Crosstalk Between the Immune System and Plant-Derived Nanovesicles: A Study of Allergen Transporting.

Background: Nanometer-sized membrane-surrounded vesicles from different parts of plants including fruits are gaining increasing attention due to their anti-inflammatory and anticancer effects demonstrated by in vitro and in vivo studies, and as nanovectors for molecular delivery of exogenous substances. These nanomaterials are very complex and contain a diverse arsenal of bioactive molecules, such as nucleic acids, proteins, and lipids. Our knowledge about the transport of allergens in vesicles isolated from plant food is limited today. Methods: Here, to investigate the allergenicity of strawberry-derived microvesicles (MVs), nanovesicles (NVs), and subpopulations of NV, we have set up a multidisciplinary approach. The strategy combines proteomics-based protein identification, immunological investigations, bioinformatics, and data mining to gain biological insights useful to evaluate the presence of potential allergens and the immunoglobulin E (IgE) inhibitory activity of vesicle preparations. Results: Immunological test showed that several proteins of strawberry-derived vesicles compete for IgE binding with allergens spotted on the FABER biochip. This includes the known strawberry allergens Fra a 1, Fra a 3, and Fra a 4, and also other IgE-binding proteins not yet described as allergens in this food, such as gibberellin-regulated proteins, 2S albumin, pectate lyase, and trypsin inhibitors. Proteomics identified homologous sequences of the three strawberry allergens and their isoforms in total protein extract (TPE) but only Fra a 1 and Fra a 4 in the vesicle samples. Label-free quantitative proteomic analysis revealed no significant enrichment of these proteins in strawberry vesicles with respect to TPE. Conclusion: Immunological tests and bioinformatics analysis of proteomics data sets revealed that MVs and NVs isolated from strawberries can carry functional allergens their isoforms as well as proteins potentially allergenic based on their structural features. This should be considered when these new nanomaterials are used for human nutraceutical or biomedical applications.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Grapefruit-Derived Micro and Nanovesicles Show Distinct Metabolome Profiles and Anticancer Activities in the A375 Human Melanoma Cell Line.

Fruit juice is one of the most easily accessible resources for the isolation of plant-derived vesicles. Here we found that micro- and nano-sized vesicles (MVs and NVs) from four Citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, specifically inhibit the proliferation of lung, skin and breast cancer cells, with no substantial effect on the growth of non-cancer cells. Cellular and molecular analyses demonstrate that grapefruit-derived vesicles cause cell cycle arrest at G2/M checkpoint associated with a reduced cyclins B1 and B2 expression levels and the upregulation of cell cycle inhibitor p21. Further data suggest the inhibition of Akt and ERK signalling, reduced intercellular cell adhesion molecule-1 and cathepsins expressions, and the presence of cleaved PARP-1, all associated with the observed changes at the cellular level. Gas chromatography-mass spectrometry-based metabolomics reveals distinct metabolite profiles for the juice and vesicle fractions. NVs exhibit a high relative amount of amino acids and organic acids whereas MVs and fruit juice are characterized by a high percentage of sugars and sugar derivatives. Grapefruit-derived NVs are in particular rich in alpha-hydroxy acids and leucine/isoleucine, myo-inositol and doconexent, while quininic acid was detected in MVs. Our findings reveal the metabolite signatures of grapefruit-derived vesicles and substantiate their potential use in new anticancer strategies.

Membrane Transporters in Citrus clementina Fruit Juice-Derived Nanovesicles.

The cellular vesicle is a fluid-filled structure separated from the surrounding environment by a biological membrane. Here, we isolated nanovesicles (NVs) from the juice of clementines using a discontinuous density gradient ultracentrifugation method. To gain information about the protein content of vesicles, mass spectrometry-based organelle proteomics and bioinformatics were applied to the exosome-like vesicle fraction isolated in the 1 mol/L sucrose/D2O cushion. Analysis of 1018 identified proteins revealed a highly complex mixture of different intra, extracellular and artificially-formed vesicle populations. In particular, clathrin-coated vesicles were significantly expressed in this sample. Membrane transporters are significantly represented in clementines nanovesicles. We have found 162 proteins associated with the transport Gene Ontology term (GO: 0006810) which includes; 71 transmembrane transport related, 53 vesicle mediated and 50 intracellular transporters. Platellin-3 like carrier protein containing a Sec14 domain is known to have a role in plant-virus interaction and that is one of the most abundant proteins in our dataset. The presence of transmembrane transporters like ATPases, aquaporins, ATP Binding Cassette (ABC) transporters and tetraspanins, regulators of protein trafficking suggests that nanovesicles of clementines can actively interact with their environment in a controlled way.

Isolation of Exosome-Like Vesicles from Plants by Ultracentrifugation on Sucrose/Deuterium Oxide (D2O) Density Cushions.

Exosomes are nanovesicles of endocytic origin that are about 30-100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fluids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fluid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide double-cushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fluid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics.

Chromatography and its hyphenation to mass spectrometry for extracellular vesicle analysis.

Extracellular vesicles (EVs), such as exosomes, microvesicles and apoptotic bodies are released by cells, both under physiological and pathological conditions. EVs can participate in a novel type of intercellular communication and deliver cargo of nucleic acids, proteins and lipids near or to distant host cells. EV research is proceeding at a fast pace; now they start to appear as promising therapeutic targets, diagnostic tools and drug delivery systems. Isolation and analysis of EVs are prerequisites for understanding their biological roles and for their clinical exploitation. In this process chromatography and mass spectrometry (MS)-based strategies are rapidly gaining importance; and are reviewed in the present communication. Isolation and purification of EVs is mostly performed by ultracentrifugation at present. Chromatography-based strategies are gaining ground, among which affinity and size exclusion chromatography (SEC) are particularly strong contenders. Their major advantages are the relative simplicity, robustness and throughput. Affinity chromatography has the added advantage of separating EV subtypes based on molecular recognition of EV surface motifs. SEC has the advantage that isolated EVs may retain their biological activity. EVs are typically isolated in small amounts, therefore high sensitivity is required for their analysis. Study of the molecular content of EVs (all compounds beside nucleic acids) is predominantly based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The chromatographic separation is mostly performed by reverse phase, nanoscale, ultra high performance LC technique. The MS analysis relying typically on nano-electrospray ionization MS/MS provides high sensitivity, selectivity and resolution, so that thousand(s) of proteins can be detected/identified/quantified in a EV sample. Beside protein identification, quantitation and characterization of protein post-translational modifications (PTMs), like glycosylation and phosphorylation are becoming feasible and increasingly important. Along with conventional LC-MS/MS, other chromatographic approaches hyphenated to MS are gaining importance for EV characterization. Hydrophilic interaction LC is used to characterize PTMs; LC-inductively coupled plasma/MS to identify metal containing molecules; while gas chromatography-MS to analyze some lipids and metabolites.

Mass spectrometry of extracellular vesicles.

The review briefly summaries main features of extracellular vesicles, a joint terminology for exosomes, microvesicles, and apoptotic vesicles. These vesicles are in the center of interest in biology and medical sciences, and form a very active field of research. Mass spectrometry (MS), with its specificity and sensitivity, has the potential to identify and characterize molecular composition of these vesicles; but as yet there are only a limited, but fast-growing, number of publications that use MS workflows in this field. MS is the major tool to assess protein composition of extracellular vesicles: qualitative and quantitative proteomics approaches are both reviewed. Beside proteins, lipid and metabolite composition of vesicles might also be best assessed by MS techniques; however there are few applications as yet in this respect. The role of alternative analytical approaches, like gel-based proteomics and antibody-based immunoassays, are also mentioned. The objective of the review is to give an overview of this fast-growing field to help orient MS-based research on extracellular vesicles.