Ultrasound Trigger Ce‐Based MOF Nanoenzyme For Efficient Thrombolytic Therapy

Abstract The inflammatory damage caused by thrombus formation and dissolution can increase the risk of thrombotic complications on top of cell death and organ dysfunction caused by thrombus itself. Therefore, a rapid and precise thrombolytic therapy strategy is in urgent need to effectively dissolve thrombus and resist oxidation simultaneously. In this study, Ce‐UiO‐66, a cerium‐based metal–organic framework (Ce‐MOF) with reactive oxygen species (ROS) scavenging properties, encapsulated by low‐immunogenic mesenchymal stem cell membrane with inflammation‐targeting properties, is used to construct a targeted nanomedicine Ce‐UiO‐CM. Ce‐UiO‐CM is applied in combination with external ultrasound stimulation for thrombolytic therapy in rat femoral artery. Ce‐UiO‐66 has abundant Ce (III)/Ce (IV) coupling sites that react with hydrogen peroxide (H2O2) to produce oxygen, exhibiting catalase (CAT) activity. The multi‐cavity structure of Ce‐UiO‐66 can generate electron holes, and its pore channels can act as micro‐reactors to further enhance its ROS scavenging capacity. Additionally, the porous structure of Ce‐UiO‐66 and the oxygen produced by its reaction with H2O2 may enhance the cavitation effects of ultrasound, thereby improving thrombolysis efficacy.

(EDX) spectroscopy on a FEI TALOS F200X transmission electron microscope with a superX-G2 energy dispersive X-ray spectrometer.The Ce-UiO-66 were dissolved in a mixture of 10 % deuterochloric acid (DCl) in D2O and deuterated dimethyl sulfoxide (d6-DMSO) (molar ratio 1:7) for 1 H and 13 C nuclear magnetic resonance (NMR) measurements recorded by Bruker Avance III-HD 500 spectrometer.The Fourier-transform infrared (FTIR) spectroscopy was used to analyze the FTIR spectrum of Ce-UiO-66 by bromide pellet method on a Fourier transform spectrometer (ThermoFisher Scientific iS50, USA).The nitrogen adsorption isotherms were recorded using a surface area and pore size analyzer (Micromeritics ASAP 2460), and the specific surface area and the pore size distribution were calculated from the adsorption data using the Brunauer-Emmett-Teller (BET) method and density functional theory (DFT) method, respectively, after degassing at 100 ℃ under vacuum for 12 h.The surface chemical composition of Ce-UiO-66 was measured using X-ray photoelectron spectroscopy (XPS) on an Escalab 250Xi X-ray photoelectron spectromater (ThermoFisher Scientific, USA).The particle sizes and zeta potentials of the nanoparticles in water were measured using a Zetasizer Pro nanometer size and potential analyzer (Malvern Panalytical, UK) at 25 ℃ for three times.The ultraviolet-visible (UV-Vis) absorption was recorded an UV-3600 UV-Vis spectrophotometer (Shimadzu, Japan).by incubation with Anti-Rabbit secondary antibody at room temperature for 1 h.The specific bands were observed using an odyssey infrared imaging system (LI-COR, CLx, USA).
In vitro thrombolysis treatment.Blood clot samples prepared from fresh mouse blood were treated with saline, Ce-UiO-66, Ce-UiO-CM, ultrasound (US), Ce-UiO-66 + US, or Ce-UiO-CM + US.The working frequency of the US was 3 MHz, the duty ratio was 20%, the sound intensity was 1.4 W•cm -2 , and the treatment time was 30 min.The aqueous dispersions of Ce-UiO-66 and Ce-UiO-CM with equivalent Ce-UiO-66 concentration (0.2 mg•mL -1 ) were prepared.The hemoglobin level in different treatment groups was assessed by measuring the absorbance of the supernatant at 540 nm using a Synergy H1 microplate reader (BioTek, USA).The weight changes of the blood clots before and after treatment in different treatment groups were recorded to evaluate the in vitro thrombolytic efficiency.All experiments were repeated for 4 times.

Cells and animals. Human umbilical vein endothelial cells (HUVECs) used in this study
were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China).were purchased from Shanghai Shengchang Biotechnology Co., Ltd.All experimental operations were carried out in accordance with the approved guidelines of Ren Ji Hospital Ethics Committee.
Cell viability.HUVECs (100 µL suspension) were seeded into a 96-well plate at the density of 1×10 4 cells/well and cultured at 37 ℃ with 5% CO2 for 24 h.These cells were treated with ultrasound (working frequency 3 MHz, duty ratio 20%) at different sound intensities (1.2, 1.3, 1.4, 1.5, 1.6, 1.7 W•cm -2 ) for 10 min, with 4 replicates for each power level.The effect of different ultrasound intensities on the viability of HUVECs was studied with the CCK-8 kit (DOJINDO, Japan), and the absorbance was measured using a Synergy H1 microplate reader.
The aqueous dispersions of Ce-UiO-66 and Ce-UiO-CM with different equivalent Ce-UiO-66 concentrations (0.0001, 0.0002, 0.001, 0.002, 0.01, 0.02 mg•mL -1 ) were used to study the effects of Ce-UiO-66 and Ce-UiO-CM on the viability of HUVECs.HUVECs cultured in DMEM only were used as control.For combinatory treatment with nanoenzyme and ultrasound, cells were additionally stimulated with ultrasound under a working frequency of 3 MHz, duty ratio of 20%, and sound intensity of 1.4 W cm -2 for 10 min.4 replicates were set up for each treatment condition.The cell viability was calculated according to the instruction of the CCK-8 kit.
Establishment of the rat femoral artery thrombosis model using the FeCl 3 injury method.SD rats were anesthetized, and their hind limbs were immobilized.A longitudinal incision was made on the medial aspect of the thigh skin, and the muscles were carefully separated to expose the femoral artery.The femoral artery was fully wrapped by a piece of filter paper (3 mm × 5 mm) pre-soaked in 5% FeCl3 solution.A plastic wrap was used to cover the filter paper to prevent damage to other tissues caused by FeCl3.After 1 min, the plastic wrap and the filter paper were removed, and the wound area was gently rinsed with saline.The incision was closed again with sutures and the rats were monitored until fully awake.
Fluorescence imaging.To evaluate the in vivo distribution of ICG-modified Ce-UiO-CM, the thrombosed rats were injected with 100 µL of ICG-modified Ce-UiO-66 or Ce-UiO-CM (containing 1 mg•mL -1 of Ce-UiO-66) via the tail vein (n = 4 for each group).Fluorescence images of live animals were collected at different time points (0, 0.5, 1, 2, 3, 4, 6, 24 h) after injection using an AniView100 system (BLT, China).The rats were sacrificed at 0.5, 2 and 24 h after injection, and major organs including heart, liver, spleen, lung, kidney, as well as the femoral artery (thrombus site) were collected for fluorescence imaging using an InVivo ART-100 (VISQUE, Korea).
Pharmacokinetic study.Four rats (4 males; weight 200 ± 10 g) were used to study the pharmacokinetic profiles of Ce-UiO-CM, which were injected with Ce-UiO-CM solution through tail vein injection (dose of Ce: 937.8 µg kg -1 ) after being fasted for 12 h.Four rats Blood samples (200 L) were collected from the ocular vein of each rat at 0.25, 1, 2, 4, 24, 48, and 72 h after intravenous administration, and the concentration of Ce was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 720ES, USA).The pharmacokinetic parameters including the area under the concentration-time curve (AUC) from zero to the last measurable plasma concentration point (AUC0−t), AUC from 0 to infinity (AUC0−∞), mean residence time (MRT0−∞), terminal elimination half-life (T1/2) and clearance (CL) were calculated by a non compartment model using MaS Studio 1.6.0.5 software.
Photoacoustic imaging.To verify the in vivo oxygen production ability of Ce-UiO-CM, the thrombosed rats were injected with 100 µL of Ce-UiO-CM (containing 1 mg•mL -1 of Ce-Uio-66) via the tail vein, and photoacoustic images of the femoral artery area were collected at different time points (0, 0.5, 1, 1.5, 2, 4 h) using Vevo 2100 LAZR and VEVO LAZR-X Imaging System (Visual Sonic, Canada) to determine the blood oxygen saturation (sO2).The thrombosed rats injected with 100 µL of Ce-UiO-66 and CM at corresponding concentrations were treated as comparison group.The sO2 was quantitatively analyzed by Vevo LAB version 5.5.1.
In vivo thrombolysis treatment.To evaluate the therapeutic effect of Ce-UiO-CM in thrombolysis treatment in vivo, the thrombosed rats were randomly divided into six treatment groups (n = 5 in each group): saline group, Ce-UiO-66 group, Ce-UiO-CM group, ultrasound (US) group, Ce-UiO-66 + US group, and Ce-UiO-CM + US group.Ultrasound with the working frequency of 3 MHz, duty ratio of 20%, and sound intensity of 1.4 W•cm -2 was used for US treatment.100 µL aqueous dispersion of Ce-UiO-66 or Ce-UiO-CM with equivalent Ce-UiO-66 concentration (1mg•mL -1 ) were injected via the tail vein 0.5 h before US treatment (10 min for 3 times).Doppler ultrasound imaging was used to detect changes in the blood flow at the site of femoral artery occlusion through a Vevo 2100 LAZR Imaging System.The femoral artery was collected for hematoxylin-eosin (H&E) and Masson's trichrome staining after treatment, and the stained sections were observed using an optical microscope.
Additionally, the femoral artery was also stained with dihydroethidium (DHE) and α-SMA antibody for fluorescence analysis.The femoral artery of the thrombus model and normal animal were stained with intercellular adhesion molecule 1 (ICAM-1) antibody.Image J software was used to evaluate the treatment effect, calculated as follows: percentage of thrombus area = area of thrombus/total area of femoral artery × 100%.
Safety evaluation.The rats treated with the above anti-thrombotic therapy were sacrificed, and blood samples were collected from the orbital socket for routine blood test, as well as analysis of serum biochemical parameters associated with liver, renal and cardiac functions.
The major organs such as heart, liver, spleen, lung, and kidney were collected for H&E staining to evaluate the organ toxicity of the treatment.

Statistical analysis.
All the statistical values in this study were displayed as mean ± standard error of mean (SEM) for n ≥ 3 independent experiments.Student's ttest was used to compare the statistical differences between two independent groups.Comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA).All statistical analyses were conducted with GraphPad Prism software (PRISM 9.0).P < 0.05 was regarded as statistically significant.The fluorescence images (A) and quantitative analysis of the major organs and the femoral artery thrombus site after injection 0.5 h (B), 2 h (C) and 24 h (D) of ICG-modified Ce-UiO-CM and Ce-UiO-66.In (A), the first row showed the femoral artery, lungs, and heart from left to right, while the second row showed the spleen, kidneys, and liver from left to right.Data are presented as mean ± standard error of mean (SEM), n = 4. * P < 0.05, student's t-test.The major organs (heart, liver, spleen, kidney and lung) of the thrombosed rats were sectioned and H&E stained after treatment.Scale bar is 100 μm.No obvious changes were observed.  a] The amount of Ce (III) and Ce(IV) was calculated from the peak area of the Ce 3d components using the following equations:

Figure S3 .
Figure S3.The TEM image of Ce-UiO-CM.The obtained Ce-UiO-CM exhibited a core-shell structure with a layer of membrane on the surface.Scale bar is 50 nm.

Figure S4 .
Figure S4.The cellular uptake of the Ce-UiO-CM in HUVECs.The HUVECs were pretreated with or without ROS for 0.5 h, and then incubated with Ce-UiO-CM for 4 h.After ROS treatment, the intercellular adhesion molecule 1 (ICAM-1) expressions were increased on HUVECs.Additionally, intense green fluorescence from CD18 on Ce-UiO-CM was notably evident in HUVECs exhibiting high ICAM-1 expression after 4 hours of incubation with Ce-UiO-CM, whereas HUVECs with low ICAM-1 expression only gave the faint fluorescence.These findings suggest enhanced cellular uptake of Ce-UiO-CM by HUVECs with elevated ICAM-1 levels, highlighting the specificity of Ce-UiO-CM for targeting inflamed endothelial cells.Scale bar is 50 μm.

Figure S8 .
Figure S8.The ultraviolet-visible (UV-Vis) absorption spectra of ICG-modified Ce-UiO-CM and Ce-UiO-66 in water.Compared with ICG (about 780nm), the absorption peaks of ICG-modified Ce-UiO-CM and Ce-UiO-66 had a slight red shift at about 800 nm, suggesting the successful assembly of ICG into Ce-UiO-66.

Figure S10 .
Figure S10.Distribution of ICG-modified Ce-UiO-CM and Ce-UiO-66 in the major organs and the femoral artery thrombus site at 0.5, 2 and 24 h after injection.

Figure S11 .Figure S12 .
Figure S11.Mean plasma concentration-time curves of Ce-UiO-CM after a single intravenous administration at a dose of 937.8 µg kg -1 of Ce in healthy rats.The concentration of Ce messured by inductively coupled plasma mass spectrometry (ICP-MS).Data are presented as mean ± standard error of mean (SEM), n = 4.

Figure S14 .
Figure S14.The fluorescence images of DHE-stained femoral artery in different treatment groups.The white circle displays the thrombus modeling blood vessels.Scale bar is 100 μm.The endothelium of the femoral artery in the control, CM and US group showed a high intensity of DHE fluorescence while the fluorescence were significantly reduced after Ce-UiO-66 and Ce-UiO-CM treatment with or without US.Additionally, the red fluorescence of DHE in these Ce-UiO-CM treated groups were weaker than these Ce-UiO-66 treated groups.

Figure S15 .
Figure S15.Immunofluorescence iamges of the femoral artery in different treatment groups stainied with smooth muscle cell marker α-smooth muscle actin (α-SMA).The number of VSMCs were significantly increased in the thrombotic area after treating with Ce-UiO-66 and Ce-UiO-CM.Scale bar is 100 μm.

Figure S16 .
Figure S16.Masson's trichrome staining diagram of the femoral artery in different treatment groups.The concentration of collagen was also greatly upregulated after Ce-UiO-66 and Ce-UiO-CM treatment with or without US.Scale bar is 100 μm.

Figure S17 .
Figure S17.The routine blood test results of different treatment groups.The levels of red blood cell count (RBC, A), hemoglobin (HGB, B), hematocrit (HCT, C), white blood cell count (WBC, D), neutrophil count (NEUT, E), lymphocyte count (LYMPH, F), platelet count (PLT, G), platelet volume distribution width (PDW, H), and monocyte count (MONO, I) in the blood samples from the treated rats were measured.Data are presented as mean ± standard error of mean (SEM), n = 4. ANOVA with Tukey's test for multiple comparisons.No obvious changes were observed.

Figure S19 .
Figure S19.The renal function related serum biochemical parameters of different treatment groups.The levels of creatinine (Crea, A), urea (Urea, B), and uric acid (UA, C) in the blood samples of the treated rats were measured.Data are presented as mean ± standard error of mean (SEM), n = 4. ANOVA with Tukey's test for multiple comparisons.No obvious changes were observed.

Figure S20 .
Figure S20.The cardiac function related serum biochemical parameters of different treatment groups.The levels of lactate dehydrogenase (LDH, A) and creatine kinase (CK, B) in the blood samples of the treated rats were measured.Data are presented as mean ± standard error of mean (SEM), n = 4. ANOVA with Tukey's test for multiple comparisons.No obvious changes were observed.

Figure S21 .
Figure S21.H&E staining of the histological sections of the major organs in different treatment groups.