Mechanisms of interaction of non-thermal plasma with living cells

252 Pages·2010·4.58 MB·English

by  Kalghatgi, Sameer Ulhas

Mechanisms of Interaction of Non-Thermal Plasma with Living Cells

A Thesis
Submitted to the Faculty
of
Drexel University
by
Sameer Ulhas Kalghatgi
in partial fulfillment of the
requirements for the degree
of
Doctor of Philosophy
March 2010
COPYRIGHT

© Copyright 2010
Sameer U. Kalghatgi. All Rights Reserved.
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DEDICATIONS

This thesis is dedicated to the following wonderful people in my life:
My wonderful parents, Ulhas and Suchita Kalghatgi who have raised me to the
person I am today, who have been my role-model for hard work, persistence and personal
sacrifices, and who instilled in me the inspiration to set high goals and the confidence to
achieve them.
My dearest wife, Sheetal, who has been my pillar of strength and stood by me
every step of the way through good times and bad, who constantly motivated me, had
confidence in me when I doubted myself, boosted my morale whenever I felt low and
who has shared the many uncertainties, challenges and sacrifices for completing my PhD.
Thank you Mom, Dad and Sheetal for your unconditional love, guidance, and
support that you have always given me, helping me to succeed and instilling in me the
confidence that I am capable of doing anything I put my mind to. Thank you for
everything. I love you!
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ACKNOWLEDGEMENTS
Though only my name appears on the cover of this dissertation, many people have
contributed towards the creation and completion. I am grateful to all those people who
have made this dissertation possible and because of whom my experience during the
course of my PhD has been one that I will cherish forever.
First, I would like to express my deepest gratitude to my advisor Dr. Gary
Friedman, for his constant encouragement, guidance and support throughout my PhD at
Drexel University. He was highly instrumental in initiating me in to the exciting world of
Plasma Medicine. He not only taught me how to express my ideas but also showed me
different ways to approach a research problem along with the virtue of persistence to
accomplish any goal. I have been fortunate to have an advisor who gave me the freedom
to explore my own ideas and at the same time his patience and support helped me
overcome many crisis situations and finish this dissertation. I really admire his
tremendous knowledge and skills and I am grateful for his assistance in editing journal
publications, peer reviewed conference publications and this thesis. He was always there
to meet and talk about my ideas, to proofread and mark up my papers and chapters, and
to help me think through my problems (whether philosophical, personal, analytical or
experimental). Gary has truly been a friend, philosopher and guide.
I would like to specially acknowledge and appreciate Dr. Alexander Fridman,
who played a highly important role in helping me complete my PhD and guiding me in
the challenging research that lies behind it. Dr. Fridman has truly been a great mentor. He
made me a better plasma physicist and brought out the good ideas in me. (More
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importantly, he taught me how to work hard and play hard, and introduced me to his
witty toasts which always ended with a shot of vodka and a slice of pickles).
I would like to acknowledge and appreciate Dr. Jane Azizkhan-Clifford who
played a pivotal role in laying the foundations of my PhD by welcoming me, an electrical
engineer, with open arms in her lab and in to the field of molecular and cell biology.
Without her support and encouragement neither would have this research been possible
nor could I have completed this dissertation. Her valuable insight, expertise and immense
wisdom helped me tide over many a challenging situation and carry out scientific
hypothesis driven research.
I would like to thank, Dr. Alisa Morss Clyne for the many fruitful discussions and
her valuable assistance she provided at all levels of the research on understanding the
effects of non-thermal plasma on proliferation of endothelial cells.
I would like to thank the members of my thesis committee, Dr. Timothy Kurzweg
and Dr. Adam Fontecchio for agreeing to serve on my committee and for reviewing my
dissertation and providing many valuable comments that improved the presentation and
contents of this dissertation.
I am grateful to all my friends at Drexel University, in particular, Shubham,
Suren, Hemang, and Sameet and fellow graduate student and medical residents, including
Crystal, Ekaterina, Behzad, Krishna, Gregory Fridman, Moogega, Rachel, Danil,
Jaqueline, Andrew Wu for many interesting and good-spirited discussions relating to my
research and about life as a graduate student which really helped me to enjoy my time at
Drexel University.
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Most importantly, none of this would have been possible without the love and
support of my parents and sister. My parents, to whom this dissertation is dedicated to,
have been a constant source of love, concern & strength; and my sister Neha, for her love
and affection and for believing in me the most. I would like to express my heart-felt
gratitude to my family over all these years.
Last but not the least; I would like to thank my wonderful and loving wife Sheetal
for her unwavering support and understanding during the course of my PhD. Her being
there made all the difference and her support, encouragement and love were in the end
what made the completion of my PhD and writing this dissertation possible.
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Table of Contents
List of Tables ................................................................................................................................. xi
List of Figures ............................................................................................................................... xii
Abstract ........................................................................................................................................ xix
Chapter 1. Introduction ............................................................................................................ 1
1.1. Plasma .............................................................................................................................. 1
1.2. Non-Thermal Dielectric Barrier Discharge Plasma ......................................................... 4
1.3. Plasma Medicine .............................................................................................................. 7
1.3.1. Overview ...................................................................................................................... 8
1.3.2. Clinical Applications of Non-Thermal Plasma ............................................................ 9
1.3.2.1. Blood Coagulation ..................................................................................................... 10
1.3.2.2. Sterilization ................................................................................................................ 10
1.3.2.3. Cancer Therapy .......................................................................................................... 11
1.3.2.4. Wound Healing .......................................................................................................... 11
1.3.2.5. Modulation of Cell Transfection ................................................................................ 12
1.3.2.6. Skin Diseases ............................................................................................................. 12
1.4. Motivation For this Work .............................................................................................. 13
1.5. Dissertation Overview and Organization ....................................................................... 14
1.6. Dissertation Contributions ............................................................................................. 18
Chapter 2. Effects Of Oxidative Stress On Mammalian Cells ............................................ 19
2.1. Overview ........................................................................................................................ 19
2.2. Reactive Oxygen Species (ROS) ................................................................................... 20
2.2.1. Biologically Relevant Reactive Oxygen Species ....................................................... 22

2.2.1.1. Hydroxyl Radical (OH ) ............................................................................................ 24
-
2.2.1.2. Superoxide Anion (O2 ) ............................................................................................ 25
2.2.1.3. Hydrogen Peroxide (H2O2) ........................................................................................ 26
1
2.2.1.4. Singlet Oxygen ( O2).................................................................................................. 27
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 
2.2.1.5. Peroxyl (ROO ) and Alkoxyl Radicals (RO ) ............................................................ 27
 -
2.2.1.6. Nitric Oxide (NO ) and Peroxynitrite (ONOO ) ........................................................ 28
2.2.2. Sources of ROS .......................................................................................................... 29
2.2.2.1. Exogenous Sources .................................................................................................... 30
2.2.2.2. Endogenous Sources .................................................................................................. 32
2.2.3. ROS signaling ............................................................................................................ 35
2.2.4. Antioxidant Systems .................................................................................................. 37
2.3. Effects of Oxidative Stress ............................................................................................. 38
2.3.1. Biological Effects of Oxidative Stress ....................................................................... 39
2.3.2. Cell Proliferation ........................................................................................................ 41
2.3.3. Cellular Adaptation .................................................................................................... 42
2.3.4. Cell Injury .................................................................................................................. 43
2.3.4.1. Effects on Lipid Membranes ...................................................................................... 44
2.3.4.2. Effects on DNA .......................................................................................................... 45
2.3.4.3. Effects on Proteins ..................................................................................................... 47
2.3.5. Cellular Senescence ................................................................................................... 48
2.3.6. Cell Death .................................................................................................................. 49
2.3.7. Role of Oxidative Stress in Diseases ......................................................................... 53
2.4. Modalities Functioning via induction of Oxidative Stress ............................................. 57
2.4.1. Ionizing Radiation ...................................................................................................... 58
2.4.1.1. Biological Effects of Ionizing Radiation.................................................................... 59
2.4.1.2. Cell Proliferation ........................................................................................................ 60
2.4.1.3. Cell Injury .................................................................................................................. 60
2.4.1.3.1. Effects on Lipids .................................................................................................... 60
2.4.1.3.2. Effects on DNA ...................................................................................................... 62
2.4.1.3.3. Effects on Proteins ................................................................................................. 63
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2.4.1.3.4. Cell Death .............................................................................................................. 66
2.4.2. Hydrogen Peroxide .................................................................................................... 69
2.4.2.1. Biological Effects of Hydrogen Peroxide .................................................................. 70
2.4.2.2. Cell Proliferation ........................................................................................................ 71
2.4.2.3. Cell Injury .................................................................................................................. 72
2.4.2.3.1. Effects on Lipids .................................................................................................... 72
2.4.2.3.2. Effects on DNA ...................................................................................................... 73
2.4.2.3.3. Effects on Proteins ................................................................................................. 73
2.4.2.4. Cell Senescence ......................................................................................................... 74
2.4.2.5. Cell Death .................................................................................................................. 75
2.4.2.6. Role of Hydrogen Peroxide in Diseases .................................................................... 76
2.4.3. Ultraviolet Radiation .................................................................................................. 77
2.4.3.1. Biological Effects of Ultraviolet Radiation................................................................ 78
2.4.3.2. Cell Injury .................................................................................................................. 78
2.4.3.2.1. Effects on Lipids .................................................................................................... 80
2.4.3.2.2. Effects on DNA ...................................................................................................... 81
2.4.3.2.3. Effects on Proteins ................................................................................................. 82
2.4.3.3. Cell Death .................................................................................................................. 83
2.4.3.4. Role of UV induced Oxidative Stress in Diseases ..................................................... 85
2.4.3.4.1. Photoaging ............................................................................................................. 86
2.4.3.4.2. Skin Cancer ............................................................................................................ 88
2.5. Summary ........................................................................................................................ 90
Chapter 3. Low Dose Non-Thermal Plasma Treatment ...................................................... 91
3.1. Introduction ........................................................................................................................ 91
3.2. Materials and Methods ....................................................................................................... 92
3.3. Results ................................................................................................................................ 96
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3.3.1. Cell Proliferation in Response to Non-Thermal Plasma ................................................ 96
3.3.2. Cell Death in Response to Non-Thermal Plasma ........................................................... 98
3.3.3. FGF-2 Release Post Non-Thermal Plasma Treatment ................................................... 99
3.3.4. Mechanisms of Release of FGF-2 Following Plasma Exposure .................................. 101
3.4. Discussion ........................................................................................................................ 102
3.5. Summary .......................................................................................................................... 108
Chapter 4. High Dose Non-Thermal Plasma Treatment ................................................... 110
4.1. Introduction .................................................................................................................. 110
4.2. Materials and Methods ................................................................................................. 111
4.3. Results and Discussion ................................................................................................ 117
4.3.1. Cell Viability ............................................................................................................ 117
4.3.1.1. Trypan Blue ............................................................................................................. 117
4.3.1.2. Colony Assay ........................................................................................................... 117
4.3.2. Apoptosis ................................................................................................................. 118
4.3.2.1. Annexin – V/PI ........................................................................................................ 118
4.3.2.2. Caspase – 3 Cleavage ............................................................................................... 120
®
4.3.2.3. TUNEL Assay ........................................................................................................ 121
4.3.3. Mechanisms of Induction of Apoptosis ................................................................... 123
4.3.3.1. Role of pH ................................................................................................................ 123
4.3.3.2. Role of ROS ............................................................................................................. 124
4.3.4. Discussion ................................................................................................................ 125
4.4. Summary ...................................................................................................................... 130
Chapter 5. Non-Thermal Plasma Treatment of Living Tissue – In Vivo Studies ........... 132
5.1. Introduction ...................................................................................................................... 133
5.2. Materials and Methods ..................................................................................................... 134
5.3. Results .............................................................................................................................. 138