A traditional Chinese medicine bionic nano preparation for treating neurodegenerative diseases, a preparation method and application thereof

Abstract

The application provides a traditional Chinese medicine bionic nano preparation for treating neurodegenerative diseases, a preparation method and application, and comprises the following: a traditional Chinese medicine active ingredient and a bionic nano delivery system, the traditional Chinese medicine active ingredient is icariin or rosmarinic acid; the bionic nano delivery system comprises: a polydopamine nano core, a microglial cell membrane, and RAGE binding peptides modified on the surface of the microglial cell membrane; and the traditional Chinese medicine active ingredient is loaded on the polydopamine nano core. The application also provides a preparation method of the traditional Chinese medicine bionic nano preparation for treating neurodegenerative diseases, which comprises the following steps: mixing the traditional Chinese medicine active ingredient with the polydopamine nano core; extracting the microglial cell membrane pretreated by lipopolysaccharide; mixing the RAGE binding peptides with the microglial cell membrane; mixing the polypeptide-modified microglial cell membrane with the drug-loaded polydopamine nano core, and treating by ultrasonic and extrusion to obtain the traditional Chinese medicine bionic nano preparation. The application can improve the brain delivery efficiency of the traditional Chinese medicine active ingredient.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic nano-preparation of traditional Chinese medicine for the treatment of neurodegenerative diseases, its preparation method, and its application. Background Technology

[0002] Due to their multi-target regulatory properties, the active ingredients of traditional Chinese medicine have unique advantages in the treatment of central nervous system (CNS) diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and brain tumors. Studies have shown that representative ingredients such as rosmarinic acid (RA) and icariin (ICA) can exert therapeutic effects through mechanisms such as inhibiting neuroinflammation, clearing β-amyloid protein (Aβ), or regulating synaptic function.

[0003] However, the clinical efficacy of the aforementioned active ingredients in traditional Chinese medicine is severely limited by their insufficient bioavailability in the brain. The blood-brain barrier (BBB) ​​is the most tightly regulated interface in the human body, protecting the brain from the influence of exogenous substances in the bloodstream, but also severely limiting the entry of most therapeutic drugs into the brain parenchyma. Even if certain treatment strategies are mechanistically feasible, limited brain delivery and low drug accumulation and penetration at the lesion site make it difficult to achieve ideal therapeutic effects. Therefore, overcoming the BBB delivery bottleneck has become a key scientific issue in advancing the treatment of brain diseases with traditional Chinese medicine.

[0004] In recent years, researchers have developed various brain-targeted nanodelivery systems to improve drug transport efficiency across the brain border (BBB). Among these, active targeting strategies mediated by highly expressed receptors on the BBB (such as transferrin receptor, LDL receptor-associated protein, and RAGE) have attracted widespread attention. In particular, the receptor for advanced glycation end products (RAGE) is significantly highly expressed on the pathological BBB of neurological diseases such as Alzheimer's disease (AD), and its expression level is upregulated with disease progression, making it an ideal lesion-specific BBB target site. However, the following technical challenges remain: existing research on brain-targeted delivery systems mainly focuses on chemical drugs; the loading efficiency, stability, and delivery synergistic effects of traditional Chinese medicine active ingredients in these carriers are still unclear; while existing RAGE targeting strategies can achieve BBB crossing, nanoparticles lack the ability to actively chemotactically target inflammatory lesions after entering the brain, resulting in relatively diffuse drug distribution within the brain; existing cell membrane coating technologies mainly focus on immune escape functions, with insufficient development and regulation of active targeting functions of membrane receptors. Summary of the Invention

[0005] To address the technical problems existing in the background art, the present invention provides a biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases, its preparation method and application, which can efficiently cross the brain blockade, evade immune clearance, and actively chemotactically attract to inflammatory lesions in the brain to improve the brain delivery efficiency of active ingredients of traditional Chinese medicine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases, comprising: an active ingredient of traditional Chinese medicine and a biomimetic nano-delivery system, wherein the active ingredient of traditional Chinese medicine is icariin or rosmarinic acid; the biomimetic nano-delivery system comprises: a polydopamine nanocore, a microglia membrane coated on the surface of the polydopamine nanocore, and a receptor for advanced glycation end products (RAGE) binding peptide modified on the surface of the microglia membrane; the active ingredient of traditional Chinese medicine is loaded onto the polydopamine nanocore.

[0007] Further specifying, the RAGE-binding peptide is KLVFFAEDC.

[0008] Further, the expression level of chemokine receptor 4 on the surface of the microglia is upregulated by lipopolysaccharide prestimulation.

[0009] Furthermore, the polydopamine nanocore is combined with the traditional Chinese medicine through a π-π stacking effect.

[0010] Further specifying, the mass ratio of the active ingredient of the traditional Chinese medicine to the polydopamine nanocore is 5-20:1.

[0011] A method for preparing a biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases includes the following steps: Step 1: Prepare an aqueous solution of polydopamine nanocore and a solution of active ingredients of traditional Chinese medicine. Mix the solution of active ingredients of traditional Chinese medicine and the aqueous solution of polydopamine nanocore in a certain proportion to obtain drug-loaded polydopamine nanocore. Step 2: Extraction of microglia pretreated with lipopolysaccharide: Microglia were seeded and cultured, pretreated with lipopolysaccharide, and collected by centrifugation. The microglia membrane was extracted using a hypotonic lysis combined with differential centrifugation method. Step 3: Mix the RAGE-binding peptide with the microglia membrane described in Step 2, so that the RAGE-binding peptide modifies the surface of the microglia membrane to obtain a peptide-modified microglia membrane. Step 4: Mix the peptide-modified microglia cell membrane described in Step 3 with the drug-loaded polydopamine nanocore described in Step 1, and obtain a biomimetic nano-formulation of traditional Chinese medicine by ultrasonication and extrusion.

[0012] Further specifying the conditions for lipopolysaccharide pretreatment, the lipopolysaccharide concentration is 0-5 μg / mL, and the treatment time is 12-48 h.

[0013] Further specifying, the mass ratio of the peptide-modified microglia to the drug-loaded polydopamine nanocore is 1:2.

[0014] Application of a biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases in the preparation of drugs for the treatment of central nervous system diseases.

[0015] Further specifying, the central nervous system diseases include Alzheimer's disease or Parkinson's disease.

[0016] The beneficial effects of this invention are: 1. This invention relates to a biomimetic nanoparticle formulation of traditional Chinese medicine (TCM) for the treatment of neurodegenerative diseases. TCM is loaded onto a biomimetic nanoparticle delivery carrier, achieving a three-tiered active targeting mechanism of "immune escape, BBB penetration, and chemotaxis" through RAGE-binding peptide-mediated active BBB crossing, microglia membrane-mediated immune escape, and CXCR4 inflammatory chemotaxis induced by lipopolysaccharide prestimulation. Compared with direct administration of TCM, this biomimetic nanoparticle formulation significantly increases the intrabrain drug concentration and prolongs the brain retention time at the same dosage, effectively improving delivery efficiency below the blood-brain barrier.

[0017] 2. The biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases of this invention achieves active inflammatory chemotaxis after the nanoparticle formulation enters the brain by pre-stimulating microglia with lipopolysaccharide to upregulate the expression of CXCR4 on the cell membrane. Compared with direct administration of active ingredients of traditional Chinese medicine, the biomimetic nanoparticle formulation of this invention significantly increases the drug accumulation at the site of inflammatory lesions in the brain, achieving precise delivery.

[0018] 3. The biomimetic nano-formulation of traditional Chinese medicine used in this invention for the treatment of neurodegenerative diseases showed significantly improved behavioral and pathological indicators in animal models of central nervous system diseases compared with the group directly administered with the same dose of active ingredients of traditional Chinese medicine. The learning and memory abilities of the model animals were significantly restored, and the survival rate of neurons was improved.

[0019] 4. Compared with existing PLGA nano-delivery systems for treating neurodegenerative diseases, the biomimetic nano-formulation of traditional Chinese medicine in this invention significantly improves the cellular uptake efficiency: Under Aβ-induced AD pathological conditions, the uptake efficiency of PDA@R@M / K by bEnd.3 cells is 3 times that of PLGA@R (p<0.01). Flow cytometry histograms show a significant rightward shift of the fluorescence peak, indicating that the cellular targeted uptake ability of the formulation of this invention is significantly superior to that of the PLGA system. The brain distribution of this invention is significantly improved: After tail vein injection in AD mice, the fluorescence signal intensity of PDA@R@M / K in brain tissue is significantly stronger than that of PLGA@R, and it co-localizes with neurons, indicating that the brain delivery efficiency of the formulation of this invention is significantly superior to that of the PLGA system. Attached Figure Description

[0020] Figure 1 The figures show the preparation and characterization results of the microglia cell membrane of the present invention, wherein (A) is a schematic diagram of the extraction process of microglia cell membrane pretreated with lipopolysaccharide; (B) is a figure of Western blot detection of CXCR4 protein expression on the surface of microglia cell membrane before and after lipopolysaccharide pretreatment; (C) is a schematic diagram of the preparation process of microglia cell membrane modified with RAGE-binding peptide; (D) is a figure of fluorescence microscopy observation of cell membrane modified with FITC-labeled RAGE-binding peptide; and (E) is a figure of statistical results of counting fluorescent positive cell membrane vesicles under a fluorescence microscope. Figure 2 The figures show the characterization results of the biomimetic nano-formulation of traditional Chinese medicine in this invention. (A) is the result of transmission electron microscopy observation of the morphology of PDA (left) and PDA@M / K (right). (B) is the result of dynamic light scattering determination of the particle size distribution of PDA, PDA@M and PDA@M / K. (C) is the result of Zeta potential determination of PDA, PDA@M and PDA@M / K. (D) is the electrophoresis diagram of SDS-PAGE detection of cell membrane, PDA@M and PDA@M / K membrane protein retention. Figure 3 This is a fluorescence imaging result of the brain distribution after treatment with the biomimetic nano-formulation of traditional Chinese medicine of the present invention. The scale bar is 50 μm. Figure 4 The diagrams show the structural formulas of the active ingredients RA and ICA from traditional Chinese medicine, as well as the HPLC detection results of the biomimetic nano-formulation of traditional Chinese medicine in this invention. (A) shows the chemical structural formula of rosmarinic acid (RA); (B) shows the HPLC standard curve and drug loading and encapsulation efficiency results of the biomimetic nano-formulation of traditional Chinese medicine (PDA@R@M / K); (C) shows the chemical structural formula of icariin (ICA); and (D) shows the HPLC standard curve and drug loading and encapsulation efficiency results of the biomimetic nano-formulation of traditional Chinese medicine (PDA@I@M / K). Figure 5The graph shows the results of flow cytometry analysis of the RAGE receptor blocking experiment in Experiment 3 of this invention, which showed the changes in nanoparticle uptake rate of bEnd.3 cells after FPS-ZM1 blocked the RAGE receptor. Figure 6 This is a fluorescence image of the uptake of the biomimetic nano-formulation by bEnd.3 cells in Experiment 3 of this invention, used to evaluate cell targeting. The scale bar is 50 μm. Figure 7 The graph shows the flow cytometry results of cellular uptake of the biomimetic nano-formulation PDA@R@M / K and the comparative formulation PLGA@R in this invention. The left graph is a statistical bar chart of average fluorescence intensity, and the right graph is a representative flow cytometry histogram (horizontal axis: fluorescence intensity, vertical axis: cell count). Figure 8 This is a fluorescence imaging image of the brain distribution of the biomimetic nano-formulation PDA@R@M / K of the present invention and the comparative formulation PLGA@R; Figure 9 The graph shows the nesting test scores of AD model mice in each group after treatment with the biomimetic nano-formulation of traditional Chinese medicine of this invention. Figure 10 The image shows the immunofluorescence staining results of Aβ plaques in the brains of AD model mice in each group after treatment with the biomimetic nano-formulation of traditional Chinese medicine of this invention. The scale bar is 50 μm. Figure 11 This is a HE staining result of the main organs of mice after treatment with the biomimetic nano-formulation of traditional Chinese medicine of the present invention. The scale bar is 50 μm. Figure 12 The image shows the results of routine blood tests in mice after treatment with the biomimetic nanoparticle formulation of traditional Chinese medicine (n = 3), where (A) is the white blood cell count (WBC); (B) is the neutrophil count (NEUT); (C) is the lymphocyte count (LYMPH); (D) is the red blood cell count (RBC); (E) is the platelet count (PLT); and (F) is the hemoglobin concentration (HGB). Figure 13 The image shows the blood biochemical results of mice after treatment with the biomimetic nano-formulation of traditional Chinese medicine (n = 3), where (A) is alanine aminotransferase (ALT); (B) is aspartate aminotransferase (AST); (C) is creatinine (CR); and (D) is blood urea nitrogen (BUN). Figure 14 The figure shows the stability test results of the biomimetic nano-formulation of traditional Chinese medicine in different media (n = 3), where (A) shows the changes in particle size and PDI in HEPES buffer; (B) shows the changes in particle size and PDI in 5% glucose solution; and (C) shows the changes in particle size and PDI in 10% fetal bovine serum (FBS). Detailed Implementation

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of methods consistent with some aspects of the invention as detailed in the appended claims.

[0023] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0024] In the description of this application, it should be understood that the numerical labels before the steps do not indicate the order of the steps, but are only used to facilitate the description of this application and to distinguish each step, and therefore should not be construed as a limitation of this application.

[0025] This invention provides a biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases. The biomimetic nano-formulation comprises: an active ingredient of traditional Chinese medicine and a biomimetic nano-delivery system. The active ingredient of traditional Chinese medicine is icariin or rosmarinic acid. The biomimetic nano-delivery system comprises: a polydopamine nanocore, a microglia membrane coated on the surface of the polydopamine nanocore, and RAGE-binding peptides modified on the surface of the microglia membrane. The active ingredient of traditional Chinese medicine is loaded onto the polydopamine nanocore.

[0026] Preferably, the RAGE-binding peptide is KLVFFAEDC.

[0027] Preferably, the expression level of chemokine receptor 4 on the microglia membrane is upregulated by lipopolysaccharide prestimulation.

[0028] Preferably, the polydopamine nanocore is combined with the traditional Chinese medicine through a π-π stacking effect.

[0029] Preferably, the mass ratio of the active ingredient of the traditional Chinese medicine to the polydopamine nanocore is 5-20:1.

[0030] This invention provides a method for preparing a biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases, comprising the following steps: Step 1: Prepare an aqueous solution of polydopamine nanocore and a solution of active ingredients of traditional Chinese medicine. Mix the solution of active ingredients of traditional Chinese medicine and the aqueous solution of polydopamine nanocore in a certain proportion to obtain drug-loaded polydopamine nanocore. Step 2: Extraction of microglia pretreated with lipopolysaccharide: Microglia were seeded and cultured, pretreated with lipopolysaccharide, and collected by centrifugation. The microglia membrane was extracted using a hypotonic lysis combined with differential centrifugation method. Step 3: Mix the RAGE-binding peptide with the microglia membrane described in Step 2, so that the RAGE-binding peptide modifies the surface of the microglia membrane to obtain a peptide-modified microglia membrane. Step 4: Mix the peptide-modified microglia cell membrane described in Step 3 with the drug-loaded polydopamine nanocore described in Step 1, and obtain a biomimetic nano-formulation of traditional Chinese medicine by ultrasonication and extrusion.

[0031] Preferably, the pretreatment conditions for lipopolysaccharide are: a concentration of lipopolysaccharide of 0-5 μg / mL and a treatment time of 12-48 h.

[0032] Preferably, the mass ratio of the peptide-modified microglia to the drug-loaded polydopamine nanocore is 1:2.

[0033] This invention also provides the application of a biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases in the preparation of drugs for treating central nervous system diseases.

[0034] Preferably, the central nervous system disease includes Alzheimer's disease or Parkinson's disease.

[0035] The present invention will be further described below with reference to embodiments.

[0036] Experimental reagents: Dopamine hydrochloride (Sigma, USA); Tris buffer (biofroxx, Germany); Icariin (ICA) reference standard (purity ≥98%, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.); Rosmarinic acid (RA) reference standard (purity ≥98%, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.); DSPE-PEG 2000-mal (Chongqing Yusi Pharmaceutical Technology Co., Ltd., China); KLVFFAEDC, FITC-KLVFFAEDC peptide (Shanghai Taopu Biotechnology Co., Ltd., China); LPS (Beijing Solarbio Science & Technology Co., Ltd., China); CXCR4 antibody (Abcam, UK); PMSF protease inhibitor, RIPA lysis buffer, BCA kit, 5 × SDS-PAGE loading buffer, Coomassie Brilliant Blue kit (Shanghai Beyotime Biotechnology Co., Ltd., China); trichromatographic pre-stained protein marker (Shanghai Yamei Biomedical Technology Co., Ltd., China); rapid gel preparation kit (Lambolid Biotechnology Co., Ltd., China); dialysis bags (Millipore, USA); chromatographic grade methanol, acetonitrile (Sigma-Aldrich, USA).

[0037] Experimental Instruments: Nanoparticle size and Zeta potential analyzer (90 Plus PALS, NanoBrook, USA); Transmission electron microscope (TEM H-600 Hitachi, Shimadzu, Japan); Time-of-flight mass spectrometer (AXIMA Performance, Shimadzu, Japan); High-performance liquid chromatograph (SHIMADZU, Japan); Gel electrophoresis apparatus (Bio-Rad, USA); Cell glass grinder (Chengdu Weilan Technology Co., Ltd., China); Polycarbonate membrane microextruder (Avanti Polar Lipids, USA); Fluorescence microscope (Thunder DMi8, Leica, Germany); Biosafety cabinet (AC2-4S1, ESCO, Singapore); Carbon dioxide incubator (CCL-170B-8, ESCO, Singapore); High-speed refrigerated centrifuge (KDC-H140R, Anhui Zhongke Zhongjia Scientific Instruments Co., Ltd., China).

[0038] Cell lines: BV2 microglia (mouse microglia, Shanghai Cell Bank, Chinese Academy of Sciences, China); bEnd.3 cells (mouse brain microvascular endothelial cells, Shanghai Cell Bank, Chinese Academy of Sciences, China).

[0039] Example 1 This embodiment provides a biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases. The preparation method includes the following steps: Step 1: Preparation of polydopamine nanocore (PDA) solution: Tris buffer solution with a pre-adjusted pH of 11.5 was added to a round-bottom flask and placed in a constant-temperature stirred water bath at 37°C and 420 rpm. After the temperature stabilized, accurately weighed dopamine hydrochloride was slowly added to a final concentration of 1 mg / mL. The mixture was allowed to react openly for 2 h to obtain the PDA solution.

[0040] Preparation of active ingredient solution of traditional Chinese medicine: Weigh 10 mg of icariin (ICA) reference standard, place it in a 10 mL volumetric flask, add 100 μL of DMSO to dissolve and prepare a stock solution, add ultrapure water to the mark, and prepare an icariin solution with a concentration of 1 mg / mL for later use.

[0041] Icariin solution and polydopamine nanocore aqueous solution were mixed at a mass ratio of 5:1, stirred at 25 °C for 24 h, and then purified by centrifugation to obtain drug-loaded polydopamine nanocore. Step 2: Extraction of lipopolysaccharide-pretreated microglia: BV2 microglia were induced for 24 h in a medium containing 2 μg / mL lipopolysaccharide. Cells were collected, washed with pre-chilled PBS, and resuspended in hypotonic lysis buffer containing 1 mM PMSF, and lysed overnight at 4°C. The lysis buffer was homogenized 20 times using a cell homogenizer, centrifuged at 3200 g for 5 min at 4°C, and the supernatant was collected. The supernatant was centrifuged at 15000 rpm for 20 min at 4°C, and the precipitate was collected, yielding the lipopolysaccharide-pretreated microglia membrane. The cell membrane precipitate was resuspended in 5% glucose and stored at -80°C for later use.

[0042] Step 3: Combine the RAGE-binding peptide KLVFFAEDC with DSPE-PEG 2000 -mal were mixed at a molar ratio of 1.5:1 and reacted at 25°C for 24 h under nitrogen protection. The reaction solution was dialyzed sequentially in dimethyl sulfoxide (DMSO) and ultrapure water (MWCO 2000 Da), and then lyophilized to obtain DSPE-PEG. 2000 Store at -K, -20℃ for later use.

[0043] The microglia membrane described in step 2 is combined with DSPE-PEG. 2000 -K was mixed at a mass ratio of 2:1 to allow RAGE-binding peptides to modify the surface of microglia, resulting in peptide-modified microglia. Step 4: Mix the peptide-modified microglia from Step 3 with the drug-loaded polydopamine nanocore from Step 1 at a mass ratio of 1:2, sonicate at 4°C for 5 min (42 kHz, 100 W), and repeatedly extrude through a 0.4 μm polycarbonate membrane using a micro extruder 10 times to obtain a biomimetic nano-formulation of traditional Chinese medicine (PDA@I@M / K).

[0044] Example 2 This embodiment provides a biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases. The preparation method is the same as that in Example 1, except that the active ingredient of traditional Chinese medicine in this embodiment is rosmarinic acid (RA), and the resulting biomimetic nano-formulation of traditional Chinese medicine (PDA@R@M / K) is obtained.

[0045] Example 3 This embodiment provides a biomimetic nano-preparation of traditional Chinese medicine for the treatment of neurodegenerative diseases. The preparation method is the same as that in Example 1, except that the mass ratio of the active ingredient of traditional Chinese medicine to the polydopamine nanocore is 20:1, the concentration of lipopolysaccharide is 5 μg / mL, and the treatment time is 48h.

[0046] Example 4 This embodiment provides a biomimetic nano-preparation of traditional Chinese medicine for the treatment of neurodegenerative diseases. The preparation method is the same as that in Example 1, except that the mass ratio of the active ingredient of traditional Chinese medicine to the polydopamine nanocore is 10:1.

[0047] To further investigate the efficacy of the biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases, the following experiments were conducted on the biomimetic nano-formulation of traditional Chinese medicine prepared in the examples.

[0048] Experiment 1: Preparation and Characterization of Biomimetic Nanoparticles of Traditional Chinese Medicine for the Treatment of Neurodegenerative Diseases 1.1 Lipopolysaccharide pretreatment for microglia extraction and RAGE-bound peptide modification Experimental Methods: Microglia prepared in Example 1 were used for the following experiments. To utilize the surface characteristics of the BV2 cell membrane, particularly its high expression of chemokine receptors, to obtain a cell membrane with targeting capabilities to inflammatory sites, microglia were pre-stimulated with 2 µg / mL lipopolysaccharide for 24 h before cell membrane extraction. The extraction procedure for the activated microglia cell membrane is as follows: Figure 1 As shown in Figure A.

[0049] Western blotting was used to detect the expression level of CXCR4 protein on the surface of microglia before and after lipopolysaccharide pretreatment. Na+ was used as the pretreatment agent. + / K + -ATPase was used as an internal control, and ECL chemiluminescence was used for imaging.

[0050] To enable the prepared biomimetic nano-formulation of traditional Chinese medicine to target the pathological BBB, DSPE-PEG was incorporated via lipid intercalation. 2000 -K modification was applied to the extracted cell membrane to prepare an engineered microglial cell membrane that could directly bind to RAGE, which is highly expressed at the pathological BBB site. The preparation process is as follows: Figure 1 As shown in C.

[0051] To further characterize the success rate of targeted peptide modification, the number of fluorescent cell membrane vesicles was counted under a microscope. The number of fluorescently positive cell membrane vesicles in 50 random fields of view were counted under a fluorescence microscope, the positivity rate was calculated, and a bar chart was plotted. Modification success rate = (number of fluorescently positive vesicles / total number of vesicles) × 100%.

[0052] The results showed that the protein immunocathode assay results were as follows: Figure 1 As shown in B. Compared with the untreated group (lipopolysaccharide-), the CXCR4 protein expression band was significantly enhanced in the lipopolysaccharide pretreated group (lipopolysaccharide+), while the internal control Na... + / K + -ATPase bands were expressed consistently in both groups.

[0053] The results are as follows Figure 1 As shown in Figure D, the FITC-labeled green fluorescent signal is uniformly distributed on the cell membrane surface.

[0054] The results are as follows Figure 1 As shown in E, the success rate of RAGE-binding peptide modification was 88 ± 6%.

[0055] The above results indicate that lipopolysaccharide stimulation can effectively upregulate the expression of CXCR4 on the microglia membrane and enhance its tropism towards inflammatory sites; the RAGE-binding peptide was successfully inserted into the microglia membrane and was evenly distributed; this method can effectively achieve efficient modification of the microglia membrane by the RAGE-binding peptide.

[0056] 1.2 Construction and Validation of Bionic Nanodelivery System To verify whether the biomimetic nano-formulation of traditional Chinese medicine in this invention has been successfully constructed, a biomimetic nano-delivery system (without loading the active ingredients of traditional Chinese medicine) was first prepared and characterized.

[0057] Experimental materials: Following the method in Example 1, without adding any active ingredients of traditional Chinese medicine in the membrane coating step, the following samples were prepared: polydopamine nanocore (PDA), unmodified microglia membrane coated on the surface of PDA (i.e., PDA@M, which was prepared separately for comparison), and microglia membrane coated on the surface of PDA with RAGE peptide (PDA@M / K). The above samples were used for the following characterization experiments.

[0058] Experimental methods: The morphology of PDA and PDA@M / K was observed using transmission electron microscopy. A suitable amount of sample was dropped onto a copper grid, allowed to dry naturally, and then observed and photographed under accelerating voltage.

[0059] The particle size and zeta potential of PDA, PDA@M, and PDA@M / K were determined using a dynamic light scattering particle size analyzer. Appropriate samples were diluted with ultrapure water to a suitable concentration and measured at 25°C. Each sample was measured three times. Results are expressed as mean ± standard deviation, and bar charts were plotted.

[0060] SDS-polyacrylamide gel electrophoresis was used to verify the retention of membrane proteins. Equal volumes of extracted cell membrane, PDA, PDA@M and PDA@M / K samples, and BV2 cell lysis buffer were taken and subjected to SDS-PAGE electrophoresis, followed by Coomassie brilliant blue staining to observe the distribution of protein bands.

[0061] The results show that: Figure 2 The transmission electron microscope (TEM) results for A show that, in the left image, the PDAs are spherical in shape, uniform in size, and evenly dispersed. (See image for example.) Figure 2 As shown in Figure A (right), PDA@M / K was prepared by encapsulating vesicles onto a PDA using ultrasound and an extruder. TEM observation revealed a spherical PDA core and a clearly defined cell membrane layer, forming a core-shell structure. The results indicate that the cell membrane was successfully coated onto the surface of the PDA nanocore.

[0062] DLS analysis shows that, Figure 2 As shown in the bar chart of average hydrated particle size, the average hydrated particle sizes of PDA, PDA@M, and PDA@M / K are 81.7 ± 0.6 nm, 133.5 ± 7.1 nm, and 138.2 ± 1.3 nm, respectively. After cell membrane coating, the average hydrated particle size of the nano-formulation increases by approximately 50 nm. Figure 2 As shown in Figure C, the surface Zeta potentials of PDA, PDA@M, and PDA@M / K are -23.2 ± 0.6 mV, -28.8 ± 0.5 mV, and -28.7 ± 0.5 mV, respectively. Due to the negative charge of the cell membrane, the coated nanoparticles have a more negative potential than the uncoated PDA core. These data confirm the successful coating of the cell membrane onto the PDA core.

[0063] like Figure 2 As shown in the D electrophoresis image, the protein profiles of PDA@M / K and PDA@M are very similar to those of the extracted cell membrane, with no obvious band loss or attenuation.

[0064] The above results demonstrate that the biomimetic nanodelivery system was successfully constructed, and the cell membrane was successfully coated on the surface of the PDA nanocore to form a core-shell structure. No loss or damage to membrane proteins was caused during the membrane modification and coating process, and the extracted proteins on the cell membrane were well preserved.

[0065] 1.3 Cell uptake validation (membrane coating effect) Experimental materials: Following the method in Example 1, without adding any active ingredients of traditional Chinese medicine in the membrane coating step, the following samples were prepared: polydopamine nanocore (PDA) and microglial cell membranes modified with RAGE peptides coated on the surface of PDA (PDA@M / K). The above samples were used for the following characterization experiments.

[0066] Experimental Methods: AD mice were randomly divided into two groups (n=3 per group) and intravenously injected with 100 μL of PDA or PDA@M / K (30 mg / kg) containing the same concentration of Cy5. 24 h after injection, mice in each group were sacrificed, their limbs were fixed, and they were perfused first with PBS, then with 4% paraformaldehyde. After perfusion, the brain was isolated in the dark and fixed in 4% paraformaldehyde for 2 days, followed by dehydration with 15% and 30% sucrose solutions for 24 h each. After dehydration, the tissue was blotted dry with absorbent paper, and hippocampal tissue blocks were excised and placed on trays pre-filled with ultrapure water and flash-frozen. The blocks were then embedded with tissue embedding medium and frozen for another 10 min. Sections were then cut to a thickness of 10 μm using a cryostat, adhered to glass slides, and stored at 4°C for later use. The sections were thoroughly washed with TBS to remove the embedding agent, blotted dry with absorbent paper, and then covered with TBS containing 5% Triton X-100 for 30 min to permeate. Afterward, they were washed three times with TBS, stained with a 1 μg / mL DAPI TBS solution for 10 min, the stain was discarded, and the sections were washed three more times with TBS. An anti-fluorescence quencher was then added. The sections were covered with coverslips, sealed with nail polish, and the distribution of nanoparticles was observed under a fluorescence microscope.

[0067] The results show that: Figure 3 As shown, DAPI (blue) represents the cell nucleus, NPs-Cy5 (red) represents the distribution of nanoparticles, and Merge represents the superposition of both. In brain slices, the fluorescence signal of PDA@M / K was stronger than that of the unmodified group, indicating that PDA@M / K can penetrate the BBB more effectively in the pathological state of AD. The results show that the uptake of PDA@M / K is significantly higher than that of PDA, indicating that membrane coating endows the nanoparticles with targeting ability.

[0068] Based on the above, the drug loading performance of the formulation of the present invention is evaluated.

[0069] Experiment 2: Determination of drug loading and encapsulation efficiency of the biomimetic nano-formulation of traditional Chinese medicine for the treatment of neurodegenerative diseases according to this invention. Experimental Methods: Traditional Chinese medicine (TCM) active ingredients possess unique advantages in AD treatment due to their multi-target regulatory properties. Studies have shown that representative components such as RA and ICA exert therapeutic effects through mechanisms such as inhibiting neuroinflammation, clearing Aβ, and regulating synaptic function. Through π-π stacking and hydrogen bonding, RA and ICA can be effectively loaded onto PDAs, enabling targeted brain delivery using this system. The chemical structures of RA and ICA are shown below. Figure 4As shown in A and 4C, the HPLC conditions for the two drugs were studied, revealing that the optimal concentrations for each drug were 330 nm and 270 nm, respectively. Different mass ratios of PDA and drug (PDA:drug = 20:1, 10:1, 5:1, 2:1, 1:1) were weighed and prepared according to the method in Example 1. The biomimetic nanoparticles of the traditional Chinese medicine were separated by ultrafiltration and centrifugation, and the filtrate was injected for analysis. The encapsulation efficiency and drug loading were calculated using the following formulas: Encapsulation efficiency (%) = (Dosage amount - Free drug amount) / Dosage amount × 100% Drug loading (%) = (Drug content in nanoparticles / Total mass of nanoparticles) × 100% The results show that the chemical structural formulas of RA and ICA are as follows: Figure 4 As shown in A and 4C. HPLC results show that RA and ICA have good linearity at 330 nm and 270 nm, respectively, and the standard curves are shown in Figure 4. Figure 4 As shown in B and 4D, the drug loading of RA increases with increasing drug dosage. At a carrier-to-drug ratio of 5:1, the drug loading reaches 16.47 ± 0.01%, but the overall encapsulation efficiency remains relatively stable, consistently above 95%. Similarly, the drug loading of ICA increases with increasing drug dosage, similar to RA, reaching 7.27 ± 0.08% at a 5:1 ratio. The encapsulation efficiency of ICA initially increases and then decreases with increasing drug dosage. At a carrier-to-drug ratio of 40:1, the encapsulation efficiency is 33.69 ± 0.95%, increasing to 50.71 ± 0.58% and 59.97 ± 0.54% at 10:1 and 20:1, respectively. At a 5:1 ratio, the encapsulation efficiency decreases slightly to 50.53 ± 0.58%. The results showed that RA and ICA can be effectively loaded onto the PDA surface through π-π stacking and hydrogen bonding, with high drug loading and encapsulation efficiency, making them suitable for brain-targeted delivery.

[0070] Table 1. Drug loading and encapsulation efficiency of rosmarinic acid (RA) at different drug loading ratios Table 2. Drug loading and encapsulation efficiency of icariin (ICA) at different drug loading ratios. After confirming that the formulation has good drug loading and release properties, we further verified its targeting mechanism.

[0071] Experiment 3: Validation of RAGE targeting mechanism 3.1 RAGE receptor blockade experiment Experimental Methods: bEnd.3 cells were seeded in 6-well plates and cultured to 80% confluence. Aβ (1 μM) was used for induction for 24 h to simulate the pathological environment of Alzheimer's disease (AD). The experimental group was pre-incubated with FPS-ZM1 (a specific RAGE inhibitor) for 1 h, while the control group received an equal volume of PBS. Cy5-labeled PDA@M or PDA@M / K nanoparticles were then added, and the cells were incubated at 37°C for 4 h. Cells were collected by trypsin digestion, washed three times with PBS, and Cy5 fluorescence intensity was detected by flow cytometry.

[0072] The results showed that, Figure 5 As shown, under the Aβ-induced simulated AD pathological environment, bEnd.3 cells showed significantly higher uptake of PDA@M / K than PDA@M; however, after the addition of the RAGE receptor blocker FPS-ZM1, the uptake of PDA@M / K decreased, returning to a level comparable to PDA@M. These results indicate that the targeted uptake of bEnd.3 cells by RAGE-binding peptide-modified nanoparticles (PDA@M / K) is mediated by the RAGE receptor.

[0073] 3.2 Cell targeting validation Experimental materials: Following the method of Example 1, without adding any active ingredients of traditional Chinese medicine in the membrane coating step, the following samples were prepared: unmodified microglia membrane coated on the surface of PDA (i.e., PDA@M, prepared separately for comparison), and microglia membrane coated on the surface of PDA with RAGE peptide (PDA@M / K). The above samples were used for the following characterization experiments.

[0074] Experimental Methods: Aβ induction was used to simulate the pathological environment of Alzheimer's disease (AD). bEnd.3 cells were divided into a normal group and an Aβ-induced group. Cy5-labeled PDA@M or PDA@M / K nanoparticles were added to each group, and the cells were incubated at 37°C for 4 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI. The cells were observed and photographed using a fluorescence microscope. The Cy5-labeled nanoparticles exhibited red fluorescence (NPs). Merge images were obtained by overlaying DAPI and NP images.

[0075] The results showed that, Figure 6As shown, DAPI staining reveals the location of cell nuclei (blue), NPs show the distribution of nanoparticles (red), and Merge indicates the superposition of the two. Normal bEnd.3 cells showed little difference in uptake of PDA@M and PDA@M / K, with similar red fluorescence intensity. However, after Aβ induction, Aβ-induced bEnd.3 cells showed a significant increase in PDA@M / K uptake, exhibiting a bright red fluorescence signal and overlapping with the DAPI-stained cell nuclei (Merge), indicating that the nanoparticles were extensively uptaken by the cells, achieving a good cytopathological targeting effect; while the PDA@M group showed no significant change. These results demonstrate that PDA@M / K has specific targeting ability for bEnd.3 cells under AD pathological conditions.

[0076] Given the positive results of the in vitro targeting mechanism verification, in order to further verify the delivery efficiency of the biomimetic nano-formulation of traditional Chinese medicine prepared in this invention, PDA@R@M / K was compared and evaluated with PLGA@R loaded with rosmarinic acid (RA) in the existing PLGA drug-loaded nanosystem.

[0077] Experiment 4: Comparative Evaluation of the Biomimetic Nanoparticle Formulation of Traditional Chinese Medicine of the Present Invention with Existing PLGA Systems 4.1 Comparison of cellular uptake Experimental materials: The formulation of this invention: Take the PDA@R@M / K prepared in Example 2; Comparative formulation PLGA@R: Prepared using the emulsification solvent evaporation method described in the reference (Acta Pharmaceutica Sinica B 14.9 (2024):4102-4117). 10 mg of PLGA (50:50, molecular weight 10 kDa, Xi'an Ruixi Biotechnology) was weighed and dissolved in 1 mL of dichloromethane, then sonicated in an ice bath (100 W, 2 min). 100 μL of RA solution was added, followed by mixing with 5 mL of 1% PVA aqueous solution. The mixture was then sonicated in an ice bath (100 W, 5 min), and the organic solvent was removed by vacuum evaporation. The mixture was centrifuged at 8000 rpm for 15 min, washed three times with ultrapure water, and lyophilized to obtain PLGA@R. DLS analysis showed that the blank PLGA nanoparticles had a particle size of 106.4 nm and a PDI of 0.218; the PLGA@R had a particle size of 110.3 nm and a PDI of 0.254.

[0078] Experimental methods: bEnd.3 cells were induced with Aβ (1 μM, 24 h) to simulate the pathological environment of Alzheimer's disease. Cy5-labeled PDA@R@M / K and PLGA@R were added respectively, and the cells were incubated at 37℃ for 4 h. After digestion and centrifugation, the cells were collected, washed three times with PBS, and the Cy5 fluorescence intensity was detected by flow cytometry to compare the efficiency of the two methods.

[0079] The results showed that the cellular uptake flow cytometry results were as follows: Figure 7 As shown on the left, compared with the PLGA@R group, bEnd.3 cells showed significantly enhanced uptake of PDA@R@M / K. The flow cytometry histogram is shown below. Figure 7 As shown on the right, the fluorescence peak of the PDA@R@M / K group is significantly shifted to the right; quantitative analysis is as follows: Figure 7 The left side shows that the average fluorescence intensity of the PDA@R@M / K group was 184597.7, which was significantly higher than the fluorescence intensity of the PLGA@R group (61107.0), and about three times that of the PLGA@R group.

[0080] The results showed that the biomimetic nano-formulation of traditional Chinese medicine described in this invention had a significantly higher targeted uptake efficiency on bEnd.3 cells under AD pathological conditions than the PLGA nanodelivery system.

[0081] 4.2 Comparison of brain distribution The formulation of this invention: Take the PDA@R@M / K prepared in Example 2; Comparative formulation PLGA@R: Prepared using the emulsification solvent evaporation method described in the reference (Acta Pharmaceutica Sinica B 14.9 (2024):4102-4117). 10 mg of PLGA (50:50, molecular weight 10 kDa, Xi'an Ruixi Biotechnology) was weighed and dissolved in 1 mL of dichloromethane, then sonicated in an ice bath (100 W, 2 min). 100 μL of RA solution was added, followed by mixing with 5 mL of 1% PVA aqueous solution. The mixture was then sonicated in an ice bath (100 W, 5 min), and the organic solvent was removed by vacuum evaporation. The mixture was centrifuged at 8000 rpm for 15 min, washed three times with ultrapure water, and lyophilized to obtain PLGA@R. DLS analysis showed that the blank PLGA nanoparticles had a particle size of 106.4 nm and a PDI of 0.218; the PLGA@R had a particle size of 110.3 nm and a PDI of 0.254.

[0082] Experimental Methods: AD mice were randomly divided into two groups and intravenously injected with 100 μL of PDA@R@M / K and PLGA@R containing the same concentration of Cy5. Twenty-four hours after injection, the mice were sacrificed, their limbs were fixed, and they were perfused first with PBS, then with 4% paraformaldehyde. After perfusion, the brain was isolated in the dark and fixed in 4% paraformaldehyde for 2 days, followed by dehydration with 15% and 30% sucrose solutions for 24 hours each. After dehydration, the tissue was blotted dry with absorbent paper, and hippocampal tissue blocks were excised and placed on trays pre-filled with ultrapure water and flash-frozen. The blocks were embedded with tissue embedding medium and frozen for another 10 minutes. Sections were then cut to a thickness of 10 μm using a cryostat, adhered to glass slides, and stored at 4°C for later use. The sections were thoroughly washed with TBS to remove the embedding medium, blotted dry with absorbent paper, and then covered with TBS containing 5% Triton X-100 for 30 minutes to permeate. The nanoparticles were then washed three times with TBS, stained with a 1 μg / mLDAPI TBS solution for 10 min, the stain was discarded, and the nanoparticles were washed three more times with TBS. An anti-fluorescence quencher was then added. The nanoparticles were covered with a coverslip, sealed with nail polish, and observed under a fluorescence microscope.

[0083] The results showed that the brain distribution measurement results were as follows: Figure 8 As shown, DAPI (blue) represents the cell nucleus, NPs-Cy5 (red) represents the distribution of nanoparticles, and Merge represents the superposition of the two. In the brain slices of the PDA@R@M / K group, the Cy5 fluorescence signal (red) was significantly stronger than that of the PLGA@R group, and it overlapped with the DAPI-stained cell nuclei (blue). These results collectively indicate that the brain delivery efficiency of this system is superior to existing delivery systems.

[0084] After confirming that the present invention can significantly improve the efficiency of drug delivery to the brain, its therapeutic effect on animal models of neurodegenerative diseases was further evaluated.

[0085] Experiment 5: In vivo pharmacodynamics experiment In vivo pharmacodynamic experiments of biomimetic nano-formulations of traditional Chinese medicine for the treatment of neurodegenerative diseases. 5.1 Nest Building Score Experimental animals and grouping: A batch of 15 AD mice were randomly divided into 5 groups: WT group (normal mouse control group), AD control group, RA group (free drug RA group), ICA group (free drug ICA group), PDA@R@M / K (prepared in Example 2) and PDA@I@M / K (prepared in Example 1).

[0086] Experimental Methods: Mice were treated with equal amounts of active ingredients from traditional Chinese medicine (RA, 3 mg / kg; ICA, 2 mg / kg). After treatment with free RA, free ICA, PDA@R@M / K, and PDA@I@M / K, mice were moved to the test room for at least 24 hours to acclimatize. Mice were shuffled and renumbered for later random scoring. Before the experiment, each mouse had fresh bedding, and 10 sheets of 5 × 5 cm thin paper were placed in the same location in the upper right corner of the mouse cage to provide nesting material. After 24 hours, nesting behavior was photographed, and scores were calculated based on random numbering and photographs. The nesting scoring criteria were as follows: 1. No obvious nesting point, and the paper was not torn, scored 1 point; 2. The paper was not obviously torn, but there was an identifiable nesting point, scored 2 points; 3. The paper was partially torn, but there was an easily identifiable nesting point, scored 3 points; 4. Most of the paper was bitten and torn, but there was an obvious nesting point, scored 4 points. All results were scored blinded.

[0087] The results show that: Figure 9 The bar chart shows the nesting scores: WT group mice scored 4.4±0.6, exhibiting intact nesting behavior; AD control group mice scored 2.8±1.0, indicating impaired nesting behavior. The scores of the free drug RA group and ICA group were 3.0±1.2 and 2.8±1.3, respectively, showing little improvement in nesting behavior compared to the AD control group. After treatment with PDA@R@M / K and PDA@R@M / K, the nesting score increased to 3.6±0.9; the PDA@I@M / K group scored 3.5±1.0. Both groups' nesting scores were closer to those of the WT group, indicating improved nesting behavior.

[0088] The above results indicate that in the nesting experiment, after treatment with PDA@R@M / K and PDA@I@M / K, the nesting behavior of AD mice improved and the scores increased, which was better than that of the AD group mice. This proves that the efficient delivery of the biomimetic nano-formulation of traditional Chinese medicine to RA and ICA in this invention can effectively improve the behavioral performance of AD mice, and the therapeutic effect is better than that of free drugs.

[0089] 5.2Aβ deposition Experimental methods: In order to investigate the improvement of brain Aβ deposition by the biomimetic nano-formulation of traditional Chinese medicine of the present invention, the standard hippocampal region of the mouse brain after the treatment in the above 5.1 nesting score was sectioned, and the number of Aβ plaques in the mouse brain was detected by Aβ immunofluorescence staining experiment.

[0090] The results show that: Figure 10As shown, Aβ immunofluorescence staining results revealed a large number of Aβ plaque deposits in the brains of AD mice, a stark contrast to WT mice. After treatment with the biomimetic nano-formula of this invention, both the number and size of the Aβ plaques decreased. These results indicate that the efficient delivery of the biomimetic nano-formula of this invention to RA and ICA can reduce the deposition of Aβ plaques in the brain.

[0091] To assess the clinical translation potential of the formulation of this invention, its in vivo safety was further investigated.

[0092] Experiment 6: In vivo safety evaluation of biomimetic nano-formulations of traditional Chinese medicine Experimental animals and grouping: A batch of 15 AD mice were randomly divided into 5 groups: WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K.

[0093] 6.1 Major Organs HE Experimental Methods: The safety of the formulation is a primary consideration for clinical use; therefore, a comprehensive safety assessment of the biomimetic nano-formulations of traditional Chinese medicine was conducted. After treatment with PDA@M / K, PDA@R@M / K, and PDA@I@M / K, the major organs of all groups, including the heart, liver, spleen, lungs, and kidneys, were isolated and stained with hematoxylin and eosin (HE).

[0094] The results are as follows Figure 11 As shown, no significant changes were observed in the AD mice across the different groups, with almost no differences between them. Myocardial fibers were neatly arranged, hepatocytes remained structurally intact, the red and white pulp boundaries of the spleen were clearly defined, alveolar structure was normal, and glomeruli and tubules exhibited normal morphology. No abnormal changes such as inflammatory cell infiltration, necrosis, or fibrosis were observed. The results indicate that the biomimetic nano-preparation of traditional Chinese medicine has no significant toxicity to major organs.

[0095] 6.2 Complete Blood Count Experimental methods: Routine blood parameters were measured in AD mice after treatment, including white blood cells, neutrophils, lymphocytes, red blood cells, platelets, and hemoglobin. A bar chart was plotted based on the test results. Figure 12 As shown.

[0096] The results show that: Figure 12 As shown in Figure A, the white blood cell counts of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 4.74 ± 1.03, 5.84 ± 1.22, 5.81 ± 0.18, 4.61 ± 0.79 and 5.43 ± 0.61 × 10⁻⁶, respectively. 9 / L, such as Figure 12As shown in Figure B, the neutrophil counts in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K, and PDA@I@M / K groups were 0.96 ± 0.25, 1.14 ± 0.47, 1.01 ± 0.30, 0.97 ± 0.46, and 0.87 ± 0.58 × 10⁻⁶, respectively. 9 / L, such as Figure 12 As shown in Figure C, the lymphocyte counts in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 4.25 ± 0.89, 4.26 ± 1.01, 3.77 ± 0.24, 4.21 ± 1.16 and 3.37 ± 1.10 × 10⁻⁶, respectively. 9 / L, such as Figure 12 As shown in Figure D, the erythrocyte counts of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 8.38 ± 0.43, 8.38 ± 0.59, 8.57 ± 0.46, 8.18 ± 0.42 and 8.76 ± 0.31 × 10⁻⁶, respectively. 12 / L, such as Figure 12 As shown in Figure E, the platelet counts of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 121.00 ± 11.53, 133.67 ± 9.71, 133.00 ± 9.54, 128.00 ± 13.11 and 133.33 ± 5.86 g / L, respectively. Figure 12 As shown in Figure F, the hemoglobin levels of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 562.67 ± 27.15, 581.33 ± 68.24, 620.33 ± 117.55, 617.00 ± 70.00, and 522.33 ± 72.60 × 10⁻⁶, respectively. 9 / L. The results showed no significant differences among the groups, and all data were within the normal range, indicating that the biomimetic nano-preparation of traditional Chinese medicine has no obvious toxicity to the blood system.

[0097] 6.3 Blood Biochemistry Experimental Methods: To further evaluate the systemic response of mice to the biomimetic nano-preparation of traditional Chinese medicine, blood biochemistry of mice in each group was measured, including ALT, AST, CR, and BUN. The results were plotted in a bar chart as shown below. Figure 13 As shown.

[0098] The results show that: Figure 13 As shown in Figure A, the ALT levels of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 53.87 ± 7.45, 41.23 ± 3.73, 50.93 ± 9.07, 60.20 ± 0.95, and 54.30 ± 5.12 U / L, respectively. Figure 13 As shown in B, the AST levels of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 144.60 ± 23.25, 133.63 ± 39.58, 132.93 ± 21.40, 123.83 ± 27.41 and 151.53 ± 10.50 U / L, respectively. Figure 13 As shown in Figure C, the CR values ​​of mice in the WT group (normal mouse control group), AD control group, PDA@M / K (bionic nanodelivery system control group), PDA@R@M / K and PDA@I@M / K groups were 4.20 ± 1.05, 1.27 ± 0.80, 2.43 ± 1.16, 1.23 ± 0.76 and 2.47 ± 0.38 U / L, respectively. Figure 13 As shown in Figure D, the BUN levels in the mice of each group were 9.93 ± 1.38, 9.56 ± 1.07, 10.53 ± 1.83, 10.00 ± 0.33, and 10.22 ± 1.27 U / L, respectively. The results indicate that ALT and AST levels were within the normal range, indicating no liver function impairment; CR and BUN levels were normal, indicating no kidney function impairment. Therefore, the biomimetic nano-formulation of traditional Chinese medicine in this invention has no significant toxicity to liver and kidney function.

[0099] The results showed that the liver and kidney functions of mice in each group were within the safe range. The biomimetic nano-formulation of traditional Chinese medicine in this invention had no obvious toxicity to major organs, blood system and liver and kidney function, and had good biosafety.

[0100] In summary, the biomimetic nano-formulation of traditional Chinese medicine of the present invention exhibits good biocompatibility and has potential for clinical application.

[0101] Furthermore, the storage stability of the biomimetic nano-formulation of traditional Chinese medicine in this invention was further investigated to evaluate its feasibility for practical application.

[0102] Experiment 7: Stability Experiment of Bionic Nanodelivery System Experimental Methods: Following the method in Example 1, without adding any active ingredients of traditional Chinese medicine in the membrane coating step, a microglial cell membrane (PDA@M / K) coated with RAGE peptide was prepared on the surface of PDA. The above PDA@M / K was divided into three groups and mixed with HEPES, 5% glucose solution (5% Glu), and 10% serum-containing culture medium, respectively. The mixtures were incubated in a constant temperature shaker at 37°C and 60 rpm. The hydrated particle size and polydispersity index (PDI) of the biomimetic nanoparticles of traditional Chinese medicine in each group were measured at 1h, 2h, 4h, 8h, 24h, 36h, 48h, and 72h to investigate the stability of the biomimetic nanoparticles of traditional Chinese medicine of the present invention.

[0103] The results showed that the HEPES group results were as follows: Figure 14 As shown in Figure A, the particle size of PDA@M / K remained around 250 nm within 48 h, and the PDI was less than 0.2, showing no significant change; the results for the 5% Glu group are as follows. Figure 14 As shown in Figure B, the particle size and PDI of PDA@M / K remained stable within 72 h, with no obvious aggregation or precipitation observed; the results for the 10% FBS group are as follows. Figure 14 As shown in Figure C, the particle size and PDI of PDA@M / K did not change significantly within 24 h.

[0104] The results showed that the hydrated particle size and PDI of PDA@M / K did not change significantly in different isotonic solutions, nor did they change much in 10% serum-containing medium, indicating that it has good stability.

[0105] In summary, the biomimetic nanoformulation of this invention can efficiently load active ingredients of traditional Chinese medicine, significantly improving their intrabrain delivery efficiency. Compared with free drugs, this formulation can effectively cross the blood-brain barrier, accumulate in inflammatory lesions in the brain, significantly improve nesting behavior in AD model mice, and effectively reduce Aβ plaque deposition in the brain. Simultaneously, this formulation exhibits good biosafety and stability. The biomimetic nanoformulation of this invention significantly improves the brain delivery efficiency and therapeutic effect of active ingredients of traditional Chinese medicine.

Claims

1. A biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases, characterized in that, The biomimetic nano-formulation of traditional Chinese medicine comprises: an active ingredient of traditional Chinese medicine and a biomimetic nano-delivery system, wherein the active ingredient of traditional Chinese medicine is icariin or rosmarinic acid; the biomimetic nano-delivery system comprises: a polydopamine nanocore, a microglia membrane coated on the surface of the polydopamine nanocore, and a receptor-binding peptide of advanced glycation end products modified on the surface of the microglia membrane; the active ingredient of traditional Chinese medicine is loaded onto the polydopamine nanocore.

2. The biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 1, characterized in that, The expression level of chemokine receptor 4 on the surface of the microglia was upregulated by lipopolysaccharide prestimulation.

3. The biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 1, characterized in that, The receptor-binding peptide for advanced glycation end products is KLVFFAEDC.

4. The biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 1, characterized in that, The polydopamine nanocore is combined with the active ingredient of the traditional Chinese medicine through π-π stacking.

5. The biomimetic nano-formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 1, characterized in that, The mass ratio of the active ingredients of the traditional Chinese medicine to the polydopamine nanocore is 5-20:

1.

6. A method for preparing a biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases as described in any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Prepare an aqueous solution of polydopamine nanocore and a solution of active ingredients of traditional Chinese medicine. Mix the solution of active ingredients of traditional Chinese medicine and the aqueous solution of polydopamine nanocore in a certain proportion to obtain drug-loaded polydopamine nanocore. Step 2: Extraction of microglia pretreated with lipopolysaccharide: Microglia were seeded and cultured, pretreated with lipopolysaccharide, and collected by centrifugation. The microglia membrane was extracted using a hypotonic lysis combined with differential centrifugation method. Step 3: Mix the advanced glycation end product receptor-binding peptide with the microglia membrane described in Step 2, so that the advanced glycation end product receptor-binding peptide modifies the surface of the microglia membrane to obtain a peptide-modified microglia membrane. Step 4: Mix the peptide-modified microglia cell membrane described in Step 3 with the drug-loaded polydopamine nanocore described in Step 1, and obtain a biomimetic nano-formulation of traditional Chinese medicine by ultrasonication and extrusion.

7. The method for preparing the biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 6, characterized in that, The conditions for lipopolysaccharide pretreatment are as follows: the concentration of lipopolysaccharide is 0-5 μg / mL, and the treatment time is 12-48 h.

8. The method for preparing the biomimetic nanoparticle formulation of traditional Chinese medicine for treating neurodegenerative diseases according to claim 7, characterized in that, The mass ratio of the peptide-modified microglia to the drug-loaded polydopamine nanocore is 1:

2.

9. The use of the biomimetic nanoparticle of traditional Chinese medicine prepared by the method of preparing the biomimetic nanoparticle of traditional Chinese medicine for treating neurodegenerative diseases according to any one of claims 1-5 or any one of claims 6-8 in the preparation of a drug for treating neurodegenerative diseases.

10. The application according to claim 9, characterized in that, The neurodegenerative diseases mentioned include Alzheimer's disease or Parkinson's disease.

Images