Butera Ester, Dupont Aurelien, Aimé Alexis, Ducarre Solène, Chiechio Regina M, Even-Hernandez Pascale, Contino Annalinda, Maccarone Giuseppe, Ravel Célia, Marchi Valérie
Institut des Sciences Chimiques de Rennes ISCR, UMR CNRS 6226, University Rennes, Campus de Beaulieu, 35042 Rennes, France.
Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Catania 95125, Italy.
ACS Appl Mater Interfaces. 2024 May 1;16(17):21643-21652. doi: 10.1021/acsami.4c02445. Epub 2024 Apr 16.
Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by both healthy and cancerous cells. They are considered as biomarkers for early cancer diagnosis and are involved in many pathologies and physiological pathways. They could serve as diagnostic tools in liquid biopsies, as therapeutics in regenerative medicine, or as drug delivery vehicles. Our aim is here to encapsulate luminescent nanoprobes in the aqueous compartment of human EVs extracted from reproductive fluids. The analysis and labeling of the EVs content with easily detectable luminescent nanoparticles could enable a powerful tool for early diagnosis of specific diseases and also for the design of new therapeutics. In this view, gold nanoclusters (AuNCs) appear as an attractive alternative as nontoxic fluorophore probes because of their luminescence properties, large window of fluorescence lifetimes (1 ns-1 μs), ultrasmall size (<2 nm), good biocompatibility, and specific ability as X-ray photosensitizers. Here, we investigated an attractive method that uses fusogenic liposomes to deliver gold nanoclusters into EVs. This approach guarantees the preservation of the EVs membrane without any breakage, thus maintaining compartmental integrity. Different lipid compositions of liposomes preloaded with AuNCs were selected to interact electrostatically with human EVs and compared in terms of fusion efficiency. The mixture of liposomes and EVs results in membrane mixing as demonstrated by FRET experiments and fusion revealed by flux cytometry and cryo-TEM. The resulting fused EVs exhibit typical fluorescence of the AuNCs together with an increased size in agreement with fusion. Moreover, the fusion events in mixtures of EVs and AuNCs preloaded liposomes were analyzed by using cryo-electron microscopy. Finally, the ratio of released AuNCs during the fusion between the fusogenic liposomes and the EVs was estimated to be less than 20 mol % by Au titration using ICP spectroscopy.
细胞外囊泡(EVs)是健康细胞和癌细胞分泌的众所周知的膜性颗粒。它们被视为早期癌症诊断的生物标志物,并参与许多病理和生理途径。它们可作为液体活检的诊断工具、再生医学的治疗手段或药物递送载体。我们的目标是将发光纳米探针封装在从生殖液中提取的人细胞外囊泡的水相区室中。用易于检测的发光纳米颗粒对细胞外囊泡内容物进行分析和标记,可以成为早期诊断特定疾病以及设计新疗法的有力工具。从这个角度来看,金纳米簇(AuNCs)因其发光特性、荧光寿命的大窗口(1纳秒 - 1微秒)、超小尺寸(<2纳米)、良好的生物相容性以及作为X射线光敏剂的特殊能力,成为一种有吸引力的无毒荧光团探针替代品。在这里,我们研究了一种利用融合脂质体将金纳米簇递送至细胞外囊泡的有吸引力的方法。这种方法保证了细胞外囊泡膜的完整性不受任何破坏,从而维持区室的完整性。选择预先装载金纳米簇的脂质体的不同脂质组成,使其与人类细胞外囊泡发生静电相互作用,并比较融合效率。脂质体和细胞外囊泡的混合物导致膜混合,这通过荧光共振能量转移(FRET)实验得到证明,融合通过流式细胞术和冷冻透射电子显微镜(cryo-TEM)揭示。所得融合的细胞外囊泡表现出金纳米簇的典型荧光,并且尺寸增大,这与融合一致。此外,通过冷冻电子显微镜分析了细胞外囊泡与预先装载金纳米簇的脂质体混合物中的融合事件。最后,使用电感耦合等离子体质谱(ICP)光谱通过金滴定法估计,在融合脂质体与细胞外囊泡融合过程中释放的金纳米簇的比例小于20摩尔%。
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