Abstract
Biomedical applications of hydrogels are rapidly increasing due to their special properties including high water absorption capacity, viscoelasticity, swelling capability, and responsiveness to environmental physical or chemical stimuli. Two major biomedical applications of hydrogels include drug delivery and tissue engineering. Knowledge of the diffusion or degree of penetration of particles in hydrogels is key to designing specific functions such as controlled release in drug delivery systems and nutrient accessibility in tissue engineering platforms. Experimental determination of solute penetration and diffusivity can be challenging depending on several factors such as the hydrogelation process, the hydrogel characteristics, and the type of diffusing particle. We describe here a simple method that uses fluorescence intensity measurements obtained with a microplate reader to determine the concentration of diffusing particles at different penetration distances in soft hydrogels. We have analyzed the diffusion behavior of three fluorescent particles of different chemical natures and various molecular weights (fluorescein and the proteins mNeonGreen and fluorophore-labeled bovine serum albumin) in agarose hydrogels of low percentages (0.05-0.2%). The diffusion coefficients were obtained by fitting the experimental data to a one-dimensional diffusion model. A good agreement between our results and previously reported diffusion coefficients of the studied particles validates our method. We demonstrate the method's capability to adapt to hydrogels of different stiffnesses and solutes of various sizes and characteristics. In addition, the combination of hydrogel sectioning with multiple simultaneous measurements in a microplate reader shows the simplicity of the experimental procedure. Finally, our data indicate the method's sensitivity to variations in diffusion conditions, which is highly relevant to studying interactions between solutes and hydrogels designed for controlled release by determining differences in penetration distances.