Combined Experimental and Molecular Simulation Study of Insulin-Chitosan Complexation Driven by Electrostatic Interactions

Abstract

Protein−polysaccharide complexes constructed via self-assembly methods are often used to develop novel 17 biomaterials for a wide range of applications in biomedicine, food, and biotechnology. The objective of this work was to 18 investigate theoretically and to demonstrate via constant-pH Monte Carlo simulations that the complexation phenomenon 19 between insulin (INS) and the cationic polyelectrolyte chitosan (CS) is mainly driven by an electrostatic mechanism. 20 Experimental results obtained from FTIR spectra and ζ-potential determinations allowed us to complement the conclusions. 21 The characteristic absorption bands for the complexes could be assigned to a combination of signals from CS amide I and INS 22 amide II. The second peak corresponds to the interaction between the polymer and the protein at the level of amide II. INS− 23 CS complexation processes not expected when INS is in its monomeric form, but for both tetrameric and hexameric forms, 24 incipient complexation due to charge regulation mechanism took place at pH 5. The complexation range was observed to be 5.5 25 < pH < 6.5. In general, when the number of INS units increases in the simulation process, the solution pH at which the 26 complexation can occur shifts toward acidic conditions. CS’s chain interacts more efficiently, i.e. in a wider pH range, with INS 27 aggregates formed by the highest monomer number. The charge regulation mechanism can be considered as a previous phase 28 toward complexation (incipient complexation) caused by weak interactions of a Coulombic nature.