A dual-function nanoprobe and its preparation method and application

Abstract

The present invention discloses a bifunctional nanoprobe, which is CICe@M-K, comprising curcumin and IR780 iodide loaded in mesoporous silicon / cerium dioxide composite nanospheres, and K peptide connected to the outer layer of the mesoporous silicon / cerium dioxide composite nanospheres; the sequence of the K peptide is CKLVFFAED. The bifunctional nanoprobe CICe@M-K provided by the present invention can synergistically treat Alzheimer's disease from two different pathways, and simultaneously inhibit Aβ aggregation and relieve oxidative stress.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dual-function nanoprobe and a preparation method and application thereof. Background Art

[0002] As the world's population ages, Alzheimer's disease (AD), a typical neurodegenerative disease, has attracted widespread attention. β-amyloid protein (Aβ) is one of the many markers of AD. It is produced by the cleavage of amyloid protein precursor (APP) by β- and γ-secretase. Under normal physiological conditions, the production and degradation of Aβ are in dynamic equilibrium; but under pathological conditions, soluble Aβ will misfold and aggregate, eventually forming insoluble senile plaques. These deposits promote the production of reactive oxygen species (ROS) by neurons, increase the oxidative stress of the brain environment, induce neuronal cell necrosis, and thus increase the risk of AD. Studies have shown that Aβ aggregation is a key step in the onset of AD, and senile plaques are an important clinical symptom. Therefore, clearing intracellular ROS and inhibiting Aβ aggregation are effective means of treating AD.

[0003] Curcumin (Cur) is a small polyphenolic molecule with excellent antioxidant, anti-inflammatory, Aβ aggregation inhibition and amyloid fibril (Aβf) properties, and is considered to be an ideal drug for the treatment of AD. However, its hydrophobicity greatly limits its application in biomedicine. 2 CeO NPs) have the properties of both superoxide dismutase (SOD) and catalytic enzyme (CAT) and are considered to be excellent biological antioxidant nanomaterials. 2 NPs can remove cytotoxic ROS (such as singlet oxygen 1 O 2 and hydrogen peroxide H 2 O 2 ) is converted into non-toxic H 2 O and O 2 . Especially small-sized CeO 2 NPs, because of their larger specific surface area, have stronger antioxidant capacity.

[0004] However, in current research on AD, the complex brain environment and blood-brain barrier (BBB) ​​limit the penetration and utilization of most drug molecules and small-particle nanomaterials in the brain, and the difficulty of drug distribution in the brain remains a difficulty in treating AD. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a dual-functional nanoprobe CICe@MK.

[0006] The technical problem that the present invention needs to solve is to provide a method for preparing the above-mentioned dual-functional nanoprobe.

[0007] The final technical problem to be solved by the present invention is to provide the application of the above-mentioned dual-functional nanoprobe in the treatment of Alzheimer's disease.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a bifunctional nanoprobe. The bifunctional nanoprobe is CICe@MK, which includes curcumin and IR780 iodide loaded in mesoporous silica / cerium dioxide composite nanospheres, and K peptide connected to the outer layer of the mesoporous silica / cerium dioxide composite nanospheres; the sequence of the K peptide is CKLVFFAED.

[0009] Mesoporous silica (MSNs), as an inorganic porous material, has excellent drug loading capacity and can load small molecule drugs into its pores, thereby improving the ability of small molecule drugs to cross the blood-brain barrier (BBB). At the same time, MSNs are non-toxic, have good biocompatibility, and are easy to functionalize on the surface.

[0010] CICe@MK can synergistically act on Alzheimer's disease through two different pathways: (1) K peptide and Cur target Aβ and inhibit Aβ aggregation; (2) CeO 2 NPs and Cur synergistically scavenge ROS and relieve brain oxidative stress.

[0011] The present invention also provides a method for preparing the above-mentioned dual-functional nanoprobe, comprising the following steps:

[0012] (1) dissolving the amino-modified mesoporous silica nanoparticles and the carboxy-modified cerium dioxide nanoparticles in a DMF solution and mixing them evenly. After the reaction is completed, a composite nanosphere solution is obtained, which is denoted as Ce@M.

[0013] In this step, the amination-treated mesoporous silicon nanoparticles and the carboxyl-treated cerium dioxide nanoparticles are compounded through an amide reaction to obtain Ce@M.

[0014] (2) Curcumin, IR780 iodide powder and Ce@M solution were mixed evenly to obtain Ce@M nanospheres loaded with drugs in the inner cavity, denoted as CICe@M;

[0015] (3) The K peptide was connected to CICe@M via Sulfo-SMCC to obtain the bifunctional nanoprobe CICe@MK; the sequence of the K peptide was CKLVFFAED.

[0016] Furthermore, step (1) also includes the steps of hydrolyzing tetraethyl silicate to prepare solid mesoporous silicon nanoparticles and aminating the same.

[0017] Furthermore, the step of preparing solid mesoporous silicon includes: the step of preparing solid mesoporous silicon includes: mixing hexadecyltrimethylammonium chloride, triethylamine and ultrapure water to obtain a mixed solution, adding tetraethyl silicate, centrifuging after the reaction is completed, removing the hexadecyltrimethylammonium chloride, and obtaining a precipitate of solid mesoporous silicon nanoparticles.

[0018] Furthermore, the reaction temperature of the mixed solution is 95°C and the reaction conditions are 1000 rpm min -1 Stir for 1 hour.

[0019] Furthermore, step (1) also includes the steps of preparing cerium dioxide nanoparticles and carboxylating the cerium dioxide nanoparticles. The cerium dioxide nanoparticles are prepared from oleylamine and have a diameter of about 3 nm.

[0020] Furthermore, the step of preparing cerium dioxide nanoparticles includes: adding sodium acetate and oleylamine to p-xylene and vigorously stirring at room temperature for 24 hours to obtain a mixed solution, heating it to 90° C., adding ultrapure water, and aging for 3 hours.

[0021] Furthermore, the mixing condition in step (2) is to react for 24 hours under light-proof and oscillating conditions.

[0022] Furthermore, the specific steps of step (3) include: adding Sulfo-SMCC to CICe@M, and obtaining CICe@MS after the reaction is completed; and then adding K peptide to CICe@MS, and obtaining CICe@MK after the reaction is completed.

[0023] The present invention also provides the use of the dual-function nanoprobe in preparing drugs or reagents for treating Alzheimer's disease.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the bifunctional nanoprobe CICe@MK provided by the present invention can synergistically treat Alzheimer's disease from two different pathways, while inhibiting Aβ aggregation and alleviating oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The characterization results of nanomaterials are shown in Figure 1, where A is the MSNs modified amino group (-NH 2 ) before and after; B is the infrared spectrum of MSNs modified with amino groups (-NH 2 ) before and after DLS analysis; C is MSNs, MSNs-NH 2 BMPA-CeO 2 , Zeta potential analysis of Ce@M; D is the HRTEM image of MSNs; E is the CeO 2HRTEM image of NPs; F is the HRTEM image of Ce@M; G is the STEM image of Ce@M; H is the EDX elemental mapping of Si element; I is the EDX elemental mapping of Ce element. Data are expressed as mean ± standard deviation.

[0026] Figure 2 The results of the in vitro validation of the nanoprobe inhibiting Aβ aggregation are shown in Figure 1, where A is the time curve of Aβf formation; B is the fluorescence spectrum of CICe@MK inhibiting Aβ aggregation; C is the ThT fluorescence intensity of CICe@MK inhibiting Aβ aggregation; D is the STEM image of CICe@MK inhibiting Aβ aggregation. Data are expressed as mean ± standard deviation. ****p<0.0001.

[0027] Figure 3 Figure 1 shows the results of the biocompatibility and antioxidant capacity tests of CICe@MK, where A is the survival rate of PC12 cells as the probe concentration changes; B is the survival rate of PC12 cells as the probe incubation time changes; C is the survival rate of PC12 cells as the probe H changes. 2 O 2 The survival rate of the cells was determined by the change of concentration; D is the fluorescence confocal image of the antioxidant capacity test of CICe@MK; E is the flow cytometry diagram for quantitative monitoring of PC12 cell apoptosis (the experimental subjects of Figure D / E were control (PC12 cell fluid), H 2 O 2 、CICe@MK、H 2 O 2 +CICe@MK and H 2 O 2 +CICe@M-K+NIR treated and incubated PC12 cells).

[0028] Figure 4 The results of the nanoprobes alleviating Aβo-induced oxidative damage in PC12 cells were measured (the experimental subjects were PC12 cells treated and incubated with control (PC12 cell fluid), Aβo, Aβo+CICe@MK, and Aβo+CICe@M-K+NIR), where A is the fluorescence confocal image of Aβo-induced ROS production in PC12 cells; B is the flow cytometry image of Aβo-induced ROS production in PC12 cells; C is the DCF fluorescence value of Aβo-induced ROS production in PC12 cells; D is the result of Aβo-induced changes in mitochondrial membrane potential in PC12 cells; E is the flow cytometry image of quantitative monitoring of PC12 cell apoptosis. The scale bar is 50μm. The data are expressed as mean ± standard deviation. ****p<0.0001.

[0029] Figure 5The results of the nanoprobe crossing the BBB are shown in Figure 1. A is the ELISA kit to determine the targeting of K peptide to Aβo; B is a schematic diagram of the blood-brain barrier constructed by simulating the BBB in vitro; C is a fluorescence confocal image of the penetration efficiency of CICe@M and CICe@MK crossing the BBB. The scale bar is 50μm. The data are expressed as mean ± standard deviation.

[0030] Figure 6 The figures are related results of in vivo Caenorhabditis elegans analysis, among which A is the ThT staining image of Aβo in the N2 wild-type strain; B is the ThT staining image of Aβo in the CL 2006 strain; C is the ThT staining image of Aβo in the CL 2006+CICe@MK strain; D is the ThT staining image of Aβo in the CL 2006+CICe@M-K+NIR strain (arrows indicate Aβo plaques); E is the survival curve of the CL2006 strain. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0032] Reagents and instruments used in this experiment:

[0033] Hexadecyltrimethylammonium chloride (CTAC), tetraethyl silicate (TEOS), 3-aminopropyltriethoxysilane (APTES), sodium acetate, p-xylene (98.5%), and 2-bromoisobutyric acid (BMPA) were purchased from Aladdin (China). Sodium acetate, sodium acetate, oleylamine, and N,N-dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co., Ltd. IR780 iodide, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC), and curcumin (Cur) were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Thioflavin T (ThT) was purchased from Sigma-Aldrich (Beijing, China). CCK-8 kit and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-pyridocarbocyanine iodide (JC-1) detection kit were provided by Biotechnology Co., Ltd. (Shanghai, China). K peptide (sequence: CKLVFFAED) was purchased from GenScript Biotech Co., Ltd. (Nanjing, China). Aβ 42 and Aβ 42 The ELISA kit was provided by Sangon Biotech Co., Ltd. (Shanghai, China) (the Aβo used in the examples is Aβ 42Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 2',7'-dichlorofluorescein diacetate (DCFH-DA) and Annexin V-FITC / PI apoptosis detection kit were purchased from Nanjing Keygene Biotechnology Development Co., Ltd.

[0034] Talos F200X high-resolution transmission electron microscope (Thermo Fisher Scientific, USA), multi-angle particle size analyzer (NanoBrook Omni, Brookhaven, USA), fluorescence spectrophotometer (fluormax-4, HORIBA Jobin Yvon, Japan), Fourier transform infrared spectrometer (NICOLET iS10, Thermo Fisher Scientific, USA), confocal laser scanning microscope (CLSM, fluviewtm FV1000, Olympus, Japan), microplate reader (Multiskan, Thermo Fisher Scientific, USA), and flow cytometer (BD Bioscience, USA).

[0035] The pH of PBS in all embodiments of the present invention is 7.4, with 137 mM NaCl, 10 mM NaH 2 PO 4 , 2.7 mM KCl, 2 mM KH 2 PO 4 The pH of the nematode growth medium (NGM) was 6.0, and 1 mM CaCl 2 、1mMMgSO 4 , 5mg L -1 Cholesterol, 250 mM KH 2 PO 4 , 17g L -1 Agar, 2.5 g L -1 Peptone and 3 g L -1 The remaining solutions were dissolved in ultrapure water.

[0036] Example 1 Preparation and characterization of dual-function nanoprobes

[0037] 1. Preparation of dual-functional nanoprobes

[0038] 25% hexadecyltrimethylammonium chloride (CTAC, 2 g), triethylamine (TEA, 0.8 g) and ultrapure water (95°C, 20 mL) were added to a round-bottom flask and placed in a 95°C water bath at 1000 rpm. -1 Stir for 1 h at a speed of 200 mg min -11.5 mL of tetraethyl silicate (TEOS) was injected at a speed of 1000 rpm, and stirring was continued for 1 hour, followed by centrifugation (1000 rpm, 10 min). The precipitate was washed three times with ethanol and ultrapure water respectively, and then dispersed with a mixed solution of hydrochloric acid and ethanol (volume ratio 1:10) and refluxed for 12 hours (80 ° C) to remove the template CTAC. Solid mesoporous silicon nanoparticles (MSNs) were prepared. 25 mg of solid MSNs and 100 μL of 3-aminopropyltriethoxysilane (APTES) were added to 25 mL of ethanol solution, refluxed for 4 hours (80 ° C), and the product was collected by centrifugation (13000 rpm, 5 min) to obtain aminated MSNs (MSNs-NH 2 ).

[0039] 0.43 g of sodium acetate and 3.2 g of oleylamine were added to 15 mL of p-xylene and stirred vigorously for 24 h. The mixed solution was slowly heated to 90 °C and 1 mL of ultrapure water was quickly added. The mixture was aged at 90 °C for 3 h. The product (CeO 2 NPs). 0.5 g 2-bromoisobutyric acid (BMPA), 0.05 g sodium acetate and 15 mg CeO 2 NPs were added to a mixed solution of 15 mL of N,N-dimethylformamide (DMF) and chloroform (volume ratio 1:1) and stirred in a 30 °C water bath for 6 h to obtain the product of carboxylated cerium dioxide nanoparticles BMPA-CeO 2 NPs.

[0040] 5 mg MSNs-NH 2 and 15 mg BMPA-CeO 2 NPs were dissolved in DMF and stirred for 3 h. The product (Ce@M) was collected by centrifugation (1000 rpm, 10 min) and washed three times with water and ethanol, respectively.

[0041] 3 mg Ce@M, 60 mL Cur (10 mg mL -1 ) and 15 mL IR780 (1 mg mL -1 ) was added to 3 mL PBS and shaken in the dark for 12-24 h. Excess Cur and IR780 were removed by centrifugation (1000 rpm, 10 min) to obtain the product CICe@M.

[0042] 5 μL Sulfo-SMCC (20 mg mL -1 ) was added to 100 μL CICe@M (1 mg mL -1 ) and after shaking for 2 h, 100 μL of K peptide solution (1 mg mL -1), and continued shaking at 4°C for 12 h. The bifunctional nanoprobe CICe@MK was obtained by centrifugation (1000 rpm, 10 min).

[0043] 2. Characterization of CICe@MK

[0044] Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) were used to characterize the 2 Characterized ( Figure 1 A and 1B). The particle size and morphology of the prepared dual-functional nanoprobes and intermediates were characterized by high-resolution transmission electron microscopy (HRTEM). 2 The diameter is about 60nm ( Figure 1 D) CeO 2 The diameter of NPs is about 3 nm ( Figure 1 E). The size of the composite nanoparticles formed is slightly larger than that of MSNs-NH 2 Size( Figure 1 F). The modification process of CICe@MK was verified by zeta potential measurement, scanning transmission electron microscopy (STEM), and EDX spectroscopy ( Figure 1 C, 1G, and 1H-I).

[0045] Example 2 Evaluation of Nanoprobes on Inhibition of Aβ Aggregation in Vitro

[0046] The K peptide CKLVFFAED used in the present invention is a peptide that can target Aβ and further inhibit Aβ aggregation.

[0047] Cur is a hydrophobic small drug molecule with antioxidant capacity that can target Aβ and further inhibit Aβ aggregation.

[0048] IR780 is a photosensitizer that can generate heat under 808 nm near-infrared (NIR) light irradiation to promote the release of Cur from MSNs.

[0049] ThT is a small fluorescent molecule with very weak fluorescence itself. It can bind to Aβ aggregates and emit strong fluorescence, but has weak binding ability to Aβ monomers and hardly emits light.

[0050] In this example, the formation time of Aβ aggregate Aβf was first measured. 42 Incubate in a 37°C water bath ( Figure 2 A), the results showed that Aβ aggregates can be formed within 48 hours. This example uses the classic thioflavin T (ThT) method to determine the ability of CICe@MK to inhibit Aβ aggregation. Aβ and Aβ+CICe@MK were co-incubated at 37°C for 48 hours, and then ThT was added. 42), CICe@MK has a significant ability to inhibit Aβ aggregation. Meanwhile, NIR irradiation is beneficial to the release of Cur, which further enhances the ability of CICe@MK to inhibit Aβ aggregation ( Figure 2 B and 2C). STEM imaging also shows that CICe@MK has the ability to inhibit Aβ aggregation, and NIR irradiation can enhance the inhibition of CICe@MK on Aβ aggregation.

[0051] Example 3 Determination of ROS scavenging by nanoprobes in vitro

[0052] In this example, PC12 cells were selected as a model to verify the scavenging effect of CICe@MK on ROS. The concentrations were 25 μg mL -1 , 50 μg mL -1 , 100 μg mL -1 , 150 μg mL -1 The Cur and CICe@MK solutions were used for cytotoxicity determination. CCK-8 was selected as a live cell indicator. The more and faster the cells proliferated, the darker the color; the greater the cytotoxicity, the lighter the color. Figure 3 As shown in A and 3B, Cur and CICe@MK were less toxic to PC12.

[0053] This embodiment also 2 O 2 The concentrations of H were screened. The concentrations of H were 50μM, 100μM, 150μM, 200μM, and 300μM. 2 O 2 , co-cultured with PC12 cells, when H 2 O 2 When the concentration reaches 200 μM, the cell survival rate will be less than 80% ( Figure 3 C).

[0054] This example also uses confocal microscopy to evaluate the ability of CICe@MK to remove ROS at the cellular level. DCFH-DA is a ROS-sensitive probe, usually used to measure the level of ROS in cells. DCFH-DA itself has no fluorescence. After entering the cell, it can be hydrolyzed by esterase in the cell to generate DCFH, and then the ROS in the cell oxidizes the non-fluorescent DCFH into fluorescent DCF. Therefore, the higher the intracellular ROS content, the greater the DCF fluorescence intensity. The experimental subjects used control (PC12 cell fluid), H 2 O 2 、CICe@MK、H 2 O 2 +CICe@MK and H 2 O 2+CICe@M-K+NIR treated and incubated PC12 cells. Figure 3 D shows that compared with the control group, H 2 O 2 The fluorescence signal of the group was significantly enhanced, but after adding CICe@MK, the green fluorescence of DCF was greatly reduced. 2 O 2 The fluorescence of the +CICe@M-K+NIR group was further reduced, indicating that the dual-functional nanoprobe CICe@MK has good ROS scavenging ability. At the same time, the cell apoptosis experiment also proved this point ( Figure 3 E). Annexin V-FITC / PI apoptosis detection kit was used to conduct apoptosis experiments. Annexin V-FITC can enter the cytoplasm of necrotic cells and bind to phosphatidylserine on the inner side of the cell membrane, thus making the necrotic cells show green fluorescence.

[0055] Example 4 Alleviating Aβo-induced neuronal cell damage

[0056] The misfolding and abnormal aggregation of Aβ (forming Aβo) will cause the generation of ROS in the brain. Conversely, the increase of ROS will aggravate the misfolding and abnormal aggregation of Aβ, thereby aggravating the occurrence of AD. In this example, DCFH-DA was also selected as an indicator of ROS. The intensity of DCF fluorescence was used to judge the amount of ROS in neuronal cells, thereby evaluating the ROS scavenging ability and Aβ aggregation inhibition ability of CICe@MK. PC12 cells were treated and incubated with control, Aβo, Aβo+CICe@MK and Aβo+CICe@M-K+NIR, respectively. From Figure 4 A It can be seen that Aβo can indeed induce an increase in intracellular ROS, while CICe@MK can inhibit this increase. After NIR irradiation, the fluorescence of DCF further decreased, indicating that near-infrared light is conducive to the release of Cur. At the same time, the measurement results of flow cytometry can also prove that CICe@MK can reduce the neuronal cytotoxicity induced by Aβo ( Figure 4 B and 4C).

[0057] In addition, mitochondrial membrane potential (MMP) can also be used to judge cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-pyridocarbocyanine iodide (JC-1) is selected as an indicator. When the cells are not damaged, MMP is in a normal state, JC-1 is aggregated and appears red; when the cells are damaged, MMP is abnormal, JC-1 exists in monomeric form and appears green. Figure 4D shows that the MMP of the control group is normal and appears red under a confocal microscope; when Aβo exists, the cell MMP is abnormal and mainly appears green; when CICe@MK is added, the green color weakens and the red color strengthens, indicating that CICe@MK can alleviate the cell damage caused by Aβo; after NIR irradiation, the damage is further alleviated, the green fluorescence is weaker and the red fluorescence is stronger. At the same time, the apoptosis data obtained by flow cytometry can also prove that CICe@MK has the ability to alleviate Aβo-induced neuronal cell damage ( Figure 4 E).

[0058] Example 5 Determination of Nanoprobe Crossing the Blood-brain Barrier (BBB)

[0059] In this example, Aβ 42 The enzyme-linked immunosorbent assay kit explored the targeting ability of K peptide CKLVFFAED to Aβ. The concentrations were 10 mg mL -1 , 20mg mL -1 , 30mg mL -1 and 40 mg mL -1 The CICe@MK and CICe@M solutions were used for the determination. Figure 5 As shown in A, the capture rate of Aβo in CICe@MK group was much higher than that in CICe@M group, and the capture rate of Aβo in CICe@MK group was significantly higher than that in CICe@M group. -1 , the capture rate of Aβo by CICe@MK continued to increase.

[0060] In this example, an in vitro BBB model was constructed using brain endothelial cells bEnd.3 and neuronal cells PC12 ( Figure 5 B), a dense layer of bEnd.3 cells was cultured in the upper layer of the Transwell chamber to simulate the BBB, and PC12 cells were co-cultured in the lower layer. FAM-labeled nanoprobes CICe@MK or CICe@M were added to the upper culture medium, and the fluorescence intensity of FAM in the PC12 cells in the lower layer was detected to determine their ability to cross the BBB. The results are shown in Figure 5 As shown in C, the fluorescence intensity of the CICe@MK group was significantly higher than that of the CICe@M group, indicating that the modification of K peptide CKLVFFAED is beneficial for the nanoprobe to cross the BBB.

[0061] Example 6 In vivo evaluation using Caenorhabditis elegans

[0062] CL2006 is a transgenic Caenorhabditis elegans that expresses human Aβ specifically in muscle tissue under 20°C temperature induction. 42 , Aβ 42The accumulation of proteins in muscle tissue eventually leads to paralysis of the nematode. All genes of Caenorhabditis elegans have been sequenced and have a 70% similarity with the human genome. It is often used as a bridge from in vitro cell level to in vivo mouse level to screen drugs.

[0063] In this example, ThT was used to perform staining experiments on C. elegans CL2006, and wild-type C. elegans N2 was used as a control group. The experimental results are shown in Figure 6 As shown, compared with the wild-type N2 strain, the CL2006 strain presented more Aβo plaques ( Figure 6 B). After adding the nanoprobe CICe@MK, Aβo plaques were significantly reduced ( Figure 6 C). After drug release under NIR irradiation, the effect of CICe@MK was enhanced and Aβo plaques were further reduced ( Figure 6 D). At the same time, the paralysis experiment results also showed that CICe@MK can extend the lifespan of CL2006 strain from 12 days to 14 days; after NIR light release, it can be further extended to 17 days. The above results show that CICe@MK nanoprobe can effectively extend the lifespan of AD nematodes.

Claims

1. A dual-function nanoprobe, characterized in that: The bifunctional nanoprobe is CICe@MK, which includes curcumin and IR780 iodide loaded in mesoporous silica / cerium dioxide composite nanospheres, and K peptide connected to the outer layer of the mesoporous silica / cerium dioxide composite nanospheres; the sequence of the K peptide is CKLVFFAED; The mesoporous silicon / cerium dioxide composite nanospheres are prepared by the following method: aminated mesoporous silicon nanoparticles and carboxylated cerium dioxide nanoparticles are dissolved in a DMF solution, mixed evenly, and after the reaction is completed, a mesoporous silicon / cerium dioxide composite nanosphere solution is obtained.

2. The method for preparing the dual-functional nanoprobe according to claim 1, characterized in that: The following steps are involved: (1) Dissolving amino-modified mesoporous silica nanoparticles and carboxy-modified cerium dioxide nanoparticles in a DMF solution and mixing them evenly, and obtaining a mesoporous silica / cerium dioxide composite nanosphere solution after the reaction, which is denoted as Ce@M; (2) Curcumin, IR780 iodide powder and Ce@M solution were mixed evenly to obtain Ce@M nanospheres loaded with drugs in the inner cavity, denoted as CICe@M; (3) The K peptide was connected to CICe@M via Sulfo-SMCC to obtain the bifunctional nanoprobe CICe@MK; the sequence of the K peptide was CKLVFFAED.

3. The preparation method according to claim 2, characterized in that: Step (1) also includes the steps of hydrolyzing tetraethyl silicate to prepare solid mesoporous silicon nanoparticles and amminating the particles.

4. The preparation method according to claim 3, characterized in that: The steps of preparing solid mesoporous silicon include: mixing hexadecyltrimethylammonium chloride, triethylamine and ultrapure water to obtain a mixed solution, adding tetraethyl silicate, centrifuging after the reaction is completed, removing the hexadecyltrimethylammonium chloride, and obtaining a precipitate of solid mesoporous silicon nanoparticles.

5. The preparation method according to claim 4, characterized in that: The reaction temperature of the mixed solution is 90-100°C, and the reaction conditions are 1000-1500 rmp min -1 Stir for 1 hour or more.

6. The preparation method according to claim 2, characterized in that: Step (1) also includes the steps of preparing cerium dioxide nanoparticles and carboxylating the nanoparticles.

7. The preparation method according to claim 6, characterized in that: The step of preparing cerium dioxide nanoparticles comprises: adding sodium acetate and oleylamine to p-xylene and vigorously stirring at room temperature for more than 24 hours to obtain a mixed solution, heating until the temperature rises to above 90° C., adding ultrapure water, and aging for 3-4 hours.

8. The preparation method according to claim 2, characterized in that: The mixing conditions described in step (2) are to react for 12-24 hours under light-proof, oscillating and room temperature conditions.

9. The preparation method according to claim 2, characterized in that: The specific steps of step (3) include: adding Sulfo-SMCC to CICe@M, and obtaining CICe@MS after the reaction is completed; and then adding K peptide to CICe@MS, and obtaining CICe@MK after the reaction is completed.

10. Use of the dual-functional nanoprobe according to claim 1 in the preparation of drugs or reagents for treating Alzheimer's disease.

Images