WO2020114460A1 - 多聚体白蛋白纳米球及其制备方法和应用、载药多聚体白蛋白纳米球及其制备方法和应用 - Google Patents

多聚体白蛋白纳米球及其制备方法和应用、载药多聚体白蛋白纳米球及其制备方法和应用 Download PDF

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WO2020114460A1
WO2020114460A1 PCT/CN2019/123329 CN2019123329W WO2020114460A1 WO 2020114460 A1 WO2020114460 A1 WO 2020114460A1 CN 2019123329 W CN2019123329 W CN 2019123329W WO 2020114460 A1 WO2020114460 A1 WO 2020114460A1
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albumin
aqueous solution
drug
solution
nanospheres
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French (fr)
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郑海荣
胡德红
盛宗海
刘新
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present application relates to the technical field of biomedical materials, in particular to a polymer albumin nanosphere and its preparation method and application, drug-loaded polymer albumin nanosphere and its preparation method and application.
  • albumin is biodegradable, non-toxic, non-antigenic, and many other features, and is considered an ideal drug carrier.
  • the size of a single albumin molecule is within a few nanometers, which is not suitable for direct drug loading.
  • Ultrasonic emulsification and desolventization can produce albumin nanospheres with a particle size of less than 1 ⁇ m, which can be used to carry drugs.
  • the protein molecule has high water solubility, and the release performance of the albumin nanosphere carrier prepared by this kind of method is not easy to control. How to make the albumin nanoparticles have good stability in water and do not dissolve under dilution conditions is currently prepared Technical difficulties.
  • Glutaraldehyde and other cross-linking agents are often used to stabilize the obtained nanospheres, but glutaraldehyde will non-selectively bind to the amino site on the surface of albumin, and will release aldehyde residues in the organism, which has Significant toxic side effects. Therefore, it is necessary to provide a safe and highly stable albumin nanosphere for drug delivery.
  • One of the objectives of the present application is to provide a polymer albumin nanosphere to alleviate the technical problems of the albumin nanosphere existing in the prior art that the stability is poor and there may be toxic side effects.
  • the polymer albumin nanospheres provided by the present application are mainly formed by aggregation of a plurality of albumin molecules, and the plurality of albumin molecules are connected by disulfide bonds, and the particles of the polymer albumin nanospheres
  • the diameter is 10 to 100 nm, preferably 30 to 50 nm.
  • the albumin molecule is at least one of human serum albumin molecule, bovine serum albumin molecule, porcine serum albumin molecule and recombinant albumin molecule.
  • the second objective of the present application is to provide a method for preparing the above-mentioned polymer albumin nanospheres, including the following steps:
  • the pH value of the aqueous albumin solution is 7-12;
  • the volume mass concentration of the aqueous albumin solution is 0.01-300 mg/mL
  • the mixing time of the aqueous solution of albumin and the thiol-containing reducing agent is 0.05-12 hours;
  • the mixing temperature of the aqueous solution of albumin and the thiol-containing reducing agent is 0-60°C;
  • step (b) the aqueous solution of reduced albumin is dispersed at 0 to 60°C;
  • the mixing temperature of the aqueous solution of reduced albumin and the organic solvent is 0-60°C;
  • the aqueous solution of reduced albumin is dispersed by an ultrasonic cell disruptor; further preferably, the power of the ultrasonic cell disruptor is 1 to 1000 W;
  • the addition rate of the organic solvent is 0.01-1000 mL/s;
  • step (b) the mixing time of the reduced albumin aqueous solution and the organic solvent is 5-240 min.
  • step (a) the molar ratio of the thiol-containing reducing agent to albumin is (10-5000): 1;
  • the thiol-containing reducing agent is selected from at least one of glutathione, dithiothreitol, mercaptoethanol, cysteine, or homocysteine.
  • step (b) the volume ratio of the organic solvent to the reduced aqueous albumin solution is (0.1-100): 1;
  • the organic solvent is selected from at least one of dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide and sulfolane.
  • step (b) after the aqueous solution of the reduced albumin and the organic solvent are uniformly mixed, an aqueous solution of polymer albumin nanospheres is obtained, and the aqueous solution of polymer albumin nanospheres is sequentially purified and dried, Multimeric albumin nanospheres were obtained.
  • dialysis is used for purification, further preferably, dialysis is performed at 0 to 60°C; further preferably, dialysis is performed under a pH value of 7 to 12; even more preferably, a pH value of 7 to 12 is used Dialysis with buffer;
  • lyophilization is used for drying, and further preferably, it is pre-frozen at -20 to 0°C for 1 to 48 hours, then frozen at -80 to -20°C for 2 to 48 hours, and then freeze dried in a freeze dryer 12 ⁇ 120h.
  • the third object of the present application is to provide a drug-loaded polymer albumin nanosphere, including a polymer albumin nanosphere and a drug, and the drug is contained in the polymer albumin nanosphere.
  • the drugs include anticancer drugs and/or contrast agents, and the particle size of the drug-loaded multiple specific albumin nanospheres is 10 to 100 nm, preferably 30 to 50 nm;
  • the anticancer drug is selected from platinum and platinum complexes, 5 ⁇ ,20-epoxy-1,2 ⁇ ,4,7 ⁇ ,10 ⁇ ,13 ⁇ -hexahydroxytaxane-11-ene-9-one- 4,10-Diacetate-2-benzoate-13[(2'R,3'S)-N-benzoyl-3-phenylisoserine], (7S:9S)-9-hydroxyl Acetyl-4-methoxy-7,8,9,10-tetrahydro-6,7,9,11-tetrahydroxy-7-0-(2',3',6',-trideoxy- 3'-Chloro-a-1-Lesothiopyranyl)-5,12-naphthalenedione, (E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione, 1,3,5,8-tetramethyl-2,4-di(a-hydroxyethyl)porphy
  • the contrast agent is selected from tetrafluoroboric acid, 4-[2-[2-chloro-3-[(2,6-diphenyl-4H-thiopyran-4-ylidene)ethylene]- 1-cyclohexen-1-yl]vinyl]-2,6-diphenylthiopyran, 2,7-bis[1,3-dihydro-1,1-dimethyl-3-( 4-sulfobutyl)-1,3,5-heptatriene monosodium salt, p-[(2,4-diaminopteridine-6)-N-methylmethylamino]benzoylglutamic acid, 3,7-bis(dimethylamino)phenothiazine-5-onium chloride, 6,6'-[(3,3'-dimethyl(1,1'-diphenyl)-4,4' -Diyl]bis(azo)]bis(4-amino-5-hydroxy-1,3-naphthalenedisulfonic acid) t
  • the fourth purpose of the present application is to provide a method for preparing the drug-loaded polymer albumin nanospheres, including the following steps:
  • (A) Provide an aqueous solution of albumin, and add a thiol-containing reducing agent to the aqueous solution of albumin to prepare an aqueous solution of reduced albumin;
  • the mass ratio of the drug to albumin is (0.0002 to 5): 1; preferably (0.0002 to 0.5): 1; further preferably (0.0008 to 0.5): 1, even more preferably (0.008 to 0.5): 1;
  • the molar ratio of the thiol-containing reducing agent to albumin is (10-5000): 1;
  • the volume ratio of the organic solvent to the reduced aqueous albumin solution is (0.1-100): 1;
  • the thiol-containing reducing agent is selected from at least one of glutathione, dithiothreitol, mercaptoethanol, cysteine or homocysteine;
  • the organic solvent is selected from at least one of dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide and sulfolane;
  • step (A) the pH value of the aqueous albumin solution is 7-12;
  • the volume mass concentration of the aqueous albumin solution is 0.01-300 mg/mL
  • the mixing time of the aqueous solution of albumin and the thiol-containing reducing agent is 0.05-12 hours;
  • step (A) the mixing temperature of the aqueous solution of albumin and the thiol-containing reducing agent is 0-60°C;
  • step (B) the aqueous solution of reduced albumin is dispersed at 0 to 60°C;
  • step (B) the mixing temperature of the aqueous solution of reduced albumin and the organic solvent is 0-60°C;
  • step (B) the aqueous solution of reduced albumin is dispersed by an ultrasonic cell disruptor; further preferably, the power of ultrasound is 1 to 1500 W;
  • step (B) the addition rate of the organic solvent is 0.01-1000 mL/s;
  • step (B) the mixing time of the reduced albumin aqueous solution and the organic solvent is 5 to 240 min.
  • step (B) after the aqueous solution of reduced albumin and the organic solvent are uniformly mixed, an aqueous solution of drug-loaded polymer albumin nanospheres is obtained, and the aqueous solution of the drug-loaded polymer albumin nanospheres is sequentially purified and dried , To obtain drug-loaded polymer albumin nanospheres;
  • dialysis is used for purification, further preferably, dialysis is performed at 0 to 60°C; further preferably, dialysis is performed under a pH value of 7 to 12; even more preferably, a pH value of 7 to 12 is used Dialysis with buffer;
  • lyophilization is used for drying, and further preferably, it is pre-frozen at -20 to 0°C for 1 to 48 hours, then frozen at -80 to -20°C for 2 to 48 hours, and then freeze dried in a freeze dryer 12 ⁇ 120h.
  • the fifth object of the present application is to provide the application of the above-mentioned polymer albumin nanospheres or drug-loaded polymer albumin nanospheres in the preparation of drugs for preventing, treating or diagnosing cancer.
  • the polymer albumin nanospheres provided in this application are connected by disulfide bonds between albumin molecules, and have good stability in water.
  • the particle diameter of the polymer albumin nanospheres is 10-100 nm, and the particle diameter Small, narrow distribution, uniform size, good dispersion, is a good carrier for drug delivery, is conducive to effective and stable drug release in the patient for a long time.
  • the polymer albumin nanospheres provided by the present application are mainly made of aggregated albumin, which has good biocompatibility, no obvious tissue toxicity, and good safety.
  • the preparation method of the polymer albumin nanospheres provided by the present application is simple, the reaction conditions are mild, the reaction reproducibility is good, and the prepared polymer albumin nanospheres have a small particle size and good dispersion.
  • the drug-loaded polymer albumin nanospheres provided in the present application encapsulate the drug in the polymer albumin nanospheres, which can stably exist in the blood system of the living body, thereby avoiding the ineffective release of the drug during the blood circulation system transmission process
  • the drug-loaded polymer albumin nanospheres provided in this application have the ability to actively target tumor tissues, and can effectively and stably release drugs in the tumor tissue area, thereby significantly improving the therapeutic effect.
  • the preparation method of the drug-loaded polymer albumin nanospheres provided by the present application has a simple process, mild reaction conditions, and good reaction reproducibility.
  • the prepared polymer albumin nanospheres have a small particle size and good dispersion.
  • Example 1 is a scanning electron microscope image of drug-loaded polymer albumin nanospheres provided in Example 13 of the present application;
  • Example 2 is a near-infrared two-region fluorescence imaging diagram of drug-loaded polymer albumin nanospheres provided in Example 13 of the present application after injection into tumor-bearing nude mice;
  • FIG. 3 is a tissue slice view of drug-loaded polymer albumin nanospheres provided in Example 13 of the present application after injection into tumor-bearing nude mice.
  • Albumin is a biologically endogenous protein, which has the advantages of biodegradability and non-toxicity. It is considered to be an ideal drug carrier, but albumin will dissolve under dilution conditions, is unstable in the organism, and cannot effectively carry drugs. The molecule enters the target organ.
  • the present application provides a polymer albumin nanosphere, which is mainly formed by the aggregation of multiple albumin molecules, and the multiple albumin molecules are connected by disulfide bonds, and the polymer white
  • the particle diameter of the protein nanosphere is 10-100 nm, preferably 30-50 nm.
  • multimeric albumin nanospheres are interconnected by albumin molecules through disulfide bonds. Since the monomer albumin molecule contains at least one sulfhydryl group or disulfide bond, if the albumin single The thiol group or disulfide bond in the body molecule does not react when forming the polymer albumin nanospheres, it may remain in the polymer albumin spheres.
  • the polymer albumin nanospheres provided in this application may contain a sulfhydryl group, a disulfide bond, or contain both a sulfhydryl group and a disulfide bond, that is, the polymer albumin nanosphere provided in this application may contain a sulfhydryl group and/or a disulfide bond.
  • the physically-aggregated albumin nanospheres are easy to disintegrate, and the drugs contained in the nanospheres are easily released prematurely, which is not conducive to the transportation of target deliverables such as drugs in the human circulatory system. That is, the controllability of the drug release performance of the albumin nanosphere carrier agglomerated by physical methods is not high, and the multimeric albumin nanospheres provided by the present application are interconnected by multiple albumin monomer molecules through intermolecular disulfide bonds
  • the chemically-stabilized polymer structure is more stable than protein nanospheres aggregated by physical methods, and is not easy to be diluted and dissolved by human body fluids and disintegrate, which helps to ensure sufficient drug concentration at the targeted site.
  • the particle size of the nano drug carrier is more important, and the metabolic pathways of different particle sizes are different.
  • the small particle size is metabolized by the kidney, and the large particle size is metabolized by the liver.
  • particles of 20 to 200 nm have passive targets for tumors.
  • the nano drug carrier in this particle size range carries the drug, it can reduce the toxic and side effects of the drug itself, and also enhance the therapeutic effect.
  • the particle size of the polymer albumin nanospheres is 10-20 nm.
  • the particle size of the polymer albumin nanospheres is 20-30 nm.
  • the particle size of the polymer albumin nanospheres is 30-40 nm.
  • the particle size of the polymer albumin nanospheres is 40-50 nm.
  • the particle size of the polymer albumin nanospheres is 50-100 nm.
  • the polymer albumin nanospheres provided in this application are connected by disulfide bonds between albumin molecules, and have good stability in water.
  • the particle diameter of the polymer albumin nanospheres is 10-100 nm, and the particle diameter Small, narrow distribution, uniform size, good dispersion, is a good carrier for drug delivery, is conducive to effective and stable drug release in the patient for a long time.
  • the polymer albumin nanospheres provided by the present application are mainly made of aggregated albumin, which has good biocompatibility, no obvious tissue toxicity, and good safety.
  • the polymer albumin nanospheres provided in this application have a small particle size, a uniform size, and good dispersibility.
  • the use of the nanospheres for drug delivery has great advantages in biomedical applications, which is beneficial for the nanospheres to enter the tumor through the EPR effect and pass The GP60 pathway targets tumor cells.
  • the multimeric albumin nanospheres provided by this application are made of multiple albumin monomer molecules connected by disulfide bonds, and the molecular weight is higher than that of free protein molecules, which is not easy to be filtered by the glomeruli, thereby effectively improving the drug, etc. Delivery efficiency of targeted deliverables.
  • the albumin molecule is at least one of human serum albumin molecule, bovine serum albumin molecule, porcine serum albumin molecule and recombinant albumin molecule.
  • the albumin molecules contain at least one sulfhydryl group or disulfide bond, so that the albumin molecules can be connected by disulfide bonds to form multimeric albumin nanospheres.
  • the present application provides a method for preparing the above-mentioned multimeric albumin nanospheres, including the following steps:
  • the preparation method of the polymer albumin nanospheres provided by the present application is simple, the reaction conditions are mild, the reaction reproducibility is good, and the prepared polymer albumin nanospheres have a small particle size and good dispersion.
  • step (a) the aqueous solution of albumin is first adjusted to a pH value of 7-12, and then a reducing agent with a thiol group is added to the aqueous solution of albumin to increase the whiteness Protein reduction efficiency.
  • the typical but non-limiting pH value of the adjusted aqueous albumin solution is 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.
  • the volume-mass concentration of the aqueous solution of albumin is 0.01-300 mg/mL, so as to facilitate the preparation of multimeric albumin nanospheres.
  • the typical but non-limiting volume mass concentration of the aqueous albumin solution is 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 , 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 295.
  • step (a) a thiol-containing reducing agent is added to the aqueous solution of albumin, and then the reaction is gently shaken at 0-60°C for 0.05-12 hours to make the albumin The reduction reaction is more complete.
  • the typical but non-limiting temperature for performing step (a) is 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60°C .
  • step (a) the molar ratio of the thiol-containing reducing agent to albumin is (10-5000): 1; to ensure that the thiol group on the albumin molecule is completely reduced.
  • the molar ratio of the thiol-containing reducing agent to albumin is 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 1000:1. 1500:1, 2000:1, 2500:1, 3000:1, 3500:1, 4000:1, 4500:1 or 5000:1.
  • the thiol-containing reducing agent is selected from at least one of glutathione, dithiothreitol, mercaptoethanol, cysteine, or homocysteine.
  • step (b) the volume ratio of the organic solvent to the reduced albumin aqueous solution is (0.1-100): 1;
  • the volume ratio of the organic solvent to the reduced aqueous albumin solution is 0.1:1, 0.5:1, 1:1, 5:1, 10:1, 20:1, 30:1. 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1.
  • the organic solvent is selected from at least one of dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylformamide and sulfolane.
  • organic solvents can precipitate protein molecules, so that the albumin molecules aggregate due to the formation of disulfide bonds to form multimeric albumin nanospheres.
  • an ultrasonic cell disruptor in step (b), can be used for dispersion.
  • the ultrasonic cell disruptor is dispersed under the condition of 0 to 60° C.
  • the power range of the ultrasonic cell disruptor is 1 ⁇ 1500W, at the same time add organic solvent to the solution processed by the ultrasonic cell disruptor at a rate of 0.01 ⁇ 1000mL/s, and react at 0 ⁇ 60°C for 5 ⁇ 240min to obtain polymer-containing albumin nanospheres The solution.
  • the typical but non-limiting temperature for performing step (a) is 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60°C .
  • the typical but non-limiting power of ultrasonic power is 1, 5, 10, 50, 100, 200, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 , 1300, 1400 or 1500W.
  • typical but non-limiting addition rates of organic solvents are 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 400, 500, 600, 700, 800, 900 or 1000mL/s.
  • the typical but non-limiting mixing time of the reduced aqueous albumin solution and the organic solvent is 5, 10, 20, 30, 40, 50, 60, 70 , 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 min.
  • the ultrasonic cell disrupter is used to increase the dispersibility of the albumin in the solution and make it fully contacted.
  • This step may use other mixing methods in the industry, such as ultrasound or stirring.
  • Controlling the amount of reducing agent, the amount of ultrasonic power and the speed of adding organic solvent can control the particle size of the prepared polymer albumin nanospheres, because when the amount of reducing agent added is small, the ultrasonic power is large , The injection speed of dimethyl sulfoxide is small, the particle size of the obtained polymer albumin nanospheres is small; conversely, when the amount of reducing agent added is large, the ultrasonic power is small, and the injection speed of dimethyl sulfoxide is large, The particle diameter of the obtained polymer albumin nanospheres is large.
  • the preparation method of the polymer albumin nanospheres provided by the present application can not only obtain the polymer albumin nanospheres, but also obtain the polymer albumin nanospheres with controllable particle sizes, and the method provided by the present application can be prepared Multimeric albumin nanospheres with a particle size between 10 and 100 nm are produced.
  • step (b) after the aqueous solution of reduced albumin and the organic solvent are mixed uniformly, an aqueous solution of multimeric albumin nanospheres is obtained, and the multimeric albumin nanospheres The aqueous solution was purified and dried in sequence to obtain polymer albumin nanospheres.
  • dialysis is used for purification, further preferably, dialysis is performed at 0 to 60°C; further preferably, dialysis is performed under a pH value of 7 to 12; even more preferably, a pH value of 7 to 12 is used Dialysis in buffer;
  • lyophilization is used for drying, and further preferably, it is pre-frozen at -20 to 0°C for 1 to 48 hours, then frozen at -80 to -20°C for 2 to 48 hours, and then freeze dried in a freeze dryer 12 ⁇ 120h.
  • the typical but non-limiting temperature for performing dialysis is 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60°C.
  • the typical but non-limiting pH of the dialysis buffer is 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.
  • the typical but non-limiting time for pre-freezing is 1, 2, 5, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35 , 38, 40, 42, 45 or 48h.
  • typical but non-limiting temperatures for pre-freezing are -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1 or 0°C.
  • typical but non-limiting temperatures for freezing are -80, -75, -70, -65, -60, -55, -50, -45, -40,- 35, -30, -25, or -20°C.
  • the typical but non-limiting time for freezing is 2, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38 , 40, 42, 45, 46 or 48h.
  • the typical but non-limiting time for freeze drying in the freeze dryer is 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40 , 42, 45, 48, 50, 52, 55, 58, 60, 62, 65, 68, 70, 72, 75, 78, 80, 85, 90, 95, 100, 105, 110, 115 or 120h.
  • the unpurified polymer albumin nanosphere solution prepared in step (b) is dialyzed to obtain a polymer albumin nanosphere purification solution;
  • the purified solution of polymer albumin nanospheres is then dried and dehydrated to obtain polymer albumin nanospheres.
  • the dialysis method is: the solution of the polymer albumin nanospheres prepared in step (b) is placed in a buffer solution with a pH value of 7-12 to dialyze to obtain Multimer albumin nanosphere purification solution.
  • the buffer used for dialysis is PBS buffer or Tris buffer.
  • the dialysis time is 10 to 300 hours.
  • the typical but non-limiting time for dialysis is 10, 20, 50, 80, 100, 120, 150, 180, 200, 220, 250, 280 or 300 h.
  • the polymer albumin nanosphere solution is dialyzed in a buffer solution with a pH value of 7 to 12, and then placed in double distilled water for dialysis.
  • the time for dialysis in double distilled water is 1-24 hours.
  • PBS buffer solution with a pH value of 7-12 is used for dialysis in this application, on the one hand, it can remove impurities such as inorganic small molecules in the solution containing polymer albumin nanospheres, more importantly Yes, the agglomerated polymer-containing albumin nanospheres can be dispersed into nanospheres with a smaller particle size under alkaline conditions, thereby obtaining dispersed and uniform particle-size polymer albumin nanospheres.
  • the particle size range of the polymer albumin nanospheres provided in this application is between 10 and 100 nm, however, this is not the particle size distribution of the polymer albumin nanospheres prepared in the same batch.
  • Each batch of polymer albumin nanospheres obtained by the preparation method of body albumin nanospheres has a narrow particle size distribution range, for example, between 20 and 30 nm. Therefore, the polymer albumin nanoparticles prepared in the present application
  • the particle size of the ball is relatively uniform and the size can be controlled.
  • the dialysis temperature is 4-60°C.
  • the method for drying and dehydrating the purified polymer albumin nano solution is freeze-drying, spray drying or reduced-pressure distillation.
  • the step of freeze drying is: the purified solution of polymer albumin nanospheres is placed at -20 to 0°C, pre-frozen for 1 to 48 hours, and then transferred to -80 to -20°C Freeze for 2 to 48 hours, and then freeze-dry for 12 to 120 hours in a freeze dryer to obtain polymer albumin nanospheres.
  • the application of the polymer nanospheres provided in the present application in the preparation of drugs for preventing, treating or diagnosing cancer.
  • cancer includes tumors.
  • polymer albumin nanospheres provided by the present application, as well as the preparation method and application thereof have the following beneficial effects:
  • the multimeric albumin nanospheres provided by this application are composed of different albumin molecules interconnected by disulfide bonds between the molecules to form nanospheres, in water, phosphate buffer, ethanol, serum, culture medium and other solvents Under dilution conditions, it has higher stability than physically bound albumin carrier;
  • albumin nanospheres Compared with the use of chemical cross-linking agents such as glutaraldehyde and other stable albumin carriers, the albumin nanospheres provided in this application use the disulfide bonds of the protein molecules to obtain stable nanospheres, so this clearly provides Of albumin nanospheres are safer;
  • the polymer albumin nanospheres provided in this application have a particle size of 10 to 100 nm, and have a uniform size and good dispersibility. It is a good carrier for the delivery of target deliverables such as drugs or contrast agents, and is beneficial to the patient Long-term effective, safe and stable release of delivery items;
  • the multimeric albumin nanospheres provided in this application can be used to prepare drugs for preventing, treating or diagnosing cancer.
  • the present application provides a drug-loaded polymer albumin nanosphere, including the polymerized albumin nanosphere and the drug provided in the present application, and the polymerized albumin nanosphere is encapsulated in
  • drugs which include anti-cancer drugs and/or contrast agents
  • the particle size of the drug-loaded multiple specific albumin nanospheres is 10 to 100 nm, preferably 30 to 50 nm.
  • the anticancer drug is selected from platinum and platinum complexes, 5 ⁇ ,20-epoxy-1,2 ⁇ ,4,7 ⁇ ,10 ⁇ ,13 ⁇ -hexahydroxytaxane -11-ene-9-one-4,10-diacetate-2-benzoate-13[(2'R,3'S)-N-benzoyl-3-phenylisoserine]( Paclitaxel), (7S: 9S)-9-hydroxyacetyl-4-methoxy-7,8,9,10-tetrahydro-6,7,9,11-tetrahydroxy-7-0-(2' ,3',6',-Trideoxy-3'-chloro-a-1-Lesothiopyranyl)-5,12-naphthalenedione (doxorubicin), (E,E)-1 ,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin), 1,3,5,
  • the contrast agent is selected from 4-[2-[2-chloro-3-[(2,6-diphenyl-4H-thiopyran-4-ylidene) tetrafluoroborate Ethylene]-1-cyclohexen-1-yl]vinyl]-2,6-diphenylthiopyran (IR1061), 2,7-bis[1,3-dihydro-1,1 -Dimethyl-3-(4-sulfobutyl)-1,3,5-heptatriene monosodium salt (indocyanine green), p-[(2,4-diaminopyridine-6)-N -Methylmethylamino]benzoylglutamic acid (methotrexate), 3,7-bis(dimethylamino)phenothiazine-5-onium chloride (methylene blue), 6,6'-[[ 3,3'-dimethyl(1,1'-diphenyl)-4,4'-di
  • Metal nanoparticles include, but are not limited to, one or more of nanogold, nanosilver, and nanocopper sulfide.
  • the drug-loaded polymer albumin nanospheres provided in the present application encapsulate the drug in the polymer albumin nanospheres, which can stably exist in the blood system of the living body, thereby avoiding the ineffective release of the drug during the blood circulation system transmission process
  • the drug-loaded multi-specific albumin nanospheres provided in this application have the ability to actively target tumor tissue, and can effectively and stably release drugs in the tumor tissue area, thereby significantly improving the therapeutic effect.
  • the particle size of the drug-loaded polymer albumin nanospheres is 20 to 200 nm, it has a passive targeting effect on the tumor, and the drug-loaded polymer within this particle size range Albumin nanospheres can reduce the toxic and side effects of the drug itself, while also enhancing the efficacy.
  • the drug-loaded polymer albumin nanospheres provided in this application are improved on the basis of the polymer albumin nanospheres provided in this application, and are prepared by encapsulating drugs in the polymer albumin nanospheres provided in this application.
  • the technical solutions and beneficial effects described in the above-mentioned multimeric albumin nanospheres also belong to this application and will not be repeated here.
  • the drug-loaded polymer albumin nanospheres provided in this application can carry both anticancer drugs and contrast agents, as well as anticancer drugs and contrast agents, so that not only can anticancer drugs be delivered to tumor tissues for effective It can be used for real-time and non-invasive monitoring of the delivery behavior of nanoparticles in the body before treatment. After treatment, the therapeutic effect can be evaluated in real time by imaging to achieve imaging-guided treatment.
  • the present application provides a method for preparing the drug-loaded polymer albumin nanospheres, including the following steps:
  • (A) Provide an aqueous solution of albumin, add a reducing agent with a thiol group to the mixed solution of albumin and drug to prepare a mixed solution of reduced albumin and drug;
  • (B) Disperse the mixed solution of reduced albumin and drug, and add an organic solvent, the reduced albumin is connected by disulfide bond, and the drug is encapsulated at the same time, that is, the drug-loaded polymer albumin nanosphere is prepared ; Among them, the drug is dissolved in the aqueous solution of albumin and/or the drug is dissolved in the organic solvent.
  • the preparation method of the drug-loaded polymer albumin nanospheres provided by the present application has a simple process, mild reaction conditions, and good reaction reproducibility.
  • the prepared polymer albumin nanospheres have a small particle size and good dispersion.
  • the preparation method of the drug-loaded polymer albumin nanospheres provided by the present application is different from the preparation method of the polymer albumin nanospheres in that a drug is dissolved in an aqueous solution of albumin and/or a drug is dissolved in an organic solvent .
  • the aqueous solution of albumin and the drug are fully mixed first, so that the albumin is in full contact with the drug before the drug is loaded, which can effectively improve the efficiency of the albumin encapsulating the drug.
  • the solubility of the drug in the aqueous solution of the reduced albumin can be improved, and the drug can be uniformly dissolved in the reduced albumin solution, so that the prepared The particle size of drug-loaded polymer albumin nanospheres is more uniform.
  • the solubility of the drug is greater and it is more uniformly dissolved in the reduced albumin solution.
  • the mass ratio of the drug to albumin is (0.0002 to 5): 1, preferably (0.0002 to 0.5): 1; further preferably (0.0008 to 0.5): 1, even further Preferably (0.008 to 0.5): 1.
  • the typical but non-limiting mass ratio of drug to albumin is: 0.0002:1, 0.0005:1, 0.008:1, 0.001:1, 0.002:1, 0.005:1, 0.008: 1. 0.01:1, 0.05:1, 0.1:1, 0.5:1, 1:1 or 5:1.
  • the drug loading rate of the polymer albumin nanospheres can be improved to reduce drug waste and enhance the therapeutic effect.
  • the present application provides the application of the drug-loaded polymer albumin nanospheres in the preparation of drugs for preventing, treating or diagnosing cancer.
  • the drug-loaded polymer albumin nanospheres provided by this application have good targeting, on the one hand, the uncontrolled growth of tumor tissue increases the selectivity, high permeability and retention of macromolecular substances and lipid particles (EPR effect) , which provides a passive targeting ability to tumor tissue for drug-loaded polymer albumin nanospheres; on the other hand, the surface of tumor tissue cell membrane is rich in albumin receptors such as GP60, GP30, GP18, etc.
  • Albumin nanospheres provide an ability to actively target tumor tissue. The combination of the two improves the targeting of drug-loaded polymer albumin nanospheres.
  • the drug-loaded polymer albumin nanospheres provided in this application are loaded with anti-cancer drugs and contrast agents, they also have near-infrared two-region fluorescence imaging capabilities, which can be used to monitor real-time, non-invasively pre-treatment drug loading The transport behavior of polymer albumin nanospheres in vivo.
  • This embodiment provides a polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add cysteine to the pH 7 albumin mixture obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 0.05 h.
  • the number of moles of cysteine is 10 times the number of moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 60°C, the power of the ultrasonic cell disruptor is 10W, and at the same time, it is injected into the ultrasonic solution at a speed of 50 ml/s 2mL of dimethyl sulfoxide solution, after the solution was reacted at 60°C for 20min, a solution containing polymer albumin nanospheres was obtained;
  • step (3) Move the solution containing polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 30°C, place the dialysis bag in 1 L of pH 7 PBS buffer and dialyze for 10 hours. During the period, the solution was changed every 12h, each time using 1L of pH 7 PBS buffer, and then dialyzing the dialysis bag in 5L double distilled water for 1h to obtain polymer albumin nanosphere solution;
  • polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -0 °C for 1 h, then transferred to -20 °C for freezing for 2 h, and then freeze-dried in a freeze dryer for 12 h to obtain polymer Body albumin nanospheres, polymer albumin nanospheres have a particle size of 40-60 nm.
  • This embodiment provides a polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add dithiothreitol to the albumin mixture solution of pH 12 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 30°C for 12 hours.
  • the number of moles of dithiothreitol is 5000 times the number of moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 30° C.
  • the power of the ultrasonic cell disruptor is 100 W, and at the same time, it is injected into the ultrasonic solution at a rate of 1000 ml/s 200mL of dimethyl sulfoxide solution, after the solution was reacted at 30°C for 240min, a solution containing polymer albumin nanospheres was obtained;
  • step (3) Move the solution containing polymer albumin nanospheres obtained in step (3) into the dialysis bag, and keep the temperature at 60°C, place the dialysis bag in 1L PBS buffer with pH 12 and dialyze for 300h. Each time, 1L of PBS buffer with a pH of 12 was used, and the solution was changed every 12h during the period, and then the dialysis bag was placed in 5L double distilled water for dialysis for 24h to obtain a polymer albumin nanosphere solution;
  • polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -4°C for 48h, then transferred to -80°C for freezing for 36h, and then freeze-dried in a freeze dryer for 96h to obtain polymer Body albumin nanospheres, polymer albumin nanospheres have a particle size of 30-40 nm.
  • This embodiment provides a polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add ⁇ -mercaptoethanol to the albumin mixture with pH 9 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 0°C for 17 hours.
  • the reaction solution the The number of moles of ⁇ -mercaptoethanol is 2500 times that of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 0°C, the power of the ultrasonic cell disruptor is 10W, and the ultrasonic solution is injected at a rate of 100 ml/s 100mL of dimethyl sulfoxide solution, after the solution was reacted at 0 °C for 5min to obtain a solution containing polymer albumin nanospheres;
  • step (3) Move the solution containing polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 0°C, place the dialysis bag in 1L PBS buffer with pH 9 and dialyze for 144h. Each time, 1L of PBS buffer with a pH of 12 was used, and the solution was changed every 12h during the period, and then the dialysis bag was placed in 5L double distilled water for dialysis for 12h to obtain a polymer albumin nanosphere solution;
  • polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -20°C for 24h, then transferred to -50°C for 48h, and then freeze-dried in a freeze dryer for 120h to obtain polymer Body albumin nanospheres, polymer albumin nanospheres have a particle size of 30-60 nm.
  • This embodiment provides a polymer albumin nanosphere.
  • the preparation method of the polymer albumin nanosphere provided in this embodiment includes the following steps:
  • step (2) Add cysteine to the aqueous solution of bovine serum albumin obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 0.05 h.
  • the cysteine The number of moles is 10 times that of albumin;
  • step (3) Move the solution containing polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 25°C, place the dialysis bag in 5L of double-distilled water and dialyze for 12h to obtain the polymer Albumin nanosphere solution;
  • polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -20°C for 2h, then transferred to -80°C for 24h, and then lyophilized in a freeze dryer for 48h to obtain polymer Body albumin nanospheres, polymer albumin nanospheres have a particle size of 10-100 nm.
  • Step (3) Treating the reduced protein reaction solution with an ultrasonic cell disruptor can make the particle size of the polymer albumin nanospheres uniform and the distribution concentration higher.
  • This embodiment provides a polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add glutathione to the pH 7 albumin mixture obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 0°C for 1 h.
  • the reaction solution all The molar number of glutathione is 10 times the molar number of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 0°C, the power of the ultrasonic cell disruptor is 10W, and at the same time, the ultrasonic solution under the conditions is 1000mL/s 4mL of dimethyl sulfoxide injected at a rate to obtain a solution, and the solution was reacted at 0°C for 5 minutes to obtain a solution containing drug-loaded polymer albumin nanospheres;
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into the dialysis bag, and keep the temperature at 0°C, place the dialysis bag in 5L of pH 10 PBS buffer for dialysis During 10h, the fluid was changed every 12h during this period, each time using 5L of PBS buffer with pH 10, and then dialyzing the dialysis bag in 5L double distilled water for 1h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -20°C for 2 hours, then transferred to -80°C for 12 hours, and then freeze-dried in a freeze dryer for 12 hours to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 20-30 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add dithiothreitol to the mixed solution of the albumin of pH 7 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 0.05 h.
  • the number of moles of dithiothreitol is 5000 times the number of moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 60°C, the power of the ultrasonic cell disruptor is 1W, and at the same time in the ultrasonic solution at a speed of 0.01ml/s 200mL of injected dimethyl sulfoxide-containing solution, which was reacted at 60°C for 20 minutes to obtain a solution containing drug-loaded polymer albumin nanospheres;
  • step (3) Move the solution containing drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 30°C, place the dialysis bag in 1L of pH 7 PBS buffer for dialysis During 300h, the solution was changed every 12h during this period, each time using 1L of pH 7 PBS buffer, and then put the dialysis bag into 5L double distilled water for dialysis for 24h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -0 °C for 1 h, then transferred to -20 °C for freezing for 2 h, and then freeze-dried in a freeze dryer for 72 h to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 40-60 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add ⁇ -mercaptoethanol to the mixed solution of albumin with pH 12 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 30°C for 12 hours.
  • the moles of ⁇ -mercaptoethanol are 100 times the moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 30° C.
  • the power of the ultrasonic cell disruptor is 1500 W, and at the same time, it is injected into the ultrasonic solution at a rate of 1000 ml/s 100mL of a solution containing dimethyl sulfoxide, the solution containing the drug-loaded polymer albumin nanospheres after reacting at 30°C for 240min;
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 60°C, place the dialysis bag in 1 L of pH 12 PBS buffer for dialysis At 144h, 1L of PBS buffer with pH 12 was used each time, and the fluid was changed every 12h during the period, and then the dialysis bag was placed in 5L double distilled water for dialysis for 12h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -4°C for 48h, then transferred to -50°C for 48h, and then freeze-dried in a freeze dryer for 96h to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 30-50 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add glutathione to the aqueous solution of the pH 9 albumin obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 0.05 h.
  • the number of moles of glutathione is 2500 times the number of moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 60° C.
  • the power of the ultrasonic cell disruptor is 100 W, and at the same time, it is injected into the ultrasonic solution at a speed of 50 ml/s 22mL of dimethyl sulfoxide-containing solution, the solution containing the drug-loaded polymer albumin nanospheres was obtained after reacting at 60°C for 30 minutes;
  • step (3) Move the solution containing drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 60 °C, place the dialysis bag in 1L pH 9 PBS buffer for dialysis During 36h, the solution was changed every 12h during this period, each time using 1L of pH 9 PBS buffer solution, and then put the dialysis bag in 5L double distilled water for dialysis for 18h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) was pre-frozen at -10°C for 24h, then transferred to -80°C for 24h, and then freeze-dried in a freeze dryer for 120h to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 50-80 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add glutathione to the aqueous albumin solution of pH 7 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 4°C for 2 hours.
  • the The moles of glutathione are 10 times the moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 4° C.
  • the power of the ultrasonic cell disruptor is 1000 W, and at the same time, it is injected into the ultrasonic solution at a rate of 1 mL/s
  • a solution of body albumin nanospheres; the volume of the dimethyl sulfoxide solution added is twice the volume of the solution after the reaction in the step (2);
  • step (3) Move the solution containing polymer albumin nanospheres obtained in step (3) into the dialysis bag, and keep the temperature at 4°C, place the dialysis bag in 1L PBS buffer with pH 9 and dialyze for 24h. During the period, the solution was changed every 8h, each time using 1L of pH 9 PBS buffer solution, and then placed in a 1L double distilled water for 12h dialysis to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -20°C for 2 hours, then transferred to -80°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 10-20 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • albumin mixed solution which contains porcine serum albumin at a concentration of 300 mg/mL (w/v) and IR1061 at a concentration of 0.1 mg/mL, and then adjust the concentration with a 10 mol/L NaOH solution The pH value of the albumin mixture reaches 12;
  • step (2) Add dithiothreitol to the aqueous solution of the pH 12 albumin obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 240 h.
  • the number of moles of dithiothreitol is 5000 times the number of moles of albumin;
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor under the condition of 60°C, the power of the ultrasonic cell disruptor is 1W, and at the same time, it is injected into the ultrasonic solution at a rate of 1000 ml/s A solution of dimethyl sulfoxide.
  • the solution of dimethyl sulfoxide contains paclitaxel with a volume concentration of 0.1 mg/mL and IR1061 with a volume concentration of 2 mg/mL.
  • the solution is reacted at 60°C for 5 minutes to obtain A solution of drug-loaded polymer albumin nanospheres; the volume of the ethanol solution added is 50 times the volume of the solution after the reaction in the step (2);
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, place the dialysis bag in 5L PBS buffer with pH 12 for 300h at room temperature, and change every 12h during the period The solution is used once, each time using 5L of pH 12 PBS buffer, and then dialyzing the dialysis bag in 5L double distilled water for 12h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -0°C for 1h, then transferred to -20°C for 2h, and then freeze-dried in a freeze dryer for 72h to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 50-100 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • albumin mixture which contains porcine serum albumin at a concentration of 200 mg/mL (w/v) and IR1061 at a concentration of 5 mg/mL, and then adjust the concentration with a 0.1 mol/L NaOH solution
  • the pH value of the aqueous albumin solution is 9;
  • step (2) Add ⁇ -mercaptoethanol to the albumin mixture with pH 9 obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 30°C for 0.05 h.
  • the number of moles of ⁇ -mercaptoethanol is 100 times the number of moles of albumin;
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 60°C, place the dialysis bag in 100 mL of pH 7 PBS buffer for dialysis 2h, 100mL of PBS buffer with pH 7 was used every time, and the fluid was changed every 1h during the period, and then the dialysis bag was placed in 100mL double distilled water for dialysis for 12h to obtain drug-loaded polymer albumin nanosphere solution;
  • step (4) Place the drug-loaded polymer albumin nanosphere solution obtained in step (4) at -4°C for 48h, transfer to -50°C for 48h, and freeze-dry in a freeze dryer for 96h to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 30-40 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • albumin mixture which contains porcine serum albumin at a concentration of 100 mg/mL (w/v), IR1061 at a concentration of 1 mg/mL, paclitaxel and DMSO at a concentration of 1 mg/mL, dimethyl
  • the volume ratio of the solvent water in the mixed solution of sulfoxide and albumin is 0.1:1, and then the pH value of the mixed solution is adjusted to 9 using 1mol/L NaOH solution;
  • step (2) Add glutathione to the mixed solution in step (1) to obtain the reaction solution, and then gently shake the reaction at 30°C for 1 h.
  • the moles of glutathione are 10 moles of albumin Times
  • step (3) The solution after the reaction in step (2) is treated with an ultrasonic cell disruptor at 4°C.
  • the power of the ultrasonic cell disruptor is 1000 W, and the ultrasonic solution is injected into the solution at a rate of 0.01 mL/s.
  • dimethyl sulfoxide a solution was obtained.
  • a solution containing drug-loaded polymer albumin nanospheres was obtained; the volume of dimethyl sulfoxide added was step (2) 50 times the volume of the solution after the reaction;
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 4°C, place the dialysis bag in 100 mL of pH 7 PBS buffer for dialysis During 2h, the fluid was changed every 1h during this period. Each time, 100mL of PBS buffer with pH 7 was used, and then the dialysis bag was placed in 100mL of double distilled water for dialysis for 12h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -20°C for 2 hours, then transferred to -80°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 10-20 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • albumin mixture which contains porcine serum albumin at a concentration of 200 mg/mL (w/v) and paclitaxel and dimethyl sulfoxide at a concentration of 1 mg/mL, dimethyl sulfoxide and The volume ratio of solvent water in the albumin mixture is 0.1:1, and then the pH value of the mixture is adjusted to 9 using 0.1mol/L NaOH solution;
  • step (2) Add glutathione to the mixed solution obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 30°C for 1 h.
  • the number of moles of glutathione is white 100 times the number of moles of protein;
  • step (3) Move the solution containing drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 0°C, place the dialysis bag in 100 mL of pH 7 PBS buffer for dialysis During 2h, the fluid was changed every 1h during this period. Each time, 100mL of PBS buffer with pH 7 was used, and then the dialysis bag was placed in 100mL of double distilled water for dialysis for 12h to obtain drug-loaded polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -20°C for 2 hours, then transferred to -80°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 30-40 nm.
  • This embodiment provides a drug-loaded polymer albumin nanosphere, and its preparation method includes the following steps:
  • step (2) Add glutathione to the albumin mixture obtained in step (1) to obtain a reaction solution, and then gently shake the reaction at 60°C for 0.05 h.
  • the glutathione The number of moles is 5000 times that of albumin;
  • step (3) Move the solution containing the drug-loaded polymer albumin nanospheres obtained in step (3) into a dialysis bag, and keep the temperature at 25°C, place the dialysis bag in 5L of double distilled water and dialyze for 12h to obtain the loaded Pharmaceutical polymer albumin nanosphere solution;
  • the drug-loaded polymer albumin nanosphere solution obtained in step (4) is pre-frozen at -20°C for 2 hours, then transferred to -80°C for 24 hours, and then freeze-dried in a freeze dryer for 48 hours to obtain
  • the drug-loaded polymer albumin nanospheres have a particle size of 10-100 nm.
  • Example 14 It can be seen from the comparison between Examples 5-13 and Example 14 that the concentration distribution of the polymer albumin nanospheres provided in Example 14 is not high, which is not conducive to its application in biomedicine. This shows that in step (3), the treatment of the reduced protein reaction solution with an ultrasonic cell disruptor can make the particle size of the drug-loaded polymer albumin nanospheres uniform and the distribution concentration higher.
  • Example 13 In order to illustrate the beneficial effects of the drug-loaded polymer albumin nanospheres provided in this application, the drug-loaded polymer albumin nanospheres provided in Example 13 were examined by scanning electron microscopy, and FIG. 1 is provided by Example 13 of this application Scanning electron microscope image of drug-loaded polymer albumin nanospheres; as can be seen from FIG. 1, the drug-loaded polymer albumin nanospheres provided in Example 13 are uniform in size and the particles are more dispersed.
  • Example 13 Using C6 in situ glioma tumor-bearing mice as a model, the aqueous solution of polymer albumin nanospheres prepared in Example 13 (concentration: 0.2 mg/mL) was injected through the tail vein, and then a fluorescent two-zone in vivo imaging system was used. To study the transport process of polymerized albumin nanospheres in mice and the specificity and sensitivity of C6 in situ glioma tissue recognition.
  • FIG. 2 is a near-infrared two-region fluorescence imaging diagram of the drug-loaded polymer albumin nanospheres provided in Example 13 of the present application after injection into tumor-bearing nude mice;
  • the fluorescent signal of the tumor site of the tumor-bearing mice with nanospheres was enhanced after 24 hours, indicating that the drug-loaded polymer albumin nanospheres provided in Example 13 have a strong targeting effect on tumor tissues.
  • the nanoparticle group in FIG. 3 is the tissue slice of the drug-loaded polymer albumin nanospheres provided in Example 13 of the present application after injection of tumor-bearing nude mice;
  • the buffer solution group is the tissue slice of PBS buffer solution after injection of tumor-bearing nude mice Figure;
  • the mice injected with the drug-loaded polymer albumin nanospheres provided in Example 13 also did not show any tissue toxicity.
  • Example 13 of the present application can be used as a near-infrared two-region fluorescent molecular imaging probe and a multi-functional targeting probe integrating chemotherapy Needle use.

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Abstract

一种多聚体白蛋白纳米球及其制备方法和应用、载药多聚体白蛋白纳米球及其制备方法和应用，涉及生物医药材料技术领域，多聚体白蛋白纳米球主要由多个白蛋白分子聚集而成，所述多个白蛋白分子之间通过二硫键连接，且所述多聚体白蛋白纳米球的粒径为10～100nm，缓解现有技术中存在的白蛋白纳米球稳定性差且可能存在有毒副作用的技术问题，达到了多聚体白蛋白纳米球由多个白蛋白分子通过二硫键连接而成，粒径小，分布窄，尺寸均一，分散性好，是运载药物的良好载体，有利于在患者体内长时间有效、安全稳定地释放药物的技术效果。

Description

多聚体白蛋白纳米球及其制备方法和应用、载药多聚体白蛋白纳米球及其制备方法和应用 技术领域
本申请涉及生物医药材料技术领域，尤其是涉及一种多聚体白蛋白纳米球及其制备方法和应用、载药多聚体白蛋白纳米球及其制备方法和应用。
背景技术
纳米技术的兴起使得基于高分子纳米微粒的药物输送得到了广泛的关注，白蛋白具有可生物降解、无毒、无抗原性等诸多特点，被认为是一个理想的药物载体。而通常单个白蛋白分子的尺寸在几个纳米以内，不适合直接用于载药，超声乳化法和去溶剂法可制得粒径小于1μm的白蛋白纳米球，可用于运载药物，但由于白蛋白分子的水溶性高，该类方法制得的白蛋白纳米球载体的释药性能不易于控制，如何使白蛋白纳米颗粒在水中有良好的稳定性，且在稀释条件下不溶解是目前制备技术上的难点。
戊二醛等交联剂常被用来稳定得到的纳米球，但戊二醛会非选择性的结合白蛋白表面的氨基位点，在生物体内会释放出醛类残基，对生物体有着显著毒副作用。因此，有必要提供一种安全、稳定性高的白蛋白纳米球以进行药物运载。
有鉴于此，特提出本申请。
发明内容
本申请的目的之一在于提供一种多聚体白蛋白纳米球，以缓解了现有技术中存在的白蛋白纳米球稳定性差且可能存在有毒副作用的技术问题。
本申请提供的多聚体白蛋白纳米球，主要由多个白蛋白分子聚集而成，所述多个白蛋白分子之间通过二硫键连接，且所述多聚体白蛋白纳米球的粒径为10～100nm，优选为30～50nm。
进一步的，所述白蛋白分子为人血清白蛋白分子、牛血清白蛋白分子、猪血清白蛋白分子和重组白蛋白分子中的至少一种。
本申请的目的之二在于提供上述多聚体白蛋白纳米球的制备方法，包括如下步骤：
(a)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂，制得还原型白蛋白的水溶液；
(b)将还原型白蛋白的水溶液分散后，加入有机溶剂混合均匀，即制得多聚体白蛋白纳米球；
优选地，步骤(a)中，白蛋白水溶液的pH值为7～12；
优选地，步骤(a)中，白蛋白水溶液的体积质量浓度为0.01～300mg/mL；
优选地，白蛋白的水溶液与带巯基的还原剂的混合时间为0.05～12h；
优选地，在步骤(a)中，白蛋白的水溶液与带巯基的还原剂的混合温度为0～60℃；
优选地，在步骤(b)中，还原型白蛋白的水溶液的在0～60℃下分散；
优选地，在步骤(b)中，还原型白蛋白的水溶液与有机溶剂的混合温度为0～60℃；
优选地，步骤(b)中，通过超声波细胞破碎仪将还原型白蛋白的水溶液进行分散；进一步优选地，超声波细胞破碎仪的功率为1～1000W；
优选地，在步骤(b)中，有机溶剂的加入速度为0.01～1000mL/s；
优选地，在步骤(b)中，还原型白蛋白水溶液与有机溶剂的混合时间为 5-240min。
进一步的，在步骤(a)中，带巯基的还原剂与白蛋白的摩尔比为(10～5000):1；
优选地，带巯基的还原剂选自谷胱甘肽、二硫苏糖醇、巯基乙醇、半胱氨酸或同型半胱氨酸中的至少一种。
进一步的，在步骤(b)中，有机溶剂与还原型白蛋白水溶液的体积比为(0.1～100):1；
优选地，所述有机溶剂选自二甲基亚砜、N-甲基吡咯烷酮、N,N-二甲基甲酰胺和环丁砜中的至少一种。
进一步的，在步骤(b)中，还原型白蛋白的水溶液与有机溶剂混合均匀后，得到多聚体白蛋白纳米球的水溶液，将多聚体白蛋白纳米球的水溶液依次进行纯化和干燥，得到多聚体白蛋白纳米球。
优选地，采用透析进行纯化，进一步优选地，在0～60℃进行透析；进一步优选地，在pH值为7～12的条件下进行透析；更进一步优选地，采用pH值为7～12的缓冲液进行透析；
优选地，采用冻干进行干燥，进一步优选地，先在-20～0℃下预冻1～48h，再在-80～-20℃下冷冻2～48h，然后再在冷冻干燥机中冷冻干燥12～120h。
本申请的目的之三在于提供一种载药多聚体白蛋白纳米球，包括多聚体白蛋白纳米球和药物，所述药物包载于所述多聚体白蛋白纳米球中，所述药物包括抗癌药物和/或造影剂，且载药多具体白蛋白纳米球的粒径为10～100nm，优选为30～50nm；
优选地，所述抗癌药物选自铂及铂的配合物、5β,20-环氧-1,2α,4,7β,10β,13α-六羟基紫杉烷-11-烯-9-酮-4,10-二乙酸酯-2-苯甲酸酯-13[(2’R,3’S)-N-苯甲酰-3-苯基异丝氨酸酯]、(7S：9S)-9-羟乙酰基-4-甲氧基-7,8,9,10-四氢 -6,7,9,11-四羟基-7-0-(2’,3’,6’,-三去氧-3’-氯基-a-1-来苏已吡喃基)-5,12-萘二酮、(E,E)-1,7-双(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮、1,3,5,8-四甲基-2,4-二(a-羟乙基)卟酚-6,7-二丙酸、4-乙基-4，12-二氧-4-羟-1H-吡喃(3'，4'，6，7)吡吲哚(1，2-6)喹啉-3，14-二酮、(2S-反式)-18-羧基-20-(羧甲基)-13-乙基-2,3-二氢3,7,12,17-四甲基-8-乙烯基-21H,23H-卟吩-2-丙酸、IR700碘化物和11-氯-1,1'-二正丙基-3,3,3',3'-四甲基-10,12-三亚甲基吲哚三碳花青碘盐中的至少一种；
优选地，所述造影剂选自四氟硼酸、4-[2-[2-氯-3-[(2,6-二苯基-4H-噻喃-4-亚基)亚乙基]-1-环己烯-1-基]乙烯基]-2,6-二苯基硫代吡喃，2,7-双[1,3-二氢-1,1-二甲基-3-(4-磺丁基)-1,3,5-庚三烯单钠盐、对-[(2,4-二氨基喋啶-6)-N-甲基甲氨基]苯甲酰谷氨酸、3,7-双(二甲氨基)吩噻嗪-5-翁氯化物、6,6'-[[3,3'-二甲基(1,1'-二苯基)-4,4'-二基]双(偶氮基)]双(4-氨基-5-羟基-1,3-萘二磺酸)四钠盐、2-((4-二乙氨基)苯)(4-(二乙氨基)环己烷-2,5-二烯)甲烷)苯基-1,4-二磺酸盐、4,4'-双(二乙氨基)三苯脱水甲醇-2”,4”-二磺酸单钠和金属纳米粒子中的至少一种。
本申请的目的之四在于提供一种上述载药多聚体白蛋白纳米球的制备方法，包括如下步骤：
(A)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂，制得还原型白蛋白的水溶液；
(B)将还原型白蛋白的水溶液分散后，加入有机溶剂混合均匀，还原型白蛋白通过二硫键连接，即制得载药多聚体白蛋白纳米球，其中，白蛋白的水溶液中溶解有药物和/或有机溶剂中溶解有药物；
优选地，药物与白蛋白的质量比为(0.0002～5):1；优选为(0.0002～0.5)：1；进一步优选为(0.0008～0.5)：1，更进一步优选为(0.008～0.5)：1；
优选地，在步骤(A)中，带巯基的还原剂与白蛋白的摩尔比为(10～5000):1；
优选地，在步骤(B)中，有机溶剂与还原型白蛋白水溶液的体积比为 (0.1～100):1；
优选地，带巯基的还原剂选自谷胱甘肽、二硫苏糖醇、巯基乙醇、半胱氨酸或同型半胱氨酸中的至少一种；
优选地，所述有机溶剂选自二甲基亚砜、N-甲基吡咯烷酮、N,N-二甲基甲酰胺和环丁砜中的至少一种；
优选地，步骤(A)中，白蛋白水溶液的pH值为7～12；
优选地，步骤(A)中，白蛋白水溶液的体积质量浓度为0.01～300mg/mL；
优选地，白蛋白的水溶液与带巯基的还原剂的混合时间为0.05～12h；
优选地，在步骤(A)中，白蛋白的水溶液与带巯基的还原剂的混合温度为0～60℃；
优选地，在步骤(B)中，还原型白蛋白的水溶液的在0～60℃下分散；
优选地，在步骤(B)中，还原型白蛋白的水溶液与有机溶剂的混合温度为0～60℃；
优选地，步骤(B)中，通过超声波细胞破碎仪将还原型白蛋白的水溶液进行分散；进一步优选地，超声的功率为1～1500W；
优选地，在步骤(B)中，有机溶剂的加入速度为0.01～1000mL/s；
优选地，在步骤(B)中，还原型白蛋白水溶液与有机溶剂的混合时间为5～240min。
在步骤(B)中，还原型白蛋白的水溶液与有机溶剂混合均匀后，得到载药多聚体白蛋白纳米球的水溶液，将载药多聚体白蛋白纳米球的水溶液依次进行纯化和干燥，得到载药多聚体白蛋白纳米球；
优选地，采用透析进行纯化，进一步优选地，在0～60℃进行透析；进一步优选地，在pH值为7～12的条件下进行透析；更进一步优选地，采用pH值 为7～12的缓冲液进行透析；
优选地，采用冻干进行干燥，进一步优选地，先在-20～0℃下预冻1～48h，再在-80～-20℃下冷冻2～48h，然后再在冷冻干燥机中冷冻干燥12～120h。
本申请的目的之五在于提供上述多聚体白蛋白纳米球或载药多聚体白蛋白纳米球在制备预防、治疗或诊断癌症的药物中的应用。
本申请提供的多聚体白蛋白纳米球，白蛋白分子之间通过二硫键连接，在水中具有良好的稳定性，同时多聚体白蛋白纳米球的粒径为为10～100nm，粒径小，分布窄，尺寸均一，分散性好，是运载药物的良好载体，有利于在患者体内长时间有效、稳定地释放药物。另外，本申请提供的多聚体白蛋白纳米球主要由白蛋白聚集而成，生物相容性好，无明显组织毒性，安全性好。
本申请提供的多聚体白蛋白纳米球的制备方法工艺简单，反应条件温和，反应重现性良好，制得的多聚体白蛋白纳米球粒径小，分散性好。
本申请提供的载药多聚体白蛋白纳米球，将药物包载在多聚体白蛋白纳米球中，能够稳定存在于生物体内血液系统中，从而避免药物在血液循环系统传输过程中无效释放，同时本申请提供的载药多聚体白蛋白纳米球具有主动靶向肿瘤组织的能力，能够在肿瘤组织区域有效稳定释放药物，从而显著提高治疗效果。
本申请提供的载药多聚体白蛋白纳米球的制备方法，工艺简单，反应条件温和，反应重现性良好，制得的多聚体白蛋白纳米球粒径小，分散性好。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请实施例13提供的载药多聚体白蛋白纳米球的扫描电子显微镜图像；
图2为本申请实施例13提供的载药多聚体白蛋白纳米球注射荷瘤裸鼠后的近红外二区荧光成像图；
图3为本申请实施例13提供的载药多聚体白蛋白纳米球注射荷瘤裸鼠后的组织切片图。
具体实施方式
下面将结合附图对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
白蛋白是一种生物内源性蛋白，具有可生物降解、无毒等优点，被认为是一个理想的药物载体，但是白蛋白在稀释条件下会溶解，在生物体内不稳定，不能有效运载药物分子进入靶器官。
根据本申请的一个方面，本申请提供了一种多聚体白蛋白纳米球，主要由多个白蛋白分子聚集而成，多个白蛋白分子之间通过二硫键连接，且多聚体白蛋白纳米球的粒径为10～100nm，优选为30-50nm。
如本申请所述的，“多聚体白蛋白纳米球”由白蛋白分子之间通过二硫键相互连接，由于单体白蛋白分子中含有至少一个巯基或二硫键，若该白蛋白单体分子中的巯基或二硫键在形成多聚体白蛋白纳米球时没有发生反应，则有可能保留在了多聚体白蛋白球中，因此，本申请提供的多聚体白蛋白纳米球可含有巯基、二硫键、或者同时含有巯基和二硫键，即本申请提供的多聚体白蛋白纳米球可含有巯基和/或二硫键。
由于白蛋白单体分子易溶性，物理方法团聚的白蛋白纳米球容易解体，纳米球中包载的药物很容易被过早地释放，不利于药物等目标投递物在人体循环系统内的运输，即物理方法团聚的白蛋白纳米球载体的释药性能的可控性不高，而本申请提供的多聚体白蛋白纳米球由多个白蛋白单体分子通过分子间的二硫键相互连接，这种化学键稳定的多聚体结构比通过物理方法聚集的蛋白纳米球更稳定，不易被人体体液稀释、溶解而解体，有助于保证靶向部位足够的给药浓度。
在生物医学医用中，纳米药物载体的粒径比较重要，不同粒径代谢途径也不一样，小粒径通过肾代谢，大粒径通过肝代谢；其中，20～200nm的粒子对肿瘤有被动靶向作用，在此粒径范围内的纳米药物载体运载药物时，可以降低药物本身的毒副作用，同时也增强了疗效。
在本申请的一种优选实施方式中，多聚体白蛋白纳米球的粒径为10～20nm。
在本申请的一种优选实施方式中，多聚体白蛋白纳米球的粒径为20～30nm。
在本申请的一种优选实施方式中，多聚体白蛋白纳米球的粒径为30～40nm。
在本申请的一种优选实施方式中，多聚体白蛋白纳米球的粒径为40～50nm。
在本申请的一种优选实施方式中，多聚体白蛋白纳米球的粒径为50～100nm。
本申请提供的多聚体白蛋白纳米球，白蛋白分子之间通过二硫键连接，在水中具有良好的稳定性，同时多聚体白蛋白纳米球的粒径为为10～100nm，粒 径小，分布窄，尺寸均一，分散性好，是运载药物的良好载体，有利于在患者体内长时间有效、稳定地释放药物。另外，本申请提供的多聚体白蛋白纳米球主要由白蛋白聚集而成，生物相容性好，无明显组织毒性，安全性好。
本申请提供的多聚体白蛋白纳米球粒径小且尺寸均一，分散性好，用该纳米球投递药物在生物医学应用方面有较大优势，有利于纳米球通过EPR效应进入肿瘤内部并通过GP60通路靶向到肿瘤细胞。
本申请提供的多聚体白蛋白纳米球由多个白蛋白单体分子通过二硫键连接而成，分子量高于自由蛋白质分子的分子量，不易被肾小球滤过，从而有效提高了药物等目标投递物的投递效率。
在本申请的一种优选实施方式中，白蛋白分子为人血清白蛋白分子、牛血清白蛋白分子、猪血清白蛋白分子和重组白蛋白分子中的至少一种。
在本申请中，白蛋白分子中至少含有一个巯基或二硫键，以使得白蛋白分子之间能够通过二硫键连接以形成多聚体白蛋白纳米球。
根据本申请的第二个方面，本申请提供了上述多聚体白蛋白纳米球的制备方法，包括如下步骤：
(a)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂，制得还原型白蛋白的水溶液；
(b)将还原型白蛋白的水溶液进行分散，并加入有机溶剂，还原型白蛋白通过二硫键连接，即制得多聚体白蛋白纳米球。
本申请提供的多聚体白蛋白纳米球的制备方法工艺简单，反应条件温和，反应重现性良好，制得的多聚体白蛋白纳米球粒径小，分散性好。
在本申请的一种优选实施方式中，在步骤(a)中，先将白蛋白的水溶液调节至pH值为7-12，再向白蛋白的水溶液中加入带巯基的还原剂，以提高白 蛋白的还原效率。
在本申请的该实施方式中，调节后的白蛋白水溶液的典型但非限制性的pH值为7、7.5、8、8.5、9、9.5、10、10.5、11、11.5或12。
在本申请的一种优选实施方式中，在步骤(a)中，白蛋白的水溶液的体积质量浓度为0.01～300mg/mL，以利于制备多聚体白蛋白纳米球。
在本申请该优选实施方式中，白蛋白水溶液的典型但非限制性的体积质量浓度为0.05、0.1、0.5、1、5、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290或295。
在本申请的一种优选实施方式中，在步骤(a)中，向白蛋白的水溶液中加入带巯基的还原剂，然后在0～60℃下轻轻摇动反应0.05～12h，以使得白蛋白的还原反应进行的更加完全。
在本申请的该优选实施方式中，进行步骤(a)的典型但非限制性的温度为0、5、10、15、20、25、30、35、40、45、50、55或60℃。
在本申请的一种优选实施方式中，在步骤(a)中，带巯基的还原剂与白蛋白的摩尔比为(10-5000):1；以保证白蛋白分子上的巯基被完全还原。
在本申请的该优选实施方式中，带巯基的还原剂与白蛋白的摩尔比为10:1、20:1、50:1、100:1、200:1、500:1、1000:1、1500:1、2000:1、2500:1、3000:1、3500:1、4000:1、4500:1或5000:1。
进一步优选地，带巯基的还原剂选自谷胱甘肽、二硫苏糖醇、巯基乙醇、半胱氨酸或同型半胱氨酸中的至少一种。
在本申请的一种优选实施方式中，在步骤(b)中，有机溶剂与还原型白蛋白水溶液的体积比为(0.1～100):1；
在本申请的该优选实施方式中，有机溶剂与还原型白蛋白水溶液的体积比为0.1:1、0.5:1、1:1、5:1、10:1、20:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1或100:1。
进一步优选地，所述有机溶剂选自二甲基亚砜、N-甲基吡咯烷酮、N,N-二甲基甲酰胺和环丁砜中的至少一种。
在本申请的该优选实施方式中，通过在还原型白蛋白的水溶液中。
加入有机溶剂能够将蛋白质分子析出，从而使得白蛋白分子间因二硫键的形成而聚集形成多聚体白蛋白纳米球。
在本申请的一种优选实施方式中，在步骤(b)中，可以采用超声波细胞破碎仪进行分散，超声波细胞破碎仪分散在0～60℃的条件下进行，超声波细胞破碎仪的功率范围为1～1500W，同时在所述超声波细胞破碎仪处理的溶液中以0.01～1000mL/s的速度加入有机溶剂，在0～60℃的条件下反应5～240min后得到含多聚体白蛋白纳米球的溶液。
在本申请的该优选实施方式中，进行步骤(a)的典型但非限制性的温度为0、5、10、15、20、25、30、35、40、45、50、55或60℃。
在本申请的该优选实施方式中，超声功率的典型但非限制性的功率为1、5、10、50、100、200、400、500、600、700、800、900、1000、1100、1200、1300、1400或1500W。
在本申请的该优选实施方式中，有机溶剂的典型但非限制性的加入速度为0.01、0.05、0.1、0.5、1、5、10、50、100、200、400、500、600、700、800、900或1000mL/s。
在本申请的该优选实施方式中，在步骤(b)中，还原型白蛋白水溶液与有机溶剂的典型但非限制性的混合时间为5、10、20、30、40、50、60、70、 80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230或240min。
在本申请的优选实施方式中，通过超声波细胞破碎仪处理以增加溶液中白蛋白的分散性，并使之充分接触，该步骤可以采用行业内其他混合方式，比如超声或搅拌。
控制好还原剂的加入量、超声功率的大小和有机溶剂的加入速度，可以控制所制备的多聚体白蛋白纳米球的粒径，这是因为，当还原剂的加入量小，超声功率大，二甲基亚砜的注入速度小，所得的多聚体白蛋白纳米球的粒径就小；反之，当还原剂的加入量大，超声功率小，二甲基亚砜的注入速度大，所得的多聚体白蛋白纳米球的粒径就大。因此，本申请提供的多聚体白蛋白纳米球的制备方法不但能获得多聚体白蛋白纳米球，而且能得到粒径可控的多聚体白蛋白纳米球，本申请提供的方法可以制备出粒径在10～100nm之间的多聚体白蛋白纳米球。
在本申请的一种优选实施方式中，在步骤(b)中，还原型白蛋白的水溶液与有机溶剂混合均匀后，得到多聚体白蛋白纳米球的水溶液，将多聚体白蛋白纳米球的水溶液依次进行纯化和干燥，得到多聚体白蛋白纳米球。
优选地，采用透析进行纯化，进一步优选地，在0～60℃进行透析；进一步优选地，在pH值为7～12的条件下进行透析；更进一步优选地，采用pH值为7～12的缓冲液中进行透析；
优选地，采用冻干进行干燥，进一步优选地，先在-20～0℃下预冻1～48h，再在-80～-20℃下冷冻2～48h，然后再在冷冻干燥机中冷冻干燥12～120h。
在本申请的一种优选实施方式中，进行透析的典型但非限制性的温度为0、5、10、15、20、25、30、35、40、45、50、55或60℃。
在本申请的一种优选实施方式中，进行透析的缓冲液的典型但非限制性的pH值为7、7.5、8、8.5、9、9.5、10、10.5、11、11.5或12。
在本申请的一种优选实施方式中，进行预冻的典型但非限制性的时间为1、2、5、10、12、15、18、20、22、25、28、30、32、35、38、40、42、45或48h。
在本申请的一种优选实施方式中，进行预冻的典型但非限制性的温度为-20、-19、-18、-17、-16、-15、-14、-13、-12、-11、-10、-9、-8、-7、-6、-5、-4、-3、-2、-1或0℃。
在本申请的一种优选实施方式中，进行冷冻的典型但非限制性的温度为-80、-75、-70、-65、-60、-55、-50、-45、-40、-35、-30、-25、或-20℃。
在本申请的一种优选实施方式中，冷冻的典型但非限制性的时间为2、5、8、10、12、15、18、20、22、25、28、30、32、35、38、40、42、45、46或48h。
在本申请的一种优选实施方式中，在冷冻干燥机中冷冻干燥的典型但非限制性的时间为12、15、18、20、22、25、28、30、32、35、38、40、42、45、48、50、52、55、58、60、62、65、68、70、72、75、78、80、85、90、95、100、105、110、115或120h。
在本申请的一种典型但非限制性的实施方式中，将步骤(b)制得的未进行纯化的多聚体白蛋白纳米球溶液进行透析，得到多聚体白蛋白纳米球纯化溶液；再将多聚体白蛋白纳米球纯化溶液进行干燥脱水处理，得到多聚体白蛋白纳米球。
在本申请的进一步优选实施方式中，进行透析的方法为：将步骤(b)中所制得的多聚体白蛋白纳米球的溶液置于pH值为7～12的缓冲液中透析，得 到多聚体白蛋白纳米球纯化溶液。
在本申请的进一步优选实施方式中，用于透析的缓冲液为PBS缓冲液或Tris缓冲液。
在本申请的更进一步优选实施方式中，透析时间为10～300h。
在本申请的该优选实施方式中，透析的典型但非限制性的时间为10、20、50、80、100、120、150、180、200、220、250、280或300h。
在本申请的更进一步优选实施方式中，多聚体白蛋白纳米球溶液先在pH值为7～12的缓冲液中透析后，再置于双蒸水内透析。
更进一步优选地，置于双蒸水内透析的时间为1～24h。
在本申请的优选实施方式中，本申请采用pH值为7～12的PBS缓冲液进行透析，一方面可以去除含多聚体白蛋白纳米球的溶液中的无机小分子等杂质，更重要的是，团聚的含多聚体白蛋白纳米球在碱性条件下能分散为粒径更小的纳米球，从而获得分散、粒径均匀的多聚体白蛋白纳米球。
本申请提供的多聚体白蛋白纳米球粒径范围在10～100nm之间，然而，这不是同一批次制备的多聚体白蛋白纳米球的粒径分布，相反，采用本申请提供多聚体白蛋白纳米球的制备方法获得的每个批次的多聚体白蛋白纳米球的粒径分布范围较窄，比如在20～30nm之间，因此，本申请制备的多聚体白蛋白纳米球的粒径比较均匀，大小可控。
在本申请的一种优选实施方式中，透析温度为4～60℃。
在本申请的一种优选实施方式中，对纯化后的多聚体白蛋白纳米溶液进行干燥脱水处理的方式为冷冻干燥、喷雾干燥或减压蒸馏。
在本申请的进一步优选实施方式中，冷冻干燥的步骤为：将纯化后的多聚体白蛋白纳米球溶液置于-20～0℃下预冻1～48h后转移至-80～-20℃下冷冻 2～48h,然后在冷冻干燥机中冷冻干燥12～120h，即可得到多聚体白蛋白纳米球。
在本申请的优选实施方式中，通过控制白蛋白和带巯基的还原剂的摩尔比，各步骤的pH，透析缓冲液的pH，以及有机相和水相混合的速度获得了粒径小、粒径分布范围较窄、粒径可控的多聚体白蛋白纳米球。
根据本申请的第三个方面，本申请提供的多聚体纳米球在制备预防、治疗或诊断癌症的药物中的应用。
如本文所用的，“癌症”包括肿瘤。
本申请提供的多聚体白蛋白纳米球及其制备方法和应用具有如下有益效果：
(1)本申请提供的多聚体白蛋白纳米球由不同的白蛋白分子通过分子之间二硫键相互连接形成纳米球团，在水、磷酸盐缓冲液、乙醇、血清、培养基等溶剂稀释条件下，比物理结合的白蛋白载体具有更高的稳定性；
(2)与使用化学交联剂如戊二醛等稳定的白蛋白载体相比，本申请提供的白蛋白纳米球采用了蛋白质分子本身的二硫键来获得稳定的纳米球，因此本分明提供的白蛋白纳米球更加安全；
(3)本申请提供的多聚体白蛋白纳米球的粒径为10～100nm，且其尺寸均一，分散性好，是运载药物或造影剂等目标投递物的良好载体，有利于在患者体内长时间有效、安全、稳定地释放投递物；
(4)本申请提供的多聚体白蛋白纳米球可用于制备预防、治疗或诊断癌症的药物。
根据本申请的第四个方面，本申请提供了一种载药多聚体白蛋白纳米球，包括本申请提供的多聚体白蛋白纳米球和药物，多聚体白蛋白纳米球中包载有 药物，所述药物包括抗癌药物和/或造影剂，且载药多具体白蛋白纳米球的粒径为10～100nm，优选为30～50nm。
在本申请的一种优选实施方式中，抗癌药物抗癌药物选自铂及铂的配合物、5β,20-环氧-1,2α,4,7β,10β,13α-六羟基紫杉烷-11-烯-9-酮-4,10-二乙酸酯-2-苯甲酸酯-13[(2’R,3’S)-N-苯甲酰-3-苯基异丝氨酸酯](紫杉醇)、(7S：9S)-9-羟乙酰基-4-甲氧基-7,8,9,10-四氢-6,7,9,11-四羟基-7-0-(2’,3’,6’,-三去氧-3’-氯基-a-1-来苏已吡喃基)-5,12-萘二酮(阿霉素)、(E,E)-1,7-双(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮(姜黄素)、1,3,5,8-四甲基-2,4-二(a-羟乙基)卟酚-6,7-二丙酸(血卟啉)、4-乙基-4，12-二氧-4-羟-1H-吡喃(3'，4'，6，7)吡吲哚(1，2-6)喹啉-3，14-二酮(喜树碱)、(2S-反式)-18-羧基-20-(羧甲基)-13-乙基-2,3-二氢3,7,12,17-四甲基-8-乙烯基-21H,23H-卟吩-2-丙酸(二氢卟吩e6)、IR700碘化物和11-氯-1,1'-二正丙基-3,3,3',3'-四甲基-10,12-三亚甲基吲哚三碳花青碘盐(IR780)中的一种或几种；
在本申请的一种优选实施方式中，造影剂选自四氟硼酸4-[2-[2-氯-3-[(2,6-二苯基-4H-噻喃-4-亚基)亚乙基]-1-环己烯-1-基]乙烯基]-2,6-二苯基硫代吡喃(IR1061)，2,7-双[1,3-二氢-1,1-二甲基-3-(4-磺丁基)-1,3,5-庚三烯单钠盐(吲哚青绿)、对-[(2,4-二氨基喋啶-6)-N-甲基甲氨基]苯甲酰谷氨酸(甲氨蝶呤)、3,7-双(二甲氨基)吩噻嗪-5-翁氯化物(美蓝)、6,6'-[[3,3'-二甲基(1,1'-二苯基)-4,4'-二基]双(偶氮基)]双(4-氨基-5-羟基-1,3-萘二磺酸)四钠盐(伊文思蓝)、2-((4-二乙氨基)苯)(4-(二乙氨基)环己烷-2,5-二烯)甲烷)苯基-1,4-二磺酸盐(异硫蓝)、4,4'-双(二乙氨基)三苯脱水甲醇-2”,4”-二磺酸单钠(专利蓝)和金属纳米粒子中的一种或几种。
金属纳米粒子包括但不限于纳米金、纳米银和纳米硫化铜中的一种或几 种。
本申请提供的载药多聚体白蛋白纳米球，将药物包载在多聚体白蛋白纳米球中，能够稳定存在于生物体内血液系统中，从而避免药物在血液循环系统传输过程中无效释放，同时本申请提供的载药多具体白蛋白纳米球具有主动靶向肿瘤组织的能力，能够在肿瘤组织区域有效稳定释放药物，从而显著提高治疗效果。
在本申请的一种优选实施方式中，当载药多聚体白蛋白纳米球的粒径为20～200nm时，对肿瘤有被动靶向作用，在此粒径范围内的载药多聚体白蛋白纳米球可以降低药物本身的毒副作用，同时也增强了疗效。
本申请提供的载药多聚体白蛋白纳米球，是在本申请提供的多聚体白蛋白纳米球基础上的改进，通过本申请提供的多聚体白蛋白纳米球包载药物制备而成，上述多聚体白蛋白纳米球所描述的技术方案和有益效果也属于本申请，在此不再赘述。
本申请提供的载药多聚体白蛋白纳米球，既可以携带抗癌药物也可以携带造影剂，还可以同时携带抗癌药物和造影剂，从而不仅能够将抗癌药物投递至肿瘤组织进行有效释放，而且能够用于实时、无创地监测治疗前纳米粒子在体内的输送行为，治疗后可通过成像对疗效进行实时评估，实现成像引导的治疗。
根据本申请的第五个方面，本申请提供了上述载药多聚体白蛋白纳米球的制备方法，包括如下步骤：
(A)提供白蛋白的水溶液，向白蛋白和药物的混合溶液中加入带巯基的还原剂，制得还原型白蛋白和药物的混合溶液；
(B)将还原型白蛋白和药物的混合溶液进行分散，并加入有机溶剂，还原型白蛋白通过二硫键连接，同时对药物进行包载，即制得载药多聚体白蛋白 纳米球；其中，白蛋白的水溶液中溶解有药物和/或有机溶剂中溶解有药物。
本申请提供的载药多聚体白蛋白纳米球的制备方法，工艺简单，反应条件温和，反应重现性良好，制得的多聚体白蛋白纳米球粒径小，分散性好。
本申请提供的载药多聚体白蛋白纳米球的制备方法，与多聚体白蛋白纳米球制备方法的不同之处在于，白蛋白的水溶液中溶解有药物和/或有机溶剂中溶解有药物。
通过将药物溶解在白蛋白的水溶液中，使得白蛋白水溶液和药物先充分混合，使得白蛋白在包载药物之前就与药物充分接触，能够有效提高白蛋白包载药物的效率。
通过将药物溶解于有机溶剂中，再加入还原型白蛋白的水溶液中，能够提高药物在还原型白蛋白水溶液中的溶解度，使药物在还原型白蛋白溶液中溶解均匀，以使得制备而成的载药多聚体白蛋白纳米球的粒径更加均一。
当采用的有机溶剂为二甲基亚砜时，药物的溶解度更大，在还原型白蛋白溶液中溶解的更均匀。
在本申请的一种优选实施方式中，药物与白蛋白的质量比为(0.0002～5):1，优选为(0.0002～0.5)：1；进一步优选为(0.0008～0.5)：1，更进一步优选为(0.008～0.5)：1。
在本申请的优选实施方式中，药物与白蛋白的典型但非限制性的质量比为：0.0002:1、0.0005:1、0.008:1、0.001:1、0.002:1、0.005:1、0.008:1、0.01:1、0.05:1、0.1:1、0.5:1、1:1或5:1。
通过控制药物与白蛋白的质量比以提高多聚体白蛋白纳米球的药物包载率，减少药物浪费，增强疗效。
根据本申请的第六个方面，本申请提供了上述载药多聚体白蛋白纳米球在 制备预防、治疗或诊断癌症的药物中的应用。
本申请提供的载药多聚体白蛋白纳米球靶向性好，一方面肿瘤组织的生长失控增加了大分子类物质和脂质粒子的选择性、高通透性和滞留性(EPR效应)，为载药多聚体白蛋白纳米球提供了一种被动靶向肿瘤组织的能力；另一方面，肿瘤组织细胞膜表面富含GP60、GP30、GP18等白蛋白受体，为载药多聚体白蛋白纳米球提供了一种主动靶向肿瘤组织的能力。二者的结合，提高了载药多聚体白蛋白纳米球的靶向性。另外，当本申请提供的载药多聚体白蛋白纳米球在包载有抗癌药物和造影剂时，其同时具有近红外二区荧光成像能力，可用于实时、无创地监测治疗前载药多聚体白蛋白纳米球在体内输送的行为。
下面结合实施例和对比例对本申请提供的技术方案做进一步的描述。
实施例1
本实施例提供了一种多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取2mL体积质量浓度为0.01(w/v，mg/mL)的猪血清白蛋白溶液，然后采用1mol/L的NaOH溶液调节所述白蛋白混合液的pH值到7；
(2)向步骤(1)所得的所述pH值为7的白蛋白混合液中加入半胱氨酸得反应液，然后在60℃下轻轻摇动反应0.05h，在所述反应液中，所述半胱氨酸的摩尔数为白蛋白摩尔数的10倍；
(3)将步骤(2)反应后的溶液在60℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为10W，同时在所述超声的溶液中以50ml/s的速度注入的2mL二甲基亚砜溶液，所述溶液在60℃的条件下反应20min后得到含多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含多聚体白蛋白纳米球的溶液移入透析袋，保持 温度为30℃的条件下，将透析袋置于1L pH为7的PBS缓冲液内透析10h，期间每12h换液1次，每次都采用1L pH为7的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析1h，得到多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的多聚体白蛋白纳米球溶液置于-0℃下预冻1h后转移至-20℃下冷冻2h,然后在冷冻干燥机中冷冻干燥12h，得到多聚体白蛋白纳米球，多聚体白蛋白纳米球的粒径为40～60nm。
实施例2
本实施例提供了一种多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取2mL体积质量浓度为300(w/v，mg/mL)的重组血清白蛋白溶液，然后采用2mol/L的NaOH溶液调节所述白蛋白混合液的pH值到12；
(2)向步骤(1)所得的所述pH值为12的白蛋白混合液中加入二硫苏糖醇得反应液，然后在30℃下轻轻摇动反应12h，在所述反应液中，所述二硫苏糖醇的摩尔数为白蛋白摩尔数的5000倍；
(3)将步骤(2)反应后的溶液在30℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为100W，同时在所述超声的溶液中以1000ml/s的速度注入的200mL二甲基亚砜溶液，所述溶液在30℃的条件下反应240min后，得到含多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含多聚体白蛋白纳米球的溶液移入透析袋，保持温度为60℃的条件下，将透析袋置于1L pH为12的PBS缓冲液内透析300h，每次都采用1L pH为12的PBS缓冲液，期间每12h换液1次，然后再将透析袋置于5L双蒸水内透析24h，得到多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的多聚体白蛋白纳米球溶液置于-4℃下预冻48h后转移至-80℃下冷冻36h，然后在冷冻干燥机中冷冻干燥96h,得到多聚体白蛋 白纳米球，多聚体白蛋白纳米球的粒径为30～40nm。
实施例3
本实施例提供了一种多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取2mL体积质量浓度为150(w/v，mg/mL)的白蛋白溶液，然后采用2mol/L的NaOH溶液调节所述白蛋白混合液的pH值到9；
(2)向步骤(1)所得的所述pH值为9的白蛋白混合液中加入β-巯基乙醇得反应液，然后在0℃下轻轻摇动反应17h，在所述反应液中，所述β-巯基乙醇的摩尔数为白蛋白摩尔数的2500倍；
(3)将步骤(2)反应后的溶液在0℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为10W，同时在所述超声的溶液中以100ml/s的速度注入的100mL二甲基亚砜溶液，所述溶液在0℃的条件下反应5min后得到含多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含多聚体白蛋白纳米球的溶液移入透析袋，保持温度为0℃的条件下，将透析袋置于1L pH为9的PBS缓冲液内透析144h，每次都采用1L pH为12的PBS缓冲液，期间每12h换液1次，然后再将透析袋置于5L双蒸水内透析12h，得到多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的多聚体白蛋白纳米球溶液置于-20℃下预冻24h后转移至-50℃下冷冻48h,然后在冷冻干燥机中冷冻干燥120h,得到多聚体白蛋白纳米球，多聚体白蛋白纳米球的粒径为30～60nm。
实施例4
本实施例提供了一种多聚体白蛋白纳米球，本实施例提供的多聚体白蛋白纳米球的制备方法包括如下步骤：
(1)取2mL体积质量浓度为0.01(w/v，mg/mL)的牛血清白蛋白溶液， 采用1mol/L的NaOH溶液调节所述白蛋白水溶液的pH值到7；
(2)向步骤(1)所得的牛血清白蛋白水溶液中加入半胱氨酸得反应液，然后在60℃下轻轻摇动反应0.05h，在所述反应液中，所述半胱氨酸的摩尔数为白蛋白摩尔数的10倍；
(3)在所述步骤(2)所得的溶液中加入的10mL二甲基亚砜溶液得到溶液，所述溶液在0℃的条件下反应10min后得到含多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含多聚体白蛋白纳米球的溶液移入透析袋，保持温度为25℃的条件下，将透析袋置于5L双蒸水内透析12h，得到多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻24h,然后在冷冻干燥机中冷冻干燥48h,得到多聚体白蛋白纳米球，多聚体白蛋白纳米球的粒径为10-100nm。
通过上述实施例1-3与实施例4的对比可以看出，实施例4提供的多聚体白蛋白纳米球粒径分布集中度不高，不利于其在生物医学上的应用，这说明在步骤(3)采用超声波细胞破碎仪处理还原蛋白反应液能够使得多聚体白蛋白纳米球粒径均一，分布集中度更高。
实施例5
本实施例提供了一种多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)1mL体积质量浓度为0.01(w/v，mg/mL)的吲哚青绿的水溶液和1mL体积质量浓度为0.02(w/v，mg/mL)的牛血清白蛋白混合，得到白蛋白混合液；
(2)向步骤(1)所得的所述pH值为7的白蛋白混合液中加入谷胱甘肽得反应液，然后在0℃下轻轻摇动反应1h，在所述反应液中，所述谷胱甘肽的 摩尔数为白蛋白摩尔数的10倍；
(3)将步骤(2)反应后的溶液在0℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为10W，同时在所述条件下超声的溶液中以1000mL/s的速度注入的4mL二甲基亚砜得到溶液，所述溶液在0℃的条件下反应5min后得到含载药多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为0℃的条件下，将透析袋置于5L pH为10的PBS缓冲液内透析10h，期间每12h换液1次，每次都采用5L pH为10的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析1h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻12h,然后在冷冻干燥机中冷冻干燥12h,得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为20～30nm。
实施例6
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取0.1mL体积质量浓度为0.1(w/v，mg/mL)阿霉素的水溶液和2mL体积质量浓度为300(w/v，mg/mL)的猪血清白蛋白混合，得到白蛋白混合液，然后采用1mol/L的NaOH溶液调节所述白蛋白混合液的pH值到7；
(2)向步骤(1)所得的所述pH值为7的白蛋白的混合液中加入二硫苏糖醇得反应液，然后在60℃下轻轻摇动反应0.05h，在所述反应液中，所述二硫苏糖醇的摩尔数为白蛋白摩尔数的5000倍；
(3)将步骤(2)反应后的溶液在60℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1W，同时在所述超声的溶液中以0.01ml /s的速度注入的200mL含二甲基亚砜溶液，所述溶液在60℃的条件下反应20min后得到含载药多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为30℃的条件下，将透析袋置于1L pH为7的PBS缓冲液内透析300h，期间每12h换液1次，每次都采用1L pH为7的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析24h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-0℃下预冻1h后转移至-20℃下冷冻2h,然后在冷冻干燥机中冷冻干燥72h,得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为40～60nm。
实施例7
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取0.5mL体积质量浓度为0.5(w/v，mg/mL)美篮的水溶液和1.5mL200(w/v，mg/mL)的重组血清白蛋白混合，得到白蛋白混合液，然后采用2mol/L的NaOH溶液调节所述白蛋白混合液的pH值到12；
(2)向步骤(1)所得的所述pH值为12的白蛋白混合液中加入β-巯基乙醇得反应液，然后在30℃下轻轻摇动反应12h，在所述反应液中，所述β-巯基乙醇的摩尔数为白蛋白摩尔数的100倍；
(3)将步骤(2)反应后的溶液在30℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1500W，同时在所述超声的溶液中以1000ml/s的速度注入的100mL含二甲基亚砜溶液，所述溶液在30℃的条件下反应240min后得到含载药多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋， 保持温度为60℃的条件下，将透析袋置于1L pH为12的PBS缓冲液内透析144h，每次都采用1L pH为12的PBS缓冲液，期间每12h换液1次，然后再将透析袋置于5L双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-4℃下预冻48h后转移至-50℃下冷冻48h，然后在冷冻干燥机中冷冻干燥96h,得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为30～50nm。
实施例8
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取1mL体积质量浓度为1(w/v，mg/mL)二氢卟吩e6溶液的水溶液和1mL 300(w/v，mg/mL)的血红蛋白溶液混合，得到白蛋白混合液，然后采用0.5mol/L的NaOH溶液调节所述白蛋白混合的pH值到9；
(2)向步骤(1)所得的所述pH值为9的白蛋白的水溶液中加入谷胱甘肽得反应液，然后在60℃下轻轻摇动反应0.05h，在所述反应液中，所述谷胱甘肽的摩尔数为白蛋白摩尔数的2500倍；
(3)将步骤(2)反应后的溶液在60℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为100W，同时在所述超声的溶液中以50ml/s的速度注入的22mL含二甲基亚砜溶液，所述溶液在60℃的条件下反应30min后得到含载药多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为60℃的条件下，将透析袋置于1L pH为9的PBS缓冲液内透析36h，期间每12h换液1次，每次都采用1L pH为9的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析18h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-10℃下预冻24h后转移至-80℃下冷冻24h,然后在冷冻干燥机中冷冻干燥120h,得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为50～80nm。
实施例9
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取体积质量浓度为50mg/mL(w/v)的牛血清白蛋白水溶液，然后采用0.1mol/L的NaOH溶液调节所述白蛋白水溶液的pH值到7；
(2)向步骤(1)所得的所述pH值为7的白蛋白水溶液中加入谷胱甘肽得反应液，然后在4℃下轻轻摇动反应2h，在所述反应液中，所述谷胱甘肽的摩尔数为白蛋白摩尔数的10倍；
(3)将步骤(2)反应后的溶液在4℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1000W，同时在所述超声的溶液中以1mL/s的速度注入二甲基亚砜溶液，该二甲基亚砜溶液中含浓度为1mg/mL的紫杉醇和浓度为0.1mg/mL的IR1061，该溶液在4℃的条件下反应10min后得到含载药多聚体白蛋白纳米球的溶液；二甲基亚砜溶液加入的体积为所述步骤(2)反应后的溶液体积的2倍；
(4)将步骤(3)所得的含多聚体白蛋白纳米球的溶液移入透析袋，保持温度为4℃的条件下，将透析袋置于1L pH为9的PBS缓冲液内透析24h，期间每8h换液1次，每次都采用1L pH为9的PBS缓冲液，然后再将透析袋置于1L双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻24h，然后在冷冻干燥机中冷冻干燥48h，得到载药 多聚体白蛋白纳米球，多聚体白蛋白纳米球的粒径为10～20nm。
实施例10
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取白蛋白混合液，白蛋白混合液含有浓度为300mg/mL(w/v)的猪血清白蛋白和浓度为0.1mg/mL的IR1061，然后采用10mol/L的NaOH溶液调节所述白蛋白混合液的pH值到12；
(2)向步骤(1)所得的所述pH值为12的白蛋白的水溶液中加入二硫苏糖醇得反应液，然后在60℃下轻轻摇动反应240h，在所述反应液中，所述二硫苏糖醇的摩尔数为白蛋白摩尔数的5000倍；
(3)将步骤(2)反应后的溶液在60℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1W，同时在所述超声的溶液中以1000ml/s的速度注入二甲基亚砜溶液，二甲基亚砜溶液中含体积质量浓度为0.1mg/mL的紫杉醇和体积质量浓度为2mg/mL的IR1061，所述溶液在60℃的条件下反应5min后得到含载药多聚体白蛋白纳米球的溶液；乙醇溶液加入的体积为所述步骤(2)反应后的溶液体积的50倍；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，室温下，将透析袋置于5L pH为12的PBS缓冲液内透析300h，期间每12h换液1次，每次都采用5L pH为12的PBS缓冲液，然后再将透析袋置于5L双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-0℃下预冻1h后转移至-20℃下冷冻2h，然后在冷冻干燥机中冷冻干燥72h，得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为50～100nm。
实施例11
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取白蛋白混合液，白蛋白混合液含有浓度为200mg/mL(w/v)的猪血清白蛋白和浓度为5mg/mL的IR1061，然后采用0.1mol/L的NaOH溶液调节所述白蛋白水溶液的pH值到9；
(2)向步骤(1)所得的所述pH值为9的白蛋白混合液中加入β-巯基乙醇得反应液，然后在30℃下轻轻摇动反应0.05h，在所述反应液中，所述β-巯基乙醇的摩尔数为白蛋白摩尔数的100倍；
(3)将步骤(2)反应后的溶液在30℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为50W，同时在所述溶液中以0.01ml/s的速度注入二甲基亚砜溶液，二甲基亚砜溶液中含体积质量浓度为1mg/mL的紫杉醇，所述溶液在30℃的条件下反应240min后得到含载药多聚体白蛋白纳米球的溶液；二甲基亚砜溶液加入的体积为步骤(2)反应后的溶液体积的50倍；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为60℃的条件下，将透析袋置于100mL pH为7的PBS缓冲液内透析2h，每次都采用100mL pH为7的PBS缓冲液，期间每1h换液1次，然后再将透析袋置于100mL双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-4℃下预冻48h后转移至-50℃下冷冻48h,然后在冷冻干燥机中冷冻干燥96h，得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为30～40nm。
实施例12
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步骤：
(1)取白蛋白混合液，白蛋白混合液含有浓度为100mg/mL(w/v)的猪血清白蛋白、浓度为1mg/mL的IR1061、浓度为1mg/mL的紫杉醇和DMSO，二甲基亚砜和白蛋白混合液中溶剂水的体积比是0.1:1，然后采用1mol/L的NaOH溶液调节混合液的pH值到9；
(2)向步骤(1)混合液中加入谷胱甘肽得反应液，然后在30℃下轻轻摇动反应1h，在反应液中，谷胱甘肽的摩尔数为白蛋白摩尔数的10倍；
(3)将步骤(2)反应后的溶液在4℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1000W，同时将超声的溶液中以0.01mL/s的速度注入到二甲基亚砜中，得到溶液，所述溶液在4℃的条件下反应10min后，得到含载药多聚体白蛋白纳米球的溶液；二甲基亚砜加入的体积为步骤(2)反应后的溶液体积的50倍；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为4℃的条件下，将透析袋置于100mL pH为7的PBS缓冲液内透析2h，期间每1h换液1次，每次都采用100mL pH为7的PBS缓冲液，然后再将透析袋置于100mL双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻24h，然后在冷冻干燥机中冷冻干燥48h，得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为10～20nm。
实施例13
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步 骤：
(1)取白蛋白混合液，白蛋白混合液含有浓度为200mg/mL(w/v)的猪血清白蛋白和浓度为1mg/mL的紫杉醇和二甲基亚砜，二甲基亚砜和白蛋白混合液中溶剂水的体积比是0.1:1，然后采用0.1mol/L的NaOH溶液调节混合液的pH值到9；
(2)向步骤(1)所得混合液中加入谷胱甘肽得反应液，然后在30℃下轻轻摇动反应1h，在所述反应液中，所述谷胱甘肽的摩尔数为白蛋白摩尔数的100倍；
(3)将步骤(2)反应后的溶液在4℃的条件下采用超声波细胞破碎仪进行处理，超声波细胞破碎仪的功率为1000W，同时向超声的溶液中以0.01mL/s的速度注入二甲基亚砜溶液，该二甲基亚砜溶液含体积质量浓度为1mg/mL的IR1061和二甲基亚砜，所述溶液在4℃的条件下反应10min后，得到含载药多聚体白蛋白纳米球的溶液；二甲基亚砜溶液加入的体积为步骤(2)反应后的溶液体积的50倍；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为0℃的条件下，将透析袋置于100mL pH为7的PBS缓冲液内透析2h，期间每1h换液1次，每次都采用100mL pH为7的PBS缓冲液，然后再将透析袋置于100mL双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻24h，然后在冷冻干燥机中冷冻干燥48h，得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为30～40nm。
实施例14
本实施例提供了一种载药多聚体白蛋白纳米球，其制备方法包括如下步 骤：
(1)取1mL体积质量浓度为1(w/v，mg/mL)的吲哚青绿和1mL300(w/v，mg/mL)的牛血清白蛋白混合，得到白蛋白混合液,然后采用0.1mol/L的NaOH溶液调节所述白蛋白混合液的pH值到8；
(2)向步骤(1)所得的白蛋白混合液中加入谷胱甘肽得反应液，然后在60℃下轻轻摇动反应0.05h，在所述反应液中，所述谷胱甘肽的摩尔数为白蛋白摩尔数的5000倍；
(3)在所述步骤(2)所得的溶液中加入的10mL二甲基亚砜溶液得到溶液，所述溶液在0℃的条件下反应10min后得到含载药多聚体白蛋白纳米球的溶液；
(4)将步骤(3)所得的含载药多聚体白蛋白纳米球的溶液移入透析袋，保持温度为25℃的条件下，将透析袋置于5L双蒸水内透析12h，得到载药多聚体白蛋白纳米球溶液；
(5)将步骤(4)所得的载药多聚体白蛋白纳米球溶液置于-20℃下预冻2h后转移至-80℃下冷冻24h,然后在冷冻干燥机中冷冻干燥48h,得到载药多聚体白蛋白纳米球，载药多聚体白蛋白纳米球的粒径为10～100nm。
通过实施例5-13与实施例14的对比可以看出，实施例14提供的多聚体白蛋白纳米球粒径分布集中度不高，不利于其在生物医学上的应用。这说明在步骤(3)采用超声波细胞破碎仪处理还原蛋白反应液能够使得载药多聚体白蛋白纳米球粒径均一，分布集中度更高。
试验例1
为了说明本申请提供的载药多聚体白蛋白纳米球的有益效果，对实施例13提供的载药多聚体白蛋白纳米球进行了扫描电子显微镜检测，图1为本申 请实施例13提供的载药多聚体白蛋白纳米球的扫描电子显微镜图像；由图1可知，实施例13提供的载药多聚体白蛋白纳米球尺寸均一，颗粒较为分散。
试验例2
以C6原位脑胶质瘤荷瘤小鼠为模型，通过尾静脉注射实施例13制备的多聚体白蛋白纳米球水溶液(浓度为0.2mg/mL)，然后利用利用荧光二区活体成像系统来研究多聚体白蛋白纳米球在小鼠体内的传输过程及对C6原位脑胶质瘤组织识别的特异性和灵敏度。
图2为本申请实施例13提供的载药多聚体白蛋白纳米球注射荷瘤裸鼠后的近红外二区荧光成像图；从图2可以看出，注射有载药多聚体白蛋白纳米球的荷瘤小鼠24h后肿瘤部位的荧光信号得以增强，说明实施例13提供的载药多聚体白蛋白纳米球对肿瘤组织具有很强的靶向性。
图3中纳米粒子组为本申请实施例13提供的载药多聚体白蛋白纳米球注射荷瘤裸鼠后的组织切片图；缓冲溶液组为PBS缓冲溶液注射荷瘤裸鼠后的组织切片图；从图3可以看出，相比于缓冲溶液组，注射了实施例13提供的载药多聚体白蛋白纳米球的老鼠也没有表现出任何组织毒性。
从图2和图3还可以看出，本申请实施例13提供的载药多聚体白蛋白纳米球能够作为一种近红外二区荧光分子成像探针、化疗于一体的多功能靶向探针使用。
最后应说明的是：以上各实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述各实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的范围。

Claims (15)

  1. 一种多聚体白蛋白纳米球，其特征在于，由多个白蛋白分子聚集而成，所述多白蛋白分子之间通过二硫键连接，所述多聚体白蛋白纳米球的粒径为10～100nm。
  2. 根据权利要求1所述的多聚体白蛋白纳米球，其特征在于，所述多聚体白蛋白纳米球的粒径为30～50nm。
  3. 根据权利要求1所述的多聚体白蛋白纳米球，其特征在于，所述白蛋白分子为人血清白蛋白分子、牛血清白蛋白分子、猪血清白蛋白分子和重组白蛋白分子中的至少一种。
  4. 根据权利要求1所述的多聚体白蛋白纳米球的制备方法，其特征在于，包括如下步骤：
    (a)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂混合均匀，制得还原型白蛋白的水溶液；
    (b)将还原型白蛋白的水溶液分散后，加入有机溶剂混合均匀，即制得多聚体白蛋白纳米球；
    步骤(a)中，白蛋白水溶液的pH值为7～12；白蛋白水溶液的体积质量浓度为0.01～300mg/mL；白蛋白的水溶液与带巯基的还原剂的混合时间为0.05～12h；白蛋白的水溶液与带巯基的还原剂的混合温度为0～60℃；
    在步骤(b)中，还原型白蛋白的水溶液在0～60℃下分散；还原型白蛋白的水溶液与有机溶剂的混合温度为0～60℃；通过细胞破碎仪将还原型白蛋白的水溶液进行分散；进一步优选地，细胞破碎仪的功率为1～1500W；有机溶剂的加入速度为0.01～1000mL/s；还原型白蛋白水溶液与有机溶剂的混合时间为 5～240min。
  5. 根据权利要求3所述的多聚体白蛋白纳米球的制备方法，其特征在于，包括如下步骤：
    (a)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂混合均匀，制得还原型白蛋白的水溶液；
    (b)将还原型白蛋白的水溶液分散后，加入有机溶剂混合均匀，即制得多聚体白蛋白纳米球；
    步骤(a)中，白蛋白水溶液的pH值为7～12；白蛋白水溶液的体积质量浓度为0.01～300mg/mL；白蛋白的水溶液与带巯基的还原剂的混合时间为0.05～12h；白蛋白的水溶液与带巯基的还原剂的混合温度为0～60℃；
    在步骤(b)中，还原型白蛋白的水溶液在0～60℃下分散；还原型白蛋白的水溶液与有机溶剂的混合温度为0～60℃；通过细胞破碎仪将还原型白蛋白的水溶液进行分散；进一步优选地，细胞破碎仪的功率为1～1500W；有机溶剂的加入速度为0.01～1000mL/s；还原型白蛋白水溶液与有机溶剂的混合时间为5～240min。
  6. 根据权利要求4所述的多聚体白蛋白纳米球的制备方法，其特征在于，在步骤(a)中，带巯基的还原剂与白蛋白的摩尔比为(10～5000):1；
    带巯基的还原剂选自谷胱甘肽、二硫苏糖醇、巯基乙醇、半胱氨酸或同型半胱氨酸中的至少一种。
  7. 根据权利要求4所述的多聚体白蛋白纳米球的制备方法，其特征在于，在步骤(b)中，有机溶剂与还原型白蛋白水溶液的体积比为(0.1～100):1；
    所述有机溶剂选自二甲基亚砜、N-甲基吡咯烷酮、N,N-二甲基甲酰胺和环 丁砜中的至少一种。
  8. 根据权利要求4所述的多聚体白蛋白纳米球的制备方法，其特征在于，在步骤(b)中，还原型白蛋白的水溶液与有机溶剂混合均匀后，得到多聚体白蛋白纳米球的水溶液，将多聚体白蛋白纳米球的水溶液依次进行纯化和干燥，得到多聚体白蛋白纳米球；
    采用透析进行纯化，采用pH值为7～12的缓冲液进行透析，在0～60℃进行透析；
    采用冻干进行干燥，先在-20～0℃下预冻1～48h，再在-80～-20℃下冷冻2～48h，然后再在冷冻干燥机中冷冻干燥12～120h。
  9. 一种载药多聚体白蛋白纳米球，其特征在于，包括权利要求1所述的多聚体白蛋白纳米球和药物，所述药物包载于所述多聚体白蛋白纳米球中，所述药物包括抗癌药物和/或造影剂，且载药多聚体白蛋白纳米球的粒径为10～100nm；
    所述抗癌药物选自铂及铂的配合物、5β,20-环氧-1,2α,4,7β,10β,13α-六羟基紫杉烷-11-烯-9-酮-4,10-二乙酸酯-2-苯甲酸酯-13[(2’R,3’S)-N-苯甲酰-3-苯基异丝氨酸酯]、(7S：9S)-9-羟乙酰基-4-甲氧基-7,8,9,10-四氢-6,7,9,11-四羟基-7-0-(2’,3’,6’,-三去氧-3’-氯基-a-1-来苏已吡喃基)-5,12-萘二酮、(E,E)-1,7-双(4-羟基-3-甲氧基苯基)-1,6-庚二烯-3,5-二酮、1,3,5,8-四甲基-2,4-二(a-羟乙基)卟酚-6,7-二丙酸、4-乙基-4，12-二氧-4-羟-1H-吡喃(3'，4'，6，7)吡吲哚(1，2-6)喹啉-3，14-二酮、(2S-反式)-18-羧基-20-(羧甲基)-13-乙基-2,3-二氢3,7,12,17-四甲基-8-乙烯基-21H,23H-卟吩-2-丙酸、IR700碘化物和11-氯-1,1'-二正丙基-3,3,3',3'-四甲基-10,12-三亚甲基吲哚三碳花青碘盐中的至少一种；
    所述造影剂选自四氟硼酸、4-[2-[2-氯-3-[(2,6-二苯基-4H-噻喃-4-亚基)亚乙基]-1-环己烯-1-基]乙烯基]-2,6-二苯基硫代吡喃，2,7-双[1,3-二氢-1,1-二甲基-3- (4-磺丁基)-1,3,5-庚三烯单钠盐、对-[(2,4-二氨基喋啶-6)-N-甲基甲氨基]苯甲酰谷氨酸、3,7-双(二甲氨基)吩噻嗪-5-翁氯化物、6,6'-[[3,3'-二甲基(1,1'-二苯基)-4,4'-二基]双(偶氮基)]双(4-氨基-5-羟基-1,3-萘二磺酸)四钠盐、2-((4-二乙氨基)苯)(4-(二乙氨基)环己烷-2,5-二烯)甲烷)苯基-1,4-二磺酸盐、4,4'-双(二乙氨基)三苯脱水甲醇-2”,4”-二磺酸单钠和金属纳米粒子中的至少一种。
  10. 根据权利要求9所述的载药多聚体白蛋白纳米球的制备方法，其特征在于，所述载药多聚体白蛋白纳米球的粒径为30～50nm。
  11. 根据权利要求9所述的载药多聚体白蛋白纳米球的制备方法，其特征在于，包括如下步骤：
    (A)提供白蛋白的水溶液，向白蛋白的水溶液中加入带巯基的还原剂，制得还原型白蛋白的水溶液；
    (B)将还原型白蛋白的水溶液分散后，加入有机溶剂混合均匀，即制得载药多聚体白蛋白纳米球；其中，白蛋白的水溶液中溶解有药物和/或有机溶剂中溶解有药物；
    药物与白蛋白的质量比为(0.0002～5):1；
    在步骤(A)中，带巯基的还原剂与白蛋白的摩尔比为(10～5000):1；
    在步骤(B)中，有机溶剂与还原型白蛋白水溶液的体积比为(0.1～100):1；
    带巯基的还原剂选自谷胱甘肽、二硫苏糖醇、巯基乙醇、半胱氨酸或同型半胱氨酸中的至少一种；
    所述有机溶剂选自二甲基亚砜、N-甲基吡咯烷酮、N,N-二甲基甲酰胺和环丁砜中的至少一种；
    步骤(A)中，白蛋白水溶液的pH值为7～12；
    步骤(A)中，白蛋白水溶液的体积质量浓度为0.01～300mg/mL；
    白蛋白的水溶液与带巯基的还原剂的混合时间为0.05～12h；
    在步骤(A)中，白蛋白的水溶液与带巯基的还原剂的混合温度为0～60℃；
    在步骤(B)中，还原型白蛋白的水溶液的在0～60℃下分散；
    在步骤(B)中，还原型白蛋白的水溶液与有机溶剂的混合温度为0～60℃；
    步骤(B)中，通过超声波细胞破碎仪将还原型白蛋白的水溶液进行分散；进一步优选地，超声波细胞破碎仪的功率为1～1500W；
    在步骤(B)中，有机溶剂的加入速度为0.01～1000mL/s；
    在步骤(B)中，还原型白蛋白水溶液与有机溶剂的混合时间为5-240min。
  12. 根据权利要求11所述的载药多聚体白蛋白纳米球的制备方法，其特征在于，在步骤(B)中，还原型白蛋白的水溶液与有机溶剂混合均匀后，得到载药多聚体白蛋白纳米球的水溶液，将载药多聚体白蛋白纳米球的水溶液依次进行纯化和干燥，得到载药多聚体白蛋白纳米球；
    采用透析进行纯化，在0～60℃进行透析，采用pH值为7～12的缓冲液进行透析；
    采用冻干进行干燥，先在-20～0℃下预冻1～48h，再在-80～-20℃下冷冻2～48h，然后再在冷冻干燥机中冷冻干燥12～120h。
  13. 根据权利要求1所述的多聚体白蛋白纳米球在制备预防、治疗或诊断癌症的药物中的应用。
  14. 根据权利要求3所述的多聚体白蛋白纳米球在制备预防、治疗或诊断癌症的药物中的应用。
  15. 根据权利要求9所述的载药多聚体白蛋白纳米球在制备预防、治疗或诊断癌症的药物中的应用。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013185032A1 (en) * 2012-06-07 2013-12-12 President And Fellows Of Harvard College Nanotherapeutics for drug targeting
CN104800855A (zh) * 2014-01-24 2015-07-29 中国科学院福建物质结构研究所 用于光学成像和光动力治疗的肿瘤靶向融合蛋白药物载体

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013185032A1 (en) * 2012-06-07 2013-12-12 President And Fellows Of Harvard College Nanotherapeutics for drug targeting
CN104800855A (zh) * 2014-01-24 2015-07-29 中国科学院福建物质结构研究所 用于光学成像和光动力治疗的肿瘤靶向融合蛋白药物载体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MU YANG ET AL: "Disulfide-Bond Scrambling Promotes Amorphous Aggregates in Lysozyme and Bovine Serum Albumin", THE JOURNAL OF PHYSICAL CHEMISTRY B, vol. 119, no. 10, 17 February 2015 (2015-02-17), pages 3969 - 3981, XP055712815, ISSN: 1520-6106, DOI: 10.1021/acs.jpcb.5b00144 *
ZHANG JIANJUN ET AL: "Albumin as Drug Carriers", PROGRESS IN CHEMISTRY, vol. 23, no. 8, 30 August 2011 (2011-08-30), pages 1747 - 1754, XP055712752 *

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