Controlled release of chondroitinase ABC in chitosan-based scaffolds and PDLLA microspheres

Spinal cord injury (SCI) can trigger inhibitory signal cascades that promote the expression of chondroitin sulfate proteoglycans (CSPGs), which are the main structures in scar tissue. The digestion of CSPGs by chondroitinase ABC (ChABC) can promote axonal re-growth after SCI. However, ChABC cannot effectively digest CSPGs because it is unstable; therefore, stable ChABC must be released in a controlled manner in the repair of SCIs. Two methods of maintaining ChABC stability and bioactivity were examined. They were the immobilization of ChABC on nerve conduits (NCs) and the encapsulation of ChABC in poly(d,l-lactic acid) (PDLLA) microspheres.Nerve conduits with variously sized pores were fabricated from chitosan and gelatin. The pore diameters of chitosan NCs were 100–160 μm, and those of chitosan/gelatin NCs were 20–40 μm. The ChABC in NCs was immobilized by ionic or covalent bonding. The experimental results reveal that immobilizing ChABC in NCs markedly improved its stability. The activity of ChABC that was immobilized in chitosan NCs by ionic bonding was 0.07 U/mg; 48% of this activity was retained at 48 h after immobilization. PDLLA microspheres, fabricated by the double emulsion method, were applied as carriers in the controlled release of ChABC. Stabilizers, including nanogold (10 nm), polylysine (Mw: 500–2000) and polylysine (Mw: 20,000–30,000), were added to microspheres to maintain the activity of ChABC. Polylysine (Mw: 500–2000) stabilized ChABC most effectively. The ChABC activity was 0.0162 U/ml after seven days of release. Experimental results indicate that ChABC activity can be preserved during release by immobilizing ChABC in chitosan NCs and encapsulating ChABC in PDLLA microspheres using an appropriate stabilizer.