Efficient cytosolic delivery is a significant hurdle when using short interfering RNA (siRNA) in therapeutic applications. Here we show that cholesterol-rich exosomes are prone to entering cancer cells through membrane fusion, achieving direct cytosolic delivery of siRNA. Molecular dynamics simulations suggest that deformation and increased contact with the target cell membrane facilitate membrane fusion. In vitro we show that cholesterol-enriched milk-derived exosomes (MEs) achieve a significantly higher gene silencing effect of siRNA, inducing superior cancer cell apoptosis compared with the native and cholesterol-depleted MEs, as well as conventional transfection agents. When administered orally or intravenously to mice bearing orthotopic or subcutaneous tumours, the cholesterol-enriched MEs/siRNA exhibit antitumour activity superior to that of lipid nanoparticles. Collectively, by modulating the cholesterol content of exosome membranes to facilitate cell entry via membrane fusion, we provide a promising approach for siRNA-based gene therapy, paving the way for effective, safe and simple gene therapy strategies.

在治疗应用中使用短干扰 RNA （siRNA） 时，高效的胞质递送是一个重大障碍。在这里，我们表明富含胆固醇的外泌体容易通过膜融合进入癌细胞，实现 siRNA 的直接胞质递送。分子动力学模拟表明，变形和与靶细胞膜接触的增加促进了膜融合。在体外，我们表明，与天然和胆固醇耗尽的 ME 以及常规转染试剂相比，富含胆固醇的牛奶衍生外泌体 （MEs） 实现了 siRNA 的显着更高的基因沉默效果，诱导了优越的癌细胞凋亡。当口服或静脉内给药给携带原位或皮下肿瘤的小鼠时，富含胆固醇的 MEs/siRNA 表现出优于脂质纳米颗粒的抗肿瘤活性。总的来说，通过调节外泌体膜的胆固醇含量以促进细胞通过膜融合进入细胞，我们为基于 siRNA 的基因治疗提供了一种有前途的方法，为有效、安全和简单的基因治疗策略铺平了道路。