Plasma needle : exploring biomedical applications of non-thermal plasmas

The plasma needle is a novel design of a radio-frequency discharge in helium/air mixtures at atmospheric pressure. The discharge contains neutral, excited and ionized particles, and emits ultraviolet (UV) light. It operates at low electric power and close to ambient temperature; it combines chemical activity with non-destructive character. Therefore it is expected that the plasma needle will be used in future in (micro) surgery, e.g. in wound healing and in controlled tissue removal through cell detachment or apoptosis, avoiding necrosis and in°ammation reactions. Focus of this study is both on optimization of needle design and on assessment of effects of plasma activity on living cells. This work is a pio- neering study of the effects of non-thermal plasma on biological samples. The design of the plasma needle was adjusted in such a way that instead of operating in a closed reactor, now the treatments could be performed in open air. Thus, larger samples could be treated and handling times were reduced. Then, a characterization of the needle was performed using electrical as well as optical diagnostics (Chapter 3). It was found that the needle operated at voltages of 140 Vrms and higher. A model was made to determine the resistance of the plasma and from this an estimation of the electron density could be made. The latter can be regarded as an indirect measure for plasma reactivity. Results from optical emission spectroscopy showed that reactive oxygen species, such as O¢ and OH¢, were produced in the plasma. Furthermore, UV emission was detected. Both the radicals and the UV are known to interact with cells and tissues. For applications, the amount of radicals that reach the sample or that are generated in the sample is important. For this reason, radicals were detected in liquid that was treated with plasma using a chemical technique (Chapter 4). It involved a fluorescent probe: the probe was dissolved in liquid and after reaction with specific radicals it became fluorescent. Radical density in the liquid depended on plasma conditions, treatment time, and amount of liquid used, but it was always in the micromolar range. These concentrations were found to be comparable with physiological concentrations that were stated in literature. Basic cell reactions after plasma treatment were determined by experiments on cultured Chinese hamster ovarian (CHO K1) cells (Chapter 5). One of these reactions was cell detachment: cells detached from their environment but remained alive after treatment. Other reactions included a small percentage of apoptosis and, when high plasma powers were used, necrosis. A comparison with the effect of UV light from UV lamps was made (Chapter 7). The main effect of UV treatment was necrosis, but only above a certain threshold value. For mammalian cells, this threshold was reasonably high. Thus, the ef- fects of plasma treatment could not be explained by the action of the UV light from the plasma. Quantitative experiments were performed on cultured bovine aortic endothelial cells (BAEC) and rat smooth muscle cells (A7r5) (Chapter 6). These two cell types constitute walls of blood vessels. It was shown that treatment times of less than one minute cause detachment of the cells if the layer thickness of the liquid that covered the cells was low (around 0.1 mm). This suggests that at short treatment times, the penetration depth of the plasma into the sample is limited. The percentage of necrotic cells was low after treatment. No difference was found in the detachment behavior of both cell types. Finally, pilot experiments were performed on carotid arteries of C57BL/6 and Swiss mice ex vivo (Chapter 8). They were studied using a two-photon laser scanning microscope (TPLSM). Cell nuclei, elastin bands, and collagen could be visualized. Preliminary results indicate that induced changes are not strongly dependent on applied energy if no heating e®ects are induced. Apparent effects were limited to the adventitia, probably due to a low penetration depth of active plasma species. In conclusion, we can state that the plasma needle is a non-destructive tool that can be ap- plied with precision. It has a superficial action and causes little damage to the tissue. The level of damage can be controlled to achieve a desired therapeutic effect. Both on cultured cells and on ex vivo arteries interesting effects were found that confirm the hypothesis that the plasma needle will have a future in surgery.