Nanoparticle and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a nanoparticle which comprises an inner core and an outer shell, the inner core is composed of a compound with a specific general formula, the outer shell is composed of 1, 2-dioleoyl-stann-glycerol-3-phosphorylcholine and a peptide-containing shell precursor, and the peptide-containing shell precursor is composed of 1, 2-dioleoyl-stann-glycerol-3-phosphorylcholine and the peptide-containing shell precursor. The peptide-containing shell precursor is composed of distearoyl phosphatidyl ethanolamine, ketal mercaptan, polyethylene glycol and a KLVFF short peptide. The nanoparticles effectively enter the brain and have the effects of resisting A beta aggregation and neuroinflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nanoparticle, its preparation method, and its uses. Background Technology

[0002] Alzheimer's disease (AD) is a common and slowly progressive neurodegenerative disease characterized by memory impairment, cognitive decline, and behavioral abnormalities. Epidemiological projections indicate that the number of AD patients worldwide will exceed 150 million by 2050, placing a heavy medical burden on families and society. The pathogenesis of AD is complex, with core pathological features including the misfolding and aggregation of β-amyloid (Aβ) protein in the brain, oxidative stress imbalance, and neuroinflammatory responses. Aβ monomers can form neurotoxic oligomers and fibrillary deposits through induction or spontaneous processes, creating senile plaques in brain tissue, directly damaging neurons and activating microglia. Activated microglia further release pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), leading to a sustained amplification of neuroinflammation, exacerbating neuronal damage, and thus driving a vicious cycle in the AD pathological process.

[0003] Currently, treatment options for Alzheimer's disease (AD) remain very limited, and overall efficacy is unsatisfactory. On the one hand, most existing drugs target only a single pathological target, making it difficult to effectively break the vicious cycle of "Aβ accumulation—neuroinflammatory—neuronal damage." For example, while the representative drug Aducanumab can target Aβ and promote its clearance, it cannot simultaneously inhibit neuroinflammatory responses, thus hindering comprehensive treatment. Traditional treatments, such as cholinesterase inhibitors, can only improve cognitive function by increasing acetylcholine levels, but cannot cure the disease or halt its progression. Furthermore, side effects such as gastrointestinal discomfort, urinary incontinence, insomnia, and nightmares may occur during drug use. On the other hand, brain drug delivery faces the natural limitation of the blood-brain barrier (BBB). The BBB has highly selective permeability, preventing almost all macromolecular drugs and 98% of small molecule drugs from entering the brain, resulting in insufficient effective drug concentrations, low bioavailability, and poor targeting. These factors collectively limit the therapeutic efficacy of AD drugs.

[0004] Therefore, providing a multi-target AD treatment drug that can effectively enter the brain and has both anti-Aβ aggregation and anti-neuroinflammatory effects has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This invention provides nanoparticles, a preparation method, and their uses to address the problem of the lack of effective AD treatment drugs that can effectively enter the brain and have both anti-Aβ aggregation and anti-neuroinflammatory effects.

[0006] In a first aspect, the present invention provides a nanoparticle comprising a core and a shell, the core being composed of a compound having general formula 1, and the shell being composed of 1,2-dioleoyl-tin-glycero-3-phosphocholine (DOPC) and a peptide-containing shell precursor. General formula 1, where a and b are 2 or 3, and a and b represent the number of hydroxyl groups on the benzene ring; The peptide-containing shell precursor is composed of distearate phosphatidylethanolamine, ketethiocyanate, polyethylene glycol, and KLVFF short peptide.

[0007] In one alternative implementation, any of the following is satisfied: The polyethylene glycol is PEG1000, PEG1000, or PEG2000; The ends of the polyethylene glycol chain are attached with KLVFF short peptides; The compound having general formula 1 is quercetin; The peptide-containing shell precursor is generated from distearate with general formula 2, phosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester, and KLVFF short peptide. Formula 2, where n is 23-150, and can be 23, 45 or 150 at will.

[0008] Secondly, the present invention also provides a method for preparing the above-mentioned nanoparticles, comprising: (1) 1,2-dioleoyl-tin-glycero-3-phosphocholine, quercetin and peptide-containing shell precursor are added to the first solvent in a certain molar ratio and mixed to obtain a mixed solution; (2) Evaporate the mixed solution to obtain a lipid membrane; (3) Add phosphate buffer to the obtained lipid membrane to obtain a suspension, and sonicate the suspension to obtain an initial nanoparticle solution; (4) Centrifuge the obtained initial nanoparticle solution, collect the supernatant, and obtain the final nanoparticle solution.

[0009] In one alternative implementation, any of the following is satisfied: The molar ratio of 1,2-dioleoyl-tin-glycero-3-phosphocholine, quercetin, and the peptide-containing shell precursor is 1:(0.1-0.3):(0.05-0.15). The first solvent is a methanol / chloroform co-solvent system; The evaporation temperature is 25-40℃, preferably 35℃, and / or the evaporation time is 30-60 minutes, preferably 45 minutes; The pH of the phosphate buffer solution is 7.0-7.4, preferably 7.4; The ultrasonic power of the ultrasonic treatment is 100-200 W, preferably 150 W, and / or the ultrasonic time is 1-10 minutes, preferably 5 minutes; The centrifugation temperature is 4-10℃, preferably 4℃, and / or the rotation speed is 3000-8000rpm, preferably 5000rpm, and / or the time is 5-15 minutes, preferably 10 minutes; The ultrasonic treatment was a probe-type ultrasonic treatment.

[0010] In one alternative implementation, any of the following is satisfied: The first solvent is a methanol / chloroform co-solvent system with a volume ratio of (3-6):1, preferably 4:1; The evaporation is rotary evaporation; After adding phosphate buffer to the obtained lipid membrane, hydration is performed; optionally, the hydration time is 30-90 minutes, preferably 60 minutes.

[0011] In one optional embodiment, the preparation method of the peptide-containing shell precursor includes the following steps: Distearate phosphatidylethanolamine-ketothiolate-polyethylene glycol-succinimide ester undergoes an amidation reaction with a KLVFF short peptide, wherein, optionally, the distearate phosphatidylethanolamine-ketothiolate-polyethylene glycol-succinimide ester has general formula 2: Formula 2, where n is 23-150, optionally 23, 45, or 150. In one alternative implementation, any of the following is satisfied: The molar ratio of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester to KLVFF short peptide is 1:(1-1.5), preferably 1:1.2; The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide was carried out at room temperature; The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide was carried out for 24 h. The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is carried out in the first organic solvent; Optionally, after the reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is completed, the step of adding a second organic solvent is also included. Optionally, the first organic solvent is dimethylformamide, and / or the second organic solvent is diethyl ether and methanol; Further optionally, the second organic solvent is diethyl ether and methanol in a volume ratio of (5-15):1, preferably 10:1.

[0012] In one alternative implementation, any of the following is satisfied: After the reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is carried out for 24 h, the reaction mixture is further heated, optionally at 50 °C, to obtain a heated reaction mixture. Optionally, it also includes concentrating the heated reaction mixture to obtain a concentrated reaction mixture; Optionally, the process further includes a step of crystallizing the concentrated reaction mixture. In one alternative implementation, any of the following is satisfied: In the step of crystallizing the concentrated reaction mixture, the temperature is -20°C; In the step of crystallizing the concentrated reaction mixture, the concentrated reaction mixture is added to the second solvent; Optionally, after adding the second solvent, the mixture is allowed to stand for 2 hours.

[0013] Thirdly, the present invention also provides the use of the above-mentioned nanoparticles or nanoparticles prepared by the above-mentioned method in the preparation of drugs for treating Alzheimer's disease, anti-Aβ aggregation drugs, anti-inflammatory drugs and / or drugs for improving cognitive impairment. In one alternative embodiment, the drug inhibits TNF-α, IL-6, and / or IL-1β pro-inflammatory factors; and / or, Clear ROS; and / or, Inhibit apoptosis, optionally inhibit HT22 cell apoptosis; and / or, Restore the phagocytic capacity of microglia, optionally restore the phagocytic capacity of BV2 cells; and / or, Improve spatial exploration ability and object recognition and memory ability; and / or, Inhibit Aβ in the brain 1-42 Deposition of the hippocampus Aβ region 1-42 Deposition.

[0014] The technical solution of this invention has the following advantages: 1. The nanoparticles provided by this invention construct a multi-target combined therapeutic strategy for Alzheimer's disease (AD), simultaneously exerting the dual functions of "inhibiting Aβ aggregation" and "regulating neuroinflammation," effectively overcoming the limitations of traditional drugs with single targets and limited efficacy. Furthermore, the nanoparticles designed in this invention possess ROS-responsive characteristics. When the nanoparticles reach lesion areas with elevated local reactive oxygen species (ROS) levels, the thioketal (TK) in their structure undergoes oxidative cleavage, thereby triggering rapid drug release. Quercetin exerts its anti-inflammatory effect after release, and the exposed KLVFF short peptide can specifically bind to Aβ oligomers, blocking their further fibrotic aggregation. This invention achieves a progressive therapeutic mechanism of "ROS triggering—drug burst release—microenvironment repair," forming a closed-loop regulatory effect, effectively breaking the vicious cycle between Aβ aggregation and neuroinflammation, and providing a new technical approach and implementation strategy for the comprehensive treatment of AD.

[0015] In the nanoparticles of this invention, quercetin encapsulated inside the nanoparticles exerts an anti-inflammatory effect; KLVFF short peptides modified on the surface of the nanoparticles inhibit Aβ aggregation; and the reactive oxygen species responsiveness is provided by TK in the shell structure.

[0016] Oxidative stress is a core factor in the pathogenesis of Alzheimer's disease (AD). Studies have shown that the brain tissue, with its high lipid content and oxygen consumption, is highly susceptible to oxidative stress. Excessive ROS (reactive oxygen species) attack neuronal cell membranes and intracellular components, causing oxidative damage and inducing apoptosis, thereby accelerating the progression of AD. TK is a typical ROS-responsive structural unit containing easily oxidized thioether bonds (-S-). Under high ROS conditions, these bonds are easily oxidized and broken, triggering structural changes. In the ROS-responsive nanoparticles of this invention, TK is used as an intermediate bridging bond connecting a hydrophilic group (PEG) and a hydrophobic group (distearylphosphatidylethanolamine, DSPE). Under normal physiological conditions, the TK structure is stable, ensuring the nanoparticles remain intact in the bloodstream; however, when the nanoparticles enter diseased tissue with high ROS levels, the TK bonds undergo oxidative breakage, leading to nanoparticle structural disintegration and triggering drug release, achieving responsive treatment at the lesion site. 2. The nanoparticles provided by this invention have good stability.

[0017] 3. The nanoparticles provided by this invention have good in vitro biocompatibility and can be efficiently taken up by cells, indicating that the nanoparticles of this invention have good cell delivery performance.

[0018] 4. The nanoparticles provided by this invention have significant ROS scavenging ability.

[0019] 5. The nanoparticles provided by this invention can effectively restore the phagocytic capacity of microglia after Aβ treatment.

[0020] 6. The nanoparticles provided by this invention inhibit cell apoptosis, especially HT22 cell apoptosis.

[0021] 7. The nanoparticles provided by this invention significantly inhibit the deposition of Aβ1-42 in the hippocampus, especially the deposition of Aβ1-42 in the hippocampus.

[0022] 8. The nanoparticles provided by this invention inhibit TNF-α, IL-6 and / or IL-1β pro-inflammatory factors. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The physicochemical properties of NPs@Qu / KLVFF in Test Example 1 of this invention are shown. A shows the particle size distribution and PDI of NPs@Qu / KLVFF; B shows the morphology of NPs@Qu / KLVFF under a transmission electron microscope.

[0025] Figure 2 This shows the quercetin release rate of NPs@Qu / KLVFF in Test Example 3 of the present invention under conditions of presence and absence of H2O2.

[0026] Figure 3 This invention demonstrates the in vitro anti-Aβ aggregation of NPs@Qu / KLVFF in Test Example 4. A shows a fluorescence microscopy image of in vitro anti-Aβ aggregation; B shows the quantitative fluorescence statistics of in vitro Aβ aggregation.

[0027] Figure 4 This invention presents a quantitative statistical analysis of the fluorescence intensity of BV2 cells phagocytosis of Aβ in test example 6 of this invention.

[0028] Figure 5 This invention demonstrates the production of inflammatory factors (IL-6, IL-1β, TNF-α) in BV2 cells after Aβ induction and NPs@Qu / KLVFF treatment in Test Example 7. Detailed Implementation

[0029] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental steps or conditions in the examples were performed in accordance with conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0031] The mice used in this invention were wild-type C57BL / 6 mice, 8 months old, male, weighing approximately 20-30 g. APP / PS1 transgenic mice, 8 months old, male, weighing approximately 20-30 g.

[0032] DOPC, with a molecular weight of 786.12 Da, was purchased from Taoshu Biotechnology. Its structural formula is as follows:

[0033] Quercetin: molecular weight 302.24 Da, purchased from: Taoshu Biotechnology, structural formula is:

[0034] DSPE-TK-PEG2000-NHS: Molecular weight 3100.52 Da, purchased from: Shenzhen Meiluo Technology Co., Ltd. n is 23-150, and can be 23, 45 or 150 at will.

[0035] The KLVFF polypeptide is composed of 5 amino acids, with a molecular weight of 652.82 Da. It is manufactured by Shenzhen Meiluo Technology Co., Ltd., and its sequence is: Lys-Leu-Val-Phe-Phe. The KLVFF structure is as follows: .

[0036] Example 1 This embodiment provides reactive oxygen species-responsive nanoparticles for the combined treatment of AD with anti-Aβ aggregation and anti-inflammation. The nanoparticles are prepared by a method comprising the following steps: 1) DSPE-TK-PEG2000-KLVFF was prepared by the following method: (1) Weigh DSPE-TK-PEG2000-NHS (300 mg, 0.0936 mmol) and dissolve it in 10 mL of anhydrous DMF; (2) Weigh out KLVFF peptide (78 mg, 0.112 mmol) and dissolve it in 10 mL of anhydrous DMF. Then, under stirring conditions, add it to the above raw material solution at a molar ratio of 0.8:1 between DSPE-TK-PEG2000-NHS and KLVFF peptide. Stir the reaction at room temperature for 24 h to obtain the reaction mixture. (3) The reaction mixture was then heated to 50°C to obtain a concentrated reaction mixture; (4) Under stirring conditions at -20°C, the concentrated reaction mixture was added to ice-cold diethyl ether / methanol (10 / 1, v / v, 150 mL). After the addition was complete, stirring was stopped, and the mixture was allowed to stand for 2 h to obtain the settled reaction mixture. (5) Finally, the reaction mixture after standing was filtered to obtain DSPE-TK-PEG2000-KLVFF.

[0037] 2) Add DOPC, quercetin and DSPE-TK-PEG2000-KLVFF to a 5 mL methanol / chloroform cosolvent (4:1 v / v) system at a molar ratio of 1:0.1:0.05, stir, and obtain a mixed solution.

[0038] 3) The mixed solution is rotary evaporated at 35°C for 30 minutes until a lipid film is formed.

[0039] 4) Add 2 mL of phosphate-buffered saline (PBS) to the obtained lipid membrane for hydration to obtain a suspension.

[0040] 5) The suspension was sonicated at 150 W for 2 minutes to obtain the initial nanoparticle solution.

[0041] 6) The obtained initial nanoparticle solution was centrifuged at 5000 rpm for 10 minutes at 4°C to remove unencapsulated quercetin. The supernatant was collected to obtain the final nanoparticle solution. The obtained nanoparticles were named NPs@Qu / KLVFF.

[0042] Comparative Examples 1 and 2 Except for NPs@Qu / KLVFF, two control systems were prepared using the same method as in Example 1: one without peptides (NPs@Qu, Comparative Example 1) and the other without quercetin (NPs@KLVFF, Comparative Example 2).

[0043] Test Example 1 Physicochemical characterization of nanoparticles Characterization of particle size, dispersion factor, and Zeta potential: The particle size, dispersion index (PDI), and zeta potential of nanoparticles were determined using a nanoparticle size and potential analyzer. 10 μL of the prepared NPs@Qu / KLVFF nanoparticle sample was diluted with 1 mL of deionized water and mixed thoroughly before being added to the particle size analysis sample cell. Dynamic light scattering (DLS) was used to determine the particle size and PDI. The test conditions were set as follows: equilibration time 1 min, measurement interval 10 s, and test temperature 25 ℃. Subsequently, diluted samples of the same concentration were transferred to the zeta potential analysis cell, and the zeta potential of the nanoparticles was measured. The results are as follows: Figure 1 As shown, the average particle size of NPs@Qu / KLVFF is 106.8 nm, and the average PDI is 0.233.

[0044] Morphological characteristics: The morphology of nanoparticles was characterized by phosphotungstic acid staining combined with transmission electron microscopy. The results are as follows: Figure 1 As shown, the nanoparticles have a spherical structure with regular morphology and good uniformity.

[0045] Quercetin encapsulation efficiency test: A standard working curve was established using high-performance liquid chromatography (HPLC) based on quercetin concentration and peak area. 50 μL of the Example 1 nanoparticle sample (NPs@Qu / KLVFF) was added to an equal volume of 2% Triton X-100 solution for demulsification, and sonicated for 5 min to ensure complete demulsification. The quercetin content was calculated using the standard curve and recorded as the total quercetin content. 200 μL of nanoparticles was centrifuged at 5000 rpm for 5 min, and 50 μL of the supernatant was collected. An equal volume of 2% Triton X-100 solution was added to demulsify, and sonicated for 5 min to ensure complete demulsification. The quercetin content was calculated using the standard curve and recorded as the quercetin encapsulation amount. Quercetin encapsulation efficiency (%) = (Quercetin encapsulation amount / Total quercetin) × 100% The encapsulation efficiency of quercetin was measured to be 78.4%.

[0046] Stability test: To evaluate the colloidal stability of the nanoparticles, their stability was monitored under simulated physiological conditions (DMEM medium containing 10% FBS, 37 °C) and in PBS buffer. Microscopic observation revealed no significant aggregation of NPs@Qu / KLVFF under simulated physiological conditions for 12 hours. DLS analysis showed no significant changes in particle size and PDI of NPs@Qu / KLVFF after 14 days of storage in PBS, verifying their storage stability and good colloidal behavior in in vitro experiments.

[0047] Test Example 2 Biocompatibility testing of nanoparticles To examine the cytotoxicity of the nanoparticles, BV2 cells were seeded into 96-well plates (10,000 cells per well) and treated with different concentrations of NPs@Qu / KLVFF for 24 hours. Cell viability was then assessed using a CCK-8 assay kit according to the manufacturer's protocol. The CCK-8 results showed that BV2 cells maintained viability above 90% after treatment with NPs@Qu / KLVFF at concentrations ranging from 1 to 200 μg / mL for 24 hours, with no significant dose-dependent differences, indicating that NPs@Qu / KLVFF possesses good biocompatibility.

[0048] Test Example 3 ROS-responsive release of nanoparticles To evaluate the ROS-responsive drug release behavior of nanoparticles, a comparative experiment was conducted under both oxidative stress-free and oxidative stress environments. The focus was on investigating the ROS responsiveness of NPs@Qu / KLVFF, simulating the oxidative stress microenvironment of Alzheimer's disease (AD) brain tissue lesions, and verifying its drug release capacity under high ROS conditions. The specific experimental method was as follows: Three different PBS buffer solutions were prepared as drug release media: a) No oxidative stress medium: PBS buffer at pH 7.4 (PBS group). b) Low oxidative stress medium: PBS buffer at pH 7.4 containing 0.2 mM H2O2 (0.2 mM H2O2 group). c) High oxidative stress medium: PBS buffer at pH 7.4 containing 1.0 mM H2O2 (1 mM H2O2 group). Equal concentrations (0.5 mg / mL) and equal volumes (5 mL) of NPs@Qu / KLVFF nanoparticle solutions were placed in dialysis bags with a molecular weight cutoff of 3500 Da. The sealed dialysis bags were completely immersed in the three release media. The entire release system was placed in a constant-temperature shaker at 37°C and constant low-speed shaking (100 rpm) in the dark to simulate the body temperature and fluid environment. At preset time points, 1 mL samples were collected from each release medium. After each sampling, an equal volume, temperature, and composition of fresh medium was immediately added to the corresponding release system to maintain a constant total volume and stable H2O2 concentration. Finally, the concentration of free quercetin in the samples was determined using high-performance liquid chromatography (HPLC).

[0049] The results are as follows Figure 2The results showed that NPs@Qu / KLVFF exhibited a low drug release rate in the absence of H2O2, with a cumulative release of approximately 23.7% after 48 hours. The release rate significantly increased under 0.2 mM H2O2 conditions, reaching approximately 61.4% cumulative release after 48 hours, and approximately 93.4% under 1 mM H2O2 conditions, demonstrating that NPs@Qu / KLVFF possesses significant ROS responsiveness. The cumulative release in the 1 mM H2O2 group was nearly four times that of the PBS group. This indicates that the TK bond is highly sensitive to ROS, enabling on-demand drug release in response to the lesion microenvironment and avoiding side effects caused by off-target release.

[0050] Test Example 4 In vitro anti-Aβ aggregation test of nanoparticles In the early stages of Alzheimer's disease (AD), Aβ gradually aggregates from monomers into oligomers and fibrils, eventually forming senile plaques deposited in brain tissue. This invention uses the Thioflavin T (ThT) fluorescence method to evaluate the anti-Aβ aggregation effect of nanoparticles. First, Aβ... 1-42 The peptide powder was thoroughly dissolved and diluted to a final concentration of 5 μM in PBS (pH=7.4). The prepared Aβ solution was then mixed with the nanoparticles (NPs@Qu / KLVFF) from Example 1 to achieve a final Aβ concentration of 20 μM. PBS was used as a negative control (final Aβ concentration of 20 μM), and Aβ was used as a positive control (final Aβ concentration of 20 μM). 10 μM of ThT fluorescent dye was added to each solution, and the solutions were aliquoted into black 96-well plates. The plates were sealed and incubated at 37 °C with gentle shaking for 6 hours to induce Aβ aggregation. After incubation, the fluorescence intensity of each well was read using a fluorescence microplate reader at an excitation wavelength of 440 nm and an emission wavelength of 485 nm. Using the fluorescence intensity of the Aβ control group as 100%, the inhibition rate of each nanoparticle against Aβ aggregation was assessed based on the percentage decrease in fluorescence intensity, thus determining its anti-Aβ aggregation activity. The aggregation morphology and degree of Aβ in different experimental groups were observed using a fluorescence microscope. The results are as follows: Figure 3 The results showed that NPs@Qu / KLVFF significantly inhibited Aβ aggregation.

[0051] Test Example 5 In vitro ROS scavenging effect of nanoparticles Microglia, as the main immune cells of the central nervous system, play a crucial role in recognizing and clearing Aβ and regulating inflammatory responses. Based on this, this test case constructed an in vitro high ROS model of BV2 cells stimulated by Aβ oligomers to systematically evaluate the response behavior and antioxidant capacity of the NPs@Qu / KLVFF nanosystem under oxidative stress. The specific experimental method was as follows: BV2 cells were seeded in 24-well plates (100,000 cells per well) and cultured overnight to allow cell adhesion. Prepared Aβ was then added... 1-42 Cells were co-cultured with a 20 μM solution for 24 hours, followed by treatment with PBS and / or different nanoparticle solutions for 24 hours. The changes in intracellular ROS levels after treatment were detected using the universal ROS probe DCFH-DA. Results showed that the Aβ+PBS group exhibited strong green fluorescence, indicating that Aβ significantly induced ROS accumulation. In contrast, NPs@Qu and NPs@KLVFF treatments inhibited ROS generation to varying degrees, with NPs@KLVFF showing the weakest effect, indicating that antioxidant activity is limited when only a ROS-responsive structure is present without active ingredients. The NPs@Qu / KLVFF treatment group exhibited the most significant ROS scavenging ability, with green fluorescence intensity almost dropping to the control group level. Quantitative results showed that NPs@Qu / KLVFF treatment reduced ROS levels by approximately 65.7%, significantly better than NPs@Qu (48.6%) and NPs@KLVFF (26.3%), suggesting that its excellent antioxidant potential may stem from a quercetin release-synergistic ROS-responsive degradation mechanism.

[0052] Test Example 6 Tests on the promoting effect of nanoparticles on microglia phagocytosis of Aβ Microglia, as resident immune cells of the central nervous system, are mainly divided into two phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2). Their activation state has a decisive influence on disease progression. M1 microglia are often accompanied by the release of inflammatory factors and impaired phagocytosis, promoting Aβ deposition and neurotoxicity, while M2 microglia exhibit strong anti-inflammatory and Aβ clearance capabilities. This study used BV2 microglia as a model to systematically evaluate the regulatory effect of the NPs@Qu / KLVFF nanosystem on the immune function of microglia, starting with the uptake of Aβ monomers. First, a FITC-labeled Aβ monomer treatment model was constructed, and the phagocytic capacity of BV2 cells for Aβ-FITC was detected using confocal microscopy. Specifically, BV2 cells were seeded in 24-well plates (100,000 cells per well) and cultured overnight to allow cell adhesion. The prepared fluorescent dye FITC-labeled Aβ monomers were then added. 1-42 The cells were co-cultured in a 20 μM solution for 24 hours. Results showed... Figure 4As shown, only a weak FITC signal appeared in BV2 cells after Aβ stimulation, suggesting that Aβ-induced phagocytic dysfunction in BV2 cells. This phenomenon may be related to the fact that toxic Aβ aggregates promote the transformation of BV2 cells to a pro-inflammatory phenotype and impair their phagocytic capacity. In contrast, the intracellular FITC signal was significantly enhanced after treatment with NPs@KLVFF, NPs@Qu, and NPs@Qu / KLVFF, with the most significant increase observed in the NPs@Qu / KLVFF group. This indicates that the nanoparticles of this invention can effectively restore the phagocytic capacity of microglia after Aβ treatment.

[0053] Test Example 7 In vitro anti-inflammatory effects of nanoparticles Aβ oligomers are a major causative factor of neurotoxicity in Alzheimer's disease (AD), inducing persistent inflammatory responses in microglia and releasing inflammatory factors, further leading to synaptic damage and neural network degeneration. To verify the inhibitory effect of the nanoparticles prepared in Example 1 on the Aβ oligomer-induced inflammatory response and their immunomodulatory ability on microglia, this invention used Aβ oligomers to induce BV2 cells to establish an in vitro AD neuroinflammation model, followed by the addition of nanoparticles for intervention. Preparation method of Aβ oligomers: Aβ… 1-42 Peptide powder was dissolved in pre-chilled hexafluoroisopropanol (HFIP) and incubated at room temperature for at least 1 hour to promote peptide monomerization and randomization of their secondary structures. The HFIP was then removed by evaporation, and the dried peptides were dissolved in DMSO to a concentration of 5 mM. The resulting solution was then diluted to 200 μM with serum-free, phenol red-free medium and aged at 4 °C for 48 hours. Afterwards, the supernatant was collected by centrifugation at 12,000 rpm for 10 minutes at 4 °C, and the soluble oligomers were used for subsequent induction experiments. BV2 cells were pretreated with Aβ oligomers to a final concentration of 20 μM for 12 hours, followed by incubation with nanoparticles for a total of 24 hours. Cells were then collected, and cytokine levels in cell lysates were measured. Results are as follows: Figure 5 As shown, NPs@Qu and NPs@Qu / KLVFF significantly inhibited the production of pro-inflammatory factors (IL-6, IL-1β, TNF-α).

[0054] Test Example 8 In vitro anti-apoptotic activity test of nanoparticles Literature reports that toxic Aβ accumulation not only activates microglia but also directly induces neuronal apoptosis, causing irreversible neurological damage. To verify the alleviating effect of NPs@Qu / KLVFF on Aβ-related neurotoxicity, the Annexin V / PI double staining assay was used to evaluate its protective effect against HT22 neuronal apoptosis. The results showed that Aβ treatment significantly induced HT22 cell apoptosis, with an apoptosis rate as high as 24.6%. The apoptosis rates of cells treated with NPs@KLVFF, NPs@Qu, and NPs@Qu / KLVFF were 20.1%, 16.9%, and 12.8%, respectively, all of which reduced the apoptosis rate to varying degrees. These results indicate that NPs@Qu / KLVFF has the strongest anti-apoptotic effect, highlighting the synergistic advantage of Qu and KLVFF.

[0055] Test Example 9 Testing the effect of nanoparticles on improving cognitive dysfunction in AD model mice To investigate the effect of the NPs@Qu / KLVFF nanoparticles described in this invention on improving cognitive dysfunction in AD model mice, APP / PS1 transgenic AD mice were used as the disease model group, and age-matched wild-type mice (WT) were used as the healthy control group. All mice were divided into 5 groups (n=8 per group): (1) WT-PBS, (2) AD-PBS, (3) AD-NPs@Qu, (4) AD-NPs@KLVFF, and (5) AD-NPs@Qu / KLVFF. PBS solution or nanoparticle solution was administered intranasally to each group. The drugs were administered intranasally every two days. After 15 administrations, behavioral tests were performed, including the open field test (OFT) and the Y-maze test. The results are shown in Table 1. After intranasal administration, NPs@Qu / KLVFF can effectively alleviate anxiety-like behavior in AD mice and enhance their spatial exploration ability and object recognition and memory ability. Moreover, the improvement effect of the NPs@Qu / KLVFF group is better than that of the NPs@Qu group and the NPs@KLVFF group, indicating that the combined efficacy of the NPs@Qu / KLVFF group is greater than that of the single drug.

[0056] Table 1 Test Results

[0057] Test Case 10 Inhibitory effect of nanoparticles on Aβ plaques in the brain of AD model mice After completing Test Example 9, mice were sacrificed, and brain tissue was collected to study the deposition of Aβ plaques in the mouse brains after nanoparticle treatment. Aβ deposition is considered a core marker of Alzheimer's disease (AD). Immunofluorescence staining and semi-quantitative analysis of mouse brain slices were performed to investigate the inhibitory effect of nanoparticles on Aβ plaques in the brains of AD model mice. Results showed abundant red fluorescence signals in the hippocampus of APP / PS1 model mice, indicating significant Aβ accumulation in the brain. While NPs@Qu treatment slightly reduced the Aβ load, accumulated plaques remained clearly visible. Both NPs@KLVFF and NPs@Qu / KLVFF treatment groups showed significant Aβ reduction and a substantial decrease in fluorescence intensity, suggesting a good effect in inhibiting Aβ deposition. Quantitative analysis of plaque area showed that the Aβ plaque area in the NPs@Qu / KLVFF group was reduced by 76% compared to the AD+PBS group, significantly better than the 65% reduction in NPs@KLVFF and the 39% reduction in the NPs@Qu group, clearly validating the key role of the KLVFF short peptide construction on the surface of the nanosystem in blocking Aβ aggregation.

[0058] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A nanoparticle, characterized in that, It comprises a core and a shell, the core being composed of a compound having general formula 1, and the shell being composed of 1,2-dioleoyl-tin-glycero-3-phosphocholine and a peptide-containing shell precursor. General formula 1, where a and b are 2 or 3; The peptide-containing shell precursor is composed of distearate phosphatidylethanolamine, ketethiocyanate, polyethylene glycol, and KLVFF short peptide.

2. The nanoparticles according to claim 1, characterized in that, Satisfy any of the following: The polyethylene glycol is PEG1000, PEG2000, or PEG5000; The ends of the polyethylene glycol chain are attached with KLVFF short peptides; The compound having general formula 1 is quercetin; The peptide-containing shell precursor is generated from distearate with general formula 2, phosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester, and KLVFF short peptide. Formula 2, where n is 23-150, and can be 23, 45 or 150 at will.

3. The method for preparing nanoparticles according to claim 1 or 2, characterized in that, include: (1) 1,2-dioleoyl-tin-glycero-3-phosphocholine, quercetin and peptide-containing shell precursor are added to the first solvent in a certain molar ratio and mixed to obtain a mixed solution; (2) Evaporate the mixed solution to obtain a lipid membrane; (3) Add phosphate buffer to the obtained lipid membrane to obtain a suspension, and sonicate the suspension to obtain an initial nanoparticle solution; (4) Centrifuge the obtained initial nanoparticle solution, collect the supernatant, and obtain the final nanoparticle solution.

4. The method for preparing nanoparticles according to claim 3, characterized in that, Satisfy any of the following: The molar ratio of 1,2-dioleoyl-tin-glycero-3-phosphocholine, quercetin, and the peptide-containing shell precursor is 1:(0.1-0.3):(0.05-0.15). The first solvent is a methanol / chloroform co-solvent system; The evaporation temperature is 25-40℃, preferably 35℃, and / or the evaporation time is 30-60 minutes, preferably 45 minutes; The pH of the phosphate buffer solution is 7.0-7.4, preferably 7.4; The ultrasonic power of the ultrasonic treatment is 100-200 W, preferably 150 W, and / or the ultrasonic time is 1-10 minutes, preferably 5 minutes; The centrifugation temperature is 4-10℃, preferably 4℃, and / or the rotation speed is 3000-8000 rpm, preferably 5000 rpm, and / or the time is 5-15 minutes, preferably 10 minutes; The ultrasonic treatment was a probe-type ultrasonic treatment.

5. The method for preparing nanoparticles according to claim 4, characterized in that, Satisfy any of the following: The first solvent is a methanol / chloroform co-solvent system with a volume ratio of (3-6):1, preferably 4:1; The evaporation is rotary evaporation; After adding phosphate buffer to the obtained lipid membrane, hydration is performed; optionally, the hydration time is 30-90 minutes, preferably 60 minutes.

6. The method for preparing nanoparticles according to any one of claims 3-5, characterized in that, The preparation method of the peptide-containing shell precursor includes the following steps: Distearate phosphatidylethanolamine-ketothiolate-polyethylene glycol-succinimide ester undergoes an amidation reaction with a KLVFF short peptide, wherein, optionally, the distearate phosphatidylethanolamine-ketothiolate-polyethylene glycol-succinimide ester has general formula 2: Formula 2, where n is 23-150, and can be 23, 45 or 150 at will.

7. The method for preparing nanoparticles according to claim 6, characterized in that, Satisfy any of the following: The molar ratio of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester to KLVFF short peptide is 1:(1-1.5), preferably 1:1.2; The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide was carried out at room temperature; The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide was carried out for 24 h. The reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is carried out in the first organic solvent; Optionally, after the reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is completed, the step of adding a second organic solvent is also included. Optionally, the first organic solvent is dimethylformamide, and / or the second organic solvent is diethyl ether and methanol; Further optionally, the second organic solvent is diethyl ether and methanol in a volume ratio of (5-15):1, preferably 10:

1.

8. The method for preparing nanoparticles according to claim 7, characterized in that, Satisfy any of the following: After the reaction of distearylphosphatidylethanolamine-ketothiol-polyethylene glycol-succinimide ester with KLVFF short peptide is carried out for 24 h, the reaction mixture is further heated, optionally at 50 °C, to obtain a heated reaction mixture. Optionally, it also includes concentrating the heated reaction mixture to obtain a concentrated reaction mixture; Optionally, the process further includes a step of crystallizing the concentrated reaction mixture.

9. The method for preparing nanoparticles according to claim 8, characterized in that, Satisfy any of the following: In the step of crystallizing the concentrated reaction mixture, the temperature is -20°C; In the step of crystallizing the concentrated reaction mixture, the concentrated reaction mixture is added to the second solvent; Optionally, after adding the second solvent, the mixture is allowed to stand for 2 hours.

10. Use of the nanoparticles according to claim 1 or 2, or nanoparticles prepared by the method of preparing nanoparticles according to any one of claims 3-9, in the preparation of medicaments for treating Alzheimer's disease, anti-Aβ aggregation drugs, anti-inflammatory drugs, and / or medicaments for improving cognitive impairment; Optionally, the drug inhibits TNF-α, IL-6 and / or IL-1β pro-inflammatory factors; and / or, Clear ROS; and / or, Inhibit apoptosis, optionally inhibit HT22 cell apoptosis; and / or, Restore the phagocytic capacity of microglia, optionally restore the phagocytic capacity of BV2 cells; and / or, Improve spatial exploration ability and object recognition and memory ability; and / or, Inhibit Aβ in the brain 1-42 Deposition of the hippocampus Aβ region 1-42 Deposition.

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