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Extracellular vesicles as vehicles for the delivery of food bioactives Agnes Reiner, and Veronika Somoza J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06369 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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Journal of Agricultural and Food Chemistry

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Extracellular Vesicles as Vehicles for the Delivery of Food Bioactives

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Agnes T. Reiner 1, Veronika Somoza 1†

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Department of Physiological Chemistry, Faculty of Chemistry, University of Vienna, Althanstraße 14 UZA II, 1090 Vienna, Austria Corresponding author: [email protected], telephone: +4314277-70611

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Keywords: Extracellular vesicles, drug delivery, bioactives, food additives

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Abstract:

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The nutritional value of foods can be improved by the addition of bioactive compounds.

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However, most of these favorable food additives demonstrate a low bioavailability because of

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their limited stability, solubility, and structural transformations upon digestion and

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absorption. One strategy to combat these limitations is to integrate bioactives into

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nanoparticles, although the mostly used artificial materials may result in immune system

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activation and fast clearing times. Therefore, novel, more biocompatible delivery systems are

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required. Extracellular vesicles are communication tools designed by evolution to transfer

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information between cells, organs, and whole organisms. Hence, these vesicles offer an

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enormous potential for targeted bioactive compound delivery.

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Introduction

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Bioactive compounds, like polyphenols, vitamins or polyunsaturated fatty acids, are often

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added to foods to increase their nutritional value. However, the effect of such bioactives can

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be impaired by their poor bioavailability due to structural transformations upon digestion and

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absorption, their limited water solubility, and metabolic transformations. For some of the

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bioactives, side effects, e.g., as interactions with drugs, are reported. For instance,

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polyphenolic compounds from bilberry extracts can inhibit the cytostatic effect of the

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anticancer drug erlotinib 1. Although the emergence of nanotechnology and the use of

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nanoparticles has markedly improved the bioavailability of food bioactives, nanoparticles may

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also demonstrate non-desired effects as they may cause immune reactions and sometimes

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even cytotoxicity. Cellular clearance of nanoparticles is also of concern since nanoparticles

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have been shown to be taken up by, e.g., intestinal cells

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mechanism has been identified yet. Within the last decades, a new class of cellular secretion

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products, namely extracellular vesicles (EVs), has emerged that demonstrates an enormous

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potential to achieve highly efficient delivery of bioactive compounds while overcoming many

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of the mentioned issues.

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Scientific Relevance of EVs

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The history of EVs started in the early 1980’s with the discovery of exosomes, one class of EVs.

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Two independent groups 4-5 identified lipid vesicles in the size of about 100 nm in diameter

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that were formed and secreted by a novel pathway described in more detail below. At this

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early stage, researchers believed that exosomes were only built as a cellular waste disposal

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system for removal of unneeded proteins. It took about another 15 years until exosomes

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gained more attention, when Raposo et al. 6 discovered that such small lipid vesicles were also

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but no cellular excretion



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secreted by B lymphocytes and were able to activate T cells via antigen presentation and MHC

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class II signaling, thereby triggering an immune response. Based on these findings, a new field

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of research developed that is still on the rise with publication records exponentially increasing

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over the last ten years. Nowadays, we know that EVs are important players in intercellular

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communication and transfer information as proteins, nucleic acids and lipids from one cell to

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another. EVs are secreted by all types of cells and are found in biofluids like blood, saliva,

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urine, cerebrospinal fluid, breast milk and others 7. EVs are also not just limited to humans and

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other mammals, they are produced by cells across all kingdoms, as they have been identified

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and isolated from bacteria, protozoa, or plants 7. Most interestingly, EVs were shown to

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transfer information, in particular small RNAs, across kingdoms, for instance from plants to

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fungi to inactivate virulence genes 8. A transfer of plant micro RNAs contained in EVs to

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mammals through food uptake has also been suggested 9. Even though such findings are

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especially interesting for the field of agriculture and food, so far, EVs are topic of only very few

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studies in this area. The majority of records on EVs found in Web of Science are categorized in

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the fields of cell biology, biochemistry, oncology, and several other fields associated to

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biomedicine, as illustrated in Figure 1, which shows the top 15 categories that comprise most

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of the records on EVs of the year 2017. Web of Science records on EVs were also found in the

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categories “Food Science Technology”, “Nutrition Dietetics”, “Plant Sciences”, “Agriculture

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Dairy Animal Science”, “Agriculture Multidisciplinary”, and “Agronomy”, which are associated

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to the field of agriculture and food, but they sum up to only 55 of a total number of 3,482

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records on EVs, which corresponds to 1.58 %.

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Besides the fact that EVs are a young research field with many open questions that are studied

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by basic science, applications of EVs are already on the rise. For instance, EVs evolved as a new

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class or source of biomarkers, since they are found in many biological fluids and therefore can 4 ACS Paragon Plus Environment

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be used as non-invasive biomarkers. For this purpose, the different constituents of EVs,

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namely proteins, small RNAs, and lipids, or simply the concentration of EVs demonstrate a

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potential as diagnostic or prognostic biomarkers for a wide range of diseases. Furthermore,

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based on the physiological functions of EVs, their application as active agents or therapeutics

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in the fields of tissue regeneration, immune modulation, cancer therapy and others are

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studied. Another application of EVs is their use for targeted delivery of compounds to cells.

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Since EVs are built naturally by cells with the purpose to deliver information to other cells,

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they are an optimal system for compound delivery. Furthermore, EVs can be harvested from

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sources like milk, which is safe for human ingestion and readily available in large quantities.

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So far, EVs have been intensely studied for drug delivery over the last years, as illustrated by

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the rising numbers of publications in Figure 1A. Most of these studies explore the potential of

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EVs in medical applications, and their findings could be translated to the field of bioactives’

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delivery via foods. This perspectives paper summarizes the key characteristics of EVs and

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compares their potential with artificially produced delivery systems like liposomes.

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Types of EVs, Biogenesis and Characteristics

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According to the International Society of Extracellular Vesicles (ISEV), EVs are categorized into

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three groups depending on their biogenesis: exosomes, microvesicles, and apoptotic bodies.

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As illustrated in Figure 2A and reviewed by van Niel et al. 10, exosomes are built through the

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inward budding of the late endosomal membrane, leading to the formation of multivesicular

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bodies, which later fuse with the plasma membrane and secrete the contained exosomes to

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the extracellular space. Microvesicles, on the other hand, form through direct budding from

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the plasma membrane. The third class of EVs are apoptotic bodies, which are generated during

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the process of controlled cell death. These vesicles are just mentioned for completeness, but 5 ACS Paragon Plus Environment


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they have no value for applications in drug delivery and are therefore not discussed further.

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In this article, the term of EVs only relates to exosomes and microvesicles.

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The size of EVs ranges from a few 10 nm to about 1000 nm in diameter, with exosomes tending

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to be smaller than microvesicles 11. Generally, all EVs are surrounded by a lipid bilayer, which,

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due to their biogenensis, has the same orientation as the cellular plasma membrane. Figure

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2B shows cryo-electron microscopy images of EVs, for illustration of the size and shape of EVs.

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Labelling with gold nanoparticles was used to better characterize the lipid bilayer of the

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imaged vesicles 12. EVs contain soluble and transmembrane proteins, as well as different types

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of nucleic acids, including small RNA, micro RNA, messenger RNA, and DNA. Their composition

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is specific for their secreting cell and the loading is performed by distinct and controlled

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pathways within the cells 10. However, the exact pathways and mechanisms responsible for

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the formation and specific loading of EVs is not yet known. The composition of EVs results in

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distinct functions of vesicles and the ability to target specific cells. The diverse physiological

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functions of EVs were extensively reviewed by Yanez-Mo et al. 7. EVs are able to exert their

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function via receptor signaling from the outside, or release of their cargo into the cytoplasm

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via endocytosis or fusion with the target cell membrane. Especially interesting is the finding

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that cargo molecules of EVs can also be transported into the cell nucleus of the recipient cells

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and it has been suggested that they are transported by a novel mechanism independent of

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the known nuclear translocation sequence pathway 13.

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As mentioned in the introduction, EVs are not only found and produced in the human body.

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They have also been identified in all kinds of animals and plants. For the field of agriculture

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and food, the presence of EVs in crop plants, life stock, and natural food products is of special

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interest. For example, EVs in milk of diverse mammalian species are studied already in great 6 ACS Paragon Plus Environment

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detail, while the identification of EVs in plants and fruits, like carrots, ginger, nuts, seeds,

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grapefruits, grapes, watermelons, and others, are still in its beginnings. More details on the

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identification of EVs in food and their potential functions can be found in the review of Perez-

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Bermudez et al. 14.

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Concerning the methods used for isolation, characterization, and functional analysis there are

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plenty of different approaches currently used in EV research, but no optimal or standard

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protocols have been identified so far. Nevertheless, a primary aim of the International Society

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of Extracellular Vesicles is to support and pursue standardization within the field. More

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information on different methods for EV research can be found in one of ISEV’s position

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papers

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researchers in planning and setting up their experiments 16.

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EVs as delivery tool for bioactives

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EVs are purposed to transfer cargo molecules between cells in close proximity but also to

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distant sites. They have been designed and built by evolution to become a perfect tool for

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cellular communication. Consequently, EVs offer significant advantages for the use as delivery

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tool compared to artificial nanoparticles. In the following paragraphs, the characteristics of

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EVs as drug delivery system are compared to those of artificial nanoparticles. Due to the great

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variety of nanoparticles and their properties, only liposomes are used for comparison, because

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these are the most similar representatives of nanoparticles to EVs. The key characteristics of

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biological nanoparticles (EVs) and artificial nanoparticles (liposomes) for the use as drug

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delivery system are summarized in Table 1 and discussed point by point in the following

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paragraphs and sections.

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and the just recently published requirements for studies of EVs that should assist



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First of all, EVs have the inherent function of protecting molecules from the extracellular

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environment and delivering them to their target cells or tissues, while liposomes need to be

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engineered and artificially synthesized to be useful for applications. Due to their composition

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of a lipophilic membrane layer and a hydrophilic core, both, EVs and liposomes, are able to

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carry lipophilic and hydrophilic compounds, allowing a wide range of applications. Moreover,

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the small biological vesicles stand out due to their extremely high stability in harsh

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environments. Besides the finding that the lipid bilayer protects their cargo from enzymatic

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degradation by nucleases or proteinases, EVs can resist low pH, high sheer stress, prolonged

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times at body temperature, all found in human digestion. This has already been proven for

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milk- 17 and grapefruit- 18 derived EVs by exposing the vesicles to a mimicked digestive system

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in vitro, including digestive juices, enzymatic activity and physical conditions. Since bioactives

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are added to foods that are taken up orally, the resistance of EVs to digestion is extremely

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important for protection of the bioactive compound. Moreover, the ability of EVs to protect

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their cargo from the environment could also be used the other way around and prevent

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interactions of the bioactive with pharmaceuticals present in the human body. However, this

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potential effect was not studied yet. Of course, liposomes protect their cargo as well, since

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this is one of their major functions, but their stability is greatly dependent on the materials

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that are used.

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The next hurdle for bioactive compound delivery systems is the uptake by cells, typically by

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intestinal cells. Several studies have shown that natural EVs, isolated from plants or milk and

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artificially loaded with drugs, are readily taken up by intestinal cells in vitro and in vivo,

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respectively

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agriculture and food, since food-derived EVs could be used as delivery vehicle, which is

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generally safe and non-toxic to humans based on the fact that humans have been exposed to 8

19-21.

These findings are of particular interest for applications in the field of

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these plant- or milk-derived EVs over centuries without experiencing negative effects. Quite

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the contrary, food-derived EVs might even have beneficial physiological functions in the

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human body, as suggested by Mu et al.

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grapefruit, ginger, or carrots had differing anti-inflammatory, antioxidant or pro-proliferative

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effects on the intestinal macrophages. Such effects could further enhance the activity of

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bioactive compounds integrated into the EVs. However, the bioactivity of EVs by themselves

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could also limit their use as drug delivery system, because many physiological functions are

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yet unknown. In this regard, liposomes are very well established delivery systems, as they have

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been studied already for several decades and their composition can be optimized for efficient

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stability and cellular uptake and low or no side effects.

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The small size in the nanoscale enables both liposomes and EVs to cross barriers within the

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human body easier compared to larger particles. This advantage of nanoparticles is critical for

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the efficient delivery of the cargo molecules to their target cells and tissues. However, certain

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highly regulated barriers, like the blood-brain barrier, still pose a big hurdle for artificially

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produced liposomes. EVs could solve these issues, because they are able to cross the blood-

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brain barrier as shown in studies by Alvarez-Erviti et al.

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exosomes to the mouse brain after intravenous injections. Hence, bioactive compounds that

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are supposed to trigger reactions in the central nervous system could be transferred from the

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digestive tract via the blood system into the brain by EVs.

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The finding that EVs present in tissues or circulating in the blood system are not recognized

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and cleared by the immune system due to their natural composition, explains the long

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circulation times and high bioavailability of EVs. Liposomes on the other hand need to be

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modified, for example by polyethylene glycol coatings, to achieve better immune-tolerance,

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who showed that EVs derived from grapes,

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who administered siRNA via



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but such coatings may negatively influence the interaction with target cells. In addition to this

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low immunogenic profile, drug delivery systems also need to be safe and non-toxic to the

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human body. For EVs, this can be very dependent on the cell of origin and the inherent

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biological functions of the vesicles. For instance, EVs derived from cancer cells bear the risk of

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transferring pro-cancerous activities, therefore they might not be safe to use for compound

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delivery. Food-derived EVs, on the other hand, could be a much safer approach. Somiya et al.

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23,

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being administered intravenously.

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Besides the efficacy of delivery systems in their function, their efficient and economic

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production is also of importance for their successful application. In this regard, the source of

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the material has to be chosen carefully. The material for encapsulation of the compound has

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to be available in large enough quantities and at affordable prices, which are both

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requirements for scale-up and commercial distribution of the encapsulated compound as food

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additive. For EVs, both can be achieved through the use of foods as source. Bovine milk has

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already been studied as a source for EVs intensively and showed promising results for drug

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and bioactive compound delivery

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great potential because of their availability and safety. Additionally, the material has to be

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compatible with the food matrix it is supposed to be added to, which again favors the use of

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food-derived EVs.

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When it comes to loading and encapsulation of the bioactive compound into EVs, several

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different approaches can be followed. Isolated EVs, that contain no bioactive material, can

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simply be mixed together with the compound of interest and the loading is performed by

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diffusion, electroporation or chemical transfection. This approach is basically the same as

for example, showed that EVs isolated from bovine milk are well tolerated by mice after

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Furthermore, EVs isolated from edible plants show


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performed for liposomes. As example, Aqil et al.

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anthocyanidines into milk-derived EVs this way and studied their anti-tumor activity in vitro

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and in vivo. In more detail, the study showed that the survival of drug-resistant ovarian cancer

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OVCA433 cells decreased when incubated with the encapsulated compounds compared to the

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free anthocyanidines. Similarly, when administering the compounds orally to mice in a

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xenograft approach using ovarian cancer A2780 cells, the resulting tumor volume was

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significantly lower in mice that had received the anthocyanidines encapsulated into EVs after

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17 days of treatment. Another approach is to engineer the cellular source of EVs in order to

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let the cell pack the compound of interest into the EVs already before secretion. This approach

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of course requires a deep understanding of gene editing and knowledge on the pathways

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involved in EV biogenesis, which can be limiting. Nevertheless, the technique also allows to

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include molecules for targeting of EVs to certain organs, tissues or cells for drug release. This

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cellular transfection-based approach to load EVs has already been applied for delivery of

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proteins 27, small RNA molecules 28 or for triggering signaling cascades like immune responses

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in the recipient cells 29. A third way to load EVs is the possibility to fuse biological vesicles with

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artificial liposomes. With this strategy, the best of both fields could be combined by using the

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biological membrane of EVs to achieve higher biocompatibility and fuse them with liposomes

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containing the targeting or tagging molecules and the active compound. Just very recently, a

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novel technique for EV-liposome fusion was demonstrated by Piffoux et al.

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polyethylene glycol to merge biological EVs derived from human umbilical vein endothelial

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cells or mesenchymal stem cells with artificially produced liposomes loaded with different

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cargoes. Furthermore, EVs that naturally contain bioactive compounds could be used, for

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instance from plants that produce polyphenols that are secreted via EVs by nature. In this way,

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successfully incorporated berry

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who used



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EVs could also be considered as a therapeutic agent themselves rather than a delivery system,

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since their inherent biological function is used without any alterations 19.

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Limitations of EVs as delivery tool

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Besides the promising potential of EVs as tool for bioactive compound delivery, there are also

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limitations. Since the field of research on EVs is rather new, many uncertainties need to be

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addressed in order to use these biological vesicles. First of all, studies are difficult to compare

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and results to interpret, because of the lack of standardized methods for isolation, qualitative

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and quantitative analysis or engineering of EVs. Hence, the optimal or best-suited methods

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for each application have to be developed, which is time and resource consuming. In addition,

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the differentiation and separation between different types and subpopulations of EVs is still a

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challenge for the field. For example, it is already known that a cell secretes a wide variety of

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vesicle subpopulations that could have different functions. Furthermore, the composition and

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function of the secreted EVs are highly dependent on the conditions of the cell of origin.

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Consequently, procedures for the production of EVs applied in compound delivery need to be

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further developed.

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Since EVs are produced by cells, their size is determined by their biogenesis, thus limiting their

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loading capacity. Theoretically, artificially produced liposomes can be synthesized in any size

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and with unlimited loading capacity. However, in most applications, the small size in the

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nanoscale is desirable, because such delivery vehicles have superior abilities in crossing

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barriers and penetrating into tissues compared to bigger particles.

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As their denomination already states, bioactive compounds are actively interacting with

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biological systems. This not only includes cells, but also EVs. Hence, bioactives encapsulated

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in EVs could also interact with the delivery system and consequently change the vesicle’s 12 ACS Paragon Plus Environment

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behavior or vice versa be inactivated or metabolized by molecules present in EVs.

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Furthermore, such changes could influence the interaction of the encapsulated compound

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with the gastro-intestinal tract, thus influencing cellular uptake. This of course is highly

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dependent on the particular type of EVs and compounds used in the particular application. Up

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to now, the knowledge on interactions of different EVs and bioactive compounds with each

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other and with the gastro-intestinal tract are very scarce, which limits the application of EVs

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as vehicle for delivery of bioactive compounds.

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Furthermore, there are still unsolved issues with regulatory affairs of the application of EVs in

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therapy, because they are new and there is no clear definition what class of drugs they belong

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to and no consensus has yet been reached on how they should be classified for approval.

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Moreover, safety is an issue, because of the diverse unknown functions, lack of standard

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procedures, and potentially harmful sources of EVs, like cancer-derived or immortalized cell

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lines, as mentioned above. However, if alternative, innocuous sources like milk or edible plants

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are used, safety concerns and approval by regulatory agencies should not be a problem.

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Besides, over time these issues will be resolved, as seen in any newly evolving field.

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Another concern regarding the safety of EVs is their potential to interact not only with

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physiological processes of the human body, but also with pharmaceuticals or drugs. Such side

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effects have not been addressed so far and need to be considered before a broad application

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of EVs as food additives can be achieved.

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Outlook

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Albeit these limitations, EVs open up new possibilities for targeted delivery of bioactive food

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compounds. Since there is urgent need for the standardization of methods of both isolation

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and analysis, their current use might not be beneficial in all applications and already 13 ACS Paragon Plus Environment


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established approaches, like the use of liposomes, could still be more effective and efficient

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for now. Nevertheless, with the growing research in the field of EVs their limitations will be

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addressed and, if solved, EVs could be applied as delivery system for food additives and drugs,

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thereby finding their way into the market.

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Besides their application as delivery tool for bioactives, EVs could also serve as active

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compounds and food additives themselves, due to their diverse physiological functions.

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Extending findings from human medical research, EVs could also be used in therapeutic

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approaches to fight animal and crop pathologies, as well as serve as biomarkers for health

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monitoring and quality assurance of livestock, since they are present in biological fluids of

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animals, like blood, saliva, or milk. Since both applications, therapeutics and biomarkers, are

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topics of medical studies, the research field of agriculture and food could make use of the

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available information and translate findings on EVs from biomedicine to veterinary medicine

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and animal health, food technology and development, and agricultural and plant science.


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Table 1: Comparison of the key characteristics of biological nanoparticles (EVs) and artificial

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nanoparticles (liposomes)

Stability Crossing barriers Immune tolerance Sources of material

Safety Methodology Loading capacity

Biological nanoparticles (EVs) Very stable by nature, resistant to digestion Able to cross complex barriers (e.g. blood-brain barrier) Dependent on source, but generally high tolerance Biological fluids from humans, animals or plants Food (milk, plants, fruits) In vitro production by cell lines Dependent on source Risks due to unknown biological functions High variability, no standardization Limited due to size

Artificial nanoparticles (liposomes) Engineered to be stable, dependent on materials Not able to cross complex barriers Needs engineering to achieve (e.g. polyethylene glycol Artificial materials Food-grade materials

Risks are well known due to many studies addressing safety Established methods of nanotechnology Variable, limit depends on application

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Figure Captions

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Figure 1. Treemap chart of the records found in the Web of Science search “extracellular

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vesicl* OR exosome* OR microvesicle*” of the year 2017, categorized in Web of Science

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categories, top 15 categories are displayed.

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Figure2. A Schematic of the biogenesis of two types of EVs, microvesicles and exosomes, by a

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cell. MVB – multivesicular body. B Cryo-electron microscopy images of EVs labelled with gold

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nanoparticles, reprinted from 12.



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Figure 2 A

B



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Table of Contents Graphic


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