A nanometer particle with magneto-acoustic response for microglial cell membrane camouflage and a preparation method and application thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a kind of microglial cell membrane camouflage acoustic magnetic response nanoparticle preparation and its application in treating ischemic stroke (cerebral infarction). Nanoparticle structure takes perfluorohexane as core, loads the PLGA shell containing Fe3O4 and anti-RGMa, and is coated with microglial cell membrane on the surface of shell. The present application provides a new type of stroke integrated vascular recanalization bridging brain protection targeted therapy scheme, after ischemic stroke, the nanoparticle is injected into vein under the action of microglial cell membrane and targets to thrombus;Exogenous administration of LIFU and magnetic field treatment, PFH releases anti-RGMa and Fe3O4 by phase change, and the phase change process and the rotation and forward effect of exogenous magnetic field on Fe3O4 jointly destroy thrombus and recanalize blood vessels;Finally, the released anti-RGMa plays a brain protection role. Nanoparticle is also a dual-mode enhanced contrast agent, and has broad and important application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of nanomedicine delivery system technology, and more specifically to an acoustomagnetic responsive nanoparticle disguised as a microglia cell membrane, its preparation method, and its application. Background Technology

[0002] Ischemic stroke is a serious disease that severely endangers human life and health, characterized by five major features: high incidence, high mortality, high disability rate, high recurrence rate, and high economic burden. The key to stroke treatment lies in destroying the thrombus as early as possible to achieve vascular recanalization, restore blood flow, and salvage damaged brain tissue. Currently, authoritative guidelines recommend the following methods for vascular recanalization: intravenous injection of tissue-type plasminogen activator (tPA) thrombolysis and endovascular thrombectomy. However, tPA thrombolysis has a low vascular recanalization rate, and endovascular thrombectomy carries a high risk of complications such as vascular injury and inguinal hematoma. Furthermore, after vascular recanalization in ischemic stroke, a series of pathophysiological reactions induced by cerebral ischemia / reperfusion, such as blood-brain barrier disruption, inflammation, and reactive astrocyte proliferation, can lead to ischemia-reperfusion injury in brain tissue, severely affecting the recovery of neurological function. Recent studies have shown that administering neuroprotective drugs after revascularization therapy can intervene in the biochemical or molecular biological processes following cerebral ischemia-reperfusion, effectively reducing cerebral ischemia-reperfusion injury. However, most current research focuses on providing neuroprotective therapy alone after revascularization, rather than integrating the two treatments. Therefore, there is an urgent need to improve current revascularization methods and design a new integrated strategy for treating ischemic stroke that combines revascularization and neuroprotective therapy.

[0003] In recent years, with the rise of nanomedicine technology, some preclinical studies have developed nanotherapeutic systems based on nanotechnology, utilizing ultrasound, magnetic fields, and other techniques to assisted in destroying arterial thrombi in various parts of the body, including the cerebral arteries, coronary arteries, and abdominal aorta, thereby achieving vascular recanalization. The use of nanomedicine technology to treat thrombosis is a hot topic and frontier in thrombotic disease research. Some studies use ultrasound-assisted thrombolysis, employing ultrasound to induce cavitation in nanoparticles based on tPA drug thrombolysis, thus assisting tPA in destroying thrombi. However, the thermal energy from ultrasound may cause serious secondary damage to brain tissue. Low-intensity focused ultrasound (LIFU) is an ultrasound technique that reduces ultrasound-induced energy damage and can precisely focus ultrasound on the target site. In recent years, studies have constructed nanoparticles loaded with perfluorohexane (PFH). LIFU irradiation caused PFH to undergo a liquid-gas phase transition, destroying abdominal aortic thrombi. However, there is currently no literature on LIFU-based destruction of cerebral vascular thrombosis. This is related to the unique structure of the brain, which is unsuitable for prolonged LIFU irradiation, as prolonged LIFU irradiation can cause vasogenic and cytotoxic cerebral edema. Recent studies have found that nanoparticles containing magnetic materials can be targeted and rotated under the manipulation of a magnetic field. Some studies have used magnetic nanoparticles containing magnetic materials to assist in the dissolution of peripheral thrombi. Under the influence of an external rotating magnetic field, the magnetic nanoparticles can target and rotate, helping thrombolytic drugs to reach the thrombus site. Simultaneously, the movement of the magnetic particles themselves can also exert a mechanical effect on thrombus destruction. Helmholtz coils in magnetic drives can generate uniform magnetic fields in different directions, manipulating the movement of the magnetically responsive material Fe3O4. It should be noted that because magnetic fields alone have low thrombus destruction efficiency and poor effects, they are not suitable as a standalone thrombus destruction method. Currently, there is also no literature on magnetic materials destroying cerebral vascular thrombosis. In summary, there are currently no safe and effective nanotechnology methods for cerebral vascular thrombosis, and current thrombus destruction techniques are limited.

[0004] Furthermore, regardless of the type of drug, non-specific delivery, limited biodistribution, and rapid clearance by the body necessitate high-dose administration to achieve therapeutic effects. Moreover, non-specific delivery can lead to drug accumulation in non-lesion sites, potentially causing toxic side effects. Therefore, precision medicine, which provides precise dosages to target areas, has been proposed in recent years, aiming to enhance drug targeting and increase drug concentration at the lesion site. The rise of nanomedicine has brought the possibility of targeted drug delivery; however, the capture and elimination of nanoparticles by the immune system remains a significant obstacle. How to improve the ability of nanoparticles to evade immune elimination and prolong their circulation time is also a problem that the nanomedicine delivery system proposed in this scheme needs to solve.

[0005] In summary, current technologies lack solutions for treating ischemic stroke using nanotechnology, the techniques for destroying thrombi are limited, and there is a lack of integrated treatment methods that address vascular recanalization, brain protection, and prevention of side effects. Furthermore, nanoparticles are easily eliminated by the immune system and lack targeting capabilities. Summary of the Invention

[0006] This invention aims to provide a microglial cell membrane-masked acoustomagnetic responsive nanoparticle to address the technical problem of the lack of nanomedicine delivery systems targeting ischemic stroke in existing technologies. Furthermore, existing nanomedicine delivery systems suffer from limitations such as limited therapeutic approaches, lack of neuroprotective efficacy, susceptibility to immune elimination, and poor targeting. To address these issues, this invention proposes a microglial cell membrane-masked acoustomagnetic responsive nanoparticle loaded with anti-RGMa (anti-RGMa monoclonal antibody). In this invention, the inventors effectively validated, using rat carotid artery embolism and mouse middle cerebral artery embolism models, that intravenous injection of this nanoparticle after ischemic stroke (cerebral infarction) resulted in targeted aggregation at the thrombus site, disrupting the thrombus and restoring blood flow. Subsequently, the anti-RGMa released by the nanoparticle exerted a neuroprotective effect, significantly reducing neurological deficits and infarct volume after ischemic stroke.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A microglia-mass-responsive nanoparticle masquerading as a microglia cell membrane comprises a perfluorohexane core and a polylactic acid-glycolic acid copolymer shell; Fe3O4 particles are encapsulated within the shell; and a microglia cell membrane is also coated on the outer side of the shell.

[0009] Furthermore, the outer shell also encapsulates anti-RGMa.

[0010] Furthermore, the ratio of polylactic acid-glycolic acid, Fe3O4 reagent, perfluorohexane, and anti-RGMa was 25 mg: 100 μl: 200 μl: 25 mg; the concentration of oleic acid-modified Fe3O4 in the Fe3O4 reagent was 25 mg / mL.

[0011] This solution also provides a method for preparing acoustomagnetic responsive nanoparticles disguised as microglial cell membranes, comprising the following steps performed sequentially:

[0012] S1: Polylactic acid-glycolic acid, Fe3O4 and dichloromethane are mixed, then perfluorohexane is added and ultrasonic treatment is performed to form a primary emulsion; then polyvinyl alcohol solution is added and ultrasonic treatment is performed; then isopropanol solution is added and the mixture is allowed to stand and washed to obtain nanoparticles that are not encapsulated in cell membranes.

[0013] S2: Microglial cell membranes are mixed with polymers (i.e., nanoparticles without cell membranes), then sonicated and washed to obtain acoustomagnetic responsive nanoparticles disguised as microglial cell membranes.

[0014] Furthermore, in S1, anti-RGMa is added along with perfluorohexane.

[0015] Furthermore, in S1, the ratio of polylactic acid-glycolic acid, Fe3O4 reagent, dichloromethane, perfluorohexane, polyvinyl alcohol solution, and isopropanol solution is 25 mg: 100 μl: 2 mL: 200 μL: 4 mL: 5 mL;

[0016] The mass percentage of solute in the polyvinyl alcohol solution was 2%, and the volume percentage of solute in the isopropanol solution was 2%; the concentration of oleic acid-modified Fe3O4 in the Fe3O4 reagent was 25 mg / mL.

[0017] Furthermore, in S1, the power of the first ultrasonic treatment is 52W and the time is 6 minutes; the power of the second ultrasonic treatment is 40W and the time is 6 minutes.

[0018] In S2, microglia are mixed with polymers (i.e., nanoparticles without cell membranes) and then sonicated at 40W for 1 minute; the mass ratio of microglia membrane proteins to polymers (i.e., nanoparticles without cell membranes) is 1:1.

[0019] This solution also provides the application of microglial cell membrane-masked acoustomagnetic responsive nanoparticles in the preparation of systems for treating ischemic stroke with vascular recanalization, brain protection, and preventing post-stroke pneumonia.

[0020] Furthermore, the system includes microglial cell membrane-masked acoustomagnetic responsive nanoparticles for intravenous administration to the site of cerebral artery embolism, a low-intensity focused ultrasound device for applying ultrasound to the nanoparticles, and a Helmholtz coil system for applying a magnetic field to the nanoparticles.

[0021] The parameters of the low-intensity focused ultrasound device are set to 3W / cm. 2 Irradiate for 3 minutes;

[0022] The parameters of the Helmholtz coil system were set to 30mT, 4Hz, and processed for 15 minutes.

[0023] This solution also provides the application of microglial cell membrane-masked acoustomagnetic responsive nanoparticles in the preparation of ultrasound or photoacoustic imaging contrast agents.

[0024] In summary, the principle of this technical solution is as follows:

[0025] This technical solution prepares a multifunctional intelligent nanoparticle for treating ischemic stroke (cerebral infarction), namely MiCM@PLGA / anti-RGMa / Fe3O4@PFH. The nanoparticle's structure consists of an ultrasound-responsive phase-change material PFH as its core, loaded with a polylactic-glycolic acid (PLGA) shell containing the magnetically responsive material Fe3O4 and the neuroprotective drug anti-RGMa. The shell surface is coated with a microglial cell membrane. After cerebral infarction, the nanoparticle is intravenously injected. Under the influence of the microglial cell membrane, the nanoparticle targets the damaged vascular endothelial cells at the thrombus site. Subsequently, an exogenous magnetic field composed of a LIFU (Liquidity-Induced Fusion) and a Helmholtz coil is applied. The LIFU causes a phase transition in PFH, releasing anti-RGMa and Fe3O4 from the PLGA shell. This phase transition process, along with the rotational and propulsive effects of the exogenous magnetic field on Fe3O4, jointly destroys the thrombus, thereby reopening the blood vessel. The released anti-RGMa exerts a neuroprotective effect on the damaged brain tissue, thus achieving targeted vascular recanalization bridging neuroprotective therapy, reducing the infarct volume, and promoting neurological function recovery. Furthermore, the phase transition process of PFH and Fe3O4 enable imaging in multiple modes, including CEUS and photoacoustic imaging, which is helpful in diagnosing thrombosis in superficial sites such as the carotid artery. This approach can be applied to target thrombi in cerebral infarction sites, achieving integrated vascular recanalization bridging and neuroprotective therapy, and has broad application prospects.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0027] (1) A new targeted therapy strategy for ischemic stroke integrating vascular recanalization and brain protection was designed.

[0028] Combining revascularization therapy with neuroprotective therapy represents the forefront of ischemic stroke treatment. A growing body of preclinical studies has shown that administering neuroprotective drugs after revascularization therapy can intervene in the biochemical or molecular biological processes following cerebral ischemia-reperfusion, effectively reducing cerebral ischemia-reperfusion injury. However, current clinical practice and most preclinical studies involve separate neuroprotective therapy following revascularization, rather than integrating the two treatments. This innovative approach organically integrates nanotechnology, magnetotherapy, ultrasound, and neurology, designing acousto-magnetic responsive microglia-inspired nanoparticles (MiCM@PLGA / anti-RGMa / Fe3O4@PFH) loaded with anti-RGMa. These nanoparticles, after targeting the thrombus, disrupt the thrombus under the influence of exogenous LIFU and a magnetic field, achieving revascularization and releasing the neuroprotective drug anti-RGMa to promote the repair of damaged brain tissue, thus achieving integrated revascularization and neuroprotective therapy.

[0029] (2) Innovatively utilize LIFU and magnetic field to destroy thrombi and achieve vascular recanalization.

[0030] Currently, authoritative guidelines recommend intravenous thrombolysis (tPA) and endovascular thrombectomy for vascular recanalization. However, the recanalization rate of tPA thrombolysis is less than 40%, therefore, the likelihood of neurological function recovery in patients treated with tPA is low. While endovascular thrombectomy has a high recanalization rate, it carries a high risk of complications such as vascular injury. Therefore, there is an urgent need to improve methods for vascular recanalization.

[0031] This project designs a method of vascular recanalization after ischemic stroke, applying MiCM@PLGA / anti-RGMa / Fe3O4@PFH followed by an exogenous application of a magnetic field composed of LIFU and a Helmholtz coil. During this process, after MiCM@PLGA / anti-RGMa / Fe3O4@PFH targets the thrombus, a phase transition occurs under the action of the exogenous LIFU, releasing Fe3O4. This phase transition, along with the rotational and propagating effects of the exogenous magnetic field on Fe3O4, jointly destroys the thrombus, thereby restoring blood flow. This recanalization method designed in this project avoids the vascular injury complications caused by endovascular interventional procedures.

[0032] (3) For the first time, anti-RGMa antibody (anti-RGMa) was encapsulated in nanoparticles for targeted drug delivery, avoiding the potential toxicity of anti-RGMa to non-target sites.

[0033] Repulsive guidance molecule α (RGMa) is a member of the repulsive guidance molecule family. RGMa plays a crucial role in the central nervous system, participating in numerous physiological and pathological processes, including repulsive guidance, cell migration, adhesion, differentiation, and axonal regeneration inhibition.

[0034] Previous research by the applicant and their project team revealed that RGMa exerts various negative effects on the ischemic sites of the brain after ischemic stroke, making it a key target for neuroprotective therapy after stroke. However, the applicant's previous research also found that RGMa exerts a positive protective effect on lung tissue after stroke, inhibiting the occurrence of post-stroke pneumonia. Exogenous administration of recombinant RGMa protein to mice after MCAO significantly reduced lung injury and significantly decreased bacterial load in lung tissue. Therefore, delivery of anti-RGMa via traditional systemic administration (peripheral intravenous injection, oral administration, etc.) may induce post-stroke pneumonia. Furthermore, traditional systemic delivery of anti-RGMa has limitations such as low drug concentration in the target area (the area of ​​cerebral ischemia-reperfusion injury) and the inability to maintain sustained drug concentration in the lesion area. Targeted therapy systems using nanomaterials to encapsulate drugs have advantages such as safety, targeting specificity, and stability. However, currently, there is no targeted drug delivery system loaded with anti-RGMa. The MiCM@PLGA / anti-RGMa / Fe3O4@PFH designed in this project will release anti-RGMa under the action of LIFU, which not only achieves targeted release of anti-RGMa, but also avoids the potential pulmonary toxicity of anti-RGMa.

[0035] (4) Innovatively using microglial cell membranes as a targeting material to target the thrombus.

[0036] In nanoparticle-targeted therapy, the capture and clearance of nanoparticles by the immune system has been a major obstacle to achieving targeted delivery for many years. Targeted delivery is crucial for vascular recanalization and neuroprotection after ischemic stroke. Cell membrane coating is a promising biomimetic targeting technology that combines a cell membrane with nanoparticles to form a "shell-core" structure. The cell membrane disguises the nanoparticles to evade or reduce immune elimination, allowing them to accumulate at the disease site and prolong circulation time. Currently, the most commonly used biomembrane for coating nanoparticles containing neuroprotective drugs in the literature is the macrophage membrane. As immune cells, macrophages can be rapidly activated and targeted to ischemic areas after stroke. The targeting ability of macrophages mainly relies on their cell membranes. Therefore, utilizing this characteristic of macrophage membranes, synthesized nanoparticles coated with macrophage membranes can target ischemic areas after stroke. The inventors attempted to use macrophage membranes to enhance the ability of their nanoparticles to treat stroke, but the results were not ideal.

[0037] Microglia are one of the main immune cells in the central nervous system, accounting for approximately 5% of the cells in the adult central nervous system. Under physiological conditions, microglia have small cell bodies with long, highly branched processes, continuously monitoring their surroundings. Comparative studies revealed that macrophages have a weaker ability to migrate to the thrombus site after ischemia than microglia. Therefore, we hypothesize that nanoparticles coated with microglia membranes have superior targeting ability compared to nanoparticles coated with macrophage membranes. Microglia membranes are excellent targeting materials for thrombus regions in stroke. However, there is currently no literature utilizing microglia membrane-coated nanoparticles for targeting ischemic areas. Therefore, this approach is the first to utilize microglia membrane (MiCM) as a targeting biomimetic material to coat nanoparticles, enabling the nanoparticles to be safely and precisely targeted to the cerebral thrombus region.

[0038] (5) The nanoparticle is also a dual-modal contrast agent with imaging potential, which is helpful in the diagnosis of thrombosis in superficial extracranial sites.

[0039] The MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles of this invention contain PFH and Fe3O4 particles, wherein the phase transition of PFH can significantly enhance the visualization of ultrasound images in b-mode and contrast-enhanced ultrasound (CEUS), and Fe3O4 can serve as a photoacoustic imaging contrast agent. These imaging modalities are helpful in the diagnosis of thrombosis, especially when the thrombus is located in an extracranial artery (such as the extracranial segment of the carotid artery).

[0040] (6) This scheme selects polylactic acid-glycolic acid copolymer (PLGA) as a unique drug delivery polymer. As one of the most widely used biodegradable materials, PLGA has good biocompatibility and can be used in practical applications to select the ratio of specific monomers according to the properties of the transported molecules.

[0041] (7) In addition to drug loading, we also need to consider the drug release problem when administering drugs to nanoparticle systems. As a form of energy conversion, ultrasound and LIFU can trigger the release of drugs to the lesion site, which is a controllable and fast-response drug release method. Attached Figure Description

[0042] Figure 1 This is a transmission electron microscope (TEM) image of the MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles from Example 2.

[0043] Figure 2 The results show the particle size and potential of the MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles in Example 2.

[0044] Figure 3The image shows a fluorescence micrograph of the nanoparticles from Example 2 (demonstrating that the microglia have successfully bound to the nanoparticles).

[0045] Figure 4 This is a comparison image of the appearance of Fe3O4-loaded nanoparticles and unloaded nanoparticles in Example 2.

[0046] Figure 5 This is an SDS-PAGE image of the nanoparticles from Example 2.

[0047] Figure 6 This is a WB image of the cell membrane surface proteins of the nanoparticles in Example 2.

[0048] Figure 7 The hysteresis curve of MiCM@PLGA / anti-RGMa / Fe3O4@PFH in Example 3 is shown.

[0049] Figure 8 This is a magnetic adsorption effect test of the thrombosis model in Example 3 (Fe3O4 moves in the direction driven by the magnetic field (white arrow)).

[0050] Figure 9 HE-stained cross-sectional images of thrombolysis in the in vitro thrombosis model of Example 3 (before and after Fe3O4 destroys the thrombus under magnetic drive).

[0051] Figure 10 The results of the in vitro thermally induced phase transition ultrasound imaging experiment of the nanoparticles in Example 4 are shown.

[0052] Figure 11 The results of the in vitro ultrasound-induced phase transition ultrasound imaging experiment of the nanoparticles in Example 4 are shown.

[0053] Figure 12 This is an in vitro comparative experiment (immunofluorescence) of the targeting effects on microglia and macrophage membranes in Example 5.

[0054] Figure 13 This is an in vivo comparative experiment (fluorescence imaging) of the targeting effects on microglia and macrophage membranes in Example 5.

[0055] Figure 14 The results are full-wavelength scans of the nanoparticles from Example 6.

[0056] Figure 15 The results show the photoacoustic signal intensity detection results for nanoparticles of different concentrations in Example 6.

[0057] Figure 16 The results are from B-mode and CEUS (contrast-enhanced imaging) experiments at the thrombus site in the rat carotid artery embolism model of Example 7.

[0058] Figure 17The results are from PA-mode (photoacoustic mode) and CEUS (contrast enhancement mode) imaging experiments at the thrombus site in the rat carotid artery embolism model of Example 7.

[0059] Figure 18 The results of the experiment on the recanalization of rat carotid artery embolism by the nanoparticles in Example 8 are shown.

[0060] Figure 19 The results of the experiment on vascular damage after recanalization of rat carotid artery embolism by nanoparticles in Example 9 are shown.

[0061] Figure 20 The results show that the nanoparticles of Example 10 exert a neuroprotective effect by inhibiting astrocyte activation.

[0062] Figure 21 The results of the experiment on the effect of nanoparticles in Example 11 on the recovery of neurological function after eMCAO in mice (*P<0.05,**P<0.01,***P<0.001,#P<0.0001, no significant difference in ns; n=3).

[0063] Figure 22 The results of the experiment on the effect of nanoparticles of Example 11 on the infarct volume after eMCAO in mice are as follows (**P<0.01, ***P<0.001, no significant difference in ns; n=3).

[0064] Figure 23 A schematic diagram showing the structure and mechanism of action of the nanoparticles MiCM@PLGA / anti-RGMa / Fe3O4@PFH prepared in this scheme.

[0065] Figure 24 The results of lung tissue staining in mice with the MCAO model of mice in Comparative Example 1 after injection of recombinant RGMa protein.

[0066] Figure 25 The results of bacterial load in lung tissue of mice in the MCAO mouse model after injection of recombinant RGMa protein are presented as comparative example 1. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0068] Example 1: Preparation of Nanoparticles

[0069] MiCM@PLGA / Fe3O4@PFH:

[0070] (1) Microglial cell membrane extraction: BV-2 microglia treated with lipopolysaccharide were collected. Cells were scraped off using a cell scraper, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in PBS buffer. The collected BV-2 cells were placed at -80℃ for 30 min and then thawed at 37℃. This process was repeated three times. The liquid containing the broken cells was then homogenized using a homogenizer. The collected liquid was centrifuged at 700g for 5 min at 4℃, and the supernatant was collected. The precipitate obtained after centrifugation at 15000g for 30 min was the microglial cell membrane (MiCM).

[0071] (2) Preparation of MiCM@PLGA / Fe3O4@PFH nanoparticles

[0072] Nanoparticles were prepared using a modified W / O / W method. 25 mg of PLGA and oleic acid-modified Fe3O4 (25 mg / mL, 100 μL, Ocean NanoTech, SOR10-02, USA) were completely dissolved in 2 mL of dichloromethane. Then, 200 μL of PFH was added. A primary emulsion was formed by ultrasonic vibration at 52 W for 6 min (cycled with 5 s on and 5 s off) using a Sonics & Materials (USA). Subsequently, 4 mL of 2% PVA solution was added to the above solution, and emulsification was performed using an ultrasonic probe at 40 W for 6 min (5 s on, 5 s off). All processes were performed in an ice bath. Then, isopropanol solution (2%, 5 mL) was added to stabilize the system, and after shaking to mix, the mixture was left to stand overnight to allow the dichloromethane to evaporate and the nanoparticle surface to solidify. The next day, the solution containing nanoparticles was centrifuged at 7,500 rpm for 5 minutes, and the supernatant was discarded. The remaining precipitate was washed with deionized water to remove any unformed particles. This centrifugation and washing process was repeated three times. Finally, the prepared nanoparticles were stored at 4°C for later use. The above steps yielded a suspension containing PLGA / Fe3O4@PFH.

[0073] Cell membranes were added to a PLGA / Fe3O4@PFH suspension (concentration 1 mg / ml). The amount of cell membrane used was based on membrane protein, and the weight ratio of PLGA / Fe3O4@PFH to membrane protein was controlled at 1:1. The mass of membrane protein in the cell membrane was determined using a BCA kit after lyophilization. The mixture was then sonicated in an ice-water bath for 1 minute (40 W energy, 5 s on, 5 s off). The resulting nanoparticles were rinsed three times with double-distilled water to remove unbound material and stored at 4°C for later use. Microglial cell membrane-coated nanoparticles MiCM@PLGA / Fe3O4@PFH were prepared according to the above method.

[0074] In addition, this technical solution also prepares MiCM@PLGA / @PFH, following the same process as described above, except that the step of adding oleic acid-modified Fe3O4 is omitted.

[0075] This technical solution also prepares PLGA / Fe3O4@PFH, following the above process, omitting the subsequent process of adding the cell membrane of microglia.

[0076] This technical solution also prepares MiCM@PLGA / anti-RGMa / Fe3O4@PFH, following the process described above, with the addition of anti-RGMa. The specific process is as follows:

[0077] PLGA (25 mg) and Fe3O4 (25 mg / mL, 100 μL) were completely dissolved in dichloromethane (2 mL). Then, PFH (200 μL) and anti-RGMa (25 mg, HA500148 Huaan Biotechnology) were added and the mixture was shaken. A brown emulsion was generated by ultrasonic vibration at 52 W for 6 minutes (5 s on, 5 s off). PVA solution (2%, 4 mL) was added to the emulsion, and emulsification was performed using an ultrasonic probe at 40 W for 6 minutes (5 s on, 5 s off). All processes were carried out in an ice bath. Isopropanol solution (2%, 5 mL) was added to the emulsion to stabilize the system. After shaking and mixing, the mixture was left overnight to allow the dichloromethane to evaporate and the nanoparticles to solidify. The next day, the solution containing nanoparticles was centrifuged at 7500 rpm for 5 minutes, and the supernatant was discarded. The remaining precipitate was washed with deionized water to remove any unspherical material. The centrifugation and washing steps were repeated three times. The final precipitate was PLGA / anti-RGMa / Fe3O4@PFH, which was stored at 4℃ for later use.

[0078] Microglial cell membranes were added to a PLGA / anti-RGMa / Fe3O4@PFH suspension. The weight ratio of the polymer (PLGA / anti-RGMa / Fe3O4@PFH) to the membrane protein was 1:1. The mixture was sonicated in an ice-water bath for 1 minute (40W energy, 5s on, 5s off), and MiCM@PLGA / anti-RGMa / Fe3O4@PFH was collected. PLGA / anti-RGMa / Fe3O4@PFH consists of anti-RGMa nanoparticles that are not coated with microglial cell membranes.

[0079] Example 2: Characterization of Nanoparticles

[0080] See electron microscopy images of MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles. Figure 1 For the test results of particle size and zeta potential, please refer to [link to relevant documentation]. Figure 2The particle size of PLGA / anti-RGMa / Fe3O4@PFH without microglia membrane coating was 194.767±7.385 nm; the particle size of MiCM@PLGA / anti-RGMa / Fe3O4@PFH coated with microglia membrane was significantly increased to 236.967±8.756 nm. The Zeta potentials of the microglia membrane (MiCM), PLGA / anti-RGMa / Fe3O4@PFH, and MiCM@PLGA / anti-RGMa / Fe3O4@PFH NPs were -30.03±0.289 mV, -5.93±0.826 mV, and -18.27±0.503 mV, respectively. This shows that coating with microglia membrane significantly altered the average potential and average particle size of the nanoparticles. MiCM and PLGA / Fe3O4@PFH were labeled with red fluorescence (DiI) and green fluorescence (DiO), respectively. After synthesis, the two components showed near-identical overlap under an inverted fluorescence microscope. Figure 3 The successful coating of microglia with nanoparticles is indicated.

[0081] Nanoparticles loaded with Fe3O4 (MiCM@PLGA / Fe3O4@PFH) and nanoparticles without Fe3O4 coating (MiCM@PLGA / @PFH) can be observed to change color from milky white to brown to the naked eye. Figure 4 ).

[0082] Next, the transfer of microglial cell membrane proteins was further investigated, and protein validation was performed. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was conducted to examine the total protein composition of the nanoparticles. The results showed that no protein was expressed on PLGA / Fe3O4@PFH (red), while the proteins in MiCM@PLGA / Fe3O4@PFH (yellow) and MiCM (blue) were identical, indicating that microglial cell membrane proteins had been successfully integrated into PLGA / Fe3O4@PFH. Figure 5 Further analysis of characteristic proteins of the microglial cell membrane revealed that proteins Tmem119 and Cx3cr1 were expressed in MiCM@PLGA / Fe3O4@PFH. These proteins are associated with microglial chemotaxis and adhesion functions, i.e., the ability of microglia to target thrombus sites after ischemic stroke, indicating that the microglial cell membrane has been successfully integrated into PLGA / Fe3O4@PFH. Figure 6 Furthermore, microglia possess the ability to target thrombus sites.

[0083] Example 3: Verification of superparamagnetism of nanoparticles and the thrombus-destructive ability of Fe3O4 under magnetic modulation

[0084] The applicant tested the hysteresis curves of MiCM@PLGA / anti-RGMa / Fe3O4@PFH. Figure 7 This indicates that these nanoparticles are superparamagnetic. Then, to explore the magnetic field-controlled motion of Fe3O4, we constructed an electromagnetic drive system consisting of two pairs of Helmholtz coils, forming X and Y axes, and tested the effect of magnetic fields in different directions on the motion of Fe3O4. This demonstrated that Fe3O4 moves in the direction driven by the magnetic field (white arrow). Figure 8 Next, the ability of magnetically controlled Fe3O4 movement to destroy thrombi was tested in vitro. Thrombi were placed in a flow chamber made of transparent PE tubes, and PBS was circulated using a peristaltic pump at a rate of 3 mL / min. Subsequently, Fe3O4 was injected into the PBS from the bottom of the tube using a syringe needle, exposing the thrombus to a rotating magnetic field for 60 minutes (30 mT, 4 Hz). The thrombus destruction effect was then assessed using hematoxylin-eosin staining. After Fe3O4 destroyed the thrombus, small channels were observed within the thrombus. Figure 9 The white arrow indicates that under the control of a magnetic field, Fe3O4 can drill holes in thrombi, thereby promoting thrombus destruction.

[0085] Example 4: In vitro ultrasound imaging experiment using MiCM@PLGA / anti-RGMa / Fe3O4@PFH

[0086] Given the potential applications of MiCM@PLGA / anti-RGMa / Fe3O4@PFH as a contrast agent, in vitro and in vivo imaging validation was conducted. The PFH core of the material possesses the ability to undergo a phase transition from a liquid state to a transformed state, with various phase transition conditions including temperature and ultrasound.

[0087] First, the temperature phase transition was tested: MiCM@PLGA / anti-RGMa / Fe3O4@PFH was diluted to 1 mg / mL, and 1 mL of nanoparticles was added to a 2 mL EP tube. The EP tube was placed in a constant temperature water bath at 20℃, 40℃, 45℃, 50℃, and 55℃. Then, 100 μL of the treated nanoparticles was transferred to a prepared 3% gel. B-mode and CEUS ultrasound imaging was performed on the liquids at different temperatures using a VevoLAZR imaging system, and the echo intensity was measured using DFY software. The results showed that at water bath temperatures of 20℃, 40℃, and 45℃, ultrasound imaging showed low echoes. As the temperature increased, more bubble echoes were visible in the gel pores, and the bubble echoes significantly increased at 55℃. DFY quantitative analysis software showed that the emulsion echo intensity was highest in the water sac at 55℃. However, 50℃ is far beyond the optimal temperature for live animals. Figure 10 Therefore, temperature-induced phase transitions must be discarded.

[0088] After the failure of temperature-induced phase transition, ultrasound-induced phase transition was explored: MiCM@PLGA / anti-RGMa / Fe3O4@PFH was diluted to 1 mg / mL, and 1 mL of nanoparticles was added to a 2 mL EP tube. A LIFU (Low Intensity Focused Ultrasound) probe (developed by the State Key Laboratory of Ultrasound Medical Engineering, Chongqing Medical University; based on this device, several papers and patents have been published, making it an existing technology) probe was filled with coupling agent. The EP tube was placed on the coupling agent. The LIFU instrument had a focal length of 1.5 cm, pulse mode, and a selection from 1 to 4 W / cm. 2 The nanoparticles were irradiated for 1, 2, 3, and 4 minutes, respectively. 100 μL of the treated nanoparticles were transferred to a prepared 3% gel, and imaging was performed using Vevo LAZR in B-mode and CEUS. The echo intensity of the region of interest (ROI) in the gel pores was quantitatively analyzed using DFY echo intensity analysis software. The results showed that at 1 W / cm²... 2 and 2W / cm 2 The echo intensity gradually increases, and 2W / cm 2 The echo intensity is consistently higher than 1 W / cm 2 At 3W / cm 2 The strongest echo intensity was observed in B-mode and CEUS imaging modes after 3 minutes of irradiation, at 4 W / cm². 2 The echo intensity gradually decreases over time, suggesting that 3W / cm 2 Irradiation for 3 minutes is the optimal ultrasonic phase transition intensity. Previous literature has confirmed that LIFU 3W / cm² is the optimal intensity. 2 Irradiation for 3 minutes is safe for the human body; therefore, LIFU 3W / cm 2 Irradiation for 3 minutes is a suitable phase transition condition for MiCM@PLGA / anti-RGMa / Fe3O4@PFH. Figure 11 ).against Figure 11 The specific data for B is shown in the table below:

[0089] Table 1: Results of in vitro ultrasound-induced phase transition ultrasound imaging of nanoparticles (for B-mode, mean ± SD, n = 3)

[0090]

[0091]

[0092] Example 5: Detection of microglial cell membrane targeting and its comparison with macrophage membrane

[0093] This embodiment investigated the targeting validation of microglia-modified nanoparticles and compared their effects with those of macrophage-modified nanoparticles. The preparation method of macrophage-coated nanoparticles (MaCM@PLGA / anti-RGMa / Fe3O4@PFH) is the same as in Example 1, except that the microglia membrane was replaced with a macrophage membrane.

[0094] In vitro validation of nanoparticle targeting: DiO-labeled nanoparticles (0.25 mg / mL, 50 μL) were added to each group, and after incubation for 3, 6, and 9 hours, the cells were fixed and observed under a fluorescence microscope to monitor the uptake of different nanoparticles by bEnd.3 cells under different conditions. After DAPI staining of the nucleus, the cell nucleus (DAPI) appeared blue under an inverted fluorescence microscope, while the nanoparticles (DiO) appeared green. At the same time interval, in the MiCM@PLGA / anti-RGMa / Fe3O4@PFH (MiCM-NPs) targeting group, more green fluorescence representing the targeted nanoparticles was observed to accumulate around bEnd.3 cells. In the PLGA / anti-RGMa / Fe3O4@PFH (uncoated-NPs) non-targeting group and the MaCM@PLGA / anti-RGMa / Fe3O4@PFH (MaCM-NPs) macrophage targeting group, less green fluorescence accumulation was observed around the cells, and the fluorescence intensity of both groups increased with the extension of incubation time. Figure 12 ).

[0095] In vivo validation of nanoparticle targeting: An embolic middle cerebral artery occlusion (eMCAO) model was established in mice. Each group was injected via tail vein with DIR-labeled nanoparticles (10 mg / kg). Two hours later, brain tissue was dissected for in vitro fluorescence analysis. In vitro fluorescence showed high fluorescence signals in the lesion areas of the targeted group compared to the non-targeted and macrophage-targeted groups. Quantitative analysis demonstrated that the targeted group exhibited better lesion targeting. Figure 13 ).

[0096] Example 6: In vitro photoacoustic imaging experiment

[0097] 100 μL of MiCM@PLGA / anti-RGMa / Fe3O4@PFH was placed inside a gel model, and photoacoustic imaging was used to scan the entire wavelength range of 680-900 nm. The excitation peak was measured to be approximately 700-710 nm. Figure 14 ), and 700nm was selected as the excitation wavelength for subsequent experiments.

[0098] 100 μL of MiCM@PLGA / anti-RGMa / Fe3O4@PFH was added to the gel model, and the concentrations of Fe3O4 were divided into 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0.0625 mg / mL. H2O and PLGA@PFH were used as control groups. Ultrasonic and photoacoustic imaging of the different liquids in B-mode and PA-mode was performed using a Vevo LAZR imager. Quantitative analysis of B-mode was performed using DFY echo intensity analysis software, and quantitative analysis of PA-mode was performed using a Vevo LAZR imager. The results showed that under 700 nm excitation, the photoacoustic signal of Fe3O4 in MiCM@PLGA / anti-RGMa / Fe3O4@PFH in the gel block increased with increasing Fe3O4 concentration, and the intensity showed a certain linear relationship with the concentration. Figure 15 ).

[0099] Example 7: In vivo ultrasound imaging and photoacoustic imaging experiments

[0100] First, a rat model of left carotid artery embolism was established: rats were anesthetized by intraperitoneal injection (tribromoethanol: 200 mg / mL). After anesthesia, the rats were fixed to the operating table in a supine position, and the neck was prepared and disinfected. The entire procedure was performed on a 37°C warming blanket. The skin was incised along the midline of the neck to expose the neck. Sealing tape was passed under the left common carotid artery to isolate adjacent tissues and other blood vessels. The distal end of the left common carotid artery was clamped with an arterial clamp. Filter paper soaked in 10% FeCl3 was placed over the proximal end of the left common carotid artery for 10 minutes. After removing the filter paper, the covered area was cleaned with physiological saline. Finally, the neck wound was sutured layer by layer.

[0101] After successful modeling, a PLGA / anti-RGMa / Fe3O4@PFH contrast agent (10 mg / kg) at a concentration of 5 mg / mL was injected via the tail vein. After 60 minutes of circulation, the contrast agent was administered at a rate of 3 W / cm². 2 The rats were irradiated with LIFU for 3 minutes, and ultrasound and photoacoustic imaging were performed on the rats.

[0102] Experimental results showed that after the carotid artery embolism model, the thrombus in the rat carotid artery exhibited a hypoechoic state. After injection of PLGA / anti-RGMa / Fe3O4@PFH, LIFU-induced CEUS imaging in the neck was significantly enhanced. Figure 16 Photoacoustic imaging also showed the same results; before the contrast agent was administered, no obvious signal was observed in the blood vessel, but after the contrast agent was injected, obvious photoacoustic signals could be seen in the thrombus and the surrounding blood vessel wall. Figure 17These results suggest that PLGA / anti-RGMa / Fe3O4@PFH has good imaging capabilities for thrombosis and is a dual-modal contrast agent with imaging potential, which may be helpful in the diagnosis of thrombosis in superficial extracranial sites.

[0103] Example 8: Detection of the vascular recanalization effect of nanoparticles

[0104] A left carotid artery embolism model was established in SD rats according to Example 7. Subsequently, MiCM@PLGA / anti-RGMa / Fe3O4@PFH (5 mg / mL, 10 mg / kg) was injected into the rats via the tail vein. One hour after injection, LIFU was administered at 3 W / cm². 2 The LIFU excitation was performed for 6 minutes (2 seconds on, 2 seconds off), followed by placing the neck in a Helmholtz coil system (Hunan Paisheng Technology) for 15 minutes (30mT, 4Hz). This LIFU excitation and neck placement in the magnetic field were repeated three times. Blood flow spectra of the left common carotid artery (LCCA) were recorded using pulses before, after, and after treatment. Results showed that after embolization, the spectrum of the distal left LCCA changed to a high-resistance arterial blood flow pattern with a low PSV of 59.897±6.690 mm / s, indicating CCA occlusion. After PLGA / anti-RGMa / Fe3O4@PFH treatment, PSV significantly increased, indicating recanalization of the occluded LCCA (LCCA PSV = 124.000±12.266 mm / s). Figure 18 These results demonstrate that MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles can effectively recanalize blocked blood vessels.

[0105] Example 9: Detection of Vascular Damage by Nanoparticles

[0106] Current clinical methods for recanalizing blood vessels can damage the vessel wall at the site of thrombus destruction, potentially leading to serious or even fatal complications such as cerebral hemorrhage. Therefore, we investigated the damage caused by nanoparticles at the thrombus destruction site.

[0107] Referring to Example 7, a carotid artery embolism model was established in SD rats. Subsequent experiments were conducted in a sham group (sham surgery) and a treatment group. The sham group did not receive tail vein injection of nanoparticles. For the treatment group, after modeling, PLGA / anti-RGMa / Fe3O4@PFH (5 mg / mL, 10 mg / kg) was injected into the rats via the tail vein. One hour after injection, LIFU was administered at 3 W / cm². 2The rats were excited for 6 minutes (2 seconds on, 2 seconds off), then the neck was placed in a Helmholtz coil system for 15 minutes (30 mT, 4 Hz). This LIFU excitation and neck placement in the magnetic field were repeated three times. After treatment, the rats were euthanized, and a specimen of the left common carotid artery was collected. Vascular damage was assessed using H&E staining, Masson staining (to reveal fibers and inflammatory factors in the tissue), resorcinol staining (elastic fiber staining), and reticular fiber staining. Results showed that the treated area of ​​the common carotid artery did not exhibit any obvious signs of damage, including deformation of the intermediate elastic plate, endothelial cell detachment, local necrosis, thermal damage, or inflammatory response. Figure 19 Currently, the most common treatment for vascular recanalization after cerebral infarction—endovascular thrombectomy—often causes significant damage to blood vessels, leading to cerebral hemorrhage and, in severe cases, even death. As shown in the figure, compared to the sham surgery group, the nanoparticles and the additional LIFU and magnetic field treatment in this protocol did not cause significant vascular damage, demonstrating ideal safety performance.

[0108] Currently, there is no existing technology for destroying cerebral vascular thrombi based on LIFU (Lipostimulation Injection). This is related to the unique structure of the brain, which is unsuitable for prolonged LIFU irradiation. Effective thrombolysis requires high-intensity or prolonged LIFU irradiation of the brain, which can cause vasogenic and cytotoxic cerebral edema. In other words, LIFU cannot achieve vascular recanalization without causing brain tissue damage. Magnetic nanoparticles can be targeted and rotated under the manipulation of a magnetic field. These magnetic nanoparticles can help thrombolytic drugs target the thrombus site, and the movement of the magnetic particles themselves can also mechanically destroy the thrombus. Helmholtz coils in magnetic drives can generate uniform magnetic fields in different directions, manipulating the movement of the magnetically responsive material Fe3O4. However, using only a magnetic field results in low thrombus destruction efficiency and poor effects, failing to achieve vascular recanalization. Currently, there are no literature reports on the destruction of cerebral vascular thrombi using only magnetic materials.

[0109] This innovative technical solution employs a treatment method combining LIFU irradiation, a magnetic field, and phase-change nanoparticles. This achieves vascular recanalization without causing significant damage to the cerebral blood vessels at the thrombus site, yielding unexpected technical results. After the phase-change nanoparticles are administered to the thrombus site, the sequential application of LIFU irradiation and a magnetic field effectively destroys the thrombus, thereby recanalizing the embolized blood vessel without damaging the vessel wall, achieving a balance between therapeutic efficacy and avoiding side effects. Furthermore, the use of a magnetic field significantly reduces the requirements for LIFU irradiation intensity and duration while achieving thrombus destruction, exceeding the expectations of those skilled in the art.

[0110] Example 10: Nanoparticles exert neuroprotective effects by inhibiting astrocyte activation.

[0111] C57BL / 6 mice were randomly divided into four groups (sham group, eMCAO group, MiCM@PLGA / anti-RGMa / Fe3O4@PFH group, and MiCM@PLGA / Fe3O4@PFH group), with three mice in each group. An eMCAO model was established in the mice, and each group received different treatments: Sham group: sham-operated control; eMCAO model group: eMCAO model established, no treatment after modeling; MiCM@PLGA / anti-RGMa / Fe3O4@PFH group: eMCAO model established, followed by intravenous administration of MiCM@PLGA / anti-RGMa / Fe3O4@PFH (5 mg / mL, 10 mg / kg); MiCM@PLGA / Fe3O4@PFH group: followed by intravenous administration of MiCM@PLGA / Fe3O4@PFH (5 mg / mL, 10 mg / kg) after modeling. Sixty minutes after intravenous drug injection, mice were subjected to a 2-week / cm² treatment. 2 LIFU stimulation was performed for 3 minutes (2 seconds on, 2 seconds off), followed by placing the thrombus site in a magnetic field system composed of Helmholtz coils for 5 minutes (30 mT, 4 Hz). The aforementioned LIFU stimulation and neck placement in the magnetic field were repeated twice. Seven days after mouse modeling, mice were sacrificed and internally fixed by perfusion with 4% paraformaldehyde. After fixation, the mice were decapitated and the brains were externally fixed in 4% paraformaldehyde for 24 hours. Subsequently, the brain tissue was sequentially soaked in 15% and 30% sucrose solutions prepared with 4% paraformaldehyde, and dehydrated in a gradient until the brain tissue settled. After settling, the brain tissue was embedded in OCT gel, flash-frozen in a cryostat, and sectioned in the coronal position. The brain slices were repaired with sodium citrate buffer and then perforated with 0.1% Triton X-100. After perforation, the slices were blocked with 10% donkey serum. After blocking, the primary antibody (anti-GFAP) was added and incubated overnight at 4°C. The next day, the secondary antibody (Alexa Fluor 488) was added and incubated at 37°C for 1 hour. Finally, DAPI was added and incubated at room temperature for 5 minutes for nucleation staining. Images were acquired using fluorescence microscopy and analyzed using ImageJ software. Results showed that compared to the Sham group, GFAP expression was significantly increased in the eMCAO group, and GFAP expression was significantly reduced after MiCM@PLGA / anti-RGMa / Fe3O4@PFH intervention. Figure 20 This suggests that MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles inhibit astrocyte activation and exert a neuroprotective effect.

[0112] Example 11: Evaluation of the overall efficacy of nanoparticle-integrated vascular recanalization and neuroprotective therapy (impact on infarct volume and neurological function)

[0113] C57BL / 6 mice were randomly divided into 6 groups (sham group, model group, MiCM-NPs group, anti-RGMa group, uncoated-NPs group, and IgG group), with 3 mice in each group. An eMCAO model was established in the mice, and each group received different treatments: Sham group: sham-operated control; Model (eMCAO model group): no treatment after modeling; MiCM-NPs group: MiCM@PLGA / anti-RGMa / Fe3O4@PFH (5mg / mL, 10mg / kg) administered via tail vein after modeling; anti-RGMa group: anti-RGMa (5mg / mL, 10mg / kg) administered via tail vein after modeling; uncoated-NPs group: PLGA / anti-RGMa / Fe3O4@PFH (5mg / mL, 10mg / kg) administered via tail vein after modeling; IgG group: IgG (a non-specific antibody used as a control for anti-RGMa, 5mg / mL, 10mg / kg) administered via tail vein after modeling. Sixty minutes after intravenous drug administration, administer 2 weeks / cm. 2 LIFU stimulation was applied for 3 minutes (2 seconds on, 2 seconds off) (the LIFU parameters were slightly lowered to control mouse mortality). The thrombus site was then placed in a magnetic field system composed of Helmholtz coils for 5 minutes (30 mT, 4 Hz). The LIFU stimulation and neck placement in the magnetic field were repeated twice. Mice were assessed for mNss scores and rotarod behavior before, on the day of, 1 day, 3 days, and 7 days after eMCAO modeling. Finally, the mice were sacrificed, and their brains were collected, frozen at -20°C, and sliced ​​into 2 mm thick sections. Fresh brain slices were stained with TTC solution at 37°C for 30 minutes, then soaked in 4% paraformaldehyde for 24 hours before photographing. Results showed that the MiCM@PLGA / anti-RGMa / Fe3O4@PFH group had the best neurological function recovery. Figure 21 The smallest volume of cerebral infarction ( Figure 22 This demonstrates that the integrated MiCM@PLGA / anti-RGMa / Fe3O4@PFH nanoparticles have a good therapeutic effect on cerebral infarction, promoting vascular recanalization and neuroprotection.

[0114] In summary, the nanoparticles of this scheme utilize perfluorohexane (PFH), an ultrasound-responsive phase change material, as their core, and polylactic-glycolic acid (PLGA) as their outer shell. The outer shell contains the magnetically responsive material Fe3O4 and the neuroprotective drug anti-RGMa, and is coated with a microglial cell membrane. After intravenous injection following cerebral infarction, the nanoparticles, under the influence of the microglial cell membrane, target the damaged vascular endothelial cells at the thrombus embolism site. Subsequently, exogenous LIFU treatment and magnetic field treatment provided by a Helmholtz coil are applied. LIFU induces a phase transition in PFH, releasing anti-RGMa and Fe3O4 from the PLGA outer shell. This phase transition process, along with the rotational and propulsive effects of the exogenous magnetic field on Fe3O4, jointly disrupts the thrombus, thereby restoring blood flow. The released anti-RGMa exerts a neuroprotective effect on damaged brain tissue, thus achieving vascular recanalization bridging neuroprotective therapy, reducing the infarct volume, and promoting neurological function recovery. For details on the nanoparticle structure and working principle, please refer to [link to relevant documentation]. Figure 23 As shown.

[0115] Comparative Example 1:

[0116] Injection of recombinant RGMa protein into mice with MCAO significantly reduced the degree of lung damage. Figure 24 ), reduced bacterial load in mouse lung tissue ( Figure 25 ).

[0117] exist Figure 24 In the study, lung injury in mice was reduced after injection of recombinant RGMa. Mouse lung tissue was stained with hematoxylin and eosin (HE) and observed under a light microscope (100x). Compared with the Sham group, the MCAO group showed diffuse hemorrhage, edema, alveolar dilation or collapse, significant thickening of alveolar septa, indistinct alveolar tissue structure, extensive leukocyte infiltration, and neutrophil accumulation in the lumen. Compared with the MCAO group, the MCAO + recombinant RGMa group showed reduced edema, a decreased number of infiltrated leukocytes, and improved alveolar dilation and collapse.

[0118] exist Figure 25 In mice, the bacterial load in lung tissue was significantly reduced after injection of rm-RGMa (recombinant RGMa). Compared with the MCAO+Vehicle group, the bacterial load in lung tissue of the MCAO+rm-RGMA group was significantly reduced (Sham group n=5, MCAO+Vehicle group n=6, MCAO+rm-RGMA group n=6; Vehicle, solvent control; rm-RGMa, recombinant RGMa. **, p<0.01).

[0119] Based on the above experimental results, if anti-RGMa (anti-RGMa monoclonal antibody) is administered directly via intravenous injection, it can reach the lung tissue through blood circulation. It binds to RGMa in the lung tissue, preventing RGMa there from fully exerting its protective effect on the lungs, thus leading to post-stroke pneumonia. Therefore, although direct intravenous administration of anti-RGMa can achieve neuroprotection, this method can cause post-stroke pneumonia as a side effect.

[0120] This technical solution encapsulates anti-RGMa in nanoparticles, which then undergo phase transition via LIFU irradiation after reaching the designated site, enabling targeted drug delivery. This enhances the neuroprotective effect of anti-RGMa and simultaneously prevents post-stroke pneumonia, thus avoiding the side effects of anti-RGMa.

[0121] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A microglial cell membrane-masked acoustomagnetic responsive nanoparticle, administered intravenously, for the treatment of ischemic stroke and simultaneous prevention of post-stroke pneumonia, characterized in that: It includes a perfluorohexane core and a polylactic acid-glycolic acid copolymer shell; Fe3O4 particles are encapsulated inside the shell; microglia are also coated on the outside of the shell; and anti-RGMa is also encapsulated inside the shell. The Fe3O4 particles are oleic acid-modified Fe3O4 particles and are derived from Fe3O4 reagent; the concentration of oleic acid-modified Fe3O4 in the Fe3O4 reagent is 25 mg / mL; The ratio of polylactic acid-glycolic acid, the Fe3O4 reagent, the perfluorohexane, and the anti-RGMa is 25 mg: 100 μl: 200 μl: 25 mg.

2. The method for preparing microglial cell membrane-masked acoustomagnetic responsive nanoparticles for treating ischemic stroke and simultaneously preventing post-stroke pneumonia via intravenous administration, as described in claim 1, is characterized in that: The steps are as follows, performed sequentially: S1: The polylactic acid-hydroxyacetic acid, the Fe3O4 reagent, and dichloromethane are mixed, then the perfluorohexane is added and ultrasonically treated to form a primary emulsion; then a polyvinyl alcohol solution is added and ultrasonically treated; then an isopropanol solution is added and the mixture is allowed to stand and washed to obtain nanoparticles that are not encapsulated in cell membranes. S2: The microglia cell membrane is mixed with the uncoated nanoparticles obtained in S1, then subjected to ultrasonic treatment, and washed to obtain acoustomagnetic responsive nanoparticles disguised as microglia cell membranes. In S1, the anti-RGMa is added simultaneously with the perfluorohexane; In S1, the ratio of the polylactic acid-glycolic acid, the Fe3O4 reagent, the dichloromethane, the perfluorohexane, the polyvinyl alcohol solution, and the isopropanol solution is 25 mg: 100 μl: 2 mL: 200 μL: 4 mL: 5 mL; The polyvinyl alcohol solution has a solute mass percentage of 2%, and the isopropanol solution has a solute volume percentage of 2%.

3. The method for preparing acoustomagnetic responsive nanoparticles disguised as microglia cell membranes according to claim 2, characterized in that: In S1, the power of the first ultrasound treatment was 52W and the time was 6 minutes; the power of the second ultrasound treatment was 40W and the time was 6 minutes. In S2, the microglia are mixed with the uncoated nanoparticles obtained in S1, and then sonicated at 40W for 1 minute; the mass ratio of the membrane proteins of the microglia to the uncoated nanoparticles obtained in S1 is 1:

1.

4. The application of the microglial cell membrane-masked acoustomagnetic responsive nanoparticles according to claim 1 in the preparation of a system for treating ischemic stroke, achieving vascular recanalization and brain protection, and simultaneously preventing post-stroke pneumonia, characterized in that: The system includes microglial cell membrane-masked acoustomagnetic responsive nanoparticles for intravenous administration to cerebral artery embolism sites, a low-intensity focused ultrasound device for applying ultrasound to the nanoparticles, and a Helmholtz coil system for applying a magnetic field to the nanoparticles. The system is used to achieve vascular recanalization without causing significant damage to the cerebral blood vessels at the site of the thrombus. The parameters of the low-intensity focused ultrasound device are set to 3W / cm. 2 Irradiate for 3 minutes; The parameters of the Helmholtz coil system were set to 30mT, 4Hz, and processed for 15 minutes.

5. The application of a microglial cell membrane-masked acoustomagnetic responsive nanoparticle according to claim 1 in the preparation of ultrasound or photoacoustic imaging contrast agents.

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