The classical model of extracellular vesicles (EVs) is a simple one. You can likely conjure the image in your mind easily enough. For me, that image is the classical cross section showing the lipid bilayer dotted with membrane proteins, all surrounding a few proteins and microRNAs in the vesicle lumen. That’s it, simple. A little too simple for biology, unfortunately. Just as EV subtype classification has had to evolve over the years, it is well past time for that classical image of an EV to evolve too.
One of the biggest changes that we will have to make to our mental image of an EV is the addition of the protein corona. This ‘shell’ of proteins cloaks the outside of EVs, and early research suggests the protein corona may have two layers.1 The first is a ‘hard corona’ which is more stably associated with the EV surface. The second is a ‘soft corona’ which is likely quite easily lost during EV isolation due to weaker associations with the EV surface. In this article, we will discuss how the EV protein corona was first identified, what proteins are likely part of the EV corona, and what impact the existence of the EV protein corona has on EV research.
The birth of a new field: The discovery of the extracellular vesicle protein corona
The idea of a protein corona in and of itself is nothing new. This phenomenon has been both vexing and delighting those working on nanoparticles for nearly 20 years. A protein corona forms spontaneously around many, if not all, nanoparticles in biological fluids. It only makes sense then that one might also form around EVs. It is somewhat fitting that the first publication which aimed to investigate whether EVs have such a corona was published in August of 2020, at a time when the whole world was highly aware of the word ‘corona’ for perhaps the first time.
Palviainen et al., compared the blood-derived EV proteome to that of the coronas surrounding synthetic nanoparticles in previous studies, and found substantial overlap.2 They also identified that the EV corona was likely altered by anticoagulant type, which was already known to alter EV properties. Of note, the researchers in this study decided to deplete albumin from their EV samples using an albumin-depleting resin. This resulted in a change in the EV proteome. The reason for depleting albumin is not immediately clear in the paper and may have stemmed from the classical view of albumin as a contaminant in EV isolates. But is albumin really a contaminant, or could it and other classical EV isolate contaminants actually be part of the EV corona?
Contaminant or corona – determining the fate of extracellular vesicle isolate proteins
Whilst the first EV corona study discussed above did not separate the protein corona from the whole proteome of EVs, subsequent studies have aimed to do just that. In a 2021 study from the lab of Edit I. Buzás, researchers took EVs from cell culture and mixed them with EV-depleted plasma, before re-isolating using different methods and comparing the resulting proteomes with those of the original cell culture EVs.3 More proteins were present after the incubation with EV-depleted plasma, and these were designated as EV corona proteins. Interestingly, different EV isolation techniques resulted in different proteins being identified as potential EV corona proteins, suggesting that perhaps some elements of the protein corona might not survive all isolation methods. However, some proteins which are not considered EV proteins, including albumin and fetuin-A, were in both datasets and were excluded. Again, likely due to concerns that they were contaminants.
Whilst subsequent studies have replicated the general themes of these results, showing that cell culture EVs pick up extra proteins in EV-depleted plasma or serum, some have shown the presence of albumin in a different light.1,4-6 Liam-Or et al., identified that albumin is part of the hard corona, and may even facilitate the corona formation.1 Furthermore, competitive inhibition of albumin receptors in vivo resulted in decreased uptake of EVs with a corona. In fact, albumin was one of the most common and abundant corona proteins identified in at least 3 studies.1,4,5 In another, exposing corona-free EVs to albumin and growth factors recapitulated a functional artificial corona.6 More recently, Singh et al., used antimicrobial peptides to disrupt the EV corona through competitive electrostatic binding. This approach allowed them to ‘spot the difference’ between treated and untreated EVs, enabling the identification of proteins that had been lost and were therefore, corona proteins.5 They identified 17 core proteins, of which albumin was one.
In addition to albumin, lipoprotein proteins such as ApoA1 and ApoB, complement proteins such as C3, and integrins were amongst the most frequently identified corona components.3-7 The presence of these proteins as integral parts of the protein corona surrounding EVs calls into question the classification of some proteins in EV isolates as contaminants. Albumin in particular looks set to become less of an undesired contaminant and more of a welcome sign of intact EV corona status.
Where – and how – does the extracellular vesicle corona form?
To date, the EV corona has mostly been discussed as forming in the blood, and this has been recapitulated by exposing EVs to EV-depleted plasma or serum. However, the presence of known corona proteins in the proteome of EVs from cell culture suggest that perhaps a corona of sorts forms in cell culture too. This seems logical as conditioned cell culture media is full of secreted proteins of all kinds. If – as it appears – the corona forms spontaneously through electrostatic and perhaps affinity interactions, then this should also occur in cell culture. Albeit, to a lesser extent due to a lower concentration of proteins than in plasma or serum. The logical result of this is that EVs produced in cell culture will have a different corona than those same EVs would have in vivo. But does this matter? Well, if the EV corona is functional, then yes.
A functional corona
Studies have shown the EV corona to indeed be highly functional. The presence of the EV corona created in plasma or serum increases EV uptake1,4, alters in vivo distribution1 and results in functional differences to immunomodulation, regeneration and angiogenesis.6,7 It is not yet known whether the difference in functionality results from differences in uptake.
Implications for EV research and applications
It will be important to determine the impact of different EV isolation techniques on the integrity of the corona. The results of this might mean that existing protocols and commercial products may need to be tweaked in order to maximise EV corona integrity in isolates. However, alongside this, our basic understanding of the protein corona on EVs and its properties must be advanced. Towards this, there are a number of questions which we feel require further investigation. Could the protein corona impact upon the lysis of EVs (by protecting the EV from detergents) and/or could the protein corona physically shield EV markers from antibody-based detection in whole EVs? Or would the affinity of antibodies for their antigen overcome the electrostatic forces (presumably) holding the EV protein corona together? Finally, how do our current buffers and storage methods impact upon the maintenance of EV corona integrity?
There are also several implications of the EV corona for research. Firstly, it is possible that future EV biomarkers may be part of the EV corona, which will have implications for designing the isolation workflow. Approaches that maintain the integrity of the corona will be essential here. Next is therapeutics. Whilst EVs may pick up some kind of protein corona in cell culture, they may not pick up a sufficient corona to optimise uptake and functionality in patients. This could be addressed by adding in a step where cell culture EVs are exposed to EV-depleted plasma or serum.
Looking ahead: harnessing the potential of the EV corona
Understanding and harnessing the potential of the EV protein corona presents an exciting avenue for future research and applications. The evolving narrative surrounding EV biology now encompasses not just the vesicles themselves but the dynamic interplay between their surface proteins and the surrounding environment. Moving forward, understanding the EV protein corona in more depth, and subsequently, deciphering the impact of isolation techniques on its preservation, will be paramount to paving the way for corona-based diagnostics, targeted drug delivery systems, and therapeutic interventions. More than anything though, the discovery of the EV corona emphasises the importance of basic EV research and never just accepting the status quo.
