Hybrid Arborescent Polypeptide-Based Unimolecular Micelles: Synthesis, Characterization, and Drug Encapsulation

This paper reports novel hybrid arborescent polypeptides based on poly(γ-benzyl l-glutamate)-co-poly(γ-tert-butyl l-glutamate)-g-polysarcosine [P(BG-co-Glu(OtBu))-g-PSar]. The synthesis is launched by ring-opening polymerization (ROP) of N-carboxyanhydride of γ-benzyl l-glutamate (BG-NCA) and γ-tert-butyl l-glutamate (Glu(OtBu)-NCA) to synthesize a random copolymer P(BG-co-Glu(OtBu)) serving as a precursor for the arborescent system, followed by deprotection of the tert-butyl (tBu) groups to afford free COOH moieties serving as coupling sites. Two copolymerization reactions were carried out to afford the side chains. One type of side chain was a random copolymer P(BG-co-Glu(OtBu)), while the other type was a triblock copolymer PGlu(OtBu)-b-PBG-b-PGlu(OtBu). The peptide coupling reactions were conducted between the COOH moieties on the precursor and the terminus amine on the chain end of the P(BG-co-Glu(OtBu)) random copolymer or the PGlu(OtBu)-b-PBG-b-PGlu(OtBu) triblock copolymer to obtain G0 polymers. Afterward, hydrolyzing the tBu moieties of the G0 substrates yielded randomly functionalized G0 and end-functionalized G0. Randomly functionalized G0 was used as a substrate for the next generation G1 (randomly functionalized and end-functionalized G1 after deprotection) or coated with polysarcosine (PSar) to gain G0-g-PSar. The G0 substrate prepared with the triblock copolymer PGlu(OtBu)-b-PBG-b-PGlu(OtBu) was only grafted with PSar after deprotection, resulting in G0-eg-PSar. Depending on the functionality mode of the G1 substrate, the PSar coating yielded two different graft polymers, G1-g-PSar and G1-eg-PSar, for randomly functionalized and end-functionalized G1, respectively. The PSar hydrophilic shell was decorated with the sequence of (arginine, glycine, and aspartic acid) tripeptides (RGD) as a targeting ligand to improve the potentiality of the arborescent unimolecular micelles as drug carriers. Preparative size exclusion chromatography (SEC) was used to fractionate these complex macromolecular architectures. Nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR), Raman spectroscopy, and SEC were used for molecular characterization of all intermediate and final products and dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) for micellar characterization. A comparison between randomly grafted (g) and end-grafted (eg) unimolecular micelles demonstrates that the former has an undefined core-shell structure, unlike its end-grafted analog. In addition, this study has proved that decoration of the shell with RGD contributed to avoiding micelle aggregation but limited chemotherapy agent encapsulation. However, more than their naked analog, the sustained release was noticeable in decorated micelles. Doxorubicin was utilized as a chemotherapy model, and loading was achieved successfully by physical entrapment.