Wang Wangxia, Huang Zhenqiu, Hu Dandan, Li Wenlong, Wu Mingyu, Feng Shun, Wan Yu
Sichuan Engineering Research Center for Biomimetic Synthesis of Natural Drugs, School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China; School of Pharmaceutical and Environmental, Chongqing Industry and Information Vocational College, Chongqing 401233, China.
Sichuan Engineering Research Center for Biomimetic Synthesis of Natural Drugs, School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China.
Acta Biomater. 2025 Jun 15;200:554-568. doi: 10.1016/j.actbio.2025.05.018. Epub 2025 May 21.
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.
细胞穿透性聚二硫化物（CPDs）是很有前景的小干扰RNA（siRNA）递送载体，利用硫醇介导的摄取来避免溶酶体滞留。然而，与其他阳离子载体一样，CPDs需要过量的正电荷来维持载体/siRNA复合物的稳定性，存在细胞毒性风险。此外，血清蛋白吸附会破坏复合物，降低转染效率。在本研究中，我们设计了一系列氟化CPDs（F-CPD-n），通过特定的亲氟效应来提高siRNA的递送效率。氟化引入了独特的疏水和亲脂特性，促进了在极性和非极性环境中的相分离。体外实验结果表明，适度氟化可改善基于CPD的载体的siRNA结合、细胞摄取和转染效率，而过度氟化则会阻碍siRNA结合并增加细胞毒性。此外，氟化略微改变了摄取机制，大多数摄取仍通过硫醇介导的途径发生。重要的是，氟化增强了血清耐受能力，在富含血清的条件下维持了有效的细胞摄取和基因沉默能力。随后，一种优化的载体F-CPDs-30有效地将siPGX4（靶向谷胱甘肽过氧化物酶4的siRNA）递送至乳腺癌细胞，在蛋白质和mRNA水平上沉默GPX4。F-CPDs-30/siGPX4通过诱导铁死亡在体外和体内均表现出显著的抗癌活性。这些效应归因于通过CPDs的二硫键骨架有效沉默GPX4和消耗谷胱甘肽（GSH）。这项工作为硫醇介导的siRNA递送载体的开发提供了有价值的见解，并为癌症治疗中基于siRNA的铁死亡诱导提供了一个有前景的平台。
在基于siRNA的治疗中，高效的胞质递送仍然是一个主要挑战。在本研究中，我们证明，与未修饰的CPDs相比，具有最佳氟密度的氟化细胞穿透性聚二硫化物（CPDs）增强了siRNA结合、细胞摄取和转染效率。氟化还提高了CPDs的血清耐受能力。此外,氟化CPDs主要通过硫醇介导的途径进入癌细胞。在体外和体内沉默siPGX4（谷胱甘肽过氧化物酶4）和诱导铁死亡方面，领先的氟化CPD显著优于天然CPD和传统转染试剂。除了有效敲低GPX4外，通过CPD二硫键骨架消耗GSH进一步增强了乳腺癌中的铁死亡。该策略为推进基于siRNA的基因治疗提供了一种有前景的方法。
