Deciphering the Role of Sugar Osmolytes in Free and Nano forms to Mitigate Protein Aggregation: Insights from Biophysical and Microscopic Studies

The native conformation of a protein is crucial for performing its function, which becomes limited due to the propensity of protein to aggregate under sub-optimal conditions. To keep protein in its compact native structure, osmolytes have always been a foremost choice but are typically effective at higher concentrations. There is a need to refine strategies that harness the protective properties of osmolytes even at lower concentrations against protein aggregation. Therefore, we aimed to provide a comparative account of the anti-aggregation potency of sugar osmolytes (glucose and trehalose) and their nano-counterparts (glucose and trehalose nanoparticles), taking catalase as a model protein. For this study, numerous biophysical techniques, microscopic visualizations and in vivo cytotoxicity assays have been conducted for comprehensive evaluation. Nanoparticles exhibited in vivo compatibility, as MTT assay showed negligible cytotoxicity in HEK9 cell lines. The turbidity measurements and light scattering studies (DLS and RLS) displayed that the nanoparticles showed better efficacy compared to the free osmolytes for alleviating aggregation. These findings were further validated by extrinsic fluorescence dye assays (ANS and ThT), confocal and TEM imaging. Circular dichroism (CD) spectra showed the mitigation of perturbation in secondary structures in the presence of these protectants. Potassium iodide (KI) and acrylamide quenching studies suggested dynamic quenching behavior and a marked decrease in Ksv with nanosugars. Henceforth, this study demonstrated that sugar nanoparticles can be used as anti-aggregating agents at a lesser concentration than free sugars.