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-targetingsiRNA 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.
背景：Canady Helios冷等离子体（CHCP）系统是一种非热、非接触式的冷大气等离子体技术，能够产生瞬态电场和活性物质，从而破坏癌细胞膜。本研究探讨了CHCP在不同电压下诱导的膜不可逆电穿孔（IRE）动力学，并针对生物学特性各异的乳腺癌亚型进行了分析。方法：选取四种乳腺癌细胞系，包括三阴性（MDA-MB-231和Hs578T）、ER+/PR+/HER2−（MCF-7）以及ER+/PR+/HER2+（BT-474），分别以25 V（约1675 V/cm PTEF）或30 V（约2010 V/cm）的电压直接暴露于CHCP或通过等离子体活化培养基（PAM）处理5分钟。通过碘化丙啶（PI）摄取在120分钟内评估细胞膜通透性变化，并利用显微镜观察形态学改变。通过靶向BCL2A1的siRNA递送和克隆形成存活实验验证功能性电穿孔效果。此外，对I期临床试验（NCT04267575）的肿瘤标本进行离体分析，以表征CHCP诱导的组织反应。结果：CHCP在所有乳腺癌细胞系中均引起电压和时间依赖性的膜通透性增加，其中30 V处理相比25 V产生的短暂效应，能引发更显著且持续的PI摄取。处理后的细胞表现出与膜破坏一致的形态学特征。CHCP成功实现了细胞内siRNA递送，并显著降低了克隆形成能力，证实了功能性孔洞的形成。离体CHCP处理选择性地损伤肿瘤细胞，同时保留邻近的非癌组织。结论：本研究证明CHCP作为一种非热（24 °C）、非接触的等离子体基IRE平台，能够诱导可控的膜通透性改变和选择性癌细胞死亡。CHCP为消除手术切缘残留肿瘤细胞、预防局部复发提供了一种转化策略，使其成为精准外科肿瘤学中一种多功能的辅助手段。
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