Platelet-Inspired Intravenous Nanomedicine for Injury-Targeted Direct Delivery of Thrombin to Augment Hemostasis in Coagulopathies

Abstract

Severe hemorrhage associated with trauma, surgery, and congenital or drug-induced coagulopathies can be life-threatening and requires rapid hemostatic management via topical, intracavitary, or intravenous routes. For injuries that are not easily accessible externally, intravenous hemostatic approaches are needed. The clinical gold standard for this is transfusion of blood products, but due to donor dependence, specialized storage requirements, high risk of contamination, and short shelf life, blood product use faces significant challenges. Consequently, recent research efforts are being focused on designing biosynthetic intravenous hemostats, using intravenous nanoparticles and polymer systems. Here we report on the design and evaluation of thrombin-loaded injury-site-targeted lipid nanoparticles (t-TLNPs) that can specifically localize at an injury site via platelet-mimetic anchorage to the von Willebrand factor (vWF) and collagen and directly release thrombin via diffusion and phospholipase-triggered particle destabilization, which can locally augment fibrin generation from fibrinogen for hemostatic action. We evaluated t-TLNPs in vitro in human blood and plasma, where hemostatic defects were created by platelet depletion and anticoagulation. Spectrophotometric studies of fibrin generation, rotational thromboelastometry (ROTEM)-based studies of clot viscoelasticity, and BioFlux-based real-time imaging of fibrin generation under simulated vascular flow conditions confirmed that t-TLNPs can restore fibrin in hemostatic dysfunction settings. Finally, the in vivo feasibility of t-TLNPs was tested by prophylactic administration in a tail-clip model and emergency administration in a liver-laceration model in mice with induced hemostatic defects. Treatment with t-TLNPs was able to significantly reduce bleeding in both models. Our studies demonstrate an intravenous nanomedicine approach for injury-site-targeted direct delivery of thrombin to augment hemostasis.

Keywords: Fibrin; Hemostasis; Nanoparticle; Platelets; Targeted delivery; Thrombin.

Figure 1.

Platelet-mediated hemostatic mechanism and platelet-inspired t-TLNP design. (A) Platelets rapidly adhere at a vascular injury site by binding to von Willebrand factor (vWF, via platelet surface GPIbα) and collagen (via platelet surface GPIa/IIa and GPVI) exposed at the site and present high amounts of an anionic phospholipid such as phosphatidylserine (PS) on the activated platelet procoagulant membrane surface to enable the assembly of coagulation factors to form tenase (FVIIa + FIXa + FX) and prothrombinase (FXa + FVa + FII) complexes, ultimately leading to the amplified generation of thrombin (thrombin burst); the thrombin locally converts fibrinogen (Fg) to fibrin that gets cross-linked by FXIIIa for hemostatic clot formation. (B) t-TLNPs can undergo platelet-mimetic adhesion at the vascular injury site by anchoring to vWF via vWF-binding peptide (VBP) and collagen via collagen-binding peptide (CBP) and release thrombin at the site via diffusion as well as injury site secreted phospholipase A₂ (sPLA₂) triggered particle destabilization; this thrombin can locally convert fibrinogen (Fg) to fibrin for hemostatic action.

Figure 2.

Manufacture and characterization of t-TLNPs. (A) Bioconjugation schematics of reacting cysteine-terminated peptides to maleimide-terminated DSPE-PEG_2K utilizing thiol–maleimide chemistry to synthesize DSPE-PEG_2K-peptide molecules. (B) Molecular components of t-TLNP manufacture. (C) Dynamic light scattering (DLS) analysis of five representative t-TLNP batches showing nanoparticle size reproducibility. (D) Cryo-transmission electron microscopy (Cryo-TEM) images of t-TLNPs (scale bar: 100 nm) showing a particle diameter of ∼175 nm. (E) Representative images from BioFlux experiments where Rhodamine B labeled control (undecorated) vs targeted nanoparticles (“VBP + CBP”-decorated) were flowed at 25 dyn/cm² over “vWF + collagen”-coated channels and targeted nanoparticles were also flowed over albumin-coated channels, showing substantially high adhesion of targeted nanoparticles to “vWF + collagen”-coated surface but not of control particles to the “vWF + collagen”-coated surface or targeted nanoparticles to the albumin-coated surface. (F) Spectrometric analysis of three representative t-TLNP batches showing a mean thrombin loading of 114.3 ±14.2 nM. (G) Thrombin release analysis showing that t-TLNPs can slowly release low amounts of thrombin by diffusion, whereas exposure to sPLA₂ significantly enhances thrombin release. *p≤ 0.05, **p≤ 0.01.

Figure 3.

Evaluation of biosafety characteristics of peptide-decorated nanoparticles. (A) Monolayers of healthy human pulmonary microvascular endothelial cells (HPMEC, nuclei stained with blue DAPI) were exposed to TNF-α (a known endothelial activator), media only, control (undecorated) nanoparticles or targeted (“VBP + CBP”-decorated) nanoparticles and vWF expression on endothelium was stained (green vWF antibody) as a marker for endothelial activation. In comparison to TNF-α-induced stimulation (high vWF staining), neither control particles nor targeted particles showed endothelial activation (low vWF staining, similar to that of the “media only” group). (B) Platelet lumi-aggregometry studies with human platelet-rich plasma (PRP) showed that addition of ADP (platelet agonist) induced significant platelet aggregation but addition of the targeted nanoparticles (t-LNPs) did not induce such aggregation (aggregation percent similar to that of the “no ADP” group); (C) ELISA-based complement C3 activation assay studies using human plasma incubated with control nanoparticles or targeted nanoparticles (t-LNPs) indicated that neither control nor targeted nanoparticles activate C3 (C3a/C3 ratio similar to the baseline of saline-incubated plasma).

Figure 4.

Evaluation of t-TLNPs in restoring fibrin generation in anticoagulated and platelet-depleted human plasma. (A) Schematic of the experimental design where human whole blood (WB) was centrifuged to obtain platelet-rich plasma (PRP) and the PRP was further centrifuged to obtain either platelet-poor plasma (PPP) or platelet-free plasma (PFP); The PPP was treated with anticoagulant Apixaban (FXa inhibitor). PFP and Apixaban-treated PPP were both subjected to spectrophotometric monitoring of fibrin generation (measuring optical density of formed/polymerized fibrin over time at 405 nm) and the onset of fibrin generation (OFG), maximum optical density (also called maximum hemostatic potential or MHP) and area under the curve (also called overall coagulation potential or OCP) was recorded. (B1–B3) Effect of adding t-TLNP vs UNP in Apixaban-treated PPP, demonstrating that thrombin released by t-TLNP can restore OCP, OFG, and MHP parameters closer to the normal plasma baseline (increased OCP, reduced OFG, increased MHP) and this effect is enhanced when sPLA₂ is added to accelerate thrombin release. (C1–C3) Effect of adding t-TLNP vs UNP in PFP, demonstrating that thrombin released by t-TLNP can restore OCP, OFG, and MHP parameters closer to the normal plasma baseline and this effect is enhanced when sPLA₂ is added to accelerate thrombin release. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 5.

Evaluation of t-TLNPs in restoring clot viscoelastic parameters as measured by rotational thromboelastometry (ROTEM). (A) Schematic of the experimental design where human whole blood (WB) was directly treated with the anticoagulant Apixaban (FXa inhibitor) or was fractionated into blood components (RBC, platelets, leukocytes, plasma) and then reconstituted with a reduced number of platelets to create thrombocytopenic whole blood (TC Blood). Anticoagulated blood and thrombocytopenic blood were analyzed in ROTEM in NATEM mode (CaCl₂-induced blood clotting resisting pin rotation), and the clot formation time (CFT), clot formation rate (also called alpha angle), and early clot amplitude at 10 min (also called A10) were monitored. (B1–B3) Effect of adding t-TLNP vs UNP in Apixaban-treated WB, demonstrating that thrombin released by t-TLNP can significantly restore CFT, alpha angle, and A10 parameters closer to the normal WB baseline (reduced CFT, increased alpha angle, increased A10) and this effect is enhanced when sPLA₂ is added to accelerate thrombin release. (C1–C3) Effect of adding t-TLNP vs UNP in thrombocytopenic blood (TC Blood) demonstrating that thrombin released by t-TLNP can partially restore CFT, alpha angle, and A10 parameters closer to the normal WB baseline, but no statistical significance was observed in this improvement without or with sPLA₂. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 6.

Evaluation of t-TLNPs in restoring fibrin generation under a simulated vascular flow environment in human plasma containing the combined hemostatic defect of platelet depletion plus anticoagulation. (A) Schematic of the BioFlux microfluidic setup and experimental design where human plasma containing fluorescently labeled platelets and fibrinogen (by Calcein and AlexaFluor647, respectively) were flowed over “vWF + collagen”-coated microchannel and fibrin formation was imaged in real time. (B) Representative fluorescence images of fibrin formation over time (0–12 min) in the microchannel with flows of platelet-rich plasma (PRP), platelet-depleted plus anticoagulated (Defective) plasma, Defective plasma treated with “t-TLNPs + sPLA₂” and Defective plasma treated with “UNPs + sPLA₂” showing that the combined defect of platelet depletion plus anticoagulation in plasma drastically reduces fibrin formation in comparison to that in PRP and treatment with “t-TLNPs + sPLA₂” is able to restore fibrin generation even when platelet numbers were low. Treatment with “UNP + sPLA₂” was unable to restore fibrin. (C) Representative dual-fluorescence images of the full microchannel surface at the experiment end point (12 min) showing a substantial number of blue platelets enmeshed in green fibrin in the channel containing PRP flow. In comparison, the channel with Defective plasma showed sparse platelets and minimal fibrin and Defective plasma treated with “t-TLNPs + sPLA₂” showed fibrin recovery even though the platelets were sparse, while treatment with “UNPs + sPLA₂” showed no such recovery. (D) Surface-averaged fluorescence intensity quantification of fibrin corroborating that treatment of Defective plasma with “t-TLNP + sPLA₂” restores fibrin generation comparably to that of PRP. (E) D-dimer ELISA based quantification of digested fibrin from the microchannels further confirming that treatment of Defective plasma with “t-TLNP + sPLA₂” restores the formation of cross-linked fibrin at concentrations comparable to those of PRP. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 7.

Evaluation of prophylactic administration of t-TLNPs in restoring hemostatic efficacy in the tail-clip model in mice with significant bleeding due to the combined effect of platelet depletion and anticoagulation. (A) Schematic of the experimental design where mice were first made thrombocytopenic (TC Mouse) by anti-CD42b dose induced platelet clearance and then further dosed with anticoagulant (Enoxaparin) to induce combine a hemostatic defect (“Defect” mouse). t-TLNP or UNP treatment was administered in the “Defect” mice via an intravenous (retroorbital) route and allowed to circulate for 15 min, and then a tail-clip injury was performed to measure the bleeding time and blood loss. (B) Bleeding time data as a percent of 15 min time period showing that normal mice stopped bleeding in 3.11 ± 0.44 min while the combined effect of thrombocytopenia and anticoagulation in “Defect” mice resulted in continuous bleeding for 15 min (and beyond). Treatment of t-TLNPs in defect mice significantly restores the hemostatic capability, with the mice stopping bleeding in 6.18 ± 3.19 min, while treatment with UNPs has no such effect (mice continue bleeding for 15 min and beyond). (C) Blood loss analysis via a spectrophotometric measurement of hemoglobin in shed blood indicating that the combined effect of thrombocytopenia and anticoagulation in “Defect” mice results in significantly increased blood loss over the 15 min time period in comparison to normal mice. Treatment of “Defect” mice with t-TLNPs significantly reduces blood loss. In contrast, treatment with UNPs did not reduce blood loss but rather exacerbated it, possibly due to a dilution effect. *≤ 0.05, **p≤ 0.01, ***p ≤ 0.001.

Figure 8.

Evaluation of emergency administration of t-TLNPs in restoring hemostatic efficacy in liver laceration bleeding model in anticoagulated mice. (A) Schematic and representative anatomic picture of liver laceration model in mice where treatment (sham saline, UNP, or t-TLNP) was administered post-injury and blood loss from the injured liver was measured by preweighed gauze. (B) Blood loss data (in grams, g) from mouse liver injury model studies showing a significant increase in bleeding from an injured liver in defect (heparinized) mice in comparison to normal (nonheparinized) mice. Treatment with UNPs was unable to reduce blood loss, but treatment with t-TLNPs was able to significantly reduce blood loss. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.