(I-341) Conjugated Conductive Polymer Nanocapacitors as Biocompatible Coatings for Neuronal Interfaces

Introduction::
The nervous system is characterized by its limited capacity for spontaneous regeneration and functional recovery after injury due to trauma or disease.[1] The increasing prevalence of degenerative neurological disorders and traumatic injuries creates an urgency for new biomaterials and treatments for neural cell repair. Due to their biocompatibility, mechanical properties and ease of functionalization, hydrogels are very appealing materials for nerve regeneration applications. However, poor hydrogel-protein interactions hinder cell attachment and further functionalization with pro-adhesive proteins such as fibronectin and collagen are often needed, increasing the complexity and cost of biomaterial obtention. Though it is known that electric fields modulate several biological processes and that neurons are electrogenic cells, electrical stimulation and conductive cues have been disregarded in tissue engineering and regenerative medicine approaches until recently.[2] In this work, biomimetically synthesized conjugated conductive nanoparticles (CCP-NPs) composed of 3,4-ethylenedioxythiophene (EDOT)-pyrrole copolymer were used as biocompatible hydrogel coatings for enhancing neuronal attachment and promoting differentiation and activity.




Materials and Methods:: Poly(ethylene glycol) diacrylate hydrogels were synthesized on top of glass coverslips via photopolymerization. Micropatterned hydrogels were obtained using a photomask. The biomimetic synthesis of EDOT-pyrrole copolymers doped with p-toluenesulfonic acid and poly(styrene sulfonate) was carried out using hematin as catalyst.[3] The obtained CCP-NPs were characterized by means of Raman spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). Electrochemical properties were characterized by galvanostatic charge discharge, cyclic voltammetry, and electrochemical impedance spectroscopy. Hydrogels were further functionalized by covalent bonding of fibronectin (FN) or adsorption of CCP-NPs. The effect pf PEGDA hydrogel functionalization on mouse neuroblastoma x rat dorsal root ganglion neuron hybrid cell line (ND7/23) viability, attachment and differentiations was evaluated. Cell attachment and differentiation were assessed by cytoskeleton staining and quantification of morphological parameters such as dendrite number and length. Cell viability was assessed using Live-Dead assay and flow cytometry.


Results, Conclusions, and Discussions::
CCP-NPs presented sizes in the range of 350-650 nm (Figure 1a), good conductivity and pseudocapacitive behavior (Figure 1b). ND7/23 cells seeded on non-coated hydrogels showed scarce attachment (Figure 1c), while cells seeded on hydrogels coated with CCP-NPs presented good attachment and were able to differentiate (Figure 1d). Moreover, cells seeded on CCP-NPs coated hydrogels presented viability values close to those found on cells seeded on conventional fibronectin-coated hydrogels (Figure 1e). Due to their size, CCP-NPs may physically adsorb on the hydrogel surface, facilitating further adsorption of proteins from the serum and promoting cell attachment. Furthermore, neurons seeded on micropatterned hydrogels presented an elongated, high-aspect ratio morphology due to alignment with the substrate.

Biomimetic synthesized CCP-NPs were used as a biocompatible coating for PEGDA hydrogels. Biomimetic synthesis was utilized to avoid cytotoxicity associated with reagents, organic solvents, and by-product of traditional wet chemistry protocols. CCP-NPs presented similar charge-transfer velocity and larger specific capacitance than PEDOT:PSS, benchmark in electroconductive material for bioengineering. The obtained coatings were able to sustain cell attachment, growth, promote neuronal differentiation and presented no toxicity towards ND7/23 cells. Adsorption of CCP-NPs is a promising alternative to traditional hydrogel surface modification techniques. Overall, biocompatible CCP-NPs fabricated here are appealing materials for obtaining biocompatible and electroconductive coatings, with promising applications as neuronal bio interfaces.




Acknowledgements (Optional): :

References (Optional): : 1) Vijayavenkataraman, S., Nerve guide conduits for peripheral nerve injury repair: A review on design, materials and fabrication methods. Acta Biomater. 2020, 106, 54-69.
2) da Silva, L. P.; Kundu, S. C.; Reis, R. L.; Correlo, V. M., Electric Phenomenon: A Disregarded Tool in Tissue Engineering and Regenerative Medicine. Trends Biotechnol. 2020, 38 (1), 24-49.
3) Martinez-Cartagena, M. E.; Muzzio, N. E.; Guntnur, R. T.; Fisher, V.; Hebbale, S.; Rodgers, T.; Romero-Garcia, J.; Romero, G., Conductive Conjugated Polymer Nanocapacitors for Localized Electrical Neurostimulation. Acs Applied Nano Materials 2022, 5 (8), 12006-12018.