A thrombus-targeting self-sensitizing hybrid nano-assembly, its preparation method and application

Abstract

The present invention discloses a thrombus-targeting self-sensitizing hybrid nano-assembly, its preparation method and application, belonging to the technical field of biomedicine. Specifically, it relates to the synthesis of antiplatelet prodrugs, the preparation method and application of a nano-assembly formed by co-assembling an antiplatelet prodrug modified with a polyethylene glycol modifier linked by a thrombus-targeting peptide and a photothermal / photodynamic bifunctional photosensitizer. The antiplatelet prodrug and the photosensitizer molecule are co-assembled to form a nano-assembly through non-covalent forces. The surface of the nano-assembly is modified with a polyethylene glycol modifier and a polyethylene glycol modifier linked by a thrombus-targeting peptide. The molar ratio of the antiplatelet prodrug to the photosensitizer is 5:1 to 1:5. The preparation method of the present invention is stable and reliable, the preparation process is simple, and the prepared nano-assembly composed of a thrombus-targeting, antiplatelet prodrug and a photothermal / photodynamic bifunctional photosensitizer has a super-high drug loading capacity, and can achieve programmed synergistic treatment of prodrug activation-antiplatelet-photothermal thrombolysis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and relates to a thrombus-targeted self-sensitizing hybrid nano-assembly, a preparation method and an application thereof, and particularly relates to a preparation method and an application of a nano-assembly formed by co-assembling an antiplatelet prodrug modified with a thrombus-targeting peptide-linked polyethylene glycol modifier and a photothermal / photodynamic bifunctional photosensitizer molecule. Background Art

[0002] Thrombosis poses a serious threat to human life and health. Although existing antithrombotic drugs can effectively reduce the formation of arterial and venous thrombi in patients with cardiovascular diseases, they also have some deficiencies, such as poor thrombus penetrability, bleeding, narrow therapeutic window, etc., which limit their applications. Currently, many new therapies have been developed for thrombus treatment. Among them, photothermal thrombolysis has attracted much attention due to advantages such as high treatment accuracy and low bleeding risk. Under laser irradiation, a photosensitizer can convert light energy into heat energy, and the high temperature can effectively loosen the blood clot by disrupting the non-covalent interactions of fibrin, not only playing a role in high-temperature thrombolysis, but also being beneficial to the deep penetration of antithrombotic drugs in the thrombus. However, a single photothermal therapy cannot eradicate thrombi, so the combined application with other therapies shows significant advantages.

[0003] Prodrugs generally refer to compounds that are inactive or have low activity in vitro after structural modification and can be converted and released in vivo to release active drugs to exert their pharmacological effects. The nano-prodrug strategy is expected to overcome the deficiencies of antithrombotic drugs such as off-target toxicity, bleeding, short drug circulation time, etc. Stimulus-responsive prodrugs have been used to design responses to hydrogen peroxide (H2O2) in the thrombus microenvironment. H2O2 is an important mediator for platelet activation, aggregation and thrombosis. However, ROS-responsive drug delivery is hindered by the heterogeneity of H2O2 and thrombus penetration. Therefore, effective thrombus-specific accumulation and deep thrombus penetration are crucial for ROS-responsive antithrombotic drug delivery. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a thrombus-targeted self-sensitizing hybrid nano-assembly, a preparation method and an application thereof, and solves the technical problem that the prodrug is not completely activated at the thrombus site, resulting in poor thrombus treatment effect.

[0005] The present invention realizes the above object through the following technical solutions:

[0006] In a first aspect, the present invention provides a thrombus-targeting self-sensitizing nano thrombolytic assembly, a nano assembly formed by non-covalent forces between an antiplatelet prodrug with an oxidation-sensitive bond bridge chain and a photothermal / photodynamic bifunctional photosensitizer; the surface of the nano assembly is modified with a polyethylene glycol modifier and a polyethylene glycol thrombin fibrin targeting peptide; the antiplatelet prodrug with an oxidation-sensitive bond bridge chain is an antiplatelet prodrug of ticagrelor with an oxidation-sensitive bond bridge chain, dipyridamole or clopidogrel; the photothermal / photodynamic bifunctional photosensitizer is IR808, DiR, ICG, IR820, IR780, IR825, zinc phthalocyanine or PPa.

[0007] Preferably, the photothermal / photodynamic bifunctional photosensitizer is IR808; the antiplatelet prodrug with an oxidation-sensitive bond bridge chain is an antiplatelet prodrug of ticagrelor with an oxidation-sensitive bond bridge chain.

[0008] Furthermore, the molar ratio of the antiplatelet prodrug to the photothermal / photodynamic bifunctional photosensitizer is 5:1 - 1:5.

[0009] Preferably, the molar ratio of the antiplatelet prodrug to the photothermal / photodynamic bifunctional photosensitizer is 1:1.

[0010] Furthermore, the oxidation-sensitive bond includes a monothiol bond, a disulfide bond, a monoselenide bond or a diselenide bond.

[0011] Preferably, the oxidation-sensitive bond is a monothiol bond.

[0012] Furthermore, the polyethylene glycol thrombin fibrin targeting peptide is CREKA or GPRPP.

[0013] Furthermore, the polyethylene glycol modifier is one or more of PCL-PEG, DSPE-PEG, PLGA-PEG, PE-PEG, wherein the molecular weight of the PEG segment is 200 - 20000.

[0014] Preferably, the polyethylene glycol modifier is DSPE-PEG by mass 2K : DSPE-PEG 2K -CREKA = 2:3.

[0015] Furthermore, the mass ratio of the nano assembly, the polyethylene glycol modifier and the polyethylene glycol thrombin fibrin targeting peptide is 75 - 80:10 - 15:15 - 20.

[0016] Preferably, the mass ratio of the nano assembly, the polyethylene glycol modifier and the polyethylene glycol thrombin fibrin targeting peptide is 75:10:15.

[0017] In a second aspect, the present invention provides a preparation method of a thrombus-targeting self-sensitizing nano thrombolytic assembly, comprising the following steps:

[0018] (1) Synthesize an antiplatelet prodrug with an oxidation-sensitive bond bridge chain by an esterification method;

[0019] (2) Dissolve the antiplatelet prodrug and the photothermal / photodynamic bifunctional photosensitizer in an organic solvent respectively, and then slowly drop them into water and stir well to form a uniform nano-assembly;

[0020] (3) Under vigorous stirring, slowly drop the mixed solution of the polyethylene glycol modifier and the polyethylene glycol thrombin fibrin targeting peptide into the nano-assembly, and remove the organic solvent to obtain the product.

[0021] Further, the organic solvent is one or any combination of two of ethanol, tetrahydrofuran, and dimethyl sulfoxide.

[0022] In the third aspect, the present invention provides an application of the thrombus-targeted self-sensitizing nano-thrombolytic assembly in the preparation of an antithrombotic drug.

[0023] Preferably, the antithrombotic drug is an antiplatelet aggregation drug.

[0024] Further, the administration method of the antithrombotic drug includes injection administration or oral administration.

[0025] Further, the thrombus-targeted self-sensitizing nano-thrombolytic assembly is used for the activation of the antithrombotic prodrug.

[0026] Further, the administration dose of the thrombus-targeted self-sensitizing nano-thrombolytic assembly is 1-5 mg / kg.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) The present invention synthesizes an antiplatelet prodrug with a monothiol bond bridge chain by an esterification method, which can release drugs in response to H2O2 at the thrombus site. Combined with the photothermal / photodynamic bifunctional photosensitizer, it synergistically promotes the activation of the prodrug and enhances the drug release.

[0029] (2) The present invention utilizes the photothermal effect and photodynamic effect of the photothermal / photodynamic bifunctional photosensitizer molecule. Under 808 nm laser irradiation, the photosensitizer will undergo photothermal energy conversion, generating a large amount of heat at the thrombus site, thereby enhancing the thrombus penetration of the nanoparticles and enabling more drug-loaded nano-assemblies to enter the deep part of the thrombus; in addition, the bifunctional photosensitizer will generate ROS under laser irradiation, which synergistically promotes the activation of the prodrug with endogenous H2O2, effectively overcoming the challenge that the lack of H2O2 in the thrombus mass leads to incomplete activation of the prodrug.

[0030] (3) By utilizing the molecular nano-assembly characteristics of antiplatelet prodrugs and photosensitizers, the present invention constructs a carrier-free nano-assembly. This molecular self-assembly nano-delivery system has the advantage of ultra-high drug loading capacity, thus realizing the efficient co-loading and synchronous delivery of two drugs.

[0031] (4) By PEGylating and targeting the surface of the nanoparticles, the present invention can improve their surface hydrophilicity, endow them with active targeting function, and prolong their circulation time in vivo. This enables the nanoparticles to efficiently accumulate at the thrombus site, achieving the efficient delivery of the co-assembled nanoparticles.

[0032] (5) In an in vitro thrombus model and a ferric chloride-induced rat carotid artery embolism model, the thrombus-targeting self-sensitizing hybrid nano-assembly shows good programmed synergistic therapeutic effects of prodrug activation-antiplatelet-photothermal thrombolysis. The prodrug strategy can reduce bleeding side effects to a certain extent and has certain potential for clinical translation. Description of the Drawings

[0033] Figure 1 Synthetic route of ticagrelor-monothiol bond-fluorene methanol (TSF) prodrug.

[0034] Figure 2 Characterization of the mass spectrometry and nuclear magnetic resonance results of the TSF prodrug; where A is the mass spectrometry result; B is the nuclear magnetic resonance result.

[0035] Figure 3 Transmission electron microscope photos of the self-sensitizing hybrid nano-assembly and the thrombus-targeting self-sensitizing hybrid nano-assembly in Examples 2 and 3 of the present invention.

[0036] Figure 4 Ultraviolet spectrum of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 4 of the present invention.

[0037] Figure 5 Fluorescence spectrum of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 4 of the present invention.

[0038] Figure 6 Investigation of the stability of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in PBS in Example 5 of the present invention.

[0039] Figure 7 In vitro drug release diagrams of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in different concentrations of H2O2 and with or without light in Example 6 of the present invention; where A is the release curve with different concentrations of H2O2 added; B is the release curve of the light and non-light groups.

[0040] Figure 8It is for the investigation of the targeting of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in two in vitro thrombus models in Example 7 of the present invention; wherein, A is the in vitro fluorescence signal of the artificial platelet thrombus; B is the quantitative analysis of the fluorescence intensity of the in vitro platelet thrombus; C is the quantitative analysis of the fluorescence intensity of the in vitro whole blood thrombus

[0041] Figure 9 It is for the temperature change of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly under 808 nm laser irradiation in Example 8 of the present invention.

[0042] Figure 10 It is for the thrombus penetration of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly under 808 nm laser irradiation in Example 9 of the present invention.

[0043] Figure 11 It is for the in vitro thrombolysis of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly under 808 nm laser irradiation in Example 10 of the present invention.

[0044] Figure 12 It is for the in vitro antiplatelet activity of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 11 of the present invention.

[0045] Figure 13 It is for the pharmacokinetic investigation of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 12 of the present invention.

[0046] Figure 14 It is for the fluorescence imaging of the embolized blood vessels using the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 13 of the present invention; wherein, A is the fluorescence imaging of the carotid artery embolized blood vessels; B is the quantitative analysis of the fluorescence signal intensity of the carotid artery embolized blood vessels.

[0047] Figure 15 It is for the in vivo photothermal conversion ability of the TSF / IRs08 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 14 of the present invention.

[0048] Figure 16 It is the H&E staining photographs of the carotid artery embolized blood vessels in each group after drug treatment in Example 15 of the present invention.

[0049] Figure 17 It is the quantitative analysis of the embolization degree of the carotid artery embolized blood vessels in each group after drug treatment in Example 15 of the present invention.

[0050] Figure 18 It is for the in vivo antiplatelet activity of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly in Example 16 of the present invention.

[0051] Figure 19 H&E staining photograph of the toxicity to normal tissues after administration of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly in Example 17 of the present invention.

[0052] Figure 20 Liver and kidney function indexes after administration of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly in Example 17 of the present invention. Detailed implementation manners

[0053] The present invention will be further described below by way of examples, but the invention is not limited to the scope of the described examples.

[0054] DSPE-PEG described in the examples 2K -CREKA is from (Suzhou Polypeptide Biotechnology Co., Ltd.).

[0055] The present invention uses prodrug synthesis technology, molecular co-assembly nanotechnology and fibrin active targeting technology to construct a thrombus-targeted nano-drug delivery system co-assembled by a photothermal / photodynamic bifunctional photosensitizer molecule, an antiplatelet prodrug, a polyethylene glycol modifier and a polyethylene glycol thrombus fibrin targeting peptide. The present invention aims to provide a novel drug delivery strategy for the combined treatment of thrombus-related cardiovascular and cerebrovascular diseases, and provide a new idea for solving the incomplete activation of prodrugs at the thrombus site and enhancing prodrug activation.

[0056] The present invention uses a photothermal / photodynamic bifunctional photosensitizer in combination with an antiplatelet prodrug for antithrombotic treatment. Under laser irradiation, the controllable temperature rise at the thrombus site can not only achieve hyperthermal thrombolysis, but also promote the deep penetration of the prodrug in the thrombus. At the same time, ROS generated by the photothermal / photodynamic bifunctional photosensitizer IR808 and endogenous H2O2 overproduced in the clot synergistically trigger site-specific prodrug activation and sensitize drug release. Antiplatelet drugs inhibit thrombus formation by preventing platelet activation and aggregation, and finally achieve photothermal / antiplatelet synergistic thrombolysis.

[0057] The preparation process of the thrombus-targeted nano-drug delivery system of the present invention is as follows: First, an antiplatelet prodrug with a monothiol bond bridge chain is synthesized by an esterification method, and a photothermal / photodynamic bifunctional photosensitizer molecule and an antiplatelet prodrug molecule are assembled into a nano-assembly by non-covalent interactions. Since the molecular self-assembly nano-drug delivery system has the advantage of ultra-high drug loading capacity, it can achieve efficient co-loading of the photosensitizer molecule and the antiplatelet prodrug to achieve the purpose of combined treatment of the two. Then, a polyethylene glycol modifier and a polyethylene glycol thrombus fibrin targeting peptide are modified on the surface of the nano-system to extend the circulation time of the nano-system in vivo and endow it with thrombus targeting ability.

[0058] Once the nanoassemblies enter the bloodstream, they can efficiently accumulate at the thrombus site. Under laser irradiation, the heat generated by the nanoassemblies can not only dissolve the thrombus but also promote their penetration into the thrombus. The reactive oxygen species generated by photodynamic action can promote the activation of antiplatelet prodrugs, releasing antiplatelet drugs, thereby exerting a therapeutic effect and simultaneously reducing the bleeding risk.

[0059] Example 1: Synthesis of Ticagrelor Prodrug with Monothiol Bond Bridge Chain (TSF)

[0060] The synthesis route of the TSF prodrug is as Figure 1 , dissolve fluorenylmethanol (Fmoc) (1 mmol) and thioacetic anhydride (1 mmol) in an appropriate amount of dichloromethane, slowly add DMAP (0.3 mmol) dropwise under stirring at 25 °C, and react for 12 h under nitrogen protection. After the reaction, the intermediate (Fmoc-S-COOH) was roughly separated by column chromatography (V dichloromethane / V methanol = 300:1, containing one-thousandth formic acid). Dissolve Fmoc-S-COOH (1 mmol) in an appropriate amount of dichloromethane under ice bath conditions, add EDCI (2 mmol) and HOBt (1 mmol), and react for 2 h under nitrogen protection. After activation, add ticagrelor (TGL) (1 mmol), then slowly add DMAP (0.3 mmol), and stir at 30 °C under nitrogen protection for 36 h. Using acetonitrile-water (75:25) as the mobile phase, the product (TGL-S-Fmoc) was separated and purified by preparative liquid chromatography. As Figure 2 shown, the results of mass spectrometry and proton nuclear magnetic resonance ( 1 1H NMR) confirmed the successful synthesis of the prodrug. 11H NMR (600 MHz, DMSO-d6) δ 9.38 (d, J = 4.1 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.69 (dd, J = 7.6, 3.6 Hz, 2H), 7.40 (q, J = 6.9 Hz, 2H), 7.38–7.34 (m, 1H), 7.33–7.30 (m, 2H), 7.29–7.26 (m, 1H), 7.08–7.04 (m, 1H), 5.59 (ddd, J = 20.8, 11.5, 5.4 Hz, 2H), 5.13 (dd, J = 5.5, 2.8 Hz, 1H), 4.97 (q, J = 9.0 Hz, 1H), 4.77 (dt, J = 8.4, 5.4 Hz, 1H), 4.65 (s, 1H), 4.46 (d, J = 6.8 Hz, 2H), 4.40–4.34 (m, 1H), 4.29 (t, J = 6.8 Hz, 1H), 3.97 (ddd, J = 8.2, 6.0, 2.8 Hz, 1H), 3.51 (tdd, J = 17.1, 9.7, 5.3 Hz, 4H), 3.46 (s, 2H), 3.15 (dq, J = 8.2, 4.1 Hz, 1H), 3.07 (dq, J = 10.2, 5.4 Hz, 1H), 2.95–2.85 (m, 1H), 2.13 (dddt, J = 15.4, 9.4, 6.2, 3.6 Hz, 2H), 2.07 (s, 1H), 1.55 (dt, J = 10.1, 5.5 Hz, 1H), 1.48 (tdd, J = 12.8, 7.3, 4.5 Hz, 2H), 0.96 (td, J = 7.5, 3.2 Hz, 1H), 0.78 (dd, J = 7.4, 4.2 Hz, 3H).

[0061] Example 2: Preparation of TSF / IR808 Self-Sensitizing Hybrid Nanostructures

[0062] Dissolve TSF and IR808 in a mixed solution of tetrahydrofuran - absolute ethanol (Vtetrahydrofuran / Vabsolute ethanol = 1:1) to prepare drug-containing solutions with a concentration of 5 mg / mL respectively. Subsequently, mix TSF solutions and IR808 solutions with different molar ratios (5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5) to 200 μL, and drop the mixture into 2 mL of deionized water at a stirring speed of 1200 rpm. After stirring for 3 min, TSF / IR808 self-sensitizing nanoassemblies are formed. Remove the organic solvent by rotary evaporation and adjust the solution volume to 2 mL. The particle size distribution of the obtained self-sensitizing hybrid nanoassemblies is shown in Table 1. Nanoassemblies can be formed when the molar ratio of IR808 to TSF is between 5:1 and 1:5. When the molar ratio of IR808 to TSF is equal to 1:1, the particle size and PDI of the nanoassemblies are the smallest and the assembly performance is the strongest. Therefore, the optimal molar ratio of IR808 to TSF is finally determined to be 1:1, and the TSF / IR808 self-sensitizing hybrid nanoassemblies described hereinafter are all prepared under this molar ratio condition.

[0063] Table 1 Proportion screening of hybrid nanoassemblies

[0064]

[0065] Example 3: Preparation of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies

[0066] DSPE-PEG 2K / DSPE-PEG 2K -CREKA mixed solution preparation: Dissolve DSPE-PEG 2K in methanol solution with a concentration of 10 mg / mL. Dissolve DSPE-PEG 2K -CREKA in methanol solution with a concentration of 1.5 mg / mL. Take 20 μL of DSPE-PEG 2K solution and 87 μL of DSPE-PEG 2K -CREKA solution and mix them to obtain. The method of modifying DSPE-PEG 2K / DSPE-PEG 2K -CREKA on the surface of nanoassemblies: As described above, first prepare TSF / IR808 self-sensitizing hybrid nanoassemblies, and then slowly drop the above DSPE-PEG 2K / DSPE-PEG 2K -CREKA mixed solution, stir for 3 min, remove the organic solvent under vacuum conditions at 30 °C, and finally adjust the solution volume to 2 mL.

[0067] The particle size distribution potential and drug loading of the prepared TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies are shown in Table 2. The particle size of the TSF / IR808 self-sensitizing hybrid nanoassemblies is 90.17 nm, and the Zeta potential is +0.233. After PEGylation and targeting modification, the particle size of the nanoparticles increases to 111.2 nm, and the Zeta potential decreases to -13.5 mV. In addition, the sizes of the above two kinds of nanoparticles are uniform and spherical ( Figure 3 ). Both kinds of nanoparticles have a very high drug loading, 52% for TSF and 48% for IR808 (TSF / IR808 self-sensitizing hybrid nanoassemblies) or 39.1% for TSF and 36.1% for IR808 (TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies).

[0068] Table 2 Characterization of TSF / IR808 series nanoassemblies

[0069]

[0070] Example 4: UV fluorescence spectra of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies

[0071] The UV spectra of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies prepared in Example 3, the TSF / IR808 self-sensitizing hybrid nanoassemblies prepared in Example 2, the TSF solution, the IR808 solution, and the TGL solution were detected by a multifunctional microplate reader respectively (the concentrations of IR808 and TGL are 20 μg / mL and 13.6 μg / mL for TGL respectively). Under the same conditions, the fluorescence spectra of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies, the TSF / IR808 self-sensitizing hybrid nanoassemblies, and the IR808 solution were scanned by a multifunctional microplate reader. Compared with the solution group, Figure 4 it shows that the UV spectra of the two kinds of nanoassemblies have a red shift, Figure 5 it shows that the peak shapes of the fluorescence spectra are the same and the fluorescence intensity decreases, proving the successful formation of the nanoassemblies.

[0072] Example 5: Physical stability experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies

[0073] The TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies prepared in Example 3 and the TSF / IR808 self-sensitizing hybrid nanoassemblies prepared in Example 2 were incubated in PBS (pH 7.4) for 12 h respectively. Then, the particle size changes were measured by dynamic light scattering method at 1, 2, 4, 8, and 12 h respectively. The results are as Figure 6As shown, the particle size of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly did not change significantly after incubation, indicating good colloidal stability.

[0074] Example 6: In vitro drug release effect of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly

[0075] Under the condition of 37 °C, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly prepared in Example 3 (equivalent to 200 μg TGL) was incubated in 30 mL of PBS (pH 7.4) release medium containing 10% tetrahydrofuran (v / v) and 10% ethanol (v / v). At regular time intervals, 200 μL of the solution was extracted and the concentration of TGL was determined by high performance liquid chromatography and its cumulative release amount was calculated. Different concentrations of H2O2 were added to the release medium to investigate its release characteristics under single oxidation conditions. Subsequently, a group with 2 mM H2O2 was set up and laser (808 nm, 2 W / cm 2 ) irradiation for 10 min was given to study the synergistic release effect of photodynamic and oxidation conditions. The results are as Figure 7 shown in A and B below. The release of TGL showed a characteristic of H2O2 concentration dependence; in addition, light irradiation could significantly increase the drug release.

[0076] Example 7: In vitro thrombus-targeting experiment of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly

[0077] Establishment of a platelet-rich plasma clot thrombus model: Platelet-rich plasma (150 μL) was taken from SD rats, mixed with thrombin (0.1 U / μL) and CaCl2 (0.3 M), and added to a 96-well plate and incubated at 37 °C for 120 min to obtain a platelet-rich plasma clot thrombus model.

[0078] Establishment of a whole blood clot thrombus model: Whole blood (150 μL) of SD rats was taken and incubated with CaCl2 (0.3 M) and thrombin (0.1 U / μL) in a 96-well plate at 37 °C for 120 min until thrombus formation.

[0079] In vitro thrombus-targeting experiment: 50 μL each of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly, the TSF / IR808 self-sensitizing hybrid nano-assembly, the IR808 solution, and PBS were added to a 96-well plate containing thrombus and incubated (37 °C, 10 min), and the thrombus mass was washed 3 times with PBS. Finally, a small animal in vivo imaging system was used to measure the fluorescence signal on the surface of the thrombus to judge its targeting ability. As Figure 8 shown in A, B, and C below, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly showed a stronger fluorescence signal, demonstrating its good thrombus-targeting property.

[0080] Example 8: In vitro photothermal effect experiment of TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies

[0081] PBS, IR808 solution, TSF / IR808 self-sensitizing hybrid nanoassemblies, and TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies (with a concentration of 0.24 mg / mL for IR808 and 0.26 mg / mL for TSF) were irradiated under an 808 nm laser for 15 min (2 W / cm 2 ), and an infrared thermal imager (Fotric 226) was used to record the temperature changes during this process. As Figure 9 shown, compared with PBS, the temperatures of the other three groups gradually increased with the increase of irradiation time, reaching up to 57 °C. Under this condition, the non-covalent bonds of fibrin in thrombus can be disrupted, further achieving the effect of photothermal thrombolysis.

[0082] Example 9: Thrombus permeability experiment of TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies

[0083] Establishment of microthrombi: Healthy SD rats were selected for orbital blood collection. 10 μL of fresh blood was placed at the bottom of an EP tube and incubated in a 37 °C incubator for 2 h to obtain microthrombi.

[0084] Preparation of coumarin-6-labeled TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies: IR808 and TSF were separately dissolved in ethanol to prepare drug solutions with a concentration of 1 mg / mL, and coumarin-6 was dissolved in ethanol to prepare a drug solution with a concentration of 10 mg / mL. Then, 100 μL of IR808 and TSF solutions and 50 μL of coumarin-6 solution were mixed. The above mixed solution was dropped into 2 mL of deionized water and stirred in the dark for 3 min. Subsequently, a DSPE-PEG 2K / DSPE-PEG 2K -CREKA mixed solution was added dropwise to the prepared TSF / IR808 self-sensitizing hybrid nanoassembly solution and stirred in the dark for 2 min. Finally, the organic solvent was removed by rotary evaporation, and the volume of the solution was adjusted to 2 mL.

[0085] Thrombus permeability experiment: Coumarin-6-labeled TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies and coumarin-6 solution were added to the EP tube containing thrombi and incubated at 37 °C for 30 min. Then, the coumarin-6-labeled TSF / IR808 thrombus-targeted self-sensitizing hybrid nanoassemblies were irradiated with an 808 nm laser (2 W / cm 2 , 15 min). A confocal microscope was used to observe the thrombus penetration effect of the nanoassemblies.

[0086] The confocal images are as follows Figure 10 As shown, the thrombus penetration ability of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly without 808 nm laser irradiation is weak, and the signal of coumarin-6 inside the thrombus is significantly enhanced after irradiation. This indicates that the photothermal effect generated by IR808 after light irradiation can increase the penetration of the nanoassembly in the thrombus mass.

[0087] Example 10: In vitro photothermal thrombolysis experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly

[0088] Establishment of thrombus model: Healthy SD rats were selected for orbital blood collection, and EP tubes coated with anticoagulant (heparin) were used as containers. At the same time, thrombin with a concentration of 0.1 U / μL and calcium chloride (CaCl2) with a concentration of 0.3 M were prepared. 500 μL of platelet plasma layer, 50 μL of thrombin and 50 μL of CaCl2 were added to a 1.5 mL EP tube respectively, and it was placed in an incubator at 37 °C for 90 min to obtain a thrombus model.

[0089] Photothermal thrombolysis experiment: Preparations of different groups (I: PBS, II: TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly, III: TSF / IR808 self-sensitizing hybrid nanoassembly, IV: IR808 solution, V: TSF solution, VI: TGL solution) were added to the thrombus mass respectively, and placed under an 808 nm laser (2 W / cm 2 , 20 min). The weights of the thrombus mass before and after thrombolysis were measured respectively, and the thrombolysis rate was calculated (thrombolysis rate = (weight of thrombus mass before thrombolysis - weight of thrombus mass after thrombolysis) / weight of thrombus mass before thrombolysis × 100%). The results are as follows Figure 11 As shown, due to only the photothermal effect taking effect, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly shows a medium thrombolysis effect.

[0090] Example 11: In vitro antiplatelet activity experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly

[0091] The in vitro antiplatelet experiment of the nano-assembly was investigated using a rat soluble CD40 ligand detection kit. 50 μL of platelet-rich plasma was activated with thrombin (0.1 U / μL), placed in the kit containing antibodies, and then H2O2, TGL solution, TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly and H2O2, TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly, TSF / IR808 self-sensitizing hybrid nano-assembly and H2O2, TSF solution and H2O2, TSF solution, IR808 solution and H2O2, TSF solution and IR808 solution mixture solution and H2O2, TSF solution and IR808 solution mixture solution and H2O2, PBS and H2O2 were incubated at room temperature for 4 h. Finally, the expression level of rat soluble CD40 ligand (sCD40L) in plasma was determined according to the kit method. The results are as Figure 12 shown that the TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly can significantly inhibit thrombin-induced platelet activation and the increase in the expression level of sCD40L, and has good in vitro antiplatelet effect.

[0092] Example 12: Pharmacokinetic study of TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly

[0093] SD rats weighing between 180 - 220 g were randomly grouped, fasted for 12 h before dosing, and allowed free access to water. IR808 solution, TSF / IR808 self-sensitizing hybrid nano-assembly and TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly (the dosing dose of IR808 was 5 mg / kg) were respectively injected into the tail vein. Rat blood (~500 μL) was collected at the specified time points (0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h), and plasma was obtained by centrifugation (8000 rpm / min, 3 min). Subsequently, IR808 was extracted from plasma by the protein precipitation method. Finally, the fluorescence intensity of IR808 in plasma samples was quantified using a multifunctional microplate reader. Pharmacokinetic parameters were calculated using DAS 2.1.1 software. The results are as Figure 13 shown that compared with the IR808 solution, the pharmacokinetic parameters such as the blood drug concentration (C1 h) and AUC of the polyethylene glycol-modified nano-assembly were significantly improved, significantly enhancing the pharmacokinetic properties of the drug.

[0094] Table 3. Pharmacokinetic parameters of the nano-assembly

[0095]

[0096] Example 13: Embolized blood vessel fluorescence imaging experiment of TSF / IR808 thrombus-targeted self-sensitizing hybrid nano-assembly

[0097] Establishment of FeCl3-induced carotid artery embolism model in rats: SD rats (male, 6 weeks old, 180 - 220 g) were anesthetized by intraperitoneal injection of 3.5% chloral hydrate. Subsequently, the rats were safely placed on the operating table to ensure the stability of the entire surgical process. A midline incision was made from the chin to the sternum, and the common carotid artery (CCA) was accessed by separating the surrounding muscles. Then, a filter paper (10×10 mm) soaked with FeCl3 (10% w / w) was applied to the CCA for 8 min to induce thrombus formation. After that, the embolized CCA was rinsed with PBS to remove any remaining FeCl3. Then, IR808 solution, TSF / IR808 self-sensitizing hybrid nanoassemblies, and TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies were intravenously injected at a dose of 5 mg / kg of IR808, respectively. The embolized blood vessels were imaged using a small animal in vivo imager at specific time intervals (5, 15, 30, 60, 120, and 180 min). The experimental results are as Figure 14 shown in A and B, and the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies exhibited strong fluorescence signals in the embolized blood vessels, which may be attributed to the good pharmacokinetic behavior after polyethylene glycol modification and also laid a solid foundation for achieving vascular fluorescence imaging.

[0098] Example 14: In vivo photothermal effect experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies

[0099] PBS, IR808 solution, TSF / IR808 self-sensitizing hybrid nanoassemblies, and TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies (the dosing dose of IR808 was 5 mg / kg) were respectively injected into FeCl3-induced carotid artery embolism rats. After administration, near-infrared laser irradiation (808 nm, 2 W / cm 2 ) was performed on the thrombus site of the model rats at the optimal drug accumulation time point (5 min for the IR808 solution group, 1 h for the TSF / IR808 self-sensitizing hybrid nanoassemblies and TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies group), and the light irradiation lasted for 15 min. An infrared thermal imager (Fotric 226) was used to record the thermal images of the above groups and measure their local temperature changes. The results are as Figure 15 shown, and compared with the IR808 solution and TSF / IR808 self-sensitizing hybrid nanoassemblies, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies exhibited more excellent heating curves and photothermal effects.

[0100] Example 15: In vivo antithrombotic experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassemblies Six-week-old male SD rats were randomly divided into 8 groups: (1) Embolism group (correspondingFigure 16 Normal saline); (2) TSF solution group; (3) IR808 solution + 808 nm laser group; (4) Mixed solution of TSF solution and IR808 solution + 808 nm laser group; (5) TGL solution group; (6) TSF / IR808 self-sensitizing hybrid nano-assembly + 808 nm laser group; (7) TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly; (8) TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly + 808 nm laser group; (9) Blank group.

[0101] As described above, a rat carotid artery embolism model was established, and various preparations were injected (the administration dose of IR808 was 5 mg / kg, and that of TGL was 3.40 mg / kg). 1 h later, all the 808 nm laser groups were irradiated with laser at the carotid artery thrombus site (2 W / cm 2 , 15 min), the rats were sacrificed and the embolized blood vessels were removed, and H&E staining was performed to check the embolism situation.

[0102] The results were as Figure 16-17 shown. The TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly + 808 nm laser group showed excellent thrombolytic effect. Compared with the TSF / IR808 self-sensitizing hybrid nano-assembly + 808 nm laser group, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly + 808 nm laser group had better blood circulation effect and thrombus-targeting ability. Similarly, although the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly had good blood circulation effect and thrombus-targeting ability, due to the lack of light, the prodrug could not be fully activated, so the thrombolytic effect was not optimistic. More notably, compared with the clinically commonly used antiplatelet drug TGL, the TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly + 808 nm laser group of the present invention showed better thrombolytic ability, verifying that this antiplatelet / photothermal combined thrombolytic therapy has excellent clinical application potential.

[0103] Example 16: In vivo antiplatelet activity experiment of TSF / IR808 thrombus-targeting self-sensitizing hybrid nano-assembly

[0104] In the pharmacodynamic experiment, blood samples were immediately collected after removing the embolized blood vessels. The samples were placed in tubes and centrifuged at 11000×g for 10 min to obtain plasma. The expression level of rat soluble CD40 ligand (sCD40L) in the plasma was measured according to the kit method. The results were as Figure 18As shown, the in vivo antiplatelet activity of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly + 808 nm laser group was significantly stronger than that of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly, mainly relying on the effective activation of the prodrug; in addition, the TSF / IR808 self-sensitizing hybrid nanoassembly + 808 nm laser group and the TGL solution group had poor blood circulation effects, resulting in worse effects than the TSF / IR808 thrombus-targeting self-sensitizing nanoassembly + 808 nm laser group.

[0105] Example 17: In vivo safety experiment of the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly

[0106] The in vivo safety of the co-assembled nanoparticles was studied using SD rats. The group settings were the same as in Example 15. After 24 h, blood was collected from the orbital cavity, and the plasma was obtained by centrifuging the blood for liver and kidney function tests. Subsequently, the rats were sacrificed, and the heart, liver, spleen, lungs, and kidneys were taken for H&E staining. As Figure 19 shown, no obvious histological damage was observed in the heart, liver, spleen, lungs, and kidneys of the rats in each treatment group. The results were as Figure 20 shown. Compared with the normal saline group, the liver and kidney functions of the rats in other groups were normal. The above results prove that the TSF / IR808 thrombus-targeting self-sensitizing hybrid nanoassembly has high therapeutic safety.

[0107] For any person skilled in the art, without departing from the scope of the technical solution of the present invention, many possible changes and modifications can be made to the technical solution of the present invention by using the technical content disclosed above, or it can be modified into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A thrombus-targeting self-sensitizing hybrid nanoassembly, characterized in that, A nano-assembly formed by the non-covalent assembly of an antiplatelet prodrug with an oxidation-sensitive bond bridge chain and a photothermal / photodynamic bifunctional photosensitizer; the surface of the nano-assembly is modified with a polyethylene glycol modifier and a polyethylene glycol thrombin fibrin targeting peptide; The antiplatelet prodrug with an oxidation-sensitive bond bridge chain is a ticagrelor prodrug with an oxidation-sensitive bond bridge chain; the photothermal / photodynamic bifunctional photosensitizer is IR808; The molar ratio of the antiplatelet prodrug to the photothermal / photodynamic bifunctional photosensitizer is 5:1 - 1:5; The oxidation-sensitive bond is a monosulfide bond; The polyethylene glycol thrombin fibrin targeting peptide is CREKA; The polyethylene glycol modifier is one or more of PCL-PEG, DSPE-PEG, PLGA-PEG, PE-PEG, and the molecular weight of the PEG segment is 200 - 20000; The mass ratio of the nano-assembly, the polyethylene glycol modifier, and the polyethylene glycol thrombin fibrin targeting peptide is 75 - 80:10 - 15:15 - 20.

2. The preparation method of the thrombus-targeting self-sensitizing hybrid nano-assembly according to claim 1, characterized in that, It includes the following steps: (1) Synthesize the antiplatelet prodrug with an oxidation-sensitive bond bridge chain by an esterification method; (2) Dissolve the antiplatelet prodrug and the photothermal / photodynamic bifunctional photosensitizer in an organic solvent respectively, then slowly drop them into water and stir well to form a uniform nano-assembly; (3) Under vigorous stirring, slowly drop the mixed solution of the polyethylene glycol modifier and the polyethylene glycol thrombin fibrin targeting peptide into the nano-assembly, and remove the organic solvent to obtain the product.

3. The preparation method according to claim 2, characterized in that, The organic solvent is one or any combination of two of ethanol, tetrahydrofuran, and dimethyl sulfoxide.

4. Use of the thrombus-targeting self-sensitizing hybrid nano-assembly according to claim 1 in the preparation of an antithrombotic drug.

5. The application according to claim 4, wherein The administration method of the antithrombotic drug includes injection administration or oral administration.

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