Rapid and bioorthogonal reactions are desirable for the construction of complex, three-dimensional (3D) tissue models. Tetrazine ligation, 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 a catalyst, 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) and octadecaethylene glycol flanked with TCO (bis-TCO), we have demonstrated the formation of hydrogel microspheres or water-filled channels via a diffusion-controlled process at the gel-liquid interface. Syringe delivery of HA-Tz to a bath of bis-TCO resulted in the instantaneous formation of a crosslinked shell, through which bis-TCO diffused inwards to introduce further crosslinking. Injection of bis-TCO into a bath of HA-Tz resulted in the instantaneous formation of a crosslinked shell, through which bis-TCO diffused outwards to create hydrogel channels. The interfacial polymerization process has permitted the creation of HA microspheres with pre-determined spatial distribution of TCO-tagged Alexa Fluor 647. Prostate cancer LNCaP cells were encapsulated in the hydrogel microspheres with 99% viability, proliferated readily in the HA matrix and formed 3D, tissue-like cell aggregates. The novel interfacial process, combined with the powerful bioothogonal reaction, will enable the construction of complex, physiologically relevant tumor models for drug screening purposes.

Interfacial bioorthogonal crosslinking for the fabrication of functional hydrogels

248th National Meeting (2014)

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