Biomedical applications of plasmas E. Stoffels, Eindhoven University of Technology The Netherlands

1. Biomedical applications of plasmasE. Stoffels, Eindhoven University of TechnologyThe Netherlands  Plasmas in material processing: thermal vs. non-thermal Plasmas in medicine: service: plasma decontamination “spare parts”: plasma coating of implants healing: plasma surgery  New: minimum-invasive surgery plasma-tissue interactions on cellular level

2. + - A+, B-, e, radicals What is plasma? • Almost everything! • Ionised gas: • thermally (fire) • electrically (gas discharge) • radiatively (ionosphere, interstellar space) • Artificial plasmas: generated by electric discharges:

3. Plasmas in material processing  Plasmas can perform various surface treatment: etching - semiconductor elements deposition - a-Si:H solar cells, hard protective coatings, optically active layers, etc. cutting, welding, spraying  Refined treatment is possible with non-thermal plasmas (no heat damage to the surface)  Also biological materials can be treated!

4. Thermal (fire) Non-thermal(vacuum reactor)

5. How to obtain non-thermal plasmas?  Electric gas discharges: electrons/ions are heated  In typical high-frequency plasmas only electrons are heated  No background gas heating when: electrons/ions in minority not enough collisions with neutrals Thermal: gas heating occurs Non-thermal: electrons hot, gas cold

6. Non-equilibrium cases low pressure: low collision frequency of electrons with gas electrons retain high energy, gas remains cold (typical situation in vacuum reactors)  short duration of the plasma pulse: not enough time for gas heating small size (micro-plasma): too much energy leaks to outside

7. Small size plasma  What is the maximum length scale (L) of a “cold” plasma?  Electron-induced heating is balanced by thermal conduction losses:  We allow DT to be at most 10 degrees.  For helium under typical conditions L 0.2 mm (in agreement with observations).

8. “Plasma needle”  RF applied to a sharp metal pin.  breakdown obtained at ca. 200 V p-p.  plasma operates in helium (most readily), argon, nitrogen, hydrogen, AIR (!)

9. Characteristics of the plasma needle

10. small size expansion Really cool! Temperature measurements using Optical Emission Spectroscopy (N2 bands) using thermocouples

11. Ar Other geometries  HF plasma pencil (Janca et al. Brno, Czech Republic) discharge created in a hollow needle/hollow cylinder in argon various surface treatments  Micro-hollow cathode discharge (Schoenbach, Norfolk, Virginia; Graves, California, Berkeley)

12. Dielectric/high resistivity material  Dielectric barrier discharge DBD (e.g. Kogelschatz, ABB Corp. Research; Chang-Jun Liu, Tianjin, China) UV radiation gas conversion (e.g. methane)  Resistive barrier discharge (Laroussi, Old Dominion Univ. Virginia) bacterial decontamination

13.  Atmospheric pressure plasma jet APPJ (Selwyn, Los Alamos) radio-frequency (13.56 MHz) material processing  One atmosphere uniform glow discharge plasma OAUGDP (Reece Roth et al. Univ. of Tennessee) kHz frequency range operates in air needs cooling used for sterilisation

14. Plasma sterilisation (medical tools) • Low temperature needed because of usage of plastic tools. • Plasma is non-toxic and efficient (seconds to minutes) • Both atmospheric (Reece Roth, Laroussi) and reduced pressure discharges (Moisan) are used. • Large-area discharges, AC, radio-frequency and microwave.

15. Air deconamination • Removal of bacteria/bacterial spores from ambient air (e.g. anthrax) • Protection from biological attacks • Example: a gas phase corona reactor design by Birmingham et al. (MesoSystems Technology Inc., Richland)

16. Water purification • Under water corona discharges (e.g. Sunka, Prague, Chech Republic; Van Veldhuizen, Eindhoven, The Netherlands) • corona streamers propagate in contaminated liquid • not only biological threats can be removed; dangerous chemicals are decomposed. pulsed corona for water cleaning (V. Veldhuizen, Eindhoven)

17. Destroying enemies • prokaryotic organisms, not very complicated • most of them well-protected by cell wall

18. Destroying enemies • Gram positive vs. gram negative single plasma membrane double membrane • Spore forming (typically more difficult to gram positive) destroy bacillus, clostridium • most of them well-protected by cell wall endospore escherichia coli

19. Mechanisms? • No sophisticated damage needed: necrosis • Atomic oxygen vs UV radiation. • Typically three phases observed. • Moisan and coworkers (Univ. of Montreal) proposed bacterial/spore de-activation mechanisms Erosion by photodesorption and O radical etching UV damage to eroded spores Direct UV absorption and DNA destruction log(number of survivors)  1 min 10 min 1 min exposure time

20. Chemical effects • Various chemistries studied argon - not very efficient N2/O2 (air) - efficient, O radicals present O radicals can also penetrate through the membrane and damage the cell interior H2O2, CO2- particularly efficient, see Hury et al. H2 - efficient, reducing agent. Maybe reducing fatty acids to aldehydes and dissolving the membrane?

21. Decontamination of other surfaces?  In vivo dental cavities using plasma needle  No temperature increase within the tooth  Mineral matrix intact, tissue-saving method  Under investigation: decontamination efficiency, penetration depth, surface activation to enhance adhesion of filling  Pain?  Others: root treatment , gingiva reattachment

22. Plasma coating  Coating of artificial implants to increase bio-compatibility  Low-pressure discharges can be used  Examples: bone prostheses: diamond-like inert coating on titanium substrate spraying of hydroxyapatite micro-patterning of surface to increase cell adhesion

23. Plasma treatment in vivo  Not always non-thermal plasmas are used, sometimes burning is desired  Techniques already implemented in medicine: electro-surgery and argon plasma coagulation  Spark erosion of atherosclerotic plaque  New trends: minimum invasive, tissue saving methods  Investigation of fine surgery using plasma needle

24. Electric methods in medicine  Electrosurgery: well established technique  High-frequency (350 kHz) cutting and coagulation: ERBE monopolar & bipolar cutting devices well-reproducible cutting little adhesion hemostasis obtained by controlled coagulation

25. From electricity to plasma First beneficial plasma-tissue interaction * non contact * self-limiting desiccation and coagulation (plasma stops when the area is dry) * no carbonization * can be applied internally * good post-operative recovery  Argon plasma coagulation

26. An APC device in action Example: treatment of hyperplasia of the nasal concha After APC treatment 10 days later

27. Spark erosion Plaque is vaporized by electric pulses, 250 kHz, 1200 V, 100 W. Restenosis in rabbits is limited. So far not applied to humans. A diseased artery Plasma-produced crater (cross-section): (lipid ablation): • Developed by dr. C.J. Slager (Erasmus Univ. Rotterdam)

28. A few words about safety Nerve stimulation by electric currents Effects of heat: hyperthermia causes cell death (> 43o C)

29. Plasma needle on tissues (a) Low-power regime: no thermal damage, possibility of refined action. (b) High-power regime: denaturation of proteins, carbonisation after long exposure. Possibility of fine surgery must be investigated on cellular level!

30. Next step: eukaryotic cells  Much more complicated structures  More interactions/effects possible  For the sake of plasma surgery, understanding plasma-induced effects on cellular level is necessary

31. Plasma-cell interactions • Cell removal: • “coarse destruction” - necrosis (damage to the membrane) caused e.g. by chemicals. • “fine works” - programmed cell death (apoptosis)

32. Apoptosis • Moderate damage to the cell, without affecting the membrane integrity. The cell shrinks, DNA in the nucleus condenses. • Cell disappears without infection, as desired in fine surgery.

33. CHO-K1 cells in culture • CHO-K1 cells (Chinese hamster ovary) , fibroblasts • Plasma treatment followed by viability assays • Fluorescent staining and observation under confocal fluorescence microscope Cell Tracker Green (CTG): stains living cells green Propidium Iodide (PI): stains dead cells red, allows to resolve DNA/RNA distribution in the cell

34. Healthy cells:  To study long term effects cells are cultured after plasma treatment  Cells are fixed and stained with PI to detect apoptosis  Intact nuclei stained by PI

35. General features of plasma treatment  High precision: influenced cells are strictly localised) detachment 50 mm alive dead

36. Plasma treated cells Example of apoptotic cells After plasma treatment

37. Plasma treated cells: dead cells Even dead cells retain the integrity! DNA damage and condensation (other than in apoptosis)

38. Cell detachment Instantaneous effect of plasma treatment  Cells round up and detach from other cells and can be removed  Better (faster) than apoptosis?

39. Long term effects Detached cells reattach after ca 1 hour, their viability is demonstrated 15 min control 4 hours 1 hour

40. Ca Ca Ca NH2 membrane COOH cytoskeleton What causes cell detachment?  cell adhesion molecules (CAMs) - trans-membrane glycoproteins  Ca2+ dependent adhesion: cadherins  Detract Ca and cause cadherin to disintegrate? (only charging)  Destroy the cadherin

41. Summary • Plasma technology finds many medical applications. • Many atmospheric sources have been developed. • Plasma de-contamination is widely studied. • Plasma needle can be applied to organic materials without thermal damage. Questions • Can plasmas perform fine surgery? • Is cell detachment valid in tissue environment in vivo tests