Enzyme and temperature dual-responsive polymer nanostructures towards the treatment of osteoarthritis

Abstract

Many diseases are associated with increased enzyme activities. Osteoarthritis (OA) is such an example where the over-expression of proteolytic enzymes leads to the erosion of the articular cartilage. These changes in the enzymatic environment offer inspiration for the design of smart drug delivery systems with triggered response to disease-specific conditions. In this context, self-assembled polymeric nanoparticles have come into focus due to their adaptable chemistry enabling the introduction of stimuli-responsive motifs to achieve triggered nanoparticle assembly and cargo release. Here, peptide-crosslinked, thermoresponsive polymer nanostructures cleavable by disease-associated enzymes are developed. More specifically, libraries of thermo-responsive N-isopropylacrylamide (NIPAM)- and N-cyclopropylacrylamide (NCPAM)-based triblock copolymers able to self-assemble across a range of temperatures were synthesized by controlled reversible addition-fragmentation chain-transfer (RAFT) polymerization. Two of the developed block copolymers were selected to be used for the construction of the enzyme-triggered delivery platform. By using copper-catalyzed click chemistry, these nanostructures were cross-linked with various peptide substrates sensitive to specific proteases. This approach confers improved system stability and controlled drug release kinetics. The morphology of both purely self-assembled and cross-linked nanostructures was investigated in detail by small angle neutron scattering (SANS) and cryogenic transmission electron microscopy (cryo-TEM). Further, loading capability towards multiple model cargos was demonstrated and enzyme-triggered release of a selected model protein was studied using fluorescence correlation spectroscopy (FCS). By accurate choice of the peptide cross-linker, nanoparticle degradation was achieved with various enzymes including MMP-13, which is the major enzyme causing cartilage degradation in OA. Finally, nanoparticle-cell interactions were studied using human OA chondrocytes and the synthesized copolymers / cross-linked nanoparticles did not reveal any cytotoxicity. Based on their fine control over the assembly temperature, chemical versatility, loading capabilities, enzyme-responsive properties and biocompatibility, these peptide cross-linked nanostructures are promising candidates as smart delivery systems for various diseases, including OA, where certain enzymes are over-expressed.