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A Versatile Fabrication Approach of Conductive Hydrogels via Copolymerization with Vinyl Monomers Lin Jiang, Carmine Gentile, Antonio Lauto, Chen Cui, Yihui Song, Tony C Romeo, Saimon Moraes Silva, Owen Tang, Poonam Sharma, Gemma A. Figtree, John Justin Gooding, and Damia Mawad ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15019 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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ACS Applied Materials & Interfaces

A Versatile Fabrication Approach of Conductive Hydrogels via Copolymerization with Vinyl Monomers Lin Jiang,1,‡ Carmine Gentile,2,3, ‡ Antonio Lauto,4 Chen Cui,1 Yihui Song,1 Tony Romeo,5 Saimon M. Silva,6,7 Owen Tang,2 Poonam Sharma,2 Gemma Figtree,2 J. Justin Gooding,6,7 Damia Mawad1,7,8* 1

School of Materials Science and Engineering, UNSW Australia, Sydney, New South Wales

2052, Australia 2

Cardiothoracic and Vascular Health, Kolling Institute, Sydney Medical School (Northern),

University of Sydney, Sydney, New South Wales 2000, Australia 3

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

4

Biomedical Engineering and Neuroscience (BENS) Research Group, University of Western

Sydney, Penrith, New South Wales 2751, Australia 5

Electron Microscopy Centre, Innovation Campus, University of Wollongong, Squires Way,

Fairy Meadow, New South Wales 2519, Australia 6

School of Chemistry, UNSW Australia, Sydney, New South Wales 2052, Australia

7

Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent BioNano

Science and Technology, UNSW Australia, Sydney, New South Wales 2052, Australia

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Centre for Advanced Macromolecular Design, UNSW Australia, Sydney, New South Wales

2052, Australia

KEYWORDS:

poly(ethylene

dioxythiophene),

hydrogel,

conducting

polymer,

copolymerization, electroactive, cardiac cells ABSTRACT

Functionalized poly(ethylene dioxythiophene) (PEDOT) was copolymerized with two vinyl monomers of different hydrophilicity, acrylic acid and hydroxyethyl methacrylate, to produce electroconductive hydrogels in a range of physical and electronic properties. These hydrogels not only possessed tailored physical properties, such as swelling ratios and mechanical properties, but also displayed electroactivity dependent on the chemical composition of the network. Raman spectroscopy indicated that the functional PEDOT in the hydrogels is in an oxidized form, most likely accounting for the good electrochemical response of the hydrogels observed in physiological buffer. In vitro cell studies showed that cardiac cells respond differently when seeded on hydrogel substrates with different compositions. This study presents a facile approach for the fabrication of electroconductive hydrogels with a range of properties paving the way for scaffolds that can meet the requirements of different electroresponsive tissues.

1. INTRODUCTION The ideal scaffold for tissue regeneration is one that has properties matching closely to the physical and functional microenvironment of the extracellular matrix of the specific tissue being regenerated.1,2 Examples of these properties include architectural cues,3 surface morphologies,4


2

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mechanical properties,5 and/or electrical conductivities.6 Electrical conductivity is important in scaffolds engineered for electroresponsive tissues, as it has been shown to enhance proliferation and differentiation of seeded cells.7 Additionally, these scaffolds can integrate better with the electroconductive network of the tissue, facilitating its physiological function.8 However, the mechanical properties of electroresponsive tissues differ widely. For instance, nerve tissue has Young’s modulus around 600 kPa, whereas that of the human myocardium is ~ 100 kPa and that of skeletal muscle cells around 24 kPa.9 Subsequently, the ideal scaffold for electroresponsive tissue will be one that is concurrently conductive and has mechanical properties matching the host tissue. Here, we report a versatile and facile fabrication approach to produce electroconductive hydrogels in a range of physical and electronic properties. We also show that the behavior of cardiac cells cultured on the surfaces of the hydrogels varied as function of the substrate. Hydrogels, three dimensional (3D) networks of cross-linked polymers, have been extensively studied in the field of tissue engineering for the regeneration of several organs due to their hydrated nature that matches the properties of human tissue.1,10 Another attractive aspect of hydrogels is the flexibility in tuning their mechanical properties by simply adjusting parameters such as the cross-linking density,11 the type of polymer precursors,12 and the solid content.13,14 However, while this could be easily achieved using conventional hydrophilic polymers or their monomeric precursors, it has been more challenging with electroconductive hydrogels that contain conjugated polymers (CPs) which are hydrophobic in nature and lacking in functionality. Fabrication methods of electroconductive hydrogels have been largely based on growing the conjugated polymer within prefabricated hydrogel structures through chemical polymerization or electro-polymerization.15-17 While both approaches result in conductive networks, developments


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needed include (i) control over the amount of conjugated polymer formed within the network,18 (ii) increase in hydrogel porosity,19,20 (iii) enhancement of low swelling ratios caused by the hydrophobic nature of the CPs,21 and (iv) a reduction in the loss of the CP chains that are physically entrapped rather than covalently bound to the hydrophilic polymeric network.17 Achieving this will result in much greater control over the mechanical properties of the produced electroconductive hydrogel. To provide some of the required improvements mentioned above, studies have looked to covalently attach the conjugated polymer to the backbone of the hydrophilic network22 or fabricate a hydrogel network from covalently bound conjugated polymers only.23,24

These

approaches require the conjugated polymer to be water soluble and contain functional groups that can react with other precursors employed to fabricate the hydrogel. To date, there are limited studies reporting electroconductive hydrogels in which the conjugated polymers are functionalized with groups that can form a covalent bond.22-24 One example is the study by Mawad et. al.22 that investigated the synthesis of a water soluble conjugated polymer based on poly (ethylene dioxythiophene) (f-PEDOT) functionalized with pendant groups bearing double bonds. The double bond was co-polymerized with vinyl monomers such as acrylic acid to produce a chemically cross-linked hydrogel that exhibited a stable electrochemical response in physiological buffer even when subject to extended cycling. Additionally, the water solubility of the f-PEDOT led to remarkably high swelling ratios well beyond values ever previously reported for electroconductive hydrogels. The stiffness of the hydrogel could be varied from ~ 8 to 45 kPa by changing its oxidation state from neutral to oxidized. Furthermore, in vitro studies have also demonstrated that these conductive hydrogels were able to support the adhesion, proliferation and differentiation of electroresponsive cells seeded on the scaffold.22


4

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Taking advantage of the flexibility of the chemistry employed, we aim to fabricate conductive hydrogels with tunable mechanical properties without compromising the electroactivity. To achieve this, we investigated using vinyl monomers with different N-substituents to highlight that the functional PEDOT can be co-polymerized with vinyl monomers of different reactivity and functionalities. We chose the vinyl monomers, hydroxyethyl methacrylate (HEMA) and acrylic acid (AA).25 HEMA is more hydrophobic in comparison to AA, and the two monomers differ in their functionality with HEMA introducing a hydroxyl group in the network whereas AA a carboxylic group. We investigated co-polymerizing AA and HEMA with the f-PEDOT at different feed ratios. Subsequently, five networks of different compositions were fabricated. Their swelling behavior and mechanical properties were investigated as a function of the two monomer ratios. The internal morphology was also assessed. To establish that the insulating components do not compromise the electroactivity of the CP within the network, cyclic voltammetry was conducted. Raman spectroscopy was used to probe the presence of the fPEDOT and investigate its oxidation state. Finally, we conducted a preliminary in vitro study that demonstrated that the phenotype of cardiac cells was dependent on the hydrogel substrate on which cells were cultured.

2. EXPERIMENTAL SECTION 2.1. Materials. All chemicals were purchased from Sigma-Aldrich. The functionalized conjugated polymer, f-PEDOT, was synthesized in our lab according to our published protocol.22 Primary antibodies: Rat antimouse CD31/PECAM (BD Pharmingen, San Diego, CA, USA), mouse monoclonal sarcomeric anti-α-actinin (Sigma-Aldrich, St. Louis, MO, USA) and mouse monoclonal AlexaFluor®488-conjugated vimentin (Abcam, Cambridge, MA, USA). Secondary


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antibodies: anti-mouse secondary fluorochrome-conjugated antibodies (Jackson Immunological Research Labs, Inc., West Grove, PA, USA). Nuclei stain: NucBlue® Live ReadyProbes® Reagent (Hoechst 33342, Invitrogen, Carlsbad, CA, USA). 2.2. Fabrication of f-PEDOT hydrogels. To fabricate f-PEDOT hydrogels with different ratios of the vinyl monomers, f-PEDOT was copolymerized with acrylic acid, hydroxyethyl methacrylate or a mixture of both, utilizing poly(ethylene glycol) diacrylate (PEGDA) (number average molecular weight = 700) as a cross-linker and 2,2′-azobis(2-methylproprionitrile) (AIBN) as the initiator. In a typical procedure, f-PEDOT (3 mg, 0.6 wt%) was dissolved in 0.5 mL dimethyl sulfoxide (DMSO) at 60 °C. The polymer solution was then cooled down to room temperature before adding 4 mg of AIBN followed by the addition of the hydrophilic monomer acrylic acid (0.297 mL) and 10 µL of PEGDA. The solution was vortexed and set in an oil bath for 10 hours at 60 °C. The inverting tube method was used to confirm the gelation of the hydrogel. The hydrogel was removed from the tube, washed thoroughly with excess deionized water (DI-H2O), freeze dried, and stored until required. Five hydrogels with different ratios of acrylic acid and HEMA monomers were fabricated as listed in Table 1. All hydrogels contained the same amount of f-PEDOT (0.6 wt%). Table 1: Feed ratios of AA and HEMA monomers employed in the fabrication of the f-PEDOT hydrogels. Compositions Hydrogel Label PEDOT_AA PEDOT_3AA_HEMA PEDOT_AA_HEMA PEDOT_AA_3HEMA PEDOT_HEMA

AA (mL) 0.297 0.223 0.216 0.108 0

HEMA (mL) 0 0.131 0.263 0.395 0.526

AA (wt%) 100 62.5 44.6 21.2 0

HEMA (wt%) 0 37.5 55.4 78.8 100

AA:HEMA Molar Ratio (%) 100:0 75:25 50:50 25:75 0:100

2.3. Characterization of the Physical Properties.


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2.3.1. Swelling Studies. Freeze-dried f-PEDOT hydrogels were weighed (md) and then incubated in phosphate buffered saline (PBS, 0.1 M, pH = 7.4) at 37 °C for three days. At predetermined time points, three swollen specimens of each type of hydrogel were weighed after being removed from PBS and gently blotted dry (ms). The percentage swelling ratio was calculated according to the following formula: % =

− × 100

2.3.2. Mechanical Testing. The Young’s moduli of swollen hydrogels were obtained by conducting compression tests using Instron 5565 with a 10 N load cell. A total of 3 specimens were tested for each composition. The samples were swollen to equilibrium in PBS at 37 °C prior to mechanical testing. The swollen samples were gently blotted to remove excess water. The diameter and the height of the samples were recorded before the test. To compensate for surface artefacts, a small load was applied until the surface of the sample was in full contact with the plates. Samples were subsequently subjected to a compression with a strain rate of 1 mm/min until 15 % of strain was reached. The Young’s modulus of the electroconductive hydrogel was calculated using the slope of the linear region in the stress strain plot at less than 5% deformation. 2.3.3. Scanning Electron Microscopy (SEM). Cryo-SEM was used to probe the internal morphology of the hydrogels. Specimens, swollen to equilibrium, were frozen for 40 secs in liquid nitrogen (LN2) and fractured with a LN2 cooled razor blade before being placed in the SEM for viewing ((JEOL 6490-LV SEM, Tokyo, Japan). The SEM was operated at 15 kV and the gels viewed in secondary electron imaging mode. The average pore sizes were estimated using the software ImageJ. 10 different pores were measured within an image and 3 images per sample were used for counting.


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2.4. Characterization of the Electronic Properties. 2.4.1. Electrochemistry. Cyclic voltammetry measurement was conducted using an Ivium Vertex potentiostat and its accompanying software (Ivium Technologies B.V., Netherlands). The counter electrode and the reference electrode were platinum mesh and Ag/AgCl in saturated KCl aqueous solution, respectively. PBS was used as the electrolyte solution. The cycling voltage was set between - 0.2 and 0.8 V and the scan rate was applied at 100 mV.s-1. The working electrode was gold mylar on which hydrogels (~ 1 mm in thickness) in their swollen state were anchored, and left to dry completely to enhance the adhesion with the electrode. Once the samples were fully dried, the gold electrode with the sample was dipped in PBS electrolyte, left for 30 minutes to stabilize before cyclic voltammetry was recorded. 2.4.2. Raman Spectroscopy. The molecular structures of the hydrogels were investigated using Raman spectroscopy (Perkin Elmer Raman microscope operating at 785 nm excitation). Swollen hydrogels were mounted on a glass slide and static scans were taken with the scanning center set at 1400 cm-1. The accumulation was set at 300 times and the laser power was set at 5% to minimize fluorescence and improve the signal to noise ratio. Attempts to record the Raman spectra of dried hydrogels failed due to the high background fluorescence of the samples. 2.5. In vitro Cell Study 2.5.1. Murine Neonatal Cardiac Cell Isolation and Cultures. Mouse primary ventricular cardiac cells were isolated from 1-2 days old neonatal hearts that were freed from atria, chopped into smaller pieces before being enzymatically digested using the Neonatal Heart Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Hydrogels were sterilized under the UV light source of the cell culture hood for 3 hours, transferred into a 96-well clear bottom polystyrene microplate (Corning®, New York, NY, USA), and the wells were filled with


8

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Dulbecco’s Minimum Essential Medium (DMEM) containing 5% foetal bovine serum (FBS), 1% Pen Strep and 1% L-glutamine presence. Isolated cardiac cells, composed primarily of cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs), were seeded and co-cultured on top of the hydrogels at a seeding density of 10,000 cells / mL. 2.5.2. Cell Morphology by Immunofluorescence Staining and Confocal Microscopy. Cardiac cells cultured on top of the hydrogels were fixed using 4% paraformaldehyde (60 min), permeabilized in PBS/0.01% sodium azide (PBSA) containing 0.02% Triton-X-100 (60 min), blocked with a 3% bovine serum albumin (BSA)/PBSA solution, and then incubated with appropriate primary (15 µg/mL) and secondary (10 µg/mL) antibodies at 4°C (18 hours). For cell behaviour analysis, fixed cardiac cells and hydrogels were exposed to primary antibodies against sarcomeric anti-α-actinin, CD31 and vimentin, to stain CMs, ECs and CFs, respectively. Nuclei were stained with Hoechst staining (Invitrogen, Carlsbad, CA, USA). Specimens were mounted using Vectashield® Mounting Medium (Vector Laboratories, Burlingame, CA, USA). Fluorescent imaging used a Zeiss LSM 800 Laser Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). Optical sectioning along the Z axis was performed and the images collapsed into a single focal plane using the manufacturer’s software. Images were processed using NIH Image 1.47v software (National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, USA). 2.5.3. Viability Assay. Following three days in cultures, Live/Dead® Viability/Cytotoxicity Kit for mammalian cells (Invitrogen, Carlsbad, CA, USA) was used according to manufacturer’s instructions to evaluate viability using the EVOS FL Auto Cell Imaging System (Invitrogen, Carlsbad, CA, USA). Cardiac cells and hydrogels were then fixed using 4% paraformaldehyde (60 min) and rinsed with PBS/0.01% sodium azide (PBSA) before being imaged using a Zeiss


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LSM 800 Laser Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany) at 530 nm and 645 nm to identify calcein-AM-positive live and ethidium homodimer-dead cells, respectively. Optical sectioning along the Z axis was performed and the images collapsed into a single focal plane using the manufacturer’s software. Images were processed using NIH Image 1.47v software (National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, USA). 2.5.4. Gene Expression Analysis. RNA was isolated from mouse cardiac cells cultured on the surfaces of tissue culture plate (TCP, control group), PEDOT_AA, PEDOT_AA_HEMA, and PEDOT_HEMA hydrogels using RNeasy Plus Mini Kit (Qiagen) following manufacturer’s instructions. Reverse transcription was performed using RT2 First Strand Kit and the resulting cDNA (20 µL) was diluted with 91 µL water and used as PCR template. Diluted cDNA was then mixed with 2xRT2 SYBR Green Mastermix (Qiagen) and water before loaded onto the Mouse Cardiovascular Disease PCR Array (PAMM-174Z, Qiagen). PCR was performed using ABI 7900HT with 384 block. Ct value was exported and analysed using Qiagen web based analysis tools. Four samples were tested on each PCR array plate and four biological repeats were performed. 2.6. Statistical Analysis. Data are expressed as mean ± standard error. One-way ANOVA with Tukey post-hoc test was used for comparisons of multiple groups. Values of p < 0.05 were considered significant. For single gene expression analysis, fold changes (calculated as 2-ΔΔCt) were compared with Welch’s t-test between groups.

3. RESULTS AND DISCUSSION


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The electroconductive hydrogels prepared herein combine tunable mechanical and swelling properties achieved by controlling the type of vinyl monomer used to form the insulating hydrophilic backbone, while retaining an excellent electrochemical response introduced by covalently linking the water soluble conjugated polymer (f-PEDOT) within the network. 3.1. Swelling Studies. To meet the requirement of a specific application, control over the swelling properties of a hydrogel is very important. Swelling behaviors of hydrogels are largely affected by the composition and amount of hydrophilic groups present within the network. To this effect, we co-polymerized our functional conjugated polymer with vinyl monomers that vary in their hydrophilicity. Freeze dried samples were soaked in PBS and their weights were collected at various time points. Figure 1 shows the percent swelling ratio for the different types of hydrogels.

Figure 1: Swelling behaviors of hydrogels with different monomer ratios between AA and HEMA. The symbol (*) denotes significant difference as compared to PEDOT_AA hydrogels at the same time point (p < 0.001). Error bars represent the standard deviation for n = 5.


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Equilibrium was reached for all hydrogels after one day swelling in PBS, with no significant difference detected in the swelling ratios of samples measured at different time points. However, the type of monomer used in the network greatly altered its water uptake capability with significant differences between the swelling ratios of hydrogels of various compositions. For instance at day 3, PEDOT_AA hydrogels exhibited the highest swelling ratio (~3300%), whereas PEDOT_HEMA showed a significantly lower swelling ratio (180%). Networks with acrylic acid and HEMA introduced at different amounts exhibited swelling ratios of ~ 2500%. This is in agreement with previous studies which have shown that incorporating acrylic acid in a copolymer network can provide higher water sorption in the system.25,26 Incorporation of the acrylic acid ionic monomer significantly increases the water uptake of hydrogels due to charge repulsion caused by its ionized carboxylic side groups on adjoining polymer chains at pH 7.4.25,27 In contrast, HEMA contains a non-polar methyl side group and as a result is hydrophobic relative to acrylic acid which accounts for the significantly lower swelling ratios obtained.26 As will be shown by the SEM micrographs and in agreement with the literature,

28,29

we found a close

correlation between the swelling ratios of the hydrogels and their internal microstructure. 3.2. Mechanical Properties. Having established that varying the chemical composition in the network significantly affects its water uptake, we investigated the mechanical properties of the fabricated conductive hydrogels by conducting compression tests on swollen samples. As shown in Figure 2, the Young’s modulus of the different types of hydrogels varied significantly demonstrating that the ratio of hydrophilic to hydrophobic monomers present in the system has large impact on the Young’s modulus of the network.


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Figure 2: Compression modulus of the different types of f-PEDOT hydrogels. The symbol (*) denotes significant difference as compared to PEDOT_AA hydrogels (p < 0.001). Error bars represent the standard deviation for n = 5.

In agreement with the swelling studies, by varying the chemical compositions of the monomers from AA to HEMA, we could significantly vary the Young’s modulus from 69.36 ± 6.97 kPa to 182.58 ± 20.87 kPa, respectively (p < 0.0001). By copolymerizing the two monomers at equal molar ratios, we achieved hydrogels with intermediate Young’s moduli (97.72 ± 9.71 kPa for PEDOT_AA_HEMA). The effect of varying the co-monomer compositions on the mechanical properties of hydrogels is well investigated in the literature.25, 27, 30-34

In our case, increasing the relative content of the more hydrophilic monomer with ionisable

groups led to increased repulsion between the polymeric chains, which in turn allowed for easier penetration of water molecules inside the network. This open structure weakens the mechanical properties

of

highly

swollen

hydrogel

systems32

such

as

our

PEDOT_AA

and

PEDOT_3AA_HEMA. On the other hand, increasing secondary chemical interactions between the components of the hydrogels, for instance hydrophobic interactions between the aromatic


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backbone of f-PEDOT and the non-polar methyl group of HEMA, will result in significant increase in the stiffness of the network, as was observed for PEDOT_AA_3HEMA and PEDOT_HEMA. Similar hydrophobic interactions have been observed for HEMA containing hydrogels assessed for the removal of aromatic dyes such as methyl violet and fuschine.26 The study reported that the presence of the HEMA component in the hydrogels resulted in a higher efficiency in removing these aromatic dyes from solutions due to these secondary interactions. Another factor that can be controlled to tune the properties of the hydrogel is the solid content. For instance in our previous work,22 we have copolymerized f-PEDOT with much lower amounts of AA (10 wt%). The swelling ratio (~5000%) and the Young’s modulus (8.2 ± 3.1 kPa) of the network were significantly different from those of the PEDOT_AA network we report here. This is an example of how facile it is to produce electroconductive hydrogels with tunable properties achieved either by changing the solid content or the hydrophilicity of the monomer. 3.3. SEM. The internal morphology of 3D networks is expected to affect the swelling behaviors and mechanical properties. Consequently, we examined the morphology of the five different hydrogels by cryo-SEM as shown in Figure 3.


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Figure 3: Cross-sectional micrographs of: A) PEDOT_AA, B) PEDOT_3AA_HEMA, C) PEDOT_AA_HEMA, D) PEDOT_AA_3HEMA and E) PEDOT_HEMA hydrogels. Scale bars: 20 µm. F) Average pore size of the different hydrogels. The symbol (*) denotes significant difference as compared to PEDOT_AA hydrogels (p < 0.001). Error bars represent the standard deviation for n = 100.

Qualitative analysis of the micrographs clearly shows a significant difference in the internal morphology as HEMA content is increased in the network. The PEDOT_AA exhibited a highly porous network with thin walls and had an average pore size of ~ 26.6 ± 3.4 µm (Figure 3A and F). As HEMA was introduced into the network, a heterogeneous pore structure was observed for PEDOT_3AA_HEMA (Figure 3B) with a significant decrease in its average pore size (17.8 ± 1.0 µm). With further increase in the HEMA content, two distinct microstructures could be discerned. PEDOT_AA_HEMA contained areas of the hydrogel made up of pores comparable to those of PEDOT_AA, and others which appear to be more condensed (smaller pores) (Figure 3C). The pore morphology of PEDOT_AA_3HEMA (Figure 3D) showed even more condensed


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cauliflower shaped pores within the hydrogel, with an average pore size of 4.0 ± 0.2 µm. Interestingly from the SEM measurements, no pores could be detected in the network containing only HEMA (Figure 3E). This exciting finding may be ascribed to a stronger attraction between the aromatic backbone of f-PEDOT and the hydrophobic side chain of HEMA, leading to a highly packed polymeric structure. The internal morphologies of the hydrogels are in agreement with the physical and mechanical properties discussed previously. PEDOT_AA hydrogels that exhibited the highest swelling ratios present with an internal structure that has the largest average pore size, which can be attributed to the ionized chains that are repulsive.33 However, as the AA content in the network decreases, the repulsive force between the chains decreases. Coupled with possible hydrophobic interaction between HEMA and f-PEDOT, this leads to a more compact structure. The closed and confined pores with thicker walls significantly decrease the mobility of the polymer chains, hence restricting the amount of water uptake of the network. Furthermore, the highly porous and thin wall structure obtained for PEDOT_AA implied a low mechanical strength. Our statistical analysis has shown that there is significant difference between the Young’s moduli observed for PEDOT_AA and all other hydrogels except for PEDOT_3AA_HEMA that still contains a significant amount of AA compared to HEMA. This can be attributed to the dense morphologies observed as HEMA content was increased in the networks. The more compact areas with thicker wall structure may have provided the hydrogel with higher Young’s modulus. Therefore, PEDOT_AA_3HEMA with the more condensed pore morphology

had

significantly

higher

Young’s

modulus

compared

to

that

of

PEDOT_3AA_HEMA. Conversely, PEDOT_HEMA with its highly compact structure displayed the highest stiffness. Hence, our designed f-PEDOT which is water soluble and contains


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functional side groups permit the fabrication of hydrogels with tunable swelling and mechanical properties never achieved before. 3.4. Electrochemistry. While the purpose of this study was to establish a versatile fabrication approach for tailoring the mechanical properties of electroconductive hydrogels, it was imperative to confirm that the electrochemical properties of the hydrogels are not compromised. The redox activity of the hydrogels was investigated using cyclic voltammetry in PBS as an electrolyte (Figure 4 and Figure S1).

Figure 4: Cyclic voltammograms of hydrogels with different compositions: A) PEDOT_AA, B) PEDOT_AA_HEMA, and C) PEDOT_HEMA in 0.1 M PBS at a scan rate of 100 mV.s-1. Red squares indicate the region where the oxidation is occurring and the oxidation potential (Eox) was calculated as the maximum peak in that area.


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All different types of hydrogels exhibited an electrochemical response typical of conjugated polymers. This indicates that the insulating polymers in the network, polyacrylic acid or poly(hydroxyethyl methacrylate), do not influence the ability of the f-PEDOT to effectively transport electrons. Nevertheless there were differences in the features of the cyclic voltammograms collected. PEDOT_AA, PEDOT_3AA_HEMA, and PEDOT_AA_HEMA hydrogels exhibited broad anodic peaks, in agreement with our previously published data.22 In contrast, the anodic peaks of PEDOT_AA_3HEMA and PEDOT_HEMA hydrogels were more defined. Moreover, the oxidation potential was dependent on the type of hydrogel investigated. PEDOT_AA and PEDOT_3AA_HEMA exhibited anodic peaks around 0.40 V, and PEDOT_AA_HEMA at 0.37 V. In contrast, a major shift in the anodic peak to lower potentials was observed for PEDOT_AA_3HEMA (Eox = 0.30 V) and PEDOT_HEMA (Eox = 0.27 V). This result indicates towards a more effective charge transport mechanism in the hydrogels containing higher amounts of HEMA. In agreement with the morphology and the physical properties discussed above, this could be attributed to the more compact structure of the network in which the polymeric chains are in closer proximity.22,23 Interestingly, the highly swollen PEDOT hydrogel with lower amounts of AA that we have previously reported22 exhibited an oxidation peak at 0.48 V, higher than the oxidation peaks of the hydrogels investigated in this study. This further points out that the more swollen the network the more potential is required to induce charge transport. This is an exciting result indicating that control over the physical properties of a conductive hydrogel network, such as morphology or swelling, has an immediate impact on the electrochemical properties. To probe why the hydrogels exhibit a good electrochemical response in an electrolyte such as PBS and without chemical doping, we used Raman spectroscopy to investigate the oxidation


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state of the f-PEDOT in the hydrogel networks (Figure S2A). The spectra revealed a significant peak around 1435 cm-1 which corresponds to the symmetric Cα = Cβ stretching vibration, a main characteristic of the doped form of PEDOT.35-38 As controls, we recorded the Raman spectra of drop-cast films of our functional PEDOT and the highly conductive and commercial PEDOTPSS and (Figure S2B). The highly doped PEDOT-PSS exhibited a band at 1439 cm-1 and in agreement with the literature.36,39 Similar to PEDOT_PSS, the spectra of f-PEDOT film revealed a significant peak at 1436 cm-1. Although the exact mechanism is not elucidated at present, the Raman data along with the cyclic voltammetry might indicate a self-doping mechanism from the ionized carboxylic side groups attached to the f-PEDOT backbone. However, further in depth study should be conducted to confirm this. 3.5. In vitro Cell Studies. The physical and electronic properties of the developed hydrogels make them suitable candidates in tissue engineering, in particular for electroresponsive tissues that require conductive scaffolds.40-42 Having established that fabrication of hydrogels with variable physical and electronic properties is feasible, we investigated whether the cell behavior cultured on the surfaces of these hydrogels will be affected by the hydrogel composition and properties. For this purpose, we investigated hydrogels with the lowest, intermediate and highest Young’s moduli, PEDOT_AA, PEDOT_AA_HEMA, and PEDOT_HEMA. The morphologies of these hydrogels were also significantly different as revealed by the SEM (Figure S3). As a model, we chose murine neonatal cardiac cells that include the main cell types found in the heart, such as cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs), and closely recapitulate the cellular composition typical of the heart microenvironment.2 Murine neonatal cardiac cells were cultured on top of PEDOT_AA, PEDOT_AA_HEMA and PEDOT_HEMA hydrogels. Hydrogels were not pre-treated with adhesion molecules, as both CFs and ECs


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present in the co-culture provide a platform of extracellular matrix (ECM) molecules fundamental for cardiomyocyte adhesion and culture.43 Following three days in culture, cells were stained with Hoescht (a blue stain for Live cells) and imaged by confocal fluorescent microscopy. All three hydrogels supported cell survival on their surfaces (Figures 5A-C), as indicated by the presence of the Hoescht stain in the nuclei. However, it is evident that the morphology of the cells varied significantly between the three hydrogels. Cells were spread on PEDOT_AA and PEDOT_HEMA surfaces, whereas they were bundled as multicellular aggregates on PEDOT_AA_HEMA. The aggregated phenotype observed for bundled cells on top of PEDOT_AA_HEMA was concerning in a first observation. Nevertheless, the bundled cells were alive and beating (see movie S1). Similarly, the same cell behavior was observed following calcein-AM staining (Figure S4). To further investigate the influence of these hydrogels on cellular phenotype, we performed confocal fluorescent microscopy using antibodies against specific markers of CMs, ECs and CFs (sarcomeric α-actinin, CD31 and vimentin, respectively) (Figures 5D-F). Qualitative confocal analysis demonstrated that different cell types adhere and grow at different ratios depending on the substrate (Figures 5D-F). PEDOT_AA substrate promoted mainly the growth of ECs and CFs, whereas PEDOT AA_HEMA and PEDOT_HEMA hydrogels favored primarily the attachment and growth of CMs (Figures 5D-F).


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Figure 5. Structural characterisation of neonatal cardiac cells cultured on the surface of PEDOT_AA, PEDOT_AA_HEMA and PEDOT_HEMA hydrogels. (A-C) Neonatal cardiac cells stained with antibodies against sarcomeric α-actinin and nuclei stained with Hoechst stain (blue). (D-F) Neonatal cardiac cells stained with antibodies against sarcomeric α-actinin (red), CD31 (green) and vimentin (blue), used as markers of cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs), respectively. Scale bars equal 50 µm.

As demonstrated by the sarcomeric α-actinin staining, CMs retain striations in all types of hydrogels, but their spreading and elongation were different depending on the hydrogel substrate (Figures 5D-F). We observed that sarcomeric α-actinin-positive CMs on PEDOT_AA hydrogels are isolated (Figure 5D), whereas they tend to grow as a group on both PEDOT_AA_HEMA and


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PEDOT_HEMA (Figures 5E and 5F). Moreover, CMs presented with an elongated phenotype when cultured on both PEDOT_AA and PEDOT_HEMA hydrogels (Figures 5D and 5F), whereas they exhibited a rounded morphology on PEDOT_AA_HEMA, suggesting that cell-cell adhesion is preferential over cell-matrix adhesion (Figures 5B and 5E). The aggregate-like structure observed on PEDOT_AA_HEMA included all three cell types clustered together (Figure 5E). The spindle-like phenotype observed for CMs on PEDOT_AA is reflecting a more mature phenotype (Figures 5A and 5D).41 On the other hand, growth of CMs is promoted on PEDOT_HEMA, as more CMs are present per area (Figures 5C and 5F). Furthermore, our qualitative confocal analysis suggests that PEDOT_AA might be more suitable for co-culturing cardiac cells including CMs with a more adult, elongated phenotype, whereas PEDOT_HEMA hydrogels may be used to preferentially expand cardiomyocytes in vitro cultures. To further assess the cell behavior observed on the different hydrogel substrates, we performed PCR analysis of gene expression of cardiac cells cultured on the surface of the hydrogels. Analysis of sarcomere-specific genes, the functional unit of cardiomyocytes and typically expressed in mature cardiomyocytes, demonstrated that sarcomere organization may have been impaired in presence of PEDOT_AA_HEMA hydrogels, as cardiac cells grown on these gels present the least expression levels of the cardiomyocyte-specific contractile protein alpha actinin (ACTC1) and cardiac myosin heavy chain (MYH6) compared to the other gels (Figure 6).


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Figure 6: Real-time PCR analysis performed using primers specific for markers: alpha actinin (ACTC1), cardiac myosin heavy chain (MYH6), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), and fibronectin (FN1). The symbol (*) denotes significant difference between groups (p < 0.001). Error bars represent the standard error of the mean for n = 4.

This effect suggests a dramatic remodeling of cardiac cells on the PEDOT_AA_HEMA hydrogels, as demonstrated by our confocal images (Figures 6D-F). Cardiac cells grown on either TCP or PEDOT_HEMA hydrogels showed very similar expression levels of these genes, whereas expression level changes measured in presence of PEDOT_AA gels were not statistically significant (Figure 6). Similarly, expression levels of other cardiac-specific structural proteins, such as cardiac troponin T (TNNT2) and I (TNNI3) was downregulated in cardiac cells


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grown on PEDOT_AA_HEMA hydrogels compared to PEDOT_HEMA hydrogels or TCP (Figure 6). Changes of TNNT2 and TNNI3 levels observed in presence of PEDOT_AA hydrogels compared to PEDOT_HEMA hydrogels and TCP were not statistically significant (Figure 6). Structural changes in the cells grown on hydrogels did not correlate with a change in expression levels of cell cycle-related genes, such as G0S2 (Figure S5), avoiding any role played by these hydrogels on cell growth. Quantitative analysis of fibronectin (FN1) expression levels demonstrated that cardiac cells grown on TCP or PEDOT_HEMA express higher levels of this extracellular matrix protein than when they are on either PEDOT_AA or PEDOT_AA_HEMA (Figure 6). As cell-matrix adhesion proteins are critical for cell attachment, viability and expansion, changes in fibronectin expression observed on these hydrogels may be correlated to the differences in morphology observed by our confocal analysis. Our gene analysis demonstrated that cells grown on PEDOT_HEMA hydrogels present features similar to those grown on tissue culture plates, where high levels of fibronectin and structural protein (ACTC1, MYH6, TNNT2 and TNNI3) favor adhesion of cardiac cells, similar to what we observed in the confocal analysis (Figure 5). Cardiac cells grown in the presence of PEDOT_AA hydrogels are exposed to lower levels of fibronectin and may therefore present more of “tethering effect” that make them attach and stretch on the surface. Downregulation of structural proteins in cells grown on PEDOT_AA_HEMA hydrogels may explain the aggregate-like phenotype observed in our confocal analysis. As a more organized structural, stretched phenotype is observed in cells expressing higher levels of ACTC1, our data suggest that cardiomyocytes grown on PEDOT_AA_HEMA hydrogels may retain their neonatal phenotype.


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Our preliminary in vitro study indicates that the cell behavior is altered as function of the hydrogel composition, although at this stage it is not clear what specific property of the hydrogel is causing this effect. We did not find a correlation between the substrate stiffness and cell phenotype, since there was no significant change in gene expression between the hydrogels presenting with the most difference in their stiffness (PEDOT_AA and PEDOT_HEMA). Owing to the complex nature of cell-matrix interactions, dedicated studies need to be conducted to establish the factors influencing the cell behavior on these hydrogels. In particular, our hydrogels contain negatively charged polymers (polyacrylic acid and f-PEDOT) copolymerized with a neutral poly(HEMA). Consequently the surface can be either tethered with neutral or negatively charged species, which will have a significant impact on cell behavior.

4. CONCLUSIONS In this study, we reported a versatile fabrication approach of conductive hydrogels composed of f-PEDOT co-polymerized with vinyl monomers with different hydrophilicity. By controlling the type and ratio of monomers used, scaffolds with tunable internal morphologies, mechanical and swelling properties could be produced. In agreement with the physical properties, cyclic voltammetry experiments revealed that the electronic properties are also linked to the chemical composition of the hydrogel. Scaffolds that had a dense internal structure exhibited a more effective charge transport as shown by the lower oxidation potential. Notably, Raman spectroscopy revealed that both the f-PEDOT in the hydrogel network or as pristine polymer is in an oxidized state, probably indicative of a self-doping mechanism. Our in vitro cell studies demonstrated that neonatal cardiac cells respond differently to hydrogels of different compositions. While the morphologies of cultured cells varied between elongated and bundled


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depending on the substrate, all hydrogels supported cardiac cells survival as revealed by the Hoechst staining and beating of the cells. Our findings demonstrate that the functional PEDOT we have synthesized offers enormous flexibility for the rational design of conductive scaffolds with the optimal physical and functional microenvironment to maximize integration with electroconductive tissues.

ASSOCIATED CONTENT Supporting

Information.

The

following

files

are

available

free

of

charge.

Cyclic voltammetry of PEDOT_3AA_HEMA and PEDOT_AA_3HEMA; Raman spectra of the hydrogel networks and films of PEDOT_COOH and the commercial PEDOT_PSS; SEM micrographs at a larger scale of the hydrogel networks; Live/dead images of cardiac cells cultured on the hydrogels; Relative expression of the G0S2 gene; Movie showing beating cardiomyocytes on the PEDOT_AA_HEMA hydrogel. AUTHOR INFORMATION Corresponding Author *Damia Mawad. Address: School of Materials Science and Engineering, UNSW Australia, Sydney, New South Wales 2052, Australia. Email: [email protected] ORCID: 0000-0002-6965-3232 Author Contributions ‡ L.J and C.G. contributed equally. L.J. and D.M. conducted all the experimental work related to the hydrogel fabrication and characterization. C.G., O.T. and P.S. did the in vitro cell studies and PCR analysis. C.C. synthesized f-PEDOT. L.J. and Y.S. recorded the Raman spectra. T.R.


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conducted the cryo-SEM. A.L. conducted the statistical analysis. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT SMS is a scholarship student from CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico and INCTBio, Instituto Nacional de Ciência e Tecnologia em Bioanalítica - Brazil. J.J.G. acknowledges funding from the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036) and the Australian Research Council for an Australian Laureate Fellowship (FL150100060). C.G. is supported by the Kick-Start Grant and a Cardiothoracic Surgery Research Grant from the University of Sydney, and by an NHMRC Project Grant (APP1129685). We would like to thank Dr. Louise Cole (University of Sydney) for her assistance with the confocal analysis. ABBREVIATIONS AA, acrylic acid; ACTC1, alpha actinin; AIBN, 2,2′-azobis(2-methyl-proprionitrile); CFs, cardiac fibroblasts; CMs, cardiomyocytes; CP, conducting polymer; ECs, endothelial cells; FN1, fibronectin; f-PEDOT, functional poly(ethylenedioxy thiophene); G0S2, G0/G1 switch gene 2; HEMA, 2-hydroxyethyl methacrylate; MYH6, cardiac myosin heavy chain; PEG-DA, poly(ethylene glycol) diacrylate; TNNT2, cardiac troponin T; TNNI3, cardiac troponin I.

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