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.

Cultivating well-being in engineering graduate students through mindfulness training.

The mental health crisis in graduate education combined with low treatment rates among engineering graduate students underscores the need for engineering graduate programs to provide effective methods to promote well-being. There is an extensive body of neuroscience research showing that contemplative practices, such as mindfulness, produce measurable effects on brain function and overall well-being. We hypothesized that a mindfulness-based training program designed for engineering graduate students would improve emotional well-being and, secondarily, enhance research capacity. An initial pilot study was conducted at a single institution (Phase 1), followed by a larger study conducted at both the original and a second institution (Phase 2) to gather additional data and show the program's transferability. The program comprised eight weekly mindfulness training sessions. Individuals in the study were randomly assigned to either an intervention group or wait-list control group. We administered pre- and post-test surveys with quantitative measures designed to assess emotional and physical well-being, as well as creativity, research satisfaction, and desire to contribute to the betterment of society. Participants also completed a summative survey to evaluate the impact of the program on their well-being and research. Analysis revealed statistically significant findings: improved emotional health, decreased neuroticism, increased positive affect, decreased negative affect, and increased mindfulness in the intervention groups compared to the control groups. Intervention groups in Phase 2 also reported statistically significant improvement in satisfaction with their research. Our findings suggest that mindfulness training has the potential to play a vital professional and personal development role in graduate engineering education.

Microwave-induced thermoacoustic signal characteristics in a dynamic temperature environment.

We conduct a simulation-based study to investigate the impact of a dynamic temperature environment on the characteristics of microwave-induced thermoacoustic signals. We investigate thermoacoustic signals that are generated using an interstitial microwave ablation antenna powered by a microsecond pulsed microwave source. Two temperature regimes are examined: first, a spatially uniform temperature throughout the medium to experimentally validate the simulation model, and second, the realistic, spatially nonuniform temperature profiles that arise during microwave ablation. We employ a multi-physics model that considers electromagnetics, heat transfer, and acoustic physics to simulate the coupled processes of microwave absorption and heating of the medium and thermoacoustic signal generation and propagation. An interstitial coaxial antenna is used to generate microsecond microwave pulses that simultaneously induce microwave heating and excite thermoacoustic signals via microwave pulse absorption. We find that thermoacoustic signal characteristics are highly temperature-dependent and thus change significantly within an environment where temperature varies through space and time. Furthermore, the temperature-dependent properties within the active region of the antenna drive the evolution of thermoacoustic signal characteristics. Temperature-dependent thermoacoustic signal characteristics can be exploited to track the progress of microwave ablation. Consequently, microwave-induced thermoacoustic imaging is a promising method for monitoring microwave ablation in real-time.

Ex Vivo Performance of a Flexible Microwave Ablation Antenna.

OBJECTIVE: In this study, we investigate the performance of a flexible microwave ablation antenna for generating localized ablation zones. METHODS: We designed a helical dipole antenna to operate at 1.9 GHz in egg white and liver. Semi-rigid prototypes of the antenna were fabricated and used to perform ablation experiments in egg white and perfused liver. Pulsed and continuous-wave power deliveries at different power levels were used. Flexible prototypes of the antenna were fabricated and used to perform ex vivo ablation experiments in perfused liver. RESULTS: Pulsing was effective in reducing the shaft heating of semi-rigid cables. The antenna was capable of producing substantial ablation zones in perfused liver. Typical diameters (perpendicular to the antenna axis) of generated ablation zones with semi-rigid antennas in egg white and perfused liver were 30 mm and 20 mm, respectively. The flexible antenna had a good impedance match while bent. Average diameter of generated ablation zones by the flexible antenna with 10-W continuous-wave experiments in perfused liver was 26 mm. No significant difference was observed between the performances of semi-rigid and flexible prototypes. CONCLUSION: The flexible helical dipole antenna is capable of maintaining its good impedance match while bent and can generate substantial ablation zones in presence of perfusion. SIGNIFICANCE: The proposed flexible antenna is promising for minimally invasive treatment of tumors that are otherwise inaccessible by rigid antennas. One example is lung where a catheter-based deployment of the flexible antenna into the tumor via airways may substantially reduce risks associated with using rigid antennas.

Multi-physics modeling of thermoacoustic pulse generation and propagation during pulsed microwave ablation of tissue.

Microwave-induced thermoacoustic (TA) imaging is a potential alternative to conventional real-time imaging methods for monitoring microwave ablation (MWA). In this study, we develop a multi-physics model for the generation and propagation of microwave-induced TA signals during pulsed MWA. Our model couples electromagnetics, heat transfer, and acoustics physics. We compare simulation and experimental results for a pulsed MWA system wherein a coaxial MWA antenna is used to heat water. The simulated and experimentally measured TA signals for this configuration are in good qualitative agreement. This multi-physics modeling tool is valuable for understanding the fundamentals of TA signal generation and propagation from within an evolving ablation zone.

A Pilot Study of the Impact of Microwave Ablation on the Dielectric Properties of Breast Tissue.

Percutaneous microwave ablation (MWA) is a promising technology for patients with breast cancer, as it may help treat individuals who have less aggressive cancers or do not respond to targeted therapies in the neoadjuvant or pre-surgical setting. In this study, we investigate changes to the microwave dielectric properties of breast tissue that are induced by MWA. While similar changes have been characterized for relatively homogeneous tissues, such as liver, those prior results are not directly translatable to breast tissue because of the extreme tissue heterogeneity present in the breast. This study was motivated, in part by the expectation that the changes in the dielectric properties of the microwave antenna's operation environment will be impacted by tissue composition of the ablation target, which includes not only the tumor, but also its margins. Accordingly, this target comprises a heterogeneous mix of malignant, healthy glandular, and adipose tissue. Therefore, knowledge of MWA impact on breast dielectric properties is essential for the successful development of MWA systems for breast cancer. We performed ablations in 14 human ex-vivo prophylactic mastectomy specimens from surgeries that were conducted at the UW Hospital and monitored the temperature in the vicinity of the MWA antenna during ablation. After ablation we measured the dielectric properties of the tissue and analyzed the tissue samples to determine both the tissue composition and the extent of damage due to the ablation. We observed that MWA induced cell damage across all tissue compositions, and found that the microwave frequency-dependent relative permittivity and conductivity of damaged tissue are lower than those of healthy tissue, especially for tissue with high fibroglandular content. The results provide information for future developments on breast MWA systems.

The Performance of Higher Frequency Microwave Ablation in the Presence of Perfusion.

OBJECTIVE: In this paper, we investigate the impact of perfusion on the performance of microwave ablation across a large frequency range. METHODS: We designed multiple microwave ablation antennas to operate in liver tissue at discrete frequencies in the range 1.9-18 GHz. We performed electromagnetic simulations to calculate microwave power absorption patterns. Five-minute, 25 W ablation experiments were performed at each frequency in perfused and nonperfused ex vivo porcine livers, and thermal lesion dimensions were measured. RESULTS: The volume of greatest microwave power absorption shrinks by two orders of magnitude as the frequency is increased from 1.9 to 18 GHz. Mean thermal lesion volumes are consistent across the frequency range for a given perfusion state and are about three times smaller under active perfusion. Typical thermal lesion diameters (perpendicular to the antenna axis) were 24 mm and 16 mm for nonperfused and perfused ablations, respectively. No significant differences in axial ratio were observed among different frequency groups in active-perfusion experiments. CONCLUSION: Higher-frequency microwave ablation produces thermal lesions with volumes comparable to those achieved at lower frequencies, even in strongly perfused environments. SIGNIFICANCE: Higher-frequency microwave ablation is appealing because it allows for more flexibility in antenna design. A critical issue concerning the feasibility of higher frequency microwave ablation, considering its strong dependence on heat diffusion to grow thermal lesions, is its performance in strongly perfused environments. This paper shows that higher frequency microwave ablation achieves thermal lesions comparable to those from microwave ablation performed at conventional frequencies in both non- and strongly perfused environments.

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.

Human Breast Phantoms: Test Beds for the Development of Microwave Diagnostic and Therapeutic Technologies.

Over the past two decades, there has been enormous growth in research activity for microwave diagnostic and therapeutic technologies that target the breast. The clinical need for new tools in the breast cancer armamentarium, combined with the promising lowcost, nonionizing nature of microwave technologies, has fueled these investigations. High-fidelity breast phantoms are essential components of computational and experimental test beds for investigating and accurately assessing the performance of new devices, algorithms, and systems related to microwave breast cancer detection and/or treatment.

The impact of frequency on the performance of microwave ablation.

PURPOSE: The use of higher frequencies in percutaneous microwave ablation (MWA) may offer compelling interstitial antenna design advantages over the 915 MHz and 2.45 GHz frequencies typically employed in current systems. To evaluate the impact of higher frequencies on ablation performance, we conducted a comprehensive computational and experimental study of microwave absorption and tissue heating as a function of frequency. METHODS: We performed electromagnetic and thermal simulations of MWA in ex vivo and in vivo porcine muscle at discrete frequencies in the 1.9-26 GHz range. Ex vivo ablation experiments were performed in the 1.9-18 GHz range. We tracked the size of the ablation zone across frequency for constant input power and ablation duration. Further, we conducted simulations to investigate antenna feed line heating as a function of frequency, input power, and cable diameter. RESULTS: As the frequency was increased from 1.9 to 26 GHz the resulting ablation zone dimensions decreased in the longitudinal direction while remaining relatively constant in the radial direction; thus at higher frequencies the overall ablation zone was more spherical. However, cable heating at higher frequencies became more problematic for smaller diameter cables at constant input power. CONCLUSION: Comparably sized ablation zones are achievable well above 1.9 GHz, despite increasingly localised power absorption. Specific absorption rate alone does not accurately predict ablation performance, particularly at higher frequencies where thermal diffusion plays an important role. Cable heating due to ohmic losses at higher frequencies may be controlled through judicious choices of input power and cable diameter.

Development and application of human breast phantoms in microwave diagnostic and therapeutic technologies.

We highlight recent progress in the development of high-fidelity numerical and physical breast phantoms. These phantoms mimic the anatomical structure and physical properties that are relevant to accurately portraying microwave interactions with the human breast. The phantoms are currently being used in numerous laboratory studies of microwave diagnostic and therapeutic technologies for a variety of potential clinical applications in breast health and disease management.

A 3-D Level Set Method for Microwave Breast Imaging.

OBJECTIVE: Conventional inverse-scattering algorithms for microwave breast imaging result in moderate resolution images with blurred boundaries between tissues. Recent 2-D numerical microwave imaging studies demonstrate that the use of a level set method preserves dielectric boundaries, resulting in a more accurate, higher resolution reconstruction of the dielectric properties distribution. Previously proposed level set algorithms are computationally expensive, and thus, impractical in 3-D. In this paper, we present a computationally tractable 3-D microwave imaging algorithm based on level sets. METHODS: We reduce the computational cost of the level set method using a Jacobian matrix, rather than an adjoint method, to calculate Frechet derivatives. We demonstrate the feasibility of 3-D imaging using simulated array measurements from 3-D numerical breast phantoms. We evaluate performance by comparing full 3-D reconstructions to those from a conventional microwave imaging technique. We also quantitatively assess the efficacy of our algorithm in evaluating breast density. RESULTS: Our reconstructions of 3-D numerical breast phantoms improve upon those of a conventional microwave imaging technique. The density estimates from our level set algorithm are more accurate than those of the conventional microwave imaging, and the accuracy is greater than that reported for mammographic density estimation. CONCLUSION: Our level set method leads to a feasible level of computational complexity for full 3-D imaging, and reconstructs the heterogeneous dielectric properties distribution of the breast more accurately than conventional microwave imaging methods. SIGNIFICANCE: 3-D microwave breast imaging using a level set method is a promising low-cost, nonionizing alternative to current breast imaging techniques.

A TSVD Analysis of the Impact of Polarization on Microwave Breast Imaging using an Enclosed Array of Miniaturized Patch Antennas.

Microwave breast imaging performance is fundamentally dependent on the quality of information contained within the scattering data. We apply a truncated singular-value decomposition (TSVD) method to evaluate the information contained in a simulated scattering scenario wherein a compact, shielded array of miniaturized patch antennas surrounds an anatomically realistic numerical breast phantom. In particular, we investigate the impact of different antenna orientations (and thus polarizations), namely two array configurations with uniform antenna orientations and one mixed-orientation array configuration. The latter case is of interest because it may offer greater flexibility in antenna and array design. The results of this analysis indicate that mixed-polarization configurations do not degrade information quality compared to uniform-polarization configurations and in fact may enhance imaging performance, and thus represent viable design options for microwave breast imaging systems.

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.

Microwave ablation at 10.0 GHz achieves comparable ablation zones to 1.9 GHz in ex vivo bovine liver.

We demonstrate the feasibility of using high-frequency microwaves for tissue ablation by comparing the performance of a 10 GHz microwave ablation system with that of a 1.9 GHz system. Two sets of floating sleeve dipole antennas operating at these frequencies were designed and fabricated for use in ex vivo experiments with bovine livers. Combined electromagnetic and transient thermal simulations were conducted to analyze the performance of these antennas. Subsequently, a total of 16 ablation experiments (eight at 1.9 GHz and eight at 10.0 GHz) were conducted at a power level of 42 W for either 5 or 10 min. In all cases, the 1.9 and 10 GHz experiments resulted in comparable ablation zone dimensions. Temperature monitoring probes revealed faster heating rates in the immediate vicinity of the 10.0 GHz antenna compared to the 1.9 GHz antenna, along with a slightly delayed onset of heating farther from the 10 GHz antenna, suggesting that heat conduction plays a greater role at higher microwave frequencies in achieving a comparably sized ablation zone. The results obtained from these experiments agree very well with the combined electromagnetic/thermal simulation results. These simulations and experiments show that using lower frequency microwaves does not offer any significant advantages, in terms of the achievable ablation zones, over using higher frequency microwaves. Indeed, it is demonstrated that high-frequency microwave antennas may be used to create reasonably large ablation zones. Higher frequencies offer the advantage of smaller antenna size, which is expected to lead to less invasive interstitial devices and may possibly lead to the development of more compact multielement arrays with heating properties not available from single-element antennas.

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.

Beamforming-Enhanced Inverse Scattering for Microwave Breast Imaging.

We present a focal-beamforming-enhanced formulation of the distorted Born iterative method (DBIM) for microwave breast imaging. Incorporating beamforming into the imaging algorithm has the potential to mitigate the effect of noise on the image reconstruction. We apply the focal-beamforming-enhanced DBIM algorithm to simulated array measurements from two MRI-derived, anatomically realistic numerical breast phantoms and compare its performance to that of the DBIM formulated with two non-focal schemes. The first scheme simply averages scattered field data from reciprocal antenna pairs while the second scheme discards reciprocal pairs. Images of the dielectric properties are reconstructed for signal-to-noise ratios (SNR) ranging from 35 dB down to 0 dB. We show that, for low SNR, the focal beamforming algorithm creates reconstructions that are of higher fidelity with respect to the exact dielectric profiles of the phantoms as compared to reconstructions created using the non-focal schemes. At high SNR, the focal and non-focal reconstructions are of comparable quality.

Multi-Band Miniaturized Patch Antennas for a Compact, Shielded Microwave Breast Imaging Array.

We present a comprehensive study of a class of multi-band miniaturized patch antennas designed for use in a 3D enclosed sensor array for microwave breast imaging. Miniaturization and multi-band operation are achieved by loading the antenna with non-radiating slots at strategic locations along the patch. This results in symmetric radiation patterns and similar radiation characteristics at all frequencies of operation. Prototypes were fabricated and tested in a biocompatible immersion medium. Excellent agreement was obtained between simulations and measurements. The trade-off between miniaturization and radiation efficiency within this class of patch antennas is explored via a numerical analysis of the effects of the location and number of slots, as well as the thickness and permittivity of the dielectric substrate, on the resonant frequencies and gain. Additionally, we compare 3D quantitative microwave breast imaging performance achieved with two different enclosed arrays of slot-loaded miniaturized patch antennas. Simulated array measurements were obtained for a 3D anatomically realistic numerical breast phantom. The reconstructed breast images generated from miniaturized patch array data suggest that, for the realistic noise power levels assumed in this study, the variations in gain observed across this class of multi-band patch antennas do not significantly impact the overall image quality. We conclude that these miniaturized antennas are promising candidates as compact array elements for shielded, multi-frequency microwave breast imaging systems.

Dielectric characterization of PCL-based thermoplastic materials for microwave diagnostic and therapeutic applications.

We propose the use of a polycaprolactone (PCL)-based thermoplastic mesh as a tissue-immobilization interface for microwave imaging and microwave hyperthermia treatment. An investigation of the dielectric properties of two PCL-based thermoplastic materials in the frequency range of 0.5-3.5 GHz is presented. The frequency-dependent dielectric constant and effective conductivity of the PCL-based thermoplastics are characterized using measurements of microstrip transmission lines fabricated on substrates comprised of the thermoplastic meshes. We also examine the impact of the presence of a PCL-based thermoplastic mesh on microwave breast imaging. We use a numerical test bed comprised of a previously reported 3-D anatomically realistic breast phantom and a multi-frequency microwave inverse scattering algorithm. We demonstrate that the PCL-based thermoplastic material and the assumed biocompatible medium of vegetable oil are sufficiently well matched such that the PCL layer may be neglected by the imaging solution without sacrificing imaging quality. Our results suggest that PCL-based thermoplastics are promising materials as tissue immobilization structures for microwave diagnostic and therapeutic applications.

A TSVD analysis of microwave inverse scattering for breast imaging.

A variety of methods have been applied to the inverse scattering problem for breast imaging at microwave frequencies. While many techniques have been leveraged toward a microwave imaging solution, they are all fundamentally dependent on the quality of the scattering data. Evaluating and optimizing the information contained in the data are, therefore, instrumental in understanding and achieving optimal performance from any particular imaging method. In this paper, a method of analysis is employed for the evaluation of the information contained in simulated scattering data from a known dielectric profile. The method estimates optimal imaging performance by mapping the data through the inverse of the scattering system. The inverse is computed by truncated singular-value decomposition of a system of scattering equations. The equations are made linear by use of the exact total fields in the imaging volume, which are available in the computational domain. The analysis is applied to anatomically realistic numerical breast phantoms. The utility of the method is demonstrated for a given imaging system through the analysis of various considerations in system design and problem formulation. The method offers an avenue for decoupling the problem of data selection from the problem of image formation from that data.