Table 2.

Summary of EV characterization methods and examples of their utilization in in vivo drug delivery studies whenever available.

Method	Principle	Advantages	Disadvantages	In vivo drug delivery examples
Scanning electron microscopy	Capturing emitted electrons	Low sample volumes, single-EV analysis, surface topography	Little informative on surface structure and membrane features, risk of agglomeration and dehydration during sample preparation	[104]
Transmission electron microscopy	Detecting diffracted electrons	Low sample volumes, single EV analysis, visualizing membranous makeup and intra-vesicular structures	Potentially biased by sample preparation, toxic chemicals	[117, 155-157]
Cryogenic electron microscopy	Detecting scattered electrons at extremely low temperature	Most native EV morphology, single EV analysis, low sample volume	Specialized sample preparation with dedicated equipment	[158, 159]
Atomic force microscopy	Scanning interaction forces between sample and detector tip	Label-free sample preparation, single-EV analysis, three-dimensional topography	Morphological changes by immobilization and by tip in contact mode possible, specialized equipment	[126, 157]
Tunable resistive pulse sensing	Measuring resistance pulses	No sample preparation required, fast	Challenging for samples with unknown size ranges, risk of pore clogging, not EV-specific	[156]
Microfluidic resistive pulse sensing	Measuring resistance pulses	No sample preparation required, quick, low sample volumes	Challenging for samples with unknown size ranges, risk of pore clogging, not EV-specific	NA
Dynamic light scattering	Detecting fluctuations in light intensities scattered by moving particles	No sample preparation required, quick	Bulk measurement, approximate size distribution, not EV-specific, heavily hampered by polydisperse samples, limited resolution	[117, 126]
Nanoparticle tracking analysis	Recording the displacement of particles that scatter light	Single particle analysis, fast, no sample preparation required, fluorescent labelling possible to increase specificity	Not EV specific, slightly hampered by polydisperse samples, limited resolution	[123, 126, 155, 157-159]
Flow cytometry	Detecting scattered light	Fast, single EV analysis and multiparametric measurement possible	Limited resolution, confounded by swarming effect and refractory index	[160]
Bead-based flow cytometry	Detecting scattered light	Quick, multiparametric measurements	Bulk analysis only, risk of aggregation, limited information on size and concentration	[123, 158]
Nanoflow cytometry	Detecting scattered light	Single-EV analysis, improved resolution, multiparametric measurement possible	Confounded by refractory index	[161]
Imaging flow cytometry	Detecting scattered light with subsequent microscopic imaging	Single-EV analysis, improved resolution, multiparametric measurement possible	Dedicated equipment	NA
Single-particle interferometric reflectance imaging sensing	Recording interferometric reflectance	Single-EV analysis, multiparametric measurement	Dedicated equipment	[159]
Immunoblotting	Detecting antibody-labelled signals	Established method, simple contamination check	Bulk analysis, high sample volume, requires pure preparations	[123, 126, 127, 155-158]
Enzyme-linked immunosorbent assay	Detecting antibody-labelled signals	Established method, commercial kits available	Bulk analysis, cross-reactivity possible	[162]
Mass spectrometry	Separation of ionized molecules by their mass to charge ratio	High resolution, high-throughput, comprehensive data output	Bulk analysis, time consuming, requires pure preparations, sophisticated data analysis	[155, 156]
Next-generation sequencing	Transcript identification with single-nucleotide resolution	High sensitivity, high-throughput, comprehensive data output	Bulk analysis, time consuming, requires pure preparations, sophisticated data analysis	[155]
Quantitative polymerase chain reaction	Real-time detection of sequences selected a priori	Established protocols, high sensitivity	Bulk analysis, requires pure preparations and a priori knowledge	[117]