Brain-targeted APP-coated Dextran-Fe3O4 nano-particles as well as preparation method and application of brain-targeted APP-coated Dextran-Fe3O4 nano-particles

Abstract

The invention belongs to the technical field of medical imaging, and particularly relates to a brain-targeted APP-coated Dextran-Fe3O4 nano particle as well as a preparation method and application of the brain-targeted APP-coated Dextran-Fe3O4 nano particle. The APP-coated Dextran-Fe3O4 nanoparticles provided by the invention can act on beta amyloid protein receptors in the brain in a non-invasive intravenous injection manner, so that blood-brain barrier-crossing transport is realized, and the nanoparticles have a higher accumulation effect in the brain of a patient suffering from the Alzheimer's disease, particularly are abnormally accumulated at the hippocampus of the main diseased region of the Alzheimer's disease, so that the Alzheimer's disease can be effectively treated. The Alzheimer's disease can be diagnosed by combining the characteristics of high resolution and high sensitivity of MPI imaging. At the same time, pathological analysis finds that the APP-coated Dextran-Fe3O4 nanoparticles have the effects of inhibiting intracerebral inflammatory response of the Alzheimer's disease and reducing neuronal cell apoptosis, and have certain benefits for alleviating the Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical imaging, and more specifically, relates to a brain-targeted APP@Dextran-Fe 3 O 4 nanoparticle and its preparation method and application. Background Art

[0002] As a neurodegenerative disease, Alzheimer's disease (AD) is mainly characterized by brain atrophy, with the most obvious atrophy in the hippocampus and temporal lobe, and is accompanied by phenomena such as β-amyloid protein deposition, hyperphosphorylated tau protein, and neurofibrillary tangles. Clinically, AD can be diagnosed by imaging techniques such as CT, MRI, and PET. However, unfortunately, no new imaging techniques have been developed in essence. The main disadvantages of CT and PET imaging are radioactive exposure, and at the same time, the diagnostic cost of PET imaging is relatively high; the main disadvantage of MRI imaging is its relatively low spatial and temporal resolution, and the renal toxicity brought by the imaging agent 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 Weizenecker developed a new imaging modality called magnetic particle imaging (MPI), which is a new radiation-free tomography technique that can provide fast, background-free, and sensitive imaging diagnostic results. However, in Alzheimer's disease, the currently FDA-approved clinical diagnostic reagent for MPI cannot be applied to brain imaging because it cannot cross the blood-brain barrier. Therefore, synthesizing a new type of brain-targeted imaging tracer is the key to realizing the application of MPI in AD diagnosis. Summary of the Invention

[0004] The purpose of the present invention is to provide a brain-targeted APP@Dextran-Fe 3 O 4 nanoparticle and its preparation method and application.

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

[0006] The present invention provides a brain-targeted APP@Dextran-Fe 3 O 4 nanoparticle, which is obtained by the positive and negative charge binding of the carboxyl group of the APP polypeptide and the hydroxyl group on the dextran on the surface of the Dextran-Fe 3 O 4 nanoparticle to modify the APP polypeptide on the surface of the Dextran-Fe 3 O 4 nanoparticle surface, and the Dextran-Fe3 O 4 The nanoparticles are Fe encapsulated by dextran 3 O 4 magnetic nanoparticles.

[0007] In the present invention, the carboxyl group of the brain-targeting APP polypeptide is combined with the magnetic nanoparticles Dextran-Fe 3 O 4 The hydroxyl group on the surface dextran of the magnetic nanoparticles Dextran-Fe 3 O 4 is modified with the brain-targeting APP polypeptide through positive and negative charge binding on the surface, thereby obtaining an APP@Dextran-Fe 3 O 4 nanoparticle with brain-targeting effect. This nanoparticle can act on the β-amyloid receptor in the brain through non-invasive intravenous injection, thereby achieving trans-blood-brain barrier transport, accurately targeting the hippocampus, the main lesion site of Alzheimer's disease, and having the characteristics of high resolution and high sensitivity in MPI imaging, and can be used for the diagnosis of Alzheimer's disease.

[0008] Furthermore, the particle size of the APP@Dextran-Fe 3 O 4 nanoparticle is 15 - 20 nm.

[0009] The present invention also provides a preparation method of the above-mentioned brain-targeting APP@Dextran-Fe 3 O 4 nanoparticle, including the following steps:

[0010] S1. Keep the dextran solution at 80 - 90 °C for 1 - 2 h, add NaOH solution and continue to react for 1 - 3 min, then add FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O, react at 90 - 100 °C for 1 - 2 h, adjust the pH to 10 - 11 and then continue to react for 5 - 15 min, cool and centrifuge to remove the supernatant solution, and the precipitate is subjected to magnetic separation, washed with water and dried to obtain Dextran-Fe 3 O 4 nanoparticle; wherein, the mass ratio of dextran, NaOH, FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O is 1 - 2 g: 1 - 3 g: 0.5 - 3 g: 0.5 - 2.5 g;

[0011] S2. Add to Dextran-Fe 3 O4 After adding acetone and epichlorohydrin to the nanoparticle solution, react for 1 to 30 minutes. After adding a NaOH solution with a mass fraction of 48% to 52%, continue stirring and reacting for 5 to 8 hours, then wash with absolute ethanol and water. After magnetic separation, discard the supernatant and retain the precipitate. Add the precipitate to the APP polypeptide solution and react at 35 to 40 °C and 200 to 400 rpm for 7 to 9 hours, and then obtain APP@Dextran-Fe by magnetic separation 3 O 4 nanoparticles; wherein Dextran-Fe 3 O 4 The mass-volume ratio of the nanoparticles, acetone, epichlorohydrin, NaOH solution and APP polypeptide is 5 to 15 mg: 1 to 5 mL: 3 to 6 mL: 0.1 to 1 ml: 0.1 to 0.5 g.

[0012] The present invention also provides the above-mentioned brain-targeting APP@Dextran-Fe 3 O 4 Application of nanoparticles in the preparation of Alzheimer's disease diagnostic products.

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

[0014] The present invention also provides the above-mentioned brain-targeting APP@Dextran-Fe 3 O 4 Application of nanoparticles in the preparation of drugs for treating Alzheimer's disease.

[0015] Furthermore, in the drug, APP@Dextran-Fe 3 O 4 nanoparticles are used as the only active ingredient.

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

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

[0018] The brain-targeting APP@Dextran-Fe provided by the present invention 3 O 4 nanoparticles cross the blood-brain barrier mediated by β-amyloid receptors in the brain and have a higher accumulation effect in the brains of patients with Alzheimer's disease, especially in the hippocampus, an important diseased site of Alzheimer's disease. It can be enriched and can be applied to the diagnosis of Alzheimer's disease using MPI imaging. At the same time, pathological analysis found that APP@Dextran-Fe 3 O 4 nanoparticles have the effect of inhibiting the inflammatory response in the brain of Alzheimer's disease and reducing neuronal apoptosis, which is beneficial to slowing down Alzheimer's disease. Description of the Drawings

[0019] Figure 1 For APP@Dextran-Fe 3 O 4 Nanoparticle characterization diagram. Among them, a is the TEM image of APP@Dextran-Fe 3 O 4 nanoparticles. b is the chromogenic experiment diagram, where 1 is APP polypeptide, 2 is Dextran-Fe 3 O 4 nanoparticles, 3 is APP@Dextran-Fe 3 O 4 nanoparticles. c is the linear relationship diagram between APP@Dextran-Fe 3 O 4 nanoparticles with different iron contents and imaging signal intensity.

[0020] Figure 2 For APP@Dextran-Fe 3 O 4 Nanoparticle application in AD mouse imaging diagnosis diagram. Among them, a is the 2D and 3D imaging schematic diagrams of the distribution of APP@Dextran-Fe 3 O 4 nanoparticles in the mouse brain. The red arrows indicate the fitting reference points provided by the combined MPI and MRI scans. WT is wild-type mice, and AD is Alzheimer's disease model mice. b is the MPI diagnosis schematic diagram of the lesions in the mouse hippocampus. c is the quantitative statistical chart of the overall signal intensity of APP@Dextran-Fe 3 O 4 nanoparticles in the brain after MPI-2D imaging. d is the quantitative statistical chart of the MPI imaging signal intensity in the hippocampus. e is the cross-sectional immunohistochemical map of the hippocampus in the brain tissue. Among them, Iba1 is the marker for microglial cell aggregation, and Aβ is amyloid deposition. f is the statistical chart of the positive rate of microglial cells in e. g is the quantitative statistical chart of the positive rate of Aβ in e.

[0021] Figure 3 For APP@Dextran-Fe 3 O 4In vivo pharmacodynamic evaluation diagram of nanoparticles for the treatment of AD. Among them, a is the diagram of observing neuronal apoptosis in the hippocampus of mice by Nissl staining. The DG area, CA1 area, and CA3 area in the hippocampus were locally magnified and observed respectively. The darker the neurons were stained, and the more blue-purple Nissl bodies were in the cells, the less neuronal damage. The red arrows indicate Nissl bodies. b is the immunohistochemical staining diagram of Aβ deposition in the hippocampus, where Aβ is stained brown, and the red arrow indicates. c is the immunohistochemical staining diagram of microglia (Iba1) in the hippocampus, and microglia are stained brown, with the red arrow indicating. d is the quantitative statistical analysis diagram of Aβ expressed in Figure b and Figure c. e is the quantitative statistical analysis diagram of Iba1 expressed in Figure b and Figure c. Detailed implementation manners

[0022] The present invention will be described in detail below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

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

[0024] Example 1: APP@Dextran-Fe 3 O 4 Synthesis and characterization analysis of nanoparticles.

[0025] Dissolve 2 g of dextran with a molecular weight of 20,000 in 20 mL of deionized water, heat and stir at 90 °C for 1 h, add 8 mL of 5 M NaOH solution and continue stirring for 2 min, then add 2.7 g of FeCl 3 ·6H 2 O and 1.9 g of FeCl 2 ·4H 2 O, react at 95 °C for 1 h, then adjust the pH to 11 and continue stirring for 10 min, cool naturally, centrifuge at 1000 rpm / min for 5 min, take the supernatant solution, and obtain Dextran-Fe 3 O 4 nanoparticles after magnetic separation and thorough washing with deionized water and drying at 45 °C. Dissolve it in an aqueous solution and assist dissolution with a 200 W water bath ultrasound for 12 hours to make it stably dispersed in the aqueous solution for further surface modification and other applications in the later stage.

[0026] Weigh 10 mg of Dextran-Fe 3 O 4The nanoparticles were dissolved in 4 mL of deionized water and ultrasonic dissolution was used. 4 mL of acetone and epichlorohydrin solution were added, and mechanical stirring was carried out at 35 °C and 200 rpm for 10 min to mix evenly. 700 μL of 50% NaOH solution was added, and stirring and reaction were continued for 6 h. The reaction was stopped, and the solution during the reaction was removed by washing 5 times with absolute ethanol and 5 times with deionized water. After magnetic separation, the supernatant was discarded to obtain the precipitate. 10 mg of APP polypeptide dissolved in 4 mL of sodium carbonate buffer with a pH of 8.5 was added to the precipitate, and the reaction was carried out in a shaker at 37 °C and 300 rpm for 8 h. The supernatant solution was recovered, and the concentration of APP polypeptide in the supernatant was calculated by the BCA protein assay method, and the reaction efficiency was calculated. The magnetic beads were separated magnetically, and the APP@Dextran-Fe was obtained after washing 3 times with deionized water and PBS solution each. 3 O 4 nanoparticles. The morphology and particle size of APP@Dextran-Fe 3 O 4 nanoparticles were observed by field emission transmission electron microscopy. The BCA protein reaction method was used to determine whether APP polypeptide was modified on its surface, and MPI was used to determine its imaging performance in vitro.

[0027] Figure 1 As shown in a of 3 O 4 the Fe in the core of APP@Dextran-Fe 3 O 4 nanoparticles observed by electron microscopy had a diameter of about 5 nm. After external modification with APP and dextran, the particle size of the formed APP@Dextran-Fe 3 O 4 nanoparticles was about 17 nm. Figure 1 As shown in b of 3 O 4 it, the detection of polypeptide modification on the nanoparticle surface was carried out by the BCA protein reaction experiment. After the reaction with APP polypeptide, it showed blue-violet color, and the Dextran-Fe 3 O 4 nanoparticles had no positive color development. The surface of APP@Dextran-Fe 3 O 4 nanoparticles was modified by APP polypeptide, and the reaction solution showed blue-violet color. The BCA protein reaction method found that compared with the unmodified Dextran-Fe Figure 1 As shown in c of 3 O 4The imaging performance of the nanoparticles shows a linear increasing trend of the MPI imaging signal with the increase of iron content, and it can be used as an MPI imaging tracer. The above results prove that we have successfully prepared a superparamagnetic nanoparticle with MPI imaging ability.

[0028] Example 2: APP@Dextran-Fe 3 O 4 The nanoparticles are used for the diagnosis of Alzheimer's disease mice.

[0029] Wild-type (WT) and 5×FAD transgenic mice (AD) were given 400 μg / mouse of APP@Dextran-Fe 3 O 4 nanoparticles via tail vein injection. 5 minutes after administration, the mouse brains were specifically scanned using MPI-2D and MPI-3D scanning modes. After the mice completed the MPI scan, T 1 -MRI-3D imaging was used to provide a structural reference image of the brain tissue. The scanning parameters were: FOV = 35 mm × 35 mm, slice thickness = 1.0 mm, TR = 439.8 ms, TE = 4 ms. The pmod4.4 software (Bruker, Germany) was used to analyze the combined MPI and MRI imaging data, and pathological analysis confirmed the accuracy of MPI diagnosis.

[0030] The experimental results are as Figure 2 shown. MPI imaging was successfully applied to the diagnosis of the brains of AD mice. MPI-2D imaging showed that APP@Dextran-Fe 3 O 4 nanoparticles had a higher brain enrichment phenomenon in AD mice, as shown in Figure 2 a, which also confirmed that the synthesized APP@Dextran-Fe 3 O 4 nanoparticles had the function of enriching in the brain through the blood-brain barrier and diagnosing. MPI-3D imaging could observe the different gradient distributions of APP@Dextran-Fe 3 O 4 nanoparticles throughout the brain, and combined with MRI imaging to provide structural image support, which could be used for the diagnosis of AD.

[0031] MPI diagnostic analysis was performed on the hippocampal injury. As shown in Figure 2 b, the signal at the hippocampus of AD mice was significantly enhanced by MPI diagnosis, indicating that the hippocampus of AD mice was damaged, ultimately leading to the decline of the learning and memory ability of the mice, while there was no damage to the hippocampus of WT mice, resulting in no MPI signal accumulation at the hippocampus. Subsequently, pathological analysis was performed on the hippocampal region of the mice. Figure 2As shown by e in [reference], there is more Aβ deposition in the hippocampus of AD mice, which causes excessive proliferation and aggregation of microglia, and a large number of microglia phagocytize APP@Dextran-Fe 3 O 4 Regarding the nanoparticles, we can observe the abnormal MPI imaging signals in the hippocampus of AD mice. Figure 2 As shown by c and d in [reference], the signal accumulation in the mouse brain and hippocampus was quantitatively statistically analyzed, and the signal in the hippocampus of MPI-diagnosed AD mice was significantly enhanced. Figure 2 As shown by f and g in [reference], the microglia and Aβ deposition in the hippocampus were quantitatively statistically analyzed, and there was more Aβ deposition in the hippocampus of AD mice. The experiments and result statistics prove that the APP@Dextran-Fe 3 O 4 nanoparticles have the function of achieving brain imaging diagnosis in AD mice by crossing the blood-brain barrier.

[0032] Example 3: APP@Dextran-Fe 3 O 4 nanoparticles alleviate the brain pathological features of AD mice.

[0033] Wild-type (WT) and 5×FAD transgenic mice (AD) received 200 μg / mouse of APP@Dextran-Fe 3 O 4 nanoparticle treatment, with the administration frequency of once every 15 days. After 30 days of treatment, the mice were euthanized, and the brain tissues of the mice were subjected to pathological analysis. Nissl staining was used to observe neuron damage, and immunohistochemical staining was used to observe Aβ deposition and microglia aggregation in the brain. The results are as Figure 3 shown. By Nissl staining, it can be observed that there is certain damage to neuron cells in the DG area, CA1 and CA3 areas of the hippocampus in AD mice, the neuron arrangement is disordered, the staining is lighter, and the Nissl bodies in the neurons are reduced; after treatment with APP@Dextran-Fe 3 O 4 nanoparticles, the staining of hippocampal neurons in AD mice deepens, and the number of Nissl bodies in the neurons increases, indicating that the neuron damage has been improved to a certain extent. And after treatment, it can significantly reduce the deposition of Aβ in the brain and the large inflammatory proliferation of microglia, and has the effect of further slowing down the brain AD pathological features. The above results confirm that APP@Dextran-Fe 3 O 4 nanoparticles have a good effect on delaying the brain AD pathological process.

[0034] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints can be selected. To prevent repetition, the present invention describes preferred embodiments.

[0035] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0036] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A brain-targeted APP@Dextran-Fe3O4 nanoparticle, characterized in that: The method 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 dextran on the surface of the Dextran-Fe3O4 nanoparticles. The Dextran-Fe3O4 nanoparticles are Fe3O4 magnetic nanoparticles coated with dextran.

2. The brain-targeted APP@Dextran-Fe3O4 nanoparticle according to claim 1, characterized in that: The particle size of the APP@Dextran-Fe3O4 nanoparticles is 15-20 nm.

3. The method for preparing brain-targeted APP@Dextran-Fe3O4 nanoparticles according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Keep the dextran solution at 80-90°C for 1-2h, add NaOH solution and continue to react for 1-3min, then add FeCl3·6H2O and FeCl2·4H2O, react at 90-100°C for 1-2h, adjust the pH to 10-11 and continue to react for 5-15min, cool and centrifuge to remove the supernatant solution, separate the precipitate by magnetic separation, wash with water and dry 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. Add acetone and epichlorohydrin to the Dextran-Fe3O4 nanoparticle solution and react for 1 to 30 minutes. Add 48% to 52% NaOH solution and continue stirring for 5 to 8 hours. Wash with anhydrous ethanol and water, discard the supernatant and retain the precipitate after magnetic separation, add the precipitate to the APP polypeptide solution, react at 35 to 40°C and 200 to 400 rpm / min for 7 to 9 hours, and then perform magnetic separation to obtain APP@Dextran-Fe3O4 nanoparticles; wherein the mass volume ratio of Dextran-Fe3O4 nanoparticles, acetone, epichlorohydrin, NaOH solution and APP polypeptide is 5 to 15 mg: 1 to 5 mL: 3 to 6 mL: 0.5 to 1 ml: 0.1 to 0.5 g.

4. Use of the brain-targeted APP@Dextran-Fe3O4 nanoparticles according to claim 1 in the preparation of Alzheimer's disease diagnostic products.

5. The use of the brain-targeted APP@Dextran-Fe3O4 nanoparticles according to claim 4 in the preparation of Alzheimer's disease diagnostic products, characterized in that: The diagnostic product is a brain-targeted imaging tracer.

6. Use of the brain-targeted APP@Dextran-Fe3O4 nanoparticles according to claim 1 in the preparation of drugs for treating Alzheimer's disease.

7. The use of the brain-targeted APP@Dextran-Fe3O4 nanoparticles according to claim 6 in the preparation of a drug for treating Alzheimer's disease, characterized in that: APP@Dextran-Fe3O4 nanoparticles are the only active ingredient in the drug.

8. The use of the brain-targeted APP@Dextran-Fe3O4 nanoparticles according to claim 7 in the preparation of a drug for treating Alzheimer's disease, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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