WO2017101653A1 - 靶向疏水性抗肿瘤药物纳米制剂及其制备方法 - Google Patents

靶向疏水性抗肿瘤药物纳米制剂及其制备方法 Download PDF

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WO2017101653A1
WO2017101653A1 PCT/CN2016/107127 CN2016107127W WO2017101653A1 WO 2017101653 A1 WO2017101653 A1 WO 2017101653A1 CN 2016107127 W CN2016107127 W CN 2016107127W WO 2017101653 A1 WO2017101653 A1 WO 2017101653A1
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hyaluronic acid
solution
albumin
preparation
drug
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PCT/CN2016/107127
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English (en)
French (fr)
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刘锋
赖树挺
曹付春
郑阳
连远发
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广州帝奇医药技术有限公司
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Priority to US16/060,103 priority Critical patent/US11090268B2/en
Priority to KR1020187016906A priority patent/KR102092407B1/ko
Priority to JP2018549383A priority patent/JP6580799B2/ja
Priority to EP16874710.3A priority patent/EP3372226B1/en
Publication of WO2017101653A1 publication Critical patent/WO2017101653A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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Definitions

  • the invention belongs to the field of medicine, and in particular relates to a nanometer preparation for targeting hydrophobic antitumor drugs and a preparation method thereof.
  • the nano drug delivery system can passively target tumors due to the EPR effect, and it has received more and more attention in the application of tumor targeted therapy.
  • Ordinary nano-dosage forms, such as liposomes and nanoparticles, are recognized in the plasma by the human reticuloendothelial system, which accelerates the plasma clearance of nano-formulations and reduces their therapeutic effects. Therefore, nano-formulations whose surface has been modified have attracted extensive research and attention.
  • Albumin is a type of protein that is abundantly present in plasma. Albumins commonly found in pharmaceutical carrier research include human serum albumin, bovine serum albumin, and ovalbumin. There are many drug binding sites in the albumin molecule, which can effectively load different kinds of drugs. At the same time, it has the characteristics of high water solubility, good stability, and biodegradability. It has high biocompatibility and has been used in US food and drug. Approved by the FDA for use in humans, albumin is an ideal carrier for drug delivery. At the same time, the amino group, the thiol group and the like present on the albumin can further modify the active targeting group to improve the targeting. The current market for injection of paclitaxel (albumin) injection has attracted widespread attention in the market.
  • Patent CN201210142991 by preparing a conjugate of hyaluronic acid and human serum albumin as a carrier, and then preparing the plant with a plant anticancer drug and a nanoparticle stabilizer under high-speed homogenization, high-pressure homogenization or sonication conditions.
  • Anti-cancer targeted nano preparations.
  • the nano-preparation has the biological targeting effect of a receptor on the surface of a tumor cell such as CD44. After intravenous injection into the body, the EPR effect of the tumor and the CD44 of the HA are highly combined, so that the drug accumulates in the tumor site, and the nano drug-loading system is extended. Cycle time in the body.
  • nanoparticle drugs In biomedical applications, the particle size of nanoparticle drugs is important, and the different particle size metabolic pathways are different. Small particle sizes are metabolized by the kidneys, and large particle sizes are passed through the liver. Among them, 20-200nm particles have passive targeting effect on tumors, and nanoparticle drugs in this particle size range can eliminate some biomedical effects caused by the size of the drug itself, thereby improving the therapeutic effect.
  • Injection is a kind of sterile preparation.
  • the sterility of the injection is achieved through a well-controlled and proven sterilization/sterilization production process and strict implementation of Good Manufacturing Practice for pharmaceutical products (GMP). of.
  • GMP Good Manufacturing Practice for pharmaceutical products
  • Formulations containing large amounts of protein, such as albumin cannot be sterilized by conventional methods such as high temperature because high temperatures can denature proteins.
  • Filtration sterilization is a final sterilization method approved by the regulatory authorities for products that are unstable and cannot be used, including heat sterilization. Therefore, filtration through a 0.22 ⁇ m filter is the preferred solution for preparing injectable nano-formulations. .
  • the nano-prepared particles of CN201210142991 have a large particle size distribution coefficient and a wide particle size distribution, and after lyophilization and reconstitution, the particles are entangled between the hyaluronic acid molecular chains of the modified human albumin, resulting in aggregation of particles. Attachment, causing particles after reconstitution of the lyophilized product As the particle size increases, it is easy to cause clogging of the filter membrane pores when the micropore filter is filtered (0.22 ⁇ m), so that the concentration of the particles in the filtrate is lowered, thereby causing a decrease in the drug content in the filtrate, thereby affecting the efficacy and reducing the product collection. rate.
  • one of the objects of the present invention is to provide a nanometer preparation for targeting hydrophobic antitumor drugs, wherein the nanometer preparation has a uniform particle size distribution, good dispersibility, stable non-agglomeration, and substantially unchanged particle size after lyophilization and reconstitution.
  • the concentration of the drug in the filtrate after filtration by the micropore filter (0.22 ⁇ m) was substantially unchanged.
  • a nanometer formulation for targeting hydrophobic antitumor drugs comprising a hydrophobic antitumor drug and a carrier having a mass ratio of 1:4-32.5, the carrier comprising 37.5-95.3 wt% albumin and 4.7-62.5 wt% transparent
  • the targeted hydrophobic anti-tumor drug nanoformulation comprises a hydrophobic anti-tumor drug and a carrier having a mass ratio of 1:4.8-19, the carrier comprising 50.0-95.0% by weight of albumin and 5.0 - 50.0 wt% of a hyaluronic acid-albumin conjugate prepared from albumin and hyaluronic acid in a molar ratio of 1:2-20.
  • the hyaluronic acid has a molecular weight of from 2 to 60 kDa.
  • the albumin is human serum albumin, bovine serum albumin, ovalbumin, and recombinant human albumin.
  • the hydrophobic anti-tumor drug is selected from the group consisting of paclitaxel, docetaxel, azithatin, doxorubicin, camptothecin, cyclosporin, rapamycin, vancomycin, and stopper Substitutes or their derivatives.
  • the targeted hydrophobic anti-tumor drug nanoformulation further comprises a nanoparticle stabilizer selected from the group consisting of polyoxyethylene-polyoxypropylene-polyoxyethylene oxime polymers, d - alpha tocopheryl succinic acid polyethylene glycol ester, povidone, the nanoparticle stabilizer is 1.0-10.0% of the mass of the hydrophobic antitumor drug.
  • Another object of the present invention is to provide a method for preparing the above-described hydrophobic antitumor drug nano preparation.
  • a method for preparing the above-mentioned nanometer formulation for targeting hydrophobic antitumor drugs comprising the following steps:
  • Hyaluronic acid and albumin are added to an aqueous medium to dissolve, the pH of the solution is adjusted to 5.0-6.0, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N- The sodium hydroxysulfosuccinimide salt is added to the above solution for 15 to 60 minutes, the pH of the solution is adjusted to 7.0-7.5, and the reaction is further stirred at room temperature for 3-24 hours. After the reaction is completed, the unbonded hyaluronic acid is removed by dialysis.
  • the hyaluronic acid is dissolved in an aqueous medium, the pH of the solution is adjusted to 5.0-6.0, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added. Hydrochloride and N-hydroxysulfosuccinimide sodium salt, stirred at room temperature for 15-60 min to obtain hyaluronic acid succinimide active lipid; then hyaluronic acid succinimide active lipid is added dropwise
  • the aqueous medium solution of albumin or the aqueous medium solution of albumin is added dropwise to the hyaluronic acid succinimide active lipid, the pH of the solution is adjusted to 7.0-7.5, and the reaction is stirred at room temperature for 3-24 hours, after the reaction is completed.
  • the albumin and the hyaluronic acid-albumin conjugate prepared in the step (1) are dissolved in an aqueous medium to obtain a carrier solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then the double emulsion is obtained under high pressure homogenization, then the organic solvent is removed, filtered, and freeze-dried to obtain Targeting hydrophobic antitumor drug nanoformulations.
  • the concentration of the carrier in the carrier solution described in the step (2) is 4.0-50.0 mg/mL
  • the concentration of the hydrophobic antitumor drug in the drug solution described in the step (3) is 16.0-345.0 mg. /mL.
  • the aqueous medium described in the step (1) is 2-(N-morpholine) ethanesulfonic acid buffer, phosphate buffer or sterile water
  • the aqueous medium described in the step (2) is a sterile water, a phosphate buffer solution, a physiological saline solution, a 5.0 wt% aqueous glucose solution or a 5.0 wt% aqueous mannitol solution
  • the organic solvent described in the step (3) is dichloromethane, chloroform, a mixture of dichloromethane and ethanol, or a mixture of chloroform and ethanol.
  • the pressure of the high pressure homogenization described in the step (4) is 10,000-40000 psi
  • the flow rate of the high pressure homogeneous material is 10.0-25.0 L/h
  • the number of cycles of high pressure homogenization is 7-20 times
  • the method of removing the organic solvent is to remove the organic solvent by rotary evaporation under reduced pressure at a temperature of 15.0 to 45.0 °C.
  • the inventors of the present invention have found in the long-term experience accumulation and experiment that the addition of unmodified albumin to the hyaluronic acid-albumin conjugate can reduce the targeted hydrophobic antitumor drug nanometer of the present invention.
  • the particle size of the preparation particles makes the particle size distribution more uniform, and the mutual aggregation and adhesion between the nanoparticles can be avoided or reduced. Therefore, the nano-targeting agent for targeting hydrophobic anti-tumor drugs of the present invention has the following advantages:
  • the nanometer preparation of the invention has a uniform particle size distribution and is mostly less than 220 nm, and the particle size does not increase after reconstitution, and has a high yield after filtration through a micropore filter (0.22 ⁇ m), which greatly reduces the cause.
  • the particle size is too large and the loss caused by the interception can improve the drug effect and enhance the inhibition of the tumor.
  • the particle size of the nano-preparation of the present invention is mostly less than 220 nm, and the particle size does not increase after reconstitution of the particles, which is beneficial to the long-term circulation of the nanoparticles in the circulatory system of the body, and is not phagocytized by mononuclear macrophages. And intercepted by the reticular system in the liver, with Active targeting helps to ensure sufficient blood concentration in the targeted site, improve drug efficacy, and reduce toxicity to non-tumor sites.
  • the nano preparation of the invention has good stability in solution after reconstitution, is stable in the body environment, and the carrier and the drug can maintain a stable complex during operation.
  • Example 1 is a particle size distribution diagram of the targeted nano-preparation particles of Example 3 before filtration
  • FIG. 3 is a schematic diagram showing the comparison of the inhibitory effects of the targeted nano preparations of Examples 1-2, Examples 5-6 and Comparative Examples 1 - 2 on tumors;
  • Figure 4 is a graphical representation of the biotargeting comparison of the targeted nanoformulations of Example 1, Example 6, and Comparative Examples I-II.
  • PEO-PPO-PEO segment polymer polyoxyethylene-polyoxypropylene-polyoxyethylene oxime segment polymer
  • TPGS d-alpha tocopherol succinic acid polyethylene glycol
  • PVP povidone
  • paclitaxel 0.1200 g was dissolved in 2.7 mL of a mixed solvent of chloroform and ethanol (9:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then The high pressure homogenization (pressure: 20000 psi, material flow rate: 10.0 L/h) was cycled 7 times to obtain double emulsion, then the organic solvent was removed by rotary evaporation under reduced pressure at 30.0-45.0 ° C, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain paclitaxel. Targeting anti-tumor nano preparations.
  • paclitaxel 0.1200 g was dissolved in 7.1 mL of a mixed solvent of chloroform and ethanol (9:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 14000 psi, material flow rate: 12.0 L/h).
  • high pressure pressure: 14000 psi, material flow rate: 12.0 L/h.
  • the double emulsion was obtained by circulating 10 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a paclitaxel-targeted antitumor nano preparation.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 17,000 psi, material flow rate: 15.0 L/h). Rehydration is obtained 12 times in a cycle, then at 15.0-25.0 ° C, -0.1 MPa
  • the organic solvent was removed by rotary evaporation under reduced pressure, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a paclitaxel-targeted antitumor nano preparation.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 20000 psi, material flow rate: 15.0 L/h).
  • high pressure pressure: 20000 psi, material flow rate: 15.0 L/h.
  • the double emulsion was obtained by circulating 14 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a paclitaxel-targeted antitumor nano preparation.
  • paclitaxel 0.6000 g was dissolved in 7.3 mL of a mixed solvent of chloroform and ethanol (9:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 25000 psi, material flow rate: 15.0 L/h).
  • high pressure pressure: 25000 psi, material flow rate: 15.0 L/h.
  • the double emulsion was obtained by circulating 10 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a paclitaxel-targeted antitumor nano preparation.
  • paclitaxel 0.8000 g was dissolved in 5.0 mL of a mixed solvent of chloroform and ethanol (9:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 30,000 psi, material flow rate: 18.0 L/h).
  • high pressure pressure: 30,000 psi, material flow rate: 18.0 L/h.
  • the double emulsion was obtained by circulating 12 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain an azithene-targeted antitumor nano preparation.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 25000 psi, material flow rate: 20.0 L/h).
  • high pressure pressure: 25000 psi, material flow rate: 20.0 L/h.
  • the double emulsion was obtained by circulating 14 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain docetaxel-targeted anti-tumor nanometer. preparation.
  • hyaluronic acid (molecular weight 25 kDa) was completely dissolved in 82.0 mL of 0.01 M MES, the pH of the solution was adjusted to 6.0, then 0.3003 g of EDCI and 0.3401 g of Sulfo-NHS were added, and the reaction was stirred at room temperature for 60 min to obtain hyaluronic acid succinyl group.
  • Imine active lipid then added hyaluronic acid succinimide active lipid to MES solution (2.4414g/49.0mL) of human serum albumin (3/8 times of moles of hyaluronic acid), adjusted The solution was stirred at room temperature for 7.5 hours at room temperature.
  • camptothecin 2.000 g was dissolved in 13.3 mL of a mixed solvent of chloroform and ethanol (11:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 30,000 psi, material flow rate: 23.0 L/h).
  • high pressure pressure: 30,000 psi, material flow rate: 23.0 L/h.
  • the double emulsion was obtained by circulating 15 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a camptothecin-targeted antitumor nano preparation.
  • hyaluronic acid (molecular weight 30 kDa) was completely dissolved in 68.0 mL of 0.01 M PBS, the pH of the solution was adjusted to 5.9, then 0.1037 g of EDCI and 0.1174 g of Sulfo-NHS were added, and the reaction was stirred at room temperature for 30 min to obtain hyaluronic acid succinyl group.
  • Imine active lipid then add human hemoglobin (molar number of hyaluronic acid 1/2 times) in PBS solution (1.4982g/25.0mL) dropwise to hyaluronic acid succinimide active lipid, adjust The solution was stirred at room temperature for 7.5 hours at room temperature.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 25000 psi, material flow rate: 25.0 L/h).
  • high pressure pressure: 25000 psi, material flow rate: 25.0 L/h.
  • the double emulsion was obtained by circulating 16 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a paclitaxel-targeted antitumor nano preparation.
  • hyaluronic acid (molecular weight 40 kDa) was completely dissolved in 300.0 mL of 0.01 M MES, the pH of the solution was adjusted to 5.1, then 0.2327 g of EDCI and 0.1845 g of Sulfo-NHS were added, and the reaction was stirred at room temperature for 45 min to obtain hyaluronic acid succinyl group.
  • An imide active lipid; the hyaluronic acid succinimide active lipid was added dropwise to a MES solution (6.0549 g / 86.0 mL) of recombinant human serum albumin (ratio of 3/5 times the number of hyaluronic acid).
  • thiotepa and 0.0120 g of TPGS were dissolved in 5.9 mL of a mixed solvent of chloroform and ethanol (7:1) to obtain a drug solution;
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 10000 psi, material flow rate: 20.0 L/h).
  • high pressure pressure: 10000 psi, material flow rate: 20.0 L/h.
  • the double emulsion was obtained by circulating 18 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.45 ⁇ m filter, and the filtrate was freeze-dried to obtain a thiotepa-targeted antitumor nano preparation.
  • hyaluronic acid (molecular weight 50 kDa) was completely dissolved in 540.0 mL of 0.01 M PBS, the pH of the solution was adjusted to 5.6, then 0.0829 g of EDCI and 0.0282 g of Sulfo-NHS were added, and the reaction was stirred at room temperature for 40 min to obtain hyaluronic acid succinyl group. Imine active lipid; then add egg white albumin (3/6 times the number of moles of hyaluronic acid) in PBS (3.5946g/45.0mL) to the hyaluronic acid succinimide active lipid, adjust the solution The reaction was stirred at room temperature for 18 hours at pH 7.4.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 35000 psi, material flow rate: 18.0 L/h).
  • high pressure pressure: 35000 psi, material flow rate: 18.0 L/h.
  • the double emulsion was obtained by circulating 19 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.22 ⁇ m filter, and the filtrate was freeze-dried to obtain a vancomycin-targeted antitumor nano preparation.
  • the drug solution prepared in the above step (3) is added to the carrier solution prepared in the step (2) to obtain colostrum, and then homogenized under high pressure (pressure: 40,000 psi, material flow rate: 15.0 L/h).
  • high pressure pressure: 40,000 psi, material flow rate: 15.0 L/h.
  • the double emulsion was obtained by circulating 20 times, and then the organic solvent was removed by rotary evaporation under reduced pressure at 15.0-25.0 ° C, -0.1 MPa, filtered through a 0.45 ⁇ m filter, and the filtrate was freeze-dried to obtain an adriamycin-targeted antitumor nano preparation.
  • hyaluronic acid active lipid 400 mg of hyaluronic acid (molecular weight 5 kDa) was dissolved in 10.0 mL of 0.1 M MES buffer, 1.2 times of hyaluronic acid moles of EDCI, and 1.2 times of hyaluronic acid moles were added. N-hydroxy sulfosuccinimide; the reaction was stirred for 30 minutes at room temperature to obtain a hyaluronic acid succinimide active ester; 500.0 mg of human serum albumin lyophilized powder was dissolved in 10.0 mL of sterile water, and the above was added.
  • Hyaluronic acid succinimide active ester solution adjust pH to 7.0-7.2, stir reaction at room temperature for 60 minutes After the reaction is completed, it is added to a dialysis bag having a molecular weight of up to 10,000, and dialysis is performed to remove unreacted reagents and reaction by-products; the hyaluronic acid-albumin solution after dialysis is freeze-dried to freeze-dried hyaluronic acid-albumin Dissolved in a phosphate buffer solution (PBS buffer) having a pH of 5.5 to obtain an albumin/hyaluronic acid solution;
  • PBS buffer phosphate buffer solution
  • paclitaxel solution 50.0 mg of paclitaxel and 50.0 mg of TPGS were weighed and dissolved in 1.0 mL of chloroform to obtain a paclitaxel solution;
  • the paclitaxel solution was added to the albumin/hyaluronic acid solution under stirring to form colostrum; the colostrum was treated with a high pressure homogenizer (9000-40000 psi) to obtain a nanoemulsion, and the nanoemulsion was transferred to a rotary evaporator at 30.0-
  • the organic solvent was quickly removed by evaporation under reduced pressure at 45.0 ° C, and lyophilized under sterile conditions to give a lyophilized powder.
  • hyaluronic acid active lipid 400.0 mg of hyaluronic acid (molecular weight 5 kDa) was dissolved in 10.0 mL of 0.1 M MES buffer, 1.2 times of hyaluronic acid moles of EDCI, and 1.2 times of hyaluronic acid moles were added. N-hydroxy sulfosuccinimide; the reaction was stirred for 30 minutes at room temperature to obtain a hyaluronic acid succinimide active ester; 500.0 mg of human serum albumin lyophilized powder was dissolved in 10.0 mL of sterile water, and the above was added.
  • Hyaluronic acid succinimide active ester solution adjust the pH to 7.0-7.2, stir the reaction at room temperature for 60 minutes; after the reaction is completed, add to the dialysis bag with molecular weight up to 10000, dialysis to remove unreacted reagents and Reaction by-product; freeze-drying the hyaluronic acid-albumin solution after dialysis, and dissolving the lyophilized hyaluronic acid-albumin in a phosphate buffer solution (PBS buffer) having a pH of 5.5 to obtain albumin/transparent Acid solution
  • PBS buffer phosphate buffer solution
  • the paclitaxel solution was added to the albumin/hyaluronic acid solution under stirring to form colostrum; the colostrum was treated with a high pressure homogenizer (9000-40000 psi) to obtain a nanoemulsion, and the nanoemulsion was transferred to a rotary evaporator at 30.0-
  • the organic solvent was quickly removed by evaporation under reduced pressure at 45.0 ° C, and lyophilized under sterile conditions to give a lyophilized powder.
  • Table 1 The drug-containing rate and drug yield of the nano-formulation before lyophilization, after lyophilization and reconstitution, and after reconstitution filtration
  • the targeted nano-preparation of the present invention still has a high drug-containing rate and drug yield after lyophilization and reconstitution through a 0.22 ⁇ m filter; the target of Comparative Example 1 and Comparative Example 2
  • the drug yield decreased sharply after filtration through a 0.22 ⁇ m filter, which was only 39.86% and 56.99% before filtration, indicating that there was more load in the filtration step.
  • the drug particles are trapped by the filter, and the particle size of this portion should be larger than the pore size of the filter membrane of 0.22 ⁇ m.
  • the particles larger than 0.22 ⁇ m have only a very small portion after lyophilization and reconstitution, and the particles of the targeted nano preparation of the present invention have better dispersibility, are more stable, and are not agglomerated. There is still a high drug yield after filtration.
  • Table 2 Average particle size and particle size range of nano-formulation before lyophilization, after lyophilization, and after reconstitution filtration
  • the particle size of the targeted nano preparation of the present invention is not more than 250 nm, and the particle size of the target nano preparation of the comparative example is up to 350 nm; It can be found from the changes in the average particle size and particle size range before and after reconstitution, although all of the examples and comparative examples of the targeted nano-formulation
  • the average particle size of the particles is less than 220 nm, but the particle size distribution of the comparative example is wider, and there are more particles with a particle diameter larger than 220 nm.
  • the result is re-dissolved with the targeted nano-preparation of the comparative example and filtered by a 0.22 ⁇ m filter. The results of lower drug yields are consistent.
  • Example 1 1.78 >72
  • Example 2 2.26 >72
  • Example 3 3..00 >72
  • Example 4 3.23 >72
  • Example 5 2.09 >72
  • Example 6 2.05 >72
  • Example 7 3.03 >72
  • Example 8 3.99 >72
  • Example 9 2.84 >72
  • Example 10 4.21 >72
  • Example 11 3.44 >72
  • Example 12 4.08 >72 Comparative example one 21.35 60 Comparative example two 19.29 48
  • the targeted nano-preparation particles of the present invention have better dispersibility and a weaker agglomeration tendency, and the results are consistent with the results that the particle size does not increase significantly before and after reconstitution.
  • the targeted nano-formulation of the present invention has a more obvious inhibitory effect on tumors at the same concentration, indicating that the drug-utilizing ratio of the targeted nano-preparation of the present invention is higher.
  • the distribution of the paclitaxel-targeted nanoformulation in vivo was determined by using 14 C-labeled paclitaxel.
  • the paclitaxel-targeted nano-preparation of Example 1, Example 6, Comparative Example 1 - Comparative Example 2 was injected into the mice after 7 days of inoculation of H22 tumor cells by a tail vein at a dose of 5.0 mg/mL.
  • the mice after the injection were sacrificed 24 hours later, and the viscera and the tumor were taken, and the radiation intensity was measured, and the concentration of paclitaxel in each tissue was calculated. The result is shown in Figure 4.
  • the targeted nano-preparation of the present invention is distributed in various tissues in the body after intravenous injection, but the accumulation of the drug-loaded particles of the present invention in the tumor is significantly improved compared to the comparative example, while in other The cumulative decrease in tissue is consistent with the above-described tumor inhibition effect. Therefore, the toxicity of the targeted nano-preparation of the present invention is lower than that of the comparative example, which is advantageous for reducing side effects and improving patient compliance.

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Abstract

一种靶向疏水性抗肿瘤药物纳米制剂，包括质量比为1：4-32.5的疏水性抗肿瘤药物和载体，所述载体由37.5-95.3wt％的白蛋白和4.7-62.5wt％的透明质酸-白蛋白共轭物组成，所述透明质酸-白蛋白共轭物由摩尔比为1：1-20的白蛋白与透明质酸制备而成。该纳米制剂的制备方法包括：制备透明质酸-白蛋白共轭物；配制载体溶液；配制药物溶液；制备纳米制剂。该纳米制剂粒径分布较均匀，分散性好，稳定不团聚，冻干复溶后粒径基本不变，微孔滤器过滤(0.22μm)后收率高，药效好。

Description

靶向疏水性抗肿瘤药物纳米制剂及其制备方法 技术领域
本发明属于医药领域，具体是涉及一种靶向疏水性抗肿瘤药物纳米制剂及其制备方法。
背景技术
大部分肿瘤内部血管生长迅速、不规则，内部淋巴系统清除慢，存在肿瘤渗透增强因子，使大分子和纳米颗粒易滞留在肿瘤内部，称为“增强渗透和滞留效应”(Enhanced Permeability and Retention，EPR)。纳米给药系统由于EPR效应可被动靶向到肿瘤，在肿瘤靶向治疗应用过程中越来受到重视。普通的纳米剂型，如脂质体、纳米粒等在血浆中，会被人体网状内皮系统识别，使纳米制剂的血浆清除加快，降低了其治疗效果。因此表面被修饰的纳米制剂引起了人们的广泛研究与关注。
白蛋白是一类在血浆中大量存在的蛋白。药物载体研究中常见的白蛋白包括人血清白蛋白、牛血清白蛋白、卵清白蛋白。白蛋白分子中存在多种药物结合位点，对不同种类的药物均能有效负载；同时其具有水溶性高、稳定性好、可降解等特点，其生物相容性高，已被美国食品药品监督管理局(FDA)批准用于人体，因此白蛋白是药物输送的理想载体。同时，白蛋白上存在的氨基、巯基等，可以进一步修饰主动靶向基团以提高靶向性。现有注射用紫杉醇(白蛋白结合型)注射剂(Abraxane)的上市引起了市场的广泛关注。
专利CN201210142991通过制备透明质酸和人血白蛋白的共轭物作为载体，然后将其与植物类抗癌药物及纳米粒子稳定剂在高速均质、高压均质或超声处理条件下制得植物类抗癌靶向纳米制剂。该纳米制剂具有肿瘤细胞表面的受体如CD44的生物靶向作用，静脉注射进入体内后依靠肿瘤的EPR效应和HA的CD44高度结合性，使药物在肿瘤部位蓄积，并且延长纳米载药系统在体内的循环时间。
在生物医学应用中，纳米粒子药物的粒径比较重要，不同的粒径代谢路径也不一样，小粒径通过肾代谢，大粒径通过肝代谢。其中，20-200nm的粒子对肿瘤有被动靶向作用，在此粒径范围内的纳米粒子药物可以消除在药物本身的尺寸大小引起的一些生物医学效应，从而可以提高疗效。
注射剂是无菌制剂的一种，注射剂的无菌是通过良好控制且经过验证的灭菌/除菌生产工艺、生产过程中严格执行药品生产质量管理规范(GMP，Good Manufacturing Practice for pharmaceutical products)实现的。含有大量蛋白质(如白蛋白)的制剂均不能用常规的方法(如高温)进行灭菌，因为高温会使蛋白变性。过滤灭菌法是监管部门认可的针对产品具有不稳定性而不能采用包括热灭菌在内的情况的最终灭菌方法，因此，通过0.22μm滤器过滤除菌是制备注射型纳米制剂的优选方案。
但是CN201210142991的纳米制剂颗粒，粒径分布系数较大，粒径分布较广，而且冻干复溶后，由于修饰人血白蛋白的透明质酸分子链之间缠绕，导致粒子之间互相聚集、附着，导致冻干品复溶后粒子 粒径增加，因此进行微孔滤器过滤(0.22μm)时容易出现滤器滤膜孔道堵塞的情况，使滤液中的粒子浓度降低，从而导致滤液中药物含量的降低，从而影响药效及降低产品收率。
发明内容
基于此，本发明的目的之一在于提供一种靶向疏水性抗肿瘤药物纳米制剂，该纳米制剂粒径分布较均匀，分散性好，稳定不团聚，冻干复溶后粒径基本不变，微孔滤器(0.22μm)过滤后滤液中的药物浓度基本不变。
实现上述目的的具体技术方案如下：
一种靶向疏水性抗肿瘤药物纳米制剂，包括质量比为1：4-32.5的疏水性抗肿瘤药物和载体，所述载体由37.5-95.3wt％的白蛋白和4.7-62.5wt％的透明质酸-白蛋白共轭物组成，所述透明质酸-白蛋白共轭物由摩尔比为1：1-20的白蛋白与透明质酸制备而成。
在其中一些实施例中，所述靶向疏水性抗肿瘤药物纳米制剂包括质量比为1：4.8-19的疏水性抗肿瘤药物和载体，所述载体由50.0-95.0wt％的白蛋白和5.0-50.0wt％的透明质酸-白蛋白共轭物组成，所述透明质酸-白蛋白共轭物由摩尔比为1：2-20的白蛋白与透明质酸制备而成。
在其中一些实施例中，所述透明质酸的分子量为2-60kDa。
在其中一些实施例中，所述白蛋白为人血白蛋白、牛血清白蛋白、卵清白蛋白以及重组人血白蛋白。
在其中一些实施例中，所述疏水性抗肿瘤药物选自紫杉醇、多西紫杉醇、阿齐他赛、阿霉素、喜树碱、环孢菌素、雷帕霉素、万古霉素、塞替派或它们的衍生物。
在其中一些实施例中，所述靶向疏水性抗肿瘤药物纳米制剂还包括纳米粒子稳定剂，所述纳米粒子稳定剂选自聚氧乙烯-聚氧丙烯-聚氧乙烯崁段聚合物，d-α生育酚琥珀酸聚乙二醇酯、聚维酮，所述纳米粒子稳定剂为疏水性抗肿瘤药物的质量的1.0-10.0％。
本发明的另一目的在于提供上述靶向疏水性抗肿瘤药物纳米制剂的制备方法。
具体技术方案如下：
一种上述靶向疏水性抗肿瘤药物纳米制剂的制备方法，包括以下步骤：
(1)制备透明质酸-白蛋白共轭物：
将透明质酸和白蛋白加入到水性介质中溶解，调节溶液pH为5.0-6.0，然后将1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐加入到上述溶液中反应15-60min，调节溶液pH为7.0-7.5，在室温下继续搅拌反应3-24h，反应完毕后，透析除去未键合的透明质酸、未反应的1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；或
将透明质酸溶于水性介质中，调节溶液pH为5.0-6.0，然后加入1-乙基-3-(3-二甲胺丙基)碳二亚胺 盐酸盐和N-羟基硫代琥珀酰亚胺钠盐，室温下搅拌反应15-60min，得到透明质酸琥珀酰亚胺活性脂；再将透明质酸琥珀酰亚胺活性脂逐滴加入到白蛋白的水性介质溶液中或将白蛋白的水性介质溶液逐滴加入到透明质酸琥珀酰亚胺活性脂中，调节溶液pH为7.0-7.5，在室温下搅拌反应3-24h，反应完毕后，透析除去未键合的透明质酸、未反应的1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将白蛋白和步骤(1)制备的透明质酸-白蛋白共轭物溶于水性介质中，得到载体溶液；
(3)配制药物溶液：
将疏水性抗肿瘤癌药物或疏水性抗肿瘤癌药物和纳米粒子稳定剂溶于有机溶剂中，得到药物溶液；
(4)制备纳米制剂：
均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质下获得复乳，然后除去有机溶剂，过滤，冷冻干燥得到靶向疏水性抗肿瘤药物纳米制剂。
在其中一些实施例中，步骤(2)所述的载体溶液中载体的浓度为4.0-50.0mg/mL，步骤(3)所述的药物溶液中疏水性抗肿瘤药物的浓度为16.0-345.0mg/mL。
在其中一些实施例中，步骤(1)所述的水性介质为2-(N-吗啡啉)乙磺酸缓冲液、磷酸盐缓冲液或无菌水；步骤(2)所述的水性介质为无菌水、磷酸盐缓冲液、生理盐水、5.0wt％葡萄糖水溶液或5.0wt％甘露醇水溶液；步骤(3)所述的有机溶剂为二氯甲烷、氯仿、二氯甲烷与乙醇的混合物、或氯仿与乙醇的混合物。
在其中一些实施例中，步骤(4)所述的高压均质的压力为10000-40000psi，高压均质的物料流量为10.0-25.0L/h，高压均质的循环次数为7-20次；所述除去有机溶剂的方法为在15.0-45.0℃的条件下减压旋转蒸发除去有机溶剂。
本发明的发明人在长期的经验积累和实验过程中发现，在透明质酸-白蛋白共轭物中加入未改性的白蛋白，可以减小本发明所述靶向疏水性抗肿瘤药物纳米制剂颗粒的粒径，使粒径分布更均匀，可以避免或减少纳米粒子之间的互相聚集与附着，因此本发明所述的靶向疏水性抗肿瘤药物纳米制剂，其具有以下的优点：
1、本发明的纳米制剂的粒径分布较均匀且绝大部分小于220nm，而且复溶后粒径没有增加，经微孔滤器(0.22μm)过滤后具有较高的收率，大大降低了因粒径过大而被截留造成的损失，从而可以提高药效，增强对肿瘤的抑制作用。
2、本发明的纳米制剂的粒径绝大部分小于220nm，而且粒子复溶后粒径没有增加，有利于纳米粒子在体内循环系统中进行较长时间循环，而且不被单核巨噬细胞吞噬和被肝脏内的网状系统拦截，具有 主动靶向性，有利于保证靶向部位足够的血药浓度，提高药效，降低对非肿瘤部位的毒性。
3、本发明的纳米制剂复溶后在溶液中的稳定性好，在体内环境中稳定，载体和药物能在运转过程中保持为稳定的复合体。
附图说明
图1为实施例3的靶向纳米制剂颗粒过滤前的粒径分布图；
图2为对比例一的靶向纳米制剂颗粒过滤前的粒径分布图；
图3为实施例1-2、实施例5-6和对比例一-二的靶向纳米制剂对肿瘤的抑制作用比较示意图；
图4为实施例1、实施例6和对比例一-二的靶向纳米制剂的生物靶向性比较示意图。
具体实施方式
以下结合具体实施例对本发明做进一步的阐述。
EDCI：1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐
Sulfo-NHS：N-羟基硫代琥珀酰亚胺钠盐
MES：2-(N-吗啡啉)乙磺酸缓冲液
PEO-PPO-PEO崁段聚合物：聚氧乙烯-聚氧丙烯-聚氧乙烯崁段聚合物
TPGS：d-α生育酚琥珀酸聚乙二醇
PVP：聚维酮
实施例1 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将2.7042g透明质酸(分子量2kDa)和4.4958g人血白蛋白(摩尔数为透明质酸的1/20倍)加入到27.0mL无菌水中溶解，调节溶液pH为5.5，然后将12.9600g EDCI和29.3584g Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.4，在室温下继续搅拌反应5h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将0.9720g人血白蛋白和0.1080g透明质酸-白蛋白共轭物溶于216.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.1200g紫杉醇溶解于2.7mL氯仿和乙醇的混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于 高压均质(压力：20000psi，物料流量：10.0L/h)下循环7次获得复乳，然后30.0-45.0℃的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例2 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将2.4575g透明质酸(分子量3kDa)和8.1711g人血白蛋白(摩尔数为透明质酸的3/20倍)加入到27.0mL 0.01M MES溶液中溶解，调节溶液pH为5.5，然后将6.2813g EDCI和14.2291g Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.4，在室温下继续搅拌反应4h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将1.2754g人血白蛋白和0.3189g透明质酸-白蛋白共轭物溶于213.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.1200g紫杉醇溶解于7.1mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：14000psi，物料流量：12.0L/h)下循环10次获得复乳，然后在15.0-25.0℃,-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例3 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.4319g透明质酸(分子量5kDa)和3.1741g人血白蛋白(摩尔数为透明质酸的1/6倍)加入到18.0mL0.01M PBS中溶解，调节溶液pH为5.4，然后将1.6470g EDCI和3.3579g Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.5，在室温下继续搅拌反应4h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将1.0594g人血白蛋白和0.4606g透明质酸-白蛋白共轭物溶于150.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.0800g紫杉醇溶解于约4.3mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：17000psi，物料流量：15.0L/h)下循环12次获得复乳，然后在15.0-25.0℃，-0.1MPa的 条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例4 阿齐他赛靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.6884g透明质酸(分子量8kDa)和2.6316g人血白蛋白(摩尔数为透明质酸的3/16倍)加入到24.0mL 0.01M MES中溶解，调节溶液pH为5.6，然后将1.0115g EDCI和2.0622g Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.4，在室温下继续搅拌反应4h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将2.16g人血白蛋白和1.44g透明质酸-白蛋白共轭物溶于290.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.4000g阿齐他赛溶解于7.2mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：20000psi，物料流量：15.0L/h)下循环14次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例5 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.8144g透明质酸(分子量10kDa)和2.5856g人血白蛋白(摩尔数为透明质酸的3/14倍)加入到30.0mL 0.01M PBS中溶解，调节溶液pH为5.3，然后将0.8696g EDCI和1.4774g Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.4，在室温下继续搅拌反应6h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将4.1800g人血白蛋白和0.2200g透明质酸-白蛋白共轭物溶于300.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.6000g紫杉醇溶解于7.3mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：25000psi，物料流量：15.0L/h)下循环10次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例6 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.6289g透明质酸(分子量15kDa)和1.8054g人血白蛋白(摩尔数为透明质酸的1/4倍)加入到33.0mL 0.01M MES中溶解，调节溶液pH为5.5，然后将0.4164g EDCI和0.7074g Sulfo-NHS加入到上述溶液中反应20min，调节溶液pH 7.3，在室温下继续搅拌反应3h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将3.8465g人血白蛋白和0.6869g透明质酸-白蛋白共轭物溶于230.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将0.8000g紫杉醇溶解于5.0mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：30000psi，物料流量：18.0L/h)下循环12次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到阿齐他赛靶向抗肿瘤纳米制剂。
实施例7 多烯紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.2221g透明质酸(分子量20kDa)和1.2191g人血白蛋白(摩尔数为透明质酸的3/10倍)加入到31.0mL 0.01M PBS中溶解，调节溶液pH为5.0，然后将0.2343g EDCI和0.3184g Sulfo-NHS加入到上述溶液中反应20min，调节溶液pH 7.0，在室温下继续搅拌反应8h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将3.7664g人血白蛋白和2.0924g透明质酸-白蛋白共轭物溶于235.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将1.2000g多烯紫杉醇溶解于4.7mL氯仿和乙醇混合溶剂(9:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：25000psi，物料流量：20.0L/h)下循环14次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到多烯紫杉醇靶向抗肿瘤纳米 制剂。
实施例8 喜树碱靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将2.4475g透明质酸(分子量25kDa)完全溶于82.0mL 0.01M MES中，调节溶液pH为6.0，然后加入0.3003g EDCI和0.3401g Sulfo-NHS，室温下搅拌反应60min，得到透明质酸琥珀酰亚胺活性脂；再将透明质酸琥珀酰亚胺活性脂逐滴加入到人血白蛋白(摩尔数为透明质酸的3/8倍)的MES溶液(2.4414g/49.0mL)中，调节溶液pH 7.5，室温下搅拌反应10h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将3.5556g人血白蛋白和4.4444g透明质酸-白蛋白共轭物溶于270.0mL无菌水中，得到载体溶液；
(3)配制药物溶液：
将2.0000g喜树碱溶解于13.3mL氯仿和乙醇混合溶剂(11:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：30000psi，物料流量：23.0L/h)下循环15次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到喜树碱靶向抗肿瘤纳米制剂。
实施例9 紫杉醇靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将1.3518g透明质酸(分子量30kDa)完全溶于68.0mL 0.01M PBS中，调节溶液pH为5.9，然后加入0.1037g EDCI和0.1174g Sulfo-NHS，室温下搅拌反应30min，得到透明质酸琥珀酰亚胺活性脂；再将人血白蛋白(摩尔数为透明质酸的1/2倍)的PBS溶液(1.4982g/25.0mL)逐滴加入到透明质酸琥珀酰亚胺活性脂中，调节溶液pH 7.5，室温下搅拌反应12h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物。
(2)配制载体溶液：
将5.7000g人血白蛋白和1.9000g透明质酸-白蛋白共轭物溶于220.0mL的0.01M PBS中，得到载体溶液；
(3)配制药物溶液：
将0.4000g紫杉醇和0.0040g PEO-PPO-PEO崁段聚合物溶解于3.3mL氯仿中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：25000psi，物料流量：25.0L/h)下循环16次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到紫杉醇靶向抗肿瘤纳米制剂。
实施例10 塞替派靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将6.60701g透明质酸(分子量40kDa)完全溶于300.0mL 0.01M MES中，调节溶液pH为5.1，然后加入0.2327g EDCI和0.1845g Sulfo-NHS，室温下搅拌反应45min，得到透明质酸琥珀酰亚胺活性脂；再将透明质酸琥珀酰亚胺活性脂逐滴加入到重组人血清白蛋白(摩尔数为透明质酸的3/5倍)的MES溶液(6.0549g/86.0mL)中，调节溶液pH 7.2，室温下搅拌反应15h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物。
(2)配制载体溶液：
将4.8500g重组人血白蛋白和8.0833g透明质酸-白蛋白共轭物溶于320.0mL生理盐水中，得到载体溶液；
(3)配制药物溶液：
将0.4000g塞替派和0.0120g TPGS溶解于5.9mL氯仿和乙醇混合溶剂(7:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：10000psi，物料流量：20.0L/h)下循环18次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.45μm滤器过滤，滤液冷冻干燥得到塞替派靶向抗肿瘤纳米制剂。
实施例11 万古霉素靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将5.4054g透明质酸(分子量50kDa)完全溶于540.0mL 0.01M PBS中，调节溶液pH为5.6，然后加入0.0829g EDCI和0.0282g Sulfo-NHS，室温下搅拌反应40min，得到透明质酸琥珀酰亚胺活性脂；再将卵清白蛋白(摩尔数为透明质酸的3/6倍)的PBS溶液(3.5946g/45.0mL)逐滴加入到透明质酸琥珀酰亚胺活性脂中，调节溶液pH 7.4，室温下搅拌反应18h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物。
(2)配制载体溶液：
将5.4000g卵清白蛋白和5.4000g透明质酸-白蛋白共轭物溶于240.0mL 5.0wt％葡萄糖水溶液中，得到载体溶液；
(3)配制药物溶液：
将1.2000g万古霉素和0.0840g TPGS溶解于4.0mL氯仿和乙醇混合溶剂(15:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：35000psi，物料流量：18.0L/h)下循环19次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.22μm滤器过滤，滤液冷冻干燥得到万古霉素靶向抗肿瘤纳米制剂。
实施例12 阿霉素靶向抗肿瘤纳米制剂的制备
(1)透明质酸-白蛋白共轭物的制备：
将0.3313g透明质酸(分子量60kDa)和0.3671g牛血清白蛋白(摩尔数为透明质酸的1倍)加入到33.0mL 0.01M MES中溶解，调节溶液pH为5.4，然后将与透明质酸等摩尔比的EDCI和Sulfo-NHS加入到上述溶液中反应15min，调节溶液pH 7.3，在室温下继续搅拌反应24h，反应完毕后，透析除去未键合的透明质酸、未反应的EDCI、Sulfo-NHS和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
(2)配制载体溶液：
将13.9683g牛血清白蛋白和0.6984g透明质酸-白蛋白共轭物溶于300.0mL 5.0wt％甘露醇水溶液中，得到载体溶液；
(3)配制药物溶液：
将2.0000g阿霉素和0.2000g PVP溶解于5.8mL氯仿和乙醇混合溶剂(5:1)中，得到药物溶液；
(4)制备纳米制剂：
高速均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质(压力：40000psi，物料流量：15.0L/h)下循环20次获得复乳，然后在15.0-25.0℃，-0.1MPa的条件下减压旋转蒸发除去有机溶剂，0.45μm滤器过滤，滤液冷冻干燥得到阿霉素靶向抗肿瘤纳米制剂。
对比例一 紫杉醇靶向抗肿瘤纳米制剂的制备
根据专利CN201210142991实施例3实施：
(1)制备透明质酸-白蛋白共轭物
先制备透明质酸活性脂：将400毫克的透明质酸(分子量5kDa)溶解在10.0mL 0.1M MES缓冲液中，加入1.2倍透明质酸摩尔数的EDCI，与1.2倍透明质酸摩尔数的N-羟基硫代琥珀酰亚胺；在室温下搅拌反应30分钟即制得透明质酸琥珀酰亚胺活性酯；将500.0mg人血白蛋白冻干粉溶解在10.0mL无菌水中，加入上述透明质酸的琥珀酰亚胺活性酯溶液中；调整pH值至7.0-7.2，在室温下搅拌反应60分 钟；反应完成后加入到分子量截至为10000的透析袋内，透析除去未反应的试剂和反应副产物；将透析后透明质酸-白蛋白溶液冷冻干燥，将冻干的透明质酸-白蛋白溶于pH为5.5的磷酸盐缓冲溶液(PBS缓冲液)中，得到白蛋白/透明质酸溶液；
(2)制备紫杉醇溶液
称取50.0mg紫杉醇、50.0mg TPGS溶于1.0mL氯仿中，得紫杉醇溶液；
(3)制备紫杉醇/TPGS/透明质酸-白蛋白纳米制剂
在搅拌条件下将紫杉醇溶液加入到白蛋白/透明质酸溶液中，形成初乳；初乳在高压均质机(9000-40000psi)处理下得到纳米乳剂，纳米乳剂转入旋转蒸发仪在30.0-45.0℃减压蒸发快速除去有机溶剂，无菌条件下冷冻干燥得到冻干粉末。
对比例二 紫杉醇靶向抗肿瘤纳米制剂的制备
根据专利CN201210142991实施例14实施：
(1)制备透明质酸-白蛋白共轭物
先制备透明质酸活性脂：将400.0mg的透明质酸(分子量5kDa)溶解在10.0mL 0.1M MES缓冲液中，加入1.2倍透明质酸摩尔数的EDCI，与1.2倍透明质酸摩尔数的N-羟基硫代琥珀酰亚胺；在室温下搅拌反应30分钟即制得透明质酸琥珀酰亚胺活性酯；将500.0mg人血白蛋白冻干粉溶解在10.0mL无菌水中，加入上述透明质酸的琥珀酰亚胺活性酯溶液中；调整pH值至7.0-7.2，在室温下搅拌反应60分钟；反应完成后加入到分子量截至为10000的透析袋内，透析除去未反应的试剂和反应副产物；将透析后透明质酸-白蛋白溶液冷冻干燥，将冻干的透明质酸-白蛋白溶于pH为5.5的磷酸盐缓冲溶液(PBS缓冲液)中，得到白蛋白/透明质酸溶液；
(2)制备紫杉醇溶液
称取50.0mg紫杉醇、50.0mg TPGS溶于1.0mL氯仿和乙醇的混合物(9:1)中得紫杉醇溶液；
(3)制备紫杉醇/TPGS/透明质酸-白蛋白纳米制剂
在搅拌条件下将紫杉醇溶液加入到白蛋白/透明质酸溶液中，形成初乳；初乳在高压均质机(9000-40000psi)处理下得到纳米乳剂，纳米乳剂转入旋转蒸发仪在30.0-45.0℃减压蒸发快速除去有机溶剂，无菌条件下冷冻干燥得到冻干粉末。
实施例13 靶向纳米制剂药物含量的比较
取实施例1-12以及对比例一-二所得纳米制剂冻干物各10.0mg，分别加入4.0mL无菌过滤后的生理盐水复溶，摇匀后取其中2.0mL以0.22μm滤器过滤。往纳米制剂溶液中分别加入2.0ml氯仿-乙醇溶液(9:1)，用漩涡振荡器充分震荡以萃取出制剂中的药物，用高压液相色谱测定过滤前后液体中的药物浓 度，并对比各实施例以及对比例中复乳除去有机溶剂后(即冻干前)的含药率和药物收率，其中，含药率(％)＝药物浓度*体积/(药物和载体质量)*100％，药物收率(％)＝含药率/理论含药率*100％。结果如表1所示。
表1纳米制剂冻干前、冻干复溶后及复溶过滤后的含药率和药物收率
Figure PCTCN2016107127-appb-000001
由表1的结果可知，本发明的靶向纳米制剂在冻干复溶以及复溶经0.22μm滤器过滤后仍具有很高的含药率和药物收率；对比例一和对比例二的靶向纳米制剂冻干前后的药物收率没有很大的区别，但是经0.22μm滤器过滤后药物收率急剧降低，仅为过滤前的39.86％和56.99％，说明了在过滤步骤有较多的载药颗粒被滤膜截留，这部分颗粒粒径应当大于0.22μm滤器滤膜的孔径。而本发明的靶向纳米制剂中，大于0.22μm的颗粒在冻干复溶后只有极少部分，也说明本发明的靶向纳米制剂的颗粒分散性更好，更稳定，不团聚，因此经过滤后仍有较高的药物收率。
实施例14 靶向纳米制剂粒径的比较
取实施例1-12以及对比例一-二所得纳米制剂冻干物各10.0mg，分别加入4.0mL无菌过滤后的生理盐水复溶，摇匀后取其中2.0mL以0.22μm滤器过滤。用美国布鲁克海文激光散射粒度分析仪(ZetaPALS)测定过滤前后纳米制剂的粒径，并对比各实施例中复乳除去有机溶剂后(即冻干前)的粒径。实施例3的靶向纳米制剂颗粒过滤前的粒径分布图如图1所示，对比例一靶向纳米制剂颗粒过滤前的粒径分布图如图2所示，所有结果如表2所示。
表2纳米制剂冻干前、冻干复溶后及复溶过滤后的平均粒径和粒径范围
Figure PCTCN2016107127-appb-000002
由表2的结果可知，所有实施例的靶向纳米制剂的颗粒粒径在冻干前和冻干复溶后并不相同，本发明的靶向纳米制剂冻干复溶后的颗粒的粒径增加幅度远小于对比例的靶向纳米制剂；本发明的靶向纳米制剂的颗粒复溶后的颗粒粒径不大于250nm，而对比例的靶向纳米制剂的颗粒粒径最大接近350nm；同时，由复溶前后的平均粒径和粒径范围变化可以发现，虽然所有实施例以及对比例的靶向纳米制剂的 颗粒的平均粒径都小于220nm，但是对比例的粒径分布范围更宽，存在较多粒径大于220nm的颗粒，该结果与对比例的靶向纳米制剂复溶并以0.22μm滤器过滤后的药物收率较低的结果是一致的。
实施例15 靶向纳米制剂稳定性的比较
(1)复溶稳定时间的比较
取实施例1-12以及对比例一-二所得纳米制剂冻干物各10.0mg，用4.0mL无菌过滤后的生理盐水复溶，于室温下观察混悬液状态、有无沉淀及出现沉淀时间。结果如表3所示。
(2)疏水性药物与白蛋白结合的稳定性比较
将50.0mg实施例1-12以及对比例一-二所得纳米制剂冻干物放入截流分子量为5000的透析袋中，置于500.0mL、37.0℃的pH＝7.4的PBS缓冲溶液(0.01M，含0.5wt％土温80)内，定时取出定量的PBS缓冲溶液，用0.22μm的滤膜过滤，然后用高压液相色谱进行测定。结果如表3所示。
表3纳米制剂复溶稳定性及24h释放率
样品 24h释放率/％ 复溶稳定时间/h
实施例1 1.78 ＞72
实施例2 2.26 ＞72
实施例3 3.00 ＞72
实施例4 3.23 ＞72
实施例5 2.09 ＞72
实施例6 2.05 ＞72
实施例7 3.03 ＞72
实施例8 3.99 ＞72
实施例9 2.84 ＞72
实施例10 4.21 ＞72
实施例11 3.44 ＞72
实施例12 4.08 ＞72
对比例一 21.35 60
对比例二 19.29 48
由表3的结果可知，本发明的靶向纳米制剂在PBS缓冲溶液中24h内只有不到5.00％的药物释放出来，远低于对比例，说明药物与载体之间结合力较强，更有利于避免或减少给药后药物在体内循环及到达靶点前的运转过程中从载体泄露出来，从而可以降低药物毒性、提高药物利用率以及有利于降低给药剂量。根据复溶分散稳定时间可知，本发明的靶向纳米制剂颗粒分散性较好，团聚倾向较弱，该结果与颗粒粒径在复溶前后没有显著增加的结果一致。
实施例16 动物实验
(1)抑瘤实验
使用BALB/c裸鼠构建人MX-1乳腺癌细胞荷瘤小鼠模型，待肿瘤生长至150-300mm3时将小鼠随机分为生理盐水阴性组，实施例1-2、实施例5-6及对比例一-二药物组，每组6只小鼠。尾静脉注射药物，剂量为10.0mg/kg，每5天注射一次，连续注射5次，在试验过程中每5天测量肿瘤体积一次，其体积计算方法如下：V＝d2D/2，其中d为短方向的长度，D为长方向的长度。结果如图3所示。
由图3可知，本发明的靶向纳米制剂在相同浓度下对肿瘤的抑制作用更加明显，说明本发明的靶向纳米制剂的药物利用率更高。
(2)药物在小鼠体内的分布比较
紫杉醇靶向纳米制剂在体内的分布的测定是通过使用14C标记的紫杉醇来完成的。将实施例1、实施例6、对比例一-对比例二的紫杉醇靶向纳米制剂以5.0mg/mL的剂量通过尾静脉注射到已接种H22肿瘤细胞7天后的小鼠体内。注射后的小鼠在24小时后处死，分别取内脏和肿瘤，测定其放射强度，计算各组织中紫杉醇的浓度。结果如图4所示。
由图4可知，本发明的靶向纳米制剂静脉注射后分布在体内的各种组织中，但相比对比例，本发明的载药颗粒在肿瘤中的累积有了显著的提高，而在其他组织中的累积相对降低，与上述肿瘤抑制效果一致。从而说明本发明的靶向纳米制剂的毒性相比对比例较低，有利于降低副作用并提高病患者依从性。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种靶向疏水性抗肿瘤药物纳米制剂，其特征在于，包括质量比为1：4-32.5的疏水性抗肿瘤药物和载体，所述载体由37.5-95.3wt％的白蛋白和4.7-62.5wt％的透明质酸-白蛋白共轭物组成，所述透明质酸-白蛋白共轭物由摩尔比为1：1-20的白蛋白与透明质酸制备而成。
  2. 根据权利要求1所述的靶向疏水性抗肿瘤药物纳米制剂，其特征在于，包括质量比为1：4.8-19的疏水性抗肿瘤药物和载体，所述载体由50.0wt％-95.0wt％的白蛋白和5.0-50.0wt％的透明质酸-白蛋白共轭物组成，所述透明质酸-白蛋白共轭物由摩尔比为1：2-20的白蛋白与透明质酸制备而成。
  3. 根据权利要求1或2所述的靶向疏水性抗肿瘤药物纳米制剂，其特征在于，所述透明质酸的分子量为2-60kDa。
  4. 根据权利要求1或2所述的靶向疏水性抗肿瘤药物纳米制剂，其特征在于，所述白蛋白选自人血白蛋白、牛血清白蛋白、卵清白蛋白以及重组人血白蛋白。
  5. 根据权利要求1或2所述的靶向疏水性抗肿瘤药物纳米制剂，其特征在于，所述疏水性抗肿瘤药物选自紫杉醇、多西紫杉醇、阿齐他赛、阿霉素、喜树碱、环孢菌素、雷帕霉素、万古霉素、塞替派或它们的衍生物。
  6. 根据权利要求1或2所述的靶向疏水性抗肿瘤药物纳米制剂，其特征在于，还包括纳米粒子稳定剂，所述纳米粒子稳定剂选自聚氧乙烯-聚氧丙烯-聚氧乙烯崁段聚合物，d-α生育酚琥珀酸聚乙二醇酯、聚维酮，所述纳米粒子稳定剂为疏水性抗肿瘤药物的质量的1.0-10.0％。
  7. 一种权利要求1-6任一项所述的靶向疏水性抗肿瘤药物纳米制剂的制备方法，其特征在于，包括以下步骤：
    (1)制备透明质酸-白蛋白共轭物：
    将透明质酸和白蛋白加入到水性介质中溶解，调节溶液pH为5.0-6.0，然后将1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐加入到上述溶液中反应15-60min，调节溶液pH为7.0-7.5，在室温下继续搅拌反应3-24h，反应完毕后，透析除去未键合的透明质酸、未反应的1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；或
    将透明质酸溶于水性介质中，调节溶液pH为5.0-6.0，然后加入1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐，室温下搅拌反应15-60min，得到透明质酸琥珀酰亚胺活性脂；再将透明质酸琥珀酰亚胺活性脂逐滴加入到白蛋白的水性介质溶液中或将白蛋白的水性介质溶液逐滴加入到透明质酸琥珀酰亚胺活性脂中，调节溶液pH为7.0-7.5，在室温下搅拌反应3-24h，反应完毕后，透析除去未键合的透明质酸、未反应的1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺钠盐和其它副产物，冷冻干燥后得透明质酸-白蛋白共轭物；
    (2)配制载体溶液：
    将白蛋白和步骤(1)制备的透明质酸-白蛋白共轭物溶于水性介质中，得到载体溶液；
    (3配制药物溶液：
    将疏水性抗肿瘤癌药物或疏水性抗肿瘤癌药物和纳米粒子稳定剂溶于有机溶剂中，得到药物溶液；
    (4)制备纳米制剂：
    均质条件下，将上述步骤(3)配制的药物溶液加入到步骤(2)配制的载体溶液中获得初乳，再于高压均质下获得复乳，然后除去有机溶剂，过滤，冷冻干燥得到靶向疏水性抗肿瘤药物纳米制剂。
  8. 根据权利要求7所述的靶向疏水性抗肿瘤药物纳米制剂的制备方法，其特征在于，步骤(2)所述的载体溶液中载体的浓度为4.0-50.0mg/mL，步骤(3)所述的药物溶液中疏水性抗肿瘤药物的浓度为16.0-345.0mg/mL。
  9. 根据权利要求7或8所述的靶向疏水性抗肿瘤药物纳米制剂的制备方法，其特征在于，步骤(1)所述的水性介质为2-(N-吗啡啉)乙磺酸缓冲液、磷酸盐缓冲液或无菌水；步骤(2)所述的水性介质为无菌水、磷酸盐缓冲液、生理盐水、5.0wt％葡萄糖水溶液或5.0wt％甘露醇水溶液；步骤(3)所述的有机溶剂为二氯甲烷、氯仿、二氯甲烷与乙醇的混合物、或氯仿与乙醇的混合物。
  10. 根据权利要求7或8所述的靶向疏水性抗肿瘤药物纳米制剂的制备方法，其特征在于，步骤(4)所述的高压均质的压力为10000-40000psi，高压均质的物料流量为10.0-25.0L/h，高压均质的循环次数为7-20次；所述除去有机溶剂的方法为在15.0-45.0℃的条件下减压旋转蒸发除去有机溶剂。
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