title: Improved local delivery of TGF-beta2 by binding to injectable fibrillar collagen via difunctional polyethylene glycol.
abstract: 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.
authors.affiliation: Research and Development, Collagen Corporation, Palo Alto, California 94303, USA.
authors.forename: H
authors.initials: H
authors.lastname: Bentz
authors.forename: J A
authors.initials: JA
authors.lastname: Schroeder
authors.forename: T D
authors.initials: TD
authors.lastname: Estridge
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D008297:Male; D008907:Mink; D011092:Polyethylene Glycols; D011485:Protein Binding; D051381:Rats; D012995:Solubility; D016212:Transforming Growth Factor beta / Q000008:administration & dosage* / Q000378:metabolism
chemical_list: D016212:Transforming Growth Factor beta; D011092:Polyethylene Glycols; D003094:Collagen