scholarly journals Cytochalasin-B-Inducible Nanovesicle Mimics of Natural Extracellular Vesicles That Are Capable of Nucleic Acid Transfer

Micromachines ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 750 ◽  
Author(s):  
Oshchepkova ◽  
Neumestova ◽  
Matveeva ◽  
Artemyeva ◽  
Morozova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nucleic Acid ◽  
Extracellular Vesicles ◽  
Cytochalasin B ◽  
Cell Communication ◽  
Extracellular Vesicle ◽  
Hek293 Cells ◽  
Nucleic Acid Delivery ◽  
Loading Capacity ◽  
Oligonucleotide Delivery

Extracellular vesicles provide cell-to-cell communication and have great potential for use as therapeutic carriers. This study was aimed at the development of an extracellular vesicle-based system for nucleic acid delivery. Three types of nanovesicles were assayed as oligonucleotide carriers: mesenchymal stem cell-derived extracellular vesicles and mimics prepared either by cell treatment with cytochalasin B or by vesicle generation from plasma membrane. Nanovesicles were loaded with a DNA oligonucleotide by freezing/thawing, sonication, or permeabilization with saponin. Oligonucleotide delivery was assayed using HEK293 cells. Extracellular vesicles and mimics were characterized by a similar oligonucleotide loading level but different efficiency of oligonucleotide delivery. Cytochalasin-B-inducible nanovesicles exhibited the highest level of oligonucleotide accumulation in HEK293 cells and a loading capacity of 0.44 ± 0.05 pmol/µg. The loaded oligonucleotide was mostly protected from nuclease action.

CSF extracellular vesicles and risk of disease activity after a first demyelinating event

2021 ◽  
pp. 135245852098754
Author(s):  
Gloria Dalla Costa ◽  
Tommaso Croese ◽  
Marco Pisa ◽  
Annamaria Finardi ◽  
Lorena Fabbella ◽  
...  
Keyword(s):  
Disease Activity ◽  
Extracellular Vesicles ◽  
Prognostic Markers ◽  
Cell Communication ◽  
Extracellular Vesicle ◽  
Intervention Strategies ◽  
Targeted Intervention ◽  
Improve Risk Stratification ◽  
Risk Of Disease ◽  
In Cis

Background: Extracellular vesicles (EVs), a recently described mechanism of cell communication, are released from activated microglial cells and macrophages and are a candidate biomarker in diseases characterized by chronic inflammatory process such as multiple sclerosis (MS). Methods: We explored cerebrospinal fluid extracellular vesicle (CSF EV) of myeloid origin (MEVs), cytokine and chemokine levels in patients with clinically isolated syndrome (CIS). Results: We found that CSF MEVs were significantly higher in CIS patients than in controls and were inversely correlated to CSF CCL2 levels. MEVs level were significantly associated with an shorter time to evidence of disease activity (hazard ratio: 1.01, 95% confidence interval: 1.00–1.02, p < 0.01) independently from other known prognostic markers. Conclusion: After a first demyelinating event, CSF EVs may improve risk stratification of these patients and allow more targeted intervention strategies.

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

Pharmaceutics ◽  
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.

scholarly journals Efficient Transfection of Large Plasmids Encoding HIV-1 into Human Cells—A High Potential Transfection System Based on a Peptide Mimicking Cationic Lipid

Pharmaceutics ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 805
Author(s):  
Christopher Janich ◽  
Daniel Ivanusic ◽  
Julia Giselbrecht ◽  
Elena Janich ◽  
Shashank Reddy Pinnapireddy ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Nucleic Acid ◽  
Fluorescent Protein ◽  
Cationic Lipid ◽  
Nucleic Acid Delivery ◽  
Molecular Clone ◽  
Green Fluorescent ◽  
Hiv 1

One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

scholarly journals Why the need and how to approach the functional diversity of extracellular vesicles

2017 ◽  
Vol 373 (1737) ◽  
pp. 20160479 ◽  
Author(s):  
Mercedes Tkach ◽  
Joanna Kowal ◽  
Clotilde Théry
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Tumour Microenvironment ◽  
Efficient Separation ◽  
The Past ◽  
Therapeutic Tools ◽  
Opinion Article ◽  
Current Stage

In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to release other types of EVs, for instance, by direct budding off their plasma membrane. Some of these EVs share with exosomes major biophysical and biochemical characteristics, such as size, density and membrane orientation, which impose difficulties in their efficient separation. Despite frequent claims in the literature, whether exosomes really display more important patho/physiological functions, or are endowed with higher potential as diagnostic or therapeutic tools than other EVs, is not yet convincingly demonstrated. In this opinion article, we describe reasons for this lack of precision knowledge in the current stage of the EV field, we review recently described approaches to overcome these caveats, and we propose ways to improve our knowledge on the respective functions of distinct EVs, which will be crucial for future development of well-designed EV-based clinical applications. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

scholarly journals Evaluation of plasmatic extracellular vesicles by size

2021 ◽  
Author(s):  
Laura Cantone ◽  
Mirjam Hoxha ◽  
Chiara Favero ◽  
Luca Ferrari ◽  
Valentina Bollati
Keyword(s):  
Plasma Membrane ◽  
Blood Plasma ◽  
Extracellular Vesicles ◽  
High Precision ◽  
Extracellular Vesicle ◽  
Nanoparticle Tracking Analysis ◽  
Research Needs ◽  
Plasma Samples ◽  
Constant Flow ◽  
Syringe Pump

Abstract Extracellular vesicles (EVs) play a key role in many physiological and pathological processes [1]. EVs are a heterogeneous group of membrane-confined particles including endosome-derived exosomes and plasma membrane-originated microvesicles. The expanding field of extracellular vesicle research needs reproducible and accurate methods to characterize EVs [2]. EV profiling can be challenging due to the small size and heterogeneity. This protocol aims to provide a method to isolate EVs and facilitate high-precision particle quantitation by Nanoparticle Tracking Analysis (NTA)[3, 4]. NTA is commonly used to determine EV concentration and diameter [5, 6]. The protocol here described refers to the isolation of EVs from blood-plasma samples by using ultracentrifugation and then quantification and sizing of EVs with NTA by NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK) provided with a syringe pump module enabling analysis in constant flow for improved sample statistics.

scholarly journals Viral Mimicry as a Design Template for Nucleic Acid Nanocarriers

2021 ◽  
Vol 9 ◽  
Author(s):  
Ina F. de la Fuente ◽  
Shraddha S. Sawant ◽  
Mark Q. Tolentino ◽  
Patrick M. Corrigan ◽  
Jessica L. Rouge
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Molecular Mechanisms ◽  
Transfection Efficiency ◽  
Delivery Systems ◽  
Endosomal Escape ◽  
Nucleic Acid Delivery ◽  
Metastable Structure ◽  
Oligonucleotide Delivery ◽  
Genome Protection

Therapeutic nucleic acids hold immense potential in combating undruggable, gene-based diseases owing to their high programmability and relative ease of synthesis. While the delivery of this class of therapeutics has successfully entered the clinical setting, extrahepatic targeting, endosomal escape efficiency, and subcellular localization. On the other hand, viruses serve as natural carriers of nucleic acids and have acquired a plethora of structures and mechanisms that confer remarkable transfection efficiency. Thus, understanding the structure and mechanism of viruses can guide the design of synthetic nucleic acid vectors. This review revisits relevant structural and mechanistic features of viruses as design considerations for efficient nucleic acid delivery systems. This article explores how viral ligand display and a metastable structure are central to the molecular mechanisms of attachment, entry, and viral genome release. For comparison, accounted for are details on the design and intracellular fate of existing nucleic acid carriers and nanostructures that share similar and essential features to viruses. The review, thus, highlights unifying themes of viruses and nucleic acid delivery systems such as genome protection, target specificity, and controlled release. Sophisticated viral mechanisms that are yet to be exploited in oligonucleotide delivery are also identified as they could further the development of next-generation nonviral nucleic acid vectors.

scholarly journals Effects of ATP9A on Extracellular Vesicle Release and Exosomal Lipid Composition

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Xiao Xu ◽  
Limei Xu ◽  
Peng Zhang ◽  
Kan Ouyang ◽  
Yin Xiao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicles ◽  
Lipid Composition ◽  
Extracellular Vesicle ◽  
Biological Processes ◽  
Vesicle Release ◽  
Endosomal Recycling ◽  
Intracellular Compartments ◽  
Intercellular Communications

Numerous biological processes are regulated by the intercellular communications arising from extracellular vesicles (EVs) released from cells. However, the mechanisms that regulate the quantity of EV discharged have yet to be understood. While it is known that ATP9A, a P4-ATPase, is involved in endosomal recycling, it is not clear whether it also contributes to the release of EVs and the makeup of exosomal lipids. This study is aimed at exploring the role of human ATP9A in the process of EV release and, further, to analyze the profiles of EV lipids regulated by ATP9A. Our results demonstrate that ATP9A is located in both the intracellular compartments and the plasma membrane. The percentage of ceramides and sphingosine was found to be significantly greater in the control cells than in the ATP9A overexpression and ATP9A knockout groups. However, EV release was greater in ATP9A knockout cells, indicating that ATP9A inhibits the release of EVs. This study revealed the effects of ATP9A on the release of EVs and the lipid composition of exosomes.

scholarly journals Extracellular vesicle-mediated cell–cell communication in haematological neoplasms

2017 ◽  
Vol 373 (1737) ◽  
pp. 20160484 ◽  
Author(s):  
Junko H. Ohyashiki ◽  
Tomohiro Umezu ◽  
Kazuma Ohyashiki
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Cell Communication ◽  
Tumour Microenvironment ◽  
Extracellular Vesicle ◽  
Emerging Issues ◽  
Haematological Neoplasms ◽  
Cell Cell

Crosstalk between bone marrow tumour cells and surrounding cells, including bone marrow mesenchymal stromal cells (BM-MSCs), endothelial cells and immune cells, is important for tumour growth in haematological neoplasms. In addition to conventional signalling pathways, extracellular vesicles (EVs), which are endosome-derived vesicles containing proteins, mRNAs, lipids and miRNAs, can facilitate modulation of the bone marrow microenvironment without directly contacting non-tumourous cells. In this review, we discuss the current understanding of EV-mediated cell–cell communication in haematological neoplasms, particularly leukaemia and multiple myeloma. We highlight the actions of tumour and BM-MSC EVs in multiple myeloma. The origin of EVs, their tropism and mechanism of EV transfer are emerging issues that need to be addressed in EV-mediated cell–cell communication in haematological neoplasms. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

scholarly journals Mechanisms of Extracellular Vesicle Biogenesis, Cargo Loading, and Release

2021 ◽  
Author(s):  
Abdel A. Alli
Keyword(s):  
Plasma Membrane ◽  
Circadian Rhythms ◽  
Nucleic Acids ◽  
Extracellular Vesicles ◽  
Extracellular Space ◽  
Extracellular Vesicle ◽  
Apoptotic Bodies ◽  
Endosomal Membranes ◽  
Vesicle Biogenesis

Extracellular vesicles (EVs) are carriers of various biomolecules including bioactive enzymes, lipids, proteins, nucleic acids, and metabolites. EVs are classified into three main types based on their size, biogenesis, and cargo. Exosomes originate from endosomal membranes and are the smallest type of EV. Microvesicles (MVs) or microparticles are larger in size, and like apoptotic bodies which represent the largest type of EVs, both of these vesicles originate from outward budding of the plasma membrane. As discussed in this chapter, cargo loading of EVs and their release into the extracellular space where they can be taken up by neighboring or distant cells plays an important role in physiology and pathophysiology. This chapter will outline specific mechanisms involved in the loading and enrichment of miRNAs, proteins, and lipids within EVs. As explained here, various external and biological stimuli play a role in EV release. Finally, recent studies have shown that the biogenesis, cargo loading, and release of EVs are governed by circadian rhythms. Although EVs were once thought to serve as garbage disposals of cells, the numerous roles they serve in physiology and pathophysiology are now being appreciated.

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