Functional siRNA Delivery by Extracellular Vesicle-Liposome Hybrid Nanoparticles

The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV-liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA

Functional siRNA Delivery by Extracellular Vesicle–Liposome Hybrid Nanoparticles

The therapeutic use of RNA interference is limited by the inability of siRNA molecules to reach their site of action, the cytosol of target cells. Lipid nanoparticles, including liposomes, are commonly employed as siRNA carrier systems to overcome this hurdle, although their widespread use remains limited due to a lack of delivery efficiency. More recently, nature's own carriers of RNA, extracellular vesicles (EVs), are increasingly being considered as alternative siRNA delivery vehicles due to their intrinsic properties. However, they are difficult to load with exogenous cargo. Here, EV–liposome hybrid nanoparticles (hybrids) are prepared and evaluated as an alternative delivery system combining properties of both liposomes and EVs. It is shown that hybrids are spherical particles encapsulating siRNA, contain EV-surface makers, and functionally deliver siRNA to different cell types. The functional behavior of hybrids, in terms of cellular uptake, toxicity, and gene-silencing efficacy, is altered as compared to liposomes and varies among recipient cell types. Moreover, hybrids produced with cardiac progenitor cell (CPC) derived-EVs retain functional properties attributed to CPC-EVs such as activation of endothelial signaling and migration. To conclude, hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA

Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs – being small and non-living – are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

New drug-strategies to tackle viral-host interactions for the treatment of influenza virus infections

The influenza virus (IV) is a highly contagious virus causing seasonal global outbreaks affecting annually up to 20% of the world's population and leading to 250,000-500,000 deaths worldwide. Current vaccines have variable effectiveness, and, in particular during a pandemic outbreak, they are probably not available in the amounts needed to protect the world population. Therefore we need effective small molecule drugs to combat an IV infection and that can be produced, in case of pandemic, rapidly and in large quantities. Unfortunately, natural occurring IV becomes more and more resistant to current anti-IV drugs. And thus, there is an urgent need for development of alternative agents with new mechanisms of action. This review provides an overview of the pharmacology and effectiveness of new anti-IV agents, focusing on inhibition mechanisms directed against virus-host interactions

New drug-strategies to tackle viral-host interactions for the treatment of influenza virus infections

The influenza virus (IV) is a highly contagious virus causing seasonal global outbreaks affecting annually up to 20% of the world's population and leading to 250,000-500,000 deaths worldwide. Current vaccines have variable effectiveness, and, in particular during a pandemic outbreak, they are probably not available in the amounts needed to protect the world population. Therefore we need effective small molecule drugs to combat an IV infection and that can be produced, in case of pandemic, rapidly and in large quantities. Unfortunately, natural occurring IV becomes more and more resistant to current anti-IV drugs. And thus, there is an urgent need for development of alternative agents with new mechanisms of action. This review provides an overview of the pharmacology and effectiveness of new anti-IV agents, focusing on inhibition mechanisms directed against virus-host interactions