Synthesis of Microgel Sensors for Spatial and Temporal Monitoring of Protease Activity

Abstract

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity , hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.

Potential of Drug Efficacy Evaluation in Lung and Kidney Cancer Models Using Organ-on-a-Chip Technology

“…Recent advances in bioengineering have allowed new possibilities for biosensor integration into systems via ECM. For instance, incorporating fluorescent microgels, including peptides cleaved by specific enzymes [ 32 ], offers the opportunity to use ECM for real-time reading of OoC assay values. The ECM is a supporting network of macromolecules that provides biochemical and structural support to surrounding cells, while also generating biochemical cues for tissue growth and maintenance, and promoting cell adhesion and cell-cell communication.…”

Hydrogel-Based Technologies for the Diagnosis of Skin Pathology

“…Comparatively, Shin et al prepared micrometer-sized PEG gels incorporating the FRET-peptide sensor through thiol-ene photoclick chemistry and embedded it in the secondary PEG/gelatin patch to evaluate collagenase activity of melanoma cells. In this manner, information obtained can have spatiotemporal resolution of tumor progression [79]. Employing similar concept to assemble an activatable chemical probe (i.e., carrying a cleavable peptide substrate and a caged near-infrared dye), we demonstrated sensitive recognition of fibroblasts activation protein α (FAP-α), a crucial biomarker of abnormal skin scarring (e.g., keloid).…”

Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels

“…[1][2][3] In particular, thiol-norbornene click reaction is advantageous in hydrogel crosslinking owing to the rapid and quantitative reactivity between thiol-and norbornene-functionalized macromers. [4][5][6][7][8][9] The modular and orthogonal reactivity of thiol-norbornene click reaction has been used to fabricate a diverse array of biomaterials, including bulk hydrogel, [10][11][12] colloidal gel, [13,14] as well as cell surface coating. [15] Current modular thiol-norbornene hydrogels are exclusively prepared from photopolymerizations initiated by ultraviolet (UV) light, visible light, or two photon irradiation.…”