Hirudin magnetic nanoparticles for treating thrombus as well as preparation method and application of hirudin magnetic nanoparticles

Abstract

The invention discloses hirudin magnetic nanoparticles for treating thrombus as well as a preparation method and application of the hirudin magnetic nanoparticles. The hirudin magnetic nanoparticles comprise hollow gold nanorods, hirudin loaded in inner cavities of the hollow gold nanorods and ferroferric oxide nanoparticles distributed on the surfaces of the hollow gold nanorods. The hollow gold nanorod, the hirudin and the ferroferric oxide particles are combined to prepare a brand-new medicine for treating thrombus, namely the hirudin magnetic nanoparticles, and the hirudin magnetic nanoparticles have a good magnetic targeting effect under the wrapping of the ferroferric oxide particles, so that the hirudin can be quickly targeted and enriched to the thrombus part, and the thrombus treatment effect is improved. The hirudin magnetic nano-particle has the advantages that the hirudin magnetic nano-particle has excellent light and heat performance of the hollow gold nano-rod, hirudin can be released at deep thrombus tissues under irradiation of laser, the clinical effect of hirudin on thrombus treatment is guaranteed, and the hirudin magnetic nano-particle is high in thrombolysis capacity, high in bioavailability and good in treatment effect.

Description

Technical Field

[0001] The present invention specifically relates to hirudin magnetic nanoparticles for treating thrombus and a preparation method and application thereof. Background Art

[0002] Thrombi are small pieces of blood formed on the surface of the peeling or repairing of the inner surface of the cardiovascular system. In the variable fluid-dependent type, thrombi are composed of insoluble fibrin, deposited platelets, accumulated white blood cells and trapped red blood cells. They are prone to form related diseases, including acute myocardial infarction, deep vein thrombosis and ischemic stroke, which are the main causes of disability or death in the global population. The most common way to treat thrombi is usually PTT, also known as photothermal therapy, which has high specificity, minimal invasiveness and precise spatiotemporal selectivity. It is more accurate and convenient in time and space, and greatly reduces the damage to surrounding healthy tissues, and has broad application prospects.

[0003] Currently, gold nanoparticles are developed and used as photothermal agents, including nanorods, nanospheres, nanoshells, nanocages, nanodisks, nanostars, etc., but only a few types can actually be tuned to have strong absorption in the NIR-II window under limited conditions;

[0004] At the same time, as a natural anticoagulant, hirudin has significant anticoagulant ability and good biocompatibility, but its half-life in vivo is short and its bioavailability is low, so it cannot be used clinically and has low effect.

[0005] Currently, among the drugs that combine gold nanoparticles with hirudin, the drugs cannot be targeted and enriched at the site of thrombus, the drugs cannot be delivered to the deep thrombus tissue, the release effect of hirudin is not ideal, and the thrombus cannot be fundamentally treated.

[0006] Therefore, it is necessary to invent hirudin magnetic nanoparticles for treating thrombosis and a preparation method and use thereof to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to provide hirudin magnetic nanoparticles for treating thrombosis, and a preparation method and use thereof. The method comprises the following steps: a hollow gold nanorod solution and a hirudin aqueous solution are stirred and centrifuged, activated ferroferric oxide nanoparticles are added to continue the reaction, and the hirudin magnetic nanoparticles are obtained by centrifugal purification. The direction and intensity of the external magnetic field are simply controlled so that the hirudin magnetic nanoparticles can be targeted to the thrombosis site. Then, under 1064nm laser irradiation, NIR-II photothermal therapy is achieved, and hirudin is slowly released to achieve the treatment and prevention of thrombosis. The method has high bioavailability and good clinical effect, so as to solve the above-mentioned shortcomings in the technology.

[0008] In order to achieve the above object, the present invention provides the following technical solution: hirudin magnetic nanoparticles for treating thrombosis, comprising:

[0009] Hollow gold nanorods,

[0010] Hirudin loaded in the inner cavity of the hollow gold nanorod,

[0011] Ferroferric oxide nanoparticles are distributed on the surface of the hollow gold nanorods.

[0012] The aforementioned hirudin magnetic nanoparticles for treating thrombosis, wherein the hollow gold nanorods have a wide diameter of 10.1 nm and a long diameter of 91.2 nm, the mass concentration of hirudin is 1.0 mg / mL, and the particle size of the ferrosoferric oxide nanoparticles is 10.0 nm;

[0013] The hirudin magnetic nanoparticles synthesized from the hollow gold nanorods, hirudin, and ferroferric oxide nanoparticles have a wide diameter of 28.2 nm and a long diameter of 91.2 nm.

[0014] A method for preparing hirudin magnetic nanoparticles for treating thrombosis comprises the following steps:

[0015] Step 1, mixing and stirring the hollow gold nanorod solution and the hirudin aqueous solution to obtain a mixed solution;

[0016] Step 2, activating the ferrosoferric oxide nanoparticles using N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0017] Step 3: adding the reactive group to the mixed solution, continuing the reaction, and then centrifuging and purifying to obtain hirudin magnetic nanoparticles.

[0018] In the aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 1, the hollow gold nanorod solution is 1.0 mL, the hirudin solution is 2.0 mg, 2.0 mL, and the mixing and stirring time is 12 hours.

[0019] In the aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 2, the ratio of N-hydroxysuccinimide ester to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is set to 1:1.

[0020] In the aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 3, the centrifugal speed is 10000 r / min, the centrifugal time is 10.0 min, and the continuous reaction time is 4.0 h.

[0021] In the aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 2, the synthesis steps of ferroferric oxide nanoparticles are specifically as follows:

[0022] 2.1. Dissolve FeCl3·6H2O and FeCl2·4H2O in HCL aqueous solution to prepare an iron salt solution for later use;

[0023] 2.2. Add the iron salt solution dropwise to the alkaline solution while stirring vigorously and wait for 30 seconds;

[0024] 2.3. Add a stabilizer, stir, centrifuge the dark suspension, and dialyze the supernatant to obtain ferrosoferric oxide nanoparticles.

[0025] The aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 2.1, wherein FeCl3·6H2O is 0.31 g, FeCl2·4H2O is 0.16 g, and HCL aqueous solution is 2.0 mL, 0.5 mol / L;

[0026] In step 2.2, the alkaline solution was prepared by dissolving 1.0 mL of 26% NH4OH in 20 mL of ultrapure water.

[0027] In the aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis, in step 2.3, the stabilizer is prepared by dissolving 0.41 g of polyacrylic acid in 5.0 mL of ultrapure water;

[0028] The stirring time was 1 h and the stirring temperature was room temperature;

[0029] The centrifugal speed was 4000 r / min and the centrifugal time was 30 min;

[0030] The dialysis bag was 3.5 kDa and the dialysis time was 3 days.

[0031] A use of hirudin magnetic nanoparticles for treating thrombosis. The hirudin magnetic nanoparticles are targeted and enriched at thrombotic sites under the action of an external magnetic field, and are used for photothermal treatment and prevention of thrombosis under 1064nm laser irradiation.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention stirs and centrifuges a hollow gold nanorod solution and a hirudin aqueous solution, adds activated ferroferric oxide nanoparticles to continue the reaction, and obtains hirudin magnetic nanoparticles by centrifugation purification. The outer side of the hirudin magnetic nanoparticles is also uniformly wrapped by ferroferric oxide nanoparticles, has good paramagnetism, can be magnetized by a magnet, and is targeted and rapidly enriched at the thrombus site under the action of an external magnetic field;

[0034] 2. The hirudin magnetic nanoparticles not only have good photothermal conversion performance, but also have good photothermal stability. After being irradiated by 1064nm laser, they can rise to a higher temperature, allowing hirudin to be better released.

[0035] 3. The thrombolytic efficiency of hirudin magnetic nanoparticles reached 56.5%, which has a good thrombus ablation effect and a good inhibitory effect on thrombus. It can be used for preventive treatment with a certain effect. At the same time, it has low toxic side effects in the body and good biological safety, which can ensure the early recovery of patients.

[0036] 4. The hirudin magnetic nanoparticles maintain the NIR-II region characteristics of hollow gold nanorods, so that the hirudin magnetic nanoparticles have good absorption effect in the NIR-II region, can penetrate deeper thrombotic tissue, achieve efficient photothermal thrombolysis, have high bioavailability, and good temporary treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0038] Figure 1 The present invention is a flow chart for preparing hirudin magnetic nanoparticles;

[0039] Figure 2 Figure a is a transmission electron micrograph of hollow gold nanorods, Figure b is a transmission electron micrograph of ferroferric oxide particles, and Figure c is a transmission electron micrograph of hirudin magnetic nanoparticles;

[0040] Figure 3 Figure a is the element distribution diagram and superposition diagram of hirudin magnetic nanoparticles, and Figure b is a schematic diagram of the relative content of each element in hirudin magnetic nanoparticles;

[0041] Figure 4 Figure a is the particle size distribution diagram of ferroferric oxide particles, and Figure b is the particle size distribution diagram of hollow gold nanorods and hirudin magnetic nanoparticles;

[0042] Figure 5 Figure a is the potential diagram of ferroferric oxide particles, hollow gold nanorods, and hirudin magnetic nanoparticles, and Figure b is the UV absorption spectrum of ferroferric oxide particles, hollow gold nanorods, and hirudin magnetic nanoparticles;

[0043] Figure 6Figure a is the XRD spectrum of ferroferric oxide particles, hollow gold nanorods, and hirudin magnetic nanoparticles, and Figure b is the Fourier transform infrared spectrum of ferroferric oxide particles, hollow gold nanorods, and hirudin magnetic nanoparticles;

[0044] Figure 7 A demonstration diagram of a magnet separating hirudin magnetic nanoparticles from an aqueous solution;

[0045] Figure 8 Figure a is a schematic diagram of separating hirudin magnetic nanoparticles from an aqueous solution by magnetic force, and Figure b is a line graph of the magnetic intensity of hirudin magnetic nanoparticles;

[0046] Fig. 9 Figure a is a temperature rise curve of hirudin magnetic nanoparticle aqueous solution with different concentrations, and Figure b is a curve of hirudin magnetic nanoparticle aqueous solution after five temperature rise and fall cycles;

[0047] Fig.10 Figure a is a schematic diagram of blood clot dissolution in vitro, and Figure b is a blood clot under light irradiation conditions;

[0048] Fig.11 Figure a is a section effect diagram of hirudin magnetic nanoparticles after H&E staining, and Figure b is a quantitative diagram of the proportion of thrombus area;

[0049] Fig.12 Figure a is a diagram showing the toxicity test of hirudin magnetic nanoparticles on cells, and Figure b is a diagram showing the toxicity test of hirudin on cells;

[0050] Fig.13 Figure a is a schematic diagram of the bleeding amount of mice, and Figure b is a schematic diagram of the bleeding time of mice;

[0051] Fig.14 H&E staining images of heart, liver, spleen, lung and kidney organs. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0053] A hirudin magnetic nanoparticle for treating thrombosis, comprising a hollow gold nanorod, hirudin loaded in the inner cavity of the hollow gold nanorod, and ferrosoferric oxide nanoparticles distributed on the surface of the hollow gold nanorod;

[0054] The hollow gold nanorods have a wide diameter of 10.1 nm and a long diameter of 91.2 nm, the mass concentration of hirudin is 1.0 mg / mL, and the particle size of the ferroferric oxide nanoparticles is 10.0 nm.

[0055] The wide diameter of hirudin magnetic nanoparticles synthesized by hollow gold nanorods, hirudin, and ferroferric oxide nanoparticles is 28.2nm, and the long diameter is 91.2nm.

[0056] A method for preparing hirudin magnetic nanoparticles for treating thrombosis comprises the following steps:

[0057] Step 1, mixing and stirring the hollow gold nanorod solution and the hirudin aqueous solution to obtain a mixed solution;

[0058] Step 2, activating the ferrosoferric oxide nanoparticles using N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0059] Step 3, adding the reactive group to the mixed solution, continuing the reaction, and centrifuging and purifying to obtain hirudin magnetic nanoparticles;

[0060] A use of hirudin magnetic nanoparticles for treating thrombosis. The hirudin magnetic nanoparticles are targeted and enriched at thrombotic sites under the action of an external magnetic field, and are used for photothermal treatment and prevention of thrombosis under 1064nm laser irradiation.

[0061] Example 1

[0062] Step 1, 1.0 mL of the hollow gold nanorod solution and 2.0 mg, 2.0 mL of the hirudin aqueous solution were mixed and stirred for 12.0 h to obtain a mixed solution;

[0063] Step 2, activating the ferrosoferric oxide nanoparticles using N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0064] Wherein, the ratio of N-hydroxysuccinimide ester to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was set to 1:1;

[0065] Step 3, adding the reactive group to the mixed solution, continuing the reaction for 4 hours, and then centrifuging at a speed of 10000 r / min for 10.0 min to purify hirudin magnetic nanoparticles;

[0066] The synthesis steps of ferroferric oxide nanoparticles are as follows:

[0067] 2.1. Dissolve 0.31 g of FeCl3·6H2O and 0.16 g of FeCl2·4H2O in 2.0 mL of 0.5 mol / L HCL aqueous solution to prepare an iron salt solution for later use;

[0068] 2.2. While stirring vigorously, add the iron salt solution dropwise to the alkaline solution prepared by dissolving 1.0 mL of 26% NH4OH in 20 mL of ultrapure water and wait for 30.0 seconds.

[0069] 2.3. Add 0.4 g of polyacrylic acid and dissolve it in 5.0 mL of ultrapure water to prepare a stabilizer. Stir it at room temperature for 1.0 h. Centrifuge the dark suspension at 4000 r / min for 30.0 min. Use a 3.5 kDa dialysis bag to dialyze the supernatant for 3 days to obtain ferrosoferric oxide nanoparticles.

[0070] Verification experiment

[0071] 1. Characterization of Hirudin Magnetic Nanoparticles

[0072] TEM was used to characterize the morphology of hollow gold nanorods, ferroferric oxide nanoparticles, and hirudin magnetic nanoparticles:

[0073] The results are as follows Figure 2 As shown in Figure a, the edges of the hollow gold nanorods are clear, their morphology is regular and uniform, they are rod-like structures, and their interiors are hollow structures;

[0074] like Figure 2 As shown in Figure b, its morphology is granular, the particle size is about 10.0nm, and the color contrast is relatively low;

[0075] like Figure 2 As shown in Figure c, the interior of the hirudin magnetic nanoparticles is a hollow structure, and the outer layer has a large number of small nanoparticles with low contrast;

[0076] At the same time, element mapping was used to further characterize the hirudin magnetic nanoparticles:

[0077] The results are as follows Figure 3 As shown in Figure a, it can be seen that Au, S, C, and N elements are evenly distributed in the hirudin magnetic nanoparticles, indicating the existence of hollow gold nanorods. In addition, Fe and O elements are evenly distributed on the outside of the hirudin magnetic nanoparticles, indicating the existence of ferroferric oxide nanoparticles, and they are evenly distributed on the outside of the hollow gold nanorods.

[0078] And if Figure 3 As shown in Figure b, X-ray energy spectrum analysis shows that the gold content in the hirudin magnetic nanoparticle material is the highest, followed by the iron content;

[0079] Wherein, the hirudin magnetic nanoparticles are hirudin magnetic nanoparticles;

[0080] At the same time, after testing the hydration particle size and Zeta potential of hollow gold nanorods, hirudin magnetic nanoparticles and ferroferric oxide nanoparticles, we obtained Figure 4 From Figure a and Figure b in the figure, we can conclude that:

[0081] The width of the hollow gold nanorods is 10.1 nm and the length is 91.2 nm, while the width of the hirudin magnetic nanoparticles is 28.2 nm and the length is 190.1 nm. The increase in particle size is due to the encapsulation of the ferroferric oxide nanoparticles.

[0082] The Zeta potential of hollow gold nanorods, hirudin magnetic nanoparticles and ferroferric oxide nanoparticles was detected by DLS. Figure 5 From Figure a in Figure 2, we can conclude that:

[0083] The potentials of hollow gold nanorods, ferroferric oxide, and hirudin magnetic nanoparticles are -36.3 mV, -40.3 mV, and -21.8 mV, respectively;

[0084] Furthermore, the UV absorption spectra of ferroferric oxide, hollow gold nanorods and hirudin magnetic nanoparticles were tested respectively. Figure 5 From Figure b in the figure, we can conclude that:

[0085] Hirudin magnetic nanoparticles have a strong absorption peak at 1064nm, which is similar to the peak shape of hollow gold nanorods, that is, hirudin magnetic nanoparticles maintain the characteristics of hollow gold nanorods, so that the hirudin magnetic nanoparticles have good absorption effect in the NIR-II region, can penetrate deeper thrombotic tissue, achieve efficient photothermal thrombolysis, high bioavailability, and good temporary treatment effect;

[0086] Finally, XRD was used to characterize hirudin magnetic nanoparticles, ferroferric oxide, and hollow gold nanorods. Figure 6 From Figure a, we can conclude that hirudin magnetic nanoparticles have a crystal structure, and loading hirudin will not affect the original crystal structure;

[0087] FTIR experiments were performed on hirudin magnetic nanoparticles, ferroferric oxide, and hollow gold nanorods. Figure 6 As shown in Figure b, it can be seen that: 1500cm -1 The enhancement of the peaks indicated that the Fe3O4 nanoparticles were grafted onto the surface of the hollow gold nanorods via amide bonds, thus indicating the successful synthesis of hirudin magnetic nanoparticles.

[0088] 2. Magnetic Characterization of Hirudin Magnetic Nanoparticles

[0089] like Figure 7 As shown in the figure, the process of separating hirudin magnetic nanoparticles from aqueous solution by magnet was recorded. Figure 7 It can be concluded that a magnet is placed on the side of the glass bottle containing hirudin magnetic nanoparticles aqueous solution. As the magnetization time increases, the color of the solution gradually becomes clear, and the color of the right glass bottle wall gradually darkens. When the magnetization time reaches 500.0s, the solution is basically clear and transparent, and the color on the right glass wall is the darkest. The results show that the magnet can separate hirudin magnetic nanoparticles from the aqueous solution, and it has paramagnetic properties.

[0090] At the same time, the adsorption of magnets on hirudin magnetic nanoparticles was recorded. The experimental results are as follows: Figure 8 As shown in Figure a, it can be seen that under the action of the magnet, a large number of hirudin magnetic nanoparticles are gathered on the right side of the glass bottle wall, and the solution is clear and transparent. The results show that the magnet can separate most of the nanoparticles from the solution. Figure 8 As shown in Figure b, hirudin magnetic nanoparticles can be strongly magnetized under the action of an external magnetic field.

[0091] 3. Characterization of Photothermal Properties of Hirudin Magnetic Nanoparticles

[0092] like Fig. 9 Figure a shows the photothermal performance of hirudin magnetic nanoparticles under different excitation power conditions. It can be seen that at 0.2, 0.5, 0.75, 1.0, and 1.5 W / cm 2 The irradiation was performed for 5.0 min at the excitation power of , and the temperature of the hirudin magnetic nanoparticle solution increased with the increase of the power;

[0093] Since hirudin magnetic nanoparticles have strong ultraviolet absorption at 1064nm, the use of 1064nm laser treatment is the best choice. Therefore, the hirudin magnetic nanoparticle solution was irradiated with 1064nm laser for 5.0min, and then the laser was turned off to record the temperature changes of five heating and cooling cycles. Fig. 9 As shown in Figure b, it can be seen that after five cycles of heating and cooling, the maximum temperature of the hirudin magnetic nanoparticle solution does not change much, and the temperature change curves are similar, indicating that the hirudin magnetic nanoparticles not only have good photothermal conversion performance, but also have good photothermal stability.

[0094] IV. In vitro thrombolytic properties of hirudin magnetic nanoparticles

[0095] The blood clots were treated under PBS, hirudin magnetic nanoparticles, PBS+L, hirudin magnetic nanoparticles+L, and hirudin magnetic nanoparticles+L+M conditions;

[0096] Among them, L means laser treatment, M means external magnet treatment;

[0097] like Fig.10 As shown in Figure a, the first group of blood clots were placed in PBS solution and irradiated with 1064 nm laser for 20.0 min;

[0098] The second group of blood clots were placed in a solution of hirudin magnetic nanoparticles and irradiated with a 1064nm laser for 20 minutes;

[0099] The third group of blood clots were placed in a solution of hirudin magnetic nanoparticles and irradiated with a 1064nm laser while being attracted by a magnet;

[0100] An infrared thermal imager was used to record the temperature changes of each experimental group. The specific experimental results are as follows: Fig.12 As shown in Figure b, the temperature of the PBS group did not change much, while the hirudin magnetic nanoparticle group after laser irradiation was able to rise to a higher temperature, and an even higher temperature could be reached after adding a magnetic field and laser irradiation. Therefore, treatment under dual conditions of 1064nm laser radiation and magnetic field is a better choice.

[0101] 5. In vivo thrombolytic properties of hirudin magnetic nanoparticles

[0102] PBS and hirudin magnetic nanoparticles were injected into the tail vein of a ferric chloride-induced thrombotic mouse model. 2 The laser radiation and magnetic field conditions are used to obtain Fig.11 ;

[0103] from Fig.11 As shown in Figure a, the H&E staining results of the hirudin magnetic nanoparticle group showed that compared with the untreated PBS group, the thrombolytic effect did not change significantly after magnetic field and laser treatment, indicating that the laser and magnetic field have good safety. Compared with the PBS group, the thrombus in the blood vessels of the hirudin magnetic nanoparticle group was ablated and the blood vessels were unblocked, indicating that the thrombus was dissolved.

[0104] Moreover, for the hirudin magnetic nanoparticle group, some thrombi were still partially dissolved even under low power conditions. Different laser powers, such as 0.1 W / cm 2 or 0.2W / cm 2 It will affect the thrombolytic effect. The higher the laser power, the better the thrombolytic effect. By calculating the area occupied by the thrombus in the entire blood vessel, the thrombolytic efficiency can be indirectly calculated.

[0105] At the same time, from Fig.11 It can be concluded from Figure b that 0.2W / cm 2 When treated with laser radiation and magnetic field, the thrombolytic efficiency of hirudin magnetic nanoparticles reached 56.5%, indicating that most of the blood clots in the blood vessels were dissolved and the blood vessels were unblocked, which had a good thrombus ablation effect compared with the PBS group.

[0106] VI. Safety performance of hirudin magnetic nanoparticles

[0107] Different concentrations of hirudin magnetic nanoparticle solutions, such as 100.0 μg / mL, 200.0 μg / mL, 300.0 μg / mL, and 400.0 μg / mL, were incubated with 4T1 cells for 24 h, and the cytotoxicity was detected by MTT assay. The results are as follows: Fig.12 As shown in a, it can be seen that:

[0108] The survival rate of 4T1 cells in the hirudin magnetic nanoparticle solution was above 98%, indicating that hirudin magnetic nanoparticles have good safety within a certain concentration range;

[0109] In addition, hirudin solutions of different concentrations, such as 100.0 μg / mL, 200.0 μg / mL, 400.0 μg / mL, and 800.0 μg / mL, were incubated with 4T1 cells for 24 h, and the cytotoxicity was detected by MTT assay. Fig.12 As shown in b, it can be seen that:

[0110] The survival rate of 4T1 cells in the hirudin solution was above 98%, indicating that hirudin has good cell safety within a certain concentration range;

[0111] At the same time, mice were treated with hirudin magnetic nanoparticle solution or hirudin solution through the tail vein of mice, and then 0.5 cm of the mouse tail was cut off. The bleeding amount and bleeding time were recorded through the laser irradiation site to conduct a tail bleeding experiment. The experimental results are as follows Fig.13 As shown, Fig.13 Figure a shows the amount of bleeding in the mouse tail hemostasis experiment after injection of different solutions. Fig.13 Figure b shows the bleeding time of the mouse tail hemostasis experiment after injection of different solutions. Fig.13 It can be seen that:

[0112] Due to the inherent hemostatic function of the blood system, coagulation occurs 5 minutes after bleeding. Compared with the blank group, the hirudin magnetic nanoparticles treatment group has more bleeding and longer bleeding time. Therefore, the hirudin magnetic nanoparticles nanomaterial has an inhibitory effect on tail thrombosis;

[0113] The safety of hirudin magnetic nanoparticles in vivo was evaluated by H&E staining of major organs. Specifically, the heart, liver, spleen, lungs and kidneys treated with PBS, hirudin magnetic nanoparticles, PBS+L, hirudin magnetic nanoparticles+L, and hirudin magnetic nanoparticles+L+M were subjected to H&E staining. The experimental results are as follows: Fig.14As shown, it can be seen that there is no inflammation or necrosis area in the H&E staining of each group of organs, indicating that hirudin magnetic nanoparticles have low toxic side effects and good biosafety in vivo.

[0114] In summary

[0115] The present invention combines hollow gold nanorods, hirudin and ferroferric oxide particles to prepare a new drug for treating thrombosis, namely, hirudin magnetic nanoparticles. The hirudin magnetic nanoparticles have good magnetic targeting effect when wrapped by ferroferric oxide particles, so that the hirudin can be quickly targeted and enriched in the thrombus site. At the same time, the present invention also has the excellent photothermal performance of the hollow gold nanorods, so that the hirudin can be released in the deep thrombus tissue under the irradiation of laser, thereby ensuring the clinical effect of hirudin on thrombus treatment. The hirudin magnetic nanoparticles have strong thrombolytic ability, high bioavailability and good temporary treatment effect.

[0116] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A hirudin magnetic nanoparticle for treating thrombosis, characterized in that: include; Hollow gold nanorods, Hirudin loaded in the inner cavity of the hollow gold nanorod, Ferroferric oxide nanoparticles are distributed on the surface of the hollow gold nanorods.

2. The hirudin magnetic nanoparticles for treating thrombosis according to claim 1, characterized in that: The wide diameter of the hollow gold nanorod is 10.1 nm, the long diameter is 91.2 nm, the mass concentration of hirudin is 1.0 mg / mL, and the particle size of the ferrosoferric oxide nanoparticle is 10.0 nm; The hirudin magnetic nanoparticles synthesized from the hollow gold nanorods, hirudin, and ferroferric oxide nanoparticles have a wide diameter of 28.2 nm and a long diameter of 91.2 nm.

3. A method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 1-2, characterized in that: The following steps are involved: Step 1, mixing and stirring the hollow gold nanorod solution and the hirudin aqueous solution to obtain a mixed solution; Step 2, activating the ferrosoferric oxide nanoparticles using N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups; Step 3: adding the reactive group to the mixed solution, continuing the reaction, and then centrifuging and purifying to obtain hirudin magnetic nanoparticles.

4. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 3, characterized in that: In step 1, the hollow gold nanorod solution is 1.0 mL, the hirudin solution is 2.0 mg, 2.0 mL, and the mixing and stirring time is 12 h.

5. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 3, characterized in that: In step 2, the ratio of N-hydroxysuccinimide ester to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was set to 1:

1.

6. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 3, characterized in that: In step 3, the centrifugal speed is 10000 r / min, the centrifugal time is 10.0 min, and the continuous reaction time is 4 h.

7. According to the method for preparing hirudin magnetic nanoparticles for treating thrombosis in claim 3, in step 2, the synthesis step of the ferrosoferric oxide nanoparticles is specifically as follows: 2.

1. Dissolve FeCl3·6H2O and FeCl2·4H2O in HCL aqueous solution to prepare an iron salt solution for later use; 2.

2. Add the iron salt solution dropwise into the alkaline solution while stirring vigorously and wait for 30.0s; 2.

3. Add a stabilizer, stir, centrifuge the dark suspension, and dialyze the supernatant to obtain ferrosoferric oxide nanoparticles.

8. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 7, characterized in that: In step 2.1, FeCl3·6H2O is 0.3 g, FeCl2·4H2O is 0.16 g, and HCL aqueous solution is 2.0 mL, 0.5 mol / L; In step 2.2, the alkaline solution is prepared by dissolving 1.0 mL of 26% NH4OH in 20.0 mL of ultrapure water.

9. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 7, characterized in that: In step 2.3, the stabilizer was prepared by dissolving 0.4 g of polyacrylic acid in 5.0 mL of ultrapure water; The stirring time was 1.0 h, and the stirring temperature was room temperature; The centrifugal speed was 4000 r / min and the centrifugal time was 30.0 min; The dialysis bag was 3.5 kDa and the dialysis time was 3 days.

10. The use of hirudin magnetic nanoparticles for treating thrombosis according to any one of claims 1-2, characterized in that: Hirudin magnetic nanoparticles are targeted and enriched at the thrombus site under the action of an external magnetic field, and are used for photothermal treatment and prevention of thrombus under 1064nm laser irradiation.

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