Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin and inherent intercellular biomolecule delivery capability.An in-depth knowledge of their in vivo biodistribution is therefore essential.This work aimed to develop a novel, reliable and universal method to radiolabel exosomes to study their in vivo biodistribution.Methods: Melanoma (B16F10) cells were cultured in bioreactor flasks to increase exosome yield.B16F10-derived exosomes (ExoB16) were isolated using ultracentrfugation onto a single sucrose cushion, and were characterised for size, yield, purity, exosomal markers and morphology using Nanoparticle Tracking Analysis (NTA), protein measurements, flow cytometry and electron microscopy.ExoB16 were radiolabelled using 2 different approaches -intraluminal labelling (entrapment of 111 Indium via tropolone shuttling); and membrane labelling (chelation of 111 Indium via covalently attached bifunctional chelator DTPA-anhydride).Labelling efficiency and stability was assessed using gel filtration and thin layer chromatography.Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled ExoB16 (1x10 11 particles/mouse) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection.Results: Membrane-labelled ExoB16 showed superior radiolabelling efficiency and radiochemical stability (19.2 ± 4.53 % and 80.4 ± 1.6 % respectively) compared to the intraluminal-labelled exosomes (4.73 ± 0.39 % and 14.21 ± 2.76 % respectively).Using the membrane-labelling approach, the in vivo biodistribution of ExoB16 in melanoma-bearing C57Bl/6 mice was carried out, and was found to accumulate primarily in the liver and spleen (~56% and ~38% ID/gT respectively), followed by the kidneys (~3% ID/gT).ExoB16 showed minimal tumour i.e. self-tissue accumulation (~0.7% ID/gT).The membrane-labelling approach was also used to study ExoB16 biodistribution in melanoma-bearing immunocompromised (NSG) mice, to compare with that in the immunocompetent C57Bl/6 mice.Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the significantly lower tumour accumulation observed in the NSG mice (~0.3% ID/gT).Conclusion: Membrane radiolabelling of exosomes is a reliable approach that allows for accurate live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells.