Dbco sulfo nhs

In order to prepare uPB-Exo, dibenzylcyclootyne (DBCO) groups were introduced on the surface of exosomes using a hetero bifunctional crosslinker [46 (link)]. Briefly, dibenzocyclooctynesulfo-N-hydroxysuccinimidyl ester (DBCO-sulfo-NHS) (Sigma, St.Louis, MO, USA) was mixed with exosomes (0.5 mg/mL) in PBS solution on a rotating mixer at room temperature (RT) for 4 h. After removing extra DBCO-sulfo-NHS was through filters (Millipore), DBCO conjugated exosomes (DBCO-Exo) obtained for linking N3@uPB via copper-free click chemistry. Therefore, uPB-Exo were obtained, and the morphology was observed by Transmission electron microscope (JEOL, Tokyo, Japan). NTA was performed using a NanoSight NS300 system (Malvern). The size distribution and zeta potentials of exosomes were measured using a Nano ZS90 device (Malvern). Ultraviolet–visible (UV–vis) absorption spectra of uPBs were obtained by a UV–vis spectrophotometer (UV-3600, Shimadzu, Japan).

Following previous research methods [39 (link)], we used a heterobifunctional cross-linking agent to introduce reactive dibenzyl ethylene oxide (DBCO) into amine-containing molecules on EVs. Specifically, 3 mM dibenzocyclooctyl-N-hydroxysuccinimide ester (DBCO-sulfo-NHS) (Sigma-Aldrich, St. Louis, MO, USA) was added to 0.5 mg/mL EVs in PBS and allowed to react in a rotary mixer at room temperature for 4 h. Unbound DBCO-sulfo-NHS was removed through four washing steps on 100 kDa ultrafiltration tubes (Millipore). Then DBCO-conjugated EVs (DBCO-EVs) were attached to azide-containing molecules through copper-free click chemistry. Specifically, 0.3 mM CAQK-N3 (SynthbioCo,. Ltd) or Cy5.5-azide was added to DBCO-EVs in PBS. The reaction was performed in a rotating mixer at 4 °C for 12 h and pH 7.4. The EVs were then floated on a 30% sucrose/D₂O cushion and centrifuged at 140,000 g for 70 min to remove unbound ligand. After being washed with PBS, the modified EVs were resuspended and stored at −80 °C until use. To assess the successful binding of EVs to azide ligands, we stained Cy5.5-bound EVs with DiO and examined them under a laser confocal microscope (Stellaris STED; LEICA, Wetzlar, Germany).

Reactive dibenzylcyclootyne (DBCO) were merged in amine-containing molecules on Exo applying a hetero-bifunctional crosslinker. 3 µM dibenzocyclooctyne-sulfo-N-hydroxysuccinimidyl ester (DBCO-sulfo-NHS; Sigma, St. Louis, MO, USA) was added to 0.5 mg/mL Exo in PBS and react at RT for 4 hrs. The DBCO-conjugated Exo (DBCO-Exo) were then ready for interact with azide-containing molecules via copper-free click chemistry. IMT peptide (CSTSMLKAC) and scrambled IMT peptide (CSKTALSMC) with an azide group on the lysine were purchased from SciLight Biotechnology Co. (Beijing, China). Cy5.5 azide was purchased from Sigma (USA). Specifically, 0.3 µM IMT peptide or scrambled IMT peptide with azide was added to DBCO-Exo in PBS, and subsequently added 0.3 µM Cy5.5 azide if needed. The reaction was performed on a rotating mixer at 4 ℃ for 12 h. Then, floated the Exo on a 30% sucrose/D2O cushion and centrifuged at 164,000 ×g for 90 min using a SW41Ti rotor (Beckman Coulter) to remove uncombined ligands. After washing with PBS, the IMT-Exo were resuspended and stored at -80 ℃ for further use. To assess the successful conjugation of Exo and IMT peptide, Cy5.5-conjugated Exo were applied to coverslips and imaged by fluorescence microscopy (Leica, USA). 0.5% BSA as an unmodified control was imaged 24 (link).