Cold Atmospheric Plasma in Biomedicine and Pharmacology: Multidisciplinary Perspectives on Therapeutic Applications

Abstract

Plasma medicine has emerged as a rapidly emerging interdisciplinary area of research that investigates the interaction of cold atmospheric plasma (CAP) with biological systems. The therapeutic power of CAP relies on the controlled generation of reactive oxygen and nitrogen species (RONS), pulsed electric fields, and ultraviolet radiation, which synergistically enable specific bioactivity. This review summarizes CAP research from the past decade, including in vitro, in vivo, and clinical studies across biomedical applications, and categorizes CAP sources by their distinctive physicochemical properties and associated medical relevance. The discussion highlights that treatment efficacy in wound healing, cancer therapy, and antimicrobial applications is strongly device-dependent, underscoring the critical role of plasma source design in clinical outcomes. CAP demonstrates significant antimicrobial effects, reduces microbial load, and preserves the integrity of healthy tissue. It promotes hemostasis and accelerates wound healing through vascularization, cell proliferation, and microcirculation. In oncology, CAP exhibits selective antitumor effects, including skin cancers and other vertebrate tumor models. Its applications in dentistry reflect its versatility in disinfection and in aiding implantation. Furthermore, CAP can stimulate the growth of stem and cultured cells via nitric oxide-mediated mechanisms. Recent studies have highlighted the potential of CAP in surface modification and transdermal drug delivery. Furthermore, plasma-activated media and solutions have garnered significant interest due to their diverse applications in plasma medicine. The review provides a comprehensive overview of all available literature, facilitating biomedical scientists in their further translational research in this field.

Plasma medicine represents a fascinating and rapidly evolving frontier in healthcare, leveraging the distinctive properties of nonequilibrium atmospheric pressure plasma to effectively treat living tissues and cells. , This interdisciplinary field intricately weaves together the principles of plasma physics, the nuances of life sciences, and the practicalities of clinical medicine, all aimed at enhancing and innovating medical practices. − The generation of plasma and its study require the knowledge of physics, chemistry, and electrical engineering, where researchers delve into its intricate composition and behavior, and scrutinize how various technical parameters, such as pressure, temperature, and gas composition, affect its physical properties. , In the life sciences, preliminary investigations examine how plasma interacts with cells, tissues, and entire organisms, aiming to understand the biological implications of these interactions. ,, The medical dimension of plasma medicine focuses on the therapeutic potential of plasma in clinical settings, aiming to develop new and effective treatments for various conditions by harnessing plasma’s unique biological effects. ,

Historically, in the twentieth century, the medical applications of plasma were largely centered on thermal effects, primarily using high temperatures to sterilize medical instruments, excise tissue, and perform cauterization or tissue dissection with devices such as argon plasma coagulators. , However, a significant paradigm shift occurred as researchers transitioned from thermal to nonthermal plasma applications in the biomedical field. This shift was driven by the desire to achieve therapeutic outcomes while minimizing collateral damage to surrounding healthy tissues, as exemplified by advancements in plasma-mediated blood coagulation and sterilization processes. , A landmark moment in plasma medicine occurred in 1996, when scientists first demonstrated the use of atmospheric-pressure plasma to effectively sterilize microorganisms, showcasing its potential as a powerful, efficient sterilizing agent. , Recognizing this breakthrough, the Physics and Electronics Directorate of the U.S. Air Force Office of Scientific Research (AFOSR) became intrigued by the biological applications of this technology, envisioning its use in treating soldiers’ wounds and in sterilizing both living and nonliving surfaces. − In a notable advancement, Russian researchers embarked on both in vitro and in vivo experiments, coining the term “plasma dynamics therapy” to describe their innovative approach to wound treatment. − The modern era of plasma medicine began in 2002, when researchers led by Stoffels et al. reported that low-intensity plasma treatments could manipulate living cells without inducing necrosis or inflammation. This pivotal discovery marked a crucial milestone, validating the feasibility of therapeutic plasma use while safeguarding cell integrity. − The groundwork in low-temperature plasma has opened the door to the development of plasma medicine as a rapidly advancing, interdisciplinary field. However, nonequilibrium CAP technology offers a range of device configurations, operating principles, and emitted reactive species that directly influence its medical applications of CAP. This review paper seeks to illuminate the various CAP sources, classified into specific categories based on their unique physicochemical properties, and how they influence the medical applications of CAP. The discussion of the various CAP sources in the treatment of wound healing, cancer, and antimicrobial resistance shows that the treatment’s efficacy depends on the device. The discussion provides a clear understanding of the position of the CAP sources in different medical applications and the importance of the device in the application of CAP in medicine.

1. A Basic Introduction to Plasma

Plasma, known as the fourth state of matter, is a quasi-neutral ionized gas composed of a variety of particles, including ions, electrons, free radicals, neutral atoms, and molecules in either their ground or excited states. Additionally, plasma electric and magnetic fields, as well as ultraviolet (UV), visible, and near-infrared radiation. Generally, plasma may exist in a wide temperature range (from 10^–2 to 10⁸ K) and a wide range of its particle densities.

1.1. Classification of Plasma

Thermodynamic variables such as temperature and pressure play a critical role in the formation of different types of plasma. Plasma can be broadly categorized into two types based on the thermodynamic equilibrium among its various constituents: thermal plasma and nonthermal plasma. − In thermal plasma, all constituents, electrons, ions, and neutral species, are at almost the same temperature and hence in a state of thermal equilibrium. An electrical arc and laser fusion plasma are examples of thermal plasma. , Thermal plasma devices are commonly used in material processing, plasma metallurgy, and waste treatment. , However, due to the very high temperatures, such plasmas are not suitable for direct biomedical use. In contrast, in nonthermal plasma, constituents are not in thermal equilibrium. The electrons in the plasma have a much higher temperature than the ions and neutrals. , This temperature difference is due to the mass difference and to insufficient collisional energy transfer between the particles. − Figure shows the classification of plasma based on the thermal equilibrium of plasma constituents. It distinguishes the key parameters, i.e., electron temperature (T_e), ion temperature (T_i), gas temperature (T_g), and electron density (n_e), which define the physicochemical properties of plasma.

1.

Schematic representation of the classification of plasma based on the thermal equilibrium of the plasma constituents.

1.2. Cold Atmospheric Plasma

Cold atmospheric plasma (CAP) is a nonthermal plasma that operates under atmospheric conditions. The term “Cold” refers to its typically operating below 40 °C. , Previously, nonthermal plasmas were generated at low gas pressures, where collisions were significantly less frequent, but this setup requires the creation of a vacuum and offers fewer biomedical advantages. Due to all the limitations, researchers have focused on CAP as a more practical alternative for biomedical applications. , CAP has been a topic of interest over the last three decades for its unique properties and potential uses in biomedicine. ,,− Accordingly, the characteristics of CAP include: ,,

• Operated in atmospheric pressure,

• mean electron temperature sufficient for electron impact dissociation, excitation, and ionization (>1 eV),

• mean gas temperature should be <40 °C,

• heat energy transfers to the target below an impairing level,

• current to the target below the significant induction of Joule heating.

2. CAP Sources

There are several techniques for producing CAP. It can be classified into three types: direct-discharge plasma, indirect-discharge plasma, and hybrid plasma. The two main approaches used widely in plasma medicine are direct-discharge and indirect-discharge plasmas. In the direct discharge plasma method, the living tissue, or cells, is used as an active part between the electrodes; the dielectric barrier discharge (DBD) is one of the most common examples. ,, In the indirect discharge method, plasma is formed in the discharge arc and transported to the target area via convection or diffusion. A common example of an indirect discharge method is the plasma jet. ,, The ionization of gas is done by any kind of energy, viz., electrical, electromagnetic, thermal, optical, radioactive, etc. Electrical and electromagnetic power sources are the most common among these energy sources. ,, The biomedical use of a plasma device depends on several key factors, including safety, effectiveness, and stability. The estimation of safety risk, plasma stability, and effectiveness has been extensively discussed in the literature. ,− The schematic diagram of primary CAP sources is shown in Figure .

2.

Schematic and pictorial representation of the different sources of cold atmospheric plasma: (a) Dielectric Barrier Discharge, (b) Plasma Jet, (c) Corona discharge.

2.1. Dielectric Barrier Discharge (DBD)

DBD has been a key source of CAP since its inception. Its first experimental investigation was conducted by Siemens in 1857 during ozone generation studies. − DBDs have been used for various applications, including gas treatment, surface cleaning and modification, sterilization, and light generation. ,,− Recent studies indicate a significant increase in the use of DBDs in plasma medicine. ,,, This growing trend highlights the increasing recognition of DBDs for their potential in various therapeutic interventions, including wound healing, sterilization, and tissue regeneration. ,,, Researchers are exploring the efficacy and safety of these technologies, reflecting a broader shift toward innovative plasma-based solutions in healthcare. , DBD features an electrode setup in which at least one electrode is covered with dielectric material, enabling self-pulsing plasma operation and nonthermal plasma generation at atmospheric pressure. , The dielectric (i.e., glass, quartz, ceramics, and plastics, etc.) barrier serves as a load resistor, limiting current density in the plasma discharge. , The concept of the discharged regime, along with the foundational principles of DBDs, has been extensively explored in a diverse array of textbooks and review articles that delve into the intricate mechanisms and theoretical frameworks. ,, Different types of DBD were designed based on the electrode shapes, sizes, voltage sources, and dielectric materials, volume DBD, surface DBD, and coplanar discharge, etc. , An in-depth exploration of these types of DBDs highlights their unique characteristics, applications, and mechanisms, emphasizing differences in their operational principles and interactions with surrounding materials. , A novel type of DBD, the FE-DBD (floating-electrode dielectric barrier discharge) has also been developed for biomedical applications, which contains a high-voltage electrode protected by a dielectric and a second electrode that is not electrically connected to the power supply, but is instead “floating”. , The difference between DBD and FE-DBD is that, in FE-DBD, the second ground electrode is absent and the biological body acts itself as the second electrode. FE-DBD applies a continuous high-voltage pulse of about 10–30 kV to a quartz-protected electrode, creating a discharge between the quartz, protects the active electrode and biological sample. The distance between the active electrode and the biological sample was kept at ∼2–3 mm. It has been used on blood coagulation, living tissue sterilization, and melanoma skin cancer cell lines. , The DBD configuration concept is also used in plasma jets, where a coaxial configuration, with a dielectric tube, serves as the barrier material, and gas flows outside the electrode setup. ,

2.2. Plasma Jets

An atmospheric-pressure plasma jet (APPJ) is a common example of an indirect-discharge plasma with a development history spanning over 50 years. In APPJ, the plasma forms in the electrode core, and a high-velocity gas flow drives it outward. The reactive species generated at the discharge exit the nozzle at high velocity. Various terms are used for the same group “APPJ” (e.g., plasma flame, plasma gun, plasma stream, plasma pencil, plasma needle, etc.), while plasma torch is used for plasmas which differ in their properties (e.g., in gas temperature). Despite differences in size, design, shape, discharge gas, frequency, and power supply, the principle behind it remains the same. In an earlier discussion on APPJs, Lu et al. classified APPJs based on the discharge gas used (noble gas plasma jet, N2 plasma jet, air plasma jet). While Winter, Brandenburg, and Weltmann provided a more exhaustive classification, including geometry and excitation frequency. , Among different APPJs, the most used carrier gas is helium, followed by air and argon. , In 1992, Koinuma et al. developed the first APPJ using a tungsten or stainless-steel needle (1 mm in diameter and 20 mm long) as a cathode connected to an RF generator (13.56 MHz) with grounded stainless-steel cylinder anode (15 mm long, 2.5 mm inner diameter) and a quartz tube barrier of 0.5 mm thick, having a 2.5 mm outer diameter and 14 mm long. The plasma gas flowed through the gap between the quartz tube and the cathode at a rate of 70 sccm.

From a practical perspective, APPJs are of particular interest in modern medical science. APPJs have the advantages of small size, lightweight, and nearly arbitrary 3D movements. The produced cold plasma can be focused into small or large treatment areas. Ma et al. and Nie et al. have discussed multineedle plasma jets for large-area treatment. , The multineedle DBD Jet developed by Ma et al. was used to study the treatment of large infected areas and their effectiveness, using psoriasis as a representative skin disease. APPJs have been shown to have antimicrobial, anti-inflammatory, and tissue-stimulating effects, which make them suitable for dermatology and wound healing treatments. − APPJs, therefore, appear to be a promising technology in plasma medicine because of their versatility and potential for treating a range of skin-related problems. ,

2.2.1. Plasma Needle

The plasma needle, a unique configuration of APPJ, plays a crucial role in medical treatments such as wound healing and dental applications. ,, It generates a low-powered CAP plume with a small diameter and minimal penetration depth, which allows precise and localized treatment. , Introduced by Stoffels et al. in 2002 and modified in 2004, it features a needle-shaped electrode within a gas-flow nozzle. , The modified version consists of a metal-alloy pin (0.3 mm in diameter, 8 cm long) that extends from a perspex tube, allowing helium gas to mix with air at the tip and creating a plasma glow about 2 mm in diameter. Initially developed for dental use, the plasma needle has since been studied for its effects on wound healing, liver cancer cell ablation, and the promotion of apoptosis and cell adhesion. ,,, Its nondestructive nature, coupled with its ability to disinfect while promoting tissue regeneration, makes it an attractive approach for various medical applications, ranging from dentistry to oncology. ,,

2.2.2. Plasma Pencil

In 2005, Laroussi et al. developed a device called the plasma pencil, which features two ring-shaped copper electrodes attached to a glass disk with a central hole (3 mm diameter). , The disk measures 2.5 cm across, and the electrodes are positioned within a dielectric cylindrical tube, separated by a gap of 0.5 to 1 cm. High-voltage pulses (1–10 kHz) generate plasma, creating a plume about 5 cm long that exits through the central hole. The temperature around the plasma was approximately 290 K, making it safe for direct contact. The plasma pencil has been used to inactivate Escherichia coli (E. coli), demonstrating its efficacy in biomedical sterilization. , Its controllability, stability, low power consumption, and room temperature operation make it appropriate for various medical applications as well as materials processing ,,

2.3. Corona Discharge

Corona discharge is a method for generating atmospheric pressure nonthermal plasma. , It occurs across asymmetric electrode pairs with sharp points at high voltages. As voltage increases, the discharge narrows and can transition into a spark discharge. − This phenomenon is characterized by a high electric field at the sharp end of the electrode, leading to gas breakdown in that localized area, while preventing breakdown in nearby regions. ,

Corona discharge can be produced using direct current (DC), alternating current (AC), or pulsed voltage, and is classified as negative or positive corona based on the electrode’s polarity. ,, Negative corona involves Townsend breakdown and secondary electron emission from the cathode, while positive corona relates to cathode-directed streamer formation. ,, Applications of corona discharge include treating fruits and vegetables with ozone, decontaminating E. coli, synthesizing ozone for purifying water, and material processing, which show its applicability in different environmental and biomedical fields. ,−

In conclusion, various CAP sources offer distinct advantages for biomedicine applications. The DBDs are simple in design and provide a large treatment area and uniformity, which are excellent for large-area wound treatment and sterilization. ,, Plasma jets offer portability, remote plasma delivery, and precise spot treatment; however, their higher gas consumption and a smaller treatment area are major drawbacks compared to DBD. ,, On the other hand, corona discharges have the simplest design, but produce nonuniform plasma with a lower yield of RONS, which makes them less preferred for biomedical applications. , The distinct characteristics of these CAP sources define their potential for different medical applications, DBD being preferable for wide-spectrum applications, and plasma jets for targeted therapies. Despite certain limitations, all CAP technologies have the potential to advance fields such as sterilization, wound healing, cancer treatment, and other medical therapies. Further research aims to optimize the characteristics of these devices for various biomedical applications.

2.4. Certified Devices of CAP

Currently, only four distinct types of plasma devices have received approval for clinical use, having undergone rigorous testing and evaluation to ensure their safety and efficacy in medical settings. , These devices work on the principles of DBD and jets, ensuring safety and effectiveness for dermatological, wound-healing, and antimicrobial applications. ,, Among them, the jet-based devices (kINPen MED) offer precise treatment in remote areas but have a disadvantage of a small treatment area coverage (1 cm²). , Whereas Adtec SteriPlas uses the concept of a plasma torch with a 6-electrode system and has a torch diameter of 3.5 cm, allowing treatment coverage of 4–5 cm². , In this case, DBD-based devices (PlasmaDerm and Plasma Care) offer flexibility in the treatment area size. A previous study by Daeschlein et al. showed that skin contamination with Staphylococcus epidermidis and Micrococcus luteus, a DBD-based plasma device, provides greater log reduction than jet-based kINPen. A detailed comparison of medically certified devices and their applications is provided in Table S1 of Supporting Information (SI). Several additional plasma devices are currently undergoing clinical trials and evaluations, with hopes of obtaining certification and becoming available for practical use in the near future. This ongoing research and development may lead to new treatments and therapies, expanding the options available to healthcare professionals and patients alike. ,

3. Interaction of CAP with Cells and Tissues

CAP initiates a cascade of biological reactions in tissues and cells through the dissociative interactions of plasma electrons with ambient gases, producing reactive oxygen and nitrogen species (RONS), along with ions and electrons. ,, Recent studies show that these reactive species and charged particles effectively inactivate bacteria upon direct plasma contact with tissue, contributing to CAP’s antimicrobial properties. , In CAP, biologically active RONS dissolve in tissues, influencing natural redox reactions that are essential in maintaining homeostasis in cells despite their short half-life on surfaces. , The deeper tissue effects of CAP arise from biological responses aimed at preserving this homeostasis through the regulation of cell proliferation, apoptosis, inflammation, and immune responses. − The biological effects of CAP on cells and tissues include the enhancement of wound healing through the promotion of cell migration and proliferation, as well as the induction of cell death in pathological cells, including cancer cells, through RONS-mediated mechanisms. ,,, Figure provides a schematic illustration of the complex biological effects of CAP on cells and tissues, highlighting its mechanism of action in sterilizing prokaryotic and eukaryotic cells and its role across the different phases of wound healing.

3.

Schematic representation of the biological effects of cold atmospheric plasma (CAP) on prokaryotic and eukaryotic cells and its role in wound healing. The figure highlights the production of reactive oxygen and nitrogen species (RONS) and their interactions with the cell membrane, which in prokaryotes consists of polysaccharides and in eukaryotes of phospholipids, leading to lipid or polysaccharide peroxidation, membrane damage, and oxidative stress. Intracellular processes include activation of oxidative stress pathways, DNA damage, apoptosis, and regulation of redox-sensitive signaling pathways. The figure further depicts CAP-mediated regulation of inflammation, angiogenesis, fibroblast proliferation, and collagen deposition across the different phases of wound healing.

The plasma’s biological effects are based on two principles:

• The alterations induced by plasma within the liquid environment surrounding tissues and cells serve as the fundamental catalyst for the range of biological effects associated with plasma exposure. ,

• In the biological responses triggered by plasma, the generation of RONS, either produced within the plasma or transferred into the surrounding liquid phases, plays a pivotal role in driving these effects. ,,

Plasma interacts with biological systems in complex ways, primarily by generating reactive species and charged particles at the cell membrane interface. The cell membrane, composed of phospholipids in eukaryotes and polysaccharides in prokaryotes, is the primary target after plasma treatment. , Initial effects occur at the membrane where chemical changes, such as lipid or polysaccharide peroxidation, depend on the medium, leading to alterations in the membrane architecture, thereby producing complex cellular responses, including the activation of intracellular signaling pathways. − Hydrogen peroxide, a type of reactive oxygen species (ROS), acts as a defense mechanism in mammalian cells and can further transform into hydroxyl radicals, initiating chain oxidation that damages cellular components and DNA. − Plasma-induced apoptosis shows typical morphological changes, including nuclear condensation and DNA fragmentation into oligonucleosomal fragments, indicating that plasma can mimic natural apoptosis pathways, regulating cellular processes to eliminate diseased tissue without causing harm to the healthy cells. ,, Plasma treatment also causes reversible cell detachment, enabling cells to be manipulated for the rearrangement or modulation of cell functions. Cell detachment from the substrate and loss of cell contact could be due to plasma-induced damage to cell adhesion molecules, such as cadherins and integrins. ,

CAP shows distinct effects on wound healing and oncologic therapy via dose-dependent production of RONS that take advantage of differences in cell redox homeostasis and antioxidant defense. , In wound healing, sublethal to moderate doses of RONS mimic physiological signals to enhance all healing phases including disinfection via lipid peroxidation. Also, inflammation modulation to a pro-healing response via macrophage polarization from M1 to M2 by regulating NF-κB pathway. ,, Fibroblast proliferation and migration are stimulated via TGF-β/Smad and HIPPO/YAP-CTGF pathways, while angiogenesis is also stimulated via VEGF. , Healthy cells tolerate these sublethal doses via Nrf2-mediated cytoprotection. , In contrast, in cancer therapy, higher doses of RONS selectively target tumor cells due to higher basal ROS levels and reduced antioxidant enzymes causing mitochondrial stress, endoplasmic reticulum stress, lipid peroxidation, and apoptosis or necrosis via JNK/caspases and immunogenic cell death. ,, Healthy cells are protected through their strong antioxidant defense. The effects on tumor cells also synergize with immunotherapy via downregulation of PD-L1 at optimal doses. ,, The key to the differential effects is that wound healing benefits from RONS signaling that stimulates regeneration, whereas tumor cells are killed by a redox imbalance. Table S2 of SI summarizes the dose-dependent impact of CAP treatment on cells and tissues. ,, The cell-type specificity of CAP action is a key feature that underlies its clinical applications in oncology and in the control of infectious diseases. These findings underscore the importance of cell-type specificity in the applications of CAP.

Plasma can be applied in two ways: direct treatment and indirect treatment. The direct approach involves applying CAP directly to cells in vitro, in vivo models, or onto human tissues. , In contrast, the indirect method uses a medium or solution activated by plasma, known as Plasma-activated media (PAM). PAM is utilized in cell culture and can also be directly injected into test subjects, such as mice, for xenograft studies. − PAM is further discussed in the latter part of the application section.

4. Biomedical Application of CAP

Plasma has emerged as a versatile tool in diverse biomedical applications over the last two decades. In this review article, we are focusing mainly on the work of the recent decade, including a few works from the previous decade. This section provides a brief discussion of different CAP applications in biomedical research, accompanied by a schematic representation.

4.1. Sterilization (Effect of CAP on Microbes)

CAP is a highly potent nonantibiotic sterilization method with strong biocidal properties. , It penetrates hard-to-reach areas such as puncture wounds, hollow needles, and crevices of surfaces by dispersing ROS into accessible volumes. , This makes CAP promising against multidrug-resistant microorganisms. Studies have shown that it has strong efficacy in inactivating spores, biofilms, viruses, Gram-positive, and Gram-negative bacteria. ,,, CAP acts as a multifaceted antimicrobial approach for treating resistant infections and improving sterilization practices. , Figure visually illustrates the antimicrobial effects of CAP against bacteria, fungi, and viruses, demonstrating its broad spectrum microbicidal potential.

4.

Schematic representation of the antimicrobial effect of CAP on (a) bacteria, (b) fungi, and (c) viruses. In bacteria, CAP-induced RONS cause membrane damage, oxidative stress, and bactericidal effects on both sensitive and antibiotic-resistant bacteria. In fungi, RONS induce cell wall and membrane damage, impair spore integrity, and trigger fungal apoptosis, thereby inhibiting skin and nail infections. In viruses, CAP-induced RONS damage viral structure and proteins, leading to viral inactivation and inhibition of viral replication, as evidenced by the degradation of the spike protein and loss of infectivity.

4.1.1. Bactericide

CAP can reduce microbial load and may potentially replace antibiotics, especially against antibiotic-resistant strains. , The bactericidal effects of CAP in sterilization were first reported in the mid-1990s in foundational research by Mounir Laroussi. While low-pressure plasma has been used in surface sterilization since the late 1960s, Laroussi used nonthermal plasma to treat bacterial cells, observing successful killing of the cells, which has been referred to as the “birth year of plasma medicine. After 1996, Laroussi developed the “plasma pencil” in the early 2000s, a hand-held, device using a pulsed electric field for the production of room-temperature plasma, safe for bactericidal treatment of wounds and surfaces. , Research in the early 2000s proved the dominance of CAP over UV and heat in sterilization, achieving a 5-log reduction in E. coli, Staphylococcus aureus, and spores through synergistic effects of different CAP components, disrupting membrane and DNA structures. ,, Nonthermal FE-DBD plasma effectively treats planktonic and biofilm forms of pathogens. , S. aureus and E. coli, key nosocomial infections, are rapidly inactivated by DBD plasma, with 100% destruction of planktonic S. aureus and E. coli in under 60 s (7.8 J/cm²) and a reduction of 90–95% and 40–45% for higher concentrations, achieving complete disinfection in about 120 s. , By 2012, DBD and plasma jets were used against clinical pathogens, including spores. , Current developments in CAP technology aim at targeting multidrug-resistant bacteria. CAP treatment resulted in a 6-log reduction of carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Klebsiella pneumoniae in 2 h, making it suitable for medical device treatment in 2025. For food safety, CAP treatment resulted in a reduction of Salmonella typhimurium and Listeria monocytogenes by up to 6.4 logs in 9–15 min. CAP technology, including hybrid jets and plasma-activated media, has shown promise in providing long-term effects, and microplasma miniaturization aims at treating internal surfaces or dental implants. , The initial mechanism of CAP treatment involved RONS-induced peroxidation, which dominated research from 1996 to 2000s. , Currently, the mechanism of inactivation of bacteria after CAP treatment has been understood as a multimodal effect, where RONS, UV, and electric fields penetrate biofilms, overcoming biofilm resistance, and time-dependent effects enhance the treatment outcome. CAP treatment has progressed from a laboratory setup to a clinical application, where kINPen MED plasma treatment has been certified for use in sterilization. CAP treatment is estimated to soon become an adjunct in the treatment of MDR pathogens. Table S3 of SI provides a list of studies that evaluate the effectiveness of different CAP sources in the inactivation of bacteria.

4.1.2. Fungicide

Fungal infections have increased in recent decades, and rising antifungal resistance highlights the need for effective treatment strategies in clinical mycology. CAP’s fungicidal potential has been well demonstrated since the early 2000s, and has shown its potential in inactivating yeasts, filamentous fungi, and spores using CAP-DBD, plasma jet, and surface microdischarge configurations. , The main dermatophytes are Trichophyton rubrum (accounting for 80 to 90% of infections) and Trichophyton interdigitale. In a study, a single treatment of 10 min of CAP resulted in slight inhibition of T. rubrum growth. However, daily treatment with 10 min of CAP for 9 days resulted in a reduction in colony sizes, with T. rubrum growth decreasing from 25.6 ± 2.7 mm to 4.3 ± 0.7 mm. , DBD and SMD configurations have shown the potential to inactivate 3–6 log10 CFU of Candida albicans and other pathogenic fungal microorganisms within seconds to minutes, and the fungicidal potential of CAP on fungal microorganisms depends on the configuration, composition, and treatment time of CAP devices. ,, Misra et al. noted that CAP inactivation involves membrane dysfunction, cellular damage, and apoptosis driven by ROS, increased vacuoles, cell wall alterations, and mitochondrial enlargement were observed with longer treatment durations, along with changes in internal structures such as the golgi apparatus and ribosomes, suggesting extensive cellular damage over time. − CAP treatment also triggers lipid peroxidation of ergosterol-containing membranes in fungi, increases membrane permeability, and induces leakage of cellular contents while also causing oxidative damage to DNA. , Spores have higher resistance due to chitinous wall, but optimized CAP treatment eventually leads to structural etching and loss of viability. , In addition to human pathogens, CAP treatment is effective in inactivating toxigenic species of fungi such as Aspergillus, Fusarium, Penicillium, and Alternaria, resulting in high levels of inhibition in the germination of their spores and in the reduction of their populations in low-moisture agri-food environments. , In the context of food safety, CAP treatment has shown inhibition of Fusarium graminearum and related species while causing partial degradation (30–50%) of mycotoxins such as deoxynivalenol (DON). , Overall, the prevailing evidence suggests that CAP has multitarget antifungal properties that induce oxidative stress, damage to membranes and DNA, and cause electroporation, allowing it to be applied in medical sterilization and food safety without the risk of any thermal damage. ,, Table S4 of SI provides a list of studies on the inactivation of fungi by CAP.

4.1.3. Virucide

Human health is at risk from harmful viruses, with adenoviruses being particularly resistant due to their protein capsid. CAP antiviral research emerged alongside foundational bacterial plasma studies, extending DBD technology to viral models such as bacteriophages λ, MS2, T4, and φX174, where early experiments demonstrated that CAP components are capable of degrading viral capsid proteins prior to nucleic acid fragmentation without thermal damage, making CAP suitable for heat-sensitive materials. ,, By the mid-2000s, DBD treatment of bacteriophage λ caused capsid protein degradation preceding detectable DNA damage, supporting oxidative etching as the dominant pathway. , CAP jets achieved complete inactivation of MS2, T4, and φX174 within minutes, with singlet oxygen and ozone identified as primary virucidal species targeting protein side chains and nucleic acids. ,− Studies between 2012–2018 demonstrated rapid 6-log reductions of feline calicivirus (FCV, a norovirus surrogate) within seconds via oxidation of capsid histidines, while adenoviruses exhibited comparatively greater resistance due to robust icosahedral protein shells, requiring extended exposure (∼240 s) to block replication in adenoviruses. , In the 2020s, CAP research intensified during viral pandemics, demonstrating that argon-air plasma and PAW inactivated bacteriophages T4, MS2, and φX174 within 80–120 s through acidification and oxidative protein damage, with PAW retaining poststorage antiviral activity. , CAP exposure reduced HSV-1 genome internalization by over 2 orders of magnitude without cytotoxicity to Vero or SH-SY5Y cells compared with aciclovir controls, highlighting selective virucidal action. Notably, argon-fed CAP achieved complete SARS-CoV-2 surface inactivation in under 180 s (≤30 s on metal), attributed to reactive species permeabilizing viral envelopes and destabilizing spike proteins. , Mechanistically, RONS, particularly singlet oxygen in FCV/T4, O₃, and H₂O₂ in influenza/RSV models, and ONOOH in phages, oxidize capsid proteins before inducing genome strand breaks. , Collectively, these findings establish CAP as a rapid, nonthermal, chemically mediated antiviral platform capable of broad-spectrum virus inactivation across surfaces, liquids, and aerosols, with expanding translational relevance from laboratory to clinically significant pathogens. Table S5 of SI highlights the antiviral properties of plasma, emphasizing its ability to rapidly inactivate Adenovirus and SARS-CoV-2 on various surfaces.

4.2. Effect of CAP on Blood Coagulation

CAP is emerging as a versatile device for controlling bleeding during surgery and wound management. Advancements in surgical therapies have significantly improved bleeding control, reducing the risk of death during surgery. , Research has demonstrated that FE-DBD plasma accelerates blood coagulation. , In an in vitro experiment by Fridman et al., a blood drop treated with FE-DBD for just 15 s coagulated within 1 min. Additionally, when cuts on organs are treated with FE-DBD, the blood coagulates without causing any visible or microscopic damage to the surrounding tissue. CAP treatment has emerged as an innovative approach to enhance blood coagulation, particularly in wound healing, through the interaction of RONS with blood components. , This significantly reduces clotting time and promotes rapid coagulation. CAP enhances platelet aggregation and decreases blood fluidity, both of which are essential for forming stable clots. − In vivo studies demonstrate CAP’s ability to achieve swift hemostasis by sealing wounds in seconds. Moreover, it improves overall wound healing by increasing microcirculation and stimulating cellular activity. Notably, the treatment’s nonthermal effects promote coagulation through biochemical interactions rather than heat, protecting surrounding tissues from damage.

4.3. Effect of CAP on Dermatology and Wound Healing

The skin is the body’s largest organ, serving as a crucial barrier against UV radiation, chemicals, and microorganisms. , When damaged, it activates healing processes involving coagulation, inflammation, and tissue remodeling. , Skin diseases are prevalent, affecting 30 to 70% of people, particularly in vulnerable groups. The early 2000s marked the beginning of the first indications that CAP could be used for the sterilization of living tissue and for blood clotting, which initiated research on plasma medicine for wound healing. , Initial studies stated that the broad antimicrobial spectrum of CAP was effective against a variety of bacteria, including antibiotic-resistant strains. , From the late 2000s onward, in vivo studies introduced another aspect: CAP not only reduced bacterial load but also accelerated wound closure in acute and chronic models, increasing re-epithelialization and granulation tissue formation. ,, This laid the groundwork for the development of CAP devices, such as MicroPlaSTER and PlasmaDerm, which were tested in clinical trials. The first clinical milestone started in 2010 with a prospective randomized controlled trial conducted by Isbary et al. with 36 patients and 38 chronic infected wounds. The trial used cold argon plasma for 5 min daily in addition to conventional treatment. The outcome was a remarkable 34% reduction in bacterial load, with excellent tolerability and no side effects, marking the safe entry of CAP for human therapy. In 2012, a randomized, placebo-controlled pilot trial on 40 skin graft donor sites demonstrated that plasma-treated areas healed significantly faster from day 2 onward, promoting re-epithelialization. By 2013–2015, devices such as kINPen and PlasmaDerm were CE-marked in Europe for chronic wounds, and meta-analyses proved faster closure rates, approximately a 40–70% reduction in healing time for leg ulcers, and also the disruption of biofilms in diabetic foot ulcers. ,, Table S6 of SI summarizes in vitro and in vivo studies to highlight the role of CAP in wound healing and dermatology.

The mechanism of action of CAP in wound healing is based on the precise release of RONS, including superoxide, hydroxyl radicals, hydrogen peroxide, nitric oxide, and peroxynitrite. These molecules are the core of redox signaling. At therapeutic doses, they selectively target microbes by damaging their membranes, proteins, and nucleic acids, thereby reducing the microbial load in the wound. At the same time, low concentrations of RONS are used as signaling molecules that trigger the growth and migration of keratinocytes and fibroblasts, which are essential for re-epithelialization. , The mechanisms depend on synergistic interaction with the various phases of the healing process. In the hemostasis and inflammation phase, RONS stimulate platelets, reduce the clotting time by modifying fibrinogen, and regulate cytokines (e.g., reducing IL-1 and TNF-α and increasing TGF-β), preparing the ground for the proliferation phase. ,, In the proliferative phase, CAP stimulates the migration of keratinocytes and fibroblasts via PI3K/AKT and MAPK/CK2 signaling pathways, increases angiogenesis by VEGF upregulation, facilitates collagen remodeling, and increases the release of growth factors (including IFN-γ). ,, Moreover, CAP also helps to modulate the inflammatory response by suppressing excessive pro-inflammatory cytokine activity while also facilitating a controlled immune response, thus preventing chronic inflammation. , In summary, the biochemical modulation of CAP provides a clear advantage over thermal methods, which not only accelerate healing but also protect tissue integrity. Table S7 of SI summarizes the clinical studies of CAP conducted on different dermal applications.

4.4. Effect of CAP on Cancer Treatment

The intricate nature of cancer cells poses significant challenges for selective treatment, as traditional therapies like chemotherapy and radiotherapy are known to harm normal tissues in addition to targeting tumor cells. The biomedical application of CAP was established in the mid-2000s. Selective cancer cell death by CAP was first demonstrated by Keidar et al. proposing that cancer cells are more susceptible due to a disrupted redox state and altered membrane properties. ,, Further studies attributed this selectivity to increased intracellular ROS production and variations in aquaporin expression and antioxidant capacity in malignant cells. Vandamme et al. demonstrated that CAP-induced apoptosis in glioma cells occurred through caspase activation and mitochondrial membrane potential depolarization, thus confirming the prevalent mode of cell death as apoptosis rather than necrosis. , Further investigations have also demonstrated the enhanced efficacy of plasma when combined with nanoparticle conjugates, resulting in notable reductions in tumor size across various in vivo xenograft models, including glioma, melanoma, bladder cancer, and hepatocellular carcinoma. These studies highlighted improved survival rates and minimal adverse effects, particularly when treatment durations were carefully optimized. ,− More recent reviews suggest the use of plasma-activated liquids as an indirect method of CAP that preserves the reactive species chemistry and maintains anticancer selectivity in both in vitro and in vivo studies, opening the door to clinical applications. ,

Mechanistically, CAP induces apoptosis predominantly by generating RONS, which trigger a cascade of cellular events leading to DNA damage, cell cycle arrest at the S and G2/M phases, and subsequent activation of apoptotic pathways. ,− ROS mediated apoptosis after CAP treatment involves lipid peroxidation, DNA strand breaks, p53 activation, MAPK signaling modulation, and activation of caspase-9 and caspase-3, all of which lead to programmed cell death. Ahn et al. reported that CAP-derived ROS activated the intrinsic pathway of apoptosis by modulating Bax/Bcl-2 ratios and inducing cytochrome-c release, emphasizing the pivotal role of mitochondrial ROS signaling. The process of CAP-induced apoptosis is intricately linked to mitochondrial dysfunction mediated by ROS. This pathway activates crucial p53-dependent mechanisms, leading to upregulation of p53 and p21, caspase-3 activation, and a notable loss of mitochondrial membrane potential. ,− Subsequent studies indicate that CAP produces both short-lived reactive species and longer-lived species, which diffuse into cells and drive oxidative stress beyond the limit cancer cells can tolerate, making them more susceptible. Cancer cells possess a constitutively elevated basal ROS level; therefore, additional RONS push these cells beyond the apoptosis threshold, accounting for CAP’s selectivity to cancer cells. , Figure shows the therapeutic uses of CAP in cancer and dermatology, focusing on the role of RONS in signaling pathways, apoptosis, and angiogenesis, as well as in skin-related disorders and skin cancer.

5.

Schematic representation of the application of CAP in cancer and dermatology. The figure shows the application of CAP in in vitro and in vivo (xenograft) tumor models, demonstrating RONS-mediated inhibition of cancer cell proliferation, mitochondrial membrane depolarization, and apoptosis, leading to reduced tumor growth and increased survival in animal models. The figure also shows the application of CAP in skin cancer and hyperproliferative skin disorders, highlighting the regulation of VEGF, MMPs, endothelial signaling, nitric oxide expression, angiogenesis, and neovascularization, emphasizing the therapeutic potential of CAP-induced reactive species in cancer and dermatological applications.

The first FDA-approved Phase I clinical trial followed in 2020–2021 (NCT04267575), using Canady Helios Cold Plasma (CHCP) in 20 patients with stage IV or recurrent solid malignancies following surgical resection. The outcome was encouraging, no treatment-related adverse events, with 26-month recurrence-free rates of 69–100% for tumors. Survival improved, with 86% at 28 months for tumors in diverse cancers, including sarcoma, breast, lung, and colon. , Collectively, CAP displays selective anticancer activity by synergistically combining cytotoxicity via oxidative stress with the unique vulnerabilities of specific phases of the cell cycle. These characteristics position CAP as a promising adjunct or alternative therapeutic option in the fight against cancer. CAP sources utilized in various cancer cells and their mechanisms of action are tabulated in Table S8 of SI.

4.5. Effect of CAP on Dentistry

The oral cavity is home to a diverse and intricate microbial ecosystem, hosting more than 700 distinct bacterial species. , Among the most common oral health issues are dental caries and periodontal disease, conditions predominantly caused by the proliferation of biofilm-forming bacteria, notably Streptococcus mutans, and various multispecies dental plaque communities that thrive in the complex environment of the mouth. , Recent advancements in oral hygiene have highlighted the promising application of CAP, which has demonstrated remarkable antibacterial and antifungal activity against a range of oral pathogens and their associated biofilms. Early studies reported that the efficacy of CAP in inactivating oral pathogens like Streptococcus mutans and Enterococcus faecalis, suggesting its potential use in caries and endodontic treatments. , CAP is also used for root canal treatments, where the RONS produced by the plasma were more effectively delivered into dentinal tubules than conventional irrigants, improving biofilm removal without damaging surrounding tissues. , Notably, treatments using plasma needle technology have demonstrated a significant reduction in both Gram-positive and Gram-negative plaque-forming bacteria within 1 to 5 min. This swift action not only disrupts bacterial structures but also causes detrimental effects, such as DNA leakage, highlighting a broader range of antimicrobial mechanisms than traditional chemical agents like chlorhexidine (CHX). − CAP demonstrated remarkable efficacy compared with CHX for eliminating Streptococcus mutans and complex multispecies saliva biofilms. The application of CAP achieved an impressive reduction of over 5 log colony-forming units (CFUs) when using DBD plasma systems. , Moreover, CAP demonstrated the ability to disrupt complex mixed fungal–bacterial biofilms while maintaining biocompatibility with oral epithelial tissues, thereby minimizing adverse effects on surrounding healthy tissues. In addition to its notable antimicrobial properties, CAP significantly enhances the hydrophilicity of dental implant surfaces, thereby fostering superior osteoblast adhesion, a crucial factor for bone tissue regeneration. − In periodontology, CAP has been found to possess anti-inflammatory and antimicrobial properties, reducing periodontopathogenic bacteria and modulating cytokine expression in gingival tissues through redox-sensitive signaling pathways. ,, Further studies included CAP in oral wound healing and implantology, where plasma treatment stimulated fibroblast proliferation and angiogenesis and improved osseointegration through controlled ROS-mediated cell signaling and growth factor activation. ,, More recently, CAP has been applied in oral oncology for its selective cytotoxicity against oral squamous cell carcinoma while sparing normal mucosa, which is attributed to the increased oxidative sensitivity of cancer cells. ,, From its initial antimicrobial applications to its current uses in regenerative dentistry and oral cancer therapy, CAP has proven to be a multifaceted dental technology. Its ability to reach and sanitize difficult-to-access areas within the oral cavity underscores its potential as a transformative tool in modern dental practices. Applications of CAP sources in dentistry are shown in Table S9 of SI.

4.6. Effect of CAP on Regenerative Medicine

Stem cells play a crucial role in tissue regeneration, due to their capacity for self-renewal and differentiation, which are regulated through their dynamic interactions with the surrounding microenvironment. , These characteristic underscores their importance in biological repair mechanisms due to their substantial therapeutic potential in regenerative medicine. , Recently, CAP has gained recognition as an innovative and effective approach for modulating stem cell behavior. ,,, CAP treatment has been shown to influence various cellular processes, including adhesion, proliferation, differentiation, and apoptosis, which are essential for effective tissue regeneration. , Prior studies stated that nitrogen oxide (NO) enriched plasma treatments significantly stimulate the proliferation and osteogenic differentiation of mesoderm-derived stem cells, as evidenced by marked increases in alkaline phosphatase activity and by the upregulation of early osteogenic markers, such as collagen type I (COL-1). ,, Remarkably, these effects are observed even in the absence of traditional osteogenic supplements, emphasizing the potency of plasma treatments in fostering osteogenic outcomes. Furthermore, exposure to plasma has been shown to enhance signaling pathways associated with osteogenesis. Specifically, it activates FOXO1- and p38-mediated pathways, leading to upregulation of crucial genes involved in bone formation, including Runx2, ALP, osteocalcin (OCN), and osterix. ,, CAP has shown remarkable efficacy in influencing neural stem cell fate, particularly by significantly increasing neuronal differentiation rates to >75%. This impressive outcome is achieved with minimal cellular damage when optimal plasma doses are used. , When intermediate plasma intensities are applied, they not only enhance neurite regeneration but also facilitate astrocyte regrowth, demonstrating a beneficial response. In contrast, exposure to excessively high plasma levels has been found to induce cytotoxic effects, thereby underscoring the crucial importance of dose-dependent responses in this context. , Furthermore, in studies involving adult stem cells, exposure to CAP has led to a notable increase in the proliferation of stem cells derived from adipose tissue, bone marrow, and hematopoietic sources. This proliferation is driven by the activation of nitric oxide (NO)-mediated signaling pathways, specifically the Akt and ERK1/2 pathways, without loss of stemness markers like Oct4 and Sox2, thereby maintaining the fundamental properties of stem cells. In addition to its biological actions on stem cells, CAP has the ability to biofunctionalize biomaterial surfaces, thus improving regenerative processes; for example, it can increase the hydrophilicity of dentin surfaces, which can improve the adhesion and migration of dental pulp stem cells. In summary, CAP is a multifaceted approach in regenerative medicine that has the ability to modulate stem cell fate and promote tissue repair through carefully controlled oxidative mechanisms. Table S10 of SI lists CAP applications across different regenerative medicines.

4.7. Effect of CAP on Surface Modification

Plasma surface modification (PSM), particularly CAP treatment, is a versatile and cost-effective technique that has gained widespread adoption for enhancing the surface properties of biomaterials. − This innovative method allows precise alteration of characteristics such as surface wettability, surface energy, nanoscale roughness, adhesion strength, and biocompatibility by incorporating reactive species while maintaining the integrity of the material’s bulk properties. , Biological responses such as cell adhesion, proliferation, and differentiation are primarily driven by surface chemistry, micro and nanoscale topography. Plasma treatments are particularly effective in optimizing implant–tissue interactions. ,

In bone tissue engineering, where scaffolds must closely mimic the structure and function of the extracellular matrix (ECM), plasma modification provides the key nanoscale surface optimization that 3D-printed scaffolds lack in bioactive surface properties. , CAP treatment introduces oxygen-containing groups and increases surface roughness and hydrophilicity, which enhances protein adsorption and improves osteoblast cell attachment. , CAP enhances osteoblast cell adhesion, proliferation, and differentiation on polycaprolactone (PCL) scaffolds by increasing surface energy and wettability. ,

CAP treatments significantly transformed scaffold surface chemistry, leading to improved hydrophilicity and enhanced cell attachment and functionality. This transformation occurs through RONS, which facilitate polymer oxidation. In the realm of cartilage tissue engineering, scaffolds composed of polycaprolactone (PCL) and cationic carboxymethyl cellulose (CMC), modified with helium plasma, showed a remarkable increase in adhesion, increase in alkaline phosphatase activity, osteocalcin expression, and chondrogenic differentiation of mesenchymal stem cells, without the application of growth factors. Collectively, plasma-based surface modification emerges as a highly versatile and effective strategy for enhancing scaffold biofunctionality across various applications in bone, cartilage, and dental tissue engineering. Applications of CAP in surface modification are shown in Table S11 of SI.

4.8. Effect of CAP on Transdermal Drug Delivery

Transdermal drug delivery (TDD) enables topical medications to reach either localized tissues or the systemic bloodstream by penetrating the stratum corneum (SC), which serves as the skin’s primary barrier to external substances. Traditional methods to enhance drug permeation through this barrier include chemical enhancers, which alter the lipid composition of the SC, and microneedles, which create microchannels in the skin. Recently, cold atmospheric plasma (CAP) has emerged as a groundbreaking, noninvasive technique that significantly enhances skin permeability without the need for physical or chemical disruption.

Ex vivo studies using Franz diffusion cell systems have shown that CAP can markedly improve the transdermal delivery of a wide range of both hydrophilic and lipophilic compounds, including small molecular weight dyes to larger, complex, pharmacologically active agents such as phenol red, rhodamine B, cyclosporine A, and various derivatives of galantamine. − CAP treatment increases the efficiency of drug absorption and broadens the possibilities for delivering a range of therapeutic agents through the skin. , The application of plasma treatment, using techniques such as DBD and APPJ, has demonstrated a remarkable ability to enhance the permeation of cyclosporine A through the skin. Notably, the peak drug flux was observed within 3 h of treatment. Furthermore, atmospheric microplasma irradiation significantly improved the penetration rates of aniline blue dye and galantamine hydrobromide in experimental models, including porcine and rat skin. This enhancement nearly doubled the cumulative transport of these drugs after a 24-h period. , The permeability enhancement effect of CAP is associated with transient, reversible modulation of skin barrier function, as evidenced by increased transepidermal water loss (TEWL) and increased stratum corneum hydration without histological evidence of irreversible damage. ,

Mechanistically, the interaction of plasma-generated reactive species with stratum corneum (SC) lipids leads to significant chemical and physical alterations. , This process increases the lipids’ hydrophilicity through oxidative modifications, enhancing their capacity to retain moisture. , Additionally, these reactive species facilitate transient pore formation in the skin via a reversible mechanism known as “plasmaporation”. , Since the CAP approach operates at room temperature and does not require thermal energy, it enables a safe, painless enhancement of drug and vaccine delivery. , Unlike traditional invasive techniques, such as electroporation, which may cause discomfort or adverse side effects, this technique offers a noninvasive alternative that promotes effective transdermal transport without compromising skin integrity. , Table S12 of SI shows the applications of CAP in transdermal drug delivery.

4.9. Plasma-Activated Media and Application

Plasma-activated solutions (PASs) encompass a range of innovative substances, notably plasma-activated water (PAW) and plasma-activated media (PAM), produced by treating aqueous solutions such as distilled water or phosphate-buffered saline (PBS) with CAP. These solutions are distinguished by their remarkable biological activities, making them highly valuable for both agricultural and biomedical applications. , These solutions exhibit strong biological effects, which are mainly mediated by RONS, with H₂O₂ and nitrite (NO₂ ^–) acting as a primary contributor. In the realm of biomedicine, PAW and PAM have shown great promise in various therapeutic interventions PASs have antimicrobial properties, biofilm disruption, wound healing, dental treatment, cancer therapy, and blood coagulation promotion. − Recent studies in 2025 confirm the effectiveness of PAW against Staphylococcus aureus, achieving bactericidal activity of up to 99.66% and biofilm efficacy in MRSA-infected wounds. These studies also show enhanced re-epithelialization of 63.2% in murine models. At the same time, PAM retains cytotoxicity against cancer cells for at least 1 week when stored at −80 °C, whereas plasma-treated PBS (pPBS) shows greater stability, indicating higher potential for use in medical sceneries. In glioblastoma cell lines (U251, LN229, U87), pPBS exerts cytotoxicity that is both concentration- and time-dependent, completely killing cells in 2.5–5 min. PAM triggers apoptosis through pathways including oxidative stress, mitochondrial damage, imbalance in Bcl-2/Bax ratio, PARP-1 activation, AIF release, NAD⁺ depletion, endoplasmic reticulum stress, and TRPM2-mediated Ca²⁺ influx. , It specifically targets cancer cells, as shown by 75% TUNEL-positive apoptosis in oral squamous cell carcinoma via MAPK pathways, and by suppression of PI3K/AKT and Ras/MAPK signaling in ovarian, melanoma, and glioblastoma models. , The anticancer potency of PAM is variable and closely linked to several treatment parameters, including the composition of the growth medium, the density of cancer cells, and the presence of RONS scavengers such as pyruvate.

Pharmaceutical applications of PASs demonstrate significant potential when used in combination therapies, notably increasing the effectiveness of small-molecule anticancer agents. They play a crucial role in selectively targeting and eliminating undifferentiated human induced pluripotent stem cells (hiPSCs), which is particularly beneficial for advancements in regenerative medicine. Additionally, PASs can modulate cell growth through mechanisms that depend on varying concentrations of nitric oxide (NO). These compelling findings underscore the versatility, selectivity, and noninvasiveness of PASs, positioning them as valuable tools in antimicrobial therapy, oncology, and regenerative applications. Table S13 of SI provides a list of PAM/PAW generation and implications on different applications.

5. Safety, Limitations, Challenges for Clinical Translations, and Future Perspectives

CAP devices in clinical use have demonstrated no side effects after rigorous testing, with temporary effects such as mild erythema or a localized temperature rise resolving quickly. , However, intense exposure can cause tissue damage or genotoxicity, underscoring the need for precise dose control and established safety guidelines. Apart from the conventional safety concerns, the primary obstacle in CAP as a pharmaceutical drug lies in an inadequate fundamental understanding of its biological, chemical, and physical mechanisms of interaction with living cells, tissues, organs, and the whole organism. Additionally, only a few studies have emphasized the long-term effects of CAP in humans and microorganisms. There is a lack of standardized protocols for device-independent plasma parameters and CAP dose variation across applications, which could lead to inconsistent RONS delivery and hinder reproducible clinical outcomes. The incomplete understanding of the molecular mechanisms underlying CAP’s biological effects also hampers clinical translation. Despite promising in vitro outcomes in cancer treatment, the limited penetration depth in deep tumors or internal sites restricts its application to surface tissues. Additional challenges include regulatory approval, device scalability, and addressing potential genotoxic or epigenetic effects in sensitive applications.

To address the limitations and challenges, many ongoing research projects focus on CAP’s fundamental understanding, − new diagnostic tools, , interactions with biological samples, numerical modeling, , and mechanisms of action. , The current researchers also focus on PAW and plasma-treated hydrogels for biomedical applications. , Integration of CAP with antibiotics, nanoparticles, and hydrogels provides promising outputs. , Despite these challenges, cold atmospheric plasma (CAP) demonstrates immense potential as the next-generation “super drug” due to its versatile applications across oncology, wound healing, microbial inactivation, and beyond.

6. Summary and Conclusions

The prominent use of CAP in the biomedical field has opened up significant opportunities in the new era of biomedicine owing to its unique ability to generate RONS, charged particles, UV photons, transient electric fields at room temperature. The nonequilibrium nature of CAP allows for selective biological responses without causing thermal damage, thus increasing its appeal for biomedical uses. Over the past decade, advances in the plasma source engineering, including DBD, plasma jets, and microwave-based systems, have enabled the controlled and reproducible delivery of bioactive plasma components for clinical translation.

The biological mechanisms underlying the action of CAP are complex and dose-dependent. CAP-induced oxidative stress modulates cellular redox homeostasis, disrupts microbial membranes, damages nucleic acids, and trigger apoptosis in cancer cells without any harm to normal tissue. The section on plasma-cell interactions highlights the importance of intracellular signaling pathways, mitochondrial damage, and immunogenic cell death in CAP mediated therapeutic effect. These mechanistic insights have improved the scientific rationale for the growing biomedical applications of CAP.

In antimicrobial treatment, CAP has a broad spectrum of activity against bacteria, fungi, virus, and biofilms, including multidrug-resistant microorganisms. As described in the CAP effect on sterilization section, the plasma-generated RONS damage microbial cell walls and biofilms, making it a promising alternative to traditional antibiotics. This has significant implications for hospital-acquired infections and the sterilization of medical devices. In cancer treatment, CAP has selective cytotoxicity against cancer cells through redox disbalance and apoptosis induction. The cancer treatment section describes that the cancer cells’ metabolic dysbalance and their increased baseline oxidative stress make them more vulnerable to plasma treatment. Preclinical trials suggest potential uses in solid cancers, either alone or as an adjunct to chemotherapy and radiation therapy, although standardized treatment protocols are yet to be developed. Applications in dentistry and oral health are an emerging field of research. CAP is effective in reducing oral pathogens, improving root canal treatments, and stimulating tissue regeneration without affecting the enamel or surrounding tissues. This makes CAP a minimally invasive complementary tool in periodontal and endodontic treatments. In the fields of wound healing and dermatology, CAP promotes angiogenesis, modulates inflammation and accelerates tissue regeneration. CAP has the ability to promote fibroblast proliferation and microcirculation, thus helping in enhanced healing responses in chronic and diabetic ulcers. Similarly, dermatological treatments can also be aided by the antimicrobial and anti-inflammatory properties of CAP, thus expanding its applications in acne, psoriasis, and other inflammatory skin conditions. In regenerative medicine, both direct and indirect applications of CAP aids in modifying the stem cell niche, have attracted attention in preclinical research. Preliminary research suggests that CAP pretreatment before topical medication administration can enhance transdermal drug delivery. Because CAP minimizes skin damage compared to other methods, it could emerge as a viable option for drug administration in the future.

However, despite these promising findings, safety and standardization are still of utmost importance. CAP has already established a role in routine medical care fields, like sterilization and wound healing, yet it remains a relatively new clinical intervention. While clinical studies are ongoing, additional research is needed in areas such as cancer treatment and regenerative medicine. Developing a deeper understanding of CAP’s effects and conducting clinical studies can help overcome current barriers in healthcare and establish CAP as a safe technique for medical practice. To date, only four devices have been certified for clinical use. These CAP devices should be designed with technical units that enable continuous, real-time monitoring of both plasma parameters and their effects on treated samples. Additionally, a control unit should be included to manage plasma parameters and treat different samples according to specific treatment requirements.

In summary, CAP is a highly versatile and innovative technology in contemporary medicine. Its multimodal action, broad-spectrum antimicrobial activity, selective anticancer activity, and regenerative properties make it one of the leading-edge technologies in plasma medicine. Further research focused on mechanistic understanding, safety validation, and standardization will help determine its eventual impact in translational healthcare.

Glossary

Abbreviations

CAP

Cold Atmospheric Plasma

RONS

Reactive Oxygen and Nitrogen Species

ROS

Reactive Oxygen Species

RNS

Reactive Nitrogen Species

K

Kelvin

C

Celsius

eV

Electron volt

kV

Kilo Volt

mm

Milli meter

AC

Alternating Current

APPJ

Atmospheric Pressure Plasma Jet

DC

Direct Current

hiPSCs

human induced pluripotent stem cells

DBD

Dielectric barrier discharge

RF

Radio frequency

MHz

Mega Hertz

Sccm

standard cubic centimeter per minute

Cm

Centimeter

SI

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.6c00042.

• Tables S1–S13 summarizing previously reported biomedical applications of cold atmospheric plasma (CAP), including medically certified devices (Table S1); dose-dependent effects of CAP on cells and tissues (Table S2); CAP inactivation of bacteria (Table S3), fungi (Table S4), and viruses (Table S5); applications in wound healing and dermatology (Table S6) and dermatological clinical studies (Table S7); cancer treatment and mechanisms of action (Table S8); dentistry applications and outcomes (Table S9); regenerative medicine applications (Table S10); surface modification for biomedical applications (Table S11); transdermal drug delivery (Table S12); and generation and applications of plasma-activated media (PAM) and plasma-activated water (PAW) (Table S13) (PDF)

P.T. and R.B.G. collected the data; conducted data curation; performed formal analysis; wrote the original draft; and reviewed and edited. S.B. supervised and reviewed the manuscript. A.B. conceptualized and performed formal analysis, and finalized the manuscript. P.T. and R.B.G. contributed equally.

The work carried out was funded by an In-house Project (IASST/R&D/ICP/IHP-23/2023-24/1338-1347) of the Institute of Advanced Study in Science and Technology (IASST) and the Indian Council of Medical Research (ICMR) (Project Sanction No. 5/3/8/85/2020-ITR dated 06/01/2021).

The authors declare no competing financial interest.