Ultramicro nano thrombolytic microbubble as well as preparation method and application thereof

Abstract

The invention discloses an ultramicro nano thrombolytic microbubble as well as a preparation method and application thereof, and belongs to the technical field of drug-loaded microbubbles. The thrombolytic microbubble comprises a core drug and a lipid shell, wherein the core drug comprises an anti-active oxygen material, urokinase and perfluoropentane; the preparation method comprises the following steps: firstly preparing the anti-active oxygen material, then encapsulating the anti-active oxygen material with liposome, adding urokinase for hydration after encapsulation, adding perfluoropentane, emulsifying, and freeze-drying to obtain the thrombolytic microbubble. The ultramicro nano thrombolysis microbubble is used for treating thrombus, phase change occurs to perfluoropentane under the ultrasonic action, so that thrombus is developed more obviously under the ultrasonic action, meanwhile, urokinase and an anti-active oxygen material are released from the microbubble through the cavitation effect, the released urokinase can deepen into the thrombus under the blasting action of the perfluoropentane, and the thrombolysis effect of the thrombus is improved. And the anti-active oxygen material can remove active oxygen and protect vascular endothelial cells and macrophages after revascularization, so that the risk of bleeding is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, in particular to ultramicro-nano thrombolytic microbubbles and a preparation method and application thereof. Background Art

[0002] Deep vein thrombosis (DVT) poses a major challenge to global public health. Therefore, it is of great significance to find effective thrombolytic treatments and reduce cellular inflammation and oxidation caused by vascular recanalization.

[0003] Thrombotic diseases involve two pathological processes: thrombosis and thromboembolism. Thrombosis is mainly composed of activated platelets and fibrin, and is closely related to the inflammatory response that destroys the vascular endothelial barrier system. At present, thrombosis treatment is roughly divided into anticoagulants and thrombolytic drugs. Anticoagulants can prevent thrombosis, such as heparin, warfarin or its derivatives. Thrombolytic drugs, also known as plasminogen activators, can dissolve pre-formed thrombus clots, mainly including urokinase, streptokinase and tissue plasminogen activator.

[0004] Urokinase is a common thrombolytic drug in clinical practice, with good thrombolytic effect and biosafety. However, its drug molecules are difficult to penetrate into the thrombus and have a short half-life. Therefore, its clinical therapeutic effect on obstructive thrombosis is poor. Summary of the invention

[0005] The purpose of the present invention is to provide an ultrafine nano-thrombolytic microbubble and a preparation method and application thereof, which can significantly improve the recanalization and post-recanalization protection of occluded blood vessels and reduce the risk of bleeding.

[0006] The objective of the present invention is achieved through the following technical solutions: An ultrafine nanometer thrombolytic microbubble comprises a core drug and a lipid shell, wherein the core drug comprises an anti-active oxygen material, urokinase and perfluoropentane.

[0007] As some possible implementation modes of the present application, the anti-active oxygen material is a material including Ce and Mn elements.

[0008] As some possible embodiments of the present application, the anti-active oxygen material has CAT enzyme and SOD enzyme activities.

[0009] As some possible implementation methods of the present application, the preparation method of the anti-active oxygen material is as follows: Ce(NO3)3･6H2O and Mn(NO3)2･6H2O are dissolved in ethanol, and after stirring for a certain period of time, ammonium hydroxide is added and stirred sufficiently, followed by centrifugation, filtration, washing and drying to obtain an anti-active oxygen material.

[0010] As some possible implementation methods of the present application, the molar ratio of Ce(NO3)3･6H2O and Mn(NO3)2･6H2O is 0.66:1.

[0011] As some possible implementation methods of the present application, the mass ratio of the anti-active oxygen material, urokinase and perfluoropentane is 1:4:8.

[0012] In addition, to achieve the above-mentioned purpose, the present application also provides a method for preparing ultrafine nano-thrombolytic microbubbles, which comprises first preparing an anti-active oxygen material, and then encapsulating it with liposomes. After the encapsulation, urokinase is added for hydration, and then perfluoropentane is added for emulsification, and then freeze-dried to obtain the ultrafine nano-thrombolytic microbubbles.

[0013] In addition, to achieve the above-mentioned purpose, the present application also provides an application of ultrafine nano-thrombolytic microbubbles in the preparation of drugs for treating thrombosis.

[0014] As some possible implementation methods of the present application, the drug for treating thrombosis is a drug for treating obstructive thrombosis, that is, the ultrafine nano thrombolytic microbubbles described in the present application are mainly used to treat obstructive thrombosis.

[0015] The beneficial effects of the present invention are: The present application provides a thrombolytic microbubble comprising a core drug and a lipid shell, wherein the core drug comprises an anti-reactive oxygen material, urokinase and perfluoropentane. The thrombolytic microbubble works in coordination with ultrasound. Under the excitation of low-frequency pulse ultrasound, perfluoropentane undergoes a phase change, making the thrombus more obvious under ultrasound. At the same time, urokinase and anti-reactive oxygen materials are released from the microbubble through the cavitation effect. The released urokinase can penetrate deeper into the thrombus under the blasting effect of perfluoropentane, thereby improving its thrombolytic efficiency and promoting vascular reperfusion. At the same time, the anti-reactive oxygen material can remove the reactive oxygen species (ROS, including hydrogen peroxide, superoxide anion and hydroxyl radicals, which are important factors in thrombosis and vascular restenosis, highlighting that oxidative stress is a key consideration in the treatment of thrombotic diseases and vascular reperfusion injury), thereby effectively treating thrombotic diseases, and at the same time, it can protect the vascular endothelial cells and macrophages after reperfusion, thereby avoiding vascular reperfusion injury.

[0016] In this scheme, the combination of ultrafine nano-thrombolytic microbubbles and ultrasound can play a role in visualized thrombolysis, significantly improve the recanalization of occluded blood vessels, protect them after recanalization, and reduce the risk of bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Synthesis and characterization of CexMnx NPs.

[0018] 1a. Synthesis of CexMnx NPs and modification process of NPU; 1b. SEM image of CexMnx NPs. 1c. Particle size of CexMnx NPs.

[0019] 1d. High-resolution TEM of CexMnx NPs crystals; 1e, 1f. Lattice analysis and FFT graphs to obtain the corresponding crystal plane spacing; 1g. Different interplanar spacings within CexMnx NPs; 1h. XRD of CexMnx NPs; 1i. EDX mapping images of Mn, Ce, and O elements in CexMnx NPs, scale bar: 10 nm; 1j. AC-HAADF-STEM image of CexMnx NPs, scale bar: 1 nm; 1k. XPS analysis of CexMnx NPs.

[0020] Figure 2 : XPS images of CexMnx NPs and CeO2.

[0021] Figure 3 : Mn and Ce metal contents in CexMnx NPs.

[0022] Figure 4 : Scavenging of superoxide anions and hydroxyl radicals by CexMnx NPs was tested by ESR.

[0023] Figure 5 : Multienzyme activity test of CexMnx NPs.

[0024] 5a. Schematic diagram of multi-enzyme activity of CexMnx NPs; 5b. CAT and SOD enzyme activities of CexMnx NPs and their ability to scavenge singlet oxygen tested by ESR; 5c. TEM image of NU; 5d. Particle size of NU; 5e. TEM image of NPU; 5f. Particle size of NPU; 5g. Zeta potential of CexMnx NPs and NPU. 5h. Encapsulation efficiency of UK in NPU; 5i. Fluorescence colocalization of NPU.

[0025] Figure 6 : In vitro imaging, thrombolysis, and reactive oxygen species scavenging of NPU.

[0026] CEUS ultrasound imaging of NPU at different powers (6a) and intensity statistics (6b); The thrombolytic effect (6c) and thrombolytic rate (6d) of different concentrations of NPU on the thrombus model; 6e. The thrombolytic effects of the NPU group and the NPU + LIPUS group were evaluated by fibrin (450 nm) in the supernatant at 2 h and 4 h; 6f. Confocal images and fluorescence intensity statistics of LPS-induced HUVECs under different treatments. ROS were labeled by oxidizing non-fluorescent DCFH (green). Scale bar 50 μm.

[0027] 6g. Confocal images and fluorescence intensity statistics of LPS-induced HUVEC under different treatments; 6h. LPS-induced intracellular IL-6, TNF-α, INF-γ and IL-10 concentrations in RAW264.7 and HUVEC under different treatments.

[0028] Figure 7 : NPU contrast-enhanced ultrasound in B-mode and CEUS modes at different powers.

[0029] Figure 8 : The thrombolytic effects of the NPU group and the NPU + LIPUS group were evaluated by hemoglobin (540 nm) in the supernatant at 2h and 4h.

[0030] Fig. 9 :Flow cytometry fluorescence intensity statistics of RAW264.7 and HUVEC induced by LPS under different treatments. ROS were labeled by oxidizing non-fluorescent DCFH (green).

[0031] Fig.10 : In vivo ultrasound imaging monitors the thrombolytic process in real time and removes reactive oxygen species.

[0032] 10a. Flow chart of the establishment and treatment of the in vivo model in SD rats; The b-mode and CEUS images (10c) and ultrasound intensity statistics (10b) of the in vivo thrombosis model after injection of NPU and saline were used after LIPUS irradiation; 10d. In vivo ultrasound imaging before injection, 5 minutes after NPU injection and 15 minutes after LIPUS irradiation; 10e. Schematic diagram of in vivo NPU treatment after LIPUS irradiation; The results of thrombolytic therapy (10f) and thrombolytic rate statistics (10g) of the in vivo thrombosis model after different treatments. The results of ROS removal (10f) and fluorescence intensity statistics of the in vivo thrombosis model after different treatments.

[0033] Fig.11 : Cytotoxicity of RAW264.7 and HUVEC treated with different concentrations of CexMnx NPs and NPU was detected using CCK8.

[0034] Fig.12 : Cytotoxicity of RAW264.7 and HUVEC treated with different concentrations of CexMnx NPs and NPU was detected using CCK8. DETAILED DESCRIPTION

[0035] In the prior art, urokinase, as a common thrombolytic drug in clinical practice, has good thrombolytic effect and biosafety, but its drug molecules are difficult to penetrate into the thrombus and have a short half-life, so the clinical treatment effect on obstructive thrombosis is poor. Based on this, the present application provides an ultrafine nano-thrombolytic microbubble, comprising a core drug and a lipid shell, wherein the core drug comprises an anti-active oxygen material (CexMnx NPs), urokinase (UK) and perfluoropentane (PFP).

[0036] The present application provides a thrombolytic microbubble comprising a core drug and a lipid shell, wherein the core drug comprises an anti-reactive oxygen material, urokinase and perfluoropentane. The thrombolytic microbubble works in coordination with ultrasound. Under the excitation of low-frequency pulse ultrasound, perfluoropentane undergoes a phase change, making the thrombus more obvious under ultrasound. At the same time, urokinase and anti-reactive oxygen materials are released from the microbubble through the cavitation effect. The released urokinase can penetrate deeper into the thrombus under the blasting effect of perfluoropentane, thereby improving its thrombolytic efficiency and promoting vascular reperfusion. At the same time, the anti-reactive oxygen material can remove the reactive oxygen species (ROS, including hydrogen peroxide, superoxide anion and hydroxyl radicals, which are important factors in thrombosis and vascular restenosis, highlighting that oxidative stress is a key consideration in the treatment of thrombotic diseases and vascular reperfusion injury), thereby effectively treating thrombotic diseases, and at the same time, it can protect the vascular endothelial cells and macrophages after reperfusion, thereby avoiding vascular reperfusion injury.

[0037] In this scheme, the combination of ultrafine nano-thrombolytic microbubbles and ultrasound can play a role in visualized thrombolysis, significantly improve the recanalization of occluded blood vessels, protect them after recanalization, and reduce the risk of bleeding.

[0038] In order to further improve the scavenging effect on the active oxygen generated during thrombosis, as some feasible embodiments of the present application, the composition of the anti-active oxidation material is further limited, that is, the anti-active oxygen material is a material including Ce and Mn elements.

[0039] In order to further improve the scavenging effect of the reactive oxygen species generated during thrombosis, as some of the embodiments of the present application, the enzyme activity of the anti-reactive oxidation material is further limited, that is, the anti-reactive oxygen material has CAT enzyme, SOD enzyme activity, and singlet oxygen and hydroxyl radical scavenging activity. When the anti-reactive oxygen material reaches the vascular endothelial cells, it can continuously catalyze the dismutation of superoxide anion radicals in the cells into hydrogen peroxide and oxygen, and convert H2O2 to O2, thereby alleviating the hypoxia of the thrombus microenvironment, protecting endothelial cells, and preventing the formation of thrombus again. In summary, the thrombolytic and antioxidant effects of ultra-micro nano-thrombolytic microbubbles provide hope for the treatment of visualized thrombolysis and ischemia-reperfusion injury.

[0040] In order to further improve the scavenging effect of the active oxygen generated during thrombosis, as some possible implementation methods of the present application, the preparation method of the anti-active oxidation material is further limited, that is, the preparation method of the anti-active oxygen material is as follows: Ce(NO3)3･6H2O and Mn(NO3)2･6H2O are dissolved in ethanol, and after stirring for a certain period of time, ammonium hydroxide is added and stirred sufficiently, followed by centrifugation, filtration, washing and drying to obtain an anti-active oxygen material.

[0041] In order to further improve the scavenging effect of the active oxygen generated during thrombosis, as some feasible embodiments of the present application, the amount of the raw materials of the anti-active oxidation material is further limited, that is, the molar ratio of Ce(NO3)3･6H2O and Mn(NO3)2･6H2O is 0.66:1.

[0042] In order to further improve the thrombolytic effect of ultrafine nano-thrombolytic microbubbles, as some feasible implementation methods of the present application, the dosage of each component in the core drug is further limited, that is, the mass ratio of the anti-active oxygen material, urokinase and perfluoropentane is 1:4:8.

[0043] In addition, to achieve the above-mentioned purpose, the present application also provides a method for preparing ultrafine nano-thrombolytic microbubbles, which comprises first preparing an anti-active oxygen material, and then encapsulating it with liposomes. After the encapsulation, urokinase is added for hydration, and then perfluoropentane is added for emulsification, and then freeze-dried to obtain the ultrafine nano-thrombolytic microbubbles.

[0044] In addition, to achieve the above-mentioned purpose, the present application also provides an application of ultrafine nano-thrombolytic microbubbles in the preparation of drugs for treating thrombosis.

[0045] For targeted treatment, as some feasible embodiments of the present application, the types of thrombi are further limited, that is, the drug for treating thrombi is a drug for treating obstructive thrombi.

[0046] Next, the specific preparation method of ultrafine nano-thrombolytic microbubbles is described in detail. Example

[0047] S1 Preparation of anti-active materials: Ce(NO3)3･6H2O (5 g) and Mn(NO3)2･6H2O (5 g) were used as initial raw materials (the molar ratio of the two was 0.66:1), dissolved in ethanol, and the solution was vigorously stirred for about 10 minutes, and then 20 mL of ammonium hydroxide was added and stirred continuously overnight; the nanoparticles were then purified by centrifugation, and washed with anhydrous ethanol and deionized water for 3 times respectively, and the brown precipitate was filtered and vacuum dried overnight to obtain the anti-active material (i.e., CexMnx NPs), as shown in FIG. Figure 1 a.

[0048] S2 is then encapsulated with liposomes. After encapsulation, urokinase is added for hydration, and then perfluoropentane (the mass ratio of the anti-active oxygen material, urokinase and perfluoropentane is 1:4:8) is added for emulsification, followed by freeze-drying to obtain the ultrafine nano-thrombolytic microbubbles.

[0049] The synthesis and structural characterization of the nanocrystals / heterojunctions of CexMnx NPs in the examples are as follows Figure 1 shown.

[0050] SEM and TEM images show that CexMnx NPs have a nearly spherical morphology with an average diameter of 20 nm, which is very similar to the existing report (Nanoceria as an Electron Reservoir: Spontaneous Deposition of Metal Nanoparticles on Oxides and Their Antiinflammatory Activities) Figure 1 b. Figure 1 d). The particle size was measured by Malvern particle size analyzer and was about 70 nm. The reason may be that the multiple nanoparticles were slightly aggregated ( Figure 1 c). The powder X-ray diffraction (PXRD) pattern of CexMnx shows a crystal structure similar to that of CeO2, indicating that the introduction of MnO2 does not change the CeO2 crystal phase or form a crystallographically separated dual phase ( Figure 1 h). In addition to the CeO2 peak, two obvious peaks appearing at 56.47° and 67.14° correspond to the (102) and (110) planes of Mn in CexMnx. At the same time, the introduction of MnO2 reduces the crystallinity of CexMnx NPs ( Figure 2). The particle morphology and crystal structure of CexMnx NPs were verified by high-resolution transmission electron microscopy (HRTEM) images. In the HRTEM images, the lattice spacing of CexMnx NPs was measured to be approximately 0.315nm, 0.196nm, and 0.311nm, respectively, which can match the (021) crystal plane of CeO2 and the (210) and (110) of MnO2 ( Figure 1 e-1g). High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images of CexMnx NPs depict the atomic resolution structure of CexMnx nanoparticles. We measured a lattice spacing of 0.222 nm corresponding to the (002) plane of MnO2, and a lattice spacing of 0.315 nm that matches the (021) plane of CeO2, which is also shown in the HRTEM image ( Figure 1 j). Elemental mapping further confirmed the successful introduction of MnO2 into CexMnx nanoparticles ( Figure 1 i). The overall Mn and Ce metal contents [defined as Mn / (O + Ce) × 100%] in CexMnx NPs were determined to be 5.54 and 8.64 atomic percent (at%), respectively, using energy dispersive X-ray spectroscopy (EDS). Figure 3 ). X-ray photoelectron spectroscopy (XPS) further provides insights into the chemical and electronic structures of the CexMnx NPs biocatalysts. Figure 1 As shown in Figure k, Ce, Mn and O signals are clearly detected, indicating that MnO2 is successfully doped into the CeO2 matrix. Compared with bare CeO2, the Ce 3 peak of CexMnx shows a positive energy shift, which is due to the increase in electron density caused by the doping of MnO2. At the same time, the peak of MnO2 also shifts in potential.

[0051] We speculated that CexMnx NPs exhibited relatively weak crystallinity due to the abundant presence of carbonaceous skeleton and the underlying loose structure. However, subsequent enzyme mimetic performance tests confirmed that the optimized performance of CexMnx NPs was superior to that of CeO2 NPs due to the introduced Mn precursor and the polycrystalline nature of the NPs.

[0052] Multienzyme activity test of CexMnx NPs ( Figure 5 ) Excessive free radicals during reperfusion can cause progressive oxidative damage and ultimately lead to cell death. The catalytic effects of CexMnxNPs on hydrogen peroxide (CAT) and superoxide dismutase (SOD) are shown in Figure 2. Figure 5a. To verify that CexMnx NPs nanoparticles have good performance in catalyzing the production of hydrogen peroxide, we used a colorimetric method [319.2 mg Ti(SO4)2 and 8.33 mLH2SO4] to detect the scavenging rate of hydrogen peroxide by CexMnx NPs. We also used the WST-8 method to detect the O2 produced by xanthine and xanthine oxidase. - , evaluate the activity of NPs in mimicking SOD.

[0053] The results showed that CexMnx NPs had the fastest hydrogen peroxide scavenging rate and O2 removal at a concentration of 200 mM compared with CeO2 and MnO2 alone. - The clearance rate was approximately 65% ​​and 89% compared with the control group ( Figure 5 b). Electron spin resonance (ESR) spectroscopy results also confirmed that CexMnx NPs reduced 1 O2, •OH, O2 - The generation of ROS, while CeO2 and MnO2 showed weak ROS scavenging function ( Figure 4 ).

[0054] Preparation and characterization of nanozymes NUP In order to improve the biosafety and dispersibility of CexMnx NPs and encapsulate UK and PFP, we modified liposomes with DPPC, DPPE-PEG2000, and Chol. We encapsulated UK and CexMnx NPs with liposomes and freeze-dried them to make NU. Transmission electron microscopy (TEM) showed that their morphology was spherical and their phospholipid bilayer ( Figure 5 c). Its shape is slightly different from the spherical shape, which may be related to the metal nanozymes encapsulated in the liposomes. The average particle size of NU liposomes is 155 nm ( Figure 5 d), joined PFP and became NUP ( Figure 5 e) The particle size increased slightly to about 345 nm ( Figure 5 f). The zeta potential of CexMnx is -23.75 mV. After adding DPPC, DPPE-PEG2000, and Chol, the zeta potential increases to +15.5 mV ( Figure 5 g). The change in charge indicated that the nanoparticles were successfully loaded into the liposomes. We then labeled the lipid layer of the liposomes with DII and labeled the UK peptide with FITC. Under a positive fluorescence microscope, the overlap of DIR and FITC fluorescence was observed, confirming that the UK drug molecules were loaded on the liposomes ( Figure 5 i). We used the BCA protein test kit to determine that the UK encapsulation rate was approximately 26% ( Figure 5 h). The above experiments prove that NUP liposomes were successfully prepared.

[0055] In vitro imaging, thrombolysis, and reactive oxygen species scavenging To verify the imaging, thrombolytic efficiency, and reactive oxygen species scavenging capabilities of NPU under ultrasound excitation, we conducted in vitro experiments ( Figure 6 ).

[0056] PFP-based liposomes are stable at body temperature and maintain their submicron size. When irradiated with ultrasound using LIPUS, they undergo a rapid phase transition from liquid to gas. When embedded in a thrombus, this explosive mechanical response disrupts the thrombus structure. When embedded in a thrombus, this explosive mechanical response disrupts the thrombus structure. In vitro ultrasound imaging examined the signal-enhancing imaging capabilities of the liposomes and their potential thrombolytic therapeutic effects. The results showed that the imaging capabilities are related to time and sound intensity, such as Figure 6 As shown in a. NPU liposomes containing physiological saline were placed in a dropper and heated at 0.8 W cm -2 and 1.6 W cm -2 The liposomes were irradiated with LIPUS for 6 min, and the changes in the liposomes were observed and recorded every 2 min during the irradiation process by a Mindray ultrasound machine. As expected, the PFH-loaded nanoparticles produced a strong signal during the vaporization process, and this change was triggered by irradiation. As time went on, the b-mode and CEUS ultrasound signal intensities of the PFP-containing liposomes gradually increased, reaching peak values ​​after 5 min of LIPUS irradiation, and continued to decrease until 9 min ( Figure 6 a, 6b, Figure 7 ). This rapid enhancement of ultrasound signal intensity was attributed to the highly echogenic microbubbles generated during the LIPUS process, verifying that the enhanced ultrasound signal presented by the liposomes successfully underwent a phase transition.

[0057] PFP-loaded NPU may contribute to real-time contrast-enhanced ultrasound (CEUS) signals or thrombolytic effects. The changes in weight and absorbance values ​​of different experimental groups at 2h and 4h indicated the thrombolytic effect. The effect of US on in vitro thrombolytic therapy of nanomedicine was evaluated by measuring the absorbance of the supernatant. When NPU+US was treated, the absorbance of fibrin (450nm) and hemoglobin (540nm) in the supernatant increased significantly, while when NPU was treated alone, the absorbance of fibrin and hemoglobin in the supernatant increased slightly ( Figure 6 e. Figure 8). The weight of the thrombus model confirmed this effect. The US group showed obvious signs of damage, while the other group without US showed less damage. Therefore, US+NPU can speed up thrombolysis, shorten recanalization time, and improve the success rate of thrombolysis. NUPNPs are concentration-dependent thrombolytic. To further verify the therapeutic effect of liposomes, we compared the thrombus weight and thrombolysis rate of each group (thrombolysis rate % = [initial weight of thrombus model-final weight of thrombus model / initial weight of thrombus model] × 100%). 1mg / ml NUP liposomes had the strongest thrombolytic effect, and the thrombolysis rate increased by 57±1.99% after 4 hours of US treatment. The 0.5mg / ml group and the 0.25mg / ml group decreased by 27±4.0% and 8±5.2%, respectively. The effect of normal saline was small, and the thrombolysis rate was only reduced by 2±2.5%. ( Figure 6 c. Figure 6 d) Vascular recanalization after thrombolytic injury generates a large amount of ROS, leading to severe cellular dysfunction. NPU contains antioxidant CexMnx NPs, which are used to eliminate reactive oxygen species and protect blood vessels. In this study, we used flow cytometry ( Fig. 9 ) and the introduction of ROS fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to detect the ROS scavenging ability of the NPU group and the NPU + US group. The production of ROS (green) in RAW264.7 cells and HUVECs was significantly changed after LPS induction. The ROS levels in the NPU group and the NPU + US group were lower than those in the positive group, especially after US treatment ( Figure 6 f, 6g). In addition, the high expression of cytokines IL-6, TNF-α, and INF-γ in LPS-induced inflammatory RAW264.7 and HUVEC cells was significantly reduced in the presence of NPU and US-triggered NPU ( Figure 6 h). On the contrary, the expression of intracellular anti-inflammatory factors such as IL-10 was upregulated. The above results show that NPU can effectively reduce cellular inflammatory response and protect blood vessels from the effects of reperfusion ( Figure 6 h).

[0058] As expected, the multifunctional nano-excavator we designed achieved greater accumulation and deeper invasive delivery to thrombotic tissue, realizing the internal explosion of thrombus, thus achieving significant effective thrombolytic efficiency in in vitro experiments. Subsequently, CexMnx NPs released by NPU disintegration could scavenge ROS induced by blood reperfusion and protect RAW264.7 and HUVEC cells from reperfusion injury. This US-triggered process provides a temporal and spatial trigger point for precise control of long-range transmission of machinery, which helps to create a convenient and non-drug diagnostic and therapeutic strategy.

[0059] In vivo ultrasound imaging to monitor the thrombolytic process and remove reactive oxygen species in real time ( Fig.10 ) The SD rat inferior vena cava thrombosis model was established. The rats were killed 7 hours after the tail vein injection of liposomes ( Fig.10 a). For decades, ultrasound has been the standard diagnostic imaging for deep vein thrombosis. However, it may not work under certain conditions, such as when thrombus formation is in its early stages. As an important supplement to conventional ultrasound, ultrasound excitation of NPU using US can provide better imaging sensitivity. This study evaluated the efficacy of US in vivo ultrasonic vaporization by real-time monitoring of the thrombosis process. After liposome injection into the tail vein, the thrombus area was examined by US at a power of 1.6 W / cm2 for 3 min. Ultrasound imaging was performed before liposome injection, 1 min after ultrasound irradiation, and 15 min after irradiation ( Fig.10 d). Before US excitation, the venous structure was clearly visible in ultrasound b-mode imaging, but the thrombus was difficult to distinguish from the surrounding tissue, and no CEUS signal was detected. After intravenous injection of liposomes and US treatment, the ultrasound b-mode and contrast mode signal intensities of NPU liposomes were significantly enhanced, indicating that NPU liposomes were effectively released into the thrombus and triggered the phase change diagram ( Fig.10 e). There was almost no increase in intensity in the control group compared with NUP liposomes ( Fig.10 c). The signal intensity of NPU in b-mode ultrasound is about 3 times that of the control group, and the signal intensity of CEUS is about 30 times that of the control group ( Fig.10 b).

[0060] The animals were killed 7 hours after US stimulation, and thrombus sections were taken and stained with HE ( Fig.10 f). The in vivo thrombolysis rate of each group was compared (thrombolysis rate % = [initial thrombus area - final thrombus area / initial thrombus area] × 100%). The thrombi in the positive group were all intact. The thrombolysis rates of the UK group, UK+US group, and NPU group decreased by 49±17.2%, 55.33±4.12%, and 60±10.5%, respectively, indicating that the treatment was partially successful. NPU liposome + US caused the greatest damage to the thrombus; the thrombus occupied the smallest area in the entire inferior vena cava section, and the thrombolysis rate was (87±5.01%), indicating that the treatment effect was good ( Fig.10 g). These in vivo measurements confirmed the previous in vitro experimental results and demonstrated that NPU + US is most effective in deep penetration of thrombus and has a good therapeutic effect.

[0061] The production of intracellular ROS after NPU+ US treatment was detected by DHE probe. The ROS produced by negative non-reperfusion was the least among all groups. Once reperfusion occurred, NPU+US could significantly reduce the level of ROS compared with NPU, because US caused NPU to release more CexMnx NPs, the key nanozyme for clearing ROS ( Fig.10 h. Fig.10 i).

[0062] In vivo and in vitro biosafety In order to investigate whether nanomedicines cause unnecessary damage to healthy cells, at the cellular level, we tested the biosafety of CexMnx NPs at different concentration gradients and CexMnx NPs (NPU) modified by liposomes using the CCK8 kit. RAW264.7 and HUVEC cells were treated with CexMnx NPs and NPU at concentrations of 20µg / ml, 40µg / ml, 60µg / ml, 80µg / ml, and 100µg / ml. Although no significant cytotoxicity was detected under the treatment of all groups, the biosafety of NPU formed by liposome encapsulation was improved ( Fig.11 The hemolysis test showed that no significant hemolysis occurred despite the NPU concentration of 150 μg / mL ( Fig.12 ).

Claims

1. An ultrafine nano-thrombolytic microbubble, characterized in that: The invention comprises a core drug and a lipid shell, wherein the core drug comprises an anti-active oxygen material, urokinase and perfluoropentane.

2. The ultramicro-nano thrombolytic microbubble according to claim 1, characterized in that: The anti-active oxygen material is a material including Ce and Mn elements.

3. The ultramicro-nano thrombolytic microbubble according to claim 2, characterized in that: The anti-active oxygen material has CAT enzyme and SOD enzyme activities.

4. The ultramicro-nano thrombolytic microbubble according to claim 2, characterized in that: The preparation method of the anti-active oxygen material is as follows: Ce(NO3)3･6H2O and Mn(NO3)2･6H2O are dissolved in ethanol, and after stirring for a certain period of time, ammonium hydroxide is added and stirred sufficiently, followed by centrifugation, filtration, washing and drying to obtain an anti-active oxygen material.

5. The ultrafine nano-thrombolytic microbubble according to claim 4, characterized in that: The molar ratio of Ce(NO3)3･6H2O to Mn(NO3)2･6H2O is 0.66:

1.

6. The ultramicro-nano thrombolytic microbubble according to claim 1, characterized in that: The mass ratio of the anti-active oxygen material, urokinase and perfluoropentane is 1:4:

8.

7. A method for preparing ultrafine and nanometer thrombolytic microbubbles according to any one of claims 1 to 6, characterized in that: The anti-active oxygen material is first prepared, and then encapsulated with liposomes. After the encapsulation, urokinase is added for hydration, and then perfluoropentane is added for emulsification, and then freeze-dried to obtain the ultrafine nano-thrombolytic microbubbles.

8. Use of the ultrafine nano-thrombolytic microbubbles according to any one of claims 1 to 6 in the preparation of drugs for treating thrombosis.

9. The use of the ultrafine nano-thrombolytic microbubbles in the preparation of a drug for treating thrombosis according to claim 8, characterized in that: The drug for treating thrombosis is a drug for treating obstructive thrombosis.

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