Potential Application of Cold Plasma in Neurodegenerative Diseases associated
with Amyloid Fibrils
Lesley Greene1, Agatha Munyanyi1, Erdinc Karakas2, Paula Mazzer1 and Mounir Laroussi2
Department of Chemistry and Biochemistry1, Laser & Plasma Engineering Institute and
Electrical & Computer Engineering Department2
Old Dominion University
Norfolk, VA 23529 USA
Abstract: Amyloid fibrils are ordered beta-sheet aggregates of proteins that are associated
with over twenty human diseases. Several are neurodegenerative disorders which include
Alzheimer’s and Parkinson’s disease. Interestingly, it has been postulated that all proteins
can be induced to form this low energy state if subjected to the right conditions. Presently
there is no cure for amyloid related diseases. The Greene and Laroussi groups have taken
an interdisciplinary approach, uniting biochemistry with engineering, in an effort to
develop potential therapeutic techniques. Our initial studies have successfully applied cold
plasma to break amyloid fibrils into smaller units in vitro. Further research is underway to
develop optimal in vitro conditions and apply this method to amyloid fibrils in cell culture.
Keywords: Amyloid fibrils, cold plasma, plasma pencil
1. Introduction
postulated that all proteins can be induced to
Amyloid fibrils are ordered beta-
form this low energy state if subjected to the
sheet aggregates that are associated with a
right conditions [3]. We are investigating
number of neurodegenerative diseases such
potential methods involving the use of cold
as
[1].
plasma that will lead to the destruction of
Amyloid fibrils can also be found in other
amyloid fibrils, formed by the protein α-
parts of the human body such as joint spaces
synuclein,
in patients undergoing prolonged renal
disease and the β-amyloid peptide which is
dialysis [2].
There are approximately
associated with Alzheimer’s disease [4]. At
twenty proteins that have been found to
present there is no cure for these progressive
form fibrils in humans and are associated
and debilitating diseases. We propose that
with disease states [1]. However, it has been
this work will also be applicable to the
Alzheimer’s
and
Parkinson’s
1
which
underlies
Parkinson’s
destruction of prion proteins and have value
High voltage pulse =7.5 kV
in decontamination processes.
Frequency: 5 kHz
Pulse width: 1 μs
2. Materials and Methods
Operating gas: Helium
α-Synuclein is the protein that forms
Flow Rate: 5 L/min
Parkinson’s
Following exposure to the cold plasma the
disease and was selected as our model
samples are placed onto 400 mesh formvar
system. We can produce this human protein
coated copper grids for analysis by electron
by expressing it in Eschericia coli from a
transmission microscopy.
amyloid
fibrils
underlying
DNA clone inserted into the bacteria. The
soluble protein is then extracted from the
3. Results and Discussion
bacterial cells and purified by ion-exchange
There are several promising applications of
and gel filtration chromatography. The α-
atmospheric
synuclein protein (4-6 mg/ml) is dissolved in
plasma jets in biomedicine. These include
a buffer solution of 0.2M NaCl in 20mM
sterilization of heat sensitive materials,
Tris Base, pH 7.5 and incubated at 37ºC in
wound healing, dental hygiene, blood
an incubator shaking at 150-190 rpm. After
coagulation and cancer therapy. Enhanced
15 days mature fibrils analogous to those in
gas phase chemistry without the need for
patient’s brain are formed in our test tubes.
elevated gas temperatures plays a key role in
pressure
low
temperature
the plasma jet’s effectiveness.
The "Plasma Pencil” is made of a 25
mm diameter hollow dielectric tube with
atmospheric
two copper ring electrodes attached to the
plasma jets, the electron energy is much
surface of centrally perforated dielectric
higher than the energies of the ions and
disks separated by 5 mm.
neutral species. Therefore, these energetic
The diameter of the hole is about 3 mm. The
electrons enter into collisions with the
diameter of the copper ring electrodes is
background gas causing enhanced level of
smaller than that of the disk but bigger than
dissociation, excitation and ionization. Two
that of the hole. During our experiments the
types of reactive species are generated.
amyoid fibrils in solution are exposed to the
These are short-lived reactive species such
cold plasma for varying lengths of time (0-
as He*, N*2, N+2 , O*, OH and long-lived
10 minutes) under the following conditions:
reactive species such as Hem, O3, NO, NO2.
2
pressure
low
In the
temperature
Figure 1. Transmission Electron
Microscopy analysis of α-synuclein fibrils
before and after exposure to the plasma
pencil. (Adapted from reference 5.)
Hence, the plasma does not cause any
thermal damage to the articles since the
heavy species (ions and neutral species) are
relatively cold.
Initial studies by the Greene and
Laroussi groups indicate that cold plasma
can break these α-synuclein fibrils into
smaller units in vitro (Figure 1) [5]. In this
study, the plasma is generated by the plasma
pencil, a device capable of emitting a plasma
plume several centimeters in length (Figure
2) [5]. The plasma pencil is powered by high
voltage
short
pulses
(nanoseconds
to
microseconds) and uses helium as a feed gas
[6].
The
plasma
plume
is
at
room
temperature but generates reactive oxygen
and nitrogen species (along with charged
particles) that interact with the fibrils and
ultimately causing their destruction.
This
avenue of research may facilitate the
development of a medical treatment.
Figure 2. The amyloid fibrils formed by αsynuclein in solution are placed into small
tubes (0.2mls) or wells and exposed to the
plasma pencil for varying lengths of time
and intensity. After exposure the fibrils are
immediately fixed onto transmission
electron microscopy grids for analysis of
breakage (see Figure 1 for TEM images).
3
4. References
Acknowledgements
[1] Chiti, F. and Dobson, C.M. (2006)
This work is partly supported by an AFOSR
Protein misfolding, functional amyloid and
grant to M.L.
human disease. Ann. Rev. Biochem. 75, 333-
(Applied Research Center, Virginia) for
366.
valuable assistance in generating the TEM
[2] Eakin, C.M. and Miranker, A.D. (2005)
images.
From chance to frequent encounters: origins
of
β2-microglobulin
fibrillogenesis.
Biochimica Biophysica Acta 1753, 92-99.
[3] Chiti, F., Webster, P., Taddei, N., Clark,
A., Stefani, M., Ramponi, G. and Dobson,
C.M. (1999) Designing conditions for in
vitro formation of amyloid protofilaments
and fibrils. Proc. Natl. Acad. Sci. USA 96,
3590-3594.
[4] Irvine, G.B., El-Anof, O.M., Shankar,
G.M. and Walsh, D.M. (2008) Protein
aggregation in the brain: the molecular basis
for Alzheimer’s and Parkinson’s disease.
Mol. Med. 14, 451-464.
[5] Karakas, E., Muyanyi, A., Greene, L.
and Laroussi, M. (2010) Destruction of αsynuclein based amyloid fibrils by a low
temperature plasma jet. Applied Physics
Letters. 97, 143702.
[6] Laroussi, M. (2009) Low Temperature
Plasmas for Medicine? IEEE Trans. Plasma
Sci. 37, 714-725.
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We thank Dr. Wei Cao