Caged Luciferin Labeling and Doxorubicin Encapsulation in Extracellular Vesicles

Five micrograms of EVs was incubated with 150 µM 1-(4,5-dimethoxy-2-nitrophenyl)ethyl ester (DMNPE)-caged-D-luciferin for 1 h, at 37°C, protected from light. Luciferin was released from the DMNPE group by UV-B (365 nm) photolysis (5 min, on ice) using an UV transilluminator (7 (link),21 (link)). Non-incorporated luciferin was removed using Exosome Spin Columns (Life Technologies). Luciferin-loaded EVs were added to 4T1^luc2 seeded in 96-well plates, or injected i.t. into mice, followed by BLI. Ten micrograms of EVs was mixed with 100 µg of dox (DOXO-cell^®) in PBS supplemented with 5 mM EDTA, and then the mixture was electroporated at 35 V and 150 µF using 0.4 cm cuvettes in a Gene Pulser II electroporator (Bio-Rad) (10 (link),21 (link)). Empty EVs were also electroporated. Non-incorporated dox was washed out with PBS by using ultracentrifugation (120,000 g, 70 min). Loaded dox was quantified by fluorescence detection in a Biotek Synergy HT microplate reader (emission, 594 nm; excitation, 480 nm) with Gen 5 software (BioTek). Fluorescence readings were compared with a standard curve of free dox. On average, yield of dox encapsulation was 10%.