(8c) Probing Hydrogel Transport Properties and Dynamic Micro-Structure with Fluorescence Correlation Spectroscopy

Knowledge of how soluble molecules diffuse through materials is important in order to efficiently design devices for controlled protein release, 3D scaffolds for cell growth, and artificial organs. Protein diffusion can be particularly impacted by the material micro-structure and thus this work has an implication for many advanced materials that are multicomponent, demonstrate dynamic phenomena (degradation, swelling) or are patterned. The purpose of this study was to apply Fluorescence Correlation Spectroscopy (FCS), a non-invasive optical technique, to study probe diffusion in cross-linked hydrogels, and then to compare the results to those derived from Fickian bulk diffusion method. The two methods, bulk diffusion and FCS, are complimentary in that they measure phenomena on disparate length-scales. The bulk diffusion method considers only the macro-environment of the gel, while FCS considers the micro-environment of the solute. Further, bulk diffusion studies are lengthy and require a large amount of protein. An average value for bulk diffusion is estimated without taking into account the heterogeneity of the system or possible solute-matrix interactions. FCS can probe very small volumes (sub to femtoliters) of samples containing nanomolar concentrations of fluorescent probes. A local diffusion coefficient is estimated which allows for probing matrix heterogeneity and the sensitivity of the measurement allows for probing protein-matrix and protein-protein interactions all of which are important both for cell growth and protein delivery.

Our hydrogel of choice is poly(ethylene glycol) (PEG), because it is biocompatible, resistant to non-specific protein adsorption, as well as, approved by FDA for biological use. In this study we use PEG vinyl sulfone gels, cross-linked with PEG-dithiol ester cross-linkers.

In this study we found that both techniques, FCS and bulk diffusion studies, yield similar diffusion coefficients. Both methods demonstrated that the diffusivity of solute decreased with increased polymer density and decreased with increased solute size. Further FCS studies were carried out to gain more detailed information on the gel structure and transport properties. The gels were found homogeneous on the micro-scale by probing BSA diffusion at various gel locations. The FCS technique also allowed for studying protein-protein and protein-matrix interactions. We demonstrated that BSA did not aggregate in the PEG gel at concentrations between 0.04% and 4%, while Ig aggregated at concentrations >1%. Further we also found that BSA did not react with the PEG hydrogel during the cross-linking. Finally, gelation and swelling behavior of the hydrogels were tested by measuring the change in BSA diffusivity as a function of time. The study revealed that the gelation occurred on the order of minutes, whereas complete swelling was reached after 2 hours of equilibration in buffer.

In this work, we showed that FCS can yield effective diffusion coefficients comparable to those derived from the bulk diffusion method. However, FCS allowed us to perform in-situ investigations of gel heterogeneity, protein-protein interactions (e.g. aggregation), protein-matrix interactions (e.g. chemical interactions during gelation) and gel dynamics (e.g. swelling behavior). These detailed characterizations of hydrogel structure dynamics and protein diffusivity are essential for the design of improved devices for protein release and cell scaffolds biomaterials for tissue engineering.