The use of exosomes as a drug delivery vehicle has gained considerable interest. To establish if exosomes could be utilized effectively for drug delivery, a better understanding of their in vivo fate must be established. Through comparisons to liposomal formulations, which have been studied extensively for the last thirty years, we were able to make some comprehensive conclusions about the fate of unmodified tumor-derived exosomes in vivo. We observed a comparable rapid clearance and minimal tumor accumulation of intravenously-injected exosomes, PC:Chol liposomes, and liposomes formulated with the lipid extract of exosomes, suggesting the unique protein and lipid composition of exosomes does not appreciably impact exosomes’ rate of clearance and biodistribution. This rapid clearance along with minimal tumor accumulation of unmodified exosomes limits their use as an anti-cancer drug delivery vehicle; however, when delivered intratumorally, exosomes remained associated with tumor tissue to a significantly greater extent than PC:Chol liposomes. Furthermore, experiments utilizing mice with impaired adaptive or innate immune systems, revealed the significance of the innate immune system along with the complement protein C5 on exosomes’ rate of clearance.

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A method for conjugation of ligands to the surface of exosomes was developed using click chemistry. Copper-catalyzed azide alkyne cycloaddition (click chemistry) is ideal for biocojugation of small molecules and macromolecules to the surface of exosomes, due to fast reaction times, high specificity, and compatibility in aqueous buffers. Exosomes cross-linked with alkyne groups using carbodiimide chemistry were conjugated to a model azide, azide-fluor 545. Conjugation had no effect on the size of exosomes, nor was there any change in the extent of exosome adherence/internalization with recipient cells, suggesting the reaction conditions were mild on exosome structure and function. We further investigated the extent of exosomal protein modification with alkyne groups. Using liposomes with surface alkyne groups of a similar size and concentration to exosomes, we estimated that approximately 1.5 alkyne groups were present for every 150 kDa of exosomal protein.

Polyethylene glycol (PEG) is widely utilized in drug delivery and nanotechnology due to its reported “stealth” properties and biocompatibility. It is generally thought that PEGylation allows particulate delivery systems and biomaterials to evade the immune system and thereby prolong circulation lifetimes. However, numerous studies over the past decade have demonstrated that PEGylation causes significant reductions in drug delivery, including enhanced serum protein binding, reduced uptake by target cells, and the elicitation of an immune response that facilitates clearance in vivo. This report reviews some of the extensive literature documenting the detrimental effects of PEGylation, and thereby questions the wisdom behind employing this strategy in drug development.