Evaluating the impact of bioinspired counterion inclusion on silk nanoparticle physicochemical attributes and physical stability

Silk fibroin is a promising material for nanocarrier-based drug delivery applications due to its biocompatibility, biodegradability, and mechanical properties, which can be fine-tuned through processing conditions. In this study, we explore the impact of Ca²⁺ and K⁺ inclusion on the morphology of silk nanoparticles and evaluate the short- and long-term stability of silk nanoparticles formed by antisolvent precipitation in deionized water and sodium phosphate buffer. Using advanced electric asymmetric flow field-flow fractionation multiplexed with online detectors (EAF4-UV-MALS-DLS) and orthogonal analytics (DLS, ELS, NTA, FE-SEM), we analyze the physicochemical attributes of silk nanoparticles. We find significant differences in nanoparticle architecture and stability in different buffers, with notable differences in particle size (<i>R</i> _g and <i>R</i> _h), charge, and shape measured over 56 days. Notably, nanoparticles formulated with 0.7 mg Ca²⁺ and 1.1 mg K⁺ maintained superior physicochemical stability, making them promising candidates for future nanocarrier-based applications.