AVS2015 Session PS+BI+SM-TuM: Plasmas for Medicine and Biological Applications

Non-equilibrium atmospheric pressure plasmas interacting with liquids offer a unique source of highly reactive chemistry beneficial for many applications in biology, medicine and advanced materials manufacturing. It has been shown that these plasma-liquid interactions can lead to inactivation of bacteria and virus and the synthesis of nanoparticles. Nonetheless the underpinning mechanisms are at least poorly understood. My group has been strongly involved in the study of the reactive chemistry of a well-characterized RF driven atmospheric pressure plasma jet and its interaction with liquids.

The presentation will highlight some examples of reaction pathways responsible for the inactivation of bacteria and virus in solution and the synthesis of silver nanoparticles from AgNO₃ solutions. I will illustrate the importance of reactive plasma chemistry induced by neutral gas phase reactive species such as OH, H₂O₂ NO, O, H, O₃ and singlet oxygen. In addition, we will show that UV emission, which is often neglected as a possible mechanism, can be important in some cases.

Low temperature plasma research directed towards biomedical applications such as sterilization, surgery, wound healing and anti-cancer therapy has seen remarkable growth in the last 3-5 years, but the mechanisms responsible for the biomedical effects have remained mysterious. It is known that CAP readily create reactive oxygen species (ROS) and reactive nitrogen species (RNS). Other potentially important plasma-generated species effects include charges, fields and photons. ROS and RNS (or RONS), in addition to a suite of other radical and non-radical reactive species, are essential actors in an important sub-field of aerobic biology termed ‘redox’ (or oxidation-reduction) biology. I will review the evidence suggesting that RONS generated by plasmas are responsible for their observed therapeutic effects. In addition, I will present several ideas about the most likely biological response mechanisms that are likely involved in therapeutic plasma biomedicine.

Two percent of the US population suffers from chronic non-healing wounds, often complicated by antibiotic-resistant bacterial infections, and the staggering cost of wound care exceeds $50 billion per year. Of increasing concern are multi-drug resistant bacteria, including methicillin-resistant Staphylococcus aureus and multi-drug resistant Pseudomonas aeruginosa infections. Within wounds, these bacteria adopt a biofilm-like state, and become notoriously recalcitrant to conventional antibiotic therapies. Currently approved products for the treatment of chronic wounds have not proven to be a panacea due to the complex nature of wound healing.

The ideal therapy for chronic, infected wounds would be non-painful, bactericidal without risk of resistance, able to break-up biofilms and enhance wound healing. Recently, there has been interest in the use of cold atmospheric plasma (CAP) technology for the treatment of infections and non-healing wounds. The technology could potentially fulfill the requirements of an ideal wound healing therapy. CAP devices producing ionized gas have been developed that can operate in ambient air and that are safe to touch without any pain sensation. CAP generates a complex mixture of reactive oxygen and nitrogen species (RONS) that are able to kill bacteria, while stimulating host cell growth. CAP has the potential to combine antiseptic and wound healing capabilities in a single treatment procedure and could eliminate the risk of cytotoxicity present in many current treatment methodologies for infected wounds.

The effects of CAP on bacteria and mammalian keratinocytes and fibroblast cells have been evaluated in vitro. Our prototype argon CAP device produces antibacterial effects on planktonic bacterial cultures of S. aureus and P. aeruginosa at a maximal treatment duration of 20 ml/min at conditions that do not impact cell viability of fibroblasts and keratinocytes in vitro. We have also recently demonstrated that CAP is effective in reducing the viability of P. aeruginosa biofilms grown in vitro. When grown on the surfaces of PVC microtitre plates for 48 h, argon-air derived CAP treatment of established biofilms showed a 95% reduction in cell viability, as determined by resazurin fluorescence, relative to untreated controls, when treated at a dose of 30min/ml, which is similar to the treatment time equivalent of mammalian cell treatment.

As part of the early investigations of the use of CAP treatment as a viable therapy for chronic-infected wounds, the presentation will focus on bacterial biofilm reduction by CAP treatment in vitro as well as in a mouse skin wound model. The effects on mouse host cells will be examined.

The surface micro-discharge (SMD), due to its scalable large area and flexibility of working gases, has great potential for many applications such as material processing and plasma medicine. The SMD normally works under ambient air conditions that contain not only N₂ and O₂ but also water vapor which can have a large impact on both the discharge behavior and plasma gas chemistry. In this study, we evaluate the effect of ambient humidity on SMD in various N₂/O₂ mixtures and the subsequent effect on the surface modification and bio-deactivation of lipopolysaccharide (LPS). Electrical behavior and optical emission spectrum (OES) of the SMD source were studied. We found that while the additional moisture did not help create strong OH (A-X) emission from SMD, it resulted in lower plasma density and extra power dissipation. We used X-ray photoelectron spectroscopy (XPS) to characterize the surface modification of LPS after treatment. We found that all SMD-treated LPS surfaces show oxygen uptake and formation of surface-bound NO₃, while the amount of these modifications was strongly dependent on the ambient gas composition. By comparing the XPS of wet-treated (50% relative humidity at 20 °C) surfaces with their dry counterparts, we find that the water vapor reduces both the oxygen uptake and surface NO₃ formation, and that the difference between wet- and dry-treated surfaces decreases with the increasing fraction of ambient N₂. When the N₂ fraction is up to 80% (synthetic air), the LPS surface shows comparable amount of modification with or without humidity. Among all the dry- and wet-N₂/O₂ mixtures, the dry 5% of N₂ ambient shows the greatest modification rate. We also evaluated the bio-deactivation efficiency of the SMD on LPS using enzyme-linked immunosorbent assay. Similar to surface modification, we found that the bio-deactivation rate of SMD in dry ambient is much higher than that of SMD in their wet counterparts, except the synthetic air condition which shows similar amount. The authors gratefully acknowledge financial support by the US Department of Energy (DE-SC0001939) and National Science Foundation (PHY-1004256 and PHY-1415353).

Atmospheric pressure, non-thermal plasma DBD is increasingly used in many processing applications. Despite their widespread usage, it remains largely unknown whether cold atmospheric plasma DBD maintains similar characteristics, such as gas temperatures and particle flux, when they breakdown while arcing or whether they possess different operating modes. It is essential for laboratory/industrial adoption of such plasmas that plasma diagnostics of the process are provided. Optical emission and absorption spectroscopy have been used as diagnostics techniques with an added avantage of their non-intrusive nature.

The type of operating gas influences the stability of atmospheric plasma discharges. In this study is used a sealed meat package filled with one of two gas mixtures: O₂-CO₂ and N₂-CO₂ . Different concentrations of nitrogen or oxygen and carbon-dioxide could cause the transition from a stable homogeneous discharge into a filamentary discharge. Atmospheric plasma discharges are affected by the surrounding ambient air, and for sealed packages from transfer between the package gas and the surrounding ambient atmosphere. In the vast majority of atmospheric plasma discharges, reactive nitrogen species dominates the ionic composition of atmospheric discharge and has an impact on the breakdown voltage. When N₂ is added/mixed with CO₂ plasma discharges, the CO₂ emission lines are significantly quenched. In the case of O₂-CO₂ chemistry, nitrogen is not a carrier gas but it still present in the package due to contaminant transfer with the surrounding ambient air, modifying the plasma chemistry in the package. The plasma’s optical spectrum in O₂-CO₂ chemistry shows molecular oxygen, nitrogen and OH peaks. Oxygen could come from the ambient air, the O₂-CO₂ gas mixture or from humidity in the package. Electron impact excitation of molecular oxygen, at low collision energies, is of particular importance because of its role in atmospheric physics and has been objective of this study. In our study we have also recorded the O₃ band-head that belongs to the Hartley Band. Ozone plays very important role for the biological aspect of this study and shows the highest change in a concentration with the processing time. Combining the results from spectral radiation in the package provides an electron energy distribution function. The study includes a detailed experimental investigation of the spatial and temporal spectroscopic data and links them with plasma kinetics.