Periodic Reporting for period 1 - NanoEXOS (Towards a Mechanistic Understanding of Nanoparticle Interactions with Exosome Secretion)
Reporting period: 2023-09-01 to 2025-08-31
CONTEXT AND OBJECTIVES
NanoEXOS was conceived to address a fundamental and still unresolved challenge in bionanoscience: understanding how engineered nanoparticles (NPs) interact with the cellular machinery that produces exosomes. Exosomes are nanosized cell-released particles enriched in biologically active molecules such as extracellular RNAs and proteins. They act as key mediators of intercellular communication and are increasingly recognised for their central roles in both physiological regulation and disease development. The latter includes cancer progression, immune alterations, and the pathogenesis of liver, metabolic, cardiovascular, infectious, and neurodegenerative disorders. Despite their growing biomedical relevance, the precise mechanisms through which exogenous NPs alter or influence exosome formation remain poorly understood. This knowledge gap limits the capacity to design safe nanotechnologies and hinders the development of therapeutic and diagnostic strategies that might harness the potential of these extracellular nanostructures. By focusing on the interaction between NPs and exosomal pathways, NanoEXOS aimed to establish a novel mechanistic framework for understanding, predicting, and ultimately controlling NP–induced cellular responses.
PATHWAY TO IMPACT
The expected impacts of NanoEXOS are scientific, technological, and societal. Scientifically, the project contributes to a deeper understanding of bio-nano interactions at the cellular level, an essential step towards the creation of predictive models of NP safety and functionality. Technologically, the knowledge gained through NanoEXOS can be used to design next-generation nanomaterials, enabling safer biomedical applications and supporting the emergence of exosome-inspired nanomedicines. Strategically, the project contributes to strengthening Europe’s position in cutting-edge nanomedicine research, with an emphasis on healthcare innovation. The potential scale of impact is significant: safer and more advanced nanomedicine will enable progress towards personalised healthcare and reduced health risks, while biomedical innovations based on exosome biology could help address major health challenges such as the treatment of cancer, neurodegeneration, and infectious diseases. In this sense, NanoEXOS contributes both to advancing scientific frontiers and to creating pathways for tangible societal benefits.
NanoEXOS was conceived to address a fundamental and still unresolved challenge in bionanoscience: understanding how engineered nanoparticles (NPs) interact with the cellular machinery that produces exosomes. Exosomes are nanosized cell-released particles enriched in biologically active molecules such as extracellular RNAs and proteins. They act as key mediators of intercellular communication and are increasingly recognised for their central roles in both physiological regulation and disease development. The latter includes cancer progression, immune alterations, and the pathogenesis of liver, metabolic, cardiovascular, infectious, and neurodegenerative disorders. Despite their growing biomedical relevance, the precise mechanisms through which exogenous NPs alter or influence exosome formation remain poorly understood. This knowledge gap limits the capacity to design safe nanotechnologies and hinders the development of therapeutic and diagnostic strategies that might harness the potential of these extracellular nanostructures. By focusing on the interaction between NPs and exosomal pathways, NanoEXOS aimed to establish a novel mechanistic framework for understanding, predicting, and ultimately controlling NP–induced cellular responses.
PATHWAY TO IMPACT
The expected impacts of NanoEXOS are scientific, technological, and societal. Scientifically, the project contributes to a deeper understanding of bio-nano interactions at the cellular level, an essential step towards the creation of predictive models of NP safety and functionality. Technologically, the knowledge gained through NanoEXOS can be used to design next-generation nanomaterials, enabling safer biomedical applications and supporting the emergence of exosome-inspired nanomedicines. Strategically, the project contributes to strengthening Europe’s position in cutting-edge nanomedicine research, with an emphasis on healthcare innovation. The potential scale of impact is significant: safer and more advanced nanomedicine will enable progress towards personalised healthcare and reduced health risks, while biomedical innovations based on exosome biology could help address major health challenges such as the treatment of cancer, neurodegeneration, and infectious diseases. In this sense, NanoEXOS contributes both to advancing scientific frontiers and to creating pathways for tangible societal benefits.
The NanoEXOS project pursued three interconnected research and innovation objectives. First, it investigated how engineered NPs interact with the endosomal pathways responsible for exosome formation. This contributed to the discovery and characterisation of a rare class of hybrid exosome-like extracellular nanostructures generated via NP–cell interactions. Similarly to exosomes, these structures originate intracellularly, carry a biologically active biomolecular cargo rich in proteins and RNAs, and are implicated in intercellular communication mechanisms. Conditionally accepted for publication in Nature Materials, these findings provide unprecedented insights into the intracellular processing of exogenous NPs. Second, NanoEXOS investigated the physicochemical and biochemical properties of these newly identified exosome-like entities using advanced biophysical and biochemical methods. A major focus was placed on omics-based analyses, which enabled detailed characterisation of the protein and RNA content within these particles. By integrating high-quality RNA sequencing with mass spectrometry-based proteomics, NanoEXOS developed a new proteogenomic workflow that simultaneously analyzes RNA and proteins, providing an integrated view of their biological interplay. This provided a more comprehensive picture of how molecules are packaged inside the particles and the potential biological roles they may play. Third, the project advanced to an in-depth analysis of the nanoscale molecular organisation of exosome-like extracellular nanostructures. To achieve this, novel methodologies were established, including single-particle fluorescent labelling analysis and nanoscale crosslinking for mapping structural RNA–protein interactions. These methodological breakthroughs are the basis of a series of new manuscripts, some already submitted to high-impact journals (e.g. ACS Nano) and others currently in preparation.
NanoEXOS generated highly innovative research results. The project revealed that exogenous nanosized particles, once internalised and processed by cells, can induce the formation of hybrid exosome-like nanostructures that are subsequently released in the extracellular environment. These nanostructures exhibit distinct RNA and protein signatures that may have relevant biological implications in vivo, including mediation of intercellular communication. Such findings redefine current understanding of NP–cell interactions and have broad implications for nanomedicine and extracellular nanoparticle research. Methodologically, the project advanced the field with two major innovations. First, the integration of long-read transcriptomics with high-throughput proteomics established a novel proteogenomic workflow that provides a comprehensive view of the protein and RNA cargo within extracellular nanostructures. Second, the project developed a pioneering nanoscale crosslinking approach to probe and resolve RNA–protein architectures at unprecedented resolution. Importantly, the scientific insights and technologies developed within NanoEXOS extend far beyond the current state of the art. They provide reference methodologies that will guide future research in bionanoscience and nanomedicine, enabling a deeper understanding of RNA-carrying extracellular nanostructures and the mechanisms of intercellular RNA delivery. In particular, they pave the way for the rational design of novel extracellular RNA nanocarriers, including advanced RNA vaccines with tangible biomedical impact. To fully unlock this potential, further research and experimental efforts are needed to refine, validate, and standardise the proteogenomic and nanoscale crosslinking workflows through ongoing omics analyses and cross-validation across diverse nanoparticle systems and biological contexts. At the same time, broad uptake and successful translation of NanoEXOS outcomes will depend on active engagement with regulatory and industrial stakeholders, ensuring that these advances move efficiently toward therapeutic and commercial applications.