Toward Tunable Protein-Driven Hydrogel Lens.

Despite the significant progress in protein-based materials, creating a tunable protein-activated hydrogel lens remains an elusive goal. This study leverages the synergistic relationship between protein structural dynamics and polymer hydrogel engineering to introduce a highly transparent protein-polymer actuator. By incorporating bovine serum albumin into polyethyleneglycol diacrylate hydrogels, the authors achieved enhanced light transmittance and conferred actuating capabilities to the hydrogel. Taking advantage of these features, a bilayer protein-driven hydrogel lens that dynamically modifies its focal length in response to pH changes, mimicking the adaptability of the human lens, is fabricated. The lens demonstrates durability and reproducibility, highlighting its potential for repetitive applications. This integration of protein-diverse biochemistry, folding nanomechanics, and polymer engineering opens up new avenues for harnessing the wide range of proteins to potentially propel various fields such as diagnostics, lab-on-chip, and deep-tissue bio-optics, advancing the understanding of incorporating biomaterials in the optical field.

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

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.

Kinetic Method of Producing Pores Inside Protein-Based Biomaterials without Compromising Their Structural Integrity.

Hydrogels made from globular proteins cross-linked covalently into a stable network are becoming an important type of biomaterial, with applications in artificial tissue design and cell culture scaffolds, and represent a promising system to study the mechanical and biochemical unfolding of proteins in crowded environments. Due to the small size of the globular protein domains, typically 2-5 nm, the primary network allows for a limited transfer of protein molecules and prevents the passing of particles and aggregates with dimensions over 100 nm. Here, we demonstrate a method to produce protein materials with micrometer-sized pores and increased permeability. Our approach relies on forming two competing networks: a covalent network made from cross-linked bovine serum albumin (BSA) proteins via a light-activated reaction and a physical network triggered by the aggregation of a polysaccharide, alginate, in the presence of Ca2+ ions. By fine-tuning the reaction times, we produce porous-protein hydrogels that retain the mechanical characteristics of their less-porous counterparts. We further describe a simple model to investigate the kinetic balance between the nucleation of alginate and cross-linking of BSA molecules and find the upper rate of the alginate aggregation reaction driving pore formation. By enabling a more significant permeability for protein-based materials without compromising their mechanical response, our method opens new vistas into studying protein-protein interactions and cell growth and designing novel affinity methods.

Cation-induced shape programming and morphing in protein-based hydrogels.

Smart materials that are capable of memorizing a temporary shape, and morph in response to a stimulus, have the potential to revolutionize medicine and robotics. Here, we introduce an innovative method to program protein hydrogels and to induce shape changes in aqueous solutions at room temperature. We demonstrate our approach using hydrogels made from serum albumin, the most abundant protein in the blood plasma, which are synthesized in a cylindrical or flower shape. These gels are then programmed into a spring or a ring shape, respectively. The programming is performed through a marked change in stiffness (of up to 17-fold), induced by adsorption of Zn2+ or Cu2+ cations. We show that these programmed biomaterials can then morph back into their original shape, as the cations diffuse outside the hydrogel material. The approach demonstrated here represents an innovative strategy to program protein-based hydrogels to behave as actuators.

Chemical unfolding of protein domains induces shape change in programmed protein hydrogels.

Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we use protein-polymer interactions to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as polyethyleneimine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels with tunable wide-range stiffness, from ~10-64 kPa, without affecting the protein mechanics and nanostructure. We use the 6-fold increase in stiffness induced by PEI to program BSA hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. The approach demonstrated here, based on protein engineering and polymer reinforcing, may enable the development and investigation of smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels enables the tethering of extremely low-volume hydrogel samples (< 5 µL) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

Harvesting Low Molecular Weight Biomarkers Using Gold Nanoparticles.

We developed and characterized a platform based on gold (Au) nanoparticles (NPs) coated with poly(acrylic acid) (PAA) for harvesting positively charged, low molecular weight (LMW) proteins. The particles are synthesized using a layer by layer (LbL) procedure: first the gold NPs are coated with positively charged polyethylenimine (PEI) and subsequently with PAA. This simple procedure produces stable PAA-PEI-Au (PPAu) NPs with high selectivity and specificity. PPAu NPs successfully harvested, separated, and detected various LMW proteins and peptides from serum containing a complex mixture of abundant high molecular weight (HMW) proteins, including bovine serum albumin (BSA) and Immunoglobulin G (IgG). In addition, PPAu NPs selectively harvested and separated LMW proteins from serum in the presence of another positively charged competing protein. Furthermore, PPAu NPs successfully harvested a LMW biomarker in a mock diseased state. This system can be applied in various biomedical applications where selective harvesting and identifying of LMW proteins is required. A particularly useful application for this system can be found in early cancer diagnosis as described hereinafter.