Serum-PEG and BSA-PEG hydrogels as advanced platforms for evaluating plasma protein binding

The binding of bioactive compounds to proteins is critical for their availability and ADME/Tox profile. Specifically, binding to serum proteins affects both the distribution and elimination of drugs, while permeation through protein-enriched matrices, such as skin, is also influenced by protein interactions. Although several methods exist to evaluate ligand-protein binding, they often fail to replicate the high protein concentrations and molecular crowding conditions found in vivo . In this study, we investigate the use of protein-PEG hydrogels with low crosslinking density as 3D matrices to quantify ligand-protein affinity. Two types of hydrogels were developed: one using bovine serum albumin (BSA) and a more physiologically relevant one using serum. BSA was chosen as a model protein due to its similarity to human serum albumin. The hydrogels were characterized for swelling, stability, mechanical properties, and porosity, and the structural integrity of BSA within the hydrogel was confirmed using circular dichroism, 1 H NMR and fluorescence spectroscopy. To assess protein functionality, we evaluated the binding affinity of various ligands, including a homologous series of fluorescent amphiphiles with different hydrophobicity (NBD-Cn, where n = 4, 6, and 8), two pesticides (malathion and chlorpyrifos), and six pharmaceutical drugs (acetaminophen, chlorpromazine, diclofenac, labetalol, salicylic acid, and verapamil). Our results demonstrated that the structural and functional properties of BSA remained intact within the hydrogel, and the large mesh size allowed for rapid and selective ligand binding. A comparison of BSA and serum hydrogels confirmed the major role of serum albumin in ligand binding, while highlighting some differences between cationic and anionic ligands. Altogether, these hydrogels offer an effective and reliable 3D platform for the fast and accurate evaluation of plasma protein binding of drugs and other bioactive compounds. • Serum and BSA-PEG hydrogels with high porosity for fast ligand-protein binding. • Protein structure and binding ability remain intact within the hydrogels. • Rapid equilibration and minimal protein leaching ensure reliable assays. • Serum-PEG hydrogels enable drug-binding evaluation under physiological conditions.