Metal–Organic‐Framework‐Derived Carbon Nanostructures for Site‐Specific Dual‐Modality Photothermal/Photodynamic Thrombus Therapy

Abstract Although near‐infrared (NIR)‐light‐mediated photothermal thrombolysis has been investigated to overcome the bleeding risk of clinical clot‐busting agents, the secondary embolism of post‐phototherapy fragments (>10 µm) for small vessels should not be ignored in this process. In this study, dual‐modality photothermal/photodynamic thrombolysis is explored using targeting nanoagents with an emphasis on improving biosafety as well as ameliorating the thrombolytic effect. The nanoagents can actively target glycoprotein IIb/IIIa receptors on thrombus to initiate site‐specific thrombolysis by hyperthermia and reactive oxygen species under NIR laser irradiation. In comparison to single photothermal thrombolysis, an 87.9% higher re‐establishment rate of dual‐modality photothermal/photodynamic thrombolysis by one‐time treatment is achieved in a lower limb thrombosis model. The dual‐modality thrombolysis can also avoid re‐embolization after breaking fibrin into tiny fragments. All the results show that this strategy is a safe and validated protocol for thrombolysis, which fits the clinical translational trend of nanomedicine.


spectrometer.
Preparation of PMCS. PMCS were prepared by carbonization of an imidazolate framework according to our earlier reports. [1,2] Firstly, 33.75 mmol 2-methylimidazole dissolved in 100 mL methanol was poured into a solution containing 8 mmol Zn(NO 3 ) 2 ·6H 2 O in (100 mL), and then stirred at room temperature for 3 h. The obtained zeolitic-imidazolate-framework (ZIF-8) nanoparticles were collected by centrifugation and washed three times with methanol. Later, ZIF-8 nanoparticles were dispersed in 240 mL 10 vol% methanol, and the mixture was adjusted to pH 11 by NaOH aqueous, and 0.2 g CTAB was further added. After stirring at 500 rpm for 30 min, 1.2 mL TEOS was added into the above solution, followed by stirring for another 30 min. The obtained ZIF-8 core/mesoporous silica shell (ZIF-8@mSiO 2 ) nanoparticles collected by centrifugation and washed three times with ethanol and then dried at 60 °C. Next, the prepared ZIF-8@mSiO 2 nanoparticles were pyrolyzed at 800 °C for 2 h with the help of flowing N 2 , and then cooled to room temperature.
Then, the pyrolyzed sample was etched by 4 M NaOH solution at 80 °C for 3 h. The final product was collected by centrifugation and washed several times with ultrapure water until the value of pH was close to neutral.
Preparation of RGD-PMCS. Firstly, 2 mg PMCS were mixed with 2 mg VE-PEG-COOH in 3 mL of ultrapure water. After vibrating at 37 °C for 4 h, the precipitates were washed three times with deionized water and collected after 10-min centrifugation at 12000 rpm. Then, 3 mL deionized water was supplemented, and the pH was adjusted to 6.0 with hydrochloric acid. Subsequently, 58 mM NHS (200 μL) and 200 mM EDC (200 μL) were added. After vibrating for 1 h at 37 °C, 2 mg RGD peptide was added into the mixture. After 3 h, the solution was centrifuged at 15000 rpm for 10 min and the precipitate was washed three times. The residual RGD concentration in the supernatant was measured by the BCA protein assay. The conjugation efficiency of RGD (%) = (RGD inputfree RGD in the supernatant)/RGD input × 100%. The final conjugation efficiency of RGD was 28.5%.

Preparation of ICG-Conjugated RGD-PMCS (ICG-RGD-PMCS). The prepared
RGD-PMCS were resuspended in 3 mL ultrapure water and adjust the pH value to 6.0 with hydrochloric acid. Subsequently, NHS (58 mM, 200 μL) and EDC (200 mM, 200 μL) were added and vibrated for 1 h at 37 °C, and ICG was further added. The mixture was then vibrated at 4 °C overnight, and ICG-RGD-PMCS were obtained by centrifugation again.
In Vitro Photothermal Measurement. A series of PMCS aqueous dispersions (1 mL) with different concentrations (0, 6.25, 12.5, 25, 50, 100 μg mL -1 ) were irradiated with near-infrared (NIR) laser (2 W cm -2 ) for 20 min, and the temperature change was recorded by a thermocouple and the photothermal images were acquired by an infrared thermal imager. In Vitro Blood Biosafety Evaluation. To demonstrate that the photosensitizers could not activate platelets in the blood, the prepared platelets were diluted with Tyrode's solution with a density of 1 × 10 7 per milliliter and divided into four groups: a) Positive control group, b) Negative control group, c) NIR group (808 nm, 2 W cm -2 , 3 min), d) RGD-PMCS group (100 μg mL -1 ). For the positive control group, the platelets were activated with 10 μM ADP (250 μL) at 37 °C for 10 min. After adding 5 mM CaCl 2 (50 μL) and 50 μg mL -1 FITC-CD41 (10 μL), all groups were incubated at room temperature for 1 h and evaluated via flow cytometry. For hemolysis evaluation, the collected red blood cells were diluted to 1/10 of their volume with PBS. Then 200 μL RGD-PMCS with different concentrations were added into 1.3 mL freshly isolated blood and then kept at 37 °C for 4 h, followed by centrifugation at 3000 rpm for 10 min. After that, the supernatant was collected and measured absorbance at 540 nm.
PBS was acted as the negative control and deionized water was adopted as the positive control. Hemolysis ratio was calculated by the following formula: hemolysis % = (sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100%. penicillin/streptomycin at 37 °C containing 5% CO 2 . For in vitro cytotoxicity test, HUVECs were seeded into 96 well plates with a density of 1 × 10 4 per well. After 24 h, cells were incubated with RGD-PMCS for another 24 h. The MTT assay was conducted following the standard protocol.
In Vitro Synergistic Therapy. Red blood cells were incubated with 100 μg mL -1 RGD-PMCS for 24 h. Subsequently, the cells were irradiated with an 808 nm laser at a power intensity of 2 W cm -2 for 3 min, and cultured for another 24 h. Then, the cells were stained with calcein-AM, followed by a Leica SP5 confocal laser scanning microscope (CLSM) observation. Quantitative analysis was also taken followed the operation as the above treatments. Then the red blood cells were stained with DCFH-DA for 30 min and evaluated through a BioTek Powerwave XS fluorescence microplate reader.
For hemolysis treatment analysis, the diluted red blood cells were incubated with various concentrations of RGD-PMCS, followed by irradiating with the 808 nm laser (2 W cm -2 ) for 3 min. After that, all operations were the same as the hemolysis evaluation described above.
Level of PF3 Detection. The prepared platelets were diluted with Tyrode's solution with a density of 1 × 10 12 per milliliter and then treated with various concentrations of RGD-PMCS. After 1 h incubation, those platelets were subjected to irradiation of NIR laser (808 nm, 2 W cm -2 , 3 min). Then, the platelets were seeded into 96-well plates and kept at 37 °C for 30 min. Subsequently, the excess solution was removed and washed with detergent for 5 times, the platelets were incubated with enzyme standard reagent, incubated and washed in the same manner as described above. In addition, platelets were further incubated with Reagent A and then with Reagent B according to the protocol. After that, the reaction was stopped with Stop solution and measured absorbance at 540 nm.
In Vitro Target Specificity. The prepared activated platelets were diluted with Tyrode's solution and incubated with 100 μg mL -1 PMCS and RGD-PMCS for 1 h.
The platelets were then washed three times with Tyrode's solution, and 200 μL aqua regia was added, the amount of Zn was measured by inductively coupled plasma mass spectrometry (X Series 2; ThermoFisher, Waltham, MA, USA).

Artificial Thrombosis Assay.
Inferior vein blood was collected from healthy rats under anesthesia. Artificial thrombosis was produced by adding 25 U mL -1 thrombin and 25 mM CaCl 2 at 37 °C for 3 h. The resulted clot was incubated with different concentrations of RGD-PMCS for 1h and further exposed to 808 nm irradiation for 20 min at a power density of 2 W cm -2 , the absorbance at 540 nm and 340 nm were monitored. In addition, the hydrodynamic size of the artificial thrombosis supernatant was monitored at 10-min intervals by dynamic light scattering.
Lower Limb Thrombosis Model. SD rats (male, weighted 250-300 g) were anesthetized with chloral hydrate. For each of rat, the left lower limb was fixed after the pre-operation skin preparation, and then an incision was made to expose the targeting lower limb artery. One piece of filter paper saturated with 10% FeCl 3 was placed on the top of the left lower limb artery for 10 min to generate vascular injury.
As for the mice (BALB/c, male, aged 6-8 weeks) thrombosis model, the lower limb artery was dealt with 5% FeCl 3 for 10 min.
In Vivo Infrared Thermal Imaging. The rats with lower limb thrombosis were intravenously injected with PBS, PMCS (10 mg kg -1 ) and RGD-PMCS (10 mg kg -1 ), respectively. The thrombotic site was irradiated by NIR light (2 W cm -2 ) for 20 min after 1 h post-injection, and the changes of temperature at the thrombotic site were monitored by an infrared thermal imager.

Magnetic Resonance Imaging. During the Magnetic Resonance Imaging (MRI)
scanning, rats in each group were continually sedated by chloral hydrate. All images were obtained from a 7-Tesla MRI scanner (Bruker, Germany). The experimental groups were shortly imaged after thrombus induction with the body coil of rats (Bruker, Germany).
After the scanning of the localizer, a 2-dimensional, time-of-flight magnetic resonance angiogram was performed from the level of iliac bifurcation to the terminal lower limbs blood vessels. Sequence parameters were as follows: repetition time/echo time = 18/4.5 ms, number of averages = 3, and excitation pulse angle = 76°. With a 256 × 256 matrix, a field of view of 55 × 55 mm was scanned with 100 continuous slices of 600 μm thickness. In addition, the saturated zone was used to reduce the impacts of breath and lightly move to image quality. We also took some measures to protect rats from damages of low body temperature before and after scanning. The In Vivo Biodistribution. The healthy mice were injected via the tail with ICG-RGD-PMCS (10 mg kg -1 ) (three mice per group). Then, the mice were sacrificed and major organs (heart, liver, spleen, lung, and kidney) were collected at 6, 12, 24, and 48 h post-injection. The fluorescence intensity organs were measured via using an IVIS Spectrum Imaging System (PerkinElmer, Waltham, MA, USA).
Tail Bleeding Assay. The mice with thrombosis were divided randomly into seven groups with the same operations as in vivo thrombosis therapy described above. A distal segment with 10 mm of the tail was cut by anatomical scissors and the injured tail was put into 37 °C saline. The bleeding time was defined as the time needed for a wound to hemostasis for at least 10 s.

Intra-Thrombosis Penetration Analysis.
Blood collected from the inferior vein of healthy rats was injected into a plastic pipette and the other end was closed. Artificial thrombosis was produced by adding 25 U mL -1 (100 μL) thrombin and 25 mM CaCl 2 at 37 °C for 3 h. The resulted clot was incubated with RGD-PMCS (1 mg mL -1 , 1 mL) and further exposed to 808 nm irradiation for 10 min at a power density of 2 W cm -2 , the PA signals pre-and post-treatment with RGD-PMCS were recorded by MSOT.