In the last 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 towards 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 over long distances via signal transduction and membrane fusion. This review will focus on methods in which EVs can be chemically or biologically modified to broaden, alter or enhance their therapeutic capability. We will 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, we will 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, we consider how EVs can be directly functionalized using strategies such as hydrophobic insertion, covalent surface chemistry and membrane permeabilization. We will 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. KeywordsExtracellular Vesicles; Exosomes; Microvesicles; Functionalization; Genetic Manipulation; Drug Loading; Membrane Modification; Cell-Free Therapy Extracellular Vesicles: Cell-Derived Nanovectors Extracellular vesicles (EVs) are a collective of small, naturally-derived particles, which, until recently, represented an overlooked and underappreciated component of the cellular secretome. Three major categories of EV have been defined, predominantly based upon vesicle biogenesis, but with notable differences in size and composition.1,2 Exosomes are formed when the peripheral membrane of multivesicular bodies (MVBs) undergo reverse budding to form small nanovesicles (30-100 nm in diameter) that are released when MVBs fuse with the cytoplasmic membrane.3 Microvesicles are larger in size (c.f. 100-1000 nm) * m.stevens@imperial.ac.uk. Europe PMC Funders GroupAuthor Manuscript ACS Nano. Author manuscript; available in PMC 2017 September 19.Published in final edited form as: ACS Nano. 2017 January 24; 11(1): 69-83. doi:10.1021/acsnano.6b07607. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts and are produced during shedding or budding of the cytoplasmic membrane.4 Exosomes and microvesicles are produced by healthy cells as part of regular membrane turnover and exocytosis. In contrast, apoptotic bodies (c.f. 500-2000 nm) are generated from outward membrane blebbing in cells undergoing apoptosis.5 Apoptotic bodies, microvesicles and exosomes are each enclosed by a phospholipid membrane bilayer, comparable to the cytoplasmic mem...

Cubosomes are highly stable nanoparticles formed from the lipid cubic phase and stabilized by a polymer based outer corona. Bicontinuous lipid cubic phases consist of a single lipid bilayer that forms a continuous periodic membrane lattice structure with pores formed by two interwoven water channels. Cubosome composition can be tuned to engineer pore sizes or include bioactive lipids, the polymer outer corona can be used for targeting and they are highly stable under physiological conditions. Compared to liposomes, the structure provides a significantly higher membrane surface area for loading of membrane proteins and small drug molecules. Owing to recent advances, they can be engineered in vitro in both bulk and nanoparticle formats with applications including drug delivery, membrane bioreactors, artificial cells, and biosensors. This review outlines recent advances in cubosome technology enabling their application and provides guidelines for the rational design of new systems for biomedical applications.

Atherosclerosis results in the narrowing of arterial blood vessels and this causes significant changes in the endogenous shear stress between healthy and constricted arteries. Nanocontainers that can release drugs locally with such rheological changes can be very useful. Here, we show that vesicles made from an artificial 1,3-diaminophospholipid are stable under static conditions but release their contents at elevated shear stress. These vesicles have a lenticular morphology, which potentially leads to instabilities along their equator. Using a model cardiovascular system based on polymer tubes and an external pump to represent shear stress in healthy and constricted vessels of the heart, we show that drugs preferentially release from the vesicles in constricted vessels that have high shear stress.