24/05/2017
1
AN INTRODUCTION TOPLASMA MEDICINE
What is Plasma medicine
Use of non-thermal plasmas for biomedical applications
Sterilization and sanification of settings, surfaces and food
Living tissues treatment for whound healing and cancer cells killing
Biopolymers surface treatment
24/05/2017
2
Non-thermal plasma – Why?Can be generated atmospheric
pressure No vacuum facilities are required
Low ionization energiesEnergy selectivity and thus an
efficient way to use it
Energetic electrons that enhance chemical processes Formation of new reactive
species
Heavy paricles low temperature
Use of these plasma in contact with heat sensitive surfaces
(skin)
Non-thermal plasmas - All in one
Conventional techniques Cold Plasmas
24/05/2017
3
Non-thermal plasmas - SourcesDielectric Barrier Discharge (DBD) Plasma Jet
Principal effects
24/05/2017
4
Charged particles: electrons• Charged particles are most abundant ones, that impact the surface under
treatment
• They are able to penetrate surfaces before interact with the matter (usually few μm).
• Electrons in water become solvated. Their mean free path becomes longer and subsequently their ‘life’ becomes longer too.
• Ifwater is present, electrons are able to produce hydroxyl radical ∙
→ ∙
• Electrons react with dissolved oxygen producing superoxide ion . This ion can be converted into hydrogen peroxide ( ) by means of a reaction called “superoxide dismutation”
2 2 ↔
• The presence of converts into hydroxyl radical or ion ( ∙, ), by means of metal ions (es: ), following the “Fenton reaction”:
→ ∙→
Charged particles: electrons
24/05/2017
5
Charged particles: ionsPositive ions: (es: )
• These particles are particularly interesting with the interaction with water (water can be found everywhere in biology)
• Charge exchange mechanism: hydroxyl radical ∙and so called ‘Plasma Acid’ are produced
→→ ∙
• Water ionization potential is quite low, thus this mechanism is easily activated. Discharge energy is used in a preferentially way to produce radical
∙.
• Ions may have catalytic effects and enhance other sterilization mechanisms.
Charged particles: ion bombardment
• Due to the high kinetic energy, ions can damage the external lipid layer of the cellular membrane. This behavior is enhanced at lower pressure, when ions energy is usually higher due to longer mean free paths.
24/05/2017
6
Electric Field• Electroporation has negligible effects in most applications, but can be useful
to transport oxidants or drugs into the cell.
• Charges deposited onto the cell membrane can create very intense electrostatic forces, able to break cell membrane itself. This effect is more evident over rough surfaces and characterized by small curvature radius (typically gram- bacteria)
• Efficacy is enhanced by localized electric field due to charges motion. This happens within DBD plasma streamers.
Oxidant neutral speciesThese species can be distinguished based on their half-life time
• Radicals ∙ and ∙, atomic oxygen and molecular oxygen exited species like metastable are extremely reactive. Despite this, their half-life time is quite low (lower than few microseconds). These species are effective only within the plasma or in regions close to it (direct treatment).
• Other neutral species like , NO, N are less reactive but their half-life times are quite longer (minutes ore more). These reactive species can be effective even outside the plasma volume (indirect or after-glow treatment). Usually these species are divided in Reactive Oxygen Species (ROS) like ozone, and Reactive Nitrogen Species (RNS) like nitrogen oxide.
24/05/2017
7
Neutral speciesCell membrane effects:
• The outer cell membrane is usually constituted by double lipid layers susceptible to oxidation. Unsaturated fatty acids are main constituents o this membrane. These acids give to the membrane a gel consistency, allowing the substances exchange between cell and outer world. Membrane proteins allow the passage of selected molecules cross the membrane. Proteins are susceptible to oxidation. Plasma action can interact with these proteins by mean of abovementioned oxidizing species..
Neutral species: oxidative damageThe main process with which cell membrane is destroyed, starts from the formation of hydroxyl radical ∙.• Ozone dissolved in water dissociates and reacts with R‐H organic molecules:
→→ R ∙
• Radical ∙ triggers a series of reactions making available new organic substrates R:
∙ → R H
• Radical ∙ reacts with substrates R producing a radical ROH ∙∙ → ROH ∙
This new radical can be oxidized by means of:• Metal ions.• A reaction between two ROH ∙ radical.
• Despite these chemical attacks, cells are able to repair limited oxidizing damages. When this damage is too significant, cells die.
24/05/2017
8
Neutral speciesDNA effects
• DNA is sensitive to oxidizing effects too. The result is the complete degradation of the cell.
Dimerizzazione della timina.SEM of S. Cerevisiae treated by ozone gas.
UV radiation• UV-C radiation owns high sterilizing properties (wavelengths between 100-
280 nm, especially in the range 200-300 nm). An energy dose of about few mJ/cm2 can induce cell death.
• Low pressure plasmas usually produce a quite high amount of UV-C radiation. On the other side, atmospheric pressure plasmas are not so efficient in UV ray production.
• Usually a synergy between UV radiation and other sterilizing agents present in the plasma takes place.
• UV radiation produces effects on DNA similar to those induced by neutral species
24/05/2017
9
Thermal effects• Thermal sterilization is one of most used sterilizing methods. Non-thermal
plasmas are characterized by low temperatures. Non-thermal plasmas do not sterilize due thermal effects.
• In non-thermal plasma microregions with high temperature can exist (1000°C).
• High temperature are usually unwanted in plasma medicine domain.
• Limited temperatures can enhance
the production of oxidizing species.
This is a typical example of plasma
synergy
Non-thermal plasma: ideal sterilizing agent• Efficacy over several typology of pathogen agents
• In-vivo application without live tissue damage
• Virtually absence of toxic/special/chemical products
• Cheap technology
• Low temperatures (T< 50 °C).
• Contact free: plasma can be used over irregular surfaces and with complex geometries.
• Scalable technology: plasma can be used in applications with large area substrates with affordable costs.
• Safe method for both operators and patients.
• High killing efficiencies
• Unlikely development of antibiotic resistance or allergic reactions.
24/05/2017
10
Sterilization and sanificationAntibiotic multiresistance of Stapphilococcus Auerus bacteria
Survival curves• Semi-logaritmic graph with survived microorganismsas a function of treatment
time (or energy dose).
• Usually sterilization is guaranteed when a 6-log reduction of pathogens population is reached (99,9999% of activation).
• Conventional sterilizing systems (steam, dry steam, chemical, UV…) applied to spores, usually produces a linear survival curve. This means that the number of survived microorganisms is an exponential function of treatment time.
24/05/2017
11
Survival curves• Plasma sterilizing treatments usually produce survival curves characterized
by more phases, generally constituted by several straight segments, with different slope.
• Treatment efficacy is still an exponential function of treatment time, but with different time constants in each sterilizing phase.
1) Destruction of outer layers and UV
damage of genetic material
2) Erosion of inactivated layers and
cell membrane due to ion
bombardment.
3) UV radiation and reactive species
penetration within survived cells
Direct VS indirect treatment• A direct treatment is operated when the plasma is in contact with the sample.
• With an indirect treatment, only plasma products reaches the sample (afterglow treatment).
• Afterglow treatment is characterized by the action of neutral species solely. Charged particles, electric fields and UV radiation are usually negligible.
Direct treatment Indirect treatment
24/05/2017
12
Bacteria treatment(E.Coli)
Control
10 min
Hueso, José L., et al. "Optical Emission Spectroscopic Evaluation of Different Microwave Plasma Discharges and Its Potential Application for Sterilization Processes." Plasma for Bio-Decontamination, Medicine and Food Security. Springer Netherlands, 2012. 121-132.
Spore treatment(G. Stearothermophilus )
Rossi, F., et al. "Low pressure plasma discharges for the sterilization and decontamination of surfaces." New Journal of Physics 11.11 (2009): 115017.
Control 30 s
80 s 120 s
24/05/2017
13
Zimmermann, Julia L., et al. "Effects of cold atmospheric plasmas on adenoviruses in solution." Journal of Physics D: Applied Physics 44.50 (2011): 505201.
(240 s)
Virus treatment(Adenovirus AdeGFPLuc)
Fungus treatment(Fungus growth on Pisum arvense seeds)
Filatova, Irina, et al. "Fungicidal Effects of Plasma and Radio-Wave Pre-treatments on Seeds of Grain Crops and Legumes." Plasma for Bio-Decontamination, Medicine and Food Security. Springer Netherlands, 2012. 469-479.
24/05/2017
14
Wound healing – live tissues treatment:treatment selectivity
• Plasma action is not the same with resect all treated cells. Healthy, diseased or bacterial cells react in a different way when subject to plasma treatment.
• Eukaryotic cells (complex organisms) present an outer and inner structure fare more complex with respect prokaryotic cells (bacteria).
• Pathogen cells own a more simplified structure and are easily attacked by the plasma action. On a parallel plane plasma do not cause lethal effects to living tissue cells.
• It is thus possible to use plasmas for wound sterilization without tissue damages.
In vivo treatment of biological tissue: sterilization and stimulation of tissueregeneration
2 treatments per week for 2 weeks
Dog: extensive ulcer
2 treatments per week for 14 weeks
Cat: extensive ulcer + diabetes mellitus
1÷2 minutes treatment time
Wound healing – live tissues treatment:treatment selectivity
24/05/2017
15
Patient with similar ulcers in both malleolus
Anti‐bacterial treatment
Anti‐bacterial treatment+
Plasma Regeneration tissue stimulation
Before After
Wound healing – live tissues treatment:treatment selectivityIn vivo treatment: sterilization and stimulation of tissue regeneration
Subletal treatments – cancer cell• Non-thermal plasmas can induce both cell apoptosis (programmed cell self-
death) and cell necrosis.
• Apoptosis is very important in plasma cancer treatment, because usually cancer cell are able to inhibit their apoptosis. These cells are thus more resistant to chemotherapy treatments.
• Usually necrosis is produced for higher energy dose levels, depending on the type of traded cell. For lower levels apoptosis is induced.
• For very low energy doses, no significant effects are detectable.
• Healthy cells are often able to repair oxidative damages better than cancer cell. Plasma treatment selectivity
Melanoma cancer cells stained following TUNEL assay. All cells are stained blue and apoptotic cells are also stained green
24/05/2017
16
In‐vitro treatment of Melanoma cells
Post‐treatment time
Treatmenttime
Efficacy and selectivity of plasma action arestrongly dependent on cancer typology andmorphology, and plasma source.
Subletal treatments – cancer cell
Biopolymer treatment: Tissue engineeringSurface treatment of biodegradable prosthesis made withbiopolymers.
Staminal cells
+ =
Cell differentiation with a well defined spatial
structure
Scaffold+
biodegradation
Biodegradable polymer
24/05/2017
17
Tissue engineeringAn ideal scaffold:
• Must be biocompatible, bioactive and biodegradable. In this way it is well accepted by the hosting living tissue;
• Must have mechanical properties compatible with replaced tissue;
• Must be workable during its fabrication
Two kind of materials can be used:
• Naturals materials are extracted by humans or animals. They are own specific cellular interaction and are quite difficult to obtain. Moreover they can be infected by pathogens.
• Synthetic materials can be produced on large scale, but they suffer about the lack of specific signals for cell recognition and diffrentiation
Tissue engineering: synthetic materials
Surface modification:
• It happens when molecular groups present in a polymer is exposed to plasma active species.
• Functional groups containing oxygen and ozone are added onto polymer surface. These new hydrophilic polar groups enhance surface wettability.
• Consequently surface cell adhesion and proliferation are improved.
• These surface modification are time dependent. These new functional groups orientate them selves and migrate toward the material bulk.
• Plasma action can induce surface modification due to ion bombardment (etching). This action must e considered for the plasma treatment efficacy.Surface etching can be limited by using pulsed discharges (μs).
24/05/2017
18
Post irradiation grafting
• Particular plasma treatments can be carried out to trigger the formation of superficial functional groups able to enhance polymerization reactions with added monomers in gaseous or solution phase.
Syn-irradiation grafting
• A monomer is adsorbed by a substrate. Subsequently plasma treatment is operated. This treatment activate the polymerization od the monomer over the substrate.
Action on tissue specificity
• Surface treatment can enhance cell differentiation (example: mechanical properties, patterning, functional groups)
Tissue engineering: synthetic materials
Tissue engineering: resultsBiopolymer surface modification and cell proliferation.
Control Plasma
Treatment efficacy is stronglydependent on cell typology,biopolymer, sample preparationand plasma source.
Vascular muscular cells
24/05/2017
19
Plasma Medicine at LIMP laboratory @ DEI Department
• Chemical/physical characterization of different discharges and plasma products
• Plasma chemical kinetic simulations
• Air purification from Volatile Organic Compounds (VOC)
• Bacteria and Yeasts sterilization
• Parasite disinfection in aqueous solution
• Biopolymer surface treatment
• Cancer cell treatment
Plasma characterizationElectrical Fluid‐dynamics
Spectroscopy
Discharge fast imaging
UV absorption
24/05/2017
20
Chemical kinetic plasma simulationPalsma diagnostics to evaluate reduced electric field, electrin number density and translational plasma temperature.
Use a zero-dimensional pen source code, ZDPlaskin, simulating air plasma chemical reactions.
Set of 602 reactions among 53 chemical species.
DBD reactor• Average power: 1-6 W• Resident time: 1-0,1 s
VOC treatment of an air flow
DBD
24/05/2017
21
Efficacy of the treatment on Toluene and Methil Ethil Keton
VOC treatment of an air flow
Bacteria and viruses sterilizationA collaboration with the Loughborough University (GB) is currentlyactive.Study focalized on surface indirect treatments operated by PlasmaSynthetic Jet Actuators (PSJAs). These devices are able to improvetransport of reactive species toward the sample.
Electro‐Fluid‐Dynamic InteractionEHD Plasma
Actuators
24/05/2017
22
Bacteria and viruses sterilization
Ozone delivered toward the sample as a funtion of electrode
diameter
Optimization of reactor geometry:• Annular 30 mm
Sample
Bacteria and yeasts sterilization
Collaborations with DIMEVET Department and a factory working onbiomedical device packaging.
Staohilococcus Candida Albicans
Sterilization of surfaces by bacteria and yeasts
‘T’ junction for intravenous tubes: sterilization of the device within its blister
24/05/2017
23
Bacteria and yeasts sterilizationCandida Albicans sterilization by using a DBD annular Jet and an helium indirect Plasma jet
Control 20 m 30 m
40 m 50 m
DBD
Jet
Bacteria and yeasts sterilizationSerilization results: CFU log reduction
24/05/2017
24
Parasite disinfection in aqueous solutionThe target is to kill coccid oocytes. These organisms arehuman/animal parasites able to survive several years in nature andquite resistant to conventional chemical agents.
10 mmEimeria spp.
Ceramic
Water
2 doubel membrnes
High voltage electrode
GND
Petri dish
Tissue engineering
Active collaboration with DICAM of Bologna and DISMI in ReggioEmilia – Modena.Treatment of biopolymers increased hydrophilicity, biodegradationrate and led to absence of cytotoxicity.
Sample 1 Sample 2
Before
After
24/05/2017
25
Drosophila larvae treatment for cancer cells death
It is active a collaboration with the Department of Pharmacology andBiotechnology of Bologna.Drosophila fruit fly larvae are treated trying to limit epithelial cancercell growth.
Epithelil cancer induction
Indirect plasma tratment
Drosophila larvae treatment for cancer cells death
Preliminary phase: healthy larvae treatment trying to obtain themaximum energy dose applicable.Three larvae samples treated for 8 minutes.A delay in larvae growth has been detected. This delay s due to theresponse of the larvae to the oxidative tress due to plasma action.Despite this stress, all larvae samples reach adult development.
50
60
70
80
90
100
%
Campioni
Total amount of developedlarvae
CTR
Camp 1
Camp 2
Camp 30
10
20
30
40
50
60
70
9 10 11 12 13 14
Per
cetu
ali
di
nas
cita
Giorni AEL
Growth curve trated larvaeCTR Camp 1 Camp 2 Camp 3
24/05/2017
26
Drosophila larvae treatment for cancer cells deathSagitta disc fluorescence technique points ou the presence ofoxidative agents (red dots).
