Modification of Bovine Serum Albumin (bsa) with A Maleimido-Functionalized, 8-Arm Polyethylene Glycol Backbone (Mal-Peg(8)) As A Drug Delivery System

Due to its ability to transport hydrophobic materials through the bloodstream, human serum albumin (HSA) has become an intriguing drug delivery system with several formulations currently available. Key residues in the two main drug binding sites, DS1 and DS2, are conserved in BSA, providing a cheaper, more readily available alternative to HSA. Additionally, BSA may be selectively modified at its lone free sulfhydryl (Cys34), requiring no additional modifications. A multi‐BSA PEG 8 complex (PEG 8 ‐BSA x ) would reduce the effect on osmotic pressure, avoid immunogenic response, and decrease the risk of drug toxicity while maintaining ligand binding capabilities. Several BSA molecules were successfully conjugated onto Mal‐PEG 8 in a one‐pot reaction, requiring only BSA, Mal‐PEG 8 and MOPS/NaCl buffer at pH 7.2. High molecular weight species (HMW) of PEG 8 ‐BSA x were separated from the reaction mixture via Size Exclusion Chromatography (SEC). Helicity was maintained following PEGylation, as determined by Circular Dichroism (CD). Using a 4–15% gradient SDS‐PAGE gel, three bands of HMW material were visualized. Two bands corresponding to molecular weights of PEG 8 complexed with two and three BSA molecules were seen in the separation zone. A heavy band was maintained in the stacking zone, indicating the presence of at least one other species of PEG 8 ‐BSA x . Thermal stability of PEGylated‐BSA complexes was evaluated using UV‐VIS and CD. Fluorimetric assays were performed with Sulfamethoxazole (SMZ), Phenytoin (PHT) and 8‐Anilino‐1‐naphthalenesulfonic acid (ANS). SMZ has shown preference for DS1 while PHT prefers DS2. ANS is a fluorescent probe commonly used in binding studies. Dissociation constants (K d ) and the number of binding sites were consistent between unmodified BSA and PEG 8 ‐BSA x . These results suggest that PEG 8 ‐BSA x is a viable delivery system for water‐insoluble pharmaceuticals. Support or Funding Information Loyola University Chicago