Exosomes are cell-derived extracellular vesicles and play important roles in mediating intercellular communications. Due to their unique advantages in transporting a variety of biomolecules, exosomes have been emerging as a new class of nanocarriers with great potential for therapeutic applications. Despite advancements in loading chemotherapeutics and interfering RNAs into exosomes, active incorporation of protein molecules into exosomes remains challenging owing to their distinctive physicochemical properties and/or a lack of knowledge of cargo sorting during exosome biogenesis. Here we report the generation of a novel type of engineered exosomes with actively incorporated membrane proteins or soluble protein cargos, named genetically infused functionally tailored exosomes (GIFTed-Exos). Through genetic fusion with exosome-associated tetraspanin CD9, transmembrane protein CD70 and glucocorticoid‐induced tumor necrosis factor receptor family‐related ligand (GITRL) could be displayed on exosome surface, resulting in GIFTed-Exos with excellent T-cell co-stimulatory activities. By genetically linking to a CD9-photocleavable protein fusion, fluorescent protein mCherry, apoptosis-inducing protein apoptin, and antioxidant enzyme catalase could be effectively packed into exosomes for light-controlled release. The generated GIFTed-Exos display notable in vitro and in vivo activities for delivering distinct types of protein cargos to target cells. As a possibly general approach, GIFTed-Exos provide new opportunities to create exosomes with new functions and properties for biomedical research.

外泌体是细胞衍生的细胞外囊泡，在介导细胞间通讯中起重要作用。由于其在运输多种生物分子方面的独特优势，外泌体已成为一类具有巨大治疗应用潜力的新型纳米载体。尽管在将化疗药物和干扰 RNA 装入外泌体方面取得了进展，但由于其独特的物理化学性质和/或在外泌体生物发生过程中缺乏货物分类知识，将蛋白质分子主动掺入外泌体仍然具有挑战性。在这里，我们报告了一种新型工程外泌体的产生，该外泌体具有积极掺入的膜蛋白或可溶性蛋白货物，命名为基因融合功能定制的exo somes ( GIFTed -Exos)。通过与外泌体相关的四跨膜蛋白CD9基因融合，跨膜蛋白CD70 和糖皮质激素诱导的肿瘤坏死因子受体家族相关配体 (GITRL) 可以展示在外泌体表面，从而产生具有优异 T 细胞共刺激活性的 GIFTed-Exos。通过与 CD9 可光裂解蛋白融合体的基因连接，荧光蛋白mCherry、凋亡诱导蛋白凋亡蛋白和抗氧化酶过氧化氢酶可以有效地包装到外泌体中进行光控释放。生成的 GIFTed-Exos在体外和体内显示显着将不同类型的蛋白质货物输送到靶细胞的活动。作为一种可能的通用方法，GIFTed-Exos 为生物医学研究创造具有新功能和特性的外泌体提供了新的机会。