A Review of Labeling Approaches Used in Small Extracellular Vesicles Tracing and Imaging

Figures & data

Figure 1 (A) Structure of the MemBright^TM markers. (B) Absorption and emission spectra of MemBright^TM probes (200 nM) in the absence (dashed lines) or presence of DOPC vesicles. (C) Turn-on mechanism of the MemBright^TM probes. (A)-(C) Reprinted from Cell Chem Biol, 26(4), Collot M, Ashokkumar P, Anton H, et al. MemBright: a family of fluorescent membrane probes for advanced cellular imaging and neuroscience. 600–614 e7, Copyright (2019), with permission from Elsevier.²⁰ (D) Confocal images of MemBright-Cy3 labeled Zmel1 EVs 3 hpi in Tg (mpeg1: GFP) (macrophage specific expression). Reprinted from Dev Cell, 48(4), Hyenne V, Ghoroghi S, Collot M, et al. Studying the fate of tumor extracellular vesicles at high spatiotemporal resolution using the zebrafish embryo. 554–572 e7, Copyright (2019), with permission from Elsevier.²¹

Figure 2 (A) Scheme of the fluorescence labelling method of sEVs surface using copper-free click chemistry with AlexaFlour^®488 (AF488)-azide. Reprinted from Xu L, Faruqu FN, Liam-Or R, et al. Design of experiment (DoE)-driven and uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling. J Extracell Vesicles. 2020;9(1):1779458. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. Creative Commons.⁴² (B)Combination the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with chemically active azide groups and biorthogonal click conjugation to modify and functionalize sEVs. Reprinted from Wang M, Altinoglu S, Takeda YS, Xu Q. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery. PLoS One. 2015;10(11):e0141860. Creative Commons.⁴³

Figure 3 (A) Schematic illustration of indirect IF labeling of CD63 and sEVs membrane stained with CM-Dil. Reprinted with permission from Chen C, Zong S, Wang Z, et al. Imaging and Intracellular Tracking of Cancer-Derived Exosomes Using Single-Molecule Localization-Based Super-Resolution Microscope. ACS Appl Mater Interfaces. 2016;8(39):25825–25833. Copyright © 2016 American Chemical Society.⁴⁶ (B) Schematic of aptamer-based DNA nanoassemblies on the surfaces of sEVs. Reprinted with permission from Wan S, Zhang L, Wang S, et al. Molecular Recognition-Based DNA Nanoassemblies on the Surfaces of Nanosized Exosomes. J Am Chem Soc. 2017;139(15):5289–5292. Copyright © 2017 American Chemical Society.⁴⁸ (C) Schematic illustration of screening for sEVs anchor peptides and functionalization on sEVs. Reprinted from Gao X, Ran N, Dong X, et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy. Sci Transl Med. 2018;10:444. Creative Commons.⁴⁹

Figure 4 (A) Determination of phase of tumor cell invasion around desmoplasia by EVs distribution. (B) Intraoperative label-free multimodal imaging system. (A) and (B) Reprinted from Sun Y, You S, Tu H, et al. Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging. Sci Adv. 2018. Creative Commons.¹¹⁹ (C)In vivo visualization of EVs from cancer tissue in rat mammary tumors by label-free multiphoton microscopy. Reprinted from You S, Barkalifa R, Chaney EJ, et al. Label-free visualization and characterization of extracellular vesicles in breast cancer. Proc Natl Acad Sci U S A. 2019;116(48):24012–24018. Creative Commons.¹²⁰ (D) EVs cluster distribution in rat mammary tissues by K-means clustering of CARS spectra. Reprinted with permission from © The Optical Society. Sun Y, Chen EW, Thomas J, Liu Y, Tu H, Boppart SA. K-means clustering of coherent Raman spectra from extracellular vesicles visualized by label-free multiphoton imaging. Opt Lett. 2020;45(13):3613–3616.¹²¹

Table 1 Fluorescent Labeling Techniques of sEVs Imaging

Technique	Agent	Wavelength (λex/λem)	Emitted Light	Modality	Target	Time	Feature	Ref
Membrane fluorescent dye	DiI	551/570 nm	Orange	FM	Lipid (M)	30min	-	[6]
	DiO	483/501 nm	Green	FM, FCM	Lipid (M)	30min	-	[7]
	DiD	646/663 nm	Red	FMT	Lipid (M)	30min	In vitro	[10]
	DiR	710/780 nm	NIR	FM	Lipid (M)	30min		[9]
	FM 4–64	558/734nm	Red	FM	Lipid (M)	15min	Low cross-staining	[11]
	PKH26	551/567nm	Orange	CLSM, RIM	Lipid (M)	10min,4min	Serious accumulation Size impact	[13,15]
	PKH67	490/502nm	Green	CLSM	Lipid (M)	10mim, -		[13,14]
	Mem	494–652 nm/512-672nm	Green to Red	CLSM	Lipid (M)	30min	No accumulation Low size influence	[18]
	CellMask^TM	522-649 nm/535-666nm	Green to Red	FM	Lipid (M)	70min	Slow internalization Cannot permeabilization	[19]
	MemBright^TM	499-689/506-713nm	Green to Red	CLSM, FM	Lipid (M)	30min	Low concentration Self-quenching	[21,22]
	3-NBD-cholesterol	470/541nm	Green	FCM	Lipid (M)	48h	For studies of cholesterol transfer	[24]
	WGA-647	650/670nm	Red	SMLM	Glycoprotein (M)	30min	Surviving from permeabilization	[26]
	R18	560/590nm	Orange	FM	Lipid (M)	30min	Membrane fusion studies	[27]
Bio-Conjugation	CFDA-SE	500nm/520nm	Green	FMi-OOC	Amino (I)	30min	Inadequacy for acid	[35]
	ExoTracker	550nm/670nm	Red	CLSM	Amino (M)	-	Suitable for acid	[37]
	TAMRA-NHS	553nm/575nm	Orange	FM	Peptide (M)	1h	-	[38]
	C5-maleimide-AF488	494nm/517nm	Green	CLSM	Tetraspanin (M)	5min	Specificity No influence	[40]
	DBCO-NHS+AF488-azide	480nm/520nm	Green	CLSM	Amino (M)	1h+4h	Flexibility Glycan analysis	[42]
	AHA/ManNAz+DBCO-Cy3	550nm/570nm	Orange	FCM, CLSM	-(M)	4h		[43]
Non-covalent	CD63/HER2-AF647	642nm/670 nm	Red	PALM/STORM	-	2h+1h	Specificity Functional influence	[46]
	TSG101/CD63/calnexin-AF594	590nm/620 nm	Red	CLSM	-	1.5h+1h		[47]
	LZH8-NA-FITC	490 nm/520 nm	Green	FCM	HepG2 (M)	30min	Specificity Biocompatibility	[48]
	MJ5C	-	Green	CLSM	PD-L1(M)	40min		[51]
	Rhodamine-CP05 AF680-CP05	525 nm/625 nm 680 nm/705 nm	Red Red	FM, FCM, IVIS	CD63(M)	12h	Specific binding to CD63.	[49]
	DPA-SCP	488nm/500-700 nm	Red	CLSM, IVIS	Charge(M)	2h	In vitro Electrostatic adsorption	[55,56]
Cell-Engineering	CD63-GFP	488 nm/507 nm	Green	CLSM	-	-	Stable labeling High technical requirements Limited clinical application	[58]
	CD63-iRFP682	663 nm/682 nm	Red	SIM	-	-		[59]
	palmNG	507 nm/514 nm	Green	CLSM	-	-		[60]
	PalmGFP PalmtdTomato	488 nm/507 nm 554 nm/581 nm	Green Red	CLSM, MP-IVM	-	-		[61]
	gLuc-LA	λem=480nm	Blue	IVIS	-	-		[64]
	Rluc	λem=460nm	Blue	CLSM, IVIS	-	-		[65]
	CD63NanoLuc	λem=420nm	Blue	IVIS	-	-		[66]
QD	InP/ZnS QD	596/615 nm	Red	FM, SPR		4h	SPR imaging; Multiplexing	[102]
	Si QD	405/420-490 nm	Green	SMLM	CD63(M)	8h	SMLM imaging; Biocompatibility	[103]
	GCDs	405/495−575 nm	Red	FM	HER2(M)	3h	Biocompatibility	[104]
	QD605-EpCAM QD655-EphA2	λem=605nm λem=655nm	Red	CLSM	EpCAM(M) EphA2(M)	-	Quantify sEVs abundance For clinical analysis	[105]
	4FB-HyNic-QD	λem=585nm	Red	CLSM	Amino(M)	2h+2h	Very stable High resolution live imaging ability	[106]

Abbreviations: CLSM, Confocal laser scanning microscope; FCM, Flow CytoMetry; FM, Fluorescence microscope; FMi-OOC, Fetal–Maternal interface Organ-on-chip; I, Inside; IVIS, In vivo optical imaging; M, Membrane; RIM, Retinal imaging microscopy; SIM, Structured Illumination Microscopy; SMLM, Single molecule localization microscopy; SPR, surface plasmon resonance; STORM, Stochastic optical reconstruction microscopy.

Table 2 Non-Fluorescent Labeling Techniques of sEVs Imaging

Technique	Agent	Injection	Modality	Target/Method	Time	Application	Ref
Radiolabeling	^131I	IV	SPECT/CT	Iodination(M)	30min	Biodistribution	[70]
	^125I-IBB	IV	γ-Counter	SAV-LA(M)	30min	Pharmacokinetics	[71]
	[^124I]NaI	IV	PET, γ-Counter	Iodination(M)	2h	Biodistribution	[72]
	[^111In] Trop	IV	SPECT/CT, γ-Counter	Shuttling (I)	20min	Biodistribution	[73]
	[^111In]DTPA			Chelation (M)
	^99mTcCl4	IV, IP, IN	SPECT/CT	Adsorption (M)	30min	Biodistribution Pharmacokinetics	[74]
	^99mTc-HMPAO	IV	SPECT/CT	Glutathione (I)	1h	Biodistribution	[75]
	^(99m) Tc-tricarbonyl	IV	SPECT/CT	Chelation (M)	30min	Biodistribution	[76]
	[^99mTc (CO)3(H2O)3]^+	IV	γ-Camera	DARPins G3(M)	1h	Biodistribution	[77]
	[^89Zr] Zr(oxinate) 4	IV	PET, γ-Counter	Lysine residues(M)	1h	Biodistribution Pharmacokinetics	[78]
	[^89Zr] Zr(oxinate) 4	IV	PET-CT	Shuttling (I)	20min	Biodistribution	[79]
	SCN-NOTA ^64Cu/^68Ga	IV, SC	PET, γ-Counter	Chelation (M)	5min,30min	Biodistribution	[80]
	^64Cu-CB-TE1A1P	IV	PET	Chelation (M)	3h	Pharmacokinetics	[81]
	Deuterium	-	SERS	Metabolism(W)	-	Tracking	[82]
Nanoparticles	Glu-GNPs	IV, IN	CT	Cell Internalization (I)	3h	Biodistribution	[83–85]
	AuNP-PEG-FA	IV	FM, SPECT/CT	Folic acid receptor(I)	2–24h	Biodistribution	[86]
	Au Nanostars	IV	PAI	Cell Internalization(I)	2d	Biodistribution, Therapy	[88]
	USPIO	IM	MRI	Cell Internalization(I)	1d,3d	Biodistribution	[92,93]
	SPIONs	IV	MRI	Electroporation(I)	1ms	Biodistribution, Therapy	[94]
	SPIONs	IV	MRI	Electroporation(I)	1ms	Biodistribution	[95]
	GdL	IV	MRI	Lipid insertion(M)	30min	Biodistribution	[98]
	Ag2Se@Mn QDs	IP	IVIS, MRI	Electroporation(I)	30min	Biodistribution	[107]
	GION	IP	MRI (PAI, CT, SERS)	Lipid insertion(I)	-	Biodistribution, Therapy	[108]
	NCs (λex/λem = 500/750 nm)	IV	FM, MRI, CT	Biosynthesis(I)	-	Biodistribution, Diagnosis	[109]
	V2C-QDs (λex/λem = 405/469nm)	IV	CLSM, PAI, MRI	RGD(I)	-	Biodistribution, Therapy	[110]
	Ag-Fe304 NCs	-	CLSM (CT, MRI)	Biosynthesis(I)	-	Uptake	[111]
Chemical contrast agent	Ce6	IV	PAI	Re-assemble(M)	1h	Biodistribution, Therapy	[113]
	FTH1	IM	MRI	Biosynthesis(I)	-	-	[115,116]
Label-free	–	–	Multimodal imaging system (MHG +MPF)	–	–	Intraoperative imaging	[119]
	NAD(P)H	–	Multiphoton Microscopy (MPF)	NAD(P)H	–	Dynamics	[120]
	–	–	Multimodal imaging system (MPF+CARS)	–	–	Biodistribution	[121]
	NAD(P)H	–	FLIM	NAD(P)H	–	Characterizing	[122]

Abbreviations: CARS, Coherent anti-Stokes Raman scattering; CLSM, Confocal laser scanning microscope; CT, Computed Tomography; FLIM, Fluorescence lifetime imaging microscopy; FM, Fluorescence microscope; I, Inside; IM, Intramuscular; IN, Intranasal; IV, Intravenous; IVIS, In vivo optical imaging; IP, Intraperitoneal; M, Membrane; MHG, Multiharmonic generation; MPF, Multiphoton autofluorescence; MRI, Magnetic Resonance imaging; PET, Positron emission tomography; SC, Subcutaneous; SERS, Surface enhanced Raman spectroscopy; SPECT, Single photon emission computed tomography SPR, Surface plasmon resonance; W, The whole sEVs.

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