Dynamics of Cell Death Due to Electroporation Using Different Pulse Parameters as Revealed by Different Viability Assays.

The mechanisms of cell death due to electroporation are still not well understood. Recent studies suggest that cell death due to electroporation is not an immediate all-or-nothing response but rather a dynamic process that occurs over a prolonged period of time. To investigate whether the dynamics of cell death depends on the pulse parameters or cell lines, we exposed different cell lines to different pulses [monopolar millisecond, microsecond, nanosecond, and high-frequency bipolar (HFIRE)] and then assessed viability at different times using different viability assays. The dynamics of cell death was observed by changes in metabolic activity and membrane integrity. In addition, regardless of pulse or cell line, the dynamics of cell death was observed only at high electroporation intensities, i.e., high pulse amplitudes and/or pulse number. Considering the dynamics of cell death, the clonogenic assay should remain the preferred viability assay for assessing viability after electroporation.

Optimal Irreversible Electroporation Combined with Nano-Enabled Immunomodulatory to Boost Systemic Antitumor Immunity.

In this work, we proposed nµPEF, a novel pulse configuration combining nanosecond and microsecond pulses (nµPEF), to enhance tumor ablation in irreversible electroporation (IRE) for oncological therapy. nµPEF demonstrated improved efficacy in inducing immunogenic cell death, positioning it as a potential candidate for next-generation ablative therapy. However, the immune response elicited by nµPEF alone was insufficient to effectively suppress distant tumors. To address this limitation, we developed PPR@CM-PD1, a genetically engineered nanovesicle. PPR@CM-PD1 employed a polyethylene glycol-polylactic acid-glycolic acid (PEG-PLGA) nanoparticle encapsulating the immune adjuvant imiquimod and coated with a genetically engineered cell membrane expressing programmed cell death protein 1 (PD1). This design allowed PPR@CM-PD1 to target both the innate immune system through toll-like receptor 7 (TLR7) agonism and the adaptive immune system through programmed cell death protein 1/programmed cell death-ligand 1 (PD1/PDL1) checkpoint blockade. In turn, nµPEF facilitated intratumoral infiltration of PPR@CM-PD1 by modulating the tumor stroma. The combination of nµPEF and PPR@CM-PD1 generated a potent and systemic antitumor immune response, resulting in remarkable suppression of both nµPEF-treated and untreated distant tumors (abscopal effects). This interdisciplinary approach presents a promising perspective for oncotherapy and holds great potential for future clinical applications.

Scheduled dosage regimen by irreversible electroporation of loaded erythrocytes for cancer treatment.

Precise control of cargo release is essential but still a great challenge for any drug delivery system. Irreversible electroporation (IRE), utilizing short high-voltage pulsed electric fields to destabilize the biological membrane, has been recently approved as a non-thermal technique for tumor ablation without destroying the integrity of adjacent collagenous structures. Due to the electro-permeating membrane ability, IRE might also have great potential to realize the controlled drug release in response to various input IRE parameters, which were tested in a red blood cell (RBC) model in this work. According to the mathematical simulation model of a round biconcave disc-like cell based on RBC shape and dielectric characteristics, the permeability and the pore density of the RBC membrane were found to quantitatively depend on the pulse parameters. To further provide solid experimental evidence, indocyanine green (ICG) and doxorubicin (DOX) were both loaded inside RBCs (RBC@DOX&ICG) and the drug release rates were found to be tailorable by microsecond pulsed electric field (µsPEF). In addition, µsPEF could effectively modulate the tumor stroma to augment therapy efficacy by increasing micro-vessel density and permeability, softening extracellular matrix, and alleviating tumor hypoxia. Benefiting from these advantages, this IRE-responsive RBC@DOX&ICG achieved a remarkably synergistic anti-cancer effect by the combination of µsPEF and chemotherapy in the tumor-bearing mice model, with the survival time increasing above 90 days without tumor burden. Given that IRE is easily adaptable to different plasma membrane-based vehicles for delivering diverse drugs, this approach could offer a general applicability for cancer treatment.

Analysis on reversible/irreversible electroporation region in lung adenocarcinoma cell model in vitro with electric pulses delivered by needle electrodes.

Irreversible electroporation (IRE) is a minimally invasive tumor therapy using pulsed electric field with high intensity while the important tissues such as blood vessel, bile duct, and nerve are preserved. In addition to ablation area, reversible electroporation (RE) region is also generated using needle electrodes for pulse delivery. The goal of this work is to study the generation of RE region and ablation region on a 2D lung adenocarcinoma cell model in vitro. The tumor model is exposed to electric pulses with various number. The calcium AM and propidium iodide (PI) are examined to detect the ablation area and electroporation area, respectively. The results show that electroporation area firstly tends to plateau after approximately 50 pulses, while the ablation area continues to increase. The percentage of IRE area in total electroporation area increases with additional pulses, which means that RE region could be gradually turned into ablation area with increased pulse number. However, the percentage of IRE area only achieves to 54% for 200 pulses, which indicates that RE region still cannot be completely removed. RE and IRE thresholds appear to converge as the number of pulses increases. An equation between pulse number and the electric field threshold of ablation including the electric field threshold of RE is also provided for lung adenocarcinoma cell ablation. This work may have the value for the optimization of IRE protocols on tumor ablation.