Different types of therapeutic payloads have been loaded into MSC-EVs, including nucleic acids, proteins and small molecules. Either endogenously, by manipulation of parental MSC, or exogenously, through manipulation of isolated EVs, cargo engineering of MSC-EVs has been shown to improve their therapeutic efficacy in particular clinical applications (Table 1).

miRs are promising new therapeutics for treating many diseases. MSC-EVs have emerged as a promising vehicle for delivering miRs, thus many researchers have been engineering MSC to express or harbour these molecules.

For instance, human BM-derived MSC (MSC(M)) were transduced with lentivirus vectors containing miR-124a which silences Forkhead box A2 expression, inducing aberrant intracellular lipid accumulation. Quantitative PCR demonstrated that the produced EVs contained approximately 60-fold higher levels of miR-124a compared to non-modified MSC-derived EVs. miR-124a-carrying EVs resulted in a significant in vitro reduction in viability and clonogenicity of glioma stem cells (GSC), and treated mice harbouring intracranial GSC xenografts after systemic administration ^[1][89]. In addition, other miRs have been endogenously loaded into MSC-EVs and demonstrated anti-cancer potential, including miR-379 ^[2][90], miR-16-5p ^[3][91] and miR-424 ^[4][92] through post-transcriptional regulation of tumour-related gene expression of ciclo-oxigenase-2, integrin α2 and transcriptional factors MYB, respectively.

In the context of skin regeneration, Li and colleagues explored the potential of EVs derived from MSCs transfected with miR-181, which has a critical role in regulating inflammation, specifically in attenuating skin-burn-induced inflammation. The results demonstrated that the engineered EVs suppressed the TLR4 signalling pathway, reducing NF-κB/p65 activation, and alleviated inflammation in burned rats more effectively than EVs produced by non-transfected MSC ^[14][102]. Furthermore, in a study by Tao et al., EVs secreted by MSCs derived from the synovial membrane (SM) engineered to overexpress miR-126 were demonstrated to be able to heal full-thickness skin defects in a diabetic rat model ^[6][94]. Interestingly, MSC(AT)-derived EVs endogenously loaded with miR-126 also showed prospective effects in the treatment of ischemic stroke ^[10][98].

MSCs have also been engineered to produce miR-containing EVs that attenuate fibrosis. In a study by Lou et al., miR-122-engineered EVs inhibited fibrosis by reducing proliferation and collagen maturation of hepatic stellate cells through miR-122-induced downregulation of target genes such as insulin-like growth factor receptor-1, cyclin G-1 and prolyl-4-hydroxylase α-1 ^[5][93]. Moreover, MSC(M) engineered to overexpress miR-let7c generated EVs that inhibited the upregulated expression of fibrotic genes in neighbouring rat kidney tubular epithelial cells and attenuated renal fibrosis in vivo in a mouse model of unilateral ureteral obstruction ^[8][96].

Envisioning the treatment of rheumatoid arthritis (RA), MSC-EVs were engineered to harbour miR-150-5p, by transfection of the parental cells. miR-150-5p-loaded EVs decreased the migration and invasion of RA synoviocytes and downregulated tube formation in vitro, by targeting matrix metalloproteinase 14 and vascular endothelial growth factor. These MSC-EVs also reduced clinical arthritic scores and joint destruction in an in vivo RA mouse model ^[12][100]. In addition, a study on EVs derived from miR-92a-3p-expressing MSCs showed enhanced cartilage development and prevented its degradation by targeting wnt5a in a collagenase-induced osteoarthritis (OA) mouse model ^[18][106]. Furthermore, EVs secreted by miR-155-5p-overexpressing SM-derived MSCs promoted ECM secretion in vitro by targeting Runx2 and effectively prevented OA in a mouse model ^[13][101].

Studies have found that MSC-EV-mediated delivery of miR showed a positive effect in neurodegenerative diseases and mitigated the damage caused by CNS injuries. Jahangard and colleagues engineered MSCs to produce EVs encapsulating miR-29, which is downregulated in Alzheimer’s disease (AD) and silences the expression β-site amyloid precursor protein cleaving enzyme 1 and Bcl-2 interacting mediator of cell death. miR-29-EVs caused a reduction in the pathological effects of amyloid-β peptides after injection into the hippocampus of a rat model of AD, namely by improving spatial learning and memory deficits ^[17][105]. Furthermore, the lentivirus-based modification of MSCs to overexpress miR-17-92 allowed the production of EVs that enhanced axon–myelin remodelling and motor electrophysiological recovery after stroke in an in vivo mouse model ^[7][95]. Spinal cord injury (SCI) recovery has been investigated using MSC-EVs endogenously loaded with miR-133b, which is a key player in the differentiation of neurons and the outgrowth of neurites. miR-133b-EVs have been shown to activate signalling pathway proteins involved in the survival of neurons and the regeneration of axons, reduce the volume of the lesion and promote the regeneration of axons after systemic injection into a rat model of SCI ^[11][99]. In addition, recent studies reported that MSC-EV-mediated delivery of miR-22 and miR-26a could represent novel therapeutic approaches for the treatment of SCI ^[15][16][103,104].

To date, only a few studies have reported developing mRNA-loaded EVs. Among these, a study by Altanerova et al. describes a strategy where MSCs from different tissue sources were modified by retrovirus transduction to overexpress the suicide gene CD-UPRT. The mRNA-CD-UPRT was incorporated into the secreted EVs and induced cell death in the presence of prodrug 5-FC upon internalization by tumour cells ^[9][97].

Therapeutic siRs have also been delivered using MSC-EVs. For example, MSC(M)-derived EVs were electroporated with siRs targeting oncogenic Kras. The modified MSC-EVs induced the suppression of oncogenic Kras and increased the survival of several mouse models with pancreatic cancer ^[25][113]. Similarly, serine/threonine protein kinase (PLK-1)-targeting siRs were electroporated into MSC(M)-derived EVs. siR-PLK-1-carrying EV delivery to bladder cancer cells resulted in the suppression of PLK-1 and contributed to cell cycle arrest and apoptosis ^[26][114]. In a different context, MSC-EVs were loaded with a siR that silences the expression of phosphatase and tensin homolog (PTEN), which is one of the major intrinsic impediments to axonal growth, aiming at improving the regenerative ability of neurons after SCI ^[29][117]. Moreover, Huang and colleagues demonstrated that siRs targeting the connective tissue growth factor (CTGF) encapsulated in MSC-EVs also has a positive effect on functional recovery after SCI ^[23][111].

Synthetic miR mimics have also been exogenously encapsulated into MSC-EVs. For instance, MSC-EVs were electroporated with the miR-132 that targets RASA1, an essential negative regulator of vascular sprouting and vessel branching. The bioengineered EVs promoted angiogenesis in vitro and enhanced neovascularization and preserved heart functions in an in vivo myocardial infarction (MI) mouse model ^[20][108]. In a study by Jing et al., MSC-EVs harbouring miR-499a-5p inhibited endometrial tumour growth and angiogenesis in vitro and in vivo, by directly targeting to upregulated gene VAV3 ^[21][109]. Using a different strategy, Wang and collaborators exogenously loaded EVs secreted by human WJ-derived MSCs (MSC(WJ)) with a miR mimic using a commercial transfection reagent. miR-326-carrying MSC-EVs suppressed the activation of the NF-κB signalling pathway and the reduced expression levels of neddylation-related enzyme molecules, inhibiting the synthesis and production of related inflammatory factors and relieving dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) in a mouse model, compared to unmodified MSC-EVs ^[30][118].

Protein loading into EVs was also investigated by the genetic manipulation of parental MSCs. For example, serine/threonine kinase Akt, which plays an important role in promoting cell proliferation and inhibiting cell apoptosis, was transduced into human MSC(WJ) using an adenovirus system. Western blot semi-quantification revealed that the produced EVs harboured significantly higher levels of Akt than the control EVs. The produced EVs harboured higher levels of Akt and demonstrated increased angiogenic effects in vitro and in vivo and promoted superior cardiac regeneration in an acute MI mouse model, compared to control EVs ^[31][119]. Similarly, angiopoietin-2 (Ang-2) loaded into MSC(WJ)-derived EVs through its lentiviral-based overexpression by parental cells. Ang-2-carrying EVs enhanced angiogenesis and accelerated cutaneous wound healing in vivo ^[32][120]. Moreover, EVs secreted by pigment epithelium-derived factor (PEDF)-overexpressing MSC(AT), were shown to ameliorate cerebral ischemia–reperfusion injury in an in vivo rat model by activating autophagy and suppressing neuronal apoptosis ^[34][122]. Furthermore, MSC(M) were transduced to overexpress osteoactivin. The produced MSC-EVs stimulated the proliferation and osteogenic differentiation of MSC(M) via the activation of Wnt/β-catenin signalling and promoted bone regeneration in an ovariectomized rat model of postmenopausal osteoporosis (OP) ^[33][121]. Using exogenous loading, Rajendran and colleagues encapsulated tyrosine kinase inhibitor (TKI) into EVs produced by human MSC(AT) by direct incubation or sonication. Sonicated TKI-EVs enhanced iodine avidity in radioactive iodine-refractory thyroid cancer compared with free-TKI treatment ^[46][133].

MSC-EVs have proven to be efficient delivery vehicles for small anti-cancer drugs. For example, MSCs incubated with PTX have been shown to secrete EVs presenting a high drug concentration as quantified by high-performance liquid chromatography (HPLC) analysis. PTX-loaded EVs and induced a dose-dependent inhibition of human pancreatic adenocarcinoma cell proliferation, reducing tumour growth by up to 50% ^[36][62]. Using an alternative approach, Kalimuthu et al. directly incorporated PTX into MSC-EVs by serial extrusion through 10-, 5- and 1-μm polycarbonate membrane filters. These vesicles demonstrated their significant therapeutic effects against breast cancer both in vitro and in vivo ^[43][130]. As a prospective approach to surpassing chemoresistance of the pancreatic ductal adenocarcinoma (PDAC), a combination therapy of gemcitabine monophosphate (GEMP) and PTX delivered by MSC-EVs was developed, using electroporation and sonication as loading methods, respectively ^[42][129]. Despite the low encapsulation efficiencies determined by HPLC (5.92% and 2.62% for GEMP and PTX, respectively), GEMP/PTX-loaded EVs showed a great anti-tumour efficacy in vitro and in vivo in a PDAC orthotopic mouse model ^[42][129]. Furthermore, the anti-cancer drug DOX was also successfully packed into MSC-EVs using different endogenous loading methods, including electroporation or dialysis ^[39][40][126,127]. UV–vis-spectroscopy-mediated quantification showed that electroporation yielded a higher DOX encapsulation efficiency with a maximum of 35% ^[40][127]. Other small anti-cancer drugs have been packed into MSC-EVs and exhibited improved therapeutic effects, including taxol (TXL) ^[37][124] and norcantharidin (NCTD) ^[41][128]. Another promising approach consists in loading other small molecule drugs into MSC-EVs to treat inflammation or tissue regeneration besides malignant tumours. For example, isolated MSC(M)-derived EVs were incubated with Cur to engineer EVs with anti-inflammatory properties. After administration into a mouse model of ischemic stroke, Cur-carrying EVs suppressed the inflammatory response and cellular apoptosis in the lesion region of an ischemic stroke mouse model more effectively than non-modified EVs or Cur alone ^[45][132].

Finally, MSC-EVs can be packed with synthetic NPs. For instance, magnetic NPs were incorporated into MSC-EVs, using an MSC-mediated assembly process. Essentially, MSCs were incubated with iron oxide NPs and the secreted EVs were loaded with the NPs. After injection and magnet guidance, the NP-harbouring EVs showed significantly enhanced accumulation at the site of injured skin, demonstrating a capacity to induce faster wound reduction with increased collagen deposition and high blood vessel density ^[35][123]. Similarly, in a study by Altanerova et al., MSC-EVs were loaded with Venofer, carbohydrate-coated ultrasmall superparamagnetic iron oxide nanoparticles (SPIONs), by incubating MSCs with a Venofer–heparin–protamine sulphate complex overnight. The secreted Venofer-carrying EVs were successfully internalized by the tumour cells and facilitated their ablation via cytotoxic hyperthermia by applying an alternating magnetic field ^[38][125]. Some studies have also reported the modification of cargo of MSC-EVs by hybrid membrane engineering strategies. Singh and colleagues assembled MSC(M)-derived EVs and liposomes containing polypyrrole (Ppy) NPs, using the freeze–thaw method. Ppy-NPs naturally possess electrical conductivity, which can promote nerve regeneration and ameliorate diabetic peripheral neuropathy (DPN). After intramuscular injection into a DPN mouse model, Ppy-NP-encapsulating hybrids in combination with electrical stimulation reduced the neural and muscular damage ^[44][131].

Apart from cargo modification, different bioengineering strategies have been used on MSC-EVs to functionalize their surface. Essentially, by genetic engineering of parental MSCs or direct chemical modification of isolated EVs, the surface of MSC-EVs has been manipulated to enhance their therapeutic properties and improve target selectivity, aiming to develop potent targeted therapies with reduced adverse effects (Table 2).

The conjugation of peptides on the surface of MSC-EVs has been shown to improve their targeting towards particular organs or tissues, demonstrating prospective effects in the treatment of different types of cancer, heart and brain diseases. Many researchers have been using genetic engineering to generate recombinant peptides that are displayed on the surface of MSC-EVs, usually by fusing a targeting ligand to an EV membrane-enriched peptide/protein. Envisioning the targeted delivery of drugs to the brain, Yang and colleagues developed neuron-specific targeting EVs by engineering MSCs to overexpress Lamp2b fused with RVG. After systemic administration into a mouse model of cortical ischemia, RVG-displaying MSC-EVs efficiently deliver the exogenously loaded miR-124 to the ischemic region and ameliorate brain injury by promoting neurogenesis ^[19][107]. Similarly, MSC(M) were transduced to overexpress Lamp2b fused with ischemic myocardium-targeting peptide (IMTP) CSTSMLKAC and produce cardiac-cell-targeting EVs. Intravenously injected IMTP-displaying EVs showed enhanced accumulation in the MI region and significantly increased capillary density, inhibited inflammatory response, reduced infarct size and preserved cardiac function, compared to naked EVs ^[50][137]. Alternatively, in a study by Wang et al., a peptide targeting cardiac troponin I (cTnI), which is highly expressed in the MI, was used as an EV membrane-displaying ligand for the targeted delivery of miR-590-3p to the ischemic area. The MSC-EVs decorated with the cTnI-targeting peptide effectively accumulated in the infarct area along the cTnI concentration gradient ^[22][110]. Gomari and collaborators improved the efficiency of MSC-EVs for targeted anti-cancer drug delivery by transducing the parental cells with a lentivirus encoding Lamp2b fused with HER2-specific designed ankyrin repeat protein (DARPin), which are synthetic peptides with high binding affinity and specificity to their target protein. The engineered EVs were preferentially uptaken by HER2-overexpressing breast cancer cells compared to normal cells, effectively delivering DOX and siR molecules ^[47][134].

Surface modification of MSC-EVs can be used not only to improve targeting but to introduce an additional therapeutic moiety. For instance, tumour necrosis factor (TNF-α)-related apoptosis-inducing ligand (TRAIL) is a widely studied anti-cancer agent that selectively triggers an extrinsic apoptotic pathway in malignant cells ^[63][149]. In this context, Yuan and colleagues found that EVs secreted by genetically engineered TRAIL-expressing MSCs selectively induced apoptosis in eleven cancer cell lines and were able to partially overcome TRAIL resistance in cancer cells ^[53][140]. In a study by Zhang et al., MSCs were transfected to overexpress a plasmid encoding fusion protein of cell-penetrating peptides (CPP) and TNF-α which resulted in the secretion of EVs with TNF-α anchored in the membrane. Compared to unmodified EVs, TNF-α-EVs significantly enhanced tumour cell growth inhibition through induction of the TNFR-I-mediated apoptotic pathway in vitro and in vivo ^[52][139]. Xu et al. proposed a platform for the treatment of autoimmune disease by developing activated immune-cell-specific targeting EVs. For that, MSCs were modified to overexpress programmed cell death ligand 1 (PD-L1), whose receptor is highly expressed in autoimmune pathological tissues and involved in the signalling pathway of inhibition of immune responses and preservation of immune homeostasis. The PD-L1-expressing MSC-EVs were recognized by various activated immune cells including T cells, macrophages and dendritic cells with high expression of PD-L1 receptor, in a DSS-induced colitis mouse model. Additionally, the engineered EVs restored tissue lesions by reconfiguring the local immune microenvironment ^[51][138]. Moreover, MSC(AT)s were engineered with lentivirus encoding interleukin 2 (IL-2), a cytokine that stimulates anti-cancer immunity, for its EV-mediated delivery, aiming to reduce systemic toxicity. IL-2-EVs were able to activate human CD8⁺ cytotoxic T cells, which effectively killed human triple-negative breast cancer cells; however, these failed to suppress the proliferation of human peripheral blood mononuclear cells (PBMCs) ^[48][135]. In a study by Conceição et al., MSCs were engineered to produce EVs displaying pro-inflammatory cytokine interleukin 6 signal transducer (IL-6ST) decoy receptors at their surface to selectively inhibit the IL-6 trans-signalling pathway, a specific mediator in chronic inflammatory responses, while not interfering with the classical signalling properties of this cytokine. IL6ST decoy receptor-decorated EVs demonstrated their decoy activity by inducing a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of a Duchenne muscular dystrophy mouse model ^[49][136].

Chemical engineering has been widely investigated in incorporating targeting moieties into the surface of MSC-EVs, including peptides, RNA/DNA aptamers and drugs. For example, Zhang et al. conjugated the surface of MSC-EVs with the c(RGDyK) peptide, known to target the ischemic brain by binding to integrin α_vβ₃ in reactive cerebral vascular endothelial cells, using bio-orthogonal copper-free click chemistry. Essentially, the reactive dibenzylcyclootyne-conjugated EVs formed a covalent bond with an azide group on the lysine of the c(RGDyK) peptide. After intravenous administration into a mouse model of ischemic stroke, the engineered EVs successfully targeted lesions within ischemic brain tissue ^[45][132]. This strategy allowed the accumulation of EV-loaded cholesterol-modified miR-210 in the lesion region and promoted microvascular angiogenesis ^[28][116]. In another study, MSC-EVs were chemically functionalized via a reaction between an aptamer-conjugated aldehyde and the amino group of EV-membrane proteins. Basically, the surface of EVs was conjugated with an MSC(M)-specific RNA aptamer to improve BM targeting. After intravenous injection, the engineered EVs successfully targeted the BM and promoted bone regeneration in OP and femur fracture mouse models, in contrast to non-functionalized EVs, which accumulated in the liver and lungs ^[54][141]. Similarly, Bagheri and colleagues engineered the surface of MSC(M)-derived EVs with the 5TR1 DNA aptamer that has a high affinity with MUC1, a transmembrane mucin glycoprotein overexpressed in different types of cancer cells. Click chemistry led to the formation of a covalent bond between carboxylate-modified 5TR1 aptamer and the amine group on the surface of EVs. After intravenous injection into a mouse model of colon adenocarcinoma, the 5TR1-aptamer-EVs exhibited higher tumour accumulation and faster liver clearance in comparison with unmodified EVs ^[40][127]. Using the same reaction, Shamili et al. conjugated MSC-EVs with the LJM-3064 DNA aptamer which has a strong affinity toward myelin, and demonstrated remyelination induction, aiming to establish a novel approach for managing multiple sclerosis (MS). LJM-3064-aptamer-EVs showed a higher affinity for the myelin basic protein-producing cells in vitro, and synergistically induced immunomodulatory and remyelination effects in the experimental mouse model of MS ^[56][143]. To overcome the immunosuppressive tumour microenvironment of PDAC, an EV-based dual DDS of siR-galectin-9 was developed to block the galectin-9/dectin-1 axis and reverse immunosuppression caused by tumour-associated macrophages, and prodrug oxaliplatin (OXA), to act as immunogenic cell death trigger and kill the tumour cells by inhibiting DNA synthesis and repair. After exogenous loading of siR-galectin-9, OXA was added to the MSC-EVs obtaining a stable maleimide–thiol conjugate through vortexing ^[24][112]. MSC-EVs cancer-targeted delivery can also be achieved with magnetism. For example, SPIONs were conjugated with transferrin (Tf) using click chemistry. Afterwards, Tf-SPIONs were assembled to the surface of MSC-EVs by transferrin–transferrin receptor-mediated interaction. The engineered EVs were used for cancer-targeted delivery of TNF-α (described above), under an external magnetic field in a mouse model of melanoma subcutaneous cancer ^[52][139].

Another chemical strategy used to engineer the surface of MSC-EVs has been lipid assembly. For instance, Gangadaran and colleagues functionalized the surface of MSC-EVs with a peptide that targets interleukin-4 receptor (IL-4R), which is upregulated in various types of tumours, using a membrane phospholipid-based linker composed of dioleylphosphatidylethanolamine (DOPE), methoxy PEG and succinyl-N-hydroxy-succinimidyl (NHS) ester. The IL-4R-targeting peptide EVs induced a faster internalization into human anaplastic thyroid cancer cells in vitro compared to EV displaying a control peptide. Additionally, engineered EVs were shown to efficiently target tumours in a xenograft mouse model, in contrast to control EVs that are predominantly localized in the liver and spleen ^[55][142]. Using a similar strategy, MSC(M)-derived EVs were conjugated with the RVG peptide using a DOPE-NHS linker. The RVG-displaying EVs enhanced their binding to the cortex and hippocampus upon intravenous administration in a mouse model of AD, ameliorating spatial learning and memory impairments ^[57][144]. In a study by Cui et al., bone-targeting EVs were developed through conjugation with the peptide SDSSD modified with a diacyl lipid tail via hydrophobic insertion. The peptide-displaying EVs specifically delivered the exogenously loaded siR targeting schnurri-3 (SHN3) to osteoblasts and bone-forming surfaces via SDSSD/periostin interactions ^[27][115].

Feng and colleagues engineered MSC-EVs with a positively charged surface by simple incubation with a novel cationic amphiphilic macromolecule ε-polylysine (εPL)-PEG-DSPE, in order to enhance EVs intra-articular bioavailability in OA therapy. In contrast with unmodified EVs, electropositive MSC-EVs demonstrated increased chondrocyte uptake and retention ability in cartilage, leading to an enhanced OA treatment ^[58][145].

Some studies have also modified the surface of MSC-EVs by engineering hybrid nanocarriers. For instance, the PEG-mediated fusion of MSC-EVs with functionalized liposomes with various liposome-to-EV ratios has allowed the manipulation of the EV membrane properties, namely cellular uptake. In fact, PEGylated liposome–EV hybrids enabled a lower internalization by macrophages in situ ^[61][84]. In another study, membrane MSC-EVs were fused with platelet membrane fractions in the presence of PEG, in order to enhance their accumulation in injured tissues. Compared to unmodified MSC-EVs, the cellular uptake of hybrid EVs was significantly enhanced in endothelial cells and cardiomyocytes, but not macrophages. Additionally, the hybrid EVs showed improved targeting to injured myocardium and enhanced therapeutic potency in a mouse model of MI ^[62][148]. Similarly, Zhang and colleagues generated monocyte-mimic–EV hybrids to improve the delivery efficiency of MSC-EVs to ischemic myocardium, by mimicking the recruitment feature of monocytes ^[60][147]. Moreover, Lee and collaborators fabricated EV hybrids by fusing the membrane of MSC(WJ) and macrophages through the serial extrusion of cells via microporous and nanoporous filters. The engineered hybrid EVs largely accumulated in the SCI area after the in vivo systemic injection, due to the increased levels of ischemic-region-targeting molecules compared to MSC-EVs ^[59][146].