Radiolabeled exosomes for theranostics: Personalized tailored therapy through imaging

Abstract

Radiolabeled exosomes have emerged as a transformative platform at the intersection of nanomedicine, molecular imaging, and precision theranostics. These nanoscale extracellular vesicles exhibit intrinsic biocompatibility, low immunogenicity, and inherent targeting capabilities, making them highly attractive for both diagnostic and therapeutic applications. The integration of radiochemistry with exosome biology enables noninvasive, real-time tracking of biodistribution, pharmacokinetics, and target engagement using advanced imaging modalities such as positron emission tomography and single-photon emission computed tomography. This minireview comprehensively summarizes current radiolabeling strategies for exosomes, including direct and indirect approaches, highlighting their advantages, limitations, and impact on vesicle integrity and imaging accuracy. Furthermore, we discuss key imaging platforms, in vivo biodistribution patterns, and pharmacokinetic profiles that influence therapeutic efficacy. Critical challenges such as rapid clearance by the mononuclear phagocyte system, labeling instability, and the lack of standardized protocols are also addressed. Finally, we outline future perspectives focusing on advanced bioengineering, multimodal imaging integration, and clinical translation frameworks. Radiolabeled exosomes represent a promising next-generation theranostic system with the potential to enable personalized, image-guided therapies across oncology and regenerative medicine.

Key Words: Radiolabeled exosomes; Extracellular vesicles; Theranostics; Positron emission tomography imaging; Single-photon emission computed tomography imaging; Biodistribution; Pharmacokinetics; Nanomedicine; Drug delivery

Core Tip: Radiolabeled exosomes offer a powerful theranostic platform that combines targeted drug delivery with real-time with noninvasive imaging. By enabling precise tracking of biodistribution and therapeutic response through positron emission tomography and single-photon emission computed tomography, they provide significant advantages over conventional nanocarriers. Despite challenges such as rapid systemic clearance and labeling instability, advances in radiochemistry and exosome engineering are accelerating their clinical translation toward personalized medicine.

INTRODUCTION

Exosomes are nanoscale extracellular vesicles (EVs) (approximately 30-150 nm) enclosed by a lipid bilayer membrane. Exosomes, also called small EVs (sEVs), are distinct from microvesicles, which are larger but overlap in size with exosomes. Collectively, all these vesicles are referred to as EVs. Their membrane is enriched with cholesterol, sphingomyelin, and phosphatidylserine, providing structural stability and facilitating membrane fusion[1]. Surface proteins such as tetraspanins (CD9, CD63, CD81), integrins, and adhesion molecules contribute to cell targeting and uptake[2]. Internally, exosomes carry a diverse cargo of proteins, lipids, nucleic acids (mRNA, miRNA, lncRNA), and metabolites, reflecting the physiological state of the parent cell[3].

Exosomes have garnered significant interest as drug delivery platforms, underscoring the need to understand their in vivo distribution and biological behavior[4]. Concurrently, precision medicine and theranostics, which integrate diagnostic imaging with targeted therapy, are revolutionizing individualized treatment strategies in oncology and regenerative medicine[5,6]. The convergence of exosome biology with nuclear imaging technologies has given rise to radiolabeled exosomes as a promising platform for theranostic applications. Radiolabeling facilitates noninvasive, real-time tracking of exosome biodistribution, pharmacokinetics, and target engagement through various imaging modalities.

In precision oncology, targeted exosome-based theranostics offer a powerful approach for personalized cancer treatment. Engineered exosomes can function as both targeted delivery vehicles and therapeutic platforms, with customizable surface ligands and cargo tailored to patient-specific tumor profiles, enabling precise targeting, reduced off-target effects, and improved therapeutic efficacy[7].

In this minireview, we comprehensively examine the diverse methodologies developed to date for the radiolabeling of exosome, enabling their in vivo tracking using positron emission tomography (PET) and single-photon emission computed tomography (SPECT). We systematically and critically assess the principal advantages and inherent limitations of each labeling strategy, with particular emphasis on radiochemical stability, labeling efficiency, and preservation of EV integrity and functionality. Furthermore, where appropriate, we contextualize these approaches in relation to their in vivo imaging performance, including biodistribution, pharmacokinetics, signal sensitivity, and translational applicability.

RADIOLABELING STRATEGIES FOR EXOSOMES

Direct radiolabeling approaches

Direct radiolabeling approach is considered as the standard method of labeling. Direct intraluminal radiolabeling of exosomes with In-111 oxine is one of the earliest and most established nuclear imaging strategies for EVs, due to in vivo stability and suitability for quantitative SPECT imaging, offering clear advantages such as deep-tissue sensitivity and accurate biodistribution assessment compared to optical methods. The method utilizes the lipophilic, membrane-permeable nature of the In-111 oxine complex, which readily diffuses across the exosomal lipid bilayer. Upon entering, oxine dissociates and In-111 binds stably to intra-vesicular metal-chelating biomolecules (proteins and nucleic acids), resulting in high radiochemical yields and durable label retention[8].

This similar approach was studied by Khan et al[9] that optimized [Zr-89]Zr(oxinate)₄ as a simple and an efficient tool for direct and intraluminal radiolabeling of sEVs, leveraging a metastable, neutral, lipophilic complex that transports through the vesicle membranes. Upon entering, the oxine ligands dissociate following protonation and Zr-89 binds to intra-vesicular metal chelating biomolecules such as nucleic acids and proteins facilitating high yield radiolabeling while preserving vesicle structure and bioactivity. The use of cryo-electron microscopic imaging, nanoparticle tracking analysis, dot blot assays, and flow cytometry, confirms radiolabeled PANC1 derived sEVs retained their morphology and surface biomolecular features, indicating that the labeling procedure does not compromise vesicle integrity. The in vivo PET-computed tomography (CT) demonstrated stable tracking up to 24 hours, distinct biodistribution for intact vs heat-damaged sEVs, and introduced the spleen: Bone uptake ratio as a biomarker of sEV integrity. The protocol was validated across multiple sEV types and builds on prior ionophore-based radiometal delivery for cells/Liposomes, positioning Zr-89 oxinate PET as a practical platform to study sEV biodistribution and to support their development as drug carriers and disease biomarkers.

Another clinically relevant and established approach of radiolabeling is the use of Tc(Technetium)-99m hexamethylpropyleneamine oxime (HMPAO) a lipophilic radiometal complex, that readily diffuses across the exosomal lipid bilayer under mild, physiologically compatible conditions, avoiding vesicle damage and preserving biological activity. Upon internalization, Tc-99m HMPAO undergoes intra-vesicular conversion to a hydrophilic form, effectively trapping the radionuclide within the lumen and yielding stable radiolabeled EVs suitable for functional tracking. This method provides a benefit in high sensitivity and quantitative accuracy of SPECT imaging[10].

A recent study by Rashid et al[11] found a brief understanding on how exosomal source derived from tumor cells (with and without pharmacological treatments), myeloid-derived suppressor cells, and endothelial progenitor cells influences biodistribution patterns in living systems, using SPECT/CT imaging for quantitative tracking. After intravenous administration in mice, they successfully tracked the labeled exosomes, identifying that the uptake patterns were strongly influenced by the protein and cytokine composition of the vesicles. This study highlighted a distinct trafficking behavior, indicating exosome surface biomolecular signatures significantly shape their in vivo fate.

González et al[12] developed a passive surface-labeling mechanism for labeling milk-derived exosomes using Tc-99m (VII) to produce more reactive Tc-99m (IV) species, in its reduced oxidation state +4, allowing the reaction with the exosomes, through an efficient coordination with phosphonate and phosphate groups naturally present on the exosomal membrane further enabling non-invasive real-time tracking of these vesicles through SPECT/CT imaging. The resulting Tc-99m (IV)-labeled milk exosomes demonstrated high labeling stability in phosphate-buffered saline, ensuring accurate and long-duration imaging, maintaining structural integrity and resisting radionuclide dissociation during in vitro assessment. This study highlighted Tc’s redox chemistry in determining labeling efficiency and stability as an insight particularly relevant for future EV-based radiopharmaceutical development.

In addition to the findings, the bifunctional chelators that contains a functional group for covalent attachment to a reactive amine, thiol, or carboxylic group onto the surface of EVs and a metal-binding moiety for radionuclide sequestration such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) has emerged as one of the most stable and versatile chelators for direct radiolabeling of exosomes with Cu-64 or Ga-68, for monitoring the biodistribution of exosomes with PET imaging under physiologically compatible pH and temperature. It provides a high radiochemical yield under mild conditions compatible with vesicle integrity alongside strong thermodynamic and kinetic stability of Cu-64 NOTA and Ga68 NOTA complexes minimizing radionuclide dissociation in vivo, enabling accurate PET quantification of exosome trafficking. Hence, it has shown to be an approach widely used for real-time, non-invasive tracking of exosome biodistribution and for studying their roles in intercellular communication, therapeutic delivery, disease progression and to track exosomes along lymphatic and hematogenous pathways[13]. Similarly, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid, enables stable coordination of diagnostic or therapeutic radionuclides such as Ga-68 and Lu-177, offering flexibility for both imaging and therapeutic applications. Another commercially available bifunctional chelator diethylene triamine pentaacetic acid (DTPA), has been used for conjugation with radiometals commonly used in basic and clinical research for imaging purposes.

Covalent surface radiolabeling represents an important approach of direct exosome labeling strategies in which radionuclides are chemically attached to surface proteins through bio-conjugation chemistries such as N-hydroxysuccinimide esters for amine groups and maleimide-thiol coupling forming stable covalent bonds. As unmodified EVs contain reactive amine/carboxylic terminated phospholipids or transmembrane proteins available for covalent binding with imaging probes, under mild conditions, thus avoiding membrane disruption or the denaturation of surface proteins. This method significantly reduces radionuclide leakage, improving label retention and quantitative accuracy during PET/SPECT imaging[14].

Limitations of direct radiolabeling of exosomes

Although direct radiolabeling exists a wide range of approaches and its applications, a major limitation is the possibility of radiotracer adsorption onto EV surfaces or co-isolated proteins, rather than true incorporation into vesicles, which can generate false biodistribution signals and misleading organ uptake patterns. Therefore, the need of rigorous purification and free-label controls, reduces the chance of this limitation[15].

In vivo label dissociation through trans-chelation, oxidative degradation, or instability of the radiometal complex may lead to radionuclide redistribution independent of EV trafficking, complicating SPECT/PET quantification[16]. To avoid this the study on direct intraluminal radiolabeling of exosomes with In-111 oxine uses post-labeling chelation (e.g., ethylenediamine tetraacetic acid) to prevent nonspecific accumulation of free radionuclide[8].

Chemical modification of EV surfaces through covalent or chelator-based approaches may also alter membrane proteins or receptor interactions, impair the native EV uptake pathways or affect biological function if not performed under mild conditions[17]. Furthermore, the approach requires careful radionuclide selection based on biological kinetics as short half-lives of certain radionuclides (e.g., Ga-68) limit imaging windows, whereas longer-lived radionuclides increase radiation exposure[18]. As illustrated in Figure 1, direct radiolabeling strategies employ intraluminal loading or surface conjugation methods to enable stable radionuclide incorporation and subsequent in vivo imaging of exosomes.

Figure 1 Schematic representation of direct radiolabeling strategies for exosomes. Exosomes released from cells contain several biological materials, such as proteins, lipids, and nucleic acids, which can be utilized as binding or entry sites for radiolabels. Intraluminal labeling employs ionophores that facilitate passive diffusion across the lipid bilayer, allowing intercellular binding to biomolecules. Lipophilic radiotracers are converted into hydrophilic forms intraluminally, resulting in their entrapment within the vesicle lumen. Surface labeling utilizes bifunctional chelators, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid or 1,4,7-triazacyclononane-1,4,7-triacetic acid, which coordinate redox reactions with membrane phospholipids. These radiolabeled exosomes enable noninvasive in vivo tracking through positron emission tomography or single-photon emission computed tomography imaging. HMPAO: Hexamethylpropyleneamine oxime; NHS: N-hydroxysuccinimide; NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid; DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; SPECT: Single-photon emission computed tomography; CT: Computed tomography; PET: Positron emission tomography. Created in BioRender, https://BioRender.com/uoiu87p.

Indirect radiolabeling approaches

In contrast to the approach of direct radiolabeling where the radionuclide is physically adsorbed onto the exosomal lipid bilayer or covalently attached to membrane-associated proteins, the indirect radiolabeling approach follows the process of radionuclide binding to an intermediate molecules, chelator, linker molecule, or carrier systems, without directly incorporating the radionuclide into the vesicle lumen or covalently binding it to the vesicle membrane[14,19]. Direct radiolabeling approaches often prioritize radiochemical robustness and high labeling efficiency, that may perturb membrane fluidity, alter protein conformation, or induce vesicle aggregation, thereby affecting EV biodistribution and cellular interactions independently of tracer stability. Radiochemical stability is best assessed through serum challenge studies, transchelation assays, and in vivo metabolite analysis, whereas EV integrity requires complementary characterization by nanoparticle tracking analysis, electron microscopy, protein marker profiling, and functional uptake or signaling assays. Indirect radiolabeling, including carrier mediated and biorthogonal approaches, are frequently selected specifically to preserve EV biology, even when radiochemical stability may be imperfect, because the radionuclide is associated with an intermediate rather than the EV itself[20,21].

The approach for metallic radionuclides (e.g., In-111, Tc-99m, Re-188), involves a stable coordination made using a bifunctional chelator, including DTPA/1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid for hard radiometals and mercaptoacetyltriglycine (MAG3)/hydrazinonicotinamide for technetium and rhenium, that is first complexed with the metal and then conjugated to the biological vector, to balance complex stability, labeling kinetics, and bioconjugation convenience.

A study by Liu et al[22] demonstrated that radiometal labeling of 18 mer morpholino oligomer labeled with Tc-99m using MAG3 and with In-111 using DTPA resulted in largely comparable biodistribution profiles in normal mice, indicating that indirect chelation-based radiolabeling does not necessarily alter in vivo pharmacokinetics for mid-sized oligomers, and shows an identical biodistribution in most organs, with an exception of intestinal clearance, where Tc-99m MAG3 showed modest intestinal excretion while In-111 DTPA showed negligible gastrointestinal accumulation. The study also identified conventional conjugation of p-isothiocyanatobenzyl-DTPA to amine derivatized morpholino oligomers could generate unstable side products, leading to radiolabel instability after In-111 complexation.

Noncovalent surface association strategies such as lipid insertion of radiolabeled amphiphiles, hydrophobic anchoring of radiolabeled prosthetic groups, and electrostatic adsorption of radiolabeled intermediates, are also considered as common approaches to indirect radiolabeling methods.

Lipid insertion of radiolabeled amphiphiles involves radiolabeled amphiphilic molecules probes consisting of a long hydrophobic lipid tail such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine or cholesterol conjugated to a hydrophilic spacer [often polyethylene glycol (PEG)] bearing the radionuclide or radiometal chelate spontaneously integrating into the EV lipid bilayer through hydrophobic interactions, driven by the favorable free energy associated with burying hydrophobic moieties within the phospholipid bilayer, under physiological temperature and buffer conditions, minimizing vesicle aggregation or cargo leakage. Hydrophobic insertion has been found to have successfully applied for both SPECT and PET imaging. A notable example of the process includes radiolabeled 1,2-distearoyl-sn-glycero-3-phosphoethanolamine PEG derivatives into tumor derived exosomes, enabling stable Tc-99m labeling for dual SPECT/near-infrared fluorescence imaging[23].

Closely related to lipid insertion is hydrophobic anchoring, using radiometal complexes and lipophilic chelates originally developed for cell or liposome labeling intercalating directly into the EV membrane without the need for a bulky phospholipid scaffold. These probes can achieve high labeling efficiencies under mild conditions, making them suitable for short lived radionuclides and time sensitive imaging studies, where the radiotracer itself possesses sufficient hydrophobic character to associate with the lipid bilayer, often through van der Waals interactions with acyl chains. Even though the process requires minimal EV manipulation, however compared with lipid PEG insertion, hydrophobic anchoring may suffer from increased tracer leakage or nonspecific transfer to serum proteins in vivo.

Electrostatic adsorption to the EV surface, exploits the intrinsically negative surface charge of EV membranes arising from phosphatidylserine, glycoproteins, and other anionic components causing radiolabeled cationic polymers, peptides, or nanoparticles to associate with the EV surface through Coulombic interactions, with surface charge density playing a dominant role in binding strength forming stable yet reversible complexes. The process is rapid and can be tuned by adjusting buffer conditions or probe charge, without the requirement of any specialized chemistry, making it suitable for short imaging, in vitro tracking, or localized administration routes. However, this approach is particularly sensitive to physiological salt concentrations and serum proteins, which can shield electrostatic interactions and promote probe dissociation in vivo[24].

Nonmetal radionuclides such as F-18 and radioiodine (I-123, I-124, I-125, and I-131), encompasses radio halogen-based strategies of indirect radiolabeling. Radio halogenated prosthetics can engage EVs via the non-covalent paradigms as discussed for radiometals’ amphiphiles depending on desired in vivo stability and biological preservation of the EV surface. For PET, F-18 is introduced through pre labeled prosthetic moieties (e.g., F-18 fluorobenzyl, F-18 fluoropropyl, activated F-18 esters), which can then be either non covalently associate with the EV membrane (hydrophobic insertion/anchoring) or coupled via mild bioconjugation to available functionalities engineered onto the EV surface. However, the approach leverages PET’s sensitivity and quantification and faces tight synthesis to labeling timelines due to F-18’s 110 minutes half-life, which can constrain EV isolation and QC windows. Studies by Almeida et al[14] and Ashique and Anand et al[19] show radioiodine incorporated via iodinated prosthetic groups (e.g., iodophenyl derivatives, Bolton-Hunter-type reagents), can be adsorbed to membranes or undergo secondary coupling under mild conditions compatible with EV integrity (I-123/I-131 for SPECT, I-124 for PET, and I-125/I-131 for preclinical quantitation/therapy).

In carrier mediated indirect radiolabeling of EVs, a significant use of carrier molecule including radiolabeled antibodies or antibody fragments directed against EV surface markers such as tetraspanins (e.g., CD9, CD63, CD81), is physically and chemically decoupled from the EV during radiochemistry, and EV association occurs only after completion of the labeling step. Here, the antibody is radiolabeled using established clinical radiochemistry workflows and subsequently incubated with purified EVs, allowing selective binding without chemical modification of the vesicles themselves.

Radio nanoparticles including liposomes, polymers, or inorganic nanomaterials, that are engineered to tether to EV membranes through affinity ligands, electrostatic interactions, or membrane fusion motifs, are also deployed during the process. The process of carrier mediated indirect labelling completely separates radiochemistry from EV handling, minimizing exposure of vesicles to harsh reaction conditions, organic solvents, elevated temperatures, or metal chelators that may compromise EV integrity, surface proteins, or biological function. Recently, bio-orthogonal adapter systems have been introduced, in which EVs are first functionalized with a nonradioactive chemical handle (e.g., azide, alkyne, tetrazine, or trans cyclooctene groups), through metabolic labeling of parent cells, enzymatic or lipid mediated insertion into the EV membrane, or mild covalent modification of surface proteins, all performed under conditions that preserve vesicle integrity and biological activity, followed by enabling highly selective post hoc radiolabeling via click chemistry using a separately radiolabeled probe. The most commonly employed reactions include strain promoted azide-alkyne cycloaddition and inverse electron demand Diels-Alder ligation between tetrazines and strained alkenes such as trans cyclooctene, characterized by exceptionally fast kinetics, high chemo selectivity, and compatibility with aqueous, physiological conditions, enabling efficient radiolabeling without the need for catalysts. As a result, the risk of EV aggregation, membrane disruption, or loss of functional surface epitopes is minimized, due to no application of elevated temperatures, or harsh solvents[25].

Limitations of indirect radiolabeling

Radioactive signals reflect the behavior of the labeling intermediate (e.g., antibody, nanoparticle, or click adapter) rather than the exosome itself, due to which any dissociation, degradation, or clearance of the intermediate can uncouple the imaging signal from the true biodistribution of intact exosomes, causing indirect reporting of exosome fate[21].

Indirect labeling often exhibits reduced radiochemical stability in vivo, and noncovalent interactions, enzymatic degradation of carriers, or metabolic processing can lead to partial or complete release of the radionuclide. Free or redistributed radionuclide may accumulate in clearance organs confounding biodistribution interpretation and limiting long term imaging studies[26,27].

Indirect radiolabeling often complicates absolute quantification of exosome dose and organ uptake and inherently prioritizes preservation of exosome biology over maximal signal permanence. It was also found, indirect radiolabeling rarely labels the entire exosome population uniformly, thereby causing variability in surface marker expression, membrane accessibility, or density of installed chemical handles that can result in heterogeneous labeling efficiency, with only a subset of exosomes contributing to the detected signal, introducing sampling bias and overrepresentation of specific exosome subpopulations[19,28]. As illustrated in Figure 2, indirect radiolabeling strategies employed in in vivo imaging of exosomes.

Figure 2 Schematic representation of indirect radiolabeling strategies. Exosomes released from cells contain several biological materials, such as proteins, lipids, and nucleic acids, which can be utilized as binding or entry sites for radiolabels. Indirect labeling incorporates radiotracers into exosomes through chelation, electrostatic binding, lipid insertion, or carrier-mediated (bio-orthogonal) methods, enabling stable, noninvasive positron emission tomography/single-photon emission computed tomography imaging tracking without compromising vesicle integrity. Created in BioRender, https://BioRender.com/278r8w1. Science Suite Inc. dba BioRender (“BioRender”) has granted Prakash Gangadaran permission to use this Completed Graphic in accordance with BioRender’s Terms of Service and Academic License Terms (“License Terms”).

IMAGING MODALITIES FOR RADIOLABELED EXOSOMES

Exosomes can now be seen non-invasively in vivo thanks to the development of radiolabeling techniques, which offer vital information about their pharmacokinetics, targeting effectiveness, and biodistribution. Because of their high sensitivity and quantitative capabilities, PET, SPECT, and multimodal imaging platforms have become the most promising of the available techniques.

PET imaging

A very sensitive imaging technique called PET enables quantitative and real-time tracking of radiolabeled biomolecules in vivo. Compared to SPECT, fewer studies have directly investigated PET-based exosome imaging; however, PET has clear advantages, including improved spatial resolution, increased sensitivity, and precise tracer distribution quantification.

Exosome pharmacokinetics and tumor targeting can be precisely assessed through radiolabeling techniques employing positron-emitting radionuclides. When compared to optical imaging, nuclear imaging techniques, such as PET, offer better tissue penetration and enable whole-body tracking of exosomes without signal attenuation[29]. Because of this, PET is especially useful for researching long-term circulation behavior and deep tissue biodistribution.

Furthermore, PET imaging can be integrated with CT or magnetic resonance imaging (MRI) to provide anatomical and functional information simultaneously, enhancing the interpretation of exosome localization in complex biological systems. Although the application of PET in exosome imaging is still evolving, it holds strong potential for future clinical translation due to its widespread use in oncology and molecular imaging.

SPECT imaging

The most popular method for imaging radiolabeled exosomes is SPECT because of its accessibility, affordability, and compatibility with commonly used radionuclides like Tc-99m. Exosomes have been successfully radiolabeled with Tc-99m for in vivo tracking in several studies. Exosome-mimetic nanovesicles labeled with Tc-99m HMPAO, for example, demonstrated high radiochemical purity (> 90%) and stability, allowing real-time SPECT/CT imaging of biodistribution in mice[10]. Organ-specific distribution patterns were highlighted by the imaging results, which showed minimal brain uptake and predominant accumulation in the liver. Similarly, Tc-99m tricarbonyl complex-labeled erythrocyte-derived EVs showed stable labelling and enabled non-invasive SPECT/CT visualization, with significant accumulation seen in the spleen and liver[30]. Another study using Tc-99m labeled red blood cell-derived exosome-mimetics confirmed these findings and further identified Kupffer cells as key mediators of hepatic uptake[31]. In addition to technetium-based labelling, radioiodine (I-125 and I-131) have been employed for SPECT imaging and gamma camera studies. These approaches enable highly stable labelling (> 98%) and allow real-time visualization of exosome trafficking and biodistribution in vivo[29]. Quantitative studies using I-125 labelling further demonstrated rapid clearance of exosomes from circulation and significant accumulation in the liver, spleen, and lungs[32]. Overall, SPECT imaging provides a robust and quantitative platform for studying exosome biodistribution, with the added advantage of well-established clinical translation pathways.

Multimodal imaging platforms

Multimodal imaging platforms have been created by combining nuclear imaging with methods like MRI, CT, and optical imaging in order to get around the drawbacks of single imaging modalities. These methods improve the accuracy of exosome tracking by enabling the simultaneous acquisition of functional, molecular, and anatomical data.

For instance, the precise localization of radiolabeled exosomes within anatomical structures is made possible by the combination of SPECT and CT imaging, which improves the interpretation of biodistribution data[10]. Similarly, MRI and fluorescence imaging have been used to successfully track exosomes labeled with gadolinium and near-infrared dyes, showing prolonged accumulation in tumor tissues[33]. Magnetic nanoparticle-based approaches further enable integration with MRI and emerging techniques such as magnetic particle imaging, allowing real-time visualization of exosome delivery to hypoxic tumor regions[34]. In addition, gold nanoparticle-labeled exosomes have been used for CT imaging, demonstrating selective targeting of pathological brain regions and inflammation-driven accumulation[35]. Recent advances also include F-19 MRI-based imaging, where fluorinated exosomes provide highly specific signals without background interference, enabling precise tracking of biodistribution while preserving exosome integrity[36]. Multimodal platforms therefore represent a significant advancement in exosome imaging by combining the high sensitivity of nuclear imaging with the spatial resolution of MRI or CT, ultimately improving both qualitative and quantitative analysis.

IN VIVO BIODISTRIBUTION, PHARMACOKINETICS AND THERAPY

Exosomes’ effectiveness as therapeutic and diagnostic nanocarriers is largely dependent on their pharmacokinetics and in vivo biodistribution. Exosome fate after systemic administration can now be precisely and quantitatively assessed thanks to radiolabeling techniques, which also reveal consistent organ distribution patterns and clearance mechanisms.

The quick removal of exosomes from the systemic circulation, which is mostly accomplished by the mononuclear phagocyte system (MPS), is a crucial finding from several investigations. After intravenous injection, circulating levels rapidly decrease, with a notable accumulation in the liver (approximately 28%), followed by the lungs and spleen within a few hours, according to quantitative analysis using I-125 labeled exosomes[32], this quick absorption emphasizes how important hepatic clearance is in controlling the pharmacokinetics of exosomes.

Similarly, research employing Tc-99m labeled exosomes consistently reports preferential accumulation in the liver and spleen, confirming their sequestration by elements of the MPS. Kupffer cells, the liver’s resident macrophages, are primarily responsible for hepatic uptake, according to additional research using red blood cell-derived exosome-mimetics, highlighting the significance of immune cell interactions in exosome clearance. All of these results suggest that phagocytic activity and innate immune recognition have a significant impact on exosome biodistribution[30,31]. Additionally, pharmacokinetic studies show that labeling strategy and origin affect exosome stability and circulation time. For instance, exosome-mimetic nanovesicles labeled with Tc-99m HMPAO demonstrated high radiochemical stability (> 90%) and preserved structural integrity throughout circulation, allowing for accurate in vivo imaging. Likewise, radioiodine labeling methods achieved high stability (> 98%) without significantly altering exosome morphology or size, ensuring accurate tracking of their biological behavior[10].

Exosomes have intrinsic and engineered targeting capabilities that go beyond passive accumulation and have a major impact on their biodistribution profiles[29]. Exosomes derived from mesenchymal stem cells have been shown to accumulate gradually in tumor tissues over the course of 24 hours to 48 hours in tumor-bearing models, suggesting improved retention in comparison to synthetic nanoparticles. Similarly, increased tumor localization was demonstrated by surface-engineered exosomes that targeted human epidermal growth factor receptor 2 receptors, indicating that receptor-mediated uptake is a crucial factor in pharmacokinetics[33].

The route of administration has a significant impact on biodistribution. Exosomes delivered intranasally have been demonstrated to accumulate at injury or disease sites within the central nervous system by avoiding systemic clearance and facilitating direct transport across the blood-brain barrier. This demonstrates how different delivery methods may enhance bioavailability and targeting effectiveness[37].

Another study reported that I-131 labeling to exosomes prepared from human embryonic kidney 293T and targeting the integrin αvβ3-positive anaplastic thyroid carcinoma tumor. Human embryonic kidney 293T expressing exosomal membrane protein (Lamp2b) fused to the αv integrin-specific iRGD peptide and tyrosine fragments. Then the exosomes were loaded doxorubicin and further radiolabeled with I-131 by chloramine-T method. SPECT/CT imaging relieved that the intravenous administration of doxorubicin@iRGD-Exos-I-131 effectively targeting the tumor and also inhibits the tumor growth. This result showed the theranostics effects of ¹I-131 labeled exosomes[38].

Overall, the pharmacokinetics of exosomes are governed by a combination of biological origin, surface characteristics, labeling methods, and administration routes. While rapid clearance by the liver and spleen remains a major limitation, advances in surface engineering and targeting strategies offer promising approaches to enhance circulation time and tissue-specific delivery[39]. Table 1 provides detailed information about the imaging modality, radiolabeling agent, labeling strategy, labeling efficiency or purity, and key findings.

Table 1 Radiolabeling and imaging strategies for in vivo tracking of exosomes and extracellular vesicle.

Imaging modality	Radiolabeling agent	Labeling strategy	Labeling efficiency/purity	Key findings	Ref.
PET	Cu-64, Ga-68	Direct or indirect (chelator-based)	98%	Enables quantitative whole-body tracking of exosome in sentinel lymph nodes	[13]
SPECT/CT	Tc-99m HMPAO	Direct	> 90%	Predominant liver accumulation, no/minimal brain uptake	[10]
SPECT	Tc-99m tricarbonyl	Direct	> 90%	Significant liver and spleen accumulation	[30]
Gamma imaging	Tc-99m	Direct	> 99%	Kupffer-cell-mediated hepatic uptake; liver and spleen accumulation	[31]
SPECT/gamma imaging	I-125, I-131	Direct surface labeling	> 98%	Rapid blood clearance; accumulation in liver, spleen, and lungs	[29,32]
MRI	Gadolinium	Surface modification/cargo loading	90%	Prolonged tumor accumulation of engineered exosomes	[33]
MPI/MRI	Magnetic nanoparticle	Nanoparticle incorporation	-	Visualization of exosome delivery to hypoxic tumors	[34]
CT	Gold nanoparticle	Nanoparticle conjugation	-	Selective accumulation in pathological brain regions and inflammatory sites	[35]
MRI	F-19	Direct membrane incorporation	-	Background-free biodistribution tracking	[36]
SPECT/CT	I-131	Chloramine-T method	50.16%-60.21%	Targeting and tumor growth inhibition	[38]

PET: Positron emission tomography; SPECT: Single-photon emission computed tomography; CT: Computed tomography; MRI: Magnetic resonance imaging; MPI: Magnetic particle imaging; Cu-64: Copper-64; Ga-68: Gallium-68; Tc-99m: Technetium-99m; HMPAO: Hexamethylpropyleneamine oxime; I-125: Iodine-125; I-131: Iodine-131; F-19: Fluorine-19.

The radiolabeled exosome studies have provided valuable insights into their in vivo behavior, demonstrating predictable biodistribution patterns alongside adaptable targeting capabilities. A deeper understanding of these pharmacokinetic properties is essential for optimizing exosome-based platforms for clinical applications in drug delivery and molecular imaging.

CLINICAL TRANSLATION AND SAFETY CONSIDERATIONS

Exosomes inherit a capability of low immunogenicity, biocompatibility and natural intercellular functions, that makes them a superior clinical application as a synthetic nanocarrier[40,41]. They have a unique capability to preserve and maintain biological stability, transport proteins, lipids and nucleic acids that has sparked a great deal of interest amongst scientists and researchers to use exosomes in targeted therapy and diagnostic imaging.

Isolation techniques, including ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation, can aid in the clinical translation thereby standardizing exosome isolation, purification, and characterization procedures[42]. However, it was noticed that the reproducibility often is impacted by variation in exosomes yield, purity and biological activity that is found due to lack of standardized protocols. The dependability of radiolabelling is also affected, when a standardized clinical translation protocol seems to be lacking, therefore, good manufacturing practice must be used to imply robust and scalable production process that adheres to safety and guarantee batch-to-batch consistency[43]. Studies have shown a major consideration to clinical safety and translation relating to the stability and efficiency of radiolabeling techniques, using nuclear imaging modalities such as PET and SPECT[9,44]. A critical step for translation is optimizing radiolabeling chemistry and validating in vivo stability. Immunogenicity, biodistribution, and toxicity require careful evaluation and investigation to maintain clinical safety, even though exosomes are considered as to carry low immunogenicity compared to synthetic nanoparticles, the biological of exosomes may introduce variability depending on the donor cell type[41,45]. For safety and clinical application, radiolabel must remain stably associated with exosomes during systemic circulation to accurately reflect their biodistribution, because a label dissociation can lead to misleading imaging results and increase off-target radiation exposure[26].

Pharmacokinetics, toxicity, immunogenicity, and radiation dosimetry is required before initiating human trials, to keep the safety protocol into consideration. Radiolabeled exosomes enable real-time tracking of therapeutic vesicles, assessment of targeted delivery efficiency, and early evaluation of treatment responses, thus clinical translation of radiolabeled exosomes is expected to progress rapidly in the coming years with continued advances in exosome engineering, radiochemistry, and imaging technologies[46].

Key clinical translation barriers specific to radiolabeled exosome platforms include the following: (1) The half-life of radiolabeling agents and their in vivo labeling stability after administration, which can lead to inaccurate measurements and require further investigation; (2) Lack of standardized protocols and quality control criteria to ensure reproducibility of radiochemical purity; (3) Challenges in dosimetry estimation due to the heterogeneous nature of EV populations; and (4) Regulatory complexities associated with radioactive theranostic agents. Despite these challenges, the integration of exosome biology with molecular imaging technologies is found to offer a significant number of opportunities for precision diagnostics and personalized medicine.

CURRENT CHALLENGES AND KNOWLEDGE GAPS

Despite significant progress in exosome-based imaging and drug delivery, several critical challenges and knowledge gaps hinder their clinical translation. One of the primary limitations is the lack of standardized methods for exosome isolation, purification, and characterization, which leads to variability in size, composition, and functional properties across studies[47]. This heterogeneity complicates reproducibility and makes it difficult to compare results between different experimental systems. Additionally, radiolabeling and imaging modifications may alter the structural integrity or biological behavior of exosomes, raising concerns about whether the observed biodistribution accurately reflects native exosome kinetics[48]. Another major challenge is the rapid clearance of exosomes by the MPS, particularly in the liver and spleen, which significantly reduces their circulation time and targeting efficiency. Furthermore, the mechanisms governing exosome uptake, trafficking, and cell-specific targeting remain incompletely understood, especially in complex in vivo environments[40]. There is also limited knowledge regarding the long-term safety, immunogenicity, and potential off-target effects of engineered or modified exosomes. Importantly, the field lacks robust quantitative pharmacokinetic models that can predict exosome behavior in humans, creating a gap between preclinical findings and clinical application. Addressing these challenges is essential for the rational design and successful translation of exosome-based imaging and therapeutic platforms.

FUTURE PERSPECTIVES

Future research in exosome-based imaging and therapeutics is expected to focus on improving target specificity, circulation time, and imaging accuracy through advanced engineering strategies. Surface modification of exosomes with targeting ligands, antibodies, or peptides offers a promising approach to enhance cell-specific delivery and reduce off-target accumulation[49]. In parallel, the development of next-generation combined radiolabeling and other imaging techniques, such as PET/MRI hybrid systems and bioorthogonal labeling approaches, may enable more precise and non-invasive tracking of exosomes in vivo without compromising their biological properties. Another promising direction is the use of genetically engineered or designer exosomes, which can be programmed to carry therapeutic cargo and exhibit controlled biodistribution profiles[40]. Advances in nanotechnology and synthetic biology may also lead to the creation of exosome-mimetic nanovesicles with improved scalability and reproducibility, overcoming current limitations in large-scale production. Furthermore, integrating computational modeling and data-driven approaches with imaging data could provide predictive insights into exosome pharmacokinetics and optimize therapeutic strategies. Importantly, future studies must prioritize clinical validation, standardized protocols, and regulatory frameworks to ensure safe and effective translation into human applications.

CONCLUSION

In conclusion, radiolabeled exosomes represent a rapidly evolving and highly promising class of nanocarriers for non-invasive imaging and targeted drug delivery. Advances in nuclear imaging modalities such as PET and SPECT, along with multimodal imaging platforms, have significantly enhanced our ability to track exosome biodistribution and understand their in vivo behavior. These studies consistently demonstrate that exosomes possess unique biological properties, including biocompatibility, intrinsic targeting ability, and capacity to transport diverse therapeutic cargo, making them advantageous over many synthetic nanoparticles[41]. However, challenges such as rapid systemic clearance, lack of standardization, and incomplete understanding of pharmacokinetics remain significant barriers to clinical translation. Continued efforts in bioengineering, imaging innovation, and mechanistic studies are essential to overcome these limitations. With the integration of advanced technologies and interdisciplinary approaches, exosome-based systems hold immense potential to revolutionize diagnostics, therapeutics, and personalized medicine, paving the way for more effective and targeted treatment strategies in the future.