Characterization and Functionalization of Iron-Oxide Nanoparticles for Use As Potential Agents for Cancer Thermotherapy at the U

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Characterization and Functionalization of Iron-Oxide Nanoparticles

for Use as Potential Agents for Cancer Thermotherapy

Nora O’Reilly

A dissertation submitted in partial fulfillment of the requirement for the degree of

Doctor of Philosophy

(Animal Science) At the University of Wisconsin-Madison 2013

Date of final oral examination: April 18th, 2013

The dissertation is approved by the following members of the Final Oral Committee:

Professor Ralph Albrecht, Animal Science, Pediatrics, Pharmaceutical Sciences

Professor Mark Cook, Animal Science

Professor Amin Fadl, Animal Science

Professor Manish Patankar, Obstetrics and Gynecology

Professor Chris Brace, Radiology, Biomedical Engineering i

Abstract

This thesis presents experimental studies of iron oxide nanoparticle synthesis, functionalization, and intracellular hyperthermal effects on murine macrophages as a model in vitro system. Colloidal suspensions of magnetic nanoparticles (MNPs) are of particular interest in Magnetic Fluid Hyperthermia (MFH). Iron oxide nanoparticles (IONPs) have garnered great interest as economical, biocompatible hyperthermia agents due to their superparamagnetic activity. Here we seek to optimize the synthetic reproducibility and in vitro utilization of IONPs for application in MFH. We compared aqueous synthetic protocols and various protective coating techniques using various analytical techniques and in vitro assays to assess the biocompatibility and feasibility of the various preparations of nanoparticles.

Using a co-precipitation of iron salts methodology, iron oxide nanoparticles (IONPs) with an average diameter of 6-8nm were synthesized and stabilized with carboxylates. By performing calorimetry measurements in an oscillating magnetic field (OMF) with a frequency of 500 kHz and field strength of 0.008Tesla the superparamagnetic behavior of these particles was confirmed. To further investigate these IONPs in a biological application, citric acid-stabilized particles, in conjunction with heat generated by these IONPs when exposed to an OMF, were assessed to determine their effects on cell viability in a RAW 267.4 murine macrophage model system.

Our results show that 91.5-97% of cells that have ingested IONPs die follow exposure to an OMF. Importantly, neither the IONPs (at applicable concentrations) nor the OMF show cytotoxic effects. These particular particles have promising preliminary results as hyperthermic agents in both the current literature and simple, proof-of-concept experiments in our laboratory setting. We present experimental results for the synthesis, characterization, and utilization of iron ii oxide nanoparticles in MFH. Our results show that while IONPs have potential in MFH, efforts to advance IONPs from laboratory to clinical contexts, where reliable generative techniques and consistent performance properties are necessary, will require an understanding of the influence of the diverse intrinsic structural and magnetic characteristics as well as surface chemistry of nanoparticles, as well as the mechanisms of particle uptake and cell death due to intracellular hyperthermia.

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Acknowledgements

There are an enormous number of people who have contributed not only to this dissertation, but my development and evolution as a student, teacher, and a scientist. Of these people who have been enormously supportive in the preparation of this thesis, I am especially thankful to my advisor, Dr. Ralph Albrecht, for all of his efforts in guiding me through to its successful completion. Dr. Albrecht has been an exceptional mentor, from the time he introduced to me to research as the professor in my undergraduate senior seminar in UW-Madison’s Animal

Science department, through my years as a graduate student in his group in the same department.

He always made himself available to provide guidance on the numerous challenges that I encountered during my studies and research. He has taught me invaluable skills as a scientific researcher and critic, especially the ability to approach scientific questions with a solid understanding of cellular biology, immunology, and nanotechnology. Again, I express my most sincere gratitude to Ralph for his support in my development as a scholarly researcher.

My special thanks to the members of my advisory committee: Professor Mark Cook,

Professor Amin Fadl, Professor Manish Patankar, and Professor Chris Brace for their valuable time, suggestions, and discussions.

My heartfelt thanks to Dr. Ian Rowland for all his valuable work with our small animal

MRI proof-of-concept feasibility studies. I am sorry that circumstances did not allow for us to continue our work together in greater depth. Ian’s enthusiasm, compassion, and sense of humor were equally appreciated as the expertise in his field that he shared so freely.

Someone who quietly carriers the lab on his shoulders is our lab manager, Joseph Heintz.

Professor Heintz taught me all the laboratory and analytical techniques that were utilized in this dissertation. As Joe’s skill as an electron microscopist far exceeds my own novice abilities, he kindly performed much of the imaging that is included in the figures for this work. I truly would have been an elephant in a china shop had it not been for Joe’s supervision, guidance, and advice.

I would also like to thank the many collaborators and lab members for their advice and suggestions throughout the years including Professor Julie Oliver, Professor Doug Steeber,

Professor Marija Gajdardziska-Josifovska, Evan Krystofiak, and Eric Mattson from the

University of Wisconsin-Milwaukee. A post-doctorate researcher under Professor Song Jin in the

Chemistry department, Chad Dooley, was immensely helpful in his explanations of the particle and surface chemistry involved in the synthesis and stabilization of our particles. His passion for chemistry was inspiring, carrying me some of the most frustrating periods of this research. While there are many undergraduate students with whom I had the pleasure of working, the current students in the Albrecht lab who have been so helpful during the completion of this dissertation are Diana Perdomo and Kaitlyn Soukup. I am also thankful for Tom Tabone, Animal Science’s resident statistician, and his ability to be bribed with baked goods. Tom was instrumental in the statistical analysis included in this dissertation.

At some point these dedications begin to take on an Oscar-night quality, so I must wrap up with thanks to all the professors from the UW Department of Neuroscience who taught me that the material professors impart is secondary to the inspiration and life they breathe into their material, igniting the minds and souls of future generations, the Delta program for providing formal training regarding teaching at the university level, and Liv Sandberg who was kind enough to allow me to flex my newly discovered teaching muscles in her classroom.

Lastly, but always first in my heart, thank you to my family and friends for listening to my excited, if slightly frenetic explanations of what I am actually doing in school and supporting me in all my academic endeavors in countless ways, both large and small. It would have been impossible for me to successfully complete my graduate education had it not been for your love.

Table of Contents

Characterization and Functionalization of Iron-Oxide Nanoparticles for Use as Potential Agents for Cancer Thermotherapy ...... i Abstract ...... i Acknowledgements ...... iii Chapter 1 ...... 1 Introduction ...... 1 Introduction to Magnetic Nanoparticles ...... 1 Biological Applications of Magnetic Nanoparticles ...... 2 Magnetic Domains ...... 3 Inductive Heating of Magnetic Nanoparticles ...... 4 Biomedical Application of the Magnetic Properties of Nanoparticles ...... 4 Diagnostic In Vivo Imaging...... 5 Therapeutics ...... 5 Introduction to Tumor Hyperthermia ...... 6 Harnessing the Potential of Superparamagnetism and the Inductive Heating Phenomena...... 6 Nanotheranostics ...... 7 Background and Motivation ...... 8 Scope of Thesis ...... 9 Organization of Thesis ...... 12 Figures ...... 13 Figure 1. Brownian Motion and Neel Relaxation...... 13 Figure 2. Employing Theranostic Nanoparticles...... 14 Figure 3. Therapeutic Gap...... 15 Figure 4. Targeting Strategies to Improve NP Delivery throughout the Tumor ...... 16 References ...... 17 Chapter 2 ...... 20 Optimization of a Synthetic Procedure for Iron Oxide Nanoparticles ...... 20 Introduction ...... 20 vii

Potential Synthetic Methods ...... 22 Experimental Methods ...... 25 Chemicals ...... 25 Synthesis of Iron Oxide Nanoparticles ...... 25 Mass Determination ...... 26 Oscillating Magnetic Field Generator ...... 26 Magnetic strength (Tesla)...... 27 Solenoid ...... 27 Thermocouple ...... 28 Measurement of Heat Production by Colloidal Magnetite Particles ...... 28 TEM Sample Preparation ...... 28 Results and Discussion ...... 29 Synthesis and Characterization of Iron Oxide Nanoparticles ...... 29 Inductive Heating of Iron Oxide Nanoparticles in an Oscillating Magnetic Field ... 32 Conclusions ...... 35 Figures ...... 36 Figure 1...... 36 Figure 2a...... 37 Figure 2b...... 38 Figure 3a...... 39 Figure 3b...... 40 Figure 4a...... 41 Figure 4b...... 42 Figure 5...... 43 Figure 6...... 44 Figure 7...... 45 Figure 8...... 46 References ...... 47 Chapter 3 ...... 49 Functionalization of Iron Oxide Nanoparticles for Biological Application ...... 49 Introduction ...... 49

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Rationale for Selection of Current Functionalization Method...... 51 Experimental Methods ...... 53 Chemicals ...... 53 Cell culture ...... 53 TEM sample prep ...... 54 Stabilization of Iron Oxide Nanoparticles with TMAOH ...... 54 Stabilization of Iron Oxide Nanoparticles with Citric Acid...... 54 Stabilization of Iron Oxide Nanoparticles with Sodium Citrate ...... 54 Stabilization of Iron Oxide Nanoparticles with Gold ...... 55 Mass Determination ...... 55 Measurement of heat production by colloidal magnetite particles ...... 56 Results and Discussion ...... 56 Stabilization of Iron Oxide Nanoparticles ...... 56 Small Organic Molecule Functionalization: TMAOH, Citric Acid, Sodium Citrate 58 Effect of Surface Modifying Molecules on the Inductive Heating Abilities of Iron Oxide ...... 58 Conclusions ...... 59 Figures ...... 60 Figure 1. Calculated surface to bulk ratios for solid metal particles versus size . .... 60 Figure 2. Core Shell Schematic...... 61 Figure 3a...... 62 Figure 3b...... 63 Figure 4...... 64 Figure 5...... 65 Figure 6...... 66 Figure 7...... 67 Figure 8...... 68 Figure 9...... 69 Figure 10...... 70 Figure 11...... 71 Figure 12...... 72

References ...... 73 Chapter 4 ...... 75 In Vitro Investigation of Iron Oxide Nanoparticles as Intracellular Mediators of Thermally-Induced Necrosis...... 75 Introduction ...... 75 Experimental Methods ...... 77 Chemicals ...... 77 Synthesis of Iron Oxide Nanoparticles ...... 77 Mass Determination ...... 78 Oscillating Magnetic Field Generator ...... 78 Solenoid ...... 79 Thermocouple ...... 79 Measurement of heat production by colloidal iron oxide nanoparticles ...... 80 Kinetics Studies of cFe Internalization using SEM ...... 80 Inductive Heating Experiments ...... 81 Light Microscopy ...... 82 Results and Discussion ...... 82 Conclusions ...... 85 Figures and Tables ...... 87 Figure 1a...... 87 Figure 1b...... 88 Figure 2...... 89 Figure 3...... 90 Figure 4...... 91 Figure 5...... 92 Figure 6a...... 93 Figure 6b...... 94 Figure 6c...... 95 Figure 7a...... 96 Figure 7b...... 97 Figure 7c...... 98

Figure 7d...... 99 Figure 7e...... 100 Table 1...... 101 Figure 8a...... 102 Figure 8b...... 103 Figure 8c...... 104 References ...... 105 Chapter 5 ...... 106 Summary and Future Directions ...... 106 References ...... 112 Appendix ...... 114 List of Abbreviations ...... 114 Co-precipitation Synthesis of Iron Oxide Nanoparticles ...... 115 Organic Synthesis of Iron Oxide Nanoparticles ...... 117 Figure 1...... 118

Chapter 1

Introduction

Introduction to Magnetic Nanoparticles

The burgeoning of nanoscale technology truly began in 1959 with physicist Richard

Feynman’s groundbreaking lecture, “There’s Plenty of Room at the Bottom.” In this exchange,

Feynman highlighted the promise of a field that focused efforts on controlling matter on smaller and smaller scale. Once materials can be synthesized in the nanometer range, the classical, physical properties that proved confounding in the macroscopic scale wane, and the quantum properties of these materials can be influenced by dictating their size and shape. Currently the field of nanotechnology is undergoing an extraordinary period of scientific and technological growth. For which the unprecedented collaboration of a wide range of physical and chemical basic science research is responsible. The enormous potential of functional systems at the nanometer (nm) scale has encouraged collaborations which include academic research, industry, and government1. Particularly exciting applications for nanomaterials have emerged in the biomedical field as their size coincides with the dimensions of important biological targets such as proteins (5-50 nm,) genes (10-100 nm,) or cellular organelles (25-2500 nm.)

Biological Applications of Magnetic Nanoparticles

One interesting sub-field utilizes the properties of magnetic nanoparticles (MNPs) in various biochemical and biomedical applications including bacterial detection, protein purification, enzyme immobilization, cell separation, drug delivery, hyperthermia, and MRI imaging. One well-established nanomaterial that offers controlled size, ability to be manipulated by an external magnetic field, and enhancement of contrast in magnetic resonance imaging is

2 colloidal magnetite or iron oxide (Fe3O4) . Synthetic control of the monodispersity of the iron oxide nanoparticles is crucial because their properties depend greatly upon the size and shape of the nanoparticles. To understand and thus fully utilize the potential of ferrofluids, careful studies that examine the physical behavior of ferrofluids in relation to stability, surfactants, particle sizes, and materials are essential3-5.

The colloidal characteristics that allow for the biological application of iron oxide nanoparticles are determined by their surfaces and not by their bulk volume6. A traditional textbook definition of a colloid is a suspension of finely divided particles of one material in a dispensing medium that do not separate on long-standing7. The phenomena of colloidal stability originates from thermal motion or Brownian motion where random particular collisions with other particles, suspending fluid, or container wall cause continuous redirecting of a particle’s trajectory that resist sedimentation. However, magnetic nanoparticles due to van-der Walls and magnetic dipole–dipole attractive forces have tendency to coagulate which results in a decline in their colloidal stability, aggregates of increasing size, and eventual gravitational sedimentation.

To achieve colloidal stability in a biological environment (pH, osmolarity) a balance must be maintained between the interparticle London and van der Waals attractive forces and the

3 electrostatic repulsion based on surface charge8. This goal can be accomplished by the formation of an electric double layer on the nanoparticle following synthesis9.

The ability to manipulate the unique magnetic properties of iron oxide nanoparticles also make them highly desirable for biomedical applications. These properties are largely determined by the chemical composition, size, and shape of the particles. Nevertheless the extent to which these factors can be completely controlled is variable. Thus the properties of the same type of magnetic nanoparticle may not be consistently reproducible3, 10.

Magnetic Domains

The magnetic property of specific interest, to this study, termed superparamagnetism, occurs in particles below 30nm in size. As constrained by their size, in theory these nanoparticles contain a single magnetic moment or domain; a summation of all the individual magnetic moments (motion) of the electrons of all the iron atoms the particle contains. A magnetic material is made up of small regions known as magnetic domains that form as the material develops its crystalline structure during synthesis. In each domain, all of the atomic dipoles are coupled together in a preferential direction. This magnetic moment will naturally orient itself in the most stable direction due to its magnetic anisotropy that is determined by the atomic crystalline structure and shape (edge regularity) of the nanoparticle. It is important to recognize that magnetic domains are not analogous to the physical, crystallographic domains, as magnetic domains cannot be viewed with non-magnetic imaging techniques11.

Inductive Heating of Magnetic Nanoparticles

When exposed to a high-frequency magnetic ﬁeld, these single magnetic domain nanoparticles generate heat through oscillation of their magnetic moments. The energy from the field drives the magnetic moments to rotate and aligns them with the magnetic field direction by overcoming the thermal energy barrier. Once the external magnetic field is removed, magnetic moments do not relax immediately but rather take some time to randomize their orientations.

This heat dissipation can be due to rotation of the entire magnetic particle within a surrounding liquid medium (Brownian relaxation) and/or to rotation of the magnetic moment within the magnetic core (Neel relaxation)12. Again the particle composition, shape, size, as well as the concentration and viscosity of the suspension medium, and the magnitude and frequency of the applied magnetic field determine the relative influence of each of these inductive heating mechanisms. It is generally inferred that the internal Neel mechanism dominates particles with diameters below 20 nm while larger particles generate heat through the external Brownian rotation mechanism13 (Figure 1.)

Biomedical Application of the Magnetic Properties of Nanoparticles

Nanomaterials inhabit the realm where the size of the largest biological molecules and the smallest manmade probes meet, allowing for nanomaterials to be utilized with in vitro

(protein and cell detection and separation) and in vivo (drug and therapy delivery and imaging) aims in interdisciplinary biomedical fields2, 14, 15. The biomedical applications of nanoparticles, specifically magnetic iron oxide particles, are diverse and include magnetic targeting for drug and gene delivery, photodynamic, photothermal, and hyperthermal therapy and imaging contrast agents16-19.

An exciting field within the magnetic nanoparticle field, termed ‘nanobiomagnetism’ takes advantage of their unique, size-dependant properties at the juncture of nanomagnetism and medicine where the magnetic-field responsive nanoparticles can be used as medical and surgical instruments20. The applications that relate specifically to the work discussed in this dissertation involve the utilization of the both the diagnostic and therapeutic potential of superparamagnetic iron oxide nanoparticles.

Diagnostic In Vivo Imaging

In the field of bio-imaging, the use of magnetic nanoparticles as contrast agents has proved useful as their longer renal clearance time and higher relaxation values are of advantage when compared to traditional gadolinium-based contrast agents. This contrast enhancement using superparamagnetic iron oxide nanoparticles (SPIONs) is a consequence of their superparamagnetism, which disturbs local magnetic field uniformities in an MRI. The water protons adjacent to the particles react to this inconsistency in the field by increasing their relaxation rate, thus generating a strong reduction of T2 relaxation time (dark, negative T2 contrast) and a relative small influence on T1 relaxation time are the consequences21, 22.

Therapeutics

SPIONs are bridging the therapeutic gap that remains due to the limitations of conventional drug delivery systems. In attempts to overcome issues inherent to conventional chemotherapy (inadequate chemotherapy dosages reaching the tumor site, severe cytotoxicity, and tumor resistance, nanoparticles are loaded or doped with biological or pharmaceutical agents. Then iron oxide particles carriers can be guided to the desired target area using an external magnetic field or by specifically-tagging the nanoparticles with tissue-specific

6 antibodies. Once the SPIONs are concentrated at the target site, they can be released through remotely-induced enzymatic activity, changes in physiological conditions, or temperature23, 24.

While these applications regard nanoparticles as secondary transporters of chemotherapeutic agents, one area of therapy focuses on the intrinsic superparamagnetic properties of the iron oxide particles a primary source of cytotoxicity.

Introduction to Tumor Hyperthermia

Cancer cells are susceptible to heat which decreases their viability and increases their sensitivity to chemotherapy and radiation. Tumor vasculature, depending on tumor type, often is inadequately developed, thus the tumor cannot sufficiently be cooled by blood flow. The cancer therapy, hyperthermia, takes full advantage of this Achilles’ heel of tumor cells, by raising the temperature of the target tissue to between 42 and 47°C25. While there are several methods that have been employed in the past to achieve this end, a discussion of the current and future state of intracellular cancer hyperthermia will best serve this dissertation.

Harnessing the Potential of Superparamagnetism and the Inductive Heating Phenomena

As mentioned earlier, magnetic fluid hyperthermia involves dispersing magnetic particles throughout the target tissue followed by the application of an alternating magnetic field of the necessary strength and frequency to cause the particles to heat by magnetic hysteresis losses or

Néel relaxation. The investigation of the application of magnetic nanomaterials for hyperthermia gained attention with Gilchrist in 1957 who studied the bulk heating of tissue samples with iron oxide nanoparticles in the 20-100nm range25.

More recently, cellular magnetic particle hyperthermia has become an attractive prospect because it offers a controlled modality by which there can be selective heating of target cell types by way of targeted nanoparticles. In addition the cellular route of administering magnetic nanoparticles could selectively heat systemically-dispersed metastases as well as bulk tumor tissue33, 34.

Nanotheranostics

Generally, theranostics combines the imaging and treatment of disease into a single formulation. Most interesting are the theranostic nanoparticles that combine imaging and treatment into a single nanomedical platform. The biocompatible and magnetic attributes of superparamagnetic iron oxide nanoaparticles make them ideal candidates to overcome biological barriers, poor biodistribution of drugs, metastatic disease, drug resistant tissues, and ineffective treatment management26. The newest frontier in nanomedicine has been coined

‘nanotheranostics’ by which the diagnostic imaging and therapeutic capabilities of iron oxide nanoparticles are used in conjunction with their abilities to act secondarily as drug-carriers or as the primary chemotherapeutic agent; exploiting their intrinsic superparamagnetic properties27.

The combined process of diagnosis and therapy into one process, a see-and-treat strategy, is at the forefront of a wave of personalized nanomedicine and the focus of this dissertation (Figure

2.) 28

Background and Motivation

Cancer is currently among the top three diseases in terms of mortality and morbidity in the developed world. However in 2009, Americans had a 20% lower risk of death from cancer than in 1991, when cancer death rates peaked. This decline can be attributed to improved early detection methods and treatment options29.

Principally, treatment for cancer includes surgery, chemotherapy, and radiation therapy, all systemic therapies which have major disadvantages including substantial toxicity to the patient and a number of other side effects. Optimal tumor destruction requires chemotherapeutic doses that exceed a patient’s tolerable toxic tolerance. One step in minimizing side effects is through the use of targeted therapy. Specifically targeting antitumor agents can reduce the total exposure so that higher levels of anti-tumor agents (either physical or chemical) can be employed at the site of the tumor.

Figure 3 shows the therapeutic gap between the critical tolerance level (toxicity) and the desired tumor destruction. Systemic therapies reduce the actual demand of further tumor destruction. However, the acceptable toxic tolerance is also reduced and the therapeutic gap is reestablished in most cases. Current treatment modalities are unable to close this gap. Therefore it is imperative that improved methods for localized cancer treatment are developed.

Scope of Thesis

One such selective yet minimally invasive approach involves the deposition of magnetic nanoparticles within the tumors mass simply by relying on what is termed an ‘EPR’effect. This relies on the ‘enhanced permeability and retention’ of small particles within the tumor cell mass due to poorly formed and leaky vasculature which is present in many aggressive, poorly differentiated tumors; however substantial limitations will be encountered in relation to multi- focal tumors and metastatic tumors30.

Another method of particle administration is via direct injection into the tumor mass. The injected particle suspension is forced along the weak links of the tissue, traveling non-uniformly through the tissue, this has proved unreliable. To avoid insufficient heating of selected areas of tumor or tumor margins, the dose of MNPs must be increased to such an extent that in parts of the tumor (and eventually healthy tissue,) temperature ‘hot spots’ may arise11. In this way, the local non-homogeneity of particle concentration for conventional IT injection makes the differentiation between hyperthermia (above 43°C) and thermoablation (above 46°C) unreasonable13. Moreover, the irregularity of particle distribution in the tumor makes the in situ temperature measurement insufficient for reliable control of therapy.

Delivery of particles systemically by specific cell (i.e. antibody or ligand) targeting may substantially increase the number of particles deposited on a cell surface. However, in order to

“bulk heat” a single cell, an unrealistic, massive amount of power, provided by an impracticable number of nanoparticles would still be required to heat a single cell in the human body31.

This work in this dissertation is designed to develop a system with the possibility of targeting cell membranes and producing cell damage and death, not by bulk heating, but through localized cell membrane damage. In this case relatively low numbers (as few as 1000) of small

(10nm to 20nm) particles can produce sufficient localized damage to the cell membrane to kill the cell in the absence of bulk heating. The use of the smaller, molecular size of particles improves their distribution and potential to effectively label the tumor cell32.

One of the reasons that MNP delivery systems have been relatively unsuccessful until recently is the deficiency in the knowledge of identified specific receptors for targeted cells.

However, in the past decade, there has been significant increase in the development of targeting delivery systems intended for selective inhibition or killing of pathogens or atypical cell types.

With targeted delivery systems, side effects of nonspecific accumulation of agents at sites other than the sites being targeted diminish. Specific targeting is accomplished through biological ligands or antibodies, which bind to specific cells33, 34. Interestingly the idea of using cell surface ligand or antibodies to target MNPs to tumor cells has not received significant attention in research to date.

Colloidal magnetite nanoparticles, Fe3O4, (cFe) are candidates for targeting at the molecular level. The targeted delivery system described in this proposal employs magnetic properties of colloidal magnetite oxides (cFe), principally magnetite (Fe3O4). The surface properties of magnetite (cFe) permit conjugation to specific targeting moieties such as ligands or antibodies. Once the targeting is accomplished, the nanoparticles can be inductively heated by exposure to an oscillating magnetic field (OMF.) For subsequent cellular specificity the colloidal nanoparticles are conjugated to antibodies or ligands specific for cell surface antigens or receptor complexes. The field is applied only to the treatment area, so any particles at non-

11 target sites are not heated33. Bulk tumor and metastases are treated simultaneously, tumor cells cannot develop resistance, non-target cells are unaffected thus limiting side effects, and the treatment can be repeated. The small size of the particles limits nonspecific accumulation to any particular tissue or cell type so nonspecific cytotoxicity is minimal.

Colloidal magnetite nanoparticles retain magnetic properties at extremely small particle size (less than 10nm) making these particles possible candidates for use in externally targeted delivery systems which use standard magnets. Synthesis of colloidal magnetite nanoparticles in the 2 to 18nm size range is achieved by an oxidation reaction using two iron chloride salts, Fe2+ and Fe3+ and the addition of a base36. Nanoparticle sized magnetite has a high specific magnetization and therefore is energized significantly when expose to an alternating current magnetic field, which make it a useful candidate for inductive heating in vitro or in vivo35.

The surface properties of magnetite (cFe) permit conjugation to specific targeting moieties such as ligands or antibodies. Once the targeting is accomplished, the nanoparticles can be inductively heated by exposure to an oscillating magnetic field (OMF.) For cellular specificity the colloidal nanoparticles are conjugated to antibodies or ligands specific for cell surface antigens or receptor molecule complexes. The field is applied only to the treatment area, so any particles at non-target sites are not heated34. With the above information, it is therefore conceivable to combine MNPs and antibody therapy together to achieve maximum tumor targeting to create a multi-functional nanotheranostic instrument (Figure 4.)

This work herein focuses on the optimization of an integrated experimental system where iron oxide nanoparticles are synthesized, characterized, and utilized in vitro for the study of intracellular hyperthermia. Using these basic principles and results of proof of concept experiments, as a foundation, work can move forward and examine the thermally-induced necrosis of tumor cells via targeted iron oxide nanoparticles.

Organization of Thesis

The primary goals of this research project were to develop iron oxide nanoparticles

(IONPs) with appropriate characteristics (size, surface chemistry) that will bind specifically to the surface of cancer cells and upon exposure to an OMF, inductively heat and cause cell death.

The model system we have previously employed in vitro has furthered our investigation into the value of the IONPs. Chapters 2 and 3 discuss the development of a reliable pathway for synthesis and functionalization of the IONPs that are used in Chapter 4. Results in Chapter 2 necessarily define NP characteristics, particularly the size, shape, and atomic lattice organization of NPs relating to the amount of heat generated by the NPs. Chapter 4 explores selective hyperthermia as a potential therapy for cancer that will lead to significant reduction in tumor cell growth. This involves the use of stabilized IONPs in RAW macrophages in cell culture conditions.

Findings from this dissertation will be applied to the investigation of the toxicity of antibody-conjugated core-shells using a murine model with in vitro and in situ OVCAR tumors.

Ultimately, results from the proposed studies are anticipated to assist in the assessment of the potential use of IONPs as nanotheranostic tools.

Figures

Figure 1. Brownian Motion and Neel Relaxation from Kelkar & Reineke, 201132.

Mechanisms of inductive heat generation by magnetic nanoparticles when exposed to a high frequency magnetic field. Neel relaxation involves the rotation of the magnetic moment within the core while Brownian motion refers to rotation of the entire particle in a liquid.

Figure 2. Employing Theranostic Nanoparticles32.

Combining the therapeutic and diagnostic capabilities in one dose, has promise to propel the biomedical field toward personalized medicine.

Figure 3. Therapeutic Gap adapted from Szasz, A. et al.(2006.) Physical Background and Technical Realizations of Hyperthermia37.

There is a therapeutic gap between the toxic tolerance if the patient and the amount of necessary toxicity required for the desired tumor destruction.

Figure 4. Targeting Strategies to Improve NP Delivery throughout the Tumor27.

I) Non-PEGylated NPs accumulate in the tumor site through the EPR effect. II) PEGylated NPs show enhanced accumulation in the tumor site through the EPR effect. III) Targeted NPs show better distribution throughout the tumor and higher cellular uptake. IV) Subcellular targeting increases NP delivery to the intracellular site of action of the drug.

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Chapter 2

Optimization of a Synthetic Procedure for Iron Oxide Nanoparticles

Introduction

Nanobiotechnology has rapidly evolved into many fields of basic and applied research.

Interesting perspectives in current technology and future applications range from environmental

1 restoration, to energy conversion, to biomedicine . Current applications of iron oxide nanoparticles in both biomedical research and clinical medicine include magnetic protein purification, enzyme immobilization, bio-contamination decorporation, magnetically targeted drug delivery and controllable release, magnetically assisted diagnostic (MRI) and therapy, and

2 magnetic fluid hyperthermia . These multifunctional iron oxide nanoparticles combine tunable surface chemistry, special optical properties, and magnetic properties, all of which heighten their potential in the emerging field of nanobiomagnetism.

Nanobiomagnetism is defined by Medeiros, S. et al. as the intersection of nanomagnetism

3 and medicine that focuses on biological systems and/or process . The unique size-dependent properties of iron oxide nanoparticles, their size compatibility with cells (10–100um) and proteins (5–50nm) sizes, and their ability to be passively or actively targeted to any desired area of the body and this characteristic makes them extremely useful as medical tools. The area of particular interest for the research presented in this dissertation is magnetic hyperthermia using iron oxide nanoparticles.

Hyperthermia as therapy takes advantages of the sensitivity of the body’s proteins and cells to heat. While the exact mechanisms are poorly understood, at elevated temperatures heat can alter the stages of the cell cycle, irreversibly denature protein and DNA, affect the viability

21 of cells, and increase susceptibility to chemotherapy and radiation. The many concerns with the direct heating of bodily tissue have highlighted the utility of targeted iron oxide nanoparticles as a potential hyperthermic therapeutic agent. Localized magnetic fluid hyperthermia employs the concept of targeted hyperthermia with the unique properties of magnetic particles on the nanometer scale that can be injected intravenously. Magnetic characteristics, discussed later in this chapter, allow for the generation of heat by iron oxide nanoparticles when exposed to an external alternating magnetic field at appropriate field strengths. Taking this model one step further, multi-functional magnetic nanoparticles that are promising nanotheranostic (diagnostic and therapeutic capabilities in one nanoparticle) agents are being developed by uniting the ability of targeted delivery with magnetic resonance imaging (MRI) contrast enhancement to image and treat cancer.

While it is not the intention of this introduction to review the basic and clinical research to date of targeted magnetic hyperthermia, it is crucial to highlight the overarching issue that has prevented nanotheranostics from making their way more rapidly into the clinic. The obvious clinical translation issues including the difference in the critical dose necessary for imaging versus therapy, and in vivo interactions, exist. However the topics that are most crucial for application of iron oxide nanoparticles, involve the lack of an understanding of the complex dynamics that influence the particles’ magnetic characteristics both in a synthetic and biological setting, and the poor reproducibility of current methods of synthesis, variation in susceptibility to inductive heating and conflicting results from in vitro experiments.

Potential Synthetic Methods

Numerous methods have been reported for the synthesis of iron oxide nanoparticles via the conventional top-down, mechanical approaches which break big objects into small ones, but also through bottom-up, chemical approaches which assemble atomic or molecular building blocks. When it comes to the fabrication of nanoparticles with uniform size, the bottom-up approaches are advantageous, as the top-down methods usually have difficulties in terms of size and size distribution control. Previously numerous physical methods have been applied to synthesis of inorganic NPs, such as laser ablation, melt spinning, sputtering, mechanical ball milling, vacuum-deposition and electro-deposition. These methods are able to produce magnetic micro- or nano-particles with high purity and in large quantities. However, they generally have

4 little control on particle size, size distribution and structural morphology .

Chemical synthetic routes to the fabrication of inorganic nanocrystals represent a type of bottom-up approach which utilizes chemical methods to initiate and control the assembly process of the atomic or molecular building blocks. Though there has not been a general method applicable to the synthesis of all kinds of nanoparticles, one widely-adopted consensus is that monodisperse nanoparticles can be synthesized in solution phase by a process with separated

5 nucleation and growth steps . A variety of these chemical or bottom-up pathways include thermal

6 decomposition, solvothermal syntheses, microemulsion methods, and metal salt co-precipitation .

The thermal decomposition reactions take advantage of the facile thermolysis of organometallic compounds and metal-surfactant complexes in high boiling solutions in the presence of surfactants to synthesize nanoparticles. Solvothermal reactions are carried out using autoclaves or high pressure reactors where the pressure can be over 2000 psi at temperatures above 200°C. Water or other solvents act as a reactant at these supercritical conditions, arising

23 greater mobility of the ionic species and accelerating the kinetics of the thermolytic reactions to fabricate various thermodynamically metastable phases.

Microemulsion or micelle methods utilize the formation of spherical micelles or reverse micelles of surfactant molecules in a solvent. The micelles serve as nano-reactors to allow the nucleation and growth of nanoparticles inside the cavity. One of the oldest techniques for the synthesis of nanoparticles, metal salt co-precipitation, uses precipitating reagents to react with a prepared solution of single or multiple precursor metal salts in the presence of surfactant

7 ligands .

Rationale for Selection of Current Synthetic Method

Recently we have shown that gold-coated iron oxide nanoparticles either taken up by macrophages or targeted to tumor cells, can be inductively heated with the attendant death of

8,9 labeled cells . For these studies a variation of the Massart method of co-precipitation of iron salts was used for the synthesis of the experimental iron oxide nanoparticles. These particles were then employed using targeting strategies designed for the different specific model systems of nanohyperthermal therapy. The most intriguing findings from these studies emphasized the importance of establishing a linear, consistently reproducible synthetic method for iron oxide nanoparticles that could be reliably inductively heated.

While the effect of various synthetic parameters have been reported in the literature, virtually all results to date relate to organic rather than an aqueous synthetic methods10 which has confounded attempts to extrapolate findings to current experimental design. Furthermore there is a bounty of conflicting experimental results due to the effects of synthetic variables on the superparamagnetic properties of iron oxide nanoparticles.

In addition to the effects of synthetic variables on the physical characteristics of the iron oxide nanoparticles, the work in this dissertation also attempts to explore the effects of various conditions that influence the inductive heating properties of iron oxide nanoparticles. As discussed in Chapter 1, the magnetic losses, the amount of magnetic field energy converted into heat during magnetization reversal, of single domain iron oxide nanoparticles are produced by

Neel relaxation, Brownian relaxation, and frictional losses in viscous suspensions (Eddy currents.) When examining iron oxide nanoparticles, Hergt, R. et al. provided substantial support for a popular hypothesis in the field11. As particle size decreases, domain walls become energetically unfavorable, the particle transitions from a multi to a single domain particle. The barriers for magnetization reversal then come into the order of magnitude of thermal energy and superparamagnetic effects dominate. A mean diameter that corresponds to the maximum coercivity and thus inductive heating properties in the single domain size range has been reported variously for 10nm11 and 12.5 nm12 iron oxide nanoparticles.

The purpose of this research is to evaluate the effect on superparamagnetism of several reaction components of an established method for the synthesis of SPIONs. For that reason it is crucial to streamline the nanoparticle synthesis and characterization in one laboratory where their inductive heating capabilities and physical structure will be studied in order to produce highly controlled and reproducible nanoparticle preparations and experimental results. The parameters that are under consideration to examine their effect on the superparamagnetic properties of the iron oxide nanoparticles for this work are the presence of oxygen in the synthetic solution and reaction temperature.

Experimental Methods

Chemicals

For particle synthesis, ferrous chloride (FeCl2) 98% reagent grade, ferric chloride (FeCl3)

97% reagent grade, ammonium hydroxide (NH4OH), citric acid monohydrate (C6H8O7*H2O) and sodium citrate (Na3C6H5O7) were purchased from Sigma-Aldrich (St. Louis, MO).

Tetramethylammonium hydroxide (CH3)4NOH 99.99% electronic grade was purchased from

Alfa Aesar (Ward Hill, MA.) All water used is brought to 17.4-17.6 MΩ-cm resistivity using a

NANOpure ultrapure water system with an organic-free filter cartridge.

Synthesis of Iron Oxide Nanoparticles

Magnetite nanoparticles were synthesized using a 2 to 1 ratio of Fe 3+ and Fe 2+ salts,

13 respectively . 1.39g FeCl2 * 4 H 2O and 0.86g FeCl3 * 6 H20 were dissolved in 40 mL of water.

To that, 5ml of 8 M NH4OH was added at room temperature, 60-70°C, or 90°C either under

100% or 20% oxygen or 100% nitrogen atmosphere while constantly stirring with a non- magnetic stir rod (Figure 1.) The precise synthetic conditions permitted examination of the effect of each reaction temperature and the presence of absence of oxygen. The pH was kept at 10 for the entirety of the synthesis. To remove salts, the precipitate was washed with distilled H20. The average particle size was obtained from measurement of 256 particles per batch. Ten µL of cFe was placed on nickel grids with formvar films and air dried for 10 minutes.

Mass Determination

Oscillating Magnetic Field Generator

An alternating magnetic field was used to heat magnetic particles (cFe). A magnetic field generator with frequencies variable from 100-500 kHz and a magnetic field strength of 0.008 to

0.01 Tesla was constructed in house. The system consists of an oscilloscope V-200F (Hitachi,

Japan), a signal generator Wavetek 178 (Parsippany, NJ), a power amplifier (1040L power amplifier, Tucker, Garland, TX), capacitors (Sangamo type, Mansfield, TX), a solenoid coil

(wound in house of small diameter copper tubing through which cooling water is passed) and a water cooling system. Suitable fixed and variable capacitors appropriate to the voltage were obtained through the University of Wisconsin High Energy Group at the Department of Physics.

Various combinations of fixed capacitors (capacitance: 1000 pF, 1800 pF, and 4000 pF) were connected to the power amplifier and to these a variable capacitor (capacitance:01000 pF) was attached to obtain the magnetic field strength required. These functioned as resistors affecting the voltage that could be delivered to the solenoid to which they are attached. The magnetic flux in the coil was calculated using Coulomb's Law.

Magnetic strength (Tesla)

A 15 gauge wire was shaped into a square, with an area of 1 cm 2 and was placed in the middle of the solenoid. The magnetic strength of the system can be converted to a real time signal which can be monitored via oscilloscope. A BNC wire was used to connect the reporter wire with the oscilloscope. The magnetic field, in Tesla, can be calculated by

Coulomb’s law (following equation.)

Coulomb’s equation

B = E/ (2 π F A)

Where,

B = magnetic field (Tesla)

E = potential (V)

F = frequency (Hz)

A = area (m2)

Solenoid

The air core solenoid or coil is the source of an oscillating magnetic field (OMF).

Different types of solenoids have been constructed and tested: wires with different diameters (17 gauge, 18 gauge and small copper tubing (hollow, for flowing liquid cooling of inside the winding themselves)), different cores of the solenoid (plexi glass vs. glass), 46 up to 134 wire turns and wires of copper or silver coated copper have been employed to achieve the desired magnetic field strength. Different types of wires and plastic tubing were kindly donated by the

UW Department of Physics.

Thermocouple

The heating capacity of magnetite was measured using a thermocouple. Temperature changes were reflected by changing potentials of the thermocouple. Thermocouples usually consist of wires of two different materials that are connected on the tip forming the sensor. The output is interpreted by a voltmeter. To attain the most accurate readings from small volumes, the thinnest wires were used to reduce dissipation of the heat produced by colloidal iron nanoparticles through the thermocouple wires and activated rosin solder (Kester, Jamestown,

NY.) A type T thermocouple, made of copper (Cu) and constantan (Cu/Ni) with 3/1000 inch, was used. A voltmeter by Omega (Japan) called the Handy-Logger OM-2041 was used to interpret the temperature readings.

Measurement of Heat Production by Colloidal Magnetite Particles

Heat production capacity of the iron oxide nanoparticles was measured using an in-house constructed calorimeter. In this system, the thermocouple wires were connected to wells made of silicone (embedding molds) which were slit at the edge to allow the insertion of the thermocouple wires into the center of the liquid. 200 µL (0.02g/ml) of iron particles were placed in the well. Controls including the same volume of water placed in adjacent wells.

TEM Sample Preparation

10 µL of nanoparticle samples were pipetted from their synthetic solution onto carbon- coated 200-mesh copper grids incubated for ten minutes then wicked dry with a Kim Wipe.

Nanoparticles were imaged using a FEI Tecnai T-12 Cryo TEM at 120 kV for amplitude contrast brightfield imaging. Histograms displaying nanoparticle size dispersity was created by measuring the longest axis of 256 individual nanoparticles per test batch using ImageJ software

(NIH.) The statistical analysis for the heating curves of iron oxide nanoparticles was generated using SAS software - Proc GLM. Copyright, SAS Institute Inc. SAS and all other SAS Institute

Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc.,

Cary, NC, USA.

Results and Discussion

Synthesis and Characterization of Iron Oxide Nanoparticles

Superparamagnetic Fe3O4 NPs can be obtained by a typical chemical co-precipitation of

Fe(II) and Fe(III) in ammonium hydroxide solution according to the following reaction:

FeCl2 + 2FeCl3 + 8NH3 + 4H2O  Fe3O4 + 8NH4Cl + 4H2O

According to the thermodynamics of this reaction, complete precipitation of Fe3O4 should be expected at a pH between 8 and 14, with a stoichiometric ratio of 2:1 (Fe3+/Fe2+) in a non- oxidizing oxygen environment. However, magnetite (Fe3O4) is not particularly stable and is sensitive to oxidation. Magnetite is transformed into maghemite (γFe2O3) in the presence of

14 oxygen .

+ 2+ Fe3O4 + 2H  γFe2O3 + Fe + H2O

Due to their crystalline structures, nanoparticles prepared via this co-precipitation reaction involve two processes, crystal formation (nucleation) and growth. For precipitation to occur, a solution must be saturated, thus the addition of excess solute will cause precipitation, and the formation of nanoparticles. For the initial nucleation to occur, the solution must be supersaturated causing a brief single burst of nucleation. Supersaturation can be reached by dissolving the solute at a high temperature, or by adding surplus solute to produce a supersaturated solution. After the short burst in nucleation, the concentration of reactants

30 declines rapidly and nucleation ceases. The maturation of the nuclei, also known as Ostwald ripening, is achieved by diffusion of solutes from the solution onto the nuclear surfaces, until an

15, 16 equilibrium concentration is reached .

Many factors can be tuned in the synthesis of iron oxide nanoparticles to control their size, magnetic characteristics, or surface characteristics. The importance of Fe II: Fe III ratio of

1:2 has been previously reported in numerous reports but has most recently been examined by

Karaagac et al. This group found that increasing the ratio of iron II:III from 1:2 to 4:1 not only increased the magnetization saturation of the nanoparticles but the relative nanoparticle diameter as well. For the purposes of future biological applications, a 1:2 ratio of iron II:III utilized in

17,18 order to maintained a population of iron oxide nanoparticles under 20nm in diameter .

Numerous reducing agents are referenced in the literature for the synthesis of iron oxide nanoparticles including sodium hydroxide, ammonium hydroxide, and ammonium acetate. While

Park et al reported an increase in crystallinity when ammonium acetate was used as reducing agent, an associated increase in nanoparticle diameter was noted as well28. To date there has been no known work with sodium hydroxide as compared to other reducing agents, combined with the positive preliminary results from the use of either sodium19 or ammonium hydroxide13 lead to the use of ammonium hydroxide as the sole reducing agent.

In an effort to lower the risk of oxidation of iron oxide nanoparticles in the reaction media, many published synthetic methods recommend the use of degassed water in the procedure and the bubbling of argon or nitrogen through the reaction flask to maintain a relatively oxygen- free environment. However there are numerous other articles that successfully produce small iron oxide nanoparticles in air; calling into question the necessity of this precaution.

The effect of reaction temperature of the aqueous co-precipitation method of preparing iron oxide nanoparticles is debatable. Several reports describe reaction temperatures of

80-90°C20, 21, while others keep the reaction at room temperature22. As with the previous experimental variable involving the oxygenation of the synthetic media, the lack of a decisive conclusion challenged the importance of synthetic temperature during the aqueous co- precipitation of iron oxide nanoparticles.

Synthetic conditions utilized here do not appear to have a significant influence on the structural and physical properties of iron oxide nanoparticles. All colloids examined have a relatively narrow size distribution. TEM micrographs of individual preparations of iron oxide particles are presented in Figures 3, 4, and 5. Iron oxide nanoparticles were synthesized at an average diameter of 6-8 nm. In these micrographs, the apparent flocculation of particles is an artifact of drying during TEM grid sample preparation. It should be noted that when comparing samples synthesized at room temperature and 60°C, that the batches produced in the presence of oxygen had a broader size distribution while the batches produced under nitrogen gas with degassed water contained fewer smaller particles. However with batches of particles produced at

90°C, there was less of a contribution of the small particles to the total population of synthesized particles.

From these studies, no significant correlation between reaction temperature, oxygen presence in synthetic media, and nanoparticle size and shape can be established. These findings are in agreement with Babes et al, who concluded that reaction temperature and the presence of oxygen in reaction media did not substantially contribute to observable morphologic or physical changes in prepared iron oxide nanoparticles23.

Inductive Heating of Iron Oxide Nanoparticles in an Oscillating Magnetic Field

To our knowledge this is the first work that attempts to establish a relationship between synthetic parameters and inductive heating capacity through calorimetric studies. As discussed earlier in this chapter, there are numerous conflicting reports regarding the effect of synthetic parameters on the crystallinity, and consequently the ability of the iron oxide nanoparticles to be inductively heated. Therefore this work hoped to establish a causal relationship between variables that would increase crystallinity (increase size but maintain a single domain limit.) As no reports to date have examined the exact variables of reaction temperature and presence of oxygen in reaction solution, a simple experimental approach that utilized one reducing agent, ammonium hydroxide, at a constant concentration, and a constant Fe II: Fe III ratio of 1:2 was used.

As the magnetization of iron oxide nanoparticles is highly size-dependent, and the TEM images of the prepared particles exhibited a small size variation between test batches. The mean particle diameter ranged from 5 nm for the particles synthesized at room temperature in the presence of oxygen, to 8nm, for the particles synthesized at 90°C with little oxygen (<20%) present in the reaction. A general trend can be observed that particles synthesized at higher temperatures, without oxygen present are about 1 nm larger in size. As shown in Figure 5, the iron oxide nanoparticles that were synthesized at room temperature and 90°C appeared to heat to higher temperatures than the particles that were synthesized at 60°C. However no statistical significance was found. While, currently, there is no complete explanation for this small increase in the inductive heating properties of the nanoparticles, the slight increase in monodispersity may have narrowed the size distribution to allow for enough of an increase in the number of particles

33 that reside closer to the optimal heating range of 10-12.5 nm (previously mentioned). This could account for the overall increase in particle sample heating.

Figure 6 demonstrates that iron oxide nanoparticles synthesized with or without oxygen in their reaction media showed no significant difference in heating as all batches heated to similar temperatures. While the presence of oxygen may pose an issue for iron oxide nanoparticles following synthesis, during storage, the absence of oxygen in the synthetic reaction does not appear to improve the size distribution or the inductive heating capacities of the iron oxide nanoparticles.

The concern over the oxidation of iron oxide nanoparticles over time may account in part for the interesting observations of the heating trends of particles we have observed over time. In

Figure 7, a phenomenon that has been observed in at least twenty batches of iron oxide nanoparticles is shown. Over time the inductive heating of the iron oxide nanoparticles is seen to rise initially following synthesis and then to drop significantly and remain low subsequently.

While further studies are needed to pinpoint the mechanism(s) responsible for these observations, it can be postulated that as the iron oxide nanoparticles are stored under oxygen, the formation of an oxide layer could account for this decline. Masoudi et al put forth an explanation that the disordered domains of the oxide layer ‘frustrate’ the movement of domains of the supposed Fe3O4 core, and thus an increase in thermal energy (stronger magnetic field) is needed to overcome the ‘frozen’ core spin, caused by the exchange coupling of the oxide layer and core domains24. Therefore as this amorphous oxide layer forms, it cannot adequately protect the metallic core, causing the foreseeable decline in magnetization and inductive heating abilities.

However, confounding this trend, prior to the decrease in susceptibility to inductive heating both preparations of particles initially demonstrate an increase in heating between a first and second measurement of their inductive heating capabilities one week apart (data not shown.)

While further trials must be completed to deduce the exact mechanism(s) that explain this behavior, this merely serves to highlight the delicate balancing act between the detrimental effects of oxygen and the additive effects of the possible aggregation. Gupta et al have proposed that iron oxide nanoparticles prepared in an aqueous solution with a similar synthetic protocol aggregate, contributing to an increase in magnetization over time25. Bordelon et al discuss the interparticle interactions that prove challenging in the theoretical and experimental study of colloidal magnetic single domain nanoparticles and their dynamic response to alternating magnetic fields26.

Lastly the effects of nanoparticle concentration on the inductive heating measurements are presented in Figure 8. As the concentration of particles decreases, there is a non-linear decline in their heating temperatures. This trend can be possibly explained by the current experimental design used to measure heating. However, there has been some discussion relative to particle aggregation by Jiang et al. This group highlights the growth of large nanostructures of different sizes allowed by less concentrated solutions. As larger clusters form, they become the epicenter of dipolar attraction and attract smaller particles and clusters, although this does not appear to be the case in the current study. This group suggests that heat conduction (rather than inductive heating) is stronger if particles exist as the individual particles in the ferrofluids. They suggest that as particle size decreases, dipolar interaction between particles becomes less significant; thus individual particles will more likely appear in colloids with a smaller particle size. Therefore it can be deduced that as the average diameter of the nanoparticle population

35 increases, so too does the potential issues with aggregation and related issues dealing with susceptibility to inductive heating. With such controversies muddying the discussion, more work must be done in order to determine the rate of particle aggregation over time and the exact relation of particle aggregation to calorimetry measurements27.

Conclusions

Here we examined the influence of reaction temperature and the presence of oxygen in the reaction media during the co-precipitation synthesis of iron oxide nanoparticles. While the presence of oxygen did not appear to contribute significantly to nanoparticle shape, the preparations of particles synthesized without oxygen in the environment seemed to be more uniform and monodisperse. The inductive heating characteristics of iron oxide nanoparticles were shown not to be dependent on the presence of oxygen, but a trend observing an increase in susceptibility to inductive heating is observed in samples prepared at room temperature and 90°C in comparison to preparation at 60°C. The inductive heating properties of iron oxide nanoparticles rely on the complex relationship of the individual iron oxide crystal properties and the collective structure and state of the nanoparticles as a ferrofluid.

Figures

Figure 1.

Experimental Set-up: components include a 3/8”, 3.1Amp, 3000rpm max, VSR electric drill,

Chicago Electric Power Tools (Harbor Freight Tools), a cell scraper utilized as a non-magnetic stirring rod, a heating mantle, and a gas line used to cover the reaction in inert gas when necessary.

Figure 2a.

TEM image at 150kx and histogram of iron oxide nanoparticles prepared at room temperature in the presence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 5nm.

Figure 2b.

TEM image at 150kx of iron oxide nanoparticles prepared at room temperature in the absence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 6nm.

Figure 3a.

TEM image at 150kx of iron oxide nanoparticles prepared at 60°C in the presence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 7 nm.

Figure 3b.

TEM image at 150kx of iron oxide nanoparticles prepared at 60°C in the absence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 6nm.

Figure 4a.

TEM image at 150kx of iron oxide nanoparticles prepared at 90°C in the presence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 7 nm.

Figure 4b.

TEM image at 150kx of iron oxide nanoparticles prepared at 90°C in the absence of oxygen. The dark particles indicate the layering of several nanoparticles on top of one another. This relatively monodisperse ferrofluid contains particles with an average diameter of 8nm.

Figure 5.

40.00 38.00 36.00

34.00

32.00 30.00 28.00

26.00 Room Temperature temperature(C) 24.00 60-70C 22.00 90C 20.00 0 20 40 60 80 100 120 140 160 180 time(s)

The effect of reaction temperature on the inductive heating capabilities of iron oxide nanoparticles. An alternating magnetic field with a frequency of 500 kHz and a field strength of 0.008 Tesla was applied to 200 µL samples of particles synthesized at room temperature (20°C,) 60-70°C, and 90°C for 3 minutes. Temperature at 180 seconds, P=0.35, not statistically significant.

Figure 6.

45.00

40.00

35.00

30.00 Average Temperature for all

temperature(C) observations with Oxygen 25.00 Average Temperature for all observations without Oxygen 20.00 0 20 40 60 80 100 120 140 160 180 time(s)

The effect of oxygen presence during the synthesis of iron oxide nanoparticles on the inductive heating capabilities of iron oxide nanoparticles. An alternating magnetic field with a frequency of 500 kHz and a field strength of 0.008 Tesla was applied to 200 µL samples of particles synthesized with either ambient air and water containing ~80% oxygen or nitrogen and degassed water containing <20% oxygen. Temperature at 180 seconds, P=0.33, not statistically significant.

Figure 7.

55 Immediately following synthesis 50 Once week after synthesis 45

Two weeks after synthesis

30 temperature(C)

20 0 20 40 60 80 100 120 140 160 180 time(s)

The effect of time on the inductive heating capabilities of iron oxide nanoparticles. An alternating magnetic field with a frequency of 500 kHz and a field strength of 0.008 Tesla was applied to 200 µL samples of particles immediately following synthesis, and at one and two weeks following the initial synthesis. Note the decline in inductive heating temperature over three test weeks.

Figure 8.

55 2:1 concentration 1:1 initial concentration 50 1:2 dilution

45 1:4 dilution

30 temperature(C)

20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 time(s)

The effect of iron oxide nanoparticle concentration on the inductive heating capabilities of iron oxide nanoparticles. An alternating magnetic field with a frequency of 500 kHz and a field strength of 0.008 Tesla was applied to 200 µL samples of particles with an initial concentration of 0.109g/ml. Observe that a reduction in the total concentration of nanoparticles by one half reduces the amount of heat generated by one third (not a linear relationship.) Note that there are individual batches of nanoparticles that are representative of inductive heating trends observed during these studies regarding the effects of time and concentration. As such, additional statistical analysis of the data was not appropriate.

References

1. Lohse, S., & Murphy, C. (2012). Applications of Colloidal Inorganic Nanoparticles: From Medicine to Energy. Journal of the American Chemical Society. 2. Huang, S.-H., & Juang, R.-S. (2011). Biochemical and biomedical applications of multifunctional magnetic nanoparticles: a review. 3. Medeiros, S. F., Santos, a M., Fessi, H., & Elaissari, a. (2011). Stimuli-responsive magnetic particles for biomedical applications. 4. Guisbiers, G., Mejía-Rosales, S., & Leonard Deepak, F. (2012). Nanomaterial Properties: Size and Shape Dependencies. 5. Gubin, S. P., Koksharov, Y. a, Khomutov, G. B., & Yurkov, G. Y. (2005). Magnetic nanoparticles: preparation, structure and properties. 6. Lu, A.-H., Salabas, E. L., & Schüth, F. (2007). Magnetic nanoparticles: synthesis, protection, functionalization, and application. 7. Hilt, J. Z. (2010). Magnetic nanoparticles in biomedicine : synthesis , functionalization and applications R eview, 5, 1401–1414. 8. Krystofiak, E. S., Matson, V. Z., Steeber, D. a., & Oliver, J. a. (2012). Elimination of Tumor Cells Using Folate Receptor Targeting by Antibody-Conjugated, Gold- Coated Magnetite Nanoparticles in a Murine Breast Cancer Model. 9. Sun, Q., Kandalam, a, Wang, Q., Jena, P., Kawazoe, Y., & Marquez, M. (2006). Effect of Au coating on the magnetic and structural properties of Fe nanoclusters for use in biomedical applications: A density-functional theory study. Physical Review B, 73(13), 2–7. 10. Lak, A., Ludwig, F., rabs, I.-M., arnweitner, ., Schilling, M., H feli, ., Sch tt, W., et al. (2010). Influence of Synthesis Parameters on Magnetization and Size of Iron Oxide Nanoparticles, 224(2010), 224–230. 11. Hergt, R., Dutz, S., & Röder, M. (2008). Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia. Journal of physics. Condensed matter : an Institute of Physics journal, 20(38), 385214. 12. Gonzales-Weimuller, M., Zeisberger, M., & Krishnan, K. M. (2009). Size- dependant heating rates of iron oxide nanoparticles for magnetic fluid hyperthermia. Journal of Magnetism and Magnetic Materials, 321(13), 1947–1950. 13. Sahoo, Y., Goodarzi, A., Swihart, M. T., Ohulchanskyy, T. Y., Kaur, N., Furlani, E. P., & Prasad, P. N. (2005). Aqueous ferrofluid of magnetite nanoparticles: Fluorescence labeling and magnetophoretic control. The journal of physical chemistry. B, 109(9), 3879–85. 14. Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Elst, L. Vander, & Muller, R. N. (2008). Magnetic Iron Oxide Nanoparticles : Synthesis , Stabilization , Vectorization , Physicochemical Characterizations , and Biological Applications, 2064–2110. 15. Lodhia, J., Mandarano, G., Ferris, N., Eu, P., & Cowell, S. (2010). Development and use of iron oxide nanoparticles (Part 1): Synthesis of iron oxide nanoparticles for MRI. Biomedical imaging and intervention journal, 6(2), e12. 16. Vervey EJW, Overbeek JTG. 1948. Theory of stability of lyophobic colloids. Amsterdam: Elsevier.

17. Karaagac, O., & Kockar, H. (2012). Iron Oxide Nanoparticles Co-Precipitated in Air Environment: Effect of [Fe ]/[Fe ] Ratio, 48(4), 1532–1536. 18. Mahmoudi, M., Azadmanesh, K., Shokrgozar, M. A., Journeay, W. S., & Laurent, S. (2011). Effect of Nanoparticles on the Cell Life Cycle, 3407–3432. 19. Massart, R. (1981). Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Transactions on Magnetics, M(2), 1980–1981. 20. Koneracka, M., Kopc, P., Antalmh, M., Timko, M., Ramchand, C. N., Lobo, D., Mehta, R. V, et al. (1999). Immobilization of proteins and enzymes to fine magnetic particles. Journal of Magnetism and Magnetic Materials, 201(1-3), 427–430.) 21. Zhu, Y., & Wu, Q. (1999). Synthesis of magnetite nanoparticles by precipitation with forced mixing. Journal of Nanoparticle Research, 1(3), 393–396. 22. Hien Pham, T. T., Cao, C., & Sim, S. J. (2008). Application of citrate-stabilized gold-coated ferric oxide composite nanoparticles for biological separations. Journal of Magnetism and Magnetic Materials, 320(15), 2049–2055. 23. Babes, L., Jacques, J., Jeune, L., & Jallet, P. (1999). Synthesis of Iron Oxide Nanoparticles sed as MRI Contrast Agents : A Parametric Study, 482, 474–482. 24. Masoudi, A., Hosseini, H. R. M., Reyhani, S. M. S., Shokrgozar, M. A., Oghabian, M. A., & Ahmadi, R. (2012). Long-term investigation on the phase stability, magnetic behavior, toxicity, and MRI characteristics of superparamagnetic Fe/Fe- oxide core/shell nanoparticles. International journal of pharmaceutics, 439(1-2), 28–40. 25. Gupta, M., & Sharma, M. (2011). Aggregation Behavior of Iron Oxide Nanoparticles Measured By Squid Magnetometry. International Journal of Nanoscience, 10(04n05), 647–651. 26. Bordelon, D. E., Corne o, C., r ttner, C., Westphal, F., DeWeese, T. L., & Ivkov, R. (2011). Magnetic nanoparticle heating efficiency reveals magneto-structural differences when characterized with wide ranging and high amplitude alternating magnetic fields. Journal of Applied Physics, 109(12), 124904. 27. Jiang, W., & Wang, L. (2010). Monodisperse magnetite nanofluids: Synthesis, aggregation, and thermal conductivity. Journal of Applied Physics, 108(11), 114311. 28. Park, JY., Oh, S.G., Ha, B.H. (2001). Characterization of iron (III) oxide nanoparticles prepared by using ammonium acetate as precipitating agent. Korean Journal of Chemical Engineering 18 (2), 215-219.

Chapter 3

Functionalization of Iron Oxide Nanoparticles for Biological Application

Introduction

Ferrites are one of the most significant and intriguing classes of magnetic materials. The ferrites discussed in this chapter assume a cubic spinel structure and consist of a presumed heterogeneous combination of formula of Fe2O3 and Fe3O4. Usually, oxygen forms a face- centered cubic close packing structure with Fe2+ and Fe3+ cations occupying either tetrahedral or octahedral interstices. The location of the iron ions in the lattice structure allows for a great versatility in structures, and dictates the physical properties and behavior that has been exhibited by various ferrites. Ferrites have long been used in high frequency-based applications, owing to their high magnetic permeability and electrical resistivity. Most recently, ferrites on the nanoscale have attracted research interests because of their potential biomedical applications, such as drug delivery, bio-magnetic sensing, magnetic resonance imaging, and ferrofluid hyperthermia. Among magnetic nanomaterials, magnetic iron oxide nanoparticles have been one of the first categories to have been investigated for biomedical applications due to their economic

1 feasibility, biocompatibility and biodegradability .

As discussed in chapter 2, ferrofluids can be commonly synthesized in aqueous phase by basic co-precipitation of iron salts2. However, for the nanoparticles to be compatible with biological systems and stable in biologically relevant conditions, they must be water-soluble and stable at pHs ranging 5-9, under salt concentrations up to a few hundred millimoles per liter, and at body temperature. To meet these physiological criteria, it is useful if the hydrophobic particles can be modified to be hydrophilic to facilitate further functionalization.

Nanoparticles have very high surface area to volume ratio because surface area is a function of the radius squared where as volume is a function of the radius cubed. For a typical

5nm nanoparticle, half or more of the total atoms will be located on the surface of the particle; compared to bulk materials, where only a relatively small fraction of atoms will be on the surface. The calculated surface to bulk ratios of atoms versus size of a spherical NP is shown

Figure 1, where around 50% of the atoms or ions are on the surface for 5 nm diameter particle3.

These atoms provide more reaction sites that influence the magnetic and physical behaviors of the nanoparticles and provide a framework that can be engineered to achieve the required biocompatible properties for potential biomedical applications. Many of these applications call for pre-clinical experiments using in vitro model systems. Pavlin et al discuss numerous studies confirming that nanoparticle stability in a particular biological media is a dynamic and delicate balancing act involving surface properties, media compositions, and ferrofluid concentrations4.

The purpose of this research is to examine several candidate molecules for the surface functionalization of the iron oxide nanoparticles, the synthesis and characterization of which is described in Chapter 2, in physiologically relevant media. Many strategies of nanoparticle surface modification are reported in the literature. Polymeric coatings, including polyethylene glycol, polyvinyl alcohol, polyacrylic acid, and dextran, involving the precipitation of inorganic particles in a cross-linked polymer matrix often prevents coagulation of particles. Organosilane groups and gold have also been used to form well-dispersed magnetic nanospheres, proving advantageous since established surface chemistry for the biomodification of these shells exists.

Magnetic iron oxide nanoparticles can also be stabilized with various carboxylates (tetramethyl ammonium hydroxide, citric acid, or sodium citrate) by the surface of the carboxylate groups,

51 leaving at least one of the functionalities exposed to the solvent, allowing for a negative surface charge and hydrophilicity5.

However there is conflicting evidence in the literature that reports either an increase or decrease of the saturation magnetization of polymer or silica coated nanoparticles, most simply explained as the ability of the nanoparticles to be inductively heated in an oscillating magnetic field, when compared to uncoated particles. While there is little discussion of a mechanism for an increase in magnetization, the decline in magnetization is thought most likely due to a non-co- linear spin structure originated from the immobilization of the nanoparticle surface spins by the coating surfactant6- 8.

As the research presented in this dissertation utilizes the magnetization and inductive heating properties of the iron oxide nanoparticles, this study focuses on the modification of particles with either gold or carboxylates.

Rationale for Selection of Current Functionalization Method.

For this work, surface functionalization of iron oxide nanoparticles was completed using different carboxylates as primary surface modifiers. Various publications have reported the coating of iron oxide nanoparticle with gold, but as with the synthesis of the magnetite NPs, these methods are highly irreproducible9-12. Results range from no coating, crystalline deposition of gold on iron, parallel production of unique gold particles, encapsulation of iron NPs in large gold nanostructures, and formation indeterminate of iron-gold nanocomplexes. Previously work in our laboratory achieved a relatively well characterized uniform gold coating. However, to date, it has proved difficult to reproduce even using the same methodology13.

An iron-gold core shell structure provides the advantage of a magnetic core and biocompatible shell that could not only protect the iron oxide core against the detrimental effects of oxidation, but can be readily conjugated to antibodies for targeted therapies. While there are reports of the synthesis of these core-shell complexes (Figure 2,) few studies provide the structural analysis necessary to confirm the existence of a true iron oxide core and a continuous gold shell in one nanoparticle. Krystofiak et al postulate that the difficulties in synthesizing true

Au-coated iron oxide nanoparticles lie not in any flaws of the published methodologies, but in a lack of complete mechanism of the growth of metals on oxide surfaces as the hydrophobic surface of iron oxide cores is not immediately conducive to the formation of a continuous crystalline gold shell14.

Following generally unsuccessful efforts to reproduce a continuous gold coating using the methodology reported by Kandela et al13 and Krystofiak et al11 a carboxylate surface modification strategy was selected for the current studies reported here. The purpose of this chapter is to evaluate several surface modification methods using various carboxylates or gold to develop a consistently reproducible method of stabilizing superparamagnetic iron oxide nanoparticles. These characterized nanoparticles would then be utilized in an effective model system for the study of targeted, inductively heated nanoparticles for further biomedical applications. See Chapter 4. The surface coatings that are under consideration for this work are gold, tetramethyl ammonium hydroxide, citric acid, and sodium citrate.

Experimental Methods

Chemicals

All reagents were of ACS grade or higher. For the synthesis ferrous chloride (FeCl 2)

98% reagent grade, ferric chloride (FeCl 3) 97% reagent grade, ammonium hydroxide (NH

4OH), citric acid monohydrate (C6H8O7 H2O), sodium citrate (Na3C6H5O7), gold chloride

(HAuCl4 3H2O), and hydrazine hydrate were purchased from Sigma-Aldrich (St. Louis, MO).

Tetramethylammonium hydroxide (CH3)4NOH 99.99% electronic grade was purchased from

Alfa Aesar (Ward Hill, MA.) All water used is brought to 17.4-17.6 Moms-cm resistivity using a

Barnstead Thermoline ultrapure water system with an organic-free filter cartridge.

Cell culture

A Mus musculus macrophage cell lines (RAW 264.7) kindly donated by Prof. Mark Cook and Prof. Charles Czuprynksi were cultured and incubated at 37º C in a 5 % CO2 atmosphere.

The cell line was cultured in Dulbecco’s modified eagle medium supplemented with 10 % fetal bovine serum. Fresh culture medium was added every 2 to 3 days. For an experiment, the macrophages were allowed to attach overnight to 35mm MatTek glass-bottom dishes (Ashland,

MA.)

TEM sample prep

10ul of nanoparticle samples were pipetted from their synthetic solution onto carbon- coated 200-mesh copper grids incubated for ten minutes then wicked dry with a Kim Wipe.

Nanoparticles were imaged using a FEI Tecnai T-12 Cryo TEM at 120 kV for amplitude contrast brightfield imaging. An FEI Titan aberration corrected STEM operating at 200kV was used for energy dispersive X-ray spectroscopy (EDS.)

Stabilization of Iron Oxide Nanoparticles with Tetramethyl Ammonium Hydroxide

The ferrofluid utilized for the production of tetramethyl ammonium hydroxide

(TMAOH,) modified iron oxide nanoparticles; citric acid modified iron oxide nanoparticles, and sodium citrate modified iron oxide nanoparticles, was obtained from the stock solution described above and previously in Chapter 2. After washing three times in distilled water, nanoparticles were suspending in 0.1M TMAOH to a concentration of 0.02g/ml and vortexed for one minute.

Stabilization of Iron Oxide Nanoparticles with Citric Acid

After washing the previously described ferrofluid twice in distilled water, the particles were resuspended to a concentration of 0.02g/ml in a 22mM citric acid solution. The solution was heated with continued stirring via a magnetic stir bar for 30 minutes at 80°C.

Stabilization of Iron Oxide Nanoparticles with Sodium Citrate

After washing the previously described ferrofluid twice in distilled water, the particles were resuspended to a concentration of 0.02g/ml in distilled water. Then, a 1.5M solution of sodium citrate was added in a ratio of 62.5 µL per 5ml ferrofluid. The solution was heated with continued stirring using a plastic (non-magnetic) stir bar for 30 minutes at 80°C.

Stabilization of Iron Oxide Nanoparticles with Gold

Two methods were utilized to coat iron oxide nanoparticles with gold. The first is a variation on the iterative seeding method proposed first by Lyon et al.11 and the second is a method reported by Zhou et al15. According to the first protocol, the ferrofluid was diluted 1:100 in 0.00275% hydrazine hydrate and the slow, iterative addition of a total volume of 120ul of 4%

HAuCl4, divided into four, 30 µL iterations, followed with rigorous stirring. The color of the synthetic solution ranged from blue, to violet and burgundy.

In the second method, iron oxide nanoparticles were prepared in the same method as described above. Rather than washing and proceeding as above sodium citrate was first added, the temperature raised to 90°C, and the reaction was completed with continuous stirring for 30 min. A 20 mL of 0.5 mM HAuCl4 solution was heated vigorously until it boiled. This was added rapidly to volumes, 10 mL or 20 mL of the ferrofluid solution prepared previously at 0.02g/ml.

The ratios of reactive volumes between nanoparticles and the HAuCl4 solution were 1:2 and 1:1.

Stirring continued for 10 min and the heating source was removed and stirring continued until the solution cooled to room temperature.

Mass Determination

To calculate the concentration of bulk nanoparticles in each synthetic batch, clean filter paper was dried in an oven at 80°C and weighed. Then 200-1,000 µL of ferrofluid was added and dried again at 80°C for thirty minutes and weighed again. No color change was observed in the dried samples.

Measurement of heat production by colloidal magnetite particles

Heat production capacity of the iron oxide nanoparticles was measured using an in-house calorimetry apparatus. In this system, 3 mil (0.003 inches thick) copper constantan thermocouple wires were inserted into wells made of silicone (embedding molds) which were slit at the edge to allow the insertion of the thermocouple wires into the center of the liquid. 200 µL (0.02g/ml) of iron particles were placed in the well. Controls including the same volume of water placed in adjacent wells.

Results and Discussion

Stabilization of Iron Oxide Nanoparticles

Iron oxide nanoparticles were prepared via an aqueous co-precipitation method discussed in detail in Chapter 2. Briefly a base was injected into a solution consisting of two iron salts, Fe2+ and Fe3+ dissolved in water in a 1:2 ratio that yields a mixture of magnetite and maghemite nanoparticles with an average size distribution of 5-8 nm. Figures 3a and 4c depict these iron oxide cores used in the gold-coating experiments. Once washed three times in water, these nanoparticles remained a stable ferrofluid for at least several days, existing as a heterogenous mixture of individual particles and small clusters of nanoparticles. Following a variation of

Lyon’s iterative seeding method, gold-coated iron oxide nanoparticles were prepared. For batches to continue to be characterized, it was deemed necessary that their color was in the red to purple range owing to the color of similarly sized colloidal gold particles and the color of previously synthesized gold-coated iron oxide nanoparticles and that did not precipitate out of solution13. This method yielded a mixture of uncoated iron oxide nanoparticles with in situ synthesized gold nanoparticles that were spherical (Figures 3b and 4b,) or amorphous

57 agglomerations (Figure 4a.) In the TEM images, the darker nanoparticles or ‘nanostructructures’ contain gold while the much lighter nanoparticles contain iron. The composition of the individual nanoparticles was further investigated using simultaneous EDS and spectrum imaging in Figure

5. The image shows a 20nm particle that proves to be gold, in the lower EDX spectrum. There is a small indication of Fe signals in the vicinity of the gold nanoparticle but it cannot be conclusively linked to an iron core-gold shell structure. Figure 6 shows another sample from the core-shell batches that appear to be a cluster of individual iron oxide nanoparticles

(approximately 2-5nm) that shows no gold signals. These images are representative of the batches of nanoparticles examined; none exhibited any variation of the core-shell nanoparticle that was intended to be synthesized using this approach.

In a second method of gold-coating reported by Zhou, H. et al, the as-synthesized iron oxide nanoparticles were left in the reaction flask and primed with a small amount of sodium citrate and heated for half an hour. These as-synthesized particles were then added to a solution of gold chloride15. The nanoparticle solution turned from black to dark brown following the addition of sodium and citrate and heat to oxidize the metal surface in an effort to make the iron oxide surface more amenable to gold-coating. However the characteristic color change to a red or purple solution, as mentioned above, was not observed and the solution remained a dark brown following the addition of the iron oxide nanoparticles into the yellow gold chloride solution. Figure 7 represents two samples from these preparations that show the coexistence of individual uncoated iron oxide nanoparticles with larger, amorphous gold ‘nanostructures.’ As results similar to these had been obtained in previous experiments, these samples were not investigated further.

Small Organic Molecule Functionalization: TMAOH, Citric Acid, and Sodium Citrate

Tetramethyl ammonium hydroxide (TMAOH) offers a free negatively charged hydroxyl group (Figure 8a) after dissociation the ammonium salt in water. Nanoparticles absorb the negatively charged ion on the hydroxyl groups and create an electrostatic repulsion between nanoparticles; diminishing interparticle interactions16. For Carboxylate functionalization, an ionic bond is created when two of the carboxylate functionalities (Figure 8b and 8c) absorb onto the iron oxide particle surface. Figures 9a, 10a, and 11a depict TEM micrographs of iron oxide core nanoparticle thinly coated with TMAOH, citric acid, and sodium citrate respectively where no difference in regards to particle aggregation is apparent. However Figures 9b, 10b, and 11b, show confocal light microscope images of the functionalized particles in cell culture. While the citric acid and sodium citrate particles appear quite similar in regards to flocculation and precipitation in the cell culture media, TMAOH-coated particles seem to aggregate in physiologically relevant conditions to a greater degree than the carboxylate-functionalized nanoparticles. These results are in agreement with reports that discuss the increased stability of particles capped with surfactants that like citric acid or sodium citrate that possess more than one hydroxyl or carboxyl functionalities. Depending upon steric necessity and the curvature of the particle surface, at least one carboxylic acid group (if not two) is exposed to the solvent, which should be responsible for making the surface negatively charged and increasingly hydrophilic5,17.

Effect of Surface Modifying Molecules on the Inductive Heating Abilities of Iron Oxide

As shown in Figure 12, the inductive heating capabilities of the TMAOH, citric acid, or sodium citrate capped iron oxide nanoparticles do not show a significant difference.

Conclusions

Attempts to stabilize the iron oxide nanoparticles with a continuous gold-shell to form a protective layer against oxidation of the core and a surface amenable to bioconjugation, were unsuccessful with existing reagents and methodology. These efforts resulted in separate particle populations of uncoated iron oxide nanoparticles in suspension with larger gold nanocomplexes

(spherical or cuboidal.) However this study revealed the efficiency of functionalization of iron oxide nanoparticles with organic molecule ligands that contain greater than one carboxyl functionality, specifically citric acid and sodium citrate and their inductive heating characteristics are unaffected by these functionalities. Compounds binding with only alcohol groups were more prone to desorption from the nanoparticle surface; leading to instability in cell culture media

(biologically relevant pH and electrolyte concentrations.)

Figures

Figure 1. Calculated surface to bulk ratios for solid metal particles versus size reproduced from Klabunde, K. et al. 199618.

Figure 2. Adapted from Leung, K. et al. 2012. Core Shell Schematic19.

Figure 3a.

TEM at 320 kx of iron oxide nanoparticle cores (approximately 6-8 nm) used in the gold- coating method in 3b. The observed layering of particles on top of one another is an artifact of drying during the sample preparation. The scale bar is 2 um in length.

Figure 3b.

TEM at 320 kx of results of Lyon’s iterative gold-coating method using iron oxide nanoparticle cores (3a.) The dark particle is an individual gold nanoparticle that is approximately 5 nm while the lighter particles are individual iron nanoparticles. The scale bar is 5 um in length.

Figure 4.

TEM of Lyon’s iterative gold-coating method with iron oxide cores a)amorphous gold

‘nanocomplexes,’ b) in situ synthesized gold nanoparticles in the foreground (dark) and uncoated iron oxide nanoparticles (light) in the background (scale bar is 5 nm in length,) and c) TEM of iron oxide cores used in the gold-coating method in 4a and b (scale bar is 2 nm in length.) a. b.

Figure 5.

EDS of iron oxide core-gold shell coating efforts. The particle observed is a a large gold nanoparticle that is approximately 140 nm in diameter. The grey area in the center of the upper half of the particle can be speculated to be iron, but the EDS data can only confirm that there is iron in the vacinity of the large gold particle in question. Perhaps a small amount of iron was encapsulated as the large gold particle nucleated during synthesis but no specific gold-coating of iron oxide nanoparticles can be definitevely reported.

Figure 6.

EDS of iron oxide core-gold shell coating efforts. The structure observed is a cluster of individual iron oxide nanoparticles (8-10 nm) that is approximately 100nm by150 nm. The

EDS data confirms that there is only iron in the vacinity of the cluster in question.

Figure 7.

TEM of Zhou’s gold-coating method with in situ synthesized gold nanoparticles in the foreground (dark) and uncoated iron oxide nanoparticles (light) in the background approximately 5-6 nm in diamter.

a. TEM at 67 kx.

b. TEM at 150 kx.

Figure 8.

Chemical Structures of the Candidate Molecules for the Functionalization of Iron Oxide

Nanoparticles.

a. Tetramethyl ammonium hydroxide (TMAOH)

b. Citric Acid

c. Sodium Citrate

Figure 9.

Transmission electron micrograph of iron oxide nanoparticles approximately 5-6 nm in diameter coated with TMAOH. The dark particles observed are several individual nanoparticles layered on top of one another as an artifact of drying during specimen preparation.

Confocal micrograph of a bright field view at 10X of raw macrophages incubated with 200 µL/ml of TMAOH stabilized nanoparticles (above.) Note the flocculation of individual nanoparticles into large clusters in the cell culture media indicating instability in biologically relevant solutions.

Figure 10.

Transmission electron micrograph of iron oxide nanoparticles approximately 5-6 nm in diameter coated with citric acid. The dark particles observed are several individual nanoparticles layered on top of one another as an artifact of drying during specimen preparation.

Confocal micrograph of a bright field view at 10X of raw macrophages incubated with 200 µL/ml of citric acid stabilized nanoparticles (above.) Observe the lack of aggregated particles in the cell culture media, indicating particle stability in biological media.

Figure 11.

Transmission electron micrograph of iron oxide nanoparticles approximately 5-6 nm in diameter coated with sodium citrate. The dark particles observed are several individual nanoparticles layered on top of one another as an artifact of drying during specimen preparation.

Confocal micrograph of a bright field view at 10X of raw macrophages incubated with 200 µL/ml of sodium citrate stabilized nanoparticles (above.) Observe the lack of aggregated particles in the cell culture media, indicating particle stability in biological media.

Figure 12.

temperature(C) Citric Acid TMAOH 22 Sodium Citrate 20 0 20 40 60 80 100 120 140 160 180 time(s)

The effect of iron oxide nanoparticle functionalization on the inductive heating capabilities of iron oxide nanoparticles. An alternating magnetic field with a frequency of 500 kHz and a field strength of 0.008 Tesla was applied to 200 µL samples of particles with an initial concentration of 0.109g/ml stabilized with citric acid, tetramethyl ammonium hydroxide

(TMAOH,) or sodium citrate. Note that there are individual batches of nanoparticles that are representative of inductive heating trends observed during these studies regarding the effects of time and surface coating. As such, additional statistical analysis of the data was not appropriate. P=0.08 at time 180 seconds, not statistically significant.

References

1. Gupta, A. K., & Gupta, M. (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials, 26(18), 3995–4021. 2. Sahoo, Y., Goodarzi, A., Swihart, M. T., Ohulchanskyy, T. Y., Kaur, N., Furlani, E. P., & Prasad, P. N. (2005). Aqueous ferrofluid of magnetite nanoparticles: Fluorescence labeling and magnetophoretic control. The Journal of Physical Chemistry B, 109(9), 3879–3885. 3. Grassian, V. H. (2008). When Size Really Matters: Size-Dependent Properties and Surface Chemistry of Metal and Metal Oxide Nanoparticles in Gas and Liquid Phase Environments. Journal of Physical Chemistry C, 18303–18313. 4. Pavlin, M., & Bregar, V. B. (2012). Stability of Nanoparticle suspensions in different biologically relevant media , 7(4), 1389–1400. 5. Laurent, S., Forge, D., Port, M., Roch, A., Robic, C., Elst, L. Vander, & Muller, R. N. (2008). Magnetic Iron Oxide Nanoparticles : Synthesis , Stabilization , Vectorization , Physicochemical Characterizations , and Biological Applications, 2064–2110. 6. Chen, W., Bardhan, R., Bartels, M., Perez-Torres, C., Pautler, R. G., Halas, N. J., & Joshi, A. (2010). A molecularly targeted theranostic probe for ovarian cancer. Molecular cancer therapeutics, 9(4), 1028–38. 7. Mahmoudi, M., Sant, S., Wang, B., Laurent, S., & Sen, T. (2011). Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Advanced drug delivery reviews, 63(1-2), 24–46. 8. Kim, E., Lee, K., Huh, Y.-M., & Haam, S. (2013). Magnetic nanocomplexes and the physiological challenges associated with their use for cancer imaging and therapy. Journal of Materials Chemistry B, 1(6), 729. 9. Hien Pham, T. T., Cao, C., & Sim, S. J. (2008). Application of citrate-stabilized gold- coated ferric oxide composite nanoparticles for biological separations. Journal of Magnetism and Magnetic Materials, 320(15), 2049–2055. 10. Banerjee, S., Raja, S. O., Sardar, M., Gayathri, N., Ghosh, B., & Dasgupta, a. (2011). Iron oxide nanoparticles coated with gold: Enhanced magnetic moment due to interfacial effects. Journal of Applied Physics, 109(12), 123902. 11. Krystofiak, E. S., Matson, V. Z., Steeber, D. a., & Oliver, J. a. (2012). Elimination of Tumor Cells Using Folate Receptor Targeting by Antibody-Conjugated, Gold-Coated Magnetite Nanoparticles in a Murine Breast Cancer Model. Journal of Nanomaterials, 2012, 1–9. 12. Xu, Z., Sun, H., Gao, F., Hou, L., & Li, N. (2012). Synthesis and magnetic property of T4 virus-supported gold-coated iron ternary nanocomposite. Journal of Nanoparticle Research, 14(12), 1267 13. Kandela, I. (2006). Development of metal nanoparticle immunoconjugates for correlative labeling in light and electron microscopy and as active targeted delivery systems. Dissertation for Doctor of Philosophy, Pharmaceutical Sciences at the University of Wisconsin.

14. Krystofiak, E. (2013). Mulitple Morpologies of Gold-Magnetite Hybrid Nanoparticles are Effectively Functionalized with Protein for Cell Targeting. Microscopy and Microanylsis, MAM-12-262. 15. Zhou, H., Lee, J., Park, T. J., Lee, S. J., Park, J. Y., & Lee, J. (2012). Ultrasensitive

DNA monitoring by Au– Fe3O4 nanocomplex. Sensors and Actuators B: Chemical, 163(1), 224–232. 16. Andrade, Â. L., Fabris, J. D., Ardisson, J. D., Valente, M. a., & Ferreira, J. M. F. (2012). Effect of Tetramethylammonium Hydroxide on Nucleation, Surface Modification and Growth of Magnetic Nanoparticles. Journal of Nanomaterials, 2012, 1–10. 17. Kotsmar, C., Yoon, K. Y., Yu, H., Ryoo, S. Y., Barth, J., Shao, S., Milner, T. E., et al. (2010). Stable Citrate-Coated Iron Oxide Superparamagnetic Nanoclusters at High Salinity, 12435–12443. 18. Klabunde, K. J., Stark, J., Koper, O., Mohs, C., Park, D. G., Decker, S., Jiang, Y., et al. (1996). Nanocrystals as Stoichiometric Reagents with Unique Surface Chemistry, 3654(Figure 1), 12142–12153. 19. Leung, K. C.-F., Xuan, S., Zhu, X., Wang, D., Chak, C.-P., Lee, S.-F., Ho, W. K. W., et al. (2012). Gold and iron oxide hybrid nanocomposite materials. Chemical Society Reviews, 41(5), 1911–28.

Chapter 4

In Vitro Investigation of Iron Oxide Nanoparticles as Intracellular Mediators of Thermally-Induced Necrosis.

Introduction

Among the potential alternatives to classical chemical or surgical methodologies for the treatment of cancer, the use of heat as a therapeutic agent, hyperthermia, is a method that dates to ancient Egypt and Greece. As the field of tumor hyperthermia evolved, the first scientific explorations began with Dr. Fulton Percy in 1914 who observed that it was unnecessary to burn cancer, a rise in the in internal temperature of the diseased tissue was sufficient to preferentially

1 induce tumor regression . Tumors have been shown to have increased susceptibility to elevated temperature compared to healthy tissue due to their increased rate of cell cycling, increased hypoxia, leaky vasculature, and poor lymphatic drainage2, 3. Whole body hyperthermia (42-

45°C), which elevates core temperatures to the level where direct thermal toxicity is observed, can cause severe side effects, which may limit its usefulness while a less extreme treatment used clinically, maintains a fever-level whole body hyperthermia (39C–41°C.) This can mitigate many of the severe side effects, but this lower heat level is presumed to stimulate the immune system and the desired effects of direct thermal cytotoxicity are diminished4. Currently under investigation are various techniques by which heat can be delivered to the target tumor tissue.

Generating localized hyperthermia directly at the cancer site could circumvent many of the side effects associated with whole body hyperthermia while still taking advantage of the thermal susceptibility of tumors. Magnetic nanoparticles in particular are potential heating sources due their superparamagnetic properties that enable them to generate heat when exposed to an oscillating magnetic field5-7.

To increase the target specificity of nanoparticles to cancer cells, two strategies, passive targeting and active targeting, are employed. Nanoparticles are passively targeted to tumor sites through the enhanced permeability and retention effect (EPR). Unlike most healthy cells, tumor cells require extra oxygen and nutrients, causing an increased development of new vessels. The lack of appropriate soluble and tissue adherent signaling species results in the disorganized growth including a leaky vascular bed. Enlarged gap junctions in cancer cells also can compromise blood flow in and lymphatic drainage from the tumor. The ERP effect results in preferential tumor accumulation of nanoparticle-delivered drugs, increased treatment efficacy, and reduced systemic toxicity8. The many variables that determine the successful delivery of chemotherapeutic agents through passive targeting have narrowed the focus of more recent research to the active targeting of magnetic nanoparticles for use in localized hyperthermal therapy.

Active targeting involves the identification of tumor biomarkers, or tumor cell-specific molecules such as antibodies or aptamers that can enhance the specificity and selectivity of the magnetic nanoparticles in circulation. Targeting particles can label unique surface antigens and also may be internalized by tumor cells via receptor-mediated endocytosis/phagocytosis. This results in in elevated particle concentrations in tumor tissue. With targeted particles located in or

9, 10 on tumor cells . The susceptibility of Fe3O4 nanoparticles to inductive heating via an oscillating magnetic field was shown in Chapter 2. Here we investigate the cell cytotoxicity of the inductively heated magnetite nanoparticles. For this determination a model system using a macrophage cell line was employed. Since particles adhere to the macrophages and can be phagocytosed by them, no specific antibody or ligand targeting system was necessary to effect

77 cell-particle attachment. Hence production of ligand or antibody coated particles was not necessary and costs and effort associated with this aspect of the targeting was reduced.

Experimental Methods

Chemicals

For particle synthesis, ferrous chloride (FeCl2) 98% reagent grade, ferric chloride (FeCl3)

97% reagent grade, ammonium hydroxide (NH4OH), citric acid monohydrate (C6H8O7*H2O) and sodium citrate (Na3C6H5O7) were purchased from Sigma-Aldrich (St. Louis, MO).

Tetramethylammonium hydroxide (CH3)4NOH 99.99% electronic grade was purchased from

Alfa Aesar (Ward Hill, MA.) All water used is brought to 17.4-17.6 MΩ-cm resistivity using a

NANOpure ultrapure water system with an organic-free filter cartridge.

Synthesis of Iron Oxide Nanoparticles

Magnetite nanoparticles were synthesized using a 2 to 1 ratio of Fe3+ and Fe2+ salts,

11 respectively . 1.39g FeCl2 * 4 H2O and 0.86g FeCl3 * 6 H20 were dissolved in 40 mL of water.

To that, 5ml of 8 M NH4OH was added at room temperature, 60-70°C, or 90°C either under

100% or 20% oxygen or 100% nitrogen atmosphere while constantly stirring with a non- magnetic stir rod. The precise synthetic conditions permitted examination of the effect of each reaction temperature and the presence of absence of oxygen. The pH was kept at 10 for the entirety of the synthesis. To remove salts, the precipitate was washed with distilled H20. For these studies, particles with comparatively good inductive heating (20°C increase in the calorimeter) were selected for the cell cytotoxicity measurements.

Mass Determination

To calculate the concentration of bulk nanoparticles in each synthetic batch, clean filter paper was dried in an oven at 80°C and weighed. Then 200-1,000 µL of ferrofluid was added and dried again for thirty minutes and weighed again. No color change was observed during this process that would indication oxidation of the iron oxide nanoparticles.

Oscillating Magnetic Field Generator

An alternating magnetic field was used to heat magnetic particles (cFe). A magnetic field generator with frequencies variable from 100-500 kHz and a magnetic field strength of 0.0 to

0.01 Tesla was constructed in house. The system consists of an oscilloscope V-200F (Hitachi,

Japan), a signal generator Wavetek 178 (Parsippany, NJ), a power amplifier (1040L power amplifier, Tucker, Garland, TX), capacitors (Sangamo type, Mansfield, TX), a solenoid coil

To obtain desired alternating magnetic field frequencies of 100-500 kHz, a signal generator is served as source of a sinusoidal waveform covering frequencies from 0.5 Hz to 50 MHz. The bias of a waveform can be adjusted with a DC offset voltage. The output power is usually limited by an internal source resistance of 50 Ω. The frequency and amplitude range can be selected with pushbuttons. Various combinations of fixed capacitors (capacitance: 1000 pF, 1800 pF, and 4000 pF) were connected to the power amplifier and to these a variable capacitor

(capacitance:01000 pF) was attached to adjust the magnetic field strength as required. These functioned as resistors affecting the voltage that could be delivered to the solenoid to which they

79 are attached. These functioned as resistors affecting the voltage that could be delivered to the solenoid to which they are attached.

Solenoid

The air core solenoid or coil is the source of an oscillating magnetic field (OMF).

Different types of solenoids have been constructed and tested: wires with different diameters (17 gauge, 18 gauge and small copper tubing (hollow, for flowing liquid cooling of inside the winding themselves), different cores of the solenoid (PMMA plexi glass vs. glass); various numbers of turns, 46-134 wire turns and wires of copper or silver coated copper have been employed to achieve the desired magnetic field strength. Different types of wires and plastic tubing were kindly donated by the UW Department of Physics.

Thermocouple

The heating capacity of magnetite was measured using a thermocouple. Temperature changes were reflected by changing potentials of the thermocouple. Thermocouples usually consist of wires of two different materials that are connected on the tip forming the sensor. The output is interpreted by a voltmeter. To attain the most accurate readings from small volumes, the thinnest wires were used to reduce dissipation of the heat produced by colloidal iron nanoparticles. Wires were mechanically connected and permanently attached with through the thermocouple wires and activated rosin solder (Kester, Jamestown, NY.) A type T thermocouple, made of copper (Cu) and constantan (Cu/Ni) with 3 mil (0.003 inch,) was used. A voltmeter by

Omega (Japan), Handy-Logger OM-2041, was used to measure temperature.

Measurement of heat production by colloidal iron oxide nanoparticles

Heat production capacity of the iron oxide nanoparticles was measured using an in-house constructed calorimeter. In this system, the 3 mil thermocouple wires were connected to wells made of silicone (embedding molds) which were slit at the edge to allow the insertion of the thermocouple wires into the center of the liquid. 200 µL (0.02g/ml) of the colloidal iron particles were placed in the well. Controls including the same volume of water placed in adjacent wells.

Kinetics Studies of cFe Internalization using SEM

Murine macrophages were allowed to attach overnight to washed glass cover slips.

Coverslips were washed and cFe colloidal nanoparticles were added and incubated with the adherent cell monolayers for 1 hour, a control was incubated without cFe. The cells were then washed with 0.1 M Phosphate Buffered Saline (PBS) and fixed in a mixture of 0.1 % tannic acid and 1.5 % glutaraldehyde in PB for 20 minutes. The samples were then dehydrated using a graded series of ethanol solutions (30%, 50%, 70%, 90%, 100%, and molecular sieved dried

100% twice) for 10 minutes per concentration. Samples were dried by the critical point procedure (Tousimis Samdri-780A, Tousimis, Rockville, MD). The dried cells were then coated with 2.5 nm platinum using a VCR IBS/TM200S Ion-Beam sputter coater (Warrendale, PA).

SEM micrographs were obtained using a Hitachi FE-SEM S-900 operated at 10 KeV accelerating voltage. Both, secondary electron images and backscattered electron images were obtained.

Inductive Heating Experiments

A Mus musculus macrophage cell lines (RAW 264.7) kindly donated by Prof. Mark Cook and Prof. Charles Czuprynksi were cultured and incubated at 37º C in a 5 % CO 2 atmosphere.

Cells were cultured in Dulbecco’s modified eagle medium supplemented with 10 % fetal bovine serum. Fresh culture medium was added every 2 to 3 days. For an experiment, the macrophages were allowed to attach overnight to 35mm MatTek glass-bottom dishes (Ashland, MA.) The cells were incubated with colloidal iron particles (cFe) in media for 1 hour. After incubation, the cells were washed with PBS twice for 2 minutes each time. The cells were exposed to an oscillating magnetic field (400 kHz, 0.008 Tesla) for 15, 30, or 45 minutes. Viability of cells was accessed with a LIVE/DEAD® Viability/Cytotoxicity Assay Kit providing a two-color fluorescence test that determines live and dead cells with two probes that measure recognized parameters of cell viability—intracellular esterase activity and plasma membrane integrity using calcein and ethidium homodimer. This fluorescence-based method of assessing cell viability can be used in place of trypan blue exclusion. Briefly 10µL of ethidium bromide and 2.5µL of calcein were suspended in 10 ml PBS. Cells were incubated in this solution for 20 minutes before washing with PBS twice for 2 minutes prior to viewing. The percentage of cell killing was calculated and compared with controls. Controls cells were incubated with equally sized iron oxide nanoparticles in media in the absence of an oscillating magnetic field or not incubated with cFe and exposed to the oscillating magnetic field for the relevant time. Controls also included cells that were not exposed to the magnetite particles or to the oscillating magnetic field. All experimental and controls were examined by brightfield imaging and stained with a fluorescent live cell/dead cell assay. Also coverslip monolayers were heated via a waterbath for 30 minutes at 46°C to 54°C and stained with the live cell/dead cell assay. This provided a control for the

82 live cell/dead cell assay (see below) and a control for the number of cells comprising the monolayer as the heated cells were strongly adherent to the substrate due to denaturation of surface proteins.

Light Microscopy

The cytotoxicity of iron oxide nanoparticles in RAW macrophages (via Live/Dead fluorescent staining) was observed using an inverted fluorescence microscope, (Zeiss Axiovert

200M), equipped with an Axiocam CCD camera (Zeiss, Oberkochen, Germany). The filters were set at Cy2 (green) and Cy3 (red) excitations. During observation, the cells were kept in PBS.

Results and Discussion

Iron oxide nanoparticles with an average diameter of 6-8 nm, were synthesized in house, methods and characterization detailed in Chapter 2, were used in cytoxocity assays using RAW

267.4 macrophages as a model in vitro system. The cytotoxic effects of the nanoparticles were observed by wide field light microscopy. Briefly cells were incubated with a range of concentrations of iron oxide nanoparticles (100-400mg/ml) for one hour, washed twice in PBS to remove excess nanoparticles, then placed under several test conditions. Following treatment cells were stained with a Live/Dead assay containing green-fluorescent calcein to indicate intracellular esterase activity and red-fluorescent ethidium bromide to indicate loss of plasma membrane integrity. Cells were then imaged using brightfield and fluorescent microscopy. Figure 1a shows macrophages that were not incubated with particles or exposed to an oscillating magnetic field

(OMF.) Cell viability or membrane integrity was not affected when cells were exposed to OMF at a frequency of 500 kHz at 0.008 Tesla for 45 minutes (Figure 1b.) Figure 1b serves as a

83 representative image of the confluency of cells in the 35mm glass-bottom flasks following treatment but before Live/Dead staining and subsequent washing. When comparing Figure 1a to

Figure 1b, a small reduction in the total number of cells can be attributed to loss of non-adherent, probably dead or dying cells, during the washing and handling of the cells during the staining procedure. Shown in Figure 2 is the establishment of a dead cell control in which macrophages were not incubated with iron oxide nanoparticles, but heated on a hot plate for 30 minutes while a media temperature of 46-54°C was maintained to mimic cell death due to hyperthermia. Nearly all cells remain on the glass cover slip (as compared to Figure 1a) due to the method of heating resulting in denaturation and subsequent coagulation of cell surface proteins during this type of bulk heating; adhering dead and dying cells to the glass surface. The hot plate heated cells are in the process of dying and show both live and dead cell imaging indicating the efficacy of the

Live/Dead assay. Macrophages incubated with iron oxide nanoparticles at a low concentration

(100 µg/ml) and treated with OMF exposure are shown in Figure 3 prior to washing so both live and loosely adherent, dead cells are present. Figure 4 shows the results of the Live/Dead staining of this cell population. The substantial reduction in the number of total adherent cells is attributed to loss of non-adherent or poorly adherent dead cells during the Live/Dead staining procedure. Remaining live adherent cells stain green and show little or no uptake of the magnetite. A few dead, red, cells or cell nucleii remain attached to the substrate and some dead cells, yellow (red + green) remain attached to the live, green, cells. An intermediate concentration of iron oxide nanoparticles (200µg/ml) was also tested with exposure to the OMF.

Figure 5 shows a brightfield image of these cells prior to Live/Dead staining to again highlight the reduction of non-adherent cells following the staining procedure. The fluorescent labeling of these cells are shown in Figure 5b where live (green) adherent cells remain attached to the

84 substrate. In efforts to establish a dose-dependent cytotoxic response, macrophages were also incubated with iron oxide nanoparticles at a high concentration (400µg/ml) and treated with

OMF exposure. Figure 6a shows a brightfield image of these cells prior to Live/Dead staining to again highlight the reduction of non-adherent cells following the staining procedure. The fluorescent signaling of these cells are shown in Figure 6b and c where live (green) adherent cells remain attached to the substrate and cell remnants from non-adherent cells stain dead (red.)

To explain the confounding results regarding the green (live) staining of macrophages that had been incubated with iron oxide nanoparticles and exposed to the OMF, when a larger red (dead) population was expected, high magnification (200X) fluorescent images of representative cell groupings were examined. Figure 7a depicts both the live and dead signals of macrophages that were incubated with 400µg/ml of iron oxide and treated in the OMF for 30 minutes, showing that mainly adherent (green) cells remain while most of the dead (red) have washed off leaving a few attached to the remaining adherent live cells. Figure 7b depicts the brightfield image showing larger spread adherent cells with surrounding small, round, dead cells attached to their surface. The larger substrate adherent, live cells are seen not to have taken up substantial amounts of particles and thus were not killed by OMF exposure. Figure 7c (green) shows surviving substrate adherent macrophages, Figure 7d (red) shows round dead cells that adherent to the live macrophages. Figure 7e demonstrates this observation even at lower concentrations (100µg/ml) of iron nanoparticles where the majority of dead cells is not attached to these surviving spreading cells and has been washed from the glass substrate during the

Live/Dead staining process. As shown in Table 1, 91.5% and 97% of macrophages are killed in the 100µg/ml colloidal magnetite and 400µg/ml treatment groups respectively. SEM images, figs 8b and 8c indicated, that for the cell lines used here, most magnetite particles were surface

85 adherent and not internalized. This reduces efficacy of the particles nevertheless, by the 30 min time point, substantial cell cytotoxicity is present.

Conclusions

This study aimed to further examine a novel approach of targeted hyperthermia first established as a set of proof-of concept experiments by Kandela (2006)10 and Kaiser et al

12 (2007) . Inductively heated colloidal iron oxide nanoparticles were investigated as a cytotoxic agent to kill macrophages. As no specific antibody or ligand targeting system was necessary to effect cell-particle attachment, efforts associated with this aspect of the targeting was concentrated on intrinsic qualities of the nanoparticles and basic in vitro cytotoxicity assays.

The amount of heat generated by the iron oxide nanoparticles in an OMF was checked prior in preliminary calorimetry tests before further in vitro experiments were completed. The heating capacity of the particles in an OMF of 500 kHz with a concentration of 0.2g/ml in a volume of 200 µL, increased the temperature of their volume on average 20-30°C within 3 minutes. Non-magnetic gold nanoparticles (are not heated by the oscillating magnetic field) of similar diameter and water controls produced no heating. A one hour incubation period of macrophages with particles was followed by OMF treatment and subsequent light microscopy and scanning electron microscopy studies in this study. Control cell macrophage monolayer cultures with no treatment, macrophage monolayers exposed to the OMF only, macrophages heated with a hot plate mimicking hyperthermia, and macrophages treated with iron oxide nanoparticles but not exposed to the magnetic field were employed to observe the cytotoxicity of the nanoparticles. When exposed to the OMF, the majority of macrophages that had ingested the particles were killed, leaving behind dead cells (with internalized iron particles) sequestered by

86 live, substrate adherent cells that had not engulfed the iron particles and hence avoided heating.

These studies demonstrated that cell death can be induced with 6-8 nm iron oxide nanoparticles inductively heated in an oscillating magnetic field with a frequency of 500 kHz and a field strength of 0.008 T. However further studies are needed to optimize the parameters and specifications of an in vitro model system to avoid the issues encountered currently. One such issue is the reduced phagocytic abilities of the RAW 267.4 macrophages employed for this assay, see Figure 8. While the exact reasons for the behavior of the subset of the macrophage population is not known, it would prove beneficial to use several different macrophage cell lines or primary monocytes to investigate the cytotoxic effects of iron oxide nanoparticles when the ingestion of particles can be optimized. Another issue involves that loss of dead, non-adherent cells following incubation with particles and OMF treatment. Referring to Table 1, the percent of

‘lost’ cells (dead, non-adherent) for cells treated with either 100µg/ml or 400µg/ml of colloidal iron is 84.5% and 90.4% respectively, a majority of the total dead cells (including adherent and non-adherent.) To further quantify this number of dead, non-adherent cells, it would be prudent to analyze the cell culture media following exposure to the OMF. Despite the challenges encountered in this work, we have validated the potential of iron oxide nanoparticles for targeted hyperthermal therapy.

Figures and Tables

Figure 1a.

Wide field light micrographs of RAW 264.7 macrophages labeled with fluorescent calcein and ethidium bromide a LIVE/DEAD® viability kit. This kit stains with green-fluorescent calcein to indicate intracellular esterase activity and red-fluorescent ethidium bromide to indicate loss of plasma membrane integrity. a.

Florescence signals of live macrophages that were not incubated with iron oxide nanoparticles or exposed to the OMF. A minor reduction in the number of total cells (as compared to Figure b is attributed to loss during the washing and handling of the cells during the Live/Dead staining procedure. Image taken at 100X magnification.

Figure 1b. b.

Bright field images of macrophages that were not incubated with iron oxide nanoparticles but were exposed to the OMF for 45 minutes. Image was taken before staining with Live/Dead assay to depict a representative population of cells prior to staining. Image taken at 100X magnification.

Figure 2.

Confocal light micrographs of RAW 264.7 macrophages labeled with fluorescent calcein and ethidium bromide a LIVE/DEAD® viability kit.

Florescence signals of macrophages that were not incubated with iron oxide nanoparticles but were heated on a hot plate for 30 minutes and maintained at a media temperature of 46-54°C. This cell population served as a ‘dead’ control in efforts to mimic cell death due to hyperthermia. The relatively high number of cells remaining on the glass cover slip (as compared to Figure 1a) is attributed to the denaturation and subsequent coagulation of cell proteins during this type of bulk heating. This results in surface adherence of dead and dying cells.

Figure 3.

Bright field images of macrophages incubated with iron oxide nanoparticles at a concentration of 100ug/ml and exposed to the OMF at a frequency of 500 kHz at 0.008T for 30 minutes. Images were taken before staining with Live/Dead assay to depict a representative population of cells prior to this treatment. Black dots indicate aggregates of iron particles in and /or on macrophages. No washing has occurred so both live and dead, non-adherent cells are present. a.

Lower magnification of image (100X.)

Higher magnification of image (200X.)

Figure 4.

Light micrographs of RAW 264.7 macrophages labeled with fluorescent calcein and ethidium bromide a LIVE/DEAD® viability kit. Fluorescence light micrographs of macrophages incubated with iron oxide nanoparticles at a concentration of 100ug/ml and exposed to the OMF at a frequency of 500 kHz at 0.008T for 30 minutes. A reduction in the number of total adherent cells (as compared to Figure 1a is attributed to loss of non- adherent or poorly adherent dead cells during the washing of the cells during the Live/Dead staining procedure. Adherent live cells remain, most of which have not taken up particles. a.

Lower magnification of image (100X.) b.

Higher magnification of image (200X.)

Figure 5.

Bright field images at a lower magnification of 10X of Raw 264.7 macrophages incubated with iron oxide nanoparticles at a concentration of 200ug/ml and exposed to the OMF at a frequency of 500 kHz at 0.008T for 30 minutes. The image in Figure 5a was taken before staining with Live/Dead assay to depict a representative population of cells prior to staining. Image recorded after washing for Live/Dead assay. Non-adherent dead cells, present in Figure 6a, have washed off leaving only live, adherent cells in Figure 5b.

Figure 6a.

Bright field images of macrophages incubated with iron oxide nanoparticles at a concentration of 400ug/ml and exposed to the OMF at a frequency of 500 kHz at 0.008T for 30 minutes. Images, taken before staining with Live/Dead assay, depict a representative population of cells prior to the staining treatment. The reduction in surface adherent cells seen following the Life/Dead staining all labeling (Figure 6b and c) is due to dead, non- adherent cells washing off the substrate during the staining process. Lower magnification of image (100X.)

Figure 6b.

Wide field light micrographs of RAW 264.7 macrophages labeled with fluorescent calcein and ethidium bromide a LIVE/DEAD® viability kit. Fluorescence light micrographs of macrophages incubated with iron oxide nanoparticles at a concentration of 400ug/ml and exposed to the OMF at a frequency of 500 kHz at 0.008T for 30 minutes. Live adherent cells remain attached to the substrate. Dead cells have washed off.

Lower magnification of image (100X.

Figure 6c. c.

Higher magnification of image (200X.)

Figure 7a.

Wide field light micrographs of RAW 264.7 macrophages labeled with fluorescent calcein and ethidium bromide a LIVE/DEAD® viability kit. Figure 7a depicts both the live (green calcein) and dead (red ethidium bromide) florescence signals of macrophages that were incubated with 400ug/ml of iron oxide nanoparticles and treated in the OMF at a frequency of 500kHz at 0.008T for 30 minutes at 200X magnification. Figure 7b is a bright field image, Figure 7c shows the green-florescent calcein image, and Figure 7d shows the red- fluorescent ethidium bromide image; all of which are included (stacked) in Figure 7a. Figure 7e shows a fluorescent image of macrophages incubated with 100ug/ml of iron oxide nanoparticles treated with the same conditions viewed at the same magnification and as the cells in Figure 7a-d.

This fluorescent image shows remaining adherent live (green) cells. The majority of dead (yellow= red+ green) have washed off. While most of the red dead cells have washed off, a few that are attached to the substrate, adherent live cells, remain.

Figure 7b. b.

This brightfield image shows larger spread adherent cells with small round attached dead cells. The larger live spreading cells have not taken up substantial amounts of iron oxide and thus were not killed by the oscillating magnetic field.

Figure 7c. c.

This fluorescent shows surviving, substrate-adherent, spread macrophages.

Figure 7d. d.

This fluorescent image shows round, dead cells that are adherent to the remaining live cells. The majority of dead cells are not attached to live cells and have been washed from the substrate during the Live/Dead staining process.

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Figure 7e. e.

This fluorescent image shows remaining adherent live (green) cells and dead (red) macrophages. The majority of dead (yellow= red+ green) have washed off. While some of the red dead cells have washed off, those that are attached to the substrate, adherent live cells, remain.

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Table 1.

Report of selected results of the cytoxicity assay for various experimental conditions and the corresponding brightfield micrographs. A total of 551 cells were counted to determine the average number of cells in a field of view for the control macrophage, untreated monolayer.

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Figure 8a. a.

Confocal florescent images of a phagocytosis assay using Raw 264.7 macrophages to demonstrate the uptake of green florescent E. coli at 200X magnification.

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Figure 8b. b.

SEM micrograph of a Raw 267.4 macrophage after incubation for 1 hour with iron oxide nanoparticles for one hour, secondary electron signals. Note the clustering of particles on the surface of the cell.

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Figure 8c. c.

SEM micrograph of a Raw 267.4 macrophage after incubation for 1 hour with iron oxide nanoparticles, backscattered electron imaging confirms the identity of particle clusters as iron-containing material.

Note: Secondary imaging demonstrates most clusters are clearly seen on the surface. Backscattered imaging does not demonstrate any internalized particles in the sample. Thus most iron oxide particles are surface adherent but not internalized.

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References

1. Moss, R. (2008). War on Cancer Hyperthermîa Moves to Fore as CAM Treatment of Cancer, (Lu), 2008–2011. 2. Chen, W., Bardhan, R., Bartels, M., Perez-Torres, C., Pautler, R. G., Halas, N. J., & Joshi, A. (2010). A molecularly targeted theranostic probe for ovarian cancer. Molecular Cancer Therapeutics, 9(4), 1028–1038. 3. Doane, T. L., & Burda, C. (2012). The unique role of nanoparticles in nanomedicine: imaging, drug delivery and therapy. Chemical Society Reviews, 41(7), 2885–911. 4. Basel, M. T., Balivada, S., Wang, H., Shrestha, T. B., Seo, G. M., Pyle, M., Abayaweera, G., et al. (2012). Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model. International journal of nanomedicine, 7, 297–306. 5. Gupta, A. K., & Gupta, M. (2005). Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials, 26(18), 3995–4021. 6. Laurent, S., & Mahmoudi, M. (2011). Superparamagnetic iron oxide nanoparticles : promises for diagnosis and treatment of cancer, 2(4), 367–390. 7. Polo-Corrales, L., & Rinaldi, C. (2012). Monitoring iron oxide nanoparticle surface temperature in an alternating magnetic field using thermoresponsive fluorescent polymers. Journal of Applied Physics, 111(7), 07B334. 8. Fernandez-Fernandez, A., Manchanda, R., & McGoron, A. J. (2011). Theranostic applications of nanomaterials in cancer: drug delivery, image-guided therapy, and multifunctional platforms. Applied Biochemistry And Biotechnology, 165(7-8), 1628– 51. doi:10.1007/s12010-011-9383-z 9. Melancon, M. P., Stafford, R. J., & Li, C. (2012). Challenges to effective cancer nanotheranostics. Journal of controlled release : official journal of the Controlled Release Society, 164(2), 177–82. 10. Kandela, I. (2006). Development of metal nanoparticle immunoconjugates for correlative labeling in light and electron microscopy and as active targeted delivery systems. Dissertation for Doctor of Philosophy, Pharmaceutical Sciences at the University of Wisconsin. 11. Sahoo, Y., Goodarzi, A., Swihart, M. T., Ohulchanskyy, T. Y., Kaur, N., Furlani, E. P., & Prasad, P. N. (2005). Aqueous ferrofluid of magnetite nanoparticles: Fluorescence labeling and magnetophoretic control. The journal of physical chemistry. B, 109(9), 3879–85. 12. Kaiser,M., Heintz, J., Kandela, I., & Albrecht, R. (2007). Tumor cell death induced by membrane melting via immunotargeted, inductively heated Core/Shell nanoparticles. Microscopy and Microanalysis, 13(S02), 18-19.

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Chapter 5

Summary and Future Directions

This dissertation presents studies that employ the use of physical, material, chemical, and biological technology in the development of innovative approaches tor targeted cancer treatment.

Key findings and conclusions arising from this work are summarized below and are discussed in the context of future applications of this technology.

Conventional chemotherapy for the treatment of cancer is far from optimal and suffers from multiple drawbacks including non-specific systemic distribution of cytotoxic agents, inadequate drug concentrations reaching the tumor site, intolerable cytotoxic side effects, limited capacity to monitor therapeutic responses, and development of drug resistance.1, 2 With any cancer therapy, the crucial objective is the achievement of tumor destruction without nor or minimal damage to surrounding healthy tissue. Therefore the development of new and innovative technologies to site-specifically deliver cancer therapeutics in a governable manner is a key issue in cancer treatment.

Magnetic iron oxide nanoparticles have been suggested as possible therapeutic agents which may prove of use in addressing several of the limitations associated with conventional therapeutic delivery systems3-8. The current interest in these particles stems from the ability to induce heat generation of the iron oxide particles by manipulating their internal magnetic domains the use of an alternating (oscillating) magnetic field9, 10, 19.

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Currently the majority of research with inductive heating focuses on bulk heating of tumor cells or the tumor cell mass to 43°C (hyperthermia) or 46°C (thermoablation.) These temperatures effect substantial damage to structural and enzymatic proteins in the cell, and induce apoptosis or necrosis. Despite some success, there are several key issues that remain problematic. The internal temperature of the bulk tissue must be elevated by at least 20°C. This involves selective deposition of substantial amounts of heatable iron particles in the tumor mass.

Often heterogenous heating of the tumor is seen and nonspecific damage to healthy surrounding tissue occurs. Insufficient heating of tumor margins or areas within the tumor results in survival and regrowth of malignant cells. It is difficult to specifically target with sufficient numbers of particles to locally heat individual or small accumulations of metastatic cells.

Another recent and auspicious application of these magnetic particles makes use of their bio-amenable surface chemistry allowing the functionalization of the particle surface to specifically target the nanoparticle to a desired location. One approach to achieve this selective- targeting aim in cancer therapy is to conjugate therapeutic drugs with monoclonal antibodies or other ligands that selectively bind to antigens or receptors that are abundantly or uniquely expressed on the tumor cell surfaces11, 12, 19. The use of iron oxide nanoparticles conjugated to antibody in combination with selective membrane toxicity offers the potential to avoid some of the shortcomings associated with the current approaches to therapy. This work in this dissertation focused on the optimization of a model system that investigates the possibility of targeting cell membranes and producing cell damage and death, not by bulk heating, but through localized cell membrane damage.

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The primary goals of this research project were to further optimize iron oxide nanoparticles (IONPs) with appropriate characteristics (size, surface chemistry) that could be functionalized to bind specifically to the surface of cancer cells and upon exposure to an OMF, inductively heat and cause cell death. The integrated model system previously employed in vitro has furthered our investigation into the value of these iron oxide nanoparticles.

The studies in Chapter 2 examined the influence of reaction temperature and the presence of oxygen in the reaction media during the co-precipitation synthesis of iron oxide nanoparticles.

While the presence of oxygen did not appear to contribute significantly to nanoparticle shape, the preparations of particles synthesized without oxygen in the environment seemed to be more uniform and monodisperse. The inductive heating characteristics of iron oxide nanoparticles were shown not to be dependent on the presence of oxygen, but a trend observing an increase in susceptibility to inductive heating is observed in samples prepared at room temperature and 90°C in comparison to preparation at 60°C. These results suggest that variable and highly complex relationship exists among individual iron oxide nanoparticles, the collective structure and state of the nanoparticles as a ferrofluid, and their inductive heating properties.

Further work is needed to determine other factors that influence the superparamagnetic characteristics of the iron oxide nanoparticles. The mean size of particles synthesized in Chapter

2 were 6-8 nm, smaller than the reported diameters that corresponds to the maximum coercivity and thus inductive heating properties in the single domain size range for 10nm13 and 12.5 nm14 iron oxide nanoparticles. Investigation into methods to increase the mean size of the particle population synthesized while maintaining the monodispersity found at higher reaction temperatures (90°C) would be beneficial for future research. Briefly, the production of monodisperse nanoparticles should ideally follow the classical LaMer mechanism beginning

109 with a short burst of nucleation from a supersaturated solution (iron salts) followed by the slow growth of particles (Oswald ripening) without the formation of additional nuclei; achieving a complete separation of nucleation and growth15. This work could involve the creating and testing of an aqueous methodology analogous to one used in the organic synthesis of iron oxide nanoparticles. Specifically, Sun et al report a method of forming iron nuclei (seeds) and subsequently forming additional iron oxide layers around the original nuclei to increase the particle diameter by controlled seed-mediated growth16.

Chapter 3 considered several different methods of surface modification of the iron oxide nanoparticles. Attempts to stabilize the iron oxide nanoparticles with a continuous gold-shell to form a protective layer against oxidation of the core and a surface amenable to bioconjugation, were unsuccessful with existing reagents and methodology. These efforts resulted in separate particle populations of uncoated iron oxide nanoparticles in suspension with larger gold nanocomplexes (spherical or cuboidal.) However this study revealed the efficiency of functionalization of iron oxide nanoparticles with organic molecule ligands that contain more than one carboxyl functionality, specifically citric acid and sodium citrate. As important as the biostability of these nanoparticles are their inductive heating characteristics which are unaffected by these functionalities. Compounds binding with only alcohol groups were more prone to desorption from the nanoparticle surface; leading to instability in cell culture media (biologically relevant pH and electrolyte concentrations.)

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As the future objective in surface functionalization is to conjugate tumor-specific antibodies to the nanoparticle surface as a method of targeted intracellular hyperthermia, work remains to be done to elucidate the mechanisms that dominate the preferential gold-coating of iron oxide nanoparticles and optimize a reproducible gold-coating procedure. Another aim that could be dually investigated is the direct conjugation of antibodies on the surface of the iron oxide nanoparticles, although this method could encounter issues regarding in vivo toxicity and systemic degradation and bioclearence17, 18.

The effects of exposure to a magnetic field on the structure and death of macrophages is presented in Chapter 4. The implications of the results were also discussed for the localized intracellular hyperthermia of cells. These studies demonstrated that cell death can be induced with 6-8 nm iron oxide nanoparticles inductively heated in an oscillating magnetic field with a frequency of 500 kHz and a field strength of 0.008 T. However further studies are needed to optimize the parameters and specifications of an in vitro model system to avoid the issues encountered currently. One such issue is the reduced phagocytic abilities of the RAW 267.4 macrophages employed for this assay. While the exact reasons for the behavior of the subset of the macrophage population is not known, it would be advantageous to use several different macrophage cell lines or primary monocytes to investigate the cytotoxic effects of iron oxide nanoparticles when the ingestion of particles can be optimized. Another issue involves that loss of dead, non-adherent cells following incubation with particles and OMF treatment. To further quantify this number of dead, non-adherent cells, it would be worthwhile to analyze the cell culture media following exposure to the OMF to not only for another direct measure of cell death, but to perhaps elucidate the mechanism of cell death (presumed necrosis via cell membrane damage) using biomarkers for apoptotic and necrotic pathways. Another interesting

111 avenue to pursue would be the imaging via SEM of non-adherent dead cell surfaces as well as embedding and sectioning of non-adherent dead cells for examination via TEM or STEM.

Examination using SEM could qualitatively establish the uptake of iron oxide nanoparticles by this population and establish visual representations of the mechanisms of cell death employed as a cellular response to intracellular hyperthermia via inductively heated iron oxide nanoparticles.

The work presented in this dissertation focused on the optimization of an integrated experimental system where iron oxide nanoparticles are synthesized, characterized, and utilized in vitro for the study of intracellular hyperthermia. Using these basic principles and results of proof of concept experiments, as a foundation, work can move forward and examine the thermally-induced necrosis of tumor cells via targeted iron oxide nanoparticles.

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References

1. Laurent, Sophie, Delphine Forge, Marc Port, Alain Roch, Caroline Robic, Luce Vander Elst, and Robert N Muller. 2008. “Magnetic Iron Oxide Nanoparticles : Synthesis , Stabilization , Vectorization , Physicochemical Characterizations , and Biological Applications.” 2064–2110. 2. Fernandez-Fernandez, Alicia, Romila Manchanda, and Anthony J McGoron. 2011. “Theranostic applications of nanomaterials in cancer: drug delivery, image-guided therapy, and multifunctional platforms.” Applied Biochemistry And Biotechnology 165(7- 8): 1628–51. 3. Weinstein, Jason S, Csanad G Varallyay, Edit Dosa, Seymur Gahramanov, Bronwyn Hamilton, William D Rooney, Leslie L Muldoon, and Edward A Neuwelt. 2012. “Magnetically vecotored platforms for the targeted delivery of therapeutics to tumors: history and current status.” Nanomedicine : nanotechnology, biology, and medicine 7(2): 289–299. 4. Comes Franchini, Mauro, Giovanni Baldi, Daniele Bonacchi, Denis Gentili, Guido Giudetti, Alessandro Lascialfari, Maurizio Corti, Patrick Marmorato, Jessica Ponti, Edoardo Micotti, Uliano Guerrini, Luigi Sironi, Paolo Gelosa, Costanza Ravagli, and Alfredo Ricci. 2010. “Bovine serum albumin-based magnetic nanocarrier for MRI diagnosis and hyperthermic therapy: a potential theranostic approach against cancer.” Small (Weinheim an der Bergstrasse, Germany) 6(3): 366–70. 5. Mahmoudi, Morteza, Shilpa Sant, Ben Wang, Sophie Laurent, and Tapas Sen. 2011. “Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy.” Advanced drug delivery reviews 63(1- 2): 24–46. 6. Masoudi, Afshin, Hamid Reza Madaah Hosseini, Seyed Morteza Seyed Reyhani, Mohammad Ali Shokrgozar, Mohammad Ali Oghabian, and Reza Ahmadi. 2012. “Long- term investigation on the phase stability, magnetic behavior, toxicity, and MRI characteristics of superparamagnetic Fe/Fe-oxide core/shell nanoparticles.” International journal of pharmaceutics 439(1-2): 28–40. 7. Weinstein, Jason S, Csanad G Varallyay, Edit Dosa, Seymur Gahramanov, Bronwyn Hamilton, William D Rooney, Leslie L Muldoon, and Edward A Neuwelt. 2012. “Magnetically vecotored platforms for the targeted delivery of therapeutics to tumors: history and current status.” Nanomedicine : nanotechnology, biology, and medicine 7(2): 289–299. 8. Arvizo, Rochelle R, Sanjib Bhattacharyya, Rachel a Kudgus, Karuna Giri, Resham Bhattacharya, and Priyabrata Mukher ee. 2012. “Intrinsic therapeutic applications of noble metal nanoparticles: past, present and future.” Chemical Society Reviews 41(7): 2943–70. 9. Latorre, Magda, and Carlos Rinaldi. 2009. “Applications of magnetic nanoparticles in medicine: magnetic fluid hyperthermia.” Puerto Rico Health Sciences Journal 28(3): 227–238. 10. Medeiros, S F, A M Santos, H Fessi, and A Elaissari. 2011. “Stimuli-responsive magnetic particles for biomedical applications.” International Journal of Pharmaceutics 403(1-2): 139–161.

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11. Kandela, I. (2006). Development of metal nanoparticle immunoconjugates for correlative labeling in light and electron microscopy and as active targeted delivery systems. Dissertation for Doctor of Philosophy, Pharmaceutical Sciences at the University of Wisconsin. 12. Krystofiak, Evan S., Vyara Z. Matson, Douglas a. Steeber, and Julie a. Oliver. 2012. “Elimination of Tumor Cells sing Folate Receptor Targeting by Antibody-Conjugated, Gold-Coated Magnetite Nanoparticles in a Murine Breast Cancer Model.” Journal of Nanomaterials 2012: 1–9. 13. Hergt, Rudolf, Silvio Dutz, and Michael Röder. 2008. “Effects of size distribution on hysteresis losses of magnetic nanoparticles for hyperthermia.” Journal of physics. Condensed matter : an Institute of Physics journal 20(38): 385214. 14. Gonzales-Weimuller, Marcela, Matthias Zeisberger, and Kannan M Krishnan. 2009. “Size-dependant heating rates of iron oxide nanoparticles for magnetic fluid hyperthermia.” Journal of Magnetism and Magnetic Materials 321(13): 1947–1950. 15. Lu, An-Hui, E L Salabas, and Ferdi Sch th. 2007. “Magnetic nanoparticles: synthesis, protection, functionalization, and application.” Angewandte Chemie (International ed. in English) 46(8): 1222–44. 16. Sun, Shouheng, Hao Zeng, David B Robinson, Simone Raoux, Philip M Rice, Shan X Wang, and uanxiong Li. 2004. “Monodisperse MFe 2 O 4 ( M ) Fe , Co , Mn ) Nanoparticles.” 4(1): 126–132. 17. Doane, Tennyson L, and Clemens Burda. 2012. “The unique role of nanoparticles in nanomedicine: imaging, drug delivery and therapy.” Chemical Society Reviews 41(7): 2885–911. 18. Taylor, Arthur, Katie M Wilson, Patricia Murray, David G Fernig, and Raphaël Lévy. 2012. “Long-term tracking of cells using inorganic nanoparticles as contrast agents: are we there yet?” Chemical Society Reviews 41(7): 2707–17. 19. Albrecht, R.M.; Kandela, I. Colloidal Magnetic Nanobioparticles for Cytotoxicityand Drug Delibery. Publication Number US 2011/0256621 A1, October 20, 2011.

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Appendix

List of Abbreviations

cFe-colloidal iron*

EPR-enhanced permeability and retention g-grams

IONP-iron oxide nanoparticle* kHz-kilohertz (1 x 103 hertz) mg-milligram (1 x 10-3 gram) ml-milliliter (1 x 10-3 liter)

MNP-magnetic nanoparticle*

MRI-magnetic resonance imaging nm- nanometer (1x10-9meter)

NP-nanoparticle

OMF- oscillating magnetic field (used interchangeably with alternating magnetic field)

SEM-scanning electron micrograph

SPION-superparamagnetic iron oxide nanoparticle*

TEM-transmission electron micrograph

T-tesla

µl-microliter (1 x 10-6 liter)

*All of these notations are used interchangeably to denote the iron oxide nanoparticles discussed throughout this dissertation.

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Co-precipitation Synthesis of Iron Oxide Nanoparticles

Method adapted from:

Sahoo, Y. et al (2005). Aqueous ferrofluid of magnetite nanoparticles: Fluorescence labeling and magnetophoretic control. The Journal of Physical Chemistry B, 109(9), 3879–3885.

1. Boil several hundred milliliters of deionized water, attach to vacuum set-up with ice traps, and pull a vacuum for at least 30 minutes. It is helpful to add a magnetic stir bar to the flask that is kept spinning at a low speed to ensure the elimination of the majority of oxygen. 2. Remove water from vacuum and cover with nitrogen before capping. Use immediately. 3. Arrange the reaction set-up that includes a clean non-magnetic stir rod (solid cell scraper will work nicely,) a three-necked flask, several glass pipettes and pipette bulb, and a 5ml plastic pipette and large, rubber pipette bulb. 4. Measure (covering with nitrogen to the best ability the circumstances allow) 40ml of degassed water into a three-necked flask. Cover in nitrogen from a glass pipette attached to the nitrogen source.

5. Weigh out 0.86 g FeCl2 and 2.35 g FeCl3 and add to the 40 mL water in the three-necked flask; vigorously stirring the reaction mixture for 1 minute to ensure the iron salts

dissolve completely. Then draw up 5 mL of NH4OH in the 5ml plastic pipette and rapidly inject into the iron salt solution. The solution with turn from a translucent brown (reminiscent of medium strength tea) to a dark, opaque black that is more viscous (sludgy.) Keep stirring the solution for 5 minutes. 6. Pour the nanoparticle solution into two 50ml conical vials (about 20-23mls in each) and fill each vial to 45ml with degassed water. Vortex well (30 seconds.) 7. Spin in a table top centrifuge at 2.5K rpm for 2 minutes. Discard the clear supernatant, and suspend the pellet of nanoparticles in 40mls of fresh degassed water. Vortex well. 8. Increase the centrifuge speed and time to 3K for 3 minutes. Discard the clear supernatant, and suspend the pellet of nanoparticles in 40mls of fresh degassed water. Vortex well. 9. Increase the centrifuge speed and time to 4.4K for 5 minutes.

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10. Now the spectator ions from the synthesis have been washed away and the nanoparticles can be functionalized. Discard the clear supernatant, and suspend the pellet of nanoparticles in 40mls of 22mM of citric acid that has been made from the degassed water prepared earlier and vortex well. Transfer to a 50 or 100ml glass beaker or flask, cover with tin foil (to prevent evaporation) and add a small stir bar. Heat on a hot plate on a low setting (about 70-80 C) while stirring at a low speed for 20 minutes. Turn off heating element and allow reaction to continue stirring and cool to room temperature. 11. Transfer stabilized nanoparticles to clean to two 50ml vials, cover with nitrogen, label, and store. Particles are best used within a week of synthesis for optimum heating.

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Organic Synthesis of Iron Oxide Nanoparticles

Method adapted from:

Sun, S. et al (2004). Monodisperse MFe2 O4 ( M ) Fe , Co , Mn ) Nanoparticles, The Journal of the American Chemical Society, 4(1), 126–132.

This synthetic method involves the use of a Schlenk line that allows a completely oxygen free atmosphere and prevents liquid reactant evaporation when heating solutions beyond their boiling points. Professor Song Jin in the Chemistry Department was kind enough to allow me to use his materials and equipment necessary for this synthetic protocol.

In brief, 6 nm Fe3O4 nanoparticles can be synthesized by mixing and magnetically stirring the following reactants in 20 ml of benzyl ether under a blanket of nitrogen.

Fe(acac)3 (2 mmol) 0.706g

Tetrahexadecanediol (10 mmol) 2.3g

Oleic acid (6 mmol) 0.5649g

Oleylamine (6 mmol) 0.535g

The reaction solution was initially heated to 100°C to remove hexanes. Then the solution heated to 200°C for 1 hour, and then heated to reflux (∼300 °C) for 30 minutes. The black-colored mixture was cooled to room temperature by removing the heat source. Following the workup procedures described in the synthesis of 4 nm particles, a black-brown hexane dispersion of 6nm iron oxide nanoparticles was produced.

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Figure 1.

TEM images of iron oxide nanoparticles synthesized using an organic synthesis described by Sun et al (2004.) The scale bar is 10 nm and the particles are approximately 6nm in diameter.