CN112137958A - Doxorubicin and immunologic adjuvant-containing combined drug liposome and preparation method thereof - Google Patents

Doxorubicin and immunologic adjuvant-containing combined drug liposome and preparation method thereof Download PDF

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CN112137958A
CN112137958A CN202010485398.8A CN202010485398A CN112137958A CN 112137958 A CN112137958 A CN 112137958A CN 202010485398 A CN202010485398 A CN 202010485398A CN 112137958 A CN112137958 A CN 112137958A
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liposome
adriamycin
doxorubicin
injection
phospholipid
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陈怀文
高洁
尹川
张莹莹
杨冬梅
谢方圆
刘俊杰
于永生
贺智明
姜普
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Shanghai Shunna Biotechnology Co ltd
University of Shanghai for Science and Technology
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Shanghai Shunna Biotechnology Co ltd
University of Shanghai for Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A

Abstract

The invention provides a drug liposome containing chemical drug adriamycin and immune adjuvant combination, the nano liposome efficiently loads adriamycin and immune antigen, can stabilize long circulation in vivo, can deliver the immune antigen to a tumor microenvironment while reducing toxic and side effects of adriamycin, can play a role in chemotherapy-immune combined treatment aiming at tumors, and achieves the purpose of inhibiting tumor growth and metastasis.

Description

Doxorubicin and immunologic adjuvant-containing combined drug liposome and preparation method thereof
Technical Field
The invention relates to a medicine liposome preparation for tumor chemotherapy immune combination therapy, in particular to a nano liposome carried by adriamycin and immune adjuvant and a preparation method thereof.
Background
The clinical treatment of tumors comprises operations, chemical therapy (chemotherapy), radiotherapy (radiotherapy), targeted therapy and immunotherapy. Chemotherapy is an indispensable tool for tumor therapy in general.
The chemical therapy usually adopts systemic administration, not only killing tumor cells; it also has definite toxic and side effects on normal cells of human body, such as nausea, alopecia, hematopoietic toxicity, hematopoietic progenitor cell mobilization from bone marrow to peripheral blood reduction, anemia, bone marrow suppression, pancytopenia, thrombocytopenia, neutropenia, lymphopenia, leukopenia. These side effects often limit the dose and frequency of administration of chemotherapeutic agents.
Doxorubicin (also called doxorubicin) is a common antitumor drug, which has a wide antitumor spectrum and is suitable for acute leukemia (lymphocytic and granulocytic), malignant lymphoma, breast cancer, bronchopulmonary carcinoma (undifferentiated small cell and non-small cell), ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer, etc. But the clinical toxic and side effects are also very obvious, and the traditional Chinese medicine composition can cause severe cardiotoxicity besides bone marrow suppression, gastrointestinal toxicity and alopecia, and shows that irreversible myocardial damage and even hematopoietic heart failure can occur when various arrhythmia and large accumulation amount are caused.
The liposome is an advanced dosage form of a targeted drug delivery system, is always a hotspot dosage form for drug development, can selectively deliver drugs to target sites to play a therapeutic role, and does not influence the functions of normal cells, tissues or organs, thereby achieving the purposes of improving the curative effect and reducing toxic and side effects. The first PEGylated long circulating doxorubicin liposome drug (Doxil) approved by the FDA in 1995 was effective in reducing the cardiotoxicity of doxorubicin. Although the liposome technology effectively reduces the toxic and side effects of adriamycin, Doxil still has the problems of insufficient drug resistance and anti-tumor efficacy and the like in tumor chemotherapy, so that the growth of tumor cells is difficult to inhibit only by a single drug or preparation. Therefore, several drugs are often selected for tumor inhibition in clinic, which is called combined chemotherapy. In addition, chemotherapy can be combined with other treatment modalities (such as surgery, radiation therapy, targeted therapy and immunotherapy) to form a combination therapy for tumors. The combined chemotherapy or the comprehensive treatment has better anti-tumor effect and lower toxic and side effect, namely synergistic effect compared with a single treatment medicament or a single treatment form.
The co-carried liposome of the chemical drug adriamycin and the immunologic adjuvant is used for carrying out combined treatment aiming at tumors, so that a definite anti-tumor synergistic effect can be generated, and the in-situ killing of tumor cells is facilitated; and generate stronger tumor specific immune response, thereby inhibiting the growth and metastasis of tumors. The development of the new technology in the aspect has important practical significance for the research and development of the anti-cancer primary medicine.
Disclosure of Invention
The invention aims to provide a medicinal liposome combining chemical medicament adriamycin and immunologic adjuvant, which has the advantages that: the liposome carried by the chemical adriamycin and the immunologic adjuvant can be used for developing chemotherapy-immunization combination treatment aiming at tumors to realize synergistic effect, namely, the immunologic adjuvant amplifies the immunogenicity of tumor-related antigens after cancer cells die while adriamycin effectively kills the tumor cells, so as to initiate stronger specific immunoreaction of human bodies to the tumors and achieve the purpose of inhibiting the growth and the metastasis of the tumors.
In addition, the invention can control the particle size of the liposome containing cationic lipid to be between 50nm and 200nm, so that the encapsulation efficiency of the liposome to the immunologic adjuvant exceeds 90 percent without influencing the in vivo application stability. Meanwhile, cholesterol and antioxidant vitamin E are added into the formula, so that the stability of adriamycin or immunologic adjuvant in the liposome is greatly improved. In addition, key steps of the adriamycin and immunoadjuvant co-carried liposome in the production process can be completed by a high-efficiency micro-jet high-pressure homogenizer, and the preparation can be prepared into a freeze-dried preparation form for storage through freeze-drying, so that the industrial production of the preparation becomes possible.
The object of the invention can be achieved by the following method:
a liposome injection carrying doxorubicin and immunoadjuvant combined medicine and a preparation method thereof are characterized in that the raw material formula of each 1000ml of the injection is as follows:
500 mg-5000 mg of adriamycin or adriamycin hydrochloride
50 mg-5000 mg of immunologic adjuvant
240 mg-30000 mg of neutral phospholipid
50 mg-20000 mg of PEG phospholipid (DSPE-PEG)
2 mg-20000 mg of positive charge phospholipid
10 mg-15000 mg of cholesterol
0.25 mg-480 mg of vitamin E
20 mg-35000 mg of alkali
28-200 g of sugar
1 g-100 g of buffering agent
The volume of the water for injection is up to 1000ml
Wherein, the weight ratio of the adriamycin or the adriamycin hydrochloride to the immunologic adjuvant is 0.1: 1-100: 1, the weight ratio of the adriamycin or the adriamycin hydrochloride to the phospholipid is 0.1: 1-2: 1, the molar ratio of the positive charge phospholipid to the neutral phospholipid is 0.05: 5-2: 5, and the molar ratio of the cholesterol to the neutral phospholipid is 0.1: 1-1: 1.
The co-carried adriamycin and immunoadjuvant combined drug liposome is characterized in that neutral phospholipid can be hydrogenated soybean lecithin, yolk lecithin, hydrogenated yolk lecithin, distearic acid lecithin, soybean lecithin, double palmitic acid lecithin or double myristic acid lecithin;
preferably, the neutral phospholipid is hydrogenated soybean lecithin.
The combined drug liposome of the co-carried adriamycin and the immunologic adjuvant, wherein the immunologic adjuvant can be at least one of negative charge immunologic adjuvants CpG, PolyIC, PolyICLC, MPL-TDM and lipopolysaccharide.
Preferably, the immune adjuvant is CpG and lipopolysaccharide in a mass ratio of 1: 1.
The co-carried adriamycin and immunoadjuvant combined drug liposome is characterized in that the positively charged phospholipid can be DOTAP, DOTMA, DOSPA, DODMA, Dlin-MC3-DMA, Dlin-KC2-DMA and c 12-200.
Preferably, the positively charged phospholipid is DOTAP.
The co-carried adriamycin and immune adjuvant combined drug liposome has the molar ratio of the vitamin E to the phospholipid of 0.1: 5-1: 5.
The liposome of the drug combination of the co-carried adriamycin and the immunologic adjuvant, wherein the alkali can be calcium carbonate, sodium bicarbonate, sodium hydroxide or potassium phosphate.
The liposome of the drug combination of the co-carried adriamycin and the immunologic adjuvant, wherein the sugar can be lactose, maltose, sucrose, glucose or trehalose.
The liposome of the drug combination of the co-carried adriamycin and the immunologic adjuvant, wherein the buffering agent can be L-alanine, histidine or tromethamine.
The formulation of the combined drug liposome of the co-carried adriamycin and the immunologic adjuvant can be injection and freeze-dried powder.
The preparation method of the combined drug liposome carrying the adriamycin and the immunologic adjuvant comprises the following steps:
step one, preparing blank liposome: according to the formula, neutral phospholipid, positive charge phospholipid, cholesterol and vitamin E are added into an ethanol solution with the volume concentration of 4-10% according to a certain mass ratio, and the temperature is raised to 60 ℃ to be dissolved uniformly to form an ethanol solution for dissolving lipid for later use; preparing 250mM ammonium sulfate aqueous solution for injection, heating to 55-65 deg.C, injecting the above lipid-dissolved ethanol solution into the aqueous solution at high speed under heating condition, and extruding the formed emulsion with high jet extrusion equipment or physical extrusion through microporous membrane with corresponding pore diameter to obtain blank liposome with particle diameter of 50-200 nm. Then the solvent system of the blank liposome is replaced by 3-20% sugar water solution.
Step two, preparing the combined drug liposome carrying the adriamycin and the immunologic adjuvant together: dissolving doxorubicin hydrochloride or doxorubicin and an immunologic adjuvant in water for injection or sugar water solution with the concentration of 3-20% according to a certain proportion, heating to 40-70 ℃, uniformly mixing with the blank liposome prepared in the step one, and preserving heat at 40-70 ℃ for a period of time to prepare the liposome carrying the doxorubicin and the immunologic adjuvant together.
Step three, adjusting a preparation liposome solvent system: adjusting water for injection containing stabilizer such as antioxidant, alkali, and sugar to pH5-7 with 0.2-10% buffer, and replacing the external solution of liposome co-loaded with adriamycin and immunoadjuvant by dialysis filtration method to disperse the co-loaded liposome in isotonic solution similar to human physiological environment.
Step four, volume fixing, sterilization, split charging and preservation: diluting with injectable water to desired volume, filtering doxorubicin or doxorubicin hydrochloride liposome suspension with microporous membrane for sterilization, and packaging to obtain the final product, which can be stored at 2-8 deg.C or lyophilized for storage.
Drawings
FIG. 1 is a transmission electron micrograph of the adriamycin and immunoadjuvant co-loaded liposome ADR/CpG & LPS-Lip of the invention.
FIG. 2 is a particle size distribution diagram of the adriamycin and immunoadjuvant co-loaded liposome ADR/CpG & LPS-Lip of the invention.
FIG. 3 is a transmission electron micrograph of the adriamycin and immunoadjuvant co-loaded liposome ADR/PolyICLC-Lip of the invention.
FIG. 4 is a distribution diagram of the particle size of ADR/PolyICLC-Lip liposome co-loaded with adriamycin and immunoadjuvant according to the present invention.
FIG. 5 is a transmission electron micrograph of the liposome ADR/MPL-TDM-Lip co-loaded with adriamycin and immunoadjuvant of the present invention.
FIG. 6 is a particle size distribution diagram of the liposome ADR/MPL-TDM-Lip co-loaded with doxorubicin and immunoadjuvant according to the present invention.
FIG. 7 is a graph of the in vitro release profile of doxorubicin in liposomes co-loaded with immunoadjuvant in accordance with an embodiment of the present invention.
FIG. 8 shows the activation state of granzyme B in the tumor microenvironment after treatment of the liposome co-loaded with doxorubicin and immunoadjuvant according to the present invention.
FIG. 9 shows the tumor growth curve of the animal model for treating liver cancer transplantable tumor by using the adriamycin and immunoadjuvant co-carried liposome of the present invention.
FIG. 10 shows the tumor growth curve of the animal model for treating breast cancer transplantable tumor by using doxorubicin and immunoadjuvant co-loaded liposome.
FIG. 11 is a graph showing the evaluation of the toxic effect of the liposome co-loaded with doxorubicin and immunoadjuvant of the present invention on tissues and organs in vivo.
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1: preparation of Adriamycin and CpG & lipopolysaccharide Co-loaded liposomes (ADR/CpG & LPS-Lip)
Formulation recipe (50 ml volume):
adriamycin hydrochloride 100mg
CpG 10mg
Lipopolysaccharide 10mg
Distearic acid lecithin (DSPC) 640mg
Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000)220mg
DOTAP 140mg
Cholesterol 190mg
Vitamin E2.4 mg
Citric acid 1120mg
Anhydrous sodium carbonate 1600mg
Sucrose is about 4500mg
Glycine about 50mg
The injection water is fixed to the required volume
The preparation process comprises the following steps:
according to the formula, DSPC, DSPE-PEG2000, DOTAP, cholesterol and vitamin E are selected and mixed evenly in 4% ethanol solution and heated to 50 ℃; dissolving doxorubicin hydrochloride, CpG and lipopolysaccharide with 250mM ammonium sulfate, heating to 55-65 deg.C, rapidly injecting lipid solution under heating to form emulsion, and extruding with high jet extruder to obtain blank liposome with particle size of 50-200 nm. Then 5% sugar water solution is dialyzed to replace the solvent system of the blank liposome. Dissolving doxorubicin hydrochloride and an immunologic adjuvant in 5% sugar water solution, heating to 50 ℃, uniformly mixing with blank liposome suspension, preserving heat at 50 ℃ for 3 hours to obtain liposome carrying doxorubicin and the immunologic adjuvant together, continuing to replace the liposome dispersion with an external solution of 1.0% glycine and ammonium bicarbonate for adjusting the pH to 6.0-7.0, fixing the volume with sugar-containing injection water and adjusting the concentration of the doxorubicin to 2.0mg/ml, filtering and sterilizing the doxorubicin and immunologic adjuvant combined drug liposome suspension by using a microporous membrane with the aperture of 0.22 micrometer, subpackaging to obtain a finished product, and storing the finished product at the temperature of 2-8 ℃ or freeze-drying and storing for use. And detecting the appearance shape and the particle size of the produced liposome preparation by using a transmission electron microscope and a Malvern laser particle sizer. The transmission electron microscope results are shown in detail in FIG. 1, and the particle size results are shown in FIG. 2.
Example 2: preparation of Doxorubicin and PolyICLC Co-carried liposomes (ADR/PolyICLC-Lip)
Formulation recipe (50 ml volume):
adriamycin hydrochloride 100mg
PolyICLC 20mg
Hydrogenated Soybean lecithin (HSPC) 760mg
Distearoylphosphatidylethanolamine-polyethylene glycol 3400 (DSPE-PEG 3400) 220mg
Dlin-MC3-DMA 220mg
Cholesterol 220mg
Vitamin E2.0 mg
Succinic acid 600mg
340mg of sodium hydroxide
Lactose about 2250mg
Glycine about 70mg
The injection water is fixed to the required volume
The preparation process comprises the following steps:
according to the formula, HSPC, DSPE-PEG3400, Dlin-MC3-DMA, cholesterol and vitamin E are selected and mixed evenly in a 4% ethanol solution and the temperature is raised to 65 ℃; and dissolving doxorubicin hydrochloride and PolyICLC in 250mM ammonium sulfate, heating to 55-65 deg.C, rapidly injecting lipid solution under heating to form emulsion, and extruding with high-jet extruder to obtain blank liposome with particle size of 150 + -10 nm. Then 5% sugar water solution is dialyzed to replace the solvent system of the blank liposome. Dissolving doxorubicin hydrochloride and an immunologic adjuvant in a 5% sugar water solution, heating to 50 ℃, uniformly mixing with the blank liposome suspension, preserving heat at 50 ℃ for 3 hours to obtain liposome carrying the doxorubicin and the immunologic adjuvant together, continuing to replace the liposome dispersion liquid with an external solution of 1.0% glycine and sodium hydroxide for adjusting the pH to 7.3, fixing the volume with sugar-containing injection water, adjusting the concentration of the doxorubicin to 2.0mg/ml, filtering and sterilizing the doxorubicin and immunologic adjuvant combined drug liposome suspension by using a microporous membrane with the pore diameter of 0.22 micrometer, and subpackaging to obtain a finished product, wherein the finished product can be stored at 2-8 ℃ or freeze-dried and stored for use. And detecting the appearance shape and the particle size of the produced liposome preparation by using a transmission electron microscope and a Malvern laser particle sizer. The transmission electron microscope results are shown in detail in FIG. 3, and the particle size results are shown in FIG. 4.
Example 3: preparation of Adriamycin and MPL-TDM Co-loaded liposomes (ADR/MPL-TDM-Lip)
Formulation recipe (50 ml volume):
adriamycin hydrochloride 100mg
MPL-TDM 35mg
Distearoyl phosphatidyl glycerol (DSPG) 160mg
Distearoylphosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG 5000) 220mg
Egg yolk phosphatidyl glycerol (EPG) 240mg
c12-200 220mg
Cholesterol 220mg
Vitamin E1.6 mg
Citric acid 1240mg
520mg of sodium hydroxide
Sucrose (about 2250 mg)
Glycine about 50mg
The injection water is fixed to the required volume
The preparation process comprises the following steps:
according to the formula, DSPG, EPG, c12-200, DSPE-PEG5000, cholesterol and vitamin E are selected and mixed evenly in 4% ethanol solution and heated to 60 ℃; and dissolving doxorubicin hydrochloride and MPL-TDM with ammonium sulfate with the concentration of 250mM, heating to 60 ℃, quickly injecting the lipid solution into the solution under a heating condition to form emulsion, and preparing blank liposome with the particle size range of 120 +/-10 nm by using microporous membrane basic equipment. Then 5% sugar water solution is dialyzed to replace the solvent system of the blank liposome. Dissolving doxorubicin hydrochloride and an immunologic adjuvant in 5% sugar water solution, heating to 50 ℃, uniformly mixing with blank liposome suspension, preserving heat at 50 ℃ for 3 hours to obtain liposome carrying the doxorubicin and the immunologic adjuvant together, continuing to replace the liposome dispersion with an external solution of 1.0% glycine and sodium hydroxide for adjusting the pH to 7.0 +/-0.5, fixing the volume with sugar-containing injection water and adjusting the concentration of the doxorubicin to 2.0mg/ml, filtering and sterilizing the doxorubicin and immunologic adjuvant combined drug liposome suspension by using a microporous membrane with the aperture of 0.22 micrometer, subpackaging to obtain a finished product, and storing the finished product at the temperature of 2-8 ℃ or freeze-drying and storing for use. And detecting the appearance shape and the particle size of the produced liposome preparation by using a transmission electron microscope and a Malvern laser particle sizer. The transmission electron microscopy results are detailed in FIG. 5 and the particle size results are detailed in FIG. 6.
Example 4: detecting the property parameters of the liposome of the adriamycin and immunologic adjuvant combined medicament
Measuring the particle size and zeta potential value of the ADR/CpG-LPS-Lip, ADR/PolyICLC-Lip and ADR/MPL-TDM-Lip solutions prepared in the examples 1-3 by using a laser particle size analyzer; performing high performance liquid chromatography (HPLC, Agilent, USA; chromatography conditions: octadecylsilane chemically bonded silica is used as filler, sodium dodecyl sulfate solution-acetonitrile-methanol is used as mobile phase, detection wavelength is 254nm, and reference reagents comprise doxorubicin hydrochloride reference substance and hydrochloric acidEpirubicin control, flow rate 1 ml/min; column temperature 25 ℃), measuring the adriamycin content in the liposome, and calculating the encapsulation rate of the adriamycin in the liposome, wherein the formula is as follows: doxorubicin encapsulation = MADR encapsulation/MADR inputX 100%, the results are detailed in table 1.
Figure 281377DEST_PATH_IMAGE001
Example 5: in-vitro drug release curve for detecting adriamycin and immunologic adjuvant combined drug liposome
2ml solutions of ADR/CpG & LPS-Lip, ADR/PolyICLC-Lip, and ADR/MPL-TDM-Lip prepared in examples 1 to 3 were placed in 300KD dialysis bags, respectively, and placed in a beaker of 1000ml of PBS solution (pH = 7.4) to be subjected to constant temperature water bath shaking (temperature 37 ℃, shaking rate 110 rpm), and the counting from zero was started. At set time points ( hours 0, 2, 5, 24, 36, 120, 240), the solution in the dialysis bag was aspirated and the content of doxorubicin was measured by high performance liquid chromatography (parameters for measurement were as in example 4). The in vitro release rate of the drug doxorubicin was calculated. The results are shown in example 7, where the drug release of various doxorubicin liposomes was more than 90% accumulated over 10 days.
Example 6: determination of Adriamycin and immune adjuvant combination drug liposome for improving the increase of the number of tumor-specific CD8+ T cells in tumor microenvironment
HepG2 liver cancer and 7721 breast cancer cells are used for establishing a liver cancer or breast cancer transplantation animal model in a C57BL6 mouse, when the tumor volume is 100 cubic millimeters, the liver cancer or breast cancer transplantation animal model is divided into groups randomly (as follows), and the groups are respectively administrated for treatment in 1, 8, 11, 15, 18, 22 and 25 days, wherein the single administration dose of the adriamycin is 2mg/kg, and the dose of the same immunologic adjuvant is the same. On day 26, mice were sacrificed and tumors were removed for flow cytometry analysis and the number of tumor-specific CD8+ T cells in the tumor microenvironment was detected by SIINFEKL-Kb tetramer staining (tumor antigen-specific T cells: CD3 +/CD 8+/SIINFEKL-H2Kb tetramer +). CD8+ T activation status in the tumor microenvironment was analyzed by staining with granzyme B:
grouping 1: physiological saline group
Grouping 2: adriamycin liposome
Grouping 3: adriamycin liposome + subcutaneous injection CpG and LPS mixed adjuvant group
Grouping 4: adriamycin liposome + subcutaneous injection PolyICLC group
Grouping 5: adriamycin liposome + subcutaneous injection MPL-TDM group
Grouping 6: ADR/CpG & LPS-Lip group
Grouping 7: ADR/PolyICLC-Lip group
Grouping 8: ADR/MPL-TDM-Lip group
By flow detection of SIINFEKLH2Kb tetramer homologous antigen, tumor-specific T cells were in a low level state in the saline group, and compared with CD8+ T cells isolated from the saline-treated control group (set as 1), CD8+ T cells extracted from the tumor microenvironment of mice in each treatment group are shown in table 2 below, and it can be seen that the liposome of the doxorubicin and immunoadjuvant combined drug significantly increased the number of CD8+ T cells in the tumor microenvironment.
Figure 541500DEST_PATH_IMAGE002
Example 7: detecting that CD8+ T cells in tumor microenvironment are in an activated state caused by adriamycin and immune adjuvant combined drug liposome
An animal model of liver cancer or breast cancer transplantable tumor is established in a C57BL6 mouse by using SiHa cervical cancer cells, when the tumor volume is about 150 cubic millimeters, the tumor volume is divided into groups randomly (as follows), and the groups are respectively administrated for treatment in 8, 11, 15, 18, 22 and 25 days, wherein the single administration dose is unified to be 2mg/kg of adriamycin, and the doses of the same immunologic adjuvant are the same. On day 26, mice were sacrificed and tumors were removed for formalin fixation. After tissue sectioning, CD8+ T activation status in the tumor microenvironment was analyzed using granzyme B staining:
grouping 1: physiological saline group
Grouping 2: adriamycin liposome
Grouping 3: adriamycin liposome + subcutaneous injection CpG and LPS mixed adjuvant group
Grouping 4: adriamycin liposome + subcutaneous injection PolyICLC and MPL-TDM mixed adjuvant group
Grouping 5: ADR/CpG & LPS-Lip group
Grouping 6: ADR/PolyICLC-Lip group
Grouping 7: ADR/MPL-TDM-Lip group
By comparing granzyme B staining after treatment of SiHa cervical cancer tissue sections for each group (fig. 8), the results show: compared with subcutaneous adjuvant-only immunization group, the liposome of the doxorubicin and immunoadjuvant combined drug can induce CD8+ T cells to be in a more strongly activated state (dense staining and darker color).
Example 9: determining the treatment effect of the adriamycin and immunologic adjuvant combined drug liposome on 7721 breast cancer cell transplantation tumor mouse model
An animal model of breast cancer transplantable tumor is established in a C57BL6 mouse by utilizing HepG2 liver cancer and 7721 breast cancer cells, when the tumor volume is 150 cubic millimeters, every 5 random (as shown below) groups are respectively administrated for treatment in 8, 11, 15, 18, 22 and 25 days, wherein the single administration dose of adriamycin is 2mg/kg, and the dose of the same immunologic adjuvant is the same. Tumor volume was then determined at different time points with a vernier caliper, the formula for tumor volume calculation: tumor volume = (length × width 2)/2. The results of the tumor growth curve are shown in fig. 9 and 10, the adriamycin and immunoadjuvant combined drug liposome shows good antitumor activity, effectively improves the number and activation state of CD8+ T cells in a tumor microenvironment, and realizes a tumor inhibition effect in an animal body:
grouping 1: physiological saline group
Grouping 2: adriamycin liposome
Grouping 3: adriamycin liposome + subcutaneous injection CpG and LPS mixed adjuvant group
Grouping 4: adriamycin liposome + subcutaneous injection PolyICLC and MPL-TDM mixed adjuvant group
Grouping 5: ADR/CpG & LPS-Lip group
Grouping 6: ADR/PolyICLC-Lip group
Grouping 7: ADR/MPL-TDM-Lip group.
Example 8: in vivo organ toxicity of adriamycin and immunologic adjuvant combined drug liposome
15 male healthy SD rats are randomly divided into three groups, each group comprises 5 rats, 1ml of ADR/CpG & LPS-Lip group (adriamycin dose is 1 mg/ml) is injected into the tail vein, the rats are killed after the adriamycin is injected once every other day and the third continuous injection, the major organs such as liver and kidney are cored, the rats are fixed by formalin, sliced and subjected to HE staining, whether the adriamycin and immunoadjuvant combined drug liposome has toxic and side effects on the important organs of the animals is observed, the specific HE result is shown in figure 11, and the toxic injury of the major organs of the animals is not found.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A liposome injection carrying doxorubicin and immunoadjuvant combined medicine and a preparation method thereof are characterized in that the raw material formula of each 1000ml of the injection is as follows:
500 mg-5000 mg of adriamycin or adriamycin hydrochloride
50 mg-2500 mg of immunologic adjuvant
240 mg-30000 mg of neutral phospholipid
10 mg-10000 mg of PEG phospholipid (DSPE-PEG)
2 mg-20000 mg of positive charge phospholipid
10 mg-15000 mg of cholesterol
0.25 mg-480 mg of vitamin E
20 mg-35000 mg of alkali
28-200 g of sugar
1 g-100 g of buffering agent
The volume of the water for injection is up to 1000ml
Wherein, the weight ratio of the adriamycin or the adriamycin hydrochloride to the immunologic adjuvant is 0.1: 1-2: 1, the weight ratio of the adriamycin or the adriamycin hydrochloride to the phospholipid is 0.1: 1-2: 1, the molar ratio of the positive charge phospholipid to the neutral phospholipid is 0.05: 5-2: 5, and the molar ratio of the cholesterol to the neutral phospholipid is 0.1: 1-1: 1.
2. The liposome of claim 1, wherein the liposome comprises a neutral phospholipid selected from the group consisting of hydrogenated soybean lecithin, egg yolk lecithin, hydrogenated egg yolk lecithin, distearoyl lecithin, soybean lecithin, dipalmitoyl lecithin and myristic acid lecithin.
3. The liposome of claim 1, wherein the liposome comprises an immunological adjuvant selected from at least one of the group consisting of immunological adjuvants CpG, PolyIC, PolyICLC, MPL-TDM, and lipopolysaccharide.
4. The liposome of claim 1, comprising a positively charged phospholipid selected from the group consisting of DOTAP, DOTMA, DOSPA, DODMA, Dlin-MC3-DMA, Dlin-KC2-DMA, and c 12-200.
5. The liposome injection of claim 1, wherein the molar ratio of vitamin E to phospholipid is 0.1: 5-1: 5.
6. The liposomal injection of doxorubicin and immunoadjuvant co-loaded pharmaceutical according to claim 1, wherein the base comprises calcium carbonate, sodium bicarbonate, sodium hydroxide, or potassium phosphate.
7. The liposome injection carrying doxorubicin and immunoadjuvant combination drug of claim 1, characterized in that the sugar is lactose, maltose, sucrose, glucose or trehalose.
8. The liposomal injection of doxorubicin and immunoadjuvant co-loaded pharmaceutical according to claim 1, characterized in that the buffer can be L-alanine, histidine or tromethamine.
9. The liposome injection carrying doxorubicin and immunoadjuvant combination drug according to claim 1, characterized in that the dosage form can be injection or lyophilized powder.
10. The method for preparing the liposome injection carrying the combination of adriamycin and immunoadjuvant of claim 1, which comprises the following steps:
the method comprises the following steps: preparing blank liposome: according to the formula, neutral phospholipid, positive charge phospholipid, cholesterol and vitamin E are added into an ethanol solution with the volume concentration of 4-10% according to a certain mass ratio, and the temperature is raised to 60 ℃ to be dissolved uniformly to form an ethanol solution for dissolving lipid for later use; preparing 250mM ammonium sulfate aqueous solution for injection, heating to 55-65 deg.C, injecting the above lipid-dissolved ethanol solution into the emulsion at high speed under heating condition, extruding the emulsion with high jet extrusion equipment or physically extruding through microporous membrane with corresponding pore diameter to obtain blank liposome with particle diameter of 50-200nm, and replacing the solvent system of the blank liposome with 3-20% sugar water solution;
step two: preparing a liposome carrying the adriamycin and the immunologic adjuvant combination drug: dissolving doxorubicin hydrochloride or doxorubicin and an immunologic adjuvant in water for injection or sugar water solution with the concentration of 3-20% according to a certain proportion, heating to 40-70 ℃, uniformly mixing with the blank liposome prepared in the step one, and preserving heat at 40-70 ℃ for a period of time to prepare liposome carrying the doxorubicin and the immunologic adjuvant together;
step three: adjusting the liposome solvent system of the preparation: adjusting water for injection containing stabilizer such as antioxidant, alkali, and sugar to pH5-7 with 0.2-10% buffer, replacing the external solution of liposome co-loaded with adriamycin and immunoadjuvant by dialysis filtration method, and dispersing the co-loaded liposome in isotonic solution similar to human physiological environment;
step four: volume fixing, sterilization, split charging and preservation: diluting with injectable water to desired volume, filtering doxorubicin or doxorubicin hydrochloride liposome suspension with microporous membrane for sterilization, and packaging to obtain the final product, which can be stored at 2-8 deg.C or lyophilized for storage.
CN202010485398.8A 2020-06-03 2020-06-03 Doxorubicin and immunologic adjuvant-containing combined drug liposome and preparation method thereof Pending CN112137958A (en)

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