Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles

Glioblastoma is one of the deadliest cancers, therefore novel efficient therapeutic approaches are urgently required. One of such are nanobodies, prospective nano-sized bio-drugs with advantageous characteristics. Nanobodies can target intracellular proteins, but to increase their efficiency, the delivery system should be applied. Here, we examined small extracellular vesicles as a delivery system for anti-vimentin nanobody Nb79. Nb79 was loaded in small extracellular vesicles either by incubation with glioblastoma cells, by passive loading into isolated small extracellular vesicles or by sonication of isolated small extracellular vesicles. Small extracellular vesicles secreted by glioblastoma cells were isolated by ultracentrifugation on sucrose cushion. The size distribution and average size of sonicated and non-sonicated small extracellular vesicles were determined by nanoparticle tracking analysis method. The loading of Nb79 into small extracellular vesicles by incubation with cells, passive loading or sonication was confirmed by Western blot and electron microscopy. The effect of small extracellular vesicles on cell survival was determined by WST-1 reagent. Loading of small extracellular vesicles by incubation of cells with Nb79 was unsuccessful and resulted in substantial cell death. On the other hand, as confirmed by Western blot and electron microscopy, sonication is a successful method for obtaining Nb79-loaded small extracellular vesicles. Small extracellular vesicles also had an effect on cell viability. Small extracellular vesicles without Nb79 increased survival of U251 and NCH644 cells for 20-25%, while the Nb79-loaded small extracellular vesicles decreased survival of NCH421k by 11%. We demonstrated that sonication is a suitable method to load nanobodies into exosome, and these small extracellular vesicles could in turn reduce cell survival. The method could be translated also to other applications, such as targeted delivery system of other proteinbased drugs.