Canady Helios Cold Plasma Induces Non-Thermal (24 °C), Non-Contact Irreversible Electroporation and Selective Tumor Cell Death at Surgical Margins

Background: The Canady Helios Cold Plasma (CHCP) system is a non-thermal, non-contact cold atmospheric plasma technology that generates transient electric fields and reactive species capable of disrupting cancer cell membranes. This study investigated the voltage-dependent membrane irreversible electroporation (IRE) dynamics induced by CHCP across biologically distinct breast cancer subtypes. Methods: Four breast cancer cell lines, triple-negative (MDA-MB-231 and Hs578T), ER+/PR+/HER2− (MCF-7), and ER+/PR+/HER2+ (BT-474), were exposed to CHCP for 5 min at 25 V (~1675 V/cm PTEF) or 30 V (~2010 V/cm), either directly or with Plasma Activated Media (PAM). Membrane permeability was assessed by propidium iodide (PI) uptake over 120 min. Morphological changes were evaluated microscopically. Functional electroporation was examined via BCL2A1-targeting siRNA delivery and clonogenic survival. Ex vivo analyses of Phase I clinical trial tumor specimens (NCT04267575) were performed to characterize CHCP-induced tissue responses. Results: CHCP produced voltage- and time-dependent membrane permeabilization in all breast cancer cell lines, with 30 V generating robust and sustained PI uptake compared to transient effects at 25 V. Treated cells exhibited morphological features consistent with membrane disruption. CHCP enabled intracellular siRNA delivery and significantly reduced clonogenic potential, confirming functional pore formation. Ex vivo CHCP treatment selectively damaged tumor cells while sparing adjacent non-cancerous tissue. Conclusions: This study demonstrates CHCP as a non-thermal (24 °C), non-contact plasma-based IRE platform which induces controlled membrane permeabilization and selective cancer cell death. CHCP offers a translational strategy to eradicate residual tumor cells at the surgical margins, and prevent local recurrence, positioning it as a versatile adjunct in precision surgical oncology.