DELETERIOUS SYNERGISTIC EFFECTS OF CONCURRENT MAGNETIC FIELD AND 

SUPERPARAMAGNETIC (Fe3O4) NANOPARTICLE EXPOSURES ON CHO‐K1 CELL LINE 

Zachary Coker, B.S. 

 

    

Thesis Prepared for the Degree of  

MASTER OF SCIENCE 

 

 

 

 UNIVERSITY OF NORTH TEXAS 

 May 2015 

 

 

  

APPROVED: Tae‐Youl Choi, Major Professor Xun Yu, Committee Member Bennett L. Ibey, Committee Member Yong X. Tao, Chair of the Department of 

Mechanical and Energy Engineering Nandika A. D’Souza, Mechanical and Energy 

Engineering Graduate Program Coordinator 

Costas Tsatsoulis, Dean of the College of Engineering and Interim Dean of the Toulouse Graduate School

Coker, Zachary. Deleterious synergistic effects of concurrent magnetic field and

superparamagnetic (Fe3O4) nanoparticle exposures on CHO-K1 cell line. Master of Science

(Mechanical and Energy Engineering), May 2015, 53 pp., 1 table, 12 illustrations, references, 90

titles.

While many investigations have been performed to establish a better understanding of

the effects that magnetic fields and nanoparticles have on cells, the fundamental mechanisms

behind the interactions are still yet unknown, and investigations on concurrent exposure are

quite limited in scope. This study was therefore established to investigate the biological impact

of concurrent exposure to magnetic nanoparticles and extremely-low frequency magnetic fields

using an in-vitro CHO-K1 cell line model, in an easily reproducible manner to establish grounds

for further in-depth mechanistic, proteomic, and genomic studies. Cells were cultured and

exposed to 10nm Fe3O4 nanoparticles, and DC or low frequency (0Hz, 50Hz, and 100Hz) 2.0mT

magnetic fields produced by a Helmholtz coil pair. The cells were then observed under confocal

fluorescence microscopy, and subject to MTT biological assay to determine the synergistic

effects of these concurrent exposures. No effects were observed on cell morphology or

microtubule network; however, cell viability was observed to decrease more drastically under

the combined effects of magnetic field and nanoparticle exposures, as compared to

independent exposures alone. It was concluded that no significant difference was observed

between the types of magnetic fields, and their effects on the nanoparticle exposed cells, but

quite clearly there are deleterious synergistic effects of these concurrent magnetic field and

nanoparticle exposure conditions.

ii

Copyright 2015

by

Zachary Coker

ii

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisers: Dr. Tae-Youl Choi for his

unwavering support and confidence in my abilities as a student and researcher. He has been an

exemplary adviser, mentor, and friend. Dr. Bennett Ibey for his guidance, support, and

suggestions in my pursuit of knowledge in a field previously unknown to me, and for his trust in

my abilities to learn, solve problems, and overcome challenges. Finally, Dr. Saber Hussain, who

after only a few conversations, put faith in my abilities, and provided me the chance to prove

myself through a summer research internship, and the opportunity to challenge myself by

branching into a new research field, and develop as a student, scientist, and engineer.

I would also like to recognize those who have helped me to learn and develop the

technical skills necessary to complete this work, or who have contributed in discussions that

have helped to advance this work, in no particular order: Dr. Gary Thompson, Mr. Larry Estlack,

Dr. Laura Braydich-Stolle, Dr. Matthew Grogg, Mrs. Elizabeth Maurer-Gardner, Ms. Madeleine

DeBrosse, Ms. Samantha Franklin, Mr. Caleb Roth, Dr. Ronald Barnes, Dr. Hope Beier, and Mr.

Cesario Cerna.

This work was supported by the United States Air Force 711th Human Performance

Wing, Human Effectiveness Directorate, Air Force Research Laboratory - Radio Frequency

(RHDR) and Molecular (RHDJ) Bioeffects Divisions.

iii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ....................................................................................................................ii LIST OF TABLES ................................................................................................................................ iv LIST OF ILLUSTRATIONS.................................................................................................................... v CHAPTER 1 INTRODUCTION ............................................................................................................ 1

1.1 Objectives................................................................................................................ 2

1.2 Organization of Thesis ............................................................................................ 3 CHAPTER 2 BACKGROUND AND LITERATURE REVIEW ................................................................... 5

2.1 Nanoparticle Toxicity and Cellular Interactions ...................................................... 5

2.2 Magnetic Fields and Cellular Interactions .............................................................. 8

2.3 Concurrent-Exposure Cellular Level Effects and In-Vitro Studies ......................... 12

2.4 Magnetic Fields and Helmholtz Coils .................................................................... 16 CHAPTER 3 MATERIALS AND METHODS ....................................................................................... 20

3.1 Cell Culture and Exposure Protocols ..................................................................... 20

3.2 Magnetic Field Exposure and Dosimetry .............................................................. 22

3.3 Nanoparticles and Characterization ..................................................................... 26

3.4 Cell Proliferation and Mitochondrial Function – MTT Assay ................................ 29

3.5 Nanoparticle Uptake and Confocal Microscopy ................................................... 31 CHAPTER 4 RESULTS AND DISCUSSION......................................................................................... 33

4.1 Cell Viability: Proliferation and Mitochondrial Function – MTT Assay ................. 33

4.2 Cell Morphology, Nanoparticle Uptake and Confocal Microscopy ...................... 35 CHAPTER 5 CONCULSIONS AND FUTURE DIRECTION ................................................................... 42

5.1 Conclusions ........................................................................................................... 42

5.2 Recommendations and Future Directions for Investigation ................................ 43 REFERENCES .................................................................................................................................. 46

iv

LIST OF TABLES

Page

Table 3.1: Zetasizer nano measurements for various Fe3O4 particle dilutions in H2O and media, with and without MF Exposure ..................................................................................................... 29

v

LIST OF ILLUSTRATIONS

Page

Figure 2.1: Sketch of Helmholtz coil, with R indicating radius of coils, with the coil pair common-axis along x-axis. Arrows indicate direction of current flow......................................................... 16

Figure 3.1: Components for magnetic field system: (a) SpinCoil-7-X Helmholtz Coil, with LakeShore probe (b) LakeShore 425 Gaussmeter (c) Kikusui PCR500M Compact Power Supply 22

Figure 3.2: FEMM simulation: Flux density plot for cross section cut of magnetic field produced by Helmholtz coil pair ................................................................................................................... 24

Figure 3.3: Flux density measurement from center of Helmholtz coil along either X or Y axis as depicted in Fig. 3.2.2 ..................................................................................................................... 24

Figure 3.4: TEM imaging of Fe3O4 nanoparticles performed to verify spherical shape and particle size ................................................................................................................................................ 27

Table 3.1: Zetasizer nano measurements for various Fe3O4 particle dilutions in H2O and media, with and without MF Exposure ..................................................................................................... 29

Figure 3.5: Zeiss LSM 710 confocal microscope used for microscopy studies ............................. 31

Figure 4.1: MTT Concentration-dependent survivability curve of cells exposed to Fe3O4 nanoparticles and no magnetic field ............................................................................................ 34

Figure 4.2: MTT Concentration-dependent survivability curves for cells exposed to Fe3O4 nanoparticles and (a) DC magnetic field, (b) 50Hz magnetic field (c) 100Hz magnetic field, and (d) the respective Δ from sham conditions. The greyed section represents the area beyond the ED50 for cells not exposed to magnetic fields. Sham exposure Δ measured against trials in an independent incubator. ................................................................................................................ 36

Figure 4.3: Select images from confocal microscopy study without DC magnetic field exposure; (a) bright field images (b) Fluorescent mEmerald tubulin expression (c) Fluorescence imaging with Rhodamine-B labeled particles and (d) Overlay results from bright field and Rhodamine-B fluorescence imaging to show particles inside the cell structures. .............................................. 39

Figure 4.4: Select images from confocal microscopy study after DC magnetic field exposure; (a) bright field images (b) Fluorescent mEmerald tubulin expression (c) Fluorescence imaging with Rhodamine-B labeled particles and (d) Overlay results from bright field and Rhodamine-B fluorescence imaging to show particles inside the cell structures. .............................................. 40

Figure 4.5: Mean fluorescence intensity of cells measured with, and without DC magnetic field exposure at 250 μg/mL nanoparticle concentration .................................................................... 41

vi

Figure 4.6: Mean fluorescence intensity of cells measured with, and without DC magnetic field exposure at 50 ug/mL nanoparticle concentration ...................................................................... 41

1

CHAPTER 1

INTRODUCTION

Despite numerous investigations since the 1970s, the effect of low frequency magnetic

fields on cells has remained unclear and the subject of much controversy, especially because

many investigators report irreproducible or even contradictory findings [1–4]. Difficulties arise

in that experiments can be very difficult to repeat as many exposure systems are custom made

for their respective experiments, and even minor variations in the design can induce moderate

to large changes in the magnetic fields produced. Additionally, the effects of magnetic field

exposures on cells are not necessarily ubiquitous, but rather may be dependent on cell line [5],

cell stage, or even age of cells or their respective donors [6,7]. Induced cellular level effects may

be also very small and difficult to quantify, and very minute variations in magnetic field

parameters such as field intensity, direction, duration of exposure, and field frequency may

have significant impacts on cell behavior [8–10]. Finally, the possible biophysical mechanisms

behind any induced cellular effects are still not well known, however many theoretical models

have been proposed [11].

Many studies have been presented on the effects of magnetic field exposures on various

cell lines, particularly at standard distribution frequencies of 50Hz and 60Hz [12–21] and DC

magnetic fields [22–25]. Additionally extensive investigations have been carried out on the

toxicological effects of nanoparticle interactions with cells, and what nanoparticle properties

impact those interactions [26–33]. Concurrent nanoparticle and magnetic field interactions

have indeed been investigated [12,23,24,34–38], however there are no consistent and

agreeable results from these studies showing specific effects of concurrent low frequency

2

magnetic fields, and magnetic nanoparticle exposure on cells. This study was therefore

designed to investigate the biological impact of concurrent DC, or low frequency magnetic

fields and magnetic nanoparticle exposures on the CHO-K1 cell line, in an easily reproducible

manner, to establish grounds for further in-depth mechanistic, proteomic, and genomic studies.

For the purpose of developing future studies, and for independent verification, the magnetic

field exposure was conducted using commercially available components, while nanoparticle

exposure procedures emulate that of well-established, previously performed toxicological

studies [32,39,40]. The exposure systems, protocols, and measurements are further detailed in

chapter 3. For the purpose of this experiment, the term “magnetic field” and “magnetic field

intensity” refer specifically to the magnitude of magnetic flux density of the induced magnetic

fields.

1.1 Objectives

The main objective of this research was to investigate the biological impact of

concurrent exposure to magnetic nanoparticles (NP) and extreme-low frequency magnetic

fields (ELF-MF) using an in-vitro CHO-K1 cell model. This objective was achieved through the

following specific aims:

Specific aims:

1. Establish in-vitro NP, ELF-MF, and concurrent NP+MF exposure system

for the CHO-K1 cell line model. Specifically DC, 50Hz, and 100Hz 2.0mT

magnetic fields and 10nm spherical superparamagnetic Fe3O4

nanoparticles were used in this study.

3

2. Investigate biological impacts and perform comparative analysis of

concurrent exposures against independent exposures, focusing

specifically on cell viability and recovery through mitochondrial activity

measurement following NP and MF treatment.

3. Investigate cell morphology and nanoparticle uptake changes in CHO-

K1 cells via confocal microscopy using mEmerald-tubulin expressing CHO

cells, and Rhodamine-B labeled nanoparticles.

1.2 Organization of Thesis

Chapter 2 presents information obtained through literature review of relevant scientific

investigations on cellular effects of nanoparticle, magnetic field, and concurrent exposures. The

first section of this chapter is dedicated specifically to current investigative efforts on

cytotoxicity and nanoparticle-cell interactions, while the second section is dedicated specifically

to magnetic field-cell interactions. As current investigations are limited with regard to

concurrent exposures, a brief discussion is presented on current investigative efforts in the final

section of this chapter. The final section of the chapter is dedicated to a brief review of

Helmholtz coils and their use to generate uniform magnetic fields.

Chapter 3 covers the methodology and materials used for the investigation, beginning

with cell culture methods for the CHO-K1 cell line, followed by a brief discussion about the

magnetic field exposure system and exposure conditions. The chapter closes with

characterization methods of the nanoparticles used and their attributes, MTT assay

methodology, and finally confocal and fluorescent microscopy methods.

4

Chapter 4 provides information related to the results of the investigation, where the

MTT assay results, as well as the confocal and fluorescent microscopy results are presented and

discussed.

Chapter 5 concludes the thesis with a summary of the investigation, and provides

suggestions for further development for studies of the mechanisms behind the cellular effects

of these exposures, and the potential future applications of the research.

5

CHAPTER 2

BACKGROUND AND LITERATURE REVIEW

This chapter briefly covers the results of a survey of relevant literature on modern

research efforts directly related to cellular interactions with nanoparticles, magnetic fields, and

concurrent nanoparticle-magnetic field exposures, and the mechanisms behind those

interactions. Following the review discussion, a brief description of Helmholtz coils is given,

along with relevant information for understanding magnetic field exposures used for this

experiment.

2.1 Nanoparticle Toxicity and Cellular Interactions

Rapid development of nanotechnology has resulted in nanomaterials being

incorporated into every day products and applications across many fields. Due to the unique

properties of nanoparticles, they have become very attractive for many wide-spread

applications, including, but not limited to commercial, medical, electronics, and military

applications. Naturally, with an increase in use and application, comes an increased risk of

incidental or unintended exposure. The reality of nanoparticle exposure is that everyone is

exposed, all the time, as particles are now found in food, beverages, clothing, cosmetics, and

various other daily-used items.

For the most part, these particles have little to no effect on health, however a growing

number of studies have shown that nanoparticle interactions with cells have the ability to

influence some of the most basic of cellular functions, such as proliferation, metabolism, and

even cell death. As nanoparticles are very small, on the order of 1x10-9m, some nanoparticles

6

have the ability to permeate through biological barriers, disrupt normal functions, and cause

substantial damage to biological systems. Several diseases have been associated with

nanoparticle-interaction and interference with basic cell processes, such as cancer,

neurodegenerative disease [28], and even Alzheimer’s disease [41]. Many investigations have

thus been performed to ascertain information about the potential risks associated with various

nanoparticle exposures [27–30,32,33,42] and their biological effects. For example, Hussain et

al. [39] reported on the in-vitro toxicity of various nanoparticles and related concentration

dependence, in BRL 3A rat liver cell model. Braydich-Stolle et al. [32] performed a similar

investigation in mammalian germline stem cells. Prabhu et al. [33] studied the size and

concentration dependence of cytotoxicity of Cu nanoparticles, and Comfort et al. [40] reported

that superparamagnetic Fe3O4 particles produced a strong alteration in epidermal growth factor

(EGF) activated gene transcription, with targeted genes influencing cell proliferation, migration,

and receptor expression in A-431 cell line model.

Magnetic nanoparticles have become particularly of interest, due to their unique

properties when exposed to magnetic fields; superparamagnetic iron oxide is already widely

implemented in the medical field as a contrast agent for MRIs. Bae et al. [43], Singh et al. [30],

and Mahmoudi et al. [27][42] have all reported findings on the cytotoxicity of

magnetic/superparamagnetic nanoparticles, and show high biocompatibility, in agreement with

the previously mentioned studies. All of these investigations together, have shown a multitude

of ways in which nanoparticle exposure can influence biological systems based on the various

properties of those particles, including size, shape, surface chemistry, chemical composition,

exposure conditions, and even synthesis methods [44–46].

7

With respect to nanoparticle-cell interactions, the size, shape and surface chemistry are

readily apparent as important factors that determine cellular response and uptake. Jiang et al.

[47] showed that dependence of cellular interaction on size and surface chemistry, which

corroborate similar reports by several others [48–51]. The reports on size dependence provide

insight into how the particles interact with the cell membrane; quantum dots, and very small

particles on the order of 10nm, appear to coat the plasma membrane before incorporation into

the cell, and must reach a critical threshold density to trigger the cell receptors to initiate

uptake, while larger particles were seen to be internalized immediately, without accumulation

on cell membranes. Likewise Jiang et al. provided their report on protein adsorption layer

impact on uptake showing results, similar to those by Nativo et al. [52]and Labhasetwar et al.

[53], that suggest surface modification strongly modulates cellular uptake. Finally, Kim et al.

[54] reported the role of cell cycle on nanoparticle uptake, and dilution amongst replicating

cells; little to no impact was reported within the first 10 hours of exposure to nanoparticles.

However, after 24 hours, concentration of nanoparticles in cells could be ranked with respect to

cell phase. An important finding in this study was that nanoparticles that are internalized by

cells are not exocytosed, but rather are split between daughter cells through cell division. Other

studies, such as the investigation by the time and space resolved uptake studies by Shapero et

al. [55] have reported negligible amounts of exocytosis through nanoparticle studies as well.

Jiang et al. [47] published a contradictory report on the endo- and exocytosis of quantum dots,

showing the particles being trafficked towards the peripheral regions of the cells for exocytosis,

though this may be a result of the very small size of the quantum dots, relative to the particles

used in Shapero’s studies. Ultimately, these contradictory reports reiterate the many variables

8

that influence cellular-level interactions with nanoparticles, and that there is still much room

for improvement in understandings of the mechanisms driving those interactions. In 2007,

Buzea et al. [28] published an extensive review covering the various sources and toxicities

associated with nanoparticle risk and exposure. Despite the extensive research efforts

conducted on nanoparticle interactions with biological systems, the true impact of these

particles, and all associated effects are yet to be clearly established or well understood. Due to

the relative ease by which nanoparticles can enter the human body, be it by inhalation,

gastrointestinal, or skin exposure, cytotoxicity of nanoparticles has grown to be a major

concern, and will continue to be so, with the constant increase in developments, and thereby

demands of nanoparticle applications.

2.2 Magnetic Fields and Cellular Interactions

In modern physics and electrodynamics, electromagnetic waves are described and

ordered by their frequency (f) or wavelength (λ), related by the equation λ = c/f, where c is

recognized as a universal constant, the speed of light in a vacuum (2.998x108 m/s). Direct

current (DC) electromagnetic fields are static electric or magnetic fields which have emission

frequencies of 0 Hz. While most real-world exposures will occur over a large range of the

electromagnetic spectrum, much research has been conducted to investigate cellular level

effects of static fields, and fields of specific frequency levels relevant to electronic appliances

and power-line distributions. The magnetic fields investigated in this thesis are in the extreme-

low frequency range of 0-300Hz; induced by current flowing through paired coils of wire at the

respective driving frequency. This is further discussed in the methods and materials section.

9

Due to rapid developments in technology and electronic communications since the late

19th century, and the industrial revolution, exposure to low frequency magnetic fields is, in-

general, an unavoidable aspect of modern life. However, in 1979, Wertheimer and Leeper

reported a correlation between living near power distribution lines, and increased risk for

childhood leukemia [11]; since their report, the concern for possible health effects of low

frequency magnetic field exposures has been a rather controversial topic, and the subject of

many investigations. In a paper published in a section of the Journal of Electroanalytical

Chemistry, Glaser [56] suggests a hierarchical view must be considered for magnetic field

interactions with biological structures, in the order of atomic, molecular, cellular, and organism

interactions. Glaser suggests that effects are possible on the basis of quantum mechanics,

dipolar orientation of molecules, double-layer structures and modifications of systems of ionic

equilibrium. Additionally, Glaser points out that, though many phenomena of field effects have

been experimentally verified, the underlying effects are still not understood. This experiment

focuses on the cellular level interactions of magnetic field exposure, with the consideration that

many of the cellular level effects are likely not directly-driven by the field exposures, but rather

by molecular-level effects.

Similar to the complexities introduced when investigating cellular-level interactions with

nanoparticles, many complexities are introduced when considering cells under magnetic field

exposure and their related interactions. Conflicting results have been reported with respect to

cellular interactions with magnetic fields between different cell types, field exposures, and even

cells of the same type, based on age or conditions [6]. The complexity of the interactions is

somewhat exemplified by the proteomic analysis performed on SH-SY5Y neuroblastoma cell

10

line model by Hasanzadeh et al. [14], where they reported 189 individual proteins altered by a

2.0mT 50Hz magnetic field exposure for 3 hours, most of these protein alterations were

associated with the cellular defense mechanisms. Tenuzzo et al. [57] also demonstrate this

complexity with their analysis of 6mT static magnetic field influences on apoptosis of

lymphocytes, where it was reported that the effects are likely not a direct result of the

magnetic field acting on the cell, but rather through Ca2+ influx in the cells. These results were

complimented when Fanelli et al. [37] reported increased cell survival by magnetic field (up to

6mT) induced Ca2+ influx inhibition of apoptosis in several cell systems. However, contradictory

evidence is presented by Ahmadianpour et al. [25] in their report of 6mT magnetic field-

induced apoptosis, and altered cell cycle of p53 mutant Jurkat cells. Goodman et al. [9,58]

showed an increased expression in several transcripts in HL60 cells under various types of

magnetic field exposures (various frequency and field intensities), and mRNA levels after only

20 minutes of exposure. Initially, they reported that the strongest responses were measured

after exposure to 1.5mT 50Hz magnetic fields; however, this same group reported conflicting

data showing that the strongest responses were actually at a much lower field intensity of only

5.6μT, and that stronger fields, and longer exposure times actually lead to a reduced cellular

response.

The effects of magnetic field interaction with cells have been reported to have a

multitude of impacts beyond those previously mentioned, and the complexity intensifies; some

reports have shown opposite effects in the same cell, with simple variations in field strength

[1,20,59] while others have shown completely contradictory reports [60] compared to other

researchers. Luukkonen et al. [18] reported that SH-SY5Y cells, pre-treated with 100μT 50Hz

11

magnetic fields experienced enhanced menadione-induced DNA damage, and micronucleus

formation, but alternatively also experienced an increase in DNA repair rate. Simkó et al. [5]

also reported on micronuclei formation, showing yet another difference in cell line response, as

they reported findings on micronuclei formation in SCL II cell line, but not in human amniotic

fluid cells, after exposure to 0.1mT – 1.0mT 50Hz magnetic fields for 24 to 72 hours. The SH-

SY5Y cell line has been of particular interest in magnetic field-cellular level interactions, as SH-

SY5Y cells are often used as in vitro models of neuronal function; Falone et al. [15] reported

increased antioxidant defenses and a shift in redox homeostasis in these cells after exposure to

1.0mT 50Hz magnetic fields for 5, 10, and 15 days, with increase in protein expression changes

related to cellular defense mechanisms, similar to the study by Hasanzadeh mentioned earlier.

Cerrato et al. [16] reported an increase in proliferation immediately following 24 hours of

2.0mT 50Hz magnetic field exposure with SH-SY5Y cell line, in addition to immediate up-

regulation of TAU mRNA gene expression, and Syp gene expression, and immediate down-

regulation of MAP2 gene expression. All three of these expressions were regulated back to

normal 24 hours after exposure had ended. This indicates that some effects of magnetic field

exposure may not be readily apparent in all investigations, if there is a prolonged delay

between field exposure and analysis. In the studies by Cerrato et al. and Falone et al., it was

also reported that a 2.0mT 50Hz magnetic field had an impact on the cell morphology,

migration, and distribution of microtubules. Falone et al. observed that the cells would cluster

together during culture process, but when exposed to magnetic field, would be observed

evenly distributed about the culture surface. Cerrato et al. observed that the microtubules were

branched radially from the perinuclear region of the control cells, while exposed cells exhibited

12

microtubule distributions organized into clusters that would transition into the neurites that

were forming. This could indicate an effect on the microtubule structures themselves, or on

molecular processes that regulate this process in the cells. Complementing these reports on

morphological change in the cells, Pozzi et al. [13] reported that 2mT 50Hz magnetic fields

interfere with the growth process of Lan-5 neuroblastoma cell line, and induces changes in cell

morphology.

Santini et al. [61,62] have published a review article on investigations of cellular effects

of low frequency magnetic fields. A general conclusion that can be drawn is that despite the

multitude of studies, there still remains debate over what, if any, health effects result from

exposure to low frequency magnetic fields.

2.3 Concurrent-Exposure Cellular Level Effects and In-Vitro Studies

While extensive research has been conducted to investigate the independent impacts of

nanoparticle and magnetic field exposures on biological systems, studies for the combined

effects of these exposures, especially at very low magnetic field frequencies are not yet well

established. Current research dedicated to the combined effects of nanoparticle and

electromagnetic field exposures on cells is mostly focused on effects induced at very high

power absorption frequencies (kHz-MHz range) [63–66] for the purpose of thermal therapies

and targeted heat generation, neglecting the lower frequency domain. A review article by

Mamiya [67] on magnetic nanoparticles and targeted hyperthermia provides a brief description

of particle response to AC magnetic fields for the purpose of these therapies.

13

As research on the combined effects of low frequency fields and nanoparticles on cells is

beginning to grow, a noticeable trend is how closely related the investigations are to the

independent-exposure investigations. For instance, Jia et al. [68] recently reported in 2014 the

combined effects of 50Hz magnetic field and magnetic nanoparticle on proliferation and

apoptosis of PC12 cells. This is not far removed from the report by McFarlane et al. mentioned

in the previous section. Jia et al., however, showed data representing no statistically significant

difference between control groups with and without magnetic fields, but reduced cell viability,

and increased apoptosis with the application of the magnetic field to nanoparticle loaded cells,

compared to cells with nanoparticles, but no applied magnetic field.

Bae et al. [43] produced a report on the effects of static magnetic fields on aggregation

and cytotoxicity of magnetic nanoparticles, demonstrating that static magnetic fields induce

aggregation of superparamagnetic iron oxide particle clusters, and also reduced viability,

apoptosis, and irregularity of cell cycle of hepatocytes in vitro and in vivo. In this report, it was

noted that the particle aggregates were the primary source of the cytotoxicity, not necessarily

an interaction with the magnetic field after uptake. As well, the apoptotic effects were

dependent upon particle uptake, and related reactive oxygen species production, but cell

viability measurement was not. Finally, it was also reported that multinuclear giant cells

became more prevalent under concurrent exposures, after long-term observation, as compared

to the control groups. Shen et al. [69] demonstrated increased particle uptake with the

application of magnetic field, as well as capabilities of lysosome and magnetic nanoparticle

relocation by the application of external magnetic field gradients generated by two antiparallel

permanent magnets. Additionally, they demonstrated the capability of these gradients to

14

impact cell migration by controlling the magnetic particles after uptake. The cells were

observed to move along the direction of the applied magnetic field gradient, and little to no

cytotoxicity was reported. The authors propose the method presented as a novel way to study

cellular mechanics and intracellular forces. Increased cellular uptake of superparamagnetic

nanoparticles induced by magnetic field gradient was also reported by Prijic et al. [23] showing

significant increase in uptake in several different cell lines by the application of two different

magnets with surface flux densities of 60mT and 403mT, and magnetic gradients of 5.5 and 38

T/m respectively, reporting that the stronger magnets were significantly more effective for

impacting the internalization process; the cellular uptake rate of the particles was cell type, and

time dependent. The authors also point out that the malignant cells tested had significantly

higher uptake rate than the normal or healthy cells tested, as in agreement with other research

referenced in their report. Comfort et al. [24] investigated the effects of concurrent 24 hour

exposure to 0.5mT and 30mT static magnetic fields and gold or iron oxide nanoparticle on the

HaCaT cell line. Contradictory to the report by Bae et al., the application of external magnetic

fields from permanent magnets were reported to reduce, or counteract the stress response of

the cells due to nanoparticle exposure, through a reduction in reactive oxygen species

production, and modified gene regulation induced by the magnetic fields. These effects were

observed independent of nanoparticle exposure.

Some more adventurous research has been conducted with attempts to take advantage

of the magnetic properties of some nanoparticles applied to biological systems, such as

González et al. [70] reporting their success in controlling magnetotactic bacteria through the

application of magnetic field gradients generated by Helmholtz coils and nanoscale permalloy

15

islands. The authors report successful manipulation of magnetotactic bacteria, and the ability to

guide the bacteria to desired locations. Dobson [71] has reported progress towards binding

magnetic nanoparticles (100nm – 2.7μm) to cell surfaces, for the purpose of manipulating

cellular function and behavior by shear stress applied by magnetic force-actuation, while giving

a brief overview of nanomagnetic actuation for targeting ion channel activation and cell

membrane receptors. By binding nanoparticles directly to ion channels, Dobson reports success

in activating single ion channels, without interfering with normal function in other membrane

channels. Finally, Zhang et al. [38] have reported on the use of dynamic or moving magnetic

fields of very low frequency (10-40Hz) to control nanoparticles in order to enhance cellular

uptake, and then for targeted cell death by forced-lysosome disruption in both rat insulinoma

tumor cells, and human pancreatic beta cells. Lysosomal disruption was accomplished through

the application of LAMP-1 antibody coated nanoparticles, which bound to the lysosome

membranes, applying torque to the membrane by particle rotation under the influence of the

external magnetic field. The authors report that the dynamic magnetic field causes a rotational

motion in the nanoparticles, causing them to roll along the surface of cell membranes,

simulating a virus-like interaction between the particles and cells’ surfaces, thus triggering

uptake mechanisms, and enhancing internalization into the cells. These research efforts show

the potential for the ever growing applications of magnetic field and nanoparticle exposures to

biological systems, highlighting the importance of further developing the underlying

mechanisms behind cellular level interaction of nanoparticles, magnetic fields, and concurrent

exposures.

16

2.4 Magnetic Fields and Helmholtz Coils

There have been many coil configurations proposed for the generation of a uniform

magnetic field [72] using anywhere from 2 to 5 circular, or square-wrapped coils. The simplest

and most common used of these designs is the Helmholtz pair configuration; a pair of circular-

wrapped conducting coils, separated by a distance equal to their radius as shown in Figure 2.1.

The coils are configured to have a common axis, such that, when current is flowing through

them, the resulting magnetic field can be obtained by the principle of superposition. In short,

for this case, the principle of superposition states that the total magnetic field produced by two

or more sources is equal to the sum of these fields; if current is flowing through the coils in the

same direction, the magnetic fields add together to generate a uniform magnetic field in one

direction, and if the current flows in opposite directions, the fields cancel out, resulting in a

R R

d = R

x = 0 Midpoint

Figure 2.1: Sketch of Helmholtz coil, with R indicating radius of coils, with the coil pair common-axis along x-axis.

Arrows indicate direction of current flow.

17

region of essentially net-zero magnetic field. The calculation of the exact magnetic field at any

point in space is mathematically complex; however, when considering positions along the

central axis, such as in this experiment, the calculations become far simpler. Cvetkovic et al.

[73] provide a generalized theory of Helmholtz coils and uniform magnetic field production, and

DeTroye et al. [74] expand by providing a comparison between experimental measurements

and analytical calculations of the field generated by a Helmholtz coil.

The magnitude of the magnetic field at any given point, along the axis of a single wound

circular coil, with radius R is given by the Biot–Savart law [75]:

𝐵𝐵(𝑥𝑥) = 𝜇𝜇0 ∙ 𝐼𝐼

2 ∙

𝑅𝑅2

(𝑥𝑥2 + 𝑅𝑅2)32 (2.1)

where x is the distance from the center of the coil to any point along its central axis. Therefore,

for two coils, placed at a distance d from one another, both carrying the same current I, the

magnetic fields measured at the midpoint between the two coils would be determined by:

𝐵𝐵1(𝑥𝑥) =

𝜇𝜇0 ∙ 𝐼𝐼2

∙ 𝑅𝑅2

��𝑥𝑥 + 𝑑𝑑2�

2+ 𝑅𝑅2�

32

(2.2)

and

𝐵𝐵2(𝑥𝑥) =

𝜇𝜇0 ∙ 𝐼𝐼2

∙ 𝑅𝑅2

��𝑥𝑥 − 𝑑𝑑2�

2+ 𝑅𝑅2�

32

(2.3)

where B1 and B2 are the magnetic fields from the first and second coils respectively, 𝜇𝜇0 is the

magnetic constant: 𝜇𝜇0 = 4𝜋𝜋 x 10−7 𝑇𝑇∙𝑚𝑚𝐴𝐴

, often referred to as the “permeability of free space”, I

18

is the current in the coils, in amperes, and R is the radius of the coils, in meters. Helmholtz coils,

however, typically consists of two coils with n-number of turns per coil; accounting for the

number of turns in the coils is rather simple, and can be done by multiplying the current term, I,

by the number of turns in the coil, n:

𝐵𝐵1 �

𝑑𝑑2� =

𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

2 ∙ �𝑅𝑅2 + �𝑅𝑅2�2�32

(2.4)

and

𝐵𝐵2 �

𝑑𝑑2� =

𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

2 ∙ �𝑅𝑅2 − �𝑅𝑅2�2�32

(2.5)

by the superposition principle mentioned earlier, the total magnetic field between the coils can

be described as a function of the distance from the midpoint of the coils x:

𝐵𝐵(𝑥𝑥) = 𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

2 ∙ ���𝑥𝑥 +

𝑑𝑑2�2

+ 𝑅𝑅2�−32

+ ��𝑥𝑥 −𝑑𝑑2�2

+ 𝑅𝑅2�−32� (2.6)

Noting that the most uniform field in a two-coil system is achieved when the distance between

the coils is equal to the radius of each coil, and through use of a minor shift – defining the

center of one coil at x = 0, and the midpoint between two coils becomes x = d/2 or x = R/2. By

utilizing symmetric relations, the equations can then be simplified to say that the magnetic field

at the center of the coil pair is equal to that of two times a single coil where the single coil B1 is

now:

𝐵𝐵1 �

𝑅𝑅2� =

𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

2 ∙ �𝑅𝑅2 + �𝑅𝑅2�2�32

(2.7)

19

and the magnetic field at the midpoint Bm is described as twice that:

𝐵𝐵𝑚𝑚 �

𝑅𝑅2� = 2 ∙ 𝐵𝐵1 ∙ �

𝑅𝑅2� =

2 ∙ 𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

2 ∙ �𝑅𝑅2 + �𝑅𝑅2�2�32

(2.8)

which simplifies to:

𝐵𝐵𝑚𝑚 �𝑅𝑅2� =

𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

�𝑅𝑅2 + 14𝑅𝑅

2�32

=𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼 ∙ 𝑅𝑅2

�54𝑅𝑅

2�32

= �45�32∙𝜇𝜇0 ∙ 𝑛𝑛 ∙ 𝐼𝐼

𝑅𝑅 (2.9)

20

CHAPTER 3

MATERIALS AND METHODS

This chapter provides a discussion about the nanoparticle and magnetic field exposure

methods and protocols, as well as descriptions of experimental instruments, setups, and

procedure for each analysis performed, from the beginning of cell culture, to the viability

assays, as well as confocal and fluorescence microscopy.

3.1 Cell Culture and Exposure Protocols

The CHO-K1 (ATCC® CCL-61™ Chinese hamster ovary, American Type Culture Collection

[ATCC], Manassas, VA) cell line was maintained in Kaighn's modification of Ham's F-12 medium

(F-12K Medium) supplemented with 10% volume fetal bovine serum (FBS) (ATCC® 30-2020™)

and 1% volume penicillin/streptomycin antibiotics (ATCC® 30-2300). Subculture procedure

followed the guidelines set forth by the manufacturer, modified slightly; cells were rinsed with

Hank’s Balanced Salt Solution (HBSS, Invitrogen-GIBCO, Waltham, MA), and the subculture ratio

used was 1:10, rather than the recommended 1:4, or 1:8 due to the very rapid proliferation rate

of the CHO-K1 cells. After the HBSS rinse, the adherent CHO cells were released from the

surface of the culture flask by incubation for 5 minutes with 1mL trypsin (Trypsin-EDTA

Solution, 1X (ATCC® 30-2101™), followed by dilution in 9mL full serum media. Cells were

incubated at 37 °C, 5% CO2 in a Heracell™150i CO2 incubator (Thermo Fisher Scientific Inc.,

Waltham, MA), in 75 cm2 cell culture flasks and 96-well plates for continued culture, and for

exposure conditions respectively. The pH level of the culture media was controlled by 5% CO2 in

the incubation chamber, in combination with sodium bicarbonate concentration (1,500mg/mL)

21

in media. For visualization of microtubule cytoskeleton, CHO cells were transfected with a

mEmerald-tubulin plasmid (a kind gift from Mr. Michael W. Davidson of the National High

Magnetic Field Laboratory at Florida State University, Tallahassee, FL) using Effectene

(#301425; Qiagen, Gaithersburg, MD) and maintained with G418 sulfate solution (#345812,

Calbiochem, Philadelphia, PA). The stable transfection of the CHO cell line was performed by Dr.

Marjorie Kuipers and provided as a generous gift from the U.S. Air Force Research Laboratories

(Fort Sam Houston, TX).

For all exposures, cells were harvested, counted, and viability assessed using the trypan

blue exclusion method using trypan blue 0.4% solution in phosphate buffered saline (PBS)

(Corning-MediaTech, Corning, NY) in an Invitrogen Countess automated cell counter (Eugene,

OR). Cells were then diluted into culture media and plated at 2 x 105 cells/mL into 96 well plates

at 50 μL/well, giving approximately 1 x 104 cells per well. Six hours after plating, the solutions in

each well were then supplemented with 50 μL of their respective nanoparticle-media dilute

solution, providing each well with a total of 100 μL solution for the duration of the exposure(s).

As pH value of cell culture media is critical to the optimal growth and development of the cell

culture [76,77], the pH value for each dilution was measured using an Orion™ 2-Star Benchtop

pH Meter (Fisher Scientific, Waltham, MA) to have values of 7.66-7.60 from complete cell

culture media, to highest nanoparticle dilution concentration, respectively. For microscopy

studies, cells were plated at 1x106cells/mL in poly-D-lysine coated glass bottom, γ-irradiated,

35mm petri dishes ((Cat# P35GC-0-10-C, MatTek, Inc., Ashland, Massachusetts) with an initial

volume of 1.5 mL cell culture media, followed by the addition of 1.5 mL dilute nanoparticle-cell

culture media solution six hours later. This is a similar protocol as used for the 96-well plates

22

with magnetic field, nanoparticle, and combined exposures. Immediately following the addition

of nanoparticle solution, the wells and dishes were placed into the exposure chamber, to begin

magnetic field exposure. Analysis of cells was then performed after 24 hours of continuous

exposure. Cytotoxicity of the exposures or cell

proliferation and viability were analyzed using a

calorimetric MTT assay; morphology, microtubule

structure, nanoparticle uptake and particle

trafficking were observed via confocal microscopy.

Throughout confocal microscopy, a custom buffer

solution (pH 7.4, ~290 mOsm) consisting of 2mM

MgCl2, 5 mM KCL, 10mM 4-(2-hydroxyethyl)-1-

piperazinee-thanesulfonic acid (HEPES), 10 mM

Glucose, 2 mM CaCl2, and 135 mM NaCl (Sigma-

Aldrich, St. Louis, MO), was used to maintain the

cells.

3.2 Magnetic Field Exposure and Dosimetry

The cell culture plates were placed in the

center of a SpinCoil-7-X Helmholtz coil system

purchased from MicroMagnetics (Fall River, MA).

Driving current was supplied, and easily regulated

(from 110V 60Hz line-source) by a variable-voltage,

Figure 3.1: Components for magnetic field system: (a) SpinCoil-7-X

Helmholtz Coil, with LakeShore probe (b) LakeShore 425 Gaussmeter (c) Kikusui PCR500M Compact Power

Supply

(a)

(b)

(c)

23

variable-frequency Kikusui PCR500M Compact AC Power Supply (Kikusui Electronics

Corporation, Yokohama, Japan) shown in figure 3.1, allowing for precise control of the system

(0-270VAC, DC, 40-400Hz). A Helmholtz coil was selected as the desired magnetic field

generation configuration due to the mostly-uniform magnetic field generated over a reasonably

large area at the center of the coils [73,74,78]. The Helmholtz coil was placed inside a NuAire

Autoflow CO2 Water-Jacketed Incubator (NuAire Plymouth, MN), maintained at 37 °C, 5% CO2,

and power-supply run through an insulated port in the side of the chamber. The magnetic flux

density was measured at multiple points across the area through which cell culture plates were

exposed, using a Lakeshore 425 Gaussmeter and LakeShore Fiberglass Transverse Probe HMNT-

4E04-VR (LakeShore Cryotronics Inc Westerville, OH). The Helmholtz coil was powered for one

hour before cell culture-exposure began, allowing any heating and resistive change in the

circuit to occur prior to executing the experiment. There was no measurable temperature rise

within the exposure chamber, or region near cell culture during operation of the Helmholtz coil

system, however there was a minor change in current measured through the coil after 15-20

minutes of operation. The power to the coil was briefly cut off when inserting and removing

each cell culture dish, to prevent any unintended magnetic field effects on the particles as they

moved into, and through the field.

In all experiments reported, field intensity was measured at 2.0mT (rms), uniform to

within ± 4% over the cell exposure area, with DC, 50Hz, and 100Hz driving current for each

respective experiment. Finite Element Method Magnetics (FEMM 4.2) and Comsol Mulitphysics

4.3 software was used to simulate the magnetic field and flux density generated by the

Helmholtz coil under these conditions shown in figures 3.2 and 3.3.

24

Figure 3.2: FEMM simulation: Flux density plot for cross section cut of magnetic field produced by Helmholtz coil pair

Figure 3.3: Flux density measurement from center of Helmholtz coil along either X or Y axis as depicted in Fig. 3.2.2

Mag

netic

Flu

x De

nsity

(T)

Length, in inches from center of coil pair

On-Axis Magnetic Flux Density

25

Magnetic field parameters of 2.0mT, DC and 50Hz fields were selected because, as

referenced earlier, they have been frequently investigated for impact on cellular behavior [13–

16,23,25,60,79], and because 50Hz and 60Hz are common frequencies at which electric power

distribution lines operate worldwide [11,80,81]. Additionally, a DC field was selected to provide

insight about field interactions with the cells and particles, as compared to alternating

frequency effects. 100Hz frequency was selected to determine any specific frequency

dependent or frequency specific effects between 0Hz (DC), 50Hz, and 100Hz exposures. In

addition, the 100Hz frequency experiment was selected in order to gleam insight towards the

magnetic field-nanoparticle interactions, and to investigate if a higher frequency magnetic field

would have an increased effect on nanoparticle uptake, and cell survival. Based on the initial

study for 50Hz magnetic field, it was conceived that the magnetic field was interacting with the

particles such that it was causing a vibratory motion, causing the particles to force their way

into the cells, or somehow enhancing the cells’ ability to interact with the particles.

For all exposure conditions, a respective parallel sham experiment was performed and used as

a control group. Shams were placed in the same incubation chamber as the magnetic field-

exposed cells, at a distance such that the magnetic field generated by the Helmholtz coil was

negligible. This was possible due to the 1/r2 relation of magnetic field strength, where r is the

distance from a magnetic field source; the magnetic field strength falls off very rapidly away

from the source of the field, and therefore only a short distance outside of the coil system, the

magnetic field strength is negligible. This procedure worked to ensure that any potential

variability between additional environmental strain between the exposed group, and the

control group was related specifically to the magnetic field exposure, and

26

3.3 Nanoparticles and Characterization

Two sets of commercially available iron oxide particles were purchased for this

experiment, 10nm Fe3O4 particles dispersed in double-distilled water at a concentration of

5mg/mL from Cytodiagnostics (Burlington, Ontario, CA), and Rhodamine-B labeled 10nm Fe2O3

particles dispersed in DI water at a concentration of 1 mg/mL (Fe) from Ocean NanoTech

(Springdale, AR). The non-labeled particles were characterized through transmission electron

microscopy (TEM) dynamic light scattering (DLS) and zeta potential. Dynamic light scattering is

a non-destructive method for measuring size and size distribution of particles in a colloidal

solution. Zeta potential measurements can be used as an indicator of electrostatic repulsion

between the particles in a colloidal solution; the greater in magnitude the zeta potential

measurement, the more stable the particles in the colloidal solution should be. DLS, Zeta

Potential, and polydispersity index (PdI) measurements were all taken using a Zetasizer Nano

(Malvern Instruments Ltd, Malvern, Worcestershire, UK), system at the University of Texas, San

Antonio, with gracious assistance from Ms. Samantha Franklin). The polydispersity index (PdI) is

a measure of particle and agglomerate size distribution. Low PdI values indicate a very small

size distribution, while larger numbers indicate a very large size distribution. Very large values,

from 0.7-1 indicate that DLS may not provide very accurate measurements of particle size or

hydrodynamic diameter, due to the large size distribution of particle agglomerates.

27

TEM imaging, shown in figure 3.4, was performed with a 120kV PC Hitachi H-7600

controlled TEM (Hitachi Ltd. Chiyoda, Tokyo, Japan) and was provided courtesy of Elizabeth

Maurer-Gardner (Research Scientist, Materials Characterization Specialist AFRL, Dayton, OH).

Particle size was observed to be 10.97 ± 0.70 nm, based on the measurement of 50

independent nanoparticles. DLS and zeta potential characterization was performed for samples

of nanoparticle-water, and nanoparticle-media dilutions at concentrations of 50, and 200

μg/mL in water, and 50, 200, and 500 μg/mL in media, under conditions with and without 24

hour DC magnetic field exposure. These dilutions were chosen to get an understanding of how

the concentration of the particle dilution impacts agglomerate behavior under various

conditions. Based on other scientific reports, it was expected that the particles would

Figure 3.4: TEM imaging of Fe3O4 nanoparticles performed to verify spherical shape and particle size

28

agglomerate, due to protein-particle interaction in cell media, and the particles’ induced

magnetization when subject to the application of a large external magnetic field [36,45,82–86].

The zeta potential of the particles dispersed in water was that of a moderately stable

colloidal solution, with magnitude ranging from 16.2 to 38.2 mV, however the PdI was very high

for the lowest concentration of particles both with and without magnetic field exposure, at

0.94-0.96, indicating that the particles had begun to sediment and settle in the solution. The

average hydrodynamic diameter was measured at 1248nm and 518.2nm for the 50 μg/mL

concentration in water with and without magnetic field exposure respectively, indicating that

the average agglomerate size is larger with magnetic field exposure, even with sedimentation.

At the higher concentration of 200μg/mL the colloidal solution appeared to be more stable,

with PdI around 0.33-0.35, and average hydrodynamic diameter measurements in the expected

range of approximately 20nm and 50nm with and without magnetic field exposure respectively.

For particles dispersed in media, the zeta potential was measured significantly closer to

zero, or near-neutral, with magnitude ranging from 0.16 to 9.0 mV. A near-neutral zeta

potential indicates that the particles may agglomerate or coagulate fairly rapidly, however the

PdI measured for all but one sample, was measured below 0.7, indicating reliable size

measurements, or small size distribution of particle agglomerates. The hydrodynamic diameter,

PdI, and Zeta potential measurements for all samples are displayed in table 3.1 for all particle

dilution conditions. Of most importance to this study, are the magnitudes of zeta potential

measurements from particle-media dilution samples, which are all less than 10, with

hydrodynamic diameters ranging from 50nm – 1 μm when exposed to magnetic field.

29

The Rhodamine-B labeled particles were used for fluorescence microscopy experiments only,

and therefore were not a focus of characterization study. The manufacturer report indicates

that the Rhodamine-B labeled particles have a near-neutral zeta potential, and particle size of

10nm ± 2.5nm, with size distribution of ~10%. Ultimately, this difference between the

Rhodamine-B labeled particles and the non-labeled particles is not of great concern in this

particular study, as all statistically significant effects were observed at very high Fe3O4

concentrations, where the respective measurements in media closely reflect this same

behavior.

3.4 Cell Proliferation and Mitochondrial Function – MTT Assay

To assess cell mitochondrial function, and thereby viability or proliferation, a

calorimetric MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was

used (ATCC ® 30-1010K™). In the MTT assay, a tetrazolium salt reagent is added to the cell

Sample Conditions ZP (mV) Size (nm) PdI

H2O 50 μg/mL No MF -16.2 518.2 0.96 H2O 50 μg/mL DC MF -32.3 1248 0.94

H2O 200 μg/mL No MF -33.4 48.78 0.355 H2O 200 μg/mL DC MF -38.2 19.75 0.333

Media 50 μg/mL No DC MF 0.167 37.44 0.524 Media 50 μg/mL DC MF -7.93 59.45 0.458

Media 200 μg/mL No DC MF -4.38 187.9 0.411 Media 200 μg/mL DC MF -7.09 146.2 0.289

Media 500 μg/mL No DC MF -9 473.5 0.656 Media 500 μg/mL DC MF -8.66 935.8 0.791

Table 3.1: Zetasizer nano measurements for various

Fe3O4 particle dilutions in H2O and media, with and without MF Exposure

30

culture, which is reduced by metabolically active cells, and converted to water-insoluble

formazan crystals, which appear dark blue or purple under inverted microscope [87]. The

microscope used for this study was the Leica DM IL inverted contrasting microscope (Leica

Microsystems Inc. Buffalo Grove, IL). After sufficient reaction time, a provided detergent

(ATCC® 30-1010K™) is added to the wells to dissolve the formazan into a solution with the cell

media. The absorbance of this solution is then measured; in this experiment a Synergy™ HT

Multi-Detection Microplate Reader (Biotek, Winooski, VT) was used. Using the aforementioned

culture methods, cells were plated at 2 x 105 cells/mL into 96 well plates at 50μl/well, giving

approximately 1 x 104 cells per well, and then exposed to each respective exposure condition

six hours later. After 24 hours of continuous exposure, the cell media and nanoparticles were

aseptically removed, and the wells were refilled with a mixture of 100μl full cell media, and

10μl MTT tetrazolium salt reagent. The cells were let sit for two hours (more than sufficient

time to observe the crystal formation under microscope) in the Heracell™150i CO2 incubator,

before 100μl of the detergent was added to each well to dissolve the formazan crystals. The

plates were then wrapped in aluminum foil, and placed on a MaxQ™ 3000 Benchtop Orbital

Shaker (Thermo Fisher Scientific Inc. Waltham, MA), at room temperature, overnight;

absorbance readings were taken the following morning. Background readings were taken from

wells that were treated identical to the experiment wells, with the exception that there were

no cells plated. These background cells allowed for analysis to consider any residual particles

that may have been left in the wells and their potential interference with the absorption

readings from the microplate reader. An effective dose (ED50) was calculated to determine the

31

concentration of the particles that successfully reduces the cell population to a fraction of half

the control value, after the exposure time is complete.

3.5 Nanoparticle Uptake and Confocal Microscopy

To observe cell morphology, microtubule formation, nanoparticle uptake and particle

trafficking, with and without magnetic field exposure, the Rhodamine-B labeled nanoparticles

were added to cells cultured in poly-D-lysine coated glass-bottom dishes six hours after the cells

were plated. Immediately following the addition of the nanoparticle solution, the culture dishes

were placed in respective exposure and sham positions in the exposure incubation chamber for

24 hours. A Zeiss LSM 710 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany)

with DIC40X 1.2NA objective (Carl Zeiss MicroImaging) was used in conjunction with the ZEN

2012 software (Zen 2012 SP1 Black Edition – Ver. 8,1,3,484, Carl Zeiss Microscopy GmbH) to

image the mEmerald-tubulin expressing CHO cells and Rhodamine-B labeled nanoparticles. The

peak excitation and emission

wavelengths for mEmerald and

Rhodamine-B are 487/509 and 540/625

respectively, and the pinhole was

adjusted for 1μm sections for all time-

series and z-stack series. For various

positions on the culture dishes, multiple

images were taken in time series - 30

cycles at a rate of one cycle every 12 Figure 3.5: Zeiss LSM 710 confocal microscope

used for microscopy studies

32

seconds, for a total period of six minutes, to observe cell-nanoparticle interaction, intracellular

trafficking, and tubulin change. Single image acquisition of CHO cells with fluorescent tubulin

expression was done with a line step of one, and line average four to acquire clear images of

microtubule formations. Likewise, several series of images were taken at multiple focal points

from beneath the cells, to above cells, in order to compile a Z-stack profile of nanoparticle

location and distribution throughout the cell. Each series was comprised of approximately 25 to

35 images, depending on cell height, with an average interval of approximately 0.5μm, using

pinhole sections of 1μm to achieve Nyquist sampling. Mean fluorescence of cells exposed to

Rhodamine-B labeled nanoparticles was measured and compared between those subjected to

DC magnetic field exposure and those of sham exposure conditions. DC magnetic field was

selected for the microscopy study based on MTT results that displayed DC magnetic field

exposure to have the most prominent effect on cell viability. As microtubules have been

identified as playing a significant role in vesicular intracellular trafficking [88], CHO cells with

both mEmerald-tubulin expression and nanoparticle exposures were imaged after magnetic

field and sham exposure conditions, in an attempt to observe any changes in tubulin-

nanoparticle interaction, tubulin expression, and microtubule structure and formation. The

public domain Java-based program Fiji [89] was used for independent image and image-series

processing.

33

CHAPTER 4

RESULTS AND DISCUSSION

This chapter presents the results of the MTT viability assay and microscopy assessments

for the various conditions of nanoparticle and magnetic field exposures. Each case is presented

and considered, from independent conditions for the viability assay, to varying particle

concentration and field effects under microscopy. A brief discussion is presented on how the

data should be interpreted for the sake of this experiment.

4.1 Cell Viability: Proliferation and Mitochondrial Function – MTT Assay

The cytotoxicity of 10nm Fe3O4 nanoparticles was assessed by means of MTT assay, as

described in the previous chapter. The measurements for nanoparticle exposure without any

applied external magnetic field are displayed in figure 4.1.1, and as sham exposures in all

graphs. These results closely resemble those of other reported toxicity assessments for Fe3O4

particles [27,39,43], showing a minor increase in mitochondrial activity at very low doses

~10μg/mL, and significant reduction at extremely high concentrations, upward of 250μg/mL

and higher. Additional assays were performed for cells cultured under nanoparticle and

concurrent (DC, 50Hz, or 100Hz) magnetic field, or respective sham exposure conditions. The

ED50 value for nanoparticle (no magnetic field) exposure was calculated at 370μg/mL, which is

also in agreement with the previously mentioned studies. Statistical significance compared to

controls was determined using Student’s unpaired t-test with two-tailed P value < 0.05.

MTT assay results for the various magnetic field exposure conditions showed a minor

increase in viability at low Fe3O4 nanoparticle concentration in most trials; however the

34

difference was not statistically significant when compared to sham conditions. At higher

nanoparticle concentration, there was observed a marked reduction in cell viability, when

compared to the respective sham exposures. These results are in agreement with those

presented by Jia et al. [68], on their assessment of PC12 cell proliferation and apoptosis with

50Hz magnetic field and SiO2-modifed magnetic nanoparticle exposures.

Initially, the data suggested a trend of reduced cytotoxicity with increased magnetic

field frequency; the DC magnetic field appeared to have the most significant reduction on cell

viability, followed by 50Hz, and finally, the least significant impact on cell viability was observed

with the 100Hz magnetic field exposure. A compilation for comparison of all exposure

conditions is displayed in figure 4.1.

Figure 4.1: MTT Concentration-dependent survivability curve of cells exposed to Fe3O4 nanoparticles and no magnetic field

35

Upon further inspection and analysis, however, it was concluded that all three magnetic field

exposures have the same effect on the cells; the apparent decrease in toxicity with 100Hz

magnetic field exposure, compared to DC and 50Hz frequency conditions is likely the results of

a small variation in dilution for those exposure conditions introduced by pipette error. This

conclusion was drawn from data shown in figure 4.2-c; a graph showing the respective

difference in survival from exposure and sham conditions. The 100Hz delta curve follows almost

exactly the curves of the DC and 50Hz exposures, with a right-shift of one concentration value,

and peaks with the same trend. The ED50 of each exposure condition was calculated by

interpolation as 370μg/mL, 217μg/mL, 237.4μg/mL, and 303.3μg/mL for no magnetic field, DC,

50Hz, and 100Hz, respectively. The differences between ED50 for each magnetic field exposure

may yet again be attributed to the right-shift in delta mentioned previously. Due to the

prominence of the DC field on cytotoxicity and cell viability measurements in the initial studies,

all fluorescence and microscopy studies were performed with DC magnetic field exposure

conditions in an attempt to investigate the cellular physiology effects of the exposures.

4.2 Cell Morphology, Nanoparticle Uptake and Confocal Microscopy

CHO cells with mEmerald-tubulin expression were observed under live-confocal

microscopy to examine cell morphology, microtubule distribution, nanoparticle uptake, and

nanoparticle trafficking after 24-hour exposure to four unique conditions. Exposures were

performed at 50 and 250 μg/mL Rhodamine-B labeled nanoparticle concentrations, with and

without DC magnetic field. The different nanoparticle concentrations were selected to

determine if there was a direct impact on cellular uptake with respect to increased particle

36

concentration, while exposure to magnetic field and sham conditions were established to

observe any possible changes in cell morphology, orientation or directionality, and microtubule

network, as a result of magnetic field exposure.

(a) (b)

(d) (c)

Figure 4.2: MTT Concentration-dependent survivability curves for cells exposed to Fe3O4 nanoparticles and (a) DC magnetic field, (b) 50Hz magnetic field (c) 100Hz

magnetic field, and (d) the respective Δ from sham conditions. The greyed section represents the area beyond the ED50 for cells not exposed to magnetic fields. Sham

exposure Δ measured against trials in an independent incubator.

37

Bright field imaging revealed no evident change in morphology between cells exposed

to DC magnetic field and sham conditions. In both cases, the cells were observed in various

shapes, sizes, and formations. Many cells, as displayed in figure 4.2.1 were observed to have

three extensions away from their nuclei, towards distal focal adhesion points, but with no

particular favor of directionality with or without magnetic field exposure. Likewise, confocal

fluorescence microscopy using the mEmerald-tubulin expression shown in figure 4.1-b and 4.2-

b revealed no change in microtubule distribution or direction between exposure conditions.

While there was no change in microtubule orientation, the images also give an idea of nuclear

height within the cells; there are very few cells with microtubules observed underneath the

perinuclear region of the cells (observed as dark circular formations toward the center of each

cell), indicating that there is no change in nuclear height between magnetic field exposure

conditions.

Mean fluorescence intensity (MFI) from Rhodamine-B in the cells was measured to

quantify nanoparticle uptake at both concentrations, under DC magnetic field and sham

exposure conditions. Cells under concurrent DC magnetic field and nanoparticle exposure were

measured to have a significantly greater MFI than that of cells exposed to nanoparticles alone,

shown in figures 4.3 and 4.4. At lower particle concentrations, the difference in MFI between

DC field exposure and sham was much greater when compared to the difference in MFI at the

higher nanoparticle concentration. Jiang, et al. have previously reported size dependent effects

of nanoparticles on cellular uptake, showing that smaller particles of ~10nm coat cell

membrane surfaces before triggering endocytosis, while larger particles (or in this case

agglomerates) of ~100nm or larger more readily trigger uptake [47,49,90]. The drastic contrast

38

at lower concentration may therefore be attributed to agglomeration effects and the size

distributions observed at lower concentrations– DLS measurements of the particle-media

solutions showed large hydrodynamic diameters up to 1μm at the concentration of 50 μg/mL.

Multiple images taken over time-series showed intracellular trafficking of nanoparticle clusters;

however, no quantification method was readily available to determine if there was a difference

in the rate of trafficking between cells exposed to magnetic field exposed cells, and those not

exposed. This is discussed further in the final section of this thesis.

39

(a)

(b)

(c)

(d)

Figure 4.3: Select images from confocal microscopy study without DC magnetic field exposure; (a) bright field images (b) Fluorescent mEmerald

tubulin expression (c) Fluorescence imaging with Rhodamine-B labeled particles and (d) Overlay results from bright field and Rhodamine-B

fluorescence imaging to show particles inside the cell structures.

40

(a)

(b)

(c)

(d)

Figure 4.4: Select images from confocal microscopy study after DC magnetic field exposure; (a) bright field images (b) Fluorescent mEmerald

tubulin expression (c) Fluorescence imaging with Rhodamine-B labeled particles and (d) Overlay results from bright field and Rhodamine-B

fluorescence imaging to show particles inside the cell structures.

41

Figure 4.6: Mean fluorescence intensity of cells measured with, and without DC magnetic field exposure at 50 ug/mL

nanoparticle concentration

0

2

4

6

8

10

12

14

16

No MF Exposure MF Exposed

Fluo

resc

ence

Uni

ts (A

rb)

Mean Fluorescence Intensity 50 μg/mL

***

0

10

20

30

40

50

No MF Exposure MF Exposure

Fluo

resc

ence

Uni

ts (A

rb)

Mean Fluorescence Intensity 250 μg/mL

**

Figure 4.5: Mean fluorescence intensity of cells measured with, and without DC magnetic field exposure at 250 μg/mL

nanoparticle concentration

42

CHAPTER 5

CONCULSIONS AND FUTURE DIRECTION

5.1 Conclusions

A study was performed to investigate the biological impact of concurrent exposure to

magnetic nanoparticles and extremely-low frequency magnetic fields (ELF-MF) using an in-vitro

CHO-K1 cell line model. Protocols were established for in-vitro nanoparticle, magnetic field, and

concurrent exposure, specifically for DC, 50Hz, and 100Hz 2.0mT magnetic fields, and 10nm

Fe3O4 nanoparticles. The investigation focused primarily on comparative analysis of concurrent

exposures against independent controls, to investigate cell viability and recovery through

mitochondrial activity measurement via MTT assay, immediately following continuous exposure

for 24 hours. The magnetic fields were generated by the application of a precisely controlled

driving current through a Helmholtz coil system, in an environment-control incubation

chamber. In all experiments reported, magnetic field intensity was measured at 2.0mT (rms),

uniform to within ± 4% over the cell exposure area. MTT assay results indicated a significant

decrease in cell viability between each magnetic field exposure condition, and respective sham

at high nanoparticle concentrations. It was concluded that there was little to no effect between

applied field frequencies, with only minor changes in cytotoxicity, or change in cell viability

measurements with each respective field. It is worth mentioning that cells exposed to the DC,

50Hz, and 100Hz magnetic fields without nanoparticle exposure did exhibit an increase in MTT

measurement, as compared to their respective sham exposures; this difference is likely due to

an increase in total cell number by magnetic-field related stimulus on proliferation rate. Further

43

investigation is most certainly required to gleam a better understanding of the mechanisms

behind this effect, as magnetic field interactions with cells are still not well known.

Confocal microscopy of the cells under concurrent nanoparticle and magnetic field

exposure revealed no apparent changes in cell morphology or cytoskeletal structure between

applied magnetic field, and no-field conditions. Fluorescence microscopy on the CHO cells with

fluorescent tubulin expression also revealed no change between exposure conditions. The

absence of effect on tubulin expression is contradictory to reports of 50Hz magnetic field effect

on tubulin expression in SH-SY5Y cell models reported by Cerrato, et al. [16]. This may be

attributed to the difference in cell line, or exposure time; additional studies are warranted to

investigate field effect on tubulin expression between cell lines.

There was a measured change in nanoparticle uptake, with regard to applied magnetic

field; a higher mean fluorescence was measured in cells subject to magnetic field exposure, as

compared to the respective no-field conditions. This effect was observed at both high and low

particle concentrations, but was more significant at lower concentrations. These results can be

used to confirm the impact of nanoparticle concentration and agglomeration effects on cellular

uptake. Intracellular trafficking of nanoparticle clusters was observed with all exposure

conditions; however no quantification method was available to determine if there was a

difference in the rate and directions of trafficking.

5.2 Recommendations and Future Directions for Investigation

With the current investigation methods, it is not possible to know the exact method of

endocytosis, or storage and trafficking of the nanoparticles once they have been taken up into

44

the cells. By changing the label on the nanoparticles, and systematically selecting alternative

photoactive tags for cell proteins, such as the lysosomal-associated membrane protein 1

(LAMP1), and Tetramethylrhodamine (TRITC), co-localization of the particles and lysosome

structures may be observed. Additionally, inhibitory drugs that interfere with specific pathways

may be systematically selected and administered to the cells to identify the particular uptake

mechanisms involved with these particles.

While there was no observed effect on cell viability between the DC, 50Hz, and 100Hz

magnetic fields, that is not to say that there are no proteomic or genomic changes within the

cells between these, and other applied external magnetic field frequencies. A brief investigation

into reactive oxygen species (ROS) or Interleukin 6 (IL-6) as an indicator of cellular stress and

inflammatory response, in addition to an assay to reveal caspase 3/7 activities could provide

some insight into the cause of changes in mitochondrial function reported via MTT assay. As the

mechanisms behind magnetic field and cell interactions are still not well known, further in-

depth studies could be developed to investigate individual proteomic and genomic expression

within the cells exposed to various magnetic field conditions, similar to the analyses reported

by Falone et al., and Hasanzadeh, et al. [14,15] with SH-SY5Y neuroblastoma cell lines exposed

to 50Hz magnetic fields for 3 hours.

Ultimately, there are near infinitely many variations available for experimentation from

field specifics such as frequency, intensity, and direction, along with dynamic and moving fields

or static field gradients, as well as nanoparticle size, shape, composition, fabrication method,

and time duration of each related exposure. An important and more impactful direction to

focus future efforts would be on bridging the gap in knowledge between the specific

45

mechanisms behind the magnetic field interactions with cells, to better understand why

different cell types have different responses to the same exposure conditions, and how to take

advantage of said mechanisms. Future applications of these advantages may be developed to

eliminate deleterious effects, and enhance desirable or salubrious effects in medical,

commercial, and military applications.

46

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