Extracellular Vesicles from Plants: Current Knowledge and Open Questions

The scientific interest in the beneficial properties of natural substances has been recognized for decades, as well as the growing attention in extracellular vesicles (EVs) released by different organisms, in particular from animal cells. However, there is increasing interest in the isolation and biological and functional characterization of these lipoproteic structures in the plant kingdom. Similar to animal vesicles, these plant-derived extracellular vesicles (PDEVs) exhibit a complex content of small RNAs, proteins, lipids, and other metabolites. This sophisticated composition enables PDEVs to be therapeutically attractive. In this review, we report and discuss current knowledge on PDEVs in terms of isolation, characterization of their content, biological properties, and potential use as drug delivery systems. In conclusion, we outline controversial issues on which the scientific community shall focus the attention shortly

The dark side of foetal bovine serum in extracellular vesicle studies

Extracellular vesicles (EVs) have been shown to be involved in cell‐cell communication and to take part in both physiological and pathological processes. Thanks to their exclusive cargo, which includes proteins, lipids, and nucleic acids from the originating cells, they are gaining interest as potential biomarkers of disease. In recent years, their appealing features have been fascinating researchers from all over the world, thus increasing the number of in vitro studies focused on EV release, content, and biological activities. Cultured cell lines are the most‐used source of EVs; however, the EVs released in cell cultures are influenced by the cell culture conditions, such as the use of foetal bovine serum (FBS). FBS is the most common supplement for cell culture media, but it is also a source of contaminants, such as exogenous bovine EVs, RNA, and protein aggregates, that can contaminate the cell‐derived EVs and influence their cargo composition. The presence of FBS contaminants in cell‐derived EV samples is a well‐known issue that limits the clinical applications of EVs, thus increasing the need for standardization. In this review, we will discuss the pros and cons of using FBS in cell cultures as a source of EVs, as well as the protocols used to remove contaminants from FBS

Emerging Insights on the Biological Impact of Extracellular Vesicle-Associated ncRNAs in Multiple Myeloma

Increasing evidence indicates that extracellular vesicles (EVs) released from both tumor cells and the cells of the bone marrow microenvironment contribute to the pathobiology of multiple myeloma (MM). Recent studies on the mechanisms by which EVs exert their biological activity have indicated that the non-coding RNA (ncRNA) cargo is key in mediating their effect on MM development and progression. In this review, we will first discuss the role of EV-associated ncRNAs in different aspects of MM pathobiology, including proliferation, angiogenesis, bone disease development, and drug resistance. Finally, since ncRNAs carried by MM vesicles have also emerged as a promising tool for early diagnosis and therapy response prediction, we will report evidence of their potential use as clinical biomarkers

Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models

Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases

Extracellular vesicle DNA from human melanoma tissues contains cancer-specific mutations

Liquid biopsies are promising tools for early diagnosis and residual disease monitoring in patients with cancer, and circulating tumor DNA isolated from plasma has been extensively studied as it has been shown to contain tumor-specific mutations. Extracellular vesicles (EVs) present in tumor tissues carry tumor-derived molecules such as proteins and nucleic acids, and thus EVs can potentially represent a source of cancer-specific DNA. Here we identified the presence of tumor-specific DNA mutations in EVs isolated from six human melanoma metastatic tissues and compared the results with tumor tissue DNA and plasma DNA. Tumor tissue EVs were isolated using enzymatic treatment followed by ultracentrifugation and iodixanol density cushion isolation. A panel of 34 melanoma-related genes was investigated using ultra-sensitive sequencing (SiMSen-seq). We detected mutations in six genes in the EVs (BRAF, NRAS, CDKN2A, STK19, PPP6C, and RAC), and at least one mutation was detected in all melanoma EV samples. Interestingly, the mutant allele frequency was higher in DNA isolated from tumor-derived EVs compared to total DNA extracted directly from plasma DNA, supporting the potential role of tumor EVs as future biomarkers in melanom

Citral-Enriched Fraction of Lemon Essential Oil Mitigates LPS-Induced Hepatocyte Injuries

Simple Summary To date, essential oil fractions are emerging as functional compounds of interest for the food and perfume industries. The aim of this study is to evaluate the ability of citral-enriched fractions obtained from lemon essential oil (Cfr-LEO) to counteract, in healthy human hepatocytes, the activity of lipopolysaccharide (LPS), a trigger of inflammation, oxidative stress, and epithelial-mesenchymal transition. In our paper, we report that the pretreatment of hepatocytes with Cfr-LEO counteracts the effects induced by LPS. The data obtained lay the basis for the development of commercial products such as food and drink aimed at preventing or alleviating chronic conditions associated with liver dysfunction.Abstract Lemon essential oil (LEO) is known for its aromatic and healthy properties; however, less consideration is given to the biological properties of the fractions obtained from LEO. This study aims to evaluate the ability of a citral-enriched fraction obtained from LEO (Cfr-LEO) to counteract lipopolysaccharide (LPS)-mediated inflammation, oxidative stress, and epithelial-mesenchymal transition (EMT) in healthy human hepatocytes. Human immortalized hepatocytes (THLE-2 cell line) were pretreated with Cfr-LEO and subsequently exposed to LPS at various time points. We report that the pretreatment with Cfr-LEO counteracts LPS-mediated effects by inhibiting inflammation, oxidative stress, and epithelial-mesenchymal transition in THLE-2. In particular, we found that pretreatment with Cfr-LEO reduced NF-kappa B activation and the subsequent proinflammatory cytokines release, ROS production, and NRF2 and p53 expression. Furthermore, the pretreatment with Cfr-LEO showed its beneficial effect in counteracting LPS-induced EMT. Taken together, these results support Cfr-LEO application in the nutraceutical research field not only for its organoleptic properties, conferred by citral enrichment, but also for its biological activity. Our study could lay the basis for the development of foods/drinks enriched with Cfr-LEO, aimed at preventing or alleviating chronic conditions associated with liver dysfunction

Proof-of-Concept Study on the Use of Tangerine-Derived Nanovesicles as siRNA Delivery Vehicles toward Colorectal Cancer Cell Line SW480

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells

Plant-RNA in Extracellular Vesicles: The Secret of Cross-Kingdom Communication

The release of extracellular vesicles (EVs) is a common language, used by living organisms from different kingdoms as a means of communication between them. Extracellular vesicles are lipoproteic particles that contain many biomolecules, such as proteins, nucleic acids, and lipids. The primary role of EVs is to convey information to the recipient cells, affecting their function. Plant-derived extracellular vesicles (PDEVs) can be isolated from several plant species, and the study of their biological properties is becoming an essential starting point to study cross-kingdom communication, especially between plants and mammalians. Furthermore, the presence of microRNAs (miRNAs) in PDEVs represents an interesting aspect for understanding how PDEVs can target the mammalian genes involved in pathological conditions such as cancer, inflammation, and oxidative stress. In particular, this review focuses on the history of PDEVs, from their discovery, to purification from various matrices, and on the functional role of PDEV-RNAs in cross-kingdom interactions. It is worth noting that miRNAs packaged in PDEVs can be key modulators of human gene expression, representing potential therapeutic agents

Emerging Insights on the Biological Impact of Extracellular Vesicle-Associated ncRNAs in Multiple Myeloma

Increasing evidence indicates that extracellular vesicles (EVs) released from both tumor cells and the cells of the bone marrow microenvironment contribute to the pathobiology of multiple myeloma (MM). Recent studies on the mechanisms by which EVs exert their biological activity have indicated that the non-coding RNA (ncRNA) cargo is key in mediating their effect on MM development and progression. In this review, we will first discuss the role of EV-associated ncRNAs in different aspects of MM pathobiology, including proliferation, angiogenesis, bone disease development, and drug resistance. Finally, since ncRNAs carried by MM vesicles have also emerged as a promising tool for early diagnosis and therapy response prediction, we will report evidence of their potential use as clinical biomarkers

LE VESCICOLE EXTRACELLULARI VEGETALI PER LO SVILUPPO DI NUOVI NUTRACEUTICI: STUDIO PRE-CLINICO E CLINICO DELLE PROPRIETÀ BIOLOGICHE DELLE VESCICOLE ISOLATE DA SUCCO DI LIMONE

Razionale dello studio: Le vescicole extracellulari (EVs) sono strutture lipoproteiche rilasciate dalle cellule animali e vegetali, che veicolano sostanze bioattive tra cui lipidi, proteine, acidi nucleici e altri metaboliti, rendendole più stabili e biodisponibili. EVs isolate da semi, foglie e frutti di svariate specie vegetali presentano attività anti-tumorale, anti-infiammatoria ed anti-ossidante. Metodi e Risultati: Il nostro gruppo di ricerca si occupa da anni della caratterizzazione strutturale e funzionale delle EVs da succo di limone (LEVs). Le LEVs esercitano effetti antinfiammatori sia in vitro, su linee di macrofagi murini, che ex vivo, su linfociti T, attraverso l’inibizione delle vie di segnalazione mediate da ERK e NF-kB. Inoltre, abbiamo recentemente condotto uno studio clinico volto a valutare gli effetti della somministrazione di un prodotto naturale, contenente LEVs, su una coorte di 20 volontari sani. I parametri antropometrici ed ematobiochimici sono stati analizzati dopo 1 e 3 mesi. I dati raccolti indicano una diminuzione significativa della circonferenza della vita dopo 1 mese di trattamento ed una riduzione dei livelli di colesterolo LDL del 18% dopo tre mesi. Conclusioni: I risultati di questo studio incoraggiano lo sviluppo di nuovi prodotti nutraceutici, contenenti LEVs, per la prevenzione delle malattie infiammatorie e come efficace strumento per gestire i fattori di rischio cardiometabolico