Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, China.
Department of Infectious Diseases, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, PR China.
Biotechnol J. 2024 May;19(5):e2400128. doi: 10.1002/biot.202400128.
Small extracellular vesicles (sEVs) are nanosized vesicles enclosed in a lipid membrane released by nearly all cell types. sEVs have been considered as reliable biomarkers for diagnostics and effective carriers. Despite the clear importance of sEV functionality, sEV research faces challenges imposed by the small size and precise imaging of sEVs. Recent advances in live and high-resolution microscopy, combined with efficient labeling strategies, enable us to investigate the composition and behavior of EVs within living organisms. Here, a modified sEVs was generated with a near infrared fluorescence protein mKate2 using a VSVG viral pseudotyping-based approach for monitoring sEVs. An observed was made that the mKate2-tagged protein can be incorporated into the membranes of sEVs without altering their physical properties. In vivo imaging demonstrates that sEVs labeled with mKate2 exhibit excellent brightness and high photostability, allowing the acquisition of long-term investigation comparable to those achieved with mCherry labeling. Importantly, the mKate2-tagged sEVs show a low toxicity and exhibit a favorable safety profile. Furthermore, the co-expression of mKate2 and rabies virus glycoprotein (RVG) peptide on sEVs enables brain-targeted visualization, suggesting the mKate2 tag does not alter the biodistribution of sEVs. Together, the study presents the mKate2 tag as an efficient tracker for sEVs to monitor tissue-targeting and biodistribution in vivo.
小细胞外囊泡 (sEVs) 是一种纳米大小的囊泡，被一层脂质膜包裹，几乎所有细胞类型都会释放这种囊泡。sEVs 被认为是诊断的可靠生物标志物和有效的载体。尽管 sEV 功能非常重要，但 sEV 研究仍面临着 sEV 尺寸小和精确成像的挑战。近年来，活细胞和高分辨率显微镜技术的进步，结合高效的标记策略，使我们能够在活生物体中研究 EV 的组成和行为。在这里，使用基于 VSVG 病毒假型化的方法，用近红外荧光蛋白 mKate2 生成了一种改良的 sEV，用于监测 sEV。结果表明，mKate2 标记的蛋白可以整合到 sEV 的膜中，而不会改变其物理性质。体内成像表明，用 mKate2 标记的 sEV 具有出色的亮度和高光稳定性，允许进行类似于使用 mCherry 标记进行的长期研究。重要的是，mKate2 标记的 sEV 显示出低毒性和良好的安全性。此外，mKate2 和狂犬病毒糖蛋白 (RVG) 肽在 sEV 上的共表达使 sEV 能够实现靶向大脑的可视化，这表明 mKate2 标签不会改变 sEV 的生物分布。总之，该研究提出 mKate2 标签是 sEV 的一种高效追踪器，可用于监测体内组织靶向和生物分布。
