Cascade antioxidant carrier-free nanomotor and preparation method and application thereof

Abstract

This invention relates to a cascaded antioxidant carrier-free nanomotor, its preparation method, and its application, belonging to the field of biomedical technology. The cascaded antioxidant carrier-free nanomotor is formed by the self-assembly of a functionally converted nitric oxide donor (BNN-NO) and a photothermal photosensitizer through non-covalent interactions, and is further modified with a polyethylene glycol modifier and a polyethylene glycol-modified lesion-targeting ligand; wherein the photothermal photosensitizer is one or both of DiR and DiD; the functionally converted nitric oxide donor (BNN-NO) has the following structure. This invention utilizes small molecule hybrid self-assembly nanotechnology, photothermal response modulation technology, and a thrombus-targeting modification strategy to construct a carrier-free nanomotor integrating "gas-driven—photothermal response—cascaded antioxidant" processes, thereby achieving synergistic optimization of thrombolysis and reperfusion protection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a cascaded antioxidant carrier-free nanomotor, its preparation method and application, specifically to a photothermal responsive nanomotor based on a nitric oxide donor with cascaded antioxidant properties and its application in the preparation of drugs for thrombotic diseases and / or ischemia-reperfusion injury. Background Technology

[0002] Thrombotic diseases are a major cause of cardiovascular and cerebrovascular events, with high mortality and disability rates. Current clinical treatment primarily involves thrombolytic drugs and mechanical thrombectomy. However, traditional thrombolytic drugs suffer from a narrow therapeutic window, high bleeding risk, short drug half-life, and poor thrombus penetration, severely limiting their clinical efficacy. Furthermore, even if the thrombus is successfully dissolved and blood flow is restored, ischemic tissue generates a large amount of reactive oxygen species (ROS) during reperfusion, inducing ischemia-reperfusion injury (IRI), leading to adverse consequences such as vascular endothelial damage, enhanced inflammation, and thrombus recurrence. Therefore, relying solely on thrombolytic therapy is insufficient to achieve ideal therapeutic effects. In recent years, nanomedicine delivery systems have provided new strategies for improving drug targeting and therapeutic efficacy. Carrier-free nanoassembly systems have attracted widespread attention due to their high drug loading capacity and good biocompatibility. However, existing systems still have the following problems: (1) they lack active motility and have limited thrombus penetration capacity; (2) most nitric oxide donors are single-function molecules, making it difficult to achieve synergistic effects; and (3) there is a lack of effective linkage mechanism between thrombolysis and reperfusion antioxidant therapy. Therefore, it is necessary to develop a nanotherapy system that combines driving capacity and cascade antioxidant function to achieve synergistic optimization of the entire thrombus treatment process. Summary of the Invention

[0003] To address the technical problems of insufficient thrombus penetration, low thrombolysis efficiency, and difficulty in effectively intervening in ischemia-reperfusion injury in existing thrombosis treatment systems, this invention provides a cascaded antioxidant carrier-free nanomotor, its preparation method, and its application. Specifically, it provides a nitric oxide donor-driven carrier-free nanomotor with cascaded antioxidant properties, its preparation method, and its application. This invention utilizes small molecule hybrid self-assembly nanotechnology, photothermal response modulation technology, and a thrombus-targeted modification strategy to construct a carrier-free nanomotor integrating "gas-driven—photothermal response—cascaded antioxidant" processes, thereby achieving synergistic optimization of thrombolysis and reperfusion protection.

[0004] In a first aspect, the present invention provides a cascaded antioxidant carrier-free nanomotor, wherein the cascaded antioxidant carrier-free nanomotor is formed by the self-assembly of a functionally converted nitric oxide donor BNN-NO and a photothermal photosensitizer through non-covalent interactions, and is modified with a polyethylene glycol modifier and a polyethylene glycol-modified lesion-targeting ligand; wherein the photothermal photosensitizer is one or both of DiR or DiD; and the functionally converted nitric oxide donor BNN-NO has the following structure:

[0005] .

[0006] The functional convertible nitric oxide donor of the present invention generates quinone diimines, imines, phenolic hydroxyl groups, phenylenediamine oxidation derivatives, or other structural units with free radical scavenging capabilities after releasing nitric oxide induced by near-infrared light.

[0007] Under near-infrared light irradiation, the cascaded antioxidant carrier-free nanomotor of the present invention can generate a photothermal effect and induce the functionally transformed nitric oxide donor to release nitric oxide; the donor residues after the nitric oxide donor releases nitric oxide are further transformed into antioxidant derivatives with reactive oxygen species scavenging ability, thereby achieving a cascaded therapeutic effect of photothermal enhancement, nitric oxide release, gas-driven, vascular protection and reactive oxygen species scavenging.

[0008] Furthermore, the non-covalent interaction force is one or more of the following: hydrophobic interaction, π-π stacking interaction, hydrogen bonding interaction, or electrostatic interaction.

[0009] Furthermore, the molar ratio of the functionally converted nitric oxide donor BNN-NO to the photothermal photosensitizer is 1:5 to 5:1; the polyethylene glycol modifier and the polyethylene glycol-modified lesion-targeting ligand account for 5% to 20% and 10% to 25% of the total mass of the system, respectively.

[0010] Furthermore, the functional convertible nitric oxide donor BNN-NO is prepared by the following method: using 5-hydroxy-2-nitrobenzaldehyde and p-phenylenediamine as raw materials, the target compound BNN-NO is obtained after condensation, reduction and nitrosation reactions.

[0011] Furthermore, the polyethylene glycol modifier is DSPE-PEG with a molecular weight of 2000.

[0012] Furthermore, the pegylated lesion-targeting ligand is one or more of the following: pegylated CREKA, pegylated polypeptide fibrin-targeting ligand, pegylated VCAM-1-targeting ligand, pegylated ICAM-1-targeting ligand, pegylated selectin-targeting ligand, or pegylated integrin-targeting ligand.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned cascaded antioxidant carrier-free nanomotor, comprising the following steps: dissolving the functionally converted nitric oxide donor BNN-NO and the photothermal photosensitizer in an organic solvent respectively, and mixing them to form an organic phase; adding the organic phase to an aqueous phase under stirring, ultrasonic or microfluidic conditions, so that the functionally converted nitric oxide donor BNN-NO and the photothermal photosensitizer self-assemble through non-covalent interactions to form a binary nanoassembly; subsequently adding a polyethylene glycol modifier and a polyethylene glycol-modified lesion-targeting ligand for surface modification, and removing the organic solvent to obtain the cascaded antioxidant carrier-free nanomotor.

[0014] Furthermore, the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, or acetonitrile.

[0015] Thirdly, the present invention provides the application of the above-mentioned cascaded antioxidant carrier-free nanomotor in the preparation of a drug delivery system.

[0016] Fourthly, the present invention provides the application of the above-mentioned cascaded antioxidant carrier-free nanomotor in the preparation of drugs for antithrombosis and / or treatment of ischemia-reperfusion injury.

[0017] Fifthly, the present invention provides the application of the above-mentioned cascaded antioxidant carrier-free nanomotor in the preparation of injection, oral or topical drug delivery systems.

[0018] Furthermore, the ischemia-reperfusion injury is thrombotic stroke.

[0019] The cascaded antioxidant carrier-free nanomotor described in this invention maintains good stability during in vivo circulation and achieves efficient accumulation at thrombus lesion sites. Under near-infrared light irradiation, the photothermal photosensitizer in the nanomotor converts light energy into heat energy, increasing the local temperature to promote thrombus structure disintegration and triggering the release of nitric oxide gas from the nitric oxide donor. The released nitric oxide acts as a driving force to propel the nanomotor into autonomous motion, thereby enhancing its penetration into the thrombus and promoting thrombus destruction. Simultaneously, the nitric oxide donor undergoes structural transformation during nitric oxide release, generating a derivative with antioxidant activity. This derivative can efficiently scavenge reactive oxygen species generated during ischemia-reperfusion, reducing oxidative stress damage.

[0020] The beneficial effects of this invention are: (1) The nitric oxide donor provided by the present invention can generate antioxidant active products while releasing nitric oxide, thereby constructing a cascade antioxidant reaction system and improving the efficiency of reactive oxygen species removal.

[0021] (2) The present invention utilizes the synergistic effect of photothermal effect and nitric oxide gas to significantly enhance the penetration ability of nanomotors in thrombi and improve thrombolysis efficiency.

[0022] (3) The present invention constructs nanostructures through a carrier-free self-assembly strategy, which has the advantages of high drug loading capacity, simple preparation process and good biocompatibility.

[0023] (4) By constructing a functional system that couples nitric oxide release with antioxidant product generation, this invention achieves a functional cascade transformation from gas-driven to antioxidant clearance, thereby forming a closed-loop treatment mode of continuous regulation of "thrombolysis-blood flow reconstruction-reperfusion antioxidant", which effectively reduces the risk of thrombosis recurrence.

[0024] (5) The cascaded antioxidant carrier-free nanomotor provided by the present invention can simultaneously alleviate ischemia-reperfusion injury while treating thrombosis, and significantly improve the overall treatment effect. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the synthesis route of BNN-NO in Embodiment 1 of the present invention.

[0026] Figure 2 This is a graph showing the time-of-flight mass spectrometry detection results of BNN-NO in Example 1 of the present invention.

[0027] Figure 3 The image shows the proton NMR spectrum of BNN-NO in Example 1 of this invention, with detection conditions of 400 MHz and DMSO-d6.

[0028] Figure 4 The image shows the carbon NMR spectrum of BNN-NO in Example 1 of this invention, with detection conditions of 400 MHz and DMSO-d6.

[0029] Figure 5 This is a high-performance liquid chromatography (HPLC) detection result of the BNN-NO methanol solution in Example 1 of the present invention.

[0030] Figure 6 The image shows the dynamic light scattering particle size detection results of the BNN-NO / DiR nanoassembly in Example 2 of this invention, where A represents B-ND NAs and B represents T-ND NAs.

[0031] Figure 7 The images shown are transmission electron microscope images of B-ND NAs and T-ND NAs in Example 2 of the present invention; where A represents B-ND NAs, B represents T-ND NAs, and the scale bar is 200 nm.

[0032] Figure 8 This is a graph showing the zeta potential detection results of B-NDNAs and T-NDNAs in Embodiment 2 of the present invention.

[0033] Figure 9This is a schematic diagram of the chemical structure of BNN-NO and DiR and the co-assembly process of the binary nano-assembly in Example 3 of the present invention.

[0034] Figure 10 The graph shows the particle size changes of B-NDNAs and T-NDNAs after treatment with urea, NaCl, and SDS, respectively, in Example 3 of this invention.

[0035] Figure 11 The results are as follows: spectral detection results of DiR solution, B-NDNAs and T-NDNAs in Example 3 of this invention; where A is the ultraviolet absorption spectrum and B is the fluorescence spectrum.

[0036] Figure 12 The results show the stability of B-NDNAs and T-NDNAs in Example 3 of this invention; where A represents the stability results after 24 h in PBS at 37°C and pH 7.4, and B represents the stability results after 13 d in the dark at 4°C.

[0037] Figure 13 The figure shows the long-term stability of the BNN-NO / DiR nanoassembly in Example 3 of this invention after being placed at 4°C in the dark for 13 days. In the figure, A represents the stability of B-ND NAs and B represents the stability of T-ND NAs.

[0038] Figure 14 The results show the in vitro photothermal performance of the BNN-NO / DiR nanoassembly under 808 nm laser irradiation in Example 4 of this invention; where A is a photothermal imaging image and B is a temperature rise curve.

[0039] Figure 15 This is a graph showing the nitric oxide release kinetics of different groups under 808 nm laser irradiation in Example 4 of the present invention.

[0040] Figure 16 The results of the nitric oxide generation performance evaluation of T-ND NAs in Example 4 of this invention are shown; the left figure shows the nitric oxide generation results at different formulation concentrations, and the right figure shows the nitric oxide generation results at different laser power densities.

[0041] Figure 17 This is a graph showing the periodic generation and variation of nitric oxide under cyclic irradiation conditions by a laser switch in Embodiment 4 of the present invention.

[0042] Figure 18 The images shown are fluorescence micrographs of the motion trajectories of T-NDNAs under different laser power densities in Example 4 of this invention. Scale bar = 50 μm.

[0043] Figure 19The results show the quantitative analysis of the motion performance of T-ND NAs under different laser power densities in Example 4 of this invention; the left figure represents the mean square displacement, and the right figure represents the motion rate.

[0044] Figure 20 The images show the in vitro thrombus-targeted fluorescence imaging and quantitative detection results of each group of preparations in Example 5 of this invention; where A is a representative thrombus-targeted fluorescence image with a scale bar of 1 mm, and B is the quantitative result of artificial whole blood clot targeting.

[0045] Figure 21 These are confocal permeation micrographs of thrombi in different formulation groups under laser irradiation in Example 5 of this invention. Scale bar = 1000 μm.

[0046] Figure 22 This is a graph showing the quantitative analysis results of the penetration depth of different preparations inside the thrombus under laser irradiation in Example 5 of the present invention.

[0047] Figure 23 These are comparative photographs of the macroscopic morphology of blood clots in each group under and without laser irradiation in Example 5 of the present invention; wherein, A is the overall morphology of each group, and B is a comparison of blood clots before and after treatment, with a scale bar of 2 mm.

[0048] Figure 24 This is a graph showing the quantitative statistical results of in vitro thrombolysis rates in different groups under laser irradiation in Example 5 of the present invention.

[0049] Figure 25 The figures show the quantitative detection results of the supernatant components under laser irradiation in Example 5 of this invention; the left figure shows the fibrin content, and the right figure shows the hemoglobin content.

[0050] Figure 26 The results of DPPH free radical scavenging ability detection of T-NDNAs and different formulations in Example 6 of the present invention are shown; where A represents the antioxidant capacity of different concentrations of T-NDNAs, and B represents the comparison of antioxidant capacity of each group of formulations.

[0051] Figure 27 This is a graph showing the results of quantitative detection of cellular oxidative stress levels using flow cytometry with the DCFH-DA fluorescent probe in Example 6 of the present invention.

[0052] Figure 28 This is a scatter plot of apoptosis distribution in Annexin V-FITC / PI double staining flow cytometry in Example 6 of the present invention.

[0053] Figure 29 This is a graph showing the results of quantitative statistical analysis of cell apoptosis rates in each group using flow cytometry in Example 6 of the present invention.

[0054] Figure 30This is a representative photograph of human umbilical vein endothelial cells JC-1 fluorescently stained in Example 6 of the present invention. Scale bar = 20 μm.

[0055] Figure 31 This is a standard curve for detecting DiR content in rat plasma in Example 7 of the present invention.

[0056] Figure 32 The results of in vivo pharmacokinetic studies of different DiR formulations in Example 7 of this invention are shown; where A is the pharmacokinetic curve and B is the quantitative result of the area under the pharmacokinetic curve from 0 to 24 h.

[0057] Figure 33 The results of in vivo fluorescence imaging and quantitative analysis of rat carotid artery thrombosis in Example 8 of the present invention are shown; where A is fluorescence imaging at different time points, B is quantitative statistical analysis of fluorescence intensity, and the scale bar is 2 mm.

[0058] Figure 34 The results are quantitative detection results of fluorescence distribution of isolated carotid artery tissue and major organs in different formulations in Example 8 of the present invention; where A represents the distribution of carotid artery tissue and B represents the distribution of major organs.

[0059] Figure 35 The images show the in vivo fluorescence imaging and quantitative analysis results of the mouse tail at different time points in Example 8 of this invention; where A is a representative fluorescence imaging image, B is the quantitative statistical analysis of fluorescence intensity, and the scale bar is 1 cm.

[0060] Figure 36 These are representative fluorescence images of mouse brains at different time points after administration of different formulations in Example 8 of this invention. Scale bar = 1 cm.

[0061] Figure 37 This is a graph showing the results of quantitative analysis of the dynamic changes in fluorescence intensity in the mouse brain at different time points in Example 8 of the present invention.

[0062] Figure 38 The images show the quantitative detection results of fluorescence distribution in major organs and the brain in different formulations in Example 8 of this invention; the left image shows the distribution of major organs, and the right image shows the distribution of brain tissue.

[0063] Figure 39 This is a macroscopic morphological photograph of the rat carotid artery and thrombus vessels under 808 nm laser irradiation in Example 8 of the present invention. Scale bar = 2 cm.

[0064] Figure 40 The images show the photothermal imaging and temperature rise curves of the rat carotid artery and thrombus under 808 nm laser irradiation in Example 8 of this invention; the left image is the photothermal imaging image, and the right image is the in vivo temperature rise curve. The scale bar is 2 cm.

[0065] Figure 41Photograph of H&E stained section of rat carotid artery tissue in Example 9 of this invention, scale bar = 100 μm.

[0066] Figure 42 This is a graph showing the quantitative analysis results of the degree of thrombolysis in each group of carotid artery vascular slices in Example 9 of the present invention.

[0067] Figure 43 This is a schematic diagram of the process for constructing a chronic inflammatory thrombosis model in Embodiment 10 of the present invention.

[0068] Figure 44 These are macroscopic photographs of thrombosis progression in mice with chronic inflammatory tail thrombosis model in Example 10 of this invention after intervention with different preparations.

[0069] Figure 45 This is a graph showing the dynamic statistical results of the changes in the length of chronic inflammatory tail thrombi in different formulation groups in Example 10 of the present invention.

[0070] Figure 46 The results are quantitative analysis of the intervention effect of the chronic thrombosis model in Example 10 of the present invention; where A is the comparison of thrombus length before and after intervention, and B is the quantitative result of tail preparation retention.

[0071] Figure 47 Macroscopic photographs comparing the thrombosis recurrence process after intervention with different treatment methods in Embodiment 10 of the present invention.

[0072] Figure 48 This is a graph showing the results of blood sCD40L inflammatory factor level detection in each group of the chronic inflammatory thrombosis model in Example 10 of the present invention.

[0073] Figure 49 The results of coagulation function index detection in each group of the chronic inflammatory thrombosis model in Example 10 of the present invention are shown; the left figure shows the activated partial thromboplastin time, and the right figure shows the fibrinogen content.

[0074] Figure 50 This is a photograph of an H&E-stained section of mouse tail tissue from Example 10 of the present invention. Scale bar = 150 μm.

[0075] Figure 51 This is a schematic diagram of the process of constructing the tMCAO model of middle cerebral artery embolism in Embodiment 11 of the present invention.

[0076] Figure 52 This is a graph showing the statistical results of neurological function scores of rats in each group of the ischemic stroke model in Example 11 of this invention.

[0077] Figure 53 The results of TTC staining and infarct volume quantification of rat brain tissue in Example 11 of this invention are shown; where A is a representative TTC staining image with a scale bar of 100 μm, and B is the statistical result of cerebral infarction volume quantification.

[0078] Figure 54 These are photographs of pathological stained sections of rat brain tissue from each group in Example 11 of this invention; where A is an H&E stained section, B is a Nissl stained section, and the scale bar is 100 μm.

[0079] Figure 55 This is a graph showing the detection results of plasma TNF-α inflammatory factor expression levels in rats using the tMCAO model in Example 11 of this invention.

[0080] Figure 56 This is a graph showing the hemoglobin detection results for biosafety evaluation in Example 12 of the present invention.

[0081] Figure 57 This is a graph showing the cell viability test results of human umbilical vein endothelial cells under and without laser irradiation in different formulations in Example 12 of the present invention.

[0082] Figure 58 The images show H&E-stained sections of the heart, liver, spleen, lungs, and kidneys of the rat thrombosis model in Example 12 of this invention. Scale bar = 100 μm.

[0083] Figure 59 This is a graph showing the results of biochemical index detection of liver and kidney function in mice after long-term intervention in Example 12 of this invention. Detailed Implementation

[0084] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0085] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0086] The DSPE-PEG described in the examples 2K -CREKA originates from (Suzhou Polypeptide Biotechnology Co., Ltd.) Example 1: Synthesis and structural confirmation of cascade antioxidant nitric oxide donor BNN-NO This embodiment prepares a nitric oxide donor, BNN-NO, with photothermal responsiveness and cascade antioxidant properties. BNN-NO can release nitric oxide under near-infrared photothermal conditions, and after releasing nitric oxide, it is converted into a diiminoquinone derivative, QDI, which has antioxidant activity, thereby achieving a cascade functional transformation from "nitric oxide release" to "reactive oxygen species scavenging".

[0087] Step 1, Aldol Condensation Reaction: 1119.00 mg (6.70 mmol) of 5-hydroxy-2-nitrobenzaldehyde and 260.10 mg (2.40 mmol) of p-phenylenediamine were weighed and dissolved completely in approximately 200 mL of anhydrous ethanol. The mixture was stirred and reacted at 85°C in an oil bath under nitrogen protection for 12 h. After the reaction was complete, the mixture was cooled, filtered, and the resulting solid residue was washed three times with anhydrous ethanol to obtain a bright yellow solid compound 1.

[0088] Step 2, reduction reaction: Compound 1 was dissolved in a methanol-tetrahydrofuran mixed solution (methanol / tetrahydrofuran = 1 / 1, v / v), vortexed until completely dissolved, with a solvent volume of approximately 40 mL. Approximately 5 mL of 10 mM sodium borohydride solution was slowly added dropwise to the reaction system, and the mixture was stirred at room temperature for 6 h, with the reaction progress monitored by thin-layer chromatography. After the reaction was complete, the organic phase was removed by rotary evaporation, and approximately 80 mL of deionized water was added to disperse the crude product, followed by extraction with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, and ethyl acetate was removed by rotary evaporation. The obtained crude product was purified by column chromatography with ethyl acetate / petroleum ether = 1 / 5, v / v, yielding an orange-yellow solid compound 2.

[0089] Step 3, nitrosation reaction: Compound 2 was fully dissolved in approximately 20 mL of anhydrous ethanol and stirred for 30 min under nitrogen protection and in the dark throughout the process. Then, approximately 16 mL of nitrogen-saturated 6 M sodium nitrite aqueous solution was added, and stirring continued for another 30 min. Next, approximately 16 mL of 6 M hydrochloric acid aqueous solution was added dropwise through a constant-pressure separatory funnel. After 12 h of reaction, the reaction solution gradually changed from orange to yellow and a precipitate formed. The precipitate was collected by filtration and thoroughly washed with anhydrous ethanol-water mixture (anhydrous ethanol / water = 1 / 1, v / v). The resulting precipitate was freeze-dried for 24 h to obtain a brownish-yellow powdery solid BNN-NO, which required no further purification.

[0090] An appropriate amount of BNN-NO powder was dissolved in methanol, and the purity of the compound was verified by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column, 4.6 × 150 mm, 5 μm; mobile phase: acetonitrile / water = 80 / 20; flow rate: 1.0 mL / min; detection wavelength: 254 nm. Separately, a BNN-NO sample was analyzed by TOF-MS in methanol, and the molecular weight was recorded. A BNN-NO sample was also dissolved in deuterated DMSO for further analysis. 1 H-NMR and 13 C-NMR detection.

[0091] The results are as follows Figures 1 to 5 As shown, BNN-NO was processed by TOF-MS, 1 H-NMR and 13 The structure was confirmed by C-NMR. BNN-NO, C20 H 16 N6O8, with a mass-to-nucleus ratio of 468.0955551, showed a result consistent with the target molecular formula. 1 The H-NMR results are as follows: 1 H NMR, 400MHz, DMSO-d6, δ 10.94, s, 2H; 8.13, d, J=9.0 Hz, 2H; 7.84, s, 4H; 6.83, dd, J=9.0, 2.6 Hz, 2H; 6.20, d, J=2.6 Hz, 2H; 5.61, s, 4H. 13 The C-NMR results are as follows: 13 C10 NMR, 101 MHz, DMSO-d6, δ 162.86, 139.94, 138.93, 132.58, 128.86, 120.38, 114.90, 113.24, 45.68. HPLC results showed that the purity of BNN-NO was approximately 95%, which meets the requirements for subsequent nanomotor preparation and efficacy evaluation.

[0092] Example 2: Preparation, formulation screening and characterization of BNN-NO / DiR carrier-free nanomotors Weigh out 2.48 mg of BNN-NO and 4.52 mg of DiR. Dissolve DiR in 903.60 μL of anhydrous ethanol and vortex for 30 s until completely dissolved. Dissolve BNN-NO in 495.0 μL of a tetrahydrofuran-anhydrous ethanol mixture (tetrahydrofuran / anhydrous ethanol = 3 / 1, v / v) and vortex for 30 s until completely dissolved. Prepare stock solutions containing 5 mg / mL of each substance.

[0093] BNN-NO solution and DiR solution were mixed at different molar ratios, including 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, and 1:5, and the volume was adjusted to 200 μL. Then, a one-step nanoprecipitation method was used, in which the mixed solution was added dropwise to 2 mL of deionized water and stirred at 1400 rpm for 3 min. After the formation of the hybrid nanoassemblies, the organic solvent was removed by rotary evaporation at 30 °C for 5 min, and the volume was adjusted to 2 mL to obtain BNN-NO / DiR hybrid nanoassemblies, i.e., B-ND NAs. The particle size and PDI of B-ND NAs at different molar ratios were determined using a Malvern particle size analyzer.

[0094] Since DiR, after exerting its photothermal effect, can promote the conversion of BNN-NO to QDI through intermolecular energy transfer, the optimal synergistic ratio was further screened by measuring the BNN-NO conversion rate. Different molar ratios of B-ND NAs (0.50 mg / mL) were subjected to 808 nm and 2 W / cm² concentrations. 2Laser irradiation was performed for 40 min. After the sample returned to room temperature, it was diluted 2-fold with acetonitrile and vortexed for 5 h to allow the nanomotors to fully disassemble and reassemble. Subsequently, the concentration of BNN-NO in the filtrate was determined by high-performance liquid chromatography, and the conversion rate of BNN-NO was calculated using the external standard method.

[0095] The formulation screening results of B-ND NAs with different DiR / BNN-NO molar ratios showed that when the DiR / BNN-NO molar ratio was 1:1, the nano-assemblies had smaller particle sizes and the highest BNN-NO conversion rate. Therefore, a DiR / BNN-NO molar ratio of 1:1 was preferred as the subsequent preparation ratio.

[0096] To endow nanomotors with better blood circulation stability and thrombus targeting ability, DSPE-PEG was used. 2K and DSPE-PEG 2K - Surface modification with CREKA. Weigh 0.61 mg of DSPE-PEG. 2K Dissolve in 61 μL methanol to obtain a 10 mg / mL stock solution; weigh 2.00 mg DSPE-PEG. 2K -CREKA, dissolved in 1333.3 μL of methanol, yielded a 1.5 mg / mL stock solution. 20 μL of DSPE-PEG was then used separately. 2K Solution and 91 μL DSPE-PEG 2K -CREKA solution is mixed and ready for use, including DSPE-PEG 2K 15% of the total drug mass, DSPE-PEG 2K -CREKA accounts for 10% of the total drug mass.

[0097] The external addition method is as follows: First, add 200 μL of a drug-containing mixed stock solution with a DiR / BNN-NO molar ratio of 1:1 dropwise to 2.00 mL of deionized water while stirring at 1400 r / min, then add DSPE-PEG. 2K / DSPE-PEG 2K - Add the CREKA mixed solution dropwise to the system and continue stirring for 3 minutes. The internal addition method is as follows: Add DSPE-PEG... 2K / DSPE-PEG 2K - The CREKA mixed solution and the drug-containing mixed stock solution were premixed and vortexed before being directly added dropwise to the aqueous phase to prepare nanoassemblies. For both methods, the organic solvent was removed by rotary evaporation at 27°C for 5 min, and the volume was adjusted to 2 mL. The results showed that the nanoparticles prepared by the internal addition method had a particle size of 377.70±4.65 nm and a PDI of 0.49±0.10; while the nanoparticles prepared by the external addition method had a particle size of 67.72±2.31 nm and a PDI of 0.17±0.20. Therefore, the external addition method is preferred for preparing thrombus-targeting nanomotor T-NDNAs.

[0098] DSPE-PEG 2K / DSPE-PEG 2K -CREKA was added to the surface of B-ND NAs via an additive method to prepare T-NDNAs. B-ND NAs and T-ND NAs were diluted 20-fold with deionized water at 0.50 mg / mL. Particle size, PDI, and Zeta potential were measured using a Malvern particle size analyzer; the results are shown in Table 1. Another 10 μL of the diluted sample was added to a carbon film copper grid. After a uniform water film formed, excess liquid was absorbed, and the sample was negatively stained with 2% phosphotungstic acid for approximately 10 s and then evaporated. Morphology was observed using TEM.

[0099] Table 1 Characterization results of B-ND NAs and T-ND Nas

[0100] The results are as follows Figures 6 to 8 As shown, both B-ND NAs and T-ND NAs are spherical structures with a particle size of approximately 60-70 nm and uniform distribution; after surface modification, the Zeta potential changed from positive to negative, suggesting that DSPE-PEG 2K / DSPE-PEG 2K -CREKA was successfully modified on the surface of nanomotors, which helps to reduce non-specific adsorption of plasma proteins and improve in vivo stability.

[0101] Example 3: Assembly mechanism, spectral characteristics and stability of BNN-NO / DiR carrier-free nanomotors Molecular docking simulations were used to investigate the interaction between BNN-NO and DiR. The chemical and three-dimensional structures of BNN-NO and DiR were plotted, energy minimization simulations were performed, and the optimal conformation was obtained through AutoDock semi-flexible docking analysis. The results are as follows: Figure 9 As shown, there are hydrophobic interactions, π-π stacking interactions, and π-cation interactions between BNN-NO and DiR. These interactions together drive the formation of a stable carrier-free hybrid nanoassembly.

[0102] To further verify the assembly forces, B-NDNAs and T-NDNAs were treated with urea, NaCl, and SDS, respectively, and the particle size changes were detected. The results are as follows: Figure 10 As shown, the particle size of the nanoassemblies changed to different degrees after treatment with different disruptors, indicating that hydrophobic interaction was the dominant force, while π-π stacking and π-cation interaction assisted in the co-assembly process.

[0103] 200 μL of B-NDNAs, T-NDNAs, and DiR Sol were placed in clear 96-well plates and black 96-well plates, respectively. The UV absorption and fluorescence emission spectra were scanned using a multi-functional microplate reader. Results are as follows: Figure 11 As shown, compared with DiR Sol, the UV absorption and fluorescence characteristics of B-ND NAs and T-ND NAs changed, proving that DiR and BNN-NO were successfully co-assembled.

[0104] Stability was further evaluated. B-NDNAs and T-NDNAs, 0.50 mg / mL, were diluted 10-fold with PBS (pH 7.4) and incubated at 37°C and 100 rpm in a shaker. Particle size changes were measured at 0, 1, 2, 4, 8, 12, and 24 h. The prepared B-NDNAs and T-NDNAs were also stored at 4°C in the dark, and particle size changes were measured at 0, 3, 5, 7, 9, 11, and 13 days. Results are as follows: Figure 12 and Figure 13 As shown, T-ND NAs exhibited better colloidal stability in PBS than B-ND NAs, and both nanomotors showed minimal particle size change after 13 days of storage at 4°C in the dark, indicating good storage stability.

[0105] Example 4: In vitro photothermal conversion, nitric oxide release, and self-driven motion capability of BNN-NO / DiR carrier-free nanomotors Take 1 mL of PBS, DiR Sol, β-NDNAs, and T-NDNAs respectively and place them in 2 mL round-necked glass bottles, where the equivalent concentration of DiR is 0.34 mg / mL. Place the samples 12 cm away from the 808 nm laser emitter and apply 2 W / cm² laser light. 2 The power density was irradiated for 15 minutes, and the temperature changes were recorded in real time using an infrared thermal imager.

[0106] The results are as follows Figure 14 As shown, the temperatures of DiR Sol, ND Sol, B-ND NAs, and T-ND NAs rapidly increased after illumination, stabilizing at approximately 50°C after 15 minutes, while the temperature of PBS remained essentially unchanged. These results indicate that the co-assembly of DiR with BNN-NO did not weaken the photothermal conversion capability of DiR, and that T-ND NAs possessed excellent near-infrared photothermal properties.

[0107] NO release was quantitatively detected using a Griess kit. 800 μL of DiR Sol, ND Sol, B-NDNAs, and T-NDNAs were added to 1.5 mL EP tubes, respectively. The equivalent concentration of DiR was 0.34 mg / mL, and the equivalent concentration of BNN-NO was 0.16 mg / mL. The wavelength was 808 nm, 2 W / cm². 2 Laser irradiation was performed for 2, 5, 10, 15 and 20 min respectively. After the samples returned to room temperature, 50 μL of the sample was transferred to a 96-well plate and the NO content was detected according to the Griess kit instructions.

[0108] The results are as follows Figure 15 As shown, after irradiation with an 808 nm laser, B-ND NAs and T-ND NAs produced a large amount of NO; ​​ND Sol released less NO due to its larger intermolecular distance and lower energy transfer efficiency; DiR Sol, which does not contain BNN-NO, released almost no NO. There was no significant difference in NO release between B-ND NAs and T-ND NAs, indicating that PEGylation and CREKA modification do not affect NO release.

[0109] The NO generation mechanism of T-NDNAs was further investigated. NO release from T-NDNAs at concentrations of 0.10, 0.50, 0.80, and 1.00 mg / mL was measured under 808 nm laser irradiation at 2 W / cm². At a T-NDNAs concentration of 1 mg / mL, irradiation was performed at power densities of 0.5, 1.0, 1.5, and 2.0 W / cm², and NO release was measured. The results are as follows: Figure 16 As shown, NO generation by T-ND NAs exhibits both concentration-dependent and laser power-dependent characteristics. T-ND NAs were placed at 808 nm and 2 W / cm². 2 Under laser illumination, the light was switched on and off alternately at 10-minute intervals, and the NO content was measured at 0, 10, 20, 30, 40, 50, and 60 minutes. The results are as follows: Figure 17 As shown, NO release varies periodically with the laser switch, indicating that the NO release of T-ND NAs has good optical controllability.

[0110] The motility of nanomotors was recorded using an inverted fluorescence microscope. T-NDNAs with a DiR concentration of 50 μg / mL were incubated in DMEM medium containing 10% FBS and then subjected to short-term irradiation at different laser power densities of 1.0, 1.5, and 2.0 W / cm². 2 Irradiation was performed for 10 seconds, and the motion trajectory was observed and recorded in bright field. The mean square displacement and motion velocity were analyzed using Fiji ImageJ.

[0111] The results are as follows Figure 18 and Figure 19 As shown, T-ND NAs mainly exhibit Brownian motion when unlit; after laser triggering, the range and speed of motion of T-ND NAs increase significantly, and this increase is enhanced with the increase of laser power density, indicating that T-ND NAs can generate self-driving force by photothermally triggering NO release, thus achieving controllable motion.

[0112] Example 5: In vitro thrombus targeting, thrombus penetration, and thrombolysis capabilities of BNN-NO / DiR carrier-free nanomotors Establishment of an in vitro thrombus clot model: Orbital blood from healthy SD rats was placed in heparin-coated EP tubes and centrifuged at 4000 rpm for 5 min to separate the layers. Platelet-rich plasma was extracted. A 0.1 U / μL thrombin-saline solution and a 0.3 M CaCl2 solution were prepared. 150 μL of platelet-rich plasma, 10 μL of thrombin solution, and 20 μL of CaCl2 solution were added to a 96-well plate and incubated at 37℃ for 90 min to form a thrombus clot.

[0113] 100 μL of T-ND NAs, B-ND NAs, ND Sol, DiR Sol, or PBS were added to a platelet-rich thrombus model and incubated together, with the equivalent concentration of DiR being 0.68 mg / mL. After incubation for 10 min, the non-specific adhering agents on the thrombus surface were removed by rinsing with PBS, and the fluorescence intensity of DiR on the thrombus surface was detected using a small animal in vivo imaging system.

[0114] The results are as follows Figure 20 As shown, the fluorescence intensity of T-NDNAs in the thrombosis model was significantly higher than that of DiR Sol, ND Sol, and B-NDNAs, indicating that DSPE-PEG 2K -CREKA modification endows nanomotors with thrombus-targeting and accumulation capabilities.

[0115] A small in vitro thrombus model was established: 10 μL of fresh whole blood from healthy SD rats was injected into the bottom of a 250 μL EP tube and incubated at 37℃ for 3 h to induce the formation of small, round thrombi. To avoid the influence of DiR photobleaching on localization, coumarin 6 (C6) was used to label the nanoassemblies. 137 μL of DiR (5 mg / mL), 63 μL of BNN-NO (5 mg / mL), and 50 μL of C6 (1 mg / mL) were mixed and vortexed for 30 s, and C6 / B-NDNAs and C6 / T-NDNAs were prepared using a one-step nanoprecipitation method. The formulations were added to the small thrombus model and incubated for 30 min, followed by incubation at 808 nm and 2 W / cm². 2 Laser irradiation was performed for 15 minutes. After washing away residual medication on the surface of the thrombus with PBS, the fluorescence signal inside the thrombus was detected using a confocal laser scanning microscope.

[0116] The results are as follows Figure 21 and Figure 22 As shown, unilluminated T-ND NAs did not exhibit significant deep thrombus penetration compared to free C6; laser-irradiated C6 / T-ND NAs displayed strong fluorescence signals within the thrombus, with a significantly increased penetration depth, superior to C6 / B-ND NAs+L. This indicates that T-ND NAs can achieve deep thrombus penetration and retention through thrombus-targeted binding and photothermal-mechanical actuation.

[0117] An in vitro whole blood thrombosis model was established: 100 μL of whole blood plasma, 10 μL of 2.5 mM CaCl2, and 10 μL of 0.1 U / μL thrombin were mixed in a 250 μL EP tube and incubated at 37°C for 3 h. After thrombus coagulation, the thrombus was slowly removed with a syringe needle and repeatedly washed with PBS. The weight of the washed thrombi was recorded, and 1.5 mL of PBS, DiR Sol, ND Sol, B-ND NAs, or T-ND NAs were added, where the equivalent concentration of DiR was 0.68 mg / mL and the equivalent concentration of BNN-NO was 0.32 mg / mL. The mixture was incubated for 30 min. Subsequently, the mixture was subjected to 808 nm, 2 W / cm² concentration. 2 Irradiate with laser for 20 minutes, repeatedly inverting the glass bottle. Collect the supernatant and measure the absorbance of fibrin and hemoglobin at 415 nm and 540 nm, respectively. After thrombolysis, remove any remaining thrombus, wash it, and weigh it again. Calculate the thrombolysis rate based on the difference in mass.

[0118] The results are as follows Figures 23 to 25 As shown, the T-ND NAs+L group exhibited the most significant changes in thrombus morphology and the highest thrombolysis rate, with a significant increase in fibrin and hemoglobin levels in the supernatant. DiR Sol+L and ND Sol+L primarily disrupted the thrombus surface, resulting in limited thrombolysis efficiency. These results indicate that T-ND NAs can achieve efficient thrombolysis through a synergistic effect of DiR-mediated photothermal activity and the mechanical drive generated by NO release from BNN-NO.

[0119] Example 6: In vitro antioxidant, ROS scavenging, anti-apoptotic and mitochondrial protective effects of BNN-NO / DiR carrier-free nanomotors The free radical scavenging ability of T-NDNAs was evaluated using the DPPH colorimetric method. A 1 mM DPPH ethanol working solution was prepared. 1.30, 1.08, 0.86, 0.65, 0.43, and 0.22 μM T-NDNAs were subjected to DPPH assay at 808 nm and 2 W / cm², respectively. 2After laser irradiation for 40 min, and once NO release was complete and the sample was cooled to room temperature, the aqueous phase was removed by rotary evaporation, and then reconstituted with ethanol and brought to a final volume. 3 mL of the treated sample was added to 1 mL of DPPH solution, mixed thoroughly, and then shaken vigorously at room temperature in the dark for 30 min. 200 μL of the solution was then measured at 517 nm to detect the absorbance, and the DPPH scavenging rate was calculated.

[0120] The results are as follows Figure 26 As shown, T-ND NAs exhibited concentration-dependent DPPH radical scavenging ability after photoirradiation; BNN-NO Sol itself did not show significant antioxidant capacity, and only after the DiR photothermal effect triggered the conversion of BNN-NO to QDI did the system possess radical scavenging ability. The scavenging efficiency of B-ND NAs and T-ND NAs was higher than that of ND Sol, indicating that co-assembly improved the photothermal-triggered conversion efficiency.

[0121] An oxygen-glucose deprivation / reperfusion (OGD / R) cell model was established. Human umbilical vein endothelial cells (HUVECs) were used and cultured in DMEM medium containing 10% FBS at 37°C, 5% CO2, 16% O2, and saturated humidity. For modeling, the medium was replaced with low-glucose DMEM, and the cells were cultured in a hypoxic environment containing 93% N2, 4% O2, and 5% CO2 for a certain period. After oxygen-glucose deprivation, the cells were restored to normal DMEM medium and cultured normally for another 24 h to induce oxidative stress.

[0122] ROS eradication experiment: HUVECs were treated at a rate of 2 × 10⁻⁶ 5 Cells / well were seeded in 12-well plates and grown to 90% confluence. After 9 h of oxygen-glucose deprivation treatment, normal culture medium, T-NDNAs, DiR Sol, ND Sol, β-NDNAs, and edaravone solution were added, with DiR equivalent concentration of 200 nM. Culture was continued for 16-24 h under normal conditions. Four h after drug administration, the groups containing DiR were subjected to 808 nm, 2 W / cm² assay. 2 Irradiate for 3 min per well. Then, dilute the DCFH-DA probe 1:1000 to 10 μM with serum-free DMEM, add it to the cells, and incubate at 37°C for 20 min. After washing three times with pre-cooled PBS, qualitative observation was performed using confocal microscopy, and quantitative detection of intracellular ROS levels was performed using flow cytometry.

[0123] The results are as follows Figure 27As shown, intracellular ROS in HUVECs significantly increased after OGD / R treatment; ND Sol+L, B-ND NAs+L, T-ND NAs+L and edaravone groups all reduced ROS levels, with the T-ND NAs+L group showing intracellular ROS levels close to physiological levels, indicating that T-ND NAs can generate QDI in situ after light-triggered exposure and effectively clear reperfusion-related ROS.

[0124] Anti-apoptosis experiment: HUVECs were subjected to 2×10 5 Cells were seeded per well in 12-well plates and cultured to 90% confluence. After 16 h of oxygen-glucose deprivation treatment, T-NDNAs, DiR Sol, ND Sol, β-NDNAs, and edaravone solution were added, and the cells were cultured for another 16-24 h. Four h after drug administration, the relevant groups were subjected to 808 nm, 2 W / cm² assay. 2 Irradiate for 3 min per well. Staining was performed using the Annexin V-FITC / PI apoptosis kit, and apoptosis was analyzed by flow cytometry.

[0125] The results are as follows Figure 28 and Figure 29 As shown, OGD / R treatment induced significant apoptosis; ND Sol+L, B-ND NAs+L, T-ND NAs+L and edaravone groups all reduced the apoptosis rate, among which T-ND NAs+L showed a significant anti-apoptotic effect.

[0126] Mitochondrial membrane potential detection: Mitochondrial damage was detected using the JC-1 kit. HUVECs were sputtered at 2 × 10⁻⁶. 5 Inoculate 12-well plates with 100 cells / well and culture until 90% confluence. After 16 h of oxygen-glucose deprivation, add normal culture medium, T-NDNAs, DiR Sol+L, ND Sol+L, B-NDNAs+L, or T-NDNAs+L, where the DiR concentration is 200 nM. After 4 h of drug administration, expose to light and continue normal culture for 16-24 h. Discard the culture medium, add an equal volume of JC-1 staining solution, mix well, incubate at 37°C for 30 min, wash, and observe under a confocal microscope.

[0127] The results are as follows Figure 30 As shown, the mitochondrial membrane potential of OGD / R model cells decreased, and the green fluorescence was significantly enhanced. The proportion of green fluorescence in the T-NDNAs+L, B-NDNAs+L, ND Sol+L and edaravone groups decreased, suggesting that in situ antioxidants can reverse the decrease in mitochondrial membrane potential caused by ischemia-reperfusion and reduce mitochondrial damage.

[0128] Example 7: In vivo pharmacokinetics of BNN-NO / DiR carrier-free nanomotors A plasma detection method was established using DiR fluorescence signals. The excitation wavelength of DiR was 748 nm, and the emission wavelength was 790 nm. 50 μL of blank rat plasma was taken, and 50 μL of DiR methanol solution of different concentrations was added. Then, 400 μL of methanol was added, and the mixture was vortexed for 5 min to precipitate proteins. After vortexing thoroughly for 3 min, the mixture was centrifuged at 10,000 rpm for 3 min. 200 μL of the supernatant was placed in a black 96-well plate, and the fluorescence intensity was detected using a microplate reader.

[0129] The results are as follows Figure 31 As shown, the standard curve for DiR blood drug concentration is y=5085.8x+0.9994, R2=0.9994, and the linear range is 0.02-8 nmol / mL.

[0130] Healthy male SD rats, 6 weeks old, 180-220 g, were randomly assigned to groups and fasted for 12 h before the experiment. DiR Sol, β-NDNAs, or T-NDNAs were injected via the tail vein, with an equivalent DiR dose of 5 mg / kg. Blood samples were collected from the orbital cavity at 0.05, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after administration. Plasma was collected by centrifugation at 10,000 rpm for 3 min. 50 μL of plasma was collected, and 400 μL of methanol was added to vortex and precipitate proteins. After centrifugation, the supernatant was collected and diluted to the detection range. DiR fluorescence intensity was measured, and blood drug concentration was calculated.

[0131] Pharmacokinetic results as follows Figure 32 As shown, the results indicated that DiR Sol was rapidly cleared after intravenous injection; B-NDNAs had a longer circulation time than DiR Sol; and T-NDNAs showed significantly improved in vivo circulation and AUC after PEGylation and CREKA modification. 0-24 h It is approximately 4.53 times that of DiR Sol and 2.59 times that of B-ND NAs, providing a basis for in vivo thrombus-targeted delivery and photothermal-mechanical thrombolysis.

[0132] Example 8: In vivo thrombus-targeting distribution and in vivo photothermal conversion of BNN-NO / DiR carrier-free nanomotors A FeCl3-induced carotid artery thrombosis model was established in rats. Healthy SD rats were anesthetized by intraperitoneal injection of 3% pentobarbital (40 mg / kg), fixed, and the neck was prepared. A 1 cm incision was made at the midline between the mandible and sternum, and the right common carotid artery and vagus nerve were bluntly dissected. 10×10 mm filter paper soaked in 10% FeCl3 was placed over the exposed common carotid artery for 30 seconds. After a clear black thrombus formed in the blood vessel, the vessel was flushed with a large amount of PBS.

[0133] After modeling, rats were intravenously injected with DiR Sol, B-NDNAs, and T-NDNAs, with an equivalent dose of DiR of 5 mg / kg. In vivo fluorescence imaging of the carotid artery thrombus site was performed at 15, 30, 60, 90, 120, and 180 min after administration. Three h later, rats were sacrificed, and the heart, liver, spleen, lung, kidney, brain, and carotid artery thrombus tissues were isolated for in vitro fluorescence imaging analysis.

[0134] The results are as follows Figure 33 and Figure 34 As shown, the fluorescence signal of T-NDNAs at the carotid artery thrombus site increased over time, reaching a high accumulation level approximately 2 hours after administration; DiR Sol and B-NDNAs showed weaker signals. The in vitro tissue distribution results further demonstrate that T-NDNAs can specifically accumulate in carotid artery thrombus tissue.

[0135] A κ-carrageenan-induced chronic inflammatory thrombosis model of the mouse tail vein was established. Healthy KM mice were selected, fasted for 12 hours, and then intraperitoneally injected with freshly prepared 1% κ-carrageenan saline solution, 35 mg / kg, while maintaining an ambient temperature of approximately 20°C to induce microthrombus formation in the terminal vessels. Twelve hours after modeling, the mouse tail turned black; the lesion length was considered successful when it exceeded 30% of the total tail length. Following modeling, DiR Sol, B-NDNAs, or T-NDNAs were injected via the tail vein. The equivalent dose of DiR was 5 mg / kg. In vivo fluorescence imaging of the tail was performed at 5, 15, 30, 60, 90, 120, 180, 240, and 300 min after administration.

[0136] The results are as follows Figure 35 As shown, the fluorescence intensity of T-NDNAs at the tail thrombus lesion was significantly higher than that of DiR Sol and B-NDNAs, and a higher accumulation was observed about 2 hours after administration, indicating that T-NDNAs also have good targeting in the venous chronic inflammatory thrombosis model.

[0137] A transient middle cerebral artery occlusion / reperfusion (tMCAO) model was established in rats. Healthy SD rats were selected, and after anesthesia, the CCA, ICA, and ECA were bluntly dissected. A silicone-containing fiber was inserted into the ICA approximately 20 mm into the ECA, allowing the silicone to be placed at the anterior end of the MCA and fixed for 90 min. The fiber was then removed, and the ECA was ligated to achieve reperfusion of the ischemic area. After successful modeling, DiR Sol or T-NDNAs were injected via the tail vein. The equivalent dose of DiR was 5 mg / kg. In vivo fluorescence imaging of the brain was performed at 15, 30, 60, 90, 120, 180, and 240 min after administration. Healthy rats were intravenously injected with T-NDNAs as controls. Four hours after reperfusion, the rats were sacrificed, and the heart, liver, spleen, lung, kidney, and brain tissues were isolated for in vitro fluorescence imaging.

[0138] The results are as follows Figures 36 to 38 As shown, in the tMCAO model, the fluorescence signal of T-NDNAs in brain tissue was significantly stronger than that of DiRSol, and significant accumulation was observed approximately 3 hours after reperfusion; no significant accumulation of T-NDNAs was observed in the brains of healthy rats. These results indicate that T-NDNAs can achieve targeted distribution in stroke-related microthrombi and reperfusion injury areas.

[0139] Further evaluation of in vivo photothermal conversion capacity was conducted. FeCl3-induced carotid artery thrombosis model rats were intravenously injected with PBS, DiRSol, β-NDNAs, or T-NDNAs, with a DiRSol equivalent dose of 5 mg / kg. Photoirradiation was performed based on the time to peak in vivo accumulation; T-NDNAs were irradiated 2 h after administration, while the other formulations were irradiated 1 h after administration. Irradiation conditions were 808 nm and 2 W / cm². 2 The temperature was measured for 15 minutes, and local temperature changes were recorded using an infrared thermal imager.

[0140] The results are as follows Figure 39 and Figure 40 As shown, the temperature rise at the thrombus site was limited after treatment with DiR Sol and ND Sol, stabilizing at approximately 40°C; B-ND NAs raised the temperature to approximately 45°C; and T-ND NAs raised the temperature more rapidly and stabilized at approximately 50°C. This indicates that T-ND NAs have a stronger in vivo photothermal conversion effect at the thrombus site due to improved in vivo circulation and thrombus-targeted delivery.

[0141] Example 9: In vivo thrombolytic effect of BNN-NO / DiR carrier-free nanomotor in a rat carotid artery thrombosis model Healthy male SD rats, 6 weeks old and weighing 180-220 g, were randomly divided into 8 groups (n=5 per group): Sham group, Thrombosis group, DiR Sol+L group, ND Sol+L group, B-NDNAs+L group, T-NDNAs group, T-NDNAs+L group, and lumbrokinase solution group. A FeCl3-induced rat carotid artery thrombosis model was established according to Example 8. Each treatment group received a single intravenous injection of the corresponding formulation; the equivalent dose of DiR was 5 mg / kg; and the dose of lumbrokinase was 8000 U / kg.

[0142] Irradiation was conducted based on the peak accumulation time of the formulation in vivo. The DiR Sol and B-NDNAs groups were irradiated 15 min after administration, while the T-NDNAs+L group was irradiated 2 h after administration. The irradiation conditions were 808 nm and 2.0 W / cm². 215 min. After the experiment, the rats were sacrificed, the carotid artery thrombus segment was collected, repeatedly washed with PBS, and then preserved in tissue fixative. Subsequently, frozen sections and H&E staining were performed, and the degree of thrombus residue was quantitatively analyzed.

[0143] The results are as follows Figure 41 and Figure 42 As shown, under the same laser conditions, the in vivo thrombolytic effects of DiR Sol+L and ND Sol+L were limited; T-ND NAs were difficult to exert a significant therapeutic effect without light irradiation, indicating that the treatment process is photocontrolled and safe; the T-ND NAs+L group showed the most significant thrombolytic effect, with a thrombus clearance rate of approximately 85%, which was significantly better than the B-ND NAs+L group. These results demonstrate that T-ND NAs can achieve highly efficient and synergistic thrombolysis in an arterial thrombosis model through thrombus-targeted delivery, photothermal effects, and NO-driven mechanical action.

[0144] Example 10: Therapeutic and relapse-preventing effects of BNN-NO / DiR carrier-free nanomotor in a chronic inflammatory tail vein thrombosis model Healthy female KM mice, 11 weeks old and weighing 35-45 g, were selected. Tail length was recorded in healthy condition, and the mice were randomly divided into 8 groups (n=6 per group): Sham group, Thrombosis group, DiR Sol+L group, ND Sol+L group, B-ND NAs+L group, T-ND NAs group, T-ND NAs+L group, and lumbrokinase-edaravone mixed solution group. A κ-carrageenan-induced chronic inflammatory thrombosis model of the mouse tail vein was established according to Example 8.

[0145] After successful modeling, each group underwent long-term, multiple-dose treatment. The drug was administered once every 2 days for a total of 5 doses, with a DiR equivalent dose of 5 mg / kg. The DiR Sol group received light exposure 90 min after drug administration; the B-NDNAs and T-NDNAs groups received light exposure 120 min after drug administration. The light conditions were 808 nm and 2 W / cm². 2 15-20 min. The length of the lesion site was measured and recorded daily, and the thrombus length was calculated using the differential method. Mice were sacrificed on day 9, and plasma was collected to detect sCD40L, APTT, FIB, and liver and kidney function indicators. The tail was cut off, fixed, frozen sectioned, and stained with H&E.

[0146] The results are as follows Figures 43 to 46As shown, the thrombosis group, DiR Sol+L group, ND Sol+L group, and unirradiated T-NDNAs group exhibited rapid tail thrombosis progression, with some mice showing tail necrosis or epidermal damage. The tail lesion length was significantly shortened in the T-ND NAs+L, B-ND NAs+L, and lumbrokinase-edaravone groups, with the T-ND NAs+L group showing the best effect, where all mice maintained a relatively intact tail morphology. This indicates that T-ND NAs+L can improve the progression of chronic inflammatory venous thrombosis through photothermal-mechanical thrombolysis and in-situ antioxidant effects.

[0147] To evaluate the relapse prevention effect, on day 10 after initial treatment, mice in the T-ND NAs+L group and the lumbrokinase-edaravone group were intraperitoneally injected again with 1% κ-carrageenan saline solution, 35 mg / kg, to induce venous thrombosis recurrence, and the length of tail lesions was continuously recorded. Four days later, the mice were sacrificed, and tail tissue was collected, fixed, sectioned, and stained with H&E.

[0148] The results are as follows Figure 47 As shown, compared with the lumbrokinase-edaravone group, the T-NDNAs+L group showed slower progression of tail lesions, suggesting that T-NDNAs can improve the thrombotic microenvironment and reduce the risk of recurrence and progression through NO release and in situ QDI antioxidant activity.

[0149] Further testing of inflammation and coagulation markers was conducted. Results were as follows: Figures 48 to 50 As shown, the sCD40L level in the T-ND NAs+L group was significantly decreased and approached physiological levels; APTT was significantly prolonged and FIB was significantly reduced, suggesting that T-ND NAs+L can reduce inflammation levels and improve coagulation status. Tail H&E staining results further confirmed that T-ND NAs+L can alleviate thrombus occlusion and tissue damage.

[0150] Example 11: Therapeutic effect of BNN-NO / DiR carrier-free nanomotor in acute ischemic stroke and ischemia-reperfusion injury A rat model of tMCAO was established. SD rats were randomly divided into Sham group, Thrombosis group, DiR Sol+L group, and T-NDNAs+L group. After reperfusion, DiR Sol or T-NDNAs were injected via the tail vein, with an equivalent dose of 5 mg / kg for DiR. The DiR Sol group was exposed to light 15 min after administration, and the T-NDNAs+L group was exposed to light 3 h after administration, with light conditions of 808 nm, 2 W / cm2, and 15 min. Sixteen h after reperfusion, neurological function was evaluated using the Zea-Longa score. Rats were then sacrificed, and brain tissue was rapidly frozen and cut into approximately 2 mm thick slices. The slices were incubated in 2% TTC solution at 37°C for 20 min, stained, fixed, and photographed for quantitative analysis. Additional brain tissue was used for H&E staining, Nissl staining, and plasma TNF-α detection.

[0151] The results are as follows Figures 51 to 53 As shown, the Thrombosis group rats had higher neurological function scores, and TTC staining showed obvious cerebral infarction foci; the DiR Sol+L group only showed slight improvement; the T-ND NAs+L group showed significantly lower neurological function scores, significantly smaller cerebral infarction volume, and infarction area of ​​less than 20%, indicating that T-ND NAs+L can improve neurological function damage after ischemic stroke and reduce the extent of cerebral infarction.

[0152] Further H&E and Nissl staining results are as follows Figure 54 As shown, the Thrombosis group exhibited significant neuronal damage, shrinkage, and deformation in brain tissue, with a reduction in Nissl bodies; the T-ND NAs+L group showed significantly reduced pathological damage in brain tissue, with better preservation of neuronal morphology in the cortex and hippocampus. Plasma TNF-α detection results are as follows... Figure 55 As shown, TNF-α levels were significantly reduced in the T-NDNAs+L group. These results indicate that T-NDNAs perform photocontrolled NO release, photothermal-mechanical thrombolysis, and QDI in-situ antioxidant activity at the thrombus site and in the ischemia-reperfusion area, thereby exerting therapeutic and neuroprotective effects on stroke.

[0153] Example 12: Biosafety Evaluation of BNN-NO / DiR Carrier-Free Nanomotors In vitro hemolysis experiment: Orbital blood was collected from healthy SD rats, and erythrocytes were separated by centrifugation at 4000 rpm for 3 min. 50 μL of erythrocytes were dispersed in 1 mL PBS to prepare a erythrocyte suspension. 50 μL each of BNN-NO Sol, DiR Sol, ND Sol, B-NDNAs, and T-NDNAs were co-incubated with the erythrocytes. The effective concentration of DiR after dilution was 200 nM. PBS served as a negative control, and pure water as a positive control. After incubation at 37℃ for 2 h, each group was subjected to either light or no-light treatment. The supernatant was collected by centrifugation, and the absorbance of hemoglobin at 540 nm was detected using a multi-mode microplate reader.

[0154] The results are as follows Figure 56 As shown, no obvious hemolysis was observed in any of the experimental groups under both light and dark conditions, suggesting that T-NDNAs do not cause significant damage to circulating erythrocytes and have good blood compatibility.

[0155] In vitro cytotoxicity experiment: HUVECs were subjected to 4 × 10⁻⁶ saturates. 3 Seeds were planted per well in 96-well plates and cultured for 12 h until 80%-90% confluence. Different concentrations of BNN-NO Sol, DiR Sol, ND Sol, B-NDNAs, and T-NDNAs were added, with DiR equivalent concentrations of 0, 25, 50, 100, and 200 nM. Four h after drug administration, the light-illuminated group was subjected to 808 nm, 2 W / cm² light. 2 Illuminate for 3 min per well, then incubate in the dark for 48 h. After incubation, add 250 μL of 0.5 mg / mL MTT solution, incubate for 4 h, discard the supernatant, add 200 μL of DMSO, shake in the dark for 15 min, and measure the absorbance at 490 nm.

[0156] The results are as follows Figure 57 As shown, within the equivalent DiR concentration range of 0-200 nM, all formulations maintained a cell viability of over 80% under both laser irradiation and non-laser irradiation conditions, indicating that T-NDNAs and corresponding light conditions have no significant toxicity to vascular endothelial cells.

[0157] Safety evaluation of a single dose: In a FeCl3-induced rat carotid artery thrombosis model, T-ND NAs were administered intravenously at a dose of 5 mg / kg (DiR), and the rats were treated according to Example 9. Fourteen days after the end of treatment, major organs of the rats, including the heart, liver, spleen, lungs, and kidneys, were collected for H&E staining to assess tissue damage.

[0158] The results are as follows Figure 58 As shown, no obvious abnormalities were found in the H&E staining sections of the heart, liver, spleen, lungs, and kidneys of rats in each group, indicating that a single administration of T-NDNAs and phototherapy did not cause significant damage to the major organs.

[0159] Long-term drug safety evaluation: Long-term treatment of chronic inflammatory thrombosis of the tail vein was performed according to Example 10. Mice were sacrificed on day 10 and plasma was collected. Liver and kidney function indicators AST, ALT, BUN and UREA were detected.

[0160] The results are as follows Figure 59 As shown, no significant abnormalities were observed in the liver and kidney function indicators of mice after long-term and repeated administration, indicating that T-NDNAs have good safety for long-term in vivo administration.

[0161] In summary, the T-ND NAs described in this invention can accumulate at the thrombus site through thrombus-targeted delivery, exert a DiR-mediated photothermal effect under 808 nm near-infrared light irradiation, and trigger BNN-NO to release NO to generate mechanical drive. At the same time, QDI is generated in situ to scavenge reactive oxygen species, thereby achieving thrombus-targeted accumulation, deep penetration, photothermal-mechanical synergistic thrombolysis, and cascade antioxidant protection. It can be used to prepare drugs for the treatment of thrombotic diseases, ischemia-reperfusion injury, and ischemic stroke.

Claims

1. A cascaded antioxidant carrier-free nanomotor, characterized in that: The cascaded antioxidant carrier-free nanomotor is formed by the self-assembly of a functionally converted nitric oxide donor BNN-NO and a photothermal photosensitizer through non-covalent interactions, and is modified with a polyethylene glycol modifier and a polyethylene glycol-modified lesion-targeting ligand; wherein the photothermal photosensitizer is one or both of DiR or DiD; the functionally converted nitric oxide donor BNN-NO has the following structure: 。 2. The cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The non-covalent interaction force is one or more of the following: hydrophobic interaction, π-π stacking interaction, hydrogen bonding interaction, or electrostatic interaction.

3. The cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The molar ratio of the functionally convertible nitric oxide donor BNN-NO to the photothermal photosensitizer is 1:5 to 5:1; the polyethylene glycol modifier and the polyethylene glycol-modified lesion-targeting ligand account for 5% to 20% and 10% to 25% of the total mass of the system, respectively.

4. The cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The functional convertible nitric oxide donor BNN-NO is prepared by the following method: using 5-hydroxy-2-nitrobenzaldehyde and p-phenylenediamine as raw materials, the target compound BNN-NO is obtained after condensation, reduction and nitrosation reactions.

5. The cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The polyethylene glycol modifier is DSPE-PEG with a molecular weight of 2000.

6. The cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The pegylated lesion-targeting ligand is one or more of the following: pegylated CREKA, pegylated polypeptide fibrin-targeting ligand, pegylated VCAM-1-targeting ligand, pegylated ICAM-1-targeting ligand, pegylated selectin-targeting ligand, or pegylated integrin-targeting ligand.

7. The method for preparing the cascaded antioxidant carrier-free nanomotor according to claim 1, characterized in that: The process includes the following steps: dissolving the functionally converted nitric oxide donor BNN-NO and the photothermal photosensitizer separately in an organic solvent, and mixing them to form an organic phase; adding the organic phase to an aqueous phase under stirring, ultrasonic, or microfluidic conditions, allowing the functionally converted nitric oxide donor BNN-NO and the photothermal photosensitizer to self-assemble into a binary nanoassembly through non-covalent interactions; subsequently adding a polyethylene glycol modifier and a polyethylene glycol-modified lesion-targeting ligand for surface modification, and removing the organic solvent to obtain the cascaded antioxidant carrier-free nanomotor.

8. The preparation method according to claim 7, characterized in that: The organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, or acetonitrile.

9. The application of the cascaded antioxidant carrier-free nanomotor as described in claim 1 in the preparation of a drug delivery system.

10. The use of the cascaded antioxidant carrier-free nanomotor of claim 1 in the preparation of drugs for antithrombosis and / or treatment of ischemia-reperfusion injury.

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