Antisense oligonucleotides (ASOs) represent a promising class of therapeutic agents; yet, their efficacy and/or toxicity profiles are heavily dependent on their tissue distribution and cellular uptake. This study employs nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to elucidate the intracellular distribution of chemically modified ASOs in liver tissue with ultra-high resolution. We demonstrated that fully phosphorothioated ASOs predominantly accumulated in the vesicular structures near nonparenchymal cells, including Kupffer cells. In contrast, partially phosphorothioated ASOs exhibit a uniform distribution throughout the liver. Notably, despite similar overall liver concentrations, ASOs with different chemical modifications exhibited markedly distinct intracellular distribution patterns. These findings highlight the critical importance of subcellular distribution in ASO drug discovery and underscore the utility of NanoSIMS in visualizing the ASO biodistribution. This approach, when combined with electron microscopy, provides invaluable insights into the chemical composition and localization of ASOs within cellular compartments. This study not only advances our understanding of ASO behavior in vivo but also highlights the potential of high-resolution imaging techniques in optimizing ASO delivery strategies. These insights are crucial for enhancing the efficacy and minimizing the adverse effects of ASO-based therapeutics, paving the way for more targeted and effective treatments.
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