Physical interactions of self-oligomerizing protein complexes are important in many biological processes such as intercellular communication and the self-assembly of biomaterials. Weakly self-associated protein complexes as physical crosslinkers are advantageous for constructing injectable and self-healing hydrogels for biomedical applications. Following injection, the weak strength of such crosslinkers result in relatively fast erosion that limits the hydrogel lifetime in vivo. Additional design strategies are necessary to improve mechanical reinforcement of injectable hydrogels.
In this study, we use streptavidin (SAv) tetramers as a model protein crosslinker and investigate the influence of biotin, a ligand known for its high affinity binding to SAv monomers at a 1:1 stoichiometric ratio, on the strength of SAv physical interactions in protein hydrogels. SAv tetramers are composed of four identical monomers with strongly associating monomer-monomer interfaces and a weaker dimer-dimer interface. Previous studies provide evidence that biotin enhances the thermal stability of SAv, however it is unknown whether biotin can reinforce SAv mechanical stability, in particular the weak dimer-dimer interface. Furthermore, biochemical and single-molecule mechanical studies indicate that correlation between protein thermal stability and mechanical stability is not guaranteed. Therefore, to evaluate whether SAv tetramers with biotin are proper physical crosslinkers for post-injection hydrogel reinforcement, we used atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) to investigate the effect of biotin on the mechanical strength of SAv tetramers. Subsequently, we engineered telechelic protein sequences, consisting of a polyelectrolyte-like protein with SAv monomer end groups, as building blocks to form protein hydrogels by SAv tetramer oligomerization. Then, we performed rheological characterization of protein hydrogels to correlate biotin-mediated SAv strengthening at single-molecule and macroscopic levels.
Characterization of individual SAv tetramers and SAv self-assembled hydrogels captured mechanical strengthening induced by biotin binding. AFM-based SMFS results reveal that increasing biotin concentrations enhance the mechanical stability of the weak SAv dimer-dimer interface, increasing its rupture force approximately 200%. At the macroscopic level, rheology results indicate that biotin, a vitamin available in over the counter dietary supplements, can modulate the stress-relaxation properties of physical hydrogels, presenting a potential post-injection mechanism for controlling the in vivo lifetime of injectable hydrogels. We propose the fabrication of telechelic proteins, consisting of proteins of interest with SAv monomer end groups, together with biotin for the customizable design of biomaterial matrices for potential applications in advanced drug delivery systems, internal wound dressing, and tissue engineering.