Amphotericin B liposome and preparation method thereof

Abstract

The invention relates to the field of biological medicine, and particularly discloses amphotericin B lipidosome and a preparation method thereof.The preparation method comprises the following steps that S1, amphotericin B, lipidosome raw materials and an organic solvent are mixed, and a mixed solution is obtained; s2, shearing the mixed solution and a freeze-drying protection solution to form lipid vesicles, then performing pre-freezing treatment, and performing low-pressure freeze-drying to obtain raw material powder; s3, dissolving the raw material powder in the step S2, hydrating, homogenizing, extruding, filtering and freeze-drying to obtain amphotericin B liposome; the invention also discloses the amphotericin B lipidosome prepared by the method. The amphotericin B lipidosome has the characteristics that the drug stability is not influenced in the process of removing the organic solvent, and the amphotericin B lipidosome with higher encapsulation efficiency is obtained.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals, and more specifically, to an amphotericin B liposome and a preparation method thereof. Background Art

[0002] Amphotericin B is a polyene antibiotic that can effectively treat deep fungal infections and is currently one of the first choice treatments for systemic fungal infections. However, polyene antibiotics can easily bind to the sterol components of host cells, leading to cell membrane damage, cell permeability and lysis, so amphotericin B may cause acute hemolysis in treated patients. In addition, due to the low solubility of amphotericin B, it is difficult to reach an effective therapeutic concentration at low doses, and high doses can cause serious adverse reactions, limiting its clinical application.

[0003] Studies have found that encapsulating amphotericin B in liposomes can reduce its systemic toxicity, reduce adverse reactions of the preparation, and improve drug stability. After administration of amphotericin B liposomes, the concentration of amphotericin B in plasma is relatively low, and the binding rate with cholesterol on the renal tubular cell membrane is reduced, thereby significantly reducing renal toxicity and increasing the clinically effective dosage, achieving better therapeutic effects, reducing the side effects of amphotericin B and prolonging patient survival.

[0004] Amphotericin B liposomes are antibiotics in the form of bilayer liposomes containing amphotericin B, the active ingredient of which is the polyene antibiotic amphotericin B. Liposomes are closed layers with a bilayer structure composed of lipid substances such as phospholipids and cholesterol. When preparing amphotericin B liposomes, phospholipid raw materials, sterol raw materials and amphotericin B are usually mixed in an organic solvent, and then the organic solvent is removed and freeze-dried to obtain amphotericin B liposomes. Currently, the main means of removing the organic solvent is spray drying, but the temperature required for spray drying is relatively high, which has a certain impact on the stability of amphotericin B original drug.

[0005] It is necessary to provide a new production method that is more suitable for industrial production, does not affect the stability of the drug, and has a higher encapsulation rate of amphotericin B liposomes. Summary of the invention

[0006] In order to obtain amphotericin B liposomes with a higher encapsulation rate and without affecting the stability of the drug during the removal of the organic solvent, the present application provides an amphotericin B liposome and a preparation method thereof.

[0007] In a first aspect, the present application provides a method for preparing amphotericin B liposomes, using the following technical scheme: A method for preparing amphotericin B liposomes, comprising the following steps: S1, mixing amphotericin B with liposome raw materials and an organic solvent to obtain a mixed solution; S2, shearing the mixed solution and the freeze-drying protection solution to form lipid vesicles, and then pre-freezing and lyophilizing to obtain raw material powder; S3, dissolving, hydrating, homogenizing, extruding, filtering and freeze-drying the raw material powder in step S2 to obtain amphotericin B liposomes.

[0008] By adopting the above technical scheme, in step S2 of the present application, a low-pressure freeze-drying method is used to remove the organic solvent. By utilizing the characteristic that the boiling point of the organic solvent decreases under low pressure, the organic solvent is removed at a lower temperature under low pressure, which can avoid the influence of high temperature on the stability of the raw material drug. In addition, the freeze-drying process is used to remove the organic solvent in the present application to achieve aseptic operation in the preparation process. Compared with the spray drying method, the cytotoxic raw material drug is released from the environment in the form of dust, which has a certain impact on the environment. In the present application, the freeze-drying method is used to remove the organic solvent to achieve aseptic operation in the preparation process. The raw material drug will not float in the air, and the material collection is also simpler, so aseptic operation of the process is achieved, which is more suitable for the industrial production process of sterile injections. Moreover, in the present application, the mixed solution is shear-mixed with the freeze-dried protective solution to form lipid vesicles, and then the subsequent freeze-drying operation is coordinated to form drug vesicles in advance, thereby improving the encapsulation efficiency of the liposomes, and obtaining liposome vesicles with smaller particle size and closer particle size distribution, especially compared with traditional spray drying, with higher encapsulation efficiency and smaller particle size. Finally, the present application obtains amphotericin B liposomes with a particle size of <100nm, PDI <0.3, qualified particle size and uniform particle size, and the finished product is a light yellow freeze-dried powder, and the product is a yellow translucent solution after reconstitution with water for injection.

[0009] Optionally, the pre-freezing processing parameters in step S2 are: First, pre-freeze the mixture at normal pressure or positive pressure of 1.05-1.3 atmospheres at 0±10°C for 1-5 hours, and then at normal pressure and -45±10°C for 1-10 hours; The low-pressure freeze-drying process is carried out in the following five stages in sequence, and the specific parameters are: The first stage is to treat at -45±20℃ and vacuum degree of 1-20Pa for 10-60min; the second stage is to treat at -20±10℃ and vacuum degree of 1-20Pa for 12-48h; the third stage is to treat at 10±5℃ and vacuum degree of 1-20Pa for 2-24h; the fourth stage is to treat at 20±10℃ and vacuum degree of 1-20Pa for 5-24h; the fifth stage is to treat at 40±20℃ and vacuum degree of 1-20Pa for 5-24h.

[0010] By adopting the above technical scheme, in the present application, a two-stage cooling method is first used for pre-freezing treatment. First, the water molecules and part of the organic solvent in the solution are frozen by treating at about 0°C, and then the remaining water and organic solvent are completely frozen into a solid state by treating at about -45°C for a long time. The pre-freezing treatment helps to stabilize the lipid vesicle structure. During the freezing process, the lipid molecules are rearranged into a more stable structure, which helps to maintain the integrity and encapsulation rate of the liposomes in the subsequent freeze-drying process. Then, the subsequent low-pressure freeze-drying operation is performed, and low-pressure conditions are maintained. The temperature is first treated at -45°C and then gradually increased. The temperature is gradually increased through five stages and maintained for a certain period of time. The frozen water and organic solvent are gradually sublimated to form gas for removal, ensuring the gradual and thorough removal of the solvent. More specifically, when the temperature is raised to about -20°C, the dichloromethane in the organic solvent is removed, and when the temperature is raised to about 10°C, the methanol in the organic solvent is removed. The organic solvent is removed more thoroughly without residue. In addition, in the present application, the speed of solvent sublimation can be slowed down by gradually raising the temperature, reducing the pressure change inside the liposome, which helps to maintain the completeness of the liposome and the encapsulation rate of the drug. Finally, in the present application, the organic solvent is removed by shearing after hydration with the solution and coordinating with pre-freezing and freeze-drying operations, so as to achieve the pre-formation of drug vesicles and improve the encapsulation rate of the liposome.

[0011] Optionally, the pre-freezing processing parameters in step S2 are: The pre-freezing processing parameters in step S2 are: First, pre-freeze the mixture at normal pressure and 0°C for 2 h, then at normal pressure and -45°C for 5 h; The low-pressure freeze-drying process is carried out in the following five stages in sequence, and the specific parameters are: In the first stage, the treatment is carried out at -45°C and a vacuum degree of 1Pa for 30 minutes; in the second stage, the treatment is carried out at -10°C and a vacuum degree of 1Pa for 20 hours; in the third stage, the treatment is carried out at 10°C and a vacuum degree of 1Pa for 20 hours; in the fourth stage, the treatment is carried out at 20°C and a vacuum degree of 1Pa for 20 hours; in the fifth stage, the treatment is carried out at 40°C and a vacuum degree of 1Pa for 20 hours.

[0012] By adopting the above technical solution and the above parameters, the encapsulation rate of the amphotericin liposomes finally obtained is higher.

[0013] Optionally, the lyophilization protection solution in step S2 is obtained by mixing a lyophilization protection agent and water in a mass ratio of 1:(2-5), wherein the lyophilization protection agent is selected from one or more of sucrose, glucose, mannitol, trehalose, and maltose.

[0014] By adopting the above technical scheme, the addition of the freeze-drying protection solution in the present application realizes the hydration process of the mixed solution on the one hand, realizes the pre-formation of drug liposome vesicles, and helps to improve the encapsulation rate of the liposomes. On the other hand, the addition of the freeze-drying protection solution helps to protect the pre-formed vesicles from rupture during the subsequent freeze-drying process to remove the solvent, and finally obtains liposomes with a higher encapsulation rate.

[0015] Optionally, in step S2, after the mixed solution and the freeze-dried protective solution are sheared to form lipid vesicles, an aeration treatment is also performed, the gas source for the aeration treatment is air or an inert gas, the aeration flow rate is 1-100 L / min, and the aeration time is 100-150 min.

[0016] By adopting the above technical scheme, in the present application, the mixed solution is mixed with the freeze-dried protective solution and sheared to form lipid vesicles, which are then aerated. The introduction of gas increases the turbulence and shear force in the solution, which helps to disperse the liposomes in the freeze-dried protective solution, promotes the further formation of liposome vesicles and improves their uniformity. The aeration treatment can also reduce the particle size of the liposomes, making them more uniform. The aeration treatment helps to rearrange the phospholipid molecules to form a more stable liposome structure, reduce the rupture of vesicles in the subsequent pre-freeze and freeze-drying process, and finally obtain a liposome product with a higher encapsulation rate.

[0017] Optionally, the amount of lyoprotectant added in step S2 is 12-15 times the mass of amphotericin B.

[0018] Optionally, the organic solvent in step S1 is a mixed solution of dichloromethane and methanol.

[0019] Optionally, the liposome raw material in step S1 includes at least phospholipid compounds and auxiliary materials, and the phospholipid compounds include a mixture of distearoylphosphatidylglycerol and hydrogenated soybean lecithin.

[0020] Optionally, the auxiliary material includes cholesterol.

[0021] Optionally, the auxiliary material also includes an antioxidant.

[0022] Optionally, the antioxidant is DL-α-tocopherol.

[0023] By adopting the above technical solution, the addition of antioxidants to the liposome raw material includes the stability of the liposome raw material and the formed vesicles during the preparation process.

[0024] Optionally, the specific operation of step S1 is: adding distearoylphosphatidylglycerol, amphotericin B and DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stirring until the mixed system is clear, adjusting the pH to 2-2.5, then adding alkali, adding acid again after it becomes turbid, repeatedly dissolving the system three times, then adding hydrogenated soybean lecithin and cholesterol to the system, and continuing to stir to obtain a mixed solution.

[0025] By adopting the above technical scheme, in the present application, the pH is adjusted to a strong acid after the phospholipids and amphotericin B are mixed in an organic solvent, which helps to change the pH value of the system, helps to change the solubility properties of the drug and the phospholipids, and makes it easier to dissolve and disperse in the solvent. Then, after adding a base, the pH value is adjusted again. After multiple dissolution treatments, the dissolution of the drug and the phospholipids can be promoted, and the interaction and arrangement between the phospholipid molecules can be affected, which helps the phospholipid molecules to form a stable double-layer structure, affects the stability and structure of the subsequent liposome formation, and finally forms vesicles in advance in the freeze-drying process and maintains more stability during the freezing process, and the encapsulation rate of the liposomes finally obtained is better.

[0026] Optionally, in step S1, the mass ratio of amphotericin B to distearoylphosphatidylglycerol is 1:(1.5-1.7), the amount of DL-α-tocopherol added is 1-1.8wt% of the amount of amphotericin B added, the amount of hydrogenated soybean lecithin added is 4-5 times the mass of amphotericin B, and the mass ratio of amphotericin B to cholesterol is 1:(0.9-1.1).

[0027] By adopting the above technical solution and selecting the above addition amount, the comprehensive performance of the liposomes is better.

[0028] Optionally, the specific operation of step S3 is as follows: the specific operation of step S3 is as follows: after re-dissolving the raw material powder in step S2 with a sucrose solution, adjusting the pH to between 2.5 and 5.0, hydrating, and then adjusting the pH to 5-6 with sodium succinate and sodium hydroxide, and then high-pressure homogenization and extrusion to a target particle size of 60-120 nm, and then keeping warm at 40-70° C. for 0.5-2 h, filtering and sterilizing, and then freeze-drying to obtain amphotericin B liposomes.

[0029] In a second aspect, the present application provides an amphotericin B liposome, which adopts the following technical solution: An amphotericin B liposome is prepared by the preparation method as described above.

[0030] By adopting the above technical solution, the amphotericin B liposomes prepared by the method provided by the present application have a higher encapsulation rate, a smaller particle size and a lower polymer dispersion coefficient than the liposomes obtained by the traditional preparation process.

[0031] In summary, this application has the following beneficial effects: 1. The present application adopts pre-freezing and freeze-drying process after shear hydration to remove organic solvents, thereby realizing the pre-formation of drug vesicles, improving the encapsulation rate of liposomes, and obtaining liposome vesicles with smaller particle size and more uniform particle size distribution; and can avoid the influence of high-temperature solvent removal on the stability of the raw material drug. Compared with the spray drying method, the aseptic operation of the process can be realized, which is more suitable for the industrial production process of sterile injections; 2. In the present application, the pre-freezing treatment is first performed by cooling in two stages. First, the water molecules and part of the organic solvent in the solution are frozen at about 0°C, and then the remaining water and organic solvent are completely frozen into a solid state at about -45°C for a long time. The pre-freezing treatment helps to stabilize the lipid vesicle structure. During the freezing process, the lipid molecules are rearranged into a more stable structure, which helps to maintain the integrity and encapsulation rate of the liposomes during the subsequent freeze-drying process. Then, the subsequent low-pressure freeze-drying operation is performed, and low-pressure conditions are maintained. First, the temperature is gradually increased after treatment at -45°C. The frozen water and organic solvent are gradually sublimated to form gas for removal through five stages of gradual heating and maintained for a certain period of time, ensuring the gradual and thorough removal of the solvent. The removal of the organic solvent is more thorough and without residue. In addition, the gradual heating in the present application can slow down the speed of solvent sublimation, reduce the pressure change inside the liposome, and help maintain the completeness of the liposome and the encapsulation rate of the drug. 3. In the present application, the mixed solution is mixed with the freeze-dried protective solution and sheared to form lipid vesicles, which are then aerated. The introduction of gas increases the turbulence and shear force in the solution, which helps to disperse the liposomes in the freeze-dried protective solution, promotes the further formation of liposome vesicles and improves their uniformity. The aeration treatment can also reduce the particle size of the liposomes, making them more uniform. The aeration treatment helps to rearrange the phospholipid molecules to form a more stable liposome structure, reduce the rupture of vesicles during the subsequent pre-freeze-drying process, and finally obtain a liposome product with a higher encapsulation rate. DETAILED DESCRIPTION

[0032] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0033] Example 1 A method for preparing amphotericin B liposomes comprises the following steps: S1. Add 1.68 g DSPG (distearoylphosphatidylglycerol), 1.0 g amphotericin B and 12.8 mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stir until the mixed system is clear, add hydrochloric acid to adjust the pH to 2, then add sodium hydroxide, add hydrochloric acid again after it becomes turbid, and repeatedly dissolve the system three times, then add 4.26 g HSPC (hydrogenated soybean phosphatidylcholine) and 1.04 g cholesterol to the system, and continue stirring to obtain an orange-yellow drug-phospholipid mixed solution; Wherein dichloromethane and methanol are mixed in a mass ratio of 1:1. S2, high-speed shearing (speed of 2000r / min) of the mixed solution and the freeze-drying protection solution for 10min to form lipid vesicles, and then aeration treatment for 2h, pre-freezing treatment, and then low-pressure freeze-drying to obtain raw material powder; The lyophilized protective solution is obtained by mixing a lyophilized protective agent and water in a mass ratio of 1:3, wherein the lyophilized protective agent is sucrose, and the amount of the lyophilized protective agent added is 13 times the mass of amphotericin B; The gas source for aeration treatment is nitrogen, the aeration flow rate is 50L / min, the aeration time is 120min, and the pre-freezing treatment parameters are: first, treat at normal pressure and 0°C for 2h, then treat at normal pressure and -45°C for 5h for pre-freezing treatment; the low-pressure freeze-drying treatment is operated in sequence through the following five stages, and the specific parameters are: the first stage is first treated at -45°C and a vacuum degree of 1Pa for 30min; the second stage is treated at -10°C and a vacuum degree of 1Pa for 20h; the third stage is treated at 10°C and a vacuum degree of 1Pa for 20h; the fourth stage is treated at 20°C and a vacuum degree of 1Pa for 20h to complete the low-pressure freeze-drying treatment to obtain the raw material powder; the fifth stage is treated at 40°C and a vacuum degree of 1Pa for 20h; S3. The raw powder in step S2 is re-dissolved in a sucrose solution with a mass concentration of 20%, and the amount of sucrose solution added is 3 times the mass of the raw powder. The pH is adjusted to between 3.5, hydrated for 1 hour, and then the pH is adjusted to 5.5 by sodium succinate and sodium hydroxide. Then, after homogenization, it is extruded to a target particle size of 60-120 nm, and then kept warm at 65° C. for 1.5 hours, filtered for sterilization, and then freeze-dried to obtain amphotericin B liposomes.

[0034] Example 2 A method for preparing amphotericin B liposomes comprises the following steps: S1. Add 1.5 g DSPG (distearoylphosphatidylglycerol), 1.0 g amphotericin B and 18 mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stir until the mixed system is clear, add hydrochloric acid to adjust the pH to 2.5, then add sodium hydroxide, add hydrochloric acid again after it becomes turbid, and repeatedly dissolve the system three times, then add 5 g HSPC (hydrogenated soybean phosphatidylcholine) and 1.1 g cholesterol to the system, and continue stirring to obtain an orange-yellow drug-phospholipid mixed solution; Wherein dichloromethane and methanol are mixed in a mass ratio of 1:1. S2, high-speed shearing (speed of 2000 r / min) of the mixed solution and the freeze-drying protection solution for 10 min to form lipid vesicles, and then aeration treatment for 100 min, pre-freezing treatment, and then low-pressure freeze-drying to obtain raw material powder; The lyophilized protective solution is obtained by mixing a lyophilized protective agent and water in a mass ratio of 1:5, wherein the lyophilized protective agent is sucrose, and the amount of the lyophilized protective agent added is 13 times the mass of amphotericin B; The gas source for aeration treatment is nitrogen, the aeration flow rate is 100L / min, the aeration time is 100min, and the pre-freezing treatment parameters are: first, treat at a positive pressure of 1.05 atmospheres and -10°C for 1h, and then treat at normal pressure and -55°C for 1h for pre-freezing treatment; the low-pressure freeze-drying treatment is sequentially operated through the following five stages, and the specific parameters are: the first stage is first treated at -65°C and a vacuum degree of 12Pa for 10min; the second stage is treated at -30°C and a vacuum degree of 12Pa for 12h; the third stage is treated at 5°C and a vacuum degree of 12Pa for 2h; the fourth stage is treated at 10°C and a vacuum degree of 12Pa for 24h; the fifth stage is treated at 20°C and a vacuum degree of 12Pa for 24h to complete the low-pressure freeze-drying treatment to obtain the raw material powder; S3. The raw material powder in step S2 is re-dissolved with a sucrose solution with a mass concentration of 20%, and the amount of sucrose solution added is 3 times the mass of the raw material powder. The pH is adjusted to between 2.5, hydrated for 1 hour, and then the pH is adjusted to 5 by sodium succinate and sodium hydroxide. Then, after homogenization, it is extruded to a target particle size of 60-120 nm, and then kept warm at 40° C. for 2 hours, filtered for sterilization, and then freeze-dried to obtain amphotericin B liposomes.

[0035] Example 3 A method for preparing amphotericin B liposomes comprises the following steps: S1. Add 1.7 g DSPG (distearoylphosphatidylglycerol), 1.0 g amphotericin B and 12.8 mg DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stir until the mixed system is clear, add hydrochloric acid to adjust the pH to 2-2.5, then add sodium hydroxide, add hydrochloric acid again after it becomes turbid, and repeatedly dissolve the system three times, then add 4.26 g HSPC (hydrogenated soybean phosphatidylcholine) and 1.04 g cholesterol to the system, and continue stirring to obtain an orange-yellow drug-phospholipid mixed solution; Wherein dichloromethane and methanol are mixed in a mass ratio of 1:1. S2, high-speed shearing (speed of 2000r / min) of the mixed solution and the freeze-drying protection solution for 10 minutes to form lipid vesicles, and then aeration treatment for 150 minutes, pre-freezing treatment, and then low-pressure freeze-drying to obtain raw material powder; The lyophilized protective solution is obtained by mixing a lyophilized protective agent and water in a mass ratio of 1:3, wherein the lyophilized protective agent is sucrose, and the amount of the lyophilized protective agent added is 13 times the mass of amphotericin B; The gas source for aeration treatment is nitrogen, the aeration flow rate is 1L / min, the aeration time is 150min, and the pre-freezing treatment parameters are: first, treat at a positive pressure of 1.3 atmospheres and 10°C for 5h, and then treat at normal pressure and -35°C for 10h for pre-freezing treatment; the low-pressure freeze-drying treatment is operated in sequence through the following five stages, and the specific parameters are: the first stage is first treated at -25°C and a vacuum degree of 20Pa for 60min; the second stage is treated at -10°C and a vacuum degree of 20Pa for 48h; the third stage is treated at 15°C and a vacuum degree of 20Pa for 24h; the fourth stage is treated at 30°C and a vacuum degree of 20Pa for 5h; the fifth stage is treated at 60°C and a vacuum degree of 20Pa for 5h to complete the low-pressure freeze-drying treatment to obtain the raw material powder; S3. The raw powder in step S2 is re-dissolved in a sucrose solution with a mass concentration of 20%, and the amount of sucrose solution added is 4 times the mass of the raw powder. The pH is adjusted to between 4.5, hydrated for 1 hour, and then the pH is adjusted to 6 by sodium succinate and sodium hydroxide. Then, the mixture is homogenized and extruded to a target particle size of 60-120 nm. After being kept warm at 70° C. for 0.5 hour, the mixture is filtered and sterilized, and then freeze-dried to obtain amphotericin B liposomes.

[0036] Example 4 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, and then hydrochloric acid is added to acidify and adjust the pH to 2, and then DSPG (distearoylphosphatidylglycerol) is added, stirred until the lipid is completely dissolved, and then sodium hydroxide is added. After turbidity, hydrochloric acid is added, and the remaining operations are the same as in Example 1.

[0037] Example 5 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, DSPG (distearoylphosphatidylglycerol) and amphotericin B are added to a mixed organic solvent of dichloromethane and methanol, and then hydrochloric acid is added to acidify and adjust the pH to 2, and then DL-α-tocopherol is added, and then sodium hydroxide is added. After turbidity, hydrochloric acid is added again, and the solution is repeatedly dissolved three times. The remaining operations are the same as in Example 1. Example 6 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that DSPG (distearylphosphatidylglycerol), HSPC (hydrogenated soybean phosphatidylcholine), amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, hydrochloric acid is added for acidification, sodium hydroxide is added for repeated dissolution, and cholesterol is added, and the remaining operations are the same as in Example 1.

[0038] Example 7 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that aeration treatment is not performed in step S2.

[0039] Example 8 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S1, DSPG (distearoylphosphatidylglycerol), amphotericin B and DL-α-tocopherol are added to a mixed organic solvent of dichloromethane and methanol, stirred until the mixed system is clarified, hydrochloric acid is added to adjust the pH to 2, and then HSPC and cholesterol are directly added without adding sodium hydroxide and repeated dissolving and clarifying operations.

[0040] Example 9 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that DL-α-tocopherol is not added to the raw materials.

[0041] Example 10 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that, after the aeration treatment in step S2, the amphotericin B liposomes are firstly treated at -45°C for 7 hours for pre-freezing treatment, and then subjected to low-pressure lyophilization treatment.

[0042] Embodiment 11 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the specific parameters of the lyophilization treatment in step S2 are: In the first stage, the mixture is treated at -45°C and a vacuum degree of 1Pa for 30 minutes; in the second stage, the mixture is treated at 0°C and a vacuum degree of 1Pa for 30 hours; in the third stage, the mixture is treated at 10°C and a vacuum degree of 1Pa for 30 hours; in the fourth stage, the mixture is treated at 20°C and a vacuum degree of 1Pa for 20 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0043] Example 12 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the specific parameters of the lyophilization treatment in step S2 are: In the first stage, the mixture was treated at -45°C and a vacuum degree of 1Pa for 30 minutes; in the second stage, the mixture was treated at 5°C and a vacuum degree of 1Pa for 40 hours; in the third stage, the mixture was treated at 15°C and a vacuum degree of 1Pa for 40 hours to complete the low-pressure freeze-drying process and obtain the raw material powder.

[0044] Comparative Example 1 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S2, a spray drying method is used after aeration treatment to obtain a raw material powder.

[0045] Comparative Example 2 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that after the aeration treatment in step S2, a rotary evaporation treatment is carried out at 65° C. for 3 hours to obtain a raw material powder.

[0046] Comparative Example 3 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that in step S2, no pre-freezing treatment is performed after the aeration treatment, and low-pressure lyophilization treatment is directly performed.

[0047] Comparative Example 4 A method for preparing amphotericin B liposomes is carried out according to the method in Example 1, except that the pre-freezing treatment and the freeze-drying treatment in step S2 are both carried out under normal pressure.

[0048] Performance Testing The encapsulation efficiency and polymer dispersion index (PDI) of the amphotericin B liposomes prepared in the examples and comparative examples of the present application were tested, and the test results are shown in Table 1 below.

[0049] Table 1: In addition, the organic solvent residue of the amphotericin B liposomes in the examples of the present application was detected, and the content was less than 0.0001%. The freeze-drying method for removing organic solvents in the present application has a good effect on removing organic solvents. In addition, the particle size of the amphotericin B liposomes in Example 1 of the present application was measured, which was less than 100 nm. Referring to the test results in Table 1 above, the PDI of the amphotericin B liposomes prepared in the examples of the present application was less than 0.3, and the liposome product had a qualified particle size and a uniform particle size, and had a higher encapsulation rate. Combining the test results of Example 1 and Example 4, in Example 1, amphotericin B, DL-α-tocopherol and DSPG are added simultaneously, while in Example 4, amphotericin B and DL-α-tocopherol are added and then acidified before adding phospholipids. It can be seen that the acidification of the mixed system before adding phospholipids helps the dissolution and dispersion of phospholipids, and the liposomes finally obtained have a better encapsulation rate and a lower polymer dispersion coefficient; combined with the test results in Example 5, the effect of adding phospholipids and amphotericin B first, then adding hydrochloric acid and then adding DL-α-tocopherol is reduced; and in Example 6, the phospholipid raw material, amphotericin B and DL-α-tocopherol are added together and then hydrochloric acid is added for acidification, and the encapsulation rate is reduced. It can be seen that the order of the phospholipid raw material, DL-α-tocopherol and repeated dissolution by acidification affects the formation and distribution of the phospholipid structure, thereby affecting the encapsulation rate of the final liposome product.

[0050] Referring to the test results of Example 1 and Example 7, when aeration treatment is not performed in step S2, the encapsulation rate of the liposome product is reduced, while the dispersion coefficient is increased. Combined with the test results of Example 8, when only hydrochloric acid is added for acidification without adding sodium hydroxide and repeated dissolution, the encapsulation rate of the obtained liposome product is reduced and the dispersion coefficient is also increased. The repeated dissolution operation is conducive to the full combination of the drug and the phospholipid. Referring to the test results of Example 9, DL-α-tocopherol (vitamin E) is not added, and its encapsulation rate is reduced. As an antioxidant, it can reduce the occurrence of lipid peroxidation, especially the influence of subsequent aeration treatment, which helps to maintain the integrity and stability of the liposome membrane and improve the encapsulation rate. Combined with the test results of Examples 10-12, it can be seen that when the two-stage pre-freezing treatment and the five-step low-pressure freeze-drying treatment in the present application are used, the obtained liposomes have a higher encapsulation rate and a lower dispersion coefficient, and the particle size is more uniform.

[0051] Referring to the test results of Example 1 and Comparative Examples 1 and 2, the present application is compared with the method of removing organic solvents by conventional spray drying or rotary evaporation, and the encapsulation rate of the liposomes finally obtained is higher. Combined with the test results of Comparative Example 3, when the low-pressure freeze-drying process is directly carried out without pre-freezing in Comparative Example 3, its encapsulation rate is significantly reduced, and its dispersion coefficient is also improved, and the residual amount of organic solvent is also increased by 8%, which is significantly reduced, and when the freeze-drying is carried out at normal pressure in Comparative Example 4, its residual amount of organic solvent is also increased by 5.6%, and the removal effect of organic solvent is significantly reduced, and its encapsulation rate is also reduced. In addition, the technical solution of removing organic solvents by freeze-drying in the present application solves the problem that the removal effect of organic solvents is reduced due to the azeotropic problem compared to conventional spray drying or rotary evaporation.

[0052] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing amphotericin B liposomes, characterized in that: The following steps are involved: S1, mixing amphotericin B with liposome raw materials and an organic solvent to obtain a mixed solution; S2, shearing the mixed solution and the freeze-drying protection solution to form lipid vesicles, and then pre-freezing and lyophilizing to obtain raw material powder; S3, dissolving, hydrating, homogenizing, extruding, filtering and freeze-drying the raw material powder in step S2 to obtain amphotericin B liposomes.

2. The method for preparing amphotericin B liposome according to claim 1, characterized in that: The pre-freezing processing parameters in step S2 are: First, pre-freeze the mixture at normal pressure or positive pressure of 1.05-1.3 atmospheres at 0±10°C for 1-5 hours, and then at normal pressure and -45±10°C for 1-10 hours; The low-pressure freeze-drying process is carried out in the following five stages in sequence, and the specific parameters are: The first stage is to treat at -45±20℃ and vacuum degree of 1-20Pa for 10-60min; the second stage is to treat at -20±10℃ and vacuum degree of 1-20Pa for 12-48h; the third stage is to treat at 10±5℃ and vacuum degree of 1-20Pa for 2-24h; the fourth stage is to treat at 20±10℃ and vacuum degree of 1-20Pa for 5-24h; the fifth stage is to treat at 40±20℃ and vacuum degree of 1-20Pa for 5-24h.

3. The method for preparing amphotericin B liposome according to claim 1, characterized in that: The pre-freezing processing parameters in step S2 are: First, pre-freeze the mixture at normal pressure and 0°C for 2 h, then at normal pressure and -45°C for 5 h; The low-pressure freeze-drying process is carried out in the following five stages in sequence, and the specific parameters are: The first stage is to treat at -45℃ and vacuum degree of 1Pa for 30min; the second stage is to treat at -10℃ and vacuum degree of 1Pa for 20h; the third stage is to treat at 10℃ and vacuum degree of 1Pa for 20h; the fourth stage is to treat at 20℃ and vacuum degree of 1Pa for 20h; The fifth stage is treated at 40°C and a vacuum degree of 1 Pa for 20 hours.

4. The method for preparing amphotericin B liposome according to claim 1, characterized in that: The lyophilization protection solution in step S2 is obtained by mixing a lyophilization protection agent and water in a mass ratio of 1: (2-5), wherein the lyophilization protection agent is selected from one or more of sucrose, glucose, mannitol, trehalose, and maltose, and the amount of the lyophilization protection agent added is 12-15 times the mass of amphotericin B.

5. The method for preparing amphotericin B liposome according to claim 1, characterized in that: In step S2, after the mixed solution and the freeze-dried protective solution are sheared to form lipid vesicles, an aeration treatment is performed. The gas source for the aeration treatment is air or an inert gas, the aeration flow rate is 1-100 L / min, and the aeration time is 100-150 min.

6. The method for preparing amphotericin B liposome according to claim 1, characterized in that: In step S1, the organic solvent is a mixed solution of dichloromethane and methanol; The liposome raw material in step S1 includes at least phospholipid compounds and auxiliary materials, and the phospholipid compounds include a mixture of distearoylphosphatidylglycerol and hydrogenated soybean lecithin; The auxiliary materials include cholesterol and DL-α-tocopherol.

7. The method for preparing amphotericin B liposome according to claim 6, characterized in that: The specific operation of step S1 is: adding distearoylphosphatidylglycerol, amphotericin B and DL-α-tocopherol to a mixed organic solvent of dichloromethane and methanol, stirring until the mixed system is clear, adjusting the pH to 2-2.5, then adding alkali, adding acid again after turbidity, repeatedly dissolving the system three times, then adding hydrogenated soybean lecithin and cholesterol to the system, and continuing to stir to obtain a mixed solution.

8. The method for preparing amphotericin B liposome according to claim 7, characterized in that: In step S1, the mass ratio of amphotericin B to distearoylphosphatidylglycerol is 1:(1.5-1.7), the amount of DL-α-tocopherol added is 1-1.8wt% of the amount of amphotericin B added, the amount of hydrogenated soybean lecithin added is 4-5 times the mass of amphotericin B, and the mass ratio of amphotericin B to cholesterol is 1:(0.9-1.1).

9. The method for preparing amphotericin B liposome according to claim 1, characterized in that: The specific operation of step S3 is: after the raw material powder in step S2 is re-dissolved with sucrose solution, the pH is adjusted to between 2.5 and 5.0, hydrated, and then the pH is adjusted to 5-6 with sodium succinate and sodium hydroxide, and then high-pressure homogenization is performed and then extruded to the target particle size, and then the mixture is kept at 40-70° C. for 0.5-2 h, filtered and sterilized, and then freeze-dried to obtain amphotericin B liposomes.

10. Amphotericin B liposomes prepared by the method according to any one of claims 1 to 9.

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