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Review  |  Open Access  |  6 Dec 2023

Research progress in in vivo tracing technology for extracellular vesicles

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Extracell Vesicles Circ Nucleic Acids 2023;4:684-97.
10.20517/evcna.2023.49 |  © The Author(s) 2023.
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Abstract

Cells have the capability to discharge extracellular vesicles (EVs) into a range of bodily fluids. Extracellular vesicles (EVs) encapsulate biological molecules such as proteins and nucleic acids, playing a role in facilitating cell-cell communication. They actively engage in a myriad of physiological and pathological processes. In vivo tracing of EVs in organisms significantly contributes to elucidating the biological mechanisms of EV-based therapy. The development of molecular imaging technology makes it possible to trace EVs in vivo. Experiments frequently employ a range of molecular imaging techniques, encompassing bioluminescence imaging, fluorescence imaging, magnetic resonance imaging, single photon emission computed tomography, positron emission tomography, photoacoustic imaging, and multimodal imaging. These methods have their own advantages and disadvantages. In this review, typical applications of in vivo tracing of EVs are reviewed.

Keywords

Extracellular vesicles, in vivo tracing, molecular imaging, multimodal

INTRODUCTION

Extracellular vesicles (EVs) are a group of nanoscale heterogeneous lipid bilayer membrane vesicles, which are released into the extracellular environment by different cell types through various stages of evolution[1]. EVs can be detected in a range of liquid body substances, including but not limited to blood, breast milk, urine, and saliva[2-5].

EVs were first observed in healthy human plasma by Chargaff et al. in 1946. Initially, they were recognized as a class of platelet microparticles with procoagulant effects[6].

In 1967, Wolf first utilized electron microscopy to examine the structure and dimensions of these particulate deposits and named them “platelet dust”[7]. Subsequently, Johnstone et al. first formalized the name of this granular material as exosomes in an in vitro culture study of sheep reticulocytes[8]. In 2011, György et al. proposed to refer to all the different types of lipid bilayer-encapsulated extracellular structures as “extracellular vesicles (EVs)”[9].

Initially, EVs were considered primarily as a means for cells to expel waste substances, and for an extended period, they remained relatively overlooked. Over the last several years, with the deepening of research, it has gradually been discovered that EVs contain other bioactive substances such as mRNA, ncRNA, miRNA, lncRNA, protein, and DNA, and have a lipid structure of information carriers, which can be used as transmitters of genetic information[10]. The surface molecules of EVs allow them to specifically target and reach receptor cells. Once attached to the target cell, EVs can activate receptor-ligand interactions or fuse with the target cell membrane through endocytosis, delivering their contents into the cytoplasm and thereby altering the physiological state of the receptor cell[11]. Diseases such as cancer can be predicted or diagnosed by detecting the presence and number of specific EVs; thus, EVs can be employed as a valuable diagnostic marker in the identification of diseases. In addition, EVs can be used as drug delivery agents. By utilizing EVs as carriers, drugs can be accurately delivered to the lesion site, thereby increasing the efficacy of the drug and reducing side effects.

According to the different sizes, biological properties, and formation processes, EVs are primarily categorized into three distinct groups: exosomes, microvesicles (MVs), and apoptotic bodies[12,13]. Exosomes emerge through the fusion of vesicles with cell membranes, exhibiting sizes ranging from 30 to 150 nm[14]. MVs are generated by the budding process of cell membranes and typically have a diameter spanning from 50 to 1,000 nm[15]. Apoptotic bodies are vesicles formed by cell shrinkage and fragmentation in the process of cell apoptosis, with a diameter of 500-2,000 nm[16]. Among them, exosomes have received more attention due to their unique generation pathway.

Studies have found that different cell types or different physiological states can cause expression differences in exosome contents. For example, exosomes derived from B cells contain B cell receptors (BCR), and exosomes derived from DC cells contain CD86, MCH-II, ICAM-1 proteins, etc.[17]. However, exosomes usually contain some common specific proteins that can be used as their markers, such as the adhesion molecule MFGE8[18], the proteins involved in membrane transport such as Annexins, Flotillins and GTPases, and the proteins engaged in the creation of the multivesicular body (MVB) encompass Alix, TSG101, and Clathrin. Additionally, four transmembrane proteins, namely CD9, CD63, CD81, and CD82, as well as heat shock proteins such as HSP90, HSP70, HSP60, and HSPA5, play crucial roles in these cellular processes[18-21]. Kowal confirmed that the protein in exosomes is different from other subtypes of vesicle structure using protein profile analysis[22,23]. Lundholm also reported that the proteins in exosomes are different from cellular structural protein components, such as cell membranes, cytoplasm, mitochondria, Golgi, and endoplasmic reticulum[24]. These data prove that exosomes are a special type of subcellular structure, which is different from cell fragments.

Currently, the most extensively studied subgroups are exosomes and MVs. However, there is some overlap in diameter size between MVs and exosomes. So far, due to the lack of specific markers and technical methods, there has not yet been a strategy to unequivocally separate these two types of EVs in complex biological fluid samples[25,26]. Therefore, EVs is uniformly used as a generic term in this review.

Recently, the study of EVs has been deepened with the continuous development of biological and medical research. In order to monitor the activities of EVs in organisms more intuitively and in real time, and to observe the biological processes such as the production, transfer and distribution of EVs in living animals, researchers have gradually expanded the scope of research from in vitro to in vivo. This shift not only contributes to a more comprehensive understanding of the biological functions of EVs, but also presents fresh viewpoints and innovative ideas to enhance the understanding and management of diseases. The method of tracing EVs in vivo is to use specific imaging methods to track EVs in animals, so as to observe the biological behavior of EVs in the body. In vivo tracing of EVs was widely used because of its intuitive results, high sensitivity, and non-invasive nature (i.e., obtaining information or data of EVs through methods that do not cause any damage or interference to EVs). Several imaging methods are currently available for tracking EVs in vivo, such as bioluminescence imaging (BLI), fluorescence imaging (FI), magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT), and photoacoustic imaging (PAI). The main current methods discussed in this review are succinctly summarized in Figure 1 and Table 1.

Research progress in <i>in vivo</i> tracing technology for extracellular vesicles

Figure 1. Strategies for tracking extracellular vesicles in vivo. Extracellular vesicles (EVs) can be extracted from the culture medium of donor cells or genetically modified cells by centrifugation. EVs can be modified to contain luciferase (NanoLuc, GFP, mCherry), fluorescent dyes (PKH67, DiD, DiR, MEMGlow, CSFE), and radionuclide labeling (99mTc-HMPAO, 99mTc-AnnV, 111Indium) for injection into animals for easy tracking. There are many imaging methods that can be used to track EVs in vivo, such as bioluminescence imaging (BLI), fluorescence imaging (FI), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), and photoacoustic imaging (PAI). Therefore, the in vivo tracing strategy of EVs is a key means to elucidate the biological mechanism of EV therapy. Created By Biorender.

Table 1

EVs labeling and in vivo tracking strategies

Imaging modalityIsolated sites/cellsIsolation methodLabeling agentConcentrationSubjectInjection siteImaging time pointsReferences
BLIProstate cancer cells (PC3)UCEGFP-CD63-Antares21 × 106 cells/100 µLBALB/c-nu/nu mice Subcutaneously
transplanting
0 d, 5 d, 10 d, 15 d, 20 d, 25 d, 30 d, 35 dHikita et al.[28] (2020)
HEK-293T cells/MSC cellsTFF/SEC/UCDiR,CD63-NanoLuc/CD63-ThermoLuc1 × 1011 cells/100 µL PBSNMRI miceIV5 min, 15 min, 30 min, 60 min, 6 h, 24 hGupta et al.[29] (2020)
Plasma/cardiomyocyte/cardiac fibroblatsExoquickTM precipitation/ UCCD63-NanoLuc-TG-αMHC-STOP-CD63NanoLuc miceIP5 minLuo et al.[30] (2020)
Fibrosarcoma cells(HT1080)Iodixanol gradient/UCpHluo_M153R-CD631 × 106 cells/mice;
1 × 105 cells/chick
NOD/SCID mice,
ex ovo chicks
Mouse mammary fat pad injection/IV1 week, 24 hSung et al.[31] (2020)
FIMilk and cancer cells (U87, B16F10)UCSCy 7.5 and BDP-FL succinimidyl ester fluorophores25 μg/150 μL PBSAlbCre mice, mT/mG miceIV1 h, 4 h, 24 h González et al.[39] (2021)
MSCUCDiR100 µg/200 µL PBSC57BL/6J mice(ischemic stroke)IV0 d, 1 d, 3 d, 5 d, 7 d, 10 d,
14 d
Xu et al.[40] (2020)
A549 cells UCDBCO-Cy5, DiD1.5 mg/kgTumor-bearing miceIV24 hSong et al.[41] (2020)
MRICESCUCPKH67, DIR40 μg/mLSD rats (CEP and IVDD)IV6 weeksLuo et al.[43] (2021)
MSCUCFTH150 µg/100 µL PBSC57BL/6 miceIMAfter injectionLiu et al.[44] (2020)
ASCsPureExo® Exosome isolation kitUSPIO5 µg and 25 µg/100 µL PBSC57BL/6 mice IMAfter injectionBusato et al.[45] (2016)
SPECTHuman umbilical vein endothelial cellsUC99mTc-AnnV2.0 ± 0.5 × 106 LEVs/2.0 ± 0.4 MBq/150 µLBALB/c miceIV30 minGiraud et al.[48] (2022)
PlasmaUC99mTc-HYNICDuramycin12 ± 3 MBq/100 μLC57BL/6IV1 hNémeth et al.[49] (2021)
HCT116 cells, ASCsUC99mTc-TEx-Cy7,99mTc-AEx-Cy737 MBq/200 µL PBSTumor-bearing miceIV1 h, 6 h, 12 h, 18 h, 24 hJing et al.[50] (2021)
Melanoma cells(B16F10) Sucrose density cushion/UC111Indium1 × 1011 particles/mouseC57BL/6 mice,NSG miceIV1 h, 4 h ,24 hFaruqu et al.[51] (2019)
ErythrocyteUC99mTc-tricarbonyl15 ± 2 MBq/200 μL volumeBALB/c mice IV1 hVarga et al.[52] (2016)
Raw 264.7 cells, HB1.F3 cellsIodixanol gradient/UC99mTc-HMPAO7.4-14.8 MBq, 11.1 MBq of 29-64 μg cellsBALB/c mice IVAfter injectionHwang et al.[53] (2015)
PET4T1 cellsUC64Cu--NOTA,64Cu--NOTA-PEG50 μCi/mouse, 200 μLBALB/c mice IV1 h, 4 h, 24 hShi et al.[54] (2019)
PAIMIA-PaCa-2 cellsUCCe6-R100 μg/mL exosomal protein and 38.4 μg/ml Ce6BALB/c mice IV6 hJang et al.[58] (2021)
Tumor cellsChina RIBOBIO Biotechnology Co., Ltd. Au Nanostars and TDSP200 μg/mL in 100 μl PBSBALB/c mice IV24 hZhu et al.[59] (2020)
NIH3T3 cells,CNE-2 cellsFacile chemical oxidation and exfoliation method DiR100 μg/mL in 100 μL volumeBALB/c nude mice IV2 h, 4 h, 8 hDing et al.[60] (2019)
Multimodal imaging4T1 cells, HeLa cells, Raw 264.7 UCCDs:Gd,Dy-TAT30 mg·kg-1·wtBALB/c miceIV6 h, 12 h, 24 hYang et al.[64] (2021)
HepG2 cells, L02 cellsUCAg ,Fe3O420 μg /mL--12 hTayyaba et al.[65] (2020)
Expi293F cells, HepG2 cellsIodixanol gradient/UCDiR,mCherry, 111indium-DTPA1 × 1011 cells in 200μL PBS; 5-10 MBq; in 200 μL BALB/c mice IV24 h; 0-30 min, 4 h, 24 h Lázaro-Ibá́ez et al.[66] (2021)
293T cells, C3H strain HCA1 cellsSucrose density cushion/UCPalmGRET100 µgC3H/HeNCrNarl miceIV5 min, 10 min, 20 min, 30 minWu et al.[67] (2020)

BIOLUMINESCENCE IMAGING

Bioluminescence imaging (BLI) mainly refers to the integration of the luciferase gene into the chromosomal DNA of the target cells, so that the cells can secrete luciferase-containing exosomes, and the luciferase gene can be stably expressed in the cell without loss. The most commonly used reporter systems for bioluminescence imaging are fluorescent protein and luciferin/luciferase bioluminescence systems[27].

Hikita et al. labeled exosomes with Antares2 and observed long-term accumulation of exosomes in vivo using bioluminescence resonance energy transfer (BRET). Implantation of prostate cancer cells expressing CD63-Antares2 into mice facilitated the quantification of exosomes originating from the primary tumors entering the bloodstream. Additionally, it enabled the visualization of the extended homing patterns of exosomes to their respective target organs or tissues over an extended period[28]. Gupta et al. investigated the bioluminescent tagging of EVs by employing various luciferase enzymes attached to CD63. Their findings revealed that the dispersion pattern of EVs is influenced by the injection route. Moreover, distinct subpopulations of EVs exhibited variations in their biodistribution patterns[29]. Luo et al. established a genetic mouse model incorporating the Nano-luciferase (NanoLuc) reporter, which was fused with the exosome surface marker CD63 to facilitate exosome labeling. The induction of CD63NanoLuc reporter expression was achieved through tamoxifen. Luciferase assays and bioluminescent imaging outcomes demonstrated the targeted labeling and discernible tissue distribution of naturally occurring exosomes released from cardiomyocytes[30]. Sung et al. introduced a stabilizing mutation, M153R, in the pHluorin moiety and released exosomes in vivo. The incorporation of a non-pH-sensitive red fluorescent tag in the exosomes enabled the observation of the complete exosome lifecycle. This included visualizing processes such as MVB trafficking, MVB fusion, exosome uptake, and endosome acidification[31].

Bioluminescence belongs to self-luminescence and does not require external light source excitation, thus avoiding the interference of natural fluorescent substances[32]. At the same time, because there is almost no endogenous light source in mammalian cells and tissues, BLI has extremely high sensitivity, specificity, and good signal-to-noise ratio. In addition, the bioluminescence system has good biocompatibility, and has non-phototoxicity caused by the excitation light during the imaging process, and it can be non-invasive, continuous and dynamic monitoring of various biological processes in the living body in real time[33-35]. However, BLI also has its own shortcomings: for example, the emitted fluorescence will be scattered and absorbed when propagating in the tissue, photons will be refracted when they meet the cell membrane and cytoplasm, and different types of cells and tissues have different ability to absorb photons. Therefore, the resolution of BLI will decrease.

FLUORESCENCE IMAGING

Fluorescence imaging (FI) refers to the implantation of experimental materials (such as nanoparticles, drugs, genes, etc.) labeled with fluorescent dyes into animals, and then use external excitation light to excite the materials with fluorescence. The in vivo imaging system collects the emitted light signals generated by the fluorescent dyes in the body.

There are two main types of fluorescent dyes commonly used for tracking EVs in vivo. One type is lipid dyes (such as PKH67, DiD, DiR, MEMGlow, etc.), which have different excitation and emission wavelengths and can effectively label EVs[33-35]. The other type of fluorescent dye is a membrane-permeable compound, such as carboxyfluorescein succinimidyl ester (CFSE), which is a lipophilic fluorescent dye that can be passively diffused into cells[36,37]. It is colorless itself, but after entering the cell, it removes the acetate group to generate carboxyfluorescein succinimidyl ester (green fluorescence), which is also a commonly used method for effectively tracking EVs[37].

González et al. documented that the secure attachment of commercially available fluorescent dyes to exosomes derived from both milk and cancer cells (specifically U87 and B16F10 cancer cells) surfaces, facilitated by covalent binding, did not alter the inherent physicochemical characteristics of the exosomes[38]. The exosomes, now fluorescently labeled, were effectively visualized using in vivo optical imaging[39]. Xu et al. engineered exosomes derived from mesenchymal stem cells (MSC-EXOs) by incorporating DiR, followed by intravenous injection into mice experiencing ischemic stroke. The near-infrared fluorescence (NIRF) images obtained indicated the migration of MSC-EXOs into the brains of mice with ischemic stroke, showcasing their ability to elicit therapeutic effects against this condition[40]. Song et al. developed exosomes labeled with DBCO-Cy5 (Cy5-Exo) derived from cancer cells. The donor cancer cells were subjected to treatment with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), and the resulting azide groups were labeled using near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5). Subsequently, non-invasive tracking and imaging of Cy5-Exo were conducted through near-infrared fluorescence (NIRF) imaging in mice with tumors[41].

The advantage of FI is that it has more selectivity of fluorescent probes, flexible labeling methods, and wide wavelength selectivity of fluorescent probes[29-31]. However, due to the scattering and absorption of light in biological tissues, coupled with the significant interference of tissue autofluorescence, the resolution and contrast of in vivo fluorescence imaging of the visible light spectrum (wavelength 400-700 nm) will decrease as the depth of the tissue increases, and resulting in a low signal-to-background ratio (SBR), and affecting the sensitivity and depth of detection.

MAGNETIC RESONANCE IMAGING

Magnetic resonance imaging (MRI), commonly known as spin imaging, is an advanced diagnostic modality that utilizes the principles of nuclear magnetic resonance to intricately visualize the internal structures within the body. The principle of MRI is that when there is an external magnetic field, radio frequency waves will act on the protons rotating around the magnetic field to increase its precession angle. When the radio frequency waves stop running, the protons will release the same signals, which are processed by a computer and converted into two-dimensional images to help the staff analyze the state of the target. Using special contrast agents or molecular probes, MRI is used to study cell activities in organisms, including proliferation, differentiation, migration, and aggregation. Superparamagnetic iron oxide (SPIO) nanoparticles exhibit biocompatibility and have demonstrated enhanced targeting capabilities when incorporated into EVs. Notably, ultrasmall SPIO (USPIO), with a size of less than 50 nm, is particularly advantageous for labeling nanoscale EVs owing to their diminutive size[42].

Luo et al. conducted an assessment of in vivo exosomes derived from normal cartilage endplate stem cells (CESC), differentiating between normal (N-Exos) and degraded exosomes (D-Exos). This evaluation was performed under the influence of intervertebral disc degeneration (IVDD) and apoptosis of nucleus pulposus cells (NPC), utilizing MRI as the diagnostic tool. The results indicated that N-Exos was more conducive to autophagy activation than D-Exos[43]. Liu et al. engineered a hybrid protein that combined the ferritin heavy chain (FTH1) with a truncated lactadherin. Utilizing FTH1 as an MRI reporter, their investigation revealed that exosomes labeled with FTH1 were observable both in vitro and in vivo through MR imaging[44]. Busato et al. introduced an innovative technique for tagging EVs derived from adipose stem cells (ASCs) using USPIO. The MRI detection limit for EVs was determined to be 3 µg in vitro and 5 µg in vivo, showcasing the effectiveness of this labeling approach[45].

Leveraging MRI for monitoring EVs offers numerous advantages, including its non-destructive, non-invasive, and non-ionizing radiation nature. MRI provides high soft tissue contrast, exceptional spatial resolution (sub-millimeter scale), and overcomes penetration depth limitations, presenting a wealth of information in the resulting images. MRI can be used not only to display the anatomical structure and morphology of tissues and organs, but also to analyze the physiology and biochemistry, tissue metabolism, organ function, etc. in the organism in a multi-dimensional and all-round way[46,47]. MRI also has some limitations of its own. A low signal-noise ratio (SNR) generally requires signal accumulation to increase SNR, which further reduces the imaging speed. Slow imaging speed can also cause problems such as insufficient time resolution of functional imaging in applications and easy to be affected by motion artifacts.

SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY

Single photon emission computed tomography imaging (SPECT) uses single-photon radionuclides as the detection object. The principle of SPECT imaging is to label exosomes with radioisotopes with a short half-life. After the exosomes are injected into the body and reach the expected position, they will emit gamma photons due to their own radioactive decay, which are located on the outer gamma camera probe. The sensitive point of the photon will detect the incoming photons, which will be converted into electrical signals by the photomultiplier tube and amplified at the same time. The amplified signals will form pulse signals. Combined with computer processing, the distribution of exosomes can be displayed in different gray scales or color scales. The probes used for tracing in living cells are mainly labeled with two radionuclides: 111In and 99mTc.

Giraud et al. used microSPECT/CT imaging to track radiolabeled endothelial EVs and quantify their whole-body distribution in vivo[48]. Németh et al. used SPECT/CT to analyze the biodistribution of Tc-HYNIC-Duramycin labeled EVs in mice, and detected an elevated circulating EV number after the high-fat diet[49]. Jing et al. employed 99mTc labeling to create radioactive exosomes derived from adipose stem cells, known as Tc-TEx-Cy7, utilizing a hydrophobic insertion mechanism. SPECT imaging results demonstrated that, in comparison to radio-labeled exosomes from adipose stem cells (Tc-AEx-Cy7), Tc-TEx-Cy7 exhibited notably enhanced tumor accumulation in mice with tumors[50]. Faruqu et al. employed two distinct methods for radiolabeling B16F10-derived exosomes (Exo): intraluminal labeling involving the entrapment of Indium tropolone shuttling, and membrane labeling through chelation of Indium covalently attached to the bifunctional chelator DTPA-anhydride. The results obtained from whole-body SPECT-CT imaging indicated that membrane-labeled Exo exhibited superior radiolabeling efficiency and radiochemical stability compared to intraluminal-labeled exosomes[51]. Varga et al. used the 99mTc-tricarbonyl complex labeling erythrocyte-derived EVs. The result of SPECT imaging showed that intravenously injected 99mTc-labeled EVs mainly accumulate in the liver and spleen[52]. Hwang et al. labeled nanovesicles mimicking macrophage-derived exosomes (ENVs) with 99mTc-HMPAO. Upon examining SPECT/CT images of mice injected with 99mTc-HMPAO-ENVs, noticeable higher uptake was observed in the liver, while there was no discernible uptake in the brain[53].

POSITRON EMISSION TOMOGRAPHY

Positron emission tomography (PET) uses radionuclides as imaging agents to label exosomes, and then uses detectors to accept photons released during the decay of the positron tracer nuclide to form tracer distribution images, thereby reflecting the presence of exosomes in the body Distribution.

Shi et al. employed in vivo PET to non-invasively monitor copper-64Cu-radiolabeled polyethylene glycol (PEG)-modified exosomes. This approach resulted in outstanding imaging quality and facilitated quantitative measurements of both blood residence and tumor retention[54].

SPECT and PET have high specificity in the monitoring of exosomes, and are easy to operate, can quickly obtain semi-quantitative data, and have good accuracy[55,56]. Compared with SPECT, PET imaging has higher sensitivity and resolution. However, it also has disadvantages, such as the need to inject radionuclides and the large radiation dose during the inspection.

PHOTOACOUSTIC IMAGING

Photoacoustic imaging (PAI) is an emerging low-cost, non-invasive imaging technology based on the absorption of light by biological tissues. This method involves capturing images by detecting acoustic signals generated from light absorption, making it an economical and minimally intrusive approach to imaging. Upon exposure to a pulsed laser, biological tissues absorb energy, undergo expansion, and generate pressure changes, thereby initiating the photoacoustic effect. This phenomenon involves the conversion of absorbed light energy into acoustic waves, contributing to a nuanced understanding of biological structures through photoacoustic imaging. The acoustic signal generated by the photoacoustic effect is called the photoacoustic signal. Under the condition of the same light source parameters, the intensity and frequency spectrum of the photoacoustic signal are closely related to the optical, thermal, and elastic properties of the tissue. Photoacoustic imaging technology obtains tissue structure and biochemical information by detecting photoacoustic signals, and realizes functional imaging while reconstructing the image of tissue structure for disease diagnosis and tissue evaluation.

Nguyen et al. successfully implemented a pH-responsive PAI-guided chemo-acoustic kinetic combination therapy. This was accomplished by incorporating indocyanine green (ICG), paclitaxel (PTX), and sodium bicarbonate (SBC) into EVs. High-resolution PA imaging showed that SBC-EVs (ICG/PTX) preferentially accumulated in tumor-bearing mice[57]. Jang et al. engineered re-assembled exosomes derived from tumors (R-Exo) by combining them with a chlorin e6 photosensitizer. The resulting chlorin e6-loaded re-assembled exosomes (Ce6-R-Exo) were observable through photoacoustic imaging and demonstrated the efficient generation of reactive oxygen species within tumor cells when subjected to laser irradiation[58]. Zhu et al. presented stellate plasmonic exosomes derived from tumor cells (TDSP-Exos). These specialized exosomes were created through the incubation of tumor cells with gold nanostars. Notably, TDSP-Exos demonstrated significant accumulation within deep tumor tissues and displayed impressive performance in the realm of PAI[59]. Ding et al. engineered nanozyme vesicles with an exosome-like structure through the biomimetic functionalization of GQDzyme/ABTS nanoparticles. The outcomes underscored the potential of GQDzyme/ABTS-based exosome-like nanozyme vesicles as an optimal nanoplatform for advancing in vivo deep-tissue tumor-targeted catalytic PAI[60].

Photoacoustic imaging technology combines optical and acoustic imaging with complementary advantages. Photoacoustic imaging has good spatial resolution. Only a very low electromagnetic radiation energy density is required to obtain a photoacoustic signal with a higher signal-to-noise ratio, thereby avoiding the ionization damage caused by high-intensity electromagnetic radiation to biological tissues[60-63]. At present, the photoacoustic imaging technology still has certain limitations. First of all, photoacoustic imaging technology uses sound waves as a carrier for imaging. Therefore, it is urgent to improve the imaging effect of bones, air-containing cavities, and other tissues where the transmission of sound waves is blocked. Thirdly, the sensitivity of the ultrasonic transducer needs to be improved to strengthen the detection of deep tissue signals and increase the imaging depth. At the same time, it is also necessary to further improve the imaging resolution.

MULTIMODAL IMAGING

There are different advantages and disadvantages in tracing in vivo cells for different molecular imaging technologies. The single imaging technology to trace EVs in vivo has some shortcomings, such as low labeling efficiency, loss of imaging probes, large signal monitoring errors, etc. Therefore, the development of new multi-functional probes and the use of multi-modal imaging technology can overcome the shortcomings of a single imaging technology, trace the EVs in the body from multiple aspects, and provide more information.

Yang et al. developed a multifaceted engineered nanoplatform using rare earth element Gd and Dy-doped carbon dots (CDs:Gd,Dy) modified with TAT peptide. These were encapsulated into exosomes engineered with RGD peptide (Exo-RGD). The application of this nanoplatform enabled the detection of tumor sites through MRI/CT imaging in mice bearing tumors[64]. Tayyaba et al. employed HepG2 cancer cells to facilitate the in situ biosynthesis of nanoclusters (NCs) composed of silver and iron oxide derived from their respective salts (AgNO3 and FeCl2). The self-assembled, biosynthesized silver and iron NCs were efficiently loaded onto exosomes as cargo. These silver NCs displayed potential as a fluorescent probe, while the iron oxide (Fe3O4) NCs served as a contrast agent for both CT and MRI[65]. Lázaro-Ibáñez et al. conducted an assessment of the impact of five distinct optical and nuclear tracers on the in vivo biodistribution of EVs. They presented a comprehensive comparison encompassing fluorescent, bioluminescent, and radioactivity methodologies. Dual labeling of EVs from Expi293F cells can be achieved by noncovalent fluorescent dyes (DiR), covalent modification of 111 indium-DTPA, or bioengineering of fluorescent (mCherry) fused with EVs-tagged CD63 or bioluminescent proteins (Firefly and NanoLuc luciferase). The outcomes highlighted that radioactivity emerged as the most precise approach for tracking EVs[66]. Wu et al. developed PalmGRET, a reporter based on BRET. This innovative creation enables the visualization, tracking, and quantification of EVs across a spectrum of resolutions, from whole-animal to nanoscopic levels, through diverse imaging modalities, including bioluminescence, BRET, and fluorescence[67].

The pivotal aspect for tracing the in vivo biological behavior of EVs lies in the development of a quantitative analysis method that boasts high sensitivity and specificity. Combining multiple detection markers into one molecule can provide complementary imaging information. Multimodal imaging-specific molecular markers have attracted the attention of researchers because they can integrate the advantages of multiple imaging methods. Targeted multimodal imaging and theoretical methods are gradually becoming research hotspots, which can provide in vivo, non-invasive, quantitative, precise, specific, and longitudinal monitoring methods for the tracing of EVs in vivo. The development of multimodal imaging and specific molecular markers will be the future research direction.

CONCLUSION

The continuous and non-invasive labeling of EVs helps us to understand the biological characteristics of EVs after entering the organism, such as migration, distribution, and survival, as well as the mechanism by which EVs mediate the recovery of cell or tissue function. The development of EV therapy for these diseases has long-term guiding significance. Efficiently monitoring EVs within living organisms poses a challenge that demands reliable techniques ensuring the preservation of membrane integrity and biological activity post-labeling with exogenous probes. Currently, various non-invasive imaging methods are at our disposal for tracking EVs within living organisms, each carrying its unique set of advantages and drawbacks. Table 2 summarizes the advantages and limitations of each method. MRI has a high spatial resolution, but its labeling specificity is still lacking, and the signal intensity may decrease as the cells proliferate. BLI/FI has a lower cost and higher sensitivity than MRI, and can support long-term real-time tracking of cells, but its spatial resolution is not as good as MRI. As the depth of the tissue increases, the intensity of the light signal decreases. SPECT/PET also has a high degree of sensitivity and can continue to detect signals during cell division. However, if researchers are exposed to radioactive internal radiation, the safety needs to be further explored and studied.

Table 2

Comparison of various in vivo imaging techniques for EVs

Imaging technologyEmission sourceDetection probeSpatial resolutionTime resolutionDepth of imagingSensitivityCosts of experiments
BLInear-infrared light sourceFluorescent protein reporter genesLowHighLowHighLow
FIlaserFluorescent dyesLowHighLowHighLow
MRIradio-frequency pulseSuperparamagnetic iron oxide (SPIO); ultrasmall SPIO (USPIO, < 50 nm)HighLowHighLowHigh
SPECTγrayradioisotopeLowHighHighHighHigh
PETγrayradioisotopeLowHighHighHighHigh
PAIlasernanoparticlesLowHighHighHighLow

At present, there is a scarcity of imaging platforms specifically designed for the dynamic tracking of EVs at the in vivo level. The present optical imaging, magnetic resonance imaging, and nuclide imaging platforms rely on cellular tracers or medical imaging equipment. Attempts to achieve compatibility with EVs involve adjusting parameters, yet this often results in experimental designs constrained by the imaging threshold of the instrument. Therefore, it is essential to construct a specialized imaging platform for the dynamic tracing of EVs. Due to the lack of imaging platforms dedicated to the dynamic tracing of EVs at the in vivo level, we strongly support the utilization of multimodal imaging techniques that offer a wide array of advantages. Multimodal imaging can combine the advantages of different imaging techniques to provide more comprehensive and accurate exosome information. For example, combining optical imaging with magnetic resonance imaging or nuclide imaging can simultaneously obtain information on the location, distribution, and dynamics of EVs. Such an integrated approach will help us to understand the biology of EVs more deeply while providing more effective tools for diagnostic and therapeutic applications of EVs. Therefore, we should actively advocate and carry out research on multimodal exosome tracking imaging.

DECLARATIONS

Acknowledgments

The authors express gratitude to Dr. Fengzhen Cui from the School of Biomedical Engineering, Shanghai Jiaotong University, for valuable insights and comments on the manuscript. Additionally, the authors recognize the creation of figures using Biorender (https://biorender.com/).

Author Contributions

Conceptualization: Tang Y, Yang N

Investigation: Shi Y, Wang X, Zhang S

The visualization: Wang X

Literature collection and collation: Shi Y, Wang X, Zhang S, Yin H, Fan H

Writing-original draft preparation: Shi Y, Wang X, Zhang S, Yin H, Fan H

Writing-review and editing: Shi Y, Wang X, Zhang S, Yin H, Fan H, Tang Y, Yang N

Financial support: Tang Y, Yang N

Having thoroughly reviewed and given their approval to the final version of the manuscript: Shi Y, Wang X, Zhang S, Yin H, Fan H, Tang Y, Yang N

Availability of data and materials

Not applicable.

Financial support and sponsorship

This research was funded by grants from the Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, 20JC1411900; the National Natural Science Foundation of China (82071284 [YT], 82371307 [YT], 81600360 [NY]); National Key R&D Program of China (2019YFA0112000 [YT]); Shanghai Rising-Star Program (21QA1405200 [YT]); Young Leading Scientists Cultivation Plan supported by Shanghai Municipal Education Commission (ZXWH108210 [YT]); the Fundamental Research Funds for the Central Universities (YG2023ZD02 [YT]); the Province Natural Science Foundation of Shandong (ZR2020KH008 [NY]).

Conflicts of interest

All authors declare that there are no conflicts of interest.

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REFERENCES

1. van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 2018;19:213-28.

2. Admyre C, Johansson SM, Qazi KR, et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol 2007;179:1969-78.

3. Li X, Jing Z, Li X, et al. The role of exosomes in cancer-related programmed cell death. Immunol Rev 2023:Online ahead of print.

4. Lozano-Andrés E, Enciso-Martinez A, Gijsbers A, et al. Physical association of low density lipoprotein particles and extracellular vesicles unveiled by single particle analysis. J Extracell Vesicles 2023;12:e12376.

5. Feng J, Xiao BL, Zhang LZ, et al. Simultaneous detection of two extracellular vesicle subpopulations in saliva assisting tumor T staging of oral squamous cell carcinoma. Anal Chem 2023;95:7753-60.

6. Chargaff E, West R. The biological significance of the thromboplastic protein of blood. J Biol Chem 1946;166:189-97.

7. Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol 1967;13:269-88.

8. Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 1987;262:9412-20.

9. György B, Szabó TG, Pásztói M, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 2011;68:2667-88.

10. Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ. Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol 2018;15:617-38.

11. Cocozza F, Grisard E, Martin-Jaular L, Mathieu M, Théry C. SnapShot: extracellular vesicles. Cell 2020;182:262-e1.

12. Jeppesen DK, Fenix AM, Franklin JL, et al. Reassessment of exosome composition. Cell 2019;177:428-45.e18.

13. Merchant ML, Rood IM, Deegens JKJ, Klein JB. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery. Nat Rev Nephrol 2017;13:731-49.

14. Mu N, Li J, Zeng L, et al. Plant-derived exosome-like nanovesicles: current progress and prospects. Int J Nanomedicine 2023;18:4987-5009.

15. Lattmann E, Levesque MP. The role of extracellular vesicles in melanoma progression. Cancers 2022;14:3086.

16. O'Brien K, Breyne K, Ughetto S, Laurent LC, Breakefield XO. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat Rev Mol Cell Biol 2020;21:585-606.

17. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 2009;9:581-93.

18. Yamauchi M, Shimizu K, Rahman M, et al. Efficient method for isolation of exosomes from raw bovine milk. Drug Dev Ind Pharm 2019;45:359-64.

19. Hegmans JP, Bard MP, Hemmes A, et al. Proteomic analysis of exosomes secreted by human mesothelioma cells. Am J Pathol 2004;164:1807-15.

20. Théry C, Regnault A, Garin J, et al. Molecular characterization of dendritic cell-derived exosomes. selective accumulation of the heat shock protein hsc73. J Cell Biol 1999;147:599-610.

21. Dang VD, Jella KK, Ragheb RRT, Denslow ND, Alli AA. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells. FASEB J 2017;31:5399-408.

22. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020;367:eaau6977.

23. Kowal J, Arras G, Colombo M, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A 2016;113:E968-77.

24. Lundholm M, Schröder M, Nagaeva O, et al. Prostate tumor-derived exosomes down-regulate NKG2D expression on natural killer cells and CD8+ T cells: mechanism of immune evasion. PLoS One 2014;9:e108925.

25. Roudi S, Rädler JA, El Andaloussi S. Therapeutic potential of extracellular vesicles in neurodegenerative disorders. Handb Clin Neurol 2023;193:243-66.

26. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 2013;200:373-83.

27. Lai CP, Tannous BA, Breakefield XO. Noninvasive in vivo monitoring of extracellular vesicles. Methods Mol Biol 2014;1098:249-58.

28. Hikita T, Miyata M, Watanabe R, Oneyama C. In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter. Sci Rep 2020;10:16616.

29. Gupta D, Liang X, Pavlova S, et al. Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging. J Extracell Vesicles 2020;9:1800222.

30. Luo W, Dai Y, Chen Z, Yue X, Andrade-Powell KC, Chang J. Spatial and temporal tracking of cardiac exosomes in mouse using a nano-luciferase-CD63 fusion protein. Commun Biol 2020;3:114.

31. Sung BH, von Lersner A, Guerrero J, et al. A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells. Nat Commun 2020;11:2092.

32. Chen L, Chen L, Dotzert M, Melling CWJ, Zhang J. Nanostructured biosensor using bioluminescence quenching technique for glucose detection. J Nanobiotechnology 2017;15:59.

33. Li JB, Wang Q, Liu HW, Yuan L, Zhang XB. A bioluminescent probe for imaging endogenous hydrogen polysulfides in live cells and a murine model of bacterial infection. Chem Commun 2019;55:4487-90.

34. Karasawa K, Sano Y, Arakawa H. Development of a novel telomerase assay using the PPDK-luciferin-luciferase detection system. Luminescence 2014;29:52-7.

35. Izumi H, Ishimoto T, Yamamoto H, Nishijo H, Mori H. Bioluminescence imaging of Arc expression enables detection of activity-dependent and plastic changes in the visual cortex of adult mice. Brain Struct Funct 2011;216:91-104.

36. Gonçalves MS. Fluorescent labeling of biomolecules with organic probes. Chem Rev 2009;109:190-212.

37. Tertel T, Schoppet M, Stambouli O, Al-Jipouri A, James PF, Giebel B. Imaging flow cytometry challenges the usefulness of classically used extracellular vesicle labeling dyes and qualifies the novel dye Exoria for the labeling of mesenchymal stromal cell-extracellular vesicle preparations. Cytotherapy 2022;24:619-28.

38. Radnaa E, Richardson LS, Sheller-Miller S, et al. Extracellular vesicle mediated feto-maternal HMGB1 signaling induces preterm birth. Lab Chip 2021;21:1956-73.

39. González MI, González-Arjona M, Santos-Coquillat A, et al. Covalently labeled fluorescent exosomes for in vitro and in vivo applications. Biomedicines 2021;9:81.

40. Xu R, Bai Y, Min S, Xu X, Tang T, Ju S. In vivo Monitoring and Assessment of Exogenous Mesenchymal Stem Cell-Derived Exosomes in Mice with Ischemic Stroke by Molecular Imaging. Int J Nanomedicine 2020;15:9011-23.

41. Song S, Shim MK, Lim S, et al. In situ one-step fluorescence labeling strategy of exosomes via bioorthogonal click chemistry for real-time exosome tracking in vitro and in vivo. Bioconjug Chem 2020;31:1562-74.

42. Liu C, Helsper S, Marzano M, et al. Human forebrain organoid-derived extracellular vesicle labeling with iron oxides for in vitro magnetic resonance imaging. Biomedicines 2022;10:3060.

43. Luo L, Jian X, Sun H, et al. Cartilage endplate stem cells inhibit intervertebral disc degeneration by releasing exosomes to nucleus pulposus cells to activate Akt/autophagy. Stem Cells 2021;39:467-81.

44. Liu T, Zhu Y, Zhao R, Wei X, Xin X. Visualization of exosomes from mesenchymal stem cells in vivo by magnetic resonance imaging. Magn Reson Imaging 2020;68:75-82.

45. Busato A, Bonafede R, Bontempi P, et al. Magnetic resonance imaging of ultrasmall superparamagnetic iron oxide-labeled exosomes from stem cells: a new method to obtain labeled exosomes. Int J Nanomedicine 2016;11:2481-90.

46. Kim H, Jin S, Choi H, et al. Target-switchable Gd(III)-DOTA/protein cage nanoparticle conjugates with multiple targeting affibody molecules as target selective T1 contrast agents for high-field MRI. J Control Release 2021;335:269-80.

47. Wang X, Chen L, Ge J, et al. Rational Constructed Ultra-Small Iron Oxide Nanoprobes Manifesting High Performance for T1-Weighted Magnetic Resonance Imaging of Glioblastoma. Nanomaterials 2021;11:2601.

48. Giraud R, Moyon A, Simoncini S, et al. Tracking radiolabeled endothelial microvesicles predicts their therapeutic efficacy: a proof-of-concept study in peripheral ischemia mouse model using SPECT/CT imaging. Pharmaceutics 2022;14:121.

49. Németh K, Varga Z, Lenzinger D, et al. Extracellular vesicle release and uptake by the liver under normo- and hyperlipidemia. Cell Mol Life Sci 2021;78:7589-604.

50. Jing B, Gai Y, Qian R, et al. Hydrophobic insertion-based engineering of tumor cell-derived exosomes for SPECT/NIRF imaging of colon cancer. J Nanobiotechnology 2021;19:7.

51. Faruqu FN, Wang JT, Xu L, et al. Membrane radiolabelling of exosomes for comparative biodistribution analysis in immunocompetent and immunodeficient mice - a novel and universal approach. Theranostics 2019;9:1666-82.

52. Varga Z, Gyurkó I, Pálóczi K, et al. Radiolabeling of extracellular vesicles with (99m)Tc for quantitative in vivo imaging studies. Cancer Biother Radio 2016;31:168-73.

53. Hwang DW, Choi H, Jang SC, et al. Noninvasive imaging of radiolabeled exosome-mimetic nanovesicle using (99m)Tc-HMPAO. Sci Rep 2015;5:15636.

54. Shi S, Li T, Wen X, et al. Copper-64 labeled PEGylated exosomes for in vivo positron emission tomography and enhanced tumor retention. Bioconjug Chem 2019;30:2675-83.

55. Lin Q, Cao C, Li T, Man Z, Cao Y, Wang H. dSPIC: a deep SPECT image classification network for automated multi-disease, multi-lesion diagnosis. BMC Med Imaging 2021;21:122.

56. Dewulf J, Adhikari K, Vangestel C, Wyngaert TVD, Elvas F. Development of antibody immuno-PET/SPECT radiopharmaceuticals for imaging of oncological disorders-an update. Cancers 2020;12:1868.

57. Nguyen Cao TG, Kang JH, Kim W, et al. Engineered extracellular vesicle-based sonotheranostics for dual stimuli-sensitive drug release and photoacoustic imaging-guided chemo-sonodynamic cancer therapy. Theranostics 2022;12:1247-66.

58. Jang Y, Kim H, Yoon S, et al. Exosome-based photoacoustic imaging guided photodynamic and immunotherapy for the treatment of pancreatic cancer. J Control Release 2021;330:293-304.

59. Zhu D, Lyu M, Huang Q, et al. Stellate plasmonic exosomes for penetrative targeting tumor NIR-II thermo-radiotherapy. ACS Appl Mater Interfaces 2020;12:36928-37.

60. Ding H, Cai Y, Gao L, et al. Exosome-like nanozyme vesicles for H2O2-responsive catalytic photoacoustic imaging of xenograft nasopharyngeal carcinoma. Nano Lett 2019;19:203-9.

61. Chen X, Ren X, Zhu Y, et al. Cathepsin B-activated fluorescent and photoacoustic imaging of tumor. Anal Chem 2021;93:9304-8.

62. Armanetti P, Flori A, Avigo C, et al. Spectroscopic and photoacoustic characterization of encapsulated iron oxide super-paramagnetic nanoparticles as a new multiplatform contrast agent. Spectrochim Acta A Mol Biomol Spectrosc 2018;199:248-53.

63. Shi B, Gu X, Fei Q, Zhao C. Photoacoustic probes for real-time tracking of endogenous H2S in living mice. Chem Sci 2017;8:2150-5.

64. Yang M, Wang X, Pu F, et al. Engineered exosomes-based photothermal therapy with MRI/CT imaging guidance enhances anticancer efficacy through deep tumor nucleus penetration. Pharmaceutics 2021;13:1593.

65. Tayyaba, Rehman FU, Shaikh S, et al. In situ self-assembled Ag-Fe3O4 nanoclusters in exosomes for cancer diagnosis. J Mater Chem B 2020;8:2845-55.

66. Lázaro-Ibáñez E, Faruqu FN, Saleh AF, et al. Selection of fluorescent, bioluminescent, and radioactive tracers to accurately reflect extracellular vesicle biodistribution in vivo. ACS Nano 2021;15:3212-27.

67. Wu AY, Sung YC, Chen YJ, et al. Multiresolution Imaging using bioluminescence resonance energy transfer identifies distinct biodistribution profiles of extracellular vesicles and exomeres with redirected tropism. Adv Sci 2020;7:2001467.

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OAE Style

Shi Y, Wang X, Zhang S, Yin H, Fan H, Tang Y, Yang N. Research progress in in vivo tracing technology for extracellular vesicles. Extracell Vesicles Circ Nucleic Acids 2023;4:684-97. http://dx.doi.org/10.20517/evcna.2023.49

AMA Style

Shi Y, Wang X, Zhang S, Yin H, Fan H, Tang Y, Yang N. Research progress in in vivo tracing technology for extracellular vesicles. Extracellular Vesicles and Circulating Nucleic Acids. 2023; 4(4): 684-97. http://dx.doi.org/10.20517/evcna.2023.49

Chicago/Turabian Style

Yanhua Shi, Xianghui Wang, Shifang Zhang, Hao Yin, Huaju Fan, Yaohui Tang, Nana Yang. 2023. "Research progress in in vivo tracing technology for extracellular vesicles" Extracellular Vesicles and Circulating Nucleic Acids. 4, no.4: 684-97. http://dx.doi.org/10.20517/evcna.2023.49

ACS Style

Shi, Y.; Wang X.; Zhang S.; Yin H.; Fan H.; Tang Y.; Yang N. Research progress in in vivo tracing technology for extracellular vesicles. Extracell. Vesicles. Circ. Nucleic. Acids. 2023, 4, 684-97. http://dx.doi.org/10.20517/evcna.2023.49

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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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