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

背景血小板缺乏细胞核，这意味着用来研究有核细胞的传统方法不能应用。因此，血小板领域已经变得严重依赖转基因小鼠模型来研究血小板功能。融合脂质体已经被用来促进水溶性货物直接进入细胞。该项目首次研究融合脂质体是否可以直接与血小板融合，以及它们是否可以作为一种递送方法将货物直接释放到血小板的细胞质中。旨在开发一种卷积神经网络(CNN)来自动化和标准化整个项目中的血小板扩散分析。目的：确定融合脂质体能否在不影响正常血小板功能的情况下与血小板联合使用。为了确定融合后包裹在融合脂质体中的货物是否可以直接输送到血小板的细胞质中。方法使用120幅差异干涉对比显微镜图像对CNN进行训练，并在独立测试集和5个手动注释器上对模型性能进行评估。通过测量P-选择素暴露、磷脂酰丝氨酸易位、血小板铺展和血小板聚集来评估融合脂质体与血小板膜融合对正常血小板行为的影响。结果CNN免去了对人和小鼠血小板进行耗时和有偏见的人工分析，提供了一种可用于在更广泛的血小板领域标准化血小板扩散分析的工具。融合脂质体可以有效地标记血小板，而不会对正常的血小板功能造成明显影响。这为体外和体内应用提供了一种替代荧光标记血小板的方法。融合脂质体将荧光标记的Lifeact多肽和全抗体载体运送到血小板细胞质中。这使得可以在活的血小板中研究细胞内的过程，有可能允许抗体介导的蛋白质在人类血小板中被敲除，从而减少使用动物模型中的血小板的需要。

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.