Separation, characterization, and standardization of extracellular vesicles for drug delivery applications

Abstract

Extracellular vesicles (EVs) are membranous nanovesicles secreted from living cells, shuttling macromolecules in intercellular communication and potentially possessing intrinsic therapeutic activity. Due to their stability, low immunogenicity, and inherent interaction with recipient cells, EVs also hold great promise as drug delivery vehicles. Indeed, they have been used to deliver nucleic acids, proteins, and small molecules in preclinical investigations. Furthermore, EV-based drugs have entered early clinical trials for cancer or neurodegenerative diseases. Despite their appeal as delivery vectors, however, EV-based drug delivery progress has been hampered by heterogeneity of sample types and methods as well as a persistent lack of standardization, validation, and comprehensive reporting. This review highlights specific requirements for EVs in drug delivery and describes the most pertinent approaches for separation and characterization. Despite residual uncertainties related to pharmacodynamics, pharmacokinetics, and potential off-target effects, clinical-grade, high-potency EV drugs might be achievable through GMP-compliant workflows in a highly standardized environment.

Keywords: Clinical pharmacology; Drug development; Exosomes; Pharmacology.

Figure 1.

Schematic representation of the biogenesis of exosomes, microvesicles, and apoptotic bodies. Exosomes are produced as intraluminal vesicles (ILV) by inward budding of early endosomes, forming a multivesicular body (MVB). MVBs can either fuse with lysosomes or with the cell membrane, thereby releasing exosomes into the extracellular milieu. Microvesicles are generated by outward budding of the plasma membrane, while apoptotic bodies are released during programmed cell death via plasma membrane blebbing. ER: endoplasmic reticulum. This figure as well as the graphical abstract were created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; www.https://smart.servier.com.

Figure 2.

Advantageous features of EVs that can be exploited for drug delivery purposes.

Figure 3.

Overview of EV characterization techniques and their capabilities. AFM: atomic force microscopy, cryo-EM: cryogenic electron microscopy; DLS: dynamic light scattering, ELISA: enzyme-linked immunosorbent assay, FCM: flow cytometry; IB: immunoblotting; IFCM: imaging flow cytometry; LC-MS: liquid chromatography mass spectrometry; MRPS: microfluidic resistive pulse sensing; MS: mass spectrometry; MS/MS: tandem mass spectrometry; NFCM: nanoflow cytometry; NGS: next-generation sequencing; NTA: nanoparticle tracking analysis; qPCR: quantitative polymerase chain reaction; SEM: scanning electron microscopy; SP-IRIS: Single-particle interferometric reflectance imaging sensing; TEM: transmission electron microscopy; TRPS: tunable resistive pulse sensing.