Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects

Abstract

Non-equilibrium atmospheric-pressure plasmas have recently become a topical area of research owing to their diverse applications in health care and medicine, environmental remediation and pollution control, materials processing, electrochemistry, nanotechnology and other fields. This review focuses on the reactive electrons and ionic, atomic, molecular, and radical species that are produced in these plasmas and then transported from the point of generation to the point of interaction with the material, medium, living cells or tissues being processed. The most important mechanisms of generation and transport of the key species in the plasmas of atmospheric-pressure plasma jets and other non-equilibrium atmospheric-pressure plasmas are introduced and examined from the viewpoint of their applications in plasma hygiene and medicine and other relevant fields. Sophisticated high-precision, time-resolved plasma diagnostics approaches and techniques are presented and their applications to monitor the reactive species and plasma dynamics in the plasma jets and other discharges, both in the gas phase and during the plasma interaction with liquid media, are critically reviewed. The large amount of experimental data is supported by the theoretical models of reactive species generation and transport in the plasmas, surrounding gaseous environments, and plasma interaction with liquid media. These models are presented and their limitations are discussed. Special attention is paid to biological effects of the plasma-generated reactive oxygen and nitrogen (and some other) species in basic biological processes such as cell metabolism, proliferation, survival, etc. as well as plasma applications in bacterial inactivation, wound healing, cancer treatment and some others. Challenges and opportunities for theoretical and experimental research are discussed and the authors’ vision for the emerging convergence trends across several disciplines and application domains is presented to stimulate critical discussions and collaborations in the future.

Introduction

Reactive atomic, molecular, and radical species play a major role in many chemical, biological, and other processes. The processes these species participate range from formation of nanometer-sized solid objects to effective control of physiological processes in living organisms. Of particular interest are reactive oxygen and nitrogen species (RONS) that are abundantly present or can easily be produced from ambient air. These species can then be delivered to the specific locations where their action is required. For example, this could be a surface of a solid nano-structured material which needs to be delicately oxidized in a microscopic localized area or a tissue or liquid culture medium containing living cells to be processed to induce the desired biological response (e.g., cell proliferation, differentiation or programmed death).

In the above two examples a significant precision is required in terms of controlled delivery of RONS (and some other) species to the surface and even into the interior of the non-living (nano-structured material) and living (cell medium or tissue) matter. This precision can be achieved using a dedicated device, which not only generates these species but also delivers them to the specified locations, with the required doses and precision.

This review focuses on the production of RONS and other reactive species, using non-equilibrium atmospheric-pressure plasma jet (N-APPJ) devices, and their delivery to liquid media that contain living cells, primarily aimed for application in the rapidly emerging field of plasma medicine. Even though many discussions are applicable to a much broader range of atmospheric-pressure plasmas, the N-APPJs have been chosen as one of the most common and effective types of the devices for which all the most important stages of the species production, transport, and interactions with liquids are well-studied and abundantly documented in the literature to allow for a critical and reasonably exhaustive review. This specific focus determines the scope and structure of this review.

This review aims to introduce the variety of processes associated with the generation of reactive species using non-equilibrium atmospheric-pressure plasma jet (N-APPJ) devices followed by the transport of these species from the point of generation in the plasma to the point of interaction with biological objects including cells and tissues as sketched in Fig. 1.

As can be seen from Fig. 1, generation and transport of the reactive species proceed in several stages. The species are produced in the plasma and then pass through the discharge afterglow before coming into contact with liquid media containing biological objects including cells, tissues, biologically relevant macromolecules such as enzymes, proteins, etc. The time scales and the prevailing elementary processes involved in the production and delivery of the reactive species vary significantly from one stage to another. For example, a typical duration of the elementary primary processes involved in the species creation, transformation, excitation, and ionization (e.g., through collisions with other species) is in the nanosecond to microsecond range, while the primary reactive species including electrons, ions, and excited species are generated. During the afterglow, some secondary reactive species such as O₃, NO, and NO₂ are generated. It typically takes microseconds to milliseconds for the reactive species to pass through the afterglow area of the plasma jet to reach the surface of the liquid medium containing biological material. During this time, some primary species disappear while other secondary species are generated.

On the other hand, when a plasma jet directly interacts with the surface of the liquid medium containing biological material, besides the relatively long-lifetime radicals mentioned above, the short-lifetime radicals including electrons, positive and negative ions, and some excited species could also play some roles in the treatment of biological material  [1].

Therefore, multiple transformations take place until the plasma-generated species reach the biological target. Importantly, on the one hand, some biological objects (e.g., cells) produce RONS through normal physiological processes and release into the medium. On the other hand, species generated by the plasma may pass through the liquid and penetrate into the cells and join the similar species that are produced inside the cells. These processes are inter-dependent making it very difficult to identify the specific channels of the species production in the liquid medium and inside the cells. Indeed, species generated by the plasma and delivered to the liquid media affect the species production inside and nearby the cells, while the species released by the cells into the medium, interact with the plasma-generated species and modify the kinetics of chemical reactions in the medium. The complexity and multitude of the chemical reactions and energy exchange processes leads to the typical duration of chemical and biochemical processes in the liquid media of approximately milliseconds to minutes. The biochemical and biological processes inside the cells and the processes of cell transformation are even longer commonly lasting from seconds to days, as sketched in Fig. 1.

The species delivered from the plasma to the biological objects interact across different regions in the gaseous and liquid states as shown in Fig. 1. This knowledge is critical to enable effective applications of such plasmas in biology, hygiene, and medicine  [2] as well as other areas including materials processing and nanotechnology  [3]. This is why in Section  1.2 we introduce non-equilibrium atmospheric-pressure plasma jets (N-APPJs) and some of their key attributes that lead to effective and controlled interactions of such plasmas with biological objects. Afterward, in Section  1.3 we briefly discuss the most common effects induced by N-APPJs and other non-equilibrium N-APPs on prokaryotic (bacterial) and eukaryotic (mammalian) cells. Wherever possible, accent is made on the specific effects of RONSs (e.g., NO, O₃, OH, H₂O₂, etc.) generated by the plasma on various types of cells such as bacterial pathogens or malignant cancer cells. These examples suggest that N-APPJs have emerged as effective tools for the production and delivery or reactive species, both in the charge-neutral and ionized forms. The N-APPJs represent a broader class of atmospheric-pressure plasma streamer discharges and are often termed guided ionization waves or guided streamers  [4].

The focus in this review lies in the diverse approaches and specific techniques for the numerical (Section  2) and experimental (Section  3) studies of the propagating plasma jets and the species generated in such jets, from the point of generation to the afterglow (experimental Section  4 and theoretical Section  5) and to the liquid media (Section  6) containing biological material. This is followed by the discussion of some of the possible mechanisms of biological effects involving reactive species of particular interest in plasma medicine applications (Section  7). Section  8 provides a brief summary of the challenges and opportunities in both the theoretical and experimental studies of N-APPJs and biological effects they cause. The review concludes with a brief discussion of the future trends and outlook for the development of this interesting and rapidly emerging field of research.

The quest to generate non-equilibrium, low-temperature plasmas at atmospheric pressure started in the early 20th century. In the 1930s, von Engel tried to generate such plasma by controlling the temperature of the cathode  [5]. However, it was only in the late 1980s and early 1990s when reports on successful generation of stable, relatively large volume, non-equilibrium, diffuse atmospheric-pressure plasmas started to emerge  [6], [7], [8]. These early works used the dielectric barrier discharge configuration and He as the operating gas. Sinusoidal excitation with voltages in the kV range and frequencies in the kHz range were used. In late 1990s and early 2000s, in order to enhance the plasma chemistry, fast rise time voltage pulses with pulse widths in the nanosecond-to-microsecond range were used. These pulses more effectively couple energy to the electron population resulting in better control of the electron energy distribution function (EEDF) and therefore better control of the plasma chemistry  [9], [10], [11], [12]. These plasma discharges are relevant to plasma processing including applications in biology and medicine that emerged in the mid-1990s  [13], [14]. It is presently commonly accepted that non-equilibrium atmospheric-pressure plasmas (N-APPs) generate reactive species that can be transported to biological targets and induce certain effects. Earlier works mostly aimed at inactivation of bacterial cells for applications in sterilization, hygiene and wound healing.

As research on the biological and medical applications of plasmas advanced, the demand has much increased for devices that can deliver reactive species well beyond the volumes limited by the electrodes or discharge tubes where the plasma is generated. This need has led to the development of plasma sources that are able to deliver the plasma plumes into the surrounding environment. These devices came to be known as non-equilibrium atmospheric-pressure plasma jets (N-APPJs)  [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31].

Various plasma jets and torches had previously been developed and used for a range of other applications such as surface modifications or gas reforming. However, the gas temperature in these jets and torches was often too high to be used on soft materials such as easy-to-melt polymers or biological cells and tissues. For instance, the RF atmospheric-pressure plasma jet developed in the late 1990s by Selwyn and co-workers was successfully used to kill bacteria  [32]. However, the gas temperature $T_g$ in this N-APPJ device was relatively high ($T_g>70°C$). While this discharge still belongs to the class of low-temperature plasmas (because of the very low electron energy compared, e.g., to high-temperature fusion plasmas), it cannot be applied to treat skin or soft tissues without causing serious thermal damage (e.g., burns).

Other plasma jet configurations, developed during the last decade, generated biologically tolerable plasmas with the gas temperatures not exceeding $40°C$   [15], [33]. Most of these devices use noble (e.g., He or Ar) operating gases with or without admixtures of oxygen or air. Using such gases or gas mixtures, low-temperature plasma plumes up to several centimeters in length have been routinely generated. This was achieved using continuous wave or pulsed power delivery modes and frequencies ranging from DC to RF and even microwave  [15]. The length of the pulse-driven plasma plumes depends on the applied voltage, the pulse width, repetition rate, pulse rise time, and gas flow rate. The plasma plumes can have small cross-sections at their tips thus producing localized effects with the precision as high as a few tens of micrometers. This is a very useful feature for high-precision biomedical treatments.

Fig. 2 shows some typical plasma jet configurations that have been used to generate plasmas that are suitable for biological applications. Some other configurations such as single-electrode devices have also been developed.

Importantly, plasma jets are not continuous plasma glows, as they appear to the naked eye, but are made of plasma bullets, propagating at very high velocities, up to ${10}^{5}m/s$   [34], [35], [36]. Over the last decade extensive experimental and modeling work has been carried out to understand how the plasma bullets are generated, why they travel at such high velocities, and why they stop and quench centimeters away from the device nozzle  [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]. The dynamics of the plasma bullets can be explained by the model that takes into account the high density of seed electrons and photo-ionization processes  [16], [35].

Further studies revealed that strong electric fields at the head of the bullet play a crucial role in their propagation. The strength of this electric field was measured by several groups to be 15–20 kV/cm in average  [49], [50], [51]. As such, the plasma bullets are fast ionization waves propagating in a guided fashion within the channel formed in the gas flow. This is why this type of electrical breakdown is commonly known as guided ionization waves  [4]. Extensive coverage of the physics of guided ionization waves can be found in a recent review  [4]. Fig. 3 shows a typical example of the plasma bullet propagation.

Because the plasma bullets are produced in a repeatable and predictable way, plasma jets can be applied with spatial and temporal controllability required for common biomedical applications that are introduced in the following section.

Plasma medicine is currently a mature and rapidly developing field of research  [52], [53], [54], [55], [56] which took its origin in early 1990s. Following earlier works on the inactivation of bacteria by N-APPs  [13], [14], [32], the US Air Force Office of Scientific Research (AFOSR) established a research program to evaluate the effects of low-temperature atmospheric-pressure plasmas on biological cells. This program aimed to study the efficacy of plasmas to destroy pathogens and the possibility of applying these plasmas to disinfect wounds and facilitate healing. The envisaged applications were decontamination of biotic and abiotic surfaces and media suitable for deployment in battlefield hospitals to disinfect and treat the wounds.

Parallel to the work done in the US were experiments conducted in Russia where the plasma was used as a source of nitric oxide (NO) for wound therapy. These experiments became known as “Plasmadynamic Therapy” of wounds  [57]. In vivo trials on mice and humans suggested that the plasma-based therapy enhanced phagocytosis and accelerated the proliferation of fibroblasts.

By the early 2000s, it was revealed that low doses of N-APP exposure can induce effects such as cell detachment or even apoptosis in some eukaryotic cell lines  [58]. The RF plasmas used in this study were produced at the tip of a thin metallic needle.

These and some other groundbreaking results have stimulated the new multidisciplinary field which is presently known as Plasma Medicine. Interested reader may refer to non-exhaustive representative publications  [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89], [90], [91].

As mentioned above, research on the biomedical applications of low-temperature atmospheric-pressure plasmas was initially focused on sterilizing harmful microorganisms (e.g., bacteria, fungi, spores) on both biotic and abiotic surfaces. It was established that N-APPs produce a mixture (often termed cocktail) of highly-reactive chemical species including reactive oxygen species (ROS) such as O, O₂⁻, O₃ and OH and reactive nitrogen species (RNS) such as NO and NO₂. These species and some of their reaction products are presently well known to exhibit strongly oxidative properties and can trigger signaling pathways in living cells. These species play important roles in cell physiology, metabolism and growth, immune responses, aging and several other cell processes. Some of these functions and the effects of the plasma exposure are discussed in Section  7.

Experiments on eukaryotic cells demonstrate that under some conditions, N-APPs appear to cause little damage to living animal and plant tissues. For example, skin fibroblast cells usually remain viable under relatively mild doses of the plasma exposure that can be lethal to bacterial cells. The proliferation of fibroblasts is a critical step in the wound healing process. The demonstrated ability of plasmas to kill antibiotic-resistant bacteria and to accelerate the proliferation of specific tissue cells opens the possibility to use plasmas to assist in chronic wound healing. Importantly, this ability has been related to the plasma-generated biologically-relevant reactive species briefly mentioned above.

N-APPs can also induce selective death of cancer cells while leaving normal cells intact. It is very likely that this process is mediated by the plasma-generated reactive species, for example through the activation of specific signaling pathways that involve a cascade of intracellular biochemical reactions eventually leading to cancer cell death. These results suggest that plasmas represent an effective and highly-promising tool in certain cancer therapies, perhaps in combination with other anti-cancer modalities such as surgery, radiation therapy and chemotherapy.

The biomedical applications of N-APPs are wide and continue experiencing rapid growth. Because the main topic of this review is on reactive species generated by N-APPJs rather than on various biomedical applications of N-APPJs, the following part of this section will only present a few selected examples of the effects of N-APPs on prokaryotic, eukaryotic, and cancer cells.

The inactivation of bacteria was the first biological application of N-APPs. Dielectric barrier discharges (DBD) were mostly used in the early trials, while N-APPJs became more and more popular with time, as the obvious benefits of the N-APPJs were recognized by the community. These and some other N-APP discharges have been successfully applied for inactivation of both gram-positive and gram-negative bacteria with various degrees of success. Fig. 4, Fig. 5 present typical results obtained by a DBD source and by a plasma jet, respectively.

Quite similar results were obtained for other types of bacteria. The level of log reduction or the size of the inactivation zone strongly depended on the type of bacteria, the initial concentration (bio-burden), and on the medium on which the cells were seeded. Importantly, sufficiently long exposure to N-APPJ leads to effective inactivation of sporulated gram-positive bacteria, which are otherwise very difficult to kill. It was also frequently observed that similar doses of plasma exposure affect planktonic bacterial cells much stronger than biofilms. This result is reasonable because biofilms are formed when a community of bacterial cells self-organizes to protect itself by forming an extracellular glue-like polysaccharide film. In this case, reasonably long exposure times (typically of the order of tens of minutes) lead to the effective (yet not always complete) biofilm destruction  [94], [95].

The sub-cellular structures of Eukaryotes are quite different compared to prokaryotes. A crucial difference is the existence of a nuclear membrane in eukaryotic cells. In addition, many sub-cellular units important for cell function are contained within membrane-bound organelles. Therefore, eukaryotes, such as mammalian cells, respond to extracellular physical and chemical stresses in a different manner than prokaryotes, such as bacteria, do. Here, we introduce two examples that illustrate the effects of N-APPJs on two eukaryotic cells: healthy fibroblast and cancerous (squamous cell carcinoma) cells.

Treatment of the fibroblast cells using different plasma exposure times (doses) has led to significant phenotypic changes  [96]. Importantly, these changes have been related to an increased flux of reactive species into the media that modify the intracellular signaling cascades leading to the altered cellular states  [96].

A quite similar treatment was applied to human bladder cancer cell line SCaBER  [97]. Fig. 6 shows the results of the SCaBER cancer cell viability. The counts immediately after plasma jet-treatment (labeled 0 h in the figure) reveal no dead cells which suggest that there were no immediate physical effects and that the interaction between the plasma-generated chemical reactive species and cancerous cells requires sufficient time to show an effect. However, at 24 h post-plasma treatment the viability of cells reduced to around 50% and to 75% for 2 min and 5 min plasma exposure, respectively. Therefore, the higher the dose of the plasma exposure the lower is the survival rate of the cells. Indeed, 5 min-long plasma treatment diminished the viability of SCaBER cells to approximately 10% observed at 48 h after the plasma treatment  [97].

The experimental results discussed above show that N-APPs affect biological cells (prokaryotes and eukaryotes) in a dose-dependent manner. The response of eukaryotic cells is not immediate but delayed, which indicates that the N-APPs do not immediately act on the cells with “brute force” (e.g., physical or chemical) but rather initiate cascades of biochemical events. These events take effect over typical time scales for relevant biological processes summarized in Fig. 1. The above examples and plentiful results of other researchers suggest that the reactive oxygen and nitrogen species and their reaction by-products are the most likely agents that cause the observed effects of N-APPs on living cells.

This is why the following sections present the details of the theoretical and experimental studies aimed to quantify the composition, concentration and fluxes of these reactive species. This knowledge is indispensable for better understanding of the complex interactions of the plasmas with liquids containing biological material.