Diffusion of macromolecules in extracellular matrix mimetic hydrogels – effect of size and charge

Subcutaneous (SC) injection is the primary alternative to oral administration for therapeutic proteins and peptides. However, bioavailability and absorption rate are often variable and difficult to predict. Therefore, there is a need for new biorelevant and predictive SC in vitro methods. In this study we systematically investigate the effect of size and charge of a macromolecule on its partitioning and diffusion within extracellular matrix (ECM) mimetic hydrogels in order to gain insight on interactions with the components of the ECM affecting the absorption of a drug after SC injection. Hydrogels consisting of either agarose, cross-linked collagen and hyaluronic acid (HA) or cross-linked HA, were made and equilibrated in solutions of FITC-dextrans of varying sizes (4 to 150 kDa) and model peptides of varying net charge (+2 to +9). Partitioning and diffusion coefficients within gel and solution were determined using confocal laser scanning microscopy and fluorescence recovery after photo bleaching (FRAP), and compared to theoretical models. Generally, the partitioning and diffusivities within the gels decreased with increasing molecular weight, which was in good agreement with models describing the effect of obstruction of the gel network corrected for heterogeneity in the gel structure. The cationic peptides were enriched in the oppositely charged gels and their diffusivities decreased with increasing peptide charge. The experimental results were in semi quantitative agreement with an electrostatic model presented in this work.