A tobacco active substance-based drug-loaded nanomicelle, a preparation method and application thereof

Abstract

The application discloses a drug-loaded nanomicelle based on tobacco active substances, a preparation method and application thereof, and the nanomicelle comprises solanols-polyethylene glycol nanomicelles, active small-molecule drugs and photosensitizers loaded in the solanols-polyethylene glycol nanomicelles, and polypeptides connected outside the solanols-polyethylene glycol nanomicelles. The drug-loaded nanomicelle based on tobacco active substances can be used for loading small-molecule drugs, improving the problems of low bioavailability and difficult crossing of the blood-brain barrier, the micelle surface is easy to be functionalized, and the drugs can jointly act from multiple channels. The application further provides a specific drug for treating Alzheimer's disease, the drug can act from two different channels, cooperatively treats Alzheimer's disease, simultaneously plays the effects of reducing Aβ plaques and reducing inflammation, and widens the application of tobacco active molecules in the treatment of neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The application relates to a tobacco active substance-based drug-loaded nanomicelle, a preparation method and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Alzheimer's disease (AD) is a multi-factorial, multi-stage neurodegenerative disease mainly characterized by cognitive, language, motor dysfunction and memory loss, and is the world's recognized death disease next to cancer, stroke and cardiovascular disease. According to the report of the International Alzheimer's Association, the number of AD patients worldwide is expected to rise to 152 million in 2050. AD is related to age and mainly occurs in the elderly over 65 years old, and the incidence of AD increases linearly with age, which seriously affects the physical health and quality of life of patients, and also brings heavy economic burden to families 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 hypotheses about the pathogenesis of AD mainly include: beta-amyloid (Aβ) hypothesis, oxidative stress hypothesis, metal ion hypothesis, etc. In recent years, based on these hypotheses, inhibiting Aβ aggregation, accelerating Aβ clearance and relieving oxidative stress have become the research focus of treating AD.

[0003] Tobacco is a Solanaceae annual herbaceous plant originally from America. In China, tobacco is mainly used as an industrial raw material for cigarette production, but with the increasingly strict measures of banning smoking in China, the development of tobacco planting industry is also restricted. In addition, about several hundred thousand tons of waste cannot be used for cigarette production during the tobacco harvesting process every year, which causes great waste of available resources. Therefore, it is of great significance to develop tobacco into food and health products, medicine, beverage, cosmetics and other industries, expand the effective utilization way of tobacco, improve the comprehensive utilization efficiency of the waste, and exert the potential value of the waste. Studies have shown that tobacco contains complex chemical components, and 5229 kinds of compounds have been found in tobacco, including alkaloids, proteins, terpenes, sugars, aromatic substances and the like, many of which are important biochemical and pharmaceutical raw materials and have wide application and high economic value.

[0004] The treatment of Alzheimer's disease depends on antioxidant active substances, and tobacco active substances such as chlorogenic acid and solanols have significant antioxidant and anti-inflammatory effects. Chlorogenic acid is an active component of tannin in tobacco, and is an effective antioxidant. Chlorogenic acid and its derivatives have stronger free radical scavenging capacity than ascorbic acid, tocopherol and caffeic acid, and can relieve oxidative stress. Solanols are widely present in tobacco leaves and can be obtained by extraction from tobacco waste. It is an unsaturated polyisoprene alcohol containing multiple non-conjugated double bonds, and therefore has excellent free radical absorption performance, and has antibacterial, anti-inflammatory and antioxidant biological activities and medicinal value. Therefore, it is of great significance to carry out research on the synergistic application of chlorogenic acid and solanols in the treatment of neurodegenerative diseases.

[0005] The water solubility of tobacco active molecules such as chlorogenic acid is poor, the bioavailability is low, it is difficult to cross the blood-brain barrier and enter the lesion area, and the active small molecules themselves cannot target damaged neurons, Aβ monomers or aggregates and other target points, 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, to improve their stability and utilization rate in the body through functional modification, and to study their synergistic application in the treatment of AD. SUMMARY

[0006] The purpose of the application is to provide a drug-loaded nanomicelle based on tobacco active substances, which is non-toxic, has good biocompatibility and is easy to functionalize on the surface.

[0007] Technical scheme: The application provides a drug-loaded nanomicelle based on tobacco active substances, which comprises a solanols-polyethylene glycol nanomicelle, an active small molecule drug and a photosensitizer loaded in the solanols-polyethylene glycol nanomicelle, and a polypeptide connected to the outside of the solanols-polyethylene glycol nanomicelle.

[0008] Further, the photosensitizer is IR780 iodide.

[0009] The application also provides a preparation method of the drug-loaded nanomicelle, comprising the following steps:

[0010] (1) dissolving solanols, amino-polyethylene glycol carboxyl, N,N-dicyclohexyl carbodiimide and 4-dimethylamino pyridine in dichloromethane, and obtaining a solanols-polyethylene glycol nanomicelle, namely Sol-PEG, after the reaction is completed;

[0011] (2) dissolving the Sol-PEG, the active small molecule drug and the photosensitizer powder in acetone, and adding dropwise into ultrapure water to obtain CI@SP;

[0012] (3) polypeptide is connected on CI@SP through Sulfo-SMCC, and drug-loaded nanomicelles CI@SPK is obtained.

[0013] Further, the reaction condition of step (1) is stirring at room temperature for 48h.

[0014] Further, the reaction condition of step (2) is stirring at 37℃ for 24h.

[0015] Further, the specific step of (3) comprises: mixing CI@SP and Sulfo-SMCC, stirring at 37℃ for 2h; adding polypeptide solution, and stirring for 12h; and obtaining CI@SPK through dialysis and freeze-drying.

[0016] The application further provides application of the drug-loaded nanomicelles in preparation of a medicament or reagent for treating Alzheimer's disease.

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

[0018] The application further provides a medicament for treating Alzheimer's disease, wherein the medicament comprises the drug-loaded nanomicelles, the active small molecule drug is chlorogenic acid, and the polypeptide is K peptide targeting Aβ.

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

[0020] Beneficial effects: compared with the prior art, the application has the following outstanding advantages: the drug-loaded nanomicelles based on tobacco active substances can be used for loading small molecule drugs, and the problems of low bioavailability and difficult crossing of blood-brain barrier are improved; the micelle surface is easy to functionalize, and the drugs can act from multiple paths. The application further provides a specific medicament for treating Alzheimer's disease, which can act from two different paths, cooperatively treat Alzheimer's disease, reduce Aβ plaque and inflammation, and widen the application of tobacco active molecules in treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Fig. 2 is a result graph for characterization and photothermal drug release performance of nanomicelles, wherein A is NH2-PEG 4000A. FTIR spectra of COOH, Sol and Sol-PEG; B. UV-Vis absorption spectra of SP and CI@SP (inset: photos of corresponding solutions); C. Efficiency of CI@SP and CI@SPK in capturing Aβo; D. STEM imaging of CI@SPK; E. Thermal imaging of PBS, C@SPK, IR780 and CI@SPK before and after 808 nm laser irradiation; F. Temperature rising curve and corresponding temperature dropping curve of CI@SPK under 808 nm laser irradiation within 180 s. Data were expressed as mean ± SD.

[0022] Figure 2 Related results of verification of the ability of nanomicelles to inhibit Aβ aggregation, wherein A. Fluorescence spectra of ThT under the action of different concentrations of CI@SPK; B. Fluorescence intensity corresponding to A; C. STEM imaging of Aβm after incubation in a 37℃ water bath for 48 h; D. STEM imaging of Aβm+CI@SPK after incubation in a 37℃ water bath for 48 h. Data were expressed as mean ± SD.

[0023] Figure 3 Related results of verification of the antioxidant ability of nanomicelles, wherein A. UV absorption spectrum for verification of the ability of CI@SPK to scavenge ·OH (inset: photo of corresponding solution); B. UV absorption spectrum for verification of the ability of CI@SPK to scavenge O2· - . Data were expressed as mean ± SD. ****p<0.0001.

[0024] Figure 4 Related results of determination of the ability of nanomicelles to cross the blood-brain barrier, wherein A. Confocal images of CI@SP and CI@SPK crossing the blood-brain barrier; B. Fluorescence intensity corresponding to the confocal images of PC12 cells; B. Fluorescence intensity corresponding to the confocal images of BV2 cells. Data were expressed as mean ± SD.

[0025] Figure 5 Related results of verification of the ability of nanomicelles to relieve Aβo-induced neuronal cell damage, wherein A. Confocal images of ROS in PC12 cells under different treatment conditions; B. Flow images of ROS in PC12 cells under different treatment conditions; C. Fluorescence intensity corresponding to B; D. Flow images of PC12 cell apoptosis after different treatment conditions; E. Results of changes in mitochondrial membrane potential of PC12 cells under different treatment conditions. Data were expressed as mean ± SD. ****p<0.0001, ***p<0.001, **p<0.01.

[0026] Figure 6The related result figures for verifying the performance of the nanomicelles in regulating the BV2 cell morphology and reducing inflammation. Among them, A is the confocal image of the BV2 cell morphology under different treatment conditions; B is the TNF-α content detection result figure in the BV2 cell culture medium under different treatment conditions; C is the IL-1β content detection result figure in the BV2 cell culture medium under different treatment conditions; D is the IL-6 content detection result figure in the BV2 cell culture medium under different treatment conditions. The data is represented by mean ± standard deviation. ***p<0.001, **p<0.01. DETAILED DESCRIPTION

[0027] The technical solutions of the present application are further described below in combination with the drawings.

[0028] Reagents and instruments used in the experiment:

[0029] N,N'-dicyclohexyl carbodiimide, 4-dimethylaminopyridine, dichloromethane, sodium bicarbonate, sodium chloride and acetone were purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd. (China, Shanghai). Solanoidol (Sol), NH2-PEG 4000 Chlorogenic acid (CHA), IR780 iodide, sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) and methylene blue were purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd. (China, Shanghai). Thioflavin T (ThT) was purchased from Sigma-Aldrich (China, Beijing). K peptide (sequence: CKLVFFAED) was purchased from Jinssr Biotechnology Co., Ltd. (China, Nanjing). Aβ42 peptide and Aβ42 enzyme-linked immunoassay (ELISA) kit were purchased from Shengong Bioengineering (Shanghai) Co., Ltd. Cell counting kit-8 (CCK-8), reactive oxygen species detection kit, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl imino carbocyanine iodide (JC-1) detection kit and total SOD activity detection (WST-8) kit were purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 2',7'-dichlorofluorescein diacetate (DCFH-DA) and Annexin V-FITC / PI apoptosis detection kit were purchased from Jiangsu Kaikai Biotechnology Co., Ltd. (China, Nanjing). Mouse tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β) and interleukin-6 (IL-6) ELISA detection kits were purchased from Beijing Solabio Technology Co., Ltd. (China, Beijing).

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

[0031] The methylene blue buffer solution in the embodiment of the present invention has a pH of 7.2 and 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 hours. After completion of the reaction, the precipitate was removed by filtration under normal pressure. Ultrapure water (10 mL) was added for extraction, and the dichloromethane layer was collected. After rotary evaporation and vacuum drying, a light yellow product was obtained. The light yellow product was redissolved in anhydrous ethanol, dialyzed, rotary evaporated, and vacuum dried to obtain a long chain of solanesol-polyethylene glycol polymer, namely Sol-PEG.

[0035] Sol-PEG (20 mg), chlorogenic acid (20 mg), and IR780 (150 μg) were solubilized in acetone (2 mL) by ultrasound and then slowly added dropwise to ultrapure water (10 mL). The mixture was evaporated at 37°C for 24 h. After dialysis and freeze-drying, drug-loaded nanomicelles CI@SP were obtained. Similarly, Sol-PEG (20 mg) was solubilized in acetone (2 mL) by ultrasound and slowly added dropwise to ultrapure water (10 mL). The mixture was evaporated at 37°C for 24 h. After dialysis and freeze-drying, unloaded nanomicelles SP were obtained.

[0036] In CI@SP solution (1 mg mL -1, 4 mL) was added Sulfo-SMCC (20 mg mL -1 , 200 μL), 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 performance determination of co-assembled nanomicelles of solanesol and chlorogenic acid

[0038] The synthesis of solanesol-polyethylene glycol polymer chains was characterized by Fourier transform infrared spectroscopy ( Figure 1 A). The UV-visible absorption spectra of drug-loaded nanomicelles CI@SP and non-drug-loaded nanomicelles SP were measured using a UV-visible spectrophotometer ( Figure 1 B). CI@SP has a clear chlorogenic acid absorption peak at 326nm, while SP does not. CI@SP is green, while the SP solution is colorless, indicating that chlorogenic acid and IR780 are successfully loaded into the nanomicelles. The modification of K peptide was characterized using an 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 morphology of the prepared drug-loaded nanomicelles CI@SPK was 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 did not affect their photothermal performance. When irradiated with an 808nm NIR laser, the temperature of the CI@SPK solution rapidly rose to approximately 50°C within 3 minutes. After the light was turned off, the temperature of the CI@SPK solution gradually decreased, demonstrating that the prepared drug-loaded nanomicelles possess 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 application is a peptide that can target Aβ and inhibit its further aggregation. The inhibition of CI@SPK on Aβ aggregation is verified by fluorescence method. Thioflavin T (ThT) can specifically bind to the beta sheet structure of Aβ aggregates, emit strong fluorescence signal at 485 nm, and is widely used as a fluorescence probe for monitoring the aggregation process of Aβ. After mixing different concentrations of CI@SPK (7.5 μL, final concentration of 0, 20 and 50 μg mL -1 ), Aβm (200 μM, 40 μL) and PBS (96.5 μL), the mixture is reacted at 37°C for 48 h. 6 μL ThT (250 μM) is mixed with the solution, and the reaction is carried out for 1 h. The fluorescence intensity of the mixed solution is determined by fluorescence spectrophotometer. As shown in Figure 2 A and B, CI@SPK has good inhibition effect on Aβ aggregation. STEM also shows that after treatment with CI@SPK (50 μg mL -1 ), the filamentous Aβ aggregates are significantly reduced, which indicates that CI@SPK has good inhibition effect on in vitro Aβ aggregation Figure 2 C and D).

[0041] Example 3 Determination of antioxidant performance of solanepentaol and chlorogenic acid co-assembled nanomicelles.

[0042] The performance of CI@SPK in vitro for removing hydroxyl radicals (·OH) and superoxide anion (O2· - ) two active oxygen (ROS) is determined. First, based on the fading of methylene blue caused by ·OH generated by Fenton reaction, the performance of CI@SPK in vitro for removing ·OH is determined. Hydrogen peroxide (4 M, 5 μL), copper chloride (1 mM, 2 μL), methylene blue (0.1 mM, 30 μL) and methylene blue buffer solution (155 μL) are added to SP, CI@SP, CI@SPK and CI@SPK solution after 808 nm light irradiation (1 mg mL -1 ), respectively. After reaction at 37°C for 2 h, the absorption peak intensity at 664 nm is recorded by ultraviolet-visible spectrometer. As shown in Figure 3 A, the unloaded solanepentaol nanomicelles SP show weak ·OH removal performance, while CI@SP and CI@SPK show excellent ·OH removal ability, indicating that the synergistic effect of chlorogenic acid and solanepentaol on ·OH removal is better than that of single solanepentaol nanomicelles. The CI@SPK solution after near-infrared light irradiation shows better ·OH removal effect than the same concentration of non-irradiated CI@SPK solution, which indicates that under the photothermal ability of IR780, more chlorogenic acid is released, further improving the ability to remove ·OH. Based on the fact that WST-8 can react with O2· -The reaction produces water-soluble methine dyes to make the solution yellow, and the total SOD activity detection kit is used to determine the CI@SPK in vitro clearance of O2· - The performance. Respectively, SP, CI@SP, CI@SPK and 808nm laser irradiation CI@SPK solution (0.5mg mL -1 , 20μL) mixed with 160μL WST-8 / enzyme working solution, then add 20μL of reaction start working solution. After 37℃ reaction for 30min, the absorbance at 450nm was detected by ultraviolet-visible spectrophotometer. As shown in Figure 3 B, the unloaded solanesol nanomicelles SP has almost no ability to scavenge O2· - , CI@SP, CI@SPK shows excellent O2· - scavenging capacity, and the CI@SPK solution after near-infrared light irradiation shows better O2· - scavenging effect than the same concentration of unirradiated CI@SPK solution. These results show that the excellent photothermal drug release performance of CI@SPK can achieve ROS scavenging in vitro.

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

[0044] First, the brain endothelial cells bEnd.3 were cultured on the upper layer of the Transwell chamber, and the neuron cells PC12 or BV2 cells were cultured on the lower layer to construct an in vitro blood-brain barrier model. When the upper layer has formed a dense monolayer, CI@SP or CI@SPK is added to the upper layer medium and continues to culture for 24h. Because IR780 itself has red fluorescence, the fluorescence intensity of IR780 in the lower layer PC12 cells or BV2 cells can be detected to determine its ability to cross the BBB. As shown in Figure 4 A-C, CI@SPK has obvious red fluorescence in the lower layer cells, and the fluorescence intensity is significantly higher than that of CI@SP. This shows that K peptide modified CI@SPK can effectively cross the BBB.

[0045] Example 5 Solanesol and chlorogenic acid co-assembled nanomicelles relieve Aβo-induced neuron cell damage determination

[0046] The stimulation of Aβo can cause an increase in intracellular ROS, eventually leading to oxidative stress and neuron cell damage. The reactive oxygen species detection kit is used to determine the ROS content in PC12 cells under different treatment conditions, in which DCFH-DA is used as a ROS indicator to emit green fluorescence. Specifically, PC12 cells are stimulated with Aβo, Aβo+CI@SPK, Aβo+CI@SPK+NIR, wherein the concentration of Aβo is 25μM, and the concentration of CI@SPK is 50μg mL-1 Aβo, Aβo+CI@SPK groups were incubated at 37℃ for 18 h, and 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. Confocal imaging was performed as described in Figure 5 A, there was strong green fluorescence in PC12 cells treated with Aβo, indicating that Aβo stimulated PC12 cells to produce a large amount of ROS. The fluorescence intensity of the Aβo+CI@SPK treatment group was significantly reduced, and the fluorescence intensity further decreased after NIR irradiation. These results show that NIR irradiation can help CI@SPK effectively remove ROS produced 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 results were obtained Figure 5 B and C). At the same time, the cell apoptosis rate under different treatments was measured by Annexin V-FITC / PI double staining cell apoptosis detection kit. As shown in Figure 5 D, after CI@SPK+NIR treatment, the cell apoptosis rate decreased from 28.32% to 8.52%. Finally, the degree of cell damage caused by Aβo stimulation was further evaluated by mitochondrial membrane potential test kit. Among them, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-pyracarbocyanine iodine (JC-1) was used as a fluorescent probe. When the cell is not damaged, the mitochondrial membrane potential (MMP) is normal, JC-1 is in an aggregated state, and emits red fluorescence; when the cell is damaged, the MMP decreases, and JC-1 exists in the form of monomer, emitting green fluorescence. The confocal imaging of MMP of PC12 cells is shown in Figure 5 E, bright green fluorescence appeared in Aβo-stimulated PC12 cells, indicating that the MMP of PC12 cells decreased and the cells were damaged. After co-incubation with CI@SPK, the green fluorescence was significantly reduced, indicating that the MMP of PC12 cells increased. After further irradiation with NIR, 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 show that CI@SPK under NIR irradiation can effectively alleviate Aβo-induced neuronal cell damage.

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

[0048] The elevated inflammation can cause microglia (BV2 cells) to be over-activated, transforming from normal phenotype (M2) to pro-inflammatory phenotype (M1), leading to impaired function of phagocytosis and degradation of Aβ; meanwhile, M1 type BV2 cells will secrete a large amount of pro-inflammatory factors, leading to damage of neuronal cells. The morphology of BV2 cells under different treatment conditions was taken by laser confocal microscope, specifically, BV2 cells were stimulated by Aβo, Aβo+CI@SPK, Aβo+CI@SPK+NIR, wherein the concentration of Aβo was 25 μM, the concentration of CI@SPK was 50 μg / mL -1 , the Aβo, Aβo+CI@SPK group was incubated at 37℃ for 18 h, and the Aβo+CI@SPK+NIR was first incubated for 6 h, then irradiated with NIR for 5 min, and then incubated for 12 h. As shown in Figure 6 A, the BV2 cells under Aβo stimulation were amoeba-like, and after CI@SPK and CI@SPK+NIR treatment, the BV2 cells gradually recovered to normal round shape. At the same time, the culture medium of BV2 cells under different treatment conditions was collected, and the content of pro-inflammatory factors in different groups of culture medium was measured by ELISA kit Figure 6 B-D). With the improvement of treatment effect, the content of pro-inflammatory factors in the culture medium was significantly reduced, indicating that CI@SPK can effectively reduce inflammation and restore the normal function of microglia.

Claims

1. A drug-loaded nanomicelle based on tobacco active substances, characterized in that: The nanomicelles include 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; the active small molecule drug is chlorogenic acid, the photosensitizer is IR780 iodide, and the polypeptide is a K peptide targeting Aβ, and the sequence of the K peptide is CKLVFFAED.

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

3. The preparation method according to claim 2, characterized in that The reaction conditions of step (1) are stirring at room temperature for 48 h.

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

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

6. Use of the drug-loaded nanomicelles according to claim 1 in the preparation of drugs or reagents for treating Alzheimer's disease.

7. The use according to claim 6, wherein the concentration of the drug-loaded nanomicelles is 20-50 μg mL -1 .

8. A drug for treating Alzheimer's disease, characterized in that: The drug comprises the drug-loaded nanomicelles according to claim 1.

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