Engineering fibrin matrices: The engagement of polymerization pockets through fibrin knob technology for the delivery and retention of therapeutic proteins

Abstract

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 (KD) obtained using surface plasmon resonance indicate that these fusions have μM 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.

Fibrin matrices: The versatile therapeutic delivery systems

“…A novel approach for retaining therapeutic proteins lacking affinity for fibrin within fibrin networks, without additional enzymatic or synthetic crosslinking factors was described by Soon et al [126]. Thrombolysis of fibrinogen exposes N-terminal fibrin knobs that bind to C-terminal pockets of the protein to form the three dimensional fibrin network.…”

Biomaterials to Prevascularize Engineered Tissues

“…Its degradation can be modulated by crosslinking with other polymers, like polyethylene glycol or adding protease inhibitors, such as aprotinin [57,58]. The major weakness of a fibrin-based construct is low mechanical stiffness, which can be improved by mixing with collagen, or crosslinking with factor XIIIa [59,60].…”

The evolution of fibrin-specific targeting strategies

“…The two knobs ‘A’ are exposed more rapidly than knobs ‘B’, and thus they are thought to play a more prominent role in protofibril formation, whereas knobs ‘B’ are thought to be more important for the lateral aggregation of protofibrils. 16,18 Activated transglutaminase factor XIII (FXIIIa) subsequently crosslinks adjacent D regions within a protofibril forming DD-Dimers. When fibrin is ultimately digested by plasmin, the resultant products are DD(E) Complexes, E-Fragments, and DD-Dimers.…”