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.

In Vitro Experimental and Numerical Studies on the Preferential Ablation of Chemo-Resistant Tumor Cells Induced by High-Voltage Nanosecond Pulsed Electric Fields.

Chemoresistance causes tumor recurrence and metastasis, resulting in poor clinical outcomes and low survival, and has been considered an obstacle to tumor therapy. The development of novel therapeutic approaches that can effectively kill chemoresistant tumor cells (CRTCs) is therefore critical to overcoming these obstacles. OBJECTIVE: Here, we introduce an emerging physical feature-based therapeutic approach based on nanosecond pulsed electric fields (nsPEFs). The goal of this study is to investigate the effect of nsPEFs on CRTCs. METHODS: The cell viability, ablation effects on a 3D-cultured scaffold, and lethal thresholds of nsPEFs were evaluated according to fluorescence staining assays. RESULTS: nsPEF treatment preferentially affected chemoresistant cells (A549/CDDP) with a higher cell viability inhibition ability/cell death rate, larger ablation area, and lower ablation threshold compared to their respective homologous tumor cells (A549). The experimental and theoretical studies suggested that nsPEFs displayed selective behavior toward intracellular structures. With this selective character, nsPEFs can induce higher electroporation effects (e.g., higher pore number, larger electroporation area, and faster fluorescence dissipation on the nuclear envelope) on CRTCs due to their larger nuclear size and cell membrane capacitance. CONCLUSION: These findings demonstrated that nsPEFs induced preferential ablation of CRTCs over their respective homologous tumor cells. SIGNIFICANCE: This study provides an experimental and theoretical basis for the study of killing CRTCs by electrical treatments and suggests potential applications in the optimization of novel anti-chemoresistance methods.

Nanosecond pulses targeting intracellular ablation increase destruction of tumor cells with irregular morphology.

The decrease in killing sensitivity of the cell membrane to microsecond pulse electric fields (µs-PEFs) is ascribed mainly to the aberrant morphology of cancer cells, with clear statistical correlations observed between cell size and shape defects and the worsening of the electrical response to the PEF. In this paper, nanosecond pulsed electric fields (ns-PEFs) inducing the nucleus effect and µs-PEFs targeting the cell membrane were combined to enhance destruction of irregular cells. The fluorescence dissipation levels of the nuclear membrane and cell membrane exposed to the µs, ns, and ns + µs pulse protocols were measured and compared, and a dynamic electroporation model of irregular cells was established by the finite element software COMSOL. The results suggest that the cell membrane disruption induced by µs-PEFs is worse for extremely irregular cells and depends strongly on cellular morphology. However, the nuclear membrane disruption induced by ns-PEFs does not scale with irregularity, suggesting the use of a combination of ns-PEFs with µs-PEFs to target the nuclear and cell membranes. We demonstrate that ns + µs pulses can significantly enhance the fluorescence dissipation of the cell and nuclear membranes. Overall, our findings indicate that ns + µs pulses may be useful in the effective killing of irregular cells.

Enhanced Antitumor Efficacy Achieved Through Combination of nsPEFs and Low-Dosage Paclitaxel.

Looking for a safe and effective cancer therapy for patients is becoming an important and promising research direction. Nanosecond pulsed electric field (nsPEF) has been found to be a potential non-thermal therapeutic technique with few side effects in pre-clinical studies. On the other hand, paclitaxel (PTX), as a common chemotherapeutic agent, shows full anti-tumor activities and is used to treat a wide variety of cancers. However, the delivery of PTX is challenging due to its poor aqueous solubility. Hence, high dosages of PTX have been used to achieve effective treatment, which creates some side effects. In this study, nsPEF was combined with low-level PTX, in order to validate if this combined treatment could bring about enhanced efficacy and allow reduced doses of PTX in clinical application. Cell proliferation, apoptosis, and cell cycle distribution were examined using MTT and flow cytometry assay, respectively. Results showed that combination treatments of nsPEF and PTX exhibited significant synergistic effects in vitro. The underlying mechanism might be that these two agents acted at different targets and coordinately enhanced MDA-MB-231 cell death.

First Human Trial of High-Frequency Irreversible Electroporation Therapy for Prostate Cancer.

Irreversible electroporation, as a nonthermal therapy of prostate cancer, has been used in clinic for several years. The mechanism of irreversible electroporation ablation is thermal independent; thus, the main structures (eg, rectum, urethra, and neurovascular bundle) in prostate are spared during the treatment, which leads to the retention of prostate function. However, various clinical trials have shown that muscle contractions occur during this therapy, which warrants deep muscle anesthesia. Use of high-frequency bipolar pulses has been proposed to reduce muscle contractions during treatment, which has already triggered a multitude of studies at the cellular and animal scale. In this study, we first investigated the efficacy and safety of high-frequency bipolar pulses in human prostate cancer ablation. There are 40 male patients with prostate cancer aged between 51 and 85 years involved in this study. All patients received 250 high-frequency bipolar pulse bursts with the repeat frequency of 1 Hz. Each burst comprised 20 individual pulses of 5 microseconds, so one burst total energized time was 100 microseconds. The number of the electrodes ranged 2 to 6, depending on tumor size. A small amount of muscle relaxant was still needed, so there were no visible muscle contractions during the pulse delivery process. Four weeks after treatment, it was found that the ablation margins were distinct in magnetic resonance imaging scans, and the prostate capsule and urethra were retained. Eight patients underwent radical prostatectomy for pathological analysis after treatment, and the results of hematoxylin and eosin staining revealed that the urethra and major vasculature in prostate have been preserved. By overlaying the electric field contour on the ablation zone, the electric field lethality threshold is determined to be 522 ± 74 V/cm. This study is the first to validate the feasibility of tumor ablation by high-frequency bipolar pulses and provide valuable experience of irreversible electroporation in clinical applications.

Synergistic combinations of short high-voltage pulses and long low-voltage pulses enhance irreversible electroporation efficacy.

Irreversible electroporation (IRE) uses ~100 µs pulsed electric fields to disrupt cell membranes for solid tumor ablation. Although IRE has achieved exciting preliminary clinical results, implementing IRE could be challenging because of volumetric limitations at the ablation region. Combining short high-voltage (SHV: 1600V, 2 µs, 1 Hz, 20 pulses) pulses with long low-voltage (LLV: 240-480 V, 100 µs, 1 Hz, 60-80 pulses) pulses induces a synergistic effect that enhances IRE efficacy. Here, cell cytotoxicity and tissue ablation were investigated. The results show that combining SHV pulses with LLV pulses induced SKOV3 cell death more effectively, and compared to either SHV pulses or LLV pulses applied alone, the combination significantly enhanced the ablation region. Particularly, prolonging the lag time (100 s) between SHV and LLV pulses further reduced cell viability and enhanced the ablation area. However, the sequence of SHV and LLV pulses was important, and the LLV + SHV combination was not as effective as the SHV + LLV combination. We offer a hypothesis to explain the synergistic effect behind enhanced cell cytotoxicity and enlarged ablation area. This work shows that combining SHV pulses with LLV pulses could be used as a focal therapy and merits investigation in larger pre-clinical models and microscopic mechanisms.

Bipolar Microsecond Pulses and Insulated Needle Electrodes for Reducing Muscle Contractions During Irreversible Electroporation.

OBJECTIVE: To minimize the effect of muscle contractions during irreversible electroporation (IRE), this paper attempts to research the ablation effect and muscle contractions by applying high-frequency IRE (H-FIRE) ablation to liver tissue in vivo. METHODS: An insulated needle electrode was produced by painting an insulating coating on the outer surface of the needle electrode tip. A series of experiments were conducted using insulated needle electrodes and traditional needle electrodes to apply H-FIRE pulses and traditional monopolar IRE pulses to rabbit liver tissues. The finite element model of the rabbit liver tissue was established to determine the lethal thresholds of H-FIRE in liver tissues. Muscle contractions were measured by an accelerometer. RESULTS: With increased constitutive pulse width and pulse voltage, the ablation area and muscle contraction strength are also increased, which can be used to optimize the ablation parameters of H-FIRE. Under the same pulse parameters, the ablation areas are similar for the two types of electrodes, and the ablation region has a clear boundary. H-FIRE and insulated needle electrodes can mitigate the extent of muscle contractions. The lethal thresholds of H-FIRE in rabbit liver tissues were determined. CONCLUSION: This paper describes the relationships between the ablation area, muscle contractions, and pulse parameters; the designed insulated needle electrodes can be used in IRE for reducing muscle contraction. SIGNIFICANCE: The study provides guidance for treatment planning and reducing muscle contractions in the clinical application of H-FIRE.

[The anti-tumor efficacy of nanosecond pulsed electric fields on the mouse with melanoma xenograft in vivo].

This study was conducted to investigate the anti-tumor efficacy of nanosecond pulsed electric fields (nsPEFs) on the mouse with A375-GFP melanoma xenograft in vivo. In vivo fluorescence image analysis system was used in this study to evaluate the effects of nsPEFs on human melanoma A375 cell xenograft. On the Day 90 af ter pulse delivery, the skin that had contained A375 cell xenograft was surgically excised and pathologically evalua ted. The changes of scar were recorded by digital camera. The experiment revealed that significant changes in fluorescence value trend and amplitude were found in the treated group from those in the control group. The fluorescence of tumor in the treated group decreased mostly 48 h after the treatment and completely disappeared 10 d after the treatment, while that in control group was increased gradually. Surgical excision of the area confirmed a complete pathologic response. Within a few days after the nsPEFs treatment, a hard scab formed at the treatment region. The scab fell off by the end of the second week. As time went on, the scar gradually became faded and all xenograft tumors were disappeared without recurrence. From the experiment, we learn that nsPEFs can bring good therapeutic effect. It may provide a new approach for the clinical treatment of superficial tumors.