A liposome preparation process of high pressure homogenization coupled with spray drying

Abstract

This invention discloses a high-pressure homogenization coupled with spray drying process for liposome preparation, belonging to the field of pharmaceutical preparation technology. The process includes the following steps: dissolving active ingredients, phospholipids, and cholesterol in an organic solvent and hydrating them to form a suspension; homogenizing the suspension under high pressure to obtain a homogenized solution; adding a glassy protective agent, chitosan, and thixotropic silica gel to the homogenized solution and mixing to obtain a composite solution; the glassy protective agent includes asiaticoside and stachyose; and subjecting the composite solution to gradient cooling spray drying to obtain liposomes. This invention, through high-pressure homogenization and the synergistic effect of the glassy protective agent composed of asiaticoside and stachyose, chitosan, and thixotropic silica gel, forms a multi-level synergistic protection during gradient cooling spray drying, significantly improving the rehydration integrity of liposomes, the long-term storage stability of active ingredients, and photostability.

Description

Technical Field

[0001] This invention relates to the field of drug preparation technology, specifically a liposome preparation process using high-pressure homogenization coupled with spray drying. Background Technology

[0002] Liposomes are effective carriers for improving the bioavailability of active ingredients such as curcumin, silymarin, vitamin K2, and glutathione. However, traditional liposomes have poor stability in the liquid state. To address this, industrial processes often employ high-pressure homogenization combined with spray drying to convert them into solid powders, thereby extending shelf life and facilitating transportation and storage.

[0003] However, the existing high-pressure homogenization coupled spray drying process for preparing liposomes still has the following drawbacks that need to be addressed:

[0004] 1. The high temperature, shear force, and gas-liquid interface interaction during spray drying can cause liposome membrane rupture and leakage of contents, resulting in low liposome integrity after rehydration.

[0005] 2. Active ingredients such as vitamin K2 and curcumin are sensitive to light, alkali, and oxidation. Ordinary liposomes cannot effectively resist oxidation and alkalization during the spray drying process.

[0006] 3. In order to improve the stability of liposomes, a large amount of freeze-drying protectant (such as trehalose and sucrose) is often added, which not only leads to a reduction in drug loading, but also makes the product more hygroscopic. Summary of the Invention

[0007] The purpose of this invention is to provide a high-pressure homogenized coupled spray drying process for liposome preparation, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0009] A high-pressure homogenization coupled with spray drying process for liposome preparation, characterized by comprising the following steps:

[0010] The active ingredients, phospholipids, and cholesterol are dissolved in an organic solvent and hydrated to form a suspension.

[0011] The suspension was homogenized under high pressure to obtain a homogenized solution;

[0012] A composite solution is obtained by adding a glassy preservative, chitosan, and thixotropic silica gel to a homogenized liquid and mixing them; the glassy preservative includes asiaticoside and stachyose.

[0013] The composite solution was subjected to gradient cooling spray drying to obtain liposomes.

[0014] Furthermore, the phospholipid is hydrogenated soybean phospholipid, and its mass ratio to cholesterol is (2-4):(0.2-1).

[0015] Furthermore, the organic solvent is anhydrous ethanol.

[0016] Furthermore, the pressure of the high-pressure homogenizer is 80-150 MPa, and the temperature is 50-65℃.

[0017] Furthermore, the mass ratio of hydroxyasiaticoside to stachyose is 1:(0.5-2).

[0018] Furthermore, the mass ratio of the vitreous protective agent, chitosan, thixotropic silica gel, and cholesterol is (1-3):(0.1-1):(0.5-2):(0.2-1).

[0019] Furthermore, the preparation method of the thixotropic silica gel includes the following steps:

[0020] Tetraalkoxysilane, deionized water, lower alcohol and volatile basic compound were mixed in a mass ratio of 1:(0.1-1):(1-3):(3-12), and a pre-dispersed halloysite nanotube suspension was added. The reaction was carried out at 20-40℃. After the reaction was completed, impurities were removed and the mixture was concentrated under reduced pressure to obtain thixotropic silica gel.

[0021] Furthermore, the tetraalkoxysilane is tetraethoxysilane; the lower alcohol is anhydrous ethanol; the volatile alkaline compound is ammonia; and the mass ratio of halloysite nanotubes to tetraalkoxysilane in the halloysite nanotube suspension is 1:(10-30).

[0022] Furthermore, the process parameters for the gradient cooling spray drying are as follows: the inlet air temperature is set to 110-130℃, the intermediate section temperature is controlled at 75-85℃, and the outlet temperature is controlled at 40-50℃; the atomization pressure is set to 0.3-0.6MPa.

[0023] Another object of the present invention is to provide a liposome prepared by the above-described liposome preparation process.

[0024] The high-pressure homogenization coupled with spray drying process for liposome preparation provided by this invention, through high-pressure homogenization treatment, and in conjunction with a glassy protective agent composed of hydroxyascorbic acid and stachyose, chitosan and thixotropic silica gel, forms a multi-level synergistic protection during gradient cooling spray drying, which significantly improves the rehydration integrity of liposomes, the long-term storage stability of active ingredients and light stability, while reducing the hygroscopicity of liposomes and improving their flowability. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] In one embodiment of the present invention, a liposome preparation process using high-pressure homogenization coupled spray drying is provided, which includes the following steps:

[0027] S1. Dissolve 0.1-2g of active ingredients (such as curcumin, silymarin, vitamin K2, glutathione, etc.), 2-4g of phospholipids (such as hydrogenated soybean phospholipids), and 0.2-1g of cholesterol in 20-40mL of organic solvent (such as anhydrous ethanol), stir, and obtain an organic phase; then, in a water bath at 35-45℃, slowly inject the above organic phase into 60-80mL of deionized water, continue stirring for 20-40min, and hydrate to obtain a suspension;

[0028] This step involves dissolving hydrogenated soybean phospholipids, cholesterol, and active ingredients together in an organic solvent, followed by hydration with an aqueous phase. This allows the lipid membrane components to self-assemble into closed bilayer vesicles, while simultaneously encapsulating the lipid-soluble active ingredients within the hydrophobic regions or lumens of the liposome membrane. The high phase transition temperature of hydrogenated soybean phospholipids and their saturated acyl chains provide a structural basis for the mechanochemical enhancement of subsequent high-pressure homogenization. The addition of cholesterol optimizes membrane fluidity, creating conditions for orderly arrangement during the high-pressure homogenization process.

[0029] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 4-8 times under the conditions of 50-65℃ and 80-150MPa to obtain a homogenized liquid.

[0030] S3. Add 1-3g of a glassy protective agent, 0.1-1g of chitosan, and 0.5-2g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing asiaticoside and stachyose in a mass ratio of 1:(0.5-2); the preparation method of thixotropic silica gel includes the following steps: adding tetraalkoxysilane (such as tetraethoxysilane), deionized water, lower alcohol (anhydrous ethanol), and volatile alkaline compound (such as ammonia water with a mass concentration of 25%-28%). Mix them at a mass ratio of 1:(0.1-1):(1-3):(3-12), and simultaneously add a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 3-7 mg / mL (the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.05:1-0.15:1). The reaction is carried out at 20-40℃. After the reaction is completed, impurities are removed, and the mixture is concentrated under reduced pressure at a temperature of 40-60℃ and a vacuum degree of 0.08-0.095 MPa to a solid content of 15%-30% to obtain thixotropic silica gel.

[0031] In this step, the triterpenoid aglycone of asiaticoside binds to the polar head group of the liposome membrane via hydrogen bonds, forming a protective layer on the membrane surface. Stachyose, with its high glass transition temperature, forms a continuous glassy matrix with asiaticoside, encapsulating the liposomes in the later stages of spray drying (after water evaporation) to prevent problems such as fusion, collapse, and leakage. In addition, the antioxidant properties of asiaticoside (triterpenoid structure) can scavenge free radicals generated during the drying process, protecting sensitive active ingredients such as vitamin K2. Stachyose's ultra-low hygroscopicity makes the powdered liposomes less prone to clumping during storage.

[0032] Thixotropic silica gel contains colloidal silica particles and halloysite nanotubes, which form a three-dimensional network in water, exhibiting shear-thinning properties. During spray drying, when the nozzle is subjected to an extremely high shear rate, this three-dimensional network deconstructs, and the viscosity drops sharply, facilitating atomization into fine droplets. After the droplets leave the nozzle, the shear is removed, and the three-dimensional network can quickly recover, maintaining liposomes within the network channels and preventing them from migrating, colliding, and fusing at the gas-liquid interface. In addition, the high aspect ratio and surface silanol groups of halloysite nanotubes can form physical entanglement and hydrogen bonds with colloidal silica particles, enhancing the network's recovery speed and strength, resulting in a better protective effect than using colloidal silica alone.

[0033] S4. The above-mentioned composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes; wherein, the process parameters for gradient cooling spray drying are: inlet air temperature set at 110-130℃, drying time 1-5s, intermediate section temperature controlled at 75-85℃, outlet temperature controlled at 40-50℃, and total drying time controlled at 15-30s; the atomization pressure of the spray dryer is set at 0.3-0.6MPa;

[0034] In this step, the surface of the droplets is first dried rapidly at a high temperature to form a solidified shell to prevent liposome aggregation; then, it is dried at a medium temperature, which, with the synergistic effect of chitosan, avoids thermal damage caused by prolonged exposure to high temperature.

[0035] In this embodiment of the invention, by synergistically combining a glassy protective agent, thermosensitive chitosan, and thixotropic silica gel, and by combining gradient cooling spray drying, effective protection of liposomes throughout the entire cycle of atomization, drying, and storage is achieved. The resulting liposomes have advantages such as high rehydration integrity, high retention rate of active ingredients, low hygroscopicity, and excellent flowability.

[0036] Example 1: This example provides a high-pressure homogenization coupled with spray drying process for preparing liposomes, which includes the following steps:

[0037] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin (CAS No.: 92128-87-5), and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension;

[0038] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0039] S3. Add 2g of glassy protective agent, 0.5g of chitosan, and 1g of thixotropic silica gel to the above homogenized solution and mix to obtain a composite solution; wherein, the glassy protective agent is prepared by mixing asiaticoside (CAS No.: 34540-22-2) and stachyose (CAS No.: 10094-58-3) at a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: adding tetraethoxysilane (CAS No.: 78-10-4), deionized water, anhydrous ethanol, and a mass concentration of... 26% ammonia water was mixed at a mass ratio of 1:0.5:2:8, and a pre-ultrasonically dispersed suspension of halloysite nanotubes (CAS No.: 1332-58-7) with a concentration of 5 mg / mL was added (wherein the mass ratio of halloysite nanotubes to tetraalkoxysilane was 0.1:1). The mixture was reacted at 30 °C for 24 h. After the reaction was completed, impurities were removed by ultrafiltration, followed by washing with deionized water. The mixture was then concentrated under reduced pressure at 50 °C and a vacuum of 0.09 MPa to a solid content of 20% to obtain thixotropic silica gel.

[0040] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes, i.e., liposome powder; wherein, the process parameters of gradient cooling spray drying are: the inlet air temperature is set to 120℃, drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, and the total drying time is controlled at 20s; the atomization pressure of the spray dryer is set to 0.5MPa.

[0041] Example 2: This example provides a high-pressure homogenization coupled with spray drying process for preparing liposomes, which includes the following steps:

[0042] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin (CAS No.: 92128-87-5), and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension;

[0043] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0044] S3. Add 1g of glassy protective agent, 0.5g of chitosan, and 1g of thixotropic silica gel to the above homogenized solution and mix to obtain a composite solution; wherein, the glassy protective agent is prepared by mixing asiaticoside (CAS No.: 34540-22-2) and stachyose (CAS No.: 10094-58-3) at a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: adding tetraethoxysilane (CAS No.: 78-10-4), deionized water, anhydrous ethanol, and a mass concentration of... 26% ammonia water was mixed at a mass ratio of 1:0.5:2:8, and a pre-ultrasonically dispersed suspension of halloysite nanotubes (CAS No.: 1332-58-7) with a concentration of 5 mg / mL was added (wherein the mass ratio of halloysite nanotubes to tetraalkoxysilane was 0.1:1). The mixture was reacted at 30 °C for 24 h. After the reaction was completed, impurities were removed by ultrafiltration, followed by washing with deionized water. The mixture was then concentrated under reduced pressure at 50 °C and a vacuum of 0.09 MPa to a solid content of 20% to obtain thixotropic silica gel.

[0045] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0046] Example 3: This example provides a high-pressure homogenization coupled with spray drying process for liposome preparation, which includes the following steps:

[0047] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin (CAS No.: 92128-87-5), and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension;

[0048] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0049] S3. Add 3g of glassy protective agent, 0.5g of chitosan, and 1g of thixotropic silica gel to the above homogenized solution and mix to obtain a composite solution; wherein, the glassy protective agent is prepared by mixing asiaticoside (CAS No.: 34540-22-2) and stachyose (CAS No.: 10094-58-3) at a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: adding tetraethoxysilane (CAS No.: 78-10-4), deionized water, anhydrous ethanol, and a mass concentration of... 26% ammonia water was mixed at a mass ratio of 1:0.5:2:8, and a pre-ultrasonically dispersed suspension of halloysite nanotubes (CAS No.: 1332-58-7) with a concentration of 5 mg / mL was added (wherein the mass ratio of halloysite nanotubes to tetraalkoxysilane was 0.1:1). The mixture was reacted at 30 °C for 24 h. After the reaction was completed, impurities were removed by ultrafiltration, followed by washing with deionized water. The mixture was then concentrated under reduced pressure at 50 °C and a vacuum of 0.09 MPa to a solid content of 20% to obtain thixotropic silica gel.

[0050] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0051] Example 4: This example provides a high-pressure homogenization coupled with spray drying process for liposome preparation, which includes the following steps:

[0052] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 2g hydrogenated soybean lecithin, and 1g cholesterol in 20mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 35℃ water bath, slowly inject the above organic phase into 80mL deionized water and continue stirring for 20min to hydrate and obtain a suspension.

[0053] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 4 times at 50℃ and 80MPa to obtain a homogenized liquid.

[0054] S3. Add 1g of glassy protective agent, 0.1g of chitosan, and 2g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:0.5; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 25% ammonia water in a mass ratio of 1:0.1:1:3, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 3mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.05:1), reacting at 20℃ for 24h, after the reaction is completed, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 40℃ and a vacuum degree of 0.08MPa to a solid content of 15% to obtain thixotropic silica gel.

[0055] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 110℃, the drying time is 5s, the intermediate temperature is controlled at 85℃, and the final outlet temperature is controlled at 50℃, with a total drying time of 15s. The atomization pressure of the spray dryer is set to 0.3MPa.

[0056] Example 5: This example provides a high-pressure homogenization coupled with spray drying process for preparing liposomes, which includes the following steps:

[0057] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 4g hydrogenated soybean lecithin, and 0.2g cholesterol in 40mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 45℃ water bath, slowly inject the above organic phase into 60mL deionized water and continue stirring for 40min to hydrate and obtain a suspension.

[0058] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 8 times at 65℃ and 150MPa to obtain a homogenized liquid.

[0059] S3. Add 3g of glassy protective agent, 1g of chitosan, and 0.5g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:2; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 28% ammonia water in a mass ratio of 1:1:3:12, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 7mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.15:1), reacting at 40℃ for 24h, after the reaction, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 60℃ and a vacuum degree of 0.095MPa to a solid content of 30% to obtain thixotropic silica gel.

[0060] S4. The above-mentioned composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 130℃, the drying time is 1s, the intermediate temperature is controlled at 75℃, and the final outlet temperature is controlled at 40℃, with a total drying time of 30s. The atomization pressure of the spray dryer is set to 0.6MPa.

[0061] Example 6: This example provides a high-pressure homogenization coupled with spray drying process for preparing liposomes, which includes the following steps:

[0062] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 2.5g hydrogenated soybean lecithin, and 0.4g cholesterol in 25mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 75mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0063] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 5 times at 55℃ and 100MPa to obtain a homogenized liquid.

[0064] S3. Add 1.5g of glassy protective agent, 0.3g of chitosan, and 1.2g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:0.8; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 26% ammonia water in a mass ratio of 1:0.5:1.5:5, and simultaneously adding a pre-ultrasonically dispersed halloysite nanotube suspension with a concentration of 4mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.08:1), reacting at 25℃ for 24h, after the reaction, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 45℃ and a vacuum degree of 0.085MPa to a solid content of 18% to obtain thixotropic silica gel.

[0065] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 115℃, the drying time is 2s, the intermediate temperature is controlled at 82℃, and the final outlet temperature is controlled at 48℃, with a total drying time of 18s. The atomization pressure of the spray dryer is set to 0.4MPa.

[0066] Example 7: This example provides a high-pressure homogenization coupled with spray drying process for liposome preparation, which includes the following steps:

[0067] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3.5g hydrogenated soybean lecithin, and 0.8g cholesterol in 35mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 42℃ water bath, slowly inject the above organic phase into 65mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0068] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 7 times at 62℃ and 130MPa to obtain a homogenized liquid.

[0069] S3. Add 2.5g of glassy protective agent, 0.8g of chitosan, and 1.5g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:1.5; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 28% ammonia water in a mass ratio of 1:0.8:2.5:10, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 6mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.12:1), and reacting at 35℃ for 24h. After the reaction, remove impurities through an ultrafiltration membrane, wash with deionized water, and concentrate under reduced pressure at a temperature of 55℃ and a vacuum degree of 0.09MPa to a solid content of 25% to obtain thixotropic silica gel.

[0070] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 125℃, the drying time is 4s, the intermediate temperature is controlled at 78℃, and the final outlet temperature is controlled at 42℃, with a total drying time of 25s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0071] Comparative Example 1: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that no vitreous protectant is added. Specifically, it includes the following steps:

[0072] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0073] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0074] S3. Add 0.5g of chitosan and 1g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid. The preparation method of the thixotropic silica gel includes the following steps: mix tetraethoxysilane, deionized water, anhydrous ethanol and 26% ammonia water at a mass ratio of 1:0.5:2:8, and simultaneously add a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 5mg / mL (wherein the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.1:1). React at 30℃ for 24h. After the reaction, remove impurities through an ultrafiltration membrane, wash with deionized water, and concentrate under reduced pressure at 50℃ and a vacuum of 0.09MPa to a solid content of 20% to obtain the thixotropic silica gel.

[0075] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0076] Comparative Example 2: This comparative example provides a high-pressure homogenization coupled spray drying process for liposome preparation. The difference between this process and Example 1 is that a larger amount of trehalose is used to replace the glassy protective agent, chitosan, and thixotropic silica gel. Specifically, it includes the following steps:

[0077] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0078] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0079] S3. Add 10g of trehalose to the above homogenized liquid and mix to obtain a composite liquid.

[0080] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0081] Comparative Example 3: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that an equal amount of trehalose is used instead of stachyose. Specifically, it includes the following steps:

[0082] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0083] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0084] S3. Add 2g of glassy protective agent, 0.5g of chitosan, and 1g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and trehalose in a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 26% ammonia water in a mass ratio of 1:0.5:2:8, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 5mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.1:1), reacting at 30℃ for 24h, after the reaction, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 50℃ and a vacuum degree of 0.09MPa to a solid content of 20% to obtain thixotropic silica gel.

[0085] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0086] Comparative Example 4: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that chitosan is not added. Specifically, it includes the following steps:

[0087] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0088] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0089] S3. Add 2g of glassy protective agent and 1g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol and 26% ammonia water in a mass ratio of 1:0.5:2:8, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 5mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.1:1), reacting at 30℃ for 24h, after the reaction is completed, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 50℃ and a vacuum degree of 0.09MPa to a solid content of 20% to obtain thixotropic silica gel.

[0090] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0091] Comparative Example 5: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that no thixotropic silica gel is added. Specifically, it includes the following steps:

[0092] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0093] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0094] S3. Add 2g of glassy protective agent and 0.5g of chitosan to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing asiaticoside and stachyose in a mass ratio of 1:1.2.

[0095] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0096] Comparative Example 6: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that halloysite nanotubes are not added. Specifically, it includes the following steps:

[0097] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0098] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0099] S3. Add 2g of glassy protective agent, 0.5g of chitosan, and 1g of silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:1.2; the preparation method of silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 26% ammonia water in a mass ratio of 1:0.5:2:8, reacting at 30°C for 24h, after the reaction is completed, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 50°C and a vacuum degree of 0.09MPa to a solid content of 20% to obtain thixotropic silica gel.

[0100] S4. The above composite liquid is placed in a spray dryer for gradient cooling spray drying to obtain powdered liposomes. The process parameters for gradient cooling spray drying are as follows: the inlet air temperature is set to 120℃, the drying time is 3s, the intermediate temperature is controlled at 80℃, and the final outlet temperature is controlled at 45℃, with a total drying time of 20s. The atomization pressure of the spray dryer is set to 0.5MPa.

[0101] Comparative Example 7: This comparative example provides a high-pressure homogenization coupled with spray drying process for liposome preparation. The difference between this process and Example 1 is that isothermal spray drying is used instead of gradient cooling spray drying. Specifically, it includes the following steps:

[0102] S1. Dissolve 0.5g curcumin, 0.5g silymarin, 0.05g vitamin K2, 3g hydrogenated soybean lecithin, and 0.5g cholesterol in 30mL anhydrous ethanol and stir for 10min to obtain an organic phase; then, in a 40℃ water bath, slowly inject the above organic phase into 70mL deionized water and continue stirring for 30min to hydrate and obtain a suspension.

[0103] S2. Place the above suspension in a high-pressure homogenizer and perform cyclic high-pressure homogenization 6 times at 60℃ and 120MPa to obtain a homogenized liquid.

[0104] S3. Add 2g of glassy protective agent, 0.5g of chitosan, and 1g of thixotropic silica gel to the above homogenized liquid and mix to obtain a composite liquid; wherein, the glassy protective agent is prepared by mixing hydroxyascorbic acid and stachyose in a mass ratio of 1:1.2; the preparation method of thixotropic silica gel includes the following steps: mixing tetraethoxysilane, deionized water, anhydrous ethanol, and 26% ammonia water in a mass ratio of 1:0.5:2:8, and simultaneously adding a pre-ultrasonic dispersed halloysite nanotube suspension with a concentration of 5mg / mL (wherein, the mass ratio of halloysite nanotubes to tetraalkoxysilane is 0.1:1), reacting at 30℃ for 24h, after the reaction, removing impurities through an ultrafiltration membrane, washing with deionized water, and concentrating under reduced pressure at a temperature of 50℃ and a vacuum degree of 0.09MPa to a solid content of 20% to obtain thixotropic silica gel.

[0105] S4. The above composite liquid is placed in a spray dryer for constant temperature spray drying to obtain powdered liposomes; wherein, the temperature of constant temperature spray drying is controlled at 120℃, the total drying time is controlled at 20s; the atomization pressure of the spray dryer is set to 0.5MPa.

[0106] Performance Testing: I. Liposome Encapsulation Efficiency: Free drug and liposomes were separated using a dextran gel G-50 column. Taking curcumin as an example, the absorbance was measured at 425 nm using ultraviolet spectrophotometry. Methanol was used as a blank, and the concentration of curcumin was calculated based on the standard curve, which is the curcumin content. Encapsulation efficiency (%) = (curcumin content in liposomes / total amount of curcumin added) × 100%.

[0107] Liposome integrity rate after rehydration: Liposome powder was rehydrated to its original volume using deionized water, and the particle size and encapsulation efficiency of the rehydrated liposomes were measured. Integrity rate (%) = (encapsulation efficiency after rehydration / encapsulation efficiency before drying) × 100%.

[0108] II. Vitamin K2 Content and Retention Rate: The vitamin K2 content was determined by HPLC (C18 column, mobile phase methanol:water = 95:5, detection wavelength 270nm). Retention rate (%) = (accelerated / illuminated vitamin K2 content / initial vitamin K2 content) × 100%; where, accelerated / illuminated vitamin K2 content refers to the vitamin K2 content of the liposome powder after accelerated or illuminated treatment, determined by the above method; initial vitamin K2 content refers to the vitamin K2 content in the liposome suspension before spray drying, converted to the theoretical initial content in the liposome powder.

[0109] Accelerated stability: Liposome powder samples were placed in a 40℃ / 75% RH constant temperature and humidity chamber, and the vitamin K2 retention rate was determined after 6 months according to the above method.

[0110] Photostability: Liposome powder samples were placed in a 4500 lx light chamber, and the retention rate of vitamin K2 was determined after 14 days according to the above method.

[0111] III. Hygroscopicity: Liposome powder samples were placed at 25℃ / 75% RH for 24 hours, and the weight gain was calculated.

[0112] IV. Angle of repose: The existing fixed funnel method is used to determine the powder flowability by measuring the angle between the inclined surface of the liposome powder accumulation cone and the horizontal plane.

[0113] The liposomes prepared in Examples 1-3 and Comparative Examples 1-7 were tested according to the above performance testing methods, and the results are shown in Table 1.

[0114] Table 1

[0115]

[0116] As can be seen from the table, the embodiments of the present invention, with the synergistic protection system of glassy protective agent, chitosan, and thixotropic silica gel, combined with gradient cooling spray drying process, can significantly improve the rehydration integrity of liposome powder and the stability of active ingredients with a much lower dosage of protective agent than traditional methods. Specifically, using the glassy protective agent alone or the chitosan and thixotropic silica gel system alone cannot achieve the desired protective effect, while the combined use of the two significantly improves the liposome integrity rate. Furthermore, the introduction of halloysite nanotubes further enhances the recovery speed of the thixotropic gel network, further improving the protective effect. Moreover, the gradient cooling spray drying process matches the temperature-sensitive gelation characteristics of chitosan, reducing thermal damage and effectively protecting the liposomes.

[0117] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A liposome preparation process using high-pressure homogenization coupled with spray drying, characterized in that, Includes the following steps: The active ingredients, phospholipids, and cholesterol are dissolved in an organic solvent and hydrated to form a suspension. The suspension was homogenized under high pressure to obtain a homogenized solution; A composite solution is obtained by adding a glassy preservative, chitosan, and thixotropic silica gel to a homogenized liquid and mixing them; the glassy preservative includes asiaticoside and stachyose. The composite solution was subjected to gradient cooling spray drying to obtain liposomes.

2. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The phospholipid is hydrogenated soybean phospholipid, and its mass ratio to cholesterol is (2-4):(0.2-1).

3. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The organic solvent is anhydrous ethanol.

4. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The pressure of the high-pressure homogenizer is 80-150 MPa, and the temperature is 50-65℃.

5. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The mass ratio of hydroxyascorbic acid to stachyose is 1:(0.5-2).

6. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The mass ratio of the vitreous protective agent, chitosan, thixotropic silica gel, and cholesterol is (1-3):(0.1-1):(0.5-2):(0.2-1).

7. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1 or 6, characterized in that, The preparation method of the thixotropic silica gel includes the following steps: Tetraalkoxysilane, deionized water, lower alcohol and volatile basic compound were mixed in a mass ratio of 1:(0.1-1):(1-3):(3-12), and a pre-dispersed halloysite nanotube suspension was added. The reaction was carried out at 20-40℃. After the reaction was completed, impurities were removed and the mixture was concentrated under reduced pressure to obtain thixotropic silica gel.

8. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 7, characterized in that, The tetraalkoxysilane is tetraethoxysilane; the lower alcohol is anhydrous ethanol; the volatile alkaline compound is ammonia; and the mass ratio of halloysite nanotubes to tetraalkoxysilane in the halloysite nanotube suspension is 1:(10-30).

9. The liposome preparation process using high-pressure homogenization coupled spray drying according to claim 1, characterized in that, The process parameters for the gradient cooling spray drying are as follows: the inlet air temperature is set at 110-130℃, the intermediate section temperature is controlled at 75-85℃, and the outlet temperature is controlled at 40-50℃; the atomization pressure is set at 0.3-0.6MPa.

10. A liposome prepared using the liposome preparation process described in any one of claims 1-9.

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