Correlating PEG-Depsipeptide Cross-Linking and Degradation Kinetics Using Ultrathin Hydrogel Networks at the Air-Water Interface

Degradable PEG-depsipeptide hydrogels are of interest to create pH-responsive nanostructures for therapeutics and biomedical applications. Thio-depsipeptides (TDP; pre-cross-linker) in particular enable pH-sensitive thiol–thioester exchange reactions to yield α,ω-dithiols (BTDP; cross-linker). BTDP can further cross-link maleimide functionalized poly(ethylene glycol) (PEG-4MAL) to form hydrogel networks via the Michael addition reaction.^[1] Yet, understanding the interplay of network formation and degradation in a complex biological environment is challenging.^[2] In this study, we use the Langmuir technique to investigate the cross-linking and degradation reactions of PEG-depsipeptide hydrogel networks in nanothickness ultrathin films. Ultrathin films as a model system enable the correlation of the effects of pH, temperature, cross-linkers, enzymes, and oxidants on the hydrogel networks behavior.<br/><br/>For preparing ultrathin hydrogel networks, chloroform solution of PEG-4MAL (20 000 g^.mol^-1) mixed with dithiol-based cross-linker (TDP, or 3,6-Dioxa-1,8-octanedithiol; DODT) was spread at the air-water interface (pH 10; 21 °C). The maleimide-to-thiol ratios of 1:1 or 1:2 used in this study were recently found effective in producing networks in aqueous solution.^[1] The spread monolayer was compressed and the resulting surface pressure - mean molecular area (π-MMA) isotherms were recorded. π-MMA isotherms show that the MMA of the highest elastic modulus for cross-linked films (PEG-4MAL + TDP or DODT) shifts to lower values when compared to the non-cross-linked sample (PEG-4MAL). Also, the cross-linked films could be compressed to higher surface pressures implying that the introduction of branching centers from the cross-linking reaction results in film stabilization. This stabilizing and contracting effect was not observed at ~ pH 6, owing to the pH sensitivity of the thiol-maleimide cross-linking reaction. Further, cross-linked films formed using a mixture of PEG-4MAL with TDP or DODT were found to desorb less than the non-cross-linked films (PEG-4MAL) when held at a constant surface pressure of 5 mN^.m^-1. This implies that cross-linked films, similar to bulk networks, were far less water-soluble than the non-cross-linked system.<br/><br/>To degrade the prepared ultrathin hydrogel network, proteinase K (enzymatically catalyzed degradation) or 3% H₂O₂(oxidative degradation) were used. Proteinase K insertion into the subphase digests the depsipeptide linkages in the cross-linked hydrogel network. This increases the water solubility of the film, which was observed by the reduction in film area under constant pressure. In terms of oxidative degradation by 3% H₂O₂, the cross-linked film (PEG-4MAL+TDP) degraded faster than the non-cross-linked film (PEG-4MAL), probably due to the oxidative cleavage of the cross-links.<br/>The present study advances the understanding of the formation mechanism of PEG-depsipeptide-based hydrogels and demonstrates that they can be degraded under physiological conditions. The described nanosystem is of further interest for preparing 2D materials using (bio)compatible materials for applications such as cell wrapping and developing bioresponsive delivery systems.<br/><br/>[1] M. Y. Folikumah, M. Behl, A. Lendlein, Biomacromolecules 2021, 22, 1875.<br/>[2] J. Ulbricht, R. Jordan, R. Luxenhofer, Biomaterials 2014, 35, 4848.