Fusogenic liposomes: the innovative delivery of compounds into human platelets to reduce animal use in platelet research

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Background Platelets lack a nucleus meaning that conventional methods, used to investigate nucleated cells, cannot be applied. As a result, the platelet field has become heavily reliant on genetically modified mouse models to investigate platelet function. Fusogenic liposomes have been used to facilitate the delivery of water-soluble cargo directly into cells. This project investigates, for the first time, if fusogenic liposomes can fuse directly with platelets and if they can be used as a delivery method to release cargo directly into the cytoplasm of platelets. Aims To develop a convolutional neural network (CNN) to automate and standardise platelet spreading analyses throughout this project. To determine if fusogenic liposomes can be used in combination with platelets without impacting on normal platelet function. To identify if cargo, encapsulated in fusogenic liposomes, can be delivered directly to the cytoplasm of platelets following fusion. MethodsA CNN was trained using 120 Differential Interference Contrast microscopy images where model performance was evaluated against an independent test set and five manual annotators. Any impact on normal platelet behaviour, due to fusion by fusogenic liposomes with the platelet membrane, was assessed by measuring P-selectin exposure, phosphatidylserine translocation, platelet spreading, and platelet aggregation. ResultsA CNN abrogates time consuming and biased manual analyses for both human and mouse platelets, delivering a tool that can be used to standardise platelet spreading assays in the wider platelet field. Platelets were efficiently labelled with fusogenic liposomes without causing significant impact to normal platelet function. This offers an alternative method to fluorescently label platelets for in vitro and in vivo applications. Fusogenic liposomes delivered fluorescently labelled Lifeact peptides and whole antibody cargo into the platelet cytoplasm. This enables intracellular processes to be studied in live platelets, with the potential to allow antibody-mediated protein knockdown in human platelets, reducing the need to use platelets from animal models.