Development of biocompatible 3-D networks by bioorthogonal crosslinking

Abstract

Due to the bioorthogonality, high selectivity and exceptional reaction rates, the inverse-electron-demand Diels-Alder cycloaddition between tetrazines and strained alkene or alkyne dienophiles has been increasingly applied to the development of novel biomaterials. Adapting established procedures, I successfully synthesized various tetrazine (Tz) and trans-cyclooctene (TCO) derivatives and conjugated them to hyaluronic acid (HA), peptides and poly(ethylene glycol) (PEG). These conjugates have been used in various tissue engineering projects in our group. ☐ Using the biorthogonal building blocks, I developed microfibrous scaffolds with a stiff poly(e-caprolactone) (PCL) core and a soft HA shell. The scaffold was prepared by electrospinning of PCL, followed by tetrazine-ligation mediated covalent layer-by-layer (cLBL) assembly. The resultant scaffolds support the attachment and growth of stiff-primed primary vocal fold fibroblasts (VFFs), and effectively suppress myofibroblast differentiation. ☐ Our collaborative team discovered that, in the presence of Si-Rhodamine (SiR) dyes, dihydrotetrazine (DHTz), a Tz precursor that is unreactive towards TCO, can be photo-catalytically activated to Tz under near infrared (NIR) light to induce rapid bioorthogonal chemistry. In the absence of light or SiR, a mixture of HA-DHTz and HA-TCO is a transparent liquid. Upon exposure to NIR light, instantaneous gelation occurred both in vitro under cell culture conditions and in vivo subcutaneously in mice. ☐ Using a slower dienophile (norbornene, Nb), I prepared a soft bulk gel by mixing HA-Tz and peptide-based bisNb with a Tz/Nb ratio of 5:2. Overlaying an aqueous solution of low molecular weight HA-TCO on the gel disk initiated a diffusion controlled interfacial biorthogonal crosslinking to stiffen the network. The time and duration of the interfacial crosslinking can be readily tuned. This platform has been successfully utilized to control stem cell function spatially and temporally. ☐ Collectively, this work demonstrates multiple novel methods to construct 3D network utilizing bioorthogonal chemistry, enabling the establishment of dynamic and cell-instructive matrices for tissue engineering applications.