Multifunctional conjugation of proteins on/into bio-nanoparticles prepared by amphiphilic poly(gamma-glutamic acid)

The present study focuses on nanoparticles composed of amphiphilic poly(gamma-glutamic acid) (gamma-PGA) as potential protein carriers. Amphiphilic graft co-polymers composed of gamma-PGA as the hydrophilic backbone and L-phenylalanine ethylester (L-PAE) as the hydrophobic segment were synthesized by grafting L-PAE to gamma-PGA using water-soluble carbodiimide (WSC). Due to their amphiphilic properties, the gamma-PGA-graft-L-PAE co-polymer formed monodispersed nanoparticles in water. The particle size of the nanoparticles composed of gamma-PGA-graft-L-PAE (gamma-PGA nanoparticles) was about 200 nm and showed a highly negative zeta potential. To evaluate their potential applications as multifunctional protein carrier, we prepared protein-entrapped gamma-PGA nanoparticles by encapsulation, covalent immobilization or physical adsorption methods. For this purpose, 11 different proteins with various molecular weights and isoelectric points (pI values) were used as model proteins. The encapsulation of the protein into the nanoparticles was observed for all tested proteins. The amount of protein covalently immobilized or adsorbed onto the nanoparticles showed different tends based on the molecular weight and pI of each protein. Positively charged proteins could be adsorbed onto the negatively charged nanoparticles by electrostatic interaction. Moreover, it was found that enzyme-encapsulated nanoparticles showed higher enzymatic activity than surface-immobilized nanoparticles. These results indicated that the enzymatic activity of the enzyme-entrapped nanoparticles was significantly affected by the conjugation method, and that encapsulation was the optimal method for the conjugation of proteins and nanoparticles. It is expected that the gamma-PGA nanoparticle will have great potential as multifunctional carriers in pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.