Antitumor application of pharmaceutical compositions having specific drug-lipid ratios

Abstract

Antitumor chemotherapeutic liposome with low drug-lipid ratio and its application. The antitumor chemotherapeutic liposome comprises an antitumor drug and liposome as a carrier, and the drug-lipid ratio of the antitumor drug to the lipid carrier is 0.01-0.15.

Description

[Technical field]

[0001] The present disclosure relates to the medical technology field, and in particular to the antitumor application of pharmaceutical compositions having specific drug-lipid ratios. [Background technology]

[0002] The application of liposome delivery system technology as a drug carrier was first proposed by Alec Bangham in 1961, and the technology has been slowly maturing. The widely used PEG-modified liposomes (stealth liposomes) can prolong the circulation time and accumulate drugs in tumor tissues through the EPR (passive targeting) effect, resulting in greater efficacy. Doxil® is a doxorubicin-loaded liposome and is the first liposome product to be marketed worldwide. There are also several doxorubicin-loaded liposome drugs in China, including Duomeisu and Libaosu. In addition, liposomes loaded with various antitumor drugs are on the market or close to being marketed, such as paclitaxel-loaded liposomes, daunorubicin-loaded liposomes, vincristine-loaded liposomes, irinotecan-loaded liposomes, mitoxantrone-loaded liposomes, daunorubicin and cytarabine combined-loaded liposomes, and their combinations.

[0003] When liposomes are administered, a key parameter in the formulation of these drug-loaded liposomes is the molar ratio of drug to lipid molecules, expressed as a mass ratio or molar ratio, or sometimes abbreviated as drug-lipid ratio. The higher the drug-lipid ratio, the more drug is loaded in each liposome and the higher the drug loading efficiency. The drug-lipid ratio varies due to differences in the molecular weight and physicochemical properties of the drugs. In the prior art, doxorubicin-loaded liposomes have a drug-lipid ratio of 0.17. In the prior art, irinotecan-loaded liposomes have a drug-lipid ratio of 0.6, and other drug-loaded liposomes, such as vincristine liposomes, have a drug-lipid ratio of 0.2. For liposomes containing two or more drugs, such as Vyxeos, which has a drug-lipid ratio of 0.12, the effective concentration of drug synergy may be reduced. It is generally believed that the higher the drug-lipid ratio, the higher the drug delivery efficiency and the better the drug efficacy.

[0004] However, through our research, we have found that the antitumor activity of the carrier liposome is rather decreased when the drug-lipid ratio exceeds a certain value. Therefore, it is an urgent task in the field to optimize the range of the drug-lipid ratio of the drug-loaded liposome to improve the antitumor activity of the pharmaceutical composition loaded in the liposome, so as to obtain the optimal antitumor effect with low toxicity. Summary of the Invention

[0005] The main objective of the present disclosure is to provide a novel range of drug-lipid ratios, within which pharmaceutical compositions loaded into liposomes have effective antitumor activity. In particular, the present disclosure provides a range of drug-lipid ratios that is lower than those commonly found in the art. That is, when the molar ratio is 0.01-0.15, pharmaceutical compositions loaded into liposomes having drug-lipid ratios within the range have better inhibitory activity against tumor cells than pharmaceutical compositions of the prior art.

[0006] In a first aspect of the present disclosure, a pharmaceutical composition is provided comprising: a1) an antitumor drug; and a2) a liposome as a carrier, wherein the molar ratio of the antitumor drug to the lipid carrier (drug-lipid ratio) is 0.01 to 0.15.

[0007] In another preferred embodiment, the drug lipid ratio ranges from 0.02 to 0.1, preferably from 0.022 to 0.085, such as 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or 0.09.

[0008] In another preferred embodiment, the encapsulation ratio of the drug in the pharmaceutical composition is greater than 90%, preferably greater than 95%, more preferably greater than 98%.

[0009] In another preferred example, the particle size of the pharmaceutical composition is 60 to 180 nm, preferably 80 to 120 nm, more preferably 85 nm.

[0010] In another preferred example, the PDI of the pharmaceutical composition is 0.01 to 0.2, more preferably 0.01 to 0.15.

[0011] In another preferred embodiment, the anti-tumor agent is selected from the following group: anthracyclines, platinum-based agents, fluorouracil, camptothecins, taxanes, immunomodulatory agents, or combinations thereof.

[0012] In another preferred embodiment, the antitumor drug is an anthracycline, preferably doxorubicin.

[0013] In another preferred embodiment, the antitumor drug is a camptothecin, preferably irinotecan.

[0014] In another preferred embodiment, the antitumor agent is a paclitaxel agent, preferably paclitaxel.

[0015] In another preferred embodiment, the antitumor agent is a platinum-based agent, preferably cisplatin.

[0016] In another preferred embodiment, the anti-tumor agent is an immunomodulatory agent, preferably resiquimod (R848).

[0017] In another preferred example, when the antitumor drug is doxorubicin, the drug lipid ratio range is less than 0.1, more preferably less than 0.9, more preferably 0.022 to 0.085.

[0018] In another preferred example, when the antitumor drug is irinotecan, the drug lipid ratio range is less than 0.2, preferably 0.1 to 0.2, more preferably 0.015.

[0019] In another preferred example, the lipid carrier is selected from the group of components consisting of: phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, polyethylene glycol glycerol fatty acid esters, and polyethylene glycol glycerol phosphatidylethanolamine.

[0020] In another preferred example, the lipid carrier comprises phosphatidylcholine and cholesterol.

[0021] In another preferred example, the phosphatidylcholine is selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (SPC), hydrogenated egg phosphatidylcholine, dilauryl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, dioleoyl phosphatidylcholine, or a combination thereof.

[0022] In another preferred embodiment, the structure of the lipid carrier is a sphere surrounded by a lipid molecular bilayer.

[0023] In another preferred embodiment, the lipid carrier is produced by injection.

[0024] In another preferred embodiment, the method for producing the lipid carrier comprises the steps of: (a) mixing liposome raw materials and a crosslinker with ethanol to obtain an alcohol phase solution of lipid raw materials; (b) providing an acidic aqueous phase solution having a pH of 3.5 to 6.0 (preferably 4.0 to 5.0); (c) Pouring the lipid raw material alcohol phase solution into the acidic aqueous phase solution, stirring and extruding, and carrying out dialysis to obtain the lipid carrier.

[0025] In another preferred example, the liposome raw material contains cholesterol and phosphatidylcholine, and the phosphatidylcholine is preferably the above-mentioned HSPC or SPC.

[0026] In another preferred example, the crosslinker is a PEG crosslinker, preferably having a molecular weight in the range of 1,000 to 5,000, more preferably DSPE-MPEG2000.

[0027] In another preferred example, the mass ratio of the liposome raw material to the crosslinking agent is 10-30:1, preferably 20:1.

[0028] In another preferred example, the molar ratio of phosphatidylcholine, cholesterol, and crosslinker is (30-80):(5-40):(0.1-10).

[0029] In another preferred example, in step (b), the acidic aqueous solution is selected from an aqueous solution of 1,3-propanedisulfonic acid to an aqueous solution of 1,3-propanedisulfonic acid triethylamine.

[0030] In another preferred example, the volume ratio of the lipid raw material alcohol phase solution to the acidic aqueous solution is 1:8-12.

[0031] In another preferred embodiment, in step (c), injecting comprises injecting at a fixed flow rate using a syringe or using a pump injection.

[0032] In another preferred embodiment, in step (c), the extruding comprises extruding through a polycarbonate filter membrane of fixed pore size.

[0033] In another preferred example, the lipid carrier has a targeting group conjugated thereon.

[0034] In another preferred example, targeting groups include, but are not limited to, polypeptides, proteins, and antibody fragments.

[0035] In another preferred example, the targeting group is an antibody fragment, and the antibody is a HER2 / neu antibody.

[0036] In another preferred example, the targeting group is selected from the following group: a Fab' fragment, a F(ab')2 fragment, a Fab fragment, a single chain Fv fragment (scFv), a single domain antibody (sdAb), a minibody, or a combination thereof.

[0037] In a second aspect of the present disclosure, there is provided a method of making the pharmaceutical composition of the first aspect, the method comprising the steps of (1) mixing and incubating an anti-tumor drug solution with a lipid carrier dispersion to obtain a pharmaceutical composition, or (2) adding an anti-tumor drug to a lipid feedstock alcohol phase solution or aqueous phase solution to obtain a pharmaceutical composition in one step during liposome preparation.

[0038] In another preferred example, in step (1), the incubation is carried out at 20 to 65°C, preferably 50 to 60°C.

[0039] In another preferred example, in step (1), the incubation time is from 10 minutes to 1 hour.

[0040] In another preferred example, the method further comprises the step of: mixing and incubating the obtained drug-loaded liposome with a modified targeting group to obtain a targeted pharmaceutical composition.

[0041] In another preferred example, the molar ratio of liposome raw material to crosslinker to targeting group is (30-120):(0.1-10):(0.01-0.1).

[0042] In a third aspect of the present disclosure, there is provided a use of the pharmaceutical composition according to the first aspect for the preparation of b1) an inhibitor that inhibits the proliferation of tumor cells; b2) an anti-tumor drug.

[0043] In another preferred embodiment, the pharmaceutical composition is administered parenterally.

[0044] In another preferred example, the dosage form of the pharmaceutical composition is selected from the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

[0045] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection solution.

[0046] In another preferred example, the injection solution is selected from the following: a liquid formulation, a suspension formulation, and a lyophilized powder for injection.

[0047] In another preferred example, the mode of administration of the injection is selected from the following: intravenous injection, subcutaneous injection, and in situ injection.

[0048] In another preferred embodiment, the tumor is selected from the following group: breast cancer, gastric cancer, ovarian cancer, liver cancer, melanoma, colorectal cancer, or a combination thereof.

[0049] In a fourth aspect of the present disclosure, there is provided a method of inhibiting proliferation of tumor cells in vitro, the method comprising the steps of: administering to the cells a medically effective amount of a pharmaceutical composition according to the first aspect.

[0050] In another preferred example, the cell is a human tumor cell.

[0051] In another preferred embodiment, the cells are chemotherapeutic agent-resistant human tumor cells.

[0052] In another preferred example, the cell is selected from the following: Sk-Br-3, BT-474, NCI-N87, MDA-MB-231, MDA-MB-453, MCF-7, A2780, MGC-803, HUVEC, and MCF-7 / Adr.

[0053] In a fifth aspect of the present disclosure, there is provided a method for preventing and / or treating a tumor, the method comprising the steps of: administering a medically effective amount of a pharmaceutical composition according to the first aspect to a subject in need thereof.

[0054] It should be understood that within the scope of the present disclosure, the various technical features described above and specifically described in the following sections (e.g., Examples, etc.) may be combined with each other to form novel or preferred technical solutions, which will not be listed in detail herein due to space limitations. [Brief description of the drawings]

[0055] [Figure 1] Figure 1 shows cryo-electron microscopy images of doxorubicin-loaded liposomes: (A) doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 (high drug-lipid ratio) and (B) doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 (low drug-lipid ratio). [Diagram 2] FIG. 2 shows pharmacological testing of doxorubicin-loaded liposomes with high drug-lipid ratio, low drug-lipid ratio, and antibody-targeted doxorubicin-loaded liposomes with low drug-lipid ratio on various cells (A) SK-Br-3, (B) NCI-N87, (C) BT474, (D) MDA-MB-453, (E) MCF-7, (F) A2780, (G) MGC-803, (H) HUVEC, (I) MDA-MB-231. [Diagram 3]FIG. 3 shows the inhibitory effects of doxorubicin-loaded liposomes with high drug-lipid ratio, low drug-lipid ratio, and antibody-targeted doxorubicin-loaded liposomes with low drug-lipid ratio on MCF-7 / Adr adriamycin-resistant breast cancer cells. [Figure 4] FIG. 4 shows plasma concentration curves following injection of doxorubicin-loaded liposomes with high, medium, and low drug-lipid ratios. [Diagram 5] FIG. 5 shows the changes in blood drug lipid concentration ratios following injection of doxorubicin-loaded liposomes with high, medium, and low drug lipid ratios. [Figure 6] FIG. 6 shows the effect of doxorubicin-loaded liposomes with high drug-lipid ratio, low drug-lipid ratio, and antibody-targeted doxorubicin-loaded liposomes with low drug-lipid ratio on tumor growth inhibition in a nude mouse BT474 breast cancer xenograft model. [Figure 7] FIG. 7 shows the effect of doxorubicin-loaded liposomes with high drug-lipid ratio, low drug-lipid ratio, and antibody-targeted doxorubicin-loaded liposomes with low drug-lipid ratio on body weight change in a nude mouse BT474 breast cancer xenograft model after administration. [Figure 8] FIG. 8 shows the pharmacological effect of antibody-targeted doxorubicin-loaded liposomes with a low drug-to-lipid ratio (0.043) on the HER2 / neu-expressing NCI-N87 gastric cancer xenograft model. [Figure 9] FIG. 9 shows the pharmacodynamic effect of antibody-targeted irinotecan-loaded liposomes with various drug-lipid ratios against NCI-N87 tumor cells. [Figure 10] FIG. 10 shows the in vivo pharmacological effects of R848-loaded liposomes with low drug-lipid ratio on the hHER2-MC38 colorectal cancer model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Through extensive and detailed research, the inventors of the present disclosure found that when the drug-lipid ratio of the antitumor drug and the lipid carrier composition was 0.01-0.15, the drug loading efficiency per lipid concentration unit was decreased, but the antitumor effect per drug concentration unit was good and had a strong antitumor effect in vivo and in vitro. Based on this result, the present invention was completed.

[0057] In particular, the present disclosure provides examples in which doxorubicin-loaded liposomes, irinotecan-loaded liposomes, paclitaxel-loaded liposomes, resiquimod-loaded liposomes, and cisplatin-loaded liposomes were prepared and tested. The examples of the present disclosure showed that the efficacy of drug-loaded liposomes was significantly improved when the drug-lipid ratio of doxorubicin-loaded liposomes was in the range of 0.02-0.08 (prior art drug-lipid ratio was 0.17), and in vivo pharmacokinetic data showed that liposomes with low drug-lipid ratios had extended circulation half-life and improved stability. In vivo antitumor efficacy was also improved. Irinotecan-loaded liposomes with drug-lipid ratios of 0.075 and 0.015 were both superior to liposomes with a drug-lipid ratio of 0.3. In particular, when the surface of liposomes is linked to polypeptide / protein / antibody molecules targeting tumor-associated antigens (TAA), the active targeted liposomes with low drug-lipid ratios have excellent efficacy against tumor cells with high, medium and low TAA expression, and also have excellent inhibitory effect against chemotherapy-resistant tumor cells.

[0058] term As used herein, the term "pharmaceutical composition of the present disclosure" refers to a pharmaceutical composition comprising an antitumor drug and a liposome, the composition having a drug-lipid ratio in the range of 0.01 to 0.15, preferably 0.02 to 0.1.

[0059] Pharmaceutical Compositions and Methods of Administration The present disclosure provides a pharmaceutical composition comprising: a1) an antitumor drug; and a2) a liposome as a carrier, wherein the molar ratio of the antitumor drug to the liposome carrier (drug-lipid ratio) is 0.01 to 0.15.

[0060] The anti-tumor agent in the pharmaceutical composition of the present disclosure is selected from the following group: anthracyclines, platinum-based agents, fluorouracil, camptothecins, taxanes, immunomodulatory agents, or combinations thereof.

[0061] The lipid molecules in the pharmaceutical compositions described herein are phospholipids and related amphipathic molecules commonly used in the art selected from phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, polyethylene glycol glycerol fatty acid esters, and polyethylene glycol glycerol phosphatidylethanolamine.

[0062] In another preferred embodiment, the liposome composition is a sphere surrounded by a bilayer self-assembled from lipid molecules.

[0063] In another preferred example, the liposome has a targeting group conjugated thereon.

[0064] In another preferred example, targeting groups include, but are not limited to, polypeptides, proteins, and antibody fragments.

[0065] In another preferred example, the targeting group is selected from the following group: a Fab' fragment, a F(ab')2 fragment, a Fab fragment, a single chain Fv fragment (scFv), a single domain antibody (sdAb), a minibody, or a combination thereof.

[0066] As used herein, the term "effective amount" or "effective dose" refers to an amount that can exert a function or activity (i.e., anti-tumor activity) on humans and / or animals and is tolerable to humans and / or animals.

[0067] As used herein, the term "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0068] The pharmaceutical composition of the present disclosure contains a safe and effective amount of the active ingredient of the present disclosure and a pharma- ceutical acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, and combinations thereof. In general, pharmaceutical preparations must be compatible with the mode of administration. The dosage form of the pharmaceutical composition of the present disclosure is selected from the following: tablets, capsules, granules, suspensions, pills, solutions, syrups, or injections.

[0069] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection solution.

[0070] In another preferred embodiment, the injection solution is selected from an injection solution, a suspension, and a lyophilized powder for injection.

[0071] In another preferred example, the mode of administration of the injection is selected from the following: intravenous injection, subcutaneous injection, and in situ injection.

[0072] Formulation is effected in the conventional manner using saline or aqueous solutions containing glucose or other adjuvants.The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0073] The effective amount of the active ingredient of the present disclosure may vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by those skilled in the art (e.g., by clinical trials) based on various factors. Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc., the severity of the disease of the patient being treated, the patient's body weight, the patient's immune status, the route of administration, etc. In general, satisfactory effects can be obtained when the active ingredient of the present disclosure is administered at a dose of about 0.00001 mg to 50 mg / kg of animal body weight (preferably 0.0001 mg to 10 mg / kg of animal body weight) per day. For example, based on the urgency of the treatment scenario, several individual doses may be given daily, or the dose may be proportionally reduced, as necessary.

[0074] Typically, when the pharmaceutical composition of the present disclosure is administered orally, the average daily dose for a subject having a body weight of 60 kg (human) is generally 10-500 mg, preferably 20-300 mg, more preferably 50-250 mg. The daily dose may be taken in one, two or more administrations.

[0075] Pharmaceutically acceptable carriers of the present disclosure include (but are not limited to) water, saline, nanogel, or combinations thereof. The choice of carrier should be compatible with the mode of administration, which is known to those skilled in the art.

[0076] Liposomes and drug-lipid ratio The application of liposome delivery system technology as a drug carrier was first proposed by Alec Bangham in 1961. Liposomes are small, spherical vesicles characterized by a lipid bilayer, composed of phospholipids or amphiphilic molecules, such as sterols (e.g., cholesterol), and contain an aqueous phase. Drugs can be loaded into the liposome or lipid membrane, altering the pharmacokinetics and pharmacology of the drug.

[0077] Among the definitions of these drug-loaded liposomes, an important parameter is the molar ratio of drug to lipid molecules (drug-lipid ratio), and doxorubicin-loaded liposomes in the prior art include Doxil, Duomesu, and Libaosu, which have a drug-lipid ratio of 0.17, and Myocet®, which has a drug-lipid ratio of 0.27. In addition, the drug-lipid ratio of irinotecan-loaded liposomes Onivyde™ is 0.6. In addition, the drug-lipid ratio of vincristine-loaded liposomes disclosed in (Cancer Chemother Pharmacol (2013) 71:555-564) is 0.2, and the total drug-lipid ratio of Vyxeos-loaded liposomes disclosed in (International Journal of Pharmaceutics 391 (2010) 248-259) is 0.12, and the drug-lipid ratio of cisplatin-loaded liposomes is 0.2.

[0078] Vincristine-loaded liposomal formulations with various drug-lipid ratios were evaluated in (Biochimica et Biophysica Acta 1758(2006)55-64) and the in vivo release half-life T1 / 2 of vincristine from egg sphingomyelin / cholesterol-loaded liposomes was found to vary with different drug-lipid ratios. The half-life was 6.1 hours when the drug-lipid ratio (D / L) was 0.025 (wt / wt), 15.6 hours when the drug-lipid ratio (D / L) was 0.1 (wt / wt), and 117 hours when the drug-lipid ratio (D / L) was 0.6 (wt / wt). This indicates that the half-life is extended with increasing drug-lipid ratio. US Patent No. 5,616,341 (High drug: lipid formulations of liposomal antineoplastic agents) discloses the preparation and application of doxorubicin-loaded liposomes and vincristine-loaded liposomes with high drug-lipid ratios.

[0079] Key advantages of the present disclosure include: (a) The antitumor activity against tumor cells of the liposomes having a low drug-lipid ratio disclosed in the present disclosure is greater than the antitumor activity of liposomes having a high drug-lipid ratio of the prior art. (b) At the same dose, liposomes with a lower drug-lipid ratio will remain in the body for a longer time and release the drug more slowly. (c) Liposomes with a lower drug-lipid ratio have better in vivo antitumor efficacy.

[0080] The present disclosure will be further described below with specific examples.It should be understood that these examples are provided only for the purpose of illustrating the present disclosure, and are not intended to limit the scope of the present disclosure.In the following examples, the experimental method without specific conditions is typically carried out under conventional conditions or as recommended by the manufacturer.Unless otherwise stated, percentage percentage and weight fraction are weight percentage and weight part.

[0081] Example 1: Preparation of liposomes encapsulating doxorubicin (adriamycin) 900mg HSPC, 300mg cholesterol, and 60mg DSPE-MPEG2000 were weighed, 2mL absolute ethanol was added, and heated to 60℃ in a water bath. A magnetic stir bar was then added and stirred until completely dissolved, resulting in a clear liquid mixture. At the same time, 3.304g ammonium sulfate was placed in a glass bottle, 100mL ultrapure water was added, stirred until dissolved, and then the pH was adjusted to 5.0 to obtain an ammonium sulfate solution with a concentration of 250mM. This solution was prepared immediately before use. The dissolved lipid mixture was injected into 19mL of ammonium sulfate aqueous solution using a syringe, then stirred, and the mixture was extruded six times to perform dialysis using a 10kD dialysis membrane in HEPES buffer (10mM, pH 6.8) to obtain empty liposomes with an external aqueous phase of HEPES solution.

[0082] In addition, 10mg / mL of adriamycin solution was added to the above empty liposome suspension according to the drug-lipid ratio set above. The total volume was adjusted to 10mL, and then incubated at 60℃ for 30min with stirring. After drug loading was completed, a 100kD dialysis membrane was used to remove the free drug, and the contents of adriamycin, HSPC, cholesterol, and DSPE-MPEG2000 were detected to confirm the molar ratio of drug molecules to total lipid molecules in adriamycin-loaded liposomes.

[0083] A Nano-S90 nanoparticle size analyzer was used for detection. The particle size of doxorubicin-loaded liposomes with various drug-lipid ratios was about 85 nm, and the polydispersity index (PDI) was less than 0.1. The cryo-electron microscopy morphology of doxorubicin-loaded liposome formulations with a drug-lipid ratio of 0.17 and doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 are shown in Figure 1A and Figure 1B. The liposomes are almost circular, with uniform size and distribution. The doxorubicin crystals in doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 were finer than those in plasma doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17.

[0084] Example 2 Efficacy studies on doxorubicin-loaded liposomes with various drug-lipid ratios In this example, the cytotoxic effects of doxorubicin-loaded liposomes with various drug-lipid ratios prepared in Example 1 on tumor cells were evaluated.

[0085] First, efficacy tests were performed on the tumor cell lines listed in Table 1, including SK-Br-3, NCI-N87, BT474, MDA-MB-453, MCF-7, MGC-803, A2780, and MDA-MB-231, and the normal cell line HUVEC. [Table 1]

[0086] The cytotoxic effects of doxorubicin-loaded liposomes with various drug-lipid ratios on tumor cells were evaluated using Promega's CellTiter-Glo® Luminescent Cell Viability Assay kit. Graph Prism 8.0 analysis software was used to fit the cytotoxicity curves and calculate the respective IC50 values. See Figure 2 and Table 2 for the results.

[0087] Experimental results showed that doxorubicin-loaded liposomes with drug-lipid ratios of 0.022, 0.043, and 0.085 were more cytotoxic than doxorubicin-loaded liposomes with a higher drug-lipid ratio (0.17). [Table 2]

[0088] Furthermore, to more fully verify the cytotoxic effects of doxorubicin-loaded liposomes with different drug-lipid ratios on different cell lines, we set up more experimental groups with different drug-lipid ratios and tested their IC50 values ​​on different cell lines.

[0089] The test results are shown in Table 3. The results show that the lower the drug-lipid ratio of the liposome, the better the cytotoxicity effect, especially the liposomes with a drug-lipid ratio of less than 0.1. [Table 3]

[0090] Example 3 Preparation of HER2 / neu antibody-targeted doxorubicin-loaded liposomes and performance in cells. 3.1 Preparation of HER2 / neu antibody-targeted doxorubicin-loaded liposomes HER2 / neu antibody-targeted doxorubicin-loaded liposomes were fabricated. The lipid composition was HSPC, cholesterol (Chol), 1,2-distearoyl-rac-glycero-3-PE-N-polyethylene glycol-2000 (DSPE-MPEG2000), and HER / neu antibody fragment conjugated lipid molecules. The molar ratio range of each component was (30–80):(5–40):(0.1–10):(0.01–0.1). The HER2 / neu antibody fragment conjugated lipid was 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]-HER2 / neu antibody fragment (HER2-DSPE-PEG2000). The HER2 / neu antibody fragment was reacted with the lipid molecule DSPE-PEG2000-mal containing a maleimide group via the free sulfhydryl group to form a thioether bond. The HER2-DSPE-PEG2000 was then incubated with the doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 prepared in Example 1 to obtain specific HER2 / neu antibody-targeted drug-loaded liposomes.

[0091] 3.2 Performance of HER2 / neu antibody-targeted doxorubicin-loaded liposomes in tumor cells In this example, the cytotoxic effects of HER2 / neu antibody-targeted doxorubicin-loaded liposomes prepared in Example 3.1 against tumor cells with different levels of HER2 / neu expression were evaluated.

[0092] Tumor cell lines: Tumor cell lines included SK-Br-3, NCI-N87, BT474, MDA-MB-453, MCF-7, MGC-803, A2780, MDA-MB-231, and normal cell line was HUVEC, with specific cell HER2 / neu expression levels shown in Table 4. [Table 4]

[0093] The cytotoxic effect of HER2 / neu antibody-targeted doxorubicin-loaded liposomes on tumor cells was evaluated using Promega's CellTiter-Glo® Luminescent Cell Viability Assay kit. Trastuzumab monoclonal antibody (Trastuzumab) was set as the control. At the same time, non-targeted doxorubicin-loaded liposomes with drug-lipid ratios of 0.043 and 0.17 were compared. Graph Prism 8.0 analysis software was used to fit the cytotoxicity curves and calculate the respective IC50 values. See Figure 5 for the results.

[0094] The results showed that the lower the drug-lipid ratio of HER2 / neu antibody-targeted doxorubicin-loaded liposomes, the better their cytotoxicity effects on tumor cells and targeted HER2 / neu-positive tumors. The cytotoxic activity of HER2 / neu antibody-targeted doxorubicin-loaded liposomes in HER2 / neu-overexpressing tumor cells was 3-10-fold higher than that of doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 and more than 10-fold higher than that of doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17. [Table 5]

[0095] 3.3 Performance of HER2 / neu antibody-targeted doxorubicin-loaded liposomes in adriamycin-resistant tumor cell lines For specific implementation procedures, see 3.2. To investigate the performance of HER2 / neu antibody-targeted doxorubicin-loaded liposomes in adriamycin-resistant tumor cell lines, MCF-7 / Adr adriamycin-resistant breast cancer lines were used in the study. Cytotoxicity results showed that the IC50 values ​​of HER2 / neu antibody-targeted doxorubicin-loaded liposomes with drug-lipid ratios of 0.043 and 0.17, doxorubicin-loaded liposomes, and trastuzumab monoclonal antibody (trastuzumab) in MCF-7 / Adr were 13.70 μg / mL, 46.41 μg / mL, and 101.30 μg / mL, respectively (shown in FIG. 3). This indicated that HER2 / neu antibody-targeted doxorubicin-loaded liposomes further had a strong cytotoxic effect on adriamycin-resistant cell lines. Thus, it has potential clinical efficacy in treating patients with adriamycin-resistant tumors.

[0096] Example 4 Pharmacokinetic performance of doxorubicin-loaded liposomes with various drug-lipid ratios The in vivo pharmacokinetic profile of doxorubicin-loaded liposomes with various drug-lipid ratios was examined in a tumor-bearing mouse model. Trace amounts of deuterated cholesterol molecular marker were added to the liposomes to detect changes in external lipid concentration.

[0097] Construction of BT474 breast cancer xenograft model in Balb / C nude mice: Female, BMS grade Balb / C-nude mice, aged 4-5 weeks and weighing approximately 18g, were purchased and acclimatized to live in the animal breeding room for approximately one week. BT474 cells were grown to the exponential growth phase, and the cells were digested with trypsin. The cells were collected by centrifugation and washed two to three times with PBS buffer. The cells were resuspended in PBS buffer, counted, and the cell density was adjusted to 1×10^8 cells / mL. Matrigel was previously removed from the -20°C freezer, thawed on ice, and mixed with equal volumes of Matrigel and cell suspension at 1:1. The corresponding volume of cell suspension was added to 5×10 6 ~1×10 7The cell volume was pipetted into a 1 mL syringe, and the cell suspension was inoculated subcutaneously into the right axilla of nude mice. After inoculation, the injection site was compressed for 30 seconds to avoid reflux of the cell solution. After inoculation, a prominent subcutaneous papule was observed. The inoculated mice were observed regularly to monitor tumor growth.

[0098] Dosing: The first group was administered doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 (high drug-lipid ratio) based on the weight of the mouse via tail vein injection. The drug dose was 5mg / kg. The second group was administered doxorubicin-loaded liposomes with a drug-lipid ratio of 0.085 (medium drug-lipid ratio) based on the weight of the mouse via tail vein injection. The drug dose was 5mg / kg. The third group was administered doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 (low drug-lipid ratio) based on the weight of the mouse via tail vein injection. The drug dose was 5mg / kg.

[0099] Blood collection: Blood was collected from four different groups receiving doxorubicin-loaded liposomes with various drug-lipid ratios at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 hours after administration. Blood was collected in EDTA anticoagulant tubes and centrifuged at 3,000 rpm for 30 minutes at 4°C on ice. The top layer of plasma was carefully pipetted into a freshly labeled centrifuge tube and subsequently tested or stored in a -80°C freezer.

[0100] Plasma sample pretreatment: 40 μL of plasma sample was accurately measured into a centrifuge tube. 160 μL of acetonitrile-methanol (1:1, v / v) was added according to a ratio of 1:4 between plasma and organic reagent, vortexed for 1-2 min to completely precipitate proteins in plasma, and centrifuged at 1,000 x g for 10 min at 4 °C. The supernatant was carefully pipetted into a new centrifuge tube, while taking care not to pipette the protein layer in this step. Centrifuged at 1,000 x g for 10 min at 4 °C. The solution was filtered using a 0.22 μm organic filter head, and 50 uL was placed into a sample vial for loading and testing.

[0101] Test: Conditions for high performance liquid chromatography (refer to the Chinese Pharmacopoeia): An Athena C18 column (120 Å, 4.6 × 150 mm, 5 μm) was used, and octadecylsilane chemically bonded to porous silica was used as the packing material. Sodium dodecyl sulfate solution (1.44 g of sodium dodecyl sulfate and 0.68 mL of phosphoric acid were taken and dissolved by adding 500 mL of water)-acetonitrile-methanol (500:500:60) was used as the mobile phase. The detection wavelength was 254 nm, the detection flow rate was 1.0 mL / min, and the loading volume was 10 μL.

[0102] Analysis: The plasma concentration results of liposomes with various drug lipid ratios in rats are shown in Figure 4. The changes in the ratio of drug concentration to deuterated cholesterol concentration in blood samples are shown in Figure 5. The changes in the ratio of drug concentration to deuterated cholesterol concentration in blood were used to demonstrate the drug release process from liposomes. It was observed that liposomes with various drug lipid ratios had different in vivo release processes. Liposomes with low drug lipid ratios also released slower, which provides good sustained release.

[0103] Example 5 In vivo pharmacological study of doxorubicin-loaded liposomes with various drug-lipid ratios 5.1. Construction of xenograft model Construction of BT474 breast cancer xenograft model in Balb / C nude mice: Female, BMS grade Balb / C-nude mice, aged 4-5 weeks and weighing approximately 18g, were purchased and acclimatized to live in the animal breeding room for approximately one week. BT474 cells were grown to the exponential growth phase, and the cells were digested with trypsin. The cells were collected by centrifugation and washed two to three times with PBS buffer. The cells were resuspended in PBS buffer, counted, and the cell density was adjusted to 1×10^8 cells / mL. Matrigel was previously removed from the -20°C freezer, thawed on ice, and mixed with equal volumes of Matrigel and cell suspension at 1:1. The corresponding volume of cell suspension was added to 5×10 6 ~1×10 7 The cell volume was pipetted into a 1 mL syringe, and the cell suspension was inoculated subcutaneously into the right axilla of nude mice. After inoculation, the injection site was compressed for 30 seconds to avoid reflux of the cell solution. After inoculation, a prominent subcutaneous papule was observed. The inoculated mice were observed regularly to monitor tumor growth.

[0104] 5.2 Dosage and Efficacy Assessment: Tumor size: 100-200mm 3 When the mice reached the target age, they were randomly divided into three groups, with at least six mice in each group: control, doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17, doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043, and HER2 / neu antibody-targeted doxorubicin liposomes with a drug-lipid ratio of 0.043.

[0105] After grouping, the control group received empty liposomes via tail vein injection. The doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 group received doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 via tail vein injection at a dose of 2 mg / kg based on the weight of the nude mice. The doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 group received doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 via tail vein injection at a dose of 2 mg / kg based on the weight of the nude mice. The HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 group also received HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 via tail vein injection at a dose of 2 mg / kg based on the weight of the nude mice. The drugs were administered once a week for a total of five times. Mouse body weight and tumor growth curves were recorded every other day, and the mice were euthanized after the end of treatment.

[0106] The inhibitory effects of the test substances on breast cancer BT474 xenograft model in mice were primarily tested.

[0107] Measurement of tumor volume and body weight of tumor-bearing mice: Tumor volume was measured every other day using a caliper, and tumor volume was calculated according to the formula V = 0.5a × b 2 The calculation was performed using the formula: where a and b represent the longest diameter and width of the tumor, respectively.

[0108] Tumor growth inhibition rate TGI (%) = [1-(D i -D0) / (C i -C0)]×100, where D i -D0>0. D0 is the average tumor volume at the time of the first administration in the treatment group (drug). D i is the mean tumor volume at a particular measurement time after the start of dosing in the treatment group. C0 is the mean tumor volume at the time of the first dose in the control group (control). C i is the mean tumor volume at a particular measurement time after the start of dosing in the control group.

[0109] The results of in vivo pharmacological tests on BT474 breast cancer xenograft models in Balb / C nude mice are shown in Figures 6 and 7. As shown in Figure 6, the tumor growth inhibition rates (TGI%) of doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17, doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043, and HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 on the 18th day after administration were 50.55%, 60.12%, and 69.66%, respectively. From these results, doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 had a strong tumor growth inhibition effect, and HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 had an even better tumor growth inhibition effect. As shown in FIG. 7, Balb / C-nude mice underwent weight loss following treatment but did not exhibit significant toxicity (weight loss <10%) and mouse weights returned to normal at the end of treatment.

[0110] Example 6: In vivo animal pharmacology studies of HER2 / neu antibody-targeted doxorubicin-loaded liposomes with low drug-lipid ratios 6.1.Establishment of NCI-N87 breast cancer xenograft model in Balb / C nude mice The NCI-N87 gastric cancer cell line, which overexpresses HER2 on the cell surface, was used to establish a xenograft tumor model in Balb / C-nude mice.

[0111] Female, BMS grade Balb / C-nude mice, aged 4-5 weeks and weighing approximately 18g, were purchased and accustomed to breeding in an animal breeding room for approximately one week. NCI-N87 cells were grown to the exponential growth phase, and the cells were digested with trypsin. The cells were collected by centrifugation and washed two to three times with PBS buffer. The cells were resuspended in PBS buffer, counted, and the cell density was adjusted to 2×10^8 cells / mL. Matrigel was previously removed from the -20°C freezer, thawed on ice, and mixed with the matrigel and cell suspension in equal volumes of 1:1. The corresponding volume of cell suspension was pipetted into a 1-mL syringe based on the 1*10^7 cell volume, and the cell suspension was inoculated subcutaneously into the right axilla of nude mice. After inoculation, the injection site was compressed for 30 seconds to avoid reflux of the cell solution. After inoculation, a noticeable subcutaneous papule was observed. Inoculated mice were observed regularly to monitor tumor growth.

[0112] 6.2. Dosage and Efficacy Assessment Tumor size: 100-200mm 3 When the mice reached 100% CI, they were randomly divided into three groups, with at least six mice in each group: a control group, a doxorubicin-loaded liposome group with a drug-lipid ratio of 0.17, and a HER2 / neu antibody-targeted doxorubicin liposome group with a drug-lipid ratio of 0.043.

[0113] After grouping, the control group was given empty liposomes via tail vein injection. The doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 group was administered with doxorubicin-loaded liposomes with a drug-lipid ratio of 0.17 via tail vein injection at a dose of 2 mg / kg based on the weight of the nude mice. The HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 group was also administered with HER2 / neu antibody-targeted doxorubicin-loaded liposomes with a drug-lipid ratio of 0.043 via tail vein injection at a dose of 0.5, 2, 5 or 10 mg / kg (mpk) based on the weight of the nude mice. At the same time, trastuzumab monoclonal antibody was set as a control and administered at 5 mg / kg once a week (qw) for a total of five times. The mouse weight and tumor growth curves were recorded every other day, and the mice were euthanized after the end of administration.

[0114] The inhibitory effects and complete cure ability of the test substances on NCI-N87 gastric cancer xenograft model in mice were mainly tested. (1) Tumor volume measurement: Tumor volume was measured every other day using a caliper. The tumor volume was calculated according to the formula V = 0.5a × b 2 The calculation was performed using the formula: where a and b represent the longest diameter and width of the tumor, respectively. (2) Tumor growth inhibition rate TGI (%) = [1-(D i -D0) / (C i -C0)]×100, where D i -D0>0. D0 is the average tumor volume at the time of the first administration in the treatment group (drug). D i is the mean tumor volume at a particular measurement time after the start of dosing in the treatment group. C0 is the mean tumor volume at the time of the first dose in the control group (control). C i is the mean tumor volume at a particular measurement time after the start of dosing in the control group. (3) The body weight of all tumor-bearing mice was measured every other day. At the same time, the change rate of body weight in mice after administration was also calculated: RCBW(%)=(BW i -BW0) / BW0×100. BW i is the average body weight at a particular measurement after the start of dosing, and BW0 is the average body weight at the time of the first dosing.

[0115] The results of in vivo pharmacology studies on NCI-N87 gastric cancer xenograft models in Balb / C nude mice are shown in Figure 8. The tumor growth curves (Figure 8) show that antibody-targeted doxorubicin-loaded liposomes with a low drug-lipid ratio (0.043) had excellent in vivo tumor growth inhibition effects on HER2 / neu-overexpressing NCI-N87 gastric cancer xenograft models, which were dose-dependent.

[0116] Example 7 Preparation of irinotecan-loaded liposomes with various drug-lipid ratios HSPC, cholesterol, and DSPE-MPEG2000 were accurately weighed and added to a glass flat-bottom flask, and the appropriate amount of absolute ethanol was added and heated in a water bath to completely dissolve the lipids. 30 mL of 500 mM 1,3-propanedisulfonic acid triethylamine was added as a hydration medium and stirred at 65°C for 45 min to allow the hydration reaction. Uniform small unilamellar vesicles were formed by successive extrusion through 100 nm and 80 nm polycarbonate filter membranes.

[0117] A 10 mM HEPES buffer, pH 7.0, was used as the dialysis medium to form a triethylamine propanedisulfonate gradient in the inner and outer aqueous phases of the liposomes. Irinotecan solutions with a concentration of 10 mg / mL were prepared and loaded with drug according to drug-lipid ratios of 0.01, 0.02, 0.04, 0.075, 0.15, and 0.3, heated and incubated in a water bath at 60° C. for 45 minutes. Then, 10 mM HEPES buffer, pH 7.0, was used as the dialysis medium to remove the free drug in the outer aqueous phase, resulting in irinotecan-loaded liposomes with various specific drug-lipid ratios of 0.01, 0.02, 0.04, 0.075, 0.15, and 0.3.

[0118] The physical parameters of irinotecan-loaded liposomes with drug-lipid ratios of 0.075, 0.15, and 0.3 are shown in Table 6. The particle sizes were similar, the PDI distributions were all less than 0.1, and the encapsulation rates were all greater than 95%. [Table 6]

[0119] The cytotoxicity of irinotecan-loaded liposomes with different drug-lipid ratios was tested in different cell models using the method according to Example 2, and the results obtained are shown in Table 7. The results showed that irinotecan-loaded liposomes with a drug-lipid ratio of 0.15 had the best cytotoxicity. [Table 7]

[0120] Example 8: Cytotoxicity testing of antibody-targeted irinotecan-loaded liposomes with various drug-lipid ratios This example uses NCI-N87 tumor cell line purchased from the Cell Bank of the Chinese Academy of Sciences. Antibody-targeted irinotecan-loaded liposomes with various drug-lipid ratios were prepared and evaluated for their cytotoxicity effects on NCI-N87. The test method was the same as that in Example 2, that is, CellTiter-Glo® Luminescent Cell Viability method was used. The results, as shown in Figure 9, show that antibody-targeted irinotecan-loaded liposomes with a drug-lipid ratio of 0.15 had excellent cytotoxicity to NCI-N87 cells.

[0121] Example 9 Preparation of R848-loaded liposomes with low drug-lipid ratio and in vivo animal pharmacological testing 9.1 Preparation of R848-loaded liposomes with low drug-lipid ratio 285.7 mg of HSPC, 95 mg of cholesterol, and 19 mg of DSPE-MPEG2000 were weighed, 1 mL of absolute ethanol was added, and heated to 60°C in a water bath. A magnetic stir bar was then added and stirred until completely dissolved, resulting in a clear liquid mixture. At the same time, 6.607 g of ammonium sulfate was placed in a glass bottle, 100 mL of ultrapure water was added, stirred until dissolved, and then the pH was adjusted to 5.0 to obtain an ammonium sulfate solution with a concentration of 500 mM. This solution was prepared immediately before use. The dissolved lipid mixture was injected into 9 mL of ammonium sulfate aqueous solution using a syringe, then stirred at 60°C for 45 minutes to hydrate, extruded six times using 100 nm and 80 nm polycarbonate filter membranes, and then dialyzed using a 10 kD dialysis membrane in HEPES buffer (10 mM, 0.9% NaCl, pH 5.5) to obtain empty liposomes.

[0122] Additionally, 6.25 mL of 1 mg / mL R848 solution was taken and added to 3.75 mL of empty liposomes with lipid concentration of 40 mg / mL to obtain a total volume of 10 mL. After stirring at room temperature for 30 minutes to load the drug, a 100 kD dialysis bag was used to remove the free drug to produce R848-loaded liposomes (R848-Lipo) with a drug-lipid ratio of 0.09. The particle size was 74.89 nm and the PDI was less than 0.026.

[0123] 9.2 Establishment of B-hHER2 MC38 colon cancer model using B-hCD3E mice and in vivo pharmacological testing of drugs 1 × 10 B-hHER2 MC38 cells resuspended in PBS 6 B-hCD3E mice were subcutaneously inoculated at 0.1 mL / mouse into the right side of the back. The average tumor volume was approximately 80–150 mm. 3When the tumor volume reached 100%, appropriate mice were selected and enrolled according to the tumor volume and mouse weight, and randomly assigned to two experimental groups, a control group and an experimental group, with six mice in each group. After grouping, the control group was administered PBS via tail vein injection, and the experimental group was administered R848-loaded liposomes with a drug-lipid ratio of 0.09 at a dose of 2 mg / kg based on the mouse's body weight via tail vein injection. The administration was performed once every three days, a total of seven times.

[0124] After grouping, tumor volumes were measured twice a week using calipers to measure the long and short diameters of the tumors. The formula for volume calculation was tumor volume = 0.5 x long diameter x short diameter. 2 It was.

[0125] The results of in vivo pharmacodynamic testing of the B-hHER2 MC38 colon cancer model in B-hCD3E mice are shown in Figure 10. The tumor growth curves showed that R848 liposomes had a significant inhibitory effect on the growth of B-hHER2 MC38 colon cancer in vivo.

[0126] Example 10 Preparation and testing of cisplatin-loaded liposomes with various drug-lipid ratios HSPC, cholesterol, and DSPE-MPEG2000 were accurately weighed and added into a glass flat-bottom flask, and an appropriate amount of absolute ethanol was added and heated in a water bath to completely dissolve the lipids. The solution was injected into 1 mg / mL, 2 mg / mL, and 5 mg / mL cisplatin solutions at 50°C with a flow rate of 30 mL / min, and the mixed solution was extruded using 100 nm and 80 nm polycarbonate filter membranes, and the unencapsulated drug was removed by dialysis to obtain cisplatin-loaded liposomes with a particle size of approximately 80 nm and drug-lipid ratios of 0.01, 0.05, and 0.08.

[0127] All documents mentioned in this disclosure are incorporated by reference in this application to the same extent as if each portion of the document was individually incorporated by reference. Also, after reading the above description of the contents of this disclosure, a person skilled in the art will understand that various changes or modifications can be made to the present disclosure, and such equivalents are also within the same scope as defined by the claims appended to this application.

Claims

1. a1) an antitumor agent, and a2) lipid-containing liposomes as carriers for antitumor drugs, A pharmaceutical composition, wherein the molar ratio of the antitumor drug to the liposome carrier (drug-lipid ratio) is in the range of 0.01 to 0.

15.

2. 2. The pharmaceutical composition of claim 1, wherein the anti-tumor agent is selected from the group of anthracyclines, platinum-based agents, fluorouracil, camptothecin, paclitaxel, or combinations thereof.

3. The pharmaceutical composition of claim 2, wherein the antitumor drug is doxorubicin, irinotecan, cisplatin or resiquimod.

4. The antitumor drug is doxorubicin and the drug lipid ratio is less than 0.1; or the antitumor drug is irinotecan and the drug lipid ratio is less than 0.2, preferably 0.075 or 0.15; or the anti-tumor drug is cisplatin and the drug lipid ratio is 0.01, 0.05 or 0.09; or the antitumor drug is resiquimod and the drug lipid ratio is 0.09; The pharmaceutical composition according to claim 3.

5. The pharmaceutical composition of claim 4, wherein the antitumor drug is doxorubicin and the drug-lipid ratio is in the range of 0.022 to 0.085, preferably 0.

43.

6. The pharmaceutical composition of claim 4, wherein the antitumor drug is irinotecan and the drug-lipid ratio is 0.

15.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition has one or more characteristics selected from the following (1) to (3): (1) The encapsulation rate of the antitumor drug in the pharmaceutical composition is more than 90%; (2) The particle size of the pharmaceutical composition is 60 to 180 nm, preferably 80 to 120 nm; (3) The polydispersity index (PDI) of the pharmaceutical composition is from 0.01 to 0.2, more preferably from 0.01 to 0.

15.

8. the liposome carrier comprises phosphatidylcholine and cholesterol; or The liposome carrier further comprises a crosslinker (e.g., 1,2-distearoyl-rac-glycero-3-PE-N-polyethylene glycol-2000 (DSPE-MPEG2000)); The pharmaceutical composition according to any one of claims 1 to 7.

9. 9. The pharmaceutical composition of claim 8, wherein the phosphatidylcholine is selected from hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (SPC), hydrogenated egg phosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, dioleoylphosphatidylcholine, or a combination thereof.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the lipid has a Her2 / neu antibody or a Her2 / neu antibody fragment conjugated thereto.

11. A method for producing the pharmaceutical composition according to claim 1, comprising the steps of: (1) mixing and incubating an antitumor drug solution and a liposome carrier dispersion to obtain the pharmaceutical composition; (2) Adding the antitumor drug to a lipid raw material alcohol phase or aqueous phase solution to obtain the pharmaceutical composition in one step during the preparation of liposome carriers.

12. A method for inhibiting the proliferation of tumor cells in vitro, comprising administering to said cells a medically effective amount of a pharmaceutical composition according to any one of claims 1 to 10.

13. A pharmaceutical composition for use in the treatment of a tumor, the pharmaceutical composition being defined in any one of claims 1 to 10.

14. The pharmaceutical composition of claim 13, for use in treating a tumor, wherein the tumor is a Her2 / neu positive tumor, wherein a Her2 / neu antibody or a Her2 / neu antibody fragment is conjugated to a liposome carrier.

15. The pharmaceutical composition of claim 13 or 14 for use in treating a tumor, wherein the tumor is selected from the group consisting of breast cancer, gastric cancer, ovarian cancer, liver cancer, melanoma and colorectal cancer.

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