A photothermal nanomedicine containing a guanidine group structure, its preparation method and application

Abstract

The present invention belongs to the technical field of biomedical materials, and specifically relates to a guanidine group-containing photothermal nano-drug, its preparation method and application. The photothermal nano-drug is a covalent organic framework (COF) containing guanidine groups BT‑TG as a carrier, loaded with a photothermal agent TS, and then modified with a fibrin-targeting peptide CA on the surface to obtain CA-COF BT‑TG @TS. The photothermal nano-drug of the present invention is a covalent organic framework material platform, which can be enriched at the thrombus site, has excellent photothermal conversion performance, can effectively remove the thrombus at the lesion site, and at the same time, the nano-drug can also improve neuron death after ischemic stroke by inhibiting ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a photothermal nano-drug containing a guanidine group structure, a preparation method thereof, and an application thereof. Background Art

[0002] Ischemic stroke is the most common type of stroke, often secondary to cerebral artery occlusion, and thrombosis is the most common cause of cerebral artery occlusion. The treatment methods for ischemic stroke include intravenous thrombolysis and arterial mechanical thrombectomy. Clinical studies have shown that intravenous thrombolysis treatment can significantly improve the functional outcome of ischemic stroke patients within 4.5 hours, and its efficacy is related to time, with the best effect when treated within the first hour. Therefore, how to efficiently remove thrombus and how to rescue neuronal injury have become major challenges in the field of ischemic stroke treatment.

[0003] Ferroptosis is a new type of cell death characterized by iron ion-catalyzed lipid peroxidation. Research has shown that after ischemic stroke occurs, the level of iron ions in the brain increases, and the overloaded iron ions cause ferroptosis, while inhibiting ferroptosis can significantly reduce the infarct area of the brain and improve neurological function. Therefore, ferroptosis has become an important target for the treatment of ischemic stroke. Drugs targeting the ferroptosis target have been continuously updated in recent years, and the development of drugs with the mechanism of iron ion chelation has received extensive attention. However, the current drugs targeting iron ion chelation still have the following limitations: (1) it is difficult to penetrate the blood-brain barrier to reach the lesion area; (2) the iron ion chelation efficiency is low, and it cannot effectively rescue ischemic neuronal injury; (3) the function is single and cannot play a role in removing thrombus.

[0004] Covalent organic frameworks (COFs) are a class of porous materials formed by covalent bonds between organic precursors, and show great potential in the nano-drug delivery system due to their high specific surface area, uniform and adjustable pore size, and easy modification and functionalization. Metformin shows anti-inflammatory and antioxidant potential due to its special guanidine group structure. Using its guanidine group as the main functional group to construct guanidine covalent organic framework materials shows great potential in the development of anti-inflammatory carriers. Photothermal therapy can enhance the activity of thrombolytic drugs by increasing the temperature at the thrombus site, thereby improving the thrombolysis efficiency.

[0005] In view of this, it is necessary to develop a drug that directly combines guanidine COFs with photothermal therapy for the treatment of ischemic stroke and the ferroptosis target. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention aims to provide a photothermal nanomedicine containing a guanidine group structure, its preparation method and application. The photothermal nanomedicine has a covalent organic framework material platform, can be enriched at the thrombus site, has excellent photothermal conversion performance, can effectively remove the thrombus at the lesion site, and at the same time, the nanomedicine can also improve neuron death after ischemic stroke by inhibiting ferroptosis.

[0007] The first object of the present invention is to provide a photothermal nanomedicine containing a guanidine group structure, and the photothermal nanomedicine is obtained by using COF BT-TG as a carrier, loading a photothermal agent (TS), and then modifying a fibrin targeting peptide (CA) on the surface to obtain CA-COF BT-TG @TS; the COF BT-TG has the structure shown in formula (Ⅰ) and is a carrier containing a guanidine group:

[0008] ;

[0009] The photothermal agent has the structure shown in formula (Ⅱ):

[0010] ;

[0011] The amino acid sequence of the fibrin targeting peptide is Cys-Arg-Glu-Lys-Ala, abbreviated as CA.

[0012] The second object of the present invention is to provide a preparation method of a photothermal nanomedicine containing a guanidine group structure, which specifically includes the following steps:

[0013] (1) Preparation of COF BT-TG : First, dissolve BT (benzo[1,2-B:3,4-B':5,6-B']trithiophene-2,5,8-tricarbaldehyde) and TG (triaminoguanidine hydrochloride) in an ortho-dichlorobenzene / n-butanol mixed solution, add a catalyst to obtain a reaction solution, then subject the reaction solution after ultrasonic treatment for 5 min to 15 min to three freeze-thaw cycles, cyclic degassing, vacuum sealing, and then heat-treat by microwave radiation for 20 min to 40 min. After cooling to room temperature, centrifuge, wash the product, vacuum dry, perform Soxhlet extraction on the crude product and then freeze-dry, and obtain COF BT-TG after sufficient grinding;

[0014] (2) Preparation of COF BT-TG @TS: First, slowly drop a THF (tetrahydrofuran) solution containing TS into COF BT-TGIn a THF solution, a mixed solution was obtained, and then it was reacted on a magnetic stirrer at room temperature for 6 h to 12 h. The temperature was raised to 60 °C, and the experiment was stopped after THF was completely volatilized. After cooling, the product was ground thoroughly and then redispersed in PBS (phosphate buffer solution) with a pH of 7.4. It was dialyzed in PBS using a 3500 Da dialysis bag for 12 h to 24 h, and the dialysis solution was changed every 6 h to 8 h. The dialysis product was collected by centrifugation and freeze-dried to obtain the product COF BT-TG @TS;

[0015] (3) Preparation of CA-COF BT-TG @TS: The fibrin-targeting peptide and COF BT-TG @TS were separately dispersed in a plastic tube and a glass bottle containing THF. After ultrasonic dispersion in an ultrasonic machine for 10 min to 30 min, the glass bottle containing COF BT-TG @TS was placed in an ice box and put into the operation chamber of an ultrasonic cell disruptor. A Φ2 mm amplitude transformer was inserted into the glass bottle. While ultrasonic treatment was carried out, the fibrin-targeting peptide solution was added drop by drop. The whole process lasted for 20 min to 30 min until the fibrin-targeting peptide solution was completely added to the COF BT-TG @TS solution. Subsequently, with the parameters unchanged, the CA-COF BT-TG @TS system was continuously ultrasonically treated for 20 min to 30 min. Then the reaction solution was collected and naturally dried in a fume hood. The powder was ground and then redispersed in PBS with a pH of 7.4, and was loaded into a 3500 Da dialysis bag and dialyzed in PBS for 12 h to 24 h. The dialysis solution was changed every 6 h to 8 h. The dialysis product was collected by centrifugation and freeze-dried to obtain the product CA-COF BT-TG @TS.

[0016] Furthermore, in step (1) of the above technical solution, the molar ratio of BT to TG is 1 to 3:1 to 4; the concentration of BT in the reaction solution is 6.01 mg / mL to 14.16 mg / mL, and the concentration of TG is 2.55 mg / mL to 8.03 mg / mL; the catalyst is 3 M acetic acid; the volume ratio of o-dichlorobenzene, n-butanol, and acetic acid is 3 to 5:3 to 5:1.

[0017] Furthermore, in step (1) of the above technical solution, the temperature of the microwave radiation heating treatment is 100 °C to 120 °C; the method for washing the product is: first, the product is centrifugally washed with THF, then centrifugally washed with acetone, repeated 4 to 6 times, and 20 mL to 25 mL of THF or acetone is consumed for each washing. Then the product is washed by the Soxhlet extraction method with THF, and 150 mL to 250 mL of THF is consumed.

[0018] Further, in step (2) of the above technical solution, the concentration of TS in the mixed solution is 0.3 mg / mL to 0.8 mg / mL; the concentration of COF BT-TG in the mixed solution is 1.0 mg / mL to 2.0 mg / mL.

[0019] Further, in step (3) of the above technical solution, the power of the ultrasonic cell disruptor is 150 W; the mass ratio of the fibrin-targeting peptide to COF BT-TG @TS is 1:2 to 4.

[0020] Further, in each step of the above technical solution, the centrifugation speed is 6000 rpm to 10000 rpm, and the time is 3 min to 5 min.

[0021] The third object of the present invention is to provide an application of the above guanidine group-containing photothermal nano-drug in the preparation of a preparation for improving the inhibition of ferroptosis and improving neuronal death after ischemic stroke.

[0022] Beneficial effects compared with the prior art:

[0023] The present invention designs a guanidine-based photothermal nano-drug CA-COF BT-TG @TS, in which guanidine-based COFs themselves have certain anti-inflammatory and antioxidant effects, which can reduce the inflammatory response and oxidative stress caused by ischemic stroke and create a more favorable microenvironment for the thrombolytic drug to exert its effect; TS, as a new type of photothermal agent, not only significantly improves the photothermal performance and has stable performance after entering COF BT-TG , but also can destroy the structure of the thrombus, making it looser, and further promoting the action of the thrombolytic drug; after surface modification with CA, the photothermal nano-drug has excellent thrombus targeting ability, can directly reach the lesion, and has high efficiency. The photothermal nano-drug obtained by the present invention can significantly improve the thrombolysis efficiency through multiple mechanisms such as photothermal effect, targeted delivery, promoting osmotic diffusion, and synergistic effect, providing new ideas and methods for the treatment of ischemic stroke, especially the development of drugs targeting ferroptosis.

[0024] It can be seen from the test results of the present invention that the MCAO model mice treated with the designed CA-COF BT-TG @TS of the present invention can inhibit ferroptosis after ischemic stroke, significantly reduce the cerebral infarction area of the mice, and the neurological function score is significantly better than that of the model group, having unique advantages and application prospects. Brief Description of the Drawings

[0025] Figure 1 It is a preparation flow chart of CA-COF BT-TG @TS of the present invention;

[0026] Figure 2CA-COF in Example 1 of the present invention BT-TG SEM images (scale bar: 1 μm) and TEM images (scale bar: 0.1 μm) during the self-assembly process of @TS, where a is COF BT-TG , b is COF BT-TG @TS, c is CA-COF BT-TG @TS;

[0027] Figure 3 CA-COF in Example 1 of the present invention BT-TG Particle size distribution during the self-assembly process of @TS;

[0028] Figure 4 CA-COF in Example 1 of the present invention BT-TG Potential diagram during the self-assembly process of @TS;

[0029] Figure 5 CA-COF in Example 1 of the present invention BT-TG Powder X-ray diffraction pattern during the self-assembly process of @TS;

[0030] Figure 6 CA-COF in Example 1 of the present invention BT-TG Fourier transform infrared spectroscopy diagram during the self-assembly process of @TS;

[0031] Figure 7 CA-COF in Example 1 of the present invention BT-TG Specific surface area test diagram during the self-assembly process of @TS;

[0032] Figure 8 COF in Example 1 of the present invention BT-TG @TS and COF BT-TG Photothermal performance characterization diagram;

[0033] Figure 9 CA-COF in Example 1 of the present invention BT-TG Photothermal conversion efficiency test analysis diagram of @TS;

[0034] Figure 10 Cy5.5-COF in Example 2 of the present invention BT-TG @TS and Cy5.5-CA-COF BT-TG @TS Fourier transform infrared spectroscopy diagram;

[0035] Figure 11 Verification in artificial thrombus in Example 2 of the present invention that CA-COF BT-TG @TS can efficiently target the fluorescence intensity of thrombus and analysis diagram;

[0036] Figure 12 CA-COF in Example 2 of the present inventionBT-TG Experimental diagram of @TS effectively dissolving artificial thrombus;

[0037] Figure 13 CA-COF in Example 2 of the present invention BT-TG Statistical chart of in vitro thrombolysis rate of @TS;

[0038] Figure 14 CA-COF in Example 2 of the present invention BT-TG Statistical chart of fibrin content in the supernatant after @TS treatment of artificial thrombus;

[0039] Figure 15 CA-COF in Example 2 of the present invention BT-TG Statistical chart of hemoglobin content in the supernatant after @TS treatment of artificial thrombus;

[0040] Figure 16 CA-COF in Example 3 of the present invention for in vivo verification BT-TG Fluorescence intensity and analysis diagram of @TS efficiently targeting thrombus;

[0041] Figure 17 CA-COF in Example 3 of the present invention BT-TG Diagram of leg temperature change in mice treated with @TS;

[0042] Figure 18 CA-COF in Example 3 of the present invention BT-TG Diagram of leg blood flow change and blood perfusion analysis in femoral artery thrombosis model mice treated with @TS;

[0043] Figure 19 CA-COF in Example 3 of the present invention BT-TG Analysis diagram of blood perfusion in femoral artery thrombosis model mice treated with @TS at 120 min;

[0044] Figure 20 CA-COF in Example 4 of the present invention BT-TG Diagram of mouse tail changes and analysis on the first and fifth days after @TS treatment;

[0045] Figure 21 CA-COF in Example 4 of the present invention BT-TG Diagram of mouse tail changes and analysis from the sixth to the eighth days after @TS treatment;

[0046] Figure 22 CA-COF in Example 5 of the present invention BT-TG Statistical chart of neurological function changes in MCAO model mice treated with @TS;

[0047] Figure 23 CA-COF in Example 5 of the present invention BT-TG@TS processed TTC staining map and infarct area analysis map of MCAO model mice;

[0048] Figure 24 This is CA-COF in Example 5 of the present invention BT-TG @TS processed immunofluorescence map of PTGS2 in brain slices of MCAO model mice;

[0049] Figure 25 This is CA-COF in Example 6 of the present invention BT-TG @TS processed HE section maps of main organs of mice;

[0050] Figure 26 This is CA-COF in Example 6 of the present invention BT-TG Hemolysis experiment map of @TS at different concentrations. Detailed implementation manners

[0051] Any of the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these examples only, and these examples do not limit the present invention in any way.

[0052] The experimental methods in the following examples are all conventional methods unless otherwise specified. The preparations involved in the following examples are all ordinary commercially available products and can be obtained through market purchase unless otherwise specified.

[0053] The carrier COF described in the present invention BT-TG Using BT and TG as raw materials, after being prepared by high-temperature microwave reaction in the system of o-dichlorobenzene / n-butanol / acetic acid, the thermosensitive agent (TS) is integrated into the COF under stirring at room temperature BT-TG in the cavity, and then its surface is modified by CA for biochemical targeting to obtain the nano-drug system CA-COF BT-TG @TS. This CA-COF BT-TG @TS can significantly improve the thrombolysis efficiency, inhibit ferroptosis after ischemic stroke, and significantly reduce the infarct area of the mouse brain through multiple mechanisms such as photothermal effect, targeted delivery, promoting osmotic diffusion and synergistic effect, providing new ideas and methods for the treatment of ischemic stroke. Among them, the preparation flow chart of CA-COF BT-TG @TS is as Figure 1 shown.

[0054] The following further describes the present invention in detail with specific examples:

[0055] Example 1: Preparation method of CA-COF BT-TG @TS

[0056] CA-COFBT-TG Preparation method of @TS, including the following specific steps:

[0057] (1) Under microwave heating conditions, a solution was prepared by using BT (66 mg, 0.2 mmol) and TG (28 mg, 0.2 mmol) in a molar ratio of 1:1 in a mixture of o-dichlorobenzene / n-butanol / 3M acetic acid (7 mL, 3:3:1) to synthesize COF BT-TG ; Then, after ultrasonic treatment for 15 min, the reaction solution was subjected to three freeze-pump thaw cycles, cyclic degassing, vacuum sealing, and heated by microwave radiation at 120 °C for 20 min using a microwave synthesizer. After cooling to room temperature, the product was first centrifugally washed with THF, and then centrifugally washed with acetone, repeating the washing 3 times. After vacuum drying, the crude product was collected, and standard Soxhlet washing was performed using THF as a solvent to remove any impurities adsorbed in the porous structure. Finally, after vacuum freeze-drying for 6 h, the product was thoroughly ground to obtain yellow powder-like COF BT-TG , with a yield of 85.6%.

[0058] (2) 1 mL of a TS solution (0.6 mg / mL) prepared with THF was slowly added dropwise to 1 mL of a THF solution of COF BT-TG (2 mg / mL). Then, after reacting for 6 h on a magnetic stirrer at room temperature, the temperature was raised to 60 °C. After the THF completely evaporated, the experiment was stopped. After cooling, the product was thoroughly ground and then redispersed in PBS with a pH of 7.4, and dialyzed in PBS for 18 h using a 3500 Da dialysis bag, changing the dialysis solution every 6 h. The dialyzed product was collected by centrifugation, and the product was thoroughly ground to obtain yellow-green powder-like COF BT-TG @TS, with a yield of 77.9%.

[0059] (3) CA (1 mg / mL) and COF BT-TG @TS (3.0 mg / mL) were respectively dispersed in a plastic tube containing 2 mL of THF and a glass bottle containing 2 mL of THF. Then, the two bottles of liquid were respectively placed in an ultrasonic machine for ultrasonic dispersion for 20 min. During this period, an ice pack was placed in the ultrasonic machine for cooling. Subsequently, the glass bottle containing COF BT-TG @TS was fixed in an ice box and placed in the operation chamber of an ultrasonic cell disruptor. A Φ2 mm amplitude transformer was inserted into the glass bottle. While ultrasonic treatment was carried out, CA was added dropwise. The whole process lasted for 30 min until CA was completely added to COF BT-TG @TS. Subsequently, with the parameters unchanged, CA-COF BT-TGThe @TS system continued ultrasonic treatment for 20 min. The reaction solution was collected and naturally dried in a fume hood. The powder was ground and redispersed in PBS with a pH of 7.4, and then loaded into a 3500 Da dialysis bag and dialyzed in PBS for 24 h, with the dialysis solution replaced every 6 h. The dialysis product was collected by centrifugation, and the product was thoroughly ground to obtain yellowish-green powdery CA-COF BT-TG @TS, with a yield of 80.2%.

[0060] For the CA-COF BT-TG @TS synthesized in the examples of the present invention and its process products were characterized, and the characterization results are as Figures 2 - 9 shown:

[0061] Prepare a CA-COF BT-TG @TS dispersion (0.5 mg / mL) in PBS. Subsequently, it was dropped on a silicon wafer and dried at room temperature to obtain a sample for scanning electron microscopy (SEM). The solution was diluted 10 times and dropped on a copper grid to obtain a sample for transmission electron microscopy (TEM). Figure 2 SEM and TEM photos of the CA-COF BT-TG @TS and its process products prepared in the examples of the present invention.

[0062] Among them, the SEM image of the COF BT-TG prepared in step (1) shows irregularly shaped granular particles (as Figure 2 shown in a), and the TEM image shows that the surface contour of the COF BT-TG is obvious (as Figure 2 shown in the inset of a). After co-loading the photothermal agent in step (2), SEM shows that the morphology of the COF BT-TG @TS has no significant change (as Figure 2 shown in b), and TEM shows that smaller particles may appear in the particles, which may be TS in the system (as Figure 2 shown in the inset of b); after surface modification in step (3), the surface contour of the CA-COF BT-TG @TS becomes blurred, and the internal granularity is obvious (as Figure 2 shown in c and the inset).

[0063] Figure 3 And Figure 4 respectively show the particle size distribution and potential of the CA-COF BT-TG @TS and its process products prepared in the examples of the present invention. Dynamic light scattering measurement shows that after steps (2) and (3), the hydrodynamic diameter of the CA-COF BT-TG @TS increases ( Figure 3 ); the zeta potential of the CA-COF BT-TG @TS changes from positive to negative, confirming the successful loading of TSFigure 4 ).

[0064] Figure 5 The CA-COF prepared in the embodiment of the present invention BT-TG @TS and the powder X-ray diffraction (PXRD) pattern of its process products. Figure 5 Shows that the COF BT-TG Exhibits peaks at 7.22°, 12.24°, 13.22°, 19.14° and 27.2°, corresponding to the (100), (110), (200), (210) and (001) planes respectively. After drug loading and surface targeting peptide modification, the crystallinity of the COF BT-TG Is reduced.

[0065] Figure 6 The CA-COF prepared in the embodiment of the present invention BT-TG @TS and the Fourier transform infrared spectroscopy (FT-IR) pattern of its process products. In the figure, the FT-IR of COF BT-TG , COF BT-TG @TS and CA-COF BT-TG @TS are observed, and it is found that the characteristic C-S and C=N stretching vibration peaks appear at 660 cm -1 And 1666 cm -1 .

[0066] Figure 7 The CA-COF prepared in the embodiment of the present invention BT-TG @TS and the specific surface area test of its process products. The nitrogen adsorption-desorption isotherm data in the figure shows that the specific surface areas of COF BT-TG , COF BT-TG @TS and CA-COF BT-TG @TS decrease in turn, being 330.39 m 2 / g, 292.62 m 2 / g and 286.19 m 2 / g respectively.

[0067] Figure 8 The COF prepared in the embodiment of the present invention BT-TG @TS and the photothermal performance characterization diagram of COF BT-TG . Among them, the characterization method is as follows: The photothermal performance of the nanomaterials was characterized using an infrared thermal imager. First, water, COF BT-TG (200 μg / mL) and COF BT-TG @TS (200 μg / mL) were placed in a 200 μL centrifuge tube, irradiated with an 808 nm (0.33 W / cm 2 ) laser, and the temperature of the centrifuge tube was recorded in real time using an infrared thermal imager. From Figure 8As can be seen from a and b in [Figure / Table], after laser irradiation, the temperatures of different systems all increased to varying degrees and reached the temperature peak at around 300 s. Among them, the centrifuge tube filled with water rose from the original 25 °C to 25.3 °C, and the COF BT-TG group rose from the original 25.3 °C to 26.3 °C, while the COF BT-TG @TS group rose from the original 25.2 °C to 61.3 °C. This indicates that after loading TS, the photothermal performance of the nanodrug is significantly improved. To explore whether the photothermal performance of COF BT-TG @TS is stable, after continuous irradiation for 300 s, the laser was turned off and cooled for 300 s. Taking each cycle of turning on the laser for 300 s and then turning it off for 300 s, five cycles were carried out to observe the stability of the photothermal performance of COF BT-TG @TS. As can be seen from c and d in [Figure / Table], after five consecutive cycles, COF Figure 8 @TS still has good photothermal performance, which shows that the photothermal performance of COF BT-TG @TS is stable. BT-TG

[0068] Figure 9 This is the photothermal conversion efficiency test of CA-COF BT-TG @TS in the embodiment of the present invention. The change of the photothermal performance of COF BT-TG @TS with concentration was explored. Different concentrations of COF BT-TG @TS were placed in 200 μL centrifuge tubes and irradiated with 808 nm (0.33 W / cm 2 ²) laser for 300 s, and a thermal imager was used to record the temperature changes of each centrifuge tube. It was found that the temperature of the centrifuge tube filled with COF BT-TG @TS increased with the increase of the concentration of COF BT-TG @TS. Finally, the photothermal conversion efficiency of COF BT-TG @TS was calculated by the following formula ( ): , where represents the heat transfer coefficient of COF BT-TG @TS, represents the laser irradiation area, represents the highest temperature of the system, represents the lowest temperature of the system, represents the power density of the laser, represents the absorbance of COF BT-TG @TS at 808 nm wavelength, represents the heat dissipation of the solvent. As shown in Figure 9 , the calculated photothermal conversion efficiency of COF BT-TG @TS is 78.1%.

[0069] Example 2: In vitro evaluation of CA-COF BT-TG @TS's ability to dissolve thrombus

[0070] In vitro evaluation of CA-COF BT-TG @TS's ability to dissolve thrombus, and compare with TS, COF BT-TG and COF BT-TG @TS as a control.

[0071] (1) Preparation of artificial thrombus: 6-week-old C57BL / 6 mice were anesthetized with isoflurane using a small animal anesthetic machine, and then fresh blood was collected by orbital bleeding. 100 μL of blood was collected into a 1.5 mL centrifuge tube. CaCl 2 (3 mM) and thrombin lyophilized powder were mixed to prepare a solution of 25 U / mL. 10 μL of the above mixed solution was added to the 1.5 mL centrifuge tube containing fresh blood and left standing at 37 °C for 2 h. After 2 h, the blood clot was taken out and repeatedly washed with physiological saline to remove the uncoagulated blood on the surface, and finally an artificial thrombus was formed.

[0072] (2) Evaluation of CA-COF BT-TG @TS's binding ability to artificial thrombus: Cyanine5.5 (Cy5.5) is a fluorescent dye used for in vivo imaging. In order to visualize the targeting ability and delivery effect of CA-COF BT-TG @TS, Cy5.5-CA-COF BT-TG @TS, and Cy5.5-COF BT-TG @TS were prepared in this example.

[0073] Among them, the preparation method of Cy5.5-CA-COF BT-TG @TS is as follows:

[0074] ① Under microwave heating conditions, a solution was prepared by mixing BT (66 mg, 0.2 mmol) and TG (28 mg, 0.2 mmol) in a molar ratio of 1:1 in a mixture of o-dichlorobenzene / n-butanol / 3 M acetic acid (7 mL, 3:3:1) to synthesize COF BT-TG ; then after ultrasonic treatment for 15 min, the reaction solution was subjected to three freeze-pump thaw cycles, cyclic degassing, vacuum sealing, and heated by microwave radiation at 120 °C for 20 min using a microwave synthesizer. After cooling to room temperature, the product was first centrifuged and washed with THF, and then centrifuged and washed with acetone, and the washing was repeated 3 times. After vacuum drying, the crude product was collected, and standard Soxhlet extraction was performed using THF as the solvent to remove any impurities adsorbed in the porous structure. Finally, after vacuum freeze-drying for 6 h, the product was thoroughly ground to obtain a yellow powdery COF BT-TG , with a yield of 85.6%.

[0075] ② Slowly add 1 mL of a solution of TS (0.6 mg / mL) and Cy5.5 (0.05 mg / mL) prepared with THF dropwise to 1 mL of a THF solution of COF BT-TG (2 mg / mL). After reacting on a magnetic stirrer at room temperature for 6 h, raise the temperature to 60 °C. Stop the experiment after THF has completely evaporated. After cooling, grind the product thoroughly and redisperse it in PBS with a pH of 7.4. Then load it into a 3500 Da dialysis bag and dialyze it in PBS for 18 h, changing the dialysis fluid every 6 h. Centrifuge to collect the dialyzed product, grind the product thoroughly to obtain dark green powdered Cy5.5-COF BT-TG @TS, with a yield of 81.2%.

[0076] ③ Disperse CA (1 mg / mL) and Cy5.5-COF BT-TG @TS (3.0 mg / mL) separately in a plastic tube containing 2 mL of THF and a glass bottle containing 2 mL of THF. Then place the two bottles of liquid in an ultrasonic machine and ultrasonically disperse them for 20 min. During this period, an ice pack needs to be placed in the ultrasonic machine to cool down. Subsequently, fix the glass bottle containing COF BT-TG @TS in an ice box, put it into the operation chamber of an ultrasonic cell disruptor, insert a Φ2 mm amplitude transformer rod into the glass bottle, and add CA dropwise while ultrasonically treating. The whole process lasts for 30 min until CA is completely added to Cy5.5-COF BT-TG @TS. Subsequently, keep the parameters unchanged and continue to ultrasonically treat the Cy5.5-CA-COF BT-TG @TS system for another 20 min. Collect the reaction solution and let it dry naturally in a fume hood. Grind the powder and redisperse it in PBS with a pH of 7.4. Then load it into a 3500 Da dialysis bag and dialyze it in PBS for 24 h, changing the dialysis fluid every 6 h. Centrifuge to collect the dialyzed product, grind the product thoroughly to obtain dark green powdered Cy5.5-CA-COF BT-TG @TS, with a yield of 87.6%.

[0077] Figure 10 The FT-IR characterization data graphs of Cy5.5-CA-COF BT-TG @TS and Cy5.5-COF BT-TG @TS prepared in the examples of the present invention. It can be observed from the figure that infrared absorption peaks derived from the sulfonic acid groups in Cy5.5 appear in both Cy5.5-CA-COF BT-TG @TS and Cy5.5-COF BT-TG @TS. The main characteristic peaks appearing in the wavenumber range of 1000 cm -1 -1300 cm -1 are 1060 cm -1 and 1180 cm-1 , corresponding to the S-O and S=O stretching vibrations in the sulfonic acid group. At 1250 cm -1 -1400 cm -1 In the wavenumber range, the main characteristic peaks that appear are 1250 cm -1 and 1350 cm -1 , corresponding to the C-S and C-O stretching vibrations of the sulfonic acid group respectively. After modifying the targeting peptide CA, the infrared characteristic peak signal weakened, but there was no obvious shift or disappearance.

[0078] The artificially prepared thrombi were respectively infiltrated into PBS, Cy5.5-COF BT-TG @TS and Cy5.5-CA-COF BT-TG @TS. Using a small animal in vivo imaging system, images were taken at 0 h, 1 h, and 2 h (as shown in Figure 11 a), and the average fluorescence intensity at each time point was statistically analyzed (as shown in Figure 11 b). It can be seen from Figure 11 the results that at 1 h, the fluorescence intensity of the artificial thrombi co-incubated with Cy5.5-CA-COF BT-TG @TS was significantly higher than that of the artificial thrombi co-incubated with Cy5.5-COF BT-TG @TS. After 2 h, this difference became more obvious. It shows that the nano-drug grafted with the CA targeting peptide has excellent in vitro thrombus targeting ability.

[0079] (3) Evaluate the ability of CA-COF BT-TG @TS to clear artificial thrombi: Take 6 3-mL transparent glass vials, place the prepared artificial thrombi in the 3-mL glass vials, add 2.5 mL of PBS solution and 0.5 mL of the test mixture to the glass vials. The test mixture includes PBS, TS (200 μg / mL), COF BT-TG (200 μg / mL), COF BT-TG @TS (200 μg / mL), CA-COF BT-TG @TS (200 μg / mL) and urokinase (UK) (1.2×10 5 IU / mL). Expose the glass vials to a laser with a wavelength of 808 nm and a power density of 0.33 W / cm 2 for 20 min, and then incubate at 37 °C for 2 h. Dry the thrombi in an oven at 37 °C for 2 h to remove surface moisture, weigh and record and take pictures. The results are as shown in Figure 12As shown, it can be seen that after laser irradiation, the mixtures in each group turned red to varying degrees. The thrombolysis rate was further calculated, and the thrombolysis rate was calculated using the following formula: Thrombolysis rate = (mass of artificial thrombus before irradiation - mass of artificial thrombus after irradiation) / mass of artificial thrombus before irradiation. The results are as Figure 13 shown, PBS, TS, COF BT-TG , COF BT-TG @TS, CA-COF BT-TG @TS and the thrombolysis rates of UK were 9.7%, 47.62%, 21.86%, 55.36%, 81.14% and 24.69% respectively. At the same time, after incubation for 2 h, 200 μL of the supernatant in the glass bottle was collected into a 96-well plate, and the absorbance of the supernatant at OD 415 and OD 540 was measured using an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the contents of fibrin and hemoglobin in the supernatant. The results are as Figure 14 and Figure 15 shown. After laser irradiation, fibrin and hemoglobin in varying degrees could be detected in the supernatant of each group. Similar to the results of the thrombolysis rate, and the contents of fibrin and hemoglobin in the supernatant of the CA-COF BT-TG @TS group were the highest. Since thrombolytic drugs activate the fibrinolytic system, fibrinogen is degraded to produce fibrin (ogen) degradation products (FDP). The change in the content of FDP can reflect the effect of thrombolytic therapy. Generally speaking, the level of FDP will increase significantly after thrombolytic therapy. And during thrombolysis, if the thrombus dissolves, it may lead to intravascular hemolysis, thus increasing the content of free hemoglobin in the plasma. Detecting the content of hemoglobin can be used as an indirect indicator to evaluate the thrombolytic effect. If thrombolysis is successful, the content of free hemoglobin in the plasma may increase. Therefore, the above results further illustrate that CA-COF BT-TG @TS can achieve good thrombolytic effects.

[0080] Example 3: In vivo evaluation of the ability of CA-COF BT-TG @TS to remove thrombus

[0081] (1) Preparation of femoral artery FeCl 3 -induced thrombus model: After 6-week-old C57BL / 6 mice were anesthetized with isoflurane using a small animal anesthesia machine, the hair on the thigh of the mice was carefully cut off with scissors, the skin surface was wiped with 75% alcohol, the skin on the thigh was carefully cut open with scissors, and the superficial fascia and soft tissues were carefully separated under a microscope. Subsequently, a filter paper (3×1 mm) soaked with 10% FeCl 3 was applied to the surface of the femoral artery. After 5 min, the filter paper was removed and the residual FeCl 3. After 10 min, white thrombus formation could be seen in the femoral artery under the microscope. The laser speckle flow imaging system was used to detect the arterial blood flow. If the femoral artery blood flow decreased significantly, it indicated the successful preparation of the thrombus model.

[0082] (2) Evaluation of CA-COF BT-TG @TS Thrombus homing ability: The thrombus homing ability is the key for this nanodrug platform to clear thrombus. To evaluate the thrombus homing ability of CA-COF BT-TG @TS, Cy5.5-COF BT-TG @TS (10 mg / kg) and Cy5.5-CA-COF BT-TG @TS (10 mg / kg) were injected into the caudal vein of normal mice and mice in the femoral artery thrombus model group. After 2 h, the main organs (heart, liver, spleen, lung, and kidney) and femoral artery of the mice were collected. The small animal in vivo imaging system was used to observe the enrichment of the nanodrug (as shown in a of Figure 16 ), and the average fluorescence intensity in each main organ was counted (as shown in b of Figure 16 ). It could be seen from the results of Figure 16 that after thrombosis, the fluorescence intensity of the blood vessels increased significantly, indicating that the drug would accumulate at the lesion site in the thrombus state. When in the same thrombus state, the blood vessels grafted with CA targeting peptide showed stronger fluorescence intensity, which further proved that the nanodrug had excellent in vivo thrombus targeting ability after grafting with CA targeting peptide.

[0083] (3) To evaluate the ability of CA-COF BT-TG @TS to clear arterial thrombus, the drugs PBS, TS (10 mg / mL), COF BT-TG (10 mg / mL), COF BT-TG @TS (10 mg / mL), CA-COF BT-TG @TS (10 mg / mL) and UK (1.2×10 5 UI / mg) were injected into the carotid artery thrombus model mice through the caudal vein. After 30 min, the carotid artery of the mice was exposed to 808 nm (0.33 W / cm 2 ) for irradiation for 10 min, and at the same time, the laser speckle flow system was used to detect the blood flow. First, the thermal imager was used to measure the temperature change of the mouse leg after irradiation with 808 nm (0.33 W / cm 2 ), and the results were as shown in Figure 17 . It could be seen that after irradiation, the temperature of the mouse leg increased to varying degrees. Among them, the temperature of the mouse leg treated with CA-COF BT-TG @TS increased the most, reaching 44.3 °C, indicating that in vivo, the nanodrug could normally exert its photothermal efficacy. Immediately afterwards, the laser speckle system was used to measure the blood flow changes in different treatment groups (as shown inFigure 18 as shown in a of Figure 18 as shown in b, c, d, e, f, g of Figure 19 shown in Figure 18 and Figure 19 From the results of BT-TG and BT-TG @TS group, the blood perfusion recovered from 16.3% to 91.7% after 120 min; compared with the UK group, although there was good blood perfusion recovery from 10 min to 30 min at the beginning, its blood perfusion gradually decreased after 30 min and finally dropped to the lowest point at 120 min. In addition, by comparing the blood flow conditions of different treatment groups at 120 min, it was found that the blood perfusion recovery of the CA-COF BT-TG @TS group was significantly better than that of the COF

[0084] Example 4: In vivo evaluation of the ability of CA-COF BT-TG @TS to prevent thrombus recurrence

[0085] Carrageenan is widely used to induce venous thrombosis in mice. Carrageenan (20 mg / kg) was injected into 6-week-old male Kunming mice via the tail vein. The next day, the end of the mouse tail turned black, indicating the formation of tail vein thrombosis. The drugs PBS, COF BT-TG (10 mg / mL), COF BT-TG @TS (10 mg / mL), CA-COF BT-TG @TS (10 mg / mL) and UK (1.2×10 5 UI / mg) were injected into mice via the tail vein. After 30 min, the mouse tails were exposed to 808 nm (0.33 W / cm 2 ) for 10 min. The drugs were injected and the mice were irradiated at the same time every day for 5 consecutive days. The length of the black tails was photographed and recorded every day. The results are as Figure 20 shown. It can be seen that on the fifth day, the average length of the black tails of the mice in the PBS group increased by 3.1 cm, while the average length of the black tails of the CA-COF BT-TG @TS group decreased by 3.1 cm, indicating that CA-COF BT-TG @TS not only has good ability to clear arterial thrombus, but also has excellent ability to clear venous thrombus. To further evaluate the effect of CA-COF BT-TG @TS against thrombus recurrence, on the fifth day, CA-COF BT-TGThe @TS group and the UK group were reinjected with carrageenan (20 mg / kg) to simulate thrombus recurrence. The length of the black tails of the mice was recorded daily for the next three days. The results are as Figure 21 shown. It can be seen that on the third day, the average length of the black tails of the mice in the UK group increased by 2.1 cm, while that of the CA-COF BT-TG @TS mice only increased by 0.1 cm on average, indicating that CA-COF BT-TG @TS has good ability to resist thrombus recurrence at the same time.

[0086] Example 5: In vivo evaluation of CA-COF BT-TG @TS on the effect of rescuing ischemic stroke

[0087] To evaluate the effect of CA-COF BT-TG @TS in the treatment of ischemic stroke, the middle cerebral artery occlusion (MCAO) model was used to simulate ischemic stroke, and the nano-drug CA-COF BT-TG @TS was injected into the tail vein.

[0088] (1) Preparation of the middle cerebral artery occlusion model: The MCAO model was prepared by the suture occlusion method. The ischemic stroke induced by this method is close to the pathological state of cerebral ischemia in patients, and the operation is simple and the repeatability is good. The specific method is as follows: Male C57BL / 6 mice at 6 weeks of age were anesthetized with isoflurane gas, and the hair on the neck of the mice was carefully cut with scissors, and the neck of the mice was disinfected with 75% alcohol. Under the microscope, the left carotid artery and the vagus nerve of the mice were carefully dissected to expose the common carotid artery, internal carotid artery and external carotid artery. The external carotid artery and the proximal end of the common carotid artery were ligated with medical sutures, and the internal carotid artery was clamped with a small surgical clip. A small incision was made at the distal end of the common carotid artery, and the suture was carefully inserted into the internal carotid artery along the common carotid artery, and the small surgical clip was released at the same time so that the suture could reach the middle cerebral artery to play an occlusive role. After 90 min, the suture was removed, the wound was sutured, and the area was disinfected again with 75% alcohol. The nano-drug CA-COF BT-TG @TS was injected into the tail vein. Neurological function was evaluated using the Longa scale 72 h later. The specific scoring rules of this scale are as follows: no neurological deficit, 0 points; the front paw on the paralyzed side cannot be fully extended, 1 point; circling towards the paralyzed side when walking, 2 points; falling towards the paralyzed side when walking, 3 points; unable to walk automatically, with loss of consciousness, 4 points. The higher the score, the more severe the behavioral disorder of the animal. The results are as Figure 22 shown. It can be seen that the neurological function score of the mice treated with CA-COF BT-TG @TS was significantly better than that of the model group.

[0089] (2) Evaluation of cerebral infarction area by TTC (2,3,5-triphenyltetrazolium chloride) staining: After the above-mentioned MCAO model was prepared, CA-COF was injected into the mice via the tail vein. BT-TG @TS (10 mg / kg). After 72 h, the mice were anesthetized with isoflurane gas, the heart was exposed, and the mice were perfused with normal saline. Subsequently, the mouse brain was carefully removed with ophthalmic scissors and forceps and placed in a -20 °C refrigerator for 20 min. After the brain was frozen, the brain was cut into slices every 2 mm along the coronal plane, a total of 5 slices were cut. The specific method is as follows: The first cut is at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm, the second cut is at the optic chiasm, the third cut is at the infundibulum, and the fourth cut is between the infundibulum and the caudal end of the posterior lobe. The cut brain slices were placed in a six-well plate containing TTC staining solution and incubated at 37 °C in the dark for 30 min, and then photographed and recorded (as shown in a of Figure 23 . At the same time, Image J software was used to analyze the infarction area (as shown in b of Figure 23 . It can be seen from the Figure 23 results that CA-COF BT-TG @TS treatment significantly reduced the cerebral infarction area in mice.

[0090] (3) Evaluation of ferroptosis level after treatment by tissue immunofluorescence: PTGS2 is considered a marker of ferroptosis. Therefore, the content of PTGS2 in the mouse brain after treatment with the nanodrug was analyzed in this example. After the above-mentioned MCAO model was prepared, CA-COF was injected into the mice via the tail vein. BT-TG @TS. One week later, the mice were anesthetized with isoflurane gas, the heart was exposed, and the mice were perfused with normal saline; then the mouse brain was carefully removed with ophthalmic scissors and forceps and fixed in 4% paraformaldehyde; then embedded in paraffin, sectioned along the coronal plane, dewaxed, and incubated with 10% goat serum at room temperature for 60 min; the goat serum was washed with PBS, and then PTGS2 and Neun primary antibodies diluted 1:200 were added and incubated overnight at 4 °C. The next day, after washing the primary antibody, a fluorescent secondary antibody diluted 1:200 was added and incubated in the dark for 60 min. The fluorescent secondary antibody was washed with PBS, DAPI (4',6-diamidino-2-phenylindole) was added to stain the cell nuclei, and then sealed with an anti-fluorescence quencher, photographed and recorded with a confocal microscope, and the results are as shown in Figure 24 . It can be seen that compared with the model group, after treatment with CA-COF BT-TG @TS, the fluorescence intensity of PTGS2 was significantly weakened, indicating that CA-COF BT-TG @TS can inhibit ferroptosis after ischemic stroke.

[0091] Example 6: Biosafety evaluation of CA-COF BT-TG @TS

[0092] To verify CA-COFBT-TG @TS's biosafety was evaluated by injecting CA-COF into MCAO model mice via the tail vein BT-TG @TS. One week later, the mice were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were removed, fixed with 4% paraformaldehyde, embedded in paraffin, deparaffinized, and stained with hematoxylin and eosin. The HE (hematoxylin-eosin) staining procedure is as follows: Stain with hematoxylin for 10-15 minutes; Decolorize with 0.5%-1.0% hydrochloric acid alcohol for several seconds to dozens of seconds, and quickly wash with distilled water; Blue with dilute ammonia water, wash with distilled water, and check with a microscope while differentiating; Rinse with running water for 3 minutes to remove alkaline moisture; Stain with 1% eosin for 5-10 minutes; Quickly wash with distilled water, dehydrate step by step with alcohol, that is, quickly wash with 70%, 80%, and 90% alcohol, 30s-60s with 95% alcohol, 3 minutes with 100% alcohol, twice; Xylene for 5 minutes, twice; Finally, mount with neutral gum and observe under a microscope. The results are as Figure 25 shown, CA-COF BT-TG @TS did not cause pathological damage to the main organs. In addition, the hemolysis experiment was further used to evaluate the biocompatibility of CA-COF BT-TG @TS. Take 10 clean EP tubes, add 900 μL of 2% red blood cell suspension to each tube, and then add 100 μL of 0.1% Triton X-100, normal saline, 12, 15, 25, 30, 50, 60, 100, 120 μg / mL of CA-COF BT-TG @TS respectively. Incubate in a 37 °C water bath for 4 hours, then centrifuge at 3000 rpm for 15 minutes, take pictures to record the hemolysis of each group, and at the same time take 200 μL of the supernatant from each group into a 96-well plate, and measure the absorbance of the sample at 540 with an enzyme-labeled instrument. Use the following formula to calculate the hemolysis rate: Hemolysis rate (%) = (OD 样本 -OD 生理盐水组 ) / (OD ddH2O -OD 生理盐水组 ). The results are as Figure 26 shown, and the hemolysis rate at each concentration is less than 5%, indicating that CA-COF BT-TG @TS has good biocompatibility.

[0093] In summary, the present invention uses a covalent organic framework material containing a guanidine group structure as a carrier platform, integrates the thermosensitive agent TS into the COF BT-TG cavity, and then performs biomimetic targeting modification on its surface with CA to obtain the photothermal nanodrug CA-COF BT-TG@TS has good biosafety and can significantly improve the thrombolysis efficiency, inhibit ferroptosis after ischemic stroke, and significantly reduce the cerebral infarction area in mice through multiple mechanisms such as photothermal effect, targeted delivery, promoting osmotic diffusion, and synergistic effect, providing new ideas and methods for the treatment of ischemic stroke.

[0094] Finally, it should be emphasized that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A photothermal nanomedicine containing a guanidine structure, characterized in that: The photothermal nanomedicine is based on COF BT-TG CA-COF was obtained by loading the photothermal agent and modifying the fibrin targeting peptide on the surface. BT-TG @TS; the COF BT-TG It has a structure as shown in formula (I): ； The photothermal agent has a structure as shown in formula (II): ； The amino acid sequence of the fibrin targeting peptide is Cys-Arg-Glu-Lys-Ala.

2. The method for preparing a photothermal nanomedicine containing a guanidine structure according to claim 1, characterized in that: The specific steps include: (1) Preparation of COF BT-TG BT and TG were first dissolved in a mixed solution of o-dichlorobenzene / n-butanol, and a catalyst was added to obtain a reaction solution. The reaction solution was then subjected to ultrasonic treatment for 5 min to 15 min and then frozen and thawed three times, degassed in cycles, and vacuum sealed. The reaction solution was then subjected to microwave radiation heating for 20 min to 40 min. After cooling to room temperature, the product was centrifuged, washed, vacuum dried, and the crude product was subjected to Soxhlet extraction and freeze-dried. COF was obtained after sufficient grinding. BT-TG ; (2) Preparation of COF BT-TG @TS: First, slowly add the THF solution containing TS to the COF BT-TG The mixture was added to a THF solution to obtain a mixed solution, which was then reacted on a magnetic stirrer at room temperature for 6 h to 12 h. The temperature was raised to 60 ° C. The experiment was stopped after THF was completely volatilized. After cooling, the product was fully ground and redispersed in PBS with a pH of 7.

4. The product was dialyzed in PBS using a 3500Da dialysis bag for 12 h to 24 h. The dialyzate was replaced every 6 h to 8 h. The dialyzed product was collected by centrifugation and freeze-dried to obtain the product COF BT-TG @TS; (3) Preparation of CA-COF BT-TG @TS: Fibrin targeting peptides and COFs BT-TG @TS was dispersed in plastic tubes and glass bottles containing THF, and then ultrasonically dispersed in an ultrasonic machine for 10 min to 30 min. BT-TG The glass bottle of @TS was placed in an ice box and placed in the operating chamber of the ultrasonic cell disruptor. A Φ2 mm amplitude rod was inserted into the glass bottle. The fibrin targeting peptide solution was added dropwise during ultrasonic treatment. The whole process lasted for 20 min to 30 min until the fibrin targeting peptide solution was completely added to COF BT-TG @TS solution, then, the parameters remain unchanged for CA-COF BT-TG The @TS system was subjected to ultrasonic treatment for 20 min to 30 min, and then the reaction solution was collected and placed in a fume hood to be naturally drained. The powder was ground and redispersed in PBS with a pH of 7.4, and then placed in a 3500Da dialysis bag and dialyzed in PBS for 12 h to 24 h. The dialysis solution was replaced every 6 h to 8 h, and the dialyzed product was collected by centrifugation and freeze-dried to obtain the product CA-COF BT-TG @TS.

3. The preparation method according to claim 2, characterized in that: In step (1), the molar ratio of BT to TG is 1-3:1-4; the concentration of BT in the reaction solution is 6.01 mg / mL-14.16 mg / mL, and the concentration of TG is 2.55 mg / mL-8.03 mg / mL; the catalyst is 3M acetic acid; and the volume ratio of o-dichlorobenzene, n-butanol, and acetic acid is 3-5:3-5:

1.

4. The preparation method according to claim 2, characterized in that: In step (1), the temperature of the microwave radiation heating treatment is 100°C to 120°C; the method for washing the product is: first centrifugally washing the product with THF, then centrifugally washing the product with acetone, repeating 4 to 6 times, each washing consuming 20 mL to 25 mL of THF or acetone, and then washing the product with THF combined with Soxhlet extraction, consuming 150 mL to 250 mL of THF.

5. The preparation method according to claim 2, characterized in that: In step (2), the concentration of TS in the mixed solution is 0.3 mg / mL to 0.8 mg / mL; the concentration of COF in the mixed solution is BT-TG The concentration is 1.0mg / mL~2.0mg / mL.

6. The preparation method according to claim 2, characterized in that In step (3), the power of the ultrasonic cell disruptor is 150 W; the fibrin targeting peptide and COF BT-TG @TS mass ratio is 1:2~4.

7. The preparation method according to claim 2, characterized in that: In each step, the centrifugal speed is 6000rpm~10000rpm, and the time is 3min~5min.

8. Use of the photothermal nanomedicine containing a guanidine structure as claimed in claim 1 or the photothermal nanomedicine containing a guanidine structure prepared by the preparation method according to any one of claims 2 to 7 in the preparation of a preparation for improving neuronal death after ischemic stroke.

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