Ternary nano self-assembled particles responsive to and regulating the arterial thrombus microenvironment and preparation method thereof

Abstract

The present invention discloses a ternary nano self-assembly particle that responds to and regulates the microenvironment of arterial thrombosis and a preparation method thereof. The ternary nano self-assembly particle uses asymmetric zinc phthalocyanine as a template and is self-assembled with a natural small molecule compound containing a para-benzoquinone mother core and a flavonoid compound through hydrophobic interaction, π-π interaction, and electrostatic interaction. The self-assembly nanoparticles provided by the present invention can adsorb plasma proteins in situ in the circulation of the organism and form a protein corona of multiple plasma protein components on the surface; and it can respond to the shear force that increases sharply at the arterial thrombosis site to achieve passive targeting and fixed-point release; in addition, the self-assembly nanoparticles can regulate the arterial thrombosis microenvironment by inhibiting the NF-κB signaling pathway, inhibiting the formation of neutrophil extracellular traps, inhibiting glycolysis, and promoting the cGMP-mediated signaling pathway, thereby significantly improving the prognosis of carotid thrombotic diseases. It also has good biosafety and provides a new nano-therapeutic drug 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 arterial blockages caused by thrombosis, commonly occurring in the coronary and cerebral arteries, leading to 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 indicates that systemic thrombolytic therapy based on plasminogen activators faces challenges with ineffective recanalization and high recurrence rates.

[0003] Vascular homeostasis plays a crucial role in the prognosis of thrombotic diseases. Unlike venous thrombosis caused by blood stasis, arterial thrombosis is closely associated with damage and dysfunction of vascular endothelial cells (ECs). Within the arterial thrombotic microenvironment, EC dysfunction, fibrinolytic resistance, and inflammation often occur simultaneously. Plasminogen activator inhibitor 1 (PAI-1) is the major endogenous inhibitor of plasminogen activators in vivo. The content of PAI-1 in arterial thrombi is two to three times that in venous thrombi, which is the main cause of fibrinolytic resistance in arterial thrombi. 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. Furthermore, increasing evidence indicates that inflammation and PAI-1-driven fibrinolytic resistance exacerbate arterial thrombosis. Therefore, inhibiting PAI-1 and excessive inflammation may be a potential strategy to synergistically regulate the arterial thrombotic microenvironment and promote the restoration of vascular homeostasis.

[0004] Natural small molecule compounds possess diverse bioactivities and are often used to modulate pathophysiological processes in vivo. Anthraquinone, a natural para-benzoquinone compound, exerts anti-inflammatory effects by inhibiting the NF-κB pathway, neutralizing free radicals, and maintaining cellular homeostasis. Isoquercetin is a flavonoid compound with significant anti-inflammatory properties. Therefore, the rational utilization of anthraquinone and isoquercetin may represent a potential strategy for modulating the arterial thrombotic microenvironment.

[0005] In addition, another key point in the study of arterial thrombosis is to use a drug delivery system based on nanocarriers to respond to the microenvironment of arterial thrombosis and increase the release of drugs at the site of arterial thrombosis. Once the nanoparticles enter the organism, 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 ultimate fate of the nanoparticles in the organism, thereby affecting the biomedical function of the nanomedicine. 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 thrombosis 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 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 pharmaceutical preparation to address the clinical need of 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. It 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 solutions:

[0008] 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. The ternary nano self-assembled particle is based on asymmetric zinc phthalocyanine as a template and is self-assembled with a natural small molecule compound of the p-benzoquinone type and a flavonoid compound that regulates the microenvironment of arterial thrombosis.

[0009] Furthermore, the asymmetric zinc phthalocyanine is easy to self-assemble through π-π stacking, and is preferably pentapolylysine-2-carbonyl zinc phthalocyanine.

[0010] Furthermore, the benzoquinone natural small molecule compound is preferably anthracene.

[0011] Furthermore, the flavonoid compound is preferably isoquercetin.

[0012] 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.

[0013] The second object of the present invention is to provide a method for preparing the ternary nano self-assembled particles, which is prepared by a nano coprecipitation method and comprises the following steps:

[0014] 1) Prepare corresponding solutions of asymmetric zinc phthalocyanine, a natural small molecule compound containing a p-benzoquinone core, and a flavonoid compound;

[0015] 2) mixing the three solutions prepared in step 1) in water, and allowing to stand in the dark for self-assembly;

[0016] 3) After self-assembly is complete, the resulting mixture is centrifuged at high speed to remove free drugs, and the precipitate is collected and resuspended.

[0017] Furthermore, in step 1), dimethyl sulfoxide is used as a solvent to prepare the three solutions.

[0018] Furthermore, the concentrations of the three solutions prepared in step 1) are all 10 mM.

[0019] 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.

[0020] Furthermore, the reaction conditions for the light-shielded self-assembly in step 2) are: temperature 25-40° C., time 15-45 min.

[0021] Furthermore, the high-speed centrifugation in step 3) is performed at a speed of 11,000-14,500 rpm, a temperature of 4-40° C., and a time of 30-45 min.

[0022] The third purpose of the present invention is to protect the use of the ternary nano self-assembled particles in the preparation of nanomedicines for preventing and / or treating thrombotic diseases.

[0023] Furthermore, the thrombotic disease is arterial thrombosis.

[0024] The main mechanisms involved in the present invention are:

[0025] (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.

[0026] (2) In the pathological 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.

[0027] (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.

[0028] Through the above technical solution, the present invention can achieve the following beneficial effects:

[0029] The self-assembled ternary nanoparticles provided by the present invention fully consider the interaction between nanomedicines and the physiological and pathological environment in the body. On the one hand, they can form a protein corona by in situ adsorption of plasma proteins, improving their stability in normal blood circulation. On the other hand, they 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 arterial thrombosis microenvironment, 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, they are of great significance for the prevention and treatment of arterial thrombosis with a high recurrence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 3 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.

[0031] Figure 2 Spectral analysis diagram of the ternary nanoparticles in Example 1, wherein A is the absorption spectrum and B is the fluorescence spectrum.

[0032] Figure 3 The spectral analysis graphs 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).

[0033] Figure 4 Figure 2 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 (A), a Native-Page gel image of the protein corona components on the surface of the ternary nanoparticles (B), and an FMT imaging image 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.

[0034] 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.

[0035] Figure 6The following are the laser speckle blood flow images of the bilateral carotid arteries of the mouse in Example 3 (A), the in vivo FMT images of the bilateral carotid artery areas of the mouse after tail vein injection of ternary nanoparticles (B), and the corresponding concentrations of pentalysine-2-carbonylphthalocyanine zinc in the ROIs on the left and right sides of the carotid artery area quantitatively calculated by TrueQuant v3.0 software (C).

[0036] Figure 7 Figure 3 shows the precise release of self-assembled ternary nanoparticles in damaged blood vessels to delay carotid artery thromboembolism after injection of different substances using real-time monitoring of laser speckle blood flow imaging (LSCI) after injection in Example 3 (A), the real-time changes in the relative blood flow velocity of the left carotid artery of each mouse (B), and the time required for the blood flow velocity of the left carotid artery of each mouse to drop to 10% of the baseline value (C). n.s. p >0.05, ** p <0.05, *** p <0.001.

[0037] 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).

[0038] Figure 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.

[0039] Figure 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, where 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.

[0040] Figure 11 This is a diagram showing the effect of self-assembled ternary nanoparticles on normal peripheral coagulation function in mice in the tail bleeding experiment of Example 6 ( n.s. p >0.05, * p <0.05, ** p <0.01, *** p <0.001).

[0041] Figure 12HE staining images of major organ tissues after intravenous injection of self-assembled ternary nanoparticles in Example 6 (scale bar 500 μm). DETAILED DESCRIPTION

[0042] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] Preparation of ternary nanoparticles

[0045] 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, with the concentration of each being 10 mM; then, a certain amount of the above-mentioned drug mother solutions were taken and mixed evenly, so that the molar ratios of pentalysine-2-carbonyl phthalocyanine zinc, isoquercetin and anthracene in the mixed solution were 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:2:1, 1:2:2, 1:2:3 and 1:2:4; then, ultrapure water (66.7 to 166.7 times the volume of the mixed solution) was added dropwise under gentle 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.

[0046] Example 1 Characterization of ternary nanoparticles

[0047] 1. Experimental Methods

[0048] 1.1 Morphology observation

[0049] 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 minutes, 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 minutes, absorb the liquid with filter paper, and test after natural drying.

[0050] 1.2 Dynamic light scattering (DLS) analysis

[0051] 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).

[0052] 1.3 Drug loading and encapsulation efficiency testing

[0053] The encapsulation efficiency and drug loading of anthracene and isoquercetin were determined and calculated by high performance liquid chromatography (HPLC), where:

[0054] (1) The HPLC analysis method of anthracene is as follows: the chromatographic column is C 18 The analytical column was 4.6 × 150 mm, 5 μm; the mobile phase was a ratio of phase A (ultrapure water containing 0.1% trifluoroacetic acid): phase B (methanol containing 0.1% trifluoroacetic acid) of 20:80; the flow rate was 1 mL / min; the injection volume was 20 μL; and the detection wavelength was 288 nm. The peak of anthracene was detected at 4.36 min.

[0055] (2) The HPLC analysis method of isoquercetin is: the chromatographic column is C 18 The analytical column was 4.6 × 150 mm, 5 μm; the mobile phase was a 50:50 ratio of phase A (ultrapure water containing 0.1% trifluoroacetic acid): phase B (methanol containing 0.1% trifluoroacetic acid); the flow rate was 1 mL / min; the injection volume was 20 μL; and the detection wavelength was 360 nm. The peak of isoquercetin was detected at 3.32 min.

[0056] (3) Linear relationship investigation and standard curve drawing: Standard solutions of anthracene (3.06~98.00 μg / mL) and isoquercetin (0.60~19.33 μg / mL) were prepared in volumetric flasks, respectively. The samples were injected according to the above chromatographic conditions and the chromatograms were recorded. The standard curve was drawn 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 3.062~98.000 μg / 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 anthracenetin and isoquercetin in the freeze-dried samples of the ternary nanoparticles were calculated according to the regression equation.

[0057] 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%.

[0058] 1.4 Analysis of self-assembly mechanism

[0059] 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). DLS and a microplate reader were used to detect changes in hydrated particle size, polydispersity index (PDI), absorption spectrum, and fluorescence spectrum.

[0060] 2. Experimental results and conclusions:

[0061] Using pentalysine-2-carbonyl phthalocyanine zinc as the 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 1 As shown, 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 24 hours at room temperature, the size and distribution of the ternary nanoparticles remain unchanged, indicating good colloidal stability (AC). This ratio was used for subsequent testing.

[0062] 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%, respectively, and the drug loading was 61.4±2.3% and 3.8±0.4%, respectively.

[0063] like Figure 2 As shown in Figure 2, the maximum absorption wavelength of the penta-lysine-2-carbonyl phthalocyanine 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 indicates that zinc pentalysine-2-carbonylphthalocyanine undergoes J-aggregation during co-assembly with isoquercetin and anthrabenin (A). Due to fluorescence quenching in the H-aggregated state, the fluorescence intensity of zinc pentalysine-2-carbonylphthalocyanine dissolved in PBS is significantly reduced. It is generally believed that J-aggregation can greatly enhance the fluorescence of fluorescent molecules. However, in the present invention, co-assembly with isoquercetin and anthrabenin through J-aggregation completely confines zinc pentalysine-2-carbonylphthalocyanine to a non-fluorescent state (B). This rare non-fluorescent J-aggregate also allows zinc pentalysine-2-carbonylphthalocyanine to serve as an indicator for the responsive release of ternary nanoparticles in vitro and in vivo.

[0064] The self-assembly mechanism of ternary nanoparticles was explored by detecting the UV-visible absorption spectrum, fluorescence spectrum and particle size changes of ternary nanoparticles resuspended in different dispersion media. Figure 3As shown, as the SDS concentration in the dispersion system increases, the characteristic absorption peak of pentalysine-2-carbonylphthalocyanine zinc gradually recovers, and the fluorescence intensity increases after excitation at 610 nm (A, B). The hydrated particle size and polydispersity index (PDI) of the ternary nanoparticles gradually increase (C). This indicates that the ternary nanoparticles disaggregate in the hydrophobic buffer, suggesting that hydrophobic forces and π-π interactions drive the self-assembly of the nanoparticles. Furthermore, different ionic strengths can produce varying degrees of charge shielding, affecting intermolecular electrostatic interactions. In 5% glucose solution, the particle size and PDI of the ternary nanoparticles remained unchanged. However, in 0.5 M or 1 M NaCl solutions, the particle size of the ternary nanoparticles increased to approximately 1.2 μm, while the PDI remained unchanged, indicating that the ternary nanoparticles further aggregated at low ionic concentrations to form relatively uniform micron-sized particles. After the addition of 2 M NaCl, the particle size decreased to approximately 500 nm, while the PDI significantly increased to 0.5, indicating that the ternary nanoparticles disaggregated at high ionic concentrations. This result suggests that electrostatic forces also play a role in the self-assembly process (D).

[0065] Example 2 Characterization of the protein corona formed by in situ adsorption of plasma proteins by ternary nanoparticles

[0066] 1. Experimental Methods

[0067] 1.1 In situ formation of protein corona on the surface of ternary nanoparticles

[0068] 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.

[0069] 1.2 Dynamic light scattering (DLS) analysis

[0070] When performing hydrated particle size detection, 50 μL of the sample solution after incubation of ternary nanoparticles with 50% plasma was placed in a microparticle size cup for measurement; when performing 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).

[0071] 1.3 Native-PAGE gel electrophoresis and fluorescence molecular tomography (FMT) imaging analysis

[0072] The sample solution after incubation of the ternary nanoparticles with 50% plasma was mixed with Native-PAGE loading buffer and loaded into the sample wells of a 12% non-denaturing polyacrylamide gel for electrophoresis (180V, 3 h). The sample was stained with Coomassie Brilliant Blue and destained before imaging.

[0073] 2. Experimental results and conclusions:

[0074] Maintaining the stability of nanosystems in the bloodstream and avoiding premature drug release are prerequisites for efficient drug delivery. With the application of nanotechnology in the biomedical field, people have gradually realized that the interaction between nanoparticles and biomolecules, especially extracellular proteins, greatly affects the behavior of nanoparticles in vivo.

[0075] like Figure 4 As shown in the figure, when the ternary nanoparticles were incubated with physiological saline containing 50% plasma, their 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 have been adsorbed on the surface of the ternary nanoparticles (A). At the same time, albumin is one of the most abundant protein species in plasma. The Native-Page test results showed that the sample after incubation of the ternary nanoparticles with 50% plasma showed protein species bands similar to those in plasma, among which albumin was the most abundant (B). The FMT imaging results showed that the sample loading hole after incubation of the ternary nanoparticles 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).

[0076] Example 3 Shear stress responsive drug release

[0077] 1. Experimental Methods

[0078] 1.1 In vitro simulation of drug release induced by different shear forces

[0079] An in vitro simulated circulation device was constructed using a peristaltic pump, a 1.6 mm inner diameter hose, a Luer connector, and an ibidi µ-slide channel. The pump speed was adjusted to control the flow rate and the resulting shear stress. The release of ternary nanoparticles was investigated after 15 minutes of circulation in a saline solution containing 50% plasma under both low and high shear stress conditions. After the circulation period, unreleased nanoparticles were recovered by high-speed centrifugation and quantified.

[0080] 1.2 Observation of localized precise drug release of self-assembled ternary nanoparticles in damaged blood vessels by FMT imaging

[0081] Eight-week-old male ICR mice were used as experimental animals. After isoflurane anesthesia, the neck skin of the mice was removed using a hair removal cream. A carotid artery thrombosis model was established as follows: the left common carotid artery (CCA) was dissected approximately 0.5 cm proximal to the bifurcation between the external and internal carotid arteries. A transparent plastic sheet was placed underneath the CCA. 30 μL of 20% ferric chloride hexahydrate solution was added to stimulate the CCA. The CCA was observed using a laser speckle blood flow imaging device until blood flow decreased by approximately 90%. The ferric chloride solution was aspirated, and normal saline was dripped to irrigate the injured vessels. The plastic sheet was removed, and the neck skin incision was sutured. The mice were placed on a 37°C warming pad until they fully regained consciousness. Ten minutes later, the ternary nanoparticles were injected through the tail vein. At 0.5 and 1 h, the mice were placed in a small animal in vivo FMT imager. The neck of the mouse was selected as the target area (ROI), and 20 to 30 source positions were scanned (the distance between adjacent scanning points was set to 2 mm). The fluorescence signal of pentapolylysine-2-carbonylphthalocyanine zinc was collected using a 680 nm laser light source for excitation. Three-dimensional reconstruction and fluorescence quantification were performed using TrueQuant v3.0 software.

[0082] 1.3 Laser Speckle Flow Imaging (LSCI) Observation of the Precise Local Drug Release of Self-assembled Ternary Nanoparticles in Injured Vascular to Delay CCA Thromboembolism

[0083] Eight-week-old healthy male ICR mice were randomly divided into three groups and received tail vein injections of normal saline, normal saline containing an equal amount of free drug (anthrabenin treatment dose of 2 μmol / kg, isoquercetin treatment dose of 80 nmol / kg), or ternary nanoparticles (1 mg / kg). Ten minutes later, the left CCA of the mice was excised and a transparent plastic sheet placed underneath. The mice were placed on the LSCI imaging platform. Immediately after the addition of 30 μL of 20% ferric chloride hexahydrate solution, blood flow velocity at the lesion was monitored in real time using LSCI. Recordings were taken every 5 seconds until the blood flow rate dropped to 10% of the baseline value. The time to occlusion (TTO) of the CCA thrombus was recorded.

[0084] 2. Experimental results and conclusions:

[0085] 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 conditions of 500 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 significantly reduced. 2 When , the cumulative release of ternary nanoparticles and ternary nanoparticle protein corona assemblies increased to nearly 60%.

[0086] like Figure 6 As shown in Figure 2, in ICR mice, local application of ferric chloride injures the left CCA, leading to thrombosis and approximately 90% occlusion of the carotid artery lumen. FMT imaging of the neck revealed significantly increased fluorescence intensity in the left CCA compared to the contralateral CCA at 0.5 and 1 hour, indicating that the ternary nanoparticles release drug at the thrombus site, with ZnPc5K visualizing the thrombus. Quantification results showed that ZnPc5K concentrations in the left carotid artery were 22.7-fold and 14.4-fold higher than those in the contralateral carotid artery at 0.5 and 1 hour, respectively.

[0087] The real-time LSCI blood flow monitoring results were obtained by pre-intravenous injection of ternary nanoparticles 10 minutes later and then irritation of the carotid artery with ferric chloride. Figure 7 As shown in the results, compared with the saline solution and saline solution containing the same amount of free drug, pre-injection of ternary nanoparticles significantly prolonged the time required for arterial occlusion (TTO). This result strongly proves that shear force can trigger the targeted release of nanoparticles in the local carotid artery where thrombus has formed, thereby delaying carotid artery thromboembolism.

[0088] Example 4 Self-assembled ternary nanoparticles significantly improve the prognosis of arterial thrombosis model mice in vivo

[0089] A carotid artery thrombosis mouse model was established as described in Item 1.2 of Example 3. Model mice were randomly divided into four groups and received tail vein injections of normal saline, alteplase (10 mg / kg), a normal saline solution containing an equal amount of free drug (anthrabenin treatment dose of 2 μmol / kg, isoquercetin treatment dose of 80 nmol / kg), and ternary nanoparticles (1 mg / kg). The alteplase-treated group received a single dose, while the normal saline solution containing an equal amount of free drug and the ternary nanoparticle-treated groups received doses once daily for a total of three doses. The survival of mice in each group was monitored, and survival curves were plotted 7 days after model establishment.

[0090] The results are as follows Figure 8 As shown in the figure, the 7-day survival rate of model mice treated with normal saline solution was about 50%; the 7-day survival rate of mice in the normal saline solution group treated with alteplase and an equal amount of free drug increased to about 70%; the 7-day survival rate of 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 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), and improved the prognosis of mice with carotid artery thrombosis model.

[0091] Example 5 Therapeutic Mechanism of Self-Assembled Ternary Nanoparticles

[0092] 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 digest DNA with DNaseI. The RNA quality was determined by measuring 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. 2 μg of total RNA was taken and the rRNA was removed using a Ribo-off rRNA removal kit and KC TM Stranded mRNA library preparation kits were used to prepare strand-specific RNA sequencing libraries. Sequences of 200–500 bp were enriched and 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 a log2-fold change > 2 and p < 0.05. Hierarchical clustering analysis of DEGs based on Pearson correlation distance was performed using the pheatmap package. Gene set enrichment analysis (GSEA) was performed using the gseGO and gseKEGG functions in the clusterProfiler package (v 4.12.0), respectively. The mouse genome was annotated using the org.Mm.eg.db package. Normalized enrichment scores (NES) were used to quantify enrichment, and gene sets with |NES| > 1 and p < 0.05 were considered statistically significant.

[0093] The results are as follows Figure 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 treated with self-assembled ternary nanoparticles, of which 778 genes were upregulated and 2187 genes were downregulated. Hierarchical cluster analysis of DEGs showed that the transcriptomes of the injured carotid arteries of mice treated with saline and self-assembled ternary nanoparticles were significantly different.

[0094] Subsequently, GO enrichment and KEGG analysis based on GSEA were used to further explore the biological functions and main enrichment pathways of these DEGs. Figure 10 As shown, the NF-κB signaling pathway, neutrophil extracellular trap (NETs) formation and glycolysis were significantly downregulated after self-assembled ternary nanoparticle treatment; on the other hand, the cGMP-mediated signaling pathway was significantly upregulated in the self-assembled ternary nanoparticle treatment group.

[0095] Example 6 Self-assembled ternary nanoparticles have good biosafety

[0096] 1. Experimental Methods

[0097] 1.1 Mouse tail bleeding experiment

[0098] The effects of self-assembled ternary nanoparticles on peripheral blood coagulation were investigated by measuring changes in tail bleeding time and hemoglobin loss before and after intravenous administration. Eight-week-old male ICR mice were randomly divided into two groups after establishing a carotid artery thrombosis model. The mice were then injected intravenously with normal saline, alteplase (10 mg / kg), a saline solution containing an equal amount of free drug (anthrabenin at a dose of 2 μmol / kg and isoquercetin at a dose of 80 nmol / kg), and ternary nanoparticles (1 mg / kg). One hour later, the distal 1 cm of the mouse tail was disinfected with 75% alcohol and cut with a sterilized blade. The tail was immediately immersed in normal saline warmed at 37°C, and the bleeding duration was recorded until hemostasis stabilized for 10 s. The saline solution containing the mouse blood sample was collected and centrifuged at 4000 rpm for 15 minutes. The supernatant was discarded, the red blood cell pellet was collected, and 2 mL of red blood cell lysis buffer was added and shaken to fully lyse the red blood cells. The sample was then sampled and the absorbance at 575 nm was measured using a microplate reader.

[0099] 1.2 Histocompatibility analysis

[0100] The acute and chronic toxicity of self-assembled ternary nanoparticles was investigated by examining the pathological morphological changes in major mouse organs before and after intravenous administration. To evaluate acute toxicity, healthy, 8-week-old male ICR mice were randomly divided into two groups and injected with saline or a double therapeutic dose of ternary nanoparticles (2 mg / kg) via the tail vein once daily for 3 days. To evaluate chronic toxicity, healthy, 8-week-old male ICR mice were randomly divided into two groups and injected with saline or a therapeutic dose of ternary nanoparticles (1 mg / kg) via the tail vein every other day for 30 days. The mice were monitored for survival, and the brain, heart, liver, spleen, lung, and kidney were harvested on days 4 (for acute toxicity) and 31 (for chronic toxicity). After fixation, paraffin sections were prepared and stained with hematoxylin and eosin (HE) for histopathological changes.

[0101] 2. Experimental results and conclusions:

[0102] The results are as follows Figure 11 As 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 significant effect on normal peripheral coagulation.

[0103] The results are as follows Figure 12 As shown, HE staining results showed that compared with the normal control group mice, there was no obvious pathological change in the brain, heart, liver, spleen, lung, kidney and other major organ tissues 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.

[0104] 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 nano self-assembly particles are self-assembled using asymmetric zinc phthalocyanine as a template, a natural small molecule compound containing a p-benzoquinone mother core, and a flavonoid compound; The molar ratio of the asymmetric zinc phthalocyanine to the flavonoid compound and the natural small molecule compound containing a benzoquinone mother nucleus is 1:1:3-4; wherein the asymmetric zinc phthalocyanine is pentalysine-2-carbonyl phthalocyanine zinc, the natural small molecule compound containing a benzoquinone mother nucleus is anthracene, and the flavonoid compound is isoquercetin.

2. A method for preparing 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 core, and a flavonoid compound are prepared into corresponding solutions; 2) mixing the three solutions prepared in step 1) in water, and allowing to stand in the dark 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.

3. The method for preparing ternary nano self-assembled particles according to claim 2, wherein: In step 1), dimethyl sulfoxide is used as a solvent to prepare three solutions.

4. The method for preparing ternary nano self-assembled particles according to claim 2, wherein: The concentrations of the three solutions prepared in step 1) are all 10 mM.

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

6. Use of the ternary nano self-assembled particles according to claim 1 in the preparation of nanomedicines for preventing and / or treating arterial thrombotic diseases.

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