Unraveling multilayered extracellular vesicles: Speculation on cause

Abstract Extracellular vesicles (EVs) are cell‐released, heterogenous nanoparticles that play important roles in (patho)physiological processes through intercellular communication. EVs are often depicted as having a single lipid bilayer, but many studies have demonstrated the existence of multilayered EVs. There has been minimal inquiry into differences between unilamellar and multilamellar EVs in terms of biogenesis mechanisms and functional effects. This commentary speculates on potential causes and roles of multilamellar EVs and serves as a call to action for the research community to unravel the complex layers of EVs.


 of 
LETTER TO THE EDITOR F I G U R E  Speculative mechanisms for the formation of multilamellar extracellular vesicles (EVs). An additional speculative mechanism, which has been proposed for lamellar bodies (secretory multilamellar organelles) is illustrated and described in a previous study (Klein et al., 2021). sucrose buffer Tian et al., 2020;Walker et al., 2022;Yang et al., 2022). Other studies reported multilamellar EVs in samples that did not undergo storage prior to cryo-TEM (Issman et al., 2013;Yuana et al., 2013). Since multilayered EVs were observed with cryo-TEM in fresh samples (Issman et al., 2013;Yuana et al., 2013) and samples stored in a cryoprotective buffer Tian et al., 2020;Walker et al., 2022;Yang et al., 2022), it is less likely that the presence of multilayered EVs can be solely attributed to storage artifacts.
Taken together, it is unlikely that isolation and storage artifacts are the sole causes of multilamellar EVs. Therefore, it is plausible that various biogenesis mechanisms result in multilayered EVs (Figure 1). For example, it can be speculated that multivesicular structures are formed inside multivesicular bodies through encapsulation of smaller intraluminal vesicles in larger ones, although evidence of this process has not been reported. An alternative mechanism for the formation of multivesicular structures was proposed for lamellar bodies, which are secretory multilamellar organelles found in certain epithelial cells. The proposed model involves flipping of phospholipids from the outer membrane leaflet to the inner one, causing the formation of perpendicular internal membrane sheets that detach, grow, and eventually form curved arrangements (Klein et al., 2021).
It can also be speculated that the presence of intracellular vesicles in close vicinity to the cell membrane could result in doublelayer EVs upon membrane budding. In certain cases, intracellular vesicles have been found to encapsulate entire membranebound organelles, such as mitochondria (Phinney et al., 2015). Such mitochondria-containing vesicles can be transported to the cell surface where outward membrane budding occurs (Phinney et al., 2015). It is also possible that additional EV layers could form through several rounds of cellular internalization, structural preservation, and release through membrane budding, although evidence of this is lacking. Gram-negative bacteria have been shown to release double-layer EVs through simultaneous budding of the cytoplasmic membrane and outer membrane ( Perez-Cruz et al., 2013;Toyofuku et al., 2019). This type of EV formation is restricted to prokaryotes with two membranes, although it is possible that eukaryotic membrane-bound organelles in close proximity to the cell surface could undergo simultaneous budding (fragmentation) with the cell membrane.
While differences between unilamellar and multilamellar EVs in terms of functional effects have not been explored, liposomes and other synthetic particles are frequently designed to have multiple layers to enable controlled and sustained drug delivery (Boyer & Zasadzinski, 2007;Chen et al., 2020;Peyret et al., 2017;Scavo et al., 2015;Seong et al., 2018;Shen, 2015). In such cases, cargo release is obtained through stimulus-sensitive or passive degradation-mediated removal of layers. Multilamellar liposomes also enable increased loading of hydrophobic drugs that are embedded in the bilayers (Kulkarni & Vargha-Butler, 1995). Therefore, if form follows function, it is reasonable to speculate that EVs have similar mechanisms for controlled/sustained delivery of cargo and increased packaging ability of membrane-embedded components.
Studies also suggest that the formation of multilamellar EVs is impacted by pathological processes and disease states. For example, Tatiana et al. noted that more multilayered EVs were found in the plasma of Gaucher disease patients (Tatiana et al., 2020). Gaucher disease is characterized by an inability to efficiently metabolize glycolipids (Tatiana et al., 2020), which could potentially impact EV biogenesis and degradation or trigger a compensation mechanism consisting of increased intercellular lipid transfer. On the contrary, it was shown that prion-infected cells have reduced abundance of multilayered EVs, potentially suggesting that prions packaged within EVs interfere with a multilamellar biogenesis process (Coleman et al., 2012).

F I G U R E  Cryogenic transmission electron microscopy images of EVs from human THP-1 monocyte-like cells grown in the absence or presence of lipopolysaccharide (LPS).
Scale bars correspond to 100 nm. These specific images have not previously been published, but the methods for cell culture, EV isolation, and imaging are described in a previous study .
Studies have also assessed EV morphology upon cell exposure to lipopolysaccharide (LPS), a bacterial endotoxin. In response to this endotoxin, immune cells release EVs with bioactive cargo that initiate inflammatory responses in recipient cells (Gebraad et al., 2018;Puhm et al., 2019;Tang et al., 2016;Wang et al., 2011). Figure 2 and a recent study demonstrate that LPS stimulation of monocytes increases the formation of multilayered EVs . Additionally, EVs from LPS-stimulated monocytes were enriched in membrane-bound glucose transporter-1 (GLUT1) , potentially enabled by the increase in multilamellar structures. GLUT-1 is known to accelerate inflammatory pathways (Peiró et al., 2016), providing a mechanism by which EVs can induce an intercellular signaling cascade triggered by LPS. The presence of multiple layers may also cause slower release of EV cargo in recipient cells, increasing the time period for intercellular communication about threatening signals in the environment. The number of layers could potentially confer time-dependent information about signals in the intra-and extracellular environment. For example, longer exposure to a stimulating agent, such as LPS, may increase EV layers. Another unanswered question regarding multilamellar EVs is whether each internal compartment separated by a lipid bilayer differs in cargo composition.
Alternatively, multilamellar EVs may be a mechanism by which to provide recipient cells with large quantities of lipid biomolecules to aid in the repair of potential pathogen-induced cell damage. It is important to note that LPS may directly cause membrane damage, as studies have shown that this agent can create holes in lipid bilayers (Adams et al., 2014(Adams et al., , 2015. Additionally, LPS-stimulated monocyte-derived EVs appeared more damaged than those from non-stimulated conditions . However, it is unclear whether potential LPS-induced membrane damage would accelerate EV fusion and/or formation of multilamellar structures. Taken together, metabolic disorders (Gaucher disease) and the presence of prions or bacterial endotoxins have been shown to impact the number of layers present in EVs. The significance of such findings in health and disease remains unknown.
This commentary highlights the lack of research into the cause and role of multilayered EVs, which have been identified in conditioned cell culture media and human biofluids. Further studies are required to determine biogenesis mechanisms, biological function, and relevance of multilayered EVs, which is likely to open opportunities for new treatment strategies, therapeutic targets, and/or biomarkers for diseases, such as those caused by inflammatory signals or metabolic disfunction.

A C K N O W L E D G E M E N T S
Partial funding was provided by The University of Queensland, Australia (JW) and the Israel Science Foundation, Israel under award 2302/20 (YT). The cryo-TEM work was performed at the Technion Center for Electron Microscopy of Soft Matter. The content is solely the responsibility of the authors and does not necessarily represent the official views of the organizations and funding agencies.