小鼠肺内雾化给药器，小鼠气管内雾化给药器

Inhalation of MSC-EVs is a noninvasive strategy for ameliorating acute 
lung injury 
Ruijing Zhao a,1
, Lina Wang a,1
, Tian Wang a,b
, Panpan Xian a
, Hongkang Wang a
, 
Qianfa Long a,c,* 
a Mini-invasive Neurosurgery and Translational Medical Center, Xi’an Central Hospital, Xi’an Jiaotong University. No. 161, West 5th Road, Xincheng District, Xi’an 
710003, China b Shaanxi Lon-EV Biotechnology Limited Company, No.9 Jiazi, Renyi village, Beilin District, Xi’an 710054, China c College of Medicine, Yan’an University, Yongxiang Road, Baota District, Yan’an 716000, China 
ARTICLE INFO 
Keywords: 
Small extracellular vesicles 
Inhalation 
Acute lung injury 
Immunomodulation 
Redox system 
ABSTRACT 
Mesenchymal stem cell-derived small extracellular vesicles (MSC-EVs) are promising nanotherapeutic agent for 
pneumonia (bacterial origin, COVID-19), but the optimal administration route and potential mechanisms of 
action remain poorly understood. This study compared the administration of MSC-EVs via inhalation and tail 
vein injection for the treatment of acute lung injury (ALI) and determined the host-derived mechanisms that may 
contribute to the therapeutic effects of MSC-EVs in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells 
(macrophage cell line) and animal models. Luminex liquid chip and hematoxylin and eosin (HE) staining 
revealed that, compared with the vehicle control, inhaled MSC-EVs outperformed those injected via the tail vein, 
by reducing the expression of pro-inflammatory cytokines, increasing the expression of anti-inflammatory 
cytokine, and decreasing pathological scores in ALI. MSC-EV administration promoted the polarization of 
macrophages towards a M2 phenotype in vitro and in vivo (via inhalation). RNA sequencing revealed that immune 
and redox mediators, including TLR4, Arg1, and HO-1, were associated with the activity MSC-EVs against ALI 
mice. Western blotting and immunofluorescence revealed that correlative inflammatory and oxidative mediators 
were involved in the therapeutic effects of MSC-EVs in LPS-stimulated cells and mice. Moreover, variable 
expression of Nrf2 was observed following treatment with MSC-EVs in cell and animal models, and knockdown of 
Nrf2 attenuated the anti-inflammatory and antioxidant activities of MSC-EVs in LPS-stimulated macrophages. 
Together, these data suggest that inhalation of MSC-EVs as a noninvasive strategy for attenuation of ALI, and the 
adaptive regulation of Nrf2 may contribute to their anti-inflammatory and anti-oxidant activity in mice. 
1. Introduction 
Acute lung injury (ALI) is characterized by impaired pulmonary gas 
exchange, bilateral infiltrates, and noncardiogenic edema, and can be 
induced by direct injury and systemic stimuli, such as mechanical 
trauma, bacteria, and viruses (e.g., SARS-CoV-2), resulting in human and 
economic burden [1,2]. If pulmonary disease is not effectively managed 
during the early stage, acute respiratory distress syndrome (ARDS) can 
develop, and is associated with high mortality [3]. ALI is associated with 
severe acute inflammation, as well as the complications of infections, 
such as increased permeability of blood vessels and the death of 
pulmonary epithelial and endothelial cells [2]. Stem cell therapy has 
demonstrated great potential in the treatment of lung injury, including 
that induced by COVID-19, owing to its immunomodulatory and tissue 
repair properties [4,5]. However, the cellular candidates, optimal 
management, and therapeutic mechanisms are not well understood. 
Increasing evidence, including data from our previous studies, suggests that small extracellular vesicles (EVs) exhibit more potential than 
their parental cells (e.g. MSCs) as therapeutics against inflammatory 
diseases, owing to characteristics such as blood-air barrier permeability, 
freeze / thaw resistance, and targeting to injured cells [6,7]. Recent 
studies have shown that EV-based therapies hold potential for the 
* Corresponding author at: Mini-invasive Neurosurgery and Translational Medical Center, Xi’an Central Hospital, Xi’an Jiaotong University, No. 161, West 5th 
Road, Xincheng District, Xi’an 710003, China. 
E-mail address: lonva@live.cn (Q. Long). 1 These authors contributed this work equally. 
Contents lists available at ScienceDirect 
Journal of Controlled Release 
journal homepage: www.elsevier.com/locate/jconrel 
https://doi.org/10.1016/j.jconrel.2022.03.025 
Received 18 November 2021; Received in revised form 21 February 2022; Accepted 14 March 2022 Journal of Controlled Release 345 (2022) 214–230
215
treatment of lung injury, such as that induced by COVID-19, as they can 
target multiple pathways and enhance tissue regeneration [8]. In addition, MSC-EVs can attenuate ALI via mitochondrial or miRNA transfer 
and modulate macrophage polarization because they contain multiple 
functional cargoes, including proteins, lipids, RNA, and metabolites 
[6,9,10]. Besides to determine the therapeutic agents carried by EVs, it 
also is important to elucidate the therapeutic mechanisms, including 
immunomodulation, antioxidation, and tissue regeneration in target 
cells or injured tissues during ALI. 
Inflammatory stimuli can evoke the excessive production of reactive 
oxygen species (ROS) in pulmonary tissue, followed by the development 
of ALI. Oxidative stress is an early contributor to ALI, and can cause 
macrophage activation, cellular infiltration, and enhanced pulmonary 
cytokine production [11,12]. Thus, crosstalk between oxidation and 
inflammation is important for regulating the initiation and progression 
of ALI. Specifically, the nuclear factor kappa beta (NF-κB) pathway is 
activated by a variety of stimuli in ALI, and activation of NF-κB downstream of Toll-like receptor 4 (TLR4) and transcription factors such as 
signal transducer and activator of transcription 3 (STAT3) mediates 
macrophage plasticity and inflammation [13]. Also, the antiinflammatory or antioxidative activity in ALI depends on the regulation of nuclear factor erythroid 2-related factor 2 (Nrf2, a key mediator 
in oxidative stress) [12,14]. Together, these mediators may orchestrate 
inflammation and oxidation during lung injury. Previously, we showed 
that MSC-EVs exert significant biological activities in models of 
inflammation and oxidative stress [15,16], which suggests that EV 
therapy may have potential to regulate the crosstalk between oxidation 
and inflammation in ALI. 
In the present study, inhalation and tail vein injection were used as 
administration methods to examine the potential activity of MSC-EVs in 
mice with ALI. This was followed by RNA sequencing (RNA-Seq), molecular pattern tests, and Nrf2 knockdown to elucidate the therapeutic 
mechanism of MSC-EVs in lipopolysaccharide (LPS)-stimulated cells or 
mice. The results showed that administration of MSC-EVs via inhalation 
has potential against acute lung inflammation and oxidation, highlighting the clinical value of MSC-EV inhalation in ALI, even in that 
induced by COVID-19. 
2. Materials and methods (Fig. S1) 
2.1. Cell preparation 
For preparation of allogeneic MSCs, informed consent was obtained 
before cell collection, and 3 donors (age 27–29) were selected from fullterm puerpera in good health. All procedures were approved by the 
Ethical Committee of the Xi’an Central Hospital, Xi’an Jiaotong University, as well as in accordance with the Guidelines of the National 
Institutes of Health. MSCs were obtained from Wharton’s jelly in the 
umbilical cord and characterized by flow cytometry, the gating strategy 
was employed by using Fluorescence Minus One control, as in our previous reports [15,17]. Primary antibodies including rabbit polyclonal 
anti-CD105, CD90, CD73, CD45, CD34 and CD11b (1:100 dilution) 
(Bioss, Wuhan, CHN), and secondary antibody Alexa Fluor 488 goat 
anti-rabbit IgG (1:500) (Invitrogen, A-21206, CA, USA) were used to 
detect the surface antigens of fifth-passage MSCs. At least three cell 
culture samples were examined on an FACS Calibur instrument (Becton 
Dickinson) and the data were analyzed using Cell Quest software (Becton Dickinson). Multi-potency of MSCs was detected by StemPro® 
Osteogenesis (Gibco, A1007201, MD, USA), Chondrogenesis (Gibco, 
A1007101, MD, USA) and Adipogenesis (Gibco, A1007001, MD, USA) 
differentiation Kits (37 ◦C, 5% CO2) according to the instructions. RAW 
264.7 cells, a murine macrophage cell line, were purchased from the Cell 
Bank of Type Culture Collection of Chinese Academy of Sciences 
(Shanghai, China) and cultured in Dulbecco’s modified Eagle medium 
(DMEM) / F12 + 10% fetal bovine serum (FBS) at 37 ◦C, 5% CO2. 
2.2. Isolation, characterization and labelling of MSC-EVs 
EVs were isolated from the supernatants of fifth-passage MSCs, as 
previously described [15]. Briefly, the ratio of live and dead MSCs was 
detected by using an automatic cell counter (Bodboge, Shenzhen, CHN), 
the batch of which contained more than 99% live cells. The MSCs were 
cultured in αMEM containing 10% EV-depleted FBS for 24 h, and then 
the supernatants were harvested and processed via a series of centrifugation steps (300 ×g for 10 min, 2000 ×g for 10 min, and 10,000 ×g for 
30 min; ST16R, Thermo Fisher, USA). Subsequently, the EVs were 
collected via ultracentrifugation at 100,000 ×g for 70 min (XPN-100, 
Beckman Coulter, USA). The nanoparticles were then characterized by 
western blotting based on the positive markers TSG101 and CD9, as well 
as negative marker calnexin, and examined via transmission electron 
microscopy (TEM) and nanoparticle tracking analysis (NTA) to evaluate 
morphology and size distribution, respectively. In addition, C5 
Maleimide-Alexa 594 (CM-A954) (Invitrogen, A10256, California, USA) 
was used to label MSC-EVs as our previous reports [17]. 
2.3. Macrophage activation and intervention 
RAW 264.7 cells were pretreated with 10 μg / mL MSC-EVs 
(Fig. S2A) for 12 h to ensure uptake, and then activated with 100 ng / 
mL lipopolysaccharide (LPS; L2880, Sigma-Aldrich, CA, USA) for 12 h, 
as previously reported [18]. ML385 (15 μM; HY-100523, Medchem 
Express, New Jersey, USA), a pharmacological inhibitor of Nrf2, was 
used to downregulate Nrf2 expression in RAW 264.7 cells. 
2.4. Animal procedures 
97 adult male (8–10-weeks old) C57BL/6 mice were purchased from 
the Experimental Animal Center of Xi’an Jiaotong University, they were 
housed in groups and were allowed a period to acclimatize to the laboratory environment before the start of the study. All animal procedures 
were performed in accordance with the ARRIVE guidelines and 
approved by the Ethics Review Board of Xi’an Central Hospital, Xi’an 
Jiaotong University. Animals were housed under a controlled environment with a 12 / 12 h light / dark cycle with food and water provided. 
Mice were intraperitoneally anesthetized using 4.0% chloralhydrate 
(10 mL / kg) and administered LPS (10 mg / kg, diluted with saline) 
intratracheally. A sham operation was performed in a similar manner 
using saline solution (Sham group, n = 5). After LPS induction for 3 h, 
50 μg MSC-EVs (diluted in 50 μL saline, Fig. S2B and C) and 50 μL saline 
(vehicle) were administered via inhalation using an atomizer (YSKD BioTec, Beijing, China) as the ALI-inh + EVs (n = 20) and ALI-inh + Veh (n 
= 15) groups, or administered by tail vein injection as the ALI-iv + EVs 
(n = 15) and ALI-iv + Veh (n = 15) groups, respectively. The mice were 
sacrificed at random via isoflurane at 24 h, 4 days (d), and 14 d after EV 
administration, and lung tissues and blood samples were collected for 
further analyses (Fig. S1). Also, to track the MSC-EVs in vitro, ALI mice 
received MSC-EVs via inhalation (negative control, n = 3), or CM-A594 
labeled MSC-EVs via inhalation (n = 3) and tail vein injection (n = 3) for 
24 h, the lung tissues were then processed as above procedures. Additionally, 8 mice were excluded due to failed injection via tail vein, and 
10 mice were died in the present experiments. 
2.5. Luminex liquid chip 
Following treatment with MSC-EVs for 24 h, whole blood was 
collected from mouse orbits and centrifuged (10,000 rpm) for 10 min. 
Supernatants from each group were assayed using a Luminex liquid chip 
(Luminex 200, USA) for interleukin (IL)-1β, macrophage chemoattractant protein-1 (MCP-1), IL-1α, tumor necrosis factor (TNF)α, IL-12, 
and IL-10 (MHSTCMAG-70 K, Mouse High Sensitivity T Cell Magnetic