Extracellular vesicles (EVs) are membrane-enclosed vesicles which play important role for cell communication and physiology. EVs are found in many human biological fluids, including blood, breast milk, urine, cerebrospinal fluid (CSF), ejaculate, saliva etc. These nano-sized vesicles contain proteins, mRNAs, microRNAs, non-coding RNAs and lipids that are derived from producing cells. EVs deliver complex sets of biological information to recipient cells thereby modulating their behaviors by their molecular cargo. In this way EVs are involved in the pathological development and progression of many human disorders, including neurodegenerative diseases. In this study EVs purified by ultracentrifugation from CSF of patients with Parkinson’s disease (PD) and individuals of the comparison group were characterized using nanoparticle tracking analysis, flow cytometry and cryo-electron microscopy. Vesicular size and the presence of exosomal marker CD9 on the surface provided evidence that most of the EVs were exosome-like vesicles. Cryo-electron microscopy allowed us to visualize a large spectrum of extracellular vesicles of various size and morphology with lipid bilayers and vesicular internal structures. Thus, we described the diversity and new characteristics of the vesicles from CSF suggesting that subpopulations of EVs with different and specific functions may exist.

Plant-derived extracellular vesicles (PEVs) have gained attention as promising bioactive nutraceutical molecules; their presence in common fruit juices has increased their significance because human interaction is inevitable. The goal of this study was to evaluate the potential of PEVs derived from grapefruit and tomato juices as functional ingredients, antioxidant compounds, and delivery vehicles. PEVs were isolated using differential ultracentrifugation and were found to be similar in size and morphology to mammalian exosomes. The yield of grapefruit exosome-like vesicles (GEVs) was higher than that of tomato exosome-like vesicles (TEVs), despite the latter having larger vesicle sizes. Furthermore, the antioxidant activity of GEVs and TEVs was found to be low in comparison to their juice sources, indicating a limited contribution of PEVs to the juice. GEVs showed a higher efficiency in being loaded with the heat shock protein 70 (HSP70) than TEVs, as well as a higher efficiency than TEV and PEV-free HSP70 in delivering HSP70 to glioma cells. Overall, our results revealed that GEVs present a higher potential as functional ingredients present in juice and that they exert the potential to deliver functional molecules to human cells. Although PEVs showed low antioxidant activity, their role in oxidative response in cells should be further addressed.

Extracellular vesicles (EV) are involved in important processes of glioblastoma multiforme (GBM), including malignancy and invasion. EV secreted by glioblastoma cells may cross the hematoencephalic barrier and carry molecular cargo derived from the tumor into the peripheral circulation. Therefore, the determination of the molecular composition of exosomes released by glioblastoma cells seems to be a promising approach for the development of non-invasive methods of the detection of the specific exosomal protein markers in the peripheral blood. The present study aimed to determine the common exosomal proteins presented in preparations from different cell lines and search potential glioblastoma biomarkers in exosomes. We have performed proteomics analysis of exosomes obtained from the conditioned culture medium of five glioblastoma cell lines. A list of 133 proteins common for all these samples was generated. Based on the data obtained, virtual two-dimensional electrophoresis (2DE) maps of proteins presented in exosomes of glioblastoma cells were constructed and the gene ontology (GO) analysis of exosome proteins was performed. A correlation between overexpressed in glial cell proteins and their presence in exosomes have been found. Thus, the existence of many potential glioblastoma biomarkers in exosomes was confirmed.

Background/Objectives: Stress protein HSP70 administered exogenously has demonstrated high potential as an efficient adjuvant in antitumor immune response. To enhance the antigen-presenting activity, bioavailability, and stability of exogenous recombinant human HSP70, we propose incorporating it into plant extracellular vesicles. Earlier, we found that grapefruit-derived extracellular vesicles (GEV) were able to store the protein with no loss of its major function, chaperone activity. Methods: In this study, we tested whether HSP70 loaded into GEV (GEV-HSP70) could elicit an antitumor immune response in cellular and animal models of colorectal cancer. Results: To test the hypothesis in vitro, human and mouse colorectal cancer cell lines were used. We have shown that the addition of HSP70, either in free form or as part of GEVs, increases the sensitivity of human (HCT-116, DLD1) or mouse (CT-26) colon cancer cells to mouse cytotoxic lymphocytes and human NK-92 cells. Moreover, the amount of protein in the form of GEV-HSP70 required to cause the same activation of antitumor immunity was 20 times less than when HSP70 was added in free form. In a colon carcinoma model in vivo, GEV-HSP70 were inoculated subcutaneously into BALB/c mice together with CT-26 cells to form a tumor node. As compared with the control groups, we observed an increase in the lifespan of animals and a decrease in the tumor size, as well as a decrease in the level of TGFB1 IL-10 factors in the blood plasma. In vitro analysis of the immunomodulatory activity of GEV-HSP70 showed that antitumor response in GEV-HSP70-treated mice was associated with the accumulation of CD8+ cells. Conclusions: These results demonstrate the high feasibility and efficacy of the new technique based on HSP70 encapsulated in plant vesicles in activation of the specific response to colon tumors.