More specific targeted drug delivery systems with low immunogenicity and toxicity are expected to increase the efficacy of therapeutic molecules. The extracellular nanovesicles have been introduced as natural delivery systems for therapeutic molecules. More recently, considerable interest has emerged in plant-derived exosomes and their natural commitment to deliver molecules of various origins. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466 nm, but its clinical use is limited by the potential systemic toxicity. In this study we investigated the ability of exosomes from Citrus Sinensis to be successfully uploaded with AO (Exo-AO). We also studied the ability of the exosomes to be uploaded into target cells, as compared to the free molecules. We found that AO was efficiently uploaded into exosomes through electroporation. In fact, Exo-AO entered into the target cells significantly better than the free molecules, leading to both a marked intracellular AO delivery into target cells and an increase in the cytotoxic effect after excitation under a fluorescence microscope. This study shows a promising new approach for a more effective and less toxic drug delivery through the use of plant-derived extracellular nanovesicles.

Plant-derived extracellular vesicles (PDEVs) are nanoscale carriers produced through conserved plant mechanisms, including multivesicular body (MVB) formation and consequent extracellular vesicle release. MVBs are formed through repeated rounds of intracellular vesicles’ fusion, thus leading to the incorporation into PDEVs of lipids, proteins, miRNAs, nucleic acids, and secondary metabolites, derived from different cellular compartments. PDEVs possess a bilayer lipid membrane, which protects their cargo from degradation and facilitates membrane–membrane fusion with target cells. Ayurvedic medicinal plants are renowned for their extensive phytochemical diversity and enduring efficacy in addressing inflammation, infections, metabolic disorders, cancer, and neurodegeneration. However, the clinical translation of traditional herbal preparation is severely bottlenecked by batch-to-batch variability, restricted compound bioavailability, mechanistic uncertainties, and limitations of conventional large-scale extractions. This perspective research study critically proposes PDEVs as an innovative interpretation for Ayurvedic medicinal plants utilization. We identify and evaluate medicinal plants with established therapeutic characteristics that remain unexamined in PDEV research, hence presenting compelling opportunities for future investigation. By establishing a synergistic bridge between ancient Ayurvedic knowledge and modern nanomedicine, this perspective provides a methodological roadmap to guide health-efficient plant selection and accelerate translational research in next-generation therapeutics.

Plant-Derived Extracellular Vesicles extracellular vesicles (PDEVs) from organic agriculture (without the use of pesticides and microbicides) contain high levels of antioxidants. Organic PDEVs have shown an increased antioxidant power compared to PDEVs from single plants, suggesting a synergistic effect of the bioactives constitutively expressed in the PDEVs from single fruits. With this study, we wanted to investigate the beneficial effects of a mix of PDEVs on human skin cells. We found detectable levels of citric acid, ascorbic acid, glutathione, catalase, and SOD in a mix of PDEVs deriving from five different fruits (grape, red orange, papaya, pomegranate, and tangerine). We then treated H2O2-conditioned fibroblasts with the mix of PDEVs. The results showed that the PDEVs’ mixture reverted the H2O2-induced redox imbalance, restoring mitochondrial homeostasis, with a strong reduction of mitochondrial anion superoxide and an increase in sirtuin levels. The antioxidant action was consistent with wound repair on a lesion produced in a fibroblast’s monolayer. This result was consistent with an increased level of vimentin and matrix metalloproteinase-9, whose expression is directly related to the efficiency of the reparative processes. These data support a beneficial role of PDEVs in both preventing and treating skin injuries through their potent antioxidant and reparative activities.

Water is a major requirement for our bodies, and alkaline water has induced an antioxidant response in a model of natural aging. A series of recent reports have shown that aging is related to reduced water intake. Hydrogen-rich water has been suggested to exert a general antioxidant effect in relation to both improving lifestyle and preventing a series of diseases. Here, we wanted to investigate the effect of the daily intake of hydrogen-rich alkaline water (HAW) in counteracting the redox imbalance induced in a model of H2O2-treated mice. Mice were treated with H2O2 for two weeks and either left untreated or supplied with HAW. The results show that HAW induced a reduction in the ROS plasmatic levels that was consistent with the increase in the circulating glutathione. At the same time, the reduction in plasmatic 8-hydroxy-2′-deoxyguanosine was associated with reduced DNA damage in the whole body. Further analysis of the spleen and bone marrow cells showed a reduced ROS content consistent with a significantly reduced mitochondrial membrane potential and superoxide accumulation and an increase in spontaneous proliferation. This study provides evidence for a clear preventive and curative effect of HAW in a condition of systemic toxic condition and redox imbalance.

The extent of both scientific articles and reviews on extracellular vesicles (EVs) has grown impressively over the last few decades [...]

Exosomes are extracellular nanovesicles (EV), that is, carriers of different biomolecules such as lipids, proteins, nucleic acids. Their composition and the fact that their release dramatically increases in cases of tumorigenesis open up different scenarios on their possible application to research into new biomarkers. The first purpose of the present review was to specifically analyze and compare different methodologies available for the use of exosomes in prostate cancer (PC). The most widely applied methodologies include ultracentrifugation techniques, size-based techniques, immunoaffinity capture-based techniques (mainly ELISA), and precipitation. To optimize the acquisition of exosomes from the reference sample, more techniques can be applied in sequence for a single extraction, thereby determining an increase in labor time and costs. The second purpose was to describe clinical results obtained with the analysis of PSA-expressing exosomes in PC; this provides an incredibly accurate method of discriminating between healthy patients and those with prostate disease. Specifically, the IC-ELISA alone method achieved 98.57% sensitivity and 80.28% specificity in discriminating prostate cancer (PC) from benign prostatic hyperplasia (BPH). An immunocapture-based ELISA assay was performed to quantify and characterize carbonic anhydrase (CA) IX expression in exosomes. The results revealed that CA IX positive exosomes were 25-fold higher in plasma samples from PC patients than in those from healthy controls. The analysis of PC-linked exosomes represents a promising diagnostic model that can effectively distinguish patients with PC from those with non-malignant prostatic disease. However, the use of exosome analysis in clinical practice is currently limited by several issues, including a lack of standardization in the analytical process and high costs, which are still too high for large-scale use.

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Citrus fruits are a natural source of ascorbic acid, and exosome-like nanovesicles obtained from these fruits contain measurable levels of ascorbic acid. We tested the ability of grapefruit-derived extracellular vesicles (EVs) to inhibit the growth of human leukemic cells and leukemic patient-derived bone marrow blasts. Transmission electron microscopy and nanoparticle tracking analysis (NTA) showed that the obtained EVs were homogeneous exosomes, defined as exosome-like plant-derived nanovesicles (ELPDNVs). The analysis of their content has shown measurable amounts of several molecules with potent antioxidant activity. ELPDNVs showed a time-dependent antiproliferative effect in both U937 and K562 leukemic cell lines, comparable with the effect of high-dosage ascorbic acid (2 mM). This result was confirmed by a clear decrease in the number of AML blasts induced by ELPDNVs, which did not affect the number of normal cells. ELPDNVs increased the ROS levels in both AML blast cells and U937 without affecting ROS storage in normal cells, and this effect was comparable to ascorbic acid (2 mM). With our study, we propose ELPDNVs from grapefruits as a combination/supporting therapy for human leukemias with the aim to improve the effectiveness of the current therapies.

Exosomes are extracellular vesicles (EVs) of nanometric size studied for their role in tumor pathogenesis and progression and as a new source of tumor biomarkers. The clinical studies have provided encouraging but probably unexpected results, including the exosome plasmatic levels' clinical relevance and well-known biomarkers' overexpression on the circulating EVs. The technical approach to obtaining EVs includes methods to physically purify EVs and characterize EVs, such as Nanosight Tracking Analysis (NTA), immunocapture-based ELISA, and nano-scale flow cytometry. Based on the above approaches, some clinical investigations have been performed on patients with different tumors, providing exciting and promising results. Here we emphasize data showing that exosome plasmatic levels are consistently higher in tumor patients than in controls and that plasmatic exosomes express well-known tumor markers (e.g., PSA and CEA), proteins with enzymatic activity, and nucleic acids. However, we also know that tumor microenvironment acidity is a key factor in influencing both the amount and the characteristics of the exosome released by tumor cells. In fact, acidity significantly increases exosome release by tumor cells, which correlates with the number of exosomes that circulate through the body of a tumor patient.

Supplementary Movie 1 from Cannibalism of Live Lymphocytes by Human Metastatic but Not Primary Melanoma Cells

Plant-derived exosomes (PDEs) are receiving much attention as a natural source of antioxidants. Previous research has shown that PDEs contain a series of bioactives and that their content varies depending on the fruit or vegetable source. It has also been shown that fruits and vegetables derived from organic agriculture produce more exosomes, are safer, free of toxic substances, and contain more bioactives. The aim of this study was to investigate the ability of orally administered mixes of PDE (Exocomplex®) to restore the physiological conditions of mice treated for two weeks with hydrogen peroxide (H2O2), compared with mice left untreated after the period of H2O2 administration and mice that received only water during the experimental period. The results showed that Exocomplex® had a high antioxidant capacity and contained a series of bioactives, including Catalase, Glutathione (GSH), Superoxide Dismutase (SOD), Ascorbic Acid, Melatonin, Phenolic compounds, and ATP. The oral administration of Exocomplex® to the H2O2-treated mice re-established redox balance with reduced serum levels of both reactive oxygen species (ROS) and malondialdehyde (MDA), but also a general recovery of the homeostatic condition at the organ level, supporting the future use of PDE for health care.

Background: Oral squamous cell carcinoma (OSCC) is characterized by an immunosuppressive tumor microenvironment. Their plasma-derived exosomes deliver immunomodulatory molecules and cargo that correlate significantly with clinical parameters. This study aims to assess the exosomal profile as a potential tool for early detection of relapse and long-term outcomes in OSCC patients undergoing conventional therapy. Methods: 27 OSCC patients with a median 38-month follow-up were included in this study. The relationship between NTA-derived parameters and clinical pathological parameters was examined, and receiver operating characteristic (ROC) curves were utilized to evaluate the diagnostic efficacy of these values in detecting cancer relapse. Results: Plasmatic levels of exosomes prior to surgery showed a drastic reduction after surgical intervention (8.08E vs. 1.41 × 109 particles/mL, p = 0.006). Postsurgical concentrations of exosomes were higher in patients who experienced relapse compared to those who remained disease-free (2.97 × 109 vs. 1.11 × 109 particles/mL, p = 0.046). Additionally, patients who relapsed exhibited larger exosome sizes after surgery (141.47 vs. 132.31 nm, p = 0.03). Patients with lower concentrations of exosomes prior to surgery demonstrated better disease-free survival compared to those with higher levels (p = 0.012). ROC analysis revealed an area under the curve of 0.82 for presurgical exosome concentration in identifying relapse. Conclusions: Presurgical exosomal plasmatic levels serve as independent predictors of early recurrence and survival in OSCC. All in all, our findings indicate that the detection of peripheral exosomes represents a novel tool for the clinical management of OSCC, with potential implications for prognosis assessment.

Supplementary Figure 1 from Proton Pump Inhibitors Induce Apoptosis of Human B-Cell Tumors through a Caspase-Independent Mechanism Involving Reactive Oxygen Species

IntroductionHow can biotechnology and organic agriculture be fused and promoted simultaneously to overcome the main challenges in drug delivery systems. The role of organic agriculture in future human health treatment still represents a binary organic-conventional question. However, exosomes-like nanoparticles define a new organic path that plants and vegetables can release. In this review, we concisely propose plant-derived exosome-like nanovesicles and discuss their most important biological and pharmacological roles, representing a new tool for drug delivery.Areas coveredPlant-derived exosomes-like nanovesicles; nature farming; green manufacturing practice; drug delivery; organic agriculture.Expert opinionThere is growing interest in the potential use of plant-derived exosomes-like nanovesicles for various diagnostic and therapeutic applications that should translate into a supplement to current nano-pharmaceuticals. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for plant-derived exosomes-like nanovesicles poses a barrier to clinical translation. An increasing number of articles are recently published on new analytical platforms to address these challenges in cross-comparison with conventional assay methods. This review also mentions two patents from ExoLab-Italia on plant-derived exosome-like nanovesicles, respectively, on plant-derived exosome-like nanovesicles' ability to naturally deliver a series of potentially therapeutic molecules and a novel approach to upload them with therapeutic molecules.

The molecular revolution could lead drug discovery from chance observation to the rational design of new classes of drugs that could simultaneously be more effective and less toxic. Unfortunately, we are witnessing some failure in this sense, and the causes of the crisis involve a wide range of epistemological and scientific aspects. In pharmacology, one key point is the crisis of the paradigm the “magic bullet”, which is to design therapies based on specific molecular targets. Drug repurposing is one of the proposed ways out of the crisis and is based on the off-target effects of known drugs. Here, we propose the microenvironment as the ideal place to direct the off-targeting of known drugs. While it has been extensively investigated in tumors, the generation of a harsh microenvironment is also a phenotype of the vast majority of chronic diseases. The hostile microenvironment, on the one hand, reduces the efficacy of both chemical and biological drugs; on the other hand, it dictates a sort of “Darwinian” selection of those cells armed to survive in such hostile conditions. This opens the way to the consideration of the microenvironment as a convenient target for pharmacological action, with a clear example in proton pump inhibitors.

Research in science and medicine is witnessing a massive increases in literature concerning extracellular vesicles (EVs). From a morphological point of view, EVs include extracellular vesicles of a micro and nano sizes. However, this simplistic classification does not consider both the source of EVs, including the cells and the species from which Evs are obtained, and the microenvironmental condition during EV production. These two factors are of crucial importance for the potential use of Evs as therapeutic agents. In fact, the choice of the most suitable Evs for drug delivery remains an open debate, inasmuch as the use of Evs of human origin may have at least two major problems: (i) autologous Evs from a patient may deliver dangerous molecules; and (ii) the production of EVs is also limited to cell factory conditions for large-scale industrial use. Recent literature, while limited to only a few papers, when compared to the papers on the use of human EVs, suggests that plant-derived nanovesicles (PDNV) may represent a valuable tool for extensive use in health care.

There is a urgent need for valuable strategy in early and less invasive diagnosis for cancer. Preliminary data have shown that the plasmatic levels of exosomes increase in cancer condition. This study investigates the relevance of plasmatic levels and size distribution of exosomes in 42 individuals with no signs of urological disease (CTR) as compared to 65 prostate cancer patients (PCa). It was used Nanoparticle Tracking Analysis (NTA), a highly reliable and sensitive method for exosomes characterization and quantification. The relation structure among the NTA-derived parameters was assessed by means of Principal Component Analysis, which allowed detecting the global discriminant power of NTA test in terms of Receiver Operating Characteristic (ROC) curve and the selection of cut-off thresholds. The results showed that PCa had significantly higher plasmatic levels of exosomes and that the exosomes were smaller in size as compared to the CTR; the values reached 89% sensitivity and 71% specificity, in distinguishing PCa from CTR. These results propose a new exosome-based non-invasive clinical approach for the clinical follow-up of prostate cancer undergoing surgical treatment; in addition this method may be developed as a new screening test for prostate cancer’s early diagnosis. While this clinical study was performed in prostate cancer, it may represent a proof of concept extendable to virtually all cancers, as it is suggested by both pre-clinical evidence and clinical data obtained with different technical approaches.