The cycloaddition of s-tetrazine (Tz) with trans-cyclooctene (TCO) derivatives is particularly attractive for the synthesis of cell-instructive hydrogel matrices. This reaction is high yielding, does not require catalysis, does not produce any toxic side products, and is more than three orders of magnitude faster than pre-existing methods for bioorthogonal ligation. Using tetrazine-modified hyaluronic acid (HA-Tz, &gt;200 kDa) and polyethylene glycol flanked with TCO (bisTCO, &lt;2 kDa), we demonstrated the formation of hydrogel spheres via a diffusion-controlled process at the gel-liquid interface. Syringe delivery of HA-Tz to a bath of bisTCO resulted in the instantaneous formation of a crosslinked shell, through which bis-TCO diffused inwards to complete the crosslinking. The interfacial diffusion-controlled process has permitted the creation of hydrogel spheres with pre-determined spatial distribution of TCO-tagged biomolecules. Through temporally controlled introduction of TCO conjugates during the crosslinking process, the enzymatic degradability, cell adhesivity, and mechanical properties of the synthetic microenvironment can be tuned with spatial precision. Alternatively, when an aqueous solution of bis-TCO is added to a reservoir of HA-Tz, a crosslinked channel wall forms instantaneously. As the reaction proceeds, the smaller bis-TCO molecule diffuses outwards across the crosslinked wall to react with HA-Tz at the interface, thereby growing the channel wall outward until bisTCO is fully exhausted. By alternating cell laden HA-Tz reservoirs during the channel formation, cells can be spatially patterned into the channel wall in a physiologically relevant spatial order. In conclusion, interfacial tetrazine ligation has enabled facile fabrication of cell-laden matrices for tissue engineering applications.

Interfacial tetrazine ligation for the fabrication of cell-instructive matrices

256th National Meeting (2018)

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