WO2016015522A1 - 脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 - Google Patents

脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 Download PDF

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WO2016015522A1
WO2016015522A1 PCT/CN2015/080977 CN2015080977W WO2016015522A1 WO 2016015522 A1 WO2016015522 A1 WO 2016015522A1 CN 2015080977 W CN2015080977 W CN 2015080977W WO 2016015522 A1 WO2016015522 A1 WO 2016015522A1
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water
albumin
liquid
drug
fatty acid
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PCT/CN2015/080977
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English (en)
French (fr)
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张旋
王洁银
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天津派格生物技术有限公司
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates

Definitions

  • the invention relates to a nanoparticle lyophilized preparation and a preparation method thereof, in particular to a fatty acid-binding albumin-drug nanoparticle lyophilized preparation and a preparation method thereof, and belongs to the technical field of medicine preparation.
  • HSA Human Serum Albumin
  • HSA Human Serum Albumin
  • the concentration of HSA in healthy human plasma is 35-55 g/L, and the half-life is about 19 days.
  • HSA plays an important role in maintaining blood colloid osmotic pressure, amino acid and energy metabolism of tissue cells, and transport of fat-soluble substances such as bilirubin, fatty acids and drugs.
  • HSA is abundant in source and can be extracted from human blood. It can also be produced by genetic recombination technology.
  • HSA is non-toxic, non-immunogenic, and has a clear metabolic mechanism. It is an ideal nanoparticle preparation material.
  • HSA may be partially accumulated in tumor tissue; HSA gp60 protein transport through the cell membrane into the cell, some tumors such as breast tissue specifically expressed SPARC protein (secreted protein, acidic And rich in cysteine), which promotes the transport of Gp60 receptor to HSA; it has been reported that 2 , albumin-bound paclitaxel (Abraxane) is more effective in chemotherapy of solid tumors with high SPARC protein expression.
  • SPARC protein secreted protein, acidic And rich in cysteine
  • Paclitaxel is a biguanide compound extracted from natural plants and has obvious anticancer effects. It prevents the mitosis and proliferation of cells by the unique action of tubulin. Paclitaxel has a very low water solubility (water solubility of 0.0005 mg/mL), while paclitaxel is almost insoluble in water by semi-synthetic docetaxel obtained by chemical modification and modification. In addition to the improvement of anticancer activity in vitro, other physical and chemical properties and paclitaxel the same.
  • paclitaxel and docetaxel intravenous injections have added a large amount of surfactant to help dissolve, such as Taxol is added with polyoxyethylene castor oil (Cremophor EL) and anhydrous ethanol to help dissolve, polyene Paclitaxel uses Tween-80 to help dissolve, while high-dose Cremophor EL or Tween 80 infusion can lead to severe allergic reactions and other side effects, or even significantly limit the increase in the dose of chemotherapy drugs, and also limit the clinical efficacy of paclitaxel. .
  • a series of patents such as ZL97199720.9 (USP 08/720,756) applied by American Biosciences Co., Ltd.
  • nab-technology utilizes only the "weak” binding of the hydrophobic region of albumin to the water-insoluble drug (hydrophobicity), but does not utilize the same a fatty acid that can be dissolved in an organic solution and that specifically binds to albumin and has a strong binding force; nor does it utilize the properties and functions of albumin that can be combined with many water-soluble drugs including proteins and peptides;
  • the encapsulated nanoscale particles are even nanoparticles smaller than 100 nm. Therefore, the aforementioned nab-technology has obvious defects in the formation mechanism of nanoparticles and
  • FA Fatty acids
  • SA serum albumin
  • the study of SA protein structure reveals the specific binding site and binding mode of FA, that is, the specific binding of FA to SA through the "salt bridge" between the basic amino acid side chain group and the fatty acid carboxyl group at the binding site (salt) Bridge interactions) and achieve 45 .
  • the new generation of long-acting insulin (Insulin Detemir, ID) approved in recent years is based on the specific binding properties of this free fatty acid and SA to achieve long-acting pharmacological effects in vivo.
  • ID is composed of Myristic acid (MA) and insulin.
  • MA Myristic acid
  • the 29-position lysine residue of the B chain is coupled at a fixed point.
  • the fatty acid on the molecule can specifically bind to the SA in the blood, thereby avoiding the rapid clearance of the small molecule insulin in the body;
  • the preparation of a fatty acid insulin analog containing a carboxyl group can be injected, and the fatty acid molecule can be specifically, effectively and stably combined with serum albumin 6 .
  • the work of Spector AA et al 7 in Ehrlich ascites cells and leukemia cells confirmed that tumor cells can rapidly take up free fatty acids (FFA), while albumin and low-density lipoprotein, which are FFA carriers, It can promote this transport mechanism of tumor cells.
  • FFA free fatty acids
  • Doxorubicin (D) is a clinically important, commonly used, broad-spectrum anti-cancer first-line drug, but its anti-cancer efficacy is limited by its toxicity to the heart; although various nanoparticle preparation techniques have been used to develop doxorubicin
  • the drug delivery system in which the stealth liposomal doxorubicin (Doxil) nanoformulation is one of the most successful development paradigms.
  • Doxil significantly reduced the cardiotoxicity of doxorubicin, but its efficacy has not been significantly improved; this is one of the reasons why "invisible liposome" technology is not widely used in other anticancer drugs.
  • Doxorubicin is a water-soluble chemotherapeutic drug that binds to HSA under certain conditions.
  • Pseudomonas exotoxin A has strong cytotoxicity and is fused with anti-tumor specific antibodies to form so-called immunotoxin (immunotoxin, IT), which is a specific target for the treatment of malignant tumors and leukemia.
  • IT immunotoxin
  • the drug known as the "biological missile”
  • Pseudomonas exotoxin (PE38) has been clinically tested for IT, and its targeting of tumor-specific antigens has been confirmed; PE38 may even cure hair.
  • the latter is a patient who cannot produce antibodies to PE38, suggesting the importance of IT molecule antigenicity for its efficacy.
  • PE38 fusion-specific antibodies can significantly enhance the anti-tumor targeting, but it can not avoid the antigenicity of PE38 toxin and its damaging effects of vascular endothelial cells, leading to severe toxicity, especially vascular leak syndrome 9. Therefore, for molecules and drugs such as PE toxins which cause serious toxic side effects in the blood circulation, it is not sufficient to improve the affinity of the antibody and/or reduce the antigenicity of the PE38 toxin only by optimization of the amino acid sequence.
  • Applicants have unexpectedly discovered that the combination of HSA and PE38 can effectively "enclose" the antigenic site of PE38, which is believed to also reduce the damage of PE38 toxin to human vascular endothelium; and the targeting of tumor tissue by albumin nanoparticles. Enrichment and localization of HSA and fatty acid transmembrane mechanisms in the tumor to achieve specific killing of tumor tissue by PE38 is a new therapeutic strategy, and similar reports have not been reported.
  • HSA Human serum albumin
  • the first object of the present invention is to overcome the limitations of the prior art nanoparticle-loaded drugs based on albumin as a main substrate, and to provide a fatty acid combination suitable for both "water-insoluble” and “water-soluble” drugs.
  • Type albumin-drug nanoparticle lyophilized preparation is to overcome the limitations of the prior art nanoparticle-loaded drugs based on albumin as a main substrate, and to provide a fatty acid combination suitable for both "water-insoluble" and "water-soluble” drugs.
  • a second object of the present invention is to overcome the deficiencies of the prior art and to provide a method for preparing a fatty acid-binding albumin-drug nanoparticle lyophilized preparation.
  • a fatty acid-binding albumin-drug nanoparticle comprising an oil phase core, albumin and a drug, said albumin being bound to a fatty acid in an oil phase core, said drug being combined with albumin and/or fatty acid
  • the drug is selected from one or more of a water-soluble drug, a water-insoluble drug, a water-soluble drug ultra-fine nanoparticle, and a water-insoluble drug ultra-microparticle.
  • the fatty acid-binding albumin-drug nanoparticles of the present invention include 0.0001% to 8% of fatty acids, 75% to 99% of albumin, and 0.1% to 25% of the drug by mass ratio. Preferably, the nanoparticles are included by mass ratio. Fatty acids 0.001% to 1%, albumin 80% to 95%, and drugs 1% to 20%.
  • the drug in combination with the fatty acid in the fatty acid-binding albumin-drug nanoparticle constitutes an oil phase core
  • the albumin binds to a fatty acid in the oil phase core.
  • the water-insoluble drug in the fatty acid-binding albumin-drug nanoparticle and the fatty acid combine to form an oil phase core
  • the albumin is combined with a fatty acid in the oil phase core.
  • the water-soluble drug ultrafine nanoparticles and/or the water-insoluble drug ultrafine nanoparticles in the fatty acid-binding albumin-drug nanoparticle and the fatty acid combine to form an oil phase core, the albumin and the oil Fatty acid binding in the phase core.
  • the drug in the fatty acid-binding albumin-drug nanoparticle binds to albumin, and the albumin binds to the fatty acid in the oil phase core.
  • the water-soluble drug in the fatty acid-binding albumin-drug nanoparticle is bound to albumin, and the albumin is bound to the fatty acid in the oil phase core.
  • the poorly water-soluble drug of the fatty acid-binding albumin-drug nanoparticle, the ultrafine nanoparticle of the poorly water-soluble drug, and/or the ultrafine nanoparticle of the water-soluble drug and the fatty acid In combination with the core of the oil phase, the water soluble drug binds to albumin, which binds to the fatty acid in the oil phase core.
  • the fatty acid of the present invention may be a fatty acid remaining in the process of preparing albumin, and it is not necessary to additionally add a fatty acid in the process of preparing the nanoparticle, in which case the albumin of the present invention and the fatty acid in the oil phase core Naturally and specifically bind. Meanwhile, the fatty acid of the present invention may also be added to the formulation according to the method of the present invention, in which case the albumin of the present invention and the fatty acid in the oil phase core are specific in the preparation process. Ground combination. In any of the above cases, the present inventors have surprisingly found that as long as the fatty acid content is within the scope of the present invention, the formed nanoparticles have higher integrity and stability in the in vitro solution and in the blood circulation in the body.
  • a pharmaceutical preparation comprising the fatty acid-binding albumin-drug nanoparticles of the present invention and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical preparation of the present invention may be an oral preparation or an injection preparation, and is preferably a suspension, an injection, or a lyophilized powder for injection.
  • the formulation is a lyophilized formulation.
  • a method for preparing a fatty acid-binding albumin-drug nanoparticle lyophilized preparation comprises the following steps:
  • Liquid A an organic solvent which is incompatible with water, or a mixture of an organic solvent insoluble in a volume ratio of 10 to 5:5 to 1 and anhydrous ethanol, said water incompatible organic
  • the solvent is chloroform, dichloromethane or ethyl acetate
  • solution B a fatty acid solution having a concentration of 0.0033-360 mg/ml is prepared with liquid A, which is oleic acid, linoleic acid, myristic acid or triglyceride
  • C An aqueous solution of albumin having a concentration of 10 to 200 mg/ml is prepared in water, and the liquid A is dropped into the albumin solution stirred at 5,000 to 15,000 rpm at a volume ratio of 1:6.67 to 333, and stirring is continued for 2 to 10 Minutes, Producing liquid C;
  • the solution B is used to dissolve the water-insoluble drug, and the liquid D having a concentration of 12.5 mg/ml to 600 mg/ml is obtained, and the liquid D is dropped into the liquid having a stirring speed of 3,000 rpm to 30,000 rpm in a ratio of 0.1% to 7.2% by volume.
  • C a fatty acid-binding albumin-water insoluble drug nanoparticle solution type suspension is prepared;
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in which the water-insoluble drug is dissolved is the oil phase, and the volume of the aqueous albumin solution in the liquid C is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is 0.4% to 17.8. %; the mass ratio of fatty acid to albumin is 0.0001% to 5%; the mass ratio of water-insoluble drug to albumin is: 1% to 20%;
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase, and the volume of the aqueous albumin solution in the liquid C is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is 0.6% to 15.6%.
  • the mass ratio of fatty acid to albumin is 0.0001% to 1%; the mass ratio of water-insoluble drug to albumin is: 1% to 25%;
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase, and the volume of the aqueous albumin solution in the liquid C is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is: 1% to 15.8. %; the mass ratio of fatty acid to albumin is 0.001% to 1.4%; the mass ratio of water-soluble drug to albumin is: 1% to 20%;
  • the ratio of the volume of the liquid A in the liquid C to the volume of the solution B is the oil phase, and the volume of the albumin-water-soluble drug binding solution is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is: 1% to 16%;
  • the mass ratio of fatty acid to albumin is 0.002% to 15%;
  • the mass ratio of water-soluble drug to albumin is: 1% to 25%;
  • the solution B is dropped into the liquid C at a stirring speed of 20,000 rpm to 25,000 rpm in a ratio of 1.4% to 3.3% by volume to prepare a fatty acid-bound albumin nanoparticle suspension;
  • the fatty acid-bound albumin nanoparticle suspension is removed from all organic solvents to obtain a translucent emulsion
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B is the oil phase, the volume of the translucent emulsion is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is: 2.0% to 16.4%; the quality of the fatty acid and albumin The ratio is 0.001% to 3.1%; the mass ratio of water-soluble drug to albumin is: 2.5% to 15%;
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • the suspension prepared by the first method, the fourth method, the sixth method and the seventh method in the step (2) is used to remove all the organic solvents in the suspension to obtain a translucent and stable nanoparticle emulsion;
  • the emulsion obtained in the step (3) is sterilized by filtration, obtained as a sterile emulsion, and freeze-dried in a vacuum to obtain a lyophilized preparation of a fatty acid-binding albumin-drug nanoparticle;
  • the albumin is human serum albumin or bovine serum albumin; the human serum albumin is prepared by blood extraction or recombinant technology;
  • a method for preparing a fatty acid-binding albumin-drug nanoparticle lyophilized preparation comprises the following steps:
  • Liquid A an organic solvent which is incompatible with water, or a mixture of an organic solvent insoluble in a volume ratio of 10 to 5:5 to 1 and anhydrous ethanol, said water incompatible organic
  • the solvent is chloroform, dichloromethane or ethyl acetate, which is a chromatographically pure reagent
  • solution B a liquid fatty acid solution having a concentration of 0.0033-360 mg/ml is prepared by using liquid A, preferably at a concentration of 0.01 mg/ml to 50 mg/ml.
  • the fatty acid for intravenous injection is oleic acid, linoleic acid, myristic acid or triglyceride, which is a component of a fat emulsion which can be intravenously infused by human body; liquid C: under sterile conditions, the concentration is 10 by using water for injection. ⁇ 200mg/ml of albumin aqueous solution, the liquid A is dropped into the albumin solution stirred at 5,000 to 15,000 rpm in a ratio of 1:6.67 to 333, and stirring is continued for 2 to 10 minutes to bind the fatty acid. Albumin is extracted from liquid A to prepare liquid C;
  • a liquid D having a concentration of 12.5 mg/ml to 600 mg/ml, preferably a concentration of 20 mg/ml to 400 mg/ml, and dropping the liquid D in a ratio of 0.1% to 7.2% by volume.
  • a liquid C having a stirring speed of 3,000 rpm to 30,000 rpm, stirring is continued for 10 to 60 minutes to prepare a fatty acid-binding albumin-water-insoluble drug nanoparticle solution type suspension;
  • the solution B is used to dissolve the water-insoluble drug, and the liquid D having a concentration of 12.5 mg/ml to 600 mg/ml is obtained, preferably at a concentration of 10 mg/ml to 400 mg/ml, and the liquid is in a ratio of 0.1% to 7.2% by volume.
  • D is dropped into liquid C at a stirring speed of 10,000 rpm to 12,000 rpm, stirring is continued for 5 ⁇ 1 minute, and added to a high pressure microjet feeding container at 4 ⁇ 1 ° C, protected from light at a pressure of 6,000 psi to 40,000 psi. , 2 to 8 cycles of high pressure homogenization treatment, to obtain a fatty acid-binding albumin-water insoluble drug nanoparticle solution type suspension;
  • the water-insoluble drug is selected from the group consisting of paclitaxel, docetaxel, rapamycin, curcumin, mitomycin, vincristine, 7-ethyl-10-hydroxycamptothecin, etoposide, and methotrexate.
  • a relatively low or low water-soluble chemotherapeutic drug or other drug soluble in at least one "organic solvent or a mixture thereof", and capable of binding to albumin; different "water insoluble” "Solubility is different from the solubility of different "organic solvents or mixtures thereof", and “solubility” is an important factor in determining the "concentration range" of "water-insoluble” drugs.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in which the water-insoluble drug is dissolved is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase.
  • the ratio of the volume of the oil phase to the volume of the aqueous phase is from 0.4% to 17.8%, preferably from 1% to 6.5%
  • the mass ratio of the fatty acid to albumin for intravenous injection is from 0.0001% to 5%, preferably from 0.001% to 1%
  • the mass ratio to albumin is from 1% to 20%, preferably from 1% to 10%.
  • the supernatant is removed to obtain ultrafine nanoparticles of water-insoluble drug; the water-insoluble drug ultrafine nanoparticles are suspended in solution B at a ratio of 40 mg/ml to 600 mg/ml to obtain liquid D', which is fatty acid-containing, water-insoluble. a suspension of drug ultrafine nanoparticles and an organic solvent;
  • the liquid D' is dropped into the liquid C at a stirring speed of 3,000 rpm to 30,000 rpm in a ratio of 0.3% to 4.0% by volume, and stirring is continued for 15 to 60 minutes to obtain a fatty acid-binding albumin-water insoluble property.
  • the drug nanoparticle suspension suspension; or the liquid D' is dropped into the liquid C at a stirring speed of 3,000 rpm to 12,000 rpm in a ratio of 0.3% to 4.0% by volume, and stirring is continued for 5 ⁇ 1 minute, and added.
  • the high-pressure micro-jet microfluidizer feed container is treated with high-pressure homogenization at 4 ⁇ 1°C in the dark at a pressure of 15,000 psi to 30,000 psi for 2 to 6 cycles to obtain a fatty acid-binding albumin-water-insoluble drug nanometer.
  • the "water-insoluble” drug is selected from paclitaxel, docetaxel, rapamycin, curcumin, mitomycin, vincristine, 7-ethyl-10-hydroxycamptothecin, etoposide and methotrexate.
  • paclitaxel docetaxel
  • rapamycin curcumin
  • mitomycin vincristine
  • 7-ethyl-10-hydroxycamptothecin etoposide and methotrexate.
  • One or more kinds of hydrazine different "water-insoluble” drugs have different densities and solubilitys, and the volume of the solution required to be suspended in the "organic solvent or its mixture” is different.
  • the definition of "suspension” is more than the concentration range. The definition of "is more important.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase
  • the ratio of volume to aqueous phase volume is: 0.6% to 15.6%
  • the mass ratio of fatty acid to albumin for intravenous injection is 0.0001% to 1%
  • the mass ratio of water-insoluble drug to albumin is: 1% to 25%, preferably 1.0% to 10%;
  • the liquid D' is dropped into the liquid C at a stirring speed of 12,000 rpm to 32,000 rpm in a ratio of 0.8% to 7.0% by volume, and stirring is continued for 5 to 30 minutes to obtain a fatty acid-binding albumin-water-soluble.
  • the drug nanoparticle suspension suspension; or the liquid D' is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 0.8% to 7.0% by volume, and stirring is continued for 5 ⁇ 1 minute, and added.
  • High-pressure micro-fluidizer feed container high-pressure homogenization treatment at 4 ⁇ 1°C, protected from light and pressure from 15,000 psi to 30,000 psi for 3-8 cycles, to produce fatty acid-bound albumin-water-soluble drug nanometer Particle suspension suspension;
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase
  • the oil The ratio of phase volume to aqueous phase volume is: 1% to 15.8%; the mass ratio of fatty acid to albumin for intravenous injection is 0.001% to 1.4%; the mass ratio of water-soluble drug to albumin is: 1% to 20%;
  • water-soluble drugs include one or more of chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, and pseudomonas exotoxin (PE38);
  • the drug is soluble in water, and the solubility in the "organic solvent or a mixture thereof” is very low; the density of the different "water-soluble” drugs is different, and the volume of the solution required to be suspended in the "organic solvent or a mixture thereof”
  • the definition of "suspension” is more important than the definition of "concentration”.
  • the solution B is dropped into the liquid C' at a stirring speed of 15,000 rpm to 25,000 rpm in a ratio of 0.8% to 7.5% by volume, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-soluble.
  • the drug nanoparticle protein-binding suspension; or the solution B is dropped into the liquid C' at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 0.8% to 7.5% by volume, and stirring is continued for 5 ⁇ 1 minute, and added.
  • High-pressure micro-jet microfluidizer feed container at 4 ⁇ 1°C, protected from light, at a pressure of 10,000 psi to 25,000 psi, continuous high-pressure homogenization for 1 to 6 cycles to produce fatty acid-bound albumin-water-soluble drug nanometers Particle protein-bound suspension;
  • Water-soluble drugs include one or more of chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, and pseudomonas exotoxin (PE38). Different "water-soluble” drugs have different binding rates to albumin in vitro. Here, the "binding" concentration of "water-soluble” drugs with albumin in liquid C and the concentration of albumin solution determine the addition of “water-soluble” drugs. Quantity or concentration.
  • the ratio of the volume of the liquid A in the liquid C to the volume of the solution B is the oil phase, and the volume of the albumin-water-soluble drug binding solution is the aqueous phase, and the ratio of the volume of the oil phase to the volume of the aqueous phase is: 1% to 16%;
  • the mass ratio of fatty acid to albumin for intravenous injection is 0.002% to 15%;
  • the mass ratio of water-soluble drug to albumin is: 1% to 25%;
  • the solution B is dropped into the liquid C at a stirring speed of 20,000 rpm to 25,000 rpm in a ratio of 1.4% to 3.3% by volume, and stirring is continued for 10 to 20 minutes to prepare a fatty acid-bound albumin nanoparticle suspension; Or in a ratio of 1.4% to 3.3% by volume, the solution B is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm, and the stirring is continued for 5 ⁇ 1 minute, and then added to the high-pressure microjet feeding container. 4 ⁇ 1°C, in the dark, at a pressure of 10,000 psi to 25,000 psi, continuous high-pressure homogenization for 2-6 cycles to prepare a fatty acid-bound albumin nanoparticle suspension;
  • the fatty acid-bound albumin nanoparticle suspension is placed in a vacuum rotary evaporator, and subjected to rotary evaporation at 35 ⁇ 2° C. and 20 mmHg at 60 to 80 rpm for 20 to 30 minutes to remove all organic solvents. Obtaining a translucent emulsion;
  • the water-soluble drug includes one or more of chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, Pseudomonas exotoxin PE38 and the like.
  • chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, Pseudomonas exotoxin PE38 and the like.
  • Different "water-soluble” drugs have different binding rates to albumin, and the “quantity" of FBA-Enp surface albumin of different particle sizes is different.
  • the "water-soluble” drug is “bound” with FBA-Enp surface albumin.
  • the concentration determines the amount of added or added concentration of the "water soluble” drug.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B is the oil phase
  • the volume of the translucent emulsion is the aqueous phase
  • the ratio of the volume of the oil phase to the volume of the aqueous phase is: 2.0% ⁇ 16.4%
  • the mass ratio of fatty acid to albumin for intravenous injection is 0.001% to 3.1%
  • the mass ratio of water-soluble drug to albumin is 2.5% to 15%;
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • Nanoparticle suspension suspension or suspension of water-soluble drug ultrafine nanoparticles with liquid D to obtain a suspension containing fatty acid, water-insoluble drug, water-soluble drug ultrafine nanoparticles, in a volume ratio of 0.1% to 7.2
  • the suspension containing fatty acid, water-insoluble drug, and water-soluble drug ultrafine nanoparticles is dropped into liquid C at a stirring speed of 10,000 rpm to 12,000 rpm, stirring is continued for 5 ⁇ 1 minute, and high pressure microfluidizer is added.
  • Nanoparticle suspension type suspension In a material container, at a temperature of 4 ⁇ 1 ° C, protected from light, at a pressure of 3,000 psi to 40,000 psi, continuous high-pressure homogenization for 2 to 8 cycles to obtain a fatty acid-binding albumin-water-insoluble and water-soluble drug.
  • Nanoparticle suspension type suspension In a material container, at a temperature of 4 ⁇ 1 ° C, protected from light, at a pressure of 3,000 psi to 40,000 psi, continuous high-pressure homogenization for 2 to 8 cycles to obtain a fatty acid-binding albumin-water-insoluble and water-soluble drug.
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • liquid D having a concentration of 10 mg/ml to 400 mg/ml
  • liquid D is dropped into the liquid C at a stirring speed of 3,000 rpm to 30,000 rpm in a ratio of 0.1% to 7.2%, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-insoluble drug.
  • Nanoparticle solution type suspension; or liquid D is dropped into liquid C at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 0.1% to 7.2%, stirring is continued for 5 ⁇ 1 minute, and high pressure microjet is added.
  • the suspension prepared by the first method, the fourth method, the sixth method and the seventh method in the step (2) is placed in a vacuum rotary evaporator, and the suspension is rotated at a temperature of 35 ⁇ 2° C. under a negative pressure for 10 to 30 minutes to remove the suspension. All organic solvents in the liquid to prepare a translucent and stable nanoparticle emulsion;
  • the emulsion obtained in the step (3) is sterilized by filtration through a 0.22 um membrane, or sterilized by filtration through a 0.45 um and 0.22 um membrane to obtain a sterile emulsion, and freeze-dried by vacuum for 24 to 36 hours to obtain a fatty acid-binding albumin- a pharmaceutical nanoparticle lyophilized preparation;
  • the albumin is human serum albumin or bovine serum albumin;
  • the human serum albumin is prepared by blood extraction or recombinant technology;
  • the volume ratio of the liquid A to the albumin solution in the step (1) is preferably 1:36 to 100.
  • the secondary structure of the albumin in the albumin mixture extracted by the organic solvent is unchanged, the albumin contains a "naked" fatty acid binding site, and the fatty acid is specifically bound at the site, white
  • the hydrophobic region of the protein molecule is bound to the water-insoluble drug by hydrophobicity, and the hydrophilic region of the albumin molecule is bound to the water-soluble drug by hydrophilicity.
  • the fatty acid-binding albumin-drug nanoparticles described in the first to eighth modes of the step (4) are each formed by combining an intravenous fatty acid dissolved in the solution B with albumin in the liquid C or the liquid C'.
  • the water-insoluble drug described in the first step, the sixth method, the seventh method or the eighth method in the step (4) is dissolved in the solution B and forms the oil phase core together with the fatty acid for intravenous injection.
  • the water-insoluble drug ultrafine nanoparticles or the water-soluble drug ultrafine nanoparticles in the second step or the third method in the step (4) are suspended in the solution B to form an oil phase core together with the fatty acid for intravenous injection.
  • the water-soluble drug in the fourth step or the fifth method of the step (4) is in the aqueous phase with the albumin in the liquid C or on the surface of the nanoparticle, and the fatty acid for intravenous injection is the oil phase core.
  • the fatty acid-binding albumin-drug nanoparticle lyophilized preparation is composed only of albumin, a drug and an intravenously-derived fatty acid, and does not contain an organic solvent or any excipient, and its components and contents are in mass percentage: intravenous injection
  • the fatty acid is 0.0001% to 8%, preferably 0.001% to 1%
  • the albumin is 75% to 99%, preferably 80% to 95%
  • the drug is 0.1% to 25%, preferably 1.0% to 20%
  • the drug is at least one of a water-insoluble drug and a water-soluble drug.
  • the fatty acid-binding albumin-drug nanoparticle lyophilized preparation has the following biological properties:
  • the lyophilized preparation and Abraxane are reconstituted, and the integrity and stability of the nanoparticles are 136 to 160 hours and 96 hours, respectively, in an in vitro solution at 4 ° C;
  • the ratio of the concentration of the drug loaded in the lyophilized preparation into the gp60-recepting cell and the concentration of the same drug into the gp60-receptor-rich cell is 2-6;
  • the LD 50 of the lyophilized preparation loading paclitaxel is 44 to 56 times that of the paclitaxel intravenous injection LD 50 ;
  • the MTD of the lyophilized preparation loading paclitaxel is 53 to 71 times that of the intravenous injection of paclitaxel;
  • the lyophilized preparation reduces the antigenicity of the encapsulated or bound Pseudomonas exotoxin PE38 protein by 20 times.
  • Plasma albumin is a process in which the fatty acid is "extracted” and “enriched” in the oil phase, because the HSA solution, which is treated by ultracentrifugation at 30,000 rpm for 5 hours in an organic solvent or a mixture thereof, is a liquid. C, the stratification of the oil phase and the water phase can be seen, and the samples of the oil phase and the water phase are taken respectively, and the free fatty acid content and the HSA content are determined. It is found that more than 60% of the free fatty acids are distributed in the oil phase; The amount of organic solvent increases the total amount of fatty acids in the oil phase.
  • Nanoparticles having an average particle diameter of less than 180 nm having a particle diameter of 80 to 150 nm and 90 to 240 nm can be obtained.
  • the average particle size is less than 160nm nanometer
  • the content of albumin free fatty acid in the core region of the nanoparticle is more than three times that of the peripheral albumin free fatty acid of the nanoparticle, and each albumin molecule in the core region is bound to an average of 2 to 5 fatty acid molecules.
  • nanoparticles by FBA-SD/Ab and FBA-SD/Eb method shows that simple fatty acids can form nanoparticles, that is, in the oil phase of preparing nanoparticles, only the fatty acid can be added to form nanoparticles, and Such nanoparticles have no or slightly better in vitro stability than "oil-insoluble" drug nanoparticles such as paclitaxel in the oil phase core.
  • the basic materials of the above fatty acid-binding albumin-drug nano-lyophilized preparation such as "long-chain or medium-chain fatty acid for intravenous injection”, “human serum albumin”, “organic solution or a mixture thereof” and “drug”, etc.
  • the basic characteristics are as follows:
  • the "long-chain or medium-chain fatty acid for intravenous injection" has the following characteristics:
  • (1) is a long-chain or medium-chain fatty acid, such as oleic acid, linoleic acid, myristic acid, ethyl acetate; (2) is a component of a fat emulsion that can be intravenously infused into the human body; (3) is an intravenous grade (4) is compatible with the organic solution, and does not undergo any structural changes or chemical reactions; (5) can specifically bind to albumin.
  • oleic acid such as oleic acid, linoleic acid, myristic acid, ethyl acetate
  • (2) is a component of a fat emulsion that can be intravenously infused into the human body
  • (3) is an intravenous grade (4) is compatible with the organic solution, and does not undergo any structural changes or chemical reactions; (5) can specifically bind to albumin.
  • the "human serum albumin” of the present invention has the following characteristics:
  • an albumin that is "extracted” by an organic solvent or an organic solvent and an anhydrous ethanol mixture that is, an HSA of a "naked” fatty acid binding site
  • an HSA that maintains a complete physiological structure
  • no immunity Proto or antigenic including HSA loaded with "water-soluble” drugs
  • HSA that maintains the gp60 transmembrane mechanism may be obtained by blood extraction or by genetic recombination techniques.
  • the "organic solvent or a mixture thereof" according to the present invention has the following characteristics:
  • (1) is an organic solvent that is incompatible with water or only slightly soluble in water; (2) can dissolve fatty acids; (3) can dissolve, slightly dissolve or not dissolve drugs, but cannot chemically react with drugs or change drugs (4) does not chemically react with fatty acids or alter the nature or structure of fatty acids; (5) organic solutions or mixtures thereof and their treatment methods cannot affect or alter the structure of HSA and various in vivo and in vitro biological properties (6) is a chromatographically pure reagent.
  • the "drug” of the present invention has the following characteristics:
  • drug must be a “drug” that binds to HSA, such as binding to the HSA hydrophobic domain by hydrophobicity, or binding to an albumin hydrophilic group by hydrophilicity;
  • nanoparticle preparation techniques may be selected depending on the solubility of the drug in the two solution media:
  • Drugs soluble in organic solvents, insoluble or slightly soluble in water such as paclitaxel, docetaxel, rapamycin, curcumin, etoposide, mitomycin, vincristine, 7-ethyl- 10-hydroxycamptothecin, methotrexate, etc., may be used in the preparation method of FBA-ID/So according to the invention;
  • the preparation method of FBA-SD/Su can also select the preparation methods of FBA-SD/Ab and FBA-SD/Eb.
  • a drug insoluble in an organic solvent and insoluble in water such as methotrexate, may be selected from the preparation method of FBA-ID/Su according to the present invention.
  • the drug exists in a dissolved state or in a state of ultrafine nanoparticles (particle diameter ⁇ 50 nm), and both physical states can prepare oil-in-water type nanoparticles.
  • Nanoparticle analysis of various nano-formulations of the present invention at room temperature and 4 ° C showed that the integrity and stability of various FBA-Drug NPs in vitro were significantly better than albumin-bound paclitaxel (Abraxane) nanometers. particle.
  • HUVEC cells on the surface of the cell membrane rich in HSA transport receptor protein gp60 showed that the intracellular transport of various FBA-Drug NPs was significantly enhanced, so that the amount of drug entering HUVEC cells was about 2-6 higher than that of the corresponding drugs. This also indicates that the preparation methods of various nanoparticles have not affected the physiological and in vivo properties of HSA.
  • FBA-SD/Ab-Drug significantly reduced the antigenicity of PE38 toxin and greatly increased the dose of PE38LD 50 by about 60 times. Therefore, FBA-SD/Ab-PE38 has potential for human application development.
  • mice with various FBA-ID/So-P and Abraxane lyophilized preparations were significantly increased by 44-56 times and 53-71 times, respectively. There was no significant difference in LD 50 and MTD values between FBA-ID/So-P and Abraxane.
  • the preparation method of the invention can be applied to both water-insoluble drugs and water-soluble drugs, and can be used as a core of an oil phase in the form of a solution, or as a core of an oil phase in the form of ultrafine nanoparticles.
  • Nanoparticles are mainly formed by the specific binding between fatty acids and albumin; therefore, the formed nanoparticles have high integrity and stability in the in vitro solution and in the blood circulation of the body, and can be enriched by nanoscale.
  • the drug enters the tumor cells through the albumin gp60 receptor channel, which greatly increases the LD 50 and MTD of the loaded drug, thereby significantly improving the anticancer effect of the chemotherapy drug.
  • Figure 1 is a transmission electron microscope scan of FBA-ID/So-P1, where the left image shows the particle size distribution and the right image shows the shape of a single particle.
  • Figure 2 is a scanning electron microscope scan of FBA-ID-Do00, where the left image shows the particle size distribution and the right image shows the shape of a single particle.
  • Figure 3 is a scanning electron microscope scan of FBA-SD/C-D25, in which the left image shows the particle size distribution and the figure shows the shape of a single particle.
  • Figure 4 is a diagram showing the albumin circular dichroism of various nanoparticle lyophilized preparations, in which: ----HSA, decomposed albumin: ⁇ Abraxane, ⁇ FBA-ID/So-P-1, FBA-ID/So- M1, FBA-ID/So-V1, FBA-ID/Su-E25, FBA-ID/So-SN381FBA-ID/Su-MTX20 ⁇ FBA-SD/Su-D25, FBA-SD/Ab-EP20, FBA -ID/So-P&C, FBA-P/So&CP/Su, FBA-Do/So&PE38/Ab, FBA-R/So&D/Eb.
  • Figure 5 is a comparison of the binding rates of various FBA-ID/So-P to Taxol HUVEC cells.
  • Figure 6 is a pharmacodynamic test of FBA-ID/So-P nanoparticles and Abraxane tumor-bearing mice.
  • Figure 7 shows the results of in vitro transmembrane experiments of MCF-7 tumor cells.
  • Figure 8 is a comparison of FBA-SD-D nanoformulations with Doxil and doxorubicin to inhibit tumor growth.
  • FBA-ID/So method Preparation of Fatty-acid Binding Albumin (FBA)-Insoluble Drug (ID) Nanoparticle/Solution (So) Lyophilized Preparation (Nanoparticle, NP)
  • the method referred to as the FBA-ID/So method, includes the following steps:
  • HSA human serum albumin
  • water for injection prepare 10 ⁇ 200mg / ml, preferably 20mg ⁇ 80mg / ml HSA solution; use water-incompatible organic solvents such as chloroform (chloroform) , dichloromethane, ethyl acetate or a mixed solution thereof with ethanol (10-5:5 ⁇ 1, v/v), liquid A, "treatment" of albumin molecule-bound fatty acids, organic solvents or mixtures thereof with HSA
  • the volume ratio of the solution is from 0.3% to 15%, preferably from 0.5% to 5.0%; the specific method is that the liquid A is slowly dropped into the continuously stirred HSA solution at 5,000 to 15,000 rpm, preferably 8,000 to 10,000 rpm, and stirring is continued for 2 to 10 minutes.
  • liquid C is obtained for 5 ⁇ 1 minute.
  • This process known as “extraction” treatment of albumin, is the process of albumin-bound fatty acid extraction and enrichment in the oil phase (see
  • medium-chain or long-chain fatty acids for intravenous injection include oleic acid, linoleic acid, myristic acid, triglyceride, etc., which are components of a fat emulsion which can be intravenously infused into the human body;
  • water-insoluble drugs include paclitaxel and polyene.
  • paclitaxel, rapamycin, curcumin, mitomycin, vincristine, 7-ethyl-10-hydroxycamptothecin, etoposide and methotrexate are relatively water soluble Lower or very low chemotherapeutic drugs or other drugs, soluble in at least one "organic solvent or a mixture thereof", and can be combined with albumin; "organic solvent or a mixture thereof” is selected from chloroform (chloroform), Methylene chloride, ethyl acetate or a mixture thereof with absolute ethanol or the like, which is a chromatographically pure reagent; wherein a medium-chain or long-chain fatty acid for intravenous injection is dissolved in an organic solvent at a concentration of 0.01 mg/ml to 50 mg/ml or In the mixed solution, the "water-insoluble" drug is dissolved in an organic solvent or a mixture thereof at a concentration of 5 mg/ml to 600 mg/ml, preferably 10 mg/ml to 400 mg/
  • the liquid D is slowly dropped into the high-speed stirring liquid C at a ratio of the liquid D to the liquid C in a volume ratio of 0.1% to 7.2%, and the stirring speed is 3000 rpm to 30,000 rpm for a duration of 10 to 60 minutes; preferably, the stirring speed is 12,000 rpm to 30,000 rpm, duration of 10 to 20 minutes, high-speed homogenization treatment, to obtain a fatty acid-binding albumin-water-insoluble drug nanoparticle solution type suspension; this is a high-speed stirring method for preparing FBA-ID/So nanometer Particles, referred to as FBA-ID/So HS method.
  • the liquid D in a ratio of 0.1% to 7.2% by volume to the liquid C, slowly drip into the high-speed stirring liquid C at a stirring speed of 10,000 rpm for a duration of 5 ⁇ 1 minute;
  • the treated mixed suspension is quickly added to the high-pressure microfluidizer (M-110P Microfluidizer) feed vessel, and is subjected to high-pressure homogenization for 2 to 8 cycles in a high pressure of 6,000 psi to 30,000 psi, protected from light and 4 ° C.
  • a fatty acid-binding albumin-water-insoluble drug nanoparticle solution type suspension is prepared; this is a high-pressure homogenization method for preparing FBA-ID/So nanoparticles, referred to as FBA-ID/So HP method.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in which the water-insoluble drug is dissolved is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase.
  • the ratio of the volume of the oil phase to the volume of the aqueous phase is from 0.4% to 10.0%; preferably from 1.0% to 6.5%.
  • the mass ratio of the long-chain or medium-chain fatty acid to albumin for intravenous injection is 0.0001% to 5%, preferably 0.001% to 1%; and the mass ratio of the water-insoluble drug to albumin is 0.1% to 20%, preferably 1.0%. 10%.
  • the nanoparticle suspension was quickly placed in a vacuum rotary evaporator and rotary evaporated under reduced pressure (20 mmHg) at 35 ° C for 10-20 minutes to remove all organic solvents to obtain a translucent emulsion.
  • the translucent emulsion is filtered through a 0.45u and/or 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is vacuum-dried for 24 hours to obtain FBA-ID/So- After the FBA-ID/So-Drug was reconstituted with physiological saline, the particle size of the obtained FBA-ID/So-Drug solution nanoparticles was the same as that before the sterilized emulsion was lyophilized.
  • the preparation method of the fatty acid-binding albumin (FBA)-"water-soluble" drug (ID) nanoparticle/suspension type (Suspension, Su) lyophilized preparation (Nanoparticle, NP) of the present invention referred to as FBA- ID/Su method, the steps include:
  • a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof Dissolving a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof to obtain a solution B, wherein the medium-chain or long-chain fatty acid includes oleic acid, linoleic acid, myristic acid, triglyceride, etc.;
  • the mass ratio of fatty acid to albumin is 0.001% to 1%.
  • Preparation of "water-insoluble” drug ultra-fine nanoparticles various techniques and methods such as mechanical pulverization, ion sputtering, freeze-drying, laser-induced gas phase chemical reaction, etc. Both can be used in the present method, wherein “ultra-microparticles” refer to nanoparticles having a particle diameter of 50 nm or less (particle diameter ⁇ 50 nm).
  • the preparation method of the “ultra-micro-nanoparticle” disclosed by the invention is a high-pressure homogenization method, and the specific step is: mixing the “water-insoluble” drug at a concentration of 1 to 10 mg/ml to the drug incompatible with the drug.
  • the "water-insoluble” drug is selected from paclitaxel, docetaxel, rapamycin, curcumin, mitomycin, vincristine, 7-ethyl-10-hydroxycamptothecin, etoposide and methotrexate.
  • paclitaxel docetaxel
  • rapamycin curcumin
  • mitomycin vincristine
  • 7-ethyl-10-hydroxycamptothecin etoposide and methotrexate.
  • One or more kinds of hydrazine different "water-insoluble” drugs have different densities and solubilitys, and the volume of the solution required to be suspended in the "organic solvent or its mixture” is different.
  • the definition of "suspension” is more than the concentration range. The definition of "is more important.
  • the liquid D' is slowly dropped into the high-speed stirring liquid C at a ratio of 0.3% to 4.0% by volume with the liquid C, and the stirring speed is 3,000 rpm to 30,000 rpm for a duration of 15 to 60. Minutes, a fatty acid-binding albumin-water-insoluble drug nanoparticle suspension suspension was prepared; this was a high-speed stirring method for preparing FBA-ID/Su nanoparticles, referred to as FBA-ID/Su HS.
  • the liquid D' is slowly dropped into the high-speed stirring liquid C at a ratio of 0.3% to 4.0% by volume with the liquid C.
  • the stirring speed is 10,000 rpm to 12,000 rpm, and the stirring is continued for 5 ⁇ 1 minute.
  • High-pressure microfluidizer M-110P Microfluidizer
  • high-pressure homogenate treatment at 20,000 psi to 30,000 psi high pressure, protected from light at 4 ° C for 2 to 6 cycles to produce fatty acid-bound albumin - A water-insoluble drug nanoparticle suspension suspension; this is a high pressure homogenization method for preparing FBA-ID/Su nanoparticles, referred to as FBA-ID/Su HP method.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase
  • the ratio of volume to aqueous phase volume is: 0.6% to 5.0%
  • the mass ratio of long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.0001% to 0.15%
  • the mass ratio of water-insoluble drug to albumin is: 1.0%. ⁇ 25%, preferably 1.0% to 10%.
  • the particle size of the obtained FBA-SD/Su-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • the preparation method of the fatty acid-binding albumin (FBA)-"water-soluble” drug (SD) nanoparticle/suspension type (Suspension, Su) lyophilized preparation (Nanoparticle, NP) of the present invention referred to as FBA-
  • the SD/Su method the steps include:
  • a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof Dissolving a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof to obtain a solution B, wherein the medium-chain or long-chain fatty acid includes oleic acid, linoleic acid, myristic acid, triglyceride, etc.;
  • the mass ratio of medium or long chain fatty acids to albumin is 0.001% to 1%.
  • Preparation of "water-soluble” drug ultra-micro nanoparticles various techniques and methods such as mechanical pulverization, ion sputtering, freeze-drying, laser-induced gas phase chemical reaction, etc. Both can be used in the present method, wherein “ultra-microparticles” refer to nanoparticles having a particle diameter of 50 nm or less (particle diameter ⁇ 50 nm).
  • the invention discloses a preparation method of “ultra-micro-nanoparticles”, that is, a high-pressure homogenization method, and the specific step is: mixing a “water-soluble” drug at a concentration of 5-10 mg/ml into a drug incompatible with the drug.
  • soybean oil with a vitamin E content of 0.4 to 2 mg/ml, it is homogenized at 1 ° to 30,000 psi for 1 to 3 times at 4 ° C and protected from light to obtain a large amount of "water-soluble" drug ultra-fine nanoparticles.
  • the soybean oil suspension is centrifuged at 4 ° C, protected from light at 5,000 to 30,000 rpm for 5 to 60 minutes to remove the soybean oil supernatant, or ultrafiltration treated with an ultrafiltration membrane with a molecular weight cut off of 30 to 100 kDa.
  • Liquid D' is a suspension of fatty acid-containing, water-soluble drug ultra-micro nanoparticles and an organic solvent
  • water-soluble drugs include one or more of chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, and pseudomonas exotoxin (PE38);
  • the drug is soluble in water, and the solubility in the "organic solvent or a mixture thereof” is very low; the density of the different "water-soluble” drugs is different, and the volume of the solution required to be suspended in the "organic solvent or a mixture thereof”
  • the definition of "suspension” is more important than the definition of "concentration”.
  • liquid C wherein the concentration of the HSA solution is 10 to 200 mg/ml, preferably 20 mg to 80 mg/ml;
  • the liquid D' is dropped into the liquid C at a stirring speed of 12,000 rpm to 22,000 rpm in a ratio of 1.5% to 2.0% by volume, and stirring is continued for 5 to 20 minutes to obtain a fatty acid-binding albumin-water-soluble drug nanometer.
  • the particle suspension type suspension is a high-speed stirring method for preparing FBA-SD/Su nanoparticles, referred to as FBA-SD/Su HS method.
  • the liquid D' is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm, stirring is continued for 5 ⁇ 1 minute, and a high pressure microfluidizer (M-110P Microfluidizer) is added.
  • M-110P Microfluidizer M-110P Microfluidizer
  • a feed container at a temperature of 4 ⁇ 1°C, protected from light at a pressure of 20,000 psi to 30,000 psi, and subjected to high-pressure homogenization for 2 to 6 cycles to obtain a fatty acid-binding albumin-water-soluble drug nanoparticle suspension type. Suspension; this is a high pressure homogenization method to prepare FBA-SD/Su nanoparticles, referred to as FBA-SD/Su HP method.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B in the liquid D' is the oil phase
  • the volume of the aqueous albumin solution in the liquid C is the aqueous phase
  • the ratio of phase volume to aqueous phase volume is: 1.0% to 7.5%
  • the mass ratio of long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.001% to 1.0%
  • the mass ratio of water-soluble drug to albumin is: 0.5 % ⁇ 20%;
  • the particle size of the obtained FBA-SD/Su-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • the preparation method of the fatty acid-binding albumin (FBA)-"water-soluble” drug (SD) nanoparticle/protein-binding type (Albumin Binding, Ab) lyophilized preparation of the present invention referred to as FBA-SD/Ab method, the steps include :
  • a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof to obtain a solution B, wherein the medium-chain or long-chain fatty acid includes oleic acid, linoleic acid, myristic acid, triglyceride, etc.;
  • the mass ratio of medium or long-chain fatty acids to albumin for intravenous injection is 0.001% to 1%.
  • doxorubicin (D) or epirubicin (Epirubicin, EP) and albumin binding solution use 0.01M ⁇ 0.1M sodium hydroxide to adjust 20 ⁇ 100mg / The pH of the ml HSA solution is 5.5-6.5; 2mg/ml doxorubicin (D) or epirubicin (Epirubicin, EP) is prepared by using water for injection to obtain a high concentration of D or EP solution; The HSA solution was slowly added to the HSA solution and magnetically stirred at 1,000 rpm ⁇ 100 rpm for 2 hours to obtain an HSA-doxorubicin or HSA-episomycin binding solution.
  • Water-soluble drugs include one or more of chemical, protein or polypeptide drugs such as doxorubicin, epirubicin, carboplatin, and pseudomonas exotoxin (PE38). Different "water-soluble” drugs have different binding rates to albumin in vitro. Here, the "binding" concentration of "water-soluble” drugs with albumin in liquid C and the concentration of albumin solution determine the amount of "water-soluble” drug added. Or concentration.
  • the solution B is dropped into the liquid C' at a stirring speed of 15,000 rpm to 25,000 rpm in a volume ratio of 0.8% to 7.5%, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-soluble drug nanometer.
  • Particle protein-bound suspension this is a high-speed stirring method for preparing FBA-SD/Ab nanoparticles, referred to as FBA-SD/Ab HS.
  • the solution B is dropped into a liquid C' at a stirring speed of 10,000 rpm to 12,000 rpm in a volume ratio of 0.8% to 7.5%, stirring is continued for 5 ⁇ 1 minute, and a high pressure microfluidizer (M-110P Microfluidizer) is added.
  • M-110P Microfluidizer M-110P Microfluidizer
  • In the feed container at a temperature of 4 ⁇ 1 ° C, protected from light, at a pressure of 10,000 psi to 25,000 psi, continuous high-pressure homogenization for 1 to 5 cycles to obtain a fatty acid-binding albumin-water-soluble drug nanoparticle protein combination Type suspension; this is a high pressure homogenization method to prepare FBA-SD/Ab nanoparticles, referred to as FBA-SD/Ab HP method.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B is the oil phase
  • the volume of the albumin-water-soluble drug binding solution is the aqueous phase, the volume of the oil phase.
  • the ratio of the volume to the aqueous phase is: 1.0% to 10.0%
  • the mass ratio of the long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.01% to 5.0%
  • the mass ratio of the water-soluble drug to albumin is 0.2%. 20%;
  • the particle size of the obtained FBA-SD/Ab-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • FBA-SD/Eb method The preparation method of the fatty acid-binding albumin (FBA)-"water-soluble" drug (SD) nanoparticle/Empty-Nanoparticle Binding (Eb) lyophilized preparation of the present invention, referred to as FBA-SD/Eb method
  • the steps include:
  • a medium-chain or long-chain fatty acid for intravenous injection in an organic solvent or a mixture thereof to obtain a solution B, wherein the medium-chain or long-chain fatty acid includes oleic acid, linoleic acid, myristic acid, triglyceride, etc.;
  • the mass ratio of medium or long-chain fatty acids to albumin for intravenous injection is 0.001% to 1%.
  • the aqueous phase is composed of liquid C, wherein the concentration of the HSA solution is 10 to 200 mg/ml, preferably 20 mg to 80 mg/ml;
  • the solution B is dropped into the liquid C at a stirring speed of 20,000 rpm to 25,000 rpm in a ratio of 1.4% to 3.3% by volume, and stirring is continued for 10 to 20 minutes to prepare a fatty acid-bound albumin nanoparticle suspension;
  • This is a high-speed homogenization method for preparing FBA-SD/Eb nanoparticles, referred to as FBA-SD/Eb HS method.
  • the solution B is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 1.4% to 3.3% by volume, and stirring is continued for 5 ⁇ 1 minute, and then added to the high-pressure microjet feeding container. , at 4 ⁇ 1°C, protected from light, at a pressure of 10,000 psi to 25,000 psi, and subjected to high-pressure homogenization for 2 to 6 cycles to obtain a fatty acid-bound albumin nanoparticle suspension; this is a high-pressure homogenization method.
  • FBA-SD/Eb HP method Preparation of FBA-SD/Eb nanoparticles
  • the fatty acid-bound albumin nanoparticle suspension is placed in a vacuum rotary evaporator, and subjected to rotary evaporation at 35 ⁇ 2° C. and 20 mmHg at 60 to 80 rpm for 20 to 30 minutes to remove all organic solvents.
  • the water-soluble drug includes doxorubicin, epirubicin, carboplatin, Pseudomonas exotoxin PE38 and the like.
  • Different "water-soluble” drugs have different binding rates to albumin, and the "quantity" of FBA-Enp surface albumin of different particle sizes is different.
  • the "water-soluble” drug is “bound” with FBA-Enp surface albumin. The concentration determines the amount of added or added concentration of the "water soluble” drug.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B is the oil phase
  • the volume of the translucent emulsion is the aqueous phase
  • the ratio of the volume of the oil phase to the volume of the aqueous phase is: 2.0% to 6.5%
  • the mass ratio of long-chain or medium-chain fatty acid to albumin for intravenous injection is 0.033% to 0.5%
  • the mass ratio of water-soluble drug to albumin is 2.5% to 15%;
  • the translucent emulsion is filtered through a 0.45u and/or 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is vacuum-dried for 24 hours to obtain FBA-SD/Eb- After the FBA-SD/Eb-Drug was reconstituted with physiological saline or water for injection, the particle size of the obtained FBA-SD/Eb-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • the preparation method of the fatty acid-binding albumin-"water-insoluble” and “water-soluble” drug nanoparticle/suspension lyophilized preparation of the present invention referred to as FBA-ID/So&SD/Su method, is the aforementioned FBA-ID/So
  • the steps include:
  • liquid C it is composed of an HSA solution of 10 to 200 mg/ml, preferably 20 mg to 80 mg/ml;
  • the drug, the water-soluble drug, the ultrafine nanoparticles are suspended in a liquid C at a stirring speed of 3,000 rpm to 30,000 rpm, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-insoluble and water-soluble drug nanoparticle.
  • Suspension suspension ie FBA-ID/So&SD/Su nanoparticle suspension;
  • the water-soluble drug ultrafine nanoparticles are suspended by the liquid D to obtain a suspension containing the fatty acid, the water-insoluble drug, and the water-soluble drug ultrafine nanoparticles, and the fatty acid is contained in a ratio of 0.1% to 7.2% by volume.
  • the water-insoluble drug, the suspension of the water-soluble drug ultra-micron particles is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm, stirring is continued for 5 ⁇ 1 minute, and added to the high-pressure microjet feeding container at 4 ⁇ 1 °C, protected from light, at a pressure of 3,000 psi to 40,000 psi, continuous 2 to 8 cycles of high-pressure homogenization to obtain a fatty acid-binding albumin-water-insoluble and water-soluble drug nanoparticle suspension suspension, ie FBA -ID/So&SD/Su nanoparticle suspension;
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B constitutes the oil phase
  • the volume of the HSA solution constitutes the aqueous phase of the system
  • the volume of the oil phase and The ratio of the volume of the aqueous phase is: 3.5%
  • the mass ratio of the long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.033%
  • the mass ratio of the "water-soluble" drug to albumin is: 3.33%
  • water-insoluble The mass ratio of drug to albumin was: 3.33%.
  • the particle size of the obtained FBA-ID/So&SD/Su-Drug solution was the same as that before the sterilized emulsion was freeze-dried.
  • the preparation method of the fatty acid-binding albumin-"water-insoluble” and “water-soluble” drug nanoparticle/protein-binding lyophilized preparation of the present invention referred to as FBA-ID/So&SD/Ab method, is the aforementioned FBA-ID/So Combined with the preparation methods of FBA-SD/Ab, the specific steps include:
  • the medium-chain or long-chain fatty acid for intravenous injection is dissolved in liquid A to obtain solution B, and then solution B is used to dissolve the water-insoluble drug, and the concentration is obtained.
  • the liquid D is dropped into the liquid C' at a stirring speed of 15,000 rpm to 25,000 rpm in a ratio of 0.1% to 7.2%, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-insoluble and water-soluble.
  • Drug nanoparticle protein-binding suspension ie FBA-ID/So&SD/Ab nanoparticle suspension;
  • the liquid D is dropped into the liquid C' at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 0.1% to 7.2%, and the stirring is continued for 2 to 5 minutes, and added to the high-pressure microjet feeding container.
  • a stirring speed 10,000 rpm to 12,000 rpm in a ratio of 0.1% to 7.2%
  • the stirring is continued for 2 to 5 minutes, and added to the high-pressure microjet feeding container.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B constitutes the oil phase of the system
  • the volume of the liquid C constitutes the aqueous phase of the system.
  • the ratio of the volume of the oil phase to the volume of the aqueous phase is: 4.5%
  • the mass ratio of the long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.083%
  • the mass ratio of the "water-soluble" drug to albumin is 0.083. %
  • the mass ratio of "water insoluble” drug to albumin is: 3.33%.
  • the particle size of the obtained FBA-ID/So&SD/Ab-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • the preparation method of the fatty acid-binding albumin-"water-insoluble” and “water-soluble” drug nanoparticle/empty particle-bound lyophilized preparation of the present invention abbreviated as FBA-ID/So&SD/Eb method, the aforementioned FBA-ID/So Combined with FBA-SD/Eb two particle preparation methods, the specific steps include:
  • solution D having a concentration of 10 mg/ml to 400 mg/ml;
  • the liquid D is dropped into the liquid C at a stirring speed of 3,000 rpm to 30,000 rpm in a ratio of 0.1% to 7.2%, and stirring is continued for 10 to 60 minutes to obtain a fatty acid-binding albumin-water-insoluble drug nanoparticle.
  • Solution type suspension
  • the liquid D is dropped into the liquid C at a stirring speed of 10,000 rpm to 12,000 rpm in a ratio of 0.1% to 7.2%, and stirring is continued for 5 ⁇ 1 minute, and added to the high-pressure microjet feeding container. 4 ⁇ 1°C, protected from light, at a pressure of 6,000 psi to 40,000 psi, and subjected to high-pressure homogenization for 2 to 8 cycles to obtain a fatty acid-binding albumin-water-insoluble drug nanoparticle solution type suspension;
  • the fatty acid-binding albumin-water-insoluble drug nanoparticle solution type suspension is placed in a vacuum rotary evaporator, and subjected to rotary evaporation at 60 ⁇ 80° C. for 20 to 30 minutes under a reduced pressure of 35 ⁇ 2° C. and 20 mmHg for 20 to 30 minutes. All organic solvents, obtained a translucent emulsion;
  • the FBA-ID/So nanoparticle surface albumin binds to produce a fatty acid-binding albumin-water-insoluble and water-soluble drug nanoparticle granule-bound emulsion, namely FBA-ID/So&SD/Eb-Drug emulsion.
  • the sum of the volume of liquid A in liquid C and the volume of solution B constitutes the oil phase of the system, and the final volume composition of the FBA-ID/So nanoparticle emulsion
  • the aqueous phase of the system wherein the ratio of the volume of the oil phase to the volume of the aqueous phase is: 7.5%, and the mass ratio of the long-chain or medium-chain fatty acid to the albumin for intravenous injection is 0.033%, "water-soluble" drug and albumin The mass ratio was: 3.33%; the mass ratio of "water insoluble” drug to albumin was: 3.33%.
  • the particle size of the obtained FBA-ID/So&SD/Eb-Drug solution nanoparticles was the same as that before the sterilized emulsion was freeze-dried.
  • Liquid D1&2; medium and long chain fatty acids include oleic acid, linoleic acid, myristic acid, triglycerides, etc.
  • water insoluble drugs include paclitaxel, docetaxel, rapamycin, curcumin, and mitogen Any combination of two kinds, such as vinorelbine, vincristine, 7-ethyl-10-hydroxycamptothecin, etoposide and methotrexate, "organic solvent or a mixture thereof” such as chloroform (chloroform), Methyl chloride, ethyl acetate or a mixed solution thereof with absolute ethanol; wherein the concentration of medium or long-chain fatty acids for intravenous injection is from 0.01 mg/ml to 50 mg/ml, and the sum of the concentrations of the two "water-insoluble” drugs It is from 5 mg/ml to 600 mg/ml, preferably from 10 mg/ml to 400 mg/ml.
  • the liquid D1 & 2 is slowly dropped into the high-speed stirring solution C at a ratio of 0.1% to 7.2% by volume to the solution C, stirred at 3,000 rpm to 30,000 rpm, and continuously stirred for 10 to 60 minutes to obtain a fatty acid-binding albumin.
  • the liquid D1 & 2 is slowly dropped into the high-speed stirring liquid C at a ratio of 5% to 6.5% by volume to the solution C at a stirring speed of 10,000 rpm for a duration of 5 ⁇ 1 minute;
  • the mixed solution is added to the feed port of the high-pressure micro-jet, and is circulated 5-8 times at 4 ° C under the pressure of 20,000 psi to 30,000 psi; the fatty acid-binding albumin-"water-insoluble" drug D1&D2 nanoparticles are obtained.
  • the sum of the volume of the liquid A in the liquid C and the volume of the solution B constitutes the oil phase of the system, and the volume composition of the aqueous albumin solution in the liquid C
  • the aqueous phase of the system, the ratio of the volume of the oil phase to the volume of the aqueous phase is: 6.5% to 11.5%; the mass ratio of the long-chain or medium-chain fatty acid for intravenous injection to albumin is 0.05% to 0.4%; two kinds of "water insoluble"
  • the mass ratio of the sum of the drug to the albumin is 5% to 10%.
  • the particle size of the obtained FBA-ID/So-D1&D2 solution nanoparticles was the same as that before the sterilized emulsion was lyophilized.
  • a preparation method of various nanoparticle lyophilized preparations such as FBA-ID/So&ID/Su method and the method thereof can be evolved or derived The products are not repeated one by one.
  • the preparation method of preparing a "drug" nanoparticle lyophilized preparation as described above is used as a basic method, and is applied to two or more kinds of the same kind of drugs alone, or combinedly applied to two or two kinds of the above two basic methods.
  • the above-mentioned similar or different types of drugs ie, "water-insoluble” and “water-soluble” drugs
  • the preparation of fatty acid-binding albumin - two or more drug nanoparticle lyophilized preparations are all applicable, and are all within the scope of the present invention.
  • the process of treating serum albumin solution with organic solvent or its mixture is the process of "extracting” and “enriching” fatty acids in the oil phase.
  • HSA or BSA extracted from human blood or bovine blood contains fatty acids, and different HSA or BSA extraction methods affect the fatty acid content.
  • the HSA solution treated with the organic solvent or a mixture thereof is subjected to ultracentrifugation at 30,000 rpm for 5 hours, and the organic phase (ie, the oil phase) and the albumin solution phase (ie, the aqueous phase) are separated, and the oil phase and the water phase are respectively taken.
  • the sample was determined by enzymatic method (Cayman product) to determine the content of free fatty acid, and the content of HSA was determined by Lowry's method.
  • HSA HSA (10% HSA, 50 ml) of Hualan Biological was weighed, and water for injection was added to prepare 10 ml of 50 mg/ml HSA solution; a mixed solution of chloroform and ethanol (8:3, v/) v) 100 ul, slowly instilled into a 50 mg/ml HSA solution at 5,000 rpm for 10 minutes.
  • Different doses of fatty acids such as myristic acid 0 (not added), 1 mg, 2 mg and 4 mg and paclitaxel 100 mg were dissolved in 200 ul of a mixture of chloroform and ethanol, and this mixture containing myristic acid, paclitaxel, chloroform and ethanol was separately added.
  • the solution was slowly dropped into a high-speed stirred "extracted” 50 mg/ml HSA solution at a stirring speed of 10,000 rpm for a duration of 5 minutes; this mixed solution was passed through a high pressure microfluidizer (M-110P Microfluidizer).
  • the fatty acid was added with 1 mg and 2 mg of myristic acid as described above, and nanoparticles having an average particle diameter of less than 180 nm having a particle diameter of 80 to 150 nm and 90 to 240 nm were obtained (see Table 2 for details).
  • the low or high fatty acid content exceeds the solubility of the organic solution and affects the formation of nanoparticles.
  • the HSA was purified by conventional ion exchange chromatography to reduce the fatty acid content of the HSA solution to less than 0.1 ug/mg HSA, and no fatty acid (such as oleic acid or linoleic acid) was added to the oil phase.
  • no fatty acid such as oleic acid or linoleic acid
  • FBA-Drug nanoparticles cannot be formed at all. This indicates that fatty acids are an indispensable and important constituent of the FBA-Drug nanoparticle formation mechanism.
  • the ultrafiltration technique was used to separate the albumin in the core region of the nanoparticle, ie albumin and nanoparticle peripheral albumin directly bound to the nanocore paclitaxel and/or fatty acid, and the content of free fatty acid was determined respectively. It was found that the nanoparticle solution having an average particle diameter of less than 160 nm prepared by the technique of the present invention was ultrafiltered by a 100 kDa molecular weight cut-off ultrafiltration tube (Millipore) at 5000 rpm for 20 minutes, and the ultrafiltrate and the filter membrane were separately collected.
  • Millipore molecular weight cut-off ultrafiltration tube
  • the concentrate was quantified by the Lowry's method and the enzymatic method (Cayman's product) to quantify albumin and free fatty acids, and the content of free fatty acids per mg of protein was calculated, and the common feature was found to be: the core region of the nanoparticles.
  • the content of free fatty acids is more than three times that of the peripheral albumin free fatty acids of the nanoparticles, and each albumin molecule in the core region binds an average of 2 to 5 fatty acid molecules.
  • FBA-ID/So-Drug and FBA-SD/Su-Drug nanoparticles show that simple fatty acids can form nanoparticles, such as implementation As shown in Example 4 (FBA), 9 (FBA-ID/So-Do00), and 19 (FBA-SD/Ab-D0), in the oil phase in which the nanoparticles are prepared, only the fatty acid is added to form nanoparticles, and The nanoparticles have no or slightly better in vitro stability than the nanoparticles formed by the core containing paclitaxel or peripheral albumin combined with doxorubicin.
  • Circular dichroism spectroscopy is a fast, simple and accurate method for studying protein conformation in dilute solutions. It can be measured in solution state and close to physiological state, and is sensitive to conformational changes. Therefore, it is currently studying the secondary structure of proteins.
  • One of the main means Comparison of Hualan biological HSA, Abraxane decomposition obtained HAS and FBA-Drug NP obtained by various preparation methods described in the present invention, such as Example 7FBA-ID/So-P7-1 and Example 15FBA-SD/Su-D25 sample.
  • the circular dichroism spectrum of the HSA obtained after decomposing did not show any significant change, indicating that different preparation methods did not significantly affect the secondary structure of albumin.
  • the scanning results are detailed in Example 27.
  • the cell membrane surface of human umbilical vascular endothelial cells is rich in gp60 receptor and is a physiological transport channel of HSA.
  • Fluorescently labeled paclitaxel was added to paclitaxel at a ratio of 1:50 to prepare various FBA-ID/So-P nanoparticles, namely F-FBA-ID/So-P; fluorescently labeled paclitaxel injection was prepared at the same ratio of addition. Liquid, F-Taxol.
  • ELISA was used to determine the concentration of various FBA-ID/So-P in the form of intact nanoparticles in the blood circulation as an indicator for determining its stability in animals, ie, the anti-embedding at the bottom of the 96-well plate.
  • Paclitaxel antibody captures FBA-ID/So-P in serum, and then the content of "FBA-ID/So-P" is detected by biotin-labeled anti-HSA antibody, and the plasma half-life is calculated. The content of paclitaxel in the tissue is determined by HPLC.
  • FBA-SD/Ab significantly reduces the antigenicity of the highly antigenic PE38 toxin, and also greatly increases the dose of PE38LD 50 by about 60 times. Therefore, FBA-SD/Ab-PE38 is developed for human body application. Potential nano-formulations.
  • the preparation method of the fatty acid-binding albumin-drug nano-lyophilized preparation (FBA-Drug NP) of the invention effectively utilizes the physiological, high-affinity and stable binding characteristics of fatty acids and albumin, in high shear force
  • the realization of "water-insoluble” or “water-soluble” drugs, in the form of "solutions” or “ultra-micro-particles", one or several "packages” or “"Bound” greatly expands the application range of nanoparticle preparation methods using albumin as the main scaffold material
  • the obtained FBA-Drug NP is composed only of albumin, drugs and fatty acids, and does not contain any other excipients or organic solvents
  • the obtained FBA-Drug NP, the integrity and stability of the blood circulation and in vitro solution in the body is significantly improved, and the content of chemotherapeutic drugs in the cells or tumor tissues rich in gp60 receptors is greatly improved by the physiological transport mechanism of H
  • FBA Fatty-binding Binding Albumin
  • ID Insoluble Drug
  • So Solution lyophilized preparation method
  • the stirring speed was 3000 rpm for a duration of 60 minutes; this yellow suspension was quickly placed in a vacuum rotary evaporator and rotary evaporated under reduced pressure (20 mmHg) for 15 minutes at 35 ° C to remove all organic solvents.
  • a yellow emulsion is obtained in which the curcumin particles have a particle size of 52 to 116 nanometers (nm) (Malvern Nano Zetasizer ZS90, UK).
  • the yellow emulsion is filtered through a 0.22u filter membrane to remove microorganisms such as bacteria in the solution to form a sterile solution; the sterile solution is freeze-dried by vacuum for 24 hours to obtain a fatty acid-binding albumin-curcumin nanoparticle. Lyophilized formulation (FBA-ID/So-C).
  • the translucent emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is vacuum-dried for 24 hours to obtain a fatty acid-binding albumin-repa Lycopene nanoparticle lyophilized preparation (FBA-ID/So-R); after reconstitution of FBA-ID/So-R plus normal saline or water for injection, the particle size of the obtained rapamycin nanoparticles is consistent with that before lyophilization .
  • FBA-ID/So-R fatty acid-binding albumin-repa Lycopene nanoparticle lyophilized preparation
  • Example 3 Preparation of paclitaxel (P) nanoparticle lyophilized preparation by FBA-ID/So method (FBA-ID/So-P)
  • the stirring speed is 20,000 rpm for 10 minutes (this is High Speed Homogenizing, HS); or the meat is contained.
  • a mixed solution of myristic acid, paclitaxel, chloroform and ethanol was slowly dropped into a 100 mg/ml HSA solution of a high-speed stirred mixed solution of chloroform and ethanol at a stirring speed of 10,000 rpm for 5 minutes, and the mixture was further mixed.
  • the high-pressure microfluidizer (M-110P Microfluidizer) was cycled 6 times at 4 ° C under a pressure of 20,000 psi (this is High Pressure Homogenizing, HP); the obtained suspension was quickly placed in a vacuum In a rotary evaporator, under a reduced pressure of 35 ° C (20 mmHg) rotary evaporation for 10 minutes, remove all organic solvents, a translucent emulsion can be obtained, wherein the paclitaxel nanoparticles have a particle size of 73 ⁇ 221 nm;
  • the translucent emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is freeze-dried by vacuum for 24 hours to obtain a fatty acid-binding albumin-paclitaxel nanometer.
  • the lyophilized preparation FBA-ID/So-P-1; after reconstitution of FBA-ID/So-P-1 plus physiological saline or water for injection, the particle size of the obtained paclitaxel nanoparticles was the same as that before lyophilization.
  • Example 4 Preparation of paclitaxel nanoparticle lyophilized preparation by FBA-ID/So method (FBA-ID/So-P)
  • the mixed solution containing triglyceride, paclitaxel, and ethyl acetate or the mixed solution containing triglyceride and ethyl acetate was slowly dropped into a high-speed stirred "ethyl acetate"-treated 200 mg/ml HSA solution.
  • the stirring speed is 30,000 rpm and the duration is 10 minutes; the obtained two suspensions are quickly placed in a vacuum rotary evaporator, and rotated under a reduced pressure (20 mmHg) at 35 ° C for 20 minutes to remove all organic solvents.
  • Two kinds of translucent emulsions wherein the paclitaxel nanoparticles have a particle diameter of 83 to 255 nm, and the paclitaxel-free nanoparticles have a particle diameter of 57 to 181 nm.
  • the two translucent emulsions are filtered through a 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is freeze-dried by vacuum for 24 hours to obtain a fatty acid-binding albumin-paclitaxel nanoparticle jelly.
  • Dry preparation FBA-ID/So-P-3
  • fatty acid-bound albumin nanoparticle lyophilized preparation FBA
  • FBA and FBA-ID/So-P-3 after reconstitution with physiological saline or water for injection, The particle size of the nanoparticles was the same as before lyophilization.
  • Example 5 Organic solvent "treatment” distribution of albumin and fatty acids in oil and water phases
  • the samples S1 to S4 in the foregoing Examples 1 to 4 were each subjected to ultracentrifugation at 30,000 rpm for 5 hours at 8 ° C, and the organic phase (ie, the oil phase) and the albumin phase (ie, the aqueous phase) were layered and sampled, respectively.
  • the free fatty acid content of the oil phase and the aqueous phase was determined by an enzymatic method (Cayman product), and the content of HSA was determined by the Lowry's method.
  • the results are shown in Table 1; the organic solvent treatment, such as the S1-1 sample, was added to the sample of Example 1S1.
  • the S1-2 sample was separately added with 450 ul of chloroform and ethanol and 600 ul (9:1, v/v).
  • the organic solvent treatment of HSA is actually a process of “extracting and enriching fatty acids” (hereinafter referred to as “extraction” treatment), and more than 60% of the treated free fatty acids are distributed in an organic solvent, ie In the oil phase; and based on the "extraction” treatment, according to the preparation method of the nanoparticles, simply adding an organic solvent (without fatty acid and paclitaxel) to "treat” HSA can increase the content or proportion of fatty acids in the oil phase. .
  • Example 6 Effect of adding different doses of fatty acids on the particle size of FBA-ID/So-P
  • the above translucent emulsion was filtered through a 0.22 u filter membrane to remove microorganisms such as bacteria in the emulsion to form a sterile emulsion; the sterile emulsion was vacuum-dried for 24 hours to obtain a FBA-ID/So-P lyophilized preparation.
  • Example 7 Relationship between albumin fatty acid content and free fatty acid addition and formation of FBA-ID/So-P nanoparticles
  • the enzymatic method (Cayman product) determined the free fatty acid content of Sigma HSA and Hualan biological HSA in 100 mg/ml albumin solution. The results showed that the difference was about 10 times, which were 5.4 ug/mg and 0.55 ug/mg albumin, respectively. .
  • the stirring speed was 10,000 rpm, and the stirring was continued for 5 minutes; the mixed solution was subjected to a high pressure micro-jet machine under a pressure of 20,000 psi at 4 ° C for 8 times; The suspension was quickly placed in a vacuum rotary evaporator and rotary evaporated at 35 ° C under reduced pressure (20 mmHg) for 10 minutes to remove all organic solvents to obtain a translucent emulsion.
  • the paclitaxel nanoparticles were 62-240 nm in average (average The particle size was 160 nm, designated as FBA-ID/So-P7-1) and 113-358 nm (average particle size 293 nm).
  • Example 8 Comparison of core and peripheral albumin free fatty acid content of FBA-ID/So-P nanoparticles
  • nanoparticle solution Z-ave ⁇ 160 nm nanoparticles having an average particle diameter of less than 160 nm, that is, FBA-ID/So-P1, FBA-ID/So-P7-1, and FBA-ID/So-P7-22 ml
  • 100kDa molecular weight cut-off ultrafiltration tube Millipore
  • the ultrafiltrate and the filter membrane concentrate were recovered, and the albumin and free fatty acid content were determined by Lowry's method and enzymatic method (Cayman product). According to this, the content of free fatty acids per mg of protein is calculated.
  • the common feature is that the content of albumin free fatty acids in the core region of FBA-ID/So-P nanoparticles exceeds FBA-ID. /So-P nanoparticles are more than 3 times higher than the peripheral albumin free fatty acids, ie Core/Outer FA is greater than 3; and the molar ratio of free fatty acids in the core region to the molar concentration of albumin (ie FA/A mol, ) is 2-5: 1, that is, each albumin molecule in the core region is bound to an average of 2 to 5 fatty acid molecules.
  • Example 9 Effect of different doses of docetaxel on particle size of FBA-ID/So-Do
  • the obtained translucent emulsion wherein the docetaxel nanoparticles have a particle diameter of 118 to 286 nm, 103 to 254 nm, 95 to 227 nm, and 81 to 157 nm, respectively. See Table 4 for details.
  • the above translucent emulsion was filtered through a 0.22 u filter membrane to remove microorganisms such as bacteria in the emulsion to form a sterile emulsion; the sterile emulsion was vacuum-dried for 24 hours to obtain a FBA-ID/So-Do lyophilized preparation.
  • Example 10 Effect of adding different volumes of organic solvents on the particle size of FBA-ID/So-mitomycin nanoparticles
  • the stirring speed was 10,000 rpm and the duration was 5 minutes; the mixed solution was subjected to a high pressure micro-jet machine and circulated 3 times at 4 ° C under a pressure of 30,000 psi; the obtained suspension was quickly placed in a vacuum rotary evaporator.
  • the organic solvent was removed by rotary evaporation under reduced pressure (20 mmHg) at 35 ° C for 30 minutes; the obtained translucent emulsion was determined, wherein the diameters of the mitomycin nanoparticles were 54 to 183 nm, 108 to 263 nm and 53 to 351 nm, respectively. .
  • Example 12 Preparation of 7-Ethyl-10-hydroxyl-camptothecin (SN38) nanoparticle lyophilized preparation by FBA-ID/So method (FBA-ID/So-SN38)
  • the stirring speed was 22,000 rpm at 4 ° C for 10 minutes in the dark; the obtained suspensions were quickly placed in a vacuum rotary evaporator and rotary evaporated under reduced pressure (20 mmHg) at 35 ° C. Minutes, all organic solvents were removed; the obtained translucent emulsion was determined, wherein the particle size of the SN38 nanoparticles was FBA-ID/So-SN 38155-153 nm and FBA-ID/So-SN 38572-213 nm, respectively.
  • the above translucent emulsion was filtered through a 0.22 u filter membrane to remove microorganisms such as bacteria in the emulsion to form a sterile emulsion; the sterile emulsion was vacuum-dried for 24 hours to obtain a FBA-ID/So-SN38 lyophilized preparation.
  • Example 13 Preparation of two "water-insoluble" drug nanoparticle lyophilized preparations of paclitaxel (P) and curcumin (Curcumin, C) by FBA-ID/So method (FBA-ID/So-P&C)
  • the translucent emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria in the emulsion to form a sterile emulsion; the sterile emulsion is freeze-dried by vacuum for 30 hours to obtain a fatty acid-binding albumin-paclitaxel- Curcumin nanoparticle lyophilized preparation (FBA-ID/So-P&C); after reconstitution of FBA-ID/So-P&C plus saline or water for injection, the particle size of the obtained paclitaxel-curcumin nanoparticles is the same as before lyophilization .
  • FBA-ID/So-P&C fatty acid-binding albumin-paclitaxel- Curcumin nanoparticle lyophilized preparation
  • Example 14 Preparation of two "water-insoluble" drug nanoparticle lyophilized preparations of paclitaxel and rapamycin (R) by FBA-ID/So method (FBA-ID/So-P&R)
  • liquid D was slowly dropped into a high-speed stirred "extracted” treated 100 mg/ml HSA solution at a stirring speed of 10,000 rpm for a duration of 5 minutes; this mixture was passed through a high pressure microfluidizer at a pressure of 30,000 psi.
  • the solution was circulated 8 times at 4 ° C; the suspension was quickly placed in a vacuum rotary evaporator, and rotary evaporated at 35 ° C under reduced pressure (20 mmHg) for 30 minutes to remove all organic solvents to obtain a translucent emulsion with a laser particle size.
  • the particle size of the nanoparticles in the obtained emulsion was measured and found to be 72 to 230 nm.
  • the emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; freeze-dried under vacuum for 36 hours to obtain a fatty acid-binding albumin-paclitaxel-rapamycin nanoparticle.
  • the lyophilized preparation FBA-ID/So-P&R
  • the obtained nanoparticles have a particle diameter of 69 to 225 nm, which is the same as before lyophilization.
  • Examples 13 and 14 are those in which the aforementioned FBA-ID/So is applied to the preparation of two "water-insoluble" drugs.
  • Example 15 Fatty acid-binding albumin (FBA)-"Soluble Drug (SD) Nanoparticle/Suspension (Su) lyophilized preparation method (referred to as FBA-SD/Su method)
  • doxorubicin (D) 5mg, 25mg and 50mg doxorubicin (D) were mixed in 8ml of pharmaceutical grade soybean oil (vitamin E content 0.4 ⁇ 2mg/ml); at 4°C, protected from light at 15,000psi
  • the slurry was treated 3 times, and the doxorubicin ultrafine nanoparticle soybean oil suspension was obtained, and then the soybean oil supernatant was removed by removing the high temperature centrifugation at 4 ° C and shaking at 20,000 rpm for 60 min to obtain doxorubicin ultrafine nanoparticles.
  • the particle size was determined using a laser particle size analyzer (Malvern Nano Zetasizer ZS90, UK), in which three translucent red emulsions FBA-SD/Su-D5, FBA-SD/Su-D25 and FBA-SD/Su-D50 were obtained by HS method.
  • the diameters are 52-155 nm, 61-167 nm and 65-192 nm, respectively; the reconstituted suspensions of the three FBA-SD/Su-D lyophilized preparations have particle sizes of 55-159 nm, 63-168 nm and 68-193 nm, respectively;
  • the "doxorubicin ultrafine nanoparticle soybean oil suspension” has an average particle diameter of 50 nm or less.
  • the experimental results show that the method of preparing fatty acid by binding fatty acid to albumin can not only prepare nano-particles by using a "water-insoluble” drug such as paclitaxel dissolved in an organic solvent, but also a water-soluble "water-soluble” drug suspended in an organic solvent. Ultrafine nanoparticles of doxorubicin can also prepare nanoparticles.
  • Example 16 Preparation method of fatty acid-binding albumin (FBA)-"insoluble drug (ID) nanoparticle/suspension type (Suspension, Su) lyophilized preparation (referred to as FBA-ID/Su method)
  • the particle size was measured by a laser particle size analyzer.
  • the particle sizes of the two translucent emulsions FBA-ID/Su-E25 and FBA-ID/Su-E50 were 62-178 nm and 61-206 nm, respectively;
  • the reconstituted suspension of the E lyophilized preparation has a particle diameter of 65 to 172 nm and 66 to 210 nm, respectively; and the "etoposide ultrafine nanoparticle soybean oil suspension" has an average particle diameter of 50 nm or less.
  • the experimental results show that the method of preparing fatty acid by binding fatty acid to albumin can not only prepare nanoparticles by using "water-insoluble” drugs such as paclitaxel dissolved in organic solvents, but also can not be completely dissolved due to solubility limitation and suspended in organic solvent. Nanoparticles can also be prepared by ultra-micro nanoparticles of "water insoluble” drugs such as etoposide.
  • Example 17 Preparation of Methotrexate (MTX) Nanoparticle Freeze-Drying Agent (FBA-ID/Su-MTX) by FBA-ID/Su Method
  • the slurry was treated once, and the methotrexate ultra-nanoparticle soybean oil suspension was obtained, and then the soybean oil supernatant was removed by a high-speed centrifugation at 4 ° C, and the rotation speed was 35000 rpm for 30 mins to obtain methotrexate ultramicron.
  • Particles take 3mg of myristic acid dissolved in 500ul and 1500ul of ethyl acetate solution (solution B), and fully suspend the collected methotrexate ultra-micro nanoparticles to obtain methotrexate ultra-micro nanoparticles Organic solvent suspension (Liquid D', sample No.
  • FBA-ID/Su-MTX20 and FBA-ID/Su-MTX500 were filtered through 0.45u and 0.22u filter membranes to remove bacteria and other microorganisms in the emulsion to become two kinds of sterile emulsions. Purified by vacuum freeze-drying for 36 hours (conditions), respectively, to obtain a FBA-ID/C-MTX lyophilized preparation.
  • the particle size was measured by a laser particle size analyzer.
  • the particle sizes of the two translucent emulsions FBA-ID/Su-MTX20 and FBA-ID/Su-MTX500 were 53-149 nm and 75-353 nm, respectively;
  • the reconstituted suspension of the MTX lyophilized preparation has a particle diameter of 55 to 153 nm and 74 to 357 nm, respectively; and the "methotrexate ultrafine nanoparticle soybean oil suspension" has an average particle diameter of 50 nm or less.
  • Methotrexate is a "water-insoluble" drug and is not soluble in organic solutions.
  • the experimental results show that the preparation method of fatty acid-bound albumin to prepare nanoparticles can also be applied to drugs insoluble in organic solvents to prepare nano-formulations.
  • HSA human serum albumin
  • curcumin (C) 180mg curcumin (C) is mixed in 6ml pharmaceutical grade soybean oil (containing vitamin E not less than 0.4 ⁇ 2mg / ml) or containing aqueous solution for injection of C (vitamin C content 50 ⁇ 100mg / ml), at 4 ° C
  • the mixture was treated with high-pressure homogenization at 15,000 psi for 3 times to obtain a suspension of curcumin ultra-micro-nanoparticles. After centrifugation at 4 ° C, high-speed centrifugation at 20,000 rpm for 60 mins, the supernatant was removed and collected.
  • Curcumin ultrafine nanoparticles 300 ⁇ l of chloroform ethanol mixed solution (9:1, v/v) mixed with curcumin ultrafine nanoparticles to obtain a suspension of curcumin ultramicron nanoparticles in chloroform ethanol; 1 ug of oleic acid Adding to the suspension, the liquid D' is obtained, and the liquid D' containing the mixture of oleic acid, curcumin ultrafine nanoparticles, chloroform and ethanol is slowly dropped into the high-speed stirred chloroform ethanol treated 10 mg/ml.
  • the stirring speed is 3000 rpm, stirring is continued for 60 minutes; or stirring at 3000 rpm for 5 minutes, and then the mixture is subjected to a high pressure micro-jet machine, and the cycle is repeated twice under a pressure of 30,000 psi, 4 ° C, and protected from light. , obtained a yellow suspension; quickly put this suspension into vacuum rotary evaporation
  • the organic solvent was removed by rotary evaporation under reduced pressure (20 mmHg) at 35 ° C for 15 minutes.
  • a yellow emulsion is obtained in which the curcumin particles have a particle size of 55 to 198 nm; and the curcumin ultrafine nanoparticle suspension has a particle size of 20 to 43 nm (Malvern Nano Zetasizer ZS90, UK).
  • the above yellow emulsion is filtered through a 0.22u filter membrane to remove microorganisms such as bacteria in the solution to form a sterile solution; the sterile solution is freeze-dried by vacuum for 24 hours to obtain a fatty acid-binding albumin-curcumin nanoparticle.
  • Lyophilized preparation FBA-ID/Su-C. As shown in Table 6, FBA-ID/Su-C was obtained from other formulation formulations and conditions.
  • Example 19 Preparation method of fatty acid-bound albumin "water-soluble” drug nanoparticle/protein-binding (Ab) lyophilized preparation (abbreviated as FBA-SD/Ab method)
  • the stirring speed is 20,000 rpm and the duration is 10 minutes, which is a high-speed homogenization method for preparing a nano preparation; or 5 mg of myristic acid is dissolved in a 350 ul of chloroform solution (solution B), and the mixed solution is slowly dropped into the high speed.
  • Stirred D25 and D50HSA-Doxorubicin binding solution or D0HSA solution The stirring speed was 11,000 rpm and the duration was 5 minutes. Then the high-speed stirring mixture was quickly placed in the feed container of the high-pressure micro-jet, and the pressure was 4 times at 4 ° C under a pressure of 20,000 psi.
  • the nano preparation was prepared by homogenization method; the obtained three suspensions were quickly placed in a vacuum rotary evaporator, and rotated under a reduced pressure (20 mmHg) at 35 ° C for 20 minutes to remove all organic solvents; three translucent reds were obtained.
  • Emulsion in which three nanoparticles of translucent red emulsions of D0, D25 and D50, namely FBA-SD/Ab-D0 (FBA), FBA-SD/Ab-D25 and FBA-SD/Ab-D50, by laser particle size analyzer
  • the measured particle diameters were 83 to 211 nm, 114 to 256 nm, and 118 to 322 nm, respectively.
  • the three translucent emulsions were filtered through a 0.22u filter membrane to remove microorganisms such as bacteria in the emulsion, and became three kinds of sterile emulsions; respectively, vacuum freeze-dried for 30 hours to obtain a D0 fatty acid-binding albumin nanoparticle freeze-dried preparation ( FBA) and D25/D50 fatty acid-binding albumin-doxorubicin nanoparticle lyophilized preparation (FBA-SD/Ab-D); after reconstitution of FBA and FBA-SD/Ab-D plus saline or water for injection, The particle size of the nanoparticles was the same as before lyophilization.
  • FBA D0 fatty acid-binding albumin nanoparticle freeze-dried preparation
  • FBA-SD/Ab-D D25/D50 fatty acid-binding albumin-doxorubicin nanoparticle lyophilized preparation
  • the particle size of the nanoparticles was the same as before lyophilization.
  • Example 20 Preparation of "Water-Soluble” Pseudomonas Exotoxin (PE38) Nanoparticle Freeze-Drying Agent (FBA-SD/Ab-PE) by FBA-SD/Ab Method
  • Liquid C 1 mg (30 kDa Pseudomonas exotoxin, PE38) 1 mg (0.5 mg/ml PBS 2 ml) was slowly added to 8 ml of 62.5 mg/ml HSA solution, and magnetically stirred at 1,00 rpm for 4 hours to obtain 10 ml of HSA- PE binding solution (liquid C'); 1 mg of oleic acid (OA) is dissolved in 150 ul of a mixed solution of chloroform and ethanol (8:5, v/v) (solution B), and the mixed solution is slowly dropped.
  • OA oleic acid
  • the stirring speed is 25,000 rpm for 10 minutes, which is a high-speed homogenization method for preparing nano preparations; or 1 mg of oleic acid is dissolved in 150 ul of chloroform and ethanol mixed solution (8:5) , v/v), the mixed solution is slowly dropped into the high-speed stirring HSA-PE binding solution at a stirring speed of 12,000 rpm for a duration of 4 minutes, and then the high-speed stirring mixture is quickly placed in the high-pressure micro-micro
  • the feed container of the jet machine is circulated once at 4 ° C under a pressure of 25,000 psi. This is a high pressure homogenate.
  • the nano preparation was prepared by the method; the obtained suspension was quickly placed in a vacuum rotary evaporator, and rotated under a reduced pressure (20 mmHg) at 35 ° C for 20 minutes to remove all organic solvents, and the obtained emulsion was determined, wherein the particle size of the PE nanoparticles was determined. It is 90 to 180 nm.
  • the emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; the sterile emulsion is freeze-dried in vacuum for 36 hours to obtain a fatty acid-binding albumin-PE nanoparticle jelly.
  • the dry preparation FBA-SD/Ab-PE38; after reconstitution of FBA-SD/Ab-PE38 plus physiological saline, the particle size of the obtained PE nanoparticles was the same as before lyophilization. As shown in Table 7, FBA-SD/Ab-PE38 was obtained from other formulation formulations and conditions.
  • Example 21 Preparation of Epirubicin (EP) Nanoparticle Freeze-Drying Agent (FBA-SD/Ab-EP) by FBA-SD/Ab Method
  • Solution (Liquid C'); Dissolve 5 mg of intravenous myristate in 450 ul of chloroform solution (solution B), and slowly mix the mixed solution into high-speed stirring EP20 and EP40HSA-doxorubicin-binding solution or EP0HSA solution. , the stirring speed is 10,000 rpm, the duration is 6 minutes, and then the high-speed stirring mixture is quickly placed into the feeding capacity of the high-pressure micro jet machine.
  • the reactor was cycled 6 times at 4 ° C under a pressure of 20,000 psi. This was prepared by high pressure homogenization. The three suspensions were quickly placed in a vacuum rotary evaporator under reduced pressure at 35 ° C (20 mmHg).
  • the three translucent emulsions were filtered through 0.45u and 0.22u filtration membranes to remove bacteria and other microorganisms in the emulsion into three kinds of sterile emulsions; respectively, vacuum freeze-dried for 24 hours to obtain EP0 fatty acid-binding albumin nanoparticles.
  • Dry preparation FBA
  • EP20/EP40 fatty acid-bound albumin-epitamycin nanoparticle lyophilized preparation FBA-SD/Ab-EP
  • FBA and FBA-SD/Ab-EP plus saline or water for injection After dissolution, the obtained nanoparticles have the same particle size as before lyophilization.
  • Example 22 Preparation method of fatty acid-binding albumin-"water-soluble" drug nanoparticle/Empty-nanoparticle Binding (Eb) lyophilized preparation (FBA-SD/Eb for short)
  • the pH of the particle emulsion is 5.5 to 6.5; 0, 25 mg, 50 mg and 150 mg of doxorubicin (D), namely D0, D25, D50 and D150 samples, dissolved in 25 ml of water for injection; 25 ml of doxorubicin solution is slowly Adding 25ml HSA nanoparticle emulsion, magnetic stirring at 250rpm for 4 hours combined with albumin, or magnetic stirring at 500rpm for 2 hours combined with albumin, can get four translucent red emulsions, of which D0, D25, D50 and D150 are translucent
  • the red emulsion nanoparticles namely FBA, FBA-SD/Eb-D25, FBA-SD/Eb-D50 and FBA-SD/Eb-D150, were determined by laser particle size analyzer to be 85-213 nm, 88-223 nm, respectively. 86 to 227 nm and 83 to 287 nm.
  • the four translucent emulsions were filtered through 0.45u and 0.22u filtration membranes to remove bacteria and other microorganisms in the emulsion into four kinds of sterile emulsions; respectively, vacuum freeze-dried for 24 hours to obtain D0 fatty acid-binding albumin nanoparticles.
  • Dry preparation FBA
  • D25/D50/D150 fatty acid-bound albumin-doxorubicin nanoparticle lyophilized preparation FBA-SD/Eb-D
  • FBA and FBA-SD/Eb-D plus saline or water for injection After reconstitution, the obtained nanoparticles have the same particle size as before lyophilization.
  • FBA-SD/Eb-D was obtained from other formulation formulations and conditions.
  • Example 23 Preparation method of fatty acid-binding albumin-"water-insoluble” and “water-soluble” drug nanoparticle/suspension lyophilized preparation (referred to as FBA-ID/So&SD/Su method)
  • carboplatin CPboplatin, CP
  • pharmaceutical grade soybean oil vitamin E content 0.4 ⁇ 2mg/ml
  • the obtained carboplatin ultrafine nanoparticles were stirred and suspended using a mixed solution of oleic acid, paclitaxel, chloroform and ethanol; the suspension containing oleic acid, paclitaxel, carboplatin, chloroform and ethanol was slowly dropped into the high-speed stirring.
  • the "extracted” treated 60 mg/ml HSA solution was shaken at 10,000 rpm, at a temperature of 4 ° C, at a stirring speed of 10,000 rpm for 5 minutes, or at a stirring speed of 12,000 rpm for 4 minutes; this mixed suspension was then added to a high pressure.
  • the feed port of the microfluidizer was cycled 3 times at 4 ° C under a pressure of 30,000 psi; the obtained suspension was quickly placed in a vacuum rotary evaporator and rotary evaporated at 35 ° C under reduced pressure (20 mmHg) for 30 minutes. The entire organic solvent was removed to obtain a translucent emulsion; the particle size of the nanoparticles in the obtained translucent emulsion was measured by a laser particle size analyzer and found to be 67 to 176 nm.
  • the translucent emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; freeze-dried under vacuum for 24 hours to obtain fatty acid-bound albumin paclitaxel-carboplatin nanoparticles.
  • the lyophilized preparation (FBA-P/So&CP/Su); after reconstitution of FBA-P/So&CP/Su plus physiological saline or water for injection, the obtained nanoparticles have a particle diameter of 67 to 178 nm, which is the same as before lyophilization.
  • FBA-P/So&CP/Su was obtained from other formulation formulations and conditions.
  • This method is a combination of the aforementioned FBA-ID/So and FBA-SD/Su two particle preparation methods.
  • Example 24 Method for preparing fatty acid-binding albumin-"water-insoluble” and “water-soluble” drug nanoparticle/protein-bound lyophilized preparation (abbreviated as FBA-ID/So&SD/Ab method)
  • PE38 toxin 0.5 mg/ml PBS 2 ml was slowly added to solution C, magnetically stirred at 1,000 rpm for 1 hour to bind albumin, or magnetically stirred at 100 rpm for 4 hours to bind albumin to obtain 12 ml of HSA-PE38 binding solution (solution C).
  • nanoparticle suspension protected from light, cycle 3 times, to obtain a nanoparticle suspension
  • This is The nano preparation was prepared by pressure homogenization method; the obtained suspension was quickly placed in a vacuum rotary evaporator, and rotated under a reduced pressure (20 mmHg) at 35 ° C for 20 minutes to remove all organic solvents to obtain a translucent emulsion; The particle diameter of the nanoparticles in the obtained translucent emulsion was measured and found to be 50 to 110 nm, 54 to 135 nm, and 58 to 169 nm.
  • the translucent emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; vacuum freeze-dried for 24 hours to obtain fatty acid-bound albumin docetaxel-PE38 toxin Nanoparticle lyophilized preparation (FBA-Do/So&PE38/Ab); after reconstitution of FBA-Do/So&PE38/Ab with physiological saline or water for injection, the obtained nanoparticles have a particle size of 53-107 nm, 58-137 nm and 55- 170 nm, the same as before lyophilization. As shown in Table 10, FBA-Do/So&PE38/Ab was obtained from other formulation formulations and conditions.
  • Example 25 Fatty acid-binding albumin-preparation method of "water-insoluble” and “water-soluble” drug nanoparticle/empty particle-bound lyophilized preparation (referred to as FBA-ID/So&SD/Eb method) Preparation of rapamycin and doxorubicin nanoparticle lyophilized preparation by FBA-ID/So&SD/Eb method (FBA-R/So&D/Eb)
  • 0.2 mg of myristic acid and 6.5 mg, 130 mg and 260 mg of rapamycin (Rapamycin, R) were co-dissolved in 650 ul of dichloromethane solution (solution D), and this containing myristic acid, rapamycin,
  • the mixed solution of methylene chloride was slowly dropped into the high-speed stirring solution C at a stirring speed of 10,000 rpm, stirring was continued for 5 minutes, or the stirring speed was 12,000 rpm, and stirring was continued for 4 minutes; this mixture was passed through a high-pressure microjet machine at Cycling 6 times at 4 ° C under 10,000 psi pressure; the suspension was quickly placed in a vacuum rotary evaporator and rotary evaporated at 35 ° C under reduced pressure (20 mmHg) for 30 minutes to remove all organic solvents.
  • Emulsion Adjust the pH of the translucent emulsion to 5.5-6.5 with 0.01M-0.1M sodium hydroxide; take 20mg doxorubicin (D), dissolve in 2ml water for injection; slowly add 2ml doxorubicin solution to 10ml three kinds
  • a translucent emulsion magnetic stirring at 250 rpm for 4 hours, or magnetic stirring at 500 rpm for 2 hours, a fatty acid-bound albumin-rapamycin-doxorubicin emulsion can be obtained, and the particle size of the nanoparticles in the obtained emulsion can be determined by a laser particle size analyzer. The results were 55 to 157 nm, 52 to 169 nm, and 56 to 193 nm.
  • the emulsion is filtered through a 0.45u and 0.22u filter membrane to remove microorganisms such as bacteria from the emulsion to form a sterile emulsion; vacuum freeze-dried for 24 hours to obtain fatty acid-bound albumin-rapamycin-Azamycin Lyophilized preparation of nano-particles (FBA-R/So&D/Eb); after reconstitution of FBA-R/So&D/Eb with physiological saline or water for injection, the obtained nanoparticles have particle sizes of 53-159 nm, 57-173 nm and 55 ⁇ 195nm, the same as before lyophilization. As shown in Table 11, FBA-R/So&D/Eb was obtained for other formulation formulations and conditions.
  • This method is a combination of the aforementioned FBA-ID/So and FBA-SD/Eb two particle preparation methods.
  • Example 26 Transmission electron microscopy scan of FBA, FBA-ID/So-P and FBA-SD/Su-D
  • FIG. 1 shows the fatty acid-binding albumin-paclitaxel nanoparticles FBA-ID/So-P obtained in the preparation of Example 6, that is, the sample FBA-ID/So-P1 transmission electron microscope scan;
  • FIG. 2 is the preparation of Example 9. The obtained fatty acid-binding albumin "empty-particle-bound" (without docetaxel) nanoparticles, namely FBA-ID/So-Do00;
  • FIG. 3 is the fatty acid-bound albumin prepared in Example 15 - Electron microscopy of doxorubicin nanoparticles, FBA-SD/Su-D25.
  • Example 27 Analysis of albumin secondary structure by circular dichroism spectroscopy
  • the HSA obtained by decomposition of Hualan biological HSA and Abraxane, the HSA obtained after decomposition of sample FBA-ID/So-P1 in Example 6, the sample FBA-ID/So-P&C in Example 13, and the sample FBA in Example 15 were respectively taken.
  • Example 28 In vitro stability of fatty acid-binding albumin-drug nanoparticles
  • HSA Umbilical Vascular Endothelial Cell
  • Fluorescently labeled FBA-ID/So-P F-FBA-ID/So-P
  • F-FBA-ID/So-P was prepared by adding the fluorescently labeled paclitaxel (invitogen, Flutax-2) to paclitaxel at a ratio of 1:50. Flutax-2 was then added to the "taxol" formulation in the same proportions as the fluorescently labeled paclitaxel injection, F-Taxol.
  • 5 ⁇ 10 4 HUVECs were seeded on 96-well plates (Costar 3614) to inoculate HUVEC cells at a dose of 5 ⁇ 10 4 cells/well per well, cultured at 37° C., 5% CO 2 cells, until the cells were adherent and overgrown.
  • F-FBA-ID/So-P and F- were added at a final concentration of 20, 40, 80, 160, 320 and 640 ug/ml PEM buffer (50 mmo/L PIPES, 2 mmol/L EGTA, 2 mmol MgCl2).
  • PEM buffer 50 mmo/L PIPES, 2 mmol/L EGTA, 2 mmol MgCl2.
  • the 96-well plate was washed three times with PEM buffer and read using an M5/M5e multi-plate reader (Molecular Devices, USA) at an excitation wavelength of 494 nm and an emission wavelength of 524 nm.
  • the data of each well was used to determine the binding rate of F-FBA-ID/So-P and F-Taxol to HUVEC cells.
  • the results are shown in Figure 5.
  • the results showed that various F-FBA-ID/So-P and HUVEC cells were obtained.
  • the binding rate is about 10 times higher than that of F
  • HUVEC cells were cultured in 6-well plates, cultured at 37 ° C, 5% CO2 cells. After the cells were attached and overgrown, various FBA-Drug NPs were added, and the same dose of the corresponding chemotherapeutic drugs such as Taxol and Ai were selected. Sudocitabine injection, Doxil and doxorubicin hydrochloride, epirubicin, vinorelbine, platinum, mitomycin for injection, etoposide injection, etc. as positive control drugs, The final concentration was 160-640 ug/ml PEM buffer. After incubating for 3 hours at 37 °C, 6-well plates were washed three times with PEM buffer. HUVEC cells were collected by centrifugation.
  • chemotherapeutic drugs such as paclitaxel and polyene were determined by HPLC.
  • the contents of paclitaxel, doxorubicin, epirubicin, vincristine, carboplatin, etc. compared the difference between FBA-Drug NP nano-preparation and common intravenous injection into HUVEC cells, as shown in Table 13, the experimental results show: FBA The drug content of the -Drug NP into the HUVEC cells is about 2 to 6 times higher than that of the corresponding drug.
  • FBA-ID/So-P, FBA-P/So&CP/Su and FBA-Do/So&PE38/Ab in the form of intact nanoparticles in the blood circulation were determined by ELISA as an indicator for determining the stability in animals.
  • Anti-Paclitaxel antibody or anti-Docetaxel antibody embedded in the bottom of 96-well plate captures three kinds of FBA-ID/So-P nanoparticles in serum, and then detects "FBA-ID/So-P" by biotin-labeled anti-HSA antibody "The plasma half-life is calculated from the content of three kinds of nanoparticles; the content of paclitaxel in the tissue is determined by HPLC to determine the aggregation characteristics of FBA-ID/So-P three kinds of nanoparticles in tumor tissues;
  • the anti-tumor efficacy of FBA-ID-P, FBA-P&CP/C and FBA-Do/So&PE38/Ab was determined by ELISA as an indicator for determining the
  • FBA-ID/So-P In vivo properties tests of FBA-ID/So-P, FBA-ID/So-P&C, FBA-P/So&CP/Su and FBA-Do/So&PE38/Ab suggest: FBA-ID/So-P, in animal blood circulation, FBA-ID/So-P&C, FBA-P/So&CP/Su and FBA-Do/So&PE38/Ab have good in vivo blood circulation stability and can be enriched in tumor tissues, especially FBA-Do/ So&PE38/Ab is more prominent, suggesting that PE38 toxin can significantly promote the enrichment of docetaxel in local tumor tissues and produce better antitumor effects.
  • doxorubicin-containing fatty acid-binding albumin-doxorubicin nanoparticle lyophilized preparation FBA-SD/Su-D
  • stealth liposome doxorubicin Doxil
  • the FBA-SD/Su-D nanoparticles in the form of intact nanoparticles in the blood circulation were determined by ELISA as an indicator for determining the stability in animals, ie, the anti-Amites embedded in the bottom of the 96-well plate.
  • the antibody detects the four nanoparticles of FBA-SD-D in serum, and then the biotin-labeled anti-HSA antibody detects the content of four kinds of nanoparticles of "FBA-SD-D", thereby calculating the plasma half-life; using HPLC Fluorescence detection was used to determine the content of doxorubicin in plasma and tissues to determine the plasma half-life of doxil and doxorubicin and the content of doxorubicin in tumor tissues; transplantation of human oral epithelial carcinoma xenografts (KB) in nude mice In the model, intravenous injection of the same dose of doxorubicin, preliminary judgment of FBA-SD/Su-D, FBA-SD/Ab-D, FBA-SD/Eb-D, FBA-R/So&D/Eb, FBA-R/So&D /Su and Doxil and doxorubicin hydrochloride anti-tumor effect; in the above animal test, FBA-SD
  • Example 32 In vivo properties and properties of FBA-SD/Ab-PE
  • PE38 is the most commonly used molecular form of PE toxin with a clear molecular mechanism of cytotoxicity. Comparing PE38 protein with FBA-SD/Ab-PE38 prepared in Example 20, the LD 50 of FBA-SD/Ab-PE38 is higher than that of PE38. The protein is about 60 times (30 mg/kg and 0.45 mg/kg, respectively). The rat PE38 antigenicity assay showed that the antigenicity of FBA-SD/Ab-PE38 was about 20 times lower than that of PE38 protein.

Abstract

一种脂肪酸结合型白蛋白-药物纳米粒子及其冻干制剂，所述纳米粒子包含油核心、白蛋白和药物，其中白蛋白与油核心中的脂肪酸结合，药物与白蛋白和/或脂肪酸结合。所述脂肪酸结合型白蛋白-药物纳米粒子的冻干制剂具有在体外溶液中和体内血液循环中纳米粒子完整性与稳定性高，能显著增加富含gp60受体细胞内或肿瘤组织中的药物含量，显著提高荷载药物的LD50和MTD。

Description

脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 技术领域
本发明涉及一种纳米粒子冻干制剂及制备方法，尤其涉及一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法，属于医药制备技术领域。
背景技术
人血清白蛋白(Human Serum Albumin，简称HSA)是人血浆中的主要蛋白，是非糖基化蛋白，由585个氨基酸组成，分子量为66kD。健康人血浆中HSA浓度为35～55g/L，半衰期约为19天。HSA对维持血液胶体渗透压、组织细胞的氨基酸与能量代谢、以及脂溶性物质如胆红素、脂肪酸和药物等的转运均具重要作用。HSA来源充足，可由人血中提取所得，也可由基因重组技术生产，HSA无毒、无免疫原性、代谢机制清楚，是理想的纳米粒子制备材料。而且，早在上世纪五十年代Babson等①就发现HSA可聚集在肿瘤组织局部；HSA通过细胞膜gp60蛋白转运至细胞内，某些肿瘤组织如乳腺癌可特异性表达SPARC蛋白(secreted protein,acidic and rich in cysteine)，后者促进gp60受体对HSA的转运作用；有报道指出②，白蛋白结合型紫杉醇(Albumin-bound paclitaxel,Abraxane)在高表达SPARC蛋白的实体肿瘤的化疗中疗效更佳。牛血清白蛋白(Bovine Serum Albumin，简称BSA)，是牛血清中的一种主要蛋白，包含583个氨基酸残基，分子量为66kDa，与HSA的氨基酸序列高度同源，且蛋白结构和性质非常相似。
紫杉醇是从天然植物中提取出的、具明显抗癌作用的双萜类化合物，通过对微管蛋白的独特作用而阻止细胞有丝分裂和增殖。紫杉醇水溶性很低(水溶解度在0.0005mg/mL)，而紫杉醇经过化学修饰与改造所获得的半合成多烯紫杉醇更是几乎不溶于水，除体外抗癌活性提高外，其它理化性质与紫杉醇相同。因此，目前临床上主要应用的紫杉醇和多烯紫杉醇静脉注射剂都添加了大量表面活性剂助溶，如泰素是以紫杉醇添加聚氧乙烯蓖麻油(Cremophor EL)和无水乙醇助溶、多烯紫杉醇采用吐温-80助溶，而大剂量Cremophor EL或吐温80输注可导致严重的过敏反应等毒副反应，甚或明显限制了化疗药给用药剂量的提高，也限制了紫杉醇的临床疗效。鉴于此，1997年美国生物科学有限公司申请的ZL97199720.9(USP 08/720,756)等系列专利，公布了“一种不溶于水的药理活性物质，如紫杉醇，以包有白蛋白包衣(作为稳定剂)的悬浮颗粒形式输运”；且这种“悬浮颗粒”是在没有表面活性剂和颗粒的聚合物材料存在的情况下，仅由白蛋白与可溶解于有机溶液中的紫杉醇、在高剪切处理的条件下形成；这种独特的输送系统，有效避免了因添加Cremophor EL或吐温80等而导致的毒副反应，而且，采用这种技术所开发出的白蛋白结合型紫杉醇注射剂(商品名：Abraxane)，在转移性乳腺癌的化疗中，取得了明显优于泰素的临床疗效。但是，这种制备白蛋白结合紫杉醇“悬浮颗粒”的技术(后称为nab-technology)，仅仅利用了白蛋白疏水区域与水不溶性药物(疏水性)的“微弱”结合力，却没有利用同样可以溶解于有机溶液中、且可与白蛋白特异性结合、且结合力较强的脂肪酸；也没有利用白蛋白还可与许多水溶性药物包括蛋白和多肽等结合的性质和功能；更没有联想到因脂肪酸与白蛋白的结合作为构成纳米粒子的主要“力量”和成分，使在有机溶剂中“悬浮”的水溶性药物超微纳米粒子如粒径小于等于50nm的粒子，也可以形成白蛋白包裹的纳米级粒子甚或是小于100nm的纳米粒子。因此，前述nab-technology存在明显的纳米粒子形成机制上的缺陷和应用限制。
脂肪酸(Fatty acids，FA)在哺乳类动物的能量代谢、细胞膜磷脂合成和信号传导中均具重要作用。但FA的水溶性很低，体内转运需FA转运蛋白，而血液中的血清白蛋白(Serum Albumin，SA)是细胞外液中最主要的FA转运蛋白。早在上世纪五十年代，Goodman DS等③就发现：SA分子上有2个FA的高亲和性结合位点、5个中等亲和性结合位点和超过20个低亲和性结合位点，FA如油酸盐(Oleate)高、中、低亲和力分别是1.1×108,4.0×106and 1×103M－1。SA蛋白结构的研究揭示了FA的特异性结合位点和结合方式，即FA与SA的特异结合通过结合位点处碱性氨基酸侧链基团与脂肪酸羧基之间的“盐桥”作用(salt bridge interactions)而实现④⑤。近年批准上市的新一代长效胰岛素(Insulin Detemir，ID)就是依据这种游离脂肪酸与SA的特异性结合性质而实现体内的长效药理作用，ID由肉豆蔻酸(Myristic acid,MA)与胰岛素B链29位赖氨酸残基定点偶联而成，注射ID后，其分子上的脂肪酸可与血液中的SA特异结合，从而避免了小分子胰岛素在体内的快速清除；这同时证实，在人体血液循环和生理条件下，注射保留羧基的脂肪酸胰岛素类似物制剂，其脂肪酸分子可与血清白蛋白特异、有效和稳定的结合⑥。早在上世纪60～70年代，Spector AA等⑦在Ehrlich腹水瘤细胞和白血病细胞的系列工作证实：肿瘤细胞可快速摄取游离脂肪酸(FFA)，而作为FFA载体的白蛋白和低密度脂蛋白，可促进肿瘤细胞的这种转运机制。
阿霉素(doxorubicin，D)是临床上重要、常用、广谱抗癌一线药，但抗癌疗效受限于其对心脏等的毒性；尽管各种纳米粒子制备技术曾经应用于开发阿霉素的给药系统，其中隐形脂质体阿霉素(Doxil)纳米制剂是最成功的开发范例之一。Doxil显著降低了阿霉素的心脏毒性，但其疗效并未因此而显著提高；这也是“隐形脂质体”技术并未广泛应用于其它抗癌药的原因之一。阿霉素是水溶性化疗药，在一定条件下可与HSA结合，Langer等⑧曾利用此性质制备以化学交联的HSA作为基架的纳米粒子，但该种纳米粒子的粒径较大(大于250nm)、载药量较低、化学交联的白蛋白纳米粒子难以白蛋白单分子形式通过gp60受体转运等。表阿霉素(Epirubicin，EP)是通过半合成途径合成的一种蒽环类抗肿瘤抗生素，与阿霉素的区别仅在氨基糖部分4'位的羟基由顺式变成反式，这种立体结构的细微变化并未影响其理化性质,故本专利所涉及的阿霉素纳米粒子的制备方法同样可以适用于表阿霉素。
假单胞菌外毒素(Pseudomonas exotoxin A,PE)具极强的细胞毒性，与抗肿瘤特异性抗体融合，而成所谓immunotoxin(免疫毒素，IT)，是治疗恶性肿瘤和白血病的特异性靶向药物，被称为“生物导弹”，其中一种称为PE38的假单胞菌外毒素(PE38)的IT经临床II试验，其肿瘤特异性抗原的靶向性得以证实；PE38甚或可以治愈毛细胞白血病的病人，后者恰是不能对PE38产生抗体的病人，这提示IT分子抗原性对其疗效的重要性。特异性抗体融合PE38，可显著提高抗肿瘤的靶向性，却不能有效避免PE38毒素的抗原性及其对血管内皮细胞损伤作用，导致严重的毒性，特别是血管渗漏综合症⑨。因此，对于PE毒素等一类在血液循环中可导致严重毒副作用的分子和药物，仅通过氨基酸序列的优化提高抗体的亲和力和/或降低PE38毒素的抗原性是不够的。申请人意外发现，通过HSA与 PE38的结合，可有效“封闭”PE38的抗原位点，相信也会降低PE38毒素对人血管内皮系统的损伤；而通过白蛋白纳米粒子对肿瘤组织的靶向与富集作用、以及在肿瘤局部利用HSA和脂肪酸的跨膜机制实现PE38对肿瘤组织的特异性杀伤，更是一种新的治疗策略，且经检索还未见有类似报道。
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发明内容
本发明的第一个目的是要克服现有技术以白蛋白为主要基架的纳米粒子荷载药物的局限性，提供一种既适用于“水不溶性”又适用于“水溶性”药物的脂肪酸结合型白蛋白-药物纳米粒子冻干制剂。
本发明的第二个目的是要克服现有技术的不足，提供一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法。
本发明的技术方案概述如下：
一种脂肪酸结合型白蛋白-药物纳米粒子，包括油相核心、白蛋白和药物，所述的白蛋白与油相核心中的脂肪酸结合，所述的药物与白蛋白和/或脂肪酸结合，所述的药物选自水溶性药物、水不溶性药物、水溶性药物超微纳米粒子和水不溶性药物超微纳米粒子中的一种或两种以上。
本发明脂肪酸结合型白蛋白-药物纳米粒子中按质量比包括脂肪酸0.0001％～8％，白蛋白75％～99％和药物0.1％～25％，优选的，所述纳米粒子中按质量比包括脂肪酸0.001％～1％，白蛋白80％～95％，药物1％～20％。
在本发明的一个实施方式中，所述的脂肪酸结合型白蛋白-药物纳米粒子中药物与脂肪酸结合共同构成油相核心，所述的白蛋白与油相核心中的脂肪酸结合。优选的，所述的脂肪酸结合型白蛋白-药物纳米粒子中水难溶性药物与脂肪酸结合共同构成油相核心，所述的白蛋白与油相核心中的脂肪酸结合。更优选的，所述的脂肪酸结合型白蛋白-药物纳米粒子中水溶性药物超微纳米粒子和/或水不溶性药物超微纳米粒子与脂肪酸结合共同构成油相核心，所述的白蛋白与油相核心中的脂肪酸结合。
在本发明的一个实施方式中，所述的脂肪酸结合型白蛋白-药物纳米粒子中药物与白蛋白结合，白蛋白与油相核心中脂肪酸结合。优选的，所述的脂肪酸结合型白蛋白-药物纳米粒子中水溶性药物与白蛋白结合，白蛋白与油相核心中脂肪酸结合。
在本发明的一个实施方式中，所述的脂肪酸结合型白蛋白-药物纳米粒子中水难溶性的药物、水难溶性药物的超微纳米粒子和/或水溶性药物的超微纳米粒子与脂肪酸结合共同构成油相核心，水溶性药物与白蛋白结合，所述的白蛋白与油相核心中的脂肪酸结合。
本发明所述的脂肪酸可以是制备白蛋白过程中存留的脂肪酸，且无需在制备纳米粒子的过程中额外添加脂肪酸，在此种情况下，本发明所述的白蛋白与油相核心中的脂肪酸天然特异性地结合。同时，本发明所述的脂肪酸也可以是如本发明所述的方法添加入制剂配方中，在此种情况下，本发明所述的白蛋白与油相核心中的脂肪酸在制备过程中特异性地结合。无论上述何种情况，本发明惊奇地发现，只要脂肪酸的含量在本发明所述的范围内，则所形成的纳米粒子在体外溶液中和体内的血液循环中的完整性和稳定性均较高，并可借纳米尺度富集于实体肿瘤局部，通过白蛋白gp60受体通道促进药物进入肿瘤细胞内，大幅提高荷载药物的LD50和MTD，进而显著提高化疗药物的抗癌药效。
一种药物制剂，包括本发明所述的脂肪酸结合型白蛋白-药物纳米粒子和药学上可接受的辅料。本发明所述的药物制剂可以为口服制剂或注射制剂，优选为混悬剂、注射液、注射用冻干粉。在本发明的优选实施方式中，所述的制剂为冻干制剂。
一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，包括如下步骤：
(1)液体A：为水不相溶的有机溶剂，或体积比为10～5:5～1的水不相溶的有机溶剂和无水乙醇的混合液，所述水不相溶的有机溶剂为氯仿、二氯甲烷或乙酸乙酯；溶液B：用液体A配制浓度为0.0033～360mg/ml的脂肪酸溶液，所述脂肪酸为油酸、亚油酸、肉豆蔻酸或甘油三酯；液体C：用水配制浓度为10～200mg/ml的白蛋白水溶液，按体积比为1:6.67～333的比例将液体A滴入以5,000～15,000rpm转速搅拌的白蛋白溶液中，继续搅拌2～10分钟， 制得液体C；
(2)脂肪酸结合型白蛋白-药物纳米粒子混悬液的制备：
用下述几种方式之一制备：
方式一：
用溶液B溶解水不溶性药物，获浓度为12.5mg/ml～600mg/ml的液体D，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
液体C中液体A的体积与溶解水不溶性药物的溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为0.4％～17.8％；脂肪酸与白蛋白的质量比为0.0001％～5％；水不溶性药物与白蛋白的质量比为：1％～20％；
方式二：
1)按1～10mg：1ml的比例将水不溶性药物与含0.4～2mg/ml维生素E的大豆油混合；或按1～10mg：1ml的比例将水不溶性药物与含50～100mg/ml维生素C的注射用水混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水不溶性药物超微纳米粒子；按40mg/ml～600mg/ml比例，将水不溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水不溶性药物超微纳米粒子和有机溶剂的悬液；
2)按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；
液体C中液体A的体积与液体D’中溶液B体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：0.6％～15.6％；脂肪酸与白蛋白的质量比为0.0001％～1％；水不溶性药物与白蛋白的质量比为：1％～25％；
方式三：
1)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml维生素E的大豆油混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；按6.6mg/ml～375mg/ml比例，将水溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水溶性药物超微纳米粒子和有机溶剂的悬液；
2)按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为12,000rpm～32,000rpm的液体C中，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；
液体C中液体A的体积与液体D’中溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：1％～15.8％；脂肪酸与白蛋白的质量比为0.001％～1.4％；水溶性药物与白蛋白的质量比为：1％～20％；
方式四：
1)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，即液体C’；
2)按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；
液体C中液体A的体积与溶液B的体积之和为油相，而白蛋白-水溶性药物结合溶液的体积为水相，油相体积与水相体积之比为：1％～16％；脂肪酸与白蛋白的质量比为0.002％～15％；水溶性药物与白蛋白的质量比为：1％～25％；
方式五：
按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为20,000rpm～25,000rpm的液体C中，制得脂肪酸结合型白蛋白纳米粒子混悬液；
将所述脂肪酸结合型白蛋白纳米粒子混悬液去除全部有机溶剂，获半透明乳液；
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水溶性药物纳米粒子空粒结合型乳液；
液体C中液体A的体积与溶液B的体积之和为油相，半透明乳液的体积为水相，油相体积与水相体积之比为：2.0％～16.4％；脂肪酸与白蛋白的质量比为0.001％～3.1％；水溶性药物与白蛋白的质量比为：2.5％～15％；
方式六：
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml维生素E的大豆油混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；
3)用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；
方式七：
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
3)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；
方式八
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
3)将脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液去除全部有机溶剂，获半透明乳液；调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子空粒结合型混悬液；
(3)乳液的制备
将步骤(2)中方式一～方式四、方式六和方式七制备的混悬液去除混悬液中全部有机溶剂，制得半透明的稳定的纳米粒子乳液；
(4)冻干制剂的制备
将步骤(3)获得的乳液过滤除菌，获无菌乳液，经真空冷冻干燥，制得脂肪酸结合型白蛋白-药物纳米粒子冻干制剂；
所述白蛋白为人血清白蛋白或牛血清白蛋白；所述人血清白蛋白由血液提取或重组技术制备；
一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，包括如下步骤：
(1)液体A：为水不相溶的有机溶剂，或体积比为10～5:5～1的水不相溶的有机溶剂和无水乙醇的混合液，所述水不相溶的有机溶剂为氯仿、二氯甲烷或乙酸乙酯，为色谱纯试剂；溶液B：用液体A配制浓度为0.0033～360mg/ml的静脉注射用脂肪酸溶液，优选浓度为0.01mg/ml～50mg/ml，所述静脉注射用脂肪酸为油酸、亚油酸、肉豆蔻酸或甘油三酯，为可人体静脉输注的脂肪乳剂的组成成分；液体C：无菌条件下，用注射用水配制浓度为10～200mg/ml的白蛋白水溶液，按体积比为1:6.67～333的比例将液体A滴入以5,000～15,000rpm转速搅拌的白蛋白溶液中，继续搅拌2～10分钟，使与脂肪酸结合的白蛋白萃取于液体A中，制得液体C；
(2)脂肪酸结合型白蛋白-药物纳米粒子混悬液的制备：
用下述几种方式制备：
方式一：
用溶液B溶解水不溶性药物，获浓度为12.5mg/ml～600mg/ml，优选浓度为20mg/ml～400mg/ml的液体D，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
或者，用溶液B溶解水不溶性药物,获浓度为12.5mg/ml～600mg/ml的液体D，优选浓度为10mg/ml～400mg/ml，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
所述水不溶性药物选紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷、甲氨蝶呤中的一种或几种；是相对水溶性较低或很低的化疗药物或其他药物，可溶解于至少一种“有机溶剂或其混合液”，并可与白蛋白结合；不同的“水不溶性”药物与不同的“有机溶剂或其混合液”的溶解度不同，“溶解度”是决定“水不溶性”药物“浓度范围”的重要因素。
在FBA-ID/So纳米粒子混悬液体系中，液体C中液体A的体积与溶解水不溶性药物的溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为0.4％～17.8％，优选1％～6.5％；静脉注射用脂肪酸与白蛋白的质量比为0.0001％～5％，优选0.001％～1％；水不溶性药物与白蛋白的质量比为：1％～20％，优选1％～10％。
方式二：
1)按1～10mg：1ml的比例将水不溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；或按1～10mg：1ml的比例将水不溶性药物与含50～100mg/ml维生素C的注射用水混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4℃下、避光、20,000～35,000rpm离心10～60min，去除上清液，获得水不溶性药物超微纳米粒子；按40mg/ml～600mg/ml比例，将水不溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水不溶性药物超微纳米粒子和有机溶剂的悬液；
2)按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌15～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；或按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光、压力为15,000psi～30,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；
其中，“水不溶性”药物选紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤等一种或几种；不同的“水不溶性”药物的密度和溶解度不同，悬浮于“有机溶剂或其混合液”中所需溶液体积不同，在此，“悬浮”的界定较“浓度范围”的界定更为重要。
在FBA-ID/Su纳米粒子混悬液体系中，液体C中液体A的体积与液体D’中溶液B体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：0.6％～15.6％；静脉注射用脂肪酸与白蛋白的质量比为0.0001％～1％；水不溶性药物与白蛋白的质量比为：1％～25％，优选1.0％～10％；
方式三：
1)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4±1℃下、避光、5,000～30,000rpm持续离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；按6.6mg/ml～375mg/ml比例，优选16.67mg/ml～166.67mg/ml比例，将水溶 性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水溶性药物超微纳米粒子和有机溶剂的悬液；
2)按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为12,000rpm～32,000rpm的液体C中，继续搅拌5～30分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；或按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光、压力为15,000psi～30,000psi下，连续3～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；
在FBA-SD/Su纳米粒子混悬液体系中，液体C中液体A的体积与液体D’中溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：1％～15.8％；静脉注射用脂肪酸与白蛋白的质量比为0.001％～1.4％；水溶性药物与白蛋白的质量比为：1％～20％；
其中，“水溶性”药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素(PE38)等化学、蛋白或多肽类药物等一种或几种；所述“水溶性”药物可溶于水，在“有机溶剂或其混合液”中的溶解度很低；所述不同的“水溶性”药物的密度不同，悬浮于“有机溶剂或其混合液”中所需溶液的体积不同，在此，“悬浮”的界定较“浓度”的界定更为重要。
方式四：
1)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，即液体C’；
2)按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；或按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为10,000psi～25,000psi下，连续1～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；
“水溶性”药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素(PE38)等化学、蛋白或多肽类药物等一种或几种。不同的“水溶性”药物与白蛋白的体外结合率不同，在此，“水溶性”药物与液体C中白蛋白的“结合”浓度以及白蛋白溶液的浓度，决定“水溶性”药物的加入量或浓度。
液体C中液体A的体积与溶液B的体积之和为油相，而白蛋白-水溶性药物结合溶液的体积为水相，油相体积与水相体积之比为：1％～16％；静脉注射用脂肪酸与白蛋白的质量比为0.002％～15％；水溶性药物与白蛋白的质量比为：1％～25％；
方式五：
按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为20,000rpm～25,000rpm的液体C中，继续搅拌10～20分钟，制得脂肪酸结合型白蛋白纳米粒子混悬液；或按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟后，加入高压微射流机进料容器中，在4±1℃下，避光，压力为10,000psi～25,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白纳米粒子混悬液；
将所述脂肪酸结合型白蛋白纳米粒子混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水溶性药物纳米粒子空粒结合型乳液；
所述水溶性药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素PE38等化学、蛋白或多肽类药物等一种或几种。不同的“水溶性”药物与白蛋白的结合率不同，不同粒径的FBA-Enp表面白蛋白的“量”不同，在此，“水溶性”药物与FBA-Enp表面白蛋白的“结合”浓度，决定“水溶性”药物的加入量或加入浓度。
在FBA-SD/Eb纳米粒子乳液体系中，液体C中液体A的体积与溶液B的体积之和为油相，半透明乳液的体积为水相，油相体积与水相体积之比为：2.0％～16.4％；静脉注射用脂肪酸与白蛋白的质量比为0.001％～3.1％；水溶性药物与白蛋白的质量比为：2.5％～15％；
方式六：
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4±1℃下、避光、5,000～30,000rpm持续离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；
3)用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；或用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为3,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；
方式七：
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
3)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～ 60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；或按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；
方式八
1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
2)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；或按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
3)将脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子空粒结合型混悬液；
(3)乳液的制备
将步骤(2)中方式一～方式四、方式六和方式七制备的混悬液放入真空旋转蒸发仪中，在35±2℃，负压下，旋转蒸发10～30分钟，去除混悬液中全部有机溶剂，制得半透明的稳定的纳米粒子乳液；
(4)冻干制剂的制备
将步骤(3)获得的乳液经0.22um膜过滤除菌，或经0.45um和0.22um膜过滤除菌，获无菌乳液，经真空冷冻干燥24～36小时，制得脂肪酸结合型白蛋白-药物纳米粒子冻干制剂；所述白蛋白为人血清白蛋白或牛血清白蛋白；所述人血清白蛋白由血液提取或重组技术制备；
步骤(1)中液体A与白蛋白溶液的体积比优选为1:36～100。
所述步骤(3)经有机溶剂萃取后的白蛋白混合液中的白蛋白的二级结构不变、白蛋白含有“裸露”的脂肪酸结合位点、并以该位点特异性结合脂肪酸，白蛋白分子的疏水区域依疏水性与水不溶性药物结合，白蛋白分子的亲水区域依亲水性与水溶性药物结合。
所述步骤(4)方式一～方式八中所述脂肪酸结合型白蛋白-药物纳米粒子均由溶解于溶液B中的静脉注射用脂肪酸与液体C或液体C’中的白蛋白结合形成。
所述步骤(4)方式一、方式六、方式七或方式八中所述水不溶性药物溶解于溶液B中与静脉注射用脂肪酸共同构成油相核心。
所述步骤(4)方式二或方式三中所述水不溶性药物超微纳米粒子或水溶性药物超微纳米粒子悬浮于溶液B中与静脉注射用脂肪酸共同构成油相核心。
所述步骤(4)方式四或方式五中所述水溶性药物与液体C中的或纳米粒子表面的白蛋白结合位于水相，静脉注射用脂肪酸为油相核心。
所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂，仅由白蛋白、药物和静脉注射用脂肪酸组成，不含有机溶剂或任何辅料，其组分及含量以质量百分比计为：静脉注射用脂肪酸为0.0001％～8％、优选为0.001％～1％，白蛋白为75％～99％、优选为80％～95％，药物为0.1％～25％、优选为1.0％～20％；所述药物为水不溶性药物和水溶性药物至少一种。
所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂，具有如下生物学性质：
(1)所述冻干制剂和Abraxane复溶后在体外溶液中4℃条件下，纳米粒子的完整性与稳定性分别在136～160小时和96小时；
(2)所述冻干制剂和Abraxane复溶后在体内血液循环中纳米粒子的完整性与稳定性分别在35～45小时和25小时；
(3)所述三种方式制备的阿霉素纳米粒子冻干制剂，如FBA-SD/Su-D25、FBA-SD/Ab-D25和FBA-SD/Eb-D25在荷瘤小鼠的血浆半衰期分别比Doxil延长8.1倍、6.1倍和2.8倍；
(4)所述冻干制剂荷载的药物进入富含gp60受体细胞内的药物浓度与同一药物静脉注射剂进入富含gp60受体细胞内的药物浓度之比为2～6；
(5)所述冻干制剂荷载紫杉醇的LD50是紫杉醇静脉注射剂LD50的44～56倍；
(6)所述冻干制剂荷载紫杉醇的MTD是紫杉醇静脉注射剂MTD的53～71倍；
(7)所述冻干制剂降低所包裹或结合的假单胞菌外毒素PE38蛋白的抗原性20倍。
本发明所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法中，脂肪酸在纳米粒子形成中的作用，如下：
1、有机溶剂或其混合液处理血浆白蛋白是一个脂肪酸“萃取”和“富集”在油相中的过程，因为超速离心30,000rpm 5小时有机溶剂或其混合液处理过的HSA溶液即液体C，可见油相与水相的分层，分别取油相和水相的样品，测定游离脂肪酸含量和HSA的含量，结果发现：60％以上的游离脂肪酸分布在油相之中；且若增加有机溶剂的量，则可增加油相中脂肪酸的总量。
2、添加不同剂量脂肪酸影响FBA-Drug NP纳米粒子的形成
配制50mg/ml HSA溶液10ml；“萃取”处理白蛋白；添加不同剂量的脂肪酸如肉豆蔻酸0(不加),1mg,2mg和4mg与紫杉醇100mg溶于200ul氯仿和乙醇混合溶液中，高速匀浆或高压匀浆法制备纳米粒子，可获半透明乳液， 经粒度分析表明：不加脂肪酸或加入过量脂肪酸，其紫杉醇纳米粒子的粒径均呈双峰，且平均粒径均大于300nm；而加入“适量”的脂肪酸如添加1mg和2mg的肉豆蔻酸，可获得了粒径在80～150nm和90～240nm之间稳定的平均粒径小于180nm的纳米粒子。
3、同样制备条件与工艺，若去除HSA溶液中的脂肪酸，且在油相中不加脂肪酸，则不能形成纳米粒子。
4、纳米粒子核心区和外周白蛋白游离脂肪酸的含量分析，进一步提供了脂肪酸对纳米粒子形成具重要作用的证据。
即采用超滤技术，以100kDa分子量截流的超滤管5000rpm离心超滤20分钟，分离纳米粒子核心区和外周白蛋白，分别测定其游离脂肪酸的含量，意外地发现：平均粒径小于160nm的纳米粒子溶液，纳米粒子的核心区白蛋白游离脂肪酸的含量超过纳米粒子外周白蛋白游离脂肪酸3倍以上，且核心区每一白蛋白分子平均结合2～5个脂肪酸分子。
5、FBA-SD/Ab和FBA-SD/Eb法制备纳米粒子的实践表明，单纯脂肪酸就可以形成纳米粒子，即在制备纳米粒子的油相中，仅加入脂肪酸，就可以形成纳米粒子，且这种纳米粒子较油相核心含有紫杉醇等“水不溶性”药物纳米粒子的体外稳定性无异甚或略优。
6、FBA-ID/Su、FBA-SD/Su和FBA-ID&SD/Su法制备纳米粒子的实践表明，由于脂肪酸与白蛋白的特异性结合，悬浮于有机溶剂中的“水不溶性”或“水溶性”药物超微纳米粒子，也可被包裹在油相核心，且FBA-ID/Su-Drug FBA-SD/Su-Drug和FBA-ID/So&SD/Su-Drugs纳米粒子在体外稳定性和体内半衰期等性质上与FBA-ID/So-Drug相当。
上述脂肪酸结合型白蛋白-药物纳米冻干制剂的基本材料，如“静脉注射用长链或中链脂肪酸”、“人血清白蛋白”、“有机溶液或其混合液”和“药物”等，其基本特征如下：
1、本发明所述“静脉注射用长链或中链脂肪酸”，具备下列特征：
(1)为长链或中链脂肪酸，如油酸、亚油酸、肉豆蔻酸、乙酸乙酯；(2)为可人体静脉输注的脂肪乳剂的组成成分；(3)为静脉注射级的脂肪酸；(4)与有机溶液相溶，且不发生任何结构改变或化学反应；(5)可与白蛋白特异性结合。
2、本发明所述“人血清白蛋白”，具备下列特征：
(1)为经有机溶剂或有机溶剂与无水乙醇混合液“萃取”处理的白蛋白，即“裸露”脂肪酸结合位点的HSA；(2)保持完整生理结构的HSA；(3)无免疫原性或抗原性，包括荷载“水溶性”药物的HSA；(4)保持gp60跨膜机制的HSA；(5)可有血液中提取获得或采用基因重组技术生产获得。
3、本发明所述“有机溶剂或其混合液”，具备下列特征：
(1)是与水不相溶或仅微溶于水的有机溶剂；(2)可以溶解脂肪酸；(3)可以溶解、微溶或不溶解药物，但不可与药物发生化学反应或改变药物的性质或结构；(4)不与脂肪酸发生化学反应或改变脂肪酸的性质或结构；(5)有机溶液或其混合液及其处理方法不能影响或改变HSA的结构和各种体内、体外生物学性质；(6)为色谱纯试剂。
4、本发明所述“药物”，具备下列特征：
(1)在有机溶剂和水溶液中稳定，不发生降解、结构与性质变化或化学反应；
(2)“药物”必需是可与HSA结合的“药物”，如依疏水性而与HSA疏水域结合、或依亲水性而与白蛋白亲水基团结合；
(3)由于所选有机溶液和水为互不相溶或仅微溶的有机溶液，根据药物在此两种溶液介质中溶解度的不同，可选择不同的纳米粒子制备技术：
①可溶于有机溶剂，不溶于或微溶于水的药物，如紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、依托泊苷、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱和甲氨蝶呤等，可选用本发明所述FBA-ID/So的制备方法；
②不溶于有机溶剂，可溶于水的药物，如药物阿霉素、表阿霉素、卡铂、假单胞菌外毒素(PE38)等化学、蛋白或多肽类药物等,首选本发明所述FBA-SD/Su的制备方法,还可选择FBA-SD/Ab和FBA-SD/Eb的制备方法。
③不溶于有机溶剂，也不溶于水的药物，如甲氨喋呤，可选择本发明所述FBA-ID/Su的制备方法。
(4)由有机溶剂构成的油相中，所述药物以溶解状态或超微纳米粒子(粒径≤50nm)状态存在，这两种物理“状态”均可制备出水包油型纳米粒子。
本发明所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的理化性质、体外和体内性质与特征
1、分析比较HSA、Abraxane分解所获HSA的圆二色光谱扫描图及其本发明所述各种制备方法所获FBA-Drug NP分解后所获HSA的圆二色光谱扫描图，均未见明显改变，说明不同的制备方法并未显著影响白蛋白的二级结构。
2、本发明所述各种纳米制剂在室温和4℃下的纳米粒子分析表明：各种FBA-Drug NP的体外溶液中的完整性和稳定性明显优于白蛋白结合型紫杉醇(Abraxane)纳米粒子。
3、细胞膜表面富含HSA转运受体蛋白gp60的HUVEC细胞实验显示，各种FBA-Drug NP的细胞内转运显著增强，使进入HUVEC细胞内的药物含量比相应药物的静脉注射剂高约2～6倍；这同时表明，各种纳米粒子的制备方法，均未影响HSA的生理功能和体内性质。
4、在荷瘤裸鼠移植瘤的模型上，初步证实：与Abraxane相比，各种FBA-ID/So-P的血浆半衰期延长50％以上、肿瘤组织内紫杉醇含量增加2～3倍、并产生了更高的抑瘤疗效。
5、在小鼠KB移植瘤上，与Doxil相比，各种FBA-SD/Su-D纳米粒子的血浆半衰期显著延长3～8倍、抑瘤效果显著增强。此外，FBA-SD/Su-D对人乳腺癌细胞的跨膜作用也明显高于Doxil。
6、FBA-SD/Ab-Drug的制备技术显著降低了PE38毒素的抗原性，大幅提高PE38LD50剂量约60倍，故FBA-SD/Ab-PE38具人体应用开发的潜力。
7、与泰素相比，各种FBA-ID/So-P和Abraxane冻干制剂的小鼠LD50和MTD或LD10均极显著增高，增幅分别为44～56倍和53～71倍；而FBA-ID/So-P与Abraxane之间LD50和MTD值未见显著性差别。
本发明的优点：
本发明所述制备方法既可以适用于水不溶性药物，也可以适用于水溶性药物，既可以以溶液形式作为油相的核心，也可以以超微纳米粒子的形式作为油相的核心，所构成纳米粒子主要依赖脂肪酸与白蛋白之间的特异性结合而形成；因此，所形成的纳米粒子在体外溶液中和体内的血液循环中的完整性和稳定性较高，并可借纳米尺度富集于实体肿瘤局部，通过白蛋白gp60受体通道促进药物进入肿瘤细胞内，大幅提高荷载药物的LD50和MTD，进而显著提高化疗药物的抗癌药效。
附图说明
图1为FBA-ID/So-P1透射电镜扫描图，其中：左图为粒子粒径分布情况，右图为单个粒子的形态。
图2为FBA-ID-Do00透射电镜扫描图，其中：左图为粒子粒径分布情况，右图为单个粒子的形态。
图3为FBA-SD/C-D25透射电镜扫描图，其中：左图为为粒子粒径分布情况，有图为单个粒子的形态。
图4为各种纳米粒子冻干制剂白蛋白圆二色谱图，其中：─---HSA，分解所得白蛋白：┄Abraxane，─FBA-ID/So-P-1，FBA-ID/So-M1,FBA-ID/So-V1,FBA-ID/Su-E25,FBA-ID/So-SN381FBA-ID/Su-MTX20──FBA-SD/Su-D25，FBA-SD/Ab-EP20，FBA-ID/So-P&C，FBA-P/So&CP/Su、FBA-Do/So&PE38/Ab，FBA-R/So&D/Eb。
图5为各种FBA-ID/So-P与泰素HUVEC细胞结合率的相比。
图6为FBA-ID/So-P类纳米粒子与Abraxane荷瘤小鼠药效试验。
图7为MCF-7肿瘤细胞体外跨膜实验结果。
图8为FBA-SD-D类纳米制剂与Doxil和多柔比星抑制肿瘤增长的比较。
具体实施方式
本发明所述脂肪酸结合型白蛋白(Fatty-acid Binding Albumin,FBA)-“水不溶性”药物(Insoluble Drug,ID)纳米粒子/溶液型(Solution,So)冻干制剂(Nanoparticle,NP)的制备方法，简称FBA-ID/So法，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
无菌条件下，取人血清白蛋白(HSA)，加注射用水，配制10～200mg/ml，优选20mg～80mg/ml HSA溶液；使用与水不相溶的有机溶剂如三氯甲烷(氯仿)、二氯甲烷、乙酸乙酯或其与乙醇的混合溶液(10～5:5～1，v/v)即液体A，“处理”白蛋白分子结合的脂肪酸，有机溶剂或其混合液与HSA溶液的体积比为0.3％～15％,优选0.5％～5.0％；具体方法是将液体A缓慢滴入5,000～15,000rpm、优选8,000～10,000rpm连续搅拌的HSA溶液中，继续搅拌2～10分钟，优选5±1分钟，获得液体C。此过程称为“萃取”处理白蛋白，是白蛋白结合的脂肪酸萃取与富集于油相的过程(详见实施例5)。
2、油相构成--------溶液B和液体D的制备
将静脉注射用中链或长链脂肪酸溶解于有机溶剂或其混合液中，获溶液B，再用溶液B溶解“水不溶性”药物，获液体D；
其中静脉注射用中链或长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等，为可人体静脉输注的脂肪乳剂的组成成分；“水不溶性”药物包括紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤等一种或几种，是相对水溶性较低或很低的化疗药物或其他药物，可溶解于至少一种“有机溶剂或其混合液”，并可与白蛋白结合；“有机溶剂或其混合液”选三氯甲烷(氯仿)、二氯甲烷、乙酸乙酯或其与无水乙醇等的混合液，为色谱纯试剂；其中，静脉注射用中链或长链脂肪酸以0.01mg/ml～50mg/ml浓度溶解于有机溶剂或其混合液中，“水不溶性”药物以5mg/ml～600mg/ml浓度、优选10mg/ml～400mg/ml浓度溶解于有机溶剂或其混合液中；不同的“水不溶性”药物与不同的“有机溶剂或其混合液”的溶解度不同，“溶解度”是决定“水不溶性”药物“浓度范围”的重要因素。
3、油相与水相的混合------FBA纳米粒子混悬液的制备
将液体D按与液体C的体积比为0.1％～7.2％的比例，缓慢滴入高速搅拌的、液体C中，搅拌速度为3000rpm～30,000rpm，持续时间为10～60分钟；优选搅拌速度为12,000rpm～30,000rpm，持续时间为10～20分钟，高速匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；此为高速搅拌法制备FBA-ID/So纳米粒子,简称FBA-ID/So HS法。
或者将液体D，按与液体C的体积比为0.1％～7.2％的比例，缓慢滴入高速搅拌的、液体C中，搅拌速度为10,000rpm，持续时间为5±1分钟；再将这种处理后的混合悬液迅速加入高压微射流机(M-110P Microfluidizer)进料容器中，在6,000psi～30,000psi高压、避光、4℃条件下，经连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；此为高压匀浆法制备FBA-ID/So纳米粒子,简称FBA-ID/So HP法。
在FBA-ID/So纳米粒子混悬液体系中，液体C中液体A的体积与溶解水不溶性药物的溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为0.4％～10.0％；优选1.0％～6.5％。静脉注射用长链或中链脂肪酸与白蛋白的质量比为0.0001％～5％，优选0.001％～1％；水不溶性药物与白蛋白的质量比为：0.1％～20％，优选1.0％～10％。
4、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
将纳米粒子混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10～20分钟，去除全部有机溶剂，可获半透明乳液。
5、无菌冻干制剂的制备
该半透明乳液经0.45u和/或0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-ID/So-Drug；FBA-ID/So-Drug加生理盐水复溶后，所获FBA-ID/So-Drug溶液纳米粒子的粒径与该无菌乳液冻干前一样。
本发明所述脂肪酸结合型白蛋白(FBA)-“水不溶性”药物(Insoluble Drug,ID)纳米粒子/悬液型(Suspension,Su)冻干制剂(Nanoparticle,NP)的制备方法，简称FBA-ID/Su法，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和液体D’的制备
将静脉注射用中链或长链脂肪酸溶解于有机溶剂或其混合液中，获溶液B，其中中链或长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等；其中，脂肪酸与白蛋白的质量比为：0.001％～1％。
“水不溶性”药物超微纳米粒子的制备：各种技术与方法如机械粉碎法、离子溅射法、冷冻干燥法、激光诱导气相化学反应法等所获“水不溶性”药物超微纳米粒子，均可用于本方法，其中“超微纳米粒子”是指粒径小于等于50nm的纳米粒子(粒径≤50nm)。本发明披露的一种“超微纳米粒子”的制备方法，为高压匀浆法，具体步骤是：将“水不溶性”药物以1～10mg/ml的浓度混合于与该药不相溶的药用级大豆油和维生素E溶液(维生素E含量在0.4～2mg/ml)中，或者与该药不相溶的注射用水和维生素C溶液(维生素C含量在50～100mg/ml)中，在4℃、避光条件下，经20,000～30,000psi高压匀浆处理1～3次，获“水不溶性”药物超微纳米粒子大豆油或注射用水混悬液，再经4℃下、避光、20,000～35,000rpm高速离心，持续10～60mins，去除上清，收集水不溶性药物超微纳米粒子；按3.0mg/ml～600mg/ml的比例，将“水不溶性药物超微纳米粒子”混悬于溶液B中，获得液体D’，为含脂肪酸、水不溶性药物超微纳米粒子和有机溶剂的悬液；
其中，“水不溶性”药物选紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤等一种或几种；不同的“水不溶性”药物的密度和溶解度不同，悬浮于“有机溶剂或其混合液”中所需溶液体积不同，在此，“悬浮”的界定较“浓度范围”的界定更为重要。
3、油相与水相的混合------FBA纳米粒子混悬液的制备
将液体D’，按与液体C的体积比为0.3％～4.0％的比例，缓慢滴入高速搅拌的、液体C中，避光，搅拌速度为3,000rpm～30,000rpm，持续时间为15～60分钟,制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；此为高速搅拌法制备FBA-ID/Su纳米粒子，简称FBA-ID/Su HS法。
或者将液体D’，按与液体C的体积比为0.3％～4.0％的比例，缓慢滴入高速搅拌的、液体C中，搅拌速度为10,000rpm～12,000rpm，持续搅拌5±1分钟，加入高压微射流机(M-110P Microfluidizer)进料容器中，在20,000psi～30,000psi高压、避光、4℃下，经连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；此为高压匀浆法制备FBA-ID/Su纳米粒子，简称FBA-ID/Su HP法。
在FBA-ID/Su纳米粒子混悬液体系中，液体C中液体A的体积与液体D’中溶液B体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：0.6％～5.0％；静脉注射用长链或中链脂肪酸与白蛋白的质量比为0.0001％～0.15％；水不溶性药物与白蛋白的质量比为：1.0％～25％，优选1.0％～10％。
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
同前FBA-ID/So法所述。
6、无菌冻干制剂的制备
同前FBA-ID/So法所述。
所述FBA-SD/Su-Drug冻干制剂加生理盐水或注射用水复溶后，所获FBA-SD/Su-Drug溶液纳米粒子的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白(FBA)-“水溶性”药物(Soluble Drug,SD)纳米粒子/悬液型(Suspension,Su)冻干制剂(Nanoparticle,NP)的制备方法，简称FBA-SD/Su法，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和溶液D’的制备
将静脉注射用中链或长链脂肪酸溶解于有机溶剂或其混合液中，获溶液B，其中中链或长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等；其中，中链或长链脂肪酸与白蛋白的质量比为：0.001％～1％。
“水溶性”药物超微纳米粒子的制备：各种技术与方法如机械粉碎法、离子溅射法、冷冻干燥法、激光诱导气相化学反应法等所获“水溶性”药物超微纳米粒子，均可用于本方法，其中“超微纳米粒子”是指粒径小于等于50nm的纳米粒子(粒径≤50nm)。本发明披露的一种“超微纳米粒子”的制备方法，即高压匀浆法，具体步骤是：将“水溶性”药物以5～10mg/ml的浓度混合于与该药不相溶的药用级维生素E含量在0.4～2mg/ml的大豆油中，在4℃、避光条件下，经15,000～30,000psi高压匀浆处理1～3次，获“水溶性”药物超微纳米粒子大豆油混悬液，再经4℃下、避光、5000～30,000rpm高速离心,持续5～60mins，去除大豆油上清；或者经截流分子量为30～100kDa的超滤膜超滤处理，离心1,000～5,000rpm,10～30分钟,去除超滤液；获得水溶性药物超微纳米粒子；按3.0mg/ml～600mg/ml比例，将水溶性药物超微纳米粒子混悬于溶液B中，获得液体D’,为含脂肪酸、水溶性药物超微纳米粒子和有机溶剂的悬液；
其中，“水溶性”药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素(PE38)等化学、蛋白或多肽类药物等一种或几种；所述“水溶性”药物可溶于水，在“有机溶剂或其混合液”中的溶解度很低；所述不同的“水溶性”药物的密度不同，悬浮于“有机溶剂或其混合液”中所需溶液的体积不同，在此，“悬浮”的界定较“浓度”的界定更为重要。
3、水相构成
由液体C构成，其中HSA溶液的浓度为10～200mg/ml，优选20mg～80mg/ml；
4、油相与水相的混合------FBA纳米粒子混悬液的制备
按体积比为1.5％～2.0％的比例，将液体D’滴入搅拌速度为12,000rpm～22,000rpm的液体C中，继续搅拌5～20分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；此为高速搅拌法制备FBA-SD/Su纳米粒子，简称FBA-SD/Su HS法。
或按体积比为1.5％～2.0％的比例，将液体D’滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机(M-110P Microfluidizer)进料容器中，在4±1℃，避光、压力为20,000psi～30,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；此为高压匀浆法制备FBA-SD/Su纳米粒子，简称FBA-SD/Su HP法。
在FBA-SD/Su纳米粒子混悬液体系中，液体C中液体A的体积与液体D’中溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：1.0％～7.5％；静脉注射用长链或中链脂肪酸与白蛋白的质量比为0.001％～1.0％；水溶性药物与白蛋白的质量比为：0.5％～20％；
5、纳米粒子去除油相中的有机溶剂-----稳定的纳米粒子乳液的制备
同前FBA-ID/So法所述。
6、无菌冻干制剂的制备
同前FBA-ID/So法所述。
所述FBA-SD/Su-Drug加生理盐水或注射用水复溶后，所获FBA-SD/Su-Drug溶液纳米粒子的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白(FBA)-“水溶性”药物(SD)纳米粒子/蛋白结合型(Albumin Binding,Ab)冻干制剂的制备方法，简称FBA-SD/Ab法，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B的制备
将静脉注射用中链或长链脂肪酸溶解于有机溶剂或其混合液中，获溶液B，其中中链或长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等；其中，静脉注射用中链或长链脂肪酸与白蛋白的质量比为：0.001％～1％。
3、水相构成-----白蛋白与药物的结合
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
例如，当配制“水溶性”药物阿霉素(doxorubicin,D)或表阿霉素((Epirubicin，EP)与白蛋白的结合溶液时，使用0.01M～0.1M氢氧化钠调节20～100mg/ml HSA溶液的pH至5.5～6.5；使用注射用水配制2mg/ml阿霉素(doxorubicin，D)或表阿霉素((Epirubicin，EP)，获得高浓度D或EP溶液；将此高浓度溶液缓慢加入HSA溶液中，1,000rpm±100rpm磁力搅拌2小时，获HSA-阿霉素或HSA-表阿霉素结合溶液。
“水溶性”药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素(PE38)等化学、蛋白或多肽类药物等一种或几种。不同的“水溶性”药物与白蛋白的体外结合率不同，在此，“水溶性”药物与液体C中白蛋白的“结合”浓度以及白蛋白溶液浓度，决定“水溶性”药物的加入量或浓度。
4、油相与水相的混合------FBA纳米粒子混悬液的制备
按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；此为高速搅拌法制备FBA-SD/Ab纳米粒子，简称FBA-SD/Ab HS法。
或者，按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌5±1分钟，加入高压微射流机(M-110P Microfluidizer)进料容器中，在4±1℃，避光，压力为10,000psi～25,000psi下，连续1～5个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；此为高压匀浆法制备FBA-SD/Ab纳米粒子，简称FBA-SD/Ab HP法。
在FBA-SD/Ab纳米粒子混悬液体系中，液体C中液体A的体积与溶液B的体积之和为油相，而白蛋白-水溶性药物结合溶液的体积为水相，油相体积与水相体积之比为：1.0％～10.0％；静脉注射用长链或中链脂肪酸与白蛋白的质量比为0.01％～5.0％；水溶性药物与白蛋白的质量比为：0.2％～20％；
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
同前FBA-ID/So法所述。
6、无菌冻干制剂的制备
同前FBA-ID/So法所述。
所述FBA-SD/Ab-Drug加生理盐水或注射用水复溶后，所获FBA-SD/Ab-Drug溶液纳米粒子的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白(FBA)-“水溶性”药物(SD)纳米粒子/空粒结合型(Empty-Nanoparticle Binding,Eb)冻干制剂的制备方法，简称FBA-SD/Eb法，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B的制备
将静脉注射用中链或长链脂肪酸溶解于有机溶剂或其混合液中，获溶液B，其中中链或长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等；其中，静脉注射用中链或长链脂肪酸与白蛋白的质量比为：0.001％～1％。
3、水相构成
水相由液体C构成，其中，HSA溶液的浓度为10～200mg/ml，优选20mg～80mg/ml；
4、油相与水相的混合------FBA纳米粒子混悬液的制备
按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为20,000rpm～25,000rpm的液体C中，继续搅拌10～20分钟，制得脂肪酸结合型白蛋白纳米粒子混悬液；此为高速匀浆法制备FBA-SD/Eb纳米粒子，简称FBA-SD/Eb HS法。
或者，按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟后，再加入高压微射流机进料容器中，在4±1℃下，避光，压力为10,000psi～25,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白纳米粒子混悬液；此为高压匀浆法制备FBA-SD/Eb纳米粒子，简称FBA-SD/Eb HP法。
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
将所述脂肪酸结合型白蛋白纳米粒子混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；其中所含纳米粒子为FBA，该粒子是由脂肪酸和白蛋白构成的“空”纳米粒子(FBA-Empty Nanoparticle，FBA-Enp)；
6、FBA-SD/Enp载药过程
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中的FBA-Enp表面白蛋白结合，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子空粒结合型乳液，FBA-SD/Eb乳液；
所述水溶性药物包括阿霉素、表阿霉素、卡铂、假单胞菌外毒素PE38等。不同的“水溶性”药物与白蛋白的结合率不同，不同粒径的FBA-Enp表面白蛋白的“量”不同，在此，“水溶性”药物与FBA-Enp表面白蛋白的“结合”浓度，决定“水溶性”药物的加入量或加入浓度。
在FBA-SD/Eb纳米粒子乳液体系中，液体C中液体A的体积与溶液B的体积之和为油相，半透明乳液的体积为水相，油相体积与水相体积之比为：2.0％～6.5％；静脉注射用长链或中链脂肪酸脂肪酸与白蛋白的质量比为0.033％～0.5％；水溶性药物与白蛋白的质量比为：2.5％～15％；
7、无菌冻干制剂的制备
该半透明乳液经0.45u和/或0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-SD/Eb-Drug；FBA-SD/Eb-Drug加生理盐水或注射用水复溶后，所获FBA-SD/Eb-Drug溶液纳米粒子的粒径与该无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/悬液型冻干制剂的制备方法，简称FBA-ID/So&SD/Su法，为前述FBA-ID/So和FBA-SD/Su两种粒子制备方法的结合，步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和液体D的制备
1)将静脉注射用中链或长链脂肪酸溶解于液体A中，获溶液B，再用溶液B溶解“水不溶性”药物使浓度为10mg/ml～400mg/ml，制得液体D；
2)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4±1℃下、避光、5,000～30,000rpm持续离心10～60min或经截留分子量为30～100kDa的滤膜超滤，去除上清液，获得水溶性药物超微纳米粒子；
3、水相构成
为液体C，由10～200mg/ml，优选20mg～80mg/ml的HSA溶液构成；
4、油相与水相的混合------FBA纳米粒子混悬液的制备
用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液，即FBA-ID/So&SD/Su纳米粒子混悬液；
或者，用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为3,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液，即FBA-ID/So&SD/Su纳米粒子混悬液；
在FBA-ID/So&SD/Su纳米粒子混悬液体系中，液体C中液体A的体积与溶液B的体积之和构成油相，而HSA溶液的体积构成本体系的水相，油相体积与水相体积之比为：3.5％，静脉注射用长链或中链脂肪酸脂肪酸与白蛋白的质量比为0.033％，“水溶性”药物与白蛋白的质量比为：3.33％；“水不溶性”药物与白蛋白的质量比为：3.33％。
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
同前FBA-ID/So法所述。
6、无菌冻干制剂的制备
同前FBA-ID/So法所述。
所述FBA-ID/So&SD/Su-Drug加生理盐水或注射用水复溶后，所获FBA-ID/So&SD/Su-Drug溶液的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/蛋白结合型冻干制剂的制备方法，简称FBA-ID/So&SD/Ab法，为前述FBA-ID/So和FBA-SD/Ab两种制备方法的结合，具体步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和液体D的制备
同前FBA-ID/So法所述。
即将静脉注射用中链或长链脂肪酸溶解于液体A中，获溶液B，再用溶液B溶解水不溶性药物，获浓度为 10mg/ml～400mg/ml的液体D；
3、水相构成：
同前FBA-SD/Ab法所述。
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
4、油相与水相的混合------FBA纳米粒子混悬液的制备：
按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液，即FBA-ID/So&SD/Ab纳米粒子混悬液；
或者，按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌2～5分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液，即FBA-ID/So&SD/Ab纳米粒子混悬液；
在FBA-ID/So&SD/Ab纳米粒子混悬液体系中，液体C中液体A的体积与溶液B的体积之和，构成本体系的油相，而液体C的体积构成本体系的水相，其中，油相体积与水相体积之比为：4.5％，静脉注射用长链或中链脂肪酸脂肪酸与白蛋白的质量比为0.083％，“水溶性”药物与白蛋白的质量比为：0.083％；“水不溶性”药物与白蛋白的质量比为：3.33％。
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备：
同前FBA-ID/So法所述。
6、无菌冻干制剂的制备：
同前FBA-ID/So法所述。
所述FBA-ID/So&SD/Ab-Drug加生理盐水或注射用水复溶后，所获FBA-ID/So&SD/Ab-Drug溶液纳米粒子的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/空粒结合型冻干制剂的制备方法，简称FBA-ID/So&SD/Eb法，前述FBA-ID/So和FBA-SD/Eb两种粒子制备方法的结合，具体步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和液体D的制备
同前FBA-ID/So法所述。
即用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的溶液D；
3、水相构成：
同前FBA-ID/So法所述。
4、油相与水相的混合------FBA纳米粒子混悬液的制备：
同前FBA-ID/So所述。
按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
或者，按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
5、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备：
将脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；
6、FBA-ID/So的载药过程
调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中FBA-ID/So纳米粒子表面白蛋白结合，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子空粒结合型乳液，即FBA-ID/So&SD/Eb-Drug乳液。
在FBA-ID/So&SD/Eb-Drug乳液体系中，液体C中液体A的体积与溶液B的体积之和，构成本体系的油相，而FBA-ID/So纳米粒子乳液的终体积构成本体系的水相，其中，油相体积与水相体积之比为：7.5％，静脉注射用长链或中链脂肪酸脂肪酸与白蛋白的质量比为0.033％，“水溶性”药物与白蛋白的质量比为：3.33％；“水不溶性”药物与白蛋白的质量比为：3.33％。
7、无菌冻干制剂的制备：
同前FBA-ID/So所述。
所述FBA-ID/So&SD/Eb-Drug冻干制剂加生理盐水或注射用水复溶后，所获FBA-ID/So&SD/Eb-Drug溶液纳米粒子的粒径与其无菌乳液冷冻干燥前一样。
本发明所述脂肪酸结合型白蛋白-两种“水不溶性”药物纳米粒子/溶液型冻干制剂的制备方法，简称FBA-ID/So-D1&D2法，即前述FBA-ID/So法应用于两种“水不溶性”药物，具体步骤包括：
1、“萃取”处理白蛋白--------液体A和液体C的制备
同前FBA-ID/So法所述。
2、油相构成--------溶液B和液体D的制备
将静脉注射用中链或长链脂肪酸溶解于液体A中，获溶液B，再用溶液B溶解“水不溶性”药物D1和D2，获 液体D1&2；其中中链和长链脂肪酸包括油酸、亚油酸、肉豆蔻酸、甘油三酯等，“水不溶性”药物包括紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤等任意两种的组合，“有机溶剂或其混合液”如三氯甲烷(氯仿)、二氯甲烷、乙酸乙酯或其与无水乙醇等的混合溶液；其中，静脉注射用中链或长链脂肪酸浓度为0.01mg/ml～50mg/ml，两种“水不溶性”药物的浓度之和为5mg/ml～600mg/ml，优选10mg/ml～400mg/ml。
3、油相与水相的混合------FBA纳米粒子混悬液的制备
按与溶液C的体积比为0.1％～7.2％的比例，将液体D1&2缓慢滴入高速搅拌溶液C中，以3,000rpm～30,000rpm搅拌、持续搅拌10～60分钟，制得脂肪酸结合型白蛋白-“水不溶性”药物D1&D2纳米粒子溶液型混悬液；
或者，按与溶液C的体积比为5％～6.5％的比例，将液体D1&2缓慢滴入高速搅拌的、液体C中，搅拌速度为10,000rpm，持续时间为5±1分钟；再将这种混合液加入高压微射流机的进料口，在20,000psi～30,000psi压力下，避光、4℃下，循环5～8次；制得脂肪酸结合型白蛋白-“水不溶性”药物D1&D2纳米粒子溶液型混悬液；
上述FBA-ID/So-D1&D2纳米粒子混悬液体系中，液体C中液体A的体积与溶液B的体积之和，构成本体系的油相，而液体C中的白蛋白水溶液的体积构成本体系的水相，油相体积与水相体积之比为：6.5％～11.5％；静脉注射用长链或中链脂肪酸脂肪酸与白蛋白的质量比为0.05％～0.4％；两种“水不溶性”药物之和与白蛋白的质量比为：5％～10％。
4、去除纳米粒子油相中的有机溶剂-----稳定的纳米粒子乳液的制备
同前FBA-ID/So法所述。
5、无菌冻干制剂的制备
同前FBA-ID/So法所述。
所述FBA-ID/So-D1&D2加生理盐水或注射用水复溶后，所获FBA-ID/So-D1&D2溶液纳米粒子的粒径与其无菌乳液冻干前一样。
依据本发明所述各种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，还可以演变或推导出FBA-ID/So&ID/Su法等多种纳米粒子冻干制剂的制备方法及其产物，不再一一赘述。
类似地，以上述制备一种“药物”纳米粒子冻干制剂的制备方法作为基本方法，单独应用于两种或两种以上同类药物，或以上述两种基本方法联合应用于两种或两种以上同类或不同类药物(即“水不溶性”和“水溶性”药物)，制备脂肪酸结合型白蛋白-两种或两种以上药物纳米粒子冻干制剂，均可行，均为本发明构思范畴。
本发明所述FBA-Drug NP的制备方法中，脂肪酸在纳米粒子形成中的作用进一步阐述如下：
1、有机溶剂或其混合液处理血清白蛋白溶液的过程是“萃取”和“富集”脂肪酸于油相中的过程
从人血或牛血中提取出的HSA或BSA均含脂肪酸，不同的HSA或BSA提取方法影响脂肪酸的含量。将有机溶剂或其混合液处理过的HSA溶液，经超速离心30,000rpm 5小时，可见有机相(即油相)与白蛋白溶液相(即水相)的分层，分别取油相和水相的样品，采用酶法(Cayman公司产品)测定游离脂肪酸含量、采用Lowry氏法测定HSA的含量，结果表明(详见实施例5)：有机溶剂处理HSA的过程实际上是一种“萃取”与“富集”脂肪酸的过程，且经此处理有60％以上的游离脂肪酸分布在有机溶剂中，即油相之中；同样的“萃取”处理条件下，若增加有机溶剂的量，则可增加油相中脂肪酸的含量或比例。
2、添加不同剂量脂肪酸影响FBA-Drug纳米粒子的形成
如实施例6所述：称取华兰生物的HSA(10％HSA,50ml)500毫克，加注射用水，配制成50mg/ml HSA溶液10ml；将氯仿和乙醇混合溶液(8:3，v/v)100ul，缓慢滴入5,000rpm的50mg/ml HSA溶液，持续10分钟。分别将不同剂量的脂肪酸如肉豆蔻酸0(不加),1mg,2mg和4mg与紫杉醇100mg溶于200ul氯仿和乙醇混合溶液中，再将这种含有肉豆蔻酸、紫杉醇、氯仿和乙醇的混合溶液缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，搅拌速度为10,000rpm，持续时间为5分钟；再将这种混合溶液经高压微射流机(M-110P Microfluidizer)，在25,000psi压力下，4℃下，循环8次；将此混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10分钟，去除全部有机溶剂，可获得半透明乳液，样品经粒度分析(马尔文Nano Zetasizer ZS90)，表明：不加脂肪酸或加入过量脂肪酸，其紫杉醇纳米粒子的粒径均呈双峰，且平均粒径均大于300nm；而加入“适量”的脂肪酸如前述添加1mg和2mg的肉豆蔻酸，均获得了粒径在80～150nm和90～240nm之间稳定的平均粒径小于180nm的纳米粒子(详见表2)。上述实验结合实施例7所述，提示：油相中脂肪酸的含量影响纳米粒子的形成和粒径的大小，脂肪酸含量过低或过高超出了有机溶液的溶解度均影响纳米粒子的形成。
3、本制备体系与工艺中，若脂肪酸含量过低，则不能形成FBA-Drug纳米粒子
如实施例7所述，采用常规的离子交换层析，纯化HSA，使HSA溶液中脂肪酸的含量低于0.1ug/mgHSA，同时，在油相中也不添加脂肪酸(如油酸、亚油酸、肉豆蔻酸等)，在其它制备条件与工艺参数不变的条件下，根本不能形成FBA-Drug纳米粒子。这说明，脂肪酸是本FBA-Drug纳米粒子形成机制中不可缺少的重要因素和主要构成成分之一。
4、纳米粒子核心区和外周白蛋白游离脂肪酸的含量分析，进一步提供了脂肪酸对纳米粒子形成具重要作用的证据。
如实施例8所述，采用超滤技术，分离纳米粒子核心区白蛋白，即直接与纳米核心紫杉醇和/或脂肪酸结合的白蛋白和纳米粒子外周白蛋白，分别测定其游离脂肪酸的含量，意外地发现：以本发明所述技术制备的平均粒径小于160nm的纳米粒子溶液，采用100kDa分子量截流的超滤管(Millipore公司)5000rpm离心超滤20分钟，分别收集超滤液和过滤膜上的浓缩液，用Lowry氏法和酶法(Cayman公司产品)定量白蛋白和游离脂肪酸，据此计算出每毫克蛋白所含游离脂肪酸的含量，结果发现其共同特征是:纳米粒子的核心区白蛋白游离脂肪酸的含量超过纳米粒子外周白蛋白游离脂肪酸3倍以上，且核心区每一白蛋白分子平均结合2～5个脂肪酸分子。
5、FBA-ID/So-Drug和FBA-SD/Su-Drug纳米粒子的形成机制表明，单纯脂肪酸就可以形成纳米粒子，如实施 例4(FBA)、9(FBA-ID/So-Do00)和19(FBA-SD/Ab-D0)所示，在制备纳米粒子的油相中，仅加入脂肪酸，就可以形成纳米粒子，且这种纳米粒子较核心含有紫杉醇或外周白蛋白结合阿霉素所形成的纳米粒子的体外稳定性无异甚或略优。
本发明所述各种FBA-Drug NP的理化性质、体外和体内活性与特征
1、圆二色光谱是研究稀溶液中蛋白质构象的一种快速、简单而准确的方法,可以在溶液状态下、接近生理状态下测定,对构象变化灵敏，所以是目前研究蛋白质二级结构的主要手段之一。比较华兰生物HSA、Abraxane分解所获HAS以及本发明专利所述各种制备方法所获FBA-Drug NP如实施例7FBA-ID/So-P7-1和实施例15FBA-SD/Su-D25样品等等分解后所获HSA的圆二色光谱扫描图，未见明显改变，说明：不同的制备方法并未显著影响白蛋白的二级结构。扫描结果详见实施例27。
2、本发明各种类型纳米制剂在室温和4℃下的稳定性试验表明：各种类型的脂肪酸结合型白蛋白-药物纳米粒子(FBA-Drug NP)的体外稳定性明显优于白蛋白结合型紫杉醇(Abraxane)，详见实施例28。
3、人脐血管内皮细胞(HUVEC)的细胞膜表面富含gp60受体，是HSA的生理转运通道。采用荧光标记的紫杉醇，按1:50比例加入紫杉醇中，制备各种FBA-ID/So-P纳米粒子，即F-FBA-ID/So-P；以相同添加比例，制备荧光标记的紫杉醇注射液，即F-Taxol。在HUVEC细胞培养孔中，加入相同紫杉醇(Paclitaxel,P)浓度的F-FBA-ID/So-P和F-Taxol，37℃下，孵育1小时后，发现：各种F-FBA-ID/So-P与HUVEC细胞的结合率较F-Taxol高约10倍左右，这说明FBA-ID/So-P可加强细胞膜表面带有gp60受体细胞紫杉醇的转运。进一步的实验表明：各种FBA-Drug NP进入HUVEC细胞内的药物含量比相应药物的静脉注射剂高约2～6倍；这表明本发明所述各种纳米粒子的制备方法，均未影响HAS的生理功能和性质，详见实施例29。
4、采用ELISA法测定血液循环中以完整纳米粒子形式存在的各种FBA-ID/So-P的浓度，作为判断其在动物体内稳定性的指标，即由包埋在96孔板底部的抗紫杉醇抗体捕获血清中FBA-ID/So-P，再由生物素标记的抗HSA抗体检测“FBA-ID/So-P”的含量，计算出血浆半衰期；采用HPLC法测定组织中紫杉醇的含量，以判断FBA-ID/So-P在肿瘤组织的聚集特征；在荷瘤裸鼠HT29移植瘤模型上，初步判断FBA-ID/So-P的抑瘤药效；结果表明：在动物血液循环中各种FBA-ID/So-P具更好的稳定性，其血浆半衰期较Abraxane长50％以上；紫杉醇在肿瘤组织内的含量也增加约2～3倍，并产生了更好的抑瘤效果。实验详细结果详见实施例30和实施例31。
5、基于前述“3”和“4”同样的方法，发现：各种FBA-SD/Su-D纳米粒子的血浆半衰期显著长于Doxil 3～8倍,且与Doxil和阿霉素注射液相比，显著增强了对KB移植瘤的抑制作用。此外，FBA-SD/Su-D对人乳腺癌细胞(MCF-7)的跨膜作用也明显高于Doxil。
6、FBA-SD/Ab的制备技术显著降低了抗原性很强的PE38毒素的抗原性，也大幅提高了PE38LD50剂量约60倍，因此，FBA-SD/Ab-PE38是具有人体应用开发的潜力的纳米制剂。
7、使用Babl/c小鼠，在标准GLP的环境下，单次静脉注射各种FBA-ID/So-P和Abraxane冻干制剂复溶液以及泰素溶液，测定小鼠的LD50和MTD或LD10，结果表明：各种FBA-ID/So-P和Abraxane冻干制剂复溶液的LD50和MTD值均显著高于泰素，增幅分别为44～56倍和53～71倍；而FBA-ID/So-P与Abraxane之间以及各种FBA-ID/So-P之间LD50和MTD值未见显著性差别，详见实施例33。
本发明所述脂肪酸结合型白蛋白-药物纳米冻干制剂(FBA-Drug NP)的制备方法，有效利用了脂肪酸与白蛋白的生理性、高亲和力与稳定的结合特性，在高剪切力的作用下，制备以脂肪酸为核心之一的纳米粒子，实现了“水不溶性”或“水溶性”药物、以“溶液”或“超微纳米粒子”形式、一种或几种“包裹”或“结合”其中，极大地扩展了以白蛋白为主要支架材料的纳米粒子制备方法的应用范围；所获FBA-Drug NP仅由白蛋白、药物和脂肪酸组成，不含其它任何辅料或有机溶剂等；所获FBA-Drug NP，体内血液循环和体外溶液的完整性与稳定性显著提高，通过HSA的gp60生理转运机制，大幅提高了富含gp60受体细胞内或肿瘤组织中的化疗药含量，并进而导致荷瘤裸鼠抑瘤率的显著提高。此外，FBA-Drug NP还可封闭所“包裹”或“结合”蛋白的抗原位点，并可显著提高荷载药物的LD50和MTD。为新型药物的开发和临床应用奠定了基础。
下面结合具体实施范例对本发明作进一步的说明与阐述，但本发明所涉及的内容与范围不仅仅限于如下范例所述。
实施例1
脂肪酸结合型白蛋白(Fatty-acid Binding Albumin,FBA)“水不溶性”药物(Insoluble Drug，ID)纳米粒子/溶液型(Solution，So)冻干制剂的制备方法(简称FBA-ID/So法)
-------FBA-ID/So法制备姜黄素(Curcumin,C)纳米粒冻干制剂(FBA-ID/So-C)
无菌条件下，取Sigma人血白蛋白(HSA)1000mg，加注射用水，配制10mg/ml HSA 100ml；将氯仿和乙醇混合液300ul(9:1,v/v)即液体A缓慢滴入10,000转/分钟(10,000rpm)的10mg/ml HSA溶液中，继续搅拌10分钟，获液体C(留样S1)。
将1ug静脉注射用油酸与1mg姜黄素(Curcumin,C)共溶于100ul氯仿乙醇混合溶液(9:1,v/v)中(液体D，其中含溶液B即1ug静脉注射用油酸/100ul氯仿乙醇混合溶液＝0.01mg/ml)，再将这种含有油酸、姜黄素、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经氯仿乙醇处理的10mg/ml HSA溶液中，搅拌速度为3000rpm，持续时间为60分钟；将这种黄色混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发15分钟，去除全部有机溶剂。可获黄色乳液，其中姜黄素粒子的粒径为52～116纳米(nm)(英国Malvern Nano Zetasizer ZS90)。
将此黄色乳液经0.22u过滤膜滤过可去除溶液中的细菌等微生物，成为无菌溶液；这种无菌溶液，经真空冷冻干燥24小时，可得脂肪酸结合型白蛋白-姜黄素纳米粒冻干制剂(FBA-ID/So-C)。
实施例2：FBA-ID/So法制备雷帕霉素(Rapamycin,R)纳米粒子冻干制剂(FBA-ID/So-R)
无菌条件下，取Sigma HSA 1800毫克，加注射用水，配制100mg/ml HSA 18ml；将二氯甲烷500ul(液体A)，缓慢滴入8,000rpm的100mg/ml HSA溶液，持续5分钟，获液体C(留样S2)。将90ug静脉注射用亚油酸与20mg 雷帕霉素(Rapamycin,R)共溶于1300ul二氯甲烷溶液中(液体D)，再将这种含有亚油酸、雷帕霉素和二氯甲烷的混合溶液即液体D缓慢滴入高速搅拌的经二氯甲烷处理的100mg/ml HSA溶液中，搅拌速度为12,000rpm，持续时间为20分钟；将这种混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂，可获半透明乳液，其中雷帕霉素纳米粒子的粒径为88～243nm。
该半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白-雷帕霉素纳米粒冻干制剂(FBA-ID/So-R)；FBA-ID/So-R加生理盐水或注射用水复溶后，所获雷帕霉素纳米粒子的粒径与冻干前一致。
实施例3：FBA-ID/So法制备紫杉醇(Paclitaxel,P)纳米粒子冻干制剂(FBA-ID/So-P)
无菌条件下，取Sigma HSA 900毫克，加注射用水，配制100mg/ml HSA 9ml；将氯仿和乙醇混合溶液(6:5，v/v)250ul(液体A)，缓慢滴入10,000rpm的100mg/ml HSA溶液，持续5分钟，获液体C(留样S3)。将9mg静脉注射用肉豆蔻酸与100mg紫杉醇(Paclitaxel,P)共溶于650ul氯仿和乙醇混合溶液中(液体D)，再将这种含有肉豆蔻酸、紫杉醇、氯仿和乙醇的混合溶液缓慢滴入高速搅拌的经上述有机溶剂处理的100mg/ml HSA溶液中，搅拌速度为20,000rpm，持续时间为10分钟(此为高速匀浆法，即High Speed Homogenizing,HS)；或者将这种含有肉豆蔻酸、紫杉醇、氯仿和乙醇的混合溶液缓慢滴入高速搅拌的经氯仿和乙醇混合溶液处理的100mg/ml HSA溶液中，搅拌速度为10,000rpm，持续时间为5分钟，再将这种混合液经高压微射流机(M-110P Microfluidizer)，在20,000psi压力下，4℃下，循环6次(此为高压匀浆法，即High Pressure Homogenizing,HP)；所获混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10分钟，去除全部有机溶剂，可得半透明乳液，其中紫杉醇纳米粒子的粒径为73～221nm；
该半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白-紫杉醇纳米粒冻干制剂(FBA-ID/So-P-1)；FBA-ID/So-P-1加生理盐水或注射用水复溶后，所获紫杉醇纳米粒子的粒径与冻干前相同。
实施例4：FBA-ID/So法制备紫杉醇纳米粒子冻干制剂(FBA-ID/So-P)
无菌条件下，取Sigma人血白蛋白(HSA)1800毫克，加注射用水，配制200mg/ml HSA 9ml；将乙酸乙酯溶液1350ul(液体A)，缓慢滴入15,000rpm的200mg/ml HSA溶液，持续2分钟，获获液体C(留样S4)。将90mg静脉注射用甘油三酯与100mg紫杉醇(Paclitaxel,P)共溶于250ul乙酸乙酯溶液中(液体D)，或仅将90mg甘油三酯溶于250ul乙酸乙酯溶液中(溶液B)，再将这种含有甘油三酯、紫杉醇、乙酸乙酯的混合溶液或含有甘油三酯和乙酸乙酯的混合溶液分别缓慢滴入高速搅拌的经“乙酸乙酯”处理的200mg/ml HSA溶液中，搅拌速度为30,000rpm，持续时间为10分钟；所得两种混悬液，迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂，可获两种半透明乳液，其中紫杉醇纳米粒子的粒径为83～255nm，不含紫杉醇的纳米粒子的粒径为57～181nm。
两种半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白-紫杉醇纳米粒冻干制剂(FBA-ID/So-P-3)和脂肪酸结合型白蛋白纳米粒冻干制剂(FBA)；FBA和FBA-ID/So-P-3加生理盐水或注射用水复溶后，所获纳米粒子的粒径与冻干前一样。
实施例5：有机溶剂“处理”白蛋白与脂肪酸在油相和水相中的分布
将前述实施例1～4中的样品S1～S4分别在8℃下、超速离心30,000rpm 5小时，可见有机相(即油相)与白蛋白相(即水相)的分层，分别取样，采用酶法(Cayman公司产品)测定油相和水相的游离脂肪酸含量、采用Lowry氏法测定HSA的含量，结果详见表1；对实施例1S1样品增加有机溶剂处理，如S1-1样品和S1-2样品为分别添加氯仿和乙醇混合液450ul和600ul(9:1,v/v)。
表1 有机溶剂“处理”白蛋白与脂肪酸在油相和水相中的分布
Figure PCTCN2015080977-appb-000001
★为sigma HSA未经处理每毫克白蛋白所含游离脂肪酸的含量。
由表1可以得出：有机溶剂处理HSA实际上是一种“萃取和富集脂肪酸”的过程(下简称“萃取”处理)，且经处理60％以上的游离脂肪酸分布在有机溶剂中，即油相之中；而且在此“萃取”处理基础上，按所述纳米粒子制备方法，单纯加入有机溶剂(不加脂肪酸和紫杉醇)“处理”HSA，则可增加油相中脂肪酸的含量或比例。
实施例6：加入不同剂量脂肪酸对FBA-ID/So-P粒径的影响
无菌条件下，取华兰生物工程股份有限公司的人血白蛋白(HSA,10％50ml)500毫克，加注射用水，配制成50mg/ml HSA 10ml；将氯仿和乙醇混合溶液(8:3，v/v)100ul(液体A)，缓慢滴入5,000rpm的50mg/ml HSA溶液，持续10分钟，获液体C。分别将0,1mg,2mg,4mg静脉注射用肉豆蔻酸与100mg紫杉醇溶于350ul氯仿和乙醇混合溶液中(为液体D，各样品编号分别为FBA-ID/So-P0,FBA-ID/So-P1,FBA-ID/So-P2,FBA-ID/So-P4)，再将这种含有肉豆蔻酸、紫杉醇、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液 中，搅拌速度为10,000rpm，持续时间为5分钟；这种混合溶液经高压微射流机，在25,000psi压力下，4℃下，循环8次；将此混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10分钟，去除全部有机溶剂，可获半透明乳液，其中紫杉醇纳米粒子的粒径分别为双峰(平均粒径大于300nm),93～247nm,85～151nm和双峰(平均粒径大于300nm),详见表2。
上述半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-ID/So-P冻干制剂。
表2 加入不同剂量脂肪酸对FBA-ID/So-P粒径测定影响
Figure PCTCN2015080977-appb-000002
实施例7：白蛋白脂肪酸含量和游离脂肪酸加量与FBA-ID/So-P纳米粒子的形成关系
酶法(Cayman公司产品)测定100mg/ml白蛋白溶液中Sigma HSA和华兰生物HSA的游离脂肪酸含量，结果表明，两者相差约10倍，分别为5.4ug/mg和0.55ug/mg白蛋白。
无菌条件下，分别取Sigma HSA和华兰生物HSA各900毫克，加注射用水，配制100mg/ml HSA 9ml,将氯仿和乙醇混合溶液(7:1,v/v)250ul(液体A)，缓慢滴入10,000rpm的100mg/ml HSA溶液，持续5分钟，获液体C。将100mg紫杉醇和5mg静脉注射用油酸溶于300ul氯仿和乙醇混合溶液中(液体D)，再将这种含有紫杉醇、油酸、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的100mg/ml HSA溶液中，搅拌速度为10,000rpm，继续搅拌为5分钟；这种混合溶液经高压微射流机，在20,000psi压力下，4℃下，循环8次；所得混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10分钟，去除全部有机溶剂，可获半透明乳液，其中紫杉醇纳米粒子的粒径分别为62～240nm(平均粒径160nm,命名为FBA-ID/So-P7-1)和113～358nm(平均粒径293nm)。
按上述方法，若将华兰生物HSA(100mg/ml)的油酸加量增加一倍，即将“100mg紫杉醇和10mg油酸”溶于300ul氯仿和乙醇混合溶液中”，其他完全同上法处理，则可获得的半透明乳液，其中紫杉醇纳米粒子的粒径为58～237nm(平均粒径160nm,命名为FBA-ID/So-P7-2)。
采用离子交换层析(Q Sepharose Fast Flow)先分离、纯化Sigma HSA和华兰生物HSA，去除HSA中的脂肪酸，使Sigma HSA和华兰生物HSA脂肪酸含量均小于0.10ug/mg白蛋白，若不加5mg油酸，按上述方法，不能制备出纳米粒子。
此实验表明，在一定的有机溶剂和紫杉醇剂量条件下，HSA结合脂肪酸的含量和油酸的使用剂量，均影响FBA-ID/So-P纳米粒子的形成；在本制备体系中，若油相和水相中脂肪酸含量过低，则不能形成纳米粒子。
实施例8：FBA-ID/So-P纳米粒子的核心和外周白蛋白游离脂肪酸的含量比较
取上述平均粒径小于160nm的纳米粒子溶液(Z-ave﹤160nm纳米粒子)即FBA-ID/So-P1、FBA-ID/So-P7-1和FBA-ID/So-P7-22ml,加入100kDa分子量截流的超滤管(Millipore)中，5000rpm离心20分钟，分别回收超滤液和过滤膜上浓缩液，分别用Lowry氏法和酶法(Cayman公司产品)测定白蛋白和游离脂肪酸的含量，据此计算每毫克蛋白所含游离脂肪酸的含量，详见表3所示,结果发现其共同特征是:FBA-ID/So-P纳米粒子的核心区白蛋白游离脂肪酸的含量超过FBA-ID/So-P纳米粒子外周白蛋白游离脂肪酸3倍以上,即Core/Outer FA大于3；且核心区游离脂肪酸的摩尔数比白蛋白含量摩尔浓度(即FA/Amol,)为2～5:1,即核心区每一白蛋白分子平均结合2～5个脂肪酸分子。
表3 Z-ave 160nm纳米粒子Core/Outer FA和FA/Amol
Figure PCTCN2015080977-appb-000003
实施例9：加入不同剂量多烯紫杉醇对FBA-ID/So-Do粒径的影响
无菌条件下，取Sigma的人血清白蛋白(HSA)500毫克，加注射用水，配制成50mg/ml HSA 10ml；将氯仿和乙醇混合溶液(5:4,v/v)150ul(液体A)，缓慢滴入8,000rpm的50mg/ml HSA溶液，持续5分钟“萃取”处理，获液体C。分别将0，20mg,40mg,80mg多烯紫杉醇(Docetaxel,Do)和100ug静脉注射用油酸溶于200ul氯仿和乙醇混合溶液中(液体D，样品编号FBA-ID/So-Do00，FBA-ID/So-Do20,FBA-ID/So-Do40和FBA-ID/So-Do80)，再将这种含有油酸、多烯紫杉醇、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，搅拌速度为12,000rpm，持续时间为4分钟；这种混合溶液经高压微射流机，在6,000psi压力下，4℃下，循环8次；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶 剂；测定所获半透明乳液，其中多烯紫杉醇纳米粒子的粒径分别为118～286nm，103～254nm，95～227nm和81～157nm。详见表4。上述半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-ID/So-Do冻干制剂。
表4 加入不同剂量多烯紫杉醇对FBA-ID/So-Do粒径的影响
Figure PCTCN2015080977-appb-000004
实施例10：加入不同体积有机溶剂对FBA-ID/So-丝裂霉素纳米粒子粒径的影响
无菌条件下，取华兰生物HAS 500毫克，加注射用水，配制成50mg/ml HSA 10ml；将乙酸乙酯80ul，缓慢滴入8,000rpm的50mg/ml HSA溶液，持续5分钟“萃取”处理白蛋白溶液。在100ul，200ul和400ul乙酸乙酯中,分别加入5mg丝裂霉素(Mitomycin C,M)和100ug油酸(液体D，样品编号FBA-ID/So-M1，FBA-ID/So-M2和FBA-ID/So-M4)，再将这种含有油酸、丝裂霉素、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，搅拌速度为10,000rpm，持续时间为5分钟；这种混合溶液经高压微射流机，在30,000psi压力下，4℃下，循环3次；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；测定所获半透明乳液，其中丝裂霉素纳米粒子的粒径分别为54～183nm，108～263nm和53～351nm。
实施例11：FBA-ID/So法制备长春新碱(Vincristine,V)纳米粒子冻干制剂(FBA-ID/So-V)
无菌条件下，取Sigma的HSA 500毫克，加注射用水，配制成50mg/ml HSA 10ml；将氯仿和乙醇混合溶液(5:1,v/v)100ul(液体A)，缓慢滴入8,000rpm的50mg/ml HSA溶液，持续5分钟，获液体C。分别将1mg,5mg,10mg和20mg长春新碱(Vincristine,V)和200ug亚油酸溶于200ul氯仿和乙醇混合溶液中(液体D，样品编号FBA-ID/So-V1，FBA-ID/So-V5,FBA-ID/So-V10和FBA-ID/So-V20)，再将这种含有亚油酸、长春新碱、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，搅拌速度为10,000rpm，持续时间为5分钟；这种混合溶液经高压微射流机，在25,000psi压力下，4℃下，循环6次；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；测定所获半透明乳液，其中长春新碱纳米粒子的粒径分别为FBA-ID/So-V155～185nm，FBA-ID/So-V562～207nm，FBA-ID/So-V1064～223nm和FBA-ID/So-V2081～254nm。上述半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-ID/So-V冻干制剂。
实施例12：FBA-ID/So法制备7-乙基-10-羟基喜树碱(7-Ethyl-10-hydroxyl-camptothecin，SN38)纳米粒子冻干制剂(FBA-ID/So-SN38)
无菌条件下，取Sigma的HSA 600毫克，加注射用水，配制成60mg/ml HSA 10ml；将氯仿和乙醇混合溶液(6:1,v/v)150ul(液体A)，缓慢滴入5,000rpm的60mg/ml HSA溶液，持续5分钟“萃取”处理，获液体C。分别将1mg和5mg 7-乙基-10羟基喜树碱(7-Ethyl-10-hydroxycamptothecin，SN38)和60ug肉豆蔻酸溶于200ul或350ul氯仿和乙醇混合溶液中(液体D，样品编号FBA-ID/So-SN381和FBA-ID/So-SN385)，再将这种含有肉豆蔻酸、SN38、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的60mg/ml HSA溶液中，在避光、4℃下搅拌速度为22,000rpm，持续时间为10分钟；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；测定所获半透明乳液，其中SN38纳米粒子的粒径分别为FBA-ID/So-SN38155～153nm和FBA-ID/So-SN38572～213nm。上述半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥24小时，获得FBA-ID/So-SN38冻干制剂。
实施例13：FBA-ID/So法制备紫杉醇(Paclitaxel,P)和姜黄素(Curcumin,C)两种“水不溶性”药物纳米粒子冻干制剂(FBA-ID/So-P&C)
无菌条件下，取Sigma HAS 500毫克，加注射用水，配制50mg/ml HSA 10ml；将氯仿和乙醇混合溶液(6:4，v/v)150ul(液体A)，缓慢滴入10,000rpm的50mg/ml HSA溶液，持续5分钟，获液体C。将2mg肉豆蔻酸、25mg紫杉醇(Paclitaxel,P)和25mg姜黄素(Curcumin,C)共溶于500ul氯仿和乙醇混合溶液中(液体D)，再将这种含有肉豆蔻酸、紫杉醇、姜黄素、氯仿和乙醇的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，4℃下、避光、搅拌，持续5分钟，搅拌速度为10,000rpm；这种混合液再经高压微射流机，在40,000psi压力、避光、4℃下，循环2次；将此混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发10分钟，去除全部有机溶剂，可获半透明乳液，其中紫杉醇和姜黄素纳米粒子的粒径为57～211nm。
该半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥30小时，获得脂肪酸结合型白蛋白-紫杉醇-姜黄素纳米粒冻干制剂(FBA-ID/So-P&C)；FBA-ID/So-P&C加生理盐水或注射用水复溶后，所获紫杉醇-姜黄素纳米粒子的粒径与冻干前一样。
实施例14：FBA-ID/So法制备紫杉醇和雷帕霉素(Rapamycin,R)两种“水不溶性”药物纳米粒子冻干制剂(FBA-ID/So-P&R)
无菌条件下，取Sigma的HSA 1000毫克，加注射用水，配制成100mg/ml HSA 10ml；将氯仿溶液500ul(液体A)，缓慢滴入5,000rpm的100mg/ml HSA溶液，持续10分钟“萃取”处理，获液体C。将0.5mg肉豆蔻酸、30mg 紫杉醇((Paclitaxel,P)和20mg雷帕霉素(Rapamycin,R)共溶于650ul氯仿溶液中(液体D)，再将这种含有肉豆蔻酸、紫杉醇、雷帕霉素、氯仿的混合溶液即液体D缓慢滴入高速搅拌的经“萃取”处理的100mg/ml HSA溶液中，搅拌速度为10,000rpm，持续时间为5分钟；这种混合液经高压微射流机，在30,000psi压力下，4℃下，循环8次；将此混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂，可获半透明乳液，用激光粒度仪测定所获乳液中纳米粒子的粒径，结果为72～230nm。
该种乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为一种无菌乳液；经真空冷冻干燥36小时，获得脂肪酸结合型白蛋白-紫杉醇-雷帕霉素纳米粒冻干制剂(FBA-ID/So-P&R)；加生理盐水或注射用水复溶FBA-ID/So-P&R后，所获纳米粒子的粒径为69～225nm，与冻干前相同。
实施例13和14为前述FBA-ID/So应用于两种“水不溶性”药物的制备方法的。
实施例15：脂肪酸结合型白蛋白(FBA)-“水溶性”药物(Soluble Drug，SD)纳米粒子/悬浮型(Suspension,Su)冻干制剂的制备方法(简称FBA-SD/Su法)
----FBA-SD/Su法制备阿霉素(doxorubicin,D)纳米粒子冻干剂(FBA-SD/Su-D)
无菌条件下，取Sigma的HSA 1000毫克，加注射用水，配制成50mg/ml HSA 20ml；将氯仿和乙醇混合溶液(5:1,v/v)150ul(液体A)，缓慢滴入5,000rpm的50mg/ml HSA溶液，持续10分钟“萃取”处理，获液体C，或滴入15,000rpm的50mg/ml HSA溶液，持续2分钟“萃取”处理，也可获液体C。分别将5mg，25mg和50mg阿霉素(doxorubicin,D)混合于药用级大豆油8ml(维生素E含量在0.4～2mg/ml)中；在4℃、避光条件下，经15,000psi高压匀浆处理3次，获阿霉素超微纳米粒子大豆油混悬液，再经4℃下、避光高速离心，转速20000rpm,持续60mins去除大豆油上清，获得阿霉素超微纳米粒子。取1mg静脉注射用油酸溶于300ul氯仿和乙醇混合溶液(5:1,v/v)中(溶液B)，加入收集的阿霉素超微纳米粒子试管中，充分搅拌，即获阿霉素超微纳米粒子有机溶剂悬液(液体D’，样品编号D5，D25和D50)；再将这种含有油酸、阿霉素、氯仿和乙醇的混合溶液即液体D’缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中(在4℃和避光条件下)，搅拌速度为22,000rpm，持续时间为20分钟(此为高速匀浆法,HS)；或者将前述搅拌速度下调至10,000rpm,持续搅拌6分钟，或调至12,000rpm,持续搅拌4分钟，再将所得混合物经高压微射流机，在20,000psi压力下、避光、4℃下，循环6次(此为高压匀浆法，HP)；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；获得半透明红色乳液，样品编号FBA-SD/Su-D5，FBA-SD/Su-D25和FBA-SD/Su-D50；将此三种半透明红色乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为三种无菌乳液；分别经真空冷冻干燥24小时(条件)，得到FBA-SD/Su-D冻干制剂，即FBA-SD/Su-D冻干制剂。
采用激光粒度仪(英国Malvern Nano Zetasizer ZS90)测定粒径，其中HS法所得三种半透明红色乳液FBA-SD/Su-D5，FBA-SD/Su-D25和FBA-SD/Su-D50的粒径分别为52～155nm，61～167nm和65～192nm；三种FBA-SD/Su-D冻干制剂的复溶悬液的粒径分别是55～159nm，63～168nm和68～193nm；而“阿霉素超微纳米粒子大豆油混悬液”的平均粒径小于等于50nm。
实验结果表明：应用脂肪酸结合白蛋白制备纳米粒子的方法，不但溶解于有机溶剂中的“水不溶性”药物如紫杉醇，可以制备纳米粒子，而且，悬浮于有机溶剂中的水“水溶性”药物如阿霉素的超微纳米粒子也可以制备纳米粒子。
其它制剂配方和条件所获FBA-SD/Su-D,详见表5所示。
表5 不同制剂配方和条件制备的FBA-SD/Su-D
Figure PCTCN2015080977-appb-000005
实施例16:脂肪酸结合型白蛋白(FBA)-“水不溶性”药物(Insoluble Drug，ID)纳米粒子/悬浮型(Suspension,Su)冻干制剂的制备方法(简称FBA-ID/Su法)
-------FBA-ID/Su法制备依托泊苷(Etoposide,E)纳米粒子冻干制剂(FBA-ID/Su-E)
无菌条件下，取Sigma的HSA 500毫克，加注射用水，配制成50mg/ml HSA 10ml；将氯仿和乙醇混合溶液(10:1,v/v)100ul(液体A)，缓慢滴入8,000rpm的50mg/ml HSA溶液，持续5分钟“萃取”处理白蛋白，获液体C。分别将20mg和50mg依托泊苷(Etoposide,E)混合于药用级大豆油(维生素E含量不低于0.4mg/ml)8ml中，在4℃、避光条件下，经25,000psi高压匀浆处理2次，获依托泊苷超微纳米粒子大豆油混悬液，再经4℃下、避光高速离心，转速35000rpm,持续10mins去除大豆油上清，获得依托泊苷超微纳米粒子；取300ug油酸溶于400ul氯仿和乙 醇混合溶液(10:1,v/v)中(溶液B)，将此液充分悬浮收集的依托泊苷超微纳米粒子，即获依托泊苷超微纳米粒子有机溶剂悬液(液体D’，样品编号E25和E50)；再将这种含有油酸、依托泊苷、氯仿和乙醇混合溶液即液体D’缓慢滴入高速搅拌的经“萃取”处理的50mg/ml HSA溶液中，搅拌速度为12,000rpm，继续搅拌4分钟；这种混合溶液经高压微射流机，在15,000psi压力下，4℃下，循环6次；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；获得半透明乳液，样品编号FBA-ID/Su-E25和FBA-ID/Su-E50；将此两种半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为两种无菌乳液；分别经真空冷冻干燥30小时(条件)，得到FBA-ID/C-E冻干制剂。
采用激光粒度仪测定粒径，其中两种半透明乳液FBA-ID/Su-E25和FBA-ID/Su-E50的粒径分别为62～178nm和61～206nm；两种FBA-ID/Su-E冻干制剂的复溶悬液的粒径分别是65～172nm和66～210nm；而“依托泊苷超微纳米粒子大豆油混悬液”的平均粒径小于等于50nm。
实验结果表明：应用脂肪酸结合白蛋白制备纳米粒子的方法，不但溶解于有机溶剂中的“水不溶性”药物如紫杉醇，可以制备纳米粒子，而且，因溶解度的限制不能完全溶解而悬浮于有机溶剂中的“水不溶性”药物如依托泊苷的超微纳米粒子也可以制备纳米粒子。
实施例17：FBA-ID/Su法制备甲氨蝶呤(Methotrexate,MTX)纳米粒子冻干剂(FBA-ID/Su-MTX)
无菌条件下，取Sigma的HSA 2000毫克，加注射用水，配制成25mg/ml HSA 80ml；将静脉注射用乙酸乙酯溶液800ul(液体A)，缓慢滴入5,000rpm的25mg/ml HSA溶液，持续10分钟“萃取”处理白蛋白，获液体C。分别将20mg和500mg甲氨蝶呤(Methotrexate,MTX)混合于药用级大豆油(维生素E含量0.4～2mg/ml)8ml和50ml中，在4℃、避光条件下，经30,000psi高压匀浆处理1次，获甲氨蝶呤超微纳米粒子大豆油混悬液，再经4℃下、避光高速离心，转速35000rpm,持续30mins去除大豆油上清，获得甲氨蝶呤超微纳米粒子；取3mg肉豆蔻酸分别溶于500ul和1500ul乙酸乙酯溶液中(溶液B)，将此液充分悬浮所收集的甲氨蝶呤超微纳米粒子，即获甲氨蝶呤超微纳米粒子有机溶剂悬液(液体D’，样品编号MTX20和MTX500)；再将这种含有肉豆蔻酸、甲氨蝶呤、乙酸乙酯的混合溶液即液体D’缓慢滴入高速搅拌的经“萃取”处理的25mg/ml HSA溶液中，在4℃、避光、搅拌速度30,000rpm条件下，搅拌15分钟；所获混悬液分别迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂；获得半透明乳液，样品编号FBA-ID/Su-MTX20和FBA-ID/Su-MTX500；将此两种半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为两种无菌乳液；分别经真空冷冻干燥36小时(条件)，得到FBA-ID/C-MTX冻干制剂。
采用激光粒度仪测定粒径，其中两种半透明乳液FBA-ID/Su-MTX20和FBA-ID/Su-MTX500的粒径分别为53～149nm和75～353nm；两种FBA-ID/Su-MTX冻干制剂的复溶悬液的粒径分别是55～153nm和74～357nm；而“甲氨蝶呤超微纳米粒子大豆油混悬液”的平均粒径小于等于50nm。
甲氨蝶呤为“水不溶性”药物，也不溶于有机溶液，实验结果表明：脂肪酸结合白蛋白制备纳米粒子的制备方法，也可应用于不溶于有机溶剂的药物，制备纳米制剂。
实施例18FBA-ID/Su法制备姜黄素(Curcumin,C)纳米粒子冻干制剂(FBA-ID/Su-C)
无菌条件下，取Sigma人血白蛋白(HSA)1000mg，加注射用水，配制10mg/ml HSA 100ml；将氯仿和乙醇混合液300ul(9:1,v/v)(液体A)，缓慢滴入15,000转/分钟(10,000rpm)的10mg/ml HSA溶液，持续2分钟，获液体C。
180mg姜黄素(C)混合于6ml药用级大豆油(含维生素E不低于0.4～2mg/ml)或含生素C注射用水溶液中(维生素C含量50～100mg/ml)，在4℃、避光条件下，经15,000psi高压匀浆处理3次，获姜黄素超微纳米粒子混悬液；再经4℃下、避光高速离心，转速20,000rpm,持续60mins去除上清，收集得到姜黄素超微纳米粒子；取300ul氯仿乙醇混合溶液(9:1,v/v)混合所获姜黄素超微纳米粒子，得氯仿乙醇混合溶液姜黄素超微纳米粒子悬液；将1ug油酸加入该悬液中，获液体D’，再将这种含有油酸、姜黄素超微纳米粒子、氯仿和乙醇的混合液即液体D’缓慢滴入高速搅拌的经氯仿乙醇处理的10mg/ml HSA溶液中，搅拌速度为3000rpm，继续搅拌60分钟；或3000rpm搅拌，持续5分钟，再将此混合液经高压微射流机，在30,000psi压力下、4℃下、避光下，循环2次，获黄色混悬液；将这种混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发15分钟，去除全部有机溶剂。可获黄色乳液，其中姜黄素粒子的粒径为55～198nm；而姜黄素超微纳米粒子悬浮液的粒径为20～43nm(英国Malvern Nano Zetasizer ZS90)。
将上述黄色乳液经0.22u过滤膜滤过可去除溶液中的细菌等微生物，成为无菌溶液；这种无菌溶液，经真空冷冻干燥24小时，可得脂肪酸结合型白蛋白-姜黄素纳米粒冻干制剂(FBA-ID/Su-C)。如表6所示，是其它制剂配方和条件所获FBA-ID/Su-C。
表6 不同制剂配方和条件制备的FBA-ID/Su-C
Figure PCTCN2015080977-appb-000006
实施例19：脂肪酸结合型的白蛋白“水溶性”药物纳米粒子/蛋白结合型(Albumin-binding,Ab)冻干制剂的制备方法(简称FBA-SD/Ab法)
----FBA-SD/Ab法制备阿霉素(doxorubicin,D)纳米粒子冻干剂(FBA-SD/Ab-D)
无菌条件下，取Sigma HAS 1000毫克，加注射用水，配制40mg/ml HSA 25ml；将氯仿150ul(液体A)，缓慢滴入5,000rpm的40mg/ml HSA溶液，持续10分钟“萃取”处理，获液体C；用0.01M～0.1M氢氧化钠调节40mg/ml HSA溶液的pH至5.5～6.5；取0,25mg和50mg阿霉素(doxorubicin,D)，即D0、D25和D50样品，溶解于25ml注射用水中；将阿霉素溶液25ml缓慢加入25ml 40mg/ml HSA溶液，1,000rpm磁力搅拌1小时,获50ml D0HSA溶液、D25HSA-阿霉素结合溶液和D50HSA-阿霉素结合溶液(液体C’)；将5mg肉豆蔻酸(Myristic acid,MA)溶于350ul氯仿溶液中(溶液B)，再将这种混合溶液缓慢滴入高速搅拌的D0HSA溶液和D25和D50HSA-阿霉素结合溶液中，搅拌速度为20,000rpm，持续时间为10分钟，此为高速匀浆法制备纳米制剂；或者将5mg肉豆蔻酸溶于350ul氯仿溶液中(溶液B)，将这种混合溶液缓慢滴入高速搅拌的D25和D50HSA-阿霉素结合溶液或D0HSA溶液中，搅拌速度为11,000rpm，持续时间为5分钟，再分别将此高速搅拌混合液迅速放入高压微射流机的进料容器，在20,000psi压力下，4℃下，循环4次，此为高压匀浆法制备纳米制剂；所获三种混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂；测定所获三种半透明红色乳液，其中D0、D25和D50三种半透明红色乳液的纳米粒子，即FBA-SD/Ab-D0(FBA)、FBA-SD/Ab-D25和FBA-SD/Ab-D50，经激光粒度仪测定粒径分别为83～211nm,114～256nm和118～322nm。
该三种半透明乳液经0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为三种无菌乳液；分别经真空冷冻干燥30小时，获得D0脂肪酸结合型白蛋白纳米粒冻干制剂(FBA)和D25/D50脂肪酸结合型白蛋白-阿霉素纳米粒冻干制剂(FBA-SD/Ab-D)；FBA和FBA-SD/Ab-D加生理盐水或注射用水复溶后，所获纳米粒子的粒径与冻干前一样。
实施例20：FBA-SD/Ab法制备“水溶性”假单胞菌外毒素(PE38)纳米粒子冻干剂(FBA-SD/Ab-PE)
无菌条件下，取Sigma HSA 500毫克，加注射用水，配制62.5mg/ml HSA溶液8ml，将氯仿150ul(液体A)，缓慢滴入8,000rpm的HSA溶液，持续4分钟“萃取”处理，获液体C；将假单胞菌外毒素38(30kDa Pseudomonas exotoxin,PE38)1mg(0.5mg/ml PBS 2ml)，缓慢加入8ml 62.5mg/ml HSA溶液，1,00rpm磁力搅拌4小时,获10ml HSA-PE结合溶液(液体C’)；将1mg油酸(Oleic acid,OA)溶于150ul氯仿和乙醇混合溶液(8:5,v/v)中(溶液B)，再将这种混合溶液缓慢滴入高速搅拌的HSA-PE结合溶液中，搅拌速度为25,000rpm，持续时间为10分钟，此为高速匀浆法制备纳米制剂；或者将1mg油酸溶于150ul氯仿和乙醇混合溶液(8:5,v/v)中，再将这种混合溶液缓慢滴入高速搅拌的HSA-PE结合溶液中，搅拌速度为12,000rpm，持续时间为4分钟，再将此高速搅拌混合液迅速放入高压微射流机的进料容器，在25,000psi压力下，4℃下，循环1次，此为高压匀浆法制备纳米制剂；所获混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂，测定所获乳液，其中PE纳米粒子的粒径为90～180nm。
该乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为无菌乳液；这种无菌乳液，经真空冷冻干燥36小时，获得脂肪酸结合型白蛋白-PE纳米粒冻干制剂(FBA-SD/Ab-PE38)；FBA-SD/Ab-PE38加生理盐水复溶后，所获PE纳米粒子的粒径与冻干前一样。如表7所示，是其它制剂配方和条件所获FBA-SD/Ab-PE38。
表7 不同制剂配方和条件制备的FBA-SD/Ab-PE38
Figure PCTCN2015080977-appb-000007
实施例21：FBA-SD/Ab法制备表阿霉素(Epirubicin，EP)纳米粒子冻干剂(FBA-SD/Ab-EP)
无菌条件下，取Sigma HSA 1000毫克，加注射用水，配制40mg/ml HSA 25ml；将氯仿150ul(液体A)，缓慢滴入15,000rpm的40mg/ml HSA溶液，持续2分钟“萃取”处理，获液体C；用0.01M～0.1M氢氧化钠调节40mg/ml HSA溶液的pH至5.5～6.5；取0,20mg和40mg表阿霉素(Epirubicin,EP)，即EP0、EP20和EP40样品，溶解于25ml注射用水中；将表阿霉素溶液25ml缓慢加入25ml 40mg/ml HSA溶液，500rpm磁力搅拌2小时,获50ml EP0HSA溶液、EP20HSA-表阿霉素结合溶液和EP40HSA-表阿霉素结合溶液(液体C’)；将5mg静脉注射用肉豆蔻酸溶于450ul氯仿溶液中(溶液B)，将这种混合溶液缓慢滴入高速搅拌的EP20和EP40HSA-阿霉素结合溶液或EP0HSA溶液中，搅拌速度为10,000rpm，持续时间为6分钟，再分别将此高速搅拌混合液迅速放入高压微射流机的进料容 器，在20,000psi压力下，4℃下，循环6次，此为高压匀浆法制备纳米制剂；所获三种混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂；测定所获三种半透明乳液，其中EP0、EP20和EP40三种半透明乳液的纳米粒子，即FBA、FBA-SD/Ab-EP20和FBA-SD/Ab-EP40，经激光粒度仪测定粒径分别为83～211nm,114～256nm和118～322nm。
该三种半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为三种无菌乳液；分别经真空冷冻干燥24小时，获得EP0脂肪酸结合型白蛋白纳米粒冻干制剂(FBA)和EP20/EP40脂肪酸结合型白蛋白-表阿霉素纳米粒冻干制剂(FBA-SD/Ab-EP)；FBA和FBA-SD/Ab-EP加生理盐水或注射用水复溶后，所获纳米粒子的粒径与冻干前一样。
实施例22：脂肪酸结合型白蛋白-“水溶性”药物纳米粒子/空粒结合型(Empty-nanoparticle Binding,Eb)冻干制剂的制备方法(简称FBA-SD/Eb)
----FBA-SD/Eb法制备阿霉素纳米粒子冻干剂(FBA-SD/Eb-D)
无菌条件下，取Sigma HSA 1000毫克，加注射用水，配制40mg/ml HSA 25ml；将氯仿150ul(液体A)，缓慢滴入5,000rpm的40mg/ml HSA溶液，持续10分钟“萃取”处理，获液体C，或滴入15,000rpm的40mg/ml HSA溶液，持续2分钟“萃取”处理，也可获液体C；将5mg肉豆蔻酸(Myristic acid,MA)溶于350ul氯仿溶液中(溶液B)，再将这种混合溶液缓慢滴入高速搅拌的40mg/ml HSA溶液中，搅拌速度为20,000rpm，持续时间为10分钟，此为高速匀浆法制备纳米制剂(FBA-SD/Eb HS)；或者将5mg肉豆蔻酸溶于350ul氯仿溶液中，将这种混合溶液缓慢滴入高速搅拌的40mg/ml HSA溶液中，搅拌速度为10,000rpm，继续搅拌6分钟，或搅拌速度为12000rpm，继续搅拌4分钟，再分别将此高速搅拌混合液迅速放入高压微射流机(M-110P Microfluidizer)的进料容器，在25,000psi压力下，4℃下，循环2次，此为高压匀浆法制备纳米制剂(FBA-SD/Eb HP)；将此悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂，获HSA纳米粒子乳液；用0.01M～0.1M氢氧化钠调节HSA纳米粒子乳液的pH至5.5～6.5；取0,25mg，50mg和150mg阿霉素(doxorubicin,D)，即D0、D25、D50和D150样品，溶解于25ml注射用水中；将阿霉素溶液25ml缓慢加入25ml HSA纳米粒子乳液中，250rpm磁力搅拌4小时与白蛋白结合，或500rpm磁力搅拌2小时与白蛋白结合，可获四种半透明红色乳液，其中D0、D25、D50和D150四种半透明红色乳液的纳米粒子，即FBA、FBA-SD/Eb-D25、FBA-SD/Eb-D50和FBA-SD/Eb-D150，经激光粒度仪测定粒径分别为85～213nm,88～223nm、86～227nm和83～287nm。
该四种半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为四种无菌乳液；分别经真空冷冻干燥24小时，获得D0脂肪酸结合型白蛋白纳米粒冻干制剂(FBA)和D25/D50/D150脂肪酸结合型白蛋白-阿霉素纳米粒冻干制剂(FBA-SD/Eb-D)；FBA和FBA-SD/Eb-D加生理盐水或注射用水复溶后，所获纳米粒子的粒径与冻干前一样。
如表8所示，是其它制剂配方和条件所获FBA-SD/Eb-D。
表8 不同制剂配方和条件制备的FBA-SD/Eb-D
Figure PCTCN2015080977-appb-000008
实施例23：脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/悬液型冻干制剂的制备方法(简称FBA-ID/So&SD/Su法)
--------FBA-ID/So&SD/Su法制备含紫杉醇和卡铂的纳米粒子冻干制剂(FBA-P/So&CP/Su)
无菌条件下，取Sigma的HSA 600毫克，加注射用水，配制成60mg/ml HSA 10ml；将氯仿和乙醇混合溶液(5:2,v/v)150ul(液体A)，缓慢滴入5,000rpm的60mg/ml HSA溶液，持续10分钟“萃取”处理白蛋白，获液体C，或15,000rpm的60mg/ml HSA溶液，持续2分钟“萃取”处理白蛋白，也可获液体C。分别将20mg紫杉醇(Paclitaxel,P)和200ug油酸溶于200ul氯仿和乙醇混合溶液中。将20mg卡铂(Carboplatin，CP)混合于药用级大豆油(维生素E含量在0.4～2mg/ml)16ml中，在4℃、避光条件下，经15,000psi高压匀浆处理3次，收集卡铂超微纳米粒子大豆油混悬液，或经30,000psi高压匀浆处理1次，收集卡铂超微纳米粒子大豆油混悬液，再经4℃下、避光高速离心，转速30000rpm,持续10mins去除大豆油上清，获得卡铂超微纳米粒子，或转速5000rpm,持续60mins去除大豆油上清，获得卡铂超微纳米粒子。使用油酸、紫杉醇、氯仿和乙醇的混合溶液搅拌、悬浮所获得的卡铂超微纳米粒子；再将这种含有油酸、紫杉醇、卡铂、氯仿和乙醇的悬液缓慢滴入高速搅拌的经“萃取”处理的60mg/ml HSA溶液中，在4℃下、避光、搅拌速度为10,000rpm，搅拌5分钟，或搅拌转速为12000rpm，搅拌4分钟；再将这种混合悬液加入高压微射流机的进料口，在30,000psi压力下，4℃下，循环3次；所获混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂，得半透明乳液；用激光粒度仪测定所获半透明乳液中纳米粒子的粒径，结果为67～176nm。
该种半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为一种无菌乳液；经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白紫杉醇-卡铂纳米粒冻干制剂(FBA-P/So&CP/Su)；FBA-P/So&CP/Su加生理盐水或注射用水复溶后，所获纳米粒子的粒径为67～178nm，与冻干前相同。如表9所示，是其它制剂配方和条件所获FBA-P/So&CP/Su。
表9 不同制剂配方和条件制备的FBA-P/So&CP/Su
Figure PCTCN2015080977-appb-000009
此法为前述FBA-ID/So和FBA-SD/Su两种粒子制备方法的联合应用。
实施例24：脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/蛋白结合型冻干制剂的制备方法(简称FBA-ID/So&SD/Ab法)
----------FBA-ID/So&SD/Ab法制备多烯紫杉醇和PE38毒素纳米粒子冻干制剂(FBA-Do/So&PE38/Ab)
无菌条件下，取Sigma的HSA 900毫克，加注射用水，配制成90mg/ml HSA 10ml；将氯仿和乙醇混合溶液(5:2,v/v)150ul(液体A)，缓慢滴入5,000rpm的60mg/ml HSA溶液，持续10分钟“萃取”处理白蛋白，获液体C，或滴入15,000rpm的60mg/ml HSA溶液，持续2分钟“萃取”处理白蛋白，也可获液体C。将PE38毒素0.5mg(0.5mg/ml PBS 2ml)，缓慢加入溶液C中，1,000rpm磁力搅拌1小时结合白蛋白,或100rpm磁力搅拌4小时结合白蛋白，获12ml HSA-PE38结合溶液(溶液C’)；分别将3mg,60mg和120mg多烯紫杉醇(Docetaxel,Do)和500ug油酸溶于300ul氯仿和乙醇混合溶液(5:2,v/v)中(溶液D)；再将这三种种混合溶液缓慢滴入高速搅拌的HSA-PE38结合溶液中，搅拌速度为25,000rpm，持续时间为10分钟，制得纳米粒子混悬液，此为高速匀浆法制备纳米制剂；或者将3mg,60mg和120mg多烯紫杉醇和500ug油酸溶于300ul氯仿和乙醇混合溶液(5:2,v/v)中，再将这种混合溶液缓慢滴入高速搅拌的HSA-PE38结合溶液中，搅拌速度为10,000rpm，继续搅拌6分钟，或搅拌速度为12,000rpm，继续搅拌4分钟，再将此三种高速搅拌混合液迅速放入高压微射流机的进料容器，在25,000psi压力下，4℃下，避光、循环3次，制得纳米粒子混悬液，此为高压匀浆法制备纳米制剂；所获混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发20分钟，去除全部有机溶剂，得半透明乳液；用激光粒度仪测定所获半透明乳液中纳米粒子的粒径，结果为50～110nm,54～135nm和58～169nm。
该种半透明乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为一种无菌乳液；经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白多烯紫杉醇-PE38毒素纳米粒冻干制剂(FBA-Do/So&PE38/Ab)；加生理盐水或注射用水复溶FBA-Do/So&PE38/Ab后，所获纳米粒子的粒径为53～107nm,58～137nm和55～170nm，与冻干前相同。如表10所示，是其它制剂配方和条件所获FBA-Do/So&PE38/Ab。
表10 不同制剂配方和条件制备的FBA-Do/So&PE38/Ab
Figure PCTCN2015080977-appb-000010
为前述FBA-ID/So和FBA-SD/Ab两种制备方法的联合应用。
实施例25：脂肪酸结合型白蛋白-“水不溶性”和“水溶性”药物纳米粒子/空粒结合型冻干制剂的制备方法(简称FBA-ID/So&SD/Eb法)----------FBA-ID/So&SD/Eb法制备雷帕霉素和阿霉素纳米粒子冻干制剂(FBA-R/So&D/Eb)
无菌条件下，取Sigma的HSA 600毫克，加注射用水，配制成60mg/ml HSA 10ml；将二氯甲烷溶液250ul(液体A)，缓慢滴入5,000rpm的60mg/ml HSA溶液，持续10分钟“萃取”处理，获液体C，或滴入15,000rpm的60mg/ml  HSA溶液，持续2分钟“萃取”处理，获液体C。将0.2mg肉豆蔻酸与6.5mg,130mg和260mg雷帕霉素(Rapamycin,R)共溶于650ul二氯甲烷溶液中(溶液D)，再将这种含有肉豆蔻酸、雷帕霉素、二氯甲烷的混合溶液缓慢滴入高速搅拌的溶液C中，搅拌速度为10,000rpm，继续搅拌5分钟，或搅拌速度为12,000rpm，继续搅拌4分钟；这种混合液经高压微射流机，在10,000psi压力下，4℃下，循环6次；将此混悬液迅速放入真空旋转蒸发仪中，在35℃减压下(20mmHg)旋转蒸发30分钟，去除全部有机溶剂，可获半透明乳液。用0.01M～0.1M氢氧化钠调节半透明乳液的pH至5.5～6.5；取20mg阿霉素(doxorubicin,D)，溶解于2ml注射用水中；将2ml阿霉素溶液分别缓慢加入10ml三种半透明乳液中，250rpm磁力搅拌4小时，或500rpm磁力搅拌2小时，可获脂肪酸结合型白蛋白-雷帕霉素-阿霉素乳液，用激光粒度仪测定所获乳液中纳米粒子的粒径，结果为55～157nm、52～169nm和56～193nm。
该种乳液经0.45u和0.22u过滤膜滤过可去除乳液中的细菌等微生物，成为一种无菌乳液；经真空冷冻干燥24小时，获得脂肪酸结合型白蛋白-雷帕霉素-阿霉素纳米粒冻干制剂(FBA-R/So&D/Eb)；加生理盐水或注射用水复溶FBA-R/So&D/Eb后，所获纳米粒子的粒径为53～159nm、57～173nm和55～195nm，与冻干前相同。如表11所示，是其它制剂配方和条件所获FBA-R/So&D/Eb。
表11 不同制剂配方和条件制备的FBA-R/So&D/Eb
Figure PCTCN2015080977-appb-000011
此法为前述FBA-ID/So和FBA-SD/Eb两种粒子制备方法的联合应用。
实施例26：FBA、FBA-ID/So-P和FBA-SD/Su-D的透射电镜扫描图
图1显示的是实施例6制备所得到的脂肪酸结合型白蛋白-紫杉醇纳米粒FBA-ID/So-P，即样品FBA-ID/So-P1透射电镜扫描图；图2是实施例9制备所得到的脂肪酸结合型白蛋白“空粒结合型”(不含多烯紫杉醇)的纳米粒子，即FBA-ID/So-Do00；图3是实施例15制备所得到的脂肪酸结合型白蛋白-阿霉素纳米粒，即FBA-SD/Su-D25的电镜扫描图。
实施例27：圆二色光谱分析白蛋白二级结构
分别取华兰生物HSA、Abraxane分解所获HSA、实施例6中样品FBA-ID/So-P1分解后所获HSA、实施例13中样品FBA-ID/So-P&C、实施例15中样品FBA-SD/Su-D25分解后所获HSA、实施例21中样品FBA-SD/Ab-EP25分解后所获HSA、实施例23中样品FBA-P/So&CP/Su分解后所获HSA、实施例24中样品FBA-Do/So&PE38/Ab分解后所获HSA和实施例25中样品FBA-R/So&D/Eb分解后所获HSA、以及FBA-ID/So-M1、FBA-ID/So-V1、FBA-ID/So-SN381、FBA-ID/Su-E25、FBA-ID/Su-MTX20等分解后所获HSA；进行圆二色光谱扫描分析，测定条件为波长190-250nm，反应1s，扫描速度20nm/min，Data acquisition 0.1nm，Accumulations 3，Bandwidth 1nm，蛋白浓度0.2mg/ml，结果如下图4所示。
实验结果显示，各种脂肪酸结合白蛋白-药物纳米粒子冻干制剂的制备方法及其组合，在圆二色光谱测定与分析白蛋白的二级结构上，均未见明显改变。
实施例28：脂肪酸结合型白蛋白-药物纳米粒子的体外稳定性
分别在4℃和25℃室温下,考察脂肪酸结合型白蛋白-药物纳米粒子(FBA-Drug NP)制冻干制剂复溶后溶液的体外稳定性，使用激光粒度测定仪(英国Malvern Nano Zetasizer ZS90)以粒径改变大于等于5％、或Zeta电位改变大于等于10％、或出现细小沉淀、或溶液透明度等改变作为指标判断，任何一个最早出现的指标即为制剂纳米粒子体外溶液完整性和稳定性改变的时间依据，观察结果详见表12所示。
表12 FBA-Drug NP的体外稳定性
Figure PCTCN2015080977-appb-000012
Figure PCTCN2015080977-appb-000013
此实验表明：各种脂肪酸结合型白蛋白-药物纳米粒子(FBA-Drug NP)冻干制剂复溶后的体外溶液在4℃和25℃下纳米粒子保持完整性与稳定性的时间分别是136～152小时和72～96小时，白蛋白结合型紫杉醇(Abraxane)冻干制剂复溶后的体外溶液在4℃和25℃下纳米粒子保持完整性与稳定性的时间分别是96小时和48小时，因此，脂肪酸结合型白蛋白-药物纳米粒子(FBA-Drug NP)冻干制剂体外溶液纳米粒子的完整性和稳定性明显优于白蛋白结合型紫杉醇(Abraxane)。
实施例29HUVEC细胞内各种FBA-Drug NP含量分析
在生理状态下，人脐血管内皮细胞(Human Umbilical Vascular Endothelial Cell,HUVEC)表面富含gp60受体，后者与HSA结合，激活小窝蛋白-1(Caveolin-1)，导致小窝(caveoli)形成，使HSA和血浆的其他组分跨过血管内皮细胞进入细胞间液。因此，通过分析HUVEC细胞内FBA-Drug NP的含量，可以判断FBA-Drug NP中的HSA是否具有正常的体内性质。
使用荧光标记的紫杉醇(invitogen,Flutax-2)，按1:50比例加入紫杉醇中，按前述相应方法制备荧光标记的FBA-ID/So-P，即F-FBA-ID/So-P。再以同样添加比例，将Flutax-2加入“泰素”制剂中，荧光标记的紫杉醇注射液，即F-Taxol。将5×104个HUVEC接种在96孔板(Costar 3614)上接种HUVEC细胞，每孔接种量为5×104个/孔，37℃、5％CO2细胞培养，待细胞贴壁、长满后，分别加入终浓度为20、40、80、160、320和640ug/ml PEM缓冲液(50mmo/L PIPES,2mmol/L EGTA,2mmol MgCl2)的F-FBA-ID/So-P和F-Taxol,37℃下，孵育1小时后，PEM缓冲液冲洗96孔板三次，使用M5/M5e多功能酶标仪(美国Molecular Devices)在激发光波长为494nm和发射光波长为524nm下，读取各孔数据，测定F-FBA-ID/So-P和F-Taxol与HUVEC细胞的结合率，结果详见图5所示，结果表明：各种F-FBA-ID/So-P与HUVEC细胞的结合率较F-Taxol高约10倍左右。
采用6孔板培养HUVEC细胞，37℃、5％CO2细胞培养，待细胞贴壁、长满后，分别加入各种FBA-Drug NP，并选相同剂量的相应化疗药静脉注射剂如泰素、艾素多西他赛注射液、Doxil和盐酸多柔比星、表柔比星，长春瑞滨、铂尔定、注射用丝裂霉素、依托泊苷注射液等等作为阳性对照药，使其终浓度为160～640ug/ml PEM缓冲液，37℃下，孵育3小时后，PEM缓冲液冲洗6孔板三次，离心收集HUVEC细胞，采用HPLC法测定细胞内各种化疗药如紫杉醇、多烯紫杉醇、阿霉素、表阿霉素、长春新碱、卡铂等的含量，比较FBA-Drug NP纳米制剂与普通静脉注射剂进入HUVEC细胞内的差别，如表13所示，实验结果表明：FBA-Drug NP进入HUVEC细胞内的药物含量比相应药物的静脉注射剂高约2～6倍。
表13 FBA-Drug NP及其相应静脉注射剂进入HUVEC细胞内的含量比较
Figure PCTCN2015080977-appb-000014
Figure PCTCN2015080977-appb-000015
上述实验结果还表明，各种FBA-Drug NP的制备技术，并未影响HSA的体内性质，而HSA显著促进了化疗药的细胞内转运过程。
实施例30FBA-ID/So-P类纳米粒子的体内性质与特性
采用ELISA法测定血液循环中完整纳米粒子形式的FBA-ID/So-P、FBA-P/So&CP/Su和FBA-Do/So&PE38/Ab，作为判断其在动物体内稳定性的指标，即由包埋在96孔板底部的抗紫杉醇抗体或抗多烯紫杉醇抗体捕获血清中FBA-ID/So-P类三种纳米粒子，再由生物素标记的抗HSA抗体检测“FBA-ID/So-P”类三种纳米粒子的含量，由此计算出血浆半衰期；采用HPLC法测定组织中紫杉醇的含量，以判断FBA-ID/So-P类三种纳米粒子在肿瘤组织的聚集特征；在荷瘤裸鼠HT29移植瘤模型上，初步判断FBA-ID-P、FBA-P&CP/C和FBA-Do/So&PE38/Ab的抑瘤药效；上述动物试验中，FBA-ID/So-P以相当于6mg/kg紫杉醇的剂量给药，FBA-P/So&CP/Su和FBA-Do/So&PE38/Ab以相当于2mg/kg紫杉醇的剂量给药，两者均为隔日尾静脉注射一次，连续给药，实验结果详见表14和图6所示。
表14 FBA-ID/So-P类纳米粒子的体内性质与特征
Figure PCTCN2015080977-appb-000016
★每100mg肿瘤组织中紫杉醇含量
FBA-ID/So-P、FBA-ID/So-P&C、FBA-P/So&CP/Su和FBA-Do/So&PE38/Ab的体内性质试验提示：在动物血液循环中FBA-ID/So-P、FBA-ID/So-P&C、FBA-P/So&CP/Su和FBA-Do/So&PE38/Ab均具较好的体内血液循环稳定性，并可富集于肿瘤组织内，其中尤以FBA-Do/So&PE38/Ab更为突出，提示PE38毒素可以显著促进多烯紫杉醇在肿瘤局部组织中的富集，并可产生更好的抑瘤效果。
实施例31FBA-SD/Su-D类纳米粒子的体外和体内性质与特点
将含等量阿霉素的脂肪酸结合白蛋白-阿霉素纳米粒冻干制剂(FBA-SD/Su-D)和隐形脂质体阿霉素(Doxil)加入处于对数增长期的人乳腺癌细胞MCF-7细胞培养液中，培养24小时，显微镜下发现，FBA-SD/Su-D进入MCF-7细胞中的量显著多于Doxil，如下图7所示。
采用ELISA法测定血液循环中完整纳米粒子形式存在的FBA-SD/Su-D类纳米粒子，以此作为判断其在动物体内稳定性的指标，即由包埋在96孔板底部的抗阿霉素抗体捕获血清中FBA-SD-D类四种纳米粒子，再由生物素标记的抗HSA抗体检测“FBA-SD-D”类四种纳米粒子的含量，由此计算出血浆半衰期；采用HPLC法荧光检测测定血浆和组织中阿霉素的含量，以判断Doxil和多柔比星血浆半衰期和在肿瘤组织阿霉素的含量；在荷瘤裸鼠人口腔上皮癌移植瘤(KB)移植瘤模型上，静脉注射同等剂量阿霉素，初步判断FBA-SD/Su-D、FBA-SD/Ab-D、FBA-SD/Eb-D、FBA-R/So&D/Eb、FBA-R/So&D/Su以及Doxil和盐酸多柔比星的抑瘤效果；上述动物试验中，FBA-SD-D以相当于3mg/kg阿霉素的剂量给药，隔日尾静脉注射一次，连续给药，实验结果详见表15和图7所示。
表15 FBA-SD-D类纳米粒子的体内性质与特征
Figure PCTCN2015080977-appb-000017
Figure PCTCN2015080977-appb-000018
★每100mg肿瘤组织中阿霉素含量
在裸鼠人口腔上皮癌移植瘤动物模型上，注射同等剂量阿霉素的各种FBA-SD-D纳米制剂和Doxil与多柔比星注射液,结果表明：由三种不同“水溶性”药物纳米粒子的制备方法所获阿霉素纳米粒子冻干制剂，即FBA-SD/Su-D25、FBA-SD/Ab-D25和FBA-SD/Eb-D25其血浆半衰期不同，但均较Doxil明显延长，分别延长8.1倍、6.1倍和2.8倍；各种FBA-SD-D纳米制剂抑瘤率显著优于Doxil和多柔比星注射液，详见图8所示。
实施例32:FBA-SD/Ab-PE的体内性质与特性
PE38是最常用的PE毒素分子形式，具明确的细胞毒性分子机制，比较PE38蛋白和实施例20所制备的FBA-SD/Ab-PE38，结果FBA-SD/Ab-PE38的LD50高于PE38蛋白约60倍(分别是30mg/kg和0.45mg/kg)。大鼠PE38抗原性测定试验表明：FBA-SD/Ab-PE38的抗原性低于PE38蛋白约20倍。
实施例33各种FBA-ID-P动物体内LD50和MTD性质测定
使用Babl/c小鼠，在标准GLP的环境下，单次静脉注射FBA-ID/So-P-1、FBA-ID/So-P00和Abraxane冻干制剂复溶液以及泰素(紫杉醇注射液)，测定各种FBA-ID/So-P的半数致死量(LD50)和最大耐受剂量(MTD)或LD10，结果详见表16。
结果表明：以白蛋白为基架的纳米紫杉醇制剂的LD50和MTD值均显著高于泰素，最低增幅分别为44倍和53倍；而FBA-ID/So-P和FBA-Do/So&PE38/Ab与Abraxane之间LD50和MTD值未见显著性差别；FBA-ID/So-P与FBA-P/So&CP/Su之间的LD50和MTD值具显著性差别，但均比紫杉醇注射液“泰素”显著增高。
表16 各种FBA-ID/So-P动物LD50的测定结果
Figure PCTCN2015080977-appb-000019

Claims (16)

  1. 一种脂肪酸结合型白蛋白-药物纳米粒子，其特征在于，所述的纳米粒子包括油相核心、白蛋白和药物，所述的白蛋白与油相核心中的脂肪酸结合，所述的药物与白蛋白和/或脂肪酸结合；所述的药物选自水溶性药物、水不溶性药物、水溶性药物超微纳米粒子和水不溶性药物超微纳米粒子中的一种或两种以上；所述的白蛋白为人血清白蛋白或牛血清白蛋白；所述人血清白蛋白由血液提取或重组技术制备；所述纳米粒子中按质量比包括脂肪酸0.0001％～8％，白蛋白75％～99％和药物0.1％～25％。
  2. 权利要求1所述的一种脂肪酸结合型白蛋白-药物纳米粒子，其特征在于，所述纳米粒子中按质量比包括脂肪酸0.001％～1％，白蛋白80％～95％，药物1.0％～20％。
  3. 权利要求1-2任一项所述的一种脂肪酸结合型白蛋白-药物纳米粒子，其特征在于，所述的药物与脂肪酸结合共同构成油相核心；所述的药物选自水不溶性药物、水溶性药物超微纳米粒子和水不溶性药物超微纳米粒子中的一种或两种以上。
  4. 权利要求1-2任一项所述的一种脂肪酸结合型白蛋白-药物纳米粒子，其特征在于，所述水不溶性药物是紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤中至少一种；所述的水不溶性药物超微纳米粒子是紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤中至少一种制备得到的超微纳米粒子。
  5. 权利要求1-2任一项所述的一种脂肪酸结合型白蛋白-药物纳米粒子，其特征在于，所述水溶性药物是阿霉素、表阿霉素、卡铂、假单胞菌外毒素中至少一种；所述的水溶性药物超微纳米粒子是阿霉素、表阿霉素、卡铂、假单胞菌外毒素中至少一种制备得到的超微纳米粒子。
  6. 一种药物制剂，其特征在于，包括权利要求1所述的脂肪酸结合型白蛋白-药物纳米粒子和药学上可接受的辅料。
  7. 权利要求6所述的药物制剂，其特征在于，所述的制剂为冻干制剂。
  8. 权利要求7所述的药物制剂，其特征是具有如下性质：
    (1)所述冻干制剂和Abraxane复溶后，在体外溶液中4℃条件下，测定纳米粒子的完整性与稳定性分别在136～160小时和96小时；
    (2)所述冻干制剂和Abraxane复溶后，测定体内血液循环中的所述纳米粒子的完整性与稳定性分别在35～45小时和25小时；
    (3)所述冻干制剂荷载的药物进入富含gp60受体细胞内的药物浓度与同一药物的静脉注射剂进入同系细胞内的药物浓度之比为2～6；
    (4)所述冻干制剂荷载的药物的LD50是同一药物静脉注射剂LD50的44～60倍；
    (5)所述冻干制剂荷载的药物的MTD是同一药物静脉注射剂MTD的53～71倍；
    (6)所述冻干制剂降低所包裹或结合的假单胞菌外毒素PE38蛋白的抗原性20倍。
  9. 一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是包括如下步骤：
    (1)液体A：为水不相溶的有机溶剂，或体积比为10～5:5～1的水不相溶的有机溶剂和无水乙醇的混合液，所述水不相溶的有机溶剂为氯仿、二氯甲烷或乙酸乙酯；溶液B：用液体A配制浓度为0.0033～360mg/ml的脂肪酸溶液，所述脂肪酸为油酸、亚油酸、肉豆蔻酸或甘油三酯；液体C：用水配制浓度为10～200mg/ml的白蛋白水溶液，按体积比为1:6.67～333的比例将液体A滴入以5,000～15,000rpm转速搅拌的白蛋白溶液中，继续搅拌2～10分钟，制得液体C；
    (2)脂肪酸结合型白蛋白-药物纳米粒子混悬液的制备：
    用下述几种方式之一制备：
    方式一：
    用溶液B溶解水不溶性药物，获浓度为12.5mg/ml～600mg/ml的液体D，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
    液体C中液体A的体积与溶解水不溶性药物的溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为0.4％～17.8％；脂肪酸与白蛋白的质量比为0.0001％～5％；水不溶性药物与白蛋白的质量比为：1％～20％；
    方式二：
    1)按1～10mg：1ml的比例将水不溶性药物与含0.4～2mg/ml维生素E的大豆油混合；或按1～10mg：1ml的比例将水不溶性药物与含50～100mg/ml维生素C的注射用水混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水不溶性药物超微纳米粒子；按40mg/ml～600mg/ml比例，将水不溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水不溶性药物超微纳米粒子和有机溶剂的悬液；
    2)按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；
    液体C中液体A的体积与液体D’中溶液B体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：0.6％～15.6％；脂肪酸与白蛋白的质量比为0.0001％～1％；水不溶性药物与白蛋白的质量比为：1％～25％；
    方式三：
    1)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml维生素E的大豆油混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；按6.6mg/ml～375mg/ml比例，将水溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水溶性药物超微纳米粒子和有机溶剂的悬液；
    2)按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为12,000rpm～32,000rpm的液体C中，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；
    液体C中液体A的体积与液体D’中溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：1％～15.8％；脂肪酸与白蛋白的质量比为0.001％～1.4％；水溶性药物与白蛋白的质量比为：1％～20％；
    方式四：
    1)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，即液体C’；
    2)按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；
    液体C中液体A的体积与溶液B的体积之和为油相，而白蛋白-水溶性药物结合溶液的体积为水相，油相体积与水相体积之比为：1％～16％；脂肪酸与白蛋白的质量比为0.002％～15％；水溶性药物与白蛋白的质量比为：1％～25％；
    方式五：
    按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为20,000rpm～25,000rpm的液体C中，制得脂肪酸结合型白蛋白纳米粒子混悬液；
    将所述脂肪酸结合型白蛋白纳米粒子混悬液去除全部有机溶剂，获半透明乳液；
    调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水溶性药物纳米粒子空粒结合型乳液；
    液体C中液体A的体积与溶液B的体积之和为油相，半透明乳液的体积为水相，油相体积与水相体积之比为：2.0％～16.4％；脂肪酸与白蛋白的质量比为0.001％～3.1％；水溶性药物与白蛋白的质量比为：2.5％～15％；
    方式六：
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml维生素E的大豆油混合；匀浆处理1～3次；离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；
    3)用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；
    方式七：
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
    3)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；
    方式八
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
    3)将脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液去除全部有机溶剂，获半透明乳液；调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子空粒结合型混悬液；
    (3)乳液的制备
    将步骤(2)中方式一～方式四、方式六和方式七制备的混悬液去除混悬液中全部有机溶剂，制得半透明的稳定的纳米粒子乳液；
    (4)冻干制剂的制备
    将步骤(3)获得的乳液过滤除菌，获无菌乳液，经真空冷冻干燥，制得脂肪酸结合型白蛋白-药物纳米粒子冻干制剂；
    所述白蛋白为人血清白蛋白或牛血清白蛋白；所述人血清白蛋白由血液提取或重组技术制备。
  10. 权利要求9所述的一种脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是包括如下步骤：
    (1)液体A：为水不相溶的有机溶剂，或体积比为10～5:5～1的水不相溶的有机溶剂和无水乙醇的混合液，所述水不相溶的有机溶剂为氯仿、二氯甲烷或乙酸乙酯；溶液B：用液体A配制浓度为0.0033～360mg/ml的静脉注射用脂肪酸溶液，所述静脉注射用脂肪酸为油酸、亚油酸、肉豆蔻酸或甘油三酯；液体C：无菌条件下，用注射用水配制浓度为10～200mg/ml的白蛋白水溶液，按体积比为1:6.67～333的比例将液体A滴入以5,000～15,000rpm转速搅拌的白蛋白溶液中，继续搅拌2～10分钟，使与脂肪酸结合的白蛋白萃取于液体A中，制得液体C；
    (2)脂肪酸结合型白蛋白-药物纳米粒子混悬液的制备：
    用下述几种方式制备：
    方式一：
    用溶液B溶解水不溶性药物，获浓度为12.5mg/ml～600mg/ml的液体D，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
    或者，用溶液B溶解水不溶性药物,获浓度为12.5mg/ml～600mg/ml的液体D，按体积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
    液体C中液体A的体积与溶解水不溶性药物的溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为0.4％～17.8％；静脉注射用脂肪酸与白蛋白的质量比为0.0001％～5％；水不溶性药物与白蛋白的质量比为：1％～20％；
    方式二：
    1)按1～10mg：1ml的比例将水不溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；或按1～10mg：1ml的比例将水不溶性药物与含50～100mg/ml维生素C的注射用水混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4℃下、避光、20,000～35,000rpm离心10～60min，去除上清液，获得水不溶性药物超微纳米粒子；按40mg/ml～600mg/ml比例，将水不溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水不溶性药物超微纳米粒子和有机溶剂的悬液；
    2)按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌15～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；或按体积比为0.3％～4.0％的比例，将液体D’滴入搅拌速度为3,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光、压力为15,000psi～30,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子悬液型混悬液；
    液体C中液体A的体积与液体D’中溶液B体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：0.6％～15.6％；静脉注射用脂肪酸与白蛋白的质量比为0.0001％～1％；水不溶性药物与白蛋白的质量比为：1％～25％；
    方式三：
    1)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4±1℃下、避光、5,000～30,000rpm持续离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；按6.6mg/ml～375mg/ml比例，将水溶性药物超微纳米粒子混悬于溶液B中，获得液体D’，为含脂肪酸、水溶性药物超微纳米粒子和有机溶剂的悬液；
    2)按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为12,000rpm～32,000rpm的液体C中，继续搅拌5～30分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；或按体积比为0.8％～7.0％的比例，将液体D’滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光、压力为15,000psi～30,000psi下，连续3～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子悬液型混悬液；
    液体C中液体A的体积与液体D’中溶液B的体积之和为油相，液体C中的白蛋白水溶液的体积为水相，油相体积与水相体积之比为：1％～15.8％；静脉注射用脂肪酸与白蛋白的质量比为0.001％～1.4％；水溶性药物与白蛋白的质量比为：1％～20％；
    方式四：
    1)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，即液体C’；
    2)按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；或按体积比为0.8％～7.5％的比例将溶液B，滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为10,000psi～25,000psi下，连续1～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水溶性药物纳米粒子蛋白结合型混悬液；
    液体C中液体A的体积与溶液B的体积之和为油相，而白蛋白-水溶性药物结合溶液的体积为水相，油相体积与水相体积之比为：1％～16％；静脉注射用脂肪酸与白蛋白的质量比为0.002％～15％；水溶性药物与白蛋白的质量比为：1％～25％；
    方式五：
    按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为20,000rpm～25,000rpm的液体C中，继续搅拌10～20分钟，制得脂肪酸结合型白蛋白纳米粒子混悬液；或按体积比为1.4％～3.3％的比例，将溶液B滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟后，加入高压微射流机进料容器中，在4±1℃下，避光，压力为10,000psi～25,000psi下，连续2～6个循环高压匀浆处理，制得脂肪酸结合型白蛋白纳米粒子混悬液；
    将所述脂肪酸结合型白蛋白纳米粒子混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；
    调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水溶性药物纳米粒子空粒结合型乳液；
    液体C中液体A的体积与溶液B的体积之和为油相，半透明乳液的体积为水相，油相体积与水相体积之比为：2.0％～16.4％；静脉注射用脂肪酸与白蛋白的质量比为0.001％～3.1％；水溶性药物与白蛋白的质量比为：2.5％～15％；
    方式六：
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)按5～10mg：1ml的比例将水溶性药物与含0.4～2mg/ml药用级维生素E的大豆油混合；在4±1℃、避光，压力为15,000～30,000psi下匀浆处理1～3次；再经4±1℃下、避光、5,000～30,000rpm持续离心10～60min，去除上清液，获得水溶性药物超微纳米粒子；
    3)用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；或用液体D混悬水溶性药物超微纳米粒子，得到含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液，按 体积比为0.1％～7.2％的比例，将含脂肪酸、水不溶性药物、水溶性药物超微纳米粒子的悬液滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为3,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子悬液型混悬液；
    方式七：
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入液体C中，在100～1000rpm下搅拌1～4小时，使水溶性药物与液体C中白蛋白结合制得白蛋白-水溶性药物结合溶液，为液体C’；
    3)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为15,000rpm～25,000rpm的液体C’中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；或按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C’中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子蛋白结合型混悬液；
    方式八
    1)用溶液B溶解水不溶性药物，获浓度为10mg/ml～400mg/ml的液体D；
    2)按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为3,000rpm～30,000rpm的液体C中，继续搅拌10～60分钟，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；或按积比为0.1％～7.2％的比例，将液体D滴入搅拌速度为10,000rpm～12,000rpm的液体C中，继续搅拌5±1分钟，加入高压微射流机进料容器中，在4±1℃，避光，压力为6,000psi～40,000psi下，连续2～8个循环高压匀浆处理，制得脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液；
    3)将脂肪酸结合型白蛋白-水不溶性药物纳米粒子溶液型混悬液放入真空旋转蒸发仪中，在35±2℃、20mmHg减压下，60～80转/分旋转蒸发20～30分钟，去除全部有机溶剂，获半透明乳液；调节水溶性药物与液体C中白蛋白结合的条件，将水溶性药物粉末或水溶性药物水溶液加入半透明乳液中，在250rpm～500rpm下搅拌2～4小时，使水溶性药物与半透明乳液中白蛋白结合制得脂肪酸结合型白蛋白-水不溶性和水溶性药物纳米粒子空粒结合型混悬液；
    (3)乳液的制备
    将步骤(2)中方式一～方式四、方式六和方式七制备的混悬液放入真空旋转蒸发仪中，在35±2℃，负压下，旋转蒸发10～30分钟，去除混悬液中全部有机溶剂，制得半透明的稳定的纳米粒子乳液；
    (4)冻干制剂的制备
    将步骤(3)获得的乳液经0.22um膜过滤除菌，或经0.45um和0.22um膜过滤除菌，获无菌乳液，经真空冷冻干燥24～36小时，制得脂肪酸结合型白蛋白-药物纳米粒子冻干制剂；所述白蛋白为人血清白蛋白或牛血清白蛋白；所述人血清白蛋白由血液提取或重组技术制备。
  11. 如权利要求9-10任一项所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征在于步骤(1)中液体A与白蛋白溶液的体积比为1:36～100。
  12. 如权利要求9-10任一项所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂按质量百分比由静脉注射用脂肪酸0.0001％～8％，白蛋白75％～99％和药物0.1％～25％组成，不含有机溶剂或任何辅料，所述药物为水不溶性药物和水溶性药物至少一种。
  13. 如权利要求12所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是所述静脉注射用脂肪酸为0.001％～1％，白蛋白为80％～95％，药物为1.0％～20％。
  14. 如权利要求9-10任一项所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是所述水不溶性药物是紫杉醇、多烯紫杉醇、雷帕霉素、姜黄素、丝裂霉素、长春新碱、7-乙基-10-羟基喜树碱、依托泊苷和甲氨蝶呤中至少一种。
  15. 如权利要求9-10任一项所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是所述水溶性药物是阿霉素、表阿霉素、卡铂、假单胞菌外毒素中至少一种。
  16. 如权利要求9所述脂肪酸结合型白蛋白-药物纳米粒子冻干制剂的制备方法，其特征是步骤(3)经液体A萃取处理后的溶液C中的白蛋白的二级结构不变、白蛋白含有“裸露”的脂肪酸结合位点、并以该位点特异性结合脂肪酸，白蛋白分子的疏水区域依疏水性与水不溶性药物结合，白蛋白分子的亲水区域依亲水性与水溶性药物结合。
PCT/CN2015/080977 2014-07-31 2015-06-08 脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 WO2016015522A1 (zh)

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