Bioprinted superparamagnetic nanoparticles for tissue engineering applications: synthesis, cytotoxicity assessment, novel hybrid printing system

Abstract

Novel technologies are required in tissue engineering to manufacture three-dimensional organs with complex architecture. While superparamagnetic nanoparticles have been widely used in medicine for magnetic resonance imaging and targeted drug delivery, they have not been extensively applied in tissue engineering. These nanoparticles would allow active patterning and non-destructive imaging during tissue growth and development. However, no inexpensive method exists for synthesis of commercial amounts of these nanoparticles with controlled morphology, chemistry and size. Furthermore, superparamagnetic nanoparticle cytotoxicity mechanisms are not well understood, which makes it difficult to control or block adverse nanoparticle effects on human health. In this dissertation, superparamagnetic iron oxide nanoparticles were produced by flame synthesis using a coflow diffusion flame. Nanoparticle flame synthesis has significant advantages, including improved nanoparticle property control and commercial production rate capability with minimal post-processing. Final iron oxide nanoparticle morphology, elemental composition, and particle size was controlled by changing flame configuration, flame temperature, and additive loading, and morphology, elemental composition, and particle size of the synthesized nanoparticles were analyzed by electron microscopy (TEM, ESEM, EDS), and Raman Spectroscopy. Then flame synthesized iron oxide nanoparticle interaction with endothelial cells was compared to commercially available iron oxide nanoparticles. Flame synthesized particles showed no statistically significant toxicity difference from commercially available nanoparticles, as measured by Live/Dead assay, Alamar blue, and lactase dehydrogenase release. Both synthesized and purchased nanoparticles localized inside the cell cytoplasm as shown by TEM images. Iron oxide nanoparticles resulted in an increase in reactive oxygen species (ROS) formation in cells within the first three hours after nanoparticle uptake, and this ROS formation contributed to actin cytoskeleton disruption. Finally, a new hybrid nano-bioprinting technique that facilitates manipulation and tracking of cells and bioactive factors within a three-dimensional tissue construct was developed. This technique combined the initial patterning capabilities of syringe-based cell deposition with the active patterning capabilities of superparamagnetic nanoparticles. Superparamagnetic iron oxide nanoparticles, either in the alginate biopolymer or loaded inside endothelial cells, were bioprinted using the hybrid solid freeform fabrication direct cell writing system and they were manipulated using an external magnet and imaged by MicroCT.

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Bioprinted Superparamagnetic Nanoparticles for Tissue Engineering Applications:
Synthesis, Cytotoxicity Assessment, Novel Hybrid Printing System


A Thesis
Submitted to the Faculty
of
Drexel University
by
Kivilcim Buyukhatipoglu
in partial fulfillment of the
requirements for the degree
of
Doctor of Philosophy
October 2009

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© COPYRIGHT 2009
KIVILCIM BUYUKHATIPOGLU. ALL RIGHTS RESERVED.

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ACKNOWLEDGEMENTS


I would like to express my deep and sincere gratitude to my supervisor, Dr Alisa Morss
Clyne. Her encouragement, guidance and support has enabled me to develop an understanding of
the subject and motivated me immensely throughout this work. Working with her was truly a
rewarding experience.
I am deeply grateful to my dissertation committee members, Dr Wei Sun, Dr Alan Lau,
Dr Michelle Marcolongo and Dr Adam Fontecchio for their detailed review and constructive
comments, and stimulating suggestions.
I wish to express my warm and sincere thanks to Dr Selcuk Guceri, Dean of Engineering
and Dr Mun Choi, former Head of Mechanical Engineering and current Dean of Engineering
University of Conneticut, who introduced me to the PhD program at Drexel University. Their
ideals and concepts have had a remarkable influence on my entire career.
I owe my sincere gratitude to Dr Alan Lau, Mechanical Engineering Graduate Advisor,
who gave me untiring help during challenging moments. His understanding, encouraging and
personal guidance have been of great value in this study.
I extend my heartfelt thanks to Dr Wei Sun, for giving me the opportunity to work with
his group, guiding me in the exciting field of tissue engineering and allowing me to use his
laboratory facilities.
During this work I have collaborated with many colleagues for whom I have great regard,
and I wish to extend my warmest thanks to all those who have helped me with my work in the

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Department of Mechanical Engineering, Department of Materials Science and Department of
Biomedical Engineering. Special thanks go to my friends and the members of Vascular Kinetics
Laboratory and Computer Aided Tissue Engineering Laboratory, in particular Dannielle
Figueroa, Steve Kemeny, Karl Reisig, Krishna Arjunan, Greeshma Manomohan, Sameer
Kalghatgi, Robert Chang and Eda Yildirim.
I have been blessed with wonderful friends at life. This thesis would not be possible
without the support of my dear friends Kate Allen and Mert Sasoglu. Thank you for always
being there when I needed you. I could not have gotten so far without Oya Okman and Asli
Yimenicioglu. Thank you for being the ones that I can tell anything to and ask anything of. As
the witnesses of endless nights in the lab, my lab mates, Andrew Bohl and Rafael Mulero, thank
you for being there and making lab a lively place to work. Also my intramural leagues sports
teammates and dear graduate school friends, Keith Sevcik, Goknur Cambaz, John Fitzpatrick,
Jamie Lane, Matt Kurman, Rodney Johnson and Yi Ma thank you for making me feel at home.
I have a family which makes me feel like the luckiest person in the world. Safak
Buyukhatipoglu, Evren Semizel, Bora Cilek and all my extended family, I can not even describe
how much I love you and how blessed I feel to have you.
Last but not least, this thesis is for my parents, Atiye and Haluk Buyukhatipoglu. Thank
you so much for never letting me settle for anything in my life and encouraging me to do my
best. Thank you for listening to me and supporting me whenever I need. Thank you for letting
me know no matter what happens you will be there for me and you love me so much. I can not
even find words to explain what a nice and secure feeling it is to know no matter how bad I fail
in life there is always somewhere called home with you guys there for me. I love you so much.

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TABLE OF CONTENTS




LIST OF TABLES ..................................................................................................................................... viii
LIST OF FIGURES ..................................................................................................................................... ix
ABSTRACT ................................................................................................................................................ xv
CHAPTER I: INTRODUCTION .................................................................................................................. 1
1.1 Thesis overview .................................................................................................................................. 1
1.2 Superparamagnetic Nanoparticles ....................................................................................................... 3
1.2.1 Nanoparticles ............................................................................................................................... 3
1.2.2 Unique Properties of Nanoparticles ............................................................................................. 4
1.2.3 Superparamagnetism .................................................................................................................... 5
1.3 Superparamagnetic Nanoparticle Synthesis ........................................................................................ 6
1.3.1 Liquid Phase Synthesis Methods ................................................................................................. 7
1.3.2 Gas Phase Synthesis Methods ...................................................................................................... 9
1.4 Applications of Superparamagnetic Nanoparticles in Biomedical Sciences and Medicine .............. 14
1.4.1 Coating and Functionalization of Superparamagnetic Nanoparticles ........................................ 14
1.4.2 Superparamagnetic Nanoparticles in MRI ................................................................................. 18
1.4.3 Superparamagnetic Nanoparticles in Hyperthermia .................................................................. 19
1.4.4 Superparamagnetic Nanoparticles in Drug and Gene Delivery ................................................. 20
1.4.5 Superparamagnetic Nanoparticles for Cell Separation and Purification .................................... 21
1.4.6 Applications of Magnetic Nanoparticles in Tissue Engineering ................................................ 21
1.5 Magnetic Nanoparticle Toxicity Mechanisms ................................................................................. 25
1.5.1 Nanoparticle Induced Reactive Oxygen Species Formation ...................................................... 25