Nanoparticles that sense thrombin activity as synthetic urinary biomarkers of thrombosis

Abstract

Thrombin is a serine protease and regulator of hemostasis that plays a critical role in the formation of obstructive blood clots, or thrombosis, that is a life-threatening condition associated with numerous diseases such as atherosclerosis and stroke. To detect thrombi in living animals, we design and conjugate thrombin-sensitive peptide substrates to the surface of nanoparticles. Following intravenous infusion, these "synthetic biomarkers" survey the host vasculature for coagulation and, in response to substrate cleavage by thrombin, release ligand-encoded reporters into the host urine. To detect the urinary reporters, we develop a companion 96-well immunoassay that utilizes antibodies to bind specifically to the ligands, thus capturing the reporters for quantification. Using a thromboplastin-induced mouse model of pulmonary embolism, we show that urinary biomarker levels differentiate between healthy and thrombotic states and correlate closely with the aggregate burden of clots formed in the lungs. Our results demonstrate that synthetic biomarkers can be engineered to sense vascular diseases remotely from the urine and may allow applications in point-of-care diagnostics.

Figure 1

(A) Schematic of approach. Synthetic biomarkers composed of NWs conjugated with a thrombin-sensitive substrate in tandem with a ligand-encoded reporter. These agents survey the vasculature for the sites of clot formation where thrombin activity cleaves and releases the reporters into urine for analysis by ELISA. (B) Schematic of fluorogenic NW assay for detecting protease activity. (C) Kinetics of fluorogenesis produced by the activity of thrombin (red) and other coagulation proteases (n = 3 per condition). Thr, thrombin; Bival, bivalirudin. (D) Kinetics of fluorogenesis in plasma after the addition of CaCl₂ to activate coagulation (n = 3 per condition).

Figure 2

Designing ligand-encoded reporters for detection by ELISA. (A) Schematic of ligand-encoded reporters R₁ and R₂ along with chemical structures of associated ligands. (B) Schematic of ELISA sandwich complex and photograph of developed 96-well plates showing specific detection of R₁ and R₂ spiked into control urine samples. (C) Absorbance values (λ = 450 nm) of wells coated with anti-Flsc antibodies used to detect serial dilutions of R₁, R₁ + R₂, and R₂ in urine (n = 3 per condition, s.d.). (D) Quantification of the level of cleaved reporters (R₁) released from NWs after incubation with increasing concentrations of thrombin (n = 3 per dose, s.d.).

Figure 3

Induction of thrombosis by thromboplastin. (A) Near-infrared fluorescent scans of excised organs to monitor the deposition of VT750-labeled fibrinogen following intravenous administration of thromboplastin (2 μL/g of body weight) or PBS. (B) Quantification of the level of VT750-fibrin(ogen) deposited in organs harvested from thrombosis and control mice (*P < 0.05, **P < 0.01, ***P < 0.005, Student’s t-test; n = 3 per group, s.d.). (C) Hematoxylin and eosin staining of lungs harvested from thrombosis and healthy mice (scale bar = 100 μm). Blue arrow denotes fibrin clot. (D) Quantification of fibrin deposited in the lungs in response to escalating doses of thromboplastin. Bival, bivalirudin (*P < 0.05, **P < 0.01, ***P < 0.005, one-way ANOVA with Tukey post-test; n = 3–5 mice, s.e.).

Figure 4

Noninvasive urinary detection of pulmonary embolism (A) Quantification of the distribution of VT750-labeled NWs in organs excised from mice treated with thromboplastin or PBS (n = 3 mice, s.d.). (B) Quantification of the fluorescent signal of organs after mice were infused mixtures of NWs conjugated with quenched substrates (labeled with VT750) and thromboplastin or PBS (**P < 0.01, Student’s t-test; n = 3 mice, s.d.). Inset shows representative fluorescent scans of the kidneys and the lungs. (C) In vivo fluorescent image after administration of NWs showing increased fluorescent signal localized to the bladders of mice challenged with thromboplastin. (D) Normalized urinary reporter levels (R₁/R₂) from healthy mice (day 0) and in response to thromboplastin and bivalirudin (day 5). Bival, bivalirudin (***P < 0.005, two-way ANOVA with Bonferroni post-test; n = 5 mice, s.e.). (E) Correlation plot of the clot burden in the lungs versus urinary biomarker levels (Pearson’s r = 0.999; n = 5–10 mice, s.e.).