Amphiphilic block or graft copolymers have been demonstrated to form a variety of self-assembled nano/microstructures in selective solvents. In this study, the self-association behavior of biodegradable graft copolymers composed of poly(γ-glutamic acid) (γ-PGA) as the hydrophilic segment and L-phenylalanine (Phe) as the hydrophobic segment in aqueous solution was investigated. The association behavior and unimer nanoparticle formation of these γ-PGA-graft-Phe (γ-PGA-Phe) copolymers in aqueous solution were characterized with a focus on the effect of the Phe grafting degree on the intra- and interpolymer association of γ-PGA-Phe. The particle size and number of polymer aggregates (N(agg)) in one particle of the γ-PGA-Phe depended on the Phe grafting degree. The size of γ-PGA-Phe with 12, 27, 35, or 42% Phe grafting (γ-PGA-Phe-12, -27, -35, or -42) was about 8-14 nm and the N(agg) was about 1, supporting the presence of a unimolecular graft copolymer in PBS. The pyrene fluorescence data indicated that γ-PGA-Phe-35 and -42 have hydrophobic domains formed by the intrapolymer association of Phe attached to γ-PGA. These results suggest that the Phe grafting degree is critical to the association behavior of γ-PGA-Phe and that γ-PGA-Phe-35 and -42 could form unimer nanoparticles. Moreover, when γ-PGA-Phe-42 dissolved in DMSO was added to various concentrations of NaCl solution, the particle size and N(agg) could be easily controlled by changing the NaCl concentration during the formation of the particles. These results suggest that biodegradable γ-PGA-Phe is useful for the fabrication of very small nanoparticles. It is expected that γ-PGA-Phe nanoparticles, including unimer particles, will have great potential as multifunctional carriers for pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.

Amphiphilic random copolymers of poly(γ-glutamic acid)-graf t-L-phenylalanine (γ-PGA-Phe), with various lengths of γ-PGA main chains (molecular weight of 70, 140, and 220 kDa) plus hydrophobic moieties of Phe groups (grafting degree 12−60%), self-assembled in aqueous media to form nanoparticles (NPs). The aggregation number (N agg ) could be adjusted according to their molecular structures as well as the preparative methods/conditions. The γ-PGA-Phe NPs were further characterized by means of dynamic and static light scattering, small-angle neutron scattering, as well as steady-state fluorescence measurements/quenching techniques. The single chain state had hydrophobic domains, and formed spherical structures, herein called unimer NPs, and were obtained using molecular weights of γ-PGA higher than 140 kDa conjugated with Phe at 27−42% due to the balance of hydrophobicity/hydrophilicity along the single polymer chain. The number of hydrophobic domains in one NP (N domain ), estimated by means of fluorescence quenching techniques and the rigidity of the inner particles detected by dipyrene fluorescence demonstrated that the N domain and the rigidity were affected by the particle size and preparative methods. In addition, the effect of pH on the stability of the unimer NPs indicated a reduction of the N domain upon the pH, supporting a loose packing due to hydrophobic association under alkaline conditions.

Stimuli-responsive unimer nanoparticles (NPs) composed of hydrophobized poly(γ-glutamic acid) were successfully prepared by a combination of hydrophobic interactions and disulfide bonds. The formation of hydrophobic associations and the existence of disulfide bonds were confirmed by UV spectroscopy and by treatment with dithiothreitol, respectively. Moreover, the release of hydrophobic drugs in response to a reducing agent was observed when compared to nonstimulus unimer NPs. These 10 nm-sized unimer NPs will be useful as reduction-responsive carriers.