High-Throughput FRAP Analysis of Solute Diffusion in Hydrogels

Abstract

Increasingly accurate mathematical models have been developed to relate solute and hydrogel properties to solute diffusion coefficients in hydrogels, primarily by comparing solute sizes and hydrogel mesh sizes. Here, we use a standardized, high-throughput method for fluorescence recovery after photobleaching (FRAP) experiments and analysis to characterize the diffusion coefficients of fluorescein, three sizes of FITC-dextran, and three sizes of FITC-conjugated poly­(ethylene glycol) (PEG) through 18 structurally varied poly­(vinyl alcohol) (PVA) hydrogel formulations. Increasing the hydrogel mesh radii increased the diffusivities of all the tested solutes within the hydrogels. While the diffusivity of FITC-dextrans in hydrogels decreased with increasing solute size, the diffusivity of FITC-PEGs increased with increasing solute size, suggesting that a generalized hydrodynamic radius-based model is not universally applicable for solute diffusion in hydrogels. The high-throughput characterization method for solute diffusion in hydrogels described here facilitates precise hydrogel design for biomedical applications.

Hydrogel Mesh Size and Its Impact on Predictions of Mathematical Models of the Solute Diffusion Coefficient

“…In our previous study, M w was measured for each FD obtained, and eq was therefore used to determine r s . Richbourg and Peppas employed 4 and 20 kDa FD and did not measure the M w of these FD but did report the D o values measured . For that data set, the r s calculated from the D o values was used to maintain internal consistency.…”

“…It should be noted that in the original equation the length of the repeating unit was given as 2­(C–C bond length) and so was applicable strictly to hydrogels prepared from vinyl polymers. However, eq has been used in this form to calculate ξ rm for hydrogels made from non-vinyl polymers such as poly­(ethylene glycol). − More recently, this expression of the mesh size has been modified to account for network cross-link functionality and topology as in which f is the cross-link functionality and the prefactor A accounts for the network topology. For tetrafunctional ( f = 4) networks A = , while other prefactors have been provided for networks with other functionalities.…”

“…In assessing the predictive nature of the obstruction and hydrodynamic models (eqs –), mesh size has been taken to be ξ c as calculated via eq or measured by using neutron scattering or as ξ rm calculated via eq . In their analysis of the MSD model, Axpe et al combined data from the literature in which the mesh size was estimated as ξ c and as ξ rm expressed in terms of eq using M c calculated via eq . , More recently, eq for mesh size for a tetrafunctional network has been employed to assess the predictive nature of the MSD model …”

“…To meet this objective, data were taken from the literature for which the necessary values for the various polymer and solute parameters could be obtained. As such, data sets for diffusion of fluorescein dextrans (FD) in hydrogels prepared from methacrylated alginate (MALG) and poly­(vinyl acohol) (PVA) cross-linked with glutaraldehyde as well as water within topologically nearly ideal hydrogel networks prepared from 4-arm poly­(ethylene glycol) (4aPEG) were used. These data cover a range of solute sizes, polymer properties, and network topologies.…”

“…These data cover a range of solute sizes, polymer properties, and network topologies. Hadjiev and Amsden as well as Richbourg and Peppas measured the diffusion coefficient of various FD in hydrogels using fluorescence recovery after photobleaching (FRAP), while Fujiyabu et al measured the diffusion coefficient of water in their hydrogels using pulsed-field gradient spin-echo proton nuclear magnetic resonance spectroscopy (PGSE-NMR) . Both techniques are considered to provide reliable measurements.…”