An ultramagnetic nanomedicine targeting pathological region of Alzheimer's disease A beta protein and its preparation method and application

Abstract

The application discloses a superparamagnetic nanomedicine targeting an Alzheimer's disease A beta protein pathological area and a preparation method and application thereof, and a structure of the superparamagnetic nanomedicine targeting the Alzheimer's disease A beta protein pathological area is Fe3O4-HS-PEG 600 -CO-NH2 complex polypeptide; the complex polypeptide is composed of a targeting A beta protein locating sequence, a transmembrane sequence, a Sema3A inhibiting peptide and a nerve protection peptide in sequence from N to C; the targeting A beta protein locating sequence is FFXXK. The application can effectively reduce hippocampal A beta protein deposition plaques by reducing the introduction of the Sema3A inhibiting peptide and the nerve protection peptide in the Alzheimer's disease A beta protein deposition pathological area, is beneficial to the repair of hippocampal nerve networks, and has great potential for treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of targeted nanomaterials technology, specifically relating to a superparamagnetic nanomedicine that targets the pathological region of Aβ protein in Alzheimer's disease, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease is a major neurodegenerative disease clinically characterized by a progressive decline in memory and cognitive abilities. Currently, approximately 50 million people worldwide suffer from Alzheimer's, placing a heavy burden on families and society. However, there is still no effective drug for Alzheimer's. Previous drugs, whether targeting Aβ protein deposition or Tau protein phosphorylation, are mostly administered systemically, lacking specific targeting, resulting in numerous side effects and limited improvement in cognitive function. Therefore, there is an urgent need to research a drug with strong targeting and fewer side effects for the treatment of Alzheimer's disease. Summary of the Invention

[0003] To address the lack of targeting in existing Alzheimer's disease treatment drugs (drugs for Aβ protein deposition or Tau protein phosphorylation), this invention provides a superparamagnetic nanomedicine targeting the pathological area of ​​Aβ protein in Alzheimer's disease, its preparation method, and its application. Under the action of guiding peptides and membrane-penetrating peptides, it can specifically target the pathological area of ​​Aβ protein deposition, while simultaneously clearing Aβ protein deposition and reducing glial scars, and has the function of repairing neural networks.

[0004] This invention is achieved through the following technical solution: A superparamagnetic nanomedicine targeting the pathological region of Alzheimer's disease Aβ protein, wherein the superparamagnetic nanomedicine targeting the pathological region of Alzheimer's disease Aβ protein has the structural formula Fe3O4-HS-PEG. 600 -CO-NH2- complex polypeptide; The composite polypeptide is composed, from N to C, of ​​a localization sequence targeting Aβ protein, a membrane-penetrating sequence, a Sema3A inhibitory peptide, and a neuroprotective peptide. The target Aβ protein localization (Starg) sequence is: FFXXK, where X is any hydrophobic amino acid.

[0005] Furthermore, the transmembrane (Sccp) sequence is YGRKKRRQRRR; the Sema3A inhibitory peptide (Score) sequence is HAVEHGFMQTLLKVTLE; and the neuroprotective peptide (Sopt) sequence is NAPVSIPQ.

[0006] Furthermore, the N-terminus of the composite polypeptide is coupled with a FITC luminescent group.

[0007] The preparation method of the superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease, as described in this invention, includes the following steps: (1) Preparation of Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles: Ferric oleate powder was added to a mixture of oleic acid and 1-octadecene, kept at 90-120℃ for 3-10 min, then heated to 300-350℃ and kept at 20-50 min, cooled, collected by a magnet and dispersed in heptane, washed to obtain Fe3O4 nanoparticle cores; the Fe3O4 nanoparticle cores were dissolved in PEG 600 During the stirring reaction, Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles were obtained. (2) Complex peptide: The complex peptide was synthesized using the Fmoc solid-phase carrier synthesis method. The complex peptide consists of a localization sequence targeting Aβ protein, a membrane-penetrating sequence, a Sema3A inhibitory peptide, and a neuroprotective peptide from N to C. (3) Fe3O4-HS-PEG 600 Preparation of -CO-NH2- complex peptide: N-ethynyl-N,4-dimethylbenzenesulfonamide and the complex peptide in step (2) were mixed, dichloromethane was added, and the reaction was stirred until the acid was exhausted. After the reaction was completed, the dichloromethane was removed under vacuum, and Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles prepared in step (1), water and DMSO were added as a mixed solvent. The reaction was stirred at room temperature until the α-acyloxamine was completely consumed. The reactants were concentrated and purified to obtain a superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease.

[0008] Further, in step (1), the feeding ratio of ferric oleate powder, oleic acid, and l-octadecene is 2 mmol: 1 mmol: 10 g; ferric oleate powder and PEG 600 The ratio was 4 mmol: 5 mL; the stirring reaction time was 1-5 h.

[0009] Furthermore, in step (3), the molar ratio of the composite peptide and the Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles is 1:1.

[0010] Furthermore, in step (3), the molar ratio of N-ethynyl-N,4-dimethylbenzenesulfonamide to the complex polypeptide is 1:1.

[0011] Further, in step (3), the volume ratio of water to DMSO in the mixed solvent of water and DMSO is 1:4 to 1:6.

[0012] In this invention, the superparamagnetic nanomedicine targeting the pathological region of Alzheimer's disease Aβ protein is used in the preparation of drugs for treating Alzheimer's disease; the transmembrane peptide in Scpp enhances the ability of nanoparticles to pass through cell membranes; the Sopt sequence contains neuroprotective peptides that enhance the protective effect on neurons; the Starg polypeptide sequence can recognize the β-amyloid deposition region of Alzheimer's disease; and the Score polypeptide sequence can specifically inhibit the effect of Sema3A. Beneficial effects

[0013] This invention utilizes thiol-based polyethylene glycol carboxyl groups (SH-PEG600-COOH) as grafting agents to link Fe3O4 via thiol groups. Furthermore, SH-PEG600-COOH forms amide bonds with the N-terminal amino groups of the complex polypeptide via carboxyl groups, thus preparing a superparamagnetic nanomedicine (Fe3O4-HS-PEG) targeting the pathological region of the Aβ protein in Alzheimer's disease. 600 The -CO-NH2- complex polypeptide can identify spatial pathological sites. The targeted polypeptide sequence, upon binding to Aβ protein in the pathological region, initiates the release of the complex polypeptide, thereby inhibiting Sema3A protein in the pathological region. This nanoparticle achieves spatial targeting and real-time tracking, representing a revolutionary approach to the in vivo regulation of Alzheimer's disease. Fe3O4-HS-PEG 600 The pathological targeting of the -CO-NH2- complex polypeptide particles allows for the regulation of Sema3A expression in pathological areas, alleviating the pathological progression of Alzheimer's disease and demonstrating great potential for its treatment. Attached Figure Description

[0014] Figure 1 Fe3O4-HS-PEG 600 High-performance liquid chromatography (HPLC) and mass spectra of the -CO-NH2- complex polypeptide: Top: HPLC chromatogram, Bottom: Mass spectrum; Figure 2 Fe3O4-HS-PEG 600 -CO-NH2- composite peptide atomic force microscopy and transmission electron microscopy images of composite nanomorphology, (a) surface morphology detection by atomic force microscopy; (b) detection results by transmission electron microscopy; (c) statistical results of composite nanodrug size; Figure 3 Fe3O4-HS-PEG 600 -CO-NH2- complex polypeptide EDS energy dispersive spectroscopy analysis diagram and Fe3O4 core particle elemental analysis table; Figure 4 Fe3O4-HS-PEG 600 Figure showing the cell membrane penetration results of the -CO-NH2- complex polypeptide; Figure 5 Fe3O4-HS-PEG600 The effect of the -CO-NH2- complex polypeptide on clearing Aβ deposits is shown in the following figures: (a) Immunofluorescence staining results of Aβ deposits in the hippocampus of mice in the control group; (b) Immunofluorescence staining results of Aβ deposits in the hippocampus of mice in the treatment group; (c) Immunofluorescence staining results of Aβ deposits in the hippocampus of mice in both groups, t-test, *** p <0.001, n=5. Detailed Implementation

[0015] Exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is only for illustrating particularly specific embodiments and is not intended to limit the present invention.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. Any modifications and improvements to equivalent or similar methods and materials without departing from the spirit of this invention are within the scope of protection of this invention.

[0017] In the following examples: the localization sequence targeting Aβ protein is: FFXXK, where X is valine, i.e., FFVVK; the membrane-penetrating sequence is YGRKKRRQRRR; the Sema3A repressor peptide sequence is HAVEHGFMQTLLKVTLE; and the neuroprotective peptide sequence is NAPVSIPQ. Example 1

[0018] Preparation of Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles: (1) Iron oxide nanoparticles were prepared by the oleic acid method: First, 80 ml of ethanol, 60 ml of deionized water and 140 ml of heptane were mixed to prepare a mixed solvent. 120 mmol of sodium oleate and 40 mmol of ferric chloride hexahydrate were added to this mixture and dissolved in an inert atmosphere at 70 °C for 4 h. The heptane layer containing ferric oleate was separated and washed with deionized water. The washed solution was evaporated with heptane to obtain ferric oleate powder. (2) 40 mmol of dried iron oleate powder was added to a mixture of 20 mmol of oleic acid and 200 g l-octadecene, and kept at 100°C for 5 min. The mixed solution was heated to 320°C and kept at 320°C for 30 min. The mixed solution was suspended in air and cooled to 25°C. It was collected by a magnet, dispersed in heptane, and washed three times with ethanol to obtain Fe3O4 nanoparticle cores. (3) The Fe3O4 nanoparticle core was dissolved in 50 mL of mercapto polyethylene glycol carboxyl group (PEG, MW 600), and stirred at room temperature for 2 h. It was then purified by washing with ethanol and PBS three times to obtain Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles. Before further modification, the Fe3O4 nanoparticle core was stored in sodium citrate buffer at pH 6.0 at 4°C with a concentration of about 4 mg Fe / mL. Example 2

[0019] The N-terminal FITC-modified complex peptide was prepared using the Fmoc solid-phase support synthesis method, specifically as follows: (1) 1 g Fmoc-lyys (Dde)-Wang resin was used as an insoluble solid support and swelled overnight with 30 ml dichloromethane; (2) Add 30 ml (30% PIPE in dimethylformamide solution) to the resin and react for 20 minutes to remove the amino moiety protected by Fmoc; (3) After washing with 30 ml of dimethylformamide and dichloromethane alternately 6 times (repeat this step before each step), add 8 times the amount of DSC and 16 times the amount of DIEA to the resin and react with the exposed amino group for 90 min to obtain the NHS structure intermediate. (4) Add 8 times the amount of resin containing an amino acid without α-amino protection (side chain protected) and 16 times the amount of resin containing DIEA to the resin, react with NHS for 24 h to form a stable peptide bond structure, and repeat the peptide bond synthesis. (5) Finally, add 8 times the amount of resin Fmoc-Ahx (aminocaproic acid)-OH, 8 times the amount of resin HOBT and 8 times the amount of resin DIC to the resin, and react for 2 h to obtain the final length peptide, which is a complex peptide consisting of a localization sequence targeting Aβ protein, a membrane-penetrating sequence, a Sema3A inhibitory peptide and a neuroprotective peptide from N to C. (6) Add 8 times the amount of FITC-NHS and 8 times the amount of succinyl iodide acetate to the resin, react with the amino group of Ahx in DMF solution, and obtain the N-terminal FITC-modified complex polypeptide after purification. Example 2

[0020] Fe3O4-HS-PEG 600 Preparation of -CO-NH2- complex polypeptide: (1) Using the mild coupling method of Ynamide to link Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles with peptides: 0.24 mM N-ethynyl-N,4-dimethylbenzenesulfonamide and 0.24 mM N-terminal FITC-modified composite peptide prepared in Example 2 were charged into a 5 ml flask, and then 1 ml of dichloromethane was added to the flask. The mixture was stirred in air at room temperature until the acid was completely consumed. After the reaction was completed, the dichloromethane was removed under vacuum. (2) After removing dichloromethane under vacuum, add 0.24 mM of Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles prepared in Example 1 and 1 ml of H2O / DMSO (1:5) mixed solvent to the flask, stir at room temperature until the α-acyloxyamine is completely consumed, and the reaction ends. (3) Add 1 ml of 20% 4-methylpiperidine (dissolved in dimethylformamide) to the above reaction product, shake well for 12 min, centrifuge at 8000 rpm for 8 min, and discard the supernatant; then add 1 ml of dimethylformamide, shake for 3 min, centrifuge at 8000 rpm for 8 min, and discard the supernatant; add 1 ml of dichloromethane, shake the system for 3 min, centrifuge at 8000 rpm for 5-10 min, and discard the supernatant; vacuum dry to remove dichloromethane for 1.5 h to obtain Fe3O4-HS-PEG. 600 -CO-NH2- complex polypeptide; dissolve the product in 200 μl of purified water and oleic acid (1:1).

[0021] Properties and Applications Testing 1. Determination of the mass, molecular weight, and solubility of superparamagnetic nanomedicines targeting the pathological region of Aβ protein in Alzheimer's disease: Determination of Fe3O4-HS-PEG by reversed-phase high-performance liquid chromatography 600 The mass of the -CO-NH2- complex peptide was measured. A C18 reversed-phase column (5 μm, 250 × 4.6 mm) was used, and the UV absorption spectrum was detected at 220 nm. The flow rate was 1 mL / min, and the mobile phase consisted of 70% solvent A (0.05% TFA + 2% acetonitrile) and 30% solvent B (0.05% TFA + 90% acetonitrile). After 16 min, the mobile phases were 54% solvent A and 46% solvent B, respectively.

[0022] 2. Mass analysis was performed using a high-performance liquid chromatography (HPLC) system coupled with mass spectrometry (MS / MS). The chromatographic peaks were then analyzed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF). Mass spectrometry analysis was performed using a MALDI-TOF-TOF AutoFlex III (Bruker Daltonics) mass spectrometer, peptide calibration standard II (Bruker Daltonics), α-cyano-4-hydroxycinnamic acid as the matrix, and FlexControl software to control the mass-to-charge ratio in positive / reflective mode. The HPLC chromatogram and mass spectrum of the peptide prepared in Example 3 are shown below. Figure 1 As shown, by Figure 1 It can be seen that the molecular weight of the polypeptide is 5439 Da, which is consistent with the theoretical value of 5439.36 Da, the purity is 99.4%, and the yield is 78.4%.

[0023] 3. Fe3O4-HS-PEG was analyzed using transmission electron microscopy (TEM, JEM-1011 TEM, 100 kV) and atomic force microscopy (AFM, FSM-precision). 600 The -CO-NH2- complex polypeptide was characterized, its morphology was examined, and its size was statistically analyzed. The results are as follows: Figure 2 As shown, (a) surface morphology detection using atomic force microscopy; (b) results from transmission electron microscopy; and (c) statistical results of the composite nanomedicine size. Figure 2 It can be seen that the composite nanoparticles are approximately spherical in shape, and the composite nanoparticles are relatively uniform in size, ranging from 30.1 ± 5.22 nm. Transmission electron microscopy results show the regular distribution characteristics of peptide formation on the surface of the composite nanoparticles, indicating that the composite nanostructure has good stability in the water:oil mixture system.

[0024] 4. Qualitative analysis (EDS) of the energy dispersive spectroscopy (EDS) spectra of the composite nanomedicine and empty metal particles was performed using transmission electron microscopy (TEM, JEM-1011 TEM, 100 kV), and the elemental composition of the Fe3O4 core particles was analyzed. The results are as follows: Figure 3 As shown, by Figure 3 The energy spectrum of the composite nanomedicine shows that the iron content is smaller than that of the empty metal nanoparticles, and the sulfur content of the composite nanomedicine (containing sulfur-containing amino acids) is significantly higher than that of the empty metal nanoparticles, verifying the successful synthesis of the composite nanomedicine.

[0025] In vitro membrane penetration test: (1) 2×10 6 Neuro2A cells were seeded in 24 mm culture dishes and grown in a humid environment at 37°C with 5% CO2 for 24 h. (2) Wash cells with PBS, 100 μg Fe3O4-HS-PEG 600-CO-NH2- complex polypeptide was added to the cell culture medium and incubated at 37°C for 8 h; (3) After incubation, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. The cells were then rinsed three more times with PBS. The cell membrane was labeled with DiL dye, and the nuclei were stained with DAPI dye. Olympus-3000 confocal fluorescence microscopy was used for detection, with excitation at 405 nm, 488 nm, and 561 nm lasers, and image acquisition was performed. The results are as follows: Figure 4 As shown, by Figure 4 It can be seen that the composite nanomedicine has passed through the cell membrane and entered the cell after 40 minutes, and is distributed in the cytoplasm.

[0026] Treatment efficacy test Experimental subjects: 10-month-old AD mice; (1) Fe3O4-HS-PEG 600 -CO-NH2- complex polypeptide was administered via nasal drops for 7 consecutive days (2.5 μl, 2.34 μM, per nostril each time). Two months after administration, both the treatment group and the control group (livestock AD mice that did not receive the drug) were deeply anesthetized with sodium pentobarbital (50 mg / kg) and perfused with 20 mL of 0.01 M phosphate-buffered saline (PBS, pH = 7.4), followed by in vivo perfusion of 100 mL of 4% paraformaldehyde 0.1 M phosphate-buffered saline (PB, pH = 7.4). (2) Brain tissue was removed, fixed in the same fixative for 3 h, and then stored at 4°C for 24 h in 0.1 M PBS containing 30% sucrose. After that, it was embedded in OCT and then frozen into sagittal sections (20 μm). (3) The sections were rinsed three times with PBS, then blocked (0.1% Triton X-100 and 2% donkey serum) for 40 min, and Aβ was added. 42 Incubate with GFAP primary antibody separately (4°C overnight). After washing with 0.1M PBS, add secondary antibody and incubate (4°C overnight). Add DAPI staining solution for nuclear counterstaining. After rinsing with 0.1M PBS, mount with anti-fluorescence quencher mounting medium and observe the staining results. Figure 5 As shown, (a) immunofluorescence staining results of Aβ deposits in the hippocampus of mice in the control group; (b) immunofluorescence staining results of Aβ deposits in the hippocampus of mice in the treatment group; (c) immunofluorescence staining results of Aβ deposits in the hippocampus of mice in both groups. The t-test was used to determine the results. p <0.001, n=5; by Figure 5Immunostaining results of Aβ deposits in the AD pathological area showed that mice treated with drugs had significantly fewer Aβ deposits in the hippocampus than age-matched mice without drug treatment, and the diameter of the deposits was also significantly reduced, indicating that the composite nanomedicine has a significant effect on clearing Aβ deposits.

Claims

1. A superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease, characterized in that, The superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease has the structural formula Fe3O4-HS-PEG. 600 -CO-NH2- complex polypeptide; The composite polypeptide is composed of a localization sequence targeting Aβ protein, a membrane-penetrating sequence, a Sema3A repressor peptide, and a neuroprotective peptide, sequentially from N to C-terminus. The targeting sequence for Aβ protein is: FFXXK, where X is valine; the transmembrane sequence is YGRKKRRQRRR; the Sema3A inhibitory peptide sequence is HAVEHGFMQTLLKVTLE; and the neuroprotective peptide sequence is NAPVSIPQ.

2. The superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease according to claim 1, characterized in that, The N-terminus of the composite polypeptide is coupled with a FITC luminescent group.

3. A method for preparing a superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease as described in any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Preparation of Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles: Ferric oleate powder was added to a mixture of oleic acid and 1-octadecene, kept at 90-120℃ for 3-10 min, then heated to 300-350℃ and kept at 20-50 min, cooled, collected by a magnet and dispersed in heptane, washed to obtain Fe3O4 nanoparticle cores; the Fe3O4 nanoparticle cores were dissolved in PEG 600 During the stirring reaction, Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles were obtained. (2) Complex peptide: The complex peptide was synthesized using the Fmoc solid-phase carrier synthesis method. The complex peptide consists of a localization sequence targeting Aβ protein, a membrane-penetrating sequence, a Sema3A inhibitory peptide and a neuroprotective peptide from N to C, and is labeled with FITC at the N end. (3) Fe3O4-HS-PEG 600 Preparation of -CO-NH2- complex peptide: N-ethynyl-N,4-dimethylbenzenesulfonamide and the complex peptide in step (2) were mixed, dichloromethane was added, and the reaction was stirred until the acid was exhausted. After the reaction was completed, the dichloromethane was removed under vacuum, and Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles prepared in step (1), water and DMSO were added as a mixed solvent. The reaction was stirred at room temperature until the α-acyloxamine was completely consumed. The reactants were concentrated and purified to obtain a superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease.

4. The method for preparing superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease according to claim 3, characterized in that, In step (1), the feeding ratio of ferric oleate powder, oleic acid, and l-octadecene is 2 mmol: 1 mmol: 10 g; ferric oleate powder and PEG 600 The ratio was 4 mmol: 5 mL; the stirring reaction time was 1-5 h.

5. The method for preparing superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease according to claim 3, characterized in that, In step (3), the molar ratio of the composite peptide and Fe3O4-HS-PEG600-carboxyl-terminated nanoparticles is 1:

1.

6. The method for preparing superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease according to claim 3, characterized in that, In step (3), the molar ratio of N-ethynyl-N,4-dimethylbenzenesulfonamide to the complex polypeptide is 1:

1.

7. The method for preparing superparamagnetic nanomedicine targeting the pathological region of Aβ protein in Alzheimer's disease according to claim 3, characterized in that, In step (3), the volume ratio of water to DMSO in the mixed solvent is 1:4 to 1:

6.

8. The use of a superparamagnetic nanomedicine targeting the pathological region of Alzheimer's disease Aβ protein as described in any one of claims 1 to 2 in the preparation of a drug for treating Alzheimer's disease.

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