A biomimetic magnetic-driven swimming nanorobot for targeting treatment of cerebral thrombosis and a preparation method thereof

Abstract

A kind of biomimetic magnetic drive swim nanorobot for targeting treatment of cerebral thrombosis and preparation method thereof belong to nanobiomedical field.The pentosan double-port bottle-shaped nanoparticles with large cavity structure are prepared by hydrothermal synthesis method, and superparamagnetic ferroferric oxide nanoparticles, thrombolytic drug and temperature-responsive fatty acid are co-loaded in the bottle-shaped cavity by vacuum perfusion technology.Under the action of alternating magnetic field, nanorobot self-organizes to form vortex cluster, and uses the local eddy current field generated by group cooperative movement to actively break through the blood laminar flow barrier around thrombus;Under near-infrared light irradiation, fatty acid undergoes solid-liquid phase transition, triggers the site-controlled release of thrombolytic drug, and realizes the efficient enrichment and deep penetration of drug in thrombus local area by the constraint effect of vortex flow field.After being navigated to the site of cerebral thrombosis by alternating magnetic field and triggered by near-infrared light, the nanorobot cluster releases high-concentration thrombolytic drug to dissolve thrombus at the lesion site, and restores cerebral blood perfusion.

Description

Technical Field

[0001] This invention relates to the fields of nanobiomedicine and nanorobotics, specifically to a method for targeted treatment of cerebral thrombosis. A biomimetic magnetically driven swimming nanorobot and its preparation method. Background Technology

[0002] Globally, approximately 13.7 million new strokes occur annually, with ischemic strokes (cerebral thrombosis) accounting for as much as 85%, characterized by high mortality and disability rates. Cerebral thrombosis disrupts local cerebral blood flow, and rapid recanalization is crucial to reducing mortality and disability. Current clinical treatments primarily involve mechanical thrombectomy and intravenous thrombolysis, but both have significant limitations, specifically: 1. Mechanical thrombectomy is difficult to safely reach distal small blood vessels with a diameter of <1 mm, resulting in a high recanalization failure rate; 2. Intravenous thrombolysis is limited to a time window of 3 to 4.5 hours. After this time, the thrombus contracts, remodels, and becomes denser, and the blood flow in the affected vessel is stagnant, forming a "laminar flow barrier" that hinders the diffusion of thrombolytic drugs into the deeper layers of the thrombus, thus significantly reducing the efficacy of thrombolysis.

[0003] Therefore, there is an urgent need to develop new, precise thrombolytic strategies that can actively overcome the laminar flow barrier of blood. Magnetically driven, mobile nanorobots can convert magnetic field energy into mechanical motion, propelling themselves efficiently in low Reynolds number fluids and actively crossing the barrier to target thrombi. While the effect of a single robot is limited, the vortex-like clusters induced by alternating magnetic fields can generate local micro-vortices, significantly enhancing drug convection mass transfer and local enrichment, overcoming the limitations of single-particle efficacy. Therefore, constructing clusters of magnetically driven nanorobots to actively overcome the laminar flow barrier provides a novel approach for the treatment of cerebral thrombosis. Summary of the Invention

[0004] This invention addresses the technical problem of low targeting efficiency and difficulty in deep penetration of existing thrombolytic drugs into the thrombus due to the laminar flow barrier in the blood. Therefore, it proposes a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis and its preparation method. This nanorobot utilizes a hydrothermal synthesis method to construct pentosan double-necked flask-shaped nanoparticles with a large cavity structure. Superparamagnetic iron oxide nanoparticles, thrombolytic drugs (urokinase uPA, alteplase tPA), and temperature-responsive fatty acids (lauric acid and stearic acid) are then co-loaded into the double-necked flask-shaped cavity using vacuum perfusion technology, forming a multifunctional nanorobot integrating magnetically controlled motion, drug loading, and near-infrared light-triggered drug release. Under the influence of an alternating magnetic field, the nanorobot can self-organize into a vortex-like cluster, generating a local vortex field through collective coordinated motion, actively breaking through the laminar flow barrier around the cerebral thrombosis lesion. Under near-infrared light irradiation, the fatty acids undergo a solid-liquid phase transition, triggering the targeted and controllable release of the thrombolytic drugs. Furthermore, the vortex flow field's confinement effect on the drug achieves efficient drug accumulation and deep penetration at the thrombus site.

[0005] The nanorobots described are uniform in size, highly magnetically responsive, and readily biodegradable. Guided by an applied alternating magnetic field, they can move against the flow of blood to the site of a cerebral thrombus, overcoming the clinical bottleneck of traditional thrombolytic drugs, which suffer from low targeting efficiency and difficulty penetrating the thrombus due to the laminar flow barrier. In vitro experiments using cerebral thrombosis models and rat middle cerebral artery embolism models demonstrate that, under the combined action of an alternating magnetic field and near-infrared light, this nanorobot cluster can significantly accelerate thrombus dissolution and restore cerebral blood flow perfusion, achieving a thrombolytic efficiency nearly ten times higher than that of free thrombolytic drugs, while simultaneously reducing systemic drug exposure and the risk of bleeding. This biomimetic, magnetically driven, mobile nanorobot offers a novel, proactive, intelligent, and controllable targeted thrombolytic strategy for ischemic stroke and has the potential for application in other thrombotic diseases and precision oncology.

[0006] The technical solution adopted by the present invention to solve the above problems is as follows: This invention provides a method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis, comprising the following steps: Step 1: Prepare pentosan double-necked bottle-shaped nanoparticles using a hydrothermal synthesis method; Step 2: Magnetic nanoparticles and thrombolytic drugs (such as urokinase uPA and alteplase tPA) are infused using a vacuum perfusion method. (etc.) and temperature-responsive fatty acids are co-loaded into the cavity of double-necked bottle-shaped nanoparticles to form a magnetically driven drug-carrying mobile nanorobot; Step 3: Disperse the magnetically driven drug-loaded nanorobots obtained in Step 2 in physiological saline or phosphate buffer. It is placed in an alternating magnetic field system for magnetic response motion control.

[0007] Furthermore, in step 1, the synthesis of the pentosan double-necked bottle-shaped nanoparticles includes the following steps: (1) Add 0.0360 g of sodium oleate (SO) and 0.0567 g of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer (P123) to a clean beaker, add 20 mL of deionized water, and stir at 300 rpm for 1 h on a magnetic stirrer to completely dissolve and form a clean and transparent solution. (2) Place 3 g of dextrorotatory ribose in another clean beaker, add 40 mL of deionized water, and stir at 500 rpm for 1 h on a magnetic stirrer to completely dissolve it and form a transparent solution; (3) Add the former solution dropwise to the latter solution at a rate of 1 ml / min, and stir at 500 rpm for 30 min on a magnetic stirrer; (4) Transfer the above mixed solution to a 100 mL reaction vessel, place it in a forced-air drying oven and react at 160 °C for 5 h, cool it to room temperature, and then heat it to 160 °C for 10 h to obtain a double-necked bottle-shaped nanoparticle dispersion. (5) The obtained double-necked bottle-shaped nanoparticle dispersion was washed with water 5 times and ethanol 5 times, dried under vacuum at 60 °C and stored at room temperature for later use.

[0008] By following the above synthesis steps and controlling the temperature and time of the hydrothermal reaction to be stable, pentosan double-necked bottle-shaped nanoparticles with large cavity structure and uniform particle size can be obtained. The cavity structure is beneficial for the efficient loading of subsequent magnetic nanoparticles and drugs.

[0009] Furthermore, the double-necked bottle-shaped nanoparticles have a large cavity structure, which is used to load magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids.

[0010] Furthermore, in step 2, loading magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids using the vacuum perfusion method includes the following steps: Step 2.1 Mix 100 mg lauric acid, 25 mg stearic acid, 5 mg Fe3O4 nanoparticles (particle size 20 nm) and 10 mg stearic acid. mg of thrombolytic drug was dissolved in 1 mL of dimethyl sulfoxide; Step 2.2 Add 2 mg of the double-necked flask-shaped nanoparticles obtained in Step 1 under vigorous stirring, and continue stirring until... The components are thoroughly mixed to obtain a mixed solution; Step 2.3 Place the mixed solution from Step 2.2 in a vacuum chamber and maintain it at a vacuum pressure of 1 bar for 15 minutes, so as to... Completely remove the air from the cavity; Step 2.4 Collect the product from the degassed mixed solution by centrifugation and wash it twice with dimethyl sulfoxide; Step 2.5 Add 1 mL of deionized water to the precipitate obtained from the last centrifugation to promote the solidification of fatty acids; Step 2.6 involves freeze-drying the solidified product to obtain the magnetically driven drug-loaded mobile nanomachine. Robot.

[0011] By following the above vacuum perfusion steps and controlling the vacuum pressure and processing time, magnetic nanoparticles, thrombolytic drugs, and fatty acids can be fully introduced into the double-necked bottle-shaped cavity, achieving integrated efficient drug encapsulation and magnetic response function.

[0012] Furthermore, the Fe3O4 nanoparticles, as a magnetic component, enable the nanorobot to respond to alternating magnetic fields; the thrombolytic drug, as a thrombolytic agent, is used to target and dissolve fibrin in thrombi; the lauric acid and stearic acid, as temperature-responsive fatty acids, undergo a solid-liquid phase transition under near-infrared light irradiation, triggering drug release.

[0013] Furthermore, in step 3, the magnetic response motion control of the magnetically driven drug-carrying nanorobot is achieved through the following methods: Step 3.1 Construct an alternating magnetic field drive system based on Helmholtz coils. This system generates a uniform magnetic field that rotates perpendicular to the XY plane. The magnetic field strength is adjustable in the range of 0-15 mT, and the frequency adjustment range is 0-100 Hz. Step 3.2 Place the magnetically driven drug-carrying nanorobots dispersed in the solution at the center of a Helmholtz coil, and observe and record the motion behavior of the nanorobots in real time using an inverted microscope and a high-resolution CCD camera. Step 3.3 By adjusting the frequency and intensity of the magnetic field, the movement speed of the individual nanorobot is precisely controlled, enabling it to move along a preset trajectory in static and flowing fluid environments; Step 3.4 Under the action of an alternating magnetic field, the nanorobots self-organize to form a vortex-like cluster. The size, shape and speed of the cluster are controlled by adjusting the magnetic field parameters.

[0014] By following the above magnetic control operation steps, the magnetically driven drug-carrying nanorobots can achieve precise navigation and cluster motion control in complex fluid environments, enabling them to target and migrate against the direction of blood flow to the site of cerebral thrombosis.

[0015] Furthermore, when the magnetic field strength of the alternating magnetic field is 10 mT and the frequency is 30 Hz, the nanorobots form a stable vortex-like cluster. The rotation of the cluster induces a ring-shaped vortex in the surrounding fluid, which is used to break through the laminar flow barrier of blood around the thrombus.

[0016] Furthermore, when the magnetically driven drug-carrying nanorobot is used for targeted treatment of cerebral thrombosis, it achieves controlled drug release and thrombolytic therapy through the following methods: 1. Driven by an alternating magnetic field, the nanorobots move in clusters to the site of cerebral thrombosis and use the local eddy current field generated by the coordinated movement of the group to break through the laminar flow barrier of the blood around the thrombus. 2. The lesion area was irradiated with near-infrared light of 808 nm wavelength at a power density of 0.2-0.6 W / cm² for 10 minutes, which triggered a solid-liquid phase transition of temperature-responsive fatty acids in the double-necked bottle-shaped cavity. 3. After the fatty acid phase transition, the thrombolytic drug is rapidly released from the double-necked bottle cavity, and with the help of the vortex flow field's confinement effect on the drug, the drug achieves efficient enrichment and deep penetration at the thrombus site. 4. The released thrombolytic drugs activate plasminogen to convert into plasmin, which degrades fibrin in the thrombus, thus achieving targeted thrombolytic therapy.

[0017] By following the above procedures, under the combined control of alternating magnetic fields and near-infrared light, precise targeting of nanorobots, targeted and controllable release of drugs, and a significant improvement in thrombolytic efficiency can be achieved.

[0018] Furthermore, the pentosan double-necked bottle-shaped nanorobots are metabolized and eliminated from the body within 96 hours after exerting their thrombolytic effect.

[0019] This invention provides a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis. The biomimetic magnetically driven mobile nanorobot is prepared according to the above-described method. The biomimetic magnetically driven mobile nanorobot can be used to prepare targeted thrombolytic drugs for the treatment of ischemic stroke, or to prepare targeted thrombolytic drugs for the treatment of myocardial infarction, pulmonary embolism and other thrombotic diseases.

[0020] The beneficial effects of this invention are: This invention constructs a magnetically driven drug-loaded mobile nanorobot with a large-cavity double-necked bottle-like structure using hydrothermal synthesis and vacuum perfusion technology. While maintaining the good biodegradability of pentosan materials, it achieves efficient co-loading of magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids. It features controllable structure, well-defined preparation process, adjustable magnetic response sensitivity, and adjustable drug release behavior, making it suitable for targeted therapy of cerebral thrombosis and related ischemic cardiovascular diseases.

[0021] This invention integrates superparamagnetic iron oxide nanoparticles with an alternating magnetic field driving system into the same system, enabling precise navigation of individual and cluster movements of nanorobots in a low Reynolds number cerebrovascular fluid environment. This solves the bottleneck problem of traditional nanomedicine delivery systems being unable to actively target in complex blood flow environments.

[0022] The magnetically driven drug-carrying nanorobots of this invention can self-organize into vortex-like clusters under the action of an alternating magnetic field. The local vortex flow field generated by their rotation can actively break through the laminar flow barrier of blood around the thrombus, significantly enhancing the convective transport and deep penetration of drugs into the thrombus. This overcomes the core defects of conventional thrombolytic drugs, such as low targeting efficiency (delivery rate <5%) and difficulty in drug accumulation in the lesion due to blood flow scouring and laminar flow barrier.

[0023] This invention further introduces a near-infrared photothermal response drug release mechanism, utilizing the solid-liquid phase transition behavior of fatty acids within the bottle-shaped cavity to achieve pulsed, targeted release of thrombolytic drugs. Combined with the confinement effect of a vortex flow field on drug molecules, this allows for sustained high-concentration accumulation of the drug at the thrombus site, significantly improving thrombolytic efficiency. Experimental results show that in a rat middle cerebral artery embolism model, the thrombolytic efficiency of the nanorobot system of this invention is nearly ten times higher than that of free thrombolytic drugs, while significantly reducing systemic drug exposure and the risk of bleeding. Furthermore, the nanorobots of this invention have a well-defined metabolic cycle in vivo, can be effectively cleared within 96 hours, exhibit no significant toxicity or immunogenicity, and demonstrate good biocompatibility.

[0024] The biomimetic magnetically driven swimming nanorobot of this invention can provide a novel, proactive, intelligent, and controllable targeted thrombolysis strategy for ischemic stroke, and has the potential to be extended to other thrombotic diseases such as myocardial infarction and pulmonary embolism, as well as precision treatment of tumors. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the fabrication process of a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to the present invention. Figure 2 The images show scanning electron microscope (SEM) images (left) and transmission electron microscope (TEM) images (right) of the double-necked bottle-shaped pentosan nanoparticles of this invention. Figure 3 The images show scanning electron microscope (SEM) images (left) and transmission electron microscope (TEM) images (right) of the biomimetic magnetically driven swimming nanorobot of this invention. Figure 4 The images show the motion delay (a) and velocity-frequency phase diagrams (b) of the biomimetic magnetically driven swimming nanorobot of this invention under different magnetic field strengths. Figure 5 This is a diagram illustrating the self-organization of a vortex-like cluster of biomimetic magnetically driven swimming nanorobots under an alternating magnetic field, as described in this invention. Figure 6 This is a drug release curve of the biomimetic magnetically driven swimming nanorobot of the present invention under near-infrared light; Figure 7 This is a diagram illustrating how the biomimetic magnetically driven swimming nanorobot of this invention overcomes the blood laminar flow barrier. Figure 8This invention presents a biomimetic magnetically driven mobile nanorobot for targeted thrombolysis to restore blood flow in blocked branches of a Y-shaped channel. Figure 9 Ultrasound images showing the targeted thrombolysis and restoration of blood supply to the rat brain by the biomimetic magnetically driven swimming nanorobot of this invention; Figure 10 Laser speckle images showing the targeted thrombolysis and restoration of blood supply to the rat brain by the biomimetic magnetically driven swimming nanorobot of this invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, parameter adjustments, or changes in functional components made based on the concept of the present invention should be covered within the scope of protection of the present invention.

[0027] Example 1: like Figure 1 As shown in the figure, this embodiment provides a method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis. The specific process is as follows: (1) Add 0.0360 g of sodium oleate and 0.0567 g of P123 to a clean beaker, and add 20 mL of deionization solution. Deionized water was stirred at 300 rpm for 1 h on a magnetic stirrer until completely dissolved to form a clean, transparent solution. Simultaneously, 3 g of dextrorotatory ribose was placed in another clean beaker, and 40 mL of deionized water was added. The mixture was stirred at 500 rpm for 1 h on a magnetic stirrer until completely dissolved to form a transparent solution. The former solution was then added dropwise to the latter solution at a rate of 1 mL / min, and stirred at 500 rpm for 30 min on a magnetic stirrer. The resulting mixture was transferred to a 100 mL reaction vessel and placed in a forced-air drying oven at 160 °C for 5 h. After cooling to room temperature, the temperature was raised to 160 °C and reacted for another 10 h to obtain a double-necked flask-shaped pentosan nanoparticle dispersion. The obtained dispersion was washed five times with water and five times with ethanol, dried under vacuum at 60 °C, and stored at room temperature for later use. (2) Mix 100 mg lauric acid, 25 mg stearic acid, 5 mg Fe3O4 nanoparticles (particle size 20 nm) and 10 mg Thrombolytic drugs were dissolved in 1 mL of dimethyl sulfoxide, and 2 mg of the double-necked flask-shaped nanoparticles obtained in step (1) were added under vigorous stirring. The mixture was placed in a vacuum chamber and maintained at a vacuum pressure of 1 bar for 15 min to fully remove air from the cavity. The product was collected by centrifugation and washed twice with dimethyl sulfoxide. 1 mL of deionized water was added to the precipitate obtained from the last centrifugation to promote fatty acid solidification. The obtained magnetically driven drug-loaded double-necked flask-shaped mobile nanorobots were freeze-dried overnight. (3) For fluorescently labeled thrombolytic drugs (such as FITC-uPA), 10 mg of FITC-uPA is combined with fatty acids. Dissolved in dimethyl sulfoxide, the subsequent steps are the same as in step (2). The resulting product needs to be washed with deionized water at least six times to completely remove the FITC-uPA molecules adsorbed on the outer surface. (4) The obtained magnetically driven drug-loaded double-necked bottle-shaped swimming nanorobots were dispersed in physiological saline or phosphate buffer. It is placed in an alternating magnetic field system for magnetic response motion control.

[0028] Figure 2 The images show scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the prepared double-necked bottle-shaped pentosan nanoparticles. As can be seen from the images, the nanoparticles exhibit a double-necked bottle-like morphology with a large cavity structure, smooth surface, and uniform particle size. Figure 3 The images show scanning electron microscope (SEM) and transmission electron microscope (TEM) images of a magnetically driven drug-loaded double-necked bottle-shaped mobile nanorobot loaded with Fe3O4 nanoparticles and thrombolytic drugs. It can be seen that the Fe3O4 nanoparticles are mainly distributed inside the bottle-shaped cavity, and the thrombolytic drugs are encapsulated in the cavity through a fatty acid matrix. This indicates that the vacuum perfusion method has successfully achieved efficient co-loading of magnetic components and thrombolytic drugs, and the bottle-shaped double-necked structure is conducive to the rapid release of drugs under triggering conditions.

[0029] Example 2: This embodiment is used to evaluate the individual motion performance of a magnetically driven, drug-loaded, two-necked bottle-shaped mobile nanorobot under an alternating magnetic field. The results are as follows: Figure 4 As shown, the specific steps are as follows: (1) Construct an alternating magnetic field drive system based on Helmholtz coils, which can generate a rotation perpendicular to the XY plane. A uniform magnetic field is generated, with the magnetic field strength adjustable from 0 to 15 mT and the frequency adjustable from 0 to 100 Hz. The system consists of an inverted microscope, a pair of Helmholtz coils, a signal generator, a power amplifier, a sample observation platform, and a system... It consists of a control platform and a cooling system; (2) The magnetically driven drug-loaded double-necked floating nanorobot sample dispersed in the solution was placed in a Helmholtz coil. On the central slide, real-time observation is performed using an inverted microscope (with selectable magnification of 20×, 40×, and 100×), and an image acquisition rate of 30 frames per second is achieved using a high-resolution CCD camera. (3) By adjusting the frequency and intensity of the alternating magnetic field, the motion speed of the nanorobot under different magnetic field parameters was recorded. Degrees, and plot the velocity-frequency phase diagram; (5) The motion trajectory of the magnetically driven drug-carrying double-necked bottle-shaped swimming nanorobot was quantitatively analyzed using ImageJ software. Analysis and characterization.

[0030] like Figure 4 As shown, the motion speed of the magnetically driven drug-carrying, two-necked bottle-shaped mobile nanorobot initially increases and then decreases with increasing magnetic field frequency, exhibiting an optimal frequency range. Within this optimal frequency range, the nanorobot can achieve high-speed directional motion, reaching speeds on the order of several micrometers per second. This result demonstrates that by adjusting the frequency and intensity of the alternating magnetic field, precise control of the individual nanorobot's motion speed can be achieved, providing a basis for its active navigation to thrombus sites within the bloodstream environment.

[0031] Example 3: This embodiment was used to verify the ability of magnetically driven drug-loaded, two-necked bottle-shaped swimming nanorobots to self-organize into vortex-like clusters under an alternating magnetic field. The results are as follows: Figure 5 As shown, the specific steps are as follows: (1) The magnetically driven drug-loaded double-necked bottle-shaped swimming nanorobots were uniformly dispersed in phosphate buffer or simulated blood flow. The body is placed in an alternating magnetic field environment (magnetic field strength 10 mT, frequency 30 Hz); (2) An inverted microscope was used in conjunction with a high-resolution CCD camera, and high-magnification observation was performed using a 100x objective lens. The self-assembly process of nanorobots under the action of an alternating magnetic field was captured in real time. (3) Use ImageJ software to process the acquired image sequences and analyze the morphological evolution and rotation speed of the cluster. and the distribution of vortex flow field; (4) A Rankine combined vortex model was established using the COMSOL Multiphysics simulation platform. The flow field-diffusion coupling model is used to simulate the flow field structure generated by the cluster under the action of an alternating magnetic field and its disturbance effect on the surrounding fluid environment.

[0032] like Figure 5As shown, under the influence of an alternating magnetic field, individual, dispersed, magnetically driven, drug-carrying, double-necked bottle-shaped nanorobots first rotate along the magnetic field direction. Subsequently, adjacent nanorobots gradually aggregate under the interaction of magnetic dipoles, self-organizing into a micrometer-scale vortex-like cluster structure. The clusters are arranged in a circular or elliptical shape, with all nanorobots rotating collaboratively around the cluster center, forming a stable vortex flow field. Fluid field simulation results show that the cluster rotation induces annular vortices in the surrounding fluid, with the flow velocity reaching its maximum at the cluster boundary. The vortex flow field can extend to a range several times the cluster diameter. This vortex flow field can effectively disturb the surrounding fluid environment, providing a physical basis for overcoming the laminar flow barrier of blood.

[0033] Example 4: This embodiment was used to investigate the drug release behavior of a magnetically driven, drug-loaded, two-necked, bottle-shaped mobile nanorobot under near-infrared light irradiation. The results are as follows: Figure 6 As shown, the specific steps are as follows: (1) The magnetically driven drug-loaded double-necked bottle-shaped mobile nanorobots were dispersed in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL; (2) A near-infrared laser with a wavelength of 808 nm was used as the light source, with power densities of 0.2, 0.4 and 0.6 W / cm², respectively, to irradiate the nanorobot suspension; (3) Samples were taken at different time points (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 min) before and after irradiation, and the supernatant was separated by centrifugation; (4) The thrombolytic drugs released in the supernatant were quantitatively analyzed by ultraviolet-visible absorption spectroscopy, and the cumulative release rate of drugs at different time points was calculated.

[0034] like Figure 6 As shown, before near-infrared light irradiation, the magnetically driven drug-loaded double-necked flask-shaped mobile nanorobot exhibits almost no drug leakage, demonstrating excellent drug retention capabilities. After near-infrared light irradiation, the internal temperature of the nanorobot rapidly increases. When the temperature-responsive fatty acids lauric acid and stearic acid reach their phase transition temperatures, a solid-liquid phase transition occurs, and the thrombolytic drug encapsulated within the cavity is rapidly released. Drug release exhibits an explosive release characteristic in the initial stage of irradiation, with a cumulative release rate reaching approximately 70%. Subsequently, the release rate gradually slows down, reaching a release plateau after approximately 20 minutes. These results indicate that the magnetically driven drug-loaded double-necked flask-shaped mobile nanorobot of this invention possesses excellent near-infrared light-responsive drug release performance, enabling targeted, on-demand, and rapid drug release, which is beneficial for creating a high-concentration drug environment at the thrombus site.

[0035] Example 5: This embodiment was used to verify the ability of a magnetically driven, drug-loaded, two-necked, bottle-shaped swimming nanorobot cluster to overcome the laminar flow barrier in an in vitro Y-channel thrombosis model. The results are as follows: Figure 7 As shown, the specific steps are as follows: (1) A Y-shaped microfluidic channel with one inlet and two outlet branches was constructed. The channel was 6.2 mm wide, 2 cm long, and 20 μm high, and fixed on a glass substrate. A mixture of thrombin solution (50 U / mL) and fresh rat whole blood (volume ratio 1:200) was injected into one of the branches and allowed to stand at 37°C for 90 min to form a stable thrombus blockage area. The other branch remained open to simulate the blood flow redistribution state after unilateral thrombus blockage at the bifurcation of cerebral blood vessels. (2) Simulated blood (physiological saline with added glycerol to adjust the viscosity to 4 cP, and red fluorescent microparticles as tracer particles) was injected from the inlet end at a flow rate of 10 cm / s using an injection pump, so that the fluid preferentially enters the unblocked branch, while the blocked branch is in a low flow rate or near-no flow rate state. (3) Inject a magnetically driven drug-carrying double-necked bottle-shaped swimming nanorobot with green fluorescence (1 mg / mL, 200 μL) into the channel inlet at a rate of 15 μL / min, and apply an alternating magnetic field (magnetic field strength 10 mT, frequency 30 Hz) to drive the nanorobot cluster movement. (4) Bright field images were acquired in real time at a rate of 30 frames per second using an inverted microscope and a high-resolution CCD camera to observe the movement path and spatial distribution of the nanorobot cluster at the bifurcation of the Y-shaped channel.

[0036] (5) The motion trajectory of fluorescent particles in the fluid was tracked using ImageJ software, and the fluid field was analyzed using particle image velocimetry (PIV) technology. The changes in the flow field distribution before and after the addition of the nanorobot cluster were compared. like Figure 7 As shown, without an alternating magnetic field, the nanorobots mainly follow the fluid into the unobstructed branch, with very few distributed within the thrombus-occluded branch. After applying the alternating magnetic field, the nanorobots self-organize into vortex-like clusters. Under the influence of the local vortices generated by the cluster's rotation, the original laminar flow state at the entrance of the occluded branch is significantly disturbed, and the vortex flow field causes some fluid to re-enter the thrombus-occluded branch. Time-lapse images show that the nanorobot clusters can deviate from the mainstream direction and actively migrate towards the thrombus-occluded branch. PIV flow field analysis indicates that the vortex clusters induce secondary flow at the entrance of the thrombus branch, effectively enhancing the convective transport of fluid from the mainstream channel to the occluded branch, and forming local microvortices on the thrombus surface. These results demonstrate that the magnetically driven drug-carrying double-necked bottle-shaped swimming nanorobot clusters of this invention can actively overcome the laminar flow barrier through the vortex flow field, redirecting fluid and drugs to the thrombus-occluded region, overcoming the problem of low targeted delivery efficiency caused by blood flow diversion in traditional drug delivery systems.

[0037] Example 6: This embodiment was used to evaluate the targeted thrombolytic effect of a magnetically driven drug-loaded, two-necked, floating nanorobot cluster in an in vitro Y-channel thrombosis model. The results are as follows: Figure 8 As shown, the specific steps are as follows: (1) A Y-shaped microfluidic channel unilateral thrombosis model was constructed according to the method in Example 5. After thrombosis, artificial cerebrospinal fluid was perfused into the channel at a flow rate of 20 μL / min for 10 min to remove unbound cells and debris. (2) An alternating magnetic field (magnetic field strength 10 mT, frequency 30 Hz) was applied in the experimental setup, and an 808 nm near-infrared laser was aimed at the branch blocked by the thrombus with a power density of 0.4 W / cm². (3) The magnetically driven drug-loaded double-necked bottle-shaped swimming nanorobot (1 mg / mL, 200 μL) was injected into the channel entrance at a rate of 15 μL / min. The nanorobots formed a vortex cluster under the drive of the alternating magnetic field and migrated directionally to the thrombus-occluded branch. (4) After the nanorobot cluster reaches the thrombus site, near-infrared laser irradiation is activated for 5 minutes to trigger drug release; (5) Acquire bright field and fluorescence channel images, and use ImageJ software to track the motion trajectory of fluorescent particles in the fluid to analyze blood flow.

[0038] like Figure 8 As shown, before the nanorobot cluster reaches the thrombus site, the thrombus maintains its structural integrity and its area remains largely unchanged within the Y-shaped channel branch. Subsequently, the magnetically driven drug-loaded, double-necked, floating nanorobot cluster of this invention can precisely target the thrombus blockage site under the guidance of an alternating magnetic field. After near-infrared light triggers drug release, the thrombolytic drug is rapidly released locally at the thrombus site and exerts a highly efficient thrombolytic effect, effectively clearing the thrombus and restoring channel patency.

[0039] Example 7: This embodiment was used to evaluate the recovery of cerebral blood flow after targeted thrombolytic therapy in a rat model of middle cerebral artery embolism using a magnetically driven, drug-loaded, two-necked bottle-shaped mobile nanorobot cluster. Ultrasound imaging was used for monitoring, and the results are as follows: Figure 9 As shown, the specific steps are as follows: (1) Six-week-old healthy male SD rats weighing 180-220 g were selected. The middle cerebral artery embolism model was established by photochemical embolization: the photosensitizer rose red (30 mg / kg) was injected into the tail vein according to the body weight. Five minutes after injection, the right cerebral cortex sensorimotor area of ​​the rat was irradiated with a 532nm laser light source (light power density of 10 mW / mm², spot diameter of 2 mm) for 10 minutes to induce local vascular endothelial damage and thrombus formation, resulting in focal cerebral ischemia. (2) After the model was established, anesthesia was maintained by inhaled isoflurane gas (induction concentration 4%, maintenance concentration 1.5%-2%). The rats were placed in an alternating magnetic field environment (magnetic field strength 10 mT, frequency 30 Hz). A magnetically driven drug-loaded double-necked bottle-shaped swimming nanorobot suspension (1 mg / mL, 200 μL) was injected via the tail vein. The nanorobots formed a vortex cluster under the drive of the alternating magnetic field and migrated directionally to the site of cerebral thrombosis. (3) Thirty minutes after drug administration, the fenestrated area of ​​the skull was irradiated with 808 nm near-infrared laser (power density 0.4 W / cm², irradiation time 5 min) to trigger drug release; (4) A high-frequency ultrasound microscopy system (Vevo®3100, FUJIFILM VisualSonics) equipped with a 40 MHz linear array probe was used to measure the blood flow velocity and vascular resistance index of the main trunk of the middle cerebral artery through the skull before modeling, after modeling and after treatment, to assess the recovery of cerebral blood flow after treatment.

[0040] like Figure 9 As shown, after the establishment of the middle cerebral artery embolism model, the blood flow velocity in the main trunk of the middle cerebral artery significantly decreased, while the vascular resistance index significantly increased. Following treatment with a magnetically driven drug-loaded, two-necked, floating nanorobot cluster combined with alternating magnetic field and near-infrared light, the blood flow velocity in the middle cerebral artery began to gradually recover within 1 hour, the vascular resistance index decreased accordingly, and the blood flow velocity recovered to over 80% of its normal level. The blood flow spectrum morphology also tended towards normal, indicating that the occluded vessel had been recanalized. These results demonstrate that the magnetically driven drug-loaded, two-necked, floating nanorobot cluster of this invention can effectively navigate to the thrombus site in the complex vascular network of the living brain. Through the synergistic effect of vortex clustering overcoming the hemodynamic barrier and near-infrared light triggering targeted drug release, it achieves targeted thrombolysis and vascular recanalization, promoting the recovery of cerebral blood flow perfusion.

[0041] Example 8: This embodiment was used to evaluate the recovery of cerebral blood flow perfusion after targeted thrombolytic therapy in a rat model of middle cerebral artery embolism using a magnetically driven, drug-loaded, two-necked, floating nanorobot cluster. Laser speckle contrast imaging was used for monitoring, and the results are as follows: Figure 10 As shown, the specific steps are as follows: (1) A photochemically induced rat middle cerebral artery embolism model was established according to the method in Example 7. Anesthesia was induced by a 5% isoflurane-oxygen mixture, and then anesthesia was maintained by 1.5%-2% isoflurane through a nasal mask. A heating pad was used to maintain the rat's body temperature at 37.0 ± 0.5°C. (2) After the model was established, the rats were placed in an alternating magnetic field (magnetic field strength 10 mT, frequency 30 Hz) environment and injected with a magnetically driven drug-loaded double-necked bottle-shaped swimming nanorobot suspension (1 mg / mL, 200 μL) via the tail vein. The nanorobots migrated directionally to the cerebral thrombosis site under the drive of the alternating magnetic field. (3) Thirty minutes after administration, the fenestrated area of ​​the skull was irradiated with 808 nm near-infrared laser (power density 1 W / cm², irradiation time 5 min) to trigger drug release; (4) Using a laser speckle contrast imaging system, the blood perfusion of the rat cerebral cortex was monitored in real time before modeling, after modeling and after treatment, and blood perfusion images of the ischemic core area and penumbra were collected.

[0042] (5) The relative blood flow value of the thrombus-related region was calculated using laser speckle imaging analysis software and compared with the blood flow perfusion of the contralateral normal brain region.

[0043] like Figure 10 As shown in the laser speckle contrast imaging monitoring results, after the establishment of the middle cerebral artery embolism model, the ischemic core area of ​​the right cerebral cortex of rats exhibited a significant hypoperfusion state, with blood flow signals decreasing by approximately 80% compared to the contralateral normal brain region. Blood flow signals in the ischemic penumbra region were also significantly reduced. After treatment with a magnetically driven drug-loaded two-necked bottle-shaped mobile nanorobot cluster combined with alternating magnetic field and near-infrared light, blood flow perfusion in the ischemic penumbra region began to recover within 1 hour. This was manifested by a gradual increase in blood flow signals in the laser speckle image, a gradual reduction in the hypoperfusion area, a significant increase in blood flow signals at the edge of the ischemic core area, and blood flow perfusion in the penumbra region recovering to over 60% of near-normal levels. The area of ​​the ischemic core area significantly decreased, and overall cortical blood flow perfusion significantly improved, with a more uniform distribution of blood flow signals. These results further demonstrate that the magnetically driven drug-loaded two-necked bottle-shaped mobile nanorobot cluster of this invention can achieve precise targeted thrombolysis in a live cerebral thrombosis model, effectively restoring cerebral tissue blood flow perfusion and exhibiting good potential for targeted thrombolytic therapy.

[0044] All the raw materials listed in this invention, as well as the upper and lower limits and ranges of the raw materials and the upper and lower limits and ranges of the process parameters (such as temperature, time, etc.), can realize this invention. Examples are not listed one by one here.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent substitutions, and improvements made to the above embodiments without departing from the scope of the present invention, based on the technical essence of the present invention and within the spirit and principles of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis, characterized in that, The method includes the following steps: Step 1: Prepare pentosan double-necked bottle-shaped nanoparticles using a hydrothermal synthesis method; Step 2: Magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids are co-loaded into the cavity of double-necked bottle-shaped nanoparticles using a vacuum infusion method to form a magnetically driven drug-carrying mobile nanorobot. Step 3: Disperse the magnetically driven drug-loaded nanorobots obtained in Step 2 in physiological saline or phosphate buffer. It is placed in an alternating magnetic field system for magnetic response motion control.

2. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 1, characterized in that, In step 1, the synthesis of the pentosan double-necked flask-shaped nanoparticles includes: Step 1.1 Add 0.0360 g sodium oleate and 0.0567 g P123 to 20 mL of deionized water and stir at 300 rpm for 1 h on a magnetic stirrer until they are completely dissolved to form a clear and transparent solution, which is denoted as solution A. Step 1.2 Add 3 g of dextrorotatory ribose to 40 mL of deionized water and stir at 500 rpm for 1 h to form a transparent solution, which is denoted as solution B; Step 1.3 Add solution A dropwise to solution B at a rate of 1 mL / min, and continue stirring at 500 rpm for 30 min to ensure thorough mixing of all components and obtain a mixed solution; Step 1.4: Transfer the above mixed solution to a 100 mL hydrothermal reactor and place it in a forced-air drying oven at 160°C. The reaction was carried out at C for 5 h, and after naturally cooling to room temperature, it was heated again to 160°C and the reaction was continued for 10 h to obtain a double-necked bottle-shaped pentosan nanoparticle dispersion. Step 1.5: Collect the nanoparticles from the obtained double-necked bottle-shaped nanoparticle dispersion by centrifugation, and then use them sequentially. The product was washed five times with deionized water and five times with ethanol to remove impurities. The washed product was then dried overnight in a vacuum drying oven at 60°C to obtain pentosan nanoparticles in the shape of a double-necked bottle. The dried nanoparticles were stored at room temperature for later use.

3. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 2, characterized in that, The double-necked bottle-shaped nanoparticles have a large cavity structure, which is used to load magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids.

4. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 3, characterized in that, In step 2, loading magnetic nanoparticles, thrombolytic drugs, and temperature-responsive fatty acids via the vacuum perfusion method includes: Step 2.1 Mix 100 mg lauric acid, 25 mg stearic acid, 5 mg Fe3O4 nanoparticles (particle size 20 nm) and 10 mg... The thrombolytic drug was dissolved in 1 mL of dimethyl sulfoxide; Step 2.2 Add 2 mg of the double-necked flask-shaped nanoparticles obtained in Step 1 under vigorous stirring, and continue stirring until the particles are fully incorporated. The components are thoroughly mixed to obtain a mixed solution; Step 2.3 Place the mixed solution from Step 2.2 in a vacuum chamber and maintain it at a vacuum pressure of 1 bar for 15 minutes to allow it to fill. Expel the air from the cavity; Step 2.4 Collect the product from the degassed mixed solution by centrifugation and wash it twice with dimethyl sulfoxide; Step 2.5 Add 1 mL of deionized water to the precipitate obtained from the last centrifugation to promote the solidification of fatty acids; Step 2.6 involves freeze-drying the solidified product to obtain the magnetically driven drug-loaded mobile nanomachine. Robot.

5. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 4, characterized in that, The Fe3O4 nanoparticles serve as a magnetic component, enabling the nanorobots to respond to alternating magnetic fields; the thrombolytic drug is used to target and dissolve fibrin in thrombi; the lauric acid and stearic acid, as temperature-responsive fatty acids, undergo a solid-liquid phase transition under near-infrared light irradiation, triggering drug release.

6. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 5, characterized in that, In step 3, the magnetic response motion control of the magnetically driven drug-carrying nanorobot is achieved through the following methods: Step 3.1 Construct an alternating magnetic field drive system based on Helmholtz coils. This system generates a uniform magnetic field that rotates perpendicular to the XY plane. The magnetic field strength is adjustable in the range of 0-15 mT, and the frequency adjustment range is 0-100 Hz. Step 3.2 Place the magnetically driven drug-carrying nanorobots dispersed in the solution at the center of a Helmholtz coil, and observe and record the motion behavior of the nanorobots in real time using an inverted microscope and a high-resolution CCD camera. Step 3.3 By adjusting the frequency and intensity of the magnetic field, the movement speed of the individual nanorobot is precisely controlled, enabling it to move along a preset trajectory in static and flowing fluid environments; Step 3.4 Under the action of an alternating magnetic field, the nanorobots self-organize to form a vortex-like cluster. The size, shape and speed of the cluster are controlled by adjusting the magnetic field parameters.

7. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 6, characterized in that, When the magnetic field strength of the alternating magnetic field is 10 mT and the frequency is 30 Hz, the nanorobots form a stable vortex-like cluster. The rotation of the cluster induces a ring-shaped vortex in the surrounding fluid, which is used to break through the laminar flow barrier of blood around the thrombus.

8. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 7, characterized in that, When the magnetically driven drug-carrying nanorobot is used for targeted treatment of cerebral thrombosis, it achieves controlled drug release and thrombolytic therapy through the following methods: S1. Driven by an alternating magnetic field, the nanorobots move in clusters to the site of cerebral thrombosis and use the local eddy current field generated by the group's coordinated motion to break through the blood laminar flow barrier around the thrombus. S2. The lesion area was irradiated with near-infrared light of 808 nm wavelength at a power density of 0.2-0.6 W / cm² for 10 min, which triggered a solid-liquid phase transition of temperature-responsive fatty acids in the double-necked bottle-shaped cavity. S3. After the fatty acid phase transition, the thrombolytic drug is rapidly released from the double-necked bottle-shaped cavity, and with the help of the vortex flow field's confinement effect on the drug, the drug achieves efficient enrichment and deep penetration at the thrombus site. S4. The released thrombolytic drugs activate plasminogen to convert into plasmin, which degrades fibrin in the thrombus, thus achieving targeted thrombolytic therapy.

9. The method for preparing a biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis according to claim 8, characterized in that, The pentosan double-necked bottle-shaped nanorobots are metabolized and eliminated from the body within 96 hours after exerting their thrombolytic effect.

10. A biomimetic magnetically driven mobile nanorobot for targeted treatment of cerebral thrombosis, characterized in that, The biomimetic magnetically driven mobile nanorobot prepared according to any one of claims 1 to 9 is used to prepare targeted thrombolytic drugs for the treatment of ischemic stroke, or to prepare targeted thrombolytic drugs for the treatment of myocardial infarction, pulmonary embolism and other thrombotic diseases.

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