An integrated nanopore–microfluidic platform for low-voltage electroporation of delivering self-amplifying RNA into dendritic cells

Microfluidic Electroporation: Recent Advances and Applications.

Localized delivery of coding nucleic acids into adherent cells by in situ electroporation: integrated impedance-based monitoring allows for loss-of-function or gain-of-function screening

The combination of ISE, specific NAs and impedance-based cell monitoring paves the way for a new class of gain-of-function or loss-of-function experiments.

A Digital Microfluidic Electroporation Platform for Low-input CRISPR Genome Editing and mRNA Transfection In Suspension T Cells and 3D Cell Models.

Digital microfluidic (DMF) electroporation enables precise, low-volume genetic manipulation of mammalian cells while minimizing cellular input by up to 100x and preserving viability. This study presents a high-throughput DMF-based transfection workflow for CRISPR-mediated knockout of the TRAC locus in primary human suspension T cells and for mRNA transfection of three-dimensional HEK293T spheroids. Using spatially deposited CRISPR guide RNAs and on- cartridge ribonucleoprotein (RNP) assembly, efficient TRAC locus disruption was achieved in both CD4⁺ and CD8⁺ T-cell populations using only 10,000 cells per condition, with post-editing viabilities exceeding 85%. Biophysical characterization using flow-induced and Taylor dispersion analyses revealed that polymer additives stabilize Cas9-sgRNA complexes under electroporation buffer conditions, supporting reproducible editing at sub microliter volumes. The workflow was further adapted for 3D applications by delivering EGFP mRNA into intact HEK293T spheroids, resulting in robust and spatially uniform fluorescence without impairing spheroid growth or morphology. Together, these results demonstrate that DMF electroporation enables efficient genome editing and mRNA delivery across both suspension immune cells and multicellular spheroids. This platform provides a scalable and low-input solution for applications in CAR-T cell therapy, functional genomics, and advanced 3D cellular models.

Synergistic enhancement of irreversible electroporation by silver nanowires in three-dimensional tumor models.

Irreversible electroporation (IRE) is an emerging tumor ablation modality that employs high-intensity pulsed electric fields to disrupt cell membranes. However, achieving complete tumor destruction while preserving adjacent critical structures remains a major challenge. In this study, we investigated whether high-aspect-ratio silver nanowires (AgNWs) could enhance IRE efficacy in a three-dimensional hydrogel-based cell model. We systematically evaluated cell viability, ablation area, conductivity, and temperature under various pulse parameters and AgNW concentrations. Increased electric field strength, longer pulse durations, and higher pulse numbers all potentiated the cytotoxic effect. Under identical pulsing conditions, the addition of 70 μg/mL AgNWs expanded the ablation zone by 42.2% ± 7.5%, allowing lower-energy pulse protocols to achieve ablation comparable to that of higher-energy settings. This enhancement was not attributable to changes in bulk conductivity; rather, the nanowires appeared to amplify the local electric field in the vicinity of cells, thereby boosting treatment sensitivity without introducing significant thermal effects. Collectively, these in vitro findings suggest that AgNWs can potentiate IRE ablation at reduced energy levels, offering a promising adjuvant strategy. Nevertheless, further validation through in vivo studies and comprehensive long-term safety assessments is essential prior to any clinical translation.

Nanosecond Pulsed Electric Fields for Extracellular Vesicle Engineering: From Electro-Exocytosis to Cargo Modulation

Extracellular vesicles (EVs), including small extracellular vesicles (sEVs) and medium/large extracellular vesicles (MVs), have emerged as promising therapeutic vectors and diagnostic biomarkers across various branches of biomedicine. However, the clinical translation of EV-based technologies remains constrained by persistent challenges in manufacturing: insufficient yield from primary cell sources, limited control over cargo composition, and the absence of scalable, standardized production platforms. Nanosecond pulsed electric fields (nsPEF) represent an emerging biophysical approach that can address several of these limitations. Unlike conventional electroporation, which targets the plasma membrane using microsecond-to-millisecond pulses, nsPEF delivers ultrashort (1–300 ns), high-amplitude (10–300 kV/cm) pulses that penetrate intracellularly to directly perturb endosomal membranes, the endoplasmic reticulum, and the multivesicular body (MVB) compartment, the very organelles where small EVs (exosome) biogenesis and cargo sorting occur. Through coordinated effects on intracellular calcium mobilization, cytoskeletal remodeling, SNARE-mediated membrane fusion, and phospholipid redistribution, nsPEF can stimulate rapid, non-lethal vesicle release, a process labeled as “electro-exocytosis.” Emerging and growing evidence suggests that nsPEF does not merely increase EV yield but actively modulates the proteomic, lipidomic, and nucleic acid composition of released vesicles, offering a potential route to cargo engineering. In addition, the same biophysical principles that drive electro-exocytosis can be exploited in reverse: nsPEF-mediated transient permeabilization of EV membranes allows for post-isolation loading of exogenous therapeutic cargo, small molecules, nucleic acids, or proteins into pre-formed vesicles without destroying their structural integrity. This review discusses current knowledge on EV biogenesis and release mechanisms, introduces the biophysical foundations of nsPEF–cell and nsPEF–membrane interactions, and, by evaluating the experimental evidence supporting nsPEF-driven EV engineering, outlines a translational roadmap for the application and development of this technology toward clinical-grade EV manufacturing.

A novel silicon-based intracellular delivery platform enables multiplexed delivery across cell types and cell states 2310229

A novel intracellular delivery platform that works through mechanoporation that unlocks scalable, gentle, and versatile intracellular delivery for sensitive immune cells, expanding the feasibility of complex genetic and molecular engineering for research and clinical cell therapies.