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 1Departamento de Física, Universidad Autónoma Metropolitana Iztapalapa, Av. Michoacán y Purísima, Col.

Vicentina-Iztapalapa, D.F., CP 09340, México. 2Departamento de Química, Universidad Autónoma Metropolitana Iztapalapa, Av. Michoacán y Purísima, Col.

Vicentina-Iztapalapa, D.F., CP 09340, México. 3Departamento de Física, Instituto Nacional de Investigaciones Nucleares, Carr. México-Toluca, km 36.5,

Ocoyoacac, Mex., CP 52750, México. 4Departamento 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

/cm

S-10151 10 1

I-20151 20 1

I-30153 30 3

I-50153 50 3

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

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

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 S-10151 (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 ball-shaped 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.

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 PC-12 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 (I-20151) and 30 W (I-30 153) than in controls (Figure 2 and Figure 3).

Conclusions

Films synthesized by low-pressure plasma from

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.

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.

a

a

b

c d