Amyloid-Based Albumin Hydrogels

Abstract

Amyloid fibrils may serve as building blocks for the preparation of novel hydrogel materials from abundant, low-cost, and biocompatible polypeptides. This work presents the formation of physically cross-linked, self-healing hydrogels based on bovine serum albumin at room temperature through a straightforward disulfide reduction step induced by tris (2-carboxyethyl) phosphine hydrochloride. The structure and surface charge of the amyloid-like fibrils is determined by the pH of the solution during self-assembly, giving rise to hydrogels with distinct physicochemical properties. The hydrogel surface can be readily functionalized with the extracellular matrix protein fibronectin and supports cell adhesion, spreading, and long-term culture. This study offers a simple, versatile, and inexpensive method to prepare amyloid-based albumin hydrogels with potential applications in the biomedical field.

Keywords: antifouling; cell mechanosensing; fibrillar hydrogels; self-assembly; sustainable materials.

Figure 1

TCEP‐pH phase diagrams for BSA gelation at room temperature. A) Photographs of BSA solutions exhibiting four different conditions. From left to right: no gelation of solution, transparent gel, phase separation, and opaque gel. The distinction between opaque and transparent gels was made based on the ability to clearly see an object through the gel. B,C) Phase diagrams for (B) 5% and 10% (C) BSA solutions with pH on the y‐axis and TCEP concentration on the x‐axis constructed on the base of conditions presented in (A). Dotted line at pH 4.7 corresponds to the isoelectric point of BSA. Red and green dashed arrows are a guide to the eye to aid the discussion in the text.

Figure 2

Rheological characterization of 5% BSA hydrogels as a function of pH and TCEP. A) Gelation kinetics were monitored using oscillatory rheology at 1% strain and angular frequency of 1 Hz. The storage (Gʹ) and loss (Gʺ) moduli were measured every minute following mixing of TCEP with BSA (t = 0). The storage modulus started to rapidly increase following a lag phase (inset). B,C) Lag phase and plateau storage modulus, determined 24 h after mixing TCEP (40 mm) with BSA (5%) as a function of solution pH. At pH values close to the isoelectric point, gelation occurred rapidly, but the resulting gels were not as stiff, compared to gels prepared at acidic pH (3.6) or physiological pH (7.4). D,E). Lag phase and plateau storage modulus as a function of TCEP concentration used for 5% BSA gels at pH 3.6. Increasing the TCEP concentration led to faster gelation. The plateau storage modulus increased when increasing TCEP concentration from 20 to 40 mm, but remained similar for 80 mm TCEP. Each data point in (B–E) corresponds to an independent experiment (N = 2 or 3).

Figure 3

TCEP‐induced denaturation of BSA leads to beta‐sheet forming amyloid‐like structures. A) Kinetics of ThT fluorescence increase after addition of 20 mm TCEP to a 2% BSA solution (t = 0) at different pH values, and negative control (2% BSA without ThT). The mean values and standard deviation from three independent experiments are presented. B) ThT fluorescence increase as a function of time after mixing 2% BSA with TCEP to reach indicated TCEP concentrations at pH 3.6. Data were normalized and presented as fold‐increase compared to the initial measurement immediately before TCEP addition. The lines correspond to best fits for the equation y = y _M − y ₀ e ^−kx. C) The values of ThT fluorescence 24 h after mixing TCEP with a 2% BSA solution at pH 3.6, as a function of the final TCEP concentration. Data from one out of three independent experiments are presented. D) Normalized FTIR spectra in the amide I′ region of BSA solutions in D₂O, in absence or presence of TCEP at pD 3.6 or 7.4. The shift of the maximum band from 1648 to 1620 cm⁻¹ for pD 3.6 and 1630 cm⁻¹ for pD 7.4 indicated the shift toward a higher beta‐sheet content. The arrow points to a shoulder peak at 1680 cm⁻¹, indicative of antiparallel β‐sheets. E) Circular dichroism spectra of a control aqueous BSA solution and BSA solutions incubated with TCEP at different pH values. The BSA control exhibits two peaks at 208 and 225 nm, characteristic of a protein rich in alpha helical structure, while TCEP‐induced denaturation of BSA results in the attenuation of these two peaks and the emergence of one peak centered ≈ 215 nm, typical of beta‐sheet structures. The secondary structure prediction of helical, β‐strand, and irregular elements shown in the inset was calculated using the web‐based server tool CAPITO. F) Zeta potential measurements of 2% BSA solutions treated with 16 mm TCEP for 24 h at room temperature as a function of the solution pH. Each data point corresponds to an independent experiment (N = 3 experiments).

Figure 4

BSA amyloid‐like fibril structure differs depending on the pH used for their preparation. A) Tapping mode AFM images under ambient conditions of BSA amyloid‐like fibrils deposited on freshly‐cleaved mica. A 2% BSA solution prepared with 20 mm TCEP at pH 7.4 was pipetted on the mica and then rinsed with water. Fibrils were observed clustered at regions of the mica surface. A height profile corresponding to the dashed white line is shown in the left, and the average height noted (mean and standard deviation from 50 measurements from two independent experiments). B) Tapping mode AFM images under ambient conditions of BSA amyloid‐like fibrils deposited on APTES‐treated mica. In this case, the BSA/TCEP mixture was adjusted to pH 3.6. The surface was homogeneously covered with short fibrils that were roughly twice as high compared to the ones prepared at basic conditions.

Figure 5

Rheological properties of BSA amyloid‐based hydrogels depend on the pH used during gelation. A) Storage (Gʹ) and loss (Gʺ) moduli for 5% hydrogels prepared with 40 mm TCEP as a function of angular frequency. The frequency dependence of Gʹ for gels prepared at pH 7.4 was slightly larger for those prepared at pH 3.6. Data from three different batches and standard deviation are presented. Red lines represent log(Y) = A + B × log(X). B) Loss tangent (tanδ = Gʺ/Gʹ) as a function of angular frequency, calculated from data presented in (A). C) Storage modulus for 5% hydrogels prepared with 40 mm TCEP as a function of strain. Gels prepared at pH 7.4 showed strain softening above a strain of ≈ 5%, whereas gels prepared at pH 3.6 strain stiffening, before breaking at 30%. One of three independent experiments is shown. Inset shows magnified the region where strain stiffening is apparent. D) Stress relaxation (normalized to maximum stress) after rapid application of 10% strain for 5% hydrogels prepared with 40 mm TCEP. The mean of three independent experiments is presented; error bars (SD) are too small to be visible. Hydrogels prepared at pH 7.4 exhibited faster stress relaxation compared to those prepared at pH 3.6.

Figure 6

Swelling behavior of BSA hydrogels depends on the pH used during preparation. A) Swelling of 5% BSA hydrogels (250 µL), prepared with 40 mm TCEP at pH 3.6 or pH 7.4, in Milli‐Q water, PBS 10 or 150 mm NaCl over time. Mean and standard deviations from three independent experiments are presented. B) The dissolution time of 5% BSA hydrogels (250 µL), prepared with 40 mm TCEP at pH 3.6 or pH 7.4, in presence of 3 or 6 m GuHCl in PBS. Each data point corresponds to an independent experiment (N = 3). Data were compared using unpaired t‐tests with Welch's correction. C) Self‐healing demonstration for a 5% BSA hydrogel prepared at pH 3.6 with 40 mm TCEP. After 24 h, the hydrogel was damaged using a syringe needle and its recovery was monitored over time. D) Two 10% BSA hydrogels, prepared at pH 3.6 with 80 mm TCEP, were cut into pieces and then placed in a Teflon mold overnight. The hydrogel pieces were joined together and formed a hydrogel that could resist force application by tweezer pulling.

Figure 7

BSA hydrogels allow for fibronectin adsorption and subsequent cell culture. A) Water contact angle measurements for 5% BSA hydrogels prepared with 40 mm TCEP at pH 3.6, 7.4 or 9.0. There was no difference between using Milli‐Q water or PBS for preparing the droplet. The contact angles for 3 independent experiments are presented on top and images of aqueous droplets on the hydrogels from one of the experiments on the bottom. B) The coating efficiency of adsorbed Fn on 5% BSA hydrogels prepared at pH 3.6 or 7.4 was estimated using a modified ELISA assay and the monoclonal anti‐Fn antibody P1H11, which recognizes the Fn central cell binding domain. A higher amount of Fn was adsorbed on hydrogels prepared at pH 7.4. The average value from 2 independent experiments is presented. Lines correspond to fits of the equation y = A(1 − e ^−kx). C) Confocal z‐stack fluorescence microscopy images of fluorescent Fn adsorbed on 5% BSA hydrogels prepared at pH 3.6 or 7.4. Fn was adsorbed homogeneously over the hydrogel surface and did not penetrate into the hydrogel interior. D) Phase contrast images of live U2OS cells 24 h after seeding on 5% BSA hydrogels prepared with 40 mm TCEP at indicated pH values, and tissue culture polystyrene (TCPS) as control. Cells adhered and spread only when hydrogels were coated with Fn. Scale bars: 100 µm. E) Epifluorescence microscopy images of live U2OS cells stained with calcein (green) and dead U2OS cells stained with ethidium homodimer (red), 20 h after cell seeding. Cells were seeded on Fn‐coated or uncoated hydrogels prepared at indicated pH or Fn‐coated glass as a control. F) Surface concentration of live U2OS cells on different substrates 20 h post‐seeding. G) Viability of U2OS cells on different substrates 20 h post‐seeding was calculated as the ratio of live to total cells. H) Confocal microscopy images of fixed U2OS cells seeded for 5 h on Fn‐coated glass or Fn‐coated 5% BSA hydrogels prepared with 40 mm TCEP at pH 3.6 or 7.4. The cells were immunostained against pY, which stains adhesion clusters, and stained with Phalloidin‐TRITC (F‐actin) and DAPI (nucleus; DNA). Focal adhesions and stress fibers were evident inside cells on both hydrogels. Data in (F) and (G) were analyzed using a Brown‐Forsythe and Welch ANOVA statistical test, comparing all data sets with each other. Only p values from statistically significant comparisons (p < 0.05) are plotted. Scale bars: 20 µm.