Functionalized exosome-traditional Chinese medicine small molecule composite preparation T-pEXOs@GB and preparation method and application thereof

Abstract

The application discloses a kind of functionalization exosome-traditional Chinese medicine small molecule composite preparation T-pEXOs@GB and its preparation method and application.The composite preparation encapsulates ginkgolide B (GB) in the exosome of healthy plasma, and is functionally modified neuron target peptide RVG (DSPE-PEG-RVG) on its surface, so that it has brain targeting and oxygen supply functions.The preparation method provided by the application can efficiently and controllably prepare the composite preparation with uniform particle size and good stability.The T-pEXOs@GB composite preparation of the application can be targeted to deliver to brain ischemia and hypoxia injury area, significantly reduce oxidative stress and inflammatory response, reduce cerebral infarction volume, improve neurological deficit score, and improve the survival rate of animal model, and can be used to prepare drugs for preventing and treating acute high altitude cerebral hypoxia, ischemic stroke, neonatal hypoxic-ischemic encephalopathy and other cerebral ischemia and hypoxia injury, and has good market prospect.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine, specifically to a functionalized exosome-traditional Chinese medicine small molecule composite preparation T-pEXOs@GB and its preparation method and application. Background Technology

[0002] Acute high-altitude hypoxia, ischemic stroke, and other hypoxia-related diseases can lead to brain tissue energy metabolism disorders, mitochondrial dysfunction, increased oxidative stress, and neuroinflammatory responses, ultimately causing neuronal damage and even death. Acute mountain sickness and reduced military operational capacity caused by hypoxia are major medical problems for troops rapidly advancing to high altitudes, severely impacting the health and combat effectiveness of soldiers stationed there. Current treatment strategies mainly rely on drug intervention and oxygen supply measures, such as acetazolamide, but these drugs have low efficacy and poor subtype selectivity, and there are no corresponding raw materials available in my country. They also commonly cause adverse reactions such as dry mouth, fever, and rash. Dexamethasone and other glucocorticoids have issues with individual variability and significant side effects, and cannot completely reverse hypoxia-induced mitochondrial dysfunction, oxidative stress, and cell damage. Pre-treatment with traditional Chinese medicine formulas and hypoxia preconditioning can temporarily alleviate hypoxia, but cannot fundamentally improve it. Furthermore, people experiencing acute mountain sickness, including soldiers, often need 2-3 days after leaving the hypoxic environment to recover. Furthermore, most drugs struggle to effectively cross the blood-brain barrier (BBB) ​​and have poor brain targeting; the treatment window is limited, making it difficult to sustainably improve brain tissue oxygenation. Drugs are prone to causing side effects such as mitochondrial dysfunction, oxidative stress, and genomic instability; and there is a lack of treatment systems that combine precise delivery, sustained oxygen supply, and multi-target regulation capabilities.

[0003] In recent years, artificial oxygen nanocarriers have attracted widespread attention from the academic community. Among them, representative oxygen-carrying systems such as hemoglobin-derived preparations and PFC emulsions provide safe and efficient new intervention strategies for acute hypoxia in early-stage stroke patients by mimicking physiological oxygen transport mechanisms.

[0004] Plasma-Derived Exosomes (pEXOs) carriers possess excellent biocompatibility and extremely low immunogenicity. Their surface retains membrane proteins and lipids from various cell sources, making them less susceptible to rapid clearance by the mononuclear phagocytic system during in vivo circulation, resulting in longer circulation times and better in vivo distribution characteristics. Furthermore, as endogenous nanocarriers, pEXOs effectively avoid immune rejection reactions caused by foreign materials, offering higher safety and suitability for repeated drug administration or long-term use. In addition, studies have shown that pEXOs have the potential to cross the brain's biological barrier (BBB), entering brain tissue via endocytosis and transcellular transport, thus providing a new technological pathway for drug delivery in central nervous system diseases. Compared to traditional drug or carrier systems, pEXOs exhibit higher efficiency and lower risk of off-target distribution in delivering small molecule drugs, proteins, peptides, and nucleic acid-based active substances. However, current pEXOs-based drug delivery strategies still face many problems: (1) efficiency issues, as the high expression of phosphatidylserine on the outer leaflets of the lipid bilayer hinders its reach to the target site, resulting in low drug loading efficiency; (2) insufficient targeting of natural exosomes, as their surfaces lack specific receptors for specific tissues or cells, making it impossible to actively recognize target cells. Therefore, constructing an efficient and safe small molecule delivery system for traditional Chinese medicine is the core key to the clinical development and translation of pEXOs drug delivery systems.

[0005] Ginkgolide B (GB) is a diterpenoid lactone compound extracted from Ginkgo biloba leaves. It exhibits significant antiplatelet-activating factor (PAF) activity, anti-inflammatory, antioxidant, and neuroprotective effects. In recent years, Ginkgolide B has shown promising potential in the treatment of cardiovascular diseases, ischemic stroke, and neurodegenerative diseases. Numerous studies have confirmed the neuroprotective effect of GB in animal models of cerebral ischemia-reperfusion injury. Oral administration of GB can alleviate hypoxia-induced hippocampal neuronal damage in rats by inhibiting oxidative stress and apoptosis, suggesting its potential as a therapeutic agent for improving ischemic stroke injury. Ginkgolide B can reduce the release of inflammatory factors (TNF-α, IL-6, IL-1β) induced by cerebral ischemia by inhibiting the CD40 / NF-κB pathway. However, the poor water solubility, poor in vivo stability, poor absorption, and lack of tissue targeting of most small molecules in traditional Chinese medicine have limited bioavailability, which has been a major drawback restricting their clinical application. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a functionalized exosome-traditional Chinese medicine small molecule composite preparation, T-pEXOs@GB, along with its preparation method and applications. This is achieved by co-encapsulating a perfluorocarbon (PFC) compound and ginkgolide B (GB) within plasma exosomes, and functionalizing the surface of these exosomes with the neuron-targeting peptide RVG, thus creating a composite exosome preparation with brain-targeting and oxygen-supplying functions. The present invention obtains natural anti-inflammatory molecular characteristics through transcriptome sequencing of healthy exosomes, aiming to enhance the effect of cerebral hypoxia through the synergistic effect of the two, delivering natural small molecule antioxidant drugs to hypoxic brain sites, and promoting the transformation of microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype to improve neuroinflammatory conditions. This further highlights the significant clinical value of T-pEXOs@GB in the treatment of cerebral hypoxia and related diseases.

[0007] To address the problems in the existing technology, the technical solution adopted by this invention is as follows: A method for preparing a functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB includes the following steps: S1. Isolation and purification of plasma-derived exosomes (pEXOs): Centrifuge at 300×g for 10 min to remove blood cells; collect the supernatant and centrifuge at 3000×g for 15 min to further remove cell residues and large particulate impurities; filter the obtained supernatant through a 0.8 μm pore size filter membrane to remove residual cell debris, apoptotic bodies and large particles; collect the supernatant and centrifuge at 100,000×g at 4℃ for 90 min; finally, resuspend in PBS to obtain plasma exosomes. S2, Preparation of targeted exosomes (T-pEXOs): The pEXOs suspension obtained in step S1 is mixed with the targeted peptide and incubated to fully fuse with the exosomes. After purification by ultrafiltration and centrifugation, T-pEXOs are obtained. S3, Assembly and purification of the compound preparation (T-pEXOs@GB): The T-pEXOs obtained in step S2 were incubated with the traditional Chinese medicine small molecule ginkgolide B (GB) and perfluorocarbon compound (PFC) under ultrasound for 20 minutes, and then extruded 12 times through a liposome extruder to collect the target components, thus obtaining T-pEXOs@GB.

[0008] Preferably, in step S2, the targeting peptide is composed of DSPE-PEG-RVG and cholesterol in a molar ratio of 55:40.

[0009] Preferably, in step S3, the mass concentration ratio of T-pEXOs to ginkgolide B is 1 mg / mL : 100 mg / kg (based on protein).

[0010] The functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB is prepared by any of the methods described above.

[0011] A pharmaceutical composition comprising the above-described functionalized exosome-traditional Chinese medicine small molecule complex T-pEXOs@GB, and a pharmaceutically acceptable carrier or excipient.

[0012] Preferably, the dosage form of the pharmaceutical composition is an injection.

[0013] The use of the above-mentioned functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB or the above-mentioned pharmaceutical composition in the preparation of a drug for the prevention and / or treatment of ischemic hypoxic-ischemic brain injury.

[0014] Preferably, the ischemic hypoxic brain injury includes brain injury caused by acute high-altitude cerebral hypoxia, ischemic stroke, or middle cerebral artery occlusion (MCAO), or brain injury caused by neonatal intrauterine distress.

[0015] Preferably, the drug is administered via intravenous injection.

[0016] Preferably, the dosage of T-pEXOs@GB in the drug is 1 mg / mL based on protein content. Beneficial effects

[0017] Compared with existing technologies, the present invention provides a functionalized exosome-traditional Chinese medicine small molecule complex formulation T-pEXOs@GB, its preparation method, and its application. Based on a brain-targeted oxygen-supplying complex biological agent derived from healthy plasma exosomes (preferably comprising a system in which perfluorocarbons (PFCs) and natural traditional Chinese medicine small molecule active ingredients such as ginkgolide B (GB) are co-encapsulated in plasma exosomes and then functionalized on the surface with RVG (such as DSPE-PEG-RVG)), the resulting functionalized exosome-traditional Chinese medicine small molecule complex formulation T-pEXOs@GB has the following advantages: This invention utilizes healthy plasma exosomes as a natural nanodelivery carrier and performs surface functionalization using brain-targeting ligands such as RVG. This allows the formulation to cross the blood-brain barrier via transcellular transport and / or endocytosis under ischemic, hypoxic, or inflammatory conditions, leveraging the increased permeability of brain microvascular endothelium. This results in higher levels of enrichment in brain tissue, significantly improving the efficiency of active ingredients entering the central nervous system and overcoming the limitation of traditional small-molecule drugs (GB) which have difficulty entering brain tissue at effective doses. The natural Chinese medicine small molecules and PFCs form a stable nanoscale composite structure encapsulated in exosomes, which improves the stability of active ingredients during in vivo circulation, reduces non-specific losses, and enables gradual release in the local hypoxic microenvironment of brain tissue, providing a basis for sustained intervention.

[0018] Compared to traditional oxygen therapy or systemic drug delivery, this invention achieves "local, continuous oxygen supply regulation" at the brain microenvironment level, providing upstream conditions for downstream anti-inflammatory, cell protection, and neurological function improvement. While improving oxygen supply, it reduces oxidative stress and inhibits the amplification of inflammatory signals, achieving multi-dimensional synergistic treatment of "oxygen support + anti-inflammation," thereby improving overall efficacy and reducing the limitations of single interventions. Hypoxia can induce the massive release of inflammatory factors and trigger apoptosis or inflammatory programmed cell death (such as pyroptosis), thus exacerbating neuroinflammation and neuronal damage. This invention improves mitochondrial oxidative phosphorylation efficiency, reduces oxidative stress, and inhibits inflammatory amplification through local oxygen supply, helping to reduce the incidence of hypoxia-related apoptosis or inflammatory cell death. This allows for more precise and effective intervention in the core pathological link of "hypoxia-inflammation-cell death," providing a new direction for the treatment of local hypoxic microenvironments in brain tissue.

[0019] Exosomes derived from healthy plasma, as natural carriers, have good biocompatibility and low immunogenicity, high overall safety, and are suitable for repeated or long-term dosing needs, showing good potential for clinical translation.

[0020] This invention, a functionalized exosome-traditional Chinese medicine small molecule complex, T-pEXOs@GB, has a wide range of applicable diseases, especially those related to central nervous system hypoxia, such as acute high-altitude cerebral hypoxia, ischemic stroke, cerebral ischemia-reperfusion injury, and perinatal hypoxic-ischemic encephalopathy in newborns. For brain diseases characterized by significant blood-brain barrier restriction, prolonged local oxygen deficiency, and prominent inflammatory responses, this invention demonstrates even greater application value. Experiments have shown that this formulation exhibits positive therapeutic effects in various cerebral ischemia-hypoxia models. Furthermore, due to its good biocompatibility, low immunogenicity, and high safety, this formulation is suitable for various populations, including personnel stationed at high altitudes, military personnel, high-altitude tourists, and other special populations at risk of hypoxia, showing promising prospects for widespread application and holding significant strategic importance in the fields of high-altitude medicine, military medicine, and translational medicine for cerebral hypoxia-related diseases. Attached Figure Description

[0021] Figure 1 The following are the characterization diagrams of the T-pEXOs@GB composite formulation, where (a) is the TEM image and (b) is the high-performance liquid chromatography encapsulation efficiency diagram of the T-pEXOs@GB composite formulation. Figure 2 The particle size distribution of the T-pEXOs@GB composite formulation is shown in the diagrams. (a) shows the particle size distribution of pEXOs, (b) shows the particle size distribution of T-pEXOs, and (c) shows the particle size distribution of T-pEXOs@GB. All three particle sizes are between 50-100 nm. Figure 3The images show the characterization of marker proteins in the T-pEXOs@GB compound formulation, where (a) is the exosome marker protein detection image and (b) is the NTA purity detection image. Figure 4 A trend graph of inflammatory factors in the plasma of acutely hypoxic mice; Figure 5 This is a map showing the in vivo targeted distribution of the T-pEXOs@GB compound formulation in hypoxic mice. Figure 6 This is a pathological staining image of brain tissue from mice with acute hypoxia. Figure 7 This is a flowchart illustrating the preparation process of the T-pEXOs@GB composite formulation of the present invention. Detailed Implementation

[0022] Example 1: Preparation of T-pEXOs@GB Formulation preparation: Plasma exosomes were separated by ultracentrifugation and size exclusion chromatography; Experimental methods: Peripheral blood was collected from healthy male C57BL / 6 mice, and whole blood was anticoagulated with EDTA. The plasma was separated by centrifugation at 800×g for 10 min at 4 ℃. Then, the plasma was centrifuged at 3000×g for 20 min at 4 ℃ and 10,000×g for 20 min at 4 ℃ to remove cell debris and macromolecular impurities. The plasma was aliquoted into 3 mL tubes and stored at -80 °C for later use. Take 3 mL of plasma, centrifuge at 100,000×g for 30 min at 4 °C, transfer the supernatant to a new centrifuge tube, filter the supernatant through a 0.8 μm pore size filter membrane, and then centrifuge at 100,000×g for 90 min to precipitate pEXOs. Resuspend the precipitate in 5 mL of pre-chilled PBS, ultracentrifuge at 100,000×g for 90 min to wash the pEXOs, and resuspend the precipitate in 200 μL of pre-chilled PBS to obtain the pEXOs suspension. Resuspend in PBS and determine the total protein concentration using the BCA method. Purity (e.g., e ... 7 above).

[0023] The pEXOs resuspension was mixed with the targeting peptide (a mixture of DSPE-PEG-RVG and cholesterol at a molar ratio of 55:40) and incubated at 37°C and 150 rpm for 2 hours on a shaker. Then, polyethylene glycol (at a molar ratio of 200:1 to pEXOs protein) was added, and the mixture was reacted at room temperature in the dark for 1 hour. The reaction solution was transferred to a 100 kDa ultrafiltration centrifuge tube, centrifuged at 3000×g for 15 minutes at 4°C, washed three times with PBS, and the retentate was collected to obtain T-pEXOs.

[0024] Weigh 21.21 mg of GB and dissolve it in 1 mL of DMSO to obtain a GB suspension (final concentration 100 mg / kg). Mix the above T-pEXOs (final concentration 1 mg / mL, based on protein) with the GB suspension and incubate at room temperature and 200 rpm in the dark for 3 hours. Sonicate the pEXOs suspension with RVG (1 mg / mL) at room temperature at a frequency of 40 kHz; sonication power: 100 W; total sonication time: 60 min. During sonication, maintain the sample temperature below 30 °C to avoid damage to the exosome structure. Add the reaction mixture to a sucrose density gradient solution (8% / 30% / 45% / 60%) and centrifuge at 4 °C and 100,000 × g for 90 minutes. Collect the milky white band at the 30%-45% interface layer, dilute with PBS, centrifuge at 4 °C and 10,000 × g for 20 minutes, and discard the precipitate. The supernatant is T-pEXOs@GB. It is resuspended in PBS to the required concentration of 1 mg / mL and stored at 4°C in the dark for later use. The concentration used in subsequent examples is 1 mg / mL.

[0025] Experimental Results and Analysis: Figure 2 This is a TEM image. As can be seen from the image, the exosomes T-pEXOs@GB exhibit a typical cup-shaped structure, with the particle size distribution concentrated in the range of 30-150 nm. Figure 3 The image shows a Western blot result, indicating that CD9, CD63, and TSG101 are positive.

[0026] Example 2: T-pEXOs@GB treatment for acute high-altitude cerebral hypoxia Ten- to twelve-week-old male C57BL / 6 mice were placed in a hypobaric chamber at an altitude of 6000 m to establish an acute high-altitude hypoxia model and to evaluate the in vivo efficacy of T-pEXOs@GB in treating acute high-altitude cerebral hypoxia. The normal control group was the Sham group; hypoxic mice were randomly divided into four groups of 14 mice each: PBS group, GB group, T-pEXOs group, and T-pEXOs@GB group. Each group of mice received a tail vein injection of 100 μL of different formulations (final concentration of T-pEXOs: 1 mg / mL; final concentration of GB: 100 mg / kg; final concentration of T-pEXOs@GB group: 1 mg / mL). After 7 days of continuous drug administration (prevention) and 1 day of hypoxia (treatment) in mice, the plasma levels of interferon-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Brain tissue from each group was subjected to HE & Nisel staining and immunofluorescence co-localization, and pathological observation was performed under an optical microscope. Brain injury and recovery were monitored through daily survival records and neurobehavioral scores. The survival time of surviving mice in each group was recorded, and brain tissue samples were collected at predetermined time points for analysis of pathological and biochemical indicators.

[0027]

[0028] In an acute high-altitude hypobaric hypoxia-induced brain injury model ( Figure 4 Mice in the PBS group showed significant neurological deficits immediately after leaving the chamber, as evidenced by a significant increase in neurological deficit scores. The T-pEXOs@GB treatment group showed a trend of functional improvement starting from day 1, and the scores decreased significantly on days 3 and 7, indicating that the overall neurological function recovery was accelerated.

[0029] Example 3: T-pEXOs@GB for the treatment of focal cerebral ischemia (MCAO) The in vivo efficacy of T-pEXOs@GB in treating focal cerebral ischemia in 10-12 week-old C57BL / 6 male mice was evaluated using a middle cerebral artery occlusion (MCAO) model. The MCAO model was established using the suture occlusion method: under anesthesia, the common carotid artery, external carotid artery, and internal carotid artery were exposed, and a silicone-coated suture was inserted through the external carotid artery to the origin of the middle cerebral artery to block blood flow, maintaining ischemia for a preset time (60 min). Mice with successful model establishment were randomly divided into four groups of 14 mice each: sham-operated group (Sham), model control group (MCAO+PBS), non-targeted agent group (MCAO+pEXOs@GB), and targeted therapy group (MCAO+T-pEXOs@GB). Except for the sham-operated group, mice in the other groups received an intravenous injection of 100 μL of the corresponding agent (final concentration of pEXOs@GB was 1 mg / mL, and final concentration of T-pEXOs@GB was 1 mg / mL) immediately after reperfusion (or model establishment) via the tail vein. The degree of injury was assessed using a neurological deficit score (Example 2) and cerebral infarction volume measurement (MRI), and survival status was recorded and molecular biological analysis was performed at preset time points.

[0030] The T-pEXOs@GB combined treatment group showed significantly better neuroprotective effects against cerebral ischemia than the GB treatment group alone and the T-pEXOs monotherapy group, indicating that the T-pEXOs@GB combined treatment can effectively reduce the infarct volume after middle cerebral artery occlusion and improve neurological deficits. Survival curve results showed that the postoperative survival rate of mice in the T-pEXOs@GB combined treatment group was significantly higher than that of other groups, indicating that this combined strategy can significantly reduce early mortality after cerebral ischemia, improve animal survival rate, and demonstrate significant neuroprotective and synergistic therapeutic effects.

[0031] Example 4

[0032] To evaluate the in vivo efficacy of T-pEXOs@GB in treating neonatal brain injury induced by intrauterine hypoxia in mice using an intrauterine hypoxia model. Bilateral uterine arteries were ligated in 17-day-gestation C57BL / 6 female mice, followed by cesarean section on day 22, resulting in the loss of neurons in the parietal cortex of newborn mice. Disordered cerebral cortical stratification was observed 7 days after birth. Stenosis of the maternal uterine arteries was induced using metal microcoils. Pups born to successfully modeled pregnant mice were randomly divided into four groups of 12 mice each: normoxic control group (Normoxia), model control group (Hypoxia+PBS), non-targeted treatment group (Hypoxia+pEXOs@GB), and targeted treatment group (Hypoxia+T-pEXOs@GB). Except for the normoxic control group, all other groups received an intravenous injection of 100 μL of the corresponding preparation (pEXOs@GB final concentration of 1 mg / mL, T-pEXOs@GB final concentration of 1 mg / mL) via the tail vein of pregnant mice (or immediately after birth) after modeling in pregnant mice (or immediately after birth). The degree of damage was assessed by pup survival rate, neurobehavioral scores, and brain tissue pathology and biochemical indicators, and growth and development were monitored and molecular biological analyses were performed at preset time points.

[0033] The T-pEXOs@GB combined treatment group showed significantly better protective effects against brain injury caused by intrauterine distress in newborns than the GB treatment group alone and the T-pEXOs monotherapy group. This indicates that the T-pEXOs@GB combined treatment can effectively reduce pathological brain tissue damage following intrauterine hypoxia and ischemia, and improve the neurobehavioral scores of newborn mice. Long-term developmental follow-up results showed that the long-term survival rate and learning and memory abilities of newborn mice in the T-pEXOs@GB combined treatment group were significantly better than those in other groups. This indicates that this combined strategy not only improves perinatal survival but also promotes long-term nervous system development, demonstrating a strong neuroprotective synergistic effect and the potential to improve prognosis.

[0034] Example 5: Validation of the "therapeutic window" of T-pEXOs@GB in cerebral ischemia-reperfusion injury (delayed dosing) The MCAO / R model was established using the suture occlusion method: Ten- to twelve-week-old C57BL / 6 female mice were anesthetized and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A silicone-coated suture was inserted through an ECA incision to the origin of the middle cerebral artery to block blood flow. Ischemia was maintained for a predetermined time (e.g., 60 min), after which the suture was removed to restore reperfusion. This reperfusion initiation point was recorded as t=0. Successful model establishment was determined by postoperative neurological deficits and / or decreased cerebral blood flow; mice that did not meet the criteria or experienced significant intraoperative complications (such as massive hemorrhage) were excluded.

[0035] MCAO / R model mice were randomly divided into four groups of four mice each: PBS group, T-pEXOs@GB group (administered at 0 h), T-pEXOs@GB group (administered at 2 h), and T-pEXOs@GB group (administered at 6 h). The injection volume for each group was uniformly 100 μL. The T-pEXOs@GB dosage was 1 mg / kg based on exosome protein. The PBS group served as an equal-volume control. Neurological function was assessed at predetermined time points (24 h post-surgery) using the method described in Example 2 (mNSS scoring), and the scoring was performed by blinded evaluators. Small animal MRI scans were performed at predetermined time points (24 h or 72 h post-surgery) to assess the high-signal area of ​​the ischemic region. The infarct area was delineated layer by layer and its volume was calculated using image analysis software.

[0036] Compared with the PBS group, T-pEXOs@GB showed a certain degree of neuroprotective effect in different administration time windows, with the most significant effect when administered within 0–2 h: (1) MRI results showed that the infarct volume of the T-pEXOs@GB group administered at 0 h and 2 h was significantly lower than that of the PBS group, and the reduction was better than that of the 6 h administration group; (2) Neurological function scores (Example 2) showed that the neurological deficits of the 0 h and 2 h administration groups were significantly improved, indicating that the functional recovery was more complete; (3) When the administration was delayed to 6 h, a certain degree of protective trend or improvement of some indicators (infarct volume reduction trend, score improvement trend) could still be observed, indicating that the system still has certain intervention value in the reperfusion-related inflammation amplification stage.

[0037] In summary, T-pEXOs@GB has an operable dosing time window in the MCAO / R model. It is preferable to administer the drug within 0–2 h after reperfusion to obtain more significant radiological and functional benefits. Under the condition of delayed dosing at 6 h, some protective effects may still be retained, providing a time window basis for subsequent clinical translation.

Claims

1. A method for preparing a functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB, characterized in that, Includes the following steps: S1, Isolation and purification of plasma-derived exosomes: Centrifuge at 300×g for 10 min to remove blood cells; After collecting the supernatant, centrifuge at 3000×g for 15 min to further remove cell residues and large particulate impurities; the obtained supernatant is filtered through a 0.8 μm pore size filter membrane to remove residual cell debris, apoptotic bodies and large particles, and the supernatant is collected and centrifuged at 100,000×g at 4℃ for 90 min; finally, plasma exosomes are obtained by resuspending in PBS. S2, Preparation of targeted exosomes: The pEXOs suspension obtained in step S1 is mixed with the targeted peptide and incubated to fully fuse with the exosomes. After purification by ultrafiltration and centrifugation, T-pEXOs are obtained. S3, Assembly and purification of the compound preparation: The T-pEXOs obtained in step S2 were incubated with the traditional Chinese medicine small molecule ginkgolide B and perfluorocarbon compound under ultrasound for 20 minutes, and then extruded 12 times through a liposome extruder to collect the target component, thus obtaining T-pEXOs@GB.

2. The method for preparing a functionalized exosome-traditional Chinese medicine small molecule complex formulation according to claim 1, characterized in that, In step S2, the targeting peptide is composed of DSPE-PEG-RVG and cholesterol in a molar ratio of 55:

40.

3. The method for preparing a functionalized exosome-traditional Chinese medicine small molecule complex formulation according to claim 1, characterized in that, In step S3, the mass concentration ratio of T-pEXOs to ginkgolide B is 1 mg / mL : 100 mg / kg (based on protein).

4. The functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB prepared by the method according to any one of claims 1-3.

5. A pharmaceutical composition comprising the functionalized exosome-traditional Chinese medicine small molecule complex formulation T-pEXOs@GB as described in claim 4, and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is an injection.

7. The use of the functionalized exosome-traditional Chinese medicine small molecule complex preparation T-pEXOs@GB according to claim 4 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for the prevention and / or treatment of ischemic hypoxic-ischemic brain injury.

8. The application according to claim 7, characterized in that, The ischemic and hypoxic brain injury mentioned includes brain injury caused by acute high-altitude cerebral hypoxia, ischemic stroke, or occlusion of the middle cerebral artery, or brain injury caused by neonatal intrauterine distress.

9. The application according to claim 7, characterized in that, Functionalized exosome-traditional Chinese medicine small molecule compound preparations or drug compositions are administered via intravenous injection.

10. The application according to claim 9, characterized in that, The dosage of T-pEXOs@GB in functionalized exosome-traditional Chinese medicine small molecule compound preparations or drug compositions is 1 mg / mL on a protein basis.

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