Pathogen-associated molecular pattern surface-modified antitumor liposome drug delivery system, and preparation method and application thereof

Abstract

The application provides a pathogen-associated molecular pattern surface modified anti-tumor liposome drug delivery system and a preparation method and application thereof, and belongs to the technical field of biological medicine. The liposome drug delivery system comprises a PAMP component, cisplatin, PAD4i and a liposome; the PAMP component is modified on the outer surface of a phospholipid bilayer of the liposome, and the cisplatin and the PAD4i are loaded in the inner part of the phospholipid bilayer of the liposome. The cisplatin and the PAD4i are used in combination to exert a synergistic anti-tumor effect. In the application, the PAMP is modified on the surface of the liposome, the phagocytosis of neutrophils is utilized to transport the nano-preparation to a tumor part, the enrichment of the nano-preparation in the liver and spleen and the clearance of the nano-preparation by the mononuclear-macrophage system are avoided, the toxic and side effects on other normal tissue cells are reduced, the drug level in the tumor part is improved, and a specific anti-tumor immune response can be activated to enhance the anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pathogen-associated molecular pattern surface modified anti-tumor liposome drug delivery system, a preparation method and application thereof. BACKGROUND

[0002] Chemotherapy is one of the main means for tumor treatment at present. However, due to the lack of specificity of most chemotherapy drugs in action and distribution in the body, the normal tissue cells are also damaged while killing tumor cells, causing serious toxic side effects such as bone marrow suppression, gastrointestinal toxicity, neurotoxicity, and immune system damage. Therefore, it is a clinical problem to be solved in tumor chemotherapy to use a safe and effective in vivo transport form, enhance the targeting of chemotherapy drugs, and improve the local drug concentration in tumors, so as to reduce the toxic side effects of drugs and improve the prognosis of patients.

[0003] Tumors have strong heterogeneity, and a single chemotherapy drug is difficult to achieve satisfactory efficacy and is prone to cause drug resistance. Therefore, the combination of multiple drugs targeting different targets is a common clinical treatment strategy. Neutrophils are important natural immune cells in the human body, which can form neutrophil extracellular traps (NETs) to capture and kill pathogens when the body is infected. Recent studies have found that NETs can capture circulating tumor cells and promote tumor liver and lung metastasis. After NETs capture tumor cells, the diffusion of chemotherapy drugs to tumor cells is blocked, causing chemotherapy drug resistance. Therefore, targeted inhibition of NETs formation has good anti-tumor effect.

[0004] Compared with traditional drug preparation modes, the nano drug delivery system has the advantages of improving the solubility of drugs, increasing the stability of drugs in the blood environment, improving the circulation time and targeting of drugs, and reducing the toxic side effects of drugs on non-target organs, and has attracted widespread attention. However, the nano drug delivery system faces the formation of protein corona, liver and spleen enrichment, monocyte-phagocyte system phagocytosis, and various biological barrier restrictions such as blood, tissue, and cells when systemically administered, which leads to little benefit in clinical trials of nano preparations. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a pathogen-associated molecular pattern surface modified anti-tumor liposome drug delivery system, a preparation method and application thereof. The liposome drug delivery system can use the hitchhiking effect to be safely transported to the tumor site by neutrophils, reduce damage to other tissues and organs, improve the local drug concentration in tumors, and the liposome drug delivery system is more easily transported into tumor cells to kill tumor cells, thereby improving the anti-tumor effect and achieving the purpose of synergistic treatment of tumors.

[0006] To achieve the above-mentioned purpose of the application, the application provides the following technical solutions.

[0007] The application provides an anti-tumor liposome drug delivery system, which comprises a PAMP component, cisplatin, PAD4i and a liposome; wherein the PAMP component is modified on the outer surface of a phospholipid bilayer of the liposome, the cisplatin and the PAD4i are loaded in the interior of the phospholipid bilayer of the liposome; the PAD4i represents a peptide arginine deiminase 4 inhibitor, and the PAMP represents a pathogen-associated molecular pattern.

[0008] Preferably, the PAMP component comprises one or more of lipopolysaccharide, mannan, peptidoglycan, lipoteichoic acid, heat shock protein, high mobility group protein B and oxidized low-density lipoprotein.

[0009] Preferably, the PAD4i comprises one of Cl-amidine, BMS-P5 and GSK199.

[0010] Preferably, the raw materials of the liposome comprise egg yolk lecithin and cholesterol.

[0011] Preferably, the mass ratio of the PAMP component, the cisplatin and the PAD4i is 0.5-1.5:5-70:300-400.

[0012] Preferably, the mass ratio of the egg yolk lecithin, the cholesterol and the PAMP component is 1900-2100:450-550:0.5-1.5.

[0013] The application further provides a preparation method of the liposome drug delivery system.

[0014] The egg yolk lecithin and the cholesterol are respectively dissolved in a first organic solvent to obtain an egg yolk lecithin solution and a cholesterol solution, and the PAMP component is dissolved in a second organic solvent to obtain a PAMP solution.

[0015] The egg yolk lecithin solution, the cholesterol solution and the PAMP solution are ultrasonically mixed to obtain a dispersed mixed solution, and the organic solvent in the dispersed mixed solution is removed to obtain a lipid film.

[0016] The cisplatin and the PAD4i are mixed and dissolved in water to obtain a solution A; the solution A is mixed with the lipid film, and then rotary evaporated, hydrated and ultrasonically dispersed to obtain a hydrated lipid film solution, i.e., a liposome solution; the liposome solution is filtered through a microporous filter membrane, and the filtrate is collected and ultrafiltered to obtain a supernatant, i.e., the liposome drug delivery system.

[0017] Preferably, the first organic solvent comprises chloroform, the second organic solvent comprises petroleum ether; the mass-volume ratio of the egg yolk lecithin to the first organic solvent is 15-20 mg: 350-450 muL; the mass-volume ratio of the cholesterol to the first organic solvent is 3-7 mg: 150-250 muL; the mass-volume ratio of the PAMP component to the second organic solvent is 5-15 mu g: 350-450 muL; the mass-volume ratio of the cisplatin, PAD4i and water is 0.5-7 mg: 30-40 mg: 4-8 mL.

[0018] The application further provides a use of the liposome nano-drug delivery system or the preparation method in preparation of an anti-tumor drug.

[0019] Preferably, the tumor comprises at least one of ovarian cancer, endometrial cancer, cervical cancer, breast cancer, lung cancer, bladder cancer and colon cancer.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The application provides a pathogen-associated molecular pattern surface modified anti-tumor liposome nano-drug delivery system, a preparation method and application thereof. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A particle size diagram determined by dynamic light scattering (DLS) for nanoparticles of the PAMP surface modified anti-tumor drug composition;

[0023] Figure 2 A transmission electron microscope diagram for nanoparticles of the PAMP surface modified anti-tumor drug composition;

[0024] Figure 3 A killing effect of the anti-tumor liposome nano-drug delivery system on ovarian cancer cells;

[0025] Figure 4The treatment effect of the anti-tumor liposome nano-drug delivery system on an ovarian cancer orthotopic model;

[0026] Figure 5 The in-vivo biological safety evaluation of the anti-tumor liposome nano-drug delivery system;

[0027] Figure 6 The treatment effect of the anti-tumor liposome nano-drug delivery system on an ovarian cancer abdominal metastasis model, wherein Figure 6 The first row from left to right in the table is I. a control group (PBS: 0.2 mL), II. an empty liposome group, III. a Cl-amidine liposome group (20 mg / kg); the second row from left to right is IV. a CDDP liposome group (2 mg / kg), V. a CDDP+Cl-amidine aqueous solution group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg), VI. an L-Lip@CC group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg);

[0028] Figure 7 The treatment effect of the anti-tumor liposome nano-drug delivery system on an ovarian cancer lung metastasis model;

[0029] Figure 8 The killing effect of the anti-tumor liposome nano-drug delivery system on cervical cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer and colon cancer cells;

[0030] Figure 9 The result of safe transportation of the anti-tumor liposome nano-drug delivery system to a tumor site by neutrophils;

[0031] Figure 10 The verification result of immune activation of the anti-tumor liposome nano-drug delivery system, wherein Figure 10 The h from left to right in the table is a Ctrl group, a CDDP+Cl-amidine aqueous solution group and an L-Lip@CC group, respectively;

[0032] Figure 11 The structural schematic diagram of the anti-tumor liposome nano-drug delivery system. DETAILED DESCRIPTION

[0033] The application provides an anti-tumor liposome nano-drug delivery system, which comprises a PAMP component, cisplatin, PAD4i and a liposome; wherein the PAMP component is modified on the outer surface of a liposome phospholipid bilayer, the cisplatin and the PAD4i are loaded in the inside of the liposome phospholipid bilayer; the PAD4i represents a peptide acylarginine deiminase 4 inhibitor, and the PAMP represents a pathogen-associated molecular pattern.

[0034] In the present application, the PAMP component modified on the outer surface of the lipid bilayer of the liposome can enable the anti-tumor liposome nanocarrier system to be safely transported to the tumor site by neutrophils, reduce damage to other tissues and organs, improve the biological safety and drug concentration in the tumor area, and after being phagocytosed by neutrophils, the activated neutrophils release various cytokines to play a cell-killing role, and can also chemotactically attract macrophages and T cells to activate specific anti-tumor immune responses, thereby improving the anti-tumor effect. The PAMP is the abbreviation of pathogen-associated molecular pattern. The PAMP component preferably includes one or more of lipopolysaccharide, mannan, peptidoglycan, lipoteichoic acid, heat shock protein, high mobility group protein B, and oxidized low-density lipoprotein, and is further preferably lipopolysaccharide, which is purchased from Solabio with the item number L-8880. The present application does not have special limitations on the source of mannan, peptidoglycan, and lipoteichoic acid, and commercially available products in the art can be used.

[0035] In the present application, PAD4i is the abbreviation of peptide acylarginine deiminase 4 inhibitor, and the PAD4i preferably includes one of Cl-amidine, BMS-P5, and GSK199, and is further preferably Cl-amidine, which is purchased from Selleck with the item number S8141. The cisplatin is purchased from Innochem with the item number A82733. The present application uses CDDP and PAD4i in combination, wherein cisplatin plays a tumor-killing effect, and PAD4i (such as Cl-amidine) inhibits tumor metastasis, and can achieve a synergistic effect in the treatment of tumors. At the same time, PAD4i can also inhibit the metastasis of tumors through blood, lymph, direct implantation and other routes. The mass ratio of the PAMP component, cisplatin, and PAD4i is preferably 0.5-1.5:5-70:300-400, further preferably 0.7-1.2:7-65:320-380, and more preferably 1:10:350 or 1:60:350.

[0036] In the present application, the raw materials of the liposome preferably include egg yolk lecithin and cholesterol. The mass ratio of the egg yolk lecithin, cholesterol, and PAMP component is preferably 1900-2100:450-550:0.5-1.5. The present application does not have special limitations on the source of egg yolk lecithin and cholesterol, and commercially available products known in the art can be used.

[0037] The present application also provides a preparation method of the above-mentioned liposome nanocarrier system, which comprises the following steps:

[0038] The egg yolk lecithin and the cholesterol are dissolved in a first organic solvent respectively to obtain an egg yolk lecithin solution and a cholesterol solution, and the PAMP component is dissolved in a second organic solvent to obtain a PAMP solution.

[0039] The egg yolk lecithin solution, the cholesterol solution and the PAMP solution are ultrasonically mixed to obtain a dispersed mixture, and the organic solvent in the dispersed mixture is removed to obtain a lipid film;

[0040] The cisplatin and the PAD4i are mixed and dissolved in water to obtain solution A; solution A is mixed with the lipid film, and then rotary evaporated, hydrated, ultrasonically dispersed to obtain a hydrated lipid film solution, i.e., a liposome solution; the liposome solution is filtered through a microporous filter, and the filtrate is collected and ultrafiltered to obtain the supernatant, i.e., the product.

[0041] In the present application, the egg yolk lecithin and the cholesterol are dissolved in a first organic solvent respectively to obtain an egg yolk lecithin solution and a cholesterol solution, and the PAMP component is dissolved in a second organic solvent to obtain a PAMP solution. The first organic solvent comprises chloroform, and the second organic solvent comprises petroleum ether; the mass-volume ratio of the egg yolk lecithin to the first organic solvent is preferably 15-25 mg: 350-450 μL, further preferably 18-22 mg: 370-420 μL, and more preferably 20 mg: 400 μL; the mass-volume ratio of the cholesterol to the first organic solvent is preferably 3-7 mg: 150-250 μL, further preferably 4-6 mg: 170-220 μL, and more preferably 5 mg: 200 μL; and the mass-volume ratio of the PAMP component to the second organic solvent is preferably 5-15 μg: 350-450 μL, further preferably 6-12 μg: 360-420 μL, and more preferably 10 μg: 400 μL, and the PAMP component is preferably a lipopolysaccharide.

[0042] After obtaining the egg yolk lecithin solution, the cholesterol solution and the PAMP solution, the egg yolk lecithin solution, the cholesterol solution and the PAMP solution are ultrasonically mixed to obtain a dispersed mixture, and the organic solvent in the dispersed mixture is removed to obtain a lipid film. The power of the ultrasonic is preferably 200-300 W, further preferably 220-280 W, and more preferably 240 W; the time of the ultrasonic is preferably 4-10 min, further preferably 5-8 min, and more preferably 5 min; and the temperature of the ultrasonic is preferably 22-30℃, further preferably 24-26℃, and more preferably 25℃. The organic solvent in the dispersed mixture is removed by rotary evaporation under a vacuum of 0.05-0.1 MPa until the organic reagent in the dispersed mixture is completely removed.

[0043] After obtaining the lipid film, the cisplatin, PAD4i and water are mixed and dissolved to obtain solution A. The mass-volume ratio of the cisplatin, PAD4i and water is preferably 0.5-7 mg: 30-40 mg: 4-8 mL, further preferably 0.5-6.5 mg: 32-38 mg: 5-7 mL, and more preferably 1 mg: 35 mg: 6 mL or 6 mg: 35 mg: 6 mL. The PAD4i is preferably Cl-amidine.

[0044] After obtaining the solution A, the solution A is mixed with the lipid film, and then rotary evaporation, hydration, ultrasonic dispersion are performed to obtain a hydrated lipid film solution, i.e. a liposome solution. The rotary evaporation time is preferably 4-10 min, further preferably 5-8 min, and more preferably 5 min. The hydration time is preferably 80-100 min, further preferably 85-95 min, and more preferably 90 min. The hydration is performed by magnetic stirring at a constant temperature of 35-40℃, and further at a constant temperature of 37℃. The ultrasonic power is preferably 200-300 W, further preferably 220-280 W, and more preferably 240 W. The ultrasonic time is preferably 4-10 min, further preferably 5-8 min, and more preferably 5 min. The ultrasonic temperature is preferably 22-30℃, further preferably 24-26℃, and more preferably 25℃.

[0045] The liposome solution is filtered by a microporous filter membrane, and the filtrate is collected. The filtrate is subjected to ultrafiltration, and the supernatant is collected to obtain the anti-tumor liposome nanocarrier drug system. The microporous filter membrane is preferably a microporous filter membrane with a pore size of 0.22 μm. The ultrafiltration is performed for 3 times, and the ultrafiltration time is preferably 80-100 min, further preferably 85-95 min, and more preferably 90 min. The supernatant is preferably the supernatant after 3 times of ultrafiltration.

[0046] The anti-tumor liposome nanocarrier drug system prepared by the method has a particle size of 47.4 ± 19.0 nm, and is a spherical nanoparticle. Experimental results show that the anti-tumor liposome nanocarrier drug system can achieve a synergistic tumor-killing effect on cervical cancer cells (such as Hela), endometrial cancer cells (such as HEC1B), breast cancer cells (such as 4T1), lung cancer cells (such as A549), bladder cancer cells (such as MB49), and colon cancer cells (such as HCT116). Moreover, the morphologies of the heart, liver, spleen, lung and kidney tissue sections of mice before and after drug treatment are normal, and there is no significant difference in the blood glutathione, glutathione, urea and creatinine, and the safety is very high.

[0047] Based on this, the application further provides a use of the above-mentioned liposome nanocarrier drug system or the above-mentioned preparation method in the preparation of an anti-tumor drug.

[0048] In the present application, the tumor includes at least one of ovarian cancer, endometrial cancer, cervical cancer, breast cancer, lung cancer, bladder cancer and colon cancer. The ovarian cancer cells include one or more of SKOV3 cells, ES2 cells, A2780 cells and ID8 cells. The cervical cancer cells include Hela, the endometrial cancer cells include HEC1B, the breast cancer cells include 4T1, the lung cancer cells include A549, the bladder cancer cells include MB49, and the colon cancer cells include HCT116. In the present application, the liposome nanocarrier system can be used in combination with active ingredients of other anti-tumor drugs to treat tumors, or the liposome nanocarrier system can be used as the only active ingredient to treat tumors. The drug also includes pharmaceutically acceptable adjuvants, and "pharmaceutically acceptable" means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in humans or animals. The active ingredient refers to the main ingredient for treating, relieving or preventing the target effect or disease. The dosage form of the drug includes injections, oral preparations or sprays.

[0049] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0050] Example 1. Preparation and characterization of an anti-tumor liposome nanocarrier system

[0051] A method for preparing an anti-tumor liposome nanocarrier system, the steps are as follows:

[0052] (1) Prepare a chloroform solution of egg yolk lecithin with a concentration of 50 mg / mL, a chloroform solution of cholesterol with a concentration of 25 mg / mL and a petroleum ether solution of lipopolysaccharide with a concentration of 0.025 mg / mL at room temperature.

[0053] (2) Take 400 μL of the chloroform solution of egg yolk lecithin prepared in step (1), 200 μL of the chloroform solution of cholesterol prepared in step (1) and 400 μL of the petroleum ether solution of lipopolysaccharide prepared in step (1), mix them uniformly at 25°C under ultrasonic waves with a power of 240 W for 5 min (i.e. 20 mg of egg yolk lecithin, 5 mg of cholesterol and 10 μg of lipopolysaccharide are put into a dry and clean round-bottom flask and mixed uniformly under ultrasonic waves), to obtain a dispersed mixture.

[0054] (3) Under the condition of 0.1 Mpa vacuum, rotary evaporate the organic solvents in the dispersed mixture to obtain a lipid film.

[0055] (4) 0.1 mg cisplatin (CDDP), 3.5 mg Cl-amidine were dissolved in 0.6 mL deionized water to obtain solution A, and solution A was added to the lipid film and rotary evaporation was continued for 5 min. After the film was removed, it was incubated in a 37°C constant temperature incubator for 90 min with magnetic stirring, and then ultrasonic dispersion was performed at 25°C for 5 min at 240W to disperse the lipid film uniformly to obtain a hydrated lipid film solution.

[0056] (5) The lipid film solution after ultrasonic hydration at 240W and 25°C for 5 min was filtered through a 0.22 μm microporous filter, and the filtrate was collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The first supernatant was collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The second supernatant was collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The third supernatant was collected, and the collected third supernatant was an antitumor liposome nanocarrier system (referred to as L-Lip@CC nanoparticles), which was stored in a 4°C refrigerator in the dark.

[0057] The particle size of the prepared L-Lip@CC nanoparticles was determined by dynamic light scattering (DLS), and the results are shown in Figure 1 The particle size of the prepared L-Lip@CC nanoparticles was 47.4 ± 19.0 nm.

[0058] The morphology of the L-Lip@CC nanoparticles was characterized by transmission electron microscopy (TEM), and the results are shown in Figure 2 Figure 2 The results show that the prepared L-Lip@CC nanoparticles are spherical nanoparticles.

[0059] Example 2

[0060] A preparation method of an antitumor liposome nanocarrier system, the steps are as follows:

[0061] (1) At room temperature, a 50 mg / mL solution of egg yolk lecithin in chloroform, a 25 mg / mL solution of cholesterol in chloroform, and a 0.025 mg / mL solution of lipopolysaccharide in petroleum ether were prepared in advance.

[0062] ​(2) Take 400 μL of the egg yolk lecithin solution in chloroform prepared in step (1), 200 μL of the cholesterol solution in chloroform prepared in step (1), and 400 μL of the lipopolysaccharide solution in petroleum ether prepared in step (1), mix them uniformly at 25°C under ultrasonic waves at 240W for 5 min (i.e., 20 mg of egg yolk lecithin, 5 mg of cholesterol, and 10 μg of lipopolysaccharide are put into a dry and clean round-bottom flask and mixed uniformly under ultrasonic waves), to obtain a mixed solution after dispersion.

[0063] (3) The organic solvents in the mixed solution after dispersion are evaporated under vacuum at 0.1 MPa to obtain a lipid film.

[0064] (4) 0.6 mg of cisplatin (CDDP) and 3.5 mg of Cl-amidine are mixed and dissolved in 0.6 mL of deionized water to obtain solution A, and solution A is added to the lipid film and continues to be evaporated for 5 min. After the film falls off, it is incubated in a thermostat incubator at 37°C for 90 min under magnetic stirring and ultrasonic hydration. The ultrasonic conditions are 25°C and 240W for 5 min, so that the lipid film is uniformly dispersed, to obtain a hydrated lipid film solution.

[0065] (5) The hydrated lipid film solution obtained in step (4) is ultrasonically treated at 240W and 25°C for 5 min, and the obtained liposome solution is filtered through a microporous filter with a pore size of 0.22 μm. The filtrate is collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The first supernatant is collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The second supernatant is collected and placed in an ultrafiltration tube (MW: 3000 Da) for ultrafiltration for 90 min. The third supernatant is collected, which is an antitumor liposome nano-drug delivery system (abbreviated as L-Lip@CC nanoparticles), and is stored in a 4°C refrigerator in the dark.

[0066] Example 3. Killing effect of the antitumor liposome nano-drug delivery system prepared in Example 2 on ovarian cancer cells

[0067] The effect of the L-Lip@CC nanoparticles prepared in Example 2 on the viability of ovarian cancer cells was detected by the CCK-8 method.

[0068] The L-Lip@CC nanoparticles prepared in Example 2 (i.e. CDDP: 6 μg / mL, Cl- amidine: 30 μg / mL) were gradiently diluted into L-Lip@CC of CDDP: 4 μg / mL, Cl- amidine: 20 μg / mL; CDDP: 2 μg / mL, Cl-amidine: 10 μg / mL; CDDP: 1 μg / mL, Cl- amidine: 5 μg / mL; and CDDP: 0.5 μg / mL, Cl-amidine: 2.5 μg / mL, respectively, and then subsequent experiments were carried out.

[0069] The ovarian cancer cell lines (SKOV3, ES2, A2780, ID8) were cultured in DMEM complete medium (containing 10% fetal bovine serum + 1% penicillin and streptomycin) at 37°C in a 5% CO2 incubator, and the medium was changed every 24 h. After the cells grew to the logarithmic growth phase, they were inoculated in a 96-well plate at a density of 3000 cells per well and cultured for 12 h. The complete medium in the 96-well plate was aspirated. The control group was set, i.e. 100 μL DMEM incomplete medium was added to each well; five nanoparticle groups were set, each with 3 wells, and 100 μL L-Lip@CC of different concentrations (① CDDP: 0.5 μg / mL, Cl-amidine: 2.5 μg / mL ② CDDP: 1 μg / mL, Cl-amidine: 5 μg / mL ③ CDDP: 2 μg / mL, Cl-amidine: 10 μg / mL ④ CDDP: 4 μg / mL, Cl-amidine: 20 μg / mL ⑤ CDDP: 6 μg / mL, Cl-amidine: 30 μg / mL) diluted with DMEM incomplete medium were added to each well. The cells were incubated with different concentrations of L-Lip@CC for 24 h and 48 h, respectively. Then, 10% CCK-8 diluted with 100 μL DMEM was added to each well. After incubation at 37°C for 1-4 h, the absorbance of the wells was tested at 450 nm by a microplate reader. The cell viability was expressed as the ratio of the absorbance of the test wells to that of the control wells.

[0070] The cell CCK-8 results are shown in Table 1; L-Lip@CC has obvious toxicity to the above ovarian cancer cell lines, and has concentration and time dependence. Figure 3

[0071] Example 4. Therapeutic effect of the anti-tumor liposome nanoparticle drug delivery system prepared in Example 1 on an ovarian cancer orthotopic model

[0072] A suitable amount of ID8 cells (1.0 x 10 7 ​(0.1 mL / animal), observe the subcutaneous tumor growth regularly, and when the tumor volume reaches 1 cm³, [the tumor is considered to be growing rapidly]. 3 The tumor was removed and set aside. The excised tumor was washed with sterile PBS and cut into 1×1×1mm pieces with a blade. 3 Tumor tissue blocks were used for orthotopic ovarian cancer transplantation. Mice were anesthetized with 5% chloral hydrate. Under a stereomicroscope, a 0.5–1 cm incision was made in the skin of the right dorsal kidney region of the mouse to expose the ovary. The tumor tissue block was then implanted into the right ovarian sac of the mouse. Two weeks later, the skin and muscle of the mouse were incised again to expose the ovary. Fifteen tumor-bearing mice (n=3 per group) with similar tumor volumes were selected for subtotal ovarian tumor resection, preserving 10 mm of the tumor. 3 Tumor tissue of varying sizes was observed. Postoperatively, mice were administered drugs via tail vein once daily for 3 consecutive days: I. Empty liposome group (dose calculated based on drug loading); II. CDDP liposome group (2 mg / kg); III. Cl-amidine liposome group (20 mg / kg); IV. CDDP + Cl-amidine aqueous solution group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg); V. L-Lip@CC group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg). Mice survival was recorded daily, and body weight was measured every 3 days. Twenty-one days after surgery, mice were sacrificed, and the uterus and ovary were removed. The length and width of the ovarian orthotopic tumor were measured and recorded using calipers, and the weight was determined using the formula: Tumor volume = (length × width) 2 The tumor volume was calculated as () / 2. H&E staining was performed on the ovarian tumor and major organs (heart, liver, spleen, lungs, and kidneys). Peripheral blood was collected for testing of key liver and kidney function indicators (alanine aminotransferase, aspartate aminotransferase, urea, and creatinine).

[0073] The synthesis method of CDDP liposomes is similar to the preparation method of L-Lip@CC in Example 1, except that: for CDDP liposomes, solution A is changed to a solution obtained by mixing and dissolving 0.1 mg cisplatin (CDDP) with 0.6 mL of deionized water, and other steps are the same as in Example 1.

[0074] The synthesis method of Cl-amidine liposomes is similar to that of L-Lip@CC in Example 1, except that solution A for Cl-amidine liposomes is changed to a solution obtained by mixing and dissolving 3.5 mg Cl-amidine with 0.6 mL of deionized water. Other steps are the same as in Example 1.

[0075] The CDDP+Cl-amidine aqueous solution is an aqueous solution obtained by mixing and dissolving 0.22 mg cisplatin (CDDP), 6 mg Cl-amidine and 4 mL of deionized water.

[0076] likeFigure 4 As shown, the tumor volume changes in each group showed different trends. The average tumor volume in the empty liposome group increased significantly; the CDDP liposome group, Cl-amidine liposome group, and CDDP+Cl-amidine aqueous solution group showed moderate tumor-suppressing effects; while the L-Lip@CC group showed a strong tumor-suppressing effect, with complete tumor disappearance. Simultaneously, there was no significant difference in the average body weight of mice in each group during the drug administration process. The morphology of tissue sections of the heart, liver, spleen, lung, and kidneys of mice was normal before and after drug treatment. There were no significant differences in blood alanine aminotransferase, aspartate aminotransferase, urea, and creatinine levels, indicating that the nanoparticles have strong safety (see...). Figure 5 ).

[0077] Example 5. Therapeutic effect of the antitumor liposome nanocarrier system prepared in Example 1 on an ovarian cancer peritoneal metastasis model.

[0078] Female C57BL mice were intraperitoneally inoculated with an appropriate amount of ID8 cells (1.0 × 10⁻⁶). 7 Four hours after surgery, 50 μg of lipopolysaccharide (PPS) was injected intraperitoneally to induce peritoneal inflammation. Twenty-four mice were randomly divided into six groups of four each. The mice received the drug via tail vein injection once daily for three consecutive days post-surgery: I. Control group (PBS: 0.2 mL); II. Empty liposome group (calculated based on drug loading from the administered dose); III. Cl-amidine liposome group (20 mg / kg); IV. CDDP liposome group (2 mg / kg); V. CDDP + Cl-amidine aqueous solution group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg); VI. L-Lip@CC group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg). Mice were fed routinely from day 4 to day 20. Body weight and abdominal circumference were measured every three days to assess treatment efficacy. Mice were sacrificed on day 21 to observe peritoneal tumor metastasis. The preparation methods for Cl-amidine liposomes, CDDP liposomes, and CDDP+Cl-amidine aqueous solutions are described in Example 4.

[0079] like Figure 6 As shown, the trends of abdominal circumference and body weight changes in mice in each group were significantly different. The changes in abdominal circumference and body weight of mice in the control group and the empty liposome group were significantly increased; the changes in abdominal circumference and body weight of the CDDP liposome group, the Cl-amidine liposome group and the CDDP+Cl-amidine aqueous solution group were moderately increased; while the L-Lip@CC group had a strong tumor-suppressing effect.

[0080] Example 6. Therapeutic effect of the antitumor liposome nanocarrier system prepared in Example 1 on an ovarian cancer lung metastasis model.

[0081] Female C57BL mice were inoculated via the tail vein with an appropriate amount of Luc-ID8 cells (1.0 × 10⁻⁶). 7 Lung inflammation was induced by inhalation of 50 μg lipopolysaccharide (PPS) 4 hours after administration (0.1 mL / mouse). Twenty-four mice were randomly divided into six groups of four each. The mice were administered the drug via tail vein once daily for three consecutive days post-surgery: I. Control group (PBS: 0.2 mL); II. Empty liposome group (calculated from the dosage based on drug loading); III. Cl-amidine liposome group (20 mg / kg); IV. CDDP liposome group (2 mg / kg); V. CDDP + Cl-amidine aqueous solution group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg); VI. L-Lip@CC group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg). Body weight was measured every three days to assess treatment efficacy. Mice were fed normally from day 4 to day 20 without any drug treatment. Mice were sacrificed on day 21 to observe peritoneal tumor metastasis. In vivo imaging of mice was performed every five days using a small animal imaging system to assess treatment efficacy. Lung tissue was harvested on day 21 to record lung metastases, and sections were stained with H&E. The preparation methods for Cl-amidine liposomes, CDDP liposomes, and CDDP+Cl-amidine aqueous solution are described in Example 4.

[0082] like Figure 7 As shown, the trends of lung lesions in mice in each group were significantly different. The lung lesions in the control group and the empty liposome group were significantly increased; the lung lesions in the CDDP liposome group, the Cl-amidine liposome group and the CDDP+Cl-amidine aqueous solution group were moderately increased; while the L-Lip@CC group had a strong tumor-suppressing effect, with only a few lesions or even the lesions disappearing.

[0083] Example 7. The killing effect of the antitumor liposome nanocarrier system prepared in Example 2 on cervical cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, and colon cancer cells.

[0084] The L-Lip@CC nanoparticles prepared in Example 2 (i.e., CDDP: 6 μg / mL, Cl-amidine: 30 μg / mL) were serially diluted to L-Lip@CC with CDDP: 4 μg / mL and Cl-amidine: 20 μg / mL; L-Lip@CC with CDDP: 2 μg / mL and Cl-amidine: 10 μg / mL; L-Lip@CC with CDDP: 1 μg / mL and Cl-amidine: 5 μg / mL; and L-Lip@CC with CDDP: 0.5 μg / mL and Cl-amidine: 2.5 μg / mL, and then subsequent experiments were carried out.

[0085] The effect of L-Lip@CC prepared in Example 2 on the viability of cervical cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, and colon cancer cells was detected using the CCK-8 assay. Cervical cancer cells (HeLa), endometrial cancer cells (HEC1B), breast cancer cells (4T1), lung cancer cells (A549), bladder cancer cells (MB49), and colon cancer cells (HCT116) were seeded at a density of 3000 cells per well in 96-well plates and cultured for 12 h. Cells were incubated with different concentrations of L-Lip@CC (①CDDP 0.5 μg / mL, Cl-amidine 2.5 μg / mL ②CDDP 1 μg / mL, Cl-amidine 5 μg / mL ③CDDP 2 μg / mL, Cl-amidine 10 μg / mL ④CDDP 4 μg / mL, Cl-amidine 20 μg / mL ⑤CDDP 6 μg / mL, Cl-amidine 30 μg / mL) for 24 h and 48 h, respectively. Then, 10% CCK-8 diluted with 100 μL DMEM was added to each well. After incubation at 37℃ for 1–4 h, the absorbance of the wells was measured at 450 nm using a microplate reader. Cell viability was expressed as the ratio of absorbance of the test wells to that of the control wells. The cell CCK-8 results are shown below. Figure 8 As shown.

[0086] Figure 8 The results showed that L-Lip@CC nanoparticles had significant toxicity to the aforementioned cervical cancer, endometrial cancer, breast cancer, lung cancer, bladder cancer, and colon cancer cells, and this toxicity was concentration- and time-dependent.

[0087] Example 8: Experiment on the safe delivery of the antitumor liposome nanocarrier system prepared in Example 1 to the tumor site via neutrophils.

[0088] First, an orthotopic ovarian cancer mouse model was established. Part of the tumor was surgically removed to create a postoperative inflammatory tumor model. Peripheral blood of the mice was collected 1 hour after administration of the drug via the tail vein.

[0089] One model of postoperative treatment for orthotopic ovarian cancer involved: ID8 tumor cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. All cells were cultured at 37°C under 5% CO2 conditions. The medium was changed daily, and cells were passaged using trypsin digestion. Cell suspensions were prepared during the logarithmic growth phase, and the number of viable cells was counted to prepare 1×10⁶ cells / cell suspensions. 7 Cell suspension of 200 μL / cell was used for inoculation. Cell viability was maintained by ice bath. Cells were inoculated into the right side of 5-week-old female C57BL mice. Subcutaneous tumor growth was observed regularly, and the tumor volume reached 1 cm³. 3The tumor was removed from both sides, and the surface membrane and necrotic tissue were carefully removed with ophthalmic scissors. It was then transferred to a clean culture dish, rinsed with PBS, and cut into fragments (1×1×1mm). 3 It is used for orthotopic ovarian cancer transplantation.

[0090] Female C57BL mice were anesthetized with 4% chloral hydrate. After a period of time, once the mice were fully anesthetized, they were placed in a lateral recumbent position on the operating table. Hair was removed from the right side of the mice using depilatory cream to facilitate the surgical procedure. The tumor tissue was implanted under a stereomicroscope. The right side of the mouse was disinfected with 75% alcohol. A 1cm incision was made in the dorsal region of the kidney area to expose the ovary. The tumor tissue block was implanted into the ovarian capsule. The ovary was gently placed back into the abdominal cavity, sutured, disinfected, and the incision was wiped clean of blood. The mice were placed on an electric blanket to keep them warm and promote recovery. The bedding and water in the cage were changed. After the mice recovered, they were returned to their cages. Two weeks after the ovarian tumor began to grow, it was surgically removed, leaving a tumor volume of 10mm. 3 One day later, Nile Red-labeled L-Lip@CC liposome nanoparticles (1 mg) and CDDP+Cl-amidine aqueous solution (i.e., Ctrl) were injected via the tail vein. One hour after injection, peripheral blood was collected and processed, and then labeled with APC-CD11b and FITC-Ly6G antibodies. The percentage of neutrophils containing liposomes in the peripheral blood was analyzed by flow cytometry. Twelve hours after injection, tumor tissue was collected and processed, and then labeled with FITC-CD45.2 and APC-Ly6G antibodies. The percentage of neutrophils containing liposomes in the tumor was analyzed by flow cytometry.

[0091] The CDDP+Cl-amidine aqueous solution is an aqueous solution obtained by mixing and dissolving 0.22 mg cisplatin (CDDP), 6 mg Cl-amidine and 4 mL of deionized water.

[0092] The experimental flowchart is shown below. Figure 9 'a' in 'a'.

[0093] Experimental results are as follows Figure 9 As shown in b and c, 70.5% of the neutrophils in peripheral blood contained liposomes. Neutrophils were rapidly mobilized and transported to the postoperative inflammatory tissue via inflammatory chemotaxis, where they accumulated. After 12 hours, 29.6% of the neutrophils in the tissue contained liposomes.

[0094] Meanwhile, the Pt content in postoperative inflammatory tissue was detected by ICP-MS to analyze the enrichment of the anti-tumor liposome nanocarrier system in postoperative inflammatory tissue.

[0095] Experimental results are as follows Figure 9As shown in d, compared to the Ctrl group, the enrichment of the antitumor liposome nanocarrier system is 4 times, demonstrating that the neutrophil-mediated delivery of the antitumor liposome nanocarrier system improves its delivery efficiency in postoperative inflammatory tumor tissues.

[0096] Example 9: Antitumor liposome nanocarrier system prepared in Example 1 activates immunity.

[0097] (1) To investigate the effect of L-Lip@CC treatment on ROS levels, intracellular ROS were labeled using the 2′-7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. Extracted neutrophils were co-incubated with PBS (Ctrl), lipopolysaccharide (LPS) (1 μg / mL), and 0.5 mg / mL L-Lip@CC for 2 h, respectively, followed by co-incubation with the DCFH-DA probe. LPS was used as a positive control, and flow cytometry was used to assess fluorescence intensity.

[0098] Compared to the control group (Ctrl), the fluorescence intensity peak of neutrophils treated with L-Lip@CC prepared in this invention shifted to the right, indicating increased ROS production. Figure 10 (a) in the middle.

[0099] (2) In order to detect the activation of NF-κB after L-Lip@CC exposure and to determine whether L-Lip@CC triggers innate immune signal transduction in neutrophils, Western blot analysis, qRT-PCR and CBA detection were performed.

[0100] ① Extracted neutrophils were co-incubated for 2 hours with PBS (Ctrl), LPS (1 μg / mL), and 0.5 mg / mL L-Lip@CC, respectively. Cell samples were lysed in RIPA buffer supplemented with a mixture of protease inhibitors and phosphatase inhibitors. Lysates were loaded and proteins were separated on a 10% polyacrylamide gel in 5×SDS-PAGE loading buffer. The separated proteins were transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TBST containing 5% milk for 1 hour, then incubated overnight at 4°C with anti-GAPDH (1:1000), anti-p-NF-KB p65 (1:1000), and anti-NF-KB p65 (1:1000). After incubation for 1 hour at room temperature with a species-specific horseradish peroxidase-conjugated secondary antibody (1:1000 dilution), protein bands were detected using a chemiluminescent substrate.

[0101] Western blot analysis confirmed that the L-Lip@CC of this invention increases p65 phosphorylation (see...). Figure 10 (b)

[0102] ② The extracted neutrophils were seeded into culture dishes pre-placed with treated coverslips and incubated for 2 hours with PBS, LPS (1 μg / mL), and 0.5 mg / mL L-Lip@CC, respectively. The cells were washed twice with PBS. Cells were fixed with 1% paraformaldehyde for 10 minutes, then washed twice with PBS and permeabilized with 0.1% Triton X-100 for 5 minutes. Cells were blocked in PBS containing 5% BSA for 30 minutes, then incubated overnight at 4°C with anti-p-NF-KB p65 (1:1000) in blocking buffer. After washing three times with PBS, the cells were incubated for 1 hour with a fluorescently conjugated secondary antibody (1:1000) and counterstained with DAPI before mounting. Observation and imaging were performed using a confocal microscope.

[0103] Based on immunofluorescence staining, the results showed that L-Lip@CC treatment for 2 hours induced p65 nuclear translocation. Figure 10 (c) in the middle.

[0104] ③ The extracted neutrophils were seeded into culture dishes pre-placed with treated coverslips and incubated for 6 hours with PBS, LPS (1 μg / mL), and 0.5 mg / mL L-Lip@CC, respectively. Total RNA was isolated and reverse transcribed using Superscript III reverse transcriptase. Real-time RT-PCR was performed using the iQ SYBR Green Supermix kit and a CFX96 Touch real-time polymerase chain reaction system.

[0105] Figure 10 The results showed that the L-Lip@CC of the present invention significantly increased the mRNA levels of cytokines (IL-6, IL-8, P<0.01; TNF-α, P<0.05).

[0106] ④ The extracted neutrophils were seeded into culture dishes pre-placed with treated coverslips and incubated for 6 hours with PBS, LPS (1 μg / mL), and 0.5 mg / mL L-Lip@CC, respectively. The cell supernatant was analyzed using a cell counting bead array, yielding results consistent with RT-PCR.

[0107] Figure 10 The results showed that L-Lip@CC significantly increased the levels of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α. In summary, these results indicate that the L-Lip@CC of the present invention activates the immune system via the TLR4 / NF-κB pathway.

[0108] (3) To demonstrate that L-Lip@CC activates anti-tumor immune cells, intratumoral cytotoxic T cells (CD8+) were evaluated in a mouse lung metastasis model (modeling scheme as in Example 6).+ The levels of T cells and tumor-associated macrophages (TAMs) were measured. Patients were divided into a PBS intervention control group, a CDDP+Cl-amidine aqueous solution treatment group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg), and an L-Lip@CC treatment group (CDDP: 2 mg / kg, Cl-amidine: 20 mg / kg). All groups received the treatment via tail vein injection. On day 16, lung tissue was removed. Lung tissue was collected and homogenized into a cell suspension, then filtered through a 200-mesh sieve and centrifuged (652 × g) for 5 min. The obtained cells were labeled with flow cytometry antibodies and analyzed by flow cytometry. The preparation method of the CDDP+Cl-amidine aqueous solution is described in Example 4.

[0109] The L-Lip@CC treatment group had CD45.2+ white blood cells with CD8+. + T cells (CD3) + CD8 + ) and TAM (F4 / 80) + The percentage of CD8+ was higher in mice than in the PBS and CDDP+Cl-amidine treatment groups. Mice treated with L-Lip@CC showed CD8+ + T cells significantly increased from 5.7% to 13.5% (P<0.01). Figure 10 In the f-g range, TAM increased from 7.08% to 63.8% (P<0.01). Figure 10 (h~i) indicates that the L-Lip@CC of this invention will cause CD8 + Increase in T cells and TAM.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antitumor liposome nanocarrier drug delivery system, characterized in that, The liposome comprises a PAMP component, cisplatin, PAD4i and a liposome; wherein the PAMP component is modified on the outer surface of the liposome phospholipid bilayer, the cisplatin and the PAD4i are loaded in the inner part of the liposome phospholipid bilayer; the PAD4i represents a peptide acylarginine deiminase 4 inhibitor, and the PAMP represents a pathogen-associated molecular pattern; The PAMP component is lipopolysaccharide. The PAD4i is Cl-amidine. The mass ratio of the PAMP component, cisplatin and PAD4i is 0.5-1.5:5-70:300-400.

2. The liposome nanocarrier drug delivery system according to claim 1, wherein, The raw materials of the liposome comprise egg yolk lecithin and cholesterol.

3. The liposome nanocarrier system according to claim 2, wherein, The mass ratio of the egg yolk lecithin, cholesterol and PAMP component is 1900-2100:450-550:0.5-1.

5.

4. A method for preparing the liposome nanocarrier system according to claim 2 or 3, characterized by, The method comprises the following steps: The egg yolk lecithin and the cholesterol are respectively dissolved in a first organic solvent to obtain an egg yolk lecithin solution and a cholesterol solution, and the PAMP component is dissolved in a second organic solvent to obtain a PAMP solution; The egg yolk lecithin solution, the cholesterol solution and the PAMP solution are ultrasonically mixed to obtain a dispersed mixture, and the organic solvent in the dispersed mixture is removed to obtain a lipid film; The cisplatin and the PAD4i are mixed and dissolved in water to obtain a solution A; The solution A is mixed with the lipid film, and then rotary evaporated, hydrated, ultrasonically dispersed to obtain a hydrated lipid film solution, i.e., a liposome solution, the liposome solution is filtered through a microporous filter membrane, and the filtrate is collected and ultrafiltered to obtain a supernatant, i.e., the liposome.

5. The preparation method according to claim 4, characterized in that, The first organic solvent comprises chloroform, and the second organic solvent comprises petroleum ether; the mass-volume ratio of the egg yolk lecithin and the first organic solvent is 15-25 mg:350-450 μL; the mass-volume ratio of the cholesterol and the first organic solvent is 3-7 mg:150-250 μL; the mass-volume ratio of the PAMP component and the second organic solvent is 5-15 μg:350-450 μL; and the mass-volume ratio of the cisplatin, the PAD4i and water is 0.5-7 mg:30-40 mg:4-8 mL.

6. The use of the liposome drug nanocarrier system of any one of claims 1 to 3 or the liposome drug nanocarrier system prepared by the preparation method of claim 4 or 5 in the preparation of an antitumor drug, characterized in that, The tumor comprises at least one of ovarian cancer, endometrial cancer, cervical cancer, breast cancer, lung cancer, bladder cancer and colon cancer.

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