Micropatterning Photoconductive Peptide Assemblies on Stiff and Soft Biomaterial Substrates

Introduction: : The human heart relies on an organized conduction pathway to provide optimal force generation of the cardiac pump and provide blood to the body. The most common methodology for simulating this pathway in vitro is by topographically aligning the cardiomyocytes. However, this fails to provide control over localization or directionality of exogenous electrical cues delivered to the cells. In previous work, we utilized conductive biomaterials that had been confined into microscale topographies to act as a “synthetic” extracellular matrix. While this strategy imparts directionality to the electrical signaling, it does not allow for the manipulation of the conductive pathway. Here, I present an approach to micropattern optoelectronic peptide-based materials to dictate cardiomyocyte organization and provide exogenous, light-induced stimulatory cues via a defined microscale biomolecular pattern using optoelectronic, π-conjugated peptides. First, the micropatterning approach and its stability will be validated for use within an aqueous environment and on glass and gelatin substrates. Then, the patterned peptide substrates will be used to provide aligned electrical cues to cardiac tissues and evaluated for its ability to generate a light-induced currents.

Materials and

Methods: : Optoelectronic biomaterials were generated by symmetrically functionalizing a known p-type semiconductor, quaterthiophene (4T), with peptides (DKK, KFKF, or DGRDKK). Ionizable amino acids were included in the peptide design to promote water-processability, as well as free primary amines for promoting stability of the pattern via (3-aminopropyl)triethoxysilane (APTES)/glutaraldehyde surface modification. Peptide-4T solutions were microcontact printed using a patterned polydimethylsiloxane (PDMS) stamp onto either APTES-coated glass or gelatin (Figure A). Peptide-4T patterns were evaluated for their stability in aqueous environment and photocurrent generation capability. The ordering of the peptide-4T was evaluated on different substrates and pH values via grazing-incidence wide-angle X-ray scattering (GIWAXS) to better understand the molecular-level changes of the peptide-4T throughout the microcontact printing process. Neonatal rat ventricular myocytes (NRVMs) were seeded onto the peptide-4T substrates and evaluated for changes in the organization of F-actin and z-lines in comparison to control substrates to establish the topographical impact of manipulating conduction pathways. Finally, isotropically-coated and anisotropically patterned peptide-4T substrates were subjected to a 415 nm light with a frequency of 2 Hz and evaluated for its photocurrent generation capacity.

Results, Conclusions, and Discussions:: The surface modification strategies rendered stability for peptide-4T micropatterns over multiple days within an aqueous environment. Our study indicates a molecular design-dependent effect for proper pattern transfer, where all three sequences could pattern on glass, but only KFKF-4T had successful pattern transfer on gelatin. Investigating KFKF-4T further, we observed substrate and pH-dependent effects on KFKF-4T ordering, as shown by UV-Vis absorption spectroscopy on the ink solutions and GIWAXS measurements on imprinted substrates. NRVMs seeded on top of anisotropically patterned KFKF-4T saw a significant increase in the organization of the F-actin and z-line cellular architecture, even compared to a microgrooved control (Figure B). Finally, we found that KFKF-4T substrates could generate photo-induced currents, allowing for its potential use as a micropatternable biomaterial platform for electrically stimulating cells using light (Figure C).
This work demonstrates the potential of an optoelectronic peptide-based platform for influencing cardiac tissues via patternable conductive biomolecular units. This work represents the first instance of direct micropatterning of conductive biomaterials and allows researchers to externally manipulate conduction pathways for excitable cells. This platform can generate photoinduced currents to provide high-spatiotemporal resolution stimulation without the need for genetic modification and avoids the need for traditional field electrodes stimulation in cardiomyocyte pacing. In the future, we hope this platform will provide an avenue to improve human induced pluripotent stem cell-derived cardiomyocyte maturation outcomes via combined topographical and optoelectronic signals.

Acknowledgements and/or References (Optional):: This work was supported by the National Heart, Lung, And Blood Institute (NHLBI) of the National Institutes of Health (NIH) under Award Number R01HL164348. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors acknowledge the use of facilities and instrumentation at the UC Irvine Materials Research Institute (IMRI), which is supported, in part, by the National Science Foundation Materials Research Science and Engineering Center: Center for Complex and Active Materials (DMR-2011967). Confocal microscopy was conducted at the Cardiovascular Innovation and Research Center (CIRC) at UC Irvine, under an S10 Equipment grant (1S10OD025064).