Flavone derivative delivery system of targeted EGFR (epidermal growth factor receptor) protein as well as preparation method and application of flavone derivative delivery system

Abstract

The invention discloses a flavone derivative delivery system of targeted EGFR (Epidermal Growth Factor Receptor) protein as well as a preparation method and application of the flavone derivative delivery system, and belongs to the technical field of medicinal chemistry. The flavonoid derivative HT11 with anti-A beta and antioxidant activity is synthesized firstly, the depolymerization effect of the flavonoid derivative HT11 on A beta 1-42 reaches up to 92.40%, and the flavonoid derivative HT11 has good blood-brain barrier permeability. Then, the HT11 is loaded by taking the graphene oxide quantum dot as a carrier to obtain the HT11 coated GOQDs, so that the defect of low solubility of the HT11 is effectively overcome, and the targeting property and the curative effect of the HT11 coated GOQDs can be enhanced. Experimental results show that the HT11-coated GOQDs effectively solve the problem of reduction of AD animal space learning and memory ability, and possibility is provided for application of the HT11-coated GOQDs in the field of biological medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and in particular relates to a flavonoid derivative delivery system targeting EGFR protein, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder often referred to as the "memory eraser." Clinically, it manifests as a progressive loss of cognitive function, profound impairment of comprehensive intelligence, behavioral disturbances, and a complete breakdown of the ability to care for oneself, resulting in a devastating impact on patients and their families. The World Health Organization predicts that by 2030, over 82 million people will be affected by this untreatable disease, with this number expected to rise to 152 million by 2050. Since Alois first described AD in 1907, its pathogenesis remains largely unknown. Current theories include the amyloid Aβ hypothesis, abnormal Tau protein phosphorylation, the cholinergic hypothesis, and the neuroinflammation hypothesis. Among these, the presence of extracellular β-amyloid protein deposits as neuritic plaques and the accumulation of intracellular hyperphosphorylated Tau protein as neurofibrillary tangles remain the primary neuropathological criteria for AD diagnosis. However, the order in which these two proteins appear in AD patients remains uncertain. In February 2024, the research team led by Jia Jianping from Xuanwu Hospital published a major research result in the New England Journal of Medicine, which accurately revealed for the first time the dynamic changes of cerebrospinal fluid and imaging biomarkers in the period from asymptomatic to symptomatic Alzheimer's disease (AD), and elaborated on the key nodes in the early stage of the disease. The study showed that the order and time point of the difference in biomarkers between the AD group and the cognitively normal group were clear: Aβ first showed differences 18 years before diagnosis, and Aβ 42 / 40 The pre-diagnosis period for AD was 14 years, for p-Tau 11 years, for t-Tau 10 years, for NFT 9 years, for hippocampal atrophy 8 years, and for cognitive decline 6 years. These findings provide a solid foundation for early diagnosis and intervention of AD.

[0003] Traditional AD treatments currently available in clinical practice primarily fall into two categories: cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. However, these drugs only improve symptoms, fail to slow disease progression, and are associated with adverse reactions. Currently, Aβ and tau inhibitors are hot candidates for AD research and development, but most have failed. Notably, in 2024, lecanemab, developed by Eisai and Biogen, was approved for marketing in my country, becoming the first anti-Aβ antibody drug approved in the country. In July of the same year, donanemab, developed by Eli Lilly and Company, received approval from the US FDA. While there has been some progress in new drug research, the slow pace of drug development has stymied the current market for a single, broadly applicable drug suitable for a diverse range of patient populations, effectively treating or even preventing AD.

[0004] Flavonoids are widely found in nature. They have a benzo-γ-pyrone nucleus with a basic structure of C6-C3-C6 and exhibit remarkable biological activities, including antioxidant, anti-inflammatory, anticancer, and cardiovascular protective properties. Accumulating evidence suggests that the consumption of flavonoid-rich foods can beneficially influence normal cognitive function. Furthermore, a growing number of flavonoids have been shown to inhibit the development of Alzheimer's disease-like pathology and reverse cognitive deficits in rodent models, suggesting their potential therapeutic applications in dementia. For example, the flavonoid hesperidin has been shown to restore non-cognitive nesting and social interaction deficits by reducing Aβ deposition and neuroinflammation. Naringin exhibits potent antioxidant, anti-apoptotic, and anti-inflammatory activities. Studies have shown that pretreatment with naringenin can improve Aβ-induced learning and memory impairments by alleviating lipid peroxidation and apoptosis. Quercetin can regulate Nrf-2 translocation from the cytoplasm to the nucleus, protect against mitochondrial dysfunction, and inhibit Aβ plaque accumulation and neurofibrillary tangle formation.

[0005] Therefore, it is very important to synthesize flavonoid derivatives with anti-Aβ and antioxidant activities, overcome the problems of low solubility and poor targeting of flavonoid derivatives, and improve their bioavailability and targeting. Summary of the Invention

[0006] The purpose of the present invention is to provide a flavonoid derivative delivery system targeting EGFR protein and its preparation method and application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A flavonoid derivative delivery system targeting EGFR protein, comprising graphene oxide quantum dots and flavonoid derivative HT11, wherein the mass ratio of the graphene oxide quantum dots to the flavonoid derivative HT11 is 1:2, and the structure of the flavonoid derivative HT11 is shown below: .

[0008] Preferably, the oxidation degree of the graphene oxide quantum dots is 25 to 35%.

[0009] The preparation steps of the flavonoid derivative HT11 are as follows: (1) Acetyl chloride and 1-bromo-3,5-dimethoxybenzene are reacted by Friedel-Crafts acylation to obtain product b, and the equivalent ratio of acetyl chloride to 1-bromo-3,5-dimethoxybenzene is 1:1; (2) reacting product b with boron tribromide in an ice bath to produce product c, wherein the equivalent ratio of product b to boron tribromide is 1:1; (3) reacting product c, 3,4-dimethoxyphenylboronic acid, tetrahydrofuran, water, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride to obtain product d, wherein the equivalent ratio of product c, 3,4-dimethoxyphenylboronic acid, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride is 1:1.1:1.5:0.1; (4) Product d and DMF-DMA were mixed and subjected to a cyclization reaction to obtain product e, with an equivalent ratio of 1:2; (5) Product e is dissolved in dichloromethane and hydrochloric acid is added. After the reaction is completed, the final HT11 is obtained by extraction, drying, and separation.

[0010] The present invention also provides a method for preparing the above-mentioned flavonoid derivative delivery system, specifically: GOQDs and HT11 are mixed and placed in the dark at room temperature with stirring for 20 to 24 hours. After the reaction is completed, the precipitate obtained by centrifugation is freeze-dried to obtain the HT11@GOQDs nanodrug.

[0011] The present invention also provides use of the flavonoid derivative delivery system in treating Alzheimer's disease.

[0012] Furthermore, the flavonoid derivative delivery system reduces the phosphorylation of EGFR, interferes with the downstream Plcg1 protein, and finally interferes with BACE1- β Secretion of secretase regulates A β The production of.

[0013] The beneficial effects of the present invention are: This invention, based on the structural modification of a flavonoid core with anti-Aβ and antioxidant activity, has yielded flavonoid derivatives with both anti-Aβ and antioxidant activities. Activity screening revealed that the flavonoid derivative HT11 exhibited a 92.40% Aβ depolymerization effect and excellent blood-brain barrier permeability. Graphene oxide quantum dots (GOQDs) were used as nanocarriers to load HT11, achieving a "1+1>2" effect. This not only effectively overcomes HT11's inherent low solubility but also enhances its targeting and therapeutic efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The H NMR spectrum of HT11 (solvent: DMSO- d 6).

[0015] Figure 2 Thioflavin T fluorescence assay for the effects of compounds HT1 to HT19 on Aβ 1-42 results in inhibition of aggregation.

[0016] Figure 3 (A) UV spectrum of HT11 and (B) standard curve of absorbance of different concentrations of HT11 at the maximum absorption wavelength of 328 nm.

[0017] Figure 4 Figure 2 shows the geometric structure of the most stable HT11@carrier (carrier = GQDs, GOQDs-1, GOQDs-2, GOQDs-3, GOQDs-4, and GOQDs-5) complexes.

[0018] Figure 5 Relationship between GOQDs with different oxidation degrees and (A) adsorption energy and (B) solvation energy.

[0019] Figure 6 IGMH plot of HT11@carrier (carrier = GQDs, GOQDs-1, GOQDs-2, GOQDs-3, GOQDs-4, and GOQDs-5) complexes with an isosurface of 0.004 au.

[0020] Figure 7 Drug loading rates of nanodrugs with different ratios of GOQDs and HT11.

[0021] Figure 8 Thioflavin T fluorescence method was used to determine the effects of nanomedicines with different ratios of GOQDs and HT11 on Aβ 1-42 Aggregation inhibition.

[0022] Figure 9(A) UV spectra and (B) IR spectra of HT11, GOQDs, and HT11@GOQDs.

[0023] Figure 10 The cell viability of HT11 (A, C) and GOQDs (B, D) cells was determined by MTT assay.

[0024] Figure 11 The cell viability of (A) BV-2 cells and (B) PC-12 cells was determined by MTT assay using HT11@GOQDs.

[0025] Figure 12 This is the inhibitory effect of HT11 on LPS-induced NO release in BV-2 cells.

[0026] Figure 13 is the potential of GOQDs and HT11@GOQDs.

[0027] Figure 14 Images and quantitative analysis of hemolysis tests after incubation of different substances with fresh red blood cells (2%) at 37°C for 4 hours.

[0028] Figure 15 In vivo biodistribution of HT11@GOQDs, including (A) observation of the accumulation of GOQDs and HT11@GOQDs in the brain using animal imaging technology; (C) biodistribution in various organs (heart, liver, spleen, lung, kidney and brain) after 48 hours; (B, D) quantitative analysis of fluorescence signal intensity.

[0029] Figure 16 Effects of oral administration of drugs (1 mg / kg) on ​​cognitive dysfunction in APP / PS1 mice; (A) Experimental design; (B) Body weight of mice during drug administration; (C) Novel object recognition parameter results; (D) Nesting test results; (E) Average speed of mice; (F) Time spent ashore by mice in the first six days (*: C57 vs. AD; #: AD vs. AD+HT11@GOQDs); (G) Number of times mice crossed the island during the test; (H) Representative trajectories of mice in the Morris water maze; (I) Representative images of nests built by mice on the third day, scale bar 4 cm.

[0030] Figure 17 Proteomic volcano plots: (A) C57 vs. AD; (B) AD vs. AD+HT11@GOQDs; (C) Venn diagrams between groups.

[0031] Figure 18(A) Proteomic box plot of Nrp2 differential proteins; (B) Proteomic box plot of PLCG1 in AD VS AD+HT11@GOQDs; (C) KEGG pathway analysis of proteomic AD VS AD+HT11@GOQDs.

[0032] Figure 19 KEGG analysis of proteomic AD VS AD+HT11@GOQDs.

[0033] Figure 20 This is the differential gene-pathway diagram of proteomics AD VS AD+HT11@GOQDs.

[0034] Figure 21 Volcano plot of metabolomics, (A) C57 VS AD, (B) AD VS AD+HT11@GOQDs; PCA classification diagram of metabolomics, (C) C57 VS AD, (D) AD VS AD+HT11@GOQDs.

[0035] Figure 22 KEGG pathway diagram of AD VS AD+HT11@GOQDs for metabolomics.

[0036] Figure 23 (A) Box plot of BACE1 differentially expressed genes in transcriptomics; (B) Violin plot of CaMP metabolites in energy metabolomics of AD VS AD+HT11@GOQDs.

[0037] Figure 24 KEGG clustering diagram of energy metabolomics AD VS AD+HT11@GOQDs. DETAILED DESCRIPTION

[0038] To illustrate the technical content, characterization and performance analysis methods of the present invention in detail, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0039] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used can be purchased commercially.

[0040] The synthesis steps of HT series compounds are as follows:

[0041] The first step is a Friedel-Crafts acylation reaction to produce product b. The methoxy protecting group is then removed under the action of boron tribromide to produce compound c. The third step is a Suzuki-Miyaura coupling reaction to obtain compound d. Cyclization then occurs using toluene as the solvent to obtain compound e. Finally, the protecting group is removed under the action of boron tribromide to obtain the final product f. Through coupling with different arylboronic acids, 19 new flavonoid derivatives were synthesized. The specific structures are as follows: .

[0042] Example 1 Synthesis of Compound HT11

[0043] Acetyl chloride (1.5 eq.) and aluminum trichloride (1.5 eq.) were added to a three-necked flask purged with nitrogen. 200 mL of dry dichloromethane (DCM) was added and stirred in an ice bath. Once the dichloromethane solution clarified, 1-bromo-3,5-dimethoxybenzene (1.5 eq.) was slowly added dropwise. The reaction was allowed to react in an ice bath for 4 hours, then the temperature was raised to room temperature and the reaction continued for 12 hours. After completion of the reaction, as monitored by thin-layer chromatography, deionized water was added to quench the reaction. The residue was extracted three times with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by concentration. The sample was mixed and separated by column chromatography to yield product b.

[0044] Add product b to a two-necked flask equipped with a magnetic stirrer and dissolve it in dry dichloromethane. Replace the nitrogen atmosphere three times and cool the reaction system to 0°C in an ice bath. Then, slowly add boron tribromide (1.0 eq.) dropwise. After reacting in an ice bath for 6 hours, warm the system to room temperature and continue reacting for 12 hours. After monitoring the reaction completion by thin-layer chromatography, quench the reaction by adding deionized water. Extract the residue three times with dichloromethane, combine the organic phases, and dry over anhydrous sodium sulfate. Concentrate to remove the solvent. Stir the sample and separate by column chromatography to obtain product c.

[0045] Under a nitrogen atmosphere, product c, 3,4-dimethoxyphenylboronic acid (1.1 eq.), 9.0 mL of tetrahydrofuran, and 1.0 mL of deionized water were added to a three-necked flask. Stirring was initiated, and potassium carbonate (1.5 eq.) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.1 eq.) were added. The nitrogen atmosphere was replaced again. The reaction was refluxed and monitored by thin-layer chromatography. After completion of the reaction, the reaction solution was cooled to room temperature. The reaction was quenched by adding 20.0 mL of deionized water. The mixture was extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by column chromatography to obtain product d.

[0046] Product d and DMF-DMA (2.0 eq.) were weighed and added sequentially to a three-necked flask equipped with a magnetic stirrer. Toluene was added to dissolve the product. After nitrogen was replaced three times, the mixture was refluxed at 90°C for 6 hours. After monitoring the reaction by thin-layer chromatography, the mixture was cooled to room temperature and quenched with deionized water. The residue was extracted three times with ethyl acetate, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. The sample was mixed and separated by column chromatography to obtain product e.

[0047] Weigh product e into a round-bottom flask equipped with a magnetic stirrer and add 20 mL of dichloromethane. Stirring was initiated until the solids were completely dissolved. The nitrogen atmosphere was replaced three times and the reaction system was cooled to 0°C in an ice bath. Boron tribromide (1.0 eq.) was then slowly added dropwise. TLC was monitored until the reaction was complete. After completion, the mixture was cooled to room temperature. Extraction was performed with deionized water and dichloromethane. The organic phases were combined and dried over anhydrous magnesium sulfate. The organic phases were concentrated, mixed, and separated by column chromatography to obtain the final product HT11.

[0048] The H NMR spectrum of product d in chloroform solvent is as follows: 1 H NMR (600 MHz, Chloroform- d ): δ 13.43 (s, 1H), 7.18 (dd, J = 8.3, 2.1 Hz, 1H), 7.10 (d, J =2.1 Hz, 1H), 6.93(d, J = 8.3 Hz, 1H), 6.76 (d, J = 1.6 Hz, 1H), 6.55-6.54 (m,1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 2.68 (s, 3H).

[0049] Figure 1 This is the H NMR spectrum of HT11 in DMSO solvent. 1 H NMR (600 MHz, DMSO- d 6): δ12.46-12.45 (m, 1H), 9.36 (s, 1H), 9.11 (s, 1H), 8.32-8.29 (m, 1H), 7.18-7.15 (m, 1H) ), 7.11-7.10 (m, 1H), 7.07-7.05 (m, 1H), 6.93-6.92 (m, 1H), 6.81-6.80 (m, 1H), 6.37 (dd, J = 5.9, 1.5 Hz, 1H).

[0050] Example 2 Synthesis of Compound HT9 The synthetic preparation process is the same as that of Example 1, except that 3-methoxyphenylboronic acid is used for Suzuki-Miyara coupling.

[0051] Example 3 Synthesis of Compound HT10 The synthetic preparation process is the same as that of Example 1, except that 2-methoxyphenylboronic acid is used for Suzuki-Miyara coupling.

[0052] Example 4 Synthesis of Compound HT18 The synthetic preparation process is the same as that of Example 1, except that 4-methoxy-3-fluorophenylboronic acid is used for Suzuki-Miyara coupling.

[0053] Example 5 Synthesis of Compound HT19 The synthetic preparation process is the same as that of Example 1, except that 3-methoxy-4-fluorophenylboronic acid is used for Suzuki-Miyara coupling.

[0054] HT series compounds against Aβ 1-42 Determination of depolymerization effect: Aβ 1-40 and Aβ 1-42 Are the two main components of amyloid plaques. 1-40 In contrast, Aβ 1-42 Therefore, we used the thioflavin T method to measure the inhibitory effects of 19 HT series compounds on Aβ 1-42 The results showed that resveratrol (Res) can effectively promote the aggregation and degradation of fibers, and was selected as a positive drug for reference. Figure 2 As shown, HT1 to HT19 were dissolved in DMSO to prepare 10mM stock solutions, and then diluted with PBS (pH = 7.4) to the test concentration. At a dosage concentration of 50 μM, compound HT11 was able to effectively inhibit Aβ 1-42 The disaggregation of HT11 was inhibited with an inhibition rate of 92.40%, which was much higher than 75.89% (Res). Therefore, HT11 was selected as the target compound in the following experiments.

[0055] Parallel Artificial Membrane Permeability Assay (PAMPA): In order to test the blood-brain barrier permeability of HT11, a parallel artificial membrane permeability experiment was used (refer to Physicochemical high throughput screening: Parallel artificial membrane permeation assay in the description of passive absorption processes, J. Med. Chem., 1998, 41(7): 1007–1010 for prediction. First, the UV spectrum of HT11 was detected ( Figure 3 A) It was found that it has the best excitation wavelength at 328 nm. HT11 solutions with concentrations of 0, 3.125, 6.25, 12.5, 25, and 50 μM were prepared using EtOH:PBS (volume ratio 3:7). The corresponding absorbance at 328 nm was measured using a microplate reader, and a standard curve was plotted ( Figure 3 B). In order to verify the reliability of the experimental method and model, the HT11 P e The value is (8.71 ± 0.052) × 10 -6 cm / s, indicating that it has the potential to passively diffuse across the blood-brain barrier.

[0056] Molecular docking of HT11: Next, molecular docking was performed with 1M17, 1I09, and 1Z0Q, targeting the relevant pathways identified through network pharmacology analysis. HT11 primarily binds to the proteins through hydrogen bonds. As shown in Table 1, HT11's binding affinity for the pathway proteins decreases in the following order: HT11-1M17 (-8.023 kcal / mol) > HT11-1I09 (-6.368 kcal / mol) > HT11-1Z0Q (-4.821 kcal / mol). These observations suggest that HT11 theoretically exhibits strong interactions with all three proteins. However, its poor water solubility and potential limitations inherent to small molecule drugs, such as poor targeting and weak binding, significantly limit its clinical application.

[0057] Table 1 Docking performance of HT11 on 1M17, 1I09, and 1Z0Q proteins (unit: kcal / mol)

[0058] Example 6 Synthesis of HT11@GOQDs Nanomedicine At the SMD-B3LYP-D3(BJ) / 6-31G computational level, the structures of the drug and five graphene oxide quantum dots (GOQDs) with different oxidation degrees, namely GOQDs-1 (19.69%), GOQDs-2 (24.09%), GOQDs-3 (27.06%), GOQDs-4 (31.03%) and GOQDs-5 (33.14%), were optimized with the help of Gaussian program, and five nanomedicines were designed (see Figure 4 It can be observed that HT11 tends to adsorb on the side of GOQDs with fewer oxidized functional groups (concave side).

[0059] The adsorption energy of the most stable configuration after calculation and optimization ( E ad ), and make a linear correlation with the degree of oxidation, from Figure 5 (A) It can be observed that the higher the degree of oxidation of GOQDs, the stronger the binding ability of HT11 with them. In addition, the interaction energy of HT11@GOQDs (-20.02 ~ -26.85 kcal / mol) is greater than that of HT11@GQDs (-17.72 kcal / mol). This can be attributed to the larger deformation energy (1.74 ~ 6.79 kcal / mol) generated by HT11 adsorbed on GOQDs, which is also the reason why HT11 and GOQDs interact strongly and bind more firmly. We also calculated the enthalpy change (Δ H ) and the Gibbs free energy change (Δ G ), are all negative, indicating that the adsorption of HT11 on the support is exothermic and can be formed spontaneously.

[0060] Then the solvation energy in 0.5% DMSO solvent was calculated ( E sol ), to evaluate the stability of the complex. HT11 adsorbed on GOQDs E sol significantly higher than that of the single HT11 drug molecule, and with the increase of the oxidation degree of GOQDs, the E sol The larger (see Figure 5 B), indicating that the loading of GOQDs significantly enhanced the solubility and stability of HT11, which is of great significance for improving its bioavailability.

[0061] The interaction between HT11 and the carrier was visualized by IGMH analysis, such as Figure 6As shown, the IGMH diagram of HT11@GQDs is characterized by distinct green isosurfaces, indicating that the drug molecules and the carrier are primarily bound via π-π interactions, with pronounced van der Waals interactions. In addition to van der Waals interactions, the IGMH diagram of HT11@GOQDs also contains several blue regions, indicating hydrogen bonding between HT11 and GOQDs. Theoretical calculations indicate that HT11@GOQDs nanomedicines exhibit numerous advantages: Firstly, their internal components exhibit strong interactions, which lays the foundation for synergistic drug effects; secondly, the nanomedicine exhibits excellent water solubility, enabling good dispersion in aqueous environments, facilitating subsequent applications; and thirdly, their excellent stability effectively ensures the long-lasting and reliable efficacy of the drug.

[0062] The specific synthesis steps of HT11@GOQDs nanomedicine are as follows: In DMSO solvent, GOQDs (purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd., with an oxidation degree of approximately 30%) and HT11 were mixed in mass ratios of 2:1, 1:1, 5:8, 1:2, 1:3, 1:4, and 1:5, and stirred at room temperature in the dark for 24 hours. The mixture was centrifuged at 12,000 rpm and 15°C for 20 minutes to obtain a yellow-brown solid, which was then freeze-dried to obtain a solid powder, namely, HT11@GOQDs nanomedicine.

[0063] The supernatant obtained by centrifugation was detected by microplate reader at 328 nm for the remaining amount of HT11 and then incorporated into the standard curve to calculate the drug loading rate of the nanomedicine. Figure 7 As shown in the figure, at a ratio of GOQDs:HT11 = 1:5, the drug has reached overload; while at 1:4, the drug is almost fully loaded. For the sake of environmental protection, the nanomedicines with the first six ratios were screened for the best through the Aβ disaggregation experiment, which is most closely related to AD disease. The experimental results are shown in the figure. Figure 8 As shown in the figure, when the carrier-drug ratio was 1:2, the inhibition rate was 76.13%, which was higher than that of the other ratios of nanodrug and Res. Therefore, the nanodrug synthesized at the ratio of GOQDs:HT11 = 1:2 was selected as the optimal HT11@GOQDs for subsequent experiments.

[0064] The UV-Vis absorption spectrum (UV-Vis) of HT11@GOQDs (GOQDs:HT11 = 1:2) is shown in Figure 2. Figure 9 As shown in (A), the free drug HT11 exhibits a sharp characteristic absorption peak, while the corresponding peak of the nano-drug complex (HT11@GOQDs) is significantly broadened, indicating that the drug interacts with the graphene oxide quantum dot carrier, resulting in a change in the electronic transition behavior. Further Fourier transform infrared spectroscopy (FTIR) analysis revealed that ( Figure 9B) Compared with the infrared characteristic peaks of free HT11 and pure GOQDs, the HT11@GOQDs composite has a peak at 3000 cm -1 A new absorption peak appears near the surface of the GOQDs, which can be attributed to the stretching vibration peak of the hydroxyl group (-OH) formed by hydrogen bonding or intermolecular forces between the drug and the carrier. The changes in the UV and IR spectra indicate that HT11 can effectively bind to GOQDs.

[0065] MTT assay to assess cytotoxicity: The toxicity of HT11, GOQDs and HT11@GOQDs to BV-2 cells (microglia) and PC-12 cells (neuronal cells) was evaluated by MTT assay. Figure 10 and Figure 11 As shown in the results, HT11 and GOQDs were not toxic to PC-12 cells at the concentrations tested. HT11 exerted over 90% viability on BV-2 cells at concentrations below 7.5 μM, while GOQDs exerted over 90% viability on BV-2 cells at a concentration of 2 μg / mL. Furthermore, HT11@GOQDs exerted over 90% viability on both BV-2 and PC-12 cells at concentrations below 7.5 μg / mL (see Table 1). Figure 11 ), which indicates that nanomedicines at a certain concentration do not have toxic effects on microglia and neurons.

[0066] Determination of anti-inflammatory activity: Inhibiting the release of NO is also an important component of anti-neuroinflammation. The present invention uses the Griess method to detect the effects of compound HT11 and GOQDs on the release of NO from LPS-induced BV-2 cells. The experimental results are as follows Figure 12 As shown, when the HT11 IC 50 The concentration of 5.273 ± 1.27 μM was observed, indicating excellent anti-inflammatory activity.

[0067] Potentiometric determination: like Figure 13 As shown in the figure, the potentials of GOQDs and HT11@GOQDs are -15.35 and -12.62 mV, respectively, both of which are electronegative, which contributes to their stability in blood circulation and in vivo delivery. Figure 14 ), the hemolysis rates of the drug, carrier, and nanodrug were all less than 5%. Therefore, HT11@GOQDs has good in vitro biocompatibility and is expected to be developed into a non-toxic and effective nanodrug for the clinical treatment of Alzheimer's disease.

[0068] Example 7 In order to explore the biodistribution of nanomedicines in vivo, the fluorescence properties of the carrier and nanomedicine themselves were used to detect the drug in vivo and with the help of small animal in vivo and in vitro imaging technology. Since the yellow fluorescence wavelength emitted by the carrier and the drug overlaps with the animal tissue band, it will be partially interfered by the fluorescence of the mouse hair. Before administration, the weight of each group of mice was weighed and recorded, and the drug was administered at 1 mg / kg according to the weight of the mice. After dissolving GOQDs and HT11@GOQDs in PBS, the drug was administered by gavage according to the above drug concentrations, such as Figure 15 As shown in (A, B), 4 to 24 hours after administration, although the nanodrug's accumulation in the brain was not as high as that of the carrier, its half-life was somewhat prolonged. Ex vivo organ imaging 48 hours later revealed that the nanodrug was still highly concentrated in the brain, exerting its therapeutic effects, and was primarily metabolized in the liver.

[0069] Then, experiments were conducted in AD model animals. The construction of the AD model was based on the reference Impaired spatial learning in the APP + PSEN1DeltaE9 bigenic mouse model of Alzheimer's disease, Genes Brain Behav., 2007, 6(1):54-65. Mice were treated with oral administration (1 mg / kg) for a total of 28 days. After 16 days of treatment, nesting, novel objects, and water maze tests were performed as shown in the figure. Figure 16 As shown in the results of the nesting experiment, novel object recognition experiment and water maze experiment, HT11@GOQDs effectively rescued the decline in spatial learning and memory ability of AD animals.

[0070] Example 8 1. Multi-omics analysis To further explore the specific action pathways of nanomedicines in vivo, we performed proteomic, transcriptomic and metabolomic tests on these three groups of animals (C57, AD and AD+HT11@GOQD). Figures 17 to 20 Omics results showed that Nrp2, a downstream protein closely associated with EGFR phosphorylation, could be effectively targeted by the nanomedicine. This reduced EGFR phosphorylation, interfered with the downstream Plcg1 protein, and ultimately, the secretion of BACE1-β secretase, thereby regulating Aβ production. KEGG analysis confirmed the same results as network pharmacology—that the nanomedicine effectively promoted Aβ clearance by activating related pathways.

[0071] 2. Energy Metabolomics Analysis Energy metabolomics was used to further detect the effect of Ca 2+ The impact of Figure 21 ~ 24, the results are consistent with expectations.

[0072] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A flavonoid derivative delivery system targeting EGFR protein, characterized by: The flavonoid derivative delivery system includes graphene oxide quantum dots and flavonoid derivative HT11, wherein the mass ratio of the graphene oxide quantum dots to the flavonoid derivative HT11 is 1:

2. The structure of the flavonoid derivative HT11 is shown below: 。 2. The flavonoid derivative delivery system according to claim 1, characterized in that: The preparation steps of the flavonoid derivative HT11 are as follows: (1) Acetyl chloride and 1-bromo-3,5-dimethoxybenzene are reacted by Friedel-Crafts acylation to obtain product b, and the equivalent ratio of acetyl chloride to 1-bromo-3,5-dimethoxybenzene is 1:1; (2) reacting product b with boron tribromide in an ice bath to produce product c, wherein the equivalent ratio of product b to boron tribromide is 1:1; (3) reacting product c, 3,4-dimethoxyphenylboronic acid, tetrahydrofuran, water, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride to obtain product d, wherein the equivalent ratio of product c, 3,4-dimethoxyphenylboronic acid, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride is 1:1.1:1.5:0.1; (4) Product d and DMF-DMA were mixed and subjected to a cyclization reaction to obtain product e, with an equivalent ratio of 1:2; (5) Product e is dissolved in dichloromethane and hydrochloric acid is added. After the reaction is completed, the final HT11 is obtained by extraction, drying, and separation.

3. The flavonoid derivative delivery system according to claim 1, characterized in that: The oxidation degree of the graphene oxide quantum dots is 25-35%.

4. A method for preparing the flavonoid derivative delivery system according to claim 1, characterized in that: The following steps are involved: After mixing GOQDs and HT11, stir them in the dark at room temperature for 20 to 24 hours. After the reaction, centrifuge the resulting precipitate and freeze-dry it to obtain the HT11@GOQDs nanomedicine.

5. Use of the flavonoid derivative delivery system according to claim 1 in the treatment of Alzheimer's disease.

6. The use according to claim 5, characterized in that: The flavonoid derivative delivery system reduces the phosphorylation of EGFR, interferes with the downstream Plcg1 protein, and finally interferes with BACE1- β Secretion of secretase regulates A β The production of.

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