Tuning Bovine Serum Albumin Nanoparticle Properties Through Desolvating Agents and Synthesis Parameters: Implications for Proteolysis and Drug Release

Bovine serum albumin nanoparticles (BSA-NPs) are widely used as model protein nanocarriers, and their physicochemical properties are strongly influenced by synthesis conditions. This study evaluated the effect of four desolvating agents (DAs), namely acetone, ethanol, isopropanol, and acetonitrile, differing in polarity and hydrogen-bonding capacity, combined with glutaraldehyde (GA) concentration, stirring speed, and DA addition rate, across 32 formulations characterized by dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). Ethanol-derived BSA-NPs were most reproducible, with hydrodynamic diameter (Dh) of 99.2 to 124.8 nm, polydispersity index (PDI) of 0.076 to 0.193, and ζ-potential of −20.78 to −23.06 mV, independent of other parameters. Acetone-derived particles were smaller but more sensitive to stirring speed, with PDI up to 0.642. Acetonitrile-derived particles showed pronounced aggregation, with PDI never below 0.404, while isopropanol-derived particles showed the strongest glutaraldehyde-dependent aggregation, with PDI up to 0.964, despite a primary size similar to ethanol. DA identity was the dominant factor governing NP properties, and its selection should precede optimization of the other parameters. DA identity further affected both resistance to trypsin digestion and drug-loading capacity. Particles produced with acetonitrile were the most resistant to proteolysis. In addition, hydrophilic Rhodamine B was better loaded into ethanol-derived carriers, and hydrophobic Nile Red was preferentially loaded into particles made via acetone desolvation.