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In situ measurement of thermodynamic partitioning in open hydrogels.

Thermodynamic partitioning dictates solute loading and release from a hydrogel. Design of drug delivery vehicles, cell and tissue matrices, and immunoassay scaffolds that utilize hydrogel materials is informed by an understanding of the thermodynamic partitioning properties of those hydrogels. We develop aberration-compensated laser scanning confocal microscopy (AC-LSCM), a technique that can be applied to all fluorescence microscope-based equilibrium partition coefficient measurements where the fluorescence is uniformly distributed in the reference material (e.g., many solutes in thermodynamic equilibrium). In this paper, we use AC-LSCM to measure the spatially-resolved in situ equilibrium partition coefficients of various fluorescently-labeled solutes in single layer and multi-layer open hydrogels. In considering a dynamic material, we scrutinize solute interactions with a UV photoactive polyacrylamide gel that incorporates a benzophenone methacrylamide backbone. We observed strong agreement with an adjusted version of Ogston's ideal size-exclusion model for spatially-resolved in situ equilibrium partition coefficients across a wide range of polyacrylamide hydrogel densities (R2 > 0.98). Partition coefficients of solutes differing in hydrodynamic radius were consistent with size-based theory in the photoactive hydrogels, but exceed those in unmodified polyacrylamide gels. This observation suggests deviation from the size-exclusion model and a shift in the thermodynamic equilibrium state of the solutes towards the gel phase. AC-LSCM also resolves differential partitioning behavior of model solute in two-layer gels, providing insight into the transport phenomena governing partitioning in multilaminate gel structures. Furthermore, AC-LSCM identifies and quantifies depth-dependent axial aberrations that could confound quantitation in multilaminate gel structures thicker than 100 μm, highlighting the need for the "aberration-compensated" aspect of AC-LSCM.