WO2011020294A1 - 具有新颖核壳结构和ph响应性的磁性纳米粒子及用途 - Google Patents

具有新颖核壳结构和ph响应性的磁性纳米粒子及用途 Download PDF

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WO2011020294A1
WO2011020294A1 PCT/CN2010/001243 CN2010001243W WO2011020294A1 WO 2011020294 A1 WO2011020294 A1 WO 2011020294A1 CN 2010001243 W CN2010001243 W CN 2010001243W WO 2011020294 A1 WO2011020294 A1 WO 2011020294A1
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nanoparticles
mpeg
composite
block
solution
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French (fr)
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刘克良
阎虎生
郭淼
阎宇
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中国人民解放军军事医学科学院毒物药物研究所
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Publication of WO2011020294A1 publication Critical patent/WO2011020294A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • A61K9/5073—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 having two or more different coatings optionally including drug-containing subcoatings
    • A61K9/5078—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 having two or more different coatings optionally including drug-containing subcoatings with drug-free core
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • 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
    • A61K9/5115—Inorganic compounds
    • 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
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/20—Compounding polymers with additives, e.g. colouring
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L53/00—Compositions of block copolymers containing at least one sequence of a polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2333/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
    • C08J2333/04—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers esters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K2201/00—Specific properties of additives
    • C08K2201/01—Magnetic additives

Definitions

  • the object of the present invention is to provide a polyethylene glycol having magnetic properties, pH sensitivity, and the outermost layer is biocompatible, the outer layer is a hydrophobically adjustable polyanion and the core is Fe 3 0 4 nanoparticles.
  • the polymer/magnetic nanoparticle composite is a composite formed by dispersing magnetic inorganic nanoparticles in a polymer matrix. Because of its combination of the advantages of polymers and magnetic inorganic nanoparticles, and the combination of magnetic responsiveness and polymer functionality, this material has broad application prospects in the fields of medicine, biochemistry, and industrial applications, including cell separation. Classification, immunoassay, immobilized enzymes, catalyst separation, nuclear magnetic resonance imaging, etc. Among them, the application of polymer/magnetic nanoparticle composites in the controlled release of drugs is the most extensive.
  • Certain therapeutic drugs such as cancer
  • various drug release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability, their research is gaining more and more attention.
  • the medicinal carrier develops rapidly, and the microspheres, liposomes and polymer nanoparticles are the most studied medicinal carriers, and each has its own advantages and disadvantages.
  • Microspheres are suitable for chemical embolization and local injection, but are not suitable for injectable drugs; liposomes can be used in various administration routes and preparations, and when used as an anticancer drug carrier, the drug can selectively kill cancer cells and improve the therapeutic effect, but Its structure is unstable and easily removed by the reticuloendothelial system.
  • polymer nanoparticles Compared with the former two, polymer nanoparticles have unique advantages as drug carriers.
  • the polymer constituting the nanoparticles has a variable structure, a stable structure, and a wide drug-loading range.
  • the surface of the polymer nanoparticles is composed of a hydrophilic and biocompatible polymer such as poly
  • ethylene glycol PEG
  • PEG ethylene glycol
  • Polyethylene glycol including derivatives such as methoxypolyethylene glycol
  • Targeted release strategies include: (1) Passive targeting: Nanoparticles do not easily penetrate the capillary wall of normal tissues, but easily penetrate into the porous vascular system of diseased tissues such as cancer, inflammation, and vascular infarction. The weak lymphatic drainage of these tissues makes it difficult for the incoming nanoparticles to diffuse out again, that is, EPR. (2) Ligand targeting: A ligand having a specific recognition function for cancer cells is coupled to the surface of the carrier to recognize cancer cells. (3) Physiological microenvironmental induction release: If pH is induced to release, the loaded drug is not released in the blood (pH 7.4).
  • Nanoparticles wherein a polyacrylic acid monoglyceride or polymethacrylic acid monoglyceride block is attached to the surface of the Fe 3 0 4 nanoparticle, and the polyacrylic acid or polymethacrylic acid block is on the surface of the composite nanoparticle, with a positive charge
  • the drug can be supported by ions on the polyanion on the surface of the composite nanoparticles.
  • the surface of the composite nanoparticles is negatively charged (carboxylate anion), which is cytotoxic and therefore not suitable as a drug carrier.
  • Polyethylene glycol and polyacrylic acid monoglyceride or polyacrylic acid monoglyceride diblock copolymer coated composite particles of Fe 3 0 4 nanoparticles, the polyethylene glycol is on the surface of the composite nanoparticles (reference Shourong Wan, Yuee Zheng, Yuanqin Liu, Husheng Yan and Kel iang Liu, J. Mater. Chem., 2005, 15, 3424 3430 ), should have good biocompatibility, but the composite nanoparticles have no drug-loading function. base.
  • the object of the present invention is to provide a polyethylene glycol having magnetic properties, pH sensitivity, and the outermost layer is biocompatible, the outer layer is a hydrophobically adjustable polyanion and the core is Fe 3 0 4 nanoparticles.
  • the nanocarrier is composed of a triblock copolymer coated with Fe 3 0 4 nanoparticles, and the size of the Fe 3 0 4 nanoparticle core is 5-20 nm, and the structure of the triblock copolymer can be represented by the following formula :
  • b represents a block copolymer
  • r represents a random copolymer.
  • the first block is a decyloxy polyethylene glycol (MPEG) having an average degree of polymerization X of 10 to 230
  • the second block is a polyacrylic acid, or polyacrylic acid, or a random copolymer of acrylic acid and acrylate. Or a random copolymer of methacrylic acid and mercapto acrylate.
  • R H or CH 3
  • m 10 ⁇ 100
  • n 0 ⁇ 30
  • R' CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 or C 6 H 13 .
  • This segment is a polyanion whose hydrophobicity is adjustable within a certain range (through the adjustment of the proportion of the ester-containing chain link).
  • the preparation method of the nano carrier includes a direct method and an indirect method.
  • 5% ⁇ 5% The direct method is to dissolve the ferrous salt and the trivalent iron salt (molar ratio 1: 2) and the triblock copolymer in deionized water, the total concentration of the iron salt (in terms of Fe) is 0. 5% ⁇ 5%
  • the mass concentration ratio of the iron salt (in terms of Fe) to the triblock copolymer is 2:1 to 1:3.
  • the solution is added with an alkaline solution such as ammonia water or NaOH aqueous solution under a nitrogen atmosphere under stirring to make the pH value of the system greater than 8, and after the reaction for a certain period of time, a composite nanoparticle of a triblock copolymer-coated FeA nanoparticle is formed.
  • the reaction time depends on the temperature, such as 12 to 24 hours at room temperature, if 80. 5 ⁇ The reaction under C only 0.5 to 1 hour. After the reaction was completed, the mixture was centrifuged (5000 rpm for 10 min), and the supernatant was transferred to a dialysis bag and dialyzed repeatedly with deionized water to obtain a nanocarrier of triblock polymer-coated Fe 3 0 4 nanoparticles dispersed in water. . After freeze-drying to give triblock polymer wrapped Fe nanocarrier solid product 304 nanoparticles.
  • the indirect method for preparing a nanocarrier of a triblock polymer-coated Fe 3 0 4 nanoparticle is as follows: firstly, a magnetic sulfur body in which an inorganic anion such as perchlorate ion is coated in an aqueous medium to encapsulate Fe 3 0 4 nanoparticles is prepared (Reference: R. Mas Sar t, IEEE Trans. Magn., 1981, MAG-17, 1247), then slowly add an aqueous solution of the polymer to the magnetic sulfur, stir at room temperature for 12 hours, and then centrifuge the mixture (5000 rpm for 10 min). the supernatant was transferred to a dialysis bag and dialyzed repeatedly with deionized water, dispersed in water to give the nanocarrier triblock polymer-wrapped Fe 3 0 4 nanoparticles.
  • the third block polyacrylic acid monoglyceride or polyacrylic acid monoglyceride in the triblock polymer in the nanocarrier of the triblock copolymer-encapsulated FeA nanoparticle is bonded to the surface of the Fe 3 0 4 nanoparticle.
  • the first block decyloxy polyethylene glycol is the most in nanocarriers
  • the outer layer provides good biocompatibility for the carrier; the second block polyanion forms the secondary outer layer of the nanocarrier, which is a drug loading layer.
  • Cationic drugs such as drugs containing amino or amine groups
  • drugs with both cationic and hydrophobic groups such as doxorubicin, epirubicin, gentamicin, mitomycin or mitoxantrone
  • the interaction of ions, or ionic and hydrophobic interactions is supported on the polyanion layer of the nanocarrier.
  • the carboxyl group of the polyanion in the nanocarrier exists as a carboxylate anion
  • the amino or amine group of the drug exists as a protonated positive ion, and the drug is loaded by ions.
  • the cationic drug may have a hydrophobic interaction with the hydrophobic group of the polyanion layer, such as the chain of the ester layer of the polyanion layer and/or the backbone of the polyanion.
  • the hydrophobic group of the polyanion layer such as the chain of the ester layer of the polyanion layer and/or the backbone of the polyanion.
  • the carboxylate anion of the polyanion in the drug-loaded nanocarrier becomes protonated and becomes a neutral carboxyl group, so that the ion interaction between the drug and the carrier disappears. If there is a hydrophobic interaction between the loaded drug and the carrier, after the ion action disappears, due to the disappearance of the chelation, the hydrophobic interaction alone has a small force, and the loaded drug is released. Achieve the pH-induced release of the loaded drug.
  • the triblock copolymer used in the present invention can be synthesized by any of the existing methods for synthesizing block copolymers, such as living radical polymerization (including atom transfer radical polymerization (ATRP), reversible addition-cleavage chain transfer radical polymerization (RAFT). ), etc., anionic polymerization, etc.
  • living radical polymerization including atom transfer radical polymerization (ATRP), reversible addition-cleavage chain transfer radical polymerization (RAFT).
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition-cleavage chain transfer radical polymerization
  • anionic polymerization etc.
  • Figure 1 Transmission electron micrograph of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 composite nanoparticles.
  • Figure 2 Particle size distribution of MPEG-b-PMAA-b-PGMA-FeA nanoparticles in water as determined by dynamic light scattering.
  • Figure 3 Hysteresis regression curve of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 nanoparticles, H is the magnetic field strength, and M is the magnetization.
  • Figure 5 Relative survival curves of mouse fibroblasts after 24 hours of incubation in the presence of different concentrations of nanoparticles.
  • Figure 6 Curve of release of doxorubicin from doxorubicin-loaded composite nanoparticles.
  • Example 1 the dried MPEG having an average molecular weight of 600, 1000, 5000, and 10000 was replaced by MPEG, respectively, except that the amount of ⁇ -bromoisobutyryl bromide used was different.
  • Example 1 The amounts of ⁇ -bromoisobutyryl bromide used were: 14. 3 mL, 8.6 mL, 3 mL, and 2 mL. A corresponding molecular weight of MPEG-Br was obtained.
  • MPEG-b-PtBMA 2 g of MPEG-Br prepared in Example 1, 150 mg of CuBr, 2. 8 g of tBMA was dissolved in 2.5 mL of cyclohexanone, and added to the system under nitrogen protection and stirring. 210 ⁇ PMDETA, the solution turned light green. After the reaction was continued for 2 hours, the reaction flask was taken out, immersed in a preheated oil bath, and the reaction was stirred for 10 hours to obtain a green viscous material. The obtained green polymer was dissolved in acetone to obtain a dark green solution.
  • the solution was passed through a 1 2 3 3 column, and the filtrate was collected, concentrated by rotary evaporation, and precipitated by a mixture of water and methanol (4:1 by volume) to obtain a milky white floc. Shape. After drying in vacuum for 24 hours, MPEG-b-PtBMA can be obtained. The NMR peak area was integrated, and the degree of polymerization of PtMBA was 18 with MPEG as a reference.
  • MPEG-b-PtBMA-b-PSMA 2 g of MPEG-bP tBMA prepared in Example 3, 48 mg CuBr, 2. 5 g SMA was dissolved in 2 mL of cyclohexanone under nitrogen protection and stirring. 69 PMDETA was added to the system, and the solution turned pale green. After the reaction was continued for 2 hours, the reaction flask was taken out, immersed in an oil bath which had been preheated, and the reaction was stirred for 12 hours. The obtained polymer was dissolved in acetone, and the solution was passed through an A1 2 0 3 column, and the filtrate was collected.
  • MPEG-b-PtBMA-b-PSMA was obtained by vacuum drying for 24 hours.
  • the NMR peak area integral, MPEG and PtMBA were used as reference, and the degree of polymerization of PSMA was 15.
  • MPEG-b-PtBA 0. 35 g MPEG-Br (obtained in MPEG from molecular weight 600 in Example 2), 150 mg CuBr, 6. 2 g tBA in 5 mL cyclohexanone, under nitrogen 210 ⁇ of PMDETA was added to the system under protection and stirring, and the solution turned light green. After continuing the reaction for 2 hours, the reaction flask was taken out, immersed in an oil bath preheated to 60, and the reaction was stirred for 10 hours to obtain a green viscous material. The obtained green polymer was dissolved in acetone to obtain a dark green solution.
  • the solution was passed through an A1 2 0 3 column, and the filtrate was collected, concentrated by rotary evaporation, and precipitated by a mixture of water and methanol (4:1, by volume) to obtain a milky white flocculent. Things. MPEG-b-PtBA can be obtained by drying in vacuum for 24 hours. The degree of polymerization of PtBA was determined by NMR peak area integration to be 75.
  • the solution was passed through an A1 2 0 3 column, and the filtrate was collected, concentrated by rotary evaporation, and precipitated by a mixture of water and methanol (4:1 by volume) to obtain a milky white floc. Shape.
  • MPEG-b-PtBMA can be obtained by drying in vacuum for 24 hours.
  • the degree of polymerization of PtMBA was determined by ⁇ NMR peak area integration to be 46.
  • the MPEG-Br, 150 mg of CuBr, 2. 3 g of tBMA, 0.6 g of nBMA was dissolved in 2. 5 mL of cyclohexanone. Add 21 0 ⁇ PMDETA to the system under nitrogen protection and stirring. The solution turns light green. After the reaction is continued for 2 hours, the reaction flask is removed, immersed in an oil bath preheated to 60, and the reaction is stirred for 10 hours to obtain green. Sticky. The resulting polymer was dissolved in acetone, and the solution through the column ⁇ 1 2 0 3, filtrate was collected as a colorless liquid. The steaming was sufficiently concentrated, and the precipitate was precipitated with water to obtain a white floc.
  • MPEG-b-(PtBMA-r-PnBMA) can be obtained by drying in vacuum for 24 hours.
  • the NMR peak area was integrated to obtain a polymerization degree of PtMBA of 13 and a polymerization degree of PnBMA of 5.
  • the obtained green polymer was dissolved in acetone, and the solution was passed through an A1 2 0 3 column, and the filtrate was collected to be a slightly yellow liquid.
  • the mixture was fully concentrated by rotary evaporation and precipitated with petroleum ether (30-60) to give a yellowish viscous material.
  • the MPEG-b-(PtBMA-r-PnBMA)-b-PSMA e was subjected to vacuum drying for 24 hours to determine the degree of polymerization of PSMA of 25 by NMR peak area integration.
  • MPEG-b-PMAA-b-PGMA Preparation of MPEG-b-PMAA-b-PGMA: Weighed 0.5 g.
  • the MPEG-b-PtBMA-b-PSMA prepared in Example 4 was dissolved in 6 mL of anhydrous dichloromethane, and then 2 mL was added. Trifluoroacetic acid was stirred at room temperature for 4 hours. Then add 2 mL of trifluoroacetic acid to the system, followed by Stirring was continued for 4 hours. 5 mL of distilled water was slowly added dropwise, and the mixture was stirred at room temperature for 2 hours. Rotary evaporation, sufficient removal of solvent and excess acid gave a viscous liquid.
  • the MPEG-b-PtBA-b-PSA, MPEG-b-PtBMA-b-PSMA and MPEG-b-(PtBMA-r-PnBMA)-b-PSMA obtained in Embodiments 6, 8, and 10, respectively, are used instead of the implementation.
  • the MPEG-b-PtBMA-b-PSMA in Example 1 1 the other operations are the same as in Embodiment 11, and the corresponding MPEG-b-PAA-b-PGA, MPEG-b-PMAA-b-PGMA and MPEG-b- ( PMAA-r-PnBMA) -b-PGMA
  • MPEG-b- PMAA-b-PGMA wrapped Fe 3 0 4 Preparation of Nano particle composite nanoparticles: MPEG-b-PMA Ab- PGMA in a round-bottomed flask 0. 25 g prepared in Example 11, 0.1 g FeCl 2 ⁇ 4H 2 0 and 0.26 g FeC l 3 ⁇ 6H 2 0 were dissolved in 10 mL of deionized water, and the flask was immersed in an oil bath preheated 80 under a nitrogen atmosphere. 2 ⁇ After stirring for 10 minutes, the flask was added dropwise 25% ammonia water 0. 2 mL. The mixture was reacted at 80 for 1 hour and then naturally cooled to room temperature.
  • TEM Transmission electron micrograph
  • Example 14 0.25 g of MPEG-b-PMAA-b-PGMA prepared in Example 11, 0.1 g of FeCl 2 ⁇ 4H 2 0 and 0.26 g of FeCl 3 ⁇ 6H 2 0 were dissolved in 10 mL of deionized water in a round bottom flask under nitrogen. After stirring for 10 minutes at room temperature under protection, 0.2 mL of 25% aqueous ammonia was added dropwise to the syringe in the flask, and the mixture was further stirred at room temperature for 24 hours.
  • the MPEG-b-PMAA-b-PGMA in the embodiment 13 was replaced by the MPEG-b-PAA-b-PGA obtained in the embodiment 12, and the other operations were the same as those in the embodiment 13, to obtain an MPEG-b-PAA-b-PGA package.
  • TEM shows that the Fe 3 0 4 core has an average diameter of 12 nm e
  • the MPEG-b-PMAA-b-PGMA in the embodiment 13 is replaced by the MPEG-b-PMAA-b-PGMA obtained in the embodiment 12, and the other operation is the same as that in the embodiment 13, to obtain the MPEG-b-PMAA-b-PGMA package.
  • TEM shows that the Fe 3 0 4 core has an average diameter of 9 nm.
  • Example 13 The MPEG-b-PMAA-b-PGMA in Example 13 was replaced with MPEG-b-(PMAA-r-PnBMA)-b-PGMA obtained in Example 12, and the other operations were the same as those in Example 13, and MPEG-b- was obtained.
  • (PMAA-r-PnBMA) -b-PGMA Composite nanoparticles encapsulating Fe 3 0 4 magnetic nanoparticles. TEM showed that the average diameter of the Fe 3 0 4 core was 7 nm.
  • Example 13 A series of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 aqueous solution obtained in Example 13 was adjusted to pH 1 with 0.1 mol/L HC1 or 0.1 mol/L NaOH. 2, 4, 6, 8, 10, 12, 13 and 14, the final composite nanoparticles were the same concentration (1 mg / mL), centrifuged for 10 minutes (2000 rpm), and then measured absorbance at 340 nm. The results show that the absorbance values of the samples with pH values 1, 2, 13 and 14 are significantly reduced, and the absorbance is less than 80% of the maximum absorbance. The absorbance values of the samples with pH values of 4, 6, 8, 10, and 12 did not change much, and the range of variation was within 10%. It is indicated that the composite nanoparticles can be stably dispersed in water in the range of pH 4-12.
  • a series of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 aqueous solution obtained in Example 13 was diluted with a NaCl solution to make the final NaCl concentration 0.5%, 1%, 2% and 5%, respectively.
  • the concentration of the nanoparticles was the same (1 mg / mL), centrifuged for 10 minutes (2000 rpm), and then the absorbance at 340 nm was measured. The results showed that the absorbance of all the samples did not change much, and the range was 5%. Inside. It indicates that the composite nanoparticles can be stably dispersed in 0.5%. ⁇ 5% NaCl solution.
  • the composite nanoparticles coated with the FeA nanoparticles prepared by the polymers prepared in Examples 15, 16, 17, and 18 were subjected to the same experiment as above, and the results showed that the composite nanoparticles were in the range of pH 4-12 and 0.5% - 5%. Stable dispersion in the NaCl solution.
  • Figure 4 shows that the addition of lead acetate to the aqueous medium has little effect on the dispersion stability of the composite particles of the triblock copolymer-coated Fe 3 0 4 nanoparticles, but when the lead acetate concentration is greater than ⁇ 0.04%, The segmented copolymer encapsulates the composite nanoparticles of Fe 3 0 4 nanoparticles to aggregate and precipitate.
  • the reason can be analyzed as follows: the di-block copolymer encapsulates the composite nanoparticles of Fe 3 0 4 nanoparticles because the polyanion is on the surface of the particles, and Pb 2+ is complexed with the carboxyl group of the polyanion to cause cross-linking and precipitation between the particles. .
  • composite nanoparticles triblock copolymer wrapped Fe 3 0 4 nanoparticles due to the surface of the protective effect of polyethylene glycol, crosslinking between the particles does not occur, and therefore not precipitated.
  • Cytotoxicity experiments composite nanoparticles: PMAA- b- PGMA- Fe 13 obtained in Example MPEG- b-PMAA-b-PGMA -Fe 3 0 4 and Example 19 mentioned in the embodiment are respectively 304
  • the cells were added to the culture medium for cell culture experiments, and then the relative survival rate of the cells was determined by the MTS method.
  • MTS method For specific experimental methods, refer to: S. Wan, J. Huang, M. Guo, H. Zhang, Y. Cao, H. Yan , K. L iu, J. B iomed. Ma ter. Res. A, 2007, 80, 946-954.
  • Figure 5 is a comparison of cytotoxicity of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 and PMAA-b-PGMA-FeA. It can be seen that the cytotoxicity of the composite nanoparticles in which the outermost layer is MPEG triblock copolymer coated with FeA nanoparticles is significantly smaller.
  • Example 15 The composite nanoparticles prepared in Example 15 were used in place of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 in Example 21, and other operating conditions were the same as in Example 21 to obtain doxorubicin-loaded composite nanoparticles.
  • the loading of themycin was 15%.
  • Example 17 The composite nanoparticle prepared in Example 17 was used instead of MPEG-b-PMAA-b-PGMA-Fe 3 0 4 in Example 21.
  • the other operating conditions were the same as in Example 21, and the composite nanoparticles loaded with Austin were obtained.
  • the loading of themycin was 23%.
  • Example 27 Loading of mitoxantrone in composite nanoparticles: To a solution of MPEG-b-PMAA-b-PGMA-Fe 3 0 7 7j prepared in Example 13 (10 mL, 2 mg/mL) was added dropwise with stirring. A solution of mitoxantrone hydrochloride (2 mg/mL, 0.4 mL) was stirred overnight and the mixture was dialyzed (up to a molecular weight of 14000) for 48 hours to obtain a composite nanoparticle loaded with mitoxantrone. The loading capacity was 23 %.
  • Controlled release of doxorubicin-loaded composite nanoparticles 4 parts of each 3 mL of the doxorubicin-loaded composite nanoparticle solution prepared in Example 21 was placed in a dialysis bag (upper molecular weight limit of 14000), respectively placed at a pH value Dialysis in phosphate buffers (20 mmol/L, 100 mL) of 7.4, 6.5, 5.5 and 4.5, and determination of the concentration of doxorubicin in the dialysate at different times (detected by UV absorption, detection wavelength 234 nm) ). The release rate is calculated. The relationship between the release rate of doxorubicin and the release time at different pH values is shown in Fig. 6.

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Description

具有新颖核壳结构和 PH晌应性
的磁性纳米粒子及用途 技术领域
本发明的目的是提供一种具有磁性、 pH敏感、 最外层为生物相容 性好的聚乙二醇、次外层为疏水性可调节的聚阴离子和核为 Fe304纳米 粒子的多层核-壳结构的复合纳米粒子 （纳米载体） 及其制备方法和 该复合纳米粒子作为药物的载体的应用。
背景技术
聚合物 /磁性纳米粒子复合材料是将磁性无机纳米粒子分散于聚 合物基体中形成的复合材料。 由于此种材料结合了聚合物和磁性无机 纳米粒子的优点， 兼具磁响应性和聚合物的功能性， 因而在医学、 生 物化学及工业应用等领域显示出广泛的应用前景， 包括细胞分离、 分 类、 免疫测定、 固定化酶、 催化剂分离、 核磁共振成像等。 其中， 对 聚合物 /磁性纳米粒子复合材料在药物控释方面的应用研究的最为广 泛。
某些用于治疗的药物如癌的化疗药通常具有毒性高、 溶解性差、 体内循环时间短等缺点， 导致应用受到了很大的限制。 减少药物的毒 性， 提高药物的生物利用度是医药学面临的一个具有挑战性的问题。 由于各种药物释放和靶向系统能够减少药物降解及损失， 降低副作 用， 提高生物利用度， 因而对它们的研究越来越受重视。 其中药用载 体发展迅速，微球、脂质体和聚合物纳米粒子是研究最多的药用载体， 它们各具优缺点。微球适合化学栓塞及局部注射，但不适于注射用药； 脂质体可用于多种给药途径和制剂， 当它作为抗癌药物载体时可使药 物选择性地杀伤癌细胞， 提高疗效， 但它的结构不稳定， 易被网状内 皮组织系统清除。 与前两者相比， 聚合物纳米粒子作为药物载体具有 独特的优势。 组成纳米粒子的聚合物的结构多变、稳定、 载药范围广。 聚合物纳米粒子的表面如果由亲水性和生物相容性好的聚合物如聚 乙二醇 （PEG)覆盖， 则其不易被网状内皮组织系统清除， 具有优良 的组织渗透性， 在体内滞留时间长。 聚乙二醇 （包括甲氧基聚乙二醇 等衍生物）具有非常好的生物相容性， 其免疫原性极弱， 已经获美国 FDA批准用作多种药物制剂的添加物或载体， 可用于口服、 静脉注射 等给药。
靶向释放策略包括： （1 )被动靶向： 纳米粒子不易透过正常组 织的毛细血管壁， 但易透过癌、 炎症和血管梗塞等病变组织的多孔性 的血管系统， 进入到这些组织中， 而这些组织的弱的淋巴引流作用又 使进入的纳米粒子不易再扩散出去， 即 EPR 作用。 （2) 配体靶向： 将对癌细胞有特异识别作用的配体偶联于载体的表面， 从而识别癌细 胞。 （ 3)生理微环境感应释放： 如 pH感应释放， 负载的药物在血液 中 （pH 7.4) 不释放， 当负载药物的载体到达癌组织并通过细胞内吞 作用进入癌细胞后， 由于细胞内较酸性的环境 （pH 4 - 6 )使药物释 放。 （4 ) 物理作用激发靶向释放： 如超顺磁性的载药纳米粒子通过 外磁场达到靶向目的。 近年来， 将多种靶向策略集中于同一体系来增 加靶向作用。如 Nasongkla等将抗癌药阿霉素和磁性 Fe304纳米粒子簇 通过疏水作用包埋于 PEG-PLA (PLA 为聚乳酸） 的胶束中， 在酸性条 件下阿霉素质子化使其水溶性增大而释放， 在 pH 7.4和 5.0时 6小 时分别释放 1.7%和 10.4%。 这一释放体系的缺点是药物负载量低和释 放速率低（参考： N. Nasongkla, E. Bey, J. Ren, H. Ai， C. Khemtong, J. S. Guthi, S. F. Chin, A. D. Sherry, D. A. Boothman and J. Gao, Nano Lett. , 2006， 6， 2427-2430 ) 0 Yang等报道了类似的多 功能体系， 所用嵌段共聚物为 PEG-PLGA (PLGA为乳酸和 2-羟基乙酸 的无规共聚物）,此载体对阿霉素的负载量也仅有 3.5% (参考： J. Yang, C. H. Lee, H. J. Ko, J. S. Suh, H. G. Yoon, K. Lee, Y. M. Huh and S. Haam, Angew. Chem. Int. Ed. , 2007, 46， 8836-8839 ) 。
因此， 开发载药量高、 释放速度快的多功能药物靶向控释载体是 十分需要的。 文献报道 (参考： Shourong Wan, Yuee Zheng, Yuanqin Liu, Husheng Yan and Kel iang Liu, J. Mater. Chem. , 2005, 15, 3424 3430 ) ， 在丙烯酸甘油单酯或曱基丙烯酸甘油单酯与丙烯酸或曱基 丙烯酸的二嵌段共聚物的存在下，碱性共沉淀 Fe27Fe3+(摩尔比为 1: 2 ) 得到分散于水中的二嵌段共聚物包裹 Fe304纳米粒子的复合纳米粒子， 其中聚丙烯酸甘油单酯或聚甲基丙烯酸甘油单酯嵌段附着于 Fe304纳 米粒子表面上， 而聚丙烯酸或聚甲基丙烯酸嵌段处于复合纳米粒子的 表面， 带正电荷的药物可通过离子作用负载于复合纳米粒子表面的聚 阴离子上。 但在中性或酸性条件下复合纳米粒子的表面带负电荷（羧 酸根负离子） ， 具有细胞毒性， 因此不适合作为药物载体。 聚乙二醇 与聚丙烯酸甘油单酯或聚曱基丙烯酸甘油单酯的二嵌段共聚物包裹 Fe304纳米粒子的复合纳米粒子， 其聚乙二醇处于复合纳米粒子的表面 (参考 = Shourong Wan, Yuee Zheng, Yuanqin Liu, Husheng Yan and Kel iang Liu, J. Mater. Chem. , 2005, 15， 3424 3430 ) ， 应该具 有好的生物相容性， 但该复合纳米粒子没有载药的功能基。
发明内容
本发明的目的是提供一种具有磁性、 pH敏感、 最外层为生物相容 性好的聚乙二醇、次外层为疏水性可调节的聚阴离子和核为 Fe304纳米 粒子的多层核 -壳结构的复合纳米粒子 （纳米载体）及其制备方法和 该复合纳米粒子作为药物的载体的应用。 具体地说， 该纳米载体是由 三嵌段共聚物包裹 Fe304纳米粒子组成， Fe304纳米粒子核的尺寸为 5 ~ 20纳米， 三嵌段共聚物的结构可用下面通式表示：
R R c=o c=o c=o
OH OR' OCH2CH-CH2 上式中 b表示嵌段共聚物， r表示无规共聚物。 第一嵌段为曱氧 基聚乙二醇 （MPEG ) , 其平均聚合度 X为 10 ~ 230， 第二嵌段为聚丙 烯酸、 或聚曱基丙烯酸、 或丙烯酸与丙烯酸酯的无规共聚物、 或曱基 丙烯酸与曱基丙烯酸酯的无规共聚物。 R=H或 CH3， m =10 ~ 100， n = 0 ~ 30, R' = CH3、 C2H5、 C3H7、 C4H9或 C6H13。 此段为疏水性在一定范围内可 调节 （通过含酯基的链节比例的调节） 的聚阴离子。 第三嵌段为聚丙 烯酸甘油单酯或聚甲基丙烯酸甘油单酯， z = 10 ~ 50。
纳米载体的制备方法包括直接法和间接法。 直接法为将二价铁盐 和三价铁盐 （摩尔比 1: 2 ) 与三嵌段共聚物溶于去离子水中， 铁盐的 总浓度（以 Fe计）为 0. 5% ~ 5%, 铁盐（以 Fe计）与三嵌段共聚物的 质量浓度比为 2: 1 ~ 1: 3。溶液在氮气保护下在搅拌下向溶液中加入碱 性溶液如氨水或 NaOH水溶液， 使体系的 pH值大于 8， 反应一定的时 间后形成三嵌段共聚物包裹 FeA纳米粒子的复合纳米粒子，所需反应 时间与温度有关， 如在室温下需反应 12 ~ 24小时， 如果在 80。C下反 应仅需 0. 5 ~ 1 小时。 待反应完全后其混合液离心 （ 5000rpm 10 min ) ， 将上清夜转移到透析袋中， 用去离子水反复透析， 得到分散 于水中的三嵌段聚合物包裹 Fe304纳米粒子的纳米载体。冷冻干燥后得 到三嵌段聚合物包裹 Fe304纳米粒子的纳米载体固体产品。间接法制备 三嵌段聚合物包裹 Fe304纳米粒子的纳米载体的方法为：首先制备在水 介质无机阴离子如高氯酸根离子包裹 Fe304纳米粒子的磁硫体（参考： R. Mas sar t, IEEE Trans. Magn. , 1981, MAG— 17， 1247 ) ， 然后向 磁硫体中慢慢加入聚合物的水溶液， 室温下搅拌 12 小时后， 混合液 离心 （ 5000 rpm χ 10 min ) 后， 将上清夜转移到透析袋中， 用去离 子水反复透析，得到分散于水中的三嵌段聚合物包裹 Fe304纳米粒子的 纳米载体。
三嵌段共聚物包裹 FeA纳米粒子的纳米载体中三嵌段聚合物中 的第三嵌段聚丙烯酸甘油单酯或聚曱基丙烯酸甘油单酯的作用是与 Fe304纳米粒子表面结合。 第一嵌段曱氧基聚乙二醇处于纳米载体的最 外层， 使载体具有好的生物相容性； 第二嵌段聚阴离子形成纳米载体 的次外层， 为药物负载层。 阳离子药物 （如含氨基或胺基的药物）或 同时带阳离子和疏水性基团的药物（如阿霉素、表阿霉素、正定霉素、 丝裂霉素或米托蒽醌等）通过离子作用、 或离子作用和疏水作用的共 同作用负载于纳米载体的聚阴离子层。 在中性条件下 （pH = 6 ~ 8 ) ， 纳米载体中的聚阴离子的羧基以羧酸根负离子的形式存在， 而药物的 氨基或胺基以质子化的正离子形式存在， 药物通过离子作用负载于聚 阴离子层。 如果阳离子药物带有疏水性的基团， 则可与聚阴离子层的 疏水基团如聚阴离子层带酯基的链节和（或）聚阴离子的主链存在疏 水作用。 根据热力学第二定律的原理， 当离子作用和疏水作用同时作 用于同一分子时， 即螯合作用， 其总的作用力大于它们单独作用时作 用力的加和 （参考： S. Cheng, H. Yan, C. Zhao, J. Chroma togr. A, 2006， 1108， 43-49 ) 。 因此离子作用和疏水作用的共同作用会大大 增大其负载作用力。 当体系的酸性增大时， 负载药物的纳米载体中的 聚阴离子的羧酸根负离子质子化而变为中性的羧基， 使药物与载体间 的离子作用消失。 如果负载的药物与载体之间还存在疏水作用， 则离 子作用消失后， 由于螯合作用的消失， 疏水作用单独的作用力较小， 使负载的药物释放。 实现使负载的药物 pH感应释放。
本发明所用三嵌段共聚物的合成可用现有任何合成嵌段共聚物的 方法， 如活性自由基聚合（包括原子转移自由基聚合（ATRP ) 、 可逆 加成 -裂解链转移自由基聚合（ RAFT ) 等） 、 阴离子聚合等。
附图说明
图 1 : MPEG-b-PMAA-b-PGMA-Fe304复合纳米粒子的透射电镜照片。 图 2 : 动态光散射测定的 MPEG- b- PMAA- b-PGMA- FeA纳米粒子在 水中的粒径分布图。
图 3: MPEG-b-PMAA-b-PGMA-Fe304纳米粒子的磁滞回归曲线， H为 磁场强度， M为磁化强度。
图 4:紫外吸收测定的复合纳米粒子在 Pb (Ac) 2溶液中的稳定性比 较。
图 5: 鼠成纤维细胞在不同浓度纳米粒子存在的条件下培养 24小 时后的相对存活率曲线。
图 6: 负载阿霉素的复合纳米粒子释放阿霉素的曲线。
具体实施方式
下面， 通过示例性的实施例具体说明本发明。 应当理解， 本发明 的范围不应局限于实施例的范围。 任何不偏离本发明主旨的变化或改 变能够为本领域的技术人员所理解。 本发明的保护范围由所附权利要 求的范围确定。
下面的实施例中所用缩写为： MPEG, 聚乙二醇单甲醚； MPEG-Br, α-溴代异丁酸 MPEG酯； tBA， 丙烯酸叔丁酯； PtBA, 聚丙烯酸叔丁酯； tBMA, 甲基丙烯酸叔丁酯； PtBMA, 聚曱基丙烯酸叔丁酯； ηΒΜΑ, 甲 基丙烯酸正丁酯； ΡηΒΜΑ,聚曱基丙烯酸正丁酯； ΡΑΑ，聚丙烯酸； ΡΜΑΑ, 聚曱基丙烯酸； SA,丙烯酸 2， 2-二甲基 -1， 3-二氧戊烷 -4-曱醇酯； PSA, 聚丙烯酸 2， 2-二曱基 -1， 3-二氧戊烷 -4-曱醇酯； SMA,曱基丙烯酸 2， 2- 二曱基 -1， 3-二氧戊烷- 4-甲醇酯； PSMA, 聚甲基丙烯酸 2，2-二甲基 - 1， 3-二氧戊烷- 4-甲醇酯； PGA, 聚丙烯酸甘油单酯； PGMA, 聚甲基 丙烯酸甘油单酯； PMDETA, 1, 1， 4， 7， 7-五曱基二乙烯三胺； b, 表示 嵌段共聚物； r， 表示无规共聚物。
实施例 1
MPEG-Br的制备： 将 10 g 干燥的 MPEG (平均分子量为 2000 ) 和 1.4 mL三乙胺溶于 70 mL四氢呋喃中， 冰水浴冷却和搅拌下緩慢滴加 4.3 mL α-溴代异丁酰溴与 20 mL 四氢呋喃的混合液， 滴加完毕后， 室温下搅拌反应 48小时。 旋转蒸发除去四氢呋喃， 加入 100 mL水溶 解所得产物。 然后以二氯甲烷萃取， 每次 30 mL, 共萃取 5次， 收集 有机相。 分别用 1 mol/L HC1 ( 30 mL χ 3) , 1 mol/L NaOH (30 mL x 3)， 饱和食盐水（ 30 mL χ 3)依次洗涤， 并收集有机相， 最后 以无水 MgS04干燥。 过滤， 除去干燥剂， 旋转蒸发除去溶剂， 所得浓 缩液以冰无水乙醚沉淀， 收集沉淀， 真空干燥， 得 MPEG-Br。
实施例 2
分别用平均分子量为 600、 1000、 5000和 10000的干燥 MPEG各 1 0 g代替实施例 1中的平均分子量为 2000的 MPEG , 除所用 α-溴代异 丁酰溴的量不同外， 其它操作同实施例 1。 所用 α-溴代异丁酰溴的量 分别为： 14. 3 mL、 8. 6 mL、 3 mL和 2 mL。 制得相应分子量的 MPEG- Br。
实施例 3
MPEG-b-PtBMA的制备： 将 2 g实施例 1制备的 MPEG- Br， 150 mg CuBr , 2. 8 g tBMA溶于 2. 5 mL环己酮中， 在氮气保护和搅拌下向体 系中加入 210 μΐ PMDETA, 溶液变为浅绿色， 继续反应 2小时后， 取 下反应瓶， 浸入预热到 的油浴中， 搅拌反应 10小时， 得绿色粘 稠物。 以丙酮溶解所得绿色聚合物， 得深绿色溶液， 溶液过人1203柱， 收集滤液， 旋转蒸发浓缩， 以水与甲醇的混合液（4: 1， 体积比） 沉 淀之， 得乳白色絮状物。 真空干燥 24小时， 即可制得 MPEG- b- P tBMA。 经 ^ NMR峰面积积分， 以 MPEG为参比得 PtMBA的聚合度为 18。
实施例 4
MPEG-b-PtBMA-b-PSMA ) 的制备： 将 2 g 实施例 3 制备的 MPEG-b-P tBMA , 48 mg CuBr , 2. 5 g SMA溶于 2 mL环己酮中， 在氮气 保护和搅拌下向体系中加入 69 PMDETA , 溶液变为浅绿色， 继续反 应 2 小时后， 取下反应瓶， 浸入已预热到 的油浴中， 搅拌反应 12 小时。 以丙酮溶解所得聚合物， 溶液过 A1203柱， 收集滤液。 旋转 蒸发浓缩， 以石油醚（30 ~ 60 ） 沉淀之， 得微黄色粘稠物。 真空 干燥 24小时， 即可制得 MPEG-b-PtBMA-b-PSMA。 经 Ή NMR峰面积积 分， 以 MPEG和 PtMBA为参比， 得 PSMA的聚合度为 15。
实施例 5
MPEG-b-PtBA的制备： 将 0. 35 g MPEG-Br (实施例 2中由分子量 为 600的 MPEG得到） ， 150 mg CuBr , 6. 2 g tBA溶于 5 mL环己酮中， 在氮气保护和搅拌下向体系中加入 210 μΐ PMDETA , 溶液变为浅绿色， 继续反应 2小时后， 取下反应瓶， 浸入预热到 60 的油浴中， 搅拌反 应 10 小时， 得绿色粘稠物。 以丙酮溶解所得绿色聚合物， 得深绿色 溶液， 溶液过 A1203柱， 收集滤液， 旋转蒸发浓缩， 以水与甲醇的混合 液（4 : 1 , 体积比） 沉淀之， 得乳白色絮状物。 真空干燥 24 小时， 即可制得 MPEG- b-PtBA。 经 NMR峰面积积分测定 PtBA的聚合度为 75。
实施例 6
MPEG- b- P tBA-b- PSA的制备：将 2 g实施例 5制备的 MPEG-b-PtBA， 30 mg CuBr , 3. 1 g SA溶于 3. 0 mL环己酮中， 在氮气保护和搅拌下 向体系中加入 42 μΐ PMDETA, 溶液变为浅绿色， 继续反应 2小时后， 取下反应瓶， 浸入已预热到 90 的油浴中， 搅拌反应 12小时。 以丙 酮溶解所得聚合物， 溶液过 Α1203柱， 收集滤液。 旋转蒸发浓缩， 以石 油醚（ 30 ~ 60 V ) 沉淀之， 得微黄色粘稠物。 真空干燥 24 小时， 即可制得 MPEG-b-PtBA- b-PSA。 经 'Η NMR峰面积积分测定 PSA的聚合 度为 45。
实施例 7
将 2 g MPEG-Br (实施例 2中由分子量为 5000的 MPEG得到） ， 60 mg CuBr , 2. 8 g tBMA溶于 2. 5 mL环己酮中， 在氮气保护和搅拌 下向体系中加入 82 L PMDETA, 溶液变为浅绿色， 继续反应 2小时后， 取下反应瓶， 浸入预热到 60 Ό的油浴中， 搅拌反应 10小时， 得绿色 粘稠物。 以丙酮溶解所得绿色聚合物， 得深缉色溶液， 溶液过 A1203 柱， 收集滤液， 旋转蒸发浓缩， 以水与甲醇的混合液（4 : 1， 体积比） 沉淀之，得乳白色絮状物。真空干燥 24小时，即可制得 MPEG- b-PtBMA。 经 ^ NMR峰面积积分测定 PtMBA的聚合度为 46。
实施例 8
将 2 g实施例 7制备的 MPEG-b-PtBMA , 25 mg CuBr , 2. 5 g SMA 溶于 2 mL环己酮中，在氮气保护和搅拌下向体系中加入 35 μΐ PMDETA, 溶液变为浅绿色，继续反应 2小时后，取下反应瓶，浸入已预热到 的油浴中， 搅拌反应 12小时。 以丙酮溶解所得聚合物， 溶液过 A 1203 柱， 收集滤液。 旋转蒸发浓缩， 以石油醚（30 X ~ 60 ) 沉淀之， 得 微黄色粘稠物。 真空干燥 24小时， 即可制得 MPEG-b-PtBMA- b- PSMA。 经 'Η NMR峰面积积分测定 PSMA的聚合度为 28。
实施例 9
MPEG-b- (PtBMA-r-PnBMA)的制备： 将 2g 实施例 1 制备的 MPEG-Br , 150 mg CuBr , 2. 3 g tBMA , 0. 6 g nBMA溶于 2. 5 mL环己 酮中， 在氮气保护和搅拌下向体系中加入 21 0 μΐ PMDETA, 溶液变为 浅绿色，继续反应 2小时后，取下反应瓶，浸入预热到 60 的油浴中， 搅拌反应 10 小时， 得绿色粘稠物。 以丙酮溶解所得聚合物， 溶液过 Α 1203柱， 收集滤液， 为无色液体。 旋蒸充分浓缩， 以水做沉淀剂沉淀 之 ， 得 白 色 絮状物 。 真 空 干 燥 24 小 时 ， 即 可制 得 MPEG-b- (PtBMA-r-PnBMA)。 经 NMR峰面积积分， 得 PtMBA的聚合度 为 1 3， PnBMA的聚合度为 5。
实施例 10
MPEG-b- (PtBMA-r-PnBMA) -b-PSMA的制备： 将 1. 9 g实施例 9制 备的 MPEG-b- (PtBMA-r-PnBMA) , 47. 4 mg CuBr , 4. 0 g SMA溶于 3. 0 mL环己酮中， 在氮气保护和搅拌下向体系中加入 69 L PMDETA , 溶液 变为浅绿色， 继续反应 2小时后， 取下反应瓶， 浸入已预热到 90 的 油浴中，搅拌反应 12小时。以丙酮溶解所得绿色聚合物，溶液过 A1203 柱，收集滤液，为微黄色液体。旋蒸充分浓缩，以石油醚（ 30 ~ 60 ) 沉淀之， 得微黄色粘稠物。 真空干燥 24 小时， 即可制得 MPEG-b- (PtBMA-r-PnBMA) -b-PSMAe 经 ^ NMR峰面积积分测定 PSMA的 聚合度为 25。
实施例 11
MPEG-b-PMAA-b-PGMA 的制备： 称取 0. 5 g 实施例 4 中制得的 MPEG-b-PtBMA-b-PSMA溶于 6 mL无水二氯曱烷中， 然后加入 2 mL三 氟乙酸， 室温搅拌 4小时。 然后向体系中再加入 2 mL 三氟乙酸， 继 续搅拌 4小时。 慢慢滴加蒸馏水 0. 5 mL, 滴加完水后完再于室温下搅 拌 2小时。 旋转蒸发， 充分除去溶剂及过量的酸， 得到粘稠液体。 加 少量水溶解后转移到透析袋中 （分子量上限为 3500 ) ， 在去离子水 中反复透析至 pH值接近 7， 得无色透明溶液。 冷冻干燥得白色粉末状 固体 MPEG-b-PMA A- b-PGMA。
实施例 12
分别用实施例 6、 8 和 1 0 中所得 MPEG-b-PtBA- b- PSA、 MPEG-b-PtBMA-b-PSMA和 MPEG-b- (PtBMA-r-PnBMA) -b-PSMA分别代替 实施例 1 1 中的 MPEG-b- PtBMA-b-PSMA， 其它操作同实施例 11， 得到 相 应 的 MPEG-b-PAA- b- PGA 、 MPEG- b-PMAA- b-PGMA 和 MPEG-b- (PMAA-r-PnBMA) -b-PGMA„
实施例 1 3
MPEG-b- PMAA-b-PGMA包裹 Fe304纳米粒子的复合纳米粒子的制备： 在圓底烧瓶中将 0. 25 g实施例 11制备的 MPEG-b-PMA A-b-PGMA, 0. 1 g FeCl2 · 4H20和 0. 26 g FeC l 3 · 6H20溶于 10 mL去离子水中， 在氮气 保护下将烧瓶浸入已预热 80 的油浴中。 搅拌 1 0分钟后向烧瓶中注 射器滴加 25%的氨水 0. 2 mL。 混合液在 80 下反应 1小时后， 自然冷 却至室温。 将混合液离心 （ 5000 rpm χ ΐ θ mi n )后， 上清液转移到 透析袋 （分子量上限为 14000 ) 中， 用去离子水反复透析， 得到 MPEG-b-PMAA-b-PGMA 包裹 Fe304磁性纳米粒子的复合纳米粒子 ( MPEG-b-PMAA-b-PGMA-Fe304 ) 水溶液。
透射电镜照片 （TEM )表明， 核心 Fe304纳米粒子的平均直径约为 8 nm (图 1 , 由于电子透射 Fe304和聚合物的能力的差别很大， 照片中 只显示 Fe304核， 而聚合物壳是显示不出来的）。 图 2为分散于水中的 复合纳米粒子的动态激光光散射测得的粒径分布图， 粒子评价直径为 23 nm; 图 3 为复合纳米粒子的磁滞回归曲线， 表明纳米粒子具有超 顺磁性。
实施例 14 在圓底烧瓶中将 0.25 g实施例 11制备的 MPEG-b-PMAA-b-PGMA, 0.1 g FeCl2 · 4H20和 0.26 g FeCl3 · 6H20溶于 10 mL去离子水中， 在氮气保护下室温搅拌 10分钟后向烧瓶中注射器滴加 25%的氨水 0.2 mL, 混合液在室温下继续搅拌 24小时。 将混合液离心 （ 5000 rpm χ lO min)后， 上清液转移到透析袋（分子量上限为 14000 ) 中， 用去 离子水反复透析， 得到 MPEG-b-PMAA-b- PGMA包裹 Fe304磁性纳米粒子 的复合纳米粒子。 TEM表明 Fe304核的平均直径为 11 nm。
实施例 15
用实施例 12 中得到的 MPEG-b-PAA-b-PGA 代替实施例 13 中的 MPEG-b-PMAA-b-PGMA,其它操作同实施例 13，得到 MPEG-b-PAA-b- PGA 包裹 Fe304纳米粒子的复合纳米粒子。 TEM表明 Fe304核的平均直径为 12 nme
实施例 16
用实施例 12 中得到的 MPEG- b-PMAA-b-PGMA代替实施例 13 中的 MPEG-b-PMAA-b-PGMA ， 其 它 操 作 同 实 施 例 13 ， 得 到 MPEG-b-PMAA-b-PGMA 包裹 Fe304磁性纳米粒子的复合纳米粒子。 TEM 表明 Fe304核的平均直径为 9 nm。
实施例 17
用实施例 12中得到的 MPEG-b-(PMAA-r-PnBMA)-b-PGMA代替实施 例 13 中的 MPEG-b- PMAA- b-PGMA , 其它操作同实施例 13， 得到 MPEG-b- (PMAA-r-PnBMA) -b-PGMA 包裹 Fe304磁性纳米粒子的复合纳米 粒子。 TEM表明 Fe304核的平均直径为 7 nm。
实施例 18
将 2 g FeCl2 · 4H20 溶于 25 mL 1 mol/L HC1 中的溶液和 5.4 g FeCl3 · 6H20溶于 25 mL去离子水中的溶液置于三口瓶内混合， 通氮气。 向三口瓶内滴加 160 mL 1.5 mol/L氨水， 溶液中产生黑色沉淀， 室温 下继续搅拌反应 24小时。通过磁铁吸住沉淀倾去上层清液并用水洗涤 3 次。 在氮气保护下向三口瓶内滴加 2 mol/L HC104 50 mL, 室温搅拌 15 分钟。 静置 10分钟， 按上法倾去上层清液， 沉淀用水洗涤 1次， 将沉 淀迅速转移到透析袋中， 用去离子水反复透析到 pH值 6~7， 得 C104— 稳定的 Fe304纳米粒子水溶液， 测得 Fe304的浓度为： 22 mg/mL。 将 0.23 mL制得的 Fe304纳米粒子溶液与 4.77 mL水混合，在搅拌下向稀释的 Fe304 纳米粒子溶液中滴加 5 mL 由实施例 12中得到的 MPEG-b-PAA-b-PGA的 水溶液（ 2 mg/mL ) , 室温搅拌 24小时。 所得混合液离心（ 5000 rpm χ lO min)后， 上清液转移到透析袋（分子量上限为 14000 ) 中， 用去离 子水反复透析， 得到 MPEG- b-PAA-b- PGA包裹 Fe304磁性纳米粒子的复合 纳米粒子。 TEM表明 Fe304核的平均直径为 9 nm。
实施例 19
复合纳米粒子稳定性的研究： 将一系列实施例 13 所得的 MPEG-b-PMAA- b-PGMA-Fe304水溶液用 0.1 mol/L HC1或 0.1 mol/L NaOH 分别调节 pH值至 1、 2、 4、 6、 8、 10、 12、 13和 14, 使最终复合纳 米粒子的浓度相同 （1 mg/mL) ， 分别离心 10 分钟（ 2000 rpm) , 然 后分别测定 340 nm处的吸光值。 结果表明， pH值为 1、 2、 13和 14 的样品的吸光值明显降低， 吸光值低于最大吸光值的 80%。 而 pH值为 4、 6、 8、 10、 和 12 的样品的吸光值变化不大， 变化范围在 10%之内。 说明复合纳米粒子在 pH 4 ~ 12的范围内在水中可稳定分散。
将一系列实施例 13 中所得的 MPEG-b-PMAA-b-PGMA-Fe304水溶液 用 NaCl溶液稀释， 使最终 NaCl 的浓度分别为 0.5%、 1%、 2%和 5%， 而复合纳米粒子的浓度相同（ 1 mg /mL )，分别离心 10 分钟（ 2000 rpm), 然后分别测定 340 nm处的吸光值， 结果表明， 所有样品的吸光值的 变化不大， 变化范围在 5%之内。 说明复合纳米粒子在 0.5%.~ 5% NaCl 溶液中可稳定分散。
用实施例 15、 16、 17和 18所制备的聚合物包裹 FeA纳米粒子的 复合纳米粒子做如上相同的实验， 结果表明这些复合纳米粒子在 pH 4- 12的范围内和在 0.5% - 5% NaCl溶液中都能稳定分散。
向一系列实施例 13中所得的 MPEG- b- PMAA-b-PGMA-Fe304水溶液加 入不同量的乙酸铅（Pb (AC) 2 )水溶液， 使最终复合纳米粒子的浓度相 同 （1 mg/mL ) , 分别离心 10 分钟 （ 2000 rpm ) ， 然后分别测定 340 nm处的吸光值， 结果如图 4所示。 用二嵌段共聚物 PMAA- b- PGMA ( PAA 的聚合度为 25， PGMA 的聚合度为 19 ) 包裹的 Fe304纳米粒子 ( PMAA-b-PGMA-Fe304 )代替 MPEG-b- PMAA-b-PGMA-Fe304做相同的实验， 如图 4所示。图 4表明，水介质中加入乙酸铅对三嵌段共聚物包裹 Fe304 纳米粒子的复合纳米粒子的分散稳定性影响不大， 但当乙酸铅浓度大 于 ~ 0. 04%后， 二嵌段共聚物包裹 Fe304纳米粒子的复合纳米粒子聚集 沉淀。其原因可做如下分析，二嵌段共聚物包裹 Fe304纳米粒子的复合 纳米粒子由于聚阴离子处于粒子的表面， Pb2+与聚阴离子的羧基络合， 使粒子间发生交联而沉淀。但三嵌段共聚物包裹 Fe304纳米粒子的复合 纳米粒子由于表面有聚乙二醇的保护作用， 不发生粒子间的交联， 因 此未发生沉淀。
实施例 20
复合纳米粒子的细胞毒性的实验： 将实施例 1 3 中所得的 MPEG- b-PMAA-b-PGMA-Fe304和实施例 19 中提到的 PMAA- b- PGMA- Fe304 分别加入到培养液中进行细胞培养实验， 然后用 MTS方法测定细胞的 相对存活率， 具体实验方法可参考： S. Wan, J. Huang, M. Guo, H. Zhang, Y. Cao, H. Yan, K. L iu, J. B iomed. Ma ter. Res . A, 2007, 80, 946-954。图 5为 MPEG- b-PMAA-b-PGMA-Fe304和 PMAA-b-PGMA-FeA 细胞毒性的比较。可以看出，最外层为 MPEG的三嵌段共聚物包裹 FeA 纳米粒子的复合纳米粒子的细胞毒性明显较小。
实施例 21
阿霉素在复合纳米粒子中的负载： 在搅拌下向实旄例 13 中制备 的 MPEG-b- PMAA- b-PGMA-Fe304水溶液 ( 1 0 mL, 2 mg/mL ) 中滴加阿霉 素盐酸盐水溶液（2 mg/mL , 0. 4 mL ) ， 搅拌反应过夜后将混合液透 析（分子量上限为 14000 ) 48小时，得到负载阿霉素的复合纳米粒子。 经分析透析外液中阿尊素的含量， 换算出复合纳米粒子中阿霉素的负 载量为 21% (阿霉素的质量除以复合纳米粒子干重的量） 。
实施例 22
用实施例 15 制得的复合纳米粒子代替实施例 21 中的 MPEG-b-PMAA-b-PGMA- Fe304， 其它操作条件同实施例 21， 得到负载阿 霉素的复合纳米粒子， 阿霉素的负载量为 15%。
实施例 23
用 实施例 17 制得的复合纳米粒子代替实施例 21 中的 MPEG-b-PMAA-b-PGMA-Fe304 , 其它操作条件同实施例 21， 得到负载阿 審素的复合纳米粒子， 阿霉素的负载量为 23%。
实施例 24
表阿霉素在复合纳米粒子中的负载： 在搅拌下向实施例 13 中制 备的 MPEG-b-PMAA-b-PGMA-Fe304水溶液（1 0 mL， 2 mg/mL ) 中滴加表 阿尊素盐酸盐水溶液（2 mg/mL , 0. 4 mL ) ， 搅拌反应过夜后将混合 液透析（分子量上限为 14000 ) 48小时， 得到负载表阿霉素的复合纳 米粒子， 负载量为 20%。
实施例 25
正定霉素在复合纳米粒子中的负载： 在搅拌下向实施例 1 3 中制 备的 MPEG-b-PMAA- b-PGMA-Fe304水溶液（10 mL， 2 mg/mL ) 中滴加正 定尊素盐酸盐水溶液（2 mg/mL , 0. 4 mL ) ， 搅拌反应过夜后将混合 液透析（分子量上限为 14000 ) 48小时， 得到负载正定霉素的复合纳 米粒子， 负载量为 22%。
实施例 26
丝裂尊素在复合纳米粒子中的负载： 在搅拌下向实施例 1 3 中制 备的 MPEG- b-PMAA-b-PGMA-Fe304水溶液（10 mL , 2 mg/mL ) 中滴加丝 裂霉素水溶液（2 mg/mL , 0. 4 mL ) , 搅拌反应过夜后将混合液透析 (分子量上限为 14000 )48小时，得到负载丝裂霉素的复合纳米粒子， 负载量为 22%。
实施例 27 米托蒽醌在复合纳米粒子中的负载： 在搅拌下向实施例 13 中制 备的 MPEG-b-PMAA-b- PGMA- Fe3047j溶液（10 mL, 2 mg/mL) 中滴加米 托蒽醌盐酸盐水溶液（2 mg/mL, 0.4 mL ) , 搅拌反应过夜后将混合 液透析（分子量上限为 14000 ) 48小时， 得到负载米托蒽醌的复合纳 米粒子， 负载量为 23%。
实施例 28
负载阿霉素的复合纳米粒子的控释： 取 4份各 3 mL实施例 21中 制备的负载阿霉素的复合纳米粒子溶液装入透析袋 （分子量上限为 14000 ) , 分别置于 pH值分别为 7.4、 6.5、 5.5和 4.5的磷酸盐緩沖 液 （ 20 mmol/L, 100 mL ) 中透析， 在不同的时间测定透析外液中阿 霉素的浓度（通过紫外吸收检测， 检测波长为 234 nm) 。 换算出释放 率， 不同 pH值下阿霉素的释放率与释放时间的关系见图 6。

Claims

权利要求
1. 一种三嵌段共聚物包裹 Fe304纳米粒子的核 -壳结构的复合纳 米粒子， 其核为 Fe304纳米粒子， 平均粒径为 5~ 20 nm, 三嵌段共聚 物的结构可用下面通式表示：
Figure imgf000018_0001
上式中 b表示嵌段共聚物， Γ表示无规共聚物， 第一嵌段为曱氧 基聚乙二醇， 其平均聚合度 X为 10~ 230， 第二嵌段为聚丙烯酸、 或 聚曱基丙烯酸、 或丙烯酸与丙烯酸酯的无规共聚物、 或曱基丙晞酸与 曱基丙烯酸酯的无规共聚物， R=H或 CH3， m =10- 100, n = 0~ 30, R' = CH3、 C2H5、 C3H7、 CJ9或 C6H13， 第三嵌段为聚丙烯酸甘油单酯或聚 曱基丙烯酸甘油单酯， z = 10~ 50， 其中第三嵌段与 FeA纳米粒子核 表面结合， 第一嵌段形成复合纳米粒子的最外层。
2. 根据权利要求 1所述的复合纳米粒子的制备方法， 其特征在于 二价铁盐、 三价铁盐和三嵌段共聚物共溶于水中， 用碱将溶液的 pH 值调节到大于 8， 经反应而得到； 或者先制备无机阴离子如高氯酸根 离子包裹 Fe304纳米粒子的磁硫体，然后向磁硫体中加入三嵌段共聚物 水溶液而得到。
3. 根据权利要求 1和权利要求 2所述的复合纳米粒子及其制备方 法，其特征在于二价铁盐和三价铁盐的摩尔比 1: 2，铁盐的总浓度（以 Fe计） 为 0.5% - 5%, 铁盐 （以 Fe计） 与三嵌段共聚物的质量浓度比 为 2: 1 ~ 1: 3。
4. 根据权利要求 1和权利要求 2所述的复合纳米粒子及其制备方 法， 其特征在于调节溶液 pH值的碱可以是无机碱如氨水、 Na0H、 0H 等， 或有机碱如曱胺、 二曱胺、 三曱胺或四甲基铵等。
5. 根据权利要求 1和权利要求 2所述的复合纳米粒子及其制备方 法， 其特征在于调节溶液 pH值后的反应温度为室温到 100°C, 反应时 间为 10分钟到 24小时。
6. 根据权利要求 1所述的复合纳米粒子的用途， 其特征在于复合 纳米粒子可以作为含氨基药物或含胺基药物的载体。
7. 根据权利要求 1和权利要求 6所述的复合纳米粒子作为药物载 体的用途， 其特征在于复合纳米粒子可以作为阿霉素、 表阿霉素、 正 定審素、 丝裂霉素或米托蒽醌的载体。
8. 根据权利要求 1、 权利要求 6和权利要求 7所述的复合纳米粒 子作为药物载体的用途， 其特征在于负载药物的复合纳米粒子可用于 pH感应药物释放。
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