An Electroactive Oligo-EDOT Platform for Neural Tissue Engineering

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, the functionality of PEDOT:PSS for neural tissue engineering is improved by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesized using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalized oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone), a block co-polymer is produced that is electroactive, processable, and bio-compatible. By combining these properties, electroactive fibrous mats are produced for neuronal culture via solution electrospinning and melt electrospinning writing. Importantly, it is also shown that neurite length and branching of neural stem cells can be enhanced on the materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

After cooling to rt the mixture was diluted with DCM (50 mL) and washed with water (2 x 50 mL) and brine (50 mL

Oligomer functionalisation
Hydrochloric acid (4 M in dioxane, 4 mL) was added to a solution of trimer 6 (350 mg, 0.34 mmol) in DCM (5 mL). After stirring for 2 hrs, the mixture was concentrated in vacuo and azeotroped with water (2 x 50 mL). The product was then dissolved in water (10 mL) and lyophilised to give the DP as a red solid. A yield of 305 mg, 0.34 mmol (99 %) was obtained.
The product was used in subsequent experiments without further characterisation.
Tetramer 7 (250 mg, 0.21 mmol) was deprotected as described above. After concentrating in vacuo the product was azeotroped with DCM (3 x 20 mL) to give the DP as a purple solid. A yield of 218 mg, 0.21 mmol (99 %) was obtained. The product was used in subsequent experiments without further characterisation.
Pentamer 8 (180 mg, 0.14 mmol) was deprotected as described above. After concentrating in vacuo the product was azeotroped with DCM (3 x 20 mL) to give the DP as a dark purple solid. A yield of 165 mg, 0.14 mmol (99 %) was obtained. The product was used in subsequent experiments without further characterisation.
Triethylamine ( give the DP as a red oil. A yield of 77 mg, 44 μmol (98 %) was obtained.
buffered and quickly became poorly soluble. Solutions were left for 6 hrs to gel after which they were inverted and gently agitated. Although partial gelation had occurred, gels were highly heterogeneous and had weak mechanical properties that led them to partially disintegrate upon addition of water (1 mL).

Hydrogelation
Solution A (10 μL, 40 nmol hub) was added to Solution B or C (10 μL, 160 nmol crosslinker) and rapidly pipetted to mix. Gelation occurred almost immediately and so the pipette was very quickly removed after mixing. Although gels could be formed using trimer 26, they were highly heterogeneous due to precipitation of the oligomer upon buffering. Gels formed with trimer 15 were observed to have a far more homogeneous structure and could be easily handled within 15 min of addition. Gel size (10-200 μL) and polymer content (5-10 wt%) could be varied by scaling volumes and concentrations accordingly.

Tetramer and pentamer
Preparation of tetramer 26 and pentamer 27 stock solutions was attempted as described above.
However, in both cases the oligomer was found to be insoluble in water and phosphate buffer.
Attempts to pre-dissolve in DMSO followed by dilution in aqueous media were unsuccessful, with precipitation occurring immediately ( Figure S5).

Oligomer leaching
Trimer 15 crosslinked PEG gels (25 μL, 5 wt %) were prepared as described above in a 96 well plate, varying the ratio of crosslinker to PEG hub (0.33-1.66 ratio thiol:maleimide). 10 minutes after gelation was initiated, 100 μL water was added and the plate agitated at room temperature. At set time points, 90 μL of supernatant was transferred to a fresh plate for absorbance measurements, and replaced with fresh water. Cumulative oligomer release was monitored over a period of 24 hrs, and compared to a standard concentration curve of oligomer 15 concentration to determine the quantities of release. Experiments were run in triplicate. It was found that a slight excess of thiol led to the most mechanically stable gels with decreased leakage ( Figure S7).

Swelling
Trimer 15 crosslinked PEG gels (100 μL, 5 wt%) were prepared as described above. Gels were washed for 1 h in water (5 mL) and this process repeated 5 times to ensure removal of all soluble factors. Gels were lyophilised and weighed to measure their dry mass. Gels were then placed in buffer (0.2 M, 10 mL) at different pHs (citrate: pH 3; phosphate: pH 7; carbonate: pH 10). After swelling for 24 hrs, gels were removed, blotted gently to remove excess water, and reweighed. Swelling ratios were calculated by dividing swollen and dry masses. Experiments were run in triplicate ( Figure S8).
Polymers were partially cooled to 80 °C to prevent solidification, and precipitated in diethyl ether (100 mL). The resultant precipitate was collected by filtration, washed with diethyl ether (2 x 50 mL), and dried in vacuo to provide the desired PCL-oligoEDOT conjugates 16a-c with a M w ~ 25 kDa.