Acta Biomaterialia 10 (2014) 2177–2186

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Nanocomposites of iridium oxide and conducting polymers as electroactive phases in biological media J. Moral-Vico a,⇑, S. Sánchez-Redondo b, M.P. Lichtenstein b, C. Suñol b, N. Casañ-Pastor a a

Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus de la UAB, E-08193, Bellaterra, Barcelona, Spain Institut d’Investigacions Biomèdiques de Barcelona (IIBB-CSIC), Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBER de Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain b

a r t i c l e

i n f o

Article history: Received 3 September 2013 Received in revised form 11 December 2013 Accepted 26 December 2013 Available online 3 January 2014 Keywords: Nanocomposites Hybrid materials Conducting polymers Iridium oxide Cytotoxicity

a b s t r a c t Much effort is currently devoted to implementing new materials in electrodes that will be used in the central nervous system, either for functional electrostimulation or for tests on nerve regeneration. Their main aim is to improve the charge capacity of the electrodes, while preventing damaging secondary reactions, such as peroxide formation, occurring while applying the electric field. Thus, hybrids may represent a new generation of materials. Two novel hybrid materials are synthesized using three known biocompatible materials tested in the neural system: polypyrrole (PPy), poly(3,4-ethylenedioxythiophene) (PEDOT) and iridium oxide (IrO2). In particular, PPy–IrO2 and PEDOT–IrO2 hybrid nanocomposite materials are prepared by chemical polymerization in hydrothermal conditions, using IrO2 as oxidizing agent. The reaction yields a significant ordered new hybrid where the conducting polymer is formed around the IrO2 nanoparticles, encapsulating them. Scanning electron microscopy and backscattering techniques show the extent of the encapsulation. Both X-ray photoelectron and Fourier transform infrared spectroscopies identify the components of the phases, as well as the absence of impurities. Electrochemical properties of the final phases in powder and pellet form are evaluated by cyclic voltammetry. Biocompatibility is tested with MTT toxicity tests using primary cultures of cortical neurons grown in vitro for 6 and 9 days. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The study of hybrid organic–inorganic materials is a recent but very fruitful and prolific enterprise. In particular, hybrid nanocomposites consist of two or more nanosized objects, resulting in materials with unique physical properties, outperforming the mere addition of the properties of the components [1,2]. The composite properties can be controlled by the synthesis procedures, as well as the features of each material involved [3]. They have a wide application. For example, hybrid nanocomposites of conducting polymers and nanoparticles (e.g. Pt, Au, Pd, Zr(HPO4)2, MoO3, MnO2, Mo3Se3, c-Fe2O3, Fe3O4 and IrO2) have applications in different fields such as electrocatalysis [4], energy storage devices such as electrochemical supercapacitors [5], sensors [6], battery cathodes [7,8], microelectronics [2], magnetic materials [9] and electrochemical devices [10]. As observed in the examples mentioned, most of these hybrids contain metals or metal oxides, which could suffer from air or moisture sensitivity if they were not entrapped in an organic material matrix, forming the core/shell nanostructures [11]. ⇑ Corresponding author. E-mail address: [email protected] (J. Moral-Vico).

Biomedical applications involving implants in soft tissues could also benefit from the use of hybrid organic–inorganic structures if the final material is also conducting and may be used as electrode. An example can be found in composites of polypyrrole (PPy) and carbon nanotubes, which have performed well as electrodes for neural interfaces in chronically implantable neural probes [12]. Iridium oxide (IrO2) is an interesting inorganic component for a hybrid material to be used in biomedical applications, since it has served as a substrate for culture, growth and electrical stimulation of neural cells [13–15] and as a coating in medical electrostimulation electrodes and sensors [15–18]. However, the organic part of a hybrid material could consist of conducting polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) and PPy, which have shown good biocompatibility [19–23]. Therefore, these phases, PEDOT, PPy and IrO2, may turn out to be the most indicated components for nanocomposites to be used in biological electrode applications. IrO2 by itself can be mechanically too rigid, so mixing it with polymers should add mechanical flexibility, making the final hybrid self-standing and more appropriate for implantation into the human body or any other living system. Even if electrochemical intensive properties are not improved, a significant charge capacity increase is expected from the presence of both components in the new material because of their faradaic

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.12.051

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behavior, which entails increased charge capacity compared with other materials such as noble metals and their alloys, permitting minor damage in biological tissues. All the cited reasons make them promising materials for implantable electrodes for recording or stimulating neurons [24,25]. Thus, a hybrid material that mixes the properties of conducting polymers and IrO2 may become a good candidate for bioelectrode applications. Other fields could also benefit from the development of new hybrid phases, whatever the final form, coating or powder, since PEDOT, PPy and IrO2 all have applications in energy devices (e.g. solar and fuel cells), electrochromic devices and sensors [18,26–36]. It has been described elsewhere how nanomaterials could induce oxidative stress and inflammation in biological systems [37]. Therefore, a study of the cytotoxic properties of this type of materials was performed. Several other research groups have described PPy and PEDOT composites and nanoparticles toxicity tests, revealing good biocompatibility of the conducting polymer [38–40], but indicating a dependence on the polymer concentration [41]. The present work presents two novel organic–inorganic nanocomposites consisting of IrO2 encapsulated in either PPy or PEDOT, and the first study on their toxicity. Their syntheses took place through a facile hydrothermal reaction using a one-pot suspension of the IrO2 and the monomer as precursor. These composites have promising properties in numerous scientific areas because of the wide range of applications of their components. This work focuses on the first stage for biological applications: the study of their biocompatibility. Thus, the toxicity of these composites for cortical neuron cells was assessed, and was compared with IrO2 and PPy–ClO4 single phase toxicity, as a prior step for the further development of these materials, and the results indicate that they are promising materials for use in the neural system.

2. Materials and methods 2.1. Materials synthesis Pyrrole (Sigma–Aldrich, 98%) was vacuum-distilled until it became a colourless liquid, and was then stored at 10 °C until use. EDOT (3,4-ethylenedioxythiophene) (Sigma–Aldrich, 97%) was used as purchased, but stored at 5 °C until use. Iridium (IV) oxide (IrO2, Sigma–Aldrich, 99.9%, 12030-49-8 CAS number) was stored at room temperature in a desiccator. In all experiments, 75 mg of IrO2 was placed in a 25 ml Pyrex bottle containing 20 ml of highpurity milli-Q water (Millipore, 18.2 MX cm resistivity). The resulting mixture was stirred for 1 h. Then, either pyrrole or EDOT was added. The resulting dispersion was stirred for 1 h more. The desired conditions for this oxidation reaction were investigated in a series of experiments using different monomer/IrO2 molar ratios, with the purpose of obtaining the maximum amount of encapsulated hybrid material: either 1.0, 4.5, 18.0 or 36.0 for pyrrole/IrO2 molar ratios or 1.0, 4.5, 5.3 or 10.0 for EDOT/IrO2 molar ratios. Finally, the optimal molar ratios regarding the results found were 18.0 for pyrrole/IrO2 and 5.3 for EDOT/IrO2, which corresponded to 415 ll of pyrrole and 185 ll of EDOT. The resulting suspensions were placed in 25 ml Pyrex bottles, using 80% of their total volume, hermetically sealed with a Teflon screw-cap, and heated at 100, 150 or 200 °C for 96, 168 and 240 h. In the case of experiments at 200 °C, a Parr pressure vessel was used instead of a Pyrex bottle, to avoid pressure problems. The resulting solid was vacuum filtered, rinsed with milli-Q water five times and dried in air at room temperature. Hybrid samples with various oxidation states were also achieved by adding reducing (Na2S2O35 H2O, sodium thiosulfate pentahydrate (ACS reagent, 99.5%, Merck)) and oxidizing (K2S2O8,

potassium persulfate (ACS reagent, P99.0%, Aldrich)) agents in molar ratio 1:1 with respect to iridium after hydrothermal treatment. This part of the reaction was carried out on previously prepared powders by mixing both the material and the reducing or oxidizing agent in water dispersions for 96 h at room temperature in a 25 ml Pyrex bottle. The resulting solid was vacuum filtered, rinsed with milli-Q water five times and dried in air at room temperature. Thus, three different types of powder were obtained for each hybrid composition: as prepared, oxidized and reduced samples. Moreover, with the purpose of comparing the cytotoxicity results of PPy–IrOx with pure IrO2 and non-hybrid PPy, the latter was synthesized in exactly the same experimental conditions as polymer–IrOx (Pyrex bottle, 150 °C, 96 h) using sodium perchlorate (NaClO4, ACS reagent, P98.0%, Sigma–Aldrich) as counterion (18.0 pyrrole/NaClO4 molar ratio), which corresponded to 41 mg of NaClO4 and 415 ll of pyrrole dissolved in the Pyrex bottle. 2.2. Electrochemical analysis Electrochemical characterization measurements of the final materials in aqueous suspensions were carried out using a PAR VMP3 potentiostat. A two-compartment electrochemical cell was used for the linear sweep voltammetry to prevent secondary reactions from the counter-electrode. A glass frit was used as separator. A platinum foil was used as the working electrode, and a platinum wire (0.5 mm diameter, 78.54 mm2 exposure) as the pseudo-reference electrode on one compartment and a coiled platinum wire (0.5 mm diameter, 78.54 mm2 exposure) in the counter-electrode compartment. The pseudo-reference platinum was previously shown to be stable vs. Ag/AgCl during cyclic voltammetries, possibly owing to the formation of an oxide at the surface, and was used successfully as such [42]. The main compartment containing the working electrode was bubbled with argon for 30 min prior to reaction, which was also performed in an argon environment. Voltammetry was performed from open circuit potential, near 0 V to 0.75 V vs. Pt. at a scan rate of 2 mV s1. The electrolyte used was a 0.1 M sodium phosphate buffer solution at a pH of 7.21, using 50 mg of the material powder for every voltammetry, and with permanent stirring at 500 rpm. Teflon™ material disks were also prepared for an additional electrochemical study. For that purpose, 85 mg of the material, 10 mg of Super P Carbon (Csp hereafter from Timcal) and 5 mg of polytetrafluoroethylene (Aldrich, 60 wt.%, dispersion in water) were mixed in ethanol, and heated at 60 °C for 5 h in a glass beaker with continuous stirring. The resulting slurry was cut into disks 1.2 cm in diameter and vacuum dried for 12 h at 80 °C. Disks voltammetries were performed in an open Swagelok-type carbon cell with the Teflon–carbon–material cylinder as the working electrode and two platinum rods as counter and reference electrodes. The same phosphate buffer was used as the electrolyte, and argon was bubbled for 10 min prior to every experiment to eliminate oxygen. The CV were performed in this case at 20 mV s1 in the 1 to 1 V vs. Pt potential window. 2.3. Physicochemical characterization Further characterization of the samples was done by scanning electron microscopy (SEM), using a Quanta FEI 200 FEG-ESEM instrument, equipped for energy-dispersive X-ray (EDX) analysis and backscattered electron imaging (BSE) analysis. Typical operating parameters were 10–20 keV accelerating voltage and 2.5–3.0 nm spot size. X-ray photoelectron spectroscopy (XPS) measurements of the samples were performed at room temperature with a SPECS EA10P hemispherical analyzer using nonmonochromatic Al Ka (1486.6 eV) radiation as the excitation source at a base pressure of 109 mbar (binding energies estimated

J. Moral-Vico et al. / Acta Biomaterialia 10 (2014) 2177–2186

error ± 0.3 eV). A Perkin Elmer Spectrum One spectrometer equipped with a Universal ATR sampling accessory was used to perform Fourier transform infrared attenuated total reflection (FTIR-ATR) analysis, and 16 scans with a resolution of 4 cm1 were performed for each spectrum. 2.4. Cellular and toxicity studies The possible cytotoxic effects of the polymer–IrOx on primary cell cultures were determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, which is a standardized method [43]. Cell viability was also assessed by phase-contrast microscopic observation (Olympus CK40) and by immunocytochemistry with fluorescent microscopic visualization (Leica DMI 4000B). Fluorescent labelling of the nuclei with Hoechst 33,258 and of the neuronal cytoskeleton with anti-tau antibody were performed as previously described [20]. Mice cultured cortical neurons were prepared according to [44], and cells were grown on poly-d-lysine (Sigma–Aldrich) coated 96-well culture plates for up to 9 days in vitro (DIV). Toxicity testing analyses were performed under the same conditions for all types of materials. The compound to be studied (PPy–IrOx or PEDOT–IrOx based) was weighed, and a water suspension was prepared with the highest concentration that would not produce flocculation. After obtaining a homogeneous suspension, more diluted suspensions were prepared, and immediately added to the wells. Each well had an exposed surface of 0.3 cm2, and had been previously filled with 100 ll of Neurobasal™ culture medium (Invitrogen/Gibco, Carlsbad, CA). To each well, 10 ll of each polymer–IrOx suspension was added, so the final material concentration was reduced one-tenth. Two sequences were performed to study the exposure of the cortical neurons to culture medium containing the polymer–IrOx compound. (1) Deposited in PDL: the culture medium containing the polymer–IrOx compound was first added to the wells coated with PDL. After 24 h, the culture medium was removed, and thereafter the cells were seeded on top of the PDL coating. Cells were grown for 6 or 9 DIV, and their viability was determined by the MTT assay. (2) Cells (100 ll of a suspension of 1.6  106 cell ml1) were first seeded on PDL-coated plastic wells. After 24 h of seeding, the culture medium containing the polymer–IrOx compound was added to the wells containing the cells. They were grown for 6 or 9 DIV, and their viability was determined by the MTT assay. 3. Results and discussion 3.1. Structural analysis The synthesis procedures described yielded black powder materials. SEM inspection showed that there were three differentiated phases: pure polymer, pure IrOx and hybrid material formed by IrOx encapsulated in a polymeric matrix (Fig. 1). The three phases could be clearly distinguished by SEM, owing to the large difference in electron density between iridium phases and polymer lighter atoms. In addition, BSE images showed that the polymer was encapsulating IrOx. Through the various temperatures tested in the synthesis, the hybrid did not form at a temperature of 100 °C, but it did at 150 °C and also at 200 °C. Since the latter temperature resulted in too great a pressure for the Pyrex bottles with a Teflon cap to resist, the temperature of 150 °C was chosen as optimal in terms of hybrid material produced and safety for all experiments. Therefore, all compounds reported herein were synthesized at 150 °C.

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Additional variables, such as monomer concentration and time of synthesis were also optimized. A molar ratio of either 1 for pyrrole/IrO2 or 1 for EDOT/IrO2 did not yield any hybrid material. However, a molar ratio of 4.5 for both monomers had satisfactory results in terms of the amount of hybrid obtained, indicating that a monomer excess is required to complete the reaction. Further experiments indicated that the optimal molar ratio was 18.0 pyrrole/IrO2 and 5.3 EDOT/IrO2. In the case of EDOT, a larger amount of the phase formed by polymer only was achieved. Doubling the amount of monomer to 10.0 EDOT/IrO2 and to 36.0 pyrrole/IrO2 molar ratio, no increase in the proportion of hybrid was achieved with respect to the isolated polymer part. A distinct morphological evolution of both materials was observed along the reaction time. When pyrrole was used, the first 96 h led to the previously described three phases of pure polymer, pure IrOx and hybrid material (Fig. 1). This reaction time span led to the formation of a film with hexagonal macro-conformation (Fig. 2a), which deformed after heating for longer times (Fig. 2b and c). However, when EDOT was used with IrO2, the three phases mentioned were also observed after 96 h of reaction, but it did not present this hexagonal conformation, and no difference in morphology was observed as a function of time (no pictures shown). The three phases for both pyrrole and EDOT cases were also studied using different times, with no change in composition or hybrid production noticed after 96, 168 or 140 h of reaction. Therefore, 96 h was used as the optimal synthesis time, as longer periods did not yield any improvements in the samples. In Fig. 1, it can be observed how pure polymer phases can form spherical nanoparticles during the synthesis performed with both pyrrole and EDOT. XPS results show the presence of the expected elements in PPy– IrOx and PEDOT–IrOx and serve to give a close approximation of the materials’ atomic composition. Fig. 3 shows the spectra of both compounds along with that of the commercial IrO2 (Sigma– Aldrich). Since the measurement of carbon can be affected by endemic carbon from the atmosphere, the polymer/IrOx atomic ratio is calculated using the ratios between iridium and either nitrogen or sulfur (N/Ir or S/Ir), which only can be attributed to the monomer pyrrole and the monomer EDOT, respectively. According to the atomic percentage values shown in Table 1, polymer/IrOx molar ratios were 3.2 monomers per iridium for PPy–IrOx and 9.5 monomers per iridium for PEDOT–IrOx. An O/Ir ratio of 2 would be expected for commercial IrO2, but a ratio of 2.8 was found in the present study (Table 1). This indicates the presence of an extra amount of oxygen, which can be attributed to the presence of carbonated species or water and hydroxyl groups adsorbed by the compound, as explained later in carbon and oxygen XPS spectra analyses. IrO2 can easily form hydrous IrO2 in contact with moisture in air, incorporating H2O and OH groups into its structure [45,46]. Fig. 4 shows the C 1s, O 1s and Ir 4f XPS spectra of commercial IrO2, PPy–IrOx and PEDOT–IrOx. In the case of carbon, all curves are very similar, and all have three binding energies of 284.8, 286.0 and 288.5 (±0.3) eV attributed to three different carbon environments present in the compound: aliphatic and/or aromatic carbon, C–O, C–N (pyrrole) or C–S (EDOT) bending bonds, and OAC@O bending bonds, respectively [47,48]. The Lorentzian–Gaussian deconvolution of these XPS binding energies and the quantification of these analyses are shown in Fig. 4 and Table 2, respectively. The C 1s and O 1s XPS spectra in commercial IrO2 show a large amount of carbon and an unexpected larger amount of oxygen, indicating the tendency of IrO2 to absorb organic volatiles, CO2 and moisture. The chemisorption of CO2 can be quantified analyzing the OAC@O bending and represents 16.7% and 10.0% of the total carbon quantity for PPy–IrOx and PEDOT–IrOx, respectively. As expected, the C 1s component derived from carbon bound to oxygen (C–O) is larger in PEDOT than in PPy, since the former

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(a)

(b)

(c)

µ

µ

(d)

(e)

µ

(f)

µ

µ

µ

Fig. 1. SEM images of polymer–IrOx synthesized at 150 °C for 96 h. (a) Spherical pure polymer balls and white pure IrOx nanoparticles. (b) IrOx nanoparticles encapsulated in polymer (hybrid material). (c) BSE image of hybrid material in which IrOx presents a lighter color, taken at the same spot as (b). (d) Pure PEDOT with the formation of spherical nanoparticles. (e) Pure PEDOT spherical forms mixed with pure IrOx nanoparticles. (f) IrOx nanoparticles encapsulated in PEDOT (hybrid) and pure PEDOT spherical forms.

(b)

(a)

(c)

µ

µ

µ

Fig. 2. SEM images of the product obtained from the reaction of PPy and IrO2 at 150 °C at different synthesis times: (a) 96 h PPy–IrOx; (b) 168 h; (c) 240 h.

XPS Intensity (a. u.)

O 1s

Table 1 Atomic percentage of elements for each compound calculated by XPS, and calculation of N/Ir and S/Ir ratios.

Ir 4f

Commercial IrO2 PPy–IrOx PEDOT–IrOx

(a) N 1s

C 1s

C

O

N

S

Ir

N/Ir

S/Ir

33.7 57.3 60.3

48.9 24.1 29.2

– 14.2 –

– – 9.5

17.4 4.4 1.0

– 3.2 –

– – 9.5

(b) (c) S 2p 1000

800

600

400

200

0

Binding Energy (eV) Fig. 3. XPS spectra: (a) commercial IrO2; (b) PPy–IrOx, 96 h synthesis at 150 °C; (c) PEDOT–IrOx, 96 h synthesis at 150 °C.

presents this C–O bond in the monomer. The presence of C–O structures in PPy–IrOx is attributed to the interaction of the polymeric chain with water and impurities. For the three cases, the largest part of carbon corresponds to aliphatic and/or aromatic bonds.

Oxygen 1s spectra analyses and atomic percentage quantification are presented in Table 3. The binding energy of 530.5 eV is attributed to O2 network oxygen in IrO2 and polymeric chains [49]. Since polymer–IrOx powders show higher binding energies for this network oxygen than for commercial IrO2, the polymer– IrOx compounds should possess a larger amount of hydroxides [Ir(OH)] or oxohydroxides [IrO2(OH)x] [13,45,48]. For all materials, the peak attributed to the latter molecules is in 1 eV higher energy than the O2 component [49]. The binding energy of 533.0 eV indicates the presence of water and hydrated groups [50]. The O 1s binding energy of PEDOT–IrOx is shifted to higher energies, because of its higher content of water compared with

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O 1s

C 1s

C1

O1

C3

Ir1 Ir2

PPy-IrOx

XPS intensity (a.u.)

O3

XPS intensity (a.u.)

XPS Intensity (a.u.)

PEDOT-IrOx

O2

C2

PPy-IrOx

IrO2

IrO2

290

Ir 4f

PEDOT-IrOx

PEDOT-IrOx

288

286

284

282

IrO2

538 536 534 532 530 528 526 524

Binding Energy (eV)

PPy-IrOx

72 70 68 66 64 62 60 58

Binding Energy (eV)

Binding Energy (eV)

Fig. 4. C 1s, O 1s and Ir 4f core-level spectra for each one of the compounds: IrO2, PPy–IrOx and PEDOT–IrOx.

Table 2 Results of the deconvolution of C 1s signal with the identification of each peak. C1, C2 and C3 are referred to the deconvoluted peaks in Fig. 4. C1: C–C/C–H

IrO2 PPy–IrOx PEDOT–IrOx

C2: C–O/C–N/C–S

C3: O@CAO

Position (eV)

Molar ratio (%)

Position (eV)

Molar ratio (%)

Position (eV)

Molar ratio (%)

284.7 284.8 284.7

84.4 77.4 64.0

286.1 286.4 286.5

7.0 5.9 26.0

288.6 287.8 288.6

3.6 16.7 10.0

Table 3 Results of the deconvolution of O 1s signal with the identification of each peak. O1, O2 and O3 are referred to the deconvoluted peaks in Fig. 4. O1: O2

IrO2 PPy–IrOx Pedot–IrOx

O2: OH/oxohydroxides

O3: H2O

Position (eV)

Molar% of O

Position (eV)

Molar% of O

Position (eV)

Molar% of O

530.5 531.0 531.4

60.6 62.5 48.1

531.6 532.2 532.5

28.1 29.9 41.0

533.1 533.0 533.2

11.3 7.6 10.9

the other species (see analysis in Table 3). In agreement with previous results, commercial IrO2 shows the presence of water and hydroxide groups resulting from moisture exposure. This is, however, lower than in polymer–IrOx compounds, which were soaked in water during the reaction. The deconvolution of the Ir 4f double peak shows two components (Fig. 4), one corresponding to oxohydroxide and the other related to the oxide. The oxohydroxides peak is shifted to 1.0 eV higher energy than the IrO2 component, as in the O 1s analysis (Table 4) [49]. EDX analyses were performed, focusing on each one of the three differentiated parts of the powder obtained: pure polymer, pure IrO2 and hybrid material. It confirmed the presence of the three different phases: there were larger quantities of iridium and oxygen in the pure IrO2 part, whereas there was more carbon in the

polymeric compounds (Table 5). Similarly, there was more carbon in the parts with the pure polymer than in the hybrid ones. It is known that the error of this technique is greater than the XPS error. However, the results of both molar quantifications are quite similar, even though in XPS the analysis is superficial, and a single phase could not be selected. Fig. 5 shows the FTIR-ATR spectra. For both materials, the broad band appearing in the range 2500–3500 cm1 indicated the presence of wide O–H stretching groups, due to water [51]. PPy–IrOx shows the typical PPy IR absorbance in the range of frequencies between 650 and 1700 cm1. The strong absorbance at 1590 cm1 is attributed to C@C stretching, the absorbances at 1340 and 1040 cm1 are attributed to @CAH in plane vibration, and the absorbance at 735 cm1 is attributed to C–H bending deformation [52,53]. For PEDOT–IrOx compound, the wavenumber region

Table 4 Results of the deconvolution of Ir 4f signal with the corresponding 4f7/2 and 4f5/2 positions for each material. Ir1 and Ir2 are referred to the deconvoluted peaks in Fig. 4. Ir1: IrO2

IrO2 PPy–IrOx Pedot–IrOx

Ir2: oxohydroxides iridium

Position 4f7/2 (eV)

Position 4f5/2 (eV)

Position 4f7/2 (eV)

Position 4f5/2 (eV)

62.5 62.0 62.2

65.4 64.9 65.1

63.5 63.0 63.3

67.0 65.9 66.2

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Table 5 Atomic percentage of elements for each part of PPy–IrOx and PEDOT–IrOx powders calculated by EDX. PPy–IrOx

C

O

N

Ir

Hybrid Pure PPy Pure IrO2

66.2 76.0 53.9

16.7 9.2 19.8

16.4 14.4 10.9

0.7 0.4 15.4

PEDOT–IrOx

C

O

S

Ir

Hybrid Pure PEDOT Pure IrO2

70.5 72.7 50.2

17.7 16.7 34.7

11.2 10.3 9.8

0.6 0.3 5.3

(a) Transmittance (a. u.)

C-H bending deformation

compounds involved [56], but the PPy reduction peak is normally in a range that varies from 0.7 to 0.5 V vs. Pt [57]. Considering that the main reduction peak of IrO2 is near 0.23 V vs. Ag/AgCl [13], it can be deduced that the polymeric part of the material is mainly responsible for the electrochemical response of the material. Similarly, the typical PEDOT reduction peak is 0.45 V vs. Pt [58], and it behaves electrochemically in a very similar way. The present authors managed to perform satisfactory voltammetries with Teflon disks in the case of the PEDOT–IrOx material, for which the results can be observed in Fig. 7. The reduction peak of the compound at around 0.4 V vs. Ag/AgCl observed in Fig. 6b appears in Fig. 7b as well. It can be noted how the presence of the hybrid increases the current response of the system compared with the blank. These voltammetries were also performed with commercial IrO2 and PPy–IrOx, but the results gave no significant electrochemical information, indicating that the larger presence of polymer in the PEDOT hybrid, as indicated before, is crucial for the electrochemical activity of the hybrid.

O-H -C-H in plane

3.3. Reaction process

C=C stretching

(b) C-S-C ring deformation

O-H

C-S ring stretching -COROCstretching C-C ring stretching

3650

3150

2650

2150

1650

Doping-induced band

1150

650

Wavelenght (cm-1)

The present authors have tried to elucidate possible reactions taking place in this synthesis procedure, considering the participating reactants and their reactive properties. First, the oxidative polymerization of both pyrrole and EDOT is clearly present in the process and can be resumed in Eq. (1), where monomer pyrrole is oxidized to PPy, and A is the counterion incorporated into the growing PPy chains to maintain the electrical neutrality of the polymer system [59]. The PEDOT mechanism of polymerization is very similar to PPy [60].

Fig. 5. FTIR-ATR spectra of (a) PPy–IrOx and (b) PEDOT–IrOx.

between 650 and 1700 cm1 shows typical PEDOT peaks: a strong absorbance at 1360 cm1 is assigned to C–C ring stretching; 1235 cm1 vibration is assigned to the doping-induced band; 1070 cm1 vibration is assigned to (–COROC–) alkylenedioxy group stretching; 850 cm1 vibration is assigned to C–S bond in the thiophene ring stretching; and 696 cm1 vibration is attributed to C–S–C ring deformation [54,55]. FTIR spectroscopy on commercial IrO2, as expected for an oxide, was not very informative. 3.2. Electrochemical analysis The electrochemical properties were investigated by linear sweep voltammetry of powder suspensions (Fig. 6). Fig. 6a and b compares different oxidation states of the PPy–IrOx and PEDOT– IrOx suspensions, respectively (as-prepared, oxidized and reduced states). When only the electrolyte (i.e. no powder in solution) is used, the intensity–potential curve is practically a straight line. However, when the as-prepared material is added, a small shoulder appears around 0.5 V, and the current increases, showing greater electrochemical activity in the electrolyte. Higher currents are reached for PEDOT–IrOx, which suggests that the polymeric part of the compound is the larger contributor to the current detected, since XPS analysis showed a minor amount of iridium in PEDOT. It is also remarkable how the more oxidized material reaches higher currents, and the reduced material presents a curve very near the blank. This confirms that, as the oxidation state increases, the conductivity is greater, as well as the electrochemical response, and demonstrates that the as-prepared material is only partly oxidized, and it requires an oxidizing agent to become more electrochemically active. The electrochemical properties of these polymers strongly depend on factors such as the polymerization conditions and the

ð1Þ The reaction needs an oxidizing agent. In the case of the polymer encapsulating IrO2 nanoparticles, the iridium phase may be the oxidizing agent. Therefore, reduction of the iridium dioxide should take place, with Ir2O3 or intermediate IrOx, with mixed valence iridium being the possible products of this reaction. The existence of iridium oxohydroxides confirmed by XPS analysis suggests Eq. (2) as the possible corresponding reduction on Ir during the process [45]:

2½IrO2 ðOHÞx 

x

reduction

þ ze ! ½Ir2 O3 ðOHÞxz ðxzÞ þ zOH

ð2Þ

XPS analysis indicated the existence of iridium oxohydroxides not only in the products present in the aqueous media, but also in commercial IrO2. Changes in pH were studied upon inmersion in water, to clarify the whole process, and an increase in its value from 6.0 to 6.4 was observed during oxide addition. On the basis of this change, one can suggest the liberation of hydroxyl groups of iridium oxohydroxides, which could be resumed in Eq. (3). þH2 O

½IrO2 ðOHÞx x  yH2 O ! ½IrO2 ðOHÞxz ðxzÞ þ zOH

ð3Þ

However, when pyrrole or EDOT is added to the IrO2 suspension, there is acidification, 0.6 and 3.0 pH units, respectively, by the end of the reaction. This agrees with the fact that the polymerization involves the liberation of H+ from the rings [59]. Hydroxyl groups may neutralize H+ and also act as counterions in the formation of polymers. The major pH difference in PEDOT is attributed to a larger amount of polymer synthesized, as pointed out previously. In the case of polymers encapsulating IrO2 nanoparticles [IrO2 (OH)x]x, [Ir2O3(OH)x]x or OH groups may act as counterions for the formation of the polymer. The formation of pure polymer

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(a)

-0.2

I(mA)

I(mA)

-0.4 -0.6 i

-0.8

ii

-1.0

iii iv

-1.2 -0.8

-0.6

-0.4

-0.2

0

0.2

0.0 -0.2 (b) -0.4 -0.6 i -0.8 -1.0 ii -1.2 iii -1.4 -1.6 iv -1.8 -0.8 -0.6

-0.4

-0.2

0

0.2

E(V) vs Pt

E(V) vs Pt

Fig. 6. Linear sweep voltammetry from OCP to 0.75 V vs. Pt at 2 mV s1 of (A) PPy–IrOx and (B) PEDOT–IrOx powders. (i) Blank curve (no powder in solution); (ii) reduced material; (iii) as-prepared material; and (iv) oxidized material.

1.4

b

1

I (mA)

0.6

a

0.2 -0.2 -0.6

30 min of argon bubbling was performed, noting no significant differences in terms of the reaction products or the amounts obtained. However, the fact that IrO2 is known as a good catalyst for water oxidation, in which O2 is produced [46,61] and the possible existence of remaining O2 after Ar bubbling, suggest that only a low O2 pressure is necessary for the oxidative polymerization. OH appears in this case as the most probable counterion. In the case of PPy–ClO4 synthesis performed for cytotoxicity comparison with hybrid materials, perchlorate together with O2 present in solution may both be the oxidizing agent.

-1 3.4. Cellular and toxicity studies

-1.4 -1

-0.5

0

0.5

1

E(V) vs Pt Fig. 7. Cyclic voltammetry of Teflon disks at 20 mV s1 of: (a) blank (no material in Teflon disk); (b) PEDOT–IrOx.

phases is, nevertheless, less clear, because of the necessary close contact of oxidizing molecules for the electron transfer to take place. Thus, IrO2 nanoparticles or its derivative molecules would not be so significative in this oxidation process. Since O2 appeared to be the most probable oxidizing agent, an experiment after

The cytotoxicity of the hybrid materials (PEDOT–IrOx and PPy– IrOx) was checked by performing the MTT assay for cell viability. Tests were performed for 6 and 9 DIV, with a concentration range of the materials. All conditions were the same for both PEDOT–IrOx and PPy–IrOx powders. Two different sequences were tested: (1) first, cell culture medium containing the polymer–IrOx; second, cell seeding; (2) first, cell seeding; second, addition of the cell culture medium containing the polymer–IrOx. A critical concentration of the materials of 50 ng ll1 was detected as the critical point from which cells were significantly

Fig. 8. MTT assay histograms for 6 days in vitro: (a) PPy–IrOx, sequence 1; (b) PPy–IrOx, sequence 2; (c) PEDOT–IrOx, sequence 1; (d) PEDOT–IrOx, sequence 2. Results are the mean value of 3–5 cultures, where each concentration was tested in triplicate. ⁄⁄p < 0.01, Dunnett’s test after significant one-way analysis of variance.

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50 ng ll1. Similarly, exposure to polymer–IrOx for 8–9 DIV showed higher cell toxicity (data not shown). For both polymer–IrOx compounds, a concentration of 10 ng ll1 was safe for the growing of the neurons up to 9 DIV. PEDOT–IrOx appears to be slightly more toxic than PPy–IrOx, since cell viability was reduced by 50% (sequence 2) when material concentration increased from 10 to 50 ng ll1 for PEDOT, against 18% in the case of PPy. When cells were seeded on top of PLD pretreated with polymer–IrOx, no significant toxic effects were observed at the tested concentrations, but PEDOT showed a trend to reduce cell viability at the highest concentration (Fig. 8a and c). The lower toxicity of PPy material compared with PEDOT material is in agreement with the improved in vivo histocompatibility of brain implanted PPy electrodes compared with PEDOT and Pt electrodes [62]. The present authors also tested whether the toxicity of PPY–IrOx composite compares with that of PPy–ClO4 and IrOx by exposing the cells to 50 ng ll1 of these materials. Cell viability values were 82 ± 8%, 87 ± 4% and 68 ± 12% for PPY–IrOx, PPY and IrOx treated cell, respectively, with respect to control cells (100 ± 5%) (n = 3). The data found in this work for the toxicity of polymer–IrOx nanocomposites in neuronal cells are in the same range as those reported for pure PPy using several types of cells in 24 h tests [41]. Although testing of materials for cell viability is very much dependent on the experimental conditions, which includes cell type, cell density and time of exposure, a concentration of 10 ng ll1 was found to be safe for neuronal growing and differentiation (this work) and for cell proliferation and adhesion of several proliferating cell types (Ref. [41]). 4. Conclusions

Fig. 9. Representative fluorescence microphotographs of cortical neurons treated with polymer–IrOx nanocomposites. Cells were processed for tau immunocytochemistry and Hoescht staining of the nuclei at day in vitro 6 (a–e) or day in vitro 2 (f–h): (a) control untreated cells; (b) 10 ng ll1 PEDOT–IrOx; (c) 100 ng ll1 PEDOT–IrOx; (d) 10 ng ll1 PPy–IrOx; (e) 100 ng ll1 PPy–IrOx; (f) control untreated cells; (g) 100 ng ll1 PEDOT–IrOx; (h) 100 ng ll1 PPy–IrOx. Scale bar = 50 lm.

affected by toxicity after being directly exposed for 6 DIV (Fig. 8b and d). Above this concentration there was a significant reduction in cell viability, i.e. in cell survival. Fig. 9 shows a representative image of neurons stained with Hoechst 33258 (nuclei) and tau antibody (cytoskeleton neurofilaments). Cells exposed to 10 ng ll1 show a dense network of neurites (Fig. 9b and d) similar to control cells (Fig. 9a) whereas those exposed to 100 ng ll1 show significantly lower staining for both nuclei and neurites (Fig. 9c and e), in agreement with the loss of cell viability determined by the MTT assay (Fig. 8). Polymer–IrOx toxicity was time-dependent: phase-contrast and fluorescence microscopic visualization of the cells revealed that they were alive after 1 DIV exposure to 100 ng ll1 (Fig. 9f–h shows fluorescent images of the nuclei and neurites of these cells) or 4 DIV exposure to

Two novel hybrid nanocomposite polymer–oxide materials, PPy–IrOx and PEDOT–IrOx, were successfully synthesized under hydrothermal conditions, in various oxidation states. In all cases, IrO2 is entrapped and encapsulated by the polymer film. The resulting material is conducting, and presents electrochemical activity in different degrees, depending on the initial oxidation state, and within the known ranges for each of the components, IrO2 and polymer. It always results as a distribution of the single components and the hybrid. Like each of the components, the hybrid materials show biocompatibility for the growing and differentiation of neurons. For suspension cases, there is a limited amount of material that may be used without toxic effects being observed. However, the combination of conducting polymers and IrO2 to form a nanocomposite material keeps the biocompatibility compared with non-hybrid conducting polymers. Those facts define a clear potential for the use of the materials as electroactive phases in biological media, used as coatings or for the delivery of nanoparticles. The final phases contain a combination of the properties of each of their components, and being conductive, they possess the capacity for charge delivery and their future use as bioelectrodes. Acknowledgements This work was supported by grants from the Spanish Ministry of Science and Education (MEC) (MAT2005-07683, MAT200806643 and MAT2011-24363) and the Instituto de Salud Carlos III (FIS10/00453), the European Commission FP6 NEST-STREP Program (Contract 028473) and La Marató project from TV3 (110130 and 110131). J.M.V. also thanks the Spanish Ministry of Education for a predoctoral fellowship. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 3–6, and 9 are difficult to interpret in black and white. The full colour images

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