A method for preparing blank liposomes using non-organic solvent

Abstract

The invention discloses a method for preparing blank liposomes by a non-organic solvent, comprising the following steps: according to the ratio: phospholipid 1%-10%, cholesterol 1%-10%, water or ammonium sulfate solution 80%-98% raw materials are taken, after phospholipid and cholesterol are mixed with water or ammonium sulfate solution, they are evenly dispersed by magnetic stirring to obtain a crude liposome suspension; the obtained crude liposome suspension is subjected to a high-speed shearing process of 10000-20000rpm; the suspension after shearing is subjected to multiple ultra-high pressure homogenization processes under a pressure condition of 30000-60000psi under low temperature conditions, i.e., blank liposomes are obtained. Blank liposomes are prepared by the method of the present invention without the use of an organic solvent, and the obtained blank liposomes have the advantages of small particle size, high efficiency, stability, safety, uniformity, etc., and can be used for further encapsulation and delivery of drugs and cosmetic active ingredients.

Description

Technical Field

[0001] The invention relates to the fields of medicine and cosmetics, and in particular to a method for preparing blank liposomes with a non-organic solvent. Background Art

[0002] Liposome is a new type of targeted drug delivery system, which forms a nanoscale spherical drug carrier system by encapsulating drugs in lipid bilayers. The liposome membrane is mainly composed of phospholipids and cholesterol and has good biocompatibility. It can encapsulate hydrophilic drugs and form a membrane with hydrophobic drugs. Liposome technology can reduce the dosage of drugs, slowly release drugs, increase the targeting of drugs, improve the efficacy and reduce the toxic side effects of drugs. Therefore, liposome technology is widely used in the fields of medicine and cosmetics.

[0003] According to the loading mechanism of the drug, the preparation methods of liposomes mainly include passive drug loading and active drug loading. The passive drug loading method is to dissolve the drug in the aqueous phase or organic phase, and then prepare the drug-containing liposomes by thin film dispersion method or ethanol injection method. This method is suitable for preparing liposomes with strong lipid solubility or water solubility. However, for some amphoteric drugs, such as some weak acid and weak base salts, their oil-water partition coefficient is easily affected by the pH or ionic strength of the medium, so it is more suitable for active drug loading. The active drug loading method requires the preparation of blank liposomes, and then uses the ion concentration gradient difference or pH gradient difference inside and outside the liposome to allow the drug to be encapsulated to enter the liposome membrane in molecular form, and form a complex precipitation with the corresponding ions, thereby completing the drug encapsulation process.

[0004] In the traditional process of preparing liposomes by active drug loading, highly toxic organic solvents such as chloroform, methanol, and ethanol are usually required. Subsequently, the organic solvent needs to be removed by dialysis and other methods to meet the organic solvent content limit standards specified in the pharmacopoeia. The use of organic solvents increases the production steps of liposomes and increases production costs. Traditional high-pressure homogenization technology faces huge challenges in homogenizing crude liposome suspensions to nanometer sizes until they are close to a dissolved state. Even if a homogenizer based on the microfluidic principle is introduced, the maximum pressure of a traditional homogenizer can usually only reach 30,000 psi. In the case where no organic solvent is added to assist dispersion, the homogenization effect is often unsatisfactory, and it is difficult to reach a nanometer or even a state close to a dissolved state.

[0005] Therefore, how to improve the preparation efficiency of liposomes in a breakthrough way and homogenize the crude liposome suspension to below the set nanoparticle size without using any organic solvents, thus creating ideal conditions for the subsequent drug loading steps and opening up new possibilities for the application of drug carriers is an urgent problem to be solved. Summary of the invention

[0006] In order to solve at least one of the above problems existing in the traditional blank liposome preparation process, the present invention provides a method for preparing blank liposomes with a non-organic solvent. The blank liposomes prepared by the method of the present invention have the advantages of small particle size, high efficiency, stability, safety, uniformity, etc., and can be used for further encapsulation and delivery of drugs and cosmetic active ingredients.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides a method for preparing blank liposomes using a non-organic solvent, comprising the following steps:

[0009] S1. Take raw materials according to the ratio of 1%-10% phospholipid, 1%-10% cholesterol, 80%-98% water or ammonium sulfate solution, mix phospholipid, cholesterol with water or ammonium sulfate solution, and disperse them evenly by magnetic stirring to obtain a crude liposome suspension;

[0010] S2, subjecting the crude liposome suspension obtained in S1 to a high-speed shearing treatment at 10000-20000 rpm;

[0011] The sheared suspensions in S3 and S2 are subjected to multiple ultrahigh pressure homogenization treatments at low temperature and pressure of 30,000-60,000 psi to obtain blank liposomes.

[0012] In some embodiments of the present invention, the ratio of the raw materials is: 5%-10% phospholipid, 1%-5% cholesterol, 85%-94% water or ammonium sulfate solution. In some embodiments of the present invention, the water is triple distilled water.

[0013] In some embodiments of the present invention, the ultra-high pressure homogenization treatment is performed 5-8 times in step S3.

[0014] In some embodiments of the present invention, the low temperature condition is 0-10°C.

[0015] In some embodiments of the present invention, the high-speed shearing time in step S2 is 1-3 minutes.

[0016] In some embodiments of the present invention, the particle size of the blank liposome is no greater than 200 nm. In some specific embodiments of the present invention, the particle size of the blank liposome is between 70-200 nm.

[0017] In some embodiments of the present invention, the phospholipids include one or more of natural lecithin, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, and PEG-modified phospholipids.

[0018] In some embodiments of the present invention, the cholesterol includes one or more of cholesterol, cholesterol PEG maleimide, amino cholesterol PEG, cholesterol sulfate, cholesterol succinate, cholesterol stearate, cholesterol nonanoate, mercapto cholesterol, cholesterol linoleate, and cholesterol arginine ethyl ester.

[0019] The second aspect of the present invention provides the use of blank liposomes prepared by the method described in the first aspect in preparing liposomes loaded with active substances, wherein the active substances in the liposomes loaded with active substances are one or more of active substances in drugs, cosmetics and substances with health care functions.

[0020] Beneficial Effects of the Invention

[0021] The present invention has the following beneficial effects: no organic solvent is used in the operation process of the present invention, which is environmentally friendly and clean, and the subsequent step of removing the organic solvent is eliminated, thereby saving production costs and improving production efficiency. The preparation process of the present invention is simple and easy to repeat, suitable for industrial production, and the particle size of the liposome can be controlled by adjusting the parameters of the formula, homogenization pressure and number of times to obtain blank liposomes with a particle size of less than 200nm. The blank liposomes obtained by the method of the present invention are translucent in appearance, have obvious opalescence, and have good stability. The preparation method is simple and easy to repeat, and can be used for encapsulation of active ingredients of medicines and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a sample image of blank liposomes prepared in Example 1 of the present invention;

[0023] Figure 2 This is a particle size distribution diagram of blank liposomes prepared in Example 1 of the present invention;

[0024] Figure 3 This is a sample image of blank liposomes prepared in Example 2 of the present invention;

[0025] Figure 4 This is a particle size distribution diagram of blank liposomes prepared in Example 2 of the present invention;

[0026] Figure 5 This is a sample image of blank liposomes prepared in Example 3 of the present invention;

[0027] Figure 6 This is the particle size distribution diagram of the blank liposomes prepared in Example 3 of the present invention;

[0028] Figure 7 This is a sample image of blank liposomes prepared in Example 4 of the present invention;

[0029] Figure 8 This is a particle size distribution diagram of blank liposomes prepared in Example 4 of the present invention;

[0030] Fig. 9 This is a sample image of blank liposomes prepared in Example 5 of the present invention;

[0031] Fig.10 This is the particle size distribution diagram of the blank liposomes prepared in Example 5 of the present invention;

[0032] Fig.11 This is a sample picture of blank liposomes prepared in Comparative Example 1 of the present invention;

[0033] Fig.12 This is a particle size distribution diagram of blank liposomes prepared in Comparative Example 1 of the present invention;

[0034] Fig.13 This is a sample picture of blank liposomes prepared in Comparative Example 2 of the present invention;

[0035] Fig.14 This is a particle size distribution diagram of blank liposomes prepared in Comparative Example 2 of the present invention;

[0036] Fig.15 This is a sample picture of blank liposomes prepared in Comparative Example 3 of the present invention;

[0037] Fig.16 This is a particle size distribution diagram of blank liposomes prepared in Comparative Example 3 of the present invention;

[0038] Fig.17 This is a sample picture of blank liposomes prepared in Comparative Example 4 of the present invention;

[0039] Fig.18 This is a particle size distribution diagram of blank liposomes prepared in Comparative Example 4 of the present invention;

[0040] Fig.19 This is a sample picture of blank liposomes prepared in Comparative Example 5 of the present invention;

[0041] Fig. 20 This is the particle size distribution diagram of the blank liposomes prepared in Comparative Example 5 of the present invention. DETAILED DESCRIPTION

[0042] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications can be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention. Those skilled in the art will recognize or will understand through routine experimentation many equivalent techniques for the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0043] Example 1

[0044] Take the following raw materials for preparing blank liposomes: egg yolk lecithin: 2.4 g, cholesterol: 0.6 g, and triple distilled water: 27 ml.

[0045] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0046] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0047] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 5 times to obtain blank liposomes.

[0048] Sample photo of blank liposomes Figure 1 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Figure 2 shown.

[0049] The results showed that the blank liposomes were translucent in appearance, had obvious opalescence, and had a particle size of 148 nm.

[0050] Example 2

[0051] Take the following blank liposome preparation materials: egg yolk lecithin: 1.8 g, cholesterol: 0.6 g, triple distilled water: 27 ml.

[0052] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0053] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0054] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 5 times to obtain blank liposomes.

[0055] Sample photo of blank liposomes Figure 3 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Figure 4 shown.

[0056] The results showed that the blank liposomes were translucent in appearance, had obvious opalescence, and had a particle size of 73 nm.

[0057] Example 3

[0058] Take the following raw materials for preparing blank liposomes: egg yolk lecithin: 2.4 g, cholesterol: 0.6 g, and triple distilled water: 27 ml.

[0059] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0060] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0061] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 8 times to obtain blank liposomes.

[0062] Sample photo of blank liposomes Figure 5 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Figure 6 shown.

[0063] The results showed that the blank liposomes were translucent in appearance, had obvious opalescence, and had a particle size of 195 nm.

[0064] Example 4

[0065] Take the following raw materials for preparing blank liposomes: 2.5 g hydrogenated soybean lecithin, 0.5 g hydrogenated phosphatidylethanolamine, 0.5 g cholesterol, and 120 ml 0.25 M ammonium sulfate.

[0066] The weighed hydrogenated soybean lecithin, hydrogenated phosphatidylethanolamine and cholesterol were added into the ammonium sulfate solution and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0067] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0068] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 8 times to obtain blank liposomes.

[0069] Sample photo of blank liposomes Figure 7 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Figure 8 shown.

[0070] The results showed that the blank liposomes were translucent in appearance, had obvious opalescence, and had a particle size of 103 nm.

[0071] Example 5

[0072] Take the following raw materials for preparing doxorubicin-containing liposomes: 2.5 g hydrogenated soybean lecithin, 0.5 g phosphatidylethanolamine, 0.5 g cholesterol, 120 ml 0.25 M ammonium sulfate, and 50 mg doxorubicin.

[0073] Preparation of blank liposomes: weighed hydrogenated soybean lecithin, phosphatidylethanolamine and cholesterol, add them into ammonium sulfate solution, and disperse them evenly by magnetic stirring to obtain a crude liposome suspension.

[0074] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0075] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 8 times to obtain blank liposomes.

[0076] Preparation of doxorubicin-containing liposomes: After dialyzing the blank liposome solution, add the doxorubicin solution, incubate at 60° C. for 1 h, and then dialyze to obtain the doxorubicin liposome solution.

[0077] Sample photo of doxorubicin liposomes Fig. 9 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig.10 shown.

[0078] The results showed that the doxorubicin liposomes had a uniform texture and a particle size of 105 nm.

[0079] Comparative Example 1

[0080] Take the following materials for preparing blank liposomes: 0.6 g of egg yolk lecithin, 0.2 g of cholesterol, and 27 ml of triple distilled water.

[0081] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0082] The crude liposome suspension was sheared by a high-speed shearing machine at 10,000 rpm for 3 minutes to obtain blank liposomes.

[0083] Sample photo of blank liposomes Fig.11 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig.12 shown.

[0084] The results showed that the appearance transparency of the blank liposomes decreased, and it was milky white with a particle size of 862nm.

[0085] Comparative Example 2

[0086] Take the following materials for preparing blank liposomes: 0.3 g of egg yolk lecithin, 0.1 g of cholesterol, and 15 ml of triple distilled water.

[0087] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0088] The crude liposome suspension was subjected to probe ultrasonic treatment at a ultrasonic time of 5 min and an ultrasonic power of 300 W to obtain blank liposomes.

[0089] Sample photo of blank liposomes Fig.13 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig.14 shown.

[0090] The results showed that the appearance transparency of the blank liposomes decreased, and it was milky white with a particle size of 650nm.

[0091] Comparative Example 3

[0092] Take the following materials for preparing blank liposomes: 0.3 g of egg yolk lecithin, 0.1 g of cholesterol, and 20 ml of triple distilled water.

[0093] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0094] The sheared crude liposome suspension was subjected to multiple uniform treatments using a Genizer microfluidizer, with the pressures being 5000 psi for 2 times and 30000 psi for 5 times, respectively, to obtain blank liposomes.

[0095] Sample photo of blank liposomes Fig.15The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig.16 shown.

[0096] The results showed that the appearance transparency of the blank liposomes decreased, and it was milky white with a particle size of 658nm.

[0097] Comparative Example 4

[0098] Take the following blank liposome preparation materials: egg yolk lecithin: 0.3 g, cholesterol: 0.1 g, triple distilled water: 20 ml.

[0099] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0100] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0101] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized once to obtain blank liposomes.

[0102] Sample photo of blank liposomes Fig.17 The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig.18 shown.

[0103] The results showed that the blank liposomes were white in appearance and had a particle size of 885 nm.

[0104] Comparative Example 5

[0105] Take the following blank liposome preparation materials: egg yolk lecithin: 0.3 g, cholesterol: 0.1 g, triple distilled water: 20 ml.

[0106] The weighed egg yolk lecithin and cholesterol were added into triple distilled water and dispersed evenly by magnetic stirring to obtain a crude liposome suspension.

[0107] The crude liposome suspension was sheared by a high-speed shearing machine at 20,000 rpm for 2 minutes.

[0108] At 4°C, the sheared crude liposome suspension was homogenized multiple times by an UltraGenizer microfluidizer: the pressures were 40,000 psi, 50,000 psi and 60,000 psi, and each pressure was homogenized 10 times to obtain blank liposomes.

[0109] Sample photo of blank liposomes Fig.19The particle size was measured by Genizer dynamic light scattering instrument, and the particle size distribution was as shown in Fig. 20 shown.

[0110] The results showed that the blank liposomes were translucent in appearance, with a particle size of 241 nm. The blank liposomes had poor stability and flocs were generated in the solution.

[0111] From the above examples 1 to 4, it can be seen that the blank liposomes prepared by the method of the present invention have a particle size of no more than 200 nm, and the appearance of the blank liposomes is translucent, has obvious opalescence, is uniform and stable in texture, has no flocculent formation and is not stratified, and the entire preparation process does not require the addition of organic substances, and the subsequent step of removing organic solvents is eliminated, saving production costs, and using 30000-60000psi ultra-high pressure for 5-8 times of homogenization treatment, with high production efficiency and strong repeatability, and can be used for further encapsulation and delivery of drugs and cosmetic active ingredients. Example 5 is a liposome containing doxorubicin prepared by the method of the present invention, the liposome has a uniform texture, and the particle size changes are not small.

[0112] It can be seen from Comparative Examples 1 to 5 that without multiple ultra-high pressure homogenization treatments, the transparency of the blank liposomes obtained decreases, and the particle size is large: 600nm-900nm. Similarly, when ultra-high pressure homogenization treatment is used, if the number of treatments is too small, the particle size of the blank liposomes obtained does not meet the requirements, and if the number of treatments is too large, the blank liposomes obtained are unstable and flocs are generated in the solution.

[0113] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing blank liposomes using a non-organic solvent, characterized in that: The steps include: S1. Take raw materials according to the ratio of 1%-10% phospholipid, 1%-10% cholesterol, 80%-98% water or ammonium sulfate solution, mix phospholipid, cholesterol with water or ammonium sulfate solution, and disperse them evenly by magnetic stirring to obtain a crude liposome suspension; S2, subjecting the crude liposome suspension obtained in S1 to a high-speed shearing treatment at 10000-20000 rpm; The sheared suspensions in S3 and S2 are subjected to multiple ultrahigh pressure homogenization treatments at low temperature and pressures of 30,000-60,000 psi to obtain blank liposomes; The number of ultra-high pressure homogenization treatments in step S3 is 5-8 times.

2. The method for preparing blank liposomes using a non-organic solvent according to claim 1, characterized in that: The ratio of the raw materials is: 5%-10% phospholipid, 1%-5% cholesterol, and 85%-94% water or ammonium sulfate solution.

3. The method for preparing blank liposomes using a non-organic solvent according to claim 1, characterized in that: The low temperature condition is 0-10°C.

4. The method for preparing blank liposomes using a non-organic solvent according to claim 1, characterized in that: The high-speed shearing time in step S2 is 1-3 minutes.

5. The method for preparing blank liposomes using a non-organic solvent according to claim 1, characterized in that: The particle size of the blank liposome is no greater than 200 nm.

6. The method for preparing blank liposomes using a non-organic solvent according to claim 1 or 2, characterized in that: The phospholipids include one or more of natural lecithin, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, and PEG-modified phospholipids.

7. The method for preparing blank liposomes using a non-organic solvent according to claim 1 or 2, characterized in that: The cholesterol includes one or more of cholesterol, cholesterol PEG maleimide, amino cholesterol PEG, cholesterol sulfate, cholesterol succinate, cholesterol stearate, cholesterol nonanoate, mercapto cholesterol, cholesterol linoleate, and cholesterol arginine ethyl ester.

8. The method for preparing blank liposomes using a non-organic solvent according to claim 1 or 2, characterized in that: The water is triple distilled water.

9. Use of the blank liposomes prepared by the method according to any one of claims 1 to 8 in preparing liposomes loaded with active substances, wherein the active substances in the liposomes loaded with active substances are one or more of active substances in drugs, cosmetics and substances with health care functions.

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