Hyaluronic acid modified oxymatrine liposome and preparation method thereof
By combining the film dispersion method and the pH gradient method, the liposome membrane constructed by hydrogenated lecithin and cholesterol and modifying hyaluronic acid, the problems of low encapsulation rate and poor stability of oxidized malpine liposomes were solved, and the preparation of high encapsulation rate and high stability of oxidized malpine liposomes was achieved.
Patent Information
- Application Number
- CN202411673246.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-06-27
AI Technical Summary
In the prior art, the encapsulation rate of oxidazine liposomes is low, and the storage stability is poor, making it difficult to effectively improve the bioavailability of oxidazine.
A preparation method combined with thin-film dispersion method and pH gradient method was used to combine the liposome membranes constructed with hydrogenated lecithin and cholesterol, and hyaluronic acid was modified on its surface to improve the encapsulation rate and stability of oxidized malpine.
The high encapsulation rate and high stability of oxidized malphinine are achieved. The encapsulation rate of oxidized malphinine is greater than 50%, and the stability is significantly improved, which can effectively exert pharmacological effects.
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Figure CN120204420A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of liposome preparation, and specifically to hyaluronic acid-modified oxymatrine liposomes and a preparation method thereof. Background Art
[0002] Liposomes are bilayer vesicles formed by the self-assembly of phospholipids in an aqueous solution. Their structure is similar to the phospholipid bilayer of biological membranes, with an aqueous phase separating the center and each layer. They are amphiphilic and can encapsulate hydrophilic and hydrophobic active substances. Since the first discovery of liposomes in 1963, liposomes have become an important research direction in the food and pharmaceutical industries due to their excellent cell membrane penetrability, targeting, and water dispersibility when encapsulating active substances. Especially in the application of functional foods and nutritional products, liposome technology can significantly improve the stability and bioavailability of active substances, greatly promoting their effective application. A large number of studies have shown that the stability and bioavailability of polyphenol compounds are significantly improved after being prepared into liposomes.
[0003] Oxymatrine is an alkaloid isolated from the roots of Sophora flavescens and has been widely studied for its protective effects on organs and tissues, such as anti-inflammatory, antiviral, anti-arrhythmic, and anti-hepatitis B. Due to the strong water solubility of oxymatrine and its easy reduction to matrine when orally administered, intestinal bacteria are more likely to metabolize oxymatrine into matrine with higher activity under anaerobic conditions, greatly reducing its bioavailability and limiting its application. Liposomes, as a nanoscale drug delivery system, have become an important carrier for improving the stability and bioavailability of active substances due to their good cell membrane penetrability, targeting, and water dispersibility. Liposomes are composed of a phospholipid bilayer and can encapsulate hydrophilic and hydrophobic active ingredients, protecting the encapsulated substances from being reduced and improving their stability and bioavailability.
[0004] The main factors affecting the encapsulation efficiency of liposomes include the selection of membrane materials, the ratio of membrane materials, and the preparation method of liposomes during liposome preparation. Oxymatrine has good water solubility, and such drugs are extremely likely to leak from the inside of liposomes into the external aqueous solvent during liposome preparation. And after the liposomes are prepared, due to the interaction between the liposome membrane and molecules such as proteins and surfactants, as well as the dynamic processes of spontaneous aggregation and fusion of the phospholipid bilayer, membrane fusion and rupture will occur, resulting in low encapsulation efficiency and poor storage stability of the prepared liposomes. Therefore, there is an urgent need in the art to provide a stable liposome that can encapsulate water-soluble oxymatrine, improve the encapsulation efficiency and storage stability of oxymatrine, so that oxymatrine can better exert its excellent efficacy in food applications. Summary of the Invention
[0005] The purpose of the present invention is to provide hyaluronic acid modified oxymatrine liposomes and a preparation method thereof, so as to solve the problems of low liposome encapsulation efficiency and poor storage performance stability mentioned in the background art.
[0006] To achieve the above object, the present invention provides the following technical solutions: A liposome containing oxymatrine, characterized in that it comprises the following raw materials: oxymatrine and liposome membrane material, wherein the liposome membrane material comprises hydrogenated lecithin, cholesterol and DSPE-MPEG1000, wherein the optimal ratio of cholesterol to hydrogenated lecithin is 1:10, the amount of cholesterol added is 20 mg, the amount of oxymatrine added is 8 mg, the pH of the external aqueous phase is 7, and the amount of HA-DEEP added is 6 mg.
[0007] On the basis of the above technical solutions, the present invention also provides the following optional technical solutions: In one optional solution: the oxymatrine is purchased as a finished product.
[0008] In an optional embodiment: the added amount of cholesterol is 20 mg.
[0009] The method for preparing the hyaluronic acid modified oxymatrine liposomes as described above comprises the following steps: Step S1: dissolving hydrogenated lecithin, cholesterol and DSPE-MPEG1000 liposome membrane material in 50 mL of anhydrous ethanol by ultrasonication, taking 5 mL of anhydrous ethanol by rotary evaporation to remove the anhydrous ethanol so that the membrane material forms a uniform transparent film on the inner wall of the round-bottom flask; Step S2: adding a citric acid buffer solution into a round-bottom flask and rotating it for complete hydration, and keeping it warm to allow the membrane material to be completely eluted and dissolved to obtain a suspension; Step S3: The solution obtained in step S2 is gently shaken, vortexed and rapidly rotated to obtain a suspension, and then placed at room temperature to obtain a blank liposome dispersion; Step S4: adjusting the pH of the external aqueous phase of the obtained blank liposome dispersion to 7 with NaOH (1 M); Step S5: Take 1 ml of OM aqueous solution (PBS with pH 7.4) and add it to the blank liposome suspension adjusted to neutrality by external aqueous phase, and then add HA-DPPE conjugate. Step S6: fully mix the two, accurately measure the volume of the final solution, and incubate in a water bath (shake slightly during the process). The finished product is stored in a refrigerator.
[0010] In an optional solution: the temperature of the rotary evaporation in step S1 is 40° C. and the rotation speed is 100-120 rpm.
[0011] After the hydration in step S2 is complete, the mixture is kept at 40° C. for 5 minutes.
[0012] In step S3, shake, vortex, and rotate rapidly and vigorously for 30 min, and leave at room temperature for 12 h.
[0013] In an alternative embodiment: In step S6, the incubation temperature of the thermostatic shaker is 50 °C, the incubation time is 15 min (with gentle shaking during this period), and the finished product is stored at 4 °C.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention adopts a preparation method combining the thin film dispersion method and the pH gradient method, enabling oxymatrine to be enriched inside the liposomes, thereby achieving the purpose of high encapsulation efficiency and high stability. At the same time, the liposome membrane constructed with hydrogenated lecithin and cholesterol has a uniform morphology. The modifier hyaluronic acid added to the liposome is a linear polysaccharide composed of a repeating structure of β-N-acetylglucosamine and D-glucuronic acid monosaccharides. This HA with a bifunctional group structure exhibits strong hydrophilicity and biocompatibility. Liposomes may be prone to aggregation, fusion, and oxidation, leading to the destruction of their integrity and thus the leakage of the encapsulated material. Depositing hyaluronic acid on the surface of the liposomes maintains their structure and increases their kinetic and mechanical stability. Thus, the problem of liposome instability during storage is solved. Compared with common liposome preparation techniques, the present invention utilizes the characteristics of oxymatrine and selects a preparation method combining the thin film dispersion method and the pH gradient method to obtain oxymatrine liposomes modified with hyaluronic acid with high encapsulation efficiency and good stability;
[0015] The encapsulation efficiency of oxymatrine in the oxymatrine liposomes modified with hyaluronic acid prepared by the present invention is greater than 50%, which can effectively encapsulate oxymatrine, and the stability is significantly improved, greatly exerting its pharmacological effect in vivo. The materials used in this liposome are highly safe, inexpensive, and easy to obtain in the market, which is also conducive to the efficient application of oxymatrine in the fields of food and medicine. Description of the Drawings
[0016] Figure 1 It is a schematic diagram of the preparation process of the oxymatrine liposomes modified with hyaluronic acid of the present invention.
[0017] Figure 2 It is the particle size and potential distribution of the oxymatrine liposomes modified with hyaluronic acid of the present invention.
[0018] Figure 3 It is a graph showing the effect of different mass ratios of cholesterol to hydrogenated lecithin on the encapsulation efficiency of the oxymatrine liposomes modified with hyaluronic acid of the present invention.
[0019] Figure 4 It is a graph showing the effect of different dosages of oxymatrine on the encapsulation efficiency of the oxymatrine liposomes modified with hyaluronic acid of the present invention.
[0020] Figure 5 This is the graph showing the effect of different pH values on the encapsulation efficiency of oxidized matrine liposomes modified with hyaluronic acid in the present invention.
[0021] Figure 6 This is the graph showing the effect of different dosages of hyaluronic acid modifiers on the encapsulation efficiency of oxidized matrine liposomes modified with hyaluronic acid in the present invention.
[0022] Figure 7 This is the TEM scanning image of oxidized matrine liposomes modified with hyaluronic acid in the present invention. Detailed implementation manners
[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with the accompanying drawings and embodiments. Each embodiment listed in the present invention is only used to illustrate the present invention, and is not used to limit the scope of the present invention. Any obvious modification or change made to the present invention does not depart from the spirit and scope of the present invention.
[0024] In one embodiment, as Figure 1 shown, a preparation method of oxidized matrine liposomes modified with hyaluronic acid is proposed. The method includes the following steps: Step 1: Synthesis of HA-DPPE conjugate (1) Weigh 14 mg of HA precisely and dissolve it in ultrapure water, and preactivate it with EDC with a pH of 4. (2) Add DPPE (1 mg) to the above-preactivated HA solution, and adjust the pH value to 8.6, and react at 37 °C for 24 h. (3) Dialyze to remove unreacted DPPE, EDC and other reaction reagents, and freeze-dry to obtain HA-DPPE. Step 2: Preparation of blank liposomes by thin film dispersion method (1) Dissolve hydrogenated lecithin, cholesterol, and DSPE-MPEG1000 liposome membrane materials completely in 50 mL of absolute ethanol under water bath heating. Take 5 mL and remove the absolute ethanol by rotary evaporation at 40 °C so that the membrane materials form a uniform and transparent thin film on the inner wall of the round-bottom flask; (2) Add 15 mL of citric acid buffer solution to the round-bottom flask and rotate to hydrate completely. Keep it at 40 °C for 5 min so that the membrane materials are completely eluted and dissolved to obtain a suspension; (3) The obtained solution is gently shaken, vortexed and rapidly rotated violently to obtain a suspension, and left at room temperature for 12 h to obtain a blank liposome dispersion; Step 3: Encapsulation of oxidized matrine by pH gradient method (4) Adjust the pH of the outer aqueous phase of the above-prepared blank liposome dispersion to 7 with NaOH (1 M); (5) Take 1 ml of 8 mg / mL OM aqueous solution (PBS pH 7.4) and add it to the blank liposome suspension adjusted to neutral external aqueous phase, and then add the hyaluronic acid modification. (6) Mix the two thoroughly, accurately measure the volume of the final solution, and incubate in a 50°C water bath for 15 min (shake slightly during the process). The finished product is stored in a refrigerator at 4°C.
[0025] The single-factor method was used to investigate the effects of the mass ratio of hydrogenated lecithin to cholesterol, the dosage of oxymatrine, pH value, and the dosage of hyaluronic acid modifier on the encapsulation efficiency and particle size of hyaluronic acid-modified oxymatrine liposomes.
[0026] Example 1: Five different mass ratios of cholesterol to hydrogenated lecithin were set at 1:6, 1:8, 1:10, 1:12, and 1:14, wherein the added amount of cholesterol was 20 mg, DSPE-MPEG1000 was 10 mg, and the added amount of oxymatrine was 8 mg.
[0027] The above four groups of hydrogenated lecithin and cholesterol mass ratio formulas were prepared according to the above preparation steps of oxymatrine liposomes, and the effect of the mass ratio of hydrogenated lecithin to cholesterol on the particle size, PDI, potential and encapsulation efficiency of oxymatrine liposomes was investigated.
[0028] The results are shown in the following table: Table 1 Effect of hydrogenated lecithin / cholesterol mass ratio on liposome particle size, PDI, potential, and encapsulation efficiency As can be seen from the above table: after the cholesterol molecules are added, they are embedded in the bilayer membrane composed of lecithin to adjust the fluidity of the membrane. The optimal ratio of hydrogenated lecithin to cholesterol is 1:10, among which the encapsulation efficiency of liposomes (EE=65.35%) is the highest at 1:6, but after a few days of preparation, obvious precipitation occurs, resulting in turbid solution, poor stability, and an average particle size of 359.07, which is a large value. It may be due to the inappropriate ratio of cholesterol to hydrogenated lecithin, resulting in uneven particle size distribution, causing liposome aggregation and precipitation. When the ratio of cholesterol to hydrogenated lecithin is between 1:10, the solution does not precipitate, the average particle size is 206.1, and the encapsulation efficiency is 61.85%, second only to 1:6, so the optimal ratio of hydrogenated lecithin to cholesterol is 1:10.
[0029] Example 2: Five groups of different dosages of oxymatrine were set, wherein the added amount of cholesterol was 20 mg and the added amount of oxymatrine was 8 mg.
[0030] Prepare the above 4 groups of oxymatrine dosage formulations according to the preparation steps of the hyaluronic acid-modified oxymatrine liposomes, and investigate the effects of the oxymatrine dosage on the particle size, PDI, zeta potential, and encapsulation efficiency of the hyaluronic acid-modified oxymatrine liposomes.
[0031] The results are shown in the following table: Table 2 Effects of Oxymatrine Dosage on Particle Size, PDI, Zeta Potential, and Encapsulation Efficiency of Liposomes As shown in the above table, as the dosage of oxymatrine increases, the PDI value is within the normal range, and the encapsulation efficiency shows a trend of first increasing and then decreasing. This is attributed to the fact that as the input ratio of oxymatrine continues to increase, while the liposomes tend to be stable, the particle size of the liposomes also becomes unstable, resulting in a decrease in the encapsulation efficiency of the liposomes after the oxymatrine is in excess. When the oxymatrine is 8 mg, the encapsulation efficiency (EE = 67.93%) is the highest.
[0032] Example 3: Set 5 groups with different pH values, where the cholesterol addition amount is 20 mg, the DSPE-MPEG1000 addition amount is 10 mg, and the oxymatrine addition amount is 8 mg.
[0033] Prepare the above 5 groups of different pH environments according to the preparation steps of the hyaluronic acid-modified oxymatrine liposomes, and investigate the effects of different pH environments on the particle size, PDI, zeta potential, and encapsulation efficiency of the hyaluronic acid-modified oxymatrine liposomes.
[0034] The results are shown in the following table: Table 3 Effects of pH on Particle Size, PDI, Zeta Potential, and Encapsulation Efficiency of Liposomes The above table shows that as the pH value increases, the encapsulation efficiency of the liposomes shows a trend of first increasing and then decreasing. However, when the pH value is between 7 - 7.5, the encapsulation efficiency of the liposomes tends to be stable, and the encapsulation efficiency is around 50.31% - 48.63%. Due to the fact that the too acidic or too alkaline environment has a certain impact on the stability of the phospholipid bilayer membrane, the toughness decreases, and the drug leakage increases, resulting in a decrease in the encapsulation efficiency. This indicates that the liposomes are most stable at around pH 7, and the optimal pH environment should be 7.
[0035] Example 4: Set 5 groups with different addition amounts of hyaluronic acid modifiers: 3 mg, 4 mg, 5 mg, 6 mg, and 7 mg. The cholesterol addition amount is 20 mg, the DSPE-MPEG1000 addition amount is 10 mg, and the oxymatrine addition amount is 8 mg.
[0036] The above 5 groups of formulations with different addition amounts of hyaluronic acid modifiers were prepared according to the above preparation steps of hyaluronic acid-modified oxymatrine liposomes, and the effects of the addition amount of hyaluronic acid modifiers on the particle size, PDI, zeta potential, and encapsulation efficiency of hyaluronic acid-modified oxymatrine liposomes were investigated.
[0037] The results are as follows in the table: Table 4 Effects of the input amount of HA-DEEP on the particle size, PDI, zeta potential, and encapsulation efficiency of liposomes In the above table, as the addition amount of HA-DEEP increases, the encapsulation efficiency of liposomes shows a trend of first increasing and then decreasing. When the addition amount of HA-DEEP is 6 mg, the encapsulation efficiency reaches the maximum value (EE = 73.89%). Therefore, the optimal addition amount of HA-DEEP is 6 mg.
[0038] The best process conditions were selected through the implementation cases: the optimal usage ratio of cholesterol to hydrogenated lecithin is 1:10, the addition amount of HA-DEEP is 6 mg, the optimal pH of the external aqueous phase is 7, and the optimal dosage of oxymatrine is 8 mg. At this time, it is in the most stable state, and the encapsulation efficiency is also relatively stable.
[0039] Morphological characteristics of hyaluronic acid-modified oxymatrine liposomes: The liposome sample appropriately diluted with ultrapure water was taken, dropped onto the copper mesh, and after drying, 2% phosphotungstic acid solution was dropped for negative staining for 3 min. After sucking off the excess phosphotungstic acid, the morphology of the liposomes was observed using a transmission electron microscope (TEM).
[0040] The appearance morphology of the liposomes, as shown in the appendix Figure 7 shown Figure 7 is the structure and morphology of the liposomes. The liposomes are round or oval. It can be clearly seen that several liposomes with uniform size and smooth surface appear in the field of view.
[0041] Particle size, Zeta potential, and polydispersity coefficient of liposomes: An appropriate amount of the suspension of hyaluronic acid-modified oxymatrine liposomes was taken, appropriately diluted with ultrapure water, and then measured using a laser particle size analyzer.
[0042] The particle size distribution diagram of hyaluronic acid-modified oxymatrine liposomes is as shown in the appendix Figure 2 shown. Under the optimal process conditions, the average particle size of hyaluronic acid-modified oxymatrine liposomes is 206.1 ± 8.88 nm, and the polydispersity coefficient PDI is 0.158 ± 0.032, indicating that the particle size of hyaluronic acid-modified oxymatrine liposomes is uniform and the distribution is homogeneous. The Zeta potential of hyaluronic acid-modified oxymatrine liposomes was measured to be -48.37 ± 1.04 mV, indicating that the hyaluronic acid-modified oxymatrine liposomes have good stability.
[0043] The schematic diagram of the encapsulation efficiency of oxymatrine liposomes modified with hyaluronic acid is as shown in the appendix Figures 3 - 6 As shown, under different cholesterol:phospholipid mass ratios, the most suitable ratio is 1:10, and the encapsulation efficiency is better, greater than 50%. When the input amount of oxymatrine is 8 mg, the encapsulation efficiency (EE = 67.93) is the highest. When the pH of the external aqueous phase is 7, the encapsulation efficiency (EE = 66.03) is the highest. When the input amount of HA-DPPE is 6 mg, the encapsulation efficiency (EE = 73.89%) is the highest. The most suitable conditions for preparing liposomes are screened through single-factor experiments, so as to prepare oxymatrine liposomes modified with hyaluronic acid with good stability.
[0044] Liposome storage stability: Take the prepared suspension of oxymatrine liposomes modified with hyaluronic acid, store it in a 4°C refrigerator, and measure the encapsulation efficiency of liposomes at 1, 3, 7, 15, and 30 days respectively to evaluate their stability during storage.
[0045] The results are as follows in the table The above table shows the change in the encapsulation efficiency of oxymatrine liposomes modified with hyaluronic acid under the storage condition of 4°C. During the 30-day storage period, the particle sizes of oxymatrine liposomes modified with hyaluronic acid are in a stable state, and the change range of particle sizes is small. The results show that oxymatrine liposomes modified with hyaluronic acid have good stability during storage at 4°C.
[0046] The present invention optimizes the mass ratio of hydrogenated lecithin and cholesterol, the dosage of hydrogenated matrine, different pH conditions, and the addition amount of HA-DEEP, which are 4 important variables in the liposome preparation process, in order to achieve the purpose of preparing liposomes with high encapsulation efficiency and good storage stability.
[0047] After the addition of cholesterol molecules, they are embedded in the bimolecular layer membrane composed of lecithin to regulate the fluidity of the membrane. The optimal ratio of cholesterol to hydrogenated lecithin is between 1:10. Among them, when it is 1:10, the encapsulation efficiency of liposomes (EE = 61.85%) is the most stable. The results show that the addition of the most suitable ratio of cholesterol to hydrogenated lecithin improves the stability of the phospholipid bimolecular membrane, increases the toughness, and reduces drug leakage.
[0048] As the pH value increases, the encapsulation efficiency of liposomes shows a trend of first increasing and then decreasing. Because the too acidic or too alkaline environment has a certain impact on the stability of the phospholipid bimolecular membrane, the toughness decreases, and the drug leakage increases. However, when the pH value is between 7 and 7.5, the encapsulation efficiency of liposomes tends to be stable, and the encapsulation efficiency is about 50.31% - 48.63%. As the pH value increases, the encapsulation efficiency shows a downward trend, indicating that the applicable regulation of this liposome should be the most stable at about 7, and the optimal pH environment should be 7.
[0049] With the increase in the input amount of oxymatrine, the PDI value is within the normal range, and the encapsulation efficiency shows a trend of first increasing and then decreasing. This is attributed to the fact that as the input ratio of oxymatrine continues to increase, the ratio of oxymatrine entering the liposome through active transport by ion gradient increases, and the encapsulation efficiency increases. However, after the oxymatrine in the liposome approaches saturation, the growth rate of the encapsulation efficiency decreases. While the liposome tends to be stable, the particle size of the liposome also shows an unstable state, resulting in a downward trend in the encapsulation efficiency of the liposome after the oxymatrine is in excess. When the oxymatrine is 8 mg, the encapsulation efficiency (EE = 67.93%) is the highest, reaching the most stable state.
[0050] With the increase in the addition amount of HA-DEEP, the encapsulation efficiency of the liposome shows a trend of first increasing and then decreasing. The results show that when the addition amount of HA-DEEP is too large, the density of the liposome modifier in the suspension is too high, which will cause it to aggregate more easily and produce precipitation, resulting in a decrease in the encapsulation efficiency. When the addition amount of HA-DEEP is 6 mg, the encapsulation efficiency is the maximum value (EE = 73.89%). Therefore, the optimal addition amount of HA-DEEP is 6 mg.
[0051] As described above, it is only the specific implementation manner of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present disclosure can easily think of changes or substitutions, which should be covered within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the protection scope of the claims.
Claims
1. An oxymatrine liposome, characterized in that: The invention comprises the following raw materials: oxymatrine and liposome membrane material, wherein the liposome membrane material comprises hydrogenated lecithin, cholesterol and DSPE-MPEG1000, wherein the optimal ratio of cholesterol to hydrogenated lecithin is 1:10, the added amount of cholesterol is 20 mg, the added amount of oxymatrine is 8 mg, the pH value of the external water phase is 7, and the added amount of HA-DEEP is 6 mg.
2. The oxymatrine liposome according to claim 1, characterized in that The oxymatrine is purchased as a finished product.
3. The oxymatrine liposome according to claim 1, characterized in that: The added amount of the hydrogenated lecithin is 200 mg.
4. A method for preparing oxymatrine liposomes according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step S1: dissolving hydrogenated lecithin, cholesterol and DSPE-MPEG1000 liposome membrane material in 50 mL of anhydrous ethanol by ultrasonication, taking 5 mL of anhydrous ethanol by rotary evaporation to remove the anhydrous ethanol so that the membrane material forms a uniform transparent film on the inner wall of the round-bottom flask; Step S2: adding a citric acid buffer solution into a round-bottom flask and rotating it for complete hydration, and keeping it warm to allow the membrane material to be completely eluted and dissolved to obtain a suspension; Step S3: The solution obtained in step S2 is gently shaken, vortexed and rapidly rotated to obtain a suspension, and then placed at room temperature to obtain a blank liposome dispersion; Step S4: adjusting the pH of the external aqueous phase of the obtained blank liposome dispersion to 7 with NaOH (1 M); Step S5: Take 1 ml of OM aqueous solution (PBS with pH 7.4) and add it to the blank liposome suspension adjusted to neutrality with external aqueous phase, and then add HA-DPPE conjugate.
5. Step S6: Mix the two thoroughly, accurately measure the volume of the final solution, and incubate in a water bath (shake slightly during the process). The finished product is stored in a refrigerator.
6. The method for preparing oxymatrine liposomes according to claim 4, characterized in that: The temperature of the rotary evaporation in step S1 is 40° C. and the rotation speed is 100-120 rpm.
7. The method for preparing oxymatrine liposomes according to claim 4, characterized in that: After the hydration in step S2 is complete, the mixture is kept at 40° C. for 5 minutes.
8. The method for preparing oxymatrine liposomes according to claim 4, characterized in that: In step S3, the mixture was shaken, vortexed and rapidly rotated for 30 min and then placed at room temperature for 12 h.
9. The method for preparing oxymatrine liposomes according to claim 4, characterized in that: In step S6, the incubation temperature of the constant temperature shaker is 50° C., the incubation time is 15 min (with slight shaking during the incubation), and the finished product is stored at 4° C.