Extracellular vesicles derived from mesenchymal stem cells (MSCs) represent a novel approach for regenerative and immunosuppressive therapy. Recently, cytochalasin B-induced microvesicles (CIMVs) were shown to be effective drug delivery mediators. However, little is known about their immunological properties. We propose that the immunophenotype and molecular composition of these vesicles could contribute to the therapeutic efficacy of CIMVs. To address this issue, CIMVs were generated from murine MSC (CIMVs-MSCs) and their cytokine content and surface marker expression determined. For the first time, we show that CIMVs-MSCs retain parental MSCs phenotype (Sca-1⁺, CD49e⁺, CD44⁺, CD45⁻). Also, CIMVs-MSCs contained a cytokine repertoire reflective of the parental MSCs, including IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, G-CSF, GM-CSF and TNF-α. Next, we evaluated the immune-modulating properties of CIMVs-MSCs in vivo using standard preclinical tests. MSCs and CIMVs-MSCs reduced serum levels of anti-sheep red blood cell antibody and have limited effects on neutrophil and peritoneal macrophage activity. We compared the immunomodulatory effect of MSCs, CIMVs and EVs. We observed no immunosuppression in mice pretreated with natural EVs, whereas MSCs and CIMVs-MSCs suppressed antibody production in vivo. Additionally, we have investigated the biodistribution of CIMVs-MSCs in vivo and demonstrated that CIMVs-MSCs localized in liver, lung, brain, heart, spleen and kidneys 48 h after intravenous injection and can be detected 14 days after subcutaneous and intramuscular injection. Collectively our data demonstrates immunomodulatory efficacy of CIMVs and supports their further preclinical testing as an effective therapeutic delivery modality.

Extracellular vesicles (EV) represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs) were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

Immune-mediated diseases are characterized by abnormal activity of the immune system. The cytochalasin B-induced membrane vesicles (CIMVs) are innovative therapeutic instruments. However, the immunomodulating activity of human mesenchymal stem cell (MSC)-derived CIMVs (CIMVs-MSCs) remains unknown. Therefore, we sought to investigate the immunological properties of CIMVs-MSCs and evaluate their effect on human peripheral blood mononuclear cells (PBMCs). We found that CIMVs-MSCs are primarily uptaken by monocytes and B-cells. Additionally, we demonstrated that CIMVs-MSCs inhibit phytohemagglutinin (PHA)-induced proliferation of PBMCs, with more pronounced effect on T-lymphocytes expansion as compared to that of B-cells. In addition, activation of T-helpers (CD4+CD25+), B-cells (CD19+CD25+), and T-cytotoxic lymphocytes (CD8+CD25+) was also significantly suppressed by CIMVs-MSCs. Additionally, CIMVs-MSCs decreased secretion of epidermal growth factor (EGF) and pro-inflammatory Fractalkine in a population of PBMCs, while the releases of FGF-2, G-CSF, anti-inflammatory GM-CSF, MCP-3, anti-inflammatory MDC, anti-inflammatory IL-12p70, pro-inflammatory IL-1b, and MCP-1 were increased. We analyzed the effect of CIMVs-MSCs on an isolated population of CD4+ and CD8+ T-lymphocytes and demonstrated their different immune response and cytokine secretion. Finally, we observed that no xenogeneic nor allogeneic transplantation of CIMVs induced an immune response in mice. Our data suggest that CIMVs-MSCs have immunosuppressive properties, are potential agents for immunomodulating treatment, and are worthy of further investigation.

The metabolism of glioma cells exhibits significant heterogeneity and is partially responsible for treatment outcomes. Given this variability, we hypothesized that the effectiveness of treatments targeting various metabolic pathways depends on the bioenergetic profiles and mitochondrial status of glioma cells. To this end, we analyzed mitochondrial biomass, mitochondrial protein density, oxidative phosphorylation (OXPHOS), and glycolysis in a panel of eight glioma cell lines. Our findings revealed considerable variability: mitochondrial biomass varied by up to 3.2-fold, the density of mitochondrial proteins by up to 2.1-fold, and OXPHOS levels by up to 7.3-fold across the cell lines. Subsequently, we stratified glioma cell lines based on their mitochondrial status, OXPHOS, and bioenergetic fitness. Following this stratification, we utilized 16 compounds targeting key bioenergetic, mitochondrial, and related pathways to analyze the associations between induced changes in cell numbers, proliferation, and apoptosis with respect to their steady-state mitochondrial and bioenergetic metrics. Remarkably, a significant fraction of the treatments showed strong correlations with mitochondrial biomass and the density of mitochondrial proteins, suggesting that mitochondrial status may reflect glioma cell sensitivity to specific treatments. Overall, our results indicate that mitochondrial status and bioenergetics are linked to the efficacy of treatments targeting metabolic pathways in glioma.

Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring extracellular vesicles and cytochalasin B-induced membrane vesicles (CIMVs) from mesenchymal stem cells (MSCs). In order to maintain accuracy in the comparative analysis, the same culture was used for the isolation of EVs and CIMVs: conditioned medium was used for EVs isolation and cells were harvested for CIMVs production. The pellets obtained after centrifugation 2300× g, 10,000× g and 100,000× g were analyzed using scanning electron microscopy analysis (SEM), flow cytometry, the bicinchoninic acid assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). We found that the use of cytochalasin B treatment and vortexing resulted in the production of a more homogeneous population of membrane vesicles with a median diameter greater than that of EVs. We found that EVs-like particles remained in the FBS, despite overnight ultracentrifugation, which introduced a significant inaccuracy in the calculation of the EVs yield. Therefore, we cultivated cells in a serum-free medium for the subsequent isolation of EVs. We observed that the number of CIMVs significantly exceeded the number of EVs after each step of centrifugation (2300× g, 10,000× g and 100,000× g) by up to 5, 9, and 20 times, respectively.

The therapeutic potential of prodigiosin as a hydrophobic anticancer agent can be enhanced by various approaches, one of which is the loading of PG into extracellular vesicles. Drug distribution and stability in aqueous media play a crucial role in targeting and accumulation, thereby enabling the attainment of therapeutically effective drug concentrations. Extracellular vesicles are nano-sized, cell-derived vesicles with a lipid bilayer membrane. Extracellular vesicles can be utilized as drug carriers for both water-soluble and non-water-soluble therapeutic agents. We hypothesized that microvesicles could effectively address the current challenges of prodigiosin delivery. Several different techniques have been developed for fabricating extracellular vesicles. These include microvesicles induction by cytochalasin B treatment as well as cell cultivation in serum depleted media. In our study, prodigiosin, like cytochalasin B, demonstrated efficacy in microvesicles formation based on protein quantification and Nanoparticle Tracking Analysis. In addition, Nanoparticle Tracking Analysis showed that vesicles from mesenchymal stem cells are more stable under ultrasound exposure. Microvesicles encapsulating prodigiosin, compared to unmodified naïve ones, demonstrated slightly increased zeta potentials and hydrodynamic diameters, which probably contributed to better stability. We demonstrated that ultrasonic treatment for the loading of prodigiosin does not significantly increase the proportion of prodigiosin-positive microvesicles in comparison with microvesicles induced with prodigiosin; moreover, this method cannot be considered as optimal due to its disadvantages, such as particle aggregation. Prodigiosin-induced and prodigiosin-loaded microvesicles from mesenchymal stem cells were significantly smaller and less polydisperse in size. Overall, prodigiosin encapsulated in extracellular vesicles might be more suitable for medical and clinical applications compared to pure forms of PG due to their cell membrane compatibility.