An indane isomer photothermal thrombolytic agent and its preparation method and application

Abstract

The present invention belongs to the technical field of biomedical materials, and specifically relates to an indane isomer photothermal thrombolytic agent, a preparation method thereof, and an application thereof. The indane isomer photothermal thrombolytic agent is co-assembled by an organic small molecule and a thrombus fusion protein peptide; the organic small molecule is 1,3-bis(dicyanomethylene)indane isomer 6DNMDD or 4DNMDD. The indane isomer photothermal thrombolytic agent obtained by the present invention has excellent photothermal conversion efficiency on the one hand, and thrombus homing ability on the other hand. When treating lower extremity artery and carotid artery embolism, 4DNMDD-C NPs exhibit higher photothermal conversion efficiency and photoacoustic imaging ability, and have better diagnostic and therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to an indane isomer photothermal thrombolytic agent, a preparation method and application thereof, and more specifically relates to a 1,3-bis(dicyanomethyl)indane isomer photothermal thrombolytic agent with photothermal conversion efficiency and photoacoustic imaging, a preparation method and application thereof. Background Art

[0002] Thrombosis-related diseases, including myocardial infarction, ischemic stroke, and venous embolism, are a major threat to human health due to their high morbidity and mortality. Currently, the clinical management of these thrombotic diseases mainly relies on drug therapy, including recombinant tissue plasminogen activator (rt-PA) and urokinase (uPA). However, the use of these thrombolytic drugs is accompanied by a series of side effects, such as potential neurotoxicity and the risk of bleeding complications, which may be life-threatening. Therefore, there is an urgent need to develop new, efficient and safe thrombolytic therapeutic strategies to reduce the heavy burden of thrombotic diseases in the global public health field.

[0003] As a treatment method, photothermal therapy rapidly converts light energy into heat energy by utilizing the Landau damping effect of photothermal agents to achieve thrombus ablation, providing precise spatiotemporal selectivity, minimal invasiveness, and efficient thrombolytic effect. It is worth noting that photothermal agents can not only play a role in thermal thrombolysis guided by thermal images, but also achieve higher-resolution diagnosis and treatment monitoring through photoacoustic imaging technology, thereby enhancing the therapeutic effect. The photothermal conversion efficiency of photothermal agents directly determines the efficacy of photothermal therapy. At present, a variety of photothermal agents have been developed to enhance the effect of photothermal therapy. Among them, organic photothermal agents have been widely studied in oncology, antibacterial therapy and other fields due to their tunable photophysical properties, high biosafety and biocompatibility, easy processing and functionalization. However, the development of efficient organic photothermal agents for photothermal thrombolysis still faces major challenges. First, molecular design requires the introduction of molecular rotors at the molecular level to promote molecular vibration, thereby inhibiting radiation inactivation and improving photothermal conversion efficiency. Secondly, enhancing the capacity of electron donors or acceptors and expanding the π-conjugated system promotes intramolecular charge transfer, resulting in a red shift in the molecular absorption spectrum and extending it to the near-infrared region, thereby improving the photothermal efficiency. These strategies involve complex chemical synthesis processes that are costly and time-consuming. At the same time, the bulky structure of photothermal agents leads to larger nanoparticle sizes, which not only increases the risk of potential biological toxicity, but also hinders their metabolic processes in biological systems. In addition, although regulating intermolecular stacking to enhance intermolecular interactions is a potential way to improve photothermal conversion efficiency, it is limited by the inherent unpredictability of molecular spatial conformation and the variability of intermolecular interactions. There is an urgent need to develop more promising thrombolytic agents with high photothermal conversion efficiency.

[0004] Isomerism is common in organic chemistry, and subtle structural differences often lead to significant changes in properties. Therefore, the use of isomer engineering technology to develop efficient organic photothermal agents for photothermal thrombolysis has practical application prospects. Based on this, it is of great significance to develop a safe thrombolytic agent with high photothermal conversion efficiency through isomer engineering technology. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide an indane isomer photothermal thrombolytic agent and a preparation method and application thereof. The photothermal thrombolytic agent can not only accurately locate the thrombus, but also efficiently promote the thrombolytic process. In the model of carotid artery and lower limb artery thrombosis treatment, the nanoagent showed significant therapeutic effects, proving its potential application value in clinical treatment.

[0006] The first object of the present invention is to provide an indane isomer photothermal thrombolytic agent, wherein the indane isomer photothermal thrombolytic agent is co-assembled by an organic small molecule and a thrombus fusion protein peptide; the organic small molecule is a 1,3-bis(dicyanomethylene)indane isomer 6DNMDD or 4DNMDD, the 6DNMDD has a chemical structure as shown in Formula I, and the 4DNMDD has a chemical structure as shown in Formula II:

[0007] .

[0008] Furthermore, in the chemical structure, R 1 and R 2 Any group.

[0009] Furthermore, the R 1 for , , , , and Any one of the R 2 for , Any one of .

[0010] Furthermore, R 1 Preferably , R 2 Preferably .

[0011] Furthermore, the preparation method of the organic small molecule is: first, the compound 1,3-bis(dicyanomethylene)indane is added to the acetic anhydride solution of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde, and the mixture is degassed by nitrogen, and then the obtained mixture is stirred for 6 hours under an inert atmosphere and at room temperature to obtain a reaction liquid, and then the reaction liquid is extracted with dichloromethane and water and the dichloromethane solution of the organic phase is retained, and after the dichloromethane solution is spin-dried, the crude product is passed through a silica gel column and eluted and separated with an eluent to obtain 6DNMDD or 4DNMDD. The chemical reaction formula is as follows:

[0012] .

[0013] Furthermore, the mass ratio of the 1,3-bis(dicyanomethylene)indane to 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde is 1-1.3:1; the mass volume (mg / mL) ratio of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde to acetic anhydride in the acetic anhydride solution of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde is 19.5-20:1; and the eluent is a mixed solution of petroleum ether and dichloromethane in a volume ratio of 3-5:1.

[0014] Furthermore, the organic small molecule is 4DNMDD.

[0015] The second object of the present invention is to provide a method for preparing an indane isomer photothermal thrombolytic agent, wherein the specific steps of the preparation method are as follows: firstly, organic small molecules and phospholipid-polyethylene glycol-thrombus fusion protein peptide (DSPP-PEG2K-CREKA) are dissolved in an appropriate amount of tetrahydrofuran, and under ultrasonic conditions, the mixed solution is added to an appropriate amount of ultrapure water, and the tetrahydrofuran in the solution is blown away by nitrogen gas, and the obtained solution is placed in a dialysis bag with a molecular weight cutoff of 2000 Daltons, and placed in deionized water for dialysis treatment, and finally the target nanoparticles are obtained, i.e., nanoparticles containing 6DNMDD (6DNMDD-C NPs) or nanoparticles containing 4DNMDD (4DNMDD-C NPs). Here, small molecule impurities in the solution can be removed by dialysis.

[0016] Furthermore, in the above technical solution, the mass ratio of the organic small molecule and the phospholipid-polyethylene glycol-thrombin fusion protein peptide is 1:1~50; the preferred mass ratio is 1:1~20; and more preferably 1:5; the ultrasonic conditions are: power 150W, time 15min.

[0017] The third object of the present invention is to provide a use of the above-mentioned indane isomer photothermal thrombolytic agent in the preparation of drugs for treating carotid artery and lower limb artery embolism.

[0018] Advantages compared to the prior art:

[0019] 1. The photothermal material designed in the present invention is an organic small molecule, and its structure is easy to modify. After being co-assembled with the thrombus-fusin peptide, it has excellent light capture ability and exhibits good photoacoustic imaging performance. With 808nm near-infrared laser as the light source, it is applied to the photoacoustic diagnosis and photothermal treatment of carotid artery and lower limb artery embolism, which can achieve a strong penetration depth and high imaging resolution, thereby obtaining a better treatment effect, with fewer side effects, showing broad clinical application potential.

[0020] 2. The present invention obtains isomers 6DNMDD and 4DNMDD by isomerization, wherein the isomer 4DNMDD photothermal thrombolytic agent has extremely high photothermal conversion efficiency and good solubility in organic solvents. After it is prepared into water-soluble nanoparticles, it can not only ensure the photothermal conversion efficiency of the nanoparticles, but also significantly improve its water solubility and biosafety; in addition, the agent has excellent biocompatibility to cells and can efficiently achieve thrombolytic effect under light conditions; the isomerization acquisition method also further provides new ideas for the development of compounds with performance advantages.

[0021] 3. The isomer photothermal reagent mentioned in the present invention has easy-to-obtain raw materials, mild synthesis conditions, simple preparation process, high repeatability, and convenient purification process, and has broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a high-resolution mass spectrum of 6DNMDD in Example 1 of the present invention, wherein the actual mass-to-charge ratio is 424.1545 and the theoretical mass-to-charge ratio is 424.1518;

[0023] Figure 2 is a high-resolution mass spectrum of 4DNMDD in Example 1 of the present invention, wherein the actual mass-to-charge ratio is 424.1558 and the theoretical mass-to-charge ratio is 424.1518;

[0024] Figure 3 Transmission electron microscopy images of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 2 of the present invention;

[0025] Figure 4 is the UV-visible absorption spectra of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 2 of the present invention, wherein the absorption peak of 6DNMDD-C NPs is at 616 nm, and the absorption peak of 4DNMDD-C NPs is at 733 nm;

[0026] Figure 5It is the time-temperature curve graph of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 2 of the present invention;

[0027] Figure 6 It is the comparison graph of photoacoustic signal intensities during photoacoustic imaging of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 2 of the present invention;

[0028] Figure 7 It is the fluorescence intensity and analysis graph of in vitro targeting experiments of Cy5.5-6DNMDD-C NPS and Cy5.5-4DNMDD-C NPs in Example 3 of the present invention;

[0029] Figure 8 It is the in vitro thrombolysis experiment graph of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 4 of the present invention;

[0030] Fig. 9 It is the statistical graph of in vitro thrombolysis rates of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 4 of the present invention;

[0031] Fig.10 It is the statistical graph of the contents of fibrin and hemoglobin in the supernatant of the glass bottle after in vitro thrombolysis of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 4 of the present invention;

[0032] Fig.11 It is the fluorescence intensity and analysis graph of in vivo thrombus targeting experiments of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 5 of the present invention;

[0033] Fig.12 It is the in vivo photoacoustic imaging and analysis graph of photoacoustic signal intensities of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 5 of the present invention;

[0034] Fig.13 It is the lower limb thermal imaging graph of 6DNMDD-C NPs and 4DNMDD-C NPs in a lower limb arterial thrombosis model mouse after irradiation with 808 nm (0.33 W / cm 2 ) for 10 min in Example 6 of the present invention;

[0035] Fig.14 It is the speckle pattern of 6DNMDD-C NPs and 4DNMDD-C NPs in a lower limb arterial thrombosis model mouse after irradiation with 808 nm (0.33 W / cm 2 ) and its corresponding statistical graph at different time points in Example 6 of the present invention;

[0036] Fig.15The 6DNMDDC-NPS and 4DNMDD C-NPS in Example 7 of the present invention were injected into the lower limb artery thrombosis model mice at 808nm (0.33W / cm 2 ) Pathological sections of mouse lower limb arteries after irradiation;

[0037] Fig.16 The 6DNMDD-C NPs and 4DNMDD-C NPs in Example 8 of the present invention were irradiated in carotid artery thrombosis model mice at 808 nm (0.33 W / cm 2 ) Thermal imaging of the neck after 10 minutes of irradiation;

[0038] Fig.17 The 6DNMDD-C NPs and 4DNMDD-C NPs in Example 8 of the present invention were irradiated in carotid artery thrombosis model mice at 808 nm (0.33 W / cm 2 ) Speckle pattern after irradiation and its corresponding statistical graphs at different time points;

[0039] Fig.18 The 6DNMDD-C NPs and 4DNMDD-C NPs in Example 9 of the present invention were irradiated in carotid artery thrombosis model mice at 808 nm (0.33 W / cm 2 ) Pathological sections of mouse carotid arteries after irradiation;

[0040] Fig.19 This is a statistical graph of cell viability detected by CCK8 after 6DNMDD-C NPs and 4DNMDD-C NPs were co-incubated with HT22 cells for 24 hours in Example 10 of the present invention;

[0041] Fig. 20 Statistical graphs of the hemolysis conditions and hemolysis rates of 6DNMDD-C NPs and 4DNMDD-C NPs in Example 10 of the present invention;

[0042] Fig.21 Pathological sections of the main organs (heart, liver, spleen, lung, and kidney) of mice injected with 6D NMDD-C NPs and 4D NMDD-C NPs via the tail vein 7 days after injection in Example 10 of the present invention. DETAILED DESCRIPTION

[0043] 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.

[0044] 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.

[0045] The present invention is further described in detail below in conjunction with specific embodiments:

[0046] Example 1: Synthesis of isomers of photothermal organic small molecules (6DNMDD and 4DNMDD)

[0047] The chemical structures of 6DNMDD and 4DNMDD are shown in Formula I and Formula II, respectively:

[0048] ,

[0049] The chemical formula R 1 and R 2 Can be any group, in this embodiment R 1 for , R 2 for , the preparation chemical reaction formula is as follows:

[0050] .

[0051] The specific synthesis steps are:

[0052] Preparation of 6DNMDD: 1,3-bis(dicyanomethylene)indane (242 mg) and 6-(dimethylamino)-2-naphthaldehyde (199 mg) were dissolved in acetic anhydride (10 mL), and the mixture was degassed again by nitrogen; then, the resulting mixture was stirred at room temperature for 6 h. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using petroleum ether and dichloromethane (volume ratio of 3:1) as eluent to obtain black solid 6DNMDD (368 mg, yield 87%). The structure was confirmed to be correct by high-resolution mass spectrometry (eg Figure 1 as shown).

[0053] Preparation of 4DNMDD: 1,3-bis(dicyanomethylene)indane (242 mg) and 4-(dimethylamino)-1-naphthaldehyde (199 mg) were dissolved in acetic anhydride (10 mL), and the mixture was degassed again by nitrogen; then, the resulting mixture was stirred at room temperature for 6 h. After evaporation of the solvent, the crude product was purified by silica gel column chromatography using petroleum ether and dichloromethane (volume ratio of 5:1) as eluent to obtain black solid 4DNMDD (372 mg, yield 88%). The structure was confirmed to be correct by high-resolution mass spectrometry (eg Figure 2 as shown).

[0054] Example 2: Preparation of isomer photothermal thrombolytic agent nanoparticles (6DNMDD-C NPs and 4DNMDD-C NPs)

[0055] The DSPE-PEG2K-CREKA described in this example was purchased from Shanghai Tuoyang Biotechnology Co., Ltd.

[0056] The specific preparation method is:

[0057] Preparation of 6DNMDD-C NPs: 6DNMDD (2 mg) and DSPE-PEG2K-CREKA (10 mg) were dissolved in 1 mL of tetrahydrofuran. Subsequently, the mixed solution was slowly added to an appropriate amount of ultrapure aqueous solution under ultrasonic stirring (power of 150 W, ultrasonic time of 15 min). The tetrahydrofuran in the solution was then blown away with nitrogen gas. The resulting solution was then placed in a dialysis bag with a molecular weight cutoff of 2000 Daltons and placed in deionized water for dialysis treatment (small molecule impurities in the solution were removed by dialysis). Finally, an aqueous solution of target nanoparticles containing 6DNMDD with photothermal and photoacoustic imaging capabilities was obtained.

[0058] Preparation of 4DNMDD-C NPs: 4DNMDD (2 mg) and DSPE-PEG2K-CREKA (10 mg) were dissolved in 1 mL of tetrahydrofuran. Subsequently, the mixed solution was slowly added to an appropriate amount of ultrapure aqueous solution under ultrasonic stirring. The tetrahydrofuran in the solution was then blown away with nitrogen gas. The resulting solution was then placed in a dialysis bag with a molecular weight cutoff of 2000 Daltons and placed in deionized water for dialysis treatment to finally obtain an aqueous solution of target nanoparticles containing 4DNMDD with photothermal and photoacoustic imaging capabilities.

[0059] The morphology, size and performance of the obtained 6DNMDD-C NPs and 4DNMDD-C NPs were verified respectively.

[0060] (1) 6DNMDD-C NPs and 4DNMDD-C NPs were dropped onto a copper mesh respectively. After the nanoparticles settled on the copper mesh and the water evaporated, the samples were sent to a transmission electron microscope to observe the morphology (e.g. Figure 3 As shown in the figure, the size of the obtained 6DNMDD-C NPs and 4DNMDD-C NPs are both spherical in shape, wherein the average size of the 6DNMDD-C NPs nanoparticles is about 109 nm, and the average size of the 4DNMDD-C NPs nanoparticles is about 115 nm. This indicates that the obtained product is uniform nanoparticles.

[0061] (2) The aqueous solutions of 6DNMDD-C NPs and 4DNMDD-C NPs (concentration of 200 µg / mL) were tested using a UV absorption instrument and the UV absorption spectra were detected. The results are as follows: Figure 4 As shown in the figure, it can be seen that the absorption peak of 6DNMDD-C NPs is at 616nm, and the absorption peak of 4DNMDD-C NPs is at 733nm. This shows that the absorption range of these two nanoparticles extends to the near-infrared absorption region and has a good ability to capture near-infrared light.

[0062] (3) 6DNMDD-C NPs and 4DNMDD-C NPs with a concentration of 100 μg / mL were exposed to 808 nm (0.5 W / cm 2 ) laser irradiation, the water temperature was monitored over time using a thermal imager. The results are as follows Figure 5 As shown in the figure, it can be seen that the photothermal conversion efficiency of 6DNMDD-C NPs is 21.29%, and the photothermal conversion efficiency of 4DNMDD-C NPs is 89.79%, indicating that 4DNMDD-CNPs have better photothermal conversion performance.

[0063] (4) 6DNMDD-C NPs and 4DNMDD-C NPs with a concentration of 100 μg / mL were exposed to 808 nm (0.5 W / cm 2 ) laser irradiation, the photoacoustic imaging effect of 6DNMDD-C NPs and 4DNMDD-C NPs was detected by photoacoustic imaging. The results are shown in Figure 6 As shown in Figure 2, it can be seen that compared with 6DNMDD-C NPs, 4DNMDD C-NPs have more excellent imaging ability.

[0064] Example 3: In vitro thrombus targeting experiment of 6DNMDD-C NPs and 4DNMDD-C NPs

[0065] (1) Artificial thrombus preparation: After 6-week-old C57BL / 6 mice inhaled isoflurane using a small animal anesthesia instrument, fresh blood was collected by orbital blood sampling. 100 μL of blood was collected into a 1.5 mL centrifuge tube. 2 (3mM) and thrombin lyophilized powder were mixed to prepare a 25 U / mL solution. 10μL of the mixed solution was added to a 1.5mL centrifuge tube containing fresh blood and allowed to stand at 37°C for 2h. After 2h, 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.

[0066] (2)Evaluating the binding ability of 6DNMDD-C NPs and 4DNMDD-C NPs to artificial thrombus: Cy5.5 (Cyanine 5.5) is a commonly used fluorescent dye. To visualize the targeting ability and delivery effect of 6DNMDD-C NPS and 4DNMDD-C NPS, Cy5.5-6DNMDD-C NPs and Cy5.5-4DNMDD-C NPs were prepared in this example. The preparation methods of Cy5.5-6DNMDD-C NPs and Cy5.5-4DNMDD-C NPs are the same as those of 6DNMDD-C NPs and 4DNMDD-C NPs respectively, except that Cy5.5 was added:

[0067] Dissolve 6DNMDD (2 mg), Cy5.5 (0.5 mg) and DSPE-PEG2K-CREKA (10 mg) in 1 mL of tetrahydrofuran. Subsequently, under the condition of ultrasonic stirring, slowly add the above mixed solution into the ultrapure aqueous solution, then blow away the tetrahydrofuran in the solution with nitrogen, and then obtain Cy5.5-6DNMDD-C NPs by dialysis.

[0068] Dissolve 4DNMDD (2 mg), Cy5.5 (0.5 mg) and DSPE-PEG2K-CREKA (10 mg) in 1 mL of tetrahydrofuran. Subsequently, under the condition of ultrasonic stirring, slowly add the above mixed solution into the ultrapure aqueous solution, then blow away the tetrahydrofuran in the solution with nitrogen, and then obtain Cy5.5-4DNMDD-C NPs by dialysis.

[0069] Incubate artificial thrombus with PBS, Cy5.5-6DNMDD-C NPs and Cy5.5-4DNMDD-C NPs respectively, and use a small animal in vivo imaging system to take pictures at 0 h, 1 h and 2 h (as shown in a of Figure 7 ), and analyze in combination with the fluorescence intensity (as shown in b of Figure 7 ). It can be seen that after 1 h, a large amount of Cy5.5-6DNMDD-C NPs and Cy5.5-4DNMDD-C NPs were enriched on the artificial thrombus; after 2 h, the fluorescence intensity on the thrombus surface was further increased, but there was no significant difference between Cy5.5-6DNMDD-C NPs and Cy5.5-4DNMDD-C NPs at both 1 h and 2 h. It shows that both 6DNMDD-C NPs and 4DNMDD-C NPs modified with the fusion protein peptide (CREKA) have excellent in vitro thrombus targeting ability.

[0070] Example 4: In vitro thrombus ablation experiment of 6DNMDD-C NPs and 4DNMDD-C NPs

[0071] In vitro evaluation of the thrombus ablation ability of 6DNMDD-C NPs and 4DNMDD-C NPs:

[0072] Take three 3 mL transparent glass vials, place the artificial thrombus prepared above 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, where the test mixture is PBS, 6DNMDD-C NPs (200 μg / mL) or 4DNMDD-C NPs (200 μg / mL). Expose the glass vial to 808 nm wavelength and a power density of 0.33 W / cm 2 Irradiate with laser for 20 min (eg Figure 8 The thrombus was then incubated at 37°C for 2 h, and then dried in a 37°C oven for 2 h to remove surface moisture, weighed, and photographed (as shown in a). Figure 8 (as shown in b in the figure). Figure 8 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. Fig. 9 As shown. The calculated results showed that the thrombolytic rates of PBS, 6DNMDD-C NPs and 4DNMDD-C NPs were 13.35%, 23.63% and 78.17%, 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 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. Fig.10 It can be seen that after laser irradiation, different levels of fibrin and hemoglobin can be detected in the supernatant of each group. Similar to the results of thrombolysis rate, the content of fibrin and hemoglobin in the supernatant of 4DNMDD-C NPs group is the highest.

[0073] Example 5: In vivo evaluation of the thrombus targeting ability of 6DNMDD-C NPs and 4DNMDD-C NPs

[0074] (1) FeCl in lower limb arteries 3 Preparation of induced thrombosis model: After 6-week-old C57BL / 6 mice inhaled isoflurane using a small animal anesthesia instrument, the hair on the thighs of the mice was carefully cut with scissors, the skin surface was wiped with 75% alcohol, and the surface skin of the legs was carefully cut with scissors. The surface fascia and soft tissue were carefully separated under a microscope, and then 10% FeCl was soaked in 3A filter paper (3 × 1 mm) was applied to the surface of the femoral artery. After 5 min, the filter paper was removed and the residual FeCl was washed with saline. 3 Ten minutes later, white thrombus formation was observed in the lower limb arteries under a microscope, and the arterial blood flow was detected using a laser speckle blood flow imaging system.

[0075] (2) In vivo evaluation of the thrombus homing ability of 6DNMDD-C NPs and 4DNMDD-C NPs: Drug enrichment at the thrombus site is a prerequisite for the drug to exert its thrombolytic effect. In order to evaluate the thrombus homing ability of 6DNMDD-C NPs and 4DNMDD-C NPs, Cy5.5-6DNMDD-C NPs (10 mg / kg) and Cy5.5-4DNMDD-C NPs (10 mg / kg) were injected into normal mice and mice in the lower limb artery thrombosis model group through the tail vein. After 2 hours, the main organs (heart, liver, spleen, lung, and kidney) and lower limb arteries of the mice were collected, and the small animal in vivo imaging system was used to observe the enrichment of nanodrugs (such as Fig.11 The fluorescence intensity of the main organs was analyzed (as shown in a). Fig.11 (as shown in b in the figure). Fig.11 It can be seen that after thrombosis occurs, the arterial vessels of the lower limbs show strong fluorescence, indicating that the drug will accumulate at the lesion in the thrombotic state.

[0076] (3) In vivo evaluation of the photoacoustic imaging capabilities of 6DNMDD-C NPs and 4DNMDD-C NPs: First, BALB / c-nu nude mice were placed in the photoacoustic imaging instrument chamber to record and photograph the photoacoustic signals of the mice in a normal state. Subsequently, the mice were treated with a lower limb artery thrombosis model using the above method, and PBS, 6DNMDD-C NPs (5 mg / kg) and 4DNMDD-C NPs (5 mg / kg) were injected into the mice through the tail vein. The subsequent photoacoustic imaging of each group of mice was recorded and photographed using a photoacoustic imaging instrument (e.g. Fig.12 The intensity of the photoacoustic signal of each group was analyzed (as shown in a Fig.12 As shown in b). It can be seen that after thrombosis, the photoacoustic signal intensity at the lower limb artery of the mouse decreased rapidly. After the injection of 4DNMDD-C NPs, the photoacoustic signal intensity of the lower limb artery of the mouse increased rapidly with the injection time, while the photoacoustic signal intensity of the lower limb artery of the mouse injected with 6DNMDD-C NPs did not change significantly, and the photoacoustic signal intensity reached the highest point at 45min to 60min. In addition, the photoacoustic signal intensity of 6DNMDD-C NPs and 4DNMDD-C NPs at 60min was analyzed (as shown in Fig.12 This further confirms that 4DNMDD-C NPs have superior photoacoustic imaging capabilities.

[0077] Example 6: Laser Speckle Evaluation of the Ability of 6DNMDD-C NPs and 4DNMDD-C NPs to Ablate Lower Limb Arterial Thrombosis

[0078] PBS, 6DNMDD-C NPs (5 mg / kg), and 4DNMDD-C NPs (5 mg / kg) were injected into C57BL / 6 mice with a lower limb artery thrombosis model via the tail vein. After 45 minutes, the lower limb arteries of the mice were exposed to 808 nm (0.33 W / cm 2 ) for 10 min, and the blood flow was detected by a laser speckle blood flow system. During the laser irradiation, 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 10 minutes of irradiation is as follows Fig.13 As shown. It can be seen that after 10 minutes of irradiation, the temperature of the mouse legs increased to varying degrees. Among them, the temperature of the mouse legs treated with 4DNMDD-C NPs increased the most, reaching 43.1°C. This shows that in vivo, nanomedicines can normally exert photothermal efficacy, and it is parallel to the in vitro results, that is, the photothermal performance of 4DNMDD-C NPs is better than that of 6DNMDD-C NPs. Then the laser speckle system was used to measure the blood flow changes in different treatment groups. The blood perfusion speckle diagram and its corresponding statistical diagram at different time points are shown as follows. Fig.14 As shown in a and b in Figure 1, it can be seen that after 120 minutes, the blood perfusion of the 4DNMDD-C NPs group recovered to 71.7%, while the blood perfusion of the 6DNMDD-C NPs group only recovered to 25.1%. This indicates that 4DNMDD-C NPs have superior lower extremity arterial thrombus ablation ability in vivo.

[0079] Example 7: Pathological section evaluation of the ability of 6DNMDD-C NPs and 4DNMDD-C NPs to clear lower limb arterial thrombus

[0080] To more intuitively evaluate the difference in thrombus removal between 6DNMDD-C NPs and 4DNMDD-C NPs in vivo, mice were euthanized after laser irradiation. The lower limb arteries of mice were removed, fixed with 4% paraformaldehyde and embedded in paraffin, and the sections were dewaxed and HE stained (hematoxylin-eosin staining). HE staining is performed as follows: stain with hematoxylin for 10 to 15 minutes; use 0.5% to 1.0% hydrochloric acid alcohol for color separation for several seconds to tens of seconds, and wash quickly with distilled water; blue with light ammonia water, wash with distilled water, and examine with a microscope while differentiating; rinse with running water for 3 minutes to wash away alkaline water; stain with 1% eosin for 5 minutes to 10 minutes; wash quickly with distilled water, and dehydrate with alcohol in ascending order, that is, wash quickly with 70%, 80% and 90% alcohol, wash with 95% alcohol for 30 seconds to 60 seconds, and examine with a microscope; wash twice with 100% alcohol for 3 minutes; wash twice with xylene for 5 minutes; finally, seal the slide with neutral gum and observe with a microscope. Fig.15 As shown. It can be seen that the 4DNMDD-C NPs group significantly reduced lower limb arterial thrombosis, while the PBS and 6DNMDD-C NPs groups had thrombi all over the lumen. This shows that 4DNMDD-C NPs have excellent ability to remove lower limb arterial thrombosis.

[0081] Example 8: Evaluation of the ability of 6DNMDD-C NPs and 4DNMDD-C NPs to ablate carotid artery thrombus

[0082] (1) Carotid artery FeCl 3 Preparation of induced thrombosis model: The preparation method of carotid artery thrombosis model is similar to that of lower limb artery thrombosis model. Specifically, 6-week-old C57BL / 6 mice were anesthetized with isoflurane using a small animal anesthesia instrument. The hair on the neck of the mice was carefully cut with scissors. The skin surface was wiped with 75% alcohol. The scissors were used to carefully cut the surface skin of the neck. The surface fascia and soft tissue were carefully separated under a microscope. Then, a 10% FeCl 3 A filter paper (3 × 1 mm) was applied to the surface of the carotid artery. After 5 min, the filter paper was removed and the residual FeCl was washed with saline. 3 Ten minutes later, white thrombus formation was observed in the femoral artery under a microscope, and the arterial blood flow was detected using a laser speckle blood flow imaging system.

[0083] (2) Laser speckle: To evaluate the ability of 6DNMDD-C NPs (5 mg / kg) and 4DNMDD-C NPs (5 mg / kg) to clear carotid artery thrombosis, PBS, 6DNMDD-C NPs, and 4DNMDD-C NPs were injected into mice with a carotid artery thrombosis model via the tail vein. After 45 minutes, the carotid arteries of the mice were exposed to 808 nm (0.33 W / cm 2) for 10 min, and a laser speckle blood flow system was used to detect blood flow. During laser irradiation, we used a thermal imager to measure the blood flow of the mouse neck at 808 nm (0.33 W / cm 2 ) The temperature change after irradiation is shown in the following figure. Fig.16 As shown. It can be seen that after 10 minutes of irradiation, the neck temperature of the mice increased to varying degrees. Among them, the neck temperature of the mice treated with 4DNMDD-C NPs increased the most, reaching 44.8°C. This shows that in vivo, nanomedicines can normally exert photothermal efficacy, and it is parallel to the in vitro results, that is, the photothermal performance of 4DNMDD-C NPs is better than that of 6DNMDD-C NPs. Then the laser speckle system was used to measure the blood flow changes in different treatment groups. The blood perfusion speckle diagram and its corresponding statistical diagram at different time points are shown as follows. Fig.17 As shown in a and b in Figure 1, it can be seen that after 120 minutes, the blood perfusion of the 4DNMDD-C NPs group recovered to 73%, and the blood perfusion of the 6DNMDD-C NPs group recovered to 22.7%. This indicates that 4DNMDD-C NPs have superior carotid artery thrombus removal ability in vivo.

[0084] Example 9: Pathological section evaluation of the ability of 6DNMDD-C NPs and 4DNMDD-C NPs to ablate carotid artery thrombus

[0085] To more intuitively evaluate the difference in thrombus removal between 6DNMDD-C NPs and 4DNMDD-C NPs in vivo, mice were euthanized after laser irradiation. The lower limb arteries of mice were removed, fixed with 4% paraformaldehyde and embedded in paraffin, and the sections were dewaxed and HE stained. The specific operation of HE staining was referred to Example 7, and the results were shown in Figure 7. Fig.18 As shown in the figure, it can be seen that the carotid artery thrombosis in the 4DNMDD-C NPs group was significantly reduced, while the thrombosis in the PBS and 6DNMDD-C NPs groups filled the lumen. This shows that 4DNMDD-C NPs have the ability to ablate carotid artery thrombosis.

[0086] Example 10: Biosafety Assessment of 6DNMDD-C NPs and 4DNMDD-C NPs

[0087] First, the safety of 6DNMDD-C NPs and 4DNMDD-C NPs was evaluated in vitro using the CCK8 assay. Approximately 10,000 HT22 cells were plated on a 96-well plate and incubated with different concentrations of 6DNMDD-C NPs and 4DNMDD-C NPs. After 24 hours, 10 μL of CCK8 reagent was added to each well and incubated for another 1 hour. The absorbance of each well at 450 nm was then measured using an ELISA reader. The absorbance was used as an indicator of cell viability, and the cell viability of each group was calculated. The results are shown in Figure 2. Fig.19 As shown. It can be seen that at different concentrations, 6DNMDD-C NPs and 4DNMDD-C NPs did not cause obvious toxic effects on cell viability, indicating that 6DNMDD-C NPs and 4DNMDD-C NPs have excellent safety.

[0088] Secondly, the hemolysis experiment was further used to evaluate the biocompatibility of 6DNMDD-C NPs and 4DNMDD-C NPs. Take 16 clean EP tubes, add 900 μL of 2% red blood cell suspension to each tube, then add 100 μL of 0.1% Triton X-100, normal saline, 20, 40, 60, 80, 100, 200 μg / mL of 6DNMDD-C NPs and 4DNMDD-C NPs, incubate in a 37°C water bath for 4 hours, centrifuge at 3000 rpm for 15 minutes, take pictures to record the hemolysis of each group, and take 200 μL of supernatant from each group into a 96-well plate, and measure the absorbance of the sample at 540 with an enzyme reader. The hemolysis rate was 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. 20 It can be seen that the hemolysis rate at each concentration is less than 5%, indicating that 6DNMDD-C NPs and 4DNMDD-C NPs have good biocompatibility.

[0089] To further verify the biosafety of 6DNMDD-C NPs and 4DNMDD-C NPs, mice were injected with 6DNMDD-C NPs (5 mg / kg) and 4DNMDD-C NPs (5 mg / kg) via the tail vein. One week later, the mice were killed, and the heart, liver, spleen, 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 staining operation was referred to Example 7. The results are shown in Figure 7. Fig.21 It can be seen that neither 6DNMDD-C NPs nor 4DNMDD-C NPs caused pathological damage to major organs and had high safety.

[0090] In summary, the 1,3-bis(dicyanomethyl)indane isomer photothermal thrombolytic reagent prepared by the present invention with high photothermal conversion efficiency and photoacoustic imaging can efficiently target thrombi, can perform photoacoustic diagnosis and photothermal treatment of thrombi, and has excellent biosafety, and is expected to provide a new treatment plan for the clinical treatment of thrombi.

[0091] 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. An indane isomer photothermal thrombolytic agent, characterized in that: The indane isomer photothermal thrombolytic agent is co-assembled by an organic small molecule and a thrombus fusion protein peptide; the organic small molecule is 1,3-bis(dicyanomethylene)indane isomer 6DNMDD or 4DNMDD; the 6DNMDD has a chemical structure as shown in Formula I, and the 4DNMDD has a chemical structure as shown in Formula II: ； The R1 is , the R2 is .

2. The indane isomer photothermal thrombolytic agent according to claim 1, characterized in that: The preparation method of the organic small molecule is as follows: firstly, the compound 1,3-bis(dicyanomethylene)indane is added to an acetic anhydride solution of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde, and the mixture is degassed by nitrogen, and then the obtained mixture is stirred for 6 hours under an inert atmosphere and room temperature to obtain a reaction liquid, and then the reaction liquid is extracted with dichloromethane and water, and the dichloromethane solution of the organic phase is retained, and after the dichloromethane solution is spin-dried, the crude product is passed through a silica gel column and eluted and separated with an eluent to obtain 6DNMDD or 4DNMDD.

3. The indane isomer photothermal thrombolytic agent according to claim 2, characterized in that: The mass ratio of the 1,3-bis(dicyanomethylene)indane to 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde is 1-1.3:1; the mass volume ratio of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde to acetic anhydride in the acetic anhydride solution of 6-(dimethylamino)-2-naphthaldehyde or 4-(dimethylamino)-1-naphthaldehyde is 19.5-20:1; and the eluent is a mixed solution of petroleum ether and dichloromethane in a volume ratio of 3-5:

1.

4. The indane isomer photothermal thrombolytic agent according to claim 1, characterized in that: The organic small molecule is 4DNMDD.

5. The method for preparing an indane isomer photothermal thrombolytic agent according to any one of claims 1 to 4, characterized in that: The specific steps of the preparation method are: firstly dissolving the organic small molecule and the phospholipid-polyethylene glycol-thrombin fusion protein peptide in an appropriate amount of tetrahydrofuran, adding the mixed solution into an appropriate amount of ultrapure water under ultrasonic conditions, blowing away the tetrahydrofuran in the solution with nitrogen gas, and then obtaining the target nanoparticles by dialysis.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the organic small molecule to the phospholipid-polyethylene glycol-thrombin fusion protein peptide is 1:1-50; the ultrasonic conditions are: power 150W, time 15min.

7. Use of the indane isomer photothermal thrombolytic agent as described in any one of claims 1 to 4 or the indane isomer photothermal thrombolytic agent prepared by the preparation method as described in any one of claims 5 to 6 in the preparation of a drug for treating carotid artery and lower limb artery embolism.

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