Compound liposome, freeze-dried powder injection as well as preparation method and application of freeze-dried powder injection

Abstract

The invention discloses a compound liposome, a freeze-dried powder injection as well as a preparation method and application of the freeze-dried powder injection. 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 gemcitabine or the salt thereof is encapsulated in the inner water phase; the compound liposome comprises the following raw materials: paclitaxel palmitate, lecithin, a polyethylene glycol derivative, gemcitabine or a salt thereof, an organic solvent and a buffer solution A; the molar ratio of the lecithin to the polyethylene glycol derivative is 1: (0.01-0.15). The compound liposome disclosed by the invention is relatively high in encapsulation efficiency (especially the encapsulation efficiency of PTX-PA is relatively good) and relatively high in safety, the preparation method is simple and easy to operate, the industrialization degree is high, and the prepared compound liposome has a relatively good long-circulation effect, a relatively good anti-tumor effect and good in-vivo tolerance.

Description

TECHNICAL FIELD

[0001] The application relates to a compound liposome, a freeze-dried powder injection and a preparation method and application thereof. BACKGROUND

[0002] Pancreatic cancer is one of common malignant tumors in the digestive tract. It is difficult to diagnose pancreatic cancer in the early stage, and most patients have spread to the surrounding organs when they are found. Moreover, pancreatic cancer has a very high degree of malignancy, and once it occurs, it progresses rapidly. The overall surgical resection rate is low, the postoperative recurrence rate or metastasis rate is high, and the prognosis is extremely poor. According to the Lancet, the five-year survival rate of pancreatic cancer after diagnosis is about 10%. Therefore, pancreatic cancer is known as the "King of Cancer" in the field of tumors.

[0003] Surgical resection is the most important method for curing pancreatic cancer. The 5-year survival rate of pancreatic cancer in the early stage can be close to 100% through surgical resection, but often the patients are diagnosed in the middle and late stages, and only about 20% of them can be surgically resected. For inoperable advanced pancreatic cancer or prevention of postoperative recurrence, adjuvant chemotherapy can be performed, mainly including fluorouracil drugs or combined chemotherapy regimen mainly with gemcitabine.

[0004] Paclitaxel (PTX) is a diterpenoid compound extracted from plants of the Taxaceae family. Its main mechanism of action is to promote the polymerization of tubulin protein, inhibit mitosis and proliferation of tumor cells, and kill tumor cells in the S phase. Gemcitabine hydrochloride (GEM) is a pyrimidine nucleic acid analogue. It mainly inhibits DNA synthesis and replication by embedding its metabolite dFdCTP into the DNA chain, causing DNA chain breakage, and can prevent the progression from G1 phase to S phase under certain conditions. Thus, tumor cells are killed.

[0005] In the face of the complex pathogenesis of cancer, single-drug immunotherapy and targeted therapy have been difficult to treat cancer and prolong the lives of patients. Therefore, the combined treatment of drugs has shown broad prospects. The combined use of PTX and GEM shows synergistic effect due to the different pharmacological mechanisms of the two drugs, no cross-resistance, and enhanced anti-tumor effect.

[0006] Liposomes are considered one of the most versatile and advanced nanoparticle delivery systems, and are the most common and well-studied drug carriers for FDA-approved nanomedicines. Liposomes are drug vesicles formed by the self-assembly of lipids into bilayers (monolayers) and / or multiple concentric bilayers (multilayers) with closed central water cavities, and are widely used as carriers for small molecule drugs, proteins, nucleic acids, and imaging agents. Because the structure of liposomes is extremely similar to that of cell membranes, they exhibit excellent biocompatibility and biodegradability. They can also be used for passive and / or active targeting to selectively deliver their encapsulated active ingredients to lesion sites, protecting the drug from enzymatic degradation before reaching the lesion site. This improves drug stability, prolongs drug half-life, controls drug molecule release, reduces systemic side effects, increases the maximum tolerated dose, and achieves better therapeutic effects. Currently marketed liposome products include doxil (a liposome injection of doxorubicin hydrochloride) and amphotericin B. Liposomes such as DaunoXome (daunorubicin) and Vyxeos (daunorubicin / cytarabine combination liposomes) are examples. These liposome products primarily focus on cancer treatment, offering more opportunities to further improve efficacy and reduce side effects in disease treatment.

[0007] The PTX and GEM co-loaded liposomes prepared in the literature (COSCO D, PAOLINO D, MAIUOLO J, et al. Liposomes as multicompartmental carriers for multidrug delivery in anticancer chemotherapy [J]. Drug Deliv Transl Res, 2011, 1(1): 66-75) have a particle size of about 100 nm. The ranking results of breast cancer MCF-7 cell toxicity test are co-loaded liposomes > free PTX + free GEM > free PTX or PTX liposomes > GEM liposomes > free GEM, indicating that co-loaded liposomes can better inhibit the activity of breast cancer cells and have certain anti-tumor activity, proving the feasibility and effectiveness of co-loading two drugs. However, PTX has poor lipid solubility and water solubility, and there are great difficulties in the development of co-loaded liposome preparations of two drugs. The encapsulation efficiency of PTX and GEM in the above preparations is only 78.3% and 88.9%. The PTX and GEM co-loaded thermosensitive magnetoliposomes in the literature (Ribeiro, R. F. L., Ferreira, R. V., Pedersoli, D. C. et al. Cytotoxic effect of thermosensitive magnetoliposomes loaded with gemcitabine and paclitaxel on human primary breast cancer cells (MGSO-3 line). J Nanopart Res 22, 172 (2020). https: / / doi.org / 10.1007 / s11051-020-04833-7) have an encapsulation efficiency of PTX and GEM of only 68% and 57%.Document (Shabana AM, Kambhampati SP, Hsia RC, Kannan RM, Kokkoli E. Thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the sustained co-delivery of gemcitabine and paclitaxel to pancreatic cancer cells. Int J Pharm. 2021;593: 120139. doi: 10.1016 / j.ijpharm.2020.120139) uses a film dispersion method and an ammonium sulfate gradient method to prepare co-loaded liposomes of PTX and GEM, and then wraps a layer of hydrogel on them, and the encapsulation efficiency of PTX and GEM is 80.1% and 93.9%, respectively.

[0008] In summary, the encapsulation efficiency of co-loaded liposomes of PTX and GEM in the prior art is not high, resulting in drug loss, and the presence of free drugs may have a greater safety risk. Moreover, most of the preparation methods of these co-loaded liposomes involve a film dispersion method, which is difficult to realize industrial production. Therefore, it is particularly important to develop a safe, simple and efficient PTX / GEM compound preparation.

[0009] Currently, there is no PTX / GEM compound preparation with high encapsulation efficiency, high safety and high industrialization degree. SUMMARY

[0010] The technical problem to be solved by the present application is to overcome the fact that there is no co-loaded liposome of PTX and GEM with high encapsulation efficiency (especially poor encapsulation efficiency of PTX-PA), high safety and high industrialization degree in the prior art, and to provide a compound liposome, a freeze-dried powder injection and a preparation method and application thereof. The compound liposome of the present application has high encapsulation efficiency (especially high encapsulation efficiency of PTX-PA), high safety, simple and easy-to-operate preparation method, high industrialization degree, and the prepared compound liposome has good long-circulating effect, good antitumor effect (for example, significant growth inhibition effect on pancreatic cancer), and good in vivo tolerance.

[0011] The present application solves the above technical problems by the following technical solutions:

[0012] The present application provides a compound liposome, which comprises a liposome membrane and an internal aqueous phase encapsulated in the liposome membrane; paclitaxel palmitate is encapsulated in the lipid bilayer of the liposome membrane; and gemcitabine or a salt thereof is encapsulated in the internal aqueous phase.

[0013] The raw material of the compound liposome comprises the following components:

[0014] 0.01%-1.6% paclitaxel palmitate;

[0015] 0.1%-3% lecithin;

[0016] 0.064%-1% polyethylene glycol derivative;

[0017] 0.01%-1% gemcitabine or salt thereof;

[0018] 10%-50% organic solvent; and,

[0019] the balance buffer A;

[0020] The molar ratio of the lecithin and the polyethylene glycol derivative is 1:(0.01-0.15);

[0021] The amount of the organic solvent is the percentage of the volume (mL) of the organic solvent to the total volume (mL) of the raw material of the compound liposome. The amount of the other components is the mass volume percentage of the mass (g) of each component to the total volume (mL) of the raw material of the compound liposome.

[0022] In the present application, the paclitaxel palmitate (PTX-PA) is obtained by esterification of palmitic acid with the 2' hydroxyl group of paclitaxel, which is a paclitaxel prodrug, and its structural formula is shown as formula I,

[0023]

[0024] In the present application, the paclitaxel palmitate is commercially available. Preferably, the amount of the paclitaxel palmitate is 0.02%-1.2%, for example, 0.03%, 0.04%, 0.07%, 0.16% or 0.5%, and the amount of the paclitaxel palmitate is expressed as the percentage of the mass (g) of paclitaxel palmitate to the total volume (mL) of the raw material of the compound liposome.

[0025] In the present application, the lecithin can be selected from one or more of high-purity egg yolk lecithin (EPCS), hydrogenated soybean lecithin (HSPC), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylcholine (DPPC), soybean lecithin, phosphatidylserine, dimyristoyl phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, for example, hydrogenated soybean lecithin (HSPC). Preferably, the amount of the lecithin is 0.5%-2%, for example, 1%. The amount of the lecithin is expressed as the percentage of the mass (g) of lecithin to the total volume (mL) of the raw material of the compound liposome.

[0026] In the present application, the polyethylene glycol derivative is commercially available, and is added mainly to improve the blood retention of the complex liposome. The polyethylene glycol derivative can be one or more of vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol modified distearoylphosphatidylethanolamine (DSPE-PEG), polyethylene glycol modified dimyristoylphosphatidylethanolamine (DMPE-PEG), and polyethylene glycol modified dipalmitoylphosphatidylethanolamine (DPPE-PEG), for example, DSPE-PEG. In the DSPE-PEG, the molecular weight of the polyethylene glycol can be 500-10,000 daltons, preferably 1,000-7,000 daltons, more preferably 2,000-5,000 daltons, for example, 2,000, 3,400, or 5,000 daltons. For example, when the molecular weight of the polyethylene glycol in the DSPE-PEG is 2,000, it is denoted as DSPE-PEG 2,000.

[0027] In the present application, the polyethylene glycol derivative can be used in an amount of 0.064%-0.8%, preferably 0.064%-0.6%, for example, 0.064%, 0.12%, 0.24%, or 0.48%. The amount of the polyethylene glycol derivative refers to the mass (g) of the polyethylene glycol derivative per 100 mL of the total volume of the complex liposome raw material.

[0028] In the present application, the gemcitabine or salt thereof refers to gemcitabine or a pharmaceutically acceptable salt of gemcitabine, for example, hydrochloride. In a preferred embodiment of the present application, hydrochloride of gemcitabine is used. The hydrochloride of gemcitabine is commercially available. The gemcitabine or salt thereof is preferably used in an amount of 0.02%-0.6%, for example, 0.04%, 0.05%, 0.055%, 0.06%, 0.1%, 0.2%, or 0.4%. The amount of the gemcitabine or salt thereof refers to the mass (g) of the gemcitabine or salt thereof per 100 mL of the total volume of the complex liposome raw material.

[0029] In the present application, the organic solvent is preferably one or more of propylene glycol, ethanol, and tert-butanol, and is preferably anhydrous ethanol. The organic solvent is preferably used in an amount of 15%-28%, for example, 27%. The amount of the organic solvent refers to the volume (mL) of the organic solvent per 100 mL of the total volume of the complex liposome raw material.

[0030] In the present application, the buffer A is preferably a PBS buffer.

[0031] In the present application, the raw material for forming the lipid bilayer of the liposome membrane preferably further comprises cholesterol. The amount of the cholesterol can be 0.03% to 0.5%, preferably 0.05% to 0.4%, for example, 0.06%, 0.12%, 0.18% or 0.24%, the amount of the cholesterol being expressed as the mass (g) of the cholesterol per 100 mL of the total volume of the raw material of the compound liposome.

[0032] In the present application, the molar ratio of the lecithin to the polyethylene glycol derivative is preferably 1:(0.015-0.14), for example, 1:0.018, 1:0.033, 1:0.067 or 1:0.135.

[0033] In the present application, when the compound liposome further comprises cholesterol, the molar ratio of the lecithin, the cholesterol and the polyethylene glycol derivative can be 1:(0.1-0.48):(0.01-0.15), preferably 1:(0.12-0.48):(0.015-0.14), for example, 1:0.24:0.018, 1:0.24:0.033, 1:0.24:0.067, 1:0.24:0.135, 1:0.129:0.067, 1:0.24:0.067, 1:0.369:0.067 or 1:0.479:0.067.

[0034] In a preferred embodiment of the present application, the raw material of the compound liposome comprises the following components:

[0035] 0.02%-1.2% paclitaxel palmitate;

[0036] 0.5%-2% lecithin;

[0037] 0.06%-0.8% polyethylene glycol derivative;

[0038] 0.02%-0.6% gemcitabine or a salt thereof;

[0039] 15%-28% organic solvent; and,

[0040] the balance of buffer A;

[0041] the molar ratio of the lecithin to the polyethylene glycol derivative is 1:(0.015-0.14);

[0042] The amount of the organic solvent is expressed as the volume (mL) of the organic solvent per 100 mL of the total volume of the raw material of the compound liposome, and the amount of the other components is expressed as the mass (g) of each component per 100 mL of the total volume of the raw material of the compound liposome.

[0043] In a preferred embodiment of the present application, the raw material of the compound liposome comprises the following components:

[0044] 0.02%-1.2% Paclitaxel Palmitate;

[0045] 0.5%-2% lecithin;

[0046] 0.06%-0.8% polyethylene glycol derivatives;

[0047] 0.02%-0.6% gemcitabine or its salts;

[0048] 15%-28% organic solvent;

[0049] 0.03%-0.5% cholesterol; and,

[0050] Remaining buffer A;

[0051] The molar ratio of the lecithin to the polyethylene glycol derivative is 1:(0.015-0.14);

[0052] The percentage of organic solvent usage is the percentage of organic solvent volume (mL) to the total volume (mL) of the compound liposome raw material; the amount of other components, excluding organic solvent, is the percentage of the mass (g) of each component to the total volume (mL) of the compound liposome raw material.

[0053] In this invention, when the gemcitabine or its salt is gemcitabine hydrochloride, the mass ratio of paclitaxel palmitate to gemcitabine hydrochloride is preferably 1:(1-11), for example 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2.75, 1:5.5 or 1:11.

[0054] As will be understood by those skilled in the art, the compound liposomes can be used as an injection.

[0055] In this invention, preferably, the compound liposome comprises a liposome membrane and an inner aqueous phase encapsulated within the liposome membrane; 0.08%-0.64% paclitaxel palmitate is encapsulated within the lipid bilayer of the liposome membrane; and 0.04%-0.4% gemcitabine or its salt is encapsulated in the inner aqueous phase.

[0056] The liposome membrane comprises 2%-20% lecithin and 0.054%-4.82% polyethylene glycol derivatives; the content of each component represents the percentage of the mass (g) of each component to the total volume (mL) of the compound liposomes.

[0057] Preferably, the content of paclitaxel palmitate in the compound liposome is 0.08%, 0.16%, 0.24%, 0.32%, 0.36%, 0.4%, 0.64%, or 0.7%, and the content of paclitaxel palmitate represents the percentage of the mass (g) of paclitaxel palmitate to the total volume (mL) of the compound liposome.

[0058] Preferably, the content of gemcitabine or its salt in the compound liposome is 0.04%, 0.05%, 0.055%, 0.06%, 0.1%, 0.2% or 0.4%, and the content of gemcitabine or its salt represents the percentage of the mass (g) of gemcitabine or its salt to the total volume (mL) of the compound liposome.

[0059] The content of lecithin in the compound liposome is preferably 2%, 4%, 6%, 8%, 9% or 10%, and the content of lecithin represents the percentage of the mass (g) of lecithin to the total volume (mL) of the compound liposome.

[0060] Preferably, the content of the polyethylene glycol derivative in the compound liposome is 0.472%, 0.64%, 0.944%, 1.18%, 1.416%, 1.888%, 2.124%, 2.36%, or 4.82%, and the content of the polyethylene glycol derivative represents the percentage of the mass (g) of the polyethylene glycol derivative to the total volume (mL) of the compound liposome.

[0061] In this invention, preferably, the compound liposomes also include cholesterol. The cholesterol content may be 0.1%-4%, preferably 0.2%-3.2%, for example 0.24%, 0.48%, 0.6%, 0.72%, 0.96%, 1.08%, 1.2%, 1.8% or 2.4%, and the cholesterol content represents the percentage of cholesterol mass (g) to the total volume (mL) of the compound liposomes.

[0062] In a preferred embodiment, the compound liposome comprises a liposome membrane and an inner aqueous phase encapsulated within the liposome membrane; 0.4% paclitaxel palmitate is encapsulated within the lipid bilayer of the liposome membrane; and 0.4% gemcitabine or a salt thereof is encapsulated in the inner aqueous phase.

[0063] The liposome membrane comprises 10% lecithin, 2.6% polyethylene glycol derivative and 1.2% cholesterol; the content of each component represents the percentage of the mass (g) of each component to the total volume (mL) of the compound liposome.

[0064] In a preferred embodiment, the compound liposome comprises a liposome membrane and an inner aqueous phase encapsulated within the liposome membrane; 0.64% paclitaxel palmitate is encapsulated within the lipid bilayer of the liposome membrane; and 0.4% gemcitabine or a salt thereof is encapsulated in the inner aqueous phase.

[0065] The liposome membrane comprises 8% lecithin, 1.888% polyethylene glycol derivative and 0.96% cholesterol; the content of each component represents the percentage of the mass (g) of each component to the total volume (mL) of the compound liposome.

[0066] In this invention, the average particle size of the compound liposomes can be 78.45-201.35 nm. The molecular weight distribution of the compound liposomes can be 0.01-0.27.

[0067] The present invention also provides a method for preparing the compound liposomes, which includes the following steps:

[0068] S1. The paclitaxel palmitate, the lecithin, the polyethylene glycol derivative, and the organic solvent are mixed to form an organic phase;

[0069] S2. Inject the organic phase obtained in S1 into the buffer A and concentrate it to obtain paclitaxel palmitate liposome concentrate.

[0070] S3. Mix the gemcitabine or its salt with the concentrate obtained in S2, incubate, mix with buffer B, dialyze, and filter.

[0071] When the amount of polyethylene glycol derivative is less than or equal to 0.12%, an extrusion step is performed after injecting the organic phase into buffer A in step S2 and before concentration; the amount of polyethylene glycol derivative is the mass-volume percentage of the polyethylene glycol derivative (g) to the total volume (mL) of the compound liposome raw material.

[0072] In this invention, the method for preparing the organic phase may include the following steps: mixing the paclitaxel palmitate, the lecithin, the polyethylene glycol derivative, and the organic solvent, and heating to dissolve them. The final heating temperature may be 50-70°C, for example, 65°C.

[0073] In this invention, the organic phase preferably also includes cholesterol. When cholesterol is present in the organic phase, the method for preparing the organic phase may include the following steps: mixing the paclitaxel palmitate, the lecithin, the cholesterol, the polyethylene glycol derivative, and the organic solvent, and heating to dissolve them.

[0074] In this invention, the buffer A is generally a buffer solution with a pH close to that of body fluids and with buffering effect, including one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane (Tris), citrate buffer, HEPES buffer and Good's buffer, preferably phosphate buffer.

[0075] In this invention, the pH of the buffer solution A can be 5-9, preferably 7-8, for example 7.4.

[0076] In this invention, preferably, the buffer solution A is heated before the organic phase is injected into it. The final heating temperature of the buffer solution A is generally the same as the temperature of the organic phase, so that no precipitation or sedimentation occurs after the organic phase and the aqueous phase are mixed. The temperature can be 50-70°C, for example, 65°C.

[0077] In this invention, the volume ratio of the organic phase to the buffer solution A can be 3:(6-10), for example 3:8.

[0078] In this invention, the organic phase is preferably injected into the buffer solution A under stirring conditions.

[0079] In this invention, concentration refers to the concentration of crude paclitaxel palmitate liposomes. The concentration factor is the ratio of the concentration of paclitaxel palmitate liposomes after concentration to the concentration of paclitaxel palmitate liposomes before concentration. The concentration method in this invention can be conventional in the art, such as nitrogen purging, vacuum evaporation, or ultrafiltration. The concentration factor in this invention can be 0-10, preferably 2-10, for example 4, 6, 8, or 9.

[0080] In this invention, the extrusion process conditions can be conventional extrusion conditions in the art.

[0081] In this invention, preferably, gemcitabine or its salt is added to the concentrate obtained in S2.

[0082] In this invention, the incubation temperature can be 50-70°C, for example, 65°C. The incubation time can be 10-30 minutes, for example, 20 minutes. In this invention, the incubation is preferably performed at 65°C for 20 minutes.

[0083] In this invention, after incubation and before dialysis, a cooling step is generally performed. The endpoint of the cooling can be room temperature, typically 20-30°C.

[0084] In this invention, buffer B is used to disperse the compound liposomes to form an external aqueous phase, and is generally an isotonic buffer. Buffer B can be PBS, 0.9% NaCl, or 9.4% sucrose-histidine buffer, preferably 9.4% sucrose-histidine buffer. Here, 9.4% refers to the mass percentage of sucrose in the buffer. In the 9.4% sucrose-histidine buffer, the molar concentration of histidine can be 10 mmol / L.

[0085] In this invention, the dialysis process can be conventional in the art, generally employing a tangential flow ultrafiltration device. The purpose of the dialysis is to remove unencapsulated drugs and other impurities from the external aqueous phase.

[0086] In this invention, the filtration process conditions can be conventional in the art, such as using a 0.22μm filter membrane for sterilization filtration.

[0087] The present invention also provides a compound liposome lyophilized powder injection, the raw materials of which include the raw materials of the compound liposome and a lyophilization protectant.

[0088] In this invention, the lyophilization protectant is preferably one or more selected from trehalose, maltose, sucrose, glucose, mannitol, lactose, sorbitol, xylitol, erythritol, and threonine, such as trehalose. The amount of the lyophilization protectant can be 5%-20%, preferably 10%, and the amount of the lyophilization protectant represents the percentage of the mass (g) of the lyophilization protectant to the total volume (mL) of the compound liposome lyophilized powder injection composition to be lyophilized.

[0089] The present invention also provides a method for preparing the compound liposome lyophilized powder injection, which includes the following steps:

[0090] S1. The paclitaxel palmitate, the lecithin, the polyethylene glycol derivative, and the organic solvent are mixed to form an organic phase;

[0091] S2. Inject the organic phase obtained in S1 into the buffer A and concentrate it to obtain paclitaxel palmitate liposome concentrate.

[0092] S3. Mix the gemcitabine or its salt with the concentrate obtained in S2, incubate, mix with buffer B, dialyze, and filter.

[0093] S4. Freeze-dry the compound liposome dispersion obtained in S3;

[0094] When the amount of polyethylene glycol derivative is less than or equal to 0.12%, an extrusion step is performed after injecting the organic phase into buffer A in step S2 and before concentration; the amount of polyethylene glycol derivative is the mass-volume percentage of the polyethylene glycol derivative (g) to the total volume (mL) of the compound liposome raw material.

[0095] In this invention, in step S4, preferably, the compound liposome dispersion and the lyophilization protectant are mixed before being freeze-dried.

[0096] In this invention, the freeze-drying preferably includes three steps: pre-freezing, sublimation drying, and desorption drying, with the temperatures used in the pre-freezing, sublimation drying, and desorption drying processes gradually increasing sequentially. The pre-freezing temperature can be (-40℃) to (-70℃), for example, -60℃. The pre-freezing time can be 300 to 400 minutes, for example, 360 minutes. The sublimation drying temperature can be (-20℃) to (-40℃), for example, -30℃ or -35℃. The sublimation drying time can be 500 to 1500 minutes, for example, 600 or 1200 minutes. The sublimation drying pressure can be 100 to 300 mtorr, for example, 200 mtorr. The desorption drying temperature can be 20℃ to 30℃, for example, 25℃. The desorption drying time can be 500 to 1500 minutes, for example, 600 minutes. The desorption drying pressure can be 100 to 300 mtorr, for example, 200 mtorr.

[0097] The present invention also provides the application of the compound liposome or compound liposome lyophilized powder injection in the preparation of antitumor drugs.

[0098] In this invention, the tumor may be pancreatic cancer.

[0099] 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.

[0100] The reagents and raw materials used in this invention are all commercially available.

[0101] The positive and progressive effects of this invention are as follows:

[0102] The compound liposomes of the present invention have a high encapsulation rate (the encapsulation rate of PTX-PA can reach more than 82%), high safety, simple and easy preparation method, high degree of industrialization, and the prepared compound liposomes have a good long-term circulation effect, good anti-tumor effect, and good in vivo tolerance. Attached Figure Description

[0103] Figure 1 The following is a schematic diagram of the structure of the compound liposomes prepared in the examples below.

[0104] Figure 2 Plasma pharmacokinetic data for PTX, PTX-PA, and GEM.

[0105] Figure 3The in vivo antitumor effect of the PANC-02 pancreatic cancer model in C57BL / 6 mice was evaluated.

[0106] Figure 4 The effect of H&E staining on mouse tumor and organ tissue sections. Detailed Implementation

[0107] The present invention will be further described below with reference to specific embodiments. However, the present invention is not limited to the embodiments. It will be understood that the present invention can be modified or altered in various ways by those skilled in the art. Such modifications or alterations are included in the present invention as long as they do not depart from its scope. Unless otherwise specified, the various reagents used in the embodiments are commercially available products.

[0108] The symbol “~” indicates that the range is defined by taking the values ​​before and after it as the minimum and maximum values, respectively.

[0109] In the following examples, unless otherwise specified, the particle size and PDI of the liposomes were determined using a Malvern laser particle size analyzer (Zetasizer Pro).

[0110] In the following examples, the encapsulation efficiency of paclitaxel palmitate was determined by dextran gel column chromatography, and the encapsulation efficiency of gemcitabine hydrochloride was determined by ultrafiltration centrifugation.

[0111] Regarding the tumor volume, the major diameter (a) and minor diameter (b) of the tumor were measured using vernier calipers and calculated using the following formula.

[0112] Formula: (a×b) 2 ) / 2 (where "a" is the major axis and "b" is the minor axis)

[0113] Figure 1 The following is a schematic diagram of the structure of the compound liposomes in the embodiments below.

[0114] Example 1: Effect of Concentration Factor on Paclitaxel Palmitate / Gemcitabine Hydrochloride Liposomes

[0115] 1. Experimental prescription

[0116] Table 1. Prescription design for experimental validation protocol

[0117]

[0118] Note: Concentration refers to the concentration of paclitaxel palmitate liposomes. The concentration factor is obtained by dividing the concentration of the concentrated paclitaxel palmitate liposomes by the concentration of the paclitaxel palmitate liposomes before concentration.

[0119] 2. Preparation process

[0120] Weigh the prescribed amounts of paclitaxel palmitate, hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and DSPE-PEG2000, add 30 mL of anhydrous ethanol, and heat (at 65°C) to dissolve, obtaining the oil phase; take 80 mL of neutral phosphate buffer (PBS, pH 7.4), heat to 65°C, and obtain the aqueous phase. Slowly inject the oil phase into the aqueous phase under stirring at 65°C, mix well, and obtain crude liposomes. Concentrate to obtain paclitaxel palmitate liposome concentrate; wherein the molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067;

[0121] The prescribed amount of gemcitabine hydrochloride was added to the above concentrate, incubated at 65°C for 20 min, cooled to room temperature, and dialyzed using a tangential flow ultrafiltration device with 9.4% sucrose-histidine buffer (in which the molar concentration of histidine is 10 mmol / L) as the ultrafiltration medium to remove unencapsulated drugs and other impurities. The mixture was then filtered through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0122] 3. Test Results

[0123] The particle size, PDI, and encapsulation efficiency were investigated, and the results are shown in Table 2:

[0124] Table 2. Effects of Concentration Factor on Compound Liposomes

[0125]

[0126]

[0127] 4. Results Analysis

[0128] The drug-to-liposome ratio is a crucial factor affecting liposome encapsulation efficiency, especially for highly water-soluble drugs like gemcitabine hydrochloride, which require high concentrations of phospholipids for encapsulation. The combination of HSPC and cholesterol results in highly rigid liposome membranes, making particle size reduction difficult. This invention focuses on simplified preparation, specifically employing concentration to improve encapsulation efficiency. Results show that with increasing concentration factor, the EE% of PTX-PA remains relatively unchanged, while the EE% of GEM increases significantly. This is because the increased phospholipid concentration per unit volume leads to an increased number of liposomes, resulting in a larger proportion of the aqueous phase within the liposome encapsulation area and consequently, a higher EE%. However, at an 11-fold increase, the liposomes adhere to the ultrafiltration membrane, preventing further concentration, indicating saturation of liposomes per unit area. Therefore, the optimal concentration factor for this invention is 10-fold.

[0129] Example 2: Effect of DSPE-PEG2000 on Paclitaxel Palmitate / Gemcitabine Hydrochloride Combination Liposomes

[0130] Liposome membranes are typically composed of phospholipids, or a combination of phospholipids and cholesterol. For paclitaxel palmitate / gemcitabine hydrochloride liposomes, a large amount of DSPE-PEG2000 must be added to the formulation; otherwise, stable liposomes cannot be prepared, and precipitation occurs within a very short time. The validation protocol is shown in Table 3.

[0131] 1. Experimental prescription

[0132] Table 3. Prescription Design for Experimental Validation Protocol

[0133]

[0134] 2. Preparation process

[0135] Weigh the prescribed amounts of paclitaxel palmitate, HSPC, cholesterol, and / or DSPE-PEG2000, add 30 mL of anhydrous ethanol, and heat to dissolve, obtaining the oil phase; take 80 mL of neutral phosphate buffer and heat to 65°C to obtain the aqueous phase. Slowly inject the oil phase into the aqueous phase under stirring at 65°C, mix well, and obtain crude liposomes. Concentrate to obtain paclitaxel palmitate liposome concentrate. When the concentration of DSPE-PEG2000 is less than or equal to 0.12% (Comparative formulations 2-1 to 2-2, and formulations 2-3 to 2-4 in Table 3), an extrusion step is required after obtaining the crude liposomes and before concentration.

[0136] The prescribed amount of gemcitabine hydrochloride was added to the above concentrate, incubated at 65°C for 20 min, cooled to room temperature, and dialyzed using a tangential flow ultrafiltration device with 9.4% sucrose-histidine buffer as the ultrafiltration medium to remove unencapsulated drugs and other impurities. The mixture was then filtered through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0137] 3. Test Results

[0138] The appearance, particle size, PDI, EE%, and permeability of 0.22 μm liposomes were investigated, and the results are shown in Table 4.

[0139] Table 4. Effects of DSPE-PEG2000 on compound liposomes

[0140]

[0141] 4. Results Analysis:

[0142] When the formulation does not contain DSPE-PEG2000, the prepared liposome solution becomes turbid, has a large particle size, and exhibits precipitation and stratification within a short period, making sterile filtration impossible (e.g., formulation 1). Furthermore, a large amount of DSPE-PEG2000 is required to obtain translucent, homogeneous, and sterile liposomes; otherwise, sterile filtration will also fail (e.g., formulations 2-4). Therefore, the addition of DSPE-PEG2000 directly affects liposome preparation. On the other hand, adding a large amount of DSPE-PEG2000 allows for the production of liposomes with a particle size less than 100 nm without an extrusion step, significantly reducing the process difficulty for large-scale industrial production.

[0143] Example 3: Effect of cholesterol on paclitaxel palmitate / gemcitabine hydrochloride liposomes

[0144] Cholesterol content has a certain impact on the fluidity, particle size, PDI, and EE% of liposomes. The validation protocol is shown in Table 5.

[0145] 1. Experimental prescription

[0146] Table 5. Prescription Design for Experimental Validation Protocol

[0147]

[0148]

[0149] 2. Preparation process

[0150] Weigh the prescribed amounts of paclitaxel palmitate, HSPC, cholesterol, and DSPE-PEG2000, add 30 mL of anhydrous ethanol, and heat to dissolve to obtain the oil phase; take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Slowly inject the oil phase into the aqueous phase under stirring at 65 °C, mix well, and obtain crude liposomes. Concentrate to obtain paclitaxel palmitate liposome concentrate.

[0151] The prescribed amount of gemcitabine hydrochloride was added to the above concentrate, incubated at 65°C for 20 min, cooled to room temperature, and dialyzed using a tangential flow ultrafiltration device with 9.4% sucrose-histidine buffer as the ultrafiltration medium to remove unencapsulated drugs and other impurities. The mixture was then filtered through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0152] 3. Test Results

[0153] The appearance, particle size, PDI and EE% of liposomes were investigated, and the results are shown in Table 6.

[0154] Table 6. Results of the effect of cholesterol on compound liposomes

[0155]

[0156] 4. Results Analysis:

[0157] Adding cholesterol to liposome membranes can regulate their fluidity and rigidity, further affecting liposome stability and drug release. Cholesterol promotes lipid chain stacking and bilayer formation, reduces membrane fluidity, and decreases transmembrane transport of water-soluble drugs. Furthermore, cholesterol reduces the interaction between liposomes and proteins in vivo, decreasing phospholipid loss and thus delaying drug release. Results on the effect of cholesterol content on compound liposomes show that adding appropriate amounts of cholesterol increases the drug's EE% (e.g., formulations 1-3), but excessive cholesterol leads to drug leakage (e.g., formulations 4-5). It is speculated that cholesterol competes with PTX-PA for phospholipid binding, and excessively high cholesterol content promotes drug release.

[0158] Example 4: Effect of phospholipid type on paclitaxel palmitate / gemcitabine hydrochloride liposomes

[0159] 1. Experimental prescription

[0160] The verification scheme is shown in Table 7:

[0161] Table 7. Prescription Design for Experimental Validation Protocol

[0162] Component Formulation 4-1 Formulation 4-2 Formulation 4-3 PTX-PA 0.04g 0.04g 0.04g GEM 0.44g 0.44g 0.44g Phospholipid EPCS 1.1 g HSPC 1.1 g DSPC 1.1 g Cholesterol 0.13g 0.13g 0.13g DSPE-PEG2000 0.26g 0.26g 0.26g Concentration fold 0 0 0

[0163] 2. Preparation process

[0164] Weigh the prescribed amounts of paclitaxel palmitate, HSPC, cholesterol, and DSPE-PEG2000, add 30 mL of anhydrous ethanol, and heat to dissolve, obtaining the oil phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Slowly inject the oil phase into the aqueous phase under stirring at 65 °C, mix well, and obtain crude liposomes. Concentrate to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0165] The prescribed amount of gemcitabine hydrochloride was added to the above concentrate, incubated at 65°C for 20 min, cooled to room temperature, and dialyzed using a tangential flow ultrafiltration device with 9.4% sucrose-histidine buffer as the ultrafiltration medium to remove unencapsulated drugs and other impurities. The mixture was then filtered through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0166] 3. Test Results

[0167] The appearance, particle size, PDI and EE% of liposomes were investigated, and the results are shown in Table 8.

[0168] Table 8. Effects of phospholipid types on liposomes (±s, n=3)

[0169]

[0170] 4. Results Analysis:

[0171] The type of phospholipid has little effect on the EE% of PTX-PA. Liposome GEMs prepared by EPCS have a lower EE%, while those prepared by HSPC and DSPC have relatively higher EE%, with particle size and PDI meeting liposome requirements. Compared to HSPC, liposomes prepared by DSPC have larger particle size and PDI, but DSPC is expensive, resulting in higher preparation costs; therefore, HSPC is preferred.

[0172] Example 5

[0173] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain a paclitaxel palmitate liposome mixture. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0174] Add 0.44 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0175] The particle size was measured to be 98.91 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine salt were 96.84% and 53.52%, respectively.

[0176] Example 6

[0177] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate twice to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0178] Add 0.22 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0179] The particle size was measured to be 100.27 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.98% and 65.35%, respectively.

[0180] Example 7

[0181] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 4 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0182] Add 0.11 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0183] The particle size was measured to be 98.62 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.15% and 74.98%, respectively.

[0184] Example 8

[0185] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 6 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0186] Add 0.07 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0187] The particle size was measured to be 99.63 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.34% and 75.00%, respectively.

[0188] Example 9

[0189] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 8 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0190] Add 0.06 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0191] The particle size was measured to be 98.32 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 96.94% and 82.72%, respectively.

[0192] Example 10

[0193] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 9 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0194] Add 0.05 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0195] The particle size was measured to be 98.83 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.47% and 89.25%, respectively.

[0196] Example 11

[0197] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0198] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0199] The particle size was measured to be 99.83 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.47% and 93.25%, respectively.

[0200] Example 12

[0201] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.13 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Extrude and concentrate 10 times to obtain paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.033.

[0202] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0203] The particle size was measured to be 106.28 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 96.36% and 81.50%, respectively.

[0204] Example 13

[0205] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.07 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Extrude and concentrate 10 times to obtain paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.018.

[0206] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0207] The particle size was measured to be 124.23 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 93.15% and 79.25%, respectively.

[0208] Comparative Example 14

[0209] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.03 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Inject the organic phase into the aqueous phase under stirring and mix well to obtain crude liposomes. Extrude and concentrate 10 times to obtain paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.007.

[0210] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0211] The particle size was measured to be 152.29 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 90.73% and 55.28%, respectively.

[0212] Comparative Example 15

[0213] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), and 0.13 g of cholesterol, add 30 mL of anhydrous ethanol, and heat at 65 °C to dissolve, obtaining the organic phase; take 80 mL of neutral phosphate buffer, heat to 65 °C, and obtain the aqueous phase; add the organic phase to the aqueous phase under stirring, mix well, and obtain crude liposomes, which are extruded and concentrated 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.

[0214] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0215] The particle size was measured to be 201.35 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 82.18% and 50.29%, respectively.

[0216] Example 16

[0217] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.53 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.135.

[0218] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0219] The particle size was measured to be 94.10 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 98.12% and 91.20%, respectively.

[0220] Example 17

[0221] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), and 0.26 g of DSPE-PEG2000, add 30 mL of anhydrous ethanol, and heat at 65 °C to dissolve, obtaining the organic phase; take 80 mL of neutral phosphate buffer, heat to 65 °C, and obtain the aqueous phase; add the organic phase to the aqueous phase under stirring, mix well, and obtain crude liposomes. Concentrate 10 times to obtain paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0:0.067.

[0222] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0223] The particle size was measured to be 74.42 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 90.34% and 36.29%, respectively.

[0224] Example 18

[0225] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.07 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.129:0.067.

[0226] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0227] The particle size was measured to be 81.20 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 92.49% and 87.94%, respectively.

[0228] Example 19

[0229] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.20 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.369:0.067.

[0230] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0231] The particle size was measured to be 105.39 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 93.69% and 91.42%, respectively.

[0232] Example 20

[0233] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.26 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.479:0.067.

[0234] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0235] The particle size was measured to be 120.57 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 85.22% and 83.85%, respectively.

[0236] Example 21

[0237] Weigh 0.04 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.240:0.067.

[0238] 0.04 g gemcitabine hydrochloride was added to the above liposomes, incubated at 65 °C for 20 min, cooled to room temperature, and dialyzed using a tangential flow ultrafiltration device with 9.4% sucrose-histidine buffer as the ultrafiltration medium to remove unencapsulated drugs and other impurities. The mixture was then sterile filtered through a 0.22 μm filter membrane, dispensed, lyophilized (with 10% (w / v) trehalose added during lyophilization), and capped to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposome lyophilized powder for injection. The lyophilization steps included pre-freezing (-60 °C, 360 min), sublimation drying (-30 °C, 1200 min, 200 mtorr), and desorption drying (25 °C, 600 min, 200 mtorr).

[0239] The particle size was measured to be 96.30 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 96.28% and 93.24%, respectively.

[0240] Example 22

[0241] Weigh 0.08 g of paclitaxel palmitate, 1.1 g of hydrogenated soybean phosphatidylcholine (HSPC), 0.13 g of cholesterol, and 0.26 g of DSPE-PEG2000. Add 30 mL of anhydrous ethanol and heat at 65 °C to dissolve, obtaining the organic phase. Take 80 mL of neutral phosphate buffer and heat to 65 °C to obtain the aqueous phase. Add the organic phase to the aqueous phase under stirring and mix well to obtain crude liposomes. Concentrate 10 times to obtain the paclitaxel palmitate liposome concentrate. The molar ratio of HSPC, cholesterol, and DSPE-PEG2000 is 1:0.24:0.067.

[0242] Add 0.04 g gemcitabine hydrochloride to the above liposomes, incubate at 65 °C for 20 min, cool to room temperature, use 9.4% sucrose-histidine buffer as ultrafiltration medium, and perform dialysis using a tangential flow ultrafiltration device to remove unencapsulated drugs and other impurities. Filter through a 0.22 μm filter membrane for sterilization to obtain paclitaxel palmitate / gemcitabine hydrochloride compound liposomes.

[0243] The particle size was measured to be 124.42 nm, and the encapsulation efficiencies of paclitaxel palmitate and gemcitabine were 97.71% and 91.89%, respectively.

[0244] Example 23: Pharmacokinetic Study in Rats

[0245] 1. Experimental Design

[0246] Fifteen SD rats were randomly divided into three groups: one group containing gemcitabine hydrochloride in sodium chloride solution (Free-GEM, 2 mg / mL), another group containing paclitaxel in sodium chloride solution (Free-PTX, 1.5 mg / mL; prepared by dissolving in DMSO and then adjusting to volume with 0.9% NaCl), and a third group containing PTX-PA / GEM-L (liposome preparation details are in Example 11). Each group consisted of five rats. Rats were fasted for 12 hours before the experiment but had free access to water. The drugs were administered via tail vein injection. The dosages of Free-GEM and Free-PTX were 4.79 mg / kg and 7.49 mg / kg, respectively, calculated as PTX, PTX-PA, or GEM. The dosages of PTX-PA and GEM in PTX-PA / GEM-L were 9.59 mg / kg and 4.79 mg / kg, respectively. The doses were measured at 0.083, 0.25, 0.50, and 0.75 mg / kg after administration.

[0247] Blood samples of 0.5 mL were collected from the orbital cavity at 1, 2, 4, 8, 12, and 24 hours. The dosage of PTX at 7.49 mg / kg was equivalent to the dosage of PTX-PA at 9.59 mg / kg.

[0248] 2. Plasma sample processing

[0249] The collected whole blood was quickly placed in a refrigerated centrifuge at 4°C and centrifuged at 12000 rpm for 10 min to separate the plasma. 100 μL of plasma was accurately measured, and 10 μL of internal standard solution was added. The mixture was vortexed for 1 min. 1 mL of acetonitrile was added, and the mixture was vortexed for 5 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was collected and evaporated to dryness at 40°C for 2 h at 1500 rpm. The resulting enrichment was dissolved in 100 μL of diluent, vortexed for 2 min, and the supernatant was injected for analysis.

[0250] 3. Results

[0251] Figure 2 Plasma pharmacokinetic diagrams for PTX, PTX-PA, and GEM (n=5). Figure 2 Part A of the figure shows the plasma concentration-time curves of PTX in Free-PTX and PTX-PA / GEM-L. Figure 2 Part B shows the plasma concentration-time curves of GEM in Free-GEM and PTX-PA / GEM-L. Figure 2 Part C represents the plasma concentration-time curves of GEM and PTX-PA in PTX-PA / GEM-L.

[0252] As shown in Table 7 and Figure 2 As shown, after tail vein injection of Free-GEM and Free-PTX, the blood concentrations of GEM and PTX in rats rose to a peak within 0.083 h and then rapidly decreased. In contrast, PTX-PA / GEM-L could simultaneously and slowly release GEM and PTX-PA. The half-lives of GEM and PTX in PTX-PA / GEM-L were 1.73 and 38.33 h, respectively, which were 4.81 and 23.09 times that of Free-GEM and Free-PTX.

[0253] Table 7. Pharmacokinetic parameters of PTX, PTX-PA, and GEM in rat plasma (±s, n=5)

[0254]

[0255] Note a: Compared with Free-GEM * P < 0.05 ** P < 0.01, *** P < 0.001;

[0256] Note b: Compared to Free-PTX # P < 0.05 ## P < 0.01, ### P < 0.001;

[0257] Note c: t 1 / 2βThe elimination half-life is represented by AUC(0-t); AUC(0-t) represents the area under the plasma concentration-time curve from time 0 to time t; AUC (0-∞) The area under the blood drug concentration-time curve from time 0 to ∞ is denoted as MRT(0-t); MRT(0-t) represents the average residence time of the drug in the body from time 0 to time t; MRT(0-∞) represents the average residence time of the drug in the body from time 0 to ∞; Cmax represents the highest concentration of the drug reached in the body.

[0258] 4. Analysis and Discussion

[0259] Pharmacokinetic studies showed that the drug in PTX-PA / GEM-L had a t-valence ratio of [missing information]. 1 / 2 The significantly higher levels of free drug indicate that PTX-PA / GEM-L has a better long-circulation effect, which is related to the DSPE-PEG2000 surface modification of the liposomes. On one hand, during liposome self-assembly, the DSPE end of DSPE-PEG2000 binds to the phospholipid membrane, while the hydrophilic end of the long PEG chain forms hydrogen bonds with the aqueous phase, forming a "water film" on the liposome surface. This enhances the "invisibility" of the liposomes, preventing them from rupturing due to binding with proteins and enzymes in the blood. On the other hand, because DSPE-PEG2000 interweaves and covers the liposomes like seaweed, it prevents the adsorption of antibodies and proteins, and "hides" the hydrophobic portion of the phospholipid molecules, helping them escape recognition by the reticuloendothelial system and be rapidly eliminated. This increases the stability of the liposomes in the blood system, prolongs the in vivo circulation time, and improves bioavailability.

[0260] Example 24: Pharmacodynamic study of subcutaneous transplantation of PANC-02 tumor-bearing mice

[0261] 1. Establishment of the PANC-02 pancreatic cancer model and experimental design

[0262] 1×10⁻⁶ PANC 02 murine pancreatic cancer cells 7 A mouse model of pancreatic cancer (PANC 02) was established by subcutaneous injection of [number] cells / mL into the right axilla of C57BL / 6 mice. Tumor volume reached 300–400 mm². 3Mice were randomly divided into 5 groups and administered single intravenous injections of normal saline (NS), GEM-L (16.30 mg / kg), PTX-PA-L (30.00 mg / kg), GEM-L+PTX-PA-L (a mixture of GEM-L and PTX-PA-L, with a dose of 16.30 mg / kg for GEM and 32.00 mg / kg for PTX-PA), and PTX-PA / GEM-L (liposome preparation details are in Example 11, with a dose of 16.30 mg / kg for GEM and 30.00 mg / kg for PTX-PA), respectively, every other day for a total of three times. Tumor volume (TV) and body weight changes were monitored and recorded daily during treatment. Two days after the last administration, mice were sacrificed, and tumors and organs were dissected and weighed. The tumor inhibition rate (TIR%) and relative tumor proliferation rate (T / C%) were calculated using the following formulas to evaluate the efficacy.

[0263]

[0264]

[0265]

[0266] Note: TV is calculated according to equation (1-1), where “a” and “b” represent the long and short diameters of the tumor, respectively; TIR% is calculated according to formula (1-2), where Wcontrol and Wtreat represent the average weight of the tumors in the control and treatment groups, respectively; T / C% is calculated according to formula (1-3), where RTV is the relative tumor volume, RTV = Vt / V0, where V0 is the tumor volume measured at the time of grouping, and Vt represents the tumor volume at each measurement.

[0267] 2. Hematoxylin-eosin staining (H&E)

[0268] Two days after the last administration, the mice were sacrificed, and the tumors, heart, liver, spleen, lungs, and kidneys were dissected and weighed. After being fixed in 4% paraformaldehyde solution, they were embedded in paraffin, sectioned, stained with H&E, and the microstructure of each tissue and tumor was observed under a microscope.

[0269] 3. Complete blood count analysis

[0270] Forty-eight hours after the last administration, 0.5 mL of whole blood was collected from the mice via orbital blood sampling and placed into a heparin sodium tube. A complete blood count was performed using a fully automated blood cell analyzer, and the following indicators were detected: white blood cells (WBC), neutrophils (Gran#), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT).

[0271] 4. Results of pharmacodynamic studies

[0272] Figure 3 The in vivo antitumor effect of the PANC-02 pancreatic cancer model in C57BL / 6 mice (n=5). Figure 3 Part A of the graph represents the curve showing the change in mouse tumor volume. Figure 3 Part B is the curve showing the change in mouse body weight; Figure 3 Part C represents the weight of the mouse tumor. Figure 3 Part D is an image of a mouse solid tumor; Figure 3 Compared with the GEM-L group, *P<0.05, **P<0.01, ***P<0.001; compared with the PTX-PA-L group, #P<0.05, ##P<0.01, ###P<0.001.

[0273] As shown in Table 8 and Figure 3 As shown, compared with mice receiving GEM-L and PTX-PA-L monotherapy and combination therapy (PTX-PA-L+GEM-L), the PTX-PA / GEM-L group showed the most significant tumor regression and the most significant growth inhibition effect on PANC-02 pancreatic cancer. Furthermore, from Figure 3 As shown in Part B, no mice in any group experienced weight loss, indicating that the mice tolerated the dosage well.

[0274] Table 8. Antitumor activity in C57BL / 6 mice (±s, n=5)

[0275]

[0276] Note a: Compared with the GEM-L group, *P<0.05, **P<0.01, ***P<0.001;

[0277] Note b: Compared with the PTX-PA-L group, #P < 0.05, ##P < 0.01, ###P < 0.001.

[0278] 5. H&E staining results

[0279] H&E staining of mouse tumors and organ tissues ( Figure 4 The mixed-L group (PTX-PA-L + GEM-L) showed that, compared with the saline group, there were no significant differences in the heart, liver, lungs, and kidneys of mice in each group, except that GEM-L had slight toxicity to the spleen. Numerous cancer cell necrosis and damage were observed in tumor sections of the PTX-PA / GEM-L group, further demonstrating the potent anti-tumor effect of PTX-PA / GEM-L in mice.

[0280] 6. Blood routine analysis results

[0281] The results of routine blood analysis are shown in Table 9. Compared with the negative control group, the blood cell levels of mice treated with PTX-PA / GEM-L decreased, but all were within the normal range and there was no statistical difference (P>0.05).

[0282] Table 9. Blood routine analysis (±s, n=5)

[0283]

[0284] Note: Compared to the NS group * P < 0.05 ** P < 0.01.

[0285] 7. Analysis and Discussion

[0286] In the PANC-02 pancreatic cancer model, compared with mice receiving GEM-L, PTX-PA-L monotherapy, and combination therapy (PTX-PA-L+GEM-L), the PTX-PA / GEM-L group showed the strongest tumor growth inhibition effect. The tumor volume and weight of each group were as follows: PTX-PA / GEM-L < PTX-PA-L+GEM-L < GEM-L < PTX-PA-L < NS. The tumor inhibition rate was as follows: PTX-PA / GEM-L (79.21%) > PTX-PA-L+GEM-L (68.28%) > GEM-L (56.33%) > PTX-PA-L (19.70%) > NS.

[0287] In terms of safety, no mice in any group experienced weight loss, and the dosage was well tolerated. Pathological sections of tumor tissue from the PTX-PA / GEM-L group showed extensive necrosis and damage of cancer cells, further demonstrating that PTX-PA / GEM-L effectively inhibited tumor growth. PTX and GEM had myelosuppressive effects, manifested as a decrease in WBC, Granules, and RBC levels. Blood routine analysis showed a slight decrease in blood cell count in the PTX-PA / GEM-L group compared to the saline group, but the difference was not statistically significant (P > 0.05), indicating that fatty acid modification and liposome encapsulation reduced the toxicity of PTX and GEM.

Claims

1. A compound liposome, characterized in that, It includes a liposome membrane and an inner aqueous phase encapsulated within the liposome membrane; paclitaxel palmitate is encapsulated within the lipid bilayer of the liposome membrane; gemcitabine or a salt thereof is encapsulated in the inner aqueous phase; The raw materials for the compound liposomes include the following components: 0.01%-1.6% paclitaxel palmitate; 0.1%-3% lecithin; 0.064%-1% polyethylene glycol derivatives; 0.01%-1% gemcitabine or its salt; 10%-50% organic solvent; and, Remaining buffer A; The molar ratio of the lecithin to the polyethylene glycol derivative is 1:(0.01-0.15); The amount of organic solvent is the percentage of the volume of organic solvent to the total volume of the compound liposome raw material; the amount of other components, excluding organic solvent, is the percentage of the mass of each component to the total volume of the compound liposome raw material.

2. The compound liposome as described in claim 1, characterized in that, The amount of paclitaxel palmitate used is 0.02%-1.2%, for example 0.03%, 0.04%, 0.07%, 0.16% or 0.5%; the amount of paclitaxel palmitate used indicates the percentage of the mass of paclitaxel palmitate to the total volume of the compound liposome raw material; And / or, the amount of lecithin used is 0.5%-2%, for example 1%; the amount of lecithin used represents the percentage of the mass of lecithin to the total volume of the compound liposome raw materials; And / or, the amount of the polyethylene glycol derivative is 0.064%-0.8%, preferably 0.064%-0.6%, for example 0.064%, 0.12%, 0.24% or 0.48%; the amount of the polyethylene glycol derivative represents the percentage of the mass of the polyethylene glycol derivative to the total volume of the compound liposome raw material; And / or, the amount of gemcitabine or its salt used is 0.02%-0.6%, for example 0.04%, 0.05%, 0.055%, 0.06%, 0.1%, 0.2% or 0.4%; the amount of gemcitabine or its salt used represents the percentage by mass of gemcitabine or its salt relative to the total volume of the compound liposome raw material; And / or, the amount of the organic solvent used is 15%-28%, for example 27%; the amount of the organic solvent used is the volume percentage of the organic solvent volume relative to the total volume of the compound liposome raw material.

3. The compound liposome as described in claim 1, characterized in that, The lecithin is selected from one or more of the following: high-purity egg yolk lecithin, hydrogenated soybean lecithin, distearyl phosphatidylcholine, distearyl phosphatidylglycerol, dipalmitoyl phosphatidylcholine, soybean lecithin, phosphatidylserine, myristoyl phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, such as hydrogenated soybean lecithin. And / or, the polyethylene glycol derivative is one or more of vitamin E polyethylene glycol succinate, polyethylene glycol-cholesterol, polyethylene glycol-modified distearylphosphatidylethanolamine, polyethylene glycol-modified dimyristoylphosphatidylethanolamine, and polyethylene glycol-modified dipalmitoylphosphatidylethanolamine, such as polyethylene glycol-modified dipalmitoylphosphatidylethanolamine; In the polyethylene glycol-modified dipalmitoylphosphatidylethanolamine, the molecular weight of polyethylene glycol is preferably 500-10000 Daltons, more preferably 1000-7000 Daltons, and even more preferably 2000-5000 Daltons, for example 2000, 3400 or 5000 Daltons. And / or, the gemcitabine or its salt is gemcitabine hydrochloride, such as gemcitabine hydrochloride; And / or, the organic solvent is one or more of propylene glycol, ethanol and tert-butanol, preferably anhydrous ethanol; And / or, the buffer A is a PBS buffer.

4. The compound liposome as described in claim 1, characterized in that, The molar ratio of the lecithin to the polyethylene glycol derivative is 1:(0.015-0.14), for example 1:0.018, 1:0.033, 1:0.067 or 1:0.135; Preferably, the raw materials for forming the lipid bilayer of the liposome membrane also include cholesterol; The amount of cholesterol used is preferably 0.03%-0.5%, more preferably 0.05%-0.4%, for example 0.06%, 0.12%, 0.18% or 0.24%; the amount of cholesterol used represents the percentage of the mass of cholesterol to the total volume of the compound liposome raw materials. Wherein, when the compound liposome also includes cholesterol, the molar ratio of the lecithin, the cholesterol and the polyethylene glycol derivative is preferably 1:(0.1-0.48):(0.01-0.15), more preferably 1:(0.12-0.48):(0.015-0.14), for example 1:0.24:0.018, 1:0.24:0.033, 1:0.24:0.067, 1:0.24:0.135, 1:0.129:0.067, 1:0.24:0.067, 1:0.369:0.067 or 1:0.479:0.067; And / or, when the gemcitabine or its salt is gemcitabine hydrochloride, the mass ratio of the paclitaxel palmitate to gemcitabine hydrochloride is 1:(1-11), for example 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2.75, 1:5.5 or 1:

11.

5. The compound liposome as described in claim 1, characterized in that, The average particle size of the compound liposomes is 78.45-201.35 nm; the molecular weight distribution of the compound liposomes is preferably 0.01-0.

27. Preferably, the compound liposome comprises a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane; 0.08%-0.64% paclitaxel palmitate is encapsulated within the lipid bilayer of the liposome membrane; 0.04%-0.4% gemcitabine or its salts are encapsulated in the internal aqueous phase; The liposome membrane comprises 2%-20% lecithin and 0.054%-4.82% polyethylene glycol derivatives; the content of each component represents the percentage of the mass of each component relative to the total volume of the compound liposome. The content of paclitaxel palmitate in the compound liposome is preferably 0.08%, 0.16%, 0.24%, 0.32%, 0.36%, 0.4%, 0.64%, or 0.7%, and the content of paclitaxel palmitate represents the percentage of the mass of paclitaxel palmitate in the total volume of the compound liposome. Preferably, the content of gemcitabine or its salt in the compound liposome is 0.04%, 0.05%, 0.055%, 0.06%, 0.1%, 0.2% or 0.4%, and the content of gemcitabine or its salt represents the percentage of the mass of gemcitabine or its salt in the total volume of the compound liposome. The content of lecithin in the compound liposome is preferably 2%, 4%, 6%, 8%, 9% or 10%, and the content of lecithin represents the percentage of the mass of lecithin in the total volume of the compound liposome. Preferably, the content of the polyethylene glycol derivative in the compound liposome is 0.472%, 0.64%, 0.944%, 1.18%, 1.416%, 1.888%, 2.124%, 2.36%, or 4.82%, and the content of the polyethylene glycol derivative represents the percentage of the mass of the polyethylene glycol derivative to the total volume of the compound liposome. Preferably, the compound liposomes further include cholesterol; the cholesterol content is preferably 0.1%-4%, more preferably 0.2%-3.2%, for example 0.24%, 0.48%, 0.6%, 0.72%, 0.96%, 1.08%, 1.2%, 1.8% or 2.4%, and the cholesterol content represents the percentage of cholesterol by mass in the total volume of the compound liposomes.

6. A method for preparing the compound liposomes as described in any one of claims 1-5, characterized in that, It includes the following steps: S1. The paclitaxel palmitate, the lecithin, the polyethylene glycol derivative, and the organic solvent are mixed to form an organic phase; S2. Inject the organic phase obtained in S1 into the buffer A and concentrate it to obtain paclitaxel palmitate liposome concentrate. S3. Mix the gemcitabine or its salt with the concentrate obtained in S2, incubate, mix with buffer B, dialyze, and filter. When the amount of polyethylene glycol derivative is less than or equal to 0.12%, an extrusion step is performed after injecting the organic phase into buffer A in step S2 and before concentration; the amount of polyethylene glycol derivative is the weight-volume percentage of the polyethylene glycol derivative relative to the total volume of the compound liposome raw material.

7. The method for preparing compound liposomes as described in claim 6, characterized in that, The method for preparing the organic phase includes the following steps: mixing the paclitaxel palmitate, the lecithin, the polyethylene glycol derivative, and the organic solvent, and heating to dissolve them; The organic phase preferably also includes cholesterol; when cholesterol is present in the organic phase, the method for preparing the organic phase includes the following steps: mixing the paclitaxel palmitate, the lecithin, the cholesterol, the polyethylene glycol derivative and the organic solvent, and heating to dissolve; The heating endpoint temperature is preferably 50-70°C, for example, 65°C; And / or, the buffer A comprises one or more of phosphate buffer, tris(hydroxymethyl)aminomethane, citrate buffer, HEPES buffer and Good's buffer, preferably phosphate buffer; And / or, the pH of the buffer solution A is 5-9, preferably 7-8, for example 7.4; Preferably, the buffer A is heated before the organic phase is injected into the buffer A; the final heating temperature of the buffer A is preferably 50-70°C, for example 65°C. And / or, the volume ratio of the organic phase to the buffer solution A is 3:(6-10), for example 3:8; And / or, the concentration factor is 0-10, preferably 2-10, for example 4, 6, 8 or 9; And / or, the incubation temperature is 50-70°C, for example 65°C; the incubation time is 10-30 min, for example 20 min; And / or, the buffer B is a phosphate buffer, 0.9% NaCl or 9.4% sucrose-histidine buffer, preferably 9.4% sucrose-histidine buffer; And / or, the dialysis is performed using a tangential flow ultrafiltration device; And / or, the filtration uses a 0.22 μm filter membrane for sterilization.

8. A compound liposome lyophilized powder injection, characterized in that, Its raw materials include the raw materials of compound liposomes as described in any one of claims 1-5, and lyophilization protectants; The freeze-drying protectant is preferably one or more of trehalose, maltose, sucrose, glucose, mannitol, lactose, sorbitol, xylitol, erythritol and threonine, such as trehalose; The amount of the lyophilization protectant is preferably 5%-20%, for example 10%, and the amount of the lyophilization protectant represents the percentage of the mass of the lyophilization protectant to the total volume of the compound liposome lyophilized powder injection composition to be lyophilized.

9. A method for preparing the compound liposome lyophilized powder injection as described in claim 8, characterized in that, It includes the following steps: S1, mixing the paclitaxel palmitate, the lecithin, the polyethylene glycol derivative and the organic solvent to form an organic phase; S2. Inject the organic phase obtained in S1 into the buffer A and concentrate it to obtain paclitaxel palmitate liposome concentrate. S3. Mix the gemcitabine or its salt with the concentrate obtained in S2, incubate, mix with buffer B, dialyze, and filter. S4. Freeze-dry the compound liposome dispersion obtained in S3; When the amount of polyethylene glycol derivative is less than or equal to 0.12%, an extrusion step is performed after injecting the organic phase into buffer A in step S2 and before concentration; the amount of polyethylene glycol derivative is the weight-volume percentage of the polyethylene glycol derivative relative to the total volume of the compound liposome raw material. The freeze-drying process preferably includes three steps: pre-freezing, sublimation drying, and desorption drying, wherein the temperature used in the pre-freezing, sublimation drying, and desorption drying processes gradually increases sequentially. The pre-freezing temperature is preferably (-40℃) to (-70℃), for example, -60℃; the pre-freezing time is preferably 300 to 400 minutes, for example, 360 minutes. The temperature of the sublimation drying is preferably (-20°C) to (-40°C), for example, -30°C or -35°C; the time of the sublimation drying is preferably 500 to 1500 min, for example, 600 or 1200 min; and the pressure of the sublimation drying is preferably 100 to 300 mtorr, for example, 200 mtorr. The temperature for the analytical drying is preferably 20°C to 30°C, for example, 25°C; the time for the analytical drying is preferably 500 to 1500 min, for example, 600 min; and the pressure for the analytical drying is preferably 100 to 300 mtorr, for example, 200 mtorr.

10. The use of a compound liposome as described in any one of 1-5 or a compound liposome lyophilized powder injection as described in claim 8 in the preparation of antitumor drugs; The tumor is preferably pancreatic cancer.

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