Crowding Effects on Insulin Aggregation Using Synthetic and Mixed Crowders

Protein aggregation is implicated in several human diseases and presents an important challenge in the development of stable protein therapeutics. Here, we examine how macromolecular crowding affects insulin amyloid aggregation using polyethylene glycol, Dextran-70, and PVP-10, including binary and ternary mixtures. Aggregation was assessed by Thioflavin-T fluorescence and supported by circular dichroism and atomic force microscopy. PEG 10K showed a biphasic response, with increased ThT intensity at low concentration and markedly reduced signals at higher concentrations, whereas PEG 1, 4, and 6K were largely inhibitory across the tested range. At higher concentration of 100 mg/mL, both Dextran-70 and PVP-10 exhibited reduced ThT intensity, although Dextran-70 showed higher beta-sheet content than PVP-10, as revealed from CD spectroscopic analysis. In mixed crowder systems, Dextran-70 containing binary mixtures produced high ThT signals among the tested binary combinations, while PVP-10-containing mixtures showed lower β-sheet content and short fibrillar structures. Among ternary mixtures, PEG 1K + PEG 10K + PVP-10 produced the lowest aggregation-associated response. Rheology was consistent with a viscosity-linked contribution at higher PEG concentrations, and docking provided supportive, qualitative comparison of crowder-insulin surface contacts. These findings demonstrate that insulin aggregation responses depend on crowder identity, molecular weight, concentration, and mixture composition.