Improved local delivery of TGF-beta2 by binding to injectable fibrillar collagen via difunctional polyethylene glycol

To overcome rapid diffusion and clearance from the implant site and to increase stability, recombinant transforming growth factor beta2 (TGF-beta2) was covalently bound to injectable bovine dermal fibrillar collagen (FC) and its activity compared to admixed TGF-beta2. Covalent binding was achieved in a two-step procedure: First, TGF-beta2 was reacted with the difunctional polyethylene glycol (PEG) linker, and then the PEG-attached TGF-beta2 (PEG-TGF-beta2) was bound to the fibrillar collagen (FC-PEG-TGF-beta2). Initial binding of TGF-beta2 to difunctional succinimidyl glutarate (D-SG-PEG) or succinimidyl propionate polyethylene glycol (D-SE-PEG) linkers was completed after reacting for 8 or 10 min as monitored by reverse-phase high-performance liquid chromatography. After reaction with injectable fibrillar collagen, extraction of unbound PEG-TGF-beta2 and Western blot analysis, using a TGF-beta specific antibody, demonstrated that at least 85% of the TGF-beta2 was bound to the fibrillar collagen. The activity of PEG-TGF-beta2 was fully stable in phosphate-buffered saline at 4 degrees C and 37 degrees C for at least up to 4 weeks. Unmodified TGF-beta2 mixed with fibrillar collagen was completely inactivated after 1 week of incubation, as measured by the mink lung epithelial cell (Mv1Lu) growth inhibition assay. Formulations of FC-PEG-TGF-beta2 containing 40 microg/ mL TGF-beta2 were implanted subcutaneously into rats and analyzed after days 7, 21, and 42. All TGF-beta2-containing formulations showed the TGF-beta typical fibroblastic response at the day 7 time point. Covalent binding of TGF-beta2 to collagen with both difunctional PEG crosslinkers resulted in a significantly stronger and longer-lasting TGF-beta2 response than that observed with admixed formulations of collagen and TGF-beta. The TGF-beta response with FC-PEG-TGF-beta2 lasted up to day 42 but was not seen after day 7 for TGF-beta2 admixed to FC. These findings clearly demonstrate that TGF-beta2 remains fully active after being covalently bound to collagen via difunctional PEG. In addition, covalent binding potentiates and prolongs in vivo TGF-beta responses and stabilizes the TGF-beta in vitro. Results suggest that this method of formulation could be useful to stabilize and deliver similar peptide growth factors or biologically active agents.