Spectroscopic and Pulse Radiolysis Studies of Water-Ethanolic Solutions of Albumins: Insight into Serum Albumin Aggregation.

Albumin-based nanoparticles are promising drug delivery systems due to their biocompatibility, biodegradability, and ability to improve targeted drug release. Among various preparation methods, radiation-induced cross-linking in the presence of ethanol has been proposed in the literature as an effective method for producing protein nanoparticles with preserved bioactivity and controlled size. However, the mechanisms by which ethanol radicals contribute to protein aggregation remain insufficiently understood. In this study, we investigate the role of ethanol in the aggregation of albumins to determine whether its presence is necessary or beneficial for nanoparticle formation. Using pulse radiolysis, spectroscopy methods, resonance light scattering (RLS), and near-infrared (NIR) spectroscopy, we examined aqueous ethanol solutions of albumins before and after irradiation. Our results show that ethanol concentrations above 40% (v/v) significantly promote both radiation-induced and spontaneous protein aggregation. Mechanistic analysis indicates that ethanol radicals react with albumin similarly to hydrated electrons, mainly targeting disulfide bridges. This reaction leads to the formation of sulfur-centered radicals and the formation of intermolecular disulfide bonds that stabilize protein nanostructures by excluding the formation of dityrosine bridges, as described in the literature. In contrast, ethanol concentration below 40% does not favor the radiation-induced aggregation compared to the solution containing t-BuOH. These results provide novel insights into the role of organic cosolvents in protein aggregation and contribute to a broader understanding of the mechanisms of formation of albumin-based nanoparticles using ionizing radiation.