The Canady Helios HERO Model: A Ventilator- Integrated Humidified Cold Atmospheric Plasma System for Lung Cancer Treatment and Respiratory Infection Applications

The Canady Helios HERO Model: A Ventilator- Integrated Humidified Cold Atmospheric Plasma System for Lung Cancer Treatment and Respiratory Infection Applications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Canady Helios HERO Model: A Ventilator- Integrated Humidified Cold Atmospheric Plasma System for Lung Cancer Treatment and Respiratory Infection Applications Saravana R.K. Murthy, Taisen Zhuang, Olivia Z. Jones, Jerome Mcqueen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8651228/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Cold atmospheric plasma (CAP) generates reactive oxygen and nitrogen species (ROS/RNS) capable of selectively destroying pathogens and malignant cells while sparing normal tissue. The Canady Helios Cold Plasma (CHCP) HERO (Humidified Electrical Reactive Oxygen) System integrates a CAP generator with a ventilator platform to enable controlled, humidified plasma delivery through the respiratory tract. Efficacy and safety were assessed using A549 human lung carcinoma cells and an in vivo swine model. CAP was delivered with humidified Air/O₂ (1:1 v/v; 2 L·min⁻¹) and helium (3 L·min⁻¹) at 35–40 V, varying helium humidity (0-100%) and discharge mode (continuous or interval). Cell viability (MTT), ozone (O₃), and ROS/RNS (H₂O₂, NO₂⁻, NO₃⁻) were quantified, and physiological and histological assessments evaluated in vivo safety. A549 viability decreased significantly with increasing helium humidity (ANOVA, F[4,10] = 1770.23, p = 3.3 × 10⁻¹⁴), with > 95% reduction within 5 min at 100% RH. Humidified Air/O₂ reduced O₃ output by 40–60% versus dry gas (p < 0.005). In swine, vital signs remained stable and lung histology showed intact alveoli without inflammation or edema; only TNF-α rose modestly (p < 0.05). The CHCP-HERO System achieved complete A549 eradication while maintaining physiological and histological safety, supporting CAP-based ventilation as a promising therapeutic gas system for respiratory infections and lung cancer. Health sciences/Diseases Health sciences/Medical research Breast cancer Cold atmospheric plasma Cancer treatment Respiratory infections COVID-19 ventilation system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Respiratory viral infections, including COVID-19 and severe acute respiratory syndrome (SARS), are primarily transmitted through aerosolized droplets that carry infectious viral particles within the respiratory tract 1 – 3 . Their global impact underscores the urgent need for novel, non-pharmaceutical interventions that directly inactivate viruses in the airways 4 . CAP has emerged as a promising bioengineering technology for biomedical applications 5 , generating reactive oxygen and nitrogen species (ROS/RNS), photons, charged particles, and electric fields at near-room temperature. Unlike thermal disinfection or chemical sterilization, CAP offers a non-thermal, non-contact, tunable oxidative environment that selectively targets pathogens and malignant cells without harming healthy tissues 6 , 7 . Numerous studies demonstrate CAP's broad-spectrum antiviral efficacy. It inactivates airborne viruses 8 , 9 , deactivate hepatitis B virus while preserving hepatocyte function 10 , inhibit HIV replication 11 , and inactivate Newcastle disease virus and avian influenza virus without compromising their antigenic determinants for vaccine development 12 . Zimmerman et al., 13 inactivated adenovirus using surface micro-discharge plasma,, while Su et al., 14 achieved potent inactivation of Newcastle disease virus with plasma-activated solutions. These results highlight CAP's potential as a versatile antiviral and antimicrobial modality. CAP-mediated inactivation arises from high oxidation-reduction potential (ORP) and electrical conductivity via abundant free radicals 15 . The ROS and RNS such as singlet oxygen (¹O₂), ozone (O₃), superoxide anion (O₂ - ˙), nitric oxide (NO˙), nitrogen dioxide (NO₂˙), and hydroxyl radicals (˙OH) induce lipid peroxidation and of fatty acid cross-linking disrupting cellular and viral membranes 15 , 16 . They also oxidize amino acids like cysteine, tyrosine, tryptophan, and histidine residues, forming disulfide bonds and hydroperoxides that cause protein aggregation and envelope destruction. Nucleic acid modifications, including guanine oxidation and guanine-lysine crosslinking, silence gene expression and inhibit replication. In biological media, short-lived species such as peroxynitrite (ONOO - ) and long-lived species such as hydrogen peroxide (H₂O₂) and nitrite (NO₂ - ), sustained oxidative activity. We hypothesized that an optimized CHCP-HERO System could safely deliver CAP for antiviral and anticancer effects. Aims included: (1) optimize system parameters, (2) evaluate antitumor efficacy in A549 cells, and (3) assess pulmonary safety in a swine preclinical model. The HERO System integrates CHCP with a ventilator for humidified, intratracheal CAP delivery. Humidification balances potency and safety: helium enhances reactivity, while Air/O₂ reduces ozone and stabilizes temperature. Separate oxygen addition controls species composition and minimizes O₃. Due to biosafety limits with live SARS-CoV-2, we used A549 lung carcinoma cells-resembling type II alveolar cells and susceptible to oxidative stress 17 , 18 , and swine, physiologically similar to humans in respiratory systems 19 . These models together provide a rigorous translational framework for evaluating the therapeutic efficacy and biosafety of the HERO System prior to clinical application. In this study, we investigated the therapeutic potential of the CHCP-HERO system for the treatment of lung cancer and the inactivation of respiratory pathogens. We optimized the CHCP-HERO system parameters which included helium and Air/O₂ humidification, discharge voltage, and interval-based plasma dosing to balance plasma potency with safety. The data demonstrate that the HERO System can effectively eradicate A549 lung cancer cells in vitro and maintain pulmonary integrity and physiological stability in the swine model. Together, these findings provide compelling evidence that, when properly tuned, the CAP-based HERO ventilator system functions as a safe and effective therapeutic gas inhalant platform with potential applications in treating respiratory diseases such as pneumonia, COVID-19, and lung cancer. 2. Results The CHCP-HERO System was designed to generate and deliver CAP through a ventilator-integrated circuit under controlled humidity and gas composition conditions (Fig. 1 ). Prior studies 20 – 22 demonstrated that ambient air is the most effective carrier gas for viral inactivation using atmospheric-pressure cold plasma, particularly at power levels between 20 W and 24 W. In head-to-head tests involving airborne MS2 bacteriophage, cold plasma generated with air resulted in significantly higher rates of viral inactivation compared to mixtures of argon-oxygen (Ar-O₂, 2% v/v) and helium-oxygen (He-O₂, 2% v/v), when discharge power was held constant. The mechanism underlying the superior performance of ambient air relates to the production of a wider spectrum of RONS in humid air environments, including ozone, singlet oxygen, and nitric oxides, which are less abundantly produced by noble gas-oxygen mixtures. These chemically reactive species disrupt viral capsids and genomic material more effectively, leading to rapid viral deactivation. Because ventilators inherently require air as an input source, air was selected as the primary carrier gas for the HERO configuration. In the present study, we systematically evaluated the influence of air/O₂ ratio, helium humidity, discharge voltage, and treatment duration on biological and chemical outcomes. Relative humidity (RH) as an important factor of the air were studied for an optimal configuration in addition to CAP treatment parameters including discharge voltage (V) and treatment time (t). The feeding gas of Air/O 2 mixture and/or He was humidified by bubbling through water. The relative humidity (RH) of the humidified gases was measured constantly with a humidity sensor. 2.1. Dose-Dependent Cytotoxicity of Humidified Cold Atmospheric Plasma on A549 Lung Cancer Cells: Effects of Oxygen Fraction and Exposure Duration To evaluate the influence of oxygen fraction and treatment duration, A549 human alveolar carcinoma cells were exposed to HERO system generated CAP using a humidified air/O₂ mixture and dry helium at 70 V. Figure 2 (A and B) shows gradual viability decline with exposure time (1–4 min), reaching ~ 60% of controls at 4 min. Increasing oxygen from 16% to 32% slightly attenuated cytotoxicity, but all conditions significantly reduced viability vs. controls (ANOVA: 16% O₂, F[4,10] = 332.59, p = 1.37 × 10 - ¹⁰; 24% O₂, F[4,10] = 290.72, p = 2.68 × 10 - ¹⁰;32% O₂, F[4,10] = 52.24, p = 1.15 × 10 - ⁶; Tukey’s post hoc in Supplementary Table S1 A-C). Prolonged 24% O₂ treatment further decreased viability time-dependently: <40% at 10 min, complete A549 elimination at 17 min (Fig. 2 B; ANOVA, F[7,14] = 685.203, P = 1.31×10 –18 ; Supplementary Table S1 D).These data confirm robust, dose-dependent cytotoxicity by the humidified CAP system via duration and gas modulation. Mechanistically, CAP treatment causes apoptosis in A549 cells, evidenced by increased ROS production, mitochondrial damage, chromatin condensation, and activation of apoptosis markers like cleaved caspases and PARP proteins. Morphologically, plasma exposure leads to cell shrinkage, detachment, and nuclear fragmentation, confirming effective programmed cell death pathways are engaged 23 . 2.2. Humidity-Dependent Enhancement of Cytotoxicity in A549 Lung Cancer Cells by the HERO Cold Atmospheric Plasma System: Morphological and Viability Assessments To assess humidity's impact on HERO System cytotoxicity, A549 lung carcinoma cells were exposed to CAP generated with 100% humidified Air/O₂ (1:1 v/v; 2 L min-1) and helium (3 L min-1) at 0%, 50%, or 100% RH. After 24 h, phase-contrast microscopy showed humidity-dependent morphological deterioration (Fig. 3 ). Controls (untreated or gas flow only) retained cobblestone morphology, intact nuclei, and adherence, confirming no gas-only effect. Dry helium (0% RH) induced shrinkage and rounding at 5 min, progressing to partial detachment and apoptotic bodies at 10 min, with some viable cells persisting. At 50% RH, 5 min caused membrane blebbing and chromatin condensation; 10 min led to extensive detachment and necrotic debris. At 100% RH, 5 min triggered catastrophic shrinkage, fragmentation, and lysis, markedly enhancing plasma efficacy. MTT assays quantified this humidity-dependent response (Fig. 3 G). Dry conditions reduced viability to ~ 60% (5 min) and 40% (10 min). At 50% and 100% RH, 5 min exposure eliminated > 95% viability. Paired t-tests confirmed significance (p = 0.0083 − 0.0013; detailed: 5 min, gas-only 100% RH vs. 0% CAP p = 0.0083, 0% vs. 50% CAP p = 0.0013, 50% vs. 100% CAP p = 0.416; 10 min, gas-only vs. 0% CAP p = 0.0025, 0% vs. 50% CAP p = 0.0098, 50% vs. 100% CAP p = 0.184). Humidified helium critically enhances CAP potency by boosting reactive species delivery, accelerating apoptosis/necrosis, membrane rupture, and DNA damage in a time- and humidity-dependent manner, selectively targeting cancer cells under optimized settings. 2.3. Humidity-Driven Optimization of Exposure Time for Complete Eradication of A549 Lung Cancer Cells Using the HERO System To determine minimum exposure for A549 eradication, cells were treated with CAP for 1–5 min using humidified/dry Air/O₂ (1:1 v/v; 2 L min - 1 ) and helium (3 L min - 1 ) at 0%, 50%, or 100% RH. Microscopy 24 h post-exposure showed time- and humidity-dependent damage (Fig. 4 ). Dry helium (0% RH) preserved morphology until 5 min (minor rounding/detachment). At 50% RH, injury emerged at 4 min (reduced density, apoptotic morphology). At 100% RH, effects were rapid: nuclear condensation/shrinkage at 2 min, disintegration by 4 min. Air/O₂ humidity variation had minimal impact, highlighting helium humidification as the key cytotoxic driver. MTT assays confirmed findings (Fig. 5 A- C). Dry conditions (0% RH) showed minimal cytotoxicity (> 80% viability at 5 min). At 50% RH, viability fell to ~ 50% at 5 min. At 100% RH, > 50% reduction at 3 min; complete ablation (0% survival) at ≥ 4 min. ANOVA verified significance (humidified Air/O₂ + He: 0% RH, F[4,10] = 4.0113, P = 0.034; 50% RH, F[4,10] = 291.143, P = 2.665×10 –10 ; 100% RH, F[4,10] = 1770.226, P = 3.343×10 –14 ; dry Air/O₂ + He: 0% RH, F[4,10] = 16.322, P = 0.00022; 50% RH, F[4,10] = 292.65, P = 2.598×10 –10 ; 100% RH, F[4,10] = 1024.67, P = 5.114×10 –13 ; Tukey’s post hoc in Supplementary Table S2 ). Fully humidified helium maximizes reactive oxygen/nitrogen species generation/transfer, enabling CHCP-HERO rapid, complete A549 destruction in a clinically feasible window. 2.4. Voltage and Gas Configuration Optimization in the HERO System: Balancing Cytotoxic Efficacy with Reduced Ozone Generation To optimize CHCP-HERO for efficacy and safety, operating voltage, gas composition, and ozone (O₃) generation were evaluated. At 70 V, robust plasma efficacy produced O₃ exceeding OSHA limits, risking respiratory irritation. Voltage was thus reduced to 35–40 V to sustain plasma while minimizing O₃. Gas delivery was refined by separating Air and O₂ inputs into the CAP mixer, reducing localized O₂ density, suppressing O₃, and preserving ROS/RNS for cytotoxicity. Figure 6 A,B illustrates the reengineered mixer and flow dynamics enabling reduced O₃ without compromising uniformity. Biological validation confirmed efficacy retention (Fig. 6 C,D). A549 cells showed < 5% viability after 8 min at 40 V (Air/O₂ mixture) or 15 min (separate Air/O₂). ANOVA revealed significant effects (Air/O2 mixture Voltage = 40V, F[9, 20] = 1174.233, P = 3.145 -25; Air/O2 mixture Voltage = 35V, F[16, 34] = 24.37, P = 4.671 -14; Air/O2 separate Voltage = 40V, F[15, 32] = 15.171, P = 1.75 -10; Air/O2 separate Voltage = 35V, F[15, 32] = 12.248, P = 2.83 -9; Tukey’s post hoc in Supplementary Table S3). Low-voltage discharge (35–40 V) with decoupled Air/O₂ streams balances potent anti-cancer activity and O₃ control, advancing HERO toward clinical respiratory/oncologic use. 2.5. Quantification and Mitigation of Ozone Generation in the HERO System: Effects of Humidity, Voltage, and Gas Configuration on Safety Optimization Ozone (O₃) concentrations were measured at the endotracheal tube distal end using a calibrated detector (Forensics Detectors, CA) across conditions: humidified/dry Air/O₂ (1:1 v/v; 24% O₂; 2 L min - 1 ) with helium (3 L min - 1 ) at 0%, 50%, 100% RH, at 70 V/114 kHz. Dry Air/O₂ yielded higher O₃ than humidified counterparts ( t- tests, p = 4.95 × 10-⁵, p = 0.0009, and p = 0.005 for 0%, 50%, and 100% RH, respectively; Supplementary Fig. S1 ). Helium humidity increases elevated O₃, correlating with cytotoxicity (Figs. 5 ), indicating O₃ as a key bioactive species driving oxidative cancer cell inactivation. Voltage dependency showed marked O₃ reduction from 50 V to 35 V (Fig. 7 ), aligning with safety optimization preserving efficacy while minimizing exposure. Premixed Air/O₂ produced higher O₃ than separate feeds into the CAP mixer; separate delivery cut O₃ by up to 40% at 35–50 V ( t -test, Humidified Air/O 2 vs Dry Air/O 2 , 0%, 50% and 100%, p = 4.95-5, p = 0.0009 and p = 0.005 respectively and Air/O 2 mixture vs Air O 2 separate at 35, 36,37,38, 39, 40, 45 and 50 voltage (V), was p = 0.06, p = 0.06, p = 0.02, p = 0.02, p = 0.02, p = 0.017, p = 0.001, and p = 0.0037 respectively). Voltage and gas configuration adjustments control O₃ without compromising performance, ensuring HERO operates within regulatory limits. 2.6. Quantification of ROS/RNS in Plasma-Activated Medium: Interval Delivery Enhances Oxidative Species Accumulation While Maintaining Safety ROS and RNS were quantified in1 mL PBS following HERO treatment in continuous and interval modes using colorimetric assays (Fig. 8 ). Continuous 8-min with an Air/O₂ mixture (1:1 v/v) at 40 V yielded 725 µM H₂O₂,11.9 µM NO₂⁻, and 3.3 µM NO₃⁻, while the corresponding interval regimen (3 + 3 + 2 min) produced 470 µM H₂O₂, 12.5 µM NO₂, and 2.2 µ NO₃⁻. When and O were supplied, 15 mins treatment generated 806 µM H₂O and 33 µM NO₂⁻, whereas the interval mode (4 + 4 + 4 + 3 min) resulted in 952 µM H₂O₂, 45 µM NO₂⁻, and 12 µM NO₃⁻, indicating more efficient ROS/RNS generation during interval, likely due to re-equilibration of reactive intermediates between pulses. Across all conditions, H₂O₂ concentrations (0.36–0.62 mg m⁻³) remained below the OSHA/NIOSH permissible exposure limit of 1.4 mg m⁻³, confirming operation within occupational safety standards. Gas-only controls produced negligible ROSNS, confirming their plasma-derived origin rather than gas flow or humidification artifacts. ANOVA revealed highly significant among treatment groups for H₂O₂ (F[9, 20] = 217.64, P = 5.89E-18; ANOVA for NO 2 - F[9, 20] = 91.06, P = 2.98E-14; ANOVA for NO 3 - F[9, 20] = 166.13, P = 8.41917E-17); Tukey’s post hoc comparisons are reported in Supplementary Table S4. Together, these findings demonstrate that the HERO System generates a balanced yet potent oxidative environment, with-derived O₃, H₂O₂, and NO₂⁻ likely serving as key mediators of cytotoxicity and oxidative damage while maintaining clinical safety and biocompatibility. 2.7. In Vivo Safety and Physiological Tolerability of Intratracheal HERO Cold Plasma Delivery Shows Excellent Physiological Tolerability in Swine Model A 55 kg anesthetized swine was used to evaluate the in vivo safety and physiological tolerability of intratracheal cold plasma delivery under mechanical ventilation. The CHCP-HERO System was operated at 40 V with 3 L min⁻¹ helium and 3 L min⁻¹ oxygen in four plasma exposure cycles (4 + 4 + 4 + 3 min), each separated by 5 minutes of recovery. Continuous monitoring over 90 minutes included core temperature, end-tidal CO₂, SpO₂, arterial blood pressure, and pulse rate (Supplementary Table S5, FS2-5). All parameters remained within normal physiological ranges during and after treatment. Core temperature rose only mildly from 33.7°C to 35.3°C by the end of the third cycle, consistent with anesthesia-related thermoregulation. End-tidal CO₂ fluctuated between 22–43 mmHg without evidence of hypoventilation or hypercapnia. Oxygen saturation was stable at 97–100%, indicating preserved gas exchange. Cardiovascular stability: systolic pressure 90–110 mmHg (median 95), diastolic 50–64 mmHg (median 55); pulse 83–102 bpm (median 92), with no hemodynamic instability, arrhythmias, respiratory distress, or desaturation events observed. Collectively, these findings demonstrate that intratracheal delivery of humidified cold plasma via the HERO System is acutely well tolerated, with no detectable respiratory, cardiovascular, or thermoregulatory compromise, supporting its translational potential for respiratory and thoracic applications. 2.8. Serum Cytokine Profiling Demonstrates Safety of HERO System in Swine Model To investigate whether intratracheal HERO exposure induces systemic immune activation or inflammatory signaling, serum cytokine levels were quantified in the swine model before and after treatment using ELISA. Blood samples were collected 1 hour prior to plasma administration and again 6 hours post-treatment to capture both immediate and delayed immune responses. The cytokine panel included interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), Angiopoietin-1, interleukin-1 beta (IL-1β), and interleukin-10 (IL-10) representing key mediators of inflammatory, vascular, and immunoregulatory pathways. Among all analytes tested, TNF-α showed a mild but statistically significant increase following CAP exposure (p < 0.05; Mann-Whitney test), while all other cytokines remained at baseline levels (Fig. 9 ). The modest rise in TNF-α is consistent with a transient local immune response to plasma-generated reactive species, reflecting limited activation of macrophage and endothelial signaling cascades without evidence of systemic inflammation or cytokine storm. Notably, IL-6, CRP, and Angiopoietin-1 biomarkers typically elevated during acute inflammatory or vascular injury states did not differ significantly between pre- and post-treatment samples, indicating no measurable systemic inflammatory or endothelial activation. Furthermore, IL-1β and IL-10 concentrations remained below the assay’s detection limit, suggesting the absence of pro-inflammatory cytokine amplification or compensatory anti-inflammatory feedback. The lack of significant changes in these parameters corroborates the favorable biocompatibility of the HERO treatment and aligns with prior observations of preserved physiological stability and normal lung histoarchitecture post-exposure. Together, these findings demonstrate that CAP delivery via the HERO System elicits only minimal systemic immune modulation, thereby reinforcing its safety and translational potential for intratracheal therapeutic applications without inducing cytokine-mediated toxicity or systemic inflammatory sequelae. 2.9. Histopathological Analysis Confirms Preserved Lung Architecture and Absence of Tissue Damage Following CHCP-HERO Treatment To evaluate the tissue-level safety and structural integrity of the lungs following intratracheal HERO treatment, formalin-fixed, paraffin-embedded lung sections were subjected to detailed histopathological examination using hematoxylin and eosin (H&E) staining. Microscopic assessment revealed well-preserved pulmonary architecture across all analyzed regions, with intact alveolar septa, bronchiolar epithelium, and vascular structures (Fig. 10 ). The alveolar sacs remained uniformly inflated, displaying a continuous and uncompromised air-blood barrier, with no evidence of alveolar collapse, hemorrhage, or congestion. The bronchiolar lining cells exhibited normal morphology and polarity, showing no signs of epithelial sloughing or necrosis. Similarly, the vascular lumen appeared with intact endothelial lining, and there were no histological signs of capillary dilation, thrombosis, or vascular injury. The absence of interstitial edema or fibrin deposition further indicates that plasma exposure did not disrupt microvascular permeability or induce inflammatory exudation. The surfactant system critical for maintaining alveolar stability and gas exchange was also morphologically intact, showing no vacuolization or lamellar body depletion. Moreover, no peribronchovascular or alveolar inflammatory infiltrates were detected, confirming that the CAP exposure did not trigger localized immune or oxidative injury responses. The overall lung parenchyma exhibited a high degree of homogeneity and structural preservation, comparable to untreated control tissue. Collectively, these findings demonstrate that the HERO system’s cold plasma stream exerts no measurable thermal, oxidative, or mechanical damage on delicate pulmonary structures. This comprehensive histological evidence validates the biocompatibility and non-destructive nature of the HERO platform and supports its clinical safety for intrapulmonary and ventilator-integrated therapeutic applications. 3. Discussion The findings from this study clearly demonstrate that the CHCP-HERO System is a safe, effective, and tunable therapeutic platform capable of achieving near-complete eradication of A549 human lung carcinoma cells while maintaining biocompatibility and safety in large-animal models. Using the A549 cell line as a representative model for human lung cancer, the study revealed a strong, humidity-dependent correlation between treatment efficacy and plasma reactivity. Increasing helium humidity from 0% to 100% resulted in a significant enhancement of plasma-induced cytotoxicity, with total loss of viability observed within a few minutes of exposure at 100% relative humidity. This humidity-driven potentiation of CAP activity underscores the critical role of water vapor as a reaction substrate, facilitating the generation of ROS/RNS such as hydroxyl radicals (·OH), H₂O₂, NO·, and ONOO- 24 . These reactive intermediates act synergistically to induce oxidative stress, leading to membrane disruption, protein denaturation, and apoptosis, thereby driving non-thermal and selective cancer cell death 7 , 23 ,25–27 28, 29 . Beyond the modulation of plasma chemistry, humidification of the Air/O₂ mixture was shown to play an essential role in enhancing the system’s safety profile 30 . The addition of moisture into the Air/O₂ stream effectively reduced ozone generation and stabilized the plasma discharge temperature, thereby lowering the risk of oxidative gas accumulation and minimizing local tissue irritation. Moreover, the system configuration allowing separate introduction of Air and O₂ rather than using a premixed gas further reduced ozone formation by limiting localized oxygen density within the discharge zone. Collectively, these adjustments enabled efficient plasma operation at lower voltages (35–40 V), preserving potent anticancer activity while ensuring that O₃ concentrations remained within OSHA-compliant safety thresholds. Moreover, Li et al. showed that humidity reshapes plasma discharge behavior by lowering electron density and shifting energy distributions toward pathways that promote controlled generation of biologically active ROS/RNS rather than damaging high-energy species 31 . This mechanistic shift directly supports our findings that humidified CAP delivered by the HERO System enhances selective lung cancer cytotoxicity while simultaneously reducing ozone and thermal stress properties that are equally critical for safe respiratory tract applications. Thus, by tuning helium and Air/O₂ humidity, the HERO System achieves a dual optimization maximizing therapeutic efficacy while minimizing environmental and physiological risk. Furthermore, theoretically the total O₃ production could be reduced by single powered ring electrode wrapped the dielectric tube and adjusting the position to an optimal configuration 32 . In addition to voltage and humidity optimization, interval-based CAP treatment was explored to further reduce potential oxidative or thermal stress. Delivering plasma in controlled pulses (e.g., 4 + 4 + 4 + 3 minutes, separated by 5-minute rest intervals) proved to be an effective strategy to maintain cellular cytotoxicity while reducing cumulative gas-phase reactivity. This pulsed dosing allows transient decay of reactive species and thermal relaxation between exposures, preventing local overheating or excessive oxidation while sustaining consistent biological effects 33 . The interval approach is therefore advantageous not only for safety and reproducibility but also for clinical scalability, particularly for ventilator-integrated or endoscopic plasma applications. These optimized plasma conditions humidified helium, moderated voltage, and interval dosing together define an operational window that achieves maximum biological efficacy at minimum toxicity, forming the basis for clinical translation of HERO mediated cold plasma therapy. The in vivo swine study provided critical validation of the system’s physiological safety and organ compatibility. Throughout the 90-minute treatment session, all monitored parameters including body temperature, oxygen saturation, blood pressure, and end-tidal CO₂ remained within normal physiological ranges, indicating no respiratory compromise or hemodynamic instability. Histopathological examination of lung tissue further confirmed intact alveolar structure, preserved bronchiolar epithelium, and absence of inflammatory infiltration or edema, verifying that the cold plasma effluent does not induce structural or oxidative damage to pulmonary tissues. Cytokine profiling supported these histological findings, showing only a mild, transient increase in TNF-α without significant changes in IL-6, CRP, or Angiopoietin-1 levels, suggesting no systemic inflammatory activation. Together, these results confirm that the HERO System is physiologically safe at optimal therapeutic doses, capable of delivering bioactive reactive species directly into the respiratory system without causing tissue injury or systemic toxicity 34 . The potential mechanism of CAP-induced inactivation of biological targets whether malignant cells or viruses is rooted in the generation of a high oxidation-reduction potential (ORP) and elevated electrical conductivity within the treated medium, driven by the abundant production of free radicals. The ROS/RNS cocktail generated by the HERO System can react with carbohydrates, lipids, proteins, and nucleic acids, leading to profound biochemical and structural damage. Lipid peroxidation initiates cross-linking of fatty acid side chains, compromising membrane integrity and cellular viability. Protein oxidation, particularly by singlet oxygen and hydroxyl radicals, modifies cysteine residues into disulfides (R-cys-S-S-cys-R), while oxidation of tyrosine, tryptophan, and histidine leads to the formation of hydroperoxides, aggregation, and conformational destabilization. These processes collectively alter protein folding, membrane stability, and enzymatic function, ultimately impairing viability. Furthermore, oxidation of nucleic acids especially guanine induces cross-links between guanine and lysine, thereby suppressing transcriptional activity and blocking replication. In viral systems, these same oxidative reactions can destroy the viral envelope, denature surface proteins, and fragment genetic material, leading to irreversible loss of infectivity and replication capability 20 , 35 – 37 . These same principles underpin the observed humidity dependence, as water vapor is a critical precursor for OH and downstream long‑lived oxidants that mediate damage in cells and virions 15 , 36 . Finally, the notion of positioning and electrode configuration to further reduce O₃ while preserving therapeutic RONS is consistent with CAP engineering literature, where discharge geometry, gas composition, and humidity jointly tune O₃ vs. HOx/NOx chemistry; continued optimization should be grounded in direct measurement of O₃ and long‑lived species alongside biological readouts to ensure compliance and efficacy 38 . Taken together with published clinical and preclinical CHCP experience 5 , these results support the HERO configuration as a non‑thermal, humidity‑tuned CAP platform with potential for oncologic use and inhalation applications when engineered to meet safety and exposure limits 39 . In conclusion, this study provides compelling evidence that the CHCP-HERO System can achieve effective eradication of lung cancer cells while maintaining a high margin of safety in vivo through precise tuning of plasma parameters. The combination of humidified helium and Air/O₂, optimized voltage, and interval-based dosing ensures both biological potency and operational safety, preventing ozone accumulation and tissue injury. The system’s ability to generate a precisely controlled spectrum of reactive species under non-thermal conditions (24 0 C) represents a significant technological advancement with broad implications for plasma-based medical therapeutics, respiratory modulation, and environmental decontamination. Collectively, the data suggest that, with its tuned configuration, the CHCP-HERO System functions as a safe and effective CAP-based ventilator, capable of serving as a therapeutic gas inhalant system for patients with respiratory diseases such as pneumonia, COVID-19, or lung cancer. This study therefore establishes a foundation for future clinical development of plasma-based respiratory therapies that combine oncologic efficacy, infectious respiratory diseases covering a wide variety of viral, bacterial, fungal, and parasitic infections, and pulmonary safety within a single, precisely engineered platform. 4. Conclusions The CHCP-HERO System achieved complete eradication of lung cancer cells in vitro and demonstrated safety in a swine model under optimized treatment conditions. By fine-tuning helium and Air/O₂ humidification, voltage, and dosing intervals, the system maintained strong therapeutic efficacy while preventing ozone accumulation and tissue injury. Its non-thermal plasma chemistry enables precise, biocompatible delivery of reactive oxygen and nitrogen species through the respiratory tract. In conclusion, the HERO System is a safe and effective CAP-based ventilator therapy with promising applications for respiratory modulations, and lung cancer. 5. Materials and Methods All in vivo experiments were performed at the Jerome Canady Research Institute for Advanced Biological and Technological Sciences JCRI-ABTS and the Swine model surgery was performed at Medical Innovation & Training Institute (Henderson, NV) Las Vegas, Nevada. 5.1. Canady Helios Cold Plasma HERO System device The CHCP-HERO System is composed of Canady Helios Cold Plasma with a mixer T piece adapter via the cable and breathing circuit tubing set. The T piece distal of the adapter is attached to the endotracheal tube. The proximal ends of the T piece are attached to the CHCP generator ventilator hoses. Helium, oxygen, and air were supplied through individual mass flow controllers to ensure precise flow regulation. Each gas stream was humidified using inline humidifiers and verified with humidity meters. Humidified gases were directed into small conditioning chambers and then delivered to the Canady Helios cold plasma generator, where a non-thermal (24 0 C) discharge ionized the gas mixture. Plasma-activated gas exited the generator through a grounded connection cable and entered a CAP mixing junction, where it was combined with ventilator airflow. The mixed CAP stream was delivered through a sterile endotracheal tube into the cell culture dish or lung airway, enabling exposure to plasma-generated reactive species during controlled ventilation. The illustration of the setup is shown in Fig. 1 A and 1 B. 5.2. Cell culture, HERO treatment and MTT Assay. The human lung adenocarcinoma cell line A549 (ATCC, Manassas, USA) was used to evaluate the efficacy of Canady Helios Cold Atmospheric Plasma HERO treatment. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, USA) and 1% penicillin-streptomycin (Pen/Strep; Thermo Fisher Scientific, Waltham, USA). Cultures were maintained at 37°C in a humidified incubator with 5% CO₂. For experiments, cells were seeded into 12-well tissue culture plates at a density of 1 × 10⁵ cells per well and allowed to adhere overnight. 5.3. HERO Treatment Protocol CAP treatments were performed using a plasma discharge generated from a helium-air gas mixture. Air flow was fixed at 4 L/min, while helium flow was varied at 2, 3, or 4 L/min. Gas humidity was adjusted between 0 to 100% relative humidity using an inline humidification module and monitored with a digital humidity sensor positioned proximal to the CAP nozzle. The CAP generator was operated at various voltage setting from 35 V to 70 V, and cells were exposed to CAP at time ranging from 3 to 17 minutes, depending on the experimental condition. For each treatment condition, a corresponding gas-only control (helium + air at the same flow rates and humidity but with plasma power turned off) was included to account for mechanical and gas-flow effects. CAP was administered using two distinct exposure modalities. For direct treatment, wells containing 1 mL of complete culture medium were positioned directly beneath the CAP nozzle at a fixed distance, and the plasma plume interacted with the cell monolayer in real time. For indirect treatment, 1 mL of culture medium was exposed to CAP under identical gas and voltage conditions, after which the treated medium was immediately transferred onto pre-seeded A549 cells. This approach allowed assessment of CAP-generated reactive species in the absence of direct plasma cell interaction. 5.4. MTT Assay. Cell viability was quantified 48 hours post-treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, USA). Briefly, MTT reagent was added to each well to a final concentration of 0.5 mg/mL and incubated for 3 hours at 37°C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA), and absorbance was measured at 570 nm using a BioTek (Winooski, USA) microplate reader. Viability was calculated relative to untreated control wells. 5.5. Swine Model A 16-week-old female Yorkshire-mix swine with a weight of 55 kg, and length of 4’2’’ was fasted overnight with free access to water prior to experimentation. All experimental protocols were approved by the veterinary inspector, State of California, department of food and agriculture, animal health and food safety services, Sacramento, CA. All animal study was conducted in compliance with applicable Good Laboratory Practice (GLP) regulation in accordance with 21 CFR Part 58 at the Medical Innovation & Training Institute (Henderson, NV). Anesthesia was induced with an intramuscular Telazol (2.4 cc) and LA Xylazine (0.3 cc) combination (22 and 2 mg/kg, respectively) and maintained with 1.5–2.5% isoflurane and oxygen delivered by facemask. Animals were intubated with a 7.5-mm cuffed endotracheal tube and mechanically ventilated to maintain end-tidal CO₂ (PetCO₂) between 35–45 mmHg. Surgery was performed by board certified General Surgeon and anesthesia protocol was planned by board certified Anesthesiologist. A multi-parameter monitor (Surgivet Advisor, Smiths Medical, Plymouth, USA) was used to measure the following physiologic parameters: Heart Rate (HR), ElectroCardioGram (ECG), En Tidal (PetCO₂), and peripheral Oxygen saturation (SpO₂). Core body temperature was maintained at 37-38.5°C using a feedback-controlled warming blanket (Cincinnati SubZero, Blanketrol II, Cincinnati, Ohio, USA). Under sterile conditions, the right carotid artery and right external and internal jugular veins were surgically cannulated to permit continuous monitoring of mean arterial pressure (MAP), pulmonary artery pressure (PAP), heart rate, and core temperature, as well as to allow arterial and mixed venous blood sampling and administration of intravenous anesthetics. CAP treatment using HERO System was delivered at a voltage of 40 V for a total of 15 minutes, administered in four intervals (4 + 4 + 4 + 3 min) with 5-minute rest periods between exposures. Helium and oxygen flow rates were set at 3 L/min each. Immediately after treatment, whole blood was collected into 10 mL K₂EDTA tubes, stored on ice, and transported to JCRI-ABTS for immediate processing. Lung tissue samples were harvested post-treatment, fixed in 10% neutral-buffered formalin, and processed for histological evaluation. Tissue morphology and potential CAP-related injury were assessed by hematoxylin staining. 5.6. ELISA Whole blood was collected in six 10-mL K₂EDTA tubes at two time points: immediately before treatment and 30 minutes after completion of the HERO cold plasma exposure. Samples were kept on ice and processed promptly for plasma separation. To obtain plasma, EDTA tubes were centrifuged at 1,500 × g for 10 minutes at 4°C, and the clear plasma layer was carefully aspirated without disturbing the buffy coat. Plasma aliquots were transferred to sterile microcentrifuge tubes and maintained on ice for downstream analysis or immediately stored at -80°C. Concentrations of IL-6, TNF-α, CRP, Angiopoietin-1, IL-1β, and IL-10 were quantified in triplicate using commercially available porcine ELISA kits (Quantikine Porcine Immunoassays, R&D Systems, Minneapolis, USA), following the manufacturer’s instructions. Assays were performed in 96-well plates, and absorbance was measured using a microplate reader. The lower limits of detection for each analyte were: IL-1β, 10 pg/mL; IL-6, 10 pg/mL; IL-10, 1.8 pg/mL; TNF-α, 2.8 pg/mL; and IFN-γ, 2.7 pg/mL. 5.7. Histology Samples of decellularized lung and tracheal tissue were processed at JCRI-ABTS’s pathology department for histological evaluation and compared directly with native, untreated controls. Tissues were fixed in 10% neutral buffered formalin for 24–48 hours, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned at 6–9 µm thickness using standard microtomy procedures. Sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) following conventional protocols, including hematoxylin staining, differentiation, eosin counterstaining, dehydration, clearing, and coverslip mounting. The primary objective was to evaluate the presence or absence of residual nuclear material in decellularized tissues in comparison with untreated controls. All slides were examined using bright-field and fluorescence microscopy (Carl Zeiss, Germany). Images were acquired using the Zeiss Imaging System under identical exposure settings to allow direct comparison between groups. 5.8. Statistical analysis All values in the figures and text are shown as mean ± SEM. Experiments were conducted across three independent days with multiple technical replicates per condition, and data assumptions were verified using Shapiro-Wilk tests for normality and Levene's tests for homogeneity of variance. All datasets met the criteria for parametric analysis (Shapiro-Wilk p > 0.05; Levene's p > 0.05), validating the statistical approaches employed throughout this study. A two-tailed Student's t ‐test or ANOVA followed by post hoc Tukey’s test was performed, considering P values less than 0.05 as statistically significant. The commercially available Microsoft Excel was used for data analysis. Statistical analysis of ELISA was performed by Mann‐Whitney test. Differences were considered significant at p values < 0.05. All box plots and 3D scatter plots were performed in Python (v3.8) using pandas, NumPy, Matplotlib, Seaborn, Statsmodels, Scikit-learn and SciPy. Three-dimensional scatter plots were generated using Matplotlib’s Axes3D module. To visualize global trends in reactive species generation, 3D interpolated surfaces were generated using triangulated surface interpolation via Matplotlib’s plot_trisurf() function. Mean analyte concentrations were plotted as a function of applied voltage (x-axis) and total treatment duration (y-axis). Color encoding of data points by gas type allowed multi-factored visualization within a single 3D space. SEM values were visualized as vertical z-axis error bars using ax.errorbar(), enabling quantitative interpretation of replicate variability. Inter-analyte associations were assessed by computing Pearson correlation coefficients among mean concentrations of H₂O₂, NO₂ - , and NO₃ - across all experimental conditions. The correlation matrix was visualized as a heatmap using Seaborn, with values ranging from − 1 (strong negative correlation) to + 1 (strong positive correlation). Declarations Supplementary Materials: The following supporting information can be downloaded at www.mdpi.com/xxx/s1: Supplemental Figures S1: Ozone production rate by the Canady Helios Cold Plasma HERO System with humidified or dry Air/O2 mixture and He + 0-100% humidity at 70V with 114 khz frequency. S2: Body temperature recording during HERO treatment; S3: End tidal CO2 recording during HERO treatment; S4: O2 saturation recording during HERO treatment; S5: Pulse recording during HERO treatment; Table S1A: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air:O 2 =3:1 O2 16%; Table S1B: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air: O 2 =1:1 O 2 24%; Table S1C: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air: O 2 =1:3 O 2 32%; S1D: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air:O 2 =1:1 O 2 24%; Table S2: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System at 24-hour post treatment with humidified Air/O 2 mixture and He + 0-100% humidity for 1-5 min.; Table S3: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with Air/O 2 mixture and Air/O 2 Separate treatment.; Table S4A: Post Hoc Tukey test for quantification of hydrogen peroxide (H₂O₂); Table S4B: Post Hoc Tukey test for quantification of Nitrite (NO 2 - ); Table S4C: Post Hoc Tukey test for quantification of Nitrate (NO 3 - ); Table S5 showing measurements of core body temperature, end-tidal CO₂, oxygen saturation (SpO₂), arterial blood pressure, and pulse rate during and after CAP treatment. Author Contributions: Conceptualization, J.C.; Methodology, J.C. and S.M.; Software, T.Z.; Validation, J.C., T.Z., S.M., and A.N.; Formal Analysis, J.C., S.M., and T.Z.; Investigation, J.C. and S.M.; Resources, J.C.; Data Curation, J.C., S.M., and O.J.; Writing-Original Draft Preparation, J.C., and S.M.; Writing-Review and Editing, J.C., S.M., O.J., M.K., and A.N.; Visualization, J.C., T.Z., and S.M.; Supervision, J.C.; Project Administration, J.C., T.Z., and S.M.; Funding Acquisition, J.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by US Medical Innovations. Conflicts of Interest: The authors declare no conflict of interest. Data Availability: All the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Peiris, J. S., Yuen, K. Y., Osterhaus, A. D. & Stohr, K. The severe acute respiratory syndrome. 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Supplementary Files SupplementaryFigures.docx SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 05 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviews received at journal 08 Feb, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers invited by journal 28 Jan, 2026 Editor assigned by journal 28 Jan, 2026 Editor invited by journal 28 Jan, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 23 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8651228","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581863988,"identity":"999f151d-7639-4dd6-a8d5-b3af700fc63a","order_by":0,"name":"Saravana R.K. 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(A) In this system, the CHCP jet was operated with Helium gas split into two lines, and it was controlled by mass flow controllers (MFC). Part of Helium gas flow (50 to 1000 mL/min) was passed through an H\u003csub\u003e2\u003c/sub\u003eO filled container. To achieve relative H\u003csub\u003e2\u003c/sub\u003eO saturation in the feed gas of 5% or 70% goal, the other lines of Helium gas was directly fed into the mixing chamber. Adjustment of these two lines flow rate will make the overall flow rate and humidity % fine tuning possible. The total feed gas flow could be varied from 0.5 L/min to 2 L/min in all cases. In this Helium humidify setup, the Helium H\u003csub\u003e2\u003c/sub\u003eO vapor content was varied during experiments. The humidity of Helium gas in the chamber was measured via calibrated High-Accuracy Humidity and Temperature Meter as show in the Figure 1A. The generator operates at a Frequency between 10-200KHz and output peak voltage from 3KV to 6KV, controlled by the touch screen operation system. At the same time, the ventilator system was fed by two un-humidified gas tank, Air and Oxygen respectively. The Ventilator will mix, adjust and measure the pressure, flowrate, i/e ratio and frequency of the patient inbreath of exhaust air, oxygen and CO\u003csub\u003e2\u003c/sub\u003e. Due to the present of the H\u003csub\u003e2\u003c/sub\u003eO, the ionization of Helium and H\u003csub\u003e2\u003c/sub\u003eO He\u003csup\u003e+\u003c/sup\u003e chemical reaction will happen simultaneously. The ionized cold plasma is mixed with dry air and oxygen subsequently transmitted to the patient via an Endotracheal tube as shown in the illustration. (B) Zoom-in view of joint CAP mixer with air and oxygen added as a mixture to the system.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/7f172ae92c87ffa499f8ebd0.png"},{"id":101752179,"identity":"6c0c1066-481d-48eb-8cb9-aae65de2cb5b","added_by":"auto","created_at":"2026-02-03 10:25:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1522833,"visible":true,"origin":"","legend":"\u003cp\u003eBox plots showing the viability of A549 cells treated with the CHCP-HERO System with (A)humidified Air/O2 mixture and dry He with various O\u003csub\u003e2\u003c/sub\u003e percentage for up to 4 minutes and (B) humidified Air/O2 mixture and dry He with 24% O2 for up to 17 minutes. The data represent the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Significance determined using paired two tailed Student t-test at (*p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/a1e4aaa17c55980df36db911.png"},{"id":101491674,"identity":"71a76594-b7dd-4548-99d0-8114e8d6eed9","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":436970,"visible":true,"origin":"","legend":"\u003cp\u003ePhase contrast images of A549 lung cancer cells at 24-hour post treatment with (A) no treatment and (B) He, Air and O2 gas mixture treatment for 10 min, (A) He + humidity at 0% for 5 min (B) He + humidity at 0% for 10 min, (C) He + humidity at 100% for 5 min (D) He + humidity at 100% for 10 min, (E) He + humidity at 100% for 5 min (F) He + humidity at 100% for 10 min (scale bar = 200 m). (G) Viability of A549 cells treated by the CHCP-HERO System for 5 or 10 minutes with humidified Air/O2 mixture (with 24% O2) and various helium humidity. The green and red arrows indicate healthy cells and dead or dying cells respectively. The data represent the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Significance determined using paired Student t-test at (*p \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/1ed3a77fa7e8cfa71e26774e.png"},{"id":101491679,"identity":"0837f417-a3cd-455e-96d6-3ac59db208b9","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":476227,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative phase contrast images of lung cancer cells A549 A) No treatment B) treated by CHCP-HERO System at 24-hour post treatment with humidified Air/O2 mixture and He + 0-100% humidity for 1-5 min. C) treated by CHCP-HERO System at 24-hour post treatment with dry Air/O2 mixture and He + 0-100% humidity for 1-5 min (scale bar = 200 m). The green and red arrows indicate healthy cells and dead or dying cells respectively.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/0fd1308ad2508328d972f62a.png"},{"id":101491681,"identity":"38f295b3-7bfb-41bd-940b-286d38ade08e","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396578,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Three-dimensional plot showing the relationship between treatment voltage, exposure time, and A549 cell viability following Canady Helios Cold Plasma HERO System at 48-hour post treatment with humidified Air/O2 mixture and dry Air/O2 mixture and He + 0-100% humidity for 1-5 mins. (B) Differentiation of humidified Air/O2 mixture and dry Air/O2 mixture for 1-5 min. (C) Correlation heatmap comparing voltage, exposure time, He humidity and viability mean all CAP treatment conditions. The data represent the mean ± SEM from n = 3 biological replicates, each containing triplicate technical measurements. Significance was set at p \u0026lt; 0.05, using one-way ANOVA followed by post-hoc Tukey’s tests for treated samples versus the helium control. All p-values \u0026lt; 0 (*), indicating they were highly significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/46000ec1ed80820b79df1de7.png"},{"id":101491678,"identity":"0074f659-8276-416c-b4c6-ad34a7700b84","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":406413,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration showing process of joint CAP mixer with air and oxygen added to the system in two ways (A) as a mixture (B) separately. (C) Three-dimensional plot showing the relationship between treatment voltage, exposure time, and cell viability following CHCP-HERO System CAP mixer joint with humidified Air/O2 mixture treatment. Each point represents the mean viability from n = 3 biological replicates; vertical capped bars indicate ±SEM, each containing triplicate technical measurements. Significance was set at p \u0026lt; 0.05, using one-way ANOVA followed by post-hoc Tukey’s tests for treated samples versus the helium control. All p-values \u0026lt; 0 (*), indicating they were highly significant. (D) Correlation heatmap comparing voltage, exposure time, viability mean, and viability SEM across all CAP treatment conditions.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/c9f901b0fae0391073b5c74f.png"},{"id":101491683,"identity":"5063d724-c192-4a4c-b3ad-d4835f351561","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96381,"visible":true,"origin":"","legend":"\u003cp\u003eOzone production rate by the CHCP-HERO System with humidified Air and O2 mixture or separately and 100% humidified `He at different voltage at 122 khz frequency. Dotted line shows the 2 mg/m-3 (1 ppm) of ozone tolerance for up to 8 min without any acute symptoms in mammalian respiratory tract. The data representing the mean ± SEM from n = 3 replicates, each containing triplicate technical measurements. Student’s t-test data are represented by *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/f2b28667e8ae89314b7f9193.png"},{"id":101491676,"identity":"0afd9bef-d500-4976-b0fe-dca121594790","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":262505,"visible":true,"origin":"","legend":"\u003cp\u003eThree-dimensional dose–response scatter plot and Correlation matrix heatmap of H₂O₂, NO₂⁻, and NO₃⁻ concentrations. This figure shows the 3D scatter distribution of HERO system generated (A) hydrogen peroxide (H₂O₂), (B) nitrite (NO₂⁻), or (C) nitrate (NO₃⁻) concentrations as functions of applied voltage (x-axis) and total treatment duration (y-axis). PBS was treated with humidified Air/O2 and humidified¬¬ He with CAP and gas mixture (He, Air and O2) for 8 or 15 min continuously and in intervals. Each point represents the mean viability from n = 3 samples; vertical capped bars indicate ±SEM, each containing triplicate technical measurements. Significance was set at p \u0026lt; 0.05, using one-way ANOVA followed by post-hoc Tukey’s tests for treated samples versus the helium control. These figures provide an intuitive overview of the chemical landscape created by varying electrical and exposure parameters. The heatmap (D) illustrates Pearson correlation coefficients between H₂O₂, NO₂⁻, and NO₃⁻ mean concentrations across all experimental conditions.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/14fda148d666ea408a96d72e.png"},{"id":101491682,"identity":"85f876b2-3fe1-4f78-8368-bdf2ca8ea9f2","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":234010,"visible":true,"origin":"","legend":"\u003cp\u003ePost-treatment detection of IL-6 (A) , TNF-α (B), CRP (C), and Angiopoietin-1(D) following CHCP exposure. Each data point represents the mean cytokine concentration derived from n = 3 biological replicates, with each biological sample measured in technical triplicate. Vertical error bars indicate the standard error of the mean (SEM). Statistical significance was defined as *p \u0026lt; 0.05, determined using the Mann-Whitney U test.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/f79672f0927726b352535a5c.png"},{"id":101752257,"identity":"1b99f284-286c-4ddb-ac23-18694891d7f9","added_by":"auto","created_at":"2026-02-03 10:26:21","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1404334,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin (H\u0026amp;E)-stained sections of porcine lung tissue after CHCP-HERO system treatment. (A)The bronchiole is lined by intact bronchiolar epithelium. (B) The distal parenchyma shows well-preserved alveolar spaces separated by thin alveolar septa and (D) encircled by organized smooth muscle bundles.(C \u0026amp; F) Surrounding peribronchiolar connective tissue contains small blood vessels and capillaries with eosinophilic erythrocytes and (E) a well-defined focus of lymph node. All these features demonstrating the characteristic architecture of healthy lung tissue. Sections were image under light microscope with a 40X objective. Scale bars = 0.2 mm.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/0313e6a4015c635b6a088727.png"},{"id":106343669,"identity":"1b9bed0f-e4db-4075-8468-230268998bfc","added_by":"auto","created_at":"2026-04-07 16:08:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7963735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/28b3fd51-f850-4933-99b7-15df50df0652.pdf"},{"id":101491677,"identity":"e85ff076-9228-40fd-b18c-a1456bb9c7bb","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":228438,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/4f8420bced1c566f5badc24d.docx"},{"id":101491675,"identity":"3fed891d-9ffd-4953-87e2-18ac1832f933","added_by":"auto","created_at":"2026-01-30 10:33:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":63673,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8651228/v1/2d1aeebcfe7bcdc3e7792923.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Canady Helios HERO Model: A Ventilator- Integrated Humidified Cold Atmospheric Plasma System for Lung Cancer Treatment and Respiratory Infection Applications","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRespiratory viral infections, including COVID-19 and severe acute respiratory syndrome (SARS), are primarily transmitted through aerosolized droplets that carry infectious viral particles within the respiratory tract\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Their global impact underscores the urgent need for novel, non-pharmaceutical interventions that directly inactivate viruses in the airways\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. CAP has emerged as a promising bioengineering technology for biomedical applications\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, generating reactive oxygen and nitrogen species (ROS/RNS), photons, charged particles, and electric fields at near-room temperature. Unlike thermal disinfection or chemical sterilization, CAP offers a non-thermal, non-contact, tunable oxidative environment that selectively targets pathogens and malignant cells without harming healthy tissues\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumerous studies demonstrate CAP's broad-spectrum antiviral efficacy. It inactivates airborne viruses \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, deactivate hepatitis B virus while preserving hepatocyte function \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, inhibit HIV replication\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and inactivate Newcastle disease virus and avian influenza virus without compromising their antigenic determinants for vaccine development \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Zimmerman et al., \u003csup\u003e13\u003c/sup\u003e inactivated adenovirus using surface micro-discharge plasma,, while Su et al., \u003csup\u003e14\u003c/sup\u003e achieved potent inactivation of Newcastle disease virus with plasma-activated solutions. These results highlight CAP's potential as a versatile antiviral and antimicrobial modality.\u003c/p\u003e \u003cp\u003eCAP-mediated inactivation arises from high oxidation-reduction potential (ORP) and electrical conductivity via abundant free radicals\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The ROS and RNS such as singlet oxygen (\u0026sup1;O₂), ozone (O₃), superoxide anion (O₂\u003csup\u003e-\u003c/sup\u003e˙), nitric oxide (NO˙), nitrogen dioxide (NO₂˙), and hydroxyl radicals (˙OH) induce lipid peroxidation and of fatty acid cross-linking disrupting cellular and viral membranes \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. They also oxidize amino acids like cysteine, tyrosine, tryptophan, and histidine residues, forming disulfide bonds and hydroperoxides that cause protein aggregation and envelope destruction. Nucleic acid modifications, including guanine oxidation and guanine-lysine crosslinking, silence gene expression and inhibit replication. In biological media, short-lived species such as peroxynitrite (ONOO\u003csup\u003e-\u003c/sup\u003e) and long-lived species such as hydrogen peroxide (H₂O₂) and nitrite (NO₂\u003csup\u003e-\u003c/sup\u003e), sustained oxidative activity.\u003c/p\u003e \u003cp\u003eWe hypothesized that an optimized CHCP-HERO System could safely deliver CAP for antiviral and anticancer effects. Aims included: (1) optimize system parameters, (2) evaluate antitumor efficacy in A549 cells, and (3) assess pulmonary safety in a swine preclinical model. The HERO System integrates CHCP with a ventilator for humidified, intratracheal CAP delivery. Humidification balances potency and safety: helium enhances reactivity, while Air/O₂ reduces ozone and stabilizes temperature. Separate oxygen addition controls species composition and minimizes O₃.\u003c/p\u003e \u003cp\u003eDue to biosafety limits with live SARS-CoV-2, we used A549 lung carcinoma cells-resembling type II alveolar cells and susceptible to oxidative stress\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and swine, physiologically similar to humans in respiratory systems\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These models together provide a rigorous translational framework for evaluating the therapeutic efficacy and biosafety of the HERO System prior to clinical application.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the therapeutic potential of the CHCP-HERO system for the treatment of lung cancer and the inactivation of respiratory pathogens. We optimized the CHCP-HERO system parameters which included helium and Air/O₂ humidification, discharge voltage, and interval-based plasma dosing to balance plasma potency with safety. The data demonstrate that the HERO System can effectively eradicate A549 lung cancer cells \u003cem\u003ein vitro\u003c/em\u003e and maintain pulmonary integrity and physiological stability in the swine model. Together, these findings provide compelling evidence that, when properly tuned, the CAP-based HERO ventilator system functions as a safe and effective therapeutic gas inhalant platform with potential applications in treating respiratory diseases such as pneumonia, COVID-19, and lung cancer.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003eThe CHCP-HERO System was designed to generate and deliver CAP through a ventilator-integrated circuit under controlled humidity and gas composition conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrior studies \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e demonstrated that ambient air is the most effective carrier gas for viral inactivation using atmospheric-pressure cold plasma, particularly at power levels between 20 W and 24 W. In head-to-head tests involving airborne MS2 bacteriophage, cold plasma generated with air resulted in significantly higher rates of viral inactivation compared to mixtures of argon-oxygen (Ar-O₂, 2% v/v) and helium-oxygen (He-O₂, 2% v/v), when discharge power was held constant. The mechanism underlying the superior performance of ambient air relates to the production of a wider spectrum of RONS in humid air environments, including ozone, singlet oxygen, and nitric oxides, which are less abundantly produced by noble gas-oxygen mixtures. These chemically reactive species disrupt viral capsids and genomic material more effectively, leading to rapid viral deactivation. Because ventilators inherently require air as an input source, air was selected as the primary carrier gas for the HERO configuration. In the present study, we systematically evaluated the influence of air/O₂ ratio, helium humidity, discharge voltage, and treatment duration on biological and chemical outcomes. Relative humidity (RH) as an important factor of the air were studied for an optimal configuration in addition to CAP treatment parameters including discharge voltage (V) and treatment time (t). The feeding gas of Air/O\u003csub\u003e2\u003c/sub\u003e mixture and/or He was humidified by bubbling through water. The relative humidity (RH) of the humidified gases was measured constantly with a humidity sensor.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.1. Dose-Dependent Cytotoxicity of Humidified Cold Atmospheric Plasma on A549 Lung Cancer Cells: Effects of Oxygen Fraction and Exposure Duration\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the influence of oxygen fraction and treatment duration, A549 human alveolar carcinoma cells were exposed to HERO system generated CAP using a humidified air/O₂ mixture and dry helium at 70 V. Figure\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e2\u003c/span\u003e (A and B) shows gradual viability decline with exposure time (1\u0026ndash;4 min), reaching\u0026thinsp;~\u0026thinsp;60% of controls at 4 min. Increasing oxygen from 16% to 32% slightly attenuated cytotoxicity, but all conditions significantly reduced viability vs. controls (ANOVA: 16% O₂, F[4,10]\u0026thinsp;=\u0026thinsp;332.59, p\u0026thinsp;=\u0026thinsp;1.37 \u0026times; 10\u003csup\u003e-\u003c/sup\u003e\u0026sup1;⁰; 24% O₂, F[4,10]\u0026thinsp;=\u0026thinsp;290.72, p\u0026thinsp;=\u0026thinsp;2.68 \u0026times; 10\u003csup\u003e-\u003c/sup\u003e\u0026sup1;⁰;32% O₂, F[4,10]\u0026thinsp;=\u0026thinsp;52.24, p\u0026thinsp;=\u0026thinsp;1.15 \u0026times; 10\u003csup\u003e-\u003c/sup\u003e⁶; Tukey\u0026rsquo;s post hoc in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eProlonged 24% O₂ treatment further decreased viability time-dependently: \u0026lt;40% at 10 min, complete A549 elimination at 17 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; ANOVA, F[7,14]\u0026thinsp;=\u0026thinsp;685.203, P\u0026thinsp;=\u0026thinsp;1.31\u0026times;10\u003csup\u003e\u0026ndash;18\u003c/sup\u003e; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).These data confirm robust, dose-dependent cytotoxicity by the humidified CAP system via duration and gas modulation. Mechanistically, CAP treatment causes apoptosis in A549 cells, evidenced by increased ROS production, mitochondrial damage, chromatin condensation, and activation of apoptosis markers like cleaved caspases and PARP proteins. Morphologically, plasma exposure leads to cell shrinkage, detachment, and nuclear fragmentation, confirming effective programmed cell death pathways are engaged\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.2. Humidity-Dependent Enhancement of Cytotoxicity in A549 Lung Cancer Cells by the HERO Cold Atmospheric Plasma System: Morphological and Viability Assessments\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo assess humidity's impact on HERO System cytotoxicity, A549 lung carcinoma cells were exposed to CAP generated with 100% humidified Air/O₂ (1:1 v/v; 2 L min-1) and helium (3 L min-1) at 0%, 50%, or 100% RH. After 24 h, phase-contrast microscopy showed humidity-dependent morphological deterioration (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Controls (untreated or gas flow only) retained cobblestone morphology, intact nuclei, and adherence, confirming no gas-only effect. Dry helium (0% RH) induced shrinkage and rounding at 5 min, progressing to partial detachment and apoptotic bodies at 10 min, with some viable cells persisting. At 50% RH, 5 min caused membrane blebbing and chromatin condensation; 10 min led to extensive detachment and necrotic debris. At 100% RH, 5 min triggered catastrophic shrinkage, fragmentation, and lysis, markedly enhancing plasma efficacy. MTT assays quantified this humidity-dependent response (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Dry conditions reduced viability to ~\u0026thinsp;60% (5 min) and 40% (10 min). At 50% and 100% RH, 5 min exposure eliminated\u0026thinsp;\u0026gt;\u0026thinsp;95% viability. Paired t-tests confirmed significance (p\u0026thinsp;=\u0026thinsp;0.0083\u0026thinsp;\u0026minus;\u0026thinsp;0.0013; detailed: 5 min, gas-only 100% RH vs. 0% CAP p\u0026thinsp;=\u0026thinsp;0.0083, 0% vs. 50% CAP p\u0026thinsp;=\u0026thinsp;0.0013, 50% vs. 100% CAP p\u0026thinsp;=\u0026thinsp;0.416; 10 min, gas-only vs. 0% CAP p\u0026thinsp;=\u0026thinsp;0.0025, 0% vs. 50% CAP p\u0026thinsp;=\u0026thinsp;0.0098, 50% vs. 100% CAP p\u0026thinsp;=\u0026thinsp;0.184). Humidified helium critically enhances CAP potency by boosting reactive species delivery, accelerating apoptosis/necrosis, membrane rupture, and DNA damage in a time- and humidity-dependent manner, selectively targeting cancer cells under optimized settings.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.3. Humidity-Driven Optimization of Exposure Time for Complete Eradication of A549 Lung Cancer Cells Using the HERO System\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo determine minimum exposure for A549 eradication, cells were treated with CAP for 1\u0026ndash;5 min using humidified/dry Air/O₂ (1:1 v/v; 2 L min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) and helium (3 L min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) at 0%, 50%, or 100% RH. Microscopy 24 h post-exposure showed time- and humidity-dependent damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Dry helium (0% RH) preserved morphology until 5 min (minor rounding/detachment). At 50% RH, injury emerged at 4 min (reduced density, apoptotic morphology). At 100% RH, effects were rapid: nuclear condensation/shrinkage at 2 min, disintegration by 4 min. Air/O₂ humidity variation had minimal impact, highlighting helium humidification as the key cytotoxic driver. MTT assays confirmed findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003eA- C). Dry conditions (0% RH) showed minimal cytotoxicity (\u0026gt;\u0026thinsp;80% viability at 5 min). At 50% RH, viability fell to ~\u0026thinsp;50% at 5 min. At 100% RH, \u0026gt;\u0026thinsp;50% reduction at 3 min; complete ablation (0% survival) at \u0026ge;\u0026thinsp;4 min. ANOVA verified significance (humidified Air/O₂ + He: 0% RH, F[4,10]\u0026thinsp;=\u0026thinsp;4.0113, P\u0026thinsp;=\u0026thinsp;0.034; 50% RH, F[4,10]\u0026thinsp;=\u0026thinsp;291.143, P\u0026thinsp;=\u0026thinsp;2.665\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e; 100% RH, F[4,10]\u0026thinsp;=\u0026thinsp;1770.226, P\u0026thinsp;=\u0026thinsp;3.343\u0026times;10\u003csup\u003e\u0026ndash;14\u003c/sup\u003e; dry Air/O₂ + He: 0% RH, F[4,10]\u0026thinsp;=\u0026thinsp;16.322, P\u0026thinsp;=\u0026thinsp;0.00022; 50% RH, F[4,10]\u0026thinsp;=\u0026thinsp;292.65, P\u0026thinsp;=\u0026thinsp;2.598\u0026times;10\u003csup\u003e\u0026ndash;10\u003c/sup\u003e; 100% RH, F[4,10]\u0026thinsp;=\u0026thinsp;1024.67, P\u0026thinsp;=\u0026thinsp;5.114\u0026times;10\u003csup\u003e\u0026ndash;13\u003c/sup\u003e; Tukey\u0026rsquo;s post hoc in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Fully humidified helium maximizes reactive oxygen/nitrogen species generation/transfer, enabling CHCP-HERO rapid, complete A549 destruction in a clinically feasible window.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.4. Voltage and Gas Configuration Optimization in the HERO System: Balancing Cytotoxic Efficacy with Reduced Ozone Generation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo optimize CHCP-HERO for efficacy and safety, operating voltage, gas composition, and ozone (O₃) generation were evaluated. At 70 V, robust plasma efficacy produced O₃ exceeding OSHA limits, risking respiratory irritation. Voltage was thus reduced to 35\u0026ndash;40 V to sustain plasma while minimizing O₃. Gas delivery was refined by separating Air and O₂ inputs into the CAP mixer, reducing localized O₂ density, suppressing O₃, and preserving ROS/RNS for cytotoxicity. Figure\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B illustrates the reengineered mixer and flow dynamics enabling reduced O₃ without compromising uniformity.\u003c/p\u003e \u003cp\u003eBiological validation confirmed efficacy retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,D). A549 cells showed\u0026thinsp;\u0026lt;\u0026thinsp;5% viability after 8 min at 40 V (Air/O₂ mixture) or 15 min (separate Air/O₂). ANOVA revealed significant effects (Air/O2 mixture Voltage\u0026thinsp;=\u0026thinsp;40V, F[9, 20]\u0026thinsp;=\u0026thinsp;1174.233, P\u0026thinsp;=\u0026thinsp;3.145 -25; Air/O2 mixture Voltage\u0026thinsp;=\u0026thinsp;35V, F[16, 34]\u0026thinsp;=\u0026thinsp;24.37, P\u0026thinsp;=\u0026thinsp;4.671 -14; Air/O2 separate Voltage\u0026thinsp;=\u0026thinsp;40V, F[15, 32]\u0026thinsp;=\u0026thinsp;15.171, P\u0026thinsp;=\u0026thinsp;1.75 -10; Air/O2 separate Voltage\u0026thinsp;=\u0026thinsp;35V, F[15, 32]\u0026thinsp;=\u0026thinsp;12.248, P\u0026thinsp;=\u0026thinsp;2.83 -9; Tukey\u0026rsquo;s post hoc in Supplementary Table S3). Low-voltage discharge (35\u0026ndash;40 V) with decoupled Air/O₂ streams balances potent anti-cancer activity and O₃ control, advancing HERO toward clinical respiratory/oncologic use.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.5. Quantification and Mitigation of Ozone Generation in the HERO System: Effects of Humidity, Voltage, and Gas Configuration on Safety Optimization\u003c/em\u003e \u003c/p\u003e \u003cp\u003eOzone (O₃) concentrations were measured at the endotracheal tube distal end using a calibrated detector (Forensics Detectors, CA) across conditions: humidified/dry Air/O₂ (1:1 v/v; 24% O₂; 2 L min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) with helium (3 L min\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e) at 0%, 50%, 100% RH, at 70 V/114 kHz. Dry Air/O₂ yielded higher O₃ than humidified counterparts (\u003cem\u003et-\u003c/em\u003etests, p\u0026thinsp;=\u0026thinsp;4.95 \u0026times; 10-⁵, p\u0026thinsp;=\u0026thinsp;0.0009, and p\u0026thinsp;=\u0026thinsp;0.005 for 0%, 50%, and 100% RH, respectively; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Helium humidity increases elevated O₃, correlating with cytotoxicity (Figs.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating O₃ as a key bioactive species driving oxidative cancer cell inactivation.\u003c/p\u003e \u003cp\u003eVoltage dependency showed marked O₃ reduction from 50 V to 35 V (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e7\u003c/span\u003e), aligning with safety optimization preserving efficacy while minimizing exposure. Premixed Air/O₂ produced higher O₃ than separate feeds into the CAP mixer; separate delivery cut O₃ by up to 40% at 35\u0026ndash;50 V (\u003cem\u003et\u003c/em\u003e-test, Humidified Air/O\u003csub\u003e2\u003c/sub\u003e vs Dry Air/O\u003csub\u003e2\u003c/sub\u003e, 0%, 50% and 100%, p\u0026thinsp;=\u0026thinsp;4.95-5, p\u0026thinsp;=\u0026thinsp;0.0009 and p\u0026thinsp;=\u0026thinsp;0.005 respectively and Air/O\u003csub\u003e2\u003c/sub\u003e mixture vs Air O\u003csub\u003e2\u003c/sub\u003e separate at 35, 36,37,38, 39, 40, 45 and 50 voltage (V), was p\u0026thinsp;=\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.017, p\u0026thinsp;=\u0026thinsp;0.001, and p\u0026thinsp;=\u0026thinsp;0.0037 respectively). Voltage and gas configuration adjustments control O₃ without compromising performance, ensuring HERO operates within regulatory limits.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Quantification of ROS/RNS in Plasma-Activated Medium: Interval Delivery Enhances Oxidative Species Accumulation While Maintaining Safety\u003c/h2\u003e \u003cp\u003eROS and RNS were quantified in1 mL PBS following HERO treatment in continuous and interval modes using colorimetric assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Continuous 8-min with an Air/O₂ mixture (1:1 v/v) at 40 V yielded 725 \u0026micro;M H₂O₂,11.9 \u0026micro;M NO₂⁻, and 3.3 \u0026micro;M NO₃⁻, while the corresponding interval regimen (3\u0026thinsp;+\u0026thinsp;3 + 2 min) produced 470 \u0026micro;M H₂O₂, 12.5 \u0026micro;M NO₂, and 2.2 \u0026micro; NO₃⁻. When and O were supplied, 15 mins treatment generated 806 \u0026micro;M H₂O and 33 \u0026micro;M NO₂⁻, whereas the interval mode (4\u0026thinsp;+\u0026thinsp;4 + 4\u0026thinsp;+\u0026thinsp;3 min) resulted in 952 \u0026micro;M H₂O₂, 45 \u0026micro;M NO₂⁻, and 12 \u0026micro;M NO₃⁻, indicating more efficient ROS/RNS generation during interval, likely due to re-equilibration of reactive intermediates between pulses. Across all conditions, H₂O₂ concentrations (0.36\u0026ndash;0.62 mg m⁻\u0026sup3;) remained below the OSHA/NIOSH permissible exposure limit of 1.4 mg m⁻\u0026sup3;, confirming operation within occupational safety standards. Gas-only controls produced negligible ROSNS, confirming their plasma-derived origin rather than gas flow or humidification artifacts. ANOVA revealed highly significant among treatment groups for H₂O₂ (F[9, 20]\u0026thinsp;=\u0026thinsp;217.64, P\u0026thinsp;=\u0026thinsp;5.89E-18; ANOVA for NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e F[9, 20]\u0026thinsp;=\u0026thinsp;91.06, P\u0026thinsp;=\u0026thinsp;2.98E-14; ANOVA for NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e F[9, 20]\u0026thinsp;=\u0026thinsp;166.13, P\u0026thinsp;=\u0026thinsp;8.41917E-17); Tukey\u0026rsquo;s post hoc comparisons are reported in Supplementary Table S4. Together, these findings demonstrate that the HERO System generates a balanced yet potent oxidative environment, with-derived O₃, H₂O₂, and NO₂⁻ likely serving as key mediators of cytotoxicity and oxidative damage while maintaining clinical safety and biocompatibility.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.7. In Vivo Safety and Physiological Tolerability of Intratracheal HERO Cold Plasma Delivery Shows Excellent Physiological Tolerability in Swine Model\u003c/em\u003e \u003c/p\u003e \u003cp\u003eA 55 kg anesthetized swine was used to evaluate the \u003cem\u003ein vivo\u003c/em\u003e safety and physiological tolerability of intratracheal cold plasma delivery under mechanical ventilation. The CHCP-HERO System was operated at 40 V with 3 L min⁻\u0026sup1; helium and 3 L min⁻\u0026sup1; oxygen in four plasma exposure cycles (4\u0026thinsp;+\u0026thinsp;4 + 4\u0026thinsp;+\u0026thinsp;3 min), each separated by 5 minutes of recovery. Continuous monitoring over 90 minutes included core temperature, end-tidal CO₂, SpO₂, arterial blood pressure, and pulse rate (Supplementary Table S5, FS2-5). All parameters remained within normal physiological ranges during and after treatment. Core temperature rose only mildly from 33.7\u0026deg;C to 35.3\u0026deg;C by the end of the third cycle, consistent with anesthesia-related thermoregulation. End-tidal CO₂ fluctuated between 22\u0026ndash;43 mmHg without evidence of hypoventilation or hypercapnia. Oxygen saturation was stable at 97\u0026ndash;100%, indicating preserved gas exchange. Cardiovascular stability: systolic pressure 90\u0026ndash;110 mmHg (median 95), diastolic 50\u0026ndash;64 mmHg (median 55); pulse 83\u0026ndash;102 bpm (median 92), with no hemodynamic instability, arrhythmias, respiratory distress, or desaturation events observed. Collectively, these findings demonstrate that intratracheal delivery of humidified cold plasma via the HERO System is acutely well tolerated, with no detectable respiratory, cardiovascular, or thermoregulatory compromise, supporting its translational potential for respiratory and thoracic applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Serum Cytokine Profiling Demonstrates Safety of HERO System in Swine Model\u003c/h2\u003e \u003cp\u003eTo investigate whether intratracheal HERO exposure induces systemic immune activation or inflammatory signaling, serum cytokine levels were quantified in the swine model before and after treatment using ELISA. Blood samples were collected 1 hour prior to plasma administration and again 6 hours post-treatment to capture both immediate and delayed immune responses. The cytokine panel included interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), Angiopoietin-1, interleukin-1 beta (IL-1β), and interleukin-10 (IL-10) representing key mediators of inflammatory, vascular, and immunoregulatory pathways. Among all analytes tested, TNF-α showed a mild but statistically significant increase following CAP exposure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Mann-Whitney test), while all other cytokines remained at baseline levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The modest rise in TNF-α is consistent with a transient local immune response to plasma-generated reactive species, reflecting limited activation of macrophage and endothelial signaling cascades without evidence of systemic inflammation or cytokine storm.\u003c/p\u003e \u003cp\u003eNotably, IL-6, CRP, and Angiopoietin-1 biomarkers typically elevated during acute inflammatory or vascular injury states did not differ significantly between pre- and post-treatment samples, indicating no measurable systemic inflammatory or endothelial activation. Furthermore, IL-1β and IL-10 concentrations remained below the assay\u0026rsquo;s detection limit, suggesting the absence of pro-inflammatory cytokine amplification or compensatory anti-inflammatory feedback. The lack of significant changes in these parameters corroborates the favorable biocompatibility of the HERO treatment and aligns with prior observations of preserved physiological stability and normal lung histoarchitecture post-exposure. Together, these findings demonstrate that CAP delivery via the HERO System elicits only minimal systemic immune modulation, thereby reinforcing its safety and translational potential for intratracheal therapeutic applications without inducing cytokine-mediated toxicity or systemic inflammatory sequelae.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Histopathological Analysis Confirms Preserved Lung Architecture and Absence of Tissue Damage Following CHCP-HERO Treatment\u003c/h2\u003e \u003cp\u003eTo evaluate the tissue-level safety and structural integrity of the lungs following intratracheal HERO treatment, formalin-fixed, paraffin-embedded lung sections were subjected to detailed histopathological examination using hematoxylin and eosin (H\u0026amp;E) staining. Microscopic assessment revealed well-preserved pulmonary architecture across all analyzed regions, with intact alveolar septa, bronchiolar epithelium, and vascular structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig20\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The alveolar sacs remained uniformly inflated, displaying a continuous and uncompromised air-blood barrier, with no evidence of alveolar collapse, hemorrhage, or congestion. The bronchiolar lining cells exhibited normal morphology and polarity, showing no signs of epithelial sloughing or necrosis. Similarly, the vascular lumen appeared with intact endothelial lining, and there were no histological signs of capillary dilation, thrombosis, or vascular injury. The absence of interstitial edema or fibrin deposition further indicates that plasma exposure did not disrupt microvascular permeability or induce inflammatory exudation.\u003c/p\u003e \u003cp\u003eThe surfactant system critical for maintaining alveolar stability and gas exchange was also morphologically intact, showing no vacuolization or lamellar body depletion. Moreover, no peribronchovascular or alveolar inflammatory infiltrates were detected, confirming that the CAP exposure did not trigger localized immune or oxidative injury responses. The overall lung parenchyma exhibited a high degree of homogeneity and structural preservation, comparable to untreated control tissue. Collectively, these findings demonstrate that the HERO system\u0026rsquo;s cold plasma stream exerts no measurable thermal, oxidative, or mechanical damage on delicate pulmonary structures. This comprehensive histological evidence validates the biocompatibility and non-destructive nature of the HERO platform and supports its clinical safety for intrapulmonary and ventilator-integrated therapeutic applications.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe findings from this study clearly demonstrate that the CHCP-HERO System is a safe, effective, and tunable therapeutic platform capable of achieving near-complete eradication of A549 human lung carcinoma cells while maintaining biocompatibility and safety in large-animal models. Using the A549 cell line as a representative model for human lung cancer, the study revealed a strong, humidity-dependent correlation between treatment efficacy and plasma reactivity. Increasing helium humidity from 0% to 100% resulted in a significant enhancement of plasma-induced cytotoxicity, with total loss of viability observed within a few minutes of exposure at 100% relative humidity. This humidity-driven potentiation of CAP activity underscores the critical role of water vapor as a reaction substrate, facilitating the generation of ROS/RNS such as hydroxyl radicals (\u0026middot;OH), H₂O₂, NO\u0026middot;, and ONOO-\u003csup\u003e24\u003c/sup\u003e. These reactive intermediates act synergistically to induce oxidative stress, leading to membrane disruption, protein denaturation, and apoptosis, thereby driving non-thermal and selective cancer cell death\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,25\u0026ndash;27 28,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Beyond the modulation of plasma chemistry, humidification of the Air/O₂ mixture was shown to play an essential role in enhancing the system\u0026rsquo;s safety profile\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The addition of moisture into the Air/O₂ stream effectively reduced ozone generation and stabilized the plasma discharge temperature, thereby lowering the risk of oxidative gas accumulation and minimizing local tissue irritation. Moreover, the system configuration allowing separate introduction of Air and O₂ rather than using a premixed gas further reduced ozone formation by limiting localized oxygen density within the discharge zone. Collectively, these adjustments enabled efficient plasma operation at lower voltages (35\u0026ndash;40 V), preserving potent anticancer activity while ensuring that O₃ concentrations remained within OSHA-compliant safety thresholds. Moreover, Li et al. showed that humidity reshapes plasma discharge behavior by lowering electron density and shifting energy distributions toward pathways that promote controlled generation of biologically active ROS/RNS rather than damaging high-energy species\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This mechanistic shift directly supports our findings that humidified CAP delivered by the HERO System enhances selective lung cancer cytotoxicity while simultaneously reducing ozone and thermal stress properties that are equally critical for safe respiratory tract applications. Thus, by tuning helium and Air/O₂ humidity, the HERO System achieves a dual optimization maximizing therapeutic efficacy while minimizing environmental and physiological risk. Furthermore, theoretically the total O₃ production could be reduced by single powered ring electrode wrapped the dielectric tube and adjusting the position to an optimal configuration\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to voltage and humidity optimization, interval-based CAP treatment was explored to further reduce potential oxidative or thermal stress. Delivering plasma in controlled pulses (e.g., 4\u0026thinsp;+\u0026thinsp;4 + 4\u0026thinsp;+\u0026thinsp;3 minutes, separated by 5-minute rest intervals) proved to be an effective strategy to maintain cellular cytotoxicity while reducing cumulative gas-phase reactivity. This pulsed dosing allows transient decay of reactive species and thermal relaxation between exposures, preventing local overheating or excessive oxidation while sustaining consistent biological effects\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The interval approach is therefore advantageous not only for safety and reproducibility but also for clinical scalability, particularly for ventilator-integrated or endoscopic plasma applications. These optimized plasma conditions humidified helium, moderated voltage, and interval dosing together define an operational window that achieves maximum biological efficacy at minimum toxicity, forming the basis for clinical translation of HERO mediated cold plasma therapy.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e swine study provided critical validation of the system\u0026rsquo;s physiological safety and organ compatibility. Throughout the 90-minute treatment session, all monitored parameters including body temperature, oxygen saturation, blood pressure, and end-tidal CO₂ remained within normal physiological ranges, indicating no respiratory compromise or hemodynamic instability. Histopathological examination of lung tissue further confirmed intact alveolar structure, preserved bronchiolar epithelium, and absence of inflammatory infiltration or edema, verifying that the cold plasma effluent does not induce structural or oxidative damage to pulmonary tissues. Cytokine profiling supported these histological findings, showing only a mild, transient increase in TNF-α without significant changes in IL-6, CRP, or Angiopoietin-1 levels, suggesting no systemic inflammatory activation. Together, these results confirm that the HERO System is physiologically safe at optimal therapeutic doses, capable of delivering bioactive reactive species directly into the respiratory system without causing tissue injury or systemic toxicity\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe potential mechanism of CAP-induced inactivation of biological targets whether malignant cells or viruses is rooted in the generation of a high oxidation-reduction potential (ORP) and elevated electrical conductivity within the treated medium, driven by the abundant production of free radicals. The ROS/RNS cocktail generated by the HERO System can react with carbohydrates, lipids, proteins, and nucleic acids, leading to profound biochemical and structural damage. Lipid peroxidation initiates cross-linking of fatty acid side chains, compromising membrane integrity and cellular viability. Protein oxidation, particularly by singlet oxygen and hydroxyl radicals, modifies cysteine residues into disulfides (R-cys-S-S-cys-R), while oxidation of tyrosine, tryptophan, and histidine leads to the formation of hydroperoxides, aggregation, and conformational destabilization. These processes collectively alter protein folding, membrane stability, and enzymatic function, ultimately impairing viability. Furthermore, oxidation of nucleic acids especially guanine induces cross-links between guanine and lysine, thereby suppressing transcriptional activity and blocking replication. In viral systems, these same oxidative reactions can destroy the viral envelope, denature surface proteins, and fragment genetic material, leading to irreversible loss of infectivity and replication capability\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These same principles underpin the observed humidity dependence, as water vapor is a critical precursor for OH and downstream long‑lived oxidants that mediate damage in cells and virions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, the notion of positioning and electrode configuration to further reduce O₃ while preserving therapeutic RONS is consistent with CAP engineering literature, where discharge geometry, gas composition, and humidity jointly tune O₃ vs. HOx/NOx chemistry; continued optimization should be grounded in direct measurement of O₃ and long‑lived species alongside biological readouts to ensure compliance and efficacy\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Taken together with published clinical and preclinical CHCP experience\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, these results support the HERO configuration as a non‑thermal, humidity‑tuned CAP platform with potential for oncologic use and inhalation applications when engineered to meet safety and exposure limits\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides compelling evidence that the CHCP-HERO System can achieve effective eradication of lung cancer cells while maintaining a high margin of safety \u003cem\u003ein vivo\u003c/em\u003e through precise tuning of plasma parameters. The combination of humidified helium and Air/O₂, optimized voltage, and interval-based dosing ensures both biological potency and operational safety, preventing ozone accumulation and tissue injury. The system\u0026rsquo;s ability to generate a precisely controlled spectrum of reactive species under non-thermal conditions (24\u003csup\u003e0\u003c/sup\u003e C) represents a significant technological advancement with broad implications for plasma-based medical therapeutics, respiratory modulation, and environmental decontamination. Collectively, the data suggest that, with its tuned configuration, the CHCP-HERO System functions as a safe and effective CAP-based ventilator, capable of serving as a therapeutic gas inhalant system for patients with respiratory diseases such as pneumonia, COVID-19, or lung cancer. This study therefore establishes a foundation for future clinical development of plasma-based respiratory therapies that combine oncologic efficacy, infectious respiratory diseases covering a wide variety of viral, bacterial, fungal, and parasitic infections, and pulmonary safety within a single, precisely engineered platform.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe CHCP-HERO System achieved complete eradication of lung cancer cells in vitro and demonstrated safety in a swine model under optimized treatment conditions. By fine-tuning helium and Air/O₂ humidification, voltage, and dosing intervals, the system maintained strong therapeutic efficacy while preventing ozone accumulation and tissue injury. Its non-thermal plasma chemistry enables precise, biocompatible delivery of reactive oxygen and nitrogen species through the respiratory tract. In conclusion, the HERO System is a safe and effective CAP-based ventilator therapy with promising applications for respiratory modulations, and lung cancer.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cp\u003eAll in vivo experiments were performed at the Jerome Canady Research Institute for Advanced Biological and Technological Sciences JCRI-ABTS and the Swine model surgery was performed at Medical Innovation \u0026amp; Training Institute (Henderson, NV) Las Vegas, Nevada.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Canady Helios Cold Plasma HERO System device\u003c/h2\u003e \u003cp\u003eThe CHCP-HERO System is composed of Canady Helios Cold Plasma with a mixer T piece adapter via the cable and breathing circuit tubing set. The T piece distal of the adapter is attached to the endotracheal tube. The proximal ends of the T piece are attached to the CHCP generator ventilator hoses. Helium, oxygen, and air were supplied through individual mass flow controllers to ensure precise flow regulation. Each gas stream was humidified using inline humidifiers and verified with humidity meters. Humidified gases were directed into small conditioning chambers and then delivered to the Canady Helios cold plasma generator, where a non-thermal (24\u003csup\u003e0\u003c/sup\u003e C) discharge ionized the gas mixture. Plasma-activated gas exited the generator through a grounded connection cable and entered a CAP mixing junction, where it was combined with ventilator airflow. The mixed CAP stream was delivered through a sterile endotracheal tube into the cell culture dish or lung airway, enabling exposure to plasma-generated reactive species during controlled ventilation. The illustration of the setup is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Cell culture, HERO treatment and MTT Assay.\u003c/h2\u003e \u003cp\u003eThe human lung adenocarcinoma cell line A549 (ATCC, Manassas, USA) was used to evaluate the efficacy of Canady Helios Cold Atmospheric Plasma HERO treatment. Cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS; ATCC, Manassas, USA) and 1% penicillin-streptomycin (Pen/Strep; Thermo Fisher Scientific, Waltham, USA). Cultures were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂. For experiments, cells were seeded into 12-well tissue culture plates at a density of 1 \u0026times; 10⁵ cells per well and allowed to adhere overnight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.3. HERO Treatment Protocol\u003c/h2\u003e \u003cp\u003eCAP treatments were performed using a plasma discharge generated from a helium-air gas mixture. Air flow was fixed at 4 L/min, while helium flow was varied at 2, 3, or 4 L/min. Gas humidity was adjusted between 0 to 100% relative humidity using an inline humidification module and monitored with a digital humidity sensor positioned proximal to the CAP nozzle. The CAP generator was operated at various voltage setting from 35 V to 70 V, and cells were exposed to CAP at time ranging from 3 to 17 minutes, depending on the experimental condition. For each treatment condition, a corresponding gas-only control (helium\u0026thinsp;+\u0026thinsp;air at the same flow rates and humidity but with plasma power turned off) was included to account for mechanical and gas-flow effects. CAP was administered using two distinct exposure modalities. For direct treatment, wells containing 1 mL of complete culture medium were positioned directly beneath the CAP nozzle at a fixed distance, and the plasma plume interacted with the cell monolayer in real time. For indirect treatment, 1 mL of culture medium was exposed to CAP under identical gas and voltage conditions, after which the treated medium was immediately transferred onto pre-seeded A549 cells. This approach allowed assessment of CAP-generated reactive species in the absence of direct plasma cell interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.4. MTT Assay.\u003c/h2\u003e \u003cp\u003eCell viability was quantified 48 hours post-treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, USA). Briefly, MTT reagent was added to each well to a final concentration of 0.5 mg/mL and incubated for 3 hours at 37\u0026deg;C. The resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA), and absorbance was measured at 570 nm using a BioTek (Winooski, USA) microplate reader. Viability was calculated relative to untreated control wells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.5. Swine Model\u003c/h2\u003e \u003cp\u003eA 16-week-old female Yorkshire-mix swine with a weight of 55 kg, and length of 4\u0026rsquo;2\u0026rsquo;\u0026rsquo; was fasted overnight with free access to water prior to experimentation. All experimental protocols were approved by the veterinary inspector, State of California, department of food and agriculture, animal health and food safety services, Sacramento, CA. All animal study was conducted in compliance with applicable Good Laboratory Practice (GLP) regulation in accordance with 21 CFR Part 58 at the Medical Innovation \u0026amp; Training Institute (Henderson, NV). Anesthesia was induced with an intramuscular Telazol (2.4 cc) and LA Xylazine (0.3 cc) combination (22 and 2 mg/kg, respectively) and maintained with 1.5\u0026ndash;2.5% isoflurane and oxygen delivered by facemask. Animals were intubated with a 7.5-mm cuffed endotracheal tube and mechanically ventilated to maintain end-tidal CO₂ (PetCO₂) between 35\u0026ndash;45 mmHg. Surgery was performed by board certified General Surgeon and anesthesia protocol was planned by board certified Anesthesiologist. A multi-parameter monitor (Surgivet Advisor, Smiths Medical, Plymouth, USA) was used to measure the following physiologic parameters: Heart Rate (HR), ElectroCardioGram (ECG), En Tidal (PetCO₂), and peripheral Oxygen saturation (SpO₂). Core body temperature was maintained at 37-38.5\u0026deg;C using a feedback-controlled warming blanket (Cincinnati SubZero, Blanketrol II, Cincinnati, Ohio, USA). Under sterile conditions, the right carotid artery and right external and internal jugular veins were surgically cannulated to permit continuous monitoring of mean arterial pressure (MAP), pulmonary artery pressure (PAP), heart rate, and core temperature, as well as to allow arterial and mixed venous blood sampling and administration of intravenous anesthetics. CAP treatment using HERO System was delivered at a voltage of 40 V for a total of 15 minutes, administered in four intervals (4\u0026thinsp;+\u0026thinsp;4 + 4\u0026thinsp;+\u0026thinsp;3 min) with 5-minute rest periods between exposures. Helium and oxygen flow rates were set at 3 L/min each. Immediately after treatment, whole blood was collected into 10 mL K₂EDTA tubes, stored on ice, and transported to JCRI-ABTS for immediate processing. Lung tissue samples were harvested post-treatment, fixed in 10% neutral-buffered formalin, and processed for histological evaluation. Tissue morphology and potential CAP-related injury were assessed by hematoxylin staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.6. ELISA\u003c/h2\u003e \u003cp\u003eWhole blood was collected in six 10-mL K₂EDTA tubes at two time points: immediately before treatment and 30 minutes after completion of the HERO cold plasma exposure. Samples were kept on ice and processed promptly for plasma separation. To obtain plasma, EDTA tubes were centrifuged at 1,500 \u0026times; g for 10 minutes at 4\u0026deg;C, and the clear plasma layer was carefully aspirated without disturbing the buffy coat. Plasma aliquots were transferred to sterile microcentrifuge tubes and maintained on ice for downstream analysis or immediately stored at -80\u0026deg;C. Concentrations of IL-6, TNF-α, CRP, Angiopoietin-1, IL-1β, and IL-10 were quantified in triplicate using commercially available porcine ELISA kits (Quantikine Porcine Immunoassays, R\u0026amp;D Systems, Minneapolis, USA), following the manufacturer\u0026rsquo;s instructions. Assays were performed in 96-well plates, and absorbance was measured using a microplate reader. The lower limits of detection for each analyte were: IL-1β, 10 pg/mL; IL-6, 10 pg/mL; IL-10, 1.8 pg/mL; TNF-α, 2.8 pg/mL; and IFN-γ, 2.7 pg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.7. Histology\u003c/h2\u003e \u003cp\u003eSamples of decellularized lung and tracheal tissue were processed at JCRI-ABTS\u0026rsquo;s pathology department for histological evaluation and compared directly with native, untreated controls. Tissues were fixed in 10% neutral buffered formalin for 24\u0026ndash;48 hours, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Paraffin blocks were sectioned at 6\u0026ndash;9 \u0026micro;m thickness using standard microtomy procedures. Sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H\u0026amp;E) following conventional protocols, including hematoxylin staining, differentiation, eosin counterstaining, dehydration, clearing, and coverslip mounting. The primary objective was to evaluate the presence or absence of residual nuclear material in decellularized tissues in comparison with untreated controls. All slides were examined using bright-field and fluorescence microscopy (Carl Zeiss, Germany). Images were acquired using the Zeiss Imaging System under identical exposure settings to allow direct comparison between groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.8. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll values in the figures and text are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Experiments were conducted across three independent days with multiple technical replicates per condition, and data assumptions were verified using Shapiro-Wilk tests for normality and Levene's tests for homogeneity of variance. All datasets met the criteria for parametric analysis (Shapiro-Wilk p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Levene's p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), validating the statistical approaches employed throughout this study. A two-tailed Student's \u003cem\u003et\u003c/em\u003e‐test or ANOVA followed by post hoc Tukey\u0026rsquo;s test was performed, considering P values less than 0.05 as statistically significant. The commercially available Microsoft Excel was used for data analysis. Statistical analysis of ELISA was performed by Mann‐Whitney test. Differences were considered significant at p values\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All box plots and 3D scatter plots were performed in Python (v3.8) using pandas, NumPy, Matplotlib, Seaborn, Statsmodels, Scikit-learn and SciPy. Three-dimensional scatter plots were generated using Matplotlib\u0026rsquo;s Axes3D module. To visualize global trends in reactive species generation, 3D interpolated surfaces were generated using triangulated surface interpolation via Matplotlib\u0026rsquo;s plot_trisurf() function. Mean analyte concentrations were plotted as a function of applied voltage (x-axis) and total treatment duration (y-axis). Color encoding of data points by gas type allowed multi-factored visualization within a single 3D space. SEM values were visualized as vertical z-axis error bars using ax.errorbar(), enabling quantitative interpretation of replicate variability. Inter-analyte associations were assessed by computing Pearson correlation coefficients among mean concentrations of H₂O₂, NO₂\u003csup\u003e-\u003c/sup\u003e, and NO₃\u003csup\u003e-\u003c/sup\u003e across all experimental conditions. The correlation matrix was visualized as a heatmap using Seaborn, with values ranging from \u0026minus;\u0026thinsp;1 (strong negative correlation) to +\u0026thinsp;1 (strong positive correlation).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eThe following supporting information can be downloaded at www.mdpi.com/xxx/s1: Supplemental Figures S1: Ozone production rate by the Canady Helios Cold Plasma HERO System with humidified or dry Air/O2 mixture and He + 0-100% humidity at 70V with 114 khz frequency. S2: Body temperature recording during HERO treatment; S3: End tidal CO2 recording during HERO treatment; S4: O2 saturation recording during HERO treatment; S5: Pulse recording during HERO treatment; Table S1A: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air:O\u003csub\u003e2\u003c/sub\u003e=3:1 O2 16%; Table S1B: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air: O\u003csub\u003e2\u003c/sub\u003e =1:1 O\u003csub\u003e2\u003c/sub\u003e 24%; Table S1C: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air: O\u003csub\u003e2\u003c/sub\u003e =1:3 O\u003csub\u003e2\u003c/sub\u003e 32%; S1D: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with humidified at Air:O\u003csub\u003e2\u003c/sub\u003e=1:1 O\u003csub\u003e2\u003c/sub\u003e 24%; Table S2: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System at 24-hour post treatment with humidified Air/O\u003csub\u003e2\u003c/sub\u003e mixture and He + 0-100% humidity for 1-5 min.; Table S3: Post Hoc Tukey test for viability of A549 cells treated with the CHCP-HERO System with Air/O\u003csub\u003e2\u003c/sub\u003e mixture and Air/O\u003csub\u003e2\u003c/sub\u003e Separate treatment.; Table S4A: Post Hoc Tukey test for quantification of hydrogen peroxide (H₂O₂); Table S4B: Post Hoc Tukey test for quantification of Nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e); Table S4C: Post Hoc Tukey test for quantification of Nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e); Table S5 showing measurements of core body temperature, end-tidal CO₂, oxygen saturation (SpO₂), arterial blood pressure, and pulse rate during and after CAP treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, J.C.; Methodology, J.C. and S.M.; Software, T.Z.; Validation, J.C., T.Z., S.M., and A.N.; Formal Analysis, J.C., S.M., and T.Z.; Investigation, J.C. and S.M.; Resources, J.C.; Data Curation, J.C., S.M., and O.J.; Writing-Original Draft Preparation, J.C., and \u0026nbsp;S.M.; Writing-Review and Editing, J.C., S.M., O.J., M.K., and A.N.; Visualization, J.C., T.Z., and S.M.; Supervision, J.C.; Project Administration, J.C., T.Z., and S.M.; Funding Acquisition, J.C.\u0026nbsp;All\u0026nbsp;authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was funded by US Medical Innovations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eAll the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePeiris, J. 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Electrode con gurations in atmospheric pressure plasma jets: production of reactive species. \u003cem\u003ePlasma Sources Science and Technology\u003c/em\u003e (2018). https://doi.org:https://doi.org/10.1088/1361-6595/aadf5b\u003c/li\u003e\n\u003cli\u003eKumar Dubey, S.\u003cem\u003e et al.\u003c/em\u003e Emerging innovations in cold plasma therapy against cancer: A paradigm shift. \u003cem\u003eDrug Discov Today\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 2425-2439 (2022). https://doi.org:10.1016/j.drudis.2022.05.014\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Cold atmospheric plasma, Cancer treatment, Respiratory infections, COVID-19, ventilation system","lastPublishedDoi":"10.21203/rs.3.rs-8651228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8651228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCold atmospheric plasma (CAP) generates reactive oxygen and nitrogen species (ROS/RNS) capable of selectively destroying pathogens and malignant cells while sparing normal tissue. The Canady Helios Cold Plasma (CHCP) HERO (Humidified Electrical Reactive Oxygen) System integrates a CAP generator with a ventilator platform to enable controlled, humidified plasma delivery through the respiratory tract. Efficacy and safety were assessed using A549 human lung carcinoma cells and an in vivo swine model. CAP was delivered with humidified Air/O₂ (1:1 v/v; 2 L\u0026middot;min⁻\u0026sup1;) and helium (3 L\u0026middot;min⁻\u0026sup1;) at 35\u0026ndash;40 V, varying helium humidity (0-100%) and discharge mode (continuous or interval). Cell viability (MTT), ozone (O₃), and ROS/RNS (H₂O₂, NO₂⁻, NO₃⁻) were quantified, and physiological and histological assessments evaluated in vivo safety. A549 viability decreased significantly with increasing helium humidity (ANOVA, F[4,10]\u0026thinsp;=\u0026thinsp;1770.23, p\u0026thinsp;=\u0026thinsp;3.3 \u0026times; 10⁻\u0026sup1;⁴), with \u0026gt;\u0026thinsp;95% reduction within 5 min at 100% RH. Humidified Air/O₂ reduced O₃ output by 40\u0026ndash;60% versus dry gas (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). In swine, vital signs remained stable and lung histology showed intact alveoli without inflammation or edema; only TNF-α rose modestly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The CHCP-HERO System achieved complete A549 eradication while maintaining physiological and histological safety, supporting CAP-based ventilation as a promising therapeutic gas system for respiratory infections and lung cancer.\u003c/p\u003e","manuscriptTitle":"The Canady Helios HERO Model: A Ventilator- Integrated Humidified Cold Atmospheric Plasma System for Lung Cancer Treatment and Respiratory Infection Applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 10:33:44","doi":"10.21203/rs.3.rs-8651228/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-17T04:46:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T11:04:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T05:48:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296073172430935880379579964819817546657","date":"2026-02-03T15:07:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80017567345428036415757483195753987731","date":"2026-01-28T13:11:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-28T12:03:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T11:53:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-28T07:13:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-23T19:09:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-23T19:01:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5e8a6c8-027a-441d-b6dd-e38f9472a1e6","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61905056,"name":"Health sciences/Diseases"},{"id":61905057,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-07T16:04:14+00:00","versionOfRecord":{"articleIdentity":"rs-8651228","link":"https://doi.org/10.1038/s41598-026-47349-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-05 15:59:00","publishedOnDateReadable":"April 5th, 2026"},"versionCreatedAt":"2026-01-30 10:33:44","video":"","vorDoi":"10.1038/s41598-026-47349-1","vorDoiUrl":"https://doi.org/10.1038/s41598-026-47349-1","workflowStages":[]},"version":"v1","identity":"rs-8651228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8651228","identity":"rs-8651228","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}