Traditional Chinese medicine nano-delivery system with function of targeted inhibition of A beta protein aggregation and preparation method and application of traditional Chinese medicine nano-delivery system

Abstract

The invention relates to the field of drug carrier design, in particular to a traditional Chinese medicine nano delivery system with a targeted A beta protein aggregation inhibition function as well as a preparation method and application of the traditional Chinese medicine nano delivery system. The traditional Chinese medicine nano-delivery system is a traditional Chinese medicine nano-carrier which is based on polypeptide A beta 16-21-r9 as shown in the amino acid sequence SEQ ID NO.1 and has the cell barrier penetrating and amyloid protein capturing capacity, and beta amyloid protein aggregation activity can be regulated in a targeted mode through the carrier. According to the traditional Chinese medicine nano delivery system prepared by the preparation method disclosed by the invention, the efficient delivery of a drug carrier can be realized, the blood brain barrier can be quickly penetrated, and the A beta protein can be accurately targeted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug carrier design, and particularly relates to a traditional Chinese medicine nano delivery system with the function of targeted inhibition of Aβ protein aggregation and a preparation method and application thereof. BACKGROUND

[0002] In the field of drug carrier design for inhibiting β amyloid protein (Aβ protein) aggregation, the prior art mainly inhibits Aβ aggregation through the hydrophobic and electrostatic effects of the side chains on the outside of the carrier, or releases inhibitor molecules to achieve the inhibition of aggregation. For example, high molecular polymers, polyoxometalates, macrocyclic compounds, two-dimensional sheet materials, etc. are used to directly bind or act on Aβ protein through their own hydrophobic and electrostatic effects, to destroy Aβ aggregation and reduce its neurotoxicity. However, the carriers designed by the above-mentioned materials have the defects of low drug delivery efficiency and poor Aβ protein targeting ability, resulting in insufficient effect of targeted regulation of Aβ protein aggregation activity. In addition, some drug carriers use polypeptides as targeting tools to interact with Aβ protein to inhibit its aggregation, but such drug carriers have poor blood-brain barrier penetration ability and cannot achieve drug brain delivery. At present, there are many drug carriers that can penetrate the blood-brain barrier, such as liposomes or nanoparticles modified with insulin, transferrin, cationized albumin, cationized immunoglobulin, etc., which can achieve brain delivery of various drugs and protect the drugs from enzyme degradation, but such drug carriers cannot target Aβ protein lesion areas after entering the brain, and the drug utilization efficiency is still insufficient. SUMMARY

[0003] In order to solve the above problems, the present application provides a traditional Chinese medicine nano delivery system with the function of targeted inhibition of Aβ protein aggregation as a drug carrier, which can quickly bind Aβ protein through Aβ targeting peptide KLVFFA in and outside cells to inhibit its aggregation and slowly release drugs, thereby achieving the effect of drug delivery into cells and outside cells. The traditional Chinese medicine nano delivery system prepared by the preparation method of the present application can achieve efficient drug carrier delivery, rapid penetration of the blood-brain barrier, and precise targeting of Aβ protein.

[0004] The present application provides a preparation method of a traditional Chinese medicine nano delivery system with the function of targeted inhibition of Aβ protein aggregation, comprising the following steps: (1) dissolving amphiphilic phospholipid and cholesterol in an organic solvent to prepare a first solution; (2) dissolving a hydrophobic drug in an organic solvent to prepare a second solution; (3) mixing the first solution and the second solution to obtain a third solution; (4) sequentially performing water bath rotary evaporation and drying on the third solution, and then performing water bath hydration with PBS buffer to obtain a fourth solution; (5) The fourth solution was ultrasonically broken and then filtered to obtain traditional Chinese medicine nanoliposomes; (6) The obtained traditional Chinese medicine nanoliposomes and polypeptide Aβ were combined 16-21 -r9 was co-incubated in PBS buffer, and after incubation, the traditional Chinese medicine nanodelivery system was obtained by dialysis purification.

[0005] Preferably, the mass ratio of the amphiphilic phospholipid, cholesterol and organic solvent in step (1) is (10~14):(4~6):(35000~40000). The amphiphilic phospholipid is composed of DSPE-PEG2000-MAL and soybean lecithin, and the mass ratio of DSPE-PEG2000-MAL to soybean lecithin is (2~4):1.

[0006] Preferably, the mass ratio of the hydrophobic drug to the organic solvent in step (2) is 1:(35000~40000). The hydrophobic drug is at least one of curcumin, ginsenoside Rg3, paclitaxel, triptolide, etc., and is a single component extracted from traditional Chinese medicinal materials.

[0007] Preferably, the mass ratio of the first solution to the second solution in step (3) is (15~25):1.

[0008] Preferably, the temperature of the water bath rotary evaporation in step (4) is 40~42℃, the time is 2~10min, and the rotation speed is 100~150rpm.

[0009] Preferably, the drying method in step (4) is vacuum drying, with a vacuum degree of 120~140Pa, a drying time of 8~12h, and a temperature of 30~40℃.

[0010] Preferably, the water bath hydration temperature in step (4) is 40~42℃, the time is 45~60min, and the rotation speed is 15~30rpm.

[0011] Preferably, the volume ratio of the third solution to the PBS buffer is 30:(15~25).

[0012] Preferably, the traditional Chinese medicine nanoliposomes and polypeptide Aβ described in step (6) 16-21 The mass ratio of -r9 to PBS buffer is (20~22):(1~3):(18000~22000).

[0013] Preferably, the polypeptide Aβ 16-21 The amino acid sequence of -r9 is shown in SEQ ID NO.1.

[0014] Preferably, the incubation temperature in step (6) is 25~37℃ and the incubation time is 4~12h.

[0015] Preferably, the molecular weight of the dialysis purified in step (6) is 30-100KD.

[0016] The present invention also provides a traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation function prepared by the above preparation method; The described traditional Chinese medicine nanodelivery system has a spherical morphology with a particle size of 100-400 nm; taking curcumin as an example, the structural schematic diagram of Cr9-LiP / curcumin is shown below. Figure 1 As shown.

[0017] The described traditional Chinese medicine nanodelivery system is self-assembled from amphiphilic phospholipids, cholesterol, and hydrophobic active ingredients of traditional Chinese medicine. A click reaction is performed on the surface of this system via maleimide groups (MAL) and cysteine ​​residues (C) at the Aβ-targeting peptide terminus, thereby modifying it with a peptide possessing both "penetration + targeting" dual effects. This system can be used to release active ingredients of traditional Chinese medicine to specific tissues, achieving therapeutic effects for certain diseases. Taking curcumin as an example, a schematic diagram of the preparation process of Cr9-LiP / curcumin is shown below. Figure 2 As shown.

[0018] Liposomes composed of DSPE-PEG2000-MAL, soybean lecithin, and cholesterol can deliver drugs by encapsulating hydrophobic active ingredients of traditional Chinese medicine in liposomes and then slowly releasing them outside and inside cells. In this traditional Chinese medicine nanodelivery system, the hydrophobic drug accounts for 1-3% of the total mass, and the system does not contain any other carriers.

[0019] The present invention also provides the application of the traditional Chinese medicine nanodelivery system prepared by the above preparation method in the preparation of drugs that target and inhibit the aggregation of Aβ protein.

[0020] In addition, the present invention also provides a method for treating related diseases using the drug delivery system, comprising the following steps: The system solution is injected into the lesion site or circulatory system of the animal. Once 50% of the drug has been released, the overall treatment effect of the system will be tested.

[0021] It contains at least the following beneficial technical effects: This invention designs a novel peptide chain composed of a cell-penetrating peptide (r9) and an Aβ-targeting peptide (CKLVFFA) in tandem (here, C represents the amino acid used to connect the nanoliposomes, and the other 6 are Aβ-targeting polypeptides; to prevent arginine degradation, the arginine used in the cell-penetrating peptide (r9) is all D-type; the peptide chain was synthesized by Jier Biochemical (Shanghai) Co., Ltd., with a purity of 95%), denoted as peptide Aβ. 16-21-r9, whose amino acid sequence is shown in SEQ ID NO.1; the cysteine ​​(C) residue at the end of the Aβ-targeting peptide reacts with the maleimide (MAL) group on the surface of the nanoliposome, so that the Aβ-targeting peptide and the nanoliposome are linked by a thioether bond (CSC), thereby modifying the peptide chain designed in this invention onto the surface of the drug-loaded nanoliposome. The cell-penetrating peptide r9 allows the entire drug carrier to cross the blood-brain barrier and reach the central nervous system in the brain; then, the Aβ-targeting peptide precisely locates the accumulation site of Aβ protein in the brain, and the binding of the Aβ-targeting peptide to Aβ protein prevents its further accumulation. Simultaneously, the penetrating effect of r9 allows the drug components in the carrier to be precisely delivered to the nerve cells at the Aβ accumulation site, achieving efficient drug delivery, rapid penetration of the blood-brain barrier, and precise targeting of Aβ protein.

[0022] This invention utilizes a thin-film dispersion method to obtain a novel traditional Chinese medicine delivery system with uniform particle size, good dispersibility, stability, and safety. The manufacturing process is simple, low-cost, and pollution-free, making it suitable for industrial production and showing promising application prospects. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of Cr9-LiP / curcumin.

[0024] Figure 2 This is a schematic diagram of the preparation process of Cr9-LiP / curcumin.

[0025] Figure 3 A schematic diagram illustrating the penetration of Cr9-LiP / curcumin across the blood-brain barrier.

[0026] Figure 4 This is a schematic diagram illustrating the role of Cr9-LiP / curcumin in inhibiting Aβ aggregation and reducing nerve damage in animals.

[0027] Figure 5 The figure shows the average particle size data of Cr9-LiP / curcumin and its different control groups. In the figure, a and e represent the average particle size determination results of Lip, Lip / Cur, r9-Lip / Cur, CA-Lip / Cur and Cr9-Lip / Cur solutions dispersed in 10.0 mM PBS buffer solution.

[0028] Figure 6 The figure shows the particle size stability data of Cr9-Lip / curcumin and its different control groups. In the figure, a and e represent the average particle size stability changes of Lip, Lip / Cur, r9-Lip / Cur, CA-Lip / Cur and Cr9-Lip / Cur solutions dispersed in 10.0 mM PBS buffer solution and stored at 4°C for 7 days.

[0029] Figure 7This is an image of the appearance of the Cr9-LiP / curcumin solution.

[0030] Figure 8 The figures show the ThT fluorescence data of Cr9-Lip / curcumin inhibiting Aβ aggregation. Figure a shows the changes in the relative ThT fluorescence intensity of different types of traditional Chinese medicine nanodelivery systems after co-incubation with 20.0 μM Aβ protein solution; and figure b shows the changes in the relative ThT fluorescence intensity of different concentrations of Cr9-Lip / Cur after co-incubation with 20.0 μM Aβ protein solution.

[0031] Figure 9 The figures show the average particle size data for Cr9-Lip / curcumin inhibiting Aβ aggregation. Figure a shows the particle size changes after different types of traditional Chinese medicine nanodelivery systems were co-incubated with 20.0 μM Aβ protein solution; figure b shows the particle size changes after co-incubating 1.0, 1.5, and 2.0 μg / mL Cr9-Lip / Cur with 20.0 μM Aβ protein solution. Detailed Implementation

[0032] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0033] The drug delivery carrier (Cr9-LiP / drug) designed in this invention can enter the bloodstream via injection. Because the carrier itself is a liposome, its time in the bloodstream can be appropriately prolonged. When the Cr9-LiP / drug circulates to the blood-brain barrier, the tissue penetration of r9 helps the entire Cr9-LiP / drug cross the blood-brain barrier and enter the central nervous system tissue. At this point, the Aβ-targeting peptide can locate the Cr9-LiP / drug in the Aβ protein accumulation area. The Aβ-targeting peptide on the carrier binds to the Aβ protein, preventing Aβ aggregation and reducing the inflammatory response caused by Aβ aggregation. Furthermore, through the penetration of r9, the entire Cr9-LiP / drug carrier can be delivered into the cells at the lesion site. The drug can be slowly released inside the cell, thereby controlling inflammation, prolonging neuronal survival time, and ultimately achieving efficient drug delivery, penetration of the blood-brain barrier, and precise inhibition of Aβ protein aggregation. (Curcumin is used as an example.) Figure 3 A schematic diagram illustrating the penetration of Cr9-LiP / curcumin across the blood-brain barrier. Figure 4 This is a schematic diagram illustrating the role of Cr9-LiP / curcumin in inhibiting Aβ aggregation and reducing nerve damage in animals.

[0034] Example 1 A method for preparing a traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation includes the following steps: (1) Dissolve amphiphilic phospholipids and cholesterol in dichloromethane to form the first solution; The mass ratio of the amphiphilic phospholipids, cholesterol, and organic solvent is 10:4:35000; The amphiphilic phospholipid is composed of DSPE-PEG2000-MAL and soybean lecithin, with a mass ratio of DSPE-PEG2000-MAL to soybean lecithin of 2:1.

[0035] (2) Ginsenoside Rg3 is dissolved in dichloromethane to form a second solution; the mass ratio of ginsenoside Rg3 to dichloromethane is 1:35000.

[0036] (3) Mix the first solution and the second solution at a mass ratio of 15:1 and then ultrasonically disperse for 20 min to obtain the third solution; (4) Pour 30 mL of the third solution into a rotary evaporation flask and evaporate at 100 rpm for 10 min at a water temperature of 40 °C; (5) Place the evaporating flask that has been spun dry into a vacuum drying oven at 120 Pa and dry at 30 °C for 12 h; (6) Add 15 mL of 10 mM PBS solution to the dried rotary evaporator flask, and then continue to rotate at 15 rpm for 60 min in a 42℃ water bath to obtain the fourth solution; (7) After the fourth solution was placed in an ultrasonic crusher and ultrasonically crushed for 15 minutes, the ultrasonically crushed solution was filtered through a 0.22 μm filter membrane to obtain the fifth solution, which is denoted as Chinese medicine nanoliposome.

[0037] (8) The fifth solution is mixed with the Aβ solution designed in this invention. 16-21 -r9 polypeptide powder was mixed and ultrasonicated in a PBS solution in a water bath for 4 min, and then reacted at 37℃ for 4 h to obtain the sixth solution; the mass ratio of the traditional Chinese medicine nanoliposomes, polypeptide powder and PBS solution was 20:1:18000.

[0038] (9) The sixth solution was placed in a dialysis bag with a molecular weight of 30KD and dialyzed for 48 hours to obtain a traditional Chinese medicine nanodelivery system.

[0039] Example 2 A method for preparing a traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation includes the following steps: (1) Dissolve amphiphilic phospholipids and cholesterol in dichloromethane to form the first solution; The mass ratio of the amphiphilic phospholipids, cholesterol, and organic solvent is 14:6:40000; The amphiphilic phospholipid is composed of DSPE-PEG2000-MAL and soybean lecithin, with a mass ratio of DSPE-PEG2000-MAL to soybean lecithin of 4:1.

[0040] (2) Paclitaxel is dissolved in dichloromethane to form a second solution; the mass ratio of paclitaxel to dichloromethane is 1:40000.

[0041] (3) Mix the first solution and the second solution at a mass ratio of 20:1 and then ultrasonically disperse for 25 min to obtain the third solution; (4) Pour 30 mL of the third solution into a rotary evaporation flask and evaporate at 150 rpm for 5 min at a water temperature of 42°C; (5) Place the evaporating flask that has been spun dry into a vacuum drying oven at 140 Pa and dry at 40 °C for 8 hours; (6) Add 25 mL of 10 mM PBS solution to the dried rotary evaporator flask, and then continue to rotate at 30 rpm for 45 min in a 42℃ water bath to obtain the fourth solution; (7) The fourth solution was placed in an ultrasonic crusher and ultrasonically crushed for 20 minutes. The resulting solution was then filtered through a 0.22 μm filter membrane to obtain the fifth solution, which was denoted as Chinese medicine nanoliposomes.

[0042] (8) The fifth solution is mixed with the Aβ solution designed in this invention. 16-21 -r9 polypeptide powder was mixed and ultrasonicated in a PBS solution in a water bath for 5 min, and then reacted at 25℃ for 12 h to obtain the sixth solution; the mass ratio of the traditional Chinese medicine nanoliposomes, polypeptide powder and PBS solution was 22:3:22000.

[0043] (9) The sixth solution was placed in a dialysis bag with a molecular weight of 50KD and dialyzed for 48 hours to obtain a traditional Chinese medicine nanodelivery system.

[0044] Example 3 A method for preparing a traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation includes the following steps: (1) Dissolve amphiphilic phospholipids and cholesterol in dichloromethane to form the first solution; The mass ratio of the amphiphilic phospholipids, cholesterol, and organic solvent is 12:5:39000; The amphiphilic phospholipid is composed of DSPE-PEG2000-MAL and soybean lecithin, with a mass ratio of DSPE-PEG2000-MAL to soybean lecithin of 3:1.

[0045] (2) Dissolve curcumin in dichloromethane to form a second solution; the mass ratio of curcumin to dichloromethane is 1:39000.

[0046] (3) Mix the first solution and the second solution at a mass ratio of 25:1 and then ultrasonically disperse for 22 min to obtain the third solution; (4) Pour 30 mL of the third solution into a rotary evaporation flask and evaporate at 120 rpm for 5-10 min at a water temperature of 40 °C; (5) Place the evaporating flask that has been spun dry into a vacuum drying oven at 133 Pa and dry at 35 °C for 10 h; (6) Add 22 mL of 10 mM PBS solution to the dried rotary evaporator flask, and then continue to rotate at 25 rpm for 50 min in a 40℃ water bath to obtain the fourth solution; (7) The fourth solution was placed in an ultrasonic crusher and ultrasonically crushed for 18 minutes. The resulting solution was then filtered through a 0.22 μm filter membrane to obtain the fifth solution, which was denoted as Chinese medicine nanoliposomes.

[0047] (8) The fifth solution is mixed with the Aβ solution designed in this invention. 16-21 -r9 polypeptide powder was mixed and ultrasonicated in a PBS solution in a water bath for 3 min, and then reacted at 20℃ for 8 h to obtain the sixth solution; the mass ratio of the traditional Chinese medicine nanoliposomes, polypeptide powder and PBS solution was 21:2:20000.

[0048] (9) The sixth solution was placed in a dialysis bag with a molecular weight of 100KD and dialyzed for 48 hours to obtain a traditional Chinese medicine nanodelivery system.

[0049] Experimental Example 1 Comparative experiments were conducted to prepare different types of traditional Chinese medicine nanodelivery systems (Lip, Lip / Cur, r9-Lip / Cur, CA-Lip / Cur, and Cr9-Lip / Cur solutions, respectively) according to the preparation method described in Example 3 of this invention. Lip represents unencapsulated liposomes; Lip / Cur represents liposomes encapsulated with curcumin; r9-Lip / Cur represents liposomes with r9 surface modification; CA-Lip / Cur represents liposomes with CKLVFFA surface modification; and Cr9-Lip / Cur represents the traditional Chinese medicine nanodelivery system obtained in Example 3 of this invention.

[0050] The above types of traditional Chinese medicine nanodelivery systems were measured using a dynamic light scattering particle size analyzer. 1. Take more than 1 mL of each solution and measure it using a dynamic light scattering particle size analyzer. The results are as follows: Figure 5 As shown, the particle size of Cr9-Lip / Cur is about 180 nm, while the particle sizes of Lip, Lip / Cur, r9-Lip / Cur, and CA-Lip / Cur are all less than 180 nm.

[0051] 2. The prepared Lip, Lip / Cur, r9-Lip / Cur, CA-Lip / Cur, and Cr9-Lip / Cur solutions were dispersed in 10.0 mM PBS buffer (pH 7.3) and incubated at 4°C. The particle size of the solution was measured every 24 hours. The results are as follows: Figure 6 As shown, the average particle size variation range of each group of traditional Chinese medicine nanodelivery systems was <20 nm within 7 days, indicating that the traditional Chinese medicine nanodelivery system prepared according to the method mentioned in this invention can be stably stored at 4℃ for 7 days.

[0052] 3. Place the Cr9-Lip / Cur solution in a glass bottle and take a picture with a camera. The results are as follows. Figure 7 As shown, the Cr9-Lip / Cur solution is a homogeneous and clear solution, indicating that the solution prepared according to the method mentioned in this invention is a stable solution for a traditional Chinese medicine nanodelivery system.

[0053] 4. The various types of traditional Chinese medicine nanodelivery systems prepared above were co-incubated with 20.0 μM Aβ protein solution (the mass ratio of traditional Chinese medicine nanodelivery system to Aβ protein was 0.4:27, where the mass of the traditional Chinese medicine nanodelivery system was calculated based on the mass of curcumin) in a shaker at 37℃. 20.0 μL of sample solution was taken at different time points, diluted with 1 mL of ultrapure water, mixed thoroughly, and the average particle size change of the liposomes and Aβ solution was measured using a dynamic light scattering instrument. Particle size change curves were plotted based on the sampling time and average particle size. The experimental results are as follows: Figure 8 As shown in Figure a, all groups of liposomes exhibited the effect of inhibiting Aβ aggregation. After the addition of liposomes, the average particle size of Aβ aggregates decreased in all groups. Among them, the Cr9-Lip / Cur group had the smallest average particle size, indicating that the degree of Aβ protein aggregation in this group was the lowest, indicating the best inhibition effect on aggregation.

[0054] Experiment Example 2 A comparative experiment was set up. The mass of curcumin in the Cr9-Lip / Cur solution of the traditional Chinese medicine nanodelivery system prepared in Example 3 was determined by UV-Vis spectrophotometry. The concentration was calculated using the mass of curcumin as a standard. The solution was diluted with PBS solution to a concentration of 1.0, 1.5, and 2.0 μg / mL. 300 μL of each diluted solution was incubated with 20.0 μM Aβ protein (i.e., 27 μg Aβ powder) in a shaker at 37°C.

[0055] 20.0 μL of sample solution was taken at different times, diluted with 1 mL of ultrapure water, mixed thoroughly, and then the average particle size change of liposomes and Aβ solution was measured using a dynamic light scattering instrument. Particle size change curves were plotted based on sampling time and average particle size. Figure 8As shown in b, Cr9-Lip / Cur showed good inhibitory effects on Aβ protein particle size growth at all concentrations, and the effect gradually improved with increasing concentration, with the 2.0 μg / mL group showing the best inhibitory effect.

[0056] Experimental Example 3 The Lip, Lip / Cur, r9-Lip / Cur, CA-Lip / Cur, and Cr9-Lip / Cur solutions prepared in Example 1 were co-incubated with 20.0 μM Aβ protein solution (the mass ratio of each solution to Aβ protein solution was 0.6:27) in a shaker at 37°C. At 0, 2, 4, 8, 24, 32, 48, and 72 h, 20.0 μL of sample solution was taken and mixed with 180.0 μL of 20.0 μM ThT fluorescent dye. The fluorescence intensity changes of ThT were measured using a fluorometer, and fluorescence intensity change curves were plotted based on the sampling time and fluorescence intensity. Figure 9 As shown in Figure a, all groups of liposomes exhibited the effect of inhibiting Aβ aggregation. Among them, the Cr9-Lip / Cur group designed in this invention had the lowest fluorescence intensity, indicating that it had the best effect in inhibiting Aβ aggregation (aggregation inhibition efficiency > 80%).

[0057] A comparative experiment was set up. The mass of curcumin in the Cr9-Lip / Cur solution of the traditional Chinese medicine nanodelivery system prepared in Example 3 was determined by UV-Vis spectrophotometry. After determining the Cr9-Lip / Cur concentration, it was diluted with PBS solution to a concentration of 1.0, 1.5, and 2.0 μg / mL. 300 μL of each diluted solution was co-incubated with 20.0 μM Aβ protein (i.e., 27 μg Aβ powder) in a shaker at 37°C. At 0, 2, 4, 8, 24, 32, 48, and 72 h, 20.0 μL of sample solution was taken and mixed with 180.0 μL of 20.0 μM ThT fluorescent dye. The fluorescence intensity change of ThT was measured using a fluorescence spectrometer, and fluorescence intensity change curves were plotted based on sampling time and fluorescence intensity. Figure 9 As shown in b, Cr9-Lip / Cur showed good inhibition efficiency of Aβ aggregation at all concentrations, with the 2.0 μg / mL group showing the best effect (inhibition efficiency greater than 80%).

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation, characterized in that, Includes the following steps: (1) Dissolve amphiphilic phospholipids and cholesterol in an organic solvent to prepare a first solution; (2) Dissolve the hydrophobic drug in an organic solvent to prepare a second solution; (3) Mix the first solution and the second solution to obtain the third solution; (4) After the third solution was successively evaporated and dried in a water bath, it was hydrated in a water bath with PBS buffer to obtain the fourth solution; (5) The fourth solution was ultrasonically broken and then filtered to obtain traditional Chinese medicine nanoliposomes; (6) The obtained traditional Chinese medicine nanoliposomes and polypeptide Aβ were combined 16-21 -r9 was co-incubated in PBS buffer, and after incubation, the traditional Chinese medicine nanodelivery system was obtained by dialysis purification.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the amphiphilic phospholipids, cholesterol and organic solvent in step (1) is (10~14):(4~6):(35000~40000). The amphiphilic phospholipid is composed of DSPE-PEG2000-MAL and soybean lecithin, and the mass ratio of DSPE-PEG2000-MAL to soybean lecithin is (2~4):

1.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the hydrophobic drug to the organic solvent in step (2) is 1:(35000~40000); The hydrophobic drug is at least one of curcumin, ginsenoside Rg3, paclitaxel, and triptolide.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the first solution to the second solution in step (3) is (15~25):

1.

5. The preparation method according to claim 1, characterized in that, The temperature of the water bath rotary evaporation in step (4) is 40~42℃, the time is 2~10min, and the rotation speed is 100~150rpm; The water bath hydration temperature is 40~42℃, the time is 45~60min, and the rotation speed is 15~30rpm.

6. The preparation method according to claim 1, characterized in that, Step (6) involves traditional Chinese medicine nanoliposomes and polypeptide Aβ. 16-21 The mass ratio of -r9 to PBS buffer is (20~22):(1~3):(18000~22000).

7. The preparation method according to claim 6, characterized in that, The polypeptide Aβ 16-21 The amino acid sequence of -r9 is shown in SEQ ID NO.

1.

8. The preparation method according to claim 1, characterized in that, The incubation temperature in step (6) is 25~37℃, and the incubation time is 4~12h.

9. A traditional Chinese medicine nanodelivery system with targeted inhibition of Aβ protein aggregation function prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the traditional Chinese medicine nanodelivery system prepared by the preparation method according to any one of claims 1 to 8 or the traditional Chinese medicine nanodelivery system according to claim 9 in the preparation of drugs that target and inhibit the aggregation of Aβ protein.

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