Effect analysis of extracellular vesicles in the treatment of bronchop

Introduction

Bronchopulmonary dysplasia (BPD) is a chronic lung disease primarily affecting preterm infants, historically associated with mechanical ventilation and oxygen therapy, underlying a long process of definitions.^1–10 Current studies on the molecular mechanisms of BPD have proposed endotype-based classifications on account of transcriptomic information (Supplementary Figure 1).¹¹ Prematurity, small for gestational age (SGA), and low birth weight (LBW), mechanical ventilation and oxygen therapy, chorioamnionitis, pregnancy-induced hypertension (PIH), genetic susceptibility, inflammation and immune response, patent ductus arteriosus (PDA), intraventricular hemorrhage (IVH)-III, inadequate nutrition, and maternal smoking are common risk factors for BPD.^12–19 However, the pathophysiology of BPD remains complex and not fully understood. Besides initial lung impairment, infants with BPD may face various clinical consequences, including pulmonary hypertension (PH), right ventricular hypertrophy (RVH), cerebral palsy, psychological disorders, and chronic pulmonary diseases like chronic obstructive pulmonary disease (COPD) (Figure 1).^20–23 Infants with BPD are often received pulmonary surfactant, caffeine, diuretics, bronchodilators, inhaled steroids to alleviate symptoms.^24,25 Thanks to current treatment of postnatal corticosteroids, less invasive surfactant administration, early non-invasive respiratory support in the delivery room, and early use of caffeine, BPD-related death or disability in preterm infants has been reduced significantly.^26–29 While vitamin A supplementation and early caffeine administration may reduce the incidence of BPD in preterm infants, therapeutic options remain limited, and further researches are needed to develop more effective and safer interventions.³⁰ The incidence of preterm infants with BPD resulting from differences in gestational age (GA) or birth weight varies from 2.4% to 71.6% in recent decades.^31–33 Preterm infants troubled by BPD often impose high financial burden on families, communities, and healthcare systems in addition to substantial morbidity. There is an urgent need for novel therapies to improve outcomes for infants with BPD.^34,35 Dini et al reported that a multiple strategy combining early non-invasive respiratory support, evidence-based pharmacologic interventions like caffeine and vitamin A, and cautious use of corticosteroids may be necessary for the effective prevention of BPD in extremely preterm infants. Stem cell-based therapy represents an emerging approach under BPD investigation.³⁶

Figure 1 Overview of major risk factors, pathophysiological mechanisms, and frequent clinical consequences of BPD. Abbreviations: PIH, pregnancy-induced hypertension; SGA, small gestational age; LBW, low birth weight; PDA, patent ductus arteriosus; IVH, intraventricular hemorrhage; COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; IDD, intellectual developmental delay; ADHD, attention deficit and hyperactivity disorder; AKI, acute kidney injury; CKD, chronic kidney disease; eGFR, glomerular filtration rate; ROP, retinopathy of prematurity; CVI, cortical visual impairment; NEC, necrotizing enterocolitis of the newborn.

Mesenchymal stem cells (MSCs) indicate potentiality as a therapeutic option for infants with BPD due to their intrinsic capacity for self-renewal, high efficiency in cell generation, and ability to replace terminally differentiated cells.³⁷ MSCs, a broad spectrum of cells derived primarily from umbilical cord, bone marrow, and adipose tissue, have significant regenerative properties, including anti-inflammatory and immunomodulatory effects, enabling allogeneic transplantation. However, there are always challenges in the direct application of human umbilical cord mesenchymal stem cells (hUC-MSCs) therapy, including ethical debates regarding MSC origins, standardization, normalization of MSC culture, high dose intravenous microvascular occlusion, tumorigenicity, and pathogen transport.^38–40 Following these difficulties, MSC-derived EVs may offer a safer therapeutic alternative for infants with BPD.

EVs are nanoscale particles secreted by all types of cells, facilitating intercellular communication through the transfer of proteins, lipids, and RNA. They have been implicated in the pathophysiology of neonatal lung disease and are increasingly recognized as a novel mechanism of cell-to-cell signaling.^41,42 Recent study has demonstrated that macrophage-derived EVs transfer monocyte-activating polypeptide-II (EMAP-II) to neutrophils, activating the PI3K/AKT/mtROS pathway and causing mitochondrial oxidative stress, which in turn promotes the formation of neutrophil extracellular traps (NETs) in lung ischemia/reperfusion injury (LIRI) pathogenesis.⁴³ Nevertheless, EVs derived from septic acute lung injury (ALI) exacerbate pulmonary inflammation by delivering miR-128-3p to macrophages, which inhibits Rab20 to promote M1 polarization, proliferation, and the production of pro-inflammatory cytokines of TNF-α/IL-6.⁴⁴ Ruan et al confirmed that human menstrual blood-derived endometrial stem cells EVs protected alveolar barrier integrity by inhibiting MAPK-mediated necroptosis, thereby attenuating lung injury and restoring epithelial permeability in ALI/ARDS.⁴⁵ Additionally, EVs influence immunomodulation by transfering miR-223-3p to suppress circPWWP2A, through NLRP3 signaling pathway, via promoting barrier integrity, reducing inflammation, then alleviating PF.⁴⁶ Furthermore, proteomic analyses of EVs verified that, through distinct expressed gene enrichment, strong and intermittent hyperoxia induced different biological processes, including an inflammatory response.⁴⁷ Due to their lower risk of transplant-related side effects, EVs are a desirable alternative for clinical use in comparison to direct MSCs transplantation.^48–50 EVs obtained from MSCs maintain comparable biological properties, such as anti-inflammatory and regenerative capabilities, but raise minimal safety concerns.^51–53 One type of corticosteroid used to treat BPD, dexamethasone, only inhibits inflammation while posing a risk of neurotoxicity, in contrast, EVs simultaneously target antioxidant, inflammatory, lung development, and therapeutic intervention.⁵⁴ Intra-tracheal administration of EVs derived from amniotic fluid could not only achieved lung-specific delivery, but also promoted pulmonary alveolar development, reduced pulmonary hypertension, and inflammation, which exhibited potential translational therapeutics for BPD.⁵⁵ As for extended durability effects, Sharma et al found that EVs offered long-lasting protections for over three months with a single dose, while sildenafil required repeated doses due to a short-acting medication.⁵⁶ EVs also have the potential to address important pathological aspects of BPD, as evidenced by studies showing that they improved alveolarization, stimulated angiogenesis, reduced inflammation and oxidative stress damage.^57,58 Research on a variety of delivery techniques, including aerosol inhalation, intraperitoneal injection, intratracheal or intravenous administration, has shown that the best possible drug delivery is essential for optimizing therapeutic efficacy while reducing the likelihood of adverse effects.^59,60 Here we review the different methods of EVs delivery in the treatment of BPD and verify efficacy before these therapies being implemented clinically.

Characterizations of EVs

EVs are delimited by a lipid bilayer and secreted by various types of cells, that do not self-replicate. EVs typically carry transmembrane proteins such as CD63, CD81, and CD9, structures that may be co-isolated from the cytosol. It was optional to use secreted proteins with EVs or lipid-bound soluble proteins with intracellular compartments or other endosomes, without source-specific, co-isolates contaminants. Isolation, identification and mechanism analysis of EVs for BPD therapy experienced a complicated process for decades. EVs were initially isolated from fresh plasma after removal of intact platelets via ultracentrifugation by Wolf P. in 1967, extruded on platelets storage, and identified as “platelet-dust”.⁶¹ Firstly named by Trams, E. in 1981, “exosomes (Exos)” were separated from exfoliated membrane vesicles and suggested to be carriers of information transfer between cells with a wide range of functions in vivo.⁶² According to Lambertsen, R., hematopoietic stem cells (HSC) use cytoplasmic processes to interact with the mature macrophages’ cell membrane. The coupling structure that results can be altered by ingesting or releasing small cell cytoplasmic vesicles.⁶³ Exos were also involved in forming during reticulocyte maturation and plasma membrane remodeling and are widely found in mammals and birds. The release of Exos was preceded by intracellular polyvesicles.⁶⁴ As for characterizations, Sokolova V. verified that nanoparticle tracking analysis (NTA) and scanning electron microscope (SEM) were effective complementary technique for evaluating Exos morphology and size. Ultracentrifugation does not alter the size of Exos, while storage conditions may largely affect their integrity and particle size.⁶⁵ Welsh et al updated MISEV 2023, giving a detailed explanation of the definition, origin, protein composition, analysis and topology of EVs (Table 1).⁴¹ The essential milestones in EVs during the last 50 years were illustrated (Figure 2).

Table 1 Recommendations of MISEV 2023 for EVs Characterization

Figure 2 Timeline of EVs milestone studies. Abbreviations: HSC, hematopoietic stem cell; sEVs, small extracellular vesicles; HILI, hyperoxygen-induced lung injury; MISEV, minimum information for studies of extracellular vesicles.

Therapeutic Mechanisms of EVs in BPD

Willis G. suggested that extracellular vesicles may protect against hyperoxia-induced neonatal BPD by delivering miRNA, or proteins that regulate the expression of target genes. These proteins can lower inflammatory factors tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion, lower inflammation and pulmonary fibrosis (PF), enhance alveolar development, and control macrophage planning to the anti-inflammatory M2 state.⁶⁶ Small extracellular vesicles (sEVs) obtained from MSCs improved experimental BPD in vivo via partial restoration of pulmonary structure and improving motor function.⁶⁷ Willis G. additionally confirmed that mesenchymal stem cell-derived small extracellular vesicles altered monocytes’ immunophenotype through epigenetic reprogramming and enhanced their immunosuppressive capacity. Additionally, it improved lung structure, reduces PF, and improves vascular remodeling by acting on bone marrow-derived myeloid cells (BMMC).⁶⁸ In recent years, EVs from mesenchymal stem cells have been extensively studied in treating BPD. Researches also demonstrated that EVs were crucial for both the positive and negative control of pulmonary injury induced by hyperoxia.^69,70

Sources, Isolations, Purifications, Identifications and Pathophysiology of EVs

Exos, microvesicles, and apoptotic bodies are the three primary subtypes of EVs that are typically distinguished by their size, contents, transmembrane proteins, and segregation parameter. Exos, microvesicles, and apoptotic bodies were compared in detail, with each type’s benefits highlighted (Table 2).^41,71–75 Studies have successfully isolated EVs from various sources, including MSCs derived from human umbilical cords, placenta, bone marrow, adipose tissue, induced pluripotent stem cells (iPSCs),^76–80 and body fluids such as amniotic fluid, cerebrospinal fluid, blood, plasma, lymphatic fluid, urine, saliva, breast milk, and vitreous body.^81–87 Additionally, EVs have been identified in fungi, bacteria, and plants.^88–90 EVs play important roles in intercellular communication, immune regulation and pathological processes. EVs derived miRNAs have signaling functions and can regulate gene expression in target cells, and specific proteins (such as SUMOylated hnRNPA2B1 and YBX-1) regulate the selective encapsulation of RNA. In addition, non-coding RNA may influence immune responses through cross-species regulations.⁹¹ Bioinformatic analyses and nanopore sequencing revealed that the transcriptome sorting mechanisms of microvesicles and Exos differed and that different EV subtypes from the same origin have different physiological functions, suggesting different clinical translation opportunities.⁹² Originated in the endosomal system, Exos are released by multivesicular bodies that range in size from 30 to 150 nm.⁹³ Heterogeneous Exos are secreted by a large range of cell types, and classified into different subtypes due to locations and functions.⁹⁴ MSCs are known for their characterizations of improving alveolar structure and angiogenesis, and suppressing inflammatory responses for treating BPD.⁹⁵ Although the effective therapy of MSCs in BPD are proven in several trials, safety concerns such as malignant transformation, aberrant osteochondral differentiation, vascular occlusion via intravenous injection, or the restricted rates of survival and differentiation occurred, which also limit their direct application in clinical trials.^96–99 Compared to MSCs, EVs have aroused huge attention for treating BPD by paracrine, non-cellular methods. The primary pathophysiological effects of EVs include reducing inflammation, apoptosis, and PF, as well as promoting the immune response, pulmonary repair, and vascular generation.

Table 2 Comparison Characteristics of Extracellular Vesicles Such as Exosomes, Microvesicles and Apoptotic Bodies

Isolation and purification of EVs is essential for clinical translations in vivo. However, the small size of Exos has led to challenges in isolation and purification. An ideal approach has all the characteristics of high yield, high specificity, high purity, low costing, as well as preservation of natural biological functions. Multiple methods have been used to isolate and purify EVs from various biological samples. Studies have indicated heterogeneity based on technologies of EVs’ concentration, purification, size distribution, proteomic content, and source composition.^100,101 Each purification and isolation technique has benefits and drawbacks. Technologies that are widely used including precipitation techniques, density gradient ultracentrifugation, high-speed ultracentrifugation, ultrafiltration, sequential filtration, size-exclusion chromatography, polyethylene glycol (PEG) precipitation, size-exclusion chromatography, and affinity binding-based separation.^102,103 To date, differential ultracentrifugation is the most popular technique for isolating EVs, due to its simple protocol, relatively high yield, and specificity.¹⁰⁴ Nevertheless, ultracentrifugation may lead to mechanical or thermal damage to EVs, and spinning speeds are variable, which could alter their composition, structure, and cause instability of biological functions in the end.¹⁰⁵ As a consequence, advanced isolation techniques are used in EVs, such as microfluidic (chip) techniques, membrane-based separation, static water filtration dialysis, reagent kit extraction, heat shock protein binding, lectin, phosphatidylserine, and immune-affinity isolations.^106–111 Different techniques have their own pros and cons as for yield, specificity, purity, and costing. Origins of EVs, amount, costing of time and money, difficulty of operation, storage condition, and requirements of subsequent experiments are the critical reasons in choosing the most appropriate isolation and purification method. Unfortunately, no single method enables optimal isolation of EVs from all species. This could be resolved by combining two or more isolation techniques, such as combining traditional and advanced technologies, however, limitations still exist.

Identification of EVs is difficult by their varieties of heterogeneity and diversity, which can be attributed to differences in origin, size, morphology, transmembrane protein, and medicine cargo. At present, the ideal identification technology of EVs mainly based on morphology, particle size, protein contents, lipid contents, nucleic acid, biomarkers, purity and costing the least. At present, transmission electron microscopy (TEM) combined with immunogold labeling to verify double-stranded DNA presentation in the lumen has been recognized as a novel method to verify associated biomarkers of EVs.¹¹² Cryogenic transmission electron microscopy (cryo-TEM), and scanning electron microscopy (SEM) are both used for the morphological assessment for their advantages of maintaining the native state of EVs and high-resolution imaging.¹¹³ Electrophoretic light scattering (ELC), NTA, and flow cytometry can be applied for primary screening EVs.¹¹⁴ Nevertheless, more refined techniques such as resistive pulse sensing (RPS), tunable resistive pulse sensing (TRPS), tandem mass spectrometry (TMS), electrochemical sensing (ES), and interferonmetric reflectance imaging (IRI) might be helpful for a more comprehensive understanding of the morphological characteristics of EVs. Besides, Western blot is often used to verify typical transmembrane proteins with relatively low costing (Figure 3). In summary, combinations of these identification techniques might mitigate EVs heterogeneity to some extent, and recognize the presence of EVs further. This lay a crucial foundation for future research on functions and mechanisms of EVs in treating BPD.

Figure 3 Sources, isolations, purifications, identifications and pathophysiology of EVs. Abbreviations: EVs, extracellular vesicles; MSC, mesenchymal stem cell; iPSC, induced pluripotent stem cells.

Biogenesis, Release, and Cellular Uptake of EVs

Biogenesis and uptake of EVs is complicated, as a result of different cell origins, cell microenvironment homeostasis in physiological state, as well as external stimulus conditions.¹¹⁵ The biogenesis of Exos is composed of three consecutive stages. Initially, endocytosis of the plasma membrane forms endocytotic vesicles. This process initiates early endosomes forming afterwards. In the second step, early endosomes are inward budding of late endosomes, then develop into multivesicular bodies. Finally, most of the multivesicular bodies degrade, and a small part of the multivesicular bodies fuse with the plasma membrane and release Exos in the extracellular region.¹¹⁶ Microvesicles, which range in size from 100 to 1000 nm, are formulated by membrane buddings from plasma membrane of the donor cell into extracellular regions. Apoptotic bodies, whose size range from 1000 to 5000 nm, are mainly formed through membrane blebbing, starting from plasma membrane to extracellular regions.¹¹⁷ Once released, EVs are able to impact on extracellular environment to different extents, and act on target cells through four mechanisms. When reaching the receptor cells, EVs can either directly interact with the cells, be phagocytized by the plasma membrane, undergo macropinocytosis via plasma membrane invagination, or get internalized through receptor mediated endocytosis. Receptors involved in the internalization are mainly including clathrin-, caveolin-, lipid raft, and heparan sulfate proteoglycan (Figure 4). ^118,119 As EVs play essential roles in transporting biological information and intercellular communication, a deep understanding of vesicular–cellular interactions is of great value to explore the pathophysiological mechanisms of targeted vesicular transport systems. For example, a progressive trajectory of relative enrichment and early increase of CD24⁺ conveyed by EVs have been reported to be connected with developing BPD.¹²⁰ These EVs effectively transported CD24⁺ proteins to target inhibiting immune overresponse induced by damage-from pathogen-associated molecular patterns (DAMPs), blocking toll-like receptors (TLRs) and Siglec-10-mediated NF-κB signaling pathways, thereby effectively inhibiting cytokine storms and reducing inflammation in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mouse model.¹²¹

Figure 4 Biogenesis, release and cellular uptake of extracellular vesicles.

Registered Clinical Trials Involving EVs for Respiratory Diseases

The transition of EV-based therapies from preclinical research to clinical translation necessitates rigorous evaluation through well-designed clinical trials. To compile relevant clinical trials, a systematic search was conducted using publicly available clinical trial registries, which was registered in the National Institutes of Health (NIH) database during the previous five years.^{105,122–126} The search strategy incorporated keywords such as “extracellular vesicles”, “Exos”, “respiratory diseases”, and “clinical trials”. Inclusion criteria comprised trials that were actively recruiting, completed, and pending results, particularly in the field of respiratory diseases. Trials involving EVs derived from MSCs, human amniotic fluid (HAF), blood, urine, human embryonic kidney (HEK), alveolar lavage fluid (ALF), T-REx™-293 cells, human adipose derived mesenchymal progenitor cell (haMPC), corona virus disease-19 (COVID-19) Specific T cells and other sources were included, whereas studies focusing on unrelated conditions were excluded. Clinical trials were stratified according to respiratory disease types, EV sources, mode of administration, age, trial phases, study type, distribution countries, and start date. The collected information was systematically analyzed to assess the current landscape of EV-based clinical research in respiratory diseases. To date, 28 clinical studies have been registered using EVs to treat respiratory diseases. Analysis of registered clinical trials revealed a growing interest in EV-based therapies for respiratory diseases, and the majority of these trials focused on conditions including coronavirus disease 2019 (COVID-19), ARDS, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pneumonia, and BPD (Table 3). Only one clinical study numbered NCT06279741, investigated the potential of MSC-derived EVs on BPD, while most of them are under the recruiting stage. Clinical trials have investigated diverse EV delivery methods to screen treatment for infants, including inhalation, intravenous injection, endotracheo-pulmonary instillation and diagnostic tests. While intravenous delivery facilitates systemic distribution, inhalation has been shown accumulated in lung tissues with optimizing therapeutic efficacy. Most extracellular vesicles are derived from mesenchymal stem cells and body fluids, including urine, blood, bronchoalveolar lavage fluid, and amniotic fluid. Geographically, clinical trials were predominantly concentrated in the United States, China, Israel, Russian Federation, and Europe (Figure 5). EVs therapeutics may offer distinct advantages over stem-cell therapies, such as lower risks of immune rejection and tumorigenicity, easier storage and administration. Compared to genetic therapy, EVs can provide a natural method of delivering therapeutic biomolecules without genetic modification, and may minimize ethical and safety concerns. However, there are still difficulties in determining the best dosage schedules, guaranteeing EV stability, and reducing possible immunogenicity.

Table 3 Registered Clinical Trials Involving EVs for Respiratory Diseases

Figure 5 The whole 28 ongoing and completed clinical trials using EVs for respiratory diseases. The number of clinical trials related to each section is exhibited inside the boxes. (A) Clinical trial status introduced by clinicaltrials website. (B) Clinical trial phase introduced by clinicaltrials website. (C) Route of administration of EVs by clinicaltrials website. (D) Source of EVs applied in each clinical trial by clinicaltrials website. (D) Countries of EVs involved in clinical trial by clinicaltrials website. Abbreviations: Recr., recruiting; N.D., not determined; EI, endotracheopulmonary instillation; ALF, alveolar lavage fluid; HAF, human amniotic fluid; HEK, human embryonic kidney cells, CSTC, COVID-19 specific T cells.

Mechanisms Analysis of Different Administration Methods, Dosages and Frequencies of Extracellular Vesicles in Treating BPD Models in Vivo

Animal experiments have revealed that EVs in BPD treatment is strongly influenced by administration method, dosage, and frequency. EVs used in BPD treatment are derived from various sources, including hUC-MSCs, BM-MSCs, AD-MSCs, as well as iPSCs. These EVs contain bioactive molecules such as microRNAs, proteins, and lipids that play crucial roles in modulating lung inflammation, apoptosis, and vascular remodeling. EVs can be delivered through multiple routes, including intratracheal mode (IT), intravenous mode (IV), intra-amniotic mode (IA), and intra-oral mode (IO). A detailed comparison of IT, IV, IA, and IO administration route is provided, alongside an evaluation of the molecular mechanisms modulated by EV therapy in BPD (Figure 6). Each method presents unique advantages and limitations in terms of targeting efficiency, systemic effects, and cellular uptake. Different administration routes are probably related to different mechanisms in BPD treatment. IT injection is widely used in preclinical BPD models primarily considering it rapid delivery into lungs, local therapeutic effects, and quick onset effects. IT administrated EVs promote vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-alpha (HIF-1α) activation, enhancing pulmonary capillary formation and alveolarization. Meanwhile, NF-κB and transforming growth factor beta (TGF-β) pathway modulation decreases pro-inflammatory cytokines and inhibits apoptosis, thereby protecting alveolar epithelial cells.¹¹⁵ IT administrated EVs attenuate fibrosis and protect against oxidative stress by reducing collagen deposition, enhancing mitochondrial integrity, and decreasing DNA oxidative damage.¹²⁷ Certain EV-derived microRNA, like miR-21-5p, target S-phase kinase-associated protein 2 (SKP2) and mediate cytoprotection by reducing inflammation, oxidative stress, and apoptosis through the SKP2/Nr2f2/CEBPα axis via intratracheal administration.⁶⁹ IT-administrated EVs increase anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, improving lung structure, enhancing neural progenitor cell (NPC) self-renewal, and modulating of Wnt/Crabp2 signaling.¹²⁸ EVs delivered via the IT shift immune cells toward an anti-inflammatory state by reprogramming gene activity.¹²⁹ Additionally, IT-administrated EVs also decrease right RVH, promote angiogenesis, alter pro-angiogenic genes such as enos, cxcr4, and restore alveolar structure.⁵⁶ Apart from these advantages, IT administration also have some disadvantages, such as invasive procedure, potential airway irritation, and risks of uneven distribution in the lung, which has impeded its clinical translation to some extent. Preclinical models demonstrated the therapeutic effects of IV-administrated EVs are preponderant as non-invasive compared to IT, systematic effects beneficial for multiple organs, and compatible with neonatal intravenous therapy. IV-administrated EVs activate VEGF-R2 signaling, reduce pulmonary inflammation, and improve lung mechanics and respiratory gas exchange.⁵⁰ IV-administrated EVs restore alveolar architecture, reduce fibrosis, and alleviate pulmonary vascular remodeling by promoting an immunosuppressive bone marrow-derived myeloid cells (BMDMy) phenotype and initiating epigenetic and phenotypic reprogramming of myeloid cells.⁶⁸ EVs derived miR-425, target phosphatase and tensin homolog (PTEN) to activate phosphoinositide 3-kinase (P_I3K)/Akt pathway, reducing in apoptosis and oxidative stress, enhancing proliferation of alveolar type II epithelial cells (TIIAECs) and pulmonary vascular endothelial cells (PVECs) also by IV administration.^130,131 IV-administrated EVs reprogram alveolar macrophages into an M2 anti-inflammatory phenotype, reduce PF and vascular muscularization and increase peripheral blood vessel count in hyperoxia-induced lung injury.⁶⁷ IV administration also has some disadvantages, such as potential clearance by the immune system before reaching the lung, and risks of off-target effects in pulmonary tissues, which has hindered its clinical translation. IA administrated EVs is noticed in preclinical BPD model because of its prenatal intervention targeting early lung injury and potentiality for preventing severe BPD in high-risk pregnancies. In addition to preserving spiral arteries and vascular architecture, IA-administrated EVs also improve alveolarization and pulmonary vascular growth, increase VEGF and surfactant protein C (SPC) expression, and decrease placental and fetal inflammation by downregulating expressions of IL-1β and nucleotide-binding oligomerization domain-containing protein-like receptor protein 3 (NLRP3).¹³² Due to its limited clinical translation, IA-administrated EVs as an invasive prenatal procedure currently lack sufficient data on efficacy and safety. The mechanism underlying the IO-administrated EVs in C57BL/6J pups with BPD is a possible modulation of the inflammatory response.¹³³ IO-administrated EVs also have some disadvantages including poor bioavailability, unpredictable systemic absorption, and unclear local effective drug concentration in pulmonary vessels. Previous studies conducted by our team showed that intranasal administration of Exos was an effective non-invasive treatment for BPD. This treatment repaired alveolar damage without causing any toxicity or tumorigenic risks, and it prevented inflammation and the epithelial–mesenchymal transition in neonatal mice induced with BPD-like pathology.¹³⁴ Overall, EVs administration without invasive procedures has shown encouraging in vivo outcomes. Further work is demanded to investigate the safety and effectiveness of non-invasive administration of EVs in vivo to discuss more suitable administrated mode to minimize the probable side effects.

Figure 6 Mechanisms analysis of different administration methods, dosages and frequencies of EVs in treating BPD. Abbreviations: IO, intraoral instillation; IT, intratracheal injection; IP, intraperitoneal injection; IA, intra-amniotic injection; IV, injection of vein; MS, media supplementation; FVB, friend leukemia virus B; TIIAECs, alveolar type II epithelial cells; PVECs, pulmonary vascular endothelial cells.

Numerous studies have shown that the BPD therapeutic dose of EVs in vivo was influenced by a variety of factors, including animal species, frequencies, local or systemic administration mode, and sources. Therapeutic dosage of MSCs-EVs varied 400 times in animal models with BPD. These doses ranged from 0.25×10⁶ to 1×10¹⁰ particles.^{55,59,132,135} The isolation and characterization of EVs are highly heterogeneous, EVs units varied widely and included EVs released over time or dosed by animal weight, as well as absolute protein amount, particle number, or amount of EVs released by a specific number of MSCs. The majority of studies provided single-dose therapy. For multiple dosing, the median study was four doses, ranging two to fifteen doses.¹³⁶ Similar to the EVs field overall, there were few studies on dose response, frequency, and administration route in BPD disease. There are currently 15 different studies that examine the effects of EVs in animal models with BPD in vivo at different dosages, frequencies, and routes of administration (Table 4). Numerous studies have verified that different EVs administration routes can achieve different extents of biological distribution in lung tissue, thus affecting BPD progression. The establishment of almost all animal models with BPD involved keeping rodents in environments with hyperoxia, which ranged from 60–90% O₂. Intraperitoneal administrated MSC-EVs fourteen times from postnatal day (PND) 1 to 14 protected alveolar growth in hyperoxia-exposed rats on the P21 and P56. Exos multiple injection promoting alveolar and blood vessel growth, and preventing the development of right ventricular hypertrophy.¹³⁷ Intratracheal administrated hUCMSC-EVs three doses on the P3, P7, and P10 at the respective dosage of 8× 10⁸, 4.5×10⁸, and 3×10⁸ particles/g enhanced increasing in total alveoli number, and decreasing in mean alveolar volume. Furthermore, hUC-MSC EVs are being explored as cell-free alternative to MSCs as better effects of alveolarization, preventing small pulmonary vessels medial thickness under hyperoxia.¹³⁸ A late single bolus intervention at P18 with the dosage of 1×10⁶ cell equivalents on newborn FVB mice under hyperoxia, partially restored alveolar simplification by alleviating mean liner intercept (MLI) induced by hyperoxia exposure. A over 4-week’s intervention of late mesenchymal stem cell-derived exosomes (MSC-MEx) intravenously may contribute to MLI and alveolar thickness, though it may not as effective as early administration. Interestingly, both the early and the late MSC-MEx intervention promoted blood vessel count, alleviated right ventricular hypertrophy to the same extent via intravenous route.⁶⁷ Amniotic fluid derived EVs (AF-EVs), which were given intravenously at P3, decreased MLI, vascular density, macrophage infiltration, and the expression of inflammatory cytokines such as macrophage inflammatory protein-1 alpha (MIP-1α), monocyte chemoattractant protein-1 (MCP-1), and IL-1β, in comparison to the hyperoxia group. Nevertheless, the late administration of AF-EVs in the same model at P7 did not clearly improve pulmonary vascular density or alveolar structure, compared to the hyperoxia group.⁵⁵ Yahui Zhou et al investigated the mechanism and efficacy of human breast milk derived exosomes (HBM-Exos) in BPD treatment via intragastric administration from P1 to P7. Their findings indicated that intragastric administrated HBM-Exos promoted the suspension of cell proliferation, the expression of TIIAECs surface marker SPC, inhibited IL-17 signalling pathway and increased apoptosis induced by hyperoxia.¹³⁹ Alison N. Abele et al examined whether antenatal treatment of MEx obtained from human bone marrow MSCs (HBM-MSCs) protect pulmonary development in rat model with BPD. Their findings verified intra-amniotic administrated MEx with the dosage of 0.25×10⁶ cell equivalents at embryonic day 20, provided one-time prenatal intervention sustaining improvements in reducing inflammatory cytokines, and preserving distal pulmonary growth in BPD.¹³² Moreover, optimal dose and route of administration of good manufacturing practices (GMP)-grade EVs, its long-term effects and how it modifies the lung transcriptome was studied by Mayank Sharma et al. Their findings indicated that EVs obtained from BM-MSCs or WJ-MSCs have comparable efficacies in restoring pulmonary vascular density, preventing the arrest of alveolarization. Interestingly, three different WJ-MSC EVs doses, including low dose (2×10⁸ particles/g), medium dose (12×10⁸ particles/g), or high dose (60×10⁸ particles/g) were administrated respectively via a single intra-tracheal injection on P3, showed no dose-dependent relationship involving pulmonary vascular density, VEGF expression, vascular remodeling, and lung structure. After long-term observation of three months, a single intra-tracheal dose of WJ-MSC EVs on P3 was reported prolonged effects in reducing RVH, PH, and vascular remodeling in BPD models. On P3, there were no discernible differences between systemically administrated MSC-EVs via intravenous administration and locally administrated MSC-EVs via intratracheal administration in terms of pulmonary vascular angiogenesis, remodeling, and structure as measured by hemodynamic or morphometric measures.⁵⁶ However, Danyang Ai et al took a different view on the route of administration and the dose-dependent relationship about MSC-EVs in treating BPD. Three different doses of MSC-EVs were administrated intraperitoneally on P4 respectively, including low (5 μg), medium (10 μg), and high (15 μg). They ameliorated hyperoxia-induced lung injury in a dose-dependent manner, and high-dose MSC-EVs attenuated alveolar simplification. MSC-EVs with high dosage of 15 μg enhanced vascular growth and mitigated pulmonary hypertension, decreased pdpn and wnt5α expression, inhibited primary fatal rat TIIAECs transdifferentiation.¹⁴⁰ Dandan Zhu et al reported that intravenously administrated term human amniotic epithelial cell MSCs derived EVs (hAEC-EVs) at a single of 10 μg on P4 effectively improved tissue-to-airspace ratio and septal crest density, increased TIIAECs in lung injury, released airway hyper-responsiveness, alleviated pulmonary hypertension, and protected from RVH, compared to the preterm hAEC-EVs.¹⁴¹ In an additional study, intravenous administrated term hUCMSC-Exos at single dose of 2 × 10¹⁰/particles/kg at E10.5 or E15.5 facilitated lamellar body development and alveolar maturation, enhanced alveolarization via activating Wnt5a/ROCK1 signaling axis, promoting ROCK1 phosphorylation, upregulating RhoA expression, and inducing autophagy in AT2 cells.¹⁴² Ou Zhou et al found that human umbilical cord mesenchymal stem cell-derived microvesicles (hUCMSC-MVs), which were given intratracheally at a single dose of 20 μg on P7 to newborn Sprague Dawley (SD) rats with BPD, showed a potential therapeutic effect for BPD by reversing the protein levels of PTEN and p-AKT, decreasing the level of lung homogenate of IL-6 and IL-10, and increasing the number of TIIAECs cells and SPC expression.¹⁴³ Marissa A. Lithopoulos et al investigated therapeutic effects of hUCMSC-EVs in newborn C57/BL6 mice with BPD. Their findings indicated that intratracheal administrated hUCMSC-EVs at a single dose of 0.005 μg/g, approximately 1×10⁶ particles/g on the P9 or P10 showed a significantly lower MLI score, improved lung vessel density, lung architecture, as well as inflammatory modulation.¹²⁸ In the BPD model among newborn FVB/NJ mice, intraperitoneal administrated umbilical cord blood-derived exosomes (UCB-Exos) at 5×10⁵ particles each time for three consecutive days on P4, P5 and P10, rescued the disrupted angiogenesis of lung tissues via increasing CD31 levels, and restoring VEGFA levels in hyperoxia-insulted mice.⁵¹ MSC-sEVs administrated preterm lambs showed lower in respiratory severity score, distal airspace wall thickness, smooth muscle thickness about bronchioles and lung arterioles, higher in radial alveolar count, alveolar capillary surface density, and secondary septal volume density. Thus, in preterm lambs with BPD, intravenous administrated two doses of MSC-sEVs respectively at 6 hour and 78 hour post-delivery improved respiratory system physiology and pulmonary alveolarization in mechanically ventilated preterm lambs.⁵⁰ In the newborn SD rats with BPD, a single 15 μg dose of AMSC-Exos on P4 intratracheally alleviated hyperoxia-induced lung injury by inhibiting NF-κB pathway, inflammation reactions, improving alveolarization and proliferation, reducing apoptosis in AECIIs.⁵⁸ Natalie Starke et al investigated inflammasome cascade molecules of the hyperoxia exposuring preterm infants’ plasma derived EVs. Their findings demonstrated that these EVs induced lung inflammation with increasing cargo of CD11b⁺/ASC⁺, CD11c⁺/ASC⁺, and CD206⁺/ASC⁺, inhibiting alveolarization and vascular development, and aggravating BPD.¹⁴⁴ These studies indicate that the ideal therapeutic dose of EVs in treating BPD is subject to comprehensive consideration of optimizing both local and systemic delivery routes, animal weight, frequencies, delivery efficiency and associated mechanisms. In the following part, we provide a thorough analysis and comparison of kinds of administration routes, their biodistribution, potential components of EVs for BPD treatments and potential mechanisms.

Table 4 Studies of Therapeutic Effects of EVs in BPD Animal Models in Vivo at Different Dosages, Frequencies, and Administration Routes

The Biodistribution, Potential Components, Efficacy Evaluations of Extracellular Vesicles of Diverse Administration Methods in Treating BPD Models in Vivo

Due to their inherent capacity for self-renewal, high efficiency in producing and replacing terminally differentiated cells, and function in pulmonary vascular regeneration, MSCs were considered as viable and promising treatment options for BPD.¹⁴⁵ EVs obtained from MSCs remain biologically similar, such as having anti-inflammatory and regenerative properties, however, they decreased the risk of thrombosis, iatrogenic tumor formation, and immunogenicity.^146,147 To date, various types of cell-derived EVs have demonstrated the therapeutic potential in experimental BPD models, such as adipose-derived MSCs, bone marrow-derived MSCs, human umbilical cord-derived MSCs, human breast milk, and plasma. EVs normally carry their bioactive cargo, including mRNAs, microRNAs, proteins and growth factors, into the recipient cells, making them candidate therapeutic agents in BPD (Table 5).¹⁰⁵

Table 5 Different Administered Routes of EVs Carrying Bioactive Cargos in Treating BPD

Intratracheal Administration

IT administration has been used as a widely used administrative route for EVs treatment in BPD. Previous study indicated that IT-administrated mice had the highest fluorescent signal in lung after 24h compared with in the liver and spleen, which was observed by IVIS analyses system.¹⁵² IT-administered EVs appeared to be effective in rat pups with BPD pathology, which was more practical as a non-systemic IV injection. IT administrated MSC-EVs on P3, P7, P10 significantly interrupted CD68+/αSMA+ macrophages in lung accumulation and decreased the expressions of Tgfβ1, Nrf2, αSMA, Col1a1, 8-oxo-dG. Additionally, by increasing Sftpc expression, MSC-EVs restored AT II cell function and decreased ROS production in mitochondria by enhancing Sod2 expression, preserving pulmonary epithelial function and delaying the onset of fibrosis.¹²⁷ The miR-34c-5p in BMSC-EVs targets ovarian tumor protease domain-containing protein (OTUD) 3 mRNA, leading to reduced OTUD3 protein expression. This subsequently decreases PTEN levels, due to loss of OTUD3-mediated stabilization, resulting in activation of the PI3K/Akt pathway which promotes angiogenesis and alveolar repair in BPD.⁵³ Upregulation of miR-21-5p in adipose-derived MSCs (ADMSCs) reduced SKP2 expression, which in turn decreased Nr2f2 ubiquitination, enhanced C/EBPα transcription, and ultimately attenuated hyperoxia-induced oxidative stress, inflammation, and apoptosis in the lungs.⁶⁹ Additionally, IT administrated MSC-EVs for 4 doses on P3, P7, P10, and P21 decreased collagen deposition and medical wall thickness, decreased fibrosis, and effectively promoted M2 polarization by increasing the density of alveolar M2 macrophages with CD163-positive and vascular endothelial cells with ki67-positive.¹²⁹ Interestingly, IT-administered EVs from hUC-MSCs also increased p-Akt and VEGF-A expressions in BPD models, decreased apoptosis, and promoted proliferation in pulmonary vascular endothelial cells that were Ki-67 positive.¹³¹

Intravenous Administration

IV administration remains the most widely used route for EVs in treating BPD in vivo. Through this pathway, EVs can enter the circulatory system directly, allowing for precise control over their dosage and concentration in the blood. EVs primarily build up in organs like the liver, spleen, lungs, and kidneys after IV.¹⁵³ Most studies investigating EV biodistribution have relied on nuclear imaging or fluorescence imaging in vivo, indicating lung accumulated concentration changes with the injection time.^154,155 Linchao Yu et al demonstrated that miR-139-3p carried by MVs of clinical plasma samples of BPD suppressed endothelial proliferation and angiogenesis through downregulating 4EBP1, which was elevated by IV administration. In mice induced with BPD-like pathology, the inhibitor of miR-139-3p reduced vascular and alveolar simplification, which served as a possible treatment strategy for MVs.¹⁴⁸ According to Gareth R. Willis et al, EVs made from human umbilical cord Wharton’s Jelly-MSCs (MEx) inhibited pro-inflammatory factors like TNFα, IL4, IL6, IL17a, and IFNγ, increased Arg1, Il-10, and CX3CR1 expressions, and changed the monocyte/macrophage phenotype toward an immunosuppressive state similar to that of MDSCs. MEx showed regenerative effects in improving exercise capacity, lung architecture, and function by restoring CD45 in a dose-dependent method.⁶⁸ Function of BMSCs-Exos in mitigating hyperoxia-induced RLE-6TN cell injury is diminished when miR-425 is silenced.¹³⁰ Xiaomeng Yi et al highlighted the possible prevention of acute lung injury (ALI) by MSC-Exos overexpressing miR-30b-3p. In the LPS-pretreated MLE-12 cells, overexpressing miR-30b-3p of MSC-Exos decreased the levels of phosphorylated NF-κB, IκB-α, ERK, MEK1/2, p38MARK, and JNK by binding to SAA3. This promoted cell proliferation by reducing inflammation and AEC apoptosis.¹⁴⁹ Gareth R. Willis et al showed that IV administration of MSC-Exos on P4 reduced inflammation, fibrosis, and vascular remodeling while suppressing the M1 phenotype, promoting M2 macrophages, and reprogramming the macrophage response.¹⁵⁰ IT administrated-Exos packaged circular RNA (circRNA)-circABPD1 targeted miR-330-3p, normalized pulmonary angiogenesis by inhibiting HIF-1α driven vascular leakage, reducing pulmonary edema by 60% in neonatal SD rats with BPD. This improved the viability of A549 cells, decreased ROS, increased GSH, SOD, and SPC expression, and lessened damage to lung epithelial cells.¹⁵¹ However, a large amount of research shows that EVs have a very short half-life and the amount of lung tissue that accumulates after IV administration is less than that of the liver and spleen.^156,157 Drug release extension has been indicated bioactive scaffolds loaded with EVs, with development of nanofiber hydrogels, nanocomposite hydrogels, and 3D printing.¹⁵⁸ For instance, CD47 has been inserted into EV membranes to evade phagocytosis, while Rltrkrglk (RLTR) peptide-functionalized Exos represent an emerging strategy for targeted delivery. However, their efficacy in BPD requires further preclinical validation, particularly for IV administration.¹⁵⁹ PEG-modified EVs exhibit improved stability by reducing immune recognition after IV injection.¹⁶⁰ Receptor-targeting peptides, including RGD, significantly increase EV retention in injured lungs versus untargeted EVs, which indicates peptide functionalization might be a valid strategy for pulmonary delivery.¹⁶¹

Intraperitoneal Administration

By utilizing the vast abdominal vasculature, IP avoids the gastrointestinal tract’s first-pass metabolism and offers a large absorption area. This administration route has been the focus of a set of researches because it can also handle higher drug volumes.^162,163 For example, IP administration of EVs derived from cord blood-derived monocytes or tracheal aspirates transmitted miR-23A-3p targeted PTEN, reducing endothelial progenitor cells (EPC) maintenance and angiogenesis, which was a candidate therapeutic target for BPD.⁷⁰ Another study indicated that IP-administrated Exos derived from human breast milk (HBK) that contained circDNAJB6 enhanced AT II cell viability and decreased damage by targeting the downstream DNAJB6 gene and promoting DNAJB6 transcription.¹⁶⁴ According to the research conducted by Juanmei Wang et al, IP-administrated BMSC Exos for 14 consecutive doses enhanced the expression of VEGF, HIF-1α, p-PI3K, and p-Akt while decreasing the expression of inflammatory cytokines like IL-1β, IL-6, IL-17, and IFN-γ. They also improved alveolarization, vascular remodeling, and oxidative stress control.¹³⁵ TNF-stimulated gene 6 (TSG-6) of Exos extracted from healthy donors’ hUC-MSCs’ preterm deliveries at 25 wks decreased the expression of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β and reversed hyperoxia-induced morphometric changes in the lung. In addition, the therapeutic effects of Exos were disrupted by TSG-6 knockdown, making it a potential option for nano non-cell therapy for BPD.¹⁶⁵ However, not all Exos from preterm infants biological samples are benefit for BPD treatment. Xinfqi Zhong et al reported that the administration of Exos from very premature BPD in mice aggravated lung damage by reducing the expression of Fabp3, Fgf9 and Cacna2d3, inhibitor of MAPK and PPAR pathways and lowering of endothelial marker secretagogue, including VEGFA and CD31 epithelial markers.¹⁶⁶ According to the research conducted by Cristiane S. R. Fonteles et al, IP-administrated Exos showed comparable accumulation in the liver, lungs, uterus, kidneys, brain, heart, and spleen 24 hours after injection.¹⁶⁷ IP route has some advantages over IV administration, including preventing cell entrapment in the pulmonary vasculature, accumulating in the surrounding tissue, and having soluble factors that affect the recipient’s immune system.¹⁶⁸ Further studies are demanded to investigate bioavailability of major components of EVs to reach pulmonary vessels by IP administration.

Intragastric Administration

The preferred method of treating BPD is still IG administration because it is affordable, simple to use, and allowing for repeated dosing. Numerous studies have shown that in simulated gastric environments, natural M-EVs lose about 85% of their protein content and face major obstacles from the mucus-epithelial barrier.¹⁶⁹ In order to improve the stability and intestinal epithelial absorption of M-EVs, researchers have used hydrophilic and zwitterionic peptides to modify their surfaces.¹⁶⁹ EVs from a variety of sources, including bacteria, plants, milk, and intestinal epithelial cells, have been shown to withstand the conditions of the gastrointestinal tract.¹⁷⁰ More research is demanded to fully understand MEVs’ potential and mechanisms for overcoming gastrointestinal barriers. It has been demonstrated that EVs originated from human milk (M-EVs) can survive pancreatic and gastric digestion simulations.^171,172 For example, Yahui Zhou et al indicated that IG administrated HBM-Exos enhanced the expression of the AT II surface marker SPC, inhibited the IL-17 signaling pathway (FADD), which was a crucial mechanism in preventing BPD, and inhibited proliferation and apoptosis caused by hyperoxia. In addition to improving the inhibition of Ki67 and the elevation of cleaved-caspase 3 (C-Caspase3) brought on by exposure to hyperoxia, they verified that DiR-labeled HBM-Exo specifically targeted lung tissue in vivo 12 hours after administration.¹³⁹

Future Directions

EVs have shown promise as nanoscale, cell-free treatments for BPD, that affects preterm infants and is characterized by vascular dysgenesis, persistent inflammation, and arrested alveolarization.^57,173 Present preclinical and early clinical research shows that EVs made from engineered sources, amniotic fluid, or MSCs provide a variety of therapeutic benefits by delivering bioactive cargo, including proteins, lipids, and miRNAs, that alter important pathways linked to the pathophysiology of BPD.^174,175 For example, amniotic fluid EVs-mediated inhibition of TGF-β1/small mother against decapentaplegic (Smad) 3 signaling reduces pulmonary fibrosis by decreasing collagen deposition and αSMA+ myofibroblast activation, thereby preserving lung elasticity and improving gas exchange. And MSC-EVs rich in miR-30b-5p inhibit hyperoxia-induced bronchial epithelial cell apoptosis through PTEN/P_I3K/Akt signaling pathway.¹⁷⁶ According to clinical trials (eg NCT04313647, NCT05624203), these results that assess EVs for respiratory conditions, which show promising safety profiles and initial effectiveness in promoting tissue repair and lowering inflammation.^177,178 Therefore, while EVs hold substantial therapeutic potential for BPD, more robust, large-scale, and well-controlled clinical trials are essential to determine their role in clinical practice. Nevertheless, there are still few BPD-specific EV trials, and the majority of research that is available focuses on proof-of-concept rodent models, leaving gaps in mechanistic specificity, dosage, and delivery optimization. However, safety dose of EVs obtained from a variety of origins remains not fully discussed. There is an urgent need for a large body of research to clarify the safety dosage of EVs from various sources and administered using different routes.

Safe dosage of EVs in the treatment of animal models with BPD is affected by many factors, such as the source of EVs, animal species, and administration routes. EV-based preclinical studies suggest that different dosages need to be considered in both animal and cell models of BPD, and the ideal therapeutic dosage is still under exploring.¹⁷⁹ IT delivery, the most researched route in preclinical models, achieves high pulmonary retention EVs localized to lung tissue within 24 hours and permits low-dose efficacy. In neonatal Sprague-Dawley rats with BPD, Exos treatment reduced pulmonary hypertension, increased survival in a non-dose-dependent way, and increased survival at a dose of 6×10⁹ particles/g via IT administration. IT administration directly targets alveolar macrophages and epithelial cells, attenuating inflammation and promoting alveolarization. However, rapid mucociliary clearance and immune cell uptake may necessitate repeated dosing. In contrast, IV administration reduces inflammation throughout the body, but higher dosages are needed to overcome the pulmonary endothelial barriers and reach therapeutic lung concentrations. The biodistribution of EVs at 30 μg intravenously in a preclinical experiment appears that once and twice administrations were non-toxic, and primarily accumulated in the liver, spleen, and lung. Additionally, EVs obtained from hUC-MSCs may reduce inflammation and lung damage brought on by LPS.¹⁸⁰ An intravenous injection of Exos at a dose of 3.85×10¹² particles did not cause any discernible adverse effects on Th1/Th2 cytokines or general clinical conditions, according to a safety dose study conducted in cynomolgus monkeys.¹⁸¹ As for IV injection, Exos at a dose of 3.85×10¹² particles did not cause any discernible adverse effects on Th1/Th2 cytokines or general clinical conditions, according to a safety dose study conducted in cynomolgus monkeys.¹⁸¹ Although IV-administered EVs have a great deal of potential to treat BPD, care should be taken into account because they may cause thrombogenicity through the angiotensin II receptor type 1 (AT1R)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidases/sodium-glucose cotransporter 2 (SGLT2) pro-oxidant pathway, which causes endothelial dysfunction, and off-target organ accumulation in the pulmonary vascular system.^161,182 IP injection of EVs is a systemic delivery method that may be useful in treating BPD. The timing of treatment and the method of IP administration produce different effects, but there were no differences in efficacy when comparing the tissue sources of MSCs or animal species.¹³⁶ IP-administered Exos at a dose of 3.4×10⁹ can reduce lung damage by regulating systemic inflammation and promoting alveolar repair, according to preclinical research in hyperoxia-induced BPD models. Mechanistically, IP-administered EVs achieve partial lung bio-distribution and engage immune cells by crossing the peritoneal lining and entering the systemic circulation, and hUC-MSCs mediate this effect in part through the secretion of TSG-6, which is an anti-inflammatory factor.¹⁸³ Dosage of 10 μg sEVs have indicated acceptable safety of temporary peritoneal irritation, ascites arousing, but no long-term organ-toxicity observed.¹⁸⁴ IG route is a potential non-invasive method of treating BPD, though harsh gastrointestinal environment and the low bioavailability. According to available data, in the severe acute pancreatitis (SAP)-associated ALI model, IG-administered Exos derived from bronchoalveolar lavage fluid (BALF) at a dose of 0.25 mg/kg contained the NOVEL-rno-miR-29a-3p, which has anti-inflammatory properties and may lessen SAP-ALI.¹⁸⁵ However, EV degradation by gastric acids and proteases necessitates encapsulation in pH-resistant carriers, such as chitosan nanoparticles or lyophilization to preserve cargo integrity.¹⁸⁶ Despite the fact that intragastric delivery eliminates the dangers of invasive routes, biodistribution studies showed that most EVs localized to the liver and intestinal epithelium, with minimal accumulation in lungs.¹⁸⁷

Additionally, we are still in the exploratory phase of the technology, considerably more needs to be learned about the biogenesis, uptake, administration, and mechanism of EVs in treating BPD, considering their heterogeneity. The variability of EVs yield, purity and cargo is due to the variation of cell sources, culture conditions including serum-free or serum-rich media, and isolation methods such as ultracentrifugation or size-exclusion chromatography.¹⁸⁸ Recently, EVs that originate from an environment that complies with GMP and has a trustworthy and traceable source are subject to quality control procedures that analyze homogeneity and purity, according to the most recent literature.¹⁸⁹ A research verified that the synergistic effects of tryptophan and trehalose allowed milk-derived extracellular vesicles (mEVs) to be stable as a powder at room temperature for up to six months without substantially altering their structure and function in vitro. This paves the way for GMP separation and end-user application of mEV-fundamental treatment.¹⁹⁰ Furthermore, inhalable dry powder development of stabilized EVs has been used in treating respiratory diseases, including pneumonia,¹⁹¹ COPD,¹⁹² PF,¹⁹³ severe COVID-19.¹⁹⁴ Nebulized EVs, which are mostly derived from MSCs or engineered sources, have been shown in recent studies to achieve localized pulmonary enrichment. In female BALB/c mice models induced by hypoxia, 60–80% of administered EVs are retained in lung tissue within 24 hours after inhalation. These EVs exhibited delivering caveolin-1 (CAV-1) to suppress expression of phosphorylated signal transducer and activator of transcription 6 (p-STAT6), and promoting airway barrier.¹⁹⁵ Nevertheless, EVs integrity during nebulization remains a critical concern, as shear stress and temperature fluctuations may compromise cargo stability. Thus, lyophilization or encapsulation in biocompatible carriers, for example, chitosan nanoparticles used for nebulization has shown great potential in preserving EVs functionality during delivery.¹⁹⁶ In a rat model of paraquat inhalation toxicity, herbicide loading on pectin (PEC)/chitosan (CS) /tripolyphosphate (TPP) nanoparticles decreased fibrosis and acute lung injury. Expressions of α-SMA, oxidative stress, and apoptosis were all decreased in the lung tissue as a result of the encapsulation.¹⁹⁷ Safety assessments in more than 180 patients with ARDS indicated that Exo-mCD24 inhalation at a dose of 5×10⁸ or 1×10⁹ for five consecutive days did not result in drug-related adverse events, which were observed up to 443–575 days.^121,198 Moreover, EXO-CD24 improved survival in an animal model of pulmonary sepsis and also showed promise in a model of bleomycin-induced pulmonary fibrosis and ovalbumin type I-induced allergic asthma.¹⁹⁹ Future studies may help investigate therapy and safety of EVs in treating BPD with non-invasive, cell-free administrations, for example, inhalation of EVs or chitosan nanoparticles. In spite of many challenges of EVs in treating BPD, it may offer great potential in cell-free nano-medicine treatment in clinical trials (Figure 7).

Figure 7 Roadmap for clinical translation of EVs in treating BPD. Abbreviation: GMP, good manufacturing practice.

Conclusions

EVs have emerged as a potential tool for treating BPD. With their ability to transport bioactive cargos, for example, miRNAs, and proteins, EVs mitigate oxidative stress, inflammation injury, and promote pulmonary alveolarization. Intra-tracheal administration targets directly to lungs, while intravenous delivery offers systematic advantages, in spite of higher dosage requirements. Future researches are aimed to develop inhalable lyophilized EVs to address the practical and ethical limitations of invasive routes. Despite prospective preclinical outcomes, clinical translation primarily depends on resolving EVs heterogeneity, optimizing delivery methods, as well as validating safety dose in neonates. Future researches are suggested to prioritize GMP-compliant production, non-invasive delivery strategies, and clinical trials based on biomarkers to completely realize the therapeutic potential of EVs.

Data Sharing Statement

All the original data presented in this research are illustrated in the manuscript and supplementary materials. Further inquiries can be contacted directly to the corresponding author named Wenbin Dong.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was supported by grants from projects of Wu Jieping Medical Foundation Clinical Research (No. 320.6750.2023-24-10) and National Natural Science Foundation of China (No. 82371710).

Disclosure

All authors in this research do not have any commercial or related interest in the work submitted.

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