Oligomer-Responsive ThT Derivative for Early Deterioration Evaluation of Insulin Injections

Abstract Insulin, an essential drug for diabetes, is prone to be aggregated due to its inherent amyloidogenicity. Particularly for insulin injections, improper handling or storage (e.g., vibration and heat) can lead to drug deterioration, namely, formation of early oligomers that are highly toxic. Compared to the cloudy amyloid fibrils, the early oligomers are highly soluble and less viscous, thus posing a great challenge for their detection. Thioflavin T (ThT) is the “gold-standard” fluorescent probe for protein aggregation and insulin fibrils but fails in recognition of the early soluble oligomers. Herein, a ThT derivative with a stronger donor unit and a larger molecular volume, namely, ThTPD, was designed with improved viscosity sensitivity and oligomer-binding affinity. In the model of insulin aggregation, ThTPD exhibited an obvious fluorescence response in the very early oligomer stage (∼30 s), whereas ThT only responded to the protofibrils and fibrils (∼30 min). For circular dichroism (CD) spectroscopy that is typically explored for protein aggregation study, a signal emerged until the protofibril formation stage (∼10 min). Real-sample analysis with commercial insulin injections demonstrated that ThTPD could achieve early deterioration evaluation, making it a promising, convenient, and rapid probe for the early deterioration monitoring of insulin injections.