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

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.