Analysis of extracellular vesicles as emerging theranostic nanoplatforms

Yanlong Xing【邢艳珑】 a, Ziyi Cheng【程子译】 a, Rui Wang【王锐】a, Chuanzhu Lv】吕传柱 a,*, Tony D. James b,*, Fabiao Yu【于法标】a,*

a Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China

b Department of Chemistry, University of Bath, Bath, UK

Coordination Chemistry Reviews 424 (2020) 213506

https://doi.org/10.1016/j.ccr.2020.213506

Abstract:

Extracellular vesicles (EVs) are nanoscale lipid membrane–bound vesicles that are secreted by cells of both prokaryotes and eukaryotes and carry bioactive cargos including proteins, nucleic acid and lipids from source cells. Given their prominent ability in transporting bioactive components, EVs are regarded as promising biomarkers for disease diagnosis and emerging therapeutic nanoparticles. However, to exert their effect in clinical applications, effective isolation and sensitive analysis of EVs from complex biofluids is required. Recent advances in EV-related research have provided feasible approaches for developing emerging therapeutic nanoplatforms using EVs. With this review, we aim to provide a comprehensive and in-depth summary of recent advances in diverse assay methods for EVs including fluorescence, Raman/Surface-enhanced Raman Spectroscopy (SERS) analysis and other methods, as well as their clinical potential in constructing EV-based theranostic nanoplatforms towards various diseases. In particular, microfluidic-assisted analysis sytems, single EV detection and the main approaches of utilizing EVs for therapeutic purposes are highlighted. We anticipate this review will be inspirational for researchers in related fields and will provide a general introduction to scientists with various research backgrounds.

Keywords: Extracellular vesicle; Fluorescence analysis; Raman analysis; Microfluidics; Theranostic Nanoplatforms

Table of Contents

1. Introduction

2. Classification, biogenesis and biological function

2.1 Classification and biogenesis of EVs

2.2 Physiological and pathological function in prokaryotes and eukaryotes

2.3 Visualizing the in vivo events of EVs

3. Isolation and characterization of EVs

3.1 Bulk method for the isolation of EVs

3.2 Microfluidic-based approaches

 3.2.1 Size-based sorting

 3.2.2 Contact-free isolation

 3.2.3 Immuno-based binding

3.3 Characterization of EVs

4. Analysis techniques of EVs

4.1 Fluorescence approach

4.1.1 Protein analysis

4.1.2 Nucleic acid analysis

4.1.3 Lipid analysis

4.2 Raman and SERS-based approaches

4.2.1 Label-free analysis of intact vesicles

4.2.2 SERS tag-labelled analysis

4.3 SPR technique

4.4 Electrochemical technique

4.5 Other methods

5. Single EV analysis

5.1 Exosome heterogeneity

5.2 Fluorescence-based analysis

  5.2.1 Flow cytometric anlaysis

  5.2.2 Fluorescence imaging-based analysis

5.3 Raman microspectroscopy and SERS

5.4 Other techniques

6. Application in diagnostics and therapeutics

6.1 Diagnosis and prognosis

6.2 Therapeutic application

6.2.1 Native EVs for therapy

6.2.2 Engineered EVs for cargo delivery

6.2.3 Challenges and considerations for EV-based therapy

7. Summary and future perspectives

Fig. 1. Schematic illustration of analysis of extracellular vesicle for emerging theranostic platforms. 

Fig. 2. Extracellular vesicle formation and release (left). Overall composition of exosomes (right). 

Fig. 3. a) In vivo tracking of breast-cancer-cell-derived exosomes in mice given intravenous injections of fluorescently labelled MCF-7-, MDA-MB-231-, and HS578T-secreted exosomes. Adapted with permission from ref [61]. Copyright 2018 American Chemical Society. b) Left: Intravital image of the central region of EL4 tumour. The white squares represent regions with the absence (1), low (2) and intermediate density (3) of detectable PalmGFPt vesicles. Right: Time-lapse recordings (min:s) highlighting a tethered vesicle. Adapted with permission from ref [62]. Copyright 2015 Nature Publishing Group. c) i) Fluorescence imaging of a 3 dpf Tg(kdrl:Hsa.HRAS-mCherry) embryo treated with ubi:CD63-pHluorin in the YSL. Box [EM] shows the region for EM analysis, indicated in (ii). Adapted with permission from ref [65]. Copyright 2019 Cell Press. d) Confocal imaging of Tg(mpeg1:GFP) embryos showing: (top) the attachment and uptake of EVs by endocytosis and (bottom) the sliding of EVs on the macrophage protrusion and its fast internalization. Adapted with permission from ref [58]. Copyright 2019 Cell Press.

Fig. 4. a) i) Cross-sectional view of the filters in the microdevice, indicating fluidics for the size-based isolation of EVs. ii) SEM images of the two filters I (pore diameter = 600 nm) and II (pore diameter = 20 nm). Adapted with permission from ref [83]. Copyright 2017 American Chemical Society. b) i) Schematic illustration showing the microfluidic chip for exosome separation from large EVs. ii) Fluorescent image showing separation of the binary mixture of 100 nm (green) and 500 nm (red) polystyrene particles. Adapted with permission from ref [87]. Copyright 2017 American Chemical Society. c) Schematic illustration of the integrated acoustofluidic device for isolating exosomes. i) Red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs) are firstly removed by the cell-removal module, and then exosomes (EXOs) are separated from other subgroups of EVs by the exosome-isolation module. ii) An optical image of the integrated acoustofluidic device. iii) Schematic illustration of the mechanism of size-based separation. Adapted with permission from ref [88]. Copyright 2017 National Academy of Sciences. 

Fig. 5. a) i) SEM image of the 3D herringbone structure of the microfluidic device (scale bar 100 μm). ii) Cells were infected with PalmtdTomato (red) or PalmGFP (green) to produce fluorescently labelled EVs. iii) Schematic illustration of red and green fluorescent EVs running through the device to capture tumour EVs. iv) Image of digitally rendered signals of captured fluorescent EVs. (Scale bar 1 μm). Adapted with permission from ref [91]. Copyright 2018 Nature Publishing Group. b) i) Workflow of the ExoSearch chip for mixing, isolation and multi-marker probing of circulating exosomes. ii)-iii) Bright-field images of the microchannels showing the mixing and isolation of exosomes by magnetic beads. iv) Image of aggregated exosome-bound immunomagnetic beads. v) TEM image of cross-sectional view of an exosome-bound immunomagnetic bead. Adapted with permission from ref [96]. Copyright 2016 The Royal Society of Chemistry. c) i) Schematic illustration of the integrated immunomagnetic exosomal RNA (iMER) platform for exosome enrichment, RNA capture and real-time analyses. ii) SEM image of magnetic beads after immunoaffinity capture. Adapted with permission from ref [108]. Copyright 2015 Nature Publishing Group.  

Fig. 6. a) Schematic illustration of the methods used for detecting circulating EVs. Adapted with permission from ref [124]. Copyright 2014 Nature Publishing Group. b) i) Schematic overview of the reconstruction of the exosome binding cavities and fluorescence-based sensing of intact exosomes. ii) Concentration-dependent signal of PC3-derived exosomes towards anti-CD9 antibody-functionalized binding cavities. iii) Response of exosomal CD9 and GGT1 on PC3-secreted and normal exosomes, and (Right) binding of tear exosomes to the cavities with the anti-CD9 antibody, anti-prostate specific antigen antibody, or control. Adapted with permission from ref [125]. Copyright 2019 John Wiley and Sons. c) i) Schematic of the workflow for multiplexed profiling of single cell EV secretion. ii) The raw data of multiplexed single-cell EV profiling showing fluorescence images. Right enlarged image indicates the fluorescent positive square spots intersecting CD63/CD81/CD9 antibody barcodes. Adapted with permission from ref [127]. Copyright 2019 National Academy of Sciences

Fig. 7. a) Working mechanism of the exosomal PD-L1 quantification analysis. b) Fluorescence imaging of PD-L1 positive HCT116 cells treated with or without deglycosylation by PNGase F and stained with DAPI (blue), the MJ5C aptamer (green) or a PD-L1 antibody (red). c) Flow cytometry assay and confocal imaging of A375 Exosome and K562 Exosome, showing the binding outcome of the MJ5C aptamer and PD-L1 antibody to PD-L1 positive) and negative exosome conjugated beads, respectively. Adapted with permission from ref [133]. Copyright 2020 John Wiley and Sons. 

Fig. 8. a) Schematic illustration of the fusion between the Vir-FVs and exosomes induced by HN and F protein. ii) Representative bivariate dot-plots of Vir-FVs showing the difference between normal and tumour exosomes based on sensing of the tumour-associated miRNAs. iii) Fluorescence response of the fused vesicles from the serum of healthy control and cancer patients (both n=5) after incubating with Vir-FVs. Adapted with permission from ref [145]. Copyright 2019 John Wiley and Sons. b) i) Schematic illustration of a thermophoretic sensor with nanoflares for in situ analysis of exosomal miRNAs. ii) Thermophoretic accumulation of nanoflare treated exosomes via localized laser heating to amplify the fluorescence response. iii) Fluorescence images of exosomes released by different breast cancer cell lines by thermophoretic sensor implemented with nanoflares. Scale bar, 50 μm. iv) Receiver operating characteristic analyses of exosomal miR-375 for differentiating ER+ BC patients from HD, as well as for early detection of ER+ BC (stages I, II). Adapted with permission from ref [147]. Copyright 2020 American Chemical Society. 

Fig. 9. a) Schematic diagram of the detection of exosomes using the nano bowl substrate and simulation of the electric field at different laser beam position. This simulation indicates that exosomes trapped inside the bowls are surrounded by uniform “hot-spots”. Adapted with permission from ref [166]. Copyright 2015 The Royal Society of Chemistry. b) i) Schematic illustration of the detection of exosomes by SERS. ii) The SERS spectra obtained for cancer cell-derived exosomes (line B, Alveolar; line C, H522; line D, H1299) and normal controls (line A). iii) Principal component scatter plot with coloured clusters of control (A, black square), alveolar (B, blue circle), H522 (C, orange star), and H1299 (D, red star) derived exosomes, respectively. iv) Principal components of PCA result in panel iii). The red area shows the Raman shifts related to NSCLC-secreted exosomes, and the blue area suggests the Raman shifts associated with the alveolar cell secreted exosomes. Adapted with permission from ref [169]. Copyright 2017 American Chemical Society. c) Schematic of the Raman scattering profiles of lung cancer cell-derived exosomes and comparison to the profiles of their potential surface protein markers. Adapted with permission from ref [170]. Copyright 2018 American Chemical Society. d) Schematic illustration of the label-free SERS analysis of exosomes and the deep learning model for early diagnosis of cancer patients. Adapted with permission from ref [171]. Copyright 2020 American Chemical Society. 

Fig. 10. a) i) Schematic overview of the Raman exosome assay. ii) and iii) are the side and top views of the interactions between exosome lipid membrane and SERS AuNR, respectively. iv) Protein profiling of exosomes derived from breast cancer cells MM231. Average SERS spectra (n=3) of exosomes targeting different surface proteins, using IgG as the control. v) Comparison of protein profiles on normal and cancer cells. Data are shown as the mean intensity of the 1497 cm^-1 peak with standard deviation (n=3). Adapted with permission from ref [179]. Copyright 2018 Ivyspring International Publisher. b) i) A schematic overview of the PDA chip and PEARL SERS tag-based exosome sensors. ii) SERS spectra of the anti-CD9, CD63, MIF and GPC1 groups for PANC-01-secreted exosomes. iii) Shapiro–Wilk analysis plots of the SERS outcome of serum samples of pancreatic cancer patients (n =71) and healthy controls (n=32) using the anti-MIF platform. The ordinate represents log values of Raman intensity. Adapted with permission from ref [180]. Copyright 2018 The Royal Chemical Society. 

Fig. 11. a) i) Schematic overview of exosomal miRNA detection using a SERS sensor. ii) From left to right, SEM images indicating upright plasmonic gold nanopillar SERS substrates, the top and side view of the plasmonic head-flocked gold nanopillar SERS substrates after solvent evaporation. iii) Cy3 intensity at 1150 cm⁻¹ was plotted against the concentration of target miRNAs to show the sensitivity of the SERS sensor for detecting target miRNAs. iv) Levels of target exosomal miRNAs were detected by the SERS sensor (blue bars) and qRT-PCR (red bars) in breast cancer cells. Adapted with permission from ref [185]. Copyright 2019 John Wiley and Sons. b) i) Schematic illustration of DSN-assisted SERS detection of microRNA. ii) SEM image of Fe3O4@Ag-SERS tags core-satellite assemblies. (iii) Determination of microRNA-10b concentration in exosome and residual supernatant plasma from three patients with PDAC. Adapted with permission from ref [186]. Copyright 2019 Elsevier.

Fig. 12. a) Schematic illustration of detection of various brain-derived subpopulations of plasma exosomes by SPR imaging Adapted with permission from ref [189]. Copyright 2018 American Chemical Society. b) i) Schematic overview of iMEX assay. Exosomes are captured on magnetic beads directly in plasma and labelled with HRP enzyme for electrochemical detection. The magnetic beads are coated with antibodies against CD63, an enriched surface marker in exosomes. The working (W) and the counter (C) electrodes are made of gold (Au), and the reference electrode (R) is made of silver/silver chloride (Ag/AgCl). HRP, horseradish peroxidase; TMB, 3, 3’, 5, 5’-tetramethylbenzidine. ii) Sensor schematic. The sensor can simultaneously measure signals from eight electrodes. Small cylindrical magnets are located below the electrodes to concentrate immunomagnetically captured exosomes. iii) Plasma samples from ovarian cancer patients (n = 11) and healthy controls (n = 5) were analysed by the iMEX assay. The levels of EpCAM and CD24 were much higher in cancer patients. The expression levels of EpCAM and CD24 (ξEpCAM vs ξCD24) were highly correlated (R² = 0.870). Adapted with permission from ref [193]. Copyright 2016 American Chemical Society. c) Illustration of the label-free electrochemical aptasensor for highly sensitive detection of exosomes. Adapted with permission from ref [194]. Copyright 2019 John Wiley and Sons. d) Reduced graphene oxide (RGO) FET biosensor. After anti-CD63 functionalization in the sensing region, exosomes can be directly bound to the CD63 antibody functionalized RGO FET biosensor for electrical and label-free detection. Adapted with permission from ref [195]. Copyright 2019 American Chemical Society. e) Principle of the electrogenerated chemiluminescence (ECL) biosensor for exosomes detection based on in situ formation of AuNPs decorated Ti₃C₂ MXenes nanoprobes. Adapted with permission from ref [196]. Copyright 2020 American Chemical Society.

Fig. 13. a) Schematic illustration of mapping subpopulations of cancer cell-derived EVs and particles by nano-flow cytometry. Adapted with permission from ref [210]. Copyright 2019 American Chemical Society. b) i) Exosomes were immobilized on a chamber slide under TIRF illumination during imaging. (Right) The principle of qPAINT-based analysis for profiling surface biomarkers of exosomes. ii) The merged image of the Cy5 and FITC channels. iii) The g value of four biomarkers on exosomes derived from breast cancer blood samples. iv)The detection results of seven unknown samples with the proposed model. Insert is the determination of cancer patients. Adapted with permission from ref [211]. Copyright 2019 John Wiley and Sons. c) Left-right: Scheme of the captured EV stained by fluorescent antibodies before imaging; Individual EV were labelled with fluorescent antibodies against conventional EV markers (CD9) as well as tumour markers (EGFR); Two-dimensional tSNE mapping of the 11-dimensional data set with an optimized clustering solution of 14 unique clusters. Adapted with permission from ref [212]. Copyright 2018 American Chemical Society. d) Schematic showing the droplet digital ExoELISA for exosome quantification. (Right) Droplet digital ExoELISA calibration results showing the dynamic range of the captured exosomes spans 5 orders of magnitude. Adapted with permission from ref [213]. Copyright 2018 American Chemical Society. 

Fig. 14. a) i) Image of an optical trap applied in RTM. ii) Cryo-TEM images of exosomes from rat hepatocytes. iii) Raman spectra of three independent vesicle groups from the same sample of rat hepatocytes. Adapted with permission from ref [217]. Copyright 2019 The Royal Society of Chemistry. b) i) Schematic illustration of the SERS measurements of AuNP coated ELVs. Each spectrum is derived from another vesicle by moving the laser to a different spatial location (e.g., 1, 2, 3). The presence of a gold coated ELV was verified by a scattering signal (location 2). Scale bar =10 μm. ii) SERS spectrum of RBC-released ELVs coated with AuNP. Red arrows illustrate peaks arising from the DMAP AuNP coating. Green arrows show presumed ELV related peaks. Adapted with permission from ref [220]. Copyright 2016 John Wiley and Sons. c). i) and ii) showed the iPM and fluorescent images of exosomes. iii) Real-time iPM signals of an exosome bonded to antibodies or that underwent Brownian motion. Adapted with permission from ref [221]. Copyright 2018 National Academy of Sciences.

Fig. 14. a) i) Image of an optical trap applied in RTM. ii) Cryo-TEM images of exosomes from rat hepatocytes. iii) Raman spectra of three independent vesicle groups from the same sample of rat hepatocytes. Adapted with permission from ref [217]. Copyright 2019 The Royal Society of Chemistry. b) i) Schematic illustration of the SERS measurements of AuNP coated ELVs. Each spectrum is derived from another vesicle by moving the laser to a different spatial location (e.g., 1, 2, 3). The presence of a gold coated ELV was verified by a scattering signal (location 2). Scale bar =10 μm. ii) SERS spectrum of RBC-released ELVs coated with AuNP. Red arrows illustrate peaks arising from the DMAP AuNP coating. Green arrows show presumed ELV related peaks. Adapted with permission from ref [220]. Copyright 2016 John Wiley and Sons. c). i) and ii) showed the iPM and fluorescent images of exosomes. iii) Real-time iPM signals of an exosome bonded to antibodies or that underwent Brownian motion. Adapted with permission from ref [221]. Copyright 2018 National Academy of Sciences.

Fig. 15. Overall illustration of the therapeutic used of EVs. Native EVs secreted by stem cells including MSC and PSC exert their ability in cell-free therapy. Engineered EVs are used as the delivery vehicles of various therapeutic entities including proteins, nucleic acids, therapeutic drugs, as well as functional nanoparticles. 

Fig. 16. a) Schematic illustration of the Pt(lau)HSA NP-loaded exosome platform (NPs/Rex) for efficient chemotherapy of breast cancer. Adapted with permission from ref [279]. Copyright 2019 American Chemical Society. b) Schematic of the design of FA-AuNR@RGD-DOX-Exos and their antitumor effect under NIR irradiation. The therapeutic efficiency of FA-AuNR@RGD-DOX-Exos was evaluated in a tumour-bearing mouse model. (Bottom) Schematic of FA-AuNR@RGD-DOX-Exos as an effective nanoplatform for targeted delivery and chemo-photothermal synergistic tumour therapy. Adapted with permission from ref [282]. Copyright 2018 John Wiley and Sons. 

Fig. 17. a) Schematic illustration of the method for preparing biomimetic EMP NPs for homotypic targeting and intracellular delivery of guest proteins. I: Caging protein cargo by self-assembly of blocks of inorganic nodes and organic ligands to synthesize MOF-protein NPs. II: Extraction of extracellular vesicle membrane (EVM) through a hypotonic treatment of EVs. III: Self-assembly of EVM on MP nanoparticle surface by ultrasonication and extrusion to form EVMOF-protein (EMP) NPs. IV: Systemic and intracellular delivery of the guest proteins by EMP NPs. Adapted with permission from ref [290]. Copyright 2018 American Chemical Society. b) Schematic illustration of EXPLOR technology. In EXPLOR-producing donor cells, CRY2 protein was fused to a cargo protein, and CIBN was conjugated with the exosomal surface biomarker, CD9 protein. The reversible PPI between CIBN and CRY2 fusion proteins can be induced by blue light illumination i. Under continuous blue light irradiation, the cargo proteins are guided to the inner surface of the cell membrane or the surface of early endosomes. MVBs then readily release cargo protein-carrying exosomes (EXPLORs) from the cells by membrane fusion with the plasma membrane. After exocytosis, EXPLORs can be easily separated and purified in vitro. Purified EXPLORs can be used to deliver the cargo proteins into target cells through membrane fusion or endocytosis. Bottom grey boxes highlight the essential steps from EXPLORs biogenesis to target cell delivery. Adapted with permission from ref [291]. Copyright 2016 Nature Publishing Group. 

Fig. 18. a) i) Exosome surface functionalization with CD9-HuR fusion protein. ii) Illustration of the procedure how miR-155 or miR-328 was encapsulated by the CD9-HuR fusion protein functionalized exosomes. iii) Schematic illustration of the study. In the packaging cells, CD9-HuR fusion protein recruits the target miRNAs or mRNAs to the exosomes via the RNA-HuR recognition. In the recipient cells, miRNAs or mRNAs of interest are released from the CD9-HuR exosomes and thus act as functional miRNAs or mRNAs. Adapted with permission from ref [304]. Copyright 2019 American Chemical Society. b) i) Representative immunofluorescent staining images of DRG neurons treated with medium, nontargeting control siRNA, MSC-Exo, PTEN-siRNA, or ExoPTEN. Neurons (green), nuclei (blue). Scale bar: 200 μm. ii) Schematic illustration of MSC-Exo loaded with PTEN-siRNA. iii) Immunofluorescent images of control and treated lesion microenvironment. Adapted with permission from ref [305]. Copyright 2019 American Chemical Society.

Fig. 19. a) i) Preparation procedure of the V2C-TAT@Ex-RGD. ii) Schematic diagram of cancer cell membrane and nucleus organelle dual-target V2C-TAT@Ex-RGD nano-agent for multimodal imaging-guided PTT in the NIR-II bio-window at low temperature. Adapted with permission from ref [318]. Copyright 2019 American Chemical Society. b) Schematic of the microfluidic sonication approach to assemble biomimetic core−shell NPs for immune evasion-mediated tumour targeting. (Left) One-step microfluidic synthesis of exosome membrane (EM)-, cancer cell membrane (CCM)-, and lipid-coated PLGA NPs via the combined effects of acoustic pulses and hydrodynamic mixing. (Right) EMPLGA NPs indicating reduced uptake by peripheral blood monocytes and extracellular matrix macrophages and superior homotypic targeting, compared to CCM-PLGA NPs and lipid-PLGA NPs. Adapted with permission from ref [320]. Copyright 2019 American Chemical Society