Cellular Output and Physicochemical Properties of the Membrane-Derived Vesicles Depend on Chemical Stimulants
Year: 2024
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide Synthetic liposomes are widely used as drug delivery vehicles in biomedical treatments, such as for mRNA-based antiviral vaccines like those recently developed against SARS-CoV-2. Extracellular vesicles (EVs), which are naturally produced by cells, have emerged as a next-generation delivery system. However, key questions regarding their origin within cells remain unresolved. In this regard, plasma membrane vesicles (PMVs), which are essentially produced from the cellular plasma membrane (PM), present a promising alternative. Unfortunately, their properties relevant to biomedical applications have not be extensively studied. Therefore, we conducted a thorough investigation of the methods used in the production of PMVs. By leveraging advanced fluorescence techniques in microscopy and flow cytometry, we demonstrated a strong dependence of the physicochemical attributes of PMVs on the chemicals used during their production. Following established protocols employing chemicals such as paraformaldehyde (PFA), N- ethylmaleimide (NEM) or dl -dithiothreitol (DTT) and by developing a modified NEM-based method that involved a hypotonic shock step, we generated PMVs from THP-1 CD1d cells. We systematically compared key parameters such as vesicle output, their size distribution, vesicular content analysis, vesicular membrane lipid organization and the mobility of a transmembrane protein. Our results revealed distinct trends: PMVs isolated using NEM-based protocols closely resembled natural vesicles, whereas PFA induced significant molecular cross-linking, leading to notable changes in the biophysical properties of the vesicles. Furthermore, our novel NEM protocol enhanced the efficiency of PMV production. In conclusion, our study highlights the unique characteristics of chemically produced PMVs and offers insights into their potentially diverse yet valuable biological functions.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: Alexander von Humboldt-Stiftung, Wellcome Trust, Friedrich-Schiller-Universität Jena, Freistaat Thüringen, International Seafood Sustainability Foundation, University of Oxford, Wolfson Foundation, Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Thüringer Ministerium für Wirtschaft, Wissenschaft und Digitale Gesellschaft, Medical Research Council, Engineering and Physical Sciences Research Council, Biotechnology and Biological Sciences Research Council, Leibniz-Gemeinschaft
Awards: G1515183235, G1781628337, G1885323389, G1937223438, G1949617376, MICA: Nanoscopy Oxford (NanO): Novel Super-Resolution Imaging Applied to Biomedical Sciences, G2205214540, G2967549892, SFB 1278: Polymerbasierte NanopartikelBibliotheken für die Entwicklung zielgerichteter antiinflammatorischer Strategien, G3056851145, G3065514043, G3423061657, G4119832957, Verbundprojekt: Hochparalleles Profiling der Wirtsantwort auf lebensbedrohliche Infektionen (LPI-BT4) - Teilvorhaben: Photonische Technologien für hochparalleles Profiling der Wirtsantwort auf lebensbedrohliche Infektionen, Verbundprojekt: Innovative molekulare und biochemische Assays für Schnelldiagnostik, Drug Development und neue Therapiekonzepte (LPI-BT5) - Teilvorhaben: Entwicklung molekularer Techniken, Probenvorbereitungsstrategien und photonischer Detektionsmethoden zum Nachweis und Charakterisierung von Mikroorganismen, G4708599237, G4900274166, G5285405813, G5290307715, G6439945554, EXC 2051: Balance of the Microverse, G738990282, G7986075241, G80795443, G8084192006, Advanced Microscopy for Chromosome and RNA Dynamics., G984412895
OpenAlex ID: W4402354980