A novel strategy is reported for sensing chemical bond breakage in hydrogels at low levels of mechanical stress using a mechanochemical reaction cascade to generate fluorescence. Hydrogels are promising substrates and frameworks for cell growth and tissue engineering, particularly for cardiovascular repair and cartilage replacement. For these applications, it is important to maintain careful control over gel mechanical properties so that these hydrogels not only match the properties of the desired tissue for replacement but also retain their integrity for extended periods. Since the failure of hydrogels begins with the breakage of cross-links within the structure, methods are needed to sense these initial events for monitoring the performance of implants. In this work, it was hypothesized that nonspecific covalent bond breakage would produce radicals that would react with water to produce reactive oxygen species, which in turn could activate fluorophores sensitive to these. A series of multiarm poly(ethylene glycol) hydrogels were synthesized with a variety of cross-links of different bond dissociation energies. It was found that gels loaded with the masked fluorophore 3′-(p-aminophenyl) fluorescein became fluorescent during compression, even with as little as 5 kPa of pressure. The effect of compression on fluorescence activation was found to depend primarily on the strength of the cross-linking functional group. Future studies include utilizing this system to image mechanical variability in heterogeneous gel structures.

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents.

This work demonstrates that hydrogen peroxide (HO) is generated in weak polyacrylamide hydrogels due to mechanochemical reactions to osmotic swelling. Hydrogels are important tools and materials for many biomedical applications, particularly for growth of stem cells. However, swollen gels are under constant tension, which makes their individual chains susceptible to mechanochemical bond breakage. In this work, an assay was developed to measure the generation of HO as a result of hydrogel swelling. Polyacrylamide hydrogels with both weak disulfide and strong PEG-diacrylate crosslinkers were synthesized and swelled. HO generation increased in the presence of weaker crosslinkers, up to 30 μM HO, whereas stronger crosslinkers reduced this to 5 μM HO. HO levels decreased when swelled in the presence of dextran to reduce osmotic stress or increased if the gels were conjugated to an acrylated surface. Finally, HO continued to form for days after the gels had reached their equilibrium sizes, independently of dissolved oxygen. The results of this work impact those working in the 3D cell culture community and demonstrate that even well-characterized systems undergo mechanochemical processes in mild environments.

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