Separation of 100 nm-sized nanoparticles using a poly-Lys-modified monolith column†

Nanoparticles (approximately 100 nm in diameter) composed of lipid layers containing drugs or biologically active substances are attracting increasing attention in various fields, including medicine, as well as for signal transduction between cells. However, the separation of such nanoparticles via conventional HPLC is challenging, often resulting in the clogging and collapse of nanoparticles, as well as a low separation efficiency. Thus far, no HPLC column capable of efficiently separating two types of 100 nm-sized nanoparticles in a short time has been reported. In this study, a poly-Lys-modified monolithic column was prepared for nanoparticle analysis via HPLC using anticancer drug-encapsulated nanoparticles (Doxil®) and small extracellular vesicles (sEVs) to examine their elution behaviors. The zeta potentials of Doxil® and the sEVs were −24.4 and −45.5 V, respectively. A column with a low surface coverage (0.96 mg mL⁻¹) of poly-Lys adsorbed the nanoparticles but did not elute them, whereas a column with a high surface coverage (2.06 mg mL⁻¹) of poly-Lys retained these nanoparticles owing to the ion-exchange effect; sEVs with highly negative charges were strongly retained in the column. Using gradient elution with different 2-amino-2-hydroxymethyl-1,3-propanediol concentrations in the mobile phase, the two types of nanoparticles (Doxil® and sEVs) were eluted and successfully separated within 10 min. Thus, the developed column is a valuable tool for evaluating the safety and performance of larger-sized nanoparticles.