Ternary nano self-assembly particles responding to and regulating arterial thrombosis microenvironment and preparation method thereof

Abstract

The invention discloses a ternary nano self-assembly particle for responding and adjusting an arterial thrombosis microenvironment and a preparation method thereof. The ternary nano self-assembled particle is formed by self-assembling asymmetric zinc phthalocyanine as a template, a natural small molecule compound containing a p-benzoquinone mother nucleus and a flavonoid compound through hydrophobic interaction, pi-pi interaction and electrostatic interaction. The self-assembled nanoparticle provided by the invention can adsorb plasma protein in situ in the circulation of an organism, and a protein crown of various plasma protein components is formed on the surface of the self-assembled nanoparticle; in addition, the system can respond to the shear force which is sharply increased at the arterial thrombus, and passive targeting and fixed-point release are realized; besides, the self-assembled nanoparticles can significantly improve prognosis of carotid thrombotic diseases by inhibiting NF-kappa B signal pathways, inhibiting formation of neutrophil extracellular netting, inhibiting glycolysis, promoting cGMP-mediated signal pathways and regulating arterial thrombosis microenvironments, also have good biological safety, and can be used for preparing the carotid thrombotic disease self-assembled nanoparticles. And a novel nano therapeutic drug is provided for arterial thrombosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a self-assembled ternary nanoparticle that responds to and regulates the microenvironment of arterial thrombosis, and a preparation method and application thereof. Background Art

[0002] Arterial thrombotic events are blockages of arterial vessels due to thrombosis, which are common in coronary arteries and cerebral arteries, causing myocardial infarction and ischemic stroke, respectively. In recent years, with the continued increase in cardiovascular risk factors such as hypertension, high cholesterol, obesity, diabetes, and metabolic syndrome, the incidence of arterial thrombotic events has also increased significantly. Clinical evidence shows that systemic thrombolytic therapy based on plasminogen activators faces the challenges of ineffective recanalization and high recurrence rate.

[0003] Vascular homeostasis plays a vital role in the prognosis of thrombotic diseases. Unlike venous thrombosis caused by blood stasis, arterial thrombosis is closely related to the damage and dysfunction of vascular endothelial cells (ECs). In the microenvironment of arterial thrombosis, ECs dysfunction, fibrinolytic resistance and inflammation often occur simultaneously. Plasminogen activator inhibitor 1 (PAI-1) is the main endogenous inhibitor of plasminogen activators in the body. The content of PAI-1 in arterial thrombosis is 2 to 3 times that in venous thrombosis, which is also the main cause of fibrinolytic resistance in arterial thrombosis. Studies have shown that proinflammatory cytokines released during inflammation, such as IL-1β, IL-6 and TNF-α, can increase the synthesis and secretion of PAI-1. In addition, increasing evidence shows that inflammatory response and PAI-1-dominated fibrinolytic resistance exacerbate the formation of arterial thrombosis. Therefore, inhibiting PAI-1 and excessive inflammatory response may be a potential strategy to synergistically regulate the microenvironment of arterial thrombosis and promote the restoration of vascular homeostasis.

[0004] Natural small molecule compounds have diverse biological activities and are often used to regulate pathophysiological processes in organisms. Anthraquinone is a natural para-benzoquinone compound that can exert anti-inflammatory effects by inhibiting the NF-κB pathway, neutralizing free radicals and maintaining cell homeostasis. Isoquercetin is a flavonoid compound with significant anti-inflammatory effects. Therefore, the rational use of anthraquinone and isoquercetin may be a potential strategy for regulating the microenvironment of arterial thrombosis.

[0005] In addition, using a nanocarrier-based drug delivery system to respond to the microenvironment of arterial thrombosis and increase the release of drugs at the site of arterial thrombosis is another key point in the study of arterial thrombosis. Once the nanoparticles enter the body, their surface will quickly adsorb protein molecules to form a "nanoprotein corona". The presence of the protein corona will affect and regulate the behavior, metabolic process and final fate of the nanoparticles in the body, thereby affecting the biomedical function of the nanodrug. The blood flow rate in the artery is fast, and the blood flow shear force at the local thrombosis site increases dramatically. The shear force of normal arterial blood flow is 10~70 dyn / cm 2 , and the shear force at the vascular stenosis caused by thrombus can reach 1000 dyn / cm 2 As shown above, when the coronary artery is blocked by more than 75%, the local shear force at the thrombus increases by 53 times. The construction of a shear-responsive drug delivery system based on the interaction between nanoparticles and the pathophysiological environment in the body can provide a new strategy from the perspective of pharmacology to solve the clinical demand for the targeted release and local enrichment of thrombotic therapeutic drugs. Summary of the invention

[0006] The purpose of the present invention is to provide a new type of nanomedicine that can simultaneously respond to and regulate the arterial thrombotic microenvironment, which can not only respond to the high shear force of the arterial thrombotic microenvironment, but also has significant anti-inflammatory and anti-PAI-1 effects, and can also effectively improve the prognosis of arterial thrombotic diseases.

[0007] To achieve the above object, the present invention adopts the following technical solution: One of the purposes of the present invention is to protect a ternary nano self-assembled particle that responds to and regulates the microenvironment of arterial thrombosis, which is self-assembled with asymmetric zinc phthalocyanine as a template and a natural small molecule compound of the benzoquinone type and a flavonoid compound that regulates the microenvironment of arterial thrombosis.

[0008] Furthermore, the asymmetric zinc phthalocyanine is easy to self-assemble through π-π stacking, and is preferably pentalysine-2-carbonyl zinc phthalocyanine.

[0009] Furthermore, the benzoquinone natural small molecule compound is preferably anthracene.

[0010] Furthermore, the flavonoid compound is preferably isoquercetin.

[0011] Furthermore, the molar ratio of the asymmetric zinc phthalocyanine to the flavonoid compound and the p-benzoquinone natural small molecule compound is 1:1-2:1-5, preferably 1:1:3-4.

[0012] The second object of the present invention is to protect the preparation method of the ternary nano self-assembled particles, which is prepared by nano coprecipitation method, comprising the following steps: 1) Asymmetric zinc phthalocyanine, a natural small molecule compound containing a p-benzoquinone mother core and a flavonoid compound are prepared into corresponding solutions respectively; 2) mixing the three solutions prepared in step 1) in water evenly, and allowing to stand in a dark environment for self-assembly; 3) After self-assembly is completed, the resulting mixture is centrifuged at high speed to remove free drugs, and the precipitate is collected and resuspended.

[0013] Furthermore, in step 1), dimethyl sulfoxide is used as a solvent to prepare the three solutions.

[0014] Furthermore, the concentrations of the three solutions prepared in step 1) are all 10 mM.

[0015] Furthermore, the volume of water used in step 2) is 66.7 to 166.7 times the total volume of the three solutions after mixing.

[0016] Furthermore, the reaction conditions of the light-shielded self-assembly in step 2) are: temperature 25-40° C., time 15-45 min.

[0017] Furthermore, the high-speed centrifugation in step 3) has a rotation speed of 11000-14500 rpm, a temperature of 4-40°C, and a time of 30-45 min.

[0018] The third purpose of the present invention is to protect the use of the ternary nano self-assembled particles in the preparation of nano drugs for preventing and / or treating thrombotic diseases.

[0019] Furthermore, the thrombotic disease is arterial thrombosis.

[0020] The main mechanisms involved in the present invention are: (1) In normal blood circulation, the self-assembled ternary nanoparticles of the present invention can enter the systemic circulation after being injected through the tail vein, in situ adsorb negatively charged plasma proteins (mainly albumin) in the plasma, and form a protein corona on the surface, thereby improving the stability of the ternary nanoparticles in the blood circulation.

[0021] (2) In the diseased blood circulation, the self-assembled ternary nanoparticles of the present invention can respond to the local surge in shear force of arterial thrombosis and have the characteristics of passive targeting and shear force responsive drug release.

[0022] (3) The self-assembled ternary nanoparticles of the present invention can play a role in treating arterial thrombosis by inhibiting the NF-κB signaling pathway, inhibiting the formation of neutrophil extracellular traps, inhibiting glycolysis, and promoting the cGMP-mediated signaling pathway.

[0023] Through the above technical solution, the beneficial effects that can be achieved by the present invention are: The self-assembled ternary nanoparticles provided by the present invention fully consider the interaction between nanomedicine and the physiological and pathological environment in the body. On the one hand, it can form a protein corona by in situ adsorption of plasma proteins to improve its stability in normal blood circulation, and on the other hand, it can respond to the local high shear force of arterial thrombosis to achieve passive targeting and fixed-point drug release. At the same time, the self-assembled ternary nanoparticles can synergistically regulate the microenvironment of arterial thrombosis, promote the repair of damaged blood vessels, and prevent excessive platelet activation and thrombosis recurrence. In addition, the use of the self-assembled ternary nanoparticles has good tissue compatibility and does not increase the risk of bleeding. Therefore, it is of great significance for the prevention and treatment of arterial thrombosis with a high recurrence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a characterization diagram of the ternary nanoparticles in Example 1, wherein A is the TEM image of the ternary nanoparticles; B is the hydrated particle size of the ternary nanoparticles; and C is the surface zeta potential of the ternary nanoparticles.

[0025] Figure 2 This is a spectrum analysis diagram of the ternary nanoparticles in Example 1, wherein A is the absorption spectrum and B is the fluorescence spectrum.

[0026] Figure 3 The spectral analysis diagrams of the ternary nanoparticles in Example 1 resuspended in 0.05-1% SDS (A, B), the hydrated particle size and PDI resuspended in 0.05-1% SDS (C), and the hydrated particle size and PDI resuspended in 0.5-2 M NaCl solution and 5% glucose solution (D).

[0027] Figure 4 The graph (A) shows the changes in hydrated particle size and surface zeta potential of the ternary nanoparticles after incubation with physiological saline containing 50% plasma in Example 2, the Native-Page gel graph of the protein corona components on the surface of the ternary nanoparticles (B), and the FMT imaging graph of the Native-Page gel (C), wherein ① human plasma albumin, ② 50% human plasma, ③ ternary nanoparticles alone, and ④ samples after incubation of ternary nanoparticles with 50% plasma.

[0028] Figure 5 This is a graph analyzing the release behavior of self-assembled ternary nanoparticles induced by different shear forces under in vitro simulation conditions in Example 3.

[0029] Figure 6 The laser speckle blood flow imaging image of the bilateral carotid arteries of the mouse in Example 3 (A), the in vivo FMT imaging image of the bilateral carotid artery area of ​​the mouse after tail vein injection of ternary nanoparticles (B), and the corresponding concentration of pentalysine-2-carbonylphthalocyanine zinc in the ROI on the left and right sides of the carotid artery area quantitatively calculated by TrueQuant v3.0 software (C).

[0030] Figure 7 The following is a diagram showing the precise release of self-assembled ternary nanoparticles in damaged blood vessels to delay carotid thromboembolism after injection of different substances in Example 3 by real-time monitoring of laser speckle blood flow imaging (LSCI) (A), the real-time change of the relative blood flow rate of the left carotid artery of each mouse (B), and the time required for the blood flow rate of the left carotid artery of each mouse to drop to 10% of the baseline value (C), wherein: n.s. p >0.05, ** p <0.05, *** p <0.001.

[0031] Figure 8 Analysis of the 7-day survival rate of mice with carotid artery thrombosis model in different treatment groups in Example 4 ( * p <0.05, ** p <0.01).

[0032] Fig. 9 This is the differentially expressed genes and cluster analysis in the damaged carotid artery tissues of mice in the normal saline control group and the self-assembled ternary nanoparticle treatment group in Example 5.

[0033] Fig.10 This is the GSEA analysis of the transcriptome of damaged carotid artery tissue in the normal saline control group and the self-assembled ternary nanoparticle treatment group of mice in Example 5, wherein A is the NF-κB signaling pathway; B is the formation of neutrophil extracellular traps (NETs); C is the glycolysis pathway; and D is the cGMP-mediated signaling pathway.

[0034] Fig.11 This is a diagram showing the effect of self-assembled ternary nanoparticles on normal peripheral coagulation function of mice in the tail bleeding experiment of Example 6 ( n.s. p >0.05, * p <0.05, ** p <0.01, *** p <0.001).

[0035] Fig.12 HE staining images of major organ tissues after intravenous injection of self-assembled ternary nanoparticles in Example 6 (scale bar 500 μm). DETAILED DESCRIPTION

[0036] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Preparation of ternary nanoparticles Self-assembled ternary nanoparticles were prepared by nanoprecipitation method, and the specific operation was as follows: first, DMSO mother solutions of pentalysine-2-carbonyl phthalocyanine zinc, isoquercetin and anthracene were prepared respectively, and the concentration was 10 mM; then, a certain amount of the above drug mother solutions were taken and mixed evenly, so that the molar ratio of pentalysine-2-carbonyl phthalocyanine zinc, isoquercetin and anthracene in the mixed solution was 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:2:1, 1:2:2, 1:2:3, 1:2:4, and then ultrapure water with a volume of 66.7 to 166.7 times of the mixed solution was added dropwise under mild stirring conditions, and the mixture was allowed to stand at 25-40°C in the dark for 15-45 min, and then centrifuged at 4-40°C and 11000-14500 rpm for 30-45 min. min to remove free drugs, collect the precipitate and resuspend it in ultrapure water to obtain the ternary nanoparticle solution.

[0039] Example 1 Characterization of ternary nanoparticles 1. Experimental Methods 1.1 Morphology observation Take a piece of carbon support membrane and place it face up on a clean sealing film surface. Use a pipette to draw 10 μL of the prepared ternary nanoparticle solution and drop it onto the carbon support membrane. After standing for 2 min, use filter paper to absorb the liquid on the surface of the carbon support membrane; then add 10 μL of 2% phosphotungstic acid solution, negatively stain for 2 min, absorb the liquid with filter paper, and test after natural drying.

[0040] 1.2 Dynamic light scattering (DLS) analysis When performing hydration particle size detection, 50 μL of the prepared ternary nanoparticle solution was placed in a micro-particle size cup for measurement; when performing surface potential detection, 1 mL of the prepared ternary nanoparticle solution was placed in a potential cup for measurement (ensuring that the solution contacts the electrode sheet).

[0041] 1.3 Drug loading and encapsulation efficiency testing The encapsulation efficiency and drug loading of anthraquinone and isoquercetin were determined and calculated by high performance liquid chromatography (HPLC), where: (1) The HPLC analysis method of anthracene is as follows: the chromatographic column is C 18 Analytical column (4.6×150 mm, 5 μm); mobile phase A (ultrapure water containing 0.1% trifluoroacetic acid): phase B (methanol containing 0.1% trifluoroacetic acid) = 20:80; flow rate 1 mL / min; injection volume 20 μL; detection wavelength 288 nm. The peak position of anthracene is at 4.36 min.

[0042] (2) The HPLC analysis method of isoquercetin is as follows: the chromatographic column is C 18 Analytical column (4.6×150 mm, 5 μm); mobile phase A (ultrapure water containing 0.1% trifluoroacetic acid): phase B (methanol containing 0.1% trifluoroacetic acid) = 50:50; flow rate 1 mL / min; injection volume 20 μL; detection wavelength 360 nm. The peak position of isoquercetin is at 3.32 min.

[0043] (3) Linear relationship investigation and standard curve drawing: Prepare standard solutions of anthracene (3.06~98.00 μg / mL) and isoquercetin (0.60~19.33 μg / mL) in volumetric flasks, inject samples according to the above chromatographic conditions and record the chromatograms. Draw the standard curve with the peak area as the ordinate and the concentration as the abscissa to obtain the regression equation and correlation coefficient. In the range of 3.062~98.000μg / mL, the concentration of anthracene showed a good linear relationship with the peak area, and the regression equation was Y=38.89X+10.41(R 2 =0.9992); in the range of 0.605~19.330 μg / mL, the linear regression equation of isoquercetin concentration and peak area was Y=22.52X-1.160 (R 2 =0.9994). The concentrations of anthracene and isoquercetin in the freeze-dried samples of ternary nanoparticles were calculated according to the regression equation.

[0044] The encapsulation efficiency (EE) and drug loading (LC) were calculated according to the following formula: EE% = W 三元纳米粒冻干样品中的药物量 / W 投药量 ×100%;LC%=W 三元纳米粒冻干样品中的药物量 / W 三元纳米粒冻干样品总质量 ×100%.

[0045] 1.4 Analysis of self-assembly mechanism The ternary nanoparticles obtained by high-speed centrifugation during the preparation process were resuspended in different dispersion media (0.05-1% SDS solution, 0.5-2 M NaCl solution and 5% glucose solution), and DLS and microplate reader were used to detect the changes in hydrated particle size, polydispersity index (PDI), absorption spectrum and fluorescence spectrum.

[0046] 2. Experimental results and conclusions: Using pentalysine-2-carbonyl phthalocyanine zinc as an assembly template, when the molar ratio of pentalysine-2-carbonyl phthalocyanine zinc, isoquercetin and anthracene is 1:1:3-4, the three can self-assemble to form nano-scale particles. Figure 1As shown in the figure, when the molar ratio of the three is 1:1:4, the ternary nanoparticles are spherical and relatively uniform, with a hydrated particle size of 122.4±2.9 nm and a surface zeta potential of 26.9±1.5 mV. After being placed at room temperature for 24 h, the size and distribution of the ternary nanoparticles did not change significantly, and they had good colloidal stability (AC), and subsequent tests were carried out at this ratio.

[0047] The HPLC method was used to determine that the encapsulation efficiency of anthracene and isoquercetin in the ternary nanoparticles was 70.0±2.7% and 11.0±0.9%, and the drug loading was 61.4±2.3% and 3.8±0.4%, respectively.

[0048] like Figure 2 As shown in Figure 2, the maximum absorption wavelength of penta-lysine-2-carbonylphthalocyanine zinc monomer dissolved in DMSO is 678 nm. In PBS, due to the π-π stacking and H-aggregation between macrocyclic molecules, λ max Blue shift to 630 nm. For ternary nanoparticles, the λ max A red shift occurs, indicating that pentalysine-2-carbonyl phthalocyanine zinc undergoes J-aggregation during the co-assembly process with isoquercetin and anthracene (A). Since pentalysine-2-carbonyl phthalocyanine zinc undergoes fluorescence quenching in the H-aggregation state, the fluorescence intensity of pentalysine-2-carbonyl phthalocyanine zinc dissolved in PBS is greatly reduced. It is generally believed that J-aggregation can greatly enhance the fluorescence of fluorescent molecules. However, in the present invention, pentalysine-2-carbonyl phthalocyanine zinc is completely restricted to a non-fluorescent state by co-assembly with isoquercetin and anthracene through J-aggregation (B). This is a rare non-fluorescent J-aggregate, which also allows pentalysine-2-carbonyl phthalocyanine zinc to serve as an indicator of the responsive release of ternary nanoparticles in vitro and in vivo.

[0049] The self-assembly mechanism of ternary nanoparticles was explored by detecting the UV-visible absorption spectra, fluorescence spectra and particle size changes of ternary nanoparticles resuspended in different dispersion media. Figure 3As shown in the figure: With the increase of SDS concentration in the dispersion system, the characteristic absorption peak of penta-lysine-2-carbonylphthalocyanine zinc in the system gradually recovered, and the fluorescence intensity increased after excitation at 610 nm (A, B); the hydrated particle size and polydispersity index (PDI) of the ternary nanoparticles gradually increased (C). This shows that the ternary nanoparticles depolymerized in the hydrophobic buffer, suggesting that hydrophobic forces and π-π interactions drive the self-assembly of the nanoparticles. In addition, different ionic strengths can produce different degrees of charge shielding, affecting the electrostatic interaction between molecules. In 5% glucose solution, the particle size and PDI of the ternary nanoparticles did not change significantly; however, in 0.5 M or 1 M NaCl solution, the particle size of the ternary nanoparticles increased to about 1.2 μm, while the PDI did not change significantly, indicating that the ternary nanoparticles further aggregated at low ion concentrations and formed relatively uniform micron-sized particles; after adding 2 M NaCl, the particle size decreased to about 500 nm, while the PDI increased significantly to 0.5, indicating that the ternary nanoparticles depolymerized at high ion concentrations. This result suggests that electrostatic forces also play a role in the self-assembly process (D).

[0050] Example 2 Characterization of the protein corona formed by in situ adsorption of plasma proteins by ternary nanoparticles 1. Experimental Methods 1.1 In situ formation of protein corona on the surface of ternary nanoparticles The ternary nanoparticles obtained by high-speed centrifugation during the preparation process were resuspended in a physiological saline solution containing 50% normal mouse plasma. After incubation at 37°C for 10 min, the sample solution after incubation of the ternary nanoparticles and 50% plasma was collected, centrifuged at 14,000 rpm, and the precipitate was recovered and resuspended in ultrapure water.

[0051] 1.2 Dynamic light scattering (DLS) analysis When conducting hydrated particle size detection, 50 μL of the sample solution after incubation of ternary nanoparticles with 50% plasma was placed in a micro-particle size cup for measurement; when conducting surface potential detection, 1 mL of the sample solution after incubation of ternary nanoparticles with 50% plasma was placed in a potential cup for measurement (ensuring that the solution contacts the electrode).

[0052] 1.3 Native-PAGE gel electrophoresis and fluorescence molecular tomography (FMT) imaging analysis The sample solution after incubation of ternary nanoparticles with 50% plasma was mixed with Native-PAGE loading buffer and added to the sample well of 12% non-denaturing polyacrylamide gel for electrophoresis (180V, 3 h). The sample was stained with Coomassie Brilliant Blue staining solution and then destained for imaging.

[0053] 2. Experimental results and conclusions: Maintaining the stability of the nanosystem in the blood circulation and avoiding premature drug release are the prerequisites for efficient drug delivery. With the application of nanotechnology in the field of biomedicine, people have gradually realized that the interaction between nanoparticles and biomolecules, especially extracellular proteins, greatly affects the behavior of nanoparticles in vivo.

[0054] like Figure 4 As shown in the figure, when the ternary nanoparticles were incubated with physiological saline containing 50% plasma, the particle size increased from 122.4±2.9 nm to 138.5±3.7 nm, and the zeta potential changed from the initial 26.9±1.5 mV to -28.5±2.0 mV, indicating that plasma proteins may be adsorbed on the surface of the ternary nanoparticles (A). At the same time, albumin is one of the most abundant protein types in plasma. The Native-Page test results showed that the samples after the ternary nanoparticles were incubated with 50% plasma showed protein type bands similar to those in plasma, among which albumin was the most abundant (B). The FMT imaging results showed that the sample loading wells after the ternary nanoparticles were incubated with 50% plasma showed obvious near-infrared fluorescence signals (λ ex = 680 nm), while the single ternary nanoparticle sample was not retained in the gel due to its positive surface charge (C).

[0055] Example 3 Shear stress responsive drug release 1. Experimental Methods 1.1 In vitro simulation of different shear stress-induced drug release An in vitro simulated circulation device was built using a peristaltic pump, a 1.6 mm inner diameter hose, a Luer connector, and an ibidi µ-slide channel. The flow rate and the resulting shear force were controlled by adjusting the pump speed. The release of the ternary nanoparticles after 15 min of circulation in a saline system containing 50% plasma under low shear and high shear conditions was investigated. After the cycle was completed, the unreleased nanoparticles were recovered by high-speed centrifugation for quantification.

[0056] 1.2 Observation of precise drug release of self-assembled ternary nanoparticles in damaged blood vessels by FMT imaging Eight-week-old male ICR mice were selected as experimental animals. After isoflurane anesthesia, the neck skin of the mice was removed with depilatory cream. The carotid artery thrombosis model was established as follows: the common carotid artery (CCA) of the left external carotid artery and the internal carotid artery was stripped about 0.5 cm proximal to the bifurcation of the mouse, and a transparent plastic sheet was placed underneath it. 30 μL of 20% ferric chloride hexahydrate solution was added to stimulate the CCA, and the blood flow rate was observed under a laser speckle blood flow imager until the blood flow rate dropped by about 90%. The ferric chloride solution was aspirated and saline was added to rinse the injured blood vessels. The plastic sheet was removed and the neck skin incision was sutured. The mice were placed on a 37 ℃ warming pad until they were fully awake. Ten minutes later, the ternary nanoparticles were injected into the tail vein. At 0.5 h and 1 h, the mice were placed in the small animal in vivo FMT imager. The neck of the mice was selected as the target area ROIs, and 20-30 source positions were scanned (the distance between adjacent scanning points was set to 2 mm). The fluorescence signal of pentalysine-2-carbonylphthalocyanine zinc was collected using a 680 nm laser light source for excitation, and three-dimensional reconstruction and fluorescence quantification were performed using TrueQuant v3.0 software.

[0057] 1.3 Observation of the precise drug release of self-assembled ternary nanoparticles in damaged blood vessels by laser speckle blood flow imaging (LSCI) to delay CCA thromboembolism Eight-week-old healthy male ICR mice were randomly divided into three groups and injected with normal saline, normal saline solution containing equal amounts of free drugs (anthraquinone treatment dose 2 μmol / kg, isoquercetin treatment dose 80 nmol / kg), and ternary nanoparticles (1 mg / kg) through the tail vein. Ten minutes later, the left CCA of the mice was peeled off and a transparent plastic sheet was placed underneath it. The mice were placed on the LSCI imaging platform, and after adding 30 μL of 20% ferric chloride hexahydrate solution, the blood flow rate at the injury site was immediately monitored in real time by LSCI, and recorded every 5 seconds until the blood flow rate dropped to 10% of the baseline value, and the time required for CCA thrombus occlusion (TTO) was recorded.

[0058] 2. Experimental results and conclusions: like Figure 5 As shown, in an in vitro simulated circulation device, under low shear stress (10 dyn / cm 2 After 15 min of circulation under the condition of 200 dyn / cm2, the cumulative release of the ternary nanoparticles was about 20%, while the release of the nanoprotein corona was significantly reduced under the same conditions. This result shows that the protein corona improves the stability of the ternary nanoparticles in circulation. However, when the shear force is increased to 300 dyn / cm2, the release of the nanoparticles is about 20%. 2 When , the cumulative release amounts of ternary nanoparticles and ternary nanoparticle-protein corona assemblies both increased to nearly 60%.

[0059] like Figure 6 As shown in the figure, in ICR mice, local application of ferric chloride injured the left CCA of the mice, resulting in thrombosis and blocking about 90% of the carotid artery lumen. FMT imaging of the mouse neck showed that the fluorescence intensity of the left CCA at 0.5 h and 1 h was significantly enhanced compared with the contralateral side, indicating that the ternary nanoparticles released drugs at the thrombus site and ZnPc5K made the thrombus visible. The corresponding quantitative results showed that at 0.5 h and 1 h, the concentration of ZnPc5K in the left carotid artery was 22.7 times and 14.4 times that of the contralateral carotid artery, respectively.

[0060] The real-time LSCI blood flow monitoring results were obtained by pre-intravenous injection of ternary nanoparticles and then irradiation of the carotid artery with ferric chloride 10 min later. Figure 7 As shown in the results, compared with the saline solution group and the saline solution group containing the same amount of free drugs, the pre-injection of ternary nanoparticles significantly prolonged the time required for arterial occlusion (TTO). This result strongly proves that shear force can cause the targeted release of nanoparticles in the local carotid artery where thrombus has formed, thereby delaying carotid thromboembolism.

[0061] Example 4 Self-assembled ternary nanoparticles significantly improve the prognosis of arterial thrombosis model mice in vivo A mouse model of carotid artery thrombosis was established according to Item 1.2 of Example 3. The model mice were randomly divided into four groups, and the tail veins were injected with normal saline, alteplase (10 mg / kg), normal saline solution containing equal amounts of free drugs (anthraquinone treatment dose 2 μmol / kg, isoquercetin treatment dose 80 nmol / kg) and ternary nanoparticles (1 mg / kg). Among them, the alteplase treatment group was given a single dose, and the normal saline solution with equal amounts of free drugs and the ternary nanoparticle treatment group were given once a day, for a total of three doses. The survival status of mice in each group was monitored, and the survival curve was drawn 7 days after modeling.

[0062] The results are as follows Figure 8 As shown in the figure, the 7-day survival rate of the model mice treated with normal saline solution was about 50%; the 7-day survival rate of the mice in the normal saline solution group with alteplase and an equal amount of free drug increased to about 70%; the 7-day survival rate of the mice in the self-assembled ternary nanoparticle group was 100%, indicating that the use of self-assembled ternary nanoparticles can significantly improve the survival status of the model mice and increase their survival rate (compared with the normal saline group p <0.01, compared with alteplase and saline solution containing an equal amount of free drug p <0.05), improving the prognosis of mice with carotid artery thrombosis model.

[0063] Example 5 Therapeutic Mechanism of Self-Assembled Ternary Nanoparticles The therapeutic mechanism of self-assembled ternary nanoparticles was analyzed by transcriptome sequencing and bioinformatics. The specific operation was to take the CCA of carotid artery thrombosis model mice after 3 days of treatment with normal saline and self-assembled ternary nanoparticles, extract total RNA from it using TRIzol, and perform DNA digestion using DNaseI. The RNA quality was determined by detecting A260 / A280 with a spectrophotometer, and the RNA integrity was confirmed by 1.5% agarose gel electrophoresis. The qualified RNA was quantified on Qubit 3.0 using a kit. Take 2 μg of total RNA and use the Ribo-off rRNA removal kit and KC TM Stranded mRNA library preparation kit was used to prepare strand-specific RNA sequencing libraries. 200-500 bp sequences were enriched, quantified, and sequenced on a DNBSEQ-T7 sequencer using the PE150 mode. Raw sequencing data were first filtered using Trimmomatic (v 0.36) to remove low-quality reads and trim reads containing adapter sequences. Valid reads were aligned to the human reference genome using STRA software (v 2.5.3a) with default parameters. Read counts were performed using featureCounts (Subread-1.5.1; Bioconductor). Data processing and visualization were performed using the R programming language (v 4.4.0) and related R packages. Differentially expressed genes (DEGs) between the two groups were identified using DESeq2 (v1.44.0) with the criteria of log2fold change > 2 and p < 0.05. Hierarchical clustering analysis of DEGs based on Pearson correlation distance was performed using the pheatmap package. GO and KEGG enrichment analysis based on gene set enrichment analysis (GSEA) was performed using the gseGO and gseKEGG functions in the clusterProfiler package (v 4.12.0), respectively. Mouse genome information was annotated using the org.Mm.eg.db package. Normalized enrichment scores (NES) were used to quantify the degree of enrichment, and gene sets with |NES| > 1 and p < 0.05 were considered statistically significant.

[0064] The results are as follows Fig. 9 As shown, compared with the saline control group, a total of 2965 differentially expressed genes (DEGs) were identified in the injured carotid arteries of mice in the self-assembled ternary nanoparticle treatment group, of which 778 genes were upregulated and 2187 genes were downregulated. Hierarchical clustering analysis of DEGs showed that the transcriptomes of the injured carotid arteries of mice in the saline control group and the self-assembled ternary nanoparticle treatment group were significantly different.

[0065] Subsequently, GO enrichment and KEGG analysis based on GSEA were used to further explore the biological functions and main enriched pathways of these DEGs. Fig.10 As shown, the NF-κB signaling pathway, neutrophil extracellular traps (NETs) formation and glycolysis were significantly downregulated after self-assembled ternary nanoparticles treatment; on the other hand, the cGMP-mediated signaling pathway was significantly upregulated in the self-assembled ternary nanoparticles treatment group.

[0066] Example 6 Self-assembled ternary nanoparticles have good biosafety 1. Experimental Methods 1.1 Mouse tail bleeding experiment The effect of self-assembled ternary nanoparticles on peripheral blood coagulation function was investigated by detecting the changes in the bleeding time and hemoglobin loss of the mouse tail before and after intravenous administration. The specific operation was to establish a carotid artery thrombosis model in 8-week-old male ICR mice and randomly divide them into groups. The tail vein was injected with normal saline, alteplase (10 mg / kg), normal saline solution containing equal amounts of free drugs (anthraquinone treatment dose 2 μmol / kg, isoquercetin treatment dose 80 nmol / kg) and ternary nanoparticles (1 mg / kg), respectively. One hour later, the end of the mouse tail was selected for 75% alcohol disinfection, and it was cut off with a disinfected blade. The mouse tail was immediately immersed in normal saline warmed at 37°C, and the bleeding time was recorded until stable hemostasis for 10 s. The normal saline containing the mouse blood sample was collected, centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, the red blood cell pellet was collected, 2 mL of red blood cell lysis solution was added, and the red blood cells were fully lysed by shaking. Then the sample was taken and the absorbance value of the sample at 575 nm was detected by an ELISA reader.

[0067] 1.2 Histocompatibility analysis The acute and chronic toxicity of self-assembled ternary nanoparticles were investigated by detecting the pathological morphological changes of the main organ tissues of mice before and after intravenous administration. The specific operation for evaluating acute toxicity was to randomly divide 8-week-old healthy male ICR mice into two groups, and inject normal saline and double therapeutic dose of ternary nanoparticles (2 mg / kg) into the tail vein, once a day for 3 days; the specific operation for evaluating chronic toxicity was to randomly divide 8-week-old healthy male ICR mice into two groups, and inject normal saline and therapeutic dose of ternary nanoparticles (1 mg / kg) into the tail vein, once every other day for 30 days. The survival status of mice was monitored, and the brain, heart, liver, spleen, lung, and kidney were taken on the 4th day (for acute toxicity evaluation) and the 31st day (for chronic toxicity evaluation), and paraffin sections and HE staining were performed after fixation to observe the changes in tissue pathology.

[0068] 2. Experimental results and conclusions: The results are as follows Fig.11As shown, in the carotid artery thrombosis model mice, compared with the normal saline group, the tail vein injection of ternary nanoparticles did not significantly change the tail bleeding time and total hemoglobin loss, indicating that the self-assembled ternary nanoparticles had no obvious effect on normal peripheral coagulation.

[0069] The results are as follows Fig.12 As shown, HE staining results showed that compared with the normal control group mice, there were no obvious pathological changes in the main organ tissues such as brain, heart, liver, spleen, lung, and kidney of the mice in the short-term high-dose group and the long-term low-dose group, indicating that the self-assembled ternary nanoparticles have good tissue compatibility.

[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A ternary nano self-assembled particle that responds to and regulates the microenvironment of arterial thrombosis, characterized in that: The ternary nanometer self-assembly particles are self-assembled by using asymmetric zinc phthalocyanine as a template, natural small molecule compounds containing a p-benzoquinone mother core and flavonoid compounds.

2. The ternary nano self-assembled particle according to claim 1, characterized in that: The molar ratio of the asymmetric phthalocyanine zinc to the flavonoid compound and the natural small molecule compound containing a p-benzoquinone mother nucleus is 1:1-2:1-5.

3. The ternary nano self-assembled particle according to claim 1 or 2, characterized in that: The asymmetric zinc phthalocyanine is pentapolylysine-2-carbonyl zinc phthalocyanine.

4. The ternary nano self-assembled particle according to claim 1 or 2, characterized in that: The natural small molecule compound containing a p-benzoquinone mother nucleus is anthracene.

5. The ternary nano self-assembled particle according to claim 1 or 2, characterized in that: The flavonoid compound is isoquercetin.

6. A method for preparing the ternary nano self-assembled particles according to claim 1, characterized in that: The following steps are involved: 1) Asymmetric zinc phthalocyanine, a natural small molecule compound containing a p-benzoquinone mother core and a flavonoid compound are prepared into corresponding solutions respectively; 2) mixing the three solutions prepared in step 1) in water evenly, and allowing to stand in a dark environment for self-assembly; 3) After the self-assembly is completed, the resulting mixture is subjected to high-speed centrifugation to remove free drugs and the precipitate is collected.

7. The method for preparing ternary nano self-assembled particles according to claim 6, characterized in that: In step 1), dimethyl sulfoxide is used as a solvent to prepare three solutions.

8. The method for preparing ternary nano self-assembled particles according to claim 6, characterized in that: The concentrations of the three solutions prepared in step 1) are all 10 mM.

9. The method for preparing ternary nano self-assembled particles according to claim 6, characterized in that: The reaction conditions of the light-shielded self-assembly in step 2) are: temperature 25-40°C, time 15-45 min.

10. Use of the ternary nano self-assembled particles as claimed in claim 1 in the preparation of nano drugs for preventing and / or treating thrombotic diseases.

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