Luciferin-Bioinspired Click Ligation Enables Hydrogel Platforms with Fine-Tunable Properties for 3D Cell Culture

Abstract

There is an increasing interest in coupling reactions for cross-linking of cell-encapsulating hydrogels under biocompatible, chemoselective, and tunable conditions. Inspired by the biosynthesis of luciferins in fireflies, here we exploit the cyanobenzothiazole-cysteine (CBT-Cys) click ligation to develop polyethylene glycol hydrogels as tunable scaffolds for cell encapsulation. Taking advantage of the chemoselectivity and versatility of CBT-Cys ligation, a highly flexible gel platform is reported here. We demonstrate luciferin-inspired hydrogels with important advantages for cell encapsulation applications: (i) gel precursors derived from inexpensive reagents and with good stability in aqueous solution (>4 weeks), (ii) adjustable gel mechanics within physiological ranges (E = 180−6240 Pa), (iii) easy tunability of the gelation rate (seconds to minutes) by external means, (iv) high microscale homogeneity, (v) good cytocompatibility, and (iv) regulable biological properties. These flexible and robust CBT-Cys hydrogels are proved as supportive matrices for 3D culture of different cell types, namely, fibroblasts and human mesenchymal stem cells. Our findings expand the toolkit of click chemistries for the fabrication of tunable biomaterials.

Redox-triggerable Luciferin-Bioinspired Hydrogels as Injectable and Cell-encapsulating Matrices

“…Additionally, the choice of precursor's molar mass was done to enable tuning of important properties of derived materials (such as crosslinking degree and gelation rate), while leading to hydrogels that are adequate for cell encapsulation applications. 27,31 Control of the disulfide cleavage reaction at the molecular level was expected to allow fine tuning of materials properties at the micro/macroscopic level. According to reported work, the rate of disulfide cleavage depends on the chemical structure of the alkyl disulfide and on the specific reductant type used.…”

“…Previously, we demonstrated that PEG-CBT macromers are stable in aqueous solution for at least 1 month. 27 Regarding the stability of PEG-Cys macromers, we noticed that solutions of these precursors had to be freshly prepared to avoid oxidation of thiols to disulfide bridges, which presumably leads to the inactivation of the macromer. Aged PEG-Cys solutions in buffer were unable to form CBT-Cys hydrogels upon mixing with PEG-CBT (see Fig.…”

“…Recently, our group reported soft biomaterials whereby crosslinking chemistry was inspired by the biochemistry of fireflies. 27 A luciferin-inspired polyethylene glycol (PEG) based hydrogel for 3D cell culture was presented. These hydrogels were crosslinked via the so-called luciferin click ligation, which involves a condensation reaction between cyanobenzothiazole (CBT) and cysteine (Cys) groups.…”

“…Remarkably, the gelation rate of the system could be regulated within the seconds range by adjusting pH within the close-to-physiological range (i.e., from 8.0 to 6.6) without impacting the final mechanical strength. 27 Despite the mentioned advantages, the CBT-Cys system presented some limitations regarding user-control over system's properties. First, it did not allow for control of the gelation onset since the crosslinking reaction starts as soon as CBT and Cys precursors are mixed.…”

“…This short gelation time proved ideal for homogeneous encapsulation of cells in the scaffold for the preparation of in vitro culture models in a culture plate. 27 However, this time is too short for other biomedical scenarios requiring slower gelation rate (e.g., within minutes range) such as those involving injection and in situ crosslinking of matrices for therapeutic delivery, tissue adhesion and engineering. 28,29 Third, we and others 30 found that under physiological media (e.g., in buffer pH 7.4) Cys precursors undergo oxidation of Cys groups to form disulfide bridges, which inactivates the reactivity of Cys groups towards CBT moieties over time.…”