Anti-neuroinflammation medicine as well as preparation method and application thereof

Abstract

The invention provides an anti-neuroinflammation medicine as well as a preparation method and application thereof. The anti-neuroinflammation medicine comprises an active component and lipidosome, the active component is an inhibitor 4-aminobenzoyl hydrazine of myeloperoxidase; the liposome is prepared from phospholipid, cholesterol and distearoyl phosphatidyl ethanolamine-thioketal-polyethylene glycol; the method comprises the following steps: weighing lipidosome and 4-aminobenzoyl hydrazine, dissolving, and carrying out rotary evaporation to obtain a lipid membrane; adding a phosphate buffer solution into the lipid membrane, stirring in a water bath, performing ultrasonic treatment, filtering, centrifuging and purifying to obtain the medicine, according to the active oxygen response-based liposome delivery system disclosed by the invention, 4-aminobenzoyl hydrazine is encapsulated in liposome to form a drug, and after the drug enters a brain inflammation microenvironment area of the Alzheimer's disease, the active oxygen can break a thioketal bond, so that local release of 4-aminobenzoyl hydrazine is triggered, targeted inhibition of myeloperoxidase is realized, and the drug delivery effect is improved. The oxidative stress level is reduced, the neuroinflammation is relieved, and the cognitive impairment is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-neuroinflammatory drug, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory impairment and cognitive decline. Its main pathological manifestations include β-amyloid (Aβ) plaque deposition in the brain, neurofibrillary tangles, synaptic loss, and neuroinflammatory responses. To date, the pathogenesis of AD is not fully understood, but the "amyloid cascade hypothesis" and the "neuroinflammatory hypothesis" are widely accepted as core theories.

[0003] In recent years, the U.S. FDA has approved several monoclonal antibody drugs targeting Aβ, including aducanumab, lecanemab, and donanemab. These antibody drugs can effectively clear Aβ plaques in the brain and delay cognitive decline, but their clinical efficacy is limited and they are often accompanied by significant adverse reactions, such as the risk of cerebral edema and hemorrhage caused by amyloid-associated imaging abnormalities (ARIA). In addition, these antibody preparations are costly, have complex administration methods, and are difficult to widely implement.

[0004] Besides Aβ-targeted therapy, neuroinflammation is considered one of the important drivers of AD progression. Studies have shown that the continuous activation of microglia and the excessive production of reactive oxygen species (ROS) create a vicious cycle, further inducing Aβ deposition and neuronal damage. Myeloperoxidase (MPO), a key oxidase secreted by activated microglia and neutrophils, can catalyze the production of strong oxidants such as hypochlorous acid (HOCl), causing irreversible damage to neural tissue. MPO is highly expressed in AD brain tissue and is closely related to cognitive decline.

[0005] Currently, several MPO inhibitors (such as 4-aminobenzoic acid hydrazine, ABAH) have demonstrated antioxidant and anti-inflammatory effects in in vitro and animal experiments. However, their clinical application is greatly limited due to their poor water solubility, rapid metabolism, and low utilization in brain tissue. To address these issues, developing a drug delivery system that can specifically release MPO inhibitors under inflammatory conditions in the brain, and possesses good biocompatibility and brain targeting, has become an important direction for precision treatment of Alzheimer's disease (AD). Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to overcome the problems of poor targeting, low brain penetration and insufficient pharmacokinetic performance of existing AD treatment drugs, and to provide an anti-neuroinflammatory drug and its preparation method and application, namely, a formulation of a ROS-responsive liposome MPO inhibitor for AD treatment.

[0007] To achieve the above objectives, the solution of the present invention is:

[0008] A ROS-responsive liposome delivery system was developed to efficiently deliver the MPO inhibitor ABAH to inflammatory areas in the brain of patients with Alzheimer's disease (AD), thereby specifically inhibiting MPO activity, reducing oxidative stress levels, and improving cognitive function.

[0009] Specifically,

[0010] An anti-neuroinflammatory drug comprises an active ingredient and liposomes; the active ingredient is ABAH, an inhibitor of MPO; the liposomes comprise phospholipids, cholesterol, and distearate phosphatidylethanolamine-thioketal-polyethylene glycol (DSPE-TK-PEG2000), wherein the molar ratio of phospholipids, cholesterol, and polyethylene glycol-modified phospholipids is (35-50):(15-25):(30-40). DSPE-TK-PEG2000 was purchased from Xi'an Ruixi Biotechnology Co., Ltd., model R-D526. DSPE-TK-PEG2000 is a carrier containing a thioketal (TK) structure, which is ROS-sensitive, i.e., the TK bond is a ROS-responsive linking group. The active ingredient ABAH is encapsulated within the liposomes to form the drug Lip-ABAH.

[0011] When liposomes enter brain regions of oxidative stress and neuroinflammation, ROS can break TK bonds, thereby disrupting the outer layer structure of the liposomes and promoting the site-directed release of ABAH, thus achieving targeted inhibition of MPO catalytic activity.

[0012] Furthermore, the phospholipid is selected from one or more of lecithin, distearate phosphatidylcholine (DSPC), and dipalmitoyl phosphatidylcholine (DPPC).

[0013] Furthermore, the liposomes also include fluorescently labeled probes selected from one or more of fluorescein isothiocyanate (FITC) and cyanine dye 5 (Cy5). The fluorescently labeled probes are used for drug tracing and release monitoring.

[0014] Furthermore, the dosage form of the drug includes an aqueous dispersion, a lyophilized powder, or a nasal spray, preferably a nasal spray, i.e., administration via the nasal cavity.

[0015] After being delivered via the nasal cavity, the drug Lip-ABAH can bypass the blood-brain barrier through the olfactory nerve and trigeminal nerve pathways, and quickly enter the brain parenchyma, thereby achieving highly efficient brain-targeted drug delivery.

[0016] A method for preparing the above-mentioned anti-neuroinflammatory drug includes the following steps:

[0017] (1) Weigh phospholipids, cholesterol, DSPE-TK-PEG2000 and ABAH accurately according to the molar ratio, and dissolve them in a mixed solvent of 10 mL of chloroform and methanol (65 / 35, v / v) to obtain a lipid solution;

[0018] (2) Gently shake the mixed lipid solution at room temperature until all lipids are completely dissolved to ensure the homogeneity of the solution. Then, transfer the dissolved lipid solution to a 50 mL rotary evaporation flask, place it on a rotary evaporator, and rotate and evaporate until the chloroform is completely evaporated to obtain a lipid membrane;

[0019] (3) Slowly add PBS buffer or distilled water preheated to 37°C to the lipid membrane to make the total volume 15 mL. Gently shake or rotate the mixture under stirring to ensure that the lipid membrane is in full contact with the aqueous phase. Then, place the mixture in a water bath at 45°C and stir continuously for 30 min to promote complete hydration of the lipid membrane and obtain primary liposomes.

[0020] (4) The hydrated primary liposomes were placed in an ice-water bath and ultrasonically treated with an ultrasonic disruptor to reduce the particle size and improve their uniformity. During the ultrasonic treatment, the solution temperature should be continuously monitored and maintained below 50°C to avoid liposome degradation due to overheating, thus obtaining a liposome solution;

[0021] (5) After ultrasonic treatment, the liposome solution was filtered through a 200 nm pore size nanofilter to remove large particles and obtain a uniform liposome dispersion. Subsequently, it was centrifuged at 4 °C to remove unencapsulated precipitates and further purify the liposomes to obtain the anti-neuroinflammatory drug Lip-ABAH.

[0022] Further, in step (1), the molar ratio of phospholipids, cholesterol, DSPE-TK-PEG2000, and ABAH is (35-50):(15-25):(30-40):(8-12).

[0023] Further, in step (2), the temperature of the rotary evaporation is 40-50℃, preferably 45℃; the rotation speed is 100-200rpm, preferably 150rpm.

[0024] Further, in step (4), the conditions for ultrasound are: ultrasound power of 25-35W, preferably 30W; cooling interval of 2s after each treatment, and continuous treatment for 10min.

[0025] Furthermore, in step (5), during filtration, the pore size of the nanofilter is 200 nm, and during centrifugation, the rotation speed is 6000-7500 rpm, preferably 7000 rpm; the time is 5-15 min, preferably 10 min.

[0026] Further, in step (5), the average particle size of the drug is about 80-170 nm, preferably 116 nm; the Zeta potential is (-76)-(-23) mV, preferably −54 mV; the encapsulation efficiency is greater than 80%, and the drug loading is ≥90% released within 12 h in the presence of H2O2 (100 μM), while the release rate is ≤5% in the absence of ROS.

[0027] The application of one of the aforementioned anti-neuroinflammatory drugs in the preparation of products for treating Alzheimer's disease (AD), which serves as a non-disease-related diagnostic and treatment method, provides a new technological approach for early intervention and disease-modifying therapy in AD.

[0028] Neuroinflammation includes other neurodegenerative diseases accompanied by neuroinflammation and oxidative stress. For example, drugs for neuroinflammation are used in the development of products for treating Alzheimer's disease, Parkinson's disease, post-stroke cognitive impairment, multiple sclerosis, and traumatic brain injury.

[0029] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0030] Highly targeted: Through the ROS response mechanism, the drug is released only in the inflammatory environment of the brain, significantly improving local efficacy and reducing peripheral side effects;

[0031] Good penetration: Nasal delivery effectively bypasses the blood-brain barrier, achieving rapid intracerebral drug distribution;

[0032] Dual anti-inflammatory and neuroprotective effects: inhibiting MPO-mediated oxidative stress and reducing microglial activation, thus mechanistically blocking the vicious cycle of AD;

[0033] High safety: Liposome materials have good biocompatibility and no significant liver or kidney toxicity with long-term administration;

[0034] Visualization and controllability: Real-time tracking of drug release and distribution can be achieved through fluorescent probe labeling.

[0035] In summary, this invention proposes a ROS-responsive liposome delivery system. Using TK bonds as the ROS-sensitive linker, the MPO inhibitor ABAH is encapsulated within liposomes to form drug Lip-ABAH. When the liposomes enter the inflammatory microenvironment of the AD brain, ROS can cleave the TK bonds, triggering the local release of ABAH. This achieves targeted inhibition of MPO, reduces oxidative stress levels, and alleviates neuroinflammation, thereby effectively improving cognitive dysfunction. This system overcomes the shortcomings of traditional MPO inhibitors, such as poor brain targeting and unsatisfactory pharmacokinetic performance, providing a new approach and technical method for the treatment of AD. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the synthesis of the drug Lip-ABAH in Example 1 of the present invention.

[0037] Figure 2 This is the Fourier transform infrared (FTIR) spectrum of the drug Lip-ABAH in Example 1 of the present invention.

[0038] Figure 3 This is the ultraviolet-visible spectrum (UV-Vis) of the drug Lip-ABAH in Example 1 of the present invention.

[0039] Figure 4 The UV-Vis spectra of different concentrations of ABAH in Example 1 of this invention are shown, along with the standard curve obtained from the absorption peak of ABAH at 272 nm.

[0040] Figure 5 This is a ROS-responsive release diagram of the drug Lip-ABAH in Example 1 of the present invention.

[0041] Figure 6 This is a diagram showing the hydration kinetic diameter (dynamic light scattering, i.e., DLS measurement) of the drug Lip-ABAH in Example 1 of the present invention.

[0042] Figure 7 This is a surface charge map of the drug Lip-ABAH in Example 1 of the present invention.

[0043] Figure 8 This is a transmission electron microscope image of the drug Lip-ABAH in Example 1 of the present invention.

[0044] Figure 9 This is a transmission electron microscope image of the drug Lip-ABAH in vitro after responding to ROS in Example 1 of the present invention.

[0045] Figure 10 This is a diagram showing the response and release of the drug Lip-ABAH in inflammatory cells in Example 2 of the present invention.

[0046] Figure 11This is a diagram showing the ROS response release in Embodiment 2 of the present invention.

[0047] Figure 12 This is a diagram of brain-targeted drug delivery in Embodiment 2 of the present invention.

[0048] Figure 13 This is a cellular toxicity evaluation diagram of the drug Lip-ABAH in Example 2 of the present invention.

[0049] Figure 14 This is a flow map of neuronal cell membranes of the drug Lip-ABAH in Example 2 of the present invention.

[0050] Figure 15 This is a diagram of the open field test of 5XFAD mice in Example 3 of the present invention.

[0051] Figure 16 This is a diagram of the Barnes maze test performed on 5XFAD mice in Example 3 of this invention.

[0052] Figure 17 This is a diagram of a novel object recognition experiment using 5XFAD mice in Example 3 of this invention.

[0053] Figure 18 This is an imaging image of Aβ plaques in 5XFAD mice in Example 3 of the present invention.

[0054] Figure 19 This is a magnetic resonance imaging image of a 5XFAD mouse in Example 3 of the present invention.

[0055] Figure 20 This is a graph showing the Aβ amyloid protein level in Example 3 of the present invention.

[0056] Figure 21 This is an Aβ immunofluorescence staining image from Example 3 of the present invention.

[0057] Figure 22 This is a flow cytometry staining image of activated microglia in Example 3 of the present invention.

[0058] Figure 23 This is a graph showing the MPO expression level in the brains of 5XFAD mice in Example 3 of this invention.

[0059] Figure 24 This is a diagram of DNA oxidative stress in the brains of 5XFAD mice in Example 3 of this invention.

[0060] Figure 25 This is a transcriptomic diagram of the hippocampus of a 5XFAD mouse in Example 3 of the present invention.

[0061] Figure 26 This is a graph showing the effect of 5XFAD mice on whole blood parameters after Lip-ABAH administration in Example 3 of the present invention.

[0062] Figure 27 This is a diagram showing the effect of Lip-ABAH administration on key tissues and organs of mice in Example 3 of the present invention. Detailed Implementation

[0063] This invention provides an anti-neuroinflammatory drug, its preparation method, and its application.

[0064] To further illustrate the technical means and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and preferred embodiments of the present invention.

[0065] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the test or detection methods involved are all conventional methods unless otherwise specified.

[0066] Example 1:

[0067] like Figure 1 As shown, the preparation method of the anti-neuroinflammatory drug in this embodiment includes the following steps:

[0068] (1) DPPC (50 mg, 40 mol%), DSPC (5 mol%), cholesterol (20 mol%), DSPE-TK-PEG2000 (35 mol%) and ABAH (10 mol%) were dissolved in 10 mL of a mixed solvent of chloroform and methanol (65 / 35, v / v) to obtain a lipid solution;

[0069] (2) Gently shake the mixed lipid solution at room temperature until all lipids are completely dissolved to ensure the homogeneity of the solution. Then, transfer the dissolved lipid solution to a 50 mL rotary evaporation flask, place it on a rotary evaporator, and evaporate it at 150 rpm at 45°C until the chloroform is completely evaporated to obtain a lipid membrane;

[0070] (3) Slowly add PBS buffer preheated to 37°C to the lipid membrane to make the total volume 15 mL. Gently shake or rotate the mixture under stirring to ensure that the lipid membrane is in full contact with the aqueous phase. Then, place the mixture in a water bath at 45°C and stir continuously for 30 min to promote complete hydration of the lipid membrane and obtain primary liposomes;

[0071] (4) The hydrated primary liposomes were placed in an ice-water bath and ultrasonically treated with an ultrasonic disruptor to reduce the particle size and improve their uniformity. The ultrasonic conditions were set to 30W power, 2s per treatment, 2s cooling interval, and 10min of continuous treatment. The solution temperature was continuously monitored during the ultrasonic process and kept below 50℃ to avoid degradation of the liposomes due to overheating, resulting in a liposome solution.

[0072] (5) After ultrasonic treatment, the liposome solution was filtered through a 200 nm pore size nanofilter to remove large particles and obtain a uniform liposome dispersion. Subsequently, the liposomes were centrifuged at 7000 rpm for 10 min at 4 °C to remove unencapsulated precipitates and further purify the liposomes to obtain the anti-neuroinflammatory drug Lip-ABAH.

[0073] Encapsulation validation and physicochemical characterization of the drug Lip-ABAH:

[0074] (1.1) As Figure 2 As shown, Fourier transform infrared (FTIR) spectra of fully dried drug Lip-ABAH, blank liposomes without ABAH loading, and ABAH powder were acquired using the KBr pellet method. The FTIR spectrum of free ABAH showed characteristic absorption peaks at 1604 cm⁻¹ and 1629 cm⁻¹, corresponding to the vibrational absorption of the –C=C– bond and the amide group, respectively. The blank liposomes without ABAH loading showed significant absorption peaks at 974 cm⁻¹, 1093 cm⁻¹, and approximately 3480 cm⁻¹, attributed to the symmetric / asymmetric stretching vibrations of the PO₂⁻ group and the –OH absorption peak. Both sets of characteristic peaks were observed in the Lip-ABAH sample, indicating that ABAH was successfully encapsulated in liposomes, yielding the drug Lip-ABAH.

[0075] (1.2) As Figure 3 As shown, the drug Lip-ABAH, blank liposomes without ABAH loading, and ABAH aqueous solution were placed in clean cuvettes and the absorbance was collected by a UV-Vis spectrometer. ABAH showed a significant absorption peak at 272 nm, and the same absorption peak was also observed in Lip-ABAH, further verifying the successful encapsulation of the drug Lip-ABAH.

[0076] (1.3) such as Figure 4 As shown, aqueous solutions of ABAH at different concentrations (0.115 ppm, 0.23 ppm, and 0.46 ppm) were placed in clean cuvettes and their absorbance was collected using a UV-Vis spectrometer. Scatter plots were plotted based on the absorbance of different concentrations of ABAH at 272 nm, and linear regression was performed to obtain a standard curve of y = 0.08854x, which was used for the quantification of drug release.

[0077] (1.4) Response release test

[0078] like Figure 5As shown, an aqueous solution of Lip-ABAH (6 mg / L) was sealed in a dialysis bag (MWC: 14,000 Da). 100 µM hydrogen peroxide was added to the ROS-responsive group, while the same volume of distilled water was added to the non-ROS-responsive control group. The dialysis bag was immersed in distilled water and gently shaken for 12 hours. At 30 min, 1 h, 2 h, 3 h, 4.5 h, and 12 h, 3 mL of peripheral fluid was collected to measure the UV-vis absorption peak. Each time 3 mL of peripheral fluid was collected, 3 mL of distilled water was added. According to the ABAH standard curve, under ROS stimulation, 99.38% of ABAH was released within 12 hours; while under ROS-free conditions, only 2.35% of ABAH was released, indicating that Lip-ABAH has significant ROS-responsive release characteristics.

[0079] (1.5) such as Figure 6 As shown, the hydration dynamic diameter of the drug Lip-ABAH aqueous solution was measured by dynamic light scattering. The average particle size was found to be approximately 116 nm based on the weighted average of the light intensity distribution. The polydispersity index (PDI) was narrow, at 0.2885, indicating good monodispersity.

[0080] (1.6) such as Figure 7 As shown, the Zeta potential of the drug Lip-ABAH aqueous solution was measured by laser Doppler electrophoresis and the average value was taken. The measured potential (surface charge) was approximately -54mV. The negative potential is beneficial to reduce liposome aggregation and improve the stability of in vivo circulation.

[0081] (1.7) such as Figure 8 As shown, the transmission electron microscopy image of the drug Lip-ABAH reveals that Lip-ABAH is a roundish vesicle with good dispersibility and relatively uniform particle size.

[0082] (1.8) such as Figure 9 As shown, the transmission electron microscopy image of the drug Lip-ABAH after its in vitro response to ROS shows that the liposome vesicles are destroyed and form sheet-like irregular fragments, and ABAH is released.

[0083] Example 2:

[0084] This embodiment uses cell-level experiments to verify the ROS-responsive release specificity, biosafety, and neuroprotective effect of the drug Lip-ABAH.

[0085] Cells: murine microglia (BV2 cells) and murine neurons (N2A cells).

[0086] Pharmacological efficacy verification:

[0087] (2.1) Response release of drug Lip-ABAH in inflammatory cells

[0088] like Figure 10 As shown, the drug Lip-ABAH was labeled with Cy5 and FITC (Cy5-Lip-ABAH-FITC) on both the surface and inside the liposomes. Drug release was observed at the cellular level in the microenvironment of non-inflammatory murine microglia (BV2 cells) and lipopolysaccharide (LPS)-induced inflamed BV2 cells (dual-fluorescently labeled drug and cells were co-cultured at 37°C for 4 h). After BV2 cells adhered to cell adhesion slides: the inflammatory group was treated with 100 ng / mL LPS and 10 µg / mL Cy5-Lip-ABAH-FITC; the non-inflammatory group was treated with an equal volume of PBS and 10 µg / mL Cy5-Lip-ABAH-FITC. After co-culturing at 37°C for 4 h, the cell adhesion slides were attached to a glass slide, mounted with an anti-fluorescent mounting medium containing the nuclear staining agent DAPI, and observed under a confocal microscope.

[0089] In a non-inflammatory BV2 microglycemic system (top row), Cy5 and FITC fluorescence overlapped and were phagocytosed by BV2 cells, indicating that the drug was not released in the non-inflammatory microenvironment. However, in an LPS-induced inflammatory BV2 microglycemic system (bottom row), Cy5 and FITC fluorescence separated, FITC was phagocytosed into the cells, and the Cy5-labeled liposome shell aggregated to form lipid droplets that adhered to the cell slide. This indicates that the drug Lip-ABAH is released responsively only in inflammatory microenvironments and not in non-inflammatory cellular microenvironments, demonstrating targeted pharmacological effects.

[0090] (2.2) ROS response release

[0091] like Figure 11 As shown, 7-month-old 5XFAD mice (AD model) were intranasally administered a dual-fluorescently labeled drug (Cy5-Lip-ABAH-FITC, volume 10 µL, concentration 0.2 mg / mL ABAH) for 6 hours, followed by tissue section observation. Results showed widely distributed fluorescent signals in the olfactory bulb, hippocampus, cortex, and cerebellum. High-magnification images revealed partial separation of Cy5 (liposome shell, red) and FITC (drug-loaded ABAH, green) signals in the hippocampus and cortex, indicating ROS-triggered drug release (shown by green and red arrows). Conversely, in the olfactory bulb and cerebellum, Cy5 and FITC fluorescent signals largely overlapped (shown by yellow), suggesting intact liposome structure and no ABAH release (shown by yellow arrows).

[0092] (2.3) Brain-targeted drug delivery

[0093] like Figure 12As shown, two-photon microscopy was used to observe the brain entry of the FITC-labeled drug (Lip-ABAH-FITC). A 5 × 5 mm² bone window was opened in the top of the mouse skull to observe the drug's entry into the brain in real time. Mouse blood vessels were labeled with dextran-Texas Red (70,000 MW, 100 mg / kg). Two-photon imaging was performed at different time points after nasal administration of the drug, with an imaging depth of 64 µm and a slice spacing of 4 µm, before administration, and at 10 min, 30 min, 1 h, and 2 h after administration. Drug distribution in brain tissue and blood vessels was observed, and the fluorescence of the brain parenchyma increased with time. This indicates that the drug can effectively enter the brain after nasal administration, possibly by directly crossing the blood-brain barrier via the nasal nerve-olfactory bulb pathway, or by absorption into the bloodstream and penetration of the blood-brain barrier.

[0094] (2.4) Evaluation of the cellular toxicity of the drug Lip-ABAH:

[0095] like Figure 13 As shown, different concentrations (0 ppm, 7.81 ppm, 15.63 ppm, 31.25 ppm, 62.5 ppm, 125 ppm, 500 ppm, 1000 ppm) of the drug were co-cultured with mouse-derived neuronal cells (N2A) and microglia (BV2) in 96-well plates for 24 h. Then, a CCK-8 assay was performed, and the absorption peak of each well was measured at 450 nm using a microplate reader. Cytotoxicity was calculated using the following formula: [OD LERAMI / OD control × 100%. No significant cytotoxicity was found (maximum concentration 1000 ppm).

[0096] (2.5) The protective effect of drug Lip-ABAH on neuronal cell membrane fluidity

[0097] like Figure 14As shown, this study investigated whether the addition of Lip-ABAH could improve cell membrane fluidity in the oxidative stress inflammatory microenvironment induced by Aβ42 in N2A neurons. N2A cells were seeded in confocal microdiscs. After cell adhesion, 1 µM Aβ42 was added to induce an oxidative stress inflammatory microenvironment. Simultaneously, 5 µg / mL Lip-ABAH was added to the experimental group, while an equal volume of sterile PBS was added to the control group. After co-culturing at 37°C for 6 h, the culture medium was removed, and the cells were washed three times with PBS. A Texas Red-labeled cell membrane fluorescent probe (4 µM) was added, and the cells were co-cultured at 37°C for 30 min. After three gentle washes with PBS, the recovery of fluorescence intensity after bleaching (FRAP) of the cell membrane was observed under a confocal microscope. Compared with the PBS control group, the Lip-ABAH group showed significantly faster and stronger fluorescence recovery (P = 0.0221), indicating that Lip-ABAH can enhance cell membrane fluidity.

[0098] Example 3: The therapeutic efficacy of the drug Lip-ABAH on AD

[0099] This embodiment uses 5XFAD mice (AD model) as the research subject, and combines behavioral, imaging and molecular biology experiments to verify the therapeutic efficacy of the drug Lip-ABAH on AD and elucidate its mechanism of action.

[0100] 5XFAD was administered to mice starting at 4 months of age via nasal drop; the dosage was 10 µL per dose (0.2 mg ABAH / mL); administration continued until the mice reached 6 months of age. Behavioral evaluations were performed at 6-7 months of age, and tissues and organs were collected at 7 months of age. Results are as follows:

[0101] (3.1) Open field test

[0102] like Figure 15 As shown, mice were placed in the test room for 30 minutes the day before the experiment to acclimatize. On the test day, mice were placed in a 40 × 40 × 40 cm white open field to explore freely for 5 minutes, and their movement speed and time spent in the central area were recorded. The results showed that there was no significant difference in average movement speed and time spent in the central area between the PBS control group (5XFAD + PBS) mice and the Lip-ABAH treatment group (5XFAD + Lip-ABAH) mice, indicating that Lip-ABAH treatment itself did not affect the mice's movement ability.

[0103] (3.2) Barnes Maze Experiment

[0104] like Figure 16As shown, the Barnes maze test apparatus consists of a circular platform with multiple holes evenly distributed along its edge, one of which is connected to a target escape box. In all experimental phases, mice were first placed in a black concealed chamber in the center of the maze for 10 seconds, after which the chamber was raised while a buzzer continuously sounded. Before formal training began, mice underwent a one-day environmental acclimatization period. The following four days constituted the acquisition training phase, with each mouse receiving four training sessions daily, each spaced 15 minutes apart. A single training session terminated when the mouse successfully entered the target hole or the 3-minute time limit expired. If the target was not found within the time limit, the mouse was gently guided to the target hole and allowed to remain in the escape box for 1 minute. Twenty-four hours after the final acquisition training session, an exploration test was conducted: the target hole was now closed, allowing the mouse to explore freely for 90 seconds. The results showed that the spatial memory ability of 5XFAD+Lip-ABAH mice was significantly improved: compared with the PBS control group 5XFAD+PBS, Lip-ABAH treatment significantly shortened the time for mice to find the target hole on day 4 of training (P = 0.0193) and significantly increased the time they spent in the target quadrant on the test day (P = 0.0336), demonstrating that the drug Lip-ABAH improves spatial memory in AD mice.

[0105] (3.3) New object recognition experiment

[0106] like Figure 17 As shown, mice were acclimatized in the test room for 30 minutes before the experiment. On the first day, mice were placed in an open field device for 5 minutes to acclimatize. After 24 hours, they underwent 8 minutes of familiarization training, where they became familiar with two identical objects. After the familiarization period, a familiar object and a novel object were placed. Six hours later, an 8-minute test was conducted to assess recognition memory, during which the mice's exploration behavior towards the familiar and novel objects was recorded. Object exploration was defined as the mouse pointing its nose toward an object at a distance of less than 2 cm, and the time spent exploring each object was recorded. The object recognition index of the mice was calculated. The novel object recognition test showed that the cognitive memory ability of 5XFAD+Lip-ABAH mice was significantly improved: compared with the PBS control group 5XFAD+PBS, the recognition index of 5XFAD+Lip-ABAH mice was significantly increased (P = 0.049), demonstrating that the drug Lip-ABAH improves recognition memory in AD mice.

[0107] (3.4) 18 F-AV45 PET-CT Aβ plaque imaging

[0108] like Figure 18 As shown, at the drug administration endpoint in mice, [the following was performed / performed]. 18 F-Florbetapir ( 18F-AV45 PET-CT imaging was used to detect plaque deposition in the mouse brain. Mice were injected with 100 ± 20 µCi via the tail vein. 18 F-AV45 tracer. One hour after administration, mice were anesthetized with 2% isoflurane and fixed in a prone position on a PET scanner. A 10-minute CT scan was performed first (80 kVp voltage, 500 µA current, exposure time 1100 ms), followed by a 10-minute static PET scan. Three-dimensional regions of interest were delineated based on CT images. Tracer uptake was measured using Inveon Research Workplace (IRW version 4.2) software. The ratio of injected activity to body weight was calculated for each individual site. 18 Quantitative analysis of F-AV45 intake was performed to obtain the average standardized intake value (SUV). 18 Representative axial, coronal, and sagittal images from F-AV45 PET-CT show the distribution of amyloid protein in the brains of healthy wild-type (WT), 5XFAD+PBS, and 5XFAD+Lip-ABAH mice. Compared with the PBS control group, Lip-ABAH treatment significantly reduced plaque volume (P = 0.0149) and the mean standardized uptake value (SUV-bw) of plaques (P = 0.0487). This demonstrates that the drug Lip-ABAH reduces Aβ deposition in the brains of AD mice.

[0109] (3.5) Magnetic resonance imaging of 5XFAD mice

[0110] like Figure 19 As shown, mouse magnetic resonance imaging (MRI) was performed at the drug administration endpoint under 2% isoflurane anesthesia. High-resolution T2-weighted imaging was used with the following parameters: repetition time (TR) = 3200 ms; echo time (TE) = 26.3 ms; averages = 8; echo spacing = 6.508 ms; rare factor = 8; slice thickness = 0.25 mm; image size = 250 × 250; field of view (FOV) = 15 × 15 mm. 2 Quantitative analysis of the imaging results was performed on each brain region. The quantitative volume analysis results showed that, compared with the PBS control group 5XFAD+PBS, the hippocampal volume of 5XFAD+Lip-ABAH mice was significantly increased (P = 0.0159), while no significant differences were found in other brain regions (cortex, cerebellum), proving that the drug Lip-ABAH protects the hippocampal structure of AD mice and delays brain atrophy.

[0111] (3.6) Western blot quantitative analysis of Aβ amyloid protein levels

[0112] like Figure 20 As shown, tissues were placed in RIPA lysis buffer containing phosphatase inhibitors, protease inhibitors, and PMSF, homogenized using an ultrasonic homogenizer, and the total protein concentration was measured. Equal volumes of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-β-amyloid (1:1000) primary antibody. After washing, the membranes were incubated with HRP-labeled secondary antibody and developed using an enhanced chemiluminescence detection system. Finally, the protein band intensities were quantitatively analyzed. Western blot analysis showed that, compared with the PBS control group (5XFAD+PBS), the Aβ levels in the hippocampus (P = 0.0354) and cortex (P = 0.0002) of 5XFAD+Lip-ABAH mice were significantly reduced.

[0113] (3.7) Aβ immunofluorescence staining

[0114] like Figure 21 As shown, OCT-embedded frozen brain tissue was sectioned using a cryostat, with the sample temperature set at -13°C and the chamber temperature at -15°C, yielding 20 µm thick sections. The sections were then permeabilized in a Tris buffer solution containing 0.25% Triton™ X-100 and blocked with 5% donkey serum at room temperature for 2 hours. After blocking, the sections were rinsed and incubated overnight at 4°C with anti-β-amyloid 1-16 antibody (1:1000). The following day, they were incubated with the corresponding secondary antibody at room temperature for 1 hour. After mounting with a DAPI-containing anti-fluorescent mounting medium, confocal imaging was performed. Aβ immunofluorescence staining results showed that, compared with the PBS control group (5XFAD+PBS mice), 5XFAD+Lip-ABAH mice exhibited significantly reduced amyloid plaque load in the hippocampus (P = 0.0013) and cortex (P = 0.0492).

[0115] (3.8) Flow cytometry staining of activated microglia

[0116] like Figure 22As shown, fresh brain tissue was stained with antibodies via flow cytometry after leukocyte separation using Percoll density gradient centrifugation: anti-CD90-PE, anti-NK1.1-PE, anti-B220-PE, anti-CD49b-PE, anti-Ly6G-PE / Cy7, anti-CD45.2-PB, anti-CD11b-APC / Cy7, and anti-TMEM119 antibodies. The secondary antibody for anti-TMEM119 was Goat anti-rabbit IgG (Alexa Fluor). ® 488). Flow cytometry results showed that, compared with the PBS control group 5XFAD+PBS mice, the number of activated microglia in 5XFAD+Lip-ABAH mice was significantly reduced (P = 0.0276).

[0117] (3.9) Level of MPO expression in the brain

[0118] like Figure 23 As shown, tissues were placed in RIPA lysis buffer containing phosphatase inhibitors, protease inhibitors, and PMSF, homogenized using an ultrasonic homogenizer, and the total protein concentration was measured. Equal volumes of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-MPO (1:1000) primary antibody. After washing, the membranes were incubated with HRP-labeled secondary antibody and developed using an enhanced chemiluminescence detection system. Finally, the protein band intensity was quantitatively analyzed. Western blot results showed that, compared with the PBS control group (5XFAD+PBS mice), the expression levels of MPO protein in the hippocampus and cortex of 5XFAD+Lip-ABAH mice were significantly reduced (P = 0.0266 in the hippocampus and P = 0.0233 in the cortex). The drug Lip-ABAH inhibited MPO activity in the brains of AD mice and reduced MPO-derived oxidation products (such as hypochlorous acid and perchlorate).

[0119] (3.10) Brain DNA oxidative stress damage

[0120] like Figure 24As shown, OCT-embedded frozen brain tissue was sectioned using a cryostat. The sections were permeabilized in a Tris buffer solution containing 0.25% Triton™ X-100 and blocked with 5% donkey serum at room temperature for 2 hours. After blocking, the sections were rinsed and incubated overnight at 4°C with anti-8-OHdG antibody (a biomarker of ROS-induced DNA oxidative stress damage, 1:1000). The next day, they were incubated with the corresponding secondary antibody at room temperature for 1 hour. After mounting with a DAPI-containing anti-fluorescent mounting medium, confocal imaging was performed. 8-OHdG immunofluorescence staining results showed that compared with the PBS control group (5XFAD+PBS mice), the green fluorescence intensity of 5XFAD+Lip-ABAH mice was significantly reduced, suggesting reduced DNA oxidative stress damage (hippocampus P=0.001, cortex P=0.002). Compared with age-matched wild-type C57BL / 6 mice, mice treated with 5XFAD+Lip-ABAH showed slightly higher 8-OHdG expression, but the difference was not statistically significant. Therefore, the drug Lip-ABAH alleviates oxidative stress damage in the brains of AD mice and reduces 8-OHdG expression.

[0121] (3.11) Transcriptomics of the hippocampus in 5XFAD mice treated with Lip-ABAH and controlled with PBS

[0122] like Figure 25 As shown, three hippocampi from each of the 5XFAD+Lip-ABAH and 5XFAD+PBS control mice were used for RNA transcriptome sequencing. First, total RNA was extracted using TRIzol reagent, and RNA purity was assessed by detecting the A260 / A280 absorbance ratio, and RNA integrity was measured. Samples meeting the quality standards were used for library construction. Results showed that, compared to the PBS control group (5XFAD+PBS mice), the transcriptome characteristics of the hippocampus in 5XFAD+Lip-ABAH mice shifted towards enhanced synaptic signaling, neuroprotection, and neurorepair, while simultaneously inhibiting microglial activation and the expression of pro-inflammatory genes.

[0123] (3.12) Effects of Lip-ABAH administration on whole blood parameters and key tissues and organs in mice

[0124] like Figure 26 and Figure 27As shown, at the experimental endpoint, blood samples and tissues from the heart, liver, spleen, lung, and kidneys were collected from 5XFAD mice in the Lip-ABAH treatment group and the PBS control group. Liver function tests included alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Complete blood count (CBC) tests covered the following parameters: white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), red blood cell distribution width-CV (RDW-CV), red blood cell distribution width-SD (RDW-SD), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), mean platelet volume (MPV), platelet distribution width (PDW), platelet hematocrit (PCT), eosinophils (EOS), basophils (BAS), neutrophils (NEU), lymphocytes (LYM), and monocytes (MON). All tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin. Histological morphological changes were observed by hematoxylin and eosin (H&E) staining. Age-matched wild-type C57BL / 6 mice without any treatment were used as a control group for histological analysis. Results showed that the drug Lip-ABAH had no significant toxic effects on whole blood parameters and key tissues and organs in mice.

[0125] In summary, the pharmacological effects of the drug Lip-ABAH include:

[0126] Inhibit MPO activity and reduce MPO-derived oxidation products (such as hypochlorous acid and perchlorate).

[0127] Reduces ROS levels, alleviates neuroinflammation and DNA oxidative damage (e.g., reduces 8-OHdG expression);

[0128] Reduce Aβ plaque deposition and microglial cell activation;

[0129] Improve cognitive behavior, including spatial memory and recognition memory functions;

[0130] It exhibits good biocompatibility and no significant toxicity was observed in cells, whole blood, or major organs.

[0131] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. An anti-neuroinflammatory drug, characterized in that: The anti-neuroinflammatory drug comprises an active ingredient and liposomes; The active ingredient is 4-aminobenzoyl hydrazine, an inhibitor of myeloperoxidase; The liposomes comprise phospholipids, cholesterol, and distearate phosphatidylethanolamine-thiophene-polyethylene glycol, wherein the molar ratio of the phospholipids, cholesterol, and distearate phosphatidylethanolamine-thiophene-polyethylene glycol is (35-50):(15-25):(30-40).

2. The anti-neuroinflammatory drug according to claim 1, characterized in that: The phospholipid is selected from one or more of lecithin, distearate phosphatidylcholine, and dipalmitoyl phosphatidylcholine.

3. The anti-neuroinflammatory drug according to claim 1, characterized in that: The liposomes also include fluorescently labeled probes selected from one or more of fluorescein isothiocyanate and cyanine dye 5.

4. The anti-neuroinflammatory drug according to claim 1, characterized in that: The dosage forms of the anti-neuroinflammatory drugs include aqueous dispersions, lyophilized powders, or nasal sprays.

5. A method for preparing an anti-neuroinflammatory drug as described in any one of claims 1-4, characterized in that: It includes the following steps: (1) Weigh out phospholipids, cholesterol, distearate phosphatidylethanolamine-thiophene-polyethylene glycol and 4-aminobenzoyl hydrazine, and dissolve them in a mixed solvent of chloroform and methanol to obtain a lipid solution; (2) The lipid solution is rotary evaporated to obtain a lipid membrane; (3) Add preheated phosphate buffer or distilled water to the lipid membrane, and then stir in a water bath to obtain primary liposomes; (4) The primary liposomes are sonicated, and the temperature of the solution needs to be continuously monitored and maintained below 50°C to obtain a liposome solution; (5) Filter the liposome solution, centrifuge, purify the liposomes, and obtain the anti-neuroinflammatory drug.

6. The preparation method according to claim 5, characterized in that: In step (1), the molar ratio of the phospholipid, cholesterol, distearate phosphatidylethanolamine-thiophene-polyethylene glycol, and 4-aminobenzoyl hydrazine is (35-50):(15-25):(30-40):(8-12); and / or, In step (2), the temperature of the rotary evaporation is 40-50℃ and the rotation speed is 100-200rpm.

7. The preparation method according to claim 5, characterized in that: In step (4), the conditions for ultrasound are: ultrasound power of 25-35W, 2s of treatment followed by 2s of cooling, and continuous treatment for 10min.

8. The preparation method according to claim 5, characterized in that: In step (5), during filtration, the filter pore size is 200 nm; during centrifugation, the rotation speed is 6000-7500 rpm, and the time is 5-15 min; and / or, In step (5), the average particle size of the drug is 80-170 nm and the Zeta potential is (-76)-(-23) mV.

9. The use of the anti-neuroinflammatory drug as described in claim 1 in the preparation of a product for treating Alzheimer's disease.

10. The use of the anti-neuroinflammatory drug as described in claim 1 in the preparation of products for treating Parkinson's disease, post-stroke cognitive impairment, multiple sclerosis, or traumatic brain injury.

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