Synthetic high-density lipoproteins loaded with an antiplatelet drug for efficient inhibition of thrombosis in mice

Figures

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  Fig. 1. Preparation and characterization of ML355-sHDL. (A) Illustration of the synthesis of ML355-sHDL composed of an ApoA1 mimetic 22-mer peptide (22A), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and ML355 using the thermal-cycling method. (B) Negative-stained transmission electron microscopy image of ML355-sHDL; scale bar, 20 nm. (C) Dynamic light scattering of blank sHDL and ML355-sHDL. (D) Gel permeation chromatography of blank sHDL and ML355-sHDL. (E) Release of ML355 from sHDL at PBS and PBS cumulative supplemented with 10% fetal bovine serum (FBS). Data are means ± SD (n = 3). (F) Platelet uptake and intracellular distribution of DiO-sHDL in platelets imaged by confocal microscopy (scale bar, 2 μm). Washed mouse platelets were suspended in 1 ml of Tyrode’s buffer at 3 × 106 platelets/ml and incubated with DiO-sHDL (sHDL at 50 μg/ml and DiO at 2.5 μg/ml) for indicated lengths of time (5, 15, 30, and 60 min) at 37°C. Platelets were then washed with Tyrode’s buffer twice to remove free DiO-sHDL and then fixed with 4% paraformaldehyde, followed by staining with Alexa Fluor 647–conjugated anti-mouse CD41 antibody. Platelet membrane is shown in red, and sHDL particle…

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  Fig. 2. ML355-sHDL treatment inhibits both human and mouse platelet aggregation. (A) Washed human platelets were incubated with different ML355 formulations for 15 min, including dimethyl sulfoxide (DMSO) (equivalent amount used to dissolve ML355), ML355 (10 μM), sHDL (100 μg/ml), or ML355-sHDL (sHDL at 100 μg/ml and ML355 at 10 μM). Untreated platelets were included as control. After different treatments, platelet aggregation was measured by the addition of thrombin at various concentrations. Compared to both control and DMSO group, both ML355 and sHDL treatment inhibited platelet aggregation. Furthermore, encapsulation of ML355 into sHDL exhibited an improved inhibitory effect on human platelet aggregation at multiple thrombin concentrations compared to treatment with either ML355 or sHDL. Inhibition of aggregation was overwhelmed for all formulations by a high concentration (1 nM) of thrombin. Data are means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and N.S. (no significant difference) relative to control or as otherwise indicated. (B) Mice were intravenously administered with saline control, ML355 (1.5 mg/kg), sHDL (50 mg/kg), or ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5…

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  Fig. 3. sHDL targets thrombus in vivo. Male mice were pretreated with DiO-sHDL via intravenous injection with sHDL (50 mg/kg) and DiO (2.5 mg/kg). After 24 hours, mice were anesthetized and surgically prepared as described in detail in Materials and Methods. DyLight 647–conjugated rat anti-mouse platelet GP1bβ antibody (0.1 μg/g, X649, EMFRET Analytics) was administered by jugular vein cannula before vascular injury. Multiple independent thrombi (8 to 10) were induced in the arterioles (30 to 50 μm diameter) per mouse (three mice in each group) by a laser ablation system as described in Materials and Methods. Images of thrombus formation at the site of injured arterioles were acquired in real time under 63× water-immersion objective with a Zeiss Axio Examiner Z1 fluorescence microscope. (A) Sequence of intravital fluorescence microscopic images recorded over 1 min showing accumulation of fluorescently labeled DiO-sHDL in a forming thrombus indicated by fluorescently labeled platelets. (B) Left: Representative three-dimensional confocal images of DiO-sHDL (green) and platelet accumulation (red) in stable thrombi recorded under confocal intravital microscopy. Right: Representative middle…

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  Fig. 4. ML355-sHDL efficiently inhibits thrombus formation in laser-induced cremaster arteriole thrombosis models. Male mice (n = 4) were pretreated intravenously with saline control, sHDL (50 mg/kg), ML355 (1.5 mg/kg), or ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5 mg/kg). After 24 hours, mice were anesthetized and surgically prepared as described in detail in Materials and Methods. Multiple independent thrombi were induced and recorded as described above. (A) Representative captured thrombi from each group at different time points. (B) Dynamics of platelet accumulation in thrombi assessed by relative mean fluorescence intensity of platelet averaged by 8 to 10 thrombi per mouse, three mice in each group. All thrombi were statistically analyzed for the change of fluorescence intensity over the course of thrombus formation after subtracting fluorescent background defined on an uninjured section of the vessel using the SlideBook program. Data are means ± SD and compared by two-way analysis of variance (ANOVA) analysis for statistical difference. ***P < 0.001.

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  Fig. 5. ML355-sHDL efficiently delays vessel occlusion in FeCl3-induced carotid artery thrombosis model. Both female and male mice (n = 6) were pretreated intravenously with saline control, sHDL (50 mg/kg), ML355 (1.5 mg/kg), or ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5 mg/kg). After 24 hours, mice were anesthetized and surgically prepared as described in detail in Materials and Methods. (A) Representative images of thrombosis in carotid artery in response to FeCl3 injury. FeCl3 (10%) was topically applied on the right carotid artery for 3 min, images of thrombi were recorded in real time, and vessel occlusion was determined by the secession of blood flow. (B) Carotid artery vessel occlusion time in each group. The mean vessel occlusion time in the control mice, ML355, sHDL, and ML355-sHDL was 7.1 ± 1.2 min, 11.0 ± 2.3 min, 11.3 ± 1.3 min, and 21.4 ± 6.4 min, respectively. Data are means ± SD (n = 6). **P < 0.01, ***P < 0.001.

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  Fig. 6. ML355-sHDL does not affect the phosphatidylserine exposure on platelet surface, coagulation system, and hemostatic action. Both female and male mice (n = 6) were pretreated intravenously with saline control, sHDL (50 mg/kg), ML355 (1.5 mg/kg), or ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5 mg/kg). All treated groups were subjected to the following different examinations. (A) At different time points after administration (1, 6, and 24 hours), the phosphatidylserine (PS) exposure over the time course in platelets from different groups was quantified by flow cytometry. Blood collected from untreated mice was taken as resting platelets (negative control), and blood stimulated with protease-activated receptor 4–activating peptide (PAR4-AP) (200 μM) was used as activated platelets (positive control). (B) Representative tracings indicating different treatments were presented. Some major coagulation parameters were analyzed. (C) R time, time to formation of the initial fibrin threads (min). (D) K time, the time until the clot reaches a certain strength (min). (E) Alpha (α) angle, the rapidity with which the clot forms (degree). (F) Maximum amplitude (MA), clot’s maximum strength (mm).…