Efficient Inhibition of TGF-β Signaling via Cytosolic Delivery of a Smad2/3-Binding Peptide Using the Cell-Penetrating PG-Surfactant <b>DKDKC</b> _<b>12</b> <b>-K</b> _<b>5</b> to Block Smad2/3 Nuclear Translocation

Targeting intracellular signaling molecules that translocate to the nucleus is a promising approach for peptide-based therapeutics. Here, we focused on Smad2/3, key mediators of TGF-β signaling that act as transcription factors upon nuclear entry. To inhibit their nuclear localization, we delivered an Smad2/3-binding SARA peptide into the cytoplasm using our previously developed cell-penetrating carrier, cpPG. A conjugate, SARA-cpPG, was synthesized by linking the SARA peptide to <b>Mal-DKDKC</b>_<b>12</b><b>-K</b>_<b>5</b>, a cpPG derivative with an N-terminal maleimide. In A549 cells, SARA-cpPG showed uptake over 20-fold higher than that of the SARA peptide alone and retained cytoplasmic localization. Functionally, SARA-cpPG suppressed TGF-β1-induced actin polymerization and cell migration, comparable to the effects of receptor kinase inhibitor LY2157299. These effects were not observed with cpPG alone or a control conjugate (SARAm-cpPG) containing a nonbinding mutant peptide. Mechanistic studies revealed that SARA-cpPG did not inhibit Smad2/3 phosphorylation but reduced TGF-β target gene expression, suggesting a blockade of nuclear translocation. This suppression was absent in treatments with SARAm-cpPG, cpPG alone, or a non-cytoplasm-specific carrier conjugate (SARA-R8). These findings demonstrate that cpPG enables efficient cytosolic delivery of functional peptides and supports a strategy for intracellular peptide therapeutics targeting nuclear signaling pathways.