Efficient Encapsulation of Insulin by Giant Macrocycle as a Powerful Approach to the Inhibition of Its Fibrillation

Abstract

Diabetes is a lifelong metabolic disease that requires frequent subcutaneous injections of insulin. However, free insulin is prone to the formation of immunogenic fibrillar aggregates in physiologic conditions which puzzles its biomedical applications. Here an inhibition approach for insulin fibrils was developed through entire encapsulation by a giant macrocyclic inhibitor agent. Negatively charged water-soluble Pentaphen[3]arene sulfate (PP[3]AS), bearing 15 benzenes on its skeleton, was designed and synthesized. In vitro and in vivo safety tests preliminary demonstrated that PP[3]AS had excellent biocompatibility. PP[3]AS could not only effectively inhibit the formation of amyloid, but also disaggregate intractable mature insulin fibrils. This macrocyclic inhibitor exhibited effective host-guest complexation toward insulin at C-terminal 11-mer peptide sequence of B chain with association constants of (5.69 ± 0.50) × 10⁶ M^-1. Such complexation behavior was distinctive to traditional macrocycles, which could only recognize the amino acid residues from the side due to their limited cavity sizes. Control experiments also proved that smaller cucurbit[7]uril and carboxylatopillar[5]arene could not prevent insulin from fibrillation under the same test conditions. Notably, co-administrated with equimolar PP[3]AS could keep normoglycemic for at least 300 min in streptozotocin-induced diabetic model mice, whereas mice received free insulin became hyperglycemic again within ~150 min