Novel Polyion Complex Micelles Entrapping Enzyme Molecules in the Core: Preparation of Narrowly-Distributed Micelles from Lysozyme and Poly(ethylene glycol)−Poly(aspartic acid) Block Copolymer in Aqueous Medium

A core−shell-type supramolecular assembly, a polyion complex micelle, was prepared in this study from chicken egg white lysozyme and poly(ethylene glycol)−poly(aspartic acid) block copolymer (PEG-P(Asp)) through electrostatic interaction in aqueous medium. Lysozyme/PEG-P(Asp) micelles thus prepared had an extremely narrow distribution (μ2/Γ2 < 0.04) with an average diameter of 47 nm in dynamic light scattering measurements. No precipitate formation was observed even after 1 month standing at ambient temperature, suggesting that the system is in a thermodynamic equilibrium state. The stoichiometry in terms of the molar ratio of Lys and Arg residues in lysozyme and Asp residues in PEG-P(Asp) was confirmed by dynamic and static light scattering as well as by laser-Doppler electrophoresis measurements. A change in the apparent molar mass of the micelle with varying PEG-P(Asp)/lysozyme ratio in the region with excess lysozyme agreed well with calculated values if a cooperative association mechanism is assumed to occur. The diffusion coefficient of lysozyme/PEG-P(Asp) micelles prepared at a stoichiometric mixing ratio showed neither angular nor concentration dependence, indicating their spherical shape with no secondary aggregate formation. A core−shell structure with a polyion complex core and a PEG corona was suggested from an extremely low absolute value of zeta-potential. The association numbers of lysozyme and PEG-P(Asp) in the stoichiometric micelle were calculated from the apparent molar mass and were determined to be 36 and 42 for lysozyme and PEG-P(Asp), respectively. Such PIC micelles entrapping enzymes in the core are expected to be useful as functional materials including carrier systems in drug delivery applications and a nanometric-scale reactor for enzymes.