Media-dependent morphology of supramolecular aggregates of β-cyclodextrin-grafted chitosan and insulin through multivalent interactions

A supramolecular protein-binding system based on multivalent interactions was investigated using β-cyclodextrin-grafted chitosan (BCC) and insulin. 1H NMR and fluorescence analyses revealed that BCC binds to insulin through electrostatic and host–guest interactions. The binding constant KBCC for the host–guest interaction between cyclodextrin (CyD) residues in BCC and Tyr and Phe residues in insulin was 478.7 M−1 in acetate buffer at pH 3.6, which was ca. 3-fold greater than the Kchi attributed to electrostatic interactions between chitosan and the Tyr residues of insulin. Furthermore, KBCC was ∼10 times greater than that of β-CyD (KCyD), suggesting that multivalent interactions composed of electrostatic and host–guest interactions strongly enhance the host–guest interaction, similar to enzymes and antibodies in living systems. Enhanced host–guest interactions resulted in effective insulin binding over a wide pH range (3.6–7.4) as well as stabilization against digestive enzymes. BCC and insulin formed supramolecular aggregates with significantly different morphologies depending on the buffer species used: a network structure in acetate buffer, nanoparticles in citrate buffer, and large aggregates in phosphate buffer. The network structure formed in acetate buffer was maintained even after dilution with phosphate buffer, a situation that mimics the environment after oral administration. In addition, the structure was fragmented easily after application of a mild force, which could be an important property for achieving absorption of protein–peptide drugs from the gastrointestinal tract. This study provides new insights for the development of CyD-based nanoarchitectures suitable for application as protein–peptide carriers for oral drug delivery.