University of Florida Thesis Or Dissertation

MAGNETOELECTRIC COMPOSITE NANOMATERIALS FOR THE STIMULATION OF NEURONAL PROLIFERATION AND DIFFERENTIATION

AMANDA MAE UHL

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2019

To my Dad

ACKNOWLEDGMENTS

Firstly, I would like to thank my family for constantly encouraging me to learn and to follow my dreams. I would like to thank both of my parents for instilling in me a sense of perseverance that can only come from facing hardship. To my father (may he rest in peace), for constantly being there and for teaching me that math, science and building things with your own two hands can be both fun and extremely rewarding. And to my mother, for being an inspiration to be both kind and patient. To my little sister, Julie, for being a built in best friend and support system.

I would like to thank the teachers I have had who encouraged me to follow my passion for math, science, and engineering. Without them, I would not have had the courage to pursue a bachelor’s degree in engineering. I would also like to thank my undergraduate research advisor,

Dr. Lara Estroff, for taking me under her wing and enabling me to discover my passion for research. Without her generosity and advice I would never have considered pursuing a Ph.D. I would also like to thank my friends Moniek and Stephanie who grounded me throughout my undergraduate studies and have greatly influenced the person I am today.

Finally, I would like to thank those people whom have supported me through the whole graduate school process. Firstly, I would like to thank my friends from the Andrew Research

Group: Emilie, Maeve, Sara, Stefan, Matt, and Prabal for creating a lab environment that fosters discussion, creativity, and team work. To Emilie, thank you for being an amazing mentor and helping me realize my strengths and grow into them. Thank you also for being a friend and source of sanity throughout our time in the lab. To Maeve, thank you for being there and supporting me through the difficulties of lab work and graduate school. To Sara, thank you for joining me in the occasional insanity that is the lab and making the last two years of graduate school infinitely better. I would also like to thank the undergraduates that I have had the pleasure

of mentoring: Camille, Maria, Camilla, Juan, Austin and Alejandra. Having had all of you in the lab has made it feel much more like a family rather than just colleagues.

Beyond the Andrew lab, I would also like to thank my friends Abigail Casey, Zach

Weinrich and Catherine Sahi, without whom I would not have survived graduate school. To

Abigail, thank you for being one of my favorite people and best friends. No matter where we are, location or life-wise, I know I can count on you. I am infinitely glad you came to UF, if only briefly. To Zach, thank you for always being there when I need you, for a laugh, a hug, or mere camaraderie. Thank you for accepting me as I am but also pushing me to be a better, more positive person. To Catherine, thank you for taking me in as a friend at a time when I needed it the most. Thank you for being there to help me get through everything, constantly accepting me and just being there providing friendship and distractions and adding a little whimsy to my life.

I would also like to thank Krista Dulany for being a writing buddy and keeping me motivated to write this document. I’m so glad to have been fortunate enough to gain a good friend through this dissertation writing process. I’m not sure either one of us would have maintained our sanity through this process without someone to go through it with.

I would also like to thank my committee members, Dr. Carlos Rinaldi, Dr. David Arnold,

Dr. Josephine Allen, and Dr. Jon Dobson for their guidance. Thank you also to Dr. Christine

Schmidt and Dr. Sahba Mobini for all of their help and knowledge for the cell work components of this dissertation. Gratitude is also extended to Dr. Thomas Angelini and Tori Ellison for their assistance with the confocal microscopy of PC12 cells.

Finally, I would like to extend my gratitude to my advisor Dr. Jennifer Andrew for her support, advice, encouragement, and overall mentorship. Your leadership through the course of my Ph.D. has constantly inspired me to strive to be the best engineer and person that I can be.

Under you advisement, I have become much more resilient, creative, and confident than I was at the start of my time at UF. Thank you.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 10

LIST OF FIGURES ...... 11

LIST OF ABBREVIATIONS ...... 16

ABSTRACT ...... 17

CHAPTER

1 BACKGROUND INFORMATION ...... 19

1.1 The Role of Electric Fields in the Peripheral Nervous System ...... 19 1.2 Multiferroic Materials ...... 21 1.3 Materials for Neuronal Stimulation ...... 24 1.3.1 Conducting Polymers for Neuronal Stimulation ...... 24 1.3.2 Piezoelectric Materials for Neuronal Stimulation ...... 26 1.3.3 Benefits of Magnetoelectric Materials Over Electronic Materials ...... 28 1.4 Ceramic Electrospinning and Electrospraying Routes for Magnetoelectric Composites ...... 29 1.4.1 Electrospinning ...... 31 1.4.2 Electrospraying ...... 35 1.4.3 Sol-Gel Chemistry ...... 38 1.5 Objective and Summary of Dissertation ...... 40

2 SOL-GEL ELECTROSPINNING OF JANUS TYPE MAGNETOELECTRIC NANOFIBERS ...... 46

2.1 Introduction ...... 46 2.2 Experimental Methods ...... 48 2.2.1 Materials ...... 48 2.2.2 Sol-Gel Precursor Solutions ...... 48 2.2.3 Nanofiber Synthesis via Electrospinning ...... 49 2.2.4 Nanofiber Characterization ...... 49 2.3 Results and Discussion ...... 50 2.3.1 Single-Phase Barium Titanate Fibers ...... 50 2.3.2 Single-Phase Cobalt Ferrite Fibers ...... 52 2.3.3 Bi-phasic Barium Titanate-Cobalt Ferrite Janus Fibers ...... 55 2.4 Summary ...... 57

3 SYNTHESIS OF BARIUM TITANATE PARTICLES VIA SOL-GEL ELECTROSPRAYING ...... 67

3.1 Introduction ...... 67 3.2 Experimental Methods ...... 68 3.2.1 Materials ...... 68 3.2.2 Sol-Gel Precursor Solutions ...... 68 3.2.3 Nanoparticle Synthesis ...... 69 3.2.4 Nanoparticle Characterization ...... 70 3.3 Results and Discussion ...... 70 3.4 Summary ...... 76

4 SYNTHESIS OF COBALT FERRITE PARTICLES VIA SOL-GEL ELECTROSPRAYING ...... 87

4.1 Introduction ...... 87 4.2 Experimental Methods ...... 91 4.2.1 Materials ...... 91 4.2.2 Sol-gel precursor solutions ...... 91 4.2.3 Electrospraying Cobalt Ferrite into a Liquid Collection Medium ...... 91 4.2.4 Characterization of Electrosprayed Cobalt Ferrite Particles ...... 92 4.3 Results and Discussion ...... 93 4.4 Summary ...... 95

5 MAGNETOELECTRIC FIBER – HYDROGEL COMPOSITES FOR NEURONAL STIMULATION ...... 100

5.1 Introduction ...... 100 5.2 Experimental Methods ...... 101 5.2.1 Materials ...... 101 5.2.2 Synthesis of Collagen Hydrogels ...... 102 5.2.3 Cell Culture ...... 103 5.2.4 Magnetoelectric Stimulation ...... 103 5.2.5 Cellular Proliferation ...... 104 5.2.6 PC12 Differentiation Studies ...... 104 5.2.7 Statistical Analysis ...... 105 5.3 Results and Discussion ...... 105 5.3.1 Effects of Magnetoelectric Stimulation on PC12 Proliferation and Cytotoxicity...... 105 5.3.2 Effects of Magnetoelectric Stimulation on PC12 Differentiation ...... 108 5.4 Summary ...... 110

6 CONCLUSIONS AND FUTURE WORK ...... 135

6.1 Summary ...... 135 6.2 Research Contributions ...... 137 6.3 Future Work ...... 138 6.3.1 Electrosprayed Ceramic Nanoparticles ...... 138 6.3.2 Magnetoelectric Nanomaterials for Neuronal Stimulation ...... 139

APPENDIX

A CALCULATIONS FOR THE MODEL OF INTERFACIAL AREA TO VOLUME RATIO FOR BOTH NANOPARTICLES AND NANOFIBERS ...... 140

B CALCULATIONS FOR COLLAGEN TYPE I HYDROGEL COMPOSITIONS ...... 142

C EFFECTS OF HUMIDITY ON BARIUM TITANATE SOL-GEL ELECTROSPRAY ....145

C.1 Experimental Methods ...... 145 C.1.1 Materials ...... 145 C.1.2 Sol-Gel Precursor Solutions ...... 145 C.1.3 Nanoparticle Synthesis ...... 145 C.1.4 Nanoparticle Characterization ...... 146 C.2 Results and Discussion...... 146 C.3 Summary and Conclusions...... 151

D SYNTHESIS OF JANUS-TYPE MAGNETOELECTRIC PARTICLES VIA SOL-GEL ELECTROSPRAYING ...... 156

D.1 Introduction ...... 156 D.2 Experimental Methods ...... 157 D.2.1 Materials ...... 157 D.2.2 Sol-Gel Precursor Solutions ...... 157 D.2.2 Electrospraying into Silicone Oil Liquid Collector ...... 158 D.2.4 Characterization of Electrosprayed Particles ...... 159 D.3 Results and Discussion ...... 160

E LDH DATA FOR PC12 CELLS CULTURED ON COLLAGEN BASED HYDROGELS FOR ALL STIMULATION CONDITIONS – PLOTS IN PERCENT CYTOTOXICITY ...... 162

F MEASUREMENT OF MAGNETIC FIELD USED FOR CELL STIMULATION ...... 165

LIST OF REFERENCES ...... 166

BIOGRAPHICAL SKETCH ...... 181

LIST OF TABLES

Table page

2-1 Citric acid concentrations and the time to complete gelation of the sol-gel precursor...... 59

2-2 Conductivity and viscosity measurements of sol-gel precursor solutions for solutions containing either no chelating agent, citric acid, or acetylacetone...... 59

5-1 Regimes for the magnetoelectric stimulation of PC12 cells on collagen based hydrogels ...... 113

5-2 Percentages of cells exhibiting neurite extension on day for each growth condition...... 113

LIST OF FIGURES

Figure page

1-1 Schematics of a bidirectional neural interface device that has been developed for electrical stimulation and recording of neuronal activity ...... 41

1-2 Diagram depicting the variety of multiferroic coupling that is possible and the related properties...... 41

1-3 Schematic of magnetoelectric coupling in composites ...... 42

1-4 Schematics of three different types of composite connectivity schemes ...... 42

1-5 Schematics for basic electrospinning and electrospraying ...... 43

1-6 SEM of PVP nanofibers made from solutions of different viscosity (based on the concentration of PVP) from Li and Xia ...... 43

1-7 Schematic of the three different possibilities for the morphology of electrospun nanofibers: randomly dispersed grains, core-shell and Janus morphologies...... 44

1-8 SEM images of as-sprayed titania particles produced by Li et al...... 44

1-9 Schematic of the process of basic sol-gel reactions ...... 45

2-1 SEM images of barium titanate fibers ...... 60

2-2 XRD pattern of calcined barium titanate fiber ...... 60

2-3 Raman spectra of barium titanate fibers...... 61

2-4 Schematic of citric acid coordinated cobalt ions ...... 61

2-5 Photos of cobalt ferrite sol-gel solutions containing citric acid at varying molar ratios of Co:Fe:citric acid...... 62

2-6 Electrospun cobalt ferrite nanoparticles from solutions containing citric acid as a chelating agent ...... 62

2-7 Schematics of cobalt and iron ions coordinated with acetylacetone...... 63

2-8 Photo of cobalt ferrite fibers electrospun onto a flat plate collector ...... 63

2-9 SEM images of cobalt ferrite fibers electrospun from solutions containing acetylacetone as the chelating agent ...... 64

2-10 XRD pattern of calcined cobalt ferrite fibers electrospun using acetylacetone as the chelating agent ...... 64

2-11 SEM images barium titanate – cobalt ferrite Janus fibers ...... 65

2-12 XRD data of calcined Janus fibers ...... 65

2-13 Shown are room temperature hysteresis loops for cobalt ferrite single-phase fibers and Janus biphasic fibers as obtained using SQUID magnetometry...... 66

2-14 Shown are magnetization versus temperature and dM/dT curves for the indirect examination of the magnetoelectric effect...... 66

3-1 SEM images of barium titanate particles electrosprayed from solutions with a Ba:Ti ratio of 1:1 ...... 78

3-2 Shown are XRD and Raman for calcined samples from precursros containing 1:1 Ba:Ti ...... 78

3-3 SEM images of as-sprayed barium titanate nanoparticles with varying Ba:Ti molar ratios ...... 79

3-4 SEM images of salt-calcined barium titanate nanoparticles with varying Ba:Ti molar ratios ...... 80

3-5 XRD data for salt-calcined barium titanate nanoparticles with varying Ba:Ti molar ratios ...... 81

3-6 Raman spectra taken of salt-calcined barium titanate nanoparticles with varying Ba:Ti molar ratios ...... 81

3-7 SEM images of calcined barium titanate samples calcined in sodium chloride at 800 °C for 3 hours with a ramp rate of 20 °C/min ...... 82

3-8 SEM images of calcined barium titanate samples calcined in sodium chloride at 750 °C at a ramp rate of 10 °C/min ...... 82

3-9 SEM image of barium titanate sample calcined in sodium chloride at 800 °C for 3 hours at a ramp of 10 °C/min in potassium sulfate ...... 83

3-10 SEM image of barium titanate sample calcined at 800 °C for 1 hour at a ramp rate of 10 °C/min in sodium chloride ...... 83

3-11 Raman spectra of barium titanate sample calcined at 800 °C for 1 hour at a ramp rate of 10 °C/min in sodium chloride ...... 84

3-12 XRD pattern for the barium titanate sample calcined at 800 °C for 1 hour at a ramp of 10 °C/min in sodium chloride ...... 84

3-13 SEM image of barium titanate sample calcined at 800 °C for 2 hours at a ramp rate of 10 °C/min in sodium chloride ...... 85

3-14 Raman spectra of barium titanate sample calcined at 800 °C for 2 hours at a ramp rate of 10 °C/min in sodium chloride ...... 85

3-15 XRD pattern for the barium titanate sample calcined at 800 °C for 2 hours at a ramp of 10 °C/min in sodium chloride ...... 86

4-1 Cobalt ferrite electrosprayed onto a flat plate collector resulting in the formation of a film containing particles ...... 97

4-2 Schematic of the electrospray setup developed for the synthesis of cobalt ferrite nanoparticles ...... 97

4-3 XRD data for salt-calcined cobalt ferrite compared to a cobalt ferrite reference ...... 98

4-4 Raman spectroscopy for salt-calcined cobalt ferrite particles ...... 98

4-5 Representative TEM of salt-calcined cobalt ferrite particles ...... 99

4-6 Room temperature hysteresis loop for the cobalt ferrite nanoparticles with an average saturation magnetization of 75.3±0.5 emu/g ...... 99

5-1 Camera images the solenoid setup created for the magnetoelectric stimulation experiments ...... 114

5-2 Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 2.3 mT magnetic field ...... 115

5-3 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 2.3 mT magnetic field...... 115

5-4 Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 3.45 mT magnetic field ...... 116

5-5 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 3.45 mT magnetic field...... 116

5-6 Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 4.6 mT magnetic field ...... 117

5-7 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 4.6 mT magnetic field...... 117

5-8 Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 3 hours in a 3.45 mT magnetic field ...... 118

5-9 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 3 hours in a 3.45 mT magnetic field...... 118

5-10 Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 4 hours in a 2.3 mT magnetic field ...... 119

5-11 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 4 hours in a 2.3 mT magnetic field...... 119

5-12 Plot showing stimulation regimes that neither hindered nor promoted cellular proliferation compared to stimulation regimes that had detrimental effects on cellular proliferation...... 120

5-13 Confocal microscopy of PC12 cells stained with fluorescent CellTracker® Green after 24 hours on the gel to allow for cell attachment...... 121

5-14 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in growth media without stimulation ...... 122

5-15 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 123

5-16 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in differentiation media without stimulation ...... 124

5-17 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 125

5-18 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in growth media without stimulation ...... 126

5-19 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 127

5-20 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in differentiation media without stimulation...... 128

5-21 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 129

5-22 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in growth media without stimulation ...... 130

5-23 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 131

5-24 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in differentiation media without stimulation...... 132

5-25 Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day ...... 133

5-26 Confocal microscopy of collagen gels with Janus fibers grown in differentiation media following 5 day of stimulation in a 2.3 mT magnetic field for 1 hour per day .....134

A-1 Table of interfacial area, volume and IA:V ratio calculated for nanoparticles of a given diameter ...... 140

C-1 Scanning electron micrographs of as-sprayed BaTiO3 particles...... 152

C-2 Scanning electron image of BaTiO3 particles post salt calcination at 750°C ...... 153

C-3 XRD scan of salt calcined BaTiO3 particles compared to a barium titanate reference pattern ...... 154

C-4 Raman spectra of BaTiO3 particles ...... 155

D-1 Model of interfacial area to volume ratio as a function of either fiber or particle diameter...... 161

D-2 SEM images of as-sprayed Janus nanoparticles ...... 161

E-1 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in 1.1 mT field presented as % cytotoxicity as compared to the collagen control hydrogel ...... 162

E-2 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 1.6 mT field presented as % cytotoxicity as compared to the collagen control hydrogel ...... 162

E-3 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 2.2 mT field presented as % cytotoxicity as compared to the collagen control hydrogel ...... 163

E-4 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 3 hours in a 1.6 mT field presented as % cytotoxicity as compared to the collagen control hydrogel ...... 163

E-5 Cytotoxic effects of the hydrogels on PC12 cells stimulated for 4 hours in a 1.1 mT field presented as % cytotoxicity as compared to the collagen control hydrogel ...... 164

F-1 Plot of the magnetic field produced by the solenoid described in Chapter 5 for an input voltage of 20 Vpp, 50 % duty cycle, and a frequency of 60 Hz ...... 165

LIST OF ABBREVIATIONS

%RH Percent relative humidity

ACHES Acetylcholinesterase

DMEM Dulbecco’ Modified Eagle Media

EDS Energy dispersive x-ray spectroscopy

LDH Lactate dehydrogenase

ME Magnetoelectric

Oe Oersted

PVP Polyvinylpyrrolidone

RH Relative humidity

RT Room temperature

SEM Scanning electron microscopy

SQUID Superconducting quantum interference device

TEM Transmission electron microscopy

VSM Vibrating sample magnetometry wt% Weight percent

XRD X-ray diffraction

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

MAGNETOELECTRIC COMPOSITE NANOMATERIALS FOR THE STIMULATION OF NEURONAL PROLIFERATION AND DIFFERENTIATION

Amanda Mae Uhl

May 2019

Chair: Jennifer S. Andrew Major: Materials Science and Engineering

Electric fields are involved in a wide variety of processes in the human body, including wound healing, muscular functions, cell division, and neuronal signaling. The potential for manipulating electric stimuli in order to aid in any of these processes has led to the development of a variety of devices for electrical stimulation. Unfortunately, devices for electrical stimulation typically require invasive procedures and the use of external electrical leads to be able to manipulate electric fields within the body. Magnetoelectric materials, materials which are capable of producing an electrical stimulus in the presence of a magnetic field, provide an opportunity to manipulate local electric stimuli via less invasive means.

Here, composite magnetoelectric nanofibers and nanoparticles were synthesized for use in biomedical applications. The composite magnetoelectric effect is a property that is dependent on the interfacial coupling of a piezoelectric and magnetostrictive material. This motivates the development of nanocomposites as they exhibit higher ratios of interfacial area to volume when compared to thin film or bulk composites, which should in turn result in enhanced magnetoelectric properties. Magnetoelectric nanofibers were synthesized via sol-gel electrospinning. In working towards the development of magnetoelectric composite

nanoparticles, sol-gel electrospraying methods were developed for the synthesis of single phase piezoelectric and magnetostrictive materials.

Finally, application of the magnetoelectric fibers for aiding neuronal growth was completed using magnetoelectric fiber-hydrogel composites to study the effects of magnetoelectric stimulation on the proliferation and differentiation of PC-12 neuronal-like cells.

In order to accomplish this, the magnetoelectric fiber-hydrogel composites were placed in a magnetic field created by applying a square wave voltage to the solenoid and compared to control empty hydrogels and magnetic control hydrogels containing magnetic nanofibers. These studies yielded the discovery of a possible upper limit of stimulation conditions (applied magnetic field and time in field) above which the applied magnetic field and thereby induced electric field is damaging the cells and causing decreased proliferation. Preliminary studies on the effects of magnetoelectric stimulation on PC12 differentiation showed the beginnings of neurite outgrowth following 5 days of stimulation, however, further studies with additional time points at longer times will be necessary to elucidate the effects of magnetoelectric stimulation.

CHAPTER 1 BACKGROUND INFORMATION

Electric fields are ubiquitous throughout the human body in a variety of functions including wound healing, angiogenesis, cell signaling, and neuronal growth and signaling.1 Of interest here is the use of electric fields to affect neuronal growth and differentiation. The existence of these electric fields within the body means that there is a potential to use applied electric fields to influence these neuronal processes. Electroactive materials provide an opportunity to create electric stimuli that will aid in the growth and differentiation of neurons.

This work is particularly interested in the development of magnetoelectric nanocomposites and their use in creating local electric fields for the stimulation of neuronal growth and differentiation. Magnetoelectric materials are capable of producing electric stimuli in response to an applied magnetic field. This property presents the possibility of using magnetoelectric materials for a minimally invasive way to create local electric stimuli within the body.

First, this chapter will present the role of electric fields in the healing injuries to the nervous system. This is followed by a discussion of the multiferroic properties of the materials that have been developed here for their use in neuronal stimulation. A review of the materials currently in use for electric stimulation of neurons are then presented. Finally, this chapter ends with an overview of the methods used for the synthesis of the multiferroic materials developed here, including an overview of sol-gel chemistry.

1.1 The Role of Electric Fields in the Peripheral Nervous System

Of specific interest for this work is the role that electric fields and stimuli play in the growth of neurons and in the repair of injured neurons. In order to understand the role electric

stimuli play in the repair of injured neurons, it is necessary to first understand the role of electric fields in signaling and growth in the nervous system.

When injuries to nerves in the peripheral nervous system occur, a series of events are triggered. First, macrophages and monocytes remove any debris caused by the injury while

Schwann cells proliferate in order to be able to create the necessary neurotrophic factors and an extracellular matrix.2 This stimulates axon regeneration which attempt to extend to bridge the gap between the two ends of the damaged nerve. Unfortunately, complete regeneration of the nerve connections is rarely complete and functionality is not fully recovered.1–4 For this reason, there has been a great deal of research devoted to developing materials that can aid in the regeneration of the nerves to better restore functionality.

Some of the materials being developed for neuronal regeneration include electroactive materials (these will discussed in further detail in section 1.3). Electroactive materials take advantage of the responsiveness of neuronal cells to electric stimuli. Work done by several groups suggests that in the case of neuronal wound healing, neuronal cells experience a constant electric field of up to 140 mV/mm.5–12 Thus, the inclusion of electroactive materials in wound healing environments can be used to guide axons to grow along specific paths.3 Given that neuronal cells are under the constant influence of an electric field during wound healing a great deal of research has been done on developing electroactive materials as well as research on applying electric stimuli in conjunction with electroactive materials. Additional electric stimuli is thought to be able to provide further cues for neuronal growth and hopefully be able to improve the healing of injuries to the nervous system and provide greater restoration of functionality.

While the inclusion of electroactive materials can be used to guide neuronal growth and differentiation, there are many challenges associated with using these materials to apply electric

fields in a non-invasive manner. Many of the methods being developed for electric stimulation of require the use of invasive electrical leads in order to be able apply the electrical stimulus. This includes microneedle arrays created from a number of different materials as well as devices such as nerve grafts with connections to an external recording device and power supply (Figure

1-1).13–15 In order to further the ability to stimulate neuronal processes such as proliferation and differentiation, non-invasive methods of accomplishing electrical stimulation need to be developed. Multiferroic materials, specifically magnetoelectric multiferroics, present an opportunity for such non-invasive stimulation.

This dissertation focuses on the development of multiferroic materials for their potential use in applying local electric fields in a non-invasive manner. The next section explains multiferroic and magnetoelectric properties and the composite magnetoelectric effect.

1.2 Multiferroic Materials

Multiferroic materials are a class of materials that combines two or more types of ferroic ordering, such as ferroelasticity, ferroelectricity, and ferromagnetism, to result in additional materials properties (Figure 1-2).16 Magnetoelectric materials are a type of multiferroic material which combine ferroelectric and ferromagnetic properties to result in a material system that exhibit changes in electrical polarization as a response to an applied magnetic field, and exhibit a change in magnetization as a response to an applied electric field. . Magnetoelectric materials are rarely naturally occurring, necessitating the use of magnetoelectric composites which combine two materials, one with ferroelectric properties and one with ferromagnetic properties. In order to obtain magnetoelectric properties from composite materials, the ferroelectric and ferromagnetic materials are typically strain coupled in order to achieve the magnetoelectric effect. Here, piezoelectric materials are used as the ferroelectric and magnetostrictive materials are used as the ferromagnetic material. Piezoelectric materials exhibit a change in polarization

upon application of a mechanical stress (Figure 1-3a). Magnetostrictive materials mechanically strain when a magnetic field is applied (Figure 1-3b).

Since both piezoelectric and magnetostrictive properties have a mechanical component, they can be elastically coupled at interfaces between the materials.17 Given that for magnetoelectric composite materials, the piezoelectric and magnetostrictive properties are coupled through strain, they are considered to be product properties. This idea was proposed by van Suchtelen in 1972.18 Thus, the magnetoelectric effect can be described by the equations 1-1 and 1-217,19, where the magnetoelectric effect induced by a magnetic field is shown in Equation

1-1 and the magnetoelectric effect (converse magnetoelectric effect) induced by an electric field is shown in Equation 1-2.

푃 = 훼퐻 (1-1)

푀 = 훼퐸 (1-2)

Where P is electric polarization, H is applied magnetic field, M is magnetization, E is electric field, and α is the magnetoelectric coefficient.

These equations show how the magnetoelectric effect is triggered by either a magnetic or electric field. In the case of an applied magnetic field, the magnetostrictive material is deformed.

Due to the interfacial coupling of the composite, the piezoelectric material is forced to strain with the magnetostrictive material resulting in an electric polarization. If instead an electric field is applied, the piezoelectric material forces the magnetostrictive material to strain through the interfacial coupling resulting in a magnetization. The direct magnetoelectric coefficient, α, can be defined as follows:

휕푃 (1-3) 훼 = = 푘 푒푚푒 퐻 휕퐻 푐

Where ∂P/∂H is the partial derivative of polarization with respect to the applied field, kc is a coupling factor between the magnetostrictive and piezoelectric phases, em is the piezomagnetic coefficient, and e is the piezoelectric coefficient.17 Piezomagnetic effects are typically weak and thus magnetostrictive materials are used instead. The magnetoelectric coefficient, α, can then be used to determine the polarization, or magnetization, give the applied magnetic field, or electric field, respectively17. The ability of magnetoelectric materials to respond to either a magnetic or electric field makes them potentially useful materials in a variety of applications, but for this dissertation, their potential for use in biomedical applications that would benefit from local electric stimuli is of particular interest.20–26 Having a material that can be used to remotely create an electric field in response to an applied magnetic field would enable local electric stimulation without the needs for invasive electrical leads.

There are a variety of ferroelectric and ferromagnetic materials that can be used to create composite magnetoelectric materials for use in the different applications listed above. The work described in this dissertation focuses on the development of magnetoelectric biomaterials. For the purposes of studying magnetoelectric biomaterials, barium titanate has been chosen as the piezoelectric material for use in the work described here over lead containing compounds.

Barium titanate is a perovskite material that exhibits piezoelectric properties in its tetragonal form.27 Of the variety of ferromagnetic materials that can be used in magnetoelectric composites, ferrimagnetic, inverse spinel cobalt ferrite has been chosen for its magnetic properties.28

The next section seeks to establish the need for magnetoelectric materials in the electrical stimulation of neuronal growth and differentiation. This is done by examining the current state of the art in electrical stimulation of neuronal growth and differentiation, namely conducting polymeric materials and piezoelectric materials.

1.3 Materials for Neuronal Stimulation

Currently there is a wide variety of research being done on the use of electronic biomaterials for neuronal stimulation including conducting polymers and piezoelectric nanomaterials. However, the use of many of these materials is limited due to the difficulty of applying electric stimuli given that the human tissues attenuate electric fields. Magnetoelectric materials present a unique opportunity for using electric stimuli within the body as it enables the local manipulation of electric fields. This is possible as the human body is permeable to magnetic fields, unlike electric fields, and therefore is not harmed by them. Though this dissertation focuses on the development of magnetoelectric biomaterials, it is first necessary to discuss the current state of the art in electrical stimulation for neuronal proliferation and differentiation utilizing conductive polymers and piezoelectric materials..

1.3.1 Conducting Polymers for Neuronal Stimulation

Conducting polymers are a popular class of materials for electric stimulation that have been studied for the last 30 years.29 These polymers conduct electricity through electrons that are loosely bound to the backbone of the polymer in conjugated π bonds. Typically, their conductive properties rely on the doping of the polymer with dopant molecules such as hydrochloric acid, lauric acid, and many others. The presence of dopants leads to a highly conductive polymer.29

Conducting polymers have been studied for their use as scaffolds by themselves as well as for electrode coatings that increase biocompatibility, charge storage capacity, charge injection limits and other properties.30 For the purposes of this work, the focus will be on the use of conducting polymers as scaffolds in order to provide a comparison for the magnetoelectric materials developed here.

There are a variety of conducting polymers that have been used for neuronal stimulation including polypyrrole (PPy)31,32,41–43,33–40, polyaniline (PANI)44–46, and poly(3,4-

ethylenedioxythiophene) (PEDOT)47–55, among others.29,30,53–56 Several studies have been performed showing that growing neuronal or neuronal-like cells on conducting polymer scaffolds without an applied current improves the proliferation of the cells, which has been attributed to the electroactivity of the scaffold.1,33,40,42,46,48,49,57,58 In addition to studying the conducting polymer materials for their effects on proliferation and differentiation in the absence of electrical stimulation, work has been done to study the effects of these materials with the addition of electrical stimulation.1,29,31,32,44,45,50,56,59 Looking at the three conducting polymers mentioned above, polypyrrole is possibly the most studied conducting polymer for electrical stimulation. Schmidt et al. studied polypyrrole nerve guidance channels for their ability to increase neurite extension in PC12 cells in 1997.32 They found that PC12 cells that were cultured on 5 mm polypyrrole disks and subjected to an electrical stimulation of 100 mV for 2 hours displayed neurites nearly double the amount of neurite extension as compared to cells cultured on tissue culture polystyrene (18.14 µm versus 9.5 µm).32 Forciniti et al. later studied the effects of electrical stimulation of Schwann cells cultured on polypyrrole substrates.31 They found that applying an electric stimulus led to an increase in Schwann cell migration.31 Along with proliferation and differentiation, cell migration is an important factor in healing injuries to the peripheral nervous system. Thus, this result points to conducting polymers as being useful for nerve regeneration in ways beyond just proliferation and differentiation of the necessary cells.

While these results are specific for polypyrrole, there are also studies that show similar results for both polyaniline and PEDOT.31,32,41–46,33–40

While there is a great deal of promising work being done to study conducting polymers in combination with electrical stimulation for in vitro stimulation, there is comparatively little for work being done in vivo. This is largely due to the difficulty associated with applying an electric

stimulus inside of the body. Electric fields and electric stimuli have the potential to cause injury or even death given the conductivity of body tissue and the tendency of tissue to attenuate electric fields.60,61 Those devices that have successfully been able to apply electric stimuli in vivo, conducting polymer based or otherwise, typically require invasive electrical leads to be attached to the implanted device.14,15,41,62,63 Developing materials that enable local electrical stimulation in vivo without the need for invasive electrical leads would vastly improve the ability to aid in repairing nerve injuries. This is where materials such as magnetoelectric materials provide immense possibility for furthering the ability to less invasively aid in repairing nerve damage.

1.3.2 Piezoelectric Materials for Neuronal Stimulation

Given that electric fields are ubiquitous throughout the human body, piezoelectric materials can potentially be used to locally manipulate electric fields using mechanical deformation from stimulation techniques such as ultrasound waves. Piezoelectric materials are materials that are capable of producing a change in the electric polarization of the material under an applied mechanical deformation. Compared to the work done on the use of conducting polymers for neuronal stimulation, piezoelectric materials have been studied far less and for a much shorter time period. Piezoelectric materials have widely been studied for use with bone regeneration given the native piezoelectric properties of bone.64–69 Recently, however, they have also garnered interest in neuronal stimulation applications.25,69,78,79,70–77 Work has been done examining the effects of both piezoelectric scaffolds and piezoelectric nanoparticles on neuronal growth.

Many of the piezoelectric scaffolds that have been studied for neuronal stimulation use the piezoelectric polymer polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) as the piezoelectric material of choice.69–71,75,79,80 PVDF-TrFE scaffolds have been studied for their

effects on the differentiation of multiple neuronal cell types, including SH-SY5Y75, PC1270,

Schwann cells and dorsal root ganglia80, and mixed spinal cord cells,71 when mechanically stimulated to trigger the piezoelectric effect. The studies mentioned here for SH-SY5Y (human neuroblastoma) and PC12 (rat pheochromocytoma) neuron-like cells made use of ultrasound waves to produce the mechanical stimulation that triggers the piezoelectric properties of the scaffolds being studied.70,75 Both research efforts found that cells that were piezoelectrically stimulated exhibited greater differentiation than those that were not stimulated. Hoop et al. also compared the piezoelectrically stimulated cells to cells grown in media containing nerve growth factor (NGF) and found that piezoelectric stimulation had similar effects on the extent of neurite outgrowth of PC12 cells to those of growth in NGF containing media.70 The work done to study mixed spinal cord cells on piezoelectric scaffolds used a different method of mechanical stimulation, namely a vibration table, to trigger the piezoelectric effect in their scaffolds. 71 Like the studies done on SH-SY5Y and PC12 cells, this work also demonstrated that piezoelectric stimulation increased the differentiation of the cells being studied. Overall, the work done using piezoelectric scaffolds presents a promising method for localized electric stimulation by applying mechanical stimuli to the piezoelectric scaffolds.

In addition to piezoelectric scaffolds, research has also been done examining piezoelectric nanoparticles for their effects on neuronal stimulation.25,72,76–78 There are two different types of piezoelectric nanoparticles that have been the focus of this research, namely, barium titanate nanoparticles and boron nitride nanotubes. Like the work done with piezoelectric scaffolds, the method of choice for stimulating the piezoelectric effect in these nanoparticles has been ultrasound stimulation.77,78 In 2010, Ciofani et al. studied the use of boron nitride nanotubes and their effects on the neurite outgrowth of PC12 cells. Their work demonstrated significantly

greater neurite outgrowth in the PC12 cells that were treated with boron nitride nanotubes and stimulated using ultrasound.77 The effects of barium titanate nanoparticles on the differentiation of SH-SY5Y cells were studied in 2015 by Marino et al. This work focused on studying the effects of ultrasound stimulation on samples with barium titanate nanoparticles on the activation of Ca2+ transients which mediate the enhancement of neurite outgrowth.78 Samples containing tetragonal barium titanate nanoparticles (tetragonal barium titanate exhibits piezoelectric properties while cubic barium titanate does not) that underwent ultrasound stimulation were

2+ 78 found to activate larger amounts of Ca transients than the unstimulated samples. More recently, interesting work by Rojas et al. used micro-electrode arrays to directly study the activation of neuronal cells due to ultrasound stimulation of barium titanate nanoparticles.72

Here, studies were carried out using cortical and hippocampal neurons obtained from rats. These cells were allowed to form a neural network on the micro-electrode arrays over the course of at least 21 days before being seeded with barium titanate nanoparticles for stimulation experiments.

The results of their experiments showed that those samples seeded with tetragonal barium titanate nanoparticles saw an increase in the firing rate of the network when stimulated with ultrasound.72 Thus, ultrasound stimulation coupled with piezoelectric barium titanate particles was shown to be an effective method of modulating activity of neural networks making it a possible method for a minimally invasive way to trigger neural responses.72

1.3.3 Benefits of Magnetoelectric Materials Over Electronic Materials

Electronic materials have been demonstrated to be useful in the monitoring and stimulation of neuronal signaling and growth. One of the major problems with electronic materials, as discussed section 1.3.1, is the need for electrical leads to be implanted within the body in order to use electric stimuli. By using magnetoelectric materials, this can be overcome due to the permeability of the human body to magnetic fields that would then trigger an electric

response. Piezoelectric materials, like magnetoelectric materials, present an opportunity for developing minimally invasive materials capable of producing local electric stimulation.

Ultrasound is one of the main ways of providing the mechanical stimulation necessary to trigger the piezoelectric effect in these materials. There are, however, limitations to using ultrasound to trigger the piezoelectric effect, namely that ultrasound waves are attenuated by body tissue.81

Unlike ultrasound, magnetic fields are not attenuated by the human body. Thus, magnetoelectric materials, which harness piezoelectric materials for their ability to create local electric stimulation, may be more suited to stimulation within the body, especially for deep tissue locations.

1.4 Ceramic Electrospinning and Electrospraying Routes for Magnetoelectric Composites

Now that the reasoning behind the use of magnetoelectric materials has been explained, it is necessary to discuss how these magnetoelectric materials have been synthesized. Traditionally, magnetoelectric materials are synthesized as either bulk materials or as thin films. However, both bulk materials and thin films present their own unique problems when it comes to optimizing the magnetoelectric effect.

Before discussing bulk magnetoelectrics and thin film magnetoelectrics, it is necessary to understand the different phase connectivities that can be used to create these composites. The idea of phase connectivity was introduced by Newnham et al. in 1978.82 Newnham defines connectivities using the number 0-3 where the number refers to the dimension of the part of the composite.82 Thus, 0-dimensional features include particles, 1-dimensional features include fibers and rods, 2-dimensional features refers to thin films, and finally 3-dimensional features refer to the bulk matrix. Common connectivities for composites include 0-3, particulates in a matrix, 2-2, laminate thin films, and 1-3, fibers or rods within in bulk matrix or thin film (Figure

1-4).

The above description of phase connectivities facilitates the discussion of traditional bulk magnetoelectric composites. Traditionally, bulk magnetoelectrics have been synthesized in one of two ways; either by unidirectional solidification of a eutectic composition melt or by conventional solid state techniques.17 Unidirectional solidification results in high quality magnetoelectric composites that can achieve magnetoelectric coefficients as high as

130 mV/cm Oe. However, this technique can be very expensive due to the need for very precise control over the solidification in order to achieve the desired results.17 Conventional solid state techniques are significantly less expensive that unidirectional solidification. However, the resultant materials typically possess a lower magnetoelectric coefficient. This is due to a variety of reasons including interdiffusion of the piezoelectric and ferromagnetic materials at high temperatures and porosity and cracking at the interfaces caused by a mismatch of thermal expansion coefficients between the two materials.17 These defects limit the transfer of strain between the materials, thus reducing the magnetoelectric response of the composite.

Magnetoelectric thin films, like bulk magnetoelectric composites, also suffer from issues that diminish their magnetoelectric properties, such a substrate clamping. T these composites are produced using a variety of deposition techniques ranging from spin-coating with a sol-gel precursor to the use of physical or chemical vapor deposition, all of which require a substrate onto which the materials are deposited.17,83–86 Substrate clamping occurs when the magnetoelectric composite is strained, either with a magnetic or an electric stimuli, but it is not capable of straining to its full extent as it is attached to the substrate material. There are two main connectivities seen in thin film magnetoelectric materials: 2-2 (laminate films) and 1-3 (rods within a film).17 Thin films with 1-3 connectivities show stronger magnetoelectric properties as compared to 2-2 connectivities due to reduced substrate clamping effects by the presence of

pillars or either the piezoelectric or magnetoelectric material.17,87,88 However, these 1-3 composites still suffer from leakage due to the low resistance of the magnetic materials, which reduces the magnetoelectric properties of the composite.17

Given the issues with both bulk and thin film magnetoelectric materials, in addition to needing nanoscale materials that can be used in the body, it is reasonable to seek out a method of developing such composites that are nanoscale but do not suffer from being clamped by a substrate. Starr et al. and Baji et al.89–91 have both produced magnetoelectric composite nanofibers that achieve this goal. These nanofibers were produced using electrospinning which is a technique that does not require a substrate by nature. For the purposes of this work, these electrospinning techniques have been used to create magnetoelectric composites. The technique has also been taken a step further towards electrospraying in order to achieve magnetoelectric nanoparticles that are free-standing.

1.4.1 Electrospinning

Electrospinning is a synthesis technique for nanofibers that was originally used to synthesize polymeric nanofibers. However, this technique has also been shown to be an effective route for producing ceramic nanofibers by combining traditional polymeric electrospinning with ceramic sol-gel chemistry.89,90,100–104,92–99 Sol-gel electrospinning has previously been used to create ceramic fibers of materials such as titania, barium titanate, lead zirconate titanate, cobalt ferrite and nickel zinc ferrite, among others.92–101

As its name suggests, electrospinning makes use of electric fields in order to create micro- or nanofibers. Specifically, a typical electrospinning setup, as shown in the schematic in

Figure 1-5, consists of a syringe of solution connected to a high voltage source placed across from a metal collector plate. The syringe of solution is placed in a syringe pump which allows for the solution to be pumped out of the syringe until it forms a droplet at the end of the syringe

needle, which when the high voltage source is applied leads to charge build up on its surface.

The repulsion of the charges building up on the surface of the droplet and the Coulombic forces from the electric field cause it to deform into a cone shape which is generally referred to as a

Taylor cone.95 In order for electrospinning to occur, the repulsion of the charges on the surface of the droplet must overcome the surface tension of the droplet. Once this happens, a jet of solution is extruded into the electric field towards the collector plate. The viscoelastic restoring force caused by the polymer content of the precursor solution prevents the solution breaking up into droplets while moving towards the collector resulting in electrospun fibers.

There are a variety of both experimental parameters and solution properties that affect the electrospinning process. Among the electrospinning parameters that can be altered are the environmental humidity, flow rate, applied voltage, and distance between the needle and collector.95 Many of these parameters are inter-related and changing one means that another must be changed. There are however some general trends for all of these parameters. For instance, higher flow rates result in fibers with a larger diameter than low flow rates. On the other hand, increasing either the applied voltage or the distance between the needle and collector has a tendency to result in fibers with a smaller diameter than those spun at lower voltages or closed distances. The humidity of the electrospinning environment affects the fiber morphology and even the ability to electrospin fibers, especially in ceramic electrospinning.

This is due in large part to the sol-gel nature of the solutions used for ceramic electrospinning.

Sol-gel precursors are extremely sensitive to the presence of water in the electrospinning environment and this can drastically affect the success of electrospinning. Briefly, too high of a humidity in the environment can lead to gelation issues with the Taylor cone thus halting electrospinning all together. The same is true of conditions where the humidity is too low as the

electrospinning solvents evaporate too quickly and the sol-gel solution gels at the needle tip.

High humidity levels can also affect fibers that have already been deposited on the grounded collector causing them to gel into something that more closely resembles a film rather than a random mat of fibers. Sol-gel chemistry and the effects of humidity will be discussed in more detail in Chapter 2.

As mentioned above, there are also properties of the precursor solutions that can be altered which affect the electrospinning process. These include the ceramic precursor concentration, viscosity, conductivity, and surface tension.95 Firstly, the concentration of the ceramic components can affect the size of the resultant fibers. Generally, if the concentration of the ceramic precursors is increased, once the fibers have been calcined it can result in fibers with a larger diameter than those spun from low concentration solutions. This is because more material remains following the burning off of the polymer binder from the as-spun fibers.

Looking next at the effects of solution viscosity, there are also some general trends that can be seen. Specifically, electrospinning solutions that have higher viscosities tend to result in fibers with a larger diameter. In the case of ceramic electrospinning, this is usually accomplished by increasing the concentration of the polymeric binder. On the other hand, electrospinning solutions that have too low of a viscosity can cause morphological issues in the resultant nanofibers. For instance, low viscosity solutions often result in fibers that exhibit undesirable beading (Figure 1-6) which means that the thickness of the fibers will not be consistent. In addition, very low viscosities will result in the electrospinning jet breaking up into droplets and particles will be obtained rather than fibers. Another important issue with electrospinning solutions that have viscosity that is very low is that they also tend to exhibit less viscoelastic response. Viscoelasticity is important for achieving nanofibers rather than

nanoparticles. Specifically, the viscoelastic restoring force is necessary to resist the breakup of the jet into droplets in order to achieve nanofibers rather than nanoparticles.

Unlike viscosity and the viscoelastic restoring force, the minimization of the surface area of the electrospinning jet serves as a driving force for changing the jet into droplets rather than for maintaining a continuous fiber.95 Surface tension effects are responsible for Rayleigh instabilities which will be discussed in more detail in the following section. For the purposes of electrospinning, it has been found that high surface tension can also lead to morphological issues such as beading.95 Thus, solutions with lower surface tensions are preferable for electrospinning.

The final solution property that can have a significant effects on electrospinning is the solution conductivity. In order to electrospin, the solutions must exhibit some amount of conductivity and increases in the solution conductivity can lead to obtaining fibers with a smaller diameter, up to a point. This is especially true for polymeric electrospinning. However, ceramic electrospinning solutions naturally have much higher conductivities than those of polymer solutions. At high conductivity levels in ceramic electrospinning solutions it can become difficult to electrospin high quality fibers which is an issue that can often occur for electrospinning ceramic nanofibers.

Sol-gel electrospinning can be used as a method for electrospinning nanofibers of a single material, but it can also be used to electrospin composite materials.89–91,95,97–99,105

Electrospun composite magnetoelectric nanofibers have previously been created with three different morphologies: a random dispersion of the magnetostrictive and piezoelectric phases, core-shell combinations of the two phases, and finally, Janus-type combinations of the two phases (Figure 1-7).89–91,105 Here, Janus refers to having the magnetostrictive and piezoelectric

phases coupled longitudinally along the length of the nanofiber.89,90 For the purposes of this work, Janus-type nanofibers are studied. This morphology was chosen over the random dispersion morphology because of the large exposed surface areas of the two phases which could lead to preferential strain relaxation over strain transfer between the two phases. Janus is chosen over core-shell morphologies due to the issues that have been seen by Xie et al. with delamination of the two separate phases during calcination caused by disparate coefficients of thermal expansion.105

1.4.2 Electrospraying

Electrospraying is being used here as a method for the synthesis of composite nanoparticles, specifically, composite nanoparticles of the Janus morphology. Magnetoelectric

Janus composite particles are desirable as they are predicted to have enhanced magnetoelectric properties as compared to fibers. The reason for using electrospraying to synthesize particles with the Janus morphology is that it would be difficult to obtain through more traditional particle synthesis routes.

Electrospraying operates similarly to electrospinning and uses an electric field that is created between a grounded collector and a syringe nozzle to shear a liquid jet into droplets.106

Typically for electrospraying, a metal syringe needle is connected to a high voltage power supply and placed opposite a grounded collector plate. The application of a voltage to the needle creates an electric field between the syringe and the collector plate. A syringe pump is used to compress the syringe and form a droplet of the precursor solution at the needle tip. The high voltage applied to the syringe needle, causes the build-up of charge on the surface of the pendant droplet at the needle tip. Repulsion between the like charges on the surface of the droplet combined with the Coulombic forces of the electric field cause the droplet to distort into a cone shape, referred to as the Taylor cone.95 In order for electrospraying to occur, the repulsion of the charges on the

surface of the droplet must overcome the surface tension of the droplet. At this point, a jet is extruded from the droplet into the electric field. In electrospraying, the lack of a viscoelastic restoring force from the presence of a polymer binder results in the jet breaking up into droplets.95,106 These charged droplets are propelled towards the collector plate by the electric field.

One of the main differences between electrospinning and electrospraying is that electrospraying solutions lack a polymer binder, which enables to formation of droplets over fibers. The variables that play a role in electrospinning also affect electrospraying. However, the effects of certain variables seen in electrospinning, such as humidity, can have far greater effects when electrospinning. Conversely, there are variables that drastically affect electrospinning, such as viscosity, which can be largely ignored in electrospraying.106,107

Electrospraying has previously been used to synthesize polymeric particles, as well as a method that can be used to make amorphous, simple oxide (i.e. zirconia or titania) particles as well.95,106–108 The synthesis of complex-oxide ceramic particles, such as barium titanate or cobalt ferrite, using this technique adds additional experimental complexity due to the increased number of ceramic ions and this charges in the precursor solutions.109 A major effort in this dissertation was in establishing the appropriate precursor solution chemistries that enabled the electrospraying of complex oxide nanoparticles, the details of which will be discussed in

Chapters 3and 4.

Like electrospinning, electrospraying makes use of an applied electric field to produce an electrified jet of solution. As this electrified jet flies towards a grounded collector plate, the jet is broken up into droplets in order to minimize the ratio of charge to surface area.95,106,107 Rayleigh instability is the breaking up of the jet into droplets, and of droplets breaking apart further as

caused by the build-up of charge on the droplet surfaces.107,110,111 The Rayleigh instability is a complex phenomenon in electrospraying that is affected by a variety of solution properties including conductivity, solution volatility, and surface tension. The theory of Rayleigh instability is based on the balance between the charge on the surface of the electrosprayed droplets (which is related to the conductivity of the solution) and the surface tension of the droplet. Specifically, a Rayleigh limit is defined as a threshold at which the accumulation of the surface charge on the droplet is no longer balanced by the surface tension of the droplet. This results in the elongation of the droplet until it breaks up into multiple smaller droplets.107 By breaking up into smaller droplets the surface charge is re-distributed over a greater surface area. This process continues as the solvent used in the precursor solution continues to evaporate, until the particles solidify, potentially forcing droplets to break up into smaller droplets multiple times.107 During this process, it is possible for morphologies such as particles with tails as shown in Figure 1-8 can be locked in as the solvent is evaporated and th-gel reaction proceeds.

The importance of Rayleigh instabilities on the final morphology of as-sprayed particles, as opposed to electrospinning, is largely due to the differences in the composition of the precursor solutions used for electrospraying versus those used for electrospinning. Namely, for electrospraying, the polymer binder that is necessary to obtain a viscoelastic restoring force for electrospinning, is omitted from the precursor solutions. This is because such large amounts of viscoelastic restoring forces as are observed with polymer binders are unnecessary and will actually hinder the production of particles in electrospraying.106,107 Removing the polymer binder from these solutions in turn makes electrospraying more sensitive to humidity due to the sol-gel reactions that take place to from the ceramic component. The details of the sol-gel reactions that occur during electrospinning and electrospraying will be discussed further in the next section.

1.4.3 Sol-Gel Chemistry

Traditionally, electrospinning and electrospraying have been used to create polymeric nanomaterials. In order to produce ceramic nanomaterials instead, the methods of electrospinning and electrospraying polymeric nanomaterials can be combined with sol-gel chemistry. Sol-gel processes are typically used to synthesize bulk nanopowders of a variety of ceramic materials. There are two main routes for sol-gel processes: gelation from colloidal solutions and hydrolysis and condensation from precursor solutions.112,113 For the purposes of this work, the focus will be on sol-gel processes involving hydrolysis and condensation from precursor solutions.

Sol-gels produced using hydrolysis and condensation from precursor solutions can use a variety of precursors including metal alkoxides, metal nitrates, and metal acetates. Regardless of what type of precursor is used, sol-gel solutions are sensitive to a variety of variables, including but not limited to, pH, precursor concentration, and the presence of water.113 In general, hydrolysis and condensation reactions occur as follows:114

퐻푦푑푟표푙푦푠푖푠: 푀(푂푅)푧 + 푥퐻2푂 → 푀(푂푅)푧−푥(푂퐻)푥 + 푥푅푂퐻 (1-4)

퐶표푛푑푒푛푠푎푡푖표푛: − 푀 − 푂퐻 + 퐻푂 − 푀 − → −푀 − 푂 − 푀 − + 퐻2푂 (1-5)

Where M represents a metal ion, O represents oxygen, H represents hydrogen, and R represents a functional group. The presence of water in both hydrolysis and condensation reactions as listed above indicates that both reactions are sensitive to water. Specifically, water acts to move hydrolysis reactions forward to the formation of M(OR)z-x(OH)x. For condensation reactions, water acts to drive the reaction in the reverse direction. Based on these reactions, the presence of water in sol-gel reactions must be controlled in order to control the rate of the reactions.

Chelating agents are one addition to the chemistry of sol-gels that can be used to help increase the homogeneity of a solution, thus improving the quality of the end product, as well as

slow down the condensation reactions of the sol-gel process.115 Chelating agents work by coordinating the metal ions in solution. This prevents OH groups that trigger condensation reactions from reaching the metal ions.116 This in turn allows for the diffusion of the coordinated metal ions such that there is an even distribution of, for example, iron and cobalt ions in solution.

This results in a precursor that is more homogenous and thus in final product that contains primarily cobalt ferrite as opposed to cobalt oxide, iron oxide, or other impurity phases. In traditional sol-gel processes, the presence of the chelating agent and delay in condensation reactions would result in an end product that is less dense than if condensation had not been impeded by the addition of chelating agents.116 The decrease in density caused by slowing down the condensation reactions can also sometimes be seen in sol-gel electrospinning with the end result being porous nanofibers.

Looking at sol-gel solutions as they are utilized for electrospinning and electrospraying solutions, we find that, like their more traditional counterparts, these solutions also remain highly sensitive to water. For both electrospinning and electrospraying, this typically means that the water vapor in the electrospinning environment can have a large impact on the final morphology of the fibers or particles. In electrospinning, high humidity levels can result in fibers melding together on the collector plate as the sol-gel reactions proceed between the fibers. Sol-gel electrospraying is even more sensitive to humidity that electrospinning. The humidity of the electrospraying environment affects the solidification of the particles in conjunction with the evaporation of the solvent which was discussed above. Li et al. have shown that varying the relative humidity of the electrospraying environment by as little as 10 % can drastically change the morphology of the as-sprayed particles (Figure 1-9).110 For sol-gel electrospraying, the solidification of the particles depends on the balance of two processes: the evaporation of the

electrospraying solvent and the sol-gel hydrolysis and condensation reactions. At low humidity levels, evaporation dominates the solidification of the droplets, while at high humidity levels, the hydrolysis and condensation reactions dominate the solidification of the droplets into particles.

By combining sol-gel chemistry with traditional electrospinning and electrospraying techniques it is possible to create ceramic nanomaterials. Electrospinning and electrospraying synthesis methods, as described here, enable the facile synthesis of standalone Janus type magnetoelectric nanocomposites. By using these methods, magnetoelectric nanofibers and nanoparticles can be developed for their use in biomedical applications such as neuronal stimulation.

1.5 Objective and Summary of Dissertation

This dissertation focuses on investigating the potential for magnetoelectric materials as a means to create local electric fields within the body. These local electric fields have the potential to beneficially affect the growth and differentiation of neuronal cells so as to improve healing of neurons following an injury.

Chapter 2 describes the use of sol-gel electrospinning to create magnetoelectric Janus fibers with a detailed examination of the sol-gel chemistry of the precursor solutions and how they affect the electrospinning process. Chapter 3 discusses the development of synthesis methods for single phase barium titanate particles via sol-gel electrospraying, with a particular focus on the effects of humidity of the final morphology of the particles. Chapter 4 instead focuses on developing synthesis methods for electrospraying single phase cobalt ferrite particles.

Chapter 5 discusses the methods for creating magnetoelectric fiber – hydrogel composites and their ability to affect the growth and differentiation of neuron-like PC12 cells in vitro when placed in a magnetic field. Finally, Chapter 6 features the conclusions and future directions of the work presented in this dissertation.

Figure 1-1. Schematics of a bidirectional neural interface device that has been developed for electrical stimulation and recording of neuronal activity. Adapted from Fraczek- Szczypta. 13

Figure 1-2. Diagram depicting the variety of multiferroic coupling that is possible and the related properties. Adapted from Spaldin et al.16

Figure 1-3. Schematic of magnetoelectric coupling in composites: A) magnetostrictive material undergoes a shape change in a magnetic field, straining the piezoelectric material, producing a spontaneous polarization, and B) a piezoelectric materials undergoes a shape change in an applied electric field, straining the magnetostrictive materials, producing a magnetization.

Figure 1-4. Schematics of three different types of composite connectivity schemes. (a) shows 0-3 connectivity, (b) show 2-2 connectivity and (c) shows 1-3 connectivity.

Figure 1-5. Schematics for (a) a basic flat plate collector electrospinning setup and (b) a flat plate collector electrospraying setup up.

Figure 1-6. SEM of PVP nanofibers made from solutions of different viscosity (based on the concentration of PVP) from Li and Xia.95 The weight percentage of PVP increases from A-C: 3 wt%, 5 wt%, and 7 wt%. Image D shows PVP fibers containing tetramethylammonium chloride. As PVP concentration increases from A-C, beading decreases as the viscosity increases.

Figure 1-7. Schematic of the three different possibilities for the morphology of electrospun nanofibers: randomly dispersed grains, core-shell and Janus morphologies.

Figure 1-8. SEM images of as-sprayed titania particles produced by Li et al.110 These particles have been electrosprayed at 4 different humidities: (A) 14 %, (B), 28 %, (C) 38 %, and (D) 55 %. The varied morphologies shown here are attributed to Rayleigh instability effects.

Figure 1-9. Schematic of the process of basic sol-gel reactions.

CHAPTER 2 SOL-GEL ELECTROSPINNING OF JANUS TYPE MAGNETOELECTRIC NANOFIBERS

2.1 Introduction

The first type of multiferroic nanocomposites that were examined were those of bi-phasic

Janus fibers. Magnetoelectric Janus fibers were developed to be used for biomedical applications, specifically for the stimulation of neuronal growth, which will be discussed in

Chapter 5. The electrospinning techniques described in Chapter 1 provide a method for creating ceramic nanofibers that exhibit 1-1 connectivity similar to the 2-2 connectivity of thin films while avoiding issues such as substrate clamping given that the fibers are free-standing. Our group has previously demonstrated the successful synthesis of both barium titanate-cobalt ferrite and cobalt ferrite-bismuth ferrite Janus nanofibers.89,90,117 The focus here was developing new, more reliable sol-gel solutions for obtaining magnetoelectric barium titanate-cobalt ferrite Janus nanofibers, where barium titanate is the piezoelectric material and cobalt ferrite is the magnetostrictive material used to obtain the magnetoelectric composite. Previous solutions for the synthesis of Janus fibers, tended to result in the formation of films or webbed fibers rather than discrete nanofibers. This required altering the chemistry of the sol-gel solutions being used to create the nanofibers. Specifically, this work focuses on the use of chelating agents to alter the precursor solutions for cobalt ferrite. Cobalt ferrite precursor solutions as previously described89 also tended to result in films or webbed fibers when electrospun for single-phase fibers. Cobalt ferrite precursor solutions are extremely sensitive to the presence of water which is a main factor in why electrospinning of these solutions can prove difficult. Barium titanate precursor solutions as described by Starr et al. were found to reliably produce good quality nanofibers and therefore the chemistry was not altered.89 In order to optimize the chemistry of the cobalt ferrite sol-gel precursors, single-fibers were first electrospun before attempting Janus fibers.

Traditionally, sol-gel chemistry has been used to synthesize bulk nanopowders of ceramic materials. Sol-gel chemistry relies on hydrolysis and condensation reactions from metal alkoxides, nitrate, acetates, or salts to create these powders and is notoriously sensitive to a number of factors including water content, concentration, and chelating agents, among others.

This holds true for sol-gel electrospinning. The concentration of the precursors and the presence or absence of chelating agents are solution parameters that were altered here in attempts to create electrospinning solutions that reliably form the desired Janus morphology. Chelating agents are commonly used in sol-gel chemistry in order to form more homogenous networks of metal ions.

Two specific chelating agents are discussed here for their use in creating sol-gel precursors of cobalt ferrite that readily and reliably electrospin: citric acid and acetylacetone. Both of these chelating agents are commonly used in traditional sol-gel syntheses of ferrites. Citric acid can act as either a monodentate or bidentate chelator.115 Monodentate and bidentate refer to the number of bonds that are formed between the chelator, or ligand, and the metal. So in the case of a monodentate ligand, one bond is formed between the ligand and the metal ion. For bidentates, there are two bonds between the chelating agent and a metal ion. Generally, metal complexes with higher dentate ligands (so bidentate versus monodentate) are considered to be more stable.116 Metal acetylacetonates, which form upon the addition of acetylacetone to the cobalt ferrite precursor solutions, are supposed to exhibit increased moisture stability.118,119 Therefore, the use of a bidentate chelating agent would likely produce sol-gel precursor solutions that are more stable and less sensitive to water. Acetylacetone is a bidentate chelating agent.116 This chapter discusses the results of using either of these two chelating agents in attempts to create cobalt ferrite electrospinning solutions.

2.2 Experimental Methods

2.2.1 Materials

Cobalt (II) nitrate hexahydrate, barium acetate, titanium (IV) isopropoxide (≥ 97.0%), glacial acetic acid, citric acid monohydrate, and polyvinylpyrrolidone (M.W. 1,000,000) were obtained from Sigma Aldrich. Ferric nitrate nonahydrate, acetylacetone, and ethanol were obtained from Fisher Scientific.

2.2.2 Sol-Gel Precursor Solutions

Barium titanate – cobalt ferrite Janus fibers were synthesized via sol-gel electrospinning.

To create the barium titanate precursor solution, 0.423 g of barium acetate was dissolved in 3 ml of glacial acetic acid under heating to promote dissolution. After cooling to room temperature,

0.493 ml of titanium (IV) isopropoxide was added to the barium acetate and acetic acid solution.

Simultaneously, a polymer binder solution was also mixed by dissolving 0.45 g of

1,000,000 MW polyvinylpyrrolidone (PVP) in 3 ml of ethanol.

Similar procedures were followed to create the precursor solutions for cobalt ferrite.

However, unlike barium titanate, it was found that the cobalt ferrite solutions required a chelating agent in order to obtain solutions that would electrospin nanofibers. The base cobalt ferrite solution consisted of 1.34 g of ferric nitrate nonahydrate and 0.48 g of cobalt nitrate hexahydrate dissolved in 3 ml of ethanol. This solution was further modified with either acetylacetone or citric acid in order to test the efficacy of the chelating agent. Simultaneously, a polymer binder solution much like the one used for barium titanate was mixed. Here, the solution consists of 0.4 mg of PVP dissolved in 3 ml of ethanol, instead of the 0.45 g PVP used for barium titanate solutions. For both cobalt ferrite and barium titanate, once the individual solutions were thoroughly mixed, they were combined to create the final sol-gel precursor.

Single phase barium titanate and cobalt ferrite fibers were synthesized in addition to the biphasic

Janus fibers.

2.2.3 Nanofiber Synthesis via Electrospinning

The electrospraying parameters used to obtain good fibers, both single phase and biphasic, are detailed below. Firstly, in order to prevent premature gelation of the solutions during electrospinning, the humidity of the electrospraying environment was kept low, typically around 35 % relative humidity. Fibers were electrospun from a distance of 20 mm from needle tip to collection plate. For barium titanate single phase fibers, an applied voltage of 11 kV is used. To spin cobalt ferrite fibers, the voltage needed to be slightly higher, around 14 kV.

Composite Janus fibers were also electrospun using an applied voltage of 14 kV. The

Janus morphology was described in Chapter 1 and can be seen in Figure 1-9. Briefly, the Janus morphology features two semi-cylinders of materials coupled longitudinally along the length of the fiber. Janus fibers were not formed below an applied voltage of 14 kV as the cobalt ferrite precursor solutions do not electrospin, and single-phase barium titanate fibers are produced. At higher applied voltages, both precursor solutions electrospin, however, they do not spin composite fibers but rather a mixture of single-phase fibers.

Following electrospinning, the fibers were placed in a vacuum oven overnight to remove any excess solvent. The fibers were then calcined in order to achieve ceramic fibers. The fibers were first heated to 250 °C for 1 hour at a ramp rate of 10 °C/min to burn off the polymer binder.

Finally the fibers were heated to 800 °C for 3 hours at a ramp rate of 2 °C/min to achieve crystalline fibers.

2.2.4 Nanofiber Characterization

Both as-spun and calcined fibers were examined using an FEI Nova 430 scanning electron microscope to determine their size and morphology. To examine the crystalline structure

of the fibers, they were examined using x-ray diffraction (PANalytical X’pert Powder

Diffractometer) and Raman spectroscopy (Renishaw InVia Raman spectrometer). Magnetic and qualitative magnetoelectric properties of the fibers were examined using superconducting quantum interference device (SQUID) magnetometry (Quantum Design MPMS3) and vibrating sample magnetometry (VSM, MicroSense EZ VSM). In order to qualitatively examine the magnetoelectric properties of the Janus fibers, the samples were heated through the Curie temperature of barium titanate while measuring magnetization as a function of temperature. It is expected that at the Curie temperature, a change in magnetization would be seen as a result of a shape change in the barium titanate being transferred to the cobalt ferrite.

2.3 Results and Discussion

Before attempting to synthesize Janus fibers, synthesis methods for both single-phase barium titanate and single-phase cobalt ferrite were first studied. This enabled any adjustments of the sol-gel precursor solutions and electrospinning conditions without having to adjust the electrospinning parameters for bi-phasic electrospinning. Understanding single-phase electrospinning before attempting to electrospin composite nanofibers will help clarify the parameters necessary for electrospinning composites.

2.3.1 Single-Phase Barium Titanate Fibers

The first material electrospun was barium titanate due to its relative ease of electrospraying as compared to cobalt ferrite. Barium titanate solutions as described in the work completed by Starr et al. did not require modification through the addition of chelating agents in order to reliably achieve barium titanate nanofibers. The only change done here to those solutions used by Starr et al. for the synthesis of barium titanate fibers was the addition of a heating step in order to better dissolve the barium acetate precursors. The addition of this heating

step improved the stability of the barium titanate solutions and prevented them from crashing during the time required for mixing the precursor solutions.

The barium titanate fibers synthesized using these solutions were first examined using

SEM to look at the morphology and size of the fibers. Figure 2-1 shows both as-spun and calcined barium titanate nanofibers. During calcination, the polymer binder is burned off at low temperatures followed by the formation of crystalline barium titanate. The removal of the polymer binder from these fibers during calcination causes a reduction in the diameter of the fibers. The average diameter of the as-spun barium titanate nanofibers is 315.0±92.6 nm, while the average diameter of the calcined fibers is 189.1±44.6 nm .

Following SEM characterization of the fibers, XRD was used to confirm the presence of barium titanate. Figure 2-2 shows XRD data for the calcined barium titanate fibers. This confirms the presence of tetragonal barium titanate in the fibers and can also be used to determine the average crystallite size of the barium titanate through the use of Scherrer’s equation shown below.

퐾휆 (2-1) 휏 = 훽 cos 휃

Here τ is the crystallite size, K is a shape factor that typically has a value of 0.9, λ is the wavelength of incident X-rays, here 1.54 Å, β is the line broadening at the full width half maximum intensity in radians, and θ is the Bragg angle.114,120 In order to determine an accurate crystallite size, the instrument broadening was obtained from XRD scans of a lanthanum hexaboride standard. This number is then subtracted from β before using Scherrer’s equation.

The average crystallite size of the barium titanate fibers was determined to be 23.2±0.004 nm using this technique. The peaks measured for the purpose of determining crystallite size were those for the (101), (111), and (200) planes.

While XRD confirmed that the fibers were indeed composed of barium titanate, it was found to be difficult to distinguish whether the fibers were composed of cubic or tetragonal barium titanate due to peak broadening cause by nanoscale crystallites. For this reason, Raman spectroscopy was done. Raman spectroscopy probes the local ordering of the material as opposed to XRD which examines the average structure. Figure 2-3 shows the Raman spectra of calcined barium titanate fibers. Raman revealed the presence of both tetragonal and cubic barium titanate in the fibers. Tetragonal barium titanate was the desired phase of barium titanate as it is the phase the exhibits piezoelectricity.

2.3.2 Single-Phase Cobalt Ferrite Fibers

While barium titanate proved to be a relatively easy to electrospin complex oxide, the same was not found to be true of cobalt ferrite. This is due to the increased conductivity of cobalt ferrite precursor solutions as well as its increased sensitivity to water. In order to achieve precursor solutions of cobalt ferrite that readily electrospin the effects of two different chelating agents on the cobalt ferrite solutions were examined. These two chelating agents were citric acid and acetylacetone. The use of chelating agents in sol-gel chemistry has been shown to improve the homogeneity of the sol-gel solutions as well as to reduce impurities in the end- products.115,116,118 In addition, some have found that the addition of chelating agents reduces the sensitivity to water and slows down the condensation reactions of sol-gel chemistry. With all of this combined, the hypothesis was that the addition of chelating agents would create more homogeneous cobalt ferrite precursor solutions that would also electrospin more readily.

The first chelating agent that was examined was citric acid. Citric acid, a schematic for which is shown in Figure 2-4, was chosen as it is a commonly used chelating agent in traditional sol-gel chemistry.121–130 A variety of concentrations of citric acid were tried in attempts to create cobalt ferrite sol-gel solutions. These are denoted as ratios of Co:Fe:citric acid (Co:Fe:CA)

where the ratio of Co:Fe always remains 1:2. The Co:Fe:CA ratios studied were 1:2:3, 1:2:2,

1:2:1, and 1:2:0.5. The ratio of 1:2:3 was chosen as the initial citric acid concentration based on

121 work done by Hwang et al. on LiMn2O4 sol-gels. Table 2-1 shows the time to gelation of the precursor solution based on the Co:Fe:CA ratio. At high ratios of citric acid, gelation occurs very rapidly making the solutions unusable for electrospinning (Table 2-1 and Figure 2-5). This could be due to the acidic environment that is created by the addition of higher concentrations of citric acid to the cobalt ferrite precursor solutions. At low pHs, such as those created by the addition of high concentrations of citric acid, complete complexation of the metal ions with citric acid does not occur.127 This in turn enables rapid hydrolysis and condensation.116 The solution with

Co:Fe:CA ratio of 1:2:0.5 remained un-gelled for more than 24 hours, so this concentration of citric acid was chosen for further work on developing cobalt ferrite electrospinning solutions. To make solutions for electrospinning, Co:Fe:CA solutions of ratio 1:2:0.5 were added to a polymer binder solution.

Attempts at electrospinning cobalt ferrite solutions containing citric acid, however, did not reliably produce nanofibers with a consistent morphology (Figure 2-6). During electrospinning, material was deposited on the aluminum foil collector (Figure 2-6a). However, when these fibers were examined in SEM, it was found that the fibers had melded together as seen in Figure 2-6b. Despite varying several other variables, including overall metal ion concentration (0.55 M reduced to 0.27 M), polymer binder concentration (0.033 g/ml, 0.038 g/ml, 0.04 g/ml or 0.05 g/ml), solution aging (1 hour, 5 hours or 24 hours), and electrospinning parameters (10-23 kV applied voltage, 14-20 cm plate to collector distance, and 15-35 % relative humidity), cobalt ferrite fibers could not be produced from solutions containing citric acid.

When citric acid did not provide promising results for the development of spinnable cobalt ferrite solutions, the second chelating agent, acetylacetone was tried. Acetylacetone is considered a bidentate ligand and coordinates to cobalt and iron as shown in Figure 2-7. The addition of acetylacetone had two noticeable effects on the sol-gel precursor solutions for cobalt ferrite: the solution changed from a brown-red color to a dark, almost black solution and the viscosity of the solution was increased. Measurements of the conductivity of the sol-gel precursor solutions also revealed that the conductivity of the solution containing acetylacetone was increased in comparison to those either without a chelating agent or those containing citric acid. Measurements of both viscosity and conductivity for a variety of cobalt ferrite precursor solutions are shown in Table 2-2. Despite the conductivity of the cobalt ferrite solutions that contained acetylacetone being higher than those with either no chelating agent or with citric acid, attempts at electrospinning these solutions successfully produced nanofibers of cobalt ferrite.

The increased conductivity of the cobalt ferrite solutions containing acetylacetone was found to affect the electrospinning of fibers from this solution, namely by causing the fibers to extend back towards the syringe needle once they had been deposited on the flat plate collector (Figure

2-8). As a result of this, fibers not only deposited on the collector but on the fibers extending back to the syringe needle as well. Some of this could be mitigated by increasing the flow rate at which the house air was being pumped into the electrospinning box. By increasing the air pressure, and thus the speed at which air was being pumped into the electrospinning box, the air was used to blow fibers back towards the collector plate and reduce their extension towards the syringe needle. While the extension of the fibers back towards the syringe needle is not ideal, it likely aided in allowing the fibers to continue to dry while electrospinning as it enabled air flow from all directions of the fibers, unlike fibers deposited onto a flat plate collector. Given that

these fibers are still extremely sensitive to the amount of water vapor present in the electrospinning environment (i.e. the humidity), this constant air flow from all sides likely minimized some of the webbing and gelation of the fibers together, enabling the production of discrete fibers. This is useful considering that if the humidity within the electrospinning environment is too low, electrospinning will not occur as the precursor will instead gel at the tip of the syringe needle as the solvent rapidly evaporates.

SEM of the as-spun cobalt ferrite fibers as well as the calcined nanofibers is shown in

Figure 2-9. SEM was used to determine the average size of the cobalt ferrite fibers in addition to confirming the morphology as discussed above. The average diameter of the as-spun cobalt ferrite fibers was found to be 2.95±0.92 µm and the average diameter of the calcined fibers was found to be 1.53±0.39 µm. Following the confirmation of the morphology of the fibers XRD was used to confirm the composition of the fibers and is shown in Figure 2-10. The XRD pattern for the calcined fibers showed that the fibers were indeed composed of cobalt ferrite and that they demonstrated an average crystallite size of 31.07±0.09 nm. Once these precursor solutions were confirmed to reliably electrospin single-phase cobalt ferrite fibers, the solutions could then be used for the synthesis of bi-phasic Janus fibers.

2.3.3 Bi-phasic Barium Titanate-Cobalt Ferrite Janus Fibers

With the solutions for electrospinning single-phase barium titanate and single-phase cobalt ferrite established, the next step was to create Janus fibers. This was done using the same solutions as those used for electrospinning single-phase nanofibers. SEM was used to examine both the as-spun and calcined Janus fibers to confirm the Janus morphology and determine the average size of the fibers. Figure 2-11 shows SEM images of both as-spun and calcined Janus fibers. SEM revealed a clear distinction between the two halves along the length of the fibers for

both as-spun and calcined fibers. This was as expected for Janus fibers. The average diameter was determined to be 639.8±371.4 nm for the as-spun Janus fibers and 335.8±161.5 nm for the calcined Janus fibers.

As with both barium titanate and cobalt ferrite single-phase fibers, XRD was used to confirm the composition of the calcined fibers. Additionally, for the Janus fibers, XRD enabled the determination of how much of each phase was present in the sample using Rietveld refinement. Figure 2-12 shows the XRD data for calcined Janus fibers. Using Rietveld refinement on the XRD data the mass ratio of barium titanate to cobalt ferrite was determined to be 55:45. This is an acceptable ratio for the use of precursor solutions with 1:1 ratios of the ceramic components between them. The average crystallite for both barium titanate and cobalt ferrite was also determined from the XRD data. These were determined to be 22.42±0.20 nm for barium titanate and 30.06±0.013 nm for cobalt ferrite which matches those found for the single- phase fibers.

Following the XRD characterization of the fibers, SQUID magnetometry was used to obtain a room temperature hysteresis loop for the Janus fibers. The hysteresis loop, and specifically the saturation magnetization of the Janus fibers is useful as an additional check for the amount of cobalt ferrite present in a fiber sample. By comparing the saturation magnetization of the biphasic Janus fibers to that of the single-phase cobalt ferrite fibers, the Janus fibers were confirmed to consist of approximately 40% cobalt ferrite by weight, which is similar to the composition found with XRD. This comparison was done simply by dividing the saturation magnetization of the Janus fibers (which is calculated using the total mass of the Janus fiber sample, including the barium titanate) by the saturation magnetization of the single-phase cobalt

ferrite fibers. The comparison of the two hysteresis loops for single-phase cobalt ferrite fibers and the Janus fibers is shown in Figure 2-13.

Once the Janus nature and the composition of the fibers were confirmed, the next step was to examine the fibers to determine if they exhibited magnetoelectric behavior. This was done qualitatively using VSM. In order to examine the magnetoelectric behavior of the fibers, the magnetic moment of the sample was monitored as it was heated through the Curie temperature of barium titanate (about 120 °C).75,90,131 What was expected from this experiment was that as barium titanate was heated through its Curie temperature, it would undergo a shape change as the crystal structure changed from tetragonal to cubic. This shape change, due to the interfacial coupling between the barium titanate and the cobalt ferrite would then have caused strain on the cobalt ferrite. Given the magnetostrictive nature of cobalt ferrite, this strain, and thus shape change, would have resulted in a change in the magnetization of the sample that could be observed with VSM. Figure 2-14 shows the results of this experiment to qualitatively examine the magnetoelectric properties of the Janus fibers. Figure 2-14a shows the plot of the magnetization versus temperature. A dip in the magnetization was seen around the Curie temperature of barium titanate. This effect was more clearly observed in Figure 2-14b, the plot of the derivative of the magnetization with respect to temperature plotted versus temperature. Here a sharp change in the slope of the magnetization was observed. This revealed that the fibers did indeed exhibit magnetoelectric behavior similarly to those fibers synthesized by Starr et al.89

2.4 Summary

Barium titanate and cobalt ferrite single phase fibers as well as biphasic Janus fibers have been successfully synthesized via electrospinning. Barium titanate precursors electrospun readily, while cobalt ferrite precursor solutions needed further alteration by the addition of a chelating agent in order to electrospin. Two chelating agents were examined for the improvement

of cobalt ferrite precursor solutions: citric acid and acetylacetone. A ratio of Co:Fe:citric acid of

1:2:0.5 was found to be the best concentration of those tried as all higher concentrations resulted in gelation of the precursor within an hour. Unfortunately, when added to a solution of polymer binder, citric acid containing precursor solutions only electrospun webbed fibers. Acetylacetone, on the other hand, proved to better aid in the electrospinning of cobalt ferrite single phase fibers, as well as the cobalt ferrite present in the Janus fibers. This could be in part caused by the additional stability that bidentate ligands (acetylacetone) impart as compared to monodentate ligands (citric acid). The increase in viscosity caused by the presence of acetylacetone in the solution could also have contributed to the success of electrospinning cobalt ferrite fibers. In addition, VSM has qualitatively proven that the magnetoelectric effect is present within these fibers. Further work is necessary to be able to complete quantitative measurements of the magnetoelectric coefficient of these fibers.

Table 2-1. Citric acid concentrations and the time to complete gelation of the sol-gel precursor. Co:Fe:citric acid molar ratio Time to gelation (hours) 1:2:3 0 1:2:2 0.05 1:2:1 0.1 1:2:0.5 >24

Table 2-2. Conductivity and viscosity measurements of sol-gel precursor solutions for solutions containing either no chelating agent, citric acid, or acetylacetone. Chelating Agent Conductivity (mS/cm) Viscosity (cP) None 5.12 60 Citric acid 3.46 110 Acetylacetone 5.5 87.5

Figure 2-1. SEM images of barium titanate fibers: (a) as-spun fibers and (b) calcined fibers.

Figure 2-2. XRD pattern of calcined barium titanate fibers. Red stars denote barium titanate.

Figure 2-3. Raman spectra of barium titanate fibers.

Figure 2-4. Schematic of citric acid coordinated cobalt ions. Iron ions would be similarly coordinated in a solution containing citric acid.

Figure 2-5. Photos of cobalt ferrite sol-gel solutions containing citric acid at varying molar ratios of Co:Fe:citric acid. Note: Co:Fe ratio is always held at 1:2. (a) 1:2:3, (b) 1:2:2, (c) 1:2:1, and (d) 1:2:0.5. In 2-4a, the orange arrow is pointing at gelled precursor solution and the blue arrow is pointing at solvent sitting on top of the gel. Solutions (a)-(c) gelled within half an hour of the solution of citric acid was added to the solution of cobalt and iron nitrates. Solution (d) remained liquid for an extended time and was chosen to be used in further experiments. Photos courtesy of author.

Figure 2-6. Electrospun cobalt ferrite nanoparticles from solutions containing citric acid as a chelating agent. (a) image of cobalt ferrite fibers electrospun onto aluminum foil and (b) SEM image of cobalt ferrite fibers from the foil in (a). The fibers shown here have webbed together during electrospinning, which is an undesirable morphology. This shows that citric acid is not the ideal chelating agent for electrospinning cobalt ferrite fibers. Photo courtesy of author.

Figure 2-7. Schematics of cobalt and iron ions coordinated with acetylacetone.132

Figure 2-8. Photo of cobalt ferrite fibers electrospun onto a flat plate collector. This image is of fibers electrospun from a cobalt ferrite solution containing acetylacetone. Photo courtesy of author.

Figure 2-9. SEM images of cobalt ferrite fibers electrospun from solutions containing acetylacetone as the chelating agent: (a) as-spun fibers and (b) calcined fibers.

Figure 2-10. XRD pattern of calcined cobalt ferrite fibers electrospun using acetylacetone as the chelating agent. Red x’s denote cobalt ferrite.

Figure 2-11. SEM images barium titanate – cobalt ferrite Janus fibers: (a) as-spun Janus fibers and (b) calcined Janus fibers. The lines running longitudinally along the length of the fiber confirm the Janus nature of the fibers.

Figure 2-12. XRD data of calcined Janus fibers. XRD confirms the presence of both barium titanate and cobalt ferrite in the nanofibers. Blue stars and green x’s mark barium titanate and cobalt ferrite, respectively.

Figure 2-13. Shown are room temperature hysteresis loops for cobalt ferrite single-phase fibers and Janus biphasic fibers as obtained using SQUID magnetometry.

Figure 2-14. Shown are a) the magnetization of biphasic Janus fibers as a function of temperature and b) the derivative of magnetization as a function of temperature plotted versus temperature.

CHAPTER 3 SYNTHESIS OF BARIUM TITANATE PARTICLES VIA SOL-GEL ELECTROSPRAYING

3.1 Introduction

The development of functional particles has increased in importance across many different technological disciplines including both the electronics and biomedical industries. Of particular interest for the work described herein is the development of functional nanoparticles for use in biomedical applications. Many areas of biomedicine, such as cellular stimulation and drug delivery, require the development of functional materials that are on the correct size scales to be used in biomedicine such that they can have the desired effect while remaining undetected by the immune system. Barium titanate is one material that has garnered interest for biomedical applications, mainly for its piezoelectric and second harmonic generation properties.

Piezoelectric barium titanate is of interest in biomedicine for the possibility of using it for local electrical stimulation of cellular growth. As discussed in Chapter 1, there has been research done using ultrasound stimulation to trigger the piezoelectric properties of barium titanate nanoparticles for the stimulation of SH-SY5Y neuronal-like cells.78,79,133 In addition, piezoelectric barium titanate nanomaterials have also been studied for use in bone regeneration due to the piezoelectric nature of bone itself.64,134

Looking instead at the use of barium titanate in second harmonic generation (SHG) applications, it has been shown that SHG enables cell tracking that has reduced limitations over conventional fluorescence tracking.133,135,136 SHG cell tracking overcomes issues such as bleaching and blinking. For this reason, researchers have started looking into using barium titanate for tracking in the place of dyes.133,135–137

There are a variety of ways to synthesize barium titanate nanoparticles to be used in the above applications including molten salt synthesis, solid state reactions, hydrothermal reactions

among others .20,21,24,126,138,139 This work, however, focuses on developing methods for synthesizing barium titanate particles using sol-gel electrospraying techniques Sol-gel electrospraying enables facile control of particle size through varying the precursor solution concentration, the solution flow rate, or the applied voltage. Finally, a salt calcination technique has been employed as an effective method for preventing agglomeration during crystallization.

The use of the salt calcination technique described herein enables easily removing the as-sprayed particles from the aluminum foil collector onto which they are deposited. However, the process of salt calcination has been found to change the resultant morphology of the particles. The final morphology was found to depend heavily on the length of time that the particles are calcined at

800 °C, varying between particles and rods.

3.2 Experimental Methods

3.2.1 Materials

Barium acetate, titanium (IV) isopropoxide (≥97.0%), and glacial acetic acid were obtained from Sigma Aldrich. Isopropanol and sodium chloride were obtained from Fisher

Scientific. All reagents were used without further modification.

3.2.2 Sol-Gel Precursor Solutions

Barium titanate particles were synthesized via sol-gel electrospraying. To create the sol- gel precursor, barium acetate was dissolved in 1.5 ml of glacial acetic acid at 70 °C. In a separate vial, 0.63 ml of titanium (IV) isopropoxide was added to 0.87 ml of isopropanol. The titanium

(IV) isopropoxide solution was then added to the barium acetate solution at room temperature.

This final solution is stirred for two hours before electrospraying. Multiple molar ratios of barium to titanium were examined for their effects on the final, calcined morphology of the particles. Specifically, molar ratios of 0.8:1, 1:1, 1.2:1, and 1.4:1 Ba:Ti were electrosprayed to examine the effects on the morphology of the calcined particles.

3.2.3 Nanoparticle Synthesis

For electrospraying, the barium titanate precursor was loaded into a syringe that was placed in a syringe pump across from a grounded copper plate electrode covered in aluminum foil. The syringe pump was set to a flow rate of 0.15ml/hr. The needle was attached to a high voltage source and the voltage was increased until an electrified jet formed at 11kV. The humidity of the electrospraying setup was monitored using a Fisher Scientific Traceable

Hygrometer/Thermometer connected to Fisher Scientific DAS-3 Data Acquisition System.

Samples were electrosprayed at 35 %RH. Humidity levels were maintained within +/-1% of the desired relative humidity level (e.g., 34-36 %RH) and were controlled by pumping dry house air into the electrospraying chamber.

The amorphous, as-sprayed particles were calcined to obtain crystalline barium titanate particles. Before calcination, particles were placed into a vacuum oven at 70°C overnight to remove excess solvent. Several calcination treatments were examined for their effects on the final, calcined morphology of the particles. For the standard salt-calcination. the as-sprayed particles were then dispersed in ~10g of sodium chloride using a mortar and pestle to remove the particles from the aluminum foil onto which they were electrosprayed. For one sample, particles were dispersed in potassium sulfate instead of sodium chloride. The particle-salt mixture was then heated to 100 °C at a rate of 10 °C/min and held for an hour before being heated to 800°C for either 1, 2 , or 3 hours at a ramp rate of 10 °C/min. One sample was calcined at a ramp rate of

20 °C/min at 800 °C for 3 hours. Following salt calcination, particles were washed out of the salt using 100,000 NMWL Amicon Ultra-15 centrifugal filters. Particles were rinsed in water 5 times to remove any salt from the sample. Particles were then dried in a vacuum oven overnight before characterization.

3.2.4 Nanoparticle Characterization

Particle morphology, both before and after salt calcination, was examined using an FEI

Nova NanoSEM 430 scanning electron microscope (SEM). ImageJ and Fiji were used in combination with the SEM images to determine an average particle size and standard deviation both before and after calcination by measuring 300 particles per sample. Energy dispersive x-ray spectroscopy (EDS) was used during SEM to examine the composition of the calcined barium titanate nanoparticles. X-ray diffraction (XRD) to confirm the particle crystal structure was completed using a PANalytical X’Pert Powder X-ray diffractometer. Raman spectroscopy using a Renishaw InVia Raman spectrometer was used to further examine the particles to detect the presence of cubic, tetragonal, or hexagonal barium titanate.

3.3 Results and Discussion

The electrospraying methods described here resulted in the successful synthesis of as- sprayed barium titanate nanoparticles. Figure 3-1a shows barium titanate particles electrosprayed from solutions with Ba:Ti ratios 1:1. These particles exhibit an average size of 305 ± 86 nm

(based on the measurement of 300 particles) and a spherical morphology. In order to achieve crystalline particles, a salt calcination technique in which the as-sprayed particles were dispersed in a powder of sodium chloride was used. While as-sprayed particles of 1:1 Ba:Ti particles were easily achieved, standard salt calcination (sodium chloride, 10 °C/min ramp, 800 °C, 3 hours) resulted in nanorods rather than spherical nanoparticles (Figure 3-1b). EDS was done on the salt calcined particles to determine which elements were present in the sample. This revealed the presence of barium, titanium, oxygen, sodium, and silicon. The presence of silicon in the EDS spectra is a result of the particles having been drop cast on a piece of silicon wafer for SEM sample preparation. The presence of sodium in the EDS spectra could possibly be attributed to the presence of salt that was not completely washed out of the sample following calcination. All

other elements present in the sample were as expected. This concurs with the XRD for the same sample which shows the presence of barium titanate and small barium carbonate impurities

(Figure 3-2a). Raman spectroscopy was also used to examine the composition of the 1:1 Ba:Ti ratio sample. Raman probes the local structure of a sample as opposed to XRD which examines the average structure. The Raman spectra for the 1:1 ratio Ba:Ti sample shows peaks that can be attributed to barium titanate, barium carbonate, rutile titania, and sodium titanate (Figure 3-2b).

The presence of titania in the sample could possibly explain the presence of nanorods following calcination instead of the particles that were expected. However, there is little titania present in the sample given its lack of appearance in XRD. This does not rule out the possibility of there having been a phase evolution during the calcination treatment. In order to determine if this was the case, experiments in which the ratio of Ba:Ti precursors were off stoichiometry were completed. The reasoning for this was that precursors rich in Ti were expected to be more likely to form rods.

For experiments examining the effects of differing Ba:Ti ratios, particles were electrosprayed from precursor solutions of Ba:Ti ratios of 0.8:1, 1.2:1, and 1.4:1. The as-sprayed particles obtained from these solutions are shown in Figure 3-3 (a), (c), and (d), respectively. As seen in Figure 3-3 particles are readily obtained from solutions of all Ba:Ti ratios. The average particle size for the three additional Ba:Ti ratios were determined to be 241 ± 76 nm, 301 ± 101 nm, and 305 ± 110 nm, respectively.

To examine if varying the ratio of Ba:Ti would affect the calcined morphology of the particles, each sample was salt calcined under the same conditions as the 1:1 Ba:Ti ratio particles. SEM images of all calcined particle samples are shown in Figure 3-4. Samples with

Ba:Ti ratios of 1:1 and 1.2:1 both exhibited primarily nanorod morphology. The particles

electrosprayed with ratios of Ba:Ti of 0.8:1 and 1.4:1 both primarily exhibited particles rather than rods. Particles electrosprayed with a Ba:Ti ratio of 0.8:1 also exhibited some nanorods, but relatively few compared to the number of particles. EDS was done on all samples during SEM.

EDS of 0.8:1 and 1.4:1 calcined samples show barium, titanium, and oxygen with few impurities, namely sodium, aluminum, silicon and gold. These can all be attributed to sample preparation: aluminum from the aluminum foil that the particles were electrosprayed onto; silicon from the piece of wafer that the particles were drop cast onto for SEM; gold from the surface coating used to prepare the SEM samples. EDS of the sample of 1.2:1 Ba:Ti, however, showed additional impurities in addition to the barium, titanium, and oxygen. These impurities include gold, silicon, aluminum, silicon, magnesium, and sulfur. Many of these can be explained by sample preparation like for the samples of other Ba:Ti ratios. The additional impurities of sulfur and magnesium are possibly explained by impurities in the sodium chloride used for the salt calcination if any salt was not completely washed out of the sample.

Given the presence of impurities in the EDS spectra for the samples, XRD and Raman were used to further check the composition of the four calcined samples. XRD for all four Ba:Ti ratios is shown in Figure 3-5. All four samples exhibit peaks characteristic of barium titanate though additional peaks can be seen in the patterns for the samples with Ba:Ti ratios of 1:1 and

1.2:1. For the samples with a Ba:Ti ratio of 1:1, additional XRD peaks corresponding to barium carbonate were seen. Barium carbonate is a commonly seen impurity in barium titanate syntheses and possesses an orthorhombic crystal structure.140 The orthorhombic nature could potentially contribute to the formation of rods during calcination. Barium carbonate was also seen in the samples with a Ba:Ti ratio of 1.2:1 in addition to barium oxide impurities. The presence of barium oxide in this sample could be attributed to the excess barium present in the precursors

which provided enough barium to form stochiometric barium titanate but also had additional barium present for the formation of impurity phases. Barium oxide possesses a cubic crystal structure and thus does not necessarily contribute to the formation of rods over particles.141

Figure 3-6 shows Raman spectroscopy for the samples of varying Ba:Ti ratios. As with XRD, samples from all Ba:Ti ratios exhibit peaks characteristic of barium titanate. However, all samples except for those from the precursors with Ba:Ti ratio of 1.4:1 exhibit additional impurities, specifically a mixture of rutile titania, barium carbonate, and sodium titanate. Both rutile and barium carbonate are impurities that can be expected in many barium titanate syntheses. On the other hand, sodium titanate and titania are both often used as precursors to synthesize barium titanate nanorods.142–144 By this method, titania or sodium titanate nanorods are exposed to barium precursors which react with the nanorods to form barium titanate.142,143

Based on these Raman spectra, it is possible that there are intermediates of either titania or sodium titanate forming during the calcination of the as-sprayed particles prior to the formation of barium titanate. Both anatase and rutile titania form at temperatures lower than barium titanate.102,145–147 Therefore, it is possible that anatase titania first crystallizes from the barium titanate precursors in the as-sprayed particles (around 250 °C),146,147 then begins converting to barium titanate around 600 °C.145,148,149 If the barium titanate that is formed during calcination of the as-sprayed particles is forming via this route of anatase converting to barium titanate then this could be an explanation for why titania impurities are present in the samples. Rutile titania is present instead of anatase as anatase converts to rutile titania above 600 °C.146,147 Similarly, the reactions to form sodium titanate from titania also occur at lower temperatures than those for the formation of barium titanate, which could account for the sodium titanate impurities in the samples.142

In addition to examining the effects of various Ba:Ti ratios, changes in the calcination conditions, such as temperature, ramp rate, and calcination salt, were also investigated.

Calcination of 1:1 Ba:Ti particles at 750 °C for 3 hours with a ramp rate of 10 °C/min resulted in a mixture of nanorods and particles as shown in Figure 3-7. Particles of 1:1 Ba:Ti calcined at 800

°C for 3 hours at ramp rate of 20 °C/min resulted in the formation of a mixture of nanorods similar to the standard calcination at 800 °C for 3 hours at a ramp rate of 10 °C/min (Figure 3-8).

Barium titanate particles with a Ba:Ti ratio of 1:1 were also calcined at 800 °C for 3 hours at a ramp rate of 10 °C/min in potassium sulfate instead of sodium chloride. The calcination in potassium sulfate also resulted in a mixture of nanorods, particles, and flakes of material rather than particles (Figure 3-9).

Calcination time was also changed in an attempt to obtain barium titanate particles rather than nanorods and to investigate if there was a phase evolution from particles to rods occurring with increased calcination time. Particles of Ba:Ti ratio of 1.2:1 were calcined at 800 °C for 1 hour at a ramp rate of 10 °C/min and at 800 °C for 2 hours at a ramp rate of 10 °C/min. Unlike the 1.2:1 particles calcined at 800 °C for 3 hours at a ramp rate of 10 °C/min, the particles calcined for 1 hour resulted in spherical particles rather than nanorods (Figure 3-10). Raman spectroscopy of these particles showed that they were composed of cubic and tetragonal barium titanate (Figure 3-11), unlike those calcined for 3 hours. SEM of the particles calcined for 2 hours showed a majority of particles of barium titanate with the presence of some nanorods

(Figure 3-12). Like the Raman spectroscopy of the sample calcined for 1 hour, the spectra of the particles calcined for 2 hours exhibited barium peaks representative of both cubic and tetragonal barium titanate (Figure 3-13). Based on the progression of the barium titanate morphology from particles to nanorods as calcination time increases from 1 hour to 3 hours, it seems that the

additional time at 800 °C is enabling the formation of nanorods. In addition, the sample calcined for 3 hours contains impurities that are seen in both Raman spectroscopy and XRD unlike the samples calcined for either 1 hour or 2 hours (Figure 3-12 and 3-15).

In the case of molten salt syntheses, the production of nanorods typically requires the addition of capping agents or surfactants such as nonylphenyl ether or ethlyenediamine.148,150

Work done by Mao et al. uses a combination of barium oxalate, anatase titania, sodium chloride and nonylphenyl ether to synthesize barium titanate nanorods.148 They attribute the production of nanorods of barium titanate over nanoparticles to the effects of preferential absorption of molecules or ions to a specific crystal facet which then impedes growth off of that facet.148

Specifically, they note that the presence of sodium chloride in the synthesis seems to be the determining factor for the production of nanorods, as experiments done without sodium chloride were said to result in barium titanate particles of random shapes.148 Work completed by Hayashi et al. gives a more in depth examination of the effects of sodium chloride presence during heat treatments on the resultant morphology of barium titanate particles.151 Hayashi et al. show that the resultant morphology of barium titanate from molten salt syntheses relies on the initial titania precursor, the amount of salt used in the synthesis, and the synthesis temperature. One key observation from this work is that synthesis at higher temperatures (900 °C versus 800 °C or

700 °C) seemed to promote the formation of equiaxed particles (1:1 aspect ratio) over rods as compared to lower temperatures.151

Based on the findings of molten salt synthesis literature it is possible to develop an explanation for what is occurring during the calcination of the amorphous, as-sprayed barium titanate particles. Given that for the synthesis described in this work, the precursor particles that are calcined already contain barium, as opposed to only titania as done with molten salt

syntheses, it is likely that at the shorter calcination times the formation of crystalline material is localized within each precursor particle. This is why at 1 and 2 hour calcinations, particles make up the majority of the end product as opposed to rods. However, as calcination time increases, the particle morphology evolves as the growth of barium titanate from the crystalline particles

(as confirmed with XRD for the 1 hour sample – Figure 3-12) is affected by the presence of sodium chloride during calcination to growing in a specific crystallographic direction.148

In order to fully clarify what is happening in regards to the formation of rods over particles, further characterization is necessary. Firstly, high resolution x-ray diffraction may be useful in helping to determine whether titania or sodium titanate intermediates are forming which cause the eventual formation of barium titanate rods. Secondly, electron diffraction patterns taken in TEM of the electrosprayed, salt-calcined particles described in this chapter may help in understanding if the presence of sodium chloride in our calcination is having similar effects on the final morphology as has been described for molten salt synthesis techniques. Electron diffraction will enable the determination of the crystallographic orientation in which the barium titanate nanorods are preferentially growing, if one exists, which would point towards sodium chloride having acted as a capping agent. Barium titanate ratios of 1:1 Ba:Ti should also be examined to see if the same effects are seen with changes in calcination time as those seen with samples of 1.2:1 Ba:Ti ratios.

3.4 Summary

This chapter demonstrates the successful electrospray synthesis of as-sprayed barium titanate nanoparticles. As-sprayed nanoparticles of all molar ratios of Ba:Ti had similar morphologies and particle sizes. However, through the process of salt calcination the majority of the resultant crystalline nanoparticles produced from solutions of Ba:Ti ratios of 1:1 and 1.2:1 lost the as-sprayed morphology. Particles produced from Ba:Ti ratios of 1:1 and 1.2:1 were

found to exhibit nanorod morphology following salt calcination. 0.8:1 Ba:Ti ratio particles showed a mixture of both spherical and nanorod particles following salt calcination. However, those particles created from solutions of a 1.4:1 Ba:Ti ratio were mainly composed of spherical particles post-calcination.

Changing the calcination time was also found to have an effect on the morphology of the resultant crystalline particles for samples produced from Ba:Ti ratios of 1.2:1. Shorter calcination times resulted in the formation of particles rather than nanorods. Increasing calcination times caused the formation of nanorods, at first in addition to particles, then instead of particles.

Further work needs to be done to determine if shorter calcination times promote particles in barium titanate samples of different ratios of Ba:Ti.

Figure 3-1. SEM images of barium titanate particles electrosprayed from solutions with a Ba:Ti ratio of 1:1: (a) as-sprayed particles and (b) calcined sample of the particles seen in (a).

Figure 3-2. Shown are two methods of characterization of the composition of the calcined nanorods shown in Figure 3-1b: (a) XRD of a sample of the same sample of calcined barium titanate particles shown in Figure 3-1b and (b) Raman spectroscopy of the same. In (b), red stars indicate barium titanate, green circles indicate sodium titanate, black crosses indicate barium carbonate, and yellow x’s indicate rutile titania.

Figure 3-3. SEM images of as-sprayed barium titanate nanoparticles with Ba:Ti molar ratios of 0.8:1 (a), 1:1 (b), 1.2:1 (c), and 1.4:1 (d).

Figure 3-4. SEM images of salt-calcined barium titanate nanoparticles with Ba:Ti molar ratios of 0.8:1 (a), 1:1 (b), 1.2:1 (c), and 1.4:1 (d).

Figure 3-5. XRD data for salt-calcined barium titanate nanoparticles with Ba:Ti molar ratios of 0.8:1 (a), 1:1 (b), 1.2:1 (c), and 1.4:1 (d). Red stars indicated barium titanate; blue crosses indicate barium carbonate; black diamonds indicate barium oxide.

Figure 3-6. Raman spectra taken of salt-calcined barium titanate nanoparticles with Ba:Ti molar ratios of 0.8:1 (a), 1:1 (b), 1.2:1 (c), and 1.4:1 (d). Red stars indicate barium titanate, black crosses indicate barium carbonate, and yellow x’s indicate rutile titania, blue circles indicate sodium titanate144,152.

Figure 3-7. SEM images of calcined barium titanate samples calcined in sodium chloride at 800 °C for 3 hours with a ramp rate of 20 °C/min.

Figure 3-8. SEM images of calcined barium titanate samples calcined in sodium chloride at 750 °C at a ramp rate of 10 °C/min.

Figure 3-9. SEM image of barium titanate sample calcined in sodium chloride at 800 °C for 3 hours at a ramp of 10 °C/min in potassium sulfate.

Figure 3-10. SEM image of barium titanate sample calcined at 800 °C for 1 hour at a ramp rate of 10 °C/min in sodium chloride.

Figure 3-11. Raman spectra of barium titanate sample calcined at 800 °C for 1 hour at a ramp rate of 10 °C/min in sodium chloride. Peaks for tetragonal barium titanate (T), cubic barium titanate (C), and witherite or barium carbonate (W) are indicated on the figure.

Figure 3-12. XRD pattern for the barium titanate sample calcined at 800 °C for 1 hour at a ramp of 10 °C/min in sodium chloride. Red stars indicate barium titanate peaks.

Figure 3-13. SEM image of barium titanate sample calcined at 800 °C for 2 hours at a ramp rate of 10 °C/min in sodium chloride.

Figure 3-14. Raman spectra of barium titanate sample calcined at 800 °C for 2 hours at a ramp rate of 10 °C/min in sodium chloride. Peaks for tetragonal barium titanate (T), cubic barium titanate (C), and witherite or barium carbonate (W) are indicated on the figure.

Figure 3-15. XRD pattern for the barium titanate sample calcined at 800 °C for 2 hours at a ramp of 10 °C/min in sodium chloride. Red stars indicate peaks that represent barium titanate.

CHAPTER 4 SYNTHESIS OF COBALT FERRITE PARTICLES VIA SOL-GEL ELECTROSPRAYING

4.1 Introduction

As with the synthesis of magnetoelectric Janus fibers, it is necessary to first understand the synthesis of the individual phases that will be used to create composite Janus nanoparticles.

Attempts at electrospraying cobalt ferrite onto a flat plate collector have resulted in the formation of films with particles embedded in the film rather than the formation of particles. Thus, different methods for electrospraying needed to be developed in order to achieve electrosprayed cobalt ferrite nanoparticles. This was done by examining both the sol-gel chemistry of the electrospraying solutions for cobalt ferrite as well as developing methods of electrospraying into liquid collectors modeled after the electrospinning of natural polymers using ionic liquids.

By changing the chemistry of the precursor solutions it is possible to control important solution parameters such as the conductivity and the reactivity to water. Examining first the solution conductivity, there are two main considerations: the conductivity of the solvent and the concentration of metal ions in solution. Therefore, it is relatively easy to change the conductivity of the electrospraying precursor solutions. One way this can be accomplished is by changing the concentration of the metal ions in solution. This method of reducing solution conductivity cannot be done indefinitely given that the presence of the metal ions is necessary in order to obtain particles. In addition, the use of less conductive solvents can help reduce the conductivity of the solution, making it easier to electrospray. Here, precursor solutions are made with ethylene glycol as the solvent as opposed to more typical electrospraying solvents, such as ethanol, which decreases the conductivity of the solutions. While both of these options decrease the conductivity of the solutions, it is important to note that ceramic precursor solutions remain significantly more conductive than polymeric precursor solutions.

Secondly, it is well known that all sol-gel precursors, whether they are used for traditional sol-gel syntheses or for sol-gel electrospraying, are extremely sensitive to water.

Hydrolysis and condensation reactions are important mechanisms of sol-gel chemistry that are both heavily dependent on the presence of water for determining the rate at which these reactions occur. In the case of sol-gel electrospraying, water vapor in the electrospraying environment is the primary source of the water. The rate at which the hydrolysis and condensation reactions occur affects the size and morphology of electrosprayed ceramic particles as well as whether or not particles can be achieved instead of films of the sprayed materials. Generally, during electrospraying when the humidity level is too high, particles will gel rapidly locking in large particles. When the humidity levels are too low, gelation is slow to occur and films of the material will be deposited as the droplets remain liquid and thus hit the flat plate collector as a liquid droplet. The addition of chelating agents is one way in which some of the adverse effects of water on sol-gel precursor solutions can be mitigated. Chelating agents achieve this by coordinating to the metal ions and helping to slow down condensation reactions.115 Chelating agents are also beneficial as they aid in the formation of a homogeneous network of the metal ions which will in turn help to create the desired end product. For example, Sanpo et al. have shown that the addition of citric acid as a chelating agent for cobalt ferrite led to increased homogeneity of the sol-gel precursor solution and which thus resulted in a lower final concentration of impurity oxides such as cobalt oxide, magnetite, maghemite, etc.115 Ethylene glycol is also a commonly used as a chelating agent in sol-gel syntheses and is often used in conjunction with acidic chelating agents such as citric acid, polyacrylic acid, or nitric acid.122,153–

155 The combination of ethylene glycol with some of these additional chelating agents has been shown to reduce the temperature at which crystalline ceramics are formed.154

While the previous paragraphs focused on the use of chelating agents and altering the solution conductivity, collection methods for electrospinning and electrospraying that could potentially aid in the successful electrospray synthesis of magnetic nanoparticles are examined.

Specifically, with the focus on the idea of developing a liquid collection medium in electrospinning or electrospraying to avoid the gelling of the as-sprayed particles into film as happens with electrospraying onto a flat plate collector (Figure 4-1). A liquid collector accomplishes this by keeping the particles separated from one another, via steric hindrance of the silicone oil, and isolated from the water vapor in the electrospraying environment as they are immersed in the silicone oil.

The area of polymeric electrospinning and electrospraying that has thus far found the most benefit from the use of liquid collectors is that of the synthesis of nanofibers of natural polymers such as cellulose and chitin.156–158 For these materials, liquid collector baths are of great use due to the insolubility of the polymers in typical electrospinning solvents. Therefore, instead of using traditionally employed organic solvents to dissolve these polymers, they are instead dissolved in an ionic liquid prior to electrospinning. Due to the nonvolatile nature of these ionic liquids, they cannot be used to electrospin onto a flat plate collector.156 To overcome this limitation, these polymers can be electrospun into liquid baths composed of a nonsolvent for these polymers that is also miscible with the ionic liquid. When the electrified jet hits the nonsolvent-based liquid bath, a solid fibers is formed, thus enabling the electrospinning of natural polymer nanofibers with limited solubility in volatile solvents. Because the ionic liquid is miscible in the collection medium, this facilitates the removal of the ionic liquid during collection without requiring additional purification steps.156–158 Here we seek to expand this concept to overcome the challenges of forming films when cobalt ferrite nanoparticles coalesce

on a flat collector plate. Unlike the use of ionic liquids and liquid collecting baths for natural polymers, this work uses a collection bath that is immiscible with the electrospraying solvent combined with heating of the collection bath to obtain discrete particles and to boil off the electrospraying solvent. The ability to maintain discrete particles as can be achieved with this method of electrospraying is important for scaling of the synthesis to achieve high yields of the nanoparticles.

There are a variety of ways to synthesize magnetic nanoparticles, including thermal decomposition, solid state, and co-precipitation, to name a few.159–163 Electrospray synthesis methods for cobalt ferrite have not previously been demonstrated, however, one advantage to the methods of electrospraying into a liquid collector presented here over other types of synthesis is the feasibility of being able to easily scale up the synthesis as electrospinning and electrospraying are considered scalable synthesis techniques.164 First, the use of a liquid collector over a flat plate collector enables the scale-up of this synthesis method. Given that the liquid collector prevents agglomeration of the particles during synthesis, as opposed to flat plates where the gelling of the particles into a film often occurs, it is possible to electrospray larger amounts into the collector without risking the loss of particle morphology. In addition, the work presented here is done using a single spinneret. In both electrospinning and electrospraying, the use of multiple spinnerets has been used to increase the yield of the desired product or to create composite mats of fibers.164–166 Therefore, electrospraying using multiple spinnerets presents a method for increasing the yield of this synthesis.

4.2 Experimental Methods

4.2.1 Materials

Cobalt (II) nitrate hexahydrate, citric acid monohydrate and 1,000 cSt silicone oil were obtained from Sigma Aldrich. Ferric nitrate nonahydrate and ethylene glycol were obtained from

Fisher Scientific. All chemicals were used without further modification.

4.2.2 Sol-gel Precursor Solutions

Like the sol-gel precursor solutions described in Chapter 2 for electrospun nanofibers, the precursor solutions used for electrospraying also included chelating agents. A combination of ethylene glycol and citric acid were the chosen chelating agents for cobalt ferrite electrospraying.

The precursor solutions here were composed of 0.875 g of ferric nitrate nonahydrate, 0.315 g of cobalt (II) nitrate hexahydrate, and 0.1 g citric acid monohydrate dissolved in 3 ml of ethylene glycol. Once the nitrates and citric acid were added to the ethylene glycol, the solutions were stirred for two hours before use.

4.2.3 Electrospraying Cobalt Ferrite into a Liquid Collection Medium

For the purposes of this work, silicone oil was chosen as the liquid collection medium.

This choice was made based on the immiscibility of the precursor solutions in silicone oil. This immiscibility means that the particles should not dissolve in the collection medium. This is necessary given that the precursors, when electrosprayed into room temperature silicone oil, must remain as liquid droplets in order to obtain particles, which would not occur if the solvents were miscible. The liquid nature of the as-sprayed droplets presents a difficulty in collecting the particles for calcination into crystalline particles. For this reason, it was decided to electrospray into silicone oil heated to 200 °C thus solidifying the particles and preventing them from dissolving in any solvents used to remove the particles from the silicone oil.

A schematic of the setup used to electrospray into 200 °C silicone oil is shown in Figure

4-2. The silicone oil was heated on a hot plate under magnetic stirring. The temperature of the silicone was monitored using a thermocouple. Once the oil equilibrated to 200 °C, the thermocouple was removed for electrospraying. To electrospray, a voltage of 11 kV was applied to the syringe needle that was placed 22cm from the surface of the silicone oil collector. A flow rate of 0.15 ml/hr was used to pump the solution into the electric field.

In order to collect the particles from the silicone oil, hexane was added to thin out the oil.

The combined silicone oil and hexane was then centrifuged at 5,000 rpm for 10 min. After configuration the supernatant was removed, and the particles were washed three times with hexane to ensure all silicone oil had been removed. The hexane washes were followed by three washes in toluene and three washes in ethanol.

Once washed, the particles were salt calcined in a manner similar to that used in Chapter

3 for the barium titanate particles. Briefly, sodium chloride was ground into a fine powder before dispersing the cobalt ferrite particles in the ground salt. The salt-particle mixture was then heated to 800 °C at a rate of 10 °C/min and held for 3 hours to allow the formation of crystalline particles. To collect the salt calcined particles, the salt-particle mixture was dissolved in water.

The particles were magnetically separated from the solution and the supernatant was removed.

This process was repeated five times to ensure the removal of all of the salt from the nanoparticles. The particles were then dried in a vacuum oven prior to characterization.

4.2.4 Characterization of Electrosprayed Cobalt Ferrite Particles

The as-sprayed and salt-calcined particles were characterized using powder X-ray diffraction (XRD), in combination with Rietveld refinement, to confirm that the particles were composed of cobalt ferrite and to examine the crystallinity of the as-sprayed particles. Scherrer’s formula was used to determine the crystallite size of the calcined cobalt ferrite particles. Raman

spectroscopy was also used to probe the local structure of the calcined particles to check for the presence of cobalt ferrite and to check whether other cobalt or iron oxides were present.

Following confirmation of the composition of the particles, transmission electron microscopy (TEM) was used to examine the size and morphology of the salt-calcined particles.

Magnetic characterization of the calcined cobalt ferrite particles was then completed using superconducting quantum interference device (SQUID) magnetometry (Quantum Design MPMS

3). SQUID was used to find the saturation magnetization of the cobalt ferrite particles.

4.3 Results and Discussion

Both the as-sprayed and calcined particles were first examined using XRD to confirm the average composition of the particles to be cobalt ferrite in the spinel phase and to check the crystallinity of the as-sprayed particles as shown in Figure 4-3. For the as-sprayed particles,

XRD reveals the development of some crystallinity following electrospraying into 200 °C silicone oil. The choice to electrospray into 200 °C silicone oil was motivated by work done by

Caruntu et al. in which crystalline metal ferrite particle were synthesized in diethylene glycol heated to 220 °C.167 In addition to the possibility of forming crystalline particles at the temperatures used for the electrospray synthesis, the heated silicone oil enabled the solidification of the droplets via sol-gel combustion of the nitrates (oxidant) and citric acid (chelator and fuel) caused by heating the precursor droplet to 200 °C.127 This reaction both begins the formation of crystalline cobalt ferrite and removes ethylene glycol from the precursor droplet. The solidification of the droplets in turn made it more feasible to retrieve the particles from the liquid collection medium without risking dissolution of the particles. To increase the crystallinity of the as-sprayed particles, it is possible that the addition of ammonia to the precursor solutions, would enable the formation of fully crystalline particles during electrospraying into 200 °C silicone oil by increasing the pH back to a more neutral range.127,168 This is because, as described by Sutka

and Mezinskis, the reaction to form cobalt ferrite from citrate-nitrate precursors only completely proceeds at 500 °C when in an acidic environment.127

While XRD confirmed the presence of some crystallinity in the as-sprayed particles, the sample only exhibits two of the peaks representative of cobalt ferrite, meaning that the 200 °C of the collector bath was not sufficient to produce fully crystalline particles. Calcination was therefore required to fully crystallize the cobalt ferrite particles. XRD of the calcined particles is also shown in Figure 4-3 and shows a significant increase in the crystallinity of the particles following calcination as well as confirming that the particles are composed of cobalt ferrite.

XRD thus confirms the need for a post-electrospraying calcination step in order to achieve crystalline cobalt ferrite. From the XRD data of the salt-calcined nanoparticles, it is possible to use Scherrer’s formula to calculate the average crystallite size of the cobalt ferrite particles.

Here, this number is calculated to be 18.02±0.04 nm. Raman spectroscopy was used to confirm the local ordering of the cobalt ferrite particles. Raman spectroscopy, as seen in Figure 4-4, shows peaks marked with red stars at 688, 600, 467, and 315 cm-1.169 These peaks correspond to the cubic inverse-spinel nature of cobalt ferrite.169 Some impurity peaks can be seen around 560 and 390 cm-1, which can be attributed to α-FeOOH.115

Figure 4-5 shows TEM of the salt-calcined cobalt ferrite particles. TEM confirms the successful synthesis of nanoparticles using the above described method of electrospraying into heated silicone oil. TEM of the calcined particles shows that the particles survived calcination while remaining discrete. This proves that the method of dispersing the particles in salt to keep them separate during calcination is an effective way in which to prevent particle agglomeration.

Particle size was calculated from TEM images such as that shown in Figure 4-5. The average

particle size was determined from measurements of 500 particles is 20.7±11.5 nm. The calcined particles are non-uniform in shape, ranging from spherical to cubic to oblong.

Following XRD and TEM examinations of the particles, characterization of the magnetic properties was completed using SQUID magnetometry. Room temperature hysteresis measurements revealed that the particles have a saturation magnetization of 75.3±.5 emu/g

(Figure 4-7a). For comparison, the bulk saturation magnetization for cobalt ferrite is 80.8 emu/g.115,170 The lower room temperature saturation magnetization of the cobalt ferrite particles can be attributed to surface spin disorders caused by broken bonds on the surface of the nanoparticles.171,172 This results in what is often referred to as a “dead” layer on the surface of particles that does not contribute to the magnetization of the particles.

4.4 Summary

This chapter demonstrates the successful electrospraying of cobalt ferrite nanoparticles.

In order to achieve nanoparticles rather than films of cobalt ferrite, a liquid collector, specifically

1,000 cSt silicone oil, was chosen over a standard grounded collector plate. By electrospraying into silicone oil, which is immiscible with the solvents used for the sol-gel precursor solutions, the sprayed particles were isolated from one another thus preventing agglomeration.

Additionally, it was shown that electrospraying into silicone heated to 200 °C better prevented particle agglomeration when compared to electrospraying into room temperature silicone oil.

However, XRD confirms the needs for an additional calcination step in order to achieve fully crystalline nanoparticles.

Transmission electron microscopy shows that cobalt ferrite nanoparticles were successfully synthesized with an average diameter of 20.7±11.5 nm. Both X-ray diffraction and

Raman spectroscopy confirm that the particles are indeed composed of cobalt ferrite. From X-ray diffraction data, the average crystallite size of the cobalt ferrite was found to be 18.02±0.04 nm

using Scherrer’s formula. Finally, the room temperature saturation magnetization of these cobalt ferrite particles was found to be 75.3±.5 emu/g using SQUID magnetometry.

Successfully obtaining cobalt ferrite particles through sol-gel electrospraying demonstrates the continued feasibility of using electrospraying techniques to synthesize complex oxides such as cobalt ferrite or barium titanate as discussed in Chapter 3. This poises electrospraying as a promising method for the scalable synthesis of functional oxide-based materials. The ability to synthesize cobalt ferrite in this manner is particularly exciting due to the implications for the potential to electrospray bi-phasic functional materials.

Figure 4-1. Cobalt ferrite electrosprayed onto a flat plate collector resulting in the formation of a film containing particles.

Figure 4-2. Schematic of the electrospray setup developed for the synthesis of cobalt ferrite nanoparticles. Here the particles are electrosprayed into a dish of silicone oil that is heated to 200 °C that is magnetically stirred in order to maintain individual particles during the electrospray process.

Figure 4-3. XRD data for salt-calcined cobalt ferrite compared to a cobalt ferrite reference.(Top) XRD of salt-calcined CFO nanoparticles, (Middle) XRD of as-sprayed cobalt ferrite particles and (Bottom) XRD reference data from ICDD. XRD shows that calcination is necessary to achieve fully crystalline nanoparticles.

Figure 4-4. Raman spectroscopy for salt-calcined cobalt ferrite particles. The peaks marked with red stars shown here at 688, 600, 467 and 315 cm-1 are indicative of cobalt ferrite. The peaks marked with black x’s at 564 and 390 cm-1 indicate α-FeOOH.115

Figure 4-5. Representative TEM of salt-calcined cobalt ferrite particles. TEM images were used to calculate an average particle size from 500 nanoparticles.

Figure 4-6. Room temperature hysteresis loop for the cobalt ferrite nanoparticles with an average saturation magnetization of 75.3±0.5 emu/g as shown in (a). (b) shows the center of the hysteresis loop at a higher magnification which shows that there may be a pinched hysteresis loop.

CHAPTER 5 MAGNETOELECTRIC FIBER – HYDROGEL COMPOSITES FOR NEURONAL STIMULATION

5.1 Introduction

This chapter will focus on the application of the magnetoelectric fibers synthesized in

Chapter 2 for their use in electric stimulation of neuronal growth and differentiation. As discussed in Chapter 1, electric fields are found throughout the human body and are involved in a wide variety of biological processes including, but not limited to, wound healing, cellular signaling, angiogenesis, neuronal signaling and neuronal growth and differentiation. For the purpose of this work, the presence of electric fields in the growth and differentiation of neuronal cells are the primary focus. The effects of magnetoelectric materials on neuronal cells was studied here using magnetoelectric materials to create local electric fields.

While there is a large variety of research being done on materials for electric stimulation, magnetoelectric biomaterials provide a direction from which to approach the manipulation of local electric fields within the body. Using electric stimuli in the human body often presents a number of difficulties as body tissues attenuate electric fields, which makes it difficult to use electrical stimuli. Many devices that make use of electric stimuli focus on applying the fields through contacts on the skin, such as transcutaneous electrical nerve stimulation, or require the implantation of devices and power supplies.173 Magnetoelectric materials enable the use of external magnetic fields to trigger localized electric responses of the materials. Given that the human body is permeable to magnetic fields, magnetoelectric biomaterials would make for far less invasive methods of manipulating cell behavior using electric fields.

In this chapter, the use of magnetoelectric nanofibers - collagen hydrogels composites are studied for the stimulation of proliferation and differentiation of PC12 neuronal-like cells.

Collagen hydrogels were chosen for their known biocompatibility and relative ease of synthesis.

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Using a magnetic field created by applying a square wave voltage input, the effects on proliferation and differentiation of a variety of stimulation regimes are examined. The preliminary work presented here on the magnetoelectric stimulation of neuronal cells provides a stepping stone for further examination of the possibility of using these materials for biological applications.

5.2 Experimental Methods

The magnetoelectric nanofibers (and magnetic cobalt ferrite nanofibers) used in this chapter were discussed in Chapter 2. The synthesis details for these fibers are detailed there. This section instead details the methods for creating the collagen hydrogel-fiber composites as well as the effects of magnetoelectric stimulation using these composites on the proliferation and differentiation of PC12 neuronal cells.

5.2.1 Materials

Cobalt (II) nitrate hexahydrate, barium acetate, titanium (IV) isopropoxide (≥ 97.0%), glacial acetic acid and polyvinylpyrrolidone were obtained from Sigma Aldrich. Ferric nitrate nonahydrate and ethanol were obtained from Fisher Scientific. All reagents were used without further modification.

HEPES (1M) was obtained from Teknova. Collagen I, high concentration, rat tail, and

Cellgro fetal bovine serum (FBS) were obtained from Corning. DMEM/F12 1:1 media was obtained from HyClone. Horse serum was obtained from ATCC. Penicillin/streptomycin 100X was obtained from Quality Biological. Nerve growth factor (NGF) 2.5S, mouse submaxillary glands was obtained from EMD Millipore.

Lactate dehydrogenase (LDH) assay kits were obtained from Thermo Scientific Pierce

Life Sciences. PicoGreen dsDNA Assay kits were obtained from Invitrogen. Triton-X 100 was

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obtained from Fisher Scientific. Mammalian cell lysis buffer and acetylcholinesterase assay kits were obtained from Abcam.

5.2.2 Synthesis of Collagen Hydrogels

Fiber-collagen hydrogel composites were used to test the effects of magnetoelectric stimulation on neuronal proliferation and differentiation. Three sets of collagen-based hydrogels were used to examine the effects of magnetoelectric stimulation on neuronal-like PC12 rat pheochromocytoma cells: control collagen hydrogels, collagen hydrogels containing magnetic cobalt ferrite single-phase fibers, and collagen hydrogels containing magnetoelectric biphasic

Janus fibers. Collagen hydrogels containing single-phase cobalt ferrite fibers are used for a mechanical control given the magnetostrictive nature of cobalt ferrite. Additionally, these gels are used as a control for the fact that magnetic material will be pulled by the magnetic field which could change the mechanical properties of the hydrogels.

The base collagen hydrogel is the same for all three gel types. The hydrogel solution has a final concentration of collagen type I of 3 mg/ml. In order to create the hydrogels solutions, collagen type I, a solution of 0.2 vol% acetic acid in deionized water, and a solution of 10 ml of

1M HEPES, 10 ml of 10X DMEM and 0.4 g of sodium bicarbonate (from here on referred to as

DMEM/HEPES solution), were mixed together. The necessary amounts of the solution components (collagen type I, 0.2 vol% acetic acid and DMEM/HEPES solution) are calculated based on the concentration of the stock collagen received from Thermo Scientific and the quantity of solution necessary. The equations used for the calculations of these amounts can be found in Appendix B. Hydrogels were made by placing 212 µl of solution into the wells of a 48- well plate. The hydrogels were placed in a 37 °C incubator for half an hour to gel prior to seeding cells on the gel surface.

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In order to incorporate fibers into the gels, the fibers are first dispersed in ethanol to sterilize them. They are then centrifuged to remove the ethanol and then re-dispersed in sterile water. The fibers in water solution are then also centrifuged and are re-dispersed in a 0.2% acetic acid solution. This solution of fibers in 0.2 vol% acetic acid is then used to create the hydrogel solutions for those hydrogels containing either cobalt ferrite or Janus nanofibers.

5.2.3 Cell Culture

PC12 rat pheochromocytoma neuronal-like cells (PC-12 Adh, ATCC CRL-1721.1) were used to examine the effects of magnetoelectric stimulation on cell growth and differentiation.

PC12 cells were cultured in DMEM/F12 media with 5 vol% fetal bovine serum, 10 vol% horse serum and 1 vol% 100X penicillin/streptomycin. Cells were seeded on the hydrogels at 15,000 cells/well.

5.2.4 Magnetoelectric Stimulation

Magnetoelectric stimulation was carried out using a solenoid developed to fit a 48-well plate. The solenoid is 6” in diameter and 16.33” in length and made with 15 gauge copper wire.

(Figure 5-1). In order to center the well plates in the center of the solenoid vertically, a holder was 3D printed such that the well plates were held in place. For the purposes of the magnetoelectric stimulation, only the center 12 wells on a 48-well plate were used for samples.

To create the desired magnetic fields, the solenoid was connected to a myDAQ (National

Instruments) board which was connected to a 1000W amplifier (Pyramid Classic PB715X 2- channel) and a 1000W power supply (EVGA G+). A square wave was used with a 50 % duty cycle at 60 Hz and an applied voltage of either 20, 30, or 40 volts peak-to-peak (Vpp).

Measurement of the resultant applied magnetic field for 20 Vpp can be found in Appendix F.

These applied voltages correspond to magnetic fields of approximately 2.3 mT, 3.45 mT, and

4.6 mT, respectively, in the wells being used. In addition to varying the voltage applied to the

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solenoid, and thus the magnetic field, the amount of time per day that the cells were stimulated was also changed. The cell stimulation regimes studied are detailed in Table 5-1. All studies were run for 5 days with time points for both assays and microscopy at 0 days, 1 day, 3 days, and

5 days.

5.2.5 Cellular Proliferation

Lactate dehydrogenase (LDH) and PicoGreen cell assays were used to examine the effects of magnetoelectric stimulation on the PC12 proliferation following standard procedures for both assays. Time points of 0 days (PicoGreen only), 1 day, 3 days and 5 days were examined. Unstimulated controls were also examined at these time points to compare the effects of the magnetic field and magnetoelectric stimulation. For PicoGreen assays, cells were prepared by first washing them in PBS and then lysing in a solution of 0.1 % Triton-X 100.

5.2.6 PC12 Differentiation Studies

The effects of magnetoelectric stimulation on PC12 differentiation was studied in addition to the effects on PC12 proliferation. For the differentiation studies, cells were seeded on hydrogels in PC12 growth media as described above. After 24 hours the media was switched to differentiation media which consists of DMEM/F12 with 0.5 vol% fetal bovine serum, 1 vol% horse serum, 1 vol% penicillin/streptomycin and 100 ng/ml of nerve growth factor. Fluorescent confocal microscopy was used to study the differentiation of the PC12 cells under magnetoelectric stimulation. Studies were run with time points of 0 days, 1 day, 3 days and 5 days. Control samples grown in differentiation media but not stimulated with the magnetic field were also collected at each time point. In addition, stimulated samples grown in growth media as opposed to differentiation media were examined for proliferation using confocal microscopy as well. Stimulation for the differentiation study was done under conditions of 20 Vpp applied to the solenoid for 1 hour per day. After treatment, the media was removed from the wells and a

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solution of CellTracker® Green CMFDA dye in dimethyl sulfoxide (DMSO) was used to dye the

PC12 cells for confocal microscopy. Cells were dyed for 30 minutes, after which, the solution of

CellTracker® Green in DMSO was removed and cell culture media was added back to the wells for imaging. Fluorescent confocal microscope images were taken with a Nikon Eclipse C2 confocal microscope using the corresponding NIS Elements software. These images were analyzed using FIJI and ImageJ image analysis software.

5.2.7 Statistical Analysis

Data obtained from LDH and PicoGreen assays were analyzed using ANOVA and two sample t-tests assuming unequal variances. Collagen, cobalt ferrite and Janus hydrogels were compared at each time point. Stimulated versus unstimulated conditions were compared against each other for each time point as well.

5.3 Results and Discussion

5.3.1 Effects of Magnetoelectric Stimulation on PC12 Proliferation and Cytotoxicity

The effects of magnetoelectric stimulation on the proliferation of PC12 cells was studied using LDH and PicoGreen assays at 5 different stimulation regimes: 1 hour of stimulation per day at 2.3 mT applied magnetic field, 1 hour of stimulation at 3.45 mT applied field, 1 hour of stimulation per day at 4.6 mT applied field, 3 hours of stimulation per day at 3.45 mT applied field, and finally, 4 hours of stimulation per day at 2.3 mT applied field (Table 5-1). The initial magnetoelectric stimulation regime that was tried was the 1 hour per day of stimulation at 20 Vpp

(2.3 mT applied magnetic field, ~200 mV/mm electric field cause by the ME effect for Janus hydrogels) applied to the solenoid. Typical electric fields used in cellular stimulation range from

1 mV/mm to 100 mV/mm applied to the cells.174–178 The choice in using an applied magnetic field that corresponds to an electric field in the range of 200 mV/mm was motivated by the fact that the cells are not sitting directly on top of the magnetoelectric nanofibers, the source of the

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electrical field. Thus, the exact amount of electric field that the cells are experiencing was assumed to be slightly less than the calculated 200 mV/mm due to the distance from the fibers. It is worth noting that the calculations for the electric field resultant from placing the magnetoelectric fibers in a magnetic field are done with a magnetoelectric coefficient for an applied signal frequency of 200 Hz. This value was found to be approximately 180 mV/cmOe for barium titanate-cobalt ferrite nanorods, courtesy of Matthew Bauer.179 This is due to difficulties in obtaining a magnetoelectric coefficient at the frequencies used for the stimulation here (60 Hz) caused by electronic noise in the measurements from the instruments used for the measurements.

Thus, the calculations done for the electric field strength likely overestimate the magnetoelectric effect that would be seen at the 60 Hz applied signal frequency that was used for the experiments. However, the experiments were still run using an applied frequency of 60 Hz given that it is relevant to neuronal processes in the body.174–178

Two different assays were used to examine the effects of the applied field on the proliferation and cytotoxicity of the hydrogels: PicoGreen assays and LDH assays. The

PicoGreen assays enables the evaluation of the effects of the applied field on the proliferation of

PC12 cells. PicoGreen quantifies the amount of DNA present in a sample which can be related to the overall amount of cells present. The LDH assay enables a comparison of the cytotoxicity of the cobalt ferrite and Janus hydrogels to the collagen hydrogels that do not contain fibers for both stimulated and unstimulated hydrogels. LDH assays are colorimetric assays that measures the amount of LDH released by damaged cells. Thus, higher amounts of LDH present in a sample means that there is an increased amount of damaged or dead cells and therefore higher cytotoxicity of the sample. These assays were chosen for the fact that they are commonly used to examine proliferation and cytotoxicity, respectively. The LDH data in this chapter is presented as

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the absorbance, or optical density, of the sample in order to include collagen samples on the graphs. To see plots of % cytotoxicity, please refer to Appendix E.

Figures 5-2 and 5-3 show the PicoGreen and LDH results of the cell studies completed on cells stimulated at 2.3 mT applied magnetic field for 1 hour/per day, respectively. The LDH data shown here is for hydrogels containing cobalt ferrite nanofibers and hydrogels containing biphasic Janus fibers as normalized to the effects of empty collagen hydrogels. The LDH data for this stimulation regime shows that overall, the cobalt ferrite and Janus hydrogels exhibit similar effects on the cells as the control collagen hydrogels, which, given that collagen is a natural material that occurs in the body, is biocompatible.180 . This tells us two things; first that the gels themselves are not exhibiting cytotoxic effects on the cells and second, that the stimulation of the cells using an applied magnetic field of 2.3 mT is additionally not exhibiting any cytotoxic effects on the cells. In addition, the LDH data shows that there is not a significant difference between the effects of the hydrogels containing cobalt ferrite fibers and the hydrogels containing

Janus fibers. Looking next at the PicoGreen Data, Figure 5-2 shows that while the applied field does not exhibit any cytotoxic effects as per LDH, it is not promoting any additional cellular proliferation as compared to the unstimulated gels. In addition, the PicoGreen data shows that the hydrogels containing Janus fibers perform similarly to both the empty hydrogels and the hydrogels containing cobalt ferrite fibers. These results prompted the studies of additional stimulation regimes in order to determine if there exists a regime that definitively aids cellular proliferation.

Looking at the whole set of stimulation regimes, there are two general categories that the results can be separated into: stimulation regimes that (1) did not promote or deter proliferation and stimulation regimes that (2) hindered cellular proliferation. Three of the stimulation regimes

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did not hinder proliferation, namely, 1 hour at 2.3 mT applied field (Figures 5-2 & 5-3) (~200 mV/mm electric field cause by the ME effect for Janus hydrogels), 1 hour at 3.45 mT applied field (Figures 5-4 & 5-5) (~300 mV/mm electric field cause by the ME effect for Janus hydrogels), and 4 hours at 2.3 mT applied field (Figures 5-6 & 5-7). All of these regimes exhibit results similar to those described above for the samples stimulated for 1 hour at 2.3 mT. The remaining two regimes, 1 hour at 4.6 mT applied field (Figures 5-8 & 5-9) (~400 mV/mm electric field cause by the ME effect for Janus hydrogels) and 3 hours at 3.45 mT applied field

(Figures 5-10 & 5-11), resulted in decreases in the proliferation of the PC12 cells.

Both the study conducted at 1 hour at 4.6 mT applied (Figures 5-8 & 5-9) and the study conducted at 3 hours at 3.45 mT applied (Figures 5-10 & 5-11) displayed harmful effects on the proliferation of the PC12 cells as compared to the cells grown on the hydrogels in the absence of stimulation. The decrease in the amount of DNA found in the samples using the PicoGreen assay implies that cells are not proliferating. The LDH studies for both of these stimulation regimes shows that the gels, both stimulated and unstimulated are not exhibiting large relative cytotoxicity as compared to the empty hydrogels (Figures 5-9 & 5-11). However, looking at the

PicoGreen data for both of these stimulation regimes, Figures 5-8 and 5-10 show that the cellular proliferation of the PC12 cells on stimulated gels of any type is decreased over the course of the

5 day experiment. The negative effects of these stimulation regimes begin to outline a possible boundary above which the combination of stimulation time and applied field result in detrimental effects on the proliferation of the PC12 cells (Figure 5-12).

5.3.2 Effects of Magnetoelectric Stimulation on PC12 Differentiation

The effects of magnetoelectric stimulation on PC12 differentiation at stimulation of

2.3 mT applied field for 1 hour per day were studied using fluorescent confocal microscopy. In order to be able to image through the hydrogels, the thickness of the hydrogel was decreased by

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about 13% (185 µl per well as opposed to the usual 212 µl per well). Collagen hydrogels were easily imaged through at either thickness, however, cobalt ferrite hydrogels and Janus hydrogels caused issues at the greater thickness due to opaqueness arising from the inclusion of dark fibers in the hydrogels. The Janus hydrogels were able to be imaged through once the thickness had been decreased, however, the autofluorescence of the barium titanate in the Janus fibers still caused some difficulty in imaging the Janus hydrogels. In order to further understand the effects of magnetoelectric stimulation, more work needs to be completed to enhance the imaging of

PC12 cells on collagen gels containing Janus nanofibers.

Figures 5-13 through 5-26 show the fluorescent images of PC12 cells cultured on collagen, collagen with cobalt ferrite, and collagen with Janus fiber hydrogels. Figure 5-13 shows day 0 PC12 cells. These cells have only been allowed to attach to the hydrogels prior to imaging. Fluorescent microscopy served to confirm that PC12 cells were present on the hydrogels. Figures 5-14 through 5-21 show fluorescent microscopy of samples on days 1 and 3, respectively. Samples from days 1 and 3 show almost no neurite extension from the PC12 cells.

The beginnings of neurite extension begin to appear in samples examined on day 5 (Figure 5-22 -

526). However, for each type of sample, less than 10 % of the cells visible in the microscope images have begun to exhibit neurite extension. Specifically, for the cells grown in growth media, unstimulated samples showed 4.0 %, 7.8 %, and 5.1 % of the cells showed neurite outgrowth for the collagen, cobalt ferrite, and Janus hydrogels, respectively. The stimulated samples in growth media showed 1.3%, 2.9%, and 3.1% of the cells exhibited neurite outgrowth for the collagen, cobalt ferrite, and Janus hydrogels, respectively. Looking instead at those samples grown in differentiation media, the unstimulated samples showed 4.4%, 8.8%, and 5.6% of the cells demonstrated neurite outgrowth for the collagen, cobalt ferrite, and Janus hydrogels,

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respectively. Finally, for the samples that underwent stimulation in differentiation media, 1.8%,

6.5%, and 4.3% of the cells were found to exhibit neurite outgrowth for the collagen, cobalt ferrite, and Janus hydrogels, respectively. Figure 2-26 shows a direct comparison of cells grown in Janus hydrogels in differentiation media for 5 days both stimulated and unstimulated. This figure clearly shows the increased number of cells exhibiting neurite outgrowth in the those gels that were not stimulated in the magnetic field. These numbers can be found in Table 5-2. Based on these numbers, it appears that, at a stimulation of 1 hour per day in a 2.3 mT applied magnetic field, the stimulated cells tend to exhibit lower amounts of neurite growth as compared to the unstimulated cells. In order to confirm these trends and ensure statistical significance the differentiation study at this stimulation regime will need to be repeated. These numbers are subject to some error due to the difficulty of imaging through the hydrogels. Further studies will need to be done, perhaps using even thinner hydrogels, in order to better understand the effects of the magnetoelectric stimulation. Based on these results, it is likely that longer studies, namely with time points at seven days or more, will be necessary in order to fully understand the effects of magnetoelectric stimulation on the differentiation of PC12 cells. It is promising, however, that some neurite extension is visible at 5 day of stimulation.

5.4 Summary

The magnetoelectric stimulation of PC12 neuronal-like cells for both proliferation and differentiation are presented here. Five different magnetoelectric stimulation regimes, with applied magnetic fields ranging from 2.3-4.6 mT corresponding to electric fields ranging from

200-400 mV/mm for magnetoelectric samples, were tested for their effects on the proliferation of

PC12 cells grown on collagen hydrogels containing magnetoelectric nanofibers. Of the five stimulation regimes, two were found to reduce the proliferation of the PC12 cells over the course of 5 days (1 hour per day at 40 Vpp applied to the solenoid and 3 hours per day at 30 Vpp applied

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to the solenoid). The remaining stimulation regimes did not increase cellular proliferation over the control collagen hydrogels without magnetoelectric fibers but also did not decrease proliferation in comparison to the collagen controls.

For the examination of the effects of magnetoelectric stimulation on the differentiation of

PC12 cells, only one stimulation regime was chosen. This regime was 1 hour per day at 30 Vpp which was shown to not harm cells after 5 days of stimulation as seen with the proliferation studies. Based on fluorescent confocal microscopy of the PC12 cells on the collagen, collagen with cobalt ferrite, and collagen with Janus fiber hydrogels, after five days of growth (an stimulation in the case of stimulated hydrogels) neurite extension had begun. However, at this point, less than 10% of the cells visible for each type of sample had begun to exhibit neurite extension. In addition, the samples that were exposed to the magnetic field seem to exhibit reduced differentiation as compared to the unstimulated samples. Based on these results, it can be concluded that longer studies will be necessary to fully assess the effects of magnetoelectric stimulation on the differentiation of PC12 cells.

The studies presented here for the effects of magnetoelectric stimulation on the proliferation and differentiation of PC12 cells on collagen based magnetoelectric composite hydrogels are preliminary in nature. Further work needs to be done in order to determine a stimulation regime that aids in the proliferation of the PC12 cells. In addition, further work needs to be done in order to determine a stimulation regime which promotes the differentiation of the

PC12 cells on the magnetoelectric composite hydrogels. Imaging methods for PC12 cells grown on these hydrogels need to be improved, specifically for those hydrogels containing Janus fibers due to the interference during imaging of the barium titanate in the Janus fibers. However, having established that it is possible to image the cells through these gels for examination of the

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differentiation of PC12 cells, these imaging methods could be expanded for examining the effects of magnetoelectric stimulation on the proliferation and cytotoxicity of PC12 cells as well.

The results shown here demonstrate that magnetoelectric materials have potential to be used in biomedical applications, specifically for aiding in neuronal healing.

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Table 5-1. Regimes for the magnetoelectric stimulation of PC12 cells on collagen based hydrogels. Electric field values from the magnetoelectric effect were completed using a magnetoelectric coefficient of 180mV/cmOe for cobalt ferrite-barium titanate Janus fibers at an applied signal frequency of 200 Hz, courtesy of Matthew Bauer. Calculated Voltage Applied Signal Electric Field Duty Cycle Magnetic Field Time in Field Applied to Frequency from ME (%) Strength (mT) (hrs) Solenoid (V) (Hz) Effect (mV/mm) 20 50 60 1.108-1.115 199-201 1 20 50 60 1.108-1.115 199-201 4 30 50 60 1.662-1.673 298-302 1 30 50 60 1.662-1.673 298-302 3 40 50 60 2.216-2.23 398-402 1

Table 5-2. Percentages of cells exhibiting neurite extension on day for each growth condition. Error on these percentages comes from counting the number of neurites by hand 3 times.

Growth Media – Growth Media – Differentiation Media - Differentiation Unstimulated Stimulated Unstimulated Media - Stimulated

Collagen 4.0±1.3 % 1.3±1.0 % 4.4±1.8 % 1.8±1.1 %

Collagen 7.8±6.2 % 2.9±1.9 % 8.8±6.9 % 6.5±5.5 % + CFO

Collagen 5.1±2.1 % 3.1±0.9 % 5.6±5.1 % 4.3±0.9 % + Janus

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Figure 5-1. Camera images the solenoid setup created for the magnetoelectric stimulation experiments. The solenoid shown here is inside of a cell culture incubator to prevent any additional shocks to the cells during stimulation. Photo courtesy of author.

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Figure 5-2. Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 2.3 mT magnetic field.

Figure 5-3. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 2.3 mT magnetic field.

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Figure 5-4. Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 3.45 mT magnetic field.

Figure 5-5. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 3.45 mT magnetic field.

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Figure 5-6. Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 1 hour in a 4.6 mT magnetic field.

Figure 5-7. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 4.6 mT magnetic field.

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Figure 5-8. Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 3 hours in a 3.45 mT magnetic field.

Figure 5-9. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 3 hours in a 3.45 mT magnetic field.

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Figure 5-10. Cellular proliferation as examined with a Picogreen assay for PC12 cells grown on magnetoelectric hydrogels stimulated for 4 hours in a 2.3 mT magnetic field.

Figure 5-11. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 4 hours in a 2.3 mT magnetic field.

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Figure 5-12. Plot showing stimulation regimes that neither hindered nor promoted cellular proliferation compared to stimulation regimes that had detrimental effects on cellular proliferation. The stimulation regimes that exhibited detrimental effects hint at the possibility of a boundary above which the applied field and length of time in the field results in decreased cellular proliferation over the course of a 5 day study.

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Figure 5-13. Confocal microscopy of PC12 cells stained with fluorescent CellTracker® Green after 24 hours on the gel to allow for cell attachment.

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Figure 5-14. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in growth media without stimulation.

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Figure 5-15. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-16. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in differentiation media without stimulation.

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Figure 5-17. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 1 day of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-18. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in growth media without stimulation

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Figure 5-19. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-20. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in differentiation media without stimulation.

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Figure 5-21. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 3 days of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-22. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in growth media without stimulation.

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Figure 5-23. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in growth media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-24. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in differentiation media without stimulation.

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Figure 5-25. Confocal microscopy of PC12 cells stained with fluorescent CellTracker Green after 5 days of growth in differentiation media with stimulation in a 2.3 mT magnetic field for 1 hour per day.

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Figure 5-26. Confocal microscopy of collagen gels with Janus fibers grown in differentiation media following 5 day of stimulation in a 2.3 mT magnetic field for 1 hour per day. Red arrows point to cells that have begun to exhibit neurite outgrowth after five days of stimulation.

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CHAPTER 6 CONCLUSIONS AND FUTURE WORK

6.1 Summary

This work developed methods for electrospinning and electrospraying magnetoelectric, ceramic nanocomposites. Sol-gel electrospinning has been shown to be a successful method for the production of bi-phasic, magnetoelectric Janus fibers. In order to achieve these fibers, the chemistry of the sol-gel precursor solutions for cobalt ferrite was altered from previously demonstrated precursor solutions by the addition of chelating agents. The addition of chelating agents creates a more homogeneous distribution of the metal ions in the precursor solutions, slows condensation reactions, and improves the reliability of the electrospinning these solutions.

Two chelating agents were studied for their ability to create solutions that would electrospin reliably, specifically, citric acid and acetylacetone. Citric acid precursor solutions resulted in webbed fibers at best, while acetylacetone solutions were found to reliably electrospin fibers.

In addition to enabling the reliable production of cobalt ferrite fibers, adding chelating agents to the precursor solutions for cobalt ferrite aided the production of biphasic Janus fibers.

These fibers were confirmed to qualitatively exhibit magnetoelectric behavior using vibrating sample magnetometry. Qualitatively confirming that the fibers exhibit magnetoelectric behavior enabled the use of the Janus fibers for applications in neuronal stimulation.

In addition, the successful electrospray synthesis of both barium titanate and cobalt ferrite nanoparticles has been demonstrated here for the first time. The electrospray synthesis of barium titanate nanoparticles was found to be sensitive to the relative humidity of the electrospraying environment as well as the concentration of metal ions in the precursor solution. Cobalt ferrite nanoparticles proved more difficult to electrospray than barium titanate nanoparticles due to the increased sensitivity to water of the precursor solutions as compared to barium titanate

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precursors as well as the increased conductivity of cobalt ferrite precursor solutions. In order to achieve cobalt ferrite nanoparticles, the use of a liquid collector bath was necessary. By using

1,000 cSt silicone oil heated to 200 °C, cobalt ferrite particles were successfully achieved. This method of electrospraying cobalt ferrite provides a scalable synthesis method for the production of magnetic nanoparticles. For both barium titanate and cobalt ferrite, salt calcination techniques were used in order to obtain crystalline nanoparticles. This involved the dispersal of as-sprayed particles in a fine powder of sodium chloride prior to calcination. This method prevented the agglomeration of the nanoparticles during calcination thus enabling the final product to remain as discrete particles rather than a large sintered mass.

Finally, this work presents preliminary data regarding the use of magnetoelectric materials for electric stimulation of neuronal proliferation and differentiation. The magnetoelectric materials described above were incorporated into collagen hydrogels and used to stimulate PC12 neuronal-like cells. Cells were stimulated using a magnetic field to trigger the magnetoelectric properties of the fibers. A variety of stimulation regimes were tested for their effects on the proliferation of PC12 cells. Two stimulation regimes, 1 hour per day of stimulation at 40 Vpp applied to the solenoid and 3 hours per day at 30 Vpp applied to the solenoid, were found to decrease the proliferation of the cells over the course of 5 days. These two regimes suggest that there is an upper boundary for magnetoelectric stimulation at which the applied field begins to damage the cells. The remaining stimulation regimes were found to have little effect of cellular proliferation, in either a positive or negative manner.

Only one regime, 1 hour of stimulation per day in a 2.3 mT magnetic field, was tested for the effects of magnetoelectric stimulation on the differentiation of PC12 cells. In order to examine the effects of this stimulation, methods of imaging cells through the hydrogels using

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fluorescent confocal microscopy were used. In order to do this, the thickness of the hydrogels was reduced to enable light to pass through the hydrogels containing fibers. The beginnings of neurite outgrowth were seen after 5 days of stimulation, though it was seen in less than 10 % of the cells for each combination of hydrogel, media, and stimulation conditions. Janus samples showed grown in growth media showed 5.1% and 3.1% of cells exhibiting neurite extension for the unstimulated and stimulated samples, respectively, while the samples in differentiation media showed 5.6% and 4.3% of cells with neurites for unstimulated and stimulated samples, respectively. Magnetic field induced magnetoelectric stimulation seems to be decreasing the amount of cells exhibiting neurite outgrowth pointing to the possibility of needing to study lower fields. However, in order to better elucidate the effects of magnetoelectric longer studies, specifically studies with time points at seven or more days, will be necessary.

6.2 Research Contributions

This work presents multiple contributions to the field of materials science and engineering. First, this work presents the development of reliable precursor solutions for cobalt ferrite electrospinning, and thus Janus electrospinning. This enables the facile synthesis of standalone magnetoelectric nanocomposites. These nanocomposite fibers can then be used for further applications such as the work done with neuronal stimulation discussed herein.

This work also presents a novel electrospray synthesis for both single-phase barium titanate and single-phase cobalt ferrite. These methods provide a basis for producing nanoparticles of complex oxide through electrospraying. In addition, this provides a foundation on which to build for the facile synthesis of composite nanoparticles. With the newfound ability to produce single-phase ceramic nanoparticles via electrospraying, it opens the door to combine the methods used for creating composite ceramic nanofibers, such as Janus ceramic

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electrospinning, and the methods presented here for the synthesis of ceramic nanoparticles via electrospraying to easily create Janus nanoparticles of functional ceramics via electrospray.

Finally, this work shows preliminary experiments for the use of magnetoelectric biomaterials. While the results do not yet show an explicit benefit of magnetoelectric stimulation, they are promising in that they also do not show that magnetoelectric stimulation is restricted to having negative effects on cellular proliferation or differentiation. This opens the door to further examinations of magnetoelectric stimulation for use in biomaterials.

6.3 Future Work

6.3.1 Electrosprayed Ceramic Nanoparticles

The novel, scalable method for electrospraying cobalt ferrite established here presents the opportunity for the development of electrospray syntheses for a wide variety of other complex oxides. Specifically, due to the difficult nature of electrospraying ferrite materials, the method of electrospraying into a silicone oil heated collector could potentially enable electrospraying of other ferrite such as bismuth ferrite and nickel zinc ferrite, among others.

Beyond extending the methods described in this document for the electrospray synthesis of other single phase complex oxide particles, these methods can also be examined for the development of composite nanoparticles. As with electrospinning, electrospraying presents a facile method for creating nanocomposites with a Janus type morphology. Using the methods of electrospraying into a liquid collector described for the synthesis of cobalt ferrite nanoparticles, it may be possible to electrospray Janus type nanoparticles. It is predicted that Janus nanoparticles would exhibit greater magnetoelectric effects as compared to Janus fibers due to their larger interfacial area to volume ratios.

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6.3.2 Magnetoelectric Nanomaterials for Neuronal Stimulation

As mentioned above, two stimulation regimes which hinder the proliferation of PC12 neuronal-like cells have been found. However, we still lack magnetoelectric stimulation regimes which definitively aid in the proliferation of PC12 cells. Further work must be done to determine if there is a stimulation regime which positively impacts cellular proliferation. This can be done by altering the strength of the field applied as well as by altering the time for which the field is applied. While the data obtained thus far shows a limit in the strength of field that can be used, currently, there is no data showing negative effects on the cells for stimulation at lower fields for longer times. Future experiments may find success in increasing the time for which the cells are stimulated past 4 hours per day at low applied fields.

Looking instead at the effects of magnetoelectric stimulation on cellular differentiation, only one regime has been studied thus far. Additional regimes should be studied in order to optimize the stimulation parameters for the differentiation of PC12 neuronal cells. However, before additional stimulation regimes are examined, stimulation using the parameters discussed above to study PC12 differentiation should be studied with additional time points of 7 or more days of stimulation. This will enable a better understanding of how many days of stimulation are necessary to influence the differentiation of PC12 neuronal-like cells.

Once an optimal stimulation regime and length of study have been determined, more in depth studies about the effects of magnetoelectric stimulation on PC12 differentiation can be completed. These may include studies of the effects of magnetoelectric stimulation on Ca2+ transients and Na+ transients which are important factors in the growth and differentiation of neuronal cells. Studies such as these would better elucidate the mechanisms through which magnetoelectric stimulation affects neuronal growth and regeneration.

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APPENDIX A CALCULATIONS FOR THE MODEL OF INTERFACIAL AREA TO VOLUME RATIO FOR BOTH NANOPARTICLES AND NANOFIBERS

Interfacial Area to Volume Ratio Calculations for Janus Nanoparticles

Assumptions:

• Perfect interface between the two phases (no defects, i.e. pores)

• Perfectly circular interface between the two phases

• Interface lies at the equator of the particle

Equations:

퐼푛푡푒푟푓푎푐푖푎푙 푎푟푒푎 = 휋푟2 (퐴 − 1)

4 푉표푙푢푚푒 = 휋푟3 (퐴 − 2) 3

퐼퐴: 푉 푟푎푡푖표 = 푖푛푡푒푟푓푎푐푖푎푙 푎푟푒푎/푣표푙푢푚푒 (퐴 − 3)

Where r is the radius of the particle.

Figure A-1. Table of interfacial area, volume and IA:V ratio calculated for nanoparticles of a given diameter.

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Interfacial Area to Volume Ratio Calculations for Janus Fibers

Assumptions:

• Perfect interface between the two phases (no defects, i.e. pores)

• Perfectly circular cross-section of the nanofibers

• Interface lies at the equator of the fiber

• Length of the fiber is equal to 1 µm

Equations:

퐼푛푡푒푟푓푎푐푖푎푙 푎푟푒푎 = 푙 × 퐷 (퐴 − 4)

푉표푙푢푚푒 = 휋푟2 × 푙 (퐴 − 5)

퐼퐴: 푉 푟푎푡푖표 = 푖푛푡푒푟푓푎푐푖푎푙 푎푟푒푎/푣표푙푢푚푒 (퐴 − 6)

Where l is the length of the fiber, D is the diameter of the fiber, and r is the radius of the cross- section of the fiber.

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APPENDIX B CALCULATIONS FOR COLLAGEN TYPE I HYDROGEL COMPOSITIONS

Materials:

• Rat tail collagen type I

• 0.2 % acetic acid in sterile water

• 10X Dulbecco’s Modified Eagle’s Medium (DMEM)

• HEPES buffer, pH 8.0

• Sodium bicarbonate

NOTE: Keep all components on ice to prevent premature gelation of collagen.

DMEM/HEPES Solution Preparation

1. Mix together 10 ml of 10X DMEM and 10 ml of HEPES

2. Add 0.4 g of sodium carbonate to the DMEM/HEPES solution and shake to dissolve

3. Solution should be bright pink

Collagen Hydrogel Pre-Solution Preparation

1. Determine the amount of hydrogel solution necessary to create the desired number of gels

(212 µl of solution is needed for each gel made in a 48-well plate). Add 3x212 µl to the

solution to ensure there is enough for the number of gels.

a. Example: 32 hydrogels desired

32 푔푒푙푠 × 212 휇푙 + 3 푔푒푙푠 × 212 휇푙 = 7.42 푚푙

2. Determine the amount of collagen necessary to obtain a solution with a final

concentration of 3 mg/ml.

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a. Example: as purchased collagen concentration: 9.41 mg/ml 푚푔 푚푔 (9.41 ) (푥 푚푙) = (3 ) (7.42 푚푙) 푚푙 푚푙

푥 = 2.366 푚푙 푐표푙푙푎푔푒푛 푡푦푝푒 퐼

3. Determine the amount of DMEM/HEPES solutions necessary. This is 20 % of the total

final volume desired.

20% × 7.42 푚푙 = 1.484 푚푙 퐷푀퐸푀/퐻퐸푃퐸푆

4. Determine the amount of 0.2% acetic acid necessary

7.42푚푙 − 1.484푚푙 − 2.366 푚푙 = 3.57 푚푙 0.2% 푎푐푒푡푖푐 푎푐푖푑

5. Measure collagen into 15ml centrifuge tube.

6. Add 0.2% acetic acid followed by DMEM/HEPES.

7. Gently mix to avoid the formation of bubbles in the solution.

8. Pipette into wells of 48-well plate.

9. Place well plate in 37 °C incubator for gels to solidify.

Additional steps for hydrogels containing CoFe2O4 or Janus fibers

1. Measure out desired amount of fibers (2.12 mg/ml CoFe2O4 or 4.65 mg/ml Janus).

2. Disperse fibers in about 8 ml of ethanol via sonicating for 20 min.

3. Centrifuge fibers and decant ethanol.

4. Re-disperse fibers in sterile water, sonication for 20 min.

5. Centrifuge and decant water.

6. Re-disperse fibers in the amount of 0.2% acetic acid necessary for the hydrogel solution

being made. Sonicate for 10 min and then cool on ice before use.

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NOTE: For hydrogels containing fibers, collagen is added to the fiber/0.2% acetic acid solution and DMEM/HEPES is the last thing added.

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APPENDIX C EFFECTS OF HUMIDITY ON BARIUM TITANATE SOL-GEL ELECTROSPRAY

C.1 Experimental Methods

The experimental methods described here closely follow those described in Chapter 3.

The main difference here is that solutions with a 1:1 molar ratio of Ba:Ti was electrosprayed.

However, this solution was electrosprayed at 7 different humidity conditions.

C.1.1 Materials

Barium acetate, titanium (IV) isopropoxide (≥97.0%), and glacial acetic acid were obtained from Sigma Aldrich. Isopropanol and sodium chloride were obtained from Fisher

Scientific. All reagents were used without further modification.

C.1.2 Sol-Gel Precursor Solutions

BaTiO3 particles were synthesized via sol-gel electrospraying. To create the sol-gel precursor, 0.55g of barium acetate was dissolved in 1.5ml of glacial acetic acid under heating. In a separate vial, 0.63ml of titanium (IV) isopropoxide was added to 0.87ml of isopropanol. The titanium (IV) isopropoxide solution was then added to the barium acetate solution at room temperature. This final solution is stirred for two hours before electrospraying.

C.1.3 Nanoparticle Synthesis

For electrospraying, the BaTiO3 precursor was loaded into a syringe placed in a syringe pump across from a grounded copper plate electrode covered in aluminum foil. The syringe pump was set to a rate of 0.15ml/hr. The needle was attached to a high voltage source and the voltage was increased until an electrified jet formed at 11kV. The humidity of the electrospraying setup was monitored using a Fisher Scientific Traceable

Hygrometer/Thermometer connected to Fisher Scientific DAS-3 Data Acquisition System.

Samples were electrosprayed at humidity levels between 20-50 %RH at intervals of 5 %RH.

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Humidity levels were maintained within +/-1% of the desired relative humidity level (e.g.,

34-36 %RH for desired 35 %RH) and were controlled by pumping dry house air into the electrospraying chamber.

The amorphous, as-sprayed particles were calcined to obtain crystalline BaTiO3 particles.

Before calcination, particles were placed into a vacuum oven at 70°C overnight to remove excess solvent. The as-sprayed particles were then dispersed in ~10g of sodium chloride using a mortar and pestle. This mixture was then calcined at 800°C for 3 hours at a ramp rate of 20°C/min.

After salt calcination, particles were washed in water 5 times to remove any salt from the sample followed by an ethanol wash.

C.1.4 Nanoparticle Characterization

Particle morphology, both before and after salt calcination, was examined using an FEI

Nova NanoSEM 430 scanning electron microscope (SEM). ImageJ was used in combination with the SEM images to determine an average particle size and standard deviation both before and after calcination by measuring 300 particles per sample. X-ray diffraction (XRD) to confirm the particle crystal structure was completed using a PANalytical X’Pert Powder X-ray diffractometer. Raman spectroscopy using a Renishaw InVia Raman spectrometer was used to further examine the particles to detect the presence of cubic, tetragonal or hexagonal BaTiO3.

C.2 Results and Discussion

Figure 1 shows as-sprayed, amorphous BaTiO3 particles that were electrosprayed at 7 different humidity conditions between 20-50% relative humidity (%RH) at intervals of 5 %RH, revealing the role of humidity on particle morphology. At low humidity levels (Figure 1a b), particles with elongated tails are observed. As the humidity increases, these elongated tails disappear and particles transition to having dimpled surfaces at intermediate humidity levels

(Figure 1c-d) before spherical particles are observed at higher humidity levels (40 and 45 %RH)

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(Figure 1e-f). The formation of the particles during the electrospraying process is driven by two main mechanisms: solvent evaporation and hydrolysis/condensation reactions. Which mechanism dominates the solidification of the particles drives the final particle morphology and is highly dependent on the relative humidity of the system during electrospraying; specifically, solvent evaporation plays a larger role at low humidity while hydrolysis and condensation dominate at higher humidity .106

The particles electrosprayed at 20 %RH (Figure 1a) reveal the presence of multiple particle morphologies including spherical particles, dimpled particles and particles with long tails as well as an occasional needle. Dimpled particles and the presence of long tails suggest that the

Rayleigh limit of the droplet was reached during the flight to the collector plate. The Rayleigh limit is the threshold at which the accumulation of surface charge on the droplet can no longer be balanced by the surface tension of the droplet, causing the droplet to elongate before breaking up

.107 The breakup of a droplet at the Rayleigh limit into several smaller droplets re-distributes the surface charge over a larger surface area, thus reducing it below the threshold. At low humidity levels the droplets are more likely to reach the Rayleigh limit as solvent evaporation occurs more quickly at low humidity. However, due to the high concentration of ceramic precursors in the droplet, hydrolysis and condensation reactions are also rapidly occurring. The combination of these two processes leads to the solidification of the droplet in an elongated shape before it has time to break up into smaller droplets.

Next, examining the particles electrosprayed at 30 %RH (Figure 1c) it can be seen that the particle morphology is composed of particles with dimpled surfaces along with spherical particles. This morphology can be described similarly to the particle morphology at 20 %RH.

However, for the 30 %RH sample, the droplets do not reach the Rayleigh limit before hydrolysis

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and condensation have solidified the droplet, resulting in dimpled particles. As explained by

Salata , as the Rayleigh limit is approached, the ratio of the electrostatic forces on the droplet to the surface tension approaches unity and shape instabilities occur.111 If one observes the particles electrosprayed at 25 %RH (Figure 1b), it is clear that the sample contains particles created by a combination of the processes occurring at 20 and 30 %RH, highlighting the evolution of morphology as a function of humidity. In addition, examining the particles electrosprayed at 35

%RH (Figure 1d), it can be seen that, while the same driving forces as those behind the particle morphology for samples at 30 %RH contribute to the morphology of the particles at 35 %RH, there is also an increased amount of particles that do not exhibit dimples. Thus, the 35 %RH sample, similar to the 25 %RH sample, likely undergoes a combination of the processes occurring at both 30 and 40 %RH, which is discussed next.

The samples electrosprayed at 40 and 45 %RH (Figure 1e-f) are spherical and no longer exhibit dimpled particles. This morphology results from the elevated humidity which causes the rate of hydrolysis and condensation to exceed the rate of evaporation. Therefore, the particles gel and solidify before they reach the Rayleigh limit and experience any surface instability.

Lastly, the samples electrosprayed at 50 %RH (Figure 1g), result in the formation of spherical particles in addition to bridged particles. Bridged particles are two sphere-like particles found at either end of an arc of solidified amorphous BaTiO3, which look like beads on a string.

These are attributed to humidity and concentration effects which result in rapid gelation of a droplet before it completely breaks away from the jet.

In comparing the trends found in the morphology of the as-sprayed BaTiO3 particles shown here with those observed by Li et al. , there are a few differences.110 For example, Li et al. observed changing particle morphologies ranging from small spherical droplets at low humidity

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to elongated, needle-like particles at intermediate humidity and finally large misshapen particles at high humidity.110 The reason for these varied morphologies is the varying rates of solvent evaporation and gelation of the sol due to the different humidity levels, with solvent evaporation dominating the resultant particle shape for particles sprayed at both low and intermediate humidity .110 Li et al. attributes the titania particle morphology to humidity effects on the

Rayleigh limit of the spraying solutions.110

In contrast, due to the greatly increased concentration of the electrospraying solutions used herein (approximately 10x concentration of the used titanium precursor v/v%) we propose that the morphology of the as-sprayed BaTiO3 particles is more affected by the hydrolysis and condensation reactions of the sol-gel constituents of the spraying solutions. As such, the determination of the morphology of the BaTiO3 particles is more of a balance between hydrolysis and condensation and Rayleigh limit effects as compared to the titania particles produced by Li et al.110 It is worth noting here that Li et al. (2007) utilized solutions of 9:1 ethanol:2-methoxyethanol versus the 1:1 acetic acid:isopropanol used herein. The difference in these solvent compositions resulted in the vapor pressure of the BaTiO3 solution being approximately half that of the solutions used by Li et al. (2007). According to Tang and Kebarle.

, the rate of solvent evaporation is directly proportional to the vapor pressure of the solvent .181

Thus, due to the lower vapor pressure of acetic acid and isopropanol compared to ethanol and 2- methoxyethanol, the solvent evaporates more slowly from the BaTiO3 precursor droplets during electrospraying than for the titania precursor droplets discussed by Li et al.110 Since the solvent evaporates more slowly in our system, it takes longer times and lower humidity levels for droplets to reach the instability causing Rayleigh limit. This allows the droplets to be further

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along in hydrolysis and condensation reactions by the time they reach the Rayleigh limit, if they do.

Following the analysis of the amorphous, as-sprayed particles, the particles were calcined to obtain crystalline BaTiO3 particles. It is worth noting that calcination was only performed on samples electrosprayed at 45 %RH as these particles exhibited a spherical morphology with a narrow size distribution (446±129 nm). After salt calcination, SEM was used to again examine the particle size and morphology (Figure 2), which revealed that the particles maintained their spherical shape and remained un-agglomerated through calcination. Following calcination, the average size of the BaTiO3 particles decreased as a dense crystalline ceramic was formed

(338±99 nm calcined versus 446±129 nm as-sprayed). These particle sizes were determined using ImageJ to obtain particle measurements for 300 particles for both the uncalcined and calcined samples.

X-ray diffraction (XRD) and Rietveld refinement were used to examine the crystal structure of the particles post salt calcination. As can be seen in Figure 3, the XRD data for the particles matches a reference pattern for BaTiO3. Unfortunately, it is difficult to discern from

XRD whether these particles are composed of tetragonal or cubic BaTiO3 due to the peak broadening arising from the small particle size. However, the XRD pattern does show that no impurities, such as barium carbonate or titania, are present in the particles. In addition, from this

XRD data the average crystallite size was calculated to be 34.8±5.4 nm using Scherrer’s formula.

This average crystallite size is greater than the 20 nm limit calculated by Fang et al. (2010). to be the minimum crystallite size at which BaTiO3 particles can exhibit piezoelectric behavior. This

20 nm size limit is due to the instability of tetragonal BaTiO3 in smaller crystallites as modeled by including domain-wall energy into Landau-Ginzburg free-energy calculations .182

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To further confirm which phases of BaTiO3 were present, Raman spectroscopy was completed using a Renishaw InVia Raman spectrometer. Raman spectroscopy is capable of distinguishing between cubic, tetragonal, or hexagonal BaTiO3. The Raman spectra of the particles is shown in Figure 4, and confirms the presence of tetragonal BaTiO3 with peaks at 306 and 710 cm-1.123,183 The spectra also shows that cubic BaTiO3 may be present with the peaks at

179 and 516 cm-1.184 In addition, Raman spectroscopy reveals that there is a small amount of barium carbonate present, likely on the particle surface, to which the peak below 200 cm-1 can be attributed .185

C.3 Summary and Conclusions

Demonstrated herein is the successful synthesis of BaTiO3 nanoparticles via electrospraying. In addition, it has been shown that the relative humidity levels during the electrospraying process have a definite effect on the resultant particle morphology. Thus, by controlling the humidity of the electrospraying environment it is possible to tune the final particle morphology to that of the desired shape. In order to achieve particles that are both spherical and monodisperse, an electrospraying humidity between 40-45 %RH was found to work best. Salt calcination has also proven to be an effective method of calcining the as-sprayed BaTiO3 particles to form a crystalline material while also preventing agglomeration.

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Figure C-1. Scanning electron micrographs of as-sprayed BaTiO3 particles at (a) 20 %RH, (b) 25 %RH, (c) 30 %RH, (d) 35 %RH, (e) 40 %RH, (f) 45 %RH, and (g) 50 %RH.

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Figure C-2. Scanning electron image of BaTiO3 particles post salt calcination at 750°C.

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Figure C-3. XRD scan of salt calcined BaTiO3 particles compared to a barium titanate reference pattern.

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Figure C-4. Raman spectra of BaTiO3 particles. Peaks are labeled as either tetragonal (T) BaTiO3, cubic (C) BaTiO3 or barium carbonate (witherite, W).

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APPENDIX D SYNTHESIS OF JANUS-TYPE MAGNETOELECTRIC PARTICLES VIA SOL-GEL ELECTROSPRAYING

D.1 Introduction

The previous two chapters introduced methods for the successful electrospray synthesis of both single-phase barium titanate nanoparticles and single-phase cobalt ferrite nanoparticles.

This chapter presents the combination of these in order to create electrosprayed bi-phasic Janus nanoparticles. Until now, composite nanoparticles typically exhibit either core-shell or randomly dispersed grains as their morphology. Janus nanoparticles, as with Janus nanofibers, is a desirable morphology as it avoids issues of preferential strain relaxation at the surface as could be an issue with randomly dispersed grains as well as delamination issues that have been seen in core-shell nanostructures.105

Chapter 2 discusses the synthesis of Janus nanofibers which benefit from a lack of substrate clamping over magnetoelectric composites such as thin films or bulk composites. Here, the motivation for the synthesis of Janus nanoparticles is the same as for Janus nanofibers with the addition of the prediction that as the composites are decreased in size, the ratio of interfacial area to volume increases which is predicted to correspond to an increase in the magnetoelectric behavior of the particles over the fibers. This is due to more of the material being located at the interface between the piezoelectric and magnetostrictive materials as opposed to in either individual hemisphere or hemi-cylinder. A model for the increase in the ratio of the interfacial area to volume ratio is shown in Figure D-1. The calculations done to obtain this model assumed perfect interfaces between the two halves of either the particle or fiber. In addition, the calculations for the fibers were done assuming a fiber length of 1 µm. The calculations also assumed that for the particles, the interface was at the equator of a perfectly spherical particle.

For the fiber calculations, fibers with a perfectly circular cross-section were assumed. Additional

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details about the calculations done to obtain this plot can be found in Appendix A. Figure D-1 shows that as the diameter of both the fibers and the particles decreases, the interfacial area to volume ratio increases as expected. However, at approximately a diameter of 200 nm, the ratio for the nanoparticle begins to exceed those for nanofibers. Thus, this simple model agrees with the hypothesis that nanoparticles will exhibit more magnetoelectric behavior than Janus nanofibers.

In this chapter, following the synthesis of Janus nanoparticles via sol-gel electrospraying the qualitative magnetoelectric behavior of the nanoparticles is compared to that of the Janus fibers to demonstrate that the particles do indeed exhibit more magnetoelectric behavior than the fibers.

D.2 Experimental Methods

D.2.1 Materials

Cobalt (II) nitrate hexahydrate, citric acid monohydrate, barium acetate, titanium (IV) isopropoxide (≥97.0%), glacial acetic acid, and 1,000 cSt silicone oil were obtained from Sigma

Aldrich. Ferric nitrate nonahydrate, ethylene glycol, and sodium chloride were obtained from

Fisher Scientific. All chemicals were used without further modification.

D.2.2 Sol-Gel Precursor Solutions

The chemistry of the sol-gel solutions used to create the cobalt ferrite half of the Janus particles was as discussed in chapter 4. Briefly, this solution consists of 0.875 g of ferric nitrate nonahydrate, 0.315 g of cobalt (II) nitrate hexahydrate, ad 0.2 g of citric acid monohydrate dissolved in 3 ml of ethylene glycol. In order to have barium titanate sol-gel solutions that would electrospin alongside the cobalt ferrite solutions, the barium titanate solutions were altered significantly as compared to the solutions described in chapter 3. For the barium titanate solutions used here, the quantities of barium acetate and titanium (IV) isopropoxide remain the

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same at 0.55 g and 0.63 ml, respectively. However, the solvents were changed in order to achieve solutions that would electrospray into a liquid collector. Here the barium titanate precursor solutions consisted of 2 ml of ethylene glycol and 1 ml of acetic acid. Citric acid as an additional chelating agent was also included in these solutions at a quantity of 0.1 g.

Like the solutions discussed in chapter 3, these barium titanate solutions needed to be heated in order to dissolve the barium acetate and citric acid. In order to do this, the ethylene glycol and acetic acid were mixed and then the citric acid and barium acetate were added. This solution was heated for half an hour. At this point, the solutions appears white as though it had crashed. However, after adding the titanium (IV) isopropoxide to the solution and allowing it to mix for an additional half an hour while being heat the solution became clear and pale yellow.

The solution was then allowed to cool to room temperature before electrospraying.

D.2.2 Electrospraying into Silicone Oil Liquid Collector

The methods used to electrospray Janus particles into a silicone oil collector are similar to the methods discussed in chapter 4 for electrospraying single phase cobalt ferrite particles into silicone oil. These particles were also electrosprayed into silicone oil heated to 200 °C in order to solidify the particles to prevent them from dissolving in the solvents used to wash them out of the silicone oil.

A schematic of the setup used to electrospray into 200 °C silicone oil is shown in Figure

4-2. The silicone oil was heated on a hot plate with magnetic stirring. The temperature of the silicone was monitored using a thermocouple. Once the oil equilibrated to 200 °C, the thermocouple was removed for electrospraying. To electrospray, a voltage of 10 kV was applied to the syringe needle that was placed 22cm from the surface of the silicone oil collector. A flow rate of 0.15 ml/hr was used to pump the solution into the electric field.

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In order to collect the particles from the silicone oil, hexane was added to thin out the oil.

The combined silicone oil and hexane was then centrifuged at 5,000 rpm for 10 min. The supernatant was removed, and the particles were washed three times with hexane to ensure all silicone oil had been removed. This was followed by three rinses in toluene and three rinses in ethanol.

Once washed, the particles were salt calcined as described in both chapters 3 and 4 for the synthesis of barium titanate and cobalt ferrite single phase particles, respectively. Briefly, sodium chloride was ground into a fine powder before dispersing the cobalt ferrite particles in the ground salt. The salt-particle mixture was then heated to 800 °C at a rate of 10 °C/min and held for 3 hours to allow the formation of crystalline particles. To collect the salt calcined particles, the salt-particle mixture was dissolved in water. The particles were magnetically separated from the solution and the supernatant was removed. This process was repeated five times to ensure the removal of the salt from the nanoparticles. The particles were then dried in a vacuum oven prior to characterization.

D.2.4 Characterization of Electrosprayed Particles

The morphology of as-sprayed cobalt ferrite particles, once they were rinsed out of the silicone oil electrospraying collection medium, was characterized using scanning electron microscopy (SEM). The primary purpose of this was to confirm the presence of particles and to confirm that they all of the silicone oil had been removed. SEM was also used to examine the morphology and size of the calcined cobalt ferrite nanoparticles. Transmission electron microscopy (TEM) was also used to examine the size and morphology of the calcined particles.

The calcined particles were also characterized to examine their crystal structure and their magnetic properties. X-ray diffraction (XRD), in combination with Rietveld refinement, was used to confirm that the particles were composed of cobalt ferrite. Scherrer’s formula was used

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to determine the crystallite size of the cobalt ferrite particles. Raman spectroscopy was also used to probe the local structure of the particles to check for the presence of cobalt ferrite and to check whether other cobalt or iron oxides were present.

Magnetic characterization of the calcined cobalt ferrite particles was completed using a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS

3). SQUID was used to find the saturation magnetization of the cobalt ferrite particles.

D.3 Results and Discussion

Using the methods described herein, electrosprayed composite nanoparticles of barium titanate and cobalt ferrite have been successfully electrosprayed (Figure D-2). SEM confirms the synthesis of as-sprayed composite nanoparticles. In addition, EDS shows that both barium titanate and cobalt ferrite are present within the particles (Figure D-3).

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Figure D-1. Model of interfacial area to volume ratio as a function of either fiber or particle diameter. Ratio calculation were done assuming perfect interfaces between the two phases. In addition, calculations for the varying particle diameter assumed the interface was at the equator of a spherical particle. Calculations for the nanofibers assumed fibers with a circular cross section.

Figure D-2. SEM images of as-sprayed Janus nanoparticles

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APPENDIX E LDH DATA FOR PC12 CELLS CULTURED ON COLLAGEN BASED HYDROGELS FOR ALL STIMULATION CONDITIONS – PLOTS IN PERCENT CYTOTOXICITY

This appendix presents the LDH data from Chapter 5 plotted as % cytotoxicity as compared to the collagen control hydrogel instead of as the absorbance of the samples.

Figure E-1. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in 1.1 mT field presented as % cytotoxicity as compared to the collagen control hydrogel.

Figure E-2. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 1.6 mT field presented as % cytotoxicity as compared to the collagen control hydrogel.

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Figure E-3. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 1 hour in a 2.2 mT field presented as % cytotoxicity as compared to the collagen control hydrogel.

Figure E-4. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 3 hours in a 1.6 mT field presented as % cytotoxicity as compared to the collagen control hydrogel.

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Figure E-5. Cytotoxic effects of the hydrogels on PC12 cells stimulated for 4 hours in a 1.1 mT field presented as % cytotoxicity as compared to the collagen control hydrogel.

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APPENDIX F MEASUREMENT OF MAGNETIC FIELD USED FOR CELL STIMULATION

This appendix shows magnetic field measurements for the magnetic field created using the solenoid described in Chapter 5 for the magnetoelectric stimulation of PC12 cells. The measurement shown here was for a field produced using an input voltage of 20 Vpp, 50% duty cycle, and 60 Hz frequency. This magnetic field was measured using a Hall probe (LakeShore

Gaussmeter Probe) to measure the magnetic fields via the Hall effect. The Hall probe was attached to a Gaussmeter (LakeShore 475 DSP Gaussmeter) to read the voltage and amplify the signal. The analog output from the Gaussmeter was observed and saved for processing using an oscilloscope (Tektronix DPO2004B) where 10 mT corresponds to 1 V.

-1 Magnetic Magnetic Field (mT)

-2

-3 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 Time (sec)

Figure F-1. Plot of the magnetic field produced by the solenoid described in Chapter 5 for an input voltage of 20 Vpp, 50 % duty cycle, and a frequency of 60 Hz.

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BIOGRAPHICAL SKETCH

Amanda Uhl was born in Syracuse, NY and graduated from Liverpool High School in

2010. She continued on to attend Cornell University where she was named a McMullen Scholar.

Amanda majored in Materials Science and Engineering in the College of Engineering and graduated in May 2014. She joined the lab of Dr. Lara Estroff in May 2013 where she studied biomineralization and crystallization in confinement. Before moving onto her graduate studies,

Amanda had the opportunity to continue studying biomineralization with Dr. Fiona Meldrum at the University of Leeds in Leeds, UK. Following this, Amanda moved to Gainesville, FL to obtain her Ph.D. in Materials Science and Engineering from the University of Florida under the advisement of Dr. Jennifer Andrew. Following graduation, Amanda joined Intel at their Portland,

Oregon location to work as a process engineer in the front end metals group.

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