Storage stability and delivery potential of cytochalasin B induced membrane vesicles

AbstractBackgroundThe cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown.ObjectivesThe objectives of this study were to analyzed the morphology, size distribution, molecular composition and angiogenic properties of CIMVs-MSCs.MethodsThe morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix.ResultsHuman CIMVs-MSCs retain parental MSCs content such as growth factors, cytokines, chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-β, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cellsin vitro. Finally, CIMVs-MSCs can induce angiogenesisin vivoafter subcutaneous injection into adult rats.ConclusionsHuman CIMVs-MSCs have similar content, immunophenotype and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.

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Angiogenic Activity of Cytochalasin B-Induced Membrane Vesicles of Human Mesenchymal Stem Cells

Cells ◽

10.3390/cells9010095 ◽

2019 ◽

Vol 9 (1) ◽

pp. 95 ◽

Cited By ~ 4

Author(s):

Marina O. Gomzikova ◽

Margarita N. Zhuravleva ◽

Vyacheslav V. Vorobev ◽

Ilnur I. Salafutdinov ◽

Alexander V. Laikov ◽

...

Keyword(s):

Cytochalasin B ◽

Therapeutic Potential ◽

Membrane Vesicles ◽

Membrane Receptors ◽

Molecular Composition ◽

Target Cells ◽

Human Mscs ◽

Angiogenic Activity ◽

Induced Membrane

The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. Objectives: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. Methods: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. Results: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-β, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. Conclusions: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.

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Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

BioMed Research International ◽

10.1155/2018/7053623 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 7

Author(s):

Marina Gomzikova ◽

Sevindzh Kletukhina ◽

Sirina Kurbangaleeva ◽

Albert Rizvanov

Keyword(s):

Targeted Delivery ◽

Cytochalasin B ◽

Membrane Vesicles ◽

Membrane Receptors ◽

Surface Proteins ◽

Vector System ◽

Target Cells ◽

Significant Preference ◽

Induced Membrane ◽

Pc3 Cells

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.

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Cytochalasin B-induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing TRAIL, PTEN and IFN-β1 Can Kill Carcinoma Cancer Cells

Tissue and Cell ◽

10.1016/j.tice.2021.101664 ◽

2021 ◽

pp. 101664

Author(s):

Daria S. Chulpanova ◽

Zarema E. Gilazieva ◽

Elvira R. Akhmetzyanova ◽

Sevindzh K. Kletukhina ◽

Albert A. Rizvanov ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cancer Cells ◽

Cytochalasin B ◽

Human Mesenchymal Stem Cells ◽

Membrane Vesicles ◽

Induced Membrane

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Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery

Pharmaceutics ◽

10.3390/pharmaceutics13111911 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1911

Author(s):

Anastasiya Oshchepkova ◽

Oleg Markov ◽

Evgeniy Evtushenko ◽

Alexander Chernonosov ◽

Elena Kiseleva ◽

...

Keyword(s):

Cancer Cells ◽

Extracellular Vesicles ◽

Mammalian Cells ◽

Cytochalasin B ◽

Drug Carrier ◽

Membrane Vesicles ◽

Nucleic Acid Delivery ◽

Isolation And Purification ◽

Cellular Origin ◽

Efficient Uptake

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

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Kinetics of glucose transport in rat skeletal muscle membrane vesicles: effects of insulin and contractions

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1992.262.5.e700 ◽

1992 ◽

Vol 262 (5) ◽

pp. E700-E711 ◽

Cited By ~ 11

Author(s):

T. Ploug ◽

H. Galbo ◽

T. Ohkuwa ◽

J. Tranum-Jensen ◽

J. Vinten

Keyword(s):

Skeletal Muscle ◽

Plasma Membrane ◽

Glucose Transport ◽

Cytochalasin B ◽

Equilibrium Distribution ◽

Initial Rate ◽

Membrane Vesicles ◽

Membrane Preparation ◽

Muscle Membrane ◽

Distribution Space

To study the mechanism of acceleration of glucose transport in skeletal muscle after stimulation with insulin and contractions, we isolated a subcellular vesicular membrane fraction, highly enriched in the plasma membrane enzyme K(+)-stimulated p-nitrophenylphosphatase and also enriched in some intracellular membranes. Protein recovery, morphology, lipid content, marker enzyme activities, total intravesicular volume, Western blot quantitation of GLUT-1, and glucose-inhibitable cytochalasin B binding were identical in membrane fractions from control, insulin-stimulated, contraction-stimulated, and insulin- and contraction-stimulated muscle. Time course of D-[3H]glucose entry in membrane vesicles at equilibrium exchange conditions showed that initial rate of transport at 30 mM of glucose was increased 19-fold and that equilibrium distribution space was increased 4-fold in vesicles from maximum stimulated muscle. The effects of insulin and contractions on initial rate of transport as well as on equilibrium distribution space were additive, and stimulation increased the substrate saturability of glucose transport. Furthermore, cytochalasin B binding to membranes prepared by using less centrifugation time than usual showed that, after stimulation with insulin and contractions, at least 35% of the total number of glucose transporters were redistributed from one kind of vesicles to a more slowly sedimenting kind of vesicles, probably reflecting translocation within the membrane preparation from intracellular vesicles to the plasma membrane upon stimulation. In the present membrane preparation the effects of insulin and/or contractions on glucose transport resemble those seen in intact muscle, and the effects are thus not dependent on cellular integrity.(ABSTRACT TRUNCATED AT 250 WORDS)

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Na+-independent D-glucose transport in rabbit renal basolateral membranes

AJP Renal Physiology ◽

10.1152/ajprenal.1988.254.5.f711 ◽

1988 ◽

Vol 254 (5) ◽

pp. F711-F718 ◽

Cited By ~ 3

Author(s):

P. T. Cheung ◽

M. R. Hammerman

Keyword(s):

Glucose Uptake ◽

Glucose Transport ◽

Brush Border ◽

Glucose Transporter ◽

Cytochalasin B ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Rabbit Kidney ◽

Basolateral Membrane Vesicles ◽

Proximal Tubular

To define the mechanism by which glucose is transported across the basolateral membrane of the renal proximal tubular cell, we measured D-[14C]glucose uptake in basolateral membrane vesicles from rabbit kidney. Na+-dependent D-glucose transport, demonstrable in brush-border vesicles, could not be demonstrated in basolateral membrane vesicles. In the absence of Na+, the uptake of D-[14C]glucose in basolateral vesicles was more rapid than that of L-[3H]glucose over a concentration range of 1-50 mM. Subtraction of the latter from the former uptakes revealed a saturable process with apparent Km of 9.9 mM and Vmax of 0.80 nmol.mg protein-1.s-1. To characterize the transport component of D-glucose uptake in basolateral vesicles, we measured trans stimulation of 2 mM D-[14C]glucose entry in the absence of Na+. Trans stimulation could be effected by preloading basolateral vesicles with D-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose, but not with L-glucose or alpha-methyl-D-glucoside. Trans-stimulated D-[14C]glucose uptake was inhibited by 0.1 mM phloretin or cytochalasin B but not phlorizin. In contrast, Na+-dependent D-[14C]glucose transport in brush-border vesicles was inhibited by phlorizin but not phloretin or cytochalasin B. Our findings are consistent with the presence of a Na+-independent D-glucose transporter in the proximal tubular basolateral membrane with characteristics similar to those of transporters present in nonepithelial cells.

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Massive Ca-induced Membrane Fusion and Phospholipid Changes Triggered by Reverse Na/Ca Exchange in BHK Fibroblasts

The Journal of General Physiology ◽

10.1085/jgp.200709865 ◽

2008 ◽

Vol 132 (1) ◽

pp. 29-50 ◽

Cited By ~ 19

Author(s):

Alp Yaradanakul ◽

Tzu-Ming Wang ◽

Vincenzo Lariccia ◽

Mei-Jung Lin ◽

Chengcheng Shen ◽

...

Keyword(s):

Cell Surface ◽

Membrane Fusion ◽

Fluorescent Protein ◽

Annexin V ◽

Membrane Vesicles ◽

Hill Coefficient ◽

Induced Membrane ◽

Binding Domains ◽

Green Fluorescent

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25–100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to ∼5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient ≈ 2). Capacitance can return to baseline in 1–3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P2 is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P2 depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P2 is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P2-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) “scrambling” between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.

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