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Effects of aspirin and its derivatives in combination with electroporation for drug delivery in cultured cells

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Title:
Effects of aspirin and its derivatives in combination with electroporation for drug delivery in cultured cells
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Book
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English
Creator:
Langham, Jennifer
Publisher:
University of South Florida
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Tampa, Fla.
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Subjects

Subjects / Keywords:
Acetylsalicylic acid
Acetic acid
Surface active drug
Membrane permeability
Salicylic acid
Melanoma cells
Dissertations, Academic -- Biomedical Engineering -- Masters -- USF   ( lcsh )
Genre:
government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The purpose of this research was to investigate the effects that aspirin (ASA) and its metabolites, salicylic acid (SA) and acetic acid (AA), have on the delivery of drugs across biological barriers when used in conjunction with electroporation. Electroporation is a technique used to enhance drug delivery across bio-membranes in which a transmembrane potential is induced into cellular membranes, resulting in the creation of aqueous pores that allow molecules to pass through the otherwise impermeable barrier. Aspirin is a widely used drug that has been used for over a century and has been proven relatively safe at normal doses as indicated by the low number of reports of poisoning cases it has been involved in. Components of aspirin are known to soften the cellular membranes by solubilizing the cell's surface proteins.
Thesis:
Thesis (MSBE)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Jennifer Langham.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains 59 pages.

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aleph - 001681131
oclc - 62793324
usfldc doi - E14-SFE0000614
usfldc handle - e14.614
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ABSTRACT: The purpose of this research was to investigate the effects that aspirin (ASA) and its metabolites, salicylic acid (SA) and acetic acid (AA), have on the delivery of drugs across biological barriers when used in conjunction with electroporation. Electroporation is a technique used to enhance drug delivery across bio-membranes in which a transmembrane potential is induced into cellular membranes, resulting in the creation of aqueous pores that allow molecules to pass through the otherwise impermeable barrier. Aspirin is a widely used drug that has been used for over a century and has been proven relatively safe at normal doses as indicated by the low number of reports of poisoning cases it has been involved in. Components of aspirin are known to soften the cellular membranes by solubilizing the cell's surface proteins.
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Effects of Aspirin and its Derivatives in Combination with Electroporation for Drug Delivery in Cultured Cells by Jennifer Langham A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering Department of Biomedical Engineering College of Engineering University of South Florida Major Professor: Mark J. Jaroszeski, Ph.D. William E. Lee, Ph.D. Michael D. Vanauker, Ph.D. Date of Approval: July 1, 2004 Keywords: acetylsalicylic acid, acetic acid, surface active drug, membrane permeability, salicylic acid, melanoma cells Copyright 2004 Jennifer Langham

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i TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT v CHAPTER 1: INTRODUCTION 1 1.1 Introduction to Drug Delivery by Electroporation 1 1.2 Aspirin and Its Derivatives 6 1.2.1 Properties of Salicylic Acid 7 1.2.2 Properties of Acetylsalicylic Acid 8 1.2.3 Properties of Acetic Acid 9 CHAPTER 2: RESEARCH GOALS 11 CHAPTER 3: MATERIALS & METHODS 13 3.1 Cell Preparation 13 3.1.1 Cell Line and Growth 13 3.1.2 Counting 14 3.1.3 Well Seeding 14 3.2 Electrode 15 3.3 Testing Conditions and Experimental Protocol 17 3.4 Fluorescence Measurements 19 3.5 Membrane Recovery 19 3.6 B16F10 Cell Size Measurements 20 3.7 pH Measurements 21

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ii 3.8 Statistical Methods 21 CHAPTER 4: RESULTS & CONCLUSIONS 22 4.1 Effects of Salicylic Acid on Calcein Delivered by E.P. 22 4.2 Effects of Acetylsalicylic Ac id on Calcein Delivered by E.P. 23 4.3 Effects of Acetic Acid on Calcein Delivered by E.P. 25 4.4 Membrane Recovery 27 4.5 B16F10 Cell Sizes Post Treatment 29 4.6 Conclusions and Discussion 30 REFERENCES 32 APPENDICES 36 Appendix A: Data Collected For Salicylic Acid Experiments 37 Appendix B: Data Collected For Acetylsalicylic Acid Experiments 41 Appendix C: Data Collected For Acetic Acid Experiments 45 Appendix D: Data Collecte d for Membrane Recovery 49 Appendix E: B16F10 Cell Sizes Post Treatment 52

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iii LIST OF TABLES Table A.1 Salicylic Acid Delivery: Experiment 1 37 Table A.2 Salicylic Acid Delivery: Experiment 2 38 Table A.3 Salicylic Acid Delivery: Experiment 3 39 Table A.4 Salicylic Acid Delivery: Average of 3 Experiments 40 Table B.1 Acetylsalicylic Acid Delivery: Experiment 1 41 Table B.2 Acetylsalicylic Acid Delivery: Experiment 2 42 Table B.3 Acetylsalicylic Acid Delivery: Experiment 3 43 Table B.4 Acetylsalicylic Acid Deli very: Average of 3 Experiments 44 Table C.1 Acetic Acid Delivery: Experiment 1 45 Table C.2 Acetic Acid Delivery: Experiment 2 46 Table C.3 Acetic Acid Delivery: Experiment 3 47 Table C.4 Acetic Acid Delivery: Average of 3 Experiments 48 Table D.1 Membrane Recovery after 1 Hour 49 Table D.2 Membrane Recovery after 2 Hours 49 Table D.3 Membrane Recovery after 3 Hours 50 Table D.4 Membrane Recovery after 4 Hours 50 Table D.5 Membrane Recovery after 24 Hours 51 Table E.1 B16F10 Cell Sizes 52

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iv LIST OF FIGURES Figure 1.1 Transmembrane Potential Induced in a Spherical Cell by an External Electric Field 2 Figure 1.2 Chemical Structures of (a) Sa licylic Acid (b) Acetylsalicylic Acid and (c) Acetic Acid 7 Figure 3.1 Untreated B16-F10 Cells in McCoy’s Media (100X) (Left) and in PBS (200X) (Right) 13 Figure 3.2 6-Prong Electrode Design 15 Figure 3.3 6-Needle Pulsing Seque nce used for Energizing Cells 16 Figure 3.4 Demonstration of Electrical Treatment 18 Figure 3.5 Fields of View for One Well at 400 Magnification 20 Figure 4.1 Electroporated Delivery with Sa licylic Acid: Mean and S.E.M. of 3 Experiments 23 Figure 4.2 Electroporated Delivery with Acetylsalicylic Acid: Mean and S.E.M. of 3 Experiments 25 Figure 4.3 Electroporated Delivery with Acetic Acid: Mean and S.E.M. of 3 Experiments 27 Figure 4.4 Membrane Recovery of Cells after Exposure: Mean and S.D. of 3 Experiments 28 Figure 3.5 Vertical and Horiz ontal Axis of B16F10 Cells 30

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v EFFECTS OF ASPIRIN AND ITS DERI VATIVES IN COMBINATION WITH ELECTROPORATION FOR DRUG DELIVERY IN CULTURED CELLS Jennifer Langham ABSTRACT The purpose of this research was to inve stigate the effects that aspirin (ASA) and its metabolites, salicylic acid (SA) and acetic acid (AA), have on the delivery of drugs across biological barriers when used in c onjunction with electropor ation. Electroporation is a technique used to enhance drug de livery across bio-membranes in which a transmembrane potential is induced into cellula r membranes, resulti ng in the creation of aqueous pores that allow molecules to pass th rough the otherwise impermeable barrier. Aspirin is a widely used drug that has been used for over a century and has been proven relatively safe at normal doses as indicated by the low number of reports of poisoning cases it has been involved in. Components of aspirin are known to soften the cellular membranes by solubilizing the cell’s surface proteins. B16F10 murine melanoma cancer cells were used in this investigation and treated with a 120M buffered solution of calcein, a fl uorescent indicator, in which the amount of delivered tracer molecules was measured using fluorescence. Identical concentrations of ASA and SA were investigated (1mM, 5mM, and 10mM) sepa rately, focusing the effects concentration has el ectroporation delivery. Dilu ted acetic acid was also

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vi investigated at pH values of 6.42, 5.36, and 4.40. The concentr ation of acetic acid that had the lowest pH and ASA with the highest concentration had the greatest impacts on the augmentation of calcein delivery. Therefore, this demonstrates th at aspirin and acetic acid have the potential to im prove targeted molecular delivery in combination with electroporation.

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1 CHAPTER 1: INTRODUCTION 1.1 Introduction to Drug Delivery by Electroporation Electroporation (E.P.) is a phenomenon in which biomembranes can enter a state of reversible electrical breakdown (REB) which allows external molecules to pass through the otherwise impermeable lipid phase of the cell me mbrane. REB is typically induced by applying an electrical potential to the cells which results in a temporary destabilization of the cell membrane and permeation through aqueous-filled pore openings [1, 2]. The transmembrane potenti al needed to rupture through a membrane bilayer is referred to as th e electroporation threshold [3]. Although the mechanism by which this phenomenon occurs is not understood in its entirety, it is known that E.P. is the direct result of an indu ced transmembrane potential th at is caused by the applied electric field [4]. Figure 1.1 shows the induced transmembrane potential in a cell exposed to electroporation. Pores form in regions of the membrane where the induced potential is large. Membrane resealing is a crucial phenomenon of electroporation. The level of membrane recovery is dependent on the amplitude and length of the applied pulses because there is a critical range in wh ich the cell can undergo lysis, often referred to as irreversible membrane permeabilization [5]. The transient aqueous pore theory describes the combined effects of the electric field across a membrane and the associated thermal fluctuations [1, 2]. During the destab ilization period, the ce ll membrane is highly permeable to exogenous molecules pr esent in the surrounding media.

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2 Figure 1.1 Transmembrane Potential Induced in a Spherical Cell by an External Electric Field [4] There are several parameters that affect electroporation as well as the recovery of the membrane such as temperature [6], waveform parameters, the composition of the bilayer membrane and ionic a nd osmotic conditions [7, 8]. One of the most important parameters is the electric field strength, E, wh ich is inversely proporti onal to the distance between the electrodes, d, with an applied voltage, V, (E = V/ d ). As the voltage is increased, the induced transmembrane potential is raised [4]. The result is the creation of pores and pore expansion due to structural alte rations. In the case of an isolated spherical membrane, this phenomenon is described by the derived equation [9, 10]: (t) = 1.5E(t) a cos where is the induced tran smembrane potential, is the angle between the applied electric field and the site on the membrane at which the potential is measured, and a is the radius of the cell. Once the membra ne becomes porous, further increase in is counteracted by the ionic current through the pores [4].

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3 Another critical parameter is the pulse time which is derived from the total circuit resistance, R, and the capac itance, C, between the electrodes ( = RC). The pulse time is a major indication of how long the pores will stay opened. For mammalian cells, optimal pulse times have been found to range from tens of microseconds to milliseconds [11, 12]. Joule heating occurs as electrical current is applied and is linearly related to the square of the field intensity a nd pulse duration [13]. This cond ition is easier to control in in vitro studies by using a low ionic cont ent pulsing buffer, whereas in in vivo studies, local heating is more difficult to avoid; therefore, by applyi ng shorter pulse lengths, this effect can be minimized. Electroporation has the potential to be us ed in biotechnological applications both in vivo and in vitro Though many in vivo applications originate from in vitro experimentation, there are a variety of in vitro examples that capital ize on the effects of electroporation. These include loading gene tic material and pharmaceutical drugs into cells, membrane protein insertion, and clinical applications such as cell-cell and celltissue fusion. Electroporation of tissue has recently become popular in the medical field due to its many possible applications such as cancer tumor thera py [14, 15], localized gene therapy [11, 16-18], and tr ansdermal drug delivery [19, 20]. Electrochemotherapy is a novel therapy approach to enhance delivery of nonpermeant chemotherapeutic agents to the cyto sol of cells, with the assistance of locally applied electric fields to te mporarily destabilize cell membrane s. This form of therapy has shown to be effective in clinical trials for the treatment of tumors including melanoma, head and neck squamous cell carcinoma, basal cell carcinoma, Kaposi’s sarcoma and adenocarcinoma [14, 15].

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4 Gene therapy is a rapidly growing biotec hnological field in wh ich a gene delivery problem exists. The development of gene transfer methods (both in vivo and in vitro ) include either the biological delivery approach of viral vect ors or non-viral methods that involve chemical techniques such as lipofect ion [21], or physical techniques such as electroporation and gene gun tran sfection. Viral vectors are co nsidered the most widely used method for transfection [9] but are often associated with immune responses; therefore, continued research in non-viral techniques is es sential in order to enhance efficiency. In one study designed to compare the efficiency of in vivo, non-viral gene transfer methods, electroporation was found to be as successful and promising as lipofection, gene gun and direct DNA injection methods [11]. Transdermal delivery of drugs is yet anot her widely explored topic of in vivo electroporation applications. Th is route of delivery is a usef ul alternative to the several conventional routes of administration such as orals or injectables because it avoids degradation in the gastrointestinal tract and first-pass hepatic metabolism. Metoprolol, (a beta blocker) used in the treatment of a ngina, has shown successful results in a study investigating the mechanisms behind improved transdermal drug delivery by electroporation [19]. Reversible skin permeability, electr ophoretic movement of the drug into the skin with applied pul ses, and drug release from the skin reservoir as a result of electroporation are examples of mechanisms th at link the linear corr elation between pulse voltage and the quantity of drug delivered [4, 20]. Examples of in vivo electroporation applications in clude clinical treatments not only for humans, but for animals also. In a recent study [22], plasmid DNA encoding mycobacterial antigens was injected into the muscles of farm animals that are frequently

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5 infected with Mycobacterium bovis. This bacterium causes bovine tuberculosis. It has been estimated that the economic losses due to tu berculosis in infected cattle alone is $3 billion annually. The DNA vaccine delivered by electroporation was reported to protect against bovine TB. The results from the st udy revealed an increas ed humoral immune response in goats and improved T-cell res ponses in cattle after the DNA vaccine was delivered using E.P. When compared to an imals that did not receive electroporation, the vaccine was much more successful. Unfortunately, there are undesirable side effects that are contributed to highvoltage permeation such as irreversible cellula r damage [23]. Troiano et al [24] showed that by altering the cell’s memb rane, excessive damage to the cell may be prevented. The authors of the study added the nonionic surfactant, octaethyleneglycol mono n -dodecyl ether (C12E8) to absorb into a lipid bilayer me mbrane and found that using various concentrations of the surface active agent al lowed electroporation at lower intensities and/or shorter pulse durations to reduce the electroporation threshold. Surfactants are typically used in pharmaceuticals to provide increasing distribution and penetration to the cell’s membrane by influenc ing the rate and extent of absorption of certain drugs. Though it has been shown that high concentrations of surfactants promote toxicity to the cell’s st ructure by causing lysis a nd other irreversible damage to the membrane, Lee et al [25] ha s provided evidence that supports the use of the nonionic surface active agent poloxamer 188 (P188) in restoring electrical damage and resealing of electropermeabilized cells. Another group of scientists [26] concluded that co-injection of P188 with plasmid DNA help ed facilitate gene tr ansfer in skeletal

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6 muscles, which supports one application of using surfactants as an excipient for intramuscular delivery of ions or water so luble species includi ng drugs or naked DNA. Just as surface-active agents are capabl e of solubilizing cell membranes, many pharmaceutical agents share the same phenomena by interacting with membranes due to their physiochemical properties. Amphi philic, or hydrophobic, molecules undergo various mechanisms of self-association w hose main site of action is by rapidly permeating the plasma membrane and/or ac cumulating in the hydrophobic interior of the lipid bilayer. They have been reported to self-associate and bind to the membranes first, causing disruption and solubilization, very si milar to the action of detergents [27]. Amphiphilic compounds contain either an ionic or non-ionic polar head group with a hydrophobic portion. These propert ies allow the compounds to organize themselves as micelles or bilayers. Th eir molecular shape a nd hydrophilic-lipophilic balance (HLB) are factors that determine thei r tendency to form other structures which often is a function of pH, temperatur e, ionic strength, a nd concentration. 1.2 Aspirin and Its Derivatives The structure of aspirin and two of its derivatives, acetic acid and salicylic acid, are shown in Figure 1.2. Aspirin and sali cylic acid are hydrophobic compounds. When aspirin is exposed to water or moisture it will begin to hydrolyze, resulting in salicylic acid and acetic acid. The rate of hydrolysis that aspirin may undergo once dissolved in a basic solution is second order because it is dependent not only on the aspirin concentration, but on the pH of the solution [28].

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7 Figure 1.2 Chemical Structures of (a) Salicylic Acid (b) Acetylsalicylic Acid and (c) Acetic Acid [29] 1.2.1 Properties of Salicylic Acid Salicylic acid is a crystalline organic carboxyl ic acid that is derived from the bark of the willow tree. Willow bark had been used for centuries in folk medicine in certain parts of the world as a pain relief treatment. It is soluble in alc ohol, but only slightly soluble in water. It was thought to be too tough on the stomach, therefore it was reacted with acetic anhydryde as a buffering agent. In 1897, the German chemist and employee of Friedrich Bayer & Co., Felix Hoffman, was the first to market the buffered form of salicylic acid called acetyls alicylic acid [30]. Salicylic acid is thought to act by solubilizing the cell proteins that keep the stratum corneum intact, resulting in desquama tion. Salicylic acid and its derivatives are toxic when consumed in large amounts, but is a popular choice as a beta hydroxy acid for acne-prone skin care, especially those with se nsitive skin. Mixtures composed of 1-2%, aids in effectively penetrating and exfoliati ng the pores in the skin due to its larger

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8 molecular size compared to its cousin, alpha hydroxy acid. At concentrations of 3-6%, it can begin to promote the skin to scale or pe el off. A 50% concentration is used to eradicate warts and calluses, due to its tendenc y to destroy tissue at higher strengths [31]. 1.2.2 Properties of Acetylsalicylic Acid Acetylsalicylic acid, commonly known as aspi rin is a derivative of salicylic acid. In the formation of ASA, the hydroxyl group of SA reacts with acetic anhydride, with sulfuric acid as a catalyst. In this cond ensation reaction, aspirin and acetic acid are the products. Repeated recrystallization is an effective purification method for impurities such as unreacted sa licylic acid [29]. Aspirin is the acetate ester of salicylic acid, therefore it will hydrolyze to form acetic acid and salicyli c acid. Both products are present to some extent in aspirin and a measure of their concentration can be indica tive of the extent of hydrolysis, which will occur when aspirin is added to basic solutions with a pH of greate r than 7.4 [20]. Its solubility in PBS (pH 7.2) is at least 3 mg/mL and is recommended to use within 30 minutes of preparation [28]. There is a wide variety of uses for aspi rin, and is continuously investigated for more uses. Some indications include, but not limited to, th e use as an analgesic, antiinflammatory, antipyretic, anticoagulant and antirheumatic. It re duces fever by causing the blood vessels in the skin to dilate, allo wing heat from the body to leave more rapidly [30]. It is also used as an additive in f ood, animal feed, drugs and cosmetics, and is now the active ingredient in more than 50 over-the-counter preparations.

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9 1.2.3 Properties of Acetic Acid Acetic acid is a clear, colorless chem ical compound that is responsible for the characteristic odor and sour taste of vinegar. Glacial, or ice-like, acet ic acid has a boiling point of 118C, with a density of 1.049 g/ml at 25C, and a flash point of 39C. Its freezing point is 16.7C, slightly lower than room temperature. It is classified as a weak acid due to its ability to not dissociate into its component io ns when dissolved in aqueous solutions. Highly concentrated solutions of acetic acid are extremely corrosive, which can result in burns if contact ed with skin surfaces [32]. Acetic acid is a derivative of aspirin, as mentioned earlier, and has several other pharmaceutical indications for both adults and children. When used topically, it can be useful for treating fungal infections, wound care (0.25% to 20% concentrations), iontophoreisis (2% to 5% concentrations ), in diagnostic testing (3% to 5% concentrations) such as duri ng a colposcopy or cervicoscopy, and for the use of otitis externa (for adults and pediatrics). At concen trations of 2%, acetic ac id is often used as a household disinfectant, and is an effec tive method for cleaning and disinfecting respiratory equipment used in the care of cystic fibrosis. Acetic acid is also often used for urinary tract irrigations at a concentrati on of 0.25% [32]. A high-dose intra-tumoral acetic acid injec tion, for the treatmen t of hepatocellular carcinoma (HCC), is another form of acetic acid therapy. This method of injecting 3 to 5 ml of a 50% concentration solution has shown to effectively treat fewer than 4 small (< 3cm diameter) lesions. This treatment has b een investigated for ove r a decade and when compared to a similar method, percutaneous ethanol injection (PE I) therapy, the acetic

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10 acid injection has shown to be superior in that it has a lo wer recurrence rate of lesions (8% vs. 37%) and a higher 2-year survival rate of patients (92% vs. 63%) [33, 34].

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11 CHAPTER 2: RESEARCH GOALS It is clear that electropora tion is an established and e ffective method of delivering molecules to cells. E.P. has proven to affect cellular membranes in many in vivo and in vitro biotechnological applications, which incl ude treatments in humans and animals alike. The need for research to enhance th e delivery of drugs, genetic material, vaccines, and other molecules beyond the established capab ilities of electroporation is essential in the medical field. The benefits of electr oporation come with some undesirable side effects. Irreversible damage to tissues and cells occur as a result of electri cal stimuli. The use of surfactants in combination with electroporation delivery has been explored because it can allow for lower electr ical thresholds and s horter pulse durations. The addition of low concentrations of su rfactants has also shown to enhance the permeation of molecules through cellular membranes. It has even been reported that certain surfactants can help restore electrica l damage to membranes. Similar properties of surfactants exist in some pharmaceutic al drugs categorized as amphiphilic, or hydrophobic compounds. These drugs have the pote ntial to assist in the treatment of electroporation by enhancing the disruption of membranes and the formation of aqueous pathways, resulting in more e fficient molecule delivery. The purpose of this study was to determine the effects that aspi rin and two of its derived components have on the delivery of molecules through the cell membrane alone

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12 and in combination with electr oporation treatment. Concentra tion and pH are factors that were investigated as well as the extent of recovery and swelling of cell membranes. The results are intended to provide insight to the potential of improving molecular delivery by electroporation. This study was designe d specifically to determine if: ASA or 2 of its derivatives could faci litate molecular uptake through the cell membrane ASA and its derivatives could augment electroporation mediated delivery of nonpermeant molecules cell membranes recover from treatment w ith ASA and its derivatives alone and when coupled with electroporation.

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13 CHAPTER 3: MATERIALS & METHODS 3.1 Cell Preparation 3.1.1 Cell Line and Growth B16-F10 mouse melanoma cells (ATCC #: CRL-6475) were obtained and grown in McCoy’s Media (Cellgro Mediatech, In c., Herndon, VA), adjusted to contain 10% fetal bovine serum (Cellgro Mediatech, Inc.) and (25mg) gentamicin 50mg/mL (Mediatech, Inc.), and were incubated in 5% CO2 at 37C. Serum was essential for many reasons. It provided hormonal factors that s timulated cell growth and function. It also contains essential proteins, amino acids, mine rals, and lipids [35]. Gentamicin is an antibiotic used as a preventative measur e to help reduce microbial growth and contamination. Figure 3.1 Untreated B16-F10 Cells in McCoy’s Media (100X) (Left) and in PBS (200X) (Right)

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14 B16F10 cells grow exponentially as a dherent monolayers (Figure 3.1), and required fluid renewal and/or sub-culturing every 2 to 3 days using 0.25% trypsin-EDTA (Sigma Chemical Co., St. Louis, MO) for detachment. 3.1.2 Counting After harvesting, the cells were washed 3 times by centrifugation. Cells were centrifuged (5810R, Eppendorf, Westbury, NY) at 220 g for 5 minutes at 37C and resuspended in approximately 2.5 ml of PBS fo r each wash. A sample of cells was then diluted in 0.9% sodium chloride (APP, Schaumburg, IL) and 0.4% trypan blue stain (Cellgro Mediatech, Inc.). Trypan blue is an indicator in which the membranes of nonviable cells are penetrated and can be distinguished from the viable cells. Using a hemacytometer (Hausser Scientific, Horsham, PA), the viable and nonviable cells were counted. The number of cells per milliliter was computed by multiplying the number of cells counted per square millimeter th e dilution (when used) 10,000 (conversion factor). Only those cultures that resulted in 80% to 100% viability were used for experimentation. 3.1.3 Well Seeding Experiments in this study were conducte d using 48-well tissue culture plates (BD Falcon, Franklin Lakes, NJ) made from polys tyrene. Each well held approximately 1.4 ml of liquid with a surface area of 0.75 cm2. The depth and diameter of each well were 18 mm and 10 mm, respectively.

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15 From experimentation, it was found that pre-treatment of the wells with a 0.1% gelatin (Acros, NJ, USA; Geel, Belgium) film coat reduced looseni ng of the cells after electrical stimulation. U nder aseptic conditions, 150 l of the sterile gelatin solution was added to each well and let stand for 1 hour. The gelatin was then aspirated from each of the wells and air dried for 5 minutes. 500l of McCoy’s Media was added to the treated wells in addition to 7.5 104 viable cells and incubated at 37C for 18 hours before treatment. 3.2 Electrode Gilbert et al [36] designed and compared several innovative el ectrodes for use in electrochemotherapy treatment of murine B16 melanoma tumors. They found that a 6needle array was the most successful when tested in vivo measured statistically by a 97.1% complete response rate. Figure 3.2 6-Prong Electrode Design The electrode shown in Figure 3.2 was speci ally designed for all experimentation in this thesis and was simila r to the design mentioned above. The electrode consisted of

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16 6 stainless steel electrodes, equally spaced at 60 intervals around a 0.7 cm diameter circle. The needles extended 1.8 cm from the electrode body to fit precisely in the wells in order to set flush against the treatment/cell growth surface. Cells in each well were treate d with a total of 12 DC pulses. Figure 3.3 illustrates the sequence pattern used to energize specific needles, where each small circle represents the location of each needle. Pulses were di rected to each needle by a rotary switch. Needles 1 & 2 were negative (represented as da rk circles) for the first two consecutive pulses, whereas opposing needles 4 & 5 (repres ented as lightly shaded circles) were positive. This pattern was repeated for the next two pulses except the pattern was rotated one-needle clockwise. This clockwise rota tion preceded each set of two pulses and was repeated until 12 pulses were delivered. This sequence was designed to treat 360 of the cell growth surface. Figure 3.3 6-Needle Pulsing Sequen ce Used for Energizing Cells [36]

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17 3.3 Testing Conditions & Experimental Protocol A hot water bath (Isotemp 105, Fisher Sc ientific, Hampton, NH) was used during the treatment of the cultures to maintain ce ll viability and was set at 50C. A sheet of Plexiglas was cut to fit over the water bath and was used as a surface for maintaining an approximate temperature of 37C. All solu tions used were allowed to reach room temperature (~22C) to preven t thermal shock to the cells. The absolute amplitude for the DC pulse generator (Transfector 800; BTX, Inc., San Diego, CA) ranged from 0-970 V, which co rresponds to electric field intensities delivered between 0-1385 V/cm. The pulse wi dth was set within optimal conditions at 99 sec (maximum). Using short electrical pulses for cellular manipulation has the advantage of resulting in neglig ible thermal heating [12]. Each set of treatment conditions was test ed in triplicate wells. 18 hours after seeding plates, each well was treated individually by aspirating the media, and quickly adding 150 l of solution, just enough to co at the bottom of th e well. For those conditions that required electr ic current, the electrode was pl aced down in the well so that the prongs set flush on the bottom to minimi ze any movement of the electrode (Figure 3.4). Electrical current was th en applied in the manner that was described in Section 3.2.

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18 Figure 3.4 Demonstration of Electrical Treatment After application of the pulse s, cells were incubated for 1 hour at 37C. This time allowed membrane resealing before the cells were carefully washed with 500 l aliquots of PBS three times. Liquid from each wash was carefully and thoroughly aspirated. Then, each well was filled w ith 500 l of PBS. The presence of delivered calcein was obs erved under the microscope with the aid of a fluorescence microscope. A 0.9% sodium dodecyl sulfate (SDS) solution was then used to lyse the cells after the completion of the washing. SDS is a detergent that dissolves hydrophobic molecules, therefore when cells are incubated with an SDS solution, the membrane proteins and lipids de nature and solubilize. 250 l of the SDS solution was added to each of the wells contai ning PBS to yield a final concentration of 0.3%. The contents of each well were transferred to a 5 ml FACS tube (BD Falcon). An additional 500 l of PBS was added to each tube to increase sample volume. All samples were then centrifuged at 220 g for 5 minutes at 37C.

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19 3.4 Fluorescence Measurements Individual readings of each sample we re taken using a fluorescence spectrometer (Perkin-Elmer LS-3B, Oakbrook, IL). The op timal excitation and emission wavelengths for calcein were found to be 488 and 515, respec tively. The readings were made in a 1 cm2 quartz cuvette, using a sample volume of 1 ml. 3.5 Membrane Recovery The membrane integrity of the cells after electroporation treatment is an essential factor in determining the applications in wh ich this procedure will be utilized. It is typically desired to preserve, as much as possi ble, the original cellular structures of these living membranes in order to obtain optimal pos t-treatment results. As mentioned earlier, cellular membranes can undergo REB in which the biological structures will eventually return to their normal state. Twenty four hours post-treatment, the fate of the cells can typically be determined. For the purpose of this study, optimal te sting conditions were used to treat B16F10 cells to conclude the ex tent of reversible permeation. Due to the large surface ar ea of the wells, the cells were physically counted by observing 7 out of 21 fields of view along th e horizontal diameter of each well at a magnification of 400 (see Figure 3.6). The r ecovery of the membranes after exposure to each treating condition was expressed as a perc entage of live cells remaining after each hour.

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20 Figure 3.5 Fields of View for One Well at 400 Magnification. The shaded circles represent the fields of view selected for counting In order to distinguish the viable cells from the dead, a 0.4% trypan blue solution in PBS was used. 150l of the solution was a dded to each well for 1 minute, and then the cells were counted using the described method. Each test condition was studied at 1 hour intervals ranging from 1 hour to 4 hours. In addition, membrane recovery was examined 24 hours post treatment. 3.6 B16F10 Cell Size Measurements The size and shape of the cells may alter when exposed to solutions that will penetrate the membranes during electroporation. Therefore so me samples in this study were further investigated in or der to determine the extent of change that occurred due to membrane swelling. Due to the variety of sh apes the cells can ta ke under the conditions of E.P., sizes of two axis were collected from cells for each treatment condition, the long axis (horizontal) and short axis (vertical). A stage micrometer was used to calibrate the

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21 reticle in the eyepiece of the microscope to a standardized measurement scale at each magnification under a white light, where each un it on the reticle is c onfigured to measure in micrometers. 3.7 pH Measurements The three concentrations of ASA, SA and AA had pH values that were determined. The pH measurements were ma de using a pH meter (Colloidal Dynamics, AZR2, Sydney, Australia), whic h was calibrated using 3 soluti ons with known pH values. The pH values of each solution were 4, 7, and 10. 3.8 Statistical Methods To determine the statistical relevance of the data, a criterion for considering the mean data of one treatment condition as more successful than another had to be established. The null hypothesis used was that no change took place when considering one set of treatment parameters over another, and the alterna tive hypothesis used was that a significant change took place that resulted in different m ean fluorescence values. The method used to test the null hypothesis was a two-tailed paired sample t test, with a level of significance of = 0.05. If the computed t -score was in between the critical values, then the null hypothesis would be accepted, whereas if the t -score was a value that lied outside of the critical value parameters, ther e would be significant evidence to allow for a conclusion that the treatments differed in their effectiveness.

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22 CHAPTER 4: RESULTS & CONCLUSIONS 4.1 Effects of Salicylic Acid on Calcein Delivered by E.P. The first set of experiment s conducted in this investig ation focused on the effects of salicylic acid on the delivery of calcein to B16F10 cells using electroporation. Three identical experiments were carried out. Fi gure 4.1 shows mean sp ectrofluorometric data for these experiments. All cells were expos ed to 120 M calcein. In addition, cells in some samples were exposed to one of three di fferent concentrations of SA (1 mM, 5 mM, or 10 mM) with pH values of 6.70, 6.32, and 3.85, respectively. Pulses with one of four different field strengths we re also applied (500, 750, 1000, or 1385 V/cm) to certain samples. SA concentration and field streng th were experimental variables; however, some cells were not exposed to applied fields or SA for comparison /control purposes. The results demonstrated that when cells were exposed to calcein and the three concentrations of SA (no app lied electric field), SA did not augment the delivery when compared to samples exposed to calcein alone. Overall, the data for samples treated with calcein and either 1, 5, or 10 mM SA and elec trical pulses were not significantly higher in fluorescent magnitude than any of the samples that were treated with calcein and pulses (no SA). Therefore, this suggests that SA is not a viable candidate for enhancing electroporative deliv ery of calcein.

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23 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 0 mM SA & Calcein1 mM SA & Calcein5 mM SA & Calcein10 mM SA & CalceinTreatmentFluorescence E500 V/cm 750 V/cm 1000 V/cm 1385 V/cm Figure 4.1 Electroporated Delivery with Salicylic Acid: Mean and S.E.M. of 3 Experiments 4.2 Effects of Acetylsalicylic Acid on Calcein Delivered by E.P. The second set of experiments conducted in this investigation focused on the delivery of calcein using aspirin. The treatmen ts were similar to those in section 4.1 in that three concentrations of aspirin were us ed (1 mM, 5 mM, or 10 mM) with pH values of 6.39, 5.40, and 4.40, respectively. The same range of pulsing conditions was used and the variables were identical for the sample treatment. Figure 4.2 shows the mean data from three replicate experiments. The data indicates that exposure to 1 mM, or 5 mM ASA (alone) did not result in a significant increase in fluorescence magnitude when compared to the treatment of 120 M calce in alone (no ASA). In addition, samples treated with 1 and 5 mM ASA that were al so exposed to electric fields had mean fluorescence magnitude that were not significantly different from those samples that were

PAGE 31

24 exposed to electric fields alone (no ASA). In contrast, samples that were exposed to calcein in 10mM ASA (no pulses) had an average fluorescent magnitude of 4.28 (SEM = 0.63) and samples that were exposed to calcei n (no ASA or pulses) had mean fluorescent magnitude 0.83 (SEM = 0.05). This 5-fold incr ease in internalized calcein that resulted from exposure to 10mM ASA was statistically significant when compared to the control sample (p < 0.0001). Figure 4.2 also demonstr ates that 10 mM AS A assisted in the delivery of calcein at each of the four electr ic fields. As the applied field was increased, the fluorescence remained at an elevated magnitude relative to samples that were exposed to 10mM ASA (alone) and to samples that were exposed to same electri c fields alone. At 500 V/cm (and 10mM ASA), there was a 3.7-fo ld increase in fluorescence magnitude relative to samples treated w ith calcein at 500 V/cm (no ASA). These same increases at 750, 1000, and 1385 V/cm were 3.82, 3.57, and 3.18-fold, respectively. The mean data with samples treated at each field were si gnificantly different from analogous samples that did not include ASA. The p-values of these comparisons were < 0.0001 (500 V/cm), 0.0011 (750 V/cm), 0.0014 (1000 V/cm), and 0.0066 (1385 V/cm). Therefore, these data clearly show that 10 mM ASA alone can augmen t calcein delivery alone to the exterior of cells and also enhance delivery as a result of electroporation.

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25 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 0 mM ASA & Calcein 1 mM ASA & Calcein 5 mM ASA & Calcein 10 mM ASA & CalceinTreatmentFluorescence E500 V/cm 750 V/cm 1000 V/cm 1385 V/cm Figure 4.2 Electroporated Delivery with Acetylsa licylic Acid: Mean and S.E.M of 3 Experiments 4.3 Effects of Acetic Acid on Calcein Delivered by E.P. The third set of experiments included the i nvestigation of calcein delivery by E.P. using acetic acid as an adjuvant. The set of 3 replicate experiments included three concentrations of AA, each with a differ ent pH (4.40, 5.36, or 6.42). The pH values of the three solutions of acetic acid were ma tched to those of ASA for the purpose of determining if AA is the derivative in ASA th at enhances the delivery of calcein. The three concentrations of the AA solutions in PBS were similar, which was about 0.15%, and varied slightly in order to establish the desired pH value. In addition, one of three electric field strengths were applied to cel ls (500, 750, or 1000 V/cm). Some cells were

PAGE 33

26 not exposed to applied fields or any concen tration of AA for purposes of comparison and control. Samples treated with acetic acid faile d to show a significant increase in fluorescence magnitude at higher pH (5.36 or 6.42) regardless of whether or not pulses were applied, when compared to samples not exposed to AA. However, Figure 4.3 shows that acetic acid had a tremendous effect on the delivery of calcein, using a low pH solution (pH 4.40). The data for samples trea ted with AA alone (no pulses with a mean fluorescence magnitude of 1.04) was significantly different in mean fluorescence when compared to control samples that were not treated with AA or pulses (mean fluorescence magnitude of 8.57). This was an 8.2-fold increas e. When electric fields were used in combination with the lowest pH solution of AA, fluorescent data was even higher than when this concentration of AA was used alone (no pulses). The data for samples treated with AA and electrical pulses was significantly higher than any of the samples that were tr eated with calcein and pul ses (no AA). At the lowest applied field, 500 V/cm, acetic acid augmented the resulting fluorescence magnitude by 3-fold. The mean fluorescence magnitudes were 12.77 for samples treated with AA (pH 4.40) and 4.19 for the samples that were not exposed to AA. This difference was significant (p < 0.0001). For the 750 V/cm samples, this same increase was 2.25-fold and was statistically significan t (p = 0.0002). Similar ily, there was a 3.5fold increase for the 1000 V/cm samples. This increase was also sign ificant (p < 0.0001). As a result of this data, a low pH ( 4.40) solution of acetic acid is clearly a candidate to augment calcein for optimal E.P. delivery of calcein in B16F10 cells at any

PAGE 34

27 of the three electric fields, though it is ofte n desired to apply lowe r field strengths to minimize the possibility of cellular damage. 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Calcein onlyAA (pH 6.42) & Calcein AA (pH 5.36) & Calcein AA (pH 4.40) & CalceinTreatmentFluorescence E500 V/cm 750 V/cm 1000 V/cm Figure 4.3 Electroporated Delivery with Acetic Acid: Mean and S.E.M of 3 Experiments 4.4 Membrane Recovery Acetic acid was chosen as the optim al for this study, therefore, further investigation of the effects it had on cell me mbranes was carried out. An ideal method of delivery would result in cell membranes that are intact after the method was performed. The trypan blue test was used to determine membrane integrity as trypan blue dye will only penetrate into cells that have porous membranes. The electric field strength of 750 V/cm was applied to triplicate wells cont aining PBS alone as well as acetic acid (pH.4.40) alone. In addition, for comparison, individual treatments of PBS and acetic

PAGE 35

28 acid (pH 4.40) without electrical pulses were investigated. The protocol used was similar to the methods described for the delivery of cal cein, after one hour of exposure at 37, the cells were washed three times with 500 l ali quots of PBS. Liquid from the last wash was carefully aspirated, and then fi lled with 500 l of growth media. 70.0 75.0 80.0 85.0 90.0 95.0 100.0 1 hr2 hrs3 hrs4 hrs24 hrs Time (Post-Treatment) % Memrane Recovery PBS, EPBS, E+ 750 V/cm AA, EAA, E+ 750 V/cm Figure 4.4 Membrane Recovery of Cells after Exposure: Mean and S.D. of 3 Experiments Figure 4.4 illustrates the time duration of membrane recovery after exposure. Treatments with PBS alone (no pulses) and w ith 750 V/cm applied electric field yielded between 90-100% membrane recovery during the 24 hours of observation. However, acetic acid alone had a greater impact on th e recovery of the cells, ranging from an average of 80-95% membrane integrity. At a time of 1 hour post-treatment for samples treated with acetic acid at 750 V/cm, there wa s a 17% decline in the number of cells with

PAGE 36

29 intact membranes, implying that some of the cells either did not survive after exposure or was permeabilized. The number of cells that excluded the trypan blue dye then increased as the hours proceeded to a valu e of 86% after 24 hours. 90% of the cells exposed to AA alone had intact membrane s 24 hours after treatment. 4.5 B16F10 Cell Sizes Post Treatment After the cells had been exposed to a ny of the treatment conditions mentioned earlier, they varied slightly in shape and si ze due to membrane swelling as determined by direct observation. This phenomenon is an indication that fluid was being delivered through the cell’s membrane. An investigati on as to how much more swelling occurred at various conditions, compared to normal B16F10 cells, was conducted. Due to the nature of the shape and size changes, both hor izontal (long axis) and ve rtical (short axis) dimensions were collected for 30 cells for each treatment condition at a magnification of 400. The treatment conditions investigated consisted of PBS (no pulses), 10mM ASA (no pulses), 10mM ASA and pul ses at 750 V/cm, 10mM SA (no pulses), 10mM SA and pulses at 750 V/cm, AA (pH 4.40) (no pul ses), and AA (pH 4.40) and pulses at 750 V/cm. For comparison and control purposes, measurements of 30 untreated cells were taken. The vertical axis of the untreated cells averaged 15m, whereas the horizontal axis averaged 44.1m. The samples of cells investigated at any of the other treatment conditions were exposed to the solutions and pulses (when applied) between approximately 1-10 minutes before the meas urements were taken. Figure 3.5 shows the mean results of the measurements as well as the corresponding standard deviations. It

PAGE 37

30 can clearly be seen that there is no significan t difference in the measured dimensions at any condition, compared to the untreated ce lls, and is confirmed using the two-tailed paired sample t test at the 0.05 si gnificance level. B16F10 Cell Sizes0 10 20 30 40 50 60 70E-E-E-750 V/cmE-750 V/cmE-750 V/cm Untreated Cells PBSASASAAA TreatmentSize (m) Vertical Axis Horizontal Axis Figure 4.5 Vertical and Horizontal Axis of B16F10 Cells 4.6 Conclusions and Discussion Aspirin and two of its deri vatives were tested in th is study, salicylic acid and acetic acid. Two of the three succeeded in delivering calcein to cells. ASA (10mM) and AA (pH 4.40) augmented the delivery of calcein at all 4 chosen electr ical field strengths, but AA (pH4.40) was clearly a better candidate. The low pH solution of AA alone (no pulses) augmented delivery, indicating a str ong effect on the membranes, and improved with the presence of pulses. When compared to any other treatment condition, including

PAGE 38

31 those using ASA and SA, with or without th e presence of electrical pulses, AA (pH 4.40) clearly resulted in higher mean fluorescence da ta. Surprisingly, SA did not show a great effect in calcein delivery, given by the significance of statistical t tests. The fact that ASA and AA augmented deliv ery to the cells over SA may not be a coincidence. As mentioned earlier, ASA will hydrolyze to AA and SA derivatives. This is why acetic acid, in addition to SA, was chos en to be investigated. After ASA showed to be successful in experimentation, it had to be established whether or not the presence of AA was the reason of such a promising result. A comparison of the data can help establish if pH alone is a de termining factor of calcein delivery. As an outcome, acetic acid (pH 4.40) resu lted in higher mean spectrofluorometric data than that of ASA and/or SA at all treatment conditions. The 0.15% concentration of AA (pH 4.40) had a hi gher pH value than 10 mM SA (pH 3.86), and was equal to the pH of 10 mM ASA (pH 4.40). This indicates that the successful delivery of calcein was not the direct effect of a low pH solution, and that 0.15% acetic acid with a pH of 4.40 inherent ly is the most optimal in delivering calcein to B16F10 cells by electroporation.

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32 REFERENCES 1. Freeman, Scott A.; Wang, Michele A.; Weaver James C. Theory of Electroporation of Planar Bilayer Membranes: Predic tions of the Aqueous Area, Change in Capacitance, and Pore-Pore Separation. Biophys. J. 1994, 67 42-56. 2. Weaver, James C. Molecular Basis for Cell Membrane Electroporation. Ann. N. Y. Acad. Sci. 1994, 141-151. 3. Weaver, J.C.; Chizmadzhev, Y.A. Theory of Electroporation: A Review. Bioelectrochem. Bioenerg. 1996, 41 135-160. 4. Hibino, Masahiro; Shigemori, Masaya; Itoh, Hiroyasu; Nagayama, Kuniaki; Kinosita, Kazuhiko, Jr. Membrane Conductance of an Electroporated Cell Analyzed by Submicrosecond Imaging of Transmembrane Potential. Biophys. J. 1991, 59 209220. 5. Gowrishankar, T.R.; Pliquett, Uwe; Lee, Rapha el C. Dynamics of Membrane Sealing in Transient Electropermeabilization of Skeletal Muscle Membranes. Ann. N. Y. Acad. Sci. 1999, 195-210. 6. Benz, R.; Zimmerman, U. The Resealing Proc ess of Lipid Bilayers after Reversible Electrical Breakdown. Biochim. Biophys. Acta 1981, 640 169-178. 7. Tung, Leslie; Troiano, Greg C.; Sharma, Vinod; Raphael, Robert M.; Stebe, Kathleen J. Changes in Electroporation Thresholds of Lipid Membranes by Surfactants and Peptides. Ann. N. Y. Acad. Sci. 1999, 249-265. 8. DeBruin, Katherine A.; Krassowska, Wanda Modeling Electroporation in a Single Cell. II. Effects of Ionic Concentrations. Biophys. J. 1999, 77 1225-1233. 9. Tsong, Tian Y. Electroporation of Cell Membranes. Biophys. J. 1991, 60 297-306. 10. Weaver, James C. Electroporation: A General Phenomenon for Manipulating Cells and Tissues. J. Cell Biochem. 1993, 51 (4), 426-435. 11. Tendeloo, VFI Van; Broeckhoven, C. Van; Berneman, ZN. Gene Therapy: Principles and Applications to Hematopoietic Cells. Leukemia 2001, 15 (4), 523-544.

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33 12. Joshi, R.P.; Schoenbach, K.H. Electr oporation Dynamics in Biological Cells Subjected to Ultrafast Electrical Pulses: A Numerical Simulation Study. Phys. Rev. E 2000, 62 (1), 1025-1033. 13. Teissi, J.; Eynard, N.; Gabriel, B.; Ro ls, M.P. Electropermeabilization of Cell Membranes. Adv. Drug Delivery Rev. 1999, 35 3-19. 14. Heller, Richard; Gilbert, Richard; Jaroszes ki, Mark J. Clinical Applications of Electrochemotherapy. Adv. Drug Delivery Rev. 1999, 35 119-129. 15. Mir, Lluis M.; Orlowski, Stphane. Mechanisms of Electrochemotherapy. Adv. Drug Delivery Rev. 1999, 35 107-118. 16. Selby, Mark; Goldbeck, Cheryl; Pertile, Terry; Walsh, Robert; Ulmer, Jeffrey. Enhancement of DNA Vaccine Pote ncy by Electroporation In Vivo. J. Biotechnol. 2000, 83 147-152. 17. Bureau, M.F.; Gehl, J.; Deleuze, V.; Mir, L.M.; Scherman, D. Importance of Association Between Permeabilization and El ectrophoretic Forces for Intramuscular DNA Electrotransfer. Biochim. Biophys. Acta 2000, 1474 353-359. 18. Zheng, Qiang; Chang, Donald C. High-E fficiency Gene Transfection by In Situ Electroporation of Cultured Cells. Biochim. Biophys. Acta 1991, 1088 104-110. 19. Vanbever, Rita; Lecouturier, Nathalie; Pr eat, Veronique. Transdermal Delivery of Metoprolol by Electroporation. Pharm. Res. 1994, 11 (11), 1660-1665. 20. Jadoul, Anne; Bouwstra, Joke; Prat, V ronique. Effects of Iontophoresis and Electroporation on the Stratum Corneum: Review of the Biophysical Studies. Adv. Drug Delivery Rev. 1999, 35 89-105. 21. Felgner, P.L.; Gadek, T.M.; Holm, M.; Ro man, R.; Chan, H.W.; Wenz, M.; Northrop, J.P.; Ringold, G.M.; Danielsen, M. Lipofecti on: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure. Proc. Natl. Acad. Sci. U.S.A. 1987, 84 7413-7417. 22. Tollefsen, S.; Vordermeier, M.; Olsen, I.; Storset, A.K.; Reitan, L.J.; Clifford, D.; Lowrie, D.B.; Wiker, H.G.; Huygen, K.; Hewinson, G.; Mathiesen, I.; Tjelle, T.E. DNA Injection in Combination with El ectroporation: A Novel Method for Vaccination of Farmed Ruminants. Scand. J. Immunol. 2003, 57 (3), 229-238. 23. Lee, Raphael C. Biophysical Mechanisms of Cell Membrane Damage in Electrical Shock. Seminars in Neurology 1995, 15 (4), 367-374.

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34 24. Troiano, Gregory C.; Tung, Leslie; Sharma, Vi nod; Stebe, Kathleen J. The Reduction in Electroporation Voltages by the Addition of a Surfactant to Planar Lipid Bilayers. Biophys. J. 1998, 75 880-888. 25. Lee, Raphael C.; River, Philip L.; Pan, Fu-Shih; Ji, Li; Wollmann, Robert L. Surfactant-Induced Sealing of Electrope rmeabilized Skeletal Muscle Membranes In Vivo Proc. Natl. Acad. Sci. U.S.A. 1992, 89 4524-4528. 26. Hartikka, Jukka; Sukhu, Loretta; Buchne r, Carol; Hazard, Dianel; Bozoukova, Vesselina; Margalith, Michal; Nishioka, Walter K.; Wheeler, Carl J.; Manthorpe, Marston; Sawdey, Michael. Electroporati on-Facilitated Delivery of Plasmid DNA in Skeletal Muscle: Plasmid Dependence of Muscle Damage and Effect of Poloxamer 188. Molecular Therapy 2001, 4 (5), 407-415. 27. Schreier, Shirley; Malheiros, Snia V.P.; Pa ula, Eneida de. Surface Active Drugs: Self-Association and Interaction with Memb ranes and Surfactants. Physiochemical and Biological Aspects. Biochim. Biophys. Acta 2000, 1508 210-234. 28. Smith, Paul K.; Smith, M.J.H. The Salicyl ates: A Critical Bibliographic Review. Interscience Publishers, New York, 1966. 29. Agreda, Victor H.; Zoeller, Joseph R. A cetic Acid and its Derivatives. Marcel Dekker, Inc New York, 1993. 30. Schindler, Paul E.; Jr., Fields; William S. Aspirin Therapy. Walker and Company, New York, 1978. 31. Page, Clive P.; Curtis, Michael J.; Sutter, Morley C.; Walker, Michael J.A.; Hoffman, Brian B. Integrated Pharmacology. Mosby, London, UK, 1997, 360-361. 32. McEvoy, G.K.; Miller, J.M.; Snow, E.K.; Welsh, O.H.; Litvak, K.; Dewey, D.R.; O’Rourke, A.; Bollinger, L.A.; Justice, L. ; Kim, J.; et al. AHFS Drug Information, 2004. American Society of Health-System Pharmacists, 2004, 2577, 2674. 33. Liang, Huei-Lung; Yang, Chien-Fang; Pan, Huay-Ban; Lai, Kwok-Hung; Cheng, JinShiung; Lo, Gin-Ho; Chen, Clement K.H. ; Lai, Ping-Hong. Small Hepatocellular Carcinoma: Safety and Efficacy of Singl e High-Dose Percutaneous Acetic Acid Injection for Treatment. Radiology 2000, 769-774. 34. Ohnishi, Kunihiko. Comparison of Perc utaneous Acetic Acid Injection and Percutaneous Ethanol Injection for Small Hepatocellular Carcinoma. HepatoGastroenterology 1998, 1254-1258.

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35 35. Freshney, R.I. Animal Cell Cult ure: A Practic al Approach. Practical Approach Series 1986, IRL Press Limited, 13-15. 36. Gilbert, Richard A.; Jaroszeski, Mark J.; Heller, Richard. Novel Electrode Designs for Electrochemotherapy. Biochim. Biophys. Acta 1997, 1334 9-14.

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36 APPENDICES

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37 Appendix A: Data Collected for Salicylic Acid Trials Table A.1 Salicylic Acid Delivery: Experiment 1 Treatment123Average Standard DeviationControl0.40.10.10.200.17 CALCEIN1.22.20.61.330.81 1mM SA&CAL0.60.61.30.830.40 5mM SA&CAL0.50.50.90.630.23 10mM SA&CAL3.22.42.12.570.57 CALCEIN0.81.811.200.53 1mM SA&CAL3.21.51.52.070.98 5mM SA&CAL2.88.84.35.303.12 10mM SA&CAL5.475.35.900.95 CALCEIN4.53.45.24.370.91 1mM SA&CAL1.72.342.671.19 5mM SA&CAL1.62.615.96.707.98 10mM SA&CAL0.91.22.31.470.74 CALCEIN33.94.43.770.71 1mM SA&CAL2.93.58.24.872.90 5mM SA&CAL2.51.51.21.730.68 10mM SA&CAL3.31.71.12.031.14 CALCEIN4.63.233.600.87 1mM SA&CAL44.13.43.830.38 5mM SA&CAL3.13.14.43.530.75 10mM SA&CAL2.913.82.571.431385 V/cm500 V/cm 750 V/cm 1000 V/cmE-Experiment 1

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38 Appendix A: (Continued) Table A.2 Salicylic Acid Delivery: Experiment 2 Treatment123Average Standard DeviationControl0.20.10.10.130.06 CALCEIN2.31.12.72.030.83 1mM SA&CAL1.10.61.41.030.40 5mM SA&CAL1.52.72.22.130.60 10mM SA&CAL0.9110.970.06 CALCEIN2.92.21.32.130.80 1mM SA&CAL2.93.12.62.870.25 5mM SA&CAL3.12.72.12.630.50 10mM SA&CAL4.32.46.14.271.85 CALCEIN1.91.72.92.170.64 1mM SA&CAL1.93.53.83.071.02 5mM SA&CAL1.81.12.51.800.70 10mM SA&CAL3.34.22.43.300.90 CALCEIN2.42.22.32.300.10 1mM SA&CAL32.53.73.070.60 5mM SA&CAL1.33.83.22.771.31 10mM SA&CAL1.62.42.62.200.53 CALCEIN3.51.22.22.301.15 1mM SA&CAL1.81.71.91.800.10 5mM SA&CAL2.51.31.61.800.62 10mM SA&CAL1.61.60.61.270.581000 V/cm 1385 V/cmExperiment 2E500 V/cm 750 V/cm

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39 Appendix A: (Continued) Table A.3 Salicylic Acid Delivery: Experiment 3 Treatment123 Average Standard DeviationControl0.30.50.50.430.12 CALCEIN1.10.60.80.830.25 1mM SA&CAL0.61.80.91.100.62 5mM SA&CAL1.40.90.71.000.36 10mM SA&CAL0.70.510.730.25 CALCEIN6.34.63.94.931.23 1mM SA&CAL1.22.93.32.471.12 5mM SA&CAL2.540.92.471.55 10mM SA&CAL3.52.42.42.770.64 CALCEIN2.62.53.12.730.32 1mM SA&CAL2.42.81.92.370.45 5mM SA&CAL4.41.82.32.831.38 10mM SA&CAL3.12.42.62.700.36 CALCEIN3.85.22.83.931.21 1mM SA&CAL3.222.92.700.62 5mM SA&CAL2.82.82.62.730.12 10mM SA&CAL1.91.92.42.070.29 CALCEIN2.92.82.82.830.06 1mM SA&CAL2.92.62.32.600.30 5mM SA&CAL0.81.91.91.530.64 10mM SA&CAL7.12.23.94.402.491000 V/cm 1385 V/cmExperiment 3E500 V/cm 750 V/cm

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40 Appendix A: (Continued) Table A.4 Salicylic Acid Deliver y: Average of 3 Experiments TreatmentAverage Standard Deviation Standard Error MeanControl, PBS0.260.170.10 CALCEIN1.400.790.46 1mM SA&CAL0.990.440.25 5mM SA&CAL1.260.770.45 10mM SA&CAL1.420.920.53 CALCEIN2.761.861.07 1mM SA&CAL2.470.830.48 5mM SA&CAL3.472.241.29 10mM SA&CAL4.311.741.00 CALCEIN3.091.150.66 1mM SA&CAL2.700.870.50 5mM SA&CAL3.784.642.68 10mM SA&CAL2.491.010.58 CALCEIN3.331.050.60 1mM SA&CAL3.541.821.05 5mM SA&CAL2.410.900.52 10mM SA&CAL2.100.650.37 CALCEIN2.910.920.53 1mM SA&CAL2.740.920.53 5mM SA&CAL2.291.110.64 10mM SA&CAL2.742.001.151385 V/cmExperiments 1, 2 & 3E500 V/cm 750 V/cm 1000 V/cm

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41 Appendix B: Data Collected for Acetylsalicylic Acid Experiments Table B.1 Acetylsalicylic Ac id Delivery: Experiment 1 Treatment123Average Standard DeviationControl, PBS0.4000.130.23 CALCEIN0.30.70.50.500.20 1mM ASA&CAL0.50.60.80.630.15 5mM ASA&CAL0.728.51.410.2015.85 10mM ASA&CAL3.95.26.45.171.25 CALCEIN1.91.21.41.500.36 1mM ASA&CAL2.32.72.82.600.26 5mM ASA&CAL2.34.44.13.601.14 10mM ASA&CAL9.912.713.912.172.05 CALCEIN3.23.33.53.330.15 1mM ASA&CAL3.72.83.43.300.46 5mM ASA&CAL1.83.34.13.071.17 10mM ASA&CAL13.815.120.916.603.78 CALCEIN6.12.41.93.472.29 1mM ASA&CAL3.24.14.53.930.67 5mM ASA&CAL5.83.85.85.131.15 10mM ASA&CAL19.411.616.915.973.98 CALCEIN44.13.83.970.15 1mM ASA&CAL3.53.93.93.770.23 5mM ASA&CAL8.13.55.65.732.30 10mM ASA&CAL16.117.618.517.401.21E-Experiment 11385 V/cm500 V/cm 750 V/cm 1000 V/cm

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42 Appendix B: (Continued) Table B.2 Acetylsalicylic Ac id Delivery: Experiment 2 Treatment123Average Standard DeviationControl, PBS0.30.30.20.270.06 CALCEIN0.71.30.60.870.38 1mM ASA&CAL0.81.80.51.030.68 5mM ASA&CAL1.81.10.81.230.51 10mM ASA&CAL2.72.543.070.81 CALCEIN2.71.32.82.270.84 1mM ASA&CAL3.12.933.000.10 5mM ASA&CAL1.91.83.62.431.01 10mM ASA&CAL4.67.276.271.45 CALCEIN2.43.22.52.700.44 1mM ASA&CAL1.63.44.53.171.46 5mM ASA&CAL5.14.32.53.971.33 10mM ASA&CAL11.17.28.38.872.01 CALCEIN1.93.52.32.570.83 1mM ASA&CAL2.433.52.970.55 5mM ASA&CAL2.22.83.72.900.75 10mM ASA&CAL11.410.89.810.670.81 CALCEIN1.92.44.22.831.21 1mM ASA&CAL3.17.12.44.202.54 5mM ASA&CAL2.53.143.200.75 10mM ASA&CAL12.16.110.29.473.07500 V/cm 750 V/cm 1000 V/cm 1385 V/cmExperiment 2E

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43 Appendix B: (Continued) Table B.3 Acetylsalicylic Ac id Delivery: Experiment 3 Treatment123 Average Standard DeviationControl, PBS0.10.30.10.170.12 CALCEIN0.40.60.60.530.12 1mM ASA&CAL1.30.51.71.170.61 5mM ASA&CAL0.610.90.830.21 10mM ASA&CAL2.54.374.602.26 CALCEIN5.621.32.972.31 1mM ASA&CAL1.1221.700.52 5mM ASA&CAL4.52.83.23.500.89 10mM ASA&CAL6.17.96.26.731.01 CALCEIN2.41.92.42.230.29 1mM ASA&CAL2.23.41.52.370.96 5mM ASA&CAL2.63.72.52.930.67 10mM ASA&CAL4.84.29.96.303.13 CALCEIN3.22.82.12.700.56 1mM ASA&CAL4.53.12.53.371.03 5mM ASA&CAL4.53.94.24.200.30 10mM ASA&CAL44.35.34.530.68 CALCEIN32.52.42.630.32 1mM ASA&CAL2.52.63.22.770.38 5mM ASA&CAL4.43.47.65.132.19 10mM ASA&CAL3.41.24.73.101.771000 V/cm 1385 V/cmExperiment 3E500 V/cm 750 V/cm

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44 Appendix B: (Continued) Table B.4 Acetylsalicylic Acid De livery: Average of 3 Experiments TreatmentAverage Standard Deviation Standard Error MeanControl, PBS0.190.070.04 CALCEIN0.630.200.12 1mM ASA&CAL0.940.280.16 5mM ASA&CAL4.095.303.06 10mM ASA&CAL4.281.090.63 CALCEIN2.240.730.42 1mM ASA&CAL2.430.670.38 5mM ASA&CAL3.180.650.37 10mM ASA&CAL8.393.281.89 CALCEIN2.760.550.32 1mM ASA&CAL2.940.500.29 5mM ASA&CAL3.320.560.32 10mM ASA&CAL10.595.363.10 CALCEIN2.910.490.28 1mM ASA&CAL3.420.490.28 5mM ASA&CAL4.081.120.65 10mM ASA&CAL10.395.723.30 CALCEIN3.140.720.42 1mM ASA&CAL3.580.740.42 5mM ASA&CAL4.691.320.76 10mM ASA&CAL9.997.164.141385 V/cmExperiments 1, 2 & 3E500 V/cm 750 V/cm 1000 V/cm

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45 Appendix C: Data Collected for Acetic Acid Experiments Table C.1 Acetic Acid Delivery: Experiment 1 Treatment123Average Standard DeviationControl0.20.10.20.170.06 CALCEIN1.11.61.41.370.25 Acetic Acid pH 6.423.32.44.73.471.16 Acetic Acid pH 5.364.84.72.43.971.36 Acetic Acid pH 4.407.5108.18.531.31 CALCEIN4.944.54.470.45 Acetic Acid pH 6.423.54.74.84.330.72 Acetic Acid pH 5.364.73.544.070.60 Acetic Acid pH 4.4012.113.110.812.001.15 CALCEIN4.72.32.83.271.27 Acetic Acid pH 6.423.54.14.74.100.60 Acetic Acid pH 5.365.73.84.74.730.95 Acetic Acid pH 4.4018.317.412.816.172.95 CALCEIN6.14.74.95.230.76 Acetic Acid pH 6.424.29.85.56.502.93 Acetic Acid pH 5.365.65.54.45.170.67 Acetic Acid pH 4.4017.51721.318.602.35E-Experiment 1500 V/cm 750 V/cm 1000 V/cm

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46 Appendix C: (Continued) Table C.2 Acetic Acid Delivery: Experiment 2 Treatment123Average Standard DeviationControl0.40.20.20.270.12 CALCEIN0.810.80.870.12 Acetic Acid pH 6.420.81.21.21.070.23 Acetic Acid pH 5.361.53.52.22.401.01 Acetic Acid pH 4.408.48.811.19.431.46 CALCEIN4.63.12.73.471.00 Acetic Acid pH 6.4224.95.14.001.73 Acetic Acid pH 5.364.94.85.35.000.26 Acetic Acid pH 4.4012.913.712.613.070.57 CALCEIN7.49.57.88.231.12 Acetic Acid pH 6.425.23.85.74.900.98 Acetic Acid pH 5.366.85.95.46.030.71 Acetic Acid pH 4.409.78.5119.731.25 CALCEIN4.84.42.63.931.17 Acetic Acid pH 6.423.622.42.670.83 Acetic Acid pH 5.363.50.73.42.531.59 Acetic Acid pH 4.4021.120.720.320.700.40750 V/cm 1000 V/cmExperiment 2E500 V/cm

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47 Appendix C: (Continued) Table C.3 Acetic Acid Delivery: Experiment 3 Treatment123 Average Standard DeviationControl0.30.50.50.430.12 CALCEIN1.10.70.90.900.20 Acetic Acid pH 6.421.51.61.41.500.10 Acetic Acid pH 5.360.81.20.80.930.23 Acetic Acid pH 4.406.2897.731.42 CALCEIN5.63.354.631.19 Acetic Acid pH 6.422.944.53.800.82 Acetic Acid pH 5.362.43.664.001.83 Acetic Acid pH 4.4010.616.11313.232.76 CALCEIN4.44.84.84.670.23 Acetic Acid pH 6.425.23.67.95.572.17 Acetic Acid pH 5.365.32.62.33.401.65 Acetic Acid pH 4.408.710.911.910.501.64 CALCEIN663.25.071.62 Acetic Acid pH 6.423.531.92.800.82 Acetic Acid pH 5.363.63.24.33.700.56 Acetic Acid pH 4.4010.59.410.510.130.641000 V/cmExperiment 3E500 V/cm 750 V/cm

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48 Appendix C: (Continued) Table C.4 Acetic Acid Deliver y: Average of 3 Experiments TreatmentAverage Standard Deviation Standard Error MeanControl, PBS0.290.150.08 CALCEIN1.040.300.17 Acetic Acid pH 6.422.011.260.73 Acetic Acid pH 5.362.431.570.91 Acetic Acid pH 4.408.571.420.82 CALCEIN4.190.980.56 Acetic Acid pH 6.424.041.050.61 Acetic Acid pH 5.364.361.090.63 Acetic Acid pH 4.4012.771.630.94 CALCEIN5.392.381.37 Acetic Acid pH 6.424.861.380.80 Acetic Acid pH 5.364.721.530.88 Acetic Acid pH 4.4012.133.542.04 CALCEIN4.741.230.71 Acetic Acid pH 6.423.992.461.42 Acetic Acid pH 5.363.801.460.84 Acetic Acid pH 4.4016.485.002.891000 V/cmExperiments 1, 2 & 3E500 V/cm 750 V/cm

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49 Appendix D: Data Collected for Membrane Recovery Table D.1 Membrane Recovery after 1 Hour Live DeadTotal Count% Dead% ViableSD 1591601.798.3 2642663.097.0 3652673.097.0 average62.71.764.32.697.40.8 1434478.591.5 26297112.787.3 31031211510.489.6 average69.38.377.710.589.52.1 15476111.588.5 2575628.191.9 3633664.595.5 average58.05.063.08.092.03.5 153126518.581.5 267168319.380.7 355167122.577.5 average58. 3 14. 7 73.020.179.92. 2 1 HOUR POST-EXPOSURE PBS (no pulses) AA (no pulses) PBS, 750 V/cm AA, 750 V/cm Table D.2 Membrane Recovery after 2 Hours Live DeadTotal Count% Dead% ViableSD 1562583.496.6 2591601.798.3 3573605.095.0 average57.32.059.33.496.61.7 136104621.778.3 263147718.281.8 358116915.984.1 average52.311.764.018.681.42.9 1442464.395.7 2501512.098.0 3632653.196.9 average52.31.754.03.196.91.2 15876510.889.2 245156025.075.0 358116915.984.1 average53. 7 11.064. 7 17. 2 82.87. 2 2 HOURS POST-EXPOSURE PBS (no pulses) AA (no pulses) PBS, 750 V/cm AA, 750 V/cm

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50 Appendix D: (Continued) Table D.3 Membrane Recovery after 3 Hours Live DeadTotal Count% Dead% ViableSD 1514557.392.7 2545598.591.5 3473506.094.0 average50.74.054.77.292.81.2 19551005.095.0 264107413.586.5 345125721.178.9 average68.09.077.013.286.88.0 1562583.496.6 2552573.596.5 33754211.988.1 average49.33.052.36.393.74.9 1572593.496.6 27498310.889.2 3665717.093.0 average65.75.371.07.192.93.7 3 HOURS POST-EXPOSURE PBS (no pulses) AA (no pulses) PBS, 750 V/cm AA, 750 V/cm Table D.4 Membrane Recovery after 4 Hours Live DeadTotal Count% Dead% ViableSD 1644685.994.1 2592613.396.7 3552573.596.5 average59.32.762.04.295.81.4 1453486.393.8 2726787.792.3 3494537.592.5 average55.34.359.77.292.80.8 1583614.995.1 2471482.197.9 3563595.194.9 average53.72.356.04.096.01.7 1554596.893.2 2574616.693.4 3736797.692.4 average61. 7 4. 7 66. 3 7.093.00.5 4 HOURS POST-EXPOSURE PBS (no pulses) AA (no pulses) PBS, 750 V/cm AA, 750 V/cm

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51 Appendix D: (Continued) Table D.5 Membrane Recovery after 24 Hours Live DeadTotal Count% Dead% ViableSD 113701370.0100.0 212231252.497.6 3892912.297.8 average116.01.7117.71.598.51.3 149105916.983.1 2625677.592.5 39551005.095.0 average68.76.775.39.890.26.3 1521531.998.1 2682702.997.1 3702722.897.2 average63.31.765.02.597.50.5 16076710.489.6 274128614.086.0 357137018.681.4 average63. 7 10. 7 74. 3 14. 3 85.74.1 24 HOURS POST-EXPOSURE PBS (no pulses) AA (no pulses) PBS, 750 V/cm AA, 750 V/cm

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52 Appendix E: B16F10 Cell Sizes Post Treatment Table E.1 B16F10 Cell Sizes VHVHVHVHVHVHVHVH 1 12.557.512.55012.55012.5451082.512.54520252040 2 22.5251562.52037.51547.512.540103522.53012.537.5 3 152512.55017.560106012.55012.56022.54017.562.5 4 12.557.517.56512.53012.55012.5751552.520552075 5 1550106020551542.5103512.542.512.56012.522.5 6 20251017.515301552.5158012.547.5204017.540 7 12.5701542.512.55512.5451552.5104512.577.512.555 8 12.515254512.517.512.54012.53512.54010802042.5 9 22.582.517.5652537.510751055107512.56512.537.5 10 15652555153522.537.512.56522.537.51557.517.525 11 1517.51572.517.5451557.51015155512.57017.575 12 17.552.53047.512.5502577.512.562.522.577.512.52012.552.5 13 1557.512.54017.577.512.56512.560156512.5402542.5 14 2035154012.552.522.5452025204512.52517.575 15 12.5152052.517.5451025206512.52517.572.51537.5 16 257012.52015302047.5102017.547.517.53512.545 17 1522.512.552.512.52517.522.512.5351030258517.557.5 18 12.53010651522.5155012.52012.5551522.522.555 19 17.5301510156515802525158012.5201575 20 12.56517.54015252072.522.57017.567.517.552.512.565 21 12.547.5152012.575154020601522.51522.517.525 22 202512.53512.56022.565154017.56527.5352030 23 1080156522.53512.54512.52512.53025252060 24 1527.512.54012.537.5103512.5501037.5206517.570 25 156012.54515251042.512.517.51547.51522.51547.5 26 12.57520801577.515201562.512.52015601522.5 27 103017.53012.572.52537.522.5253042.52062.52535 28 1055104012.562.52022.512.5352022.512.5701525 29 12.5402017.512.5353062.517.522.5305512.552.512.547.5 30 101517.5451557.5154012.57512.54022.52522.535 Avg. 15.0044.0815.7545.6715.1746.0816.1748.2514.4246.0015.4247.0016.9247.0817.0047.17 S.D.3.9921.264.7917.433.2817.585.2416.154.1420.815.3016.334.7220.803.7916.93 10mM SA (no pulses) 10mM SA 750 V/cm AA (pH4.40) (no pulses) AA (pH4.40) 750 V/cm Untreated Cells PBS (no pulses) 10mM ASA (no pulses) 10mM ASA 750 V/cm