Changing mechanical properties of photopolymerized, dityrosine-crosslinked protein-based hydrogels

Abstract

Hydrogels based on renewable resources are a promising class of materials for future applications in pharmaceutics, drug delivery and personalized medicine. Thus, optional adjustments of mechanical properties such as swelling behavior, elasticity and network strength are desired. In this context, hydrogels based on the biological raw materials bovine serum albumin and casein were prepared by dityrosine-crosslinking of their tyrosine residues through visible light-induced photopolymerization. Changing the tyrosine accessibility by urea addition before photopolymerization increased the storage modulus of the hydrogels by 650% while simultaneously being more elastic. Furthermore, contributions of the buffer system composition, variation of protein concentration and storage medium towards mechanical properties of the hydrogel such as storage moduli, elasticity, fracture strain, compressive strength and relative weight swelling ratio are discussed. It could be shown, that changes in precursor solution and storage medium characteristics are crucial parameters towards tuning the mechanical properties of protein-based hydrogels.

Mechanical Properties of Protein-Based Hydrogels Derived from Binary Protein Mixtures—A Feasibility Study

“…The third buffer component, sodium citrate, is known to increase the solubility of casein [ 44 , 45 ], which may increase the surface availability of potential crosslinking sites. As previously described [ 22 ], the visible crack formation directly after polymerization for hydrogels containing a casein share ≥75% may thus be related to the non-stabilizing buffer formulation conditions, which may also explain the lower structural strength of homopolymeric casein hydrogels prepared in SPB compared to the copolymers formed with a 75% casein share in SPB. In addition to the different buffer components, the MCB formulation has higher ionic strength, as evidenced by the fact that a total of 96 mM of salt was were instead of 20 mM in the case of the SPB.…”

“…The buffer components MOPSO and TAPS used in the MCB are known to stabilize the native structure of BSA [ 42 , 43 ] and, thus, possibly stabilize the second protein investigated with an ordered tertiary structure (α-amylase) as well. This may result in a lower crosslinking density due to a lower degree of protein unfolding which was previously shown to be critical for structural strength [ 22 ]. The third buffer component, sodium citrate, is known to increase the solubility of casein [ 44 , 45 ], which may increase the surface availability of potential crosslinking sites.…”

“…If necessary, Vivaspin ® ultrafiltration units (Sartorius Stedim Biotech, Göttingen, Germany) with a molecular weight cut-off of 10 kDa were used according to the manufacturer’s recommendation to reach higher stock solution concentrations. Protein concentrations were determined with a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) using the extinction coefficients ε BSA,280 nm = 0.67 L/(g·cm) [ 33 ], ε α-Amylase,280 nm = 2.60 L/(g·cm) [ 34 ], ε ELP[V2Y-45],280 nm = 0.799 L/(g·cm) [ 32 ], and ε Casein,280 nm = 0.73 L/(g*cm) [ 22 ].…”

“…Hydrogels with a total protein concentration of 80 mg/mL composed of a single protein (homopolymeric hydrogels) or a binary protein mixture (copolymeric hydrogels), 0.25 mM Ru(bpy) 3 Cl 2 and 100 mM APS were prepared as previously described [ 22 ]. Briefly, this was performed through the following steps: (1) Formulation buffer, protein, and photoinitiator stock solutions were mixed in the DAC.…”

“…One of these hydrogelation methods is based on the crosslinking of phenolic hydroxy groups, which are naturally present in tyrosine, to form dityrosines under mild reaction conditions. This processing pathway can be mediated by a ruthenium-containing photoinitiator which is induced by visible light [ 15 , 16 , 17 ] and has been applied to several proteins, including bovine serum albumin (BSA), casein, tyrosine-enriched gelatin, maltose-binding protein, I27, protein L, anegen, ELPs, and silk [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. An alternative photoinitator system would be riboflavin, which is limited by a slower crosslinking speed and efficiency and is thus less favorable with regard to potential applications in 3D printing or for large volumes [ 29 , 30 ].…”