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Elaborately Engineering a Self‐Indicating Dual‐Drug Nanoassembly for Site‐Specific Photothermal‐Potentiated Thrombus Penetration and Thrombolysis

Abstract Thrombotic cardio‐cerebrovascular diseases seriously threaten human health. Currently, conventional thrombolytic treatments are challenged by the low utilization, inferior thrombus penetration, and high off‐target bleeding risks of most thrombolytic drugs, resulting in unsatisfactory treatment outcomes. Herein, it is proposed that these challenges can be overcome by precisely integrating the conventional thrombolytic strategy with photothermal therapy. After co‐assembly engineering optimization, a fibrin‐targeting peptide‐decorated nanoassembly of DiR (a photothermal probe) and ticagrelor (TGL, an antiplatelet drug) is prepared for thrombus‐homing delivery, abbreviated as FT‐DT NPs. The elaborately engineered nanoassembly shows multiple advantages, including simple preparation with high drug co‐loading capacity, synchronous delivery of two drugs with long systemic circulation, thrombus‐targeted accumulation with self‐indicating function, as well as photothermal‐potentiated thrombus penetration and thrombolysis with high therapeutic efficacy. As expected, FT‐DT NPs not only show bright fluorescence signals in the embolized vessels, but also perform photothermal/antiplatelet synergistic thrombolysis in vivo. This study offers a simple and versatile co‐delivery nanoplatform for imaging‐guided photothermal/antiplatelet dual‐modality thrombolysis.

Other chemical reagents were analytical grade.

Preparation and characterization of nanoassemblies
The naked nanoassembly (DT NPs), PEGylated nanoassembly (PEG-DT NPs) and PEGylated fibrin-homing nanoassembly (FT-DT NPs) were prepared using a one-step nano-precipitation approach [1] . Briefly, 10 mg of DiR and 10 mg of TGL were dissolved in absolute ethyl alcohol (1 mL), respectively. Then, series mixtures of the above DiR Sol and TGL Sol with different mass ratios (3:1, 2:1, 1:1, 1:2, 1:3) of DiR and TGL were dripped respectively into purified water (2 mL) under intense stirring (1500 rpm, 3min) to obtain DT NPs. The optimal formulation of DT NPs was screened out by evaluating the particle size and colloidal stability of the nanoassembly.
Finally, the organic solvent in these nanoassembly systems was removed in vacuum at 33 °C.
The hydrodynamic diameters and zeta potentials of DT NPs and FT-DT NPs were measured by a Zetasizer (Nano ZS, Malvern Co., UK). The morphology of DT NPs and FT-DT NPs was observed by Transmission electron microscopy (TEM) (HITACHI, HT7700, Japan) after staining with phosphotungstic acid (2%, w/v).

Ultraviolet and Fluorescence Spectra
The ultraviolet (UV) spectra of DiR Sol, TGL Sol, DT NPs and FT-DT NPs and the fluorescence spectra of DiR Sol, DT NPs and FT-DT NPs (DiR and TGL 25 μg/mL) were acquired at an equivalent DiR or TGL concentration of 25 μg/mL using Varioskan Flash multimode microreader (Thermo Scientific, USA).

Co-Assembly Simulation
Molecular docking simulation method was used to investigate the intermolecular interactions of DiR and TGL. Moreover, the Autodock Vina software was utilized to construct the 3-dimentional structures of DiR and TGL. The runtime environment and optimized parameters have corresponded with our previous study [2] .

Stability
To evaluate the stability of nanoassemblies, DT NPs, PEG-DT NPs and FT-DT NPs (50 µg/mL of DiR and TGL) were incubated in pure water or PBS (pH 7.4) for 30 min. Furthermore, to further explore the stability of nanoassemblies in a simulated S5 physiological environment, PEG-DT NPs and FT-DT NPs were incubated in PBS (pH 7.4) containing 10% FBS for 12 h at 37 ℃. The particle sizes of PEG-DT NPs and FT-DT NPs were measured by a Zetasizer. Moreover, the drugs leakage from nanoassemblies during the incubation process was explored by an ultrafiltration method. Briefly, 1 mL of PEG-DT NPs or FT-DT NPs of (TGL and DiR of 1 mg/mL) were incubated in PBS (pH 7.4) containing 10% FBS (10 mL) at 37 ℃. At pre-set time intervals, the samples (1 mL) were taken out, and the unencapsulated drugs were removed from the nanoassemblies by passing through an ultrafiltration membrane with a retention molecular weight of 30 kDa after centrifugation (4000 r/min, 10 min).
Then, the concentrations of TGL in the filtrates were determined by HPLC. The chromatographic condition was as follows: C18 chromatographic column (4.6 × 150 mm, 5 μm); mobile phase: acetonitrile: water = 50: 50. The flow rate was set to 1.0 mL/min; the wavelength was set to 254 nm for TGL detection (n=3). And the concentrations of DiR in the filtrates were determined by Varioskan Flash multimode microreader (Thermo Scientific, USA). Additionally, the long-term stability of DT NPs, PEG-DT NPs and FT-DT NPs (0.5 mg/mL) were explored by determining the particle sizes and zeta potentials of nanoassemblies stored at 4 °C for one week.

S6
DT NPs, PEG-DT NPs and FT-DT NPs with an equivalent concentration of DiR (0.5 mg/mL) and TGL (0.5 mg/mL) were added on the top of clots and incubated for 10 min, respectively. Finally, the clots were washed three times with fresh PBS (pH 7.4), and the fluorescent signals were measured using an in vivo imaging system (IVIS Lumina Series III) (n=3).

In vitro light-triggered TGL release
The release behaviors of TGL from FT-DT NPs were determined using the dialysis method, and PBS (pH=7.4) containing 20% ethanol was utilized as the release medium. The nanoassemblies (TGL 0.5 mg/mL) were placed into dialysis bags and were immersed into the release medium at 37 ℃. The laser-treated group was exposed to 808 nm laser irradiation (2.0 W/cm 2 , 15 min) beforehand. At pre-set time points, 150 μL of the samples were taken out to determine the concentrations of TGL using HPLC, and the equivalent volume of fresh media was replenished.

In vitro shear-responsive TGL release
The release behaviors of TGL from FT-DT NPs were determined using the dialysis method, and PBS (pH=7.4) containing 20% ethanol was selected as the release medium. To simulate the high hemodynamic shear stress at the thrombus site, FT-DT NPs (TGL 0.5 mg/mL) and a small rotor were put into dialysis bags to exert a slight stirring effect (300 rpm) on the nanoassemblies. The dialysis bag without stirring was set as the control group. At pre-set time points, 150 μL of the samples were taken out S7 to determine the concentrations of TGL using HPLC, and equivalent volume of fresh media was replenished.

In vitro photothermal conversion efficiency
To investigate the in vitro photothermal conversion efficiency, PBS, DiR Sol, DT NPs and FT-DT NPs with an equivalent DiR concentration of 0.5 mg/mL were exposed to laser irradiation (808 nm, 2.0 W/cm 2 ) for 15 min. An infrared thermal imaging camera (Fotric 226) was used to measure the temperature variations (n=3).

In vitro photothermal thrombolysis
An artificial blood clot was prepared to evaluate the photothermal thrombolysis effect.
In brief, platelet-poor plasma (100 μL), CaCl 2 (2.5 mM), and thrombin (1 U/mL) were mixed and incubated in EP tubes (1.5 mL) for 1 h at 37 °C [4] . Then, PBS, DiR Sol, DT NPs and FT-DT NPs with an equivalent DiR concentration of 0.5 mg/mL was added into the artificial blood clot under laser irradiation (808 nm, 2.0 W/cm 2 ), respectively. After irradiation for 15 min, the EP tubes were put upside down several times for preliminary evaluation of the thrombolysis effect. Moreover, the supernatants in tubes were collected, and the absorbance of fibrin in the supernatants was measured at 450 nm using a multimode microreader (Thermo Scientific, USA).
A small red thrombus model was prepared to evaluate the thrombus penetration ability of FT-DT. Fresh blood was collected from Sprague-Dawley rats without adding anticoagulants. Then 5 μL blood were added to the bottom of 200 μL EP tube and incubated at 37 ℃ for 3 h to induce thrombosis. The formed blood clot was incubated with free C-6 and C-6-FT-DT NPs (C-6, 25 μg/mL) of 5 μL at 37℃ for 30 min and C-6-FT-DT NPs were further exposed to 808 nm irradiation for 10 min at a power density of 2 W/cm 2 . Finally, the fluorescence signal in the thrombus interior was observed by a confocal laser scanning microscope (CLSM, C2, Nikon, Japan). following the manufacturer's instructions (n=3) [5] .

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All the animal protocols were conformed to the Animal Laboratory Ethics Committee of Shenyang Pharmaceutical University (accreditation number: No. 19169).

Tail bleeding assay
After anesthetization by intraperitoneal injection of a chloral hydrate (3.5%, 10 μL/g), wound to stop bleeding for at least 10 s. To determine the bleeding volume, the blood samples were centrifuged at 4000g for 5 min, and the supernatants were discarded to obtain the blood cells. Then, the blood cells were resuspended in 2 mL of lysis buffer.
The absorbance of hemoglobin in the lysates at 550 nm was analyzed using a multimode microreader (Thermo Scientific, USA). The bleeding volume was evaluated by comparing the measured ultraviolet absorption to that of the standard blood samples (n=5) [6] .
Moreover, to further verify the targeting ability of FT-DT NPs, the hemostatic amputated tails were obtained and soaked into DT NPs and FT-DT NPs for 3 min.
Then, the amputated tails were taken out and washed by PBS three times, and their fluorescent signal was detected using an in vivo imaging system (IVIS Lumina Series III). S10

Pharmacokinetics
To investigate the pharmacokinetics behaviors of co-delivery nanoassemblies, DiR Sol, TGL Sol, DT NPs, and FT-DT NPs were intravenously injected into Sprague-Dawley rats (180-220 g) at an equivalent dose with DiR (5 mg/kg) and TGL

Thrombus-targeting fluorescence imaging
Sprague-Dawley rats (180-220 g) were anaesthetized by intraperitoneal injection of chloral hydrate (3.5%, 10 µL/g). After scraping the fur on the neck, the rats were placed and fixed on a surgical table using strings. To expose the carotid arteries, a midline incision was made between the chin and sternum, and the peripheral muscles were separated using fine forceps. Then, a filter paper (10 × 10 mm) saturated with 10% FeCl 3 was wrapped on the right carotid artery for 10 min to thrombosis. Finally, PBS was used to wash the residual FeCl 3 for three times [7] . After thrombus formation, DiR Sol, DT NPs and FT-DT NPs were intravenously injected into rats at a DiR equivalent dose of 5 mg/kg, respectively. The embolic vessel fluorescence images were observed S11 using a noninvasive optical imaging system (IVIS Lumina Series III) at 5, 15, 30, 60 and 90 min post-administration, respectively (n=5).

In vivo photothermal efficacy
A FeCl 3 -induced arterial thrombosis rat model was established by using the same method described previously. To investigated the in vivo photothermal efficiency, PBS, DiR Sol, DT NPs, and FT-DT NPs were intravenously injected into rats at a DiR equivalent dose of 5 mg/kg, respectively. The rat necks were exposed to laser (808 nm, 2 W/cm 2 ) for 15 min at 1 h post-administration (n=5). The infrared thermographic images and local temperature variations were recorded by infrared thermal imaging camera (Fotric 226).

In vivo photothermal-facilitated antithrombic effect
The Sprague-Dawley rats (180-220 g) were randomly divided into ten groups: 1) untreated group; 2) embolized group; 3) laser group; 4) DiR Sol group; 5) TGL Sol group; 6) DiR Sol + laser group; 7) DT NPs group; 8) DT NPs + laser group; 9) FT-DT NPs group; 10) FT-DT NPs + laser group (n=6). The drug-treated rats received an equivalent dosage of DiR and/or TGL (5mg/kg). As previously described, a FeCl 3 -induced arterial thrombosis rat model was established by using the same method. The neck of rats was exposed to laser (808 nm, 2.0 W/cm 2 ) for 15 min at 1 h post-administration. One hour later, the rats were sacrificed, and the carotid vessels were harvested and dried for measurement of the dry weight of the thrombus.
(Thrombus therapy rate = (thrombus weight before therapy-thrombus weight after therapy)/ S12 thrombus weight before therapy) The photographs of carotid artery embolization in rats with or without FT-DT NPs treatment under laser irradiation were recorded. In addition, the embolismic vessels were washed by PBS and fixed in the 4% paraformaldehyde, and stained with hematoxylin and eosin (H&E) to evaluate the thrombus treatment effect.

Preliminary evaluation of therapeutic safety
For hemolysis evaluation, the collected erythrocytes (50 μL) were diluted into PBS (1 mL). Then the TGL Sol, DiR Sol, DT NPs and FT-DT NPs (50 μL) with the same concentrations (DiR and TGL 0.1 mg/mL) were added into erythrocytes PBS Sol and then kept at 37 °C for 3 h, followed by centrifugation at 10000 rpm for 3 min (n=3).
Finally, the supernatant was collected and its absorbance was measured at 540 nm with PBS (pH 7.4) as the negative control and pure water as the positive control.
Healthy Sprague-Dawley rats were intravenously administrated with DT NPs and FT-DT NPs (5 mg/kg for both DiR and TGL), and the neck of rats was exposed to laser (808 nm, 2.0 W/cm 2 ) for 15 min at 1 h post-administration. One day later, the plasma was collected for hepatic and renal function measurements (n=5). Moreover, the main organs (heart, liver, spleen, lung and kidney) were washed by PBS, fixed in the 4% paraformaldehyde, and stained with H&E to evaluate the pathological changes.

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The data were calculated and expressed as mean value ± standard deviation. Student's T-test and one-way analysis of variance (ANOVA) were employed to analyze the differences. The difference was considered as statistically significant when the p values were less than 0.05.  S28 Figure S16. H&E staining of heart, liver, spleen, lung and kidney of rats. Scale bar represents 100 μm.

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Supporting Tables   Table S1. Characteristics of DT NPs with different prescriptions.