WO2019223728A1 - 甘露糖靶向的纳米制剂及其制备和应用 - Google Patents

甘露糖靶向的纳米制剂及其制备和应用 Download PDF

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WO2019223728A1
WO2019223728A1 PCT/CN2019/088001 CN2019088001W WO2019223728A1 WO 2019223728 A1 WO2019223728 A1 WO 2019223728A1 CN 2019088001 W CN2019088001 W CN 2019088001W WO 2019223728 A1 WO2019223728 A1 WO 2019223728A1
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targeting
mannose
nano
targeted
carrier
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PCT/CN2019/088001
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English (en)
French (fr)
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宋相容
魏于全
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成都瑞博克医药科技有限公司
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Priority to JP2021515269A priority Critical patent/JP2021525277A/ja
Priority to CN201980034892.8A priority patent/CN112292154B/zh
Priority to EP19806493.3A priority patent/EP3845249A4/en
Priority to US17/058,533 priority patent/US20210196832A1/en
Publication of WO2019223728A1 publication Critical patent/WO2019223728A1/zh

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    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to the field of medicine, in particular to a mannose-modified targeting nano preparation, a composition for preparing a nano preparation, a targeting element, a targeting carrier, and a prepared targeting drug, and a preparation method and application thereof.
  • the lectin receptor is a type of glycoprotein, glycolipid, or sugar complex distributed on the surface of cell membranes, and can specifically recognize and bind to some glycosyl groups.
  • the mannose receptor is the most important and highly efficient endocytic lectin receptor. Its functions include elimination of endogenous molecules, promotion of antigen presentation, and regulation of cell activation and transport. It is also closely related to tumor immune escape and metastasis. It is mainly expressed in Macrophages, dendritic cells and tumor cells can specifically recognize mannose glycosyl molecules.
  • mannosyl has many advantages such as non-toxicity, non-immunogenicity, good biocompatibility and biodegradability, and can be widely used for glycosylation modification of drug delivery systems.
  • liposomes and nanoparticles are based on the selective uptake of microparticles by different organs and selectively accumulate in the lung and other lymphoid tissues, thereby improving Therapeutic effects of antitumor drugs in various organs. Its preparation is simple, it has the characteristics of controlled release, non-immunogenicity, and improved efficacy. However, after liposomes and nanoparticles rely on the endocytosis of macrophages to achieve targeting, the stability during targeted transportation and the local uptake efficiency of the lesion are relatively low, and their targeting needs to be further improved. Continuous improvement.
  • the physical targeting liposomes include pH-sensitive liposomes (PLP), light-sensitive liposomes, magnetic liposomes, and heat-sensitive liposomes.
  • PBP pH-sensitive liposomes
  • light-sensitive liposomes As an example, Yang et al. Used a light-sensitive cell penetrating peptide pcCPP and a short peptide containing an aspartic acid-glycine-arginine residue (Asn-Gly-Arg, NGR) to attach to the liposome.
  • NGR aspartic acid-gly-Arg
  • This light-sensitive liposome can promote cell uptake, and effectively silence the c-mycc gene, and delay the growth of human fibrosarcoma.
  • PH-sensitive liposomes have failed to solve the problem of liver and spleen accumulation toxicity of liposomes.
  • the issues of viability and long-term storage stability of the release of photosensitive and magnetic liposome organisms still need to be solved urgently.
  • Heat-sensitive liposomes can directly kill tumor cells, but too long heating time can also cause damage to normal tissues.
  • Molecular targeting liposomes also have problems such as the targeted delivery of drugs and poor absorption of target organs in the body.
  • nanocarriers remain stable during transportation without being destroyed.
  • Ordinary nanoparticles or liposomes do not have active targeting (some may have passive targeting) It is easy to be removed by the endothelial system, which reduces the utilization rate and may cause unnecessary toxic and side effects.
  • the second is that after the drug reaches the target cell, it is the key to study the drug effect through more effective ligands, faster and direct entry into the cell.
  • One of the objectives of the present invention is to provide a nano preparation, which is modified by mannose and its derivatives to improve the targeting of the nano preparation.
  • Another object of the present invention is to provide the application of the above-mentioned targeted nano preparation.
  • the invention provides a mannose and its derivative modified targeted nano preparation, which is composed of a targeting ligand mannose and its derivative, a nano preparation and a main drug component, and can be used for vaccine delivery, tumor targeted therapy, Treatment of arteriosclerosis and various inflammatory diseases.
  • the mannose derivative is one or more of mannoside, mannosamine, and mannan.
  • the mannose derivative is methyl-D-mannoside, 1- ⁇ formylmethyl-mannopyranoside, 4-aminophenyl- ⁇ -D-pyran Mannoside, 4-nitrophenyl- ⁇ -D-pyranoside, 4-methylumbelliferyl- ⁇ -D-pyranoside, mannose-6-phosphate, carbamoyl-D- One or more of mannose, mannosamine, mannan and the like.
  • the targeted nano preparation provided by the present invention is one or more of liposomes, emulsions, nanogels, core-shell nanoparticles, HDL nanoparticles, solid lipid nanoparticles, polymer micelles, and the like.
  • the main drug component is one or more of a small molecule drug, a protein peptide drug or a gene drug.
  • the small-molecule drug with the main drug component can be encapsulated in the nano-formulation and used for tumor targeted therapy, arteriosclerosis treatment, and various inflammatory diseases.
  • the antitumor drugs taxanes (paclitaxel, docetaxel, harringtonine, 7-epitaxel), camptothecins (camptothecin, SN38, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, etc.), vinblastine (vinblastine, vincristine, vincristine, vinorelbine, vinblastine, etc.), doxorubicin, epirubicin, daunorubicin, epirubicin Rubicin, amphotericin, methotrexate, cytarabine, 5-fluorouracil, mitoxantrone, or a derivative thereof, gefitinib, noscapine, cisplatin, carb
  • the main medicine component protein polypeptide drugs are interleukins (such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, etc.), and various growth factors (such as fibroblast growth factor, liver growth factor, vascular endothelial growth factor, hematopoietic growth factor, etc.), interferon (such as IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factor (such as TNF ⁇ , TNF ⁇ ), integrin, monoclonal antibodies, Enzymes, insulin, etc.
  • the main medicine component gene medicine is DNA, plasmid, mRNA, siRNA, shRNA, microRNA and the like.
  • the main pharmaceutical ingredient can also be coated with an immune adjuvant to enhance the immune effect.
  • the content of the targeting ligand mannose and its derivative is 0.05% -40%.
  • the content of the targeting ligand mannose and its derivative is 1% -10%.
  • the content of the targeted ligand mannose is 2% -5%.
  • the targeting ligand mannose and its derivative are modified in the following manner: after preparing the nano preparation, mannose and its derivative are adsorbed or coupled to the surface of the nano preparation; Or, a mannose and a derivative thereof are first coupled to a carrier material for preparing the nano preparation, and then the nano preparation is prepared.
  • the manner in which the mannose and its derivative provided by the present invention are adsorbed on the surface of the nano-formulation is: the manner of preparing the nano-formulation by using a solvent volatilization method, a high-pressure homogenization method, or a microemulsion method, and adsorbing the nano-formulation on the surface .
  • mannose and its derivative of the modified nano preparation provided by the present invention are composed of a single mannose and its derivative and / or 2 to 10 mannose and its derivative.
  • the manner in which mannose and its derivatives are adsorbed on the surface of the nano preparation is: a solvent volatilization method, a high-pressure homogenization method, or a microemulsion method.
  • the coupling manner of the targeted nano preparation provided by the present invention, mannose and its derivative, and the nano preparation or the carrier material for preparing the nano preparation is: direct coupling or coupling through a spacer.
  • mannose and its derivative are coupled with the nano preparation or a carrier material for preparing the nano preparation through a spacer
  • the spacer is glutaraldehyde, ethylenediamine, and malonic acid.
  • One or more of short-chain alkyl chains (such as -CH2-CH2-), PEG, amino acids, dipeptides, oligopeptides, polypeptides, stearoyl, palmitoyl, and the like.
  • the spacer PEG is a PEG having a molecular weight of 100-5000 and having hydroxyl groups at both ends or at least one end being aminated.
  • the molecular weight of the spacer PEG is 100-2000.
  • the spacer amino acids are arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, and tyrosine.
  • the amino acids at the two ends of the dipeptide, oligopeptide and polypeptide are selected from the following amino acid composition: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, casein Amino acids; intermediate amino acids are selected from 20 arbitrary amino acids (such as RGD).
  • the present invention provides the targeted nano preparation.
  • the material for preparing the nano preparation is pharmaceutically acceptable preparation micelles, emulsions, nanogels, core-shell nanoparticles, HDL nanoparticles with active amino groups and / or hydroxyl groups. , Solid lipid nanoparticles, liposomes, polymer micelles and other carrier materials.
  • the carrier material is lipid, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polylysine (PLL), polyethyleneimine (PEI) , One or more of hyaluronic acid (HA), chitosan, gelatin, poloxamer, stearyl alcohol, etc.
  • the lipid materials used to prepare liposomes and core-shell nanoparticles are cholesterol, egg yolk lecithin, soybean lecithin, cerebrolysin, sphingomyelin, PC (phosphatidylcholine), EPG (lecithin), SPG (soybean phosphatidylglycerol), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) , Distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dilinoleylphosphatidylcholine (DLPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphospholipid Glycerol (DPPG), Dimyristylphosphatidylcholine (DM
  • the solid lipid materials for preparing solid lipid nanoparticles are stearic acid, cholesterol, glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl tristearate, glyceryl laurate, glycerin Palmitic stearate, monoglyceryl behenate, diglyceryl behenate, triglyceryl behenate, glyceryl trimyristate, glyceryl citrate, stearyl alcohol, palm One or more of acids, myristic acid, trimellitic acid, and lauric acid.
  • a third object of the present invention is to provide a material composition for preparing a specific targeted drug-loading carrier, which can be used for preparing a highly targeted drug-loading carrier.
  • a composition for preparing a nano preparation with a targeting function includes a targeting material and a basic nano preparation material, and the targeting material is mannose and / or a mannose derivative.
  • mannose derivative is mannoside and / or mannosamine and / or mannan.
  • mannose derivative is methyl-D-mannoside, 1- ⁇ formylmethyl-mannopyranoside, 4-aminophenyl- ⁇ -D-mannopyranoside, 4-nitro Phenyl- ⁇ -D-pyranoside, 4-methylumbelliferyl- ⁇ -D-pyranoside, mannose-6-phosphate, carbamoyl-D-mannose.
  • the weight percentage of the targeting material in the composition is 0.05-40%.
  • the weight percentage of the targeting material in the composition is 1-10%.
  • the composition is composed of the targeting material, the basic nano-formulation material, and a spacer material that distances the targeting material from the basic nano-formulation material, and the spacer material is glutaraldehyde, ethyl One or more of diamine, malonic acid, short-chain alkyl chain, PEG, amino acid, dipeptide, oligopeptide, polypeptide, stearoyl, and palmitoyl.
  • the spacer is one of glutaraldehyde, ethylenediamine, malonic acid, short-chain alkyl chain (such as -CH2-CH2-), PEG, amino acid, dipeptide, oligopeptide, and polypeptide. Or several.
  • the spacer amino acids are arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, and tyrosine.
  • amino acids at the two ends of the dipeptide, oligopeptide and polypeptide are selected from the following amino acid composition: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, casein Amino acids; intermediate amino acids are selected from 20 arbitrary amino acids (such as RGD).
  • the basic nano preparation material is a composition for preparing a material of liposome, emulsion, nanogel, core-shell nanoparticle, HDL nanoparticle, solid lipid nanoparticle, and polymer micelle.
  • the basic nano preparation material is a composition for preparing a nano preparation material, and may be a nano preparation material mentioned in "Modern Pharmaceutical Preparation Technology".
  • the basic nano preparation material is lipid, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polylysine (PLL), and polyethyleneimine.
  • PLGA polylactic acid-glycolic acid copolymer
  • PLA polylactic acid
  • PCL polycaprolactone
  • PLL polylysine
  • PEI amine
  • PEI hyaluronic acid
  • chitosan gelatin
  • poloxamer poloxamer
  • stearyl alcohol stearyl alcohol
  • the lipid materials used to prepare liposomes and core-shell nanoparticles are cholesterol, egg yolk lecithin, soybean lecithin, cerebrolysin, sphingomyelin, PC (phosphatidylcholine), EPG (lecithin), SPG (soybean phosphatidylglycerol), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) , Distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dilinoleylphosphatidylcholine (DLPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphospholipid Glycerol (DPPG), Dimyristylphosphatidylcholine (DM
  • the solid lipid material for preparing the solid lipid nanoparticles is stearic acid, cholesterol, glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl tristearate, glyceryl laurate Esters, glyceryl palmitate stearate, behenyl monoglyceride, behenyl diglyceride, behenyl triglyceride, glyceryl trimyristate, glyceryl citrate, eighteen One or more of alcohol, palmitic acid, myristic acid, trimellitic acid, and lauric acid.
  • the n is 200, 400, 1000, or 2000.
  • it is composed of the following components based on parts by weight: 1 part of mannose; 10-25 parts of PEG, and 4-8 parts of cholesterol.
  • it is composed of the following components on a molar basis: 1 part of mannose; 1-3 parts of PEG, and 1-3 parts of cholesterol.
  • the fourth object of the present invention is to provide a targeting carrier and a targeting drug, which can transport a variety of medicinal active ingredients to a target cell and have strong targeting properties.
  • the present invention adopts the following scheme:
  • a drug-targeting carrier prepared by the composition.
  • the drug is a small molecule drug and / or a protein peptide drug and / or a gene drug.
  • the small-molecule drug is a functional small molecule that can be wrapped in a nano-formulation for tumor-targeted therapy, arteriosclerosis therapy, and various inflammatory diseases.
  • mRNA encoding a specific tumor antigen can be encapsulated in a targeting nano-formulation carrier, and then introduced into a somatic cell, and an antigen protein is synthesized by the expression system of the host cell to induce the host immune system to produce the tumor antigen.
  • the immune response can achieve the function of preventing and treating tumors, and the targeted nano-formulations containing mRNA have better tumor targeting, which is beneficial to the treatment of tumors.
  • anti-tumor chemical and biological drugs, and drugs for treating arteriosclerosis and inflammatory diseases can also be wrapped in the carrier of targeted nano-formulations to play a more accurate targeted therapeutic role.
  • the small molecule drugs include, but are not limited to, PTX, DOX, and quercetin
  • the protein polypeptide drugs include, but are not limited to, albumin
  • the gene drugs include, but are not limited to, mRNA and siRNA.
  • the small-molecule drug is a functional small molecule that can be wrapped in a nano-formulation for tumor-targeted therapy, arteriosclerosis therapy, and various inflammatory diseases.
  • the antitumor drugs taxanes (paclitaxel, docetaxel, harringtonine, 7-epitaxel), camptothecins (camptothecin, SN38, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, etc.), vinblastine (vinblastine, vincristine, vincristine, vinorelbine, vinblastine, etc.), doxorubicin, epirubicin, daunorubicin, epirubicin Rubicin, amphotericin, methotrexate, cytarabine, 5-fluorouracil, mitoxantrone, or a derivative thereof, gefitinib, noscapine, cisplatin, carboplatin, oxali Platinum, carmustine,
  • the main medicine component protein polypeptide drugs are interleukins (such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, etc.), and various growth factors (such as fibroblast growth factor, liver growth factor, vascular endothelial growth factor, hematopoietic growth factor, etc.), interferon (such as IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factor (such as TNF ⁇ , TNF ⁇ ), integrin, monoclonal antibodies, Enzymes, insulin, etc.
  • the main medicine component gene medicine is DNA, plasmid, mRNA, siRNA, shRNA, microRNA and the like.
  • the main pharmaceutical ingredient can also be coated with an immune adjuvant to enhance the immune effect.
  • the drug is encapsulated by the targeting vector.
  • the drug is a small molecule drug and / or a protein peptide drug and / or a gene drug.
  • the small molecule drugs include, but are not limited to, PTX, DOX, and quercetin
  • the protein polypeptide drugs include, but are not limited to, albumin
  • the gene drugs include, but are not limited to, mRNA and siRNA.
  • the medicament may be in any pharmaceutically acceptable dosage form.
  • the pharmaceutically acceptable dosage form includes one or more pharmaceutically acceptable carriers, diluents, or excipients and is added in a suitable step in the above preparation process.
  • pharmaceutically acceptable refers to such compounds, raw materials, compositions and / or dosage forms, which are within the scope of sound medical judgment and are suitable for contact with patient tissues without excessive toxicity or irritation , Allergies or other problems and complications commensurate with a reasonable benefit / risk ratio, and effectively used for the intended purpose.
  • compositions are suitable for administration by any suitable route, such as by oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual, or transdermal), vaginal or parenteral (including subcutaneous, intradermal) , Intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous or subdermal injection or infusion) route.
  • Such formulations may be prepared by any method known in the pharmaceutical arts, for example by mixing the active ingredient with a carrier or excipient.
  • Oral, topical or injection administration is preferred.
  • Pharmaceutical preparations suitable for oral administration are provided in separate units, such as solutions or suspensions in aqueous or non-aqueous liquids; capsules or tablets; powders or granules; edible foam preparations or foaming preparations, and the like.
  • a fifth object of the present invention is to provide a method for preparing a targeting vector, which can be used for industrially preparing a targeting vector.
  • the present invention adopts the following scheme:
  • the method for preparing the targeting carrier is: preparing the basic nano preparation material into a nano drug delivery carrier, and then attaching the targeting material to the surface of the nano drug delivery carrier.
  • the method for attaching the targeting material to the surface of the nano-medicine carrier includes, but is not limited to, preparation using a solvent evaporation method, a thin film dispersion method, an ultrasonic dispersion method, an injection method, a reverse evaporation method, a high-pressure homogenization method, and a microemulsion method.
  • the targeting material solution is mixed with it to make the targeting material adhere to its surface.
  • the above method for preparing a nano-drug carrier can refer to "Modern Pharmaceutical Preparation Technology"
  • the solvent volatilization method is a method for preparing microspheres.
  • the simple process is to first configure a polymer solution of an appropriate concentration, and then emulsify the polymer solution in a continuous phase, and gradually evaporate the solvent to make microspheres. Preparation, and adding a targeting material to the microsphere preparation, so that the targeting material is adsorbed on the surface of the microsphere preparation.
  • the high-pressure homogenization method is that the material passes through a homogenizing valve under high pressure and is ejected at a very high flow rate, hits a collision ring, and ultra-fines and disperses and emulsifies the material through cavitation, impact, and shear effects to form nanoparticles.
  • This process The surface area of the Chinese material increases, and the targeting material can be attached to the surface after the targeting material is added thereto.
  • the thin film evaporation method is to dissolve the phospholipid material in an organic solvent, and then evaporate the organic solvent under a reduced pressure to form a thin film on the inner wall of the bottle, and then add water or PBS solution to stir repeatedly.
  • the nanoparticle preparation is obtained by mass and ultrasonic treatment, and the targeting material can be attached to the surface after the targeting material is added thereto.
  • a sixth object of the present invention is to provide a method for improving the targeting of a targeting vector, which significantly improves the targeting of the targeting vector.
  • the present invention adopts the following scheme:
  • the method for improving the targeting property of the targeting vector includes the following steps:
  • the basic nano preparation material is prepared into a nano preparation, and then the targeting element obtained in step A is connected with the nano drug delivery carrier to obtain a targeting carrier or the spacer material is first synthesized with the basic nano preparation material and then with the target. The material is further ligated to a targeting carrier. .
  • the distance between the targeting element and the nano-drug carrier is 0.2-100 nm.
  • the distance between the targeting element and the nano-medicine carrier is 0.3-10 nm.
  • a seventh object of the present invention is to provide a method for delivering medicines, which can efficiently carry medicines to a destination.
  • the present invention adopts the following scheme:
  • the method for transporting a medicinal ingredient to a target cell is: placing the medicinal ingredient in the targeting carrier and delivering the medicinal ingredient.
  • the target cell is a cell containing a mannose receptor.
  • target cells include, but are not limited to, macrophages, dendritic cells and tumor cells.
  • An eighth object of the present invention is to provide a composition with a targeting element, which can be used to prepare a highly targeted drug-loaded carrier.
  • a composition with a targeting element is composed of a targeting material and a spacer material, and the targeting material is mannose and / or a mannose derivative.
  • mannose derivative is mannoside and / or mannosamine and / or mannan.
  • the mannose derivative is a mannose derivative which is methyl-D-mannoside, 1- ⁇ formylmethyl-mannopyranoside, 4-aminophenyl- ⁇ -D-mannopyranoside , 4-nitrophenyl- ⁇ -D-pyranoside, 4-methylumbelliferyl- ⁇ -D-pyranoside, mannose-6-phosphate, carbamoyl-D-mannose .
  • the spacer material is one or more of glutaraldehyde, ethylenediamine, malonic acid, short-chain alkyl chain, PEG, amino acid, dipeptide, oligopeptide, polypeptide, stearoyl, and palmitoyl. .
  • the spacer is one of glutaraldehyde, ethylenediamine, malonic acid, short-chain alkyl chain (such as -CH2-CH2-), PEG, amino acid, dipeptide, oligopeptide, and polypeptide. Or several.
  • the spacer amino acids are arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, and tyrosine.
  • amino acids at the two ends of the dipeptide, oligopeptide and polypeptide are selected from the following amino acid composition: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, casein Amino acids; intermediate amino acids are selected from 20 arbitrary amino acids (such as RGD).
  • the molar ratio of the spacer material to the targeting material is 10-1: 1.
  • the molar ratio of the spacer material to the targeting material is 5: 1.
  • a ninth object of the present invention is to provide a targeting element, which can be used to prepare a highly targeted drug-loaded carrier.
  • the present invention adopts the following scheme:
  • a targeting element prepared using the described composition.
  • R1 is -C 5 H 8 O 2 , -C 2 H 7 N 2 , -C 3 H 3 O 4 , -O (CH 2 CH 2 O) nH , -C 2 H 3 O 2 NR 'or -C 2 H 3 ONR', -C 4 H 5 O 3 N 2 R 1 'R 2 ' or -C 4 H 5 O 2 N 2 R 1 'R 2 ' , -C 3 n + 1H 3 n + 2On + 2Nn + 1R 1 'R 2 ' or -C 3 n + 1H 3 n + 2On + 1Nn + 1R 1 'R 2 ', -C 18 H 35 O, C 16 H 31 O, -H, -CHO, -C 6 H 4 NH 2 , -C 6 H 4 NO 2 , -CONH 2, -CH 2 C 6 CCHCO 2 CH 3 , both R 1 ′ and R 2 ′
  • a tenth object of the present invention is to provide a targeting vector that can transport a drug to a target cell.
  • the present invention adopts the following scheme:
  • a targeting vector containing said targeting element is provided.
  • lipid nanoparticles formed by linking a targeting element and a nano-formulation, which is used for preparing liposomes, emulsions, nanogels, core-shell nanoparticles, HDL nanoparticles, solid lipid nanoparticles, and polymer micelles.
  • the material of the nano preparation may be the material of the nano preparation mentioned in "Modern Pharmaceutical Preparation Technology".
  • the material of the nano preparation is lipid, polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polylysine (PLL), and polyethyleneimine.
  • PLGA polylactic acid-glycolic acid copolymer
  • PLA polylactic acid
  • PCL polycaprolactone
  • PLL polylysine
  • PEI amine
  • PEI hyaluronic acid
  • chitosan gelatin
  • poloxamer poloxamer
  • stearyl alcohol stearyl alcohol
  • the lipid materials used to prepare liposomes and core-shell nanoparticles are cholesterol, egg yolk lecithin, soybean lecithin, cerebrolysin, sphingomyelin, PC (phosphatidylcholine), EPG (lecithin), SPG (soybean phosphatidylglycerol), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) , Distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dilinoleylphosphatidylcholine (DLPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphospholipid Glycerol (DPPG), Dimyristylphosphatidylcholine (DM
  • the solid lipid material for preparing the solid lipid nanoparticles is stearic acid, cholesterol, glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl tristearate, glyceryl laurate Esters, glyceryl palmitate stearate, behenyl monoglyceride, behenyl diglyceride, behenyl triglyceride, glyceryl trimyristate, glyceryl citrate, eighteen One or more of alcohol, palmitic acid, myristic acid, trimellitic acid, and lauric acid.
  • the single targeting vector is connected with 1-100000 of the targeting elements.
  • From 1 to 1000 of said targeting elements are connected to said single targeting vector.
  • the distance between the target of the targeting element and the nano-formulation is 0.2-100 nm.
  • the distance between the target of the targeting element and the nano-formulation is 0.3-10 nm.
  • the target is mannoside and / or mannosamine and / or mannan.
  • An eleventh object of the present invention is to provide a method for connecting the targeting element to a nano-formulation.
  • the method has universality, can synthesize a variety of targeted nano-formulations, and facilitates purification and characterization.
  • the present invention adopts the following scheme:
  • the method for connecting the targeting element to the nano preparation is characterized in that the targeting material and the spacer material are connected and then connected with the material of the nano preparation to make a nano preparation.
  • diethylene glycol, p-toluenesulfonyl chloride and triethylamine are taken, dissolved in DCM, reacted at room temperature for 24 hours, and the product TosOC 2 H 4 OC 2 H 4 OH (compound 1) is separated by column chromatography. Take compound 1 and pentaacetylated mannose and its derivative and boron trifluoride ether (BF 3 ⁇ Et 2 O) in DCM and react at room temperature for 24 hours. The product Aco-M-OC 2 H 4 is separated by column chromatography. OC 2 H 4 OTos (compound 2), M is mannose and its derivatives.
  • a twelfth object of the present invention is to provide a method for improving the drug delivery ability of the targeted carrier according to the claims.
  • the present invention adopts the following scheme:
  • the method for improving the drug delivery ability of the targeted carrier according to claim is to encapsulate the drug in the targeted carrier for delivery.
  • the drug is a small molecule drug and / or a protein peptide drug and / or a gene drug.
  • the small-molecule drug is a functional small molecule that can be wrapped in a nano-formulation and used for tumor targeted therapy, arteriosclerosis therapy, and various inflammatory diseases.
  • mRNA encoding a specific tumor antigen can be encapsulated in a targeting nano-formulation carrier, and then introduced into a somatic cell, and an antigen protein can be synthesized through the expression system of the host cell to induce the host immune system to The immune response can achieve the function of preventing and treating tumors, and the targeted nano-formulations containing mRNA have better tumor targeting, which is beneficial to the treatment of tumors.
  • anti-tumor chemical drugs and biological drugs, and drugs for treating arteriosclerosis and inflammatory diseases can be wrapped in the carrier of targeted nano-formulations to play a more precise targeted therapeutic role.
  • the small molecule drugs include, but are not limited to, PTX, DOX, and quercetin
  • the protein polypeptide drugs include, but are not limited to, albumin
  • the gene drugs include, but are not limited to, mRNA and siRNA.
  • the small-molecule drug is a functional small molecule that can be wrapped in a nano-formulation for tumor-targeted therapy, arteriosclerosis therapy, and various inflammatory diseases.
  • the antitumor drugs taxanes (paclitaxel, docetaxel, harringtonine, 7-epitaxel), camptothecins (camptothecin, SN38, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, etc.), vinblastine (vinblastine, vincristine, vincristine, vinorelbine, vinblastine, etc.), doxorubicin, epirubicin, daunorubicin, epirubicin Rubicin, amphotericin, methotrexate, cytarabine, 5-fluorouracil, mitoxantrone, or a derivative thereof, gefitinib, noscapine, cisplatin, carboplatin, oxali Platinum, carmustine,
  • the main medicine component protein polypeptide drugs are interleukins (such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, etc.), and various growth factors (such as fibroblast growth factor, liver growth factor, vascular endothelial growth factor, hematopoietic growth factor, etc.), interferon (such as IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factor (such as TNF ⁇ , TNF ⁇ ), integrin, monoclonal antibodies, Enzymes, insulin, etc.
  • the main medicine component gene medicine is DNA, plasmid, mRNA, siRNA, shRNA, microRNA and the like.
  • the main pharmaceutical ingredient can also be coated with an immune adjuvant to enhance the immune effect.
  • the medicament may be in any pharmaceutically acceptable dosage form.
  • the pharmaceutically acceptable dosage form includes one or more pharmaceutically acceptable carriers, diluents or excipients and is added in a suitable step in the above preparation process.
  • pharmaceutically acceptable refers to such compounds, raw materials, compositions and / or dosage forms, which are within the scope of sound medical judgment and are suitable for contact with patient tissues without excessive toxicity or irritation , Allergies or other problems and complications commensurate with a reasonable benefit / risk ratio, and effectively used for the intended purpose.
  • compositions are suitable for administration by any suitable route, such as by oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual, or transdermal), vaginal or parenteral (including subcutaneous, intradermal) , Intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous or subdermal injection or infusion) route.
  • Such formulations may be prepared by any method known in the pharmaceutical arts, for example by mixing the active ingredient with a carrier or excipient.
  • Oral, topical or injection administration is preferred.
  • Pharmaceutical preparations suitable for oral administration are provided in separate units, such as solutions or suspensions in aqueous or non-aqueous liquids; capsules or tablets; powders or granules; edible foam preparations or foaming preparations, and the like.
  • the targeting vector can deliver the drug to a target cell containing a mannose receptor target.
  • a thirteenth object of the present invention is to provide a complex containing the targeting vector.
  • the present invention adopts the following scheme:
  • the complex is a complex formed by the targeting vector and a receptor.
  • the targeting carrier carries a drug.
  • the receptor is a mannose receptor.
  • a fourteenth object of the present invention is to provide a method for improving the transfection efficiency of a transfection reagent, which can significantly improve the transfection efficiency of the transfection reagent.
  • the present invention adopts the following scheme:
  • the method for improving the transfection efficiency of the transfection reagent is to connect the targeting element with a carrier for preparing the transfection reagent to prepare a transfection reagent with high transfection efficiency.
  • Transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of foreign DNA.
  • the development of DNA transfection technology has had a huge impact on modern molecular biology.
  • Gene transfection technology is not only an important tool for studying transgenes and gene expression, but also a key step in current gene therapy.
  • the ideal gene transfection reagent should have the following characteristics: efficient transfection; safety; low cytotoxicity; simple method; time-saving and economical.
  • a fifteenth object of the present invention is to provide an application of the targeting element.
  • the present invention adopts the following scheme:
  • the targeted nano-formulations, nano-formulation compositions, targeting elements, targeting carriers, and targeted drugs provided by the present invention all have good targeting properties of the mannose receptor, and can efficiently target the target. Mannose receptor binding on cells;
  • the targeting element and the targeting carrier provided by the present invention have low preparation cost, are easy to synthesize, and have universality, can synthesize a variety of targeted nano preparations, and are beneficial to purification and characterization;
  • the spacer material provided by the present invention can make the targeting material and the basic nano-formulation material have a certain distance, so that the targeting material can be exposed on the surface of the targeting nano-formulation, thereby having high-efficiency manna. Sugar receptor targeting ability;
  • the prepared targeted nano preparations show good profile, small size, close to spherical shape, good serum stability, low cytotoxicity, and can be applied to targeted drug delivery systems for drugs (chemical and biological drugs)
  • the transfection efficiency is significantly higher than the commercial Lipo 3K, which can be used as a transfection reagent and applied to scientific research and commerce.
  • Figure 1 shows the results of NMR verification of mannose-PEG 100 -Chol.
  • FIG. 2 is a particle size chart of a liposome of Formula 1.
  • FIG. 3 is a potential diagram of a liposome of Formula 1.
  • FIG. 4 is a TEM image of a prescription 1 liposome.
  • FIG. 5 shows the results of liposome transfection.
  • Fig. 6 is a fluorescent microscope for observing the expression of GFP in DC cells.
  • Figure 7 is a histogram of transfection efficiency analysis.
  • Figure 8 shows the stability analysis of mannose-PEG 1000 -Chol.
  • Figure 9 shows the transfection efficiency of mannose-PEG 1000 -Chol under 4 ° C storage.
  • Figure 10 shows the stability analysis of mannose-PEG 1000 -Chol in serum.
  • Figure 11 Flow cytometry to detect the toxicity of mannose-PEG 1000 -Chol and Lipo 3K on DC cells.
  • FIG. 12 Histogram of statistical analysis of data of flow cytometry to detect the toxicity of mannose-PEG 1000 -Chol and Lipo 3K on DC cells.
  • Figure 13 is a study of targeted nanoparticles uptake by DC2.4 cells.
  • FIG. 14 is a fluorescence microscope for observing the uptake of mRNA-targeted liposome complexes in BMDCs cells.
  • FIG. 15 is a data analysis of fluorescence microscope observation of uptake of mRNA-targeted liposome complexes in BMDCs cells.
  • FIG. 16 is an in vivo antitumor effect study of a vaccine prepared by liposome-loaded mRNA prepared from mannose polyethylene glycol 400 cholesterol.
  • FIG. 17 is a mouse body weight monitoring of an in vivo antitumor study of a vaccine prepared by liposome-loaded mRNA prepared with mannose polyethylene glycol 400 cholesterol.
  • FIG. 18 shows the tumor volume of mice on the 28th day of the in vivo antitumor study of the vaccine prepared by liposome-loaded mRNA prepared with mannose polyethylene glycol 400 cholesterol.
  • Figure 19 shows the antitumor effect of m / MP400-LPX groups in vivo.
  • Figure 20 shows the survival time of mice after m / MP400-LPX immunization.
  • Figure 21 shows the uptake of different ligand-chain-targeted liposome complexes by mRNA in DC2.4 cells.
  • Figure 22 is a data analysis of the uptake of different ligand-chain-targeted liposome complexes carrying mRNA in DC2.4 cells.
  • FIG. 23 shows the transfection effect of DC2.4 cells on different ligand chain length targeting liposome complexes.
  • Figure 24 is a data analysis of the transfection effect of DC2.4 cells on different ligand chain length targeting liposome complexes.
  • Figure 25 shows the transfection effect of BMDCs cells on different ligand chain length targeting liposome complexes.
  • Figure 26 is a data analysis of the effect of BMDCs cells on transfection of different ligand chain length targeting liposome complexes.
  • FIG. 27 shows the antitumor effect in liposome complexes with different ligand chain lengths.
  • FIG. 28 is a mouse body weight monitoring for antitumor studies in liposome complexes targeting different ligand chain lengths.
  • Figure 29 shows the tumor volume of mice on anti-tumor studies in liposome complexes targeting different ligand chain lengths on day 28.
  • FIG. 30 is an analysis of antitumor effect data in liposome complexes targeting different ligand chain lengths.
  • Figure 31 is the survival time of mice after immunization with different ligand chain length targeting liposome complexes.
  • the nano preparation is composed of a targeting ligand mannose and its derivative, a nano preparation and a main drug component.
  • the targeting ligand is mannose.
  • the targeting ligand may be a mannose derivative, and may be one or more of the above-mentioned mannoside, mannosamine, mannan, and the like.
  • the mannose derivatives are methyl-D-mannoside, 1- ⁇ formylmethyl-mannanoside, 4-aminophenyl- ⁇ -D-pyranoside, 4-nitrophenyl - ⁇ -D-pyranoside, 4-methylumbelliferone- ⁇ -D-pyranoside, mannose-6-phosphate, carbamoyl-D-mannose, mannamine, mannose One or more of sugar.
  • the nano preparations are liposomes, HDL nanoparticles, solid lipid nanoparticles, and polymer micelles.
  • the nano-formulation may be one or more of an emulsion, a nanogel, a core-shell type nanoparticle, and the like.
  • the main drug component contained in the nano preparation is one or more of a small molecule drug, a protein peptide drug, or a gene drug.
  • the nano preparation is modified by a single mannose and its derivative and / or 2 to 10 mannose and its derivative.
  • the nano-formulation is prepared after coupling the targeting ligand mannose and its derivative with the carrier material for preparing the nano-formulation.
  • the targeting ligand mannose and its derivatives can be prepared and then the mannose and its derivatives can be adsorbed or coupled to the surface of the nano-formulations.
  • the mannose and its derivative are coupled with the nano preparation or a carrier material for preparing the nano preparation through a spacer PEG.
  • the mannose and its derivatives are directly coupled to the nano-formulation or the carrier material for preparing the nano-formulation or are coupled through other spacers.
  • the other spacers may be glutaraldehyde, ethylenediamine, and malondialdehyde.
  • the spacer amino acids are arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, and tyrosine.
  • amino acids at the two ends of the dipeptide, oligopeptide and polypeptide are selected from the following amino acid composition: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, casein Amino acids; intermediate amino acids are selected from 20 arbitrary amino acids (such as RGD).
  • the PEG may be an ordinary PEG with unmodified both ends having hydroxyl groups, or may be an PEG with at least one end being aminated, and one end of the PEG is connected to DSPE, cholesterol, and palmitic acid.
  • one end of the PEG may be connected to other carrier materials having active amino groups or hydroxyl groups, which are pharmaceutically acceptable for preparing liposomes, HDL nanoparticles, solid lipid nanoparticles, polymer micelles, and the like.
  • the carrier material is selected from the group consisting of polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polylysine (PLL), polyethyleneimine (PEI), and hyaluronic acid (HA ), Chitosan, gelatin, poloxamer, stearyl egg yolk lecithin, soy lecithin, brain phospholipid, sphingomyelin, PC (phosphatidylcholine), EPG (lecithin), SPG (soy phospholipid) Acylglycerol), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), dimyristyl Phospholipidylcholine (DMPC), Dilinoleylphosphatidylcholine (DLPC), Dioleylphosphati
  • the prepared nano preparation is delivered into the body by intravenous injection or subcutaneous injection.
  • the prepared nano-formulation can also be delivered into the body by intraperitoneal injection or intramuscular injection.
  • mRNAs involved in the examples were obtained under conventional conditions, such as those described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook, et al.), Or purchased or implemented according to the conditions recommended by the manufacturer.
  • Mannose-PEGn-Chol Take mannose-PEG100-Chol as an example. Other PEGs of different lengths are also synthesized according to this synthetic route. Diethylene glycol, p-toluenesulfonyl chloride and triethylamine were dissolved in DCM and reacted at room temperature for 24 hours. The product TosOC2H4OC2H4OH (compound 1) was separated by silica gel column chromatography.
  • Mannose-PEG2000-DSPE DSPE-PEG200-NH2 can be purchased directly. Weigh 5mg of mannose and add 5ml of ethanol to dissolve. Weigh 5mg DSPE-PEG-NH2, dissolve in 5ml chloroform-ethanol mixed solvent (9: 1, V / V), and add 0.5ml mannose ethanol solution dropwise under nitrogen protection, stir and mix, then add 100 ⁇ l of triethylamine was stirred at 40 ° C. for 12 h. After the reaction, the organic solvent was removed by rotary evaporation under reduced pressure, deionized water was added for reconstitution, dialysis was performed to remove unreacted mannose, and the product was mannose-PEG2000-DSPE by freeze-drying.
  • the main drug of prescriptions 1 to 5 is GFP-mRNA.
  • Preparation method 2 (prescription 8): According to the above formula, weigh different proportions of phospholipids, add chloroform / ether to dissolve, then add an aqueous solution of albumin, ultrasonically obtain an emulsion, remove all organic solvents by rotary evaporation, and then add PBS7. 4 Buffers rehydrate the liposomes.
  • Solid lipid nanoparticles are nano-structured carriers prepared from lipids as the backbone material, and have the characteristics of physiological compatibility, cell affinity, and targeting.
  • the mannose-modified lipid is added to improve the targeting of the solid lipid nanoparticles.
  • Preparation method Precisely weigh a prescribed amount of a drug, a lipid material dissolved in ethanol, and stir in a constant temperature water bath to form an oil phase; weigh a prescribed amount of a surfactant to dissolve in purified water, and heat in a constant temperature water bath to form an aqueous phase; under stirring conditions , The oil phase is injected into the water phase, and the emulsion is concentrated by constant temperature stirring, emulsification and concentration. After the emulsion is concentrated to a certain volume, it is poured into cold water at a constant temperature of 4 ° C, stirred and solidified, and obtained by passing through a 0.45 ⁇ m filter membrane.
  • HDL is a class of lipoproteins with uneven composition, density, and particle size.
  • the most abundant apolipoprotein in HDL is ApoA-I, whose hydrophobic core transports cholesterol in the body.
  • Recombinant high-density lipoprotein has endogenous isolation or in vitro synthesis of ApoA-I and phospholipids, cholesterol, etc., which are reconstituted in vitro, and have similar biochemical characteristics and functions to endogenous HDL.
  • High-density lipoprotein has many excellent characteristics: it has a high degree of safety, high-efficiency carrying capacity (can carry drugs in three ways: core embedding, and lipophilic drug embedding and recombination in the core of high-density lipoprotein; surface Embedding, and insertion of lipophilic drugs into the phospholipid monolayer; protein bonds and), targeting.
  • a mannose ligand is added to the prepared HDL nanoparticles to further improve the targeting of the nanoparticles.
  • Preparation method Similar to the preparation method of liposomes, all methods suitable for the preparation of liposomes can be used for the preparation of HDL nanoparticles.
  • Preparation method 1 According to the above formula, weigh different proportions of phospholipids (the fat-soluble main drug DOX is added at the same time), add them to a round-bottom flask, add chloroform to dissolve, and then remove the organic solvent by rotary evaporation under reduced pressure to form a layer on the inner wall of the bottle.
  • the membrane was hydrated by adding PBS 7.4 buffer solution and sonicated at 100 W for 3 min to obtain liposomes. Weigh the prescribed amount of ApoA-I slowly into the liposome suspension, incubate at 4 ° C for 24 hours, and obtain it by dialysis.
  • Preparation method 2 Weigh different proportions of phospholipids according to the above prescription, add them to a round bottom flask, add chloroform to dissolve, and then remove the organic solvent by rotary evaporation under reduced pressure. The suspension was sonicated at 100 W for 3 min to obtain liposomes. Weigh the prescribed amount of ApoA-I slowly into the liposome suspension, incubate at 4 ° C for 24 hours, and obtain it by dialysis.
  • Preparation method Take the carrier material and the main medicine and dissolve them in chloroform (you can choose other solvents, such as ethanol, or a mixed solvent of ethanol and chloroform according to the dissolution), remove the organic solvent by rotary evaporation under reduced pressure, and form a thin film on the inner wall of the bottle. Hydration of PBS7.4 buffer solution.
  • the material of the lipid is cholesterol, egg yolk lecithin, soybean lecithin, cerebral phospholipid, sphingomyelin, PC (phosphatidylcholine), EPG (lecithinyl glycerol), SPG (soybean phosphatidyl glycerol), Distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dilinoleylphosphatidylcholine (DLPC), dioleylphosphatidyl glycerol (DOPG), dipalmitoylphosphatidyl glycerol (DPPG), dimeat Legumylphosphatidylcholine (DMPC),
  • the amino acid of the spacer material is one of arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, and tyrosine.
  • the peptide is a dipeptide, an oligopeptide, a polypeptide, and the amino acids at both ends of the peptide are selected from the following amino acid composition: arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine Amino acid, tyrosine; intermediate amino acid is selected from 20 arbitrary amino acids (such as RGD).
  • the basic nano preparation material of Example 7 is prepared into a nano drug delivery carrier, and the targeting material of Example 7 is attached to the surface of the prepared nano drug delivery carrier to obtain a targeting carrier.
  • the targeting material and the spacer material of Example 7 were synthesized.
  • the synthesis of mannose and PEgn is taken as an example.
  • the targeting element of Example 9 is further synthesized with the basic nano-formulation material.
  • the synthesis of mannose, PEGn, and cholesterol is taken as an example.
  • Example 11 Improving the targeting of a targeting vector
  • Method for improving the targeting carrier of Example 8 A. synthesizing the targeting element and spacer material of Example 7 into a targeting element; B. preparing the basic nano-formulation material of Example 7 into a nano-formulation, and then preparing the The targeting element is connected to the nano-drug carrier of Example 8.
  • Mannose-PEGn-Chol Take mannose-PEG100-Chol as an example. Other PEGs of different lengths are also synthesized according to this synthetic route. Diethylene glycol, p-toluenesulfonyl chloride and triethylamine were dissolved in DCM and reacted at room temperature for 24 hours. The product TosOC2H4OC2H4OH (formula I) was separated by silica gel column chromatography. Take the compound of formula I, pentaacetylated mannose and boron trifluoride ether (BF3 ⁇ Et2O), dissolve it in DCM, and react at room temperature for 24 hours.
  • BF3 ⁇ Et2O boron trifluoride ether
  • the product Aco-Mannose-OC2H4OC2H4OTos (formula II) is separated by silica gel column chromatography.
  • the compound of formula II and sodium azide were dissolved in DMF and reacted at 60 ° C for 24 hours.
  • the solid product Aco-Mannose-OC2H4OC2H4N3 (formula III) was separated by silica gel column chromatography.
  • the compound of formula III is dissolved in a methanol solution of sodium methoxide, reacted at room temperature for 3 hours, and concentrated to obtain the product Mannose-OC2H4OC2H4N3 (formula IV).
  • Cholesterol, bromopropyne and sodium hydrogen were dissolved in a mixed solution of ether and DMF and reacted at room temperature for 24 hours.
  • the solid product Chol-CH2CCH was isolated by silica gel column chromatography (formula V).
  • the compound of formula IV, the compound of formula V and cuprous iodide were dissolved in DMF and reacted at room temperature for 24 hours.
  • the solid product Mannose-Chol (formula VI) was obtained by silica gel column chromatography.
  • Mannose-PEG2000-DSPE DSPE-PEG200-NH2 can be purchased directly. Weigh 5mg of mannose and add 5ml of ethanol to dissolve. Weigh 5mg DSPE-PEG-NH2, dissolve in 5ml chloroform-ethanol mixed solvent (9: 1, V / V), and add 0.5ml mannose ethanol solution dropwise under nitrogen protection, stir and mix, then add 100 ⁇ l of triethylamine was stirred at 40 ° C. for 12 h. After the reaction, the organic solvent was removed by rotary evaporation under reduced pressure, deionized water was added for reconstitution, dialysis was performed to remove unreacted mannose, and the product was mannose-PEG2000-DSPE by freeze-drying.
  • the drug was encapsulated in the targeting vector prepared in Example 8.
  • the drug is a small molecule drug and / or a protein peptide drug and / or a gene drug.
  • the small-molecule drugs are anti-tumor drugs taxanes (paclitaxel, docetaxel, harringtonine, 7-epitaxel), camptothecins (camptothecin, SN38, irinotecan, 9- Aminocamptothecin, 9-nitrocamptothecin, etc.), vinblastine (vinblastine, vincristine, vincristine, vinorelbine, vinblastine, etc.), doxorubicin, epirubicin, roucine Erythromycin, epirubicin, amphotericin, methotrexate, cytarabine, 5-fluorouracil, mitoxantrone or derivatives thereof, gefitinib, noscapine, cisplatin, card Platinum, oxaliplatin, carmustine, quercetin, etc.
  • taxanes paclitaxel, docetaxel, harringtonine, 7-epitaxel
  • camptothecins camptothecins
  • the main medicine component protein polypeptide drugs are interleukins (such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, etc.), and various growth factors (such as fibroblast growth factor, liver growth factor, vascular endothelial growth factor, hematopoietic growth factor, etc.), interferon (such as IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factor (such as TNF ⁇ , TNF ⁇ ), integrin, monoclonal antibodies, Enzymes, insulin, etc.
  • interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, etc.
  • various growth factors such as fibroblast growth factor, liver growth factor, vascular endothelial growth factor, hematopoietic growth factor, etc.
  • interferon such as IFN ⁇ , IFN ⁇ , IFN ⁇
  • tumor necrosis factor such as TNF ⁇ , TNF ⁇
  • the main medicine component gene medicine is DNA, plasmid, mRNA, siRNA, shRNA, microRNA and the like.
  • the main pharmaceutical ingredient can also be coated with an immune adjuvant to enhance the immune effect.
  • the main drug of prescriptions 1 to 5 is GFP-mRNA, (purchased from trilink, batch number: WOTL6647).
  • the obtained liposomes were characterized. The results showed that the particle size of the liposomes was 132.93 ⁇ 4.93nm and the zeta potential was 37.93 ⁇ 2.95mV. It was observed that the liposomes had a clear lipid membrane structure and a shape close to a sphere ( Figure 2-4).
  • Preparation method 2 (prescription 8): According to the above formula, weigh different proportions of phospholipids, add chloroform / ether to dissolve, then add an aqueous solution of albumin, ultrasonically obtain an emulsion, remove all organic solvents by rotary evaporation, and then add PBS7. 4 Buffers rehydrate the liposomes.
  • Solid lipid nanoparticles are nano-structured carriers prepared from lipids as the backbone material, and have the characteristics of physiological compatibility, cell affinity, and targeting.
  • the mannose-modified lipid was added to improve the targeting of the solid lipid nanoparticles.
  • Preparation method Precisely weigh a prescribed amount of a drug, a lipid material dissolved in ethanol, and stir in a constant temperature water bath to form an oil phase; weigh a prescribed amount of a surfactant to dissolve in purified water, and heat in a constant temperature water bath to form an aqueous phase; under stirring conditions , The oil phase is injected into the water phase, and the emulsion is concentrated by constant temperature stirring, emulsification and concentration. After the emulsion is concentrated to a certain volume, it is poured into cold water at a constant temperature of 4 ° C, stirred and solidified, and obtained by passing through a 0.45 ⁇ m filter membrane.
  • HDL is a class of lipoproteins with uneven composition, density, and particle size.
  • the most abundant apolipoprotein in HDL is ApoA-I, and its hydrophobic core transports cholesterol in the body.
  • Recombinant high-density lipoprotein has endogenous isolation or in vitro synthesis of ApoA-I and phospholipids, cholesterol, etc., which are reconstituted in vitro, and have similar biochemical characteristics and functions to endogenous HDL.
  • High-density lipoprotein has many excellent characteristics: it has a high degree of safety, high-efficiency carrying capacity (can carry drugs in three ways: core embedding, and lipophilic drug embedding and recombination in the core of high-density lipoprotein; surface Embedding, and insertion of lipophilic drugs into the phospholipid monolayer; protein bonds and), targeting.
  • a mannose ligand is added to the prepared HDL nanoparticles to further improve the targeting of the nanoparticles.
  • Preparation method Similar to the preparation method of liposomes, all methods suitable for the preparation of liposomes can be used for the preparation of HDL nanoparticles.
  • Preparation method 1 According to the above formula, weigh different proportions of phospholipids (the fat-soluble main drug DOX is added at the same time), add them to a round-bottom flask, add chloroform to dissolve, and then remove the organic solvent by rotary evaporation under reduced pressure to form a layer on the inner wall of the bottle.
  • the membrane was hydrated by adding PBS 7.4 buffer solution and sonicated at 100 W for 3 min to obtain liposomes. Weigh the prescribed amount of ApoA-I slowly into the liposome suspension, incubate at 4 ° C for 24 hours, and obtain it by dialysis.
  • Preparation method 2 Weigh different proportions of phospholipids according to the above prescription, add them to a round bottom flask, add chloroform to dissolve, and then remove the organic solvent by rotary evaporation under reduced pressure to form a thin film on the inner wall of the bottle. The suspension was sonicated at 100 W for 3 min to obtain liposomes. Weigh the prescribed amount of ApoA-I slowly into the liposome suspension, incubate at 4 ° C for 24 hours, and obtain it by dialysis.
  • Preparation method Take the carrier material and the main medicine and dissolve them in chloroform (you can choose other solvents, such as ethanol, or a mixed solvent of ethanol and chloroform according to the dissolution), remove the organic solvent by rotary evaporation under reduced pressure, and form a film on the inner wall of the bottle. Hydration of PBS 7.4 buffer solution, that is, obtained.
  • DC2.4 cells were co-incubated with the prepared nano-targeting preparation in a 24-well plate, where the N / P ratio of the nano-targeting preparation was 5.
  • Flow cytometry was used to detect the transfection efficiency and mean fluorescence intensity (MFI) of GFP-positive DC2.4 cells.
  • DC2.4 cells are captured by forward scattering (FSC) and side scattering (SSC).
  • FSC forward scattering
  • SSC side scattering
  • Viable DC2.4 cells are shown in region 1 (R1) and GFP positive cells are shown in region 2 (R2).
  • Transfection efficiency is automatically displayed on R2.
  • MFI for GFP expression in GFP-positive cells was obtained using FlowJo software. Calculating MFI requires subtracting the background value of untreated DC2.4 cells.
  • mannose-PEG1000-Chol group induced the most GFP-positive cells, with a ratio as high as 52%, which was significantly higher than any other group.
  • the transfection efficiency of mannose-PEG1000-Chol was calculated as 52.09 ⁇ 4.85%, far exceeding the commercial transfection reagent Lipo 3K (11.47 ⁇ 2.31%).
  • the stability of mannose-PEG1000-Chol modified nano-targeted formulations was studied. The stability was determined by the particle size, zeta potential and transfection efficiency. The results showed that the mannose-PEG1000-Chol modified nano-targeting preparation was stored at 4 ° C, and the particle size of the mannose-PEG1000-Chol modified nano-targeting preparation was slightly reduced, but the zeta potential was not reduced ( Figure 8). When stored at 4 ° C for 3 days, the transfection efficiency is about 50% ( Figure 9). See the table below for details. Also in the serum, the mannose-PEG1000-Chol-incubated mRNA was not dissociated ( Figure 10). Similarly, the mannose-PEG100-Chol and mannose-PEG2000-Chol modified nano-targeted formulations also had good stability.
  • BMDCs cells in the plate were blown up, the medium was resuspended, and counted.
  • 1 mL of cell suspension was inserted into each well of the 24-well plate with a density of 10 ⁇ 10 4 cells / mL in an incubator. Incubate for about 12 hours and set aside. Discard the culture medium, and add lipopolysaccharide (LPS) with a final concentration of 100ng / mL to each well, and incubate at 37 ° C and 4 ° C for about 12 hours to stimulate BMDCs cells.
  • LPS lipopolysaccharide
  • the culture medium was discarded, and the cells cultured at 37 ° C were divided into a control group, a cytochalasin D group, a philippine group, a wortmannin group, and a chlorpromazine group, and a final concentration of 25ng / mL was added to each group.
  • MP 1000 -LPX added to the cells incubated at 4 °C MP 1000 -LPX, 37 [deg.] C or more packets respectively dark at 4 °C and incubated for about 2 hours at different temperatures, the uptake of the different drugs investigated BMDCs cell. After reaching the incubation time point, discard the medium and wash it twice with cold PBS; collect the cells in the 24-well plate with PBS, wash the cells once with PBS (1200 rpm, 5 min), and finally analyze the results in a flow cytometer .
  • MP400-LPX and methoxypolyethylene glycol 400 cholesterol were synthesized, which contained the main component E6 / E7-mRNA to form the E6 / E7-mRNA of the mannose target. Liposomal complex. Mice were injected with 30ug of the complex on day 0, day 3, day 10, and day 17, and the results are shown in Figure 16-20. Compared with the control group and the unrelated mRNA group, the E6 / E7-mRNA liposome complex-treated mice had significantly reduced tumor growth data.
  • DC2.4 cells had the best uptake of MP400-LPX.
  • the uptake efficiency of DC2.4 cells with different ligand-chain-targeted liposome complexes carrying mRNA is about 100%, but the uptake of each preparation by DC2.4 cells is different, and the specific expression is the intracellular fluorescence intensity Differently, MP100-LPX has significantly increased intracellular accumulation compared to LPX, MP400-LPX has significantly increased intracellular accumulation compared to MP100-LPX, and MP1000-LPX has significantly reduced intracellular accumulation compared to MP400-LPX. MP1000-LPX and MP2000-LPX accumulate in cells to a similar degree.
  • DC2.4 cells transfected MP400-LPX the best.
  • DC2.4 cells have different transfection efficiencies of different ligand-chain-targeting liposome complexes carrying mRNA, as shown by the percentage of GFP-positive cells.
  • MP400-LPX is different from LPX, MP100-LPX, and MP1000-LPX.
  • GFP-positive cells were significantly increased, and MP2000-LPX was significantly decreased in GFP-positive cells compared with other preparations.
  • the transfection results of BMDCs are basically similar to DC2.4, but different from DC2.4, the transfection efficiency of MP2000-LPX and other preparations is significantly higher than that of Lipo 3K, and BMDCs are primary cells. This transfection result shows that Targeted formulations have good application prospects.
  • Example 25 In vivo antitumor test of different ligand-chain-targeted liposome complexes carrying mRNA
  • MP100-LPX, MP400-LPX, MP1000-LPX, and MP2000-LPX were synthesized according to the methods of Example 7 and Example 9, containing the main component E6 / E7-mRNA to form E6 / E7-mRNA liposomes of the mannose target Complex. Mice were injected with 30 ug of complex on day 0, day 3, day 10, and day 17, and the results are shown in Figure 27-31. MP400-LPX had the best effect.
  • polyethylene glycol 400's mannose cholesterol targeting ligand exhibits the best antitumor effect in vivo, specifically: mice have the highest tumor cure rate, up to 60% Right and left, the tumor growth rate of uncured mice is slow, and at the same time, the survival time of tumor-bearing mice can be prolonged.

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Abstract

一种甘露糖修饰的靶向纳米制剂、制备纳米制剂的组合物、靶向元件、靶向载体和制备的靶向药物及其制备方法和应用。所述带有靶向功能的纳米制剂由靶向配体甘露糖及其衍生物、纳米制剂和主药成分组成，所述靶向材料和间隔材料连接后再与所述纳米制剂的材料连接制成纳米制剂。所述靶向纳米制剂具较好的甘露糖受体的靶向性，可以高效的与靶细胞上的甘露糖受体结合，且制备方法具有通用性，可以合成多种靶向纳米制剂，并有利于纯化和表征。

Description

甘露糖靶向的纳米制剂及其制备和应用
优先权申请
本申请要求2018年5月25提交的中国发明专利申请201810511368.2“一种甘露糖修饰的靶向纳米制剂”的优先权，该优先权发明专利申请以引用方式全文并入。
技术领域
本发明涉及医药领域，具体涉及一种甘露糖修饰的靶向纳米制剂、制备纳米制剂的组合物、靶向元件、靶向载体和制备的靶向药物及其制备方法和应用。
背景技术
近年来，基于药物受体介导的靶向药物传递系统已被广泛研究，靶向药物传递系统可提高其特异靶向性，克服药物体内分布广、毒副作用严重的问题。其中，凝集素受体是一类分布于细胞膜表面的糖蛋白、糖脂或糖复合物，能特异性识别并结合部分糖基。甘露糖受体是最重要的高效内吞凝集素受体，其功能包括清除内源性分子、促进抗原呈递、调节细胞激活和运输，其也与肿瘤的免疫逃逸和转移密切相关，主要表达于巨噬细胞，树突细胞和肿瘤细胞，可特异性识别甘露糖糖基分子。甘露糖基作为最有潜力的靶向基团，具有无毒、无免疫原性、生物相容性和生物可降解性良好等诸多优点，可广泛用于对药物传递系统的糖基化修饰。
脂质体、纳米粒作为最经典的纳米靶向给药载体，其靶向性的作用基础基于不同器官对微粒粒径大小摄取情况不同而选择性的聚集在肺等淋巴组织器官中，从而提高抗肿瘤药物的在各个器官的治疗效果。其制备简单 具有控释、无免疫原性，及提高疗效等特点。但是脂质体、纳米粒依赖巨噬细胞的内吞作用实现靶向性后，在靶向性运输过程中的稳定性及病灶局部的摄入效率还相对较低，其靶向性还需要进一步的不断的提高。为此随着生物药剂学、材料科学、纳米技术的不断发展，提高纳米粒靶向性和新型的靶向载体开发逐渐成为抗肿瘤领域的研究热点，现阶段出现了多种不同形式的纳米靶向递药类型。
以脂质体作为药物的靶向载体为例，根据其靶向作用发挥的原理，目前研究得比较成熟的可分为物理靶向性和分子靶向性脂质体。其中物理靶向性脂质体包括pH敏感脂质体(PLP)、光敏感脂质体、磁性脂质体、热敏感脂质体等。以光敏感性脂质体为例，Yang等利用光敏感细胞渗透肽pcCPP和含有天冬氨酸-甘氨酸-精氨酸残基(Asn-Gly-Arg，NGR)的短肽连接在脂质体表面，构建pcCPP-NGR-LP，该光敏感性脂质体可促进细胞摄取，并有效的沉默c-mycc基因，延缓人纤维肉瘤的生长。然而，以上不同形式的脂质体虽然在一定程度上加强了靶向作用，但仍有不足之处，pH敏感脂质体未能解决脂质体的肝脾蓄积毒性问题。光敏和磁性脂质体生物体释放的可行和长期存放稳定性等问题仍然亟待解决。热敏脂质体可直接杀伤肿瘤细胞，但加热时间过长也可造成正常组织损伤。分子靶向性脂质体也存在药物的定向输送和体内靶器官的吸收不好等问题。
综上，研究开发靶向药物，首要解决的问题就是纳米载体在运输过程中保持其稳定而不被破坏，普通的纳米粒或脂质体由于不具有主动靶向性(有些可能具有被动靶向性)，易被内皮系统清除，降低了利用率，还可能引起不必要的毒副反应。其次就是药物到达了靶细胞后，通过更有效的配体、更快速和直接的进入细胞发挥药效是研究的关键所在。
发明内容
本发明的目的之一是提供一种纳米制剂，通过甘露糖及其衍生物修饰，提高纳米制剂的靶向性。
本发明的目的之二是提供上述靶向纳米制剂的应用。
本发明提供的一种甘露糖及其衍生物修饰的靶向纳米制剂，其由靶向配体甘露糖及其衍生物、纳米制剂和主药成分组成，可用于疫苗递送、肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗。
具体地，本发明提供的靶向纳米制剂，甘露糖衍生物为甘露糖苷、甘露糖胺、甘露聚糖等中的一种或几种。
具体地，本发明提供的靶向纳米制剂，甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖、甘露糖胺、甘露聚糖等中的一种或几种。
具体地，本发明提供的靶向纳米制剂为脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束等中的一种或几种。
具体地，本发明提供的靶向纳米制剂，所述主药成分为小分子药物、蛋白多肽类药物或基因药物等中的一种或几种。
具体地，本发明提供的靶向纳米制剂，所述主药成分小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。比如抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等)，各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等)，干扰素(比如IFNα、IFNβ、IFNγ)，肿瘤坏死因子(比如TNFα、TNFβ)，整合素，单克隆抗体，酶，胰岛素 等。所述主药成分基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时，所述主药成分除用于免疫治疗的药物外，还可同时包裹免疫佐剂以增强免疫效果。
具体地，本发明提供的靶向纳米制剂，所述靶向配体甘露糖及其衍生物的含量为0.05％-40％。
优选地，本发明提供的靶向纳米制剂，所述靶向配体甘露糖及其衍生物的含量为1％-10％。
更优地，本发明提供的靶向纳米制剂，所述靶向配体甘露糖修的含量为2％-5％。
具体地，本发明提供的靶向纳米制剂，所述靶向配体甘露糖及其衍生物的修饰方式为：制备纳米制剂之后再将甘露糖及其衍生物吸附或偶联到纳米制剂表面；或先将甘露糖及其衍生物与制备纳米制剂的载体材料偶联之后再制备纳米制剂。
具体地，本发明提供的所述甘露糖及其衍生物吸附在纳米制剂表面的方式为：采用溶剂挥发法、高压均质法或微乳法制备纳米制剂的方式，使其吸附在纳米制剂表面。
具体地，本发明提供的靶向纳米制剂，修饰纳米制剂的甘露糖及其衍生物由单个甘露糖及其衍生物和/或2～10个甘露糖及其衍生物组成。
具体地，本发明提供的靶向纳米制剂，甘露糖及其衍生物吸附在纳米制剂表面的方式为：采用溶剂挥发法、高压均质法或微乳法。
具体地，本发明提供的靶向纳米制剂，甘露糖及其衍生物与纳米制剂或制备纳米制剂的载体材料偶联方式为：直接偶联或通过一个间隔基偶联。
具体地，本发明提供的靶向纳米制剂，甘露糖及其衍生物与纳米制剂或制备纳米制剂的载体材料通过间隔基偶联，所述间隔基为戊二醛、乙二胺、丙二酸、短链烷基链(比如-CH2-CH2-)、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰等中的一种或几种。具体地，间隔基PEG为分子量为100-5000，双端具有羟基或至少一端氨基化的PEG。较优地，间隔基PEG 的分子量为100-2000。间隔基氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸。二肽、寡肤、多肽的两端氨基酸选自以下的氨基酸组成：精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸；中间氨基酸选自20种任意氨基酸(比如RGD)。
具体地，本发明提供的靶向纳米制剂，制备纳米制剂的材料为具有活性氨基和/或羟基的药学上可以接受的制备胶束、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、脂质体、聚合物胶束等的载体材料。具体地，载体材料为脂质、聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、多聚赖氨酸(PLL)、聚乙烯亚胺(PEI)、透明质酸(HA)、壳聚糖、明胶、泊洛沙姆、硬脂醇等中的一种或几种。具体地，制备脂质体、核壳型纳米粒的脂质材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)等中的一种或多种。制备固脂纳米粒的固体脂质材料为硬脂酸、胆固醇、单硬脂酸甘油酯、双硬脂酸甘油酯、三硬脂酸甘油酯、三嵛酸甘油酯、月桂酸甘油酯、甘油棕榈酸硬脂酸脂、二十二酸单甘油酯、二十二烷酸双甘油酯、二十二烷酸三甘油酯、三豆蔻酸甘油酯、枸橼酸甘油酯、十八醇、棕榈酸、豆蔻酸、三嵛酸、月桂酸中的一种或几种。
本发明的目的之三在于提供用于制备带有特异性靶向载药载体的材料组合物，该组合物能够用于制备靶向性强的载药载体。
为实现上述目的，本发明的技术方案为：
用于制备带有靶向功能的纳米制剂的组合物，所述组合物包括靶向材料和基础纳米制剂材料，所述靶向材料为甘露糖和/或甘露糖衍生物。
进一步，所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
更进一步，所述甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖。
进一步，所述靶向材料占所述组合物的重量百分比为0.05-40％。
优选的，所述靶向材料占所述组合物的重量百分比为1-10％。
进一步，所述组合物由所述靶向材料、所述基础纳米制剂材料、使所述靶向材料与所述基础纳米制剂材料产生距离的间隔材料组成，所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
具体的，所述间隔基为戊二醛、乙二胺、丙二酸、短链烷基链(比如-CH2-CH2-)、PEG、氨基酸、二肽、寡肽、多肽等中的一种或几种。具体地，间隔基氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸。二肽、寡肤、多肽的两端氨基酸选自以下的氨基酸组成：精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸；中间氨基酸选自20种任意氨基酸(比如RGD)。
进一步，所述基础纳米制剂材料为制备脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束的材料的组合物。
具体的，所述基础纳米制剂材料为制备纳米制剂材料的组合物，可以是《现代药物制剂技术》中提到的纳米制剂材料。
具体的，所述基础纳米制剂材料为脂质、聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、多聚赖氨酸(PLL)、聚乙烯亚胺(PEI)、透明质酸(HA)、壳聚糖、明胶、泊洛沙姆、硬脂 醇中的一种或几种。
具体地，制备脂质体、核壳型纳米粒的脂质材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)中的一种或多种。
具体的，制备固脂纳米粒的固体脂质材料为硬脂酸、胆固醇、单硬脂酸甘油酯、双硬脂酸甘油酯、三硬脂酸甘油酯、三嵛酸甘油酯、月桂酸甘油酯、甘油棕榈酸硬脂酸脂、二十二酸单甘油酯、二十二烷酸双甘油酯、二十二烷酸三甘油酯、三豆蔻酸甘油酯、枸橼酸甘油酯、十八醇、棕榈酸、豆蔻酸、三嵛酸、月桂酸中的一种或几种。
进一步，所述间隔材料为PEGn,其中n＝100-5000。
优选的，所述n为200、400、1000或2000。
进一步，按照重量份计由以下组分组成：甘露糖1份；PEG 0.5-56份，胆固醇2-11份。
优选的，按照重量份计由以下组分组成：甘露糖1份；PEG 10-25份，胆固醇4-8份。
进一步，按照按照摩尔份计由以下组分组成：甘露糖1份；PEG 1-5份，胆固醇1-5份。
优选的，按照摩尔份计由以下组分组成：甘露糖1份；PEG 1-3份，胆固醇1-3份。
本发明的目的之四在于提供一种靶向载体及靶向药物，该靶向载体能 运送多种药效成分到达靶细胞，且靶向性强。
为实现上述目的，本发明采用以下方案：
所述的组合物制备的运载药物的靶向载体。
进一步，所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。
某些实施例中，将编码特定肿瘤抗原的mRNA可包裹于靶向纳米制剂载体中，再导入体细胞内，并通过宿主细胞的表达系统合成抗原蛋白，诱导宿主免疫系统产生对该肿瘤抗原的免疫应答可实现防治肿瘤的功能，且载有mRNA的靶向纳米制剂具有更好的肿瘤靶向性，有利于肿瘤的治疗。同样将抗肿瘤的化药和生物药、治疗动脉硬化和炎症性疾病的药物也可包裹于靶向纳米制剂载体中，起到更精准的的靶向治疗作用。
具体的，所述小分子药物包括但不限于PTX、DOX、槲皮素，所述蛋白多肽类药物包括但不限于白蛋白，所述基因药物包括但不限于mRNA、siRNA。
更具体的，所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。比如抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等)，各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等)，干扰素(比如IFNα、IFNβ、IFNγ)，肿瘤坏死因子(比如TNFα、TNFβ)，整合素，单克隆抗体，酶，胰岛素等。所述主药成分基因药物为DNA、质 粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时，所述主药成分除用于免疫治疗的药物外，还可同时包裹免疫佐剂以增强免疫效果。
被所述的靶向载体包裹的药物。
进一步，所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
具体的，所述小分子药物包括但不限于PTX、DOX、槲皮素，所述蛋白多肽类药物包括但不限于白蛋白，所述基因药物包括但不限于mRNA、siRNA。
进一步，所述药物可以为任意药学可接受剂型。
具体的，所述药学可接受剂型包一种或多种药学上可接受的载体、稀释剂或赋形剂并在上述制备过程中合适的步骤加入。本发明所使用的术语“药学上可接受的”是指这样的化合物、原料、组合物和/或剂型，它们在合理医学判断的范围内，适用于与患者组织接触而无过度毒性、刺激性、变态反应或与合理的利益/风险比相对称的其他问题和并发症，并有效用于既定用途。
药物制剂适于通过任何合适的途径给药，例如通过口服(包括口腔或舌下)、直肠、鼻、局部(包括口腔、舌下或经皮)、阴道或胃肠外(包括皮下、皮内、肌内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或者真皮下注射或输注)途径。可按药剂学领域的任何已知方法制备这类制剂，例如通过将活性成分与载体或赋形剂混合。优选口服给药、局部给药或注射给药。适于口服给药的药物制剂按独立的单位提供，例如水性或非水性液体中的溶液剂或混悬剂；胶囊剂或片剂；散剂或颗粒剂；可食用泡沫制剂或起泡制剂等。
本发明的目的之五在于提供一种靶向载体的制备方法，该方法可以用于工业化的制备靶向载体。
为实现上述目的，本发明采用以下方案：
制备所述的靶向载体的方法为：将所述基础纳米制剂材料制备成纳米运药载体，再将所述靶向材料附于所述纳米运药载体表面。
将所述靶向材料附于所述纳米运药载体表面的方法包括但不限于采用溶剂挥发法、薄膜分散法、超声波分散法、注入法、逆向蒸发法、高压均质法和微乳法制备纳米药物载体后，再将靶向材料溶液与其混合，使靶向材料附于其表面。上述制备纳米药物载体方法可以参照《现代药物制剂技术》
具体的，所述溶剂挥发法是制备微球的一种方法，简单的过程就是，先配置合适浓度的高分子溶液，然后将高分子溶液在连续相乳化，逐渐挥发掉溶剂，制成微球制剂，再向该微球制剂中加入靶向材料，使其靶向材料吸附在微球制剂表面。
高压均质法是物料在高压下通过均质阀，以极高的流速喷出，撞击碰撞环，通过空穴、撞击、剪切效应使物料超微细化和分散乳化，形成纳米微粒，该过程中物料的表面积增大，向其中加入靶向材料后，所述靶向材料便可附于表面。
薄膜蒸发法是将磷脂类材料溶于有机溶剂中，然后在减压状态下，将有机溶剂蒸发，在瓶内壁上形成一薄膜后，再加入水或PBS溶液将反复搅拌，洗下薄膜经均质、超声处理，即得纳米微粒制剂，向其中加入靶向材料后，所述靶向材料便可附于表面。
本发明的目的之六在于提供一种提高靶向载体靶向性的方法，该方法显著的提升的靶向载体的靶向性。
为实现上述目的，本发明采用以下方案：
提高所述的靶向载体的靶向性的方法，包括以下步骤：
A合成靶向元件
将所述靶向材料与所述间隔材料合成靶向元件；
B制备靶向载体
将基础纳米制剂材料制备成纳米制剂，再将步骤A所得的靶向元件与所述纳米运药载体连接得靶向载体或先将所述间隔材料与基础纳米制剂材料合成，再与所述靶向材料进行进一步连接得靶向载体。。
进一步，所述靶向元件与所述纳米运药载体的距离为0.2-100nm。
优选的，所述靶向元件与所述纳米运药载体的距离为0.3-10nm。
本发明的目的之七在于提供一种运送药物的方法，该方法能高效的运载药物到目的地。
为实现上述目的，本发明采用以下方案：
运送药效成分到达靶细胞的方法为：将药效成分置于所述靶向载体内，并运送。
进一步，所述靶细胞为含有甘露糖受体的细胞。
更进一步，所述靶细胞包括但不限于巨噬细胞、树突细胞和肿瘤细胞。
本发明目的之八在于提供一种带有靶向元件的组合物，所述组合物能用于制备靶向性强的载药载体。
为实现上述目的，本发明的技术方案为：
带有靶向元件的组合物，由靶向材料和间隔材料组成，所述靶向材料为甘露糖和/或甘露糖衍生物。
进一步，所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
更进一步，所述甘露糖衍生物为甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖。
进一步，所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
具体的，所述间隔基为戊二醛、乙二胺、丙二酸、短链烷基链(比如-CH2-CH2-)、PEG、氨基酸、二肽、寡肽、多肽等中的一种或几种。具体地，间隔基氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、 赖氨酸、丝氨酸、苏氨酸、酪氨酸。二肽、寡肤、多肽的两端氨基酸选自以下的氨基酸组成：精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸；中间氨基酸选自20种任意氨基酸(比如RGD)。
进一步，所述间隔材料与所述靶向材料的摩尔比为10-1:1。
优选的，所述间隔材料与所述靶向材料的摩尔比为5:1。
进一步，甘露糖与PEG100的摩尔比为1:1。
本发明的目的之九在于提供一种靶向元件，所述靶向元件能用于制备靶向性强的载药载体。
为实现上述目的，本发明采用以下方案：
运用所述的组合物制备的靶向元件。
进一步，所述靶向元件如式Ⅰ所示：其中R1为-C 5H 8O 2、-C 2H 7N 2、-C 3H 3O 4、-O(CH 2CH 2O)nH、-C 2H 3O 2NR＇或-C 2H 3ONR＇、-C 4H 5O 3N 2R 1＇R 2＇或-C 4H 5O 2N 2R 1＇R 2＇、-C 3n+1H 3n+2On+2Nn+1R 1＇R 2＇或-C 3n+1H 3n+2On+1Nn+1R 1＇R 2＇、-C 18H 35O、C 16H 31O、-H、-CHO、-C 6H 4NH 2、-C 6H 4NO 2、-CONH 2、-CH 2C 6CCHCO 2CH 3，所述R 1＇和R 2＇均为任意天然氨基酸侧链R＇基团，R 2为-CH 3或-PO 3H 2、R 3为-H，R 4为-H，R 5是-H或-NHCOCH 3，
Figure PCTCN2019088001-appb-000001
进一步，R1为聚乙二醇，其结构如下所示：R1为聚乙二醇，其结构为
Figure PCTCN2019088001-appb-000002
其中n＝1～5000，其对应的分子量为：40*n。
本发明的目的之十在于提供一种靶向载体，所述靶向载体可以运送药 物到达靶细胞。
为实现上述目的，本发明采用以下方案：
含有所述的靶向元件的靶向载体。
进一步，由靶向元件和纳米制剂连接而成，所述纳米制剂为制备脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束。
具体的，所述纳米制剂的材料可以是《现代药物制剂技术》中提到的纳米制剂材料。
具体的，所述纳米制剂的材料为脂质、聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、多聚赖氨酸(PLL)、聚乙烯亚胺(PEI)、透明质酸(HA)、壳聚糖、明胶、泊洛沙姆、硬脂醇中的一种或几种。
具体地，制备脂质体、核壳型纳米粒的脂质材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)中的一种或多种。
具体的，制备固脂纳米粒的固体脂质材料为硬脂酸、胆固醇、单硬脂酸甘油酯、双硬脂酸甘油酯、三硬脂酸甘油酯、三嵛酸甘油酯、月桂酸甘油酯、甘油棕榈酸硬脂酸脂、二十二酸单甘油酯、二十二烷酸双甘油酯、二十二烷酸三甘油酯、三豆蔻酸甘油酯、枸橼酸甘油酯、十八醇、棕榈酸、豆蔻酸、三嵛酸、月桂酸中的一种或几种。
进一步，所述单个靶向载体上连接有1-100000个所述的靶向元件。
优选的，所述单个靶向载体上连接有1-1000个所述的靶向元件。
进一步，所述靶向元件的靶头与所述纳米制剂的距离为0.2-100nm。
进一步，所述靶向元件的靶头与所述纳米制剂的距离为0.3-10nm。
进一步，所述靶头为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
本发明的目的之十一在于提供一种所述靶向元件连接至纳米制剂的方法，该方法具有通用性，可以合成多种靶向纳米制剂，并有利于纯化和表征。
为实现上述目的，本发明采用以下方案：
将所述的靶向元件连接至纳米制剂的方法，其特征在于，将所述靶向材料和间隔材料连接后再与所述纳米制剂的材料连接制成纳米制剂。
进一步，具体包括以下步骤：
1)取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应，通过柱层析分离得化合物1；
2)取化合物1与五乙酰化甘露糖及其衍生物和三氟化硼乙醚溶于DCM中，室温反应，柱层析分离得化合物2；
3)取化合物2与叠氮钠，溶于DMF中，硅胶柱层析分离得固体化合物3；
4)取化合物3溶于甲醇钠的甲醇溶液中，室温反应，浓缩得化合物4；
5)取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应，硅胶柱层析分离得固体化合物5；
6)取化合物4、化合物5和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得到靶向载体固体化合物6。
某些实施例中，取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应24h，通过柱层析分离得产物TosOC 2H 4OC 2H 4OH(化合物1)。取化合物1与五乙酰化甘露糖及其衍生物和三氟化硼乙醚(BF 3·Et 2O)溶于DCM中，室温反应24h，柱层析分离得产物Aco-M-OC 2H 4OC 2H 4OTos(化 合物2)，M为甘露糖及其衍生物。取化合物2与叠氮钠，溶于DMF中，60℃反应24h，硅胶柱层析分离得固体产物Aco-M-OC 2H 4OC 2H 4N 3(化合物3)。取化合物3溶于甲醇钠的甲醇溶液中，室温反应3h，浓缩得产物M-OC 2H 4OC 2H 4N 3(化合物4)。取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应24h，硅胶柱层析分离得固体产物Chol-CH 2CCH(化合物5)。取化合物4、化合物5和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得到固体产物M-PEG n-Chol(化合物6)。
本发明的目的之十二在于提供一种提高权利要求所述靶向载体运送药物能力的方法。
为实现上述目的，本发明采用以下方案：
提高权利要求所述靶向载体运送药物能力的方法为将药物包裹于所述靶向载体中运送。
进一步，所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
具体的，所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。
某些实施例中，可将编码特定肿瘤抗原的mRNA包裹于靶向纳米制剂载体中，再导入体细胞内，并通过宿主细胞的表达系统合成抗原蛋白，诱导宿主免疫系统产生对该肿瘤抗原的免疫应答可实现防治肿瘤的功能，且载有mRNA的靶向纳米制剂具有更好的肿瘤靶向性，有利于肿瘤的治疗。同样也可将抗肿瘤的化药和生物药、治疗动脉硬化和炎症性疾病的药物包裹于靶向纳米制剂载体中，起到更精准的的靶向治疗作用。
具体的，所述小分子药物包括但不限于PTX、DOX、槲皮素，所述蛋白多肽类药物包括但不限于白蛋白，所述基因药物包括但不限于mRNA、siRNA。
更具体的，所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。比如抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱 类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等)，各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等)，干扰素(比如IFNα、IFNβ、IFNγ)，肿瘤坏死因子(比如TNFα、TNFβ)，整合素，单克隆抗体，酶，胰岛素等。所述主药成分基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时，所述主药成分除用于免疫治疗的药物外，还可同时包裹免疫佐剂以增强免疫效果。
进一步，所述药物可以为任意药学可接受剂型。
具体的，所述药学可接受剂型包括一种或多种药学上可接受的载体、稀释剂或赋形剂并在上述制备过程中合适的步骤加入。本发明所使用的术语“药学上可接受的”是指这样的化合物、原料、组合物和/或剂型，它们在合理医学判断的范围内，适用于与患者组织接触而无过度毒性、刺激性、变态反应或与合理的利益/风险比相对称的其他问题和并发症，并有效用于既定用途。
药物制剂适于通过任何合适的途径给药，例如通过口服(包括口腔或舌下)、直肠、鼻、局部(包括口腔、舌下或经皮)、阴道或胃肠外(包括皮下、皮内、肌内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或者真皮下注射或输注)途径。可按药剂学领域的任何已知方法制备这类制剂，例如通过将活性成分与载体或赋形剂混合。优选口服给药、局部给药或注射给药。适于口服给药的药物制剂按独立的单位提供，例如水性或非水性液体中的溶液剂或混悬剂；胶囊剂或片剂；散剂或颗粒剂；可食用 泡沫制剂或起泡制剂等。
进一步，所述靶向载体可将药物运送到含有甘露糖受体靶点的靶细胞中。
本发明的目的之十三在于提供一种包含了所述靶向载体的复合物。
为实现上述目的，本发明采用以下方案：
所述复合物为所述靶向载体与受体形成的复合物。
进一步，所述靶向载体运载了药物。
进一步，所述受体为甘露糖受体。
本发明的目的之十四在于提供一种提高转染试剂转染效率的方法，该方法可显著的提高转染试剂的转染效率。
为实现上述目的，本发明采用以下方案：
提高转染试剂转染效率的方法为将所述的靶向元件与用于制备转染试剂的载体连接制备高转染效率的转染试剂。
转染(transfection)指真核细胞由于外源DNA掺入而获得新的遗传标志的过程。DNA转染技术的发展对现代分子生物学产生了巨大的影响。基因转染技术不仅是研究转基因和基因表达的重要工具，而且是目前基因治疗的关键步骤。理想的基因转染试剂应该具有如下特点：高效转染；安全；低细胞毒性；方法简单；省时、经济。
本发明的目的之十五在于提供所述靶向元件的应用。
为实现上述目的，本发明采用以下方案：
所述靶向元件作为靶向油相的运用。
所述靶向元件作为靶向水相的运用。
具体的，根据相似相溶原理。不易溶于水的属于油相，易溶于水的属于水相。再简单一点则是把该物质与水混溶，结果能成透明溶液的属水相；分层或浑浊的属油相。
本发明的有益效果在于：
1)本发明所提供的所述靶向纳米制剂、纳米制剂的组合物、靶向元件、 靶向载体、靶向药物均具较好的甘露糖受体的靶向性，可以高效的与靶细胞上的甘露糖受体结合；
2)本发明所提供的所述靶向元件、靶向载体的制备成本低廉，易于合成，且具有通用性，可以合成多种靶向纳米制剂，并有利于纯化和表征；
3)本发明提供的所述间隔材料可使所述靶向材料与所述基础纳米制剂材料产生一定距离，因此可使所述靶向材料暴露于靶向纳米制剂的表面，从而具有高效的甘露糖受体靶向能力；
4)所制备的靶向纳米制剂显示出良好的廓形，尺寸小，接近球形，有着良好的血清稳定性，细胞毒性小，可适用于药物(化药和生物药)的靶向给药系统，且转染效率显著高于商品化的Lipo 3K，可作为转染试剂使用，应用到科研以及商业中。
附图说明
图1为甘露糖-PEG 100-Chol核磁验证结果图。
图2为处方1脂质体的粒径图。
图3为处方1脂质体的电位图。
图4为处方1脂质体的TEM图。
图5为脂质体转染结果。
图6为荧光显微镜观察DC细胞中GFP的表达情况。
图7为转染效率分析柱状图。
图8为甘露糖-PEG 1000-Chol的稳定性分析。
图9为4℃存放下甘露糖-PEG 1000-Chol的转染效率。
图10为甘露糖-PEG 1000-Chol在血清中的稳定性分析。
图11流式细胞术检测甘露糖-PEG 1000-Chol和Lipo 3K对于DC细胞的毒性。
图12流式细胞术检测甘露糖-PEG 1000-Chol和Lipo 3K对于DC细胞的毒性的数据统计分析柱状图。
图13为DC2.4细胞摄取靶向纳米粒研究。
图14为荧光显微镜观察BMDCs细胞中mRNA靶向脂质体复合物的摄取情况。
图15为荧光显微镜观察BMDCs细胞中mRNA靶向脂质体复合物的摄取情况的数据分析。
图16为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤效果研究。
图17为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤研究的小鼠体重监测。
图18为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤研究的小鼠第28天的肿瘤体积。
图19为m/MP400-LPX各组体内抗肿瘤效果。
图20为m/MP400-LPX免疫后小鼠生存期。
图21为DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取。
图22为DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取的数据分析。
图23为DC2.4细胞对不同配体链长靶向脂质体复合物的转染效果。
图24为DC2.4细胞对不同配体链长靶向脂质体复合物的转染效果数据分析。
图25为BMDCs细胞对不同配体链长靶向脂质体复合物的转染效果。
图26为BMDCs细胞对不同配体链长靶向脂质体复合物的转染效果数据分析。
图27为不同配体链长靶向脂质体复合物体内抗肿瘤效果。
图28为不同配体链长靶向脂质体复合物体内抗肿瘤研究的小鼠体重监测。
图29为不同配体链长靶向脂质体复合物体内抗肿瘤研究的小鼠第28 天的肿瘤体积。
图30为不同配体链长靶向脂质体复合物体内抗肿瘤效果数据分析。
图31为不同配体链长靶向脂质体复合物免疫后小鼠生存期。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1
针对现有纳米制剂的不足，设计出一种甘露糖及其衍生物修饰的具有靶向性的纳米制剂。
本实施方式中，纳米制剂由靶向配体甘露糖及其衍生物、纳米制剂和主药成分组成。
本实施方式中，靶向配体为甘露糖。
在其他实施方式中，靶向配体可以是甘露糖衍生物，可以是上述的甘露糖苷、甘露糖胺、甘露聚糖等中的一种或几种。具体地，甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖、甘露糖胺、甘露聚糖等中的一种或几种。
本实施方式中，纳米制剂为脂质体、HDL纳米粒、固脂纳米粒、聚合物胶束。
在其他实施方式中，纳米制剂可以是乳剂、纳米凝胶、核壳型纳米粒等中的一种或几种。
本实施方式中，纳米制剂包载的主药成分为小分子药物、蛋白多肽类药物或基因药物等中的一种或几种。
本实施方式中，由单个甘露糖及其衍生物和/或2～10个甘露糖及其衍 生物修饰纳米制剂。
本实施方式中，靶向配体甘露糖及其衍生物与制备纳米制剂的载体材料偶联之后再制备纳米制剂。
在其他实施方式中，靶向配体甘露糖及其衍生物可以制备纳米制剂之后再将甘露糖及其衍生物吸附或偶联到纳米制剂表面。
本实施方式中，甘露糖及其衍生物与纳米制剂或制备纳米制剂的载体材料通过间隔基PEG偶联。
在其他实施方式中，甘露糖及其衍生物与纳米制剂或制备纳米制剂的载体材料直接偶联或通过其他间隔基偶联，其他间隔基可以是上述的戊二醛、乙二胺、丙二酸、短链烷基链(比如-CH2-CH2-)、PEG、氨基酸、二肽、寡肽、多肽等中的一种或几种。具体地，间隔基氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸。二肽、寡肤、多肽的两端氨基酸选自以下的氨基酸组成：精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸；中间氨基酸选自20种任意氨基酸(比如RGD)。
在本实施方式中，PEG可以是未修饰的双端均为羟基的普通PEG，也可以是至少一端氨基化的PEG，PEG一端连接DSPE、胆固醇、棕榈酸。
在其他实施方式中，PEG一端也可以连接其他上述的具有活性氨基或羟基的，药学上可以接受的制备脂质体、HDL纳米粒、固脂纳米粒、聚合物胶束等的载体材料。具体地，所述载体材料选自聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、多聚赖氨酸(PLL)、聚乙烯亚胺(PEI)、透明质酸(HA)、壳聚糖、明胶、泊洛沙姆、硬脂醇蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆 碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)、硬脂酸、胆固醇、单硬脂酸甘油酯、双硬脂酸甘油酯、三硬脂酸甘油酯、三嵛酸甘油酯、月桂酸甘油酯、甘油棕榈酸硬脂酸脂、二十二酸单甘油酯、二十二烷酸双甘油酯、二十二烷酸三甘油酯、三豆蔻酸甘油酯、枸橼酸甘油酯、十八醇、豆蔻酸、三嵛酸、月桂酸等中的一种或几种。
在本实施方式中，制备的纳米制剂，采用静脉注射、皮下注射的方式送入体内。
在其他实施方式中，制备的纳米制剂，也可以采用腹腔注射、肌肉注射的方式送入体内。
在实施例中所涉及到的mRNA按照常规条件，例如分子克隆实验指南(第三版，J.萨姆布鲁克等著)中所述的条件，或购买或按照制造厂商所建议的条件实施得到。
以下为具体实施例。
实施例1 载体材料的合成
甘露糖-PEGn-Chol：以甘露糖-PEG100-Chol合成为例，其他不同长度PEG也按此合成路线合成。取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应24h，硅胶柱层析分离得产物TosOC2H4OC2H4OH(化合物1)。取化合物1与五乙酰化甘露糖和三氟化硼乙醚(BF3·Et2O)，溶于DCM中，室温反应24h，硅胶柱层析分离得产物Aco-Mannose-OC2H4OC2H4OTos(化合物2)。取化合物2与叠氮钠，溶于DMF中，60℃反应24h，硅胶柱层析分离得固体产物Aco-Mannose-OC2H4OC2H4N3(化合物3)。取化合物3溶于甲醇钠的甲醇溶液中，室温反应3h，浓缩得产物Mannose-OC2H4OC2H4N3(化合物4)。取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应24h，硅胶柱层析分离得固体产物Chol-CH2CCH (化合物5)。取化合物4、化合物5和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得到固体产物Mannose-Chol(化合物6)，合成路线如反应式Ⅰ所示(甘露糖-PEG1000-Chol的合成路线如反应式Ⅱ所示)。分析附图1中各特征氢质子的化学位移δ(ppm)：5.30-5.42ppm(1个)分别为胆固醇中＝CH-氢的位移；7.92ppm(1个)为N-CH＝C中氢的位移，这些特征峰的存在，说明化合物4连接到化合物5片段上，通过1H-NMR核磁结果可知甘露糖-PEG100-Chol已经成功偶联。
甘露糖-PEG2000-DSPE：DSPE-PEG200-NH2可以直接购买，称取5mg甘露糖，加入5ml乙醇使溶解。称取5mg DSPE-PEG-NH2，溶于5ml氯仿-乙醇的混合溶剂(9:1，V/V)，在氮气保护条件下，滴加入0.5ml甘露糖的乙醇溶液，搅拌混匀后，加入100μl三乙胺，于40℃搅拌反应12h。反应结束后，减压旋蒸除去有机溶剂，加入去离子水使复溶，透析，除去未反应的甘露糖，冷冻干燥即得产物甘露糖-PEG2000-DSPE。
甘露糖-PEG2000-棕榈酸的合成：FmocNH-PEG-COOH溶于水，加入EDC·HCl和sulfo-NHS,室温搅拌10分钟，2-氨基乙基-α-D-吡喃甘露糖苷水溶解后加入上述反应体系中，室温反应48h，硅胶柱层析分离得产物(化合物1)。将化合物1溶于DCM，加入1-辛二醇，反应数小时，硅胶柱层析分离得产物(化合物2)。将化合物2与棕榈酸加入乙醚和DMF的混合溶剂中，室温反应数小时，硅胶柱层析分离得固体产物甘露糖-PEG2000-棕榈酸，其结构式为
Figure PCTCN2019088001-appb-000003
Figure PCTCN2019088001-appb-000004
Figure PCTCN2019088001-appb-000005
实施例2 脂质体的制备
处方组成
Figure PCTCN2019088001-appb-000006
Figure PCTCN2019088001-appb-000007
注：处方1～处方5的主药为GFP-mRNA。
制备方法1：按上述处方称取不同配比的磷脂(主药为PTX时，与磷脂一同加入)，加入圆底烧瓶中，加入氯仿/乙醇＝1:1(v/v)溶解后，减压旋 蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS7.4缓冲溶液水化，100W超声3min，得到脂质体。包裹pGFP时，可以将空白脂质体与pGFP在室温孵育得到。对得到的脂质体进行表征，结果见图2-4。
制备方法2(处方8)：按上述处方称取不同配比的磷脂，加入氯仿/乙醚使溶解，再加入白蛋白的水溶液，超声得到乳浊液，旋蒸除尽有机溶剂，再加入PBS7.4缓冲液重新水化脂质体。
实施例3 固脂纳米粒的制备
固体脂质纳米粒是以脂质为骨架材料制备而成的纳米结构载体，具有生理相容性、细胞亲和性、靶向性等特点。本实施例中，加入经甘露糖修饰的脂质，提高了固体脂质纳米粒的靶向性。
处方组成
Figure PCTCN2019088001-appb-000008
制备方法：精密称取处方量的药物、脂质材料溶于乙醇中，恒温水浴搅拌形成油相；称取处方量的表面活性剂溶于纯净水中，恒温水浴加热形成水相；在搅拌条件下，将油相注入水相中，恒温搅拌乳化浓缩，乳化液浓缩到一定体积后，将其倾入恒温4℃冷水中搅拌固化，过0.45μm滤膜即得。
实施例4 HDL纳米粒的制备
HDL是一类组成、密度、颗粒大小不均一的脂蛋白。HDL中含量最多的 载脂蛋白是ApoA-I，其疏水性的内核在体内运输胆固醇。重组高密度脂蛋白有内源性分离或体外合成的ApoA-I与磷脂、胆固醇等在体外重组形成，在生化特性和功能上与内源性HDL类似。高密度脂蛋白具有许多优良的特性：具有高度的安全性，高效的运载性(能够以三种方式载药：核心包埋，及亲脂性药物包埋与重组高密度脂蛋白的核心内；表面嵌入，及亲脂性药物插入到磷脂单层中；蛋白键和)，靶向性。本实施例中，制备的HDL纳米粒中加入了甘露糖配体，进一步提高了纳米粒的靶向性。
处方组成
Figure PCTCN2019088001-appb-000009
制备方法：与脂质体的制备方法类似，适用于制备脂质体的方法均可以用于HDL纳米粒的制备。
制备方法1：按上述处方称取不同配比的磷脂(脂溶性的主药DOX同时加入)，加入圆底烧瓶中，加入氯仿溶解后，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS7.4缓冲溶液水化，100W超声3min，得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中，4℃静置孵育24h，再透析即得。
制备方法2：按上述处方称取不同配比的磷脂，加入圆底烧瓶中，加入 氯仿溶解后，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PLGA/PTX的纳米粒混悬液，100W超声3min，得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中，4℃静置孵育24h，再透析即得。
实施例5 聚合物胶束的制备
处方组成
处方 甘露糖-PEG-DSPE DSPE DOPC DSPC DMPC 主药
处方26 20 60 20 0 0 槲皮素
处方27 20 60 0 20 0 DOX
处方28 20 60 35 0 20 PTX
处方29 20 60 10 35 0 DOX
处方30 50 0 50 0 0 DOX
制备方法：取载体材料和主药溶于氯仿(根据溶解情况可以选择其他溶剂，如乙醇，或乙醇与氯仿的混合溶剂)，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS7.4缓冲溶液水化，即得。
实施例6 靶向纳米粒的摄取实验
转染前12小时，将平皿中Raw264.7细胞吹散，培养基重悬，计数，在24孔板的每个孔中接入1mL细胞悬液，密度为10×104个/mL，于培养箱中孵育约12小时，备用。弃掉培养基，向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS)，于37℃孵育约12小时刺激巨噬细胞。弃掉培养基，向各孔中分别加入终浓度为25ng/mL的不同制剂(处方1和处方3)，于37℃避光孵育约2小时，考察巨噬细胞对不同制剂的摄取情况。到达孵育时间点后，弃去培养基，并以冷的PBS洗2次；PBS收集24孔板中细胞，再以PBS洗细胞一次(1200rpm、5min)，最后置于流式细胞仪中分析结果。结果见图5，其从左到右分别为：Control，处方3，处方1。可见，加入了靶向配体甘露糖-PEG1000-Chol后，能显著增加细胞的转染效率。
实施例7 材料
Figure PCTCN2019088001-appb-000010
在本实施例中脂质的材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)中的一 种或多种。
在本实施例中，所述间隔材料氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸中的一种或多种。肽为二肽、寡肽、多肽，肽的两端氨基酸选自以下的氨基酸组成：精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸；中间氨基酸选自20种任意氨基酸(比如RGD)。
实施例8 制备靶向载体
将实施例7的所述基础纳米制剂材料制备成纳米运药载体，再将实施例7的所述靶向材料附于制备成的所述纳米运药载体表面，即得靶向载体。
实施例9 靶向元件的制备
将实施例7的靶向材料与间隔材料进行合成，此处以甘露糖与PEGn的合成为例。
取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应24h，通过柱层析分离得产物TosOC 2H 4OC 2H 4OH(化合物1)。取化合物1与五乙酰化甘露糖及其衍生物和三氟化硼乙醚(BF 3·Et 2O)溶于DCM中，室温反应24h，柱层析分离得产物Aco-M-OC 2H 4OC 2H 4OTos(化合物2)，M为甘露糖及其衍生物。
实施例10 制备靶向载体
将实施例9的靶向元件与基础纳米制剂材料进行进一步合成，此处以甘露糖、PEGn和胆固醇的合成为例。
取实施例2制得的化合物与叠氮钠，溶于DMF中，60℃反应24h，硅胶柱层析分离得固体产物Aco-M-OC 2H 4OC 2H 4N 3(化合物3)。取化合物3溶于甲醇钠的甲醇溶液中，室温反应3h，浓缩得产物M-OC 2H 4OC 2H 4N 3(化合物4)。取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应24h，硅胶柱层析分离得固体产物Chol-CH 2CCH(化合物5)。取化合物4、化合物5和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得 到固体产物M-PEG n-Chol(化合物6)。
实施例11 提高靶向载体的靶向性
提高实施例8靶向载体的方法：A.将实施例7的靶向材料与间隔材料合成靶向元件；B.将实施例7的基础纳米制剂材料制备成纳米制剂，再将步骤A所得的靶向元件与实施例8的纳米运药载体连接。
实施例12 靶向载体的合成
甘露糖-PEGn-Chol：以甘露糖-PEG100-Chol合成为例，其他不同长度PEG也按此合成路线合成。取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应24h，硅胶柱层析分离得产物TosOC2H4OC2H4OH(式Ⅰ)。取式Ⅰ的化合物与五乙酰化甘露糖和三氟化硼乙醚(BF3·Et2O)，溶于DCM中，室温反应24h，硅胶柱层析分离得产物Aco-Mannose-OC2H4OC2H4OTos(式Ⅱ)。取式Ⅱ的化合物与叠氮钠，溶于DMF中，60℃反应24h，硅胶柱层析分离得固体产物Aco-Mannose-OC2H4OC2H4N3(式Ⅲ)。取式Ⅲ的化合物溶于甲醇钠的甲醇溶液中，室温反应3h，浓缩得产物Mannose-OC2H4OC2H4N3(式Ⅳ)。取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应24h，硅胶柱层析分离得固体产物Chol-CH2CCH(式Ⅴ)。取式Ⅳ的化合物、式Ⅴ的化合物和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得到固体产物Mannose-Chol(式Ⅵ)。分析附图1中各特征氢质子的化学位移δ(ppm)：5.30-5.42ppm(1个)分别为胆固醇中＝CH-氢的位移；7.92ppm(1个)为N-CH＝C中氢的位移，这些特征峰的存在，说明化合物式Ⅳ连接到化合物式Ⅴ片段上，通过1H-NMR核磁结果可知甘露糖-PEG100-Chol已经成功偶联，具体见图1，合成路线如下：
Figure PCTCN2019088001-appb-000011
甘露糖-PEG2000-DSPE：DSPE-PEG200-NH2可以直接购买，称取5mg甘露糖，加入5ml乙醇使溶解。称取5mg DSPE-PEG-NH2，溶于5ml氯仿-乙醇的混合溶剂(9:1，V/V)，在氮气保护条件下，滴加入0.5ml甘露糖的乙醇溶液，搅拌混匀后，加入100μl三乙胺，于40℃搅拌反应12h。反应结束后，减压旋蒸除去有机溶剂，加入去离子水使复溶，透析，除去未反应的甘露糖，冷冻干燥即得产物甘露糖-PEG2000-DSPE。
甘露糖-PEG2000-棕榈酸的合成：FmocNH-PEG-COOH溶于水，加入EDC·HCl和sulfo-NHS,室温搅拌10分钟，2-氨基乙基-α-D-吡喃甘露糖苷水溶解后加入上述反应体系中，室温反应48h，硅胶柱层析分离得产物。将化合物1溶于DCM，加入1-辛二醇，反应数小时，硅胶柱层析分离得产物。将化合物2与棕榈酸加入乙醚和DMF的混合溶剂中，室温反应数小时，硅胶柱层析分离得固体产物甘露糖-PEG2000-棕榈酸，其结构式为
Figure PCTCN2019088001-appb-000012
实施例13 制备靶向药物
将药物包裹于实施例8所制备的靶向载体中。
本实施例中，药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
所述小分子药物为抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。
所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等)，各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等)，干扰素(比如IFNα、IFNβ、IFNγ)，肿瘤坏死因子(比如TNFα、TNFβ)，整合素，单克隆抗体，酶，胰岛素等。
所述主药成分基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时，所述主药成分除用于免疫治疗的药物外，还可同时包裹免疫佐剂以增强免疫效果。
实施例14 制备靶向药物
Figure PCTCN2019088001-appb-000013
Figure PCTCN2019088001-appb-000014
注：处方1～处方5的主药为GFP-mRNA，(购自trilink公司，批号为：WOTL6647)。
制备方法1：按上述处方称取不同重量配比的磷脂(主药为PTX时，与磷脂一同加入)，加入圆底烧瓶中，加入氯仿/乙醇＝1:1(v/v)溶解后，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS7.4缓冲溶液水化，100W超声3min，得到脂质体。包裹pGFP时，可以将空白脂质体与pGFP在室温孵育得到，由甘露糖-PEG1000-Chol得到的纳米粒可记为MP1000-LPX。对得到的脂质体进行表征，结果显示脂质体的粒径大小为132.93±4.93nm、zeta电势为37.93±2.95mV，并观察到脂质体具有明显的脂膜结构，形状接近球形(图2-4)。
制备方法2(处方8)：按上述处方称取不同配比的磷脂，加入氯仿/乙醚使溶解，再加入白蛋白的水溶液，超声得到乳浊液，旋蒸除尽有机溶剂，再加入PBS7.4缓冲液重新水化脂质体。
实施例15 制备靶向药物
固体脂质纳米粒是以脂质为骨架材料制备而成的纳米结构载体，具有生理相容性、细胞亲和性、靶向性等特点。本实施例中，加入经甘露糖修 饰的脂质，提高了固体脂质纳米粒的靶向性。
Figure PCTCN2019088001-appb-000015
制备方法：精密称取处方量的药物、脂质材料溶于乙醇中，恒温水浴搅拌形成油相；称取处方量的表面活性剂溶于纯净水中，恒温水浴加热形成水相；在搅拌条件下，将油相注入水相中，恒温搅拌乳化浓缩，乳化液浓缩到一定体积后，将其倾入恒温4℃冷水中搅拌固化，过0.45μm滤膜即得。
实施例16 制备靶向药物
HDL是一类组成、密度、颗粒大小不均一的脂蛋白。HDL中含量最多的载脂蛋白是ApoA-I，其疏水性的内核在体内运输胆固醇。重组高密度脂蛋白有内源性分离或体外合成的ApoA-I与磷脂、胆固醇等在体外重组形成，在生化特性和功能上与内源性HDL类似。高密度脂蛋白具有许多优良的特性：具有高度的安全性，高效的运载性(能够以三种方式载药：核心包埋，及亲脂性药物包埋与重组高密度脂蛋白的核心内；表面嵌入，及亲脂性药物插入到磷脂单层中；蛋白键和)，靶向性。本实施例中，制备的HDL纳米粒中加入了甘露糖配体，进一步提高了纳米粒的靶向性。
Figure PCTCN2019088001-appb-000016
制备方法：与脂质体的制备方法类似，适用于制备脂质体的方法均可以用于HDL纳米粒的制备。
制备方法1：按上述处方称取不同配比的磷脂(脂溶性的主药DOX同时加入)，加入圆底烧瓶中，加入氯仿溶解后，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS7.4缓冲溶液水化，100W超声3min，得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中，4℃静置孵育24h，再透析即得。
制备方法2：按上述处方称取不同配比的磷脂，加入圆底烧瓶中，加入氯仿溶解后，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PLGA/PTX的纳米粒混悬液，100W超声3min，得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中，4℃静置孵育24h，再透析即得。
实施例17 制备靶向药物
Figure PCTCN2019088001-appb-000017
Figure PCTCN2019088001-appb-000018
制备方法：取载体材料和主药溶于氯仿(根据溶解情况可以选择其他溶剂，如乙醇，或乙醇与氯仿的混合溶剂)，减压旋蒸去除有机溶剂，在瓶内壁形成一层薄膜，加入PBS 7.4缓冲溶液水化，即得。
实施例18 靶向纳米粒的摄取实验
转染前12小时，将平皿中Raw264.7细胞吹散，培养基重悬，计数，在24孔板的每个孔中接入1mL细胞悬液，密度为10×10 4个/mL，于培养箱中孵育约12小时，备用。弃掉培养基，向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS)，于37℃孵育约12小时刺激巨噬细胞。弃掉培养基，向各孔中分别加入终浓度为25ng/mL的不同制剂(所有处方均已展开了实验，这里以处方1和处方3为例)，于37℃避光孵育约2小时，考察巨噬细胞对不同制剂的摄取情况。到达孵育时间点后，弃去培养基，并以冷的PBS洗2次；PBS收集24孔板中细胞，再以PBS洗细胞一次(1200rpm、5min)，最后置于流式细胞仪中分析结果。结果见图5，其从左到右分别为：Control，处方3，处方1。可见，加入了靶向配体甘露糖-PEG1000-Chol后，能显著增加细胞的转染效率。
实施例19 纳米靶向制剂的转染效率
将DC2.4细胞与制备好的纳米靶向制剂在24孔板中同共孵育，其中纳米靶向制剂的N/P比为5。采用流式细胞术检测GFP阳性的DC2.4细胞的转染效率与平均荧光强度(MFI)。简而言之，DC2.4细胞是通过正向散射(FSC)和侧散射(SSC)捕获的。存活的DC2.4细胞显示在区域1(R1)，GFP阳性细胞显示在区域2(R2)。转染效率自动显示在R2。获得GFP阳性细胞中GFP表达的MFI使用FlowJo软件。计算MFI需要扣除未处理DC2.4细胞的背 景值。进一步详细了解主药mRNA的体外转染动力学，所述甘露糖-PEG1000-Chol在DC2.4细胞上的转染效率在12～72h的表达也得到证实。
如图6-7结果显示，甘露糖-PEG1000-Chol组诱导的GFP阳性细胞最多，比例高达52％，明显高于任何其他组别，通过计算得出甘露糖-PEG1000-Chol的转染效率为52.09±4.85％，远远超过商业使用的转染试剂Lipo 3K(11.47±2.31％)。
实施例20 稳定性测试
对甘露糖-PEG1000-Chol修饰的纳米靶向制剂进行了稳定性研究，其稳定性由粒径大小、zeta电势和转染效率共同决定。结果显示，甘露糖-PEG1000-Chol修饰的纳米靶向制剂储存与4℃，甘露糖-PEG1000-Chol修饰的纳米靶向制剂的颗粒尺寸略有减小，但zeta电位没有减小(图8)，在4℃条件下保存3天，转染效率约为50％(图9)，详见下表。同样在血清中，甘露糖-PEG1000-Chol孵育的mRNA未解离(图10)，同样，甘露糖-PEG100-Chol和甘露糖-PEG2000-Chol修饰的纳米靶向制剂同样具有良好的稳定性。
甘露糖-PEG1000-Chol修饰的纳米靶向制剂转染稳定性测试结果：
时间 转染效率
DAY 0 52.4±3.6
DAY 1 61.7±3.8
DAY 3 53.5±3.2
实施例21 细胞毒性实验
采用指定配方孵育24h以后，采用流式细胞术进行细胞毒性分析。其结果如图11-12所示，对照组、甘露糖-PEG1000-Chol组和Lipo 3K组的活细胞率分别为86.7±3.6％、86.7±1.7％和90.1±1.2％，早期和晚期凋亡无明显差异，总体来说表现良好，无明显细胞毒性，详见下表：
Figure PCTCN2019088001-appb-000019
Figure PCTCN2019088001-appb-000020
实施例22 DC2.4摄取靶向纳米粒研究
转染前12小时，将平皿中DC2.4细胞细胞吹散，培养基重悬，计数，在24孔板的每个孔中接入1mL细胞悬液，密度为10×10 4个/mL，于培养箱中孵育约12小时，备用。弃掉培养基，向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS)，分别于37℃和4℃孵育约12小时刺激DC2.4细胞。弃掉培养基，向各孔中分别加入终浓度为25ng/mL的不同制剂(LPX和MP 1000-LPX)，分别于37℃和4℃避光孵育约2小时，考察DC2.4细胞在不同温度、不同制剂下的摄取情况。到达孵育时间点后，弃去培养基，并以冷的PBS洗2次；PBS收集24孔板中细胞，再以PBS洗细胞一次(1200rpm、5min)，最后置于流式细胞仪中分析结果。
结果如图13所示，不论是mRNA普通脂质体复合物还是mRNA靶向脂质体复合物，当摄取温度由37℃降低到4℃时，摄取均降低，提示DC2.4细胞对mRNA脂质体复合物的摄取存在能量依赖性，即随着温度降低，摄取降低。对比37℃的数据可以看出，DC2.4细胞可以更多的摄取mRNA靶向脂质体复合物，对比是否加入甘露糖的摄取数据可以看出，加入甘露糖后细胞对mRNA普通脂质体复合物的摄取无明细变化，而加入甘露糖后细胞对mRNA靶向脂质体复合物的摄取显著降低，说明DC2.4细胞更多的摄取mRNA靶向脂质体复合物主要时因为甘露糖受体介导的，结果详见下表：
Figure PCTCN2019088001-appb-000021
实施例22 BMDCs细胞摄取靶向纳米粒研究
转染前12小时，将平皿中BMDCs细胞吹散，培养基重悬，计数，在 24孔板的每个孔中接入1mL细胞悬液，密度为10×10 4个/mL，于培养箱中孵育约12小时，备用。弃掉培养基，向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS)，分别于37℃和4℃孵育约12小时刺激BMDCs细胞。弃掉培养基，37℃培养的细胞分为对照组、细胞松弛素D组、菲律平组、渥曼青霉素组和氯丙嗪组，并向各组中分别加入终浓度为25ng/mL的MP 1000-LPX；在4℃孵育的细胞中加入MP 1000-LPX，以上分组分别于37℃和4℃避光孵育约2小时，考察BMDCs细胞在不同温度、不同药物作用下的摄取情况。到达孵育时间点后，弃去培养基，并以冷的PBS洗2次；PBS收集24孔板中细胞，再以PBS洗细胞一次(1200rpm、5min)，最后置于流式细胞仪中分析结果。
如图14-15所示，当摄取温度由37℃降低到4℃时，摄取降低，提示BMDCs对mRNA靶向脂质体复合物的摄取存在能量依赖性，即随着温度降低，摄取降低，此数据与DC2.4结果吻合。对比是否加入内吞抑制剂的摄取数据可以看出，加入细胞松弛素D和菲律平后细胞对mRNA靶向脂质体复合物的摄取无明显变化，而加入渥曼青霉素和氯丙嗪后细胞对mRNA靶向脂质体复合物的摄取均显著降低，说明BMDCs摄取mRNA靶向脂质体复合物除了具有能量依赖性还受胞饮(渥曼青霉素)和网格蛋白(氯丙嗪)介导内吞的影响，相关结果详见下表：
试验条件 摄取结果(×104)
Control 2.52±0.16
Cytochalasin-D 2.73±0.15
Filipin 2.61±0.15
4° 1.29±0.25
Wortmannin 1.47±0.17
Chloropromazine 0.42±0.10
实施例23 MP400-LPX体内抗肿瘤结果
按照实施例7和实施例9的方法合成MP400-LPX和甲氧基聚乙二醇400胆固醇(mP400-LPX)，包含主要成分E6/E7-mRNA，形成甘露糖靶头的E6/E7-mRNA脂质体复合物。在第0天、第3天、第10天、第17天给小鼠注射30ug的复合物，结果如图16-20所示，与对照组和不相关mRNA组相比，甘露糖靶头的E6/E7-mRNA脂质体复合物治疗组小鼠的肿瘤生长数据显著降低。值得关注的是：与甲氧基聚乙二醇400胆固醇的对照材料相比，甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗小鼠生存期延长，且有一只小鼠的肿瘤完全消退，靶向制剂制备的疫苗体现出较好的免疫治疗效果。
实施例24 DC2.4细胞对MP400-LPX的摄取
转染前12小时，将平皿中DC2.4和BMDCs细胞分别吹散，培养基重悬，计数，在24孔板的每个孔中接入1mL细胞悬液，密度为10×10 4个/mL，于培养箱中孵育约12小时，备用。弃掉培养基，向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS)，于37℃孵育约12小时刺激DC2.4和BMDCs细胞。弃掉培养基，向各孔分为六组，向中DC2.4细胞分别加入终浓度为25ng/mL的不同载了Cy5-H-mRNA的制剂(对照、LPX、MP 100-LPX、MP 400-LPX、MP 1000-LPX和MP 2000-LPX)，于37℃避光孵育约2小时，考察DC2.4细胞不同制剂下的摄取情况；同时向DC2.4和BMDCs细胞中分别加入终浓度为25ng/mL的不同载了GFP-mRNA的制剂(LPX、MP 100-LPX、MP 400-LPX、MP 1000-LPX、MP 2000-LPX和Lipo 3K)，于37℃避光孵育约2小时，考察DC2.4和BMDCs细胞不同制剂下的转染效果。到达孵育时间点后，弃去培养基，并以冷的PBS洗2次；PBS收集24孔板中细胞，再以PBS洗细胞一次(1200rpm、5min)，最后置于流式细胞仪中分析结果。
结果如图21-22所示，DC2.4细胞对MP400-LPX的摄取最好。DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取效率均为100％左右，但是DC2.4细胞对各制剂的摄取量不同，具体的表现在细胞内荧光 强度不同，MP100-LPX与LPX相比在细胞内的蓄积显著增加，MP400-LPX与MP100-LPX相比在细胞内的蓄积显著增加，MP1000-LPX与MP400-LPX相比在细胞内的蓄积显著降低，MP1000-LPX与MP2000-LPX在细胞内的蓄积程度类似。MP400-LPX与LPX，MP100-LPX，MP1000-LPX和MP2000-LPX等其他制剂相比在细胞内的蓄积均显著增加。上述结果提示：DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取受配体链长的影响，存在一个合适的链长或者链长范围使得DC2.4细胞对其摄取更多。
如图23-26所示，DC2.4细胞对MP400-LPX的转染最好。DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的转染效率不同，具体的表现在GFP阳性细胞的百分比不同，MP400-LPX与LPX、MP100-LPX、MP1000-LPX和MP2000-LPX相比在GFP阳性的细胞显著增加，MP2000-LPX与其他制剂相比，其GFP阳性的细胞显著降低。BMDCs的转染结果与DC2.4基本类似，但是与DC2.4不同的是，MP2000-LPX与其他制剂一样的转染效率显著高于Lipo 3K，BMDCs为原代细胞，这一转染结果说明靶向制剂具有较好的应用前景。
实施例25 载mRNA的不同配体链长靶向脂质体复合物的体内抗肿瘤试验
按照实施例7和实施例9的方法合成MP100-LPX、MP400-LPX、MP1000-LPX和MP2000-LPX，包含主要成分E6/E7-mRNA，形成甘露糖靶头的E6/E7-mRNA脂质体复合物。在第0天、第3天、第10天、第17天给小鼠注射30ug的复合物，结果如图27-31所示，MP400-LPX的效果最好。与其他链长的靶向配比相比，聚乙二醇400的甘露糖胆固醇靶向配体表现出最好的体内抗肿瘤效果，具体表现为：小鼠的肿瘤治愈率最高，高达60％左右，未治愈的小鼠肿瘤的生长速度较低，同时能够延长荷瘤小鼠的生存期。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (66)

  1. 一种甘露糖及其衍生物修饰的靶向纳米制剂，其特征在于，其由靶向配体甘露糖及其衍生物、纳米制剂和主药成分组成，可用于疫苗递送、肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗。
  2. 根据权利要求1所述的靶向纳米制剂，其特征在于，甘露糖衍生物为甘露糖苷、甘露糖胺、甘露聚糖等中的一种或几种。
  3. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述纳米制剂为脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束等中的一种或几种。
  4. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述主药成分为小分子药物、蛋白多肽类药物或基因药物等中的一种或几种。
  5. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述靶向配体甘露糖及其衍生物的含量为0.05％-40％。
  6. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述靶向配体甘露糖及其衍生物的含量为1％-10％。
  7. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述靶向配体甘露糖修的含量为2％-5％。
  8. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述靶向配体甘露糖及其衍生物的修饰方式为：制备纳米制剂之后再将甘露糖及其衍生物吸附或偶联到纳米制剂表面；或先将甘露糖及其衍生物与制备纳米制剂的载体材料偶联之后再制备纳米制剂。
  9. 根据权利要求8所述的靶向纳米制剂，其特征在于，所述甘露糖及其衍生物吸附在纳米制剂表面的方式为：采用溶剂挥发法、高压均质法或微乳法。
  10. 根据权利要求1所述的靶向纳米制剂，其特征在于，所述修饰纳米制剂的甘露糖及其衍生物为单个甘露糖及其衍生物或2～10个甘露糖及其衍生物。
  11. 根据权利要求8所述的靶向纳米制剂，其特征在于，所述甘露糖及其 衍生物与纳米制剂或制备纳米制剂的载体材料偶联方式为：直接偶联或通过一个间隔基偶联。
  12. 根据权利要求11所述的靶向纳米制剂，其特征在于，所述间隔基为戊二醛、乙二胺、丙二酸、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰等中的一种或几种。
  13. 根据权利要求1-12所述的靶向纳米制剂，其特征在于，制备纳米制剂的材料为具有活性氨基或羟基中的至少一种，药学上可以接受的制备胶束、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、脂质体、聚合物胶束等的载体材料，采用药学上可以接受的纳米制剂制备方法制得。
  14. 用于制备带有靶向功能的纳米制剂的组合物，其特征在于，所述组合物包括靶向材料和基础纳米制剂材料，所述靶向材料为甘露糖和/或甘露糖衍生物。
  15. 根据权利要求14所述的组合物，其特征在于，所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
  16. 根据权利要求15所述的组合物，其特征在于，所述甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖。
  17. 根据权利要求14或15或16所述的组合物，其特征在于，所述靶向材料占所述组合物的重量百分比为0.05-40％。
  18. 根据权利要求14或15或16所述的组合物，其特征在于，所述靶向材料占所述组合物的重量百分比为1-10％。
  19. 根据权利要求14所述的组合物，其特征在于，所述组合物由所述靶向材料、所述基础纳米制剂材料、使所述靶向材料与所述基础纳米制剂材料产生距离的间隔材料组成，所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
  20. 根据权利要求14所述的组合物，其特征在于，所述基础纳米制剂材料 为制备脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束的材料的组合物。
  21. 根据权利要求19所述的组合物，其特征在于，所述间隔材料为PEGn,其中n＝100-5000。
  22. 根据权利要求21所述的组合物，其特征在于，所述n为100、200、300、400、500、600、800、900、1000或2000。
  23. 根据权利要求19所述的组合物，其特征在于，按照重量份计由以下组分组成：甘露糖1份；PEG 0.5-56份，胆固醇2-11份。
  24. 根据权利要求19所述的组合物，其特征在于，按照重量份计由以下组分组成：甘露糖1份；PEG 10-25份，胆固醇4-8份。
  25. 权利要求14所述的组合物制备的运载药物的靶向载体。
  26. 根据权利要求25所述的靶向载体，其特征在于，所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
  27. 根据权利要求26所述的靶向载体，其特征在于，所述小分子药物为紫杉烷类、喜树碱类、长春碱类、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀和槲皮素中的任一中或多种。
  28. 根据权利要求26所述的靶向载体，其特征在于，所述蛋白多肽类药物为白细胞介素、生长因子、干扰素、整合素，单克隆抗体，酶和胰岛素。
  29. 根据权利要求28所述的靶向载体，其特征在于，所述白细胞介素包括但不限于IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6，所述生长因子包括但不限于成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子，所述干扰素包括但不限于IFNα、IFNβ、IFNγ，所述肿瘤坏死因子包括但不限于TNFα、TNFβ。
  30. 根据权利要求26所述的靶向载体，其特征在于，所述基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA中的一种或几种。
  31. 被权利要求25所述的靶向载体包裹的药物。
  32. 权利要求31所述的靶向载体的制备方法，其特征在于，将所述基础纳米制剂材料制备成纳米运药载体，再将所述靶向材料附于所述纳米运药载体表面。
  33. 提高权利要求25所述的靶向载体的靶向性的方法，其特征在于：
    A 合成靶向元件
    将所述靶向材料与所述间隔材料合成靶向元件；
    B 制备靶向载体
    将基础纳米制剂材料制备成纳米运药载体，再将步骤A所得的靶向元件与所述纳米运药载体连接得靶向载体；或先将所述间隔材料与基础纳米制剂材料合成，再与所述靶向材料进行进一步连接得靶向载体。
  34. 根据权利要求33所述的方法，其特征在于，所述靶向材料具体为甘露糖及其衍生物，所述间隔材料具体为PEG。
  35. 根据权利要求33所述的方法，其特征在于，所述靶向元件与所述纳米运药载体的距离为0.2-100nm。
  36. 根据权利要求35所述的方法，其特征在于，所述靶向元件与所述纳米运药载体的距离为0.3-10nm。
  37. 运送药效成分到达靶细胞的方法，其特征在于，将药效成分置于权利要求25所述的靶向载体内，并运送。
  38. 根据权利要求37所述的方法，其特征在于，所述靶细胞为含有甘露糖受体的细胞。
  39. 根据权利要求37所述的方法，其特征在于，所述靶细胞包括但不限于巨噬细胞、树突细胞和肿瘤细胞。
  40. 制备靶向元件的组合物，其特征在于，所述组合物由靶向材料和间隔材料组成，所述靶向材料为甘露糖和/或甘露糖衍生物。
  41. 根据权利要求40所述的组合物，其特征在于，所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
  42. 根据权利要求40所述的组合物，其特征在于，所述甘露糖衍生物为甲 基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖中的任一中或多种。
  43. 根据权利要求40所述的组合物，其特征在于，所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
  44. 根据权利要求40所述的组合物，其特征在于，所述间隔材料与所述靶向材料的摩尔比为1-10:1。
  45. 根据权利要求44所述的组合物，其特征在于，所述间隔材料与所述靶向材料的摩尔比为5:1。
  46. 根据权利要求40所述的组合物，其特征在于，甘露糖与PEG的摩尔比为1:5。
  47. 根据权利要求46所述的组合物，其特征在于,甘露糖与PEG的摩尔比为1:1。
  48. 运用权利要求40所述的组合物制备的靶向元件。
  49. 根据权利要求48所述的靶向元件，其特征在于，所述靶向元件如式Ⅰ所示：其中R1为-C 5H 8O 2、-C 2H 7N 2、-C 3H 3O 4、-O(CH 2CH 2O)nH、-C 2H 3O 2NR＇或-C 2H 3ONR＇、-C 4H 5O 3N 2R 1＇R 2＇或-C 4H 5O 2N 2R 1＇R 2＇、-C 3n+1H 3n+2On+2Nn+1R 1＇R 2＇或-C 3n+1H 3n+2On+1Nn+1R 1＇R 2＇、-C 18H 35O、C 16H 31O、-H、-CHO、-C 6H 4NH 2、-C 6H 4NO 2、-CONH 2、-CH 2C 6CCHCO 2CH 3，所述R 1＇和R 2＇均为任意天然氨基酸侧链R＇基团，R 2为-CH 3或-PO 3H 2、R 3为-H，R 4为-H，R 5是-H或-NHCOCH 3，
    Figure PCTCN2019088001-appb-100001
  50. 根据权利要求49所述的靶向元件，其特征在于，R1为聚乙二醇，其结构为
    Figure PCTCN2019088001-appb-100002
    其中n＝1～5000，其对应的分子量为：40*n。
  51. 含有权利要求48所述的靶向元件的靶向载体。
  52. 根据权利要求51所述的靶向载体，其特征在于，由靶向元件和纳米制剂连接而成，所述纳米制剂为脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束。
  53. 根据权利要求51所述的靶向载体，其特征在于，单个靶向载体上连接有1～100000个所述的靶向元件。
  54. 根据权利要求53所述的靶向载体，其特征在于，所述单个靶向载体上连接有20-1000个所述的靶向元件。
  55. 根据权利要求52所述的靶向载体，其特征在于，所述靶向元件的靶头与所述纳米制剂的距离为0.2-100nm。
  56. 根据权利要求55所述的靶向载体，其特征在于，所述靶向元件的靶头与所述纳米制剂的距离为0.3-10nm。
  57. 根据权利要求55或56所述的靶向载体，其特征在于，所述靶头为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
  58. 将权利要求48所述的靶向元件连接至纳米制剂的方法，其特征在于，将所述靶向材料和间隔材料连接后再与所述纳米制剂的材料连接制成纳米制剂.
  59. 将权利要求48所述的靶向元件连接至纳米制剂的方法，其特征在于，具体包括以下步骤：
    1)取二甘醇、对甲苯磺酰氯与三乙胺，溶于DCM中，室温反应，通过柱层析分离得化合物1；
    2)取化合物1与五乙酰化甘露糖及其衍生物和三氟化硼乙醚溶于DCM中，室温反应，柱层析分离得化合物2；
    3)取化合物2与叠氮钠，溶于DMF中，硅胶柱层析分离得固体化合物3；
    4)取化合物3溶于甲醇钠的甲醇溶液中，室温反应，浓缩得化合物4；
    5)取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中，室温反应，硅胶柱层析分离得固体化合物5；
    6)取化合物4、化合物5和碘化亚铜溶于DMF中，室温反应24h，硅胶柱层析得到靶向载体固体化合物6。
  60. 提高权利要求51所述靶向载体运送药物能力的方法，其特征在于，将药物包裹于所述靶向载体中运送。
  61. 根据权利要求60所述的方法，所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
  62. 根据权利要求60所述的方法，所述靶向载体可将药物运送到含有甘露糖受体靶点的靶细胞中。
  63. 一种包含了权利要求51所述靶向载体的复合物，其特征在于，所述复合物为所述靶向载体与受体形成的复合物，所述靶向载体运载了药物，所述受体为甘露糖受体。
  64. 提高转染试剂转染效率的方法，其特征在于，将权利要求40所述的靶向元件的组合物与用于制备转染试剂的载体连接制备高转染效率的转染试剂。
  65. 权利要求48所述靶向元件作为靶向油相的运用。
  66. 权利要求48所述靶向元件作为靶向水相的运用。
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