PB0781 - Liposome-assisted uptake of procoagulant proteins by platelets

Background: Platelets are gaining interest as a drug delivery vehicle. Clinically important areas in this context include the treatment of bleeding and cancer, and the killing of bacteria. However, research has not yet led to a clinically applicable treatment. One of the reasons for this is lack of knowledge about effective platelet loading and how platelets subsequently deal with this cargo.

Aims: To investigate the preparation and utility of liposome-assisted procoagulant drug-loaded platelet concentrates for transfusion.

Methods: We used coagulation factor IX (FIX) as a model cargo to study the effectiveness of different liposomes in supporting drug loading of washed platelets and how platelets subsequently cope with this artificial cargo.

Results: FIX is not taken up spontaneously, but platelets can be loaded with FIX when encapsulated in liposomes with a composition of DOPC/DOPE/cholesterol in a 23/4/23/molar ratio (8%DOPE). Confocal microscopy demonstrated endocytosis of complete FIX-8%DOPE liposomes. FACS analysis revealed rapid uptake kinetics. However, adjustment of the liposome composition was required as stability experiments revealed that our 8%DOPE liposomes tend to aggregate at physiological conditions. We therefore examined the following liposomes with a more positive or negative charge: DOPC/DOPE/cholesterol/DOTAP 23/4/11/12 molar ratio (24%DOTAP) and DOPC/DOPE/cholesterol/DOPS 23/4/11/12 molar ratio (24%DOPS). Suspensions of 24%DOTAP liposomes and 24%DOPS liposomes are stable. 24%DOTAP liposomes are fusogenic with its FIX load localizing to the platelet membrane. 24%DOTAP liposome-loaded platelets have lost their ability to respond to a PAR-1 selective agonist. Uptake behaviour of FIX-containing 24%DOPS liposomes was similar as for 8%DOPE liposomes. Platelets loaded with 8%DOPE or 24%DOPS liposomes responded normally to the PAR-1 selective agonist. Follow-up studies with other liposome compositions are ongoing.

Conclusion(s): Platelets are able to engulf liposomes. Liposome uptake, cargo localization, and response of loaded platelets to activation triggers are liposome composition dependent.