Development of Conductive Gelatine-Methacrylate Inks for Two-Photon Polymerisation

Abstract

Conductive hydrogel-based materials are attracting considerable interest for bioelectronic applications due to their ability to act as more compatible soft interfaces between biological and electrical systems. Despite significant advances that are being achieved in the manufacture of hydrogels, precise control over the topographies and architectures remains challenging. In this work, we present for the first time a strategy to manufacture structures with resolutions in the micro-/nanoscale based on hydrogels with enhanced electrical properties. Gelatine methacrylate (GelMa)-based inks were formulated for two-photon polymerisation (2PP). The electrical properties of this material were improved, compared to pristine GelMa, by dispersion of multi-walled carbon nanotubes (MWCNTs) acting as conductive nanofillers, which was confirmed by electrochemical impedance spectroscopy and cyclic voltammetry. This material was also confirmed to support human induced pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) viability and growth. Ultra-thin film structures of 10 µm thickness and scaffolds were manufactured by 2PP, demonstrating the potential of this method in areas spanning tissue engineering and bioelectronics. Though further developments in the instrumentation are required to manufacture more complex structures, this work presents an innovative approach to the manufacture of conductive hydrogels in extremely low resolution.

Keywords: GelMa; carbon nanotubes; conductive hydrogels; nano-fabrication; two-photon polymerization.

Figure 1

Mechanisms of (a) modification of gelatine with methacrylate groups to obtain gelatine methacrylate (GelMa) and (b) photo-polymerisation of GelMa groups to obtain a cross-linked gel. GelMa pre-polymer hydrogel (5 wt. %) with 1 mg mL⁻¹ multi-walled carbon nanotubes (MWCNTs-GelMa) (c) before and (d) after centrifugation and (e) ink formulation after addition of photoinitiator. Stability of MWCNTs-GelMa solutions (f) 3 and (g) 45 h after MWCNTs dispersion. SEM imaging of lyophilised (h) GelMa and (i) MWCNTs-GelMa structures.

Figure 2

Electrochemical characterisation of gels. (a) Impedance spectroscopy measurements of GelMa and MWCNTs-GelMa (1 mg mL⁻¹) compared to gold surfaces. (b) Typical cyclic voltammograms (CVs) of gels developed in situ on gold surfaces. Potassium hexacyanoferrate (II) (1 mM) in phosphate buffer saline (PBS) was used as an electrolyte. CVs were measured from 0.8 to −0.2 at 100 mV s⁻¹ vs. an Ag/AgCl reference electrode. Arrow indicates the direction of the CV. All measurements were performed in triplicate. Inset CV corresponds to GelMa, shown for clarity.

Figure 3

Viability studies performed on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) on (a) 5% and 10% GelMa and (b) 0.5 mm mL⁻¹ and 1 mg mL⁻¹ MWCNTs in 5% GelMa. Fluorescence images of immunostained hPSC-CMs after 7 days of culture in (c) 5% GelMa and (d) 1 mg mL⁻¹ MWCNTs in 5% GelMa. Nuclei were stained with a Hoechst probe (blue), actin fibres were stained with a phalloidin probe (red), and sarcomeres were immunostained with anti-alpha actinin (green). Scale bar 200 µm.

Figure 4

Hydrogel GelMa structures developed by 2PP. (a) Optical microscopy and (b) SEM imaging of hydrated and dehydrated GelMa ultra-thin films, respectively. (c) Optical microscopy and (d) SEM imaging of 120 µm × 100 µm complex GelMa scaffolds with 10 µm channels developed by 2PP.

Figure 5

(a) Optimisation conditions of MWCNTs-GelMa inks to produced thin-films by 2PP. Laser power was varied between 50, 40 and 30 mW and the scan rate was varied between 500, 1000, 2000, 5000, 10,000, 20,000, 30,000, 40,000 and 50,000 µm/s. Layer distance corresponds to 0.3 µm. Hatch spacing corresponds to 0.2 µm. Yellow arrow indicates our selected optimal conditions. (b) SEM image of dehydrated MWCNTs-GelMa thin-film manufactured by 2PP.