Assembly of Bioactive Peptide–Chitosan Nanocomplexes

The assembly of nanocomplexes from bioactive peptides, namely, caseinophosphopeptides (CPPs) and chitosan (CS), at physiological conditions and various CS/CPP mass ratios has been systematically studied using a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), turbidimetric titration, dynamic light scattering (DLS), electrophoretic mobility (ζ-potential) measurements, transmission electron microscopy (TEM), and fluorescence spectroscopy. Peptides incorporated with CS forming nanoparticles were prepared and identified using LC-MS/MS. They were characterized by different amounts of clusters of phosphorylated seryl residues. At low salt concentrations, an increase in CS/CPP mass ratio shifted the critical pH(φ1) value, which was designated as the formation of CS/CPP nanocomplexes, as well as pH(max), which represents the neutralization of positive and negative charges at higher pH values. The sizes, charges, morphologies, binding mechanisms, and binding constants of the bioactive peptide-chitosan nanocomplexes were analyzed, and our results suggest that three processes are involved in nanocomplex formation: First, negatively charged CPPs absorb to positively charged CS molecular chains to form intrapolymer nanocomplexes saturated with CPPs (CPPNPs). Subsequently, the negatively charged CPPNPs are bridged by the addition of positively charged CS, resulting in the formation of nearly neutral associative biopolymer complexes. Finally, further addition of excess chitosan breaks down the bridges of associative complexes and causes the formation of positively charged isolated spherical nanocomplexes. The binding between the peptides and CS is mainly driven by electrostatic interactions with a binding constant of K(cs) = 4.6 × 10(4) M(-1). Phosphorylated groups and other negatively charged amino acids, such as aspartic acid (Asp) and glutamic acid (Glu), in the CPPs might be the dominant sites for interaction with -NH(3)(+) groups on the CS molecular chains.