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EWMA 2013 - Ep449 - Variable Frequency Atmospheric Plasma Source for Skin Treatment Applications

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Ahmed Chebbi, Claire Staunton, Vic Law, and Denis Dowling

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EWMA 2013 - Ep449 - Variable Frequency Atmospheric Plasma Source for Skin Treatment Applications

  1. 1. Variable Frequency Atmospheric Plasma Source for Skin Treatment Applications Ahmed Chebbi, Claire Staunton, Vic Law, and Denis Dowling University College Dublin
  2. 2. What is Plasma? • Defined as the fourth state of matter. • When adding energy (DC, microwave, radiofrequency) to a gas, the electrons separate from the nucleus and move around freely producing ionized gas (plasma). • 99% of all matter in the visible universe is in the state of plasma. • Atmospheric plasma has been used extensively in industry for surface treatment, activation and modification.
  3. 3. Plasma Medicine • Plasma medicine is the intersection of plasma science and technology with biology and medicine for: – Plasma based sterilization (e.g. for medical devices) – Direct therapeutic plasma applications (e.g. for wound healing) – Plasma modification of biomedical surfaces (e.g. for hip implants) •Cancer treatment •Blood coagulation •Dentistry •Orthopedics •Wound healing •Skin disease
  4. 4. Plasma Medicine for Wound Healing • What’s in plasma? UV, heat, Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS)… • The plasma wound healing effect is obtained through a reduction of the bacterial load count on the wound surface, and in some case through the enhancement of critical phases in the wound healing cascade. • Reactive oxygen and nitrogen species produced by the plasma are mainly responsible for this bactericidal effect. • Plasma processing parameters affect the production of reactive species. • An optimal treatment regime needs to be identified in order to obtain the highest bactericidal effect. A 61-year-old patient with venous ulcers: wounds before plasma treatment (a), after 7 (b) and after 11 treatments (c). With a daily plasma therapy (MicroPlaster®) of 2 min. At the beginning of plasma treatment Klebsiella oxytoca and Enterobacter cloacae were detectable, after 11th treatment (23 days later) swabs were sterile*. * Plasma Medicine: Possible Applications in Medicine, Journal of German Society of Dermatology, 2010-8
  5. 5. Volumedischarge 20 kV power supply Teflon tube (72 mm x 15 mm) Work surface Gap distance Needle electrodes HDPE HV Probe Plasma plume Atmospheric Plasma Jet at University College Dublin Variable frequency power Quartz tube Glass Processing parameters: Voltage: 0-300 V Frequency: 0-500 kHz Helium Flow rate: 0-20 l/min Helium Gas • Study objectives: • Using a novel variable frequency plasma source, to identify an adequate plasma processing window for the treatment of skin (wound healing applications) without causing damage,. • To demonstrate the effect of plasma treatment on bacterial cells.
  6. 6. Optimal Plasma Treatment Regime • Optical Emission Spectroscopy (OES) is a qualitative elemental analysis used to detect chemical species in the plasma. 0 5000 10000 15000 20000 25000 0 50 100 150 200 250 300 350 400 450 500 Intensity(a.u.) Frequency kHz OH NO2 O He OES Probe • The frequency of the plasma power supply was varied from 0 to 500 kHz in order to identify the treatment regime which yields the highest production of active species. • The highest production of NO and OH was found at a frequency of 160 kHz (with 100 V and 10 l/min of helium flow rate). 160 kHz
  7. 7. Effect of Plasma Treatment on E.Coli 1 10 100 1000 Control 140 kHz 160 kHz 180 kHz Plasma for 2 Minutes at 140, 160, and 180 kHz CFU1013/ml 1 10 100 1000 10000 100000 1000000 Untreated 2 min 4 min 6 min CFU/mlx106 Plasma at 160 kHz for 2, 4, 6 minutes 140 kHz 180 kHz160 kHzControl Untreated 2 min 4 min 6 min The highest production of active species resulted in the highest reduction of bacterial load count in vitro. Longer plasma exposure results in higher reduction of bacterial load count ex vivo on pig skin samples. In order to correlate the production intensity of plasma species with the bactericidal effect: different frequencies were used to treat solutions containing the same amount of E.Coli. After plasma treatment and serial dilution, 100 µl was spread on agar plates and left overnight. In vitro Ex vivo An ex vivo pig skin model was used to investigate the effect of plasma exposure time on the bactericidal effect. Fresh pig skin sample were used and a known amount of bacteria was placed on the surface. After plasma treatment, skin samples were PBS washed and a 100µl dilution spread on agar plates.
  8. 8. Sensitivity of Wound Pathogens to Plasma Treatment Order of susceptibility to atmospheric plasma treatments Klebsielle (G-) > E. coli (G-) > P. Aeruginosa (G-) > S. Aureus (G+) > B. Subtilis (G+) • A comparison was made on the sensitivity of both gram negative and gram positive bacteria to the plasma treatment under the same processing conditions. • Gram negative bacteria were found to be far more susceptible to the treatments (e.g. E.coli (Gram negative) and B. subtillis (Gram positive)). • The relative lack of structural damage observed for Gram-positive bacteria is due to their thicker murein layer, making them more rigid and thus increasing their tensile strength.
  9. 9. Flow Cytometry Analysis L) Untreated control sample, no dye uptake (healthy cells) R) 2 min plasma treatment: 3 populations corresponding to healthy cells, depolarised membrane (BOX) and permeabilised membrane (PI, full cell death) • The bacterial cell (E.coli) is exposed to a fluorescent dye, which is adsorbed only when damage occurs. Two flourescent dyes were used during this study: Bisoxonol (BOX) for the detection of a depolarised membrane and Propidium Iodide (PI) for the detection of a fully permeabilised membrane. • With increasing plasma intensity the progression towards cell death was clearly evident. The mechanism observed was initially a decrease in cell membrane potential culminating in full membrane permeabilisation as shown by the initial uptake of the dye BOX, followed by the uptake of PI.
  10. 10. Conclusions  We demonstrated an antibacterial activity of the variable frequency helium plasma system in vitro (E coli solution) and ex vivo (pig skin).  We optimized the plasma treatment for maximal bacterial load reduction while minimising damage to pig skin.  A once-off plasma treatment for 120 seconds led to a 1 log reduction of bacterial count load in vitro and on pig skin samples inoculated with E.coli.  Higher treatment times of up to 6 minutes led to a 4 log reduction in bacterial count load ex vivo (on pig skin).  Gram negative bacteria were more susceptible to plasma exposure than gram positive bacteria.  Flow cytometry data showed that the cell breakdown pathway consists of membrane depolarisation and eventual permeabilisation.

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