Modulating STAT3 pathway in breast cancer: Improved activity of T40214 aptamer by peptide-based nanofibers delivering

G-quadruplex (G4)-forming aptamers are highly selective oligonucleotides with significant potential in cancer therapy, acting as inhibitors of oncogenic proteins such as Signal Transducer and Activator of Transcription 3 (STAT3). Among them, the G4-forming aptamer T40214 (STAT) effectively modulates STAT3 signaling; however, its clinical translation is currently hindered by poor cellular internalization. In this study, we developed a multifunctional peptide amphiphile (PA)-based nanofiber (NF) to enhance the intracellular delivery and tumor selectivity of the G4 STAT aptamer in breast cancer cells. The nanofiber integrates a poly-arginine domain for aptamer binding, the cell-penetrating peptide gH625 to promote internalization, and the EGFR-targeting peptide P22 for selective tumor cell recognition. The aptamer-decorated NFs exhibited an average fiber length of 100 ± 30 nm and a diameter of 25 ± 5 nm and remained stable under physiological conditions. Aptamer binding was confirmed by agarose gel electrophoresis and circular dichroism. The resulting STAT-NF complex showed stability over time and varying temperature and ionic strength effects. A markedly enhanced antiproliferative activity of the STAT was observed in multiple triple-negative breast cell models, proved by an efficient and selective uptake in a triple negative breast cancer cell line. Mechanistic studies revealed that STAT-NF internalization occurs predominantly through an endocytosis-independent translocation mechanism, and the activation of apoptotic pathways by the released STAT was confirmed. These results highlight PA-based nanofibers as an effective and versatile platform for tumor-targeted delivery of G4 aptamers.