A nitric oxide-empowered carrier-free self-driven nanorobot, its preparation method and application

Abstract

This invention discloses a carrier-free, self-driven nanorobot empowered by nitric oxide, its preparation method, and its applications, belonging to the field of biomedical technology. This invention involves forming a hybrid nanoassembly by driving a nitric oxide donor drug and a photothermal photosensitizer molecule through non-covalent interactions. The surface of the hybrid co-assembled nanoassembly is modified with one or both of polyethylene glycol (PEG) modifiers and PEG thrombofibrin-targeting peptides. The molar ratio of the nitric oxide donor drug to the photothermal photosensitizer molecule is 1:10 to 10:1. The preparation method of this invention is stable, convenient, simple, and practical. The resulting thrombotargeting, nitric oxide donor drug, and photothermal photosensitizer hybrid nanoassembly exhibits extremely high drug loading capacity, enabling efficient co-loading of the hybrid nanoassembly and efficient photothermal-nitric oxide thrombolysis. This provides a novel and efficient drug delivery strategy for the treatment of cerebrovascular diseases such as thrombosis and ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to fibrin-targeting peptide-modified nitric oxide / photothermal hybrid nanorobots for the treatment of thrombosis and ischemic stroke, their preparation methods, and applications. Background Technology

[0002] Current drug treatments for cardiovascular and cerebrovascular diseases, primarily acute myocardial infarction and stroke, still fall short of expectations. For example, due to the short half-life of thrombolytic drugs like plasminogen activator (PAA), frequent high-dose administration is often required, leading to side effects such as bleeding and allergic reactions. Furthermore, due to the lack of target sites, less than 5% of the drug reaches the patient's site, resulting in low drug utilization. In addition to traditional treatments, photothermal therapy has attracted widespread attention from thrombosis researchers. It uses laser irradiation to raise the temperature of the thrombus lesion, breaking the interfibrillary bonds and destroying the thrombus under high shear force, thus achieving precise thrombus treatment while addressing safety concerns. However, studies have shown that photothermal therapy alone is still insufficient to completely dissolve thrombi, and the resulting thrombus fragments can easily cause secondary embolism, limiting its application.

[0003] Nanomotors, as an emerging and non-invasive treatment approach, have the potential to provide novel therapeutic advantages. They can be driven by converting various energy sources, such as chemical fuels, light, magnetic fields, and sound fields, into mechanical energy, overcoming biological barriers and improving therapeutic efficacy. Among various types of nanomotors, gas-driven nanorobots have attracted much attention because gases (such as O2 and H2) not only provide the driving force but also play a crucial role in endogenous regulation. Nitric oxide, as a key molecule, plays a vital role in the treatment of cardiovascular and cerebrovascular diseases, possessing multiple biological functions. It plays an important role in dilating blood vessels, inhibiting platelet adhesion and aggregation, inhibiting leukocyte-endothelial cell adhesion and smooth muscle cell proliferation, promoting the growth of vascular endothelial cells to repair damaged blood vessels, and protecting nerve cells. To date, no research has been reported on the design of nanorobots driven by nitric oxide to achieve efficient thrombolysis of thrombi and ischemic stroke using photothermal imaging agents for photothermal thrombolysis. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a carrier-free, self-driven nanorobot powered by nitric oxide, its preparation method, and its applications. This invention utilizes small-molecule hybrid assembly nanotechnology, thrombus fibrin-targeting peptide modification technology, and nanorobot driving technology to construct a thrombosis-targeting nanodelivery system composed of a photothermal imaging agent, a nitric oxide donor drug, a polyethylene glycol modifier, and a polyethylene glycol thrombus fibrin-targeting peptide hybrid assembly. First, a hybrid nanoassembly is prepared based on the molecular interactions between the photothermal imaging agent and the nitric oxide donor drug. This invention employs a simple, high-capacity, small-molecule self-assembled nano-drug delivery system to achieve efficient co-loading of the photothermal imaging agent and the nitric oxide donor drug, enabling combined delivery and efficient treatment. The photothermal imaging agent, as a fluorescent probe, can not only diagnose embolic vessels through fluorescence imaging but also provide energy for the nanorobot and treat thrombosis through photothermal action. Under photothermal triggering, the nitric oxide donor drug can not only achieve mechanical thrombolysis through movement but also release nitric oxide molecules for functional regulation. Furthermore, we modified the surface of the nanoassemblies with polyethylene glycol (PEG) modifiers and PEG thrombus fibrin-targeting peptides to increase their in vivo circulation time and endow them with thrombus lesion targeting capabilities. After entering the bloodstream, the hybrid nanoassemblies can efficiently accumulate at the thrombus lesion. Upon laser irradiation, the heat generated by the hybrid nanoassemblies not only dissolves the thrombus but also promotes the transformation of the nanoassemblies into nanorobots, further enabling secondary penetration of the thrombus and pathway regulation, thus achieving thrombus treatment and regulation. This invention provides a novel and efficient drug delivery strategy for the treatment of cerebrovascular diseases such as thrombosis and ischemic stroke.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a carrier-free self-driven nanorobot powered by nitric oxide. The carrier-free self-driven nanorobot is a hybrid nanoassembly formed by the co-assembly of a photothermal photosensitizer and a nitric oxide donor molecule through non-covalent interactions (hydrophobic interactions, π-π stacking, electrostatic interactions). The surface of the hybrid co-assembled nanoassembly is modified with one or two of polyethylene glycol modifiers and polyethylene glycol thrombofibrin targeting peptides.

[0007] Based on the above technical solution, the molar ratio of the nitric oxide donor molecule to the photothermal photosensitizer is further 1:10 to 10:1.

[0008] Based on the above technical solution, the photosensitizer is further selected from one or more combinations of DiR, IR808, IR780, ZnPC, and ICG; the nitric oxide donor molecule is selected from one or a combination of two of N,N'-disec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6) and S-nitrosothiol (SNAP).

[0009] Based on the above technical solution, the thrombus fibrin targeting peptide is further defined as CREKA or GPRPP.

[0010] Based on the above technical solution, the polyethylene glycol modifier is one or a combination of two or more of PCL-PEG, DSPE-PEG, PLGA-PEG, and PE-PEG, wherein the molecular weight of the PEG segment is 200-20000.

[0011] Another aspect of the present invention provides a method for preparing the above-mentioned nitric oxide-empowered carrier-free self-driving nanorobot, comprising the following steps:

[0012] (1) The photothermal photosensitizer and nitric oxide donor molecules were dissolved in organic solvents and mixed evenly in proportion. Under stirring conditions, the resulting mixed solution was added dropwise to water to spontaneously form a uniform hybrid nano-assembly.

[0013] (2) Prepare a polyethylene glycol modifier and / or a polyethylene glycol thrombofibrin-targeting peptide solution. Under stirring conditions, add the polyethylene glycol modifier and / or the polyethylene glycol thrombofibrin-targeting peptide solution dropwise to the hybrid nano-assembly obtained in step (1). Remove the organic solvent by rotary evaporation to obtain the final product.

[0014] Based on the above technical solution, further, the organic solvent in step (1) is one or more of methanol, ethanol, tetrahydrofuran, acetone, and dimethyl sulfoxide.

[0015] Based on the above technical solution, further, the concentration of the photothermal photosensitizer in the mixed solution in step (1) is 0.1 to 50 mg / mL.

[0016] Based on the above technical solution, further, the photothermal photosensitizer in step (1) is DiR; the nitric oxide donor molecule is BNN6.

[0017] Based on the above technical solution, further, the molar ratio of photothermal photosensitizer and nitric oxide donor molecules in the hybrid nano-assembly described in step (1) is 1:3.

[0018] Based on the above technical solution, further, the concentration of the hybrid nano-assembly in step (2) is 0.1 to 50 mg / mL.

[0019] Based on the above technical solution, further, in step (2), the amount of polyethylene glycol modifier and / or polyethylene glycol thrombofibrin targeting peptide added is 0.1 to 10% of the mass of the hybrid nanoassembly.

[0020] The present invention also provides the application of the above-mentioned nitric oxide-empowered carrier-free self-driven nanorobots in the preparation of drug delivery systems.

[0021] Based on the above technical solution, the drug delivery system further includes an oral drug delivery system and an injectable drug delivery system.

[0022] Based on the above technical solution, the drug delivery system is further used to prepare antithrombotic drugs, ischemic stroke drugs, antitumor drugs, and anti-inflammatory drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) By utilizing the molecular engineering nano-assembly characteristics of photothermal photosensitizers and nitric oxide donor drugs, carrier-free hybrid nanoassemblies are constructed. Because molecular assembly engineering nano-drug delivery systems have the advantage of ultra-high drug loading, they can achieve efficient co-loading and synchronous delivery of the two drugs.

[0025] (2) By utilizing the photothermal effect of photothermal photosensitizers, they can not only enhance the drug thrombus penetration by achieving high heat in thrombus lesions through energy conversion, but also trigger nitric oxide donor molecules to release fuel nitric oxide to power nanorobots.

[0026] (3) Due to the ultra-high drug loading capacity and efficient co-loading of two drugs, nanorobots have better fuel driving force and movement ability than traditional encapsulated nano-formulations.

[0027] (4) In various thrombosis models (rat carotid artery embolism model, mouse tail vein embolism model and rat stroke model), the photothermal-nitric oxide synergistic diagnosis and treatment effect of nitric oxide-empowered thrombosis-targeting therapy nanorobot is good, with good biosafety, can reduce bleeding side effects, and has clinical translation potential. Attached Figure Description

[0028] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0029] Figure 1 Transmission electron microscopy (TEM) images of the nitric oxide-enabled hybrid nanoassembly DiR / BNN6(A), the thrombosis-targeting nitric oxide-enabled hybrid nanoassembly DiR / BNN6(B), and the thrombosis-targeting non-nitric oxide-enabled hybrid nanoassembly DiR / BPA(C) in Examples 1 and 2 of this invention.

[0030] Figure 2 The image shows the ultraviolet spectrum of the DiR / BNN6 thrombus-targeting nitric oxide-enabling hybrid nanoassembly in Example 3 of this invention.

[0031] Figure 3The fluorescence spectrum of the DiR / BNN6 thrombus-targeting nitric oxide-enabling hybrid nanoassembly in Example 3 of this invention is shown.

[0032] Figure 4 This study investigates the stability of the DiR / BNN6 thrombus-targeting nitric oxide-enriched hybrid nanoassembly in PBS in Example 4 of this invention.

[0033] Figure 5 This study investigates the in vitro thrombosis targeting properties of the DiR / BNN6 thrombosis-targeting nitric oxide-enriched hybrid nanoassembly in Example 5 of this invention.

[0034] Figure 6 The temperature change of the DiR / BNN6 thrombus-targeting nitric oxide-enhanced hybrid nanoassembly in Example 6 of this invention under 808nm laser irradiation.

[0035] Figure 7 The image shows the motion of the DiR / BNN6 thrombus-targeting nitric oxide-empowered hybrid nanoassembly under different powers of 808nm laser irradiation in Example 7 of this invention. A: The motion trajectory of the DiR / BNN6 thrombus-targeting nitric oxide-empowered hybrid nanoassembly; B: Quantitative analysis of the motion rate of the DiR / BNN6 thrombus-targeting nitric oxide-empowered hybrid nanoassembly.

[0036] Figure 8 This document compares the motion of the DiR / BNN6 thrombus-targeting nitric oxide-enriched hybrid nanoassemblies under 808nm laser irradiation with that of PLGA nanoparticles encapsulated on a conventional carrier in Example 8 of this invention. A: Comparison of the motion of the DiR / BNN6 thrombus-targeting nitric oxide-enriched hybrid nanoassemblies with that of nanoparticles encapsulated on a conventional carrier.

[0037] B: Quantitative analysis of the movement rates of DiR / BNN6 thrombosis-targeting nitric oxide-empowered hybrid nanoassemblies and conventional carrier-encapsulated PLGA nanoparticles under light and non-light conditions.

[0038] Figure 9 This is an example of the in vitro thrombolysis of the DiR / BNN6 thrombus-targeting nitric oxide-enhanced hybrid nanoassembly under 808nm laser irradiation in Example 9 of the present invention, where A: qualitative analysis and B: quantitative analysis.

[0039] Figure 10 This image shows the thrombus penetration of the DiR / BNN6 thrombus-targeting nitric oxide-enriched hybrid nanoassembly in Example 10 of this invention under 808nm laser irradiation.

[0040] Figure 11This study investigates the pharmacokinetic properties of the DiR / BNN6 thrombosis-targeting nitric oxide-enhanced hybrid nanoassemblies in Example 11 of this invention.

[0041] Figure 12 This is a carotid artery embolism vascular fluorescence imaging of the DiR / BNN6 thrombus-targeting nitric oxide-enabled hybrid nanoassembly in Example 12 of the present invention, wherein A: carotid artery embolism vascular fluorescence imaging, and B: quantitative analysis of the fluorescence signal intensity of carotid artery embolism vascular.

[0042] Figure 13 This refers to the in vivo photothermal conversion capability of the DiR / BNN6 thrombus-targeting nitric oxide-enabling hybrid nanoassembly in Example 13 of the present invention.

[0043] Figure 14 These are H&E stained photographs of carotid artery embolism vessels in each group after drug treatment in Example 14 of the present invention.

[0044] Figure 15 This is a quantitative analysis of the degree of carotid artery embolism in each group after drug treatment in Example 14 of the present invention.

[0045] Figure 16 These are H&E stained images of tail vein thrombosis vessels in each group after drug treatment in Example 15 of this invention.

[0046] Figure 17 This invention presents the drug efficacy of each group of ischemic stroke patients after drug treatment in Example 16 of this invention. A is a brain tissue diagram of each group of ischemic stroke patients after treatment, and B is an enlarged view of A.

[0047] Figure 18 These are H&E staining images of the heart, liver, spleen, lung, and kidney tissues after administration of the DiR / BNN6 thrombosis-targeting nitric oxide-enabled hybrid nanoassemblies in Example 17 of this invention. Detailed Implementation

[0048] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0049] The DSPE-PEG described in the examples 2K -CREKA was purchased from Suzhou Polypeptide Biotechnology Co., Ltd.

[0050] Example 1: Preparation of DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies

[0051] First, DiR and BNN6 were dissolved in methanol to prepare a 10 mg / mL stock solution. Then, 100 μL of the DiR methanol solution was mixed with methanol solvents containing BNN6 in different molar ratios to prepare a mixed drug solution, which was then diluted with methanol to 200 μL. Under a magnetic stirrer at 1200 rpm, 200 μL of the mixed drug solution was slowly added dropwise to 2 mL of deionized water. The mixture was stirred for 3 minutes in the dark. The nano-formulation was then removed by rotary evaporation to remove the organic solvent, and the volume was brought to 2 mL with deionized water.

[0052] The particle size distribution of the resulting hybrid nanoassemblies is shown in Table 1. Hybrid nanoassemblies can be formed when the mass ratio of DiR to BNN6 is between 10:1 and 1:10. However, when the molar ratio of DiR to BNN6 is 1:3, the hybrid nanoassemblies exhibit the smallest particle size and polydispersity index (PDI), the best assembly performance, and the highest efficiency in generating nitric oxide (NO) (see Table 1). Therefore, the preferred ratio of DiR to BNN6 is 1:3.

[0053] Table 1. Ratio screening of hybrid nanoassemblies

[0054]

[0055] Example 2: Preparation of DiR / BNN6 thrombus-targeting nitric oxide-enhanced and DiR / BPA thrombus-targeting non-nitric oxide-enhanced hybrid nanoassemblies

[0056] DSPE-PEG 2K / DSPE-PEG 2K Preparation of CREKA mixed solution: DSPE-PEG 2K Dissolved in ethanol solution (10 mg / mL), DSPE-PEG 2K -CREKA dissolves in methanol (1.5 mg / mL). Of this, take...

[0057] DSPE-PEG 2K (15wt%) and DSPE-PEG 2K -CREKA (10wt%) is obtained by mixing the two.

[0058] Surface modification of hybrid nanoassemblies with DSPE-PEG 2K / DSPE-PEG 2K -CREKA method: make it first

[0059] DiR / BNN6 hybrid nanoassemblies were first prepared by dissolving DiR and BNN6 separately in methanol (10 mg / mL). Then, 109.6 μL of DiR and 90.4 μL of BNN6 were added (DiR / BNN6 = 1:3). 200 μL of the mixed drug solution was slowly added dropwise to 2 mL of deionized water using a magnetic stirrer at 1200 rpm, and stirred in the dark for 3 min. Subsequently, while stirring, DSPE-PEG was slowly added dropwise to the DiR / BNN6 hybrid nanoassembly system. 2K / DSPE-PEG 2K -CREKA mixed solution, continue stirring in the dark for 2 min. Take out the nano-formulation and remove the organic solution by rotary evaporation, then make up to 2 mL with deionized water to obtain the nitric oxide-powered thrombus-targeting targeted hybrid nanoassembly.

[0060] To further achieve the preparation of a thrombus-targeting, non-nitric oxide-powered hybrid nanoassembly for the control group, we used BPA, which has a similar structure to BNN6 but lacks nitric oxide-generating ability, for our experiments. The experimental process only requires...

[0061] By replacing BNN6 with BPA and following the same procedure, a thrombosis-targeting non-nitric oxide-powered hybrid nanoassembly can be obtained.

[0062] The particle size distribution, potential, and drug loading of the prepared DiR / BNN6 series hybrid nanoassemblies are shown in Table 2. The particle size of the DiR / BNN6 hybrid nanoassemblies is 74.1 nm, and the Zeta potential is +18.33. The particle sizes of both polyethylene glycol / targeted modified nitric oxide-powered nanoassemblies and non-nitric oxide-powered hybrid nanoassemblies are increased (105.0 nm, 118.7 nm), while the Zeta potentials are decreased (-6.68 mV, -5.80 mV). The morphology of all three types of nanoparticles is uniformly spherical. Figure 1 Surprisingly, all three nanoassemblies exhibited extremely high drug loading: DiR = 54.8%, BNN6 = 45.2% (DiR / BNN6 hybrid nanoassemblies) or DiR = 41.1%, BNN6 = 33.9% (DiR / BNN6 thrombus-targeting nitric oxide-powered co-assembled nanoparticles).

[0063] Table 2. Characterization of DiR / BNN6 series hybrid nanoassemblies

[0064]

[0065] Example 3: Ultraviolet fluorescence spectrum of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0066] The DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies prepared in Example 2, along with DiR and BNN6 solutions, were scanned using a multi-mode microplate reader (DiR concentration: 20 μg / mL, BNN6 concentration: 16.5 μg / mL). Under the same conditions, the fluorescence spectra of the DiR / BNN6 hybrid nanoassemblies, the DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies, and the DiR solution were scanned using a multi-mode microplate reader. Compared to the solvent group, the UV spectra of both nanoassemblies showed a redshift. Figure 2 The fluorescence spectrum peaks are aligned, but the fluorescence intensity decreases. Figure 3 This demonstrates the successful preparation of hybrid nanoassemblies.

[0067] Example 4: Colloidal stability experiment of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0068] The DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies prepared in Example 2 were incubated in PBS (pH 7.4). The nanoassemblies were removed at 1, 2, 4, 6, and 8 hours, and their particle size changes were measured using a dynamic light scattering particle size analyzer. The results showed that the DiR / BNN6 hybrid nanoassemblies exhibited a large particle size variation and poor colloidal stability. However, after modification with polyethylene glycol / targeting peptides, the DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies and the DiR / BNN6 thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies showed no significant particle size change after incubation, demonstrating good colloidal stability. Figure 4 ).

[0069] Example 5: In vitro thrombosis targeting experiment of DiR / TGL thrombosis-targeting co-assembled nanoparticles

[0070] Construction of an in vitro thrombosis model: Blood was collected from the orbits of SD rats, and whole blood was centrifuged to obtain platelet-rich plasma. 150 μL of the plasma was added to a 96-well plate along with CaCl2 (0.3 mol / L) and thrombin (0.3 U / μL) and mixed well. The plate was then incubated in a constant temperature incubator (37℃) for 120 min.

[0071] In vitro thrombus targeting validation experiment: PBS, DiR solution, DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassemblies were co-incubated with thrombus blocks in 96-well plates (37℃, 10 min). The thrombus blocks were washed three times with PBS. Finally, a small animal in vivo imaging system was used to measure the DiR fluorescence on the thrombus surface to determine its targeting ability. Figure 5 As shown, the groups involving thrombosis-targeting peptides (DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies) all exhibited strong fluorescence signals, demonstrating their good thrombosis-targeting ability.

[0072] Example 6: In vitro photothermal experiment of DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies

[0073] PBS, DiR solution, DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies (DiR concentration 0.548 mg / mL, BNN6 concentration 0.452 mg / mL) were irradiated for 15 min under an 808 nm laser (Changchun New Industries Optoelectronic Technology Co., Ltd., MDL-N-5W) at 2.0 W / cm². 2 This process uses an infrared thermal imager (Fotric 226) to record temperature changes. For example... Figure 6 As shown, compared to PBS, the temperatures of the other four solutions gradually increased with increasing irradiation time, reaching as high as 50°C or more. Under these conditions, the non-covalent bonds of fibrin in the thrombus can be disrupted, further achieving the effect of photothermal thrombolysis.

[0074] Example 7: Motion experiment of DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies under different power irradiation

[0075] The DiR / BPA thrombus-targeting non-nitric oxide-powered hybrid nanoassemblies (DiR concentration 0.01 mg / mL) were placed under an 808 nm laser (Changchun New Industries Optoelectronic Technology Co., Ltd., MDL-N-5W) at different power levels: 2.0 W / cm². 2 1.0W / cm 2 1.5W / cm 2 2.0W / cm 2 After irradiation for 60 seconds, the motion trajectory of the nanoassemblies was recorded using a Nikon (40×) positive fluorescence microscope. The results are as follows: Figure 7As shown, the DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies exhibit power-dependent mobility.

[0076] Example 8: Motion Comparison Experiment of DiR / BNN6 Thrombus-Targeting Nitric Oxide-Enabled Hybrid Nanoassemblies and PLGA Nanoparticles Encapsulated in Traditional Carriers

[0077] Preparation of PLGA nanoparticles: PLGA nanoparticles loaded with BNN6 and DiR were prepared by emulsion evaporation. PLGA (60 mg), DiR (2.35 mg), and BNN6 (2.19 mg) were dissolved in 4 mL of a mixed solvent (dichloromethane / tetrahydrofuran = 3:1, v / v). The mixed solvent (2 mL) was added to an aqueous solution (4 mL) containing 1% PVA, followed by sonication in an ice bath for 6 min. The organic solvent in the emulsion was removed by vacuum rotary evaporation at 37 °C for 3 h. The nanoparticle suspension was centrifuged at 4200 r / min for 30 min, washed three times, and finally dispersed in purified water to obtain PLGA-loaded DiR / BNN6 nitric oxide hybrid-enabled nano-hybrid assemblies.

[0078] PLGA-encapsulated DiR / BNN6 nitric oxide hybrid nanoassemblies and DiR / BNN6 thrombus-targeting nitric oxide hybrid nanoassemblies (DiR concentration 0.01 mg / mL) were placed under an 808 nm laser (2.0 W / cm²). 2 The motion trajectory of the nanoassemblies was recorded using a Nikon (40×) positive fluorescence microscope (60s). The results are as follows: Figure 8 As shown, the carrier-free DiR / BNN6 nitric oxide-enriched hybrid nanoassemblies exhibit more efficient motion capabilities compared to traditional PLGA carrier materials.

[0079] Example 9: In vitro photothermal thrombolysis using DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies

[0080] Establishment of the thrombosis model: Blood was collected from the orbital cavity of healthy SD rats, and EP tubes coated with anticoagulant (heparin) were used as containers. Thrombin at a concentration of 0.1 U / μL and calcium chloride (CaCl2) at 0.3 M were prepared. 500 μL of platelet-rich plasma, 50 μL of thrombin, and 50 μL of CaCl2 were added to 1.5 mL EP tubes, and the tubes were incubated at 37°C for 90 min to obtain the thrombosis model.

[0081] Photothermal thrombolysis experiment: Different groups of formulations (I: PBS, II: BNN6 solution, III: DiR solution, IV: mixed BNN6 and DiR solution, V: DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, VI: DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassemblies, VII: DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassemblies) were added to the thrombus and placed under an 808nm laser (2W / cm²). 2 (15 min), the weight of the thrombus before and after thrombolysis was measured, and the thrombolysis rate was calculated (thrombolysis rate = (weight of thrombus before thrombolysis - weight of thrombus after thrombolysis) / weight of thrombus before thrombolysis × 100%). The results are as follows: Figure 9 As shown, the DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies exhibit good photothermal thrombolytic effects.

[0082] Example 10: Validation of the thrombus permeability of the DiR / BNN6 thrombus-targeting hybrid nanoassembly

[0083] Establishment of microemboli: Blood was collected from the orbit of healthy SD rats, and 10 μL of fresh blood was placed at the bottom of an EP tube and incubated in a 37°C incubator for 2 hours.

[0084] Preparation of coumarin-6-labeled DiR / BNN6 thrombus-targeting hybrid nanoassemblies: DiR and BNN6 were dissolved in methanol to prepare a drug-containing solution with a concentration of 1 mg / mL. Coumarin-6 was prepared using ethanol as a solvent to prepare a drug-containing solution with a concentration of 10 mg / mL. Then, 100 μL of the DiR and BNN6 mixed solution and 50 μL of the coumarin-6 solution were vortexed together. Under light-protected conditions, the above mixed solution was added dropwise to deionized water (2 mL) and stirred for 3 min to obtain the coumarin-6-labeled DiR / BNN6 thrombus-targeting hybrid nanoassemblies. Subsequently, DSPE-PEG was added dropwise to the prepared coumarin-6-labeled DiR / BNN6 hybrid nanoassemblies. 2K / DSPE-PEG 2K -CREKA solution, stirred for 2 min, yields coumarin-6 labeled DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassemblies. Alternatively, replacing BNN6 with BPA, using the same method, yields coumarin-6 labeled thrombus-targeting non-nitric oxide-energized hybrid nanoassemblies. All three formulations require removal of organic solvents using a rotary evaporator, and are finally diluted to 2 mL with deionized water.

[0085] Validation of thrombus permeability experiment: The three preparations were added to the prepared microthrombi and incubated at 37°C for 30 min. Subsequently, they were irradiated with an 808nm laser (2W / cm²).2 (15 min), and the emboli were washed three times with PBS. Finally, the penetration effect of the drug in the microemboli was observed using laser confocal microscopy.

[0086] Confocal microscopy results as follows Figure 10 Compared to coumarin-6 solution, the coumarin-6 labeled thrombus-targeting nitric oxide-powered hybrid nanoassemblies exhibit stronger green fluorescence, indicating that the photothermal effect and released nitric oxide generated after DiR irradiation can effectively increase drug penetration in thrombi.

[0087] Example 11: Pharmacokinetics of DiR / BNN6 Thrombus-Targeting Hybrid Nanoassemblies

[0088] Administration: Healthy male SD rats weighing 170g-220g were randomly divided into four groups (n=5), and their weights were recorded. Food intake was prohibited for 12 hours prior to administration, but water was allowed free access. DiR solution, DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies were injected, respectively, at a dose of DiR 5mg / kg.

[0089] Blood collection: Blood was collected from the orbital cavity of rats at specified time points (3 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h) and plasma was obtained by centrifugation (8000 rpm / min, 3 min).

[0090] Sample testing: DiR was extracted using the protein precipitation method for measurement. Specifically, 450 μL of methanol was added to 50 μL of the blood sample to be tested, vortexed thoroughly (1 min), and centrifuged again (8000 rpm / min, 3 min). The supernatant was collected and measured using a multi-functional microplate reader. Results are as follows: Figure 12 As shown, compared with DiR solution, the plasma drug concentration (C1) of PEGylated modified nanoassemblies is significantly higher. h The pharmacokinetic parameters such as AUC were significantly improved, resulting in a substantial enhancement of the drug's pharmacokinetic properties.

[0091] Table 3. Pharmacokinetic parameters of hybrid nanoassemblies.

[0092]

[0093] Example 12: Fluorescence imaging of carotid artery embolism vessels using DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0094] Establishment of a rat carotid artery embolism model: Healthy SD rats were first selected, randomly divided into groups, and anesthetized with 3.5% chloral hydrate. The rats were then immobilized, and the carotid artery was isolated from the neck of the SD rat. A 1cm wide filter paper strip soaked in 10% FeCl3 was used to incubate the blood vessel for 5 minutes. After incubation, the filter paper was removed, and the blood vessel and surrounding tissue were washed three times with PBS to establish the embolism model.

[0095] After successful modeling, rats were immediately injected via tail vein with the following drugs: DiR solution, DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassemblies (dose: 5 mg / kg). Subsequently, vascular fluorescence signals were recorded under in vivo imaging at preset time points (5 min, 15 min, 30 min, 60 min, and 120 min). Experimental results are as follows: Figure 13 As shown, both the DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassembly and the DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassembly exhibit strong fluorescence signals in embolized vessels. This may be attributed to the good pharmacokinetic behavior after PEG modification, and it also lays a solid foundation for realizing vascular fluorescence imaging.

[0096] Example 13: In vivo photothermal effect verification of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0097] The in vivo photothermal effect was verified using photothermal imaging: Healthy SD rats were randomly divided into groups and anesthetized (n=5). A carotid artery embolism model was established according to previous procedures. Subsequently, PBS, DiR solution, DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassemblies, and DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassemblies (dose: 5 mg / kg) were injected, respectively. One hour later, the embolized carotid artery was placed under an 808 nm laser (2 W / cm²). 2 (15 min), while simultaneously recording temperature changes in the embolized carotid artery of the rats using a photothermal imaging system. Results are as follows: Figure 14 As shown, compared with DiR solution and DiR / BNN6 nitric oxide-energized hybrid nanoassemblies, DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassemblies and DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassemblies exhibit superior heating curves and photothermal effects.

[0098] Example 14: In vivo carotid artery antithrombotic experiment of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0099] Healthy SD rats weighing 170g-220g were randomly divided into the following 8 groups: (1) normal blood vessel group; (2) embolized blood vessel group; (3) DiR solution + 808nm laser group; (4) mixed DiR solution and BNN6 solution + 808nm laser group; (5) DiR / BNN6 nitric oxide-energized hybrid nanoassembly + 808nm laser group; (6) DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassembly + 808nm laser group; (7) DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassembly + 808nm laser group; (8) lumbrokinase group.

[0100] Following the above procedures, a carotid artery thrombosis model was established. Immediately afterwards, drugs were administered via tail vein injection at a dose of DiR 5 mg / kg and lumbrokinase 8000 U / mL. One hour later, all groups in the 808 nm laser group underwent laser irradiation at the carotid artery thrombosis site (2.0 W / cm²). 2 After 15 minutes, the rats were euthanized and the embolized blood vessels were removed. H&E staining was performed to examine the embolism.

[0101] The results are as follows Figure 14-15 As shown, the DiR / BNN6 thrombus-targeting nitric oxide-energized hybrid nanoassembly + 808nm laser group exhibited excellent thrombolytic effects. Compared to the DiR / BNN6 nitric oxide-energized hybrid nanoassembly + 808nm laser group, the optimal formulation group demonstrated superior blood circulation and thrombus-targeting capabilities. Similarly, although the DiR / BPA thrombus-targeting non-nitric oxide-energized hybrid nanoassembly + 808nm laser group possessed good blood circulation and thrombus-targeting capabilities, its inability to generate nitric oxide resulted in less than satisfactory thrombolytic effects. More notably, compared to the clinically commonly used fibrinolytic drug lumbrokinase, our optimal formulation group exhibited superior thrombolytic ability, validating the excellent clinical application potential of this nitric oxide-energized thrombus-targeting therapeutic nanorobot.

[0102] Example 15: In vivo tail vein antithrombotic experiment of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0103] Establishment of a tail vein thrombosis model: Carrageenan was selected as the inflammatory drug for tail thrombosis. 1.5% carrageenan (10mg / kg) was injected intraperitoneally one night in advance, and the mice were placed in an indoor environment with a temperature of 20℃. The tail thrombosis was observed after 12 hours. KM mice weighing 18-22g were randomly divided into the following 8 groups: (1) normal blood vessel group; (2) embolized blood vessel group; (3) DiR solution + 808nm laser group; (4) DiR solution and BNN6 solution mixture + 808nm laser group; (5) DiR / BNN6 nitric oxide-energized hybrid nanoassembly + 808nm laser group; (6) DiR / BPA thrombosis-targeting non-nitric oxide-energized hybrid nanoassembly + 808nm laser group; (7) DiR / BNN6 thrombosis-targeting nitric oxide-energized hybrid nanoassembly + 808nm laser group; (8) lumbrokinase group.

[0104] According to the grouping, drugs were administered via tail vein injection on days 1, 3, 5, and 7, with the dosage of DiR 5 mg / kg and lumbrokinase 8000 U / mL. After administration, the drugs were irradiated with an 808 nm laser (2.0 W / cm²). 2 (15 min). Mice were sacrificed on day 9, and their tails were collected for H&E staining to check for embolism.

[0105] The results are as follows Figure 16 As shown, the DiR / BNN6 thrombus-targeting nitric oxide-enhanced hybrid nanoassembly combined with an 808nm laser exhibited excellent thrombolytic effects. Furthermore, this result is consistent with that of the carotid artery thrombosis model, where the optimal formulation demonstrated thrombolytic efficacy comparable to the clinically used drug lumbrokinase.

[0106] Example 16: Thrombolytic Experiment of DiR / BNN6 Thrombus-Targeting Hybrid Nanoassemblies in Ischemic Stroke

[0107] Establishment of a MCAO (chemo-angiopathic stroke) model: SD rats weighing 230g-270g were anesthetized and immobilized. First, the common carotid artery was isolated, and a suture was prepared and knotted at the proximal end. Then, a suture was prepared at the distal end, and an arterial clamp was used below the suture to prevent blood flow. At the proximal ligation site, a V-shaped incision was made with ophthalmic scissors, and the suture plug was inserted into the common carotid artery along the incision. The suture prepared at the distal end was immediately tied to prevent the suture plug from slipping out of the common carotid artery. The arterial clamp was removed, and the suture plug was inserted into the internal carotid artery. Two hours after successful insertion, the suture plug was removed, the blood vessel was ligated with fine suture, and the wound was sutured.

[0108] After establishing the MCAO model, drugs (DiR solution and DiR / BNN6 thrombus-targeting nitric oxide-enhanced hybrid nanoassemblies) were immediately injected via the tail vein and the timing was recorded. One hour later, the heads of the SD rats were exposed to an 808 laser (2.0 W / cm²) for irradiation. 2 (15 min). After 3 h, the rats were sacrificed, and their brains were removed for TTC and H&E staining to check for embolism.

[0109] The results are as follows Figure 17 As shown, the DiR / BNN6 thrombus-targeting nitric oxide-empowered hybrid nanoassemblies exhibit superior thrombolytic properties compared to DiR solutions.

[0110] Example 17: In vivo safety evaluation of DiR / BNN6 thrombus-targeting hybrid nanoassemblies

[0111] In vivo safety studies of co-assembled nanoparticles were conducted using SD rats. Rats were intravenously injected with DiR solution, a mixture of DiR and BNN6 solution, DiR / BNN6 nitric oxide-enabled hybrid nanoassemblies, DiR / BNN6 thrombus-targeting nitric oxide-enabled hybrid nanoassemblies, DiR / BPA thrombus-targeting non-nitric oxide-powered hybrid nanoassemblies, and lumbrokinase. The control group consisted of SD rats injected with PBS. One hour after administration, the rats were exposed to 2.0 W / cm². 2 Rats were subjected to 808nm laser light for 15 minutes. After 24 hours, they were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were separated for H&E staining. The results are as follows: Figure 18 As shown, no obvious histological damage was observed in the heart, liver, spleen, lungs, and kidneys, demonstrating that the DiR / BNN6 thrombus-targeting hybrid nanoassembly has good therapeutic safety.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A carrier-free, self-driving nanorobot powered by nitric oxide, characterized in that, The carrier-free self-driven nanorobot is a hybrid nanoassembly formed by the co-assembly of a photothermal photosensitizer and a nitric oxide donor molecule through non-covalent interactions. The surface of the hybrid nanoassembly is modified with a polyethylene glycol modifier and a polyethylene glycol thrombofibrin targeting peptide. The molar ratio of the nitric oxide donor molecule to the photothermal photosensitizer is 1:8 to 10:1; The photothermal photosensitizer is DiR; the nitric oxide donor molecule is N,N'-disec-butyl-N,N'-dinitroso-1,4-phenylenediamine BNN6; The polyethylene glycol modifier is one or a combination of two or more of PCL-PEG, DSPE-PEG, PLGA-PEG, and PE-PEG, wherein the molecular weight of the PEG segment is 200-20000; the polyethylene glycol thrombofibrin targeting peptide is DSPE-PEG. 2K -CREKA; The method for preparing the nitric oxide-empowered carrier-free self-driving nanorobot includes the following steps: (1) The photothermal photosensitizer and nitric oxide donor molecules were dissolved in organic solvents and mixed evenly in proportion. Under stirring conditions, the resulting mixed solution was added dropwise to water to spontaneously form a uniform hybrid nano-assembly. (2) Prepare polyethylene glycol modifier and polyethylene glycol thrombofibrin targeting peptide solution. Under stirring conditions, add polyethylene glycol modifier and polyethylene glycol thrombofibrin targeting peptide solution dropwise to the hybrid nano-assembly obtained in step (1). Remove organic solvent by rotary evaporation to obtain the final product.

2. The carrier-free self-driving nanorobot empowered by nitric oxide according to claim 1, characterized in that, The organic solvent mentioned in step (1) is one or more of methanol, ethanol, tetrahydrofuran, acetone, and dimethyl sulfoxide; the concentration of the photothermal photosensitizer in the mixed solution is 0.1~50 mg / mL.

3. The carrier-free self-driving nanorobot empowered by nitric oxide according to claim 1, characterized in that, The concentration of the hybrid nanoassembly in step (2) is 0.1~50 mg / mL; the amount of polyethylene glycol modifier and polyethylene glycol thrombofibrin targeting peptide added is 0.1~10% of the mass of the hybrid nanoassembly.

4. The application of the nitric oxide-empowered carrier-free self-driven nanorobot of claim 1 in the preparation of a drug delivery system, wherein the drug delivery system is used to prepare antithrombotic drugs and ischemic stroke drugs.

5. The application according to claim 4, characterized in that, The drug delivery systems described include oral drug delivery systems and injectable drug delivery systems.

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