Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells.

Previous studies have shown greater fluorophore uptake during electroporation on the anode-facing side of the cell than on the cathode-facing side. Based on these observations, we hypothesized that hyperpolarizing a cell before electroporation would decrease the requisite pulsed electric field intensity for electroporation outcomes, thereby yielding a higher probability of reversible electroporation at lower electric field strengths and a higher probability of irreversible electroporation (IRE) at higher electric field strengths. In this study, we tested this hypothesis by hyperpolarizing HL-60 cells using ionomycin before electroporation. These cells were then electroporated in a solution containing propidium iodide, a membrane integrity indicator. After 20 min, we added trypan blue to identify IRE cells. Our results showed that hyperpolarizing cells before electroporation alters the pulsed electric field intensity thresholds for reversible electroporation and IRE, allowing for greater control and selectivity of electroporation outcomes.

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells.

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Toward contrast-enhanced microwave-induced thermoacoustic imaging of breast cancer: an experimental study of the effects of microbubbles on simple thermoacoustic targets.

Microwave-induced thermoacoustic tomography (MI-TAT) is an imaging technique that exploits dielectric contrast at microwave frequencies while creating images with ultrasound resolution. We propose the use of microbubbles as a dielectric contrast agent for enhancing the sensitivity of MI-TAT for breast cancer detection. As an initial investigation of this concept, we experimentally studied the extent to which the microwave-induced thermoacoustic response of a dielectric target is modified by the presence of air-filled glass microbubbles. We created mixtures of ethylene glycol with varying weight percentages of microbubbles and characterized both their microwave properties (0.5-6 GHz) and thermoacoustic response when irradiated with microwave energy at 3 GHz. Our data show that the microbubbles considerably lowered the relative permittivity, electrical conductivity and thermoacoustic response of the ethylene glycol mixtures. We hypothesize that the interstitial infusion of microbubbles to a tumor site will similarly create a smaller thermoacoustic response compared to the pre-contrast-agent response, thereby enhancing sensitivity through the use of differential imaging techniques.

Microwave-induced mass transport enhancement in nano-porous aluminum oxide membranes.

Experiments were conducted to compare the annealing of nano-porous aluminum oxide membranes by 2.45 GHz microwave radiation and by conventional (resistive element) furnace heating. The starting material was Al2O3 membranes that were 60 microm thick, 13 mm in diameter, and containing pores of approximately 200 nm diameter. Changes in the porosity and morphology were recorded from digital processing of scanning electron microscope (SEM) images. The data indicates that both microwave and conventionally-heated annealing resulted in a decrease of surface porosity and an apparent increase in the number of pores. However, microwave annealing consistently resulted in a 4-5% greater reduction in porosity and a greater increase in the number of (small) pores than conventionally-heated annealing. These results are consistent with a non-thermal mechanism for microwave-enhanced surface diffusion, although the complex morphology of the pores precluded a quantitative theoretical analysis.

An examination of athermal photonic effects on boron diffusion and activation during microwave rapid thermal processing.

This work examines the role of 672 nm optical illumination on the diffusion and activation of B in Si as a function of various factors. The factors studied include length of anneal, maximum temperature of anneal, type of co-implanted and pre-amorphizing species and ambient oxygen concentration. The anneal conditions fell into one of three categories: (1) high-temperature (> 900 degrees C) spike and 10-second anneals; (2) low temperature (550 degrees C) 30-minute anneals; and (3) room-temperature long-term "anneals". Implanted species included BF2 implants, pre-amorphized B-only implants, and pre-amorphized BF2 implants. Finally, the ambient oxygen concentration was varied from atmospheric pressure to 100 ppm. The results show that illumination: (1) affects the diffusion of B on spike anneal time frames; (2) has limited effect on diffusion over 10-second time frames; and (3) fails to enhance the activation of B during low-temperature solid phase epitaxy and at room temperature. Additionally, illumination has no effect on B-diffusion when oxygen is present in ambient concentrations but does show an effect when the presence of oxygen is restricted. Finally, the presence of F affects both the net diffusion of B in Si and the relative effect of illumination on the diffusion of B.

A large-scale study of the ultrawideband microwave dielectric properties of normal breast tissue obtained from reduction surgeries.

The efficacy of emerging microwave breast cancer detection and treatment techniques will depend, in part, on the dielectric properties of normal breast tissue. However, knowledge of these properties at microwave frequencies has been limited due to gaps and discrepancies in previously reported small-scale studies. To address these issues, we experimentally characterized the wideband microwave-frequency dielectric properties of a large number of normal breast tissue samples obtained from breast reduction surgeries at the University of Wisconsin and University of Calgary hospitals. The dielectric spectroscopy measurements were conducted from 0.5 to 20 GHz using a precision open-ended coaxial probe. The tissue composition within the probe's sensing region was quantified in terms of percentages of adipose, fibroconnective and glandular tissues. We fit a one-pole Cole-Cole model to the complex permittivity data set obtained for each sample and determined median Cole-Cole parameters for three groups of normal breast tissues, categorized by adipose tissue content (0-30%, 31-84% and 85-100%). Our analysis of the dielectric properties data for 354 tissue samples reveals that there is a large variation in the dielectric properties of normal breast tissue due to substantial tissue heterogeneity. We observed no statistically significant difference between the within-patient and between-patient variability in the dielectric properties.

A large-scale study of the ultrawideband microwave dielectric properties of normal, benign and malignant breast tissues obtained from cancer surgeries.

The development of microwave breast cancer detection and treatment techniques has been driven by reports of substantial contrast in the dielectric properties of malignant and normal breast tissues. However, definitive knowledge of the dielectric properties of normal and diseased breast tissues at microwave frequencies has been limited by gaps and discrepancies across previously published studies. To address these issues, we conducted a large-scale study to experimentally determine the ultrawideband microwave dielectric properties of a variety of normal, malignant and benign breast tissues, measured from 0.5 to 20 GHz using a precision open-ended coaxial probe. Previously, we reported the dielectric properties of normal breast tissue samples obtained from reduction surgeries. Here, we report the dielectric properties of normal (adipose, glandular and fibroconnective), malignant (invasive and non-invasive ductal and lobular carcinomas) and benign (fibroadenomas and cysts) breast tissue samples obtained from cancer surgeries. We fit a one-pole Cole-Cole model to the complex permittivity data set of each characterized sample. Our analyses show that the contrast in the microwave-frequency dielectric properties between malignant and normal adipose-dominated tissues in the breast is considerable, as large as 10:1, while the contrast in the microwave-frequency dielectric properties between malignant and normal glandular/fibroconnective tissues in the breast is no more than about 10%.

Ultrawideband temperature-dependent dielectric properties of animal liver tissue in the microwave frequency range.

The development of ultrawideband (UWB) microwave diagnostic and therapeutic technologies, such as UWB microwave breast cancer detection and hyperthermia treatment, is facilitated by accurate knowledge of the temperature- and frequency-dependent dielectric properties of biological tissues. To this end, we characterize the temperature-dependent dielectric properties of a representative tissue type-animal liver-from 0.5 to 20 GHz. Since discrete-frequency linear temperature coefficients are impractical and inappropriate for applications spanning wide frequency and temperature ranges, we propose a novel and compact data representation technique. A single-pole Cole-Cole model is used to fit the dielectric properties data as a function of frequency, and a second-order polynomial is used to fit the Cole-Cole parameters as a function of temperature. This approach permits rapid estimation of tissue dielectric properties at any temperature and frequency.

FDTD analysis of a gigahertz TEM cell for ultra-wideband pulse exposure studies of biological specimens.

Gigahertz transverse electromagnetic (GTEM) transmission cells have been previously used to experimentally study exposure of biological cells to ultra-wideband (UWB), monopolar, electromagnetic pulses. Using finite-difference time-domain (FDTD) simulations we examine the time-dependent electric field waveforms and energy dose spatial distributions within a finite volume of biological cell culture medium during these experiments. The simulations show that when one or more flasks containing cell culture media are placed inside the GTEM cell, the uniform fields of the empty GTEM cell are significantly perturbed. The fields inside the cell culture medium, representing the fields to which the biological cells are exposed, are no longer monopolar and are spatially highly nonuniform. These effects result from a combination of refraction and distortion of the incident wave, combined with excitation of resonant eigenmodes within the cell culture medium volume. The simulations show that these distortions of the incident waveform may be mitigated by supporting the sample on a high permittivity pedestal and modifying the incident waveform to more closely approximate a Gaussian pulse. Under all simulated conditions, the estimated maximum temperature rises are completely negligible, ensuring that any experimentally observed unusual cell function or histopathology can be associated with nonthermal effects.

Mechanisms for phase distortion in a traveling wave tube.

We present a view of the physics of phase distortion in a traveling wave tube (TWT) based on unique insights afforded by the MUSE models of a TWT [IEEE Trans. Plasma Sci. 30, 1063 (2002)]]. The conclusion, supported by analytic theory and simulations, is that prior to gain compression phase distortion is due to harmonic frequencies in the electron beam and the resulting "intermodulation" frequency at the fundamental, and not the often cited "slowing down of electrons in the electron beam." We draw these conclusions based on MUSE simulations that allow explicit control of electron beam frequency content, an analytic solution to the S-MUSE model [IEEE Trans. Plasma Sci. 30, 1063 (2002)]] that reveals that phase distortion is due to the fact that the fundamental frequency is an intermodulation product of itself, and large signal LATTE [IEEE Trans. Plasma Sci. 30, 1063 (2002)]] simulations that are modified to remove the effect of the slowing down of electrons in the electron beam. As applications of the theory we compare S-MUSE simulations to an amplitude phase model using the analytic phase transfer curve, we study dependence of phase distortion on circuit dispersion and electron beam parameters at the second harmonic with large signal LATTE simulations for narrow and wide band TWT designs, and we consider the phase distortion theory in the context of TWT linearization.

Eulerian method for computing multivalued solutions of the Euler-Poisson equations and applications to wave breaking in klystrons.

We provide methods of computing multivalued solutions to the Euler-Poisson system and test them in the context of a klystron amplifier. An Eulerian formulation capable of computing multivalued solutions is derived from a kinetic description of the Euler-Poisson system and a moment closure. The system of the moment equations may be closed due to the special structure of the solution in phase space. The Eulerian moment equations are computed for a velocity modulated electron beam, which has been shown by prior Lagrangian theories to break in a finite time and form multivalued solutions. The results of the Eulerian moment equations are compared to direct computation of the kinetic equations and a Lagrangian method also developed in the paper. We use the Lagrangian formulation for the explicit computation of wave breaking time and location for typical velocity modulation boundary conditions.

Clinically relevant CNT dispersions with exceptionally high dielectric properties for microwave theranostic applications.

We present a formulation for achieving stable high-concentration (up to 20 mg/ml) aqueous dispersions of carbon nanotubes (CNTs) with exceptionally high microwave-frequency (0.5-6 GHz) dielectric properties. The formulation involves functionalizing CVD-synthesized CNTs via sonication in nitric and sulfuric acid. The overall chemical integrity of the CNTs is largely preserved, as demonstrated via physical and chemical characterizations, despite significant shortening and functionalization with oxygen-containing groups. This is attributed to the protected inner walls of double-walled CNTs in the samples. The resulting CNT dispersions show greatly enhanced dielectric properties compared to a CNT-free control. For example, at 3 GHz, the average relative permittivity and effective conductivity across several 20 mg/ml CNT samples were increased by ∼ 70% and ∼ 400%, respectively, compared to the control. These CNT dispersions exhibit the stability and extraordinary microwave properties desired in systemically administered theranostic agents for microwave diagnostic imaging and/or thermal therapy.

Cationic peptide exposure enhances pulsed-electric-field-mediated membrane disruption.

BACKGROUND: The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF's ability to disrupt plasma membranes. METHODOLOGY/PRINCIPAL FINDINGS: We exposed leukemia cells to 40 µs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell's PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1-2 kV/cm, exposure to >50 µg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. CONCLUSIONS/SIGNIFICANCE: Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired.

Toward carbon-nanotube-based theranostic agents for microwave detection and treatment of breast cancer: enhanced dielectric and heating response of tissue-mimicking materials.

The experimental results reported in this paper suggest that single-walled carbon nanotubes (SWCNTs) have the potential to enhance dielectric contrast between malignant and normal tissue for microwave detection of breast cancer and facilitate selective heating of malignant tissue for microwave hyperthermia treatment of breast cancer. In this study, we constructed tissue-mimicking materials with varying concentrations of SWCNTs and characterized their dielectric properties and heating response. At SWCNT concentrations of less than 0.5% by weight, we observed significant increases in the relative permittivity and effective conductivity. In microwave heating experiments, we observed significantly greater temperature increases in mixtures containing SWCNTs. These temperature increases scaled linearly with the effective conductivity of the mixtures. This work is a first step towards the development of functionalized, tumor-targeting SWCNTs as theranostic (integrated therapeutic and diagnostic) agents for microwave breast cancer detection and treatment.