WO2017092088A1 - 一种磁共振成像造影剂及其制备方法与应用 - Google Patents

一种磁共振成像造影剂及其制备方法与应用 Download PDF

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WO2017092088A1
WO2017092088A1 PCT/CN2015/098234 CN2015098234W WO2017092088A1 WO 2017092088 A1 WO2017092088 A1 WO 2017092088A1 CN 2015098234 W CN2015098234 W CN 2015098234W WO 2017092088 A1 WO2017092088 A1 WO 2017092088A1
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mass
contrast agent
solution
polylysine
raw material
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周琦冰
魏裕双
杨祥良
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华中科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear 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
    • A61K49/1821—Nuclear 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 coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1863—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear 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
    • A61K49/1821—Nuclear 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 coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • A61K49/1872—Nuclear 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 coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid coated or functionalised with a polyamino acid, e.g. polylysine, polyglutamic acid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the field of magnetic resonance imaging contrast agents, and more particularly to a magnetic resonance imaging contrast agent and a preparation method and application thereof.
  • Superparamagnetic nanoscale ferroferric oxide nanoparticles are a contrast enhancer for magnetic resonance imaging and have been widely used in the in vivo clinical diagnosis and prognosis of tumors.
  • the superparamagnetic nano-scale ferroferric oxide nanoparticles are mainly used for the in vivo diagnosis of liver cancer.
  • Liver cancer is a major disease in China. There are 350,000 new cases each year, and its fatality rate ranks second in China.
  • Superparamagnetic nanoscale triiron tetroxide contrast agents have been reported to contribute to the discovery of tumors smaller than 1 cm, thus significantly increasing the survival rate of currently only 10% of patients with liver cancer in 5 years. In recent years, superparamagnetic nanoscale triiron tetroxide contrast agents have been further applied to the diagnosis of hepatic focal lesions and functional levels of immune Kupffer cells in steatohepatitis and liver fibrosis.
  • the liver is the main organ of lipid synthesis and metabolism.
  • the iron peroxidation product caused by iron overload not only accelerates the fibrosis process of tissues in nonalcoholic fatty liver, but also causes cirrhosis, and it is significantly improved in patients with cirrhosis.
  • the risk of deterioration in an inflammatory environment Therefore, how to reduce the safety risk caused by the retention of nano-sized ferroferric oxide particles in the liver and spleen and iron overload is a problem in the clinical application of nano-scale triiron tetroxide contrast agent.
  • the present invention provides a magnetic resonance imaging contrast agent and a preparation method and application thereof, the object of which is to coat a ferroferric oxide particle with a right-handed polylysine, thereby solving Technical problems of residual ferroferric oxide particles in the body.
  • a magnetic resonance imaging contrast agent which is a solution or a lyophilized powder, and includes a super-shun having a mass ratio of 1:5 to 1:15.
  • Magnetic nanoparticles and hydroxyethyl starch the superparamagnetic nanoparticles having a particle diameter of 100 nm to 140 nm, and consisting of ferroferric oxide particles, a citric acid layer and a surface right-handed polylysine layer from the inside to the outside,
  • the mass of the citric acid is 6% to 13% of the mass of the triiron tetroxide particles, and the mass of the right-handed polylysine is 6% to 20% of the mass of the triiron tetroxide particles;
  • the citric acid layer is coated on the surface of the ferroferric oxide particles by coordination and adsorption to increase the solubility of the ferroferric oxide particles in an aqueous solution; the right-handed polylysine layer is bonded by ionic bonding On the surface of the citric acid layer, for reducing the concentration of free iron ions, thereby reducing the damage of iron ions to cells; the average molecular weight of the hydroxyethyl starch is 110KDa to 150KDa for increasing the magnetic resonance imaging contrast agent Solubility.
  • the contrast agent is a lyophilized powder, and further comprises mannitol having a mass of 10 to 30 times the mass of the superparamagnetic nanoparticles for preserving the magnetic resonance imaging contrast agent under low temperature conditions.
  • the contrast agent is a solution, wherein the content of the superparamagnetic nanoparticles is 10 -3 g / L - 10 2 g / L, and the liquid contrast agent is more convenient to use than the lyophilized contrast agent.
  • the mass of the right-handed polylysine is 10% to 15% of the mass of the triiron tetroxide particles, and at the mass ratio, the right-handed polylysine is more uniformly coated with the triiron tetroxide. .
  • a method of preparing the above contrast agent comprising the steps of:
  • the raw material powder includes triiron tetroxide having a mass fraction of 6% to 12% Granules, 0.6% to 1.2% citric acid and 86.8% to 93.4% hydroxyethyl starch, the ferroferric oxide particles having a particle diameter of 60 nm to 75 nm, and the citric acid coated on the triiron tetroxide Particle surface
  • the mass of the right-handed polylysine is 6% to 20% of the mass of the triiron tetroxide particles, and 0.01 to 0.05 times of the volume of the aqueous solution in the step (1) is added to 0.04% to 0.2 times.
  • the % dextran polylysine solution is such that the dextran polylysine is completely uniformly dispersed in the solution and is ionically bonded to the surface of the citric acid to obtain the magnetic resonance imaging contrast agent.
  • the preparation method of the raw material powder is:
  • ferroferric oxide particles having a particle diameter of 60 nm to 75 nm, citric acid and N,N-dimethylformamide, uniformly mixed at a mass ratio of 1:0.1:10 to 1:1:100, 60 ° C Heating at ⁇ 90° C. until the ferroferric oxide particles are completely dissolved, and citric acid is coated on the surface of the ferroferric oxide particles; the agglomerated ferroferric oxide particles are removed to obtain a triiron tetroxide particle solution;
  • free iron ions and small molecule compounds in the aqueous solution are removed by tangential flow ultrafiltration.
  • the step (1) is specifically: placing the aqueous solution in a storage container of a tangential flow ultrafiltration device, performing tangential flow filtration purification through an ultrafiltration membrane block until the tangential flow filtration
  • the volume of the liquid in the filter container of the device is between 2:1 and 2:3, so that the free iron ions and the small molecule compound are completely removed, and the volume is not caused. Excessive loss of the raw material powder, at which point the liquid in the filter storage container is collected.
  • the method further comprises the step (3) of adding 1-2 times the mass of the raw material powder to the magnetic resonance imaging contrast agent obtained in the step (2).
  • the mixture was uniformly mixed, sterilized, and lyophilized to obtain the magnetic resonance imaging contrast agent in the form of a lyophilized powder.
  • the contrast agent in magnetic resonance imaging and the use as a low retention iron agent.
  • Magnetic resonance imaging contrast agent can reduce the concentration of free iron ions by coating the right-handed polylysine layer on the surface of the ferroferric oxide particles, effectively reducing the adsorption of iron ions by cells and reducing tissue damage;
  • the triiron tetroxide particles coated with the right-handed polylysine layer are not absorbed by the human body, and can be rapidly metabolized after magnetic resonance imaging, and the tissue residual amount is low;
  • the magnetic resonance imaging contrast agent of the present invention is used for magnetic resonance imaging of the liver, and the low residual tissue tissue not only prevents liver cirrhosis caused by accelerating the fibrosis process of tissues in nonalcoholic fatty liver, but also reduces The risk of exacerbation of cirrhotic patients in an inflammatory environment;
  • D-poly-polylysine has antifungal activity and ensures the quality of the magnetic resonance imaging contrast agent under long-term storage conditions
  • the magnetic resonance imaging contrast agent is prepared as a lyophilized powder, which is easier to store for a long time.
  • Embodiment 2 is a transmission electron microscope diagram of Embodiment 2 of the present invention.
  • FIG. 2 is a Prussian blue staining analysis of non-natural dextran polylysine-coated nano-scale iron oxide particles adsorbed on a cell strain according to the present invention
  • FIG. 2a is a nano-scale oxidation without dextro-polylysine-coated Iron has a large amount of adsorption on the cell line, and the positive distribution of Prussian blue staining is high
  • Fig. 2b shows the adsorption of the cell strain on Example 2, and the results show that the nano-scale iron oxide coated with dextran polylysine is in the cell. There was only a very weak positive result of Prussian blue staining on the strain;
  • Example 3 is a magnetic resonance T2 image imaging analysis of a mouse liver cirrhosis orthotopic liver cancer tumor according to Example 2 of the present invention
  • FIG. 4 is a distribution analysis of Prussian blue staining in subcutaneous tumor tissue according to Example 2 of the present invention
  • FIG. 4a is a subcutaneous tumor tissue of pancreatic cancer
  • FIG. 4b is a subcutaneous tumor tissue of liver cancer
  • an arrow indicates a positive distribution of Prussian blue staining
  • nano-scale iron oxide coated with dextran polylysine has a certain distribution at the interface of subcutaneous tumor tissue, and it has potential application prospects.
  • a magnetic resonance imaging contrast agent comprising a superparamagnetic nanoparticle having a mass ratio of 1:5 to 1:15 and hydroxyethyl starch, the superparamagnetic property.
  • the nanoparticle has a particle diameter of 100 nm to 140 nm, and is composed of a ferroferric oxide particle, a citric acid layer and a surface right-handed polylysine layer from the inside to the outside, and the citric acid is adsorbed on the surface of the ferroferric oxide particle.
  • the dextrorotatory polylysine is bound to the surface of the citric acid by an ionic bond, the mass of the citric acid being 6% to 13% by mass of the ferroferric oxide particles, the right-handed polylysine The mass is 6% to 20% of the mass of the triiron tetroxide particles, wherein when the mass of the right-handed polylysine is 10% to 15% of the mass of the triiron tetroxide particles, the right-handed polylysine
  • the coating effect of acid on triiron tetroxide is the most uniform.
  • the magnetic resonance imaging contrast agent is a solution or a lyophilized powder, and when the contrast agent is a lyophilized powder, mannitol having a mass of 10 to 30 times the mass of the superparamagnetic nanoparticles may be added to the contrast agent, so that The magnetic resonance imaging contrast agent is more convenient to be stored in a powder state under low temperature conditions.
  • the contrast agent is a solution
  • the superparamagnetic nanoparticles are stored in a content of 10 -3 g/L to 10 2 g/L.
  • Solution or lyophilized powdered contrast agents have different uses, lyophilized powders are easier to transport and long-term storage, and solutions are more convenient to use.
  • the preparation method of the above contrast agent comprises the following steps:
  • the raw material powder includes a mass fraction of 6%. 12% of ferroferric oxide particles, 0.6% to 1.2% of citric acid and 86.8% to 93.4% of hydroxyethyl starch, the ferroferric oxide particles having a particle diameter of 60 nm to 75 nm, the citric acid coating On the surface of the ferroferric oxide particles;
  • the mass of the right-handed polylysine is 6% to 20% of the mass of the triiron tetroxide particles, and 0.01 to 0.05 times of the volume of the aqueous solution in the step (1) is added to 0.04% to 0.2 times. % of the right-handed polylysine solution, so that the right-handed polylysine is completely dispersed uniformly in the solution, and is bonded to the surface of the citric acid by ionic bonding to obtain the magnetic resonance imaging contrast agent.
  • the reaction time is mainly related to the reaction time. The temperature and the total amount of the reactants are related. When the total mass of the solution is about 2500 ml at normal temperature, the reaction can be combined for 30 minutes;
  • the shelf life of the magnetic resonance imaging contrast agent prepared by the method is as follows: mass fraction of ferric oxide 2.0% to 5.0%, citric acid 0.2% to 0.5%, and dextran polylysine 0.2% to 5.0. %, hydroxyethyl starch 25% to 40% and mannitol 50% to 70%.
  • the raw material powder in the step (1) is prepared according to the method of the patent document CN103316361A, and the specific steps are as follows:
  • ferroferric oxide particles having a particle diameter of 60 nm to 75 nm, citric acid and N,N-dimethylformamide, uniformly mixed at a mass ratio of 1:0.1:10 to 1:1:100, 60 ° C Heat at ⁇ 90 °C until the place
  • the ferroferric oxide particles are completely dissolved, and the citric acid is coated on the surface of the ferroferric oxide particles; the agglomerated ferroferric oxide particles are removed to obtain a ferroferric oxide particle solution;
  • the specific method of the tangential flow ultrafiltration method in the step (1) is: placing the aqueous solution in a storage container of the tangential flow ultrafiltration device, and performing tangential flow filtration purification through the ultrafiltration membrane block.
  • the volume of liquid in the filtrate container of the tangential flow filtration device is between 2:1 and 2:3, the liquid in the storage device is collected.
  • the volume of the liquid in the filtrate container and the volume of the liquid in the storage container are between 2:1 and 2:3, so that the free iron ions and the small molecule compound are completely removed without causing excessive loss of the raw material powder.
  • the recommended amount per kg of body weight of the magnetic resonance imaging contrast agent is 0.2 mg to 1 mg based on the iron content.
  • the raw material powder in the step (1) is described in the Chinese patent "a stable nano-scale superparamagnetic nano-ferric oxide solution and its preparation method and application” (patent number: ZL201310284215.6) Prepared by a method for preparing a superparamagnetic nano-scale triiron tetroxide solid containing hydroxyethyl starch.
  • Example 2 R-polylysine coated nano-scale ferric oxide lyophilized powder injection
  • the liquid obtained in the step (2) of the above Example 1 was filtered through a 0.2 ⁇ m pore size filter, and then dispensed into a 5 ml volume glass bottle, and then subjected to freeze vacuum drying, and the powder obtained in the glass bottle was passed through a nitrogen gas.
  • the protective and gland seal is a R-polylysine coated nano-scale ferric oxide lyophilized powder injection. When using a powder injection, inject 3.5 ml of clinical saline into a glass bottle, shake and dissolve to prepare a solution. The recommended injection dose is 0.5 ml per 10 kg body weight.
  • Example 1 was repeated in the same manner as described, except that in step (1), when the volume of the liquid in the filtration container of the tangential flow filtration device was 1700 ml, the storage capacity of the filtration device was collected. Liquid in the device;
  • step (1) repeating Example 1 in the same manner as described, except that in step (1), when the liquid volume in the filtration container of the tangential flow filtration device is 3000 ml, the liquid in the filtration device storage container is collected;
  • Example 1 was repeated in the same manner as described except that the iron ion and the small molecule compound in the solution were removed by dialysis in the step (1).
  • the raw material powder (containing 9.2 wt% of superparamagnetic nano-ferric oxide particles, 0.8 wt% of citric acid, 88.5% of hydroxyethyl starch, and other parts as impurities in the raw material powder). 5 g of solid was dissolved in 5.0 liters of pure water, the raw material powder solution was obtained; 1 g of L-polylysine solid was weighed, and 9% g of ultrapure water was added to prepare a 0.1% L-polylysine solution; the raw material powder solution was stirred and gradually added. The above L-polylysine solution was 50 ml and stirred for 30 minutes.
  • Main equipment freeze dryer (labconco), Nano-ZS90 dynamic laser scatterometer (Malvern), transmission electron microscope (H-7000FA, Hitachi, Japan), magnetic resonance imager (Siemens Magnetom Trio Tim 3.0T), SpectrAA- 40 atomic absorption spectrometer (US Country VARIAN company) and so on.
  • Example 1 5.0 ml of the solution obtained in the step (1) in Example 1 was placed in an ultrafiltration apparatus of Amicon Ultra-15 of Millpore Co., Ltd., and centrifuged at 4000 g ⁇ 10 minutes, and the ferric ions present in the collected filtrate passed through hydrochloric acid. Hydroxylamine is reduced to a ferrous iron by a reducing agent, reacted with an o-morpholine chromogenic reagent in the range of pH ⁇ 5, and then measured at a wavelength of 530 nm. As a result, it is found that it is opposite to the standard curve containing ferric ions.
  • the total concentration of divalent and ferric ions in the solution obtained by tangential flow filtration in the step (1) of Example 1 is lower than the lowest detection line of the measurement method (mass ratio of ⁇ 50 ppm), but not cut
  • the amount of iron ions in the solution filtered to the flow was 1.15% of the total mass. This result indicates that the free iron ions have been effectively removed by tangential flow filtration.
  • Inventive Example 2 provides a transmission electron microscopic analysis of nano-sized particles in a solution prepared by R-polylysine-coated nano-sized ferric oxide lyophilized powder:
  • the superparamagnetic nano-scale ferroferric oxide solid prepared by the R-poly-lysine preparation prepared in Example 1 can be calculated. It is composed of the following components in terms of mass ratio: triiron tetroxide 3.3%, citric acid 0.3%, non-natural dextran polylysine 0.5%, hydroxyethyl starch 31.8%, mannitol: 63.1%.
  • the experiment was repeated as many times as in Example 1 - Example 5, and the mass fraction of each component in the obtained magnetic resonance imaging contrast agent was: 2.0% to 5.0% of triiron tetroxide, 0.2% to 0.5% of citric acid, and right-handed polycondensation. Lysine is 0.2% to 5.0%, hydroxyethyl starch is 25% to 40%, and mannitol is 50% to 70%.
  • the mouse liver cirrhosis model was established according to the method reported by Chang ML et al (World Journal of Gastroenterology 2005, 11, 4167). After the cirrhosis model was established, 150 mg of the lyophilized powder solid of Example 2 was accurately weighed and added to the clinic. After 5.0 ml of physiological saline was used, the prepared solution was intravenously injected at a dose of 0.5 mg of iron per kilogram of body weight according to the body weight of the mouse, and then the liver of the serum before and after the injection, 1, 3 and 5 days after the injection. The renal function index was measured, and the reference comparison experiment of the experiment was the liver and kidney function index of normal mice, and the results are shown in Table 4.
  • liver function index of the liver cirrhosis model mice was slightly after 24 hours after the injection of the R-polylysine coated nanometer ferritic ferric oxide lyophilized powder provided in Example 2 of the present invention. Elevated, but consistent with liver and kidney function indicators of normal mice after 5 days of injection, this result indicates that the injection of R-polylysine coated nano-sized ferric oxide lyophilized powder in the liver cirrhosis model It will not cause further damage.
  • the mouse model of liver cirrhosis orthotopic liver cancer tumor was transplanted into the liver cirrhosis liver of mice by surgery.
  • 150 mg of lyophilized powder solids of Example 2 was accurately weighed, and clinical saline was added.
  • the injection uses the R-polylysine provided in the second embodiment of the invention to encapsulate the nano-sized tetraoxide
  • the T2 image of the tumor in the magnetic resonance imaging shows the location, boundary and size of the tumor (as indicated by the arrow in Figure 3); therefore, the invention is used for injection.
  • the injection prepared by the R-polylysine coated nano-sized ferric oxide lyophilized powder provided in Example 2 can help to find the orthotopic liver cancer tumor in cirrhosis, and the imaging effect is remarkable.
  • the model of subcutaneous tumor was transplanted into the skin of mice by surgery, mainly including pancreatic cancer and subcutaneous tumor of liver cancer.
  • 150 mg of lyophilized powder solid of Example 2 was accurately weighed, and 5.0 ml of clinical saline was added.
  • the prepared solution was intravenously injected at a dose of 0.5 mg of iron per kilogram of body weight according to the body weight of the mouse, and after 24 hours of injection, the tumor tissues were collected and subjected to tissue sectioning and Prussian blue staining, and then images were taken with a microscope. Analysis, the results are shown in Figure 4. 4a is a subcutaneous tumor tissue of pancreatic cancer, and FIG.
  • the R-polylysine-coated nano-sized ferroferric oxide provided by the present invention has a certain distribution at the interface of the subcutaneous tumor tissue, and has potential application prospects.

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Abstract

公开了一种磁共振成像造影剂，包括质量比为1:5～1:15的超顺磁性纳米颗粒以及羟乙基淀粉，所述超顺磁性纳米颗粒的粒径为100nm～140nm，从内至外由四氧化三铁颗粒，柠檬酸以及右旋聚赖氨酸组成，所述柠檬酸吸附于四氧化三铁颗粒表面，所述右旋聚赖氨酸通过离子键与所述柠檬酸相结合，且所述柠檬酸的质量为所述四氧化三铁颗粒质量的6％～13％，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的8％～20％，所述磁共振成像造影剂为溶液或冻干粉末。还公开了该磁共振成像造影剂的制备方法与应用。

Description

一种磁共振成像造影剂及其制备方法与应用 【技术领域】
本发明属于磁共振成像造影剂领域，更具体地，涉及一种磁共振成像造影剂及其制备方法与应用。
【背景技术】
超顺磁性的纳米级四氧化三铁纳米颗粒是一种用于磁共振成像的对比增强剂，在肿瘤的体内临床诊断和预后中已有广泛的应用。超顺磁性的纳米级四氧化三铁纳米颗粒目前主要用于肝癌的体内诊断，肝癌在中国属重大疾病，每年新发病例有35万人次，其致死率在中国高居第二。超顺磁性的纳米级四氧化三铁对比剂已有报道能有助于小于1厘米肿瘤的发现，因此会显著提高现有仅10％的5年肝癌病人生存率。近年来，超顺磁性的纳米级四氧化三铁对比剂被进一步应用于肝脏灶性病变的诊断和脂肪性肝炎以及肝纤维化中免疫库普弗细胞的功能水平。
但是，由于超顺磁性的纳米级四氧化三铁颗粒在体内的滞留和缓慢的代谢转化，存在着铁过载引起的安全性问题。在动物研究中，注射的纳米级四氧化三铁颗粒主要在肝脏和脾脏中滞留，持续累积时间超过3周。即使是美国FDA批准的由Berlex实验室开发的菲立磁纳米级四氧化三铁对比剂(Feridex IV)，肝脏和脾脏中的铁含量在注射30天后，依然显著高于正常值。尽管铁过载在健康的模型中对肝功能的损伤很小，但是在肝脏和脾脏中由于持续的铁过载所产生的氧化应激和脂质过氧化产物，却是加重癌症的高风险因素。特别是肝脏是脂质合成和代谢的主要器官，铁过载所导致脂质过氧化产物不仅在非酒精性脂肪肝中加快组织的纤维化进程而导致肝硬化，而且在肝硬化患者会显著提高其在炎症环境中恶化的风险。因此，如何降低纳米级四氧化三铁颗粒在肝脏和脾脏中滞留和铁过载引起的安全性风险，是纳米级四氧化三铁对比剂在临床应用面临的问题。
【发明内容】
针对现有技术的以上缺陷或改进需求，本发明提供了一种磁共振成像造影剂及其制备方法与应用，其目的在于以右旋聚赖氨酸包覆四氧化三铁颗粒，由此解决四氧化三铁颗粒在体内残留的技术问题。
为实现上述目的，按照本发明的一个方面，提供了一种磁共振成像造影剂，所述磁共振成像造影剂为溶液或冻干粉末，包括质量比为1:5～1:15的超顺磁性纳米颗粒以及羟乙基淀粉，所述超顺磁性纳米颗粒的粒径为100nm～140nm，从内至外由四氧化三铁颗粒，柠檬酸层以及表面的右旋聚赖氨酸层构成，所述柠檬酸的质量为所述四氧化三铁颗粒质量的6％～13％，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％；
其中，所述柠檬酸层通过配位和吸附方式包覆于四氧化三铁颗粒表面，用于增加四氧化三铁颗粒在水溶液中的溶解度；所述右旋聚赖氨酸层通过离子键结合于所述柠檬酸层表面，用于降低游离的铁离子浓度，从而减少铁离子对细胞的损伤；所述羟乙基淀粉的平均分子量为110KDa～150KDa，用于增加所述磁共振成像造影剂的溶解性。
优选地，所述造影剂为冻干粉末，还包括质量为所述超顺磁性纳米颗粒10倍～30倍的甘露醇，用于使所述磁共振成像造影剂在低温条件下保存。
优选地，所述造影剂为溶液，其中，超顺磁性纳米颗粒的含量为10-3g/L～102g/L，液态的造影剂较冻干的造影剂使用起来更方便。
优选地，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的10％～15％，在该质量比下，右旋聚赖氨酸对四氧化三铁包覆更加均匀。
按照本发明的另一方面，提供了一种上述造影剂的制备方法，包括以下步骤：
(1)将原料粉配制为0.04％～0.2％的水溶液，并去除其中的游离铁离子以及小分子化合物；所述原料粉包括质量分数为6％～12％的四氧化三铁 颗粒，0.6％～1.2％的柠檬酸以及86.8％～93.4％的羟乙基淀粉，所述四氧化三铁颗粒的粒径为60nm～75nm，所述柠檬酸包覆于所述四氧化三铁颗粒表面；
(2)以右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％计，加入所述步骤(1)中水溶液体积的0.01倍～0.05倍的0.04％～0.2％的右旋聚赖氨酸溶液，使得右旋聚赖氨酸在溶液中完全分散均匀，并通过离子键结合于所述柠檬酸表面，得到所述磁共振成像造影剂。
优选地，所述原料粉的制备方法为：
(1)取粒径为60nm～75nm的四氧化三铁颗粒、柠檬酸和N，N-二甲基甲酰胺，以1:0.1:10～1:1:100的质量比混合均匀，60℃～90℃加热，直至所述四氧化三铁颗粒完全溶解，且柠檬酸包覆于四氧化三铁颗粒表面；除去团聚的四氧化三铁颗粒，得到四氧化三铁颗粒溶液；
(2)将步骤(1)得到的所述四氧化三铁颗粒溶液、羟乙基淀粉溶液以及N，N-二甲基甲酰胺，以1:0.1:5～1:1:20的质量比混合，在60℃～90℃搅拌反应，直至完全分散均匀，得到原料混合液；其中，羟乙基淀粉溶液的质量分数为5％～20％；
(3)向所述步骤(2)得到的原料混合液中，加入所述原料混合液2倍～5倍溶液体积的甲基叔丁基醚，使得所述原料混合液中的四氧化三铁颗粒和羟乙基淀粉成为沉淀；
(4)离心并干燥所述步骤(3)得到的沉淀后，得到所述原料粉。
优选地，在所述步骤(1)中，利用切向流超滤法去除所述水溶液中的游离铁离子以及小分子化合物。
作为进一步优选地，所述步骤(1)具体为，将所述水溶液置于切向流超滤装置的储存容器中，通过超滤膜块进行切向流过滤纯化，直至所述切向流过滤装置的滤出容器中的液体体积与储存容器中的液体体积为2:1～2:3之间，使得游离铁离子以及小分子化合物去除较为完整，同时不至于造成 原料粉的过多损失，此时收集过滤装置储存容器中的液体。
优选地，在所述步骤(2)之后还包括步骤(3)：向所述步骤(2)得到的所述磁共振成像造影剂中加入所述原料粉质量的1倍～2倍的甘露醇混合均匀，除菌并冻干后，得到冻干粉末状的所述磁共振成像造影剂。
按照本发明的另一方面，还提供了一种该造影剂在磁共振成像中的应用以及作为低滞留的铁剂的应用。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，由于右旋聚赖氨酸包覆四氧化三铁颗粒，能够取得下列有益效果：
1、磁共振成像造影剂通过在四氧化三铁颗粒表面包覆右旋聚赖胺酸层，能降低游离的铁离子浓度，有效降低细胞对铁离子的吸附作用，减少了组织损伤；
2、包覆了右旋聚赖胺酸层后的四氧化三铁颗粒不为人体吸收，磁共振成像后能快速代谢，组织残留量低；
3、将本发明的磁共振成像造影剂用于肝脏的磁共振成像，由于组织残留量低，不仅防止了在非酒精性脂肪肝中加快组织的纤维化进程而导致的肝硬化，而且减少了肝硬化患者在炎症环境中恶化的风险；
4、右旋聚赖胺酸具有抗真菌活性，能确保所述磁共振成像造影剂在长期保存状况下的质量；
5、优选将磁共振成像造影剂制备为冻干粉剂，更易于长期保存。
【附图说明】
图1为本发明实施例2的透射电子显微镜图；
图2为本发明提供的非天然右旋多聚赖胺酸包裹纳米级氧化铁颗粒在细胞株上吸附的普鲁士蓝染色分析；图2a为没有经右旋多聚赖胺酸包裹的纳米级氧化铁在细胞株上有大量的吸附，普鲁士蓝染色的阳性分布高；图2b为实施例2在细胞株上的吸附，其结果显示经右旋多聚赖胺酸包裹的纳米级氧化铁在细胞株上只有非常微弱的普鲁士蓝染色的阳性结果；
图3为本发明实施例2在小鼠肝硬化原位肝癌肿瘤的磁共振T2图像成像分析；
图4为本发明实施例2在皮下肿瘤组织中的普鲁士蓝染色分布分析；图4a为胰腺癌皮下肿瘤组织，图4b为肝癌皮下肿瘤组织；箭头所指为普鲁士蓝染色的阳性分布；其结果显示经右旋多聚赖胺酸包裹的纳米级氧化铁在皮下肿瘤组织的界面有一定的分布，存在着潜在的应用前景。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
为实现上述目的，按照本发明的一个方面，提供了一种磁共振成像造影剂，包括质量比为1:5～1:15的超顺磁性纳米颗粒以及羟乙基淀粉，所述超顺磁性纳米颗粒的粒径为100nm～140nm，从内至外由四氧化三铁颗粒，柠檬酸层以及表面的右旋聚赖氨酸层构成，所述柠檬酸吸附于四氧化三铁颗粒表面，所述右旋聚赖氨酸通过离子键与结合于所述柠檬酸表面，所述柠檬酸的质量为所述四氧化三铁颗粒质量的6％～13％，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％，其中，当右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的10％～15％时，右旋聚赖氨酸对四氧化三铁的包覆效果最为均匀。
所述磁共振成像造影剂为溶液或冻干粉末，当所述造影剂为冻干粉末，还可以在造影剂中加入质量为所述超顺磁性纳米颗粒10倍～30倍的甘露醇，使所述磁共振成像造影剂更便于在低温条件下以粉末状态保存。当所述造影剂为溶液时，超顺磁性纳米颗粒的保存含量为10-3g/L～102g/L。溶液或者冻干粉末状的造影剂具有不同的用途，冻干粉末更便于运输和长期 保存，而溶液使用起来更方便。
上述造影剂的制备方法包括以下步骤：
(1)将原料粉配制为0.04％～0.2％的水溶液，并用切向流超滤法或透析法等方法去除其中的游离铁离子以及小分子化合物；所述原料粉包括质量分数为6％～12％的四氧化三铁颗粒，0.6％～1.2％的柠檬酸以及86.8％～93.4％的羟乙基淀粉，所述四氧化三铁颗粒的粒径为60nm～75nm，所述柠檬酸包覆于所述四氧化三铁颗粒表面；
(2)以右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％计，加入所述步骤(1)中水溶液体积的0.01倍～0.05倍的0.04％～0.2％的右旋聚赖氨酸溶液，使得右旋聚赖氨酸在溶液中完全分散均匀，并通过离子键结合于所述柠檬酸表面，得到所述磁共振成像造影剂，当右旋聚赖氨酸加入量过低时，对四氧化三铁颗粒的包覆不完整，而加入量过高时，则容易使四氧化三铁颗粒过大，从而团聚而产生沉淀；其结合反应时间主要与温度以及反应物的总量有关，当常温下，溶液总质量为2500ml左右时，反应30min即可结合完全；
(3)将该磁共振成像造影剂除菌后直接保存，即得到溶液状的造影剂，冻干后保存，即得到冻干粉状的造影剂；当以冻干粉形式保存时，可先向磁共振成像造影剂中加入原料粉质量的0.5倍～2倍的甘露醇混合均匀，除菌后再冻干后，这样得到的造影剂由于加入了甘露醇，从而在低温条件下具有更长的保存期限，用该方法制备的磁共振成像造影剂按质量百分比的组成成分如下：四氧化三铁2.0％～5.0％，柠檬酸0.2％～0.5％，右旋聚赖胺酸0.2％～5.0％，羟乙基淀粉25％～40％以及甘露醇50％～70％。
其中，步骤(1)中的原料粉按照专利文献CN103316361A的方法制备，其具体步骤为：
(1)取粒径为60nm～75nm的四氧化三铁颗粒、柠檬酸和N，N-二甲基甲酰胺，以1:0.1:10～1:1:100的质量比混合均匀，60℃～90℃加热，直至所 述四氧化三铁颗粒完全溶解，且柠檬酸包覆于四氧化三铁颗粒表面；除去团聚的四氧化三铁颗粒，得到四氧化三铁颗粒溶液；
(2)将步骤(1)得到的所述四氧化三铁颗粒溶液、羟乙基淀粉溶液以及N，N-二甲基甲酰胺，以1:0.1:5～1:1:20的质量比混合，在60℃～90℃搅拌反应，直至完全分散均匀，得到原料混合液；其中，羟乙基淀粉溶液的质量分数为5％～20％，所述羟乙基淀粉的平均分子量为110KDa～150KDa，用于增加所述四氧化三铁颗粒的溶解性；
(3)向所述步骤(2)得到的原料混合液中，加入所述原料混合液2倍～5倍溶液体积的甲基叔丁基醚，使得使得所述原料混合液中的四氧化三铁颗粒和羟乙基淀粉成为沉淀；
(4)离心并干燥所述步骤(3)得到的沉淀后，得到所述原料粉。
其中，所述步骤(1)中用切向流超滤法的具体方法为，将所述水溶液置于切向流超滤装置的储存容器中，通过超滤膜块进行切向流过滤纯化，直至所述切向流过滤装置的滤出容器中的液体体积与储存容器中的液体体积为2:1～2:3之间，此时收集过滤装置储存容器中的液体。其中，滤出容器中的液体越多，则表示切向流过滤纯化的循环次数越多，则滤去铁离子和小分子的程度越彻底，但过多容易造成原料粉中的有效成分有损失；将滤出容器中的液体体积与储存容器中的液体体积为2:1～2:3之间，使得游离铁离子以及小分子化合物去除较为完整，同时不至于造成原料粉的过多损失。
该磁共振成像造影剂的每公斤体重建议使用量按铁含量计为0.2mg～1mg。
以下内容为实施例：
实施例1右旋多聚赖胺酸包裹纳米级四氧化三铁颗粒的制备方法
(1)称取原料粉(其中含有9.2wt％的超顺磁性纳米四氧化三铁颗粒，0.8wt％的柠檬酸，88.5％的平均分子量为130KDa的羟乙基淀粉，其它部分 为原料粉中的杂质)固体5克溶解于5.0升纯水中，置于切向流超滤装置(Millipore公司的Pellicon 2装置)的储存容器中，通过超滤膜块(Millipore公司的Pellicon 2的5K分子量的纤维膜)进行切向流超滤纯化，切向流流速设置为切向流速线性压差与饱和压差的临界点时的流速(10毫升/分钟)，当切向流过滤装置的滤出容器中的液体体积在2500毫升时，收集过滤装置储存容器中的液体；
(2)称取R-聚赖氨酸固体1克，加入999克超纯水配制得到0.1％的R-聚赖氨酸溶液；对所述步骤(1)所得的液体进行搅拌，并逐渐加入上述R-聚赖氨酸溶液50ml，并搅拌30分钟；
(3)再加入4.2克的甘露醇；
(4)对上述步骤中所得的液体通过0.22微米的孔径滤膜进行过滤，所得的溶液进行冷冻形成固体，然后进行冷冻真空干燥，得到的粉末即为R-聚赖氨酸包裹制备的超顺磁性纳米级四氧化三铁固体；
其中，步骤(1)中所述原料粉是参照中国专利“一种稳定的纳米级超顺磁性纳米四氧化三铁溶液及其制备方法和应用”(专利号：ZL201310284215.6)中所描述的制备含羟乙基淀粉的超顺磁性纳米级四氧化三铁固体的方法所制备的。
实施例2 R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末针剂
以上实施例1的步骤(2)中所得的液体通过0.2微米的孔径滤膜进行过滤后，分装于5毫升容积的玻璃瓶中，然后进行冷冻真空干燥，玻璃瓶中得到的粉末，经氮气保护并压盖密封，即为R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末针剂。使用粉末针剂时，往玻璃瓶中注入临床用生理盐水3.5毫升，摇匀溶解配制成溶液，建议的注射剂量为0.5毫升每10千克体重。
实施例3
(1)以所述的相同步骤重复实施例1，区别在于，步骤(1)中当切向流过滤装置的滤出容器中的液体体积在1700毫升时，收集过滤装置储存容 器中的液体；
(2)加入0.04％的R-聚赖氨酸溶液67ml，并搅拌30分钟；
(3)直接过滤除菌后分装在20ml的玻璃瓶中密封保存。
实施例4
(1)以所述的相同步骤重复实施例1，区别在于，步骤(1)中当切向流过滤装置的滤出容器中的液体体积在3000毫升时，收集过滤装置储存容器中的液体；
(2)加入0.2％的R-聚赖氨酸溶液45ml，并搅拌30分钟；
(3)加入2.5g的甘露醇，过滤除菌后分装在5ml的玻璃瓶中密封保存。
实施例5
(1)以所述的相同步骤重复实施例1，区别在于，步骤(1)中用透析法除去溶液中的铁离子以及小分子化合物。
(2)加入0.2％的R-聚赖氨酸溶液45ml，并搅拌30分钟；
(3)加入10g的甘露醇，过滤除菌后分装在5ml的玻璃瓶中密封保存。
对比例
称取原料粉(其中含有9.2wt％的超顺磁性纳米四氧化三铁颗粒，0.8wt％的柠檬酸，88.5％的羟乙基淀粉，其它部分为原料粉中的杂质)固体5克溶解于5.0升纯水中，得到原料粉溶液；称取L-聚赖氨酸固体1克，加入999克超纯水配制得到0.1％的L-聚赖氨酸溶液；搅拌原料粉溶液，并逐渐加入上述L-聚赖氨酸溶液50ml，并搅拌30分钟。
实验结果分析
主要化学药品和试剂：美国sigma-Aldrich公司，百灵威科学公司，阿拉丁试剂公司，国药试剂公司等。
主要仪器设备：冷冻干燥机(labconco)，Nano-ZS90动态激光散射仪(Malvern)，透射电子显微镜(H-7000FA，日本日立公司)，磁共振成像仪(西门子Magnetom Trio Tim 3.0T)，SpectrAA-40型原子吸收光谱仪(美 国VARIAN公司)等。
本发明实施例1所提供样品中游离铁离子的含量分析：
取实施例1中步骤(1)中所得溶液5.0毫升，置于Millpore公司的Amicon Ultra-15的超滤装置中，离心4000g×10分钟，所收集的滤液中存在的三价铁离子通过以盐酸羟胺为还原剂还原成为二价铁，在pH～5的范围内，与邻啡罗啉显色剂反应，然后在530纳米的波长下测吸收值，其结果发现，与含有三价铁离子标准曲线相对比，实施例1的步骤(1)中通过切向流过滤所得溶液中二价和三价铁离子的总浓度低于该测定方法的最低检测线(<50ppm的质量比)，而未通过切向流过滤的溶液中铁离子的含量是占总质量的1.15％，这一结果表明游离的铁离子已有效地通过切向流过滤被清除了。
本发明实施例2所提供R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末配制的溶液中纳米级颗粒粒径分析：
准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，再用纯水稀释10倍，取2.0毫升稀释液在25℃条件下使用Nano-ZS90动态激光散射仪测量其粒径、分布系数和zeta电位，其结果如表1所示。
表1
  zeta平均粒径 分布系数
样品1 126.3nm 0.114
样品2 124.9nm 0.161
样品3 127.4nm 0.159
样品4 126.5nm 0.141
样品5 122.4nm 0.126
粒径分析的结果表明，R-聚赖氨酸包裹的纳米颗粒的粒径分布呈正态分布，平均粒径在125.5nm。
本发明实施例2提供R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制溶液中纳米级颗粒的透射电镜分析：
准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，取部分溶液制作做电镜样品，然后在透射电子显微镜(H-7000FA，日本日立公司)下观察；样品分散性良好，透射电镜图如图1。透射电子显微镜分析显示，纳米颗粒的分布呈多个聚集体分布，与动态激光散射仪的结果一致；因此，本发明提供的新型R-聚赖氨酸包裹四氧化三铁的纳米级颗粒。
本发明实施例2所提供R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末中铁含量分析：
准确称取实施例2中冻干粉末固体样品150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，再用纯水稀释1000倍，取25.0毫升稀释液通过SpectrAA-40型原子吸收光谱仪测定其中铁的含量，其结果如表2所示。
表2
Figure PCTCN2015098234-appb-000001
原子吸收光谱仪测定的结果表明，平均每个样品中含铁的质量百分比为2.44％。
根据以上测量结果，以及制备过程中的原料比例可以计算得出，实施例1里制备得到的R-聚赖氨酸包裹制备的超顺磁性纳米级四氧化三铁固体 按质量比由以下成分组成：四氧化三铁3.3％，柠檬酸0.3％，非天然右旋多聚赖胺酸0.5％，羟乙基淀粉31.8％，甘露醇：63.1％。按实施例1-实施例5多次重复实验，得到的磁共振成像造影剂中各成分的质量分数范围为：四氧化三铁2.0％～5.0％，柠檬酸0.2％～0.5％，右旋聚赖胺酸0.2％～5.0％，羟乙基淀粉25％～40％以及甘露醇50％～70％。
细胞对本发明实施例2所提供的冻干粉末来配制的溶液中纳米级四氧化三铁颗粒的吸附分析：
准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，取100微升溶液加入含有贴壁细胞株BEL-7402的培养液中，孵育一小时后，吸取细胞培养液，贴壁的细胞用生理盐水轻轻冲洗二次后用多聚甲醛固定，细胞所吸附的四氧化三铁颗粒用普鲁士蓝染色，然后用显微镜拍摄图像进行分析，该实验的参照对比实验是没有经右旋聚赖氨酸包裹的纳米级四氧化三铁在细胞株上的吸附，其结果如图2所示。结果显示，没有经右旋聚赖氨酸包裹的纳米级四氧化三铁在细胞株上的吸附有大量的普鲁士蓝染色的阳性分布(图2a)，而经右旋聚赖氨酸包裹的纳米级四氧化三铁在细胞株上只有非常微弱的普鲁士蓝染色的阳性结果(图2b)，因此，本发明提供的R-聚赖氨酸包裹纳米级四氧化三铁的方法能有效降低非特异性的细胞吸附。
小鼠注射本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂后，各器官的铁含量的测定分析：
准确称取实施例2冻干粉末粒固体150mg，加入临床用生理盐水5.0ml，待完全溶解后，依照小鼠体重(每组三只)，按铁含量2.0mg/kg体重注射剂量静脉注射所配制的溶液，然后对注射后0.5、3以及24小时的血清、肝和脾脏中铁的含量进行测定和分析，并计算出各样品中的铁的质量分布，其结果如表3所示。
表3
Figure PCTCN2015098234-appb-000002
结果表明：用R-聚赖氨酸包裹纳米级四氧化三铁在脾脏中有极为快速的代谢，注射3小时后已恢复到正常值，而天然左旋聚赖氨酸包裹制备纳米级四氧化三铁在脾脏中在注射3小时后依然显著地高于正常值；在肝脏中，R-聚赖氨酸包裹的纳米级四氧化三铁在注射3小时后也低于然左旋聚赖氨酸包裹的样品；因此，R-聚赖氨酸包裹纳米级氧化在内脏器官中有其独特的快速代谢的特点。
在小鼠肝硬化模型试验中，注射本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂后，血清学的肝和肾功能指标的测定分析：
小鼠肝硬化模型是依照Chang ML et al报道的方法建立的(World Journal of Gastroenterology 2005，11,4167)，肝硬化模型建立后，准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，依照小鼠体重，按0.5毫克铁每千克体重注射剂量静脉注射所配制的溶液，然后对注射前和注射后1、3和5天的血清进行肝和肾功能指标进行测定，该实验的参照对比实验是正常小鼠的肝和肾功能指标，其结果如表4所示。
表4
Figure PCTCN2015098234-appb-000003
测定的结果表明,注射本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂后，肝硬化模型小鼠的肝功能指标在24小时后略有升高，但在注射5天后与正常小鼠的肝和肾功能指标相一致，这一结果表明，R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂在肝硬化模型中是不会引起进一步的损伤。
本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂在肝硬化原位肝癌肿瘤模型中的磁共振造影分析：
肝硬化原位肝癌肿瘤的小鼠动物模型是通过手术移植肝癌肿瘤组织到小鼠的肝硬化肝脏上；模型建立后，准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，依照小鼠体重，按0.5毫克铁每千克体重注射剂量静脉注射所配制的溶液，然后在磁共振仪器上进行T2图像扫描并进行图像分析；磁共振扫描分为注射前，注射后两批 次实验；结果如图3所示。
在注射对比剂之前，如图3a，图3b，肿瘤在磁共振造影中模糊，难以确定肿瘤存在以及其大小；注射使用本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂后，如图3c，3d，肿瘤在磁共振造影中T2图像显示了包括肿瘤的位置、边界和大小(如图3中箭头所示)；因此，注射使用本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂后能有助于发现肝硬化中原位肝癌肿瘤，其成像效果显著。
本发明实施例2提供的R-聚赖氨酸包裹纳米级四氧化三铁冻干粉末所配制的针剂在其他肿瘤模型中的分布分析：
皮下肿瘤的模型是通过手术移植到小鼠的皮下，主要包括胰腺癌和肝癌皮下肿瘤；模型建立后，准确称取实施例2冻干粉末粒固体150毫克，加入临床用生理盐水5.0毫升，待完全溶解后，依照小鼠体重，按0.5毫克铁每千克体重注射剂量静脉注射所配制的溶液，在注射24小时后，将肿瘤组织收集并进行组织切片和普鲁士蓝染色，然后用显微镜拍摄图像进行分析，其结果如图4所示。其中，图4a为胰腺癌皮下肿瘤组织，图4b为肝癌皮下肿瘤组织；箭头所指为普鲁士蓝染色的阳性分布。其结果显示本发明提供的R-聚赖氨酸包裹的纳米级四氧化三铁在皮下肿瘤组织的界面有一定的分布，存在着潜在的应用前景。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种磁共振成像造影剂，其特征在于，所述磁共振成像造影剂为溶液或冻干粉末，包括质量比为1:5～1:15的超顺磁性纳米颗粒以及羟乙基淀粉，所述超顺磁性纳米颗粒的粒径为100nm～140nm，从内至外由四氧化三铁颗粒，柠檬酸层以及表面的右旋聚赖氨酸层构成，所述柠檬酸的质量为所述四氧化三铁颗粒质量的6％～13％，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％。
  2. 如权利要求1所述的造影剂，其特征在于，所述造影剂为冻干粉末，还包括质量为所述超顺磁性纳米颗粒10倍～30倍的甘露醇。
  3. 如权利要求1所述的造影剂，其特征在于，所述造影剂为溶液，其中，超顺磁性纳米颗粒的含量为10-3g/L～102g/L。
  4. 如权利要求1所述的造影剂，其特征在于，所述右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的10％～15％。
  5. 如权利要求1-4中任意一项所述的造影剂的制备方法，其特征在于，包括以下步骤：
    (1)将原料粉配制为0.04％～0.2％的水溶液，并去除其中的游离铁离子以及小分子化合物；所述原料粉包括质量分数为6％～12％的四氧化三铁颗粒，0.6％～1.2％的柠檬酸以及86.8％～93.4％的羟乙基淀粉，所述四氧化三铁颗粒的粒径为60nm～75nm，所述柠檬酸包覆于所述四氧化三铁颗粒表面；
    (2)以右旋聚赖氨酸的质量为所述四氧化三铁颗粒质量的6％～20％计，向所述步骤(1)得到的水溶液中加入右旋聚赖氨酸溶液，使得右旋聚赖氨酸在溶液中完全分散均匀，并通过离子键结合于所述柠檬酸表面，得到所述磁共振成像造影剂。
  6. 如权利要求5所述造影剂的制备方法，其特征在于，所述原料粉的 制备方法为：
    (1)取粒径为60nm～75nm的四氧化三铁颗粒、柠檬酸和N，N-二甲基甲酰胺，以1:0.1:10～1:1:100的质量比混合均匀，60℃～90℃加热，直至所述四氧化三铁颗粒完全溶解，且柠檬酸包覆于四氧化三铁颗粒表面；除去团聚的四氧化三铁颗粒，得到四氧化三铁颗粒溶液；
    (2)将步骤(1)得到的所述四氧化三铁颗粒溶液、羟乙基淀粉溶液以及N，N-二甲基甲酰胺，以1:0.1:5～1:1:20的质量比混合，在60℃～90℃搅拌反应，直至完全分散均匀，得到原料混合液；其中，羟乙基淀粉溶液的质量分数为5％～20％；
    (3)向所述步骤(2)得到的原料混合液中，加入所述原料混合液2倍～5倍溶液体积的甲基叔丁基醚，使得四氧化三铁颗粒和羟乙基淀粉溶液成为沉淀；
    (4)离心并干燥所述步骤(3)得到的沉淀后，得到所述原料粉。
  7. 如权利要求5所述的制备方法，其特征在于，在所述步骤(1)中，利用切向流超滤法去除所述水溶液中的游离铁离子以及小分子化合物。
  8. 如权利要求7所述的制备方法，其特征在于，所述步骤(1)具体为，将所述水溶液置于切向流超滤装置的储存容器中，通过超滤膜块进行切向流过滤纯化，直至所述切向流过滤装置的滤出容器中的液体体积与储存容器中的液体体积为2:1～2:3之间。
  9. 如权利要求5所述的制备方法，其特征在于，在所述步骤(2)之后还包括步骤(3)：向所述步骤(2)得到的所述磁共振成像造影剂中加入所述原料粉质量的1倍～2倍的甘露醇混合均匀，除菌并冻干后，得到冻干粉末状的所述磁共振成像造影剂。
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