WO2023040037A1 - 氧化铁纳米粒子在制备甲状旁腺和/或***显影剂中的应用 - Google Patents

氧化铁纳米粒子在制备甲状旁腺和/或***显影剂中的应用 Download PDF

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WO2023040037A1
WO2023040037A1 PCT/CN2021/131855 CN2021131855W WO2023040037A1 WO 2023040037 A1 WO2023040037 A1 WO 2023040037A1 CN 2021131855 W CN2021131855 W CN 2021131855W WO 2023040037 A1 WO2023040037 A1 WO 2023040037A1
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ionps
thyroid
iron oxide
parathyroid
oxide nanoparticles
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French (fr)
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郑伟慧
郭鹏
覃江江
程向东
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中国科学院大学附属肿瘤医院
中国科学院肿瘤与基础医学研究所
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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
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/225—Microparticles, microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • 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 technical field of surgical navigation imaging and relates to a parathyroid negative contrast agent and its preparation and application, in particular to an iron oxide nanoparticle and its preparation and application in the parathyroid negative contrast agent.
  • GLOBOCAN reported that thyroid cancer ranks ninth among all cancers in the world, and fifth among female cancers. According to the statistics of 25 countries in the world by The Lancet, the incidence rate of all major thyroid cancer subtypes in China has increased significantly; at the same time, low-risk papillary thyroid cancer is rapidly increasing in the world, including China.
  • the treatment of thyroid cancer mainly relies on surgery, and with the rapid increase in incidence, the amount of surgery has increased significantly.
  • the parathyroid gland is small, the short-term and long-term complications caused by its damage are extremely serious. It is extremely important for patients if the parathyroid glands can be better visualized during the operation.
  • parathyroid glands in the current operation mainly includes two aspects: positive development and negative development.
  • Positive developers include methylene blue, aminolevulinic acid, indocyanine green, and the like.
  • the positive contrast agent lacks the tissue specificity of parathyroid glands. Usually, the staining rate of normal parathyroid gland tissue is low, the effect is not good, and there are side effects: such as arrhythmia, neurotoxicity, iodine allergy, etc.
  • the negative image is mainly carbon nanomaterials, which are black carbon nanoparticles.
  • the function of the contrast agent is to negatively develop the parathyroid glands by staining the normal thyroid body black. At present, it has been used clinically, but its application in non-tumor surgery is limited, and it is more difficult to apply in secondary surgery.
  • carbon nanomaterials have important potential safety issues, which will cause harm to the human body: because carbon nanomaterials cannot be degraded normally in the human body, they will damage DNA during cell replication and may induce tumors, and their human safety has always existed dispute.
  • Carbon black can also reduce the photosensitive ability of zebrafish visual cells, cause toxicity to glial cells in the nervous system of zebrafish, and cause disorder in the movement behavior of zebrafish.
  • Zhao Dandan's master's thesis described the effects of nano-carbon black particles combined with cold stimulation on the respiratory system and energy metabolism of mice.
  • Zhou Lixiao's master's thesis showed that nano-carbon black has a toxic effect on human bronchial epithelial cells.
  • materials such as carbon nanotubes and graphene in carbon nanomaterials have also been reported to have reproductive toxicity, cause vascular endothelial dysfunction and epithelial cell damage, and affect human pleural mesothelial cells. Therefore, finding a non-toxic, harmless and biodegradable parathyroid contrast agent has become an urgent clinical need.
  • the object of the present invention is to provide iron oxide nanoparticles (Iron oxide nanoparticles, IONPs) and its preparation and application in negative imaging parathyroid glands.
  • the IONPs can help better preserve the parathyroid glands and maintain the function of the parathyroid glands during the operation.
  • iron oxide nanoparticles can be degraded and metabolized into iron ions through the lysosome of normal human cells, and then enter the normal iron metabolism of the human body after being combined with ferritin, and are completely absorbed and degraded by the human body, so they are very safe;
  • Ferumoxytol iron oxide nanoparticles have been approved by the US FDA for the clinical treatment of adult iron deficiency anemia (IDA).
  • the present invention provides a kind of iron oxide nanoparticles (IONPs), the physicochemical characteristics of the IONPs are nanoparticle structure, the particle size range is 1-1000nm, polydispersity index ⁇ 0.3, zeta- The potential range is -50 to 50mV, and the UV-Vis absorption spectrum peak is 100-500nm.
  • IONPs iron oxide nanoparticles
  • the particle diameter is 1-1000nm; 5-200nm, 10-50nm, 10-100nm, 10-200nm, 10-20nm, 20-30nm, 30-40nm, 40-50nm, 50-60nm, 60-70nm, 70-80nm, 80-90nm, 90- 100nm, 100-110nm, 110-120nm, 120-130nm, 130-140nm, 140-150nm, 150-200nm, 200-250nm, 250-300nm, 350-400nm, 450-500nm, 550-600nm, 650-700nm, 750-800nm, 850-900nm, 900-950nm, 950-1000nm.
  • it is 1 to 200 nm. More preferably, it is 1 to 100 nm. More preferably, it is 1-30nm. Even more preferably, it is 10-30nm. Even more preferably, it is 10-20nm. More preferably, it is 10 nm.
  • the zeta-potential range is -50 to 50mV; it can be -50 to 40mV, -40 to -30mV, -30 to -20mV, -20 to -10mV, -20 to 20mV, -10 to 0mV, 0 to 10mV, 10 to 20mV, 20 to 30mV, 30 to 40mV, 40 to 50mV; preferably, -35 to 20mV; more preferably, -35 to 0mV; more preferably , is -30.57mV.
  • the peak value of the ultraviolet-visible absorption spectrum is 100-500nm; 200-300nm,220-240nm,240-260nm,260-280nm,280-300nm,300-320nm,320-340nm,340-360nm,360-380nm,380-400nm,400-420nm,420-440nm,440- 460nm, 460-480nm, 480-500nm.
  • it is 180-300nm. More preferably, it is 200-260nm. Even more preferably, it is 200-220nm.
  • the IONPs can also have a specific polydispersity index, preferably, the polydispersity index ⁇ 0.3; it can be 0.01, 0.02 , 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28. More preferably, it is 0.01-0.10. More preferably, it is 0.01-0.05. More preferably, it is 0.01-0.03. Still more preferably, it is 0.01, 0.02 or 0.03. More preferably, it is 0.03.
  • the present invention also provides the application of the iron oxide nanoparticles (IONPs) in the preparation of parathyroid negative contrast agent.
  • the present invention also provides the application of the iron oxide nanoparticles (IONPs) in the preparation of a lymph node positive contrast agent.
  • IONPs iron oxide nanoparticles
  • the present invention also provides a developer, the negative developer comprising the above-mentioned iron oxide nanoparticles (IONPs).
  • the contrast agent can be used as a positive contrast agent for parathyroid glands, a negative contrast agent for parathyroid glands, or a positive contrast agent for lymph nodes.
  • the IONPs of the present invention can be preliminarily screened for their efficacy in diagnosis through in vivo or in vitro tests, and other methods are also obvious to those skilled in the art.
  • the present invention can adopt any iron oxide nanoparticles (IONPs) satisfying the above properties, and can also be prepared by other methods.
  • the method includes a thermal decomposition method, and its general process is to heat iron oxide powder and oleic acid in octadecene at high temperature to form IONP, then mix IONP in the organic solvent with the aqueous phase solvent and reflux, and convert the IONP to the aqueous phase solvent , get IONPs.
  • the IONPs can be administered alone or in the form of a composition.
  • the composition may be prepared as a contrast agent or diagnostic tracer for diagnostic purposes, or the composition may be prepared as a pharmaceutical composition for therapeutic purposes.
  • the composition may further include one or more pharmaceutically acceptable carriers or media.
  • the carrier and medium must be compatible with the IONPs.
  • the acceptable carrier, medium such as sterile water or physiological saline, stabilizer, excipient, antioxidant (ascorbic acid, etc.), buffer (phosphoric acid, citric acid, other organic acids, etc.), preservative, surface Active agents (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders, etc.
  • polypeptides such as serum albumin, gelatin or immunoglobulin; amino acids such as glycine, glutamine, asparagine, arginine and lysine; carbohydrates such as polysaccharides and monosaccharides or Carbohydrates; sugar alcohols such as mannitol or sorbitol.
  • aqueous solution for injection such as physiological saline
  • isotonic solution containing glucose or other auxiliary drugs such as D-sorbitol, D-mannose, D-mannitol, sodium chloride
  • appropriate Solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (Tween 80, HCO-50) and the like.
  • the present invention further provides the use of said composition in the diagnosis of thyroid and/or lymph node related diseases or conditions, or for thyroid or lymph node tissue imaging or intraoperative navigation.
  • the diagnostic tracer composition can also be prepared as a detection kit.
  • the IONPs are prepared together with the drug in the form of a pharmaceutical composition for tracking the targeting and therapeutic effect of the drug.
  • the content of IONPs is usually a safe and effective amount, and the safe and effective amount should be adjustable for those skilled in the art, for example, the administration amount of the IONPs usually depends on the patient's body weight, application type of disease, condition and severity of the disease, for example, the administration amount as IONPs can usually be 0.1-1 mg/kg/time.
  • composition provided by the present invention can be applied to any form of administration, which can be oral or parenteral administration, for example, can be through the lungs , nasally, rectally and/or intravenously, more specifically can be intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or Parenteral administration; injection administration includes intravenous injection, intramuscular injection, subcutaneous injection, etc., transdermal administration, etc.
  • the dosage form of the composition is selected from: injections, sterile powders for injections, tablets, pills, capsules, lozenges, retorts, powders, granules, syrups, solutions, tinctures, aerosols , powder spray, or suppository.
  • the preparation form suitable for oral administration can include but not limited to pills, tablets, chewables, capsules, granules, solutions, drops, etc. formulations, syrups, aerosols or powder sprays, etc.
  • formulations suitable for parenteral administration may include but not limited to solutions, suspensions, reconstituted dry formulations or sprays, etc.
  • those suitable for rectal administration can usually be suppositories
  • those suitable for injection administration can be injections, sterile powders for injection, and the like.
  • the meaning of "diagnosis” includes but not limited to the diagnosis or detection of thyroid disease, or intraoperative real-time imaging through imaging. Surgical navigation or observation of drug targeting, etc.
  • the IONPs of the present invention can be used in various diagnostic imaging methods of the thyroid, including ultrasound, magnetic resonance and x-ray imaging. Use them for ultrasound diagnostic imaging and magnetic resonance imaging.
  • additives such as emulsifiers, coating agents, plasticizers, fillers, cryoprotectants and/or antioxidants can also be added to the developer or diagnostic tracer of the present invention, for example to improve its stability, Dispersion, aggregation tendency, biological properties, etc., or improve the flexibility and/or polarity of the film.
  • the present invention also provides a method for negative parathyroid development, the method comprising: administering the IONPs of the present invention to an individual.
  • the present invention also provides a method of visualizing lymph nodes, the method comprising: administering the IONPs of the present invention to an individual.
  • parathyroid negative imaging in the present invention is that the parathyroid glands are small, have large anatomical variation, are located on the dorsal side of the thyroid gland, and are closely related to the thyroid gland.
  • IONPs By injecting IONPs through the thyroid region, the parathyroid glands close to the thyroid gland can be negatively imaged.
  • lymph nodes at the lower pole of the thyroid can be black-stained and developed, thereby negatively developing the parathyroid glands at the lower pole.
  • lymph node visualization After local injection (lower lip) IONPs, IONPs are transported to the lymph nodes in the submandibular region through the anterior cervical lymphatic vessels. lymph nodes.
  • the present invention further provides a method for diagnosing the thyroid gland and/or lymph nodes using the IONPs described above, comprising administering the IONPs or the composition described herein, the IONPs or the composition being used as a tissue imaging agent, and after administering the IONPs or Images of the mammal were acquired after composition.
  • the method further comprises evaluating the signal level after administration of a diagnostic tracer or imaging agent (comprising IONPs, compositions described herein) with the signal level of a control.
  • the present invention also provides a method for preventing and/or treating diseases or conditions related to thyroid and/or lymph nodes, comprising administering to mammals an effective amount of IONPs or compositions described herein, and may also include administering said IONPs or combinations Images of mammals were acquired after the experiment.
  • the methods can also be in vitro or non-therapeutic.
  • the present invention also provides a method for screening effective drugs for thyroid and/or lymph node treatment.
  • IONPs and drug molecules on the same carrier, such as microspheres, IONPs can positively develop the thyroid gland, negatively develop the parathyroid gland, and/or positively develop the lymph nodes, which can be used for drug molecules. Evaluate the targeting of thyroid and/or lymph node diseases, and screen effective drugs for thyroid and/or lymph node treatment.
  • the method of the invention has excellent operability, good accuracy and the like.
  • iron oxide nanoparticles As used herein represent the same meaning.
  • treatment and prevention are to be understood in their broadest sense.
  • the term “treatment” does not necessarily imply that the mammal is treated until fully recovered.
  • prevention does not necessarily mean that the subject will not eventually contract the disease condition.
  • treatment and prevention include alleviation of the symptoms of a particular condition or preventing or reducing the risk of a particular condition developing.
  • prevention is understood as reducing the severity of the onset of a particular condition. Treatment may also reduce the severity of pre-existing conditions or the frequency of exacerbations.
  • the subject or individual for therapeutic or preventive treatment is preferably a mammal, such as but not limited to humans, primates, livestock (such as sheep, cows, horses, donkeys, pigs), pets (such as dogs, cats) , laboratory test animals (such as mice, rabbits, rats, guinea pigs, hamsters) or captured wild animals (such as foxes, deer).
  • the subject is preferably a primate.
  • the subject is most preferably a human.
  • the iron oxide nanoparticles (IONPs) of the present invention and its preparation and application have the following beneficial effects:
  • iron oxide nanoparticles (IONPs) of the present invention can accurately negatively develop parathyroid glands and trace lymph nodes; compared with positive control carbon nanoparticles, IONPs of the present invention are used as a developer, which is not easy to ooze out and does not pollute The surgical field, the development is clearer.
  • the iron oxide nanoparticles of the present invention are degradable, highly safe, metabolizable by the human body, do not affect normal cells, and do not affect secondary operations; they can be prepared as reagents for intravenous injection.
  • Adopt iron oxide nanoparticles of the present invention to reduce complications such as numbness of hands and feet, general convulsions, epilepsy, neurasthenia, spasm of diaphragm, and permanent hypoparathyroidism caused by surgery.
  • the iron oxide nanoparticles (IONPs) of the present invention help solve unresolved problems that have been eager to be solved, and have broad application prospects and clinical transformation value.
  • the preparation method of the iron oxide nanoparticles (IONPs) of the present invention is simple in operation, low in price, uniform in product quality and good in repeatability, and is suitable for mass production.
  • Figure 1 is a scene diagram of the clinical application of carbon nanoparticles in thyroid cancer surgery, in which the histogram shows the gray scale comparison before and after injection of nano carbon in human thyroid cancer surgery.
  • Fig. 2 is the ultraviolet-visible absorption spectrum figure of iron oxide nanoparticle; Wherein, Fig. 2A is the ultraviolet absorption spectrum of 10nm iron oxide nanoparticle, Fig. 2B is the ultraviolet absorption spectrum of 50nm iron oxide nanoparticle, and Fig. 2C is 100nm iron oxide nanoparticle
  • FIG. 2D is the ultraviolet absorption spectrum of 150nm carbon nanoparticles (nanocarbon, positive reference). The abscissa is the wavelength, and the ordinate is the absorbance value.
  • FIG. 3 Pathological diagram of parathyroid glands of SD rats stained with HE; A is the laryngotracheal diagram of the parathyroid gland cross-section, B is a partially enlarged diagram of the thyroid and parathyroid glands, and the principal cells and phagocytosis of the rat parathyroid glands can be seen under the microscope acid cells.
  • FIG. 4 Gross anatomy of the thyroid gland and parathyroid glands in rats, A: The thyroid gland and parathyroid glands were judged by the naked eye, which were transparent; B: The parathyroid glands were negatively developed after the injection of iron oxide nanoparticles, and the thyroid gland was stained black. There is no black staining of the parathyroid glands, which can be clearly judged by the naked eye.
  • FIG. 5 Experiment of observing iron oxide nanoparticles and carbon nanoparticles of different sizes in rats.
  • A, B, C, D, E represent carbon nanoparticles, 10nm, 50nm, 100nm iron oxide nanoparticles and 0.9% saline group respectively.
  • Figure 6 is a histogram of quantitative analysis of the gray scale change rate of iron oxide nanoparticles and carbon nanoparticles of different sizes before and after injection in the thyroid region.
  • the carbon nanoparticle group was 44.02%
  • the 10nm IONPs group was 43.32%
  • the 50nm group was 21.25%
  • the 100nm group was 4.28%
  • the normal saline group was 4.05%.
  • Figure 7 The experiment of comparing iron oxide nanoparticles and carbon nanoparticles in rats; A, B, C, and D represent four SD rats, A: the right thyroid and subcutaneous injection of iron oxide nanoparticles, and the left injection of carbon nanoparticles Materials; B: left thyroid and subcutaneous injection of iron oxide nanoparticles, right injection of carbon nanomaterials; C: bilateral thyroid and subcutaneous injection of iron oxide nanoparticles; Group D: bilateral thyroid and subcutaneous injection of carbon nanomaterials.
  • Figure 8 The in vitro experiment and esophageal contamination of the injection of iron oxide nanoparticles and carbon nanoparticles in rats, in which, picture A shows the injection of carbon nanoparticles and iron oxide nanoparticles into the left and right thyroid glands respectively, and it can be seen that both thyroid glands are uniform color. ; The left picture of Figure B shows that after 2.5 hours, the carbon nanoparticle group contaminates the esophagus, and the right picture of Figure B shows that after 2.5 hours, the iron oxide nanoparticles do not pollute the esophagus, and there is no black staining in the adjacent esophagus, indicated by circles.
  • FIG. 9 Anatomical diagram of the thyroid and parathyroid glands of New Zealand rabbits and a comparison diagram of the in vivo injection of iron oxide nanoparticles and carbon nanoparticles
  • A Anatomical diagram of the thyroid and parathyroid glands of New Zealand rabbits, the thyroid glands are located on both sides of the trachea, and the parathyroid glands are located Not constant, scattered on the medial surface of the anterior cervical muscle
  • B carbon nanoparticles were injected into the right thyroid, and iron oxide nanoparticles were injected into the left thyroid.
  • FIG. 10 In vitro comparison of New Zealand rabbit thyroid injected with iron oxide nanoparticles and carbon nanoparticles, A: Bilateral thyroid glands were exchanged and injected with carbon nanoparticles and iron oxide nanoparticles, and the developing effect was equivalent. B: After 2.5 hours, the normal saline is wet. It is observed that the leakage of the carbon nanoparticle side is more obvious than that of the iron oxide side, and the phenomenon of contamination of the surgical area is more likely to occur, marked with +.
  • Fig. 11 is a graph showing the results of developing lymph nodes with 10nm iron oxide nanoparticles.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology.
  • Carbon nanoparticles particle size, 150nm; manufacturer, Chongqing Laimei Pharmaceutical Co., Ltd. (batch number: Z200603; production date: 2020-06-12); black suspension liquid.
  • Iron oxide nanoparticles particle size, 10, 50, 100nm; magnetic nanoparticle solution.
  • iron oxide nanoparticles prepared by other methods can also be used, such as prepared by thermal decomposition: heat iron oxide powder and oleic acid in octadecene at high temperature to form IONP, and then mix IONP in the organic solvent with the aqueous phase solvent and reflux , transforming IONP into an aqueous solvent to obtain iron oxide nanoparticles.
  • Nano particle size and potential analyzer Zetasizer Nano ZS (Malvern, ZEN3600);
  • UV/Vis/NIR Spectrophotometer PerkinElmer, LAMBDA1050+
  • Morphological structure polydispersity index (dispersion or polydispersity index (PDI)), particle size, Zeta potential (surface charge (Dynamic Light Scattering, DLS)), ultraviolet-visible absorption spectrum (Ultraviolet–Visible Spectroscopy, UV-Vis) .
  • Embodiment 1 iron oxide nanoparticle, carbon nanoparticle characterization
  • the particle size and Zeta potential were measured by Zetasizer Nano ZS (Malvern, ZEN3600), a nanoparticle size and potential analyzer: the suspension containing 10nm IONPs was diluted with normal saline to a concentration of IONPs of 50ug/mL, and then the IONPs sample was directly placed in the sample cell Determination in middle; The measurement result is -30.57 ⁇ 0.59mV.
  • Ultraviolet-visible absorption spectrum was measured by ultraviolet-visible absorption spectrometer: the suspension containing IONPs was diluted with physiological saline to a concentration of (IONPs) of 10 ⁇ g/mL, and then the IONPs sample was directly put into the sample cell for measurement.
  • Figure 2A is the UV-visible absorption spectrum of 10nm IONPs, with a peak of 200-220nm, which is close to the peak of the UV-visible absorption spectrum of carbon nanoparticles
  • Figure 2B is the UV-visible absorption spectrum of 50nm IONPs Absorption spectrum
  • Figure 2C is the UV-Vis absorption spectrum of 100nm IONPs.
  • the morphology, structure and polydispersity index were measured by transmission electron microscopy: IONPs with a concentration of 50 ⁇ g/mL was dropped onto the copper grid and kept for 30 minutes, then the remaining liquid was blotted dry, and the copper grid was completely dried naturally, and then put into the electron microscope for observation.
  • the measurement results are: 10nm, 50nm, and 100nm IONPs are all spherical, and the electron microscope size (Fe 3 O 4 core diameter) of 10nm IONPs is about 7nm.
  • the measurement method of carbon nanoparticles is the same as above.
  • the Zeta potential of carbon nanoparticles is -4.44 ⁇ 0.25;
  • Figure 2D is the ultraviolet absorption spectrum curve;
  • the particle size is 150nm;
  • the morphology is spherical;
  • the hydration diameter is 171.50 ⁇ 0.85;
  • the polydispersity index (PDI) is 0.135.
  • Embodiment 2 carry out pathological HE staining to the isolated thyroid gland and parathyroid gland of SD rat
  • Figure 3A is a view of the larynx and trachea in a cross-section through the parathyroid glands. It can be seen that the parathyroid glands are close to the thyroid gland and located outside and above the thyroid gland.
  • Figure 3B is a partially enlarged view of the thyroid and parathyroid glands. The characteristic principal cells and eosinophils (gold standard) of the parathyroid glands can be seen under the microscope.
  • Embodiment 3 SD rat thyroid and parathyroid anatomy and negative imaging
  • Experimental groups A, B, and C corresponding to IONPs with three different particle sizes of 10nm, 50nm, and 100nm respectively;
  • Figure 4A is an anatomical diagram of the rat thyroid and parathyroid glands, where the thyroid and parathyroid glands are judged by the naked eye as indicated by the arrows, and the parathyroid glands are usually transparent, and the parathyroid glands are not obvious. Judgment is more difficult.
  • the SD rats (marked as M) were photographed on the side of the thyroid gland, and the results are shown in Figure 4B. It can be seen that the thyroid gland appears black, and the parathyroid glands have no black staining. The parathyroid glands can be clearly judged by the naked eye, and the effect is good.
  • the determination method of the gray level before and after clinical nanocarbon injection is applied, as follows: take pictures during the operation after the operation cavity is exposed, and compare the gray level changes of the thyroid gland before and after injection; Above, make an adjacent circle of equal size around the border of the parathyroid gland, and the software calculates the gray value before and after the injection. The diameter of the circle taken each time is the same, and the change rate of the gray value before and after the thyroid injection is obtained ( The gray value before injection minus the gray value after injection is divided by the gray value before injection), and the results are shown in Figure 6. It can be seen from Figure 6 that the 10nm IONPs (44.02%) are close to the carbon nanoparticles (43.32%), and the effect is the best. Therefore, 10nm IONPs with the best developing effect were used for subsequent experiments.
  • Embodiment 4 SD rat thyroid and parathyroid anatomy and negative imaging
  • A The right thyroid and subcutaneous injection of 10nm IONPs, the left Injection of carbon nanoparticles
  • B left thyroid and subcutaneous injection of 10nm IONPs, right injection of carbon nanoparticles
  • C bilateral thyroid and subcutaneous injection of 10nm IONPs
  • D bilateral thyroid and subcutaneous injection of carbon nanoparticles.
  • Figures 7A-D include 7 pictures (marked as A1-A7, B1-B7, C1-C7, D1-D7 respectively) from left to right, among which, the first, second, Image 3 is the blank front view of the rat before injection (X1, X3, X5 times); images 4, 5, and 6 are the images after injection of nanomaterials (X1, X3, X5 times).
  • A7, B7, C7, and D7 were incisions made in the middle of the front of the neck, and the left and right sides were injected according to the nanoparticle injection method of the group to illustrate the comparative degradation of 10nm IONPs and carbon nanoparticles in the skin and subcutaneous tissue.
  • A7 is the subcutaneous injection of 10nm IONPs and carbon nanoparticles on the left and right sides of the anterior median incision
  • B7 is the subcutaneous injection of carbon nanoparticles and 10nm IONPs on the left and right sides of the anterior median incision
  • C7 is the subcutaneous injection of 10nm IONPs on the left and right sides of the anterior median incision
  • D7 is the subcutaneous injection of carbon nanoparticles on the left and right sides of the anterior median incision.
  • Embodiment 5 the infiltration situation of SD rat isolated thyroid after injecting nanomaterials
  • the esophagus on the side injected with carbon nanoparticles was stained black (the range shown in the circle), while the esophagus on the side injected with 10nm IONPs was not polluted and white in color. (The range shown in the circle), without obvious penetration into the parathyroid gland area, the developing effect is better.
  • Example 6 New Zealand rabbit thyroid and parathyroid anatomy and negative imaging
  • the lymph nodes were selected to be traced, and the parathyroid glands were negatively developed.
  • Figure 9A shows the anatomical diagram of the thyroid gland and parathyroid glands of New Zealand rabbits. It can be seen that the thyroid gland is located on both sides of the trachea, and the parathyroid glands are not constant, scattered on the medial surface of the anterior neck muscle.
  • Example 7 the infiltration situation of the isolated New Zealand rabbit thyroid after injection of nanomaterials
  • the extravasation of the carbon nanoparticle injection side is obvious, and the surgical area is polluted, the color is black, and the "+" is marked in the figure; the IONPs injection side has a clear border and no pollution extravasation Phenomena (wherein, the thyroid gland on the left side of Fig. 10A corresponds to the thyroid gland above Fig. 10B, and the thyroid gland on the right side of Fig. 10A corresponds to the thyroid gland below Fig. 10B).
  • lymph node visualization After local injection (lower lip) of 10nm IONPs, IONPs are transported to the submandibular lymph nodes through the anterior cervical lymphatic vessels. The submandibular lymph nodes of rats are relatively superficial, and black-stained lymph nodes can be observed by cutting the skin .
  • lymphatic vessels draining from the lower lip to the lymph nodes are slender, dark brown, and accompany the blood vessels;
  • the normal lymph nodes were darker, appearing dark brown; the normal lymph nodes of the control group without injection were white.
  • IONPs can be used to visualize lymph nodes, and can be used as a tracer of lymph nodes or a delivery vehicle for delivering related drugs to lymph nodes.

Abstract

一种氧化铁纳米粒子IONPs在制备甲状旁腺或***显影中的应用，IONPs的粒径为1-1000nm，多分散指数<0.3，zeta-potential为-50至50mV，紫外-可见吸收光谱峰值为100-500nm。该IONPs可以准确地负显影甲状旁腺，示踪***。

Description

氧化铁纳米粒子在甲状旁腺和/或***显影剂中的应用 技术领域
本发明属于手术导航影像学技术领域，涉及甲状旁腺负显影剂及其制备和应用，特别是涉及一种氧化铁纳米粒子及其制备和在甲状旁腺负显影剂中的应用。
背景技术
2021年GLOBOCAN报道全球甲状腺癌在所有癌症发病率中排第九，女性癌症发病率第五。柳叶刀子刊统计全球25个国家的数据显影，中国所有主要甲状腺癌亚型发病率都显著增加；与此同时，低风险甲状腺***状癌在全球包括中国在内快速增加。
甲状腺癌的治疗主要依靠手术，随着发病率迅速增加，手术量随之增加显著。甲状旁腺虽然微小，但其损伤后所引起近期和远期的并发症却是极其严重的。如果术中可以更好的显影甲状旁腺，对患者是极其重要的。甲状腺的手术中，由于甲状旁腺自身的解剖特点和与甲状腺之间的紧密关系，甲状腺手术量增加的同时，甲状旁腺功能受损的基数增加；由于甲状旁腺从胚胎发育的过程中来源于不同的咽囊，存在诸多的解剖变异，尤其是下位旁腺，可以异位至下颌角、前纵膈、甚至心包，还有包埋在胸腺内或甲状腺内。甲状旁腺原位保留仍是极具挑战性的难题。因此，如何更好地术中保护甲状旁腺是亟需解决的临床问题之一。迄今为止，这仍是一个尚未被满足的临床重要需求问题。
目前临床甲状旁腺术中显影存在的局限性：
当前术中甲状旁腺的显影，主要包括正显影和负显影两方面。正显影剂包括亚甲蓝、氨基乙酰丙酸、吲哚菁绿等。但正显影剂，缺乏甲状旁腺的组织特异性，通常正常甲状旁腺组织染色率低，效果不佳，并且存在副反应：比如心律紊乱、神经毒性、碘过敏等。
负显影主要是碳纳米材料，其一种黑色碳纳米颗粒。该显影剂的功能是通过对正常甲状腺体进行染黑，来负显影甲状旁腺。目前在临床上已应用，但其在非肿瘤类手术中应用受到限制，二次手术应用中更加困难。另外，碳纳米材料存在重要的潜在安全问题，对人体会造成危害：碳纳米材料由于无法在人体内正常降解，在细胞复制过程中会损伤DNA，有可能诱导肿瘤发生，其人体安全性一直存在争议。碳纳米材料通过甲状腺区的***渗透至侧颈部***，常规的甲状腺微小癌手术时侧颈部***不做清扫，碳纳米材料将长期残留于该区域***。或因腔镜甲状腺手术时，皮肤注射的针眼而致皮肤长期残留。
近年，多位硕博士毕业论文探究碳纳米材料的毒理研究。2020年，程文婷硕士论文中报道长期暴露炭黑导致的外周血淋巴细胞基因组不稳定性。2019年，山东大学张瑞博士毕业论文《分子、细胞和动物水平上超细炭黑诱发氧化应激和基因毒性》中详细的探讨了纳米炭黑 在分子、细胞和动物层面上的毒性影响。发现炭黑可诱导小鼠原代肝细胞凋亡，能进入小鼠原代肝细胞，抑制细胞活力，并诱发毒性效应。炭黑也能使斑马鱼视觉细胞的感光能力下降，对斑马鱼的神经***的胶质细胞造成毒性，导致斑马鱼的运动行为发生紊乱。2018年，赵丹丹硕士论文阐述了纳米炭黑颗粒复合冷刺激对小鼠呼吸***及机体能量代谢的影响。2017年，周丽晓硕士论文中表明纳米炭黑对人支气管上皮细胞有毒性作用。此外，碳纳米材料中的碳纳米材料管、石墨烯等材料，同样报道具有生殖毒性、引起血管内皮功能障碍和上皮细胞损伤、影响人的胸膜间皮细胞等。因此，寻找无毒、无害、生物体内可降解的甲状旁腺显影剂成为临床的迫切需要。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的是提供氧化铁纳米粒子(Iron oxide nanoparticles,IONPs)及其制备和在负显影甲状旁腺中的应用。所述IONPs在术中能够帮助更好的保留甲状旁腺，维持甲状旁腺的功能。氧化铁纳米粒子已经被证实可以通过人体正常细胞的溶酶体降解代谢为铁离子，与铁蛋白结合后继而进入人体正常的铁代谢中，完全被人体吸收降解，因此具有很高的安全性；如Ferumoxytol氧化铁纳米粒子已经被美国FDA批准用于临床治疗成人缺铁性贫血(IDA)。
为实现上述目的及其他相关目的，本发明提供一种氧化铁纳米粒子(IONPs)，所述IONPs的物理化学特性为纳米粒子结构，粒径范围为1-1000nm，多分散指数<0.3，zeta-potential范围为-50至50mV，紫外-可见吸收光谱峰值为100-500nm。
本发明所述氧化铁纳米粒子(IONPs)中，粒径为1-1000nm；可以为1-5nm、1-10nm、1-50nm、1-100nm、1-200nm、5-50nm、5-100nm、5-200nm、10-50nm、10-100nm、10-200nm、10-20nm、20-30nm、30-40nm、40-50nm、50-60nm、60-70nm、70-80nm、80-90nm、90-100nm、100-110nm、110-120nm、120-130nm、130-140nm、140-150nm、150-200nm、200-250nm、250-300nm、350-400nm、450-500nm、550-600nm、650-700nm、750-800nm、850-900nm、900-950nm、950-1000nm。优选地，为1～200nm。进一步优选地，为1～100nm。更进一步优选地，为1-30nm。更进一步优选地，为10-30nm。更进一步优选地，为10-20nm。更进一步优选地，为10nm。
本发明所述氧化铁纳米粒子(IONPs)中，zeta-potential范围为-50至50mV；可以为-50至40mV、-40至-30mV、-30至-20mV、-20至-10mV、-20至20mV、-10至0mV、0至10mV、10至20mV、20至30mV、30至40mV、40至50mV；优选地，为-35至20mV；进一步优选地，为-35至0mV；进一步优选地，为-30.57mV。
本发明所述氧化铁纳米粒子(IONPs)中，紫外-可见吸收光谱峰值为100-500nm；可以为100-120nm、120-140nm、140-160nm、160-180nm、180-200nm、200-220nm、200-300nm、220-240nm、240-260nm、260-280nm、280-300nm、300-320nm、320-340nm、340-360nm、360-380nm、380-400nm、400-420nm、420-440nm、440-460nm、460-480nm、480-500nm。优选地，为180-300nm。进一步优选地，为200-260nm。更进一步优选地，为200-220nm。
进一步地，为实现本发明所述氧化铁纳米粒子(IONPs)在显影中的优异性能，所述IONPs还可以具有特定的多分散指数，较优地，多分散指数<0.3；可以为0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.12、0.14、0.16、0.18、0.20、0.22、0.24、0.26、0.28。进一步优选地，为0.01-0.10。进一步优选地，为0.01-0.05。进一步优选地，为0.01-0.03。更进一步优选地，为0.01、0.02或0.03。更进一步优选地，为0.03。
本发明还提供了所述氧化铁纳米粒子(IONPs)在制备甲状旁腺负显影剂中的应用。
本发明还提供了所述氧化铁纳米粒子(IONPs)在制备***正显影剂中的应用。
本发明还提供了一种显影剂，所述负显影剂包含上文所述的氧化铁纳米粒子(IONPs)。所述显影剂可以作为甲状旁正显影剂，甲状旁腺负显影剂，或***的正显影剂。
本发明所述IONPs可通过体内或体外试验初步筛选其在诊断中的功效，其它方法对于本领域技术人员也是显而易见的。
本发明可以采用满足上述性质的任何氧化铁纳米粒子(IONPs)，也可以通过其他方法制备获得。所述方法包括热分解法，其一般过程是将氧化铁粉末和油酸在十八烯中高温加热形成IONP，然后将有机溶剂中的IONP与水相溶剂混合回流，将IONP转化到水相溶剂中，得到IONPs。
为了实践本文所公开的应用，所述IONPs可以单独施用或通过制备成组合物的形式进行施用。
所述组合物可以制备成用于诊断目的的显影剂或诊断示踪剂，或所述组合物可以被制备成用于治疗目的的药物组合物。所述组合物除包含上述IONPs之外，还可以进一步包括一种或多种药学上可接受的载体或介质等。所述载体、介质必须与该IONPs相容。所述可接受的载体、介质例如无菌水或生理盐水、稳定剂、赋形剂、抗氧化剂(抗坏血酸等)、缓冲剂(磷酸、枸橼酸、其它的有机酸等)、防腐剂、表面活性剂(PEG、Tween等)、螯合剂(EDTA等)、粘合剂等。而且，也可含有其它低分子量的多肽；血清白蛋白、明胶或免疫球蛋白等蛋白质；甘氨酸、谷酰胺、天冬酰胺、精氨酸和赖氨酸等氨基酸；多糖和单糖等糖类或碳水化物；甘露糖醇或山梨糖醇等糖醇。当制备用于注射的水溶液时，例如生理盐水、含有葡萄糖或其它 的辅助药物的等渗溶液，如D-山梨糖醇、D-甘露糖、D-甘露糖醇、氯化钠，可并用适当的增溶剂例如醇(乙醇等)、多元醇(丙二醇，PEG等)、非离子表面活性剂(吐温80，HCO-50)等。
本发明进一步提供了所述组合物在诊断甲状腺和/或***相关的疾病或病症中的应用，或用于甲状腺或***组织成像或术中导航。
在一实施方式中，所述诊断示踪剂组合物还可以被制备成检测试剂盒。
在一实施方式中，所述IONPs与药物一起制备成药物组合物的形式，用于跟踪药物的靶向性和治疗效果。
本发明药物组合物中，IONPs的含量通常为安全有效量，所述安全有效量对于本领域技术人员来说应该是可以调整的，例如，所述IONPs的施用量通常依赖于患者的体重、应用的类型、疾病的病情和严重程度，例如，作为IONPs的施用量通常可以为0.1-1mg/kg/次。
本发明所提供的组合物(即包含显影剂或诊断示踪剂、或药物组合物等)可以适用于任何形式的给药方式，可以是口服或胃肠外给药，例如，可以是经肺、经鼻、经直肠和/或静脉注射，更具体可以是真皮内、皮下、肌内、关节内、腹膜内、肺部、口腔、舌下含服、经鼻、经皮、***、口服或胃肠外给药；注射给药包括静脉注射、肌内注射和皮下注射等，经皮给药等。
如本文所用，所述组合物的剂型选自：注射剂、注射用无菌粉末、片剂、丸剂、胶囊、锭剂、醑剂、散剂、颗粒剂、糖浆剂、溶液剂、酊剂、气雾剂、粉雾剂、或栓剂。本领域技术人员可根据给药方式，选择合适的制剂形式，例如，适合于口服给药的制剂形式可以是包括但不限于丸剂、片剂、咀嚼剂、胶囊剂、颗粒剂、溶液剂、滴剂、糖浆、气雾剂或粉雾剂等，再例如，适合于胃肠外给药的制剂形式可以是包括但不限于溶液、悬浮液、可复水的干制剂或喷雾剂等，再例如，适合于直肠给药的通常可以是栓剂，再例如，适合注射给药的可以是注射剂、注射用无菌粉末等。
进一步地，所述显影剂或诊断示踪剂在用于诊断时，所述“诊断”的含义包括但不限于用于对是否患有甲状腺疾病进行的诊断或检测，或通过成像进行术中实时手术导航或观察药物的靶向性等。
本发明的IONPs可以用于甲状腺各种诊断显影方法，包括超声、磁共振和x-射线成像。将它们用于超声诊断成象和磁共振成像。
本发明的显影剂或诊断示踪剂中还可以加入各种添加剂，如乳化剂、涂层剂、增塑剂、填充剂、低温防护剂和/或抗氧剂，例如以改进其稳定性、分散性、聚集倾向、生物性能等， 或改进膜的柔韧性和/或极性。
本发明还提供了一种对甲状旁腺负显影的方法，所述方法包括：向个体中施用本发明所述的IONPs。
本发明还提供了一种对***显影的方法，所述方法包括：向个体中施用本发明所述的IONPs。
本发明甲状旁腺负显影的原理：甲状旁腺微小、解剖变异大，位于甲状腺背侧，与甲状腺关系密切，通过甲状腺区注射IONPs，可负显影紧贴甲状腺的甲状旁腺。结合***正显影的功能，可以黑染显影甲状腺下极处的***，从而负显影下极的甲状旁腺。
本发明***显影的原理：局部注射(下唇)IONPs后，通过颈前***，将IONPs输送至颌下区***，大鼠颌下***较表浅，切开皮肤即可观察到黑染的***。
本发明进一步提供了采用上文所述IONPs对甲状腺和/或***进行诊断的方法，包括给予本文所述的IONPs或组合物，所述IONPs或组合物用作组织成像剂，并且在给予IONPs或组合物之后获取哺乳动物的图像。在一些实施方式中，所述方法还包括用对照的信号水平评价给予诊断示踪剂或成像剂(包含本文所述的IONPs、组合物)之后的信号水平。
本发明还提供了预防和/或治疗与甲状腺和/或***相关的疾病或病症的方法，包括向哺乳动物给予有效量的本文所述的IONPs或组合物，还可以包含给予所述IONPs或组合物之后获取哺乳动物的图像。所述方法也可以是体外的或非治疗性的。
本发明还提供了筛选对甲状腺和/或***治疗有效的药物的方法。通过将IONPs与药物分子构建到同种载体，如微球上，通过IONPs对甲状腺的正显影、对甲状旁腺的负显影、和/或对***的正显影效果，可以用于对药物分子对甲状腺和/或***疾病的靶向性进行评价，筛选对甲状腺和/或***治疗有效的药物。本发明的方法具有优异的可操作性和良好的准确率等。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
本文述及“包含”、“含有”等应理解为是包括性的意思，而没有排他性或穷尽的意思；即“包括但不限于”的意思。
本文所述“氧化铁纳米粒子”、“氧化铁纳米颗粒”、“纳米氧化铁”代表相同的含义。
本文所述“碳纳米粒子”、“碳纳米颗粒”、“纳米碳”代表相同的含义。
本文述及“治疗”和“预防”应理解为其最宽的意义。术语“治疗”不一定暗示哺乳动 物接受治疗直至完全恢复。类似地，“预防”不一定表示对象最终不会感染疾病病症。因此，治疗和预防包括缓解具体病症的症状或防止或降低具体病症产生的风险。术语“预防”可理解为降低具体病症发作的严重程度。治疗也可降低已有病症的严重程度或急性发作的频率。
本发明中，进行治疗性或预防性治疗的对象或个体优选哺乳动物，例如但不限于人、灵长类、牲畜(如绵羊、牛、马、驴、猪)、宠物(如狗、猫)、实验室试验动物(如小鼠、家兔、大鼠、豚鼠、仓鼠)或被捕获的野生动物(如狐狸、鹿)。所述对象优选灵长类。所述对象最优选人。
通过说明书、附图和权利要求书，不难了解其它的特征、目的和优点。
本发明氧化铁纳米粒子(IONPs)及其制备和应用，具有以下有益效果：
(1)采用本发明的氧化铁纳米粒子(IONPs)可以准确地负显影甲状旁腺，示踪***；相比阳性对照碳纳米粒子，本发明的IONPs用作显影剂，不易渗出，不污染术野，显影更清晰。
(2)本发明的氧化铁纳米粒子可降解、安全性高，人体可代谢，不影响正常细胞，不影响二次手术；可制备成如静脉注射用试剂。
(3)采用本发明的氧化铁纳米粒子(IONPs)，降低因为手术原因，引起手足麻木，全身抽搐，癫痫，神经衰弱，膈肌痉挛，永久性的甲状旁腺功能低下等并发症。
(4)本发明的氧化铁纳米粒子(IONPs)帮助解决一直渴望解决而未解决的难题，具有广阔应用前景和临床转化价值。
(5)本发明的氧化铁纳米粒子(IONPs)的制备方法操作简单，价格低廉，产品质量均一且重复性好，适合大批量生产。
附图说明
图1为临床上甲状腺癌手术应用碳纳米粒子的场景图，其中柱状图为人甲状腺癌术中注射纳米碳前后的灰度对比。
图2为氧化铁纳米粒子的紫外-可见吸收光谱图；其中，图2A为10nm氧化铁纳米粒子的紫外吸收光谱，图2B为50nm氧化铁纳米粒子的紫外吸收光谱，图2C为100nm氧化铁纳米粒子的紫外吸收光谱，图2D为150nm碳纳米粒子(纳米碳，正参比)的紫外吸收光谱。横坐标为波长，纵坐标为吸光度数值。
图3HE染色SD大鼠甲状旁腺的病理图；A为经过甲状旁腺横切面的喉气管图，B为局部放大甲状腺及甲状旁腺图，显微镜下可见大鼠甲状旁腺的主细胞和嗜酸细胞。
图4大鼠甲状腺及甲状旁腺大体解剖图，A：肉眼判断为甲状腺和甲状旁腺的部位，呈透明状；B：注射氧化铁纳米粒子后甲状旁腺负显影结果，甲状腺被黑染，甲状旁腺无黑染，通过肉眼可明显判断。
图5大鼠中观察不同尺寸大小的氧化铁纳米粒子与碳纳米粒子的实验。A、B、C、D、E分别代表碳纳米粒子、10nm、50nm、100nm的氧化铁纳米粒子和0.9％生理盐水组。每组6只大鼠的右侧甲状腺注射相应材料后的侧面图。
图6定量分析不同尺寸大小的氧化铁纳米粒子与碳纳米粒子在甲状腺区注射前后的灰度变化率的柱状图。碳纳米粒子组44.02％、10nm IONPs组43.32％、50nm组21.25％、100nm组4.28％、生理盐水组4.05％。
图7大鼠中对比观察氧化铁纳米粒子与碳纳米粒子的实验；A、B、C、D代表四只SD大鼠，A：右侧甲状腺及皮下注射氧化铁纳米粒子，左侧注射碳纳米材料；B：左侧甲状腺及皮下注射氧化铁纳米粒子，右侧注射碳纳米材料；C：双侧甲状腺及皮下注射氧化铁纳米粒子；D组：双侧甲状腺及皮下注射碳纳米材料。
图8大鼠中注射氧化铁纳米粒子和碳纳米粒子的离体实验及食管污染图，其中，A图为左、右两侧甲状腺分别注射碳纳米粒子和氧化铁纳米粒子，可见双侧甲状腺均显色。；B图左图为经过2.5小时后，碳纳米粒子组出现污染食管的现象，B图右图为经过2.5小时后，氧化铁纳米粒子不污染食管，邻近食管无黑染，圆圈表示。
图9新西兰兔甲状腺及甲状旁腺的解剖图和注射氧化铁纳米粒子和碳纳米粒子的活体对比图，A：新西兰兔甲状腺及甲状旁腺的解剖图，甲状腺位于气管两侧，甲状旁腺位置不恒定，散在颈前肌内侧面；B：右侧甲状腺注射碳纳米粒子，左侧甲状腺注射氧化铁纳米粒子。
图10新西兰兔甲状腺注射氧化铁纳米粒子和碳纳米粒子的离体对比图，A：双侧甲状腺分别交换注射碳纳米粒子和氧化铁纳米粒子，显影效果相当。B：经过2.5小时后，生理盐水湿润，观察碳纳米粒子侧渗漏较氧化铁侧明显，更易出现污染术区的现象，+号标示。
图11为10nm氧化铁纳米粒子显影***的结果图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
实验材料及方法
1、实验材料
碳纳米粒子：粒径，150nm；生产厂商，重庆莱美药业股份有限公司(批号：Z200603；生产日期：2020-06-12)；黑色混悬液体。
氧化铁纳米粒子(IONPs)：粒径，10，50，100nm；磁性纳米粒子溶液。
以下实施例中，采用的是商业购买的氧化铁纳米粒子。也可以采用其他方法制备的氧化铁纳米粒子，如按照热分解法制备获得：将氧化铁粉末和油酸在十八烯中高温加热形成IONP，然后将有机溶剂中的IONP与水相溶剂混合回流，将IONP转化到水相溶剂中，得到氧化铁纳米粒子。
2、实验仪器
纳米粒度及电位分析仪Zetasizer Nano ZS(Malvern,ZEN3600)；
紫外-可见吸收光度仪UV/Vis/NIR Spectrophotometer(PerkinElmer,LAMBDA1050+)；
透射电子显微镜(Transmission Electron Microscope，TEM)(JEOL,JEM-2100plus)。
3、纳米粒子表征指标
形貌结构，多分散指数(分散度或polydispersity index(PDI))，粒径，Zeta电位(表面电荷(Dynamic Light Scattering,DLS))，紫外-可见吸收光谱(Ultraviolet–Visible Spectroscopy，UV-Vis)。
实施例1、氧化铁纳米粒子、碳纳米粒子表征
1、对氧化铁纳米粒子(IONPs)进行表征
采用纳米粒度及电位分析仪Zetasizer Nano ZS(Malvern,ZEN3600)测定粒径和Zeta电位：将含有10nm IONPs的悬浮液用生理盐水稀释至IONPs浓度为50ug/mL，然后将IONPs 样品直接放入样品池中测定；测定结果为-30.57±0.59mV。
采用紫外-可见吸收光度仪测定紫外-可见吸收光谱：将含有IONPs的悬浮液用生理盐水稀释至(IONPs)浓度为10μg/mL，然后将IONPs样品直接放入样品池中测定。测定结果如图2所示，其中，图2A为10nm IONPs的紫外-可见吸收光谱图，峰值为200-220nm，与碳纳米粒子的紫外可见吸收光谱峰值接近；图2B为50nm IONPs的紫外-可见吸收光谱图，图2C为100nm IONPs的紫外-可见吸收光谱图。
采用透射电镜测定形貌结构、多分散指数：将浓度为50μg/mL IONPs滴一滴到铜网上并保持30分钟，然后将剩余液体吸干，铜网彻底自然干燥后，放入电镜中观察。测定结果为：10nm、50nm、100nm的IONPs均为球形，10nm IONPs的电镜尺寸(Fe 3O 4内核直径)约7nm。
碳纳米粒子的测定方法同上。
其中，碳纳米粒子的Zeta电位-4.44±0.25；图2D为紫外吸收光谱曲线；粒径为150nm；形貌结构为球形；水合直径171.50±0.85；多分散指数(PDI)为0.135。
实施例2、对SD大鼠离体的甲状腺及甲状旁腺进行病理HE染色
选择重量在240g-260g左右，6周龄的1只SD大鼠，按实施例1步骤1)-7)处理后，将喉、气管、甲状腺和甲状腺旁腺行手术切除，切除范围上至甲状软骨上缘，下至气管5环，采用苏木素(Hematoxylin)-伊红(Eosin)染色(简称HE染色)，显微镜下观察大鼠甲状旁腺的病理结果。
如图3所示，其中，图3A为经过甲状旁腺横切面的喉气管图，可见甲状旁腺紧贴甲状腺，位于甲状腺外上方。图3B为局部放大的甲状腺及甲状旁腺图，显微镜下可见甲状旁腺特征性的主细胞和嗜酸细胞(金标准)。
实施例3、SD大鼠甲状腺及甲状旁腺解剖及负显影
选择重量在240g-260g左右，6周龄的雌性、雄性SD大鼠。根据甲状旁腺的血供特点，选择示踪***，负显影甲状旁腺。实验分为5组，每组6只大鼠，共计5组*6只＝30只。
实验组A、B、C：分别对应10nm、50nm、100nm 3种不同粒径的IONPs；
正参比：150nm碳纳米粒子；
负参比：PBS溶液。
1、活体SD大鼠正常甲状腺及甲状腺旁腺解剖
1)对5组30只大鼠异氟烷吸入麻醉；
2)腹腔水合氯醛麻醉；
3)面罩持续麻醉；
4)脱毛、消毒、铺巾；
5)颈前正中切口，纵形切开；
6)分离颈前肌；
7)显露双侧甲状腺；
8)对10nm IONPs实验组中的1只SD大鼠(标记为M)进行活体甲状腺侧面拍照。
如图4A所示为大鼠甲状腺及甲状旁腺解剖图，其中，通过肉眼判断为甲状腺和甲状旁腺的部位如箭头所示，通常甲状旁腺呈透明状，甲状旁腺不明显，通过肉眼判断较为困难。
2、注射不同粒径氧化铁纳米粒子对甲状腺及甲状旁腺的显影
向5组30只大鼠甲状腺分别按上述分组进行注射，负显影结果如图5所示，每一行代表一种材料的6只SD大鼠甲状腺局部注射后的结果；其中，图5A为使用碳纳米粒子的阳性对照，图5B至5D分别为10nm、50nm和100nm的IONPs实验组，图5E为0.9％的生理盐水空白对照组。由图5可知，10nm的IONPs显影效果最明显。
注射后，对上述SD大鼠(标记为M)甲状腺侧面拍照，结果如图4B所示，可知甲状腺呈现黑色，甲状旁腺无黑染，肉眼可明确判断为甲状旁腺，效果佳。
根据图5，应用临床纳米碳注射前后的灰度下降测定方法，具体如下：显露术腔后术中拍照，对比注射前后的甲状腺的灰度变化；应用Image J软件中的灰度计算，在甲状腺上，与甲状旁腺边界紧邻的周围做一相邻的大小相等的圆，软件计算注射前后的灰度值，每次所取的圆直径相同，得出甲状腺注射前后的灰度的变化率(注射前灰度值减去注射后灰度值的差，除以注射前的灰度值)，结果如图6所示。由图6可知，10nm的IONPs(44.02％)和碳纳米粒子(43.32％)接近，效果最好。因此，采用显影效果最优的10nm的IONPs进行后续实验。
实施例4、SD大鼠甲状腺及甲状旁腺解剖及负显影
选择重量在240g-260g左右，6周龄的4只大鼠，按实施例1步骤1)-7)处理后，分别 按如下方式进行注射：A：右侧甲状腺及皮下注射10nm IONPs，左侧注射碳纳米粒子；B：左侧甲状腺及皮下注射10nm IONPs，右侧注射碳纳米粒子；C：双侧甲状腺及皮下注射10nm IONPs；D：双侧甲状腺及皮下注射碳纳米粒子。
显影结果如图7所示，其中图7A～D从左至右依次包括7幅图(分别标记为A1～A7、B1～B7、C1～C7、D1～D7)，其中，第1、2、3图像为注射前大鼠(X1、X3、X5倍)的空白正面图；第4、5、6图像为注射纳米材料后的显影(X1、X3、X5倍)。由图7A～D第1～6图像结果可见，IONPs和碳纳米粒子的显影效果相近，无明显差别；具体地，图7B结果与图7A结果相近，图7C中，无论是左侧还是右侧注射IONPs，均能获得较好的显影效果，图7D中，无论是左侧还是右侧注射碳纳米粒子，均能获得较好的显影效果。
A7、B7、C7、D7分别为在颈前正中进行切口，左右两侧分别按照所在组的纳米粒子注射方式进行注射，以说明10nm IONPs和碳纳米粒子在皮肤及皮下组织中的对比降解情况，即A7是颈前正中切口左右两旁皮下分别注射10nm IONPs和碳纳米粒子，B7是颈前正中切口左右两旁皮下分别注射碳纳米粒子和10nm IONPs，C7是颈前正中切口左右两旁皮下分别注射10nm IONPs，D7是颈前正中切口左右两旁皮下分别注射碳纳米粒子。由A7或B7可知，同一只大鼠间颈前切口左右注射不同纳米粒子，显色无明显差异。由C7或D7可知，大鼠左右两侧注射同一种纳米粒子，显色无明显差异。
实施例5、SD大鼠离体甲状腺注射纳米材料后的渗透情况
选择重量在240g-260g左右，6周龄的1只大鼠，按实施例1步骤1)-7)处理后，对SD大鼠切除颈前部位(包括喉、气管、食管、甲状腺、甲状腺旁腺)，左侧甲状腺注射碳纳米粒子，右侧甲状腺注射10nm IONPs，侧面拍照，如图8A所示。观察纳米材料渗漏情况，经过2.5小时后，结果如图8B所示，碳纳米粒子注射一侧食管被染及黑色(圆圈内所示范围)，而10nm IONPs注射一侧食管未被污染，色白(圆圈内所示范围)，无明显渗透至甲状旁腺区域，显影效果更佳。
综合上述，图4～8可以看出，在离体甲状腺或在体甲状腺显影中，10nm的IONPs效果与碳纳米粒子相近，但10nm的IONPs更不易渗漏，具有显著优于碳纳米粒子的性能。
实施例6、新西兰兔甲状腺及甲状旁腺解剖及负显影
根据甲状旁腺的血供特点，选择示踪***，负显影甲状旁腺。
实验组：注射10nm IONPs：
1、新西兰兔甲状腺及甲状旁腺解剖
1)称重，选择重量在3kg-3.5kg左右的新西兰兔；
2)腹腔水合氯醛麻醉；
3)颈前正中切口，纵形切开；
4)分离颈前肌；
5)显露双侧甲状腺及甲状旁腺；
6)对活体甲状腺正面活体拍照。
如图9A所示为新西兰兔甲状腺及甲状旁腺的解剖图，可见，甲状腺位于气管两侧，甲状旁腺位置不恒定，散在颈前肌内侧面。
2、注射纳米材料后，新西兰兔活体中甲状腺及甲状腺旁腺显影效果
7)分别向上述新西兰兔的左侧甲状腺局部注射10nm IONPs，右侧甲状腺注射碳纳米粒子(通过IONPs和碳纳米粒子左右自身对照进行显影)；
8)然后，对活体新西兰兔甲状腺拍照，两者均显影，结果如图9B所示，在新西兰兔动物模型中，10nm IONPs和碳纳米粒子的显色效果均较明显，效果接近。
实施例7、离体新西兰兔甲状腺注射纳米材料后的渗透情况
选择重量在3kg-3.5kg左右的2只新西兰兔，按实施例6步骤1)-5)处理后，切除新西兰兔甲状腺、甲状旁腺、喉及气管，新西兰兔左右两侧甲状腺分别注射碳纳米粒子和10nm IONPs后，结果如图10A所示，可见10nm IONPs和碳纳米粒子对甲状腺染色效果接近。经过2-3小时后，如图10B所示，碳纳米粒子注射一侧外渗明显，污染术区，色黑，图中“+”标示；IONPs注射一侧，边界较清，无污染外渗现象(其中，图10A左侧甲状腺对应图10B上方甲状腺，图10A右侧甲状腺对应图10B下方甲状腺)。
由图9、10显影结果图可知，本发明10nm IONPs在对新西兰兔甲状腺负显影方面与碳纳米粒子等效，但碳纳米粒子组更易出现周围污染情况。
实施例8、大鼠颌下***IONPs正显影情况
***显影的原理：局部注射(下唇)10nm IONPs后，通过颈前***，将IONPs输送至颌下区***，大鼠颌下***较表浅，切开皮肤即可观察到黑染的***。
1)选择6周龄的SD大鼠，称重，选择重量在240g-260g左右的大鼠；
3)异氟烷吸入麻醉；
4)腹腔注射10％的水合氯醛麻醉(300ul/g计算)；
5)面罩持续麻醉；
6)脱毛、消毒、铺巾；
7)大鼠下唇注射60ul的10nm IONPs，分3个点注射，每个点20ul，浓度为20mg/ml；
8)颈前正中切口，纵形切开，长约3-4cm；
9)观察颌下区***染色情况，如图11所示，可见从下唇引流至***的***，细长，黑褐色，与血管相伴行；相应***，与之相通的***，颜色较正常***更黑，呈现黑棕色；无注射对照组的正常***色白。可见IONPs可用于显影***，可作为***的示踪剂或相关药物递送至***的输送载体。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 氧化铁纳米粒子IONPs在制备显影剂中的应用，其特征在于，所述IONPs为纳米粒子结构，粒径范围为1-1000nm，zeta-potential范围为-50至50mV，紫外-可见吸收光谱峰值为100-500nm。
  2. 如权利要求1所述的应用，其特征在于，所述IONPs的粒径为1-200nm，zeta-potential为-35至20mV，紫外-可见吸收光谱峰值为180至300nm。
  3. 如权利要求1所述的应用，其特征在于，所述IONPs的粒径为1-100nm中的一种或几种，zeta-potential为-35至0mV，紫外-可见吸收光谱峰值为200-220nm。
  4. 如权利要求1～3之任一项所述的应用，其特征在于，所述显影剂为甲状旁腺负显影剂，和/或***正显影剂。
  5. 如权利要求1～3之任一项所述的应用，其特征在于，所述显影剂用于甲状腺、甲状旁腺和/或***组织成像或术中导航。
  6. 一种显影剂，其特征在于，所述显影剂包含如权利要求1～3之任一项所述的应用中所述的氧化铁纳米粒子IONPs。
  7. 一种诊断示踪剂，其特征在于，所述诊断示踪剂包含如权利要求1～3之任一项所述的应用中所述的氧化铁纳米粒子IONPs。
  8. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1～3之任一项所述的应用中所述的氧化铁纳米粒子IONPs以及药学上可接受的载体或介质。
  9. 如权利要求8所述的药物组合物在制备对甲状腺和/或***进行诊断和/或治疗的产品中的应用。
  10. 一种筛选对甲状腺和/或***治疗有效的药物的方法，其特征在于，通过将IONPs与药物分子构建到同种载体，通过IONPs对甲状腺的正显影、对甲状旁腺的负显影、和/或对***的正显影效果，对药物分子的有效性进行评价，筛选对甲状腺和/或***治疗有效的药物。
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