Preparation method of nano-liposome

Abstract

The invention provides a preparation method of a nano-liposome, and belongs to the technical field of liposome preparation. The preparation method comprises the following steps: (1) mixing phospholipid and polyhydric alcohol, heating at 80-100 DEG C for 10-20 minutes under the condition of being filled with protective gas or vacuum, and uniformly mixing to obtain a mixture; and (2) cooling the mixture, and dispersing the mixture in a water phase according to the mass ratio of the mixture to the water phase of 1: (15-25) to obtain the nano-liposome. The preparation process is simple, green, efficient and easy to implement, use and residues of organic reagents in a traditional liposome preparation means are avoided, the problem that in a traditional preparation method, the requirement for preparation equipment is strict is solved, and the prepared nano-liposome is uniform in particle size distribution and good in stability and has good application and popularization prospects. The method provided by the invention can also be used for preparing a drug-loaded nano-liposome, and the drug-loaded nano-liposome obtained by adopting the method provided by the invention can obviously improve the encapsulation efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome preparation, and in particular relates to a method for preparing nano liposome. Background Art

[0002] Liposomes are bilayer lipid vesicles made of lipid materials that can be used as drug carriers. Phospholipids are the main lipid materials for preparing liposomes. Liposomes are one of the most widely used nano-delivery systems in clinical practice. Since its first discovery in 1965, liposome technology has undergone continuous development, and in 1995, the first liposome-based drug Doxil was approved by the FDA for marketing. By 2020, mRNA vaccines encapsulated by lipid nanoparticles have become the focus of global attention. In the medical field, liposomes can change the release performance of drugs in the body and the resulting increase in efficacy and reduction in toxic side effects. Its characteristics include targeting and lymphatic orientation, prolonging the blood circulation time of drugs, reducing drug toxicity, and improving stability. In the food industry, liposomes also show their unique advantages and can effectively encapsulate, protect and control the release of various nutrients and bioactive substances. Through liposome technology, the bioavailability of nutrients such as vitamins, minerals and antioxidants can be significantly improved, thereby enhancing their absorption and utilization efficiency. The encapsulation function of liposomes can also protect sensitive substances such as ω-3 polyunsaturated fatty acids, fat-soluble vitamins and plant compounds, preventing them from being affected by heat, light and oxygen during processing and storage, and avoiding degradation or oxidation. In addition, as a controlled release system, liposomes can release encapsulated substances in specific parts of the digestive tract to improve efficacy or reduce side effects. Liposome encapsulation can also be used to mask the unpleasant flavor of certain substances, such as fish oil and certain herbal extracts, to improve the taste and acceptability of food. In short, from the laboratory to commercialization, liposomes have become an important component of drug delivery systems, and liposome technology has broad and far-reaching application prospects in both the medical field and the food industry.

[0003] The preparation of liposomes requires the conversion of phospholipids that are crystalline at room temperature into a phospholipid bilayer membrane structure with certain fluidity and rigidity. This requires changing the phase state of phospholipids and promoting the re-swelling and arrangement of phospholipid molecules in the aqueous phase. Traditional methods for preparing liposomes include thin film dispersion, organic reagent injection, extrusion, and heating; emerging methods for preparing liposomes include supercritical CO2, thin film-ultrasound, and high-pressure homogenization. Thin film dispersion is the most common liposome preparation technology and is suitable for embedding fat-soluble substances. First, phospholipids and drugs are dissolved in an organic solvent, and the solvent is removed by rotary evaporation to form a phospholipid film. Then, an aqueous phase above the phase transition temperature of phospholipids is added, and the phospholipid film absorbs water and expands during rotation, self-assembling into a liposome suspension with a double-layer structure. The prepared liposomes have a large particle size, and usually overcome energy barriers by ultrasound or high-pressure homogenization to reduce the size. The organic reagent injection method is to dissolve phospholipids and drugs in ethanol, and then inject it into the water phase. After the alcohol solution of phospholipids is exposed to the water phase, liposomes can be spontaneously formed. Finally, the ethanol in the water phase is removed by rotary evaporation. The liposomes generated by this method have small particle size and high dispersion. Emerging liposome preparation methods such as ultrasound or high-pressure homogenization use the impact and cavitation caused by high energy input such as ultrasound / high-pressure homogenization to break the swollen phospholipid liquid crystals in the water phase into extremely small phospholipid fragments. These phospholipid fragments bend and fuse to form liposome structures.

[0004] The use of organic reagents in traditional methods, the requirements of new methods for special equipment and high energy consumption have kept liposome preparation mostly in the laboratory stage and have not been mass-produced and applied. In addition, when liposome solutions are exposed to an aqueous environment, phospholipid molecules are prone to oxidation and rancidity, resulting in the destruction of the liposome structure and leakage of the contents. Its storage stability is also an urgent problem to be solved. Therefore, it is crucial to propose a new liposome preparation process for the production and application promotion of liposomes. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing nanoliposomes. The preparation method of the present invention does not require the use of organic reagents throughout the process, achieves the requirements of green environmental protection, and is simple and easy to operate, has low requirements on equipment, overcomes the requirements of traditional methods on equipment, and can easily realize large-scale production and preparation of liposomes.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing nanoliposomes, comprising the following steps:

[0008] (1) mixing the phospholipid and the polyol, heating them at 80 to 100° C. for 10 to 20 minutes in a protective gas atmosphere or under vacuum conditions, and mixing them to obtain a mixture;

[0009] (2) After cooling the mixture, the mixture is dispersed in the aqueous phase at a mass ratio of the mixture to the aqueous phase of 1:15 to 25 to obtain nanoliposomes.

[0010] Preferably, the mass ratio of the phospholipid to the polyol is 1:0.1-10.

[0011] Preferably, the phospholipids include any one of soybean lecithin, sunflower lecithin, egg lecithin, shrimp lecithin and hydrogenated lecithin.

[0012] Preferably, the polyol is a liquid polyol, including any one of glycerol, propylene glycol and polyethylene glycol.

[0013] Preferably, the protective gas is an inert gas.

[0014] Preferably, the aqueous phase comprises water, PBS, sodium chloride solution or sucrose solution.

[0015] Preferably, the pH of the PBS is 3-8, the concentration of the sodium chloride solution is 50-1000 mmol / L, and the concentration of the sucrose solution is 1-20 wt%.

[0016] The invention provides nano liposomes obtained by the preparation method.

[0017] The present invention also provides a method for preparing drug-loaded nanoliposomes, comprising the following steps:

[0018] (1) mixing the phospholipid, polyol and drug, heating at 80-100° C. for 10-20 min in a protective gas or vacuum environment, and mixing to obtain a drug-containing mixture;

[0019] (2) After cooling the drug-containing mixture, the drug-containing mixture is dispersed in the aqueous phase at a mass ratio of the drug-containing mixture to the aqueous phase of 1:15 to 25 to obtain drug-loaded nanoliposomes.

[0020] The invention also provides the drug-loaded nano-liposome obtained by the preparation method of the drug-loaded nano-liposome, wherein the drug loaded is curcumin.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for preparing nanoliposomes, which can effectively isolate air and moisture by mixing phospholipids with liquid polyols, and protecting with inert gas or mixing under vacuum conditions, thereby overcoming the shortcomings of traditional liposome solutions that are easily unstable and easily oxidized, and the preparation method of the present invention is simple, and nanoliposomes can be obtained by simply heating and stirring. The preparation process of the present invention is simple, green, efficient and easy to implement, avoiding the use and residue of organic reagents in traditional liposome preparation methods, solving the problem of strict requirements on preparation equipment in traditional preparation methods, and the prepared nanoliposomes have uniform particle size distribution, good stability, and good prospects for promotion and application. The method of the present invention can also be used to prepare drug-loaded nanoliposomes, and the drug-loaded nanoliposomes obtained by the method of the present invention can significantly improve the encapsulation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the technical roadmap of the present invention;

[0023] Figure 2 This is an appearance diagram of the proliposomes and nanoliposomes formed in Example 1;

[0024] Figure 3 The particle size distribution diagram of the nanoliposomes formed in Example 1;

[0025] Figure 4 TEM image of nanoliposomes formed in Example 1;

[0026] Figure 5 The appearance and polarized light microscope images of the proliposomes prepared in Examples 1 to 5 and Control Group 1, wherein the scale bar is 500 μm;

[0027] Figure 6 DSC graphs of proliposomes prepared in Examples 1 to 5 and Control Group 1;

[0028] Figure 7 FTIR images of the proliposomes prepared in Examples 1 to 5 and Control Group 1;

[0029] Figure 8 This is the appearance of the liposomes obtained in Comparative Example 2;

[0030] Fig. 9 The XRD patterns of curcumin, proliposomes and curcumin-loaded nanoproliposomes in Example 6;

[0031] Fig.10 The appearance of the curcumin-loaded proliposome (left) and the curcumin-loaded nanoliposome (right) formed by spontaneous hydration in Example 6;

[0032] Fig.11This is the particle size distribution diagram of curcumin-loaded nanoliposomes prepared in Example 6;

[0033] Fig.12 TEM image of curcumin-loaded nanoliposomes prepared in Example 6;

[0034] Fig.13 This is the appearance of the curcumin-loaded liposomes prepared in Comparative Example 3. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing nanoliposomes, comprising the following steps:

[0036] (1) mixing the phospholipid and the polyol, heating them at 80 to 100° C. for 10 to 20 minutes in a protective gas atmosphere or under vacuum conditions, and mixing them to obtain a mixture;

[0037] (2) After cooling the mixture, the mixture is dispersed in the aqueous phase at a mass ratio of the mixture to the aqueous phase of 1:15 to 25 to obtain nanoliposomes.

[0038] In the present invention, phospholipids and polyols are mixed, placed in a protective gas or vacuum condition, heated at 80-100°C for 10-20 minutes, and mixed to obtain a mixture; phospholipids and polyols with a mass ratio of 1:0.1-10 are mixed, added to a heated stirring container, filled with protective gas or heated at 80-100°C for 10-20 minutes under vacuum conditions, stirred and mixed to fully dissolve the phospholipids in the polyols to obtain a mixture (proliposome); the mass ratio of the phospholipids to the polyols is preferably 1:0.5-8, more preferably 1:5, and the phospholipids include soybean lecithin, sunflower lecithin, egg lecithin, shrimp lecithin, Any one of phospholipids and hydrogenated phospholipids; the polyol is a liquid polyol, including any one of glycerol, propylene glycol, and polyethylene glycol; the protective gas is an inert gas, including one or more of nitrogen, helium, argon, and xenon, and the filling of the protective gas can prevent the oxidation of the phospholipids; under vacuum conditions, there is no need to add protective gas; the heating temperature is preferably 85-95°C, more preferably 90°C, the heating time is preferably 12-18min, more preferably 15min; the stirring speed is 300-500rpm, preferably 350-450rpm, more preferably 400rpm.

[0039] In the present invention, after cooling the mixture, the mixture is dispersed in the aqueous phase at a mass ratio of 1:15 to 25 to obtain nanoliposomes; the mixture is cooled to 20 to 30°C, the mixture is added to the aqueous phase at a mass ratio of 1:15 to 25 to obtain nanoliposomes; the mixture is cooled to 20 to 30°C, the mixture is added to the aqueous phase at a mass ratio of 1:15 to 25 to obtain nanoliposomes; the cooling temperature is preferably 22 to 28°C, and more preferably 25°C; the mass ratio of the mixture to the aqueous phase is preferably 1:18 to 22, and more preferably 1:20, and the aqueous phase includes water, PBS, sodium chloride solution or sucrose solution; the water is one of distilled water, ultrapure water and tap water, and is preferably ultrapure water; the pH of the PBS is 3 to 8, the concentration of the sodium chloride solution is 50 to 1000 mmol / L, the concentration of the sucrose solution is 1 to 20 wt%, and the temperature of the aqueous phase is preferably 22 to 28°C, and more preferably 25°C.

[0040] In the present invention, phospholipids mainly refer to glycerophospholipids, in which the two hydroxyl groups of glycerol form ester bonds with fatty acids, and the third hydroxyl group is phosphated. The phase transition temperature (Tm) of phospholipids is the temperature at which the acyl chains that make up the phospholipids transition from a crystalline state to a liquid state. When the temperature is lower than Tm, the acyl chains in the phospholipid molecules are tightly stacked, and at this time the phospholipids are crystalline and not easily hydrated; when the temperature is higher than Tm, the acyl chains in the phospholipid molecules change from a crystalline state to a fluid state, and at this time the phospholipids exhibit better fluidity. In the preparation process of traditional liposomes, it is necessary to use an aqueous phase with a temperature higher than the Tm of the phospholipids to swell the phospholipid membrane and promote the formation of liposomes. The present invention utilizes the hydroxyl group (-OH) in the polyol and the phosphate group (-PO4 - ) to open the tightly packed state of acyl groups in phospholipid molecules, thereby reducing the Tm of phospholipid molecules, making the acyl chains flowable at room temperature. Therefore, at room temperature, hydration can form a stable liposome structure after simple stirring. At the same time, due to the decrease in the Tm of phospholipids, the target drug can be more easily dissolved in the phospholipid / polyol mixture during heating. After stirring and hydration, the target drug can be efficiently loaded into the formed liposomes to obtain drug-loaded liposomes.

[0041] The invention provides nano liposomes obtained by the preparation method.

[0042] The present invention also provides a method for preparing drug-loaded nanoliposomes, comprising the following steps:

[0043] (1) mixing the phospholipid, polyol and drug, heating at 80-100° C. for 10-20 min in a protective gas or vacuum environment, and mixing to obtain a drug-containing mixture;

[0044] (2) After cooling the drug-containing mixture, the drug-containing mixture is dispersed in the aqueous phase at a mass ratio of the drug-containing mixture to the aqueous phase of 1:15 to 25 to obtain drug-loaded nanoliposomes.

[0045] In the present invention, phospholipids, polyols and drugs are mixed, placed in a protective gas or vacuum condition, heated at 80-100°C for 10-20 minutes, and mixed to obtain a drug-containing mixture; phospholipids and polyols are mixed at a mass ratio of 1:0.1-10, and then the drug is added at a mass ratio of phospholipids to drugs of 1:0.01-0.02, added to a heated and stirred container, filled with protective gas or heated at 80-100°C for 10-20 minutes under vacuum conditions, stirred and mixed to fully dissolve the phospholipids in the polyol to obtain a drug-containing mixture (drug-containing preliposomes); the mass ratio of the phospholipids to the polyols is preferably 1:0.5-8, more preferably 1:5, and the mass ratio of the phospholipids to the drugs is preferably 1:0.013-0.018, more preferably 1:0.019-0.020. The ratio of the phospholipids to the phospholipids is 1:0.015, the phospholipids include any one of soybean phospholipids, sunflower phospholipids, egg phospholipids, shrimp phospholipids, and hydrogenated phospholipids; the polyols are liquid polyols, including any one of glycerol, propylene glycol, and polyethylene glycol; the drug is preferably curcumin; the protective gas is an inert gas, including one or more of nitrogen, helium, argon, and xenon, and the filling of the protective gas can prevent the oxidation of the phospholipids; under vacuum conditions, there is no need to add protective gas; the heating temperature is preferably 85-95°C, more preferably 90°C, the heating time is preferably 12-18min, more preferably 15min; the stirring speed is 300-500rpm, preferably 350-450rpm, more preferably 400rpm.

[0046] In the present invention, after cooling the drug-containing mixture, the drug-containing mixture is dispersed in the aqueous phase at a mass ratio of 1:15 to 25 to obtain drug-loaded nanoliposomes; the drug-containing mixture is cooled to 20 to 30°C, the mixture is added to the aqueous phase at a mass ratio of 1:15 to 25 at 20 to 30°C, and a glass rod is used to completely disperse the drug-containing mixture in the aqueous phase until no precipitation is visible to the naked eye, thereby obtaining drug-loaded nanoliposomes; the cooling temperature is preferably 22 to 28°C, and more preferably 25°C; the mass ratio of the mixture to the aqueous phase is preferably 1:18 to 22, and more preferably 1:20, and the aqueous phase includes water, PBS, sodium chloride solution or sucrose solution; the water is one of distilled water, ultrapure water and tap water, and is preferably ultrapure water; the pH of the PBS is 3 to 8, the concentration of the sodium chloride solution is 50 to 1000 mmol / L, the concentration of the sucrose solution is 1 to 20wt%, and the temperature of the aqueous phase is preferably 22 to 28°C, and more preferably 25°C.

[0047] The invention also provides the drug-loaded nano-liposome obtained by the preparation method of the drug-loaded nano-liposome, wherein the drug loaded is curcumin.

[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] according to Figure 1 Nanoliposomes were prepared by the following method:

[0051] (1) Weigh 1 g of sunflower lecithin (PC>90%) and 5 g of glycerol, mix them evenly and place them in a sample bottle, the mass ratio of lecithin to glycerol is 1:5, fill with nitrogen as protective gas, heat in a water bath at 90°C for 15 min, and use a magnetic stirrer at a speed of 400 rpm to ensure that the sample is evenly fused to obtain a mixture (proliposome);

[0052] (2) The mixture was cooled to room temperature (25±2°C), and the mixture was added to ultrapure water (room temperature, 25±2°C) at a mass ratio of 1:20, and gently stirred with a glass rod until the proliposomes were completely dispersed in the ultrapure water without visible precipitation, thereby forming nanoliposomes.

[0053] Example 2

[0054] Nanoliposomes were prepared in the same manner as in Example 1, except that the mass ratio of phospholipid to glycerol was 1:10.

[0055] Example 3

[0056] Nanoliposomes were prepared in the same manner as in Example 1, except that the mass ratio of phospholipid to glycerol was 1:2.

[0057] Example 4

[0058] Nanoliposomes were prepared in the same manner as in Example 1, except that the mass ratio of phospholipid to glycerol was 1:1.

[0059] Example 5

[0060] Nanoliposomes were prepared in the same manner as in Example 1, except that the mass ratio of phospholipid to glycerol was 2:1.

[0061] Example 6

[0062] according to Figure 1 The drug-loaded nanoliposomes were prepared by the following method:

[0063] (1) Weigh 1 g of sunflower lecithin (PC>90%) and 5 g of glycerol, mix them evenly and place them in a sample bottle, the mass ratio of lecithin to glycerol is 1:5, and add 15 mg of curcumin powder (>98%), fill with nitrogen as a protective gas, heat in a water bath at 90° C. for 15 min, and use a magnetic stirrer to stir at a speed of 400 rpm to ensure that the curcumin powder is fully and evenly integrated in the system to obtain a drug-containing mixture (drug-containing preliposomes);

[0064] (2) The drug-containing mixture was cooled to room temperature (25±2°C), and the drug-containing mixture was added to ultrapure water (room temperature, 25±2°C) at a mass ratio of 1:20, and gently stirred with a glass rod until the proliposomes were completely dispersed in the ultrapure water to form curcumin-loaded nanoliposomes.

[0065] Example 7

[0066] Nanoliposomes were prepared in the same manner as in Example 1, except that sunflower lecithin was replaced with soybean lecithin.

[0067] Example 8

[0068] Nanoliposomes were prepared in the same manner as in Example 1, except that sunflower lecithin was replaced with hydrogenated lecithin.

[0069] Example 9

[0070] Nanoliposomes were prepared in the same manner as in Example 1, except that sunflower lecithin was replaced with self-extracted shrimp oil lecithin (the preparation method of self-extracted shrimp oil lecithin is as follows: krill oil is dissolved in acetone at a ratio of 1:5 (w / v), heated to 60°C until completely dissolved, and then placed in a -30°C refrigerator for 24 h to promote the precipitation of lecithin. The precipitate layer was taken out and fully spin-dried to obtain self-extracted shrimp oil lecithin, with a lecithin purity of 93.53%).

[0071] Example 10

[0072] Nanoliposomes were prepared in the same manner as in Example 1, except that glycerol was replaced with propylene glycol.

[0073] Embodiment 11

[0074] Nanoliposomes were prepared in the same manner as in Example 1, except that glycerol was replaced with polyethylene glycol 400 (PEG-400).

[0075] Example 12

[0076] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 3.0 and a concentration of 20 mmol / L.

[0077] Example 13

[0078] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 4.0 and a concentration of 20 mmol / L.

[0079] Embodiment 14

[0080] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 5.0 and a concentration of 20 mmol / L.

[0081] Embodiment 15

[0082] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 6.0 and a concentration of 20 mmol / L.

[0083] Example 16

[0084] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 7.0 and a concentration of 20 mmol / L.

[0085] Embodiment 17

[0086] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 8.0 and a concentration of 20 mmol / L.

[0087] Embodiment 18

[0088] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 50 mmol / L.

[0089] Embodiment 19

[0090] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 100 mmol / L.

[0091] Embodiment 20

[0092] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 200 mmol / L.

[0093] Embodiment 21

[0094] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 400 mmol / L.

[0095] Embodiment 22

[0096] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 800 mmol / L.

[0097] Embodiment 23

[0098] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sodium chloride solution having a pH of 7.0 and a concentration of 1000 mmol / L.

[0099] Embodiment 24

[0100] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 1 wt%.

[0101] Embodiment 25

[0102] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 2 wt%.

[0103] Embodiment 26

[0104] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 5 wt%.

[0105] Embodiment 27

[0106] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 10 wt%.

[0107] Embodiment 28

[0108] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 15 wt%.

[0109] Embodiment 29

[0110] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with a sucrose solution having a pH of 7.0 and a concentration of 20 wt%.

[0111] Comparative Example 1

[0112] Nanoliposomes were prepared in the same manner as in Example 1, except that glycerol was not added.

[0113] Comparative Example 2

[0114] The liposomes prepared by the thin film hydration method are as follows:

[0115] (1) Weigh 1 g of sunflower lecithin (PC>90%) into a beaker, add 100 mL of anhydrous ethanol, and heat in a 60°C water bath until completely dissolved;

[0116] (2) placing the sample obtained in step (1) in a round-bottom rotary evaporator, and rotary evaporating in a 50° C. water bath until the anhydrous ethanol is completely removed and the phospholipid forms a uniform film in the rotary evaporator;

[0117] (3) Weigh 100 mL of pure water and pour it into the rotary evaporator described in step (2), turn off the vacuum, and wash the film by rotating it in a 50°C water bath until the film in the bottle is completely washed into the rotary evaporator, thereby obtaining liposomes ( Figure 8 ).

[0118] Comparative Example 3

[0119] The thin film hydration method was used to prepare curcumin-loaded liposomes, as follows:

[0120] (1) Weigh 1 g of sunflower lecithin (PC>90%) and 15 mg of curcumin powder (>98%) in a beaker, add 100 mL of anhydrous ethanol, and heat in a water bath at 60°C under light protection until completely dissolved;

[0121] (2) placing the sample in step (1) in a round-bottom rotary evaporator, and rotary evaporating in a 50° C. water bath until the anhydrous ethanol is completely removed and the phospholipids and curcumin form a uniform film in the rotary evaporator;

[0122] (3) Weigh 100 mL of pure water and pour it into the rotary evaporator described in step (2), turn off the vacuum, and wash the film by rotating it in a 50° C. water bath until the film in the bottle is completely washed into the rotary evaporator, thereby obtaining curcumin-loaded liposomes ( Fig.13 ).

[0123] Comparative Example 4

[0124] Nanoliposomes were prepared in the same manner as in Example 1, except that glycerol was replaced with polyethylene glycol 800 (PEG-800).

[0125] Comparative Example 5

[0126] Nanoliposomes were prepared in the same manner as in Example 1, except that glycerol was replaced with sorbitol.

[0127] Comparative Example 6

[0128] Nanoliposomes were prepared in the same manner as in Example 1, except that ultrapure water was replaced with PBS having a pH of 2.0 and a concentration of 20 mmol / L.

[0129] Experimental Example 1

[0130] The nanoliposomes obtained in Example 1 were used as samples, and the appearance of the obtained nanoliposomes ( Figure 2 ), particle size distribution and microstructure.

[0131] Particle size distribution: The particle size distribution of nanoliposomes was measured using a Malvern Nano-Sizer laser nanoparticle size analyzer. The liposomes were diluted with purified water to a phospholipid concentration of 1 mg / mL and placed in a four-way dish for measurement. Figure 3 shown.

[0132] Microstructure: The microstructure of the nanoliposomes obtained in Example 1 was observed using a transmission electron microscope (TEM) to verify the liposome structure. The nanoliposomes were diluted with purified water to a phospholipid concentration of 1 mg / mL. A drop of the sample was placed on a copper grid and allowed to stand for 4 minutes, and then excess liquid was removed with filter paper. After drying for 1 minute, the sample was stained with a 1% phosphotungstic acid solution for 1 minute, and excess staining liquid was removed with filter paper. After drying in air for 4 hours, the sample was placed under a transmission electron microscope for observation. The microstructure of the nanoliposomes is shown in FIG. Figure 4 shown.

[0133] Depend on Figure 2 It can be seen that the phospholipid is waxy at room temperature, and presents a uniform flow state after heating and fusing with 5 times the mass of glycerol. After further dispersion in water and hydration, a clear and transparent liposome solution is obtained without precipitation or flocculation, indicating that the nanoliposome obtained by the method of the present invention has good dispersibility and stability.

[0134] Depend on Figure 3 It can be seen that the particle size distribution of the nanoliposomes presents a narrow single-peak distribution, indicating that the liposomes have a relatively uniform particle size distribution, and the average particle size of the nanoliposomes is 80.08 nm.

[0135] Depend on Figure 4 It can be seen that the nanoliposomes obtained in Example 1 present a typical liposome bilayer membrane structure.

[0136] Experimental Example 2

[0137] The mixtures and liposomes obtained in Examples 1 to 5, Comparative Example 1 (Control Group 1) and Comparative Example 2 (Control Group 2) were used as samples, and the state, phase transition temperature, and enthalpy change value of the mixtures were measured, and the particle size of the liposomes was detected. The results are shown in Table 1.

[0138] The mixture (preliposome) was examined using a polarized light microscope. Figure 5 shown.

[0139] The phase transition temperature (Tm) and thermal enthalpy change (ΔH) of the mixture (proliposome) were determined using DSC. 10 mg of the mixture sample was placed in an aluminum crucible, the test temperature was -60 to 90°C, the heating rate was 5°C / min, and the protective gas was 50 mL / min of nitrogen. The Tm and thermal enthalpy change (ΔH) of the sample were analyzed using TRIOS software. The results are as follows: Figure 6 shown.

[0140] The mixture (proliposome) was tested by FTIR, and the results were as follows Figure 7 shown.

[0141] Table 1 Physicochemical properties and liposome particle size of different samples

[0142]

[0143] As shown in Table 1, the mass ratio of phospholipids to polyols has an important influence on the state of pre-liposomes and the formation of liposomes. As the proportion of glycerol increases, phospholipids gradually dissolve in glycerol, the fluidity of the formed mixture (pre-liposomes) gradually increases, and the particle size of liposomes formed by spontaneous hydration by stirring shows a decreasing trend. In the absence of sugar alcohol (glycerol), the phase transition temperature of sunflower phospholipids is higher than room temperature. At room temperature, it is waxy, and the acyl tails are tightly stacked to show a crystalline state. It cannot spontaneously hydrate to form liposomes when added to water. From Examples 1 to 5 and Control Group 2 ( Figure 8 ) It can be seen from the comparison that the traditional thin film hydration method can also form liposomes, but the particle size of the obtained liposomes is 2143.14nm, which is much larger than the particle size of the liposomes formed in Examples 1 to 5. This shows that compared with the traditional thin film hydration method, the method of the present invention can form nanoliposomes with smaller particle size.

[0144] Depend on Figure 5 It can be seen that with the increase of the proportion of glycerol, the crystal structure of phospholipids gradually disappears under a polarizing microscope, indicating that the addition of glycerol can destroy the crystal structure of phospholipids and transform the crystal structure into a liquid structure.

[0145] From Table 1 and Figure 6 It can be seen that with the increase of the proportion of glycerol, the phase transition temperature (Tm) and enthalpy change value (ΔH) of phospholipids decrease rapidly, indicating that the destruction of the phospholipid crystal structure by glycerol leads to a decrease in the phase transition temperature of phospholipids, which is conducive to the hydration of phospholipids to form liposomes at room temperature.

[0146] Depend on Figure 7 It can be seen that with the addition of glycerol, the phosphate group (-PO4 - ) by 1239cm -1 Red shifted to 1229 cm -1This indicates that the phosphate group in the phospholipid molecule can form a hydrogen bond with the hydroxyl group (-OH) in the glycerol molecule. At the same time, the characteristic absorption peak of the acyl chain (-CH2, -CH3) of the phospholipid molecule changes from 2925cm -1 and 2854cm -1 Blue shift to 2933cm -1 and 2884cm -1 This indicates that the acyl chains of the phospholipid molecules are opened by glycerol and the degree of accumulation is reduced.

[0147] Experimental Example 3

[0148] The crystal structure of curcumin in the curcumin-loaded preliposomes was determined using an X-ray diffractometer (XRD) to verify the solubility of curcumin. The sample was evenly spread on a glass slide and scanned between 5-70° at a scanning rate of 5° / min.

[0149] According to the determination method of Experimental Example 1, the appearance, particle size and microstructure of the curcumin-loaded nanoliposomes obtained in Example 6 were determined. The results are shown in Figures 9 to 12 .

[0150] Depend on Fig. 9 It can be seen that curcumin has an obvious crystal peak, while blank pre-liposomes do not have a crystal peak. There is no curcumin crystal peak in the curcumin-loaded pre-liposomes, indicating that curcumin has been fully dissolved in the pre-liposomes.

[0151] Depend on Fig.10 It can be seen that curcumin is fully dissolved in the pre-liposome system after heating, and can form yellow and transparent curcumin-loaded liposomes after hydration, indicating that curcumin is successfully embedded in the formed liposomes.

[0152] Depend on Fig.11 and Fig.12 It can be seen that the particle size of the curcumin-loaded liposome is 156.70 nm, showing a unimodal distribution. The curcumin-loaded liposome presents a typical double-layer membrane structure.

[0153] Experimental Example 4

[0154] The encapsulation efficiency and loading amount of the curcumin-loaded nanoliposomes obtained in Example 6 were measured. The results are shown in Table 2. Comparative Example 3 (Control Group 3) was used as a control.

[0155] Method for determining the encapsulation efficiency of curcumin: 0.1 g of pre-liposomes loaded with curcumin were dispersed in 1 mL of ultrapure water, vortexed thoroughly until completely dispersed to obtain a curcumin suspension, and centrifuged at 12,000 g for 10 min at 4°C to remove the unencapsulated curcumin precipitate. 0.1 mL of the supernatant or suspension was added to 0.9 mL of anhydrous ethanol, and the absorbance was measured at 425 nm after vortexing thoroughly. The curcumin concentration was calculated by comparing the absorbance of the curcumin ethanol solution (90%, v / v) at 425 nm.

[0156] The encapsulation efficiency of curcumin in liposomes was calculated by the following formula:

[0157] Encapsulation efficiency = supernatant curcumin concentration / suspension curcumin concentration*100%;

[0158] The effective loading amount of curcumin in the proliposomes was calculated by the following formula:

[0159] Loading amount = encapsulation efficiency * 15 mg (amount of curcumin added) / 6 g (weight of liposomes containing pre-drug).

[0160] Table 2 Physicochemical properties of different curcumin-loaded liposomes

[0161]

[0162] From Table 2 and Fig.13 It can be seen that the particle size of the curcumin-loaded liposome obtained by the thin film hydration method is much larger than the curcumin-loaded liposome prepared by the proliposome method, indicating that the proliposome method in the present invention can effectively prepare nano-scale drug-loaded liposomes. The encapsulation efficiency (72.14%) of the curcumin-loaded liposomes obtained by the thin film hydration method is lower than that of the curcumin-loaded liposomes (93.62%) prepared by the proliposome method. This may be because the coarse liposome particle size is larger, which easily causes the leakage of the encapsulated curcumin. The curcumin-loaded liposomes obtained by the method of the present invention have an encapsulation efficiency of 93.62% in the nano-liposomes, and the effective carrying capacity of curcumin in the proliposomes is 2.34 mg / g.

[0163] Experimental Example 5

[0164] The liposomes obtained in Example 1, Examples 7 to 11, Comparative Examples 4 and Comparative Example 5 were used as samples to test their appearance, whether they can be spontaneously hydrated by stirring to synthesize liposomes, and the particle size of the liposomes. The results are shown in Table 3.

[0165] Table 3 Physicochemical properties and particle size of different samples

[0166]

[0167]

[0168] As shown in Table 3, different types of phospholipids (soy phospholipids, hydrogenated phospholipids and self-extracted shrimp oil phospholipids) can form uniform proliposomes with glycerol, and can spontaneously form nanoliposomes after adding ultrapure water and stirring, indicating that the method of the present invention is applicable to most phospholipids. And sunflower phospholipids and different types of liquid polyols (glycerol, propylene glycol, PEG-400) can form uniform proliposomes, and can spontaneously form nanoliposomes after adding ultrapure water and stirring, indicating that the method of the present invention is applicable to most liquid sugar alcohols. For polyols (PEG-800, sorbitol) that are solid at room temperature, they cannot be stirred and dissolved with phospholipids after heating, cannot form a mixture, and cannot spontaneously form nanoliposomes. It shows that the method of the present invention is applicable to polyols that are liquid at room temperature.

[0169] Experimental Example 6

[0170] The liposomes obtained in Examples 12 to 29 and Comparative Example 6 were used as samples to test whether they could spontaneously hydrate and synthesize liposomes by stirring and the particle size of the liposomes. The results are shown in Table 4.

[0171] Table 4 Physicochemical properties and particle size of different samples

[0172]

[0173] As shown in Table 4, the proliposomes can spontaneously hydrate to form nanoliposomes in PBS with a pH of 3.0 to 8.0 (Examples 12 to 17), and the particle size is in the range of 82.14 to 87.52 nm, indicating that the proliposomes of the present invention can be used in aqueous phases with a wide pH range. When the pH is 2.0 (Comparative Example 6), the particle size of the hydrated liposomes rises to 1025.43 nm, which may be because the phospholipids are hydrolyzed in this strongly acidic pH range, resulting in the destruction of the liposome structure. As shown in the results of Examples 18 to 23, the proliposomes can spontaneously hydrate to form nanoliposomes in aqueous solutions with a sodium chloride concentration of 50 to 1000 mmol / L, and the particle size is in the range of 75.12 to 79.68 nm, indicating that the proliposomes of the present invention can be used in a wide range of salt solutions. From the results of Examples 24 to 29, it can be seen that the proliposomes can spontaneously hydrate to form nanoliposomes in 1-20wt% sucrose solutions with a particle size ranging from 77.35 to 78.07nm, indicating that the proliposomes of the present invention can be used in solutions with different sugars.

[0174] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing nanoliposomes, characterized in that: The following steps are involved: (1) mixing the phospholipid and the polyol, heating at 80 to 100° C. for 10 to 20 minutes in a protective gas atmosphere or under vacuum conditions, and mixing to obtain a mixture; (2) After cooling the mixture, the mixture is dispersed in the aqueous phase at a mass ratio of the mixture to the aqueous phase of 1:15 to 25 to obtain nanoliposomes.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the phospholipid to the polyol is 1:0.1-10.

3. The preparation method according to claim 1, characterized in that: The phospholipids include any one of soybean lecithin, sunflower lecithin, egg lecithin, shrimp lecithin and hydrogenated lecithin.

4. The preparation method according to claim 1, characterized in that: The polyol is a liquid polyol, including any one of glycerol, propylene glycol and polyethylene glycol.

5. The preparation method according to claim 1, characterized in that: The protective gas is an inert gas.

6. The preparation method according to claim 1, characterized in that: The aqueous phase includes water, PBS, sodium chloride solution or sucrose solution.

7. The preparation method according to claim 6, characterized in that: The pH of the PBS is 3-8, the concentration of the sodium chloride solution is 50-1000 mmol / L, and the concentration of the sucrose solution is 1-20 wt %.

8. The nanoliposome obtained by the preparation method according to any one of claims 1 to 7.

9. A method for preparing drug-loaded nanoliposomes, characterized in that: The following steps are involved: (1) mixing the phospholipid, polyol and drug, heating at 80-100° C. for 10-20 min in a protective gas or vacuum environment, and mixing to obtain a drug-containing mixture; (2) After cooling the drug-containing mixture, the drug-containing mixture is dispersed in the aqueous phase at a mass ratio of the drug-containing mixture to the aqueous phase of 1:15 to 25 to obtain drug-loaded nanoliposomes.

10. The drug-loaded nanoliposome obtained by the method for preparing drug-loaded nanoliposome according to claim 9, characterized in that: The drug contained is curcumin.

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