Characterization of resilin-like proteins with tunable mechanical properties

Abstract

Resilin is an elastomeric protein abundant in insect cuticle. Its exceptional properties, which include high resilience and efficient energy storage, motivate its potential use in tissue engineering and drug delivery applications. Our lab has previously developed recombinant proteins based on the resilin-like sequence derived from Anopheles gambiae and demonstrated their promise as a scaffold for cartilage and vascular engineering. In this work, we describe a more thorough investigation of the physical properties of crosslinked resilin-like hydrogels. The resilin-like proteins rapidly form crosslinked hydrogels in physiological conditions. We also show that the mechanical properties of these resilin-like hydrogels can be modulated simply by varying the protein concentration or the stoichiometric ratio of crosslinker to crosslinking sites. Crosslinked resilin-like hydrogels were hydrophilic and had a high water content when swollen. In addition, these hydrogels exhibited moderate resilience values, which were comparable to those of common synthetic rubbers. Cryo-scanning electron microscopy showed that the crosslinked resilin-like hydrogels at 16 wt% featured a honeycomb-like structure. These studies thus demonstrate the potential to use recombinant resilin-like proteins in a wide variety of applications such as tissue engineering and drug delivery due to their tunable physical properties.

Keywords: Biomaterials; Hydrogels; Protein engineering; Resilience; Tissue engineering.

Figure 1

RZ10-RGD protein design and successful production. (A) The design scheme and protein sequence of recombinant RZ10-RGD. There are a total of 10 resilin repeats (gray rectangles) with one lysine residue (black line) introduced into every two repeats. Lysines serve as crosslinking sites to create a crosslinked RZ10-RGD network. Near the C-terminus there is a cell-binding domain (RGD) to promote cell interactions. (B) SDS-PAGE gel showed successful purification of RZ10-RGD (1 mg/mL in Milli-Q water). The expected molecular weight of RZ10-RGD is 18.3 kDa, and the purity of RZ10-RGD was calculated to be >95%.

Figure 2

(A, B) Swelling ratio and (C, D) water content of RZ10-RGD hydrogels. (A,C) Welch’s ANOVA coupled with a Games-Howell post hoc analysis were performed for hydrogels with a stoichiometric crosslinking ratio of 5× and different protein concentrations. (B,D) ANOVA and Tukey’s honestly significant difference post hoc tests were performed for 12 wt% hydrogels with various crosslinking ratios. Letters indicate groups that are not significantly different from each other (p > 0.05).

Figure 3

Rheological properties of 8 wt% and 14 wt% crosslinked RZ10-RGD. (A) Dynamic oscillatory time sweeps were used to monitor gelation of crosslinked RZ10-RGD. Both moduli reached equilibrium after 30 min with G’ >> G’’, indicating formation of steady-state gels. (B) Graphing the first 10 min of gelation revealed that fast gelation occurred for both 8 wt% and 14 wt% hydrogels. Gelation occurs when the storage modulus (G’) crosses over the loss modulus (G’’). (C) Oscillatory frequency sweeps and (D) oscillatory strain sweeps showed that for both 8 wt% and 14 wt% RZ10-RGD hydrogels, G’ was not a function of frequency or strain within the frequency range of 0.1 – 10 rad/s and the strain range of 0.1 – 10%.

Figure 4

Rheological properties of crosslinked RZ10-RGD with varying protein concentrations and stoichiometric crosslinking ratios. The shear moduli of crosslinked RZ10-RGD can be modulated from 75 Pa to 22 kPa by increasing the protein concentration and the stoichiometric crosslinking ratio. (A) Welch’s ANOVA coupled with a Games-Howell post hoc analysis were performed for hydrogels with a stoichiometric crosslinking ratio of 5× and different protein concentrations. (B) ANOVA and Tukey’s honestly significant difference post hoc tests were performed for 12 wt% hydrogels with various stoichiometric crosslinking ratios. Letters indicate groups that are not significantly different from each other (p > 0.05).

Figure 5

Cyclic compression tests on hydrated 16 wt% RZ10-RGD hydrogels at a stoichiometric crosslinking ratio of 5×. Five strain loading and unloading cycles were performed up to 10% (black) and 20% (gray) strain. Resilience was calculated as the ratio of the area under the unloading curve to the area under the loading curve at each cycle.

Figure 6

Cryo-SEM images of (A) 8 wt% and (B) 14 wt% RZ10-RGD hydrogels. Hydrogels at 8 wt% showed rounded protein particles with little connectivity. On the other hand, 14 wt% hydrogels showed well-connected networks. Scale bar represents 10 μm in images on the left and 5 μm in images on the right.

Scheme 1

Crosslinking scheme in which the primary amine groups of RZ10-RGD react with the hydroxyl groups of THP. The reaction happens under physiological conditions and is biocompatible. Crosslinked RZ10-RGD is opaque and forms a free-standing 3D hydrogel.