Plasma-activated water: a dual-action disinfectant and wound-healing therapy for diabetes-related foot infections

Abstract

Globally, approximately 18.6 million individuals develop diabetes-related foot ulcers each year, with 50%–60% of these cases subsequently becoming infected. Diabetes-related foot infections (DFI) are a serious complication often resulting in gangrene, lower extremity amputation and death—outcomes exacerbated by multidrug-resistant and biofilm-forming pathogens that render standard antibiotics and antiseptics inadequate, driving up healthcare costs and morbidity. Addressing this challenge necessitates the development of novel, dual-action therapeutic strategies capable of eradicating infection while simultaneously promoting tissue regeneration. Plasma-activated water (PAW) is an emerging solution, produced by treating water with cold plasma; an ionized gas that generates a complex mixture of reactive oxygen and nitrogen species. PAW elicits potent broad-spectrum antimicrobial and antibiofilm activity against a wide range of pathogens implicated in chronic wound infections, including DFI. PAW has also been shown to accelerate wound healing by modulating immune cell activity and promoting epithelial cell proliferation and migration. In this review, we summarize DFI prevalence and recurrence, PAW generation and physicochemical properties, antimicrobial and antibiofilm efficacy against relevant pathogens, effects on cellular responses, therapeutic potential and current limitations. This review positions PAW as a dual-action, resistance-refractory therapeutic strategy for DFIs and other chronic wounds and outlines the translational pathway towards clinical implementation.

This review evaluates plasma-activated water as an emerging dual-action therapy with potential to combat drug-resistant infection and promote wound healing in people with diabetes-related foot infections.

Introduction

Chronic wounds represent a significant global health challenge, affecting millions of individuals and imposing substantial economic and social burdens. Specifically in Australia, more than 450 000 Australians suffer from chronic wounds which cost the health and aged care systems $6.6 billion yearly, with each patient spending an average of $4000 in out-of-pocket cost (Wounds Australia 2024). Diabetes-related foot ulcers (DFUs) are among the most serious form of chronic wounds, as they recur frequently, are prone to persistent microbial infections, and often culminate into lower extremity amputation (LEA) (Armstrong et al. 2017, Narres et al. 2017, Armstrong et al. 2023, Ezzatvar and García-Hermoso 2023). Each year, an estimated 18.6 million individuals worldwide develop DFUs, with infection occurring in ∼50%–60% of cases (Armstrong et al. 2023). In North America alone, diabetes-related foot infections (DFIs) affect ∼13% of DFU patients (Zhang et al. 2017), highlighting both global prevalence and disproportionate regional burden. Healing is often delayed by biofilm infections and rising antimicrobial resistance, limiting treatment options. Nevertheless, mortality rates following LEA are comparable to those of several major cancers like breast and lung cancers (McDermott et al. 2023), carrying a 5-year mortality risk of 50%–70% (Armstrong et al. 2020). Patients with DFU, arterial ulcers, and venous leg ulcers typically suffer from underlying immunodeficiency, peripheral neuropathy, and vascular disease, which compromise the healing process and predispose them to chronic, recurrent infections that lead to LEA (Australian Journal of General Practice 2020). The rise of antibiotic- and multidrug-resistant (MDR) pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), further undermines treatment success and increases the urgency for innovative, non-antibiotic therapies (Schaper et al. 2024).

Current wound care strategies face significant limitations because of the reliance on painful and possibly damaging debridement, unsuccessful and improper dressings, systemic antibiotics, and antiseptic solutions (Sood et al. 2014, Jeffcoate et al. 2024). However, most antiseptics in clinical use are cytotoxic, impair wound healing, and are not recommended for chronic wounds (Wilkins and Unverdorben 2013, Norman et al. 2017). Despite this, silver-based dressings remain widely used in wound care and management due to the broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains, primarily through membrane disruption and oxidative damage (Obagi et al. 2019, Haidari et al. 2020, May et al. 2022, Yousefian et al. 2023). Preclinical and clinical studies report reduced microbial burden and improved healing outcomes, particularly in burn wounds (Davies et al. 2017, May et al. 2022). However, cytotoxicity concerns remain with prolonged use (AshaRani et al. 2009, Haidari et al. 2020), and silver-based dressings remain costly with environmental deposition concerns (Fahim et al. 2024). Polyhexamethylene biguanide (PHMB) is another antiseptic wound care solution, as a cationic polymer that disrupts negatively charged bacterial membrane integrity and penetrates membranes to disrupt bacterial DNA to kill a broad spectrum of microbes, whilst also showing some evidence of improved wound healing outcomes, but there is a lack of high quality, large RCT of PHMB (Worsley et al. 2019, Curtin University Wound Healing and Management Unit 2020).

Recently, a systematic review of topical antiseptics benzalkonium chloride, chlorhexidine, hydrogen peroxide, hypochlorous acid, sodium hypochlorite, octenidine, PHMB and povidone-iodine in chronic wound care was conducted (Geng et al. 2024). The therapeutic index (ratio comparing of minimum mammalian cytotoxicity concentration: minimum bactericidal concentration) of these topical antiseptics against human keratinocytes and fibroblasts were found to be generally low, meaning effective antimicrobial activity was generally cytotoxic. Among the antiseptics tested, the highest therapeutic index was achieved by hypochlorous acid against Escherichia coli, S. aureus, and Pseudomonas aeruginosa, while PHMB was greatest against MRSA, and povidone-iodine, sodium hypochlorite and benzalkonium chloride had the poorest therapeutic indices (Geng et al. 2024). The therapeutic index of plasma-activated water (PAW) is study-dependent and complex, since PAW can define a range of physical properties and chemical compositions that change over time, limiting comparisons to other topical antiseptics and is still a current avenue for research. Further, retrospective observational studies or clinical trials for direct comparisons between PAW and other topical antiseptics are lacking. These limitations, together with the rise of MDR pathogens and biofilm-mediated tolerance, highlight the need for alternative strategies such as PAW that combine antimicrobial efficacy with tissue-regenerative capacity. Emerging antimicrobial modalities such as antimicrobial photodynamic therapy and antimicrobial peptides are also being explored for chronic wound infection, though direct comparative data against PAW are similarly lacking (Murthy et al. 2026).

PAW made from cold plasma (CP) technology, presents a promising next-generation therapy capable of overcoming several challenges associated with antibiotics and alternative therapeutics. For instance, PAW generates a mixture of reactive oxygen and nitrogen species (RONS) that exert potent antimicrobial and antibiofilm effects. Moreover, PAW has low cytotoxicity and minimal risk of promoting resistance, highlighting its potential as an effective adjunct therapy.

Cold plasma technology

CP, also known as non-thermal or atmospheric pressure plasma, is a rapidly evolving technology that uses ionized gases for diverse applications in healthcare, materials science, agriculture, and electronics (Tanaka et al. 2021, Okyere et al. 2022, Reema et al. 2022, Rao et al. 2023, Kolbe et al. 2024). CP is produced by ionizing gases with strong electric fields, resulting in plasma below 40°C being generated (Laroussi 2020, Tabares and Junkar 2021). The non-thermal nature makes CP safe for direct application to biological tissues for several minutes without causing heat damage (Woedtke et al. 2020). CP devices typically contain electrodes and a power source that generate high-voltage fields to ionize gases such as air, O₂, N₂, He, or Ar (Starikovskiy 2015, Nwabor et al. 2022). The resulting plasma is a complex mixture of radicals, free electrons, ultraviolet radiation, charged ions, and excited molecules (Woedtke et al. 2020). This unique composition confers broad antimicrobial activity, the ability to regulate inflammation, and stimulation of epithelial tissue growth (Gan et al. 2021, Abdo et al. 2022, Dubey et al. 2022). Several CP devices are now clinically approved in Europe for wound therapy and oncology (Stratmann et al. 2020, Faramarzi et al. 2021, Min et al. 2022, Nguyen et al. 2022, Strohal et al. 2022, Abu Rached et al. 2023, Bolgeo et al. 2023, Sabrin et al. 2024, Apelqvist et al. 2024).

PAW is a promising derivative that extends the application of CP. When CP is discharged into water, it produces PAW, an acidic and oxidative liquid enriched with RONS. PAW has demonstrated potent antimicrobial, antibiofilm, anti-cancer, and wound-healing properties (Kim and Kim 2021, Woedtke et al. 2025, Long et al. 2026). Its liquid form offers practical advantages over direct CP, such as easier storage, transport, and integration into conventional wound cleansing protocols. In preclinical studies, PAW has been shown to eradicate biofilms, including those formed by MDR pathogens like MRSA, while also promoting cellular proliferation, immune modulation, and tissue regeneration (Kaushik et al. 2018, Xu et al. 2020, Kim and Kim 2021, Mai-Prochnow et al. 2021, Abdo et al. 2022, Oliveira et al. 2022, Abdo et al. 2023, Lee et al. 2023, Vyas et al. 2023, Abdo et al. 2025). Despite these advances, the clinical efficacy of PAW remains untested in humans, and further research is required to optimize generation methods, dosing, and safety protocols (Cheng et al. 2020, Li et al. 2022, Woedtke et al. 2022). Notably, the world’s first Phase I clinical trial of PAW for diabetes-related foot ulcers (PAW-DFU I; Australian New Zealand Clinical Trials Registry: ACTRN12625000902493) has recently commenced at The Queen Elizabeth Hospital, Adelaide, Australia, with the trial protocol now published, marking a defining translational milestone for this emerging therapy (Abdo et al. 2026).

This review summarizes current evidence and future directions for PAW in chronic wound management, particularly in regard to DFU/DFI, highlighting antimicrobial and antibiofilm efficacy, effects on wound healing, and limitations to be addressed for clinical translation.

Plasma-activated water generation and physicochemical properties

Generally, CP is generated by applying an electric field to a gas, such as air, N₂, O₂, He or Ar, or a pure gas spiked with O₂ or air, at atmospheric pressure (Tendero et al. 2006, Woedtke et al. 2022). The specific method is dependent on the desired RONS formed, the application requirements, and device design (Hadinoto et al. 2023). As such, CP devices may adjust parameters such as voltage, frequency, operating gas, gas flow rate, and treatment duration to optimize plasma generation and treatment outcomes (Laroque et al. 2022). CP generates RONS in PAW, including short-lived species like hydroxyl radicals (OH^•), superoxide (O₂^•-), nitric oxide (NO^•), singlet oxygen (¹O₂) and peroxynitrite/peroxynitrous acid (ONOO^-/ONOOH), and long-lived species like hydrogen peroxide (H₂O₂), ozone (O₃) and nitrite/nitrate ions (NO₂^-/NO₃^-) which influence the pH, conductivity and oxidation-reduction potential of PAW (Chauvin et al. 2017, Jager et al. 2017, Rahman et al. 2022, Borkar et al. 2023, Pandey et al. 2023). However, spiking noble gases with other molecular gases (e.g. O₂, N₂, air) can result in increased production of ROS compared with RNS (Lietz and Kushner 2018). Analogous to CP, the choice of method (i.e. direct, or indirect) for generating PAW also depends on factors such as the desired chemical properties and specific application requirements (Bradu et al. 2020, Mirpour et al. 2020, Jungbauer et al. 2021). In the direct method, CP is discharged straight into water, whereas the indirect method generates reactive species in the gas phase, which subsequently dissolve into the liquid or react at the gas-liquid interphase to create PAW (Milhan et al. 2022).

CP devices typically utilize one of several methods to generate plasma, including dielectric barrier discharge (DBD), corona discharge, plasma jet, gliding arc discharge, microwave plasma, and piezoelectric direct discharge (Dasan et al. 2017, Šimončicová et al. 2019, Korzec et al. 2021, Bae et al. 2022, Khan et al. 2023). Using DBD to generate PAW uses a high voltage alternating current, passes between two electrodes, separated by a dielectric barrier (Fig. 1a). During PAW production, water itself is dielectric and so acts as the dielectric barrier (Wang et al. 2022, Dhakal et al. 2023). Corona discharge involves applying a high-voltage electric field to a fine-tipped point of an electrode, creating an electrically conductive region of air around the electrode tip that becomes ionized and interacts with the water (Fig. 1b) (Korachi et al. 2010, Lazra et al. 2020). Plasma jets generate plasma using gas flowing through a nozzle with high-voltage electrodes and typically utilize one of the aforementioned CP generator designs to ionize a gas under high flow rate through a narrow channel to discharge CP into the liquid (Rupf et al. 2010, Bae et al. 2022). The plasma jet can be submerged or directed onto the surface of the water to create PAW (Fig. 1c). The gliding arc discharge method involves creating a high-voltage electric arc that slides along from the narrowest to the widest ends of a pair of diverging electrodes, ionizing the gas to plasma to create PAW (Fig. 1d) (Elaragi 2015, Marcinauskas et al. 2024). An operating gas can also be introduced into a microwave cavity resonator that produces microwave radiation to generate CP directly in contact with water or above the water surface (Fig. 1e) (Lebedev 2010, Mallick et al. 2020). The piezoelectric direct discharge uses a piezoelectric material to produce high voltage when mechanically stressed with pressure or vibration, creating an electric discharge that ionizes the surrounding gas or directly ionizes the water to form PAW (Fig. 1f) (Korzec et al. 2021).

Figure 1.

Schematic representation of different plasma generation systems used to produce plasma-activated water (PAW). (a) Dielectric barrier discharge (DBD), showing a high-voltage electrode and grounded electrode separated by dielectric barriers, with carrier gas introduced into the discharge region. (b) Corona discharge generated between a pointed high-voltage cathode towards the anode electrode. (c) Atmospheric-pressure plasma jet system, in which gas passes through the plasma-generating region and the resulting plasma plume interacts directly with deionized water. (d) Gliding arc discharge, where plasma is generated between diverging electrodes in the presence of a flowing gas. (e) Electromagnetic-field-based plasma generation using microwave excitation, with plasma generated within the chamber beneath a quartz plate. (f) Piezoelectric direct discharge, in which gas flow around an insulated needle electrode facilitates plasma formation and produces a plasma plume at the outlet. Created in BioRender (https://BioRender.com). Abbreviations: AC; alternating current, DC; direct current.

A recent systematic review and meta-analysis have comprehensively compared the five most common PAW production methods (plasma jet, DBD, surface DBD, corona, and gliding arc) on RONS concentrations and physical properties (Montalbetti et al. 2024). Synthesis of the 358 studies from 2017–2023 found that RONS concentration tends to be higher in pure water rather than tap water, and plasma jets produced the greatest median concentration of RONS (measured by NO₂^-, NO₃^-, and H₂O₂) compared to DBD, gliding arc, corona and other discharges. Although plasma jet and DBD were the most energy efficient compared to gliding arc and corona discharges, the overall efficiency was still low, between 0.01 and 1 mol RONS/kWh (Montalbetti et al. 2024). This has significant implications in terms of plant design, energy sourcing, and scalability of PAW production that is greater than lab-scale, with current “large scale” production being described as between 5 and 20 L but having lower activity than smaller volumes (Rathore et al. 2024, Ferreyra et al. 2025). However, this systematic review could not compare the five common PAW generation methods to less common ones (e.g., piezoelectric), due to lack of data, presenting further avenues for research. Given that different CP designs yield PAW with distinct physicochemical properties, which precludes the establishment of standardized protocols, including acceptable thresholds for batch consistency, and the lack of large-scale production and clinical trials of PAW, determining which PAW generation method is most advantageous, scalable and efficacious for clinical wound management is yet to be determined. Production-scale and engineering considerations for PAW have been addressed in detail elsewhere (Long et al. 2026), and clinical evaluation of PAW in DFU management is now underway (Abdo et al. 2026), though a dedicated regulatory pathway for PAW as a therapeutic agent remains to be established.

Depending on the PAW generation conditions, different physicochemical characteristics can be observed. The RONS composition and concentrations in PAW depend on the CP design, gas feed composition, gas flow rate, power, voltage, exposure time and liquid volume (Rotondo et al. 2025). RONS reduces the pH and increases the oxidation-reduction potential and conductivity of PAW (Zhao et al. 2020), which combined make PAW a potent antimicrobial and oxidative agent (Guo et al. 2022). Once generated, PAW can be used immediately or stored prior to application, with stability depending on factors such as storage temperature, exposure to light, degree of CP activation and the intended end-use (Shen et al. 2016, Risa et al. 2019, Smet et al. 2019, Tsoukou et al. 2020).

Freshly generated PAW contains a heterogeneous mixture of predominantly short-lived RONS with markedly different biological activities. Antimicrobial efficacy is thought to arise from the synergistic interactions among these species rather than from any single stable component (Oehmigen et al. 2010, Machala et al. 2013, Zhou et al. 2018, Rotondo et al. 2025). Short-lived species, like OH^•, O₂^•-, NO^{•, 1}O₂, and ONOO^- are associated mainly with freshly generated PAW and can decay within microseconds to minutes, whereas long-lived species like H₂O₂, O₃, and NO₂^-/NO₃^- may persist for hours to days and increasingly define the chemistry of stored PAW (Lukes et al. 2014, Shen et al. 2016, Zhou et al. 2018, Kutasi et al. 2019, Dou et al. 2025, Lunder et al. 2026). This meaning fresh and stored PAW should not be treated as chemically equivalent materials and the efficacy of fresh PAW cannot be extrapolated to its aged counterpart untested. Therefore, storage has important biological consequences. While stored PAW does retain antimicrobial activity, it may exhibit reduced potency and a narrower antimicrobial spectrum than freshly generated PAW, particularly against micro-organisms possessing robust antioxidant defence systems such as catalase and peroxidase, including S. aureus and Candida spp. (Dong et al. 2022a,b, Krewing et al. 2019, He et al. 2025). Reactive-species decay is strongly temperature-dependent, generally following Arrhenius kinetics; refrigeration or freezing improves retention of longer-lived species and preserves antibacterial activity, both of which are central to defining PAW shelf life, preserved antimicrobial efficacy, and the realistic translational deployment of PAW-based therapeutics (Ikawa et al. 2016, Shen et al. 2016, Tsoukou et al. 2020, Cheng et al. 2020, Wang and Salvi 2021, Volkov et al. 2021, Wang et al. 2024, Dou et al. 2025).

General applications of plasma-activated water

PAW can be typically applied for medical surface or equipment disinfection and sanitization (Park et al. 2017, Bălan et al. 2018, Sakudo et al. 2019, Soni et al. 2021), as demonstrated by the potent antimicrobial/antibiofilm activity against a wide range of infectious pathogens (Darmanin et al. 2020, Lin et al. 2020, Lee et al. 2022, Rahman et al. 2022, Abdo et al. 2023, Abdo et al. 2025). The mechanism of action involves disrupting microbial cell membranes, inhibiting enzyme activity, impairing QS, preventing or disrupting biofilm formation, and inducing oxidative stress, which together lead to microbial inactivation or cell death (Zhang et al. 2013, Royintarat et al. 2019, Zhang et al. 2024). PAW also shows promise as a disinfectant for medical equipment (e.g. endoscopes, dental waterlines) and surfaces (Abuzairi et al. 2018, Bălan et al. 2018, Noopan et al. 2019, Lee et al. 2023). It may also be useful for oral hygiene (Milhan et al. 2022, Qiao et al. 2022), and disinfection and irrigation of wounds (Busco et al. 2020, Xu et al. 2020, Wang et al. 2021, Fink et al. 2023).

The prospect to combine antimicrobial properties, immune activation and tissue regeneration potencies of PAW may enhance wound healing outcomes. This makes PAW a potentially valuable preliminary treatment or adjuvant to the current therapeutic arsenal for DFU. There is ongoing in vitro and in vivo research that continue to refine PAW generation, application, and efficacy by exploring the optimal chemical conditions against biofilm-infected wounds, and long-term safety and effectiveness in wound care.

Plasma-activated water antibiofilm efficacy in diabetes-related foot infections

A major challenge in DFI management is the increasing prevalence of MDR pathogens and biofilm-associated infections (Scalise et al. 2015, López-Montesinos et al. 2020, Wozniak et al. 2022). Biofilms protect micro-organisms from host immune responses and antimicrobial therapies, contributing to chronic infection, delayed healing, and treatment failure (Hussain et al. 1993, Costerton et al. 1995, Munita and Arias 2016, Kragh and Richter 2022). Although debridement remains the cornerstone of biofilm management, residual micro-organisms can rapidly recolonize the wound and re-establish biofilms within days (Wolcott et al. 2010, Schwartz et al. 2014, Tian et al. 2023). Combined with patient comorbidities and impaired tissue repair, these factors highlight the need for novel therapeutic approaches that can simultaneously control infection, disrupt biofilms, and promote wound healing (Armstrong et al. 2017, Schaper et al. 2024).

PAW is garnering interest particularly in treating chronic and hard-to-heal wounds like DFU due to broad spectrum antimicrobial activity and the ability to enhance various aspects of the wound healing process (Shen et al. 2016, Wang et al. 2021, Lee et al. 2023, Abdo et al. 2025). RONS produced in PAW have a diverse role in disrupting biofilm formation to help prevent and treat wound infection (Fig. 2). The composition of PAW has been reported in various studies to impede biofilms of several micro-organisms in vitro (e.g. E. coli, P. aeruginosa, and MRSA) that are relevant to chronic wound infections like DFU (Pereira et al. 2017, Wu et al. 2019, Su et al. 2022). This reactive species ‘cocktail’ in PAW can disrupt biofilms by penetrating the biofilm matrix and inducing oxidative stress in microbial cells, resulting in damage to cell membranes, proteins, and DNA and eventually cell death (Charoux et al. 2020, Fasnacht and Polacek 2021, Xia et al. 2025). Some studies also demonstrated that CP solutions can disrupt early stages of biofilm formation, by downregulating genes involved in bacterial motility and adherence (Joshi et al. 2010, 2015, Han et al. 2016). This broad-spectrum activity of PAW is also beneficial in treating polymicrobial biofilms since they can interrupt QS between microbial cells (Cáp et al. 2012, Ali et al. 2023).

Figure 2.

Application of plasma-activated water (PAW) in wound healing. PAW treatment exposes microbes to reactive oxygen and nitrogen species, that have antibiofilm and antimicrobial effects, interrupting quorum sensing, decreasing pH, encouraging angiogenesis, and killing planktonic cells to prevent new colonization of wounds. By chemically disrupting biofilms, PAW may help reduce the reliance on antibiotics. Direct application of PAW to the wound site, such as soaking or dressing wounds with PAW, may diminish and ultimately eliminate microbial biofilms to maintain a biofilm-free environment. Created in BioRender (https://BioRender.com).

Moreover, the interplay between RONS and water can result in the generation of acids and oxidants, which are responsible for the pH decrease of water (Lukes et al. 2014, Tibbits et al. 2022). The physiological pH of the human epidermis is slightly acidic (pH ∼5) and gradually becomes neutral (pH 6.8) towards the outermost stratum corneum (Fluhr and Elias 2002). Contrastingly in a wound, the pH becomes more alkaline as tissue damage removes the acidic microenvironment and exposes the alkali underlying tissue and blood that is physiological pH of 7.4 (Schneider et al. 2007). Wound pH depends mainly on the type of wound, but has been reported at pH 7.5–9 which is favourable for bacterial growth, and wounds that develop infection are shown to increase in alkalinity (Power et al. 2017). Furthermore, better healing outcomes has been associated with lower pH, which could be due to lower pH promoting healing directly, formation of the new skin barrier restoring pH homeostasis, or both (Power et al. 2017, Rembe et al. 2026). As such, PAW has low pH that would promote an acidic environment to aid the healing process and inhibit many skin pathogenic microbes (Qi et al. 2018, Wallace et al. 2019, Derwin et al. 2022, Sim et al. 2022). Yet, there is still contention around a causative role of pH in wound healing due to lack of high-quality evidence and requires further study. Despite its acidity, PAW can be made safe for application on the skin (Wang et al. 2021, Abdo et al. 2025).

Cellular effects of plasma-activated water-derived reactive species

Physiological RONS have a direct active role in promoting cell survival, proliferation and migration of keratinocytes to promote wound healing (Abdo and Kopecki 2024). In addition to antibiofilm properties, PAW-derived RONS play crucial roles in cellular signalling pathways, influencing processes such as cell migration, proliferation, self-killing (apoptosis), self-eating (autophagy), and release of growth factors and both pro- and anti-inflammatory cytokines/chemokines (Hsieh and Yang 2013, Redza-Dutordoir and Averill-Bates 2016, Checa and Aran 2020, Zarkovic 2020). RONS can enhance collagen synthesis, and the proliferation and migration of keratinocytes and fibroblasts involved in tissue repair/regeneration (Wende et al. 2014, Schmidt et al. 2017, Bhartiya et al. 2021, Khorsandi et al. 2022). The skin defends itself against redox-active elements (e.g. ultraviolet, ionizing radiation) that can induce DNA damage directly or stimulate intracellular reactive species generation. However, supratherapeutic CP exposure could modify or destroy the structure and function of lipids, proteins and DNA (Xu et al. 2017). As a result, downstream signalling cascades are triggered, leading to altered cytokine release and aggravating inflammation that can impede wound healing (Xu et al. 2017, Khorsandi et al. 2022). For instance, chronic wounds like DFU exhibit impaired phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling, an important, redox-sensitive intracellular signalling pathway with diverse regulatory functions in cell survival and angiogenesis (Kasowanjete et al. 2024), resulting in reduced cell survival and growth factor release that would otherwise stimulate wound healing (Jere et al. 2019). Plasma-activated media (cell media exposed to CP with similar chemical properties to PAW) was shown to promote PI3K/Akt/mTOR cell signalling in keratinocytes in vitro (Wu et al. 2023), while direct CP exposure promoted Akt activity in vivo (Zhu et al. 2025).

Other cell signalling pathways, such as the nuclear factor erythroid-2 related factor 2 (Nrf2), mitogen-activated protein kinase and the unfolded protein response pathways could also play a role in wound healing stimulated by PAW, as has been the case with direct CP therapy [recently reviewed in (Abdo and Kopecki 2024)]. Particularly, the redox-sensitive Nrf2 and unfolded protein response have been shown to be the most enriched signalling pathways induced in epithelial cells in response to CP exposure, promoting survival by inhibiting apoptosis (Scharf et al. 2019). These pro-survival pathways in epithelial cells could help build an adaptive response to oxidative stress in the chronic wounds and possibly transition them to acute wounds that heal. Unfortunately, a similar analysis of cell signalling pathways involved in PAW therapy for wound healing is yet to be reported and is an open avenue for research.

Mechanistically, the molecular pathways underlying PAW-mediated wound repair remain incompletely understood, and much of the current mechanistic framework is inferred from applications of CP rather than PAW (Schmidt and Bekeschus 2018, Schmidt et al. 2019, Wang et al. 2021, Rajić et al. 2025). In direct CP models, therapeutic exposure of CP-derived RONS activate redox-sensitive signalling pathways, particularly the Nrf2/ARE axis, resulting in increased expression of antioxidant and cytoprotective genes, including HMOX1, NQO1, CAT, TRX, and glutathione-related enzymes (Schmidt et al. 2019, 2026). CP has also been shown to modulate inflammatory signalling, stimulate fibroblast proliferation and migration, promote angiogenesis through increased expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, keratinocyte growth factor, and CD31, and regulate transforming growth factor β (TGF-β)-mediated extracellular matrix remodelling and collagen maturation (Arndt et al. 2013, Schmidt et al. 2019, He et al. 2020, Schmidt et al. 2021, Cheng et al. 2026). In contrast, direct mechanistic evidence for PAW remains comparatively limited, demonstrating improved wound-healing outcomes, including reduced bacterial burden, attenuation of inflammation and oxidative stress, enhanced re-epithelialization, increased collagen deposition, elevated VEGF/CD31 expression, and favourable modulation of extracellular matrix remodelling, including reductions in matrix metalloproteainase-9 (MMP-9) (Wang et al. 2021, Rajić et al. 2025, Abdo et al. 2025, Wei et al. 2026).

This distinction is biologically important because direct CP and PAW differ fundamentally in mode of delivery and physicochemical properties. Direct CP exposes tissues to short-lived gaseous RONS together with electric fields, ultraviolet radiation and charged particles, whereas PAW delivers predominantly longer-lived aqueous RONS like H₂O₂, HNO₂/NO₂^- and HNO₃/NO₃^- (Haertel et al. 2014, Duchesne et al. 2019, Bekeschus et al. 2021, Kim and Kim 2021, Li et al. 2023). Consequently, although PAW is likely to engage overlapping redox-sensitive and wound-repair pathways, these effects probably occur indirectly through modulation of the wound microenvironment and with different kinetics and exposure dynamics than direct CP. Therefore, while CP studies provide valuable mechanistic insight, proposed activation of Nrf2, TGF-β, and MMP-regulated signalling by PAW are hypothesized for future mechanistic investigation.

Wound healing studies of plasma-activated water

To date, only a few studies have explored the general application of PAW produced with various CP designs in wound healing and the treatment of wound infections (Xu et al. 2020, Wang et al. 2021, Lee et al. 2023, Abdo et al. 2025, Rajić et al. 2025), which are summarized in Table 1. In a study by Xu et al. (2020), PAW eradicated common wound infection pathogens P. aeruginosa, E. coli, S. aureus, and Salmonella Paratyphi-B in vitro and significantly accelerated wound healing compared to the vehicle control group in a mouse model with full-thickness skin wounds. Wang and colleagues also tested wound healing potency in a mouse model with PAW compared to vehicle control (water) and medical alcohol-treated groups (Wang et al. 2021). PAW significantly accelerated wound closure as compared to both the alcohol-treated and control groups, reaching wound closure ∼4–5 days faster than the control and comparison groups (Wang et al. 2021). This was linked to PAW suppressing the expression of pro-inflammatory interleukin-1β (IL-1β), IL-6 and promoting anti-inflammatory IL-10 cytokines and VEGF, leading to faster blood vessel formation and wound recovery (Wang et al. 2021).

Table 1.

Overview of PAW as an antibiofilm and/or antimicrobial agent in in vivo wound healing studies.

PAW experiment	Treatment groups (n*)	Outcome	Ref
Pseudomonas aeruginosa, E. coli, S. aureus and S. paratyphi-B killing in vitro. Mouse full-thickness skin wounds with P. aeruginosa infection. 10 mL treatment applied on days 0, 4, 7 and 10.	vehicle control (5) PAW (5)	PAW effectively inactivated all micro-organisms in vitro. Compared to control group, PAW group had no pus after 7 days and healed faster (cf. 17 vs 22 days), with significant reduction in inflammatory cell infiltration.	(Xu et al. 2020)
Mouse full-thickness skin wound model. Treatment applied on days 0, 1, 2, 4 and 8.	vehicle control (10) PAW (10) Medical alcohol (10)	Time to complete healing faster in PAW compared to alcohol and control groups (cf. 12, 16, and 17 days, respectively). PAW inhibited IL-1β and IL-6 release over 5 days. Anti-inflammatory IL-10 release was initially higher over first 2 days. 1.8–2-fold proangiogenic VEGF expression in PAW vs. vehicle.	(Wang et al. 2021)
Keratinocytes exposed to 25% PAW in growth media in vitro. Full-thickness skin wounds on backs of rats. Treatment applied for 15 min every two days for 14 days.	PBS-soaked gauze (10) PAW-soaked gauze (10)	PAW promoted keratinocyte migration: increased extracellular NOX3, ROS, ECM integrin β1, and MMP2 expression, and phosphorylation of FAK and paxillin. PAW group significantly increased NOX3, integrin β1, p-FAK and paxillin expression in wounds. Maximum wound closure reached faster with PAW compared PBS group (cf. 11 vs 14 days).	(Lee et al. 2023)
Full-thickness excisional wounds in non-diabetes and streptozotocin-induced diabetes rat model. Treatment applied twice/day over 15 days.	ND + vehicle spray (3) ND + PAW spray (3) D + vehicle spray (3) D + PAW spray (3)	Wound closure significantly faster in PAW group compared to vehicle in D mice. No difference between treatments in ND mice. WBC infiltration, IL-1β, IL-6, TNF-α, and MMP9 mRNA significantly lower in PAW vs vehicle group in D mice. No difference between treatments in ND mice.	Rajić et al. 2025
Balb/c mice with bioluminescent MRSA-infected burn wounds. Treatment applied twice-daily for 7 days.	Uninfected control (17) infected + vehicle (17) infected + PAW (18)	Biofilm infection significantly reduced from day 4–7 (bioluminescence), confirmed by a 1.33 log10(CFU g-1) reduction by day 8 compared to vehicle. Histological analysis of wound re-epithelialization was significantly improved in PAW (63.2%) compared to vehicle (49.2%).	(Abdo et al. 2025)

Abbreviations: IL; interleukin, FAK; focal adhesion kinase, MMP; matrix metalloproteinase, TNF; tumour necrosis factor, ECM; extracellular matrix, VEGF; vascular endothelial growth factor, NOX; NADPH oxidase, ROS; reactive oxygen species, ND; non-diabetes, D; diabetes. WBC: white blood cell.

^* In vivo sample size within each group.

PAW as a pre-treatment for biofilm infected wounds in vitro demonstrated that PAW can significantly enhance the effectiveness of topical antiseptics povidone-iodine, PHMB and medical-grade manuka honey, against E. coli biofilms (Vyas et al. 2023), which are notoriously difficult to eradicate in chronic wounds. PAW alone exhibited bactericidal activity on both plastic and keratinocyte monolayers but demonstrated more biofilm-eradicating power when combined with the other antiseptics, reducing the effective biofilm-killing concentration of these agents. Additionally, PAW exposure caused membrane blebbing and flattening; morphological changes that indicate severe membrane disruption (Vyas et al. 2023).

Another study assessed the effects of PAW produced by a microwave-generated low-pressure plasma system on skin cell migration and wound healing in vitro and in vivo (Lee et al. 2023). PAW treatment enhanced keratinocyte and fibroblast migration without harming cell viability, while increasing MMP-2 activity involved in tissue remodelling in vitro. PAW also boosted integrin β-subunits expression, and phosphorylation of FAK and paxillin to promote cytoskeletal reorganization and cell migration of the skin cells (Lee et al. 2023). These cellular responses to PAW were completely ablated by preloading cells with the ROS-scavenger N-acetylcysteine, indicating that intracellular ROS generated by mitochondria and NADPH oxidase were involved (Lee et al. 2023). In a similar study, normal Wistar rats and rats with streptozotocin-induced diabetes with full thickness incisional wounds were treated with PAW or water spray twice-daily over 15 days (Rajić et al. 2025). PAW treatment moderately accelerated wound healing among the diabetes group (though not the non-diabetes group), but also inflammatory cell infiltration and gene expression of IL-1β, IL-6, tumour necrosis factor-α and MMP-9 were significantly suppressed by PAW treatment (Rajić et al. 2025).

Another study has investigated both the antimicrobial activity and wound healing effects of PAW in a wound infection model in vivo (Abdo et al. 2025). PAW significantly reduced bacterial infection from day 4–7, with biofilm bacteria count at day 8 finding a 1.33 log reduction in viable bacteria compared to vehicle treatment (Abdo et al. 2025). Macroscopic wound healing rates were similar between PAW and control groups, however, histological analysis showed significantly greater re-epithelialization in PAW-treated wounds compared to vehicle (Abdo et al. 2025). In contrast, most antiseptic solutions currently in clinical use impair healing and are therefore not recommended for chronic wounds (Wilkins and Unverdorben 2013, Norman et al. 2017), highlighting PAW’s potential advantages over conventional antiseptics.

Overall, the studies above conclude that PAW is a promising, effective, and safe approach for promoting wound healing, with no reported side effects and a lower risk of driving antimicrobial resistance. However, of the five studies, only one study (Abdo et al. 2025) described any a priori sample size analysis and statistical power, assessment of statistical test assumptions (e.g. data and residual distribution, homoscedasticity), and reported the results quantitatively as well as qualitatively in the text. Consequently, most of the animal studies were underpowered, and hence the overall quality of animal in vivo evidence for the efficacy of PAW in wound healing remains low and more appropriately powered studies are needed. Further, highly controlled animal study conditions do not reflect wound complexity, heterogeneity, and microenvironment; these wounds are in fact acute and there is still no validated animal model of chronic wounds. Additionally, chronic wound infections including in DFI are typically polymicrobial, whereas animal studies have only investigated wounds that are non-infected or inoculated with a single species. Given the lack of chronic wound models and apparent tolerability and effectiveness in acute models in vivo, PAW efficacy in treating chronic wounds like DFU/DFI need to be clinically trialled.

Cytocompatibility and therapeutic window of plasma-activated water

Despite PAW often being described as having low cytotoxicity, its safety profile should be interpreted cautiously because the biological effects of RONS are inherently dose-dependent (Kim and Kim 2021). The therapeutic window between antimicrobial efficacy and therapeutic stimulation (or at least preservation) of new granulation tissue and epithelialization is likely narrow and is determined primarily by the physicochemical composition of the formulation (Kim and Kim 2021, Tsoukou et al. 2022). Plasma operating parameters, including feed gas composition, power input, activation duration, plasma–liquid interaction conditions and storage influence the concentrations and relative abundance of RONS (Machala et al. 2018, Kim and Kim 2021, Rathore and Nema 2022). Air-based plasma systems generally produce stronger acidification and higher amounts of nitrogen-oxides, whereas argon- and helium-based systems generate distinct ROS/RNS profiles, leading to formulation-specific differences in antimicrobial activity and biological response (Machala et al. 2018, Ghimire et al. 2021, Rathore and Nema 2022, Lin et al. 2023). Similarly, prolonged plasma activation increases oxidant loading and lowers pH, which can enhance antimicrobial potency, but sacrifices tissue tolerability (Tsoukou et al. 2022, Ferreira et al. 2026).

Current evidence suggests that PAW can achieve strong antimicrobial activity while remaining within accepted mammalian cell biocompatibility limits. For example, air DBD microbubble-generated PAW activated for 14 min inhibited 99.15% of S. aureus, 99.68% of E. coli, and 97.45% of Candida albicans, while maintaining 86.54% L929 fibroblast viability, above the ISO 10993–5 cytocompatibility threshold of 70% (Ferreira et al. 2026). Likewise, a neutral-pH PAW formulation was reported to remain non-cytotoxic to L929 fibroblasts up to 20% concentration and, at that concentration, reduced bacterial burden and improved wound closure in an infected murine wound model (Mukherjee et al. 2025. However, relatively small changes in solvent chemistry can markedly reduce host-cell tolerance. In plasma-activated saline, strong antimicrobial effects were accompanied by pronounced cytotoxicity toward HaCaT keratinocytes, with viability largely preserved only after diluting PAW to 40%–50% (Tsoukou et al. 2020).

The selective antimicrobial effects of PAW are also commonly attributed to differences in oxidative-stress tolerance between microbial and mammalian cells. Keratinocytes and fibroblasts can mount antioxidant and cytoprotective responses, including catalase-, glutathione-, and Nrf2-associated systems to bolster intracellular antioxidant capacity, whereas bacterial cells are generally more vulnerable to oxidative damage (Schmidt and Bekeschus 2018, Schmidt et al. 2019). Despite this, plasma-based antimicrobials are considered less prone to inducing resistance than conventional antibiotics because they deliver a complex mixture of RONS that target multiple cellular components simultaneously, intrinsic bacterial defence mechanisms may still influence susceptibility to treatment (Krewing et al. 2019). Krewing et al. identified 87 E. coli mutants that were hypersensitive to CP and demonstrated that several oxidative stress-related genes, including cysB, mntH, rep, and iscS, contribute to bacterial tolerance by protecting against H₂O₂, O₂^•- and NO^• (Krewing et al. 2019), hence these bacterial defence pathways may likewise influence the antimicrobial efficacy of PAW. However, there is currently no evidence that repeated PAW exposure selects for stable clinical resistance, and future studies investigating bacterial adaptive responses and the potential for PAW tolerance development, particularly in chronic wound biofilms are warranted.

Nevertheless, no universal safety threshold exists for PAW formulations, particularly in chronic or diabetic wounds containing fragile granulation tissue (Kim and Kim 2021, Tsoukou et al. 2022). PAW should therefore be regarded as a formulation-dependent therapeutic in which pH, buffer capacity, carrier liquid, reactive-species composition, and activation parameters must be optimized to balance antimicrobial and anti-biofilm efficacy against preservation of fibroblast and keratinocyte viability (Machala et al. 2018, Kim and Kim 2021, Tsoukou et al. 2022, Ferreira et al. 2026). Future clinical trials should be designed as dose-finding studies in the phase I/II stages to determine clinically useful efficacy and safety thresholds for wound-care applications (Kim and Kim 2021, Ferreira et al. 2026).

Considerations for integrating plasma-activated water into wound management protocol

The International Working Group on Diabetic Foot (IWGDF) publishes evidence-based guidelines for the diagnosis and treatment of DFI (Schaper et al. 2024, Senneville et al. 2024). In these current guidelines (updated 2023), prophylactic use of systemic or local antibiotics, topical antiseptics (including silver, honey, bacteriophages or negative pressure wound therapy), and topical application of antibiotics at the site of infection is not recommended (Recommendation 11, 23, and 24). Therefore, there are limited non-surgical strategies recommended to perform to complement the surgical debridement of wounds. The overarching rationale of these recommendations is based on low quality of evidence, high risk of bias, and that most topical treatments either do not benefit or impede wound healing (Senneville et al. 2024). As a result, the IWGDF conclude that a key area of concern for the management of wound healing is the potential of topical antimicrobial administration to limit the use of systemic antibiotics (Senneville et al. 2024).

PAW could benefit patients with DFU both therapeutically and prophylactically, helping reduce reliance on systemic antibiotics and addressing the IWGDF’s concerns regarding antibiotic-related adverse effects, cost, and resistance propagation. This clinical direction is now supported by our recently published phase I protocol evaluating PAW as a wound-rinse solution for DFU in two Australian hospitals (Abdo et al. 2026).

Currently, all evidence of PAW therapy for wound healing is in cell and animal models; the efficacy of PAW in treating DFI and in healing DFU has not been reported in clinical trials.

As mentioned, PAW contains a heterogeneous mixture of RONS that differ fundamentally in half-lives and antimicrobial mechanisms. Although PAW offers greater logistical flexibility than direct CP treatment, its translational scalability may be constrained by the time-dependent evolution of its reactive chemistry in certain clinical use cases.

On the other hand, practical implementation of PAW also faces challenges related to scalability, stability, shelf-life, and safety. Variability in the generation and composition of PAW can affect consistency and reliability. More research is needed to fully understand the mechanisms through which PAW exerts effects on wound healing (Konchekov et al. 2023, Abdo and Kopecki 2024). Although PAW is generally considered safe for human cells at certain exposure times or frequency, excessive levels of RONS can be cytotoxic and may damage healthy tissues if not carefully controlled (Balzer et al. 2015, Girard et al. 2016, Abdo and Kopecki 2024). Hence, wound treatment with PAW will be acute and intermittent.

Additionally, since PAW is a surface disinfectant, it may not be able to penetrate deeper tissues to clear deep tissues or bone infections. However, it may have bystander effects on surrounding tissues by regulating inflammatory responses which will help create a more favourable healing environment around the wound. Despite PAW treatment alone being likely limited in cases of advanced infection, the favourable safety profile of PAW also makes it useful as a prophylactic treatment in preventing infection without damaging wounded or surrounding healthy tissue. These observations have important implications for the clinical translation of PAW. Claims regarding its scalability should distinguish between immediate application, short-delay off-site use, and longer-term transportation and storage, as liquid portability alone does not guarantee preservation of antimicrobial efficacy (Shen et al. 2016, Wang et al. 2024). Consequently, storage conditions, shelf life, and intended clinical deployment should be clearly defined, and PAW may not be a stand-alone treatment; rather, it represents a valuable addition to the clinician’s toolbox, complementing rather than replacing established wound care and infection management strategies.

Chemically engineered water compared to PAW: Promise and limitations

Parenthetically, chemically engineered water (CEW) formulated from low pH, H₂O₂, NO₂^-, and NO₃^- is an important comparator to PAW, because it may reproduce a meaningful facsimile of PAW, and may offer advantages in cost, controlled acidification, batch reproducibility, and quality control (Oehmigen et al. 2010). Studies that experimentally simulated PAW with HCl or HNO₃ instead of CP activation had similar but lower bactericidal activity in some instances (Wang and Salvi 2021), and that pH and ORP are integral properties dictating the antimicrobial effect (Zhang et al. 2013, Tian et al. 2014).

PAW-mimicking formulations reproduce limited PAW-like activity and may not fully capture the complex redox chemistry and synergistic reactive species interactions characteristic of PAW (Oehmigen et al. 2010, Zhou et al. 2018, Wang et al. 2024). Oehmigen et al. demonstrated complete inactivation of E. coli and S. aureus in non-buffered liquids following indirect CP treatment, while HNO₃ alone failed to reproduce the same antimicrobial effect, showing that antimicrobial activity arises from the synergistic interaction between acidification and CP-derived ROS/RNS, rather than acidification alone (Oehmigen et al. 2010). Similarly, the antimicrobial activity of PAW against equivalent concentrations of H₂O₂, NO₂⁻, and NO₃⁻, with PAW achieving > 4-log reduction in E. coli, S. aureus, and P. aeruginosa (Zhou et al. 2018, Trebulová et al. 2025). However, none of the individual chemical controls produced comparable inactivation, with PAW efficacy attributed to the formation of ONOO^- under acidic conditions (Zhou et al. 2018), which supports similar E. coli-killing efficacy with PAW attributed to ONOO^-/ONOOH (Pogoda et al. 2026). Likewise, Wang et al. evaluated off-site generated PAW against MRSA and E. coli alongside conventional disinfectants and chemical controls, including 3% H₂O₂, 0.5% NaClO, 75% ethanol, 0.5% iodophor, and acidified HNO₃ (pH 2). PAW exhibited greater antimicrobial efficacy than both 3% H₂O₂ and 0.5% NaClO at equivalent contact times, whereas the acidified and O₃-related controls were considerably less effective, concluding that PAW potency was primarily driven by high-valence nitrogen oxides, rather than by acidity alone (Wang et al. 2024). Moreover, PAW compared to different CEW types were not equivalent in antimicrobial performance against planktonic Listeria monocytogenes or Hafnia alvei; while PAW treatment reached below detection limits in both studies (>5.8 log reductions), pH matched and unmatched CEWs of H₂O₂, NO₂^-, NO₃^- individually or in combination had mixed but lesser results (Naïtali et al. 2010, Fernández-Gómez et al. 2023).

In general, the more CEW chemically resembled PAW, the better it performed, with both studies showing that mixtures of H₂O₂, NO₂^- and NO₃^- pH- and concentration-matched to PAW were closest to reaching PAW performance for microbial reduction (Naïtali et al. 2010, Fernández-Gómez et al. 2023). However, there is no in vitro or in vivo evidence (refer to Table 1) for whether wound healing is significantly different between PAW and equivalent CEW. Thus, CEW may be a suitable application-specific alternative, but it should not be assumed to be universally equivalent to PAW. We recommend that future research explicitly benchmark PAW (fresh and stored) against matched CEW formulations to delineate which therapeutic applications genuinely require PAW versus which could be adequately served by a cheaper, better-characterized chemical equivalent for honest translational benchmarking and for defining where CP technology offers irreplaceable value.

Concluding remarks

Key priorities for advancing PAW toward clinical adoption include the standardization of generation methods and RONS characterization to enable reproducible, dose-controlled treatment protocols across clinical sites. Validated, point-of-care physicochemical metrics, particularly pH, H₂O₂ concentration, and oxidation-reduction potential, will be essential for ensuring batch-to-batch consistency and regulatory compliance. Delivery format optimization, including PAW-impregnated dressings and controlled-release formulations, represents a practical avenue to integrate PAW seamlessly into existing wound care pathways.

Beyond DFI, the dual antimicrobial and tissue-regenerative profile of PAW warrants investigation in other chronic wound contexts where biofilm infection and impaired healing converge. Long-term safety data, particularly regarding repeated topical exposure and systemic RONS absorption, will be critical for regulatory submission. Addressing these gaps will determine whether PAW fulfils its potential as a low-cost, resistance-refractory, next-generation therapeutic for the growing global burden of chronic wound infections. The recent commencement of a Phase I clinical trial of PAW for diabetes-related foot ulcers (ACTRN12625000902493), now formalized in a published trial protocol (Abdo et al. 2026), marks an important first step in this translational pathway, offering an immediate bridge from the preclinical evidence summarized here to rigorous efficacy testing in patients with diabetes-related foot ulcers.

Author contributions

Angela Boahen (Conceptualization, Visualization, Original manuscript draft), Adrian Issa Abdo (Conceptualization, Original manuscript draft, Supervision, Manuscript review), Neil McMillan (Manuscript review), Guilherme Pena (Manuscript review), and Katharina Richter (Conceptualization, Supervision, Manuscript review). All authors read and agreed to the final version of the manuscript.

Conflicts of interest

K.R. is a non-financial board member of RIBU Plasma Pty. Ltd., Australia, and inventor on patent (Buske, C., & Richter, K. (2024). Process, apparatus and use of an apparatus for producing a plasma-activated liquid. Patent No. WO2024013069. World Intellectual Property Organisation). All other authors declare no conflicts of interest.

Funding

This work was supported by Adelaide University, Australia; the Medical Research Future Fund, Department of Health and Aged Care, Australia, Early to Mid-Career Researchers Grant [grant number 2022/MRF2023153]; the Hospital Research Foundation Group, Australia, BHI TQEH Supplementary Scholarship [grant number 2025-CS-BHI-300-86100]; and the Australian Centre for Accelerating Diabetes Innovations, Australia, Health Tech Innovation Challenge [grant number 2025HTIC29].