Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles

Meng-Meng Shi, Qing-Yuan Yang, Antoine Monsel, Jia-Yang Yan, Cheng-Xiang Dai, Jing-Ya Zhao, Guo-Chao Shi, Min Zhou, Xue-Mei Zhu, Su-Ke Li, Ping Li, Jing Wang, Meng Li, Ji-Gang Lei, Dong Xu, Ying-Gang Zhu, Jie-Ming Qu, Meng-Meng Shi, Qing-Yuan Yang, Antoine Monsel, Jia-Yang Yan, Cheng-Xiang Dai, Jing-Ya Zhao, Guo-Chao Shi, Min Zhou, Xue-Mei Zhu, Su-Ke Li, Ping Li, Jing Wang, Meng Li, Ji-Gang Lei, Dong Xu, Ying-Gang Zhu, Jie-Ming Qu

Abstract

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) turn out to be a promising source of cell-free therapy. Here, we investigated the biodistribution and effect of nebulized human adipose-derived MSC-EVs (haMSC-EVs) in the preclinical lung injury model and explored the safety of nebulized haMSC-EVs in healthy volunteers. DiR-labelled haMSC-EVs were used to explore the distribution of nebulized haMSC-EVs in the murine model. Pseudomonas aeruginosa-induced murine lung injury model was established, and survival rate, as well as WBC counts, histology, IL-6, TNF-α and IL-10 levels in bronchoalveolar lavage fluid (BALF) were measured to explore the optimal therapeutic dose of haMSC-EVs through the nebulized route. Twenty-four healthy volunteers were involved and received the haMSC-EVs once, ranging from 2 × 108 particles to 16 × 108 particles (MEXVT study, NCT04313647). Nebulizing haMSC-EVs improved survival rate to 80% at 96 h in P. aeruginosa-induced murine lung injury model by decreasing lung inflammation and histological severity. All volunteers tolerated the haMSC-EVs nebulization well, and no serious adverse events were observed from starting nebulization to the 7th day after nebulization. These findings suggest that nebulized haMSC-EVs could be a promising therapeutic strategy, offering preliminary evidence to promote the future clinical applications of nebulized haMSC-EVs in lung injury diseases.

Keywords: extracellular vesicles; healthy volunteers; lung injury; mesenchymal stromal cells; nebulization.

Conflict of interest statement

The authors indicate no potential conflicts of interest.

© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.

Figures

FIGURE 1
FIGURE 1
The manufacture and characterization of haMSC‐EVs. (a) haMSC‐EVs manufacture. (b) Representative electron microscopic photograph of haMSC‐EVs, scale bar = 100 nm. (c) The concentration and size distribution of haMSC‐EVs were determined by NTA. (d) Representative western blots showing the expression of EV markers, including CD9, CD81, CD63, TSG101, and CANX. haMSC‐EVs: human adipose‐derived MSC‐Extracellular vesicles; NTA: Nanoparticle Tracking Analysis
FIGURE 2
FIGURE 2
Mesh nebulizer set‐up and biodistribution of haMSC‐EVs. (a‐b) Vibrating mesh nebulizer set‐up. (c) Biodistribution of DiR‐labelled haMSC‐EVs in vivo continuously until 28 days post‐nebulization. (d) Biodistribution of DiR‐labelled haMSC‐EVs in vitro continuously until 28 days post‐nebulization. The list of terms represents the layout of isolated organs. haMSC‐EVs: human adipose‐derived MSC‐Extracellular vesicles
FIGURE 3
FIGURE 3
Therapeutic effects of haMSC‐EVs in P. aeruginosa‐induced murine lung injury model. (a) Kaplan‐Meier survival curves of P. aeruginosa‐induced murine lung injury model (N = 10 per group). (b‐c) Aerosol inhalation of haMSC‐EVs post‐infection decreased the influx of white blood cells (P = 0.0026, ** indicates P < 0.01, 2.0 ± 0.3 for PA + NS, 0.8 ± 0.1 for PA + haMSC‐EVs, N = 5) and neutrophils (P = 0.0029, ** indicates P < 0.01, 1.9 ± 0.3 for PA, 0.7 ± 0.1 for PA + haMSC‐EVs, N = 5) in BALF at 24 h. (d) The BALF levels of IL‐6 (**** indicates P < 0.0001, 990.1 ± 12.3 for PA + NS, 316.3 ± 18.9 for PA + haMSC‐EVs, N = 5), TNF‐α (P = 0.0044, ** indicates P < 0.01, 589.9 ± 68.4 for PA + NS, 294.8 ± 31.7 for PA + haMSC‐EVs, N = 5) were decreased in haMSC‐EVs group. The level of IL‐10 expression was increased (P = 0.0032, ** indicates P < 0.01, 17.2 ± 3.1 for PA + NS, 34.4 ± 2.7 for PA + haMSC‐EVs, N = 5) in haMSC‐EVs group. (e) The histology showed less inflammatory cells infiltrating interalveolar septa and respecting alveolar space and lung architecture. (f) Aerosol inhalation of haMSC‐EVs post‐infection also reduced histological severity of lung injury better than other groups (**** indicates P < 0.0001, 0.908 ±0.03 for PA + NS, 0.284 ± 0.03 for PA + haMSC‐EVs, N = 5). haMSC‐EVs: human adipose‐derived MSC‐Extracellular vesicles; PA: P. aeruginosa; L‐929‐Exos: NCTC clone 929 cells derived exosomes; BALF: bronchoalveolar lavage fluid; WBC: white blood cell; IL‐6: interleukin 6; TNF‐α: tumour necrosis factor‐α; IL‐10: interleukin 10
FIGURE 4
FIGURE 4
Flow diagram for MEXVT. WBC: white blood cell; haMSC‐EVs: human adipose‐derived MSC‐Extracellular vesicles
FIGURE 5
FIGURE 5
Clinical characters of healthy volunteers. (a) Vital sign parameters before and after haMSC‐EVs nebulization in MEXVT. (b) Level of IgE before and after haMSC‐EVs nebulization in MEXVT. haMSC‐EVs: human adipose‐derived MSC‐Extracellular vesicles; IgE: ImmunoglobulinE

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