WO2018033118A1 - 一种全反式维甲酸脂质体制剂及其制备与应用 - Google Patents

一种全反式维甲酸脂质体制剂及其制备与应用 Download PDF

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WO2018033118A1
WO2018033118A1 PCT/CN2017/097869 CN2017097869W WO2018033118A1 WO 2018033118 A1 WO2018033118 A1 WO 2018033118A1 CN 2017097869 W CN2017097869 W CN 2017097869W WO 2018033118 A1 WO2018033118 A1 WO 2018033118A1
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liposome
trans retinoic
retinoic acid
preparation
calcium acetate
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PCT/CN2017/097869
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French (fr)
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徐宇虹
郑安杰
陈晓龙
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上海交通大学
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Priority to EP17841090.8A priority patent/EP3501500B1/en
Priority to ES17841090T priority patent/ES2919999T3/es
Publication of WO2018033118A1 publication Critical patent/WO2018033118A1/zh

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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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • 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
    • 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

Definitions

  • the invention belongs to the technical field of biopharmaceuticals, and particularly relates to an all-trans retinoic acid liposome preparation and preparation and application thereof.
  • Retinoic acid is a metabolite of vitamin A in the body.
  • All-trans retinoic acid (ATRA) is used as a drug to treat acne, and is also an important drug for the clinical treatment of acute promyelocytic leukemia (APL).
  • All-trans retinoic acid (ATRA) affects gene expression by binding to specific receptors (RARs, RXRs and RORs) in cells, and promotes APL cell differentiation and PML/RAR ⁇ gene in the treatment of acute promyelocytic leukemia Degradation, to achieve the effect of treatment.
  • all-trans retinoic acid drugs is limited by the following aspects: 1 all-trans retinoic acid has very low water solubility (4.77e-03g/l); 2 all-trans retinoic acid has a short half-life plasma To exert its efficacy, it is necessary to maintain a certain blood concentration for a long period of time, and the concentration of the drug in the target organ. Therefore, it is particularly important to choose a drug delivery method suitable for all-trans retinoic acid.
  • Liposomes have been used as a nano drug delivery vehicle for more than 30 years.
  • a variety of anticancer drugs based on liposome delivery systems have been widely used in clinical treatment of tumors.
  • the most successful drug is doxorubicin liposome.
  • the advantage of liposome as a drug delivery system is that it changes the biodistribution of the drug, reduces the systemic toxicity of the drug, and can be achieved by specific modification on the liposome.
  • the long circulation/targeting effect increases the drug concentration of the target tissue.
  • all-trans retinoic liposome formulations incorporate all-trans retinoic acid into the phospholipid bilayer. Due to the physical properties of all-trans retinoic acid, the all-trans retinoic acid liposome produced by this method is not ideal in terms of drug loading and stability in vivo.
  • an all-trans retinoic acid liposome formulation comprising all-trans retinoic acid and a liposome carrier.
  • the all-trans retinoic liposome formulation further contains a solubilizing molecule for increasing the solubility of all-trans retinoic acid.
  • the solubilizing molecule is selected from any one or a combination of PVP, HPMC, cyclodextrin, PEG.
  • the molar ratio between the solubilizing molecule and the all-trans retinoic acid ranges from (38 to 0.075):1.
  • the cyclodextrin is selected from hydroxypropyl- ⁇ -cyclodextrin, sulfobutyl- ⁇ -cyclodextrin or methyl- ⁇ -cyclodextrin.
  • the PEG is selected from PEG-400.
  • the molar ratio between the solubilizing molecule and the all-trans retinoic acid may range from (20 to 2):1.
  • the molar ratio between the solubilizing molecule and the all-trans retinoic acid may range from (1.5 to 0.075):1.
  • the molar ratio between the solubilizing molecule and the all-trans retinoic acid may range from (38 to 9.5):1.
  • the molar ratio of the drug to the all-trans retinoic acid and the liposome carrier ranges from 1: (5 to 20).
  • the raw material of the liposome contains phospholipids, cholesterol and pegylated phospholipids.
  • the molar ratio between the phospholipid, cholesterol and the pegylated phospholipid in the raw material of the liposome is: (30 to 80): (0.1 to 40): (0.1 to 30).
  • the liposome contains HSPC, CHOL and DSPE-PEG in the raw material.
  • the molar ratio between HSPC, CHOL, and DSPE-PEG in the raw material of the liposome ranges from (30 to 80): (0.1 to 40): (0.1 to 30). More preferably, the molar ratio between HSPC, CHOL, DSPE-PEG in the raw material of the liposome ranges from 57:38:5.
  • the molecular weight of PEG in the DSPE-PEG ranges from 50 to 10,000. More preferably, the molecular weight of PEG in the DSPE-PEG is 2000.
  • the liposome is a single compartment liposome.
  • the liposome has a particle size ranging from 30 nm to 200 nm. More preferably, the liposome has a particle size ranging from 50 to 150 nm. More preferably, the liposome has a particle diameter of from 70 nm to 130 nm.
  • the liposome has a narrow particle size distribution, and its D95 (having a particle size smaller than 95% of the total amount of the liposome) is 120 nm or less. More preferably, D95 is less than or equal to 110 nm, and more preferably, D95 is less than or equal to 95 nm.
  • the concentration of the all-trans retinoic acid is 0.1 mg/ml or more. Further preferably, in the all-trans retinoic liposome preparation, the concentration of the all-trans retinoic acid is 0.5 mg/ml or more. More preferably, in the all-trans retinoic liposome formulation, the concentration of the all-trans retinoic acid ranges from 0.5 to 5 mg/ml.
  • the all-trans retinoic liposome formulation is an injectable formulation.
  • the injection administration preparation is selected from the group consisting of a subcutaneous injection dosage form, an intravenous injection dosage form, an intramuscular injection dosage form or a pelvic injection dosage form.
  • a method of preparing an all-trans retinoic acid liposome formulation which is an active drug loading method.
  • the preparation method is a calcium acetate gradient method.
  • sodium acetate gradient method is a calcium acetate gradient method.
  • the calcium acetate gradient method comprises the steps of:
  • the inner and outer aqueous phases prepared in step (3) are all aqueous calcium acetate aqueous liposomes are placed in an isotonic liquid having a pH of 6.0 to 7.0, and a blank of calcium acetate gradient between the inner and outer aqueous phases is obtained.
  • step (4) adding all-trans retinoic acid suspension to the blank liposome with calcium acetate gradient between the inner and outer aqueous phases obtained in step (4), incubating, removing free all-trans retinoic acid, ie obtaining all-trans dimension Formate liposome preparation.
  • the concentration of calcium acetate in the aqueous calcium acetate solution is 120 mM to 360 mM.
  • the calcium acetate aqueous solution contains a solubilizing molecule.
  • a calcium acetate gradient exists between the inner and outer aqueous phases of the liposome obtained in step (4).
  • the inner aqueous phase of the liposome obtained in the step (4) is an aqueous calcium acetate solution having a pH of 7.0 to 11.0
  • the external aqueous phase is an isotonic solution having a pH of 6.0 to 7.0.
  • the isotonic solution is selected from the group consisting of HEPEs buffer solution containing 0.9% NaCl or 10% sucrose at a concentration of 10 mM.
  • the sodium acetate gradient method comprises the steps of:
  • the inner and outer aqueous phases prepared in step (3) are all aqueous sodium acetate aqueous liposomes are placed in an isotonic liquid having a pH of 6.0 to 7.0, and a blank of sodium acetate gradient between the inner and outer aqueous phases is obtained.
  • step (4) adding all-trans retinoic acid suspension to the blank liposome with sodium acetate gradient between the inner and outer aqueous phases obtained in step (4), incubating, removing free all-trans retinoic acid, ie obtaining all-trans virgin Formate liposome preparation.
  • the concentration of calcium acetate in the aqueous solution of sodium acetate is 120 mM to 360 mM.
  • the sodium acetate aqueous solution contains a solubilizing molecule.
  • a gradient of sodium acetate is present between the inner and outer aqueous phases of the liposomes obtained in step (4).
  • the inner aqueous phase of the liposome obtained in the step (4) is an aqueous calcium acetate solution having a pH of 7.0 to 11.0
  • the external aqueous phase is an isotonic solution having a pH of 6.0 to 7.0.
  • the isotonic solution is selected from the group consisting of HEPEs buffer solution containing 0.9% NaCl or 10% sucrose at a concentration of 10 mM.
  • the present inventors have found that the method can be used to increase the solubility or drug concentration of all-trans retinoic acid in all-trans retinoic liposome formulations.
  • the present inventors have also discovered that the method can be used to increase the effect of all-trans retinoic acid on tumor-associated macrophages in all-trans retinoic liposome formulations.
  • the present inventors have also found that the method can be used to enhance the effect of all-trans retinoic acid on myeloid suppressor cells in all-trans retinoic liposome formulations.
  • a third aspect of the invention there is provided the use of the aforementioned all-trans retinoic acid preparation for the preparation of a medicament for the treatment of a tumor.
  • the tumor therapeutic agent is a drug for abnormal myeloid suppressor cells, differentiation of myeloid suppressor cells, inhibition of tumor proliferation and recurrence in a tumor patient.
  • the myeloid suppressor cells are breast cancer, colon cancer, ovarian cancer, lung cancer, kidney cancer, stomach cancer, liver cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer, colorectal cancer, Myeloid suppressor cells of basal cell carcinoma, melanoma, follicular lymphoma or small lymphoma.
  • the invention also provides the use of all-trans retinoic acid or the aforementioned all-trans retinoic acid in the preparation of a medicament selected from any one or more of the following:
  • a method of treating a tumor comprising the step of administering to a patient an all-trans retinoic acid formulation as described above.
  • the particular dosage administered will be within the purview of those skilled in the art.
  • the present invention has the following beneficial effects:
  • the present invention is the first application of an active drug loading method to load all-trans retinoic acid to form an all-trans retinoic acid liposome preparation having a high drug loading amount and stable in vivo.
  • the all-trans retinoic acid drug is embedded in a liposome phospholipid bilayer membrane or bound to a liposome phospholipid bilayer membrane or encapsulated in a liposome capsule.
  • the all-trans retinoic acid drug is embedded in the liposome phospholipid bilayer membrane, meaning that all-trans retinoic acid is partially or completely encapsulated in the hydrophobic layer of the phospholipid bilayer membrane by hydrophobic action; the all-trans dimension
  • the combination of formic acid on the liposome phospholipid bilayer membrane means that the all-trans retinoic acid is bound to the liposome phospholipid bilayer membrane by interaction with a phospholipid bilayer through charge, hydrophobic, physical or chemical adsorption;
  • the all-trans retinoic acid drug is encapsulated in the liposome capsule. It refers to all-trans retinoic acid in the form of a soluble or all-trans retinoic acid mono- or all-trans-metamate precipitate.
  • the layer is encapsulated in a liposome capsule.
  • the all-trans retinoic acid lipid prepared by the present invention has higher drug loading and better in vivo stability than other types of all-trans retinoic acid lipid. Moreover, the all-trans retinoic acid lipid prepared by the invention has a solubility of all-trans retinoic acid increased by at least 100 times from 0.01 mg/ml in the prior art by at least 0.1 mg/ml.
  • Figure 1A Formulation No. 1, the particle size of liposomes before and after drug delivery.
  • Figure 1B Formulation No. 6, the particle size of the liposomes before and after drug delivery.
  • Figure 2 Final drug loading achieved after removal of free drug by calcium acetate aqueous liposomes and non-calcium acetate aqueous liposomes.
  • Figure 4A MDSC inhibition assay of No. 1 preparation all-trans liposome preparation on mouse tumor site cells.
  • Figure 4B MDSC inhibition assay of No. 6 preparation all-trans liposome preparation on mouse tumor site cells.
  • Figure 5A Formulation No. 1 All-trans liposome formulation promotes MDSC differentiation into mature DCs in mouse tumor site cells.
  • Figure 5B Formulation No. 1 All-trans liposome formulation promotes MDSC differentiation into mature DCs in mouse tumor site cells.
  • Figure 5C Formulation No. 6 All-trans liposome formulation promotes MDSC differentiation into mature DCs in mouse tumor site cells.
  • Figure 5D Formulation No. 6 All-trans liposome formulation promotes MDSC differentiation into mature DCs in mouse tumor site cells.
  • Fig. 6A Formulation No. 1 of the present invention All-trans retinoic acid liposome promotes differentiation of MDSC into DC cells in the blood of patients with head and neck mucosal squamous cell carcinoma.
  • Figure 6B Formulation No. 6 of the present invention All-trans retinoic acid liposome promotes differentiation of MDSC into DC-type cells in the blood of patients with head and neck mucosal squamous cell carcinoma.
  • Fig. 7A The all-trans retinoic acid liposome of the present invention is applied to tumor tissues in tumor patients, which can significantly reduce the number of CD33+HLA-DR-MDSC in bladder cancer cells infiltrating into the myeloid cells.
  • Fig. 7B The all-trans retinoic acid liposome of the invention No. 6 preparation acts on tumor tissues in tumor patients, and can significantly reduce the number of CD33+HLA-DR-MDSC in the infiltrating myeloid cells of bladder cancer tumors.
  • Fig. 8A The all-trans retinoic acid liposome of the present invention is applied to tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • Fig. 8B The No. 6 preparation of the present invention has all-trans retinoic acid liposome acting on tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • Figure 9 PK profile of rats in Example 6 administered by tail vein injection.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman And AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • Hydrogenated soybean phosphatidylcholine was purchased from NOF Corporation, pegylated phospholipid: distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and cholesterol purchased from Avanti Polar Lipids, USA; All-trans retinoic acid was purchased from Sigma.
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • the liposome obtained in the step (3) is dialyzed through a 10000 pore size dialysis membrane in a 10% by mass aqueous solution of sucrose having a pH of 6 to 7, and the outer aqueous phase of the liposome is replaced with 10%.
  • a sucrose aqueous solution having a mass fraction and a pH of 6 to 7 gives a calcium acetate liposome having a phospholipid bilayer membrane and a bilateral pH inside and outside aqueous phase having a certain pH gradient and an ion gradient.
  • the aqueous phase in the bilayer membrane is an aqueous solution of calcium acetate (pH 9.0, concentration: 200 mM), and the aqueous phase of the bilayer membrane is an aqueous solution of sucrose (pH). It is 6 to 7 and the mass fraction is 10%).
  • the all-trans retinoic acid liposome was prepared by the above method by using a molar ratio of all-trans retinoic acid to the liposome carrier of 1:5.
  • the step (1) is:
  • the all-trans retinoic acid liposome obtained by the blank liposome prepared under this condition and 8.175 mg of all-trans retinoic acid was recorded as the No. 2 preparation.
  • the all-trans retinoic acid liposome was prepared by the above method by using a molar ratio of all-trans retinoic acid to the liposome carrier of 1:20.
  • the step (1) is:
  • the all-trans retinoic acid liposome obtained by preparing blank liposome prepared under this condition and 8.175 mg of all-trans retinoic acid was recorded as No. 3 preparation.
  • the all-trans retinoic acid preparation having a drug-to-lipid ratio (molar ratio) of 0.08 was prepared by referring to the above preparation method, and was designated as No. 4 preparation.
  • the all-trans retinoic acid preparation having a drug-to-lipid ratio (molar ratio) of 0.12 was prepared by referring to the above preparation method, and was designated as No. 5 preparation.
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • the liposome obtained in the step (3) is passed through a 0.2 ⁇ m sterile filtration membrane and then dialyzed in a 10% by mass aqueous solution of sucrose having a pH of 7 to obtain a certain ion having a water phase inside and outside the phospholipid membrane.
  • Gradient calcium acetate liposomes Specifically, the aqueous phase in the two-layer membrane was an aqueous solution of calcium acetate (pH 9.0, concentration: 200 mM), and the aqueous phase of the two-layer membrane was an aqueous solution of sucrose (mass fraction: 10%).
  • the all-trans retinoic acid preparation having a drug-to-lipid ratio (molar ratio) of 0.20 is prepared, and specifically includes the following steps:
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to obtain an aqueous phase having an average particle diameter of about 80 nm. Liposomes;
  • the liposome obtained in the step (3) is dialyzed through a 10000 pore size dialysis membrane in a 10% by mass aqueous solution of sucrose having a pH of 6 to 7, and the outer aqueous phase of the liposome is replaced with 10%.
  • a sucrose aqueous solution having a mass fraction and a pH of 6 to 7 gives a calcium acetate liposome having a phospholipid bilayer membrane and a bilateral pH inside and outside aqueous phase having a certain pH gradient and an ion gradient.
  • the aqueous phase in the two-layer membrane is an aqueous solution of calcium acetate (pH 9.0, concentration: 200 mM), and the aqueous phase of the two-layer membrane is an aqueous solution of sucrose (pH of 6 to 7, mass fraction of 10%).
  • the encapsulation efficiency of the all-trans retinoic liposome of the preparation No. 3 was about 95%.
  • the encapsulation efficiency of the all-trans retinoic liposome of the preparation No. 4 was about 92%.
  • the encapsulation efficiency of the all-trans retinoic liposome of the preparation No. 5 was about 93%.
  • the encapsulation efficiency of the all-trans retinoic liposome of the preparation No. 6 was about 95%.
  • the preparation of the No. 1 preparation all-trans retinoic liposome preparation had an average particle diameter of about 70 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • Formulation No. 2 all-trans retinoic acid liposome preparation has an average particle size of about 75 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • PDI particle size distribution
  • the preparation of the all-trans retinoic liposome preparation No. 3 has an average particle diameter of about 75 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • the preparation of the all-trans retinoic liposome preparation No. 4 has an average particle diameter of about 70 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • the preparation of the all-trans retinoic liposome preparation No. 5 has an average particle diameter of about 70 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • the preparation of the all-form retinoic acid liposome preparation No. 6 has an average particle diameter of about 70 nm and a particle size distribution (PDI) of ⁇ 0.3.
  • Example 2 Preparation of all-trans retinoic acid liposomes by active drug-loading preparation of all-trans retinoic acid liposome and passive drug delivery
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to obtain an aqueous phase having an average particle diameter of about 80 nm. Liposomes;
  • the liposome prepared in the step (3) was dialyzed through a 10000 pore size dialysis membrane to replace the aqueous phase with 10% mass.
  • Calcium acetate liposome having a certain pH gradient and an ion gradient in the aqueous phase of the phospholipid bilayer membrane with a fractional pH of 6-7;
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to obtain an aqueous phase having an average particle diameter of about 90 nm, which is non-acetic acid.
  • Calcium liposome
  • the drug loading of the all-trans retinoic acid liposome prepared by the active drug loading method is about 1.2 mg/ml, and the drug loading of the all-trans retinoic acid liposome prepared by the passive drug loading method. It is about 0.6 mg/ml.
  • the results are shown in Fig. 3.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • the all-trans retinoic acid liposome was prepared by the corresponding passive drug loading method and compared.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • all-trans retinoic acid liposomes were prepared by the corresponding passive drug loading method for comparison.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • all-trans retinoic acid liposomes were prepared by the corresponding passive drug loading method for comparison.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • the all-trans retinoic acid liposome was prepared by the corresponding passive drug loading method for comparison.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • the corresponding passive drug loading method was used to prepare all-trans retinoic acid liposome, and the method was carried out. Comparison.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release. Specifically, the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • the all-trans retinoic acid liposome was prepared by the corresponding passive drug loading method for comparison.
  • the all-trans retinoic acid liposome prepared by the active drug loading method and the all-trans retinoic acid liposome prepared by the passive drug loading method are more advantageous in sustained release.
  • the non-calcium acetate aqueous phase all-trans retinoic acid liposome was substantially released in the in vitro simulated release at 24 h, while the all-trans retinoic acid liposome in the aqueous calcium acetate phase remained 50% unreleased at 48 h. .
  • CT-26 cells were cultured to logarithmic growth phase using trypsin digestion, and the digested cells were collected and centrifuged at 300 g for 5 min in a centrifuge, the supernatant was decanted, and the cells were resuspended in sterile PBS. Count the cells and adjust the cell concentration to 1*10 7 cells/ml;
  • mice Purchase a 6-week-old Balb/c mouse, and shave the side of the skin inoculated beforehand.
  • the mice were anesthetized with 4% chloral hydrate 200 ⁇ l. After anesthesia, the mice were subcutaneously injected into the right lower arm.
  • the CT-26 suspension has a cell inoculation amount of 5*10 5 -1*10 6 /piece, and continues to be reared after inoculation;
  • the experiment can be performed at any time.
  • mice were sacrificed by cervical dislocation, and the tumor was removed from the skin using tweezers and scissors, and cut on a 40 ⁇ m cell strainer. Care should be taken to avoid damage to the tumor cells by shearing the tumor tissue while cutting the tissue. Rinse the tissue continuously with 5% PBS;
  • the digested cells were again passed through a 40 ⁇ m cell strainer, and the cells were washed with PBS to remove residual tumor tissue digestive juice and cell debris and dead cells (conditions for centrifugation after washing: rotation speed: 1000 rpm centrifugation time: 5 min), washing 2-3 times.
  • the cells were finally resuspended in PBS, at which time a single cell suspension of the tumor was obtained.
  • the tumor single cell suspension of the incubated magnetic beads is added from above the MS column, and the MS column is washed with 1 ml of Buffer to wash the cells of the unbound magnetic beads to the column;
  • Formulation No. 1 As shown in Figure 4A, we can observe that a significant decrease in the population of Gr-1 hi by administering different doses of the drug, in the high-dose administration group (+50 ⁇ l all-trans retinoic acid lipid The proportion of the body decreased from 17.5% to 9.10% in the control group, while the proportion of Gr-1 int cells did not seem obvious, but it may be due to the high expression of Gr-1 in all-trans retinoic acid. The transformation is induced to moderate to low expression. However, the proportion of cells in the group of Gr-1 low increased with the dose of dosing, further indicating that all-trans retinoic acid induced differentiation of MDSC, resulting in a decrease in the expression of Gr-1, so Gr-1 low was at a high dose.
  • the proportion of the drug is gradually increased. Since most of the infiltrating MDSCs at the tumor site are characterized by Gr-1 hi or Gr-1 int , we can conclude that all-trans retinoic liposomes can induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 6 As shown in Figure 4B, by administering different doses of the drug, we observed that the CD11b + Gr-1 + group of cells showed a significant decrease in the high-dose administration group (+100 ⁇ l all-trans retinoic acid) The proportion of liposomes decreased from 44.5% to 32.60% in the control group, indicating that all-trans retinoic acid induced differentiation of MDSC, resulting in decreased expression of Gr-1. Since most of the infiltrating MDSCs at the tumor site are characterized by CD11b+Gr-1+, we can conclude that all-trans retinoic liposomes can induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 2 The results of the experiment consistent with the No. 6 preparation were obtained, and all-trans retinoic liposomes were able to induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 3 An experimental result consistent with No. 6 preparation was obtained, and all-trans retinoic liposomes were able to induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 4 The results of the experiment consistent with the No. 6 preparation were obtained, and all-trans retinoic liposomes were able to induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 5 The results of the experiment consistent with the No. 6 preparation were obtained, and all-trans retinoic liposomes were able to induce differentiation of MDSC at the tumor site, resulting in a decrease in the number of MDSCs.
  • Formulation No. 1 As shown in Fig. 5A, further we can see from the expression of MHC-II, CD11c, CD80, CD86 on the surface markers of mature DC cells, the proportion of cells of mature DCs increased significantly after administration, among which CD80 and CD86 double positive cells increased from 18.85% in the control group to 39.39%. As shown in FIG. 5B, MHC-II and CD11c double positive cells increased from 21.34% to 30.08%.
  • Formulation No. 6 As shown in Fig. 5C, further we can see from the expression of MHC-II, CD11c, CD80, CD86 on the surface markers of mature DC cells, the proportion of cells of mature DCs increased significantly after administration, among which CD80 and CD86 double positive cells increased from 18.85% in the control group to 39.39%. As shown in FIG. 5D, MHC-II and CD11c positive cells increased from 16.75% and 45.83% to 22.65% and 49.27%, respectively.
  • Formulation No. 2 The results of the experiment consistent with the preparation of No. 6 were obtained. From the expression of the surface markers MHC-II, CD11c, CD80, and CD86 of mature DC cells, the proportion of cells of mature DCs increased significantly after administration. .
  • Formulation No. 3 The results of the experiment consistent with the preparation of No. 6 were obtained. From the expression of the surface markers MHC-II, CD11c, CD80, and CD86 of mature DC cells, the proportion of cells of mature DCs increased significantly after administration. .
  • Formulation No. 4 The results of the experiment consistent with the No. 6 preparation were obtained. From the expression of the surface markers MHC-II, CD11c, CD80, and CD86 of mature DC cells, the proportion of mature DC cells increased significantly after administration. .
  • Formulation No. 5 The results of the experiment consistent with the preparation of No. 6 were obtained. From the expression of the surface markers MHC-II, CD11c, CD80, and CD86 of mature DC cells, the proportion of cells of mature DCs increased significantly after administration. .
  • Example 4 all-trans retinoic acid liposome promotes T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC
  • the myeloid cells in the PBMC cells were isolated by magnetic beads labeled with CD33 antibody, referring to the magnetic bead separation apparatus operating manual.
  • the cells were cultured in a RPMI1640 complete medium (addition of 10% FBS) to a 12-well cell plate at 5 x 10 5 cells per well, and the prepared all-trans retinoic acid liposome obtained in the preparation of Example 1 was added for 24 hours. Flow cytometry was then used to detect the percentage of HLA-DR + CD11c + phenotypic DC cell population in myeloid cells.
  • the No. 2 preparation obtained the experimental results consistent with the No. 6 preparation, and the PtMC in the blood of the head and neck mucosal squamous cell carcinoma patients, the all-trans retinoic liposome of the present invention promoted the differentiation of MDSC into DC-type cells.
  • the No. 3 preparation obtained the experimental results consistent with the No. 6 preparation, and the PtMC in the blood of the head and neck mucosal squamous cell carcinoma patients, the all-trans retinoic liposome of the present invention promoted the differentiation of MDSC into DC-type cells.
  • the No. 4 preparation obtained the experimental results consistent with the No. 6 preparation, and the PtMC in the blood of the head and neck mucosal squamous cell carcinoma patients, the all-trans retinoic liposome of the present invention promoted the differentiation of MDSC into the DC line cells.
  • the No. 5 preparation obtained the experimental results consistent with the No. 6 preparation, and the PtMC in the blood of the head and neck mucosal squamous cell carcinoma patients, the all-trans retinoic liposome of the present invention promoted the differentiation of MDSC into DC-type cells.
  • the myeloid cells in the tumor tissue infiltrating cells were obtained by separation with magnetic beads labeled with CD33 antibody, referring to the magnetic bead separation device operating manual.
  • the cells were cultured in a RPMI1640 complete medium (addition of 10% FBS) to a 12-well cell plate at 5 x 10 5 cells per well, and the all-trans retinoic acid liposome prepared in Example 1 was added for 24 hours. Flow cytometry was then used to detect the percentage of CD33 + HLA-DR-MDSC cell population in myeloid cells.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer cells infiltrating into bladder cells. Quantity. As shown in Fig. 8A, the all-trans retinoic liposome of the present invention acts on tumor tissues in tumor patients and promotes T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer infiltrating myeloid cells. Quantity. As shown in Fig. 8B, the all-trans retinoic liposome of the present invention acts on tumor tissues in tumor patients and promotes T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer cells infiltrating into bladder cells.
  • the amount of the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer cells infiltrating into bladder cells.
  • the amount of the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer infiltrating myeloid cells.
  • the amount of the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can significantly reduce CD33+HLA-DR-MDSC in bladder cancer cells infiltrating into bladder cells.
  • the amount of the all-trans retinoic acid liposome of the present invention acts on tumor tissues in tumor patients, and can promote T cell proliferation in head and neck mucosal squamous cell carcinoma PBMC.
  • Example 6 When administered by intravenous injection or intravenous drip, the plasma terminal half-life of the all-trans retinoic liposome of the invention can reach 8-12h.
  • the experimental SD rats were purchased from Weitong Lihua Experimental Animal Co., Ltd., a total of 3, numbered IV-1, IV-2, IV-3. One day before the experiment, SD rats were fasted overnight. On the day of the experiment, group A SD rats were injected with 10 mg ⁇ kg -1 all-trans retinoic liposome in the tail vein, before, and after 5, 15, 30, 1, 2, 4, 8, 24 after administration. At 48 hours, 0.20 mL of blood was collected from the jugular vein and placed in a heparinized test tube after drying. After centrifugation (5500 rpm) for 10 min, the plasma was separated and stored in a refrigerator below -20 °C. SD rats can resume eating after 4 hours of administration, and they can drink water freely during the experiment.
  • the plasma terminal half-life of Formulation No. 1 can reach 8-12 h.
  • the plasma terminal half-life of No. 2 preparation, No. 3 preparation, No. 4 preparation, No. 5 preparation and No. 6 preparation can reach 8-12 h.
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm and 50 nm, respectively, to finally obtain an aqueous phase having an average particle diameter of about 90 nm.
  • the liposome obtained in the step (3) is dialyzed through a 10000 pore size dialysis membrane in a 10% by mass aqueous solution of sucrose having a pH of 6 to 7, and the outer aqueous phase of the liposome is replaced with 10
  • a sucrose aqueous solution having a mass fraction of 6 to 7 and having a pH of 6 to 7 gives a calcium acetate liposome having a phospholipid bilayer membrane and having a pH gradient and an ion gradient in the aqueous membrane inside and outside the membrane.
  • the aqueous phase in the two-layer membrane is an aqueous solution of calcium acetate (pH 9.0, concentration: 200 mM), and the aqueous phase of the two-layer membrane is an aqueous solution of sucrose (pH of 6 to 7, mass fraction of 10%).
  • the step (2) is: adding the ethanol mixture obtained in the step (1) 6.4 ml of calcium acetate buffer (pH 9.0, consisting of 200 mM calcium acetate, hydroxypropyl- ⁇ -cyclodextrin and water) containing hydroxypropyl- ⁇ -cyclodextrin (4%, w/v) And placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) 6.4 ml of calcium acetate buffer (pH 9.0, consisting of 200 mM calcium acetate, hydroxypropyl- ⁇ -cyclodextrin and water) containing hydroxypropyl- ⁇ -cyclodextrin (40%, w/v) And placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the all-trans retinoic acid liposome is prepared by the above method, that is,
  • step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of acetic acid containing HPMC (1%, w/v).
  • Calcium buffer pH 9.0, which consists of 200 mM calcium acetate, HPMC and water, and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of acetic acid containing PVP (2%, w/v).
  • Calcium buffer pH 9.0, which consists of 200 mM calcium acetate, PVP and water, and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the PVP containing (1%). , w/v) calcium acetate buffer (pH 9.0, It consists of 200 mM calcium acetate, PVP and water) and is placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of PVP containing (20%). , w/v) calcium acetate buffer (pH 9.0, which consists of 200 mM calcium acetate, PVP and water), and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • step (2) is: adding the ethanol mixture obtained in step (1) to 6.4 ml of PEG-400 (17%, w/).
  • v) calcium acetate buffer (pH 9.0, which consists of 200 mM calcium acetate, PEG-400 and water), and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the PEG-containing substance.
  • 400 (5%, w/v) calcium acetate buffer (pH 9.0, which consists of 200 mM calcium acetate, PEG-400 and water), and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the PEG-containing substance.
  • 400 (20%, w/v) calcium acetate buffer (pH 9.0, which consists of 200 mM calcium acetate, PEG-400 and water), and placed in a water bath at 70 ° C for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml.
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the hydroxy group.
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the hydroxy group.
  • Propyl- ⁇ -cyclodextrin (12%, w/v) + PVP (0.5%, w/v) calcium acetate buffer (pH 9.0, which consists of 200 mM calcium acetate, hydroxypropyl- ⁇ -cyclodextrin Fine, PVP and water composition) and placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles.
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the sulfobutyl group.
  • - ⁇ -cyclodextrin (10%, w/v) calcium acetate buffer (pH 9.0 consisting of 200 mM calcium acetate, sulfobutyl- ⁇ -cyclodextrin and water) and placed at 70 ° C Water bath for 30 minutes, Liposomal vesicles are obtained.
  • the step (2) is: adding the ethanol mixture obtained in the step (1) 6.4 ml of calcium acetate buffer containing sulfobutyl- ⁇ -cyclodextrin (4%, w/v) (pH 9.0 consisting of 200 mM calcium acetate, sulfobutyl- ⁇ -cyclodextrin and water) And placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) 6.4 ml of calcium acetate buffer containing sulfobutyl- ⁇ -cyclodextrin (40%, w/v) (pH 9.0 consisting of 200 mM calcium acetate, sulfobutyl- ⁇ -cyclodextrin and water) And placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the methyl-containing compound.
  • - cyclodextrin (20%, w/v) calcium acetate buffer pH 9.0 consisting of 200 mM calcium acetate, methyl- ⁇ -cyclodextrin and water
  • pH 9.0 consisting of 200 mM calcium acetate, methyl- ⁇ -cyclodextrin and water
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4.
  • ML of calcium acetate buffer pH 9.0, consisting of 200 mM calcium acetate, methyl- ⁇ -cyclodextrin and water
  • methyl- ⁇ -cyclodextrin 4%, w/v
  • juxtaposed Liposomal vesicles were obtained by a water bath at 70 degrees Celsius for 30 minutes;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4.
  • ML of calcium acetate buffer pH 9.0, consisting of 200 mM calcium acetate, methyl- ⁇ -cyclodextrin and water) containing methyl- ⁇ -cyclodextrin (40%, w/v), juxtaposed Liposomal vesicles were obtained by a water bath at 70 degrees Celsius for 30 minutes;
  • the step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of the hydroxypropyl group.
  • - ⁇ -cyclodextrin (12%, w/v) calcium acetate buffer (pH 9.0 consisting of 200 mM calcium acetate, hydroxypropyl- ⁇ -cyclodextrin and water) and placed at 70 ° C The water bath was incubated for 30 minutes to obtain liposome vesicles.
  • the step (2) is: adding the ethanol mixture obtained in the step (1) 6.4 ml of calcium acetate buffer (pH 9.0, consisting of 200 mM calcium acetate, hydroxypropyl- ⁇ -cyclodextrin and water) containing hydroxypropyl- ⁇ -cyclodextrin (4%, w/v) And placed in a water bath at 70 degrees Celsius for 30 minutes to obtain liposome vesicles;
  • Step (2) is: adding the ethanol mixture obtained in the step (1) to 6.4 ml of a calcium acetate buffer (pH 9.0, containing 200 mM acetic acid) containing hydroxypropyl- ⁇ -cyclodextrin (40%, w/v). Calcium, hydroxypropyl- ⁇ -cyclodextrin and water), and placed in a water bath at 70 ° C for 30 minutes to obtain liposome vesicles;
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm, and 50 nm for 8 times, and finally an aqueous phase having an average particle diameter of about 90 nm is obtained as sodium acetate.
  • a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm, and 50 nm for 8 times, and finally an aqueous phase having an average particle diameter of about 90 nm is obtained as sodium acetate.
  • the liposome obtained in the step (3) is dialyzed through a 10000 pore size dialysis membrane in a 10% by mass aqueous solution of sucrose having a pH of 6 to 7, and the outer aqueous phase of the liposome is replaced with 10%.
  • a sucrose aqueous solution having a mass fraction and a pH of 6 to 7 obtained a liposome having a phospholipid bilayer membrane and having a certain concentration of sodium acetate and cyclodextrin in the aqueous phase inside and outside the membrane.
  • the purified all-trans retinoic liposome preparation was destroyed with 9 volumes of methanol, and the encapsulation efficiency was determined by high performance liquid chromatography using a UV detector. Measurement conditions: ODS column (Diamonsil, 5 ⁇ m, 250*4.6 mm); detection temperature was 25 ° C; detection wavelength was 340 nm; flow rate was 1.0 ml/min; mobile phase was acetonitrile/methanol (volume ratio: 95:5).
  • Wtotal is the mass of all-trans retinoic acid before dialysis separation of free drug and the same volume as all-trans retinoic acid after dialysis.
  • the results showed that the entrapment efficiency of the all-trans retinoic acid liposome prepared in the present Example was 85.6%.
  • the particle size before the drug loading was 71.57 nm and the PdI was 0.083.
  • the particle size after drug loading was 90.25 nm and the PdI was 0.125.
  • the liposome vesicles obtained in the step (2) are sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm, and 50 nm, respectively, to finally obtain a liposome having an average particle diameter of about 90 nm;
  • the liposome obtained in the step (3) is dialyzed through a 10000 pore size dialysis membrane in a 10% by mass aqueous solution of sucrose having a pH of 6 to 7, and the outer aqueous phase of the liposome is replaced with 10%.
  • a sucrose aqueous solution having a mass fraction and a pH of 6 to 7 obtained a liposome having a phospholipid bilayer membrane and having a certain HPMC and HEPE concentration gradient in the aqueous membrane inside and outside the membrane.
  • the purified all-trans retinoic liposome preparation was destroyed with 9 volumes of methanol, and the encapsulation efficiency was determined by high performance liquid chromatography using a UV detector. Measurement conditions: ODS column (Diamonsil, 5 ⁇ m, 250*4.6 mm); detection temperature was 25 ° C; detection wavelength was 340 nm; flow rate was 1.0 ml/min; mobile phase was acetonitrile/methanol (volume ratio: 95:5).
  • Wtotal is the mass of all-trans retinoic acid before dialysis separation of free drug and the same volume as all-trans retinoic acid after dialysis.
  • the results showed that the entrapment efficiency of the all-trans retinoic acid liposome prepared in the present Example was 57.5%.
  • the particle diameter before drug loading was 77.39 nm and PdI was 0.101 as measured by the above method.

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Abstract

一种全反式维甲酸脂质体制剂及其制备方法。所述全反式维甲酸脂质体制剂包括全反式维甲酸和脂质体载体。其制备方法为主动载药法，包括醋酸钙梯度法或者醋酸钠梯度法。所制备的全反式维甲酸制剂载药量高、体内稳定，提高了全反式维甲酸的血药浓度，延长了半衰期。

Description

一种全反式维甲酸脂质体制剂及其制备与应用 技术领域
本发明属于生物制药技术领域，具体涉及一种全反式维甲酸脂质体制剂及其制备与应用。
背景技术
维甲酸为体内维生素A代谢产物。全反式维甲酸(ATRA)作为药物被用来治疗粉刺，同时也是临床上治疗急性早幼粒细胞白血病(acute promyelocytic leukemia，APL)的重要药物。全反式维甲酸(ATRA)通过结合至细胞内的特定受体(RARs，RXRs和RORs)从而影响基因表达，在急性早幼粒细胞白血病治疗中，可以促进APL细胞分化和PML/RARα gene的降解，达到治疗的效果。
然而，全反式维甲酸的药物的临床应用受到以下几个方面的限制：1全反式维甲酸的水溶性极低(4.77e-03g/l)；2全反式维甲酸血浆半衰期较短，而发挥其药效需要在一个较长的时间内维持一定的血药浓度，和在靶器官中的药效浓度。因此，选择一种适于全反式维甲酸的药物输送方式尤为重要。
脂质体作为一种纳米药物输送载体，自从提出至今已有30余年。多种基于脂质体输送系统的抗癌药物已广泛应用于临床治疗肿瘤中。其中最为成功的药物便是阿霉素脂质体，脂质体作为药物输送系统优势主要在于其改变了药物的生物分布，降低药物的系统毒性，并可通过脂质体上的特定修饰而达到长循环/靶向的效果，提高了靶组织的药物浓度。
此前报道的全反式维甲酸脂质体制剂大多将全反式维甲酸嵌合于磷脂双脂层中。受全反式维甲酸的物理性质限制，此方法所制成的全反式维甲酸脂质体在载药量与体内稳定性方面表现并不理想。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种全反式维甲酸制剂及其制备与应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种全反式维甲酸脂质体制剂，所述全反式维甲酸脂质体制剂包括全反式维甲酸和脂质体载体。
优选地，所述全反式维甲酸脂质体制剂还含有用于增加全反式维甲酸溶解度的助溶分子。
进一步优选地，所述助溶分子选自PVP、HPMC、环糊精、PEG中的任一种或多种的组合。
进一步优选地，所述助溶分子与全反式维甲酸之间的摩尔比例范围是：(38～0.075)∶1。
本发明一优选例中，所述环糊精选用羟丙基-β-环糊精、磺丁基-β-环糊精或甲基-β-环糊精。
本发明一优选例中，所述PEG选用PEG-400。
当助溶分子选用环糊精时，所述助溶分子与全反式维甲酸之间的摩尔比例范围可以是(20～2)∶1。
当助溶分子选用PVP时，所述助溶分子与全反式维甲酸之间的摩尔比例范围可以是(1.5～0.075)∶1。
当助溶分子选用PEG-400时，所述助溶分子与全反式维甲酸之间的摩尔比例范围可以是(38～9.5)∶1。
优选地，所述全反式维甲酸和脂质体载体之间的药脂摩尔比范围是1∶(5～20)。
优选地，所述脂质体的原料含有磷脂、胆固醇和聚乙二醇化的磷脂。
进一步优选地，所述脂质体的原料中含有磷脂、胆固醇和聚乙二醇化的磷脂之间的摩尔比例范围是：(30～80)∶(0.1～40)∶(0.1～30)。
进一步优选地，所述脂质体的原料中含有HSPC、CHOL和DSPE-PEG。
进一步优选地，所述脂质体的原料中HSPC、CHOL、DSPE-PEG之间的摩尔比例范围是(30～80)∶(0.1～40)∶(0.1～30)。更优选地，所述脂质体的原料中HSPC、CHOL、DSPE-PEG之间的摩尔比例范围是57∶38∶5。
优选地，所述DSPE-PEG中PEG的分子量范围是50～10000。更优选，所述DSPE-PEG中PEG的分子量是2000。
优选地，所述脂质体为单室脂质体。
优选地，所述脂质体的粒径范围是30nm～200nm。更优选地，所述脂质体的粒径范围是50～150nm。更优选地，所述脂质体的粒径是70nm～130nm。
优选地，所述脂质体的粒径分布窄，其D95(粒径小于其占脂质体总量的95％)为小于等于120nm。更优选地，D95小于等于110nm，更优选地，D95小于等于95nm。
优选地，所述全反式维甲酸脂质体制剂中，所述全反式维甲酸的浓度大于等于0.1mg/ml。进一步优选地，所述全反式维甲酸脂质体制剂中，所述全反式维甲酸的浓度大于等于0.5mg/ml。更优选地，所述全反式维甲酸脂质体制剂中，所述全反式维甲酸的浓度范围是0.5～5mg/ml。
优选地，所述全反式维甲酸脂质体制剂为注射给药制剂。
进一步优选地，所述注射给药制剂选自皮下注射剂型、静脉注射剂型、肌肉注射剂型或盆腔注射剂型。
本发明的第二方面，提供了全反式维甲酸脂质体制剂的制备方法，为主动载药法。
优选地，所述制备方法为醋酸钙梯度法。或者，醋酸钠梯度法。
进一步优选地，所述醋酸钙梯度法包括步骤：
(1)按配比取用于制备脂质体的各原材料，用乙醇溶解，获得乙醇混合物；
(2)将步骤(1)中的乙醇混合物中加入pH为7.0～11.0的醋酸钙水溶液，获得脂质体囊泡；
(3)将步骤(2)中所得脂质体囊泡依次挤压通过不同孔径的聚碳酯膜，获得粒径均一的空白脂质体；
(4)将步骤(3)所制得的内外水相均为醋酸钙水溶液空白脂质体置于pH为6.0～7.0的等渗透析液中，得到内外水相之间存在醋酸钙梯度的空白脂质体；
(5)在步骤(4)所得内外水相之间存在醋酸钙梯度的空白脂质体中添加全反式维甲酸混悬液，孵育，去除游离全反式维甲酸，即得全反式维甲酸脂质体制剂。
优选地，步骤(2)中，醋酸钙水溶液中醋酸钙的浓度是120mM～360mM。
优选地，当全反式维甲酸脂质体制剂含有助溶分子时，步骤(2)中，醋酸钙水溶液中含有助溶分子。
优选地，步骤(4)所得脂质体的内外水相之间存在醋酸钙梯度。
优选地，步骤(4)所得脂质体的内水相为pH为7.0～11.0的醋酸钙水溶液，外水相为pH为6.0～7.0的等渗溶液。
进一步优选地，所述等渗溶液选自：含有0.9％NaCl或10％蔗糖的浓度为10mM的HEPEs缓冲溶液。
此外，所述醋酸钠梯度法包括步骤：
(1)按配比取用于制备脂质体的各原材料，用乙醇溶解，获得乙醇混合物；
(2)将步骤(1)中的乙醇混合物中加入pH为7.0～11.0的醋酸钠水溶液，获得脂 质体囊泡；
(3)将步骤(2)中所得脂质体囊泡依次挤压通过不同孔径的聚碳酯膜，获得粒径均一的空白脂质体；
(4)将步骤(3)所制得的内外水相均为醋酸钠水溶液空白脂质体置于pH为6.0～7.0的等渗透析液中，得到内外水相之间存在醋酸钠梯度的空白脂质体；
(5)在步骤(4)所得内外水相之间存在醋酸钠梯度的空白脂质体中添加全反式维甲酸混悬液，孵育，去除游离全反式维甲酸，即得全反式维甲酸脂质体制剂。
优选地，步骤(2)中，醋酸钠水溶液中醋酸钙的浓度是120mM～360mM。
优选地，当全反式维甲酸脂质体制剂含有助溶分子时，步骤(2)中，醋酸钠水溶液中含有助溶分子。
优选地，步骤(4)所得脂质体的内外水相之间存在醋酸钠梯度。
优选地，步骤(4)所得脂质体的内水相为pH为7.0～11.0的醋酸钙水溶液，外水相为pH为6.0～7.0的等渗溶液。
进一步优选地，所述等渗溶液选自：含有0.9％NaCl或10％蔗糖的浓度为10mM的HEPEs缓冲溶液。
本发明发现，所述方法能够用于提高全反式维甲酸脂质体制剂中全反式维甲酸的溶解度或药物浓度。
本发明还发现，所述方法能够用于提高全反式维甲酸脂质体制剂中全反式维甲酸作用于肿瘤相关巨噬细胞的效果。
本发明还发现，所述方法能够用于提高全反式维甲酸脂质体制剂中全反式维甲酸作用于髓系抑制细胞的效果。
本发明的第三方面，提供前述全反式维甲酸制剂在制备肿瘤治疗药物中的用途。
优选地，所述肿瘤治疗药物为针对肿瘤病人中异常的髓系抑制性细胞、诱导髓系抑制性细胞分化、抑制肿瘤增殖和复发的药物。
进一步优选地，所述髓系抑制性细胞为乳腺癌、结肠癌、卵巢癌、肺癌、肾癌、胃癌、肝癌、宫颈癌、子宫内膜癌、膀胱癌、前列腺癌、胰腺癌、大肠癌、基底细胞癌、黑色素瘤、滤泡性淋巴癌或小淋巴细胞瘤的髓系抑制性细胞。
本发明还提供了全反式维甲酸或前述全反式维甲酸在制备药物中的用途，所述用途选自以下任一项或多项：
(1)促进肿瘤部位细胞MDSC分化为成熟DC；
(2)促进肿瘤部位T细胞增殖；
(3)减少肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；
(4)减少肿瘤部位MDSC的数量。
进一步地，所述用途选自以下任一项或多项：
(1)促进肿瘤部位细胞MDSC分化为成熟DC；
(2)促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖；
(3)减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；
(4)减少肿瘤部位MDSC的数量。
本发明的第四方面，提供一种治疗肿瘤的方法，包括步骤：给患者施用前述全反式维甲酸制剂。具体给药剂量在本领技术人员所熟知的范围内。
与现有技术相比，本发明具有如下有益效果：
(1)本发明首次应用主动载药法装载全反式维甲酸，形成载药量高、体内稳定的全反式维甲酸脂质体制剂。根据本发明，其中所述全反式维甲酸药物被嵌入脂质体磷脂双层膜中或者结合在脂质体磷脂双层膜上或者包裹于脂质体内囊之中。所述全反式维甲酸药物被嵌入结合在脂质体磷脂双层膜上是指全反式维甲酸通过疏水作用部分或全部包裹在磷脂双层膜的疏水层中；所述全反式维甲酸结合在脂质体磷脂双层膜上是指全反式维甲酸通过电荷作用，疏水作用，物理或化学吸附作用与磷脂双分子层相互作用而结合在脂质体磷脂双层膜上；所述全反式维甲酸药物被包裹在脂质体内囊之中是指全反式维甲酸以溶解或全反式维甲酸单分子或全反式维甲酸盐沉淀形式被脂质体磷脂双分子层包裹在脂质体内囊之中。
(2)通过长期实验发现并证明本发明所制备的全反式维甲酸脂质相比与其他类型的全反式维甲酸脂质具有更加高的载药量及更好的体内稳定性。并且，本发明所制备的全反式维甲酸脂质将全反式维甲酸的溶解度由现有技术中的0.01mg/ml，提升至少0.1mg/ml以上，提高了至少100倍。
附图说明
图1A：1号制剂，载药前后，脂质体的粒径。
图1B：6号制剂，载药前后，脂质体的粒径。
图2：乙酸钙水相脂质体及非乙酸钙水相脂质体在去除游离药物后所达到的最终载药量。
图3：比较2种处方的累计释放率。
图4A：1号制剂全反式脂质体制剂对小鼠肿瘤部位细胞MDSC抑制实验。
图4B：6号制剂全反式脂质体制剂对小鼠肿瘤部位细胞MDSC抑制实验。
图5A：1号制剂全反式脂质体制剂促进小鼠肿瘤部位细胞MDSC分化为成熟DC。
图5B：1号制剂全反式脂质体制剂促进小鼠肿瘤部位细胞MDSC分化为成熟DC。
图5C：6号制剂全反式脂质体制剂促进小鼠肿瘤部位细胞MDSC分化为成熟DC。
图5D：6号制剂全反式脂质体制剂促进小鼠肿瘤部位细胞MDSC分化为成熟DC。
图6A：本发明的1号制剂全反式维甲酸脂质体促进头颈部黏膜鳞状细胞癌病人血液中MDSC向DC系细胞分化。
图6B：本发明的6号制剂全反式维甲酸脂质体促进头颈部黏膜鳞状细胞癌病人血液中MDSC向DC系细胞分化。
图7A：本发明1号制剂全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量。
图7B：本发明6号制剂全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量。
图8A：本发明1号制剂全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
图8B：本发明6号制剂全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
图9：实施例6尾静脉注射给药时大鼠的pK曲线图。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外， 根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
实施例1 全反式维甲酸脂质体的制备和鉴定
氢化豆磷脂(HSPC)购自日本油脂公司(NOF Corporation)，聚乙二醇化磷脂∶二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及胆固醇购自美国Avanti Polar Lipids公司；全反式维甲酸购自美国西格玛公司。
一、全反式维甲酸脂质体的制备
(一)1号制剂的制备
(1)称取121.742毫克氢化豆磷脂(HSPC，分子量783.8)、38.22毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及40.04毫克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入6.4毫升的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的水相为乙酸钙的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜置于10％质量分数且pH为6～7的蔗糖水溶液中透析，将脂质体的外水相置换为10％质量分数且pH为6～7的蔗糖水溶液，得到具有磷脂双层膜、且双侧膜内外水相具有一定pH梯度和离子梯度的乙酸钙脂质体。具体地，双层膜内水相为乙酸钙水溶液(pH9.0，浓度200mM)、双层膜外水相为蔗糖水溶液(pH 为6～7，质量分数10％)。
(5)往步骤(4)中所得乙酸钙脂质体中添加4mg/ml的全反式维甲酸的混悬液，加入的全反式维甲酸的混悬液与乙酸钙脂质体的体积比为1∶1，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体，记为1号制剂。
(二)2号制剂的制备
以全反式维甲酸和脂质体载体之间的药脂摩尔比为1∶5，参照上述方法制备全反式维甲酸脂质体，亦即：
当全反式维甲酸和脂质体载体之间的药脂摩尔比为1∶5时，步骤(1)为：
称取60.87092毫克氢化豆磷脂(HSPC，分子量783.8)、19.1094毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及20.01967毫克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物。
此条件制备的空白脂质体与8.175mg全反式维甲酸制备获得的全反式维甲酸脂质体，记为2号制剂。
(三)3号制剂的制备
以全反式维甲酸和脂质体载体之间的药脂摩尔比为1∶20，参照上述方法制备全反式维甲酸脂质体，亦即：
当全反式维甲酸和脂质体载体之间的药脂摩尔比为1∶20时，步骤(1)为：
称取243.48369毫克氢化豆磷脂(HSPC，分子量783.8)、76.43760毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及80.07870毫克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物。
此条件制备的空白脂质体与8.175mg全反式维甲酸制备获得的全反式维甲酸脂质体，记为3号制剂。
(四)4号制剂的制备
参照上述制备方法制得药脂比(摩尔比)为0.08的全反式维甲酸制剂，记为4号制剂。
(五)5号制剂的制备
参照上述制备方法制得药脂比(摩尔比)为0.12的全反式维甲酸制剂，记为5号制剂。
(1)称取0.097克氢化豆磷脂(HSPC，分子量783.8)、0.031克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及0.031克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入6.4毫升的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的水相为乙酸钙的脂质体；
(4)将步骤(3)所制得的脂质体通过0.2um的无菌过滤膜后置于10％质量分数且pH为7的蔗糖水溶液中透析，得到具有磷脂膜内外水相具有一定离子梯度的乙酸钙脂质体。具体地，双层膜内水相为乙酸钙水溶液(pH9.0，浓度200mM)、双层膜外水相为蔗糖水溶液(质量分数10％)。
(5)往步骤(4)中所得乙酸钙脂质体中添加20mg/ml的全反式维甲酸混悬液，并于60摄氏度中孵育45分钟，最终得到全反式维甲酸脂质体。
(六)6号制剂的制备
制备药脂比(摩尔比)为0.20的全反式维甲酸制剂，具体包括如下步骤：
(1)称取243.484毫克氢化豆磷脂(HSPC，分子量783.8)、76.44毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及80.08毫克胆固醇，用1毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入5毫升的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为80nm左右的水相为乙酸钙的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜置于10％质量分数且pH为6～7的蔗糖水溶液中透析，将脂质体的外水相置换为10％质量分数且pH为6～7的蔗糖水溶液，得到具有磷脂双层膜、且双侧膜内外水相具有一定pH梯度和离子梯度的乙酸钙脂质体。具体地，双层膜内水相为乙酸钙水溶液(pH9.0，浓度200mM)、双层膜外水相为蔗糖水溶液(pH为6～7，质量分数10％)。
(5)往步骤(4)中所得乙酸钙脂质体中添加3.3mg/ml的全反式维甲酸的混悬液，加入的全反式维甲酸的混悬液与乙酸钙脂质体的体积比为2∶1，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体，记为6号制剂。
二、全反式维甲酸脂质体的表征
1、全反式维甲酸脂质体包封率的检测
将纯化后的全反式维甲酸脂质体制剂用9倍体积甲醇破坏，用紫外检测器的高效液相色谱测定包封率。测定条件：ODS柱(Diamonsil，5μm，250*4.6mm)；检测温度为25摄氏度；检测波长为340nm；流速为1.0ml/min；流动相为pH＝4三乙胺盐酸缓冲液/乙腈/甲醇(体积比为17.5∶57.5∶25)。全反式维甲酸包封率(EE)按以下公式计算：EE＝(Wi/Wtotal)*100％，其中，Wi是纯化后被甲醇破坏的脂质体制剂中全反是维甲酸的质量，Wtotal是透析分离游离药物前并与透析后全反式维甲酸相同体积的全反式维甲酸质量。结果显示1号制剂全反式维甲酸脂质体的包封率约为80％。2号制剂全反式维甲酸脂质体的包封率约为90％。3号制剂全反式维甲酸脂质体的包封率约为95％。4号制剂全反式维甲酸脂质体的包封率约为92％。5号制剂全反式维甲酸脂质体的包封率约为93％。6号制剂全反式维甲酸脂质体的包封率约为95％。
2、全反式维甲酸脂质体的粒径
全反式维甲酸脂质体制剂的粒径实验重复三次取平均值±标准差。
结果，如图1A，显示1号制剂全反式维甲酸脂质体制剂平均粒径为70nm左右，粒度分布(PDI)均＜0.3。
2号制剂全反式维甲酸脂质体制剂平均粒径为75nm左右，粒度分布(PDI)均＜0.3。
3号制剂全反式维甲酸脂质体制剂平均粒径为75nm左右，粒度分布(PDI)均＜0.3。
4号制剂全反式维甲酸脂质体制剂平均粒径为70nm左右，粒度分布(PDI)均＜0.3。
5号制剂全反式维甲酸脂质体制剂平均粒径为70nm左右，粒度分布(PDI)均＜0.3。
如图1B，显示6号制剂全反式维甲酸脂质体制剂平均粒径为70nm左右，粒度分布(PDI)均＜0.3。
实施例2、主动载药制备全反式维甲酸脂质体与被动载药制备全反式维甲酸脂质体比较
一、主动载药法及被动载药法制备全反式维甲酸脂质体
(1)称取121.742毫克氢化豆磷脂(HSPC，分子量783.8)、38.22毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及40.04毫克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入2.5毫升的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为80nm左右的水相为乙酸钙的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜透析将水相置换为10％质量 分数且pH为6～7的蔗糖溶液中，得到具有磷脂双层膜内外水相具有一定pH梯度和离子梯度的乙酸钙脂质体；
(5)往步骤(4)中所得乙酸钙脂质体中添加4mg/ml的全反式维甲酸的混悬液，加入的全反式维甲酸的混悬液与乙酸钙脂质体的体积比为1∶2，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体。
2.使用被动载药法制备维甲酸脂质体
(1)称取121.742毫克氢化豆磷脂(HSPC，分子量783.8)、38.22毫克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及40.04毫克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入2.5毫升纯水溶液，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的水相为非乙酸钙的脂质体；
(4)往步骤(3)所得乙酸钙脂质体中添加4mg/ml的全反式维甲酸的混悬液，加入的全反式维甲酸的混悬液与乙酸钙脂质体的体积比为1∶1，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体。
3.主动载药法与被动载药法载药量对比
将纯化后的全反式维甲酸脂质体用9倍体积甲醇破坏，用紫外检测器的高效液相色谱测定包封率。测定条件：ODS柱(Diamonsil，5μm，250*4.6mm)；检测温度为25摄氏度；检测波长为340nm；流速为1.0ml/min；流动相为pH＝4三乙胺盐酸缓冲液/乙腈/甲醇(体积比为17.5∶57.5∶25)。全反式维甲酸包封率(EE)按以下公式计算：EE＝(Wi/Wtotal)*100％，其中，Wi是纯化后被甲醇破坏的复合物中全反是维甲酸的质量，Wtotal是透析分离游离药物前并与透析后全反式维甲酸相同体积的全反式维甲酸质量。结果如图2所示，主动载药法制备的全反式维甲酸脂质体的载药量约为1.2mg/ml，被动载药法制备的全反式维甲酸脂质体的载药量约为0.6mg/ml。
由此可见，对比不同水相条件下制备获得的脂质体的载药效率，除去游离药物后，最终得到的全反式维甲酸脂质体在载药量上存在着显著的差别。结果说明乙酸钙水相脂质体在载药量上具有明显的优势。
4.主动载药法与被动载药法体外模拟累计释放对比
(1)配置含10％血清的PBS透析液；
(2)将上步提及的2种不同方法制备的全反式维甲酸脂质体各量取400μl装入透析管(选用10000透析袋)，并置于1000ml透析液内，在37℃、200rmp条件下放于溶出仪透析；
(3)分别在0.5h、2h、4h、8h、12h、24h、48h时间点取20μl透析管内脂质体溶液，加入180μl色谱纯甲醇，于votex上放置5min破膜萃取全反式维甲酸；
(4)将萃取过的甲醇溶液置于离心机中，10000rmp条件下离心30min以沉淀破乳后沉淀的脂质材料，取40μl上清进行按上步HPLC条件进行检测，比较2种处方的累计释放率。
结果如图3所示，主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
此外，参照上述方法，对于1号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
参照上述方法，对于2号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
参照上述方法，对于3号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
参照上述方法，对于4号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
参照上述方法，对于5号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行 比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
参照上述方法，对于6号制剂，采用对应的被动载药法制备全反式维甲酸脂质体，进行比较。主动载药法制备获得的全反式维甲酸脂质体与被动载药法制备的全反式维甲酸脂质体在缓释上更有优势。具体地，非乙酸钙水相全反式维甲酸脂质体在体外模拟释放中在24h基本释放完毕，而乙酸钙水相的全反式维甲酸脂质体在48h时仍保持50％未释放。
实施例3、全反式维甲酸脂质体体外诱导肿瘤髓系抑制性细胞分化
1.Balb/c小鼠的肿瘤模型建立
(1)CT-26细胞培养至对数生长期时使用胰酶消化，将消化下的细胞收集并于离心机中以300g转速离心5min，倒去上清液，并用无菌PBS重悬细胞，细胞计数，并调整细胞浓度为1*107个细胞/毫升；
(2)购买6周大小的Balb/c白鼠，将皮下接种的一侧皮毛事先剃干净，用4％的水合氯醛200μl腹腔注射麻醉小鼠，麻醉后于右侧腋下部位皮下注射消化下来的CT-26悬液，细胞接种量为5*105-1*106/只，接种后继续饲养；
(3)大约饲养2-3周后，使用游标卡尺测量肿瘤长径及短径，通过肿瘤体积计算公式计算肿瘤大小：V＝1/2*长径*短径2，肿瘤体积约生长至100mm3时即可进行实验。
2.肿瘤相关淋巴细胞MDSC表征和分选
(1)断颈处死小鼠，使用镊子与剪刀从皮下去除肿瘤，并于40μm细胞滤网上剪碎，注意剪碎肿瘤组织时应小心避免剪切力对肿瘤细胞的损伤，同时在剪碎组织的过程中用含有5％的PBS不断冲洗组织；
(2)剪碎组织后将所有组织及冲洗PBS液一起离心，倒去上清后将所有组织一起转移至含1ml肿瘤组织消化液的15ml离心管中，于37℃、200rpm/min的摇床条件下消化1h；
(3)消化后的细胞再次通过40μm细胞滤网，使用PBS清洗细胞以去除残留的肿瘤组织消化液及细胞碎片与死细胞(清洗后离心时的条件为转速：1000rpm离心时间：5min)，清洗2-3遍。最终使用PBS重悬，此时得到肿瘤单细胞悬液。
(4)以每107个细胞/90μl体积的比例加入分选buffer并以每107个细胞/10μl体积的比例加入CD11b磁珠；
(5)将磁珠与细胞充分混匀，于4℃避光条件下孵育30min；孵育结束后按每107个细 胞/1ml体积的比例加入90μl Buffer，1000rpm条件下离心5min，离心结束后使用Buffer继续清洗2遍；
(6)最终加入500μl Buffer重悬制得孵育磁珠的肿瘤单细胞悬液；
(7)将MS柱放入配套的磁铁中，预先使用Buffer进行润洗，使MS柱得到充分饱和；
(8)润洗结束后将孵育磁珠的肿瘤单细胞悬液从MS柱上方加入，并用1ml Buffer冲洗MS柱以将未结合磁珠的细胞冲洗下柱子；
(9)反复冲洗3-5遍后取下MS柱，放入15ml离心管中，加入1ml Buffer并用MS柱配套仪器将其快速推下，此时收集到的是停留在MS柱上的CD11b阳性细胞。
(10)将上一步得到的CD11b阳性细胞悬液进行细胞计数，将细胞浓度调整至107/ml，制备流式细胞术样品；
(11)流式细胞样品分为：阴性组、Gr-1单阳性组、CD11b单阳性组、待测样品组。每组设置2-3个平行管。其中阴性组不加任何荧光抗体以设置阴性条件，2个单阳性分别加入单一荧光抗体以便后续荧光补偿，待测样品组加入待测的荧光抗体；
(12)所有流式管中各加入100μl体积使用流式Staining Buffer重悬的细胞(约106个)，阴性对照组中不加入任何荧光抗体，Gr-1单阳性组加入Gr-1抗体，CD11b单阳性组加入CD11b抗体，待测样品组同时加入Gr-1与CD11b抗体；
(13)于4℃避光条件下孵育30min，孵育结束后加入1ml Staining Buffer离心以洗去未结合至细胞上的抗体，并接着使用Staining Buffer清洗细胞2次，最后用500μl体积Staining Buffer进行细胞重悬；流式细胞仪检测。
3.全反式维甲酸脂质体对外周血中淋巴细胞及肿瘤部位淋巴细胞的诱导分化作用
(1)按实施例1的方法，制备全反式维甲酸脂质体(包括：1号制剂、2号制剂、3号制剂、4号制剂、5号制剂、6号制剂)；
(2)分选肿瘤组织的中的MDSC细胞，细胞计数后将细胞浓度调整为调整107/ml，将分选后的收集到的细胞接种到24孔版中，使得每孔最终细胞浓度为106/孔；
(3)向铺好细胞的孔板中依次加入20μl PBS、20μl ATRA-Liposome、50μl ATRA-Liposome，37℃中孵育24h；
(4)孵育1天后使用Gr-1及CD11b、CD11c、CD80、CD86、MHC-II抗体对细胞进行孵育，并通过流式细胞仪以观察细胞中MDSC及DC的变化，在流式样品处理过程中注意加入同型对照及做好荧光补偿。
1号制剂：如图4A所示，通过给予不同剂量的药物我们可以观察到，Gr-1hi这一群细 胞发生了显著的下降，在高剂量给药组(+50μl全反式维甲酸脂质体)中比例从对照组中17.5％下降至9.10％，而Gr-1int这一群细胞在比例上变化看起来并不明显，但可能是由于高表达的Gr-1在全反式维甲酸的诱导作用下转变为了中低表达。而Gr-1low这一群在的细胞比例随着给药剂量的增加而上升，进一步说明全反式维甲酸诱导MDSC发生分化，从而导致Gr-1表达量下降，所以Gr-1low在高剂量的给药下比例逐渐增高。由于肿瘤部位大部分浸润的MDSC具有Gr-1hi或Gr-1int的表征，因此我们可以推断全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
6号制剂：如图4B所示，通过给予不同剂量的药物我们可以观察到，CD11b+Gr-1+这一群细胞发生了显著的下降，在高剂量给药组(+100μl全反式维甲酸脂质体)中比例从对照组中44.5％下降至32.60％，说明全反式维甲酸诱导MDSC发生分化，从而导致Gr-1表达量下降。由于肿瘤部位大部分浸润的MDSC具有CD11b+Gr-1+的表征，因此我们可以推断全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
2号制剂：获得了与6号制剂一致的实验结果，全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
3号制剂：获得了与6号制剂一致的实验结果，全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
4号制剂：获得了与6号制剂一致的实验结果，全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
5号制剂：获得了与6号制剂一致的实验结果，全反式维甲酸脂质体能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
1号制剂：如图5A所示，进一步我们可以从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升，其中CD80、CD86双阳性细胞由对照组18.85％上升至39.39％。而如图5B所示，MHC-II、CD11c双阳性细胞由21.34％上升至30.08％。
6号制剂：如图5C所示，进一步我们可以从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升，其中CD80、CD86双阳性细胞由对照组18.85％上升至39.39％。而如图5D所示，MHC-II、CD11c阳性细胞分别由16.75％、45.83％上升至22.65％、49.27％。
2号制剂：获得了与6号制剂一致的实验结果，从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升。
3号制剂：获得了与6号制剂一致的实验结果，从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升。
4号制剂：获得了与6号制剂一致的实验结果，从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升。
5号制剂：获得了与6号制剂一致的实验结果，从成熟DC细胞的表面标记MHC-II、CD11c、CD80、CD86中的表达情况看出，通过给药以后成熟DC的细胞比例发生显著上升。
实施例4、全反式维甲酸脂质体促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖
取2ml抗凝血加入2ml PBS稀释一倍，将4ml稀释血液沿试管内壁缓缓加入3ml人淋巴细胞分离液内，使稀释血液标本叠加在人淋巴细胞分离液上。室温每分钟300g离心力30min(加速2，减速1)，用吸管将单个核细胞层全部吸出，用PBS液稀释至10ml，300g离心洗涤2次。弃上清后加入少量PBS，得到大量PBMC，4℃备用。参照磁珠分离设备操作手册，用CD33抗体标记的磁珠分离获得PBMC细胞中的髓源性细胞。按照每孔5x105个细胞，用RPMI1640完全培养基(添加10％FBS)培养细胞至12孔细胞板，加入实施例1制备获得的制备好的全反式维甲酸脂质体药物培养24小时。然后运用流式细胞仪检测HLA-DR+CD11c+表型DC细胞群在髓源性细胞中所占百分比。
1号制剂，结果如图6A所示，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
6号制剂，结果如图6B所示，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
2号制剂获得了与6号制剂一致的实验结果，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
3号制剂获得了与6号制剂一致的实验结果，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
4号制剂获得了与6号制剂一致的实验结果，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
5号制剂获得了与6号制剂一致的实验结果，与头颈部黏膜鳞状细胞癌病人血液中的PBMC作用，本发明的全反式维甲酸脂质体促进其中MDSC向DC系细胞分化。
实施例5、全反式维甲酸脂质体明显减少膀胱癌肿瘤浸润髓源细胞中的 CD33+HLA-DR-MDSC数量
尽量剔除周围的坏死组织以减少实验影响，无菌盐水冲洗干净后放入4℃含10％胎牛血清的RPMI 1640(含青霉素100μg/ml，链霉素100μg/ml)细胞培养液中，送入实验室处理。将肿瘤置于2ml培养基中冰上切碎组织，转移至含2ml酶消化液(胶原蛋白酶I和IV消化液，0.6～1mg/ml)的15ml离心管中，轻轻的涡旋混匀。37℃恒温摇床，200rpm，2h。然后用无菌PBS缓慢冲洗，过40μm膜至50ml离心管。300g离心10min后用PBS重悬细胞并计数。参照磁珠分离设备操作手册，用CD33抗体标记的磁珠分离获得肿瘤组织浸润细胞中的髓源性细胞。按照每孔5x105个细胞，用RPMI1640完全培养基(添加10％FBS)培养细胞至12孔细胞板，加入实施例1制备好的全反式维甲酸脂质体药物培养24小时。然后运用流式细胞仪检测CD33+HLA-DR-MDSC细胞群在髓源性细胞中所占百分比。
1号制剂，结果，如图7A所示，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量。如图8A所示，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
6号制剂，结果，如图7B所示，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量。如图8B所示，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
2号制剂，结果，与6号制剂一致，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
3号制剂，结果，与6号制剂一致，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
4号制剂，结果，与6号制剂一致，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
5号制剂，结果，与6号制剂一致，本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；本发明全反式维甲酸脂质体作用于肿瘤病人体内的肿瘤组织，能够促进头颈部黏膜鳞状细胞癌PBMC中T细胞增殖。
实施例6、通过静脉注射或静脉滴注给药时，该发明全反式维甲酸脂质体的血浆终末半衰期可达到8-12h
1.制备实施例1中制得的全反式维甲酸制剂，先取1号制剂，测定其药物浓度；
2.实验用SD大鼠购买自维通利华实验动物有限公司，共3只，分别编号为IV-1、IV-2、IV-3号。实验前一天，SD大鼠禁食过夜。实验当天，A组SD大鼠分别尾静脉注射10mg×kg-1全反式维甲酸脂质体制剂，于给药前及给药后5、15、30min、1、2、4、8、24和48h，由颈静脉采血0.20mL，置于烘干后的肝素化试管中。离心(5500转/分)10min后分离血浆，保存在低于-20℃冰箱。SD大鼠给药4h后可恢复进食，实验过程中可自由饮水。
3.血药浓度的生物分析根据苏州圣苏新药开发有限公司SOP-RS-04-001/01(Bioanalytical Method Validation for Quantification of Early Drug Discovery Compounds Using LC-MS/MS Methods)要求，建立测定SD大鼠血浆中全反式维甲酸的LC-MS/MS分析方法，用于本实验获得的生物样品的浓度测定。采用Pharsight Phoenix 6.3中的非房室模型计算药代动力学参数。
表1-1 1号制剂尾静脉注射给药时大鼠的pK数据
Figure PCTCN2017097869-appb-000001
Figure PCTCN2017097869-appb-000002
由表1-1和图9所示，1号制剂的血浆终末半衰期可达到8-12h。
采用上述方法，获得2号制剂、3号制剂、4号制剂、5号制剂和6号制剂尾静脉注射给药时大鼠的pK数据，如表1-2所示。
表1-2
Figure PCTCN2017097869-appb-000003
由表1-2所示，2号制剂、3号制剂、4号制剂、5号制剂和6号制剂的血浆终末半衰期可达到8-12h。
实施例7 含有助溶分子的全反式维甲酸制剂
一、内水相含有羟丙基-β-环糊精的全反式维甲酸脂质体
(1)称取121.742克氢化豆磷脂(HSPC，分子量783.8)、38.22克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及40.04克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(12％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的水相为乙酸钙的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜置于10％质量分数且pH为6～7的蔗糖水溶液中透析，将脂质体的外水相置换为含有10％质量分数且pH为6～7的蔗糖水溶液，得到具有磷脂双层膜、且双侧膜内外水相具有一定pH梯度和离子梯度的乙酸钙脂质体。具体地，双层膜内水相为乙酸钙水溶液(pH9.0，浓度200mM)、双层膜外水相为蔗糖水溶液(pH为6～7，质量分数10％)。
(5)往步骤(4)中所得乙酸钙脂质体中添加5mg/ml的全反式维甲酸的混悬液，加入的全反式维甲酸的混悬液与乙酸钙脂质体的体积比为1∶1，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体。
(6)通过紫外检测器的高效液相色谱测定全反式维甲酸的浓度。
当全反式维甲酸和脂质体载体之间的含有的羟丙基-β-环糊精与药物摩尔比为2∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(4％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的羟丙基-β-环糊精与药物摩尔比为20∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(40％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
此外，当脂质体载体内水相之间含有其他不同种类的增溶剂中的一种或多种时，参照上述方法制备全反式维甲酸脂质体，亦即
二、当全反式维甲酸和脂质体载体之间的含有HPMC时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有HPMC(1％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、HPMC和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
三、当全反式维甲酸和脂质体载体之间的含有PVP时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PVP(2％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、PVP和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的PVP与药物摩尔比为0.075∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PVP(1％，w/v)的乙酸钙缓冲液(pH为9.0， 其由200mM乙酸钙、PVP和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的PVP与药物摩尔比为1.5∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PVP(20％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、PVP和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
四、当全反式维甲酸和脂质体载体之间的含有PEG-400时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PEG-400(17％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、PEG-400和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的PEG-400与药物摩尔比为9.5∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PEG-400(5％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、PEG-400和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的PEG-400与药物摩尔比为37.5∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有PEG-400(20％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、PEG-400和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
五、当全反式维甲酸和脂质体载体之间的含有羟丙基-β-环糊精与PEG-400时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(12％，w/v)+PEG-400(5％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精、PEG-400与水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
六、当全反式维甲酸和脂质体载体之间的含有羟丙基-β-环糊精与HPMC时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(12％，w/v)+HPMC(0.5％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精、HPMC与水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
七、当全反式维甲酸和脂质体载体之间的含有羟丙基-β-环糊精与PVP时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-β-环糊精(12％，w/v)+PVP(0.5％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-β-环糊精、PVP与水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡。
八、当全反式维甲酸和脂质体载体之间的含有磺丁基-β-环糊精时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有磺丁基-β-环糊精(10％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、磺丁基-β-环糊精与水组成)，并置于70摄氏度中水浴30分钟， 得脂质体囊泡。
当全反式维甲酸和脂质体载体之间的含有的磺丁基-β-环糊精与药物摩尔比为2∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有磺丁基-β-环糊精(4％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、磺丁基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的磺丁基-β-环糊精与药物摩尔比为20∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有磺丁基-β-环糊精(40％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、磺丁基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
九、当全反式维甲酸和脂质体载体之间的含有甲基-β-环糊精时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有甲基-β-环糊精(20％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、甲基-β-环糊精与水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡。
当全反式维甲酸和脂质体载体之间的含有的甲基-β-环糊精与药物摩尔比为2∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有甲基-β-环糊精(4％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、甲基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的甲基-β-环糊精与药物摩尔比为20∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有甲基-β-环糊精(40％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、甲基-β-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
十、当全反式维甲酸和脂质体载体之间的含有羟丙基-γ-环糊精时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-γ-环糊精(12％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-γ-环糊精与水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡。
当全反式维甲酸和脂质体载体之间的含有的羟丙基-γ-环糊精与药物摩尔比为2∶1时，步骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-γ-环糊精(4％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-γ-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
当全反式维甲酸和脂质体载体之间的含有的羟丙基-γ-环糊精与药物摩尔比为20∶1时，步 骤(2)为：将步骤(1)所得乙醇混合物加入6.4毫升的含有羟丙基-γ-环糊精(40％，w/v)的乙酸钙缓冲液(pH为9.0，其由200mM乙酸钙、羟丙基-γ-环糊精和水组成)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
表2
Figure PCTCN2017097869-appb-000004
实施例8 内水相为乙酸钠的全反式维甲酸脂质体的制备
(一)制备
(1)称取0.097克氢化豆磷脂(HSPC，分子量783.8)、0.031克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及0.031克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入6.4毫升的内含20％质量分数的环糊精分子的200mM乙酸钠缓冲液(pH9.0)，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的水相为乙酸钠的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜置于10％质量分数且pH为6～7的蔗糖水溶液中透析，将脂质体的外水相置换为10％质量分数且pH为6～7的蔗糖水溶液，得到具有磷脂双层膜、且双侧膜内外水相具有一定乙酸钠和环糊精浓度梯度的脂质体。
(5)往步骤(4)中所得乙酸钠脂质体中添加10mg/ml的全反式维甲酸的混悬液，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体，记为7号制剂。
(二)全反式维甲酸脂质体包封率的检测
将纯化后的全反式维甲酸脂质体制剂用9倍体积甲醇破坏，用紫外检测器的高效液相色谱测定包封率。测定条件：ODS柱(Diamonsil，5μm，250*4.6mm)；检测温度为25摄氏度；检测波长为340nm；流速为1.0ml/min；流动相为乙腈/甲醇(体积比为95∶5)。全反式维甲酸包封率(EE)按以下公式计算：EE＝(Wi/Wtotal)*100％，其中，Wi是纯化后被甲醇破坏的脂质体制剂中全反是维甲酸的质量，Wtotal是透析分离游离药物前并与透析后全反式维甲酸相同体积的全反式维甲酸质量。结果显示本实施例中所制备的全反式维甲酸脂质体包封率为85.6％。参照上述方法检测，载药前的粒径为71.57nm，PdI为0.083；载药后的粒径为90.25nm，PdI为0.125。
实施例9 内水相为HEPES缓冲液的全反式维甲酸脂质体的制备
(一)制备
(1)称取0.097克氢化豆磷脂(HSPC，分子量783.8)、0.031克二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及0.031克胆固醇，用1.6毫升的乙醇溶解，并于70摄氏度水浴锅中水浴以溶解混合，获得乙醇混合物；
(2)将步骤(1)所得乙醇混合物加入6.4毫升的10％质量百分比环糊精分子和0.1MHEPES的缓冲液，并置于70摄氏度中水浴30分钟，得脂质体囊泡；
(3)将步骤(2)所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的脂质体；
(4)将步骤(3)所制得的脂质体通过10000孔径透析膜置于10％质量分数且pH为6～7的蔗糖水溶液中透析，将脂质体的外水相置换为10％质量分数且pH为6～7的蔗糖水溶液，得到具有磷脂双层膜、且双侧膜内外水相具有一定HPMC和HEPE浓度梯度的脂质体。
(5)往步骤(4)中所得脂质体中添加20mg/ml的全反式维甲酸的混悬液，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体，记为8号制剂。
(二)全反式维甲酸脂质体包封率的检测
将纯化后的全反式维甲酸脂质体制剂用9倍体积甲醇破坏，用紫外检测器的高效液相色谱测定包封率。测定条件：ODS柱(Diamonsil，5μm，250*4.6mm)；检测温度为25摄氏度；检测波长为340nm；流速为1.0ml/min；流动相为乙腈/甲醇(体积比为95∶5)。全反式维甲酸包封率(EE)按以下公式计算：EE＝(Wi/Wtotal)*100％，其中，Wi是纯化后被甲醇破坏的脂质体制剂中全反是维甲酸的质量，Wtotal是透析分离游离药物前并与透析后全反式维甲酸相同体积的全反式维甲酸质量。结果显示本实施例中所制备的全反式维甲酸脂质体包封率为57.5％。参照上述方法检测，载药前的粒径为77.39nm，PdI为0.101。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (17)

  1. 一种全反式维甲酸脂质体制剂，所述全反式维甲酸脂质体制剂包括全反式维甲酸和脂质体载体。
  2. 根据权利要求1所述的全反式维甲酸脂质体制剂，其特征在于，所述全反式维甲酸和脂质体载体之间的药脂摩尔比范围是1∶(5～20)。
  3. 根据权利要求1所述的全反式维甲酸脂质体制剂，其特征在于，所述全反式维甲酸脂质体制剂还含有用于增加全反式维甲酸溶解度的助溶分子。
  4. 根据权利要求3所述的全反式维甲酸脂质体制剂，其特征在于，所述助溶分子选自PVP、HPMC、环糊精或PEG中的任一种或多种的组合。
  5. 根据权利要求3所述的全反式维甲酸脂质体制剂，其特征在于，所述助溶分子与全反式维甲酸之间的摩尔比例范围是：(38～0.075)∶1。
  6. 根据权利要求1所述的全反式维甲酸脂质体制剂，其特征在于，所述脂质体的原料含有磷脂、胆固醇和聚乙二醇化的磷脂。
  7. 根据权利要求6所述的全反式维甲酸脂质体制剂，其特征在于，所述脂质体的原料中含有磷脂、胆固醇和聚乙二醇化的磷脂之间的摩尔比例范围是：(30～80)∶(0.1～40)∶(0.1～30)。
  8. 根据权利要求6所述的全反式维甲酸脂质体制剂，其特征在于，所述脂质体的原料中含有HSPC、CHOL和DSPE-PEG。
  9. 根据权利要求1～8任一权利要求所述的全反式维甲酸脂质体制剂，其特征在于，所述脂质体的粒径范围是30nm～200nm。
  10. 根据权利要求1～8任一权利要求所述的全反式维甲酸脂质体制剂，其特征在于，所述全反式维甲酸的浓度大于等于0.1mg/ml。
  11. 如权利要求1～10任一权利要求所述全反式维甲酸脂质体制剂的制备方法，为主动载药法。
  12. 根据权利要求11所述的方法，其特征在于，所述制备方法为醋酸钙梯度法或者醋酸钠梯度法。
  13. 根据权利要求11所述的方法，其特征在于，所述方法包括步骤：
    (1)按配比取用于制备脂质体的各原材料，用乙醇溶解，获得乙醇混合物；
    (2)将步骤(1)中的乙醇混合物中加入pH为7.0～11.0的醋酸钙水溶液，获得脂质体囊 泡；
    (3)将步骤(2)中所得脂质体囊泡依次挤压通过不同孔径的聚碳酯膜，获得粒径均一的空白脂质体；
    (4)将步骤(3)所制得的空白脂质体置于pH为6.0～7.0的等渗透析液中，得到内外水相之间存在醋酸钙梯度的空白脂质体；
    (5)在步骤(4)所得内外水相之间存在醋酸钙梯度的空白脂质体中添加全反式维甲酸混悬液，孵育，去除游离全反式维甲酸，即得全反式维甲酸脂质体制剂。
  14. 根据权利要求13所述的方法，其特征在于，步骤(2)中，醋酸钙水溶液中醋酸钙的浓度是120mM～360mM。
  15. 根据权利要求13所述的方法，其特征在于，步骤(4)所得脂质体的内外水相之间存在醋酸钙梯度。
  16. 如权利要求1～15任一权利要求所述全反式维甲酸脂质体制剂在制备肿瘤治疗药物中的用途。
  17. 全反式维甲酸或如权利要求1～15任一权利要求所述全反式维甲酸脂质体制剂在制备药物中的用途，所述用途选自以下任一种或多种：
    (1)促进肿瘤部位细胞MDSC分化为成熟DC；
    (2)促进肿瘤部位T细胞增殖；
    (3)减少肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量；
    (4)减少肿瘤部位MDSC的数量。
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