A two-dimensional nanothrombolytic agent and its preparation method and application

Abstract

The present invention discloses a two-dimensional nanothrombolytic agent and a preparation method and application thereof, wherein the two-dimensional nanothrombolytic agent is specifically composed of two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme. The two-dimensional nanothrombolytic agent of the present invention can remain stable in the blood circulation, and after reaching the thrombus site, the fibrinogen is cut into insoluble fibrin, thereby promoting the deposition of the nanothrombolytic agent at the thrombus site, and slowly releasing the thrombolytic enzyme and hydrogen to achieve thrombolysis and anti-inflammation. The nanothrombolytic agent provided by the present invention can not only increase the in vivo circulation time and thrombus targeting of the thrombolytic enzyme, play a better thrombolytic effect and reduce the risk of bleeding, but also can achieve active oxygen scavenging and endothelial tissue repair through hydrogen treatment, regulate the thrombus microenvironment, prevent thrombus recurrence, and play a combined anti-thrombotic effect.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and specifically relates to a two-dimensional nano thrombolytic agent and a preparation method and application thereof. Background Art

[0002] Thrombosis-related diseases seriously threaten human health and life, and are the main cause of diseases such as acute myocardial infarction, stroke and pulmonary embolism. Thrombi are mainly composed of insoluble fibrin, red blood cells, activated platelets and other blood components, which can cause obstruction or even complete occlusion of the circulatory system. Thrombi can cause obstruction of distal blood return, leading to severe symptoms such as swelling and pain of the distal limbs, ischemia of the corresponding organs, limb necrosis, and even threaten the patient's life. One of the main causes of thrombosis is endothelial damage, which leads to local platelet recruitment and upregulation of coagulation factor levels, resulting in blood coagulation and vascular blockage. In addition, the high level of reactive oxygen at the site of thrombosis will further aggravate platelet activation, leading to the continuous aggravation of thrombosis.

[0003] Commonly used clinical treatments for thrombosis include thrombolytic therapy, anticoagulant therapy, and interventional therapy. Among them, the most commonly used strategy for already formed thrombi is emergency thrombolysis through intravenous infusion of plasminogen activators and other drugs. This type of drug mainly includes alteplase, urokinase, streptokinase, etc. This type of drug can activate plasminogen and then act on the fibrinolytic system to play a thrombolytic effect. However, this type of drug has a short half-life and poor specificity, making it difficult to continuously exert a thrombolytic effect, and increases the risk of systemic bleeding, bringing great side effects. Therefore, it is of great significance to develop a thrombolytic agent with good effect and low side effects. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a two-dimensional nanothrombolytic agent to achieve thrombus-targeted delivery of thrombolytic enzymes and hydrogen-mediated thrombus microenvironment regulation, which can improve the half-life and specificity of the drug, exert a sustained thrombolytic effect, and reduce the risk of systemic bleeding; it solves the problems of poor targeting, large side effects, and easy recurrence of thrombus in thrombolytic therapy.

[0005] The present invention also provides a method for preparing the two-dimensional nano thrombolytic agent.

[0006] The present invention also provides an application of the two-dimensional nano thrombolytic agent.

[0007] According to one aspect of the present invention, a two-dimensional nanothrombolytic agent is provided, comprising: two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme.

[0008] In some embodiments of the present invention, the thrombolytic enzyme includes but is not limited to urokinase, streptokinase, alteplase, reteplase and other drugs that can activate the thrombolytic system in the body.

[0009] In some embodiments of the present invention, the mass ratio of the two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme is 1: (0.2-2): (0.01-1).

[0010] In some preferred embodiments of the present invention, the mass ratio of the two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme is 1:1:0.1.

[0011] In some embodiments of the present invention, the structure of the two-dimensional hydrogen silicene nanosheet is: a single-layer nanosheet with a diameter of 200 to 1000 nm, the main part of which is composed of silicon atoms arranged in a hexameric warped structure.

[0012] According to another aspect of the present invention, a method for preparing the above-mentioned two-dimensional nano thrombolytic agent is provided, comprising the following steps:

[0013] S1, preparation of two-dimensional hydrogen silicene nanosheets;

[0014] S2. Dispersing fibrinogen and thrombolytic enzyme in the two-dimensional hydrogen silane nanosheet to prepare the nano thrombolytic agent.

[0015] In some embodiments of the present invention, the step S1 specifically includes the following steps: mixing calcium silicide and concentrated hydrochloric acid, reacting at a temperature of -30°C to -10°C for 3 to 10 days to obtain the two-dimensional hydrogen silicene nanosheet.

[0016] In some preferred embodiments of the present invention, step S1 more specifically includes the following steps: dispersing calcium silicide in concentrated hydrochloric acid, and vigorously stirring the mixture at -20°C under argon protection for 3-10 days; after the reaction is completed, centrifugation and washing are performed, and probe ultrasound is performed in anhydrous ethanol for 2-16 hours to obtain uniformly dispersed hydrogen silicene nanosheets.

[0017] In some embodiments of the present invention, step S2 specifically includes the following steps: after dispersing the two-dimensional hydrogen silene nanosheets, adding fibrinogen and thrombolytic enzyme, precipitating and washing, and then dispersing in physiological saline to obtain the two-dimensional nano thrombolytic agent.

[0018] In some preferred embodiments of the present invention, step S2 more specifically includes the following steps:

[0019] S21. Take 1 mg of the prepared two-dimensional hydrogen silene nanosheets and disperse it in 1 mL of anhydrous ethanol. Take an appropriate amount of fibrinogen and urokinase and dissolve them in 1 mL of ultrapure water. Add the aqueous solutions of fibrinogen and urokinase dropwise into the two-dimensional hydrogen silene nanosheet dispersion under vigorous stirring and continue stirring for 30 minutes.

[0020] S22. Centrifuge the above product at 12000 rpm for ten minutes, take out the precipitate, and wash it three times with ultrapure water. The washed sample is dispersed in 1 mL of normal saline and ultrasonicated for 1 min to completely disperse it, thereby obtaining a dispersion of the two-dimensional nano thrombolytic agent.

[0021] According to another aspect of the present invention, the use of the above-mentioned two-dimensional nano thrombolytic agent in the preparation of a drug for treating thrombosis is proposed.

[0022] In some embodiments of the present invention, the dosage form of the drug includes tablets, capsules, syrups, suspensions or injections.

[0023] In some preferred embodiments of the present invention, the dosage form of the drug is an injection.

[0024] The present invention relates to a responsive two-dimensional nanothrombolytic agent, which is specifically composed of two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme. The nanothrombolytic agent can remain stable in the blood circulation, and after reaching the thrombus site, the fibrinogen is cut into insoluble fibrin, thereby promoting the deposition of the nanothrombolytic agent at the thrombus site, and slowly releasing the thrombolytic enzyme and hydrogen to achieve thrombolysis and anti-inflammation. The nanothrombolytic agent provided by the present invention can not only increase the in vivo circulation time and thrombus targeting of the thrombolytic enzyme, play a better thrombolytic effect and reduce the risk of bleeding, but also can achieve active oxygen scavenging and endothelial tissue repair through hydrogen treatment, regulate the thrombus microenvironment, prevent thrombus recurrence, and play a combined anti-thrombotic effect.

[0025] The present invention has at least the following beneficial effects:

[0026] 1) Targeted delivery of thrombus-targeted thrombus-dissolving enzymes is achieved: by coating fibrinogen on the surface of the material, it can respond to the hypercoagulable state at the thrombus site, thereby achieving the conversion of soluble fibrinogen to insoluble fibrin, and promoting the deposition of nano-thrombolytic agents at the thrombus site.

[0027] 2) Effectively prolong the half-life of thrombolytic enzymes: thrombolytic enzymes are protein-based bioactive drugs with small molecular weight, short half-life, and are easily metabolized and excreted from the body. By loading thrombolytic enzymes on two-dimensional nanomaterials, they can have the long circulation characteristics of nanomaterials.

[0028] 3) Reduced side effects of thrombolytic enzymes: Thrombolytic enzymes are non-specific and are often accompanied by the risk of systemic bleeding during clinical use. By loading two-dimensional nanomaterials and modifying fibrinogen, thrombolytic enzymes will not activate the fibrinolytic system during circulation, but will be slowly released after being enriched at the thrombus site, achieving continuous thrombolysis and thus reducing systemic side effects.

[0029] 4) Hydrogen therapy prevents recurrence of thrombosis: One of the main causes of thrombosis is the damage of the vascular endothelium, which activates the autologous coagulation system. The local high-active oxygen environment will further promote the recruitment and activation of platelets, leading to the aggravation of thrombosis. Single thrombolytic therapy cannot fundamentally solve this problem. Local oxidative stress and vascular damage will still induce recurrence of thrombosis. Hydrogen has the function of resisting oxidative stress and promoting tissue repair, so it can play a combined anti-thrombotic role to prevent the aggravation and recurrence of thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 The transmission electron microscopy images of the thrombin response aggregation experiment of the two-dimensional nano-thrombolytic agent in Example 2 of the present invention are shown, wherein the left image is when no thrombin is added, and the right image is after thrombin is added.

[0032] Figure 2 The scanning electron microscope images are of the thrombus targeting ability experiment of the two-dimensional nano thrombolytic agent in Example 3 of the present invention, wherein the left image is a control group without the addition of the two-dimensional nano thrombolytic agent, and the right image is an experimental group with the addition of the two-dimensional nano thrombolytic agent.

[0033] Figure 3 This is a graph showing the experimental results of an in vitro thrombolytic effect experiment of the two-dimensional nanothrombolytic agent in Example 4 of the present invention.

[0034] Figure 4 This is a graph showing the experimental results of the in vivo thrombolytic effect experiment of the two-dimensional nanothrombolytic agent in Example 5 of the present invention. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0036] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0037] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0038] Since conventional thrombolytic drugs have a short half-life, they cannot work continuously; they have poor specificity, which can easily lead to systemic coagulation disorders and increase the risk of bleeding; they are difficult to continuously exert thrombolytic effects, cannot solve the problems of vascular endothelial damage and inflammatory microenvironment, and cannot avoid the recurrence of thrombosis. Therefore, the present invention develops a responsive two-dimensional nanothrombolytic agent that can effectively respond to thrombin to send aggregation and deposition, improve the drug's thrombus targeting and enrichment efficiency; prolong the drug's half-life and increase the in vivo circulation time; and simultaneously achieve anti-inflammatory and vascular repair to prevent the recurrence of thrombosis.

[0039] One embodiment of the present invention prepares a two-dimensional nano thrombolytic agent, which includes the following components: two-dimensional hydrogen silene nanosheets, fibrinogen, and thrombolytic enzyme in a mass ratio of 1: (0.2-2): (0.01-1). In a preferred embodiment of the present invention, two-dimensional hydrogen silene nanosheets, fibrinogen, and urokinase are used in a mass ratio of 1:1:0.1.

[0040] Another embodiment of the present invention is a method for preparing the two-dimensional nano thrombolytic agent:

[0041] S1. Preparation of two-dimensional hydrogen silicene nanosheets:

[0042] Calcium silicide is dispersed in concentrated hydrochloric acid, and the mixture is vigorously stirred for 3-10 days at -20°C under argon protection. After the reaction is completed, the mixture is centrifuged and washed, and probe ultrasonication is performed in anhydrous ethanol for 2-16 hours to obtain uniformly dispersed hydrogen silene nanosheets.

[0043] S2, Fibrinogen and Urokinase loading:

[0044] Take 1 mg of the prepared two-dimensional hydrogen silene nanosheets and disperse it in 1 mL of anhydrous ethanol. Take an appropriate amount of fibrinogen and urokinase and dissolve them in 1 mL of ultrapure water. Add the aqueous solutions of fibrinogen and urokinase dropwise into the two-dimensional hydrogen silene nanosheet dispersion under vigorous stirring and continue stirring for 30 minutes.

[0045] The above product was centrifuged at 12000 rpm for ten minutes, and the precipitate was taken and washed three times with ultrapure water. The washed sample was dispersed in 1 mL of physiological saline and ultrasonicated for 1 min to completely disperse it, thereby obtaining a dispersion of the two-dimensional nano-thrombolytic agent.

[0046] The following further examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description of this article, and are not necessarily limited to the specific values ​​​​exemplified below.

[0047] Example 1: Preparation of two-dimensional nanothrombolytic agent

[0048] Dissolve 1g of calcium silicide in 100mL of concentrated hydrochloric acid, react at -20°C under argon protection for 7 days, centrifuge at 10000rpm for 10 minutes after the reaction, wash the precipitate twice with acetone and ethanol respectively, and resuspend it in ethanol. Ultrasonic probe (600W) for 6 hours to obtain hydrogen silene nanosheets. Disperse hydrogen silene nanosheets in ethanol (1mg / mL), dissolve fibrinogen (1mg / mL) and urokinase (100U / mL) in deionized water, and mix dropwise with the hydrogen silene dispersion. The obtained mixed solution is washed 3 times with deionized water and resuspended in physiological saline to obtain a dispersion of a two-dimensional nanothrombolytic agent.

[0049] Example 2: Thrombin response aggregation of two-dimensional nanothrombolytic agents

[0050] Thrombin (1 U / mL) was added to the two-dimensional nanothrombolytic agent, and after incubation at 37°C for 5 minutes, the two-dimensional nanothrombolytic agent before and after treatment was dropped onto a copper grid and observed by transmission electron microscopy. Figure 1 As shown in the figure (where the left picture is when no thrombin is added, and the right picture is after thrombin is added), the two-dimensional nanothrombolytic agent before treatment is basically in a monodisperse state, while the material treated with thrombin shows large-area aggregation, indicating that the two-dimensional nanothrombolytic agent can effectively respond to thrombin to send aggregation and deposition.

[0051] Example 3: Thrombus targeting ability of two-dimensional nanothrombolytic agents

[0052] The whole blood of mice was mixed with coagulation buffer (containing calcium chloride, sodium chloride, Tris HCl, and thrombin) and incubated at 37°C for 30 minutes to prepare blood clots. The blood clots were incubated with the dispersion of the two-dimensional nanothrombolytic agent at 37°C for 10 minutes, fixed, dehydrated, and observed with a scanning electron microscope. The blood clots not treated with the two-dimensional nanothrombolytic agent were used as the control group. Figure 2 As shown (wherein the left picture is a control group without the addition of two-dimensional nano-thrombolytic agents, and the right picture is an experimental group with the addition of two-dimensional nano-thrombolytic agents), compared with the blood clots of the control group, the distribution of two-dimensional nanosheets can be observed in the blood clots treated with the two-dimensional nano-thrombolytic agents (the position indicated by the arrows), indicating that the nano-thrombolytic agents can be quickly enriched and deposited at the thrombus site.

[0053] Example 4: In vitro thrombolytic effect of two-dimensional nanothrombolytic agents

[0054] The whole blood of mice was mixed with coagulation buffer (containing calcium chloride, sodium chloride, Tris HCl, and thrombin) and incubated at 37°C for 30 minutes to prepare a blood coagulation ring. The blood coagulation ring was co-incubated with nanothrombolytic dispersion containing different concentrations of urokinase (including 0μg / mL, 0.2μg / mL, 0.5μg / mL, 1μg / mL and 2μg / mL) and urokinase solution, and observed at different time points (recording the blood coagulation ring status after 2 hours and 12 hours respectively) to determine its thrombolytic ability. Figure 3 As shown ( Figure 3 In the video, different concentrations of urokinase nanothrombolytics were taken, as well as photos of coagulation rings at different observation points. The two-dimensional nanothrombolytics showed a thrombolytic effect that was not inferior to that of urokinase, indicating that the nanothrombolytics had a good ability to dissolve blood clots.

[0055] Example 5: In vivo thrombolytic effect of two-dimensional nanothrombolytic agents

[0056] Filter paper soaked in 10% ferric chloride solution was applied to the mouse carotid artery for 10 minutes to establish a carotid artery thrombosis model. After modeling, the two-dimensional nanothrombolytic agent was injected into the tail vein, and the thrombus dissolution was observed after 1 day. Figure 4 As shown in the figure (left picture is before administration, right picture is after administration), the local color of the mouse carotid artery deepened and turned dark red before administration, indicating the presence of local thrombus. After the tail vein injection of the two-dimensional nanothrombolytic agent, the carotid artery returned to bright red, indicating that the thrombolytic agent has good thrombus dissolving ability.

[0057] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A two-dimensional nanothrombolytic agent, characterized in that: include: Two-dimensional hydrogen silicene nanosheets, fibrinogen, and thrombolytic enzymes; The two-dimensional hydrogen silicene nanosheet is prepared by the following method: Calcium silicide is dispersed in concentrated hydrochloric acid and reacted at a temperature of -30°C to -10°C for 3 to 10 days to obtain the two-dimensional hydrogen silicene nanosheet.

2. The two-dimensional nanothrombolytic agent according to claim 1, characterized in that: The thrombolytic enzyme is urokinase, streptokinase, alteplase or reteplase.

3. The two-dimensional nano thrombolytic agent according to claim 1, characterized in that: The mass ratio of the two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme is 1: (0.2-2): (0.01-1).

4. The two-dimensional nano thrombolytic agent according to claim 3, characterized in that: The mass ratio of the two-dimensional hydrogen silene nanosheets, fibrinogen and thrombolytic enzyme is 1:1:0.

1.

5. The two-dimensional nano thrombolytic agent according to claim 1, characterized in that: The structure of the two-dimensional hydrogen silicene nanosheet is: a single-layer nanosheet with a diameter of 200-1000 nm, the main part of which is composed of silicon atoms arranged in a hexavalent warped structure.

6. A method for preparing the two-dimensional nano thrombolytic agent according to claim 1, characterized in that: The following steps are involved: S1, dispersing calcium silicide in concentrated hydrochloric acid, reacting at a temperature of -30°C to -10°C for 3 to 10 days to obtain the two-dimensional hydrogen silicene nanosheet; S2. Dispersing fibrinogen and thrombolytic enzyme in two-dimensional hydrogen silene nanosheets to prepare the nano thrombolytic agent.

7. The preparation method according to claim 6, characterized in that: The step S2 specifically includes the following steps: After the two-dimensional hydrogen silene nanosheets are dispersed, fibrinogen and thrombolytic enzyme are added, and after precipitation and washing, the precipitates are dispersed in physiological saline to obtain the two-dimensional nano thrombolytic agent.

8. Use of the two-dimensional nano thrombolytic agent as claimed in claim 1 in the preparation of a drug for treating thrombosis.

9. The use according to claim 8, characterized in that The dosage form of the drug includes tablets, capsules, syrups, suspensions or injections.

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