(93d) Immobilization of Nanoparticles through Protease Degradable Tethers for Controlled and Cell Specific Release

Non-viral gene delivery is an ideal approach to guide tissue formation through the delivery of genes that encode for bioactive signals that otherwise would not be present. Unfortunately, gene delivery efficiency and controlled release of DNA nanopartilces from tissue engineering scaffolds have proven to be major limitations in their widespread application. One possible solution to address both of these limitations is to immobilize DNA through protease degradable tethers near the proximity of the targeted cells to enhance gene delivery and initiate release through the action of cell-released proteases. We developed a 2D model system in which polystyrene nanoparticles, similar in size to DNA polyplexes, are immobilized to a cell binding surface through either protease sensitive peptide tethers or poly(ethylene glycol) polymer chains. Peptide-modified particles were shown to release over a 5-day period with the rate of release being determined by both the peptide sensitivity to proteases in addition to the degree of particle modification and tethering to the surface. For in vitro studies, two cell types that express either high or low levels of proteases were incubated on top of previously bound particles and particle internalization was assessed using fluorescence microscopy and flow cytometry. No extensive particle internalization was observed for either cell type when particles were modified solely with polymer chains, while peptide-modified particles were internalized to a significantly greater extent by cells which express high levels of proteases. Flow cytometry data showed that protease-expressing cells internalized approximately 10-fold more particles when compared to non-protease expressing cells, suggesting that nanoparticle internalization could be controlled by the protease expression profile of the seeded cells. Immobilization of nanoparticles through protease sensitive tethers is effective in controlling release rate and internalization, which has applications for controlled release of DNA polyplexes and other gene delivery vectors.