Variable Frequency Atmospheric Plasma Source for Skin Treatment Applications

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

University College Dublin

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.

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

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

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isch

arge

20 kV power supply

Teflon tube(72 mm x 15 mm)

Work surfaceGap distance

Needleelectrodes

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.

Optimal Plasma Treatment Regime

• Optical Emission Spectroscopy (OES) is a qualitative elemental analysis used to detect chemical species in the plasma.

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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

Effect of Plasma Treatment on E.Coli

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Control 140 kHz 160 kHz 180 kHz

Plasma for 2 Minutes at 140, 160, and 180 kHz

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Plasma at 160 kHz for 2, 4, 6 minutes

140 kHz 180 kHz 160 kHz Control 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.

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.

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.

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.