Improved local delivery of TGF-β2 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 β2 (TGF‐β2) was covalently bound to injectable bovine dermal fibrillar collagen (FC) and its activity compared to admixed TGF‐β2. Covalent binding was achieved in a two‐step procedure: First, TGF‐β2 was reacted with the difunctional polyethylene glycol (PEG) linker, and then the PEG‐attached TGF‐β2 (PEG‐TGF‐β2) was bound to the fibrillar collagen (FC‐PEG‐TGF‐β2). Initial binding of TGF‐β2 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‐β2 and Western blot analysis, using a TGF‐β specific antibody, demonstrated that at least 85% of the TGF‐β2 was bound to the fibrillar collagen. The activity of PEG‐TGF‐β2 was fully stable in phosphate‐buffered saline at 4°C and 37°C for at least up to 4 weeks. Unmodified TGF‐β2 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‐β2 containing 40 μg/mL TGF‐β2 were implanted subcutaneously into rats and analyzed after days 7, 21, and 42. All TGF‐β2–containing formulations showed the TGF‐β typical fibroblastic response at the day 7 time point. Covalent binding of TGF‐β2 to collagen with both difunctional PEG crosslinkers resulted in a significantly stronger and longer‐lasting TGF‐β2 response than that observed with admixed formulations of collagen and TGF‐β. The TGF‐β response with FC‐PEG‐TGF‐β2 lasted up to day 42 but was not seen after day 7 for TGF‐β2 admixed to FC. These findings clearly demonstrate that TGF‐β2 remains fully active after being covalently bound to collagen via difunctional PEG. In addition, covalent binding potentiates and prolongs in vivo TGF‐β responses and stabilizes the TGF‐β in vitro. Results suggest that this method of formulation could be useful to stabilize and deliver similar peptide growth factors or biologically active agents. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 539–548, 1998.