Biocompatible EDOT−Pyrrole Conjugated Conductive Polymer Coating for Augmenting Cell Attachment, Activity, and Differentiation

Abstract

Developing scaffolds supporting functional cell attachment and tissue growth is critical in basic cell research, tissue engineering, and regenerative medicine approaches. Though poly(ethylene glycol) (PEG) and its derivatives are attractive for hydrogels and scaffold fabrication, they often require bioactive modifications due to their bioinert nature. In this work, biomimetic synthesized conductive polypyrrole-poly(3,4-ethylenedioxythiophene) copolymer doped with poly(styrenesulfonate) (PPy−PEDOT:PSS) was used as a biocompatible coating for poly(ethylene glycol) diacrylate (PEGDA) hydrogel to support neuronal and muscle cells’ attachment, activity, and differentiation. The synthesized copolymer was characterized by Raman spectroscopy and dynamic light scattering. Its electrochemical properties were studied using galvanostatic charge−discharge (GCD) and voltammetry. PPy−PEDOT:PSS-coated hydrogels were characterized by Raman spectroscopy and atomic force microscopy, and protein adsorption was assessed using a quartz crystal microbalance with dissipation monitoring. Attachment and differentiation of the ND7/23 neuron hybrid cell line and C2C12 myoblasts were evaluated by cell cytoskeleton staining and quantification of morphological parameters. Viability was assessed by live/dead staining using flow cytometry. Cortex neural activity was studied by calcium ion influx that could be detected through the dynamic fluorescence changes of Fluo-4. The PPy−PEDOT:PSS coating supported cell attachment and differentiation and was nontoxic to cells. Primary neurons attached and remained responsive to electrical stimulation. Altogether, the biocompatible copolymer PPy−PEDOT:PSS is a simple yet effective alternative for hydrogel coating and presents great potential as an interface for nervous and other electrically excitable tissues.

Graphical Abstract

1. INTRODUCTION

Nervous system injuries and diseases are leading causes of ill health and disability worldwide. As the nervous system presents a limited capacity for spontaneous regeneration and functional recovery after injury due to trauma or disease,¹ developing new technologies and therapies for recovering nervous tissue function is very appealing. Although it is well-known that bioelectricity, i.e., an electrical phenomenon that is actively generated by cells,² plays a crucial role in the functional and structural organization of the nervous system,³ electrical stimulation (ES) and conductive cues have been disregarded in nerve tissue engineering and regenerative medicine until recently.⁴ Scaffold-based approaches to evoke neural activity or to promote nerve growth usually coat or embed matrices with conductive materials such as metallic or polymeric films and nanoparticles to obtain conductive biomaterials that interface with cells.⁵ Though the effectiveness of electroconductive scaffolds and ES has been studied for a few decades, their mechanisms of action are poorly understood.⁶ It has been suggested that ES could directly act on neurons via the activation of ion channels in the plasma membrane.⁷ These channels are responsible for triggering signaling pathways that can lead, for example, to increasing the expression of nerve growth factor (NGF).⁸ Moreover, conductive biomaterials can maintain the integrity of physiological electrical pathways and enhance the interactions between neurons. Electroactive materials and ES can also mediate nervous tissue regeneration by modulating the immune microenvironment.⁹ Conductive biomaterials are also very appealing as scaffolds for tissue engineering of other electrically excitable tissues such as muscle as they can promote muscle tissue formation.

Biomaterial physicochemical properties such as stiffness, roughness, surface charge, and wettability are important in regulating cell and tissue function.¹⁰ Hydrogels are porous networks obtained by chemical or physical cross-linking of hydrophilic polymers. Due to their resemblance to nervous tissue extracellular matrix, porosity, viscoelastic properties, and biocompatibility, hydrogels are widely used as scaffolds in neural tissue engineering.¹¹ Poly(ethylene glycol) (PEG) and its modifications such as poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) dimethacrylate (PEGD-MA) are among the most biocompatible and widely used synthetic polymer hydrogels approved by the FDA. However, due to their bioinert nature, PEG hydrogels cannot support cell adhesion and tissue formation and need to be further modified with cell adhesion motifs, polymers, or biomolecules.¹²

Conductive polymers (CPs) such as polypyrrole (PPy), polyaniline (PANI), and poly(3,4-ethylenedioxythiophene) (PEDOT) offer several advantages compared to traditional inorganic conducting materials. While cells use ionic conduction mechanisms and metallic materials use electronic conduction mechanisms, CPs present a mix of both, allowing optimal bidirectional communication between biological tissues and devices.¹³ CPs’ stability, biocompatibility, and easy tunability of their structural and physicochemical properties make them very appealing for biomedical applications. For instance, Ren et al. developed highly conductive PPy−PEDOT doped with poly(styrenesulfonate) (PSS) hybrid hydrogels by a solution-mixing method. The hydrogels presented tissue-like mechanical properties and were able to sustain PC12 cell attachment and growth.¹⁴ In a different approach, Zheng et al. used N-(3-(dimethylamino)-propyl)-N′-ethylcarbodiimide hydrochloride (EDC) to cross-link PEDOT:PSS and multiwalled carbon nanotubes to gelatin hydrogels. The obtained mechanically flexible, electroactive, and self-healable hydrogels facilitated ES and promoted cell proliferation and healing of a full-thickness skin defect model.¹⁵ Goestenkors et al. studied the influence of ionic liquid concentration (gelling agent in the hydrogel precursor mixture) in PEDOT:PSS cross-linking to optimize hydrogel conductivity.¹⁶ Song et al. used electroplated PPy 14G nickel−chromium alloy wire tubes as conductive nerve guides. ES enhanced the efficacy of human neural progenitor cells in treating sciatic nerve transection in rats.¹⁷ Ritzau-Reid et al. used an end-capping strategy to synthesize EDOT oligomers. These oligomers were polymerized with poly(caprolactone), and conductive scaffolds for neural stem cell culture were obtained by electrospinning.¹⁸ In a simpler approach, Magaz et al. coated silk fibroin electrospun scaffolds with PEDOT−PSS by means of electrostatic interactions.¹⁹ NG108-15 neuronal cells were already able to attach and differentiate on silk fibroin scaffolds, but metabolic activity and proliferation were enhanced with the PEDOT−PSS coating.

The synthesis of conducting polymers, especially PEDOT, PPy, or PANI, is carried out through two main approaches: electrochemical and chemical. In chemical synthesis, highly oxidizing chemical species, such as ammonium persulfate or ferric chloride, are used. These conditions enable the polymerization of the aromatic monomers EDOT or pyrrole. The use of small molecules or nanoparticles that resemble the active site of oxidoreductases as green catalysts allows the polymerization of electrically conductive and conjugated polymers with a controlled structure and are free of toxic products. In these enzyme-mimetic or biomimetic synthesis approaches, the reaction mechanism involved is very similar to the mechanism observed in peroxidase-mediated synthesis. Ferrocene, an organometallic compound that contains Fe, and hematin, a porphyrin with an Fe core that closely resembles the prosthetic center found in horseradish peroxidase, are two common catalysts used in biomimetic synthesis. Wang et al. reported the biomimetic synthesis of microsphere polyaniline using the peroxidase-like catalytic activity of ferrocene.²⁰ Bruno et al. used poly(ethylene glycol)-modified hematin (PEG-hematin) to catalyze the synthesis of homo and copolymers of pyrrole and EDOT²¹ and polyaniline.²² We have reported the use of hematin as a catalyst to synthesize PPy, PEDOT, and PANI homopolymers and copolymers.²³ The copolymer obtained using a mole fraction of EDOT of 0.9 in the EDOT−pyrrole copolymer presented the optimal balance of properties such as conductivity, suspension stability, size, and charge storage capacity.

We have recently developed a platform for wireless neuronal stimulation using biomimetically synthesized PPy−PE-DOT:PSS.²⁴ The interaction of PPy−PEDOT:PSS with a neuron hybrid cell line was able to enhance neural differentiation. This effect was more pronounced when PPy−PEDOT:PSS was electrically charged right before cell exposure. Furthermore, the administration of charged PPy−PEDOT:PSS to primary rat cortical networks largely increased the neural activity. Moreover, electrodes coated with PPy−PEDOT:PSS by electrophoretic deposition showed enhanced galvanotaxis compared to conventional electrodes and, unlike their counterparts, do not release toxic species into the cell medium or disrupt cell viability after several cycles of ES.²⁵

Here, we report the use of biomimetically synthesized PPy−PEDOT:PSS as a coating to promote neuron attachment, activity, and differentiation on eosin-photopolymerized PEGDA hydrogels. Previous research has employed complex procedures to fabricate PEDOT-derived hydrogels, whereas, in our approach, PEGDA hydrogels are coated with PPy−PEDOT:PSS by physical adsorption, simplifying the process significantly to obtain PEG-PP. Noncoated hydrogels (PEG-NC) and hydrogels functionalized with fibronectin (PEG-FN) were used as controls. To compare PPy−PEDOT:PSS to another conductive but nonpolymeric material, hydrogels incubated with gold nanoparticles (PEG-GN) were also used. FN was covalently linked to the PEGDA hydrogels via standard sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)-hexanoate (sulfo-SANPAH)-mediated succinimide cross-linking.²⁶ The synthesized PPy−PEDOT:PSS copolymer chemical composition was confirmed by Raman spectroscopy, and dynamic light scattering was used to characterize the colloidal suspension, which showed a negative charge of −37 mV and hydrodynamic diameters in the range of 300−700 nm. Galvanostatic charge−discharge and cyclic voltammetry experiments revealed that the copolymer displays a pseudocapacitive behavior and large mass-specific capacitance. Polymer-coated hydrogels were characterized by Raman spectroscopy and atomic force microscopy (AFM). The increase in surface roughness and the appearance of 20−50 nm peaks and 200−500 nm structures confirmed the stable adsorption of PPy−PEDOT:PSS on the hydrogels. Protein adsorption was assessed by using a quartz crystal microbalance with dissipation monitoring. PEGDA hydrogels resulted in rather antifouling surfaces, and the copolymer coating enhanced protein adsorption. While PEGDA hydrogels prevented neuron attachment, the PPy−PEDOT:PSS coating promoted neuron adhesion to the same degree as FN-coated hydrogels. PPy−PEDOT:PSS-coated surfaces were nontoxic and supported ND7/23 neuron hybrid cells and C2C12 myoblast attachment and differentiation. Primary cortex neural activity assessed by calcium ion influx through the dynamic fluorescence changes of Fluo-4 showed active cells on the coated surfaces that respond to ES. In summary, we present a straightforward and efficient coating made from biomimetically synthesized CPs, which holds significant promise for neural and biointerface applications.

2. MATERIALS AND METHODS

2.1. PSS RAFT Synthesis.

PSS was obtained via Reverse Addition−Fragmentation Chain-Transfer (RAFT) polymerization as reported previously.²³ Briefly, 4 g of monomer sodium 4-vinyl-benzenesulfonate (Sigma-Aldrich, 94904) was dissolved in 24 mL of ultrapure water (Thermo Scientific, Barnstead, Smart2Pure Water Purification System). Then, 2 mL of methanol (Sigma-Aldrich, 34860) containing 34 mg of S-(thiobenzoyl) thioglycolic acid as the CTA agent (Sigma-Aldrich, 157880) and 11.3 mg of 4,4′-Azobis (4-cyanovaleric acid) as the initiator (Sigma-Aldrich, 11590) were added. The solution was placed in a round-bottom flask with a magnetic stirrer and sealed with a rubber septum. The solution pH was brought to 1 by dropping 2N H₂SO₄ (Thermo Scientific, 42452–5000). The reactor was purged with nitrogen gas for 30 min at room temperature. Then, the flask was immersed in an oil bath at 70 °C with magnetic stirring at 600 rpm. The reaction mixture was left to react for 15 h in the dark. The polymer was purified by precipitation in 80 mL of cold acetone (Fisher, A949–4) and centrifugation for 10 min at 10 000 rpm and 4 °C. The supernatant was discarded; the pellet was dissolved in 5 mL of ultrapure water, and the polymer was precipitated in 35 mL of acetone and centrifuged. This process was repeated 5 times. A translucent, pinkish-colored rigid wafer was obtained.

2.2. Biomimetic Synthesis of EDOT−Pyrrole Copolymer Doped with PSS.

Biomimetic synthesis of EDOT−PPy copolymer doped with p-toluenesulfonic acid and PSS was carried out as reported previously.²⁴ Pyrrole was distilled before use. Briefly, 1 mL of a 100 mg mL⁻¹ hematin (Sigma-Aldrich, H3281) solution in dimethyl sulfoxide (DMSO) (Fisher, D128–1), 297 μL of 3,4-ethylenedioxythiophene (EDOT) (Sigma-Aldrich, 483028), and 22 μL of pyrrole (Sigma-Aldrich, 131709) were vortex mixed in a 50 mL tube and placed in an ice bath. Then, 20 mL of a 1:40 mass ratio of poly(styrenesulfonate) (PSS)/toluene sulfonic acid (TSA) (Sigma-Aldrich, 402885) solution at pH 2 was added, mixed thoroughly, and left in the ice bath. PSS with varying molecular weights were used. Polymerization is initialized by adding 1 mL of H₂O₂ (Fisher, BP2633–500) and vortex mixing for 10 min. Then, the tube was left at −80 °C overnight. After that, the copolymer is lyophilized for 72 h at −84 °C (FreeZone; Labconco, Houston, TX, USA), resuspended in cold acetone, and filtered on a 0.22 μm polytetrafluoroethylene (PTFE) (AMTAST, AMPT02247R) membrane, and multiple acetone washes were run over the product until a colorless filtered liquid was obtained. Finally, the product was dried at 70 °C for 12 h.

2.3. Glass Cleaning and Functionalization.

Fifteen mm diameter glass coverslips (Matsunami, C015001) were cleaned by submerging them in basic piranha, 5:1:1 water:NH₄OH (Acros Organics, 42330–5000):H₂O₂ at 80 °C for 30 min. Then, the coverslips were thoroughly rinsed with distilled water, dried with air, and submerged in a 1% (3-aminopropyl)triethoxysilane (APTES) (Beantown Chemical, 123580) solution in ethanol (Thermo Scientific Chemicals, AC615095000) for 30 min. After rinsing with ethanol and drying, the coverslips were submerged in a 25 μg mL⁻¹ Eosin-5-isothiocyanate (Chemodex, CDX-E0013) solution in phosphate-buffered saline (PBS, Gibco, 10010–023) for 15 min, rinsed with Milli-Q water, and dried.

2.4. Hydrogel Photopolymerization.

PEGDA hydrogels were obtained by photopolymerization. The polymerization solution consisted of 500 mM PEGDA M_n 575 (Sigma-Aldrich, 437441), 250 mM triethanolamine (TEA) (Sigma-Aldrich, 471283), and 10 μg mL⁻¹ Eosin-5-isothiocyanate in PBS. This solution was filtered using a 0.2 μm PTFE syringe filter (VWR, 28145–491) and purged with nitrogen gas for 3 min. Functionalized coverslips were placed on top of a glass slide, 35 μL of polymerization solution were added on top of the functionalized coverslips, and another glass slide was placed on top using 200 μm spacers (Scheme 1a). This assembly was held together with binder clips. The coverslips and polymerization solution were irradiated with 530 nm light (ThorLabs, M530L3-C1) with a power of 25 mW (measured with a PM16–140, ThorLabs) for 15 min.

Scheme 1. a) Hydrogel Photopolymerization Scheme; b) Scheme of the Main Coatings Used in This Work: Noncoated Hydrogels (PEG-NC), Gold Nanoparticle-Coated Hydrogels (PEG-GN), Hydrogels Functionalized with Fibronectin (PEG-FN), and PPy−PEDOT:PSS-Coated Hydrogels (PEG-PP).

For Fluo-4 calcium indicator experiments, hydrogels were obtained using 2-hydroxy-2-methylpropiophenone as a photoinitiator instead of eosin. Clean coverslips were functionalized in a solution containing 100 μL of 3-(trimethoxysilyl)propyl methacrylate (TCL, M0725), 600 μL of acetic acid (Sigma-Aldrich, 695092), and 20 mL of ethanol for 10 min and rinsed with ethanol to facilitate covalent attachment of PEGDA polymers. Initiator stock was prepared in methanol at 200 mg mL⁻¹. The polymerization solution contained 10 mg mL⁻¹ of initiator and 500 mM PEGDA M_n 575 in PBS. 35 μL of polymerization solution were added to the functionalized coverslips and photo-polymerized using a High Intensity 100 W Longwave UV Lamp (B-100AP, Blak-Ray).

2.5. Hydrogel Coating.

The obtained hydrogels were functionalized by either covalent bonding of fibronectin or the adsorption of PPy−PEDOT:PSS or gold nanoparticles (Scheme 1b). Fibronectin was covalently bound to the hydrogel surface by the heterobifunctional cross-linker sulfosuccinimidyl 6-(4′-azido-2′ - nitrophenylamino)hexanoate (Sulfo-SANPAH) following standard protocols.²⁷ Briefly, a 25 mg mL⁻¹ Sulfo-SANPAH (Thermo Scientific, 22589) stock solution in DMSO was prepared, aliquoted, and stored at −80 °C. An extended parafilm was placed in the laminar flow hood, and the hydrogels were placed on top. Sulfo-SANPAH stocks were diluted in PBS to a final concentration of 0.5 mg mL⁻¹ immediately before use, and 100 μL were used to cover the hydrogels. The hydrogels were left under UV light for 15 min and rinsed with PBS. Then, Sulfo-SANPAH-functionalized hydrogels were covered with 100 μL of a 0.1 mg mL⁻¹ human plasma fibronectin (Sigma-Aldrich, FC010) solution, left under UV light for 60 min, and rinsed with sterile PBS. For coating with PPy−PEDOT:PSS or 400 nm gold nanoparticles (ThermoFisher, J67106.AC), hydrogels were covered with 100 μL of 25 and 250 μL mL⁻¹ suspensions in PBS, left under UV light for 60 min, and rinsed with sterile PBS. This nanoparticle size was selected as it is similar to the PPy−PEDOT:PSS size we previously reported.²⁴ Noncoated hydrogels were covered with 100 μL of PBS and sterilized with UV light for 60 min before cell seeding.

2.6. Raman Spectroscopy.

PSS, NPs, and PEGDA hydrogels were characterized by using Raman spectroscopy. Raman spectra were acquired on a LabRAM HR Evolution confocal Raman microscope, using a 100× objective, a 532 nm laser, 10 accumulations, a 20 s acquisition time, and a 400−1800 cm⁻¹ range (grating 600 (500 nm)).

2.7. Gel Permeation Chromatography.

GPC measurements were carried out using a 1260 Infinity II Liquid Chromatography System (Agilent) with a PL aquagel−OH MIXED-M, 4.6 mm × 250 mm, 8 μm column (PL1549−5801, Agilent). Polymer solutions were prepared at 5 mg mL⁻¹ in Na₂HPO₄ 67 mM (BP332, Fisher) using HPLC-grade water (W5SK−1, Fisher). Polystyrenesulfonate sodium salt standards (PSS−PSSKIT, Agilent) were used for calibration.

2.8. Transmission Electron Microscopy (TEM).

TEM images of PPy−PEDOT:PSS were taken on a JEOL JEM-2010F high-resolution transmission electron microscope. The samples were prepared by dropping 10 μL of a PPy−PEDOT:PSS suspension in water (10 μg mL⁻¹) on the TEM copper grid and leaving it to dry at room temperature before imaging.

2.9. Electrochemical Characterization.

Electrochemical characterization was performed using a Pine Research potentiostat (WaveDriver 200). A three-electrode configuration was used to perform cyclic voltammetry (CV) and chronopotentiometry to measure the stable electrochemical window and capacitance of the polymer complex (PPy−PEDOT:PSS) in PBS. Glassy carbon was used as a disc (working electrode) in a rotating disk electrode (RDE) setup with a spiral platinum electrode and silver/silver chloride (Ag/AgCl) serving as counter and reference electrodes, respectively. To prepare the ink dispersion, 4 mg of PPy−PEDOT:PSS was mixed with 1 mL of ethanol:water mixture (1:1) along with 10 μL of 5 wt % Nafion serving as the binder. The resulting ink solution was sonicated for 15 min for complete dispersion. The ink solution was coated over a glassy carbon surface multiple times, such that the final weight of the PPy−PEDOT:PSS reached 0.11 mg. The glassy carbon electrode was rotated at 400 rpm for a uniform coating of the PPy−PEDOT:PSS during deposition.

The cyclic voltammetry was performed under a nitrogen environment with a scan rate ranging from 20 to 100 mV s⁻¹ in a potential window between −200 and 900 mV vs Ag/AgCl reference. The nitrogen saturation of the PBS was carried out for 15 min before performing cyclic voltammetry. The capacitance measurements were quantified using the galvanostatic charge−discharge cycles. The charging was carried out from −200 to 900 mV, and discharge was carried out from 900 to −200 mV at various rates: 0.1, 1, 3, and 5 A g⁻¹. Since the value of capacitance is directly proportional to the quantity of electrode material and the area, the capacitance is represented with respect to loading as described in the following:

$$C_{mp}=\frac{I·∆t}{m·∆V}$$ (1)

where C_mp is mass-specific capacitance (F g⁻¹), and I, Δt, m, and ΔV correspond to the applied current, time of discharge, loading mass, and electrochemical window of operation, respectively.²⁸

2.10. Atomic Force Microscopy (AFM).

Bare and NP-coated hydrogels were examined under an atomic force microscope (Nanoscope Multimode V, Bruker). Topography data were acquired using the ScanAsyst imaging technique, and mechanical properties were assessed by force−volume imaging. SCANASYST-FLUID+ (Bruker) probes with a nominal spring constant (k) of 0.7 N m⁻¹ were used. The probe was calibrated before use. The spring constant was determined using the thermal tuning method, and the deflection sensitivity was determined using freshly cleaved mica as a stiff reference material. For indentation experiments, 64 (8 × 8) force−distance curves (FDCs) in a 15 × 15 μm² area were obtained at three random positions of the sample. All measurements were performed in PBS at room temperature.

To obtain Young’s modulus values, the FDC data were processed with AtomicJ software using a pyramid model and a half angle of 18°. A value of 0.5 of Poisson’s ratio of the hydrogel was assumed, as has been done previously in the literature.²⁹

2.11. Protein Absorption Evaluated by Quartz Crystal Microbalance with Dissipation Monitoring.

QCM-D measurements were performed on a QSense Explorer microbalance with a single channel (Biolin Scientific, Gothenburg, Sweden) using QSense silicon dioxide sensors (QSX 301). Sensors were cleaned, following the manufacturer’s protocols, and placed on a Parafilm sheet supported by a Petri dish bottom, silicon dioxide-coating side up. A 14 mm diameter rubber O-ring was placed on top of the sensor to contain the APTES and Eosin-5-isothiocyanate solutions used for functionalization. The concentrations and times used were as described previously. To obtain hydrogels thin enough to be used in the QCM-D, 35 μL of polymerization solution without Eosin-5-isothiocyanate were placed on top of the functionalized sensor and illuminated with 530 nm light for 75 s. Bovine serum albumin (BSA) (0332, VWR) and fetal bovine serum (FBS) (35–010-CV, Corning) adsorption on bare and NP-coated hydrogels was evaluated. For BSA adsorption, a baseline of PBS was recorded, and then, a 5 mg mL⁻¹ BSA solution in PBS was injected into the system. This value is in the range of reported BSA concentration in 10% FBS-supplemented media. A wash with PBS was performed to remove loosely bound BSA. For FBS adsorption, a baseline of CO₂ Independent Medium (18045088, Gibco) supplemented with 1% penicillin-streptomycin (15140–122, Gibco) was recorded. CO₂ Independent Medium was used as it does not require a CO₂ environment to maintain physiological pH. Then, a 10% FBS-supplemented CO₂ Independent Medium was injected into the system. CO₂ Independent Medium with 1% penicillin-streptomycin was used for washing. Proteins were injected into the system for 20 min at a 10 μL min⁻¹ rate and then incubated for another 40 min without flow, followed by rinsing.

Changes in the resonance frequency (ΔF) and dissipation (ΔD) were monitored. The relationship between frequency changes monitored by QCM-D and mass was calculated using the Sauerbrey equation,³⁰

$$m_{\mathrm{Q}\mathrm{C}\mathrm{M}-\mathrm{D}}=-C\frac{∆f_i}{i}$$ (2)

with the mass sensitivity constant of the crystal C = −17.7 ng cm⁻² Hz⁻¹ and i the overtone number. The ratio of dissipation and normalized frequency shifts, ΔD_i/(−ΔF_i/i), was smaller than 0.4 × 10⁻⁶ Hz⁻¹, fulfilling the conditions to use the Sauerbrey equation.³¹

2.12. ND7/23 Cell Culture and Differentiation.

ND7/23 cells, a mouse neuroblastoma × rat dorsal root ganglion neuron hybrid cell line, were used as a dorsal root ganglion cell model (Sigma, 92090903). Cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, 10–014-CV, Corning) supplemented with 10% fetal bovine serum (FBS, 35–010-CV, Corning) and 1% penicillin-streptomycin (15140–122, Gibco), hereinafter referred to as complete DMEM. For differentiation experiments, DMEM supplemented with 0.5% FBS and 1% penicillin-streptomycin, hereinafter referred to as differentiation DMEM, was used.

Hydrogels were placed in 12-well plates (07–200-82, Corning), and 15 000 cells were seeded in 1.5 mL of complete DMEM. After 24 h of incubation to allow attachment, the media were replaced with differentiation DMEM for 1 day to inhibit mitosis. Then, the media were replaced by differentiation DMEM supplemented with 50 ng mL⁻¹ of recombinant rat β-nerve growth factor (β-NGF) (556-NG-100/CF, R&D Systems) and 1 mM dibutyryl-cAMP (dbcAMP) (S7858, Selleckchem). The media were refreshed every other day.

2.13. C2C12 Cell Culture and Differentiation.

C2C12 mouse myoblast cells were cultured in complete DMEM at 37 °C and 5% CO₂. Hydrogels were placed in 12-well plates, and 30 000 cells per well were seeded in 1.5 mL of complete DMEM. After 4 days, cells reached confluency, and the media were replaced with DMEM containing 0.25% FBS and 1% penicillin-streptomycin. The media were refreshed every other day.

2.14. Isolation and Maintenance of Primary Rat Cortical and Hippocampal Neurons.

All experiments in this study were approved by the UTSA Institutional Animal Care and Use Committee (approval no. MU-RA007). The cortex and hippocampus were extracted from neonatal rats, dissociated, and plated on 35 mm collagen-coated glass-bottom dishes. Primary neurons were maintained in Neurobasal Plus Medium (A3582901, Gibco) supplemented with 1% penicillin-streptomycin, 2% B-27 Plus Supplement (A3582801, Gibco), and 1% Glutamax-I (35050–061, Gibco) at 37 °C and 5% CO₂. Glial inhibition was performed after 3 days with a solution of 5-fluoro-2′-deoxyuridine and uridine (FURD, Sigma-Aldrich). Half of the media were replaced with fresh media every other day.

2.15. Immunohistochemistry and Confocal Imaging.

Primary neurons were stained for microtubules, presynaptic vesicles, and the cell nucleus. Samples were fixed with 4% paraformaldehyde in PBS. After washing 3 times with washing solution, i.e., PBS containing 0.05% Tween-20 (BP337, Fisher Bioreagents), cells were permeabilized with 0.25% Triton X-100 (T8787, Sigma-Aldrich) for 10 min followed by three washes. A blocking solution, i.e., 1% bovine serum albumin (0332, VWR) in PBS, was applied for 1 h. Anti-Tubulin β-III Antibody (clone TU-20, Alexa Fluor 555 Conjugate, CBL412A5 from Sigma-Aldrich) and Mouse Monoclonal Synaptophysin Antibody (Alexa Fluor 647 Conjugate, NBP147483AF647 from Novus Biologicals) were diluted to a working concentration in blocking solution, added to the samples, and incubated overnight at 4 °C. Cells were washed 3 times for 10 min each, and cell nucleus counterstaining was performed by incubating with DAPI for 5 min (90229, Merck Millipore). Acti-Stain 555 Phalloidin was used for actin cytoskeleton staining of ND7/23 and C2C12 cells (PHDH1-A, Cytoskeleton). After 1 h of incubation at room temperature with actin staining solution, cells were washed 3 times, followed by nucleus counterstaining. The samples were washed, and PBS was added to the Petri dish before visualization in a Leica TCS SP8 Confocal Microscope. Image processing was completed using the open-source software ImageJ 1.52p (National Institutes of Health, USA). ND7/23 cells were stained for the actin cytoskeleton and nucleus. C2C12 cells were stained for the actin cytoskeleton, nucleus, and myosin. Myosin 4 monoclonal antibody (MF20, 14–6503-82, Invitrogen) and goat antimouse antibody conjugated with DyLight 650 (NBP1–75965, Novus Biologicals) were used as primary and secondary antibodies, respectively.

2.16. Sample Preparation for Scanning Electron Microscopy.

Seeded hydrogels were fixed with 4% paraformaldehyde in PBS. Cells were gradually dehydrated in a serial change of ethanol in Milli-Q water for 5 min each (0%, 5%, 10%, 25%, 35%, 50%, 65%, 75%, 90%, 100%). Then, 100% ethanol was removed, and samples were exposed to 2:1 and 1:2 solutions of ethanol:hexamethyldisilazane (AAA15139AC, Fisher), for 20 min each. Finally, samples were left with pure hexamethyldisilazane for 20 min, then hexamethyldisilazane was removed, leaving a thin layer covering the cells, and samples were left to dry. All the steps were performed carefully, as dehydration generated cracks in the hydrogel. The samples were mounted on aluminum pin stubs using double-sided carbon tape and gold-coated (PELCO SC-7 Sputter Coater) before imaging in a Zeiss Crossbeam 340.

2.17. Quantification of Cell Adhesion and Differentiation.

The density of adhered cells was calculated by counting cell nuclei from five 1162 × 1162 μm² images from three different samples. Cell morphologic parameters were quantified to evaluate adhesion and differentiation. For adhesion, at least 100 contours from cells in three samples were manually traced and analyzed using ImageJ. For ND7/23 differentiation, aspect ratio and solidity were chosen as shape descriptors. The aspect ratio is the quotient between the major and minor axes of the best-fitted ellipse to a given object. This parameter is higher in elongated cells. Solidity is the area of an object divided by its convex area. The more ramified the object, the larger the convex area, and thus the smaller the solidity. Cell axon length was quantified using NeuronJ (an ImageJ plugin). Cell differentiation stages were manually tagged according to their shape in stages 0−1 (mostly rounded cells or with circumferential lamellipodia and filopodia), stage 2 (cells with dendrites), and stage 3 (cells with axon).²⁴ For C2C12 cells, the percentage of area covered by cells and by myosin was obtained from the actin cytoskeleton and myosin staining, from at least three 2326 × 2326 μm² images from at least two replicates.

2.18. Neuron Activity Experiments.

Following the manufacturer’s protocol, cells were labeled before stimulation experiments with the calcium indicator Fluo-4 AM (F10471, Invitrogen). The experiments were performed on healthy cultures between 8 and 12 days after isolation. Coverslips supporting the hydrogels were transferred to a Petri dish containing a Tyrode buffer. Cells were imaged at a 4 Hz frame rate for 90−120 s under a stereomicroscope (Leica M205 FCA) equipped with 2x and 5x Plan Apo objectives and an sCMOS camera (Leica DFC9000). Electrical stimulation was applied using platinum−iridium wires (AA10056BS, Thermo Scientific Chemicals). Cells were electrically stimulated with pulsed DC voltage of 1 V cm⁻¹, 100 Hz frequency, 10 ms period.

2.19. Analysis of Calcium Imaging.

The analysis was performed using ImageJ functions to subtract background and measure fluorescence intensity.³² The time resolution of the fluorescence microscopy was insufficient to be interpreted as resolving individual spikes. Instead, the method might be taken to show time-integrated levels of neuronal activity. An automated code was generated in MATLAB to analyze the fluorescence intensity traces of neurons in each video. Three different videos containing 50−100 neurons each were analyzed for each condition. Videos from nonstimulated neurons were used to calculate the mean fluorescence intensity standard deviation (SD). Neurons were considered active when their fluorescence intensity increased more than 2 SD.

2.20. Live−Dead Assay.

To assess the effect of the PEGDA hydrogel and PPy−PEDOT:PSS exposure on cell viability, a Live/Dead assay was used to quantify live and dead cell populations by using flow cytometry. ND7/23 cells were seeded as aforementioned, and after 48 h of incubation, the coverslip containing the hydrogel was transferred to a 24-well plate, and 100 μL of TrypLE Express (12604–013, Gibco) was added to detach the cells from the hydrogel. The cells were suspended in 400 μL of fluorescence-activated cell-sorting buffer (FACS buffer) containing 0.8 μM Calcein (C34852, Invitrogen) and 0.5 μM Sytox (S34859, Invitrogen) to stain the live and dead cells, respectively. FACS buffer was prepared by mixing 20 μL of 0.5 M EDTA and 20 μL of FBS in 20 mL of PBS. The live and dead cell populations were then quantified using flow cytometry (BD Accuri C6 Plus, Becton-Dickinson, Franklin Lakes, NJ, USA).

2.21. Statistical Analysis.

Experiments were performed in triplicate unless otherwise stated. All data were reported as the mean ± standard error unless otherwise stated. One-way analysis of variance (ANOVA), followed by Fisher’s least significant difference (LSD) test, was performed for means comparison unless otherwise stated (significance level of 0.05, OriginPro 2016).

3. RESULTS AND DISCUSSION

3.1. Nanoparticle and Hydrogel Characterization.

3.1.1. PPy−PEDOT:PSS Characterization.

Even though PEDOT and PPy are known for their high electrical conductivity, their poor aqueous solubility hinders their biomedical applications. PSS is a polymer surfactant that serves as a dopant and helps in dispersing and stabilizing PEDOT in aqueous environments.³³ We obtained PSS via RAFT polymerization and characterized it by using GPC and Raman spectroscopy (Figure S1). PSS presented a peak in its molecular weight (MW) distribution at 42 kDa with a polydispersity index (PDI) of 1.45. The Raman spectrum of PSS matches with previously reported data.³⁴ The Raman shift at 1600 cm⁻¹ is associated with aromatic ring quadrant stretching. Peaks at 1040 and 1130 cm⁻¹ indicate the υ(SO₃) and υ(SO) vibrational modes, respectively. Peaks at 740 and 800 cm⁻¹ are associated with the υ(CS) vibrational modes.

The PPy−PEDOT:PSS copolymer obtained via biomimetic synthesis was characterized by dynamic light scattering, Raman spectroscopy, TEM, and UV−vis spectroscopy (Figures 1a,b and S2). The copolymer showed a negative charge of −37 mV and hydrodynamic sizes in the 300−700 nm range (Figure 1a). The size of the dehydrated PPy−PEDOT:PSS observed by TEM was in a similar range to the hydrodynamic size found in DLS measurements (gray−black quasicircular structures in Figure S2). It has been reported that PEDOT:PSS complexes form colloidal dispersions in an aqueous environment with core−shell structures where hydrophobic PEDOT is in the center of the colloids surrounded by hydrophilic PSS that acts as a counterion and stabilizer.³⁵ The Raman spectrum of PPy−PEDOT:PSS shows spectral characteristics closer to those of PEDOT than to those of PPy. The bands around 1572 and 1503 cm⁻¹ are related to antisymmetric stretching of Cα═Cβ, while the most intense peak at around 1438 cm⁻¹ relates to symmetric stretching of Cα#x2550;Cβ of the thiophene ring.³⁶ The peaks at 1367, 988, and 573 cm⁻¹ correspond to Cβ−Cβ interring stretching, C−C antisymmetrical stretching mode, and oxyethylene ring deformation, respectively. The peak at 439 cm⁻¹ corresponds to SO₂ bending and indicates doping of PEDOT by the SO³⁻ ion from PSS units. The small peak at 1224 cm⁻¹ is related to the Cα−N stretching from the pyrrole ring.³⁷

Figure 1.

PPy−PEDOT:PSS nanoparticle characterization. a) Hydro-dynamic size distribution determined by DLS. b) Raman spectrum. c) Cyclic voltammetry. d) Galvanostatic charge−discharge measurements of PPy−PEDOT:PSS ink drop-cast on a glassy carbon electrode. Data at short times are zoomed-in in the inset. e) Mass-specific capacitance vs current density calculated from panel (d).

3.1.2. PPy−PEDOT:PSS Electrochemical Characterization.

The electrochemical window for capacitance characterization was selected in a potential range with minimal faradaic processes, as observed from the cyclic voltammetry. The electrochemical window of PPy−PEDOT:PSS is shown in Figure 1c, where the cyclic voltammetry was carried out at scan rates ranging from 20 to 100 mV s⁻¹ between the potentials of −200 to +900 mV vs Ag/AgCl reference. Five CV cycles were performed at each scan rate, and only the last cycle is represented. The electrodes were stable in the electrochemical window of our interest. The current recorded during the cyclic voltammetry is the result of the electrical double layer developed due to the applied potential bias and is used to store electrical charge. GCD curves of PPy−PEDOT:PSS are shown in Figure 1d in the potential window ranging between −200 and +900 mV vs Ag/AgCl reference, where the capacity was assessed at controlled current conditions, i.e., at various charge rates. The PPy−PEDOT:PSS charge curves were symmetrical to their corresponding discharge curves, suggesting high reversibility. This symmetry and reversibility are characteristic of nonfaradaic, high Coulombic efficiency charging and discharging of an electrical double layer. Note that the GCD carried out at 0.1 A/g does not reach the maximum potential value of 900 mV. A possible explanation could be that at this low current density, enough time is provided for charge redistribution within the conducting PPy−PEDOT:PSS polymer, which limits the potential from reaching 900 mV. The discharge curves and eq 1 were used to calculate the C_mp, which ranged between 50 and 40 F cm⁻². The application of a charging current causes the ions in the electrolyte to arrange themselves in ordered electrical double layers (layers of oppositely charged ions) at the electrode surface. Thus, the diffusive and migratory ion transport rate to the surface ultimately decides the capacitance at the electrode.

Charging currents (responsible for rapid charging/response) and capacitance (an indicator of the quantity of stored charge) were found to be anticorrelated (Figure 1e), indicating the need to balance these two parameters to ensure optimal performance.

3.1.3. PEGDA Hydrogel Characterization.

Hydrogel chemistry and topography were characterized by using Raman spectroscopy and AFM, respectively (Figure 2). The Raman spectrum coincides with what has been previously reported for PEGDA hydrogels (Figure 2a).³⁸ Peaks at 1285 and 1475 cm⁻¹ correspond to CH₂ twisting and CH₂ scissors, respectively. The peak at 1634 cm⁻¹ is assigned to C=C bond stretching from the eosin and the PEGDA monomer and can be related to some extent to the degree of cross-linking of the polymer meshwork.³⁸ The peak around 1720 cm⁻¹ corresponds to the carbonyl group (>C=O) and signals with strong-medium intensities at 1130, 1038, 854, and 815 cm⁻¹, which should be related to C−C skeletal stretching.³⁹

Figure 2.

PEGDA hydrogel characterization. a) Raman spectra. b) and c) AFM three-dimensional height images of PEGDA hydrogels before (PEG-NC) and after incubation with PPy−PEDOT:PSS (PEG-PP), respectively. d) Root-mean-square roughness (R_q) of PEG-NC and PEG-PP. *Indicates statistically significant differences (one-way ANOVA, followed by Fisher’s LSD test, p-value < 0.05).

AFM shows that PEGDA hydrogels present a rather smooth surface (Figure 2b) with peaks in the 10−30 nm range and a root-mean-square roughness (R_q) of 9.4 ± 1.4 nm (Figure 2d). Hydrogel stiffness obtained by force−volume indentation was 258 ± 11 kPa. Mechanical properties of PEG hydrogels can be tuned in the kPa−MPa range by adjusting photopolymerization parameters such as monomer and initiator concentration, monomer molecular weight, light intensity, etc.⁴⁰ Though substrate stiffness is an important material property regulating cellular function, this work does not investigate hydrogel stiffness as a variable. After incubation with PPy−PEDOT:PSS and rinsing with PBS, the R_q increases to 14.9 ± 0.9 nm, and the surface presents 20−50 nm peaks (Figure 2c) and a few 200−500 nm structures (Figure S3). These results indicate the presence of the PPy−PEDOT:PSS coating on the hydrogel surface.

3.1.4. Protein Adsorption.

Proteins play a crucial role in the adhesion of cells to surfaces. Cells require the adsorption of specific adhesion proteins, such as fibronectin, laminin, and collagen, or peptides that mimic part of those proteins to attach to surfaces.¹³ Several factors, such as protein coating concentration, protein conformation, and binding strength or exchangeability, determine whether cells will attach.⁴¹ Proteins interact with material surfaces by van der Waals, hydrophobic, and electrostatic forces. Hydration layers on very hydrophilic surfaces, such as PEG or alginate, can act as an energetic barrier that prevents nonspecific protein adsorption. It has been reported that hydrophobic PEDOT improves protein adsorption on alginate scaffolds,⁴² and PSS promotes protein adsorption and may influence protein orientation and conformation.⁴³ We hypothesized that PEGDA hydrogel hydrophilicity and inert nature prevent protein adsorption, but the PPy−PEDOT:PSS copolymer coating that remains after the rinsing steps supports protein adsorption. We used QCM-D to study BSA (Figure S4) and FBS (Figure 3) interactions with PEG-NC and PEG-PP hydrogel surfaces.

Figure 3.

FBS adsorption studied by QCM-D on noncoated (PEG-NC) and PPy−PEDOT:PSS-coated (PEG-PP) PEGDA hydrogels. a) Frequency change (ΔF) vs time. b) Mass change during FBS injection and after rinsing with CO₂-independent medium. *Indicates statistically significant differences (one-way ANOVA, followed by Fisher’s LSD test, p-value < 0.05).

The BSA concentration used was within the range of BSA concentrations in 10% FBS-supplemented media. When BSA was injected into the QCM-D chamber, there was a decrease in frequency that can be related mainly to the adsorption of BSA and to a lesser extent to changes in solution viscosity.⁴⁴ Frequency changes were correlated with adsorbed mass by using eq 2. PEG-NC presented a mass adsorption of 36 ± 12 ng cm⁻² while incubated with BSA, and just 36% (13 ± 7 ng cm⁻²) remained after rinsing loosely bound BSA with PBS. PEG-PP presented mass adsorptions of 110 ± 20 and 79 ± 14 ng cm⁻² (72% of initial adsorbed mass), before and after rinsing with PBS, respectively.

FBS contains approximately 1800 different proteins, and some of them, such as fibronectin or vitronectin, are essential for components required for cell attachment.⁴⁵ Our FBS adsorption experiments followed a similar trend to BSA experiments, though the adsorbed mass was around 5 times lower. PEG-NC presented an adsorbed mass of 7 ± 3 and 2 ± 1 ng cm⁻² (29% of the initial adsorbed mass), before and after rinsing with media, respectively. On PEG-PP, there was an adsorbed mass of 26 ± 7 ng cm⁻² during FBS incubation, and after rinsing with media, 65% of the mass (17 ± 5 ng cm⁻²) remained. Adsorption in the range of tens of ng cm⁻² has been reported to be enough to support cell adhesion.⁴⁶ The PPy−PEDOT:PSS copolymer coating increases protein adsorption and reduces the mass of proteins lost after rinsing, from two-thirds to one-third; thus, stronger substrate−protein interactions are expected.

3.2. Neuron and Myoblast Attachment.

Cell adhesion is crucial for anchorage-dependent cells to survive and is the first step for other cellular processes such as cell migration, proliferation, activity, and differentiation.⁴⁷ Depending on substrate mechanical and physicochemical properties, different cell types may exhibit distinct adhesion strengths and spreading characteristics.⁴⁸ Neuronal cells are particularly sensitive to extracellular microenvironments and substrates, often requiring substrate modifications to promote their adhesion.⁴⁹

Adhesion between neural cells and their microenvironment is crucial in processes like axon outgrowth, synapse formation, and nerve regeneration.⁵⁰ On the other hand, other cell types such as myoblasts and fibroblasts may exhibit stronger adhesion characteristics. The capacity of the PPy−PE-DOT:PSS coating to promote cell attachment was tested on ND7/23 cells, primary rat cortical and hippocampal neurons, and C2C12 mouse myoblast cells.

3.2.1. ND7/23 Cells’ Attachment.

ND7/23 cells are often used as a model of dorsal root ganglion neurons, and their adhesion was evaluated 48 h after seeding on the different samples (Figures 4 and S5).⁵¹ When seeded on PEGDA hydrogels, ND7/23 cells barely adhere, finding only 4−5 cells per mm². Coating with gold nanoparticles increased the number of cells found to ∼45 per mm². On PEG-PP, ∼130 cells per mm² were found 48 h after seeding. The largest cell density was found on PEG-FN, with 220 cells per mm² (Figure 4b). Though around 40 cells per mm² were seeded (15 000 cells in 3.5 cm² of the well), ND7/23 cells’ proliferation is rather fast, with doubling times reported in the range of 12−19.⁵² Cells may attach faster and start proliferating sooner on PEG-FN, leading to more cells 48 h postseeding. Adhered cells presented mean areas of around 600 μm² on all samples (Figure 4c). Cells on PEG-PP present a solidity distribution significantly different from the distribution found on PEG-FN, with a mean solidity of 0.86 which is lower than the 0.88 found on the other samples (Figure 4d). These results indicate more ramified cells, as can be observed in microscopy images. It is worth noting that coating levels play an important role in the performance of PEGDA hydrogels coated with PPy−PEDOT:PSS. While ND7/23 cells adhere on hydrogels coated with PPy−PEDOT:PSS suspensions of 25 and 250 μg mL⁻¹, on samples coated with 1000 μg mL⁻¹ suspensions, the cells look rounded and aggregated (Figure S5). This may be related to increased levels of loosely attached polymer to the hydrogel, which may affect the formation of stable focal contacts. Thus, optimizing the coating protocol regarding polymer suspension concentration, coating time, rinsing, etc., is an important step to maximize the PPy−PEDOT:PSS coating performance.

Figure 4.

Adhesion and morphology characteristics of ND7/23 cells 48 h after seeding on noncoated hydrogels (PEG-NC) and hydrogels coated with gold nanoparticles (PEG-GN), fibronectin (PEG-FN), and PPy−PEDOT:PSS (PEG-PP). a) Confocal images. Cell actin cytoskeleton (orange); nuclei (blue). b) Cell density bar plot. Cell area (c) and cell aspect ratio (d) box plots. Box plots represent the median; 5th, 25th, 75th, and 95th percentiles; and mean (white square). One-way ANOVA followed by Fisher’s LSD test was used to compare the data on b). ^#Indicates that at the 0.05 level, population means are significantly different from PEG-FN’s mean. The Wilcoxon signed-rank test was used to compare the sample data on c) and d). *Indicates that at the 0.05 level, the distribution is significantly different from PEG-FN’s distribution.

The live/dead assay was performed 48 h after seeding to assess ND7/23 cells’ viability on the samples that supported good cell adhesion, PEG-FN, and PEG-PP. Cells seeded on both samples exhibited 90% viability, similar to what was found on the control cell culture Petri dish (Figure S6).

3.2.2. Primary Neuron Attachment.

Long-term stable cell culture is crucial for studying cell function. As many primary cells, and neuronal cells in particular, do not sufficiently adhere to the glass or plastic surfaces of cell culture vessels, ECM components such as fibronectin, collagen, and laminins, and ECM extracts like Matrigel are used to support their attachment.⁵³ However, the high cost⁵⁴ and interbatch variations in the mechanical and biochemical properties⁵⁵ of these ECM components pose a challenge, and developing alternative coatings is very appealing. As the ND7/23 cell line presents scarce adhesion on PEG-NC or PEG-GN, we tested primary neuron attachment on PEG-FN and PEG-PP. Adhesion of primary cortical and hippocampal neurons to PEG-FN and PEG-PP surfaces was evaluated 8−10 days after cell extraction and seeding. Both surfaces supported neuronal survival and neurite extension for several days (Figures 5 and S7). However, neurons seeded on PEG-FN tended to form axon clusters in defined regions (yellow arrows), which translates to weaker cell−substrate interactions.⁵⁶ On PEG-PP, neurons tend to present extensive arborization and reduced cell aggregation. Hydrogel samples were also analyzed through scanning electron microscopy (SEM), confirming the cell−substrate interaction patterns observed during confocal imaging. Sample preparation procedures for SEM imaging produced observable cracks in the hydrogels. The PEG-FN surface is rather smooth, while globular structures can be observed on the PEG-PP surface (Figure S8).

Figure 5.

Adhesion of rat cortical primary neurons seeded on hydrogels coated with fibronectin (PEG-FN) and PPy−PEDOT:PSS (PEG-PP) coating. Cell cytoskeleton microtubules were stained with antitubulin β-III (magenta); presynaptic vesicles were labeled with a synaptophysin antibody (green); nuclei counterstaining was performed with Hoechst (blue). Yellow arrows indicate areas with clusters of axons.

3.2.3. C2C12 Attachment.

C2C12 myoblast cell line has been utilized extensively as an in vitro muscle model for studying metabolic disease progression,⁵⁷ muscle atrophy,⁵⁸ and muscle tissue engineering.⁵⁹ These studies require a substrate that provides the necessary mechanical and biochemical stimuli to support attachment and then promote myogenic differentiation.⁶⁰ Figure 6 shows the confocal images and morphological characteristics of C2C12 cells 48 h after seeding on the different hydrogel samples. Unlike ND7/23 cells, C2C12 cells could adhere to some extent to the PEG-NC hydrogels. Density ranged from 40 and 50 cells mm⁻² for PEG-NC and PEG-GN, respectively, to 80 cells mm⁻² for PEG-FN and PEG-PP. Cells seeded on PEG-NC presented a mean area of 610 μm² and a mean aspect ratio of 2.55. Though some cells presented a normal C2C12 tapered morphology, a large population of rounded, low-area cells indicates that this surface is not optimal for promoting adhesion. Coating with gold nanoparticles improved adhesion characteristics, with a mean area and aspect ratio of 695 μm² and 2.75, respectively. Several cells presented a very elongated morphology with aspect ratios above 4 and small areas. Fibronectin functionalization enhanced the C2C12 cell spreading to a mean area of 800 μm² and an aspect ratio of 2.75. Finally, cells on PEG-PP adhered and exhibited a mean area of 980 μm² and an aspect ratio of 2.35, similar to the mean area of 930 μm² and an aspect ratio in the 2.2−2.5 range that has been reported for C2C12 cells seeded on glass.⁶¹

Figure 6.

Adhesion and morphology characteristics of C2C12 cells 48 h after seeding on noncoated hydrogels (PEG-NC) and hydrogels coated with gold nanoparticles (PEG-GN), fibronectin (PEG-FN), and PPy−PEDOT:PSS (PEG-PP). a) Confocal images. Cell actin cytoskeleton (orange); nuclei (blue). b) Cell density bar plot. Cell area (c) and cell aspect ratio (d) box plots. Box plots represent the median; 5th, 25th, 75th, and 95th percentiles; and mean (white square). One-way ANOVA followed by Fisher’s LSD test was used to compare the data on b). ^#Indicates that at the 0.05 level, population means are significantly different from PEG-FN’s mean. The Wilcoxon signed-rank test was used to compare the sample data on (c) and (d). *Indicates that at the 0.05 level, the distribution is significantly different from PEG-FN’s distribution.

3.3. Cell Differentiation.

Differentiation, the process by which nonspecialized cells become specialized to perform distinct functions, is crucial during development and tissue regeneration. The capacity of a biomaterial to support or guide cell differentiation depends on its physical, chemical, and mechanical properties.⁶² As cell adhesion to the substrate is a crucial and important differentiation regulator, differentiation experiments were performed on PEG-FN and PEG-PP, where the cells exhibited the best adhesion characteristics.

3.3.1. ND7/23 Differentiation.

ND7/23 cells extend projections of their bodies, i.e., neurites, when they differentiate, which can subsequently become polarized into axons or dendrites.⁶³ Cells were seeded and incubated for 24 h to allow adhesion and growth. Then, they were primed in DMEM containing 0.5% FBS for another 24 h to inhibit mitosis. After adding the media containing 50 ng mL⁻¹ of β-NGF and 1 mM of dbcAMP, cells went through morphological changes, and some cells became rounded and detached. ND7/23 cells’ detachment from the culture substrate after a couple of days of incubation in differentiation media has already been reported.⁶⁴ Cells were incubated for 7 days, and their morphology was evaluated to assess differentiation (Figure 7). The changes in cell morphology during neuronal morphogenesis can be divided into different stages.⁶³ We grouped the cells into three stages. As differentiation progresses, spherical neurons extend circumferential lamellipodia and filopodia (stages 0−1). Then, short neurites are formed (stage 2). Eventually, one of these neurites elongates faster and becomes an axon (stage 3). Four 581 × 581 μm² images from 3 different samples were analyzed, with a total of 143 and 303 cells for PEG-FN and PEG-PP, respectively. More cells detach from PEG-FN than from PEG-PP, indicating better cell−substrate interactions in the latter. On PEG-FN, 59%, 34%, and 7% of the cells were on stages 0−1, stage 2, and stage 3, respectively (Figure 7b). On PEG-PP, the percentage of cells in stages 0−1 decreased, and more cells were on more advanced differentiation stages (stages 2 and 3). ND7/23 cells on different stages of differentiation presented different trends in their shape factors (Figure 7c). Stage 0−1 cells presented solidity values between 0.8 and 1 and aspect ratio values in the 1−3 range. Stage 2 cells presented aspect ratios between 1 and 4 but solidity values in the 0.4−0.8 range. Finally, most of the cells in stage 3 had solidity values in the 0.1−0.6 range and variable aspect ratio values from 1.5 to 10. The mean axon lengths were 85 ± 22 and 106 ± 12 μm for stage 3 neurons on PEG-FN and PEG-PP, respectively (Figure 7d). With more cells and in more advanced stages, ND7/23 differentiation appears to be enhanced on PEG-PP.

Figure 7.

Differentiation of ND7/23 cells after 7 days. a) Confocal images of ND7/23 cells (orange, actin cytoskeleton; blue, cell nucleus). b) Percentage of cells that exhibited stages 0−1, 2, and 3 morphologies. c) Solidity vs aspect ratio scatter plots. Each symbol represents a cell (n is 143 and 303 for PEG-FN and PEG-PP, respectively). d) Axon length box plot. Box plots represent the median; 5th, 25th, 75th, and 95th percentiles; and mean (white square).

3.3.2. C2C12 Differentiation.

3.4. C2C12 cells were seeded on PEG-FN and PEG-PP, allowed to proliferate for 4 days to reach confluency, and differentiation was induced by replacing the complete DMEM with 0.25% FBS DMEM. Myogenesis is characterized by cell fusion to form multinucleated myotubes and the expression of muscle-specific proteins such as myosin.⁶⁵ One day after inducing cell differentiation, C2C12 detachment was observed, with some adhered cells forming star-shaped aggregates. This was more pronounced 2 and 4 days after inducing differentiation when 52 ± 8% and 45 ± 2% of the area remained covered by cells, respectively (Figures S9 and 8a,b). Though some cell detachment was also observed on PEG-PP, 86 ± 4% and 78 ± 4% of the area remained covered by cells 2 and 4 days after induced differentiation, respectively. Aggregation and detachment of cells is a common challenge for cultured myotube studies.⁶⁶ It has been found that C2C12 cells’ traction forces on the underlying substrate monotonically increase during differentiation.⁶⁷ The force balance between cell−cell and cell-matrix interactions is necessary to maintain the cells adhered. Several strategies such as tuning differentiation media, enhancing adhesion using Matrigel or collagen, or substrates with patterning or tuned stiffness have been used to reduce the likelihood of detachment.⁶⁶ The percentage area covered by myosin 2 days after inducing differentiation was 2.7 ± 0.4% on PEG-FN and 3.0 ± 0.3% on PEG-PP (Figure 8c). The 5.5 ± 0.6% myosin area on PEG-PP 4 days after induced differentiation was significantly larger than the 4.6 ± 0.8% obtained on PEG-FN. However, the myosin area/cell area ratio was similar in both hydrogels (Figure 8d), indicating the larger myosin area on PEG-PP was due to less cell detachment.

Figure 8.

C2C12 differentiation. a) Confocal images of cells 4 days after induced differentiation on PEG-FN and PEG-PP (red: myosin; orange: actin cytoskeleton; cyan/blue: cell nucleus). Percentage area covered by cells (b) and differentiated cells expressing myosin (c). d) Percentage ratio between myosin area and cell area. *Indicates statistically significant differences (one-way ANOVA, followed by Fisher’s LSD test, p-value < 0.05).

3.4. Primary Neuron Activity.

As neural activity causes rapid changes in intracellular free calcium, calcium imaging is an effective way to track the activity and excitation of neurons.⁶⁸ Cortical neuron activity was studied before and after ES. The excitation and emission spectra of eosin and Ca²⁺-bound Fluo-4 overlap; thus, hydrogels obtained with 2-hydroxy-2-methylpropiophenone as a photoinitiator were used for neuron activity studies. To increase the stability during image acquisition, the system consisting of the Petri dish containing the hydrogel in Tyrode buffer and the electrodes was assembled under the microscope objective and left still for 10 min before video recording. An increase in fluorescence intensity can be correlated to increased Ca²⁺ concentration and thus neural activity (Figure 9a). Neurons seeded on PEG-FN and PEG-PP showed similar activity patterns. Neurons before ES showed some activity, with an average of 1−6% of active cells for a certain 250 ms frame (Figure 9b and Video S1). During ES, the percentage of active cells increases to 11 ± 1.5% (Figure 9b,c, and Video S2). These results indicate active cells with working connections and signaling. More experiments varying ES parameters (e.g., frequency, voltage, and alternating vs direct current) and coating conditions (e.g., PPy−PEDOT:PSS concentration) are necessary to elucidate the possible contribution of conductive PPy−PEDOT:PSS on primary neural activity and response to ES.

Figure 9.

Cortical neuron activity was studied by calcium indicators. a) Fluorescence images from representative videos of cortical neurons seeded on PEG-PP in control conditions and during ES. Areas of increased intracellular fluorescence intensity (increased calcium concentration) are indicated with orange arrows. b) Average percentage of active cells per 250 ms frame of 90−120 s videos. c) Percentage of active cells as a function of time per 250 ms.

4. CONCLUSIONS

In this work, we present a straightforward and efficient PPy−PEDOT:PSS coating for introducing bioactivity into bioinert PEGDA hydrogels. Conductive PPy−PEDOT:PSS copolymer doped with PSS was obtained via biomimetic synthesis to avoid possible cytotoxicity associated with traditional wet chemistry protocols. PEGDA hydrogels obtained by photo-polymerization were relatively flat and antifouling, and after PPy−PEDOT:PSS coating, the surface became rougher and the amount and stability of adsorbed proteins increased. ND7/23 neuron hybrid cells do not attach to nonfunctionalized PEGDA hydrogels, barely adhered to PEG-GN, and adhered to PEG-PP with similar characteristics to those found on standard fibronectin-functionalized hydrogels. As anticipated, hydrogels coated with FN or PPy−PEDOT:PSS showed no toxicity toward ND7/23 cells in our live/dead assays. C2C12 cells adhered to all the samples but covered larger areas on PEG-PP and PEG-FN. The addition of differentiation media produced cell detachment, an effect more pronounced on PEG-FN, and better differentiation characteristics were observed on PEG-PP. Future studies include optimizing differentiation media composition, investigating longer differentiation times, fine-tuning the electrochemical properties of the polymer, including substrate patterning, and providing electrical stimulation to enhance cell maturation.⁶⁹ Fibronectin and PPy−PEDOT:PSS-coated hydrogels supported primary neuron attachment and activity, though cell clusters were found on PEG-FN. Neuron activity assessed by calcium dynamics indicates working neuron connections and signaling. PPy−PEDOT:PSS can serve as a simple, versatile, and robust biocompatible coating for PEGDA hydrogels and other materials in the interface with neurons and other electrically excitable cells.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.4c01647.

Calcium imaging of cortical neurons seeded on PEG-PP. The 90 s video has been sped up 30 times. The video is 4 × 4 mm² (MP4)

Data of PSS characterization (GPC and Raman), BSA adsorption studied by QCM-D, ND7/23 cells’ viability, adhesion of rat hippocampal primary neurons (Confocal), attachment of cortical and hippocampal primary neurons (SEM), and C2C12 differentiation (PDF)

Calcium imaging of cortical neurons seeded on PEG-PP and under electrical stimulation. The 90 s video has been sped up 30 times. The video is 4 × 4 mm² (MP4)

ABBREVIATIONS

AFM

Atomic Force Microscopy

APTES

(3-aminopropyl)-triethoxysilane

BSA

bovine serum albumin

CPs

conductive polymers

DMEM

Dulbecco’s modified Eagle’s medium

ES

electrical stimulation

FBS

fetal bovine serum

FDCs

force−distance curves

FN

fibronectin

PBS

phosphate-buffered saline

PEDOT

poly(3,4-ethylenedioxythiophene)

PEG

poly-(ethylene glycol)

PEGDA

poly(ethylene glycol) diacrylate

PEG-FN

hydrogels functionalized with fibronectin

PEG-GN

hydrogels incubated with gold nanoparticles

PEG-NC

noncoated hydrogels

PEG-PP

PPy−PEDOT:PSS-coated hydrogels

PPy

polypyrrole

PSS

poly(styrenesulfonate)

QCM-D

Quartz Crystal Microbalance with Dissipation Monitoring

Sulfo-SANPAH

sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate