Molecular sampling with vacuum-assisted tissue electroporation is a novel, minimally invasive method for molecular profiling of solid lesions. In this paper, we report on the design of the ...battery-powered pulsed electric field generator and electrode configuration for an electroporation-based molecular sampling device for skin cancer diagnostics. Using numerical models of skin electroporation corroborated by the potato tissue phantom model, we show that the electroporated tissue volume, which is the maximum volume for biomarker sampling, strongly depends on the electrode’s geometry, needle electrode skin penetration depths, and the applied pulsed electric field protocol. In addition, using excised human basal cell carcinoma (BCC) tissues, we show that diffusion of proteins out of human BCC tissues into water strongly depends on the strength of the applied electric field and on the time after the field application. The developed numerical simulations, confirmed by experiments in potato tissue phantoms and excised human cancer lesions, provide essential tools for the development of electroporation-based molecular markers sampling devices for personalized skin cancer diagnostics.
Introduction: The aim of this study was to review the effect of irreversible electroporation parameter settings on the size of the ablation zone and the occurrence of thermal effects. This insight ...would help to optimize treatment protocols and effectively ablate a tumor while controlling the occurrence of thermal effects. Methods: Various individual studies report the influence of variation in electroporation parameters on the ablation zone size or occurrence of thermal effects. However, no connections have yet been established between these studies. With the aim of closing the gap in the understanding of and personalizing irreversible electroporation parameter settings, a systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A quality assessment was performed using an in-house developed grading tool based on components of commonly used grading domains. Data on the electroporation parameters voltage, number of electrodes, inter-electrode distance, active needle length, pulse length/number/protocol/frequency, and pulse interval were extracted. Ablation zone size and temperature data were grouped per parameter. Spearman correlation and linear regression were used to define the correlation with outcome measures. Results: A total of 7661 articles were screened, of which 18 preclinical studies (animal and phantom studies) met the inclusion criteria. These studies were graded as moderate (4/18) and low (14/18) quality. Only the applied voltage appeared to be a significant linear predictor of ablation zone size: length, surface, and volume. The pulse number was moderately but nonlinearly correlated with the ablation zone length. Thermal effects were more likely to occur for higher voltages (≥2000 V), higher number of electrodes, and increased active needle length. Conclusion: Firm conclusions are limited since studies that investigated and precisely reported the influence of electroporation parameters on the ablation zone size and thermal effects were scarce and mostly graded low quality. High-quality studies are needed to improve the predictability of the combined effect of variation in parameter combinations and optimize irreversible electroporation treatment protocols.
The influence of pulsed electric fields with different waveforms on the electroporation of Chlorella vulgaris was investigated in this research. A pulsed power generator that can produce high-voltage ...pulses with arbitrary waveforms was developed. This device was utilized to generate pulsed electric fields of nine different waveforms (rectangle, bipolar rectangle, sine, half-sine, exponential decay, exponential increase, oscillation superimposed on rectangle, two-step rectangle, and triangle) with the same amplitude (17.5 kV/cm) and energy (0.27 J/pulse) for the electroporation of Chlorella vulgaris. The results showed that the pulse waveform played a significant role in the electroporation, which could be explained by the duration of transmembrane potential above threshold Δφth and by the frequent and rapid changes in the electric field above threshold Eth. Among the nine pulses, the oscillating superimposed rectangular pulse and half-sinusoidal pulse had the highest electroporation rate, with an improvement of about 40% compared to the conventional rectangular pulse.
•A pulse generator that generates pulses with arbitrary waveforms is reported.•Electroporation experiments using pulses with unconventional waveforms.•The unconventional pulse works about 40% better than the rectangular pulse.•A numerical modeling of electroporation was established to analyze the results.
•IRE is generally considered to be a non-thermal ablation modality.•Treatments with 1–8 µs pulses demonstrate a significant temperature dependence.•Significantly larger ablations were produced at ...physiological temperatures.•Clinically relevant treatment zones possible with microsecond duration pulses.
Irreversible electroporation (IRE) is generally considered to be a non-thermal ablation modality. This study was designed to examine the relative effect of temperature on IRE ablation sizes for equivalent dose treatments with constitutive pulses between 1 and 100 µs. 3D in-vitro brain tumor models maintained at 10 °C, 20 °C, 30 °C, or 37 °C were exposed to 500 V treatments using a temperature control algorithm to limit temperature increases to 5 °C. Treatments consisted of integrated energized times (doses) of 0.01 or 0.1 s. Pulse width, electrical dose, and initial temperature were all found to significantly affect the size of ablations and the resulting lethal electric field strength. The smallest ablations were created at 10 °C and ELethal were calculated to be 1729, 1359, 929, 777, 483 V/cm for 0.01 s treatments with 1, 2, 4, 8, and 100 µs pulses, respectively. At 37 °C these values decreased to 773, 614, 507, 462, and 394 V/cm, respectively. Increasing the dose from 0.01 to 0.1 s at 37 °C resulted in statistically significant decreases (p < 0.001) in ELethal for all treatments except for the 100 µs group. This study found that IRE is a thermally mediated, dose-dependent ablation modality for pulses on the order of one microsecond. Tissue temperatures are not accounted for when determining ablative boundaries in treatment planning algorithms. This work demonstrates that data generated at room temperature may not be predictive of ablation volumes in-vivo and that local temperatures should be accounted for in treatment planning.
Chimeric antigen receptor (CAR)‐T cell therapy has emerged as a promising cell‐based immunotherapy approach for treating blood disorders and cancers, but genetically engineering CAR‐T cells is ...challenging due to primary T cells’ sensitivity to conventional gene delivery approaches. The current viral‐based method can typically involve significant operating costs and biosafety hurdles, while bulk electroporation (BEP) can lead to poor cell viability and functionality. Here, a non‐viral electroactive nanoinjection (ENI) platform is developed to efficiently negotiate the plasma membrane of primary human T cells via vertically configured electroactive nanotubes, enabling efficient delivery (68.7%) and expression (43.3%) of CAR genes in the T cells, with minimal cellular perturbation (>90% cell viability). Compared to conventional BEP, the ENI platform achieves an almost threefold higher CAR transfection efficiency, indicated by the significantly higher reporter GFP expression (43.3% compared to 16.3%). By co‐culturing with target lymphoma Raji cells, the ENI‐transfected CAR‐T cells’ ability to effectively suppress lymphoma cell growth (86.9% cytotoxicity) is proved. Taken together, the results demonstrate the platform's remarkable capacity to generate functional and effective anti‐lymphoma CAR‐T cells. Given the growing potential of cell‐based immunotherapies, such a platform holds great promise for ex vivo cell engineering, especially in CAR‐T cell therapy.
An electroactive nanoinjection (ENI) platform consisting of arrays of vertically configured conductive nanotubes, which mediate the loading and efficient nanoinjection of CAR construct into primary human T cells, with negligible cellular damage, is developed. The engineered ENI platform provides a non‐viral, low‐voltage (10 V), scalable, and reusable intracellular delivery system that can generate functional and effective anti‐lymphoma CAR‐T cells.
Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as ..."bipolar cancellation," enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing the electric field in the pairs (180° direction change) caused 2-fold (1 Hz) or 20-fold (833 kHz) weaker electroporation than the train of single nsEPs. Reducing the angle between pulse directions in the pairs weakened cancellation and replaced it with facilitation at angles <160° (1 Hz) and <130° (833 kHz). Facilitation plateaued at about three-fold stronger electroporation compared to single pulses at 90-100° angle for both nsEP frequencies. The profound dependence of the efficiency on the angle enables novel protocols for highly selective focal electroporation at one electrode in a three-electrode array while avoiding effects at the other electrodes. Nanosecond-resolution imaging of cell membrane potential was used to link the selectivity to charging kinetics by co- and counter-directional nsEPs.
High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of ...such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100μs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1μs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses.
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•Real-time imaging and permeability analysis were performed post-electroporation treatment.•Rapid bipolar electrical pulses induce lower membrane permeability than longer monopolar pulses.•Transmembrane transfer of molecules may be achieved using trains of short pulse widths.•Rapid bipolar pulses need larger amplitudes to disrupt cells similar to longer monopolar pulses.
Label‐Free Electroporation
In article number 2310221, Ruiguo Yang and co‐workers propose a porous substrate‐based electroporation platform for high‐throughput and controlled delivery using ...commercially available cell culture inserts. Transepithelial electrical impedance (TEEI) monitoring was incorporated to the platform, providing insight into the intermediate steps of the electroporation process. The TEEI changes were found to correlate with delivery efficiency, enabling label‐free delivery.
Objectives
To determine the safety, quality of life (QoL) and short‐term oncological outcomes of primary focal irreversible electroporation (IRE) for the treatment of localized prostate cancer (PCa), ...and to identify potential risk factors for oncological failure.
Patients and Methods
Patients who met the consensus guidelines on patient criteria and selection methods for primary focal therapy were eligible for analysis. Focal IRE was performed for organ‐confined clinically significant PCa, defined as high‐volume disease with Gleason sum score 6 (International Society of Urological Pathology ISUP grade 1) or any Gleason sum score of 7 (ISUP grades 2–3). Oncological, adverse event (AE) and QoL outcome data, with a minimum of 6 months’ follow‐up, were analysed. Patient characteristics and peri‐operative treatment variables were compared between patients with and without oncological failure on follow‐up biopsy. Wilcoxon's signed rank test, Wilcoxon's rank sum test and the chi‐squared test were used to assess statistically significant differences in paired continuous, unpaired continuous and categorical variables respectively.
Results
A total of 63 patients met all eligibility criteria and were included in the final analysis. No high‐grade AEs occurred. QoL questionnaire analysis demonstrated no significant change from baseline in physical (P = 0.81), mental (P = 0.48), bowel (P = 0.25) or urinary QoL domains (P = 0.41 and P = 0.25), but there was a mild decrease in the sexual QoL domain (median score 66 at baseline vs 54 at 6 months; P < 0.001). Compared with baseline, a decline of 70% in prostate‐specific antigen level (1.8 ng/mL, interquartile range 0.96–4.8 ng/mL) was seen at 6–12 months. A narrow safety margin (P = 0.047) and system errors (P = 0.010) were identified as potential early risk factors for in‐field oncological failure. In‐field and whole‐gland oncological control on follow‐up biopsies was 84% (38/45 patients) and 76% (34/45 patients); this increased to 97% (38/39 patients) and 87% (34/39 patients) when patients treated with a narrow safety margin and system errors were excluded.
Conclusion
Our data support the safety and feasibility of focal IRE as a primary treatment for localized PCa with effective short‐term oncological control in carefully selected men.
This paper proposes a new model of membrane electropermeabilisation that combines the water content of the membrane and the transmembrane voltage. Interestingly, thanks to a well defined free-energy ...of the membrane, we somehow generalise the seminal approach of Chizmadzhev, Weaver and Krassowska, getting rid of the geometrical cylindrical assumption upon which most of the current electroporation models are based. Our approach is physically relevant and we recover a surface diffusion equation of the lipid phase proposed by Leguèbe et al. in a previous phenomenological model. We also perform a fine analysis of the involved nonlocal operators in two simple configurations (a spherical membrane and a flat periodic membrane) that enables us to compare the time constants of the phenomenon in spherical and flat membranes. An accurate splitting scheme combined with Fast Fourier Transforms is developed for efficient computations of the model. Our numerical results enable us to make a link between the molecular dynamics simulations of membrane permeabilisation and the experimental observations on vesicles and cells.
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