Neuronal Cells Cultured on Polypyrrole Thin Films Polymerized by Glow
Discharge: Effect of the Plasma Variables
Roberto Olayo1, Elizabeth Pérez-Tejada1, 2, R. Godinez4, G.J. Cruz3, G. Olayo3, Juan Morales-Corona1
1
2
Departamento de Física, Universidad Autónoma Metropolitana Iztapalapa, Av. Michoacán y Purísima, Col.
Vicentina-Iztapalapa, D.F., CP 09340, México.
Departamento de Química, Universidad Autónoma Metropolitana Iztapalapa, Av. Michoacán y Purísima, Col.
Vicentina-Iztapalapa, D.F., CP 09340, México.
3
4
Departamento de Física, Instituto Nacional de Investigaciones Nucleares, Carr. México-Toluca, km 36.5,
Ocoyoacac, Mex., CP 52750, México.
Departamento de Ingeniería Electrica, Universidad Autónoma Metropolitana Iztapalapa, Av. Michoacán y
Purísima, Col. Vicentina-Iztapalapa, D.F., CP 09340, México.
Abstract: The loss of organs or tissue due to accident or illness can be treated in many cases with
biological implants; therefore, the development of new synthetic biomaterials that may help to
produce this implants has been presented as an alternative. In particular, polymeric materials with
potential biomedical applications have been in constant evolution.
In this work we present polypyrrole (PPPy) thin films obtained by plasma polymerization at
different power and reaction times with RF glow discharges The PPPy are use as substrates for
nervous cell cultures. The effect of the synthesis parameters on the properties of films is analyzed.
The films were studied by thermogravimetric analysis, infrared spectroscopy and scanning
electron microscopy to determine the structure and oxidation states of PPPy. We used contact
angle to verify that the PPPy was present even at short deposit periods. The solubility of the films
was studied in distilled water, saline solution, and culture medium before they were use as
substrates for cell culture. Three separate cell lines with neuronal model behavior were used for in
vitro essays (PC12, NG108-15, and N1E 115). The samples were observed under a phase contrast
microscope coupled to a digital camera and SEM was used on some samples.
Keywords: Plasma, polypyrrole, neuronal model.
1. Introduction
Is estimated that in the U.S. 12,000 new cases every
year of traumatic injuries that cause damage in
spinal cord and peripheral nerves. To try to recoup
some of the damaged functions, transplants with
tissues of the patient or cadaveric donor are used, but
these techniques have drawbacks such as
incompatibility, lack of availability of adequate and
timely tissue, possible rejection, etc.; is therefore of
interest to produce alternative solutions.
Polymers allow the creation of materials and
coatings capable of work as promoters of cell
regeneration in damaged areas.
Specifically in nerve regeneration and neural
damage, polymers have been used as scaffold or
coatings capable of functioning efficiently as
substrates and promoting cell regeneration in
damaged areas.
The surface properties of a material are directly
related to their performance in contact with cells.
Plasma surface treatment and deposition of polymers
by plasma can produce a uniform surface change on
any type of substrate as a polymer coating on it or
directly produce materials for cellular support or
drug delivery, obtaining regeneration characteristics
difficult to obtain by other methods.
Polymer films with potential biological and medical
applications are tested in cellular cultures of cell
lines with similar characteristics to those of the
lessened areas, it seeks to replace or regenerate. In
vitro studies using PC12 (rat pheochromocytoma,
ATCC
CRL-1721),
N1E-115
(mouse
neuroblastoma, ATCC CRL-2263) and NG108-15
(hybridoma cell line of a rat neuroblastoma and a
mouse glioma, ATCC No. HB-12317) have been
used to assess toxicity, biocompatibility, nerve fiber
regeneration,
electrical
activity,
neuronal
communication and interaction between biomaterials
and neurons.
In this paper we present the procedure for obtaining
thin films by plasma polymerization of pyrrole,
PPPy, with RF glow discharges. We analyzed the
influence of the synthesis parameters on the
properties of PPPy thin films. The films were
deposited at different reaction times, because the
reaction time is directly related to the thickness of
the films [3]. The films were studied by
thermogravimetry, infrared spectroscopy, FT-IR,
and scanning electron microscopy to determine the
structure and oxidation states of PPPy. We use the
contact angle to verify that the plasma polymer was
present even short periods of deposit; we studied the
solubility of the films in distilled water, saline
solution, and culture medium before they were used
as substrates for cell culture. Three separate cell
lines were used as neuronal behavior models for in
vitro essays: PC12, NG108-15, and N1E-115, they
have been used to study their interaction with
polymers. Samples were observed under a phase
contrast microscope coupled to a digital camera. We
used scanning electron microscopy on some
samples.
2. Experimental
Plasma deposition process
The experimental setup consists of a stainless steel
reactor of 28 cm in diameter and 30 cm in high with
four detachable windows. The RF electrode is
connected to Dressler CESAR-1500 amplifier with a
resistive coupling mechanism at 13.56 MHz, the RF
electrode can be slid along the y-axis to adjust the
distance between both electrodes, 1 to 3 cm, this
electrode is electrically isolated from the body
reactor through a Teflon mechanism that allows it to
slid. The pyrrole (Aldrich reactive grade) monomer
was introduced to the reactor in vapor phase for an
access port located in the upper lid, the pyrrole vapor
is carried out to the RF electrode center through a
rubber hose to prevent electrical contact with the RF
electrode, thus ensuring that the monomer vapor is
distributed throughout the glow discharge area. The
grounded electrode is connected to the metal body of
the reactor and to the RF source. The reactor is
coupled to system vacuum that consist of cold
particles tramp and mechanical pumps. The pressure
into the reactor was of 1-6 x10-2Torr (Edwards
Pirani gauge).
Before starting the reactor operation, on the
grounded electrode were placed glass coverslips
(Corning, 22 mm x 22 mm), this coverslips are used
for physic-chemical analysis of the polymeric
material and carry out the cell cultures.
Figure 1. Experimental setup
In all experiments the reactor was operated for 10
min without feeding monomer to the reaction
chamber purge oxygen and clean the surface of the
coverslips and then delivered the monomer (Pyrrole,
Aldrich 99%) without carrier gas. Samples were
synthesized at reaction times of 15 min, varying the
power and the distance between the electrodes (See
Table 1). After the scheduled deposit time was
finished the flow of monomer was interrupted and
subsequently the reactor was opened
Table 1. The experimental conditions of PPPy thin films. The
capital letter S is applied to soluble film and the letter I is
applied to insoluble film. The first two numbers are indicated of
polymerization power, the follow two numbers is the reaction
time and the last number is the separation between electrodes in
cm.
Sample
Power/
W
Separation between electrodes
S-10151
10
1
I-20151
20
1
I-30153
30
3
I-50153
50
3
/cm
atmosphere in a tissue culture incubator. Samples
were kept in a culture medium prepared to promote
cell proliferation that was changed every 48 hours
after washing with saline solution PBS (GIBCO
13151-014). The film surface and the cells surface
were observed every 24 hours using an inverted
microscope (IROSCOPE MG-20IF), we used a 10X
ocular and objectives 10X, 25X and 40X and
photographs were taken using a digital camera
(Panasonic GP 244) attached to the microscope and
image management program (Studio 9).
3. Results
The contact angle was measured using one drop of
distilled water and is the average of 5 measured. A
picture was taked with a FujiFilm Digital Camera
Fine Pix F480, after, the photograph was analyzed
with Image-Pro program. The IR spectra of the
films were taken with a Perkin-Elmer spectrum GX
FT-IR System spectrophotometer of 400-4000 cm-1
range sampled on KBr tablets coated with PPPy film
to interpret which functional groups are present in
the samples.
The solubility of the films was studied in distilled
water, saline solution, and culture medium, a partial
solubility can incapacitate a surface to allow
attachment of the cells and/or release compounds
into the culture medium. To observe the possible
solubility weight change of samples was recorded,
samples were weighed, placed in distilled water,
rinsed and dried recording his weight again. The
micrographs were taken with a Philips XL 30
scanning electron microscope on the surface of the
films.
Nerve cell cultures
In sterile conditions the coverslips with PPPy films
were placed inside commercial petri dishes (SARST
35 mm) and were used as substrates to cultivate
different cellular lines types: PC12, N1E-115 and
NG108-15; in all the samples the same initial
number of well-dispersed cells (2 x 105 viable
cells/ml) were used, and were provided with the
necessary conditions for the proliferation. All cell
cultures were maintained at 37°C in a 5% CO2
Solubility and contact angle.
The coverslips with the deposited PPPy films were
immersed separately in distilled water, culture
medium and saline solution for 48 hours. The
insoluble samples, I-20151, I-30153, and I-50153,
are not altered by the immersion in fluids tests. The
soluble sample, S-10151, have a contact angle of
60o, and is partially soluble in distilled water. The
insoluble samples have a contact angle of 49o.
FT-IR-ATR analysis
In the IR-spectrum shows the typical behavior of
plasma-synthesized materials, broad and complex
bands. The FT-IR analysis of partially soluble S10151 (two peaks around 3400 cm-1 primary amines,
and the signal of the carbon nitrogen triple bonds
2100 cm-1) while for insoluble polymer film the
spectrum shows a single signal in the region that is
assigned to secondary amines. The signal assigned
to carbon-nitrogen triple bonds is presented at 2100
cm-1, this signal is more intense in the films
polymerized at 30W. The IR spectrum is not shown.
Cells culture
Fig 2 (a) shows the cell line N1E-115 on a
commercial culture dish. It shows only few ballshaped cells on the attached to the surface and only
some of them are releasing axons. Fig 2(b)
corresponds to Sample I-20151with N1E-115 cells
almost all the nuclei emit long axons and some of
this axons are communicating cells.
a
c
b
pyrrole retained properties in short times synthesis,
if the same power and separation of electrodes is
kept. Polymerizing at 20 W and 30 W increases the
presence of amino groups and insoluble films are
obtained, they promote cell proliferation and
differentiation. PPPy films are stable, non toxic and
allow adherence and proliferation of PC12, N1E115
and NG108-15 cells.
d
Fig 2. Cell line N1E-115 in culture after 120 hrs, in all pictures
the amplification is 10x: a) commercial culture dish, and the
insoluble films b), I-20151, c) I-30153, d) I-50153. The
proliferation and the length of the axon terminals is more about
the films sintered at 20 W and 30 W. The white bar is 0.01mm.
Fig 2(c) shows the sample I-30153 with the same
cell line, there are greater concentration of
polygonal-shaped nuclei and they are emitting axons
that communicate to form an axons network. This
sample has a better cell proliferation, it is possible
that at these conditions of plasma (30W and 3cm)
there is some monomer destruction giving a PPPy
with moderate crosslinking and some amine and
nitilus exposure and but that allow cells of N1E-115
remain functional on the surface. In Fig 2 (d) sample
I-50153 shows fewer cell nuclei and only some of
them grow axons and they are smaller as compared
to samples 2(b) and 2(c), the PPPy film was
synthesized at 50W and is possible that the high
plasma energy gives heavy crosslinkin of the
polymer.The insoluble PPPy films allows
proliferation and differentiation of three cells lines
PC12, N1E-115 and NG108-15. Culture cells of PC12 and NG108-15 lines are not shown, but all are
good viable cells lines. In N1E-115 and NG108-15
lines the axonal length and the dendritic terminals
length is higher in the films synthesized at 20 W (I20151) and 30 W (I-30 153) than in controls (Figure
2 and Figure 3).
Conclusions
Films synthesized by low-pressure plasma from
a
Fig 3. Differentiated cultures of N1E-115,120 hrs, 10x: a)
commercial culture dish, and the films insoluble b), I-2015, The
white bar is 0.01mm.