CN113456593A

CN113456593A - Preparation method of liposome emulsion

Info

Publication number: CN113456593A
Application number: CN202110746127.8A
Authority: CN
Inventors: 卢永杰; 程路诗; 张炽坚; 张兵; 江桦; 关焕敏; 艾勇; 何廷刚
Original assignee: Hua An Tang Biotech Group Co ltd
Current assignee: Hua An Tang Biotech Group Co ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-10-01

Abstract

The invention relates to the technical field of liposome, in particular to a preparation method of liposome emulsion. The invention discloses a preparation method of liposome emulsion, which comprises the steps of mixing phospholipid, insoluble drugs and water to obtain suspension, dispersing at a proper temperature through high-speed shearing, and crushing the phospholipid and the insoluble drugs to form a micron scale. Under the micron scale, after long-time shearing, the insoluble drug particles and the phospholipid particles interact to emulsify, and then self-assemble to form the micron-scale liposome. The preparation method does not use toxic and harmful organic solvents, the production process is safe and environment-friendly, and the product safety is high; the preparation equipment and the process are simple, and the method is suitable for large-scale production and improves the production efficiency.

Description

Preparation method of liposome emulsion

Technical Field

The invention relates to the technical field of liposome, in particular to a preparation method of liposome emulsion.

Background

In recent years, liposome has attracted much attention and is widely used in the fields of biomedicine, cosmetics, health foods and the like.

There are many methods for preparing liposomes of fat-soluble drugs, such as: the film method, the reverse phase evaporation method, the injection method and the like, and most preparation methods involve the use of organic solvents. The introduction of organic solvents may cause risks such as environmental pollution and product solvent residue, and directly affect the quality of products, so strict control and management are required in industrial production. Besides the thin film method, other traditional liposome preparation methods are generally not suitable for large-scale industrial production, thereby limiting the popularization and application of the liposome in industrialization.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing a nanoliposome emulsion, which can prepare a lipid-soluble drug liposome simply and rapidly without using an organic solvent.

The specific technical scheme is as follows:

the invention provides a preparation method of a nanoliposome emulsion, which comprises the following steps:

step 1: mixing phospholipid, cholesterol, medicine and water to form suspension; the drug comprises a fat-soluble drug; the water is deionized water, pure water or ultrapure water;

step 2: shearing the suspension at a high speed under a heating and heat-preserving state to obtain emulsion;

the invention mixes phospholipid, insoluble drug and water to obtain suspension, which is dispersed by high speed shearing at proper temperature to pulverize phospholipid and insoluble drug to form micron scale. Under the micron scale, after long-time shearing, the insoluble drug particles and the phospholipid particles interact to emulsify, and then self-assemble to form the micron-scale liposome.

The method does not use toxic and harmful organic solvents in the process of preparing the fat-soluble drug liposome, and has safe and environment-friendly production process and high product safety; the preparation equipment and the process are simple, and the method is suitable for large-scale production and improves the production efficiency.

In step 1 of the present invention, the fat-soluble drug comprises: one or more of glabridin, ceramide, resveratrol and fat-soluble vitamins.

The phospholipid comprises one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, dilauroyl phosphatidylcholine, dimyristoyl phosphorylcholine, dipalmitoyl phosphorylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine and dioleoyl phosphatidylethanolamine.

The medicament further comprises: a water-soluble drug. The water-soluble medicine comprises one or more than two of sodium hyaluronate, nano metal colloid dispersion liquid and water-soluble vitamins.

Before mixing, the method also comprises the following steps: adding a surfactant. The addition of a surfactant can facilitate the solubilization of the fat-soluble drug.

The surfactant is ionic surfactant and/or nonionic surfactant, wherein the cationic surfactant comprises quaternary ammonium salt, the anionic surfactant comprises higher fatty acid salt or sulfate salt, and the nonionic surfactant comprises one or more than two of tween, span, PEG fatty acid ester and polyglycerol-10-hard fatty acid ester.

Before mixing, the method also comprises the following steps: the polyol is added. The addition of the polyol can further facilitate the dissolution of a portion of the fat-soluble drug. Glabridin has low solubility in polyhydric alcohol.

The polyhydric alcohol comprises one or more of glycerol, propylene glycol, butylene glycol, pentylene glycol, and isoprene glycol.

In the invention, according to the mass percentage,

2-15% of phospholipid;

0.01-5% of a medicament;

0-5% of cholesterol;

the balance being water.

Preferably, the amount of the surfactant is, in mass percent,

2-10% of phospholipid;

0.01-4% of a medicament;

0-4% of cholesterol;

the balance being water.

More preferably, the amount of the surfactant is, in mass percent,

2-8% of phospholipid;

0.1-3% of a medicament;

0-3% of cholesterol;

when the medicament is a fat-soluble medicament and a water-soluble medicament, the dosage ratio of the fat-soluble medicament and the water-soluble medicament is not particularly limited.

In the invention, the weight percentage of the surfactant is 0.1-20%, preferably 0.1-15%, and more preferably 0.1-8%; 5 to 60% of a polyol, preferably 5 to 45%, more preferably 5 to 30%.

In step 2 of the invention, the heating temperature of the suspension liquid needs to be higher than the phase transition temperature of the phospholipid. The heating temperature is 50-100 ℃, and preferably 50-70 ℃; then, maintaining the heating temperature for shearing, wherein a shearing disperser is adopted for shearing; the shearing rate is 8000-20000 rpm (rotor diameter is 13mm), the shearing time is more than 30min, preferably more than 60min at the shearing rate of 8000-15000 rpm (rotor diameter is 13mm), more preferably 60-90 min. The liposome obtained after shearing is micron-sized.

The nano-scale liposome has better dispersibility and stability and better skin permeability. Therefore, after the high-speed shearing is performed in step 2 of the present invention, the method further comprises: homogenizing under high pressure. The high-pressure homogenization can crush and thin the micron-sized liposome to obtain the nano-sized liposome. The high-pressure homogenization specifically comprises the following steps: each cycle was carried out at a pressure of 400bar, 600bar, 800bar, 1000bar for 2 or more times.

According to the technical scheme, the invention has the following advantages:

the invention provides a preparation method of a nanoliposome emulsion, which comprises the steps of mixing phospholipid, an insoluble drug and water to obtain a suspension, dispersing at a proper temperature through high-speed shearing, and crushing the phospholipid and the insoluble drug to form a micron scale. Under the micron scale, after long-time shearing, the insoluble drug particles and the phospholipid particles interact to emulsify, and then self-assemble to form the micron-scale liposome. The preparation method can dissolve the fat-soluble medicine only by mechanical shearing action, and has unexpected effect. In addition, the preparation method does not use toxic and harmful organic solvents, the production process is safe and environment-friendly, and the product safety is high; the preparation equipment and the process are simple, and the method is suitable for large-scale production and improves the production efficiency.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 1 of the present invention;

FIG. 2 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 2 of the present invention;

FIG. 3 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 3 of the present invention;

FIG. 4 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 4 of the present invention;

FIG. 5 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 5 of the present invention;

FIG. 6 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 6 of the present invention;

FIG. 7 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 7 of the present invention;

FIG. 8 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 8 of the present invention;

FIG. 9 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 9 of the present invention;

FIG. 10 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 10 of the present invention;

FIG. 11 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 11 of the present invention;

FIG. 12 is a graph showing the average particle size of liposomes in the emulsion obtained in step 1 of example 1 of the present invention;

FIG. 13 is a graph showing the average particle size of liposomes in the emulsion obtained in step 1 of example 2 of the present invention;

FIG. 14 is a graph showing the average particle size of liposomes in the emulsion obtained in step 1 of example 3 of the present invention;

FIG. 15 is a graph showing the mean particle size of liposomes in an emulsion obtained in step 1 of example 4 of the present invention;

FIG. 16 is a cryo-transmission electron micrograph of a liposome sample (scale on the left is 50nm and scale on the right is 100nm) of example 6 according to the present invention;

FIG. 17 is a distribution diagram of liposome particle sizes in example 6 of the present invention;

FIG. 18 is a graph showing the mean particle size of liposome emulsions obtained at different shear times (shear rate 10000rpm, shear temperature 50 ℃) in step 1 of example 6, example 9 and example 10 of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The reagents in the examples of the present invention are all commercially available.

Wherein, the high-speed homogeneous dispersion machine of experimental type: IKA, Germany.

Experimental high-pressure homogenizer: suzhou ATS Inc.

Zeta nanometer particle size analyzer: malvern, uk.

Reagent:

soybean lecithin: PC > 95%, Sigma-Aldrich Sigma Aldrich trade company, Shanghai.

Glabridin: HPLC purity is more than or equal to 90 percent, Qinghai lake pharmaceutical industry Co.

Cholesterol: purity > 96%, Sigma-Aldrich Sigma Aldrich trade ltd.

Polyglycerol-10-stearate: heliochemical trade (shanghai) limited.

Butanediol: purity > 98%, manufactured by Nippon Daiillo Co., Ltd.

Example 1

The preparation formula of glabridin liposome in this example is shown in table 1, and the specific preparation steps are as follows:

(1) adding phospholipid (PC 95%), glabridin and cholesterol into water, heating in water bath at 60 deg.C, and high-speed shearing with experiment type IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

(2) And (2) homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure respectively to obtain uniform glabridin liposome.

As shown in fig. 12, the particle size of the liposome in the emulsion obtained in step (1) of this example is about 10 to 20 μm, which indicates that the high-speed shearing sample does not reach the nanometer level, and the fat-soluble drug is emulsified and dissolved in water.

As can be seen in FIG. 1, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Example 2

As can be seen in FIG. 2, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

As shown in fig. 13, the particle size of the liposome in the emulsion obtained in step (1) of this example is about 4-10 μm, which indicates that the high-speed shearing sample does not reach nanometer level, and the fat-soluble drug is emulsified and dissolved in water.

Example 3

As can be seen in FIG. 3, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion.

As shown in fig. 14, the particle size of the liposome in the emulsion obtained in step (1) of this example is about 4-8 μm, which indicates that the high-speed shearing sample does not reach nanometer level, and the fat-soluble drug is emulsified and dissolved in water.

Example 4

(1) adding phospholipid (PC 95%), glabridin and cholesterol into water, heating in 50 deg.C water bath, and high-speed shearing with experiment type IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

As can be seen in FIG. 4, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion. As shown in fig. 15, the particle size of the liposome in the emulsion obtained in step (1) of this example is about 4-10 μm, which indicates that the high-speed shearing sample does not reach nanometer level, and the fat-soluble drug is emulsified and dissolved in water.

Example 5

The preparation formula of glabridin liposome in this example is shown in table 2, and the specific preparation steps are as follows:

(1) adding phospholipid (PC 95%), glabridin and cholesterol into a mixture of water and butanediol at a certain amount, heating in water bath at 50 deg.C, and high-speed shearing for 30min at 10000rpm by using an experimental IKA high-speed shearing disperser to obtain uniform emulsion.

As can be seen in FIG. 5, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Example 6

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in a water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

(2) And (2) homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure to obtain transparent and uniform glabridin liposome.

As can be seen in FIG. 6, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion.

As can be seen from fig. 16, glabridin was successfully encapsulated in liposomes.

As can be seen from FIG. 17, the mean particle size of glabridin liposome was 78 nm.

Example 7

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in water bath at 50 deg.C, and high-speed shearing at 8000rpm for 30min with an experimental IKA high-speed shearing disperser to obtain uniform emulsion.

As can be seen in FIG. 7, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion.

Example 8

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in water bath at 60 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

As can be seen in FIG. 8, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion.

Example 9

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in a water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

As can be seen in fig. 9, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Example 10

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in a water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 90min to obtain uniform emulsion.

As can be seen in fig. 10, after high shear in step 1, a multi-lamellar globular structure resembling liposomes can be observed in the step 1 emulsion.

As can be seen from FIG. 18, the particle size of the liposomes in the emulsion tended to be stable and maintained around 5-6 μm with increasing shearing time, which indicates that the high-speed sheared sample did not reach nanometer level and the particle size was uniform, and the fat-soluble drug was emulsified and dissolved in water.

Example 11

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into water of a prescribed amount, heating in water bath at 50 deg.C, and high-speed shearing for 30min at 10000rpm by using an experimental IKA high-speed shearing disperser to obtain uniform emulsion.

As can be seen in FIG. 11, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the step 1 emulsion.

Comparative example 1

The comparative example is the preparation of glabridin liposome, the formula is shown in table 2, and the preparation steps are as follows:

(1) phospholipid (PC 95%), glabridin and cholesterol were dissolved in methanol-chloroform (62:38) to give a clear and transparent solution, and the organic solvent was removed by rotary evaporation to give a film.

(2) And (2) placing the film obtained in the step (1) in a water bath at 60 ℃, and adding a mixed solution of water and butanediol in a prescription amount while stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure to obtain glabridin liposome.

Comparative example 2

(1) dissolving phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol in methanol-chloroform (62:38) to obtain clear and transparent solution, and rotary evaporating to remove organic solvent to obtain film.

Comparative example 3

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in water bath at 50 deg.C, and high-speed shearing at 5000rpm for 30min with an experimental IKA high-speed shearing disperser to obtain uniform emulsion.

Comparative example 4

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in water bath at 30 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

TABLE 1 examples 1-4 formulations

(g)	Example 1	Example 2	Example 3	Example 4
					Soybean lecithin	15	8	2	5
Cholesterol	5	3	0	1.5
					Glabridin	5	3	0.1	0.5
Water (W)	75	86	97.9	93

TABLE 2 examples 4-11 and comparative examples 1-4 formulations

Test examples

Dispersing the glabridin liposome prepared in examples 1 to 11 by using PBS as a dispersing agent, and measuring the average particle size by using a Zeta nanometer particle size analyzer; separating the encapsulated and free glabridin by an ultrafiltration centrifugal tube, detecting the glabridin content by HPLC and calculating the encapsulation rate. Test results table 3 and table 4.

As is clear from the results in tables 3 to 4, glabridin liposome was successfully obtained in examples 1 to 11. Compared with the comparative example 1 and the comparative examples 6-10, the glabridin liposome prepared in the example 5 has no difference on appearance, average particle size and glabridin content basically, which shows that the effect of the liposome prepared in the example is not changed along with the process, and the liposome can replace the traditional preparation method. The glabridin liposome prepared by the liposome preparation method of the comparative example 3 is in a yellow turbid state in appearance, and insoluble particles exist; the sample has larger grain diameter, the encapsulation efficiency of the glabridin medicament is obviously reduced, which shows that the glabridin and other fat-soluble medicaments are difficult to fully react with phospholipid molecules only at a lower shearing speed, so that the grain diameter is increased, the encapsulation efficiency is reduced, and the quality of a liposome preparation is reduced. The glabridin liposome prepared by the liposome preparation method of comparative example 4 is in a yellow turbid state in appearance, and insoluble particles exist; the sample has larger particle size, the encapsulation efficiency of the glabridin medicament is obviously reduced, which shows that when the temperature is insufficient, the phospholipid phase cannot be fully converted into the liquid crystal phase, and fat-soluble medicaments such as glabridin and the like are difficult to fully react with phospholipid molecules, so that the particle size is increased, the encapsulation efficiency is reduced, and the quality of a liposome preparation is reduced.

TABLE 3 characterization of glabridin liposomes of examples 1-4

TABLE 4 characterization of glabridin liposomes of examples 1-10 and comparative examples 1-4

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a liposome emulsion, comprising the steps of:

step 2: and shearing the suspension at a high speed under the heating and heat preservation state to obtain the liposome emulsion.

2. The method according to claim 1, wherein the shearing rate in step 2 is 8000rpm or more and the time is 30min or more.

3. The method according to claim 1 or 2, wherein the temperature of the heating and holding in step 2 is not lower than the phase transition temperature of the phospholipid.

4. The process according to claim 1, wherein the fat-soluble drug comprises: one or more of glabridin, ceramide, resveratrol and fat-soluble vitamins;

5. The method of claim 1, wherein the medicament further comprises: a water-soluble drug.

6. The method according to claim 5, wherein the water-soluble drug includes one or more of sodium hyaluronate, nano-metal colloid dispersion, and water-soluble vitamin.

7. The method according to claim 1, wherein the reaction mixture is, in mass percent,

2-15% of phospholipid;

0.01-5% of a medicament;

0-5% of cholesterol;

the balance being water.

8. The method of claim 1, wherein the step 2 further comprises, before obtaining the liposome emulsion: homogenizing under high pressure;

the high-pressure homogenization specifically comprises the following steps: each cycle was carried out at a pressure of 400bar, 600bar, 800bar, 1000bar for 2 or more times.

9. The method according to claim 1, wherein the step 1 further comprises, before the mixing: adding a surfactant.

10. The method according to claim 1 or 9, wherein the step 1, before the mixing, further comprises: the polyol is added.