Influence of formulation parameters on the physicochemical properties and cellular uptake of human serum albumin nanocarriers.

, was developed, in which GSH transiently reduces intramolecular disulfide bonds and subsequent oxidation forms stabilising intermolecular disulfides. GA-crosslinked and GSH-mediated disulfide-crosslinked HSA nanoparticles were compared in terms of colloidal and serum stability, cytotoxicity and cellular uptake in 4T1 cells, and curcumin was encapsulated as a model hydrophobic drug. Both strategies produced physically stable nanoparticles, curcumin-loaded nanoparticles with good drug loading. In carrier-focused evaluations, GA-crosslinked HSA nanoparticles exhibited smaller sizes, negative charge, and high biocompatibility, with sustained cellular uptake and minimal serum-induced aggregation. In contrast, GSH-based HSA NPs were strongly cationic, exhibited faster initial cellular uptake, and underwent pronounced serum-mediated charge reversal, while showing comparable carrier cytocompatibility to GA-crosslinked NPs under the tested conditions. Using curcumin as a model hydrophobic payload, both formulations achieved effective drug encapsulation (GA-crosslinked 17.89% and GSH-based 14.6%). While GA-crosslinked CCM-HSA NPs showed a smaller particle size, lower PdI, and demonstrated enhanced antiproliferative activity compared with free curcumin. These results provide design guidelines for engineering albumin-based nanocarriers with tailored stability and drug delivery performance.