Piccarducci Rebecca, Germelli Lorenzo, Falleni Alessandra, Luisotti Lucrezia, Masciulli Benedetta, Signore Giovanni, Migone Chiara, Fabiano Angela, Bizzarri Ranieri, Piras Anna Maria, Giacomelli Chiara, Marchetti Laura, Martini Claudia
Department of Pharmacy, University of Pisa, 56126 Pisa, Italy.
Department of Experimental and Clinical Medicine, University of Pisa, 56126 Pisa, Italy.
ACS Appl Bio Mater. 2024 Dec 16;7(12):8305-8318. doi: 10.1021/acsabm.4c01112. Epub 2024 Dec 5.
Exosomes are small extracellular vesicles (EVs) constituting fully biological, cell-derived nanovesicles with great potential in cell-to-cell communication and drug delivery applications. The current gold standard for EV labeling and tracking is represented by fluorescent lipophilic dyes which, however, importantly lack selectivity, due to their unconditional affinity for lipids. Herein, an alternative EV fluorescent labeling approach is in-depth evaluated, by taking advantage of green fluorescent protein (GFP) farnesylation (GFP-f), a post-translational modification to directly anchor GFP to the EV membrane. The performance of GFP-f is analyzed, in terms of selectivity and efficiency, in several typical EV experimental setups such as delivery in recipient cells, surface engineering, and cargo loading. First, the capability of GFP and GFP-f to label exosomes was compared, showing significantly higher GFP protein levels and fluorescence intensity in GFP-f- than in GFP-labeled exosomes, highlighting the advantage of directly anchoring the GFP to the EV cell membrane. Then, the GFP-f tag was further compared to Vybrant DiD lipophilic dye labeling in exosome uptake studies, by capturing EV intracellular fluorescence in a time- and concentration-dependent manner. The internalization assay revealed a particular ability of GFP-f to monitor the uptake of tagged exosomes into recipient cells, with a significant peak of intensity reached 12 h after administration by GFP-f but not Vybrant-labeled EVs. Finally, the GFP-f labeling capability was challenged in the presence of a surface modification of exosomes and after transfection for siRNA loading. Results showed that both procedures can influence GFP-f performance compared to naïve GFP-f exosomes, although fluorescence is importantly maintained in both cases. Overall, these data provide direct insight into the advantages and limitations of GFP-f as a tagging protein for selectively and accurately tracking the exosome route from isolation to uptake in recipient cells, also in the context of EV bioengineering applications.
外泌体是小型细胞外囊泡（EVs），是具有完全生物学特性、源自细胞的纳米囊泡，在细胞间通讯和药物递送应用中具有巨大潜力。目前用于EV标记和追踪的金标准是亲脂性荧光染料，然而，由于它们对脂质具有无条件亲和力，因此严重缺乏选择性。本文深入评估了一种替代性的EV荧光标记方法，该方法利用绿色荧光蛋白（GFP）的法尼基化（GFP-f），这是一种翻译后修饰，可将GFP直接锚定到EV膜上。在几种典型的EV实验设置中，如递送至受体细胞、表面工程和货物装载等方面，从选择性和效率方面分析了GFP-f的性能。首先，比较了GFP和GFP-f标记外泌体的能力，结果显示，与GFP标记的外泌体相比，GFP-f标记的外泌体中GFP蛋白水平和荧光强度显著更高，突出了将GFP直接锚定到EV细胞膜上的优势。然后，在囊泡摄取研究中，将GFP-f标签与亲脂性染料Vybrant DiD标记进一步进行比较，通过以时间和浓度依赖性方式捕获EV细胞内荧光。内化试验显示，GFP-f具有监测标记的外泌体被受体细胞摄取的特殊能力，在通过GFP-f给药后12小时达到显著的强度峰值，而Vybrant标记的EVs则未达到。最后，在对外泌体进行表面修饰以及转染以加载siRNA后，对GFP-f标记能力进行了挑战。结果表明，与未处理的GFP-f外泌体相比，这两种操作都会影响GFP-f的性能，尽管在两种情况下荧光都得到了显著保留。总体而言，这些数据直接深入了解了GFP-f作为一种标记蛋白在选择性和准确追踪外泌体从分离到被受体细胞摄取的过程中的优势和局限性，这也是在EV生物工程应用背景下的情况。
