Voltage–Frequency Tuned Argon Plasma Jets Enable Targeted H₂O₂ Delivery to Overcome Redox Resistance in Colorectal Cancer Cells

Voltage–Frequency Tuned Argon Plasma Jets Enable Targeted H₂O₂ Delivery to Overcome Redox Resistance in Colorectal Cancer Cells | 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 Research Article Voltage–Frequency Tuned Argon Plasma Jets Enable Targeted H₂O₂ Delivery to Overcome Redox Resistance in Colorectal Cancer Cells Afshin Eftekharinasab, Hassan Mehdian, Ali Hasanbeigi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6791551/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Colorectal cancer (CRC) poses a significant therapeutic challenge due to intrinsic chemoresistance and molecular heterogeneity. This study introduces a tunable voltage and frequency argon plasma jet, powered by kHz AC (1–20 kV, 18–28 kHz) as a novel platform for spatially targeted hydrogen peroxide (H₂O₂)-mediated cytotoxicity in chemoresistant HT29 CRC cells. By precisely modulating voltage and frequency, we achieved precise control over extracellular H₂O₂ concentrations (291–371 µM) in the culture medium, which correlated linearly (R² = 0.995, p < 0.001) with dose-dependent cell death. Optimized parameters (10.5 kV, 28 kHz, 3 min) induced near-complete cytotoxicity (9.2% ± 3.6% viability), surpassing conventional plasma therapies. Morphological analysis revealed hallmark apoptotic phenotypes—rounding, membrane blebbing, and detachment—consistent with H₂O₂-driven oxidative stress overwhelming HT29’s antioxidant defenses. Notably, this tunable system bypasses resistance mechanisms observed in helium plasma-treated cells, where Nrf2/Srx upregulation neutralizes reactive species. Our findings establish spatially controlled H₂O₂ delivery as a potent strategy to overcome CRC chemoresistance, positioning argon plasma jets as a scalable, non-thermal modality for precision oncology. Argon plasma jet Hydrogen peroxide (H₂O₂) Colorectal cancer Chemoresistance Oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. INTRODUCTION Colorectal cancer (CRC) is the third most common malignancy globally, accounting for approximately 1.9 million new cases and 0.9 million deaths in 2020 [1]. By 2030, projections indicate an annual global burden of 2.2 million new cases and 1.1 million deaths annually from this disease [2]. Despite advances in surgery, chemotherapy, and targeted therapies, CRC faces high recurrence rates, driven by therapeutic resistance and molecular heterogeneity [3, 4]. These persistent challenges highlight the urgent need for novel therapeutic strategies capable of overcoming resistance mechanisms and improving clinical outcomes [5]. Cold atmospheric plasma (CAP), a nonthermal partially ionized gas, generates a complex mixture of short- and long-lived reactive oxygen and nitrogen species (RONS), including hydroxyl radicals (•OH), singlet oxygen (¹O 2 ), nitric oxide (NO), hydrogen peroxide (H 2 O 2 ), and nitrite (NO 2 ⁻). These species exploit cancer cells’ dysregulated redox homeostasis to induce oxidative stress and apoptosis [6]. Among these, H₂O₂ has emerged as a key long-lived effector molecule in CAP-mediated anticancer activity. H₂O₂ anticancer effects is significantly amplified by the common overexpression of aquaporins in cancer cells, which may facilitate its preferential uptake and enhance targeted cytotoxicity [7]. Among CAP delivery platforms, dielectric barrier discharges (DBDs) and plasma jets have been most widely explored in biomedical applications, with plasma jets accounting over 70% of oncological studies [8, 9]. The predominant working gases include Helium (35.8%), air (26.3%), and argon (22.1%), each offering distinct RONS profiles efficiencies [9]. However, conventional plasma jet systems typically operate with fixed voltage and frequency settings, or utilize radiofrequency (RF) power supplies,, thereby limiting the dynamic control of RONS dosage [10]. This constraint is particularly significant when targeting chemoresistant CRC phenotypes, such as the HT29 cell line, which upregulate antioxidant defense pathways (e.g., Nrf2/Srx) to neutralize ROS-induced damage [11]. In this study, we address this limitation by developing a kHz AC-driven argon plasma jet with independently tunable voltage (1–20 kV) and frequency (18–28 kHz). We hypothesize that precision tuning of these parameters enables spatially controlled H₂O₂ delivery to overwhelm HT29’s antioxidant defenses, bypassing chemoresistance. Accordingly, we (1) quantifies voltage/frequency-dependent H₂O₂ generation, (2) evaluates cytotoxicity via viability assays and morphological observations, and (3) construct a predictive model linking H₂O₂ concentration to observed cell death. 2. MATERIALS AND METHODS 2.1. Plasma Jet Design and Power Supply A custom-fabricated argon plasma jet was developed to generate a spatially uniform and stable nonthermal plasma plume for RONS delivery. The jet body, constructed from acrylonitrile butadiene styrene (ABS), housed a quartz dielectric tube (3 mm inner diameter, 5 mm outer diameter). A stainless-steel rod (1.8 mm diameter), serving as the high-voltage electrode, was inserted into the quartz tube. A 5 mm-long grounded copper ring electrode was positioned on the tube’s exterior, such that its distal edge was 4 mm from the tube exit. This coaxial electrode configuration, with the central electrode aligned within the axial plane of the ring, ensured a symmetrical and consistent plasma discharge. The system operated with 99.999% pure argon, regulated at 2 standard liters per minute (SLM) using a mass flow controller. Plasma excitation was achieved using a custom-built kHz AC power supply (1–20 kV, 18–28 kHz) with independent voltage and frequency tuning. Electrical diagnostics identified a resonant frequency range of 20–24 kHz, corresponding to maximum energy transfer to the plasma column. 2.2. Cell Culture The human colorectal adenocarcinoma cell line HT29 (IBRC, C10097) was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a humidified incubator at 37°C with 5% CO 2 . For experiments, cells in the logarithmic growth phase were detached using 0.25% trypsin-EDTA, and seeded in 35 mm culture dishes at a density of 2×10 5 cells/dish in 2 mL of complete medium. After 24 hours of incubation, cultures reached 70–80% confluency. 2.3. Plasma Treatment Prior to treatment, HT29 cells cultured in DMEM with 10% heat-inactivated FBS were washed twice with Phosphate-buffered saline (PBS). For viability assays, cells were incubated in 2 mL of serum-free DMEM to avoid FBS-mediated scavenging of plasma-generated reactive species. For H₂O₂ quantification, 2 mL of DMEM (without FBS) was treated under identical conditions in cell-free dishes. The plasma jet nozzle was positioned 10.0 mm vertically above the liquid surface (Fig. 1 ). Cells were exposed to argon plasma at seven distinct voltage-frequency combinations during the screening phase, including (9.5 kV, 18 kHz), (10.5 kV, 18 kHz), (9.5 kV, 25.5 kHz), (10.5 kV, 25.5 kHz), (11.1 kV, 25.5 kHz), (9.5 kV, 28 kHz), and (10.5 kV, 28 kHz), each applied for 3 minutes. For time-dependency analysis, the optimized parameters of (10.5 kV, 28 kHz) were applied for 1 to 4 minutes. Argon (99.999% purity) continuously supplied at 2 SLM. Four hours after plasma exposure, 0.22 mL of heat-inactivated FBS was added to restore 10% serum concentration, standardizing post-treatment conditions for viability assays. All experiments were conducted in triplicate biological replicates, with untreated serum-free controls processed in parallel. 2.4. Cell Viability Assay (MTT) Cell viability was assessed 24 hours post-treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After plasma exposure, cells were incubated with 0.5 mg/mL MTT in PBS for 4 hours at 37°C under 5% CO₂. The supernatant was gently removed, and the resulting formazan crystals were solubilized in 1 mL dimethyl sulfoxide (DMSO). Absorbance at 570 nm was recorded using a spectrophotometric microplate reader. All measurements were performed in triplicate. 2.5. H 2 O 2 Concentration Measurement H 2 O 2 concentrations in cell-free DMEM (2 mL) were quantified using a colorimetric hydrogen peroxide assay kit (ZellBio GmbH) following the manufacturer’s protocol. All plasma treatment conditions were replicated identically in the quantification assays. Absorbance at 546 nm was measured using a microplate reader. 2.6. Morphological Analysis Morphological alterations in HT29 cells were assessed 24 hours post-treatment using an inverted light microscope. Images were captured with a digital camera., and characteristic apoptotic features—including cell rounding, membrane blebbing, and detachment—were quantified in five randomly selected fields per dish by two independent, blinded investigators. 2.7. Statistical Analysis All data are reported as mean ± standard deviation (SD) from three independent replicates. Statistical analyses were performed using SciPy (v1.10.0) and NumPy (v1.24.0) libraries in Python. The linear correlation between extracellular H₂O₂ concentration and cell viability was assessed via simple linear regression, with model parameters estimated using the 'linregress' function. The coefficient of determination (R²) was calculated to quantify the proportion of the variance explained by the model. The statistical significance was determined at a threshold of p < 0.001. The IC50 (CAP exposure time yielding 50% inhibition) was estimated using non-linear regression. Data visualization and curve fitting were performed using the Matplotlib (v3.7.0) and Seaborn (v0.12.2) libraries. 3. RESULTS 3.1. Voltage- and Frequency-Dependent Cytotoxicity Systematic modulation of voltage (9.5–11.1 kV) and frequency (18–28 kHz) revealed distinct cytotoxic profiles in HT29 cells following 3- minute exposure to the argon plasma jet (Fig. 2 ). At 18 kHz, increasing the voltage from 9.5 kV to 10.5 kV reduced viability from 25.16% ± 3.9–19.87% ± 3.2%, demonstrating voltage-dependent enhancement in cytotoxicity. In contrast, at 25.5 kHz, all tested voltages (9.5, 10.5, 11.1 kV) yielded comparatively higher viability rates (viability: 35–48%), suggesting frequency-mediated attenuation of reactive species generation. Notably, Maximum cytotoxicity (9.2% ± 3.6% viability) was observed at 10.5 kV and 28 kHz, while under identical frequency, reducing the voltage to 9.5 Kv resulted in minimal cytotoxicity (54.3% ± 1.2% viability). These findings underscore the critical interplay between voltage and frequency in modulating CAP-induced cell death. 3.2. Time-Dependent Cytotoxicity at Optimal Parameters Time-course experiments conducted at the optimized conditions (10.5 kV, 28 kHz) demonstrated rapid and saturable cytotoxic kinetics in HT29 cells (Fig. 3 a). Cell viability declined exponentially from 91.4% ± 3.9% after 1 minute of plasma exposure to 9.2% ± 3.6% after 3 minutes, with an IC₅₀ of 124 s (Fig. 3 b). Extending the exposure to 4 minutes resulted in 8.5% ± 1.5% viability, showing no statistically significant increase in cytotoxicity beyond the 3-minute mark. This plateau suggests that H₂O₂ accumulation may reach saturation, limiting additional cytotoxic effects despite prolonged treatment duration. 3.3. Tunable H₂O₂ Generation via Voltage-Frequency Modulation Extracellular H₂O₂ concentrations measured in cell-free DMEM closely mirrored the observed cytotoxicity patterns. At 10.5 kV, H₂O₂ production peaked reached its peak value of 371.0 ± 12.7 µM (at 28 kHz), surpassing the levels obtained at 25.5 kHz (319.15 ± 12.09 µM) and 18 kHz (347.75 ± 14.50 µM) (Fig. 4 ). Frequency modulation produced distinct H₂O₂ generation profiles that were strongly dependent on the applied voltage (Fig. 5 ). Specifically, at 10.5 kV, H₂O₂ concentrations followed a parabolic response, with a clear maximum at 28 kHz (Fig. 6 a). In contrast, at 9.5 kV, H₂O₂ output exhibited an exponential decay with increasing frequency, declining from 335.94 ± 16.59 µM (at 18 kHz) to 291.50 ± 9.19 µM (at 28 kHz) (Fig. 6 b). Notably, operation at 11.1 kV, 25.5 kHz yielded suboptimal H₂O₂ levels (310.55 ± 13.36 µM), possibly due to reactive species quenching under excessively energetic plasma conditions. 3.4. Temporal Kinetics of H₂O₂ Accumulation Under optimized plasma conditions (10.5 kV, 28 kHz), H₂O₂ accumulation in cell-free DMEM exhibited time-dependent saturation behavior (Fig. 7 ). The concentration increased progressively, reaching 371.0 ± 12.7 µM at 3 minutes, which corresponded closely with the observed IC₅₀ value of 314 µM for HT29 viability. Extending plasma exposure to 5 minutes resulted in only a modest further increase in H₂O₂ levels (385.0 ± 16.5 µM), indicating a plateau phase. 3.5. H₂O₂ Drives Argon Plasma Jet-Induced Cytotoxicity A strong, statistically significant linear correlation (R² = 0.995, p < 0.001) was observed between extracellular H₂O₂ concentrations (ranging from 291 to 371 µM) and HT29 cell viability across all tested voltage–frequency conditions (Fig. 8 ). Regression analysis confirmed that H₂O₂ levels alone could reliably predict treatment-induced cytotoxicity, highlighting the potential for dose-controlled oxidative stress induction via electrical parameter tuning. 3.6. Morphological Hallmarks of Apoptosis Phase-contrast microscopy revealed distinct morphological alterations consistent with apoptotic cell death, with clear variation depending on the plasma treatment conditions (Fig. 9 ). Untreated control cells exhibited a typical epithelial morphology, with approximately 80% confluency and well-maintained cell–cell contacts (Fig. 9 a). Exposure to sublethal doses (e.g., 9.5 kV, 28 kHz) resulted in partial cell rounding and reduced adhesion (Fig. 9 b). In contrast, lethal conditions (e.g., 10.5 kV, 25.5 kHz) induced membrane blebbing and cytoplasmic shrinkage, indicative of apoptotic initiation (Fig. 9 e). Under maximally cytotoxic parameters (10.5 kV, 28 kHz), cells displayed extensive detachment, nuclear fragmentation, and formation of apoptotic bodies (Fig. 9 h), confirming late-stage apoptosis. 4. DISCUSSION This study demonstrates the potential of voltage- and frequency-tunable argon plasma jets as a precision platform for H₂O₂- mediated therapy in CRC. By optimizing electrical parameters to (10.5 kV, 28 kHz), we achieved near-complete cytotoxicity in chemoresistant HT29 cells (9.2% ± 3.6% viability) through extracellular accumulation of H₂O₂ reaching (371.0 ± 12.7 µM). A robust linear correlation (R² = 0.995, p < 0.001) between H₂O₂ concentration and cell death confirms its dose-dependent biological activity and reinforces its role as the dominant cytotoxic agent in this system. These findings position the described CAP approach beyond traditional fixed-parameter plasma systems, offering spatiotemporal control over reactive species delivery and presenting a strategic means of overcoming chemoresistance in CRC. 4.1. Voltage-Frequency Synergy in H₂O₂ Generation The experimental data revealed a clear interplay between applied voltage and driving frequency in modulating H₂O₂ production within the argon plasma jet. Voltage dependence was evident, as higher voltages (e.g., 10.5 kV compared to 9.5 kV) promoted electron-impact reactions, leading to an increased •OH radical density (Ar + e⁻ → Ar* + •OH) and subsequent H₂O₂ formation (•OH + •OH → H₂O₂) [12]. Frequency tuning further influenced the system's output: operating beyond the resonant range (20–24 kHz) ), specifically at 28 kHz, maximized H₂O₂ generation (371.0 ± 12.7 µM), likely by increasing pulse repetition rates, thereby surpassing H₂O₂ decomposition processes [13]. Interestingly, while increasing voltage at a fixed frequency (25.5 kHz) initially led to elevated H₂O₂ levels, further voltage escalation beyond a critical threshold (11.1 kV) resulted in a decline. This phenomenon may attributable to enhanced decomposition pathways of H₂O₂, driven by the more intense plasma conditions at higher energy inputs [14]. 4.2. H₂O₂ as the Dominant Cytotoxic Mediator The strong linear correlation observed between extracellular H₂O₂ concentration and HT29 cell viability (R² = 0.995, p < 0.001) highlights H₂O₂ as the primary effector of cytotoxicity in our argon plasma jet. This finding aligns with Bekeschus et al., who reported H₂O₂-dominated cytotoxicity in CT26 CRC cells when using the kINPen argon jet, compared to DBD [15]. In contrast to argon plasma jets, the cytotoxicity of DBDs often relies on short-lived species (e.g., •OH, O₃), electric fields and UV radiation [15]. Moreover, the correlation implies that H₂O₂ overload may directly overwhelm intracellular antioxidant defenses, including Nrf2/Srx-mediated redox buffering, thus bypassing resistance mechanisms previously observed in helium plasma jet-treated HT29 cells [11]. These insights underscore the therapeutic potential of long-lived RONS-based interventions, particularly in redox-adapted malignancies. 4.3. Advantages Over Conventional CAP Systems HT29 cells are known for their inherent resistance to standard chemotherapeutic agents, including oxaliplatin and 5-fluorouracil (5-FU), primarily due to upregulation of the Nrf2/Srx antioxidant axis, low basal ROS levels, and metabolic reprogramming, particularly under hypoxia [16–22]. Ishaq et al. (2014) reported HT29 resistance to a helium plasma jet due to elevated Nrf2/Srx levels, which protect against ROS-induced stress; siRNA-mediated silencing of Nrf2/Srx sensitized these cells and reduced viability [11]. In contrast, the present study shows that electrically tunable argon plasma jets can induce potent cytotoxicity in HT29 cells via controlled extracellular H₂O₂ accumulation, potentially circumventing the redox-buffering mechanisms that limit the efficacy of helium plasma [11]. While some DBDs have achieved cytotoxicity in HT29 cells without adjunct therapies [23–26], the lack of spatial resolution and potential thermal risks associated with certain DBDs may limit their translational utility in clinical settings [27–29]. 4.4. Considerations and Future directions Future applications of H₂O₂-mediated cytotoxicity require further validation and optimization to enhance therapeutic efficacy. Studies using 3D cultures and in vivo patient-derived organoids and orthotopic CRC models are essential for understanding H₂O₂ penetration and its interactions with the stromal environment. Additionally, exploring synergistic therapies—such as combining sublethal H₂O₂ doses (e.g., ~ 100 µM) with standard chemotherapeutics like 5-fluorouracil (5-FU) or oxaliplatin—could improve treatment outcomes while minimizing off-target toxicity. Mechanistic investigations, including caspase-3/7 activation assays and PI3K/AKT pathway profiling, are necessary to confirm H₂O₂-driven apoptotic signaling. Furthermore, device scaling efforts, such as miniaturizing plasma jets for laparoscopic compatibility, hold promise for facilitating clinical translation and expanding therapeutic applications. 5. CONCLUSION This study demonstrates that voltage- and frequency-tunable argon plasma jets can effectively overcome chemoresistance in HT29 colorectal cancer cells through controlled extracellular delivery of H₂O₂. Optimal operating parameters (10.5 kV at 28 kHz) yielded near-complete cell death (9.2% ± 3.6% viability), supported by a highly predictive linear correlation (R² = 0.995, p < 0.001) between H₂O₂ concentration and cytotoxicity. Morphological evidence of apoptosis further corroborates H₂O₂ as the dominant effector in this CAP system. These findings distinguish tunable argon plasma jets from other CAP platforms such as helium plasma jets or DBDs, which often rely on short-lived species and exhibit limited spatial precision. While current results are based on in vitro models, the demonstrated tunability and therapeutic specificity provide a solid framework for future in vivo validation, further elucidation of the apoptotic pathways, combinatorial regimens, and device miniaturization toward clinical translation. Overall, this work offers a blueprint for harnessing dose-controlled plasma chemistry to target redox-adapted, treatment-resistant colorectal tumors. Declarations Competing interests The authors declare no competing interests. Author Contribution AE conducted the laboratory experiments, collected the data, and prepared the first draft of the manuscript. HM and AH contributed to data analysis and critically revised the manuscript. All authors read and approved the final manuscript. 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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-6791551","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484094810,"identity":"ef9374e2-7dfd-4b87-8be4-cf7ae3a9d95b","order_by":0,"name":"Afshin Eftekharinasab","email":"","orcid":"","institution":"Kharazmi university","correspondingAuthor":false,"prefix":"","firstName":"Afshin","middleName":"","lastName":"Eftekharinasab","suffix":""},{"id":484094811,"identity":"2aebfdec-d7cc-471f-9747-a56da01ff9b0","order_by":1,"name":"Hassan Mehdian","email":"","orcid":"","institution":"Kharazmi university","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Mehdian","suffix":""},{"id":484094812,"identity":"5be20b79-12b0-49e3-834f-5304daf670ae","order_by":2,"name":"Ali Hasanbeigi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYBACAwkGhgMMBjaJGyB8CaK1pCUbkKQFCA6nGRDtMHPp5oMHvxSczzGXSGD88IPBIp+gFss5xxIOyxjcrrCckcAs2cMgYdlA0GE3cgwOSwC1GNxIYJAG+oWwAw1u5H8AajmXA9TC/JtILTkMBz8YHEgDamEj0pY7xwwOMxgkJxucedhm2WNAjJbbzY8//vhjl7jhePLhGz8q6ogLbGYeMMXYADSBKA1AtT+IVDgKRsEoGAUjFAAAlj090zeMUG4AAAAASUVORK5CYII=","orcid":"","institution":"Kharazmi university","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Hasanbeigi","suffix":""}],"badges":[],"createdAt":"2025-05-31 14:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6791551/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6791551/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86756519,"identity":"94545392-7aa7-4be0-9bdb-a0eae1d3070e","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261765,"visible":true,"origin":"","legend":"\u003cp\u003eArgon plasma jet treatment in a 35 mm culture dish\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/2922767a68c53dcd8346c227.jpeg"},{"id":86756520,"identity":"c4569f79-5b1c-4884-84d1-808dc7d8df17","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46713,"visible":true,"origin":"","legend":"\u003cp\u003eVoltage- and frequency-dependent cytotoxicity in HT29 cells after 3-minute plasma treatment.\u003cbr\u003e\nMTT assay results show cell viability (%) at different voltage-frequency combinations (9.5–11.1 kV, 18–28 kHz). Maximum cytotoxicity (9.2% viability) occurred at 10.5 kV, 28 kHz. Data: mean ± SD (n=3)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/cfae9b8dd96d3f382fe499ee.png"},{"id":86756890,"identity":"fb287e46-8586-4171-b406-37039fc7156e","added_by":"auto","created_at":"2025-07-15 09:36:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40387,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent cytotoxicity at 10.5 kV, 28 kHz. \u003cstrong\u003ea\u003c/strong\u003e. Viability decreased progressively with exposure time (1–4 minutes). Data: mean ± SD (n=3). \u003cstrong\u003eb\u003c/strong\u003e. Correlation between H₂O₂ concentration and IC50 at 10.5 kV, 28 kHz. The IC50 treatment time (124 seconds) corresponds to 314.8 µM H₂O₂\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/97407f31ccf6858c69cee1c6.png"},{"id":86756522,"identity":"834913cf-95e9-4252-8dcf-ae4a3460e991","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44443,"visible":true,"origin":"","legend":"\u003cp\u003eH₂O₂ generation across voltage-frequency combinations. H₂O₂ concentrations (µM) in cell-free DMEM after 3-minute exposure. Highest H₂O₂ (371.0 ± 12.7 µM) at 10.5 kV, 28 kHz. Data: mean ± SD (n=3)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/e128bc055272b4db25b11b01.png"},{"id":86756526,"identity":"da599815-581f-45cc-bf52-6004d85656dc","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127913,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration for 9.5 kV and 10.5 kV across frequency. At 10.5 kV, H₂O₂ generally exceeded 9.5 kV. Data: mean ± SD (n=3)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/27f87cd78e04984f09d12daa.png"},{"id":86756528,"identity":"10a23de7-9727-4fa6-ac42-128bc4707b8e","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":96678,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency-dependent H₂O₂ trends. \u003cstrong\u003ea\u003c/strong\u003e At 9.5 kV, exponential asymptote at higher frequencies .\u003cstrong\u003eb\u003c/strong\u003e At 10.5 kV, parabolic. Data: mean ± SD (n=3)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/5007dd2630912f0d3c1a2fe6.png"},{"id":86756535,"identity":"be79d3a7-4442-43c8-a234-7b1fa0a296e6","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52692,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent H₂O₂ accumulation at 10.5 kV, 28 kHz. Saturating increase in H₂O₂ (1–5 minutes). Plateau was observed after 3 minutes. Data: mean ± SD (n=3).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/1d409f29b6018a16bf4a7dbd.png"},{"id":86756530,"identity":"21a78056-3f48-41b7-9943-07e1a7241d8e","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":89685,"visible":true,"origin":"","legend":"\u003cp\u003eLinear correlation between H₂O₂ concentration and HT29 cell viability. Strong inverse relationship (R² = 0.995, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/44a8009fe3ec78b6fafd8086.png"},{"id":86756536,"identity":"1be0ad89-d892-4423-8090-ded7b37a2818","added_by":"auto","created_at":"2025-07-15 09:28:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1367072,"visible":true,"origin":"","legend":"\u003cp\u003ePlasma-induced morphological changes in HT29 cells (24 hours post-treatment). \u003cstrong\u003e(a)\u003c/strong\u003e Untreated control: epithelial morphology, 80% confluency. \u003cstrong\u003e(b)\u003c/strong\u003e 9.5 kV, 28 kHz: Minimal changes.\u003cstrong\u003e (c)\u003c/strong\u003e 9.5 kV, 25.5 kHz: Moderate detachment.\u003cstrong\u003e (d)\u003c/strong\u003e 11.1 kV, 25.5 kHz: Reduced cytotoxicity vs. 10.5 kV. \u0026nbsp;\u003cstrong\u003e(e)\u003c/strong\u003e 10.5 kV, 25.5 kHz: Widespread shrinkage. \u003cstrong\u003e(f)\u003c/strong\u003e 9.5 kV, 18 kHz: Initial rounding and reduced cell-cell contact. \u003cstrong\u003e(g)\u003c/strong\u003e 10.5 kV, 18 kHz: Shrinkage and membrane blebbing. \u003cstrong\u003e(h)\u003c/strong\u003e 10.5 kV, 28 kHz: Severe apoptosis (fragmentation, detachment). Treatment time: 3 minutes; DMEM volume: 2 ml; argon flow: 2 SLM; distance: 10 mm.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/f40d3a278cb4a938f64b6ba7.png"},{"id":89083054,"identity":"01d3e099-0993-425c-934e-5f984d63c619","added_by":"auto","created_at":"2025-08-14 13:32:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2889621,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6791551/v1/e9d7b007-e361-4731-83e3-795e8af4c713.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eVoltage–Frequency Tuned Argon Plasma Jets Enable Targeted H₂O₂ Delivery to Overcome Redox Resistance in Colorectal Cancer Cells\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eColorectal cancer (CRC) is the third most common malignancy globally, accounting for approximately 1.9\u0026nbsp;million new cases and 0.9\u0026nbsp;million deaths in 2020 [1]. By 2030, projections indicate an annual global burden of 2.2\u0026nbsp;million new cases and 1.1\u0026nbsp;million deaths annually from this disease [2]. Despite advances in surgery, chemotherapy, and targeted therapies, CRC faces high recurrence rates, driven by therapeutic resistance and molecular heterogeneity [3, 4]. These persistent challenges highlight the urgent need for novel therapeutic strategies capable of overcoming resistance mechanisms and improving clinical outcomes [5].\u003c/p\u003e\u003cp\u003eCold atmospheric plasma (CAP), a nonthermal partially ionized gas, generates a complex mixture of short- and long-lived reactive oxygen and nitrogen species (RONS), including hydroxyl radicals (\u0026bull;OH), singlet oxygen (\u0026sup1;O\u003csub\u003e2\u003c/sub\u003e), nitric oxide (NO), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and nitrite (NO\u003csub\u003e2\u003c/sub\u003e⁻). These species exploit cancer cells\u0026rsquo; dysregulated redox homeostasis to induce oxidative stress and apoptosis [6]. Among these, H₂O₂ has emerged as a key long-lived effector molecule in CAP-mediated anticancer activity. H₂O₂ anticancer effects is significantly amplified by the common overexpression of aquaporins in cancer cells, which may facilitate its preferential uptake and enhance targeted cytotoxicity [7].\u003c/p\u003e\u003cp\u003eAmong CAP delivery platforms, dielectric barrier discharges (DBDs) and plasma jets have been most widely explored in biomedical applications, with plasma jets accounting over 70% of oncological studies [8, 9]. The predominant working gases include Helium (35.8%), air (26.3%), and argon (22.1%), each offering distinct RONS profiles efficiencies [9]. However, conventional plasma jet systems typically operate with fixed voltage and frequency settings, or utilize radiofrequency (RF) power supplies,, thereby limiting the dynamic control of RONS dosage [10]. This constraint is particularly significant when targeting chemoresistant CRC phenotypes, such as the HT29 cell line, which upregulate antioxidant defense pathways (e.g., Nrf2/Srx) to neutralize ROS-induced damage [11].\u003c/p\u003e\u003cp\u003eIn this study, we address this limitation by developing a kHz AC-driven argon plasma jet with independently tunable voltage (1\u0026ndash;20 kV) and frequency (18\u0026ndash;28 kHz). We hypothesize that precision tuning of these parameters enables spatially controlled H₂O₂ delivery to overwhelm HT29\u0026rsquo;s antioxidant defenses, bypassing chemoresistance. Accordingly, we (1) quantifies voltage/frequency-dependent H₂O₂ generation, (2) evaluates cytotoxicity via viability assays and morphological observations, and (3) construct a predictive model linking H₂O₂ concentration to observed cell death.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plasma Jet Design and Power Supply\u003c/h2\u003e\u003cp\u003eA custom-fabricated argon plasma jet was developed to generate a spatially uniform and stable nonthermal plasma plume for RONS delivery. The jet body, constructed from acrylonitrile butadiene styrene (ABS), housed a quartz dielectric tube (3 mm inner diameter, 5 mm outer diameter). A stainless-steel rod (1.8 mm diameter), serving as the high-voltage electrode, was inserted into the quartz tube. A 5 mm-long grounded copper ring electrode was positioned on the tube\u0026rsquo;s exterior, such that its distal edge was 4 mm from the tube exit. This coaxial electrode configuration, with the central electrode aligned within the axial plane of the ring, ensured a symmetrical and consistent plasma discharge. The system operated with 99.999% pure argon, regulated at 2 standard liters per minute (SLM) using a mass flow controller. Plasma excitation was achieved using a custom-built kHz AC power supply (1\u0026ndash;20 kV, 18\u0026ndash;28 kHz) with independent voltage and frequency tuning. Electrical diagnostics identified a resonant frequency range of 20\u0026ndash;24 kHz, corresponding to maximum energy transfer to the plasma column.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Cell Culture\u003c/h2\u003e\u003cp\u003eThe human colorectal adenocarcinoma cell line HT29 (IBRC, C10097) was cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. For experiments, cells in the logarithmic growth phase were detached using 0.25% trypsin-EDTA, and seeded in 35 mm culture dishes at a density of 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/dish in 2 mL of complete medium. After 24 hours of incubation, cultures reached 70\u0026ndash;80% confluency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Plasma Treatment\u003c/h2\u003e\u003cp\u003ePrior to treatment, HT29 cells cultured in DMEM with 10% heat-inactivated FBS were washed twice with Phosphate-buffered saline (PBS). For viability assays, cells were incubated in 2 mL of serum-free DMEM to avoid FBS-mediated scavenging of plasma-generated reactive species. For H₂O₂ quantification, 2 mL of DMEM (without FBS) was treated under identical conditions in cell-free dishes. The plasma jet nozzle was positioned 10.0 mm vertically above the liquid surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Cells were exposed to argon plasma at seven distinct voltage-frequency combinations during the screening phase, including (9.5 kV, 18 kHz), (10.5 kV, 18 kHz), (9.5 kV, 25.5 kHz), (10.5 kV, 25.5 kHz), (11.1 kV, 25.5 kHz), (9.5 kV, 28 kHz), and (10.5 kV, 28 kHz), each applied for 3 minutes. For time-dependency analysis, the optimized parameters of (10.5 kV, 28 kHz) were applied for 1 to 4 minutes. Argon (99.999% purity) continuously supplied at 2 SLM. Four hours after plasma exposure, 0.22 mL of heat-inactivated FBS was added to restore 10% serum concentration, standardizing post-treatment conditions for viability assays. All experiments were conducted in triplicate biological replicates, with untreated serum-free controls processed in parallel.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cell Viability Assay (MTT)\u003c/h2\u003e\u003cp\u003eCell viability was assessed 24 hours post-treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After plasma exposure, cells were incubated with 0.5 mg/mL MTT in PBS for 4 hours at 37\u0026deg;C under 5% CO₂. The supernatant was gently removed, and the resulting formazan crystals were solubilized in 1 mL dimethyl sulfoxide (DMSO). Absorbance at 570 nm was recorded using a spectrophotometric microplate reader. All measurements were performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Concentration Measurement\u003c/h2\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations in cell-free DMEM (2 mL) were quantified using a colorimetric hydrogen peroxide assay kit (ZellBio GmbH) following the manufacturer\u0026rsquo;s protocol. All plasma treatment conditions were replicated identically in the quantification assays. Absorbance at 546 nm was measured using a microplate reader.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Morphological Analysis\u003c/h2\u003e\u003cp\u003eMorphological alterations in HT29 cells were assessed 24 hours post-treatment using an inverted light microscope. Images were captured with a digital camera., and characteristic apoptotic features\u0026mdash;including cell rounding, membrane blebbing, and detachment\u0026mdash;were quantified in five randomly selected fields per dish by two independent, blinded investigators.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent replicates. Statistical analyses were performed using SciPy (v1.10.0) and NumPy (v1.24.0) libraries in Python. The linear correlation between extracellular H₂O₂ concentration and cell viability was assessed via simple linear regression, with model parameters estimated using the 'linregress' function. The coefficient of determination (R\u0026sup2;) was calculated to quantify the proportion of the variance explained by the model. The statistical significance was determined at a threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. The IC50 (CAP exposure time yielding 50% inhibition) was estimated using non-linear regression. Data visualization and curve fitting were performed using the Matplotlib (v3.7.0) and Seaborn (v0.12.2) libraries.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Voltage- and Frequency-Dependent Cytotoxicity\u003c/h2\u003e\u003cp\u003eSystematic modulation of voltage (9.5\u0026ndash;11.1 kV) and frequency (18\u0026ndash;28 kHz) revealed distinct cytotoxic profiles in HT29 cells following 3- minute exposure to the argon plasma jet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 18 kHz, increasing the voltage from 9.5 kV to 10.5 kV reduced viability from 25.16% \u0026plusmn; 3.9\u0026ndash;19.87% \u0026plusmn; 3.2%, demonstrating voltage-dependent enhancement in cytotoxicity. In contrast, at 25.5 kHz, all tested voltages (9.5, 10.5, 11.1 kV) yielded comparatively higher viability rates (viability: 35\u0026ndash;48%), suggesting frequency-mediated attenuation of reactive species generation. Notably, Maximum cytotoxicity (9.2% \u0026plusmn; 3.6% viability) was observed at 10.5 kV and 28 kHz, while under identical frequency, reducing the voltage to 9.5 Kv resulted in minimal cytotoxicity (54.3% \u0026plusmn; 1.2% viability). These findings underscore the critical interplay between voltage and frequency in modulating CAP-induced cell death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Time-Dependent Cytotoxicity at Optimal Parameters\u003c/h2\u003e\u003cp\u003eTime-course experiments conducted at the optimized conditions (10.5 kV, 28 kHz) demonstrated rapid and saturable cytotoxic kinetics in HT29 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Cell viability declined exponentially from 91.4% \u0026plusmn; 3.9% after 1 minute of plasma exposure to 9.2% \u0026plusmn; 3.6% after 3 minutes, with an IC₅₀ of 124 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Extending the exposure to 4 minutes resulted in 8.5% \u0026plusmn; 1.5% viability, showing no statistically significant increase in cytotoxicity beyond the 3-minute mark. This plateau suggests that H₂O₂ accumulation may reach saturation, limiting additional cytotoxic effects despite prolonged treatment duration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Tunable H₂O₂ Generation via Voltage-Frequency Modulation\u003c/h2\u003e\u003cp\u003eExtracellular H₂O₂ concentrations measured in cell-free DMEM closely mirrored the observed cytotoxicity patterns. At 10.5 kV, H₂O₂ production peaked reached its peak value of 371.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 \u0026micro;M (at 28 kHz), surpassing the levels obtained at 25.5 kHz (319.15\u0026thinsp;\u0026plusmn;\u0026thinsp;12.09 \u0026micro;M) and 18 kHz (347.75\u0026thinsp;\u0026plusmn;\u0026thinsp;14.50 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Frequency modulation produced distinct H₂O₂ generation profiles that were strongly dependent on the applied voltage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, at 10.5 kV, H₂O₂ concentrations followed a parabolic response, with a clear maximum at 28 kHz (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In contrast, at 9.5 kV, H₂O₂ output exhibited an exponential decay with increasing frequency, declining from 335.94\u0026thinsp;\u0026plusmn;\u0026thinsp;16.59 \u0026micro;M (at 18 kHz) to 291.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.19 \u0026micro;M (at 28 kHz) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Notably, operation at 11.1 kV, 25.5 kHz yielded suboptimal H₂O₂ levels (310.55\u0026thinsp;\u0026plusmn;\u0026thinsp;13.36 \u0026micro;M), possibly due to reactive species quenching under excessively energetic plasma conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Temporal Kinetics of H₂O₂ Accumulation\u003c/h2\u003e\u003cp\u003eUnder optimized plasma conditions (10.5 kV, 28 kHz), H₂O₂ accumulation in cell-free DMEM exhibited time-dependent saturation behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The concentration increased progressively, reaching 371.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 \u0026micro;M at 3 minutes, which corresponded closely with the observed IC₅₀ value of 314 \u0026micro;M for HT29 viability. Extending plasma exposure to 5 minutes resulted in only a modest further increase in H₂O₂ levels (385.0\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5 \u0026micro;M), indicating a plateau phase.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5. H₂O₂ Drives Argon Plasma Jet-Induced Cytotoxicity\u003c/h2\u003e\u003cp\u003eA strong, statistically significant linear correlation (R\u0026sup2; = 0.995, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed between extracellular H₂O₂ concentrations (ranging from 291 to 371 \u0026micro;M) and HT29 cell viability across all tested voltage\u0026ndash;frequency conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Regression analysis confirmed that H₂O₂ levels alone could reliably predict treatment-induced cytotoxicity, highlighting the potential for dose-controlled oxidative stress induction via electrical parameter tuning.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Morphological Hallmarks of Apoptosis\u003c/h2\u003e\u003cp\u003ePhase-contrast microscopy revealed distinct morphological alterations consistent with apoptotic cell death, with clear variation depending on the plasma treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Untreated control cells exhibited a typical epithelial morphology, with approximately 80% confluency and well-maintained cell\u0026ndash;cell contacts (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Exposure to sublethal doses (e.g., 9.5 kV, 28 kHz) resulted in partial cell rounding and reduced adhesion (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). In contrast, lethal conditions (e.g., 10.5 kV, 25.5 kHz) induced membrane blebbing and cytoplasmic shrinkage, indicative of apoptotic initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee). Under maximally cytotoxic parameters (10.5 kV, 28 kHz), cells displayed extensive detachment, nuclear fragmentation, and formation of apoptotic bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh), confirming late-stage apoptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis study demonstrates the potential of voltage- and frequency-tunable argon plasma jets as a precision platform for H₂O₂- mediated therapy in CRC. By optimizing electrical parameters to (10.5 kV, 28 kHz), we achieved near-complete cytotoxicity in chemoresistant HT29 cells (9.2% \u0026plusmn; 3.6% viability) through extracellular accumulation of H₂O₂ reaching (371.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 \u0026micro;M). A robust linear correlation (R\u0026sup2; = 0.995, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between H₂O₂ concentration and cell death confirms its dose-dependent biological activity and reinforces its role as the dominant cytotoxic agent in this system. These findings position the described CAP approach beyond traditional fixed-parameter plasma systems, offering spatiotemporal control over reactive species delivery and presenting a strategic means of overcoming chemoresistance in CRC.\u003c/p\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Voltage-Frequency Synergy in H₂O₂ Generation\u003c/h2\u003e\u003cp\u003eThe experimental data revealed a clear interplay between applied voltage and driving frequency in modulating H₂O₂ production within the argon plasma jet. Voltage dependence was evident, as higher voltages (e.g., 10.5 kV compared to 9.5 kV) promoted electron-impact reactions, leading to an increased \u0026bull;OH radical density (Ar\u0026thinsp;+\u0026thinsp;e⁻ \u0026rarr; Ar* + \u0026bull;OH) and subsequent H₂O₂ formation (\u0026bull;OH + \u0026bull;OH \u0026rarr; H₂O₂) [12]. Frequency tuning further influenced the system's output: operating beyond the resonant range (20\u0026ndash;24 kHz) ), specifically at 28 kHz, maximized H₂O₂ generation (371.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 \u0026micro;M), likely by increasing pulse repetition rates, thereby surpassing H₂O₂ decomposition processes [13]. Interestingly, while increasing voltage at a fixed frequency (25.5 kHz) initially led to elevated H₂O₂ levels, further voltage escalation beyond a critical threshold (11.1 kV) resulted in a decline. This phenomenon may attributable to enhanced decomposition pathways of H₂O₂, driven by the more intense plasma conditions at higher energy inputs [14].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.2. H₂O₂ as the Dominant Cytotoxic Mediator\u003c/h2\u003e\u003cp\u003eThe strong linear correlation observed between extracellular H₂O₂ concentration and HT29 cell viability (R\u0026sup2; = 0.995, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) highlights H₂O₂ as the primary effector of cytotoxicity in our argon plasma jet. This finding aligns with Bekeschus et al., who reported H₂O₂-dominated cytotoxicity in CT26 CRC cells when using the kINPen argon jet, compared to DBD [15]. In contrast to argon plasma jets, the cytotoxicity of DBDs often relies on short-lived species (e.g., \u0026bull;OH, O₃), electric fields and UV radiation [15]. Moreover, the correlation implies that H₂O₂ overload may directly overwhelm intracellular antioxidant defenses, including Nrf2/Srx-mediated redox buffering, thus bypassing resistance mechanisms previously observed in helium plasma jet-treated HT29 cells [11]. These insights underscore the therapeutic potential of long-lived RONS-based interventions, particularly in redox-adapted malignancies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Advantages Over Conventional CAP Systems\u003c/h2\u003e\u003cp\u003eHT29 cells are known for their inherent resistance to standard chemotherapeutic agents, including oxaliplatin and 5-fluorouracil (5-FU), primarily due to upregulation of the Nrf2/Srx antioxidant axis, low basal ROS levels, and metabolic reprogramming, particularly under hypoxia [16\u0026ndash;22]. Ishaq et al. (2014) reported HT29 resistance to a helium plasma jet due to elevated Nrf2/Srx levels, which protect against ROS-induced stress; siRNA-mediated silencing of Nrf2/Srx sensitized these cells and reduced viability [11]. In contrast, the present study shows that electrically tunable argon plasma jets can induce potent cytotoxicity in HT29 cells via controlled extracellular H₂O₂ accumulation, potentially circumventing the redox-buffering mechanisms that limit the efficacy of helium plasma [11]. While some DBDs have achieved cytotoxicity in HT29 cells without adjunct therapies [23\u0026ndash;26], the lack of spatial resolution and potential thermal risks associated with certain DBDs may limit their translational utility in clinical settings [27\u0026ndash;29].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Considerations and Future directions\u003c/h2\u003e\u003cp\u003eFuture applications of H₂O₂-mediated cytotoxicity require further validation and optimization to enhance therapeutic efficacy. Studies using 3D cultures and in vivo patient-derived organoids and orthotopic CRC models are essential for understanding H₂O₂ penetration and its interactions with the stromal environment. Additionally, exploring synergistic therapies\u0026mdash;such as combining sublethal H₂O₂ doses (e.g., ~\u0026thinsp;100 \u0026micro;M) with standard chemotherapeutics like 5-fluorouracil (5-FU) or oxaliplatin\u0026mdash;could improve treatment outcomes while minimizing off-target toxicity. Mechanistic investigations, including caspase-3/7 activation assays and PI3K/AKT pathway profiling, are necessary to confirm H₂O₂-driven apoptotic signaling. Furthermore, device scaling efforts, such as miniaturizing plasma jets for laparoscopic compatibility, hold promise for facilitating clinical translation and expanding therapeutic applications.\u003c/p\u003e\u003c/div\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThis study demonstrates that voltage- and frequency-tunable argon plasma jets can effectively overcome chemoresistance in HT29 colorectal cancer cells through controlled extracellular delivery of H₂O₂. Optimal operating parameters (10.5 kV at 28 kHz) yielded near-complete cell death (9.2% \u0026plusmn; 3.6% viability), supported by a highly predictive linear correlation (R\u0026sup2; = 0.995, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) between H₂O₂ concentration and cytotoxicity. Morphological evidence of apoptosis further corroborates H₂O₂ as the dominant effector in this CAP system. These findings distinguish tunable argon plasma jets from other CAP platforms such as helium plasma jets or DBDs, which often rely on short-lived species and exhibit limited spatial precision. While current results are based on in vitro models, the demonstrated tunability and therapeutic specificity provide a solid framework for future in vivo validation, further elucidation of the apoptotic pathways, combinatorial regimens, and device miniaturization toward clinical translation. Overall, this work offers a blueprint for harnessing dose-controlled plasma chemistry to target redox-adapted, treatment-resistant colorectal tumors.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAE conducted the laboratory experiments, collected the data, and prepared the first draft of the manuscript. HM and AH contributed to data analysis and critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoshandel, G., F. Ghasemi-Kebria, and R. 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Li, \u003cem\u003eMT2A promotes oxaliplatin resistance in colorectal cancer cells.\u003c/em\u003e Cell Biochemistry and Biophysics, 2020. \u003cstrong\u003e78\u003c/strong\u003e(4): p. 475-482.\u003c/li\u003e\n\u003cli\u003eKitahara, T., et al., \u003cem\u003eIdentification and characterization of CD107a as a marker of low reactive oxygen species in chemoresistant cells in colorectal cancer.\u003c/em\u003e Annals of surgical oncology, 2017. \u003cstrong\u003e24\u003c/strong\u003e: p. 1110-1119.\u003c/li\u003e\n\u003cli\u003eCheraghi, O., et al., \u003cem\u003eThe effect of Nrf2 deletion on the proteomic signature in a human colorectal cancer cell line.\u003c/em\u003e BMC cancer, 2022. \u003cstrong\u003e22\u003c/strong\u003e(1): p. 979.\u003c/li\u003e\n\u003cli\u003eKim, H.G., et al., \u003cem\u003eQuinacrine-mediated inhibition of Nrf2 reverses hypoxia-induced 5-fluorouracil resistance in colorectal cancer.\u003c/em\u003e International journal of molecular sciences, 2019. \u003cstrong\u003e20\u003c/strong\u003e(18): p. 4366.\u003c/li\u003e\n\u003cli\u003eBarrera, J.C.A., et al., \u003cem\u003eMetabolomic and lipidomic analysis of the colorectal adenocarcinoma cell line HT29 in hypoxia and reoxygenation.\u003c/em\u003e Metabolites, 2023. \u003cstrong\u003e13\u003c/strong\u003e(7): p. 875.\u003c/li\u003e\n\u003cli\u003eTouil, Y., et al., \u003cem\u003eColon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis.\u003c/em\u003e Clinical cancer research, 2014. \u003cstrong\u003e20\u003c/strong\u003e(4): p. 837-846.\u003c/li\u003e\n\u003cli\u003eHe, Y., et al., \u003cem\u003eCold atmospheric plasma stabilizes mismatch repair for effective, uniform treatment of diverse colorectal cancer cell types.\u003c/em\u003e Scientific Reports, 2024. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 3599.\u003c/li\u003e\n\u003cli\u003eWang, Y., et al., \u003cem\u003eCold atmospheric plasma induces apoptosis in human colon and lung cancer cells through modulating mitochondrial pathway.\u003c/em\u003e Frontiers in Cell and Developmental Biology, 2022. \u003cstrong\u003e10\u003c/strong\u003e: p. 915785.\u003c/li\u003e\n\u003cli\u003eSchneider, C., et al., \u003cem\u003eCold atmospheric plasma treatment inhibits growth in colorectal cancer cells.\u003c/em\u003e Biological chemistry, 2018. \u003cstrong\u003e400\u003c/strong\u003e(1): p. 111-122.\u003c/li\u003e\n\u003cli\u003eHan, D., et al., \u003cem\u003eAntitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor.\u003c/em\u003e Scientific reports, 2017. \u003cstrong\u003e7\u003c/strong\u003e(1): p. 43081.\u003c/li\u003e\n\u003cli\u003eAyan, H., et al., \u003cem\u003eHeating effect of dielectric barrier discharges for direct medical treatment.\u003c/em\u003e IEEE Transactions on Plasma Science, 2008. \u003cstrong\u003e37\u003c/strong\u003e(1): p. 113-120.\u003c/li\u003e\n\u003cli\u003eKogelheide, F., et al., \u003cem\u003eCharacterisation of volume and surface dielectric barrier discharges in N2\u0026ndash;O2 mixtures using optical emission spectroscopy.\u003c/em\u003e Plasma Processes and Polymers, 2020. \u003cstrong\u003e17\u003c/strong\u003e(6): p. 1900126.\u003c/li\u003e\n\u003cli\u003eChoi, M., et al., \u003cem\u003eOn encapsulated dielectric barrier discharge plasma sources for radar cross section reduction in mobile environments.\u003c/em\u003e Sensors, 2023. \u003cstrong\u003e23\u003c/strong\u003e(22): p. 9170.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Argon plasma jet, Hydrogen peroxide (H₂O₂), Colorectal cancer, Chemoresistance, Oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-6791551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6791551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) poses a significant therapeutic challenge due to intrinsic chemoresistance and molecular heterogeneity. This study introduces a tunable voltage and frequency argon plasma jet, powered by kHz AC (1\u0026ndash;20 kV, 18\u0026ndash;28 kHz) as a novel platform for spatially targeted hydrogen peroxide (H₂O₂)-mediated cytotoxicity in chemoresistant HT29 CRC cells. By precisely modulating voltage and frequency, we achieved precise control over extracellular H₂O₂ concentrations (291\u0026ndash;371 \u0026micro;M) in the culture medium, which correlated linearly (R\u0026sup2; = 0.995, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with dose-dependent cell death. Optimized parameters (10.5 kV, 28 kHz, 3 min) induced near-complete cytotoxicity (9.2% \u0026plusmn; 3.6% viability), surpassing conventional plasma therapies. Morphological analysis revealed hallmark apoptotic phenotypes\u0026mdash;rounding, membrane blebbing, and detachment\u0026mdash;consistent with H₂O₂-driven oxidative stress overwhelming HT29\u0026rsquo;s antioxidant defenses. Notably, this tunable system bypasses resistance mechanisms observed in helium plasma-treated cells, where Nrf2/Srx upregulation neutralizes reactive species. Our findings establish spatially controlled H₂O₂ delivery as a potent strategy to overcome CRC chemoresistance, positioning argon plasma jets as a scalable, non-thermal modality for precision oncology.\u003c/p\u003e","manuscriptTitle":"Voltage–Frequency Tuned Argon Plasma Jets Enable Targeted H₂O₂ Delivery to Overcome Redox Resistance in Colorectal Cancer Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 09:28:34","doi":"10.21203/rs.3.rs-6791551/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ac0a024-530b-4160-a158-33275cb099fa","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-14T13:23:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 09:28:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6791551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6791551","identity":"rs-6791551","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}