In the past decade, extracellular vesicles (EVs) have emerged as a key cell-free strategy for the treatment of a range of pathologies, including cancer, myocardial infarction, and inflammatory diseases. Indeed, the field is rapidly transitioning from promising in vitro reports toward in vivo animal models and early clinical studies. These investigations exploit the high physicochemical stability and biocompatibility of EVs as well as their innate capacity to communicate with cells via signal transduction and membrane fusion.
Researchers from Imperial College London review methods in which EVs can be chemically or biologically modified to broaden, alter, or enhance their therapeutic capability. They examine two broad strategies, which have been used to introduce a wide range of nanoparticles, reporter systems, targeting peptides, pharmaceutics, and functional RNA molecules. First, they explore how EVs can be modified by manipulating their parent cells, either through genetic or metabolic engineering or by introducing exogenous material that is subsequently incorporated into secreted EVs. Second, the researchers consider how EVs can be directly functionalized using strategies such as hydrophobic insertion, covalent surface chemistry, and membrane permeabilization. They discuss the historical context of each specific technology, present prominent examples, and evaluate the complexities, potential pitfalls, and opportunities presented by different re-engineering strategies.
Strategies for EV modification
(a) Genetic engineering can be used to introduce coding and noncoding oligonucleotides into cells. There it can be packaged into EVs to promote gene expression or regulate transcription in recipient cells. Alternatively, transgenic proteins can be incorporated into EVs, for instance, as fluorescent reporters or targeting moieties. (b) Metabolic labeling, in which metabolite analogues are incorporated into cell biosynthesis, has been widely used to introduce non-native moieties into cells. This approach can be used to introduce functional groups, such as azides, to EVs, which allows subsequent bio-orthogonal reactions to be performed. (c) Exogenous material may be introduced to EVs via liposomes or micelles that fuse with cytoplasmic membranes. (d) Alternatively, the process of packaging endocytosed material into EVs as part of normal membrane turnover and exocytosis can be hijacked to introduce exogenous species to EVs. (e) A direct EV modification strategy is to permeabilize the vesicle membrane to allow the active loading of molecules into the EV interior, an approach that has been exploited for drug delivery. (f) A similar approach uses lipophilic or amphiphilic molecules that can insert into the EV membrane via hydrophobic interactions with the phospholipid bilayer. (g) Chemical reactions may also be performed directly on the vesicle membrane, for instance, carbodiimides can be used to modify native amines in order to present azide groups for click chemistry reactions.