pH-Responsive Theranostic Colloidosome Drug Carriers Enable Real-Time Imaging of Targeted Thrombolytic Process with Near-Infrared-II for Deep Venous Thrombosis

Abstract

Thrombosis can cause life-threatening disorders. Unfortunately, current therapeutic methods for thrombosis using injecting thrombolytic medicines systemically resulted in unexpected bleeding complications. Moreover, the absence of practical imaging tools for thrombi raised dangers of undertreatment and overtreatment. This study develops a theranostic drug carrier, Pkr(IR-Ca/Pda-uPA)-cRGD, that enables real-time monitoring of the targeted thrombolytic process of deep vein thrombosis (DVT). Pkr(IR-Ca/Pda-uPA)-cRGD, which is prepared from a Pickering-emulsion-like system, encapsulates both near-infrared-II (NIR-II) contrast agent (IR-1048 dye, loading capacity: 28%) and urokinase plasminogen activators (uPAs, encapsulation efficiency: 89%), pioneering the loading of multiple drugs with contrasting hydrophilicity into one single-drug carrier. Upon intravenous injection, Pkr(IR-Ca/Pda-uPA)-cRGD considerably targets to thrombi selectively (targeting rate: 91%) and disintegrates in response to acidic thrombi to release IR-1048 dye and uPA for imaging and thrombolysis, respectively. Investigations indicate that Pkr(IR-Ca/Pda-uPA)-cRGD enabled real-time visualization of targeted thrombolysis using NIR-II imaging in DVT models, in which thrombi were eliminated (120 min after drug injection) without bleeding complications. This may be the first study using convenient NIR-II imaging for real-time visualization of targeted thrombolysis. It represents the precision medicine that enables rapid response to acquire instantaneous medical images and make necessary real-time adjustments to diagnostic and therapeutic protocols during treatment.

Fig. 1.

Schematic and theranostic mechanism of Pkr(IR-Ca/Pda-uPA)-cRGD. (A) Preparation and pH-responsive disintegration. (B) Targeted thrombolysis in a rat venous thrombosis model.

Fig. 2.

(A) Preparation of CaCO₃-PDA-uPA. (B) TEM images of CaCO₃-PDA. (C) Size distribution of CaCO₃-PDA. (D) Thermogravimetric analysis. (E) Self-assembly of Pkr(IR-Ca/Pda-uPA)-cRGD. (F) TEM images of Pkr(Ca/Pda-uPA). (G) SEM image of Pkr(Ca/Pda-uPA). (H) SEM images of partially broken Pkr(Ca/Pda-uPA). (I) Zeta potential. (J) NIR absorbance spectra of IR-1048. (K) Standard curve of IR-1048. (L) UV-vis-NIR absorbance. (M) Fluorescence spectra. All error bars depicted the mean ± SD of at least 3 independent experiments.

Fig. 3.

(A) Schematic of cRGD ligands targeted to α_IIbβ₃ integrins from AP. (B) FLI of artificial blood clots incubated with various samples. (C) Fluorescence intensity of artificial blood clots. (D) CLSM of CD41a-eFluor 450-labeled AP incubated with FITC-labeled materials (scale bar: 10 μm). (E) Mean fluorescence intensity of FITC-labeled materials. (F) Binding rate to AP and (G) the corresponding fluorescence-activated cell sorting analysis. Statistical analysis for (C) was performed using the ANOVA (multiple comparisons) test, while the Student t test for (E) and (G). Data are presented as the mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001, and ns represents no significant difference between 2 groups.

Fig. 4.

(A) Schematic of the microfluidic system. (B) Fluorescence intensity of FITC-labeled drug carriers bond to stimulated thrombi. (C) Fluorescence images of FITC-labeled drug carriers bond to stimulated thrombi (scale bar: 10 μm). All error bars depicted the mean ± SD of at least 3 independent experiments.

Fig. 5.

(A) pH-responsive disintegration of Pkr(IR-Ca/Pda-uPA)-cRGD and dissolution of CaCO₃-PDA. (B) Disintegration of Pkr(IR-Ca/Pda-uPA)-cRGD at different pH. (C) Time-dependent disintegration of Pkr(IR-Ca/Pda-uPA)-cRGD. (D) Time-dependent absorbance of CaCO₃-PDA. (E) Decomposition of CaCO₃-PDA at different pH. (F) Release of uPA by Pkr(Ca/Pda-uPA)-cRGD after incubation with resting platelets or APs. All error bars depicted the mean ± SD of at least 3 independent experiments.

Fig. 6.

(A) Schematic of the agar plate model. (B) Representative photos of fibrin clots after 240 min of treatment (scale bar: 1 cm). (C) Time-dependent area of fibrin clot lysis zone. (D) Comparison of area of fibrin clot lysis zone after 240 min of treatment. (E) Schematic of the halo blood clot model. (F) Clots treated with various uPA formulation. (G) Time-dependent of clot lysis. (H) Comparison of clot lysis ratio after 200 min of treatment. Statistical analysis for (D) and (H) was performed using the ANOVA (multiple comparisons) test. Data are presented as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns represents no significant difference between 2 groups.

Fig. 7.

(A) FLI of mouse organs after intravenous injection (1.5 h later). (B) Fluorescence intensity of organs. (C) Cell viability of HUVECs. (D) Pharmacokinetic profiles of uPA. (E) Half-life times (t_1/2) of uPA. (F) Schematic of tail bleeding risk assay. (G) I. Hemorrhage assay performed by truncating the tail tip, followed by immediate immersion in 37 °C saline solution. II. Beginning of bleeding. III. End of bleeding. (H) Bleeding time. (I) Bleeding volume. (J) Hemolytic activity of Pkr(IR-Ca/Pda)-cRGD. Statistical analysis for (H) and (I) was performed using the ANOVA (multiple comparisons) test, while the Student t test for (E). Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 8.

(A) Establishment of the tail thrombus model. (B) Fluorescent images of tail thrombi (scale bar: 100 μm). (C) Mean fluorescence intensity of tail thrombi. (D) Schematic to establish DVT model by IVC stenosis. (E) IVC stenosis-induced DVT model (the black circle spotlighted the stenosis site, scale bar: 5 mm). (F) Isolated thrombotic IVC (scale bar: 5 mm). (G) Hematoxylin and eosin-stained histological sections of thrombotic IVC (scale bar: 1 mm). (H) In vivo real-time NIR-II FLI of thrombi of DVT with Pkr(IR-Ca/Pda)-cRGD. The white dashed lines depict the corresponding part of thrombotic IVC, and red circles spotlight thrombi in thrombotic IVC (scale bar: 5 mm). (I) Fluorescence intensity of thrombi in thrombotic IVC (treated with Pkr(IR-Ca/Pda)-cRGD) and the nearby vessels. (J) In vivo real-time NIR-II FLI of thrombi of DVT during thrombolysis with Pkr(IR-Ca/Pda-uPA)-cRGD. The white dashed lines depict thrombotic IVC, and red circles spotlight thrombi in corresponding part of thrombotic IVC (scale bar: 5 mm). (K) NIR-II fluorescence intensity of thrombi in thrombotic IVC. Statistical analysis for (C) was performed using the ANOVA (multiple comparisons) test. Data are presented as the mean ± SD (n = 3). ***P < 0.001 and ****P < 0.0001.