Tracking Exosomes in Vitro and in Vivo to Elucidate their Physiological

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Tracking Exosomes in Vitro and in Vivo to Elucidate their Physiological Functions: Implications for Diagnostic and Therapeutic Nanocarriers Li-Ming Shen, Linglai Quan, and Jing Liu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00601 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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ACS Applied Nano Materials

Tracking Exosomes in Vitro and in Vivo to Elucidate their Physiological Functions: Implications for Diagnostic and Therapeutic Nanocarriers Li-Ming Shen1, 2, Linglai Quan1, 2, Jing Liu1, 2* 1 Regenerative Medicine Center, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China 2 Stem Cell Clinical Research Center, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China * Correspondence: [email protected]; Tel.: +86-411-83635963-2170

Abstract Exosomes are nano sized extracellular vesicles secreted by a variety of cell types and widely distributed in body fluids. With bilayer lipid membrane encapsulating genetic and proteomic information, exosomes play an important role in cell-to-cell communication. As newly emerged extracellular vesicles, exosomes has gathered wide scientific and clinical research interest owing to their important functions under both physiological and pathological conditions. Furthermore, because some bioactive cargo, e.g. mRNA, miRNA and proteins, is specifically sorted into exosomes from parent cells, noninvasive analysis of the molecular profiles of circulating exosomes may reveal potential biomarkers for disease diagnosis and prognosis. Moreover, inherent features of stability, low immunogenicity, targeted delivery as well as the ability to overcome natural barriers facilitate the potential for exosomes to serve as drug delivery vehicles for cancer, neurodegenerative diseases and regenerative medicine. In practice, the further investigation and application of exosomes requires accurate knowledge of their specific metabolic pathways in vitro and in vivo, such as, their release from parent cells and uptake by recipient cells, tissue distribution and body liquids levels. The establishment of exosomes tracking protocols can effectively solve this problem. Using cells or other enclosed vesicles as reference, exosomes labeling and tracking strategies first adopted fluorescence microscopy, following which bioluminescence imaging, magnetic resonance imaging and computed tomography were also successively applied to investigate exosomes function in

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realistic pathophysiological contexts. In this review, we first introduce the biogenesis and compositions of exosomes together with two other kinds of extracellular vesicles, microvesicles and apoptotic bodies, and then discuss their applications in the field of diagnosis and therapeutics. Subsequently, the latest developments of exosomes tracking techniques are reviewed. Finally, we present the remaining challenges and further perspectives in this exciting and promising field. Keywords: Extracellular vesicle, exosome, fluorescence, bioluminescence, MRI, CT


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1. INTRODUCTION Extracellular vesicles (EVs) are cell-derived vesicles with nuclear acids and proteins packaged in phospholipid bilayer membrane. EVs carry information from their progenitor cells and serve as messengers for intercellular communication. As a membrane enveloped structure, multiple phospholipid compositions have been identified in EVs, including cholesterol, phosphatidylcholine, phosphatidylserine, ceramide, and saturated fatty acids [1, 2]. EVs have also been confirmed to load a variety of proteins, such as transcriptional factors, surface receptors, and heat shock proteins [3, 4], along with nucleic acids, including microRNA, mRNA [5, 6] and non-coding RNAs (ncRNA), as well as DNA [7]. The three types of EVs, exosomes, microvesicles (MVs), and apoptotic bodies (Abs), are usually discriminated by their biogenesis, size and membrane composition. Recently, exosomes have attracted a wide interest in diagnostic and therapeutic field, therefore, profiling exosome half-life, circulation, and cargo delivery in vivo is urgently needed to elucidate the associated intricate intercellular communication functions. In this review, we will first introduce the biogenesis, composition, biological behavior and functions of EVs, which served as foundation for constructing imaging methodologies, and then analyze the latest development in tracking exosomes in vitro and in vivo. Finally, the remaining challenges and perspectives of exosomes imaging technique are presented. 1.1 Overview of EVs: Biogenesis and Compositions Exosomes were first discovered as vesicles discharging transferrin receptors from maturing blood reticulocytes in 1983 [8, 9]. The morphology of exosomes shows a cup shaped appearance with diameters ranging from 30 to 150 nm [10]. The buoyant density of exosomes in sucrose is 1.13-1.19 g/mL; and they precipitate at over 100 000g [11]. Exosomes originate from early endosomes which are products of plasma membrane internalization. Then inward budding of endosomes produce multiple intraluminal vesicles (ILVs) therein, whereupon endosomes turn into multivesicular bodies (MVBs). With twice membrane invagination, the membrane orientations of ILVs are in accord with the cellular plasma membrane. A small number of MVBs escaped the lysosomal degradation pathway, fused with the plasma membrane and



released internal ILVs which are thereafter termed as exosomes (Figure 1A) [12]. The endocytotic origin and small size of exosomes are recognized as their most significant difference from MVs and Abs. According to the biogenesis, it is obvious that contents in cytoplasm, such as microRNA, cytoskeleton components, and signal transducers are packaged into exosomes [11]. In addition, transmembrane protein tetraspanins, such as CD9, CD63 and CD81, are identified on the surface of exosomes, which would facilitate the directional transportation and adherence to their target [13]. Moreover, tetraspanins are widely utilized as markers to verify the identity of exosomes after isolation from cell culture medium or body fluids. In summary, most exosomal proteins are found in the cytosol, the membrane of endocytic compartments, or the plasma membrane. However, the biogenesis of exosomes is intricate and delicate, investigation on the pattern of cell sorting of contents into exosomes is still underway. Furthermore, considering that almost all cell types can secrete exosomes, exosomes are consequently widely distributed in biological fluids, including the blood, urine, saliva, and breast milk. The outward budding of plasma membrane and fission generates MVs, with this process resulting in the heterogeneous morphology and highly variable diameters (100 to 1000 nm) of MVs. Unlike exosomes, MVs sediment at relative lower speed at 10000 g. MVs are usually formed in membrane regions, termed lipid rafts of originating cells, which are characterized by high levels of cholesterol and signalling complexes (Figure 1A) [14]. The outer membrane of MVs contains phosphatidylserine residues and other markers, which are dependent on cell type, e.g., β1 integrin [15, 16], matrix metalloproteases and their activators[17], P selectin glycoprotein ligand 1 [18] , cytokines and chemokines [15, 19] , vascular endothelial growth factor and fibroblast growth factor 2 [20]. Generally, cells secret a certain amount of MVs in resting state, whileas the yield can increase significantly under stimulation, such as increased Ca2+ [21], cytokine exposure [15], and anticancer drug treatment [22]. Thus the heterogeneous and larger sizes together with payload of distinctive molecules comprise the main features of MVs. Abs are by definition products of apoptotic cell disassembly. Apoptotic cell surface


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grew plasma membrane blebs which became apoptotic membrane protrusions afterwards. Fragmentation of surface protrusions formed individual Abs (Figure 1B). Abs are ultimately taken up by tissue phagocytes or neighboring cells [23]. Similar to MVs, Abs are also heterogeneous with sizes ranging from 50 to 5000 nm, and they sediment between 1200 and 100,000 g [24]. The generation of Abs is regulated by specific molecular factors, such as the Rho-associated protein kinase ROCK1 and the plasma membrane channel pannexin 1 [25]. According to their biogenesis, Abs possess the same membrane as their parenting cells. Compared with exosomes and MVs, Abs are characterized by the presence of intact organelles within the vesicles.

Figure 1 Biogenesis of EVs. (A) Vesiculation of endosome formed MVBs which subsequently fused with plasma membrane to release exosomes. The orientations of transmembrane protein on exosomes are the same as plasma membrane. Outward budding of plasma membrane and fission generated MVs. (B) Fragmentation of irregular protrusions on the surface of apoptotic cells generates Abs. Reprinted with permission from Lee et al [14].

1.2 Applications of Exosomes Exosomes were initially recognized as scavenger disposing unwanted proteins from cells; however, in-depth research revealed their physiological functions as intracellular communication nanocarriers. The contents of exosomes and their biological functions depend on the cell of origin. Exosomes containing functional mRNA, miRNA and proteins transport the information to neighboring or distant cells, then modulate their microenvironment and the recipient cells. The physiological



functions of exosomes indicated their clinical significance as diagnostic and therapeutic nanocarriers. 1.2.1 Diagnostic Nanocarriers It is reported that tumor cells secret more exosomes than healthy cells, and that tumor derived exosomes carry specific cellular proteins, mRNAs, and miRNAs of the originating cells, which may further affect ambient normal cells to stimulate metastases [26], immune system modulation (activation/suppression)[27, 28], and drug resistance [22]. Accordingly, circulating exosomal miRNAs and proteins in body fluids constitute promissing noninvasive diagnostic and screening tool for cancer detection, discrimination between histotypes and prognosis [29]. For example, Melo et al discovered that circulating exosomes enriched in glypican-1 have potential for the prognosis of patients with pancreatic cancer. Based on this finding, they established informative early detection technique of pancreatic cancer using flow cytometry-based isolation of exosomes from the serum of affected patients [30]. 1.2.2 Therapeutic Nanocarriers As natural endogenous nano vesicles that can function as a protective barrier against premature transformation, exosomes outperform their counterparts, i.e., liposomes and synthetic nanoparticles, as therapeutic nanocarriers. The transmembrane proteins and ligands on exosomes surface likely promote tissue-specific endocytosis. In particular, their intrinsic features, such as stability, immune-tolerance in circulation systems, the capacity to cross natural barriers and inherent targeting properties are beneficial for them working as drug carriers. On the one hand, exosomes derived from certain cell types are intrinsically therapeutic. Mesenchymal stem cells (MSCs) derived exosomes still possess certain functions of parent MSCs, and work through paracrine signaling to promote repair of damaged tissues, such as skeletal muscle regeneration [31] and brain damage recovery[32, 33]. Moreover, exosomes possess better biocompatibility than their progenitor cells. Unlike engrafted stem cells, exosomes circumvent the shortcomings of limited survival and the reduced regenerative capacity caused by immune-mediated rejection [32]. Exosomes secreted by dendritic cells transfer functional MHC class I/peptide complexes between each


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other and are promising as potent cell-free peptide-based vaccine [34]. On the other hand, as enclosed biological vesicles, exosomes can load therapeutic agents which are unstable or insoluble in body fluids into their lipophilic outer membrane or hydrophilic interior cavity. Besides, exosomal membrane can be further modified to enhance tissue-specific homing which would facilitate targeting drug delivery. Drug molecules can be preloaded into exosomes both in vitro and in vivo. In vitro drug loading usually requires purifying exosomes from cell culture medium or body fluids in advance, and then incorporate drugs into exosomes through incubation, sonication or electroporation procedures. Combined with the superior performance of exosomes in bypassing the blood-brain barrier, drug loaded exosomes can ultimately deliver their payload into the brain. Accordingly, it has been suggested that exosome protection might constitute a solution for the dilemma that many drugs are unable to pass through the blood-brain barrier [33]. For example, T cells derived exosomes encapsulated anti-inflammatory agents curricum via incubation at 22 °C for 5 minutes. Intranasal administration of exosome-curcumin was selectively taken up by brain macrophages, microglial cells, in lipopolysaccharide-induced brain inflammation mouse model. This induced apoptosis of microglial cells and inhibited brain inflammation [35]. Kamerkar employed normal fibroblast-like mesenchymal cells derived exosomes to deliver siRNA or shRNA targeting oncogenic KrasG12D specifically to pancreatic cancer cells. CD47 on exosomes inhibited phagocytosis by monocytes and macrophages, leading to their enhanced uptake by tumor cells. The RNA loaded exosomes administered by mouse through intraperitoneal injection suppressed pancreatic tumor growth and improved their survival significantly [36]. Except for inherent surface ligands, exosomes can also be modified by expressing targeting moiety to deliver drugs into specific cells. Dendritic cells derived exosomes were engineered by fusing exosomal membrane protein Lamp2b to neuron-specific RVG peptide. Subsequently, exogenous siRNA were successfully loaded into purified exosomes by electroporation. Due to the targeting moiety, intravenously injected exosomes transported BACE1 (a therapeutic target in Alzheimer’s disease) siRNA exclusively to neurons, microglia and oligodendrocytes in the brainand induced



notable gene knockdown in mice [37]. As for in vivo therapeutic agents loading, human bone marrow mesenchymal stem cells (hBMSCs) derived exosomes overexpress siFas and anti-miR-375 after transfecting cells with plasmid encoding these factors. hBMSCs derived exosomes silenced Fas and miR-375 in transplanted islets, and suppressed immune rejection simultaneously, which had a synergistic promoting effect on islet transplantation [38]. 1.3 Cellular Uptake of Exosomes Circulating exosomes perform intercellular communication function by entry into the recipient cells and release of containing genetic materials and signaling protein. Binding of exosomes to target cells rely on ligands on cell surface which will then trigger intracellular signaling pathways. Uptake of exosomes through different endocytosis pathways have been elucidated, including clathrin-mediated endocytosis, micropinocytosis [39] and lipid raft-dependent endocytosis [40]. Heusermann claimed the internalization of exosomes is similar to bacteria and viruses. Exosomes travel along filopodia and enter the cells through endocytosis at the filopodial base. After internalization, intact exosomes shuttle within endocytic vesicles to interact with the endoplasmic reticulum before settling in the lysosome finally [41]. Direct fusion with membrane of recipient cell is another possible pathway for internalization of exosomes. This process requires both of them possessing similar fluidity, physical conditions, such as acidic pH and the physiologic state of the recipient cells [42]. It’s also reported that surface glycosylation patterns have an impact on exosomes uptake, and heparin sulfate proteoglycans is involved, hence the uptake efficiency of exosomes can be obviously suppressed by heparin [43]. The elaborate metabolic pathway of exosomes in vitro and in vivo is critical to investigate their applications in diagnostic and therapeutic field. Models to study the interactions between exosomes and cells usually relay on imaging the movement of isolated exosomes in the recipient cells upon incubation. Exosomes were first labeled with optical reporter and then tracked under optical microscopy. Tracing the movement of exosomes from donor cells to recipient cells, or biodistribution and pharmacokinetics of exogenously administered exosomes, are more complicated and


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requires versatile imaging techniques. 2. IMAGING OF EXOSOMES In this review, we classify the present imaging techniques according to the detection method, i.e., fluorescence labeling, bioluminescence imaging, MRI, and CT. The reporters, advantages, disadvantages, and specific application scopes of each technique are summarized in Table 1. Table 1 Summary of exosomes imaging techniques Imaging technique

Fluorescence

Reporters

Advantages

Organic dyes

Strong and long lasting fluorescence signal, multiple colors

Genetically encoded fluorescent proteins Immunofluorescent reporters Fluorescent nanomaterials

Bioluminescence

Gluc

MRI

SPIONs, USPIO

CT

99mTc-HMPAO GNPs

Disadvantages

Scopes

Ref

In vitro

26, 44-53

Multidirectional exosomes transport among cells

Staining other lipid entities and providing false positive signal Limited labeling efficiency

In vitro and in vivo

54-58

High specificity

High cost

In vitro

59

Tedious synthesis process

In vitro

60,61

Transient signal unable to track in the long term

In vivo and ex vivo

57,62

Limited labeling efficiency

In vivo

63, 64

Labeling purified exosomes

In vivo

65, 66

Excellent optical properties, functionalization with targeting moiety High sensitivity, low background signal, no need of excitation source Deep penetration, high-quality 3D imaging of tissues with anatomical details High spatial and temporal resolution

2.1 Fluorescence Labeling Optical imaging represents the most popular tracing technique in molecular and cellular biology, among which fluorescence microscopy constitutes a powerful tool to investigate cellular and subcellular metabolic activity, especially in living cells. Fluorescent probe-labeled analytes emit fluorescence signal upon excitation which is recorded by fluorescent microscopy. Fluorescent microscopy is easy to operate, able to trace analytes in real time and noninvasively, and is compatible with multiform samples. In particular, fluorescent microscopy can monitor the behaviors of a single cell in real time, which is beneficial to observe the interaction between exosomes and cells. To date, organic dyes, genetically encoded fluorescent proteins,



immunofluorescent reporters and fluorescent nanomaterials have already been utilized for labeling exosomes. 2.1.1 Organic Dyes Optical imaging of exosomes was inspired by plasma membrane labeling strategies owing to their similar membrane structure. Lipophilic fluorescent membrane dyes emit strong fluorescence signal after insertion of their long aliphatic tails into the lipid bilayer of exosomes. Commonly used reporters include PKH67 (λex/λem=490/502 nm) [44-46], PKH26 (λex/λem=551/567 nm) [26, 47], DiO (λex/λem=484/501 nm) [48-50], DiI (λex/λem=549/565 nm) [51-53], DiR (λex/λem=750/780 nm) [50], and FM 4-64 (558/734 nm) [50]. These organic dyes are commercially available and emit stable and long lasting fluorescent signals in the exosomal membrane which facilitate the observation of interactions between exosomes and recipient cells, especially in vitro. The labeling mechanism is the same for organic dyes, but they differ in fluorescence quantum yiled, color and half-life etc. The emission wavelength of organic dyes ranges from 502 to 734 nm, and this provides researchers multiple choices when picking suitable probes to label exosomes. In practice, exosomes lipid labeling with lipophilic dyes may not reflect their exact half-life, because organic dyes can also stain other lipid entities and be retained for long periods, thereby providing false positive signal and interfering with accurate spatiotemporal assessments of exosomes fate [54]. On the other hand, fluorescent chemical labeling method is only applicable for purified exosomes from conditioned culture medium or body fluids, but is unable to stain exosomes in originating cells and then track their cell-to-cell movement. 2.1.2 Genetically Encoded Fluorescent Proteins To track the movement of exosomes from donor to recipient cells, gene editing technology has been adopted to fuse a fluorophore (e.g., green fluorescent protein, GFP; red fluorescent protein, RFP) with the marker protein on the exosomal membrane. By monitoring the fluorescence of membrane protein, direct visualization of exosomes transfer in live culture and in vivo is accomplished. Moreover, as the fluorophore is specifically conjugated onto the exosomes, this labeling strategy avoids


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the false positive signal from labeling other lipid entities. As CD63 from tetraspanin family is recognized as general exosomal membrane marker, fluorescent proteins fused to CD63 were reliable reporters to track pathway of exosomes in tumor, immune system, and neurogenesis. Breast cancer cells were engineered to stably expressing CMV-driven GFP-tagged CD63 using a lentivector system, and the cells produced GFP labeled exosomes [55]. Co-culture of GFP exosome-producing breast cancer cells with RFP-labeled breast cancer cells or lung tissue cells both led to recipient cells incorporating GFP-exosomes in their cytoplasm. Furthermore, tumor cell derived GFP-exosomes were found to spread into the surrounding environment and then incorporate into host cells in a mouse model. GFP-exosomes were also detected in blood, which verified the circulation of exosomes in host mice. Owing to the genetically encoded fluorescent proteins on exosomes, the profile of exosomes metabolism in mouse models of breast cancer is studied comprehensively. According to the in vivo and ex vivo imaging results of GFP-exosomes, it could be concluded that tumor-derived exosomes were dispersed in the tumor microenvironment, tumor-associated cells and blood, and that this wide distribution might play a role in promoting tumor growth and metastasis. Similarly, T cell derived exosomes were labeled with CD63-GFP to investigate exosomes mediating antigen-driven unidirectional transfer of miRNAs from T cells to antigen-presenting cells (APC). The yield of exosomes produced by T cells was diminished upon addition of neutral sphingomyelinase-2, and this further hindered the transport of miRNAs to APCs accompanied with an upregulation of target gene expression in recipient cells. The exosomal transfer of genetic material during immune synapsis could thus explain the mediator role of exosomes in immune system [56]. Involvement of exosomal transfer in neurogenesis was also investigated by imaging GFP labeled exosomes. The pathway of exosomes in brain like structure was studied in a microfluidic device with convective interstitial fluid and ECM microenvironment. The design of microfluidic device was depicted in Figure 2A and B. According to high-resolution real-time imaging, donor cell derived exosomes entered the ECM during the first 4 min 30 s, then passed through the ECM and



approached the recipient cells during the next 8 min. Finally, exosomes were taken up by recipient cells within 3 min (Figure 2C, D and E) [57].

Figure 2 Real time imaging of exosomes transport of from differentiated to undifferentiated cells in the microfluidic assay. (A) Illustration of microfluidic chip design. (B) Operating procedures of microfluidic assay. (C, D, E) High-resolution real-time imaging tracked the movements of exosome from GFP-exosome donor F11 cells to recipient F11 cells. Scale bar, 50 µm. Reprinted with permission from Oh et al [57].

Aside from the fusion of fluorescent protein with exosomal membrane marker, optical reporters can also be expressed in cells to selectively bind plasma membrane for visualization and tracking of EVs. Conjugation of palmitic acid with the sulfhydryl group on fluorescent proteins produced reporters which can associate with bilayer lipid membrane, including cells and all subpopulation of EVs. In practice, 293T cells were transduced to express palmitoylated enhanced GFP and tandem dimer Tomato (PalmGFP, PalmtdTomato) [54].Under live-cell confocal microscopy, 293T-PalmGFP cells and budlike structure on their surface emit strong green fluorescence, and


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PalmGFP reporters labeled EVs distributed around cells. As the optical reporters were synthesized in cells and then labeled both plasma membranes together with EVs, multidirectional EVs transport among cells could be imaged without an intermediate purification step. PalmtdTomato-EVs were found to pass through nanotube like projections from 293T to glioblastoma cells. The same group also tracked glioblastoma-derived palmtdTomato+ EVs in mouse using intravital 2-photon microscopy, and revealed EVs uptake by microglia and monocytes/macrophages in the brain. Notably, miRNA carried by EVs were functional in recipient cells and caused decrease of target mRNA and corresponding encoded proteins, presumably as a novel pathway for the tumor to modulate its microenvironment [58]. 2.1.3 Immunofluorescent Reporters Immuno fluorescent labeling adopts organic dyes tagged antibody to label the analytes, thus possessing high specificity. Although exosomes are below 150 nm, they are imaged as 200-300 nm light spot under laser confocal fluorescence microscopy owing to the diffraction limit of light. Thus, the accurate localization of a single exosome requires higher resolution. Currently, single molecule localization microscopy (SMLM) based on single molecule detection and localization possesses lateral imaging resolutions of 20 to 50 nm, and therefore is suitable for the visualization of ultrasmall subcellular structures. In turn, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are powerful tools for investigating the association of proteins with various structures inside cells. With the successive excitation and photobleaching of photoswitchable fluorophores, accurate localization of a single fluorophore was acquired, followed by the reconstruction of thousands of images of a single fluorophore into super resolution images. The high resolution of PALM and STORM well suits the size of exosomes. Indirect immune-fluorescent labeling with rabbit anti-CD63 and Alexa Fluor 647 tagged goat anti-rabbit IgG was also employed, wherein the exosomal membrane was simultaneously stained with CM-Dil to colocalize exosomes (Figure 3A and B). The PALM/STORM image clearly presents a much higher spatial resolution as compared with the TIRF image (Figure 3C). The full width at half-maximum of exosome is 70



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nm (Figure 3F), whereas the TIRF image of an individual exosome is much larger (Figure 3D and F). Moreover, an outstanding advantage of fluorescence microscopy is the ease of simultaneously imaging multiple constituents following labeling with spectrally distinct fluorophores. In particular, surface markers on C-erbB-2/HER2-postive tumor cell derived exosomes, CD63 and HER2, were immune fluorescently labeled with Alexa Fluor 647 and Alexa Fluor 488 respectively (Figure 3G, H and I). Super resolution imaging technique applied to investigate the intracellular distribution of exosomes after endocytosis showed that exosomes localizes in the interior of lysosomes. This clearly demonstrated that PALM can provide more reliable location information for exosomes intracellular visualization as compared to conventional TIRF imaging [59]. A

D

G

C

B

F

E

H

I

Figure 3 PALM/STORM and TIRF imaging of exosomes. (A) Schematic illustration of indirect IF labelingand membrane dye staining of exosomes. (B) TIRF image of exosomes stained with CM-Dil. (C) Exosomes were labeled with Alexa Fluor 647, then imaged under TIRF (upper right) together with PALM/STORM (low left). (D) Single exosome was labeled with Alexa Fluor 647 and observed with TIRF. (E) The same exosome as in (D) was characterized with PALM.(F) Cross-sectional profiles of exosome shown in (D, E). (G), (H), (I) Dual-color super-resolution imaging of CD63


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and HER2 on exosomes. (G) Alexa Fluor 647 channel. (H) Alexa Fluor 488 channel. (I) Merged image of the two super-resolution channels. Reproduced from Chen et al [59] with permission. 2.1.4 Fluorescent Nanomaterials Fluorescent nanomaterials not only have excellent optical properties, but also can be further functionalized with recognition elements to specifically label targets. The small size of nanoparticles is beneficial for binding exosomes and doesn’t change the original physiological functions of exosomes. For example, anti-HER2 was conjugated with gold-carbon quantum dots (GCDs) to fabricate a specific nanoprobe for tumor-derived exosomes [60]. Intracellular tracking by fluorescence microscopy revealed that the majority of exosomes were endocytosed by cells, finally settled in lysosomes. However, the as-prepared GCDs are restricted to moderate fluorescence intensity and may not represent the optimal choice for in vivo exosomes tracking. Alternatively, silicon quantum dots (Si QDs) with the feature of fluorescence blinking comprise promising probes for SMLM [61]. Accordingly, nanoprobes for exosomes were prepared by Si QDs conjugated with CD63 aptamers which are small oligonucleotides (20-60 nt) that can interact with their ligands with high affinity and specificity. Compared to antibodies, aptamers are much smaller and more suitable for accurate localization by SMLM. Si QDs nanoprobes labeled exosomes were absorbed onto the bottom of an eight chambered slide, and then subjected to SMLM imaging. The sizes of exosomes detected by SMLM (65 nm and 104 nm) were consistent with those measured from TEM images. This work utilized Si QDs as fluorescent nanoprobes for superresolution imaging of exosomes for the first time; however further tracking Si QDs labeled exosomes in living cells was not mentioned. The exploration of versatile fluorescent nanomaterials is still underway, especially near infrared fluorescent nanoprobes with deep tissue penetration and low autofluorescence background facilitating in vivo tracing of exosomes are in high demand.



Figure 4 In vivo bioluminescence and fluorescence imaging of EVs. (A) GlucB and biotin ligase (sshBirA) were delivered via lentivectors to HEK 293T cells for stable expression. (B) The sshBirA tags the BAP sequence of GlucB with a single biotin moiety at a specific lysine residue. Purified EVs were injected intravenously (iv) via tail or retro-orbital veins into nude mice for bioluminescence and fluorescence-mediated tomography imaging. For bioluminescence imaging, coelentrazine, a Gluc substrate, was iv-administered immediately prior to imaging. For FMT imaging, isolated EVs were conjugated with streptavidin-Alexa680 prior to administration into nude mice. (C) and (D) Bioluminescence imaging of EVs-GlucB in athymic nude mice at dorsal and ventral regions. (E) Quantitation of EVs-GlucB bioluminescence signal at ventral regions in liver and spleen at 60 min after EVs administration. Reproduced from Lai et al [54] with permission.

2.2 Bioluminescence Imaging As another widely used imaging method for studying biological activity, bioluminescence possesses high sensitivity, low background signal and does not require an excitation source to emit light. In practice, luciferases emit


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bioluminescence through reaction of their respective substrate with oxygen alone. Together, these traits make luciferase an ideal reporter for in vivo studies. Accordingly, Lai et al fabricated a membrane-bound (mb) variant of gaussia princeps luciferase (Gluc) fused to a biotin acceptor peptide (BAP), termed mbGluc-BAP, for EVs labeling. EVs derived by 293T cells transduced with mbGluc-BAP were administered in athymic nude mice. Bioluminescence images of nude mice suggested that the spleen and liver exhibited a prominent optical signal when compared to the control at 30 min post-EVs injection [62]. Furthermore, Lai et al also constructed a multimodal imaging reporter with mbGluc-BAP (Figure 4A) [54]. For bioluminescence imaging, coelentrazine, a Gluc substrate, was intravenously-administered immediately prior to imaging. For fluorescence-mediated tomography imaging, isolated EVs were conjugated with streptavidin-Alexa680 prior to administration into nude mice (Figure 4B). Mice with intravenously injected EVs displayed noticeable signal in the spleen followed by the liver as depicted in bioluminescence and fluorescence-mediated tomography imaging (Figure 4C, D and E). According to the ex vivo analysis of EVs in the organs, blood and urine, EVs first rapidly spread in the whole body rapidly, and then were eliminated through hepatic and renal routes in the following 6 h. Notably, this metabolism process revealed by bioluminescence detection is faster than that of fluorescent dye-labeled EVs. In a word, in vivo and ex vivo assessment of EVs using a bioluminescence reporter can provide accurate spatiotemporal distribution of EVs while it is impossible to study exosomes in a long term, because bioluminescence is transient signal. 2.3 Magnetic Resonance Imaging Magnetic resonance imaging (MRI) represents a powerful tool for deep penetration and high-quality 3D imaging of tissues with anatomical details. The image contrast in MRI primarily arises from the relaxation time differences of hydrogen nuclei in different environments of the studied tissues. In order to improve the sensitivity, chemical contrast agents were adopted in MRI, such as superparamagnetic iron oxide nanoparticles (SPIONs). Because electroporation can effectively load exosomes with RNA cargo, Hu et al prepared SPION loaded exosomes by electroporation of 5 nm



SPION5 and melanoma exosomes. SPION5 labeled exosomes and free SPION5 were both injected into the left food pad of C57BL/6 mice to study their movement to local lymph nodes. In vivo MRI analysis suggested that even though the intake amount of SPION5 was the same, the deposition of SPION5 in the ipsilateral node was significantly greater for SPION5 delivered by exosomes than for free SPION5 at 48 h. In ex vivo histological examination, Prussian blue iron staining indicated that SPION5 exosomes distributed predominantly to the subcapsule instead of homogeneously distribution all over the entire lymph node as in the case of free SPION5 [63]. Busato modified the labeling strategy by incubating ultrasmall superparamagnetic iron oxide nanoparticles (USPIO, 4-6 nm) with adipose stem cells which secreted USPIO loaded exosomes. However, only a small fraction of exosomes were labeled with nanoparticles in this indirect labeling method, which may further have reduced imaging sensitivity [64]. 2.4 Computed Tomography (CT) Tracing of Exosomes CT is a widely used medical imaging technique that has high spatial and temporal resolution. The working principle is based on differential absorption of X-rays by tissues, with three dimensional images being made up of multiple cross-sectional images. An important advantage of CT is that it can be used to perform fusion imaging with functional imaging methods such as positron emission tomography (PET) and single photon emission computed tomography (SPECT). Like MRI, contrast agents and nanomaterials are commonly used to enhance CT signal. Exosome-mimetic nanovesicles (ENVs) were utilized as substitutes for exosomes owing to high yield and similar characteristics. Hwang et al developed a novel SPECT/CT imaging methodology for monitoring the in vivo distribution of macrophage-derived ENVs after labeled with 99mTc-HMPAO, a radiotracer widely adopted in the clinic for cell labeling [65]. Intracellular glutathione converts 99m

Tc-HMPAO to the hydrophilic form that is trapped inside exosomes.

99m

Tc-HMPAO-ENVs distributed mainly in the liver and spleen of mice 30 min after

administration, whereas they accumulated in the salivary glands at 3 h. Conversely, no brain accumulation was observed until 5 h. As a control, mice injected with only


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99m

Tc-HMPAO displayed intense uptake in the brain and intestine (Figure 5).

Alternatively, gold nanoparticles (AuNP/GNPs) are popular as experimental CT contrast agents, as they attenuate x-rays strongly. In order to circumvent the inherent defect of restricted tissue penetration depth as observed for fluorescent and optical imaging modalities, especially in the deep brain, Betzer et al employed glucose-coatedGNPs to label purified exosomes which were then visualized by CT in vivo [66]. Glucose-coated GNPs were incorporated into MSC-derived exosomes via an active, energy-dependent pathway dominated by the glucose transporter GLUT-1. In a focal brain ischemia mouse model, more intranasal administered GNP-labeled exosomes settled in lesion site over 24 h, unlike the nonspecific migration and clearance observed from control brains over the same period (Figure 6). Exosomes distribution in the ex vivo brain was detected by both CT imaging and spectral fluorescence imaging after double-labeling with GNPs and the fluorescent dye PKH26, with both showing identical localization. Since contrast agents need to be loaded into exosomes first, so CT tracing method are only applicable to label purified exosomes.

Figure 5 In vivo SPECT/CT images of 99mTc-HMPAO-ENVs (a) and 99mTc-HMPAO (b) 30 min, 3 h, and 5 h after intravenous injection in BALB/c mice. Reproduced from



Hwang et al [65] with permission.

Figure 6 In vivo coronal and sagittal 3D CT imaging of exosomes in ischemic mouse model (a-f): (a,d) 1 h, (b,e) 3 h, and (c,f) 24 h post-exosome administration (the ischemic region is demarcated in yellow circle). (g-i) In vivo CT imaging of exosomes in control brain: (g) 1 h, (h) 3 h, and (i) 24 h post-exosome administration. Reproduced from Betzer et al [66] with permission.

3. CONCLUSIONS and PERSPECTIVES In summary, an ideal exosomes labelling method for imaging should meet the following criteria: stable and sufficient signal intensity; confer biocompatibility; free of unfavorable influence on biofunction; and high specificity and sensitivity. Compared with the single imaging model, multi-modal imaging that combines the advantages of different imaging methods to provide more reliable imaging effects is eagerly anticipated. Exosomes secreted by different cells are confronted with diverse fate in vivo, however, the exploration of effective protocols to illustrate the in vivo behavior of exosomes, is still in the early stage, which restricts the wide biomedical application of exosomes. Currently, optical imaging has generally been adopted to investigate the distribution and accumulation of exosomes. Although optical imaging is a versatile implement for both in vitro and in vivo study, it has inherent restrictions in terms of difficulties in quantification and limited tissue penetration depth. Thus, the


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use of innovative reporters in combination with appropriate imaging methodology to trace the elaborate metabolic pathway of exosomes is still urgently desired. Acknowledgments This work is financially supported by the Natural Science Foundation of China (Grant no. 81471308, 81601202) and Stem Cell Clinical Research Project (Grant no. CMR-20161129-1003).

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Table of Contents


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Figure 1 Biogenesis of EVs. (A) Vesiculation of endosome formed MVBs which subsequently fused with plasma membrane to release exosomes. The orientations of transmembrane protein on exosomes are the same as plasma membrane. Outward budding of plasma membrane and fission generated MVs. (B) Fragmentation of irregular protrusions on the surface of apoptotic cells generates Abs. Reprinted with permission from Lee et al [14]. 216x80mm (96 x 96 DPI)



Figure 2 Real time imaging of exosomes transport of from differentiated to undifferentiated cells in the microfluidic assay. (A) Illustration of microfluidic chip design. (B) Operating procedures of microfluidic assay. (C, D, E) High-resolution real-time imaging tracked the movements of exosome from GFP-exosome donor F11 cells to recipient F11 cells. Scale bar, 50 µm. Reprinted with permission from Oh et al [57]. 151x159mm (96 x 96 DPI)


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Figure 3 PALM/STORM and TIRF imaging of exosomes. (A) Schematic illustration of indirect IF labelingand membrane dye staining of exosomes. (B) TIRF image of exosomes stained with CM-Dil. (C) Exosomes were labeled with Alexa Fluor 647, then imaged under TIRF (upper right) together with PALM/STORM (low left). (D) Single exosome was labeled with Alexa Fluor 647 and observed with TIRF. (E) The same exosome as in (D) was characterized with PALM.(F) Cross-sectional profiles of exosome shown in (D, E). (G), (H), (I) Dualcolor super-resolution imaging of CD63 and HER2 on exosomes. (G) Alexa Fluor 647 channel. (H) Alexa Fluor 488 channel. (I) Merged image of the two super-resolution channels. Reproduced from Chen et al [59] with permission. 155x124mm (96 x 96 DPI)



Figure 4 In vivo bioluminescence and fluorescence imaging of EVs. (A) GlucB and biotin ligase (sshBirA) were delivered via lentivectors to HEK 293T cells for stable expression. (B) The sshBirA tags the BAP sequence of GlucB with a single biotin moiety at a specific lysine residue. Purified EVs were injected intravenously (iv) via tail or retro-orbital veins into nude mice for bioluminescence and fluorescencemediated tomography imaging. For bioluminescence imaging, coelentrazine, a Gluc substrate, was ivadministered immediately prior to imaging. For FMT imaging, isolated EVs were conjugated with streptavidin-Alexa680 prior to administration into nude mice. (C) and (D) Bioluminescence imaging of EVsGlucB in athymic nude mice at dorsal and ventral regions. (E) Quantitation of EVs-GlucB bioluminescence signal at ventral regions in liver and spleen at 60 min after EVs administration. Reproduced from Lai et al [54] with permission. 186x156mm (96 x 96 DPI)


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Figure 5 In vivo SPECT/CT images of 99mTc-HMPAO-ENVs (a) and 99mTc-HMPAO (b) 30 min, 3 h, and 5 h after intravenous injection in BALB/c mice. Reproduced from Hwang et al [65] with permission. 161x122mm (96 x 96 DPI)



Figure 6 In vivo coronal and sagittal 3D CT imaging of exosomes in ischemic mouse model (a-f): (a,d) 1 h, (b,e) 3 h, and (c,f) 24 h post-exosome administration (the ischemic region is demarcated in yellow circle). (g-i) In vivo CT imaging of exosomes in control brain: (g) 1 h, (h) 3 h, and (i) 24 h post-exosome administration. Reproduced from Betzer et al [66] with permission. 122x121mm (96 x 96 DPI)


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Tracking Exosomes in Vitro and in Vivo to Elucidate their Physiological

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