Low-intensity focused ultrasound response nanoparticles for enhancing thrombus targeting and penetration, preparation method of low-intensity focused ultrasound response nanoparticles and application of low-intensity focused ultrasound response nanoparticles in anti-thrombus treatment

Abstract

The invention relates to the technical field of nano-drugs, in particular to low-intensity focused ultrasound response nanoparticles for enhancing thrombus targeting and penetration, a preparation method of the nanoparticles and application of the nanoparticles in anti-thrombus treatment. The low-intensity focused ultrasound response nanoparticle for enhancing thrombus targeting and penetration comprises a platelet membrane shell, and a core is formed by wrapping perfluoropentane and L-arginine with polylactic acid-glycolic acid copolymer-polyethylene glycol. The platelet membrane ensures that the nanoparticles specifically target a thrombus part; under the stimulation of LIFU, the nanobubbles penetrate through the deep layer of thrombus, L-arginine is oxidized into L-citrulline, and nitric oxide is formed. Meanwhile, the LIFU triggers liquid-gas phase change and nitric oxide release, the ultrasonic cavitation effect is enhanced through the synergistic effect, thrombus structures including red blood cells, blood platelets and fibrin are specifically attacked, and efficient thrombolysis treatment is achieved. According to the technical scheme, the technical problem that the thrombolysis effect of ultrasonic nanoparticles is not ideal can be solved, and good clinical application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to low-intensity focused ultrasound responsive nanoparticles capable of enhancing thrombus targeting and penetration, a preparation method thereof, and an application thereof in anti-thrombotic treatment. Background Art

[0002] Thrombotic diseases such as acute myocardial infarction, ischemic stroke, and pulmonary embolism are caused by thrombosis and thromboembolism, posing a major threat to human life and health. At present, thrombolytic therapy is mainly divided into two stages: the early use of thrombolytic drugs to restore vascular patency and the later use of anticoagulants to prevent thrombosis. However, due to the short half-life and poor targeting of thrombolytic drugs, frequent high-dose systemic administration in a short period of time is required, resulting in a serious risk of bleeding side effects. Achieving safe and effective antithrombotic therapy remains a daunting challenge in the management of thrombotic diseases.

[0003] In recent years, ultrasound thrombolysis has become an alternative technology for the treatment of thrombotic diseases due to its non-invasiveness and high penetration depth. Clinical trials have shown that ultrasound combined with local thrombolytic therapy can reduce the incidence of complications in patients with venous occlusive disease. However, in the absence of thrombolytic drugs, local ultrasound energy alone may not be sufficient to significantly reduce the weight of thrombus. Fortunately, due to the enhanced cavitation effect of microbubbles under ultrasound stimulation, they have been widely used to synergistically enhance the ultrasound thrombolytic potential. As a non-invasive ultrasound treatment method, low-intensity focused ultrasound (LIFU) mainly uses periodic vibration waves to induce vibration, expansion, and collapse of microbubbles in the sound field, produce mechanical effects, and promote the physical dissolution of thrombi, which has considerable potential for clinical application.

[0004] However, most microbubbles remain on the surface of the thrombus, and small nanoparticles can more easily penetrate the thrombus. Perfluoropentane (PFP) is an inert organic compound with good biocompatibility and can be emulsified into smaller nanoparticles. And PFP-based nanoparticles can respond to LIFU radiation, produce a strong cavitation effect, and exert the characteristics of microbubbles through liquid-gas phase transition, thereby producing a mechanical burst effect for thrombolytic therapy. It is worrying that such LIFU-responsive nanoparticles may cause harmful effects when they nonspecifically destroy the thrombus structure. In addition to PFP, nitric oxide (NO) also has the potential to be used in gas-mediated thrombolysis. However, due to the short half-life of nitric oxide and its limited diffusion distance, the delivery of NO in vivo remains a major challenge.

[0005] Therefore, for diseases caused by thrombosis and thromboembolism, how to improve the targeting of LIFU-responsive nanoparticles and the ability of nanoparticles to deliver nitric oxide and enhance the thrombolytic effect of nanoparticles are technical issues that need to be solved urgently. Summary of the invention

[0006] The present invention is intended to provide a low-intensity focused ultrasound responsive nanoparticle that enhances thrombus targeting and penetration, so as to solve the technical problem that the thrombolytic effect of ultrasound nanoparticles used to treat diseases caused by thrombus is not ideal.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A low-intensity focused ultrasound responsive nanoparticle for enhancing thrombus targeting and penetration comprises a core and a platelet membrane shell, wherein the core is formed by poly(lactic-co-glycolic acid)-polyethylene glycol encapsulating perfluoropentane and L-arginine.

[0009] The technical principle and beneficial effects of adopting the above technical solution are:

[0010] Safe and effective antithrombotic therapy remains challenging due to the short half-life, limited targeting ability and high risk of bleeding of thrombolytic drugs. Effective treatment requires precise targeting, thrombus penetration and non-invasive destruction of thrombus structure. In this technical solution, an air core containing perfluoropentane (PFP) and L-arginine (L-Arg) is encapsulated in a polylactic-co-glycolic acid (PLGA) shell coated with a platelet membrane (PM) to form nanobubbles (PAF@M) to specifically target damaged vascular endothelial cells. More specifically, in view of the key role of platelets in thrombosis, this solution uses PM-coated nanoparticles to achieve the purpose of thrombus targeting and prolonged blood flow circulation. Perfluoropentane achieves a certain therapeutic effect after low-intensity focused ultrasound (LIFU) irradiation. On this basis, this solution further supplements the gas supply (nitric oxide, NO), enhances the cavitation effect, shortens the treatment time, and achieves a more effective therapeutic effect. Nanobubbles specifically target the thrombus site, and under the optimal spatial and temporal stimulation of low-intensity focused ultrasound, the nanobubbles penetrate the deep layer of the thrombus. Subsequently, perfluoropentane undergoes a liquid-gas phase transition, and L-arginine releases nitric oxide under low-intensity focused ultrasound stimulation to synergistically enhance the ultrasonic cavitation effect and achieve efficient thrombolytic therapy. The low-intensity focused ultrasound-responsive nanobubbles specifically attack thrombotic structures, including red blood cells, platelets, and fibrin. Both in vivo carotid artery thrombosis and black tail models showed good thrombolytic effects and significantly delayed the progression of thrombosis. In summary, these low-intensity focused ultrasound-responsive nanobubbles showed specific accumulation and deep penetration at the thrombus site as well as safe and efficient thrombolytic effects. This approach provides a promising prospect for the non-drug treatment of thrombotic diseases.

[0011] The technical solution also provides a method for preparing low-intensity focused ultrasound responsive nanoparticles that enhance thrombus targeting and penetration, comprising the following steps performed in sequence:

[0012] S1: dissolving poly(lactic acid-co-glycolic acid)-polyethylene glycol, perfluoropentane and polyvinyl alcohol in dichloromethane, then adding L-arginine solution, and obtaining a first emulsion after ultrasonic treatment;

[0013] S2: After adding polyvinyl alcohol to the first emulsion, ultrasonic treatment is performed to obtain a second emulsion;

[0014] S3: adding isopropanol to the second emulsion, stirring until the dichloromethane evaporates, and separating to obtain PAF nanoparticles;

[0015] S4: The solution containing PAF nanoparticles and platelet membranes are mixed, and the mixture is subjected to ultrasonic treatment and extrusion by a liposome extruder to obtain PAF@M nanoparticles.

[0016] Furthermore, in S1, the dosage ratio of polylactic acid-co-glycolic acid-polyethylene glycol, perfluoropentane, polyvinyl alcohol solution, L-arginine solution and dichloromethane is 10 mg: 100 μL: 100 μL: 200 μL: 2 mL; the concentration of L-arginine solution is 10 mg / mL, and the concentration of polyvinyl alcohol solution is 4%.

[0017] Furthermore, the volume ratio of the polyvinyl alcohol solution used in S1 and S2 was 100 μL:3 mL.

[0018] Furthermore, in S1, the parameters of the ultrasonic treatment are set as: power 78 W, time 3 min; in S2, the parameters of the ultrasonic treatment are set as: power 65 W, time 3 min.

[0019] Further, in S4, the parameters of the ultrasonic treatment were set as follows: power 65 W, time 2 min; the volume ratio of the solution containing PAF nanoparticles and the platelet membrane was 1:1, and the concentration of the solution containing PAF nanoparticles was 1 mg / mL.

[0020] Further, the amount of isopropanol used in step S3 is 5 times that of dichloromethane in step S1; the concentration of isopropanol is 2%.

[0021] The technical principle and beneficial effects of the above technical solution (preparation method) are:

[0022] L-arginine is a natural source of nitric oxide produced in the human body by nitrous oxide synthase deguanidine. L-arginine can be oxidized to L-citrulline under ultrasonic irradiation and release nitric oxide. Using L-arginine as a nitric oxide supplement source and applying ultrasound to control the directional release of nitric oxide in the lesion site is a safe and effective gas supplement strategy, which has the effect of enhancing ultrasonic cavitation to specifically destroy thrombus structure. This technical solution adds L-arginine to the nanoparticles as an alternative delivery agent for nitric oxide. This component is one of the keys to achieving the technical effect of this solution. How to ensure the effective load of this substance on the nanodrug presentation system is a key problem that this technology needs to solve.

[0023] In this technical solution, polyvinyl alcohol is used to increase the loading effect of nanoparticles on L-arginine, and the dosage ratio of polyvinyl alcohol and L-arginine is very critical for improving the drug loading effect of L-arginine. L-arginine is a small molecule compound. Generally speaking, in the process of preparing nanoparticles, polyethanol does not have obvious interaction with small molecule compounds. However, under the special application environment of this solution and the conditions of the composition of nanoparticle materials, adding polyvinyl alcohol in a specific way has a significant effect on improving the loading effect of L-arginine, which exceeds the inventor's expectations.

[0024] The technical solution also provides an application of low-intensity focused ultrasound-responsive nanoparticles that enhance thrombus targeting and penetration in the preparation of a system for treating thrombus.

[0025] Furthermore, the system includes the aforementioned PAF@M nanoparticles and a LIFU device; the PAF@M nanoparticles are used at the thrombus site, and the LIFU device is used to apply low-intensity focused ultrasound irradiation to the PAF@M nanoparticles located at the thrombus site.

[0026] Furthermore, the low-intensity focused ultrasound parameter was 1 W / cm 2 ,1MHz,50% duty cycle.

[0027] The technical principle and beneficial effects of adopting the above technical solution are:

[0028] Nanobubbles specifically target the thrombus site due to the characteristics of the platelet membrane. Under optimal temporal and spatial conditions, the physical shear stress triggered by low-intensity focused ultrasound helps the nanobubbles penetrate into the deep layer of the thrombus. Subsequently, perfluoropentane undergoes a liquid-gas phase transition, and L-arginine releases nitric oxide, which synergistically enhances the ultrasonic cavitation effect, produces local burst microflows, and attacks red blood cells, fibrin, and activated platelets within the thrombus structure. In summary, these LIFU-responsive spatiotemporal catalytic nanobubbles show site-specific accumulation and deep penetration at the thrombus site, and have a safe and efficient anti-thrombotic effect. This approach offers good prospects for non-drug treatment of thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The characterization experimental results of the nanobubbles of Example 1 of the present invention (a: TEM image of PAF; b: TEM image of PAF@M; c: TEM image of PAF@M after LIFU irradiation; d: sizes of PAF, PAF@M and PAF@M+LIFU; e: Zeta potential of PAF, PAF@M and PAF@M+LIFU; f: protein blot analysis of PAF, PM and PAF@M; g: F-NMR spectra of PFP and PAF@M; h: optical microscope images of LIFU responsiveness of PAF@M nanobubbles at different times; n=3, *: P<0.05, **: P<0.01, ***: P<0.001).

[0030] Figure 2 The results of the cell uptake, platelet adhesion and NO detection experiments of Example 2 of the present invention (a: CLSM image of RAW264.7 cells incubated with nanobubbles; b: quantitative analysis of fluorescence intensity of a; c: CLSM image of activated HUVECs incubated with nanobubbles; d: quantitative analysis of fluorescence intensity of c; e: CLSM image of activated platelets incubated with nanobubbles; f: flow cytometry analysis of platelet targeting ability; g: CLSM image of platelets treated with nanobubbles binding to HUVECs; h: CLSM image of NO level (DAFFM DA) in HUVECs; i: quantitative analysis of fluorescence intensity of NO; ​​n=3, *: P<0.05, **: P<0.01, ****: P<0.0001).

[0031] Figure 3 The results of in vitro and in vivo thrombus targeting and penetration experiments of Example 3 of the present invention (a: fluorescence images and fluorescence intensity quantitative analysis of artificial blood clots; b: frozen sections of blood clots after different treatments; c: fluorescence images of carotid thrombi at different times; d: fluorescence intensity quantitative analysis of carotid thrombi; e: fluorescence images of blood samples at different times; f: changes in fluorescence intensity in blood samples; g: frozen sections of carotid thrombi; h: fluorescence intensity quantitative analysis of frozen sections of carotid thrombi; n=3, *: P<0.05, **: P<0.01, ***: P<0.001).

[0032] Figure 4 These are the experimental study results of the in vitro thrombolytic effect of Example 4 of the present invention (a: supernatant and digital photographs of blood clots after different treatments; b: absorbance of hemoglobin in the supernatant; c: absorbance of fibrin in the supernatant; d: change in weight of blood clots after different treatments; e: H&E staining of blood clots after different treatments; f: SEM images of blood clots after different treatments; n=3).

[0033] Figure 5 These are electron microscope images of the damage of red blood cells, fibrin and platelets in Example 5 of the present invention (a: SEM images of red blood cells after different treatments; b: CLSM images of fibrin network structure; c: SEM images of platelets after different treatments).

[0034] Figure 6 The experimental results of the thrombolytic effect of carotid thrombosis in Example 6 of the present invention (a: schematic diagram of the treatment of carotid thrombosis; b: photograph of the thrombosis model; c: MRA imaging of the thrombus site after treatment; d: H&E staining of the carotid thrombus on the 4th day after treatment; e: thrombus clearance rate, n=4; f: tail bleeding time, n=3).

[0035] Figure 7 These are the in vivo biosafety evaluation results of Example 6 of the present invention (a: H&E staining images of the heart, liver, spleen, lung, and kidney of different groups; B: ALT, AST, UREA, and CREA levels of SD rats in each group; c: H&E staining of carotid artery thrombosis on the 10th day after PAF@M+LIFU treatment, n=3).

[0036] Figure 8 The experimental results of thrombolytic effect in the black tail model of Example 6 of the present invention (a: schematic diagram of black tail thrombosis treatment; b: changes in black tail disease after different treatments; c: loss of black tail length of mice after different treatments; d: number of cases of tail amputation; e: H&E staining of black tail, n=3).

[0037] Fig. 9 Electron micrograph of the nanoparticles obtained after increasing the amount of platelet membrane. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0039] Embodiment 1:

[0040] (1) Materials, cells, and animals

[0041] Poly(lactic acid-co-glycolic acid)-polyethylene glycol 2000 (PLGA-PEG2000-COOH, lactic acid: glycolic acid 50:50) was purchased from Jinan Daigang Biomaterial Co., Ltd. (Shandong). Perfluoropentane (PFP) and L-arginine (L-Arg) were purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Dichloromethane (CH2Cl2), polyvinyl alcohol (PVA) and isopropanol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China. DiI, DiR, DAPI, DCFH-DA and DAF-FM DA were purchased from Shanghai Bio-Technology Co., Ltd., China.

[0042] RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) were provided by Chongqing Medical University. The SD rats (8-10 weeks old) used in this study were provided by the Experimental Animal Center of Chongqing Medical University, and all animal experiments were approved by the Animal Ethics Committee and Animal Protection Agency of Chongqing Medical University.

[0043] (2) Preparation of PAF@M

[0044] Gather fresh whole blood of SD rats, centrifuge to obtain the plasma of platelet-rich organisms by conventional means of prior art, add EDTA and prostaglandin E1 to prevent platelet activation. The above solution is centrifuged to obtain platelets, then frozen and thawed at-80°C. This process is repeated three times to obtain platelet membrane (PM). More specifically, gather fresh whole blood of SD rats, 800g is centrifuged for 20min, and the upper layer is taken to obtain platelet-rich plasma. Then, EDTA and prostaglandin E1 are added to prevent platelet activation, wherein, the addition (final concentration in platelet-rich plasma) of EDTA and prostaglandin E1 is respectively 0.2mM EDTA, 1mM prostaglandin E1. Then, the above solution 100g is centrifuged for 20min, and the lower layer is taken to obtain platelets, then frozen and thawed at-80°C, and after repeated freezing and thawing three times, platelet membrane (PM) is obtained.

[0045] The preparation method of PLGA-PFP-L-Arg (PAF) is as follows: 10 mg PLGA-PEG2000-COOH, 100 μL PFP and 100 μL PVA (in solution state, its concentration is 4%) are completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) is added, and the mixture is ultrasonically emulsified in an ice-water bath (about 0-4°C) for 3 min (preferably: ultrasonic power 130 W×60%) using an ultrasonic probe (sonic Materials, Inc., USA). 3 mL PVA is added to the above emulsified solution, and ultrasonically emulsified again in an ice-water bath for 3 min (preferably: ultrasonic power 130 W×50%). Then, 10 ml 2% isopropanol is added to the solution, mechanically stirred for 3 h, and CH2Cl2 is volatilized. The solution is centrifuged to obtain PAF. Subsequently, the platelet membrane PM obtained according to the above method was mixed with the PAF suspension (PAF concentration was 1 mg / mL) in a volume ratio of 1:1, ultrasonically treated for 2 min (preferably: ultrasonic power 130 W×50%), and finally extruded with a liposome extruder (preferably, extrusion times 50 times, pore size 300 nm) to obtain PLGA-L-Arg-PFP@PM (PAF@M).

[0046] The material dosage is summarized as follows: the dosage ratio of polylactic acid-co-glycolic acid-polyethylene glycol, perfluoropentane, 4% polyvinyl alcohol solution (first addition), 10 mg / mL L-arginine solution, dichloromethane, 4% polyvinyl alcohol solution (second addition) and 2% isopropanol is 10 mg: 100 μL: 100 μL: 200 μL: 2 mL: 3 mL: 10 mL.

[0047] The volume ratio of platelet membrane PM to PAF suspension was maintained at 1:1, which can effectively improve the encapsulation effect of platelet membrane on PAF, improve the utilization rate of platelet membrane, and reduce background contamination under electron microscope observation. The inventors tried to adjust the ratio of platelet membrane and PAF in PAF@M. The volume ratio of platelet membrane PM to PAF suspension was maintained at 2:1 (see Fig. 9 ) or 3:1, the electron microscope image shows that there are many platelet membrane impurities in the background, and the background is dirty, which is not conducive to the effective observation and detection of nanoparticles.

[0048] The preparation process of PLGA-PFP (PF) is the same as that of PAF, except that L-Arg solution is not added to wrap the platelet cell membrane to obtain PA@M.

[0049] (3) Characterization of PAF@M

[0050] The LIFU (1 W / cm 2 , 1MHz, 50% duty cycle) after 30min stimulation of PAF, PAF@M and PAF@M+LIFU. Particle size, particle size stability, polydispersity index (PDI) and surface zeta potential were characterized by dynamic light scattering (DLS, Zetasizer Nano 1090, UK). PM coating was confirmed by SDS-PAGE and Western blot analysis. The chemical shifts of the characteristic protons of PFP and PAF@M were verified by fluorine nuclear magnetic resonance (F-NMR, Bruker 600MHzAVANCE III, Germany). According to the literature, the loading efficiency of L-Arg in PAF was determined by 1-naphthol-diacetyl colorimetry. Briefly, 3 mL of L-Arg samples of different masses (1.25, 2.5, 5, 10, and 20 mg) were added to 0.5 mL of 14 wt% NaOH, 1 mL of n-propanol, and 1.5 mL of a standard colorimetric solution consisting of α-naphthol (50 mg / mL) and diacetyl (0.25 μL / mL). Then, the absorbance of the mixture was measured by UV-visible spectrophotometry after 15 min at 30 ° C, and a standard curve was established to calculate the amount of L-Arg in the PAF supernatant and the loading efficiency of L-Arg. The phase transition ability of PAF after LIFU stimulation of different durations (5, 10, 15, 20, 25, and 30 min) was observed using an optical microscope (Olympus DP 70, Canada). The nanobubbles were then imaged by ultrasound after 25 and 30 min of LIFU irradiation.

[0051] TEM shows that PAF nanobubbles are spherical and uniform in size ( Figure 1 a). PAF@M nanobubbles were synthesized by modifying PAF with PM. TEM images show the characteristic core-shell structure with a uniform outer membrane ( Figure 1 b). After PM modification, the average size of PAF@M increased from 196.7±.04nm to 226.4±9.32nm (Figure Id), and the surface ζ potential changed from -5.93±0.54mV to -7.73±0.23mV ( Figure 1 e), confirming that PM was successfully coated on PAF nanobubbles. In addition, PAF and PAF@M nanobubbles have good PDI, and the size does not change much within 9 days, indicating that the nanobubbles have good stability. After LIFU irradiation for 30 min, TEM shows obvious debris around PAF@M nanobubbles ( Figure 1 c). In addition, the particle size of PAF@M nanobubbles after LIFU irradiation was similar to that of PAF nanobubbles, and a significant decrease in zeta potential and a significant increase in PDI were observed ( Figure 1 e), which may be attributed to the release of contents and membrane fragmentation after the collapse of PAF@M nanobubbles.

[0052] CD47 is a self-recognition protein on PM that can escape macrophage phagocytosis. CD41 and CD61 together form GPIIb / IIIa, which plays a key role in platelet adhesion and activation. Western blotting and SDS-PAGE were performed to analyze the specific protein markers and total protein content of PAF@M nanobubbles. The results showed that major protein markers, including CD47, CD41, and CD61, were present on both PAF@M and PM, and the protein profile of PAF@M was similar to that of PM ( Figure 1 f). F-NMR spectrum shows that PAF@M has characteristic chemical peaks of PFP, indicating the successful loading of PFP ( Figure 1 g). According to the standard curve, the L-Arg loading efficiency of PAF was 17.95%.

[0053] Subsequently, the LIFU responsiveness of PAF@M nanobubbles was directly observed by optical microscopy. Figure 1 As shown in h, the diameter of the nanobubbles increases with the increase of LIFU irradiation duration (1 W / cm 2 , 1MHz and 50% duty cycle). After 25min of LIFU exposure, the nanobubbles underwent liquid-gas phase transition and expanded in volume, and gradually ruptured after 30min. The gas supply capacity of PAF@M nanobubbles was compared with that of PF@M nanobubbles. Ultrasound imaging was performed after 25min of LIFU irradiation. In both B-ultrasound and CEUS modes, the ultrasonic signals of PAF@M nanobubbles were significantly stronger than those of PF@M nanobubbles, indicating that L-Arg loading in nanobubbles enhanced the gas supply. After 30min of LIFU irradiation, the nanobubbles collapsed and ruptured due to cavitation, resulting in a significant decrease in the ultrasonic signal. The results show that PAF@M nanobubbles have a strong ultrasonic cavitation effect, which lays the foundation for mechanical thrombolysis.

[0054] This technical solution is the first to encapsulate L-Arg in nanoparticles with specific structures and components of this solution (PLGA is used as the shell membrane, and PLGA is used to encapsulate PFP and L-Arg in the same step). L-Arg is a key functional ingredient. How to effectively improve the encapsulation rate of L-Arg is the key to achieving an increase in NO carrying capacity. In the process of preparing nanoparticles, the following method is adopted: PLGA-PEG2000-COOH, PFP and PVA are completely dissolved in CH2Cl2, L-Arg (10 mg / mL) is added, and ultrasonic emulsification is performed. PVA is then added to the above emulsified solution and then emulsified. Among them, adding a small amount of PVA (100 μl) before the first emulsification and adding a large amount of PVA (3 mL) after the first emulsification are very critical to improving the encapsulation rate of L-Arg. Using this technical solution, the L-Arg loading efficiency in PAF can reach 17.95%.

[0055] Generally speaking, when using PLGA to encapsulate small molecule compounds, it is usually not necessary to add PVA during the first emulsification process. PVA has no significant effect on the encapsulation effect of small molecule drugs. In the research of other projects, the inventors tried to use PLGA to encapsulate other small molecule drugs (for example, paclitaxel). For paclitaxel, a similar method to this scheme was used to prepare drug-loaded nanoparticles, and the problem of the use of PVA affecting the drug loading efficiency did not occur. That is, referring to the preparation method of PAF in the previous "(2) Preparation of PAF@M", only L-Arg was replaced with paclitaxel in equal amounts, and PVA was added or not before the first emulsification step for comparative experiments. The results showed that the target drug loading of the nanoparticles obtained was almost the same when PVA was added or not before the first emulsification step (the encapsulation rate with or without PVA was less than 1%, which determined that the loading efficiency and encapsulation rate of paclitaxel were basically the same). Although L-Arg is also a small molecule drug, it presents a completely different pattern, for example:

[0056] 10 mg PLGA-PEG2000-COOH and 100 μL PFP were completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) was added, and the mixture was ultrasonicated in an ice-water bath for 3 min. 3 mL PVA was added to the above emulsified solution and emulsified again in an ice-water bath for 3 min. That is, PVA was not added before the first emulsification, and other operation methods were the same as the best method (refer to the above "(2) Preparation of PAF@M"). Since L-Arg is a small molecule drug, the inventor initially believed that PVA had little effect on the loading effect of L-Arg. In order to save operating steps and introduce as few non-therapeutic ingredients as possible, PVA was not added before the first emulsification. The L-Arg loading efficiency in the PAF obtained by this method was only 5%, and the L-Arg loading efficiency decreased by about 13%.

[0057] The above experimental results show that L-Arg is different from other small molecule drugs. In the nanoparticle composition environment of this scheme, the addition of PVA before the first step of emulsification plays a key role in improving its loading effect. Based on the common knowledge of those skilled in the art, there are reports in the prior art that attempt to use PVA to perform surface functional modification on iron-containing nanoparticles (e.g., ferroferric oxide, Prussian blue), thereby improving the dispersion performance and stability of iron-containing nanoparticles. However, iron-containing nanoparticles and small molecule compounds are two completely different substances, and the latter are not in granular form and there is no condition for surface modification. Experiments have also shown that for ordinary small molecule drugs (such as paclitaxel), PVA has no significant effect on improving the loading effect of drugs. Therefore, it is difficult for those skilled in the art to think of applying PVA to the preparation of L-Arg nanoparticles to increase the loading effect of L-Arg.

[0058] In order to independently study how to improve the loading effect of L-Arg, the inventors eliminated the influence of PFP and conducted the following experiments:

[0059] Test A: 10 mg PLGA-PEG2000-COOH was completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) was added, and the mixture was ultrasonicated in an ice-water bath for 3 min. 3 mL PVA was added to the emulsified solution, and the mixture was emulsified again in an ice-water bath for 3 min. That is, PVA and PFP were not added before the first emulsification, and the other operation methods were the same as the best method (refer to the above "(2) Preparation of PAF@M").

[0060] Test B: 10 mg PLGA-PEG2000-COOH and 100 μL PVA were completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) was added, and the mixture was ultrasonicated in an ice-water bath for 3 min. 3 mL PVA was added to the emulsified solution, and the mixture was emulsified again in an ice-water bath for 3 min. That is, PVA was added before the first emulsification, but PFP was not added, and the other operation methods were the same as the best method (refer to the above "(2) Preparation of PAF@M").

[0061] After testing, the loading efficiency of L-Arg in the nanoparticles of test A and test B (the amount of target drug loaded into the nanoparticles / the total amount of target drug × 100%) is basically the same (the difference in the loading efficiency of test A and test B is within 1%, so it is determined that the loading efficiency of the two methods is consistent), and the loading efficiency is relatively ideal. This shows that in the absence of PFP, there is no significant problem with the efficiency of using PLGA to encapsulate L-Arg, and it is not affected by whether PVA is added before the first step of emulsification. In this case (PLGA encapsulates nanoparticles formed by L-Arg, in the absence of PFP), PVA cannot play a role in improving the loading effect of L-Arg. The main purpose of this technical solution is to add a new gas source L-Arg on the basis of the original PFP gas source, thereby increasing the ultrasonic cavitation effect under the action of LIFU, and achieving a synergistic effect. The addition of two gas sources is the key point to realize this solution. However, in the actual operation process, it was found that the joint loading of PFP and L-Arg affected the loading efficiency of the second gas source L-Arg. This is the first time that the inventors have encountered such a technical problem, and they finally solved the problem by adding a small amount of PVA before the first step of emulsification. That is, adding a small amount of PVA before the first step of emulsification increases the loading amount of L-Arg in the PLGA nanoparticles containing both PFP and L-Arg. The effect of PVA can only be realized under specific nanoparticle composition conditions (PLGA encapsulates PFP and L-Arg). The above effect was not predicted by the inventors before the actual test.

[0062] The inventors also conducted research on the addition mode and amount of PVA, as follows:

[0063] Test C: 10 mg PLGA-PEG2000-COOH, 100 μL PFP, 3 mL PVA were completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) was added, and the mixture was ultrasonicated in an ice-water bath for 3 min. After a short interval, the mixture was emulsified again in an ice-water bath for 3 min. That is, a large amount of PVA was added before the first emulsification, and no PVA was added after emulsification. The other operation methods were the same as the best method. The L-Arg loading efficiency in the PAF obtained by this method was only 8%.

[0064] Test D: 10 mg PLGA-PEG2000-COOH, 100 μL PFP, 1.5 mL PVA were completely dissolved in 2 mL CH2Cl2, 200 μL L-Arg (10 mg / mL) was added, and the mixture was ultrasonicated in an ice-water bath for 3 min. 1.5 mL PVA was added to the above emulsified solution and emulsified again in an ice-water bath for 3 min. That is, an equal amount of PVA was added before and after the first emulsification, and the other operation methods were the same as the best method. The L-Arg loading efficiency in the PAF obtained by this method was only close to 13%.

[0065] The above operation methods will reduce the encapsulation effect of L-Arg in nanoparticles. Under the same dosage, the drug loading of L-Arg will be reduced, which will affect the drug delivery effect of the nano drug delivery system. Due to the reduction of the loading of the active ingredient, the therapeutic effect of the overall nanoparticles will also be reduced. In order to ensure the most ideal drug loading effect, PVA needs to be added twice, with a smaller amount added the first time and a larger amount added the second time (the ratio of the two additions is 100μl:3mL, and the amount of L-Arg is 200μL, 10mg / mL remains unchanged, that is, 2mg).

[0066] In addition to the above attempts, the inventors have also conducted a large number of studies on the ratio of the amount of PVA added twice. For example: the amount of PVA added for the first and second times is 100μl / 5mL, or 100μl / 10mL, or 300μl / 3mL, or 25μl / 1.25mL, or 25μl / 3mL; the rest of the operation methods are the same as the aforementioned nanoparticle preparation method ("(2) Preparation of PAF@M", in which the amount of L-Arg is 200μL, 10mg / mL, unchanged). The L-Arg loading efficiency in the PAF obtained by detection is difficult to exceed 13%; even when the first PVA addition is too little (25μl / 1.25mL, 25μl / 3mL), the L-Arg loading efficiency is difficult to exceed 8%. It further illustrates that the method and specific ratio of adding PVA for the first and second times are very critical to improving the L-Arg loading efficiency (encapsulation rate) of nanoparticles. The optimal choice is L-Arg: PVA added for the first time: PVA added for the second time = 2 mg: 100 μL: 3 mL.

[0067] It can be seen from the above experimental results that encapsulating L-Arg in the nanoparticles of this solution (PLGA encapsulates PFP and L-Arg at the same time) and ensuring a good encapsulation rate has certain technical difficulties. The key to solving the above technical problems lies in the addition of PVA twice, and the first addition amount and the second addition amount are maintained at about 100μL:3mL (L-arginine: PVA added for the first time: PVA added for the second time = 2mg: 100μL: 3mL), and emulsification treatment is performed separately after addition. Such a technical effect has not been reported in the prior art, is the result of the invention's experimental attempts based on specific circumstances, and is also one of the key technical points for realizing this technical solution.

[0068] Example 2: Cellular uptake, platelet adhesion and NO detection

[0069] (1) Experimental methods

[0070] Cells and animals: RAW264.7 cells and human umbilical vein endothelial cells (HUVECs) were provided by Chongqing Medical University. SD rats (8-10 weeks old) used in this study were provided by the Experimental Animal Center of Chongqing Medical University.

[0071] Cytotoxicity: PAF@M nanobubbles at different concentrations (12.5, 25, 50, 100, 200, and 400 μg / mL) were incubated with HUVECs for 24 h, and the absorbance was measured at 450 nm using a CCK-8 kit.

[0072] Cell uptake: RAW264.7 cells (5×10 4 ) and incubated with DiI-labeled PAF or PAF@M (200 μg / mL) for 2 h, then the cell nuclei were stained with DAPI and observed by confocal laser scanning microscopy (CLSM, Nikon A1, Japan).

[0073] Cell adhesion: HUVEC (5×10 4 ) and treated with TNF-α (50 ng / mL) for 24 h. Subsequently, fresh culture medium containing DiI-labeled PAF or PAF@M was added and incubated for 2 h. The cell nuclei were stained with DAPI and observed using CLSM.

[0074] Platelet adhesion: The collected platelets were mixed with 8×10 -9 M CaCl2 and 2U / mL thrombin in PBS were activated for 30 min. Then, the platelets were incubated with DiI-labeled PAF and PAF@M for 2 h and analyzed by CLSM and flow cytometry. 4) and treated with TNF-α (50 ng / mL) for 24 h. Then, calcein-AM-labeled platelets were added and activated for 0.5 h. Subsequently, DiI-labeled PAF or PAF@M were added and incubated for 4 h. The cell nuclei were stained with DAPI, and the samples were observed by CLSM.

[0075] NO detection: For in vitro NO release, 1 mL of PAF@M solution was added to 1 mL of PBS and then LIFU (1 W / cm 2 , 1MHz, 50% duty cycle) for 30min. 50uL samples were collected at different time points (0, 1, 3, 5, 10, 15, 20, 25 and 30min) during irradiation. After treatment with Griess reagent, the absorbance of each sample was measured at 540nm, and a standard curve of NaNO2 was prepared, from which the NO concentration was calculated. For intracellular NO production, HUVEC (5×10 4 ), incubated with PAF@M (200 μg / mL) for 2 h, and stimulated with LIFU for 10 min (1 W / cm 2 , 1 MHz, 50% duty cycle). DAF-FM-DA probe was used to detect intracellular NO, and DAPI was added for nuclear staining.

[0076] (2) Experimental results

[0077] Cellular uptake: For cell experiments, we used CCK-8 to evaluate the cytotoxicity of PAF@M nanobubbles. No obvious cellular abnormalities were observed after HUVECs were incubated with PAF@M for 24 h. Platelet membrane protein CD47, a "don't eat me" signal, can prevent PAF@M nanobubbles from being recognized by the mononuclear phagocyte system. Figure 2 As shown in Figures 2a and 2b, RAW264.7 cells incubated with DiI-labeled PAF@M for 2 h showed significantly lower uptake than cells incubated with PAF alone. For activated HUVECs, PAF@M exhibited greater adhesion than PAF due to the natural targeting properties of PM ( Figure 2 c, d).

[0078] Platelet adhesion: The specific binding of PAF@M to activated platelets was evaluated. As shown in the CLSM images ( Figure 2 e), compared with PAF, activated platelets showed greater fluorescence intensity after incubation with DiI-labeled PAF@M for 2 h, and PAF@M nanobubbles significantly surrounded platelets. Flow cytometry data ( Figure 2f) shows a similar trend to the CLSM data. Finally, HUVECs were co-incubated with platelets to simulate thrombosis in the vascular microenvironment. After 4 h, PAF@M showed more obvious red fluorescence and stronger colocalization with platelet-bound HUVECs compared with PAF ( Figure 2 g). These results indicate that PAF@M nanobubbles can effectively evade the surveillance and clearance of the immune system while targeting damaged endothelial cells and platelets, providing a solid basis for enhanced thrombus targeting in vivo.

[0079] NO detection: NO is a highly reactive chemical with a short diffusion radius (40-200 μm). The delivery of NO to the thrombus site by LIFU triggering can greatly improve the safety and efficiency of gas-mediated thrombolysis. Therefore, we investigated the ability of nanobubbles to release NO in vitro by using Griess reagent as an indicator. According to the standard curve of NO detection, the concentration of NO released by PAF@M increased with the increase of LIFU irradiation time. This is attributed to the oxidation of L-Arg to L-citrulline and the release of NO by LIFU irradiation. We further investigated the intracellular NO release ability of PAF@M. After incubation of PAF@M nanobubbles with HUVECs, no fluorescence signal was observed. However, after LIFU irradiation, strong green fluorescence was detected in HUVECs co-treated with PAF@M nanobubbles, indicating that LIFU can serve as a trigger for NO production ( Figure 2 h, i). In conclusion, PAF@M has the potential to be used as a gas supplement source to enhance ultrasonic cavitation.

[0080] Example 3: Thrombus Targeting and Penetration In Vitro and In Vivo

[0081] (1) Experimental methods

[0082] In vitro targeting and penetration ability: Fresh whole blood was collected from SD rats and incubated at room temperature to prepare blood clots. Next, the blood clots were incubated with DiR-labeled PAF or PAF@M (2 mg / mL) for 2 h and irradiated with LIFU for 30 min. Subsequently, the blood clots were observed using an in vivo imaging system (IVIS), and frozen sections were prepared for observation by fluorescence microscopy.

[0083] In vivo targeting distribution and circulation time: SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The left carotid artery was exposed, and a 3mm filter paper was soaked with 10% FeCl3 solution and fixed on the surface of the blood vessel for 10 minutes to establish a thrombosis model. After modeling, DiR-labeled PAF or PAF@M (2mg / mL, 1ml) were injected respectively. At different times after injection (0.25, 0.5, 1, 2 and 4h), the thrombosis site of the left carotid artery was removed and imaged by IVIS. In addition, fresh blood was collected from the tail at different times after injection (0.5, 1, 2, 4, 8, 12 and 24h), and the blood samples were imaged by IVIS to evaluate the circulation time of the nanobubbles in vivo.

[0084] In vivo targeting and penetration ability: SD rats were treated with DiR-labeled PAF or PAF@M after modeling. One hour later, the thrombus site of SD rats in the PAF@M group was irradiated with LIFU for 30 minutes, and the left carotid artery was frozen and stained with DAPI and observed under a fluorescence microscope.

[0085] (2) Experimental results

[0086] In vitro thrombus targeting and penetration: Artificial blood clots were prepared to evaluate the in vitro thrombus targeting ability. Fluorescence images showed that the fluorescence signal in the blood clots incubated with DiR-labeled PAF@M was stronger than that in the blood clots incubated with PAF alone. In addition, the blood clots treated with PAF@M and irradiated with LIFU showed significantly enhanced fluorescence signals ( Figure 3 a). Subsequently, the blood clots were prepared into frozen sections and observed under a fluorescence microscope. Deeper thrombus penetration was observed in the PAF@M+LIFU group compared with the PAF@M group and the PAF alone group ( Figure 3 b), which is attributed to the good targeting ability of PAF@M and the further deep penetration of nanobubbles promoted by the mechanical force of LIFU.

[0087] In vitro thrombus targeting and penetration experiments: To confirm the in vivo thrombus targeting ability of nanobubbles, we established a FeCl3-induced left carotid artery thrombosis model and explored the optimal time for nanobubbles to target the thrombus site. After nanobubbles were administered to rats for different durations, the fluorescence images of the ex vivo blood vessels showed that the fluorescence signal of the PAF@M group was significantly stronger than that of the PAF group, and the fluorescence signal was strongest about 1 hour after injection, and then gradually weakened over time ( Figure 3 c, d). Blood samples were collected from the tail for fluorescence imaging to evaluate the blood circulation time of nanobubbles in vivo. The fluorescence signal in the blood samples decreased over time. The fluorescence signal of the PAF@M group was significantly stronger than that of the PAF group. This result may be due to the fact that the platelet membrane-modified nanobubbles (PAF@M) escaped the phagocytosis of the endothelial reticular system, thereby prolonging the blood circulation time ( Figure 3e, f).

[0088] According to the optimal targeting time of PAF@M nanobubbles, DiR-labeled PAF@M was injected via the tail vein. One hour later, LIFU was applied to the thrombus site for 30 min, and then the left carotid artery was removed for frozen section preparation. Fluorescence microscopy showed that the fluorescence signal of the PAF@M group was stronger than that of the PAF group, which was attributed to the natural chemotaxis of PM. Due to the mechanical effect of LIFU, the fluorescence intensity of the PAF@M+LIFU group was significantly higher than that of the PAF@M group. These results highlight the important role of PM and LIFU in enhancing thrombus targeting and penetration ( Figure 3 g, h).

[0089] Example 4: In vitro thrombolysis

[0090] In vitro thrombolytic therapy: Fresh whole blood from SD rats was collected and incubated at room temperature to prepare thrombi. The blood clots were then treated with PBS, LIFU, PF (2 mg / mL) + LIFU, PAF (2 mg / mL) + LIFU, or PAF@M (2 mg / mL) + LIFU. The LIFU parameters were as follows: 1 W / cm 2 , 1 MHz, 50% duty cycle, 30 min. After treatment, the blood clots were weighed, and the absorbance of hemoglobin and fibrin in the supernatant was measured at 540 nm and 405 nm, respectively. Finally, the blood clots were stained with hematoxylin-eosin (H&E) and observed under a scanning electron microscope (SEM).

[0091] Based on the LIFU responsiveness and targeted penetration ability of nanobubbles, the in vitro thrombolytic effect of nanobubbles was evaluated. Figure 4 As shown in a, after LIFU, PF+LIFU, PAF+LIFU or PAF@M+LIFU treatment, the supernatant of the solution gradually became turbid, and the contents of hemoglobin and fibrin gradually increased ( Figure 4 b, c). As the nanobubbles underwent liquid-gas phase transition, many bubbles were observed in the supernatant, and the PAF+LIFU group had significantly more bubbles than the PF+LIFU group, indicating that the production of NO increased the gas supply ( Figure 4 a). Compared with the PBS, LIFU, PF+LIFU and PAF+LIFU groups, the reduction in clot weight in the PAF@M+LIFU group was more significant ( Figure 4 d). In addition, some small holes were observed on the blood clot surface in the PAF@M+LIFU group, and the clot structure collapsed significantly ( Figure 4 a).

[0092] H&E staining of the remaining blood clots showed that the thrombus structure remained intact after PBS treatment, but many cracks were observed within the thrombus after LIFU treatment. It is worth noting that the thrombus structure became loose after PF+LIFU treatment, but the thrombus was destroyed and formed large pieces after PAF+LIFU treatment. The degree of destruction in the PF+LIFU group was less than that in the PAF+LIFU group, indicating that the generated NO further enhanced the cavitation effect induced by LIFU. In addition, after treatment with PAF@M+LIFU, only a small amount of thrombus fragments were left due to the accumulation of more nanobubbles around the blood clot ( Figure 4 e). The SEM image of the blood clot is consistent with the H&E staining results and more directly shows the fibrin rupture and collapse of the thrombus structure after PAF@M+LIFU treatment ( Figure 4 f). The above results indicate that PAF@M nanobubbles exhibit excellent thrombus targeting and penetration capabilities after LIFU irradiation, along with enhanced thrombus dissolving ability due to the ultrasonic cavitation effect of phase transition and NO generation.

[0093] Example 5: Damage to red blood cells, fibrin and platelets

[0094] (1) Experimental methods

[0095] Hemolysis analysis: Fresh whole blood from SD rats was centrifuged at 3000 rpm for 5 min to obtain red blood cells. The red blood cell pellet was diluted 10 times with PBS and treated with double distilled water, PBS, L-Arg, PF, PAF or PAF@M for 4 h, and then centrifuged at 3000 rpm for 10 min to obtain the supernatant. Finally, the absorbance at 540 nm was measured to evaluate the blood safety of the nanobubbles.

[0096] Erythrocyte injury: Erythrocytes were treated with PBS, PAF@M (200 μg / ml), LIFU (10 min), or PAF@M+LIFU (0.5, 1, 3, 5, and 10 min). Next, the erythrocytes were centrifuged and observed by SEM.

[0097] Fibrinogen injury: Fibrinogen networks were prepared by incubating FITC-labeled fibrinogen with thrombin (25 U / mL) and CaCl2 (2.5 mM) for 2 h. The networks were then treated with PBS, PAF@M (200 ug / ml), LIFU (10 min), or PAF@M+LIFU (0.5, 1, 3, 5, and 10 min). The fibrin networks were then washed with PBS and observed by CLSM.

[0098] Platelet injury: Platelets were treated with PBS, PAF@M (200ug / ml), LIFU (10min), thrombin (1U / mL, 30min), or PAF@M+LIFU (1, 3, 5, and 10min). 2 LIFU was applied at 100 MHz, 1 MHz and 50% duty cycle. Then, the platelets were centrifuged and observed by SEM.

[0099] (2) Experimental results

[0100] Red blood cell damage

[0101] Based on the excellent thrombolytic effect of LIFU-responsive nanobubbles in vitro, we further explored the effect of nanobubbles on thrombus structure at the cellular level. We evaluated the blood compatibility of nanobubbles by incubating them with red blood cells, and the hemolysis rate of PAF@M nanobubbles was less than 5%, indicating no hemolysis risk in vivo. The red blood cells were then placed in PAF@M solution. Scanning electron microscopy showed that the biconcave disc morphology of red blood cells in PAF@M solution was the same as that in the PBS group, and no abnormal red blood cells were observed. However, in the LIFU group, red blood cells showed protrusions and a jagged appearance, which was mainly caused by the mechanical effect of LIFU. When red blood cells were placed in PAF@M solution and treated with LIFU for 0.5min, concave holes began to appear on the surface of red blood cells. After 3 minutes of LIFU irradiation, the number of concave holes gradually increased, which may be due to the mechanical action that caused the PAF@M nanobubbles to expand and release energy, inducing sonopores on the surface of red blood cells and increasing their fragility. After 5min of LIFU irradiation, some red blood cells began to fragment, and the fragility of red blood cells further increased. When the irradiation time was increased to 10 min, the energy released by the PAF@M nanobubbles was significantly enhanced, resulting in only a few red blood cells remaining intact and many cell fragments were observed by SEM ( Figure 5 a), which visually demonstrates the specific mechanical effect of LIFU-responsive PAF@M nanobubbles on RBCs in thrombus structures.

[0102] Fibrin damage

[0103] CLSM images showed regular and dense network structures in the PBS and PAF@M groups. However, after 10 min of LIFU irradiation, the fibrin network structure was partially destroyed and shrunk, and many small fragments aggregated. After 0.5 min of PAF@M and LIFU treatment, only a small number of small fragments were observed, but these fragments became significantly larger after 1 min. Pores began to appear in the fibrin network structure after 3 min of PAF@M+LIFU treatment, and these pores became more obvious after 5 min of irradiation. With continuous LIFU irradiation, the pores of the network structure became significantly larger, and the structure loosened and broke after 10 min ( Figure 5b) These results intuitively indicate that PAF@M nanobubbles can destroy fibrin and loosen its structure by perforating it under LIFU treatment.

[0104] Platelet damage

[0105] Scanning electron microscopy showed that the platelets in the PBS group were round and smooth. In addition, no obvious abnormalities were observed when the platelets were incubated with PAF@M, indicating the biosafety of the nanobubbles. When the platelets were irradiated with LIFU for 10 minutes, the platelet surface became relatively wrinkled, but no obvious pseudopodia were observed, indicating that the LIFU parameters we used did not cause platelet activation. Then, after 30 minutes of thrombin stimulation, the platelets were activated and pseudopodia appeared. After PAF@M treatment and LIFU irradiation, as the irradiation time increased, the platelet pseudopodia elongated and entangled tightly, and many micropores gradually appeared on the platelet surface. After 5 to 10 minutes, the number of micropores increased, the platelet structure showed more severe rupture, and there were fewer activated platelets ( Figure 5 c) These results indicate that LIFU-responsive PAF@M nanobubbles can promote the aggregation of activated platelets and mechanically destroy activated platelets within a limited range.

[0106] In summary, the above results directly indicate that PAF@M nanobubbles have excellent thrombolytic effect by specifically and comprehensively destroying the thrombus structure.

[0107] Example 6: In vivo experimental study

[0108] (1) Experimental methods

[0109] A carotid artery thrombosis model was established and the modeling process was photographed. After modeling, the cells were treated with PBS, LIFU, PF+LIFU, PAF+LIFU or PAF@M+LIFU. Nanoparticle concentration: 2 mg / ml; LIFU parameter: 1 W / cm 2 , 1MHz, duty cycle 50%, 30min. After 3d of treatment, magnetic resonance angiography (MRA) was performed once a day to examine vascular recanalization, and the animals were killed on the 4th day for H&E staining. Serum was also collected, and blood biochemical analysis was performed by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine (CREA) levels to evaluate liver and kidney function. Heart, liver, spleen, lung, and kidney were obtained for H&E staining. In addition, in order to observe the long-term effect of thrombolytic therapy and thrombosis recurrence, the left carotid artery of SD rats was obtained for H&E staining on the 10th day after PAF@M+LIFU treatment.

[0110] After starvation for 8 hours, Kunming mice were intraperitoneally injected with carrageenan (20 mg / kg), and the black-tailed mouse model was successfully established 24 hours later. After modeling, PBS, LIFU, PF+LIFU, PAF+LIFU, and PAF@M+LIFU were used for treatment. The LIFU parameters were the same as before. The changes in the tail were observed on the 4th day after treatment, and H&E staining was performed.

[0111] Tail bleeding experiment: 6-8 week old Kunming mice were treated with PBS, L-Arg, PF, PAF or PAF@M. After 2 hours, the mice were anesthetized, and a 1 cm fragment was cut from the end of the tail and soaked in PBS. Next, the hemostasis time of the tail was observed and recorded.

[0112] (2) Experimental results

[0113] Thrombolytic therapy and safety in carotid artery thrombosis

[0114] Based on the above studies on targeted penetration and excellent antithrombotic effect in blood clots, the antithrombotic effect of PAF@M nanobubbles was evaluated in a SD rat model of FeCl3-induced carotid artery thrombosis ( Figure 6 a). Figure 6 As shown in b, the color of the blood vessels deepened after thrombosis, and thrombosis in the lumen led to vascular embolism. The model rats were treated with different doses of LIFU, including PBS, LIFU, PF+LIFU, PAF+LIFU, and PAF@M+LIFU. The LIFU parameters were 1W / cm2, 1MHz, 50% duty cycle, and 30min. In healthy rats, the blood flow in the bilateral carotid arteries was stable. After thrombosis, there was no blood flow signal in the left carotid artery of the PBS group. However, the left carotid artery of the LIFU group and the PF+LIFU group also did not show obvious blood flow signals, which may be due to the low vascular patency, slow blood flow velocity, and limited sensitivity of MRA in collecting blood flow signals ( Figure 6 c). It is exciting that in the PAF+LIFU group, better thrombolysis resulted in lower vascular embolism and improved vascular imaging; however, stenosis caused by residual thrombus was still visible in the vessel lumen. In the PAF@M+LIFU group, the blood flow signal was more obvious, and the vessel lumen was only mildly narrowed. This imaging provides a safe and effective method to evaluate vascular recanalization without the use of contrast agents.

[0115] Then, the SD rats were killed and the carotid arteries were taken for H&E staining. The blood vessels in the PBS group were completely blocked. The overall thrombus area in the LIFU group did not decrease significantly, but the internal structure was loose, indicating that the thrombolytic effect of mechanical force alone still needs to be improved. After PF+LIFU treatment, the thrombus edge was slightly dissolved, while the thrombus area in the PAF+LIFU group was reduced by about 44.23%, indicating that the LIFU-induced phase change and L-Arg-mediated NO release can synergistically enhance the thrombolytic effect. In addition, due to its targeting ability, the thrombus area in the PAF@M+LIFU group was reduced by 68.18%, showing an excellent thrombolytic effect ( Figure 6 d, e). In addition, tail bleeding experiments showed that nanobubble treatment did not increase the risk of bleeding ( Figure 6 f)

[0116] After the above treatments, organ toxicity was evaluated by H&E staining. No tissue damage or pathological changes were detected in the heart, liver, spleen, lung, or kidney after PAF@M+LIFU treatment ( Figure 7 a), indicating that PAF@M nanobubbles have good tissue biocompatibility. In addition, hematological indices were used to evaluate liver and kidney function. After PAF@M+LIFU treatment, ALT, AST, UREA, and CREA levels were not significantly different from those in the PBS group ( Figure 7 b), indicating that PAF@M nanobubbles do not damage liver or kidney function. Considering the risk of thrombosis recurrence after short-term thrombolytic therapy, we performed H&E staining of carotid artery thrombi on day 10 after PAF@M+LIFU treatment ( Figure 7 c), the results showed no significant increase in thrombus size (n=4).

[0117] Thrombolytic therapy in the blacktail model

[0118] The Kunming mouse blacktail model was established by intraperitoneal injection to visually observe the progression of thrombosis ( Figure 8 a). Figure 8 As shown in 8b and 8c, the length of the black tail changed significantly before and after treatment. Both the PAF+LIFU group and the PAF@M+LIFU group could significantly inhibit thrombosis, and the treatment effect of the PAF+LIFU group was better than that of the PF+LIFU group, which was due to the gas-enhanced cavitation effect. In addition, during the treatment period, the number of cases of tail amputation due to ischemic necrosis was significantly reduced ( Figure 8 d). After treatment, the tail was taken for H&E staining ( Figure 8 e), the results showed that the treatment effect was Figure 8 b. PAF@M nanobubbles significantly inhibited thrombosis and delayed the progression of thrombosis under mechanical force-driven gas therapy. These results indicate that PAF@M nanobubbles have excellent in vivo thrombolytic effect under LIFU irradiation.

[0119] In summary, this technical solution successfully developed a LIFU-responsive PAF@M nanobubble for enhancing the depth of thrombus targeting and achieving efficient and precise thrombolytic therapy. LIFU not only promotes the penetration of nanobubbles, but also triggers the liquid-gas phase transition of nanobubbles. In addition, nanobubbles produce NO, which enhances the ultrasonic cavitation effect and improves the therapeutic effect. At the cellular level, nanobubbles destroy fibrin by drilling to loosen the thrombus structure, especially destroying red blood cells and activated platelets. This LIFU-responsive PAF@M nanobubble comprehensively attacks the thrombus structure and achieves efficient thrombolytic therapy without the risk of bleeding. It is a safe and effective non-drug alternative therapy for the treatment of thrombotic diseases and has good clinical application prospects.

[0120] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A low-intensity focused ultrasound-responsive nanoparticle for enhancing thrombus targeting and penetration, characterized in that: The invention comprises a core and a platelet membrane shell. The core is formed by poly(lactic acid-co-glycolic acid)-polyethylene glycol encapsulating perfluoropentane and L-arginine.

2. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 1, characterized in that: The method includes the following steps in sequence: S1: dissolving poly(lactic acid-co-glycolic acid)-polyethylene glycol, perfluoropentane and polyvinyl alcohol in dichloromethane, then adding L-arginine solution, and obtaining a first emulsion after ultrasonic treatment; S2: After adding polyvinyl alcohol to the first emulsion, ultrasonic treatment is performed to obtain a second emulsion; S3: adding isopropanol to the second emulsion, stirring until the dichloromethane evaporates, and separating to obtain PAF nanoparticles; S4: The solution containing PAF nanoparticles and platelet membranes are mixed, and the mixture is subjected to ultrasonic treatment and extrusion by a liposome extruder to obtain PAF@M nanoparticles.

3. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 2, characterized in that: In S1, the dosage ratio of poly(lactic-co-glycolic acid)-poly(ethylene glycol), perfluoropentane, poly(vinyl alcohol) solution, L-arginine solution and dichloromethane is 10 mg: 100 μL: 100 μL: 200 μL: 2 mL; The concentration of the L-arginine solution was 10 mg / mL, and the concentration of the polyvinyl alcohol solution was 4%.

4. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 2, characterized in that: The volume ratio of the polyvinyl alcohol solution used in S1 and S2 was 100 μL:3 mL.

5. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 2, characterized in that: In S1, the parameters of the ultrasonic treatment were set as: power 78 W, time 3 min; in S2, the parameters of the ultrasonic treatment were set as: power 65 W, time 3 min.

6. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 5, characterized in that: In S4, the parameters of the ultrasonic treatment were set as follows: power 65 W, time 2 min; the volume ratio of the solution containing PAF nanoparticles and the platelet membrane was 1:1, and the concentration of the solution containing PAF nanoparticles was 1 mg / mL.

7. The method for preparing low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 6, characterized in that: The amount of isopropanol used in step S3 is 5 times that of dichloromethane in step S1; the concentration of isopropanol is 2%.

8. Use of the low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 1 in the preparation of a system for treating thrombus.

9. The use of low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 8 in the preparation of a system for treating thrombus, characterized in that: The system includes PAF@M nanoparticles prepared by the preparation method described in any one of claims 2-6, and also includes a LIFU device; the PAF@M nanoparticles are used at the thrombus site, and the LIFU device is used to apply low-intensity focused ultrasound irradiation to the PAF@M nanoparticles located at the thrombus site.

10. The use of low-intensity focused ultrasound responsive nanoparticles for enhancing thrombus targeting and penetration according to claim 9 in preparing a system for treating thrombus, characterized in that: Low-intensity focused ultrasound parameters were 1 W / cm 2 ,1MHz,50% duty cycle.

Images