Enhanced anti-tumor efficacy of electroporation (EP)-mediated DNA vaccine boosted by allogeneic lymphocytes in pre-established tumor models.

BACKGROUND: Tumor-reactive T cells play a crucial role in anti-tumor responses, but T cells induced by DNA vaccination are time-consuming processes and exhibit limited anti-tumor efficacy. Therefore, we evaluated the anti-tumor effectiveness of reactive T cells elicited by electroporation (EP)-mediated DNA vaccine targeting epidermal growth factor receptor variant III (pEGFRvIII plasmid), in conjunction with adoptive cell therapy (ACT), involving the transfer of lymphocytes from a pEGFRvIII EP-vaccinated healthy donor. METHODS: The validation of the established pEGFRvIII plasmid and EGFRvIII-positive cell model was confirmed through immunofluorescence and western blot analysis. Flow cytometry and cytotoxicity assays were performed to evaluate the functionality of antigen-specific reactive T cells induced by EP-mediated pEGFRvIII vaccines, ACT, or their combination. The anti-tumor effectiveness of EP-mediated pEGFRvIII vaccines alone or combined with ACT was evaluated in the B16F10-EGFRvIII tumor model. RESULTS: EP-mediated pEGFRvIII vaccines elicited serum antibodies and a robust cellular immune response in both healthy and tumor-bearing mice. However, this response only marginally inhibited early-stage tumor growth in established tumor models. EP-mediated pEGFRvIII vaccination followed by adoptive transfer of lymphocytes from vaccinated healthy donors led to notable anti-tumor efficacy, attributed to the synergistic action of antigen-specific CD4+ Th1 cells supplemented by ACT and antigen-specific CD8+ T cells elicited by the EP-mediated DNA vaccination. CONCLUSIONS: Our preclinical studies results demonstrate an enhanced anti-tumor efficacy of EP-mediated DNA vaccination boosted with adoptively transferred, vaccinated healthy donor-derived allogeneic lymphocytes.

Non-viral approaches in CAR-NK cell engineering: connecting natural killer cell biology and gene delivery.

Natural Killer (NK) cells are exciting candidates for cancer immunotherapy with potent innate cytotoxicity and distinct advantages over T cells for Chimeric Antigen Receptor (CAR) therapy. Concerns regarding the safety, cost, and scalability of viral vectors has ignited research into non-viral alternatives for gene delivery. This review comprehensively analyses recent advancements and challenges with non-viral genetic modification of NK cells for allogeneic CAR-NK therapies. Non-viral alternatives including electroporation and multifunctional nanoparticles are interrogated with respect to CAR expression and translational responses. Crucially, the link between NK cell biology and design of drug delivery technologies are made, which is essential for development of future non-viral approaches. This review provides valuable insights into the current state of non-viral CAR-NK cell engineering, aimed at realising the full potential of NK cell-based immunotherapies.

Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.

The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.

The Effects of Bipolar Cancellation Phenomenon on Nano-Electrochemotherapy of Melanoma Tumors: In Vitro and In Vivo Pilot.

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3-7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca2+ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.

Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells.

Excitable cells such as neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could be efficiently evaluated.

Highly efficient CRISPR-mediated genome editing through microfluidic droplet cell mechanoporation.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based editing tools have transformed the landscape of genome editing. However, the absence of a robust and safe CRISPR delivery method continues to limit its potential for therapeutic applications. Despite the emergence of various methodologies aimed at addressing this challenge, issues regarding efficiency and editing operations persist. We introduce a microfluidic gene delivery system, called droplet cell pincher (DCP), designed for highly efficient and safe genome editing. This approach combines droplet microfluidics with cell mechanoporation, enabling encapsulation and controlled passage of cells and CRISPR systems through a microscale constriction. Discontinuities created in cell and nuclear membranes upon passage facilitate the rapid CRISPR-system internalization into the nucleus. We demonstrate the successful delivery of various macromolecules, including mRNAs (~98%) and plasmid DNAs (~91%), using this platform, underscoring the versatility of the DCP and leveraging it to achieve successful genome engineering through CRISPR-Cas9 delivery. Our platform outperforms electroporation, the current state-of-the-art method, in three key areas: single knockouts (~6.5-fold), double knockouts (~3.8-fold), and knock-ins (~3.8-fold). These results highlight the potential of our platform as a next-generation tool for CRISPR engineering, with implications for clinical and biological cell-based research.

Analysis of magnetic resonance contrast agent entrapment following reversible electroporation in vitro.

BACKGROUND: Administering gadolinium-based contrast agent before electroporation allows the contrast agent to enter the cells and enables MRI assessment of reversibly electroporated regions. The aim of this study was evaluation of contrast agent entrapment in Chinese hamster ovary (CHO) cells and comparison of these results with those determined by standard in vitro methods for assessing cell membrane permeability, cell membrane integrity and cell survival following electroporation. MATERIALS AND METHODS: Cell membrane permeabilization and cell membrane integrity experiments were performed using YO-PRO-1 dye and propidium iodide, respectively. Cell survival experiments were performed by assessing metabolic activity of cells using MTS assay. The entrapment of gadolinium-based contrast agent gadobutrol inside the cells was evaluated using T1 relaxometry of cell suspensions 25 min and 24 h after electroporation and confirmed by inductively coupled plasma mass spectrometry. RESULTS: Contrast agent was detected 25 min and 24 h after the delivery of electric pulses in cells that were reversibly electroporated. In addition, contrast agent was present in irreversibly electroporated cells 25 min after the delivery of electric pulses but was no longer detected in irreversibly electroporated cells after 24 h. Inductively coupled plasma mass spectrometry showed a proportional decrease in gadolinium content per cell with shortening of T1 relaxation time (R 2 = 0.88 and p = 0.0191). CONCLUSIONS: Our results demonstrate that the contrast agent is entrapped in cells exposed to reversible electroporation but exits from cells exposed to irreversible electroporation within 24 h, thus confirming the hypothesis on which detection experiments in vivo were based.

Protocol for in vivo nucleic acid delivery utilizing the rolling microneedle electrode array.

Electroporation temporarily enhances cell membrane permeability and promotes the absorption of external molecules. We have developed a device termed the rolling microneedle electrode array (RoMEA) that combines a densely arranged microneedle array of electrodes with rolling structures. Use RoMEA to create uniform skin micropores for efficient, low-damage transfection of nucleic acids over extended areas of the body. We describe in detail the design, fabrication, and assembly of the device and the application of in vivo electroporation of nucleic acids. For complete details on the use and execution of this protocol, please refer to Tongren Yang et al. 1.

Contribution and advances of robotics in percutaneous oncological interventional radiology.

The advent of robotic systems in interventional radiology marks a significant evolution in minimally invasive medical procedures, offering enhanced precision, safety, and efficiency. This review comprehensively analyzes the current state and applications of robotic system usage in interventional radiology, which can be particularly helpful for complex procedures and in challenging anatomical regions. Robotic systems can improve the accuracy of interventions like microwave ablation, radiofrequency ablation, and irreversible electroporation. Indeed, studies have shown a notable decrease of an average 30% in the mean deviation of probes, and a 40% lesser need for adjustments during interventions carried out with robotic assistance. Moreover, this review highlights a 35% reduction in radiation dose and a stable-to-30% reduction in operating time associated with robot-assisted procedures compared to manual methods. Additionally, the potential of robotic systems to standardize procedures and minimize complications is discussed, along with the challenges they pose, such as setup duration, organ movement, and a lack of tactile feedback. Despite these advancements, the field still grapples with a dearth of randomized controlled trials, which underscores the need for more robust evidence to validate the efficacy and safety of robotic system usage in interventional radiology.

Simulation study on electroporation of cancer cells in multicellular system.

Electroporation (EP) of the normal cell and cancer cell both in single-cell and multicellular models was investigated by the meshed transport network method (MTNM) in this paper. The simulation results suggest that the cancer cell undergoes faster and more significant local EP than that of the corresponding normal cell induced by nanosecond pulsed electric fields (nsPEFs) both in single-cell and multicellular models. Furthermore, the results of the multicellular model indicate that there is a unidirectional neighboring effect in the multicellular model, meaning that cells at the center are affected and their pore formation is significantly reduced, but this effect is very weak for cells at the edges of the system. This means that the electric field selectively kills cells in different distribution locations. This work can provide guidance for the selection of parameters for the cancer cell EP process.