Hirudin magnetic nanoparticle for treating thrombosis and preparation method and use thereof

Abstract

This invention discloses a hirudin magnetic nanoparticle for treating thrombosis, its preparation method, and its uses. The hirudin magnetic nanoparticle comprises hollow gold nanorods, hirudin loaded within the cavities of the hollow gold nanorods, and iron oxide nanoparticles distributed on the surface of the hollow gold nanorods. This invention combines hollow gold nanorods, hirudin, and iron oxide particles to prepare a novel drug for treating thrombosis: hirudin magnetic nanoparticles. Encapsulated by iron oxide particles, it exhibits excellent magnetic targeting, allowing hirudin to rapidly target and accumulate at the thrombus site. Simultaneously, it possesses the excellent photothermal properties of hollow gold nanorods, enabling hirudin to be released at deep thrombus tissue under laser irradiation, ensuring the clinical efficacy of hirudin in treating thrombosis. These hirudin magnetic nanoparticles exhibit strong thrombolytic ability, high bioavailability, and good therapeutic effects.

Description

Technical Field

[0001] This invention specifically relates to a hirudin magnetic nanoparticle for treating thrombosis, its preparation method, and its uses. Background Technology

[0002] A thrombus is a small mass that forms on the surface of a blood vessel in the cardiovascular system at a site of rupture or repair. In variable fluid-dependent thrombi, thrombi consist of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells. Thrombi are prone to causing related diseases, including acute myocardial infarction, deep vein thrombosis, and ischemic stroke, which are leading causes of disability or death worldwide. The most common treatment for thrombi is PTT, or photothermal therapy, which has high specificity, is minimally invasive, and has precise spatiotemporal selectivity. It is more precise and convenient in time and space and greatly reduces damage to surrounding healthy tissues, showing broad application prospects.

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

[0004] Meanwhile, hirudin, as a natural anticoagulant, has significant anticoagulant ability and good biocompatibility, but its short half-life and low bioavailability prevent it from being used in clinical applications and have a low efficacy.

[0005] Currently, in drugs combining gold nanoparticles and hirudin, the drugs cannot be targeted and enriched at the thrombus site, nor can they be delivered to deep thrombus tissue. The hirudin release effect is not ideal, and the thrombus cannot be fundamentally treated.

[0006] Therefore, it is necessary to invent a hirudin magnetic nanoparticle for treating thrombosis, its preparation method, and its uses to solve the above problems. Summary of the Invention

[0007] The purpose of this invention is to provide a hirudin magnetic nanoparticle for treating thrombosis, its preparation method, and its uses. The method involves stirring and centrifuging a hollow gold nanorod solution with a hirudin aqueous solution, adding activated iron oxide nanoparticles for further reaction, and then purifying the solution by centrifugation to obtain hirudin magnetic nanoparticles. By simply controlling the direction and intensity of an external magnetic field, the hirudin magnetic nanoparticles can target the thrombus site. Then, under 1064nm laser irradiation, NIR-II photothermal therapy is achieved, and hirudin is slowly released to treat and prevent thrombosis. This method exhibits high bioavailability and good clinical efficacy, thus overcoming the aforementioned shortcomings in the technology.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a hirudin magnetic nanoparticle for treating thrombosis, comprising:

[0009] Hollow gold nanorods

[0010] Hirudin, loaded within the cavity of the hollow gold nanorods,

[0011] Iron oxide nanoparticles 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 width of 10.1 nm and a length of 91.2 nm, the mass concentration of hirudin is 1.0 mg / mL, and the particle size of the iron oxide nanoparticles is 10.0 nm.

[0013] Furthermore, the hirudin magnetic nanoparticles synthesized from hollow gold nanorods, hirudin, and iron oxide nanoparticles have a width of 28.2 nm and a length of 91.2 nm.

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

[0015] Step 1: Mix and stir the hollow gold nanorod solution with the hirudin aqueous solution to obtain a mixed solution;

[0016] Step 2: Activate the iron oxide nanoparticles with N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0017] Step 3: Add the reactive groups to the mixture, continue the reaction, and then centrifuge to purify and 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, and the mixing and stirring time is 12 h.

[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, step 3 involves centrifugation at 10,000 r / min for 10.0 min and a further reaction time of 4.0 h.

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

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

[0023] 2.2 While stirring vigorously, add the iron salt solution dropwise to the alkaline solution and wait 30 seconds;

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

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

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

[0027] The aforementioned method for preparing hirudin magnetic nanoparticles for treating thrombosis involves dissolving 0.41 g of polyacrylic acid in 5.0 mL of ultrapure water as a stabilizer in step 2.3.

[0028] The stirring time was 1 hour, and the stirring temperature was room temperature.

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

[0030] The dialysis bag has a capacity of 3.5 kDa, and the dialysis duration is 3 days.

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

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] 1. This invention involves stirring and centrifuging a hollow gold nanorod solution with a hirudin aqueous solution, adding activated iron oxide nanoparticles to continue the reaction, and then purifying the solution by centrifugation to obtain hirudin magnetic nanoparticles. These nanoparticles are uniformly coated with iron oxide nanoparticles on the outside and have good paramagnetism. They can be magnetized by a magnet and rapidly accumulate at the thrombus site under the action of an external magnetic field.

[0034] 2. These hirudin magnetic nanoparticles not only possess excellent photothermal conversion performance but also good photothermal stability. After being irradiated with a 1064nm laser, they can reach a relatively high temperature, allowing for better release of hirudin.

[0035] 3. Hirudin magnetic nanoparticles have a thrombolytic efficiency of 56.5%, exhibiting good thrombus ablation effect and good thrombus inhibition effect, which can be used for certain preventive treatment. At the same time, they have low toxicity and side effects in vivo and good biosafety, which can ensure the early recovery of patients.

[0036] 4. The hirudin magnetic nanoparticles retain the NIR-II region characteristics of hollow gold nanorods, which gives them good absorption in the NIR-II region. They can penetrate deeper thrombus tissue to achieve efficient photothermal thrombolysis, high bioavailability, and good temporary therapeutic effect. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0038] Figure 1 This is a flowchart illustrating the preparation process of hirudin magnetic nanoparticles according to the present invention.

[0039] Figure 2 Figure a is a transmission electron microscope (TEM) image of hollow gold nanorods, Figure b is a TEM image of iron oxide particles, and Figure c is a TEM image of hirudin magnetic nanoparticles.

[0040] Figure 3 Figure a shows the elemental distribution and overlay diagram of hirudin magnetic nanoparticles, and Figure b shows the relative content of each element in hirudin magnetic nanoparticles.

[0041] Figure 4 Figure a shows the particle size distribution of iron oxide particles, and Figure b shows the particle size distribution of hollow gold nanorods and hirudin magnetic nanoparticles.

[0042] Figure 5 Figure a shows the potential diagrams of magnetite particles, hollow gold nanorods, and hirudin magnetic nanoparticles, while Figure b shows the ultraviolet absorption spectra of magnetite particles, hollow gold nanorods, and hirudin magnetic nanoparticles.

[0043] Figure 6Figure a shows the XRD patterns of magnetite particles, hollow gold nanorods, and hirudin magnetic nanoparticles, while Figure b shows the Fourier transform infrared spectra of magnetite particles, hollow gold nanorods, and hirudin magnetic nanoparticles.

[0044] Figure 7 This is a demonstration of how a magnet separates hirudin magnetic nanoparticles from an aqueous solution.

[0045] Figure 8 Figure a shows a schematic diagram of the separation of hirudin magnetic nanoparticles from the aqueous solution by magnetic force, and Figure b is a broken line graph of the magnetic strength of hirudin magnetic nanoparticles.

[0046] Figure 9 Figure a shows the heating curves of aqueous solutions of hirudin magnetic nanoparticles at different concentrations, and Figure b shows the curves of aqueous solutions of hirudin magnetic nanoparticles after five heating and cooling cycles.

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

[0048] Figure 11 Figure a shows the cross-section of hirudin magnetic nanoparticles after H&E staining, and Figure b shows the quantitative map of the proportion of thrombus area.

[0049] Figure 12 Figure a shows the toxicity detection of hirudin magnetic nanoparticles on cells, and Figure b shows the toxicity detection of hirudin on cells.

[0050] Figure 13 Figure a in the diagram shows the amount of bleeding in the mice, and Figure b shows the time of bleeding in the mice.

[0051] Figure 14 H&E staining images of the heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0052] 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 type of hirudin magnetic nanoparticle for treating thrombosis includes hollow gold nanorods, hirudin loaded in the inner cavity of the hollow gold nanorods, and iron oxide nanoparticles distributed on the surface of the hollow gold nanorods.

[0054] The hollow gold nanorods have a width of 10.1 nm and a length of 91.2 nm, the hirudin has a mass concentration of 1.0 mg / mL, and the iron oxide nanoparticles have a particle size of 10.0 nm.

[0055] Furthermore, the hirudin magnetic nanoparticles synthesized from hollow gold nanorods, hirudin, and iron oxide nanoparticles have a width of 28.2 nm and a length of 91.2 nm.

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

[0057] Step 1: Mix and stir the hollow gold nanorod solution with the hirudin aqueous solution to obtain a mixed solution;

[0058] Step 2: Activate the iron oxide nanoparticles with N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0059] Step 3: Add the reactive groups to the mixture, continue the reaction, and then centrifuge to purify and obtain hirudin magnetic nanoparticles;

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

[0061] Example 1

[0062] Step 1: Mix 1.0 mL of hollow gold nanorod solution with 2.0 mg and 2.0 mL of hirudin aqueous solution and stir for 12.0 h to obtain a mixed solution;

[0063] Step 2: Activate the iron oxide nanoparticles with N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups;

[0064] The ratio of N-hydroxysuccinimide ester to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was set to 1:1.

[0065] Step 3: Add the reactive group to the mixture and continue the reaction for 4 hours. Then, centrifuge at 10,000 r / min for 10.0 min to collect the leech magnetic nanoparticles after purification.

[0066] The specific steps for synthesizing iron(III) oxide nanoparticles are as follows:

[0067] 2.1 Dissolve 0.31g of FeCl3·6H2O and 0.16g of FeCl2·4H2O in 2.0mL of 0.5mol / 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 1.0 mL of an alkaline solution prepared by dissolving 26% NH4OH in 20 mL of ultrapure water, and wait for 30.0 s;

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

[0070] Verification Experiment

[0071] I. Characterization of Hirudin Magnetic Nanoparticles

[0072] The morphology of hollow gold nanorods, iron oxide nanoparticles, and hirudin magnetic nanoparticles were characterized by TEM.

[0073] The results are as follows Figure 2 As shown in Figure a, the hollow gold nanorods have clear edges, regular and uniform morphology, and a rod-shaped structure, with a hollow internal structure.

[0074] like Figure 2 As shown in Figure b, its morphology is granular with a particle size of approximately 10.0 nm and relatively low color contrast.

[0075] like Figure 2 As shown in Figure c, the hirudin magnetic nanoparticles have a hollow internal structure and a large number of low-contrast nanoparticles on the outer layer.

[0076] Meanwhile, the hirudin magnetic nanoparticles were further characterized by elemental mapping:

[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 uniformly distributed in the hirudin magnetic nanoparticles, indicating the presence of hollow gold nanorods. In addition, Fe and O elements are uniformly distributed on the outside of the hirudin magnetic nanoparticles, indicating the presence of iron(III) oxide nanoparticles, which are uniformly distributed on the outside of the hollow gold nanorods.

[0078] And such as Figure 3 As shown in Figure b, X-ray energy dispersive spectroscopy analysis shows that the hirudin magnetic nanoparticle material has the highest gold content, followed by iron content.

[0079] Among them, hirudin magnetic nanoparticles are hirudin magnetic nanoparticles;

[0080] Simultaneously, after detecting the hydration particle size and zeta potential of hollow gold nanorods, hirudin magnetic nanoparticles, and iron oxide nanoparticles, the following results were obtained. Figure 4 From graphs a and b, we can conclude that:

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

[0082] The zeta potentials of hollow gold nanorods, hirudin magnetic nanoparticles, and iron oxide nanoparticles were detected by DLS. Figure 5 From graph a in the diagram, we can conclude that:

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

[0084] Furthermore, the ultraviolet absorption spectra of iron oxide, hollow gold nanorods, and hirudin magnetic nanoparticles were tested separately, as detailed below. Figure 5 From graph b in the diagram, we can conclude that:

[0085] Hirudin magnetic nanoparticles have a strong absorption peak at 1064 nm, which is similar to the peak shape of hollow gold nanorods. In other words, hirudin magnetic nanoparticles retain the characteristics of hollow gold nanorods, which makes them have good absorption in the NIR-II region. They can penetrate deeper thrombus tissue to achieve efficient photothermal thrombolysis, with high bioavailability and good temporary treatment effect.

[0086] Finally, XRD was used to characterize the hirudin magnetic nanoparticles, iron oxide, and hollow gold nanorods, and the results are as follows: Figure 6 Figure a shows that the hirudin magnetic nanoparticles have a crystal structure, and loading hirudin does not affect the original crystal structure.

[0087] Furthermore, FTIR experiments were performed on hirudin magnetic nanoparticles, iron oxide, and hollow gold nanorods, and the results are as follows: Figure 6 As shown in Figure b, it can be seen that: 1500cm -1 The enhanced peak indicates that the iron oxide nanoparticles were grafted onto the surface of the hollow gold nanorods via amide bonds, thus demonstrating the successful synthesis of hirudin magnetic nanoparticles.

[0088] II. Magnetic Characterization of Hirudin Magnetic Nanoparticles

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

[0090] Simultaneously, the adsorption of hirudin magnetic nanoparticles by magnets was recorded, and the experimental results are as follows: Figure 8 As shown in Figure a, it can be seen that under the influence of a magnet, a large number of hirudin magnetic nanoparticles accumulated on the right side of the glass bottle wall, and the solution became clear and transparent. The results indicate that a magnet can separate most of the nanoparticles from the solution. Furthermore, [the following text appears to be unrelated and possibly a separate excerpt:] ...by... Figure 8 As shown in Figure b, the hirudin magnetic nanoparticles can be strongly magnetized under the influence of an external magnetic field.

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

[0092] like Figure 9 Figure a shows the photothermal properties of hirudin magnetic nanoparticles under different excitation powers. It can be seen that at excitation powers of 0.2, 0.5, 0.75, 1.0, and 1.5 W / cm², the photothermal performance of the nanoparticles is significantly improved. 2 Irradiation was performed for 5.0 min at an excitation power of [value missing], and the temperature of the hirudin magnetic nanoparticle solution increased with increasing power.

[0093] Furthermore, since hirudin magnetic nanoparticles exhibit strong ultraviolet absorption at 1064nm, using a 1064nm laser is the optimal choice for treatment. Therefore, the hirudin magnetic nanoparticle solution was irradiated with a 1064nm laser for 5.0 min, followed by laser shutdown, and the temperature changes were recorded during five heating and cooling cycles. Specifically... Figure 9 As shown in Figure b, it can be seen that after five cycles of heating and cooling treatment, the maximum temperature of the hirudin magnetic nanoparticle solution did not change much, and the temperature change curves were similar. On the surface, the hirudin magnetic nanoparticles not only have good photothermal conversion performance, but also good photothermal stability.

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

[0095] Blood clots were treated under the following conditions: PBS, hirudin magnetic nanoparticles, PBS+L, hirudin magnetic nanoparticles+L, and hirudin magnetic nanoparticles+L+M.

[0096] Wherein, L indicates laser processing and M indicates external magnet processing;

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

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

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

[0100] The temperature changes of each experimental group were recorded using an infrared thermal imager. Specific experimental results are as follows: Figure 12 As shown in Figure b, the temperature change of the PBS group is not significant, while the hirudin magnetic nanoparticle group exposed to laser radiation can reach a higher temperature. The addition of a magnetic field and laser irradiation can achieve an even higher temperature. Therefore, treatment under the dual conditions of 1064nm laser radiation and magnetic field is a better option.

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

[0102] In a mouse model of thrombosis induced by ferric chloride, PBS and hirudin magnetic nanoparticles were injected via the tail vein, respectively, while simultaneously being subjected to a 0.2 W / cm² concentration. 2 Processed under laser radiation and magnetic field conditions, to obtain Figure 11 ;

[0103] from Figure 11 Figure a shows that the H&E staining results of the hirudin magnetic nanoparticle group indicate that, compared with the untreated PBS group, the thrombolytic effect did not change significantly after magnetic field and laser treatment, indicating that 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 dissolved and the blood vessels were cleared, indicating thrombus dissolution.

[0104] Furthermore, even under low-power conditions, some thrombi were dissolved in the hirudin magnetic nanoparticle array, with varying laser powers, such as 0.1 W / cm². 2 Or 0.2W / cm 2 It will affect the thrombolysis effect. The higher the laser power, the better the thrombolysis effect. By calculating the area occupied by the thrombus in the entire blood vessel, the thrombolysis efficiency can be indirectly calculated.

[0105] At the same time, from Figure 11 Figure b shows that, simultaneously using 0.2 W / cm²... 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 cleared, showing a better thrombolysis effect compared to 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 co-incubated with 4T1 cells for 24 h. Cytotoxicity was detected by the MTT assay. The results are as follows: Figure 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 the hirudin magnetic nanoparticles have good safety within a certain concentration range.

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

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

[0111] Simultaneously, mice were treated by intravenous injection of hirudin magnetic nanoparticle solution or hirudin solution in the tail vein. Then, 0.5 cm of the mouse tail was cut off. The amount and time of bleeding were recorded at the laser irradiation site to conduct a tail bleeding experiment. The experimental results are as follows: Figure 13 As shown, where Figure 13 Figure a shows the amount of bleeding in the tail hemostasis experiment of mice after injection of different solutions. Figure 13 Figure b shows the bleeding time in the mouse tail hemostasis experiment after injection of different solutions, from the entire... Figure 13 It can be seen from this:

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

[0113] Furthermore, the safety of hirudin magnetic nanoparticles in vivo was assessed 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: Figure 14As shown, no inflammatory or necrotic areas were observed in the H&E staining images of any of the organs, indicating that hirudin magnetic nanoparticles have low toxicity and good biocompatibility in vivo.

[0114] In summary

[0115] This invention combines hollow gold nanorods, hirudin, and iron oxide particles to prepare a novel drug for treating thrombosis: hirudin magnetic nanoparticles. Encapsulated by iron oxide particles, these nanoparticles exhibit excellent magnetic targeting, allowing hirudin to rapidly accumulate at the thrombus site. Simultaneously, they possess the superior photothermal properties of hollow gold nanorods, enabling hirudin to be released into deep thrombus tissue under laser irradiation, ensuring the clinical efficacy of hirudin in treating thrombosis. These hirudin magnetic nanoparticles demonstrate strong thrombolytic ability, high bioavailability, and good temporary therapeutic effects.

[0116] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, 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 foregoing 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 type of hirudin magnetic nanoparticle for treating thrombosis, characterized in that: include; Hollow gold nanorods; Hirudin loaded in the inner cavity of the hollow gold nanorods; Iron oxide nanoparticles distributed on the surface of the hollow gold nanorods; The hollow gold nanorods are connected to the iron oxide nanoparticles via amide bonds; The hollow gold nanorods have a width of 10.1 nm and a length of 91.2 nm, the hirudin has a mass concentration of 1.0 mg / mL, and the iron oxide nanoparticles have a particle size of 10.0 nm. Furthermore, the hirudin magnetic nanoparticles synthesized from hollow gold nanorods, hirudin, and iron oxide nanoparticles have a width of 28.2 nm and a length of 91.2 nm. The specific steps for synthesizing the iron oxide nanoparticles are as follows: Step 2.1: Dissolve FeCl3·6H2O and FeCl2·4H2O in an aqueous HCl solution to prepare an iron salt solution for later use; Step 2.2: While stirring vigorously, add the iron salt solution dropwise to the alkaline solution and wait for 30.0 seconds; Step 2.3: Add stabilizer, stir, centrifuge the dark suspension, dialyze the supernatant to obtain iron oxide nanoparticles; The stabilizer was prepared by dissolving 0.4 g of polyacrylic acid in 5.0 mL of ultrapure water.

2. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 1, characterized in that: Includes the following steps: Step 1: Mix and stir the hollow gold nanorod solution with the hirudin aqueous solution to obtain a mixed solution; Step 2: Activate the iron oxide nanoparticles with N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to form reactive groups; Step 3: Add the reactive groups to the mixture, continue the reaction, and then centrifuge to purify and obtain hirudin magnetic nanoparticles.

3. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 2, characterized in that: In step 1, the hollow gold nanorod solution was 1.0 mL, the hirudin solution was 2.0 mg, and the mixing time was 12 h.

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

1.

5. The method for preparing hirudin magnetic nanoparticles for treating thrombosis according to claim 2, characterized in that: In step 3, the centrifugation speed is 10000 r / min, the centrifugation time is 10.0 min, and the reaction time is 4 h.

6. The hirudin magnetic nanoparticles for treating thrombosis according to claim 1, characterized in that: In step 2.1, FeCl3·6H2O is 0.3g, FeCl2·4H2O is 0.16g, and HCl aqueous solution is 2.0mL, 0.5mol / L; In step 2.2, the alkaline solution is prepared by dissolving 26% NH4OH in 20.0 mL of ultrapure water, which is 1.0 mL in volume.

7. The hirudin magnetic nanoparticles for treating thrombosis according to claim 1, characterized in that, In step 2.3: The stirring time was 1.0 h, and the stirring temperature was room temperature; The centrifugation speed was 4000 r / min, and the centrifugation time was 30.0 min; The dialysis bag has a capacity of 3.5 kDa, and the dialysis duration is 3 days.

8. The use of hirudin magnetic nanoparticles for treating thrombosis according to claim 1, characterized in that: Hirudin magnetic nanoparticles are targeted and enriched at thrombus sites under the action of an external magnetic field, and can be used to prepare drugs for photothermal therapy and prevention of thrombosis under 1064nm laser irradiation.

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