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Studies of nanosecond pulsed power for modifications of biomaterials and nanomaterials (SWCNT)
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Studies of nanosecond pulsed power for modifications of biomaterials and nanomaterials (SWCNT)
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STUDIES OF NANOSECOND PULSED POWER FOR MODIFICATIONS OF BIOMATERIALS AND NANOMATERIALS (SWCNT) By Meng-Tse Chen A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MATERIALS SCIENCE) December 2009 Copyright 2009 Meng-Tse Chen ii Acknowledgments With sincere gratitude, I thank all those who made this work possible. First, I would like to thank my advisor Dr. Martin A. Gundersen for his unlimited support and continuous guidance, support along the way. He is not only a knowledgeable professor but also a great mentor. It’s my honor to have Dr. Edward Goo and Dr. Chongwu Zhou as members in my dissertation committee and also Dr. Florian B. Mansfeld and Dr. Steve R. Nutt in my qualify committee. In addition, I thank Dr. Chunqi Jiang and Dr. P. Thomas Vernier for taking care of me in my PhD study. They provided invaluable inspiration, persistent patience and thoughtful discussion. I also thank Dr. Andrus Kuthi for sharing his knowledge in pulsed power Also, I sincerely thank people who work with me during my Ph.D. study. Dr. Christoph Schaudinn and Amita Gorur shared their knowledge on microbiology and biofilms. Lewis Gomez De Arco eagerly and persistently cooperated with me on single- walled carbon nanotubes project. Dr. Yinghua Sun taught me how to design the biology experiments and set up imaging system. Suet Ying Christin Chong helped me on cell culture. I would also like to thank all other lab members in the USC pulsed power group: Yu-San Liu, Chi-Hui James Liang, Yu-Hsuan Wu, Hao Jessica Chen, Daniel Singleton, and Jason Sanders for assistance and corporation. Without your kind help, I would not be able to successfully finish this work. Finally, I thank my family for their endless support through all these years, especially for my sister, for her hearty encouragement during all my difficult time. I also thank all the friends I know in Los Angeles for their company through all these years. iii Table of Contents Acknowledgments ii List of Tables iv List of Figures v Abbreviations viii Abstract ix Chapter 1 Introduction 1 Chapter 2 Nanosecond electric pulse induced calcium entry into chromaffin cells 14 Chapter 3 Two-Dimensional Nanosecond Electric Field Mapping Based on Cell Electropermeabilization 24 Chapter 4 Pulsed Atmospheric-Pressure Cold Plasma for Endodontic Disinfection 44 Chapter 5 pH-sensitive intracellular photoluminescence of carbon nanotube- fluorescein conjugates in human ovarian cancer cells 59 Chapter 6 Laser-Induced Hyperthermia of Cells with Carbon Nanotubes 72 Chapter 7 Brain Cancer Cell Migration Reduction with Carbon Nanotubes 82 Chapter 8 Future Work 87 Bibliography 90 Appendix: Cell Culture Information 103 iv List of Tables 3.1 Maximum electric field intensity and maximum fluorescence intensity of different electrode configurations 38 3.2 Energy/per pulse and temperature change of different electrode configurations 40 A.1 Cell lines catalogue 103 v List of Figures 1.1 Intracellular SWCNTs-induced YO-PRO-1 permeabilization reduction to nanoelectropulses 10 2.1 Fluorescence imaging of [Ca 2+ ] i in chromaffin cells exposed to a single 4 ns, 8MV/m pulse 16 2.2 Response of chromaffin cells to multiple applications of 4 ns pulses 17 2.3 Response of chromaffin cells to 4 ns, 8 MV/m pulses 20 2.4 Effect of single and multiple applications of 4 ns, 8 MV/m pulses on the influx of YO-PRO-1 into chromaffin cells 23 3.1 Schematic of the experimental setup for nanosecond pulsed electric field mapping 25 3.2 Three types of electrode configurations designed for nanoelectropulse treatment and cancer therapy 27 3.3 Temperature measurement setups 31 3.4 YO-PRO-1 permeabilization of Jurkat T lymphoblast cells exposed to nanoelectropulses with different electric field amplitudes 32 3.5 Voltage and current pulse waveforms 33 3.6 Fluorescence images of the PC3 cell monolayer exposed to nanoelectropulses with three different electrode configurations 35 3.7 Electric field distributions modeled with the COMSOL Multiphysics electrostatics module 37 vi 3.8 Fluorescence images of U251 GBM cell monolayer and keratoacanthoma cell monolayer after nanoelectropulse exposure 39 3.9 The temporal development of the temperature at the tips of electrodes 40 4.1 Schematic of a plasma dental probe version 1 46 4.2 Typical voltage and current pulse waveforms 48 4.3 Images of the plasma plumes 49 4.4 Experimental setup for the B. atrophaeus growth inhibition treated with PDP on nutrient medium plates 50 4.5 Treatment with He and He/(1%)O 2 plasmas for different exposure times 51 4.6 Treatment with He and He/(1%)O 2 plasmas at different peak pulse voltages 52 4.7 SEM image of control root canals 54 4.8 SEM images of PDP treated root canals 55 4.9 Emission spectrum of the non thermal atmospheric-pressure plasma 56 5.1 Internalization of SWCNT-FC conjugates in SKOV-3 cells 61 5.2 SEM and 3D z-stack images of intracellular SWCNT-FC in SKOV-3 cells 62 5.3 Fluorescence images of intracellular SWCNT-FC in SKOV-3 cells 64 5.4 Background fluorescence of SWCNT-FC in growth medium 65 vii 5.5 pH sensitivity of SWCNT-FC within SKOV-3 cells 66 5.6 Fluorescence emission intensity measurements 69 5.7 Fluorescence intensity integration 70 6.1 Diagram of near-infrared laser setup 74 6.2 Optical and thermal properties of SWCNTs 75 6.3 Translocation of SWCNT-FC across U251 human GBM cell membrane 77 6.4 Intracellular detection of SWCNT-FC conjugates 78 6.5 NIR irradiation of U251 human GBM cells and astrocytes normal brain cells with and without intracellular SWCNTs 80 7.1 Aggregation and clustering effects of SRB12 cells 84 7.2 SWCNTs induced migration reduction effect of rat C6 GBM cell monolayer 85 7.3 SWCNTs induced migration reduction effects of in U251 human GBM cell monolayer 85 viii Abbreviations [Ca 2+ ] i –Intracellular calcium concentration CHX–Chlorhexidine DBDs–Dielectric barrier discharges DIC–Differential interference contrast DMEM–Dulbecco’s Modified Eagle’s Medium DMPP–Dimethylphenylpiperazinium DNA–Deoxyribonucleic acid ECT–Electrochemotherapy FBS–Fetal bovine serum FC–Fluorescein GBM–Glioblastoma Multiforme NIR–Near infrared NMR–Nuclear magnetic resonance spectroscopy NaOCl–Sodium hypochlorite PBS–Phosphate buffered saline PDP–Plasma dental plume PI–Propidium iodide pH i –Intracellular pH PMT–Photomultiplier tube VN–Vicrostatin SWCNTs–Single-walled carbon nanotubes SWCNT-FC–Single-walled carbon nanotubes fluorescein ix Abstract This work investigates the modification of biological materials through the applications of modern nanosecond pulsed power, along with other forms of nanotechnologies. The work was initially envisaged as a study of the effect of intense nanosecond pulsed electric fields on cancer cells. As the work progressed, the studies suggested incorporation of additional technologies, in particular, cold plasmas, and carbon nanotubes. The reasons for these are discussed below, however, they were largely suggested by the systems that we were studying, and resulted in new and potentially important medical therapies. Using nanosecond cold plasmas powered with nanosecond pulses, collaboration with endodontists and biofilm experts demonstrated a killing effect on biofilms deep within root canals, suggesting a fundamentally new approach to an ongoing problem of root canal sterilization. This work derived from the application of nanosecond pulsed power, resulting in effective biofilm disinfection, without excessive heating, and is being investigated for additional dental and other medical applications. In the second area, collaboration with medical and nanotube experts, studies of gliomamultiforme (GBM) led to the incorporation of functionalized carbon nanotubes. Single-walled carbon nanotube-fluorescein carbazide (SWCNT-FC) conjugates demonstrated that the entry mechanism of the single-walled carbon nanotubes (SWCNTs) was through an energy-dependent endocytotic pathway. Finally, a monotonic pH sensitivity of the intracellular fluorescence emission of SWCNT-FC conjugates in human ovarian cancer cells suggests these conjugates may serve as intracellular pH sensors. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular x pH changes are proposed as mechanisms. The use of SWCNTs for cancer therapy of gliomas, resulting in hyperthermia effect after 808 nm infrared radiations, absorbed specifically by SWCNTs but not by biological tissue. Heat was only observed to kill cells containing intracellular SWCNTs. Furthermore, intracellular SWCNTs also cause aggregation and clustering of the cells, and a reduced ability of the cells to attach to and migrate over a substrate. This phenomenon has the potential to reduce the multiplication, migration, and invasion of brain cancer cells into the surrounding tissue. 1 Chapter 1 Introduction In this dissertation, we start on studying nanosecond pulsed power technology in intracellular effects and cell membrane permeabilization for biological applications. To explore our research more widely, nanosecond pulsed power technology is applied to generate atmospheric-pressure plasma for endodontic disinfection. Modifications of biomaterials and nanomaterials such as single-walled carbon nanotubes (SWCNTs) integrated with nanosecond pulsed power technology is an interesting area to explore for potential biomedical and therapeutic applications. The following research contains the potential utilization of SWCNTs in brain cancer therapy development as an extension. 1.1 Nanosecond Pulsed Power Technology Pulsed power refers to a technology of collecting energy over a relatively long period of time and releasing it immediately thus increasing the instantaneous power. Electroporation of the cell membrane is one classical example in biomedical applications of pulsed power technology by applying electric pulses with pulse widths in millisecond (ms) to microsecond (µs) range and electric field amplitudes of a few kV/cm [Neumann et al 1982; Weaver 2000; Zimmerman et al 2000; Gehl 2003]. The electroporation process transports extracellular ions or molecules into cells, thus allows local drug or gene delivery for cancer therapies, for instance, Electrochemotherapy (ECT) has reached clinical trials [Belehradek et al 1993; Heller et al 1996]. When electric pulses with pulse widths reduce to nanosecond (ns) range and with electric field intensity enhance to a few MV/m, nanoelectropulses induce intracellular 2 responses in the absence of conventional electroporation of the plasma membrane [Sher et al1970; Schoenbach et al 2001]. Most notable is the release of calcium ions from intracellular organelles [Vernier et al 2003; White et al 2004; Beebe et al 2004], which can sequentially stimulate cellular processes such as apoptosis, which is significant for cancer therapy [Nuccitelli et al 2006; Garon et al 2007]. Although the exact mechanism is remaining unclear, the nanoelectroperturbation (ns; MV/m) based cancer treatment has likely advantages compared to the conventional electroporation (ms to µs; kV/m) based cancer therapy for the following reasons. It needs no assistance of drugs and it delivers a less amount of energy, typically a few mJ to 100 mJ per pulse, into biological samples, therefore, causes less pain in patient therapies. However, detailed experimental studies, which are necessary to understand the nanoelectroperturbation phenomenon, rise up a great challenge for nanosecond pulsed power technology in biological and biomedical applications. We start on investigating the entry mechanism of calcium ions induced by nanoelectropulse in bovine adrenal chromaffin cells. 1.1.1 Nanoelectroperturbation-Induced Intracellular Effect The pulse rise time, pulse duration, number of pulses and electric field intensity are important factors which influence the response of particular cell types to nanoelectropulses [Hair et al 2004]. The delivery parameters of nanoelectropulses potentially could be modified to achieve selectivity not only with respect to the cellular response desired but also the cell type being targeted [Polevaya et al 1999]. However, a better understanding of how diverse cell types respond to nanoelectropulses is 3 indispensable to realize this possibility. The blueprint of sensitivity and selectivity of diverse cell types to nanoelectropulses parameters needs to be built up. Our short term goal is to study the mechanism of nanoelectropulses-induced calcium release on electrically excitable chromaffin cells, which release catecholamines primarily triggered by calcium ions entering the cell through voltage-gated calcium channels, rather than by calcium releasing from intracellular organelles. In chapter 2, the details are described about how nanoelectropulses perturb the cell membrane permeability to calcium ions then in turn result in intracellular organelle releasing on electrically excitable cells. 1.1.2 In-Vitro Electric Field Distributions of Needle-Array Electrodes Nanoelectropulses have been demonstrated to kill a wide variety of human cancer cells, including pancreatic cancer and basal cell carcinoma, in vitro, and to induce tumor regression in vivo [Nuccitelli et al 2006; Garon et al 2007]. The nanoelectroperturbation based therapy is under development for skin cancer treatment. Some studies of nanoelectropulses effects on tumors have been carried out with parallel-plate electrodes, like those in commercial electroporation cuvettes, where fringing effects are negligible and the electric field distribution can be assumed to be homogeneous. In published [Nuccitelli et al 2006; Garon et al 2007] and ongoing efforts directed at tumor therapy, however, needle-array electrodes are employed, for which the distribution of the electric field is not as simple as parallel-plate electrodes. Therefore, the electric field distribution of needle-array electrodes designed for nanoelectropulses delivery of clinic purpose is essential to find out. Magnetic resonance current density imaging in combination with 4 three-dimensional finite modeling were employed to qualitatively evaluate the electric field distribution in different electrode configurations during in vivo electroporation study [Miklavcic et al 1998]. Here, we demonstrate a “qualitative” mapping of the electric field, which does show the distribution of the electric field around the electrodes. However, quantitatively mapping the electric field distribution in these more complex electrode configurations need to be investigated. It will not only increase our understanding of the in vivo electroporation process but also contribute to evaluations of the efficacy of nanoelectropulse exposure during clinical trials. In chapter 3, we describe the details about how the nanosecond electric pulse-induced electropermeabilization is used to map the electric field distribution of needle-array electrodes on cell monolayers. 1.1.3 Nanosecond Electric Pulse-Powered Cold Plasma in Endodontic Disinfection Postprocedure infections in the root canal of teeth usually happened caused by the surviving bacteria often gathered in the form of what is known as biofilms. Bacteria in such a film are embedded in a polymer matrix, which makes them harder to kill than isolated individuals. One of the primary goals in root canal treatment is to reduce the bacterial population in the root canals of infected teeth. This is usually accomplished by mechanical preparation along with the use of irrigants. Chemomechanical instrumentation is often the first method of bacterial reduction during endodontic treatment of infected root canals [Orstavik et al 1991; Dalton et al 1998]. Although several mechanical cleaning instrumentations cause a marked reduction in bacterial colony numbers in the root canal, a total removal of bacteria content could not be achieved by mechanical preparation [Colak et al 2005]. Mechanical cleaning techniques 5 can’t completely reach the whole infected areas of the root canal system by the instruments [Davis et al 1972]. Since residual bacteria is one of the major factors responsible for post treatment disease [Nair et al 1990], therefore, an endodontic irrigants with powerful antimicrobial activity is needed to aid in the debridement of the root canal system. Sodium hypochlorite (NaOCl) is a widely used endodontic irrigant because of its bleaching, antimicrobial ability and tissue dissolving properties. It reacts with fatty acids and amino acids hence liquefying the organic tissue [Estrela et al 2002]. Its main disadvantages are the toxicity to vital tissues and corrosion of metals in endodontic treatment [O'Hoy et al 2003]. Without careful isolation of NaOCl, chemical burns and tissue necrosis happen when it spread out from the root canal into the peri-radicular tissues and result in upper airway obstruction of the patient. A 0.5% solution of NaOCl with reduced toxicity still can dissolve necrotic but not vital tissues [Spångberg et al 1973]. However, the antimicrobial effect of NaOCl is further reduced after dilution, when its concentration varies down from 5.25%. Several other limitations of using NaOCl irrigant in endodontic treatment are as follow: It does not consistently disinfect the root canal [Byström et al 1981]; and it does not remove the smear layer from the dentin walls [McComb et al 1975]. At the mean time, the more biocompatible Chlorhexidine (CHX) has been recommended as an alternative irrigant in root canal treatment. CHX targets inner membrane cells, thereby causing generalized membrane damage to the phospholipids bilayers. It affects the cell membrane integrity but its activity is pH dependent and is greatly reduced in the presence of organic matter [McDonnell et al 1999]. 6 Although nontoxicity to periapical tissues is one of the requirements of an ideal irrigating substance, antimicrobial activity, water solubility and the capacity to dissolve organic matter are other factors need to be concerned [Kuruvilla et al 1998; Okino et al 2004]. Except for its toxicity NaOCl meets those properties, while CHX is less toxic but not effective in dissolving organic matter. Most important thing is if there still exist bacteria residues inside the root canal after treatment. Conventional methods [Heling et al 1998; Menezes et al 2004; Colak et al 2005, Ruddle et al 2006] such as mechanical cleaning and irrigation with antibacterial compounds result in rates of postprocedure infection exceeding 10%. Based on these limitations, a search for a better root canal therapy continues. Surgical laser, e.g. Nd:YAG laser, emitted at 1.34 µm is an alternative tool used in endodontics since its antibacterial ability has been shown to be more effective than the use of irrigants in complex canal system [Folwaczny et al 2002; Schoop et al 2002; Perin et al 2004]. Although surgical laser provides equal or improved treatment over conventional care, sterilization of root canals by lasers is also problematical. There exists a potential for spreading bacterial contamination from the root canal to the patient and the dental team via the smoke produced by the laser, which can cause bacterial dissemination [Hardee et al 1994]. To make the using of surgical laser in root canal treatment successful, the thermal injury on periodontal tissues need to be considered. Water-cooling system combined with appropriate laser irradiation parameters reduces possible thermal damages in the root canal dentine [White et al 1994; Armengol et al 1999] but too much water also absorb and reduce the ablation rate of lasers [Lee et al 2004]. It was reported that the Nd:YAG laser treatment is not effective in removing debris and smear layer which are 7 harmful to successful root canal therapy [Setlock et al 2003]. In summary, near infrared laser-based disinfection techniques are still problematic mainly due to the treatment time and cost/benefit ratio of instrument, their high capital cost and laser-induced tissue trauma [Dederich et al 2004]. Therefore, a new strategy to effectively and safely sterilize root canals is needed. Here, we report a hollow-electrode-based, nanosecond pulsed plasma dental probe (PDP) that generates a room temperature plasma plume in ambient atmosphere. The plasma plume causes minimal heating of biological materials and is safe to touch with bare hands without causing burning pain. We have applied the plasma to disinfect root canals of human teeth, thereby to investigate a new strategy based on the pulsed PDP as a safe and effective alternative or adjunct for endodontic treatment. While laser systems costing up to 25,000 dollars, the pulsed plasma dental probe system could retail for as little as 1,000 dollars, provided it passes official clinical trials. The details of this nanoelectropulse-powered plasma application for endodontic disinfection are discussed in chapter 4. 1.2 The modifications of biomaterials and nanomaterials in biomedical and therapeutic applications Biomaterial refers to a nonviable material used in a medical device, intended to interact with biological systems [1st Biomaterials Consensus Conference, 1986, Chester, UK] to evaluate, treat, augment, or replace any tissue, organ, or function of the body [2nd Biomaterials Consensus Conference, 1992, Chester, UK]. For example, fluorochromes, such as organic fluorescent dyes, are biomaterials which have been widely used as probes 8 in monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity [Stephens et al 2003; Weijer 2003]. In the continuous research of this dissertation, we study biomaterials which have structured components with at least one dimension less than 100 nm as nanomaterials for biomedical and therapeutic applications. Single-walled carbon nanotube (SWCNT) is a good candidate of bio- and nano- materials to modify and integrate with different kinds of technologies. SWCNTs are well-ordered; all-carbon hollow graphitic nanosized materials with a high aspect ratio, lengths diverse from several hundred nanometers to several micrometers and diameters change from 0.4 to 2 nanometers. This one dimensional carbon structure is of high surface area, high mechanical strength but ultra-light weight, rich electronic properties, and excellent chemical and thermal stability [Ajayan 1999]. Ever since the discovery of carbon nanotubes, researchers have been exploring their potential in biological and biomedical applications [Baughman et al 1999; Mattson et al 2000]. The recent expansion and availability of chemical modification and bio-functionalization methods have made it possible to generate a new class of bioactive carbon nanotubes which are conjugated with proteins, carbohydrates, or nucleic acids. Biocompatibility and the availability of accessible methods for functionalization with bioactive molecules make SWCNTs promising candidates for intracellular delivery agents and for other applications in bio- sensing and biomedicine [Sotiropoulou et al 2003; Lin et al 2004; Pastorin et al 2006; Zeni et al 2008]. 9 1.2.1 The Integration of SWCNTs and Nanosecond Pulsed Power Technology- Electropermeabilzation Reduction Effect To achieve the goal of specifically or selectively kill the cancer cells but not the normal cells, we propose an idea of adding intracellular SWCNTs which are assumed to enhance the localized electric field on cell to nanoelectropulses. This proposed idea links our research from nanosecond pulsed power technology to modifications of biomaterials and nanomaterials. To observe the cell membrane integrity, SKOV-3 human ovarian cancer cells are mixed with and without intracellular SWCNTs. Nanoelectropulses were delivered to the cells in the presence of the fluorochrome YO-PRO-1 (Molecular Probes, Invitrogen; λ ex = 491 nm, λ em = 509 nm), a non-permeant fluorochrome which is only weakly fluorescent in the extracellular medium. A permeabilized cell can be identified by the greatly increased fluorescence of YO-PRO-1 bound to nucleic acid material in the cell interior. YO-PRO-1 has been used as an indicator of plasma membrane permeabilization and early apoptosis after nanosecond pulse exposure [Idziorek et al 1995; Vernier et al 2006]. The integration of YO-PRO-1 fluorescence intensity inside SKOV-3 cells with and without intracellular SWCNTs to nanoelectropulses is shown as Figure 1.1. The cell membrane permeabilization induced by nanoelectropulses to cells with intracellular SWCNTs is lower than without intracellular SWCNTs. This is not a positive result to support our assumption that intracellular SWCNTs exposed to nanoelectropulses related to the localized electric field enhancement then results in stronger cell killing effect. However, the mechanism of YO-PRO-1 uptake reduction effect is not clear and worthy to investigate. 10 Figure 1.1 Intracellular SWCNTs-induced YO-PRO-1 permeabilization reductions to nanoelectropulses. Fluorescence microscopic images of SKOV-3 human ovarian cancer cells in growth medium containing YO-PRO-1 (1.0 µM) were captured immediately after exposure to 50, 30 ns pulses at 50 Hz with electric field values of 3 MV/m. The fluorescence intensity change for each condition was measured by photometric integration. At higher amplitudes, permeability of the cells to YO-PRO-1 increases. Over 300 of cells are counted for each pulsing condition. At the mean time, we find some interesting results of functionalized SWCNT-FC conjugates when demonstrating the internalization of SWCNTs to SKOV-3 human ovarian cancer cells. This potential application of using functionalized SWCNT-FC as intracellular pH sensor is described in the following section. 1.2.2 Functionalized SWCNTs Conjugates as an Intracellular pH Probes Many studies have focused on the use of SWCNTs as vehicles for direct delivery of anti-cancer drugs across the cell membrane of cancer cells [Lin et al 2005; Klumpp et al 2006]. Utilization of the optical and electronic properties of carbon nanotubes for intracellular sensing has been less explored [Barone et al 2005; Heller et al 2005]. For 11 example, Heller and colleagues found that DNA-encapsulated nanotubes could be used to sense intracellular cationic concentrations. After the addition of divalent cations such as Hg 2+ , the transition of DNA secondary structure from a B to Z conformation modulated the dielectric environment of SWCNTs and decreased their near-IR emission energy, thus yielding observable characteristic shifts in the SWCNTs luminescence spectrum [Heller et al 2006]. Intracellular pH (pH i ) is a key parameter in most biochemical and biological processes [Graber et al 1986]. Conventional methods for measuring pH i include the use of radioactively labeled weak organic acids and bases [Kashket 1985; Rottenberg 1989], nuclear magnetic resonance spectroscopy (NMR) [Moon et al 1973; Chacko et al 1993], and fluorescent dyes [Schwiening 1999]. However, as many disease states are associated with abnormal chemical environments at a subcellular level; new probes are needed that measure pH in localized environments inside living cells in real time. The development of nanoparticle-based fluorescent probes represents a new trend that offers the advantages of localized and continuous pH i monitoring with high time resolution and sensitivity [Al-Hilli et al 2006; Sun et al 2006; Liu et al 2007]. Although SWCNTs are widely used as intracellular carriers, little has been reported about utilizing them as pH i probes. Two hypotheses are proposed to explain such observations: First, dequenching of fluorescein fluorescence could result from light-stimulated intracellular hydrolysis of the amide linkage between SWCNTs and fluorescein molecules. Second, light-induced intracellular pH changes may cause fluorescence intensity variations of the SWCNT-FC conjugates, since fluorescein is a pH- sensitive dye. Either of the above-mentioned processes, or both, would produce a fluorescence intensity increase upon light stimulation. Chapter 5 describes that nanosized 12 SWCNT-FC conjugates have the potential to be used as localized pH i sensors and trackers in studies of uptake mechanisms, endocytotic degradation, and photo-induced phenomena in living cells. 1.2.3 Laser-Induced Hyperthermia Effect of Intracellular SWCNTs In chapter 6, we report a potential brain tumor therapy developed from intracellular SWCNTs together with near infrared laser exposure. Glioblastoma multiforme tumors (GBM) can rapidly infiltrate the normal brain tissue and are difficult to remove surgically. In this work, we investigate the use of SWCNTs for hyperthermic therapy of gliomas. SWCNTs were delivered through endocytosis to U251 human GBM cells and to astrocytes form normal brain tissue. Successful SWCNTs uptake was demonstrated by using 3-D fluorescence imaging, which clearly shows intracellular SWCNT-FC conjugates. 808 nm infrared radiations, absorbed specifically by SWCNTs but not by biological tissue, heats and kills only cells containing intracellular SWCNTs. We are currently investigating methods for targeting brain tumors with SWCNTs selectively relative to normal brain cells. 1.2.4 Intracellular SWCNTs-Induced Migration Reduction Effect of Brain Cancer Cell In chapter 7, the observations of migration reduction effect of brain caner cells induced by intracellular SWCNTs were discussed. Our experiments show that intracellular SWCNTs can significantly reduce the migration rate of rat C6 GBM and U251 human GBM cells. The mechanism of brain cancer cell migration inhibition caused 13 by intracellular SWCNTs may be related to the metabolic state of the tumor cells or to SWCNTs-induced modifications of the cell membrane, hypotheses that are under investigation. To conclude, the potential of using the functionalized SWCNTs to specific target the brain tumors as cancer therapeutics materials, and the possibility of using cold plasma as a post procedure cleaning tool to dissolve the residues of SWCNTs, are proposed. Finally, future work for cancer therapy development and bacterial disinfection applications related to nanosecond pulsed power technology and functionalized SWCNTs conjugates are proposed in chapter 8. 14 Chapter 2 Nanosecond electric pulse-induced calcium entry into chromaffin cells The release of calcium ions from intracellular organelles is the most notable effect of nanoelectropulses which can sequentially stimulate apoptosis and is significant for the cancer therapy development. In this chapter, to understand the mechanism of nanoelectropulses-induced calcium release on electrically excitable bovine adrenal chromaffin cells, we monitor intracellular calcium concentration [Ca 2+ ] i in cells exposed to 4 ns duration electric pulses with field intensities ranging from 2 MV/m to 8 MV/m. The intracellular calcium levels ([Ca 2+ ] i ) are monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca 2+ ] i , with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca 2+ ] i could be triggered. The pulse-induced rise in [Ca 2+ ] i was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co 2+ , or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca 2+ ] i that involves entry of calcium via L-type calcium channels. The result involves a different mechanism from that described to date for non-excitable cells [Craviso et al 2006]. 15 2.1 Preparation and fluorescence microscopy of chromaffin cells The Adrenal Chromaffin cells information was in Appendix. Chromaffin cell aggregates that form in suspension culture were dissociated into clusters of ten cells or less, including single cells [Hassan et al 2002; Craviso 2004], and the cells were loaded with the calcium fluorescent indicator dye Calcium Green-1-AM (Invitrogen; Ex 480 nm and Em 535 nm) at 1 μM in RPMI 1640 medium (Irvine Scientific) for 1 h at 37 °C. After resuspension in dye-free RPMI 1640, the cells were transferred to a microfabricated electrode chamber [Sun et al 2005] that was positioned on the stage of a Zeiss Axiovert 200M epifluorescence microscope. Images were captured and analyzed with a LaVision Imager QE camera and software and a MOSFET, saturable-core, transformer-switched, fast-recovery diode pulse generator [Kuthi et al 2005] that was mounted on the microscope stage delivered 4 ns pulses (200–800 V) directly to the microchamber electrodes in ambient atmosphere at room temperature. Details of this experimental apparatus are given in Sun et al [Sun et al 2006]. Fluorescence intensity curves were generated from a set of sequential images of cells from each experiment. All fluorescence data have been normalized to the intensity value at the point where the first pulse was applied, and each fluorescence curve represents the integrated response over the area of an individual cell. Background fluorescence was measured in several cell-free regions of the visual field. Because direct addition of reagents to the cells in the microelectrode chamber was not feasible, the response of the cells to addition of the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP; Sigma) was obtained separately by introducing 0.4 μL of a 5mM solution of the drug into a cover glass chamber containing 200 μL of the cell suspension. YO-PRO-1 was obtained from Invitrogen. 16 2.2 Effect of 4 ns pulses on [Ca 2+ ] i As shown in Figure 2.1, application of a single 4 ns, 8 MV/m pulse induced a rapid, marked rise in [Ca 2+ ] i that was maximal by 1 to 2 seconds and transient, with [Ca 2+ ] i returning to baseline values typically by 15 to 45 seconds. Figure 2.1 Fluorescence imaging of [Ca 2+ ] i in chromaffin cells exposed to a single 4 ns, 8MV/m pulse. (A) Photomicrographs of cells before (bright field and 0 seconds) and 6 seconds after the pulse was applied. (B) Calcium traces for the cells shown in (A); (C) calcium traces for cells from a separate, similar experiment. In (B) and (C), and in subsequent figures, the lines with symbols are mean values, and the arrows indicate when the pulse was applied. 17 A second 4 ns, 8 MV/m pulse delivered before [Ca 2+ ] i reached baseline values produced only a slight additional elevation of [Ca 2+ ] i (Figure 2.2(A)) and a maximal rise in [Ca 2+ ] i was again observed when another pulse was delivered more than 30 seconds after the calcium response from the previous pulse had returned to baseline (Figure 2.2(B)). Figure 2.2 Response of chromaffin cells to multiple applications of 4 ns pulses. (A) A second 8 MV/m pulse was applied 8 s after the first 8 MV/m pulse; (B) A second 8 MV/m pulse was applied 40 s after the first 8 MV/m pulse, with cell illumination discontinued between pulses. 18 Consistent with this latter finding was that multiple pulses delivered at repetition rates from 0.5 Hz to 1 kHz did not elicit a greater rise in [Ca 2+ ] i than that elicited by a single pulse (data not shown). These results indicate that delivery of only a single 4 ns pulse is sufficient to elicit a maximal increase in [Ca 2+ ] i and that multiple pulses delivered to the cells at high repetition rates are no more effective for increasing [Ca 2+ ] i than a single pulse. They further show that a second increase in [Ca 2+ ] i can be elicited after [Ca 2+ ] i returns to resting levels, suggesting that calcium clearance mechanisms most likely involving both cell extrusion and uptake into internal storage sites are functional and that as long as a sufficient period of time elapses between pulses, the cells can respond fully to another nanoelectropulse stimulus. The response of chromaffin cells to a nanoelectropulse was highly reproducible across experiments that included cells at different times in culture as well as cells from different preparations, and was also observed in both single and aggregated cells. In addition, the magnitude of the response was dependent on field intensity, with the rise in [Ca 2+ ] i progressively decreasing as field intensity decreased from 8 MV/m to 2 MV/m (data not shown). Although direct comparisons of the calcium response of the cells to a single nanoelectropulse and to the nicotinic receptor agonist DMPP were not feasible, the magnitude of the increase in [Ca 2+ ] i elicited by a pulse at a field intensity of 8 MV/m was at least as great in magnitude as that elicited by nicotinic receptor activation. 2.3 Inability of calcium store depletion to block the rise in [Ca 2+ ] i Experiments to determine whether the pulse-stimulated increase in [Ca 2+ ] i was due to release of calcium from internal stores consisted of incubating chromaffin cells prior to 19 and during pulsing with drugs that cause a depletion of stored calcium. These drugs include caffeine (10mM),which empties calcium from ryanodine-sensitive endoplasmic reticulum stores [Cheek et al 1993], and thapsigargin (2 μM), which prevents calcium from accumulating in stores by blocking endoplasmic reticulum Ca 2+ -ATPase [Thastrup et al 1990]. As shown in Figure 2.3(A), neither the response to a single 4 ns, 8 MV/m pulse nor to a subsequent pulse applied 40 s after the first pulse was prevented by caffeine-induced calcium store depletion. However, the rise time of the pulse-induced increase in [Ca 2+ ] i appeared to be faster and the calcium response was more sustained when caffeine was present. Treatment of the cells with thapsigargin also did not prevent the increase in [Ca 2+ ] i elicited by the nanoelectropulse (data not shown). Thus, a single nanosecond duration electric pulse elicits a rapid rise in [Ca 2+ ] i in chromaffin cells that does not involve mobilization of calcium from internal stores. This finding contrasts with that observed for non-excitable cells, such as Jurkat and HL-60 cells, where application of a single nanosecond electric pulse elicits a rise in [Ca 2+ ] i that can be prevented by agents that deplete calcium stores [Vernier et al 2003; Beebe et al 2004; White et al 2004; Sun et al 2005]. Taken together, these results suggest that for excitable chromaffin cells, internal organelle membranes are not the primary target of the nanoelectropulse with respect to increasing [Ca 2+ ] i . 20 Figure 2.3 Response of chromaffin cells to 4 ns, 8 MV/m pulses. In (A) the cells were incubated with 10mMcaffeine before and during the application of a single 4 ns, 8 MV/m pulse. A second 8 MV/m pulse was applied 40 s after the first 8 MV/m pulse, with cell illumination discontinued between pulses. In (B), 20 M nitrendipine was present in the extracellular medium. 2.4 Chelating extracellular calcium or blocking plasma membrane calcium channels prevents the rise in [Ca 2+ ] i In contrast to the results observed with calcium store depleting agents, chelating calcium in the extracellular medium by adding EGTA to a final concentration of 5 mM prevented the rise in [Ca 2+ ] i (data not shown), indicating that the source of calcium was 21 from outside the cell. To explore this result further, cells were pulsed in the presence of Co 2+ (1 mM), a non-selective blocker of calcium channels. The response of the cells to the nanoelectropulse was again blocked (data not shown). Pulsing the cells in the presence of nitrendipine (20 μM), a dihydropyridine that is a selective antagonist of L- type voltage-sensitive calcium channels, also prevented the rise in [Ca 2+ ] i (Figure 2.3(B)), suggesting that L-type calcium channels are involved in the pulse-induced entry of calcium into the cells. These results indicate not only that the plasma membrane appears to be the immediate target of the pulse but also that calcium entry into the cell occurs via a specific type of plasma membrane voltage-sensitive calcium channel. Our findings therefore broaden the range of responses that have been identified for nanoelectropulse effects on biological cells to include, as a primary response, changes in the conductance of a specific dihydropyridine-sensitive, voltage-dependent plasma membrane ion channel that gates calcium. With respect to bovine chromaffin cells, this observation is particularly intriguing since these cells express three other types of plasma membrane voltage-sensitive calcium channels (N- and P/Q-type channels [Artalejo et al 1994; Albillos et al 1996] that account for 80% of the whole cell calcium current [Garcia et al 2006]. Therefore, if the pulse-induced rise in [Ca 2+ ] i results from a pulse-driven depolarization of the chromaffin cell plasma membrane [Frey et al 2006] that activates the voltage dependent L-type channels, then one might expect N- and P/Q-type channels to open as well since these channels are not only present in much greater abundance relative to L-type channels but also activate at membrane potentials similar to those for L-type channels [Hille 2001]. Also worthy of note is that our pulse exposures appear to have no effect on a constitutively active, voltage-insensitive plasma membrane cation 22 channel that is responsible for influx of calcium into bovine chromaffin cells under resting conditions [Cheek et al 2006]. One possible explanation for the apparent selectivity of the pulse-induced effects on calcium entry into the cells is that the very short duration of the high intensity electric field has a direct electroperturbating effect only on L-type channels. This possibility is currently being explored. 2.5 Lack of evidence of plasma membrane electroporation after a single 4 ns pulse The observation that application of multiple pulses to chromaffin cells does not result in additive increases in [Ca 2+ ] i suggests that nanoelectropulse treatment does not produce significant poration of the chromaffin cell plasma membrane, or that any pores produced are not calcium-conductive. To assess plasma membrane integrity directly, nanoelectropulses were delivered to the cells in the presence of the fluorochrome YO- PRO-1 (5 μM), a compound that is excluded from cells with intact membranes [Idziorek et al 1995] and which has been used as an indicator of plasma membrane permeabilization after nanosecond pulse exposure [Vernier et al 2006]. As shown in Figure 2.4, detectable uptake of the dye into the cells occurred after the application of fifty 4 ns, 8 MV/m pulses, but not after single pulses. Thus, plasma membrane electroporation does not appear to be a significant factor in the chromaffin cell response to the application of a single pulse. 23 Figure 2.4 Effect of single and multiple applications of 4 ns, 8 MV/m pulses on the influx of YO- PRO-1 into chromaffin cells. A single pulse was delivered at 0 seconds and at 5 seconds, and 50 pulses were delivered at 10 seconds, as indicated by the arrows. 2.6 Conclusion This study is the first to show that for an electrically excitable cell, a single 4 ns electric pulse causes entry of calcium into the cell via voltage-sensitive L-type channels. Because L-type channels in chromaffin cells are associated with the exocytotic machinery that is located at the plasma membrane and responsible for triggering catecholamine release, an obvious question is whether the calcium that enters the cells with each nanoelectropulse attains sufficiently high subplasmalemmal levels to have physiological significance. A determination of whether catecholamine release is stimulated in response to a single pulse is underway since the results of these experiments will have implications for predicting effects of nanosecond electric pulse exposure on the behavior of other excitable cells that use calcium entry via L-type calcium channels for eliciting and controlling cellular responses. 24 Chapter 3 Two-Dimensional Nanosecond Electric Field Mapping Based on Cell Electropermeabilization Nanosecond, megavolt-per-meter electric pulses cause permeabilization of cells to small molecules, programmed cell death (apoptosis) in tumor cells, and are under evaluation as a treatment for skin cancer. We use nanoelectroporation and fluorescence imaging to construct two-dimensional maps of the electric field associated with delivery of 15 ns, 10 kV pulses to monolayers of the human prostate cancer cell line PC3 from three different electrode configurations: single-needle, five-needle, and flat-cut coaxial cable. Influx of the normally impermeant fluorescent dye YO-PRO-1 serves as a sensitive indicator of membrane permeabilization. The level of fluorescence emission after pulse exposure is proportional to the applied electric field strength. Spatial electric field distributions were compared in a plane normal to the center axis and 15-20 μm from the tip of the center electrode. Measurement results agree well with models for the three electrode arrangements evaluated in this study. This live-cell method for measuring a nanosecond pulsed electric field distribution provides an operationally meaningful calibration for electrode designs for biological applications and permits visualization of the relative sensitivities of different cell types to nanoelectropulse stimulation. 25 3.1 Experimental setup The experimental setup consists of a pulse generator, a voltage and current diagnostic system, and an optical stage for accurately positioning a cell culture plate, as shown in Figure 3.1. Figure 3.1 Schematic of the experimental setup for nanosecond pulsed electric field mapping. 3.1.1 Pulse generation and measurement A solid-state, opening-switch-based pulse generator, generating 15 ns, 10 kV pulses at repetition rates up to 50 Hz was designed and fabricated at the University of Southern California [Tang et al 2007]. A built-in resistive voltage divider based on cascaded attenuation stages with a total attenuation of -54 dB (ratio: 1:500) was used to measure the pulse voltage delivered to the load. The architecture is similar as in [Kuthi et al 2005]. 26 A current transformer with a ratio of 1 to 5 was used to measure the pulse current. A high saturation flux density Finemet ® Metglas core (ID = 0.8 cm, OD = 1.5 cm, h = 0.6 cm) was used to ensure fast response and linearity for the current measurement. Attenuated pulse current was converted to a voltage signal with a 50 ohm, surface-mount, low-inductive resistor, terminated at the secondary winding of the transformer, to give a total current conversion of 20 V/A. A 50 ohm terminated digital oscilloscope (Tektronix TDS 5104) was connected with 50 ohm coaxial cables to record the output from the voltage divider and the current sensor. A 50 ohm SHV coaxial cable assembly was used to deliver nanosecond electric pulses to the electrodes. The losses in the cable are about 3%, single transit. Since the pulse generator represents an open circuit for the reflected pulses and the impedance of the electrodes is 100 ohms or less, depending on the electrode configuration, the reflected pulse amplitude is always less than 50%. Because of the complexity and variable impedance of biological loads precise matching is not possible, but since nanosecond biolectric effects are primarily dependent on the applied electric field, power transfer is not a critical consideration. 3.1.2 Electrode configurations Three types of electrode assemblies, single-needle, five-needle, and a flat-cut coaxial cable, as shown in Figure 3.2, were tested. All the electrodes are in center- symmetrical configurations. Stainless steel needles, 0.2 mm in diameter and 5 mm long, are used for the single-needle and five-needle electrodes. The needle tips are cut off and the edges rounded by polishing, leaving a cylindrical profile. For the five-needle array, 27 one needle is at the center, and the other four needles, equally spaced, are located on a 3.5 mm-diameter circle and laser-welded to the coaxial outer shield. For the flat-cut cable, the center stainless steel electrode is 0.2 mm in diameter, and the end surfaces of the electrodes and the insulator are in the same plane. The separation between center and outer conductors is 1.7 mm. The insulating material separating the center electrode and the outer grounding shield is Teflon. The other end of these electrodes is an SHV adapter to facilitate connections with SHV cable assemblies. Figure 3.2 Three types of electrode configurations designed for nanoelectropulse treatment and cancer therapy: (A) single-needle, (B) five-needle array, and (C) flat-cut cable. 3.1.3 Adjustable stage An adjustable stage, consisting of a spiral micrometer with a minimum reading of 25 µm, was used to adjust the spacing distance between the cells and the electrodes. The culture dish for treatment was placed on a horizontal plane holder, calibrated with a spirit level meter. The electrode applicator was fixed to an arm of the optical stage, and its 28 center electrode was adjusted with the center axis normal to the bottom surface of the culture dish. The applied cell monolayer has a thickness of 5–10 µm. After identifying the location where the electrodes touches the bottom of the culture dish, we retracted the electrodes for 25 µm to obtain a monolayer-to-electrode tip spacing of 15–20 µm. 3.2 Cell lines and cell preparations Human Jurkat T lymphoblasts (ATCC TIB-152) were cultured in suspension with RPMI 1640 medium (Irvine Scientific, Irvine, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA), 2 mM L-glutamine (Gibco), 45 units/mL penicillin (Gibco), and 45 μg/mL streptomycin (Gibco). Human prostate cancer PC3 cells (ATCC CRL-1435), U251 human glioblastoma cells (RCB-0461, RIKEN CELL BANK), and human keratoacanthoma cells (skin, mixed morphology, ATCC CRL-7630) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC) with 4 mM L- glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate, 10% FBS, 45 units/mL penicillin, and 45 µg/mL streptomycin. All cells were grown at 37 ºC in a humidified, 5% CO 2 atmosphere. Before an experiment, the PC3, U251, and keratoacanthoma cells were detached with 0.05% trypsin/0.53 mM EDTA in Hank’s Buffered Salt Solution (HBSS) without sodium bicarbonate, calcium and magnesium (Cellgro, Herndon, VA) and washed with DMEM growth medium. 1 mL of PC3, U251, and keratoacanthoma cell suspensions (1×10 6 cells/mL) was added to appropriate flat- bottomed wells of a 24-well culture plate, and the cells were incubated until they reached confluence (about 24 hours). 29 3.3 Fluorescence microscopy and imaging processing YO-PRO-1 (Molecular Probes, Invitrogen; λ ex = 491 nm, λ em = 509 nm) is a membrane-impermeant fluorescent probe. A permeabilized cell can be identified by the greatly increased fluorescence resulting from YO-PRO-1 influx and binding to nucleic acid material in the cell interior. A Zeiss AxioVert 200 M fluorescence microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY) and AxioVision 3.1 imaging software were used to capture and analyze fluorescence images. Low-power (10X objective) images of cell monolayers were taken 15 minutes after pulse exposure. Since the total area of the pulse-exposed cells is greater than the imaging region of the 10X objective, composite images were generated from a sequence of overlapping images that covered the entire area under and around the electrodes. Each experiment was performed three times, with similar results in each case. 3.4 Electrostatic calculation of the electric field distribution using COMSOL Multiphysics The wavelength, λ, and the skin depth, d, of an electromagnetic wave propagating in a medium with zero magnetic susceptibility (relative permeability = 1) depend on the frequency, f, and on the dielectric constant, ε r , and conductivity, σ, of the medium, as in the following equations (1) and (2): 2 / 1 2 1 1 2 2 ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ + ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + = εω σ ε ω π λ r c (1) and 30 2 / 1 2 1 1 2 1 ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ − ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + = εω σ ε ω r c d (2), where c is the speed of light in vacuum, ω=2 πƒ is the angular frequency, and ε is the permittivity of free space. The rise and fall times of the 15 ns voltage pulses are 5 ns or longer so that the primary frequency of the pulses is expected to be 200 MHz or lower. For DMEM (conductivity: σ = 1.4 S/m, and dielectric constant: ε r ≈ 80 [Arnold et al 1994]), the minimum wavelength and the skin depth of the electromagnetic fields are 14 cm and 4 cm, respectively. Since both the wavelength and the skin depth are large compared to the geometry of interest, we can use an electrostatic model, the commercial COMSOL Multiphysics (http://www.comsol.com/) electrostatics module, for the electric field distribution calculation. 3.5 Temperature measurement To evaluate the thermal effect caused by electric pulses, we have conducted experiments to measure the localized temperature change at the tips of electrodes with a resistive temperature detector (RTD) (OMEGA, HSRTD-3-100-A-40-E). The RTD, in a shape of a 2 mm-OD wire, is flexible, hermetic-sealed and PFA-insulated and was fixed on the bottom of a culture dish. Tips of electrodes were adjusted normal to the wire and slightly in contact with the detector. All of them were immersed in DMEM solution (5 mL) as shown in Figure 3.3. 31 Figure 3.3 Temperature measurement setups at the tips of single-needle, five-needle array, and flat-cut cable electrodes powered with continuous electric pulses (10 kV, 15 ns @ 50 Hz). 3.6 Results and Discussions 3.6.1 Nanoelectropulse-induced membrane permeabilization depends on pulse amplitude It has been reported that cellular permeabilization in Jurkat T lymphoblasts with ultra-short (< 10 ns), high-field (MV/m) electric pulses is a function of pulse count [Vernier et al 2006]. To quantify the YO-PRO-1 uptake of cells in suspension exposed to nanoelectropulses at different electric field amplitudes, pulses were delivered to Jurkat cells in standard electroporation cuvettes with a 1 mm electrode gap. Cells (10 7 cells/ mL) in growth medium containing 1 µM YO-PRO-1 were exposed to 50, 15 ns pulses at 50 Hz with electric field intensities of 0, 2, 4, and 6 MV/m. Treated cells were transferred to 8-well cover glass chambers. After 15 minutes fluorescence images of the cells were generated with the 20X objective. Experiments were repeated twice, with more than 1000 cells analyzed for integrated fluorescence intensity for each test condition. The results are summarized in Figure 3.4, which shows that cell permeabilization to YO-PRO-1 depends on the magnitude of the applied pulsed electric field. In experiments in which other 32 variables are held constant, YO-PRO-1 fluorescence intensity is an indicator of the strength of the local electric field. Figure 3.4 YO-PRO-1 permeabilization of Jurkat T lymphoblast cells exposed to nanoelectropulses with different electric field amplitudes. Fluorescence microscopic images of Jurkat cells in growth medium containing YO-PRO-1 (1.0 µM) were captured 15 minutes later after exposure to 50, 15 ns pulses at 50 Hz with electric field values of 0, 2, 4 and 6 MV/m. The fluorescence intensity change for each condition was measured by photometric integration. 3.6.2 Electrical measurement Nanoelectropulses were applied with each of the three electrode configurations to cell monolayers with an electrode-monolayer spacing distance at 15-20 µm. Voltage and current waveforms are shown in Figure 3.5. Different peak currents — 900 A, 600 A, and 500 A — were observed for the five-needle, single-needle, and flat-cut cable electrodes, respectively. The observed current pulses are a summation of the displacement current associated with the capacitance of the electrodes and the resistive current due to charge migration in the medium. The energy per pulse was calculated by integrating the product 33 of the voltage and current pulse waveforms over a complete pulse period. The energy per pulse for the flat-cut cable, 1.5 mJ, is ten times less than that for the single-needle electrode, 19.1 mJ, and the five-needle array, 20.5 mJ. Thus the pulsed energy delivered to the biological load can vary over a wide range, depending on the electrode configuration. Figure 3.5 Typical voltage and current pulse waveforms delivered to the PC3 cell monolayer in DMEM growth medium with three types of electrode configurations: (A) single-needle, (B) five- needle array, and (C) flat-cut cable. 3.6.3 Electric field mapping 3.6.3.1 Fluorescence images of three electrode configurations PC3 cell monolayers were exposed to nanoelectropulses (1000, 15 ns, 10 kV pulses at 50 Hz) with three different electrode configurations in fresh DMEM containing 1 µM YO-PRO-1. To exclude mechanical damages to cells, the cell monolayers were treated 34 with three electrode configurations at spacing distance 15-20 µm without pulse doses as the negative control. Figure 3.6(A), (B), and (C) show fluorescence images of permeabilized PC3 cells after nanoelectropulse exposures with the single-needle, five- needle, and flat-cut cable electrode configurations, respectively. No YO-PRO-1 uptake was observed in un-pulsed PC3 cell monolayers treated three electrodes. Figure 3.6(D) show the fluorescence image of PC3 cell monolayers treated with flat-cut cable but no pulse exposure was applied. The fluorescence brightness and pattern for each image indicate the level of YO-PRO-1 permeabilization and the affected area of cells for each electrode arrangement. The concentric shape of the fluorescence pattern from cells exposed to the single-needle and flat-cut cable electrodes corresponds to the geometry of the electrode configuration (Figure 3.6(A) and 3.6(C)). The fluorescence brightness and pattern for the five-needle array are in between of the single-needle and flat-cut cable electrodes. A square-shape fluorescence pattern centered at the center needle electrode is discernible for the five-needle array. The slight asymmetry might be due to many factors including evenness of cell monolayer surfaces and electrode surfaces. The location of center electrode was determined by comparing the brightness and contrast with the imaging software between the darker center region and the surrounding brighter regions. For lower field intensity, a circle area with 0.2 mm in diameter which corresponds to the center electrode is expected at the axial center compared to that at its nearest vicinity. Once the axial center is determined, we mapped the electrodes according to the scale. The fluorescence intensity is strongest near the center conductor of the flat-cut cable electrode. The five-needle array produces fluorescence of intermediate intensity; the single-needle has the weakest effect. 35 Figure 3.6 Fluorescence images of the PC3 cell monolayer exposed to nanoelectropulses with three different electrode configurations, (A) single-needle, (B) five-needle array, and (C) flat-cut cable, based on YO-PRO-1 permeabilization. 1000, 15 ns, 10 kV pulses at 50 Hz were delivered to cell monolayer. (D) Fluorescence image of un-pulsed PC3 cell monolayer with flat-cut cable as CTRL sample. To generate the fluorescence images of the area between the ground and high voltage electrodes, a series of 640 µm × 710 µm images are combined. The needles and the inner boundary of the ground electrodes are indicated with a small red dashed circle and outer red dashed circles, respectively. 3.6.3.2 Comparisons of electric field distribution between electrostatic simulation and fluorescence integration analysis Three-dimensional electrostatic models for each of the three electrode configurations (Figure 3.2) immersed in water were generated, considering the electrodes 36 as perfect conductors. Gauss’s law, (with ), where V is the voltage potential, was solved for a 1 V potential difference between the center electrodes and the ground electrodes. Zero-space charge and zero-electric displacement were assumed at the dielectric boundaries far enough from the active electrodes. The radial distributions of the electric field for the three electrode configurations in a plane perpendicular to the center axis and 10 µm above the center electrodes are shown in Figure 3.7(A). The flat-cut cable electrode has a higher electric field at the center of the exposure plane than the five-needle and single-needle electrodes. The maximum electric field appears at the edges of the center electrodes for all the electrode configurations. At regions with radius higher than 0.125 mm, the electric field decays rapidly with the radial distance. When 10 kV electric pulses are applied to the center electrodes, the electric field decays to <10 MV/m at a radius of 0.4 mm for all three electrode configurations. Note that the five-needle array electrode is not a cylindrically symmetrical arrangement, which results in difference between electric fields of regions near the ground electrode and away from the ground electrode. If we define a radial line connecting the center needle with one of the ground needles at an angle of zero, at angle of 45°, the radial line is cross the middle of two ground needles. The electric fields of the five-needle array along radius at angle = 0° and 45° are compared, as shown in the insert plot of Figure 3.7(A), and implies minimum difference in the field intensity for a radial distance <1 mm. 37 Figure 3.7 Electric field distributions modeled with the COMSOL Multiphysics electrostatics module (A) and fluorescence intensity distributions of electropermeabilized PC3 cell monolayers (B) for three different electrode configurations. To better compare the calculated electric field distribution with the measured permeabilization effect, the radial distribution of the relative fluorescence intensities for the three electrode configurations are shown in Figure 3.7(B). The relative fluorescence intensity was calculated by averaging the total intensity over 10 µm wide concentric rings spaced 50 µm apart. The integration was done in a full concentric circle within 1 mm radius. Again, the flat-cut cable electrode shows a higher fluorescence intensity at the center of the electrode than the other two electrodes. All three electrode configurations have a maximum fluorescence intensity near the edge of the center electrode (r = 0.125 mm). These results agree with the simulated behavior of the electric field distributions with these electrode configurations. For each electrode configurations, Table 1 shows the maximum electric field intensity of electrostatic simulation and the maximum fluorescence intensity of permeabilized PC3 cell monolayers. 38 Table 3.1 Maximum electric field intensity and maximum fluorescence intensity of different electrode configurations. 3.6.3.3 Fluorescence images of various cell types at different spacing distances Different cell lines may show different sensitivities to the same nanoelectropulse dose with same electrode configuration. To demonstrate this, we applied pulses (1000, 15 ns, 10 kV pulses at 50 Hz) with the flat-cut cable electrode to cell monolayers (U251 GBM and keratoacanthoma) in fresh DMEM containing 1 µM YO-PRO-1. Two different separation distances of 15-20 µm and 200 µm between the monolayer of cells and the electrode tips are applied here. Comparing the two cell lines when the monolayer is 15-20 µm from the tip of the center electrode demonstrates that the U251 GBM cells (Figure 3.8(A)) are more nanoelectropulse-sensitive than keratoacanthoma cells (Figure 3.8(C)). When the spacing distance between monolayer cells and electrode increases from 15-20 µm to 200 µm, the influx of YO-PRO-1 into the permeabilized cells decreases for both U251 GBM cells and keratoacanthoma cells shown as Figure 3.8(B) and Figure 3.8(D), respectively. Although the fluorescence intensity between the U251 GBM and keratoacanthoma cell monolayers are not shown here, the fluorescence images obviously indicate this method can be used to sense the nanoelectropulses-induced permeabilization of various cell types. It also reveals that the electric field distribution changes with different spacing distance. The more detail exploration included pulse number; pulse Table 3.1 Electrode Configurations Maximum Electric Field Intensity (MV/m) Maximum Fluorescence Intensity(Arbitrary Unit) Single needle electrode Five-needle array electrode Flat-cut cable 5.0 6.5 11.5 3.3 ± 0.8 6.5 ± 0.8 15.1 ± 3.0 39 duration; repetition rate of different electrode configurations to nanoelectropulses on cell monolayer can be assigned as the future work. Figure 3.8 Fluorescence images of U251 GBM cell monolayer [(A) and (C)] and keratoacanthoma cell monolayer [(B) and (D)] after nanoelectropulse exposure (1000, 10 kV, 15 ns pulses at 50 Hz) with the flat-cut cable electrode at a distance of < 25 µm [(A) and (B)] and 200 µm [(C) and (D)], respectively. 3.7 Thermal effect induced by electric pulses 3.7.1 Experimental measurement To exclude possible influence on heat dissipation brought by the RTD as well as the temperature response time (5 seconds), continuous pulses (10 kV, 15 ns @ 50 Hz) were 40 applied for 5 minutes and temperature was recorded every 20 seconds (1000 pulses per data point, 15K pulses in total). The increases of the localized temperature measured by RTD for three electrode configurations are below 1 ° C, as shown in Figure 3.9. The highest temperature increase was found in the single-needle array compared to the five- needle array and the flat-cut electrodes. Table 2 shows the temperature increase and energy per pulse of three different electrode configurations form measurement. Table 3.2 Energy/per pulse and temperature change of different electrode configurations. Figure 3.9 The temporal development of the temperature at the tips of single-needle, five-needle array, and flat-cut cable electrodes powered with continuous electric pulses (10 kV, 15 ns @ 50 Hz). Table 3.2 Electrode Configurations Energy(mJ)/ per pulse Temperature Change (°C/15K pulses) Single needle electrode Five-needle array electrode Flat-cut cable 19.1 20.5 1.5 0.9 0.7 0.3 41 3.7.2 Numerical calculation The measurement results suggest that heating induced by nanosecond electric pulses is negligible and is not sufficient to produce permeabilization in cell membranes or to induce hyperthermal effects [Tanabe et al 2004; Haemmerich et al 2005]. It can be expected that joule heating produced by each 15 ns pulse is near the surfaces of stainless steel needles and can be completely or partly conducted away with heat diffusion before the 2 nd pulse arrives (after 20 ms). For simplicity, we first calculated a model in which a 10 µm gap filled with water between a heat insulator plate at left and a stainless steel plate at right. In addition, considering the thermal diffusivity of stainless steel is 4 × 10 -6 m 2 /s, 26 times greater than water, 1.45 × 10 -7 m 2 /s, temperature of stainless steel plates can be assumed constant at room temperature, 273 K. For estimation, the 1-dimensional Fourier’s equation for heat diffusion was derived by using separation-variable solution. According to 1-dimensional Fourier’s equation for heat diffusion 2 2 ) , ( ) , ( x t x T D t t x T ∂ ∂ = ∂ ∂ (3) where T(x,t) indicates the temperature distribution, and D is thermal diffusivity of the medium. With separation of variable solution, we obtain ) exp( ) cos sin ( ) , ( 2 0 Dt L x n L x n b L x n a t x T n n n ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ − + = ∑ ∞ = π π π (4) Assuming the initial condition: T (x, 0) = T 0 ; 0<x<L, and the boundary condition: T (L, t) = 0; and 0 0 = ∂ ∂ = x T x , for t>0. Equation (4) can be solved to be: 42 () ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ + − + + − = ∑ ∞ = 2 2 2 0 0 4 ) 1 2 ( exp ) 1 2 ( cos 1 2 1 4 ) , ( L Dt n L x n n T t x T n n π π π (5) or () ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ − + + − = ∑ ∞ = n n n t L x n n T t x T τ π π exp ) 1 2 ( cos 1 2 1 4 ) , ( 0 0 (6) where () D n L n 2 2 2 1 2 4 π τ + = , and 9 / 1 0 = τ τ ( 1 0 τ τ >> ). A time constant, D L 2 2 4 π τ = = 280µs, can be assumed for the time required to conduct the electric pulse-induced heat away. This means that the time for temperature to drop to one third of maximum temperature (e -1 =0.368) is 280 µs. After 20 ms, the increased temperature induced by the first electric pulse will be is negligible. For 2D and 3D calculations, we expect the time constants will be in the same order of magnitude. In addition, there are other forms of heat dissipation including convection and radiation which will help equalize the temperature even faster. Therefore, based on the above experimental measurements and thermal conduction calculation, the thermal effect induced by the nanosecond pulses are negligible. 3.8 Conclusion Electric field distributions for three different electrode configurations have been evaluated based on nanoelectropulse-induced YO-PRO-1 influx and electrostatic models. The measurement method was also used to gauge the electropermeabilization sensitivity of different cell lines. The visualization of the two-dimensional pattern of 43 permeabilization in living cell monolayer allows us to map the electric field distribution with nanoelectropulses in a biological system for different kinds of electrode configurations. More important, we have proved that a diagnostic tool based on electropermeabilization of cells can be used to test invasive, minimum invasive and noninvasive electrodes for nanoelectropulse therapy. This method can be expected to test the sensitivity of tissues from patients, animals or plants to nanoelectropulses for ex-vivo studies. It also has potential to construct a three-dimensional nanosecond electric field distribution mapping by combining a series of fluorescence images taken with sequential spacing distance between the cell monolayers and electrode. 44 Chapter 4 Pulsed Atmospheric-Pressure Cold Plasma for Endodontic Disinfection To investigate a new strategy as a safe and effective alternative or adjunct for endodontic treatment, we designed a plasma dental probe (PDP), powered with 4–6 kV, 100 ns electric pulses at repetition rates of up to 2 kHz, generates a room-temperature > 2 cm long, ~2 mm diameter plasma plume at ambient atmospheric pressure. Plasma, which is generated by an electrical discharge in a gas, contains free electrons and ions, various active species (e.g., atomic radicals and excited molecules) and energetic UV photons [Moisan et al 2001]. The attractive characteristic of non-equilibrium, atmospheric- pressure plasmas is their ability to enhance chemical and biochemical reactions with minimal heating. The electrons in these plasmas, which have much higher energies than the heavier ions and neutral species, collide with background atoms and molecules causing enhanced levels of dissociation, excitation, and ionization. Reactive neutral species and charged particles, generated by these collisions, interact with the materials under treatment, while the bulk gas remains near room temperature. Because the ions and the neutrals remain relatively cold, this relatively “cold” plasma causes no or minimal thermal damage to the substrate it might contact with. These properties make it possible to use these plasmas for the treatment in heat-sensitive environment of biomedical applications, including low-heat surface modification of polymers, [Akishev et al 2002; Borcia et al 2005; Bhoj et al 2007] clinical instrument sterilization, [Lee et al 2006; Deng 45 et al 2007] tissue engineering, [Brown et al 2003] food processing, [Vleugels et al 2005] and dental cavity treatment [Sladek et al 2004; Sladek et al 2007]. Many different types of plasma devices, including nanosecond pulsed plasma pencils, [Laroussi et al 2005; Laroussi et al 2006; Walsh et al 2006; Walsh et al 2006] radio-frequency plasma needles, [Stoffels et al 2002; Stoffels et al 2006] direct-current (dc) plasma brushes [Duan et al 2005; Yu et al 2006], and plasma jets [Kolb et al 2008] have been developed for non-thermal atmospheric-pressure plasma generation. The common challenge for generating these plasmas is the inhibition of the glow-to-arc transition at one atmosphere [Kunhardt 2000]. Different types of discharges have used different schemes to achieve this. Dielectric barrier discharges (DBDs), typically driven by sine-wave high voltages at several kHz, cover at least one of the two electrodes with dielectric materials to limit the discharge current [Kanazawa et al 1988]. Laroussi et al. replaced the dielectric barrier with a resistive layer to extend the DBD operating frequency down to DC [Laroussi et al 2002]. RF ‘‘plasma needles’’ use needle electrodes excited with relatively low voltage (600–900 V) RF sources in the MHz range [Stoffels et al 2002; Goree et al 2005]. For RF voltage amplitude higher than 700 V, arcing between the high voltage electrode and the sample was observed [Goree et al 2005]. DC-driven microhollow cathode discharges employ sub-millimeter hollow-cathode structures to stabilize non-equilibrium plasma at one atmosphere [Schoenbach et al 1996; Stark et al 1999]. The discharge current is limited by ballast resistors in series. When the excitation electric pulses have duration less than the critical time for the development of glow-to-arc transition, weakly ionized non-equilibrium plasmas can also be generated stably in one atmosphere. Examples include the microhollow cathode discharge-sustained, 10 ns, 46 atmospheric pressure glow discharges [Schoenbach et al; 2001] and the submicrosecond pulsed, kilohertz, dielectric barrier-free, glow discharges [Walsh et al 2006]. 4.1 Design and Schematic of Plasma Dental Probe The plasma dental probe (PDP) (Figure 4.1(A)) is based on a coaxial tubular structure with a maximum envelope OD of 34 mm. The center, high-voltage electrode, a 6.35 mm OD, 12.7 mm long copper tube, is separated by a ceramic cylindrical structure from a 12.7 mm ID, stainless steel, conflate flange that is at ground potential. The inner diameter of the gas flow channel is 3 mm. The high-voltage electrode is recessed 5 mm from the outer surface of the ceramic, which is flush with the exit surface of the stainless steel flange. The grounded flange envelope shields the high voltage electrode, reducing the propagation of electromagnetic interference, and enables handheld operation without the danger of electric shock. 47 Figure 4.1 Schematic of a plasma dental probe version 1. 4.2 Nanosecond Pulse Generator A custom-designed high-voltage pulse generator was used to drive the PDP. The inductive adder-based pulse generator is able to generate up to 10-kV 100-ns pulses at a rate from single shot to 5 kHz. Current and voltage were measured with a current monitor (Pearson 2877) and a high voltage probe (Tektronix 6015A) in conjunction with a digital oscilloscope. A flow meter (Omega, FL-3839ST) delivers He or a premixed He/(1%)O 2 gas mixture to the PDP at flow rates in the range of 1 to 12 SLPM (standard liters per minute). Typical voltage and current waveforms of the plasma plume, excited with 6-kV, 100 ns electric pulses at 1 kHz and with premixed He/(1%)O 2 flow exiting the PDP nozzle at 5 SLPM, are shown in Figure 4.2. The pulsed current is a summation of a displacement current induced by the capacitance of the electrodes and a discharge current. Integrating the product of the voltage and current pulses gives values for the energy per pulse in the range of 1.2-1.8 mJ. The average power at the aforementioned conditions is 48 less than 2 W. The shape of the plasma plume strongly depends on the peak pulse voltage and the gas flow rate. At a constant gas flow rate, e.g., 5 SLPM, the length of the plasma plume increases from 1.0 to 2.8 cm when the peak voltage increases from 4 to 6 kV. When the peak voltage exceeds 7 kV, turbulence occurs in the plasma plume, the plume splits downstream, and its length decreases. Figure 4.2 Typical voltage and current pulse waveforms of He/(1%)O 2 plasma operating at 1 kHz and with a gas flow rate of 5 SLPM. 4.3 Images of Plasma Plume In addition, the visible light intensity of the plasma plume increases with voltage. At a constant peak pulse voltage, the plasma plume reaches a maximum length at a certain flow rate and becomes shorter when the flow rate is smaller or larger than the optimum. Figure 4.3 shows that the shape of the plasma plume changes with flow rates at a constant pulse voltage (6 kV peak voltages at 1 kHz). We have also investigated the 49 influence of pulse repetition rates on the plasma. The visible emission intensity increases with the pulse repetition rate (0.05 to 2 kHz) when other pulse conditions are the same. However, the length and the diameter of the plasma plume are not affected by the pulse repetition rate. In addition, the plasma can be sustained at single shot or higher repetition rates. With increasing pulse repetition rate (> 3 kHz), higher gas flow rates (> 5 SLPM) are required to generate cold and stable plasmas without local arcing. Figure 4.3 Images of the plasma plumes. Plasma plumes were generated from the PDP powered with 6-kV 100-ns 1-kHz pulses at different He/(1%)O 2 flow rates: (a) 1 SLPM, (b) 3 SLPM, (c) 6 SLPM, and (d) 12 SLPM. Turbulence was observed when flow rates are higher than 5 SLPM. The plasma plume reaches 3 cm long at a flow rate of 6 SLPM. 50 4.4 Cold Plasma for Bacillus atrophaeus Growth Inhibition and Experimental Setup The experimental setup, as shown in Figure 4.4, consists of a gas flow system, the custom pulse generator, and plasma exposure stage. The gas flow system delivers He or premixed He and 1% O 2 to the PDP at a controlled flow rate. Figure 4.4 Experimental setup for the B. atrophaeus growth inhibition treated with PDP on nutrient medium plates. (Left) A photograph of plasma plume impinging on B. atrophaeus on a nutrient agar plate. A series of sterilization tests was designed to investigate the plasma bactericidal effects and identify optimal treatment parameters with B. atrophaeus, a standard sterilization monitor bacterial species. Tryptic soy agar plates of 100 mm were spread uniformly with 0.2 mL of B. atrophaeus in tryptic soy broth at approximately 2.5 × 10 8 cells/mL. The agar plates were treated with both He and He/(1%)O 2 plasmas with varying plasma exposure time, peak pulse voltage, pulse repetition rate, gas flow rate, and spacing distance between the plasma nozzle and the agar surface. Two plates were subjected to each treatment condition to ensure repeatability. Controls were performed by 51 flowing the gas mixture onto the sample with the plasma generator switched off. After the treatment, all of the plates were incubated at 37 ºC for 24 h. For treatments with both He and He/(1%)O 2 plasmas, effective growth inhibition of B. atrophaeus was observed. A typical control result and ones under He and He/(1%)O 2 plasma treatment for different durations are shown in Figure 4.5. The plasma-treated area shows effective B. atrophaeus growth inhibition. With longer plasma exposure time (up to 180 s), a larger area of bacterial killing was observed [Figure. 4.5(C) and (F)]. Figure 4.5 Treatment with He and He/(1%)O 2 plasmas for different exposure times. B. atrophaeus on nutrient agar plates treated with (upper row) He and (lower row) He/(1%)O 2 plasmas for different exposure times: (a) He flow at 5 SLPM for 180 s with plasma off (the control); growth inhibition after (b) 60 seconds and (c) 180 seconds He plasma exposure; (d) He/(1%)O 2 flow at 5 SLPM for 180 s with plasma off (the control); growth inhibition after (e) 60 seconds and (f) 180 seconds He/(1%)O 2 plasma exposure. Other treatment conditions are constant: peak voltage = 6 kV, pulse repetition rate = 1 kHz, gas flow rate = 5 SLPM, and nozzle-sample spacing = 5 mm. For the same treatment condition, He/(1%)O 2 plasma shows a stronger bacterial inactivation effect—larger area of bacterial killing—than He plasma. Dependence of B. atrophaeus growth inhibition on pulse voltage was also observed for both He and 52 He/(1%)O 2 plasma treatment, as shown in Figure 4.6 Larger areas of bacterial inactivation were observed after treatment with 6 kV pulsed plasmas compared to 4 kV pulsed plasmas. At the same peak pulse voltage of 4 kV, treatment with He/(1%)O 2 plasma inactivates bacteria more extensively than He plasma, as shown in Figure 4.6(B) and (E). Effects of other parameters including gas flow rate, nozzle-sample spacing, and pulse repetition rate on the bactericidal effect were also observed. Figure 4.6 Treatment with He and He/(1%)O 2 plasmas at different peak pulse voltages. B. atrophaeus on nutrient agar plates treated with (upper row) He and (lower row) He/(1%)O 2 plasmas at different peak pulse voltages: (a) He flow at 5 SLPM for 180 s with plasma off (the control); growth inhibition after He plasma exposure at the peak voltage of (b) 4 kV and (c) 6 kV; (d) He/(1%)O 2 flow at 5 SLPM for 180 seconds with plasma off (the control); growth inhibition after He/(1%)O 2 plasma exposure at the peak voltage of (e) 4 kV and (f) 6 kV. The other treatment conditions are kept constant: pulse repetition rate = 1 kHz, gas flow rate = 5 SLPM, nozzle-sample spacing = 5 mm, and plasma exposure time = 180 seconds. Preliminary results show that, for an area density of 7 × 10 5 CFUs/cm 2 , > 99.9% killing of B. atrophaeus after plasma treatment (6 kV, 1 kHz, flow rate of 5 SLPM, and 5 mm nozzle-sample spacing) for 3 min was achieved for a defined area of 1.1 cm 2 , which is 9 times the plasma plume cross section. The interaction between the plasma and the 53 nutrient agar and its impact on the plasma bactericidal process are not completely understood. Plasma inactivation kinetics with bacteria supported with different bio- logical media needs to be studied. 4.5 Cold Plasma for Root Canal Disinfection Bacterial biofilms, consisting of bacteria covered by an extracellular polymeric matrix, are known to be a cause of numerous oral infections such as dental caries, periodontitis, and pulpitis [Costerton et al 1999]. They can resist intracanal disinfection procedures and hence are implicated in the failure of root canal treatments [Stewart et al 2001; Chávez de Paz et al 2005]. To approximate a worst case scenario, we applied the PDP to dense salivary biofilms cultured in root canals. Efficacy against this heavy bacterial concentration can be extrapolated to a reasonable assurance of success in disinfecting a thin smear layer that might be found in a real root canal. Before the treatment, standard endodontic instrumentation using files and irrigation was applied to two extracted teeth. Saliva was collected from a volunteer and incubated in Todd-Hewitt media for 24 hours at 37 ºC. After autoclaving (30 minutes, 121 ºC), the teeth were placed in a six-well plate and filled with 4-mL Todd-Hewitt media. The six- well plate containing the teeth was inoculated with 1 mL of pre-cultured saliva biofilm and incubated for four days at 37 ºC with daily change of media. For treatment, teeth were placed 5 mm below the PDP nozzle. The standard plasma treatment conditions as described earlier were applied to one of the teeth for 5 minutes. The other tooth, serving as the control, was subjected to He/(1%)O 2 at the same flow rate for the same amount of time, but with the plasma switched off. After the treatment, the treated and the control 54 teeth were split longitudinally and transversely with a dental burr. The tooth slices were then dehydrated in a graded ethanol line, critical-point dried with carbon dioxide, and mounted on a stub. The samples were sputter-coated with 25 nm platinum and examined with a scanning electron microscope (XL 30 S, FEG, Philips). SEM images of split-tooth roots revealing the root canals for each case were taken at different locations and with different magnifications (ranging from 33 × to 9000 ×). The typical SEM images for a control case (gas flow only while the plasma was off) are shown in Figure 4.7. For the control tooth, biofilms covered the entire root canal surface, as shown in Figure 4.7(A). A microcolony with predominant morphotypes of fusiform bacteria and cocci is shown in Figure 4.7(B). Figure 4.7 SEM images of control root canals. (A) SEM image of part of the split root canals after He/(1%)O 2 flow at 5 SLPM with plasma switched off for 5 min (control), and (B) a magnified SEM image of the saliva-derived biofilm on the canal. The biofilm is composed of fusiform bacteria (recognizable by their characteristic tapered ends) and cocci. In many cases, biofilms were observed to be thickly embedded in an exopolymeric matrix which protects the bacterial biofilms against disinfection agents. In the plasma- treated root canal, a visible contrast line distinguishes the zones for plasma-treated and non-plasma treated surfaces, as shown in Figure 4.8(A). The plasma-treated surface extends 1 mm into the root canal. A typical SEM image with a higher magnification 55 shows a predominantly clean surface revealing open dentinal tubules [Figure 4.8(B)] for this area. In the same canal, biofilms occupied the regions not reached by the plasma (the non-plasma-treated surface). Figure 4.8 SEM images of PDP treated root canals. (A) SEM images of the upper half of the root canal after He/(1%)O 2 plasma treatment for 5 min, revealing a distinct zone of clearance (1 mm in depth) with a visible line horizontally below which biofilms are still present where the plasma failed to reach, and (B) a SEM micrograph of the plasma treated root canal surface, revealing open dentinal tubules [16]. We have tested four other root canals in different tooth roots inoculated with endodontic biofilms with the plasma at different treatment parameters, including plasma exposure time and nozzle-tooth spacing. Although disinfection of biofilms in the root canals was observed consistently, the levels of biofilm removal (e.g., dispersal depth in the root canal, and root canal and dentinal tubule surface clearance) are different for different treatment parameters. Currently, we are in the process of optimizing plasma treatment parameters and probe nozzle designs to extend the plasma into the entire root canal. Importantly, this study represents a necessary first step in demonstrating the effectiveness of plasma in treating biofilm-contaminated surfaces such as root canals, with the potential for application to the treatment of many biofilm-mediated diseases. 56 4.6 Plasma Emission Spectroscopy Plasma emission spectroscopy was conducted to identify reactive chemical species contributing to the plasma sterilization process. With the plasma impinging on B. atrophaeus-spread agar plates under standard treatment conditions, emission spectra were scanned with a monochromator (Acton SpectraPro 300i) coupled with a photomultiplier tube (PMT; Hamamatsu, R7400P-01) at an optical resolution of 0.5 nm (grating:1200 lines/mm; slit width: 200 μm). A UV-visible lens system was used to focus the plasma emission into the entrance slit of the monochromator. Prior to the plasma emission measurement, the optical system was aligned and calibrated with a HeNe laser (JDS Uniphase1508P-0). The emission spectrum (Figure 4.9) in the 300-800 nm wavelength range was obtained for the He/(1%)O 2 plasma plume at 5 mm below the probe nozzle. Emission lines from excited nitrogen molecules, excited atomic oxygen, excited helium, and nitrogen ions were observed. Among them, atomic oxygen, the most reactive species, is able to inactivate cells and cause cell lysis by oxidation [Laroussi et al 2002]. OH emission was not observed. 57 Figure 4.9 Emission spectrum of the non-thermal atmospheric-pressure plasma. Plasma was powered by 1 kHz 6 kV 100 ns electric pulses. Spectral lines associated with excited and ionized species are indicated in the plot. Note the line at 777 nm associated with atomic oxygen, a highly reactive species. 4.7 Conclusion In this chapter, we have demonstrated a safe and novel technique for endodontic disinfection based on a nanosecond pulsed plasma dental probe. The handheld device offers flexible control of the length of the plasma plume up to 2.5 cm in ambient atmosphere. Due to the coaxial tubular design of the electrodes, the device generates negligible electromagnetic noise; the low EMI noise level allows a resistive 130 mA pulse peak current of the plasma plume, excited at 6 kV, to be measured (at SNR < 20%) with a Rogowski coil just below the device nozzle. Biological specimen temperatures did not exceed 35 °C even after 5 min of plasma exposure under ambient conditions. In our preliminary test, the plasma was successfully applied to an endodontic biofilm-inoculated root canal in a human tooth with complete biofilm removal in the region reached by the 58 plasma. Greater sterilization depth and surface coverage will be achieved by optimizing the width and length of the plasma plume. Growth of B. atrophaeus on nutrient agar plates is completely inhibited by exposure to both He and He/(1%)O 2 plasmas for 60 seconds. A 300-s treatment with the He/(1%)O 2 plasma disinfected a human tooth root canal colonized with saliva-derived biofilms, demonstrating the potential of the PDP as a simple, safe, and effective tool or adjunct for the disinfection of root canals during endodontic therapy. He/(1%)O 2 plasma is more effective than He alone for B. atrophaeus growth inhibition, and the emission spectrum of the He/O 2 plasma supports the identification of atomic oxygen as the likely primary reactive chemical species contributing to bacterial inactivation. The plasma emission spectrum suggests that atomic oxygen may be one of the plasma species contributing to the inactivation of bacteria. Other chemical species including O 3 and NO x may also play roles in the bactericidal effect, although no significant odors of O 3 or NO x were detected during the experiments. We are in the process of measuring the concentrations of these chemical species and the results will be reported in the future. In addition, effects such as surface detachment [Stoffels et al 2006] and electrostatic disruption [Mendis et al 2000] on bacterial membranes induced by the non-thermal plasma may also play roles in endodontic biofilm disinfection. Further investigation into plasma bactericidal mechanisms is currently under progress in our laboratories. 59 Chapter 5 pH-sensitive intracellular photoluminescence of carbon nanotube-fluorescein conjugates in human ovarian cancer cells In this chapter, to add to our understanding of the properties of functionalized carbon nanotubes in biological applications, we report a monotonic pH sensitivity of the intracellular fluorescence emission of SWCNT-FC conjugates in human ovarian cancer cells. Our results demonstrate that nanosized conjugates like SWCNT-FC have the potential to be used as localized pH i sensors and trackers in studies of uptake mechanisms, endocytotic degradation, and photo-induced phenomena in living cells. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. SWCNT-FC conjugates may serve as intracellular pH sensors. 5.1 Synthesis of carbon nanotubes-fluorescein (SWCNT-FC) conjugates HiPco SWCNTs (Carbon Nanotechnology Inc) were shortened and carboxylated by acid reflux treatment [Chen et al 1998; Hamon et al 2001; Kam et al 2006] to yield highly functionalized nanotubes about 200 nm long with carboxylic acid groups around the open ends and at defect sites in the sidewalls. The mole percent concentration of carbon atoms belonging to carboxyl groups in oxidized SWCNTs by this method has been reported to be between 3.6% and 8.0% [Kam et al 2006]. Oxidized nanotubes were reacted with fluorescein-5-thiosemicarbazide (FC) (Sigma Aldrich) and 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide (Sigma-Aldrich) to form covalent conjugates 60 through an amide linkage. SWCNT-FC conjugates were then filtered, rinsed thoroughly to remove unreacted materials, and re-dispersed in Dulbecco’s Phosphate Buffered Saline (PBS 1X) solution without calcium and magnesium salts (Irvine Scientific, Irvine, CA) to a SWCNTs concentration of 0.25 mg/mL. 5.2. Cell lines and fluorescence microscope White light and fluorescence microscopy were employed to evaluate the uptake of SWCNT-FC conjugates by SKOV-3 cells cultured in RPMI 1640 medium. The detail information of cell culture is in Appendix. Cells were incubated with solutions of: FC [0.025 mg/mL], SWCNT [0.025 mg/mL] mixed with FC [0.025 mg/mL], and SWCNT- FC [0.025 mg/mL] conjugates for 17 hours in RPMI 1640 culture medium. Cells were washed to remove extracellular nanotubes and dye molecules prior to fluorescence imaging. A Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY, USA) was used for imaging. Filter sets for SWCNT-FC 480 nm excitation and 540 nm emission, respectively. 5.3 Internalization of SWCNT-FC conjugates Figures 5.1(A) 1(B) and 1(C) compare the FC uptake levels by SKOV-3 cells incubated with free FC, SWCNTs mixed with FC, and SWCNT-FC conjugates, respectively. Unlike their unbonded counterparts where the uptake is minimal, SWCNT- FC conjugates were able to readily cross the cell membrane and exhibit strong fluorescence from the cytoplasm and intracellular organelles. The SWCNT-FC conjugates entry mechanism was evaluated by comparing uptake at low (4 °C) and 61 normal growth temperatures (37 °C). After 1 hour, only cells incubated at 37 °C (Figure 5.1(E)) show strong fluorescence due to internalized SWCNT-FC conjugates; no detectable emission was observed in cells incubated at 4 °C (Figure 5.1(D)). This result is consistent with an energy-dependent endocytotic pathway as the SWCNT-FC conjugates uptake mechanism [Schwartz et al 1995]. Figure 5.1 Internalization of SWCNT-FC conjugates in SKOV-3 human ovarian caner cells. Differential interference contrast (DIC) (upper panels) and fluorescence (lower panels) images of SKOV-3 cells incubated in RPMI 1640 for 17 hours with (A) FC, (B) a mixture of SWCNTs and FC, and (C) SWCNT-FC conjugates, and for 1 hour with SWCNT-FC conjugates at 4 °C (D) and 37 °C (E). Figure 5.2(A) depicts a SEM image of fixed SKOV-3 cells incubated with SWCNT-FC conjugates. Interestingly, nanotube-like structures were found across the cell membrane. The micro-Raman spectrum of one of these structures reveals a strong G- band peak at 1592 cm -1 , which is characteristic of SWCNTs (Figure 5.2(B)). In order to gather more information in regards to intracellular SWCNT-FC, the cell was chosen inside square on the left white-light image of Figure 5.2(C). Fluorescence images were taken at different focal planes along the z-axis. The distance between each focal plane is 1 µm. The resulting 3D fluorescence image is shown in Figure 5.2(D) with Zeiss 62 AxioVision 3.1 imaging software. Vesicle-like intracellular fluorescence emission sources were found to be localized in the perinuclear region in the cytoplasm. Figure 5.2 SEM and 3D z-stack images of intracellular SWCNT-FC in SKOV-3 cells. (A) SEM image of fixed intracellular SWCNT-FC SKOV-3 cells; (B) Micro Raman spectrum of intracellular SWCNTs showing a G band peak at 1592.4 cm-1; (C) Left: Optical microscopy image of SKOV-3 cell culture previously incubated with SWCNT-FC conjugates, Right: Fluorescence microscopy images taken at different focus planes in Z-direction from top to bottom. The inter plane distance is 1 μm; (D) Three-dimensional representation of the data collected in (D). 5.4 Photoluminescence of SWCNT-FC within cells Interestingly, we observed that intracellular SWCNT-FC conjugates exhibit a fluorescence emission enhancement after light stimulation. SKOV-3 cells incubated with SWCNT-FC were exposed for 2.5 seconds to the 480 nm band-pass-filtered emission of a mercury lamp (source power density = 0.04 W/m 2 ), after which fluorescence microscopy images were taken at 0, 5 and 10 minutes (Figure 5.3(A)). We integrated and averaged the fluorescence intensity of cells from each image in Figure 5.3(A). Comparison of the 63 averaged fluorescence intensity at 0 and 10 minutes reveals a 50% increase in fluorescence intensity (Date were analyzed in Figure 5.7). To further confirm that the intracellular fluorescence intensity increase was caused by light stimulation, we used a 63X objective to irradiate a small area 300 µm in diameter, and then we imaged a larger concentric area 1 mm in diameter with a 10X objective. Figure 5.3(B) clearly shows that there is a stronger fluorescence emission area which corresponds to the region irradiated with the 63X objective. The regions photoexcited later with the 10X objective show much lower fluorescent emission. This result demonstrates that exposing intracellular SWCNT-FC conjugates to light stimulation produces a strong increase in fluorescence intensity and suggests an intracellular photoactivated process that was sensed by the SWCNT-FC conjugates. To understand the nature of the observed photoactivation phenomenon, the time evolution of the intracellular SWCNT-FC fluorescence intensity after being photo-activated was further studied. We first photo-exposed SKOV-3 cells with intracellular SWCNT-FC conjugates for 2.5 seconds, then the cells were placed in fresh growth medium for 36 hours. After incubation, cells were separated into two samples, washed thoroughly and photo-exposed again for either 2.5 seconds (Figure 5.3(C)) or 1.5 seconds (Figure 5.3(D)). Note that figures 5.3(A) and 5.3(C) reveal that although the fluorescence intensity of SWCNT-FC conjugates increases with time in all areas within the cells, it becomes stronger within intracellular organelles than in the cytoplasm. This behavior may be attributed to the activation of a photo-stimulated process that affects the endosomal membranes. Disruption/perturbation of the endosomal membranes may allow an increase in the endosomal pH and/or facilitate the diffusion of the fluorescence emitters from organelles to the cytoplasm, where they find a higher pH 64 and therefore exhibit stronger fluorescence [Ma et al 2004]. Comparisons of figures 3(c) and 3(d) reveal decreased fluorescence intensity when the photo-activation time was reduced from 2.5 to 1.5 seconds (110% and 60%, respectively, after 10 minutes) (Date were analyzed in figure 7). Figure 5.3 Fluorescence images of intracellular SWCNT-FC in SKOV-3 cells (A) after 2.5 seconds light exposure (480 nm), (B) with 10 X objective after photoactivation of with 63 X objective, (C) after PBS washing and 2.5 seconds light exposure, and (D) after PBS washing and 1.5 seconds light exposure. We found strong background fluorescence in growth medium after SWCNT-FC- loaded SKOV-3 cells (light-stimulated (480 nm)) were incubated for another 36 hours (Figure 5.4(A)), although the cells themselves retained their fluorescence after replacement of the medium (Figure 5.4(B)). The presence of fluorescent material in the growth medium may be due to diffusion or expulsion of SWCNT-FC conjugates from the SKOV-3 cells to the external medium, or from endosomal hydrolysis of the conjugates that yields free and dequenched FC molecules which are then expelled to the extracellular medium. 65 Figure 5.4 Background fluorescence of SWCNT-FC in growth medium: (A) A strong background fluorescence in the medium from SKOV-3 cells containing intracellular SWCNTs after 36 hours incubation was observed. (B) Same cells after washing with PBS. 5.5 pH sensitivity of SWCNT-FC within cells To explain the observed photoactivation of SWCNT-FC in SKOV-3 cells, we considered the pH sensitivity of FC fluorescence. FC photoluminescence increases with pH monotonically over the pH range 3 to 10 [Ma et al 2004]. We confirmed this behavior for our SWCNT-FC conjugates by measuring the fluorescence intensity of aqueous solutions of SWCNT-FC conjugates at different pH values. Figure 5.5(A) shows that the fluorescence intensity of SWCNT-FC conjugates increases by two orders of magnitude from pH 4.1 to 7.0. An additional 20% increase occurs between pH 7.0 and 10.0. To investigate whether the intracellular photoluminescence of SWCNT-FC conjugates corresponds to changes in pHi, we used chloroquine to modulate pHi. Chloroquine is a weakly basic amine which concentrates in acidic intracellular compartments (lysosomes, Golgi complex) and increases the intracellular pH [Maxfield 1982; Oda et al 1985; Strous et al 1985; Oda et al 1986; Caplan et al 1987]. Figure 5.5(B) shows that the fluorescence intensity of intracellular SWCNT-FC conjugates increases after chloroquine is added to 66 the medium (60% increase after 10 minutes) (Date were analyzed in Figure 5.7). Moreover, chloroquine-treated, SWCNT-FC-loaded SKOV-3 cells photo-excited and imaged with 63X and 10X objectives display a uniform distribution of fluorescence emission intensity throughout the imaged sample (Figure 5.5(C)), contrary to what is observed for photoactivated samples without chloroquine (Figure 5.3(B)). Figure 5.5(D) shows fluorescence spectra of intracellular and extracellular SWCNT-FC conjugates before and after photoexcitation. Emission from extracellular SWCNT-FC conjugates is observed to decrease slightly (photobleaching) after excitation while photoexcited (480 nm) intracellular conjugates exhibit a strong fluorescence intensity increase. Figure 5.5 pH sensitivity of SWCNT-FC within SKOV-3 cells. (A) Normalized fluorescence intensity of free SWCNT-FC conjugates increases with an increasing pH value within the range of 4.1 to 10.0. Fluorescence images of intracellular SWCNT-FC in (B) chloroquine-treated SKOV-3 cells after 2.5 seconds light exposure (480 nm), (C) with 10 X objective after photoactivated with 63 X objective. (D) Fluorescence emission intensity of free SWCNT-FC conjugates decreases after 480 nm exposure (photo-bleaching), while fluorescence emission intensity of intracellular SWCNT-FC conjugates increases after 480 nm light exposure. 67 If SWCNT-FC internalization into SKOV-3 cells occurs via endocytosis, we may expect that the conjugates are localized in endosomal compartments, where they find a pH of around 6.0-6.5, before being delivered to lysosomes to be degraded. Lysosomes have an acidic internal pH of about 5.0 [Sipe et al 1987; Yamashiro et al 1987]. In this environment the fluorescence of internalized SWCNT-FC conjugates will be substantially quenched. On the other hand, for internalized SWCNT-FC conjugates to increase their emission intensity, i.e. to be exposed to a higher pH environment, they must either cross the endosomal or lysosomal membrane into the cytoplasm, or the pH of the lysosomal compartment must increase. SWCNT-FC in the cytoplasm sees pH values around 7, which would correspond to a two- or four-fold increase in fluorescence intensity for endosomal and lysosomal conjugates, respectively [Ma et al 2004]. Integration of the fluorescence intensities in Figure 5.3 reveals a two-fold intensity increase for all photoactivated samples after 10 minutes, which matches closely the predicted intensity increase of SWCNT-FC conjugates due to endosome-to-cytoplasm transfer (Date were analyzed in Figure 5.7) or an increase in vesicle pH. Although cationic conjugates possibly disrupt endosomal membranes [Ma et al 2004], unreacted carboxyl groups in the SWCNT-FC conjugates would make them anionic at intracellular pH values. 5.6 Hydrolysis of SWCNT-FC within cells An additional issue of concern in assessing the photoactivation process is the possibility of a photo-induced hydrolysis of the amide linkage between nanotubes and FC molecules. Fluorophores such as FC undergo strong fluorescence quenching when 68 conjugated to carbon nanotubes due to energy transfer by π- π interaction between the nanotube and the dye molecules [Wang et al 2004]. Therefore, hydrolysis of SWCNT-FC conjugates would in principle release de-quenched FC molecules, which in turn could also contribute to the photo-activated fluorescence intensity increase in SKOV-3 cells. Figure 5.5(D) shows fluorescence spectra of SWCNT-FC conjugates outside and inside SKOV-3 cells before and after photoexcitation. Emission from extracellular conjugates is observed to decrease slightly after photoexcitation while photoexcited intracellular conjugates underwent a strong fluorescence intensity increase. Hydrolysable amide bonds that undergo photocleavage to give a free carboxyl group in significant yields are almost unknown [Amit et al 1976]. Because the UV radiation required for amide photolysis falls in the range between 190-300 nm, therefore a pure photolysis of this bond during photoactivation (with light of 480 nm in wavelength) would not be favored. The fact that only intracellular conjugates yield a substantial fluorescence increase under light excitation clearly demonstrates the activation of one or several light-induced intracellular processes that may disrupt endosomal membranes and/or lead to the enzymatic hydrolysis of SWCNT-FC conjugates. Lysosomes carry hydrolases, which become active in the acidic intra-lysosomal environment and could cleave FC from nanotubes via hydrolysis. However, for large non-viral vectors, transfer from endosomes to lysosomes may be inhibited due to their size [Wattiaux et al 2000], therefore SWCNT-FC of comparable size may be hydrolyzed in endosomal compartments. This is consistent with the results shown in figure 5.3, for which the (two-fold) fluorescence increase matched better an endosome-to-cytoplasm rather than a lysosome-to-cytoplasm fluorophore transfer. To demonstrate that hydrolysis of SWCNT-FC conjugates could lead to 69 fluorescence de-quenching we measured the fluorescence of free FC and SWCNT-FC conjugates before and after hydrolytic treatment. Briefly, free FC and SWCNT-FC conjugates were mixed with a 1:2 mixture of acetic and hydrochloric acids in a sealed tube at 110 ºC for 48 hours, for amide bond cleavage [Christensen et al 1945]. Figure 5.6(A) shows that the fluorescence intensity remains unchanged for FC solutions after the hydrolysis process. De-quenching due to hydrolysis of SWCNT-FC conjugates resulted in a nearly two-fold enhancement in fluorescence intensity (Figure 5.6(B)), similar to the experimental data obtained with cells and consistent with the mechanisms proposed for the observations of internalized, photoexcited carbon nanotube conjugates in SKOV-3 cells. Figure 5.6 Fluorescence emission intensity measurements. (A) Fluorescence emission of an FC solution remains unchanged after acid hydrolysis. (B) Fluorescence emission intensity of an SWCNT-FC solution increases after hydrolysis. 70 Figure 5.7 Fluorescence intensity integration: Intracellular fluorescence intensity from SWCNT- FC SKOV-3 cells was integrated and averaged with Zeiss AxioVision 3.1 imaging software. (A) After 2.5 seconds light exposure (488 nm), (B) after PBS washing and 2.5 seconds light exposure, (C) after PBS washing and 1.5 seconds light exposure, (D) after 2.5 seconds light exposure with chloroquine added (figure 5 (B)). 5.7 Conclusion The monotonic pH sensitivity of SWCNT-FC conjugates allows us to monitor the fate of SWCNT-FC conjugates in SKOV-3 cells. We find that SWCNT-FC conjugates are more likely taken up by an endocytotic process, for which the conjugates remain in endosomes. Two hypotheses are proposed to explain such observations: First, dequenching of fluorescein fluorescence could result from light-stimulated intracellular hydrolysis of the amide linkage between SWCNTs and fluorescein molecules. Hydrolysis of endosomal SWCNT-FC conjugates yielding free de-quenched FC molecules; 71 permeation of the endosomal membrane to allow SWCNT-FC conjugates into the cytosol; neutralization of the acidic environment of the endosome. Second, light-induced intracellular pH changes may cause fluorescence intensity variations of the SWCNT-FC conjugates, since fluorescein is a pH-sensitive dye. Either of the above-mentioned processes, or both, would produce a fluorescence intensity increase upon light stimulation. Although an unambiguous explanation of the photoactivation process remains to be determined, we have demonstrated the potential use of SWCNT-FC conjugates as localized intracellular probes. 72 Chapter 6 Near-Infrared Induced Hyperthermia of Brain Cancer Cells with Carbon Nanotubes Glioblastoma Multiforme (GBM) is the most common and most aggressive of the primary brain tumors. It is notoriously successful at evading all types of treatments and the prognosis is poor, with the average individual surviving only 8 to 11 months after the tumor is identified [Holland 2000]. The primary therapy for most solid tumors is surgical resection, followed by a combination of radiation and chemotherapy. Radiation is the most effective therapy. However, normal brain can only tolerate a certain amount of ionizing radiation. GBM is characterized by a highly invasive potential. It divides at a very rapid rate and its cells readily spread to other locations in the brain by extensive infiltration, displaying a wide diversity of histological features and preventing the success of conventional treatment [Rust et al 2002; Deshane et al 2003]. In this chapter, we demonstrate the internalization of SWCNTs in U251 human GBM cells and astrocytes normal brain cells with SWCNT-FC conjugates described in chapter 5. Functionalized SWCNTs were delivered through endocytosis to both malignant and normal brain cells. The high thermal conductivity of SWCNTs is of significant interest in cancer therapy due to the strong absorption exhibited in the near infrared (NIR) region of the spectrum where human tissue and biological fluids are particularly transparent [König 2000; Shi Kam et al 2005]. Once within the cell interior, the hyperthermic effect of SWCNTs with near infrared (NIR) laser exposure of 700-1100 nm wavelength are used to kill the cells containing intracellular SWCNTs. Propidium Iodide (PI) was employed as an indicator to 73 determine the number of cells with compromised plasma membrane. This study reveals new insights upon the influence of SWCNTs optical properties in the physicochemical processes within cells and how they can be used to develop therapy for cancerous brain tumors. 6.1 Cell lines and fluorescence microscope White light and fluorescence microscopy were employed to evaluate the uptake of SWCNT-FC conjugates by U251 human GBM cells cultured in DMEM medium. Human astrocytes and the astrocyte culture medium, in which astrocytes were grown, were purchased from ScienCell Research Laboratories. The detail information of cell culture is in Appendix. A Zeiss Axiovert 200 M fluorescence microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY, USA) was used for imaging. Filter sets for SWCNT-FC 480 nm excitation and 540 nm emission, respectively. 6.2 Near infrared laser setup Typical exposure times were varied from 1-10 min and intensities were in the range of 0.8 to 1.5 W/cm 2 . NIR irradiation effect on GBM cells was monitored by Propidium Iodide (PI) staining. Laser diode (SDL 2372, JDS Uniphase Corporation, CA, and USA) with wavelength (808 ± 3 nm) was chosen to irradiate cancer cells in this chapter. The output of the diode laser is a divergent beam, which diverges at 32 degree perpendicular to the junction and 12 degree parallel with the junction. In order to achieve a homogeneous intensity distribution at the sample, a 10X objective was first placed in front of the laser to focus the beam. The laser beam profile was then smoothened by a 74 ground glass diffuser (transmission ~70%). A plano-convex lens was used to collimate the output beam. The laser beam was finally delivered to the sample by a 45° folding mirror highly reflective at near-infrared range. The setup is shown in Figure 6.1. The resultant laser intensity can be as high as 1.5 W/cm 2 . After laser irradiation, the cells were incubated with PI (final concentration 5 μM) at 37°C to check the cell viability. Figure 6.1 Diagram of near-infrared laser setup. The divergence of the output beam from the laser diode is reduced first by the objective, and then by the plano-convex lens. The resultant slowly-diverging beam is then directed to the sample by a mirror. Typical beam size at the sample location is about 7 mm. The intensity on the cells can reach 1.5 W/cm 2 . 6.3 Absorption spectrum measurement of SWCNTs Optical absorption spectrum of HiPco SWCNTs is characterized by discrete inter band transitions related to van Hove singularities in their density of states [Connell et al 2002]. Figure 6.2(A) shows an absorption spectrum of sodium dodecyl sulfate (SDS) 75 encapsulated HiPco SWCNTs in aqueous suspension. This spectrum reveals the second van Hove transitions of the semiconducting nanotubes (S22) in the 600 to 900 nm region of the spectrum while the first band gap transitions for metallic nanotubes are seen in the 400 to 600 nm range. Carbon nanotubes have a high absorption cross section. Once excited, they can radiatively relax to their fundamental state. Radiative relaxation requires fundamentally individual and semiconducting nanotube with weak inter-tube interactions. However, presence of approximately one third metallic nanotubes in a typical sample plus frequently present nanotube bundles can lead to non radiative decay times in the sub-picoseconds and picoseconds ranges for metallic and semiconducting nanotubes, respectively [Berciaud et al 2007]. This makes non radiative decay an important relaxation route for excited SWCNTs aqueous dispersions. Figure 6.2 Optical and thermal properties of SWCNTs: (A) Absorption spectrum of a HiPco SWCNTs suspension in aqueous SDS solution. (b) Temperature profile of HiPco SWCNTs aqueous dispersions versus exposure time to NIR radiation. Figure 6.2(B) shows the temperature profile of two different nanotube dispersions when exposed to a continuous wave optical source in the NIR region. Temperature profile of deionized water under identical irradiation conditions is used as a reference. 76 SWCNTs dispersed in deionized water exhibited a temperature increase of approximately 10 °C after 30 seconds of continuous irradiation and reached a saturation temperature increase of 14 °C after 2 minutes. On the other hand, temperature of an aqueous solution of DNA-wrapped nanotubes of similar concentration increased around 27 °C after irradiating for 30 seconds, and reached a constant increased temperature of 35 °C after 2 minutes. These results are consistent with heat generation due to non radiative decay and a magnified thermal conductivity from the carbon nanotube dispersions due to percolation effects [Huxtable et al 2003]. Lower heat generation by SWCNTs may be related to lower absorption cross section due to their lower structural conjugation. A model structure of a SWCNT-FC conjugate is depicted in Figure 6.3(A). As can be seen, SWCNT-FC contains outfacing functionalities that makes them soluble in water. Figure 6.3(B) shows an image of the actual aqueous solution employed in experiments. In order to test the importance of SWCNTs in translocating the cell membrane we incubated U251 human GBM cells with FC and SWCNT-FC conjugates. Results from these experiments are shown in Figures 6.3(C) and 6.3(D). Figure 6.3(C) shows a representative fluorescence image taken from GBM cells after being incubated in the presence of FC. This image reveals that FC molecules were unable to efficiently stain GBM cells. On the other hand, fluorescence image of U251 human GBM cells after being incubated in presence of SWCNT-FC conjugates (Figure 6.3(D)) showed efficient fluorescence emission when excited with a light source of 480 nm. These results confirm the effectiveness of carbon nanotubes as highly efficient intracellular transporters of molecules that otherwise could not penetrate across the cell membrane. 77 Figure 6.3 Translocation of SWCNT-FC conjugates across U251 human GBM cell membrane. (A) Model structure of a SWCNT-FC conjugate showing the presence of hydrophilic groups on SWCNTs sidewalls. (B) Optical image of a SWCNT-FC solution. Fluorescence image of U251 human GBM cells incubated in the presence of FC (C) and SWCNT-FC (D). 6.4 Internalization of SWCNT-FC conjugates to U251 human GBM and astrocytes normal brain cells Z-stack images are shown in Figure 6.4. Successive fluorescence images were taken at different planes of cells in the Z direction in intervals of 2 µm to cover a total vertical distance of 18 µm. Images were then combined to make a 3D representation of the localized fluorescence signal displayed by GBM cells. Figure 6.4(A) and (D) shows a white light image of U251 human GBM cells and astrocytes normal brain cells which were incubated with SWCNT-FC conjugates, respectively. Figure 6.4(B) and (E) presents fluorescence images at different focal planes from top to bottom corresponding to the cell marked on Figure 6.4(A) and (D), respectively. Single-plane fluorescence images on 78 figure 6.4(B) and (E) show that the fluorescence signal seen in U251 human GBM cells and astrocytes normal brain cells are not localized in the outside of the cell membrane but distributed all along the vertical direction in the cytosol. These results suggest as a solid evidence of the presence of intracellular SWCNT-FC conjugates in U251 human GBM cells and astrocytes normal brain cells. Figure 6.4(C) and (F) show 3D images constructed from Z-stack images of Figure 6.4(B) and (E), respectively. Close observation of the distribution of intracellular nanotubes suggests that SWCNT-FC conjugates seem to localize in the surrounding region outside the nucleus. Figure 6.4 Intracellular detection of SWCNT-FC conjugates. (A) White light image of U251 human GBM cells mixed with SWCNT-FC conjugates. (B) Single plane fluorescence images corresponding to the marked cell on part A. Numbering corresponds to different planes in the Z direction from top of the cell to the bottom. (C) 3D image showing localized fluorescence detection from inside U251 human GBM cells. (D) White light image of astrocytes normal brain cell mixed with SWCNT-FC conjugates. (E) Single plane fluorescence images corresponding to the marked cell on part D. Numbering corresponds to different planes in the Z direction from top of the cell to the bottom. (F) 3D image showing localized fluorescence detection from inside astrocytes normal brain cell. 79 6.5 Cell viability examination with Propidium Iodide U251 human GBM cells and astrocytes normal brain cells with intracellular SWCNTs were exposed to near infrared (NIR) radiation of 808 nm in wavelength. Typical exposure times were varied from 1 to10 minutes and intensities were in the range of 0.8 to 1.5 W/cm 2 . PI is a membrane-impermeant nucleic acid stain with a broad emission spectrum that exhibits a maximum at 620 nm. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20 to 30 folds. PI can be used to detect necrotic cells in culture when the cell membrane is disrupted, leaks into the cell and binds to DNA and RNA, so only necrotic cells fluoresce red [Cevik et al 2003]. Therefore, we employed PI as a dead cell indicator for cells under NIR exposure. Figures 6.5 shows white light images combined with fluorescence images of cells without and with intracellular SWCNTs respectively after NIR exposure for 9 minutes. Red fluorescence indicates PI uptake which indicates the dead cells. Figure 6.5(A) and (C) shows that only ~5 % of U251 human GMB cancer cells and astrocytes normal brain cells without intracellular SWCNTs have a compromised membrane due to NIR exposure, respectively. On the other hand, Figure 6.5(B) and (D) reveals a highly extended necrosis in which only 10 to 20 % of U251 human GBM cells and astrocytes normal brain cells with intracellular SWCNTs survived. With no intracellular SWCNTs, both U251 human GBM cells and astrocytes normal brain cells can survive NIR exposure for 9 minutes. Most U251 human GBM cells and astrocytes normal brain cells with intracellular SWCNTs were killed after 9 minutes NIR exposure. These observations confirm a much higher damage to cells with than without intracellular SWCNTs under the same NIR exposure. The remarkable difference in results for these 80 samples provides strong evidence on the capability of SWCNTs, provided selectivity, to be used as an efficient therapy for GBM brain tumors. Figure 6.5 NIR irradiation of U251 human GBM cells and astrocytes normal brain cells with and without intracellular SWCNTs. (A) and (B) White light combined with fluorescence images after 9 min exposure to NIR radiation of U251 human GBM cells without and with intracellular SWCNTs, respectively. (C) and (D): White light combined with fluorescence images after 9 min exposure to NIR radiation of astrocytes normal brain cells without and with intracellular SWCNTs, respectively. 6.6 Conclusion We demonstrate that SWCNTs can transport covalently attached molecules such as FC, across normal and brain tumor cell membranes. A cell surviving ratio of 10 to 20% was found for both normal and brain tumor cells with intracellular SWCNTs after only 9 minutes exposure to NIR radiation. Taking advantage of the fact that SWCNTs absorb 808 nm NIR laser radiation much more strongly than brain tissue, we demonstrated 81 conditions under which only cells with intracellular SWCNTs are killed under near- infrared laser exposure. Ongoing work is aimed to target selectivity of SWCNTs moieties towards GBM brain tumor cells. It is obvious that the next generation of therapeutic agents needs to be designed to target the discriminating destruction of cancer cells but not of normal cells. 82 Chapter 7 Brain Cancer Cell Migration Reduction with Single-Walled Carbon Nanotubes Life expectancy for a patient with an aggressive glioblastoma is one year or less. In this chapter, we propose a new brain tumor therapy by using SWCNTs to reduce or stop the multiplication, migration, and invasion of brain cancer cells into the surrounding tissue. This therapy has the potential to increase patient survival time and improve their quality of life. The cytotoxicity of SWCNTs has not yet been clearly defined, and is a topic of active research. One report shows that SWCNTs can reduce the viability of osteoblasts [Zhang et al 2007], but other studies demonstrate that well-prepared SWCNTs without remaining amorphous carbon, nickel, iron, and other heavy metals are not cytotoxic [Worle-Knirsch et al 2006]. However, a selective targeting method of SWCNTs towards brain tumor cells can reduce the concern of cytotoxicity of SWCNTs. In Chapter 8, vicrostatin (VN), a brain cancer-targeted ligand and pharmacological agent, is proposed to be functionalized with SWCNTs as a combination therapy for anti-invasion. The removal of the SWCNTs from the tumor area can be solved by bio-compatible helium-oxygen plasma which can dissolve SWCNTs possibly by activated atomic oxygen. 7.1 Preparation of water-solubilized SWCNTs High-pressure carbon monoxide-derived (HiPco) SWCNTs (Carbon Nanotechnology Inc.) are water-solubilized by dispersing them in deionized water with 83 the surfactant sodium dodecylbenzene sulfonate, then filtering and re-dispersing them in phosphate buffer solution (PBS) to a SWCNT concentration of 0.25 mg/mL, as previously described [Islam et al 2003]. Irrigating the cavity remaining after tumor excision with the SWCNT suspension will inhibit migration of any remaining tumor cells into healthy tissue. 7.2 Cells lines preparation SRB12 squamous cell carcinoma cells, Rat C6 GBM cells and U251 human GBM cells were grown in DMEM medium. The detail information of cell culture is in Appendix. All the cells were grown at 37 ºC in a humidified, 5% CO 2 atmosphere. Before an experiment, the SRB12, U251 human GBM, and rat C6 GBM cells were detached with 0.05% Trypsin/0.53 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium (Cat. No. 25-052-CL, Cellgro, Herndon, VA) and washed with DMEM growth medium. 1 mL of SRB12, U251 human GBM, and rat C6 GBM cell suspensions (1×10 6 cells/mL) was added to appropriate wells of a 24-well culture dish, and the cells were incubated until they reached 80% confluence (about 12 hours). 7.3 SWCNTs-induced cell clustering effect When SWCNTs [25 µg/mL] are added to SRB12 squamous cell carcinoma cells, we observe aggregation and clustering of the cells and a reduced ability of the cells to attach to and migrate over a substrate in Figure 7.1. This phenomenon is observed in various cell lines such as U251 human GBM cells, rat C6 GBM cells, SKOV-3 human ovarian cancer cells, U87 GBM cells, brain cancer stem cells. The SWCNTs adhere to 84 the cell membrane and are internalized. The aggregation and clustering of cells may be facilitated by the hydrophobicity of the SWCNTs. Figure 7.1 Aggregation and clustering effects of SRB12 cells. (A) Detached SRB12 cells without intracellular SWCNTs spread homogeneous and start to attach to the substrate of culture plates within 2 hours. At the mean time, (B) SRB12 cells with intracellular SWCNTs tends to aggregate together to form a cluster without attaching to the substrate of culture plate. 7.4 Intracellular SWCNTs-induced cell migration reduction effect Furthermore, to understand if SWCNTs can affect the migration rate of rat C6 and U251 human GBM cells. We mix rat C6 and U251 human GBM cells with [25 µg/mL] SWCNTs for 24 hours in 24-well culture plates. Make ~800 µm gaps in the monolayers by scratching the attached cells with 100 µL pipette tips. Image the same location at 0 hours, 8 hours, 20 hours, and 32 hours for each condition. Our experiments (three times) show that carbon nanotubes can significantly reduce the migration rate of rat C6 GBM and U251 human GBM cells (Figure 7.2 and 7.3). 85 Figure 7.2 SWCNTs induced migration reduction effect of rat C6 GBM cell monolayer. (A) Without intracellular SWCNTs, rat C6 GBM cell monolayer migrates about 190 and 350 µm after 8 and 16 hours, respectably. (B) With intracellular SWCNTs, the migration of rat C6 GBM cells reduces to 80 and 230 µm after 8 and 16 hours, respectively. Figure 7.3 SWCNTs induced migration reduction effects of in U251 human GBM cell monolayer. (A) Without intracellular SWCNTs, U251 human GBM cell monolayer migrates about 140 and 400 µm after 8 and 16 hours, respectably. (B) With intracellular SWCNTs, the migration of U251 human GBM cells reduces to 70 and 200 µm after 8 and 16 hours, respectively. 86 7.5 Conclusion The migration speed of rat C6 and U251 human GBM cells mixed with SWCNTs is about half that of untreated cells. This migration reduction effect is expected to be observed in other cell lines. The inhibition of brain cancer cell migration is still not clear and is under investigation. 87 Chapter 8 Future work In this chapter, future works are proposed. Some results of the continuous researches are undergoing and will be published soon in the future. 8.1 Lipid peroxidation in living cells promotes membrane electropermeabilization In order to optimize pulsing protocols for specific applications such as gene and drug delivery or tumor ablation, a better understanding of the mechanisms of nanoelectropulse permeabilization and of the factors affecting the susceptibility of cells to nanoelectropulse exposure is needed. Our group continues the research on the effect of membrane oxidation, an important variable in healthy and diseased tissues and in laboratory cultures of different cell types, on electropermeabilization sensitivity. Molecular dynamics studies have shown that oxidized lipids increase the frequency of water defects in phospholipid bilayers [Wong-Ekkabut et al 2007], suggesting that peroxidation of cell membranes may facilitate nanoelectropermeabilization. To investigate this hypothesis, human Jurkat T lymphoblasts are treated with ferrous sulfate and hydrogen peroxide to peroxidize membrane lipids through the Fenton reaction. After peroxidation, cell suspensions are placed in electroporation cuvettes and exposed to nanoelectropulses in medium containing YO-PRO-1 to see if the cell membrane is permeabilized. In addition to these experimental observations our group applies electric fields during molecular dynamics simulations of phospholipid bilayers containing varying concentrations of oxidized lipid species. Molecular dynamics results are consistent with the experimental results, showing that systems with higher concentrations 88 of oxidized lipids form hydrophilic electropores in significantly shorter times and at lower electric fields than do systems with lower oxidized lipid concentrations. 8.2 Cold Plasma Jets for Microbial Disinfection A new designed PDP powered with nanoelectropulses generates a 2.5 cm long plasma plume with 1 mm diameter in width at ambient atmosphere. In our recent research, growth inhibition of six microorganisms, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus atrophaeus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, on nutrient agar plates have been observed after exposure He/(1%)O 2 plasmas for 180 seconds. Log reduction values of all treated microorganisms were measured to be above 4 (99.99% bacterial reduction). These results demonstrate the potential of the PDP as a simple, safe, and effective tool for the biomedical and dental disinfection. 8.3 Specific targeting of brain tumor cells with functionalized SWCNT-VN conjugates SWCNTs functionalized with brain cancer-targeted ligands and pharmacological agents such as vicrostatin (VN), a recombinant version of contortrostatin [Swenson et al 2005], could be used in combination therapy with SWCNTs for anti-invasion. Vicrostatin targets the cell membrane integrins and the actin cytoskeleton, and inhibits glioma cell migration (Pyrko). Vicrostatin also induces glioma cytotoxicity. Linkage of SWCNTs to VN would facilitate SWCNTs binding and uptake by brain cancer cells. Tumor cell killing by the VN and reduction of subsequent tumor cell migration by SWCNT-VN into normal brain tissue would make this agent a doubly effective adjunct to surgical tumor removal. 89 8.4 Post-operative removal of SWCNTs by Pulsed Atmospheric-Pressure Cold Plasma Removal of the SWCNTs from the tumor area could be a concern. We recently find that bio-compatible helium-oxygen plasma can dissolve SWCNTs in a few seconds to several minutes. This atmospheric pressure, room-temperature plasma, which also serves as a disinfectant, can be used to remove the SWCNTs residue in the recession area after surgery. A plasma surgical probe using this technology delivers a tapered cylindrical plasma plume approximately 2 cm long and 2 mm in diameter. The plasma is generated in ambient atmosphere with 4-6 kV, 100 ns electric pulses at repetition rates up to 2 kHz when a flow of He or He/(1%)O 2 mixture exits the probe nozzle at 1-6 SLPM. 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No. 30-2002), 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA), 45 units/mL penicillin (Gibco, Carlsbad, CA), and 45 µg/mL streptomycin (Gibco, Carlsbad, CA). II Cell Lines Catalogue Types Cell Lines Source Culture Medium Bovine Adrenal chromaffin cells Note 1 RPMI 1640 medium Rat C6 GBM cells ATCC CCL-107 DMEM medium Human Jurkat T lymphoblasts cells ATCC TIB-152 RPMI 1640 medium Human PC3 prostate cancer cells ATCC CRL-1435 DMEM medium Human U251 GBM cells ATCC RCB-0461 DMEM medium Human keratoacanthoma skin, mixed morphology cells ATCC CRL-7630 DMEM medium Human SKOV-3 ovarian cancer cells ATCC HTB-77 RPMI 1640 medium Human Astrocytes normal brain cells Note 2 DMEM medium Human SRB12 squamous cell carcinoma cells Note 3 DMEM medium Table A.1 Cell lines catalogue Note 1, Chromaffin cells were isolated from fresh bovine adrenal medullas using the method described by Waymire et al. [Waymire et al 1983] and maintained in suspension culture as previously described [Hassan et al 2002]. Note 2, Human astrocytes and the astrocyte medium in which astrocytes were grown, were purchased from ScienCell Research Laboratories. Note 3, SRB-12 cell line was derived from cells taken from an epidermal lesion on a patient undergoing skin cancer treatment at the hospital of University of Southern California.
Abstract (if available)
Abstract
This work investigates the modification of biological materials through the applications of modern nanosecond pulsed power, along with other forms of nanotechnologies. The work was initially envisaged as a study of the effect of intense nanosecond pulsed electric fields on cancer cells. As the work progressed, the studies suggested incorporation of additional technologies, in particular, cold plasmas, and carbon nanotubes. The reasons for these are discussed below, however, they were largely suggested by the systems that we were studying, and resulted in new and potentially important medical therapies. Using nanosecond cold plasmas powered with nanosecond pulses, collaboration with endodontists and biofilm experts demonstrated a killing effect on biofilms deep within root canals, suggesting a fundamentally new approach to an ongoing problem of root canal sterilization. This work derived from the application of nanosecond pulsed power, resulting in effective biofilm disinfection, without excessive heating, and is being investigated for additional dental and other medical applications. In the second area, collaboration with medical and nanotube experts, studies of gliomamultiforme (GBM) led to the incorporation of functionalized carbon nanotubes. Single-walled carbon nanotube-fluorescein carbazide(SWCNT-FC) conjugates demonstrated that the entry mechanism of the single-walled carbon nanotubes(SWCNTs)was through an energy-dependent endocytotic pathway. Finally, a monotonic pH sensitivity of the intracellular fluorescence emission of SWCNT-FC conjugates in human ovarian cancer cells suggests these conjugates may serve as intracellular pH sensors. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. The use of SWCNTs for cancer therapy of gliomas, resulting in hyperthermia effect after 808 nm infrared radiations, absorbed specifically by SWCNTs but not by biological tissue.
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Creator
Chen, Meng-Tse
(author)
Core Title
Studies of nanosecond pulsed power for modifications of biomaterials and nanomaterials (SWCNT)
School
Viterbi School of Engineering
Degree
Doctor of Philosophy
Degree Program
Materials Science
Publication Date
04/22/2010
Defense Date
09/21/2009
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
brain tumor therapy,carbon nanotubes,cold plasma,migration reduction,nanosecond pulsed power,OAI-PMH Harvest,pH sensor,root canal disinfection
Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Gundersen, Martin A. (
committee chair
), Goo, Edward K. (
committee member
), Zhou, Chongwu (
committee member
)
Creator Email
mengtsec@usc.edu,mongao5429@hotmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m2681
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UC1163091
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etd-Chen-3275 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-272365 (legacy record id),usctheses-m2681 (legacy record id)
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272365
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Chen, Meng-Tse
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texts
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University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
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Libraries, University of Southern California
Repository Location
Los Angeles, California
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cisadmin@lib.usc.edu
Tags
brain tumor therapy
carbon nanotubes
cold plasma
migration reduction
nanosecond pulsed power
pH sensor
root canal disinfection