Poster X7 - Microfluidic Synthesis of Hyaluronic Acid-Based Hydrogel Fibers via Bioorthogonal Tetrazine Ligation

Introduction: Situated within the larynx, the human vocal folds are driven into oscillations by air from the lung to produce sound at frequencies of 20 Hz to 3 kHz. Vocal folds can be damaged by various chemical and mechanical insults, resulting in vocal fold scarring that is difficult to treat.[1] Preliminary studies have shown that hepatocyte growth factor (HGF), an antifibrotic cytokine, was effective in modulating vocal fold fibrogenesis when injected into the vocal folds. Because HGF has a short in vivo half-life and poor tissue retention, its long-term therapeutic efficacy is not guaranteed.[2] The long-term goal of this study is to develop an injectable hyaluronic acid (HA)-based fibrous hydrogel with controlled HGF release capacity to treat vocal fold scarring. Here, we demonstrate a microfluidic approach to the synthesis of HA microfibers employing the fast cycloaddition reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) derivatives.[3]

Materials and

Methods: Hydrogel precursors, including tetrazine-modified HA (HA–Tz), TCO-tagged peptidic crosslinker (SMV–bisTCO) and TCO-conjugated collagen-binding peptide (CBP-TCO), were synthesized following established methods.[4] A customized parallel laminar flow microfluidic device was made using the standard soft lithography. The microfluidic device features a central inlet and two side inlets (Fig. A). Using separate syringe pumps, the flow rates of the inlet solutions were modulated to achieve a laminar flow profile inside the microfluidic spinning channel.

Results, Conclusions, and Discussions: Results and Discussions:
The Tz/TCO reaction features fast reaction kinetics (with a second order rate constant of 10^3 -10^6 M^(-1)s^(-1)), high selectivity at low concentration, biocompatibility, and exceptional yield.[3] We have demonstrated the utility of tetrazine ligation in producing poly(ethylene glycol) (PEG)-based microfibers via interfacial bioorthogonal polymerization at the oil-liquid interface.[5] For this method to be successful, the polymer needs to be semicrystalline. Although proteins can be incorporated into the microfibers, exposure to the oil phase during the fiber pulling process leads to significant protein denaturation.[6] The microfluidic device can establish stable flow profiles of two aqueous streams, thus eliminating the need for the oil phase. The laminar flow can also introduce chain alignment, facilitating the generation of amorphous HA-based microfibers. In our study, high molecular weight HA-Tz was introduced to the central channel and the low molecular weight SMV-bisTCO was introduced to the two side channels to enable efficient cross-linking at the liquid-liquid interface (Fig. A). The resultant microfiber was collected in a TCO reservoir to allow for the interfacial reaction to complete. Fibers with an average diameter of 10-30 µM were produced and injectability was confirmed using a 25G needle. Work is underway to encapsulate NanoLuc luciferase in HA microfibers and its biological activity will be confirmed by luminescence assay.

Conclusions:
Using a customized microfluidic device, we demonstrated the utility of tetrazine ligation in producing HA-based protein-loaded microfibers. Future work involves synthesis of HGF-loaded HA microfibers and testing the applicability in treating vocal fold scarring.

Acknowledgements (Optional): Acknowledgment: This work was supported by National Institutes of Health (R01 DC014461).

References:
1. Hirano M., Folia phoniat.,1974, 26, 89−94.
2. Xu H. et al. The Laryngoscope, 2021, 131(1), 139-145.
3. Scinto S. et al. Nat Rev Methods Primers, 2021, 1, 30.
4. Pol M. et al. Biomaterials, 2023, 299, 122180.
5. Shuang L. et al. Adv. Mater., 2015, 27 (17), 2783-2790.
6. Ramaraj P., 2024, Manuscript in preparation.