Well-Ordered Porous Conductive Polypyrrole as a New Platform for

ARTICLE pubs.acs.org/Langmuir

Well-Ordered Porous Conductive Polypyrrole as a New Platform for Neural Interfaces Grace Kang,† Richard Ben Borgens,†,‡ and Youngnam Cho*,† †

Center for Paralysis Research, Department of Basic Medical Sciences, School of Veterinary Medicine Weldon School of Biomedical Engineering Purdue University, West Lafayette, Indiana 47907, United States

‡

ABSTRACT: We present the preparation of electrically conductive, porous polypyrrole surfaces and demonstrate their use as an interactive substrate for neuronal growth. Nerve growth factor (NGF)-loaded porous conducting polymers were initially prepared by electrochemical deposition of a mixture of pyrrole monomers and NGF into two- or three-dimensional particle arrays followed by subsequent removal of a sacrificial template. Morphological observation by scanning electron microscopy (SEM) revealed these to possess high regularity and porosity with well-defined topographical features. A four-point probe study demonstrated remarkable electrical activities despite the presence of voids. In addition, we investigated the effects of these surfaces on cellular behaviors using PC 12 cells in the presence and absence of electrical stimulation. Our results suggest that the surface topography as well as an applied electrical field can play a crucial role in determining further cell responses. Indeed, surface-induced preferential regulation leads to enhanced cellular viability and neurite extension. Establishing the underlying cellular mechanisms in response to various external stimuli is essential in that one can elicit positive neuronal guidance and modulate their activities by engineering a series of electrical, chemical, and topographical cues.

’ INTRODUCTION Recently, there has been a tremendous effort to develop cell interfacing biomaterials to improve cell-to-cell or cell-to-substrate interactions.1 3 In particular, since the optimization of an interface between neural prostheses and tissues strongly governs cell behavior and subsequently promotes electrical signals to stimulate neurological functions, a deeper understanding of these issues is necessary to improve device performance. In general, damage to the nervous system generally entails a series of complex processes that progressively results in even more complex pathological disturbances and functional deficits. Abnormalities in cellular signaling and the functioning of biomaterials could be sufficient to cause undesired side effects and even device failure. For these and other reasons, the use of conducting polymers to adapt drug delivery systems in a controlled way has been explored.4 8 In these electrically responsive systems, sitespecific drug delivery can be manipulated by adjusting the released concentration of dopants (e.g., drugs) deposited inside the conductive membrane.9 13 However, the mismatch in the interaction between cells and conductive surfaces often causes a limited regulation of cellular response. This in turn has led us to extend current strategies by combining various guidance cues meant to enhance cell survival and useful processes, but simultaneously suppressing deleterious processes such as the inflammatory reaction. With this approach, we have adapted a r 2011 American Chemical Society

methodology to create three-dimensional well-ordered pores in the conductive surfaces. The self-assembled colloidal arrays serve as a template for large-area electrochemical deposition of conductive polymers, such as polypyrrole (Ppy). Subsequent removal of colloidal beads results in a well-organized and closely packed porous Ppy surface with multiple layers while retaining structural stability. The construction of porous Ppy surfaces imparts several advantageous factors over conventional flat surfaces: (i) as an open pore architecture, it enables relatively free diffusion of neurotrophic factors and nutrients through the voids with enhanced accessibility, (ii) as a multilayered structure, it affords enhanced entrapment and release of NGF that is more effective in inducing neurite outgrowth, (iii) as a nanostructured surface analogous to the topography of native extracellular matrix (ECM), it provides favorable cell adherence with superior contact at the cell substrate interface, and (iv) as an electrically conductive surface, it fully elicits a cellular response positively influencing the adhesion, proliferation, and neurite extension (see Figure 1).14,15 Thus, it is recognized that a combination of structural, electrical, and chemical guidance can be used as an organizer Received: October 18, 2010 Revised: February 23, 2011 Published: April 18, 2011 6179

dx.doi.org/10.1021/la104194m | Langmuir 2011, 27, 6179–6184

Langmuir

ARTICLE

Figure 1. Schematic illustration for (a) the preparation of NGF-doped porous polypyrrole surfaces and (b) cellular interaction with a porous surface in the presence of electrical stimulation.

for controlling neuronal process outgrowth. In this paper, we demonstrate the preparation and characterization of electrically conductive porous NGF-doped Ppy surfaces and investigate how the surface porosity coupled with electrical stimulation influences the neuronal activities of PC 12 cells.

’ EXPERIMENTAL SECTION Preparation of Polystyrene Colloids Arrays. The well-ordered construction of polystyrene (PS) beads with diameters of 200 nm and 1 μm was fabricated according to protocol.16 In brief, aqueous suspension of PS spheres (Spherotech, Inc., IL) was adjusted to 5% w/v by adding equal volume of ethanol solution before use. The clean ITO (Indium tin oxide, Delta Technologies) surfaces were incubated in the PS solution to induce the self-assembly of particles into three-dimensional layered constructs. The typical process to achieve a uniform deposition of PS nanoparticles takes ∼5 days at room temperature by slow ethanol evaporation. Finally, the template was airdried for at least 1 day and kept in vacuum desiccators until use. Electrochemical Deposition of Polypyrrole on Colloidal Crystal Construction and the Preparation of Porous NGFDoped Polypyrrole. Polypyrrole (Ppy) was electrochemically deposited on the resulting templates using 604 model potentiostat (CH Instruments). A template, platinum gauze, and saturated calomel electrode were employed as a working, a counter, and a reference electrode, respectively. For the preparation of NGF-loaded polypyrrole, the electrochemical deposition was conducted on colloidal templates in an aqueous mixture of 0.1 M pyrrole (Py) monomer, 0.1 M sodium salt of poly (styrene sulfonate) (PSS) as a dopant ion, and 100 μg/mL of NGF 2.5 S (Invitrogen) by applying a constant cathodic potential of 0.9 V. These films were immediately rinsed with deionized water and dried under nitrogen to avoid any further deposition. To create the porosity within Ppy films, NGF-doped Ppy template was incubated in the tetrahydrofuran (THF) for 24 h, where polystyrene core was dissolved leaving hollow composites. The resulting porous film was air-dried and stored in vacuum desiccators until use. Characterization of Porous Polypyrrole. The analysis of various types of Ppy films was achieved using a FEI NOVA nanoSEM (FEI Company) with a 5 kV acceleration voltage. Prior to these measurements, all samples were sputtered with gold palladium. Ppy films were further analyzed with four-point probes (micromanipulator’s

model 6000) to evaluate the conductance values depending on the surface porosity at room temperature. Prior to taking conductance measurements, Ppy films were peeled off using tape. As a constant current was passed through the surface, the voltage induced was measured and further correlated to determine sheet resistance. The electrical conductivity is directly determined by Van der Pauw equation. The surface wettability as a result of porosity was studied using contact angle analyzer (Ahtech Lts Co.). The thickness of the electrodeposited film was determined by averaging several measurements using an Alpha step 500 stylus profilometer. The conductive Ppy surfaces using a polystyrene bead of 200 nm diameter had a 0.5 1.4 μm average thickness, whereas Ppy surfaces using a polystyrene bead of 1 μm diameter had a 2.3 5.4 μm average thickness, indicating the dependence of the diameter of polystyrene beads, as well as the number of deposition layers. Indeed, the film thickness could be controllable by varying the layer thickness of PS beads. All measurements were performed four times in different directions from the center of the film. NGF Release Studies from Ppy Films. The NGF release profile was assessed using a commercially available sandwich ELISA kit (Millipore). For electrical stimulation, NGF-doped Ppy films were incubated in the PBS solution at 37 °C and subjected to a constant electric voltage of 0.1 V for 2 h to induce the release of the drug incorporated within Ppy. Subsequently, an aliquot was taken from the suspension with time intervals. The aliquots collected were evaluated with ELISA immunoassay in undiluted aqueous samples. The intracellular NGF content was calculated based on the absorbance at 450 nm. Each NGF “release” experiment was performed in triplicate. For the natural release of NGF, the same procedures were carried out without electrical stimulation. PC 12 Cell Culture. PC 12 cells (density of 1  106 cells/mL) were grown in Dulbecco’s modified eagle’s medium (DMEM; Invitrogen) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/ mL penicillin, and 5 mg/mL streptomycin at an incubator setting of 5% CO2 and 37 °C. After trypsinization and centrifugation, cell pellets were resuspended in tissue culture dishes containing Ppy films to observe both cell proliferation and neurite extension as a function of time. The media were changed every 2 days. Cells were observed and photographed using phase-contrast microscopy.

Evaluation of Cell Viability, Proliferation, and Morphology in the Absence and Presence of Electrical Stimulation. To induce electrical stimulation of the PC 12 cells grown on various types of 6180

dx.doi.org/10.1021/la104194m |Langmuir 2011, 27, 6179–6184

Langmuir Ppy films, Ppy substrates were placed in custom-built coverglass chambers,17 where Ppy film served as the anode, a platinum (Pt) wire attached to the chamber served as the cathode, and a silver wire (Ag/ AgCl) served as a reference electrode. A constant voltage of 0.1 V for 2 h was applied to the Ppy films to induce the release of NGF as well as a neurite extension from the composite as a result of electrical stimulation of the films. Neurite outgrowth was analyzed after 1 day of stimulation. All experiments were performed in triplicate. To evaluate the viability of PC 12 cells on various Ppy films, MTT assay was used. The color change from pale yellow to dark blue would be direct evidence indicating the cleavage of tetrazolium rings of MTT by the activity of a mitochondrial dehydrogenase enzyme from viable cells. MTT reconstituted in PBS was added to each suspension containing Ppy films. An MTT solubilization solution was added to break formazan crystals and measured the absorbance at 570 nm. Cell proliferation was observed using optical microscopy upon rinsing with PBS to remove nonadherent cells, where ten random fields per surface was selected and averaged. All the experiments were repeated four times. The cellular morphology was observed using a fluorescent microscopy. For immunostaining of cells, Ppy films were washed in PBS for 30 min, and then fixed with 4% paraformaldehyde (20 min). Cell membranes were permeabilized with 0.1% Triton X-100 (5 min) and washed with PBS three times. DNA was stained with propidium iodide (0.05 mg/mL, Sigma), and finally, actin microfilaments were labeled with the exposure of phalloidin conjugated AlexaFluor 488 (Invitrogen) by incubating for 20 min. Statistical Analysis. Unless otherwise specified, the unpaired Student’s t test (for comparison of 2 groups) or one-way ANOVA and Post Hoc Newman Keul’s test (for more than 2 groups) were used for statistical analyses (InStat software). Normality was tested for by the Shapiro-Wilk test (STATA). Equal variances were tested by the method of Barlett for n g 5 (InStat), and by less than 2-fold difference in SD for n < 5. Results are expressed as the mean ( SD. P < 0.05 was considered statistically significant.

’ RESULTS AND DISCUSSION The typical SEM images of NGF-doped flat Ppy (a), assembled colloidal arrays (b), and well-ordered porous Ppy surfaces (c and d) are shown in Figure 2. In particular, the three-dimensional multilayered colloidal constructs should be noted as shown in Figure 2b. The assembly of colloidal particles relies on a template-based methodology to achieve closely packed three-dimensional structures. We used 200 nm and 1 μm polystyrene beads to generate reliable particle templates using a conventional colloidal selfassembly technique of the charged particles. Further, the resulting colloidal surfaces were employed as a template to obtain NGF-incorporated conductive polypyrrole (Ppy) surfaces through efficient electrochemical deposition. Ppy coated colloidal layers were immediately dissolved with aqueous THF. The corresponding porous surfaces showed similar morphologic features to those of the original templates with structural regularity and interconnected channels as shown in Figure 2c, d. The geometrical distortion or defects in the pore channels were often observed that might have occurred from either the shrinkage generated during the removal process when sacrificing the template—or the lack of control in infiltration of pyrrole monomers into a colloidal crystal template.18 20 The thickness of porous Ppy film corresponded not only to the number of particle layers, but also to the amount of total electrical charge passing through the surface during the deposition. These Ppy films were further explored to verify the effect of porosity on the electrical conductivity and surface wettability (Figure 2e). The

ARTICLE

Figure 2. High-resolution SEM images of (a) flat NGF-doped Ppy, (b) colloidal arrays with multiple layers, (c) NGF-doped porous Ppy with 200 nm pores, and (d) NGF-doped porous Ppy with 1 μm pores. (e) shows the comparison of electrical conductance and water contact angle of various types of Ppy films.

measurement of the sheet resistance of each surface under the same conditions experimentally confirmed that these porous structures still retained their electrical features, regardless of the presence of voids. There was a subtle statistical difference when comparing the electrical conductivities measured from 200 nm and 1 μm porous Ppy. To observe the influence of porosity in the interaction between the liquid and the surface, contact angle measurements were performed. According to Redon et al.’s experimental results, the water molecules tend to preferentially occupy only the very top of the nanopores instead of filling the pore wells.21 For this reason, the surface wettability was increased with porosity, or water molecules showed hydrophilic nature on porous Ppy by immediately spreading over the surface. Thus, we found that surface porosity slightly reversed its intrinsic hydrophobic nature. We consecutively investigated the influence of the applied potentials and surface morphology on the NGF release characteristics. As a dopant, NGF was electrochemically deposited in Ppy films using electrostatic interactions to be utilized as a chemical guidance cue for neuronal cells. As expected, high surface area and the presence of nanometer-sized pores gave rise to significant changes in release behavior as shown in Figure 3. We point out that electrically stimulated porous surfaces show a significant improvement in the release profile over conventional flat Ppy. This suggests that the conventional flat surfaces have a limited mobility for drug release whereas the increased permeability associated with porous topology ensures effective drug transport. Further, as multilayered structures, this design is likely to offer higher loading capacity as well as advanced release ability of entrapped molecules. Indeed, this open structure, produced in part by a combination of electrical stimulation, would be more likely to affect the stability and activity in an enhanced drug delivery. We expanded this study to observe whether porous scaffolds can support enhanced cellular growth and attachment 6181

dx.doi.org/10.1021/la104194m |Langmuir 2011, 27, 6179–6184

Langmuir

ARTICLE

Figure 3. Cumulative NGF release from Ppy films in the absence and presence of electrical stimulation.

Figure 5. Effect of Ppy surfaces on cellular processes: (a) cell proliferation, (b) percentage of cells with neurites, and (c) neurite length grown in the absence and presence of electrical potential. The cells were exposed to a constant voltage of 0.1 V for 2 h.

Figure 4. Viability of PC 12 cells grown on various types of Ppy films. The metabolic activity of cells was assessed regarding to the porosity. * p < 0.05, *** p < 0.001.

in combination with electrical potential in a controlled way. Initially, we observed the effect of surface porosity on PC 12 cell behaviors through cell viability studies as described in Figure 4. No obvious difference in the metabolic activity of the cells was observed between flat and porous surfaces at 1 day. However, an increased rate of effective optical density was observed at 4 days when PC12 cells were cultured on porous substrate. Likely, three-dimensional architectures might offer analogous environments for extracellular matrix by mimicking endogenous chemical and physical architectures that benefit positive cellular developmental responses.22 25 In addition, in recent findings it has been well-established that the conductive Ppy surfaces can induce desired cellular responses from specified cell types, such as fibroblasts, osteoblasts, astrocytes, and neuronal cells, by efficiently promoting neuronal survival and neurite extension with applied electrical fields.18,26 28 Clearly, this unique electrically conducting property of Ppy would be suitable for tissue engineering applications providing remarkable improvements in directing neuronal activities. Figure 5 shows the overall trends of PC 12 cells cultured on flat and porous Ppy surfaces. We

quantified these cellular responses based on the neuronal morphological characteristics by light microscopy. Interestingly, PC12 cells showed slightly enhanced attachment on the porous matrix (Figure 5a). Electrical stimulation by itself was not likely to alter cellular viability or attachment, as is consistent with our preliminary results.29 We continue to evaluate neuronal differentiation and neurite outgrowth by combining electrical, chemical, and surface topographical cues. Several previous studies have reported the cellular growth on the aligned fibers or linearly microfabricated scaffolds that provide neuronal adhesion and extension as a structural contact guidance cue.30 32 Notably, these features provide engineered scaffolds that could closely mimic the architecture of the extracellular matrix (ECM). This interest has prompted us to explore the neuronal processes using unique well-ordered porous Ppy matrix as an ideal platform. As a consequence, the porosity within Ppy surfaces has offered significant advantages over conventional flat Ppy in not only the number of cells with neurites but also the average length of neurites (Figure 5b,c). In particular, the pores with a diameter of 200 nm showed an obvious effect on cell attachment and processes that most likely relied on a synergistic effect by the correlation of electrical, chemical, and topographical characteristics. We attempted to visualize the neurite outgrowth microscopically through staining of the cell nucleus and the actin cytoskeleton to clearly observe morphological activity along a neurite as shown in Figure 6a c. We noticed that much longer neurite extensions have been observed on PC 12 cells cultured on porous surfaces, particularly 6182

dx.doi.org/10.1021/la104194m |Langmuir 2011, 27, 6179–6184

Langmuir

ARTICLE

Figure 6. Representative fluorescence images of PC 12 cells cultured for 2 days. (a) Cells grown on stimulated flat NGF-doped Ppy surface. (b) Cells cultured on electrically stimulated NGF-doped porous Ppy surface with 200 nm pores. (c) Cells grown on stimulated NGF-doped porous Ppy surface with 1 μm pores. The scale bar represents 20 μm. (d f) SEM images of PC 12 cells grown on electrically stimulated porous NGF-doped Ppy surfaces with a diameter of 1 μm for 1 day, showing the initiation of microspike and filopodia extension.

200 nm pores, in the presence of electrical stimulation. This was quite consistent with the NGF release profiles, demonstrating the sufficient supply of NGF to sustain neurite outgrowth in a substrate-dependent way. The cells grown on flat surfaces showed analogous morphological changes, but some of cells displayed more spherical features with a limited number of focal adhesions when compared to those from porous surfaces. On the other hand, high-resolution SEM images give more details for their interaction with the surfaces (Figure 6d f). After 1 day in culture, cells grown on stimulated porous NGFdoped Ppy with a diameter of 1 μm showed neuronal process preferentially sprouting filopodial and microspikes extensions toward the pore channels. This observation indicated neuronal morphology and branching through preferential association with pores, indicating that the porous topography could regulate various growth factors and promote neurite outgrowth.

’ CONCLUSIONS In this study, we demonstrated the preparation of porous NGF-doped Ppy surfaces using self-assembly of polystyrene beads as a sacrificing template. The open pore structure of Ppy resulted in dramatically amplified actions in NGF release profiles, especially when combined with the electrical fields. Using a combination of electrical, chemical, and topographical guidance cues, porous Ppy has presented its enhanced cellular behaviors such as adhesion, metabolic activity, and neurite extension over conventional flat supports. This approach can be simply adapted to conventional electrode devices with controllable micro- and nanoarchitecture. ’ AUTHOR INFORMATION Corresponding Author

*To whom correspondence should be addressed. E-mail: [email protected].

’ ACKNOWLEDGMENT We gratefully acknowledge Judy Grimmer for her expert assistance in vitro cell culture. Debbie Sherman was responsible

for expert SEM assistance. We appreciate the excellent illustrations and graphics of Michel Schweinsberg and the assistance of Jennifer Danaher for manuscript preparation. We thankfully acknowledge the financial support from the General Funds of the Center for Paralysis Research, The State of Indiana, and a generous endowment from Mrs. Mari Hulman George.

’ REFERENCES (1) Chung, S. H.; Son, S. J.; Min, J. Nanotechnology 2010, 21, 125104. (2) Lopez, C. A.; Fleischman, A. J.; Roy, S.; Desai, T. A. Biomaterials 2006, 27, 3075. (3) Low, S. P.; Williams, K. A.; Canham, L. T.; Voelcker, N. H. Biomaterials 2006, 27, 4538. (4) Abidian, M.; Kim, D.; Martin, D. Adv. Mater. 2006, 18, 405. (5) Cui, X.; Wiler, J.; Dzaman, M.; Altschuler, R.; Martin, D. Biomaterials 2003, 24, 777. (6) Kim, D.; Martin, D. Biomaterials 2006, 27, 3031. (7) Thompson, B.; Moulton, S.; Ding, J.; Richardson, R.; Cameron, A.; O’Leary, S.; Wallace, G.; Clark, G. J. Controlled Release 2006, 116, 285. (8) Wallace, G.; Spinks, G. Soft Matter 2007, 3, 665. (9) Edell, D. J.; Toi, V. V.; Mcneil, V. M.; Clark, L. D. IEEE Trans. Biomed. Eng. 1992, 39, 635. (10) Gautier, C.; Cougnon, C.; Pilard, J.-F.; Casse, N.; Chenais, B. Anal. Chem. 2007, 79, 7920. (11) Parthasarathy, R. V.; Martin, C. R. Nature 1994, 369, 298. (12) Schmidt, C.; Leach, J. Annu. Rev. Biomed. Eng. 2003, 5, 293. (13) Turner, J. N.; Shain, W.; Szatowski, D. H.; Andersen, M.; Martins, S.; Isaacson, M. Exp. Neurol. 1999, 156, 33. (14) Frey, M. T.; Tsai, I. Y.; Russell, T. P.; Hanks, S. K.; Wang, Y. Biophys. J. 2006, 90, 3774. (15) Harley, B. A. C.; Kim, H.; Zaman, M. H.; Yannas, I. V.; Lauffenburger, D. A.; Gibson, L. J. Biophys. J. 2008, 95, 4013. (16) Zang, F.; Sun, Z.; Wang, C.; Ren, B.; Liu, X.; Tong, Z. Langmuir 2002, 18, 9116. (17) Wood, M.; Willits, R. K. Bioelectromagnetics 2006, 27, 328. (18) Cho, Y.; Borens, R. B. Nanotechnology 2010, 21, 205102. (19) Jiang, P.; Hwang, K. S.; Mittleman, D. M.; Bertone, J. F.; Colvin, V. L. J. Am. Chem. Soc. 1999, 121, 11630. (20) Kotov, N. A.; Liu, Y.; Wang, S.; Cumming, C.; Eghtedari, M.; Vargas, G.; Motamedi, M.; Nicols, J.; Cortiella, J. Langmuir 2004, 20, 7887. 6183

dx.doi.org/10.1021/la104194m |Langmuir 2011, 27, 6179–6184

Langmuir

ARTICLE

(21) Redon, R.; Vazquez-Olmos, A.; Mata-Zamora, M. E.; OrdonezMedrano, A.; Rivera-Torres, F.; Saniger, J. M. J. Colloid Interface Sci. 2005, 287, 664. (22) Karlsson, M.; Johansson, A.; Tang, L.; Boman, M. Microsc. Res. Techniq. 2004, 63, 259. (23) Luo, X.; Cui, X. T. Electrochem. Commun. 2009, 11, 1956. (24) O’Brien, F. J.; Harley, B. A.; Yannas, I. V.; Gibson, L. J. Biomaterials 2005, 26, 433. (25) Yan, W.; Hsiao, V.; Zheng, Y. B.; Shariff, Y. M.; Gao, T.; Huang, T. J. Thin Solid Films 2009, 517, 1794. (26) Cho, Y.; Shi, R.; Ivanisevic, A.; Borgens, R. Nanotechnology 2009, 20, 275102. (27) Ateh, D. D.; Navsaria, H. A.; Vadgama, P. J. R. Soc. Interface 2006, 3, 741. (28) Mihardja, S. S.; Sievers, R. E.; Lee, R. J. Biomaterials 2008, 29, 4205. (29) Au, H.; Cheng, I.; Chowdhury, M.; Radisic, M. Biomaterials 2007, 28, 4277. (30) Foley, J. D.; Grunwald, E. W.; Nealey, P. F.; Murphy, C. J. Biomaterials 2005, 26, 3639. (31) Neeley, W. L.; Redenti, S.; Klassen, H.; Tao, S.; Desai, T.; Young, M. J.; Langer, R. L. Biomaterials 2008, 29, 418. (32) Xie, J.; MacEwan, M. R.; Willerth, W. M.; Li, X.; Moran, D. W.; Sakiyama-Elbert, S. E.; Xia, Y. Adv. Funct. Mater. 2009, 19, 2312.

6184

dx.doi.org/10.1021/la104194m |Langmuir 2011, 27, 6179–6184