Delivery of AuNCs into eXtracellular Vesicles by Fusion Using Ionizable Liposomes Cargos

Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by healthy and cancerous cells. EVs have heterogeneous size and three subtypes are described depending on the location of secretion: microvesicles, myelinosomes and exosomes. EVs are identified in human follicular fluid as a mode of communication in the ovarian follicle.¹ In addition EVs involved in cell-cell communication are considered as biomarkers for early cancer diagnosis. The analysis of their content and their labeling with easily detectable nanoparticles could enable the development of a powerful tool for the early diagnosis of specific diseases<br/><br/>In this view, gold nanoclusters (AuNCs) appear as a recent class of non-toxic fluorophores. Their brightness, their ultrasmall size (&lt; 2 nm) and large window of fluorescence lifetime (1ns - 1μs) and their good biocompatibility make them an attractive alternative as fluorescent probes for biological labeling and bioimaging.² In addition, their ultra-small size facilitates their clearance when they are injected into the body. Their small size makes them attractive to encapsulate them into liposomes without damaging the compartment integrity and then to be delivered into the extracellular vesicles. We previously demonstrate that their direct attractive electrostatic interaction with extracellular vesicles result into the labelling of their membrane or disruption of the EVs into multilamellar structures.³<br/><br/>We investigated encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. The efficiency of the process was correlated to the structural and morphological aspect of the AuNCs encapsulating vesicles via complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. We confirmed the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Finally, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.⁴<br/><br/>Herein, we investigate the interaction of fusogenic liposomes encapsulating the AuNCs with EVs extracted from human seminal fluids. The composition of the membrane was optimized to improve the fusion properties of the liposomes. The mixture of the liposomes and EVs results in lipid exchange as demonstrated by FRET experiments and in increased size revealed by flux cytometry. The resulting fused hybridosomes were successfully isolated by size exclusion chromatography. They exhibit typical fluorescence of the AuNCs together with an increased size in agreement with the expected size of the fused exosomes. Besides their interest to analyze the EVs content, such hybridosomes could serve as drug or macromolecule carriers or nanosensors in living organisms.<br/><br/><br/>[1] A.S. Neyroud et al. Basic and Clinical Andrology. 2021, 31, 25.<br/>[2] R. M. Chiechio et al. ACS Appl. Nano Mater. 2023, 6, 10, 8971–8980.<br/>[3] R. M. Chiechio et al. J. Phys. Chem. Lett. 2022, 13, 30, 6935–6943<br/>[4] R. M. Chiechio et al. Nanomaterials. 2022,12, 21, 3875.