Diffusion of Affibody molecules in extracellular matrix mimetic hydrogels and the effect of albumin binding

Affibody molecules are protein ligands, that due to their small size (6-19 kDa) and high target affinity exhibit favourable properties for tumour uptake valuable in diagnostic imaging and therapeutic applications. Fusion to a high affinity albumin binding domain (ABD) has been shown to improve circulatory half-life and biodistribution. However, the effect of molecular design is not obvious to predict and in vitro methods to evaluate their transport properties in physiologically relevant environment are needed. In this work we investigated the diffusivities (D) of Affibody molecules, with systematically varied molecular design, in solution and within extracellular matrix mimetic hydrogels composed of either agarose or collagen and hyaluronic acid (COL-HA) using fluorescence recovery after photobleaching. Furthermore, the effect of presence of human serum albumin (HSA) was evaluated. The correlation between D of the tested Affibody molecules in solution and their molecular weight (Mw) was weak, indicating that propensity to form reversible oligomers and the size of the oligomers are more important for their diffusion properties than Mw of the monomer. Positively charged Affibody molecules were enriched in polymer-rich domains of the COL-HA gel accompanied by a decrease in D as a result of electrostatic interactions. Binding to HSA by Affibody molecules containing an ABD was evident as a decrease of D when HSA was present. In COL-HA gels HSA-binding reduced the effect of electrostatic interactions effectively facilitating the transport of those compounds. In conclusion, molecular design especially inclusion of an ABD affected the transport properties of the tested Affibody molecules.