Cerebral neuron nanometer diagnosis and treatment integrated system with early Alzheimer disease diagnosis and treatment property, preparation method and application

Abstract

The invention discloses a brain neuron nano diagnosis and treatment integrated system with early-stage Alzheimer disease diagnosis and treatment properties, a preparation method and application. The brain neuron targeted nano diagnosis and treatment integrated system comprises nanoparticles and baicalein, wherein the baicalein is connected to the surfaces of the nanoparticles by forming phenylboronic acid ester bonds with phenylboronic acid; wherein the nanoparticles are formed by entrapment of fluorescent probes aniline tetramer and anthracene derivative by polyethylene glycol-polyaspartic acid-phenylboronic acid modified by brain neuron targeting peptide. The brain neuron nanometer diagnosis and treatment integrated system is simple in production process, green and low in economic cost, has active oxygen sensitive response and rapid drug release and fluorescence opening response capabilities and a brain neuron active targeting function, can be used for diagnosis and treatment of early Alzheimer's disease and real-time monitoring of treatment cycle and effect, and has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the field of materials and relates to a nano-biomaterial, and specifically to a brain neuron-targeted nano-diagnosis and treatment integrated system with early Alzheimer's disease diagnosis and treatment properties, a preparation method and an application. Background Art

[0002] Over the past three decades, the aging of the world's population has intensified and is accelerating, and the number of patients with neurodegenerative diseases has increased significantly, especially Alzheimer's disease (AD). The incidence and mortality of AD have risen rapidly, and it has now become the leading cause of death. AD patients often require treatment and care for several years, more than ten years or even longer, which brings a heavy economic burden and huge costs to individuals, families and society. However, nowadays, due to the complex etiology and unclear mechanism of AD, there is a lack of effective treatment methods in clinical practice.

[0003] This is mainly due to: ① The diagnosis of AD is not timely. Usually, when the diagnosis is confirmed, it is already in the middle and late stages of the disease. At this time, treatment is difficult. The root cause is the lack of early diagnostic pathological markers for AD. At present, the main diagnostic pathological markers of AD are amyloid plaques and neurofibrillary tangles in brain tissue, which mainly appear in the late stages of AD and have little significance for the effective treatment of further diseases. In addition, the diagnostic means are single, and the diagnosis and treatment methods are mostly separated; ② Due to the existence of a special protective barrier in brain tissue—the blood-brain barrier, most drugs are prevented from entering the brain; even if the drug enters the brain, it is distributed throughout the brain, and the actual drug concentration in the AD lesion site such as the cerebral cortex, hippocampus and other parts is extremely low, and it is difficult to produce a therapeutic effect; ③ The release of the drug is uncontrollable, or it is released before reaching the lesion site, or it is released too slowly at the lesion site, which affects the therapeutic effect of AD.

[0004] To solve the above problems, we have developed a brain neuron nano-diagnosis and treatment system for early AD that integrates diagnosis and treatment, which is used for the diagnosis of early Alzheimer's disease and real-time monitoring of the treatment cycle and effect. Based on this technical solution and technical effect, the present invention is specially proposed. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a brain neuron targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response and early Alzheimer's disease diagnostic and therapeutic properties; the second purpose is to provide a preparation method of the brain neuron targeted nano-diagnosis and treatment integrated system; and the third purpose is to provide the application of the brain neuron targeted nano-diagnosis and treatment integrated system.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response and diagnostic and therapeutic properties for early Alzheimer's disease, comprising nanoparticles and baicalein connected to the surface of the nanoparticles by forming a phenylboronic acid ester bond with phenylboronic acid; wherein the nanoparticles are formed by encapsulating fluorescent probe aniline tetramer and anthracene derivatives with polyethylene glycol-polyaspartic acid-phenylboronic acid modified with brain neuron targeting peptides;

[0008] The chemical structural formula of the aniline tetramer is as follows:

[0009]

[0010] The chemical structural formula of the anthracene derivative is as follows:

[0011]

[0012] Preferably, the polyethylene glycol-polyaspartic acid-phenylboronic acid is PEG2000-polyaspartic acid 5000-phenylboronic acid.

[0013] Preferably, the brain neuron targeting peptide is RVG29 polypeptide or Tet1 polypeptide.

[0014] RVG29 polypeptide sequence: YTIWMPENPRPGTPCDIFTNSRGKRASNG;

[0015] Tet1 polypeptide sequence: HLNILSTLWKYR.

[0016] A method for preparing the above-mentioned brain neuron targeted nano-diagnosis and treatment integrated system comprises the following steps:

[0017] Step S1, preparing reagent A: dissolving appropriate amounts of aniline tetramer and anthracene derivative in DMSO;

[0018] Step S2, preparing reagent B: dissolving an appropriate amount of brain neuron targeting peptide-modified polyethylene glycol-polyaspartic acid-phenylboronic acid in a PBS buffer at pH 7.4;

[0019] Step S3, injecting reagent A into a certain volume of reagent B, stirring for self-assembly, and then ultrasonically dispersing in an ice bath to obtain a nanoparticle solution carrying a fluorescent probe;

[0020] Step S4, dissolving baicalein in DMSO to prepare a baicalein solution of a certain concentration, adding the baicalein solution to a certain volume of the above-mentioned nanoparticle solution loaded with fluorescent probes, stirring, connecting baicalein to the surface of the nanoparticles through the reaction of the vicinal diol in the baicalein structure with the phenylboronic acid in the polymer structure to form a phenylboronic acid bond, ultrafiltration and concentration, removing free baicalein through a microporous filter membrane, and preparing a brain neuron targeted nano-diagnosis and treatment integrated system.

[0021] Preferably, in step S1, the mass ratio of the aniline tetramer to the anthracene derivative is 1:1, and the sum of their concentrations in DMSO is 5-10 mg / mL.

[0022] Preferably, the concentration of the brain neuron targeting peptide modified with polyethylene glycol-polyaspartic acid-phenylboronic acid in step S2 is 1-2 mg / mL.

[0023] Preferably, in step S3, the volume ratio of reagents A to B is 1:20 to 1:40.

[0024] Preferably, in step S4, baicalein is dissolved in DMSO to prepare a baicalein solution with a concentration of 5 to 10 mg / mL.

[0025] Preferably, in step S4, the volume ratio of the baicalein solution to the fluorescent probe-carrying nanoparticle solution is 1:10 to 1:30.

[0026] The above-mentioned brain neuron targeted nano-diagnosis and treatment integrated system is used in the preparation of nanomaterials for diagnosing and treating Alzheimer's disease and / or real-time monitoring of the progress of Alzheimer's disease treatment.

[0027] Beneficial effects:

[0028] The present invention provides a brain neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response and early Alzheimer's disease diagnosis and treatment properties. The brain neuron-targeted nano-diagnosis and treatment integrated system comprises nanoparticles and baicalein connected to the surface of the nanoparticles by forming a phenylboronic acid ester bond with phenylboronic acid. The beneficial effects thereof include:

[0029] 1. Under the triggering of reactive oxygen species sensitive response, the phenylboronic acid ester bond on the nanoparticles breaks, and baicalin can be quickly released within 5 minutes, exposing the nanoparticle core loaded with fluorescent probes. The exposed particle core and baicalin oxidized to quinone structure then turn on dual-channel fluorescence at 425nm and 500nm respectively. After the reactive oxygen species are eliminated, the dual-channel fluorescence turns off. Its ROS detection limit reaches 1μM, showing sensitive reactive oxygen species fluorescence response capability in vivo and in vitro.

[0030] 2. The integrated nano-diagnosis and treatment system has the ability to target brain neurons, and can deliver drugs and fluorescent probes to brain neurons, thereby achieving diagnosis and treatment under the triggering of reactive oxygen species, one of the pathological characteristics of early Alzheimer's disease. Based on the sensitive response of the integrated nano-diagnosis and treatment system to reactive oxygen species, the disease treatment cycle and effect can be monitored in real time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Anthracene derivative (R)-3-amino-4-(anthracen-2-amino)-4-oxobutanoic acid 1HNMR spectra;

[0032] Figure 2 This is a transmission electron microscope photo of the brain neuron targeted nano-diagnosis and treatment integrated system of the present invention;

[0033] Figure 3 The cytotoxicity results of the brain neuron targeted nano-diagnosis and treatment integrated system of the present invention;

[0034] Figure 4 The fluorescence spectrum of the brain neuron targeted nano-diagnosis and treatment integrated system of the present invention that is sensitive to active oxygen;

[0035] Figure 5 The drug release curve of the brain neuron targeted nano-diagnosis and treatment integrated system of the present invention in response to active oxygen sensitivity;

[0036] Figure 6 Verification of the brain neuron targeting of the integrated system for brain neuron targeted nano-diagnosis and treatment of the present invention;

[0037] Figure 7 To verify the in vitro diagnostic capability of the brain neuron-targeted nano-diagnosis and treatment integrated system of the present invention for early AD;

[0038] Figure 8 To investigate the in vivo diagnostic capability of the brain neuron-targeted nano-diagnosis and treatment integrated system of the present invention for early AD by using a living microscope;

[0039] Fig. 9 Immunofluorescence was used to investigate the in vivo diagnostic capability of the brain neuron-targeted nano-diagnosis and treatment integrated system of the present invention for early AD;

[0040] Fig.10 The brain neuron targeted nano-diagnosis and treatment integrated system of the present invention reduces the level of reactive oxygen in vitro; #p<0.05, ##p<0.01, ####p<0.0001, compared with the Aβ25-35 group; **p<0.01, comparison between the two groups. DETAILED DESCRIPTION

[0041] The following is a detailed description of the substantive content of the present invention in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. In the following embodiments, the instruments and reagents not specifically explained are all commercially available conventional instruments and reagents.

[0042] 1. Preparation of aniline tetramer

[0043] The synthesis method of aniline tetramer is reported in the literature (SP Surwade, SR Agnihotra, V. Dua, N. Manohar, S. Jain, S. Ammu, SK Manohar, J Am Chem Soc 2009, 131, 12528-9), as follows:

[0044] 0.2 g of aniline dimer (N-phenyl-1,4-phenylenediamine) was added to a hydrochloric acid solution (100 mL, 1 M HCl) of sodium chloride (20 g). After magnetic stirring for 5 minutes, 2 mL of H2O2 (35 wt%) was added and stirred for 2 hours. After 60 minutes of reaction, the reaction mixture turned dark green and solid green precipitates appeared in the reaction flask. After 2 hours, the reaction mixture was filtered and washed 10 times with 1 M hydrochloric acid and acetone until the filtrate was colorless. The generated green aniline salt precipitate was air-dried in the filter funnel for 10 minutes and then placed in a conical flask filled with 0.1 M NH4OH (1 L) and sealed with a sealing film to exclude air. Stir for 24 hours to ensure that the aniline salt was completely converted into aniline. The black blue precipitate was suction filtered in the air, washed with water, air-dried in the filter funnel for 10 minutes, and then dried in a dynamic vacuum oven for 24 hours. The structural formula of aniline tetramer is as follows:

[0045]

[0046] 2. Preparation of anthracene derivative (R)-3-amino-4-(anthracene-2-amino)-4-oxobutyric acid

[0047] The synthetic route and steps are as follows:

[0048]

[0049] Step 1:

[0050] Compound 1 (300 mg, 0.93 mmol), 2-aminoanthracene (179 mg, 0.93 mmol) and HATU (600 mg, 1.58) were added to a DMF (10 mL) solution, and DIPEA (311 mg, 2.40 mmol) was added and stirred at room temperature overnight. LC-MS verified that the reaction was complete, and the reaction was quenched with water (20 mL) and extracted with ethyl acetate EA 3 times, 20 mL each time. The EA layer was washed with brine 3 times, 30 mL each time, and dried over Na2SO4. The solvent was removed to obtain a crude product, which was chromatographed on flash silica gel. Purification of crude product (25 g Silica gel flash column, eluent 0-100% ethyl acetate / petroleum ether gradient, 30 mL / min) to obtain compound 2 (371 mg, yield 80%).

[0051] Step 2:

[0052] LiOH was added to a solution of compound 2 (2.0 g, 4.01 mmol) in MeOH / THF / H2O (48 mL, 1:1:1). The mixture was stirred at room temperature for 4 h, most of the solvent was removed under reduced pressure, water and concentrated HCl were added to acidify to pH = 1-2, and compound 3 (1.47 g, yield 90%) was obtained by extraction with EA.

[0053] Step 3:

[0054] 4N HCl dioxane (5 mL) was added to a dioxane (10 mL) solution of compound 3 (1.47 g, 3.60 mmol), stirred at room temperature for 4 h, most of the solvent was removed under reduced pressure, and vacuum dried to obtain compound 4 (1.0 g, yield 90%), i.e., (R)-3-amino-4-(anthracene-2-amino)-4-oxobutanoic acid. 1 H NMR (400 MHz, DMSO-d6) signals and their attributions are as follows Figure 1 shown.

[0055] 3. Preparation of RVG29 peptide modified PEG-PAsp-PBA polymer material (RVG-PEG-PAsp-PBA)

[0056] RVG29-PEG2000-polyaspartic acid 5000-phenylboronic acid was commissioned to an outsourcing company (Xi'an Ruixi Biotechnology Co., Ltd.) for synthesis. NHS-PEG2000-BOC and Fmoc-RVG29 used in the synthesis process are commercial products provided by Xi'an Ruixi Biotechnology Co., Ltd. The specific steps are as follows:

[0057] 1. Weigh 500 mg of NHS-PEG2000-BOC and dissolve it in 6 mL of dry DMF. Add Fmoc-RVG29 (0.9 eq.) and triethylamine (3.0 eq.) to dissolve completely and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure, add 2 mL of dichloromethane to disperse the product, add 0.6 mL of trifluoroacetic acid and react at room temperature for 0.5 h. Remove the solvent by distillation under reduced pressure, add 1 mL of dry DMF to redissolve and transfer to acetone, centrifuge, and dry in vacuo to obtain Fmoc-RVG29-PEG2000-NH2.

[0058] 2. Weigh 400 mg of Fmoc-RVG29-PEG2000-NH2 and dissolve it in 5 mL of dry DMF. Weigh N-carboxylic anhydride (NCA, 600 mg, 1.5 eq.) and dissolve it in 10 mL of DMF. Heat and stir under N2 environment and react at 30°C for 72 h. After the reaction is completed, add the product dropwise into a large amount of ice ether, centrifuge, and vacuum dry to obtain

[0059] Fmoc-RVG29-PEG2000-PASP (benzyl ester protection).

[0060] 3. Weigh 500 mg of Fmoc-RVG29-PEG-PASP (benzyl ester protection) and completely dissolve it in trifluoroacetic acid solvent. Add 33% HBr / AcOH solution while stirring in an ice bath. After reacting for 2 hours, precipitate in a large amount of ice ether to obtain a crude product, then dissolve it in water, adjust the pH to about 7 with NaOH solution, put it into a dialysis bag for dialyzation (molecular weight cutoff 3500Da), dialyze it in deionized water for 24 hours, and finally freeze-dry it to obtain Fmoc-RVG29-PEG2000-PASP.

[0061] 4. Weigh 300 mg of Fmoc-RVG29-PEG2000-PASP and dissolve it in 5 mL of pure water. Add EDC (10.0 eq.) and NHS (10.0 eq.) and react at room temperature for 3 h. Then add 2 mL of DMF solution of 4-aminomethylphenylboronic acid (10.0 eq.) and triethylamine (12.0 eq.). Continue to react at room temperature for 12 h. Transfer to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze in deionized water for 24 h. Finally, freeze-dry to obtain Fmoc-RVG29-PEG2000-polyaspartic acid 5000-phenylboronic acid.

[0062] 5. Weigh 200 mg of Fmoc-RVG29-PEG2000-polyaspartic acid 5000-phenylboronic acid and dissolve it in 5 mL of DMF. Add 2 mL of piperidine and react at room temperature for 0.5 h. Transfer it to a large amount of icy ether for precipitation to obtain RVG29-PEG2000-polyaspartic acid 5000-phenylboronic acid (RVG-PEG-PAsp-PBA).

[0063] 4. Tet1 peptide modified PEG-PAsp-PBA polymer materials

[0064] Tet1-PEG2000-polyaspartic acid 5000-phenylboronic acid was commissioned to an outsourcing company (Xi'an Ruixi Biotechnology Co., Ltd.) for synthesis. NHS-PEG2000-BOC and Fmoc-Tet1 used in the synthesis process are commercial products provided by Xi'an Ruixi Biotechnology Co., Ltd. The specific steps are as follows:

[0065] 1. Weigh 500 mg of NHS-PEG2000-BOC and dissolve it in 6 mL of dry DMF. Add Fmoc-Tet1 (0.9 eq.) and triethylamine (3.0 eq.) to dissolve completely and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure, add 2 mL of dichloromethane to disperse the product, add 0.6 mL of trifluoroacetic acid and react at room temperature for 0.5 h. Remove the solvent by distillation under reduced pressure, add 1 mL of dry DMF to redissolve and transfer to acetone, centrifuge and dry in vacuo to obtain Fmoc-Tet1-PEG2000-NH2.

[0066] 2. Weigh 400 mg of Fmoc-Tet1-PEG2000-NH2 and dissolve it in 5 mL of dry DMF. Weigh N-carboxylic anhydride (NCA, 600 mg, 1.5 eq.) and dissolve it in 10 mL of DMF. Heat and stir under N2 environment and react at 30°C for 72 h. After the reaction is completed, add the product dropwise into a large amount of ice ether, centrifuge, and vacuum dry to obtain Fmoc-Tet1-PEG2000-PASP (benzyl ester protection).

[0067] 3. Weigh 500 mg of Fmoc-Tet1-PEG-PASP (benzyl ester protection) and completely dissolve it in trifluoroacetic acid solvent. Add 33% HBr / AcOH solution while stirring in an ice bath. After reacting for 2 hours, precipitate in a large amount of ice ether to obtain a crude product, then dissolve it in water, adjust the pH to about 7 with NaOH solution, put it into a dialysis bag for dialyzation (molecular weight cutoff 3500Da), dialyze it in deionized water for 24 hours, and finally freeze-dry it to obtain Fmoc-Tet1-PEG2000-PASP.

[0068] 4. Weigh 300 mg of Fmoc-Tet1-PEG2000-PASP and dissolve it in 5 mL of pure water. Add EDC (10.0 eq.) and NHS (10.0 eq.) and react at room temperature for 3 h. Then add 2 mL of DMF solution of 4-aminomethylphenylboronic acid (10.0 eq.) and triethylamine (12.0 eq.). Continue to react at room temperature for 12 h. Transfer to a dialysis bag (molecular weight cutoff 3500 Da) and dialyze in deionized water for 24 h. Finally, freeze-dry to obtain Fmoc-Tet1-PEG2000-polyaspartic acid 5000-phenylboronic acid.

[0069] 5. Weigh 200 mg of Fmoc-Tet1-PEG2000-polyaspartic acid 5000-phenylboronic acid and dissolve it in 5 mL of DMF. Add 2 mL of piperidine and react at room temperature for 0.5 h. Transfer it to a large amount of icy ether for precipitation to obtain Tet1-PEG2000-polyaspartic acid 5000-phenylboronic acid (Tet-PEG-PAsp-PBA).

[0070] Example 1: Preparation of a brain neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response

[0071] Prepare as follows:

[0072] (1) Preparation of reagent A: Dissolve fluorescent probe O (aniline tetramer) and fluorescent probe N (anthracene derivative) in DMSO at a mass ratio of 1:1, and the concentrations of fluorescent probe O and fluorescent probe N are both 5 mg / mL;

[0073] (2) Preparation of reagent B: RVG29 peptide-modified PEG-PAsp-PBA polymer material (RVG-PEG-PAsp-PBA) was completely dissolved in PBS buffer (pH 7.4) at a concentration of 1 mg / mL;

[0074] (3) Reagent A was injected into 30 times the volume of reagent B, stirred for 30 min, and then ultrasonically dispersed in an ice bath (400 W, 30 s) to obtain a nanoparticle solution loaded with a fluorescent probe;

[0075] (4) Baicalein was dissolved in DMSO to prepare a baicalein solution with a concentration of 10 mg / mL. The baicalein solution was added to 20 times the volume of the above-mentioned nanoparticle solution loaded with fluorescent probes, stirred for 12 h, concentrated by ultrafiltration (10 kDa, 5000 rpm×20 min), and filtered through a 0.45 μm filter membrane to remove free baicalein, thereby preparing a brain neuron targeted nanodiagnostic and therapeutic system.

[0076] Example 2: Preparation of a brain neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response

[0077] Prepare as follows:

[0078] (1) Preparation of reagent A: Dissolve fluorescent probe O (aniline tetramer) and fluorescent probe N (anthracene derivative) in DMSO at a mass ratio of 1:1, and the concentrations of fluorescent probe O and fluorescent probe N are both 2.5 mg / mL;

[0079] (2) Preparation of reagent B: Tet1 peptide-modified PEG-PAsp-PBA polymer material (Tet-PEG-PAsp-PBA) was completely dissolved in PBS buffer (pH 7.4) at a concentration of 1 mg / mL;

[0080] (3) Reagent A was injected into 30 times the volume of reagent B, stirred for 30 min, and then ultrasonically dispersed in an ice bath (400 W, 30 s) to obtain a nanoparticle solution loaded with a fluorescent probe;

[0081] (4) Baicalein was dissolved in DMSO to prepare a baicalein solution with a concentration of 5 mg / mL. The baicalein solution was added to 20 times the volume of the above-mentioned nanoparticle solution loaded with fluorescent probes, stirred for 12 h, concentrated by ultrafiltration (10 kDa, 5000 rpm×20 min), and filtered through a 0.45 μm filter membrane to remove free baicalein, thereby preparing a brain neuron targeted nanodiagnostic and therapeutic system.

[0082] Example 3: Preparation of a brain neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response

[0083] Prepare as follows:

[0084] (1) Preparation of reagent A: Dissolve fluorescent probe O (aniline tetramer) and fluorescent probe N (anthracene derivative) in DMSO at a mass ratio of 1:1, and the concentrations of fluorescent probe O and fluorescent probe N are both 5 mg / mL;

[0085] (2) Preparation of reagent B: RVG29 peptide-modified PEG-PAsp-PBA polymer material (RVG-PEG-PAsp-PBA) was completely dissolved in PBS buffer (pH 7.4) at a concentration of 2 mg / mL;

[0086] (3) Reagent A was injected into 30 times the volume of reagent B, stirred for 30 min, and then ultrasonically dispersed in an ice bath (400 W, 30 s) to obtain a nanoparticle solution loaded with a fluorescent probe;

[0087] (4) Baicalein was dissolved in DMSO to prepare a baicalein solution with a concentration of 10 mg / mL. The baicalein solution was added to 20 times the volume of the above-mentioned nanoparticle solution loaded with fluorescent probes, stirred for 12 h, concentrated by ultrafiltration (10 kDa, 5000 rpm×20 min), and filtered through a 0.45 μm filter membrane to remove free baicalein, thereby preparing a brain neuron targeted nanodiagnostic and therapeutic system (expressed as RVG-NP / ONB).

[0088] Example 4: Characterization of the integrated system for brain neuron targeted nanodiagnosis and treatment prepared in Example 1

[0089] DLS (dynamic light scattering) was used to measure the particle size and potential of the brain neuron targeted nano-diagnosis and treatment integrated system of Example 1; and a transmission electron microscope was used to observe the morphology of the nano-diagnosis and treatment integrated system.

[0090] DLS results show that the particle size of the brain neuron targeted nano-diagnosis and treatment integrated system is 229.4nm, PDI is 0.18, and the potential is -15.3mV. TEM results are as follows Figure 2 As shown, the integrated system for brain neuron targeted nanodiagnosis and treatment has a round appearance and a particle size slightly smaller than the DLS result, at around 200 nm.

[0091] The brain neuron targeted nano-diagnosis and treatment integrated system prepared in Examples 2 and 3 is basically the same as that in Example 1.

[0092] Example 5: CCK8 method to determine the cytotoxicity of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 2

[0093] PC12 neuronal cells in the logarithmic growth phase were plated in a 96-well plate. After 24 hours of growth, the cell confluence was 70-80%. The culture medium was discarded, and the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 2 was administered according to a concentration gradient. After 48 hours, 10 μL of CCK8 solution was added to each well, and the incubation was continued for 1 hour. The absorbance was measured at 450 nm by a microplate reader to calculate the cell survival rate.

[0094] The experimental results are as follows Figure 3 As shown, when the concentration of the nano-diagnosis and treatment integrated system is 0.1-2 mg / mL, the cell survival rate is above 80%, indicating that the brain neuron targeted nano-diagnosis and treatment integrated system of the present invention has no cytotoxicity.

[0095] The brain neuron targeted nano-diagnosis and treatment integrated system prepared in Examples 1 and 3 is basically the same as that in Example 1.

[0096] Example 6: Detection of the fluorescence sensitive response ability of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 3 to reactive oxygen species by using an ELISA instrument

[0097] The brain neuron targeted nano-diagnosis and treatment integrated system RVG-NP / ONB prepared in Example 3 was mixed with hydrogen peroxide of different concentrations and incubated for 5 min and 4 h, and then the fluorescence spectrum was scanned (400-700 nm) by a microplate reader at an excitation wavelength of 372 nm.

[0098] The experimental results are as follows Figure 4 As shown in the figure, after 5 minutes of incubation, the fluorescence intensity of the nano-diagnosis and treatment system at 500nm gradually increased, while there was no change at 425nm. After 4 hours of incubation, the fluorescence spectrum showed that the fluorescence intensity of the two emission channels of 425nm and 500nm of the nano-diagnosis and treatment system was significantly enhanced. This shows that the integrated nano-diagnosis and treatment system targeting brain neurons has excellent ROS fluorescence response ability, and its fluorescence responsive H2O2 concentration can reach 1μM.

[0099] Example 7: Drug release based on active oxygen sensitive response of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 3

[0100] The dialysis method was used to investigate the drug release ability of the brain neuron-targeted nanodiagnosis and treatment integrated system in response to active oxygen species. The release medium was H2O2 of different concentrations (PBS pH 7.4, 37°C, 100rpm). 400 μL of the release medium was aspirated at different time points, and the same volume of fresh release medium was supplemented at the same time.

[0101] The experimental results are as follows Figure 5 As shown in the data, the brain neuron targeted nano-diagnosis and treatment integrated system has almost no release in PBS, and the average cumulative release percentage in 10 hours is only 6.2%; but it shows the characteristics of rapid response drug release in H2O2. In the release media of 1mM, 5mM and 10mM H2O2, the cumulative release percentages within 10 hours are 53.7%, 62.7% and 76.0%, respectively, indicating that the brain neuron targeted nano-diagnosis and treatment integrated system has excellent ROS-triggered rapid response drug release capability.

[0102] Example 8: Investigation of the brain neuron targeting of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 3

[0103] The tri-transgenic 3×TgAD mice were injected with the DiR-loaded brain neuron-targeted nano-diagnosis and treatment integrated system through the tail vein, and the brain was taken out 1 hour later to prepare brain slices. After being placed at room temperature, the cells were washed 3 times with PBS, incubated at room temperature for 10 minutes with 0.25% Triton X-100, washed 3 times with PBS, added with immunofluorescence blocking solution, incubated at room temperature for 1 hour, aspirated the blocking solution, added with primary anti-NeuN antibody (1:100 dilution), anti-Iba1 antibody (1:100 dilution) and GFAP antibody (1:200 dilution), incubated at 4°C overnight, washed 3 times with PBS, added with AlexaFluor 594-labeled goat rabbit IgG (secondary antibody, 1:200 dilution), incubated at room temperature for 1 hour, washed 3 times with PBS, sealed with anti-fluorescence quenching sealing agent, and observed with laser confocal microscope.

[0104] The experimental results are as follows Figure 6 As shown, the brain neuron targeted nano-diagnosis and treatment integrated system RVG-NP / ONB has good co-localization with neurons after entering the brain, and a smaller amount is around microglia and astrocytes, and its fluorescence intensity in the hippocampus and cortical areas is significantly stronger than that of the unmodified nano-diagnosis and treatment system, confirming that the brain neuron targeted nano-diagnosis and treatment integrated system has excellent brain neuron targeting.

[0105] Example 9: Investigation of in vitro diagnostic effects of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 3

[0106] PC12 cells were cultured at 1×10 5The cells were inoculated at a high density in a 12-well plate and cultured for 24 hours. The culture medium was aspirated, washed 3 times with PBS, and then 25 μM Aβ25-35 was added to stimulate the cells for 48 hours. The culture medium was discarded, and the brain neuron targeted nano-diagnosis and treatment integrated system RVG-NP / ONB was added to incubate the cells for 4 hours and 48 hours, and observed under a fluorescence microscope. In this experiment, normal cells incubated with the brain neuron targeted nano-diagnosis and treatment integrated system were used as controls.

[0107] The experimental results are as follows Figure 7 As shown, compared with normal PC12 cells treated with RVG-NP / ONB, the fluorescence signals of PC12 cells stimulated by Aβ25-35 after treatment increased significantly in blue and green channels. In addition, after RVG-NP / ONB was incubated with cells for 48 hours, the fluorescence microscope images showed that in the cell group treated with Aβ25-35, blue fluorescence emitted at 425nm was visible, but the green fluorescence signal at 500nm was greatly reduced, close to the background of normal cells. It shows that after 48h incubation, ROS was eliminated and the green fluorescence signal was turned off, showing its potential real-time AD diagnostic ability.

[0108] Example 10: Investigation of the in vivo diagnostic effect of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 2

[0109] After opening the skull window of 6-month-old triple transgenic 3×TgAD mice, they rested for a week and then received tail vein injection of the brain neuron-targeted nano-diagnosis and treatment integrated system. One hour later, they were intraperitoneally anesthetized with 1% sodium pentobarbital and the fluorescence in the brain was observed under an intravital microscope (excitation wavelength: 405nm, emission wavelengths 425-465nm and 503-558nm).

[0110] The experimental results are as follows Figure 8 As shown in the figure, at emission wavelengths of 425-465nm and 503-558nm, AD mouse brains showed obvious fluorescence. This result shows that the integrated system of brain neuron-targeted nanodiagnosis and treatment has excellent early AD diagnosis capabilities.

[0111] Example 11: Investigation of the in vivo diagnostic effect of the brain neuron targeted nano-diagnosis and treatment integrated system prepared in Example 3

[0112] Wild-type mice and triple transgenic 3×TgAD mice were injected with the brain neuron-targeted nano-diagnosis and treatment integrated system through the tail vein, and brain tissue was taken 1 hour later to prepare frozen sections. After being placed at room temperature, the sections were washed 3 times with PBS, incubated at room temperature for 10 minutes with 0.25% Triton X-100, washed 3 times with PBS, added with immunofluorescence blocking solution, incubated at room temperature for 1 hour, aspirated the blocking solution, added with primary anti-NeuN antibody (1:100 dilution), incubated at 4°C overnight, washed 3 times with PBS, added with Alexa Fluor 594-labeled goatrabbit IgG (secondary antibody, 1:200 dilution), incubated at room temperature for 1 hour, washed 3 times with PBS, sealed with anti-fluorescence quenching sealing agent, and observed with laser confocal microscope.

[0113] The experimental results are as follows Fig. 9 As shown, there is no fluorescence in the brain of wild-type mice. In the triple transgenic mouse group, the dentate gyrus, CA1, CA3 and cortex of the hippocampus all showed strong blue and green fluorescence signals, which were concentrated around the neurons. This result shows that the integrated system of nanodiagnosis and treatment targeting brain neurons has excellent AD in vivo diagnostic capabilities, and once again confirms that the integrated system of nanodiagnosis and treatment targeting brain neurons has excellent brain neuron targeting.

[0114] Example 12: Evaluation of the in vitro reduction of reactive oxygen species levels by the integrated system for brain neuron-targeted nanodiagnosis and treatment prepared in Example 3

[0115] PC12 cells were cultured at 1×10 5 The cells were inoculated in a 96-well plate at a density of 24 h. The culture medium was aspirated, washed 3 times with PBS, and then 25 μM Aβ25-35 and free baicalin drug solution (BAI), non-targeted nano-diagnosis and treatment integrated system NP / ONB (with PEG2000-polyaspartic acid 5000-phenylboronic acid without peptide modification as raw material, the preparation method is referred to Example 3), RVG-NP / BAI without fluorescent probe and only connected to baicalin (the preparation method is referred to Example 3) or brain neuron targeted nano-diagnosis and treatment integrated system solution RVG-NP / ONB mixture was added, wherein the concentrations of BAI, NP / ONB, RVG-NP / BAI, and RVG-NP / ONB were all 10 μM, and the solution was discarded after incubation at 37°C for 48 h. The cells were washed 3 times with PBS, and the intracellular reactive oxygen level was detected using a reactive oxygen detection kit. In this experiment, cells cultured in serum-free medium (Blank, normal control group) and cells incubated with 25 μM Aβ25-35 (Aβ25-35, model control group) were used as controls.

[0116] The experimental results are as follows Fig.10As shown in the figure, compared with the normal control group, the cell reactive oxygen level increased to 237.4% after Aβ25-35 treatment, indicating that it would cause oxidative damage to neuronal cells. After administration of free drug BAI solution and non-targeted nano-diagnosis and treatment integrated system NP / ONB, the damage reduction effect was weak, and the reactive oxygen level was still 183.2% and 165.3%, while the brain neuron targeted nano-diagnosis and treatment integrated system RVG-NP / ONB had a significant improvement effect, and the reactive oxygen level dropped to the normal control level. The improvement effect of RVG-NP / BAI without fluorescent probe and only connected to baicalin was basically the same as that of RVG-NP / ONB, indicating that the fluorescent probe will not affect the loading and release of the therapeutic drug baicalin while providing fluorescent signals.

[0117] In summary, the present invention provides a neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response and early Alzheimer's disease diagnosis and treatment properties, the neuron-targeted nano-diagnosis and treatment integrated system comprising nanoparticles and baicalein connected to the surface of the nanoparticles by forming a phenylboronic acid ester bond with phenylboronic acid, and has the following functional characteristics:

[0118] 1. Under the triggering of reactive oxygen species sensitive response, the phenylboronic acid ester bond on the nanoparticles breaks, and baicalin can be quickly released within 5 minutes, exposing the nanoparticle core loaded with fluorescent probes. The exposed particle core and baicalin oxidized to quinone structure then turn on dual-channel fluorescence at 425nm and 500nm respectively. After the reactive oxygen species are eliminated, the dual-channel fluorescence turns off. Its ROS detection limit reaches 1μM, showing sensitive reactive oxygen species fluorescence response capability in vivo and in vitro.

[0119] 2. The integrated nano-diagnosis and treatment system has the ability to target brain neurons, and can deliver drugs and fluorescent probes to brain neurons, thereby achieving diagnosis and treatment under the triggering of reactive oxygen species, one of the pathological characteristics of early Alzheimer's disease. Based on the sensitive response of the integrated nano-diagnosis and treatment system to reactive oxygen species, the disease treatment cycle and effect can be monitored in real time.

[0120] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A neuron-targeted nano-diagnosis and treatment integrated system with active oxygen sensitive response and diagnostic and therapeutic properties for early Alzheimer's disease, characterized by: The brain neuron targeted nano-diagnosis and treatment integrated system comprises nanoparticles and baicalein connected to the surface of the nanoparticles by forming a phenylboronic acid ester bond with phenylboronic acid; wherein the nanoparticles are formed by encapsulating fluorescent probe aniline tetramer and anthracene derivatives with polyethylene glycol-polyaspartic acid-phenylboronic acid modified with brain neuron targeting peptides; The chemical structural formula of the aniline tetramer is as follows: The chemical structural formula of the anthracene derivative is as follows:

2. The brain neuron targeted nano-diagnosis and treatment integrated system according to claim 1 is characterized by: The polyethylene glycol-polyaspartic acid-phenylboronic acid is PEG2000-polyaspartic acid 5000-phenylboronic acid.

3. The brain neuron targeted nano-diagnosis and treatment integrated system according to claim 1 or 2, characterized in that: The brain neuron targeting peptide is RVG29 polypeptide or Tet1 polypeptide.

4. A method for preparing the brain neuron targeted nano-diagnosis and treatment integrated system according to any one of claims 1 to 3, characterized in that: The steps include: Step S1, preparing reagent A: dissolving appropriate amounts of aniline tetramer and anthracene derivative in DMSO; Step S2, preparing reagent B: dissolving an appropriate amount of brain neuron targeting peptide-modified polyethylene glycol-polyaspartic acid-phenylboronic acid in a PBS buffer at pH 7.4; Step S3, injecting reagent A into a certain volume of reagent B, stirring for self-assembly, and then ultrasonically dispersing in an ice bath to obtain a nanoparticle solution carrying a fluorescent probe; Step S4, dissolving baicalein in DMSO to prepare a baicalein solution of a certain concentration, adding the baicalein solution to a certain volume of the above-mentioned nanoparticle solution loaded with fluorescent probes, stirring, connecting baicalein to the surface of the nanoparticles through the reaction of the vicinal diol in the baicalein structure with the phenylboronic acid in the polymer structure to form a phenylboronic acid bond, ultrafiltration and concentration, removing free baicalein through a microporous filter membrane, and preparing a brain neuron targeted nano-diagnosis and treatment integrated system.

5. The method according to claim 4, characterized in that: The mass ratio of the aniline tetramer to the anthracene derivative in step S1 is 1:1, and the sum of their concentrations in DMSO is 5-10 mg / mL.

6. The method according to claim 4, characterized in that: In step S2, the concentration of the midbrain neuron targeting peptide modified with polyethylene glycol-polyaspartic acid-phenylboronic acid is 1-2 mg / mL.

7. The method according to claim 4, characterized in that: In step S3, the volume ratio of reagents A to B is 1:20 to 1:

40.

8. The method according to claim 4, characterized in that: In step S4, baicalein is dissolved in DMSO to prepare a baicalein solution with a concentration of 5 to 10 mg / mL.

9. The method according to claim 4, characterized in that: In step S4, the volume ratio of the baicalein solution to the fluorescent probe-carrying nanoparticle solution is 1:10 to 1:

30.

10. Use of the brain neuron targeted nano-diagnosis and treatment integrated system according to claim 1 in the preparation of nanomaterials for diagnosing and treating Alzheimer's disease and / or real-time monitoring of the progress of Alzheimer's disease treatment.

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