Salt-Mediated Electro-Deformation of AAV Capsids Revealed by Nanopipette-Based Single-Particle Analysis

Adeno-associated viruses (AAVs) are leading gene therapy vectors; however, maintaining their structural integrity and cargo stability remains a major challenge for consistent therapeutic performance. Although solid-state nanopores can discriminate AAVempty, AAVssDNA, and AAVscDNA capsids based on genomic content, the ionic regulation of their mechanical deformability remains poorly understood. In this study, we employ quartz nanopipettes, a mechanically robust subset of solid-state nanopores, to directly probe bias-induced electro-deformation of individual AAV9 particles in two monovalent electrolytes, LiCl and KCl. In LiCl, AAV9 capsids exhibited higher cargo-dependent deformability and approximately 3-fold slower translocation kinetics than in KCl, reflecting extended residence times and increased structural compliance. However, only small differences in deformability were observed for genome-containing AAVs (AAVssDNA and AAVscDNA) across both electrolytes. This likely reflects intrinsic heterogeneity in preparation, with coexisting particle subpopulations that span partially filled to fully packaged capsids. Voltage-dependent current blockade analysis revealed a substantial reduction in ΔI/I0 in LiCl, indicative of enhanced capsid deformation promoted by the chaotropic Li+ ions that weaken interprotein cohesion within the viral shell. Integrating nanopipette sensing with a self-supervised machine learning framework enabled quantitative discrimination of full and defective capsids based on their electromechanical fingerprints. Together, these results elucidate how electrolyte composition governs nanoscale capsid mechanics and establish nanopipette-based single-particle analysis combined with data-driven classification as a powerful tool for rapid and quantitative assessment of AAV stability and vector quality in gene therapy manufacturing.