Matthew R. DeWitt

In-vitro bipolar nano- and microsecond electro-pulse bursts for irreversible electroporation therapies

Under the influence of external electric fields, cells experience a rapid potential buildup across the cell membrane. Above a critical threshold of electric field strength, permanent cell damage can occur, resulting in cell death. Typical investigations of electroporation effects focus on two distinct regimes. The first uses sub-microsecond duration, high field strength pulses while the second uses longer (50 μs+) duration, but lower field strength pulses. Here we investigate the effects of pulses between these two extremes. The charging behavior of the cell membrane and nuclear envelope is evaluated numerically in response to bipolar pulses between 250 ns and 50 μs. Typical irreversible electroporation protocols expose cells to 90 monopolar pulses, each 100 μs in duration with a 1 second inter-pulse delay. Here, we replace each monopolar waveform with a burst of alternating polarity pulses, while keeping the total energized time (100 μs), burst number (80), and inter-burst delay (1s) the same. We show that these bursts result in instantaneous and delayed cell death mechanisms and that there exists an inverse relationship between pulse-width and toxicity despite the delivery of equal quantities of energy. At 1500 V/cm only treatments with bursts containing 50 μs pulses (2×) resulted in viability below 10%. At 4000 V/cm, bursts with 1 μs (100×), 2 μs (50×), 5 μs (20×), 10 μs (10×), and 50 μs (2×) duration pulses reduced viability below 10% while bursts with 500 ns (200×) and 250 ns (400×) pulses resulted in viabilities of 31% and 92%, respectively.

Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth.

Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.

Influence of hyperthermia on efficacy and uptake of carbon nanohorn-cisplatin conjugates

Single-walled carbon nanohorns (SWNHs) have significant potential for use in photothermal therapies due to their capability to absorb near infrared light and deposit heat. Additionally, their extensive relative surface area and volume makes them ideal drug delivery vehicles. Novel multimodal treatments are envisioned in which laser excitation can be utilized in combination with chemotherapeutic-SWNH conjugates to thermally enhance the therapeutic efficacy of the transported drug. Although mild hyperthermia (41-43 °C) has been shown to increase cellular uptake of drugs such as cisplatin (CDDP) leading to thermal enhancement, studies on the effects of hyperthermia on cisplatin loaded nanoparticles are currently limited. After using a carbodiimide chemical reaction to attach CDDP to the exterior surface of SWNHs and nitric acid to incorporate CDDP in the interior volume, we determined the effects of mild hyperthermia on the efficacy of the CDDP-SWNH conjugates. Rat bladder transitional carcinoma cells were exposed to free CDDP or one of two CDDP-SWNH conjugates in vitro at 37 °C and 42 °C with the half maximal inhibitory concentration (IC50) for each treatment. The in vitro results demonstrate that unlike free CDDP, CDDP-SWNH conjugates do not exhibit thermal enhancement at 42 °C. An increase in viability of 16% and 7% was measured when cells were exposed at 42 deg compared to 37 deg for the surface attached and volume loaded CDDP-SWNH conjugates, respectively. Flow cytometry and confocal microscopy showed a decreased uptake of CDDP-SWNH conjugates at 42 °C compared to 37 °C, revealing the importance of nanoparticle uptake on the CDDP-SWNH conjugates efficacy, particularly when hyperthermia is used as an adjuvant, and demonstrates the effect of particle size on uptake during mild hyperthermia. The uptake and drug release studies elucidated the difference in viability seen in the drug efficacy studies at different temperatures. We speculate that the disparity in thermal enhancement efficacy observed for free drug compared to the drug SWNH conjugates is due to their intrinsic size differences and, therefore, their mode of cellular uptake: diffusion or endocytosis. These experiments indicate the importance of tuning properties of nanoparticle-drug conjugates to maximize cellular uptake to ensure thermal enhancement in nanoparticle mediated photothermal-chemotherapy treatments.

High-Frequency Irreversible Electroporation: Safety and Efficacy of Next-Generation Irreversible Electroporation Adjacent to Critical Hepatic Structures:

Irreversible electroporation (IRE) is a nonthermal ablation modality employed to induce in situ tissue-cell death. This study sought to evaluate the efficacy of a novel high-frequency IRE (H-FIRE) system to perform hepatic ablations across, or adjacent to, critical vascular and biliary structures. Using ultrasound guidance H-FIRE electrodes were placed across, or adjacent to, portal pedicels, hepatic veins, or the gall bladder in a porcine model. H-FIRE pulses were delivered (2250 V, 2-5-2 pulse configuration) in the absence of cardiac synchronization or intraoperative paralytics. Six hours after H-FIRE the liver was resected and analyzed. Nine ablations were performed in 3 separate experimental groups (major vessels straddled by electrodes, electrodes placed adjacent to major vessels, electrodes placed adjacent to gall bladder). Average ablation time was 290 ± 63 seconds. No electrocardiogram abnormalities or changes in vital signs were observed during H-FIRE. At necropsy, no vascular damage, coagulated-thermally desiccated blood vessels, or perforated biliary structures were noted. Histologically, H-FIRE demonstrated effective tissue ablation and uniform induction of apoptotic cell death in the parenchyma independent of vascular or biliary structure location. Detailed microscopic analysis revealed minor endothelial damage within areas subjected to H-FIRE, particularly in regions proximal to electrode insertion. These data indicate H-FIRE is a novel means to perform rapid, reproducible IRE in liver tissue while preserving gross vascular/biliary architecture. These characteristics raise the potential for long-term survival studies to test the viability of this technology toward clinical use to target tumors not amenable to thermal ablation or resection.

Vascularized microfluidic platforms to mimic the tumor microenvironment: MICHNA et al.

Microfluidic technology has led to the development of advanced in vitro tumor platforms that overcome the challenges of in vivo animal and in vitro two dimensional models. This paper presents platform designs and methods used to develop complex vascularized in vitro models to mimic the tumor microenvironment. Features of these platforms include a continuous, aligned endothelium that allows for cell‐cell interactions between vasculature and tumor cells. A novel platform for fabrication of a single endothelialized microchannel encased within a collagen platform hosting breast cancer cells was developed and utilized to study the influence of cellular interaction on transport phenomenon through vasculature in a hyperpermeable tumor microenvironment. This platform relies on subtractive tissue engineering fabrication techniques. Through confocal imaging we have demonstrated that the platform produces enhanced vessel leakiness recapitulating physiological features of the tumor microenvironment. The influence of tumor endothelial interactions on transport of particles was also demonstrated. Additionally, we designed two more complex and intricate endothelialized microfluidic networks by combining lithographic techniques with additive tissue engineering methods. We created a network platform consisting of interconnected microchannels to model a highly vascularized system and successfully perfused the system with fluorescent particles. Finally, we developed a physiologically representative in vitro microfluidic platform with vasculature patterned from in vivo data showing the versatility of these systems to replicate the complex geometries of tumor microvasculature and dynamically measured particle transport. Overall, we have shown the ability to develop functional microfluidic vascular tumor platforms of varying complexities and demonstrated their utility for studying spatial particle transport within these systems.

Burst and continuous high frequency irreversible electroporation protocols evaluated in a 3D tumor model

High frequency irreversible electroporation (H-FIRE) is an emerging cancer therapy which uses bursts of alternating polarity pulses to target and destroy the membranes of cells within a predictable volume. Typically, 2 µs pulses are rapidly repeated 24-50 times to create a 48-100 µs long energy burst. Bursts are repeated 100×  at 1 Hz, resulting in an integrated energized time of 0.01 s per treatment. A 3D in vitro tumor model was used to investigate H-FIRE parameters in search of optimal energy timing protocols. Monopolar IRE treatments (100  ×  100 µs positive polarity pulses) resulted in a lethal electric field threshold of 423 V cm-1. Baseline H-FIRE treatments (100  ×  100 µs bursts of 2 µs pulses) resulted in a lethal threshold of 818 V cm-1. Increasing the number of H-FIRE bursts from 100×  to 1000×  reduced the lethal threshold to 535 V cm-1. An alternative diffuse H-FIRE protocol, which delivers 4 µs pulse cycles (one positive and one negative 2 µs pulse) continuously at 100 Hz, resulted in the lowest H-FIRE lethal threshold of 476 V cm-1. Finite element simulations using 5 kV pulses predict an IRE ablation volume of 3.9 cm3 (1.7 cm diameter) and a maximum H-FIRE ablation volume of 5.3 cm3 (2.4 cm diameter) when a clinical electrode and grounding pad configuration is used. Ablations as large as 15.7 cm3 (3.3 cm diameter) are predicted for H-FIRE treatments with 10 kV pulses. These results combine to demonstrate the importance of electrode geometry, pulse timing, and clinical delivery protocols for the creation of large clinically meaningful ablations.

Tunable Collagen Microfluidic Platform to Study Nanoparticle Transport in the Tumor Microenvironment

This chapter describes the motivation and protocol for creating a perfused 3D microfluidic in vitro platform representative of the tumor microenvironment to study nanoparticle transport. The cylindrical vascularized tumor platform described consists of a central endothelialized microchannel surrounded by a collagen hydrogel matrix containing cancer cells. This system can be employed to investigate key nanoparticle transport events in the tumor such as extravasation, diffusion within the extracellular matrix, and nanoparticle uptake. This easily manufactured tumor platform can be used for novel nanoparticle refinement focused on optimizing nanoparticle features such as size, shape, and functionalization method. This can yield ideal nanoparticles with properties that facilitate increased transport within the tumor microenvironment, leading to more effective nanoparticle-based treatments for cancer including nanoparticle-based drug delivery systems.

Spatial Measurement of Viability in Tissue Phantoms and Ex Vivo Bladder Tissue in Response to Photothermal Therapy and Single Walled Carbon Nanohorns

Cancer is one of the most deadly diseases and leading cause of death. Laser based photothermal therapy can provide a minimally invasive alternative to surgical resection. The selectivity and effectiveness of laser therapy can be greatly enhanced when photoabsorbing nanoparticles such as nanoshells, single walled carbon nanotubes, multi-walled carbon nanotubes, or single wall carbon nanohorns (SWNHs) are introduced into the tissue[1]. Quantitative methods for measuring tumor response to nanoparticle enhanced laser therapies are critical for determining appropriate laser parameters and nanoparticle properties needed to achieve maximum therapeutic benefit. We have previously reported a new method for measuring two dimensional (2D) spatial viability distributions in cell monolayers in response to laser irradiation and nanoparticles. This method has been refined to allow determination of cell viability in three dimensions (3D) within a more physiologically representative tumor volume. This refined method was used to determine the viability of breast cancer cells suspended within sodium alginate tissue phantoms following treatment with SWNHs and external laser irradiation. The tumor treatment volume was accurately quantified in response to varying laser treatment parameters and nanoparticle concentrations. Spatial cellular viability was also measured in ex vivo pig bladders in response to SWNHs and laser irradiation to provide a more anatomically relevant environment. These new measurement methods enable quantification of spatial viability and therapeutic effectiveness, using 3D tumor environments which are more representative than cell monolayers.Copyright