Thiol-Ene Photo-Click Collagen-PEG Hydrogels: Impact of Water-Soluble Photoinitiators on Cell Viability, Gelation Kinetics and Rheological Properties.

Thiol-ene photo-click hydrogels were prepared via step-growth polymerisation using thiol-functionalised type-I collagen and 8-arm poly(ethylene glycol) norbornene-terminated (PEG-NB), as a potential injectable regenerative device. Type-I collagen was thiol-functionalised by a ring opening reaction with 2-iminothiolane (2IT), whereby up to 80 Abs.% functionalisation and 90 RPN% triple helical preservation were recorded via 2,4,6-Trinitrobenzenesulfonic acid (TNBS) colorimetric assay and circular dichroism (CD). Type, i.e., either 2-Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and concentration of photoinitiator were varied to ensure minimal photoinitiator-induced cytotoxicity and to enable thiol-ene network formation of collagen-PEG mixtures. The viability of G292 cells following 24 h culture in photoinitiator-supplemented media was largely affected by the photoinitiator concentration, with I2959-supplemented media observed to induce higher toxic response (0.1 → 0.5% (/) I2959, cell survival: 62 → 2 Abs.%) compared to LAP-supplemented media (cell survival: 86 → 8 Abs.%). In line with the in vitro study, selected photoinitiator concentrations were used to prepare thiol-ene photo-click hydrogels. Gelation kinetics proved to be largely affected by the specific photoinitiator, with LAP-containing thiol-ene mixtures leading to significantly reduced complete gelation time (: 187 s) with respect to I2959-containing mixtures (: 1683 s). Other than the specific photoinitiator, the photoinitiator concentration was key to adjusting the hydrogel storage modulus ('), whereby 15-fold ' increase (232 → 3360 Pa) was observed in samples prepared with 0.5% (/) compared to 0.1% (/) LAP. Further thiol-ene formulations with 0.5% (/) LAP and varied content of PEG-NB were tested to prepare photo-click hydrogels with porous architecture, as well as tunable storage modulus (': 540⁻4810 Pa), gelation time (: 73⁻300 s) and swelling ratio (: 1530⁻2840 wt %). The photoinitiator-gelation-cytotoxicity relationships established in this study will be instrumental to the design of orthogonal collagen-based niches for regenerative medicine.