Intracellular delivery crossing the endomembrane barrier is the “last mile to target” for nano delivery systems carrying biomacromolecules, including genetic medicines. Nevertheless, a mass of nanomedicines is currently restricted by their equivocal safety and delivery efficiency. Here, we establish a universal strategy independent of nanomaterials. Such a policy broadly facilitates the intracellular delivery of all kinds of tested nanomedicines, subtly by inducing ARF6 GTPases to their overactivated GTP-bound state. ARF6, one member of ARF subfamily in small GTPases, is verified to regulate intracellular vesicle transport and lipid metabolism through GTP/GDP conversion. ARF6 biased to GTP-bound state causes the increased endocytosis and reduced exocytosis of eleven types of nanoparticles. This universal effect is derived from the formation of a hybrid type of endosomes triggered by overactivated ARF6 via regulating cholesterol-associated vesicles and lipid raft/caveolae pathways. Due to the mild microenvironment in hybrid endosomes, the internalized protein and nanoparticles are steadily delivered to the cytoplasm, avoiding the intensive degradation in lysosomes. Based on these findings, we identify QS11, a safe small molecule inhibitor of ARF GTPase-activating proteins, significantly enhances the antitumor efficacy of siEGFR-loaded nanoparticles by inducing ARF6 overactivation. In sum, these findings reveal that the tactics of tuning ARF6 GTPases to GTP-bound form will widely benefit cellular nano delivery.

穿过内膜屏障的细胞内递送是携带生物大分子（包括基因药物）的纳米递送系统的“目标的最后一英里”。然而，大量纳米药物目前受到其模棱两可的安全性和递送效率的限制。在这里，我们建立了一个独立于纳米材料的通用策略。这种策略通过巧妙地将 ARF6 GTPases 诱导到其过度激活的 GTP 结合状态，广泛地促进了各种经过测试的纳米药物的细胞内递送。ARF6 是小 GTPases 中 ARF 亚家族的一员，经证实可通过 GTP/GDP 转化调节细胞内囊泡转运和脂质代谢。偏向 GTP 结合状态的 ARF6 导致 11 种纳米粒子的内吞作用增加和胞吐作用减少。这种普遍效应源自过度激活的 ARF6 通过调节胆固醇相关小泡和脂筏/小窝通路触发的混合型核内体的形成。由于混合核内体中温和的微环境，内化的蛋白质和纳米粒子被稳定地输送到细胞质，避免了溶酶体中的强烈降解。基于这些发现，我们确定 QS11 是一种安全的 ARF GTPase 激活蛋白小分子抑制剂，通过诱导 ARF6 过度激活显着增强载有 siEGFR 的纳米颗粒的抗肿瘤功效。总之，这些发现表明将 ARF6 GTPases 调整为 GTP 结合形式的策略将广泛有益于细胞纳米递送。