Macromolecular Solute Transport in Supramolecular Hydrogels Spanning Dynamic to Quasi-Static States

Abstract

Hydrogels prepared from supramolecular cross-linking motifs are appealing for use as biomaterials and drug delivery technologies. The inclusion of macromolecules (e.g., protein therapeutics) in these materials is relevant to many of their intended uses. However, the impact of dynamic network cross-linking on macromolecule diffusion must be better understood. Here, hydrogel networks with identical topology but disparate cross-link dynamics are explored. These materials are prepared from cross-linking with host-guest complexes of the cucurbit[7]uril (CB[7]) macrocycle and two guests of different affinity. Rheology confirms differences in bulk material dynamics arising from differences in cross-link thermodynamics. Fluorescence recovery after photobleaching (FRAP) provides insight into macromolecule diffusion as a function of probe molecular weight and hydrogel network dynamics. Together, both rheology and FRAP enable the estimation of the mean network mesh size, which is then related to the solute hydrodynamic diameters to further understand macromolecule diffusion. Interestingly, the thermodynamics of host-guest cross-linking are correlated with a marked deviation from classical diffusion behavior for higher molecular weight probes, yielding solute aggregation in high-affinity networks. These studies offer insights into fundamental macromolecular transport phenomena as they relate to the association dynamics of supramolecular networks. Translation of these materials from in vitro to in vivo is also assessed by bulk release of an encapsulated macromolecule. Contradictory in vitro to in vivo results with inverse relationships in release between the two hydrogels underscores the caution demanded when translating supramolecular biomaterials into application.

Keywords: bioinspired materials; injectable gels; materials chemistry; self-assembly; soft matter.

Figure 1.

(a) Schematics and molecular structures of 8-arm PEG macromers used to form hydrogels, including the monovalent molecular-scale binding affinities of the various motifs explored. (b) Variable frequency dynamic oscillatory rheology at 2% strain displaying the disparate dynamics of CB[7]–guest hydrogels arising from the affinity of the crosslinking motif used. (c) Stress relaxation by oscillatory rheology at 2% strain of more dynamic Xyl-based hydrogels and (d) less dynamic Ada-based hydrogels at different temperatures. (e) Transition state theory performed via Eyring analysis of stress relaxation data at relevant temperatures, wherein k_off of each sample is the inverse of τ_R at G(t)/G₀ = e⁻¹, R is the gas constant, k_B is the Boltzmann constant, and h is the Planck constant. (f) Energy diagram depicting the thermodynamic parameters arising from Eyring analysis on the transition state for dynamic crosslinks. (g) Table of thermodynamic parameters governing conversion from the bound to transition state (ΔG^‡) for Xyl and Ada hydrogels obtained from Eyring analysis.

Figure 2.

(a) Fluorescence recovery after photobleaching (FRAP) data for normalized ROI fluorescence of free (PBS) vs. Xyl-encapsulated FITC-dextran probes, showing an average of 3 experiments per trace. (b) Select representative images of hydrogels immediately following photobleaching, t₀, and at the study endpoint, t_f, with the scale bar shown applicable to all images. (c) Table displaying (left) applicable models of calculating mean mesh size of the hydrogel and (right) calculated hydrodynamic diameters of FITC-dextran macromolecule solutes. Models adapted according to references listed in superscript (refs ^–).

Figure 3.

(a) Fluorescence recovery after photobleaching (FRAP) data averages (n=3) after 16 d equilibration for the 70 and 250 kDa probe encapsulated in Xyl and Ada hydrogels. (b) Select representative images displaying time-dependent hydrogel equilibration with aggregation of high molecular weight solutes by Ada hydrogel networks. (c) Analysis of diffusivity ratio versus the solute hydrodynamic diameter scaled to network pore size, exhibiting local maxima in the diffusivity ratio for solutes with dimensions in excess of the pore size.

Figure 4.

(a) Bulk release studies of a 70 kDa FITC-dextran probe from hydrogels in vitro (n=3 gels/group); data reproduced with permission (ref ). (b) In vivo release of 70 kDa Cy5-dextran solute from subcutaneously injected hydrogels, quantified as the percent reduction in signal at the depot site using in vivo imaging (n=5 mice/group). (c) Representative in vivo fluorescent images of Cy5-dextran solute encapsulated within subcutaneous Xyl and Ada hydrogels, overlaid onto mouse photographs.