Acetic Acid Enables Precise Tailoring of the Mechanical Behavior of Protein-Based Hydrogels

Abstract

Engineering viscoelastic and biocompatible materials with tailored mechanical and microstructure properties capable of mimicking the biological stiffness (<17 kPa) or serving as bioimplants will bring protein-based hydrogels to the forefront in the biomaterials field. Here, we introduce a method that uses different concentrations of acetic acid (AA) to control the covalent tyrosine-tyrosine cross-linking interactions at the nanoscale level during protein-based hydrogel synthesis and manipulates their mechanical and microstructure properties without affecting protein concentration and (un)folding nanomechanics. We demonstrated this approach by adding AA as a precursor to the preparation buffer of a photoactivated protein-based hydrogel mixture. This strategy allowed us to synthesize hydrogels made from bovine serum albumin (BSA) and eight repeats protein L structure, with a fine-tailored wide range of stiffness (2-35 kPa). Together with protein engineering technologies, this method will open new routes in developing and investigating tunable protein-based hydrogels and extend their application toward new horizons.

Keywords: Biomaterials; Dynamic hydrogels; Protein folding transitions; Protein-based hydrogels; Responsive biomaterials.

Figure 1

Reaction mechanism of suppressing tyrosine-tyrosine covalent cross-links during protein-based hydrogel synthesis using a photoactivated reaction in the presence of acetic acid in the preparation buffer. (a) A photoactive reaction mixture containing protein, APS, Ru (II), and acetic acid is exposed to white light at room temperature. Ru (II) is photolyzed due to exposure to visible light in the presence of APS, which leads to Ru (III) and sulfate radical generation. Then, Ru (III) oxidizes tyrosine amino acids on surrounding proteins. A nearby carboxymethyl radical (·CH₂COOH and H atoms) produced during the photoinitiated protein cross-linking reaction may attack the radical and attach to the tyrosine amino acid, suppressing the tyrosine-tyrosine formation and decreasing the cross-linking density within the hydrogel network. In the absence of carboxymethyl radical, a covalent cross-link forms between two adjacent exposed tyrosine amino acids as previously reported, promoting the synthesis of the protein-based hydrogel.^, This scheme represents a hypothetical mechanism for the effect of AA on the tyrosine–tyrosine cross-linking mechanism. Further experiments are still needed to determine the exact mechanism. (b) Stress–strain curves of BSA-based hydrogel samples were prepared (i) in the absence of acetic acid and (ii) in the presence of acetic acid 1% (v/v) (175 mM). The hydrogel prepared in a buffer containing AA showed lower stiffness and higher extension than the hydrogel sample prepared in a buffer without AA.

Figure 2

Investigating the effect of AA on the BSA native structure before and after gelation. (a) FTIR spectra of (i) 2 mM BSA solution dissolved in phosphate buffer containing various AA concentrations and in 6 M GuHCl denaturing solution; (ii) 2 mM BSA-based hydrogel samples with different AA concentrations in TRIS and 6 M GuHCl solutions. The region of the amide I band is labeled. (b) The secondary structure of BSA protein pre- and post- gelation determined by the Fourier deconvolution of each amide I band for each sample. The deconvolution of the amide I band of BSA solution and hydrogels showed that BSA protein has three secondary structures, including intramolecular β-sheet, α-helix, and β-turn. The BSA preserved its secondary structure in all solutions, which indicates that AA does not affect its native structure before and after gelation. (c) Summary of the major conformation of the secondary structure of BSA protein pre- and postgelation. The conformation content of the secondary structure was estimated by calculating the area under the curve of each peak through the amide I deconvolution.

Figure 3

Characterizing the effect of AA on the mechanical behavior of BSA-based hydrogels. (a) Quantification of di-tyrosine bonds in BSA-based hydrogels formed with different AA concentrations. (b) Stress–strain curves of 2 mM BSA-based hydrogels with various AA concentrations. (c) Average Young’s modulus and energy dissipation vs AA concentrations as calculated from stress–strain curves. (d) CryoSEM images of BSA-based hydrogel with different AA concentrations: (i) 0% (v/v) (0 mM), (ii) 0.5% (v/v) (87 mM), (iii) 0.75% (v/v) (131 mM), (iv) 1% (v/v) (175 mM), and (v) 1.5% (v/v) (262 mM). (e) Swelling ratio and average pore-size area of the BSA-based hydrogel with different AA concentrations.

Figure 4

Studying the effect of cross-linking density and protein folding transitions on BSA-based hydrogel mechanical behavior. (a–c) Stress–strain curves on BSA-based hydrogels formed in the presence of various AA concentrations immersed in TRIS solution. (d–f) Stress–strain curves of BSA-based hydrogels at different AA concentrations submerged into 6 M GuHCl solution. (g–i) Average Young’s modulus of BSA-based hydrogels immersed into TRIS or 6 M GuHCl solutions as a function of loading rates. Due to the viscoelasticity resulting from the protein domains’ (un)folding mechanics, the hydrogel samples exhibit varying stiffness in response to different loading rates when characterized in TRIS solution. However, when the mechanical behavior of hydrogel samples was investigated in 6 M GuHCl, the Young’s modulus did not change at different loading rates.

Figure 5

Using AA as a di-tyrosine cross-linking inhibitor to manipulate protein-based hydrogel mechanical behavior to mimic biological tissue functional dynamics. (a) Stress–strain curves of pL-8-based hydrogels with various AA concentrations. (b) Stiffness of biological tissues compared to the Young’s modulus of BSA and pL-8-based hydrogels formed in various concentrations of AA (indicated with red and blue markers, respectively).