AP39 delivery system and preparation method and application thereof

Abstract

The invention discloses an AP39 delivery system and a preparation method and application thereof, and belongs to the technical field of molecular diagnosis and molecular biology. The AP39 delivery system AP39 (at) Lip is prepared by a film hydration method, and the AP39 delivery system AP39 (at) Lip is subjected to characterization detection, so that the AP39 delivery system AP39 (at) Lip has general characteristics of liposomes and can realize slow release of drugs. The AP39 delivery system prepared by the invention can improve the H2S level of the damaged brain region of an HI mouse through nasal delivery, inhibit neuronal apoptosis and activation of microglial cells, relieve neuroinflammation and oxidative stress response, relieve mitochondrial injury, play an effective neuroprotective role in hypoxic ischemic brain injury, and can be used for treating hypoxic ischemic brain injury. And a new thought is provided for application of small molecule drugs in clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis and molecular biology, and particularly relates to an AP39 delivery system and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Neonatal hypoxic-ischemic encephalopathy (HI) is a brain injury caused by perinatal asphyxia and is a common disease that leads to neonatal death and long-term neurological dysfunction in survivors. Currently, the clinical treatment for this disease is mainly comprehensive treatment, combining mild hypothermia, hyperbaric oxygen, magnesium sulfate, and gangliosides. However, the treatment effect is not significant and is very likely to cause adverse consequences such as tissue damage and infection. Oxidative stress is one of the important factors that aggravate HI brain damage. Mitochondria are the main site of reactive oxygen species production. Reducing the production of reactive oxygen species can effectively alleviate HI brain damage. However, due to the special nature of the mitochondrial membrane structure, it is difficult to achieve targeted treatment of mitochondria. Therefore, there is an urgent need to improve our understanding of the HI brain damage process in order to explore various new therapies for brain damage caused by hypoxia and ischemia.

[0004] Research has shown that hydrogen sulfide (H2S), an endogenous gaseous signaling molecule, possesses a wide range of biological activities, including vasodilatory, cardioprotective, anti-inflammatory, antioxidant, and anti-tumor effects. Hydrogen sulfide inhibits oxidative stress and effectively alleviates HI brain damage by directly scavenging reactive oxygen species (ROS) within mitochondria, where they are produced. However, the diffusion of H2S within the body is not specifically targeted, making it difficult to precisely control its distribution and concentration when using H2S donors, potentially leading to uncertainty in therapeutic effects and potential safety risks. Furthermore, the use of H2S donors makes it difficult to determine the threshold level of H2S in the body, and subtle changes in H2S levels may lead to adverse effects on specific functions. Therefore, targeted delivery and controlled release of H2S are crucial for achieving effective treatment and ensuring biosafety. These limitations indicate that the key challenge in the development of H2S donor drugs lies in improving the selectivity of H2S donor molecules and releasing appropriate concentrations of H2S at the target site (typically the lesion site) to exert therapeutic effects while limiting adverse reactions. Current research focuses on the design and development of hydrogen sulfide donor drugs with controlled release potential to provide new therapeutic strategies and ideas.

[0005] AP39, a mitochondrially targeted H2S donor, specifically releases H2S within mitochondria, and has been shown to alleviate oxidative stress and mitochondrial damage, exerting a protective effect. However, in existing animal studies, AP39 requires multiple intravenous or intraperitoneal injections over several months, making clinical application difficult due to the pain it causes patients and the need for expensive specialized treatment. Therefore, the development of new delivery methods for AP39 is urgently needed. Summary of the Invention

[0006] In order to address the deficiencies of the prior art, the present invention aims to provide an AP39 delivery system, a preparation method, and an application thereof. The present invention utilizes liposomes to encapsulate AP39, thereby preventing the drug from being cleared or degraded before taking effect in the human physiological environment. Intranasal administration can bypass the blood-brain barrier and allow the drug to directly enter the central nervous system, thereby having the advantages of avoiding the first-pass effect and improving bioavailability. The system causes less damage and irritation to the body, making it more feasible for clinical treatment applications.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides an AP39 delivery system comprising AP39, soybean lecithin and cholesterol.

[0009] Among nanoparticle drug delivery systems, liposomes are the most widely used drug carriers due to their favorable physicochemical properties, ability to protect drugs from degradation, and high biocompatibility. They are also the only nanoparticle system approved for clinical use by the U.S. Food and Drug Administration (FDA). Using liposomes for drug delivery can improve the safety and efficacy of therapeutic molecules such as drugs, nucleic acids, vaccines, and proteins. Liposome encapsulation of drugs can prevent clearance or degradation before the drugs can take effect in the human physiological environment, providing a drug delivery method more suitable for clinical applications.

[0010] In some embodiments of the present invention, the mass ratio of AP39, soybean lecithin and cholesterol is 1:(40-80):(10-20).

[0011] In some embodiments of the present invention, the mass ratio of AP39, soybean lecithin and cholesterol is 1:(40-60):(10-15).

[0012] In some embodiments of the present invention, the mass ratio of AP39, soybean lecithin and cholesterol is 1:50:12.5.

[0013] In some embodiments of the present invention, the particle size of the AP39 delivery system is 35-40 nm.

[0014] In a second aspect, the present invention provides a method for preparing the above-mentioned AP39 delivery system, comprising:

[0015] AP39, soybean lecithin and cholesterol are dissolved in an organic solvent, mixed and evaporated to dryness to obtain a liposome film;

[0016] Phosphate buffer was added to the liposome film for hydration to obtain the AP39 delivery system.

[0017] In some embodiments of the present invention, the organic solvent is a volatile organic solvent, preferably an alcohol, and more preferably ethanol.

[0018] In some embodiments of the present invention, the concentration of the phosphate buffer is 0.005-0.015M, preferably 0.01M.

[0019] The third aspect of the present invention provides a use of the above-mentioned AP39 delivery system or the AP39 delivery system prepared by the above-mentioned preparation method in the preparation of a drug for treating hypoxic-ischemic brain damage.

[0020] In some embodiments of the present invention, the administration method of the drug for treating hypoxic-ischemic brain damage is nasogastric administration.

[0021] The beneficial effects of the present invention are:

[0022] The present invention prepared AP39-encapsulated liposomes (AP39@Lip), or the AP39 delivery system, by a thin film hydration method. Characterization and testing demonstrated that the system possessed the general properties of liposomes and could achieve slow drug release, making it suitable for subsequent experiments. AP39@Lip was administered via nasogastric tube feeding at 2, 24, and 48 hours after HI surgery, while a control group received intraperitoneal injection of AP39 at the same dose. Three days after surgery, cortical tissue samples from the injured side were examined. The results showed that nasal delivery of AP39@Lip and intraperitoneal injection of AP39 upregulated H2S levels in the injured cortex, mitigated cerebral infarction and brain tissue loss caused by HI brain injury, alleviated HI-induced neuronal apoptosis, inhibited microglial activation, downregulated inflammatory cytokines, reduced total reactive oxygen species (ROS) levels and mitochondrial ROS levels in the injured cortex, reversed the decrease in mitochondrial membrane potential and ATP content, upregulated the expression of mitochondrial-related proteins, alleviated oxidative stress and mitochondrial damage caused by HI brain injury, and exerted a neuroprotective effect. Based on the above experimental results, the AP39 delivery system prepared by the present invention can be delivered nasally to increase the H2S level in the damaged brain area of ​​HI mice, inhibit neuronal apoptosis and microglial activation, reduce neuroinflammation and oxidative stress response, alleviate mitochondrial damage, and play an effective neuroprotective effect in hypoxic-ischemic brain damage, providing new ideas for the application of small molecule drugs in clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 Characterization and identification of AP39@Lip prepared in Example 1 of the present invention, wherein A is an electron micrograph of blank liposomes and the AP39 delivery system, B is a particle size analysis of blank liposomes, C is a chromatogram of AP39, D is a standard curve of AP39, and (E) is the drug release curve of AP39@Lip from 0 to 50 hours;

[0025] Figure 2 Figure 1 shows that AP39@Lip prepared in Example 1 of the present invention increases H2S levels in the damaged cortex of neonatal mice after HI brain injury and reduces HI-induced cerebral infarction and neural tissue loss. Figure A shows H2S content in the damaged cortex of mice 72 hours after HI, Figure B shows representative brain images of each group, Figure C shows quantitative analysis of cerebral infarction area in each group, and Figure D shows Nissl staining of brain tissue in each group.

[0026] Figure 3Figure 1 shows the AP39@Lip prepared in Example 1 of the present invention reducing apoptosis of neurons in the damaged cortex after HI brain injury in neonatal mice. Figure A shows a TUNEL staining image of neurons in the cortex and hippocampus of the right damaged area 72 hours after HI. Figure B shows a statistical graph of the number of apoptotic neurons in the right damaged cortex. Figure C shows a statistical graph of the number of apoptotic neurons in the right damaged hippocampus. Figure D shows the Western blotting results of cleaved-caspase-3 and caspase-3 in the damaged cortex. Figure E shows a quantitative analysis of changes in protein expression of cleaved-caspase-3 and caspase-3 in the damaged cortex.

[0027] Figure 4 Figure 1 shows the AP39@Lip prepared in Example 1 of the present invention alleviating the activation of microglia and the release of inflammatory factors in the damaged cortex of neonatal mice after HI brain injury. Figure A shows a representative Iba-1 staining of the right damaged cortex 72 hours after HI injury, Figure B shows the Western blotting results and quantitative analysis of iNOS in the isolated right damaged cortex 72 hours after HI injury, and Figure C shows the Western blotting results and quantitative analysis of TNF-α in the isolated right damaged cortex 72 hours after HI injury.

[0028] Figure 5 Figure 3 shows that the AP39@Lip prepared in Example 1 of the present invention reduces the ROS level in the damaged cortex of neonatal mice after HI brain injury and alleviates mitochondrial damage caused by HI brain injury. A is the total ROS content in the fresh tissue of the damaged cortex of the mouse, B is the mitochondrial ROS content in the fresh tissue of the damaged cortex of the mouse, C is the mitochondrial membrane potential in the fresh tissue of the damaged cortex of the mouse, D is the ATP content in the fresh tissue of the damaged cortex of the mouse, E is the Western blotting results and quantitative analysis of SOD2 in the isolated right damaged cortex 72 hours after HI injury, and F is the Western blotting results and quantitative analysis of TOMM20 in the isolated right damaged cortex 72 hours after HI injury. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0030] Example 1

[0031] 1. Experimental process:

[0032] 1.HI model:

[0033] Experimental animals were 7-day-old (postnatal day, P7) C57BL / 6J suckling mice weighing 4 to 6 g. Animals were purchased from the Experimental Animal Center of Shandong University. Animal experiments were conducted in accordance with the International Guidelines for Animal Research provided by the Council for International Organizations of Medical Sciences and approved by the Animal Ethics and Welfare Committee of Shandong University. All experimental personnel were trained in accordance with the guidelines of the Animal Care and Use Committee.

[0034] The HI model was established in 7-day-old C57BL / 6J neonatal mice using the Rice–Vannucci method. Specifically, C57BL / 6J mice were anesthetized with 2% isoflurane and the right common carotid artery was ligated. After resting for 30 minutes, the mice were placed in a hypoxic environment (9.7% O₂) for 60 minutes. In the sham group, the right common carotid artery was exposed after anesthesia, but the artery was not ligated, and the skin was sutured without hypoxia.

[0035] 2. Preparation of AP39 Liposomes

[0036] 20 mg of soybean lecithin and 5 mg of cholesterol were weighed and ultrasonically dispersed in 10 mL of ethanol. Then, 0.4 mL of AP39 (1 mg / mL) was added and stirred for 5 minutes. The mixture was then rotary evaporated (30°C, 100 rpm) until the ethanol was evaporated to dryness and a thin film of liposomes formed at the bottom of the round-bottom flask. 0.01 M phosphate buffered saline (PBS) was added and the mixture was hydrated by rotating on a rotary evaporator at room temperature. The liposome solution was removed and stored in a refrigerator at 4°C. The resulting AP39 liposomes are the AP39 delivery system, designated AP39@Lip.

[0037] The dosage ratio used in this example is the optimal ratio of the present invention. The AP39 liposomes prepared under this ratio have good stability, a particle size suitable for nasal delivery, an encapsulation efficiency of up to 71.8%, and achieve slow release of the drug. Compared with other dosage ratios, it has a more excellent therapeutic effect.

[0038] 3. Experimental Grouping

[0039] Neonatal mice were randomly divided into four groups: sham, HI, HI+AP39, and HI+AP39@Lip. Drugs were administered via intraperitoneal injection in the HI+AP39 group and via nasogastric administration in the HI+AP39@Lip group, respectively, at 2, 24, and 48 hours after HI injury. No drug was administered to the sham and HI groups. Pathology and gene protein expression were assessed 72 hours after HI injury.

[0040] 4. Detection of H2S content

[0041] The mice were sacrificed 72 hours after HI injury, and the damaged cortex of the mice of the same weight was collected. The samples were tested using an endogenous hydrogen sulfide kit, and the H2S content was recorded.

[0042] 5. Determination of Cerebral Infarction Area

[0043] Mice were sacrificed 72 hours after HI injury and TTC staining was performed to observe the degree of brain edema and cerebral infarction area. Infarction area (%) = (contralateral hemisphere area - ipsilateral hemisphere healthy area) / contralateral hemisphere area × 100%.

[0044] 6. Nissl staining

[0045] 72 hours after HI injury, the mice were sacrificed and Nissl staining was performed to observe the tissue loss in the damaged brain area of ​​the mice.

[0046] 7. TUNEL staining

[0047] 72 hours after HI injury, mice were sacrificed and apoptotic cells were labeled using a TUNEL apoptosis detection kit. Apoptotic cells were counted using Image J. Apoptosis rate (%) = number of apoptotic cells / total number of cells × 100%.

[0048] 8. Western blotting analysis

[0049] EVs, cells, or ipsilateral cortex were homogenized in RIPA buffer containing protease / phosphatase inhibitors and PMSF for 15 min and then centrifuged at 12,000 rpm / 10 min (4°C). Protein concentration was determined using a BCA protein assay kit. Chemiluminescent signal was generated using an ECL kit and detected using a Tanon imaging system.

[0050] 9. Immunohistochemical Staining

[0051] The mice were sacrificed 72 h after HI injury, and immunohistochemical staining was performed using a microglia-specific antibody against ionized calcium channel protein (Iba1) to specifically mark microglia.

[0052] 10. Detection of reactive oxygen species levels

[0053] Mice were sacrificed 72 hours after HI injury, and fresh tissues of the damaged cortex of mice of the same mass were collected to prepare single-cell suspensions. After centrifugation, the cell pellets were incubated with fluorescent probes (DCFH-DA or MitoSOX) for 30 minutes (37°C), and fluorescence quantitative detection was performed using a microplate reader.

[0054] 11. Mitochondrial Membrane Potential Detection

[0055] Mice were sacrificed 72 hours after HI injury, and fresh tissues of the damaged cortex of mice of the same mass were collected to prepare single-cell suspensions. After centrifugation, the cell pellets were incubated with the fluorescent probe JC-1 for 30 min (37°C). Fluorescence quantitative detection was performed using a microplate reader, and the Red / Green ratio was calculated.

[0056] 12. Mitochondrial ATP content detection

[0057] The mice were sacrificed 72 hours after HI injury, and fresh tissues of the damaged cortex of the mice of the same weight were collected. Samples were prepared using an ATP detection kit, and a standard curve was prepared. The samples were detected using a microplate reader.

[0058] 2. Experimental results:

[0059] 1. Characterization and Identification of AP39-loaded Liposomes

[0060] Figure 1 (A) is an electron micrograph of blank liposomes and AP39-loaded liposomes (AP39@Lip). The liposomes appear as hollow vesicles and are evenly distributed. Scale bar = 50 nm. Figure 1 (B) is the particle size analysis of blank liposomes, with the fitted peak at 37 nm, which is consistent with the results shown by electron microscopy. Figure 1 (C) is the chromatogram of AP39, Figure 1 (D) is the standard curve of AP39, Figure 1 (E) Drug release curve of AP39 from AP39@Lip within 0-50 h.

[0061] The above results show that encapsulating AP39 has no effect on the morphology of liposomes. The size of the prepared AP39@Lip can be used for nasogastric administration, and AP39@Lip can be rapidly released within 0-20 hours and continuously released within 50 hours, indicating that liposomes can be used as a carrier to load and transport AP39 and achieve slow release.

[0062] Note: The difference between the preparation method of blank liposomes and "2. Preparation of AP39 liposomes" in Example 1 is that AP39 is not added, and the other steps are the same.

[0063] 2. AP39@Lip can increase H2S levels in the damaged cortex of neonatal mice after HI brain injury and alleviate HI brain injury-induced cerebral infarction and neural tissue loss Figure 2 (A) H2S content was measured in the right damaged cortex of mice 72 hours after HI. Results showed decreased H2S content in the damaged cortex compared with the sham group. Administration of AP39 or AP39@Lip increased H2S levels in the damaged tissue, with AP39@Lip producing even higher levels. N = 4 / group. Figure 2 (B) and Figure 2 (C) Representative brain images of each group and quantitative analysis of infarct size. TTC-stained white areas represent infarct size. Measured 72 hours after HI, the average infarct size in the injured hemisphere was 71.15%, compared with 56.89% in the AP39 group and 45.23% in the AP39@Lip group. As can be seen, infarct size was significantly reduced in the nasal AP39@Lip delivery group (i.e., the HI+AP39@Lip group). N = 4 / group. Figure 2 (D) Nissl staining of brain tissue from each group shows that administration of AP39 and AP39@Lip can reduce brain tissue loss caused by HI. The results show that H2S levels in the damaged cortex decrease after HI. Administration of AP39 or AP39@Lip can upregulate H2S levels in the damaged cortex and improve HI-induced cerebral infarction, reducing infarct size and brain tissue loss. The effect is greater with AP39@Lip. Statistics = mean ± SD; **p < 0.01, ***p < 0.001. Data were analyzed using one-way ANOVA with Bonferroni correction.

[0064] 3. AP39@Lip can alleviate neuronal apoptosis in the damaged cortex of neonatal mice after HI brain injury

[0065] Figure 3 (A) TUNEL staining images of neurons in the cortex and hippocampus of the right damaged area after HI for 72 hours. Cells labeled with red fluorescence are neurons specifically labeled with NeuN, and cells labeled with green fluorescence are TUNEL-positive cells. Administration of AP39 and AP39@Lip can reduce the number of TUNEL-positive neurons in the hippocampus and cortex. Figure 3 (B) Quantitative analysis of the number of apoptotic neurons in the cortex, N = 27 / group. Approximately 50% of neurons in the injured cortex underwent apoptosis, while the apoptotic rate decreased to 20% and 13% after administration of AP39 and AP39@Lipo, respectively. Figure 3 (C) Quantitative analysis of the number of apoptotic neurons in the hippocampus, N = 9 / group. Approximately 50% of neurons in the injured hippocampus underwent apoptosis, which decreased to 28% and 19% after administration of AP39 and AP39@Lipo, respectively. Figure 3 (D) shows the changes in protein expression of Cleaved-caspase-3 and Caspase-3 in the damaged cortex, which were quantitatively analyzed using Image-Pro Plus 6.0. The results are expressed as the ratio of Cleaved-caspase-3 / Caspase-3. Figure 3(E) N = 4 / group. Results show that AP39 and AP39@Lip administration can reduce HI-induced cell apoptosis. Statistics = mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed using one-way ANOVA with Bonferroni correction.

[0066] 4. AP39@Lip can alleviate the activation of microglia and the release of inflammatory factors in the damaged cortex after HI brain injury in neonatal mice

[0067] Figure 4 (A) is a representative Iba-1 staining of the right damaged cortex 72 hours after HI injury. The second row is a local magnification of representative Iba-1 positive cells. It can be seen that microglia in the cortex are activated 72 hours after HI, with enlarged cell bodies and shortened processes. The administration of AP39 and AP39@Lip can reverse the morphological changes of microglia and alleviate microglia activation. Figure 4 (B) and Figure 4 (C) Western blotting analysis of iNOS and TNF-α in the right injured cortex 72 hours after HI injury. Quantitative analysis was performed using Image-Pro Plus 6.0. This figure shows that AP39 and AP39@Lip reduced the expression of proinflammatory cytokines after HI. N = 4 / group. The results indicate that administration of AP39 and AP39@Lip alleviated microglial activation and reduced the inflammatory response after HI. Statistics = mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed using one-way ANOVA with Bonferroni correction.

[0068] 5. AP39@Lip can reduce ROS levels in the damaged cortex of neonatal mice after HI brain injury and alleviate mitochondrial damage caused by HI brain injury

[0069] Figure 5 (A) Fluorescence quantitative analysis of total ROS levels in fresh mouse cortical tissue using the DCFH-DA fluorescent probe. It shows that administration of AP39 and AP39@Lip can reduce the elevated total ROS levels induced by HI brain injury. N = 4 / group. Figure 5 (B) Fluorescence quantitative analysis of mitochondrial ROS content in fresh mouse cortical tissue using the MitoSOX fluorescent probe. It can be seen that administration of AP39@Lip can reduce mitochondrial ROS content. N = 4 / group. Figure 5 (C) Fluorescence quantitative analysis of mitochondrial membrane potential in fresh mouse cortical tissue using the JC-1 fluorescent probe (Red / Green). It can be seen that AP39@Lip administration can upregulate the HI-induced decrease in mitochondrial membrane potential. N = 4 / group. Figure 5 (D) Determination of ATP content in fresh tissue of damaged mouse cortex. HI brain injury reduces ATP content in tissue, while administration of AP39@Lip increases ATP content. N = 3 / group. Figure 5 (E) and Figure 5 (F) Western blotting results of SOD2 and TOMM20 in the right damaged cortex 72 hours after HI injury. Quantitative analysis was performed using Image-Pro Plus 6.0. This figure shows that AP39 and AP39@Lip upregulated the expression of the antioxidant enzyme SOD2 and the mitochondrial protein TOMM20. The results indicate that administration of AP39 and AP39@Lip alleviated oxidative stress and reduced mitochondrial damage caused by HI. Statistics = mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed using one-way ANOVA with Bonferroni correction.

[0070] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An AP39 delivery system, characterized in that: Includes AP39, soybean lecithin and cholesterol.

2. The AP39 delivery system according to claim 1, characterized in that The mass ratio of the AP39, soybean lecithin and cholesterol is 1:(40-80):(10-20).

3. The AP39 delivery system according to claim 2, characterized in that The mass ratio of the AP39, soybean lecithin and cholesterol is 1:(40-60):(10-15).

4. The AP39 delivery system according to claim 2, wherein: The mass ratio of the AP39, soybean lecithin and cholesterol is 1:50:12.

5.

5. The AP39 delivery system according to claim 1, wherein: The particle size of the AP39 delivery system is 35-40 nm.

6. A method for preparing the AP39 delivery system according to any one of claims 1 to 5, characterized in that: include: AP39, soybean lecithin and cholesterol are dissolved in an organic solvent, mixed and evaporated to obtain a liposome film; Phosphate buffer was added to the liposome film for hydration to obtain the AP39 delivery system.

7. The preparation method according to claim 6, characterized in that: The organic solvent is a volatile organic solvent, preferably an alcohol, and more preferably ethanol.

8. The preparation method according to claim 6, characterized in that: The concentration of the phosphate buffer is 0.005-0.015M, preferably 0.01M.

9. Use of the AP39 delivery system according to any one of claims 1 to 5 or the AP39 delivery system prepared by the preparation method according to any one of claims 6 to 8 in preparing a drug for treating hypoxic-ischemic brain damage.

10. The use according to claim 9, characterized in that The administration method of the drug for treating hypoxic-ischemic brain damage is nasogastric administration.

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