Drug-loaded nano-micelle based on tobacco active substances as well as preparation method and application of drug-loaded nano-micelle

Abstract

The invention discloses a drug-loaded nano-micelle based on tobacco active substances as well as a preparation method and application of the drug-loaded nano-micelle. The nano-micelle comprises a solanesol-polyethylene glycol nano-micelle as well as an active small molecule drug and a photosensitizer which are loaded in the solanesol-polyethylene glycol nano-micelle, and the polypeptide is connected outside the solanesol-polyethylene glycol nano-micelle. The drug-loaded nano-micelle based on the tobacco active substance can be used for loading small molecule drugs, the problems that the bioavailability is low, blood brain barriers are difficult to pass through and the like are solved, the micelle is easy to functionalize, and the drugs can act together from multiple ways. The invention further provides a specific medicine for treating the Alzheimer's disease, the medicine can act in two different ways to treat the Alzheimer's disease in a synergistic mode, meanwhile, the effects of reducing A beta plaques and reducing inflammation are achieved, and the application range of tobacco active molecules in treatment of neurodegenerative diseases is widened.

Description

Technical Field

[0001] The invention relates to a drug-loaded nano micelle based on tobacco active substances, a preparation method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Alzheimer's disease (AD) is a multi-cause, multi-stage neurodegenerative disease with cognitive, language, and motor dysfunction and memory loss as its main clinical features. It is a world-recognized death disease second only to cancer, stroke, and cardiovascular disease. According to the International Alzheimer's Association, the number of AD patients worldwide is expected to climb to 152 million in 2050. AD is related to age and mainly occurs in the elderly population over 65 years old. With the increase of age, the incidence of AD increases linearly, which seriously affects the physical health and quality of life of patients, and also brings a heavy economic burden to the family and society. Therefore, AD has always been a research hotspot in the field of international neurological diseases. Due to the complexity of pathogenic factors, the pathogenesis of AD has not yet been determined. At present, the main hypotheses about the pathogenesis of AD are: β-amyloid protein (Aβ) hypothesis, oxidative stress hypothesis, metal ion hypothesis, etc. In recent years, based on these hypotheses, inhibiting Aβ aggregation, accelerating Aβ clearance, and alleviating oxidative stress have become the research focus of AD treatment.

[0003] Tobacco is an annual herbaceous plant of the Solanaceae family native to America. In my country, tobacco is mainly used as an industrial raw material for cigarette production, but as my country's anti-smoking measures become increasingly stringent, the development of the tobacco planting industry is also restricted. In addition, about hundreds of thousands of tons of waste are produced each year during tobacco harvesting and cannot be used for cigarette production, causing a huge waste of available resources. Therefore, it is of great significance for tobacco to develop in the direction of food, health products, medicine, beverages, cosmetics and other industries, expand the effective utilization of tobacco, improve the comprehensive utilization efficiency of its waste, and give play to the potential value of these wastes. Studies have shown that the chemical composition of tobacco is relatively complex. At present, 5,229 compounds have been found in tobacco, including alkaloids, proteins, terpenes, sugars, aromatics and other substances, many of which are important biochemical and pharmaceutical raw materials with a wide range of uses and high economic value.

[0004] The treatment of Alzheimer's disease mostly relies on antioxidant active substances, and tobacco active substances such as chlorogenic acid and solanesol have significant antioxidant and anti-inflammatory effects. Chlorogenic acid is a tannin active ingredient currently found in tobacco in high content and is an effective antioxidant. Chlorogenic acid and its derivatives have stronger free radical scavenging ability than ascorbic acid, tocopherol and caffeic acid, and can relieve oxidative stress. Solanesol is widely present in tobacco leaves and can be extracted from tobacco waste. It is an unsaturated polyisoprene alcohol containing multiple non-conjugated double bonds, so it has very excellent free radical absorption performance, and has biological activities and medicinal value such as antibacterial, anti-inflammatory and antioxidant. Therefore, it is of great significance to carry out research on the synergistic application of chlorogenic acid and solanesol, antioxidant active molecules in tobacco, in the treatment of neurodegenerative diseases.

[0005] Tobacco active molecules such as chlorogenic acid have poor water solubility and low bioavailability, making it difficult for them to cross the blood-brain barrier and enter the lesion area. Moreover, these active small molecules themselves cannot target damaged neuronal cells, Aβ monomers or aggregates, etc., which greatly limits the application of tobacco active molecules in the treatment of neurodegenerative diseases. Therefore, it is of great significance to prepare multifunctional nanomaterials for delivering these active molecules, improve their stability and utilization in the body through functional modification, and study their synergistic application in the treatment of AD. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a drug-loaded nano-micelle based on tobacco active substances which is non-toxic, has good biocompatibility and can be easily functionalized on the surface.

[0007] Technical solution: The present invention provides a drug-loaded nanomicelle based on tobacco active substances, wherein the nanomicelle comprises solanesol-polyethylene glycol nanomicelles, active small molecule drugs and photosensitizers loaded in the solanesol-polyethylene glycol nanomicelles, and polypeptides connected to the outside of the solanesol-polyethylene glycol nanomicelles.

[0008] Furthermore, the photosensitizer is IR780 iodide.

[0009] The present invention also provides a method for preparing the above-mentioned drug-loaded nanomicelles, comprising the following steps:

[0010] (1) Solanesol, aminopolyethylene glycol carboxyl, N,N-dicyclohexylcarboximide and 4-dimethylaminopyridine are dissolved in dichloromethane, and after the reaction is completed, the solvent-polyethylene glycol nano-micelles, i.e., Sol-PEG, are obtained;

[0011] (2) Sol-PEG, active small molecule drugs and photosensitizer powder were dissolved in acetone and added dropwise into ultrapure water to obtain CI@SP;

[0012] (3) The peptide was connected to CI@SP via Sulfo-SMCC to obtain the drug-loaded nanomicelle CI@SPK.

[0013] Furthermore, the reaction conditions of step (1) are stirring at room temperature for 48 hours.

[0014] Furthermore, the reaction conditions of step (2) are stirring and volatilizing at 37° C. for 24 hours.

[0015] Furthermore, the specific steps of (3) include: mixing CI@SP and Sulfo-SMCC, stirring at 37°C for 2 hours; adding the polypeptide solution, and stirring for another 12 hours; and obtaining CI@SPK by dialysis and freeze-drying.

[0016] The present invention also provides the use of the drug-loaded nano-micelles in preparing drugs or reagents for treating Alzheimer's disease.

[0017] Furthermore, the concentration of the drug-loaded nanomicelles is 20 to 50 μg mL -1 .

[0018] The present invention also provides a drug for treating Alzheimer's disease, the drug comprises the above-mentioned drug-loaded nano micelles, the active small molecule drug is chlorogenic acid, and the polypeptide is K peptide targeting Aβ.

[0019] Furthermore, the sequence of the K peptide is CKLVFFAED.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the drug-loaded nano-micelles based on tobacco active substances of the present invention can be used to load small molecule drugs, improve the problems of low bioavailability and difficulty in crossing the blood-brain barrier, and the surface of the micelles is easy to functionalize, which can make the drugs act together from multiple pathways. The present invention also provides a specific drug for treating Alzheimer's disease, which can act from two different pathways to synergistically treat Alzheimer's disease, and at the same time play the role of reducing Aβ plaques and reducing inflammation, broadening the application of tobacco active molecules in the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The characterization and photothermal drug release performance results of nanomicelles, where A is NH2-PEG 4000-COOH, Sol and Sol-PEG infrared spectra; B is the UV-visible absorption spectra of SP and CI@SP (the inset is the photo of the corresponding solution); C is the efficiency of CI@SP and CI@SPK in capturing Aβo; D is the STEM imaging of CI@SPK; E is the thermal imaging of PBS, C@SPK, IR780 and CI@SPK before and after 808nm laser irradiation; F is the heating curve and corresponding cooling curve of CI@SPK within 180s under 808nm laser irradiation. Data are expressed as mean ± standard deviation.

[0022] Figure 2 The results of the nanomicelles inhibiting Aβ aggregation are shown in Figure 1, where A is the fluorescence spectrum of ThT under the action of different concentrations of CI@SPK; B is the fluorescence intensity corresponding to Figure A; C is the STEM imaging of Aβm after incubation in a 37°C water bath for 48 hours; D is the STEM imaging of Aβm+CI@SPK after incubation in a 37°C water bath for 48 hours. Data are expressed as mean ± standard deviation.

[0023] Figure 3 Figure 1 is a graph showing the relevant results of the antioxidant capacity verification of nanomicelles, where A is the UV absorption spectrum for verifying the removal of ·OH by CI@SPK (the inset is a photo of the corresponding solution); B is the UV absorption spectrum for verifying the removal of O2· - UV absorption spectrum of (the inset is a photograph of the corresponding solution). Data are expressed as mean ± standard deviation. ****p<0.0001.

[0024] Figure 4 The results of the nanomicelles crossing the blood-brain barrier are shown in Figure 1. A is the confocal image of CI@SP and CI@SPK crossing the blood-brain barrier; B is the fluorescence intensity of the corresponding confocal image of PC12 cells; B is the fluorescence intensity of the corresponding confocal image of BV2 cells. The data are expressed as mean ± standard deviation.

[0025] Figure 5 The results of the nano-micelles in alleviating Aβo-induced neuronal cell damage are shown in Figure 1. A is a confocal image of ROS in PC12 cells under different treatment conditions; B is a flow cytometry image of ROS in PC12 cells under different treatment conditions; C is the fluorescence intensity corresponding to Figure B; D is a flow cytometry image of PC12 cell apoptosis after different treatment conditions; E is a result of the change of mitochondrial membrane potential of PC12 cells under different treatment conditions. Data are expressed as mean ± standard deviation. ****p<0.0001, ***p<0.001, **p<0.01.

[0026] Figure 6The results of the verification of the ability of nano-micelles to regulate BV2 cell morphology and reduce inflammation are shown in Figure 1. A is a confocal image of BV2 cell morphology under different treatment conditions; B is a graph showing the results of TNF-α content detection in BV2 cell culture medium under different treatment conditions; C is a graph showing the results of IL-1β content detection in BV2 cell culture medium under different treatment conditions; D is a graph showing the results of IL-6 content detection in BV2 cell culture medium under different treatment conditions. Data are expressed as mean ± standard deviation. ***p<0.001, **p<0.01. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] Reagents and instruments used in this experiment:

[0029] N,N'-Dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, sodium bicarbonate, sodium chloride, and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Solanesol (Sol), NH2-PEG 4000 -COOH, chlorogenic acid (CHA), IR780 iodide, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), and methylene blue were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Thioflavin T (ThT) was purchased from Sigma-Aldrich (Beijing, China). K peptide (sequence: CKLVFFAED) was purchased from GenScript Biotech Co., Ltd. (Nanjing, China). Aβ42 peptide and Aβ42 enzyme-linked immunosorbent assay (ELISA) kit were purchased from Sangon Biotech (Shanghai) Co., Ltd. Cell counting kit-8 (CCK-8), reactive oxygen species detection kit, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyliminocyanine iodide (JC-1) detection kit, and total SOD activity detection (WST-8) kit were purchased from Shanghai Biotech Co., Ltd. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 2',7'-dichlorofluorescein diacetate (DCFH-DA), and AnnexinV-FITC / PI apoptosis detection kit were purchased from Jiangsu KeyGen Biotech Co., Ltd. (Nanjing, China). Mouse tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin-6 (IL-6) ELISA detection kits were purchased from Beijing Solebao Technology Co., Ltd. (Beijing, China).

[0030] Talos F200X high-resolution transmission electron microscope (Thermo Fisher Scientific, USA), fluorescence spectrophotometer (Fluoromax-4, Horiba, Japan), UV-visible spectrophotometer (Cary 100, Agilent, Singapore), laser confocal scanning microscope (FluoViewTM FV1000, Olympus, Japan), Fourier transform infrared spectrometer (NICOLET iS10, Thermo Fisher Scientific, USA), Vacuo-Temp small vacuum heating dryer (Selecta, Xunran Instruments, China), FDU-1200 freeze dryer (EYELA, Japan), rotary evaporator (IKA, Germany), flow cytometer (BD FACSCalibur, BD Biosciences, USA), and thermal imager (UTi260A, UNI-T, Fluke, USA).

[0031] The pH of the methylene blue buffer solution in the embodiment of the present invention is 7.2, and it is prepared by mixing sodium bicarbonate (25 mM) and sodium chloride (100 mM). The remaining solutions are all dissolved in ultrapure water.

[0032] Example 1 Preparation and characterization of co-assembled nanomicelles of solanesol and chlorogenic acid

[0033] 1. Preparation of co-assembled nanomicelles of solanesol and chlorogenic acid

[0034] Aminopolyethylene glycol carboxyl NH2-PEG 4000 -COOH (400 mg), solanesol (126.24 mg), N, N-dicyclohexylcarboximide (41.28 mg) and 4-dimethylaminopyridine (12 mg) were dissolved in dichloromethane (10 mL) and stirred at room temperature for 48 h. After the reaction was completed, the precipitate was removed by filtration under normal pressure. Ultrapure water (10 mL) was added for extraction, and the dichloromethane layer was taken. After rotary evaporation and vacuum drying, a light yellow product was obtained. The light yellow product was redissolved in anhydrous ethanol, and after dialysis, rotary evaporation and vacuum drying, a long chain of solanesol-polyethylene glycol polymer, i.e., Sol-PEG, was obtained.

[0035] Sol-PEG (20 mg), chlorogenic acid (20 mg) and IR780 (150 μg) were dissolved in acetone (2 mL) by ultrasound, then slowly added to ultrapure water (10 mL), evaporated at 37 ° C for 24 h, dialyzed and freeze-dried to obtain drug-loaded nanomicelles CI@SP. Similarly, Sol-PEG (20 mg) was dissolved in acetone (2 mL) by ultrasound, slowly added to ultrapure water (10 mL), evaporated at 37 ° C for 24 h, dialyzed and freeze-dried to obtain drug-free nanomicelles SP.

[0036] In CI@SP solution (1 mg mL -1, 4 mL) was added with Sulfo-SMCC (20 mg mL -1 , 200 μL) and stirred at 37°C for 2 h. Add K peptide solution (2 mg mL -1 , 2 mL), and stirred for 12 h. After dialysis and freeze-drying, the solanesol and chlorogenic acid co-assembled nanomicelles CI@SPK were obtained.

[0037] 2. Characterization and photothermal properties of co-assembled nanomicelles of solanesol and chlorogenic acid

[0038] The synthesis of solanesol-polyethylene glycol polymer long chain was characterized by Fourier transform infrared spectroscopy ( Figure 1 A). The UV-visible absorption spectra of drug-loaded nanomicelles CI@SP and unloaded nanomicelles SP were measured using a UV-visible spectrophotometer ( Figure 1 B). CI@SP has an obvious chlorogenic acid absorption peak at 326nm, while SP does not. CI@SP is green and SP solution is colorless, indicating that chlorogenic acid and IR780 are successfully loaded into the nanomicelles. The modification of K peptide was characterized by Aβ42 enzyme-linked immunosorbent assay (ELISA) kit ( Figure 1 C), with the increase of concentration, the capture efficiency of Aβo by the drug-loaded nanomicelles CI@SPK modified with K peptide gradually increased, while the capture efficiency of Aβo by the drug-loaded nanomicelles CI@SP without K peptide modification remained basically unchanged, indicating that K peptide has been successfully modified on CI@SPK. The morphological characteristics of the prepared drug-loaded nanomicelles CI@SPK were characterized by high-resolution transmission electron microscopy ( Figure 1 D), CI@SPK is spherical and well dispersed. The above characterization results show that CI@SPK has been successfully prepared. In addition, the photothermal performance of drug-loaded nanomicelles CI@SPK was verified ( Figure 1 E and F). In the present invention, IR780 is a photosensitizer that can generate heat under 808nm near-infrared (NIR) light to promote the release of chlorogenic acid from the nanomicelles. Figure 1 As shown in Figure E, loading IR780 into the nanomicelles does not affect their photothermal properties. When CI@SPK was irradiated with an 808nm NIR laser, the temperature of the CI@SPK solution quickly rose to about 50°C within 3 minutes. After the light was turned off, the temperature of the CI@SPK solution gradually decreased. This shows that the prepared drug-loaded nanomicelles have excellent photothermal properties.

[0039] Example 2 Determination of the inhibitory effect of solanesol and chlorogenic acid co-assembled nanomicelles on Aβ aggregation in vitro

[0040] The K peptide CKLVFFAED used in the present invention is a peptide that can target Aβ and inhibit its further aggregation. The inhibition of Aβ aggregation by CI@SPK was verified by fluorescence method. Thioflavin T (ThT) can specifically bind to the β-folded structure of Aβ aggregates and emit a strong fluorescence signal at 485nm. It is widely used as a fluorescent probe to monitor the Aβ aggregation process. Different concentrations of CI@SPK (7.5μL, final concentrations of 0, 20 and 50μg mL -1 ), Aβm (200μM, 40μL) and PBS (96.5μL) were mixed and reacted at 37°C for 48h. 6μL ThT (250μM) was mixed with the solution and reacted for 1h. The fluorescence intensity of the mixed solution was measured using a fluorescence spectrophotometer. Figure 2 As shown in A and B, CI@SPK has a good inhibitory effect on Aβ aggregation. STEM also shows that CI@SPK (50 μg mL -1 ) treatment, the filamentous Aβ aggregates were significantly reduced. These results indicate that CI@SPK has a good inhibitory effect on Aβ aggregation in vitro ( Figure 2 C and D).

[0041] Example 3 Determination of the antioxidant properties of solanesol and chlorogenic acid co-assembled nanomicelles.

[0042] The scavenging effects of CI@SPK on hydroxyl radicals (·OH) and superoxide anions (O2· - ) were tested for their performance in removing two reactive oxygen species (ROS). First, based on the fact that ·OH produced by the Fenton reaction can fade methylene blue, the in vitro ·OH scavenging performance of CI@SPK was tested. SP, CI@SP, CI@SPK, and CI@SPK solution irradiated with 808 nm (1 mg mL -1 Hydrogen peroxide (4M, 5μL), copper chloride (1mM, 2μL), methylene blue (0.1mM, 30μL) and methylene blue buffer solution (155μL) were added to the solution. After reacting at 37℃ for 2h, the absorption peak intensity at 664nm was recorded by UV-visible spectrometer. Figure 3 As shown in A, the unloaded solanesol nanomicelles SP showed weak ·OH scavenging performance, while CI@SP and CI@SPK showed excellent ·OH scavenging ability, indicating that the synergistic scavenging effect of chlorogenic acid and solanesol on ·OH was better than that of single solanesol nanomicelles. The CI@SPK solution after near-infrared light irradiation showed better ·OH scavenging effect than the unirradiated CI@SPK solution of the same concentration, which showed that more chlorogenic acid was released under the photothermal capacity of IR780, further improving the scavenging ability of ·OH. Based on the fact that WST-8 can react with the O2· generated by xanthine oxidase -The reaction produced a water-soluble formazan dye that turned the solution yellow. The total SOD activity detection kit was used to measure the in vitro clearance of O2· - SP, CI@SP, CI@SPK and CI@SPK solution irradiated with 808 nm laser (0.5 mg mL -1 , 20 μL) was mixed with 160 μL of WST-8 / enzyme working solution, and then 20 μL of reaction start working solution was added. After reacting at 37°C for 30 min, the absorbance at 450 nm was measured using a UV-visible spectrophotometer. Figure 3 As shown in B, the unloaded solanesol nanomicelles SP barely scavenged O2· - capability, while CI@SP and CI@SPK showed excellent O2 - The CI@SPK solution after near-infrared light irradiation showed better O2 scavenging ability than the CI@SPK solution without light irradiation of the same concentration. - These results indicate that the excellent photothermal drug release performance of CI@SPK can achieve ROS scavenging in vitro.

[0043] Example 4 Determination of the performance of co-assembled nanomicelles of solanesol and chlorogenic acid in crossing the blood-brain barrier (BBB)

[0044] First, brain endothelial cells bEnd.3 were cultured in the upper layer of the Transwell chamber, and neuronal cells PC12 or BV2 cells were cultured in the lower layer to construct an in vitro blood-brain barrier model. When a dense monolayer of cells had formed in the upper layer, CI@SP or CI@SPK was added to the upper culture medium and cultured for 24 hours. Because IR780 itself has red fluorescence, its ability to cross the BBB can be determined by detecting the fluorescence intensity of IR780 in the lower layer PC12 cells or BV2 cells. Fluorescence confocal images and fluorescence intensity are shown in Figure 2. Figure 4 As shown in AC, CI@SPK has obvious red fluorescence in the lower layer cells, and the fluorescence intensity is significantly higher than that of CI@SP. This indicates that CI@SPK modified with K peptide can effectively cross the BBB.

[0045] Example 5 Determination of the effect of co-assembled nanomicelles of solanesol and chlorogenic acid on alleviating Aβo-induced neuronal cell damage

[0046] Aβo stimulation will increase intracellular ROS, eventually leading to oxidative stress and neuronal cell damage. A reactive oxygen species detection kit was used to measure the ROS content in PC12 cells under different treatment conditions. DCFH-DA was used as a ROS indicator and emitted green fluorescence. Specifically, PC12 cells were stimulated with Aβo, Aβo+CI@SPK, and Aβo+CI@SPK+NIR, respectively. The Aβo concentration was 25 μM and the CI@SPK concentration was 50 μg mL-1 The Aβo and Aβo+CI@SPK groups were incubated at 37°C for 18 h, and the Aβo+CI@SPK+NIR group was first incubated for 6 h, then irradiated with NIR for 5 min, and then incubated for another 12 h. Figure 5 As shown in A, there is strong green fluorescence in the PC12 cells of the Aβo-treated group, indicating that Aβo stimulates PC12 cells to produce a large amount of ROS. The fluorescence intensity of the Aβo+CI@SPK-treated group was significantly reduced, and the fluorescence intensity further decreased after NIR irradiation. These results show that NIR irradiation can assist CI@SPK in effectively removing ROS generated by Aβo stimulation. In addition, the lower layer cells were collected and the fluorescence intensity of each group was measured by flow cytometry, and similar conclusions were obtained ( Figure 5 B and C). At the same time, the cell apoptosis rate under different treatments was determined using the Annexin V-FITC / PI double staining cell apoptosis detection kit. Figure 5 As shown in D, after CI@SPK+NIR treatment, the cell apoptosis rate dropped from 28.32% to 8.52%. Finally, the mitochondrial membrane potential test kit was used to further evaluate the degree of cell damage caused by Aβo stimulation. Among them, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-pyridocarbocyanine iodide (JC-1) was used as a fluorescent probe. When the cells were not damaged, the mitochondrial membrane potential (MMP) was normal, JC-1 was aggregated, and emitted red fluorescence; when the cells were damaged, MMP decreased, and JC-1 existed in monomeric form, emitting green fluorescence. Confocal imaging of MMP in PC12 cells is shown in Figure 1. Figure 5 As shown in E, bright green fluorescence appeared in PC12 cells stimulated by Aβo, indicating that the MMP of PC12 cells decreased and the cells were damaged. After co-incubation with CI@SPK, the green fluorescence was significantly weakened, indicating that the MMP of PC12 cells increased. After further NIR irradiation, the red fluorescence of JC-1 was further enhanced, indicating that JC-1 existed in an aggregated form, indicating that CI@SPK+NIR can alleviate Aβo-induced mitochondrial dysfunction. These results all indicate that CI@SPK under NIR irradiation can effectively alleviate Aβo-induced neuronal cell damage.

[0047] Example 6 Evaluation of the performance of co-assembled nanomicelles of solanesol and chlorogenic acid in regulating BV2 cell morphology and reducing inflammation

[0048] Increased inflammation can lead to overactivation of microglia (BV2 cells), which change from a normal phenotype (M2) to a pro-inflammatory phenotype (M1), resulting in impaired phagocytosis and degradation of Aβ. At the same time, M1 BV2 cells secrete a large amount of pro-inflammatory factors, leading to damage to neuronal cells. The morphology of BV2 cells under different treatment conditions was photographed using a laser confocal microscope. Specifically, BV2 cells were stimulated with Aβo, Aβo+CI@SPK, and Aβo+CI@SPK+NIR, respectively. The Aβo concentration was 25μM and the CI@SPK concentration was 50μgmL -1 The Aβo and Aβo+CI@SPK groups were incubated at 37°C for 18 h, and the Aβo+CI@SPK+NIR group was first incubated for 6 h, then irradiated with NIR for 5 min, and then incubated for 12 h. Figure 6 As shown in A, BV2 cells stimulated by Aβo were amoeba-like. After treatment with CI@SPK and CI@SPK+NIR, BV2 cells gradually recovered to their normal rounded shape. At the same time, the culture medium of BV2 cells under different treatment conditions was collected, and the contents of pro-inflammatory factors in the culture medium of different groups were measured using ELISA kits ( Figure 6 BD). As the therapeutic effect improved, the content of pro-inflammatory factors in the culture medium decreased significantly, indicating that CI@SPK can effectively reduce inflammation and restore the normal function of microglia.

Claims

1. A drug-loaded nano-micelle based on tobacco active substances, characterized in that: The nano micelles include solanesol-polyethylene glycol nano micelles, active small molecule drugs and photosensitizers loaded in the solanesol-polyethylene glycol nano micelles, and polypeptides connected outside the solanesol-polyethylene glycol nano micelles.

2. The drug-loaded nanomicelle based on tobacco active substances according to claim 1, characterized in that: The photosensitizer is IR780 iodide.

3. The method for preparing the drug-loaded nanomicelles according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Solanesol, aminopolyethylene glycol carboxyl, N,N-dicyclohexylcarboximide and 4-dimethylaminopyridine are dissolved in dichloromethane, and after the reaction is completed, the solvent-polyethylene glycol nano-micelles, i.e., Sol-PEG, are obtained; (2) Sol-PEG, active small molecule drugs and photosensitizer powder were dissolved in acetone and added dropwise into ultrapure water to obtain CI@SP; (3) The peptide was connected to CI@SP via Sulfo-SMCC to obtain the drug-loaded nanomicelle CI@SPK.

4. The preparation method according to claim 3, characterized in that: The reaction conditions of step (1) are stirring at room temperature for 48 hours.

5. The preparation method according to claim 3, characterized in that: The reaction conditions of step (2) are stirring and volatilizing at 37° C. for 24 h.

6. The preparation method according to claim 3, characterized in that: The specific steps of (3) include: mixing CI@SP and Sulfo-SMCC, stirring at 37°C for 2 h; adding the polypeptide solution, and stirring for another 12 h; and obtaining CI@SPK by dialysis and freeze-drying.

7. Use of the drug-loaded nanomicelles according to any one of claims 1 to 2 in the preparation of drugs or reagents for treating Alzheimer's disease.

8. The use according to claim 7, wherein the concentration of the drug-loaded nanomicelles is 20 to 50 μg mL -1 .

9. A drug for treating Alzheimer's disease, characterized in that: The drug comprises the drug-loaded nanomicelle according to any one of claims 1 to 2, the active small molecule drug is chlorogenic acid, and the polypeptide is K peptide targeting Aβ.

10. The drug according to claim 4, characterized in that The sequence of the K peptide is CKLVFFAED.

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