Stimuli-responsive smart gels realized via modular protein design

Abstract

Smart gels have a variety of applications, including tissue engineering and controlled drug delivery. Here we present a modular, bottom-up approach that permits the creation of protein-based smart gels with encoded morphology, functionality, and responsiveness to external stimuli. The properties of these gels are encoded by the proteins from which they are synthesized. In particular, the strength and density of the network of intermolecular cross-links are specified by the interactions of the gels' constituent protein modules with their cognate peptide ligands. Thus, these gels exhibit stimuli-responsive assembly and disassembly, dissolving (or gelling) under conditions that weaken (or strengthen) the protein-peptide interaction. We further demonstrate that such gels can encapsulate and release both proteins and small molecules and that their rheological properties are well suited for biomedical applications.

Figure 1. Modularity of TPR proteins allows the design of smartgels with predetermined structures

(A) Ribbon and cartoon representations of the structure of the 3TPR module used as either a spacer or a binding unit in the gel designs (based on PDB 1NA0). Each component 34 amino acid repeat is shown in a different color in the ribbon representation. The thermodynamic stability of both the spacer and binding modules can be varied greatly – from a melting temperature of less than 37° C to over 100° C^,. The specificity and affinity of the binding module can also be manipulated. (b) Ribbon and cartoon representations of the structure of an 18TPR array, side and end on views (based on PDB 2FO7). The 3TPR modules are colored as in 1A. The superhelical rise is 1 nm per repeat, and 8TPR repeats form one turn of the superhelix: The 18TPR array is 18nm long with an outer diameter of 5nm. (C) Cartoon representation of TPR gel formation. The PEG-peptide cross-linker is represented by black lines for the PEG component and red ovals for the peptide. The cartoon is drawn approximately to scale – each arm of the 10kD four-arm star PEG has a contour length of ~ 18nm.

Figure 2. TPR-based smart-gels are self-supporting, soft yet elastic materials

(A) Photograph of a gel cast in the bottom of a microcentrifuge tube. A ruler is shown alongside for scale - 1 tick corresponds to 1mm. (B) Microrheology measurements tracking the Brownian motion of fluorescent probe particles. The mean square displacement is plotted as a function of time. The data for individual gel components is shown as red squares, with a line of slope = 1 drawn for comparison. The data for the gel, 21 days post mixing, is shown as open black squares. (C) Frequency dependence of the elastic modulus (G’ – closed symbols) and the viscous modulus (G’’ – open symbols). Behavior at the onset of gelation (diamonds, 243 h post-mixing) and close to the end of gelation (triangles, 292 h post-mixing) is shown. Defining characteristics of a gel are (i) the value of G’ is independent of frequency and (ii) the value of G’ is larger than G’’. Both these conditions are clearly met by the TPR smart-gels. (D) Iterative strain sweeps, (black, blue, red in order of acquisition: Elastic modulus (G’, solid diamonds) and viscous modulus (G’’, open diamonds). The strain-hardening followed by yielding at 1000% strain is evident. The elastic modulus recovers to its initial value after repeated strain sweeps.

Figure 3. Erosion and content-release by TPR smart-gels

(A) A piece of gel was placed into a solution of 0.01M sodium phosphate pH 7.4, at different ionic strengths, 25 °C and removed and weighed at various times following immersion. The percent gel mass remaining is plotted versus time for the different ionic strength solutions. (B) An aliquot of fluorescent protein (mVFP) or (C) the small molecule rhodamine (MW 422 Da) was entrapped during gelation. The release of entrapped molecule was monitored as a function of time, following the increase in fluorescence of the solution in which the gel was immersed. These measurements were made in DMEM (Dulbecco/Vogt modified Eagle's minimal essential medium), a commonly used tissue culture medium, which has an ionic strength of 166 mM, pH 7.4, at 37°C. Release of the 26 kDa mVFP protein from the gel clearly mirrors gel erosion. Note that there is virtually no release in the 30 mM ionic strength solution (grey circles). (C) Data are shown for 0.01M sodium phosphate pH 7.4, plus 0.01M NaCl (light red) or plus 0.5M NaCl (dark red) at 25oC. Release of rhodamine is far more rapid than the erosion of the gel. The lines through the points in all plots are included as a guide for the eye.