In the past years, nanotechnology has been increasingly applied in modern medicine owing to their advantages including high drug loading, increased drug stability, longer circulation time, EPR effect, and lower systemic toxicity compared to small molecular drugs. While most nanomaterials are fabricated synthetically, extracellular vesicles (EVs) present a special class as they are naturally produced by mammalian cells during inward budding of endosomes, which seems beneficial regarding stability, immunotolerance, capacity to cross natural barriers, and inherent targeting properties in vivo compared to synthetically prepared nanomaterials. Although EV-based cancer vaccines are currently tested in clinical phases I and II, a huge gap, preventing broad application of EVs, remains the lack of detailed information concerning the in vivo behavior of EVs. To understand the role of EVs and to optimize their application in future, long-term tracking of EVs is crucial.Single-walled carbon nanotubes (SWCNTs), tube-like, carbon-based nanomaterials with a diameter of around 1 nm and a length of around 100 nm, holding great promise as imaging probes for medical diagnostics owing to their optical properties in the short-wave infrared region. The fluorescent properties of SWCNTs are caused by excitons (bound electron-hole pairs) those energetically favorable dark states result naturally in a low photoluminescence yield. A way to circumvent this effect is oxygen-doping of SWCNT or introduction of sp3 defects which lead to lower energetic states which trap excitons and circumvent dark states. This results in a prolonged photoluminescence lifetime and an emission in the short-wave infrared (SWIR) window allowing imaging with deep optical penetration, reduced scattering, and low autofluorescence. In addition, SWCNTs possess a high photostability, low photobleaching, and negligible fluorescence blinking. The high photostability of SWCNTs in combination with an emission in the SWIR window as well as a size in the nanometer regime, mark SWCNTs as excellent imaging probes for in vivo tracking of extracellular vesicles.To achieve specific labeling of EVs with SWCNTs, we are using bioorthogonal chemistry. Metabolic engineering of cells is an elegant method to equip membrane proteins with bioorthogonal reactive groups. To this end, cells are treated with unnatural sugars which are metabolically incorporated into the glycan chain of proteins. Recently, it was revealed that EVs from metabolically engineered cells also carry unnatural sugars in their membrane proteins. We successfully isolated EVs from MCF-7 cells, which were treated with azide-bearing mannose. Using Cy5-DBCO, we could prove that azide groups present on EVs were able to react with the dye. EVs isolated from metabolically engineered HeLa cells seemed to possess a too low number of azide groups. Hence, EVs were modified successfully with DSPE-PEG-azide. Modification of EVs with Cy5-DBCO enabled in vitro imaging.We followed several strategies to introduce DBCO groups onto SWCNTs to link those to EVs. We were able to successfully functionalize SWCNTs with ssDNA carrying DBCO as an end-group as well as DPSE-PEG-DBCO. However, due to lack of specificity we embarked to use sp3 defects as reactive sites for modification of SWCNTs. Diazonium salt chemistry was used to introduce sp3 defects with a carboxylic acid group which allows further modification using a DBCO end-functionalized PEG linker.Ultimately, we will conjugate DBCO-modified SWCNTs with azid-bearing EVs to enable long-term tracking in vivo.