Traditional Chinese medicine compound preparation based on hybrid vesicles and application thereof

Abstract

The invention designs a medicinal preparation, and relates to a traditional Chinese medicine compound preparation based on extracellular vesicles and application thereof. According to the preparation method, extracellular vesicles are utilized, hybrid vesicles are prepared, compound effective components of the rhizoma coptidis detoxification decoction are further jointly delivered, the designed targeted compound preparation can effectively penetrate through a blood brain barrier to efficiently target a nervous system so as to achieve better druggability, stronger targeting and more excellent drug effect, the scheme preparation process is simple, the in-vivo and in-vitro drug effect is remarkable, and the preparation method is suitable for large-scale popularization and application. And the method has ideal popularization and application prospects.

Description

Technical Field

[0001] The invention designs a drug preparation and relates to a traditional Chinese medicine compound preparation based on extracellular vesicles and an application thereof. Background Art

[0002] Huanglian Jiedu Decoction is one of the classic prescriptions of traditional Chinese medicine, which was first recorded in "Wai Tai Mi Yao". The prescription is mainly composed of four Chinese herbal medicines, namely, coptis chinensis, scutellaria baicalensis, phellodendron amurense and gardenia jasminoides. Among them, coptis chinensis mainly contains alkaloids, mainly berberine, bamipine, coptis chinensis alkaloids, etc. The main active ingredients of scutellaria baicalensis are flavonoid compounds, including baicalin, baicalein and wogonin. The main ingredients of phellodendron amurense include phellodendron amurense alkaloids, berberine and bamipine. The main ingredients of gardenia jasminoides are gardenia glycosides, gardenia acid, etc. In the clinical practice of traditional Chinese medicine, Huanglian Jiedu Decoction is usually used to treat diseases related to heat and heat toxins. For example, it can be used to treat hot surface infections, such as skin sores, acne, etc. It can also be used for some hot fevers and infections. In addition, it is used for inflammatory diseases, such as acute tonsillitis and inflammation of the nervous system. Although Huanglian Jiedu Decoction has certain effects in clinical applications, it has some limitations in terms of drugability. Some active ingredients have poor bioavailability and permeability, so they may be limited in gastrointestinal absorption and passing through the blood-brain barrier, affecting their effective concentration in the body and the ultimate therapeutic effect. In addition, its ingredients are complex and have different properties. How to effectively combine multiple ingredients in a stable preparation and ensure the release and absorption of these ingredients in the body is a difficult problem in the preparation technology of Huanglian Jiedu Decoction.

[0003] Exosomes are extracellular vesicles with a size of 40-160nm secreted by intracellular multivesicular bodies. They have a lipid bilayer membrane structure, so they can carry a variety of drugs. In terms of drug delivery, exosomes are naturally released by cells and have better biocompatibility and biosafety; the lipid bilayer structure of exosomes gives them a high degree of stability, which can protect the internal loaded drugs from biodegradation, enzymatic hydrolysis and immune system clearance, and improve the bioavailability of drugs; exosomes have a small size and are composed of special lipids, which enable them to effectively cross biological membranes, including cell membranes and blood-brain barriers; the surface membrane of exosomes contains a variety of proteins and sugars, which can bind to specific cell receptors to achieve targeted delivery to specific cells or tissues; exosomes are derived from their own cells and have low immunogenicity. Compared with other exogenous nanodrug carriers, exosomes are less likely to cause immune system responses and rejection reactions, which helps to improve the stability of drugs and the feasibility of long-term application. The exosome-liposome hybrid system combines the characteristics of both exosomes and liposomes, providing a novel carrier system for drug delivery. This hybrid system combines exosomes and liposomes, taking advantage of the advantages of both and overcoming their respective limitations, thereby improving the efficiency and specificity of drug delivery. Liposomes have a stable phospholipid bilayer structure and are physically stable. The exosome-liposome hybrid system combines the stability of liposomes with the biocompatibility of exosomes to ensure that the drug remains stable during transportation and storage. The surface of exosomes contains a variety of biomolecules that can be targeted to specific cells or tissues. The hybrid system obtained by combining with liposomes can obtain better cell targeting, thereby achieving precise drug delivery. The exosome-liposome hybrid system is multifunctional and can not only be used for drug delivery, but also can achieve functions such as immunomodulation and signal transmission by modifying surface molecules.

[0004] Alzheimer's disease is a progressive neurodegenerative disease. The main pathological features of the disease include β-amyloid deposition and Tau protein tangles. These abnormal changes lead to neuronal damage and brain structural degeneration. As a traditional Chinese medicine prescription, Huanglian Jiedu Decoction may have a potential role in reducing neuroinflammation, protecting neurons and inhibiting β-amyloid deposition. At present, the study of Huanglian Jiedu Decoction on neuroinflammation, especially its role in Alzheimer's disease, has been a research hotspot in recent years. The lesions of Alzheimer's disease and most brain diseases depend largely on the type and stage of progression of the disease. In Alzheimer's disease, the patient's brain gradually loses neurons and synaptic connections, mainly in the cerebral cortex and hippocampus. Due to the particularity of the lesions, current preparations for brain diseases face the problem of difficulty in penetrating the blood-brain barrier, which affects the efficacy of the drug. Even if the drug successfully crosses the blood-brain barrier, its concentration in the brain may be limited, and brain diseases often cause changes in the structure and function of brain tissue, affecting the distribution of drugs in the brain.

[0005] In summary, it is feasible to prepare hybrid vesicles based on extracellular vesicles-exosomes and load the compound preparation of the active ingredients of Huanglian Jiedu Decoction to treat Alzheimer's disease. Summary of the invention

[0006] Purpose of the Invention

[0007] The present invention provides a traditional Chinese medicine compound preparation based on hybrid vesicles for efficient co-delivery of the compound ingredients of Huanglian Jiedu Decoction and its application

[0008] Technical Solution

[0009] In order to achieve efficient co-delivery of the ingredients of Huanglian Jiedu Decoction, the specific technical solutions of the present invention are as follows:

[0010] A compound preparation based on hybrid vesicles, characterized in that the hybrid vesicles encapsulate the effective ingredients of the Huanglian Jiedu Decoction compound, and the hybrid vesicles are hybrid vesicles formed by exosomes and liposomes.

[0011] The compound preparation is characterized in that the exosomes are derived from microglia or astrocytes.

[0012] The compound preparation is characterized in that the active ingredients of the Huanglian Jiedu Decoction compound include berberine, palmatine, baicalin, magnolone or gardenia glycoside.

[0013] The compound preparation is characterized in that the membrane material ratio of liposomes in the hybrid vesicles is 4:1-20:1, and the drug-lipid ratio is 1:10-1:30; the number of exosomes and liposomes in the hybrid vesicles can be 1:1-1:4.

[0014] The compound preparation is characterized in that the ratio of liposome membrane material in the hybrid vesicle is 8:1.

[0015] The drug-lipid ratio is 1:30; the number of exosomes and liposomes in the hybrid vesicles can be 1:2.

[0016] The compound preparation is characterized in that the liposomes include PC, DSPE-PEG2000, Tf-DSPE-PEG2000 and CHOL, and are prepared by a thin film hydration method.

[0017] The compound preparation is characterized in that the liposomes are prepared by the following steps: fully dissolving PC, CHOL, Tf-DSPE-PEG2000 or DSPE-PEG2000 in chloroform, removing chloroform by rotary evaporation at 40°C for 30 minutes to obtain a dry translucent lipid film, then adding an aqueous solution of the active ingredients of the Huanglian Jiedu Decoction compound and 10 mL of PBS to hydrate the dry lipid film, and fully mixing the hydrated solution by vortex oscillation, then performing probe ultrasound, starting for 3 seconds, stopping for 3 seconds, using an ultrasound power of 20%, and ultrasound for 30 minutes.

[0018] The compound preparation is used in preparing medicines for treating brain diseases.

[0019] The application is characterized in that the brain disease includes Alzheimer's disease, Parkinson's disease or glioma.

[0020] The preparation method of the compound preparation is as follows:

[0021] 1) Prepare liposomes by thin film dispersion method, encapsulate one of the effective ingredients in Huanglian Jiedu Decoction, and set aside for use.

[0022] 2) Extract exosomes by size exclusion method.

[0023] 3) The liposomes and exosomes obtained in step 1) and step 2) are hybridized in a water bath at a certain ratio, and another effective ingredient in Huanglian Jiedu Decoction is added at the same time.

[0024] Beneficial Effects

[0025] The present invention is based on microglial exosomes, and the active ingredients of Huanglian Jiedu Decoction are loaded into liposomes and exosomes through membrane fusion to form hybrid vesicles to deliver the active ingredients of Huanglian Jiedu Decoction. The particle size of the prepared compound preparation is about 200nm, PdI < 0.3, and the dispersibility is good. At the same time, the homing effect of exosomes and transferrin modification are used to achieve efficient targeting. Combining the above advantages, the final compound preparation has the advantages of targeting, high biocompatibility, high drug loading, etc., and can efficiently deliver the active ingredients of Huanglian Jiedu Decoction into the brain to achieve the purpose of treating diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an investigation of the hEVs-Ber / Pal preparation process described in Example 1; wherein: A is the change in particle size after hybridization of exosomes and liposomes according to different particle number ratios in Example 1; B is: after hybridization of exosomes and liposomes according to different particle number ratios in Example 1; C is: the change in Zate potential of the transmission electron microscopy image of the hybrid vesicles obtained in Example 1; D is: the optical resonance energy transfer obtained by different hybridization ratios of different exosomes and liposomes in Example 1.

[0027] Figure 2 The in vitro anti-inflammatory effect of hEVs-Ber / Pal described in Example 2; wherein A is the change of the inflammatory indicator interleukin-1β (L-1β); B is the change of the inflammatory indicator interleukin-10 (IL-10); C is the change of the inflammatory indicator tumor necrosis factor (TNF-α); D is the change of the inflammatory indicator interleukin-4 (IL-4); E is the change of nitric oxide (NO); * is the comparison between the model group (Lps) and the blank group (con), *** is p<0.001; # is the comparison between each drug administration group and the model group (Lps), # is p<0.05, ## is p<0.01, and ### is p<0.001.

[0028] Figure 3 The in vivo brain targeting of Tf-hEVs-Ber / Pal described in Example 3; A is a live imaging picture after tail vein injection, B is a quantitative analysis of fluorescence intensity of the live imaging picture; C is a volume imaging picture of the brain; D is the result of brain fluorescence data analysis, from which it can be seen that the preparation has brain targeting;

[0029] Figure 4 It is the in vivo anti-Alzheimer's disease efficacy of Tf-hEVs-Ber / Pal described in Example 4; wherein A is the escape latency of the water maze of each group of mice after one course of treatment of mice with the hybrid vesicles obtained in Example 4; B is the platform crossing time of the water maze of each group of mice after one course of treatment of mice with the hybrid vesicles obtained in Example 4; C is the target quadrant occupancy rate of the water maze of each group of mice after one course of treatment of mice with the hybrid vesicles obtained in Example 4; D is the average movement speed of mice in the water maze of each group of mice after one course of treatment of mice with the hybrid vesicles obtained in Example 4; E is the total distance traveled by mice in the water maze of each group of mice after one course of treatment of mice with the hybrid vesicles obtained in Example 4. DETAILED DESCRIPTION

[0030] Abbreviations: berberine (Ber), palmatine (Pal), hybrid vesicles (hEVs), hybrid vesicles encapsulating berberine (hEVs-Ber), hybrid vesicles encapsulating palmatine (hEVs-Pal), hybrid vesicles encapsulating berberine and palmatine hEVs-Ber / Pal, lipopolysaccharide (Lps), magnolia alkaloids (Mag), baicalin (Bai), Tf-DSPE-PEG2000 (DSPE-PEG2000 modified with transferrin) Example 1 Preparation process of hybrid vesicles and composite preparation hEVs-Ber / Pal based on hybrid vesicles

[0031] 1. Investigation of the preparation process of hybrid vesicles

[0032] The process of liposomes was explored. First, the membrane material ratio of liposomes was investigated. The molar ratio of PC: (DSPE-PEG2000+CHOL) was set to 4:1, 8:1, 12:1, 16:1, and 20:1 to prepare liposomes. The encapsulation rate and drug loading of the drug were determined by ultraviolet spectrophotometer. Then the drug-lipid ratio was investigated. The mass ratio of drug and membrane material (drug-lipid ratio) was set to 1:10, 1:20, and 1:30 respectively. After the liposomes were prepared, the encapsulation rate and drug loading of the drug were determined by ultraviolet spectrophotometer. Finally, different ultrasonic times and different ultrasonic powers were investigated. Finally, the optimal process for the preparation of liposomes (Lip) was obtained, with the optimal membrane material ratio of 8:1, the optimal drug-lipid ratio of 1:30, the optimal ultrasonic power of 20%, and the ultrasonic time of 30min.

[0033] At the same time, microglia were cultured in a special culture medium prepared with serum that had removed exosomes, and the cell supernatant was collected for gradient centrifugation. After centrifugation, the filtrate was filtered through a 0.22 μm microporous filter membrane, and then concentrated using a 100 kDa ultrafiltration tube. The concentrate was further purified by a size exclusion chromatography column to finally obtain an exosome (Exo) solution.

[0034] The fusion mode of exosomes and liposomes was investigated, and the water bath method and the repeated freeze-thaw method were investigated respectively. Compared with the repeated freeze-thaw method, the hybrid vesicles obtained by the water bath method had a smaller change in particle size within 48 hours, and PDI was less than 0.3, indicating that the hybrid vesicles obtained by the water bath method were more stable, so the water bath method was selected to prepare the hybrid vesicles.

[0035] The hybridization ratio of Exo and Lip was explored, and the Exo and Lip particle number ratios were set to 1:1, 1:2, 1:4, 1:8, 1:16, etc. The fusion efficiency was used as an indicator to obtain the optimal fusion ratio. Too few exosomes or too many liposomes will cause self-fusion rather than exosome-liposome hybridization. Therefore, we finally found that 1:2 is the best fusion ratio, with the highest hybrid fusion efficiency. The changes in particle size and potential are shown in Figure 2. Figure 1 A and B.

[0036] Table 1: Investigation of different liposome preparation processes in Example 1.

[0037]

[0038]

[0039] 2. Preparation of hybrid vesicle-based compound preparation hEVs-Ber / Pal:

[0040] prescription:

[0041] pH 7.4 PBS

[0042] PC: 9mg

[0043] DSPE-PEG2000: 1mg

[0044] CHOL: 0.73 mg

[0045] Berberine: 0.36mg

[0046] Bamipine: 0.36 mg

[0047] Liposomes were prepared according to the conditions obtained from the above investigation steps. The molar ratio of PC: (DSPE-PEG2000+CHOL) was 8:1 and chloroform was added to fully dissolve. The chloroform was removed by rotary evaporation at 40°C for 30 minutes to obtain a dry translucent lipid film. Then 0.36 mg berberine (Ber) and 10 mL PBS were added to hydrate the dried lipid film. The hydrated solution was fully mixed by vortex oscillation and probe ultrasound was performed (on 3s, off 3s, ultrasound power 20%, ultrasound 30min), and finally Lip-Ber aqueous solution was obtained. At the same time, exosomes were extracted for use.

[0048] The Exo and Lip-Ber particle number ratio was 1:2, and the hybrid fusion was carried out by water bath method, vortexed to make it fully mixed, and 0.36 mg of bamipine (Pal) was added at the same time, and then the mixture was water bathed at 37°C for 1 hour to finally prepare the composite preparation (hEVs-Ber / Pal) described in this patent, with a total drug loading of 3.5%, and characterized. The results are as follows Figure 1 As shown, transmission electron microscopy observation ( Figure 1 C) and FRET images ( Figure 1 D) Demonstration of fusion of exosomes and liposomes.

[0049] Example 2 Study on the anti-inflammatory effect of the composite preparation of hybrid vesicles hEVs-Ber / Pal at the cellular level

[0050] hEVs, hEVs-Ber, hEVs-Pal and hEVs-Ber / Pal were prepared with reference to Example 1. In order to verify its anti-inflammatory effect in vitro, microglia were cultured and administered in groups. The groups were: blank control group, LPS, Ber, Pal, Ber / Pal physical mixture, hEVs, hEVs-Ber, hEVs-Pal, hEVs-Ber / Pal group. The cells were pre-treated with LPS for 24 hours to induce microglial inflammation, and different drugs were added and incubated for 24 hours. Three replicate wells were set up for each group, and the supernatant of cells from each group was collected to determine the concentrations of TNF-α, IL-1β, IL-4, IL-10, and NO. The absorbance was measured using an enzyme reader. The results are shown in the attached figure. Figure 2 As shown, hEVs-Ber / Pal had the best anti-inflammatory effect compared with the other groups.

[0051] Example 3 Construction of Tf-targeted modified hybrid vesicles encapsulating berberine and palmatine Tf-hEVs-Ber / Pal and in vivo imaging experiments using Tf-DSPE-PEG2000 as one of the raw materials

[0052] prescription:

[0053] exosomes

[0054] pH 7.4 PBS

[0055] PC: 9mg

[0056] Tf-DSPE-PEG2000: 1mg

[0057] CHOL: 0.73 mg

[0058] DiR: 1mg

[0059] Berberine: 0.36mg

[0060] Bamipine: 0.36 mg

[0061] Tf-Lip was prepared by thin film hydration method. PC, CHOL, Tf-DSPE-PEG2000 and DiR were fully dissolved in chloroform, and the chloroform was removed by rotary evaporation at 40°C for 30 min to obtain a dry translucent lipid film, followed by adding 0.36 mg Ber aqueous solution and 10 mL PBS to hydrate the dry lipid film, and the hydrated solution was fully mixed by vortex oscillation and then probe ultrasonication (on 3s, off 3s, ultrasonic power 20%, ultrasonication 30min), and finally Tf-Lip-Ber aqueous solution (transferrin modified liposome solution) was obtained. At the same time, microglial exosomes were extracted for use.

[0062] The ratio of Exo to Tf-Lip-Ber particles was 1:2. The hybrid fusion was carried out by water bath method, vortexed to mix thoroughly, and 0.36 mg Pal was added. Then, it was in a water bath at 37°C for 1 hour to finally obtain the desired targeted modified compound preparation (Tf-hEVs-Ber / Pal). In order to study its in vivo targeting, the preparation was labeled with DiR dye. The mice were divided into three groups according to the drug administration: DiR free dye group, hEVs-Ber / Pal group, and Tf-hEVs-Ber / Pal group. The drugs were administered by tail vein injection, and the qualitative analysis was performed using in vivo imaging technology, and the fluorescence intensity of the mouse brain was quantitatively analyzed. The results are shown in Figure 3 As shown in the figure, hEVs-Ber / Pal has a certain targeting effect due to the homing effect of Exo, and with the targeted modification of Tf (Tf-hEVs-Ber / Pal), its targeting is further enhanced.

[0063] Example 4: In vivo anti-Alzheimer's disease efficacy of Tf-hEVs-Ber / Pal using Tf-DSPE-PEG2000 as one of the raw materials to construct Tf-targeted modified hybrid vesicles encapsulating berberine and palmatine

[0064] Tf-hEVs-Ber / Pal was prepared as in Example 3, and the in vivo anti-Alzheimer's efficacy of the compound preparation was studied. Aβ protein was injected into the mouse hippocampus to establish an Alzheimer's disease mouse model. The mice in the experimental group were generally anesthetized using mouse anesthetics. After the mice were anesthetized, they were fixed on a mouse brain stereotaxic instrument. According to the mouse brain atlas, the location of the hippocampus was determined. The skin on the mouse head was cut open to expose the skull, and the injection needle was positioned in the mouse hippocampus using a stereotaxic instrument. Aβ protein was slowly injected into the mouse hippocampus using a microinjection pump. After the injection, the skin on the mouse head was sutured and recovered for one week.

[0065] The drug administration groups were wild-type mice, model mice, Ber, Pal, Ber / Pal, Tf-hEVs, Tf-hEVs-Ber, Tf-hEVs-Pal, hEVs-Ber / Pal, and Tf-hEVs-Ber / Pal. The drug was administered once every two days for a total of 5 times.

[0066] The water maze is the most commonly used method to evaluate spatial learning and memory function, which is closely related to hippocampal synaptic plasticity. The learning and memory ability of mice was tested in two parts: the positioning navigation experiment and the spatial exploration experiment. The mice first received water maze training, and four experiments were conducted every day from four different fixed starting positions until most animals quickly reached the platform. Each training lasted 60 seconds or until the animal found the platform. After each training, all animals adapted to the platform for 10 seconds (including animals that did not find the platform). The positioning navigation experiment lasted for 5 days. On the sixth day, the platform was removed from the pool and a spatial exploration experiment was conducted. Each mouse was placed in a position far away from the quadrant where the platform was located, and the time spent in the platform quadrant within 60 seconds and the number of times the mouse crossed the platform were observed. The experimental results are shown in the attached. Figure 4 (AC and E) Compared with the control group, hEVs-Ber / Pal and Tf-hEVs-Ber / Pal significantly increased the time mice stayed in the target quadrant and the number of times they crossed the platform, and the Tf-targeted modified preparation had a better therapeutic effect.

[0067] The novel object localization experiment uses the natural tendency of mice to interact with new objects to reveal hippocampal-dependent learning and memory. Mice were fixed in an open field 6×6 cm away from their respective walls in front of two different objects. The mice were placed diagonally facing the wall and allowed to freely investigate the field and objects for 5 minutes. After the mice rested for 20 minutes, one of the two objects was replaced with a new location, and the animals were placed in the same way and allowed to freely investigate the field and objects for 5 minutes. The investigation time of each mouse on each object was recorded and analyzed. The final experimental results are shown in the attached figure. Figure 4 As shown in (D), compared with the control group, hEVs-Ber / Pal and Tf-hEVs-Ber / Pal significantly increased the proportion of mice's exploration time of new objects, and the Tf-targeted modified preparation had a better therapeutic effect.

[0068] Example 5: Preparation of hybrid vesicles loaded with baicalin and bamipine Tf-hEVs-Bai / Pal

[0069] prescription:

[0070] pH 7.4 PBS

[0071] PC: 9mg

[0072] Tf-DSPE-PEG2000: 1mg

[0073] CHOL: 0.73 mg

[0074] Baicalin: 0.44mg

[0075] Bamipine: 0.36 mg

[0076] Lip was prepared by thin film hydration method. PC, CHOL and Tf-DSPE-PEG2000 raw materials were fully dissolved in chloroform, and the chloroform in the rotary evaporation bottle was removed by rotary evaporation at 40°C for 30 minutes to obtain a dry translucent lipid film, followed by adding 0.44 mg baicalin and 10 mL PBS to hydrate the dry lipid film, and the hydrated liposome solution was vortexed and subjected to probe ultrasound (on 3s, off 3s, ultrasound power 20%, ultrasound 30 minutes), and finally a baicalin-loaded Lip aqueous solution was obtained.

[0077] Microglia were cultured in a special culture medium prepared with serum that had been stripped of exosomes. The cell supernatant was collected for gradient centrifugation. After centrifugation, the filtrate was filtered through a 0.22 μm microporous filter membrane and then concentrated using a 100 kDa ultrafiltration tube. The concentrate was further purified by a size exclusion chromatography column to finally obtain an exosome solution.

[0078] Lip and Exo were fused by water bath method, and according to the particle number ratio of the two, they were vortexed to mix them thoroughly, and 0.36 mg of bamipine was added, and then the mixture was in a water bath at 37°C for 1 hour to finally obtain a composite preparation containing the active ingredients of Huanglian Jiedu Decoction. Example 6: Preparation of hybrid vesicles encapsulating baicalin and magnolia alkaloids Tf-hEVs-Bai / Mag

[0079] prescription:

[0080] pH 7.4 PBS

[0081] PC: 9mg

[0082] Tf-DSPE-PEG2000: 1mg

[0083] CHOL: 0.73 mg

[0084] Baicalin: 0.44mg

[0085] Magnolia alkaloids: 0.34 mg

[0086] Lip was prepared by thin film hydration method. PC, CHOL and Tf-DSPE-PEG2000 raw materials were fully dissolved in chloroform, and chloroform was removed by rotary evaporation at 40°C for 30 min to obtain a dry translucent lipid film, followed by adding 0.44 mg baicalin and 10 mL PBS to hydrate the dry lipid film. The hydrated liposome solution was vortexed and then probe ultrasonicated (on for 3 s, off for 3 s, ultrasonic power of 20%, ultrasonic for 30 min), and finally a baicalin-loaded Lip aqueous solution was obtained.

[0087] Microglia were cultured in a special culture medium prepared with serum that had been stripped of exosomes. The cell supernatant was collected for gradient centrifugation. After centrifugation, the filtrate was filtered through a 0.22 μm microporous filter membrane and then concentrated using a 100 kDa ultrafiltration tube. The concentrate was further purified by a size exclusion chromatography column to finally obtain an exosome solution.

[0088] The water bath method was used for the hybridization of Lip and Exo. According to the particle number ratio of the two, they were mixed and 0.34 mg of magnolia alkaloids was added. Then, the mixture was in a water bath at 37°C for 1 hour. Finally, a compound preparation containing the active ingredients of Huanglian Jiedu Decoction was obtained.

Claims

1. A compound preparation based on hybrid vesicles, characterized in that: The hybrid vesicles encapsulate the effective ingredients of the Huanglian Jiedu Decoction compound, and the hybrid vesicles are hybrid vesicles formed by exosomes and liposomes.

2. The compound preparation according to claim 1, characterized in that: The exosomes are derived from microglia or astrocytes.

3. The compound preparation according to claim 1, characterized in that: The effective ingredients of the Huanglian Jiedu Decoction compound include berberine, palmatine, baicalin, magnolamine or gardenia glycoside.

4. The compound preparation according to claim 1, characterized in that: The membrane-to-material ratio of liposomes in hybrid vesicles is 4:1-20:

1. The drug-lipid ratio is 1:10-1:30; the number of exosomes and liposomes in the hybrid vesicles can be 1:1-1:

4.

5. The compound preparation according to claim 4, characterized in that: The ratio of liposome membrane to material in the hybrid vesicles is 8:

1. The drug-lipid ratio is 1:30; the number of exosomes and liposomes in the hybrid vesicles can be 1:

2.

6. The compound preparation according to any one of claims 1 to 5, characterized in that: The liposomes include PC, DSPE-PEG2000, Tf-DSPE-PEG2000 and CHOL, and are prepared by thin film hydration method.

7. The compound preparation according to claim 6, characterized in that: The liposomes are prepared by the following steps: fully dissolving PC, CHOL, Tf-DSPE-PEG2000 or DSPE-PEG2000 in chloroform, removing the chloroform by rotary evaporation at 40°C for 30 min to obtain a dry translucent lipid film, then adding an aqueous solution of the active ingredients of the Huanglian Jiedu Decoction compound and 10 mL of PBS to hydrate the dry lipid film, and then performing probe ultrasound to fully mix the hydrated solution by vortex oscillation, starting for 3 s, stopping for 3 s, using an ultrasound power of 20%, and ultrasound for 30 min.

8. Use of the compound preparation according to claims 1-7 in the preparation of drugs for treating brain diseases.

9. The use according to claim 8, characterized in that: The brain diseases include Alzheimer's disease, Parkinson's disease or glioma.

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