Denaturation-renaturation strategy for fabrication of biocompatible BSA-based nanoparticles with dual interaction capability for efficient intracellular delivery

The development of biocompatible and efficient nanocarriers remains a critical challenge in drug delivery. In this study, we introduce a denaturation-assisted strategy for the preparation of bovine serum albumin (dBSA)-based nanoparticles (NPs) capable of encapsulating both hydrophilic (Rhodamine B) and hydrophobic (8-anilino-1naphthalenesulfonic acid, ANS) model compounds. Controlled protein denaturation prior to complexation, followed by renaturation, enabled the formation of stable NPs with high encapsulation efficiencies (64.7 % for RdB and 69.6 % for ANS) and minimal alterations in particle size and morphology. Structural characterization by ATR-FTIR and circular dichroism (CD) spectroscopy, together with fluorescence analyses, confirmed the successful incorporation of both model molecules into the dBSA matrix. Denaturation-induced conformational changes and partial structural recovery after renaturation were confirmed by CD spectroscopy. In vitro cytotoxicity assays revealed high in vitro biocompatibility rather than complete biosafety, maintaining over 95 % cell viability in cancer (MCF-7, HeLa) and normal (HEK 293) cell lines after 24 h exposure. Furthermore, flow cytometry and confocal microscopy analyses demonstrated efficient cellular internalization, with NPs predominantly localized in the cytoplasmic region. Hydrophobic ANS-loaded NPs exhibited significantly higher cellular uptake compared to RdB-loaded systems, indicating a membrane-affinity-driven internalization mechanism. These findings establish a simple and versatile denaturation-renaturation approach for fabricating BSA-based nanocarriers as a promising platform for intracellular delivery of bioactive compounds.