Compound liposome as well as preparation method and application thereof

Abstract

The invention discloses a compound liposome as well as a preparation method and application thereof. The compound lipidosome comprises a lipidosome membrane and an inner water phase encapsulated in the lipidosome membrane, the paclitaxel palmitate is encapsulated in a lipid bilayer of the liposome membrane; the adriamycin is encapsulated in the inner water phase; the raw materials of the paclitaxel palmitate sustained-release tablet comprise the following components: 0.1-1% of paclitaxel 0.05 to 0.5 percent of adriamycin; 1-10% of a phospholipid; and 0.05% to 1% of DSPE-PEG2000 (Distearoyl 5-40% of an internal water phase freeze-drying protective agent; 0.5-5% of a salt substance; and water for injection; the percentage represents the percentage of the mass of each component in the total volume of all the raw materials. The compound liposome provided by the invention is high in encapsulation efficiency and uniform in particle size, and has no obvious difference before and after freeze-drying and redissolving; during practical application, the drug release proportion can be effectively controlled, the curative effect is improved, the toxicity is reduced, and the acting time of active ingredients in vivo is prolonged.

Description

Technical Field

[0001] This invention relates to a compound liposome, its preparation method and application, specifically to a paclitaxel palmitate and doxorubicin compound liposome, its preparation method and application. Background Technology

[0002] Cancer seriously threatens human health and is one of the diseases that people cannot effectively control. Chemotherapy is a common treatment for cancer; however, due to drug resistance and severe side effects, the efficacy of single-drug chemotherapy is limited. Therefore, in clinical practice, combination chemotherapy with two or more drugs is commonly used. Combination chemotherapy can act on tumor cells through multiple pathways and routes, while reducing the dosage of each drug and mitigating the severe side effects caused by high doses of individual drugs. Combination chemotherapy has become the mainstream approach to cancer treatment.

[0003] Paclitaxel (PTX) is a common anticancer drug that acts on tubulin to inhibit the mitosis of tumor cells, thereby effectively preventing the proliferation of cancer cells. However, paclitaxel has poor drug-like properties. Clinically used paclitaxel injections use a large amount of polyoxyethylene castor oil as a solubilizer, which has caused severe allergic reactions during use. It also has drawbacks such as easy development of drug resistance and short half-life. Doxorubicin (DOX) is an anthracycline antibiotic antitumor drug that mainly exerts its antitumor effect by inhibiting DNA synthesis and RNA transcription. Clinically, doxorubicin injections are prone to causing severe cardiotoxicity, bone marrow suppression, and other toxicities.

[0004] In clinical practice, the combination of paclitaxel and doxorubicin injections in chemotherapy remains an important treatment option for various cancers, especially breast cancer. However, traditional combination chemotherapy has the following problems: ① The different half-lives of the drugs after systemic administration lead to inconsistent tumor accumulation, which means that the two drugs cannot exert their optimal synergistic effect, because different proportions of the drugs combined may be synergistic, additive, or even antagonistic; ② The toxic side effects of the two drugs are not truly improved, and serious adverse reactions still occur after medication, resulting in poor patient compliance. These problems, to some extent, limit the clinical application of the combined use of paclitaxel and doxorubicin.

[0005] Given the shortcomings of traditional paclitaxel and doxorubicin combination chemotherapy, scholars both domestically and internationally have conducted extensive research on nano-formulations co-loaded with paclitaxel and doxorubicin, such as nanoparticles, polymer micelles, and nanohydrogels. However, the following problems are generally present: ① The provided co-loaded nano-formulations cannot control the optimal synergistic ratio of the two drugs; ② The preparation process is complex, the conditions are harsh, and the conversion to mass production is difficult, making it unsuitable for large-scale industrial production; ③ The formulation process has not been optimized, the drug encapsulation rate is low, and product quality is difficult to control.

[0006] Liposomes are a common nanodelivery carrier. Due to their advantages such as targeting, sustained release, and good biocompatibility, they are now widely used in fields such as cancer, fungal treatment, and viral infection.

[0007] Literature search revealed reports both domestically and internationally on the co-loading of paclitaxel and doxorubicin liposomes, such as patents CN107753434A and CN103622912A, and literature (Roque MC, Franco MS, Viela JMC, et al. Development of Long-Circulating and Fusogenic Liposomes Co-encapsulating Paclitaxel and Doxorubicin in Synergistic Ratio for the Treatment of Breast Cancer[J]. Curr Drug Del, 2019, 16(9): 829-838.). However, the following problems are commonly encountered: ① During the preparation process, the membrane material needs to be dissolved in one or more organic solvents and the solvent removed under reduced pressure using a rotary evaporator to form a lipid film. This characteristic is difficult to achieve in process scale-up, resulting in poor industrial operability, and the residue of organic solvents may also pose a risk to human health; ② Due to the poor lipid solubility of paclitaxel, a large amount of lipids is usually required to encapsulate it within the lipid membrane, leading to low encapsulation efficiency and drug loading, which not only affects its drug-like properties but also its in vivo efficacy; ③ Paclitaxel is released rapidly on the liposome membrane and cannot achieve synchronous release with doxorubicin in the aqueous phase to achieve the best synergistic antitumor effect. Even though existing technologies have reported ways to improve the stability, tumor-site responsiveness, and targeting of compound liposomes by modifying liposomes, cross-linking multilayer liposomes, or linking paclitaxel and doxorubicin through chemical bonds to form a new compound before encapsulating it in liposomes, there are still drawbacks such as low encapsulation efficiency, difficulty in controlling the drug ratio, easy leakage, and complex processes.

[0008] Therefore, it is of great significance to develop a paclitaxel and doxorubicin combination preparation with simple process, high encapsulation rate, stable quality and controllable drug release ratio, which can lay a solid foundation for the effectiveness, safety and convenience of combined use of paclitaxel and doxorubicin. Summary of the Invention

[0009] To address the shortcomings of the existing technology, this invention provides a compound liposome, its preparation method, and its application. This compound liposome simultaneously loads two active ingredients, paclitaxel palmitate and doxorubicin, exhibiting high encapsulation efficiency, uniform particle size, and no significant difference before and after lyophilization and reconstitution. Furthermore, in practical applications, it can effectively control the drug release ratio, increase efficacy, reduce toxicity, and prolong the duration of action of the active ingredients in vivo.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] This invention provides a compound liposome comprising a liposome membrane and an inner aqueous phase encapsulated within the liposome membrane; paclitaxel palmitate (PTX-PA) is encapsulated within a lipid bilayer of the liposome membrane; doxorubicin is encapsulated in the inner aqueous phase; the raw materials of the compound liposome comprise the following components:

[0012] 0.1-1% paclitaxel palmitate; 0.05-0.5% doxorubicin; 1-10% phospholipids; 0.05-1% DSPE-PEG2000; 5-40% internal aqueous phase lyophilization protectant; 0.5-5% salts; and water for injection;

[0013] All percentages above represent the percentage of the mass (g) of each component to the total volume (mL) of all raw materials.

[0014] In this invention, the total volume of raw materials refers to the total volume after all raw materials have been prepared to the final volume.

[0015] In this invention, the paclitaxel palmitate is obtained by esterification of palmitic acid with the 2'-hydroxyl group of paclitaxel, and is a paclitaxel prodrug, the structural formula of which is shown in Formula I.

[0016]

[0017] In some implementations, the paclitaxel palmitate is commercially available and meets pharmaceutical grade requirements.

[0018] In some embodiments, the amount of paclitaxel palmitate is 0.2-0.7%, for example 0.25%, 0.3%, 0.4%, 0.45%, 0.5%, 0.64%, or 0.65%.

[0019] In some implementation schemes, the doxorubicins mentioned are all commercially available and meet pharmaceutical grade requirements.

[0020] In some embodiments, the dosage of doxorubicin is 0.1-0.4%, for example 0.15%, 0.2%, 0.25% or 0.35%.

[0021] In some embodiments, the mass ratio of paclitaxel palmitate to doxorubicin is 1:(0.08-0.8), preferably 1:(0.16-0.8), for example 1:0.2, 1:0.25, 1:0.3, 1:0.33, 1:0.38, 1:0.4, 1:0.44, 1:0.5, 1:0.54 or 1:0.67.

[0022] In some embodiments, the phospholipid is one or more selected from high-purity egg yolk lecithin, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, sphingomyelin, phosphatidylserine, myristoyl phosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine; preferably, it is one or more selected from high-purity egg yolk lecithin, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, and sphingomyelin. The high-purity protein lecithin may be, for example, EPCS or PC-98T.

[0023] In some embodiments, the amount of phospholipid used is 2-9%, for example 3%, 3.5%, 3.6%, 5%, 6%, 7% or 8%.

[0024] In some embodiments, the amount of DSPE-PEG2000 used is 0.1-0.5%, for example 0.15% or 0.3%.

[0025] In some implementations, the raw material also includes cholesterol.

[0026] In some embodiments, the amount of cholesterol used is no more than 2%, for example 0.2%, 0.23%, 0.68%, 0.72%, or 1.5%.

[0027] In some embodiments, the ratio of the phospholipid to the cholesterol is 1:(0-0.4), but not 1:0, preferably 1:(0-0.2), but not 1:0, for example 1:0.06, 1:0.07 or 1:0.19.

[0028] In some embodiments, the phospholipid bilayer is formed of the phospholipid and DSPE-PEG2000, or the phospholipid, DSPE-PEG2000 and cholesterol.

[0029] In some embodiments, the internal aqueous phase freeze-drying protectant includes one or more of sucrose, lactose, maltose, trehalose, mannitol, glucose, threonine, xylitol, sorbitol, and erythritol; preferably one or more of sucrose, lactose, maltose, trehalose, mannitol, and sorbitol.

[0030] In some embodiments, the amount of the internal aqueous phase freeze-drying protectant is 10-30%, for example 12% or 15%.

[0031] In some embodiments, the salt substance includes one or more of ammonium sulfate, sucrose octasulfate triethylamine, and copper gluconate, preferably ammonium sulfate.

[0032] In some embodiments, the amount of the salt substance used is 0.5-2.5%, for example 1%, 1.2%, 1.5% or 2%.

[0033] In some preferred embodiments, the formulation of the compound liposomes is as follows:

[0034] 0.2-0.7% paclitaxel palmitate; 0.1-0.4% doxorubicin; 2-9% phospholipids; 0-2% cholesterol; 0.1-0.5% DSPE-PEG2000; 10-30% internal aqueous phase lyophilization protectant; 0.5-2.5% salts; and water for injection.

[0035] The present invention also provides a method for preparing the compound liposomes as described above, the method comprising the following steps:

[0036] S1. The organic phase containing the paclitaxel palmitate, the phospholipid, the DSPE-PEG2000 and solvent A is mixed with the aqueous phase containing the salt, the internal aqueous phase freeze-drying protectant and solvent B to obtain crude liposomes.

[0037] S2. The crude liposomes obtained in step S1 are emulsified, replaced, and pH adjusted. The doxorubicin and buffer are added to the mixture, which is then incubated. An external aqueous phase freeze-drying protectant is added, the mixture is brought to a final volume, sterilized, and freeze-dried to obtain the compound liposomes.

[0038] In step S2, the volume adjustment is performed using the water for injection.

[0039] In some embodiments, the raw materials for the compound liposomes also include cholesterol, and step S1 includes the following steps: mixing an organic phase containing paclitaxel palmitate, phospholipids, cholesterol, DSPE-PEG2000 and solvent A with an aqueous phase containing salts, an internal aqueous phase freeze-drying protectant and solvent B to obtain crude liposomes.

[0040] In some embodiments, in step S1, solvent A includes one or more of anhydrous ethanol, ethylene glycol, propylene glycol, and tert-butanol, preferably propylene glycol.

[0041] In some embodiments, in step S1, the amount of solvent A is 1-10%, preferably 3-8%, for example 5% or 6%; the percentage represents the mass of solvent A as a percentage of the total volume of all raw materials.

[0042] In some embodiments, step S1, the preparation of the organic phase includes the following steps: dissolving the paclitaxel palmitate, phospholipids, and DSPE-PEG2000 in solvent A, heating, to obtain the organic phase; or, dissolving the paclitaxel palmitate, phospholipids, cholesterol, and DSPE-PEG2000 in solvent A, heating, to obtain the organic phase. Preferably, the endpoint temperature of the heating is 25°C-75°C, for example, 40°C, 50°C, 55°C, 60°C, or 70°C.

[0043] In some implementations, in step S1, solvent B is water for injection.

[0044] In some embodiments, step S1 includes the following steps: dissolving the salt substance and the internal aqueous phase freeze-drying protectant in solvent B, heating to obtain the aqueous phase; preferably, the endpoint temperature of the heating is 25°C-75°C, for example 40°C, 45°C, 55°C, 60°C, 65°C or 70°C.

[0045] In some implementations, the mixing step in step S1 is carried out under stirring conditions.

[0046] In some implementations, in step S1, the mixing step involves injecting the organic phase into the aqueous phase, preferably under stirring conditions.

[0047] In some implementations, step S2, the emulsification step includes homogenization and / or extrusion.

[0048] In some implementations, step S2, the emulsification step includes homogenization followed by extrusion.

[0049] In some implementations, in step S2, the homogenization is performed in a high-pressure homogenizer at a pressure of 10,000-22,000 psi, such as 12,000 psi, 15,000 psi, 18,000 psi, or 20,000 psi.

[0050] In some implementations, the homogenization is performed 2-9 times in step S2.

[0051] In some implementations, step S2, the homogenization step includes homogenizing 2-3 times at 10000 psi, homogenizing 3 times each at 10000 psi, 15000 psi, and 20000 psi, or homogenizing 3 times each at 12000 psi, 18000 psi, and 22000 psi.

[0052] In some embodiments, in step S2, the extrusion is performed through an extrusion membrane, the pore size of which is preferably 0.05-0.8 μm, for example 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, 0.1 μm, or 0.08 μm or 0.05 μm.

[0053] In some implementations, the number of extrusions in step S2 is 4-8.

[0054] In some implementations, in step S2, the extrusion step is to first extrude from a large-pore extrusion membrane and then from a small-pore extrusion membrane. Preferably, when extruding from either a large-pore or small-pore extrusion membrane, the pore size remains unchanged.

[0055] In some implementations, step S2, the extrusion step, includes any of the following:

[0056] Extrusion of a 0.08μm extrusion film 4-5 times;

[0057] Extrusion of 0.1μm film 4-6 times;

[0058] First, extrude the membrane with a 0.2μm membrane 2-3 times, then extrude the membrane with a 0.1μm membrane 3-5 times.

[0059] First, extrude the membrane twice with a 0.4 μm extrusion membrane, then extrude it five times with a 0.1 μm extrusion membrane;

[0060] First, extrude the membrane with a 0.4μm membrane 2-3 times, then extrude it with a 0.08μm membrane 3 times;

[0061] First, extrude the membrane twice with a 0.8 μm extrusion membrane, then extrude it four times with a 0.2 μm extrusion membrane;

[0062] First, extrude twice with a 0.2μm extrusion membrane, then extrude four times with a 0.08μm extrusion membrane.

[0063] In some implementations, in step S2, the displacement step may displace the solution in the external aqueous phase, remove solvent A from step S1, and form a transmembrane gradient to complete the active drug loading of doxorubicin.

[0064] In some embodiments, step S2, the displacement step includes adding water for injection, preferably an equal volume of water for injection, to the emulsion obtained in the emulsification step, and displacing the external aqueous phase using any of the following methods:

[0065] Ultrafiltration replacement: The resulting emulsion is placed in an ultrafiltration system and ultrafiltration is performed with water for injection; the specific ultrafiltration steps can be routine in the field.

[0066] Dialysis replacement: The resulting emulsion is placed in a dialysis bag and dialyzed with water for injection; the specific dialysis procedure can be routine in this field.

[0067] Glucose gel column replacement: The obtained emulsion is placed in a glucose gel column and passed through the column with water for injection; the specific steps of the column passing can be routine in the field.

[0068] In some implementations, the number of replacements in step S2 is 5.

[0069] In some implementations, step S2, the step of adjusting pH, includes adjustment with a pH adjuster.

[0070] In some implementations, in step S2, the pH adjuster can adjust the pH to 4.5-7.5, preferably 5.5-7, such as 6 or 6.5.

[0071] In some embodiments, in step S2, the pH adjuster is selected from one or more of sodium hydroxide, dipotassium hydrogen phosphate, phosphoric acid, disodium hydrogen phosphate, hydrochloric acid, disodium phosphate, citric acid, disodium citrate, and trisodium citrate.

[0072] In some embodiments, step S2, the step of adjusting pH, is, for example, the step of adding the pH adjuster to the solution obtained in the displacement step to adjust the pH.

[0073] In some embodiments, in step S2, the buffer includes one or more of histidine, morpholine-ethyl sulfonate (MES), tartrate, citrate, and hydroxyethylpiperazine-ethyl sulfonate (HEPES).

[0074] In some embodiments, in step S2, the amount of buffer used is 0.1-0.75%, for example 0.12%, 0.13%, 0.2% or 0.3%.

[0075] In some embodiments, in step S2, the external aqueous phase freeze-drying protectant includes one or more of sucrose, lactose, maltose, trehalose, mannitol, glucose, threonine, xylitol, sorbitol, and erythritol; preferably one or more of sucrose, lactose, maltose, trehalose, mannitol, and sorbitol.

[0076] In some embodiments, in step S2, the amount of the external aqueous phase freeze-drying protectant is 5-40%, preferably 10-30%, for example 12%, 15%, 18% or 20%; here the percentage represents the mass (g) of the external aqueous phase freeze-drying protectant relative to the total volume (mL) after volume adjustment.

[0077] In some preferred embodiments, the type of the internal aqueous phase freeze-drying protectant in step S2 is the same as that in step S1.

[0078] In some implementations, in step S2, the incubation temperature is 40°C-75°C, for example 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C.

[0079] In some implementations, the incubation time in step S2 is 15-90 min, for example 25 min, 30 min, 35 min, 40 min, 45 min, 60 min, 65 min or 70 min.

[0080] In some implementations, in step S2, the incubation temperature is 40°C-75°C and the time is 15-90 min.

[0081] In some implementations, step S2, the incubation step, includes any of the following schemes:

[0082] Incubation temperature: 60℃; Incubation time: 45 min;

[0083] Incubation temperature: 60℃; Incubation time: 30 min;

[0084] Incubation temperature: 70℃; Incubation time: 30 min;

[0085] Incubation temperature: 60℃; Incubation time: 60 min;

[0086] Incubation temperature: 70℃; Incubation time: 35 min;

[0087] Incubation temperature: 65℃; Incubation time: 70 min;

[0088] Incubation temperature: 70℃; Incubation time: 25 min;

[0089] Incubation temperature: 55℃; Incubation time: 40 min;

[0090] Incubation temperature: 65℃; Incubation time: 45 min;

[0091] Incubation temperature: 50℃; Incubation time: 60 min;

[0092] Incubation temperature: 45℃; Incubation time: 45 min;

[0093] Incubation temperature: 50℃; Incubation time: 65 min;

[0094] Incubation temperature 65℃, incubation time 70min.

[0095] In some implementations, step S2, the sterilization step is performed using a 0.22 μm filter membrane.

[0096] In some embodiments, step S2 includes a cooling step after the incubation step and before the addition of the external aqueous phase freeze-drying protectant. The cooling may be, for example, cooling to room temperature.

[0097] In some implementations, step S2, after the step of adding the external aqueous phase freeze-drying protectant and before the step of volume adjustment, further includes a stirring and dissolving step.

[0098] In some implementations, step S2 includes a dispensing step after the sterilization step and before the freeze-drying step.

[0099] In some implementations, step S2 further includes sealing and capping steps after the freeze-drying step.

[0100] In some preferred embodiments, the preparation method includes the following steps:

[0101] Weigh out paclitaxel palmitate, phospholipids, and DSPE-PEG2000, add solvent A, and heat to dissolve to obtain the organic phase; weigh out a mixed aqueous solution containing salts and an internal aqueous phase freeze-drying protectant, heat to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes.

[0102] The crude liposomes obtained are homogenized and / or emulsified by extrusion, and then subjected to ultrafiltration replacement in an ultrafiltration system, dialysis replacement in a dialysis bag, or column replacement with a dextran gel column. The pH is adjusted with a pH adjuster, and then doxorubicin and a buffer are added. The mixture is incubated, cooled, and an external aqueous phase freeze-drying protectant is added. The mixture is then dissolved, brought to a final volume, sterilized, dispensed, freeze-dried, sealed, and capped to obtain the compound liposomes.

[0103] The present invention also provides a compound liposome, which is prepared by the preparation method described above, wherein the compound liposome exists in the dosage form of a lyophilized powder for injection.

[0104] In some embodiments, the particle size of the compound liposomes is 50-200 nm, preferably 80-180 nm.

[0105] The present invention also provides the application of the compound liposomes as described above, or the compound liposomes prepared by the preparation method described above, in the preparation of anticancer drugs.

[0106] In some implementations, the anticancer drug is one or more of the following: anti-breast cancer drugs, anti-ovarian cancer drugs, anti-thyroid cancer drugs, and anti-lymphoma drugs.

[0107] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0108] The reagents and raw materials used in this invention are all commercially available.

[0109] The positive and progressive effects of this invention are as follows:

[0110] The paclitaxel palmitate and doxorubicin compound liposomes of the present invention can simultaneously load two active ingredients, paclitaxel palmitate and doxorubicin; and possess the following excellent properties:

[0111] 1. High encapsulation efficiency and good stability, with the drug encapsulation efficiency, particle size, and PDI remaining basically unchanged before and after lyophilization and reconstitution.

[0112] 2. After administration, the synergistic ratio of the two drugs can be maintained for a long time to exert anti-tumor effects. This solves the shortcomings of traditional combination therapy of paclitaxel and doxorubicin, which has limited efficacy and large toxic side effects due to the different stability and half-life of the two drugs. It has better anti-tumor effects and lower toxic side effects.

[0113] 3. It has a significant sustained-release effect, which greatly slows down the elimination rate of the drug in the body, resulting in a higher drug concentration at the tumor site.

[0114] 4. The process is simple, safe, and efficient, laying the foundation for further research and application of the combined use of paclitaxel and doxorubicin. Attached Figure Description

[0115] Figure 1 This is the CI-Fa diagram of the combined use of paclitaxel and doxorubicin in Example 1.

[0116] Figure 2 This is a schematic diagram of the structure of the compound liposome in Example 16.

[0117] Figure 3 The pharmacokinetic data for PTX, PTX-PA, and DOX in Example 33 are presented. Detailed Implementation

[0118] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0119] In the following examples, unless otherwise specified, all paclitaxel palmitate used was provided by Yancheng Kaili Pharmaceutical Co., Ltd.

[0120] In the following examples, unless otherwise specified, paclitaxel palmitate liposomes were prepared by the following steps: 0.64 g of paclitaxel palmitate, 8 g of high-purity egg yolk lecithin, and 0.05 g of DSPE-PEG2000 were weighed and 6 g of propylene glycol were added. The mixture was heated at 55°C to dissolve the organic phase. 85.31 g of water for injection was weighed and heated to 55°C to obtain the aqueous phase. The organic phase was injected into the aqueous phase under stirring and mixed to obtain crude liposomes. The crude liposomes were placed in an extruder and extruded twice through a 0.2 μm extrusion membrane, and then four times through a 0.1 μm extrusion membrane to obtain a liposome solution. An equal volume of water for injection was added and ultrafiltered five times to replace the in vitro aqueous phase solution of the liposomes. The volume was adjusted to 100 mL with water for injection. The solution was then filtered through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate liposomes.

[0121] In the following examples, unless otherwise specified, DSPE-PEG2000 was purchased from Lipoid GmbH, Germany.

[0122] Example 1: Cytotoxicity study to investigate the synergistic effect of combined use of paclitaxel and doxorubicin

[0123] 1. Experimental Procedure

[0124] 4T1 breast cancer cells cultured in T75 flasks were digested and centrifuged. The cell pellet was resuspended in 10 mL of Umbro-Cell Resin, and 10 μL of the cell suspension was transferred to a hemocytometer for observation and counting under a microscope. The hemocytometer was then diluted 1 × 10⁻⁶ with Umbro-Cell Resin. 5 Cells / mL were collected, homogenized, and seeded into plates. 100 μL of the cell suspension was seeded into each well of a 96-well plate and incubated for 24 h. After cell attachment, a series of gradient concentrations of paclitaxel and doxorubicin were prepared using DMEM. The old culture medium was removed. 100 μL of DMEM solution and the drug solution prepared with DMEM were added to each well of the control and experimental groups, respectively. Six parallel wells were set up for each concentration. Wells containing only DMEM served as the control group, and wells without cells served as the blank group. Forty-eight hours after drug administration, the drug-containing culture medium was removed. 100 μL of 10% CCK-8 DMEM solution was added to each of the blank, control, and experimental groups in the dark. The plates were shaken for 30 s using a microplate reader and incubated for 2 h. The absorbance of each well in the 96-well plate was measured at 450 nm using a microplate reader, and the cell inhibition rate (Fa) was calculated.

[0125] Cell inhibition rate (Fa) = (Ac-As) / (Ac-Ab) * 100%

[0126] As: Experimental wells (containing cell culture medium, CCK-8 reagent, and drug); Ab: Blank wells (containing no cells or drug, and no CCK-8 reagent); Ac: Control wells (containing cell culture medium and CCK-8 reagent).

[0127] The combination index (CI) of paclitaxel and doxorubicin at different ratios was calculated using the median pharmacodynamic method and CompuSyn software. The evaluation criteria were: CI>1 for antagonistic effect, CI=1 for additive effect, and CI<1 for synergistic effect.

[0128] 2. Experimental Results

[0129] CI-Fa diagram of paclitaxel and doxorubicin combination therapy Figure 1 As shown in Table 1, when the cell inhibition rate is 50%, the CI values ​​of different ratios of paclitaxel and doxorubicin combined are as follows. The results of converting paclitaxel into its prodrug paclitaxel palmitate at an equimolar ratio are as follows.

[0130] Table 1. CI values ​​(Fa = 50%) for the combined use of paclitaxel and doxorubicin.

[0131]

[0132]

[0133] Results Analysis: Different ratios of paclitaxel and doxorubicin showed varying degrees of inhibitory effects on 4T1 breast cancer cells. This verifies that the appropriate drug ratio in combined therapy is crucial for tumor efficacy. To avoid antagonistic effects from the combined use of paclitaxel and doxorubicin, extensive experimental verification has shown that the mass ratio of paclitaxel to doxorubicin should be controlled at 1:(0.1-1). When paclitaxel is converted to paclitaxel palmitate at an equimolar ratio, the equivalent mass ratio of paclitaxel palmitate to doxorubicin is 1:(0.08-0.8).

[0134] Example 2: The Key Role of Inner Aqueous Phase Lyophilization Protectant in the Development of Paclitaxel Palmitate and Doxorubicin Liposome Lyophilized Powder Injection

[0135] 1. Experimental prescription

[0136] Table 2. Recipe Design for Experimental Validation Scheme

[0137]

[0138] 2. Preparation process

[0139] Weigh the prescribed amounts of paclitaxel palmitate, high-purity egg yolk lecithin, and DSPE-PEG2000, add 10g of propylene glycol, and heat at 60℃ to dissolve, obtaining the organic phase; weigh the aqueous solution containing 2.5g of ammonium sulfate for injection (a salt) or the prescribed amount of sucrose (an internal aqueous phase freeze-drying protectant), and heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then extrude five times through a 0.1μm extrusion membrane to obtain liposome solution. The solution was prepared by ultrafiltration five times with an equal volume of water for injection to replace the in vitro aqueous phase of the liposome solution; the pH was adjusted to 6.50 with sodium hydroxide; the prescribed amount of doxorubicin powder and histidine (buffer) were weighed and placed in the above liposome solution, incubated at 60°C for 60 min, cooled to room temperature, and then 20 g of sucrose (external aqueous phase lyophilization protectant) was weighed and placed in the above liposome solution, stirred to dissolve, and diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder for injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0140] 3. Experimental Results

[0141] Changes in particle size, PDI, and drug encapsulation efficiency (the particle size and PDI were measured using a Malvern laser particle size analyzer, and the encapsulation efficiency of the two active ingredients was determined using the dextran gel column method) before and after reconstitution of lyophilized liposome injections are important indicators for evaluating their quality. Since doxorubicin encapsulated in the aqueous phase is prone to leakage after reconstitution, this invention focuses on investigating the above-mentioned liposome particle size, PDI, and doxorubicin encapsulation efficiency. The results are shown in Table 3.

[0142] Table 3. Effects of internal aqueous phase lyophilization protectants on the lyophilized powder of paclitaxel palmitate and doxorubicin compound liposome injection.

[0143]

[0144] Results Analysis: When no lyophilization protectant was added to the aqueous phase of the liposome in the formulation, the lyophilized liposome powder injection showed turbidity after reconstitution, and the liposome particle size, PDI, and doxorubicin encapsulation efficiency all changed significantly. This may be because the liposome structure was destroyed during reconstitution, leading to drug leakage (as in formulations 1-3). However, when a certain amount of lyophilization protectant was added to the aqueous phase of the liposome, the particle size, PDI, and doxorubicin encapsulation efficiency of the lyophilized liposome powder injection remained almost unchanged after reconstitution. Therefore, the addition of the lyophilization protectant to the aqueous phase is crucial for this lyophilized liposome powder injection and will directly affect the drug-likeness of the liposomes. This is the most core technical feature of this invention.

[0145] Example 3: Key Effects of Aqueous Phase pH on the Development of Paclitaxel Palmitate and Doxorubicin Liposomes

[0146] 1. Experimental prescription

[0147] Table 4. Recipe Design for Experimental Validation Scheme

[0148]

[0149] 2. Preparation process

[0150] Weigh out the prescribed amounts of paclitaxel palmitate, high-purity egg yolk lecithin, and DSPE-PEG2000, add 10g of propylene glycol, and heat to 60℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 2.5g of ammonium sulfate (a salt for injection), heat to 60℃, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude five times through a 0.1μm extrusion membrane to obtain a liposome solution; add an equal volume of injection... The in vitro aqueous phase solution of the liposome was replaced five times by ultrafiltration with water; the corresponding pH value of the in vitro aqueous phase was adjusted; the prescribed amount of doxorubicin powder and histidine (buffer) were weighed and placed in the above liposome solution, incubated at 60℃ for 55 min, cooled to room temperature, 15 g of sucrose (lyophilization protectant of the in vitro aqueous phase) was weighed and placed in the above liposome solution, stirred to dissolve, and diluted to 100 mL with water for injection; sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposome.

[0151] 3. Experimental Results

[0152] Encapsulation efficiency is an important indicator for evaluating liposome quality. Therefore, this invention primarily uses encapsulation efficiency as the main evaluation indicator. This invention conducted parallel comparisons of paclitaxel palmitate and doxorubicin compound liposomes with varying external aqueous phase pH values. The results showed that external aqueous phase pH has a significant impact on the encapsulation efficiency of both drugs. When the external aqueous phase pH is less than 4.5, it is unfavorable for the encapsulation of doxorubicin, with an encapsulation efficiency of less than 90%. When the external aqueous phase pH is between 4.5 and 7.5, the encapsulation efficiency of both drugs is greater than 95%. When the pH is greater than 7.5, it is unfavorable for the encapsulation of paclitaxel palmitate, with an encapsulation efficiency of less than 95%. The results are shown in Table 5.

[0153] Table 5. Effects of external aqueous phase pH on the drug-likeness of paclitaxel palmitate and doxorubicin compound liposomes.

[0154]

[0155] Results Analysis: Through extensive experimental verification, the present invention obtained the above experimental results. The present invention found that the pH of the external aqueous phase directly affects the encapsulation of paclitaxel palmitate and doxorubicin. Their encapsulation efficiency is closely related to the pH value of the external aqueous phase. It can be seen that the adjustment of the pH of the external aqueous phase is crucial for the liposomes and will directly affect the drug-likeness of the liposomes.

[0156] Example 4: Comparative evaluation of paclitaxel palmitate and doxorubicin compound liposomes prepared with different phospholipid to cholesterol ratios

[0157] 1. Preparation of liposomes containing paclitaxel palmitate and doxorubicin with different phospholipid to cholesterol ratios

[0158] Table 6. Recipe Design for Experimental Validation Scheme

[0159]

[0160]

[0161] 2. Preparation process

[0162] Weigh out the prescribed amounts of paclitaxel palmitate, high-purity egg yolk lecithin, cholesterol, and DSPE-PEG2000, add 9g of propylene glycol, and heat to dissolve at 60℃ to obtain the organic phase; weigh out an aqueous solution containing 2.5g of ammonium sulfate (a salt for injection), heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude through a 0.2μm extrusion membrane 3 times, and then through a 0.1μm extrusion membrane 4 times to obtain the liposome solution; add an equal amount of... The liposome in vitro aqueous phase solution was replaced 5 times by ultrafiltration with water for injection; the corresponding pH value of the external aqueous phase was adjusted; the prescribed amount of doxorubicin powder and histidine (buffer) were weighed and placed in the above liposome solution, incubated at 55℃ for 55 min, cooled to room temperature, 15 g of sucrose (external aqueous phase lyophilization protectant) was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposome.

[0163] 3. Experimental Results

[0164] The effects of different phospholipid to cholesterol ratios on paclitaxel palmitate and doxorubicin liposomes are shown in Table 7.

[0165] Table 7. Effects of different phospholipid to cholesterol ratios on paclitaxel palmitate and doxorubicin liposome combination.

[0166]

[0167] Results analysis: Through a series of parallel comparisons of experimental results, it was found that the ratio of lecithin to cholesterol has little effect on the encapsulation efficiency of doxorubicin. However, when the ratio is too small, the extrusion pressure of the formulation is too high, and it cannot be extruded smoothly. It also directly affects the encapsulation efficiency of paclitaxel palmitate, which decreases as the ratio of the two decreases. At the same time, it leads to an increase in particle size and PDI, which affects the stability of the formulation.

[0168] Example 5: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0169] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 10g of propylene glycol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude four times through a 0.1μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection to... The aqueous phase of the liposome was replaced by filtration five times; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60 °C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0170] The particle size was measured to be 119.1 nm, the PDI was 0.153, the encapsulation efficiency of paclitaxel palmitate was 99.13%, and the encapsulation efficiency of doxorubicin was 98.68%.

[0171] Example 6: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0172] Weigh out 0.64g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 6g of propylene glycol, and heat at 55℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 1.5g of ammonium sulfate for injection and 10g of sucrose, heat to 55℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude four times through a 0.1μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection and ultrafilter. The aqueous phase of the liposome was replaced 5 times; the pH was adjusted to 6.50 with sodium hydroxide; a solution containing 0.2g doxorubicin and 0.1g histidine were weighed and placed in the above liposome solution, incubated at 60℃ for 45min, cooled to room temperature, and then 20g sucrose was weighed and placed in the above liposome solution. The solution was stirred to dissolve and then diluted to 100mL with water for injection; the solution was sterilized by filtering through a 0.22μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0173] The particle size was measured to be 106.7 nm, the PDI was 0.131, the encapsulation efficiency of paclitaxel palmitate was 99.26%, and the encapsulation efficiency of doxorubicin was 98.35%.

[0174] Example 7: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0175] Weigh out 0.4g of paclitaxel palmitate, 6g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 8g of propylene glycol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 12g of trehalose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; homogenize the crude liposomes three times each at 10000psi, 15000psi, and 20000psi using a high-pressure homogenizer to obtain liposome solutions; add an equal volume of injectable lecithin... The aqueous phase of the liposome was replaced five times by ultrafiltration with water; the pH was adjusted to 6.0 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.05 g of histidine were weighed and placed in the above liposome solution, incubated at 60 °C for 30 min, cooled to room temperature, 12 g of trehalose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0176] The particle size was measured to be 178.1 nm, the PDI was 0.181, the encapsulation efficiency of paclitaxel palmitate was 99.51%, and the encapsulation efficiency of doxorubicin was 99.16%.

[0177] Example 8: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0178] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.08g of DSPE-PEG2000 according to the prescription, add 8g of propylene glycol, and heat at 70℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of injectable sucrose octasulfate triethylamine and 10g of sucrose, heat to 70℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.4μm extrusion membrane, and then extrude five times through a 0.1μm extrusion membrane to obtain the liposome solution; use water for injection... The liposome solution was replaced by dialysis; the pH was adjusted to 7.0 with sodium hydroxide; 0.25 g of doxorubicin powder and 0.2 g of histidine were weighed and placed in the above liposome solution, incubated at 70 °C for 30 min, cooled to room temperature, 10 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin liposome compound.

[0179] The particle size was measured to be 108.6 nm, the PDI was 0.104, the encapsulation efficiency of paclitaxel palmitate was 99.34%, and the encapsulation efficiency of doxorubicin was 98.87%.

[0180] Example 9: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0181] Weigh out 0.4g of paclitaxel palmitate, 9g of high-purity egg yolk lecithin EPCS, and 0.08g of DSPE-PEG2000 according to the prescription, add 5g of propylene glycol, and heat to 60℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 1g of injectable copper gluconate and 10g of maltose, heat to 55℃, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; homogenize the crude liposomes three times at 10000psi using a high-pressure homogenizer, then place the liposomes in an extruder and extrude them four times through a 0.1μm extrusion membrane to obtain a liposome solution; use injection... The aqueous phase of the liposome was replaced with water for injection through a dextran gel column; the pH was adjusted to 6.0 with disodium hydrogen phosphate; 0.1 g of doxorubicin and 0.3 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 60 min, cooled to room temperature, 15 g of maltose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder for injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0182] The particle size was measured to be 142.7 nm, the PDI was 0.184, the encapsulation efficiency of paclitaxel palmitate was 98.73%, and the encapsulation efficiency of doxorubicin was 98.39%.

[0183] Example 10: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0184] Weigh out 0.8g of paclitaxel palmitate, 10g of high-purity egg yolk lecithin PC-98T, and 0.05g of DSPE-PEG2000 according to the prescription, add 10g of propylene glycol, and heat at 50℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 10g of sucrose, heat to 55℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder and extrude through a 0.08μm extrusion membrane 5 times to obtain a liposome solution; add an equal volume of water for injection and ultrafilter 5 times to replace the lipids. An in vitro aqueous solution was prepared; the pH was adjusted to 7.50 with sodium hydroxide; 0.1 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 70°C for 35 min, cooled to room temperature, 15 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0185] The particle size was measured to be 88.1 nm, the PDI was 0.122, the encapsulation efficiency of paclitaxel palmitate was 99.42%, and the encapsulation efficiency of doxorubicin was 98.83%.

[0186] Example 11: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0187] Weigh out 0.2g of paclitaxel palmitate, 3g of hydrogenated soybean lecithin, and 0.05g of DSPE-PEG2000 as prescribed, add 8g of anhydrous ethanol, and heat at 50℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 1g of ammonium sulfate for injection and 15g of sucrose, heat to 40℃, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; homogenize the crude liposomes three times each at 10000psi, 15000psi, and 20000psi using a high-pressure homogenizer to obtain liposome solutions; add an equal volume of water for injection and ultrafilter five times to replace the lipids. The liposome was prepared in an in vitro aqueous phase solution; the pH was adjusted to 5.5 with sodium hydroxide; a solution containing 0.1 g doxorubicin and 0.05 g histidine were weighed and placed in the above liposome solution, incubated at 65°C for 70 min, cooled to room temperature, and then 25 g sucrose was weighed and placed in the above liposome solution. The solution was stirred to dissolve and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0188] The particle size was measured to be 162.7 nm, the PDI was 0.175, the encapsulation efficiency of paclitaxel palmitate was 98.49%, and the encapsulation efficiency of doxorubicin was 99.17%.

[0189] Example 12: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0190] Weigh out 0.65g of paclitaxel palmitate, 3.5g of hydrogenated soybean lecithin, and 0.15g of DSPE-PEG2000 according to the prescribed dosage, add 5g of anhydrous ethanol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2.5g of injectable sucrose octasulfate triethylamine, 2g of maltose, and 8g of sucrose, and heat to 45℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; homogenize the crude liposomes twice at 10000psi using a high-pressure homogenizer, and then place the liposomes in an extruder and extrude through a 0.08μm diameter. The membrane was extruded four times to obtain a liposome solution. The aqueous phase of the liposome was replaced with water for injection through a dextran gel column. The pH was adjusted to 6.0 with dipotassium hydrogen phosphate. 0.07 g of doxorubicin powder and 0.12 g of histidine were weighed and placed in the above liposome solution. The mixture was incubated at 70 °C for 25 min. After cooling to room temperature, 8 g of maltose and 10 g of sucrose were weighed and placed in the above liposome solution. The mixture was stirred to dissolve and then diluted to 100 mL with water for injection. The mixture was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain a lyophilized powder injection of paclitaxel palmitate and doxorubicin liposomes.

[0191] The particle size was measured to be 92.4 nm, the PDI was 0.128, the encapsulation efficiency of paclitaxel palmitate was 99.38%, and the encapsulation efficiency of doxorubicin was 99.47%.

[0192] Example 13: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0193] Weigh out 0.4g of paclitaxel palmitate, 3g of soybean lecithin, and 0.06g of DSPE-PEG2000 according to the prescribed dosage, add 6g of anhydrous ethanol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 1.5g of ammonium sulfate for injection, 10g of mannitol, and 10g of lactose, and heat to 65℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder and extrude three times through a 0.4μm extrusion membrane, then extrude three times through a 0.08μm extrusion membrane to obtain the liposome solution; replace the liposomes five times with water for injection via dialysis. An aqueous solution was prepared; the pH was adjusted to 6.50 with sodium hydroxide; 0.15 g of doxorubicin powder and 0.12 g of histidine were weighed and placed in the above liposome solution, incubated at 55°C for 40 min, cooled to room temperature, 10 g of mannitol and 15 g of lactose were weighed and placed in the above liposome solution, stirred to dissolve, and diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0194] The particle size was measured to be 130.6 nm, the PDI was 0.141, the encapsulation efficiency of paclitaxel palmitate was 98.73%, and the encapsulation efficiency of doxorubicin was 99.28%.

[0195] Example 14: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0196] Weigh out 0.3g of paclitaxel palmitate, 2g of hydrogenated soybean lecithin, and 0.3g of DSPE-PEG2000 as prescribed, add 6g of anhydrous ethanol, and heat at 40℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 0.5g of ammonium sulfate for injection and 10g of sucrose, heat to 60℃, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude through a 0.2μm extrusion membrane 3 times, and then extrude through a 0.1μm extrusion membrane 3 times, obtaining the liposome solution; add an equal volume of water for injection and ultrafilter 5 times to replace the lipids. The liposome was prepared in an in vitro aqueous phase solution; the pH was adjusted to 5.0 with hydrochloric acid; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 65°C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0197] The particle size was measured to be 127.1 nm, the PDI was 0.161, the encapsulation efficiency of paclitaxel palmitate was 99.45%, and the encapsulation efficiency of doxorubicin was 96.17%.

[0198] Example 15: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0199] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 8g of tert-butanol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.8μm extrusion membrane, and then extrude four times through a 0.2μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection and ultrafilter. The aqueous phase of the liposome was replaced 5 times; the pH was adjusted to 6.50 with sodium hydroxide; 0.1 g of doxorubicin powder and 0.05 g of histidine were weighed and placed in the above liposome solution, incubated at 50 °C for 60 min, cooled to room temperature, 20 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0200] The particle size was measured to be 187.4 nm, the PDI was 0.214, the encapsulation efficiency of paclitaxel palmitate was 99.61%, and the encapsulation efficiency of doxorubicin was 98.94%.

[0201] Example 16: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0202] Weigh out 0.65g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin (EPCS), 1.5g of cholesterol, and 0.05g of DSPE-PEG2000 as prescribed. Add 8g of propylene glycol and heat at 65℃ to dissolve, obtaining the organic phase. Weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of trehalose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Homogenize the crude liposomes three times each at 12000psi, 18000psi, and 22000psi using a high-pressure homogenizer to obtain liposome solutions. Add an equal volume of water for injection and ultrafilter five times to replace the lipids. The liposome was prepared in an in vitro aqueous phase solution; the pH was adjusted to 6.50 with sodium hydroxide; 0.35 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 45 °C for 45 min, cooled to room temperature, 15 g of trehalose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0203] A schematic diagram of the structure of the compound liposomes obtained in this embodiment is shown below. Figure 2 As shown.

[0204] The particle size was measured to be 138.4 nm, the PDI was 0.183, the encapsulation efficiency of paclitaxel palmitate was 98.59%, and the encapsulation efficiency of doxorubicin was 98.73%.

[0205] Example 17: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0206] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin PC-98T, and 0.05g of DSPE-PEG2000 according to the prescription, add 8g of anhydrous ethanol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude four times through a 0.08μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection to... The aqueous phase of the liposome was replaced by filtration five times; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60 °C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0207] The particle size was measured to be 81.1 nm, the PDI was 0.119, the encapsulation efficiency of paclitaxel palmitate was 99.48%, and the encapsulation efficiency of doxorubicin was 99.36%.

[0208] Example 18: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0209] Weigh out the prescribed amounts of paclitaxel palmitate (0.45g), distearate (3.6g), cholesterol (0.2g), and DSPE-PEG2000 (0.05g), add 6g of anhydrous ethanol, and heat at 60℃ to dissolve, obtaining the organic phase. Weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 12g of sucrose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then four times through a 0.1μm extrusion membrane to obtain a liposome solution. Add an equal volume of water for injection and ultrafilter five times to replace the liposomes. An aqueous phase solution was prepared; the pH was adjusted to 6.0 with sodium hydroxide; 0.15 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60 °C for 45 min, cooled to room temperature, 10 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0210] The particle size was measured to be 137.3 nm, the PDI was 0.124, the encapsulation efficiency of paclitaxel palmitate was 98.42%, and the encapsulation efficiency of doxorubicin was 98.61%.

[0211] Example 19: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0212] Weigh out the prescribed amounts of 0.3g paclitaxel palmitate, 3.6g hydrogenated soybean lecithin, 0.72g cholesterol, and 0.05g DSPE-PEG2000, add 6g tert-butanol, and heat at 60℃ to dissolve, obtaining the organic phase. Weigh out an aqueous solution containing 2g ammonium sulfate for injection and 12g sucrose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then extrude five times through a 0.1μm extrusion membrane to obtain a liposome solution. Add an equal volume of water for injection and ultrafilter five times to replace the liposomes. An aqueous solution was prepared; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0213] The particle size was measured to be 112.4 nm, the PDI was 0.153, the encapsulation efficiency of paclitaxel palmitate was 99.18%, and the encapsulation efficiency of doxorubicin was 96.53%.

[0214] Example 20: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0215] Weigh out 0.60g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 8g of propylene glycol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 1.8g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.4μm extrusion membrane, and then extrude five times through a 0.1μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection to... The liposome in vitro aqueous phase solution was replaced by filtration five times; the pH was adjusted to 6.50 with sodium hydroxide; 0.19 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60 °C for 45 min, cooled to room temperature, 15 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0216] The particle size was measured to be 104.7 nm, the PDI was 0.103, the encapsulation efficiency of paclitaxel palmitate was 99.62%, and the encapsulation efficiency of doxorubicin was 98.55%.

[0217] Example 21: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0218] Weigh out the prescribed amounts of paclitaxel palmitate 0.25g, distearate phosphatidylcholine 3.6g, cholesterol 0.68g, and DSPE-PEG2000 0.05g, add 6g of anhydrous ethanol, and heat at 60℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 12g of sucrose, and heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; homogenize the crude liposomes three times at 10000psi using a high-pressure homogenizer, then place the liposomes in an extruder and extrude them five times through a 0.08μm extrusion membrane to obtain a liposome solution; add an equal volume of water for injection and ultrafilter for 5 minutes. The in vitro aqueous phase solution of the liposomes was replaced; the pH was adjusted to 6.50 with disodium hydrogen phosphate; 0.2g of doxorubicin powder and 0.1g of histidine were weighed and placed in the above liposome solution, incubated at 60℃ for 45min, cooled to room temperature, 20g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100mL with water for injection; the solution was sterilized by filtering through a 0.22μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0219] The particle size was measured to be 91.6 nm, the PDI was 0.081, the encapsulation efficiency of paclitaxel palmitate was 97.73%, and the encapsulation efficiency of doxorubicin was 98.29%.

[0220] Example 22: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0221] Weigh out 0.4g of paclitaxel palmitate, 7g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 10g of tert-butanol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of copper gluconate for injection and 10g of maltose, and heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder and extrude through a 0.1μm extrusion membrane 6 times to obtain the liposome solution; add an equal volume of water for injection and ultrafilter 5 times to replace the lipids. An in vitro aqueous solution was prepared; the pH was adjusted to 6.50 with sodium hydroxide; 0.1 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, 20 g of maltose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0222] The particle size was measured to be 100.5 nm, the PDI was 0.102, the encapsulation efficiency of paclitaxel palmitate was 98.84%, and the encapsulation efficiency of doxorubicin was 99.06%.

[0223] Example 23: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0224] Weigh out 0.4g of paclitaxel palmitate, 7g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 5g of propylene glycol and 3g of tert-butanol, and heat to 60℃ to dissolve, obtaining the organic phase; weigh out an aqueous solution containing 2g of injectable copper gluconate and 10g of sucrose, and heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude five times through a 0.1μm extrusion membrane to obtain the liposome solution; use injectable... Water was used to replace the aqueous phase of the liposome in vitro solution via dialysis; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, and then 12 g of sucrose was weighed and placed in the above liposome solution. The solution was stirred to dissolve and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0225] The particle size was measured to be 109.8 nm, the PDI was 0.149, the encapsulation efficiency of paclitaxel palmitate was 99.37%, and the encapsulation efficiency of doxorubicin was 99.54%.

[0226] Example 24: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0227] Weigh out 0.4g of paclitaxel palmitate, 7g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription. Add 5g of anhydrous ethanol and 3g of tert-butanol, and heat at 60℃ to dissolve to obtain the organic phase. Weigh out an aqueous solution containing 2g of injectable copper gluconate and 12g of trehalose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, and then extrude four times through a 0.1μm extrusion membrane to obtain the liposome solution. Add an equal volume of injectable... The aqueous phase of the liposome was replaced five times by ultrafiltration with water; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.12 g of histidine were weighed and placed in the above liposome solution, incubated at 50 °C for 65 min, cooled to room temperature, 12 g of trehalose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0228] The particle size was measured to be 124.3 nm, the PDI was 0.151, the encapsulation efficiency of paclitaxel palmitate was 97.74%, and the encapsulation efficiency of doxorubicin was 98.51%.

[0229] Example 25: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0230] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 10g of propylene glycol, and heat at 55℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 8g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes; place the crude liposomes in an extruder and extrude through a 0.1μm extrusion membrane 5 times to obtain the liposome solution; add an equal volume of water for injection and ultrafilter 5 times to replace the liposomes. Aqueous solution; adjust pH to 6.50 with sodium hydroxide; weigh 0.16g of doxorubicin powder and 0.1g of histidine, place them in the above liposome solution, incubate at 60℃ for 45min, cool to room temperature, weigh 25g of sucrose, place it in the above liposome solution, stir to dissolve, and make up to 100mL with water for injection; filter through a 0.22μm filter membrane for sterilization, dispense, freeze dry, seal, and cap to obtain paclitaxel palmitate and doxorubicin compound liposome lyophilized powder for injection.

[0231] The particle size was measured to be 105.8 nm, the PDI was 0.116, the encapsulation efficiency of paclitaxel palmitate was 99.32%, and the encapsulation efficiency of doxorubicin was 97.49%.

[0232] Example 26: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0233] Weigh out 0.5g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription, add 10g of propylene glycol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.4μm extrusion membrane, and then extrude three times through a 0.08μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection to... The aqueous phase of the liposome was replaced by filtration six times; the pH was adjusted to 7.50 with dipotassium hydrogen phosphate; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0234] The particle size was measured to be 126.8 nm, the PDI was 0.161, the encapsulation efficiency of paclitaxel palmitate was 98.62%, and the encapsulation efficiency of doxorubicin was 98.37%.

[0235] Example 27: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0236] Weigh out the prescribed amounts of paclitaxel palmitate (0.15g), dipalmitoylphosphatidylcholine (3.5g), cholesterol (0.23g), and DSPE-PEG2000 (0.05g), add 10g of propylene glycol, and heat at 60℃ to dissolve, obtaining the organic phase. Weigh out an aqueous solution containing 1.2g of ammonium sulfate for injection and 5g of sucrose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then extrude five times through a 0.1μm extrusion membrane to obtain a liposome solution. Add an equal volume of water for injection and ultrafilter five times to replace the lipids. An in vitro aqueous solution was prepared; the pH was adjusted to 7.50 with sodium hydroxide; 0.1g of doxorubicin and 0.1g of histidine were weighed and placed in the above liposome solution, incubated at 65℃ for 70 min, cooled to room temperature, 8g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100mL with water for injection; the solution was sterilized by passing through a 0.22μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0237] The particle size was measured to be 131.9 nm, the PDI was 0.154, the encapsulation efficiency of paclitaxel palmitate was 97.44%, and the encapsulation efficiency of doxorubicin was 97.16%.

[0238] Example 28: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0239] Weigh out 0.5g of paclitaxel palmitate, 6g of high-purity egg yolk lecithin PC-98T, and 0.05g of DSPE-PEG2000 according to the prescription, add 8g of propylene glycol, and heat at 60℃ to dissolve to obtain the organic phase; weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, heat to 60℃ to obtain the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude five times through a 0.1μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection and ultrafilter for 5 minutes. The in vitro aqueous phase solution of the liposomes was replaced; the pH was adjusted to 6.50 with disodium citrate; 0.2g of doxorubicin powder and 0.13g of histidine were weighed and placed in the above liposome solution, incubated at 60℃ for 45min, cooled to room temperature, 12g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100mL with water for injection; the solution was sterilized by filtering through a 0.22μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0240] The particle size was measured to be 111.5 nm, the PDI was 0.116, the encapsulation efficiency of paclitaxel palmitate was 99.04%, and the encapsulation efficiency of doxorubicin was 98.75%.

[0241] Example 29: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0242] Weigh out 0.5g of paclitaxel palmitate, 3g of egg yolk lecithin (98% purity), 2g of sphingomyelin, and 0.05g of DSPE-PEG2000 according to the prescribed dosage. Add 9g of anhydrous ethanol and heat at 60℃ to dissolve, obtaining the organic phase. Weigh out an aqueous solution containing 2g of ammonium sulfate for injection and 15g of sucrose, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then extrude four times through a 0.1μm extrusion membrane to obtain the liposome solution. Add an equal volume of water for injection and ultrafilter five times to replace the liposomes. An aqueous solution was prepared; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, 12 g of sucrose was weighed and placed in the above liposome solution, stirred to dissolve, and then diluted to 100 mL with water for injection; the solution was sterilized by passing through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0243] The particle size was measured to be 153.1 nm, the PDI was 0.167, the encapsulation efficiency of paclitaxel palmitate was 97.30%, and the encapsulation efficiency of doxorubicin was 96.81%.

[0244] Example 30: Preparation of paclitaxel palmitate and doxorubicin compound liposomes

[0245] Weigh out 0.45g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin EPCS, and 0.05g of DSPE-PEG2000 according to the prescription. Add 6g of propylene glycol and 3g of tert-butanol, and heat at 50℃ to dissolve, obtaining the organic phase. Weigh out a mixed aqueous solution containing 2g of injectable copper gluconate, 10g of maltose, and 5g of sorbitol, and heat to 60℃ to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Place the crude liposomes in an extruder and extrude twice through a 0.2μm extrusion membrane, then extrude four times through a 0.1μm extrusion membrane to obtain a liposome solution. Add an equal volume of injectable... The in vitro aqueous phase of the liposome was replaced five times with ultrafiltration with water; the pH was adjusted to 6.50 with sodium hydroxide; 0.2 g of doxorubicin powder and 0.1 g of histidine were weighed and placed in the above liposome solution, incubated at 60°C for 45 min, cooled to room temperature, 10 g of maltose and 5 g of sorbitol were weighed and placed in the above liposome solution, stirred to dissolve, and diluted to 100 mL with water for injection; the solution was sterilized by filtering through a 0.22 μm filter membrane, dispensed, lyophilized, sealed, and capped to obtain the lyophilized powder injection of paclitaxel palmitate and doxorubicin compound liposomes.

[0246] The particle size was measured to be 128.1 nm, the PDI was 0.159, the encapsulation efficiency of paclitaxel palmitate was 99.43%, and the encapsulation efficiency of doxorubicin was 99.20%.

[0247] Example 31: Pharmacodynamic study of paclitaxel palmitate and doxorubicin compound liposome for injection

[0248] 1. Sample preparation

[0249] Free paclitaxel and doxorubicin mixture: prepared by diluting paclitaxel injection and doxorubicin according to their respective drug instructions and mixing them evenly.

[0250] Paclitaxel palmitate liposomes: Weigh 0.64g of paclitaxel palmitate, 8g of high-purity egg yolk lecithin, and 0.05g of DSPE-PEG2000 according to the prescribed amount, add 6g of propylene glycol, and heat to 55℃ to dissolve, obtaining the organic phase; weigh 85.31g of water for injection, heat to 55℃, obtaining the aqueous phase; inject the organic phase into the aqueous phase under stirring, mix well, and obtain crude liposomes; place the crude liposomes in an extruder, extrude twice through a 0.2μm extrusion membrane, and then extrude four times through a 0.1μm extrusion membrane to obtain the liposome solution; add an equal volume of water for injection and ultrafilter five times to replace the in vitro aqueous phase solution of the liposomes; make up to 100mL with water for injection; filter through a 0.22μm filter membrane for sterilization to obtain paclitaxel palmitate liposomes.

[0251] Doxorubicin liposomes: Weigh the prescribed amounts of HSPC 0.958g, DSPE-PEG2000 0.319g, and Chol... 0.319 g of ethanol and 20 g of sodium sulfate were placed in a 100 mL beaker and heated in a 65 °C water bath to dissolve them, yielding an organic phase. 3.30 g of ammonium sulfate for injection was added to 75.10 g of water for injection and heated to 65 °C to obtain an aqueous phase. The organic phase was injected into the aqueous phase under stirring and mixed to obtain crude liposomes. The crude liposomes were placed in an extruder and extruded 6 times through a 0.1 μm extrusion membrane to obtain a liposome solution. An equal volume of water for injection was added and ultrafiltered 5 times to replace the aqueous phase solution. The ethanol and ammonium sulfate in the aqueous phase of the liposomes were removed by ultrafiltration with water for injection. 0.2 g of doxorubicin powder, 0.155 g of histidine, and 10 g of sucrose were weighed and added to the above liposome solution and mixed. The mixture was incubated at 65 °C for 60 min, cooled to room temperature, and then diluted to 100 mL with water for injection. The mixture was sterilized by filtering through a 0.22 μm filter membrane to obtain doxorubicin liposomes.

[0252] Paclitaxel palmitate and doxorubicin compound liposomes: The liposomes prepared in Examples 6, 20 and 25 were used as the compound liposome group in this invention.

[0253] 2. Experimental Methods

[0254] 4T1 cells in logarithmic growth phase (purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were resuspended in PBS and the cell density was adjusted to 1×10⁻⁶. 7 0.2 mL of cell suspension was injected into the right axilla of mice. Ten days after inoculation, mice were randomly divided into four groups based on tumor volume: ① saline group; ② free paclitaxel and doxorubicin mixed control group; ③ paclitaxel palmitate liposome and doxorubicin liposome mixed control group; ④ paclitaxel palmitate and doxorubicin compound liposome group prepared in Examples 6, 20, and 25. Eight mice were administered the drugs via tail vein injection at a dose of 15 mg / kg paclitaxel (equivalent to 20 mg / kg paclitaxel palmitate) and 6 mg / kg doxorubicin. Mice in the saline group received saline injections under the same conditions. The drugs were administered every 5 days for a total of 3 times. The mice were sacrificed the day after drug withdrawal, and the tumors were harvested and weighed to calculate the tumor inhibition rate.

[0255] Tumor inhibition rate = (tumor weight in the saline group - tumor weight in the drug-treated group) / tumor weight in the saline group × 100%.

[0256] 3. Experimental Results: The pharmacodynamic comparison results are shown in Table 8.

[0257] Table 8 Comparison of the efficacy results of paclitaxel palmitate and doxorubicin liposome combination therapy

[0258]

[0259] Note: Compared with the saline group: * P<0.05, ** P<0.01; Compared with the control group containing free paclitaxel and doxorubicin: # P<0.05

[0260] Results Analysis: Experimental results showed that the antitumor effect of the paclitaxel palmitate and doxorubicin compound liposomes was significantly higher than that of the traditional combination drug administration method (i.e., mixed administration of free paclitaxel and doxorubicin). To verify the importance of the ratio of the two drugs within a certain range in antitumor activity, this invention found that the antitumor effect of the paclitaxel palmitate and doxorubicin compound liposomes was still higher than that of the two single-drug liposome mixture. This may be because the ratio of the two single-drug liposome mixture to the initial drug changes significantly when it reaches the tumor site. The above indicates that the compound liposomes prepared by this invention have a good synergistic antitumor effect.

[0261] Example 32: Acute toxicity study of paclitaxel palmitate and doxorubicin compound liposomes

[0262] 1. Experimental Methods

[0263] One hundred ICR mice were randomly divided into 10 groups of 10 mice each (half male and half female). Five groups were administered a mixture of free paclitaxel and doxorubicin at doses of 30.00 mg / kg (meaning the total dose of paclitaxel and doxorubicin was 30.00 mg / kg, with a mass ratio of paclitaxel:doxorubicin = 1:0.4; the same applies below), 34.09 mg / kg, 38.73 mg / kg, 44.01 mg / kg, and 50.00 mg / kg, respectively. The other five groups... The paclitaxel palmitate and doxorubicin compound liposomes prepared in Example 6 of this invention were administered at the following doses per group: 60.00 mg / kg (the dose here means the total dose of paclitaxel and doxorubicin is 60.00 mg / kg, with a mass ratio of paclitaxel:doxorubicin = 1:0.4; the total dose of paclitaxel palmitate and doxorubicin can be obtained after conversion; the same applies below), 69.82 mg / kg, 81.24 mg / kg, 94.54 mg / kg, and 110.00 mg / kg. After animal grouping and dose calculation, the animals were administered via tail vein injection. Animals were observed for 14 days after administration, and the number of deaths was recorded. The LD50 of each group was calculated using the Bliss method. 50 .

[0264] 2. Experimental Results

[0265] The results of the acute toxicity test are shown in Table 9.

[0266] Table 9 shows the mortality rate of mice in each group.

[0267]

[0268]

[0269] Results Analysis: Calculations showed that the LD50 of the free paclitaxel and doxorubicin mixture was [missing value]. 50 The LD50 was 38.58 mg / kg, while the LD50 of the paclitaxel palmitate and doxorubicin liposome combination group was... 50 The toxicity of the paclitaxel palmitate-doxorubicin compound liposome was 84.63 mg / kg, which was significantly lower than that of the traditional free paclitaxel and doxorubicin mixture, indicating that the compound liposome of the present invention has higher safety.

[0270] Example 33: Pharmacokinetic Study of Paclitaxel Palmitate and Doxorubicin Liposome Compound

[0271] 1. Experimental Methods

[0272] Twelve male SD rats were randomly divided into two groups of six each. Before the experiment, rats were fasted for 24 hours but allowed free access to water. The following were administered via tail vein: ① the paclitaxel palmitate and doxorubicin compound liposome prepared in Example 6 of this invention; ② a mixture of free paclitaxel and doxorubicin. Both groups were administered 6.0 mg / kg paclitaxel (equivalent to 7.7 mg / kg paclitaxel palmitate) and 2.40 mg / kg doxorubicin. Blood samples of 0.5 mL were collected from the orbital cavity at 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h after administration.

[0273] Sample processing and analysis: Plasma samples were centrifuged at 4000 rpm for 15 min. 100 μL of the supernatant was collected, and 10 μL of carbamazepine internal standard solution (0.05 mg / mL) was added. After vortexing for 30 s, 1 mL of methyl tert-butyl ether was added for extraction. The mixture was vortexed for 5 min and centrifuged at 10000 rpm for 10 min. The upper organic phase was collected in another EP tube, and the extraction was repeated once. The extract was collected and the organic phase was evaporated to dryness using a vacuum concentrator. 100 μL of methanol was added to reconstitute the extract, and the mixture was vortexed for 5 min and centrifuged at 10000 rpm for 5 min. The supernatant was collected, and the drug content was determined by HLPC. The blood drug concentration was calculated, and pharmacokinetic fitting was performed using DAS2.0 software.

[0274] 2. Experimental Results

[0275] The plasma concentration-time curves of SD rats injected with paclitaxel palmitate and doxorubicin liposome compound (PTX-PA-DOX-L) and free paclitaxel and doxorubicin mixture (Free PTX / DOX) are shown below. Figure 3 As shown in the figure, from left to right, the blood concentration-time curves of PTA-PA and DOX in PTX-PA-DOX-L, the blood concentration-time curves of PTX in PTX-PA-DOX-L and Free PTX / DOX, and the blood concentration-time curves of DOX in PTX-PA-DOX-L and Free PTX / DOX are respectively. The pharmacokinetic elimination curves of paclitaxel palmitate and doxorubicin in the compound liposomes are basically consistent in vivo and can be maintained for up to 48 hours, indicating that after encapsulation by liposomes, the two drugs can maintain a fixed ratio for a long time to achieve a better anti-tumor synergistic effect. In contrast, in the mixture of free paclitaxel and doxorubicin, the two drugs are metabolized rapidly, and the drugs are basically undetectable by HLPC after 6 hours. Moreover, due to the difference in half-lives, the ratio of the two drugs differs greatly and cannot maintain a certain synergistic effect.

[0276] The main pharmacokinetic parameters are shown in Table 10. The results show that the drug in the compound liposome has a larger AUC and a significantly longer half-life compared with the free drug mixture. This indicates that the compound liposome prolongs the in vivo action time, which is more conducive to the accumulation of the drug at the tumor site and has a certain sustained-release effect.

[0277] Table 10. Main pharmacokinetic parameters of compound liposomes and free drug mixture.

[0278]

[0279] Note a: Comparison with PTX in the free drug mixture group: * P<0.05, ** P<0.01, *** P<0.001

[0280] Note b: Comparison with DOX in the free drug mixture group: # P<0.05, ## P<0.01, ### P<0.001

[0281] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A complex liposome, characterized by, It comprises a liposome membrane, and an inner water phase enclosed inside the liposome membrane; paclitaxel palmitate is enclosed in the lipid bilayer of the liposome membrane; doxorubicin is enclosed in the inner water phase; the raw materials of the compound liposome comprise the following components: 0.1-1% paclitaxel palmitate; 0.05-0.5% doxorubicin; 1-10% phospholipid; 0.05-1% DSPE-PEG2000; 5-40% inner water phase freeze-drying protective agent; 0.5-5% salt substance; and water for injection; The above percentages all represent the percentage of the mass of each component in the total volume of all raw materials.

2. The complex liposome according to claim 1, wherein The inner water phase freeze-drying protective agent comprises one or more of sucrose, lactose, maltose, trehalose, mannitol, glucose, threonine, xylitol, sorbitol and erythritol; preferably one or more of sucrose, lactose, maltose, trehalose, mannitol and sorbitol; And / or, the amount of the inner water phase freeze-drying protective agent is 10-30%; for example, 12% or 15%.

3. The complex liposome according to claim 1, wherein The amount of paclitaxel palmitate is 0.2-0.7%, for example, 0.25%, 0.3%, 0.4%, 0.45%, 0.5%, 0.64% or 0.65%; And / or, the amount of doxorubicin is 0.1-0.4%, for example, 0.15%, 0.2%, 0.25% or 0.35%; And / or, the mass ratio of paclitaxel palmitate to doxorubicin is 1:(0.08-0.8), preferably 1:(0.16-0.8), for example, 1:0.2, 1:0.25, 1:0.3, 1:0.33, 1:0.38, 1:0.4, 1:0.44, 1:0.5, 1:0.54 or 1:0.67; And / or, the phospholipid is one or more of high-purity egg yolk lecithin, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, sphingomyelin, phosphatidylserine, dimyristoyl phosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine; preferably one or more of high-purity egg yolk lecithin, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine and sphingomyelin; And / or, the amount of phospholipid is 2-9%, for example, 3%, 3.5%, 3.6%, 5%, 6%, 7% or 8%; And / or, the amount of DSPE-PEG2000 is 0.1-0.5%, for example, 0.15% or 0.3%; And / or, the salt substance comprises one or more of ammonium sulfate, sucrose octasulfate triethylamine and copper gluconate, preferably ammonium sulfate; And / or, the amount of salt substance is 0.5-2.5%, for example, 1%, 1.2%, 1.5% or 2%.

4. The complex liposome according to claim 1, wherein The raw materials further comprise cholesterol; Preferably, the amount of cholesterol is not higher than 2%, for example, 0.2%, 0.23%, 0.68%, 0.72% or 1.5%; Preferably, the amount of cholesterol is not higher than 2%, for example, 0.2%, 0.23%, 0.68%, 0.72% or 1.5%; Preferably, the phospholipid and the cholesterol are used in a ratio of 1:(0-0.4) and not 1:0, preferably 1:(0-0.2) and not 1:0, for example 1:0.06, 1:0.07 or 1:0.

19.

5. The complex liposome according to any one of claims 1 to 4, wherein The formula of the complex liposome is as follows: 0.2-0.7% paclitaxel palmitate; 0.1-0.4% doxorubicin; 2-9% phospholipid; 0-2% cholesterol; 0.1-0.5% DSPE-PEG2000; 10-30% inner aqueous phase lyoprotectant; 0.5-2.5% salt substance; and water for injection.

6. A method for preparing the complex liposome according to any one of claims 1 to 5, characterized by, The preparation method comprises the following steps: S1, mixing an organic phase containing the paclitaxel palmitate, the phospholipid, the DSPE-PEG2000 and solvent A with an aqueous phase containing the salt substance, the inner aqueous phase lyoprotectant and solvent B to obtain a liposome crude product; S2, emulsifying, replacing, adjusting the pH of the liposome crude product obtained in step S1, adding the doxorubicin and the buffer to the liposome crude product, incubating, adding the outer aqueous phase lyoprotectant to the liposome crude product, adjusting the volume, sterilizing and lyophilizing to obtain the complex liposome; In step S2, the volume is adjusted with the water for injection.

7. The method of claim 6, wherein the liposome is a complex liposome. The raw materials of the complex liposome further comprise cholesterol, and step S1 comprises the following steps: mixing an organic phase containing the paclitaxel palmitate, the phospholipid, the cholesterol, the DSPE-PEG2000 and solvent A with an aqueous phase containing the salt substance, the inner aqueous phase lyoprotectant and solvent B to obtain a liposome crude product; And / or, in step S1, the solvent A comprises one or more of anhydrous ethanol, ethylene glycol, propylene glycol and tert-butyl alcohol, preferably propylene glycol; And / or, in step S1, the amount of the solvent A is 1-10%, preferably 3-8%, for example 5% or 6%; the percentage indicates the mass of the solvent A accounting for the percentage of the total volume of all raw materials; And / or, in step S1, the preparation of the organic phase comprises the following steps: dissolving the paclitaxel palmitate, the phospholipid and the DSPE-PEG2000 in the solvent A, heating to obtain the organic phase; or dissolving the paclitaxel palmitate, the phospholipid, the cholesterol and the DSPE-PEG2000 in the solvent A, heating to obtain the organic phase; wherein the end point temperature of the heating is preferably 25-75°C, for example 40°C, 50°C, 55°C, 60°C or 70°C; And / or, in step S1, the solvent B is water for injection; And / or, in step S1, the preparation of the aqueous phase comprises the following steps: dissolving the salt substance and the inner aqueous phase lyoprotectant in the solvent B, heating to obtain the aqueous phase; preferably, the end point temperature of the heating is 25-75°C, for example 40°C, 45°C, 55°C, 60°C, 65°C or 70°C; And / or, in step S1, the mixing step is carried out under stirring; And / or, in step S1, the mixing step is that the organic phase is injected into the aqueous phase, preferably under stirring.

8. The method of claim 6, wherein the liposome is a complex liposome. In step S2, the emulsifying step comprises homogenization and / or extrusion; wherein the homogenization is preferably performed in a high pressure homogenizer; the pressure of the homogenization is preferably 10000-22000 psi, such as 12000 psi, 15000 psi, 18000 psi or 20000 psi; the number of times of the homogenization is preferably 2-9 times; wherein the extrusion is preferably performed by an extrusion membrane; the pore size of the extrusion membrane is preferably 0.05-0.8 pm, such as 0.8 pm, 0.6 pm, 0.4 pm, 0.2 pm, 0.1 pm or 0.08 pm or 0.05 pm; the number of times of the extrusion is preferably 4-8 times; the step of the extrusion is preferably first extruded by an extrusion membrane with a large pore size, and then extruded by an extrusion membrane with a small pore size, preferably the pore size is unchanged when extruded by an extrusion membrane with a large pore size or a small pore size; and / or, in step S2, the step of replacing comprises adding water for injection to the emulsion obtained in the step of emulsifying, preferably adding equal amount of water for injection, and performing the replacement of the outer aqueous phase by any one of the following methods: ultrafiltration replacement: placing the obtained emulsion in an ultrafiltration system, and performing ultrafiltration with water for injection; dialysis replacement: placing the obtained emulsion in a dialysis bag, and performing dialysis with water for injection; glucose gel column replacement: placing the obtained emulsion in a glucose gel column, and performing column chromatography with water for injection; and / or, in step S2, the number of times of the replacement is 5 times; and / or, in step S2, the step of adjusting pH comprises adjusting with a pH adjusting agent; wherein the pH adjusting agent preferably adjusts the pH to 4.5-7.5, more preferably 5.5-7, such as 6 or 6.5; wherein the pH adjusting agent is preferably selected from one or more of sodium hydroxide, dipotassium hydrogen phosphate, phosphoric acid, disodium hydrogen phosphate, hydrochloric acid, disodium phosphate, citric acid, disodium citrate and trisodium citrate; and / or, in step S2, the buffer comprises one or more of histidine, morpholine-ethyl sulfonate, tartrate, citrate and hydroxyethylpiperazine-ethyl sulfonate; and / or, in step S2, the amount of the buffer is 0.1-0.75%, such as 0.12%, 0.13%, 0.2% or 0.3%, the percentage indicating the mass of the buffer relative to the total volume of all raw materials; and / or, in step S2, the outer aqueous phase lyophilization protectant comprises one or more of sucrose, lactose, maltose, trehalose, mannitol, glucose, threonine, xylitol, sorbitol and erythritol; preferably, the inner aqueous phase lyophilization protectant in step S2 and the outer aqueous phase lyophilization protectant in step S1 are the same; and / or, in step S2, the amount of the outer aqueous phase lyophilization protectant is 5-40%, preferably 10-30%, such as 12%, 15%, 18% or 20%; the percentage here indicates the mass of the outer aqueous phase lyophilization protectant relative to the total volume after being made up to volume; and / or, in step S2, the temperature of the incubation is 40-75°C, such as 45°C, 50°C, 55°C, 60°C, 65°C or 70°C; and / or, in step S2, the time for the incubation is 15-90 min, such as 25 min, 30 min, 35 min, 40 min, 45 min, 60 min, 65 min or 70 min; and / or, in step S2, the sterilization is performed by a 0.22 μm filter.

9. A complex liposome, characterized by comprising, which is prepared by the method of claim 6-8, and is in the form of a lyophilized powder injection; The particle size of the complex liposome is preferably 50-200 nm, more preferably 80-180 nm.

10. Use of the complex liposome of any one of claims 1-5, or claim 9, in the preparation of an anticancer drug. Preferably, the anticancer drug comprises one or more of an anti-breast cancer drug, an anti-ovarian cancer drug, an anti-thyroid cancer drug and an anti-lymphoma drug.

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