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Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.This protocol describes an atomic force microscopy infrared spectroscopy (AFM-IR) approach for nanometer-resolution characterization of the structure and composition of single extracellular vesicles.

A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19.A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19.

Aging is a complex feature and involves loss of multiple functions and nonreversible phenotypes. However, several studies suggest it is possible to protect against aging and promote rejuvenation. Aging is associated with many factors, such as telomere shortening, DNA damage, mitochondrial dysfunction, and loss of homeostasis. The integrity of the cytoskeleton is associated with several cellular functions, such as migration, proliferation, degeneration, and mitochondrial bioenergy production, and chronic disorders, including neuronal degeneration and premature aging. Cytoskeletal integrity is closely related with several functional activities of cells, such as aging, proliferation, degeneration, and mitochondrial bioenergy production. Therefore, regulation of cytoskeletal integrity may be useful to elicit antiaging effects and to treat degenerative diseases, such as dementia. The actin cytoskeleton is dynamic because its assembly and disassembly change depending on the cellular status. Aged cells exhibit loss of cytoskeletal stability and decline in functional activities linked to longevity. Several studies reported that improvement of cytoskeletal stability can recover functional activities. In particular, microtubule stabilizers can be used to treat dementia. Furthermore, studies of the quality of aged oocytes and embryos revealed a relationship between cytoskeletal integrity and mitochondrial activity. This review summarizes the links of cytoskeletal properties with aging and degenerative diseases and how cytoskeletal integrity can be modulated to elicit antiaging and therapeutic effects.

Correspondence to Dr Sally Yunsun Kim; sally.kim@kcl.ac.uk Extracellular vesicles (EVs) are membranous nanosized particles that contain proteins, nucleic acids and lipids and are generated by nearly all cell types that participate in intercellular communication.1 The roles of EVs in pulmonary diseases are increasingly being recognised, and EVs have emerged as promising biomarkers for diagnosis and prognosis of cancer and acute and chronic respiratory diseases.2–4 In the field of regenerative medicine, stem/progenitor cell-derived EVs are proving revolutionary owing to their potential as cell-free strategies for tissue regeneration in many organs, including the lungs.5 6 More recently, EVs have been established as highly promising drug delivery vehicles with targeting abilities for specific tissues and cells, achieved through methods such as functionalisation of EV surfaces and modifying parent cells to obtain EVs that target particular cell types.7 The work by Kim et al in this issue of Thorax presents the development of surfactant protein A-coated extracellular vesicles (SP-A-EVs) as a novel drug delivery system that targets alveolar macrophages.8 SP-A is a hydrophilic protein, which is a major component of pulmonary surfactant secreted by alveolar type II cells and is known to modulate innate immune response to pathogens.9 SP-A has been previously shown to protect against interleukin (IL)-13-induced lung inflammation in bronchial epithelial cells from asthmatic patients by inhibiting IL-13-induced STAT3 phosphorylation.10 The high affinity of SP-A for receptors expressed on alveolar macrophages is well known, having been recognised for more than three decades11 and indeed, during the development of SP-A-EVs, Kim et al confirmed that SP-A uptake was significantly higher in alveolar macrophages compared with lung epithelial cells and fibroblasts.8 Kim et al developed SP-A-EVs by binding SP-A to mouse bronchoalveolar lavage fluid (BALF)-derived EVs through a simple co-incubation process.8 Increased internalisation of fluorescently labelled SP-A-EVs was observed in alveolar macrophages obtained from mouse lungs compared with standard EVs (BALF-derived EVs without SP-A), and thus demonstrated successful targeted delivery to alveolar macrophages in vitro. The therapeutic potential of SP-A-EVs as a drug delivery system was demonstrated by loading with let-7b microRNA which has potent anti-inflammatory effects in the lungs.12 Delivery of let-7b-loaded SP-A-EVs intratracheally to lipopolysaccharide-injured mouse lungs resulted in an attenuation of lung inflammation, shown by significant downregulation of inflammatory pathways and genes in alveolar macrophages. Unlike the many studies into the intrinsic immunomodulatory properties of natural EVs for therapeutic effects, the development of an SP-A-EV drug delivery system achieved more specific goals by modifying EVs to both increasing their uptake and avoiding lysosomal trafficking. In particular, immunogenicity of EVs may be due to the biomolecular corona of EVs which could include proteins, immunoglobulins, nucleic acids, lipoproteins or apolipoproteins.13 Prior to progressing SP-A-EVs towards clinical translation, it would be necessary to analyse the changes in the composition of biomolecular corona in response to SP-A coating.

Many adult lung diseases involve dysregulated lung repair. Deciphering the molecular and cellular mechanisms that govern intrinsic lung repair is essential to develop new treatments to repair/regenerate the lungs. Aberrant Wnt signalling is associated with lung diseases including emphysema, idiopathic pulmonary fibrosis and pulmonary arterial hypertension but how Wnt signalling contributes to these diseases is still unclear. There are several alternative pathways that can be stimulated upon Wnt ligand binding, one of these is the Planar Cell Polarity (PCP) pathway which induces actin cytoskeleton remodelling. Wnt5a is known to stimulate the PCP pathway and this ligand is of particular interest in regenerative lung biology because of its association with lung diseases and its role in the alveolar stem cell niche. To decipher the cellular mechanisms through which Wnt5a and the PCP pathway affect alveolar repair we utilised a 3-D ex-vivo model of lung injury and repair, the AIR model. Our results show that Wnt5a specifically enhances the alveolar epithelial progenitor cell population following injury and surprisingly, this function is attenuated but not abolished in Looptail (Lp) mouse lungs in which the PCP pathway is dysfunctional. However, Lp tracheal epithelial cells show reduced stiffness and Lp alveolar epithelial cells are less migratory than wildtype (WT), indicating that Lp lung epithelial cells have a reduced capacity for repair. These findings provide important mechanistic insight into how Wnt5a and the PCP pathway contribute to lung repair and indicate that these components of Wnt signalling may be viable targets for the development of pro-repair treatments.

VANGL2 is a component of the planar cell polarity (PCP) pathway, which regulates tissue polarity and patterning. The mutation causes lung branching defects due to dysfunctional actomyosin-driven morphogenesis. Since the actomyosin network regulates cell mechanics, we speculated that mechanosignaling could be impaired when VANGL2 is disrupted. Here, we used live-imaging of precision-cut lung slices (PCLS) from mice to determine that alveologenesis is attenuated as a result of impaired epithelial cell migration. tracheal epithelial cells (TECs) and alveolar epithelial cells (AECs) exhibited highly disrupted actomyosin networks and focal adhesions (FAs). Functional assessment of cellular forces confirmed impaired traction force generation in TECs. YAP signaling in airway epithelium was reduced, consistent with a role for VANGL2 in mechanotransduction. Furthermore, activation of RhoA signaling restored actomyosin organization in , confirming RhoA as an effector of VANGL2. This study identifies a pivotal role for VANGL2 in mechanosignaling, which underlies the key role of the PCP pathway in tissue morphogenesis.

The benefits of using silk fibroin, a major protein in silk, are widely established in many biomedical applications including tissue regeneration, bioactive coating and in vitro tissue models. The properties of silk such as biocompatibility and controlled degradation are utilized in this study to formulate for the first time as carriers for pulmonary drug delivery. Silk fibroin particles are spray dried or spray-freeze-dried to enable the delivery to the airways via dry powder inhalers. The addition of excipients such as mannitol is optimized for both the stabilization of protein during the spray-freezing process as well as for efficient dispersion using an in vitro aerosolisation impactor. Cisplatin is incorporated into the silk-based formulations with or without cross-linking, which show different release profiles. The particles show high aerosolisation performance through the measurement of in vitro lung deposition, which is at the level of commercially available dry powder inhalers. The silk-based particles are shown to be cytocompatible with A549 human lung epithelial cell line. The cytotoxicity of cisplatin is demonstrated to be enhanced when delivered using the cross-linked silk-based particles. These novel inhalable silk-based drug carriers have the potential to be used as anti-cancer drug delivery systems targeted for the lungs.

Abstract Background: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were used in this study due to their potential use in inflammatory and fibrotic conditions of the lung. This study aimed at demonstrating that hAMSCs can be atomized with high viability, for the purpose of achieving a more uniform distribution of cells throughout the lung. Another aim of this study was to set ground for future application to healthy and diseased lungs by demonstrating that hAMSCs were able to survive after being sprayed onto substrates with different stiffness. Methods: Two methods of atomization were evaluated, and the LMA MAD780 device was selected for atomizing hAMSCs for optimized delivery. To mimic the stiffness of healthy and diseased lungs, gelatin gel (10% w/v) and tissue culture plastic were used as preliminary models. Poly-l-lysine (PLL) and collagen I coatings were used as substrates on which the hAMSCs were cultured after being sprayed. Results: The feasibility of atomizing hAMSCs was demonstrated with high cell viability (81 ± 3.1% and 79 ± 11.6% for cells sprayed onto plastic and gelatin, respectively, compared with 85 ± 4.8% for control/nonsprayed cells) that was unaffected by the different stiffness of substrates. The presence of the collagen I coating on which the sprayed cells were cultured yielded higher cell proliferation compared with both PLL and no coating. The morphology of sprayed cells was minimally compromised in the presence of the collagen I coating. Conclusions: This study demonstrated that hAMSCs are able to survive after being sprayed onto substrates with different stiffness, especially in the presence of collagen I. Further studies may advance the effectiveness of cell therapy for lung regeneration.

Background: Extracellular vesicles (EVs) are specialized, nanoscale messengers that deliver biological signals. The evidence shows that within populations of EVs, important properties including morphology, composition and content vary substantially. Thus, measuring EV heterogeneity is paramount to our understanding of how EVs influence physiological and pathological functions of their target cells. Thus far, devising effective methods for measuring EV heterogeneity remains a global challenge. Methods: We present, for the first time, a study of the molecular and structural composition of individual EVs, subpopulations of EVs and whole populations of EVs using resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR). This approach is labelfree, has ultra-high sensitivity and has the power to measure EV heterogeneity. EVs were isolated from placenta stem cells using ultrafiltration and after further purification using the additional size-exclusion chromatography column and both methods were compared. Results: We demonstrated for the first time the possibility to characterise individual EV at nanoscale, EV populations and showed the critical differences in their composition depending on extraction protocols - heterogeneity. Ultra-high resolution of AFM-IR that allows probing of multiple points on individual EVs is key to develop new extraction and separation protocols for EVs and to unlock their full therapeutic and diagnostic potential. Our approach outperforms other methods for vesicles characterization providing unmatched resolution (single vesicle) and is \"probe free\", thus it avoids bias and resolution limitations of molecular probes. Summary/Conclusion: The AFM-IR is advancing the EV field forward by revealing their molecular constituents and structures, as well as enabling purity assessment of EV preparations. The data presented in this study suggest AFM-IR can transform existing protocols for interrogating EV composition and structures, and assessing EV purity. This nanoscale technique can be developed into a powerful screening tool for detecting specific EV \"fingerprints\" that are associated with pathology by correlating the structural differences to biomarkers, addressing unmet clinical needs in diseases where early diagnosis is critical, for example multiple sclerosis or cancer.