Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents

Figures

• 

  Figure 1.. Preparation and physicochemical characterization of platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs). Schematic illustration of PLT-PFCs as universal reversal agents of antiplatelet agents (APAs). PLT-PFCs competitively bind to APAs at their corresponding specific platelet membrane receptors to prevent the bleeding risk, while the substitution of the intraplatelet matrix with an inactive PFC core diminishes the thrombotic risk. A, Diameter and zeta potential of PLT-PFCs at the indicated PFC to platelet membrane ratios 7:1 (PFC:platelet, w/w) was the optimal ratio (indicated by the red frame), with an appropriate size and zeta potential (n=3). B, Average hydrodynamic size of PLT-PFCs at around 150 nm (n=3) and their morphology (transmission electron microscopy). Scale bar = 200 nm.

• 

  Figure 2.. Functional protein characterization of platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs). A, Nano-flow cytometry validation of key platelet receptors presented on the surface of the PLT-PFC nanoparticles, including αIIbβ3 and P2Y12 receptors. B, Protein profiles of PLT-PFC, platelet nanovesicles (PNV), and platelet lysates, as analyzed by SDS-PAGE. C and D, Differential analysis of cytoplasmic (C) and membrane (D) proteins between platelets and PLT-PFCs (n=3). E, Semiquantitative western blot results showed the preservation of membrane surface receptors, CD41, CD61, and P2Y12, together with the loss of the platelet intracellular protein marker GAPDH, analyzed by ImageJ software (n=3). GAPDH indicates glyceraldehyde phosphate dehydrogenase.

• 

  Figure 3.. In vitro binding affinity of platelet-mimicking perfluorocarbon-based nanospones (PLT-PFCs) to antiplatelet agents (APAs) and the reversal of APA activity. A, Ticagrelor inhibits the platelet P2Y12 receptor, which can be competitively reversed by PLT-PFC supplementation. B, Isothermal titration calorimetry (ITC) assay of the adsorbing affinity (Ka) of PLT-PFCs for ticagrelor. C, Microscale thermophoresis (MST) assay of the dissociation ability (Kd) between PLT-PFCs and ticagrelor. D, Fitting analysis of flow cytometry results of ticagrelor inhibiting platelet P2Y12 receptors in the presence of a series of PLT-PFCs amounts. Experimental plots applied for the calculations originated from the flow cytometry results. E, PLT-PFC supplementation restored platelet aggregation ability, which had been inhibited by ticagrelor. F, Human platelet aggregation rate with series concentrations of ticagrelor and PLT-PFC supplementation was quantified. Low, medium, and high dose of PLT-PFCs indicated equivalent doses of 6.7, 13.3, and 26.7×108 platelet counts/mL, respectively (n=6; P-values were assessed by Kruskal-Wallis tests followed by Dunn-Bonferroni post hoc tests. For 4 independent groups…

• 

  Figure 4.. Blood pharmacokinetics and biodistribution of platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs). A, Representative fluorescence images (left) and quantification (right) of blood samples derived from mice treated with Cy5.5-labeled PLT-PFCs, platelet nanovesicles (PVNs), or BSA-PFCs administered at predetermined time points (n=6). B and C, Representative images (left) and quantification (right) of the biodistribution of PLT-PFC (B) and PNV (C) nanoparticles in the major organs, including heart, brain, spleen, lung, liver, and kidney, at the indicated time points. Quantitative results were presented in Figure S27.

• 

  Figure 5.. In vivo blood elimination and reversal of ticagrelor activity by platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs). A, High performance liquid chromatography (HPLC) quantification of ticagrelor in plasma derived from mice treated with ticagrelor, ticagrelor+PLT-PFC transfusion, ticagrelor + high-dose platelet transfusion, or ticagrelor+standard-dose platelet transfusion at predetermined time points post ticagrelor injection. B, The pharmacokinetic behavior of ticagrelor was analyzed, including its half-life and area under the curve (AUC), calculated using a linear 1-compartment model. High dose (HD), 1×109 platelet counts/kg; standard dose (SD), 2×108 platelet counts/kg (n=6; P-values were assessed by Kruskal-Wallis tests [multiple groups] followed by Dunn-Bonferroni post hoc tests. For 4 independent groups comparison in the left, the overall ANOVA P-value=0.12; for 4 independent groups comparison in the right, the overall ANOVA P-value=0.0012.). C, Tail bleeding time of mice treated with saline, ticagrelor, ticagrelor+PLT-PFC transfusion, ticagrelor+high-dose platelet transfusion, or ticagrelor+standard-dose platelet transfusion at the indicated time points. The…

• 

  Figure 6.. In vivo reversal of antiplatelet agent (APA) activity in the hemorrhagic transformation mouse model. A, Experimental timeline for the administration of various drug formulations in the middle cerebral artery occlusion (MCAO) mouse model pretreated with APA and rt-PA. B, Magnetic resonance imaging of hemorrhage and thrombosis in brains from platelet-mimicking perfluorocarbonbased nanosponge (PLT-PFC)-treated mice (n=6 for N group; n=4 for Tr group; n=5 for Tc group; n=6 for Tr/P group; n=5 for Tc/P group, respectively, as shown in Figures S13 and S14; Scale bar = 5 mm). Susceptibility weighted imaging (SWI) was applied to identify the hemorrhagic spots (red arrows); T2-weighted imaging was applied to identify infarctions (green arrows). C, Evans blue (EB) extravasation in brains from PLT-PFC-treated mice (n=3; Scale bar = 2 cm). D, Bilateral comparison of EB extravasation in brains from PLT-PFC-treated mice (n=18). P-values between 2 groups were assessed by Kruskal-Wallis test followed by Dunn-Bonferroni post hoc tests. For multiple-group comparison, the overall ANOVA P-value=3.8×10−5. E, Triphenyl tetrazolium chloride (TTC) staining of brains from mice treated with the indicated…

• 

  Figure 7.. Loss of thrombogenic potential of platelet-mimicking perfluorocarbon-based nanosponges (PLT-PFCs) due to the inactive core compared to living platelets. A, Schematic illustration of the differential aggregation behavior of platelets and PLT-PFCs. Upon attachment to the damaged endothelium, platelets are activated and can further secrete thrombogenic substances to activate more resting platelets. PLT-PFCs cannot secrete thrombogenic substances to activate more platelets due to the inactive core. B, Fibrinogen adhesion test of platelets, PLT-PFCs, and BSA-PFCs. Scale bar = 40 μm. C, Representative images of blood samples subjected to the clot retraction assay with platelet poor plasma (PPP), platelet-rich plasma (PRP), and PRP+PLT-PFCs. D through G, Coagulation indicators of plasma from mice (activated partial thromboplastin time [aPTT], fibrinogen concentration[FIB], prothrombin time [PT] and thrombin time [TT]) post injection with the indicated drug formulations (n=6; all groups passed Shapiro-Wilks normality tests [α=0.05]. For comparisons among multiple groups, 1-way ANOVA tests followed by Bonferroni post hoc tests were carried out.). D, For multiple-group comparison, the…