Nanoscale Interfaces of a Semiconducting

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Biological and Medical Applications of Materials and Interfaces

Photoconductive micro/nano-scale interfaces of a semiconducting polymer for wireless stimulation of neuron-like cells Yingjie Wu, Yanfen Peng, Hassan Bohra, Jianping Zou, Ranjan Vivek Damodar, Yilei Zhang, Qing Zhang, and Mingfeng Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19631 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Materials & Interfaces

Photoconductive micro/nano-scale interfaces of a semiconducting polymer for wireless stimulation of neuron-like cells Yingjie Wu†, Yanfen Peng†, Hassan Bohra†, Jianping Zou‡, Ranjan Vivek Damodar§, Yilei Zhang§, Qing Zhang‡, Mingfeng Wang*† †School

of Chemical and Biomedical Engineering, Nanyang Technological University, 62

Nanyang Drive, 637459, Singapore ‡School

of Electrical and Electronic Engineering, Nanyang Technological University, 50

Nanyang Avenue, 639798, Singapore §School

of Mechanical & Aerospace Engineering, Nanyang Technological University, 50

Nanyang Avenue, 639798, Singapore

ABSTRACT: We report multiscale structured fibers and patterned films based on a semiconducting polymer, poly(3-hexylthiophene) (P3HT), as photoconductive biointerfaces to promote neuronal stimulation upon light irradiation. The micro/nano scale structures of P3HT used for neuronal interfacing and stimulation include nanofibers with an average diameter of 100 nm, microfibers with an average diameter of about 1 µm, and the lithographically patterned stripes width of 3, 25 and 50 μm, respectively. The photoconductive effect of P3HT upon light irradiation provide electrical stimulation for neuronal differentiation and directed growth. Our results demonstrate that neurons on P3HT nanofibers showed a significantly higher total number of

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branches while neurons grown on P3HT microfibers had longer and thinner neurites. Such a combination strategy of topographical and photoconductive stimulation can be applied to further enhance neuronal differentiation and directed growth. These photoconductive polymeric micro/nano structures demonstrated their great potential for neural engineering and development of novel neural regenerative devices. KEYWORDS: polymers, semiconductors, nanostructures, neurons, tissue engineering INTRODUCTION The nervous system plays a central and complex role in human biological processes interacting in physiological ones. Damage to the nerve may impose tremendous consequences and its recovery can be difficult under serious damage.1,2 It is well known that regeneration of damaged tissue begins with the neuron differentiation and axon growth.3-6 The potential for harnessing the electric fields to induce enhanced neurite outgrowth in biological systems arouses intensive interest and research efforts.7 To repair or restore damaged neurological functions, majority of the neural scaffolds are electroactive biomaterials, so that electric current can pass electroconductive scaffolds and stimulate neuronal differentiation to repair nerve lesions. Stable and corrosion resistant metals (gold,8 platinum,9 iridium,10 platinum-iridium alloy,11 titanium,12 and stainless steel13), carbon nanotubes,14,15 graphene16-18 and conducting polymers19,20 have been popular choices for neural scaffolds. Carbon nanotubes or conducting polymers such as polypyrrole,21 poly(3,4-ethylenedioxythiophene) (PEDOT)22,23 and polyaniline24, are considered as a newgeneration neural interfacing material, and could optimize the neural adhesion, differentiation and neurite outgrowth. However, these electronically conducting scaffolds require an additional external power source and complicated wiring to provide effective electrical stimulation against


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cells or tissues. Moreover, a normal electrical stimulator connected to a high-voltage power source attached to the body for nerve regeneration is uncomfortable, inconvenient, and unsafe. Therefore, a long-lasting self-powered new electrical stimulation system with suitable topography is urgently required for nerve regeneration devices.25,26 Compared to purely electrical stimulation, optoelectrical stimulation that converts light into an electrical signal, provides a less invasive and wireless option for neural interfacing. For instance, Goda and coworkers have reported noninvasive depolarization of neurons via photoconductive stimulation on a silicon wafer.27 But the clinical application of silicon-based materials and devices for neural interfacing could be limited by their relatively low light-absorption efficiency, the mechanical mismatch between silicon (hard and rigid) and neural cells and tissues (soft), and the poor biostability of silicon under physiological conditions.28 In contrast to conventional inorganic semiconductor such as silicon, semiconducting polymers as a new class of soft materials often show high efficiency of light absorption and tunable optical, electronic, mechanical properties that are promising for applications such as photovoltaic and light emitting devices.29,30,31 These organic polymers have several advantages over inorganic semiconductors such as easy processability and compatibility with low-temperature processes, large-scale fabrication, and improved mechanic and biological compatibility.32 For instance, some semiconducting polymers, due to their easy processability from common organic solvents, have been processed into films via different coating methods, and micro/nano-scale fibers by continuous melt-drawing33 or electrospinning34. Among the various organic photovoltaic polymers, poly(3-hexylthiophene-2,5-diyl) (P3HT) has received considerable attention because of its good solubility, favorable optoelectronic properties and its ability to form crystalline structures via strong π-π interactions between the aromatic backbones of adjacent extended polymer chains.35-39 In 2013, Ghezzi et al. reported photoexciation


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and electrochemical recording of single neurons on surfaces of P3HT thin films spin-cast on indium-tin-oxide (ITO) coated glass substrates.40 The photostimulation of primary neurons and retinal explants was mainly attributed to the generation of photocurrent that could be detected using a patch pipette in voltage-clamp mode at the P3HT/electrolyte interface under light irradiation. But photostimulated neuronal differentiation and growth was not studied in that system. On the other hand, the topographical features of the substrate have been widely used to influence the sub-cellular behaviors, particularly to guide the direction of neuronal differentiation and growth.41,42 Previous studies have demonstrated that substrates with a certain size of microgrooved structure or other micro-topological cues significantly promote nerve cell alignment and accelerate growth longitudinally.43-45 For instance, Hsiao et al. reported the selective adhesion of neuron-like pheochromocytoma (PC-12) cells on patterned PEDOT)microelectrode arrays on ITO-coated glass.46 The outgrowth of neurites on the PEDOT microelectrodes was promoted by ca. 60% upon applied electrical stimulation. Nevertheless, as the PEDOT involved were electrochemically doped and were used as organic metallic electrodes, the neuronal stimulation still relied on external electric field and power resources in which extra electrical wiring was needed. Herein, we report a strategy of wireless photostimulation to control the neuronal differentiation and growth on surfaces of a semiconductor polymer, using P3HT as an example, with well-defined structures and topographies, including self-assembled fibers, electro-spun micrometer-scale fibers and lithographically patterned stripes, versus homogeneous films as control. We chose rat pheochromocytoma (PC12) cell as an example of primary neuron, and studied how these cells respond, upon light irradiation, on the surface of P3HT with different structures and dimensions.


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One of our research objectives is to establish the relationship between the micro/nano structures of the P3HT substrates and the neuronal response. The knowledge that we gain from these studies will be important to engineer other semiconducting polymers towards noninvasive photostimulation of a broad scope of neural cells and tissues. RESULTS AND DISCUSSION

Figure 1 (A) Schematic illustration of the preparation of P3HT micro/nanofibers and P3HT patterned surface for PC12 cell culture. (B) UV-vis absorption spectra of P3HT polymer in anisole solution, P3HT micro/nanofiber and homogenous films. (C-D) Digital photographs of P3HT in hot anisole (100 °C) (C) and after being cooled down to 25 °C.

Scaffolds in tissue engineering provide a microenvironment for cells to adhere, proliferate, and differentiate.47 In this work, we studied neural cells cultured on P3HT homogeneous film,


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nanofibers, e-spun microfibers and lithographically patterned micro-scale stripes. (Figure 1A) UVvis absorption spectra of P3HT in anisole showed the maximum absorption peak at 502 nm (Figure 1B). P3HT micro/nanofiber and patterned films both show strong vibronic bands at 550 and 600 nm, which are mainly attributed the enhanced π-π stacking in the solid states.38,48-50

Figure 2. AFM height (A, C) and phase (B, D) images of P3HT in solid states with different morphologies and structures: (A, B) homogeneous film, (C, D) nanofibers and (E, F) SEM images of P3HT e-spun microfibers.


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The dissolution of P3HT in anisole resulted in a transparent orange solution at a temperature above 70 °C (Figure 1C). During cooling, the P3HT solution displayed a dramatic color change into purple and gelled at 20 °C (Figure 1D). Such gelation is attributed to the formation of P3HT nanofibers in the gradual cooling process, which is further proved by AFM imaging (Figure 2CD) of the nanofiber dispersion dried on glass substrates. The nanofiber formation of P3HT is mainly driven by the strong π-π interactions between the adjacent aromatic backbones.48,50 We adopted a spin-coating method to prepare P3HT homogenous film for the following cell study. Because P3HT is well soluble in chloroform,51 here we dissolved P3HT in chloroform to prepare P3HT homogeneous film as the control to study the additional effects of film morphology on the growth and differentiation of neurons. AFM images in Figure 2A and B showed that a featureless uniform film was obtained after the P3HT solution with a concentration of 1 mg/mL in chloroform was spin-cast on a flat glass substrate without other treatment. Self-assembled P3HT nanofibers were prepared from a solution in anisole with a polymer concentration of 1 mg/mL. More details of the sample preparation are described in Experimental Section. The dispersion of the P3HT nanofibers was diluted to 0.01 mg/mL and drop-cast on a silicon substrate for further characterization by AFM (Figure 2C and D), or on a copper grid coated with a thin carbon film for TEM characterization (Figure S1A). We found that P3HT nanofibers could not be obtained by rapid cooling (e.g. at a cooling rate of 5 oC/min), probably because rapid cooling of P3HT solution to room temperature could disturb the self-organization of the polymer chains. P3HT nanofiber can only be produced by slow cooling (i.e. a cooling rate of 1 oC/min). We obtained well-dispersed nanofibers by spin coating a 100-fold diluted dispersion on a flat glass substrate. As shown in Figure S2, two different concentrations of P3HT anisole solution were


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compared when quick cooling or slowly cooling to room temperature. The results showed that highly dispersed and well-defined nanofibers can be achieved when P3HT with a concentration of 1 mg/mL in anisole is slowly cooled to room temperature. AFM images showed one-dimensional nano-fibrillar structures on glass substrates where the solutions were spin coated at room temperature. The P3HT nanofibers had the diameter of about 50 ± 5 nm and the length of several micrometers. Composite microfibers formed by P3HT/poly(ε-caprolactone) (PCL) were prepared by electrospinning. Because the viscosity of P3HT solution was too low for electrospinning, here we chose biocompatible PCL as the fiber matrix, mixed with P3HT as the photoconductive component at a weight ratio of 1:1. SEM and TEM images of the microfibers showed that the microfibers were generally uniform, with an average diameter of 1.0 ± 0.2 µm (Figure 2E, F and Figure S1B). These results are consistent with what Jeong and coworkers observed in their electrospun fibers of P3HT/PCL with the same weight ratio as ours, in which a continuous P3HT phase formed in the composite fibers.52 Moreover, the dimensions of microfibers could be changed by controlling PCL concentration. The experiments were carried out on various PCL concentrations of 10%, 12% and 14% (w/v) with the optimized constant voltage of 8 kV, constant tip-to-collector distance of 12 cm and flow rate of 1 mL/h. As shown in Figure S3, at the PCL concentration of 12%, smooth and uniform fibrillar structure was observed. Thus, we chose PCL concentration of 12% for the following studies.


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Figure 3. (A-H) Laser confocal microscopy images of PC12 cells culture on blank glass substrate, P3HT homogeneous film, P3HT nanofiber substrate and P3HT/PCL microfiber substrate with/without LED irradiation. (I) The average total neurite extension per cell after 3 days of cell culture on four kinds of substrates. (J) The longest neurite extension per cell after 3 days of cell culture on different kinds of substrates.

To promote cell adhesion, the surfaces of the polymer films were pre-treated with poly-D-lysine. A green LED device (power: 2 W) was used for light stimulation (Figure S4). The cellular distribution and morphology on scaffolds were measured by fluorescence images via F-actin staining. Both laser-scanning confocal fluorescence microscopy and white-contrast optical microscopy were used to image the cells and measure the neurite extension after a certain period of the cell culturing (Figure 3A-H and Figure S5). After three days of culturing, most of the PC12


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cells changed from their originally round shape to elongated and spindle-like shape. LEDstimulated PC12 cells on P3HT polymer substrate showed new axon growth. In contrast, unstimulated PC12 cells and cells stimulated on blank glass showed only small protrusions and no formation of neurites. Moreover, neurons on P3HT nanofiber substrate showed a significantly higher total number of branches, with dendritic axons spreading from the cell soma. Neurons grown on P3HT microfibers with LED irradiation had longer and thinner neurites. After three days of cell culture, the average neurite extensions of PC12 cells on different substrates were statistically analyzed (Figure 3I). Here twenty cells were analyzed for each substrate. Among them, there was no apparent difference between cells with and without stimulation by LED light irradiation on blank glass substrates. The average total neurite extensions per cell on P3HT homogeneous film was 22 ± 2 µm, which was significantly longer than the control group (8 ± 1 µm) of cells cultured on the blank substrate. The average total neurite extensions per cell on P3HT microfibers and nanofibers were 32 ± 3 and 38 ± 2 µm, respectively, significantly longer than the control group of homogeneous film. The neurite extension of cells cultured under LED irradiation was significantly longer than the control group without LED irradiation. From the statistical analysis of the longest neurite from each neuron, as shown in Figure 3J, one can see that the P3HT substrate with LED irradiation supported longer neurite extension of PC12 cells compared to the control group without LED irradiation. All the results show that neurons cultured on P3HT substrate under LED light irradiation led to longer neuritis outgrowth compared to those grown on blank substrate, which could be mainly attributed to the photoconductive effect of P3HT under light irradiation that dramatically promotes neurite outgrowth35,53. Furthermore, it was found that the morphology of P3HT film can affect cell


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differentiation. P3HT micro/nanofibers can enhance PC12 cell adhesion and differentiation and can serve as effective substrates to enhance nerve regeneration.

Figure 4. (A-F) Laser scanning confocal fluorescence microscopy images of PC12 cells cultured on ITO-coated glass surfaces patterned with 3-, 25-, 50-μm wide stripes of P3HT with/without LED irradiation. (G) The average total neurite extension per cell after 3 days of cell culture on three kinds of patterned substrates. (H) The average longest neurite extension per cell after 3 days of cell culture on P3HT region of three kinds of patterned substrates.


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We investigated further the cell morphology by scanning electron microscopy (SEM) on the third day after cell seeding. The SEM images (Figure S6) show that, without LED irradiation, cells grown on the four kinds of substrates appeared little differentiated as the typical non-spreading morphology of PC12. In contrast, after LED irradiation more dispersive and elongated axons were observed, as shown in Figure S6 D, F, H. The microfiber, meanwhile, supports the growth of new axons along fibers. These results further declared that green LED irradiation can effectively stimulate new axon growth and promote the differentiation of PC-12 cells on P3HT based substrates. Previous studies, including ours,54 have demonstrated that topographical stimulation by micro and nanopatterned surfaces can promote neuron differentiation via the assembly of focal adhesion related proteins including vinculin, and paxillin.33 In this study we furthermore found that the substrates coated with P3HT nanofibers led to enhanced neuronal differentiation compared to substrates coated with P3HT homogeneous films or microfibers. Microscale grooves are among the most commonly fabricated topographical features that have been employed to control cell behaviour, but have been largely limited to electrically insulating or conducting substrates.55 As a consequence, we further studied the photoconductive stimulation of neurons on well-defined microscale-wide P3HT stripes fabricated by a photolithography process which is described in detail in Supporting Information. Alexa Fluor® 633 phalloidin was used to stain filamentous actin (F-actin) cytoskeleton. In the meantime, P3HT patterned stripes also adsorbed fluorescent dyes and showed red-fluorescence under the confocal laser-scanning fluorescence microscopy. Figure 4 demonstrates the observed morphologies of the PC12 cells and neurite extension on different P3HT-patterned ITO surfaces. The ITO underlying P3HT was used mainly to promote the photoconductive effect while minimize the photothermal effect, as previously reported by Lanzani and coworkers.56 The widths of the patterned P3HT stripes


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(thickness: 300 nm) were 3, 25 and 50 μm, respectively. Fluorescence imaging (Figure 4A-F) and measurement of the neurite length (Figure 4G-H) clearly showed that light irradiation can support PC12 adhesion and differentiation. Statistical analysis of the neurite lengths (Figure 4G-H, and Figure S7-8 in Supporting Information) showed that LED irradiation group (Figure 4D-F) led to significantly longer cell neurite outgrowth in comparison with the control group without light irradiation (Figure 4A-C). The neurite elongation, alignment, and neuronal differentiation properties of the PC12 cells were found to be strongly related to P3HT photoconductive stimulation. We also found that the width of the P3HT stripes affects the neurite’s outgrowth. Specifically, the PC12 cells irradiated on the surface of 3-μm wide P3HT stripes tend to show larger neurite extension in average compared to those on 25- and 50-μm P3HT stripes.(Figure 4H) Such difference could be attributed to the effect of integrin clustering which is enhanced on the patterned substrates with a smaller size of grooves.57 In addition, we used histograms and Gaussian fit curves to analyse the neurite length of PC12 cells incubated on P3HT-patterned surface with 25- and 50μm stripes, respectively. A total number of 50 cells in each region were selected randomly and analysed. The distribution of the neurite length of PC12 cells incubated on P3HT patterned ITO surface with 25-μm stripes is shown in Figure S7 in Supporting Information. Without light irradiation, the average neurite length of PC12 cells incubated on ITO region was 14.84 µm. Under light irradiation, the average neurite length of PC12 cells incubated on P3HT-patterned ITO region was 26.44 µm, which was much longer than those without light irradiation or the cells with light irradiation but grown on bare ITO region. As PC12 cells incubated on P3HT-patterned ITO surface with 50 μm stripes, under light irradiation the average neurite extension of PC12 cells incubated on P3HT region and ITO region was 25.49 and 26.34 µm, respectively (Figure S8). The neurite


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extension that we observed on bare ITO regions under light irradiation could be attributed to the photoconductive effect of ITO itself. But other possibilities such as the migration of the cells across different regions during the cell culturing and light irradiation cannot be excluded here. Further investigation of the neural differentiation, growth and migration in situ by optical imaging is needed in the future study. Overall, these results suggest that a combination of surface topography and photoconductive stimulation could lead to a remarkable promotion of PC12 neuronal differentiation, and that small-scale groove structures can promote axon regeneration. Previous studies have shown that the electrical stimulation could induce the plasma membrane of a neuron depolarization, which in turn, prompting membrane channels open and trigger calcium ions (Ca2+) influx. The upsurge in intracellular Ca2+ stimulates the release of neurotransmitters and triggers downstream signaling pathways. The formation of the Ca2+/calmodulin (CaM) complexes activates CaM kinase, by virtue of up- or down-regulating the expression levels of the neurite’s development related proteins that promotes the axonal regeneration.58,59 The related schematic diagram is shown in Figure S9A. Therefore, we verified the photoconductive effect of P3HT on the neuronal response based on a calcium indicator Fluo-4 AM. Fluo-4 AM is a membranepermeable, Ca2+-dependent dye and exhibits a large increase of fluorescence intensity upon binding of free Ca2+.60 If the voltage pulse stimuli on a neuron could open calcium ion channels and increased the calcium ion concentration of cell, it will result in the enhanced fluorescence intensity of Fluo-4 AM dye in the neuron. To examine this hypothesis, we studied the effect of photostimulation on P3HT micro/nanofiber substrates by monitoring the changes of the intracellular Ca2+ using a membrane permeable Ca2+ indicator dye, Fluo-4 AM. Figure S9B and C clearly showed a fluorescence boundary. The boundary shows that PC12 cells in the laser exposed section turned green due to intracellular calcium ion concentration enrichment stained by Fluo-4


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AM dye, as compared to the unexposed part, in which most cells stayed transparent nonfluorescence. These results demonstrate that the electrical stimulation caused by photoelectric conversion effect of the P3HT polymer under light irradiation could activate calcium channels and lead to Ca2+ concentration enrichment. From Figure S9D and E, we not only observed the growth of new axons up and down the microfiber but also detected fluorescence from cells. Moreover, similar phenomenon of Ca2+ fluorescence was observed in homogenous film group (Figure S10). These results imply that P3HT with photoconductive property is a promising strategy to achieve neuronal activation with light. CONCLUSIONS We have presented an effective approach of wireless photostimulation of neuron-like cells using photoconductive effect of a semiconducting polymer P3HT with multiscale ordered structures and patterns. Specifically, self-assembled nanofibers with an average diameter of about 100 nm, electrospun microfibers with an average diameter of 1 µm, and lithographically patterned grooves were fabricated. The differentiation and growth of PC-12 neuron-like cells adhered on these P3HT substrates, upon light irradiation, were studied. Contributed by the photoconductive effect of P3HT, the P3HT-based substrate can provide electrical stimulation for neuron growth. Moreover, the topographical features of the substrate also have a significant effect in tissue engineering. It has been demonstrated that neurons on P3HT nanofibers showed a significantly higher total number of branches while neurons grown on P3HT microfibers had longer and thinner neurites. Our results suggest the potential applications of semiconductor polymers for the stimulation of neuronal cells exhibiting functionally mature neuronal phenotypes and repairing injured peripheral nerves.


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ASSOCIATED CONTENT Supporting Information Materials and methods; TEM images of P3HT nanofibers and P3HT/PCL microfibers; AFM images of P3HT nanofibers prepared under different conditions; SEM images of P3HT/PCL microfibers with different ratios; digital photographs of the light stimulation setup for PC12 cells; bright-field optical microscopy and SEM images of PC12 cells cultured onto various substrates; histograms and Gaussian fit curves statistical analyses of neurite length of PC12 cells incubated on P3HT patterned ITO surfaces; bright-field optical and fluorescence microscopy images of PC12 cells. The Supporting Information is available free of charge on the ACS Publications website at DOI: XX. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT M.W. is grateful to the funding support by a start-up grant of Nanyang Assistant Professorship (M4080992) from Nanyang Technological University, and AcRF Tier 2 (ARC 36/13: M4020172) from the Ministry of Education, Singapore. REFERENCES


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Nanoscale Interfaces of a Semiconducting

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