A polysaccharide double-layer modified liposome and preparation and application thereof

Abstract

The present application relates to the technical field of multilayer liposome, and particularly relates to a polysaccharide double-layer modified liposome and preparation and application thereof. The polysaccharide double-layer modified liposome takes the oleanolic acid (OA) liposome as a core, and is coated with chitosan (CHI) and polygalacturonic acid (PGA) two kinds of polysaccharides on the surface in sequence to form a double-layer modified oleanolic acid liposome (PGA-CHI-OA liposome) with a core-shell structure. The modifiers chitosan and polygalacturonic acid used are food raw materials, and are low in cost and rich in resources. Under certain conditions, the two can be self-assembled on the surface of the liposome by mutual attraction of positive and negative charges, the preparation method is simple, the PGA-CHI-OA liposome particles formed are uniform in dispersion, and the PGA-CHI-OA liposome particles are high in safety, stability and bioavailability, and can be applied to food and health products.

Description

Technical Field

[0001] This invention relates to the field of multilayer liposome technology, specifically to a polysaccharide bilayer modified liposome and its preparation and application. Background Technology

[0002] Oleanolic acid (OA) is a pentacyclic triterpenoid compound consisting of six isoprene units linked together to form five closed rings. It has been found in approximately 1600 plant species, including fruits, vegetables, and traditional Chinese medicines, and possesses therapeutic effects for cardiovascular diseases, diabetes, and cancer. However, OA exhibits poor water solubility, is easily oxidized, and is unstable in acidic or alkaline environments, resulting in low oral bioavailability and limiting its widespread application in food and pharmaceuticals.

[0003] Liposomes are vesicles primarily composed of phospholipids, possessing a cell-like bilayer membrane structure. In water, the hydrophilic heads of phospholipid molecules insert into the aqueous phase, while the hydrophobic tails aggregate inward. They range in diameter from 25 to 1000 nm and can simultaneously transport both water-soluble and lipid-soluble nutrients. Combining oxytocin (OA) with liposomes can solve the problem of OA's poor water solubility, broadening its applications. For example, Chinese patent CN106491535A designs an octreotide-modified gold-shelled nanoliposome; the nanoliposomes in this design are oleanolic acid nanoliposomes. The resulting octreotide-modified gold-shelled nanoliposomes can be used to treat cancer.

[0004] However, traditional liposomes are susceptible to the effects of light, oxygen, and gastrointestinal digestive enzymes, and are easily cleared from the bloodstream by the lymphatic and reticuloendothelial systems. Current single oleanolic acid liposomes often suffer from being cleared before reaching their target site, affecting their efficacy. Summary of the Invention

[0005] The present invention aims to overcome the defect of oleanolic acid liposomes being easily cleared in the prior art, and provides a polysaccharide bilayer modified liposome, its preparation and application to overcome the above defects.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A polysaccharide bilayer modified liposome is formed by using oleanolic acid liposome as the core and sequentially coating its surface with two polysaccharides, chitosan (CHI) and polygalacturonic acid (PGA), to form a bilayer modified oleanolic acid liposome with a core-shell structure.

[0008] To address the problem of easy clearance of single oleanolic acid liposomes, the inventors of this application designed a composite liposome by coating a negatively charged oleanolic acid liposome with a positively charged chitosan layer, and then coating the chitosan layer with a negatively charged polygalacturonic acid layer. The entire composite liposome design follows the principle of electrostatic adsorption, and the positively charged chitosan and the negatively charged polygalacturonic acid layer self-assemble into a bilayer modification of the negatively charged oleanolic acid liposome.

[0009] Modifying liposomes into bilayers not only enhances their stability during storage but also improves their protective effect against osteocalcin (OA) in the stomach and allows for slow release in the intestine, thus increasing bioavailability. Furthermore, based on OA's hepatoprotective effects against α-naphthyl isothiocyanate-induced cholestatic liver injury and its simultaneous activation of the NRF2 signaling pathway (a fundamental signaling cascade responsible for resistance to metabolism, oxidative stress, inflammation, and anticancer effects), this study verified that oleanolic acid liposomes modified with chitosan and polygalacturonic acid bilayers (PGA-CHI-OA liposomes) showed a more significant effect on the repair of acute liver injury compared to free OA.

[0010] Therefore, it can be confirmed that after bilayer modification of oleanolic acid liposomes with chitosan layer and polygalacturonic acid layer, oleanolic acid liposomes can be effectively and stably protected, thereby enhancing the application effect.

[0011] Preferably, the polysaccharide bilayer modified liposomes have a uniform particle size of 280–320 nm.

[0012] The particle size of liposomes is crucial for drug dispersibility and bioavailability. Smaller particle sizes are generally more readily absorbed by cells, thus improving the dispersibility and bioavailability of the encapsulated functional ingredients. Furthermore, smaller particle size and uniform dispersion also contribute to improved liposome stability. Therefore, ensuring small particle size and uniform dispersion is essential when preparing liposome carriers.

[0013] A method for preparing polysaccharide bilayer modified liposomes includes the following steps:

[0014] S1. Oleanolic acid, phospholipids and PBS buffer were mixed and homogenized twice to obtain oleanolic acid liposomes (OA liposomes);

[0015] S2. Mix the chitosan solution with oleanolic acid liposomes to obtain chitosan-coated oleanolic acid liposomes (CH-OA liposomes);

[0016] S3. Mix the chitosan-coated oleanolic acid liposomes obtained in S2 with the polygalactose solution to obtain polygalacturonic acid and chitosan bilayer modified oleanolic acid liposomes (PGA-CHI-OA liposomes).

[0017] By using simple solution compounding, bilayer modified oleanolic acid liposomes can be obtained after two-step mixing. The preparation method is simple and easy to promote. After mixing OA and phospholipids, microfluidic treatment was performed to make the particle size uniform and stable, which can enhance the encapsulation effect, reduce the release of encapsulated substances, and extend the shelf life of liposomes.

[0018] Preferably, in step S1, the mass ratio of oleanolic acid to phospholipid is 1:(6-10).

[0019] More preferably, in step S1, the mass ratio of oleanolic acid to phospholipid is 1:(8-10).

[0020] More preferably, in step S1, the mass ratio of oleanolic acid to phospholipid is 1:8.

[0021] The mass ratio of oleanolic acid to phospholipids affects the particle size of oleanolic acid liposomes, which in turn affects the particle size of the bilayer-modified liposomes with oleanolic acid as the core. The liposome particle size, in turn, influences the absorption and utilization of functional components. The inventors of this application designed to regulate the particle size of liposomes by using different mass ratios of oleanolic acid to phospholipids, ensuring that the resulting liposomes are more suitable for application. Verification revealed that when the mass ratio of oleanolic acid to phospholipids is 1:(6-10), the resulting bilayer-modified liposomes have a uniform particle size and are relatively small. Furthermore, when the ratio is 1:8, the resulting liposomes are small, and further increasing the amount of phospholipid has little effect on the size change. Considering the cost of phospholipids, a ratio of 1:8 is preferred here.

[0022] Preferably, in step S1, oleanolic acid is added in the form of an oleanolic acid solution with a concentration of 1 mg / mL.

[0023] Preferably, in step S1, the phospholipid is added in the form of a phospholipid solution with a concentration of 60-100 mg / mL.

[0024] To ensure rapid and uniform fusion of phospholipids and oleanolic acid, they can be prepared as solutions beforehand and mixed together in solution form. The concentration of the solution can be adjusted according to actual needs. The reason for controlling the concentration is to control the amount of liquid introduced and avoid excessive liquid affecting the overall liposome system.

[0025] Preferably, in step S1, the mixing method of oleanolic acid, phospholipids and PBS buffer is as follows: after mixing oleanolic acid, phospholipids and PBS buffer, ethanol is added, the ethanol is evaporated, and then PBS buffer is added, mixed and brought to a final volume.

[0026] Preferably, in step S1, the secondary homogenization is performed by two cycles at a dynamic high-pressure microjet pressure of 1200–1500 bar.

[0027] More preferably, the dynamic high-pressure microjet pressure is 1500 bar.

[0028] Preferably, in step S2, the solute in the chitosan solution is chitosan, the solvent is glacial acetic acid solution, and the pH of the chitosan solution is 3.5.

[0029] Chitosan needs to be fully dissolved in an acidic solution beforehand.

[0030] Preferably, in the method:

[0031] S1. Oleanolic acid, phospholipids, and 100 mL of PBS buffer were mixed and homogenized twice to obtain oleanolic acid liposomes.

[0032] S2. Mix 0.05-0.1 wt% chitosan solution with oleanolic acid liposomes at a volume ratio of 1:1 to obtain chitosan-coated oleanolic acid liposomes.

[0033] More preferably, the concentration of the chitosan solution is 0.1 wt%.

[0034] The concentration of chitosan affects the amount of chitosan added, and the amount of chitosan added has a significant impact on the particle size of liposomes. When the concentration of the chitosan solution is 0.05–0.1 wt%, the average particle size of the liposomes is smaller and more stable.

[0035] Preferably, in step S3, the solute in the polygalactose solution is polygalactose and the solvent is PBS buffer.

[0036] Preferably, in the method:

[0037] S1. Oleanolic acid, phospholipids, and 100 mL of PBS buffer were mixed and homogenized twice to obtain oleanolic acid liposomes.

[0038] S2. Mix 0.05-0.1 wt% chitosan solution with oleanolic acid liposomes at a volume ratio of 1:1 to obtain oleanolic acid liposomes coated with a chitosan layer.

[0039] S3. The oleanolic acid liposomes obtained in S2 are mixed with 1-2 mg / mL polygalactose solution at a volume ratio of 1:1 to obtain oleanolic acid liposomes modified with chitosan and polygalacturonic acid bilayer.

[0040] More preferably, the concentration of the polygalactose solution is 1 to 1.5 mg / mL.

[0041] More preferably, the concentration of the polygalactose solution is 1.5 mg / mL.

[0042] The concentration of polygalactose affects the amount of polygalactose added, and the amount of polygalactose added has a significant impact on the particle size of liposomes. When the concentration of the polygalactose solution is 1–2 mg / mL, the average particle size of the liposomes is smaller and more stable.

[0043] Preferably, in step S3, the pH of the oleanolic acid liposome solution modified with polygalacturonic acid and chitosan bilayer is 6.5 to 7.5.

[0044] The solution pH is close to that of the human body. This design helps ensure that the liposomes maintain good compatibility with the surrounding environment after entering the human body, and is conducive to improving their stability and bioavailability in the human body.

[0045] The present invention also provides that the above-mentioned polysaccharide bilayer modified liposomes or the polysaccharide bilayer modified liposomes prepared by the above method can be used to study the prevention or improvement of acute liver injury.

[0046] By establishing a mouse model of acute liver injury, it was found that polysaccharide bilayer modified liposomes have a significant effect on the repair of acute liver injury and are expected to be applied to the prevention or improvement of acute liver injury.

[0047] Therefore, the present invention has the following beneficial effects:

[0048] (1) The polysaccharide modifiers chitosan and polygalacturonic acid used are both food raw materials with low cost and abundant resources. Under certain conditions, the two can be modified layer by layer on the surface of liposomes by mutual attraction of positive and negative charges. The preparation method is simple, and the PGA-CHI-OA liposome particles formed are uniformly dispersed, with high safety, stability and bioavailability, and can be applied to food and health products.

[0049] (2) PGA-CHI-OA liposomes can maintain structural stability in gastric acid and the modified material is removed in the small intestine to expose the liposomes. Subsequently, the liposomes are hydrolyzed by pancreatic enzymes to release the embedded material, which has a certain small intestine-targeted release capability and improves the bioavailability of the delivered substance.

[0050] (3) PGA-CHI-OA liposomes have obvious functional characteristics. Compared with free OA, animal experiments have shown that this liposome formulation has a better repair effect on mice with acute liver injury, and has broad application prospects. Attached Figure Description

[0051] Figure 1 Flowchart of the preparation process of oleanolic acid liposomes (PGA-CHI-OA liposomes) modified with a bilayer of chitosan (CHI) and polygalacturonic acid (PGA).

[0052] Figure 2 Flowchart for the mouse experiment on the repair of acute liver injury by PGA-CHI-OA liposomes.

[0053] Figure 3 The TEM microstructure of liposomes is shown, with a scale bar of 200 nm.

[0054] Figure 4 OA liposomes, CHI-OA liposomes, and PGA-CHI-OA liposomes were stored at 4℃ for 28 days, and their particle size distribution was measured at 7 days, 14 days, and 28 days.

[0055] Figure 5 The malondialdehyde (MDA) values ​​of OA liposomes, CHI-OA liposomes, and PGA-CHI-OA liposomes were measured at 7d, 14d, and 28d after being stored at 4℃ for 28d.

[0056] Figure 6 The images show pathological sections of liver tissue from mice with acute liver injury after administration of PGA-CHI-OA liposomes. F-OA: free OA administered by gavage + liver injury; PGA-CHI-OA liposomes: bilayer-modified oleanolic acid liposomes administered by gavage + liver injury; PO-C (positive control): biphenyl diester administered by gavage + liver injury; NE-C (negative control): saline administered by gavage + liver injury; NO-C (normal group): saline administered by gavage.

[0057] Figure 7 The effect of PGA-CHI-OA liposomes on serum aspartate aminotransferase (AST) in mice with acute liver injury was investigated. F-OA: free OA administered by gavage + liver injury; PGA-CHI-OA liposomes: bilayer-modified oleanolic acid liposomes administered by gavage + liver injury; PO-C (positive control): biphenyl diester administered by gavage + liver injury; NE-C (negative control): normal saline administered by gavage + liver injury; NO-C (normal group): normal saline administered by gavage.

[0058] Figure 8 The effect of PGA-CHI-OA liposomes on the malondialdehyde (MDA) value, an intermediate product of lipid peroxidation, in mice with acute liver injury. F-OA: free OA administered by gavage + liver injury; PGA-CHI-OA liposomes: bilayer-modified oleanolic acid liposomes administered by gavage + liver injury; PO-C (positive control): biphenyl diester administered by gavage + liver injury; NE-C (negative control): normal saline administered by gavage + liver injury; NO-C (normal group): normal saline administered by gavage.

[0059] Figure 9The effect of PGA-CHI-OA liposomes on the inflammatory factor IL-1β in blood tissue of mice with acute liver injury was investigated. F-OA: free OA administered by gavage + liver injury; PGA-CHI-OA liposomes: bilayer-modified oleanolic acid liposomes administered by gavage + liver injury; PO-C (positive control): biphenyl diester administered by gavage + liver injury; NE-C (negative control): normal saline administered by gavage + liver injury; NO-C (normal group): normal saline administered by gavage. Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0061]

Example

[0062] Example 1

[0063] (1) Add 100 mg of oleanolic acid and 600 mg of phospholipid (m:m = 1:6) to 100 mL of ethanol and vortex to dissolve them thoroughly to obtain a mixed solution.

[0064] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0065] Example 2

[0066] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0067] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0068] Example 3

[0069] (1) 100 mg of oleanolic acid and 1000 mg of phospholipid (m:m = 1:10) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0070] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0071] Example 4

[0072] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0073] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0074] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.05 wt% CHI solution.

[0075] (4) CHI-OA liposomes: Mix 0.05wt% CHI solution with the OA liposomes obtained in (2) at a volume ratio of 1:1 at 500rpm for 90min at room temperature to obtain CHI-OA liposome solution.

[0076] Example 5

[0077] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0078] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0079] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.075 wt% CHI solution.

[0080] (4) CHI-OA liposomes: 0.075wt% CHI solution and the OA liposomes obtained in (2) were mixed at a volume ratio of 1:1 at 500rpm for 90min at room temperature to obtain CHI-OA liposome solution.

[0081] Example 6

[0082] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0083] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0084] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.1 wt% CHI solution.

[0085] (4) CHI-OA liposomes: Mix 0.1 wt% CHI solution with the OA liposomes obtained in (2) at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature to obtain CHI-OA liposome solution.

[0086] Example 7

[0087] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0088] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0089] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.1 wt% CHI solution.

[0090] (4) CHI-OA liposomes: Mix 0.1 wt% CHI solution with the OA liposomes obtained in (2) at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature to obtain CHI-OA liposome solution.

[0091] (5) Preparation of polygalacturonic acid (PGA) solution: At room temperature, polygalacturonic acid (PGA) was fully dissolved in 20mM PBS solution to obtain a 1mg / mL PGA solution.

[0092] (6) PGA-CHI-OA liposomes: CHI-OA liposomes and 1 mg / mL PGA solution were mixed at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature, and then treated in an ultrasonic instrument for 20 min to obtain PGA-CHI-OA liposomes.

[0093] Example 8

[0094] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0095] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0096] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.1 wt% CHI solution.

[0097] (4) CHI-OA liposomes: Mix 0.1 wt% CHI solution with the OA liposomes obtained in (2) at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature to obtain CHI-OA liposome solution.

[0098] (5) Preparation of polygalacturonic acid (PGA) solution: At room temperature, polygalacturonic acid (PGA) was fully dissolved in 20mM PBS solution to obtain a 1.5mg / mL PGA solution.

[0099] (6) PGA-CHI-OA liposomes: CHI-OA liposomes were mixed with 1.5 mg / mL PGA solution at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature, and then treated in an ultrasonic instrument for 20 min to obtain PGA-CHI-OA liposomes.

[0100] Example 9

[0101] (1) 100 mg of oleanolic acid and 800 mg of phospholipid (m:m = 1:8) were added to 100 mL of ethanol and vortexed to dissolve them completely to obtain a mixed solution.

[0102] (2) Preparation of oleanolic acid (OA) liposomes: The mixed solution obtained in (1) was mixed with 50 mL of 20 mM PBS solution and subjected to sonication. The mixed suspension was then placed in a rotary evaporator to remove ethanol. 50 mL of 20 mM PBS solution was added, mixed well, and the volume was adjusted to obtain crude OA liposomes. Two cycles of dynamic high-pressure microfluidic treatment at 1500 bar were performed to obtain OA liposome solution.

[0103] (3) Preparation of chitosan (CHI) solution: At room temperature, CHI was dissolved in glacial acetic acid and the pH was adjusted to 3.5 to obtain a 0.1 wt% CHI solution.

[0104] (4) CHI-OA liposomes: Mix 0.1 wt% CHI solution with the OA liposomes obtained in (2) at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature to obtain CHI-OA liposome solution.

[0105] (5) Preparation of polygalacturonic acid (PGA) solution: At room temperature, polygalacturonic acid (PGA) was fully dissolved in 20mM PBS solution to obtain a 2mg / mL PGA solution.

[0106] (6) PGA-CHI-OA liposomes: CHI-OA liposomes and 2 mg / mL PGA solution were mixed at a volume ratio of 1:1 at 500 rpm for 90 min at room temperature, and then treated in an ultrasonic instrument for 20 min to obtain PGA-CHI-OA liposomes.

[0107] [Performance Testing]

[0108] 1. TEM characterization

[0109] The OA, CHI-OA, and PGA-CHI-OA liposomes obtained in Examples 2, 6, and 8 were subjected to TEM testing, and the results are as follows: Figure 3 As shown, OA liposomes have a uniform and small particle size. After being coated with a CHI layer, their particle size remains uniform, and the morphology of CHI-OA liposomes is unchanged compared to OA liposomes. This indicates that CHI coating does not affect the structure of the OA liposomes themselves. Furthermore, PGA coating on this basis also has no significant impact on particle size uniformity or liposome structure.

[0110] 2. Particle size measurement

[0111] The particle size of the liposomes obtained in Examples 1-9 was measured, and the results are shown in Table 1. Observation of the data from Examples 7-9 shows that the PGA-CHI-OA liposomes prepared at different OA to phospholipid ratios have similar particle sizes, ranging from approximately 290 to 300 nm; and the liposomes exhibit uniform particle size. Smaller liposome size ensures better drug dispersibility and higher bioavailability because smaller liposomes are more easily taken up by cells. Furthermore, uniform particle size also contributes to stability and consistency of efficacy.

[0112] Observation of the data from Examples 1-3 and Examples 4-6 shows that the particle size of CHI-OA liposomes is significantly larger than that of OA liposomes. This increased particle size is due to the formation of a thicker coating layer of CHI on the surface of the OA liposomes, increasing the overall size of the liposomes. However, after further modification with PGA, the size of PGA-CHI-OA liposomes actually decreases. This is because the charge interaction between CHI and PGA alters the original CHI coating layer structure, resulting in a denser coating layer on the surface of the CHI-OA liposomes. This leads to a decrease in the total coating layer thickness after the double-layer modification, thus reducing the size of the liposomes after the double-layer modification.

[0113] Table 1 Particle Size Statistics

[0114] Average particle size (nm) Example 1 202.47 Example 2 188.42 Example 3 185.82 Example 4 374.67 Example 5 353.62 Example 6 340.72 Example 7 297.52 Example 8 294.42 Example 9 319.34

[0115] 3. Stability, such as Figures 4-5 As shown, the particles were stored at 4℃ for 28 days, and the particle size and MDA value were measured at 7, 14, and 28 days. The results showed that the prepared PGA-CHI-OA liposomes were more stable and less prone to oxidation than single OA liposomes and CHI-OA liposomes.

[0116] 4. Mouse model of acute liver injury

[0117] Acute liver injury in mice: Naphthyl α-isothiocyanate induces cholestatic liver injury in mice.

[0118] like Figure 2 As shown, PGA-CHI-OA liposomes (Example 8), OA liposomes (Example 2), biphenyl diester (hepatoprotective drug), and an equal volume of physiological saline were administered to mice by gavage for 21 days. On day 22, CCl4 was administered by gavage. 12 hours later, blood was collected from the eyeballs of the mice and they were euthanized by cervical dislocation. The livers were removed and key indicators were detected.

[0119] Control group mice were given an equal volume of physiological saline by gavage for 22 days. At the same time as the experimental group mice, blood was collected from the eyeballs and the mice were euthanized by cervical dislocation. The livers were then removed and key indicators were detected.

[0120] Relevant test results are as follows Figures 6-9 As shown in the figure, the data indicates that, based on the hepatoprotective effect of oleanolic acid (OA) on cholestatic liver injury induced by α-naphthyl isothiocyanate and its simultaneous activation of the NRF2 signaling pathway (a fundamental signaling cascade responsible for metabolism, oxidative stress, inflammation, and resistance to anticancer effects), PGA-CHI-OA liposomes have a more significant effect on the repair of acute liver injury compared to free OA.

Claims

1. A polysaccharide bilayer modified liposome, characterized in that, Using oleanolic acid liposomes as the core, chitosan and polygalacturonic acid polysaccharides are sequentially coated on their surface to form a bilayer modified oleanolic acid liposome with a core-shell structure.

2. The polysaccharide bilayer modified liposome as described in claim 1, characterized in that, The polysaccharide bilayer modified liposomes have a uniform particle size of 280–320 nm.

3. The method for preparing polysaccharide bilayer modified liposomes as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Oleanolic acid, phospholipids, and PBS buffer are mixed and homogenized twice to obtain oleanolic acid liposomes; S2. Chitosan solution is mixed with oleanolic acid liposomes to obtain chitosan-coated oleanolic acid liposomes; S3. The chitosan-coated oleanolic acid liposomes obtained in S2 are mixed with polygalacturonic acid solution to obtain polygalacturonic acid and chitosan bilayer modified oleanolic acid liposomes.

4. The method as described in claim 3, characterized in that, In step S1, the mass ratio of oleanolic acid to phospholipid is 1:(6-10).

5. The method as described in claim 3, characterized in that, In step S2, the solute in the chitosan solution is chitosan, the solvent is glacial acetic acid solution, and the pH of the chitosan solution is 3.

5.

6. The method as described in claim 3, characterized in that, In step S3, the solute in the polygalacturonic acid solution is polygalacturonic acid, and the solvent is PBS buffer.

7. The method as described in claim 5, characterized in that, In step S3, the pH of the oleanolic acid liposome solution modified with chitosan and polygalacturonic acid bilayer is 6.5 to 7.

5.

8. The method according to any one of claims 3 to 7, characterized in that, S1. Oleanolic acid, phospholipids, and 100 mL of PBS buffer were mixed and homogenized twice to obtain oleanolic acid liposomes; S2. 0.05-0.1 wt% chitosan solution was mixed with oleanolic acid liposomes at a volume ratio of 1:1 to obtain chitosan-coated oleanolic acid liposomes.

9. The method as described in claim 8, characterized in that, S3. The chitosan-coated oleanolic acid liposomes obtained in S2 are mixed with a 1-2 mg / mL polygalactose solution at a volume ratio of 1:1 to obtain polygalacturonic acid and chitosan bilayer modified oleanolic acid liposomes.

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