WO2015039555A1 - 基于氧化石墨烯材料的造影剂及其制备方法 - Google Patents

基于氧化石墨烯材料的造影剂及其制备方法 Download PDF

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WO2015039555A1
WO2015039555A1 PCT/CN2014/085611 CN2014085611W WO2015039555A1 WO 2015039555 A1 WO2015039555 A1 WO 2015039555A1 CN 2014085611 W CN2014085611 W CN 2014085611W WO 2015039555 A1 WO2015039555 A1 WO 2015039555A1
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contrast agent
molecule
graphene oxide
group
bridging
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French (fr)
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张智军
张萌欣
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中国科学院苏州纳米技术与纳米仿生研究所
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Publication of WO2015039555A1 publication Critical patent/WO2015039555A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/06Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1887Agglomerates, clusters, i.e. more than one (super)(para)magnetic microparticle or nanoparticle are aggregated or entrapped in the same maxtrix
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/06Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/06Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10Organic compounds
    • A61K49/101Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
    • A61K49/103Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA
    • A61K49/105Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA the metal complex being Gd-DTPA
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01BNON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00Carbon; Compounds thereof
    • C01B32/20Graphite
    • C01B32/21After-treatment
    • C01B32/23Oxidation

Definitions

  • the present invention relates to the field of medical nanomaterials, and in particular, to a functionalized graphene oxide material having superior magnetic resonance imaging properties, and a method of preparing the same. Background technique
  • Graphene is composed of a single atomic layer in which carbon atoms are sp 2 hybridized, and its basic structural unit is the most stable benzene six-membered ring in organic materials.
  • graphene has developed rapidly in the field of biomedicine.
  • one of the most important derivatives of graphene, graphene oxide, due to its good biocompatibility, high specific surface area, chemically functional modification, can be widely used in biomedical applications for targeted drug delivery. , cell imaging, bioassay, tumor therapy, etc. (Liu, Z.; Robinson, JT; Sun, X.; Dai, HJ Am. Chem. Soc. 2008, 130, 10876. Zhang, L.; Xia, J.
  • Magnetic resonance imaging is a non-invasive imaging method that utilizes magnetic resonance phenomena and is imaged by computer technology and two-dimensional image reconstruction. It has the advantages of no ionizing radiation, multi-core, multi-parameter imaging. Medically, it is necessary to obtain accurate diagnostic images and data. However, some of the diseased tissues of the human body sometimes have little difference with the signals of normal tissues, so it is necessary to use contrast agents to help obtain a magnetic resonance image with clear contrast. There are reports that by 2009 global sales of various contrast agents have exceeded $18 billion and are growing globally at a rate of 6-8% per year. According to relevant data, in the domestic Chinese pharmaceutical market, various contrast agents increase at a rate of 25-30% per year.
  • the contrast agent on the market is mainly DTPA-Gd, but it has problems such as low relaxation rate, fast metabolism in the body, short retention time and poor stability. Therefore, there is still no contrast agent in the art which has high relaxation rate, slow body metabolism, long retention time and good stability. Summary of the invention
  • a contrast agent having a high relaxation rate and good stability and a preparation method thereof.
  • a contrast agent having the structure of Formula I:
  • R is a ruthenium metal complex formed by complexing a complexing molecule Ra with a ruthenium metal;
  • B is a bridging molecule, and the bridging molecule is bonded to the surface of the graphene oxide by a chemical bond, and is connected to the rhodium metal complex by a chemical bond;
  • n is greater than 1.
  • the functional group is selected from the group consisting of a carboxyl group, a sulfonic acid group, a hydroxyl group, a folic acid group, or the like, or a combination thereof.
  • the ruthenium metal complex comprises a complexing molecule and a ruthenium ion complexed with the complexing molecule.
  • the graphene oxide comprises modified or unmodified graphene oxide.
  • the modified graphene oxide may be graphene oxide having a surface modified with a targeting group or a targeting molecule.
  • the targeting molecule comprises a protein, a polypeptide, a nucleic acid, a polysaccharide, a small molecule compound.
  • the small molecule compound is selected from the group consisting of folic acid, lactobionic acid, or a combination thereof.
  • the targeting molecule comprises an antibody, a ligand, folic acid.
  • the bridging molecule is selected from the group consisting of a bifunctional molecule, a polyfunctional molecule, or a combination thereof.
  • the bis/polyfunctional molecule is selected from the group consisting of functionalized polyethylene glycol, functionalized polyethyleneimine, functionalized dendrimer, hyaluronic acid, and shell poly Sugar, dextran, polylactic acid
  • the complexing molecule is selected from the group consisting of: diethylenetriaminepentaacetic acid (DTPA) or a derivative thereof, 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetra Acetic acid (DOTA) or a derivative thereof, or a combination thereof.
  • DTPA diethylenetriaminepentaacetic acid
  • DOTA diethylenetriaminepentaacetic acid
  • the functional group is selected from the group consisting of hydroxyl, amino, carboxyl, aldehyde, decyl, azide, isocyanate, silane, maleimide, succinimide carbonate Ester, succinimide acetate, succinimide propionate, succinimide succinate, succinimide valerate, or a combination thereof.
  • the ester is an ester formed with a substituted or unsubstituted C1-C20 alkyl group or a C3-C20 cycloalkyl group.
  • the bridging molecule is a biocompatible molecule. In another preferred embodiment, the bridging molecule is further modified with a group capable of specifically interacting with the cell.
  • the diethylenetriaminepentaacetic acid derivative or the 1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid derivative is selected from the group consisting of Group: Diethylenetriaminepentaacetic acid-bisdicarboxamide (DTPA-BDMA), diethylenetriaminepentaacetic acid-bis(isoniazid) (DTPA-BIN), diethylenetriaminepentaacetic acid-bisformamide (DTPA) -BMA), 1, 4, 7, 10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 10-(2-hydroxypropyl)-1,4,7, 10- Tetraazacyclododecane-1,4,7-triacetic acid (HPDO3A), 2-methyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- Acetic acid (MCTA), ( ⁇ , ⁇ ', ⁇ " , ⁇ " ' )-tetramethyl-1,4,
  • the mass ratio of the bridging molecule to graphene is 1:
  • the contrast agent is a contrast agent that can be used for cell imaging.
  • the contrast agent is a non-targeting contrast agent.
  • the contrast agent has the following properties:
  • the relaxation rate is determined as S mM ⁇ S relaxation rate r 1 20 mM ⁇ 1 S ⁇ 1 ; preferably 8 mM- 1 S- 1 relaxation rate r 1 12 mM- 1 S - 1 ; and/or
  • the physiological solution is selected from the group consisting of fetal calf serum, DMEM cell culture fluid, or a combination thereof.
  • the "non-causation phenomenon” means that the contrast agent of the present invention is mixed with a physiological solution to form a solution having a concentration of 0.25 mM, and after being left at room temperature for 48 hours, a contrast of coagulation occurs. Ratio of agents Lwt%), based on the total weight of the contrast agent.
  • a contrast agent according to the first aspect of the invention comprising the steps of:
  • A-(B-Ra) n (la) (iii) complexing a graphene oxide having a complex molecule on the surface of the formula la and a phosphonium salt and/or a phosphonium salt solution to obtain a contrast agent of the formula I according to the first aspect of the invention. ;
  • A, B, R, n are as defined above;
  • Ra is a complex molecule and m is greater than 1.
  • n m In another preferred embodiment, n m .
  • the steps (ii) and (iii) further comprise a dialysis step.
  • the dialysis step is for removing unreacted ruthenium ions, ruthenium metal complexes, bridging molecules, and the like.
  • the graphene oxide having a surface-modified bridge molecule is prepared by a method comprising the following steps:
  • the method further includes the following features:
  • the sulfonium salt is a water-soluble cerium salt, preferably selected from the group consisting of cerium chloride, cerium nitrate, or a combination thereof; the bridging molecule is selected from the group consisting of: functionalized polyethylene glycol, functional group Polyethyleneimine, functionalized dendrimer, hyaluronic acid, chitosan, dextran, polylactic acid-glycolic acid, or a combination thereof.
  • a contrast agent composition comprising a diluent and a contrast agent according to the first aspect of the invention.
  • the contrast agent is a magnetic resonance imaging contrast agent.
  • the contrast agent composition is in the form of a solution.
  • the composition further comprises a pharmaceutical component selected from the group consisting of: a therapeutic drug, a tracer molecule, or a combination thereof; and/or
  • the composition further comprises a component selected from the group consisting of cerium ions, manganese ions, cerium nanoparticles, manganese nanoparticles, small size ferroferric oxide nanoparticles, or combinations thereof.
  • the therapeutic drug is selected from the group consisting of doxorubicin, camptothecin, Iressa, blood root, and the like, or a combination thereof.
  • the tracer molecule is selected from the group consisting of fluorescein isothiocyanate, rhodamine, Cy series dyes, AlexaFluor series dyes, doxorubicin, or combinations thereof.
  • the small-sized ferroferric oxide nanoparticles have a particle diameter d of ld 5 nm.
  • the contrast agent composition according to the first aspect of the invention or the third aspect of the invention is applied to a subject or sample to be contrasted and subjected to contrast.
  • the contrast method is non-diagnostic and non-therapeutic.
  • the sample is a cell sample.
  • the contrast condition is measured at a magnetic field strength of 0.5-20 T, preferably at a magnetic field strength of 0.5-3 T.
  • a pharmaceutical composition comprising:
  • contrast agent according to the first aspect of the invention, wherein the contrast agent is used as a drug carrier;
  • the contrast agent is a targeted drug carrier.
  • the drug is combined with the contrast agent by adsorption.
  • the pharmaceutically active ingredient is selected from the group consisting of a therapeutic drug, a tracer molecule, or a combination thereof.
  • the therapeutic drug is selected from the group consisting of doxorubicin, camptothecin, Iressa, blood root, or a combination thereof.
  • the tracer molecule is selected from the group consisting of fluorescein isothiocyanate, rhodamine, Cy series dyes, AlexaFluor series dyes, doxorubicin, or combinations thereof.
  • a contrast agent intermediate having the structure la:
  • A is graphene oxide or graphene oxide modified by a functional group
  • Ra is a complexing molecule, and the complexing molecule can form a complex with a base metal
  • B is a bridging molecule, and the bridging molecule is linked to the surface of the graphene by a chemical bond, and is connected to the complexing molecule through a chemical bond;
  • n is greater than 1.
  • the functionalized modified graphene oxide is selected from the group consisting of carboxyl modified oxygen oxides Motenol, sulfonate-modified graphene oxide, folic acid-modified graphene oxide, or a combination thereof.
  • a contrast agent intermediate according to the sixth aspect of the invention for the preparation of a contrast agent according to the first aspect of the invention.
  • FIG. 1 is a schematic view showing the preparation of a graphene oxide-based contrast agent of the present invention
  • Figure 2 is a diagram showing the enhanced imaging effect of the magnetic resonance imaging contrast agent of the present invention.
  • Figure 3 is a graph showing the effect of the magnetic resonance imaging contrast agent of the present invention on cell imaging
  • Figure 4a is a UV-Vis spectrum of a magnetic resonance imaging contrast agent of the present invention for loading a drug
  • Figure 4b is a graph showing the cell survival rate of a magnetic resonance imaging contrast agent of the present invention acting on a cancer cell after loading the drug.
  • a non-specific magnetic resonance imaging contrast agent can be prepared by covalently modifying diethylenetriaminepentaacetate-oxime on graphene oxide.
  • the magnetic resonance imaging contrast agent has good imaging effect, wide application range and good stability. Based on the above findings, the inventors completed the present invention.
  • bifunctional molecule refers to a molecule having two functional groups within the molecule, said functional group being a group that can covalently link graphene oxide or diethylenetriamine pentaacetic acid.
  • the two functional groups may be the same or different.
  • substituted means that the hydrogen atom on the group is substituted by one or more substituents selected from the group consisting of halogen, phenyl, C1 to C4 alkyl; wherein the phenyl group includes an unsubstituted phenyl group or A substituted phenyl group having 1 to 3 substituents selected from a hydroxyl group, a C1 to C3 alkyl group, and a halogen.
  • C1-C20 alkyl refers to a straight or branched alkyl group having from 1 to 20 carbon atoms, such as methyl, Ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
  • C3-C20 cycloalkyl refers to a cycloalkyl group having 3 to 20 carbon atoms, such as a cyclopropyl group, a methylcyclopropane group, or the like.
  • iridium metal complex refers to a complex molecule and a ruthenium ion complexed with said complex molecule, preferably, said complex is in a chelated form.
  • the ruthenium metal complex may be used alone as a contrast agent.
  • the ruthenium metal complex is linked to the bridged molecule of the graphene oxide surface by chemical bonding.
  • the chemical bond connection mode includes a covalent bond, a coordination bond and the like.
  • the ruthenium metal complex is linked to the bridged molecule modified graphene oxide by a non-physical adsorption means.
  • Bridge molecule
  • bridged molecule refers to a molecule used to attach a graphene surface to a ruthenium metal complex, said bridging molecule being attached to said graphene surface by a chemical bond.
  • the bridging molecule is linked to the base metal complex by a chemical bond such as covalent bonding or coordinate bonding.
  • the bridging molecule is generally a molecule having two or more functional groups, such as a polyethylene glycol molecule having a functional group at both ends.
  • the bridging molecule is a biocompatible molecule.
  • the bridging molecule is further modified with a group capable of specifically interacting with cells.
  • a group capable of specifically interacting with cells Such as amino acids, peptides, antibodies, nucleic acids, monosaccharides, polysaccharides, vitamins.
  • Magnetic resonance imaging contrast agent such as amino acids, peptides, antibodies, nucleic acids, monosaccharides, polysaccharides, vitamins.
  • the present invention provides a contrast agent having the following structure:
  • A is graphene oxide or graphene oxide modified by a functional group
  • R is a ruthenium metal complex formed by complexing a complexing molecule Ra with a ruthenium metal
  • B is a bridging molecule, and the bridging molecule is connected to the surface of the graphene oxide by a chemical bond, and is connected to the rhodium metal complex by a chemical bond;
  • n is greater than 1.
  • the R in another preferred embodiment, may be directly connected to the surface of the graphene, or may be connected to the surface of the graphene through the bridging molecule B, preferably through the bridge.
  • Molecular B is attached to the surface of the graphene.
  • the graphene oxide may be oxy graphene having a surface modified with a targeting group or a targeting molecule to improve binding of the contrast agent to the target cell line.
  • the graphene oxide may also be graphene oxide without modification of a targeting group or a targeting molecule to obtain a non-specific contrast agent.
  • the targeting molecule can be any targeting molecule capable of increasing the binding of a contrast agent to a cell line, such as a protein, a polypeptide, a nucleic acid, a polysaccharide, a small molecule compound, and the like.
  • the targeting molecule comprises an antibody, a ligand, folic acid.
  • the complexing molecule may be any oxidestone which is capable of complexing with cerium ions and capable of being modified with a bridging molecule by a chemical bond such as a coordinate bond or a covalent bond.
  • the complexing molecule is selected from the group consisting of: diethylenetriaminepentaacetic acid and derivatives thereof, 1,4,7, 10-tetraazacyclododecane-1, 4, 7, 10- Tetraacetic acid and its derivatives, or a combination thereof.
  • the derivative refers to diethylenetriaminepentaacetic acid-bisdic dimethylformamide (DTPA-BDMA;), diethylenetriaminepentaacetic acid-bis(isoniazid) (DTPA-BIN), diethylenetriamine Acetic acid-bisformamide (DTPA-BMA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 10-(2-light propyl)-1 ,4,7, 10-tetraazacyclododecane-1,4,7-triacetic acid (HPDO3A), 2-methyl-1,4,7, 10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (MCTA), ( ⁇ , ⁇ ', ⁇ " , ⁇ " ' )-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4, 7, 10-tetraacetic acid
  • the contrast agent can be used alone as a contrast agent or as a contrast agent together with other components.
  • the contrast agent further comprises a component selected from the group consisting of cerium ions, manganese ions, cerium nanoparticles, manganese nanoparticles, small-sized ferroferric oxide nanoparticles, or a combination thereof.
  • the small size refers to a particle diameter d of l d 5 nm.
  • the contrast agent can be used as an intracellular contrast agent for intracellular imaging and detection.
  • the contrast agent may also be used in combination with other components, such as a drug loaded together for human or extracorporeal tissue.
  • the above drugs may be therapeutic drugs or tracer molecules, such as A. , camptothecin, Iressa, hematoxylin, paclitaxel, fluorescein isothiocyanate, rhodamine, Cy series dyes, AlexaFluor series dyes, etc., or combinations thereof.
  • the drug is combined with the contrast agent in an adsorption manner. Since the ruthenium metal complex is chemically bonded to the surface of the modified graphene oxide in the contrast agent, the contrast agent of the present invention can provide a larger The amount of adsorption.
  • the drug delivery vehicle has a drug loading rate of 50%, preferably 60%, more preferably 70%.
  • the contrast agent can also be used to prepare a pharmaceutical composition, the pharmaceutical composition comprising:
  • the contrast agent of the first aspect of the invention as a drug carrier
  • the contrast agent is a drug delivery vehicle.
  • the drug is combined with the contrast agent by adsorption.
  • the drug is not particularly limited and may be doxorubicin, camptothecin, Iressa, blood base, paclitaxel or the like.
  • the contrast agent of the present invention can be prepared by the following method:
  • A, B, R, n are as defined in the first aspect of the invention.
  • Ra is a complex molecule and m is greater than 1.
  • n m In another preferred embodiment, n m .
  • the steps (ii) and (iii) further comprise a dialysis step.
  • the graphene oxide having a surface modified with a bridging molecule can be obtained by a commercially available route or by a conventional method. Method preparation. In another preferred embodiment, the graphene oxide having a surface modified with a bridging molecule can be prepared by a method comprising the following steps:
  • the onium salt is a water soluble onium salt, preferably selected from the group consisting of ruthenium chloride, ruthenium nitrate, or combinations thereof.
  • the main advantages of the invention include:
  • a material suitable for preparing a magnetic resonance imaging contrast agent is provided, and the magnetic resonance imaging contrast agent prepared by the invention has good biocompatibility, and is similar to the existing magnetic resonance imaging contrast agent of the same type. Compared to, it has a better imaging effect.
  • the contrast agent of the invention not only significantly improves the contrast effect, but also is suitable for imaging of various human tissues or cells, does not require special processing according to the corresponding tissue or cells, and can be non-specifically Or widely applicable to a variety of different human tissues or cells.
  • the contrast agent of the present invention has a long circulation time in the body and is suitable for intracellular imaging.
  • the contrast agent of the present invention is easy to load a drug, and has a high drug loading rate of 70% or more.
  • the preparation of the magnetic resonance imaging contrast agent is divided into three steps, including preparation of graphene oxide, modification of graphene oxide by polyethylene glycol molecules, covalent attachment of diethylenetriamine pentaacetic acid to complex ruthenium ions, Specific steps are as follows:
  • the first step the preparation of graphene oxide:
  • the lg flake graphite, 0.5 g potassium persulfate, and 0.5 g phosphorus pentoxide were dissolved in 1.5 mL of concentrated sulfuric acid, heated to 8 CTC for 6 hours, cooled to room temperature, and then washed with water. Neutral, naturally dried into a powder. Add the above powder to 23 mL of concentrated sulfuric acid pre-cooled to 0 ° C, and add 3 g of permanganic acid with stirring. Potassium, keep the temperature below 20 °C. The temperature of the mixture was raised to 35 ° C, and after stirring for 2 h, 46 mL of three times of water was added and stirred for 15 min.
  • the second step the preparation of polyethylene oxide molecularly modified graphene oxide:
  • the third step is the preparation and application of graphene oxide-diethylenetriaminepentaacetic acid-ruthenium system:
  • the polyethylene glycol-modified graphene oxide obtained in the second step reaction was dispersed in an organic phase, 5 mL was added and 0.1 g of diethylenetriamine pentaacetic anhydride was added, and then EDAC 1 mL was added, followed by stirring overnight. The resulting product was dispersed by dialysis into the aqueous phase. GdCl 3 solution was then added, stirred for 4 child removing unreacted GdCl 3, i.e., to obtain the desired contrast agent. Contrast effect test:
  • the solution of the contrast medium (concentration: 0.5 mM) and the physiological solution (the fetal bovine serum, and the DMEM cell culture solution, respectively) were mixed in an equal volume ratio to form a solution having a concentration of 0.25 mM, and no significant precipitation was observed by the naked eye.
  • concentration of cerium ions remaining on the graphene after being mixed with the fetal bovine serum for 24 hours is 90% or more of the initial concentration of the mixed solution (when the mixing is completed).
  • the HepG2 cells were seeded in a cell culture dish, and contrast medium materials of different concentration gradients were added, and after 24 hours of incubation, the cells were digested, and the cells were fixed with agarose solution, and the cell imaging test was performed under the magnetic field strength of 11.7 T ( The result is shown in Figure 3.
  • Loaded drug test - Dispensing doxorubicin into an aqueous solution at a concentration of 1 mg/mL adding an equal volume to the contrast solution prepared above, adjusting the pH of the solution, and stirring for about 4 hours in the dark, dialysis removes excess Mycin.
  • Step 1 and Step 2 are the same as Embodiment 1.
  • the third step is the preparation and application of graphene oxide-(1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid)-ruthenium system:
  • the polyethylene glycol-modified graphene oxide obtained in the second step reaction is dispersed in the organic phase, and 1 ml of a concentration of 100 mg/mL is added to 5 ml of the polyethylene glycol-modified graphene oxide dispersion. 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid solution, then add EDAC lml, stir overnight, and dialyze off unreacted molecules. Thereafter, a cerium chloride salt solution was added, and after stirring for 4 hours, unreacted Gd ions were removed by dialysis to obtain a desired contrast agent.
  • the results show that the contrast agent produced can significantly enhance the contrast effect compared with the commercially available Magenville, which is 2-3 times higher at 1 1. 7T.
  • the specific preparation process described above is given by way of example only, and the molecules of the modified graphene oxide mentioned are not limited to the polyethylene glycol, the diethylenetriamine pentaacetic acid, the phosphonium ion mentioned in the above examples, Other molecules such as polyethyleneimine, dendrimer, hyaluronic acid, chitosan, dextran, polylactic acid-glycolic acid, 1, 4, 7, 10-tetraazacyclododecane may be used.

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Abstract

一种基于氧化石墨烯材料的造影剂及其制备方法,所述造影剂具有式I:A-(B-R) n的结构,其中,A为氧化石墨烯,R为钆金属络合物,B为桥联分子,且A、B、R之间通过化学键相互结合;该造影剂具有驰豫高、稳定性好、非特异性等特征。

Description

基于氧化石墨烯材料的造影剂及其制备方法
技术领域
本发明涉及医用纳米材料领域, 具体地, 本发明涉及一种具有优越磁共振造 影性能的功能化氧化石墨烯材料, 及其制备方法。 背景技术
石墨烯由碳原子以 sp2杂化连接的单原子层构成, 其基本结构单元为有机材料中 最稳定的苯六元环。近两年来石墨烯在生物医学领域的研究发展迅速。特别是石墨烯 最重要的衍生物之一, 氧化石墨烯, 由于其良好的生物相容性, 超高的比表面积, 能 够化学功能化修饰,在生物医学上可广泛应用于靶向药物输运、细胞成像、生物检测、 肿瘤治疗等方面(Liu, Z.; Robinson, J. T.; Sun, X.; Dai, H. J. Am. Chem. Soc. 2008, 130, 10876. Zhang, L.; Xia, J.; Zhao, Q.; Liu, L.; Zhang, Z. Small. 2010, 6, 537. Gon alves, G.; Vila, M.; Portoles, M. T.; Vallet-Regi, M.; Gracio, J.; Marques, P. A. Adv. Healthcare Mater. 2013, 2, 1072.) 0
磁共振成像是利用磁共振现象, 借助计算机技术和二维图像重建方法进行成 像, 是一种无创的影像手段。 其具有无电离辐射、 可实现多核、 多参数成像等优 点。 医学临床上需要得到精确的诊断学图像和数据, 但人体的某些病变组织有时 与正常组织之间的信号差异不大, 故需要使用造影剂帮助得到对比度清晰的磁共 振图像。有报告显示至 2009年全球各类造影剂销售额已超过 180亿美元, 且以每 年 6-8%的速度在全球范围内增长。 根据有关数据显示, 在中国国内医药市场上, 各种造影剂每年以 25-30%的速率递增。
目前市场上的造影剂主要是 DTPA-Gd, 但其存在着弛豫率偏低, 在体内代谢 快, 存留时间短, 稳定性差等问题。 因此, 本领域尚缺乏一种弛豫率高, 体内代 谢慢, 存留时间长, 稳定性好的造影剂。 发明内容
本发明的目的是提供一种弛豫率高, 稳定性好的造影剂及其制备方法。 在本发明的第一方面, 提供了一种造影剂, 所述的造影剂具有式 I结构:
A-(B-R)n (I) 式中, A为氧化石墨烯, 或被功能化基团修饰的氧化石墨烯;
R为钆金属络合物, 所述钆金属络合物由络合分子 Ra与钆金属络合形成;
B为桥联分子, 所述的桥联分子通过化学键与所述的氧化石墨烯表面相连, 并通 过化学键与所述的钆金属络合物相连;
"-"表示化学键;
n大于 1。
在另一优选例中 , 所述的功能化基团选自下组: 羧基、 磺酸基、 羟基、 叶酸等基 团或分子, 或其组合
在另一优选例中 ,所述的钆金属络合物包括络合分子和与所述的络合分子络合的 钆离子。
在另一优选例中 , 所述的氧化石墨烯包括经修饰的或未经修饰的氧化石墨烯。 在另一优选例中,所述的经修饰的氧化石墨烯可以是表面修饰有靶向基团或靶向 分子的氧化石墨烯。
在另一优选例中 , 所述的靶向分子包括蛋白、 多肽、核酸、 多糖、小分子化合物。 在另一优选例中, 所述的小分子化合物选自下组: 叶酸、 乳糖酸, 或其组合。 在另一优选例中, 所述的靶向分子包括抗体、 配体、 叶酸。
在另一优选例中 , 所述的桥联分子选自下组: 双官能团分子、 多官能团分子, 或 其组合。
在另一优选例中 , 所述的双 /多官能团分子选自下组: 官能团化的聚乙二醇、 官 能团化的聚乙烯亚胺、 官能团化的树状大分子、 透明质酸、 壳聚糖、 葡聚糖、 聚乳酸
-羟基乙酸, 或其组合; 和 /或
所述的络合分子选自下组: 二乙三胺五乙酸 (DTPA)或其衍生物, 1, 4, 7, 10-四氮 杂环十二烷 -1, 4, 7, 10-四乙酸(D0TA)或其衍生物, 或其组合。
在另一优选例中, 所述的官能团选自下组: 羟基、 氨基、 羧基、 醛基、 巯基、 叠 氮基、 异氰酸基、 硅烷基、 马来酰亚胺、 琥珀酰亚胺碳酸酯、 琥珀酰亚胺乙酸酯、 琥珀酰亚胺丙酸酯、 琥珀酰亚胺琥珀酸酯、 琥珀酰亚胺戊酸酯, 或其组合。
在另一优选例中, 所述酯为与取代或未取代的 C1-C20烷基或 C3-C20的环烷基 所形成的酯。
在另一优选例中, 所述的桥联分子是生物相容性分子。 在另一优选例中,所述的桥联分子上还修饰有能够特异性地与细胞相互作用的基 团。
在另一优选例中, 所述的二乙三胺五乙酸衍生物或 1, 4, 7, 10-四氮杂环十二烷 -1, 4, 7, 10-四乙酸衍生物选自下组: 二乙三胺五乙酸 -双二甲酰胺 (DTPA-BDMA)、 二乙三胺五乙酸-双 (异烟肼) (DTPA-BIN)、二乙三胺五乙酸-双甲酰胺 (DTPA-BMA)、 1, 4, 7, 10- 四氮杂环十二烷 -1, 4, 7- 三乙酸 (DO3A)、 10-(2-羟丙基 )-1,4,7, 10- 四氮杂 环十二烷 -1,4,7- 三乙酸 (HPDO3A)、 2- 甲基 -1,4,7,10- 四氮杂环十二烷 -1,4,7,10- 四乙 酸 (MCTA)、 (α,α',α " ,α " ' )- 四甲基 -1,4,7,10- 四氮杂环十二烷 -1,4,7,10- 四乙酸 (DOTMA)等, 或其组合。
在另一优选例中, 在所述的造影剂中, 所述的桥联分子与石墨烯的质量比为 1 :
0.2〜5。
在另一优选例中, 所述的造影剂是可用于细胞成像的造影剂。
在另一优选例中, 所述的造影剂是非靶向性造影剂。
在另一优选例中, 所述的造影剂具有以下性能:
在 11.7 T磁场强度下, 弛豫率 测定为 S mM^S 弛豫率r1 20 mM·11 ; 较佳 地为 8 mM-1S-1 弛豫率r1 12 mM-1S-1 ; 禾口 /或
在生理性溶液中无聚沉现象出现。
在另一优选例中, 所述的生理性溶液选自下组: 胎牛血清、 DMEM细胞培养液, 或其组合。
在另一优选例中, 所述的 "无聚沉现象"指, 将本发明造影剂与生理性溶液混合 后形成浓度为 0.25 mM的溶液, 在室温下放置 48小时后, 发生聚沉的造影剂比例
Figure imgf000005_0001
lwt%), 按造影剂的总重量计。
本发明的第二方面, 提供了一种如本发明第一方面所述的造影剂的制备方法, 所 述的方法包括以下步骤:
(i) 提供一表面连接有桥联分子的氧化石墨烯 A-(B)m, 其中所述桥联分子 B通过 化学键连于氧化石墨烯 A的表面;
(ϋ) 用络合分子 Ra与所述的表面连接有桥联分子的氧化石墨烯 - ^进行反应, 形成表面连接有络合分子的、 式 la所示的氧化石墨烯;
A-(B-Ra)n (la) (iii) 将式 la所示的表面连接有络合分子的氧化石墨烯与钆盐和 /或钆盐溶液进行 络合反应, 得到如本发明第一方面所述的式 I所示的造影剂;
其中, A、 B、 R、 n的定义如上文所述;
Ra为络合分子, m大于 1。
在另一优选例中, n m。
在另一优选例中, 对所述步骤 (ii)和 (iii)还包括透析步骤。
在另一优选例中, 所述的透析步骤用于去除未反应的钆离子、 钆金属络合物、 桥 联分子等反应物。
在另一优选例中,所述的表面修饰有桥联分子的氧化石墨烯通过包括以下步骤的 方法制备:
(a) 提供一氧化石墨烯;
(b) 将桥联分子与所述的氧化石墨烯反应, 得到表面修饰有桥联分子的氧化石墨 烯。
在另一优选例中, 所述方法还包括以下特征:
所述的钆盐为水溶性钆盐, 较佳地选自下组: 氯化钆、 硝酸钆, 或其组合; 所述的桥联分子选自下组: 官能团化的聚乙二醇、 官能团化的聚乙烯亚胺、 官能 团化的树状大分子、 透明质酸、 壳聚糖、 葡聚糖、 聚乳酸 -羟基乙酸, 或其组合。
本发明的第三方面, 提供了一种造影剂组合物, 所述的造影剂包括稀释剂以及如 本发明第一方面所述的造影剂。
在另一优选例中, 所述的造影剂是磁共振成像造影剂。
在另一优选例中, 所述的造影剂组合物为溶液形式。
在另一优选例中, 所述的组合物还包括选自下组的药物组分: 治疗性药物、 示踪 性分子, 或其组合; 和 /或
所述组合物还包括选自下组的组分: 钆离子、 锰离子、 钆纳米粒子、 锰纳米粒 子、 小尺寸四氧化三铁纳米粒子, 或其组合。
在另一优选例中, 所述的治疗性药物选自下组: 阿霉素、 喜树碱、 易瑞沙、 血根 碱等, 或其组合。
在另一优选例中, 所述的示踪性分子选自下组: 异硫氰酸荧光素、 罗丹明、 Cy 系列染料、 AlexaFluor系列染料、 阿霉素, 或其组合。 在另一优选例中, 所述的小尺寸四氧化三铁纳米粒子的粒径 d为 l d 5nm。 本发明的第四方面, 提供了一种造影方法, 所述方法包括步骤:
将本发明第一方面或本发明第三方面所述的造影剂组合物施用于待造影的对象 或样品, 并进行造影。
在另一优选例中, 所述的造影方法是非诊断和非治疗性的。
在另一优选例中, 所述的样品是细胞样品。
在另一优选例中, 所述造影条件为在 0.5-20T磁场强度下测定, 优选在 0.5-3T磁 场强度下测定。
本发明的第五方面, 提供了一种药物组合物, 所述的药物组合物包括:
©如本发明第一方面所述的造影剂, 所述造影剂作为药物载体;
(ii)负载于所述造影剂上的药物活性成分; 和 (iii)药学上可接受的其他载体。
在另一优选例中, 所述的造影剂为靶向性药物运载体。
在另一优选例中, 所述的药物通过吸附方式与所述的造影剂结合。
在另一优选例中, 所述的药物活性成分选自下组: 治疗性药物、 示踪性分子, 或 其组合。
在另一优选例中, 所述的治疗性药物选自下组: 阿霉素、 喜树碱、 易瑞沙、 血根 碱, 或其组合。
在另一优选例中, 所述的示踪性分子选自下组: 异硫氰酸荧光素、 罗丹明、 Cy 系列染料、 AlexaFluor系列染料、 阿霉素, 或其组合。
本发明的第六方面, 提供了一种造影剂中间体, 所述的造影剂中间体具有式 la 结构:
A-(B-Ra)n (la)
式中, A为氧化石墨烯, 或被功能化基团修饰的氧化石墨烯;
Ra为络合分子, 所述络合分子可与钆金属形成络合物;
B为桥联分子, 所述的桥联分子通过化学键与所述的石墨烯表面相连, 并通过化 学键与所述的络合分子相连;
"-"表示化学键;
n大于 1。
在另一优选例中, 所述的功能化修饰的氧化石墨烯选自下组: 羧基修饰的氧化石 墨烯、 磺酸基修饰的氧化石墨烯、 叶酸修饰的氧化石墨烯, 或其组合。
本发明的第七方面,提供了一种本发明第六方面所述的造影剂中间体用于制备如 本发明第一方面所述的造影剂的用途。 应理解, 在本发明范围内中, 本发明的上述各技术特征和在下文 (;如实施例) 中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。 限于篇幅, 在此不再一一累述。 附图说明
图 1是本发明的基于氧化石墨烯的造影剂的制备示意图;
图 2是本发明的磁共振成像造影剂增强成像效果图;
图 3是本发明的磁共振成像造影剂用于细胞成像效果图;
图 4a是本发明的磁共振成像造影剂用于负载药物的紫外-可见光谱图; 图 4b是本发明的磁共振成像造影剂负载药物后作用于癌细胞的细胞存活率 图。 具体实施方式
本发明人经过长期而深入的研究, 意外地发现, 通过在氧化石墨烯上共价修 饰二乙三胺五乙酸 -钆, 可以制备得到非特异性的磁共振成像造影剂。 所述的磁共 振成像造影剂成像效果好, 适用范围广, 稳定性好。 基于上述发现, 发明人完成 了本发明。 术语
如本文所用, 术语 "双官能团分子" 指分子内具有两个功能性基团的分子, 所述的功能性基团是可以共价连接氧化石墨烯或二乙三胺五乙酸的基团。 所述的 两个功能性基团可相同或不同。
术语 "取代" 是指基团上的氢原子被一个或多个选自下组的取代基取代: 卤 素、 苯基、 C1〜C4烷基; 其中所述的苯基包括未取代的苯基或具有 1-3个选自羟 基、 C1〜C3烷基、 卤素的取代基的取代苯基。
术语 " C1-C20烷基" 指具有 1-20个碳原子的直链或支链烷基, 例如甲基、 乙基、 丙基、 异丙基、 丁基、 异丁基、 仲丁基、 叔丁基、 或类似基团。 术语 " C3-C20环烷基" 指具有 3-20个碳原子的环烷基, 例如环丙烷基、 甲 基环丙烷基、 或类似基团。 礼金属络合物
如本文所用, 术语 "钆金属络合物"指络合分子以及与所述的络合分子络合的钆 离子, 较佳地, 所述的络合是螯合形式。
所述的钆金属络合物可以单独作为造影剂使用, 在本发明中, 所述的钆金属络合 物通过化学键连接方式与氧化石墨烯表面修饰的桥联分子连接。其中, 所述的化学键 连接方式包括共价键, 配位键等。
特别地,所述的钆金属络合物通过非物理吸附方式与桥联分子修饰的氧化石墨烯 连接。 桥联分子
如本文所用, 术语 "桥联分子"指用于连接石墨烯表面与钆金属络合物的分子, 所述的桥联分子通过化学键与所述的石墨烯表面相连。
特别地, 所述的桥联分子通过共价结合、配位键结合等化学键连接方式与钆金属 络合物相连。
所述的桥联分子一般为具有两个或两个以上功能性基团的分子,如双端具有官能 团的聚乙二醇分子等。
在另一优选例中, 所述的桥联分子是生物相容性分子。
在另一优选例中,所述的桥联分子上还修饰有能够特异性地与细胞相互作用的基 团。 如氨基酸、 肽、 抗体、 核酸、 单糖、 多糖、 维生素。 磁共振成像造影剂
本发明提供了一种造影剂, 所述的造影剂具有以下结构:
A-(B-R)n (I)
式中, A为氧化石墨烯, 或被功能化基团修饰的氧化石墨烯;
R为钆金属络合物, 所述钆金属络合物由络合分子 Ra与钆金属络合形成; B为桥联分子, 所述的桥联分子通过化学键与所述的氧化石墨烯表面相连, 并通 过化学键与所述的钆金属络合物相连;
"-"表示化学键;
n大于 1。
在另一优选例中, 在所述的造影剂中, 所述的 R可以直接与石墨烯表面相连, 也可以通过所述的桥联分子 B与石墨烯表面连接, 优选通过所述的桥联分子 B与石 墨烯表面连接。
在另一优选例中,所述的氧化石墨烯可以是表面修饰有靶向基团或靶向分子的氧 化石墨烯, 以改善造影剂与目标细胞系的结合。所述的氧化石墨烯也可以是未经修饰 有靶向基团或靶向分子的氧化石墨烯, 以得到非特异性的造影剂。
所述的靶向分子可以是任何能够增加造影剂和细胞系的结合的靶向分子, 如蛋 白、 多肽、 核酸、 多糖、 小分子化合物等。
在另一优选例中, 所述的靶向分子包括抗体、 配体、 叶酸。
所述的络合分子可以是任意能够与钆离子络合,且能够与桥联分子修饰的氧化石 墨烯通过化学键 (如配位键或共价键)相连。 较佳地, 所述的络合分子选自下组: 二乙 三胺五乙酸以及其衍生物、 1,4,7, 10-四氮杂环十二烷 -1, 4, 7, 10-四乙酸以及其衍 生物,或其组合。其中,所述的衍生物指二乙三胺五乙酸 -双二甲酰胺 (DTPA-BDMA;)、 二乙三胺五乙酸-双 (异烟肼 )(DTPA-BIN)、 二乙三胺五乙酸-双甲酰胺 (DTPA-BMA)、 1,4,7,10- 四氮杂环十二烷 -1,4,7- 三乙酸 (DO3A)、 10-(2- 轻丙基 )-1,4,7, 10- 四氮杂环 十二烷 -1,4,7- 三乙酸 (HPDO3A)、 2- 甲基 -1,4,7, 10- 四氮杂环十二烷 -1,4,7,10- 四乙酸 (MCTA)、 (α,α',α " ,α " ' )- 四甲基 -1,4,7,10- 四氮杂环十二烷 -1,4,7, 10- 四乙酸
(DOTMA)等, 或其组合。
所述的造影剂可以单独作为造影剂使用, 也可以和其他组分共同作为造影剂使 用。 在另一优选例中, 所述的造影剂还包括选自下组的组分: 钆离子、 锰离子、 钆 纳米粒子、 锰纳米粒子、 小尺寸四氧化三铁纳米粒子,或其组合。 其中, 所述的小 尺寸指粒径 d为 l d 5 nm。
特别地, 所述的造影剂可以用作细胞内造影剂, 用于进行细胞内成像和检测。 在另一优选例中, 所述的造影剂还可以与其他组分共同使用, 如负载药物共 同用于人体或体外组织等。 上述的药物可以是治疗性药物或示踪性分子等, 如阿霉 素、 喜树碱、 易瑞沙、 血根碱、 紫杉醇、 异硫氰酸荧光素、 罗丹明、 Cy系列染料、 AlexaFluor系列染料等, 或其组合。
所述的药物与所述的造影剂以吸附方式结合, 由于所述造影剂中, 钆金属络合物 以化学键方式与修饰的氧化石墨烯表面结合, 因此, 本发明的造影剂可以提供较大的 吸附量。在另一优选例中, 所述的药物递送载体的载药率为 50%, 较佳地为 60%, 更佳地为 70%。
在另一优选例中, 所述的造影剂还可以用于制备药物组合物, 所述的药物组 合物包括:
«如本发明第一方面所述的造影剂, 所述造影剂作为药物载体;
(ii)负载于所述造影剂上的药物活性成分; 和
(iii)药学上可接受的其他载体。
在另一优选例中, 所述的造影剂为药物递送载体。
在另一优选例中, 所述的药物通过吸附方式与所述的造影剂结合。
所述的药物没有特别限制, 可以是阿霉素、喜树碱、 易瑞沙、血根碱、紫杉醇等。 造影剂的制备
本发明所述的造影剂可以通过以下方法制备:
(i) 提供一表面连接有桥联分子的氧化石墨烯 A-(B)m, 其中所述桥联分子 B通过 化学键连于氧化石墨烯 A的表面;
(ii) 用络合分子 Ra与所述的表面连接有桥联分子的氧化石墨烯 A-(B)m进行反 应, 形成表面连接有络合分子的、 式 la所示的氧化石墨烯;
A-(B-Ra)n (la)
(iii) 将式 la所示的表面连接有络合分子的氧化石墨烯与钆盐和 /或钆盐溶液进行 络合反应, 得到如本发明第一方面所述的式 I所示的造影剂;
其中, A、 B、 R、 n的定义如本发明第一方面所述;
Ra为络合分子, m大于 1。
在另一优选例中, n m。
在另一优选例中, 对所述步骤 (ii)和 (iii)还包括透析步骤。
所述的表面修饰有桥联分子的氧化石墨烯可以通过市售途径获得,或通过常规方 法制备。在另一优选例中, 所述的表面修饰有桥联分子的氧化石墨烯可以通过包括以 下步骤的方法制备:
(a) 提供一氧化石墨烯;
(b) 用桥联分子与所述的氧化石墨烯反应, 得到表面修饰有桥联分子的氧化石墨 烯。
所述的钆盐为水溶性钆盐, 较佳地选自下组: 氯化钆、 硝酸钆, 或其组合。 本发明的主要优点包括:
(1) 提供了一种适用于制备磁共振成像造影剂的材料, 本发明所制备的磁共 振成像造影剂具有良好的生物相容性, 且与现有的同类型磁共振成像造影剂马根 维显相比, 具有更好的成像效果。
(2) 相较于现有技术, 本发明的造影剂不仅显著改善了造影效果, 而且适用 于多种人体组织或细胞的成像, 不需要根据相应的组织或细胞进行特殊加工, 能 够非特异性地或广泛地适用于多种不同的人体组织或细胞。
(3) 本发明的造影剂体内循环时间长, 并适合用于进行细胞内成像。
(4) 本发明的造影剂易于负载药物, 且载药率高, 可达到 70%或更高。
下面结合具体实施例, 进一步阐述本发明。 应理解, 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法, 通 常按照常规条件, 或按照制造厂商所建议的条件。 除非另外说明, 否则百分比和 份数按重量计算。 实施例 1 磁共振成像造影剂的制备
本实施例中, 磁共振成像造影剂的制备分为三个步骤, 包括氧化石墨烯的制 备, 聚乙二醇分子修饰氧化石墨烯, 共价连接二乙三胺五乙酸以络合钆离子, 具 体步骤如下:
第一步, 氧化石墨烯的制备:
将 l g片状石墨、 0. 5 g过硫酸钾、 0. 5 g五氧化二磷溶于 1. 5 mL浓硫酸中, 加热至 8CTC反应 6小时, 冷却到室温后, 再用水将其洗成中性, 自然干燥成粉末。 将上述粉末加入到 23 mL事先预冷至 0°C的浓硫酸中, 边搅拌边加入 3 g高锰酸 钾, 保持温度在 20 °C以下。 将混合物的温度升至 35 °C, 搅拌 2 h后, 加入 46 mL 三次水, 搅拌 15 min。 为中止氧化反应, 加入 140 mL三次水和 2 mL浓度为 30% 的过氧化氢。 将混合物用 250mL 10%的盐酸离心清洗, 以除去溶液中的金属离子。 将上述氧化石墨用超声 2 h, 10000转 /分钟离心 30 min到 1 h, 上清液即为氧化 石墨烯, 进一步超声可制备出纳米尺度的氧化石墨烯。
第二步, 聚乙二醇分子修饰的氧化石墨烯的制备:
取第一步反应中得到的氧化石墨烯 5 mL预先超声半小时, 充分分散后, 调 pH值约为 8,加入聚乙二醇分子(PEG)水溶液 lmL,在室温搅拌下,加入 EDAC 0. 15 mL, 继续搅拌, 再次加入 EDAC 0. 35 mL , 搅拌过夜。 随后透析得到 PEG修饰的氧 化石墨烯。
第三步, 氧化石墨烯-二乙三胺五乙酸-钆体系的制备及应用:
将第二步反应中得到的聚乙二醇修饰的氧化石墨烯分散到有机相中, 取 5 mL 并加入二乙三胺五乙酸酸酐 0. 1 g, 再加入 EDAC l mL, 之后搅拌过夜。 得到的产 物经透析分散到水相中。随后加入 GdCl3溶液,搅拌 4小时候除去未反应的 GdCl3, 即得到所需的造影剂。 造影效果测试:
该造影剂的制备示意图如图 1所示, 其与市售马根维显相比, 能显著增强造 影效果(图 2所示), 在 11. 7 T场强下约 2. 4倍, 为 10. 8 m TS— 稳定性测试:
该造影剂水溶液(浓度为 0.5 mM)与生理性溶液 (分别为胎牛血清、 和 DMEM 细胞培养液) 等体积比混合后, 形成浓度为 0. 25 mM的溶液, 肉眼均未见明显沉 淀。 其中, 与胎牛血清混合 24h后留存在石墨烯上的钆离子浓度为该混合液的初 始浓度(完成混合时)的 90%以上。 细胞内成像测试:
将 HepG2细胞接种于细胞培养皿内, 分别加入不同浓度梯度的造影剂材料, 共同孵育 24小时后, 消化收集细胞, 并用琼脂糖溶液固定细胞, 在 11. 7 T磁场 强度下进行细胞成像测试(结果如图 3所示)。 负载药物测试- 将阿霉素配置成浓度为 1 mg/mL的水溶液, 将其等体积加入上述制备的造影 剂溶液中, 调节溶液 pH值, 避光搅拌约 4小时后, 透析除去多余的阿霉素。 结果 显示, 本发明的造影剂能够负载阿霉素(图 4a所示), 载药率可达到 70%, 并能有 效地杀伤癌细胞(图 4b所示)。 实施例 2 磁共振成像造影剂的制备
步骤一、 步骤二同实施例 1。
第三步,氧化石墨烯- (1, 4, 7, 10-四氮杂环十二烷 -1, 4, 7, 10-四乙酸) -钆体系 的制备及应用:
将第二步反应中得到的聚乙二醇修饰的氧化石墨烯分散到有机相中, 在 5ml 聚乙二醇修饰的氧化石墨烯分散液中, 加入 1 ml浓度为 100 mg/mL的 1, 4, 7, 10- 四氮杂环十二烷 -1,4, 7, 10-四乙酸溶液, 再加入 EDAC lml , 搅拌过夜, 透析出去 未反应的分子。 之后加入氯化钆盐溶液, 搅拌 4小时后, 透析除去未反应的 Gd 离子, 即得到所需的造影剂。
结果显示,制得的造影剂与市售马根维显相比能显著增强造影效果,在 1 1. 7T 下提高 2-3倍。 上述具体的制备工艺仅是作为示例给出, 其中所提到的修饰氧化石墨烯的分 子不仅仅局限于上述例子中所提到的聚乙二醇、 二乙三胺五乙酸、 钆离子, 也可 以选用其它分子, 如聚乙烯亚胺、 树状大分子、 透明质酸、 壳聚糖、 葡聚糖、 聚乳 酸-羟基乙酸、. 1, 4, 7, 10-四氮杂环十二烷 -1, 4, 7, 10-四乙酸、锰离子、锰纳米粒子、 小尺寸四氧化三铁纳米粒子或其组合。 在本发明提及的所有文献都在本申请中引用作为参考, 就如同每一篇文献被 单独引用作为参考那样。 此外应理解, 在阅读了本发明的上述讲授内容之后, 本 领域技术人员可以对本发明作各种改动或修改, 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
1、 一种造影剂, 其特征在于, 所述的造影剂具有式 I结构:
A-(B-R)n (I)
式中, A为氧化石墨烯, 或被功能化基团修饰的氧化石墨烯;
R为钆金属络合物, 所述钆金属络合物由络合分子 Ra与钆金属络合形成;
B为桥联分子, 所述的桥联分子通过化学键与所述的氧化石墨烯表面相连, 并通 过化学键与所述的钆金属络合物相连;
"-"表示化学键;
n大于 1。
2、 如权利要求 1所述的造影剂, 其特征在于, 所述的桥联分子选自下组: 双官 能团分子、 多官能团分子, 或其组合。
3、 如权利要求 1所述的造影剂, 其特征在于, 所述的经修饰的氧化石墨烯是表 面修饰有靶向基团或靶向分子的氧化石墨烯; 较佳地, 所述的靶向分子选自下组: 蛋 白、 多肽、 核酸、 多糖、 小分子化合物。
4、如权利要求 1所述的造影剂, 其特征在于,所述的双 /多官能团分子选自下组: 官能团化的聚乙二醇、 官能团化的聚乙烯亚胺、 官能团化的树状大分子、 透明质酸、 壳聚糖、 葡聚糖、 聚乳酸 -羟基乙酸, 或其组合; 和 /或
所述的络合分子选自下组: 二乙三胺五乙酸 (DTPA)或其衍生物, 1, 4, 7, 10-四氮 杂环十二烷 -1, 4, 7, 10-四乙酸(D0TA)或其衍生物, 或其组合。
5、 如权利要求 1所述的造影剂的制备方法, 其特征在于, 所述的方法包括以下 步骤:
(i) 提供一表面连接有桥联分子的氧化石墨烯 A-(B)m,其中所述桥联分子 B通过 化学键连于氧化石墨烯 A的表面;
(ii) 用络合分子 Ra与所述的表面连接有桥联分子的氧化石墨烯 A-(B)m进行反 应, 形成表面连接有络合分子的、 式 la所示的氧化石墨烯;
A-(B-Ra)n (la)
(iii) 将式 la所示的表面连接有络合分子的氧化石墨烯与钆盐和 /或钆盐溶液进行 络合反应, 得到如权利要求 1所述的式 I所示的造影剂;
其中, A、 B、 R、 n的定义如权利要求 1所述; Ra为络合分子, m大于 1。
6、 如权利要求 5所述的制备方法, 其特征在于, 所述的表面修饰有桥联分子的 氧化石墨烯通过包括以下步骤的方法制备:
(a) 提供一氧化石墨烯;
(b) 将桥联分子与所述的氧化石墨烯反应, 得到表面修饰有桥联分子的氧化石墨 烯。
7、 如权利要求 5所述的制备方法, 其特征在于, 还包括以下特征:
所述的钆盐为水溶性钆盐, 较佳地选自下组: 氯化钆、 硝酸钆, 或其组合; 所述的桥联分子选自下组: 官能团化的聚乙二醇、 官能团化的聚乙烯亚胺、 官能 团化的树状大分子、 透明质酸、 壳聚糖、 葡聚糖、 聚乳酸 -羟基乙酸, 或其组合。
8、 一种造影剂组合物, 其特征在于, 所述的造影剂包括稀释剂以及权利要求 1 所述的造影剂。
9、 如权利要求 8所述的造影剂组合物, 其特征在于, 所述的组合物还包括选自 下组的药物组分: 治疗性药物、 示踪性分子, 或其组合; 和 /或
所述组合物还包括选自下组的组分: 钆离子、 锰离子、 钆纳米粒子、 锰纳米粒 子、 小尺寸四氧化三铁纳米粒子, 或其组合。
10、 如权利要求 9所述的造影剂组合物, 其特征在于, 所述的治疗性药物选自下 组: 阿霉素、 喜树碱、 易瑞沙、 血根碱等, 或其组合; 和 /或
所述的示踪性分子选自下组: 异硫氰酸荧光素、 罗丹明、 Cy系列染料、 AlexaFluor系列染料、 阿霉素, 或其组合。
11、 一种造影方法, 其特征在于, 包括步骤:
将权利要求 1或权利要求 6所述的造影剂组合物施用于待造影的对象或样品,并 进行造影。
12、 一种药物组合物, 其特征在于, 所述的药物组合物包括:
©如权利要求 1所述的造影剂, 所述造影剂作为药物载体;
(ii)负载于所述造影剂上的药物活性成分; 和 (iii)药学上可接受的其他载体。
13、 如权利要求 12所述的药物组合物, 其特征在于, 所述的药物活性成分选自 下组: 治疗性药物、 示踪性分子, 或其组合。
14、 一种造影剂中间体, 其特征在于, 所述的造影剂具有式 la结构: A-(B-Ra)n (la)
式中, A为氧化石墨烯, 或被功能化基团修饰的氧化石墨烯;
Ra为络合分子, 所述络合分子可与钆金属形成络合物;
B为桥联分子, 所述的桥联分子通过化学键与所述的石墨烯表面相连, 并通过化 学键与所述的络合分子相连;
"-"表示化学键;
n大于 1。
15、 一种权利要求 14所述的造影剂中间体的用途, 其特征在于, 用于制备权利 要求 1所述的造影剂。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102397563A (zh) * 2010-09-16 2012-04-04 同济大学 一种磁共振成像造影剂用石墨烯纳米载体的制备方法
CN102657872A (zh) * 2012-05-05 2012-09-12 上海师范大学 氧化石墨烯/PAMAM/DTPA-Gd/PSCA抗体多功能材料及其制备方法和应用
US20130079503A1 (en) * 2011-09-08 2013-03-28 Research & Business Foundation Sungkyunkwan University Go-gd-dtpa complex, preparation method thereof, and mri contrast agent comprising the same

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101670108A (zh) * 2009-08-13 2010-03-17 苏州纳米技术与纳米仿生研究所 基于纳米氧化石墨烯的载药体系

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102397563A (zh) * 2010-09-16 2012-04-04 同济大学 一种磁共振成像造影剂用石墨烯纳米载体的制备方法
US20130079503A1 (en) * 2011-09-08 2013-03-28 Research & Business Foundation Sungkyunkwan University Go-gd-dtpa complex, preparation method thereof, and mri contrast agent comprising the same
CN102657872A (zh) * 2012-05-05 2012-09-12 上海师范大学 氧化石墨烯/PAMAM/DTPA-Gd/PSCA抗体多功能材料及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHEN HE ET AL.: "Application of Graphene in Biomedical Fields", JOURNAL OF SOUTHEAST UNIVERSITY (MEDICAL SCIENCE EDITION, vol. 30, no. 1, 28 February 2011 (2011-02-28) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017066268A1 (en) * 2015-10-12 2017-04-20 Ohio University Supercapacitor-based biosensors and methods of detecting a biomarker
CN108042809A (zh) * 2018-02-01 2018-05-18 南京林业大学 一种抑菌功能化氧化石墨烯的制备方法

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