Co-loading versus post-loading reveals maturation-controlled methylene blue organization and glutathione-triggered optical remodeling in polydopamine nanoparticles

Polydopamine nanoparticles (PDA NPs) provide catechol/quinone-rich adaptive interfaces for molecular adsorption, yet the role of PDA maturation in controlling payload organization remains unclear. Here, methylene blue (MB) was used as an organization-sensitive cationic probe to compare co-loading during dopamine polymerization (CL, 1–240 h) with post-loading onto pre-formed PDA NPs (PL, 24 h incubation). CL produced a strong maturation-dependent increase in loading efficiency, from 11.09 ± 1.12% at 1 h to 80.88 ± 2.59% at 240 h, whereas PL remained nearly time-independent (25.96 ± 1.30–31.10 ± 1.55%). UV–Vis–NIR spectroscopy showed that CL promoted broad long-wavelength MB contributions associated with aggregate-like and/or electronically coupled states, while PL retained sharper features consistent with surface-associated adsorption. Representative 24 h Raman spectroscopy provided local route-comparative evidence of distinct MB interfacial arrangements, without being used to infer time-resolved behavior across the full series. Upon glutathione exposure (10 mM GSH in PBS, 37 °C, 24 h), the aggregation index decreased in a formulation- and maturation-dependent manner. ΔIj was interpreted as an integrated optical descriptor of MB/PDA nanoassembly remodeling rather than as a direct release metric. These findings identify PDA maturation and loading route as coupled design parameters for regulating molecular incorporation, aggregate-like optical coupling, and reducing-triggered optical response in dye-loaded PDA nanoassemblies.