Screening and Identification of Novel DNA Aptamer for Targeted Delivery to Injured Podocytes in Glomerular Diseases

Abstract

Selective drug delivery to podocytes remains a challenge. Aptamers, nucleic acids that bind specific cells, offer a potential solution, though podocyte-targeting aptamers have not yet been developed. Podocytes stimulated with adriamycin, puromycin aminonucleoside, and high glucose are used to screen an single-stranded DNA (ssDNA) library (10¹⁵ sequences). High-throughput sequencing identifies nucleotide sequences, and the aptamer's affinity, stability, cytotoxicity, uptake, biodistribution (especially to podocyte), target protein and ability to deliver siRNA are evaluated. After 11–14 rounds of selection, high-affinity pools are identified. Sequencing reveals 23,848 unique sequences, narrowed down to 12 candidates. Aptamer S7 is specifically bound to podocytes, and its truncated version, RLS-2, demonstrates superior affinity (50–70 nM) and improved stability with phosphorothioate modifications. RLS-2 exhibits no significant cytotoxicity, is internalized by podocytes, and localized to lysosomes. In adriamycin-induced and diabetic nephropathy mice, RLS-2 preferentially accumulates within glomeruli. Its specificity to podocyte is verified by colocalization examination and quantitated via flowcytometry. EPB41L5 is identified as a target protein. Aptamer-siRNA chimeras based on RLS-2 successfully downregulate gene expression without the need for transfection reagents in vitro. These findings underscore the potential of RLS-2 as a promising agent for the development of podocyte-targeted drug delivery systems.