Antisense oligonucleotides (ASOs) are promising therapeutic agents targeting intracellular RNA, yet their clinical application is limited by poor membrane permeability. To overcome this challenge, we investigated hydrophobic cell-penetrating peptides (CPPs) as alternative delivery vectors. Ten hydrophobic CPPs were synthesized and screened for cellular uptake using live-cell fluorescence imaging. Selected CPPs were conjugated to a chemically modified ASO via click chemistry, and their intracellular delivery and antisense efficacy were evaluated using a splicing reporter assay in HeLa 705 cells. While certain CPPs, such as MPG, showed high membrane permeability, conjugation with ASOs did not always translate to enhanced antisense activity. Notably, among the evaluated CPP-ASO conjugates, SP-ASO exhibited the most potent functional activity despite moderate uptake. This finding suggests that factors beyond membrane permeability, such as endosomal escape, intracellular trafficking, or nuclear delivery efficiency, may critically influence the overall efficacy. Fluorescence microscopy confirmed lysosomal entrapment of both naked and CPP-conjugated ASOs. These findings emphasize the importance of rational design strategies that address endosomal release to maximize the therapeutic potential of CPP-ASO conjugates.
