A brain-targeting app-dextran-fe3o4 nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of medical imaging, and particularly relates to brain-targeting APP@Dextran-Fe3O4 nanoparticles as well as a preparation method and application thereof. The APP@Dextran-Fe3O4 nanoparticles provided by the application can act on the beta amyloid protein receptor in the brain through a non-invasive intravenous injection mode, thereby realizing the transport across the blood-brain barrier, and having a higher accumulation in the brain of Alzheimer's disease patients, especially in the hippocampus which is the main lesion site of Alzheimer's disease. In combination with the high resolution and high sensitivity characteristics of MPI imaging, the diagnosis of Alzheimer's disease can be realized. Meanwhile, pathological analysis shows that the APP@Dextran-Fe3O4 nanoparticles have the effects of inhibiting the inflammatory reaction in the brain of Alzheimer's disease and reducing the apoptosis of neuron cells, and have certain benefits for slowing down Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical imaging, and more particularly relates to a brain-targeting APP@Dextran-Fe3O4 nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease, and its pathological features mainly include brain atrophy, especially hippocampal and temporal lobe atrophy, and deposition of beta-amyloid protein, excessive phosphorylation of tau protein and neurofibrillary tangles. Clinically, CT, MRI and PET imaging techniques can be used to diagnose Alzheimer's disease. Unfortunately, no new imaging technology has been developed in essence. The main shortcomings of CT and PET imaging are radioactive exposure, and the high cost of PET imaging in diagnosis. The main shortcomings of MRI imaging are low spatial and temporal resolution, and the kidney toxicity caused by imaging agents cannot be ignored. Therefore, there is an urgent need for innovative, safe, efficient and economical diagnostic methods for the diagnosis of Alzheimer's disease.

[0003] From 2001 to 2005, Gleich and Weichert developed a new imaging method (MPI) called magnetic particles. This imaging is a new non-radiation tomography technology, which can provide fast, background-free and sensitive imaging diagnostic results. However, in Alzheimer's disease, the clinical diagnostic reagent approved by FDA for MPI cannot be applied to brain imaging due to its inability to pass through the blood-brain barrier. Therefore, the synthesis of a new brain-targeting imaging tracer is the key to the application of MPI in AD diagnosis. SUMMARY

[0004] The purpose of the present application is to provide a brain-targeting APP@Dextran-Fe3O4 nanoparticle and a preparation method and application thereof.

[0005] To achieve the above purpose, the technical solution adopted by the present application is as follows:

[0006] The present application provides a brain-targeting APP@Dextran-Fe3O4 nanoparticle, which is obtained by modifying the APP polypeptide on the surface of Dextran-Fe3O4 nanoparticles through the positive and negative charge combination of the carboxyl group of the APP polypeptide and the hydroxyl group on the surface of the Dextran-Fe3O4 nanoparticles. The Dextran-Fe3O4 nanoparticles are Fe3O4 magnetic nanoparticles wrapped by dextran.

[0007] This invention modifies the surface of Dextran-Fe3O4 nanoparticles by binding the carboxyl groups of the brain-targeting APP peptide to the hydroxyl groups on the surface of the magnetic nanoparticles through positive and negative charge bonding, thereby obtaining APP@Dextran-Fe3O4 nanoparticles with brain-targeting activity. These nanoparticles can act on the β-amyloid receptors in the brain via non-invasive intravenous injection, achieving cross-blood-brain barrier transport and accurately targeting the hippocampus, a major lesion site in Alzheimer's disease. They also feature high resolution and high sensitivity in MPI imaging, making them suitable for the diagnosis of Alzheimer's disease.

[0008] Furthermore, the particle size of the APP@Dextran-Fe3O4 nanoparticles is 15–20 nm.

[0009] This invention also provides a method for preparing the above-mentioned brain-targeting APP@Dextran-Fe3O4 nanoparticles, comprising the following steps:

[0010] S1. Incubate the dextran solution at 80-90℃ for 1-2 hours, add NaOH solution and continue the reaction for 1-3 minutes, then add FeCl3·6H2O and FeCl2·4H2O, react at 90-100℃ for 1-2 hours, adjust the pH to 10-11 and continue the reaction for 5-15 minutes, cool and centrifuge to remove the supernatant, the precipitate is separated magnetically, washed with water and dried to obtain Dextran-Fe3O4 nanoparticles; wherein the mass ratio of dextran, NaOH, FeCl3·6H2O and FeCl2·4H2O is 1-2g: 1-3g: 0.5-3g: 0.5-2.5g;

[0011] S2. Acetone and epichlorohydrin were added to the Dextran-Fe3O4 nanoparticle solution, and the reaction was carried out for 1–30 min. Then, a 48%–52% NaOH solution was added, and the reaction was continued with stirring for 5–8 h. The mixture was washed with anhydrous ethanol and water, and after magnetic separation, the supernatant was discarded and the precipitate was retained. The precipitate was added to the APP peptide solution and reacted at 35–40 °C and 200–400 rpm / min for 7–9 h. Then, the APP@Dextran-Fe3O4 nanoparticles were obtained by magnetic separation. The mass-volume ratio of Dextran-Fe3O4 nanoparticles, acetone, epichlorohydrin, NaOH solution and APP peptide was 5–15 mg: 1–5 mL: 3–6 mL: 0.1–1 mL: 0.1–0.5 g.

[0012] This invention also provides the application of the above-mentioned brain-targeting APP@Dextran-Fe3O4 nanoparticles in the preparation of Alzheimer's disease diagnostic products.

[0013] Furthermore, the diagnostic product is a brain-targeted imaging tracer.

[0014] This invention also provides the application of the above-mentioned brain-targeting APP@Dextran-Fe3O4 nanoparticles in the preparation of drugs for treating Alzheimer's disease.

[0015] Furthermore, the APP@Dextran-Fe3O4 nanoparticles are the sole active ingredient in the drug.

[0016] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0017] The present invention has the following beneficial effects:

[0018] The brain-targeting APP@Dextran-Fe3O4 nanoparticles provided in this invention cross the blood-brain barrier via β-amyloid receptor-mediated transport and exhibit higher accumulation in the brains of Alzheimer's disease patients, particularly in the hippocampus, a key site of Alzheimer's disease. MPI imaging can be used for the diagnosis of Alzheimer's disease. Pathological analysis also revealed that APP@Dextran-Fe3O4 nanoparticles inhibit inflammatory responses in the brain of Alzheimer's patients and reduce neuronal apoptosis, suggesting potential benefits in alleviating the progression of Alzheimer's disease. Attached Figure Description

[0019] Figure 1 Characterization images of APP@Dextran-Fe3O4 nanoparticles are shown below. Image a is an electron microscopy image of the APP@Dextran-Fe3O4 nanoparticles. Image b is a colorimetric experiment image, where 1 represents the APP peptide, 2 represents the Dextran-Fe3O4 nanoparticles, and 3 represents the APP@Dextran-Fe3O4 nanoparticles. Image c shows the linear relationship between APP@Dextran-Fe3O4 nanoparticles with different iron contents and the imaging signal intensity.

[0020] Figure 2Images show the application of APP@Dextran-Fe3O4 nanoparticles in the imaging diagnosis of Alzheimer's disease (AD) mice. Figure a shows 2D and 3D imaging diagrams of the distribution of APP@Dextran-Fe3O4 nanoparticles in the mouse brain; the red arrows indicate the fitting reference points provided by the combined MPI and MRI scans. WT represents wild-type mice, and AD represents Alzheimer's disease model mice. Figure b shows an MPI diagnostic diagram of lesions in the hippocampus of mice. Figure c shows a quantitative statistical graph of the overall signal intensity of APP@Dextran-Fe3O4 nanoparticles in the brain after MPI-2D imaging in figure a. Figure d shows a quantitative statistical graph of the signal intensity of MPI imaging in the hippocampus. Figure e shows an immunohistochemical image of a cross-section of the hippocampus, where Iba1 is a marker of microglia aggregation, and Aβ is a marker of amyloid protein deposition. Figure f shows the statistical graph of the positive rate of microglia in figure e. Figure g shows the quantitative statistical graph of the positive rate of Aβ in figure e.

[0021] Figure 3 This image shows the in vivo pharmacodynamic evaluation of APP@Dextran-Fe3O4 nanoparticles in the treatment of Alzheimer's disease (AD). Image a shows Nissl staining of neurons in the hippocampus of mice, with magnified observations of the DG, CA1, and CA3 regions. The darker the staining of neurons and the greater the number of blue-purple Nissl bodies within the cells, the less neuronal damage; red arrows indicate Nissl bodies. Image b shows immunohistochemical staining of Aβ deposition in the hippocampus, where Aβ is stained brown (marked by red arrows). Image c shows immunohistochemical staining of microglia (Iba1) in the hippocampus, where microglia are stained brown (marked by red arrows). Image d shows the quantitative statistical analysis of Aβ expression in images b and c. Image e shows the quantitative statistical analysis of Iba1 expression in images b and c. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0023] The APP polypeptide of this invention is derived from the amino acid sequence in the Aβ amyloid domain, which is: cysteine-histidine-leucine-aspartic acid-isoleucine-isoleucine-tryptophan, and was synthesized by GeneCopeia Biotechnology.

[0024] Example 1: Synthesis and characterization of APP@Dextran-Fe3O4 nanoparticles.

[0025] 2g of dextran with a molecular weight of 20,000 was dissolved in 20mL of deionized water and heated and stirred at 90℃ for 1h. 8mL of 5M NaOH solution was added and stirring continued for 2min. Then, 2.7g of FeCl3·6H2O and 1.9g of FeCl2·4H2O were added, and the reaction was carried out at 95℃ for 1h. The pH was then adjusted to 11, and stirring continued for 10min. After natural cooling, the mixture was centrifuged at 1000rpm / min for 5min. The supernatant was collected, and after magnetic separation and thorough washing with deionized water, it was dried at 45℃ to obtain Dextran-Fe3O4 nanoparticles. These nanoparticles were dissolved in an aqueous solution and ultrasonically dissolved in a 200W water bath for 12 hours to ensure stable dispersion in the aqueous solution, facilitating further surface modification and other applications.

[0026] 10 mg of Dextran-Fe3O4 nanoparticles were weighed and dissolved in 4 mL of deionized water. The solution was sonicated to aid dissolution. 4 mL of acetone and epichlorohydrin solution were added, and the mixture was mechanically stirred at 35 °C and 200 rpm for 10 min to mix thoroughly. 700 μL of 50% NaOH solution was added, and the reaction was continued for 6 h. The reaction was then stopped. The mixture was washed 5 times with anhydrous ethanol and 5 times with deionized water to remove the solution from the reaction process. After magnetic separation, the supernatant was discarded to obtain the precipitate. 10 mg of APP peptide was dissolved in 4 mL of sodium carbonate buffer (pH 8.5) and added to the precipitate. The mixture was reacted at 37 °C and 300 rpm in a shaker for 8 h. The supernatant was recovered, and the concentration of APP peptide in the supernatant was calculated using the BCA protein assay. The reaction efficiency was calculated. The magnetic beads were then magnetically separated, and the mixture was washed 3 times each with deionized water and PBS solution to obtain APP@Dextran-Fe3O4 nanoparticles. The morphology and particle size of APP@Dextran-Fe3O4 nanoparticles were observed using field transmission electron microscopy, the BCA protein reaction method was used to determine whether the surface was modified with APP peptides, and the in vitro imaging performance was determined by MPI.

[0027] Figure 1 As shown in Figure a, under electron microscopy, the Fe3O4 core of the APP@Dextran-Fe3O4 nanoparticles has a diameter of approximately 5 nm, while the APP@Dextran-Fe3O4 nanoparticles formed after external modification with APP and dextran have a particle size of approximately 17 nm. Figure 1 As shown in b, the surface peptide modification of nanoparticles was detected by BCA protein reaction experiment. APP peptides showed a blue-purple color after reaction, while Dextran-Fe3O4 nanoparticles showed no positive color development. The reaction solution of APP@Dextran-Fe3O4 nanoparticles modified with APP peptides showed a blue-purple color. The BCA protein reaction method showed that compared with unmodified Dextran-Fe3O4 nanoparticles, it can turn the BCA reaction solution purple, which is consistent with the positive control color of APP peptides. Figure 1As shown in c, the imaging performance of APP@Dextran-Fe3O4 nanoparticles was measured in vitro. With increasing iron content, the MPI imaging signal showed a linear enhancement trend, suggesting its potential as an MPI imaging tracer. These results demonstrate that we have successfully prepared a superparamagnetic nanoparticle with MPI imaging capability.

[0028] Example 2: APP@Dextran-Fe3O4 nanoparticles for the diagnosis of Alzheimer's disease in mice.

[0029] Wild-type (WT) and 5×FAD transgenic mice (AD) were administered 400 μg / mouse of APP@Dextran-Fe3O4 nanoparticles via tail vein injection. Five minutes after administration, specific brain scans were performed using MPI-2D and MPI-3D scanning modes. Following the MPI scan, structural reference images of the brain tissue were obtained using combined T1-MRI-3D imaging with the following parameters: FOV = 35 mm × 35 mm, slice thickness = 1.0 mm, TR = 439.8 ms, TE = 4 ms. The combined MPI and MRI imaging data were analyzed using pmod4.4 software (Bruker, Germany), and pathological analysis confirmed the accuracy of the MPI diagnosis.

[0030] Experimental results are as follows Figure 2 As shown, MPI imaging was successfully applied to the diagnosis of AD mouse brain disease. MPI-2D imaging revealed that APP@Dextran-Fe3O4 nanoparticles exhibited higher brain enrichment in AD mice, such as... Figure 2 As shown in Figure a, the synthesized APP@Dextran-Fe3O4 nanoparticles can cross the blood-brain barrier to achieve intracerebral accumulation and diagnostic effects. MPI-3D imaging can observe the different gradient distributions of APP@Dextran-Fe3O4 nanoparticles throughout the brain, and combined with MRI imaging, it provides structural imaging support, which can be used for the diagnosis of AD.

[0031] MPI diagnostic analysis was performed on the hippocampal lesions, such as Figure 2 As shown in b, the MPI signal in the hippocampus of AD mice was significantly enhanced, indicating hippocampal damage in AD mice, ultimately leading to decreased learning and memory abilities. In contrast, the hippocampus in WT mice showed no damage, resulting in no MPI signal accumulation in the hippocampus. Pathological analysis of the mouse hippocampus was subsequently performed. Figure 2 As shown in e, AD mice have more Aβ deposition in the hippocampus, causing excessive proliferation and aggregation of microglia. Microglia engulf a large number of APP@Dextran-Fe3O4 nanoparticles. We can observe abnormal MPI imaging signals in the hippocampus of AD mice. Figure 2As shown in c and d, quantitative statistical analysis of signal accumulation in the mouse brain and hippocampus showed that MPI-diagnosed AD mice showed significant signal enhancement in the hippocampus. Figure 2 As shown in f and g, quantitative statistics on microglia and Aβ deposition in the hippocampus revealed that AD mice had more Aβ deposition in the hippocampus. Experimental results and statistical analysis demonstrate that the APP@Dextran-Fe3O4 nanoparticles synthesized in this invention can cross the blood-brain barrier to achieve brain imaging diagnosis in AD mice.

[0032] Example 3: APP@Dextran-Fe3O4 nanoparticles alleviate brain pathological features in AD mice.

[0033] Wild-type (WT) and 5×FAD transgenic mice (AD) were treated with 200 μg / mouse of APP@Dextran-Fe3O4 nanoparticles every 15 days. After 30 days of treatment, the mice were euthanized, and their brain tissue was analyzed pathologically. Nissl staining was used to observe neuronal damage, and immunohistochemical staining was used to observe Aβ deposition and microglia aggregation in the brain. Results are as follows: Figure 3 As shown, Nissl staining revealed some damage to neurons in the DG, CA1, and CA3 regions of the hippocampus in AD mice. Neuronal arrangement was disordered, staining was lighter, and Nissl bodies within neurons were reduced. After treatment with APP@Dextran-Fe3O4 nanoparticles, hippocampal neuronal staining in AD mice deepened, and the number of Nissl bodies within neurons increased, indicating some improvement in neuronal damage. Furthermore, treatment significantly reduced Aβ deposition and the extensive inflammatory proliferation of microglia in the brain, further mitigating the pathological features of AD in the brain. These results confirm that APP@Dextran-Fe3O4 nanoparticles have a good effect on delaying the pathological progression of AD in the brain.

[0034] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0035] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0036] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of brain-targeting APP@Dextran-Fe3O4 nanoparticles in the preparation of MPI magnetic particle imaging diagnostic products for Alzheimer's disease or in the preparation of drugs for treating Alzheimer's disease, characterized in that, The amino acid sequence of the APP polypeptide is: cysteine-histidine-leucine-aspartic acid-isoleucine-isoleucine-tryptophan, and the particle size of the APP@Dextran-Fe3O4 nanoparticles is 15~20nm. The method for preparing the brain-targeting APP@Dextran-Fe3O4 nanoparticles includes the following steps: S1. Incubate the dextran solution at 80-90℃ for 1-2 hours, add NaOH solution and continue the reaction for 1-3 minutes, then add FeCl3•6H2O and FeCl2•4H2O, react at 90-100℃ for 1-2 hours, adjust the pH to 10-11 and continue the reaction for 5-15 minutes, cool and centrifuge to remove the supernatant, the precipitate is separated magnetically, washed with water and dried to obtain Dextran-Fe3O4 nanoparticles; wherein the mass ratio of dextran, NaOH, FeCl3•6H2O and FeCl2•4H2O is 1-2g: 1-3g: 0.5-3g: 0.5-2.5g; S2. Acetone and epichlorohydrin were added to the Dextran-Fe3O4 nanoparticle solution, and the reaction was carried out for 1-30 min. Then, a 48%-52% NaOH solution was added, and the reaction was continued with stirring for 5-8 h. The mixture was washed with anhydrous ethanol and water, and after magnetic separation, the supernatant was discarded and the precipitate was retained. The precipitate was added to the APP peptide solution and reacted at 35-40℃ and 200-400 rpm / min for 7-9 h. Then, the APP@Dextran-Fe3O4 nanoparticles were obtained by magnetic separation. The mass-volume ratio of Dextran-Fe3O4 nanoparticles, acetone, epichlorohydrin, NaOH solution and APP peptide was 5-15 mg: 1-5 mL: 3-6 mL: 0.5-1 mL: 0.1-0.5 g.

2. The application according to claim 1, characterized in that, The diagnostic product is a brain-targeted imaging tracer.

3. The application according to claim 1, characterized in that, In the drug, APP@Dextran-Fe3O4 nanoparticles are the only effective ingredient.

4. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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