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

Abstract

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.