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

Nanoplataformas derivadas de células para o tratamento de câncer

Nanomaterials are promising platforms for cancer therapy due to their innate passive targeting. The success of nanomaterials into the clinics depends on their blood circulation time and accumulation in the target tissue, factors related with their ability to evade the immune system. Cell-derived nanoplatforms are an emerging technology to enhance the delivery by active targeting the tumor site, without the perks of chemical conjugations. In this thesis we report the development of biomimetic novel platforms using the cell-derived technology and their in vitro interaction in cells from tumor microenvironment. To understand of the cell-derived nanoplatforms, two different nanomaterials were synthesized and further coated with extracellular vesicles and cell membrane extract from two different cell lines. First, gold nanorods (AuNRs) were coated with two macrophage derived vesicles, cell membrane extract and extracellular vesicles. Cell membrane-coated AuNRs interacted more with the metastatic cancer cells and the extracellular vesicles interacted more with the source cells. The main difference evaluated among the coatings was the presence of the tetraspanin CD47, an immunosuppressive marker for phagocytosis. Furthermore, we developed a paclitaxel-loaded polymeric nanoparticle carrier coated with metastatic breast cancer cell membrane. All cell lines showed a preferential uptake for the nanoparticles coated with the cell membrane, with stronger interaction with the source cell and the fibroblasts. Our results pointed to the role of adhesion molecules in the homotypic bind to cancer cells and the interaction with stroma cells as a heritage of the tumor progression pathways. As a consequence of the enhanced interaction of the nanocarriers with fibroblasts, the nanoparticles were significantly cytotoxic. We also explored the tunability of the plasmonic band in relation to their composition and size and evaluated basic culture parameters for extracellular vesicles isolation by means of size distribution and concentration.Os nanomateriais são plataformas promissoras para a terapia do câncer devido ao seu inato acúmulo passivo em tumores. O sucesso dos mesmos na clinica depende do seu tempo dentro da circulação sanguínea e da sua acumulação no tecido alvo, fatores relacionados com a capacidade de escapar ao sistema imunológico. As nanoplataformas derivadas de células são uma tecnologia emergente para melhorar estas propriedades através do acúmulo ativo no local do tumor. Nesta tese relatamos o desenvolvimento de plataformas biomiméticas inovadoras avaliando suas interações in vitro em células modelo do microambiente tumoral. Para compreendermos melhor as nanoplataformas derivadas de células, dois nanomateriais diferentes foram sintetizados e posteriormente revestidos. Inicialmente, os nanorods de ouro (AuNRs) foram revestidos com duas vesículas derivadas de macrófagos, vesículas de membrana celular e vesículas extracelulares. Os AuNRs revestidos de membrana celular interagiram mais com a linha celular metastática de câncer e as vesículas extracelulares interagiram com a célula de origem (macrofágos). A principal diferença avaliada entre os revestimentos foi a presença da tetraspanina CD47, imunossupressor para a fagocitose. Além disso, desenvolvemos nanopartículas poliméricas com paclitaxel, revestidas com membrana celular metastática de câncer da mama. Todas as linhagens celulares mostraram uma interação preferencial para as nanopartículas revestidas com membrana celular, tendo uma interação mais pronunciada com a célula de origem e os fibroblastos. Este resultado indica o papel das moléculas de adesão nas interações homotípicas das nanopartículas às células cancerosas, além da herança da interação da célula tumoral com as células do estroma para a progressão do tumor. Como consequência, para uma maior interação com os fibroblastos, as nanopartículas foram significativamente citotóxicas. Os resultados da tese mostram como estas novas classes de nanomateriais são desenvolvidas e as suas interações com o microambiente tumoral. Além disso, estudamos mudanças na banda plasmonica dos nanorods de ouro em relação à sua composição e tamanho. Por fim, avaliamos os parâmetros de isolamento das vesículas extracelulares por distribuição de tamanho e concentração

Extracellular vesicles: innovative cell-free solutions for wound repair

Chronic non-healing wounds are often associated with conditions such as diabetes and peripheral vascular disease, pose significant medical and socioeconomic challenges. Cell-based therapies have shown promise in promoting wound healing but have major drawbacks such as immunogenicity and tumor formation. As a result, recent research has shifted to the potential of extracellular vesicles (EVs) derived from these cells. EVs are nanosized lipid bilayer vesicles, naturally produced by all cell types, which facilitate intercellular communication and carry bioactive molecules, offering advantages such as low immunogenicity, negligible toxicity and the potential to be re-engineered. Recent evidence recognizes that during wound healing EVs are released from a wide range of cells including immune cells, skin cells, epithelial cells and platelets and they actively participate in wound repair. This review comprehensively summarizes the latest research on the function of EVs from endogenous cell types during the different phases of wound healing, thereby presenting interesting therapeutic targets. Additionally, it gives a critical overview of the current status of mesenchymal stem cell-derived EVs in wound treatment highlighting their tremendous therapeutic potential as a non-cellular of-the-shelf alternative in wound care.The author(s) declare that financial support was received for the research and/or publication of this article. This research was supported by the research fund “BOF Grand Challenges” provided by Hasselt University (grant number: 21GP08BROA) to AB and EP and following research grants of the “FWO- Research Fund Flanders” 1S28025N to SW and grant 1269322N to PL

Manufacture of extracellular vesicles derived from mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are a promising therapy for various diseases ranging from ischemic stroke to wound healing and cancer. Their therapeutic effects are mainly mediated by secretome-derived paracrine factors, with extracellular vesicles (EVs) proven to play a key role. This has led to promising research on the potential of MSC-EVs as regenerative, off-the-shelf therapeutic agents. However, the translation of MSC-EVs into the clinic is hampered by the poor scalability of their production. Recently, new advanced methods have been developed to upscale MSC cultivation and EV production yields, ranging from new cell culture devices to priming procedures. This review gives an overview of these innovative strategies for manufacturing MSC-EVs

The amount of dextran in PLGA nanocarriers modulates protein corona and promotes cell membrane damage

Polymeric nanocarriers (NCs) are efficient vehicles to prevent drug unspecific biodistribution and increase the drug amounts delivered to tumor tissues. However, some toxicological aspects of NCs still lack a comprehensive assessment, such as their effects on cellular processes that lead to toxicity. We evaluate the interaction of poly(lactic-co-glycolic acid) (PLGA) NCs prepared using dextran (Dex) and Pluronic®-F127 as stabilizing agents with myocardial cells (H9C2), breast adenocarcinoma cells (MCF-7) and macrophages (RAW 264.7) to address the effect of Dex in PLGA NC formulations. By an emulsion diffusion method, doxorubicin-loaded NCs were prepared with no Dex (PLGA-DOX), 1% (w/v) Dex (Dex1/PLGA-DOX) and 5% (w/v) Dex (Dex5/PLGA-DOX). Uptake analyses revealed a significant reduction in Dex5/PLGA-DOX NC uptake by H9C2 and MCF-7, as in the case of Dex1/PLGA-DOX NCs in the absence of in vitro protein corona, revealing an effect of dextran concentration on the formation of protein corona. RAW 264.7 cells presented a greater uptake of Dex5/PLGA-DOX NCs than the other NCs likely because of receptor mediated endocytosis, since C-type lectins like SIGN-R1, mannose receptors and scavenger receptor type 1 that are expressed in RAW 264.7 can mediate Dex uptake. Despite the lower uptake, Dex5/PLGA-DOX NCs promote the generation of reactive oxygen species and oxidative membrane damage in MCF-7 and H9C2 even though cellular metabolic activity assessed by MTT was comparable among all the NCs. Our results highlight the importance of an in-depth investigation of the NC–cell interaction considering additional mechanisms of damage apart from metabolic variations, as nanoparticle-induced damage is not limited to imbalance in metabolic processes, but also associated with other mechanisms, e.g., membrane and DNA damage

Outer membrane vesicles (OMVs) and their therapeutic potential as anti-infectious agents

Outer membrane vesicles (OMVs) derived from Gram-negative bacteria have emerged as promising therapeutic agents for combating infectious diseases. These small, spherical structures carry diverse cargo molecules, including virulence factors, antigens, and immunomodulatory molecules. OMVs derived from pathogenic bacteria can be engineered to deliver antimicrobial peptides, antibiotics, or specific antigens, eliciting targeted immune responses against bacterial pathogens. Similarly, OMVs can deliver viral antigens, facilitating immune recognition and clearance of viral infections. Furthermore, OMVs can be engineered to encapsulate antifungal agents or fungal antigens, enabling targeted delivery and immune activation against fungal pathogens. The cargo-carrying capacity and immunostimulatory properties of OMVs make them valuable tools for developing effective treatments against infectious diseases. This review also discusses the challenges and future directions in applying OMVs as therapeutic agents, highlighting the need for further research and development to harness their full potential in clinical applications. Overall, OMVs represent a promising avenue for developing novel therapeutic strategies against infectious diseases, offering targeted and immunomodulatory strategies for combating bacterial, viral, and fungal infections