Open access peer-reviewed chapter

Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting

Written By

Stefan Vogt, Gerhard Stadlmayr, Johannes Grillari, Florian Rüker and Gordana Wozniak-Knopp

Submitted: September 5th, 2018 Reviewed: December 13th, 2018 Published: January 28th, 2019

DOI: 10.5772/intechopen.83537

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Extracellular vesicles (EVs) have in the recent decades gained an important stand as vehicles enabling cell-to-cell transport and communication. With the advanced development towards their clinical use and increasing versatility of potential applications, improving their tissue-specific targeting in order to enhance their functionality in drug delivery opened as a challenging engineering field. In the past, the question of specific intercellular contact has been addressed by decoration of the EV surface with agents able of specific target recognition. An attractive possibility here is the modification of strongly overexpressed EV surface marker proteins towards recognition of target cells. As these proteins are involved in a plethora of biological functions in EV biogenesis, cargo targeting and intercellular transfer, a minimal impact on protein architecture upon modifications is desirable, which would also increase the stability of the exosomal preparation intended for therapeutic use. This chapter focuses on the possibilities of engineering of the EV marker proteins towards antigen-recognition units broadly applicable to endow EVs with tissue-targeting functionality.


  • extracellular vesicles
  • exosomes
  • tetraspanin
  • tissue-specific targeting
  • exosomal drug delivery

1. Introduction

The transfer of extracellular vesicles (EVs) has emerged in the last two decades as a novel mechanism for intercellular communication. Nomenclature of EVs has by now been agreed to be based on biogenesis pathway. Therefore, EVs budding from plasma membranes are termed ectosomes or microvesicles, while exosomes are formed via the endosomal compartment within multivesicular bodies (MVB) which then are released by their fusion with the plasma membrane. Finally, when blebbing from apoptotic cells, the term apoptotic bodies has been retained [1]. Biomarkers to differentiate these EVs are still questionable, and in general, size is used to differentiate exosomes from ectosomes, while apoptotic bodies are considered to present phosphatidylserine on the outside and thus can be stained by annexin V. Exosomes (40–150 nm in diameter) are produced by formation of endosomal intraluminal vesicles (ILVs) in multivesicular bodies (MVBs) and are secreted by fusion of these vesicles with the plasma membrane [2, 3] (Figure 1).

Figure 1.

Biogenesis of EVs.

Since they have been known to mediate directional intercellular transfer of their content, typically proteins, mRNAs, microRNAs (miRNAs) and a variety of non-coding RNAs [4], several studies were oriented towards their application as drugs per se or as therapeutic tool for drug delivery [5]. Their membrane is strongly enriched in sphingomyelin and cholesterol, which contributes to their unique buoyancy coefficient and enables practical isolation from other particles of cellular origin using differential centrifugation methods. Another discernible feature of exosome membranes is that it reflects the composition of the MVB membrane and has a high density of endosomal membrane proteins, such as proteins involved in MVB biogenesis (Alix and TSG101), membrane transport, in particular the components of the endosomal sorting complex required for transport (ESCRT) and most prominently tetraspanin proteins (CD9, CD37, CD53, CD63, CD81, CD82 and CD151) [6, 7]. Some tetraspanins such as CD9, CD81 and CD151 are more broadly expressed, while others are restricted to specific EV subsets [8, 9]. The overexpression of tetraspanins, as shown for CD9, can in several production cell lines enhance the exosome production and, in addition, cause a reduced overall size of the vesicles [10].

Tetraspanins play an important role in several physiological processes [11] and have been discovered to cooperate in states of health and disease in signal transduction, cellular activation, polarization, motility, adhesion, tissue differentiation, angiogenesis, tumorigenesis and metastasis [12, 13, 14], both by regulating cellular interactions as cell-membrane bound molecules and indirectly through exosomes. They are involved in each step of the metastatic cascade due to their ability to interact with cell surface receptors, adhesion molecules, matrix-remodeling proteases and signaling molecules. In this pathological state, they hence regulate cell proliferation, participate in epithelial-mesenchymal transition, modulate integrin-mediated cell adhesion and mediate the invasion through modulation of angiogenesis, tumor-endothelial cell interactions and regulation of cancer cell migration through the regulation of tumor microenvironment, as well as direct influence on extracellular matrix [15]. In this chapter, we introduce the characteristics of EVs and the engineering approaches aimed at their surface proteins to achieve tissue-specific targeting.


2. Structure of the tetraspanin proteins

The rod-shaped structure of a tetraspanin consists of four transmembrane helices that connect the two extracellular loops [16] (Figure 2). The short extracellular loop (SEL), which has not yet been reported to contain any element of a particular secondary structure, is of 12–31 amino acids in length. In the studies oriented towards tetraspanin engineering, the EC1 domain has until now been less addressed due to its lower degree of structural organization. Its replacement for a strand of glycine-serine residues has led to an unusual cell surface expression and a distribution dissimilar to the wild-type parental molecule, which may suggest the importance of this structural subunit for the stability of the membrane-bound tetraspanin [17].

Figure 2.

Organization of tetraspanin CD81: 4 transmembrane helices (TM) span 2 extracellular loops. Positions of cysteine residues are indicated with yellow dots. Crystal structure of EC2 domain is presented as a cartoon diagram of the dimeric form (PDB: 1G8Q); the conserved three-helix bundle is black and the variable domains C and D are surfaced in blue or green for each protomer. The figure was prepared using PyMOL (PyMOL molecular graphics system, version 1.3 Schrödinger, LLC).

The large extracellular loop (LEL) or extracellular domain 2 (EC2) has on the other hand been well characterized. The crystal structure of CD81 [18] demonstrated the four invariant cysteine resides within EC2 to form the 2 disulfide bridges, as a hallmark stability feature of the conserved tetraspanin fold [8, 19] (Figure 2). Moreover, the topology of several other tetraspanins was predicted by molecular modeling studies in the absence of an available crystal structure, using the CD81 EC2 structure as a template: these were the tetraspanins CD37, CD53, CD82 and CD151 [20, 21]. It has been early established that binding of CD81-specific antibodies depends on the formation of the disulfide bonds [22]. One class of tetraspanins harbors the EC2 composed of five α-helical elements, forming stalk and head elements of a mushroom-like structure, and the second class of tetraspanins is postulated to contain an additional helical element, stabilized with another pair of cysteine residues. The EC2 fold structure is evolutionarily conserved across species for any particular tetraspanin in spite of a high degree of variability at the amino acid level [23], with a subdomain consisting of a three-helix-bundle fold and a second subdomain variable in size among members of the tetraspanin family [18, 20].

The presence of an energetically unfavorable hydrophobic patch and the dimeric form of CD81 LEL in a crystal hinted at the high likelihood of tetraspanin assembly into dimers or multimers in the cell membrane. As shown later, clustering of tetraspanins leads to the organization of tetraspanin-associated proteins in a tetraspanin web or tetraspanin-enriched microdomain (TEM) [24, 25]. Biologically, such domains induce partitioning of the complexes into lipid rafts and clustering of the lipid rafts [26].

Apart from the hydrophobic interactions of the extracellular domains, the membrane-proximal cysteines residing in transmembrane domains contribute to the dimer formation and stability of tetraspanins. For the CD9 tetraspanin, it has been demonstrated that these residues are positioned close to the dimerization interface and influence homotypic and heterotypic tetraspanin association depending on their reversible palmitoylation status [27]. Similarly, the mutation of N-terminal and C-terminal transmembrane cysteines to serine eliminated palmitoylation of CD151, which turned out to be deleterious for the assembly with other cell surface proteins, including tetraspanins CD9 and CD63, their organization to TEMs and subsequently their subcellular distribution and cell morphology [28]. At the same time, it had minimal influence on the density of tetraspanin protein complexes and was dispensable for CD151-α3β1 integrin association. Depalmitoylation of CD81 did not impact its surface expression and stability, but rendered it less available for contact with its natural interaction partner CD9 and the relevant epitopes less accessible for binding of structurally dependent antibodies [29].

The four transmembrane helices of tetraspanin proteins form two largely separated pairs of antiparallel helices: one pair comprises TM1/TM2 and the other TM3/TM4. The two pairs of helices only converge close to the cytoplasmic side of the membrane through contacts between TM2 and TM3. In the recently solved crystal structure of full-length CD81, this cone-like structure has been shown to harbor a binding pocket for cholesterol [30], and mutations within transmembrane domains in certain tetraspanins have been connected with pathological states [31]. The short cytoplasmic tails show no obvious functional significance in signaling processes, suggesting that their signaling competence relies on association with other molecules [32]; nevertheless, its mutation can lead to different assembly with association partners as shown for CD9 [33].


3. Natural ligands of tetraspanin proteins

3.1. The tetraspanin web and intertetraspanin contacts

The ability of members of the tetraspanin family to assemble into a unique biological feature known as tetraspanin-enriched microdomain (TEM) is due to their mutual interactions; however, these structures include also receptors, integrins and signaling molecules such as phosphatidyl-kinase C (PKC) and phosphatidylinositol-4-kinase (PI4K) [9]. These interactions are fundamental for cellular functions such as cell adhesion, proliferation and motility. Interactions between tetraspanin members are important in maintaining the integrity and stability of the tetraspanin web and providing binding sites for different ligands. The multimers of newly synthesized proteins are formed in the Golgi apparatus. The predominantly cross-linked tetraspanin species are homodimers, but also higher order complexes and low amounts of heterodimeric tetraspanins (CD81/CD9, CD9/CD151, CD81/CD151) were identified [27]. It has been suggested that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks in the assembly of higher organized tetraspanin protein complexes. Interestingly, the exosomes originating from cell lines overexpressing CD9 are believed to be enriched in more stable TEMs [10]. Overall, although most tetraspanins can interact with most other tetraspanins, and similarly engage with several other proteins, the nature of these interactions has been until recently classified only according to their stability in the presence of detergents of different stringency, which does not necessarily reflect their significance in the cellular milieu [34]. A thorough characterization of strength and abundance of the interactions between the members participating in a tetraspanin web in a particular cell and physiological situation is therefore needed and will support the understanding of its mediated biological effects. Similarly, most data on tetraspanin functionality come from studies on their localization on cell membranes, while functional data in vesicles are still scarce. Therefore, we here summarize the known cellular functions, while speculating how this might translate to EVs.

An important step towards the understanding of the specificity of the tetraspanin interactions in TEM has been achieved by delineation of the involved tetraspanin regions by dissecting the model tetraspanin into domains, differently amenable for modification. Early experiments that addressed the relatively unstructured and at the same time antigen-binding competent regions that appeared attractive for mutagenesis resulted in a protein that showed aberrant clustering involving both homo- and heterodimerization of resulting full-length tetraspanins [35], albeit the mutagenesis method employed in this study was a complete deletion of targeted domains. The CD81 D-region was studied in more detail: the CD9 and CD151 tetraspanins were more competent of clustering with CD81 when homologously engrafted with CD81 D-region [36]. When the mutagenized CD81 EC2 molecular subunits were transplanted to other tetraspanins, the extremely flexible conformation of the solvent-exposed D-segment of CD81 EC2 was sufficient to overcome the orientational restrictions to initiate the homotypic contact for dimerization, and this finding has been corroborated with both wet-lab data and the insights from molecular dynamic simulation of the cell membrane-embedded protein [37].

3.2. Interaction of tetraspanins with integrins and matrix-degrading enzymes: role of tetraspanins in cancer and metastasis

Important association partners of tetraspanins are the integrins. The role of such complexes in invasive growth in vivo as well as the effect of integrin-mediated binding events on cell proliferation and invasion is well established. Especially, the laminin-binding integrins (α6β4, α3β1, α6β1 and α7β1) exhibit extensive interactions with tetraspanin proteins [12, 38]. The functionality of integrins may depend critically on their interaction of tetraspanins: it has early been described that the remarkably stable association of the tetraspanin CD151 and the integrin α3β1 leads to a high level of activation of cellular PI4K [39]. Further, CD151 interacts directly with the α3 subunit and links it to other tetraspanins, CD9 and CD81. Loss of CD151 abrogates the α3β1 mediated mobility on its ligands, laminin-332 and laminin-551. CD9/CD81 complex may even regulate the integrin-mediated functions independently of CD151 by forming a complex with the integrin and directing the PKCα-α3β1 association [40]. Another example of tetraspanin-integrin association reveals its proangiogenic role through VEGF induction, mediated by cooperation between TM4SF5 and integrin α5 of epithelial cells [41]. Interestingly, removal of CD151 palmitoylation sites did not disrupt the CD151–α6β4 complex in epithelial cells but strongly influenced α6β4-integrin–dependent cell morphology [42]. The rat tetraspanin D6.1A (human homolog is CO-029) was able to induce systemic angiogenesis by initiation of an angiogenic loop that reached organs distant from the tumor, probably due to the abundance of D6.1A in tumor-derived exosomes. This is in line with reports claiming that EVs prepare niches for metastatic tumor cells at tissues distant from the primary tumor [43]. This tetraspanin associates with integrins α3β1, α6β1 and α6β4, as well as with tetraspanins CD9 and CD81, and is similarly to CD151 linked to tumor-promoting activities [44].

Active complexes of tetraspanins and integrins influence biological processes other than cellular signaling by interacting with cellular metalloproteinases, important players in the remodeling of extracellular matrix. A study of MDA-MB-231 cells, a breast cancer cell line, has indicated that the α3β1-tetraspanin protein complex may be linked to an invasive phenotype of tumor cells via modulation of various signaling pathways, including activation of membrane metalloproteinase-2 (MMP-2), an enzyme associated with invasive migration of the cells, and affecting phosphatidylinositol-3-kinase (PI3K) signaling pathways, which control actin cytoskeleton dynamics [45]. By the incorporation of the members of a disintegrin and metalloproteinase (ADAM) family members the tetraspanins are able to influence the cellular ectodomain cleavage and release activity of these enzymes [46]. The tetraspanins of the TspanC8 group (tetraspanins with 8 cysteines) have a significant impact on the cellular exit and catalytic activity of ADAM10 [47], in particular the activity of Tspan15/ADAM10 promoted N-cadherin cleavage [48, 49, 50]. Different TspanC8/ADAM10 complexes seem to have different substrate specificities [51]. The silencing of CD9 enhanced shedding of ADAM17-substrates TNF-α and ICAM-1 [52].

Important discovery of the possible consequences of fine differences in composition of TEMs has been delivered by the study of exosomes enriched in Tspan8-α4 complex that were preferentially taken up by the endothelial and pancreatic cells [53]. The fact that such modifications can allow selective targeting in vitro and in vivo holds promise to achieve improved exosomal delivery by engineering of their membrane components.

3.3. The role of tetraspanins in immune complexes

In antigen-presenting cells (APCs), tetraspanins integrate into TEMs protein-recognition receptors binding to conserved repeated motifs of microbes, such as Toll-like receptors, and MHCII molecules into tetraspanin web platforms, as well as Fcγ receptor I in phagocytic cells, Fcγ receptor IIb and III upon the activation of macrophages and Fcε receptor I in monocytes and skin-derived dendritic cells [54].

The particular role of CD81 protein in the formation of specialized microdomains in the plasma membrane of the cells of the immune system was discovered by elucidating its function of recruiting various adhesion molecules, receptors and signaling proteins to the central zone of the immune synapse in T-lymphocytes and APCs [55]. Therefore, it has early been proposed for CD81 to play a key role during antigenic presentation, since it colocalizes with the T-cell receptor/CD3 [56], and CD81 indeed turned out to be a regulator of CD3 clustering and sustained CD3 signaling [57].

Further, the T-cell side of the immune synapse is densely populated by tetraspanins CD9 and CD151. The abolishment of their expression reduces markers of activation of T-lymphocytes conjugated to the APCs, such as IL-2 secretion and expression of CD69 [58].

Another role in the immune response of tetraspanin CD81 is amplifying and sustaining B-cell receptor signaling from lipid rafts by ligation to the co-receptor CD19/CD21 complex. The signaling through a variety of cell surface protein complexes implies a role of lipid rafts, again highlighting the ability of tetraspanin to facilitate raft association [9].

3.4. Tetraspanins in pathogen infection

Several studies have been oriented towards the research of tetraspanins as ligand molecules for pathogen entry. CD81 has been identified as a ligand for hepatitis C virus (HCV) recognition and viral entry [59]. The ligand for viral glycoprotein E2 is the D-domain of the LEL, a dynamic region positioned within the triple-bundle helix, whose conformation in solution differs substantially from the one suggested by crystal structure. Challenging for structure-based design, this region nevertheless presents an attractive target for design of therapeutically relevant ligands with methods such as NMR [60]. Apart of the EC2 domain, other regions of CD81 have proven important for virus infection. Experimental evidence here was based on the exchange of the structural domains of the molecule with the ones of tetraspanins of different degrees of homology, and it was found that closely related substitutions were more efficient at functional complementation of CD81. Viral entry has been shown to correlate with surface expression of the chimeric protein and to depend on the presence of the cholesterol-coordinating glutamate residue [61]. EWI-2wint, a cleavage form of EWI-2, a member of the immunoglobulin superfamily, has an inhibitory effect on HCV infection by obstructing the interaction between CD81 and HCV E2 [62]. The related factor EWI-F inhibits Plasmodium infection, whereas its silencing increases infection efficiency [63].

Tetraspanin microdomains have been described to regulate HIV-1 entry, assembly and transfer between the cells [64, 65]. CD81 influences importantly the early stages of virus replication by controlling the stability of HIV-1 restriction factor and consequently the activity of viral reverse transcriptase [66], while CD63 facilitates endocytosis of the HIV receptor CXCR4 [67] as well as supports the replication steps in macrophages [68, 69]. Also Coronaviruses and low-pathogenicity Influenza A viruses utilize TEM domains as entry portals to co-engage with cellular receptors and proteases, which enable viral proteolytic priming [70]. As shown in an in vivo model with CD151 null-mice, this tetraspanin is a critical novel host factor of nuclear export signaling of Influenza A virus, used complementary to the viral nuclear export proteins [71].

Tspan9 modulates the early endosome compartment to make it more permissive for membrane fusion of early-penetrating viruses, and its depletion strongly inhibits infection by alphaviruses that fuse in early endosomes but does not alter the delivery of virus to early endosomes or change their pH or protease activity [72]. It is unclear, what function then EV-based tetraspanins might have in the context of viral infection, and it might be speculated that cells use EVs to titrate away virus into membrane structures that are unable to provide replication and protein synthesis machineries for the virus. This is supported by the findings that EVs might have anti-influenza infection activity in vitro [73].


4. Extracellular vesicles as mediators of cell-cell interaction

4.1. Biological basis for therapeutic applications: EVs as mediators of intercellular interactions

EVs are secreted by most cell types and are taken up by recipient cells, where their cargo consisting of a cocktail of proteins, mRNAs and non-coding RNAs alters the behavior of the recipient cells in a way that might be even considered similar to hormones or cytokines [74], e.g. in the context of skin or bone cell paracrine signaling [75, 76, 77]. siRNAs (small interfering RNAs) and miRNA-based inhibitors have been recognized as potent novel drug candidates for many years. As EVs can be loaded with different drugs in vitro, they qualify as an attractive drug delivery system. The specificity of the recipient cell targeting in vivo is understood in a limited way only, although there is evidence of accumulation of specific EVs [43]. For example, EVs from human mesenchymal stem cells accumulated in the liver, spleen and sites of acute kidney injury [78]. Such tropism for a specific cell type, a requirement for targeted drug delivery, appears to be determined by surface proteins of the source cells. The composition of EV membrane reflecting the one of their source cell makes these particles non-immunogenic, and their small size allows them to pass the immune surveillance of the host organism [79, 80]. The reported engagement of exosomes in physiological processes in normal and diseased central nervous system makes them attractive vehicles for delivering neurotherapeutic agents across the blood-brain barrier [81, 82, 83]. Nevertheless, their delivery in humans seems so far limited to liver and kidney as they are reported not to reach therapeutic amounts in brain, heart and other tissues due to lack of specific targeting and thus low enrichment of the intended therapeutic ingredient in the target tissue. Modifications of the EV surface membrane to achieve enhanced targeting of a specific cell type are hence a common strategy embodied in several different engineering approaches.

4.2. Mechanisms of EV entry into the target cell

When an EV attaches to the target cell surface, it can in some cases activate the cognate receptors without internalization or transfer of the content to the recipient cell via its fusion with the target cell membrane or via endocytosis [84]. Endocytosis is an active process that requires cytoskeletal remodeling dependent on actin dynamics and includes clathrin-dependent endocytosis, phagocytosis and macropinocytosis. The clathrin-dependent endocytosis has been established as cellular entry for EVs based on the experiments with specific inhibitors of this pathway. Additionally, endocytic uptake of EVs can involve lipid rafts, sometimes dependent on caveolin proteins. The size of EVs may be a limiting factor for cellular entry via endocytosis [85]. The EV uptake by phagocytosis was monitored by their high level of accumulation in phagocytic cells and localization into the phagolysosome [86], as well as the identification of the crucial role of the phosphatidylserine binding T-cell immunoglobulin and mucin domain containing (TIM4) receptor for the uptake of exosomes into macrophages [87, 88]. The contribution of macropinocytosis pathway was revealed with studies where exosomal uptake was decreased by the inhibition of cytoskeletal rearrangements that normally lead to membrane ruffles [89], as well as with its promotion caused by the activation of the agonistically acting epidermal growth factor [90].


5. Molecular engineering to facilitate EV labeling and delivery to target cells

5.1. Modifications of EV membrane with non-covalent and chemical modifications

The role of EVs as encapsulated intercellular messengers makes them attractive for development into nanoscale therapeutic agents [91], and therefore, the need of augmenting the interaction with the recipient cell has been widely recognized. The higher rigidity of the membrane of EVs in comparison with their source cells does not appear to obstruct the efficient application of common hydrophobic insertion strategies to EVs. The introduction of small lipophilic ligands, such as membrane dyes, works effectively for their labeling and aids in monitoring in in vitro and in vivo experiments. Furthermore, hydrophobic loading is used for encapsulation of certain drugs and leads to their increased stability and therapeutic effect [92, 93].

Due to the negative charge on the recipient cells, binding and uptake of EVs were enhanced by increasing the charge interactions with an artificially introduced positive surface potential, as exemplified in their derivatization with cationic lipids [94]. A downside of this method can be that the extremely charged cationic reagents can cause cytotoxicity and the cellular uptake of the modified particles can proceed differently from the usual pathways, which results in an unpredictable cellular fate of the particle and its cargo and possibly its undesired degradation. The innate slightly negative electrostatic potential exhibited by unmodified EVs should contribute to the longer half-life of such particles in vivo as inferred from liposomal studies [95]. Further, the extremes of unbalanced positive charge may negatively impact the storage stability and the application of such reagents in high concentrations.

As an alternative to other methods, labeling of EV surfaces with fluorescent probes has been achieved using click chemistry without an apparent effect on the size and function of the particles [96]. Nevertheless, there is an immense complexity behind the engineering and downstream methods developed for other biologicals conjugated with small molecules, especially when intended for therapeutic purposes [97, 98].

The idea of decoration of EV membranes with functional ligands further led to the consideration of receptor binding strategies. Such EV functionalization can readily be achieved by modification of the source cell. A resulting opportunity harvesting the specificity of this approach could also be used as a strategy to eliminate vesicles implicated in pathological processes, such as cancer metastasis, or to neutralize the undesired activity of therapeutically applied vesicles. Transferrin-conjugated superparamagnetic nanoparticles, reactive with surface-expressed transferrin receptor of exosomes, enabled their isolation from blood and endowed the vesicles with superior targeting properties [99]. A robust labeling approach of microvesicles was the expression of biotin acceptor peptide-transmembrane domain (BAP-TM) receptor on the source cells in combination with biotinylation in vivo [100].

Another example of employing an EV-localizing protein for efficient presentation on the EV surface involved a peptide or a protein fused with the C1C2 domain of lactadherin, which binds to EV outer membrane due to its affinity to phosphatidylserine, strongly enriched in exosome membranes. This method was used to generate antibodies against tumor biomarkers [101] and to increase the host immune response to tumor-associated antigens [102, 103].

Strong binding of lactadherin to the exosome membrane was also the basis of an efficient labeling protocol utilizing overexpression of a fusion protein composed of Gaussia luciferase and a truncated lactadherin in source melanoma cells. After harvest by ultracentrifugation, the labeled exosomes were successfully used for in vivo biodistribution studies [104].

5.2. Engineering of tetraspanin proteins for detection and monitoring of EVs

Regarding the tetraspanins not only as very abundant, but as prominent structural and stability elements of the exosomal membrane, it is sensible to engineer genetic fusions of these proteins enriched in EVs to ensure a high density of the expressed product and optimize the derivatization for the different purposes of engineering: cognate ligand binding, labeling for visualization or isolation, modifying cargo uptake and transfer and stability (Figure 3). Most practically, transgene expression in the parent cell would be induced to deliver EVs enriched in the modified protein. The success of such set-up depends on the understanding of both molecular biology and protein architecture of the targeted species.

Figure 3.

Main engineering goals utilizing the modification of tetraspanins.

In one of the pioneering studies, certain sites on the tetraspanin CD63 have been chosen to allow the integration of fluorescent fusion proteins on the extra- and intravesicular side of the exosomal membrane [105]. CD63-GFP fusions have proven valuable reporters in the elucidation of the role of immune synapse in secretion of exosomes from T-cells to APCs and their fusion with recipient cells [106], in determining differential uptake properties of different immune cell subsets for EVs originating from a cancer cell line [107] and in an in vivo study imaging the fate of EVs produced by breast cancer cell line in nude mice [108].

5.3. Engineering of EV surface proteins for enhanced cell-type specific targeting: introduction of target specificity, stability and improved cargo delivery

In the pioneering example of derivatization of a protein enriched in exosomal membrane to achieve specific targeting [109], the rabies virus glycoprotein (RVG)-peptide fused with lysosome-associated membrane glycoprotein 2b (Lamp-2b) was used to target exosomes to the central nervous system in an in vivo mouse model. Immature dendritic cells-derived exosomes, enriched in an N-terminal fusion of an αv integrin-specific internalizing RGD-sequence containing peptide with this scaffold, could internalize efficiently into target-positive breast cancer cells [110]. The display of such constructs could be efficiently enhanced by introducing a protective glycosylation motive, which improved the surface expression of exosome-bound N-terminally fused peptides by preventing their acid-mediated proteolysis during endosomal passage and indeed led to a more efficient specific cellular uptake of exosomes engineered in this way [111]. Specific targeting of IL3-receptor overexpressing chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells has been shown for exosomes armed with a fusion protein of IL3 and Lamp2b and loaded with Imatinib, a tyrosine kinase inhibitor, and led to a the reduction of tumor size in vivo [112]. Exosomes engineered in this way achieved a higher abundance at the tumor site and were hence able to inhibit xenograft growth more efficiently than the active ingredient alone or than loaded control exosomes.

Glycosylphosphatidylinositol (GPI)-mediated anchoring of the specific targeting unit was demonstrated to be a stable alternative to Lamp2b fusions. EVs expressing GPI-anchored nanobodies, specific to epidermal growth factor receptor (EGFR), displayed enhanced binding to the EGFR-overexpressing cancer cells. This, however, did not lead to an increased uptake, and it was suggested that in this particular biological system not only the affinity but also the density of the targeting ligand must be high enough to induce receptor clustering and subsequent internalization [113]. Exosomes derived from a HEK cell line transfected with a construct of platelet-derived growth factor (PDGF)-anchoring sequence and an EGFR-binding peptide were on the other hand efficient in targeting EGFR-expressing tumors in vivo and reducing their size with delivery of microRNA Let7 [114]. Enhanced uptake has been achieved for EVs enriched in a fusion protein of tetraspanin CD63 and stearylated octaarginine, a representative cell-penetrating peptide, by their ability to induce active macropinocytosis [115].

The concept of enhancing the stability of the targeted exosomal surface protein intended for fusion with a targeting agent to allow a higher degree of versatility for their modification for an improved target recognition has raised interest in their engineering at the protein level. A significant increase in thermal stability has been achieved by introduction of additional disulfide bonds in the EC2 of tetraspanin CD81, with the best variant exhibiting a positive shift in the melting temperature for 45°C comparing with the wild-type protein [116]. When engrafted with a human transferrin-receptor specific peptide, the stabilized scaffold exhibited significantly better biophysical properties than the analogously engrafted wild-type protein. In the same study, a mutant has been discovered that exhibits reversible unfolding behavior up to a temperature of 110°C in contrast to the wild-type CD81 EC2, which presents another option for extensive engineering required for directed evolution of tetraspanin proteins towards novel antigen binding.

Not only can the overexpression of tetraspanins in source cell lines increase the production and stability of exosomes, but also the exosomes engineered to contain adeno-associated viruses (AAVs), designed for improved delivery of genetic material to target cell [100], were superior in their yield and functionality when CD9 was overexpressed in AAV-producer cells [10]. CD9 overexpression has also increased the speed and transduction efficiency of lentiviral gene delivery into numerous cell lines, confirming the important role of this tetraspanin in gene transfer [117].

The current scope of EV engineering reaches beyond receptor targeting systems and aspires towards modifications with complex modules, assigning them simultaneously with multiple novel functionalities, such as specific recognition as well as enzymatic activity, to enhance their potential therapeutic effect. Recently, regression of orthotopic Her2-positive tumors has been achieved by applying exosomes, able of specific targeting via their surface decoration with a fusion protein of high-affinity anti-Her2 single-chain Fv, and containing mRNA encoding a bacterial enzyme Hchr6, which in the strongly Her2-positive cells catalyzed the conversion of the prodrug CNOB to cytotoxic MCHB [118]. Tetraspanin engineering could support a sophisticated concept to aid intracellular delivery of exosomal cargo proteins, directionally incorporated during vesicle biogenesis [119]. Genes encoding two recombinant proteins, a fusion of photoreceptor cryptochrome 2 (CRY2) and the protein of interest and a fusion of tetraspanin protein CD9 and CRY-interacting basic-helix–loop–helix 1 (CIB1), were co-transfected into a single cell line. The blue light-induced binding of CRY2 and CIB1 enabled docking of CRY2-fused target proteins into nascent exosomes, and in the absence of the blue light, the cargo protein was released into the exosomal lumen. Transfer of Cre recombinase confirmed the efficiency of this system in vitro and in vivo.


6. Conclusions

The tetraspanins, well established as the biomarker proteins of extracellular vesicles, have been addressed for increasing EV stability and improving their function as delivery vehicles, both by assigning them with target recognition properties and modulating their cargo transfer. From the current point of view, the complexity of the tetraspanin-mediated interactions and signaling networks formed in a cell is yet to be discerned. Tetraspanins are known to interact naturally with a plethora of cell surface-bound ligands, which results in potent biological effects conveyed through different pathways; however, systematic evaluation of the affinity of the association with the interaction partners would assist in prediction of the consequential cellular processes as well as in determining optimal choice of the tetraspanin targeted for modification. There are recent reports describing modified tetraspanins mediating both surface protein interactions and an intracellular fusion-mediated enzymatic activity, which underline the feasibility of engineering versatile functions into tetraspanin proteins as fusion partners. The structural details on tetraspanins modified in this way, as well as the read-out revealing their actual behavior in the foreseen role and the influence of such modifications on the fate of an EV preparation, will pave the way into the design and production of EV-based reagents as therapeutics.



The financial support by the Austrian Federal Ministry for Digital and Economic Affairs and the National Foundation for Research, Technology and Development is gratefully acknowledged. S.V. was also supported by the PhD program BioToP (Biomolecular Technology of Proteins) funded by the Austrian Science Fund (FWF W1224).


Conflict of interest

The authors declare no conflict of interest.


  1. 1. Wickman G, Julian L, Olson MF. How apoptotic cells aid in the removal of their own cold dead bodies. Cell Death & Differentiation. 2012;19:735-742. DOI: 10.1038/cdd.2012.25
  2. 2. Harding C, Heuser J, Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. The Journal of Cell Biology. 1983;97(2):329-339. DOI: 10.1083/jcb.97.2.329
  3. 3. Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. The Journal of Cell Biology. 1985;101(3):942-948. DOI: 10.1083/jcb.101.3.942
  4. 4. Keerthikumar S, Chisanga D, Ariyaratne D, Al Saffar H, Anand S, Zhao K, et al. ExoCarta: A web-based compendium of exosomal cargo. Journal of Molecular Biology. 2016;428(4):688-692. DOI: 10.1016/j.jmb.2015.09.019
  5. 5. Lässer C, Jang SC, Lötvall J. Subpopulations of extracellular vesicles and their therapeutic potential. Molecular Aspects of Medicine. 2018;60:1-14. DOI: 10.1016/j.mam.2018.02.002
  6. 6. Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, Geuze HJ. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. The Journal of Biological Chemistry. 1998;273(32):20121-20127. DOI: 10.1074/jbc.273.32.20121
  7. 7. Kalra H, Drummen GPC, Mathivanan S. Focus on extracellular vesicles: Introducing the next small big thing. International Journal of Molecular Sciences. 2016;17:170. DOI: 10.3390/ijms17020170
  8. 8. Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: Molecular facilitators. The FASEB Journal. 1997;11(6):428-442. DOI: 10.1109/TNN.2011.2169808
  9. 9. Wright MD, Moseley GW, Van Spriel AB. Tetraspanin microdomains in immune cell signalling and malignant disease. Tissue Antigens. 2004;64:533-542. DOI: 10.1111/j.1399-0039.2004.00321.x
  10. 10. Schiller LT, Lemus-Diaz N, Rinaldi Ferreira R, Böker KO, Gruber J. Enhanced production of exosome-associated AAV by overexpression of the Tetraspanin CD9. Molecular Therapy–Methods & Clinical Development. 2018;9:278-287. DOI: 10.1016/j.omtm.2018.03.008
  11. 11. Yáñez-Mó M, Mittelbrunn M, Sánchez-Madrid F. Tetraspanins and intercellular interactions. Microcirculation. 2001;8(3):153-168. DOI: 10.1038/
  12. 12. Berditchevski F. Complexes of tetraspanins with integrins: More than meets the eye. Journal of Cell Science. 2001;114:4143-4151. DOI: 10.1091/MBC.12.2.351
  13. 13. Ang J, Fang BL, Ashman LK, Frauman AG. The migration and invasion of human prostate cancer cell lines involves CD151 expression. Oncology Reports. 2010;24(6):1593-1597. DOI: 10.3892/or-00001022
  14. 14. Yáñez-Mó M, Siljander PRM, Andreu Z, Zavec AB, Borràs FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of Extracellular Vesicles. 2015;4:1-60. DOI: 10.3402/jev.v4.27066
  15. 15. Detchokul S, Williams ED, Parker MW, Frauman AG. Tetraspanins as regulators of the tumour microenvironment: Implications for metastasis and therapeutic strategies. British Journal of Pharmacology. 2014;171:5462-5490. DOI: 10.1111/bph.12260
  16. 16. Min G, Wang H, Sun TT, Kong XP. Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-Å resolution. The Journal of Cell Biology. 2006;173(6):975-983. DOI: 10.1083/jcb.200602086
  17. 17. Masciopinto F, Campagnoli S, Abrignani S, Uematsu Y, Pileri P. The small extracellular loop of CD81 is necessary for optimal surface expression of the large loop, a putative HCV receptor. Virus Research. 2001;80(1-2):1-10. DOI: 10.1016/S0168-1702(01)00245-3
  18. 18. Kitadokoro K, Bordo D, Galli G, Petracca R, Falugi F, Abrignani S, et al. CD81 extracellular domain 3D structure: Insight into the tetraspanin superfamily structural motifs. The EMBO Journal. 2001;20(1-2):12-18. DOI: 10.1093/emboj/20.1.12
  19. 19. Tomlinson MG, Hanke T, Hughes DA, Barclay AN, Scholl E, Hünig T, et al. Characterization of mouse CD53: Epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection. European Journal of Immunology. 1995;25(8):2201-2205. DOI: 10.1002/eji.1830250813
  20. 20. Seigneuret M, Delaguillaumie A, Lagaudrière-Gesbert C, Conjeaud H. Structure of the tetraspanin main extracellular domain: A partially conserved fold with a structurally variable domain insertion. The Journal of Biological Chemistry. 2001;276(43):40055-40064. DOI: 10.1074/jbc.M105557200
  21. 21. Seigneuret M. Complete predicted three-dimensional structure of the facilitator transmembrane protein and hepatitis C virus receptor CD81: Conserved and variable structural domains in the tetraspanin superfamily. Biophysical Journal. 2006;90(1):212-227. DOI: 10.1529/biophysj.105.069666
  22. 22. Imai T, Yoshie O. C33 antigen and M38 antigen recognized by monoclonal antibodies inhibitory to syncytium formation by human T cell leukemia virus type 1 are both members of the transmembrane 4 superfamily and associate with each other and with CD4 or CD8 in T cells. Journal of Immunology. 1993;151(11):6470-6481
  23. 23. Bienstock RJ, Carl Barrett J. KAI1, a prostate metastasis suppressor: Prediction of solvated structure and interactions with binding partners; integrins, cadherins, and cell-surface receptor proteins. Molecular Carcinogenesis. 2001;32(3):139-153. DOI: 10.1002/mc.1073
  24. 24. Rubinstein E, Le NF, Lagaudrière-Gesbert C, Billard M, Conjeaud H, Boucheix C. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. European Journal of Immunology. 1996;26(11):2657-2665. DOI: 10.1002/eji.1830261117
  25. 25. Hemler ME. Tetraspanin functions and associated microdomains. Nature Reviews Molecular Cell Biology. 2005;6:801-811. DOI: 10.1038/nrm1736
  26. 26. Claas C, Stipp CS, Hemler ME. Evaluation of prototype Transmembrane 4 superfamily protein complexes and their relation to lipid rafts. The Journal of Biological Chemistry. 2001;276(11):7974-7984. DOI: 10.1074/jbc.M008650200
  27. 27. Kovalenko OV, Yang X, Kolesnikova TV, Hemler ME. Evidence for specific tetraspanin homodimers: Inhibition of palmitoylation makes cysteine residues available for cross-linking. The Biochemical Journal. 2004;377(Pt 2):407-417. DOI: 10.1042/BJ20031037
  28. 28. Yang X, Kovalenko OV, Tang W, Claas C, Stipp CS, Hemler ME. Palmitoylation supports assembly and function of integrin-tetraspanin complexes. The Journal of Cell Biology. 2004;167(6):1231-1240. DOI: 10.1083/jcb.200404100
  29. 29. Delandre C, Penabaz TR, Passarelli AL, Chapes SK, Clem RJ. Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface. Experimental Cell Research. 2009;315(11):1953-1963. DOI: 10.1016/j.yexcr.2009.03.013
  30. 30. Zimmerman B, Kelly B, McMillan BJ, Seegar TCM, Dror RO, Kruse AC, et al. Crystal structure of a full-length human tetraspanin reveals a cholesterol-binding pocket. Cell. 2016;167(4):1041-1051.e11. DOI: 10.1016/j.cell.2016.09.056
  31. 31. Sung CH, Davenport CM, Nathans J. Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. The Journal of Biological Chemistry. 1993;268(35):26645-26649
  32. 32. Fitter S, Sincock PM, Jolliffe CN, Ashman LK. Transmembrane 4 superfamily protein CD151 (PETA-3) associates with beta 1 and alpha IIb beta 3 integrins in haemopoietic cell lines and modulates cell-cell adhesion. The Biochemical Journal. 1999;338(Pt 1):61-70. DOI: 10.1042/0264-6021:3380061
  33. 33. Wang H-X, Kolesnikova TV, Denison C, Gygi SP, Hemler ME. The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization. Journal of Cell Science. 2011;124(16):2702-2710. DOI: 10.1242/jcs.085449
  34. 34. van Deventer SJ, Dunlock V-ME, van Spriel AB. Molecular interactions shaping the tetraspanin web. Biochemical Society Transactions. 2017;45(3):741-750. DOI: 10.1042/BST20160284
  35. 35. Homsi Y, Schloetel JG, Scheffer KD, Schmidt TH, Destainville N, Florin L, et al. The extracellular δ-domain is essential for the formation of CD81 tetraspanin webs. Biophysical Journal. 2014;107(1):100-113. DOI: 10.1016/j.bpj.2014.05.028
  36. 36. Homsi Y, Lang T. The specificity of homomeric clustering of CD81 is mediated by its δ-loop. FEBS Open Bio. 2017;7(2):274-283. DOI: 10.1002/2211-5463.12187
  37. 37. Schmidt TH, Homsi Y, Lang T. Oligomerization of the tetraspanin CD81 via the flexibility of its δ-loop. Biophysical Journal. 2016;110(11):2463-2474. DOI: 10.1016/j.bpj.2016.05.003
  38. 38. Hemler ME. Integrin associated proteins. Current Opinion in Cell Biology. 1998;10:578-585. DOI: 10.1016/S0955-0674(98)80032-X
  39. 39. Yauch RL, Berditchevski F, Harler MB, Reichner J, Hemler ME. Highly stoichiometric, stable, and specific association of integrin alpha 3beta 1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Molecular Biology of the Cell. 1998;9(10):2751-2765. DOI: 10.1091/mbc.9.10.2751
  40. 40. Gustafson-Wagner E, Stipp CS. The CD9/CD81 Tetraspanin complex and Tetraspanin CD151 regulate α3β1 integrin-dependent tumor cell behaviors by overlapping but distinct mechanisms. PLoS One. 2013;8(4):e61834. DOI: 10.1371/journal.pone.0061834
  41. 41. Choi S, Lee SA, Kwak TK, Kim HJ, Lee MJ, Ye SK, et al. Cooperation between integrin α 5 and tetraspan TM4SF5 regulates VEGF-mediated angiogenic activity. Blood. 2009;113(8):1845-1855. DOI: 10.1182/blood-2008-05-160671
  42. 42. Yang X, Claas C, Kraeft SK, Chen LB, Wang Z, Kreidberg JA, et al. Palmitoylation of tetraspanin proteins: Modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. Molecular Biology of the Cell. 2002;13(3):767-781. DOI: 10.1091/mbc.01-05-0275
  43. 43. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527(7578):329-335. DOI: 10.1038/nature15756
  44. 44. Gesierich S, Paret C, Hildebrand D, Weitz J, Zgraggen K, Schmitz-Winnenthal FH, et al. Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: Impact on cell motility. Clinical Cancer Research. 2005;11(8):2840-2852. DOI: 10.1158/1078-0432.CCR-04-1935
  45. 45. Sugiura T, Berditchevski F. Function of α3β1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2). The Journal of Cell Biology. 1999;146(6):1375-1389. DOI: 10.1083/jcb.146.6.1375
  46. 46. Yáñez-Mó M, Gutiérrez-López MD, Cabañas C. Functional interplay between tetraspanins and proteases. Cellular and Molecular Life Sciences. 2011;68:3323-3335. DOI: 10.1007/s00018-011-0746-y
  47. 47. Dornier E, Coumailleau F, Ottavi JF, Moretti J, Boucheix C, Mauduit P, et al. Tspanc8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote notch activation in flies and mammals. The Journal of Cell Biology. 2012;199(3):481-496. DOI: 10.1083/jcb.201201133
  48. 48. Prox J, Willenbrock M, Weber S, Lehmann T, Schmidt-Arras D, Schwanbeck R, et al. Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10. Cellular and Molecular Life Sciences. 2012;69(17):2919-2932. DOI: 10.1007/s00018-012-0960-2
  49. 49. Jouannet S, Saint-Pol J, Fernandez L, Nguyen V, Charrin S, Boucheix C, et al. TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, notch activation and ADAM10 membrane compartmentalization. Cellular and Molecular Life Sciences. 2016;73(9):1895-1915. DOI: 10.1007/s00018-015-2111-z
  50. 50. Noy PJ, Yang J, Reyat JS, Matthews AL, Charlton AE, Furmston J, et al. TspanC8 tetraspanins and a disintegrin and metalloprotease 10 (ADAM10) interact via their extracellular regions: Evidence for distinct binding mechanisms for different TspanC8 proteins. The Journal of Biological Chemistry. 2016;291(7):3145-3157. DOI: 10.1074/jbc.M115.703058
  51. 51. Matthews AL, Szyroka J, Collier R, Noy PJ, Tomlinson MG. Scissor sisters: Regulation of ADAM10 by the TspanC8 tetraspanins. Biochemical Society Transactions. 2017;45(3):719-730. DOI: 10.1042/BST20160290
  52. 52. Gutiérrez-López MD, Gilsanz A, Yàñez-Mó M, Ovalle S, Lafuente EM, Domínguez C, et al. The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9. Cellular and Molecular Life Sciences. 2011;68(19):3275-3292. DOI: 10.1007/s00018-011-0639-0
  53. 53. Rana S, Yue S, Stadel D, Zöller M. Toward tailored exosomes: The exosomal tetraspanin web contributes to target cell selection. The International Journal of Biochemistry & Cell Biology. 2012;44(9):1574-1584. DOI: 10.1016/j.biocel.2012.06.018
  54. 54. Saiz ML, Rocha-Perugini V, Sánchez-Madrid F. Tetraspanins as organizers of antigen-presenting cell function. Frontiers in Immunology. 2018;9:1074. DOI: 10.3389/fimmu.2018.01074
  55. 55. Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nature Reviews Immunology. 2005;5:136-148. DOI: 10.1038/nri1548
  56. 56. Mittelbrunn M, Yanez-Mo M, Sancho D, Ursa A, Sanchez-Madrid F. Cutting edge: Dynamic redistribution of Tetraspanin CD81 at the central zone of the immune synapse in both T lymphocytes and APC. Journal of Immunology. 2002;169(12):6691-6695. DOI: 10.4049/jimmunol.169.12.6691
  57. 57. Rocha-Perugini V, Zamai M, Gonzalez-Granado JM, Barreiro O, Tejera E, Yanez-Mo M, et al. CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses. Molecular and Cellular Biology. 2013;33(18):3644-3658. DOI: 10.1128/MCB.00302-13
  58. 58. Rocha-Perugini V, González-Granado JM, Tejera E, López-Martín S, Yañez-Mó M, Sánchez-Madrid F. Tetraspanins CD9 and CD151 at the immune synapse support T-cell integrin signaling. European Journal of Immunology. 2014;44(7):1967-1975. DOI: 10.1002/eji.201344235
  59. 59. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, et al. Binding of hepatitis C virus to CD81. Science. 1998;282(5390):938-941. DOI: 10.1126/science.282.5390.938
  60. 60. Rajesh S, Sridhar P, Tews BA, Feneant L, Cocquerel L, Ward DG, et al. Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81. Journal of Virology. 2012;86(18):9606-9616. DOI: 10.1128/JVI.00559-12
  61. 61. Banse P, Moeller R, Bruening J, Lasswitz L, Kahl S, Khan AG, et al. CD81 receptor regions outside the large extracellular loop determine hepatitis C virus entry into hepatoma cells. Viruses. 2018;10(4):207. DOI: 10.3390/v10040207
  62. 62. Rocha-Perugini V, Montpellier C, Delgrange D, Wychowski C, Helle F, Pillez A, et al. The CD81 partner EWI-2wint inhibits hepatitis C virus entry. PLoS One. 2008;3(4):e1866. DOI: 10.1371/journal.pone.0001866
  63. 63. Charrin S, Yalaoui S, Bartosch B, Cocquerel L, Franetich JF, Boucheix C, et al. The Ig domain protein CD9P-1 down-regulates CD81 ability to support plasmodium yoelii infection. The Journal of Biological Chemistry. 2009;284(46):31572-31578. DOI: 10.1074/jbc.M109.057927
  64. 64. Florin L, Lang T. Tetraspanin assemblies in virus infection. Frontiers in Immunology. 2018;9:1140. DOI: 10.3389/fimmu.2018.01140
  65. 65. Sims B, Farrow AL, Williams SD, Bansal A, Krendelchtchikov A, Matthews QL. Tetraspanin blockage reduces exosome-mediated HIV-1 entry. Archives of Virology. 2018;163(6):1683-1689. DOI: 10.1007/s00705-018-3737-6
  66. 66. Rocha-Perugini V, Suárez H, Álvarez S, López-Martín S, Lenzi GM, Vences-Catalán F, et al. CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels. Nature Microbiology. 2017;2(11):1513-1522. DOI: 10.1038/s41564-017-0019-0
  67. 67. Yoshida T, Kawano Y, Sato K, Ando Y, Aoki J, Miura Y, et al. A CD63 mutant inhibits T-cell tropic human immunodeficiency virus type 1 entry by disrupting CXCR4 trafficking to the plasma membrane. Traffic. 2008;9(4):540-558. DOI: 10.1111/j.1600-0854.2007.00700.x
  68. 68. Chen H, Dziuba N, Friedrich B, von Lindern J, Murray JL, Rojo DR, et al. A critical role for CD63 in HIV replication and infection of macrophages and cell lines. Virology. 2008;379(2):191-196. DOI: 10.1016/j.virol.2008.06.029
  69. 69. Li G, Endsley MA, Somasunderam A, Gbota SL, Mbaka MI, Murray JL, et al. The dual role of tetraspanin CD63 in HIV-1 replication. Virology Journal. 2014;11(1):23. DOI: 10.1186/1743-422X-11-23
  70. 70. Earnest JT, Hantak MP, Park J-E, Gallagher T. Coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains. Journal of Virology. 2015;89(11):6093-6104. DOI: 10.1128/JVI.00543-15
  71. 71. Qiao Y, Yan Y, Sen TK, Tan SSL, Seet JE, Arumugam TV, et al. CD151, a novel host factor of nuclear export signaling in influenza virus infection. The Journal of Allergy and Clinical Immunology. 2018;141(5):1799-1817. DOI: 10.1016/j.jaci.2017.11.032
  72. 72. Stiles KM, Kielian M. Role of TSPAN9 in alphavirus entry and early endosomes. Journal of Virology. 2016;90(9):4289-4297. DOI: 10.1128/JVI.00018-16
  73. 73. Jantaratrirat S, Boonarkart C, Ruangrung K, Suptawiwat O, Auewarakul P. Microparticle release from cell lines and its anti-influenza activity. Viral Immunology. 2018;31(6):447-456. DOI: 10.1089/vim.2017.0201
  74. 74. Bayraktar R, Van Roosbroeck K, Calin GA. Cell-to-cell communication: microRNAs as hormones. Molecular Oncology. 2017;11:1673-1686. DOI: 10.1002/1878-0261.12144
  75. 75. Weilner S, Schraml E, Wieser M, Messner P, Schneider K, Wassermann K, et al. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells. Aging Cell. 2016;15(4):744-754. DOI: 10.1111/acel.12484
  76. 76. Weilner S, Keider V, Winter M, Harreither E, Salzer B, Weiss F, et al. Vesicular Galectin-3 levels decrease with donor age and contribute to the reduced osteo-inductive potential of human plasma derived extracellular vesicles. Aging (Albany NY). 2016;8(1):16-33. DOI: 10.18632/aging.100917
  77. 77. Terlecki-Zaniewicz L, Lämmermann I, Latreille J, Bobbili MR, Pils V, Schosserer M, et al. Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype. Aging (Albany NY). 2018;10(5):1103-1132. DOI: 10.18632/aging.101452
  78. 78. Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, et al. Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. Journal of the American Society of Nephrology. 2009;20(5):1053-1067. DOI: 10.1681/ASN.2008070798
  79. 79. György B, Hung ME, Breakefield XO, Leonard JN. Therapeutic applications of extracellular vesicles: Clinical promise and open questions. Annual Review of Pharmacology and Toxicology. 2015;55(1):439-464. DOI: 10.1146/annurev-pharmtox-010814-124630
  80. 80. Zhu X, Badawi M, Pomeroy S, Sutaria DS, Xie Z, Baek A, et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. Journal of Extracellular Vesicles. 2017;6(1):1324730. DOI: 10.1080/20013078.2017.1324730
  81. 81. Wood MJA, O’Loughlin AJ, Lakhal S. Exosomes and the blood-brain barrier: Implications for neurological diseases. Therapeutic Delivery. 2011;2:1095-1099. DOI: 10.1002/star.19940460805
  82. 82. Robbins PF, Kassim SH, Tran TLN, Crystal JS, Morgan RA, Feldman SA, et al. A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: Long-term follow-up and correlates with response. Clinical Cancer Research. 2015;21(5):1019-1027. DOI: 10.1158/1078-0432.CCR-14-2708
  83. 83. EL Andaloussi S, Lakhal S, Mäger I, Wood MJA. Exosomes for targeted siRNA delivery across biological barriers. Advanced Drug Delivery Reviews. 2013;65:391-397. DOI: 10.1016/j.addr.2012.08.008
  84. 84. Mulcahy LA, Pink RC, Carter DRF. Routes and mechanisms of extracellular vesicle uptake. Journal of Extracellular Vesicles. 2014;3:24641. DOI: 10.3402/jev.v3.24641
  85. 85. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature. 2003;422:37-44. DOI: 10.1038/nature01451
  86. 86. Feng D, Zhao WL, Ye YY, Bai XC, Liu RQ, Chang LF, et al. Cellular internalization of exosomes occurs through phagocytosis. Traffic. 2010;11(5):675-687. DOI: 10.1111/j.1600-0854.2010.01041.x
  87. 87. Zakharova L, Svetlova M, Fomina AF. T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor. Journal of Cellular Physiology. 2007;212(1):174-181. DOI: 10.1002/jcp.21013
  88. 88. Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature. 2007;450(7168):435-439. DOI: 10.1038/nature06307
  89. 89. Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G, Dibaj P, Bakhti M, et al. Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. Journal of Cell Science. 2011;124(3):447-458. DOI: 10.1242/jcs.074088
  90. 90. Nakase I, Kobayashi NB, Takatani-Nakase T, Yoshida T. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes. Scientific Reports. 2015;5:10300. DOI: 10.1038/srep10300
  91. 91. Armstrong JPK, Stevens MM. Strategic design of extracellular vesicle drug delivery systems. Advanced Drug Delivery Reviews. 2018;130:12-16. DOI: 10.1016/j.addr.2018.06.017
  92. 92. Sun D, Zhuang X, Xiang X, Liu Y, Zhang S, Liu C, et al. A novel nanoparticle drug delivery system: The anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Molecular Therapy. 2010;18(9):1606-1614. DOI: 10.1038/mt.2010.105
  93. 93. Zhuang X, Xiang X, Grizzle W, Sun D, Zhang S, Axtell RC, et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Molecular Therapy. 2011;19(10):1769-1779. DOI: 10.1038/mt.2011.164
  94. 94. Nakase I, Futaki S. Combined treatment with a pH-sensitive fusogenic peptide and cationic lipids achieves enhanced cytosolic delivery of exosomes. Scientific Reports. 2015;5:10112. DOI: 10.1038/srep10112
  95. 95. Zhao W, Zhuang S, Qi XR. Comparative study of the in vitro and in vivo characteristics of cationic and neutral liposomes. International Journal of Nanomedicine. 2011;6:3087-3098. DOI: 10.2147/IJN.S25399
  96. 96. Smyth T, Petrova K, Payton NM, Persaud I, Redzic JS, Graner MW, et al. Surface functionalization of exosomes using click chemistry. Bioconjugate Chemistry. 2014;25(10):1777-1784. DOI: 10.1021/bc500291r
  97. 97. Drake PM, Rabuka D. Recent developments in ADC technology: Preclinical studies signal future clinical trends. BioDrugs. 2017;31:521-531. DOI: 10.1007/s40259-017-0254-1
  98. 98. Vezina HE, Cotreau M, Han TH, Gupta M. Antibody–drug conjugates as Cancer therapeutics: Past, present, and future. Journal of Clinical Pharmacology. 2017;57:S11-S25. DOI: 10.1002/jcph.981
  99. 99. Qi H, Liu C, Long L, Ren Y, Zhang S, Chang X, et al. Blood exosomes endowed with magnetic and targeting properties for Cancer therapy. ACS Nano. 2016;10(3):3323-3333. DOI: 10.1021/acsnano.5b06939
  100. 100. Maguire CA, Balaj L, Sivaraman S, Crommentuijn MHW, Ericsson M, Mincheva-Nilsson L, et al. Microvesicle-associated AAV vector as a novel gene delivery system. Molecular Therapy. 2012;20(5):960-971. DOI: 10.1038/mt.2011.303
  101. 101. Delcayre A, Estelles A, Sperinde J, Roulon T, Paz P, Aguilar B, et al. Exosome display technology: Applications to the development of new diagnostics and therapeutics. Blood Cells, Molecules, and Diseases. 2005;35(2):158-168. DOI: 10.1016/j.bcmd.2005.07.003
  102. 102. Zeelenberg IS, Ostrowski M, Krumeich S, Bobrie A, Jancic C, Boissonnas A, et al. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Research. 2008;68(4):1228-1235. DOI: 10.1158/0008-5472.CAN-07-3163
  103. 103. Zeelenberg IS, van Maren WWC, Boissonnas A, Van Hout-Kuijer MA, Den Brok MHMGM, Wagenaars JAL, et al. Antigen localization controls T cell-mediated tumor immunity. Journal of Immunology. 2011;187(3):1281-1288. DOI: 10.4049/jimmunol.1003905
  104. 104. Takahashi Y, Nishikawa M, Takakura Y. In vivo tracking of extracellular vesicles in mice using fusion protein comprising Lactadherin and Gaussia luciferase. Methods in Molecular Biology. 2017;1660:245-254. DOI: 10.1007/978-1-4939-7253-1_20
  105. 105. Stickney Z, Losacco J, McDevitt S, Zhang Z, Lu B. Development of exosome surface display technology in living human cells. Biochemical and Biophysical Research Communications. 2016;472(1):53-59. DOI: 10.1016/j.bbrc.2016.02.058
  106. 106. Mittelbrunn M, Gutiérrez-Vázquez C, Villarroya-Beltri C, González S, Sánchez-Cabo F, González MÁ, et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nature Communications. 2011;2(1):282. DOI: 10.1038/ncomms1285
  107. 107. Sadovska L, Zandberga E, Sagini K, Jekabsons K, Riekstiņa U, Kalniņa Z, et al. A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication. Biochemical and Biophysical Research Communications. 2017;495(2):1930-1935. DOI: 10.1016/j.bbrc.2017.12.072
  108. 108. Suetsugu A, Honma K, Saji S, Moriwaki H, Ochiya T, Hoffman RM. Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models. Advanced Drug Delivery Reviews. 2013;65:383-390. DOI: 10.1016/j.addr.2012.08.007
  109. 109. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJA. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology. 2011;29(4):341-345. DOI: 10.1038/nbt.1807
  110. 110. Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials. 2014;35(7):2383-2390. DOI: 10.1016/j.biomaterials.2013.11.083
  111. 111. Hung ME, Leonard JN. Stabilization of exosome-targeting peptides via engineered glycosylation. The Journal of Biological Chemistry. 2015;290(13):8166-8172. DOI: 10.1074/jbc.M114.621383
  112. 112. Bellavia D, Raimondo S, Calabrese G, Forte S, Cristaldi M, Patinella A, et al. Interleukin 3- receptor targeted exosomes inhibit in vitro and in vivo chronic myelogenous leukemia cell growth. Theranostics. 2017;7(5):1333-1345. DOI: 10.7150/thno.17092
  113. 113. Kooijmans SAA, Aleza CG, Roffler SR, van Solinge WW, Vader P, Schiffelers RM. Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting. Journal of Extracellular Vesicles. 2016;5(1):31053. DOI: 10.3402/jev.v5.31053
  114. 114. Ohno SI, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, et al. Systemically injected exosomes targeted to EGFR deliver antitumor microrna to breast cancer cells. Molecular Therapy. 2013;21(1):185-191. DOI: 10.1038/mt.2012.180
  115. 115. Nakase I, Noguchi K, Aoki A, Takatani-Nakase T, Fujii I, Futaki S. Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery. Scientific Reports. 2017;7(1):1991. DOI: 10.1038/s41598-017-02014-6
  116. 116. Vogt S, Stadlmayr G, Stadlbauer K, Sádio F, Andorfer P, Grillari J, et al. Stabilization of the CD81 large extracellular loop with De novo disulfide bonds improves its amenability for peptide grafting. Pharmaceutics. 2018;10(3):138. DOI: 10.3390/pharmaceutics10030138
  117. 117. Böker KO, Lemus-Diaz N, Rinaldi Ferreira R, Schiller L, Schneider S, Gruber J. The impact of the CD9 Tetraspanin on Lentivirus infectivity and exosome secretion. Molecular Therapy. 2018;26(2):634-647. DOI: 10.1016/j.ymthe.2017.11.008
  118. 118. Wang J-H, Forterre AV, Zhao J, Frimannsson DO, Delcayre A, Antes TJ, et al. Anti-HER2 scFv-directed extracellular vesicle-mediated mRNA-based gene delivery inhibits growth of HER2-positive human breast tumor xenografts by prodrug activation. Molecular Cancer Therapeutics. 2018;17(5):1133-1142. DOI: 10.1158/1535-7163.MCT-17-0827
  119. 119. Yim N, Ryu SW, Choi K, Lee KR, Lee S, Choi H, et al. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module. Nature Communications. 2016;7:12277. DOI: 10.1038/ncomms12277

Written By

Stefan Vogt, Gerhard Stadlmayr, Johannes Grillari, Florian Rüker and Gordana Wozniak-Knopp

Submitted: September 5th, 2018 Reviewed: December 13th, 2018 Published: January 28th, 2019