This PDF file contains the front matter associated with SPIE Proceedings Volume 9326, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

The development of minimally invasive medical devices, which employ hyperthermic and/or cryothermic modalities to treat a variety of organ system diseases, is a rapidly expanding field. Consultation with a knowledgeable pathologist during the development of these devices is crucial, as characterization of the treated tissues can support the device’s safety and future efficacy. The properties of thermally treated tissues often share a set of overlapping histopathologic characteristics, regardless of organ system. Several methods for optimally evaluating these thermal changes have been developed that depend on the tissue’s post-treatment time interval. For devices associated with hyperthermic collagen denaturation, bright field or polarized light microscopy of hematoxylin and eosin stained sections can be utilized to assess thermal spread within the tissue. For applications resulting in collagen denaturation with associated desiccation, trichrome staining may provide additional information. For cryothermic devices, these collagen-based methods are generally less informative. Tetrazolium-based tissue viability staining (triphenyltetrazolium chloride, TTC; nitroblue tetrazolium, NBT; NADH/NADPH staining) can be used to assess for the presence of associated tissue necrosis within either hyperthermically or cryothermically treated tissues. The advantages and limitations of several of these methods will be discussed.

The recent discovery of the various specific" triggers" and mechanisms of cell life and death responses suggests that certain non-ionizing irradiation spectra and other physical signals could be coupled with specific cellular "triggers" to improve diagnostic or treatment effects. Genetic, chemical and/or physical modifications of specific extrinsic cellular "triggers" have centered on chemical binding of specific molecules to specific receptors, i.e. chemical " triggers" thus allowing development of specific drugs to counteract the initiating function of the “triggers” in single cells. Investigations of non-ionizing irradiation effects on cells and tissues indicate that their “triggers” involve intimate interactions of linked components of the extracellular matrix and the cytoskeleton in tissues. Therefore, the search for single “triggers” that may work well for chemical “triggers” in single cells in culture may not be effective for discovery of the mechanisms that initiate sensing of the stimulations of non-ionizing irradiation and mechanical force.

For more than 50 years, hyperthermia-based cancer researchers have utilized mathematical models, cell culture studies and animal models to better understand, develop and validate potential new treatments. It has been, and remains, unclear how and to what degree these research techniques depend on, complement and, ultimately, translate accurately to a successful clinical treatment. In the past, when mathematical models have not proven accurate in a clinical treatment situation, the initiating quantitative scientists (engineers, mathematicians and physicists) have tended to believe the biomedical parameters provided to them were inaccurately determined or reported. In a similar manner, experienced biomedical scientists often tend to question the value of mathematical models and cell culture results since those data typically lack the level of biologic and medical variability and complexity that are essential to accurately study and predict complex diseases and subsequent treatments. Such quantitative and biomedical interdependence, variability, diversity and promise have never been greater than they are within magnetic nanoparticle hyperthermia cancer treatment. The use of hyperthermia to treat cancer is well studied and has utilized numerous delivery techniques, including microwaves, radio frequency, focused ultrasound, induction heating, infrared radiation, warmed perfusion liquids (combined with chemotherapy), and, recently, metallic nanoparticles (NP) activated by near infrared radiation (NIR) and alternating magnetic field (AMF) based platforms. The goal of this paper is to use proven concepts and current research to address the potential pathobiology, modeling and quantification of the effects of treatment as pertaining to the similarities and differences in energy delivered by known external delivery techniques and iron oxide nanoparticles.

The clinical utility of current thermal ablation planning tools is severely limited by treatment variability. We discuss the development of an open platform for evidence-based thermal ablation treatment planning and validation. Improved predictive treatment modeling and consistent outcome analysis are crucial components for useful planning and guidance tools.

Plasma is referred to as the fourth state of matter and is frequently generated in the environment of a strong electric field. The result consists of highly reactive species--ions, electrons, reactive atoms and molecules, and UV radiation. Plasma Medicine unites a number of fields, including Physics, Plasma Chemistry, Cell Biology, Biochemistry, and Medicine. The treatment modality utilizes Cold Atmospheric Plasma (CAP), which is able to sterilize and treat microbes in a nonthermal manner. These gas-based plasma systems operate at close to room temperature and atmospheric pressure, making them very practical for a range of potential treatments and are highly portable for clinical use throughout the health care system. The hypothesis is that gas based plasma kills bacteria, fungus, and viruses but spares mammalian cells. This paper will review systematic work which shows examples of systems and performance in regards to antimicrobial effects and the sparing of mammalian cells. The mechanism of action will be discussed, as well as dosing for the treatment of microbial targets, including sterilization processes, another important healthcare need. In addition, commercial systems will be overviewed and compared, along with evidence-based, patient results. The range of treatments cover wound treatment and biofilms, as well as antimicrobial treatment, with little chance for resistance and tolerance, as in drug regimens. Current clinical studies include applications in dentistry, food treatment, cancer treatment, wound treatment for bacteria and biofilms, and systems to combat health care related infections.

Coblation® is an electrosurgical technology which employs electrically-excited electrodes in the presence of saline solution to produce a localized and ionized plasma that can cut, ablate, and otherwise treat tissues for many different surgical needs. To improve our understanding of how Coblation plasmas develop from devices made from different electrode materials we describe several experiments designed to elucidate material effects. Initial experiments studied simple, noncommercial cylindrical electrode test devices operating in buffered isotonic saline without applied suction. The applied RF voltage, approximately 300 V RMS, was sufficient to form glow discharges around the active electrodes. The devices exhibited significantly different operating characteristics, which we ascribe to the differing oxidation tendencies and other physical properties of the electrode materials. Parameters measured include RMS voltage and current, instantaneous voltage and current, temporally-resolved light emission and optical emission spectra, and electrode mass-loss measurements. We correlate these measured properties with some of the bulk characteristics of the electrode materials such as work functions, standard reduction potentials and sputter yields.

INTRODUCTION: The ABC® D-Flex Probe utilizes argon beam coagulation (ABC) technology to achieve hemostasis during minimally invasive surgery. A handle on the probe allows for integration with robotic surgical systems and introduces ABC to the robotic toolbox. To better understand the utility of D-Flex, this study compares the performance of the D-Flex probe to an existing ABC laparoscopic probe through ex vivo tissue analysis. METHODS: Comparisons were performed to determine the effect of four parameters: ABC device, tissue type, activation duration, and distance from tissue. Ten ABC D-Flex probes were used to create 30 burn samples for each comparison. Ex vivo bovine liver and porcine muscle were used as tissue models. The area and depth of each burn was measured using a light microscope. The resulting dimensional data was used to correlate tissue effect with each variable. RESULTS: D-Flex created burns which were smaller in surface area than the laparoscopic probe at all power levels. Additionally, D-Flex achieved thermal penetration levels equivalent to the laparoscopic probe. CONCLUSION: D-Flex implements a small 7F geometry which creates a more focused beam. When used with robotic precision, quick localized superficial hemostasis can be achieved with minimal collateral damage. Additionally, D-Flex achieved equivalent thermal penetration levels at lower power and argon flow-rate settings than the laparoscopic probe.

INTRODUCTION: Argon Beam Coagulation (ABC^®) technology is used in conjunction with the ConMed ABCFlex^® Probe to provide non-contact hemostasis, coagulation, and tissue devitalization during endoscopic procedures. ABC provides a superficial tissue effect; however, there is a risk of bowel perforation. To better understand the settings that lead to perforation, this study reviews the literature and provides an ex vivo characterization of the ABCFlex Probe tissue effect at different settings when used at small distances. METHODS: Depth of thermal tissue effect was characterized to determine the effect of three parameters: power (W), distance from probe tip to tissue (mm) and application duration (s). 3 ABCFlex Probes were used to create 15 samples on ex vivo porcine small intestine for each combination of parameters. The depth of tissue effect for each sample was measured using a light microscope. RESULTS: Depth of tissue effect increases as power and application time increases. An increase of distance from the probe tip to the tissue results in a decrease in depth of tissue effect from a near contact to 1mm distance. Depth of tissue effect doesn’t significantly change from 1mm to 3mm distance. CONCLUSION: ABCFlex Probe can be used to achieve hemostasis in endoscopic procedures. Increasing power and application time increases the depth of thermal effect while increasing distance from the probe time to the surface of the tissue decreases the depth of tissue effect.

Several clinically successful clinical radio frequency vessel-sealing devices are currently available. The dominant thermodynamic principles at work involve tissue water vaporization processes. It is necessary to thermally denature vessel collagen, elastin and their adherent proteins to achieve a successful fusion. Collagens denature at middle temperatures, between about 60 and 90 C depending on heating time and rate. Elastin, and its adherent proteins, are more thermally robust, and require temperatures in excess of the boiling point of water at atmospheric pressure to thermally fuse. Rapid boiling at low apposition pressures leads to steam vacuole formation, brittle tissue remnants and frequently to substantial disruption in the vessel wall, particularly in high elastin-content arteries. High apposition pressures substantially increase the equilibrium boiling point of tissue water and are necessary to ensure a high probability of a successful seal. The FDM numerical models illustrate the beneficial effects of high apposition pressures.

Energy-based vessel sealing remains a common alternative to traditional mechanical ligation procedures, despite considerable uncertainty as to the origin and stability of vascular adhesion forces. Evidence of conformal changes in Collagen IA has fostered support of denatured collagen as the origin of tissue adhesion; experimental observation suggests that while pure collagen fails to adhere, remaining vascular constituents play a critical adhesive role. This study initiates a constitutive model of adhesion forces in thermal fusion by determining the effects of glycosaminoglycan (GAG) content on the bursting pressure of thermally sealed vessels. GAG content of porcine splenic arteries was progressively altered via pre-fusion treatment in Chondroitinase ABC (ChABC) for 0-5h at 1U/mL (n=10/gp.), followed by fusion with the ConMed ALTRUS® thermal fusion device and subsequent strength testing. Sulfated GAG (sGAG) concentrations as quantified by the Dimethylmethylene Blue (DMMB) assay were reduced in ChABC-treated vessels (5h) by 73.8 ± 4.2 % as compared with untreated tissue. Bursting pressures of ChABC-treated vessels (5h) were significantly greater than those of control vessels (800.33 ± 54.34 mmHg and 438.40 ± 51.81 mmHg respectively, p=2.0e-04). Histology enabled qualitative visualization of the treated arterial cross-section and of the bonding interface. The negative correlation between GAG content and arterial seal strengths suggests that by resisting water transport, arterial GAG presence may inhibit adhesive interactions between adjacent cellular tissue layers during energy-based vessel sealing. By elucidating the components which facilitate or inhibit adhesion in thermal vessel sealing, this study provides an important step towards understanding the chemistry underlying fusion and evaluating its potential for expansion to avascular tissues.

Tissue fusion devices apply heat and pressure to ligate or ablate blood vessels during surgery. Although this process is widely used, a predictive finite element (FE) model incorporating both structural mechanics and heat transfer has not been developed, limiting improvements to empirical evidence. This work presents the development of a novel damage parameter, which incorporates stress, water content and temperature, and demonstrates its application in a FE model. A FE model, using the Holzapfel-Gasser-Ogden strain energy function to represent the structural mechanics and equations developed by Cezo to model water content and heat transfer, was created to simulate the fusion or ablation of a porcine splenic artery. Using state variables, the stresses, temperature and water content are recorded and combined to create a single parameter at each integration point. The parameter is then compared to a critical value (determined through experiments). If the critical value is reached, the element loses all strength. If the value is not reached, no change occurs. Little experimental data exists for validation, but the resulting stresses, temperatures and water content fall within ranges predicted by prior work. Due to the lack of published data, additional experimental studies are being conducted to rigorously validate and accurately determine the critical value. Ultimately, a novel method for demonstrating tissue damage and fusion in a FE model is presented, providing the first step towards in-depth FE models simulating fusion and ablation of arteries.

The first bipolar vessel sealing system was developed nearly 15 years ago and has since become standard of care in surgery. These systems make use of radio frequency current that is delivered between bipolar graspers to permanently seal arteries, veins and tissue bundles. Conventional vessel sealing generators are based off traditional electrosurgery generator architecture and deliver high power (150-300 Watts) and high current using complex control and sense algorithms to adjust the output for vessel sealing applications. In recent years, a need for small-scale surgical vessel sealers has developed as surgeons strive to further reduce their footprint on patients. There are many technical challenges associated with miniaturization of vessel sealing devices including maintaining electrical isolation while delivering high current in a saline environment. Research into creating a small, 3mm diameter vessel sealer revealed that a highly simplified generator system could be used to achieve excellent results and subsequently a low power vessel sealing system was developed. This system delivers 25 Watts constant power while limiting voltage (≤ Vrms) and current (≤ Amps) until an impedance endpoint is achieved, eliminating the use of complicated control and sensing software. The result is optimized tissue effect, where high seal strength is maintained (> 360mmHg), but seal times (1.7 ± 0.7s versus 4.1 ± 0.7s), thermal spread (<1mm vs ≤2mm) and total energy delivery are reduced, when compared to an existing high power system.

As the use of hyperthermic ablation technologies has increased, so too has the need to understand their effects on tissue and their healing responses. This study was designed to characterize tissue injury and healing following hyperthermic vapor ablation in the in vivo porcine longissimus muscle model. The individual ablations were performed using the NxThera Vapor Delivery System (NxThera Inc., Minneapolis, MN). To assess the vapor ablation’s evolution, the swine were euthanized post-treatment on Day 0, Day 3, Day 7, Day 14, Day 28, Day 45 and Day 90. Triphenyltetrazolium chloride viability staining (TTC staining) was used to macroscopically assess the extent of each vapor ablation within the tissue. The ablation associated healing responses were then histologically evaluated for acute inflammation, chronic inflammation, foreign body reaction and fibrosis. Two zones of tissue injury were initially identified in the ablations: 1) a central zone of complete coagulative necrosis and 2) an outer “transition zone” of viable and non-viable cells. The ablations initially increased in size from Day 0 to Day 7 and then progressively decreased in size though Day 45. The initial Day 3 healing changes originated in the transition zone with minimal acute and chronic inflammation. As time progressed, granulation tissue began to form by Day 7 and peaked around Day 14. Collagen formation, deposition and remodeling began in the adjacent healthy tissue by Day 28, replaced the ablation site by Day 45 and reorganized by Day 90. In conclusion, this vapor ablation technology provided a non-desiccating form of hyperthermic ablation that resulted in coagulative necrosis without a central thermally/heat-fixed tissue component, followed a classical wound healing pathway, and healed with minimal associated inflammation.

An ultrasound applicator for endoluminal thermal therapy of pancreatic tumors has been introduced and evaluated through acoustic/biothermal simulations and ex vivo experimental investigations. Endoluminal therapeutic ultrasound constitutes a minimally invasive conformal therapy and is compatible with ultrasound or MR-based image guidance. The applicator would be placed in the stomach or duodenal lumen, and sonication would be performed through the luminal wall into the tumor, with concurrent water cooling of the wall tissue to prevent its thermal injury. A finite-element (FEM) 3D acoustic and biothermal model was implemented for theoretical analysis of the approach. Parametric studies over transducer geometries and frequencies revealed that operating frequencies within 1-3 MHz maximize penetration depth and lesion volume while sparing damage to the luminal wall. Patient-specific FEM models of pancreatic head tumors were generated and used to assess the feasibility of performing endoluminal ultrasound thermal ablation and hyperthermia of pancreatic tumors. Results indicated over 80% of the volume of small tumors (~2 cm diameter) within 35 mm of the duodenum could be safely ablated in under 30 minutes or elevated to hyperthermic temperatures at steady-state. Approximately 60% of a large tumor (~5 cm diameter) model could be safely ablated by considering multiple positions of the applicator along the length of the duodenum to increase coverage. Prototype applicators containing two 3.2 MHz planar transducers were fabricated and evaluated in ex vivo porcine carcass heating experiments under MR temperature imaging (MRTI) guidance. The applicator was positioned in the stomach adjacent to the pancreas, and sonications were performed for 10 min at 5 W/cm² applied intensity. MRTI indicated over 40⁰C temperature rise in pancreatic tissue with heating penetration extending 3 cm from the luminal wall.

An intra-luminal ultrasound catheter system (ReCor Medical’s Paradise System) has been developed to provide circumferential denervation of the renal sympathetic nerves, while preserving the renal arterial intimal and medial layers, in order to treat hypertension. The Paradise System features a cylindrical non-focused ultrasound transducer centered within a balloon that circulates cooling fluid and that outputs a uniform circumferential energy pattern designed to ablate tissues located 1-6 mm from the arterial wall and protect tissues within 1 mm. RF power and cooling flow rate are controlled by the Paradise Generator which can energize transducers in the 8.5-9.5 MHz frequency range. Computer simulations and tissue-mimicking phantom models were used to develop the proper power, cooling flow rate and sonication duration settings to provide consistent tissue ablation for renal arteries ranging from 5-8 mm in diameter. The modulation of these three parameters allows for control over the near-field (border of lesion closest to arterial wall) and far-field (border of lesion farthest from arterial wall, consisting of the adventitial and peri-adventitial spaces) depths of the tissue lesion formed by the absorption of ultrasonic energy and conduction of heat. Porcine studies have confirmed the safety (protected intimal and medial layers) and effectiveness (ablation of 1-6 mm region) of the system and provided near-field and far-field depth data to correlate with bench and computer simulation models. The safety and effectiveness of the Paradise System, developed through computer model, bench and in vivo studies, has been demonstrated in human clinical studies.

The development and in vivo testing of a high-intensity ultrasound thermal ablation catheter for epicardial ablation of the left ventricle (LV) is presented. Scar tissue can occur in the mid-myocardial and epicardial space in patients with nonischemic cardiomyopathy and lead to ventricular tachycardia. Current ablation technology uses radiofrequency energy, which is limited epicardially by the presence of coronary vessels, phrenic nerves, and fat. Ultrasound energy can be precisely directed to deliver targeted deep epicardial ablation while sparing intervening epicardial nerve and vessels.

The proof-of-concept ultrasound applicators were designed for sub-xyphoid access to the pericardial space through a steerable 14-Fr sheath. The catheter consists of two rectangular planar transducers, for therapy (6.4 MHz) and imaging (5 MHz), mounted at the tip of a 3.5-mm flexible nylon catheter coupled and encapsulated within a custom-shaped balloon for cooling.

Thermal lesions were created in the LV in a swine (n = 10) model in vivo. The ultrasound applicator was positioned fluoroscopically. Its orientation and contact with the LV were verified using A-mode imaging and a radio-opaque marker. Ablations employed 60-s exposures at 15 – 30 W (electrical power). Histology indicated thermal coagulation and ablative lesions penetrating 8 – 12 mm into the left ventricle on lateral and anterior walls and along the left anterior descending artery.

The transducer design enabled successful sparing from the epicardial surface to 2 – 4 mm of intervening ventricle tissue and epicardial fat. The feasibility of targeted epicardial ablation with catheter-based ultrasound was demonstrated.

To overcome the limitations of currently available clinical hyperthermia systems which are based on rigid waveguide antennas, a wearable microwave hyperthermia system is presented. A light wearable system can improve patient comfort and be located in close proximity to the breast, thereby enhancing energy deposition and reducing power requirements. The objective of this work was to design and assess the feasibility of a conformal patch antenna element of an array system to be integrated into a wearable hyperthermia bra. The feasibility of implementing antennas with silver printed ink technology on flexible substrates was evaluated. A coupled electromagnetic-bioheat transfer solver and a hemispheric heterogeneous numerical breast phantom were used to design and optimize a 915 MHz patch antenna. The optimization goals were device miniaturization, operating bandwidth, enhanced energy deposition pattern in targets, and reduced Efield back radiation. The antenna performance was evaluated for devices incorporating a hemispheric conformal groundplane and a rectangular groundplane configuration. Simulated results indicated a stable -10 dB return loss bandwidth of 88 MHz for both the conformal and rectangular groundplane configurations. Considering applied power levels restricted to 15 W, treatment volumes (T>41⁰C) and depth from the skin surface were 11.32 cm³ and 27.94 mm, respectively, for the conformal groundplane configuration, and 2.79 cm³ and 19.72 mm, respectively, for the rectangular groundplane configuration. E-field back-radiation reduced by 85.06% for the conformal groundplane compared to the rectangular groundplane configuration. A prototype antenna with rectangular groundplane was fabricatd and experimentally evaluated. The groundplane was created by printing silver ink (Metalon JS-B25P) on polyethylene terephthalate (PET) film surface. Experiments revealed stable antenna performance for power levels up to 15.3 W. In conclusion, the proposed patch antenna with conformal groundplane and prined ink technology shows promising performance to be integrated in a clinical array system.

The use of hyperthermia to treat cancer is well studied and has utilized numerous delivery techniques, including microwaves, radio frequency, focused ultrasound, induction heating, infrared radiation, warmed perfusion liquids (combined with chemotherapy), and recently, metallic nanoparticles (NP) activated by near infrared radiation (NIR) and alternating magnetic field (AMF) based platforms. It has been demonstrated by many research groups that ablative temperatures and cytotoxicity can be produced with locally NP-based hyperthermia. Such ablative NP techniques have demonstrated the potential for success. Much attention has also been given to the fact that NP may be administered systemically, resulting in a broader cancer therapy approach, a lower level of tumor NP content and a different type of NP cancer therapy (most likely in the adjuvant setting). To use NP based hyperthermia successfully as a cancer treatment, the technique and its goal must be understood and utilized in the appropriate clinical context. The parameters include, but are not limited to, NP access to the tumor (large vs. small quantity), cancer cell-specific targeting, drug carrying capacity, potential as an ionizing radiation sensitizer, and the material properties (magnetic characteristics, size and charge). In addition to their potential for cytotoxicity, the material properties of the NP must also be optimized for imaging, detection and direction. In this paper we will discuss the differences between, and potential applications for, ablative and non-ablative magnetic nanoparticle hyperthermia.

The use of nanotechnology for the treatment of cancer affords the possibility of highly specific tumor targeting and improved treatment efficacy. Iron oxide magnetic nanoparticles (IONPs) have demonstrated success as an ablative mono-therapy and targetable adjuvant therapy. However, the relative therapeutic value of intracellular vs. extracellular IONPs remains unclear. Our research demonstrates that both extracellular and intracellular IONPs generate cytotoxicity when excited by an alternating magnetic field (AMF). While killing individual cells via intracellular IONP heating is an attractive goal, theoretical models and experimental results suggest that this may not be possible due to limitations of cell volume, applied AMF, IONP concentration and specific absorption rate (SAR). The goal of this study was to examine the importance of tumor size (cell number) with respect to IONP concentration. Mouse mammary adenocarcinoma cells were incubated with IONPs, washed, spun into different pellet sizes (0.1, 0.5 and 2 million cells) and exposed to AMF. The level of heating and associated cytotoxicity depended primarily on the number of IONPs /amount Fe per cell pellet volume and the relative volume of the cell pellet. Specifically, larger cell pellets achieved greater relative cytotoxicity due to greater iron amounts, close association and subsequently higher temperatures.

Iron oxide nanoparticles (IONP) have therapeutic potential to deliver a thermal dose to tumors when activated in an alternating magnetic field (AMF). Through various targeting methods such as antibody labeling or injection site choice, delivery of IONPs to tumors yields enhanced treatment accuracy and efficacy. Despite this advantage, delivery an AMF, which is sufficient to result in clinically relevant IONP heating, can result in nonspecific tissue heating via the generation of eddy currents and tissue permeated by local electric fields (joule heating). The production of eddy current heating is a function of tissue size, geometry and composition as well as coil design and operation. The purpose of this research is to increase the level of energy deposited into the IONPs versus the non-target tissue (power ratio/PR)1 in order to improve target heating and reduce nonspecific tissue damage. We propose to improve the PR using two primary concepts: (1) reduce power deposition into non-target tissue by manipulating the fields and eddy current flow and (2) enhance heat removal from non-target tissue. We have shown that controlling tissue placement within the AMF field, accounting for tissue geometry, utilizing external cooling devices, and modifying the field properties can decrease non-target heating by more than 50%, at clinically relevant AMF levels, thereby allowing for an increase in thermal dose to the tumor and increasing the therapeutic ratio.

Iron oxide nanoparticles (IONP) are being developed for use as a cancer treatment. They have demonstrated efficacy when used either as a monotherapy or in conjunction with conventional chemotherapy and radiation. The success of IONP as a therapeutic tool depends on the delivery of a safe and controlled cytotoxic thermal dose to tumor tissue following activation with an alternating magnetic field (AMF). Prior to clinical approval, knowledge of IONP toxicity, biodistribution and physiological clearance is essential. This preliminary time-course study determines the acute toxicity and biodistribution of 110 nm dextran-coated IONP (iron) in mice, 7 days post systemic, at doses of 0.4, 0.6, and 1.0 mg Fe/ g mouse bodyweight. Acute toxicity, manifested as changes in the behavior of mice, was only observed temporarily at 1.0 mg Fe/ g mouse bodyweight, the highest dose administered. Regardless of dose, mass spectrometry and histological analysis demonstrated over 3 mg Fe/g tissue in organs within the reticuloendotheilial system (i.e. liver, spleen, and lymph nodes). Other organs (brain, heart, lungs, and kidney) had less than 0.5 mg Fe/g tissue with iron predominantly confined to the organ vasculature.

Iron oxide nanoparticles (IONPs) are one of several high-Z materials currently being investigated for their ability to enhance the cytotoxic effects of therapeutic ionizing radiation. Studies with iron oxide, silver, gold, and hafnium oxide suggest radiation dose, radiation energy, cell type, and the type and level of metallic nanoparticle are all critical factors in achieving radiation enhancement in tumor cells. Using a single 4 Gy radiation dose, we compared the level of tumor cell cytotoxicity at two different intracellular iron concentrations and two different radiation energies in vitro. IONPs were added to cell culture media at concentrations of 0.25 mg Fe/mL and 1.0 mg Fe/mL and incubated with murine breast adenocarcinoma (MTG-B) cells for 72 hours. Extracellular iron was then removed and cells were irradiated at either 662 keV or 10 MV. At the 0.25 mg Fe/mL dose (4 pg Fe/cell), radiation energy did not affect the level of cytotoxicity. However with 1.0 mg Fe/mL (9 pg Fe/cell), the higher 10 MV radiation energy resulted in 50% greater cytotoxicity as compared to cells without IONPs irradiated at this energy. These results suggest IONPs may be able to significantly enhance the cytotoxic effects of radiation and improve therapeutic ratio if they can be selectively associated with cancer cells and/or tumors. Ongoing in vivo studies of IONP radiation enhancement in a murine tumor model are too immature to draw conclusions from at this time, however preliminary data suggests similar effectiveness of IONP radiation enhancement at 6 MV and 18 MV energy levels. In addition to the IONP-based radiation enhancement demonstrated here, the use of tumor-localized IONP with an externally delivered, non-toxic alternating magnetic field affords the opportunity to selectively heat and kill tumor cells. Combining IONP-based radiation sensitization and heat-based cytotoxicity provides a unique and potentially highly effective opportunity for therapeutic ratio enhancement.

Although preliminary clinical trials are ongoing, successful the use of iron-oxide magnetic nanoparticles (IONP) for heatbased cancer treatments will depend on advancements in: 1) nanoparticle platforms, 2) delivery of a safe and effective alternating magnetic field (AMF) to the tumor, and 3) development of non-invasive, spatially accurate IONP imaging and quantification technique. This imaging technique must be able to assess tumor and normal tissue anatomy as well as IONP levels and biodistribution. Conventional CT imaging is capable of detecting and quantifying IONPs at tissue levels above 10 mg/gram; unfortunately this level is not clinically achievable in most situations. Conventional MRI is capable of imaging IONPs at tissue levels of 0.05 mg/gm or less, however this level is considered to be below the therapeutic threshold. We present here preliminary in vivo data demonstrating the ability of a novel MRI technique, Sweep Imaging with Fourier Transformation (SWIFT), to accurately image and quantify IONPs in tumor tissue in the therapeutic concentration range (0.1-1.0 mg/gm tissue). This ultra–short, T2 MRI method provides a positive Fe contrast enhancement with a reduced signal to noise ratio. Additional IONP signal enhancement techniques such as inversion recovery spectroscopy and variable flip angle (VFA) are also being studied for potential optimization of SWIFT IONP imaging. Our study demonstrates the use of SWIFT to assess IONP levels and biodistribution, in murine flank tumors, following intra-tumoral and systemic IONP administration. ICP-MS and quantitative histological techniques are used to validate the accuracy and sensitivity of SWIFT-based IONP imaging and quantification.

Real-time hyperpolarized (HP) ¹³C MR can be utilized during high-intensity focal ultrasound (HIFU) therapy to improve treatment delivery strategies, provide treatment verification, and thus reduce the need for more radical therapies for lowand intermediate-risk prostate cancers. The goal is to develop imaging biomarkers specific to thermal therapies of prostate cancer using HIFU, and to predict the success of thermal coagulation and identify tissues potentially sensitized to adjuvant treatment by sub-ablative hyperthermic heat doses. Mice with solid prostate tumors received HIFU treatment (5.6 MHz, 160W/cm², 60 s), and the MR imaging follow-ups were performed on a wide-bore 14T microimaging system. ¹³C-labeled pyruvate and urea were used to monitor tumor metabolism and perfusion accordingly. After treatment, the ablated tumor tissue had a loss in metabolism and perfusion. In the regions receiving sub-ablative heat dose, a timedependent change in metabolism and perfusion was observed. The untreated regions behaved as a normal untreated TRAMP prostate tumor would. This promising preliminary study shows the potential of using ¹³C MR imaging as biomarkers of HIFU/thermal therapies.

Nanosecond pulsed electric fields (nsPEF) cause the formation of small pores, termed nanopores, in the membrane of cells. Current nanoporation models treat nsPEF exposure as a purely electromagnetic phenomenon, but recent publications showing pressure transients, ROS production, temperature gradients, and pH waves suggest the stimulus may be physically and chemically multifactorial causing elicitation of diverse biological conditions and stressors. Our research group's goal is to quantify the breadth and participation of these stressors generated during nsPEF exposure and determine their relative importance to the observed cellular response. In this paper, we used advanced imaging techniques to identify a possible source of nsPEF-induced acoustic shock waves. nsPEFs were delivered in an aqueous media via a pair of 125 μm tungsten electrodes separated by 100 μm, mirroring our previously published cellular exposure experiments. To visualize any pressure transients emanating from the electrodes or surrounding medium, we used the Schlieren imaging technique. Resulting images and measurements confirmed that mechanical pressure waves and electrode-based stresses are formed during nsPEF, resulting in a clearer understanding of the whole exposure dosimetry. This information will be used to better quantify the impact of nsPEF-induced acoustic shock waves on cells, and has provided further evidence of non-electrical-field induced exposures for elicitation of bioieffects.

Second Harmonic Generation (SHG) imaging is a useful tool for examining the structure of interfaces between bulk materials. Recently, this technique was applied to detecting subtle perturbations in the structure of cellular membranes following nanosecond pulsed electric field (nsPEF) exposure. Monitoring the cell’s outer membrane as it is exposed to nsPEF via SHG has demonstrated that nanoporation is likely the root cause for size-specific, increased cytoplasmic membrane permeabilization. It is theorized that the area of the membrane covered by these pores is tied to pulse intensity or duration. The extent of this effect along the cell’s surface, however, has never been measured due to its temporal brevity and minute pore size. By enhancing the SHG technique developed and elucidated previously, we are able to obtain this information. Further, we vary the pulse width and amplitude of the applied stimulus to explore the mechanical changes of the membrane at various sites around the cell. By using this unique SHG imaging technique to directly visualize the change in order of phospholipids within the membrane, we are able to better understand the complex response of living cells to electric pulses.

Microwave ablation is a minimally invasive modality increasingly being used for thermal treatment of cancer in various organs. During ablation procedures, treatment planning is typically restricted to vendor specifications of expected ablation zone volumes based on experiments in unperfused ex vivo tissues, presuming parallel insertion of antennas. However, parallel antenna implants are not always clinically possible due to the restricted control of flexible antennas and presence of intervening organs. This paper aims to quantify the effect of non-parallel antenna implants on the ablation volume. 3D electromagnetic-bioheat transfer models were implemented to analyze ablation zone profiles created by dual antenna arrays. Parallel and non-parallel implants spaced 10-25 mm with antenna tips deviated to create converging or diverging configurations were analyzed. Volumetric Dice Similarity Coefficients (DSC) were calculated to compare ablation zone volumes for parallel and non-parallel configuration. Antenna tip displacements of 3 mm/antenna yielded an average DSC of 0.78. Tip displacements of 5 mm/antenna yielded a DSC of 0.78 and 0.64 for 15 mm and 20 mm antenna spacing, respectively. For ablation with dipole antennas as the frequency of operation decreases from 2.45 GHz to 915 MHz the similarity between the ablation zones for parallel and angled cases increased significantly. In conclusion, ablation volumes with non-parallel antenna implants may differ significantly from the parallel configuration. Patient-specific treatment planning tools may provide more accurate predictions of 3D-ablation volumes based on imaging data of actual implanted antenna configurations. Methods to compare ablation zone volumes incorporating uncertainty in antenna positions and experimental results to validate the numerical modelling are also presented.

Radio-frequency ablation (RFA) is a minimally invasive surgical procedure to thermally ablate the targeted diseased tissue. There have been many finite-element method (FEM) studies of cardiac and hepatic RFA, but hardly find any FEM study on endometrial ablation for abnormal uterine bleeding. In this paper, a FEM model was generated to analyze the temperature distribution of bipolar RF global endometrial ablation with three pairs of bipolar electrodes placed at the perimeter of the uterine cavity. COMSOL was utilized to calculate the RF electric fields and temperature fields by numerically solving the bioheat equation in the triangle uterine cavity range. The 55^°C isothermal surfaces show the shape of the ablation dimensions (depth and width), which reasonably matched the experimental results.

The underlying mechanism(s) responsible for nanoporation of phospholipid membranes by nanosecond pulsed electric fields (nsEP) remains unknown. The passage of a high electric field through a conductive medium creates two primary contributing factors that may induce poration: the electric field interaction at the membrane and the shockwave produced from electrostriction of a polar submersion medium exposed to an electric field. Previous work has focused on the electric field interaction at the cell membrane, through such models as the transport lattice method. Our objective is to model the shock wave cell membrane interaction induced from the density perturbation formed at the rising edge of a high voltage pulse in a polar liquid resulting in a shock wave propagating away from the electrode toward the cell membrane. Utilizing previous data from cell membrane mechanical parameters, and nsEP generated shockwave parameters, an acoustic shock wave model based on the Helmholtz equation for sound pressure was developed and coupled to a cell membrane model with finite-element modeling in COMSOL. The acoustic structure interaction model was developed to illustrate the harmonic membrane displacements and stresses resulting from shockwave and membrane interaction based on Hooke’s law. Poration is predicted by utilizing membrane mechanical breakdown parameters including cortical stress limits and hydrostatic pressure gradients.

A treatment planning platform for interstitial microwave hyperthermia was developed for practical, free-hand clinical implants. Such implants, consisting of non-parallel, moderately curved antennas with varying insertion depths, are used in HDR brachytherapy for treating locally advanced cancer.

Numerical models for commercially available MA251 antennas (915 MHz, BSD Medical) were developed in COMSOL Multiphysics, a finite element analysis software package. To expedite treatment planning, electric fields, power deposition and temperature rises were computed for a single straight antenna in 2D axisymmetric geometry. A precomputed library of electric field and temperature solutions was created for a range of insertion depths (5-12 cm) and blood perfusion rates (0.5-5 kg/m³/s). 3D models of multiple antennas and benchtop phantoms experiments using temperature-sensitive liquid crystal paper to monitor heating by curved antennas were performed for comparative evaluation of the treatment planning platform.

A patient-customizable hyperthermia treatment planning software package was developed in MATLAB with capabilities to interface with a commercial radiation therapy planning platform (Oncentra, Nucleotron), import patient and multicatheter implant geometries, calculate insertion depths, and perform hyperthermia planning with antennas operating in asynchronous or synchronous mode. During asynchronous operation, the net power deposition and temperature rises were approximated as a superposition sum of the respective quantities for one single antenna. During synchronous excitation, a superposition of complex electrical fields was performed with appropriate phasing to compute power deposition. Electric fields and temperatures from the pre-computed single-antenna library were utilized following appropriate non-rigid coordinate transformations. Comparison to 3D models indicated that superposition of electric fields around parallel antennas is valid when they are at least 15 mm apart. Phantom experiments with curved antennas produced temperature profiles quite similar to those created using the planning system.

The hyperthermia planning software allowed users to select power and phasing, assess the corresponding 3D contours of energy and temperature, and optimize treatment parameters through gradient search techniques. The system produces fairly accurate temperature distributions in cases when the antennas are at least 15 mm apart.

A major challenge for image guided tumor ablation is the high treatment variability due to heterogeneous tissue characteristics and thermal sinks. In this work, we present a methodology to analyze the geometry of the treatment zones and treatment zone variability. Our first contribution is an applicator centric co-ordinate system which enables us to compare treatment zones and vendor specifications across patients. Our second contribution is the analysis of the shape of the ablation zone using applicator centric longitudinal 2D cross sections. We present initial results of applying this methodology to analyze the geometry and variability in synthetic examples like ellipsoid, sphere and real microwave ablation zones in lung and liver.

As an increasing number of studies use gold nanoparticles (AuNPs) for potential medicinal, biosensing and therapeutic applications, the synthesis and use of readily functional, bio-compatible nanoparticles is receiving much interest. For these efforts, the particles are often taken up by the cells to allow for optimum sensing or therapeutic measures. This process typically requires incubation of the particles with the cells for an extended period. In an attempt to shorten and control this incubation, we investigated whether nanosecond pulsed electric field (nsPEF) exposure of cells will cause a controlled uptake of the particles. NsPEF are known to induce the formation of nanopores in the plasma membrane, so we hypothesized that by controlling the number, amplitude or duration of the nsPEF exposure, we could control the size of the nanopores, and thus control the particle uptake. Chinese hamster ovary (CHO-K1) cells were incubated sub-10 nm AuNPs with and without exposure to 600-ns electrical pulses. Contrary to our hypothesis, the nsPEF exposure was found to actually decrease the particle uptake in the exposed cells. This result suggests that the nsPEF exposure may be affecting the endocytotic pathway and processes due to membrane disruption.

Phosphatidylinositol_4,5-biphosphate (PIP₂) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP₂ releases inositol-1,4,5-triphosphate (IP₃) from the membrane, activating IP₃ receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP₂ in the cellular membrane, activating the IP₃ signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP₂ are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP₂ hydrolysis), PIP₂ depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP₃/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP₂ in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

Exposure of cells to very short (<1 μs) electric pulses in the megavolt/meter range have been shown to cause disruption of the plasma membrane. This disruption is often characterized by the formation of numerous small pores (<2 nm in diameter) in the plasma membrane that last for several minutes, allowing the flow of ions into the cell. These small pores are called nanopores and the resulting damage to the plasma membrane is referred to as nanoporation. Nanosecond electrical pulse (nsEP) exposure can impart many different stressors on a cell, including electrical, electro-chemical, and mechanical stress. Thus, nsEP exposure is not a “clean” insult, making determination of the mechanism of nanoporation quite difficult. We hypothesize that nsEP exposure creates acoustic shock waves capable of causing nanoporation. Microarray analysis of primary adult human dermal fibroblasts (HDFa) exposed to nsEP, indicated several genes associated with mechanical stress were selectively upregulated 4 h post exposure. The idea that nanoporation is caused by external mechanical force from acoustic shock waves has, to our knowledge, not been investigated. This work will critically challenge the existing paradigm that nanoporation is caused solely by an electric-field driven event and could provide the basis for a plausible explanation for electroporation.