Wireless electrical-molecular quantum signalling for cancer cell apoptosis

The study addresses how to achieve targeted, on-demand electrical–molecular communication within living cells to modulate specific biochemical pathways. Biological electron transfer, including in cytochrome c (Cyt c) and photosynthesis, can involve quantum mechanical effects such as electron tunnelling (QBET). Cyt c’s redox state (Fe2+/Fe3+) regulates mitochondrial electron transport and apoptosis via APAF-1 binding. Prior tools lacked the capability to interface at the spatiotemporal and energetic scales of native intracellular redox processes. The authors hypothesize that bifunctionalized gold nanoparticles acting as intracellular bipolar nanoelectrodes, stimulated by alternating current electric fields (a.c. EFs), can wirelessly switch Cyt c redox state via nanoscale electrochemistry and QBET, thereby inducing selective apoptosis in glioblastoma (GBM) cells.

Previous work has shown gold nanoparticles and carbon nanotubes can operate as bipolar nanoelectrodes under applied EFs, enabling wireless electrochemistry, though nanoscale wireless electrochemistry in the presence of cells was considered challenging. High-frequency a.c. EFs lower membrane impedance, facilitating cytoplasmic access and potentially enabling bipolar electrochemistry at low voltages. Carbon nanotube porins have exhibited redox reactions in cells without inducing direct cell death. Cytochrome c electron transfer and roles in apoptosis are well established, with evidence for electron tunnelling mechanisms. Tumor-treating fields (TTFields) are an approved modality but act via different mechanisms; the present approach seeks greater molecular specificity by directly modulating Cyt c redox through wireless electrochemistry and QBET.

Design and synthesis: 100 nm carboxylic-PEG-coated gold nanoparticles (GNP100; PEG 1–5 kDa) were bifunctionalized with reduced cytochrome c (r.Cyt c, Fe2+) and zinc porphyrin mediator (Z; zinc 5-(4-aminophenyl)-10,15,20-(tri-4-sulfonatophenyl)porphyrin triammonium) via EDC/NHS carbodiimide coupling to yield GNP100@r.Cyt c@Z bio-nanoantennae. Controls included GNP100, GNP100@r.Cyt c and GNP100@Z. Reduced Cyt c was prepared by ascorbic acid reduction of oxidized Cyt c and dialysis. Bifunctionalization was also performed on GNPs of 20, 50, and 100 nm with PEG linkers of 1, 2, 3.5, and 5 kDa to modulate tunnelling barrier length. Characterization: TEM, DLS, zeta potential, and UV–vis confirmed conjugation; circular dichroism probed conformational/redox changes. Cyclic voltammetry (ITO working electrode, Pt counter, Ag/AgCl reference; 25 µg mL−1 in 10 mM PBS) assessed redox behavior and heterogeneous electron transfer rates (k0). Cellular models: Patient-derived GBM cells from invasive margin (GIN 28, GIN 31) and core (GCE 28, GCE 31), commercial GBM line U251, and non-tumorigenic human cortical astrocytes; additional normal cells (liver bile duct and cerebellar astrocytes) in supplements. Uptake and association: Confocal z-stacks verified internalization; ICP-MS quantified particles per cell. Biocompatibility and metabolic activity: PrestoBlue HS assays evaluated cytotoxicity (up to 100 µg mL−1) and metabolic activity changes. Electrical stimulation (ES): Cells incubated with bio-nanoantennae (25 µg mL−1, 8 h), washed, then exposed to a.c. EFs using steel electrodes (10 mm gap) driven by a function generator. Optimal condition identified: 3 MHz, 0.65 V cm−1 (peak), applied for 2 or 12 h. Voltage and frequency were optimized; 0.65 V cm−1 was chosen to avoid water electrolysis concerns. Temperature and ROS were monitored (no significant changes observed). Cell death and apoptosis: Live/dead assays (calcein AM/propidium iodide), flow cytometry with CellEvent Caspase-3/7 Green and Zombie NIR dyes, and confocal microscopy for caspase 3/7 activation. Localization studies: Endosomal/lysosomal markers (CellLight Late Endosomes-GFP; LysoTracker Green) assessed localization and endosomal escape after ES. Transcriptomics: RNA-seq performed (Qiagen QIAseq UPX 3' kit; Illumina NextSeq). Differential gene expression analyzed with edgeR/limma frameworks; variance-stabilized data used for clustering; gene-set enrichment analysis (GSEA) on GO biological processes. Quantum tunnelling evidence: Systematically varied GNP size and PEG linker length; metabolic activity vs barrier length analyzed. Mathematical model related metabolic activity M(t) to rate of Cyt c donor charging r_a via ln(M(t))/t, expecting exponential dependence on barrier length consistent with tunnelling. Plasmon resonance scattering (dark-field microscopy, spectra over 1000 frames) probed plasmon resonance energy transfer and quantized spectral dips indicating redox state changes (PRET), with and without ES. Statistical analysis: Two-way ANOVA with Tukey post-test; significance at P ≤ 0.05; results reported as mean ± s.e.m. or s.d. as specified.

• Electrical parameters: A resonant a.c. EF at 3 MHz and 0.65 V cm−1 maximally reduced GBM cell metabolic activity when combined with GNP100@r.Cyt c@Z; 0.65 and 1.0 V cm−1 produced similar effects (P = 0.23), 0.65 V cm−1 chosen for studies. - Selective cytotoxicity and apoptosis: In GIN/GCE GBM cells, ES (3 MHz, 0.65 V cm−1) for 12 h after 8 h nanoparticle incubation led to ~50% decrease in metabolic activity (versus ~20% after 2 h). Effects were significantly greater than controls (GNP100, GNP100@r.Cyt c, GNP100@Z) and larger than 24 h tumor-treating fields benchmarks. Cortical astrocytes showed a weaker effect (~20% decrease; P = 0.011). Live/dead assays and flow cytometry revealed caspase-3/7 activation and apoptosis; confocal imaging corroborated caspase activation. - Intracellular behavior: Confocal microscopy indicated bio-nanoantennae undergo endosomal escape and localize to the cytosol after ES; no increases in ROS or temperature were detected. - Redox switching and spectroscopy: Circular dichroism and UV–vis showed ES-induced redox switching of Cyt c on nanoantennae (blueshift consistent with r.Cyt c to o.Cyt c). - Electrochemistry: CV-derived heterogeneous electron transfer rates: k0 ≈ 9.6 × 10−3 cm s−1 for GNP100@r.Cyt c and 3.75 × 10−3 cm s−1 for GNP100@r.Cyt c@Z. - Quantum tunnelling evidence: Varying nanoparticle size (20/50/100 nm) and PEG linker length (1/2/3.5/5 kDa) revealed resonant biological effects (greatest metabolic decreases) with 1–2 kDa linkers and 50–100 nm GNPs under ES. Mathematical modelling showed an exponential dependence of the donor charging rate on barrier length, consistent with electron tunnelling. Plasmon resonance scattering detected quantized dips; without ES, peaks at ~465 nm and ~568 nm and an LSPR shifted to 711 nm; with ES, a quantized dip at 536 nm appeared, consistent with o.Cyt c formation, and a ~20 mV LSPR shift indicative of charge transfer. - Uptake and safety: Bio-nanoantennae internalized across GBM and astrocyte cells; PrestoBlue assays indicated biocompatibility up to 100 µg mL−1 in the absence of ES. - Transcriptomics: Hierarchical clustering and GSEA indicated substantial modulation of apoptosis, proliferation, angiogenesis, ER stress, UPR, and starvation pathways in GBM cells treated with GNP100@r.Cyt c@Z plus ES, with minimal transcriptomic changes in astrocytes, supporting selective targeting.

The findings demonstrate that intracellular bio-nanoantennae can transduce an external a.c. EF into molecular redox actuation by wirelessly switching Cyt c from Fe2+ to Fe3+, thereby triggering APAF-1-mediated caspase-3/7 activation and apoptosis in GBM cells. The selective impact on GBM versus astrocytes is supported by both phenotypic assays and transcriptomics, suggesting differential sensitivity of cancer cell redox/apoptotic pathways to this electrical–molecular input. The electrical parameters (3 MHz, 0.65 V cm−1) and nanoscale design (GNP size and PEG linker length) are critical; the resonance behavior, exponential dependence of charging rate on barrier length, and PRET quantized dips collectively support EF-induced quantum biological electron tunnelling (QBET) as the operative mechanism. Absence of ROS or thermal effects indicates the response is not due to nonspecific stress. This approach provides a route for precise bioelectric control over intracellular redox signaling, distinct from TTFields, and highlights the potential of quantum-informed electroceuticals for targeted cancer therapy.

Applying a.c. electric fields to bifunctionalized gold bio-nanoantennae enables wireless nanoscale electrochemistry that switches cytochrome c redox state, selectively inducing apoptosis in patient-derived GBM cells. Evidence from spectroscopy, modelling, and size/linker-length dependencies supports EF-induced quantum tunnelling (QBET) as the mechanism of electron transfer. Transcriptomics confirms selective modulation of cancer-related pathways with minimal impact on healthy astrocytes. This wireless electrical–molecular communication platform represents a quantum functional medicine tool with potential for developing quantum nanomedicines and targeted cancer treatments.

The study is conducted in vitro; in vivo efficacy, biodistribution, and safety remain to be established. While multiple lines of evidence support QBET, classical contributions cannot be entirely excluded, and further mechanistic studies are needed to fully elucidate the electron transfer pathway and endosomal escape mechanisms. The apoptotic response and selectivity were characterized in GBM and a limited set of normal cells; broader cell-type validation and parameter optimization (frequency, voltage, linker length, nanoparticle size) are required for generalization and translational development.