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This article belongs to the Special Issue Optical Nanoparticles for Biomedicine The vertiginous increase in the use of extracellular vesicles and especially exosomes for therapeutic applications highlights the necessity of advanced techniques for gaining a deeper knowledge of their pharmacological properties. Herein, we report a novel chemical approach for the robust attachment of commercial fluorescent dyes to the exosome surface with covalent binding. The applicability of the methodology was tested on milk and cancer cell-derived exosomes (from U87 and B16F10 cancer cells). We demonstrated that fluorescent labeling did not modify the original physicochemical properties of exosomes. We tested this nanoprobe in cell cultures and healthy mice to validate its use for in vitro and in vivo applications. We confirmed that these fluorescently labeled exosomes could be successfully visualized with optical imaging. This study was supported by the Comunidad de Madrid, projects: “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/BMD-3867 (RENIM-CM)”; it was also co-funded by the European Structural and Investment Fund. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and it is a Severo Ochoa Center of Excellence (SEV-2015-0505). JV was supported by grants from Instituto de Salud Carlos III (PI18/01833), co-funded by European Regional Development Fund (ERDF) and from Comunidad de Madrid, project “S2017/BMD2737 (ExoHep-CM)”, co-funded by European Structural and Investment Fund. A. Santos-Coquillat is grateful for the financial support from Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III Sara Borrell Fellowship grant CD19/00136.

Exosomes are cell-derived nanovesicles with a proven intercellular signaling role in inflammation processes and immune response. Due to their natural origin and liposome-like structure, these nanometer-scale vesicles have emerged as novel platforms for therapy and diagnosis. In this work, goat milk exosomes are isolated and fully characterized in terms of their physicochemical properties, proteomics, and biochemical profile in healthy mice, and used to detect inflammatory processes by optical imaging. For the in vitro and in vivo experiments, the exosomes are covalently labeled with the commercial fluorophores sulfo-Cyanine 5 and BODIPY-FL to create nanoprobes. In vitro studies using confocal imaging, flow cytometry, and colorimetric assays confirm the internalization of the nanoprobes as well their lack of cytotoxicity in macrophage populations RAW 264.7. Optical imaging in the mouse peritoneal region confirms the in vivo ability of one of the nanoprobes to localize inflammatory processes. In vivo imaging shows exosome uptake in the inflamed peritoneal region, and flow-cytometric analysis of peritonitis exudates confirms the uptake by macrophage and neutrophil populations. These results support the promising use of goat milk exosomes as natural probes in the detection of inflammatory processes. This study has been funded by Instituto de Salud Carlos III, through the project "PI20/01632", co-funded by European Regional Development Fund (ERDF), "A way to make Europe" and by Comunidad de Madrid, project "Y2018/NMT-4949 (NanoLiver-CM)" and "S2017/BMD-3867 (RENIM-CM)", co-funded by European Structural and Investment Fund. This work has been also supported by "Diagnosis and treatment follow-up of severe Staphylococcal Infections with Anti-Staphylococcal antibodies and Immune-PET - Grant Fundación BBVA a Equipos de Investigación Científica 2018". A. Santos-Coquillat is grateful for financial support to Consejería de Educación e Investigación, co-financed by European Social Fund (ESF) grant PEJD-2018-POST/BMD-9592. A. Santos Coquillat is also funded by Instituto de Salud Carlos III, co-funded by European Social Fund "Investing in your future" (grant CD19/00136). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation. The CNIO Proteomics Unit is funded by the H2020 project EPIC-XS (ref. 823839). Biomedical Imaging has been conducted at the Advanced Imaging Unit of the CNIC (Centro Nacional de Investigaciones Cardiovasculares Carlos III), Madrid, Spain.