Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes

Abstract

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

1 Introduction

To maintain organisms’ homeostasis, intercellular communication is the key event to control multiple biological processes including mediator secretion, cellular proliferation, differentiation, and apoptosis. Cells located remotely communicate each other via soluble factors including neurotransmitters, hormones, cytokines/chemokines, lipid mediators, and extracellular vesicles (EVs) [1,2,3]. EVs, cell-secreted natural nanoparticles, are classified into three subtypes including exosomes, microvesicles, and apoptotic bodies, which exhibit different biological characteristics in terms of biogenesis, content, morphology and size (exosomes: 30 ~ 200 nm, microvesicles: 100 ~ 1,000 nm, and apoptotic bodies: 1 ~ 5 μm) [3, 4]. Exosomes are single-membrane lipid bilayer vesicles generated either by vesicle budding into endosomes that mature into multivesicular bodies or by direct vesicle budding from the plasma membrane [5]. Exosomes are secreted by all living cell types and have been found in various body fluids such as plasma, urine, saliva, semen, and breast milk [6,7,8,9,10,11]. Microvesicles (or ectosomes) are formed by direct outward budding of the plasma membrane with size typically ranging from 100 to 1000 nm [12]. Apoptotic bodies are relatively larger lipid vesicles released by dying cells which contain fragments of apoptotic cells such as micronuclei, chromatin remnants, and intact organelles [13]. EVs have traditionally been defined and sorted based on their different densities and sizes which enables separation by various methods such as differential centrifugation, filtration, and size exclusion chromatography [14]. It should be noted, however, that due to the overlap** size and density between EVs such as exosomes and microvesicles, current EV isolation techniques have limitation regarding precise purification without completely excluding other groups of EVs. Within the EVs, microvesicles and exosomes are considered as delivery vehicles of diverse biological molecules for intercellular communication including delivery of nucleic acid (e.g. DNAs, RNAs), proteins, lipids, and carbohydrates. Of note, in vivo circulating exosomes isolated from body fluids (e.g. urine and blood) carry biological materials (e.g. proteins and nucleic acid) and represent current physiological conditions, which suggests the diagnostic value of exosomes as novel biomarkers for multiple pathophysiological conditions including cancer [15, 16].

Due to their biological and functional characteristics, the therapeutic potential of exosomes is also being investigated as either natural or engineered form for different therapeutic purposes, including drug delivery tools, biological targeting agents, and vaccination [17,18,19,20,23,24,25].

Here, we will review the current knowledge of biodistribution and PK of systemically administered exosomes, and various active targeting strategies to improve target specificity with better clinical outcome.

2 Biodistribution and pharmacokinetics (PK) of in vivo administered exosomes

Numerous biocompatible and nontoxic nanoparticles have been employed as drug delivery systems including liposomes, polymeric nanoparticles, and exosomes. The biodistribution and PK profile of nanoparticles represent the in vivo behavior of administered nanoparticles and determining these two parameters are the key for successful nanoparticles-mediated novel therapeutics development. Whereas this review paper is mainly focused on exosomes, readers can find recent advances regarding other EVs, such as microvesicles, on review papers cited here [12, 26].

Focusing on exosome therapeutics, the major tissues distribution of systemically administered exosomes generally include liver, spleen, kidney, lung and gastrointestinal tract, which can be altered by various factors such as cellular origin of exosomes, exosomal membrane composition (e.g., protein, lipid, and glycan) and pathophysiological condition of host [27,28,29,30,31,32,33,34,35]. Exosome engineering for targeted delivery of therapeutic exosomes to various tissues including brain, placenta, heart, spinal cord, and cartilages is also being investigated [36,37,38,39,40,41]. Once the exosomes are administered systemically, they show rapid clearance from the blood with less than a few minutes of half-life in the circulation of healthy animals, which is primarily due to the rapid clearance by circulating phagocytic cells including macrophages and neutrophils [29, 32, 42, 43]. In contrast with the blood PK, exosomes display prolonged retention in the tissues such as the liver and spleen, showing sustained retention longer than 24 h [27, 29, 32]. Nonetheless, careful interpretation is needed for analyzing the tissue PK of exosomes, since most exosome imaging techniques utilize methods to label the lipid bilayer of exosomes with various imaging dyes which may lead to tracking of the cell-ingested phospholipids and not the exosome itself.

3 Factors modulating biodistribution and PK of in vivo administered exosomes

Recent studies begin to identify molecules displayed on the exosomal membrane which determine their cellular or organ tropism (Fig. 1) [44, 45]. If the molecular mechanisms generating target cell tropism of exosomes is fully decoded, the potential of exosomes as a therapeutic vehicle would be greatly expanded, especially to the most challenging disease areas including the central nervous system (CNS) related diseases [46]. In this part, various factors that determine biodistribution and targetability of exosomes are discussed.

Fig. 1

Targeting and biodistribution/PK analysis strategies of exosome therapeutics. Targeting of exosomes to specific organs or cells could be achieved via modification of the composition of exosomal membrane proteins including tetraspanins and integrins. Exosomal surface engineering by displaying targeting peptides conjugated with exosomal membrane-associated domains such as lysosome-associated membrane glycoprotein 2b (Lamp2b) or C1C2 domain of lactadherin (LA) is another approach for active tissue targeting. Both glycan and lipid compositions of exosomal membrane also contribute to the biodistribution of administered exosomes. Biodistribution/PK analysis of administered exosomes can be conducted via various exosome labeling methods (i.e., bioluminescence, fluorescence, and radio isotope-labeling methods)

3.1 Cellular origin

One of the main factors that determine the biodistribution of exosomes is its cellular origin. Exosomes from different cellular sources were observed to have an asymmetric biodistribution [27], and subsequent studies found an inclination that exosomes tend to have different tropism based on their cells of origin, which could be further utilized for organ-targeted delivery. For instance, targeted delivery of exosomes to the brain could be achieved by using neural stem cell (NSC)-derived EVs, as it showed preferential brain targeting compared to mesenchymal stem cell (MSC)-derived EVs in a murine stroke model [47]. Tumor-derived exosomes were shown to be efficient in targeting its parental tumor for delivering anti-cancer drugs [29, 30, 48,49,50]. In a HT1080 xenograft mouse model, systemically injected HT1080-derived exosomes were targeted more efficiently to HT1080 tumor burden compared to HeLa-derived exosomes, with HT1080-derived exosomes delivered twice as much as HeLa-derived exosomes [30]. In zebrafish model, exosomes derived from either brain endothelial cells or brain tumor cells crossed the blood–brain barrier (BBB) and successfully deliver anti-cancer drugs to the brain tumors, with 4 nl of 0.2 mg/ml doxorubicin loaded in 200 μg/ml of brain endothelial cell-derived exosomes inhibiting expression of VEGF RNA more than half compared to doxorubicin-only injected brain tumor model of zebrafish [48]. Moreover, prostate cancer cell lines LNcaP- and PC-3-derived EVs loaded with Paclitaxel (PTX) have been shown to be effective carriers for delivering PTX to their parental cells [49]. However, Smyth et al. suggested that tumor-derived exosomes showed the tumor targeting property only when the exosomes were injected locally into the tumors [29]. In their study, they observed a minimal tumor targeting of systemically injected breast and prostate tumor-derived exosomes [29]. Jung et al. also showed that hypoxic cancer cell-derived exosomes are targeted to hypoxic cancer cells only in vitro but not in vivo [50]. More in-depth mechanistic studies are required regarding targeting ability of tumor cell-derived exosomes to their parental cancers. Even with tumor-targeting benefits for utilizing tumor-derived exosomes, they may have safety issues when administered systemically: tumor-derived exosomes may deliver tumorigenic factors to healthy cells and moreover, promote tumor metastasis by initiating pre-metastatic niche formation in healthy tissues [51,52,53]. Therefore, utilizing tumor-derived exosomes for tumor therapeutics may not be feasible. Instead, mechanistic insight for understanding tumor tropism of tumor-derived exosome can be applied to design targeting approach for tumor therapeutics with exosomes.

3.2 Membrane composition of exosomes (e.g., proteins, lipids, and glycans)

Cellular or organ targeting of exosomes is influenced by various membrane compositions of exosomes, such as proteins, lipids, and glycans. Membrane protein composition of exosomes is determined by their cellular origin as well as the physiological state of parental cells during exosome biogenesis [54]. For instance, exosomes originated from antigen-presenting cells, including dendritic cells, macrophages, and B cells, tend to display immune regulatory proteins and antigens similar to that of their parental cells [54]. Exosomes from mature dendritic cells were found to express mature phenotype markers such as major histocompatibility complex (MHC) class I, class II molecules, CD40, CD86 and ICAM-1/CD54 [55]. Similarly, exosomes released by B cells or T cells carry B-cell or T-cell receptor subunits, respectively [56, 57], and those derived from natural killer cells contain the NK cell marker CD56 [58], which partially resemble the features of the originated cells.

The major proteins that constitute the exosomal membranes are proteins such as tetraspanins (e.g., CD9, CD63, CD81, CD82), integrins and MHC molecules, of which various composition of these proteins could influence organotropism of exosomes [5]. For instance, exosomes expressing integrin α₆β₄ and α₆β₁ are targeted to laminin-enriched lung microenvironments, especially to the S100-A4-positive fibroblasts and surfactant protein C-positive epithelial cells in the lungs [59]. In contrast, exosomes displaying integrin α_vβ₅ preferentially interacts with fibronectin in the liver microenvironments, which is specifically targeted to F4/80 positive Kupffer cells [59]. Also, Qiao et al. identified eight different integrins (integrin αv, α3, α5, α6, β1, β4, β5, β6) in tumor-derived exosomes by proteome profiler array with receptor proteins, suggesting that these integrins contribute to the tumor tropism of tumor-derived exosomes [30]. Tetraspanins, which are abundant on the membrane of exosomes, also contribute to the organotropism of exosomes by forming a complex with other tetraspanins and integrins: exosomes with tetraspanin Tspan-8 and integrin α₄ complex were readily targeted to endothelial and pancreas cells [60]. CD47 is the ligand for signal regulatory protein alpha (SIRPα), which upon binding initiates the ‘don’t eat me’ signal that inhibits phagocytosis [61]. Kamerkar et al. showed that CD47 expressed on the exosomes mediated protection from phagocytosis by monocytes and macrophages, which showed that engineering surface of exosomes with CD47 could induce prolonged circulation time [62].

The lipid and glycan composition of the surface of exosomes may also contribute to tissue tropism by modulating cellular uptake of exosomes [33, 63]. In vivo administered exosomes are rapidly up-taken by circulating phagocytic cells within several minutes after systemic administration [42, 43], and Matsumoto et al. found that the rapid uptake of intravenously administered B16-BL6 melanoma cell-derived exosomes by macrophages is mediated via recognizing negatively charged phosphatidylserine (PS) displayed on the membrane of exosomes [33]. Exosomal uptake could also be mediated by glycans on the membrane of exosomes. The uptake of glioblastoma (GBM) cell-derived EVs to the recipient GBM cells were shown to involve a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18, which in turn promoted GBM cell proliferation and resistance to the alkylating agent temozolomide [63].

3.3 Pathophysiological conditions of host

The biodistribution and PK parameters of exosomes could be affected by the pathophysiological conditions of host. Grange et al. observed the biodistribution of MSC-derived EVs in a model of acute kidney injury (AKI) after intravenous injection and found significant accumulation of EVs in the kidney of AKI-induced mouse 15 min after exosome injection, whereas accumulation of EVs in kidney appeared 5 h after injection in healthy mouse [64]. BBB crossing can also be achieved at certain pathological conditions. Under the circumstances of brain inflammation, Yuan et al. showed that macrophage-derived exosomes expressing LFA-1 and C-type lectin receptors can penetrate the BBB by interacting with inflamed brain microvascular endothelial cells, with showing over three times more accumulation of exosomes in brain of inflamed mice compared to healthy mice [65]. An in vitro trans-well assay study demonstrated that unmodified exosomes can cross the BBB through endocytosis by brain microvascular endothelial cells which occurred only under stroke-like, inflamed conditions induced by TNF-α [66]. Mirzaaghasi et al. investigated the biodistribution and blood PK of HEK293T cell-derived exosomes in sepsis-induced mouse. They found that substantial number of exosomes were delivered to the lung compared to healthy mouse after intravenous injection, of which more than 30% of exosomes were delivered to the lung in sepsis-induced mouse after 1 h of injection whereas almost none were detected in the lung of healthy mouse. Also, prolonged retention of exosomes in the blood circulation were observed due to liver dysfunction [28]. In other disease models, clearance of fluorophore labeled exosomes (10 nmol, intravenous) from blood in normal mice were 0.0054–0.0154 mL/min [67], whereas Gaussia luciferase (gLuc)-lactadherin (LA) labeled exosomes (5 µg, intravenous) in macrophage-depleted mice [42] and ¹²⁵I labeled exosomes (4 × 10⁵ cpm, intravenous) in Parkinson’s disease mouse model [65] were 0.651 ± 0.157 mL/h and 0.016 mL/min, respectively.

4 Imaging methods for determining biodistribution and PK of exosomes

Currently, bioluminescence and fluorescence imaging are the most commonly use methods for monitoring in vivo behavior of administered exosomes. However, with the recent technological advances for deep tissue penetration imaging, other clinical imaging methods including magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT) are also being utilized for biodistribution and PK studies of exosomes (Fig. 1) [27, 62,

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Acknowledgement

This work was supported by a grant (2016M3A9B6945931) from Ministry of Science and ICT, Republic of Korea.

Author information

1. Hojun Choi, Yoorim Choi authors have been contributed equally.

Authors and Affiliations

1. ILIAS Biologics Incorporated, 40-20, Techno 6-ro, Yuseong-gu, Daejeon, 34014, Republic of Korea

   Hojun Choi, Yoorim Choi, Hwa Young Yim, Jae-Kwang Yoo & Chulhee Choi

2. Department of Bio and Brain Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

   Amin Mirzaaghasi & Chulhee Choi

Contributions

HC, YC, HY and AM wrote, and edited the manuscript. JY and CC supervised, reviewed, edited, and approved the manuscript.

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Correspondence to Jae-Kwang Yoo or Chulhee Choi.

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CC is the founder and shareholder, and JY, HC, HY are minor shareholders of ILIAS Biologics Inc. The authors have no additional financial interests.

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Choi, H., Choi, Y., Yim, H.Y. et al. Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes. Tissue Eng Regen Med 18, 499–511 (2021). https://doi.org/10.1007/s13770-021-00361-0

• Received: 12 May 2021

• Revised: 03 June 2021

• Accepted: 09 June 2021

• Published: 14 July 2021

• Issue Date: August 2021

• DOI: https://doi.org/10.1007/s13770-021-00361-0

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