Capillary‐Based Physicochemical Characterization of Lipid Nanoparticles

Lipid nanoparticles (LNPs) are widely used for the delivery of nucleic acid (NAs), most notably in gene therapy and messenger ribonucleic acid (mRNA)-based vaccines. Understanding their physicochemical properties is essential, yet current analytical approaches often fall short in capturing their complexity. Here, we introduce an analytical strategy using capillary zone electrophoresis (CZE) and pressure-driven Taylor dispersion (TD) analysis beside the combination of both separation principles. This novel separation mode of electrophoretic TD or electrohydrodynamic coupling (termed here as eTD) can be used to characterize deoxyribonucleic acid (DNA)-loaded LNP formulations using standard capillary electrophoresis (CE) instrumentation. eTD is a new separation approach that combines electrophoretic and hydrodynamic movement in micro-scaled capillaries for the analysis of drug carriers as LNPs. Focusing on critical quality attributes (CQAs), TD provided information on the hydrodynamic radius of LNPs and the distribution of NAs across different chemical environments. CZE enabled the estimation of ζ-potential and localization of DNA within distinct particle populations. The novel eTD mode offers deeper insight into LNP structure and morphological aspects, yielding characteristic profiles for individual formulations and revealing the presence of unencapsulated DNA. To contextualize LNP measurements, we also analysed free NAs and their mixtures with LNPs under identical conditions. The method distinguished between encapsulated and unencapsulated species, revealing individual electrophoretic and dispersion profiles for single-stranded mRNA and double-stranded DNA. These findings demonstrate the potential of capillary techniques for the advanced physicochemical characterization of NA-loaded LNPs. Further investigations are warranted to expand their analytical utility and deepen our understanding of LNP structural features.