Macrophage membrane coated plateau encephaledema drug carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a macrophage membrane coated plateau encephaledema drug carrier and a preparation method and application thereof, and the macrophage membrane coated plateau encephaledema drug carrier is composed of a nanoparticle core and a macrophage membrane shell layer wrapping the core; the nanoparticle core comprises an ROS-responsive carrier material and carbon monoxide release molecules MnCO entrapped in the ROS-responsive carrier material; the content of phosphatidylserine in the shell layer of the macrophage membrane is not less than 5 mol% of the total phospholipid of the membrane. According to the invention, a bionic delivery system of ROS response type MnCO nanoparticles coated with a macrophage membrane is constructed, phosphatidylserine on the surface of the membrane is utilized to actively activate a microglial cell Trem2 receptor, a positive feedback cycle of targeting phagocytosis-CO release-Trem2 up-regulation is formed, and at the same time, fatty acid oxidative metabolism reprogramming is promoted by means of degraded membrane lipid, so that the biomimetic delivery system is used for preparing the ROS response type MnCO nanoparticles. And multi-mechanism synergistic efficient brain-targeted therapy is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a macrophage membrane-coated high-altitude cerebral edema drug carrier, a preparation method and application thereof. BACKGROUND

[0002] High altitude cerebral edema (HACE) is a critical manifestation of acute high altitude disease, which is characterized by rapid onset, rapid progression and high mortality, and seriously threatens the life safety of personnel entering high altitude areas. When the human body rapidly enters an area above 2500 meters above sea level, the low-pressure hypoxic environment will cause a series of pathophysiological changes. Studies have shown that hypoxia-inducible factor-1 alpha (HIF-1 alpha) as a key protein of cellular oxygen sensing regulation cannot be normally ubiquitinated and degraded when high altitude acclimatization is poor, resulting in overexpression, which in turn causes changes in vascular permeability, blood brain barrier (BBB) damage and overexpression of inflammatory factors, and ultimately induces HACE.

[0003] At present, the clinical treatment of HACE is very limited, mainly including oxygen inhalation, transfer to low altitude areas and the use of diuretics, but these methods have low efficiency, are strongly restricted by the environment, and are difficult to meet the urgent needs. Therefore, the development of a new drug delivery system that can precisely target brain lesions, efficiently relieve inflammation and repair the blood brain barrier has become an urgent need in the field of high altitude medicine.

[0004] In recent years, nano drug delivery systems have provided new ideas for the treatment of brain diseases. Traditional nano carriers, such as liposomes and polymer nanoparticles, can improve drug solubility, but still face challenges such as poor targeting, rapid clearance by the mononuclear phagocyte system (MPS), and difficulty in effectively crossing the blood brain barrier. In particular, after systemic administration, nano carriers tend to non-specifically accumulate in tissues such as the liver and spleen, resulting in a significant lack of drug reaching the lesion site.

[0005] In order to improve brain targeting, researchers have developed a variety of functionalized nano carriers. For example, a patent (CN118453514A) discloses a ROS-responsive intelligent drug carrier, which uses hydrophobic borate ester grafted to hydrophilic dextran to form an amphiphilic block copolymer as a carrier, and loads carbon monoxide releasing molecule MnCO, which can respond to the high reactive oxygen species (ROS) environment in the HACE lesion site and release the drug. However, such carriers still mainly rely on passive targeting (such as EPR effect or environmental response) at the lesion site, and their active targeting ability is limited, and the penetration efficiency of the blood brain barrier is insufficient, which affects the final treatment effect.

[0006] Macrophages, as the core members of the immune system, have been considered as ideal drug delivery carriers in recent years due to their natural pathological tissue tropism, phagocytic ability and immune regulation function. Macrophage and its derivative delivery systems (MDDSs) mainly include intact cell drug loading, cell membrane biomimetic, extracellular vesicles (EVs) and cell "hitchhiking" strategies. Among them, the macrophage membrane biomimetic nanosystem extracts the macrophage membrane to wrap the synthetic nanoparticles, which not only retains the natural proteins and receptors (such as integrins, chemokine receptors, etc.) on the surface of the source cell membrane, endows the nanoparticles with excellent active targeting ability and immune evasion ability, but also avoids the activity instability problem that may exist in the active cell drug loading. Studies have shown that the macrophage membrane-wrapped nanoparticles have higher biocompatibility and targeting therapeutic effect than traditional nanometer drug delivery systems in various disease models such as malignant tumors, Alzheimer's disease, liver ischemia-reperfusion injury, etc.

[0007] Although the macrophage membrane biomimetic technology has shown great potential in targeted delivery, its research is currently mainly concentrated in the field of anti-tumor, and its application in the treatment of high altitude cerebral edema is still blank. In addition, the existing ROS-responsive carrier and macrophage membrane biomimetic technology have not been organically combined, which lacks the active intervention ability to key immune cells such as microglia cells in the lesion site, and also fails to form a synergistic therapeutic effect, limiting its comprehensive therapeutic effect in complex neuroinflammatory environment.

[0008] Therefore, it is necessary to develop a new type of drug carrier integrating ROS-responsive drug release, macrophage membrane active targeting and immune expression regulation, which has important theoretical significance and clinical value for efficient prevention and treatment of high altitude cerebral edema. SUMMARY

[0009] The purpose of the present application is to provide a macrophage membrane-coated high altitude cerebral edema drug carrier, its preparation method and application. By constructing a biomimetic delivery system of ROS-responsive MnCO nanoparticles coated with macrophage membrane, the phosphatidylserine on the membrane surface is used to actively activate the Trem2 receptor of microglia cells, forming a positive feedback loop of "targeted phagocytosis-CO release-Trem2 upregulation", and the degraded membrane lipids are used to promote fatty acid oxidative metabolism reprogramming, realizing efficient brain-targeted therapy with multiple mechanisms.

[0010] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: A macrophage membrane-coated high altitude cerebral edema drug carrier, which is composed of a nanoparticle core and a macrophage membrane shell wrapped outside the core; The nanoparticle core comprises a ROS-responsive carrier material and a carbon monoxide release molecule MnCO loaded therein; The content of phosphatidylserine in the macrophage membrane shell is not less than 5 mol% of the total phospholipid of the membrane; the phosphatidylserine can specifically bind to the Trem2 receptor on the surface of microglial cells, and the CO released by the MnCO can up-regulate the expression of the Trem2 receptor of the microglial cells, forming a positive feedback loop of "Trem2 activation-enhanced phagocytosis-CO release-up-regulation of Trem2 expression".

[0011] The ROS-responsive borate-dextran copolymer is used as a carrier core to encapsulate the MnCO. The carrier can be specifically degraded in the high ROS environment of the HACE lesion area, achieving site-specific and controllable release of the drug, so that the CO with anti-inflammatory and anti-apoptotic effects is accurately delivered to the brain injury site, reducing the systemic toxicity.

[0012] The macrophage membrane wrapped in the outermost layer is used as a biomimetic shell. The phosphatidylserine (PS) rich on the membrane surface can be used as a natural ligand and be specifically recognized and combined with the Trem2 receptor on the surface of microglial cells. This "eat me" signal actively initiates and greatly enhances the phagocytosis of the microglial cells to the nano system, realizing the transition from passive enrichment to active uptake.

[0013] After the carrier is phagocytosed, it is rapidly degraded to release CO under the intracellular ROS environment. The released CO not only can play a direct therapeutic effect, but also can up-regulate the expression of the Trem2 receptor of the microglial cells. The increase of the expression level of Trem2 will further promote the phagocytosis of the cells to the subsequent nanoparticles, thereby forming a self-enhancing positive feedback loop of "Trem2 activation-enhanced phagocytosis-released CO-up-regulation of Trem2", continuously amplifying the therapeutic effect.

[0014] The phagocytosed macrophage membrane is degraded in the lysosome, and the lipid components thereof can be used as a substrate to provide fuel for the fatty acid beta-oxidation (FAO) of the microglial cells, driving the reprogramming of the cell metabolism from the pro-inflammatory glycolysis to the anti-inflammatory oxidative phosphorylation (OXPHOS), thereby synergistically promoting the polarization of the microglial cells to the reparative M2 phenotype, achieving anti-inflammation and tissue repair.

[0015] Preferably, the ROS-responsive carrier material is an amphiphilic block copolymer; the amphiphilic block copolymer is formed by grafting a hydrophobic borate onto a hydrophilic dextran skeleton.

[0016] Preferably, the macrophage membrane is derived from RAW 264.7 cells or obtained from primary macrophages derived from bone marrow.

[0017] Preferably, the average particle size of the nanoparticle core is 80-200 nm, and the average particle size of the nanoparticle drug delivery system after the macrophage membrane is coated is 120-240 nm.

[0018] Preferably, the ROS-responsive carrier material is degraded in an environment with a hydrogen peroxide concentration of 0.1-10 mM.

[0019] The application provides a preparation method of the macrophage membrane-coated drug carrier for treating high-altitude cerebral edema.

[0020] The preparation method can mildly retain phosphatidylserine (a natural ligand of the Trem2 receptor) on the surface of the macrophage membrane and the activity of functional proteins, ensures that the carrier can actively activate the Trem2 signal pathway of microglial cells, realizes close and stable combination of the membrane shell and the nanoparticle core, strengthens immune camouflage to prolong the in-vivo circulation time, can accurately regulate the particle size of the carrier through the liposome extrusion method, adapts to the blood-brain barrier crossing and microglial cell uptake, does not need to introduce harmful chemical reagents, guarantees the biocompatibility of the carrier, can synergistically strengthen the ROS-responsive release efficiency, matches the MnCO release rate and the carrier degradation rate, and provides support for the targeted treatment, safe application and treatment mechanism landing of the drug carrier.

[0021] The application also provides application of the macrophage membrane-coated drug carrier for treating high-altitude cerebral edema in preparation of a drug for preventing and / or treating high-altitude cerebral edema.

[0022] Preferably, the prevention and / or treatment mechanism comprises up-regulating the Trem2 expression of microglial cells in the brain, promoting the M2 phenotype polarization of microglial cells, and / or enhancing the fatty acid beta-oxidation metabolic level of microglial cells.

[0023] A pharmaceutical composition comprises a therapeutically effective amount of the macrophage membrane-coated drug carrier for treating high-altitude cerebral edema and a pharmaceutically acceptable carrier or excipient.

[0024] The application has the following beneficial effects: (1) By organically combining the ROS-responsive nanoparticle core and the macrophage membrane biomimetic shell layer, dual targeting capability of passive targeting (EPR effect and ROS response) and active targeting (membrane protein-mediated homing) is created. The macrophage membrane provides natural immune camouflage, effectively prolongs the in-vivo circulation time of the nanoparticle system, and significantly improves the enrichment concentration of the drug at the high-altitude cerebral edema lesion site by using the inherent inflammatory tropism.

[0025] (2) The most prominent effect is that the macrophage membrane shell is not only a targeting tool, but also the phosphatidylserine (PS) on its surface as a natural ligand of the Trem2 receptor actively triggers the phagocytic behavior of microglia. This makes the delivery system change from a passive aggregated particle to a signaler that can actively activate key immune signaling pathways, greatly improving its intracellular delivery efficiency.

[0026] (3) The application discloses and applies the new finding that CO release can up-regulate Trem2 expression, and constructs a self-amplifying positive feedback loop: Trem2 activation—enhanced phagocytosis—release of CO—up-regulation of Trem2. This cycle enables the therapeutic effect to be enhanced in a cycle, producing a non-linear, unique synergistic effect, which is far superior to the simple addition of the functions of each component.

[0027] (4) The carrier system simultaneously realizes the synergy of three major frontier treatment strategies of immune regulation (Trem2 phagocytosis), gas treatment (CO anti-inflammatory), and metabolic regulation (FAO reprogramming) on one platform. The released CO directly plays an anti-inflammatory and anti-apoptotic role; at the same time, the degraded macrophage membrane lipid provides a substrate for the fatty acid beta-oxidation (FAO) of microglia, driving its polarization to the reparative M2 phenotype, which fundamentally relieves inflammation from the energy metabolism level, and achieves a comprehensive effect of anti-inflammatory and repair.

[0028] (5) The ROS-responsive core ensures that MnCO mainly releases CO in the high-ROS environment of the lesion site, minimizing the potential toxicity to normal tissues. At the same time, the natural composition of the macrophage membrane endows the system with excellent biocompatibility and low immunogenicity. Through dosage form design (such as freeze-dried powder injection), the stability of the product and the convenience of clinical administration are further ensured. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic diagram of the mechanism of the drug carrier in Example 1 of the application in the animal body.

[0030] Figure 2 It is a MnCO release curve diagram of the drug carrier in Example 1 of the application in the ROS simulation environment.

[0031] Figure 3 It is a DLS release kinetic curve diagram of the drug carrier in Example 1 of the application.

[0032] Figure 4 It is a NPs residual release kinetic curve diagram of the drug carrier in Example 1 of the application.

[0033] Figure 5 It is a NPs ROS-responsive release diagram of the drug carrier in Example 1 of the application.

[0034] Figure 6 The images show the distribution of the drug carrier in the major organs of the animal in Experimental Example 2 of this invention, along with statistics on fluorescence intensity.

[0035] Figure 7 This is from Experiment 2 of the present invention, where CLSM was used to observe the localization of the drug carrier in the mouse cortex and hippocampus.

[0036] Figure 8 This is from Experiment Example 2 of the present invention, which uses TEM to observe the biological processes of drug carrier crossing the blood-brain barrier and being phagocytosed by microglia.

[0037] Figure 9 This is a polarization fluorescence staining image of BV2 cells in Experiment Example 3 of this invention.

[0038] Figure 10 This is a bar chart showing the expression of microglial M1 / M2 phenotype-related markers in Experiment Example 3 of this invention.

[0039] Figure 11 This refers to the uptake efficiency of the drug carrier by BV2 cells observed by CLSM in Experimental Example 3 of this invention.

[0040] Figure 12 This refers to the uptake efficiency of drug carriers by BV2 cells as shown by flow cytometry in Experimental Example 3 of this invention.

[0041] Figure 13 In Experiment 4 of this invention, HE staining was used to observe the effects of the drug carrier on various major organs.

[0042] Figure 14 The results of biochemical index detection of liver, kidney and heart function after the drug carrier was applied in Experiment Example 4 of this invention.

[0043] Figure 15 This is a bar chart showing the effect of different concentrations of MM-MnCO-NPs on the viability of mouse microglia lines BV2 and Bend.3 in Experimental Example 4 of this invention.

[0044] Figure 16 This refers to the microglia subset of the single-cell transcriptome in Experiment Example 5 of this invention. Trem2 Pseudo-temporal expression trend diagram of genes.

[0045] Figure 17 The ROS content gradient of BV2 cells as shown by flow cytometry in Experiment Example 5 of this invention.

[0046] Figure 18 This invention illustrates the effects of LPS+Hypoxia and MM-MnCO-NPs on the expression of various factors in the oxidative stress response pathway in Experimental Example 5.

[0047] Figure 19The four-quadrant analysis diagram of BV2 cell mitochondrial membrane potential polarization under the treatment of LPS+Hypoxia and MM-MnCO-NPs in Experimental Example 5 of the present application.

[0048] Figure 20 The four-quadrant analysis diagram of BV2 cell mitochondrial membrane potential depolarization under the treatment of LPS+Hypoxia and MM-MnCO-NPs in Experimental Example 5 of the present application.

[0049] Figure 21 The data distribution diagram of metabolic function indicators of microglial cells under the treatment of LPS+Hypoxia and MM-MnCO-NPs in Experimental Example 5 of the present application.

[0050] Figure 22 The expression level changes of FAO key genes affected by MM-MnCO-NPs in Experimental Example 5 of the present application.

[0051] Figure 23 The expression level changes of fatty acid β-oxidation key genes of microglial cells after drug carrier treatment in Experimental Example 5 of the present application. DETAILED DESCRIPTION

[0052] The specific embodiments are described below in conjunction with the accompanying drawings.

[0053] Example 1 This embodiment provides a macrophage membrane-coated drug carrier for high-altitude cerebral edema, and the preparation process is as follows: (1) Materials and instruments Cell line: mouse monocyte macrophage leukemia cells (RAW 264.7).

[0054] Chemical reagents: dextran (Dextran, MW 40kDa), 4-carboxyphenylboronic acid pinacol ester, N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), manganese monoxide carboxylate (MnCO), dialysis bag (MWCO 3.5 kDa), phosphate buffer (PBS, pH 7.4), cell membrane extraction kit.

[0055] Instruments and equipment: ultrasonic cell pulverizer, liposome extruder (equipped with 100 nm, 200 nm polycarbonate membrane), high-speed low-temperature centrifuge, dynamic light scattering (DLS) particle size potential analyzer, transmission electron microscope (TEM).

[0056] (2) Synthesis of ROS-responsive polymer (PHB-Dextran) Dissolve 1.0 g of dextran in 100 mL of anhydrous dimethyl sulfoxide (DMSO) and stir until completely dissolved.

[0057] 0.6 g EDC HCI and 0.36 g NHS were added to activate the carboxyl group for 2 hours at room temperature under nitrogen protection.

[0058] 1.2 g of 4-carboxyphenylboronic acid pinacol ester was dissolved in a small amount of DMSO and added dropwise to the above activated solution.

[0059] The reaction system was continuously stirred at 25°C for 24 hours under nitrogen protection and light protection.

[0060] After the reaction was completed, the mixed solution was transferred to a dialysis bag and dialyzed with ultrapure water for 48 hours, and the dialysate was replaced every 6 hours to remove unreacted raw materials and byproducts.

[0061] The dialyzed solution was freeze-dried to obtain a white flocculent solid, which was the hydrophobic borate ester grafted hydrophilic dextran amphiphilic copolymer (PHB-Dextran), and stored at -20°C for standby.

[0062] (3) Preparation of MnCO-loaded nanoparticles (MnCO-NPs) Nano-precipitation method was used: 20 mg of the above synthesized PHB-Dextran copolymer and 2 mg of MnCO molecules were dissolved in 2 mL of organic solvent (acetone / dimethylformamide, v / v=1:1) as the organic phase.

[0063] The above organic phase was slowly injected into 10 mL of ultrapure water under magnetic stirring (600 rpm) at a rate of 1 mL / min using a microsyringe.

[0064] Continue stirring for 4 hours to completely volatilize the organic solvent, and the nanoparticles self-assemble.

[0065] The obtained crude nanoparticle suspension was centrifuged at 4°C and 12000 rpm for 30 minutes, and the supernatant was discarded to remove unencapsulated MnCO.

[0066] The precipitate was resuspended with PBS (pH 7.4) and sterile filtered through a 0.22 μm microporous filter to obtain a purified MnCO-NPs suspension, which was stored at 4°C.

[0067] (4) Extraction of macrophage membrane (MM) RAW 264.7 cells were expanded and cultured in DMEM complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator.

[0068] When the cells grew to 80%-90% confluence, the cells were trypsinized and collected.

[0069] The cells were washed with pre-chilled PBS three times.

[0070] The operation was performed according to the instructions of the commercialized cell membrane protein extraction kit: the cell pellet was resuspended in a hypotonic buffer containing protease inhibitors, and incubated on ice for 30 minutes to swell the cells.

[0071] Subsequently, the cell suspension was transferred to a glass homogenizer and homogenized thoroughly under ice bath conditions.

[0072] The homogenate was subjected to low-speed centrifugation (1000 x g, 10 min, 4°C) to remove the nuclei and unbroken cells, and then the supernatant was subjected to ultracentrifugation (100,000 x g, 45 min, 4°C), and the obtained precipitate was the crude cell membrane fragments.

[0073] The cell membrane precipitate was resuspended in an appropriate amount of PBS, and repeatedly passed through a 400 nm polycarbonate membrane 11 times using a liposome extruder to obtain a uniform macrophage membrane vesicle suspension.

[0074] (5) Construction of macrophage membrane-coated nanoparticles (MM-MnCO-NPs) The MnCO-NPs suspension prepared in step 3 was mixed with the macrophage membrane vesicle suspension prepared in step 4 in PBS at a membrane protein mass ratio of 1:1, and the total volume was 5 mL.

[0075] The mixed suspension was placed in an ice bath and subjected to ultrasonic fusion using an ultrasonic cell crusher. The parameters were set as follows: working power 200 W, ultrasonic on for 1 second, intermittent for 2 seconds, and total processing time 5 minutes.

[0076] After ultrasonication, the mixture was immediately transferred to a liposome extruder for sequential extrusion. It was passed through a 400 nm polycarbonate membrane 3 times and then through a 200 nm polycarbonate membrane 7 times.

[0077] Finally, the final product after extrusion was centrifuged at 4°C and 12000 rpm for 30 minutes, and the precipitate was washed once with PBS to remove the un-fused free membrane components and nanoparticles, and then resuspended in an appropriate amount of PBS to obtain the macrophage membrane-coated high-altitude cerebral edema drug carrier (MM-MnCO-NPs), which was stored at 4°C in the dark for later use.

[0078] Experimental Example 1 Carrier degradation kinetics and ROS-responsive release experiment ROS-responsive degradation test experimental method The prepared MM-MnCO-NPs were dispersed in PBS buffer (pH 7.4) containing different concentrations of H2O2 (0 mM, 0.025 mM, 0.1 mM, 0.5 mM, 1.0 mM) respectively, and incubated at 37°C with constant shaking to simulate the ROS environment from normal to abnormal increase in the focal area of high altitude cerebral edema (HACE). At the preset time points (0, 2, 6, 12, 24 hours), samples were taken and the changes of hydrodynamic particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS).

[0079] Results and discussion The above experimental results show that the MM-MnCO-NPs have excellent ROS concentration-dependent degradation and drug controlled release ability.

[0080] As shown in the accompanying Figure 2 , in PBS without H2O2, the MM-MnCO-NPs carrier particle size distribution is stable, indicating that the carrier has good storage stability in normal physiological environment. As shown in the accompanying Figure 3 , in PBS without H2O2, the particle size distribution of MM-MnCO-NPs remains stable within 24 hours, indicating that the carrier has good storage stability in normal physiological environment. However, in the presence of H2O2, as shown in the accompanying Figure 4 and accompanying Figure 5 , the particle size of the carrier decreases significantly with the increase of H2O2 concentration and the extension of the action time, and the Zeta potential moves to the negative value, which indicates that ROS triggers the chemical bond rupture of the carrier material, leading to the dissociation of nanostructure.

[0081] Experimental Example 2 Evaluation of brain targeting and blood brain barrier crossing ability of drug carrier Experimental method To evaluate the brain targeting distribution and the ability of crossing blood brain barrier of the drug carrier of the present application, we carried out the following animal experiments.

[0082] Animal model: Establish a high altitude cerebral edema (HACE) mouse model.

[0083] Grouping and administration: The model mice were randomly divided into groups, and were injected with DiR fluorescent dye labeled macrophage membrane coated nanocarrier (MM-MnCO-NPs) and its non-membrane coated control nanoparticles (MnCO-NPs) through the tail vein respectively.

[0084] In vivo distribution tracking: Using a small animal live imaging system, at 1, 2, 4, 6, 24 hours after injection, the overall distribution of fluorescent signal in the live mouse body was observed and recorded.

[0085] Ex vivo tissue quantification analysis: At 24 h time point, mice were sacrificed, major organs (heart, liver, spleen, lung, kidney, brain) were isolated, ex vivo fluorescence imaging was performed, and the fluorescence intensity of each organ was semi-quantitatively analyzed using the system software.

[0086] Microscopic localization observation in brain: Confocal laser scanning microscopy observation: Brain tissues were sectioned, FITC-dextran was used to label the blood vessel lumen, and DiI was used to label MM-MnCO-NPs. CLSM was used to observe the distribution of nanoparticles in the cortex and hippocampus regions of brain tissue and their colocalization with Iba1 positive microglia cells.

[0087] Transmission electron microscopy observation: Brain tissue samples were extracted, ultrathin sections were prepared, and the interaction of nanoparticles with brain vascular endothelial cells and the ultrastructure of phagocytosis by microglia cells were directly observed by TEM.

[0088] Experimental results In vivo and ex vivo distribution confirmed brain targeting: As shown in the attached Figure 6 Small animal in vivo imaging showed that the MM-MnCO-NPs group exhibited significant fluorescence signal enhancement in the brain region 4 hours after injection, and the signal remained stable and accumulated within 24 hours. In contrast, the fluorescence signal in the brain of the MnCO-NPs group was always weak. Ex vivo organ imaging and semi-quantitative analysis further confirmed that the fluorescence enrichment intensity of MM-MnCO-NPs in brain tissue was more than 3.5 times that of the control group (MnCO-NPs), clearly demonstrating that the macrophage membrane modification endowed the carrier with excellent active brain targeting ability.

[0089] Intracerebral localization and cellular uptake: The attached Figure 7 confocal images showed that DiI-labeled MM-MnCO-NPs had successfully crossed the FITC-dextran-labeled blood vessels, distributed in the brain parenchyma, and exhibited obvious colocalization with the microglia marker Iba1, indicating that the carrier could be effectively taken up by microglia cells, the key effector cells in the lesion area.

[0090] Ultrastructural evidence of crossing the BBB and phagocytosis process: The attached Figure 8 (TEM observation of the biological process of drug carriers crossing the blood-brain barrier and being phagocytosed by microglia cells) provides more direct morphological evidence. TEM images clearly captured the process of nanoparticles crossing the vascular endothelial cells and the images of nanoparticles being phagocytosed by microglia cells and existing in intracellular vesicles.

[0091] Experimental Example 3 Experimental Methods To explore the effect of the drug carrier on microglial cell polarization and its mechanism, we conducted the following in vitro cell experiments.

[0092] Cell line and grouping: The mouse microglial cell line BV2 was used, and the following experimental groups were set up: Control group: Normal BV2 cells without treatment.

[0093] LPS model group: Lipopolysaccharide (LPS) was used to stimulate BV2 cells to establish an inflammatory model.

[0094] MM-MnCO-NPs treatment group: MM-MnCO-NPs were added for intervention while LPS was stimulated.

[0095] Trem2-KO mechanism verification group: Trem2 gene knockout (Trem2-KO) BV2 cells were used to repeat the above LPS stimulation and MM-MnCO-NPs treatment process.

[0096] Detection index and method: Cell uptake efficiency: Flow cytometry: DiI fluorescent dye was used to label the nanocarrier, and after co-incubation with BV2 cells, the mean fluorescence intensity of the cells was quantitatively detected by flow cytometry to evaluate the uptake efficiency.

[0097] Confocal laser scanning microscope observation: Similarly, DiI was used to label the carrier, and after co-incubation with cells, the distribution of the carrier in the cells was directly observed and photographed by CLSM.

[0098] Cell phenotype polarization analysis: qPCR and Western Blot: Total RNA and total protein were extracted, and the expression of M1 markers (iNOS, CD86) and M2 markers (Arg1, CD206) at the mRNA and protein levels was detected by quantitative real-time fluorescent PCR and Western Blot, respectively.

[0099] Inflammatory factor detection: Cell culture supernatant was collected, and the secretion levels of pro-inflammatory factors IL-6 and TNF-α were detected using an ELISA kit.

[0100] Fluorescent staining: Immunofluorescent staining was performed on the cells, and the protein expression and localization of M1 / M2 phenotype-specific markers were observed by CLSM.

[0101] Experimental results Trem2-mediated enhanced cell uptake: as shown in Figure 11 and Figure 12As shown, the uptake efficiency of MM-MnCO-NPs was significantly higher than that of the control group without membrane coating in wild-type BV2 cells. However, this enhanced effect was significantly weakened in Trem2-KO cells, indicating that the macrophage membrane on the surface of the carrier mediates an active phagocytosis process through the Trem2 receptor.

[0102] Promoting microglia polarization to M2 phenotype: attached Figure 9 immunofluorescence images and attached Figure 10 qPCR and Western Blot data consistently showed that, compared with the LPS model group, MM-MnCO-NPs treatment significantly reduced the expression of M1 markers (iNOS, CD86) while enhancing the expression of M2 markers (Arg1, CD206). In addition, the secretion levels of IL-6 and TNF-α in the cell supernatant were also significantly reduced due to MM-MnCO-NPs treatment.

[0103] Key role of Trem2 in polarization process: In Trem2-KO BV2 cells, the promotion of M2 polarization and the inhibition of inflammatory factor secretion by MM-MnCO-NPs were significantly weakened, which confirmed the core role of the Trem2 receptor in the immune regulation function of the carrier.

[0104] This experimental example demonstrates that MM-MnCO-NPs can enhance the phagocytosis of microglia cells through a Trem2-mediated pathway, effectively reverse the LPS-induced inflammatory state, promote the polarization of microglia cells from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and thus play a role in relieving neuroinflammation.

[0105] Experimental Example 4 Experimental Methods In vivo acute toxicity experiment: Animals and grouping: healthy ICR mice were selected and randomly divided into two groups (n=6): saline group (as negative control) and MM-MnCO-NPs administration group.

[0106] Dosing regimen: The MM-MnCO-NPs group was administered at twice the effective therapeutic dose through tail vein injection, with administration every 3 days, and continuous observation for 14 days.

[0107] Observation index: General state observation: The activity behavior, food and water intake, hair condition and body weight changes of the mice were observed and recorded every day.

[0108] Biochemical analysis of blood: 24 hours after the last administration, blood was collected from the inner canthus, and serum was separated. The key indicators of liver function, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the key indicators of renal function, urea nitrogen (BUN), creatinine (Cr), were detected using an automatic biochemical analyzer.

[0109] Histopathological examination: After the mice were sacrificed, the important organs such as heart, liver, spleen, lung, kidney, etc. were completely taken out, fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E). The histomorphology was observed by a pathologist under an optical microscope in a blind manner.

[0110] In vitro cytotoxicity experiment: Methods and detection: The mouse microglial cell line BV2 was co-cultured with different concentrations of MM-MnCO-NPs (0 - 200 μg / mL) for 24 hours. Then the cell viability was detected by CCK-8 method, and the absorbance value at 450 nm was measured by a microplate reader to calculate the cell survival rate.

[0111] Experimental results In vivo safety results: General state: During the entire 14-day observation period, there was no abnormal difference in the activity, food intake, water intake, and body weight gain of the MM-MnCO-NPs group mice compared with the saline group, and no obvious symptoms of poisoning or abnormal behavior were observed.

[0112] As shown in the accompanying Figure 14 , the levels of ALT, AST, BUN, and Cr in the serum of the mice in the administration group were not statistically significantly different from those in the saline control group, indicating that the experimental dose of the carrier did not cause observable damage to the liver and kidney functions.

[0113] The accompanying Figure 13 shows that the important organ tissue structures such as heart, liver, spleen, lung, and kidney of the mice in the administration group were complete and clear, and the cell morphology was normal, and no obvious pathological changes such as inflammatory cell infiltration, tissue necrosis, or fibrosis occurred.

[0114] The accompanying Figure 15 shows that even at a concentration as high as 200 μg / mL, the survival rate of BV2 cells co-cultured with MM-MnCO-NPs for 24 hours remained above 90%, demonstrating that the carrier has extremely low cytotoxicity in vitro.

[0115] The experimental example confirmed through systematic in vivo and in vitro safety evaluation that under the experimental conditions, MM-MnCO-NPs did not cause significant acute toxicity reactions and major organic damage, showing good biocompatibility and in vivo safety, providing an important safety basis for its further medical application.

[0116] Experimental Example 5 To further explore the cellular targets and potential molecular mechanisms of the drug carrier of the application in treating high altitude cerebral edema (HACE) at the transcriptome level, we performed the following single-cell sequencing and bioinformatics analysis.

[0117] Animal model and grouping: HACE mouse models were established, and model control and MM-MnCO-NPs treatment groups were set up.

[0118] Sample preparation and sequencing: After the mice were sacrificed, brain tissue was taken to prepare single nucleus suspension. 10x Genomics platform was used to perform single nucleus RNA sequencing on the two groups of samples.

[0119] Bioinformatics analysis: Cell clustering and annotation: The obtained sequencing data was subjected to quality control, standardization, dimensionality reduction and clustering analysis, and the cell clusters were annotated according to known cell marker genes, and the main brain cell types such as neurons, oligodendrocytes, astrocytes, endothelial cells and microglia cells were identified.

[0120] Differential expression and pseudotemporal analysis: The differential expression genes of microglia cells between groups were compared. Pseudotemporal analysis tools (such as Monocle2) were used to infer the state transition trajectory of microglia cells under different treatment conditions.

[0121] qPCR verification: Microglia cells in mouse brain tissue were isolated, and the mRNA expression levels of fatty acid beta-oxidation key genes (such as CPT1A) and Trem2 were detected by real-time fluorescent quantitative PCR to experimentally verify the bioinformatics analysis results.

[0122] Experimental results The experimental results are shown in Figure 16 to Figure 19 .

[0123] Cell target identification and state transition: Single-cell analysis successfully resolved the cell composition of brain tissue. Differential expression analysis showed that microglia cells were the most significantly changed cell type in gene expression. As shown in Figure 16 , pseudotemporal analysis clearly showed that after MM-MnCO-NPs intervention, microglia cells showed a trajectory of transition to a steady or repair state, and the expression of Trem2 gene showed a significant upward trend along this trajectory.

[0124] By constructing a BV2 microglial cell inflammation model combined with LPS and hypoxia (LPS+Hypoxia), the effects of MM-MnCO-NPs on oxidative stress, inflammatory polarization and metabolic function were explored, and the role of Trem2 gene in it was analyzed.

[0125] like Figure 17 As shown, in terms of oxidative stress, LPS+Hypoxia treatment significantly increased intracellular ROS levels in BV2 cells. MM-MnCO-NPs treatment, on the other hand, exhibited a clear antioxidant trend, reducing ROS accumulation in both Trem2-KO and wild-type cells. Further mechanistic studies are expected in... Figure 18 As shown, LPS+Hypoxia inhibited Trem2 expression and activated the oxidative stress response factor Nrf1; MM-MnCO-NPs further activated the classical antioxidant axis NRF2–HO-1 and partially restored Trem2 expression levels. These results suggest that Trem2 affects the ability of MM-MnCO-NPs to scavenge intracellular oxidative stress, and its deficiency may weaken the antioxidant effect of nanoparticles.

[0126] Regarding inflammatory polarization, such as Figure 19 As shown, flow cytometry analysis revealed that LPS+Hypoxia induced BV2 cells to convert to the M1 genotype (CD86). + Polarization and reduction of M2 type (CD206) + The Trem2 deficiency makes cells more prone to the M1 phenotype under inflammatory stimulation. After treatment with MM-MnCO-NPs, M1 polarization was significantly inhibited and the proportion of the M2 phenotype increased, indicating that the nanoparticles can exert anti-inflammatory effects by regulating the inflammatory polarization state.

[0127] Mitochondrial function test results as follows Figure 20 As shown, LPS+Hypoxia induced mitochondrial membrane potential (Δψm) depolarization, resulting in an increased proportion of green fluorescence (Q3) in JC-1 staining. Trem2-deficient cells exhibited a stronger tendency for depolarization. MM-MnCO-NPs treatment partially restored the membrane potential, increasing the proportion of Q2 (high red, normal membrane potential), and this trend was observed in both Cas9 and Trem2-KO cells.

[0128] Cellular energy metabolism analysis, such as Figure 21 As shown, LPS+Hypoxia impaired the basal and maximal respiratory capacity of microglia. Treatment with MM-MnCO-NPs enhanced maximal and reserve respiratory capacity and partially restored basal respiratory levels. This effect was more pronounced in intact Trem2 cells, suggesting that Trem2 may be involved in the reconstruction of metabolic function.

[0129] Further analysis was conducted using RT-qPCR to detect the expression of key genes involved in fatty acid oxidation (FAO), such as... Figure 22As shown, LPS+Hypoxia down-regulated CPT1A and other FAO-related genes such as ACADL, while MM-MnCO-NPs treatment up-regulated the expression of these genes. However, in Trem2-KO cells, the restoration of FAO metabolism was inhibited, indicating that Trem2 plays an important role in regulating microglial lipid metabolism and energy supply.

[0130] As shown in the accompanying Figure 23 As shown, qPCR verified that compared with the model control group, the transcription levels of the rate-limiting enzyme CPT1A of fatty acid β-oxidation and Trem2 in microglial cells in the MM-MnCO-NPs treatment group of mice were significantly up-regulated, which was highly consistent with the prediction results of bioinformatics.

[0131] In this experimental example, through high-resolution single-cell transcriptome sequencing and multi-level bioinformatics analysis, it is revealed that the cell target of MM-MnCO-NPs is mainly microglial cells from the whole genome level, and the potential molecular mechanism of regulating microglial metabolic reprogramming (activating fatty acid β-oxidation and oxidative phosphorylation) through Trem2 as the core hub is elucidated, providing deep molecular biology evidence for the multi-mechanism synergistic treatment of the carrier system.

Claims

1. A macrophage membrane-coated drug carrier for high-altitude cerebral edema, characterized in that, It consists of a nanoparticle core and a macrophage membrane shell surrounding the core; The nanoparticle core contains a ROS-responsive carrier material and carbon monoxide-releasing molecules MnCO encapsulated in the carrier material. The content of phosphatidylserine in the macrophage membrane shell is not less than 5 mol% of the total phospholipids in the membrane; the phosphatidylserine can specifically bind to the Trem2 receptor on the surface of microglia, and the CO released by MnCO can upregulate the expression of Trem2 receptor in microglia, forming a positive feedback loop of "Trem2 activation - enhanced phagocytosis - CO release - upregulation of Trem2 expression".

2. The macrophage membrane-coated high-altitude cerebral edema drug carrier according to claim 1, characterized in that, The carrier material for the ROS response is an amphiphilic block copolymer; the amphiphilic block copolymer is formed by grafting a hydrophobic borate ester onto a hydrophilic dextran backbone.

3. The macrophage membrane-coated high-altitude cerebral edema drug carrier according to claim 1, characterized in that, The macrophage membranes are derived from RAW 264.7 cells or from primary macrophages derived from bone marrow.

4. The macrophage membrane-coated high-altitude cerebral edema drug carrier according to claim 1, characterized in that, The average particle size of the nanoparticle core is 80-200 nm, and the average particle size of the macrophage membrane-coated nanodrug delivery system is 120-240 nm.

5. The macrophage membrane-coated high-altitude cerebral edema drug carrier according to claim 1, characterized in that, The ROS-responsive support material degrades in environments with hydrogen peroxide concentrations of 0.1–10 mM.

6. A method for preparing a macrophage membrane-coated drug carrier for high-altitude cerebral edema as described in any one of claims 1-5, characterized in that, Macrophage membrane shells were prepared by ultrasonic fusion and liposome extrusion.

7. The use of a macrophage membrane-coated high-altitude cerebral edema drug carrier as described in any one of claims 1-5 in the preparation of a medicament for the prevention and / or treatment of high-altitude cerebral edema.

8. The use of the macrophage membrane-coated high-altitude cerebral edema drug carrier according to claim 7 in the preparation of a drug for the prevention and / or treatment of high-altitude cerebral edema, characterized in that, The preventive and / or therapeutic mechanisms include: upregulating Trem2 expression in microglia, promoting microglia polarization toward the M2 phenotype, and / or enhancing the fatty acid β-oxidation metabolism level of microglia.

9. A pharmaceutical composition, characterized in that, The drug carrier for high-altitude cerebral edema, comprising a therapeutically effective amount of the macrophage membrane-coated drug carrier as described in any one of claims 1-5, and a pharmaceutically acceptable carrier or excipient.

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