PB0871 - Fusogenic liposomes label human platelets without impacting normal platelet function

Background: Platelets play a critical role at sites of vascular injury to arrest bleeding, yet hyper-responsive platelets can be causative to heart attacks and stroke. Understanding platelet molecular mechanisms is therefore important to identify novel drug targets and improved therapeutics. However, since platelets lack a cell nucleus, conventional methods used to identify processes in nucleated cells cannot directly be applied.

Aims: This project aims to use biocompatible liposomes with a unique fusogenic nature to deliver cargo directly into the cytoplasm of platelets. Enabling biological processes and molecular mechanisms to be studied directly in human platelets. The first step towards this goal is to determine if fusogenic liposomes can fuse with human platelets without impacting function.

Methods: Granule secretion was measured using P-selectin as a marker of platelet activation. Annexin V binding was used as a measure of apoptotic platelets. Platelet adhesion and spreading on Fibrinogen was quantified using a convolutional neural network (CNN). Confocal microscopy assessed the extent of cell labelling and cargo delivery by fluorescently labelled fusogenic liposomes.

Results: Fluorescently labelled fusogenic liposomes efficiently fuse with the membrane of human platelets. Spontaneous fusion with platelets does not lead to platelet activation in the presence of PGI2, with no significant increase in P-selectin surface expression, or the induction of phosphatidylserine translocation to the outer platelet membrane as measured by Annexin V binding. Normal platelet behaviour is unaffected by liposome fusion as measured by agonist induced P-selectin exposure and platelet spreading.

Conclusion(s): These results demonstrate that fusogenic liposomes can label human platelets without impacting normal cell behaviour, revealing a method to deliver cargo directly into the cytoplasm of human platelets to interrogate both known and novel molecular mechanisms in vitro, and in real time.