Delivery of siGPX4 through a thiol-mediated pathway using a fluorinated cell-penetrating poly(disulfide) for inducing ferroptosis in breast cancer.

Cell-penetrating poly(disulfides) (CPDs) are promising siRNA delivery vectors, utilizing thiol-mediated uptake to avoid lysosomal retention. However, like other cationic vectors, CPDs require excess positive charge for vector/siRNA complexes stability, risking cytotoxicity. Additionally, serum protein adsorption can disrupt complexes, diminishing transfection efficiency. In this study, we designed a series of fluorinated CPDs (F-CPD-n) to enhance siRNA delivery efficiency through the specific fluorophilic effect. Fluorination introduces unique hydrophobic and lipophobic characteristics, promoting phase separation in polar and non-polar environments. In vitro results demonstrated that moderate fluorination improved siRNA binding, cellular uptake, and transfection efficiency of the CPD-based vectors, while excessive fluorination hindered siRNA binding and increased cytotoxicity. Moreover, fluorination slightly altered the uptake mechanism, with most uptake still occurring via the thiol-mediated pathway. Importantly, fluorination enhanced serum tolerance capacity, maintaining effective cellular uptake and gene silencing capacity in serum-rich conditions. Subsequently, an optimized vector, F-CPDs-30, effectively delivered siPGX4 (siRNA targeting glutathione peroxidase 4) to breast cancer cells, silencing GPX4 at both the protein and mRNA levels. F-CPDs-30/siGPX4 exhibited significant anticancer activity in vitro and in vivo by inducing ferroptosis. These effects were attributed to efficient GPX4 silencing and GSH depletion via the disulfide backbone of CPDs. This work provides valuable insights into the development of thiol-mediated siRNA delivery vectors and offers a promising platform for siRNA-based ferroptosis induction in cancer therapy. STATEMENT OF SIGNIFICANCE: Efficient cytosolic delivery remains a major challenge in siRNA-based therapeutics. In this study, we demonstrated that fluorinated cell-penetrating poly(disulfide)s (CPDs), with optimal fluorine density, enhanced siRNA binding, cellular uptake, and transfection efficiency compared to unmodified CPDs. Fluorination also improved the serum tolerance capacity of CPDs. Moreover, the fluorinated CPDs predominantly entered cancer cells via a thiol-mediated pathway. The leading fluorinated CPD significantly outperformed both native CPDs and conventional transfection agents in silencing siPGX4 (glutathione peroxidase 4) and inducing ferroptosis in vitro and in vivo. In addition to efficient GPX4 knockdown, GSH depletion through the CPD disulfide backbone further potentiated ferroptosis in breast cancer. This strategy presents a promising approach for advancing siRNA-based gene therapy.