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Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production., Journal of Extracellular Vesicles, 10 (6), ISSN:2001-3078

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Introduction: VES4US is a new European project funded by the Horizon 2020-Future and Emerging Technology Open programme, which aims to develop an innovative platform for the efficient production of extracellular vesicles (EVs) from a renewable biosource, enabling their exploitation as tailor-made products in the fields of nanomedicine, cosmetics and nutraceutics (https://ves4us.eu). A core aspect of the project is to focus on an identified natural source to constitute a cost-effective and sustainable source of EVs. The project will run for the next three years with six organizations from six European countries. Methods: In the first phase, the focus will be the selection of the natural source and the optimization of culture condition at pre-industrial scale. Then, the isolation and physiochemical characterization of the EVs will be realized. In the second phase, the functionalization and load of the EVs will be approached. Finally, the biological activity of the EVs will be explored both in vitro and in vivo. Results: The selection of the best EV-producing natural source strain/s is ongoing and it will get to the production of the EVs needed to develop the natural nanocarriers. Before that happens, it exists the need of applying good research practices that include personnel training on Standard Operating Procedures to control major experimental activities for harvesting, manipulating, storing, characterizing and treating EVs, as well as for key related activities. Summary/Conclusion: Safe, efficient and specific nano-delivery systems are essential to current therapeutic medicine, cosmetic and nutraceutics sectors. The ability to optimize the bioavailability, stability and targeted cellular uptake of a bioactive molecule while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. VES4US aims at creating a fundamentally new bioprocessing approach to generate and functionalize EVs from a renewable biological source. Funding: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 801338.