate was incubated at 37°C with 5% CO2 for 30 minutes.

11. After the 30-minute incubation, the absorbance of each well at 570 nm was read

using a Synergy 4 BioTek microplate reader.

2.6.2 – Stress ELISAs

Purpose: There are multiple ways that cells can exhibit symptoms of stress. Besides the cytokines tested, NF-κB and HSP27 also can be indicators of stress. Heat shock proteins, which are generally present in cells under normal physiological conditions, have been known to have rapidly increased levels when cells are exposed to a known cytotoxin, like

AgNPs. NF-κB plays a role in the cellular stress response [9]. It has been proven to be involved in the mediation of inflammation caused by ROS and other such inflammatory stimuli [9]. As such, ELISA kits were run to determine if the activation of these two proteins was altered when U937s were exposed to AgNPs. Phosphorylated protein levels, which are indicative of the activated state, were normalized by the total protein content of

57 each protein. All ELISAs were purchased from Cell Signaling Technologies and run in accordance with the manufacturer’s instructions, which are described briefly below.

pNF-κB and pHSP27 Kit Procedure:

1. Detection Antibody Solution was prepared by reconstituting the lyophilized

sample with the provided Detection Antibody Diluent.

2. The solution was allowed to sit for 5 minutes and was occasionally gently mixed

to ensure full dissolution. The entirety of the reconstituted solution was then

diluted with the remaining 10.0 mL of Detection Antibody Diluent and gently

mixed.

3. HRP-Linked Antibody Solution was prepared by reconstituting the lyophilized

antibody with the included HRP Diluent.

4. The solution was allowed to sit for 5 minutes and was occasionally gently mixed

to ensure complete dissolution. The entirety of the reconstituted solution was then

diluted with the remaining 10.0 mL of HRP Diluent and gently mixed.

5. 100 µL of properly diluted experimental lysate was pipetted into one well of the

provided, pre-coated ELISA strips. The plate was then covered and incubated at

37°C for 2 hours.

6. After incubation, the wells were washed four times with prepared 1x wash buffer.

7. After washing, 100 µL of previously prepared Detection Antibody Solution was

pipetted into to each well. The plate was then covered and incubated at 37°C for 1

hour.

58 8. Following one-hour incubation, the wells were washed again four times with the

prepared 1x wash buffer.

9. Next, 100 µL of the previously prepared HRP-Linked Secondary Antibody

Solution was pipetted into each well. The plate was then incubated at 37°C for 30

minutes.

10. After incubation, the wells were washed four times with 1x wash buffer.

11. After the third wash, 100 µL of TMB substrate was pipetted into each well. The

plate was incubated at 37°C for 10 minutes.

12. 100 µL of the included Stop Solution was then added to each well and the plate

was lightly tapped to mix.

13. The absorbance was read at 450 nm using a Synergy 4 BioTek microplate reader.

14. Phosphorylation levels of both NF-κB and HSP27 were determined by

normalizing the results from the phosphorylated ELISAs by the results of the total

ELISAs for each respective protein.

2.7 – Statistical Analysis

All data is presented as the mean ± the standard error of the mean. For all experiments, three independent trials were performed. For AgNP characterization and internalization, a one-way ANOVA with Bonferroni post-test was carried out using GraphPad Prism. A two-way ANOVA with Bonferroni post-test was performed for cellular evaluations. For all experimentation, a p-value threshold of 0.05 was used to determine statistical significance.

59 2.8 – References

[1] R. F. Uhrig, “The Impact of a Dynamic Environment on Deposition and Cellular

Response to Silver Nanoparticles,” University of Dayton, 2017.

[2] S. A. Berger, W. Goldsmith, and E. R. Lewis, Introduction to Bioengineering.

Oxford ; New York : Oxford University Press, 1996, 1996.

[3] “IL-1 Beta Human Platinum ELISA Product Information and Manual,” Thermo

Fisher Scientific, 2017. [Online]. Available: www.thermofisher.com/order/catalog/

product%0A/BMS224-2TEN?SID=srch- srp-BMS224-2TEN%0A.

[4] “Human IL-6 ELISA Kit User Guide,” Thermo Fisher Scientific, 2017. [Online].

Available: www.thermofisher.com/order/catalog/product/BMS213-2?SID=srch-

srp-BMS213-2.

[5] “Human IL-8 Coated ELISA Kit Product Information & Manual,” Thermo Fisher

Scientific, 2017. [Online]. Available: www.thermofisher.com/order/catalog/

product%0A/BMS204-3TEN?SID=srch-srp-BMS204-3TEN%0A.

[6] “Human IL-10 Platinum ELISA Product Information and Manual,” Thermo Fisher

Scientific, 2017. [Online]. Available: www.thermofisher.com/order/catalog/product

%0A/BMS215-2TEN?SID=srch-srp-BMS215-2TEN%0A.

[7] “Human TNF Alpha Total Coated ELISA Kit Product Information & Manual,”

Thermo Fisher Scientific, 2017. [Online]. Available: www.thermofisher.com/order

/%0Acatalog/product/BMS2034?SID=srch-srp-BMS2034%0A.

[8] W. Liao, J. X. Lin, and W. J. Leonard, “IL-2 family cytokines: New insights into

the complex roles of IL-2 as a broad regulator of T helper cell differentiation,”

Current Opinion in Immunology, vol. 23, no. 5. pp. 598–604, 2011.

60 [9] O. Straume et al., “Suppression of heat shock protein 27 induces long-term

dormancy in human breast cancer.,” Proc. Natl. Acad. Sci. U. S. A., vol. 109,

no. 22, pp. 8699–704, 2012.

61 CHAPTER 3

RESULTS, DISCUSSION, AND CONCLUSIONS

3.1 – Introduction

The overall goal of this work was to determine the degree to which fluid dynamics affects the transport and internalization of AgNPs of various primary particle size. While previous studies have explored the impact of dynamic flow on NP transport and cellular distribution, this study is one of the first to examine NP internalization and subsequent biological responses within a suspension cell model. In this study, the human monocytic,

U937, cell line was implemented as the immune system is the predominant mechanism through which in vivo systems protect against exposure to foreign material, such as NPs.

The following chapter shows the results obtained throughout the course of experimentation, as well as a thorough discussion of the significance and implications of these results.

3.2 – Results

3.2.1 – NP Characterization Results

As stated in the previous chapter, the experimental AgNPs used in this study were concurrently used by undergraduate students in Dr. Kristen Comfort’s lab on a related

62 project. As such, the characterization assessments were previously carried out by these students, with the results presented below. [1]. The characterization assessments, which are summarized here, verify that the AgNPs used throughout the entirety of the experimentation were of the size and morphology stated by the manufacturers, and were of high quality. A summary of all characterization data can be found in Tables 3.1 and

3.2.

For this study, AgNPs were specifically selected owing to their well-documented biological responses, which provide a benchmark for comparison. In order to include an analysis with regards to primary particle size, two different AgNPs were employed in this study; 5 nm and 50 nm particle stocks. In order to eliminate as many variables as possible, both of these particle sets were spherical in nature and functionalized with PVP, which is known to promote particle stability.

TEM was employed to verify spherical morphology and primary size of the AgNPs, as well as their uniformity. Representative TEM images of both the 5 nm and 50 nm particles are shown in Figure 3.1. These images clearly demonstrate a spherical shape and a uniform size as well. The average sizes of the 5 nm and 50 nm AgNPs were found to be

5.3 ± 1.4 nm and 52.6 ± 6.9 nm, respectively.

63

Figure 3.1: TEM Images of a) 5 nm AgNPs and b) 50 nm AgNPs confirming spherical morphology and uniformity [1].

After uniformity and morphology were confirmed, UV-Vis was employed to further confirm the size and purity of the AgNP stocks, as well as to visualize the unique plasmonic profiles of the experimental particles. Figure 3.2 shows the absorbance spectra for both the 5 nm and 50 nm AgNPs. The figure shows two distinctive peaks, both quite sharp, with the 50 nm peak showing a slight red shift, precisely as anticipated. As previously discussed increasing primary size will cause a shift in the visible spectrum, which shifts the UV-VIS peak to the right. Furthermore, the distinctive, sharp peaks are indicative of pure, non-contaminated AgNP stocks.

64

1 .2 )

. 5 n m

u 1 .0

. 5 0 n m

a (

0 .8

e c

n 0 .6

a b

r 0 .4

o s

0 .2

b A 0 .0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 W a v e le n g th (n m )

Figure 3.2: Plot showing the absorbance of both the 5 nm and 50 nm AgNPs using UV- Vis. The two peaks are sharp, and the 50 nm peak is shifted about 50 nm to the right, as expected [1].

Next, the degree of AgNP agglomeration, in both water and cell culture media, was carried out using dynamic light scattering (DLS), the results of which are shown in Table

3.1. As the surrounding fluid has the ability to significantly impact the extent of inter- particle association it was important to run DLS analysis in all experimental fluids.

Additionally, as the U937 exposure was carried out in both static and dynamic environments, AgNP characterization was performed under both these conditions. The agglomerate sizes in water are quite similar to the primary AgNP sizes as determined by

TEM, due to the PVP surface chemistry. In culture media, the NPs agglomerated much more, as expected, due to both the high ionic content and the formation of a protein corona surrounding the AgNPs. The agglomerate size of both the 5 nm and 50 nm AgNPs did not change in a statistically relevant manner in either water or media when studied within a dynamic environment. While the presence of shear stress does have the ability to disrupt particle agglomeration, the pump operated at a low, physiological rate, which makes the consistent agglomerate size under static and dynamic conditions unsurprising.

65

Table 3.1: A Summary of all size-related data obtained from AgNP Characterization analysis. This includes primary and agglomerate sizes [2]. Size (nm) Agglomerate Size (nm) Static Dynamic

Water Media Water Media 5 nm 5.3 ± 1.4 8.7 ± 0.9 19.3 ± 2.5 8.9 ± 1.2 18.6 ± 1.7

50 nm 52.6 ± 6.9 78.6 ± 3.0 89.3 ± 2.2 76.7 ± 2.2 90.8 ± 2.9

Next, zeta potential analysis was conducted to determine the surface charge of the AgNPs and the extent to which the culture media altered this key physicochemical property. This analysis was performed on both sizes of the NPs in water and culture media under both static and dynamic conditions, as shown in Table 3.2. In water, the AgNPs displayed a negative surface charge, in agreement with the PVP coating. Following dispersion in media, the formation of a protein corona resulted in a shift in surface charge to approximately -10 mV, the innate charge of proteins. Moreover, the addition of the fluid dynamics was shown to not alter the surface charge in a statistically significant manner.

Table 3.2: A Summary of all Zeta Potential Data for both 5 nm and 50 nm AgNPs [2].

Zeta Potential (mV) Static Dynamic Water Media Water Media 5 nm -27.7 ± 1.1 -10.6 ± 0.8 -25.4 ± 1.7 -9.9 ± 0.5 50 nm -30.4 ± 1.7 -9.2 ± 0.6 -32.2 ± 2.4 -10.5 ± 1.0

Based on the results from each of these characterization experiments, it was determined that these AgNPs were within acceptable tolerances to continue with the planned

66 experimentation, as they demonstrated a high degree of uniformity and characterization reproducibility. Additionally, these assessments determined that the presence of fluid dynamics did not significantly impact the physicochemical properties of the AgNPs, meaning that any changes to the nano-cellular interface and biological response are due to environmental alterations and not changes to NP parameters.

3.2.2 – Cellular Association Results

The overall goal of this study was to determine the effect of fluid dynamics on the interaction of AgNPs with mammalian cells grown in suspension in an environment which incorporated fluid dynamics, and any cellular response that may arise due to changes in the exposure environment. As a control for comparison, the U937s were first cultured in a static environment with the AgNPs for 24 hours. This analysis examined to what degree the AgNPs were associating with the U937 cells; either through an internalization process or through tight binding to the external cellular membrane. The 5 nm particles were found to be taken up by the cells much more readily than the 50 nm particles, an unsurprising result as it is well known that cellular uptake is often related to particle size, which can be seen in Figure 3.3 [3]. Additionally, as the larger particles are associated with an augmented sedimentation force, a greater number of 5 nm AgNPs remained in solution, increasing contact potential with the U937 cells.

67

Figure 3.3: The percentage of NPs taken up or bound by the U937 cells after a 24-hour exposure in either a static or dynamic environment. The dynamic exposures of both the 5 and 50 nm particles show a significant increase in internalization when compared with their static counterpart. *Denotes statistical significance between static and dynamic conditions.

After a successful static culture, a venture into a culture incorporating fluid dynamics came next. After a 24-hour exposure to AgNPs within a dynamic culture, the same internalization pattern which was seen as within the static culture. The monocytes took up the 5 nm AgNPs more readily than the 50 nm particles. More importantly, when directly comparing the static versus dynamic conditions for both experimental NPs, the cellular- particle association rates within the dynamic conditions were significantly higher.

3.2.3 – Cellular Response Results

Once a full understanding of the cellular internalization/association was elucidated, cellular responses could be studied and correlated back to the degree of AgNP internalization by the U937 cells. As AgNPs are renowned for their cytotoxic potential, numerous cellular responses focusing on cellular death and stress were selected.

68 Specifically, this work included analysis of cellular death, cell stress through several mechanisms, and inflammation: all of which have been previously shown to occur in mammalian cells after exposure to AgNPs [4] – [6].

A reliable sign of cellular stress is the production of reactive oxygen species (ROS), as it is a well-documented cellular response following AgNP exposure. While a basal level of

ROS continually exists, an elevated amount of ROS in cells is a telltale sign of intracellular stress and a predictor of apoptosis; making it a critical nanotoxicological endpoint. As shown in Figure 3.4, all four experimental conditions were associated with an increase in intracellular ROS levels, though not to an equal extent.

Figure 3.4: A comparison of ROS produced by the monocytes after 24 hours of exposure to 2.5 µg/mL of each NP size and environmental condition compared to a static, untreated control. *Denotes statistical significance from untreated control. †Denotes statistical significance between static and dynamic conditions.

69 Within both the static and dynamic exposures, the 50 nm AgNPs evoked a much higher

ROS response than the 5 nm NPs did. When looking at each size of NP, the dynamic exposure elicited a statistically significant elevation in ROS production versus static conditions. This shows that the addition of the fluid dynamics to the culture environment increases the production of ROS, corresponding with the increased internalization rate of

AgNPs. Controls demonstrated that untreated U937s within dynamic conditions exhibited equivalent ROS levels to untreated static conditions (data not shown).

The next cellular target evaluated was NF-κB, an upstream regulator for ROS. As shown in Figure 3.5, the 5 nm AgNPs were able to substantially activate the NF-κB pathway, though this response was not detected with the 50 nm AgNPs. It appears that the 5 nm exposures experienced a significantly larger activation of the transcription factor than the

50 nm exposures did, aligning with the smaller amounts of ROS seen from those exposures. A challenging aspect of NF-κB activation is that it is difficult to delineate its direct correlation to ROS activation, as it is both an activator and a repressor of the formation of oxidative stress. However, this work clearly shows that the presence of dynamic flow within a cellular model does target and diminish NF-κB activation levels, regardless of levels of activation under static conditions.

70

Figure 3.5: The activation of NF-κB of each experimental exposure compared to a static, untreated control. The static exposures showed a higher activation of this transcription factor than the dynamic exposures did. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions.

Another cellular stress response following AgNP exposure is heat shock protein 27 (HSP

27) activation. Beyond AgNPs, activation of HSP 27 has been shown to be triggered by radiation and other cytotoxic substances as well [7]. Compared to the untreated control, all four experimental exposure conditions resulted in a substantial increase in HSP27 activation within U937 cells, as shown in Figure 3.6. Aligning with the AgNP internalization and ROS levels, HSP27 activation was greater under dynamic flow conditions for both nanoparticle sets.

71

Figure 3.6: A comparison of the activation of HSP 27 by AgNP treated U937s to a static, untreated control. The extent of activation appears to correspond to both the NP size and the extent to which the AgNPs were taken up by the U937s. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions.

As the static exposures likely offer fewer opportunities for the NPs to interact with the cells, owing to a fluid-dynamic dependent reduction in AgNP sedimentation, this result makes sense. The 50 nm particles are shown to have the ability to increase the HSP27 activation more than the 5 nm particles, demonstrating that, when it comes to nanotoxicology, more than one attribute of the NPs or the environment in which they are used, have an effect on how the cells respond to the NPs.

Another useful marker of cytotoxicity is LDH secretion, which is known to be proportional to cell death, as it is released during apoptosis. In the lower experimental dose of 2.5 µg/mL of 5 nm AgNPs, little LDH leakage was seen outside of what was

72 found in the untreated control, as shown in Figure 3.7. This result indicates that at this low concentration the U937 cells are exhibiting extensive cellular stress, but not undergoing active cellular death.

Figure 3.7: A comparison of 5 nm AgNP exposures to an untreated, static control. A small concentration did not seem to influence the LDH leakage of U937s, but a larger concentration induced LDH production, which correspond to the internalization of the AgNPs within the U937s. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions.

However, at the higher AgNP concentration of 25 µg/mL, a significant increase in the

LDH production for the static exposure was uncovered. LDH levels were further augmented under dynamic conditions, consistent with the increase in U937 internalization. Taken together the observed increase in AgNP internalization under dynamic conditions led to a corresponding rise in both cellular stress and cytotoxicity.

73 Monocytes are a critical part of the immune system, which is responsible for initiating a protective, inflammatory response following the detection of a foreign pathogen or material. Therefore, investigating the activation of the inflammatory response following

AgNP exposure is crucial to fully understanding the negative effects NPs can induce on a biological system. Multiple inflammatory responses were investigated, however, two cytokines showed results above basal levels: IL-6 and IL-8. Both of these cytokines are key modulators of the pro-inflammatory response, frequently triggered by cellular stress.

Both the IL-6 and IL-8 responses were activated in a similar manner. The IL-6 response, which can be seen in Figure 3.8, shows that the inflammatory response was not influenced by the addition of the fluid dynamics, but only by the size of the AgNP. The 5 nm particles were unable to elicit an inflammatory response from the U937s. However, the 50 nm particles were able to largely increase the amount of IL-6 secreted. While not a cytotoxic result, an inflammatory response is still a negative cellular response to the presence of AgNPs.

74

Figure 3.8: A chart showing the comparison of the secretion of IL-6 across all four experimental conditions with a static, untreated control. The 5 nm particles appear not to have influenced the secretion of the inflammatory response, only the larger particles were able to do so. *Denotes statistical significance from untreated control.

As discussed previously, the IL-8 secretion was similar to the IL-6 secretion in this study.

The 5 nm particles induced a similar or lesser secretion of IL-8 by the U937 cells, as can be seen in Figure 3.9. The 5 nm dynamic exposure actually had a smaller amount of the cytokine present than the static, untreated control did, an anomaly which was found difficult to explain. The 50 nm particles, however, induced an increase in the amount of

IL-8 secreted by the cells, without seeing a statistically significant difference between the static and dynamic exposures. The 50 nm AgNPs induced a slightly higher secretion of the IL-8 than they did the IL-6, indicating that each inflammatory response reacts differently to the presence of AgNPs.

75

Figure 3.9: IL-8 secretion induced by the four experimental AgNP exposures compared to a static, untreated control. This plot shows little to no difference occurred between the static and dynamic exposures to the same NP. *Denotes statistical significance from untreated control †Denotes statistical significance between static and dynamic conditions.

3.3 – Discussions

3.3.1 – Discussion of NP Characterization Results

The characterization of the NPs is an important beginning step in any experiment involving NPs. As discussed in Chapter 1, there are numerous tunable properties of NPs, and confirming that the NPs have the properties desired for the experiment at hand is key to ensuring accurate and repeatable results. Additionally, as any modification to physicochemical properties can be directly correlated to observed cellular outcomes, it is critical to ascertain if dispersion within cell culture media or circulation within a dynamic environment altered these parameters. The AgNPs used for this particular study were synthesized to have a spherical morphology and have individual sizes of 5 nm and 50 nm.

These properties were confirmed through the use of TEM. In addition to TEM, particle

76 uniformity was determined via UV-Vis, in addition to obtaining the unique spectral signature of the AgNPs.

In order to eliminate the variable of surface chemistry, both AgNPs were coated with

PVP. The PVP surface moiety was verified with the initial negative surface charge assessment. Following dispersion in cell culture media, the proteins instantaneously adsorb onto the AgNP surface, thereby creating a protein corona and masking the PVP functionalization. This was verified by the shift in zeta potential measurement to a value of -10 mV, which is the innate protein charge.

While the confirmation of individual particle size was confirmed via TEM, a study of the agglomerate size of the NPs was also necessary, as agglomerates dictate behavior and determine particle stability in solution. When NPs aggregate, they have been known to act like particles of their agglomerate size, rather than their individual size, making it more difficult to elucidate the responses which smaller NPs can induce [8]. The agglomerate sizes of the AgNPs were studied in both water and culture media, in order to understand how they act in a neutral environment, as well the environment they would be exposed to during cellular experimentation. While the culture media increased the final agglomerate size of both AgNP sets, due to the formation of a protein corona, this increase was not so extensive that the particles became unstable within solution.

77 Finally, all of the agglomerate size and surface charge characterization was performed on

AgNPs that had been exposed to environments which incorporated fluid dynamics, as well as static environments. These control experiments were necessary in order to verify that under dynamic conditions, the physiological flow did not alter AgNP physicochemical properties. The presented results confirmed that the surface charge and agglomeration tendencies of both the 5 and 50 nm AgNPs were unaltered during circulation within a dynamic environment, meaning that any alterations to the nano- cellular interface were due to changes in biotransport mechanisms and not the particles themselves.

3.3.2 – Discussion of Cellular Association Results

Previously, most studies which incorporate fluid dynamics into in vitro models have utilized only cell lines which grow adherent to a plate. This study sought to understand the influence of fluid dynamics on both AgNP behavior and the circulating cells. This makes this work unique, as fluid dynamics is known to alter the transport mechanisms of

NPs. As one could hypothesize, when cells grown in suspension are allowed to grow in a static environment, some settling occurs, and many of the cells end up sitting on the bottom of the culture flask. When this happens, the uneven distribution disrupts NP- cellular interactions. This phenomenon results in a relatively low uptake of the AgNPs into the U937 cells and introduces error due to uneven cellular clumping and particle distribution.

78 However, when fluid dynamics were introduced to the environment, both the U937 cells and the AgNPs were constantly in motion, were better maintained within the core of the exposure environment and were evenly distributed; due to a disruption of sedimentation effects. This constant motion further increases the likelihood of interaction between the

AgNPs and the cells, as fluid dynamics increases the kinetic energy of the system. These increased interactions were confirmed in this study by the significant rise in AgNP internalization within U937 cells in environments which incorporated fluid dynamics

(Figure 3.3).

Furthermore, the size of the NP is seen to affect the extent to which they are taken up by the U937 cells. The smaller 5 nm AgNPs show a much higher internalization than the 50 nm particles. This is likely due to the fact that the 5 nm particles have a much smaller mass, making them more susceptible to the fluid motion, and less susceptible to sedimentation forces; thereby increasing U937-NP interactions. This is just the first example of how any sort of cellular response to the NPs is related to more than just one aspect of their physical properties or surroundings.

3.3.3 – Discussion of Cellular Response Results

When studying nanotoxicity, often times only cellular stress or cell death are included in the study [3]. However, more studies are beginning to look at cellular inflammation as well, as it may indicate negative side effects that can a cause systemic impact [9], [10].

Inflammatory responses, while not deadly to the cell, could be responsible for long-term

79 effects, and thus, are being included in this study as a cellular response to be concerned about. Moreover, since U937s are a part of the immune response, two key goals of this work were to ascertain if AgNPs activated an immune/inflammatory pathway from these cells and if the presence of dynamic flow modified these responses.

The typical first signal studied for cell stress is ROS. ROS is always present within a cell, and an augmentation of intracellular ROS levels is directly proportional to cellular stress levels. The 5 nm static exposure shows no statistical difference from the untreated control, showing that the small particle size and lower internalization did not significantly induce a stress response within the U937s. However, when looking at the 5 nm dynamic exposure, it is clear that the higher internalization of the AgNPs into the cells caused a significant increase in the cellular stress.

The 50 nm particles, however, show even a significant increase in the cellular stress in the static exposure. While the pattern of higher internalization resulting in higher stress levels remains between the two 50 nm exposures, it is clear, that the size of the NP also has a part to play in the resulting cellular stress. If delivered silver content, and not particle size, were the predominant factor in stress activation, the 5 nm dynamic exposure would show the highest levels of ROS. However, as Figure 3.4 clearly shows, the larger particles were associated with higher ROS levels, likely due to a difference in internalization mechanisms. This is the second example of numerous NP and environmental properties affecting the cellular response. Regardless of primary particle

80 size, the ROS levels increased when AgNPs were introduced within a dynamic environment, aligning with augmented NP internalization.

While ROS levels exhibited a stepping-up pattern across the exposures, a regulator for

ROS, NF-κB exhibited a stepping-down pattern, resulting in an almost exactly mirror- imaged chart. The NF-κB is at its highest activation of about 150% of the control in the 5 nm static exposure, the exposure which had the lowest ROS level. The lowest NF-κB activation was seen in the 50 nm dynamic exposure, an even lower activation than was observed in the untreated control. Once again, the amount of NF-κB activation is likely related to both the AgNP size as well as the dynamic environment, as the 5 nm particles activate the transcription factor more than the 50 nm particles do and there is a statistically significant difference between the static and dynamic exposures of both particle sizes. The correlations between NF-κB, ROS, and AgNP internalization are very difficult to delineate owing to the complex nature and multiple roles of NF-κB. However, what this work does demonstrate is a link between exposure condition and NF-κB activation, as the addition of dynamic flow impacted the activation of the other.

HSP27 is a protein that is a member of a cellular signal transduction cascade activated following stress induction, making it another excellent marker to evaluate the levels of cellular stress. This is a cellular response which seems to rely much more heavily on the fluid dynamics. While each of the four exposures activates the HSP27 in a statistically significant way, the addition of the fluid dynamics seems to increase the activation much

81 more significantly. That being said, the 50 nm particles do raise the activation a bit more than the 5 nm particles, especially in the dynamic exposure. This cellular response seems to rely on both NP size and environmental conditions, with a preference for physiologically relevant conditions. The HSP27 results are in excellent agreement with the ROS data and the AgNP internalization results.

Another commonly looked at cellular response is LDH, which is released during apoptosis, a cellular process which is well regulated. If an increase of LDH leakage is seen, it would mean that the AgNPs are interfering with the pathways which regulate apoptosis. While at a low concentration (2.5 µg/mL) 5 nm AgNPs did not change the amount of LDH leakage from levels seen in U937s grown in a static environment without exposure to AgNPs, a high concentration (25 µg/mL) of the same sized AgNPs elicited changes in the apoptosis pathway. The static exposure increased the LDH leakage in a small but significant way, while the dynamic environment increased the LDH leakage to over 200% of what was observed in the untreated cells. Clearly, at higher concentrations, the addition of fluid dynamics drastically increases cell death, likely due to the fact that the AgNPs had the opportunity to interact more with the cells, increasing the likelihood that they could interfere with the cells’ signaling and regulation.

As discussed earlier, this study endeavored to not only investigate commonly researched cytotoxicity but other cellular responses which may cause unintended consequences or long-term effects without being toxic to the cell itself. The inflammatory responses of

82 cytokines have been shown to cause damage without killing the cells. Two cytokines showed increased secretion when exposed to the larger 50 nm AgNPs. Both IL-6 and IL-

8, which activate inflammatory responses, were affected by this [11]. For IL-6, the 5 nm particles did not alter the secretion at all, suggesting that the inflammatory response has less to do with the internalization and more to do with a physical property of the NP, in this case, likely size. The 50 nm particle significantly increases the secretion of this cytokine, but there is also no significant difference between the static and dynamic exposures, once again, leading the investigators to believe that the inflammatory response, in this case, is not related to the environment, but tied to the size of the particle itself.

The story is a little bit different for the other cytokine that was secreted following AgNP exposure, IL-8. The dynamic exposure of the 5 nm particles showed a significant decrease in the IL-8 secretion, dropping lower than the static, untreated control. The static exposure secreted the IL-8 similarly to the IL-6, about equal to the amount secreted by the control. Also, similarly to the IL-6 secretion, the 50 nm particles in both the static and dynamic exposures increased the IL-8 secretion to over 150% of what was observed from the control. This data paired with the IL-6 data suggests that when it comes to these two inflammatory responses, primary particle size has more effect on the extent of the response than the environment does, which differentiates it from most of the other cellular responses observed.

83 3.4 – Conclusions

In Chapter 1, a need for more physiologically relevant means to investigate the potential toxicity of NPs was established. This investigation further solidifies the need to continue developing enhanced in vitro models in order to truly understand the full physiological outcome of using a particular NP for any application that may come into contact with a human. Both the internalization of the NPs as well as many of the cellular responses were influenced by the presence of physiologically relevant flow.

While the observed results are likely due to a combination of core composition, particle size, and the type of exposure environment, it is quite clear, that the addition of physiologically relevant flow changes the way that the U937 cells interact with AgNPs.

The fluid dynamics increased the internalization rates of the AgNPs by the suspended monocytic cell line. Overall, the inflammatory response data seems to suggest that they are not influenced by the addition of fluid dynamics, but by particle size, however, they were the only cellular responses which follow that pattern. The cellular stress responses of ROS, NF-κB, and HSP27 were all altered in some way by the addition of the fluid dynamics as well, likely due to the increase in internalization, with some influence of particle size as well. The cellular death results also suggest that at higher concentrations of AgNPs the fluid dynamics will increase cell death.

As previously stated in both this chapter as well as Chapter 1, the cellular response to

NPs is often related to a combination of physical properties of the specific NP as well as

84 the environmental conditions of the cell culture. This study confirms the beliefs of many who study nanotoxicology that it is necessary to develop and utilize in vitro models which better mimic the human body. As NPs continue to be utilized more frequently, the implementation of enhanced in vitro models to better evaluate NP safety and efficacy will only become more important.

3.5 – References

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