(L-441) Engineering strategies for hydrogel bioelectronics stabilization, miniaturization and multi functionalization for in vivo neural application.

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 441

Presenting Author(s)

Sizhe Huang, PhD (he/him/his)

Research Assistant
Binghamton University
Vestal, New York, United States

Primary Investigator(s)

Siyuan Rao

Assistant Professor
Binghamton University, United States

Last Author(s)

Qianbin Wang

Assistant propfessor
Binghamton University, United States

Introduction:: Neural probe technologies have been instrumental in studying complex neural activities. Despite diverse applications of neural interfaces such as deep brain stimulation and spinal cord functional recovery, current soft neural devices lack multifunctionality and struggle to adapt to the complex signaling and mechanical conditions within the nervous system. To enhance the multifunctionality of soft neural probes and ease the mechanical mismatch between neural interfaces and tissues, we developed a fabrication strategy via the control of metamorphic polymers’ amorphous-crystalline transition (COMPACT) with three aspects: (i) polymer chain folding with multiple cross-linkers, (ii) modulating intermolecular interactions in the hydrogel, and (iii) inducing nanocrystalline domain growth (Fig. 1A). Consequently, this strategy can achieve a high shrinking ratio (80% in diameter) of hydrogel fibers, which prevents polymer chains’ expansion from their designed nanocrystalline structure and maintain hydrogels’ volumes under a solvated state. Our COMPACT hydrogel fibers, miniaturized by polymeric crystallization approaches, have lower bending stiffness owing to the diminished diameter thus mitigating tissue damage during in vivo applications. Additionally, to improve the dimensional stability and prolong the long-term functionality of hydrogels, we fabricated SiO₂-coated PVA hydrogel fiber through atomic layer deposition (ALD) which were applied for in vivo optogenetic experiments to control locomotor behaviors in transgenic mice expressing Channelrhodopsin-2 (ChR2, Fig. 2). To expand the multifunctionality of hydrogel scaffolds, we used COMPACT strategy to introduce conductive carbon nanotubes (CNTs) in hydrogel matrix, creating an optrode. These were employed for muscle electromyographic and brain electrophysiological recordings of light-triggered neural activities in Thy1::ChR2 mice.

Materials and Methods::

COMPACT PVA hydrogel were fabricated via chemical cross-linking (under acidic condition) with two main cross-linkers: glutaraldehyde (GA) and tetraethyl orthosilicate (TEOS). A molding-extrusion method was used to fabricate hydrogel fibers, the mixed hydrogel pre-solution (with cross-linkers) was infused into silicone tubing and cross-linked at room temperature (RT). The hydrogel fibers were then eluted from the tubing and followed by incubation (2 hours) in hydrochloric acid (HCl, 12 mM) solution, stretching (100~200% strain) and drying (RT, 12 hours), and high temperature annealing (100 ^oC, 20 mins). COMPACT hydrogel optical fibers were utilized for concurrent photometric recording in VTA regions of AAV9-hSyn::GCaMP6s-injected mice with social interaction assays. A novel mouse was introduced for interaction while GCaMP fluorescence changes were recorded. Using an overhead camera, interactions were videoed and analyzed via DeepLabCut algorithms, correlating interaction epochs with GCaMP signals. SiO₂-coated hydrogel fibers were applied to optogenetics stimulation in mouse cortex for motor studies. Mice were habituated in a testing chamber before undergoing three optogenetic sessions, divided into light-OFF (first and third) and light-ON periods (second). During optogenetic modulation, mouse behaviors were recorded and analyzed for speed, distance, trajectories, and rotation behaviors. COMPACT electrode was implanted in mouse gastroonemius muscles for EMG recording and COMPACT optrode was implanted in mouse VTAs for electrophysiological recordings. EMG signals were recorded using the COMPACT hydrogel electrodes, under transdermal optical stimulation from a 473 nm laser. Electrophysiological recordings utilized optrode devices and a DAM50 system, with data analyzed via a MATLAB algorithm.

Results, Conclusions, and Discussions::

The COMPACT methodology universally enabled the miniaturization of PVA hydrogels across membranes, fibers, and bulks. We used Fourier transform infrared spectroscopy (FTIR) to examine the chemical bonds, and X-ray scattering to investigate the amorphous-crystallin transition. X-ray patterns suggested that the lamellae crystal domains of hydrogel fibers were re-oriented via COMPACT strategy. COMPACT hydrogel fibers exhibited refractive indices 1.48-1.60 (desiccated) and 1.37-1.40 (hydrated), suitable for core-cladding optics. The optimized fiber showed a 34 MPa modulus, better suiting nervous tissue than silica and polymer fibers, reducing neural damage. We tested COMPACT hydrogel fibers for in vivo optical recording during mouse social behaviors. Through fiber photometry, we detected increased GCaMP fluorescence (in mouse VTA) correlating with social interactions. To increase the stability of hydrogels for biomedical applications, we leveraged the stable nature of SiO₂ via ALD process to coat a uniform SiO₂ coating layer on PVA hydrogel fibers. We used energy dispersive spectrum (EDS) to confirm the existence of silicon on hydrogel fibers. Since the degree of PVA crystallinity contributes to hydrogels’ stability, we conducted X-ray scattering to analyze the PVA polymeric nanocrystals. The SiO₂-coated PVA hydrogel fibers exhibited a higher degree of crystallinity compared to uncoated PVA hydrogel fibers. To investigate the stability and functionality of PVA hydrogel fibers for in vivo optogenetic stimulation, we implanted SiO₂-coated hydrogel fibers in the M2 cortex region of Thy1::ChR2 mice and performed motor control behavioral assays. We observed optogenetically modulated mouse locomotor behaviors, including increased contralateral rotation, mobility speeds, and travel distances. To enhance the functionality of hydrogel fiber probes, we also developed an optoelectrical device, the optrode, enabling concurrent optical modulation and electrophysiological recording. The optrodes captured repeatedly neural activities in VTA of Thy1::ChR2 mice.

In conclusion, COMPACT provides a controllable fabrication method for micro-structured hydrogel fibers. These hydrogels provide a platform for functional interfaces through loadings of additional nanomaterials, which can be used for neural modulation and recordings concurrent with behavioral assays. The SiO₂-coated hydrogel fibers displayed enhanced crystallinity, improved dimensional stability under various physiological conditions which presents an advancement for the stability of hydrogel-based optical fibers for in vivo optogenetics stimulation.

Acknowledgements (Optional): : This work was funded in part by the UMass Amherst Faculty Research Grant (P1FRG0000000295), the Brain&Behavior Research Foundation Young Investigator Grant (29878) and the National Institutes of Health (R00MH120279).

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