Design and Characterization of Thioester Networks with Adaptable and Enzymatically Degradable Cross-Links

Viscoelastic properties of the extracellular matrix (ECM) impact cell processes including proliferation, spreading, and migration. During these basic cellular processes, cells remodel the ECM by secreting enzymes and applying cytoskeletal tension to the network. To design cell delivery platforms that mimic physical ECM properties, new designs incorporate viscoelasticity and moieties that enable cell-mediated network remodeling. In this work, we design and characterize networks with two different types of cross-links, covalent adaptable and enzymatically degradable. Our networks consist of 8-arm poly(ethylene glycol) (PEG)-thiol, PEG-thioester norbornene, and a norbornene functionalized matrix metalloproteinase (MMP)-degradable peptide, KKGPQG↓IWGQKK. We characterize three network compositions with a ratio of 1:1, 3:1, and 4:1 adaptable to MMP-degradable cross-links. We characterize network mechanical properties using bulk rheology. Using multiple particle tracking microrheology (MPT), we measure the evolving microstructure of the network during degradation. MPT measures Brownian motion of fluorescently labeled probe particles, which can be used to calculate rheological properties. Our results show that the elastic modulus increases with an increasing ratio of adaptable to MMP-degradable cross-links, and all networks have the same extent of stress relaxation. We then measure degradation of these networks by incubating in l-cysteine, which degrades only the adaptable cross-links by the thioester exchange reaction. We measure complete degradation of all three compositions using bulk rheology. Networks with 4:1 adaptable to MMP-degradable cross-links are the slowest to degrade and networks with 3:1 adaptable to MMP-degradable cross-links are the fastest to degrade. MPT measurements during degradation show networks with 1:1 and 4:1 adaptable to MMP-degradable cross-links rearrange multiple times before complete degradation. In networks with 3:1 adaptable to MMP-degradable cross-links, we measure fewer network rearrangements prior to degradation. Using time-cure superposition (TCS), we measure the network structure at the phase transition. Networks with 1:1 and 4:1 adaptable to MMP-degradable cross-links are elastic and tightly cross-linked and networks with 3:1 adaptable to MMP-degradable cross-links can range from elastic to open networks. The most open network structure, networks with 3:1 adaptable to MMP-degradable cross-links, degrade on the shortest time scale. We also measure ≥70% hMSC viability in each network after 3D encapsulation. In this work, we characterize different compositions of hybrid networks that incorporate both adaptable and enzymatically degradable cross-links. This work can enable design that specifies the mechanical properties and degradation behavior of the material to better mimic aspects of the native ECM.