Potential of Mesenchymal Stromal Cell-Derived Extracellular Vesicles as Natural Nanocarriers: Concise Review

Intercellular communication, through direct and indirect cell contact, is mandatory in multicellular organisms. These last years, the microenvironment, and in particular, transfer by extracellular vesicles (EVs), has emerged as a new communication mechanism. Different biological fluids and cell types are common sources of EVs. EVs play different roles, acting as signalosomes, biomarkers, and therapeutic agents. As therapeutic agents, MSC-derived EVs display numerous advantages: they are biocompatible, non-immunogenic, and stable in circulation, and they are able to cross biological barriers. Furthermore, EVs have a great potential for drug delivery. Different EV isolation protocols and loading methods have been tested and compared. Published and ongoing clinical trials, and numerous preclinical studies indicate that EVs are safe and well tolerated. Moreover, the latest studies suggest their applications as nanocarriers. The current review will describe the potential for MSC-derived EVs as drug delivery systems (DDS) in disease treatment, and their advantages. Thereafter, we will outline the different EV isolation methods and loading techniques, and analyze relevant preclinical studies. Finally, we will describe ongoing and published clinical studies. These elements will outline the benefits of MSC-derived EV DDS over several aspects.


Introduction
Intercellular communication is essential in multicellular organisms. It occurs through direct and indirect cell contact. In these last years, the microenvironment, and in particular, the transfer of extracellular vesicles (EVs), was highlighted as a new communication mechanism. Different biological fluids and cell types are common sources of EVs [1][2][3]. MSC-derived EVs display several advantages: biocompatibility, immunological inertness, and the ability to cross biological barriers [4]. In recent years, different roles have been suggested for EVs as signalosomes, biomarkers, and therapeutic agents [5,6]. EVs have been used in several conditions or diseases: cancers (brain, breast, lung, colorectal, and liver cancers and lymphoma), cardiovascular diseases (infarction and stroke), neurological diseases (Parkinson's and Alzheimer's), inflammatory diseases (arthritis and allergic cutaneous contact dermatitis), infectious diseases (HIV-1 and tuberculosis), obesity, diabetes, and others (kidney disease, liver disease, muscular disease, cutaneous wounds, and immunomodulation) [7]. Furthermore, EVs have emerged as a novel drug delivery  favorable miRNAs responsible for promoting angiogenesis, fibrosis, and cell proliferation; (2) the potential for "off the shelf" availability and for repetitive transplantation; (3) cell-free biological products that may be utilized as drug carrier systems in the pharmaceutical industry; and (4) reduced immunogenicity, which allows for allogeneic transplantation. In recent years, different roles have been attributed to EVs, such as signalosomes, biomarkers, and therapeutic agents [5,6]. Furthermore, EVs have emerged as a novel drug delivery vehicle [5,6].

Mesenchymal Stromal Cell-Derived Extracellular Vesicles (MSC-Derived EVs) for Drug Delivery Systems (DDS)
Several types of DDS have been considered for drug-targeting applications. Synthetic lipid nanoparticles (liposomes) are the most biocompatible and the least toxic artificial systems. They are composed of phospholipids and cholesterol, which are both components of cell membranes. Liposomes can entrap drugs in both aqueous and lipid phases, and thus deliver hydrophilic and hydrophobic drugs. They can load multiple drugs to increase drug delivery, and consequently, potentially reduce toxicity and increase the treatment effectiveness [54]. Liposomes can be endowed with specific targeting ligands to enhance the accumulation and intracellular uptake into target cells expressing the specific receptor [55]. However, these nanoparticles suffer from poor biocompatibility and biodegradability. In addition, immunogenicity limits their therapeutic applicability. EV-based DDS could resolve these drawbacks.
In contrast to liposomes, EVs share the lipid asymmetry of the parent cells, allowing for optimal interaction with their target cells [56]. Different studies have reported that EVs may be taken up more efficiently into target cells than the liposomes, leading to an enhanced delivery of the cargo contained in EVs [57,58].
EV-based DDS present multiple advantages ( Figure 2): (1) their structure is composed of an aqueous core and a rich lipid bilayer surface structure, allowing for the compartmentalization and solubilization of both hydrophilic and lipophilic agents [59]. (2) EVs carry various biomolecules, such as proteins, lipids, and DNA and RNA species, depending on the producer cell types; the surface structure consists of fatty acids, high concentrations of cholesterol, sphingomyelin, ceramides, and other lipids; and interestingly, this surface also contains proteins that are implicated in adhesion, such as tetraspanins and αβ integrins, conferring on EVs an endogenous homing and targeting capacity [60]. (3) EVs are considered to be non-immunogenic, with a lower risk of allogeneic immune rejection from the host [61]. (4) Their surface composition can be modified through different engineering approaches [62,63]. (5) EVs can efficiently cross biological tissue, cellular and intracellular barriers (i.e., the gastrointestinal barrier and blood-brain barrier), and induce functional changes in the target cell [64]. Moreover, EVs have fewer off-target effects, due to the natural tendency to act on specific target cells.
EVs can also be produced by plant cells (PEVs) [65], or by bacteria (BEVs) [66] and fungi (FEVs) [67], and they contain bioactive molecules, displaying multiple functions. These EVs can deliver exogenous and endogenous agents to mammalian cells in the majority of organs, and they have also a great potential to become novel drug delivery systems. As human EVs, they display advantageous properties such as low immunogenicity, tissue-specific targeting, safety, negative zeta potential, and the ability to load many biomolecules [68]. However, the therapeutic potential of these EVs is still in its infancy, due to the absence of a comprehensive understanding of the biogenesis mechanism, internalization and packaging processes, cargo identification, and the comparison with liposomes-based methods [69].
Due to their cell-based biological structures and functions, EVs represent an ideal natural material for the development of nanomedicine. However, there are different challenges to face before any clinical application of EV-based drug delivery systems. Indeed, EV preparations are highly heterogeneous due to the difficult purification of a specific EV population. Moreover, the isolation and purification methods are not uniform, limiting standardization. Due to their cell-based biological structures and functions, EVs represent an ideal natural material for the development of nanomedicine. However, there are different challenges to face before any clinical application of EV-based drug delivery systems. Indeed, EV preparations are highly heterogeneous due to the difficult purification of a specific EV population. Moreover, the isolation and purification methods are not uniform, limiting standardization.

The Isolation of Extracellular Vesicles
The process of large-scale EV production includes the expansion of MSCs, the collection of conditioned medium, and the isolation of EVs. Numerous EV isolation methods have been described, from differential ultracentrifugation (UC) to immuno-isolation by different surface molecules through density gradients, polymer-based precipitation, microfiltration, and size-exclusion-based chromatography [70] (Table 1). Differential UC is the most commonly used method. The process is based on the separation of particles according to their buoyant density. This procedure includes several substeps: centrifugation at 300-400× g for 10 min to sediment cells, at 2000× g to remove cell debris, and at 10,000× g to remove the aggregates and apoptotic bodies. Thereafter, the EV pellet is obtained via UC (100,000-200,000× g for 2 h). Filtration can replace the low-speed centrifugal steps for the large-scale preparation of exosomes in specific cases [71]. The EV isolation efficiency after differential UC depends on many factors: acceleration, rotor type, and sample viscosity. Sucrose density gradients (sucrose, iohexol, and iodixanol) and UC can be applied to increase the efficiency of particle separation to obtain highly purified EVs [72].

The Isolation of Extracellular Vesicles
The process of large-scale EV production includes the expansion of MSCs, the collection of conditioned medium, and the isolation of EVs. Numerous EV isolation methods have been described, from differential ultracentrifugation (UC) to immuno-isolation by different surface molecules through density gradients, polymer-based precipitation, microfiltration, and size-exclusion-based chromatography [70] (Table 1). Differential UC is the most commonly used method. The process is based on the separation of particles according to their buoyant density. This procedure includes several substeps: centrifugation at 300-400× g for 10 min to sediment cells, at 2000× g to remove cell debris, and at 10,000× g to remove the aggregates and apoptotic bodies. Thereafter, the EV pellet is obtained via UC (100,000-200,000× g for 2 h). Filtration can replace the low-speed centrifugal steps for the large-scale preparation of exosomes in specific cases [71]. The EV isolation efficiency after differential UC depends on many factors: acceleration, rotor type, and sample viscosity. Sucrose density gradients (sucrose, iohexol, and iodixanol) and UC can be applied to increase the efficiency of particle separation to obtain highly purified EVs [72]. However, both methods are expensive and time-consuming. Moreover, EV aggregation and rupture due to high shear forces have been reported [73].
Polymer-based precipitation is another isolation method. The method is based on EV precipitation in polymer solutions, due to changes in EV solubility and aggregation. The reagents used for polymer-based exosome isolation mainly include protamine, acetate, protein organic solvent precipitation (PROSPR), and polyethylene-glycol (PEG). PEG is the most commonly used polymer [74]. Dash  display high stability and good quality [75]. This operation is simple, fast, and suitable for large-volume samples, and it preserves the bioactivity of isolated EVs. However, potential contamination with copurifying protein aggregates or residual polymers isolated with EVs may occur. The EV size is comparable between the UC and precipitation methods. Nevertheless, the EV count is higher with polymer precipitation [76]. Recently, Jia et al. reported that the PEG-based method isolated more EVs, proteins, and RNA than the UC method [77].
Ultrafiltration is a size method that is used to isolate EVs. It employs membrane filters with different pore sizes to allow smaller particles to penetrate and to pass through the membrane, while larger particles are excluded. Depending on the driving force, ultrafiltration can be classified as electric charge, centrifugation, and pressure. This method is efficient and simple, and allows for high-purity exosome isolation [78]. Ultrafiltration is a time-and cost-effective alternative to the gold-standard UC method [79]. Indeed, this EV isolation method is 50 times more efficient, and it removes smaller-sized proteins from EV suspension. However, one of the disadvantages is membrane pore blockage leading to low EV yield. Lamparski et al. demonstrated for the first time the possibility of isolating EVs for clinical application using the association of ultrafiltration and density gradient UC [80].
Size-exclusion chromatography (SEC) is an isolation process that is based on EV size. This column chromatographic approach offers a quicker method of vesicle enrichment and better standardization using commercially available columns, as recently highlighted by Böing et al. [81]. Guan Sheng et al. compared SEC and UC, and demonstrated that the recovery rate, structural integrity, and biological activity of EVs isolated using SEC were higher than those isolated via UC [82]. Moreover, the EV purity obtained is sufficient for proteomic and functional analyses [83]. However, the harvested EVs are severely diluted, and the elution processes are time-consuming. Nevertheless, no specific equipment is needed. UC and SEC methods could be used together for large-scale clinical applications such as drug delivery purposes.
Immunoselection is based on specific interactions between EV membrane proteins and the corresponding antibodies, allowing for EV separation from other molecules. Lipids, proteins, and polysaccharides are exposed on the EV surface, and they are thus potential ligands for selection. Antibodies to these surface proteins bind specific targeted EV populations via positive selection, and they remove irrelevant EVs, allowing for the isolation of a specific subclass of EVs [84]. The most commonly used targets are tetraspanins (CD9, CD63, and CD81), which allow for the isolation of total EVs [85]. Antibodies can be covalently attached to plates, beads, filters, or other matrices. This method requires a small number of samples, allows for the isolation of EVs with high purity, and induces no modifications in structure and morphology, but it is not adapted for clinical applications, and the cost of immunoselection is high.
These EV purification methods are not always mutually exclusive, and they can be combined to enhance the effectiveness of isolation and purification. Indeed, UC that is used to enrich EVs can be followed by SEC to remove proteins and contaminants [86].

Methods for Loading Drugs into EVs
A major challenge in applying EVs to DDS is to achieve an efficient loading of drugs/molecules into EVs [63]. Many different loading methods (Table 2), either endogenous or exogenous approaches, have been developed to promote the EVs drug delivery. The endogenous approach involves the modification of parental cells through transfection [9], lipofection [10], or coincubation with a drug, before the purification of these modified MSC-derived EVs [11]. Exogenous methods consist of loading drugs or molecules in EVs after their purification.
Several parameters may influence the incorporation of drugs into EVs: the structure of the EV, the drug properties, and the ratio of EV/drug. A wide range of drugs with different molecular weights can be loaded into EVs, but the choice of the loading method and the efficiency of encapsulation are very dependent on the properties of the drugs, in terms of their relative hydrophilicities/hydrophobicities [94,95]. Importantly, the analysis of physicochemical features, morphological appearance, and cellular uptake demonstrated that the EV integrity can be affected by the loading method [96]. The mean diameter of loaded EVs mostly increased, but the alteration was dependent on the drug properties, and the loading method confirming that the EV characterization before and after drug loading is essential [97].
Drugs can be coincubated with EVs, and they diffuse into the EVs along a concentration gradient. This passive method allows for the loading of hydrophobic drugs interacting with the lipid membrane of EVs. Through this method, small-molecule drugs such as curcumin [58], paclitaxel [98], and doxorubicin [99] have been effectively loaded in EVs. This method is simple, requires no additional stimulation, and preserves EV integrity, but the drug loading efficiency is low and is dependent on the hydrophobicity of molecules [100]. The efficiency can be increased by optimizing the incubation temperature, time, volume of buffer, and EV ratio.
EV donor cells can be treated with drugs (small molecules such as paclitaxel or doxorubicin and different types of RNA) to obtain drug-loaded EVs [101]. Two methods can be applied: transfection and coincubation. These allow for the loading of both hydrophilic and hydrophobic molecules. The efficiency of drug packaging into EVs depends on its concentration inside cells [102]. Interestingly, cells can be exposed to ultraviolet light and/or heat to stimulate the formation of drug-loaded EVs.
The exogenous method refers to the artificial incorporation of therapeutic molecules within EVs after their isolation and purification. This active loading requires EV permeabilization via different methods, such as electroporation, sonication, extrusion, freeze/thaw cycles, hypotonic dialysis, chemical methods, and incubation with membrane permeabilizers [103]. The molecules' physiochemical properties determine which method best fits their encapsulation in EVs.
Electroporation is a simple and fast method. It consists of creating temporary small pores in the EV membrane under the action of an electric field, which increases membrane permeability. Molecules enter EVs through diffusion, and the membrane quickly recovers its integrity after drug loading. Nevertheless, electroporation requires specific equipment and the testing of optimum working conditions before the experiment. This approach has been used for loading EVs with curcumin or paclitaxel [58,104], and for encapsulating siRNAs or miRNAs. Some studies have shown that RNA and EVs can aggregate, resulting in a low loading capacity [105]. However, Bendix Johnsen et al. successfully optimized the use of a trehalose-containing buffer as a way of maintaining the structural integrity of EVs [106]. Recently, Liang et al. loaded a microRNA-21 inhibitor and chemotherapy drugs in EVs via electroporation [107].
The ultrasound method or sonication involves multiple ultrasonic treatments of a mix of EVs with the intended cargo. The mechanical shear force produced using the ultrasound probe compromises the integrity of the EV membrane and allows for drug encapsulation. This method is often used to load chemotherapeutic drugs in EVs [98], and provides superior drug loading compared with electroporation or coincubation. However, during sonication, a drug may adhere to the outer membrane layer, affecting its release. Some disadvantages of sonication include membrane integrity destruction and stability.
Mechanical extrusion can be used to encapsulate chemotherapeutic drugs. EVs are mixed with a drug, and the mixture is loaded into a syringe-based lipid extruder with 100-400 nm porous membranes under a controlled temperature. During extrusion, the EV membrane is disrupted and is vigorously mixed with the drug. This method allows for a high cargo loading efficiency, but this intensive extrusion process can change the EV membrane properties [15].
A freeze/thaw cycle strategy can be used to load drugs into EVs. It is a simple process [14]. Drugs are incubated with EVs at room temperature (RT) for a fixed amount of time, and then by performing at least three cycles of rapid freeze/thawing (−80 • C or in liquid nitrogen/RT), efficient EV drug loading is obtained. This method can induce EV aggregation, and the particle size increase. Moreover, the drug loading efficiency is lower than that obtained with sonication or extrusion [14]. Lee et al. used this method to prepare EVs containing miR-140 [108]. Recently, this strategy was used to create exosome-mimetic particles via membrane fusion between exosomes and liposomes [109].
Another approach for loading drugs into EVs is a hypotonic dialysis method based on the formation of drug transmembrane channels using osmotic pressure. This hypotonic environment allows for the penetration of small molecular substances via the opening of membrane pores. Fuhrmann et al. reported that porphyrin transfer in EVs can be drastically increased through hypotonic dialysis [15]. However, this method may induce EV size and charge changes.
Saponin has been described as an efficient permeabilization agent for cellular plasma membranes. Saponin can also increase the loading of different cargos in EVs. It creates pores in EV lipid bilayers through selective cholesterol removal. The EV loading of a small hydrophilic molecule, porphyrin, with saponin, allowed for an increased degree of loading (11-fold), in comparison with passive loading [15]. When compared with the simple incubation method, saponin was shown to enhance the loading of catalase into EVs and to preserve its activity [110].
Different methods result in varying loading efficiencies for the delivery of the same cargo. Chen et al. evaluated six commonly used drug-loading strategies (coincubation, electroporation, extrusion, freeze/thawing, sonication, and surfactant treatment) to incorporate Doxo into EVs at the single-particle level via nanoflow cytometry. The authors observed that the Doxo-loaded EVs prepared via coincubation and electroporation possessed a higher encapsulation ratio and a greater Doxo content than the EVs loaded with a single method. These Doxo-loaded EVs prepared via these two procedures resulted in a higher level of cellular uptake and induced more significant apoptosis for tumour cells, compared with EVs prepared with other drug-loading strategies [111].

Loaded EVs for Therapy in Preclinical Studies
EVs have a great potential for drug delivery [5]. Different EV isolation protocols and loading methods have been tested and compared. In 2013, the first preclinical study used MSC-derived EVs transfected with miR-146 to treat glioma in an animal model [112]. The authors observed that MSC-derived EVs loaded with miR-146 elicited an antitumour effect in the rat brain. The authors demonstrated that the miRNA could be loaded into extracellular EVs, and that the plasmid-expressed miRNA was efficiently packaged into MSC-derived EVs via endogenous mechanisms. These findings suggest that miR-146b, delivered via MSC-derived EVs, is functionally active in acceptor tumour cells. Since then, numerous preclinical studies, both in vivo and in vitro, have indicated that MSC-derived EVs are safe and well tolerated. However, standardization is still lacking. A majority (61%) of the studies have used differential centrifugation and UC as the gold standard method, with or without a sucrose gradient, or under GMP production conditions. A commercial kit, with all types combined, was used in 39% of the studies (Figure 3). The electroporation loading method and plasmid transfection were the principal loading methods. The other loading methods were used more rarely (i.e., incubation or lentivirus transfection). There is great interest in MSC-derived EVs for oncological diseases and other conditions. MSC-derived EVs have been used in applications for glioma, breast cancer, melanoma, pancreatic cancer, hepatocellular carcinoma, colorectal cancer, acute myocardial infarction, myocardial ischemia-reperfusion injury, cerebral ischemia, and inflammatory diseases.
EVs have a great potential for drug delivery [5]. Different EV isolation protocols and loading methods have been tested and compared. In 2013, the first preclinical study used MSC-derived EVs transfected with miR-146 to treat glioma in an animal model [112]. Th authors observed that MSC-derived EVs loaded with miR-146 elicited an antitumour ef fect in the rat brain. The authors demonstrated that the miRNA could be loaded into ex tracellular EVs, and that the plasmid-expressed miRNA was efficiently packaged into MSC-derived EVs via endogenous mechanisms. These findings suggest that miR-146b delivered via MSC-derived EVs, is functionally active in acceptor tumour cells. Since then numerous preclinical studies, both in vivo and in vitro, have indicated that MSC-derived EVs are safe and well tolerated. However, standardization is still lacking. A majority (61% of the studies have used differential centrifugation and UC as the gold standard method with or without a sucrose gradient, or under GMP production conditions. A commercia kit, with all types combined, was used in 39% of the studies (Figure 3). The electroporation loading method and plasmid transfection were the principal loading methods. The othe loading methods were used more rarely (i.e., incubation or lentivirus transfection). Ther is great interest in MSC-derived EVs for oncological diseases and other conditions. MSC derived EVs have been used in applications for glioma, breast cancer, melanoma, pancre atic cancer, hepatocellular carcinoma, colorectal cancer, acute myocardial infarction, my ocardial ischemia-reperfusion injury, cerebral ischemia, and inflammatory diseases.

In Vitro Studies
In vitro, there have been many preclinical studies of EV-based therapy for patholog ical conditions, especially those in oncology. These studies aim to improve the treatmen of these diseases using MSC-derived EVs loaded with proteins, miRNA, and drugs. Th beneficial results have led to the development of most of the in vivo preclinical studie described in the next paragraph. Several studies have focused on fibrosis, ischemia, and inflammation reduction, but also on an increase in proliferation, migration, neurogenesis and ageing prevention, through miRNA regulation mediated via EVs in cell line

In Vitro Studies
In vitro, there have been many preclinical studies of EV-based therapy for pathological conditions, especially those in oncology. These studies aim to improve the treatment of these diseases using MSC-derived EVs loaded with proteins, miRNA, and drugs. The beneficial results have led to the development of most of the in vivo preclinical studies described in the next paragraph. Several studies have focused on fibrosis, ischemia, and inflammation reduction, but also on an increase in proliferation, migration, neurogenesis, and ageing prevention, through miRNA regulation mediated via EVs in cell lines [113,114]. Other studies have described that EVs have the ability to improve endothelial cell remodeling and angiogenesis, or decrease ischemia when cell lines are treated with EVs carrying specific proteins or drugs [115][116][117]. These studies support the emerging role of EVs as a drug delivery system (Table 3a).
Exosome-loaded miRNA (Exo-miRNA) or exosome-loaded drugs can act as a better delivery system, enhancing their effects on cancer cells. Li and colleagues reported on the synergistic effect of chemo-phototherapy by treating glioma cell lines with Exos CUR + ICG, combined with photothermal NIR radiation [113]. Katakowski et al. and Lang et al., respectively, observed the positive effects of Exo-miR-124 and Exo-miR-146 on glioma cells [112,118]. Melzer et al. reported that EVs loaded with paclitaxel (PTX) showed interesting outcomes in the treatment of breast cancer, with more efficient tumour-targeting properties [119], bonding receptor activity [120], and transcriptional regulation [121]. There are multiple benefits of EVs as nanocarriers in therapy, and Bagheri et al. reported on their potential to inhibit tumour growth in vitro [122]. Interestingly, Lou et al. described an increase in hepatocellular carcinoma (HCC) cell sensitivity to EVs containing doxorubicin, in comparison to free drugs [123]. Moreover, Yang et al. demonstrated interesting achievements in targeted therapy mediated by encapsulating doxorubicin in desialylated MSC-derived EVs on HCC cell lines [124]. Tumour growth inhibition induced by the apoptotic pathway can also be initiated by exosomes derived from TNFα plasmid-transfected MSCs [125]. The apoptotic pathway seems to play a key role when B16F0 melanoma cell lines are treated with Exo-TRAIL [126]. Cancer cell migration and proliferation are major points to be elucidated for a better understanding of oncology. Exosomes incubated with doxorubicin, recently described by Wei and colleagues, suppressed the migration and proliferation of osteosarcoma cells [117]. The last cancer in preclinical study is pancreatic cancer. This cancer is very resistant, and is considered as being almost "undruggable", due to intense fibrosis and the immunosuppression of the TME [127]. Recent studies have shown promising results by targeting the specific mutation, KRAS G12D that is involved in pancreatic ductal adenocarcinoma (PDAC). Both Mendt and collaborators, and Kamerkar and colleagues have shown that the use of engineered exosomes derived from MSCs in Panc-1 cells resulted in the upregulation of genes associated with lysosome, proteasome, or phagosome pathways, and in cell death [128,129]. Moreover, a better efficiency was observed, due to higher phosphorylated-ERK protein levels in Panc-1 cells treated with iExo sh/siRNA-KRAS G12D . In parallel, Ding et al. demonstrated E-cadherin and Bax upregulation, and Smad3, Bcl-2, and N-cadherin downregulation when pancreatic cells were treated with EV-miR-145 [130]. The following diagrams summarize the main effect in vitro (Figure 4).
Exosome-loaded miRNA (Exo-miRNA) or exosome-loaded drugs can act as a better delivery system, enhancing their effects on cancer cells. Li and colleagues reported on the synergistic effect of chemo-phototherapy by treating glioma cell lines with Exos CUR + ICG, combined with photothermal NIR radiation [113]. Katakowski et al. and Lang et al., respectively, observed the positive effects of Exo-miR-124 and Exo-miR-146 on glioma cells [112,118]. Melzer et al. reported that EVs loaded with paclitaxel (PTX) showed interesting outcomes in the treatment of breast cancer, with more efficient tumour-targeting properties [119], bonding receptor activity [120], and transcriptional regulation [121]. There are multiple benefits of EVs as nanocarriers in therapy, and Bagheri et al. reported on their potential to inhibit tumour growth in vitro [122]. Interestingly, Lou et al. described an increase in hepatocellular carcinoma (HCC) cell sensitivity to EVs containing doxorubicin, in comparison to free drugs [123]. Moreover, Yang et al. demonstrated interesting achievements in targeted therapy mediated by encapsulating doxorubicin in desialylated MSC-derived EVs on HCC cell lines [124]. Tumour growth inhibition induced by the apoptotic pathway can also be initiated by exosomes derived from TNFα plasmid-transfected MSCs [125]. The apoptotic pathway seems to play a key role when B16F0 melanoma cell lines are treated with Exo-TRAIL [126]. Cancer cell migration and proliferation are major points to be elucidated for a better understanding of oncology. Exosomes incubated with doxorubicin, recently described by Wei and colleagues, suppressed the migration and proliferation of osteosarcoma cells [117]. The last cancer in preclinical study is pancreatic cancer. This cancer is very resistant, and is considered as being almost "undruggable", due to intense fibrosis and the immunosuppression of the TME [127]. Recent studies have shown promising results by targeting the specific mutation, KRAS G12D that is involved in pancreatic ductal adenocarcinoma (PDAC). Both Mendt and collaborators, and Kamerkar and colleagues have shown that the use of engineered exosomes derived from MSCs in Panc-1 cells resulted in the upregulation of genes associated with lysosome, proteasome, or phagosome pathways, and in cell death [128,129]. Moreover, a better efficiency was observed, due to higher phosphorylated-ERK protein levels in Panc-1 cells treated with iExo sh/siRNA-KRAS G12D . In parallel, Ding et al. demonstrated E-cadherin and Bax upregulation, and Smad3, Bcl-2, and N-cadherin downregulation when pancreatic cells were treated with EV-miR-145 [130]. The following diagrams summarize the main effect in vitro ( Figure 4).      Exosomes derived from TIMP2-modified ucMSCs repaired the ischemia injuries by inhibiting apoptosis and promoting angiogenesis, and ECM remodeling in cardiomyocytes. [116] Acute myocardial infarction Myocardial and endothelial cells ("homemade" isolation)
[132]   Interesting reports have demonstrated the added value of EVs as nanocarriers in the therapy of non-oncological conditions (Table 3b). These studies have focused on acute myocardial infarction, myocardial ischemia-reperfusion injury, cerebral ischemia, or inflammatory diseases such as intestinal fibrosis, rheumatoid, and osteoarthritis, as well as vascular dysfunction that is ageing-associated [7]. The in vitro studies have shown encouraging results in all of these different conditions. Luo et al. described the preventive effect of Exo-miR-126 on myocardial damage [114]. Ma and colleagues reported that endothelial cell proliferation was promoted by Exo-Akt loading, which affected different signaling pathways [131]. Moreover, ischemia injury was repaired by the treatment of cardiomyocytes with EVs loaded with TIMP2 protein [116]. Indeed, microvascular regeneration was obtained via the overexpression of SDF-1 mediated by the EV preparation [115]. Similar results in cerebral ischemia and ageing-induced vascular dysfunction were reported by Zhao et al., Tian et al.,and Tao et al. [133,134,136]. Encouraging studies on inflammatory diseases have shown improvements through the use of EV-based therapy. Groups studying osteoarthritis and rheumatoid arthritis have established the key role of miRNAs in vitro: miR-140-5p promoted the proliferation and migration of osteoarthritis cells treated with SMSC-140-Exos, and miR-150-5p encapsulated in exosomes led to a downregulation of tube formation by HUVECs via the VEGF and MMP4 signaling pathways [136][137][138].The following diagrams summarize ( Figure 5) the main active pharmaceutical ingredients (API) in EVs derived from MSCs as drug delivery systems, and their effects in preclinical studies in the oncological and non-oncological fields. Interesting reports have demonstrated the added value of EVs as nanocarriers in the therapy of non-oncological conditions (Table 3b). These studies have focused on acute myocardial infarction, myocardial ischemia-reperfusion injury, cerebral ischemia, or inflammatory diseases such as intestinal fibrosis, rheumatoid, and osteoarthritis, as well as vascular dysfunction that is ageing-associated [7]. The in vitro studies have shown encouraging results in all of these different conditions. Luo et al. described the preventive effect of Exo-miR-126 on myocardial damage [114]. Ma and colleagues reported that endothelial cell proliferation was promoted by Exo-Akt loading, which affected different signaling pathways [131]. Moreover, ischemia injury was repaired by the treatment of cardiomyocytes with EVs loaded with TIMP2 protein [116]. Indeed, microvascular regeneration was obtained via the overexpression of SDF-1 mediated by the EV preparation [115]. Similar results in cerebral ischemia and ageing-induced vascular dysfunction were reported by Zhao et al., Tian et al.,and Tao et al. [133,134,136]. Encouraging studies on inflammatory diseases have shown improvements through the use of EV-based therapy. Groups studying osteoarthritis and rheumatoid arthritis have established the key role of miRNAs in vitro: miR-140-5p promoted the proliferation and migration of osteoarthritis cells treated with SMSC-140-Exos, and miR-150-5p encapsulated in exosomes led to a downregulation of tube formation by HUVECs via the VEGF and MMP4 signaling pathways [136][137][138].The following diagrams summarize ( Figure 5) the main active pharmaceutical ingredients (API) in EVs derived from MSCs as drug delivery systems, and their effects in preclinical studies in the oncological and non-oncological fields.

In Vivo Animal Studies
Li et al. have shown in a mouse model that exos-based therapy can significantly abrogate glioma. Exos-based therapy consists of EVs loaded via electroporation with curcumin and indocyanine green [113]. Lang et al. showed the growth inhibition of glioblastoma stem cells (GSCs), also in an animal model, after treatment with EVs loaded with a supraphysiological level of miR-142a by plasmid transfection before EV isolation [118]. Interestingly, Katakowski and collaborators reported on a tumour growth decrease after rat treatment with EVs derived from MSCs transfected with a miR-146b plasmid [112]. In breast cancer, chemotherapeutic molecules such as paclitaxel or doxorubicin are loaded in EVs through incubation and electroporation, respectively. Gomari et al. observed a better inhibition of tumour growth with drug-loaded EVs than with free drugs [120]. Melzer et al. reported a 60% reduction in the subcutaneous primary tumour and distant organ metastases in mice with MDA-hy1 breast cancer after treatment with EVs incubated with paclitaxel [119]. In the same context, Naseri and colleagues reduced the expression levels of miR-142-3p and miR-150 associated with cancer stem cell tumourigenicity via the electroporation of LNA-antimiR-142-3p-loaded exosomes [121]. In the study of Bagheri et al.,

In Vivo Animal Studies
Li et al. have shown in a mouse model that exos-based therapy can significantly abrogate glioma. Exos-based therapy consists of EVs loaded via electroporation with curcumin and indocyanine green [113]. Lang et al. showed the growth inhibition of glioblastoma stem cells (GSCs), also in an animal model, after treatment with EVs loaded with a supraphysiological level of miR-142a by plasmid transfection before EV isolation [118]. Interestingly, Katakowski and collaborators reported on a tumour growth decrease after rat treatment with EVs derived from MSCs transfected with a miR-146b plasmid [112]. In breast cancer, chemotherapeutic molecules such as paclitaxel or doxorubicin are loaded in EVs through incubation and electroporation, respectively. Gomari et al. observed a better inhibition of tumour growth with drug-loaded EVs than with free drugs [120]. Melzer et al. reported a 60% reduction in the subcutaneous primary tumour and distant organ metastases in mice with MDA-hy1 breast cancer after treatment with EVs incubated with paclitaxel [119]. In the same context, Naseri and colleagues reduced the expression levels of miR-142-3p and miR-150 associated with cancer stem cell tumourigenicity via the electroporation of LNA-antimiR-142-3p-loaded exosomes [121]. In the study of Bagheri et al., a single intra-venous (IV) injection of EVs-DOXO significantly suppressed tumour growth in a mouse model of colorectal cancer [122]. In orthotopic mouse models of hepatocellular carcinoma (HCC), the IV injection of EVs-miR-199a loaded by the lentivirus transfection of adiposetissue derived MSCs improved HCC chemosensitivity to doxorubicin via mTOR pathway targeting [123]. Yang et al. showed enhanced cytotoxicity efficiency using Doxo-loaded desialylated MSC-derived EVs for better targeting efficiency in Balb/c nude mice injected with HCC cells [124]. TNF-α and TRAIL protein can also be transfected using a plasmid in MSCs before EV isolation to obtain better drug delivery with increased antitumour activity, and less toxicity, in a melanoma mouse model [125]. Moreover, EXO-TRAIL reduced tumour progression by enhancing necrosis [126]. In pancreatic cancer, the deadliest disease, with a five-year overall survival rate of only 11%, Mendt and Kamerkar and their respective colleagues reported that EVs electroporated with siRNA-KRASG12D induced a robust antitumour effect in the PDAC model, and in multiple pancreatic cancer mouse models [128,129]. Kamerkar et al. reported on an increase in overall survival (OS) after the treatment of mice with EVs loaded with siRNA/pLKO.1-shRNA [128]. The following diagrams summarize the most relevant in vivo effects ( Figure 6).
Pharmaceutics 2023, 15, x FOR PEER REVIEW a single intravenous (IV) injection of EVs-DOXO significantly suppressed tumour in a mouse model of colorectal cancer [122]. In orthotopic mouse models of hepato carcinoma (HCC), the IV injection of EVs-miR-199a loaded by the lentivirus tran of adipose-tissue derived MSCs improved HCC chemosensitivity to doxorub mTOR pathway targeting [123]. Yang et al. showed enhanced cytotoxicity efficienc Doxo-loaded desialylated MSC-derived EVs for better targeting efficiency in Balb mice injected with HCC cells [124]. TNF-α and TRAIL protein can also be transfe ing a plasmid in MSCs before EV isolation to obtain better drug delivery with in antitumour activity, and less toxicity, in a melanoma mouse model [125]. Moreove TRAIL reduced tumour progression by enhancing necrosis [126]. In pancreatic can deadliest disease, with a five-year overall survival rate of only 11%, Mendt and Ka and their respective colleagues reported that EVs electroporated with siRNA-KRA induced a robust antitumour effect in the PDAC model, and in multiple pancreatic mouse models [128,129]. Kamerkar et al. reported on an increase in overall surviv after the treatment of mice with EVs loaded with siRNA/pLKO.1-shRNA [128]. lowing diagrams summarize the most relevant in vivo effects ( Figure 6). There has also been great interest in the therapeutic role of EVs in non-onc conditions (Table 4b). In acute myocardial infarction (AMI), EVs loaded with m AKT, or TIMP2 protein via transfection allowed for recovery by decreasing the in area through a decrease in proinflammatory cytokines and a reduction in cardiac [114,116,131]. Ma et al. treated AMI rats with AKT-loaded EVs, and observed an im ment in cardiac function through angiogenesis promotion [131]. Ni and col demonstrated, in a rat model treated with exosomes derived from TIMP2-overexp MSCs, the apoptosis inhibition of cardiomyocytes and the promotion of angiogene ECM remodeling in the context of AMI [116]. In a myocardial infarction (MI) model, EVs collected from MSCs transfected with an SDF-1 plasmid inhibited cell agy and promoted microvascular endothelial cell production [115]. Many therape fects of EVs as nanocarriers have been discovered in recent decades. Both in rat and models, and in different pathological contexts, EVs display multiple therapeutic ac In cerebral ischemia, miR-223-3p-or miR-124-loaded EVs have been used for is cortex and hippocampus treatment in animal models, and they attenuate ischemi by stimulating neurogenesis [133,134,139]. Effects on senescence in ageing-induc cular dysfunctions, chondrocyte proliferation in osteoarthritis, synovial inflam and joint damage in rheumatoid arthritis or oedema edema reduction in cerebra mia-reperfusion injury, attest to the great diversity of potential applications of EV ing miRNAs or molecules, bringing benefits for treatment and recovery from ma eases [135,137,140]. The following diagrams (Figure 7) summarize the main APIs derived from [135,137,140] MSCs as drug delivery systems, and their effects in pre studies in the fields of oncological and non-oncological diseases. There has also been great interest in the therapeutic role of EVs in non-oncological conditions (Table 4b). In acute myocardial infarction (AMI), EVs loaded with miR-126, AKT, or TIMP2 protein via transfection allowed for recovery by decreasing the infarction area through a decrease in proinflammatory cytokines and a reduction in cardiac fibrosis [114,116,131]. Ma et al. treated AMI rats with AKT-loaded EVs, and observed an improvement in cardiac function through angiogenesis promotion [131]. Ni and colleagues demonstrated, in a rat model treated with exosomes derived from TIMP2-overexpressing MSCs, the apoptosis inhibition of cardiomyocytes and the promotion of angiogenesis and ECM remodeling in the context of AMI [116]. In a myocardial infarction (MI) mouse model, EVs collected from MSCs transfected with an SDF-1 plasmid inhibited cell autophagy and promoted microvascular endothelial cell production [115]. Many therapeutic effects of EVs as nanocarriers have been discovered in recent decades. Both in rat and mouse models, and in different pathological contexts, EVs display multiple therapeutic activities. In cerebral ischemia, miR-223-3p-or miR-124-loaded EVs have been used for ischemic cortex and hippocampus treatment in animal models, and they attenuate ischemia injury by stimulating neurogenesis [133,134,139]. Effects on senescence in ageing-induced vascular dysfunctions, chondrocyte proliferation in osteoarthritis, synovial inflammation, and joint damage in rheumatoid arthritis or oedema edema reduction in cerebral ischemia-reperfusion injury, attest to the great diversity of potential applications of EVs carrying miRNAs or molecules, bringing benefits for treatment and recovery from many diseases [135,137,140]. The following diagrams (Figure 7) summarize the main APIs in EVs derived from [135,137,140] MSCs as drug delivery systems, and their effects in preclinical studies in the fields of oncological and non-oncological diseases.  Doxorubicin loaded in desialylated MSC-derived EVs as a drug delivery system targeted hepatoma cells in mouse model. [124] Melanoma Mouse MSCs TNF-α Transfection (plasmid-based/before isolation) Coupled SPIONs and CTNF-α anchored exosomes delivered peptide drugs to the cytomembrane better than to the cytoplasm, and resulted in an increase in antitumour activity and lower toxicity.
[125] Exo-DOXO displayed higher cytotoxicity than free drug, and was efficient as a drug delivery system. [117] Pancreatic cancer Mouse MSCs siKRAS G12D Electroporation (after isolation) Both BM-MSCs-and BJ-MSCs-derived exosomes loaded with siKRAS G12D showed a robust antitumour efficiency in PDAC models. [129] Pancreatic cancer Mouse MSCs siKRASG12D and pLKO.  Electroporation (after isolation) Exosomes derived from mouse skin fibroblast were used as a nanocarrier to specifically target pancreatic cancer cells in multiple mouse models of pancreatic cancer. EV injection drastically increased OS. [128] Pancreatic cancer Mouse hucMSCs miRNA-145-5p Transfection reagent (after isolation) Intratumour injection of miR145-5p UC-MSCs-derived exosomes reduced xenograft tumour growth in a BALB/c mouse model of Panc-1 cells. [130] (b) Acute myocardial infarction Rat Adipose stem cells miRNA-126 Transfection (miRNA-based/before isolation) The treatment of AMI rats with miR-126-enriched exosomes decreased the infarction area in myocardial injury, inflammatory cytokine expression, and cardiac fibrosis.

Loaded EVs for Therapy in Clinical Trials
More than 150 clinical studies involving EVs are in progress [141]. These studies aim to treat numerous conditions: bronchopulmonary dysplasia, dystrophic epidermolysis bullosa, acute ischemic stroke, chronic graft-versus-host diseases, macular holes, metastatic pancreatic cancer, and type I diabetes mellitus. However, only a few studies have been published. In 2014, the first patient suffering from steroid-refractory graft-versushost disease (GvHD) was treated with hBMMSC-derived EVs [142]. The clinical GvHD symptoms improved briefly but significantly after the onset of MSC-derived EV therapy. The patient was stable for a few months. The obtained results suggested that MSC-derived EVs may provide a potentially new and safe tool to treat refractory GvHD and other inflammation-associated diseases. Nassar and colleagues also observed that EV therapy could ameliorate inflammatory immune reactions [143]. Interestingly, the authors demonstrated that the administration of cell-free cord-blood MSC-derived EVs was safe and could improve the inflammatory immune reaction and improve the overall kidney function in grade III-IV CKD patients.
Currently, no studies using MSC-derived EVs as nanocarriers have been published. Several preclinical studies have been published, which have shown optimistic results [144]. Munoz et al. demonstrated that the delivery of functional anti-miR-9 by MSC-derived EVs to glioblastoma multiforme cells conferred chemosensitivity [145]. Melzer et al. observed that the systemic intravenous application of Taxol-loaded MSC-derived EVs induced a greater than 60% reduction in subcutaneous breast tumours [119]. Moreover, the number of distant organ metastases observed in the lung, liver, spleen, and kidney was reduced by 50% with Taxol-loaded MSC-derived EVs, similar to the effects observed with Taxol. However, the Taxol concentration in EVs was reduced by approximately 1000-fold. Few clinical studies are in progress (Home-ClinicalTrials.gov). Kamerkar et al. evaluated the best dose and side effects of MSC-derived EVs with KrasG12D siRNA (iEVs) to treat metastatic pancreatic cancer patients harboring the KrasG12D mutation, in a phase I trial (NCT03608631) [146]. This clinical trial is in the recruitment phase, and 28 participants will be included. These patients will receive EVs IV over 15-20 min on Days 1, 4, and 10. The treatment will be repeated every 14 days for up to 3 courses. The study should be completed in March 2023.
MSC-derived EVs are also currently studied in non-oncological conditions such as homozygous familial hypercholesterolemia or acute ischemic stroke. The first trial (NCT05043181) aims to create an LDL receptor-expressing virus vector to generate LDLR mRNA-enriched EVs derived from BM-MSCs and purified via filtration and ultracentrifugation. EVs will be injected through abdominal puncture to evaluate their safety and efficacy for the treatment of homozygous familial hypercholesterolemia patients present-

Loaded EVs for Therapy in Clinical Trials
More than 150 clinical studies involving EVs are in progress [141]. These studies aim to treat numerous conditions: bronchopulmonary dysplasia, dystrophic epidermolysis bullosa, acute ischemic stroke, chronic graft-versus-host diseases, macular holes, metastatic pancreatic cancer, and type I diabetes mellitus. However, only a few studies have been published. In 2014, the first patient suffering from steroid-refractory graft-versus-host disease (GvHD) was treated with hBMMSC-derived EVs [142]. The clinical GvHD symptoms improved briefly but significantly after the onset of MSC-derived EV therapy. The patient was stable for a few months. The obtained results suggested that MSC-derived EVs may provide a potentially new and safe tool to treat refractory GvHD and other inflammationassociated diseases. Nassar and colleagues also observed that EV therapy could ameliorate inflammatory immune reactions [143]. Interestingly, the authors demonstrated that the administration of cell-free cord-blood MSC-derived EVs was safe and could improve the inflammatory immune reaction and improve the overall kidney function in grade III-IV CKD patients.
Currently, no studies using MSC-derived EVs as nanocarriers have been published. Several preclinical studies have been published, which have shown optimistic results [144]. Munoz et al. demonstrated that the delivery of functional anti-miR-9 by MSC-derived EVs to glioblastoma multiforme cells conferred chemosensitivity [145]. Melzer et al. observed that the systemic intravenous application of Taxol-loaded MSC-derived EVs induced a greater than 60% reduction in subcutaneous breast tumours [119]. Moreover, the number of distant organ metastases observed in the lung, liver, spleen, and kidney was reduced by 50% with Taxol-loaded MSC-derived EVs, similar to the effects observed with Taxol. However, the Taxol concentration in EVs was reduced by approximately 1000-fold. Few clinical studies are in progress (Home-ClinicalTrials.gov). Kamerkar et al. evaluated the best dose and side effects of MSC-derived EVs with KrasG12D siRNA (iEVs) to treat metastatic pancreatic cancer patients harboring the KrasG12D mutation, in a phase I trial (NCT03608631) [146]. This clinical trial is in the recruitment phase, and 28 participants will be included. These patients will receive EVs IV over 15-20 min on Days 1, 4, and 10. The treatment will be repeated every 14 days for up to 3 courses. The study should be completed in March 2023.
MSC-derived EVs are also currently studied in non-oncological conditions such as homozygous familial hypercholesterolemia or acute ischemic stroke. The first trial (NCT05043181) aims to create an LDL receptor-expressing virus vector to generate LDLR mRNA-enriched EVs derived from BM-MSCs and purified via filtration and ultracentrifugation. EVs will be injected through abdominal puncture to evaluate their safety and efficacy for the treatment of homozygous familial hypercholesterolemia patients presenting a functional loss due to the mutation of the LDLR. Thirty patients will be included, and the study will be finalized in December 2026 [147]. The second study (NCT03384433) aims to assay the administration of MSC-derived EVs on the improvement of disability in patients with acute ischemic stroke. Five patients will receive allogenic MSC-derived exosomes transfected by miR-124, 1 month after the attack via stereotaxis/intraparenchymal injection. This randomized, single-blind, placebo-controlled phase I/II trial was supposed to be completed in December 2021, but no data have been published [148].

Challenges
Even though the results of preclinical studies have been positive, different steps are needed to overcome quality control and procedure standardization. Indeed, different protocols of EV purification, quantification, and characterization coexist [144]. The lack of standardized isolation and purification methods for EVs, the limited drug delivery efficiencies of EVs, the isolation of EVs contaminated with impurities (cell debris, non-exosomal vesicles, and proteins) and insufficient production are still major challenges. For drug delivery, the evaluation of storage conditions, pharmacokinetics, and the biodistribution of loaded EVs is needed. In addition, the culture of MSCs that produce EVs must also be considered. The bioreactors for cell expansion should provide sufficient EV quantities for clinical-grade production.

Conflicts of Interest:
The authors declare no conflict of interest.