Engineering fibrin matrices: the engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins.
Engineering extracellular matrices that utilize the body's natural healing capacity enable the progression of regenerative therapies. Fibrin, widely used as a surgical sealant, is one such matrix that may be augmented by the addition of protein factors to promote cell infiltration and differentiation. The thrombin-catalyzed conversion of fibrinogen to fibrin exposes N-terminal fibrin knobs that bind to C-terminal pockets to form the fibrin network. Here, we have created a platform system for the production of therapeutic proteins that capitalize on these native knob:pocket interactions for protein delivery within fibrin matrices. This system enables the retention of therapeutic proteins within fibrin without additional enzymatic or synthetic crosslinking factors. Using an integrin-binding fibronectin fragment as a model protein, we demonstrate that engineered knob-protein fusions bind consistently and specifically to fibrin(ogen). Equilibrium dissociation constants (K(D)) obtained using surface plasmon resonance indicate that these fusions have mum binding affinities, comparable to the native knob-containing fibrin fragments. The specificity of these interactions was verified by ELISA in the presence of molar excess of competing knob mimics. Release profiles and real-time confocal imaging demonstrate that the fusions were retained within fibrin matrices, even under the stringent continuous perfusion conditions used in the latter. In summary, this work explores the benefits and limitations of engaging native, biologically-inspired, non-covalent knob:pocket interactions within fibrin(ogen) for the retention of therapeutic proteins in fibrin matrices and provides insight into the stability of native knob:pocket interactions within fibrin networks.
Soon
Allyson S C
AS
The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Stabenfeldt
Sarah E
SE
Brown
Wendy E
WE
Barker
Thomas H
TH
D012103:Batroxobin / Q000494:pharmacology; D001704:Biopolymers / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D026122:Factor XIIIa / Q000378:metabolism; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry; D010477:Perfusion; D011485:Protein Binding / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000627:therapeutic use*; D020449:Repetitive Sequences, Amino Acid; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000494:pharmacology
D001704:Biopolymers; D009842:Oligopeptides; D011994:Recombinant Proteins; D005337:Fibrin; D005340:Fibrinogen; D026122:Factor XIIIa; D012103:Batroxobin; D013917:Thrombin