Bioelectricity Buzz

Recent Bioelectricity-Related Articles Selected by Ann M. Rajnicek, Media Editor of Bioelectricity

Spring is finally here and new ideas in the Bioelectricity sphere are truly buzzing. This installment covers electrically facilitated transportation of flower mites, synthetic G-protein-coupled receptor proteins designed to drive specific cell behaviors, pain relief via targeted inhibition of voltage-gated sodium channel activity, mechanosensitive channels that drive swallowing, a wireless electrical neural scaffold material, and using a charged substrate that enhances T cell-mediated cancer immunotherapy.

A Mitey Fine Ride: Tropical Flower Mites Use Electrical Forces to Locate Hummingbirds and Hitch a Lift to Distant Flowers

It seems apt to open this spring installment of Buzz with a captivating story about interactions between hummingbirds, flowers, and insects, where electrical forces mediate both detection of an avian host and electrotaxis of phoretic mites toward it.

Insects and birds generate electric charge during flight. Pollinators tend to be positively charged relative to the atmosphere but the flowers they visit are generally negatively charged, so this difference in charge can facilitate interactions between pollinators and flowers.

Hummingbirds can accumulate charges from 66 pC to 800 pC, with wingbeat movements yielding fundamental frequencies between ∼20 and 80 Hz. Other movements, including oscillations due to aerodynamics generate second harmonic frequencies between ∼40 and 160 Hz. Therefore, when a hummingbird hovers at a flower, there is an associated local electrical environment with superimposed modulated and unmodulated components.

Hummingbird flower mites feed on nectar and pollen, and they are phoretic. When a hummingbird inserts its beak into a mite-infested flower, the mites are attracted immediately to the hummingbird’s beak. They run up the beak to enter the bird’s nostrils and when the bird inserts its beak into the next flower the mites emerge rapidly from the nostrils, running down the beak toward the flower where they feed, complete their life cycle, and colonize the flower.

Given the established complex electrical milieu associated with hummingbirds García-Robledo and colleagues tested the hypothesis that flower mites use electroreception as a cue to detect the presence and location of the hummingbird, with the electric forces aiding attraction and rapid attachment of the mites to the beak surface. Because the mechanism of electroreception in flower mites was unknown, the team further hypothesized the existence of electroreceptors in the mite’s front legs to drive this behavior.

The first experiments explored the sensitivity of mites to electrical cues. A spherical aluminum electrode 1.5 cm in diameter was suspended 1.5 mm above a grounded copper plate to create electric fields (EFs) that were either modulated or static. At its maximum potential (550 V), the EFs generated were within the range of those reported for hummingbirds and the modulated frequencies were in the range of higher harmonics reported previously for hummingbirds. Mites anesthetized with CO₂ were seeded onto the copper plate. Immediately upon waking, they were exposed for 10 s to EFs of 550 V direct current (DC) without any modulation or to 550 V DC EFs modulated with an AC component (388.91 V_RMS at 120 Hz). Controls had no EF applied for 10 s.

When no voltage was applied to the electrode, the mites all walked away from the electrode position and, under an unmodulated DC electric field (550 V), all mites walked away except one individual. However, under EFs with 120 Hz modulation, 86% of the mites remained under the electrode while tapping their front legs slowly until the EF was terminated. Interestingly, a different phoretic mite species associated with herbivorous beetles that feed on the same plants used by hummingbirds showed no responsiveness to these EF conditions, suggesting that electroreception is not a universal property of phoretic mites and that this mite species evolved electroreception to facilitate interaction with specific bird hosts.

Next, the team explored the mite’s sensitivity to modulated EF cues, which simulates the AC component that would arise due to vibrations and movements (e.g., wingbeats) of an electrically charged hummingbird. The same mite seeding and electrode configurations were used for the DC EF experiments, but here the DC voltage had a 120 Hz modulated component. The initial voltage of 550 V was decreased by 50 V every 5 s until reaching 100 V. Mite responses were recorded at each 50 V interval. The team specifically reported questing behavior (moving front legs toward the electrode) and the minimum voltage that elicited attraction to the electrode.

Under 550 V and 120 Hz stimulation, 100% of mites (n = 29) stayed under the electrode, but they became more active and began tapping their front legs as the voltage was reduced, with 5–20% retreating from the electrode position at each 50 V increment. At 200–250 V (120 Hz), the mites approached the electrode and stood on their back legs, projecting their front legs toward the electrode. All mites stopped approaching the electrode and walked away at 100 V (120 Hz), indicating a threshold for the response.

To test whether mites are attracted to a particular polarity and whether the EF needs to be modulated, a single mite was placed in a “choice arena” comprising a glass tube with a copper wire inserted at each end. Touching the external segments of the wire for 1 s to either the positive aluminum electrode or the negative copper plate charged the system under DC and modulated conditions. The charge applied simulated that of a hummingbird at either 40% relative humidity (400 pC) or at 95% tropical humidity (105 pC), which is relevant to the tropical hummingbird mite species studied here. Mites detect electric fields immediately at both charges but only if they were induced by a modulated voltage (DC, V_RMS, 120 Hz). Mites were attracted toward the positive charge even at 105 pC, suggesting the relevance of electroreception cues in tropical environments.

The mechanism for electroreception in mites was not known, but the questing behavior implicated the front legs. Scanning electron microscopy of front legs revealed structures in both tarsi that resembled Haller’s organs, which are sensory structures found in insects that detect chemical, mechanic, and infrared environmental signals. The requirement for front legs in electroreception was tested by removing one or both tarsi and placing the mites under the spherical electrode and then applying 550 V and 120 Hz EFs as soon as they recovered from CO₂ anesthesia and started tapping their legs. Locomotion itself was not impacted by leg removal so all mites with both tarsi intact retreated from the electrode but most mites with only one leg stayed under the electrode and extended that leg toward the electrode (questing), retreating from the electrode once it was switched off.

Collectively, the data demonstrate the importance of electroreception and electroattraction for hummingbird mite long-distance transportation and for the rapid transfer of mites by electrostatic attraction to the bird’s beak. This phenomenon is important to ensure the survival of this tiny insect, permitting it to extend its reach broadly within its ecosystem.

The demonstration of electroreceptors on the mite’s front legs resolves the question of how electrical cues are detected, but the anatomy of the innervation network remains unclear. The ability to understand how animals read and interpret complex electrical cues is becoming increasingly important, given the growing number of electroreception behaviors identified in aerial and land animals and the increasing electrical environmental conditions introduced globally by technology.

You’re Making That Up: Synthetic Membrane Receptors Designed to Control Cell Behavior

Being able to control cell behavior is central to many therapeutic advances using bioengineered devices. Kalogriopoulos and team have developed a platform for engineering synthetic G-protein-coupled receptors (GPCRs), demonstrating that this important class of cell signaling receptors can be precisely tuned to achieve desired cellular outputs in response to defined inputs.

GPCRs are the largest and most versatile class of cell-surface receptors mediating cellular responses to a wide range of natural extracellular signals, including neurotransmitters, peptides, and hormones. However, their structural complexity (e, g. lack of structural modularity) and GPCR signaling promiscuity have historically limited their use in synthetic biology aimed at precision control of cell behavior.

Kalogriopoulos and coworkers addressed this limitation by fusing a nanobody and a receptor auto-inhibitory domain to the extracellular N terminus of the GPCR scaffold. The binding of the auto-inhibitory domain to the GPCR and the binding of the nanobody to an antigen are mutually exclusive. This design allows antigen binding to relieve auto-inhibition, thereby enabling agonist activation of the receptor. These modular systems are known as programmable antigen-gated G-protein-coupled engineered receptors (PAGERs). PAGERs have the potential to modulate diverse cellular functions, including transgene expression, real-time fluorescence, and endogenous G-protein activation, offering programmable control over cell behavior in response to user-defined antigens.

The authors began by identifying structural motifs critical for ligand recognition and G-protein coupling in native GPCRs. Using a combination of rational design and directed evolution, they constructed receptor chimeras that replaced natural ligand-binding domains with synthetic modules capable of responding to non-natural orthogonal ligands. These chimeric receptors were further modified to incorporate engineered intracellular domains that coupled efficiently to chosen downstream effectors, such as cyclic AMP (cAMP) or calcium signaling pathways, which are central to many bioelectric cell responses. This modular architecture allowed the receptor to act as a “plug-and-play” device, permitting both input (ligand specificity) and output (signaling cascade) to be tailored to specific applications.

Functional assays using cultured mammalian cells demonstrated that the synthetic GPCRs exhibited high specificity and sensitivity to their designated ligands. Upon ligand binding, the receptors reliably activated downstream signaling pathways leading to quantifiable changes in second messenger levels. Importantly, the engineered receptors showed minimal crosstalk with endogenous GPCRs, suggesting that PAGERs would ensure minimal off-target effects if used therapeutically. High-throughput screening methods fine-tuned receptor performance, optimizing both ligand affinity and signal transduction efficiency.

Beyond basic signal transduction, the work highlights the utility of synthetic GPCRs for the programmable control of complex cellular behaviors. The team integrated the synthetic receptors into cellular circuits that govern processes such as cell proliferation, migration, and differentiation. PAGERs facilitated antigen-dependent control of T cell migration along a gradient of soluble ligand, macrophage differentiation, and secretion of therapeutic antibodies. In a neuroscience context, PAGERs enabled selective inhibition of neuronal activity, providing a novel tool for investigating bioelectrical signaling and therapeutic interventions for neurological disorders.

By providing a means to control cellular responses precisely through engineered receptors, PAGERs pave the way for next-generation cell-based therapies that can be dynamically regulated in vivo. The ability to customize both the input and output of GPCR signaling cascades holds promise for targeted drug delivery, immunomodulation, and real-time control of cellular processes, including bioelectrical signaling.

A Real Pain: Suzetrigine Inhibition of Na_V1.8 Pain Signaling as an Alternative to Opioids

The opioid crisis has highlighted the urgent unmet need for pain medications that are effective, yet not addictive. Osteen and coworkers have explored the mechanism and promising efficacy of suzetrigine, a selective voltage-gated sodium channel inhibitor that sidesteps this issue.

Pain is a natural response to injury, but it causes considerable suffering and is therefore an important target for drug discovery. Voltage-gated sodium channels (Na_V) are of considerable interest in this sense. There are nine Na_V subtypes in mammals, with Na_V1.7, Na_V1.8, and Na_V1.9 identified as potential pain targets because they are expressed predominantly in peripheral pain-sensing neurons (nociceptors).

Suzetrigine (4-[[(2R,3S,4S,5R)-3-(3,4- difuoro-2-methoxy-phenyl)-4,5-dimethyl-5-(trifluoromethyl)tetrahydrofuran-2-carbonyl] amino]pyridine-2-carboxamide) is a novel, non‐opioid analgesic designed to target pain at its source by selective inhibition of the voltage‐gated sodium channel Na_V1.8, which is expressed in peripheral pain-sensing neurons but not in the central nervous system (CNS). This strategic targeting addresses the critical need for effective pain relief while avoiding the CNS-related side effects or the addictive potential of opioids. However, it has proved challenging to identify a specific Na_V1.8 channel inhibitor for pain due to the structural similarity of the Na_V channel subtypes.

Initial experiments using RNA-seq confirmed the absence of Na_V1.8 channels in more than 1000 human postmortem tissues and over 190 CNS tissues. Electrophysiological studies in human dorsal root ganglia neurons (DRG) demonstrated that suzetrigine exhibits sub‐nanomolar potency against Na_V1.8 channels with an IC₅₀ of approximately 0.68 nM, demonstrating >31,000-fold selectivity over other Na_V isoforms. This exceptional selectivity was further confirmed by extensive off-target screening, where suzetrigine did not significantly interact with over 180 other molecular targets, including 44 targets associated with abuse potential.

Next, Osteen and coworkers probed the compound’s mechanism of action through a series of in vitro electrophysiology and radioligand binding assay studies. In chimeric channel experiments, researchers attributed the selectivity of suzetrigine to its binding to the voltage-sensing domain 2 (VSD2) of Na_V1.8. A unique KKGS motif within the S3–S4 loop of VSD2 was found to be critical for drug binding. By attaching to this site, suzetrigine stabilizes Na_V1.8 in its closed state, a mechanism distinct from that of traditional local anesthetics, which block the channel pore non-selectively. This allosteric inhibition prevents the channel from opening in response to depolarizing stimuli, thereby reducing the propagation of pain signals.

Electrophysiological assessments using both automated and manual patch clamp techniques revealed that suzetrigine produced tonic inhibition of Na_V1.8, maintaining its potency even under repetitive stimulation that mimics physiological action potential firing. Notably, in primary human DRG neurons, the drug significantly reduced the number of action potentials at concentrations as low as 1 nM, with maximal inhibition observed around 10 nM. Together these findings validate the therapeutic potential of suzetrigine to modulate peripheral pain signaling effectively.

The preclinical safety profile of suzetrigine is equally promising. In repeat-dose toxicity studies in rats and cynomolgus monkeys, suzetrigine was well-tolerated at exposures many-fold higher than those predicted to be clinically effective. Importantly, no adverse effects on CNS, cardiovascular, or respiratory functions were observed. Additionally, in a dedicated physical dependence study in rats, abrupt withdrawal of suzetrigine failed to produce signs of dependence, contrasting sharply with the withdrawal effects seen with opioid administration.

These promising preclinical results translated into clinical efficacy. Systematic analysis of adverse event data from phase 2 and phase 3 trials involving more than 2400 participants undergoing surgeries such as abdominoplasty and bunionectomy, suzetrigine significantly reduced moderate to severe acute pain compared with placebo. Furthermore, the incidence of adverse events related to abuse or dependence was negligible, underscoring the safety of its non-opioid mechanism.

This work represents a breakthrough in pain management by offering potent, selective inhibition of Na_V1.8 with a unique allosteric mechanism that curtails peripheral pain signaling. Its robust preclinical and clinical safety profiles, combined with effective analgesia without sedation or the risk of addiction make it attractive as a new option for the treatment of moderate to severe pain.

Taking the Plunge: Mechanotransduction by PIEZO Channels Regulates Rhythmic Pharyngeal Swallowing Movements in Caenorhabditis elegans

Mechanotransduction is important for a variety of biological functions but the extent to which it controls organ function is an area of active research. Park and colleagues shed some light on this for the digestive tract by demonstrating a role for PIEZO ion channels. Specifically, their role in detecting the distention that results from food accumulation and driving rhythmic contractions in an esophagus-like structure in worms.

Caenorhabditis elegans is a familiar organism in many laboratories, where it is a genetically accessible model for developmental biology studies. Its digestive tract comprises mouth, pharynx, intestine, and a pharyngeal-intestinal valve (PI valve), a group of six epithelial cells that connects the posterior pharynx to the anterior intestine. The worm’s bacterial meal is pumped by peristalsis to the intestine, where nutrient absorption occurs. In an anatomical sense, the PI is analogous to the vertebrate esophagus, but its role in the C. elegans digestive system is unknown. The PI valve lacks muscle fibers and synaptic innervation, leading to the suggestion that it passively mediates food passage.

Although mechanically activated ion channels are found in the C. elegans digestive tract, it isn’t known whether they contribute to coordinated food transport, especially a rhythmic movement termed the “pharyngeal plunge” that propels ingested food from the pharynx into the intestine. Therefore, the team focused on pezo-1, a C. elegans ortholog of PIEZO channels.

Park and colleagues first characterized 14 putative multiple isoforms of pezo-1 by sorting them into four groups based on the location of the translational start codon, which may share promoter regions called p1, p2, p3, and p4. The p1 promoter was found to specifically drive expression in the PI valve, a finding that was consistent across various experimental approaches. The CRISPR-Cas9 system was used to generate pezo-1 deletion mutants that lacked the C-terminus of all isoforms and thus could be a null allele. Allowing worms to feed on fluorescent microspheres for 20 min demonstrated that in wild-type worms the particles accumulated in the anterior intestinal lumen near the PI valve, gradually being defecated 10 min later. Despite normal food ingestion, pezo-1 mutants exhibited significant delays in food transit. In these mutants, food particles accumulate in the anterior intestine due to impaired movement, highlighting the critical role of PIEZO-1 in clearing food from this region.

Detailed kinematic analyses aimed to explore the dynamics of pharyngeal plunge movements. To quantify this, the relative distance was measured from the posterior end of the pharynx to the anterior end of the intestine (“plunge length”). In wild-type animals, the pharyngeal plunge occurred approximately every 4 s with a typical plunge length of about 5 μm. In contrast, pezo-1 mutants displayed reduced frequency and reduced plunge length. Rescue experiments further pinpointed the function of PEZO-1 to the PI valve. Normal pharyngeal plunge dynamics and food transit were rescued fully by the expression of a pezo-1 cDNA under the control of pezo-1p1 or the PI valve-specific promoter, but not under the control of a body wall-specific promoter, confirming that pezo-1 is acting specifically at the PI to regulate food movement.

There is a high degree for homology in terms of protein sequence and topology between PEZO-1 and mammalian PIEZO1 and PIEZO2. Therefore, to explore the evolutionary conservation of this mechanism, the team expressed mouse Piezo1 or Piezo2 cDNA in the PI valve of pezo-1 mutants and monitored pharyngeal plunge dynamics. Remarkably, mouse Piezo1, but not mouse Piezo2, fully rescued the swallowing defects, indicating that the functional properties of the PIEZO channel are conserved across these species, and suggesting a potential relevance to human esophagus function. Moreover, mechanical stimulation experiments, in which buffer solution was microinjected into the anterior intestine to induce distension, elicited a rapid pharyngeal plunge in wild-type worms. This response was absent in pezo-1 mutants, directly demonstrating that PEZO-1 acts as a mechanosensory, detecting the internal pressure generated by food accumulation.

Optogenetic approaches further underscored the importance of the PI valve. Activation of the valve using channelrhodopsin led to an immediate and sustained pharyngeal plunge, while genetic ablation of the valve mimicked the defects seen in pezo-1 mutants. Calcium imaging experiments revealed that activation of PEZO-1 in the PI valve causes transient increases in intracellular Ca²⁺, which are tightly correlated with the initiation and termination of the pharyngeal plunge. These calcium signals are absent in the pezo-1 mutants, suggesting that PEZO-1 mediates a critical Ca²⁺ influx necessary for triggering the muscle contractions that drive the plunge.

The final set of experiments linked this valve-mediated sensing mechanism to downstream motor control, specifically testing roles for the two types of motor neurons that innervate the head and neck muscles of C. elegans (SMB and SMD neurons). When compared with pezo-1 mutants, animals in which the SMB and SMD neurons had been ablated exhibited defects in pharyngeal plunge and microsphere movement. This indicates that the contraction of head and neck muscles stimulated by these motor neurons generates the force required for the pharyngeal plunge.

Collectively, these findings not only define a novel role for PEZO-1 in internal mechanosensation and swallowing in C. elegans but also suggest that similar processes may be conserved in higher organisms, offering valuable insights into gastrointestinal physiology and the fundamental mechanisms of mechanotransduction.

This work is relevant beyond C. elegans in that the evolutionary conservation demonstrated by rescuing the swallowing defects with mouse Piezo1 suggests these principles are shared across species, potentially informing therapeutic strategies for human disorders linked to mechanosensation and bioelectrical dysfunction. Future exploration of specific PEZO-1 isoforms may prove beneficial in this context.

“Look Ma, No Wires”: Promoting Nerve Repair with a Flexible, Wireless Implantable Scaffold Battery

Using electrical stimulation to improve tissue repair is on the increase, including nervous system applications but there are significant limitations to existing strategies. Li and colleagues seek to remove the wires from the equation by developing a biocompatible, self-powered electrostimulation material suitable for stimulating nerve repair.

Peripheral nerve injuries can result in long-term disability, with electrical stimulation (ES) emerging as a promising strategy for nerve repair by enhancing neuronal differentiation, axonal growth, and neurotrophic factor expression. However, conventional ES approaches typically rely on bulky external power sources and invasive wired electrodes, limiting their practicality and increasing the risk of infection. Some recent strategies have leveraged photovoltaic or piezoelectric effects, but these need external devices for energy conversion. To address these challenges, Li and colleagues devised a wireless, self-powered neural scaffold designed to provide localized and sustained bioelectric stimulation for nerve regeneration. The scaffold was engineered to match the mechanical properties of native nerve tissue, ensuring optimal tensile strength and flexibility.

The scaffold was fabricated using laser additive manufacturing with a composite structure comprising poly-l-lactide (PLLA) as the base material, zinc (Zn) as the negative electrode, silver oxide (Ag₂O) as the positive electrode, and PLLA/Polypyrrole (PLLA/PPy) particles as a conductive interlayer. Scanning and transmission electron microscopy revealed a microsphere structure of PLLA particles, with smooth surface contours and diameters ranging from 10 to 80 μm. PLLA/PPy particles had a uniform distribution of PPy on the PLLA surface and were 70 μm in diameter.

When the electrical performance of the material was tested in simulated body fluids, Zn underwent oxidation, releasing electrons that traveled through the conductive PPy layer to the Ag₂O electrode, generating a continuous microcurrent. This bioelectric field was intended to mimic endogenous electrical signals. The initial voltage generated by the PLLA/Zn-Ag₂O scaffold was ∼0.477 mV, degraded gradually but stabilized at 0.462 mV after 12 min. The initial current produced by the scaffold was 17.2 μA, reaching 5.7 μA by 12 min.

Biocompatibility was assessed using live/dead staining of bone marrow mesenchymal stem cells (BMSCs) grown on scaffold materials for 1, 3, or 5 days. There was no difference in the number of live cells on PLLA/Zn, PLLA/Ag₂O, and PLLA/PPy groups compared with PLLA alone. Interestingly, there was an increase in cell number and viability in the PLLA/Zn-Ag₂O group, which may be related to the wireless electrical stimulation generated by the PLLA/Zn-Ag₂O scaffold itself. After 5 days, the PLLA/Zn-Ag₂O scaffold also had the highest cell BMSC cell number. Similarly, optical density assays for cell proliferation revealed high optical density after 3 days for cultures on the PLLA/Zn-Ag₂O scaffold and after 5 days the density increased sharply, indicating that the active scaffold promoted sustained cell proliferation.

Neuronal differentiation of BMSCs was explored through immunofluorescence staining for nestin and microtubule-associated protein 2 (MAP2). The intermediate protein nestin is a marker of early neural development whereas MAP2 is a marker of more mature neurons. Immunostaining revealed significantly higher MAP2 fluorescence (and lower nestin) for cells on electrically active scaffolds. Additionally, quantitative RT-PCR results indicate a substantial increase in nestin expression during early differentiation, followed by enhanced MAP2 expression in later stages (especially at 10 days), supporting the scaffold’s role in promoting progressive neuronal maturation.

To explore the mechanism that encourages neural differentiation, Fluo-4 imaging of intracellular calcium was performed. The calcium ion (Ca²⁺) influx increased 14-fold on the PLLA/Zn-Ag₂O scaffold relative to the inactive scaffolds, which may promote signaling pathways critical for neurite extension and synapse formation. Gene expression analysis showed 24-fold upregulation of MAP2, confirming that the scaffold effectively stimulated neurogenesis.

Beyond nerve regeneration, the scaffold exhibited potent antibacterial effects, attributed to the controlled release of Zn²⁺ and Ag⁺ ions. In situ, the body fluids would act as the electrolyte and as a consequence of electrochemical reactions the PLLA/Zn-Ag₂O would gradually degrade, releasing Zn²⁺ and Ag⁺. These metal ions disrupt bacterial membranes, inhibit enzymatic activity, and induce oxidative stress, leading to bacterial cell death, thereby suggesting an antimicrobial effect as a desirable side effect. Indeed, the scaffold achieved antibacterial rates of 92.6% against Escherishia coli and 91.9% against Staphylococcus aureus, which has the potential to reduce significantly the risk of post-implantation infections.

This study introduces a novel self-powered bioelectric scaffold, advancing the field of bioelectronic medicine by eliminating the need for an external power source. The scaffold’s ability to generate sustained microcurrents, enhance neuronal differentiation, and prevent infection offers a minimally invasive and potentially clinically relevant scheme for nerve repair. Future research should focus on in vivo validation, long-term stability, and potential integration with biodegradable energy storage systems to optimize performance for broader neurological applications.

Charged with Killing: Using Charged Substrates to Enhance Cancer T Cell Immunotherapy

Innovations for the efficient killing of tumor cells are always welcome, and the Bioelectricity community is striding boldly into this arena. Here, Song and coworkers investigated the use of charged substrate treatments to potentiate T cell-mediated cancer immunotherapy. The authors hypothesized that modulating the physical microenvironment of T cells using electroactive nanocomposites could enhance immune cell activation, migration, and tumor cell recognition, thereby improving the efficacy of T cell immunotherapies.

Precision targeting and killing of cancer cells can be achieved using adoptive cellular transfer (ACT) therapy but its efficacy can be reduced by impaired CD8⁺ T cell function, insufficient T cell activation, and the limited lifespan of tumor-reactive T cells. Typically, after T cell receptor (TCR) engagement, formation of TCR clusters at the cell membrane reinforces antigen recognition, leading to formation of an immune synapse and activation of a series of transcription factors that drive T cell effectors, including early growth response 1 (EGR1), nuclear factor of activated T cells (NFAT), and activator protein 1 (AP-1).

Depolarizing the T cell membrane potential in tumor microenvironments affects receptor mobility and oscillations occur at immunological synapses, with electrostatic interactions occurring at TCR signaling. Unanswered questions are how electrical signals are converted into appropriate biochemical signals in T cells and whether external electrical stimulation can modulate T cell activity in cancer therapies. Therefore, the research team employed a multidisciplinary approach combining in vitro assays, advanced imaging techniques, and in vivo murine tumor models to address these questions.

Experiments used CD8⁺ T cells cultured on electroactive substrates engineered as a ferroelectric nanocomposite membrane that could be tuned with distinct surface charges. The membrane incorporated BaTiO₃ nanoparticles into a poly(vinylidene fluoridetrifluoroethylene) (P(VDF-TrFE)) matrix. Tweaking parameters permitted tuning the electrical charge density of the membrane, referred to as non-charged (NC, 55°C, no poling), low-charged (LC, 55°C, poling), mid-charged (MC, 90°C, poling), and high-charged (HC, 120°C, poling). These substrates were designed to mimic the charged microdomains found in physiological settings, enabling a detailed analysis of how electrostatic interactions influence T cell behavior.

In vitro cytotoxicity assays demonstrated a significant decrease in target cell viability when co-cultured with electrically stimulated T cells. A follow-up in vivo study used T cells that were electrically activated using the composite surfaces with increasing charge densities (low charge (LC); medium charge (MC); high charge (HC)) (controls were plated on uncharged materials). Treated cells were subsequently expanded, induced, and then adoptively transferred to mice with tumors. Tumors were controlled best by cells stimulated using the HC material, with reduced efficacy as the surface charge was reduced. Additionally, there was a similar dose-response with increasing amounts and percentages of CD8⁺ T cells isolated from tumor-infiltrating lymphocytes when using substrates with increasing charge densities.

T cells exposed to charged surfaces exhibited significant enhancements in immune cell activation markers, including increased expression of CD69 and CD25 (cluster of differentiation 69 and 25), as well as augmented secretion of key cytokines, such as interferon-γ (IFN-γ) and interleukin-2 (IL-2). This enhanced cytokine production indicates a more robust effector function following antigen recognition due to electrical stimulation.

Furthermore, the charged substrates were found to modulate TCR signaling pathways. Biochemical assays revealed that exposure to the charged surfaces led to increased phosphorylation of proximal signaling molecules, such as zeta-chain associated protein kinase 70 (ZAP-70) and linker for activation of T cells (LAT). This upregulation of intracellular signaling cascades is thought to underlie the improved functional responses observed in T cells. Confocal microscopy studies supported these findings by showing enhanced formation of immunological synapses, suggesting that charged substrates facilitate better T cell-tumor cell interactions.

The in vivo component of the study involved the treatment of tumor-bearing mice with charged substrate-modified scaffolds, in combination with adoptive T cell transfer. Results indicated that this combinatorial approach led to a marked improvement in T cell infiltration into tumor tissues. Notably, treated mice exhibited a significant reduction in tumor burden and prolonged survival compared with control groups receiving conventional immunotherapy. Histological analyses of tumor sections revealed not only an increased density of activated T cells but also a concomitant decrease in immunosuppressive cell populations, such as regulatory T cells and myeloid-derived suppressor cells (MDSCs).

Mechanistic studies suggested that the charged substrates might also alter the tumor cell-surface properties, potentially enhancing antigen presentation and the expression of adhesion molecules, further promoting T cell engagement. The alteration in the electrostatic landscape at the tumor site appears to create a more favorable niche for T cell activity, overcoming some of the inherent barriers posed by the tumor microenvironment.

Collectively, the findings present a compelling case for the integration of charged substrate treatment as an adjuvant to existing T cell-based immunotherapies. By improving T cell activation, enhancing synapse formation, and promoting effective tumor infiltration, this physical modulation strategy offers a new avenue for augmenting anti-tumor immune responses. Future investigations should focus on optimizing substrate design, understanding long-term effects, and translating these preclinical findings into clinical protocols.

Beyond the Buzz: Recurring Items and Reviews Important to the Bioelectricity Community

Ion channel ligands in clinical development: The quarterly blog by Marc Rogers https://www.ionchannellibrary.com/expert_opinions/ion-channel-ligands-in-clinical-development-quarterly-review-q3-2024/

Drug repurposing patent applications: Including molecules that impact bioelectrical dynamics in cells and tissues

Well, that’s the first Buzz for 2025. Here’s to another year filled with exciting bioelectrical discoveries.