Pathway-Dependent Formation of Compact Polypeptide-Based Single-Chain Nanoparticles

Abstract

Single-chain nanoparticles (SCNPs) are synthetic macromolecules that undergo intramolecular collapse to form protein-sized structures (5–20 nm), making them attractive as protein mimics whose peptide backbone is expected to confer susceptibility to enzymatic and hydrolytic degradation. While most reported SCNPs rely on controlled radical polymerization techniques such as RAFT, ATRP, and NMP, these approaches yield nonbiodegradable materials that limit biological applicability. Polypeptide-based SCNPs offer a compelling alternative: their amide backbone impart biodegradability and biocompatibility, and they retain the capacity to adopt ordered secondary structures. Here, we report a series of SCNPs derived from poly-l-glutamic acid, formed via intramolecular crosslinking using 2,2′-(ethylenedioxy)bis(ethylamine) and stabilized by grafting of poly(ethylene glycol) methyl ether amine (Mn ∼2000). By systematically varying the sequence of PEG grafting and crosslinking, we establish that PEGylation is critical for suppressing intermolecular aggregation and enabling controlled single-chain collapse. Characterization by dynamic light scattering (DLS), size exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and circular dichroism (CD) consistently demonstrates that a minimum degree of crosslinking is required to induce moderately compact nanoparticle formation. The optimal strategy, namely, PEG grafting prior to crosslinking, yields well-defined SCNPs with a hydrodynamic diameter decreasing from 27 nm (uncrosslinked PEGylated precursor) to 18.6 nm (40% crosslinked). SAXS confirms a reduction in the radius of gyration from 13.5 nm (uncrosslinked) to 9.0 nm (40% crosslinked), while the Porod exponent increases from 1.7 to 2.4, reflecting enhanced compactness. CD analysis further reveals a transition from random coil conformation at low crosslinking degrees to increasingly ordered helical structures at 40% crosslinking. These findings establish clear design principles for structurally controlled, biodegradable polypeptide-based SCNPs, paving the way for better-controlled nanoparticles across applications in biomedicine, catalysis, and related fields.