Photo-thermal nano-drug containing guanidyl structure as well as preparation method and application of photo-thermal nano-drug

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a photothermal nano-drug containing a guanidyl structure as well as a preparation method and application of the photothermal nano-drug, and the photothermal nano-drug is CA-COFBT-TG (at) TS obtained by taking COFBT-TG containing guanidyl as a carrier, loading a photothermal agent TS and then modifying a fibrin targeting peptide CA on the surface. The photo-thermal nano-drug is a covalent organic framework material platform, can be enriched at a thrombus part, has excellent photo-thermal conversion performance, can effectively remove thrombus at a focus part, and can improve neuronal death after cerebral arterial thrombosis by inhibiting ferroptosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a photothermal nanomedicine containing a guanidine structure, and a preparation method and application thereof. Background Art

[0002] Ischemic stroke is the most common type of stroke, often secondary to cerebral artery occlusion, of which thrombosis is the most common cause. Treatments for ischemic stroke include intravenous thrombolysis and mechanical arterial thrombectomy. Clinical studies have shown that intravenous thrombolysis can significantly improve the functional outcomes of ischemic stroke patients within 4.5 hours. Its efficacy is time-related, and the best effect is achieved within the first hour. Therefore, how to efficiently remove thrombi and how to save neuronal damage have become major challenges in the treatment of ischemic stroke.

[0003] Ferroptosis is a new type of cell death catalyzed by iron ions and characterized by lipid peroxidation. Studies have shown that after an ischemic stroke, the iron ion level in the brain increases, and the overload of iron ions causes ferroptosis. Inhibiting ferroptosis can significantly reduce the area of ​​cerebral infarction and improve neurological function. Therefore, ferroptosis has become an important target for the treatment of ischemic stroke. Drugs targeting ferroptosis have been continuously updated in recent years, among which the development of drugs based on iron ion chelation has received widespread attention. However, 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 efficiency of iron ion chelation is low and cannot effectively rescue post-ischemic neuronal damage; (3) The action is single and cannot play a role in clearing blood clots.

[0004] Covalent organic frameworks (COFs) are a class of porous materials formed by covalent bonds between organic precursors. They show great potential in nano drug delivery systems due to their high specific surface area, uniform and adjustable pore size, and easy modification and functionalization. Metformin has anti-inflammatory and antioxidant potential due to its special guanidine structure. Using its guanidine 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 thrombolytic efficiency.

[0005] Therefore, it is necessary to develop a drug that directly combines guanidine COFs with photothermal therapy for the treatment of ischemic stroke and ferroptosis targets. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a photothermal nanodrug containing a guanidine structure and a preparation method and application thereof. The photothermal nanodrug has a covalent organic framework material platform, can be enriched at the thrombus site, has excellent photothermal conversion performance, and can effectively remove thrombus at the lesion site. At the same time, the nanodrug can also improve neuronal death after ischemic stroke by inhibiting ferroptosis.

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

[0008] The second object of the present invention is to provide a method for preparing a photothermal nanomedicine containing a guanidine structure, which specifically comprises the following steps: (1) Preparation of COF BT-TG : First, BT (benzo[1,2-B:3,4-B':5,6-B']terthiophene-2,5,8-trialdehyde) and TG (triaminoguanidine hydrochloride) were 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, cyclically degassed, vacuum sealed, and then heated by microwave radiation 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, and fully ground to obtain COF. BT-TG ; (2) Preparation of COF BT-TG @TS: First, slowly add the THF (tetrahydrofuran) 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 (phosphate buffer) 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.

[0009] Furthermore, in step (1) of the above technical solution, 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.

[0010] Furthermore, in step (1) of the above technical solution, the temperature of the microwave radiation heating treatment is 100°C~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 20mL~25mL of THF or acetone, and then washing the product with THF combined with Soxhlet extraction, consuming 150mL~250mL of THF.

[0011] Furthermore, 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 in the mixed solution is BT-TG The concentration is 1.0mg / mL~2.0mg / mL.

[0012] Furthermore, in step (3) of the above technical solution, 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.

[0013] Furthermore, in the above technical solution, in each step, the centrifugal speed is 6000rpm~10000rpm, and the time is 3min~5min.

[0014] The third object of the present invention is to provide an application of the above-mentioned photothermal nanomedicine containing a guanidine structure in the preparation of a preparation for improving the inhibition of ferroptosis and improving neuronal death after ischemic stroke.

[0015] Advantages compared to the prior art: The present invention designs a guanidine-based photothermal nanomedicine CA-COF BT-TG @TS, guanidine 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 thrombolytic drugs to exert their effects; TS, as a new type of photothermal agent, enters COF BT-TG After that, not only the photothermal performance is significantly improved and the performance is stable, but also the structure of the thrombus can be destroyed, making it looser, further promoting the effect of thrombolytic drugs; after surface modification with CA, the photothermal nanomedicine has excellent thrombus targeting ability, can reach the lesion directly, and has high efficiency. The photothermal nanomedicine obtained by the present invention can significantly improve the thrombolytic efficiency through multiple mechanisms such as photothermal effect, targeted delivery, promotion of penetration and diffusion, and synergistic effect, providing new ideas and methods for the treatment of ischemic stroke, especially the development of ferroptosis target drugs.

[0016] It can be seen from the experimental structure of the present invention that the CA-COF designed by the present invention BT-TG @TS-treated MCAO model mice can inhibit ferroptosis after ischemic stroke and significantly reduce the area of ​​cerebral infarction in mice. The neurological function scores are significantly better than those in the model group, which has unique advantages and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 CA-COF of the present invention BT-TG @TS preparation flow chart; Figure 2 CA-COF in Example 1 of the present invention BT-TG @TS SEM images (scale bar: 1 μm) and TEM images (scale bar: 0.1 μm) during self-assembly, where a is COF BT-TG , b is COF BT-TG @TS, c is CA-COF BT-TG @TS; Figure 3 CA-COF in Example 1 of the present invention BT-TG @TS particle size distribution during self-assembly; Figure 4CA-COF in Example 1 of the present invention BT-TG @TS potential diagram during self-assembly; Figure 5 CA-COF in Example 1 of the present invention BT-TG @TS powder X-ray diffraction pattern during self-assembly; Figure 6 CA-COF in Example 1 of the present invention BT-TG @TS Fourier transform infrared spectra during self-assembly; Figure 7 CA-COF in Example 1 of the present invention BT-TG @TS surface area test diagram during self-assembly process; Figure 8 COF in Example 1 of the present invention BT-TG @TS and COF BT-TG Characterization diagram of photothermal performance; Fig. 9 CA-COF in Example 1 of the present invention BT-TG @TS photothermal conversion efficiency test analysis chart; Fig.10 Cy5.5-COF in Example 2 of the present invention BT-TG @TS and Cy5.5-CA-COF BT-TG Fourier transform infrared spectrum of @TS; Fig.11 The CA-COF was verified in the artificial thrombus in Example 2 of the present invention. BT-TG @TS can effectively target the fluorescence intensity and analysis of thrombus; Fig.12 CA-COF in Example 2 of the present invention BT-TG @TS can effectively dissolve artificial blood clots; Fig.13 CA-COF in Example 2 of the present invention BT-TG @TS In vitro thrombolysis rate statistics; Fig.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; Fig.15 CA-COF in Example 2 of the present invention BT-TG @Statistical graph of hemoglobin content in the supernatant after TS treated artificial thrombus; Fig.16 In Example 3 of the present invention, CA-COF was verified in vivo BT-TG @TS can effectively target the fluorescence intensity and analysis of thrombus; Fig.17CA-COF in Example 3 of the present invention BT-TG @Temperature changes in the legs of mice treated with TS; Fig.18 CA-COF in Example 3 of the present invention BT-TG @Chart of blood flow changes and blood perfusion analysis in the legs of mice with femoral artery thrombosis model treated with TS; Fig.19 CA-COF in Example 3 of the present invention BT-TG @Analysis of blood perfusion in TS-treated femoral artery thrombosis model mice at 120 minutes; Fig. 20 CA-COF in Example 4 of the present invention BT-TG @Changes and analysis of mouse tails on the first and fifth days after TS treatment; Fig.21 CA-COF in Example 4 of the present invention BT-TG @Changes and analysis of mouse tails from the sixth to eighth day after TS treatment; Fig. 22 CA-COF in Example 5 of the present invention BT-TG @Statistical chart of changes in neurological function in MCAO model mice treated with TS; Fig.23 CA-COF in Example 5 of the present invention BT-TG @TTC staining and infarct area analysis of MCAO model mice treated with TS; Fig.24 CA-COF in Example 5 of the present invention BT-TG @PTGS2 immunofluorescence images of brain sections of MCAO model mice treated with TS; Fig.25 CA-COF in Example 6 of the present invention BT-TG @HE sections of the main organs of TS-treated mice; Fig.26 CA-COF in Example 6 of the present invention BT-TG @TS hemolysis experiment diagram at different concentrations. DETAILED DESCRIPTION

[0018] The above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0019] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0020] The carrier COF of the present invention BT-TG Using BT and TG as raw materials, after the preparation was completed by high-temperature microwave reaction in the system of o-dichlorobenzene / n-butanol / acetic acid, the thermosensitizer (TS) was integrated into the COF under stirring at room temperature. BT-TG The surface of the cavity was then biomimetic-targeted modified with CA to obtain the guanidine-containing nanodrug system CA-COF. BT-TG @TS. The CA-COF BT-TG @TS can significantly improve the efficiency of thrombolysis, inhibit ferroptosis after ischemic stroke, and significantly reduce the area of ​​cerebral infarction in mice through multiple mechanisms such as photothermal effect, targeted delivery, promotion of penetration and diffusion, and synergistic effect, providing new ideas and methods for the treatment of ischemic stroke. BT-TG The preparation flow chart of @TS is as follows Figure 1 shown.

[0021] The present invention is further described in detail below in conjunction with specific embodiments: Example 1: CA-COF BT-TG Preparation method of @TS CA-COF BT-TG The preparation method of @TS comprises the following specific steps: (1) COF was synthesized by preparing a solution with BT (66 mg, 0.2 mmol) and TG (28 mg, 0.2 mmol) in a 1:1 molar ratio in a mixture of o-dichlorobenzene / n-butanol / 3 M acetic acid (7 mL, 3:3:1) under microwave heating conditions. BT-TG ; Then after ultrasonic treatment for 15 minutes, 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 minutes using a microwave synthesizer. After cooling to room temperature, the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated 3 times. The crude product was collected after vacuum drying, and THF was used as a solvent for standard Soxhlet washing to remove any impurities adsorbed in the porous structure. Finally, after vacuum freeze drying for 6 hours, the product was fully ground to obtain a yellow powder COF BT-TG , with a yield of 85.6%.

[0022] (2) Slowly add 1 mL of TS solution (0.6 mg / mL) prepared in THF to 1 mL of COF BT-TGThe product was then added to a THF solution (2 mg / mL) and then reacted on a magnetic stirrer at room temperature for 6 h. The temperature was then 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 placed in a 3500 Da dialysis bag and dialyzed in PBS for 18 h. The dialysate was replaced every 6 h. The dialyzed product was collected by centrifugation and fully ground to obtain a yellow-green powdered COF. BT-TG @TS, the yield is 77.9%.

[0023] (3) CA (1 mg / mL) and COF BT-TG @TS (3.0 mg / mL) was dispersed in a plastic tube containing 2 mL of THF and a glass bottle containing 2 mL of THF, and then the two bottles of liquid were placed in an ultrasonic machine for ultrasonic dispersion for 20 minutes. During this period, an ice bag was placed in the ultrasonic machine to cool it down. BT-TG The glass bottle of @TS was fixed 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. CA was added dropwise during ultrasonic treatment. The whole process lasted for 30 minutes until CA was completely added to COF. BT-TG @TS, then, the parameters remain unchanged for CA-COF BT-TG The @TS system was ultrasonically treated for 20 min. The reaction solution was collected and placed in a fume hood to dry naturally. The powder was ground and redispersed in PBS with a pH of 7.4. The powder was placed in a 3500Da dialysis bag and dialyzed in PBS for 24 h. The dialysis solution was replaced every 6 h. The dialyzed product was collected by centrifugation and fully ground to obtain a yellow-green powdery CA-COF. BT-TG @TS, the yield is 80.2%.

[0024] The CA-COF synthesized in the embodiment of the present invention BT-TG @TS and its process products are characterized, and the characterization results are as follows Figure 2~Figure 9 As shown: Preparation of PBS-dispersed CA-COF BT-TG @TS dispersion (0.5 mg / mL) was then dropped on a silicon wafer and dried at room temperature to obtain a scanning electron microscope (SEM) sample. The solution was diluted 10 times and dropped on a copper grid to obtain a transmission electron microscope (TEM) sample. Figure 2 CA-COF prepared in the embodiment of the present invention BT-TG @SEM and TEM photos of TS and its process products.

[0025] Among them, the COF prepared in step (1) BT-TG The SEM images show irregular granular morphology (e.g. Figure 2 (a) TEM image shows COF BT-TGThe surface contour edges are obvious (such as Figure 2 After the photothermal agent is co-loaded in step (2), SEM shows that COF BT-TG @TS No significant changes in appearance (such as Figure 2 As shown in b in the figure, TEM shows that the smaller particles in the particles may be TS in the system (such as Figure 2 As shown in the inset in b); after the surface modification in step (3), CA-COF BT-TG @TS's surface contours become blurred, and the internal granularity is obvious (such as Figure 2 c and illustration).

[0026] Figure 3 and Figure 4 The CA-COF prepared in the embodiment of the present invention are BT-TG @TS and its process products. Dynamic light scattering measurements show that after steps (2) and (3), CA-COF BT-TG @TS hydrodynamic diameter increases ( Figure 3 );CA-COF BT-TG @The zeta potential of TS changes from positive to negative, confirming the successful loading of TS ( Figure 4 ).

[0027] Figure 5 CA-COF prepared in the embodiment of the present invention BT-TG Powder X-ray diffraction (PXRD) patterns of @TS and its process products. Figure 5 Shows COF BT-TG Peaks appeared at 7.22°, 12.24°, 13.22°, 19.14°, and 27.2°, corresponding to the (100), (110), (200), (210), and (001) planes, respectively. The COF was reduced after drug loading and surface targeting peptide modification. BT-TG of crystallinity.

[0028] Figure 6 CA-COF prepared in the embodiment of the present invention BT-TG Fourier transform infrared spectra (FT-IR) of @TS and its process products. COF was observed in the figure. BT-TG 、COF BT-TG @TS and CA-COF BT-TG @TS FT-IR shows that the characteristic CS and C=N stretching vibration peaks appear at 660 cm -1 and 1666cm -1 .

[0029] Figure 7 CA-COF prepared in the embodiment of the present invention BT-TG@TS and its process product specific surface area test. The nitrogen adsorption-desorption isotherm data in the figure show COF BT-TG 、COF BT-TG @TS and CA-COF BT-TG The specific surface areas of @TS decrease successively, which are 330.39m 2 / g, 292.62m 2 / g and 286.19m 2 / g.

[0030] Figure 8 COF prepared in the embodiment of the present invention BT-TG @TS and COF BT-TG The photothermal performance characterization diagram of the nanomaterials is shown in Figure 1. The characterization method is as follows: the photothermal performance of the nanomaterials is characterized using a thermal imager. First, water, COF BT-TG (200 μg / mL) and COF BT-TG @TS (200 μg / mL) was placed in a 200 μL centrifuge tube and the 808 nm (0.33 W / cm 2 ) laser irradiation, and use a thermal imager to record the temperature of the centrifuge tube in real time. Figure 8 As can be seen from a and b in the figure, after laser irradiation, the temperature of different systems increased to varying degrees and reached a peak temperature around 300s. The temperature of the centrifuge tube containing water increased from the original 25℃ to 25.3℃, and COF BT-TG The temperature of the group increased from 25.3℃ to 26.3℃, while COF BT-TG The temperature of the @TS group increased from 25.2℃ to 61.3℃. This shows that the photothermal performance of the nanomedicine was significantly improved after TS was encapsulated. BT-TG @TS photothermal performance is stable. After continuous irradiation for 300s, turn off the laser and cool for 300s. Perform five cycles with 300s of laser on and 300s of laser off as one cycle. Observe the COF BT-TG @TSThe stability of photothermal performance. Figure 8 As can be seen from c and d in Figure 5, after five consecutive cycles, COF BT-TG @TS still has good photothermal performance, which shows that COF BT-TG @TS's photothermal performance is stable.

[0031] Fig. 9 CA-COF according to the present invention BT-TG @TS's photothermal conversion efficiency test. BT-TG @TS photothermal performance varies with concentration. BT-TG @TS was placed in a 200μL centrifuge tube and the 808nm (0.33W / cm2 ) laser irradiation for 300s, and the temperature changes of each centrifuge tube were recorded using a thermal imager. BT-TG @TS centrifuge tube temperature increases with COF BT-TG Finally, COF was calculated by the following formula: BT-TG @TS photothermal conversion efficiency ( ): ,in Representing COF BT-TG @TS heat transfer coefficient, represents the laser irradiation area, represents the maximum temperature of the system, represents the lowest temperature of the system, represents the power density of the laser, Representing COF BT-TG @TS absorbance at 808 nm, represents the heat dissipation of the solvent. Fig. 9 As shown, the COF is calculated BT-TG @TS's photothermal conversion efficiency is 78.1%.

[0032] Example 2: In vitro evaluation of CA-COF BT-TG @TS's ability to clear blood clots In vitro evaluation of CA-COF BT-TG @TS's ability to clear thrombus, and TS, COF BT-TG and COF BT-TG @TS for comparison.

[0033] (1) Preparation of artificial thrombus: After 6-week-old C57BL / 6 mice inhaled isoflurane using a small animal anesthesia instrument, fresh blood was collected through the eye sockets and 100 μL of blood was collected into a 1.5 mL centrifuge tube. CaCl2 (3 mM) and lyophilized thrombin powder were mixed to prepare a 25 U / mL solution. 10 μL of the above mixed solution was added to a 1.5 mL centrifuge tube containing fresh blood and allowed to stand at 37°C for 2 h. After 2 h, the blood clot was removed and repeatedly washed with saline to remove the uncoagulated blood on the surface, and finally an artificial thrombus was formed.

[0034] (2) Evaluation of CA-COF BT-TG @TS binding ability to artificial thrombus: Cyanine5.5 (Cy5.5) is a fluorescent dye used for in vivo imaging. BT-TG @TS targeting ability and delivery effect visualization, this example prepared Cy5.5-CA-COF BT-TG @TS, and Cy5.5-COF BT-TG @TS.

[0035] Among them, Cy5.5-CA-COF BT-TG @TS preparation method is: ①COF was synthesized by preparing a solution with BT (66 mg, 0.2 mmol) and TG (28 mg, 0.2 mmol) in a 1:1 molar ratio in a mixture of o-dichlorobenzene / n-butanol / 3 M acetic acid (7 mL, 3:3:1) under microwave heating. BT-TG ; Then after ultrasonic treatment for 15 minutes, 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 minutes using a microwave synthesizer. After cooling to room temperature, the product was first centrifuged and washed with THF, and then with acetone, and the washing was repeated 3 times. The crude product was collected after vacuum drying, and THF was used as a solvent for standard Soxhlet washing to remove any impurities adsorbed in the porous structure. Finally, after vacuum freeze drying for 6 hours, the product was fully ground to obtain a yellow powder COF BT-TG , with a yield of 85.6%.

[0036] ② Slowly add 1 mL of TS solution (0.6 mg / mL) and Cy5.5 (0.05 mg / mL) prepared in THF to 1 mL of COF BT-TG The mixture was added to a THF solution (2 mg / mL) and then reacted on a magnetic stirrer at room temperature for 6 h. The temperature was then 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 placed in a 3500 Da dialysis bag and dialyzed in PBS for 18 h. The dialyzate was replaced every 6 h. The dialyzed product was collected by centrifugation and fully ground to obtain dark green powdered Cy5.5-COF BT-TG @TS, the yield is 81.2%.

[0037] ③CA (1 mg / mL) and Cy5.5-COF BT-TG @TS (3.0 mg / mL) was dispersed in a plastic tube containing 2 mL of THF and a glass bottle containing 2 mL of THF, and then the two bottles of liquid were placed in an ultrasonic machine for ultrasonic dispersion for 20 minutes. During this period, an ice bag was placed in the ultrasonic machine to cool it down. BT-TG The glass bottle of @TS was fixed 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. CA was added dropwise during ultrasonic treatment. The whole process lasted for 30 minutes until CA was completely added to Cy5.5-COF. BT-TG @TS, then, the parameters remain unchanged for Cy5.5-CA-COF BT-TGThe @TS system was ultrasonically treated for 20 min. The reaction solution was collected and placed in a fume hood to dry naturally. The powder was ground and redispersed in PBS with a pH of 7.4. The powder was placed in a 3500Da dialysis bag and dialyzed in PBS for 24 h. The dialysate was replaced every 6 h. The dialyzate was collected by centrifugation and fully ground to obtain dark green powdered Cy5.5-CA-COF BT-TG @TS, the yield is 87.6%.

[0038] Fig.10 Cy5.5-CA-COF prepared in the embodiment of the present invention BT-TG @TS and Cy5.5-COF BT-TG @TS FT-IR characterization data diagram. From the diagram, we can see that Cy5.5-CA-COF BT-TG @TS and Cy5.5-COF BT-TG @TS all show infrared absorption peaks originating from the sulfonic acid group in Cy5.5. -1 -1300cm -1 The main characteristic peak in the wave number range is 1060cm -1 and 1180cm -1 , corresponding to the SO and S=O stretching vibrations in the sulfonic acid group. -1 -1400cm -1 In the wave number range, the main characteristic peak appears at 1250cm -1 and 1350cm -1 , corresponding to the CS and CO stretching vibrations of the sulfonic acid group, respectively. After modification of the targeting peptide CA, the infrared characteristic peak signal weakened, but there was no obvious shift or disappearance.

[0039] Artificially prepared thrombi were immersed in 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 0h, 1h, and 2h (e.g. Fig.11 The average fluorescence intensity at each time point was calculated (as shown in a Fig.11 (as shown in b in the figure). Fig.11 The results show that in 1h, with Cy5.5-CA-COF BT-TG The fluorescence intensity of artificial thrombus co-incubated with @TS was significantly higher than that of Cy5.5-COF BT-TG For artificial thrombi co-incubated with @TS, the difference was more obvious after 2 hours, indicating that the nanomedicine grafted with CA targeting peptide has excellent in vitro thrombus targeting ability.

[0040] (3) Evaluation of CA-COFBT-TG @TS ability to remove artificial thrombus: Take 6 3 mL transparent glass vials, place the prepared artificial thrombus in a 3 mL glass vial, add 2.5 mL of PBS solution and 0.5 mL of the test mixture into the glass vial, the test mixture includes PBS, TS (200 μg / mL), COF BT-TG (200μg / 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). The glass vial was exposed to 808 nm wavelength and a power density of 0.33 W / cm 2 The thrombus was irradiated with laser for 20 min and then incubated at 37°C for 2 h. The thrombus was dried in a 37°C oven for 2 h to remove surface moisture, weighed, and recorded and photographed. The results are shown in Fig.12 As shown. It can be seen that after laser irradiation, each group of mixtures turned red to varying degrees. The thrombolysis rate was further 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 shown in Fig.13 As shown, PBS, TS, COF BT-TG , COF BT-TG @TS, CA-COF BT-TG The thrombolytic rates of TS and 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 and placed in a 96-well plate. The supernatant was measured at OD using an enzyme reader. 415 and OD 540 The absorbance at 400 nm was used to evaluate the content of fibrin and hemoglobin in the supernatant. The results were as follows: Fig.14 and Fig.15 As shown in Figure 2, after laser irradiation, different levels of fibrin and hemoglobin were detected in the supernatant of each group, which was similar to the results of thrombolysis rate, and CA-COF BT-TG @The TS group had the highest levels of fibrin and hemoglobin in the supernatant. Since thrombolytic drugs activate the fibrinolytic system, degrade fibrinogen, and produce fibrin (ogen) degradation products (FDP), changes in FDP content can reflect the effect of thrombolytic therapy. Generally speaking, the FDP level will increase significantly after thrombolytic therapy, and during the thrombolytic process, if the thrombus dissolves, it may cause intravascular hemolysis, thereby increasing the free hemoglobin content in the plasma. Detecting hemoglobin content can be used as an indirect indicator to evaluate the effect of thrombolysis. If thrombolysis is successful, the free hemoglobin content in the plasma may increase. Therefore, the above results further illustrate that CA-COFBT-TG @TS can achieve better thrombolytic effect.

[0041] Example 3: In vivo evaluation of CA-COF BT-TG @TS's ability to clear blood clots (1) Preparation of the femoral artery FeCl3-induced thrombosis model: After 6-week-old C57BL / 6 mice inhaled isoflurane using a small animal anesthesia apparatus, the hair on the mouse thighs was carefully cut with scissors, the skin surface was wiped with 75% alcohol, and the scissors were carefully cut open the superficial skin of the back. The surface fascia and soft tissue were carefully separated under a microscope, and then a filter paper (3×1mm) soaked in 10% FeCl3 was applied to the surface of the femoral artery. After 5 minutes, the filter paper was removed and the residual FeCl3 was washed off with saline. After 10 minutes, white thrombus formation was visible in the femoral artery under a microscope. The arterial blood flow was detected using a laser speckle blood flow imaging system. If the femoral artery blood flow decreased significantly, it indicated that the thrombosis model was successfully prepared.

[0042] (2) Evaluation of CA-COF BT-TG @TS Thrombus homing ability: Thrombus homing ability is the key to the thrombus removal of this nanodrug platform. BT-TG @TS's thrombus homing ability, Cy5.5-COF BT-TG @TS (10 mg / kg) and Cy5.5-CA-COF BT-TG @TS (10 mg / kg) was injected into normal mice and mice in the femoral artery thrombosis model group through the tail vein. The main organs (heart, liver, spleen, lung, and kidney) and femoral artery of the mice were collected 2 hours later, and the enrichment of nanodrugs was observed using a small animal in vivo imaging system (such as Fig.16 The average fluorescence intensity of each major organ was calculated (as shown in a Fig.16 (as shown in b in the figure). Fig.16 The results show that after thrombosis occurs, the fluorescence intensity of the blood vessels increases significantly, indicating that the drug will accumulate at the lesion in the thrombotic state. When in the thrombotic state, the blood vessels grafted with CA targeting peptides show stronger fluorescence intensity, which further proves that when nanomedicines are grafted with CA targeting peptides, they have excellent in vivo thrombotic targeting ability.

[0043] (3) To evaluate CA-COF BT-TG @TS's ability to clear arterial thrombus, drug PBS, TS (10 mg / mL), COF BT-TG (10mg / mL)、COF BT-TG @TS (10 mg / mL), CA-COF BT-TG @TS (10 mg / mL) and UK (1.2×10 5UI / mg) was injected into the carotid artery thrombosis model mice through the tail vein. After 30 minutes, the carotid artery of the mice was exposed to 808nm (0.33W / cm 2 ) for 10 min, and the blood flow was detected using a laser speckle blood flow system. First, a thermal imager was used to measure the blood flow of the mouse legs at 808 nm (0.33 W / cm 2 ) The temperature change after irradiation is shown in the following figure. Fig.17 As shown in Figure 2, after irradiation, the temperature of the mouse legs increased to varying degrees. BT-TG The leg temperature of mice treated with @TS rose the most, reaching 44.3°C, which shows that the nanomedicine can normally exert its photothermal effect in vivo. Then, the laser speckle system was used to measure the blood flow changes in different treatment groups (such as Fig.18 The blood perfusion of different treatment groups was also statistically analyzed (as shown in a in Figure 2). Fig.18 The blood perfusion of the femoral artery thrombosis model mice in different treatment groups at 120 min was also counted (as shown in b, c, d, e, f, and g in Figure 1). Fig.19 ). Fig.18 and Fig.19 The results show that after 120 min, CA-COF BT-TG @The blood perfusion of the TS group recovered from 16.3% to 91.7%; while compared with the UK group, although there was a good blood perfusion recovery in the first 10 minutes to 30 minutes, its blood perfusion gradually decreased after 30 minutes and finally fell to the lowest point at 120 minutes. In addition, by comparing the blood flow of different treatment groups at 120 minutes, it was found that CA-COF BT-TG @The blood perfusion recovery of the TS group was significantly better than that of the COF group BT-TG @TS, this may be attributed to the former's excellent thrombus targeting ability.

[0044] Example 4: In vivo evaluation of CA-COF BT-TG @TS's ability to prevent recurrent thrombosis Carrageenan is widely used to induce venous thrombosis in mice. Carrageenan (20 mg / kg) was injected into 6-week-old Kunming male mice through the tail vein. The next day, the end of the mouse tail turned black, indicating tail vein thrombosis. BT-TG (10mg / mL)、COF BT-TG @TS (10 mg / mL), CA-COF BT-TG @TS (10 mg / mL) and UK (1.2×10 5 UI / mg) was injected into mice through the tail vein. After 30 min, the tails of mice were exposed to 808 nm (0.33 W / cm 2) for 10 minutes, inject the drug and irradiate at the same time every day for 5 consecutive days, take pictures every day and record the length of the black tail. The results are as follows Fig. 20 As shown in Figure 2, the length of the black tail of mice in the PBS group increased by an average of 3.1 cm on the fifth day, while the length of the black tail of mice in the CA-COF group increased by an average of 3.1 cm on the fifth day. BT-TG The average length of the black tail in the TS group decreased by 3.1 cm, which indicates that CA-COF BT-TG @TS has not only good ability to clear arterial thrombus, but also excellent ability to clear venous thrombus. BT-TG @TS's effect on preventing thrombosis recurrence on CA-COF on the fifth day BT-TG @TS group and UK group were injected with carrageenan (20 mg / kg) again to simulate thrombosis recurrence. The length of the black tail of mice was recorded every day for three days thereafter. The results are as follows Fig.21 As shown in Figure 2, the length of the black tail of mice in the UK group increased by an average of 2.1 cm on the third day, while that of mice in the CA-COF group increased by an average of 2.1 cm on the third day. BT-TG The length of the black tail of @TS mice increased by only 0.1 cm on average, indicating that CA-COF BT-TG @TS also has a good ability to fight against recurrence of thrombosis.

[0045] Example 5: In vivo evaluation of CA-COF BT-TG @TS's effect on rescuing ischemic stroke To evaluate CA-COF BT-TG @TS treatment of ischemic stroke, using the middle cerebral artery occlusion (MCAO) model to simulate ischemic stroke, and the nanodrug CA-COF was injected into the tail vein BT-TG @TS.

[0046] (1) Preparation of the middle cerebral artery occlusion model: The MCAO model was prepared by the suture 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 reproducible. The specific method is as follows: After 6-week-old male C57BL / 6 mice were anesthetized with isoflurane gas, the hair on the mouse neck was carefully cut with scissors, and the neck of the mouse was disinfected with 75% alcohol. The left carotid artery and vagus nerve of the mouse were carefully separated under a microscope 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. At the same time, the small surgical clip was released so that the suture could reach the middle cerebral artery to occlude it. After 90 minutes, the suture was pulled out, the wound was sutured, and it was disinfected again with 75% alcohol. Tail vein injection of nanomedicine CA-COF BT-TG@TS, after 72 hours, the Longa scale was used to evaluate neurological function. The specific scoring criteria of this scale are: no neurological deficit, 0 points; the paralyzed front paw cannot be fully extended, 1 point; turning in circles toward the paralyzed side when walking, 2 points; leaning toward the paralyzed side when walking, 3 points; unable to walk automatically and having loss of consciousness, 4 points. The higher the score, the more severe the animal's behavioral disorder. The results are as follows: Fig. 22 As shown. You can view, through CA-COF BT-TG The neurological function scores of mice treated with TS were significantly better than those of the model group.

[0047] (2) TTC (2,3,5-triphenyltetrazolium chloride) staining to evaluate cerebral infarction area: After the above MCAO model was prepared, CA-COF was injected into mice through the tail vein. BT-TG @TS (10mg / kg). After 72h, the mice were anesthetized with isoflurane gas, the heart was exposed, and the mice were irrigated with normal saline. The mouse brain was then carefully removed with ophthalmic scissors and forceps and placed in a -20°C refrigerator for 20min. After the brain was frozen, the brain was cut into 5 slices every 2mm along the coronal position. The specific method is: 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 caudate of the posterior lobe. The cut brain slices were placed in a six-well plate containing TTC staining solution, incubated at 37°C in the dark for 30min, and then photographed and recorded (such as Fig.23 Image J software was used to analyze the infarct area (as shown in a). Fig.23 (as shown in b in the figure). Fig.23 The results show that CA-COF BT-TG @TS treatment significantly reduced the area of ​​cerebral infarction in mice.

[0048] (3) Tissue immunofluorescence evaluation of ferroptosis after treatment: PTGS2 is considered a marker of ferroptosis, so this example analyzed the content of PTGS2 in the mouse brain after nanomedicine treatment. After the above MCAO model was prepared, CA-COF was injected into the mice through the tail vein. BT-TG@TS. One week later, the mice were anesthetized with isoflurane gas, the hearts were exposed, and the mice were irrigated with normal saline; the mouse brains were then carefully removed with ophthalmic scissors and forceps and fixed in 4% paraformaldehyde; they were then embedded in paraffin, sectioned along the coronal plane, dewaxed, and incubated with 10% goat serum at room temperature for 60 minutes; 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 minutes. The fluorescent secondary antibody was washed with PBS, and DAPI (4',6-diamidino-2-phenylindole) was added to stain the cell nuclei, and then the slides were sealed with an anti-fluorescence quencher, and photographed and recorded using a confocal microscope. The results are as follows Fig.24 As shown. It can be seen that compared with the model group, the CA-COF BT-TG After @TS treatment, the fluorescence intensity of PTGS2 was significantly weakened, indicating that CA-COF BT-TG @TS can inhibit ferroptosis after ischemic stroke.

[0049] Example 6: CA-COF BT-TG @TS Biosafety Assessment To verify CA-COF BT-TG Biosafety of @TS, CA-COF was injected into MCAO model mice via tail vein route BT-TG @TS. One week later, the mice were killed, and the hearts, livers, spleens, lungs, and kidneys were removed. They were fixed with 4% paraformaldehyde and embedded in paraffin. The sections were dewaxed and stained with hematoxylin-eosin. The HE (hematoxylin-eosin) staining procedure was as follows: Use hematoxylin staining for 10 min to 15 min; use 0.5% to 1.0% hydrochloric acid alcohol for color separation for a few seconds to tens of seconds, and quickly wash with distilled water; blue with light ammonia water, wash with distilled water, and examine under a microscope while differentiating; rinse with running water for 3 min to wash away alkaline water; 1% eosin staining for 5 min to 10 min; quickly wash with distilled water, and dehydrate with alcohol in a step-by-step manner, that is, quickly wash with 70%, 80%, and 90% alcohol, 95% alcohol for 30s to 60s, 100% alcohol for 3 min, twice; xylene for 5 min, twice; finally, use neutral gum to seal the slides and observe under a microscope. The results are as follows Fig.25 As shown, CA-COF BT-TG @TS did not cause pathological damage to major organs. In addition, hemolysis experiments were further used to evaluate the BT-TG @TS biocompatibility. 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, saline, 12, 15, 25, 30, 50, 60, 100, 120 μg / mL CA-COF BT-TG@TS, incubate in a 37℃ water bath for 4 hours and centrifuge at 3000rpm for 15 minutes. Take photos to record the hemolysis of each group. Take 200μL of supernatant from each group to a 96-well plate and measure the absorbance of the sample at 540 with an ELISA reader. The hemolysis rate is calculated using the following formula: Hemolysis rate (%) = (OD 样本 -OD 生理盐水组 ) / (OD ddH2O -OD 生理盐水组 ). The results are as follows Fig.26 As shown in the figure, the hemolysis rate at each concentration was less than 5%, indicating that CA-COF BT-TG @TS has good biocompatibility.

[0050] In summary, the present invention uses a covalent organic framework material containing a guanidine structure as a carrier platform, and integrates the thermosensitizer TS into the COF BT-TG The surface of the nanostructured CA-COF was modified by biomimetic targeting with CA. BT-TG @TS has good biological safety and can significantly improve the thrombolytic efficiency through multiple mechanisms such as photothermal effect, targeted delivery, promotion of penetration and diffusion, and synergistic effect, inhibit ferroptosis after ischemic stroke, and significantly reduce the area of ​​cerebral infarction in mice, providing new ideas and methods for the treatment of ischemic stroke.

[0051] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection 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 nanodrug containing a guanidine structure as claimed in claim 1 or the photothermal nanodrug 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 the inhibition of ferroptosis and improving neuronal death after ischemic stroke.

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