ALI and ARDS are devastating and rapidly progressive respiratory disorders that are characterized by disruption of the integrity of alveolar and vascular endothelial barriers (34–36). In response to inflammatory stimuli, EVs and microparticles (MPs) are released from circulating inflammatory cells, damaged PVECs, and epithelial cells (37). In preclinical models, PVEC-derived EVs induce significant lung injury, as demonstrated by alveolar-capillary barrier failure, lung edema, and neutrophil infiltration in mice (38). These pathological effects are linked to and presumably at least in part mediated by the detrimental effects of PVEC-derived EVs on endothelial function. In ALI models, PVEC-derived EVs induce a reduction in endothelial nitric oxide (NO) production and an increased release of lung inflammatory cytokines (39).

Alveolar macrophage derived EVs are also abundant in the bronchoalveolar lavage fluid (BALF) in animal models of ALI. They are capable of inducing inflammatory responses both in vivo and in vitro (37, 40–42). Alveolar macrophage derived EVs trigger EV release by epithelial cells and neutrophils and deliver high concentrations of TNF-α to alveolar epithelial cells, leading to increased production of keratinocyte-derived chemokine and intercellular adhesion molecule-1 (37, 40–42), inducing a vicious cycle of inflammatory injury.

Alveolar epithelial cell derived EVs are also important mediators of ALI. In hyperoxia-induced ALI, alveolar epithelial cell-derived EVs are increased in BALF and serum (43) and they activate proinflammatory responses in systemic and pulmonary macrophages leading to disease progression (44).

COPD is characterized by severe airway inflammation and subsequent lung parenchymal damage. Mononuclear/macrophage-derived EVs rich in inflammatory mediators such as cytokines, chemokines, adhesion molecules, and proteases have been linked to alveolar wall destruction and emphysema, the hallmarks of COPD (11, 45). Endothelial-derived microparticles can promote the progression of COPD by inducing apoptosis of neighboring health endothelial cells upon delivery of inflammatory cargo (46). Epithelial-derived EVs have also been linked to the pathogenesis of COPD. Cigarette smoke stimulates human bronchial epithelial cells to release EVs enriched in full-length CYR61/CTGF/NOV family 1 (CCN1) protein that not only mediates IL-18 induced inflammation but also helps maintain lung homeostasis by increasing the levels of vascular endothelial growth factor (VEGF) (47). Cigarette smoke extract-induced human bronchial epithelial cell-derived EVs promote myofibroblast differentiation of lung fibroblasts, leading to the development of fibrosis (48). Cigarette smoke-exposed lung epithelial cells also release EVs that contain pro-inflammatory cytokines and Wnt-5a into the circulation, and these EVs can reach distant cells and organs (49).

EVs are also implicated in the pathogenesis of pulmonary hypertension. Patients with pulmonary arterial hypertension (PAH) have increased endothelial-derived CD62e microparticles in their pulmonary arterial blood (50). PAH patients also have increased microparticles positive for endothelial PECAM and VE-cadherin in their plasma samples (51). In monocrotaline-induced PAH, lung- and plasma-derived small-sized EVs isolated from monocrotaline-exposed mice induce PAH in healthy mice (52). EVs from PAH mice and patients contain elevated levels of miR-19b, miR-20a, miR-20b, and miR-145, known to target bone morphogenesis protein receptor signaling, apoptosis, and cell proliferation. EVs from the lungs of PAH mice reduce apoptosis of PVECs (53). Furthermore, EVs released by PVECs from PAH mice convert healthy bone marrow-derived endothelial progenitor cells into a pathological progenitor phenotype. These cells induce pulmonary vascular remodeling when injected into the lungs of healthy mice (54).

BPD is the most common adverse outcome of extreme prematurity (55). It is the result of antenatal injury to the developing lung combined with repetitive and multiple post-natal insults, including oxygen therapy and ventilation, leading to alveolar simplification and vascular rarefaction (55). Not much is, however, known about the role of EVs in BPD pathogenesis. Genschmer and collaborators compared the function of EVs derived from BALF from BPD and non-BPD infants in a murine model (56). Intriguingly, mice that received intranasal BPD-derived EVs had significant alveolar hypoplasia and right ventricular hypertrophy, suggesting a role for EVs in BPD pathogenesis (56).

Recently, Lal et al. also demonstrated that the tracheal aspirate of infants with severe BPD had higher EV particle concentrations as compared to control infants, and the majority of these EVs were derived from epithelial cells (57). EVs shed from hyperoxia and LPS-exposed epithelial cells had reduced miR-876-3p. Gain of miR-876-3p in murine models attenuated hyperoxia and LPS-induced alveolar simplification, highlighting a potential critical role of lung epithelial cell-derived EV-miRNAs in the pathogenesis of BPD (57). miRNAs are non-coding RNAs that bind to sequences in the 3′ untranslated region (3′UTR) of target mRNA, resulting in the destruction of target mRNA or its repression (58).

Recently, our laboratory investigated the critical role of circulating EVs from hyperoxia-exposed and mechanical ventilated newborn rats in inducing brain injury in healthy newborn rats (59, 60). In the hyperoxia model, newborn rats were exposed to room air or 85% oxygen for two weeks, and circulating EVs were isolated from the plasma of these rats. Fluorescence activated cell sorting (FACS) and Western blot analyses demonstrated that the EVs from hyperoxia-exposed rats contain increased levels of both surfactant C (SPC) and gasdermin D (GSDMD), a key executor of inflammasome-induced cell pyroptosis. When these EVs were adoptively transferred into healthy newborn rats by intra-tail vein injection, they were taken up by the lung and brain. In the lung, the EVs from the hyperoxia-exposed rats induced inflammation, indicated by increased inflammatory cell infiltration in the alveolar airspaces and expression of inflammatory cytokines and chemokines. Furthermore, alveolarization and vascular density were drastically reduced in the lungs that received EVs from hyperoxia-exposed rats. In vitro experiments with PVECs demonstrated reduced cell proliferation and increased cell death when cultured with EVs from hyperoxia-exposed rats (59). Upon examining the brain, EVs from hyperoxia-exposed rats induced brain inflammation by activating microglia and increasing expression of pro-inflammatory cytokines. These changes were associated with increased cell death in the cortex, subventricular zone, and subgranular zone. Additionally, in vitro experiments showed that neural stem cells (NSC) had decreased proliferation and increased cell death when cultured with EVs from hyperoxia-exposed rats (59). EVs from cultured hyperoxia-exposed lung epithelial cells induced pyroptosis in NSC (59). This data revealed a novel lung-brain crosstalk mediated by lung epithelial-derived EVs in both lung and brain injury.

This EV-mediated lung-brain crosstalk was further investigated in mechanical ventilation-associated brain injury in newborn rat models (60). We demonstrated that injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as IL-1β and activated caspase-1/GSDMD in both lung and brain, in addition to inducing microglial activation and cell death in the brain (60). EVs isolated from neonatal rats with ventilator-induced lung injury had increased caspase-1. Adoptive transfer of these EVs into healthy newborn rats led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain, similar to that observed in neonatal rats that were mechanically ventilated (60). Thus, circulating EVs can contribute to brain injury and possibly poor neurodevelopmental outcomes in preterm infants exposed to hyperoxia and mechanical ventilation (60).

MSCs are efficacious in neonatal lung injury models (67–69). The pleiotropic properties of these cells make them particularly attractive and given their paracrine-mediated mechanism of action, MSC-derived EVs have been investigated as potential therapies.

In an experimental model of chorioamnionitis, antenatal administration of MSC-EVs reduced placental inflammation, and preserved lung structure, suggesting that antenatal MSC-EVs are efficacious in alleviating the deleterious effects of intrauterine inflammation. In experimental pre-eclampsia, MSC-EVs restore placental vascularity and preserve neonatal lung structure (70). In experimental BPD models, MSC-derived EVs restore alveolar structure, prevent lung vascular rarefaction, and alleviate PH by altering macrophage polarization, reprogramming bone marrow myeloid cells and increasing pro-angiogenic signaling pathways (70–77).

We recently compared the therapeutic efficacy of intra-tracheal (IT) and intravenously (IV) delivered MSC-EVs in a preclinical model of BPD. We demonstrated that systemically and IT delivered MSC-EVs have similar beneficial effects in experimental BPD (78). This finding is promising as IV MSC-EVs may also have beneficial effects on the developing brain (79). Another important question which we recently sought to address is the duration of MSC-EV therapeutic effects in experimental BPD. We administered MSC-EVs to neonatal pups with hyperoxia-induced BPD on postnatal day 3 and followed the pups into young adulthood (78). We found that one dose of MSC-EVs at postnatal day 3 had persistent beneficial effects at three month follow up (78). Importantly, late administration of MSC-EVs in an established BPD model was also found to partially reverse lung injury (79, 80). Clinical trials are now on the horizon but identifying the ideal patient will be critical.

EVs are also being investigated as “drug vehicles” (81). The ability of EVs to target a particular tissue or cell could be used to deliver drugs to intended targets while avoiding off-targets selectively (81). The “drug cargo” is selectively loaded into the EVs and the EVs are engineered to have specific properties to enhance their targeting and biomimetic features (82, 83). The lower number of transmembrane proteins, such as MHC complexes on their surface, make EVs less immunogenic than their parental source (84, 85). In addition, EVs do not replicate after injection. Thus, EVs are less likely to transfer latent viral pathogens or enable tumor generation (86). Compared to synthetic drug carriers, the intrinsic ability of EVs to cross cell barriers and penetrate tissues gives them an advantage (87). Synthetic drug carriers such as polymeric micelles and lipid nanoparticles cause high toxicity and immunogenicity compared to EVs (88). As therapeutic EVs are derived from benign biological or autologous sources, they are less likely to induce adverse effects.

Harnessing these unique properties of EVs to develop smart drug delivery systems with enhanced targeting, safety and pharmacokinetics has however been challenging (89). One study showed that that after intravenous injection, EVs are rapidly distributed and retained in the liver, spleen, gastrointestinal tract and lungs (90). Another study however showed rapid clearance of plasma-derived EVs following intravenous administration, with a half-life of approximately 7 min (91). Moving forward, more studies will be needed to understand EV circulation kinetics, biodistribution, cell tropism, and intracellular trafficking routes as the cellular origin, dose and route of administration may affect EV biodistribution pattern (92).

Other obstacles such as low isolation yield, the lack of purification protocols, large-scale clinical grade production, parental cell-dependent composition, and inefficient drug payload of the EVs continue to hamper the therapeutic ability of EVs (93). To improve de novo EV yield and therapeutic efficacy, re-engineering of the parental cell has been done through genome modification, stimulation with exogenous biomolecules and specific environmental factors (93). Bioreactors are also being extensively used to scale up the production of cell-based therapy and EVs. Bioreactors provide well-controlled nutrients, uniform culture conditions and biomimetic stimuli to regulate cell growth, differentiation and tissue development (94). While bioengineering of the parental cell predictably loads only a small proportion of the modified content into EVs, direct modification of isolated EVs may be another strategy to enrich EVs (95). For example, hydrophobically modified small interfering RNAs efficiently load into EVs upon coincubation, without altering EV size or integrity (96). Active EV loading can also be done by electroporation, sonication, extrusion, freeze-thawing and by surfactant-assisted loading, where surfactant saponin disrupts the membrane and increases its permeability (97).

Another option currently being investigated is the development of artificial EVs, namely the top-down and bottom-up approaches. The top-down approach is based on the disruption of the cultured cells to produce membrane fragments that will be used to form vesicles, while retaining the same membrane features of the initial cell (98). The bottom-up approach starts from small components of molecular building blocks to create complex structures, namely synthetic EVs (99).