Mitigation of Oxidative Stress in Idiopathic Pulmonary Fibrosis Through Exosome-Mediated Therapies

Figures & data

Figure 1 Exosome biogenesis involves the interaction of these vesicles with target cells. These vesicles originate from the endocytic pathway, arising from the inward budding of the MVB membrane. Reproduced from Xie X, Xiong Y, Panayi AC, et al. Exosomes as a Novel Approach to Reverse Osteoporosis: a Review of the Literature. Front Bioeng Biotechnol. 2020;8:594247. Creative Commons.²³

Table 1 Summary of the Methods for Exosome Isolation and Purification

Method	Principle	Advantages	Disadvantages
Ultracentrifugation	Differential sedimentation	High purity, widely used	Time-consuming, may co-pellet contaminants
Density gradient centrifugation	Buoyant density separation	Highly pure exosome fractions	Time-consuming, requires optimization
Size exclusion chromatography	Size-based separation	Gentle, maintains exosome integrity	May not yield highly concentrated exosomes
Immunocapture affinity chromatography	Antibody-based capture	Selective isolation, specific for markers	Bias towards targeted markers, may be costly
Ultrafiltration	Membrane-based size separation	Rapid, scalable, suitable for pre-concentration	Limited purity, may require additional steps
Precipitation methods	Chemical-induced precipitation	Simple, quick	May co-precipitate contaminants
Microfluidics-based isolation	Microfluidic devices	Automated, rapid, low sample volume	Evolving technology, requires validation
Commercial kits	Various (eg, precipitation, affinity)	Convenient, standardized, reproducible	Cost, variable performance across kits
Combination methods	Sequential use of multiple methods	Improved purity and yield	Increased complexity, potential for sample loss

Figure 2 (A) The augmented neurogenesis effect of engineered exosome-encapsulated hydrogel was examined through immunofluorescence staining. The scale bar is set at 50 µm. (B) The schematic representation of the tissue collection timeline. (C) General images depicting wound healing in various treatment groups. (D) Immunofluorescence images illustrating the expression of CGRP, NGF, and CRH in skin tissues on day 7 post-wounding, accompanied by corresponding statistical results. Scale bar: 100 µm. Reproduced from Xiong Y, Lin Z, Bu P, et al. A Whole-Course-Repair System Based on Neurogenesis-Angiogenesis Crosstalk and Macrophage Reprogramming Promotes Diabetic Wound Healing. Adv Mater. 2023;35(19):e2212300. © 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.⁵⁸

Figure 3 Schematic illustration of preparation of engineered exosome as targeted lncRNA MEG3 delivery vehicles for OS therapy. Reprinted from J Control Release, volume: 343, Huang X, Wu W, Jing D, et al. Engineered exosome as targeted lncRNA MEG3 delivery vehicles for osteosarcoma therapy. 107–11, Copyrihgt 2022, with permission from Elsevier.⁶²

Table 2 Mechanisms of Exosomes in Counteracting Oxidative Stress

Mechanism	Description	References
Antioxidant enzymes	SOD catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen, enhancing the cellular capacity to neutralize harmful free radicals.	[77,78]
ncRNAs	Encapsulation of miRNAs and lncRNAs in exosomes. These molecules modulate gene expression, particularly targeting genes involved in oxidative stress pathways.	[79,80]
Antioxidant molecules	Inclusion of antioxidant molecules in exosomes, such as GSH, vitamins, and cofactors essential for the activity of antioxidant enzymes.	[81–83]
Maintenance of mitochondrial quality	Presence of mitochondrial components, including mtDNA, in exosomes. This cargo contributes to maintaining mitochondrial integrity and preventing the excessive generation of ROS within mitochondria.	[84,85]
DNA Repair Machinery	Transport of proteins involved in DNA repair mechanisms by exosomes. These proteins aid in the restoration of oxidative stress-induced DNA damage, supporting the repair of genomic instability caused by ROS.	[86]

Figure 4 (A) Schematic representation of the experimental design for the exosome study in SD rats, with a total of 12 biological independent animals per group. (B) Representative H&E staining images, with the upper panel showing a scale bar of 100 μm and the lower panel showing a scale bar of 50 μm. (C) Representative Gomori’s trichrome staining images, illustrating muscle fibers (red), collagen (blue), nuclei (black-purple), and erythrocytes (red). The scale bar is set at 100 μm for the upper panel and 50 μm for the lower panel. (D) Representative picrosirius red staining images depicting collagen types I and III in red. The scale bar is set at 50 μm. (E) Quantification of fibrosis using the Ashcroft score. Each data point represents information from one animal, with a total of 12 biological independent animals. The Ashcroft score was determined by averaging the scores provided by one blinded and one non-blinded scorer. (F) Quantification of pulmonary hydroxyproline levels. Each data point represents information from one animal, with a total of 4 biological independent animals. Statistical significance is denoted by P < 0.05. Reproduced from Dinh PC, Paudel D, Brochu H, et al. Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis. Nat Commun. 2020;11(1):1064. Creative Commons.¹¹³

Figure 5 (A) Transmission electron microscopy (TEM) image capturing the morphology of the exosome. (B) Determination of the size and size distribution of L-929 cell-derived exosomes using dynamic light scattering (DLS). (C) Western blot analysis depicting the protein levels of CD9, HSP70, calnexin, and β-actin in the exosomes secreted by L-929 cells. (D) Schematic representation of the procedure employed to produce the EL-CLD hybrid, involving the hybridization of exosomes with L-CLD using membrane extrusion. (E) TEM image illustrating the morphology of the EL-CLD hybrid, showing the successful integration of exosomes with clodronate-loaded liposomes through the described procedure. (F) Micro-CT images depicting the condition of the lung following the respective treatments. (G) Lung Index quantifying pulmonary fibrosis in mice post-treatments, with an inset providing visual comparisons between healthy lungs and those affected by fibrosis at the conclusion of the treatments. The study includes a sample size of n=5. *p<0.05, **p<0.01. Bar: 200 nm. Reproduced from Biomaterials, volume 271, Sun L, Fan M, Huang D, et al. Clodronate-loaded liposomal and fibroblast-derived exosomal hybrid system for enhanced drug delivery to pulmonary fibrosis. 120761, Copyright 2021, with permission from Elsevier.⁵⁹

Xie X, Xiong Y, Panayi AC, et al. Exosomes as a Novel Approach to Reverse Osteoporosis: a Review of the Literature. Front Bioeng Biotechnol. 2020;8:594247. doi:10.3389/fbioe.2020.594247

 PubMed Web of Science ®Google Scholar

Xiong Y, Lin Z, Bu P, et al. A Whole-Course-Repair System Based on Neurogenesis-Angiogenesis Crosstalk and Macrophage Reprogramming Promotes Diabetic Wound Healing. Adv Mater. 2023;35(19):e2212300. doi:10.1002/adma.202212300

 PubMed Web of Science ®Google Scholar

Huang X, Wu W, Jing D, et al. Engineered exosome as targeted lncRNA MEG3 delivery vehicles for osteosarcoma therapy. J Control Release. 2022;343:107–117. doi:10.1016/j.jconrel.2022.01.026

 PubMed Web of Science ®Google Scholar

Yan Y, Jiang W, Tan Y, et al. hucMSC Exosome-Derived GPX1 Is Required for the Recovery of Hepatic Oxidant Injury. Mol Ther. 2017;25(2):465–479. doi:10.1016/j.ymthe.2016.11.019

 PubMed Web of Science ®Google Scholar

Yao J, Zheng J, Cai J, et al. Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate rat hepatic ischemia-reperfusion injury by suppressing oxidative stress and neutrophil inflammatory response. FASEB J. 2019;33(2):1695–1710. doi:10.1096/fj.201800131RR

 PubMed Web of Science ®Google Scholar

Guo L, Chen Y, Feng X, et al. Oxidative stress-induced endothelial cells-derived exosomes accelerate skin flap survival through Lnc NEAT1-mediated promotion of endothelial progenitor cell function. Stem Cell Res Ther. 2022;13(1):325. doi:10.1186/s13287-022-03013-9

 Web of Science ®Google Scholar

Liu X, Yuan W, Yang L, Li J, Cai J. miRNA Profiling of Exosomes from Spontaneous Hypertensive Rats Using Next-Generation Sequencing. J Cardiovasc Transl Res. 2019;12(1):75–83. doi:10.1007/s12265-017-9784-7

 PubMed Web of Science ®Google Scholar

Vats S, Galli T. Introducing secretory reticulophagy/ER-phagy (SERP), a VAMP7-dependent pathway involved in neurite growth. Autophagy. 2021;17(4):1037–1039. doi:10.1080/15548627.2021.1883886

 PubMed Web of Science ®Google Scholar

Wojnacki J, Nola S, Bun P, et al. Role of VAMP7-Dependent Secretion of Reticulon 3 in Neurite Growth. Cell Rep. 2020;33(12):108536. doi:10.1016/j.celrep.2020.108536

 Web of Science ®Google Scholar

Hu X, He C, Zhang L, et al. Mesenchymal stem cell-derived exosomes attenuate DNA damage response induced by cisplatin and bleomycin. Mutat Res Genet Toxicol Environ Mutagen. 2023;889:503651. doi:10.1016/j.mrgentox.2023.503651

 Web of Science ®Google Scholar

Dinh PC, Paudel D, Brochu H, et al. Inhalation of lung spheroid cell secretome and exosomes promotes lung repair in pulmonary fibrosis. Nat Commun. 2020;11(1):1064. doi:10.1038/s41467-020-14344-7

 PubMed Web of Science ®Google Scholar

Sun L, Fan M, Huang D, et al. Clodronate-loaded liposomal and fibroblast-derived exosomal hybrid system for enhanced drug delivery to pulmonary fibrosis. Biomaterials. 2021;271:120761. doi:10.1016/j.biomaterials.2021.120761

 PubMed Web of Science ®Google Scholar