Thrombus-targeted diagnosis and treatment integrated nano probe and preparation method and application thereof

Abstract

The application provides a thrombus-targeting diagnosis and treatment integrated nano probe, which comprises a core-shell structure, the core of the core-shell structure is bismuth, and the shell is expanded pore silicon dioxide wrapping bismuth; a chemical medicine for treating thrombus is loaded in the pore channel of the expanded pore silicon dioxide, and a thrombus-targeting polypeptide is connected to the surface of the expanded pore silicon dioxide; the amino acid sequence of the thrombus-targeting polypeptide is GPRPVTSEIHLK. The application not only improves the drug loading capacity of the core-shell nano probe by forming the expanded pore Bi@SiO2 particle with Bi as the core and SiO2 as the shell, but also makes the probe capable of enriching at the thrombus site by using the targeting effect of the thrombus-targeting polypeptide, so as to construct a targeted nano drug delivery system with targeted imaging and photo-thermal-chemical synergistic thrombolysis, accurately treat the thrombus, and realize the diagnosis and treatment integration of the thrombus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a thrombosis-targeting diagnostic and therapeutic integrated nanoprobe, its preparation method, and its application. Background Technology

[0002] Deaths caused by thrombotic diseases such as atherosclerosis due to vascular embolism account for 25% of all deaths worldwide, posing a significant threat to human health. The formation of abnormal thrombi in the body can block blood vessels, slowing or even halting blood flow, thus affecting blood and oxygen supply to ischemic areas, leading to tissue ischemia and necrosis, and ultimately threatening human life. Therefore, developing and obtaining effective methods and means to treat vascular embolism is not only crucial for curbing cardiovascular complications but also essential for the healthy and stable development of society.

[0003] The rational design of targeted nanomedicine delivery systems is a key research focus in the integrated diagnosis and treatment of thrombosis. To this end, three key issues need to be considered: 1) how to specifically target the thrombus site; 2) how to quickly identify and accurately locate the thrombus site using the targeted nanomedicine delivery system; and 3) how to achieve highly efficient thrombolysis; ultimately realizing the integrated diagnosis and treatment of thrombosis.

[0004] First, specific targeting of thrombi is a crucial component of targeted nanomedicine delivery systems. Fibrin, as a major component of thrombi, is abundant at the site of vascular embolism and can serve as a recognition substrate for targeted nanomedicine delivery systems. Based on this, to quickly distinguish the specific targeting affinity between the targeted nanomedicine delivery system and the thrombus, and to ensure accurate diagnosis of the thrombus site, it is necessary to select appropriate in vivo imaging techniques and corresponding contrast agents.

[0005] Photothermal therapy for thrombosis has attracted widespread attention from researchers. This treatment method raises the temperature of the thrombus lesion site through local laser irradiation, thereby disrupting the non-covalent bonds of fibrin and destroying the thrombus under high shear force. It has good therapeutic precision and extremely low toxicity. The efficient combined delivery of photothermal and antithrombotic drugs, along with in vivo imaging technology, holds promise for further improving the safety and efficacy of thrombosis treatment. Summary of the Invention

[0006] To address the problems existing in the background art, the present invention provides a thrombosis-targeted diagnostic and therapeutic integrated nanoprobe, its preparation method and application. The thrombosis-targeted diagnostic and therapeutic integrated nanoprobe is used to construct a targeted nanodrug delivery system that combines targeted imaging with photothermal-chemical synergistic thrombolysis, enabling precise treatment of thrombosis and achieving integrated thrombosis diagnosis and treatment.

[0007] The specific technical solution is as follows:

[0008] This invention provides a thrombosis-targeting diagnostic and therapeutic integrated nanoprobe, comprising a core-shell structure;

[0009] The core-shell structure has bismuth as the core and porous silica encapsulating bismuth as the outer shell; chemical drugs for treating thrombosis are loaded in the pores of the porous silica, and thrombosis-targeting peptides are attached to the surface of the porous silica.

[0010] The amino acid sequence of the thrombosis-targeting polypeptide is GPRPVTSEIHLK.

[0011] Furthermore, the chemical drug is urokinase.

[0012] Furthermore, the thrombosis-targeting polypeptide is linked to the porous silica via an amide bond; the chemical drug is loaded into the pores of the porous silica via electrostatic interaction.

[0013] Furthermore, based on the thrombosis-targeting diagnostic and therapeutic integrated nanoprobe, the loading amount of the chemical drug is 120-130 μg / mg; and the loading amount of the thrombosis-targeting peptide is 150-200 μg / mg.

[0014] This invention also provides a method for preparing the aforementioned thrombosis-targeting and therapeutic integrated nanoprobe, comprising the following steps:

[0015] (1) Using bismuth nitrate as a raw material, elemental Bi nanoparticles were prepared;

[0016] (2) Using elemental Bi nanoparticles as the core, SiO2 is grown in situ on the surface of elemental Bi nanoparticles by ethyl silicate to form Bi@SiO2 nanoparticles.

[0017] (3) Using Bi@SiO2 nanoparticles as raw materials, large pores are formed on the surface of SiO2 by the pore-forming agent hexadecyltrimethylammonium chloride to obtain Mp-Bi@SiO2;

[0018] (4) The thrombus-targeting polypeptide is activated by an activator and linked to the surface of Mp-Bi@SiO2 to form Mp-Bi@SiO2-GK;

[0019] (5) Chemical drugs are loaded into the pores of the expanded silica of Mp-Bi@SiO2-GK by a one-pot blending method to form an integrated nanoprobe for thrombosis-targeted diagnosis and treatment, Mp-Bi@SiO2-GK / UK.

[0020] Further, step (1) includes: steps (1-1) to (1-3);

[0021] Step (1-1): Using Bi(NO3)3·5H2O as raw material, dissolve it in dodecanethiol, stir, and continuously pump nitrogen gas at 50-60℃ to remove air;

[0022] Steps (1-2): After evacuating the air 3-5 times, heat the solution to 170-175℃ using a gradient heating method. The solution will start to produce yellow bubbles and gradually turn black. Maintain the temperature at 170-175℃ for 1-3 minutes, and then stop heating.

[0023] Steps (1-3): Centrifuge to remove the supernatant, wash the precipitate several times with anhydrous ethanol, then wash with cyclohexane to collect Bi nanoparticles.

[0024] Furthermore, step (2) includes: steps (2-1) to (2-5);

[0025] Step (2-1): While stirring continuously, add the emulsifier to cyclohexane to obtain mixture I; then slowly add the cyclohexane solution containing Bi nanoparticles dropwise to mixture I to obtain mixture II;

[0026] Step (2-2): Sonicate and continuously stir mixture II, add concentrated ammonia dropwise, and stir to obtain mixture III;

[0027] Step (2-3): Add ethyl silicate to mixture III, stir, and obtain mixture IV;

[0028] Step (2-4): Add anhydrous ethanol to mixture IV, sonicate, and then add acetone to obtain mixture V;

[0029] Steps (2-5): Centrifuge the mixture V, resuspend it in ethanol, add acetone, centrifuge again, and obtain the precipitate Bi@SiO2.

[0030] Furthermore, step (3) includes: steps (3-1) to (3-2);

[0031] Step (3-1): Disperse Bi@SiO2 nanoparticles in water containing the pore-forming agent hexadecyltrimethylammonium chloride and trimethylbenzene, and sonicate to form an emulsion;

[0032] Step (3-2): Add an aqueous solution of ethyl silicate and L-arginine to the emulsion, heat and stir, wash and centrifuge to obtain Mp-Bi@SiO2.

[0033] Furthermore, step (4) includes: steps (4-1) to (4-3);

[0034] Step (4-1): The Mp-Bi@SiO2 obtained in step (3) is washed with anhydrous dimethyl sulfoxide and then resuspended in anhydrous dimethyl sulfoxide solution to obtain solution i;

[0035] Step (4-2): Add 3-aminopropyltriethoxysilane to solution i to generate -NH2, and stir to obtain a precipitate;

[0036] Step (4-3): Resuspend the precipitate in dimethylformamide, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and thrombus-targeting peptide, stir and resuspend the precipitate particles in ethanol to form Mp-Bi@SiO2-GK.

[0037] This invention also provides the application of the aforementioned thrombosis-targeting diagnostic and therapeutic integrated nanoprobe in the preparation of thrombosis detection reagents or thrombosis drugs.

[0038] Furthermore, the thrombus-targeted diagnostic and therapeutic integrated nanoprobe is visualized under CT technology to accurately locate the thrombus. After being irradiated with near-infrared light with high skin transmittance, it can undergo photothermal conversion, causing the local temperature of the thrombus to rise and the thrombus to undergo thermal ablation.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1) This invention enables the probe to accumulate at the thrombus site through the targeting effect of the targeting peptide, thereby reducing the side effects of systemic administration.

[0041] 2) In this invention, Bi@SiO2 particles with Bi as the core and SiO2 as the shell are used to form larger pores on the SiO2 surface through the pore-forming agent CTAC, thereby increasing the drug loading capacity of the core-shell nanoprobe.

[0042] 3) This invention utilizes Bi, a high atomic number element, to achieve a high X-ray attenuation coefficient and excellent biocompatibility. Combined with targeting technology, the nanoprobe can specifically perform CT imaging of the thrombus site and the free detached thrombus tissue during treatment, realizing dynamic visualization of the real-time treatment process.

[0043] 4) This invention utilizes the photothermal effect of Bi to convert near-infrared light energy into heat energy, enabling the nanoprobe to generate photothermal capacity under 808nm laser irradiation, thereby removing thrombi through photothermal therapy.

[0044] 5) This invention improves the efficiency of thrombus clearance treatment by combining the chemical drug urokinase with photothermal therapy, and the drug has a better therapeutic effect under elevated temperature conditions.

[0045] 6) In in vitro thrombosis models and ferric chloride-induced mouse carotid artery embolism models, the thrombosis-targeting and therapeutic integrated nanoprobe can image the thrombosis site in real time. At the same time, the chemo-photothermal therapy of the nanoprobe has a good synergistic therapeutic effect and can reduce bleeding side effects to a certain extent, showing certain clinical translation potential. Attached Figure Description

[0046] Figure 1 Transmission electron microscopy (TEM) images of Bi nanoparticles, Bi@SiO2 nanoparticles, Mp-Bi@SiO2 nanoparticles, and Mp-Bi@SiO2-GK / UK nanoparticles in Example 2 of this invention.

[0047] Figure 2 This is the EDS elemental analysis spectrum of the Mp-Bi@SiO2-GK / UK nanoparticles in Example 2 of this invention.

[0048] Figure 3 The release curve of drug UK loaded onto Mp-Bi@SiO2-GK nanoparticles in Example 3 of this invention.

[0049] Figure 4 The images show in vitro CT images of Mp-Bi@SiO2-GK / UK nanoparticles at different concentrations in Example 4 of this invention.

[0050] Figure 5 This image shows the in vitro thrombolytic effect of Mp-Bi@SiO2-GK / UK nanoparticles and other particles in Example 5 of this invention.

[0051] Figure 6 This is an in vivo CT effect image of Mp-Bi@SiO2-GK / UK nanoparticles and other particles in Example 6 of the present invention.

[0052] Figure 7 This is an in vivo thrombolysis diagram of Mp-Bi@SiO2-GK / UK nanoparticles and other particles in Example 7 of the present invention.

[0053] Figure 8 The image shows the fluorescence intensity results after washing following the affinity reaction of the GK peptide loaded with fluorescence with the thrombus in Example 8.

[0054] Figure 9 This is to verify the ability of the GK peptide in Example 8 to target thrombi in vivo. Detailed Implementation

[0055] The present invention will be further described below with reference to the embodiments and accompanying drawings. The following embodiments are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0056] Example 1: Preparation of a thrombus-targeted diagnostic and therapeutic integrated nanoprobe

[0057] The preparation method of the thrombosis-targeted diagnostic and therapeutic integrated nanoprobe is as follows:

[0058] 1) Synthesis of Bismuth Nanoparticles: Bi(NO3)3·5H2O was used as the raw material, dissolved in dodecanethiol, and magnetically stirred in a three-necked flask. Nitrogen gas was continuously pumped in at 55℃ to remove air. After 3-5 rounds of evacuation, the flask was heated to 172℃ with a gradient temperature increase. The solution began to produce yellow bubbles and gradually turned black. The temperature was maintained at 172℃ for 1 min, and then heating was stopped. The supernatant was removed by centrifugation at 12000 rpm for 10 min. The precipitate was washed three times with anhydrous ethanol and then once with cyclohexane. The collected Bi nanoparticles were stored in cyclohexane for later use.

[0059] 2) Synthesis of Bismuth-Silica Nanoparticles (Bi@SiO2): First, silica was coated onto the surface of Bi nanoparticles to form Bi@SiO2. 25 mL of cyclohexane was added to a 50 mL three-necked flask, followed by 1.5 mL of emulsifier Igepal CO-520 while stirring. Then, 1 mL of cyclohexane solution containing the Bi nanoparticles was slowly added dropwise to the flask. After sonication for 30 s, the mixture was stirred continuously at 750 rpm for 5 min. 160 μL of 33% (wt / vol) concentrated ammonia was added dropwise, and the mixture was magnetically stirred for 30 min. 80 μL of TEOS was added dropwise, and the mixture was magnetically stirred at 700 rpm for 24 h. The resulting solution was divided equally into two 50 mL centrifuge tubes. 10 mL of anhydrous ethanol was added to each tube, and the tubes were sonicated for 1 min. Then, 10 mL of acetone was added to each tube, and the tubes were sonicated for another 1 min. Centrifuge at 6000 rpm for 10 min to obtain a precipitate. Add 10 mL of ethanol to each tube to resuspend the precipitate. After vortexing and sonicating for 10 min, combine the solutions from the two centrifuge tubes. Add 15 mL of acetone to the combined solution and centrifuge at 10400 g for 20 min. Remove the supernatant to obtain the precipitate Bi@SiO2.

[0060] 3) Synthesis of bismuth-porous silica nanoparticles (Mp-Bi@SiO2): Precipitated Bi@SiO2 was resuspended and redispersed in 120 mL of water containing CTAC (480 mg) and trimethylbenzene (1.2 mL). After sonication for 30 min, an emulsion was formed. Then, 1 mL of an aqueous solution containing 160 μL TEOS and 24 mg L-arginine was added. The mixture was heated and stirred at 45 °C for 24 h, and after washing and centrifugation, Mp-Bi@SiO2 was obtained.

[0061] 4) Thrombosis-targeting peptide GK (amino acid sequence GPRPVTSEIHLK) reacts with Mp-Bi@SiO2 via an amino-carboxyl condensation reaction under EDC / NHS activation to form Mp-Bi@SiO2-GK. 3 mg of Mp-Bi@SiO2 was washed three times with anhydrous dimethyl sulfoxide (DMOS) and resuspended in 10 mL of DMSO solution, then sonicated for 15 min. 20 μL of APTES was added to generate -NH2. After stirring for 4 h, the precipitate was resuspended in 5 mL of dimethylformamide (DMF), and 4 mg of EDC, 3 mg of NHS, and 1 mL of 0.5 mg / mL GK peptide solution were added. After stirring for 4 h, the precipitate was resuspended in ethanol solution to form Mp-Bi@SiO2-GK.

[0062] 5) Drug loading: Dissolve 2 mg of Mp-Bi@SiO2-GK particles in 20 mL of deionized water, add 1 mg of UK drug, and stir for 2 h to obtain Mp-Bi@SiO2-GK / UK.

[0063] Example 2

[0064] The morphology of each component of the Mp-Bi@SiO2-GK / UK thrombus-targeted diagnostic and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1 was characterized.

[0065] The specific method is as follows: Bi, Bi@SiO2-GK, Mp-Bi@SiO2, and Mp-Bi@SiO2-GK / UK prepared in Example 1 are respectively prepared into 2 mg / mL solutions. Then, 2 μL of each solution is dropped onto a copper grid and left to stand overnight at room temperature, or dried at 70°C for 3 hours. Then, transmission electron microscopy is performed to take pictures.

[0066] The results are as follows Figure 1 As shown, the synthesized Bi consists of uniform, regular black spherical nanoparticles with a particle size of approximately 50 nm. A thin SiO2 film, approximately 5 nm thick, is formed on the Bi@SiO2-GK morphology surface. The image shows that Mp-Bi@SiO2-GK / UK are uniformly sized and regularly shaped nanoparticles with a particle size of approximately 100 nm. Elemental analysis using EDS (Electronic Data Separation) confirms this. Figure 2 The distribution of each element within the particle can be observed, with Bi distributed in the core and O and Si uniformly distributed throughout the particle. This further demonstrates the core-shell structure of the nanoprobe Mp-Bi@SiO2-GK / UK.

[0067] Example 3

[0068] The drug loading function of the Mp-Bi@SiO2-GK / UK thrombus-targeted diagnostic and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1 was characterized.

[0069] The specific method is as follows: Mp-Bi@SiO 2- GK was suspended in PBS solution, and the thrombolytic drug UK was added and stirred for 12 h to form Mp-Bi@SiO2-GK / UK. The supernatant was removed by centrifugation, and the solution was washed three times to remove free UK. The absorbance of the solution at 280 nm was measured. The encapsulation efficiency was calculated by measuring the UK content in the solution. For the near-infrared group, after near-infrared irradiation for 10 min and stirring for 1, 2, 4, 8, 12, and 24 h, the supernatant was collected, the UK concentration was measured, and the release rate was calculated.

[0070] The results are as follows Figure 3 As shown, by detecting the supernatant containing UK, the release efficiency of Mp-Bi@SiO2-GK / UK at pH 5.5 is higher than that at pH 7.0, indicating that Mp-Bi@SiO2-GK / UK has pH-responsive release in the acidic environment of thrombus.

[0071] Example 4

[0072] This embodiment is an in vitro imaging evaluation of the thrombosis-targeting and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1.

[0073] Specifically as follows:

[0074] 1) Prepare a 1% agarose solution in a beaker, heat the beaker to melt the agarose, and use a pipette to transfer the agarose to a 200μL centrifuge tube containing Mp-Bi@SiO2-GK / UK, so that the final concentrations of Mp-Bi@SiO2-GK / UK are 0.5, 1, 2, 3, 4, 6, 8, and 10 mg / mL, respectively. Continuously blow the particle solution to make the particles evenly distributed in the agarose solution, and place it at room temperature to allow the agarose to cool and solidify.

[0075] 2) Place the centrifuge tube under an X-ray scanner to measure the CT signal. For example... Figure 4 As shown, Mp-Bi@SiO2-GK / UK at concentrations of 0.5-10 mg / mL can all be visualized under CT scans. The brightness of the white area reflects the CT effect; as the concentration increases, the imaging effect becomes more pronounced.

[0076] Example 5

[0077] This embodiment evaluates the in vitro thrombolysis of the thrombus-targeted diagnostic and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1.

[0078] Specifically as follows:

[0079] 1) Seven groups were established: PBS treatment group, NIR treatment group, UK treatment group, Mp-Bi@SiO2-GK treatment group, Mp-Bi@SiO2-GK+NIR treatment group, Mp-Bi@SiO2-GK / UK treatment group, and Mp-Bi@SiO2-GK / UK+NIR treatment group. Blood was obtained from C57 mice by orbital blood collection. 400 μL of blood was added to each 1.5 mL centrifuge tube and incubated at 37°C for 2 hours to allow the blood to coagulate into clots.

[0080] 2) After removing the blood clot and washing it three times with PBS solution, place it into a clear vial containing 5 mL of physiological saline. Add different groups of particle solutions to each clear vial, with a final particle concentration of 0.2 mg / mL.

[0081] 3) Irradiate using NIR. The power of the NIR is 1W cm⁻¹. After standing at 37℃ for 30 minutes, observe the color change in the bottle.

[0082] The blood clot supernatants before and after treatment in different treatment groups were compared, and the results are as follows: Figure 5 As shown, Mp-Bi@SiO2-GK / UK has a good thrombolytic effect, and the effect is even better when combined with photothermal therapy.

[0083] Example 6

[0084] This embodiment is an in vivo imaging evaluation of the thrombosis-targeting and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1.

[0085] Specifically as follows:

[0086] 1) Construction of carotid artery thrombosis in mice: Carotid artery thrombosis was modeled using FeCl3. FeCl3 can cause lipid peroxidation in blood vessels and damage endothelial cells, thereby inducing vascular damage and the expression of various adhesion molecules, leading to the adhesion and aggregation of platelets and leukocytes. This process plays a key role in the activation of the coagulation cascade, ultimately resulting in thrombus formation.

[0087] 2) Mice were injected with Mp-Bi@SiO2 / UK, Mp-Bi@SiO2-GK / UK and random polypeptide sequence GV: Mp-Bi@SiO2-GV / UK, respectively.

[0088] 3) Four hours later, the mice were placed under a small animal X-ray CT scanner for CT imaging of the thrombus site. The imaging was magnified using "ultra-focus" mode and scanned in "accurate mode". The beam energy was 55kV and the current was 0.17mA.

[0089] 4) The obtained data were reconstructed into three dimensions using MILabs Rec 10.16 and the images were analyzed using IMALYTICS Preclinical 2.1 software.

[0090] like Figure 6 The results showed that white plaques were observed near the cervical spine in the CT images of the Mp-Bi@SiO2-GK / UK group. The tubular appearance near the cervical spine in the cross-sectional images corresponded to the location of the left carotid artery. The 3D reconstruction images revealed that the white plaques exhibited a slight triangular bifurcation, consistent with the branching point from the common carotid artery to the left carotid artery. These white plaques represent the location of the thrombus, indicating that under the action of the targeting peptide GK, Mp-Bi@SiO2-GK / UK successfully accumulated at the carotid thrombus site and reached the CT imaging threshold, enabling CT imaging.

[0091] Example 7

[0092] This embodiment evaluates the in vivo thrombolysis of the thrombus-targeted diagnostic and therapeutic integrated nanoprobe (Mp-Bi@SiO2-GK / UK) prepared in Example 1.

[0093] Specifically as follows:

[0094] 1) Construction of carotid artery thrombosis in mice: Carotid artery thrombosis was modeled using FeCl3. FeCl3 can cause lipid peroxidation in blood vessels and damage endothelial cells, thereby inducing vascular damage and the expression of various adhesion molecules, leading to the adhesion and aggregation of platelets and leukocytes. This process plays a key role in the activation of the coagulation cascade, ultimately resulting in thrombus formation.

[0095] 2) Forty mice were divided into eight groups (control, PBS treatment group, NIR treatment group, UK treatment group, Mp-Bi@SiO2-GK treatment group, Mp-Bi@SiO2-GK+NIR treatment group, Mp-Bi@SiO2-GK / UK treatment group, and Mp-Bi@SiO2-GK / UK+NIR treatment group). Each mouse was anesthetized by injecting 200 μL of 4% chloral hydrate. The left carotid artery was bluntly dissected, and a 0.5 cm * 0.5 cm filter paper was thoroughly soaked in 10% FeCl3·6H2O and wrapped around the outside of the blood vessel for 5 minutes. Each group was injected with 100 μL of sample. After 5 days, the carotid artery was dissected, washed with PBS solution, and then immersed in 4% paraformaldehyde solution. PBS refers to phosphate buffered saline solution; NIR refers to 808 nm near-infrared laser light.

[0096] 3) Embed and section the vascular tissue, stain it with hematoxylin and eosin (H&E), and observe the condition inside the tube.

[0097] 4) After preparing the slices, the blockage of thrombi in the blood vessels was analyzed using PS and Image-J software. The antithrombotic level was assessed by using the pixel ratio of the blank part and the blood vessel space. The following formula was used for calculation: Antithrombotic clearance rate = pixel value of blank part / total pixel value of blood vessel part.

[0098] A transverse section of the carotid artery can reveal the extent of blockage within the thrombus. Figure 7 The slicing results show that normal blood vessels are hollow, while those with thrombi are almost completely closed. The Mp-Bi@SiO2-GK+NIR group exhibited the highest thrombolytic efficiency, with almost all blood clots being removed. This is because Mp-Bi@SiO2-GK+NIR combines targeted drug delivery and photothermal therapy; the synergistic effect of these two methods significantly enhances the ability to clear thrombi. The photothermal effect dissociates the blood clot, allowing for better drug penetration and greater efficacy, resulting in a thrombus clearance rate as high as 90%.

[0099] Example 8

[0100] The thrombosis-targeting peptides used above were obtained through screening using the following method.

[0101] 1. Construction of a phage 12-peptide library

[0102] Using a phage 12 peptide library constructed through phage display technology, four rounds of in vitro screening were conducted targeting fibrin-specific positive peptides that are present in large numbers at vascular embolism sites.

[0103] (1) Preparation of fibrin substrate: 3 mg / μL fibrinogen solution was mixed with 10 U thrombin at the bottom of a 1.5 mL centrifuge tube. After incubation for 1 h, fibrinogen was converted to fibrin, and the fibrin was washed three times with TBST.

[0104] (2) Background removal of phage library: 10 μL of phage (the first round of screening uses the ph.D.-12 phage library, and the subsequent rounds of screening use the library obtained from the previous round of amplification) was diluted into 500 μL of TBST solution and added to a brand new 1.5 mL ep tube and incubated at 4 °C for 1 h.

[0105] (3) Aspirate all the liquid from step (2) and add it to the fibrin from step (1). Incubate at room temperature for 1 hour with a shaking environment of 200 rpm.

[0106] (4) Aspirate the supernatant from step (3) and wash the fibrinogen 10 times with PBST (5000 rpm, 10 min) to remove the phages that have not bound to the fibrinogen.

[0107] (5) Phage elution: Add 400 μL of Gly-HCl buffer (pH=2.2) to step (3), incubate at room temperature for 10 min, add 70 μL of 1M Tris-HCl (pH=9.1) neutralization solution for neutralization, centrifuge at 5000 rpm for 10 min, and then take out the supernatant and store it in a 4℃ refrigerator.

[0108] (6) Titer counting: Take 10 μL of the eluted phage from (5), dilute it 10-fold with TBS, and add it to 90 μL of EER2738 Escherichia coli solution. Infect for 5 min. Then plate the infected E. coli solution onto LB agar plates containing IPTG and X-gal. Invert the plates in the dark and incubate them in a 37℃ constant temperature incubator for 12 h.

[0109] The phage titer is calculated using the following formula: Titer = 1000 × N × M pfu / mL, where N is the dilution factor and M is the number of blue phage plaques.

[0110] (7) Phage amplification: Add the remaining phage solution from step (5) to 100 mL of activated ER2738 bacterial culture, incubate at room temperature for 15 min, and then incubate in a shaker at 200 rpm and 37 °C for 8 h.

[0111] (8) Phage purification: Centrifuge the phage obtained from (7) at 8000 rpm for 20 min, transfer the supernatant to a 250 mL Erlenmeyer flask, add 9 mL of 16.7% PEG / NaCl, shake to mix, and let it settle overnight at 4 °C; the next day, centrifuge the solution at 12000 g for 30 min, remove the supernatant, resuspend the precipitate in 1 mL of PBS, and determine the concentration to use as the input library for the next round of screening.

[0112] (9) Repeat the above steps for the next round of screening. After four rounds of screening, phages that target fibrin are obtained.

[0113] (10) Take 10 μL of the phage obtained in rounds 2-4 and perform the phage titer experiment according to step (6) to obtain blue phage plaques.

[0114] 2. Phage sequencing

[0115] Sixty blue phage plaques were randomly selected and placed into 5 mL LB medium containing tetracycline in shake tubes. The culture was incubated overnight at 37°C and 150 rpm. The culture was then sequenced using sequencing primer 5'-CCCTCATAGTTAGCGTAACG-3' to obtain the exogenous polypeptide sequences and their number distribution. The most abundant polypeptide was GK, with 28 plaques. The amino acid sequence of GK was GPRPVTSEIHLK, and the nucleotide sequence was ggtcctcgtcctgtgacttcggagattcatctgaaa.

[0116] 3. Affinity verification of the target peptide was performed by constructing an in vitro fibrin model.

[0117] (1) The highest frequency phage clones obtained from screening and the wild M13 phage were amplified and purified respectively.

[0118] (2) Take 100 μL of phage solution (10 10 Add pfu / mL to a test tube containing fibrin and incubate for 1 hour.

[0119] (3) Wash fibrinogen 10 times with PBST (5000 rpm, 10 min) to remove phages that have not bound to fibrinogen.

[0120] (4) Add 400 μL of Gly-HCl buffer (pH=2.2), incubate at room temperature for 10 min, and add 70 μL of 1M Tris-HCl (pH=9.1) neutralization solution for neutralization. Centrifuge at 5000 rpm for 10 min.

[0121] (5) Take 10 μL of the bacteriophage eluted in (4), dilute it 10-fold with TBS, and add it to 90 μL of EER2738 Escherichia coli solution. Infect for 5 min. Then plate the infected E. coli solution onto LB agar plates containing IPTG and X-gal. Incubate the plates upside down in a 37℃ incubator for 12 h in the dark.

[0122] The phage titer is calculated using the following formula: Titer = 1000 × N × M pfu / mL, where N is the dilution factor and M is the number of blue phage plaques.

[0123] (6) The amino acid sequence of the bacteriophage was sequenced, and the sequence with the highest affinity was found to be GK, with an output quantity of 2.27 × 10⁻⁶. 7 pfu.

[0124] (7) Different peptides (GK) were linked to Rhodamine B, and their affinity was verified by fluorescence imaging. 100 μL of fibrinogen and 10 u of thrombin were added to a 96-well plate to form fibrin.

[0125] (8) After washing with PBS three times, add the rhodamine-modified peptide and fibrin and incubate for 1 hour. After incubation, wash with PBS five times and test the fluorescence intensity using a fluorescence imaging device.

[0126] 4. The affinity of the target peptide was verified by constructing a mouse in vivo thrombosis model.

[0127] The specific steps are as follows:

[0128] (1) The high-frequency phage clones and wild M13 phage obtained from screening were amplified and purified respectively.

[0129] (2) Add 100 μL of phage solution (10¹⁰ pfu / mL) to a test tube containing fibrin and incubate for 1 hour.

[0130] (3) Wash fibrinogen 10 times with PBST (5000 rpm, 10 min) to remove phages that are not bound to fibrinogen.

[0131] (4) Add 400 μL of Gly-HCl buffer (pH=2.2), incubate at room temperature for 10 min, and add 70 μL of 1M Tris-HCl (pH=9.1) neutralization solution for neutralization. Centrifuge at 5000 rpm for 10 min.

[0132] (5) Take 10 μL of the phage eluted in (4), dilute it 10-fold with TBS, and add it to 90 μL of EER2738 E. coli solution. Infect for 5 min. Then plate the infected E. coli solution onto LB agar plates containing IPTG and X-gal. Incubate the plates upside down in a 37℃ incubator for 12 h in the dark. Calculate the phage titer using the following formula: Titration = 1000 × N × M pfu / mL, where N is the dilution factor and M is the number of blue phage plaques.

[0133] (6) Sequencing the amino acid sequence of the bacteriophage yielded the sequence with the highest affinity.

[0134] (7) The peptide (GK) was linked to Rhodamine B and injected into mice with a carotid artery thrombosis model via the tail vein. The mice were anesthetized 1 hour later, and the carotid artery was removed after cardiac perfusion for fluorescence intensity observation.

[0135] (8) Anesthetize the mice, cut open the skin of the mouse's chest, separate the muscles above the heart, cut the ribs above the heart, and use surgical instruments to open the chest cavity to expose the heart. Insert a syringe into the heart through the apex, cut open the right atrial appendage with surgical scissors, and simultaneously open the saline valve to allow saline to be pumped into the body through the heart. Blood flows out from the right atrial appendage until the mouse's limbs turn white. Then switch the saline to 4% paraformaldehyde and perfuse again in the same way. After perfusion, separate the carotid artery, cut off the thrombus, and soak it in 4% paraformaldehyde solution.

[0136] (9) Place the carotid artery under a fluorescence imaging device and test its fluorescence intensity by fluorescence intensity test.

[0137] The results are as follows Figure 8 , 9 As shown.

Claims

1. A thrombosis-targeting and therapeutic integrated nanoprobe, comprising a core-shell structure, characterized in that, The core-shell structure has bismuth as the core and porous silica encapsulating bismuth as the outer shell; chemical drugs for treating thrombosis are loaded in the pores of the porous silica, and thrombosis-targeting peptides are attached to the surface of the porous silica. The amino acid sequence of the thrombosis-targeting polypeptide is GPRPVTSEIHLK.

2. The thrombosis-targeting and therapeutic integrated nanoprobe as described in claim 1, characterized in that, The chemical drug is urokinase.

3. The thrombosis-targeting and therapeutic integrated nanoprobe as described in claim 1, characterized in that, The thrombosis-targeting polypeptide is linked to the porous silica via amide bonds; the chemical drug is loaded into the pores of the porous silica via electrostatic interaction.

4. The thrombosis-targeting and therapeutic integrated nanoprobe as described in claim 1, characterized in that, Based on the thrombosis-targeted diagnostic and therapeutic integrated nanoprobe, the loading amount of the chemical drug is 120-130 μg / mg; the loading amount of the thrombosis-targeting peptide is 150-200 μg / mg.

5. The method for preparing the thrombosis-targeting and therapeutic integrated nanoprobe as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Using bismuth nitrate as a raw material, elemental Bi nanoparticles were prepared; (2) Using elemental Bi nanoparticles as the core, SiO2 is grown in situ on the surface of elemental Bi nanoparticles by ethyl silicate to form Bi@SiO2 nanoparticles. (3) Using Bi@SiO2 nanoparticles as raw materials, large pores are formed on the surface of SiO2 by the pore-forming agent hexadecyltrimethylammonium chloride to obtain Mp-Bi@SiO2; (4) The thrombus-targeting polypeptide is activated by an activator and linked to the surface of Mp-Bi@SiO2 to form Mp-Bi@SiO2-GK; (5) Chemical drugs are loaded into the pores of the expanded silica of Mp-Bi@SiO2-GK by a one-pot blending method to form an integrated nanoprobe for thrombosis-targeted diagnosis and treatment, Mp-Bi@SiO2-GK / UK.

6. The preparation method according to claim 5, characterized in that, Step (1) includes: steps (1-1) to (1-3); Step (1-1): Using Bi(NO3)3·5H2O as raw material, dissolve it in dodecanethiol, stir, and continuously pump nitrogen gas at 50-60℃ to remove air; Steps (1-2): After evacuating the air 3-5 times, heat the solution to 170-175℃ using a gradient heating method. The solution will start to produce yellow bubbles and gradually turn black. Maintain the temperature at 170-175℃ for 1-3 minutes, and then stop heating. Steps (1-3): Centrifuge to remove the supernatant, wash the precipitate several times with anhydrous ethanol, then wash with cyclohexane to collect Bi nanoparticles.

7. The preparation method according to claim 5, characterized in that, Step (2) includes: steps (2-1) to (2-5); Step (2-1): While stirring continuously, add the emulsifier to cyclohexane to obtain mixture I; then slowly add the cyclohexane solution containing Bi nanoparticles dropwise to mixture I to obtain mixture II; Step (2-2): Sonicate and continuously stir mixture II, add concentrated ammonia dropwise, and stir to obtain mixture III; Step (2-3): Add ethyl silicate to mixture III, stir, and obtain mixture IV; Step (2-4): Add anhydrous ethanol to mixture IV, sonicate, and then add acetone to obtain mixture V; Steps (2-5): Centrifuge the mixture V, resuspend it in ethanol, add acetone, centrifuge again, and obtain the precipitate Bi@SiO2.

8. The preparation method according to claim 5, characterized in that, Step (3) includes: steps (3-1) to (3-2); Step (3-1): Disperse Bi@SiO2 nanoparticles in water containing the pore-forming agent hexadecyltrimethylammonium chloride and trimethylbenzene, and sonicate to form an emulsion; Step (3-2): Add an aqueous solution of ethyl silicate and L-arginine to the emulsion, heat and stir, wash and centrifuge to obtain Mp-Bi@SiO2.

9. The preparation method according to claim 5, characterized in that, Step (4) includes: steps (4-1) to (4-3); Step (4-1): The Mp-Bi@SiO2 obtained in step (3) is washed with anhydrous dimethyl sulfoxide and then resuspended in anhydrous dimethyl sulfoxide solution to obtain solution i; Step (4-2): Add 3-aminopropyltriethoxysilane to solution i to generate -NH2, and stir to obtain a precipitate; Step (4-3): Resuspend the precipitate in dimethylformamide, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and thrombus-targeting peptide, stir and resuspend the precipitate particles in ethanol to form Mp-Bi@SiO2-GK.

10. The use of the thrombosis-targeting diagnostic and therapeutic integrated nanoprobe as described in any one of claims 1 to 4, or the thrombosis-targeting diagnostic and therapeutic integrated nanoprobe prepared by the preparation method described in claim 5, in the preparation of thrombosis detection reagents or thrombosis treatment drugs.

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