Bio-responsive hydrogels for an in vitro brain cancer cell model: self-controlled inhibition of matrix metalloproteinase activity

Abstract

In nature, an unbalanced level of enzyme activity (e.g. proteases) is correlated to various disease states, particularly cancer. Among them, several studies have established that medulloblastoma (MB), a brain cancer in children, is associated with an overexpression of matrix metalloproteases (MMPs) (predominantly MMP-2), which is the consequence of the downregulation of their natural inhibitors tissue inhibitors of matrix metalloproteases (TIMPs) (e.g. TIMP-2). Although MMP inhibition remains a relevant therapeutic approach, MMP synthetic inhibitors have not converted to clinical application due to their dose-limiting side effects following systemic administration. As a result, the controlled delivery of endogenous MMP inhibitors directly in situ, could therefore provide the re-establishment of the MMP/TIMP equilibrium. The main goal of this work is to develop a MMP-2 responsive hydrogel that could provide an on-demand controlled release of the recombinant tissue inhibitor of MMP-2 (rTIMP-2) in response to elevated MMP-2 activity. This could be beneficial to restore the enzyme/inhibitor equilibrium and provide reduction of tumour growth and metastasis. MMP-2 responsive hydrogels were fabricated through thiol-ene step-growth polymerization of multiarm PEG-norbornene (PEG4-NB) with a MMP-2 recognised peptide sequence. Hydrogels demonstrated their MMP-2 sensitivity through the release of an encapsulated fluorescent dextran probe, as crosslinks are degraded by the enzyme. Following mesh size optimisation using a model dithiothreitol (DTT) cross-linked PEG4-NB hydrogel, MMP-2 responsive hydrogels were found to proficiently encapsulate and retain rTIMP-2, a necessary condition to maintain an exclusively enzyme triggered release. rTIMP-2 loaded hydrogels demonstrated their ability to efficiently inhibit different levels of MMP-2 and to modulate enzyme activity on the basis of the different rTIMP-2 payloads. These outcomes were obtained by monitoring both the residual MMP-2 activity and the subsequent release of a co-encapsulated fluorescent dextran upon MMP-2 action. Lastly, in a preliminary study, rTIMP-2 containing hydrogels proved to be cell biocompatible and able to inhibit MMP-2 secreted by MB cells. These findings promote the potential ability of this bio-responsive hydrogel to promote the renewal of the imbalance between MMP-2 and TIMP-2, an essential condition to reduce medulloblastoma growth