Endothelium‐Derived Engineered Extracellular Vesicles Protect the Pulmonary Endothelial Barrier in Acute Lung Injury

Abstract Acute lung injury (ALI) is a severe respiratory disease with a high mortality rate. The integrity of the pulmonary endothelial barrier influences the development and prognosis of ALI. Therefore, it has become an important target for ALI treatment. Extracellular vesicles (EVs) are promising nanotherapeutic agents against ALI. Herein, endothelium‐derived engineered extracellular vesicles (eEVs) that deliver microRNA‐125b‐5p (miRNA‐125b) to lung tissues exerting a protective effect on endothelial barrier integrity are reported. eEVs that are modified with lung microvascular endothelial cell‐targeting peptides (LET) exhibit a prolonged retention time in lung tissues and targeted lung microvascular endothelial cells in vivo and in vitro. To improve the efficacy of the EVs, miRNA‐125b is loaded into EVs. Finally, LET‐EVs‐miRNA‐125b is constructed. The results show that compared to the EVs, miRNA‐125b, and EVs‐miRNA‐125b, LET‐EVs‐miRNA‐125b exhibit the most significant treatment efficacy in ALI. Moreover, LET‐EVs‐miRNA‐125b is found to have an important protective effect on endothelial barrier integrity by inhibiting cell apoptosis, promoting angiogenesis, and protecting intercellular junctions. Sequencing analysis reveals that LET‐EVs‐miRNA‐125b downregulates early growth response‐1 (EGR1) levels, which may be a potential mechanism of action. Taken together, these findings suggest that LET‐EVs‐miRNA‐125b can treat ALI by protecting the endothelial barrier integrity.

The "Pathological lung injury score" was performed by a pathologist who was unaware of the study group.The pathologist scored pathological damage on H&E-stained lung tissue sections, with 20 randomly selected 400× high magnification fields of view in each group, in which alveoli should occupy at least 50% of each field of view and areas consisting mainly of lumens of large airways or blood vessels should be excluded.Pathologists assigned values to five independent variables: neutrophils in the alveolar space, neutrophils in the interstitial space, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening and weighted them to obtain the final lung injury score.

Figure S2 .
Figure S2.Delivery of Cy5 labeled miRNA-125b to HUVECs by LET-EVs.The Cy5 labeled miRNA-125b was loaded into DIO labeled LET-EVs.The labeled LET-EVs-miRNA-125b were co-cultured with HUVECs for 2 h.HUVECs were fixed with paraformaldehyde and stained with DAPI.The localization of Cy5 labeled miRNA125b and DIO labeled LET-EVs was observed by fluorescence microscopy, suggesting LET-EVs could deliver miRNA125b into HUVECs.Stains used are as follows: DIO labeled EVs (Green), Cy5 labeled miRNA125b (Red), and DAPI (blue) (Scale bar: 200 μm).

Figure S3 .
Figure S3.Distribution of DIR labeled LET-EVs and EVs in healthy mice.(A) Imaging of healthy mice after 0, 2 and 48 h of administration of DIR, DIR-EVs and DIR -LET-EVs.Compared with DIR and DIR-EVs group, the fluorescence signal of LET-EVs mainly accumulated in epigastric after 2 h.(B) Fluorescence imaging of organs of healthy mice in DIR, DIR-EVs and DIR-LET-EVs group after 48 h.Compared with DIR and DIR-EVs group, signal in DIR-LET-EVs groups mainly gathered in lung tissues.

Figure
Figure S4 Establishment and evaluation of acute lung injury animal models (A) Representative images of H&E-stained lung tissue sections after LPS stimulation for 4h and 48h(Scale bar: 200 μm).(B) The morphology evaluation of the lung tissues by reducing the number of alveoli/hpf and increasing the alveolar septal thickness and the mean linear intercept after LPS after LPS stimulated for 4 h and 48 h in compared with the control group.(C) Wright-Giemsa staining and cell count of inflammatory cell.A large number of neutrophils were aggregated in the alveoli/interstitium after giving LPS.(D)The protein concentration in BALF was increased in LPS treatment group compared to the control group.(E)The concentration of TNF-α, IL-6, and IL-1β in BALF was increased after LPS stimulation.Data were presented as Mean ± SD (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with control group by unpaired Student's t-tests.

Figure S5 .
Figure S5 .Standard pulmonary function test in ALI mice (A) Minute volume and (B) Frequency of breathing in different groups detected by the small animal respiratory physiological detection system of EMKA Company.All the data were presented as Mean ± SD (n = 3).*P < 0.05, ***P < 0.001, compared with PBS treatment group by unpaired Student's t-tests.# P < 0.05, ## P < 0.01, compared with LET-EVs-miRNA-125b treatment group by unpaired Student's t-tests.

Fig S6 .
Fig S6.Comparison of the efficacy between EVs and LET-EVs.(A) Representative images of H&E-stained lung tissue sections between EVs and LET-EVs group(Scale bar: 100 μm).(B) Representative images of lung tissues after staining with Evans blue.(C) Protein concentrations in BALF in EVs group compared to those of the LET-EVs group.(D) Wright-Giemsa staining and cell count of inflammatory cells (Scale bar: 100 μm).

Figure S8 .
Figure S8.The rate of apoptosis in HUVECs was determined using Annexin V/PI flow cytometry.