Doxorubicin liposome and application thereof in preparation

Abstract

The invention discloses doxorubicin lipidosome, a preparation method thereof and application of the doxorubicin lipidosome in a preparation, and belongs to the technical field of lipidosome drugs and preparations. The doxorubicin liposome comprises doxorubicin hydrochloride, hydrogenated soybean lecithin, cholesterol, fructo-oligosaccharide and glycine, the preparation method comprises the steps of premixing, blank liposome preparation, ammonium sulfate gradient establishment, multi-stage temperature incubation drug loading and post-treatment, and gradient temperature regulation is adopted in multi-stage incubation. According to the doxorubicin liposome, through the synergistic effect of all the components in the formula and the optimized preparation process, the performance of the liposome is improved, and the doxorubicin liposome can be used for preparing injections and has application value in the field of tumor treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome medicines and preparations, and particularly relates to doxorubicin liposomes and application thereof in preparations. Background Art

[0002] Doxorubicin, an anthracycline antitumor drug, has a broad antitumor spectrum and is effective against hypoxic cells, making it a crucial treatment for cancer. However, doxorubicin exhibits a potent cytotoxic effect and poor selectivity, damaging both normal and tumor cells, limiting its clinical application. Furthermore, the original doxorubicin is unstable and is typically marketed as doxorubicin hydrochloride, which is neurotoxic.

[0003] With the advancement of nanomolecular medicine and molecular biology, liposomes, with their smectic liquid crystal structure, similar to the bilayer of biological membranes, have become a promising microparticle-based targeted drug delivery vehicle. Liposome formulations of doxorubicin can leverage the targeting properties of liposomes to increase drug concentration at tumor sites, improving efficacy and reducing toxicity to normal tissues. Liposomes also increase drug solubility and stability, and their lipid structure is biodegradable in vivo, resulting in a high safety profile.

[0004] To overcome these adverse reactions, researchers are committed to modifying the structure of existing anticancer drugs, adopting new drug delivery systems, or combining them with substances that can interfere with the toxicity production mechanism, in order to achieve targeted drug delivery, improve patients' tolerance to drugs, and expand the clinical application range of existing drugs. Chinese patent publication number CN116549394A discloses a doxorubicin hydrochloride liposome injection and its preparation method. Doxorubicin hydrochloride meglumine salt is prepared using meglumine and doxorubicin hydrochloride, and is encapsulated by a phospholipid membrane to form liposomes. The lipid phase components are hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, the internal aqueous phase component is a meglumine solution, and the external aqueous phase component is a sugar solution.

[0005] Chinese patent publication number CN101897667A discloses a doxorubicin hydrochloride liposome injection and its preparation process. The weight percentages of the components are as follows: doxorubicin hydrochloride 0.05-0.5%, hydrogenated soy lecithin 0.025-3%, cholesterol 0.001-1.5%, PEGylated lipid 0.01-1%, organic acid or ammonium sulfate 0.0025-2.5%, sugar 2.8-20%, buffer 0.1-10%, and the remainder water for injection. The preparation process includes the following steps: 1) lyophilization of the lipid phase; 2) hydration of the lipid phase; 3) liposome granulation; 4) creation of an inner and outer transmembrane gradient; 5) drug loading of the liposomes; and 6) sterilization, packaging, and storage.

[0006] However, existing methods for preparing doxorubicin liposomes still present several challenges. For example, some preparation methods utilize large amounts of organic solvents, resulting in high residual organic solvent content and high toxicity. Furthermore, most existing methods produce doxorubicin liposomes with poor storage stability, and the phospholipids in the liposomes are easily oxidized, leading to high drug leakage rates. This can reduce the drug loading within the liposomes and affect the clinical dosage.

[0007] The main technologies for drug delivery of doxorubicin to liposomes include active transmembrane gradient drug delivery and double emulsion methods. Since doxorubicin is an amphiphilic weakly basic drug, the ammonium sulfate gradient method can take advantage of the fact that NH3 generated by the ionization and hydrolysis of ammonium sulfate can quickly diffuse into the external aqueous phase, while H⁺ can hardly pass through the phospholipid bilayer and remains in the internal aqueous phase, indirectly forming an "inner acid and outer alkaline" pH gradient. After the amphiphilic weakly basic drug doxorubicin enters the liposome's internal aqueous phase, the ionization process consumes a large amount of H⁺, prompting more NH3 to overflow and promote drug loading into the liposome. At the same time, with the rapid overflow of NH3 from the internal aqueous phase, a transmembrane diffusion potential of "inner positive and outer negative" will be formed, which can also drive doxorubicin to cross the membrane and accumulate in the internal aqueous phase.

[0008] Therefore, this patent aims to develop a better doxorubicin liposome preparation and its preparation method to solve the above problems, improve drug efficacy and stability, and better meet clinical needs. Summary of the Invention

[0009] To overcome the deficiencies of the prior art, the present invention provides a doxorubicin liposome and a preparation method thereof, aiming to solve the problems of low encapsulation efficiency, high leakage rate, and poor storage stability of the existing doxorubicin liposome, and to improve the efficacy and safety of the drug.

[0010] Specifically, the doxorubicin liposomes of the present invention comprise the following key components: Doxorubicin hydrochloride: As the active ingredient, it exerts anti-tumor effects; hydrogenated soybean lecithin (HSPC): The main membrane material for the liposome bilayer, with excellent biocompatibility and stability; cholesterol, oligofructose (content ≥ 95.0%), and glycine: Regulate membrane fluidity, enhance liposome structural stability, inhibit drug leakage, and improve liposome storage stability. The formulation includes 40-60 parts doxorubicin hydrochloride, 40-60 parts hydrogenated soybean lecithin, 15-25 parts cholesterol, 5-15 parts oligofructose, and 3-7 parts glycine. The preferred ratio is: 50 parts doxorubicin hydrochloride, 50 parts hydrogenated soybean lecithin, 20 parts cholesterol, 10 parts oligofructose, and 5 parts glycine.

[0011] The present invention also provides a method for preparing the above-mentioned doxorubicin liposomes, comprising the following steps: (1) Premixing: Dissolve oligofructose and glycine in 25%-35% ethanol aqueous solution and stir at 28-32°C for 25-35 minutes to prepare a premix.

[0012] (2) Preparation of blank liposomes: HSPC and cholesterol were dissolved in anhydrous ethanol to form a lipid phase solution, and the premixed solution of step (1) was added and mixed, and then an ammonium sulfate solution at 60-64°C was injected to form colostrum. After homogenization and extrusion, a blank liposome suspension was obtained.

[0013] (3) Establishing a gradient: Transfer the blank liposomes into a dialysis bag and dialyze them in a sodium chloride solution containing sucrose for 4-6 hours to make the concentration ratio of ammonium sulfate to the internal and external phase ≥1400:1.

[0014] (4) Drug loading incubation: Doxorubicin hydrochloride solution (prepared with pH 7.2-7.6 phosphate buffer) was mixed with blank liposome suspension at a volume ratio of 5:1-7:1, and incubated at three temperatures (40-50°C for 25-35 minutes → 25-35°C for 20-30 minutes → 55-65°C for 30-40 minutes) to achieve efficient drug loading.

[0015] (5) Post-processing: Remove free drugs through ultrafiltration purification, sterilize, package and seal to obtain the finished product.

[0016] The doxorubicin liposomes of the present invention can be used to prepare injections.

[0017] Compared with the prior art, the present invention has the following significant advantages: (1) High encapsulation efficiency and low leakage rate: The present invention enhances the drug encapsulation ability of liposomes through the synergistic effect of oligofructose, glycine and cholesterol, and the encapsulation efficiency can reach more than 94%. At the same time, it inhibits drug leakage and improves the storage stability of liposomes. The leakage rate is ≤1.8% after storage at 4°C for 30 days, which is significantly better than the existing technology.

[0018] (2) Excellent stability: Accelerated tests (40°C / 75%RH, 6 months) show that the content retention rate of the liposome injection of the present invention is ≥99%, which is much higher than that of commercially available preparations, ensuring the efficacy of the drug within its shelf life.

[0019] (3) Process optimization: Multi-stage temperature incubation is used to regulate the transmembrane movement of drugs, combined with the ammonium sulfate gradient method to reduce the use of organic solvents and reduce residual toxicity. The process has good reproducibility and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Encapsulation efficiency and leakage rate of doxorubicin liposomes of Examples 1-3 and Comparative Examples 1-10. DETAILED DESCRIPTION

[0021] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited to these embodiments, and the embodiments are only used to illustrate the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0022] Example 1 formula: Doxorubicin hydrochloride 50g Fructooligosaccharides 10g HSPC 50g Cholesterol 20g Glycine 5g Preparation method: (1) Premixing of oligofructose and glycine: Weigh the prescribed amount of oligofructose and glycine, add 30% ethanol aqueous solution as premix solvent (the solvent volume is 10 times the total mass of oligofructose and glycine), place in a 30°C constant temperature water bath, and stir magnetically (300 rpm) for 30 minutes. If there is a small amount of undissolved glycine, the water bath temperature can be appropriately increased to 35°C and the stirring time can be extended until it is completely dissolved.

[0023] (2) Preparation of blank liposomes: Weigh the prescribed amount of HSPC and cholesterol, place them in a 500 mL round-bottom flask, add 8 times the volume of anhydrous ethanol of the total mass of lipids, and stir magnetically (400 rpm) in a constant temperature water bath at 35°C until completely dissolved to form a clear lipid phase solution; slowly add the oligofructose-glycine premix prepared in step (1) to the lipid phase solution, and continue stirring for 20 minutes to fully mix the premix and lipid phase. At this time, the solution should remain clear (if turbidity occurs, the stirring speed can be appropriately increased to 500 rpm and the stirring time can be extended for 10 minutes to ensure uniform mixing). The mixed solution was slowly injected into a 250 mM ammonium sulfate solution (lipid to aqueous phase volume ratio of 1:10) preheated to 62°C through a constant flow pump (flow rate of 1.5 mL / min), while mechanical stirring (1200 rpm) was started and stirring was continued for 18 minutes to form colostrum; the colostrum was processed by a high-pressure homogenizer with a set pressure of 80 MPa, and the homogenization cycle was repeated 4 times. Subsequently, the colostrum was extruded through 0.8 μm, 0.45 μm, and 0.2 μm polycarbonate membranes in sequence, 3 times each, to finally obtain a blank liposome suspension with an average particle size of 140-160 nm and a PDI ≤ 0.20.

[0024] (3) Establishment of ammonium sulfate gradient: transfer the blank liposomes into a dialysis bag and place them in 50 volumes of 0.9% sodium chloride solution (containing 1.0% sucrose to maintain osmotic pressure). Dialyze at 25°C with magnetic stirring (200 rpm) for 5 hours, replacing the external solution every 1.5 hours. Use ion chromatography to detect the concentration of ammonium sulfate in the external phase. When the internal and external phase concentration ratio is ≥1400:1 (internal phase 230-270 mM, external phase ≤0.19 mM), the gradient establishment is confirmed to be complete.

[0025] (4) Doxorubicin hydrochloride loading: Weigh doxorubicin hydrochloride and prepare an 8 mg / mL drug solution with a phosphate buffer solution of pH=7.4. Filter and sterilize through a 0.22 μm filter membrane for later use. Mix the liposome suspension and drug solution at a volume ratio of 6:1 and incubate in three stages. The first stage is a medium temperature stage, in which the mixed solution is placed in a 45°C constant temperature shaking water bath (100 rpm) and incubated for 30 minutes. The second stage is a low temperature stage, in which the temperature is lowered to 30°C and incubated at the same shaking speed for 25 minutes. The third stage is a high temperature stage, in which the temperature is raised to 60°C and incubated at a shaking speed of 100 rpm for 35 minutes.

[0026] (5) Post-treatment of liposomes: Ultrafiltration was performed using an ultrafiltration device (molecular weight cut-off 100 kDa) at 6°C and a pressure of 0.20 MPa. After each ultrafiltration, an equal amount of sucrose-glycine buffer solution was added (4.5-5.5 g of sucrose and 0.4-0.6 g of glycine were dissolved in water for injection, and the pH was adjusted to 6.3-6.7 with sodium dihydrogen phosphate and sodium hydrogen phosphate, and then diluted to 100 mL with water for injection and shaken well). Ultrafiltration was performed 4 times in total until the free drug content was ≤1% (HPLC detection). The liposome suspension was filtered through a 0.22 μm sterile filter membrane and packaged into vials in a Class A clean area. The vials were sterilized, filled with nitrogen for protection, and then stoppered and sealed with aluminum caps.

[0027] Example 2 formula: Doxorubicin hydrochloride 40g Fructooligosaccharides 5g HSPC 40g Cholesterol 15g Glycine 3g Preparation method: (1) Premixing of oligofructose and glycine: Weigh the prescribed amount of oligofructose and glycine, add the premixed solvent 25% ethanol aqueous solution (the solvent volume is 8 times the total mass of oligofructose and glycine), place in a constant temperature water bath at 28°C, and stir magnetically (280 rpm) for 25 minutes. If there is a small amount of undissolved glycine, the water bath temperature can be appropriately increased to 33°C and the stirring time can be extended until it is completely dissolved.

[0028] (2) Preparation of blank liposomes: Weigh the prescribed amount of HSPC and cholesterol, place them in a 500 mL round-bottom flask, add 7 times the volume of anhydrous ethanol to the total mass of the lipids, and stir magnetically (380 rpm) in a constant temperature water bath at 33°C until completely dissolved to form a clear lipid phase solution; slowly add the oligofructose-glycine premix prepared in step (1) to the lipid phase solution, and continue stirring for 18 minutes to fully mix the premix and lipid phase. At this time, the solution should remain clear (if turbidity occurs, the stirring speed can be appropriately increased to 480 rpm and the stirring time can be extended by 8 minutes to ensure uniform mixing). The mixed solution was slowly injected into a 230 mM ammonium sulfate solution (lipid to aqueous phase volume ratio of 1:9) preheated to 60°C via a constant flow pump (flow rate of 1.2 mL / min), while mechanical stirring (1100 rpm) was started and stirred for 16 minutes to form colostrum. The colostrum was processed by a high-pressure homogenizer at a set pressure of 70 MPa and circulated for homogenization three times. The colostrum was then extruded through 0.8 μm, 0.45 μm, and 0.2 μm polycarbonate membranes twice each, to obtain a blank liposome suspension with an average particle size of 140-160 nm and a PDI ≤ 0.20.

[0029] (3) Establishment of ammonium sulfate gradient: transfer the blank liposomes into a dialysis bag and place them in 45 times the volume of 0.8% sodium chloride solution (containing 0.8% sucrose to maintain osmotic pressure). Dialyze the liposomes at 23°C with magnetic stirring (180 rpm) for 4 hours, replacing the external solution every hour. Use ion chromatography to detect the concentration of ammonium sulfate in the external phase. When the internal and external phase concentration ratio is ≥1400:1 (internal phase 230-270 mM, external phase ≤0.19 mM), the gradient establishment is confirmed to be complete.

[0030] (4) Doxorubicin hydrochloride loading: Weigh doxorubicin hydrochloride and prepare a 7 mg / mL drug solution with pH = 7.2 phosphate buffer solution. Filter sterilize through a 0.22 μm filter membrane and set aside. Mix the liposome suspension and drug solution at a volume ratio of 5:1 and incubate in three stages. The first stage is the medium temperature stage, in which the mixed solution is placed in a 40°C constant temperature shaking water bath (90 rpm) and incubated for 25 minutes. The second stage is the low temperature stage, in which the temperature is lowered to 25°C and incubated at the same shaking speed for 20 minutes. The third stage is the high temperature stage, in which the temperature is raised to 55°C and incubated at 90 rpm for 30 minutes.

[0031] (5) Post-treatment of liposomes: Ultrafiltration was performed using an ultrafiltration device (molecular weight cut-off 100 kDa) at 5 °C and a pressure of 0.18 MPa. After each ultrafiltration, an equal amount of sucrose-glycine buffer solution was added (4.5-5.5 g of sucrose and 0.4-0.6 g of glycine were dissolved in water for injection, and the pH was adjusted to 6.3-6.7 with sodium dihydrogen phosphate and sodium hydrogen phosphate, and then diluted to 100 mL with water for injection and shaken well). Ultrafiltration was performed three times in total until the free drug content was ≤1% (HPLC detection). The liposome suspension was filtered through a 0.22 μm sterile filter membrane and packaged into vials in a Class A clean area. The vials were sterilized, filled with nitrogen for protection, and then stoppered and sealed with aluminum caps.

[0032] Example 3 formula: Doxorubicin hydrochloride 60g Fructooligosaccharides 15g HSPC 60g Cholesterol 25g Glycine 7g Preparation method: (1) Premixing of oligofructose and glycine: Weigh the prescribed amount of oligofructose and glycine, add 35% ethanol aqueous solution as premix solvent (the solvent volume is 12 times the total mass of oligofructose and glycine), place in a 32°C constant temperature water bath, and stir magnetically (320 rpm) for 35 minutes. If there is a small amount of undissolved glycine, the water bath temperature can be appropriately increased to 37°C and the stirring time can be extended until it is completely dissolved.

[0033] (2) Preparation of blank liposomes: Weigh the prescribed amount of HSPC and cholesterol, place them in a 500 mL round-bottom flask, add 9 times the volume of anhydrous ethanol to the total mass of the lipids, and stir magnetically (420 rpm) in a constant temperature water bath at 37°C until completely dissolved to form a clear lipid phase solution; slowly add the oligofructose-glycine premix prepared in step (1) to the lipid phase solution, and continue stirring for 22 minutes to fully mix the premix and lipid phase. At this time, the solution should remain clear (if turbidity occurs, the stirring speed can be appropriately increased to 520 rpm and the stirring time can be extended by 12 minutes to ensure uniform mixing). The mixed solution was slowly injected into a 270 mM ammonium sulfate solution (lipid to aqueous phase volume ratio of 1:11) preheated to 64°C via a constant flow pump (flow rate of 1.8 mL / min), while mechanical stirring (1300 rpm) was started and stirred continuously for 20 minutes to form colostrum. The colostrum was processed by a high-pressure homogenizer at a set pressure of 90 MPa and homogenized five times. The colostrum was then extruded through 0.8 μm, 0.45 μm, and 0.2 μm polycarbonate membranes four times each, to obtain a blank liposome suspension with an average particle size of 140-160 nm and a PDI ≤ 0.20.

[0034] (3) Establishment of ammonium sulfate gradient: transfer the blank liposomes into a dialysis bag and place them in 55 volumes of 1.0% sodium chloride solution (containing 1.2% sucrose to maintain osmotic pressure). Dialyze at 27°C with magnetic stirring (220 rpm) for 6 hours, replacing the external solution every 2 hours. Use ion chromatography to detect the concentration of ammonium sulfate in the external phase. When the internal and external phase concentration ratio is ≥1400:1 (internal phase 230-270 mM, external phase ≤0.19 mM), the gradient establishment is confirmed to be complete.

[0035] (4) Doxorubicin hydrochloride loading: Weigh doxorubicin hydrochloride and prepare a 9 mg / mL drug solution with pH = 7.6 phosphate buffer solution. Filter and sterilize through a 0.22 μm filter membrane and set aside. Mix the liposome suspension and drug solution in a volume ratio of 7:1 and incubate in three stages. The first stage is the medium temperature stage, in which the mixed solution is placed in a 50°C constant temperature shaking water bath (110 rpm) and incubated for 35 minutes. The second stage is the low temperature stage, in which the temperature is lowered to 35°C and incubated at the same shaking speed for 30 minutes. The third stage is the high temperature stage, in which the temperature is raised to 65°C and incubated at a speed of 110 rpm for 40 minutes.

[0036] (5) Post-treatment of liposomes: Ultrafiltration was performed using an ultrafiltration device (molecular weight cut-off 100 kDa) at 7°C and a pressure of 0.22 MPa. After each ultrafiltration, an equal amount of sucrose-glycine buffer solution was added (4.5-5.5 g of sucrose and 0.4-0.6 g of glycine were dissolved in water for injection, and the pH was adjusted to 6.3-6.7 with sodium dihydrogen phosphate and sodium hydrogen phosphate, and then diluted to 100 mL with water for injection and shaken well). Ultrafiltration was performed 5 times in total until the free drug content was ≤1% (HPLC detection). The liposome suspension was filtered through a 0.22 μm sterile filter membrane and packaged into vials in a Class A clean area. The vials were sterilized, filled with nitrogen for protection, and then stoppered and sealed with aluminum caps.

[0037] Comparative Example 1 formula: Doxorubicin hydrochloride 50g HSPC 50g Cholesterol 20g Glycine 5g Preparation method: Same as Example 1.

[0038] Comparative Example 2 formula: Doxorubicin hydrochloride 50g Fructooligosaccharides 10g HSPC 50g Cholesterol 20g Preparation method: same as Example 1.

[0039] Comparative Example 3 formula: Doxorubicin hydrochloride 50g Mannooligosaccharide 10g HSPC 20g Cholesterol 20g Glycine 5g Preparation method: same as Example 1.

[0040] Comparative Example 4 formula: Doxorubicin hydrochloride 50g Fructooligosaccharides 10g HSPC 50g Cholesterol 20g Proline 5g Preparation method: same as Example 1.

[0041] Comparative Example 5 formula: Doxorubicin hydrochloride 50g 2g fructooligosaccharides HSPC 50g Cholesterol 30g Glycine 12g Preparation method: same as Example 1.

[0042] Comparative Example 6 Recipe: Same as Example 1.

[0043] Preparation method: In step (4), the incubation step is as follows: the mixed solution is placed in a constant temperature shaking water bath (100 rpm) at 50° C. and incubated for 90 minutes. Other steps are the same as those in Example 1.

[0044] Comparative Example 7 Recipe: Same as Example 1.

[0045] Preparation method: In step (4), the incubation is as follows: placing the mixed solution in a 30°C constant temperature shaking water bath, shaking and incubating at a speed of 100 rpm for 45 minutes; raising the temperature to 50°C, and continuing to shake and incubate at a speed of 100 rpm for 45 minutes. Other steps are the same as those in Example 1.

[0046] Comparative Example 8 Recipe: Same as Example 1.

[0047] Preparation method: In step (4), the incubation step is as follows: the mixed solution is placed in a constant temperature shaking water bath (100 rpm) at 45°C for 45 minutes; the temperature is raised to 60°C, and the shaking incubation is continued at 100 rpm for 45 minutes. Other steps are the same as those in Example 1.

[0048] Comparative Example 9 Recipe: Same as Example 1.

[0049] Preparation method: In step (4), the incubation is as follows: the mixed solution is placed in a constant temperature shaking water bath (100 rpm) at 30°C for 45 minutes; the temperature is adjusted to 40°C and the shaking speed is maintained for 25 minutes; the temperature is adjusted to 60°C and the shaking speed is continued at 100 rpm for 35 minutes. Other steps are the same as those in Example 1.

[0050] Comparative Example 10 Recipe: Same as Example 1.

[0051] Preparation method: The incubation in step (4) is carried out in three stages: the first stage is a medium temperature stage, in which the mixed solution is placed in a 55°C constant temperature shaking water bath (100 rpm) and incubated for 45 minutes. The temperature is then adjusted to 45°C and the incubation is continued at a shaking speed of 100 rpm for 45 minutes. Other steps are the same as those in Example 1.

[0052] Doxorubicin liposome encapsulation efficiency and leakage rate Encapsulation efficiency and leakage rate are key indicators for evaluating the quality of liposome formulations, directly impacting a drug's efficacy, stability, and safety. Encapsulation efficiency refers to the percentage of drug encapsulated by liposomes to the total drug content, reflecting the liposome's drug-carrying capacity. A higher encapsulation efficiency can reduce the toxic side effects of free drug on normal tissues and increase drug concentration at the target site. In this study, encapsulation efficiency was determined using high-performance liquid chromatography (HPLC) by separating free drug from liposome-encapsulated drug and calculating the peak area ratio between the two.

[0053] The leakage rate measures the extent of drug leakage from liposomes during storage or in vivo transport, expressed as the percentage of drug leakage under specific conditions (storage at 4°C for 30 days) to the initial encapsulated drug amount. A low leakage rate is crucial for ensuring the stability of liposome formulations, ensuring that the drug maintains an effective concentration before reaching the site of action, thereby preventing premature drug release that could lead to decreased efficacy or increased adverse reactions. The leakage rate in this invention is also measured using HPLC.

[0054] Table 1 Encapsulation efficiency and leakage rate of doxorubicin liposomes in Examples 1-3 and Comparative Examples 1-10 From the comparison of the data in Table 1, it can be seen that the doxorubicin liposomes and the preparation method thereof provided by the present invention have significant advantages. (1) Synergistic effect of key excipients: Examples 1-3 all contain doxorubicin hydrochloride, hydrogenated soybean phospholipids (HSPC), cholesterol, oligofructose and glycine, and the proportion of each component is within the specified range. The encapsulation efficiency of Examples 1-3 is more than 94%, and the leakage rate is less than 1.8%, which is significantly better than that of Comparative Example 1 lacking oligofructose and Comparative Example 2 lacking glycine. (2) Necessity of multi-stage temperature incubation: Examples 1-3 adopt a three-stage incubation process of "medium temperature (40-50°C) → low temperature (25-35°C) → high temperature (55-65°C)". Through gradient temperature control, the drug transmembrane movement is gradually promoted, the transmembrane gradient is used to drive drug enrichment, and the membrane structure is stabilized, so that the encapsulation efficiency and leakage rate of Examples 1-3 are higher than those of the comparative examples. In the comparative examples, single temperature incubation or stage missing or temperature abnormality processes all lead to performance degradation. The present invention successfully solves the problems of low encapsulation efficiency and high leakage rate in the prior art by optimizing the formula composition (synergy of oligofructose and glycine) and the preparation process (multi-stage temperature incubation).

[0055] Example 4 Doxorubicin liposome injection Prescription composition: Preparation method: Take 800 mL of water for injection, add appropriate amount of sucrose, stir and dissolve, adjust the pH to 6.5±0.2 with sodium dihydrogen phosphate / disodium hydrogen phosphate, add additional sucrose to adjust the osmotic pressure to 280-320 mOsmol / kg, make up to 1000 mL, filter through a 0.22 μm filter membrane, divide into packages, sterilize by autoclaving at 121°C for 15 minutes, and bottle.

[0056] Example 5 Doxorubicin liposome injection Prescription composition: Preparation method: Same as Example 4.

[0057] Example 6 Doxorubicin liposome injection Prescription composition: Preparation method: Same as Example 4.

[0058] Commercially available preparation: Doxorubicin hydrochloride liposome injection (Kailai) Stability verification The content of doxorubicin hydrochloride for injection in the 2020 edition of the Chinese Pharmacopoeia was determined by high performance liquid chromatography (General Chapter 0512). (Accelerated test: 40°C / 75% RH, 6 months) Table 2 Content stability of doxorubicin hydrochloride for injection The stability verification results for doxorubicin liposome injections in Table 2 indicate that the doxorubicin liposome injections prepared according to the present invention (Examples 4, 5, and 6) exhibited excellent stability under accelerated test conditions (40°C / 75% RH, 6 months). After 6 months of accelerated storage, the drug content remained at a high level, with minimal change compared to the initial level. In contrast, the drug content of commercially available formulations exhibited a significant decrease after 6 months under the same accelerated test conditions. This demonstrates that the doxorubicin liposome injections of the present invention can better maintain drug content stability during storage, helping to ensure the drug's efficacy and safety throughout its shelf life, demonstrating significant advantages.

Claims

1. A doxorubicin liposome, characterized in that: The doxorubicin liposomes include: doxorubicin hydrochloride, hydrogenated soybean lecithin, cholesterol, oligofructose and glycine.

2. The doxorubicin liposome according to claim 1, wherein The doxorubicin liposomes comprise: 40-60 parts by weight of doxorubicin hydrochloride, 40-60 parts by weight of hydrogenated soybean lecithin, 15-25 parts by weight of cholesterol, 5-15 parts by weight of oligofructose, and 3-7 parts by weight of glycine.

3. The doxorubicin liposome according to claim 1, wherein The doxorubicin liposomes comprise: 50 parts by weight of doxorubicin hydrochloride, 50 parts by weight of hydrogenated soybean lecithin, 20 parts by weight of cholesterol, 10 parts by weight of oligofructose, and 5 parts by weight of glycine.

4. A method for preparing the doxorubicin liposome according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Premixing: Dissolve oligofructose and glycine in a premixed solvent containing ethanol to prepare a premixed solution; (2) Preparation of blank liposomes: dissolve the lipid material in anhydrous ethanol to form a lipid phase solution, add the premixed solution of step (1) and mix, inject preheated ammonium sulfate solution to form colostrum, and obtain blank liposome suspension by homogenization and extrusion; (3) Gradient establishment: dialyze blank liposomes to establish an ammonium sulfate gradient; (4) Drug loading incubation: The doxorubicin hydrochloride solution was mixed with the blank liposome suspension and incubated at multiple temperatures to achieve drug loading; (5) Post-processing: ultrafiltration purification, sterilization, packaging and sealing of drug-loaded liposomes.

5. The preparation method according to claim 4, wherein The preparation method comprises the following steps: (1) Premixing: Dissolve oligofructose and glycine in 25%-35% ethanol aqueous solution and stir at 28-32°C for 25-35 minutes to prepare a premix solution; (2) Preparation of blank liposomes: HSPC and cholesterol were dissolved in anhydrous ethanol, added to the premix solution and stirred evenly, and then injected with 60-64°C ammonium sulfate solution to form colostrum. Blank liposomes were obtained by homogenization and extrusion. (3) Gradient establishment: dialyze blank liposomes to remove ammonium sulfate from the external phase until the internal-external phase concentration ratio is ≥1400:1; (4) Drug-loaded incubation: The drug solution was mixed with the liposome suspension and incubated at 40-50°C for 25-35 minutes, 25-35°C for 20-30 minutes, and 55-65°C for 30-40 minutes. (5) Post-processing: ultrafiltration, sterile filtration.

6. The preparation method according to claim 4, wherein In step (3), the dialysis conditions are as follows: blank liposomes are transferred into a dialysis bag, placed in 45-55 times the volume of 0.8%-1.0% sodium chloride solution (containing 0.8%-1.2% sucrose), and dialyzed at 23-27°C with magnetic stirring at 180-220 rpm for 4-6 hours, with the external solution replaced every 1-2 hours.

7. The preparation method according to claim 4, wherein In step (4), the multi-stage incubation is as follows: the first stage is incubation in a constant temperature shaking water bath at 40-50°C at 90-110 rpm for 25-35 minutes; the second stage is cooled to 25-35°C and incubated at the same speed for 20-30 minutes; the third stage is heated to 55-65°C and incubated at the same speed for 30-40 minutes.

8. The preparation method according to claim 4, wherein In step (4), the doxorubicin hydrochloride solution is prepared by using a phosphate buffer solution having a pH of 7.2-7.6 to prepare a 7-9 mg / mL drug solution, which is then sterilized by filtration through a 0.22 μm filter membrane; and the volume ratio of the liposome suspension to the drug solution is 5:1-7:

1.

9. Use of the doxorubicin liposomes according to claim 1 in the preparation of anti-tumor preparations.

10. The use according to claim 9, characterized in that The preparation is an injection.

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