WO2017045508A1 - 多聚糖醛酸为载体的活体染色造影剂及制备方法和应用 - Google Patents

多聚糖醛酸为载体的活体染色造影剂及制备方法和应用 Download PDF

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WO2017045508A1
WO2017045508A1 PCT/CN2016/095825 CN2016095825W WO2017045508A1 WO 2017045508 A1 WO2017045508 A1 WO 2017045508A1 CN 2016095825 W CN2016095825 W CN 2016095825W WO 2017045508 A1 WO2017045508 A1 WO 2017045508A1
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reaction
group
ligand
dmt
acid
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江涛
万升标
张南
赵洺良
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中国海洋大学
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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

Definitions

  • the invention relates to a medicinal contrast agent, in particular to a living body dyeing contrast agent using a polyglycolic acid as a carrier, a preparation method and application thereof.
  • the lymphatic system is a network of lymph nodes and lymphatic vessels. Its distribution is roughly parallel to the capillaries and venous vessels, and it spreads throughout the body. As an important part of the human immune system, the lymphatic system plays a key role in the spread of pathogens and the transfer of cancer. Studies have shown that when breast cancer cells and melanoma cells metastasize, the sentinel lymph node (SLN) is the first to be found to be the most likely to metastasis. Metastatic cancer cells use SLN as a reservoir and spread along the lymphatic vessels to invade the surrounding lymph nodes, eventually completing the spread of cancer cells throughout the body. Therefore, lymphatic angiography as a medical detection method is of great significance for the prevention and diagnosis of cancer metastasis, the definition and treatment of various periods.
  • SSN sentinel lymph node
  • lymphatic magnetic resonance imaging has been widely used as a high resolution non-invasive technique. Due to the unique open structure of the lymphatic system and the one-way flow of lymph fluid, the development of a highly targeted contrast agent for the lymphatic system to obtain a high sensitivity and clarity of the lymphatic system MRI image has been a research hotspot in medical imaging.
  • the commonly used contrast agents for clinical use are mainly small molecule strontium-containing contrast agents (such as Magnevist), which can be used for vascular enhanced imaging, but can not specifically develop sentinel lymph nodes and lymphatic vessels; clinically use another tissue-specific magnetic Resonance imaging contrast agent is ultra-small superparamagnetic iron oxide nanoparticle (USPIO) feride magnetic (feridex), mainly used for liver and spleen magnetic resonance imaging; so so far, there is no clinically specific drug for lymphatic system And sentinel lymph nodes.
  • a series of new lymphatic targeted MR contrast agents can improve the quality of lymphatic mapping, and provide great help for lymph node localization, evaluation of metastatic tumor staging, and functional diagnosis of lymphatic vessels such as lymphedema.
  • a lipid- ⁇ complex disclosed in CN101637612 is used as a respiratory magnetic resonance spray contrast agent, and CN101433726 discloses one.
  • a magnetic resonance contrast agent using carbon nanotubes as a carrier, and a tree-shaped macromolecule with 1,4,7,10-tetraazacyclododecane as a core disclosed by CN1943791A is used as a carrier for the preparation of a paramagnetic metal.
  • Tumor-targeting macromolecular contrast agent is used as a carrier for the preparation of a paramagnetic metal.
  • the macromolecules containing the ruthenium chelate are passively pressed into the lymphatic vessels through the different osmotic pressures inside and outside the lymphatic vessels, without active targets.
  • the tropism causes the relaxation time of the contrast agent in the lymphatic system, the signal intensity, and the duration of the contrast to be unsatisfactory.
  • Mannose-binding protein is a type of foreign lectin-like receptor protein.
  • Such receptors highly present in mammalian lung phagocytic cells, hepatic parenchyma cells and non-thin parenchyma cells, serum and lymphoid tissues can bind to glycoproteins with mannose at the end and introduce mannose groups into lymphoid targeting contrast agents.
  • the group can make the contrast agent have the function of actively targeting lymphoid tissue, and has broad application prospects.
  • lymphatic targeted nuclear magnetic resonance contrast agents have important requirements in clinical medicine, in the past decade, lymphatic contrast agents have been prepared using dextran, polyamide polymers, polyethylene glycol polymers or peptides as carriers. None of them entered the new drug approval stage. The main reason is that the carrier used above can only be targeted to enter the lymphatic system by the particle size of the material, but does not have the binding effect on lymphocyte receptors, and cannot be derived from lymphoid specific receptors. An angled active agent with fast targeting, strong specificity, and long residence time is obtained.
  • lymphoid targeting contrast agent needs to meet three conditions, a fast injection site clearance rate, a long lymphatic dwell time, and a high lymph node contrast agent accumulation concentration.
  • a lymph-specific contrast agent with both active and passive targeting can satisfy the above characteristics.
  • the lymphoseek approved by the US FDA for clinical use in 2013 is a target protein that actively targets the mannose receptor of lymphoid macrophages. It is a mannose fragment and a radioisotope ⁇ Tc on the molecule of dextran. Lymphatic targeting scintillation radionuclide contrast agent, which maps and localizes lymph nodes and lymphatic vessels.
  • the contrast agent specifically binds to lymphoid tissue, indicating the mannose group in the contrast agent and the nectar rich in lymphoid macrophages.
  • MBP glycoconjugate protein
  • the radionuclide contrast agent has been clinically applied, it has the disadvantages of low sensitivity and high toxicity of radioactive elements compared with nuclear magnetic resonance contrast agents.
  • CN101862461.B discloses a bismuth-containing macromolecular contrast agent based on hyaluronic acid, it is desirable to pass the lymphatic vessel hyalkane receptor-1LYVE-1 (Lymphatic vascular). Endothelial hyaluronan receptor-1), which achieves the active targeting of the lymphatic system.
  • this patent only discloses the preparation of a cerium-containing macromolecular contrast agent with hyaluronic acid as a carrier, and there is no data to support that the contrast agent can be actively targeted. There is no data in the lymphatic system that supports the specific binding of hyaluronic acid to lymphatic hyaluronan receptor-1 (LYVE-1).
  • LYVE-1 lymphatic hyaluronan receptor-1
  • lymphocyte surface receptors can bind to a variety of acidic polysaccharides (Parish CR, Cell Immunol. 1985 Mar; 91(1): 201-14.), marine polysaccharides, oligosaccharides are rare immune active molecules, oceans.
  • the polysaccharide-derived polymannuronic acid and polyguluronic acid have strong immunity and activity, and the brown algae acidic polysaccharide can enhance the phagocytic ability of lymphoid macrophages (acid phosphatase ACP) by immunological activity.
  • These marine fucoidan polysaccharides can enhance the mannose receptor binding ability and the phagocytic ability of macrophages, and provide a possibility to study the nuclear magnetic resonance contrast agent that actively targets the lymphatic system.
  • the contrast agent of the invention has a mannose receptor recognition fragment, a paramagnetic metal chelating agent and a living body dye group, and has lymphatic active targeting.
  • the animal experiments in vivo can accurately determine the tumor sentinel lymph node condition.
  • Anterior lymph node imaging is helpful for intraoperative sentinel lymph node localization and lymphadenectomy during tumor fine surgery. It can be widely used for preoperative sentinel lymph node localization such as breast cancer, head and neck cancer, and melanoma. It can meet the requirements of biopsy and magnetic resonance imaging at the same time, and is an excellent dual-function lymphatic targeted contrast agent.
  • the present invention is directed to the prior art that there is a problem that the active targeting, the slow entry, the poor transferability, and the long residence time are too long, and the invention provides a living body staining contrast agent with a polyglycolic acid as a carrier, a preparation method thereof and application thereof .
  • Lymphocyte surface receptors can be combined with a variety of acidic polysaccharides.
  • Marine polysaccharides and oligosaccharides are rare immune active molecules.
  • Marine polysaccharide-derived polymannuronic acid and polyguluronic acid have strong immunity and
  • the brown algae acidic polysaccharide can enhance the phagocytic ability of lymphoid macrophages (acid phosphatase ACP) by immunological activity.
  • lymphoid macrophages (acid phosphatase ACP) by immunological activity.
  • These polysaccharides are capable of enhancing mannose receptor binding capacity and The phagocytic ability of macrophages provides a possibility for the study of nuclear magnetic resonance contrast agents that actively target the lymphatic system.
  • a living body dyeing contrast agent containing a polyglycolic acid as a carrier which has the following structure: a carboxyl group-containing polysaccharide aldehyde acid as a carrier, and a 6-position carboxyl group on the carrier passes through an alkyl group, an aryl group or a heterocyclic ring
  • the group is a linking arm which is respectively combined with the mannose receptor MBP recognition group ligand A, the paramagnetic metal chelate ligand B or the living dye ligand C, and has the following formula:
  • n 1 is an integer, n 2 , n 3 and n 4 are positive integers;
  • X is O, N or S;
  • linker is an alkyl group, an aryl group or a heterocyclic group; and
  • ligand A is a mannose receptor MBP recognition group
  • Ligand B is a paramagnetic metal chelate;
  • ligand C is a living dye.
  • the molar content of the ligand A accounts for 0-30% of the original carboxyl group of the polyglycolic acid
  • the molar content of the ligand B accounts for 0 of the original carboxyl group of the polyglycolic acid.
  • the molar content of the ligand C is 1-30% of the original carboxyl group of the polyglycolic acid, wherein the molar content of the ligand A, the molar content of the ligand B, the molar content of the ligand C, and The sum of the molar contents of the unreacted carboxyl groups in the original carboxyl group of the polyglycolic acid is 100%.
  • the polyglycolic acid carrier is polymannuronic acid PM, polyguluronic acid PG, polyglucuronic acid, polygalacturonic acid and block copolymers thereof and includes corresponding ones thereof.
  • the molecular weight is from 100 to 10 8 Da.
  • the Linker bonded to the 6-position carboxyl group of the polysaccharide via an amide bond is an alkyl, aryl or heterocyclic structure having an atomic number of 1 to 20, and further with the mannose receptor MBP recognition group ligand A, a paramagnetic metal chelate The ligand B or the living dye ligand C is combined.
  • the uronic acid cyclic carboxyl group of the polyglycolic acid is further linked to the mannose receptor MBP recognition group ligand A by an amide bond, and the mannose and its derivative in the ⁇ or ⁇ configuration, the specific structure as follows:
  • R is a hydrocarbon group, a carboxyl group, an amino group, a hydroxyl group or a halogen.
  • the paramagnetic metal chelate ligand B is formed by chelation of a metal chelating agent with a paramagnetic metal ion, and the metal chelating agent is an amino group-containing metal chelating agent, and the structure is as follows:
  • the metal chelating agent is an amino-free metal chelating agent, and the structure is as follows:
  • the paramagnetic metal ion used is a divalent or trivalent ion of Gd, Mn, Cr, Fe, Co, Ni, La, Tc, Dy or Cu.
  • the living dye ligand C is toluidine blue, Evans blue or p-aminoazobenzene.
  • Another object of the present invention is to disclose a method for preparing a living body staining contrast agent comprising a polyglycolic acid as a carrier, comprising the steps of:
  • the first step the polyglycolic acid and the acylating reagent are dissolved in the solvent, and then the alkyl diamine compound and the living dye are sequentially added to stir the reaction for 1-24 hours, the reaction is completed, the reaction is quenched, and the solution is dialyzed through a dialysis bag. , removal of small molecular impurities, to obtain intermediates, living dyes, alkyl diamines, acylating agents, polysaccharides, aldehydes, all carboxyl groups, the molar ratio of the mixture is 0-0.999: 0.001-100: 0.001-100:1 ;
  • the second step dissolving the intermediate and the acylating reagent in the above step in deionized water, adding a metal chelating agent to stir the reaction for 1-24 hours, quenching the reaction after completion of the reaction, quenching the reaction, adding an aqueous solution of metal ions thereto,
  • the final product is obtained after dialysis and freeze-drying treatment, and the molar ratio of the metal chelating agent, the acylating agent, the metal ion and the polycarboxylate aldehyde to all the carboxyl groups is 0.001-100: 0.001-100: 0.001-100:1.
  • the solvent for the two-step acylation reaction is water or DMSO, DMF polar aprotic solvent, and the reaction temperature is between 0 and 150 °C.
  • the acylating agent used in the acylation reaction is selected from the group consisting of DMT-MM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride), EDC ( 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine), DMT-Pip (4-(4,6-dimethoxytriazin-2-yl)-4-methylpiperidine hydrochloride), DMT-EMM (4-(4,6-dimethoxy) Oxazin-2-yl)-N-ethyl-N,N-dimethylhydrochloride) or DMT-TMM (4-(4,6-dimethoxytriazin-2-yl)-N,N Any one of N-trimethyl hydrochloride, wherein the structure of DMT-MM is as shown in formula (I), the structure of
  • DMT-Pip As shown, the structure of DMT-Pip is as shown in formula (IV), the structure of DMT-EMM is as shown in formula (V), and the structure of DMT-TMM is as shown in formula (VI): Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI).
  • a third object of the present invention is to disclose another method for preparing a living body staining contrast agent comprising a polyglycolic acid as a carrier to take the following steps:
  • the first step the polyglycolic acid and the acylating reagent are dissolved in a solvent, and then the mannose or mannose derivative is added and stirred for 1-24 hours. After the reaction is completed, the reaction is quenched, and the solution is dialyzed through a dialysis bag to remove small molecules. Impurity, using freeze-drying method to obtain intermediate 1, mannose or mannose derivative, acylating reagent, all of the carboxyl groups of uronic acid, the molar ratio of the mixture is 0-0.999: 0.001-100:1;
  • the second step the intermediate 1 and the acylating reagent described in the above step are dissolved in deionized water, and the metal chelating agent with an amino group is added to stir the reaction for 1-24 hours, the reaction is quenched, the reaction is quenched, and the solution is dialyzed. Bag dialysis, removal of small molecular impurities, using freeze-drying method to obtain intermediate 2;
  • Step 3 Dissolve the intermediate 2 and the acylating reagent in the above step in deionized water, slowly add the living dye to stir the reaction for 1-24 hours, quench the reaction after completion of the reaction, quench the reaction, and dialyze the solution through the dialysis bag to remove Small molecular impurities, to which an aqueous solution of metal ions is added, and subjected to dialysis and freeze-drying to obtain a final product, wherein the molar ratio of the metal chelating agent, the acylating agent, the metal ion and the polycarboxylate aldehyde to all carboxyl groups is 0.001- 100: 0.001-100: 0.001-100:1.
  • the solvent for the three-step acylation reaction is water or DMSO, DMF polar aprotic solvent, and the reaction temperature is between 0 and 150 °C.
  • the acylating agent used in the acylation reaction is selected from the group consisting of DMT-MM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride), EDC ( 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), CDMT (2-chloro-4,6-dimethoxy-1,3,5-triazine), DMT-Pip (4-(4,6-dimethoxytriazin-2-yl)-4-methylpiperidine hydrochloride), DMT-EMM (4-(4,6-dimethoxy) Oxazin-2-yl)-N-ethyl-N,N-dimethylhydrochloride) or DMT-TMM (4-(4,6-dimethoxytriazin-2-yl)-N,N Any one of N-trimethyl hydrochloride, wherein the structure of DMT-MM is as shown in formula (I), the structure of
  • DMT-Pip As shown, the structure of DMT-Pip is as shown in formula (IV), the structure of DMT-EMM is as shown in formula (V), and the structure of DMT-TMM is as shown in formula (VI): Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI).
  • a fourth object of the present invention is to disclose a living body staining contrast agent for preparing a diagnostic lymphoid system. The application of the disease reagents.
  • the contrast agent of the invention has a mannose receptor recognition fragment, a paramagnetic metal chelating agent and a living body dye group, and has lymphatic active targeting.
  • the animal experiments in vivo can accurately determine the tumor sentinel lymph node condition.
  • Anterior lymph node imaging is helpful for intraoperative sentinel lymph node localization and lymphadenectomy during tumor fine surgery. It can be widely used for preoperative sentinel lymph node localization such as breast cancer, head and neck cancer, and melanoma. It can meet the requirements of biopsy and magnetic resonance imaging at the same time, and is an excellent dual-function lymphatic targeted contrast agent.
  • a nuclear magnetic resonance contrast agent containing a paramagnetic metal ion and a living body dye prepared by using a polyglycolic acid such as marine fucoidan, polyguluronic acid, polymannuronic acid or polyglucuronic acid as a carrier. It has the function of targeting lymphatic capillaries and sentinel lymph nodes on the scale. It also has the same function as the mannose receptor of lymphoid macrophages. It has the same efficacy as the FDA-approved radionuclide contrast agent Lymphoseek and can be used for lymphoid tissue. The biopsy is obviously superior to the patented CN101862461.B invention contrast agent HA-DTPA-Gd.
  • the key point of macromolecular nanoscale nuclear magnetic resonance contrast agent is the high water solubility of the drug.
  • the invention develops a method for amidating the aqueous phase in the preparation method, and uses a high-efficiency aqueous phase condensing agent to conveniently prepare a glycosylamide in a high yield, and introduces a chelating reagent to obtain a water-soluble contrast agent.
  • the present invention also develops a nanoparticle purification method, which uses a water phase dialysis membrane method to obtain a water-soluble contrast agent of 70-80 nm by molecular weight control.
  • Figure 1 shows a bilateral MR imaging of the lower limbs and an animal anatomy of the New Zealand rabbit after subcutaneous injection of the macromolecular developer PM-TB-GdDTPA in Example 1 of the present invention
  • A anatomical images of lower extremity lymph nodes 1 h after injection of contrast agent
  • B MR images of lower limb lymphatic system 1 h after injection of contrast agent.
  • polyglycolic acid is polymannuronic acid
  • specific molecular formula is as follows:
  • n 1 is an integer, n 2 , n 3 and n 4 are positive integers, X is O, N or S; and linker is an alkyl group, an aryl group or a heterocyclic group.
  • Ligand A is a mannose or mannose derivative.
  • Ligand B is a paramagnetic metal chelate fragment.
  • Ligand C is a living body dye.
  • polyglycolic acid is polyguluronic acid
  • specific molecular formula is as follows:
  • n 1 is an integer, n 2 , n 3 and n 4 are positive integers, X is O, N or S; and linker is an alkyl group, an aryl group or a heterocyclic group.
  • Ligand A is a mannose or mannose derivative.
  • Ligand B is a paramagnetic metal chelate fragment.
  • Ligand C is a living body dye.
  • polyglycolic acid is polygalacturonic acid
  • specific molecular formula is as follows:
  • n 1 is an integer, n 2 , n 3 and n 4 are positive integers, X is O, N or S; and linker is an alkyl group, an aryl group or a heterocyclic group.
  • Ligand A is a mannose or mannose derivative.
  • Ligand B is a paramagnetic metal chelate fragment.
  • Ligand C is a living body dye.
  • the polymannuronic acid PM 3.52g and the acylating reagent EDC (1.92g, 10mmol) were dissolved in 100ml of distilled water, stirred at 60 ° C for 5h, then cooled to room temperature, and slowly added with the alkyl diamine compound ethylenediamine and stirring.
  • the living dye was toluidine blue (0.306 g, 1 mmol) and stirred at room temperature overnight.
  • the reaction solution was placed in a dialysis bag (retention amount 3500), dialyzed against 3 L of distilled water, and water was changed every 4 hours, and water exchange was repeated 5 times. After the dialysis was completed, the solution in the dialysis bag was concentrated to 100 ml to obtain a PM-TB-N solution.
  • New Zealand white rabbit has a body weight of 3.2 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • the macromolecular bifunctional developer PM-TB-GdDTPA (30 mg/L) was then injected subcutaneously into the first, second and third toe of the feet, and 0.2 ml was injected each time for 3D enhanced scanning.
  • the relevant parameters of the enhanced scan sequence are the same as those for the flat scan, and are scanned every 15 minutes for a total of 5 scans. After the end of the scan, animal anatomy was performed based on the MR image to obtain stained lymph nodes.
  • FIG. 1 The bilateral lung MR imaging images and animal anatomy images of the New Zealand rabbit subcutaneously injected with the macromolecular developer PM-TB-GdDTPA in Example 1 are shown in Fig. 1.
  • A anatomical images of lower limb lymph nodes 1 hour after injection of contrast agent
  • B MR image of the lower limb lymphatic system 1 h after injection of contrast agent.
  • New Zealand white rabbit has a body weight of 3.1 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • the PM-M 3.0 g obtained by the previous reaction and the acylating reagent EDC (1.92 g, 10 mmol) were dissolved in 100 ml of deionized water, and 1-(p-aminobenzyl)-DTPA hydrochloride was added thereto with stirring.
  • the salt (1.93 g, 3 mmol) was stirred at room temperature for 12 h.
  • K 2 CO 3 (8.3 g, 60 mmol) was added to the solution and stirred for 0.5 h.
  • the solution was placed in a dialysis bag with a cut-off of 3,500, dialyzed against 5 L of deionized water, and water was changed every 6 h for a total of 5 changes. After the end of the dialysis, the solution was concentrated, and after lyophilization, a white solid compound, PM-M-NDTPA, 3.1 g was obtained.
  • the PM-M-NDTPA 3g obtained by the reaction of the previous step and the acylation reagent EDC (1.92 g, 10 mmol) were dissolved in 100 ml of distilled water, and toluidine blue (0.306 g, 1 mmol) was slowly added thereto with stirring, and stirred at room temperature overnight.
  • the reaction solution was placed in a dialysis bag (retention amount 3500), dialyzed against 3 L of distilled water, and water was changed every 4 hours, and water exchange was repeated 5 times.
  • the solution in the dialysis bag was concentrated to 100 ml to obtain a PM-M-TB-NDTPA solution.
  • the PM-M-TB-NDTPA solution prepared by the reaction was added to GdCl 3 ⁇ 6H 2 O (0.93 g, 2.5 mmol) in portions, and the pH was adjusted to 5-6 with a 5% NaOH solution, and stirred at room temperature for 1 hour.
  • the solution was then placed in a dialysis bag with a cut-off of 3,500, dialyzed against 5 L of deionized water, and water was changed every 6 h for a total of 5 changes. After completion of the dialysis, the solution was concentrated, and after lyophilization, 2.66 g of a blue solid compound PM-M-TB-GdNDTPA was obtained.
  • New Zealand white rabbit has a body weight of 3.2 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • lymph nodes and primary and secondary lymphatic vessels remained clear. Animal anatomy was performed based on MR images to obtain stained lymph nodes.
  • New Zealand white rabbit has a body weight of 3.2 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • lymph nodes and primary and secondary lymphatic vessels remained clear. Animal anatomy was performed based on MR images to obtain stained lymph nodes.
  • the PM-M 3.0 g obtained by the previous reaction and the acylating agent DMT-Pip (1.81 g, 10 mmol) were dissolved in 100 ml of deionized water, and 1-(p-aminobenzyl)-DTPA was added thereto with stirring.
  • Hydrochloride (0.64 g, 1 mmol) was stirred at room temperature for 12 h.
  • K 2 CO 3 (8.3 g, 60 mmol) was added to the solution and stirred for 0.5 h.
  • the solution was placed in a dialysis bag with a cut-off of 3,500, dialyzed against 5 L of deionized water, and water was changed every 6 h for a total of 5 changes. After completion of the dialysis, the solution was concentrated, and after lyophilization, 3.2 g of a white solid compound PM-M-NDTPA was obtained.
  • the PM-M-NDTPA 3g obtained by the previous reaction and the acylating agent DMT-Pip (1.81 g, 10 mmol) were dissolved in 100 ml of distilled water, and toluidine blue (0.306 g, 1 mmol) was slowly added with stirring, and stirred at room temperature overnight. .
  • the reaction solution was placed in a dialysis bag (retention amount 3500), dialyzed against 3 L of distilled water, and water was changed every 4 hours, and water exchange was repeated 5 times. After the dialysis was completed, the solution in the dialysis bag was concentrated to 100 ml to obtain a PM-M-TB-NDTPA solution.
  • the PM-M-TB-NDTPA solution prepared by the reaction was added to GdCl 3 ⁇ 6H 2 O (0.93 g, 2.5 mmol) in portions, and the pH was adjusted to 5-6 with a 5% NaOH solution, and stirred at room temperature for 1 hour.
  • the solution was then placed in a dialysis bag with a cut-off of 3,500, dialyzed against 5 L of deionized water, and water was changed every 6 h for a total of 5 changes. After the end of the dialysis, the solution was concentrated, and after lyophilization, 2.87 g of a blue solid compound PM-M-TB-GdNDTPA was obtained.
  • New Zealand white rabbit has a body weight of 3.1 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • lymph nodes and primary and secondary lymphatic vessels remained clear. Animal anatomy was performed based on MR images to obtain stained lymph nodes.
  • the acylation reagent DMT-EMM (1.42 g, 10 mmol) was added to the above GA-EB-N solution, and stirred until dissolved.
  • DTPA 11.82 g, 30 mmol
  • GdCl 3 ⁇ 6H 2 O 5.58 g, 15 mmol
  • the pH of the reaction solution was adjusted to 7 with a saturated sodium hydrogencarbonate solution, and then placed in a dialysis bag with a cut-off amount of 3,500, and dialyzed against 3 L of distilled water. Change the water 4h and repeat the water change 5 times.
  • the solution was concentrated to 50 ml, and 50 ml of ethanol was added to concentrate to dryness to remove water, and the blue solid powder GA-EB-GdDTPA 3.68 g was obtained twice.
  • New Zealand white rabbit has a body weight of 3.1 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • the New Zealand rabbits were injected subcutaneously with the first, second and third toes of the hind limbs, 0.1 ml each time, and the sputum concentration was 0.03 mmol/ml. After the developer was injected, it was scanned every 15 to 60 minutes in 5.5 hours for a total of 10 scans.
  • New Zealand white rabbit has a body weight of 3.1 kg.
  • New Zealand white rabbits were anesthetized by intramuscular injection with ketamine (80 mg/kg, 1.6 ml) and diazepam (5 mg/kg, 1 ml). It was fixed on a rabbit operating table and subjected to MRI plain scanning.
  • the New Zealand rabbits were injected subcutaneously with the first, second and third toes of the hind limbs, 0.1 ml each time, and the sputum concentration was 0.03 mmol/ml. After the developer was injected, it was scanned every 15 to 60 minutes in 5.5 hours for a total of 10 scans.
  • the polymannuronic acid polyglucuronic acid (PGlu) (3.12 g, 20 mmol) and the acylating agent DMT-TMM (2.32 g, 20 mmol) were dissolved in 50 ml of deionized water, and the 1-O was slowly added in portions at room temperature with stirring.
  • -Aminoethylmannose (2.84 g, 10 mmol) stirred at room temperature for 12 h.
  • K 2 CO 3 (8.3 g, 60 mmol) was added to the solution and stirred for 0.5 h; the solution was placed in a dialysis bag with a cut-off of 3500, dialyzed against 5 L of deionized water, and water was changed every 6 h. Change water 5 times.
  • the solution was concentrated, and after lyophilization, a white solid compound PGlu-M 3.34 g was obtained.
  • the PGlu-M 3g obtained by the reaction of the previous step and the acylating agent DMT-TMM (2.32 g, 10 mmol) were dissolved in 100 ml of distilled water, and toluidine blue (0.306 g, 1 mmol) was slowly added with stirring, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was placed in a dialysis bag (retention amount 3500), and dialyzed against 3 L of distilled water. Change the water every 4 hours and repeat the water change 5 times. After the dialysis was completed, the solution in the dialysis bag was concentrated to 100 ml to obtain a PGlu-M-TB solution.
  • the PM-M-TB solution prepared by the reaction was adjusted to pH 5-6, stirred at room temperature for 1 h, and then placed in a dialysis bag with a cut-off amount of 3500, and dialyzed against 5 L of deionized water, every Change water once in 6h and change water a total of 5 times. After completion of the dialysis, the solution was concentrated, and after lyophilization, 2.42 g of a blue solid compound PGlu-M-TB was obtained.
  • the in vivo staining contrast agent showing the polyglycolic acid as the carrier described in Examples 1-8 has the ability of biological living body staining and nuclear magnetic resonance dual function lymphatic imaging, and achieves clear mapping and precise positioning of lymph nodes and lymphatic vessels (Table 1). ).

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Abstract

一种大分子活体染色造影剂及其制备方法和应用。所述造影剂以多聚糖醛酸为载体，甘露糖或甘露糖衍生物作为甘露糖受体（MBP）识别基团、顺磁性金属离子螯合物作为核磁共振造影基团，用于乳腺癌、头颈癌、黑素瘤等术前前哨淋巴结定位，并满足活体检查以及核磁共振造影，为双功能淋巴靶向造影剂。

Description

多聚糖醛酸为载体的活体染色造影剂及制备方法和应用 技术领域
本发明涉及医药学造影剂，具体涉及一种以多聚糖醛酸为载体的活体染色造影剂及其制备方法和应用。
背景技术
淋巴系统是由淋巴结和淋巴管组成的网状结构，其分布与毛细血管和静脉血管大致并行，遍布人体全身各部位。作为人体免疫系统重要组成部分，淋巴系统在病菌的扩散和癌症的转移上起了关键作用。研究表明，乳腺癌细胞、黑色素瘤细胞转移时，前哨淋巴结(SLN)首先发现转移癌细胞的可能性最大。转移癌细胞将SLN作为蓄积地，沿淋巴管扩散侵入周围各级淋巴结，最终完成整个身体的癌细胞扩散。因此，淋巴系统造影作为一种医疗检测手段，对于癌转移的预防、诊断、各时期的评估界定和治疗有着重要意义。
近年来，淋巴核磁共振成像(MRI)作为一种分辨率高的非创伤性技术获得了广泛应用。由于淋巴系统的独特的开放式结构和淋巴液的单向流动，研制淋巴系统的靶向性高的造影剂从而获得高灵敏度和清晰度的淋巴系统MRI图一直是医学影像学的研究热点。目前临床使用的常用造影剂主要为小分子含钆造影剂(如马根维显)，它可以用于血管强化显影，但不能对前哨淋巴结和淋巴管特异显影；临床上使用另外一种组织特异性磁共振成像造影剂是超小型超顺磁性氧化铁纳米颗粒(USPIO)菲立磁(feridex)，主要用于肝脾磁共振造影；所以至今，临床上还没有一种药物特异性的用于淋巴系统和前哨淋巴结。一系列新型淋巴靶向性MR造影剂的研究可以提高淋巴系统绘图质量，为淋巴结的定位、转移瘤分期的评估、淋巴管道功能性诊断如淋巴水肿提供巨大帮助。
近10年来，肝、肿瘤、脑部等特殊部位的磁共振造影剂的研究取得了长足的发展，如CN101637612公开的一种脂质-钆复合物作为呼吸道磁共振喷雾造影剂，CN101433726公开的一种以碳纳米管为载体的钆磁共振造影剂，CN1943791A公开的以1，4，7，10-四氮杂环十二烷为核的树形大分子为载体连接顺磁性金属制备的用于肿瘤靶向性大分子造影剂。
在淋巴靶向造影的造影机理研究方面，申请号201010023087.6公开了以二氧化硅纳米微球包载的钆磁共振造影剂，由于其粒径范围在50-200nm之间， 可以被动地被毛细淋巴管吸收而不透过毛细血管。另外，以多糖为载体的淋巴靶向造影剂的研究也是此类造影剂开发的重要方面，如以右旋糖酐(即葡聚糖)为载体共价连接含有钆的螯合物(鄢国平等，多糖类大分子顺磁性金属配合物及其合成方法，中国专利申请号，200910063629.X，江涛等，靶向淋巴系统造影剂的制备及其动物实验.中国科学:化学,2011(11):第1712-1718页)，大分子磁共振造影剂之所以没有用于临床，主要是因为二氧化硅纳米微球和右旋糖酐(葡聚糖)对淋巴细胞和巨噬细胞都没有特异性结合，它们是依据毛细淋巴管的上皮细胞裂隙大于毛细血管的上皮细胞裂隙，皮下注射后，通过淋巴管内外渗透压的不同，被动的将含有钆螯合物的大分子压到淋巴管中，不具有主动靶向性，从而导致该造影剂在淋巴系统内的弛豫时间、信号强度和造影的持续时间不尽如意。甘露糖结合蛋白(MBP)属于外源凝集素类受体蛋白的一种。这种高度存在于哺乳动物肺吞噬细胞、肝薄壁细胞和非薄壁细胞、血清和淋巴组织中的受体可以结合末端为甘露糖的糖蛋白，在淋巴靶向造影剂中引入甘露糖基团，可以使得造影剂具有主动靶向淋巴组织的功能，具有广阔的应用前景。
虽然淋巴靶向核磁共振造影剂在临床医学上有重要需求，但近十年的研究，用右旋糖苷、聚酰胺高分子、聚乙二醇高分子或多肽为载体的制备的淋巴系统造影剂都没有进入新药审批阶段，主要原因是上述使用的载体只能依靠材料的粒径大小靶向进入淋巴系统，却不具备与淋巴细胞受体的结合作用，还不能从淋巴的特异性受体的角度获得主动靶向、快速进入、专一性强且停留时间长的造影剂。
理想的淋巴靶向性造影剂需要满足三个条件，快的注射位点清除速率、长的淋巴驻留时间以及高的淋巴结造影剂积累浓度。主动靶向和被动靶向兼具的淋巴特异性造影剂才能够满足以上特性。2013年美国FDA批准用于临床的lymphoseek是一种以淋巴巨噬细胞的甘露糖受体为主动靶向的靶点蛋白，是在右旋糖酐的分子上接有甘露糖片段和含有放射性同位素锝Tc的淋巴靶向闪烁核素造影剂，实现了淋巴结和淋巴管的绘图与定位，该造影剂特异性结合于淋巴组织中，表明了造影剂中甘露糖基团与淋巴巨噬细胞中富含的甘露糖结合蛋白(MBP)受体结合策略的成功应用。但是，该核素造影剂虽然已应用临床，但与核磁共振造影剂相比，仍然具有灵敏度低、放射性元素毒性大的缺点。虽然，CN101862461.B公开了一种以透明质酸为载体的含钆大分子造影剂，希望通过淋巴管具有的淋巴管透明质烷受体-1LYVE-1(Lymphatic vascular  endothelial hyaluronan receptor-1),达到主动靶向淋巴系统的作用，但是，该专利仅仅公开了透明质酸为载体的含钆大分子造影剂的制备方法，并没有数据支持该造影剂可以主动靶向淋巴系统，也没有数据支持透明质酸与淋巴管透明质烷受体-1(LYVE-1)特异性结合。青岛大学医学院附属医院刘凤桐(新型特异性染料SW作为头颈部淋巴走行示踪剂的实验研究,[硕士学位论文]:青岛大学，2010.)于新西兰大白兔舌下粘膜分别注射甲苯胺蓝(TB)及右旋糖酐(40kDa)结合甲苯胺蓝的大分子染料(SW)。解剖结果显示，二者均可使双侧颈部淋巴结和淋巴管清晰染色，而SW的染色时间更长。组织切片检查显示SW在一小时后大部分由淋巴窦进入巨噬细胞内。这种直观精确的活体染料检查极大便利了淋巴清扫术。但是该显影剂依然有代谢清除速率过高，不具有淋巴主动靶向性等缺点。
已经研究发现淋巴细胞表面受体可以与多种酸性多糖结合(Parish CR,Cell Immunol.1985Mar；91(1):201-14.)，海洋多糖、寡糖是不可多得的免疫活性分子，海洋多糖来源的聚甘露糖醛酸和聚古罗糖醛酸具有强的提高免疫力和活性，而且，褐藻酸性多糖可以通过免疫活性增强淋巴巨噬细胞的吞噬能力(酸性磷酸酶ACP)。这些海洋褐藻多糖能够增强甘露糖受体结合能力和巨噬细胞的吞噬能力，为研究发明主动靶向淋巴系统的核磁共振造影剂提供了可能。
本发明所述造影剂，同时具有甘露糖受体识别片段，顺磁性金属螯合剂以及活体染剂基团，具有淋巴主动靶向性，动物体内实验说明，它能准确确定肿瘤前哨淋巴结状况，术前淋巴结显像，有助于术中前哨淋巴结定位和肿瘤精细外科手术过程中的淋巴清扫术，可以广泛用于乳腺癌、头颈癌、黑素瘤等术前前哨淋巴结定位上。可以同时满足活体检查以及核磁共振造影的要求，是优秀的双功能淋巴靶向造影剂。
发明内容
本发明针对现有技术中存在不能主动靶向、进入慢、转移性不强以及停留时间过长的问题，提供一种以多聚糖醛酸为载体的活体染色造影剂及其制备方法和应用。
淋巴细胞表面受体可以与多种酸性多糖结合，海洋多糖、寡糖是不可多得的免疫活性分子，海洋多糖来源的聚甘露糖醛酸和聚古罗糖醛酸具有强的提高免疫力和活性，而且，褐藻酸性多糖可以通过免疫活性增强淋巴巨噬细胞的吞噬能力(酸性磷酸酶ACP)。这些多聚糖醛酸能够增强甘露糖受体结合能力和 巨噬细胞的吞噬能力，为本发明研究主动靶向淋巴系统的核磁共振造影剂提供了可能。
本发明的技术方案是：
一种多聚糖醛酸为载体的活体染色造影剂，其具有以下结构：以带有羧基的多聚糖醛酸为载体，所述载体上的6位羧基通过烷基、芳基或杂环基团为连接臂，分别与甘露糖受体MBP识别基团配体A、顺磁性金属螯合物配体B或活体染剂配体C结合，具有以下通式：
其中：n1是整数、n2、n3和n4是正整数；X为O、N或S；linker为烷基、芳香基或杂环基；配体A为甘露糖受体MBP识别基团；配体B为顺磁性金属螯合物；配体C为活体染剂。
优选的，所述的配体A的摩尔含量占所述的多聚糖醛酸原有羧基的0-30％，配体B的摩尔含量占所述的多聚糖醛酸原有羧基的0-40％，配体C的摩尔含量占所述的多聚糖醛酸原有羧基的1-30％，其中配体A的摩尔含量、配体B的摩尔含量、配体C的摩尔含量以及多聚糖醛酸原有羧基中未反应羧基的摩尔含量总和为100％。
优选的，所述的多聚糖醛酸载体为聚甘露糖醛酸PM、聚古罗糖醛酸PG、聚葡萄糖醛酸、聚半乳糖醛酸及它们的嵌段共聚物并包括其相应的羧酸盐形式，分子量为100-108Da。
优选的，与多糖6位羧基通过酰胺键结合的Linker为原子数目1到20的烷基、芳香基或杂环基结构，再与甘露糖受体MBP识别基团配体A、顺磁性金属螯合物配体B或者活体染剂配体C结合。
优选的，所述多聚糖醛酸的糖醛酸环羧基还以酰胺键连接有甘露糖受体MBP识别基团配体A，为α或β构型的甘露糖及其衍生物，具体结构如下：
Figure PCTCN2016095825-appb-000002
其中，R为烃基、羧基、氨基、羟基或卤素。
优选的，所述顺磁性金属螯合物配体B由金属螯合剂与顺磁性金属离子螯合而成，金属螯合剂为含氨基的金属螯合剂，结构如下：
Figure PCTCN2016095825-appb-000003
Figure PCTCN2016095825-appb-000004
或金属螯合剂为不含氨基的金属螯合剂，结构如下：
Figure PCTCN2016095825-appb-000005
所用顺磁性金属离子为Gd、Mn、Cr、Fe、Co、Ni、La、Tc、Dy或Cu的二价或三价离子。
优选的，所述的活体染剂配体C为甲苯胺蓝、伊文思蓝或对氨基偶氮苯。
本发明的另一个目的在于公开一种制备多聚糖醛酸为载体的活体染色造影剂的方法，包括以下步骤：
第一步：将多聚糖醛酸和酰化试剂溶解于溶剂中，随后依次加入烷基二胺类化合物、活体染剂搅拌反应1-24h，反应完毕，淬灭反应，溶液经透析袋透析，除去小分子杂质，得到中间体，活体染剂、烷基二胺类化合物、酰化试剂、多聚糖醛酸所有羧基的投料摩尔比值为0-0.999：0.001-100：0.001-100：1；
第二步：将上步所述中间体和酰化试剂溶解于去离子水中，加入金属螯合剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，向其加入金属离子的水溶液，经透析、冷冻干燥处理后得到最终产品，所述金属螯合剂、酰化试剂、金属离子与多聚糖醛酸所有羧基的投料摩尔比值为0.001-100：0.001-100：0.001-100：1。
优选的，两步酰化反应的溶剂为水或DMSO、DMF极性非质子溶剂，反应温度在0至150℃之间。
优选的，酰化反应所用到的酰化试剂选自DMT-MM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、CDMT(2-氯-4,6-二甲氧基-1,3,5-三嗪)、DMT-Pip(4-(4,6-二甲氧基三嗪-2-基)-4-甲基哌啶盐酸盐)、DMT-EMM(4-(4,6-二甲氧基三嗪-2-基)-N-乙基-N,N-二甲基盐酸盐)或DMT-TMM(4-(4,6-二甲氧基三嗪-2-基)-N,N,N-三甲基盐酸盐)中的任意一种，其中，DMT-MM的结构如式(Ⅰ)所示，EDC的结构如式(Ⅱ)所示，CDMT的结构如式(Ⅲ)所示，DMT-Pip的结构如式(Ⅳ)所示，DMT-EMM的结构如式(Ⅴ)所示，DMT-TMM的结构如式(Ⅵ)所示：
Figure PCTCN2016095825-appb-000006
式(Ⅰ)，
Figure PCTCN2016095825-appb-000007
式(Ⅱ)，
Figure PCTCN2016095825-appb-000008
式(Ⅲ)，
Figure PCTCN2016095825-appb-000009
式(Ⅳ)，
Figure PCTCN2016095825-appb-000010
式(Ⅴ)，
Figure PCTCN2016095825-appb-000011
式(Ⅵ)。
本发明的第三个目的在于公开另一种制备多聚糖醛酸为载体的活体染色造影剂的方法，以采取以下步骤：
第一步：将多聚糖醛酸和酰化试剂溶解于溶剂中，随后加入甘露糖或甘露糖衍生物搅拌反应1-24h，反应完毕，淬灭反应，溶液经透析袋透析，除去小分子杂质，使用冷冻干燥法处理得到中间体1，甘露糖或甘露糖衍生物、酰化试剂、多聚糖醛酸所有羧基三者的投料摩尔比值为0-0.999：0.001-100：1；
第二步：将上步所述中间体1和酰化试剂溶解于去离子水中，加入带氨基基团的金属螯合剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，溶液经透析袋透析，除去小分子杂质，使用冷冻干燥法处理得到中间体2；
第三步：将上步所述中间体2和酰化试剂溶解于去离子水中，缓慢加入活体染剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，溶液经透析袋透析，除去小分子杂质，向其加入金属离子的水溶液，经透析、冷冻干燥处理后得到最终产品，所述金属螯合剂、酰化试剂、金属离子与多聚糖醛酸所有羧基的投料摩尔比值为0.001-100：0.001-100：0.001-100：1。
优选的，三步酰化反应的溶剂为水或DMSO、DMF极性非质子溶剂，反应温度在0至150℃之间。
优选的，酰化反应所用到的酰化试剂选自DMT-MM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、CDMT(2-氯-4,6-二甲氧基-1,3,5-三嗪)、DMT-Pip(4-(4,6-二甲氧基三嗪-2-基)-4-甲基哌啶盐酸盐)、DMT-EMM(4-(4,6-二甲氧基三嗪-2-基)-N-乙基-N,N-二甲基盐酸盐)或DMT-TMM(4-(4,6-二甲氧基三嗪-2-基)-N,N,N-三甲基盐酸盐)中的任意一种，其中，DMT-MM的结构如式(Ⅰ)所示，EDC的结构如式(Ⅱ)所示，CDMT的结构如式(Ⅲ)所示，DMT-Pip的结构如式(Ⅳ)所示，DMT-EMM的结构如式(Ⅴ)所示，DMT-TMM的结构如式(Ⅵ)所示：
Figure PCTCN2016095825-appb-000012
式(Ⅰ)，
Figure PCTCN2016095825-appb-000013
式(Ⅱ)，
Figure PCTCN2016095825-appb-000014
式(Ⅲ)，
Figure PCTCN2016095825-appb-000015
式(Ⅳ)，
Figure PCTCN2016095825-appb-000016
式(Ⅴ)，
Figure PCTCN2016095825-appb-000017
式(Ⅵ)。
本发明的第四个目的在于公开所述的活体染色造影剂在制备诊断淋巴系 统疾病的试剂中的应用。
本发明所述造影剂，同时具有甘露糖受体识别片段，顺磁性金属螯合剂以及活体染剂基团，具有淋巴主动靶向性，动物体内实验说明，它能准确确定肿瘤前哨淋巴结状况，术前淋巴结显像，有助于术中前哨淋巴结定位和肿瘤精细外科手术过程中的淋巴清扫术，可以广泛用于乳腺癌、头颈癌、黑素瘤等术前前哨淋巴结定位上。可以同时满足活体检查以及核磁共振造影的要求，是优秀的双功能淋巴靶向造影剂。
本发明的有益效果是：
1、以多聚糖醛酸如海洋褐藻多糖、聚古罗糖醛酸、聚甘露糖醛酸、聚葡萄糖醛酸为载体制备的含有顺磁金属离子以及活体染剂的核磁共振造影剂，不仅在尺度上具有靶向淋巴毛细管和前哨淋巴结的作用，同样具有与淋巴巨噬细胞的甘露糖受体结合作用，与目前美国FDA批准上市的核素造影剂Lymphoseek有相同的功效且可用于淋巴组织活体检查，明显优于专利CN101862461.B发明造影剂HA-DTPA-Gd。
2、大分子纳米级核磁共振造影剂的关键点在于药物的高水溶性。本发明在制备方法上发展了一种水相进行酰胺化反应的方法，利用高效水相缩合剂，方便高产率地制备糖基酰胺，引入螯合试剂，获得水溶性的造影剂。
3、本发明还发展了一种纳米颗粒纯化方法，采用水相透析膜方法，通过分子量控制，获得70-80nm的水溶性造影剂。
附图说明
图1本发明实施例1中新西兰兔皮下注射大分子显影剂PM-TB-GdDTPA后的双侧下肢淋巴MR显影图片及动物解剖图；
其中，A：注射造影剂1h后下肢淋巴结解剖图像；B：注射造影剂1h后下肢淋巴系统MR图像。
具体实施方式
本发明的具体实施方式如下：
以下结合附图和实施例对本发明的技术方案做进一步的说明。
当所述多聚糖醛酸为聚甘露糖醛酸时，具体分子式如下：
Figure PCTCN2016095825-appb-000018
n1为整数、n2、n3和n4为正整数，X为O、N或S；linker为烷基、芳香基或杂环基。配体A为甘露糖或甘露糖衍生物。配体B为顺磁金属螯合物片段。配体C为活体染剂。
当所述多聚糖醛酸为聚古罗糖醛酸时，具体分子式如下：
Figure PCTCN2016095825-appb-000019
n1为整数、n2、n3和n4为正整数，X为O、N或S；linker为烷基、芳香基或杂环基。配体A为甘露糖或甘露糖衍生物。配体B为顺磁金属螯合物片段。配体C为活体染剂。
当所述多聚糖醛酸为多聚半乳糖醛酸时，具体分子式如下：
Figure PCTCN2016095825-appb-000020
n1为整数、n2、n3和n4为正整数，X为O、N或S；linker为烷基、芳香基或杂环基。配体A为甘露糖或甘露糖衍生物。配体B为顺磁金属螯合物片段。配体C为活体染剂。
实施例1：
多聚糖醛酸载体为聚甘露糖醛酸(PM)；n1＝0，n2＝18，n3＝61，n4＝21；X为氮原子(N)；linker1、linker2为二亚甲基氨基，无linker3；甘露糖受体(MBP) 识别基团(配体A)，摩尔含量为0；配体B为金属螯合剂DTPA，摩尔含量为18％；配体C为活体染剂甲苯胺蓝，摩尔含量为21％；顺磁性金属为钆(Gd)；得到造影剂PM-TB-GdDTPA。包括以下步骤:
Figure PCTCN2016095825-appb-000021
将聚甘露糖醛酸PM 3.52g和酰化试剂EDC(1.92g,10mmol)溶于100ml蒸馏水中，60℃搅拌5h后降温至室温，搅拌下缓缓加入烷基二胺类化合物乙二胺和活体染剂甲苯胺蓝(0.306g,1mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到PM-TB-N溶液。
向以上的PM-TB-N溶液中加入酰化试剂EDC(1.92g,10mmol)，搅拌至溶解后缓慢加入DTPA(5.91g,15mmol)，室温搅拌过夜后用饱和碳酸氢钠溶液调pH值至6。搅拌下向溶液中加入GdCl3·6H2O(5.58g,15mmol)，并将反应液pH用饱和碳酸氢钠溶液调至7后装入截留量3500的透析袋，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，浓缩溶液至50ml，加入乙醇50ml浓缩至干带走水分，重复两次得蓝色固体粉末PM-TB-GdDTPA2.9g。
健康的新西兰大白兔1只，体重为3.2kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后于双脚第一、二、三趾蹼处皮下注射大分子双功能显影剂PM-TB-GdDTPA(30mg/L),每次注射0.2ml，进行3D增强扫描。增强扫描序列的相关参数与平扫时一致，每隔15分钟扫描一次，总共扫描5次。扫描结束后，根据MR图像进行动物解剖，获取染色的淋巴结。
实施例1中新西兰兔皮下注射大分子显影剂PM-TB-GdDTPA后的双侧下肢淋巴MR显影图片及动物解剖图片如图1所示，图中A：注射造影剂1h后下肢淋巴结解剖图像；B：注射造影剂1h后下肢淋巴系统MR图像。
结果显示，注射造影剂之前的双侧腘窝淋巴结无显影，定位淋巴结困难。皮下注射PM-TB-GdDTPA 10min后，双侧一级淋巴管和淋巴结信号迅速强化。60min后双侧淋巴结仍显影清晰，动物解剖过程中发现的染色淋巴结位置形状与MR造影实验一致。表明PM-TB-GdDTPA具有生物活体染色和核磁共振双功能淋巴显影的能力。
实施例2：
多聚糖醛酸载体为聚半乳糖糖醛酸(GA)；n1＝11，n2＝16，n3＝63，n4＝10；X为氮原子(N)；linker1、linker2为二亚甲基氨基，无linker3；甘露糖受体(MBP)识别基团(配体A)，摩尔含量为0；配体B为金属螯合剂DTPA，摩尔含量为16％；配体C为活体染剂伊文思蓝，摩尔含量为10％；顺磁性金属为钆(Gd)；得到造影剂GA-EB-GdDTPA。包括以下步骤:
Figure PCTCN2016095825-appb-000022
将多聚半乳糖醛酸GA 3.12g和酰化试剂EDC(1.92g,10mmol)溶于100ml蒸馏水中，60℃搅拌5h后降温至室温，搅拌下缓缓加入烷基二胺类化合物 乙二胺和活体染剂甲苯胺蓝(0.306g,1mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到GA-EB-N溶液。
向以上的GA-EB-N溶液中加入酰化试剂EDC(1.92g,10mmol)，搅拌至溶解后缓慢加入DTPA(5.91g,15mmol)，室温搅拌过夜后用饱和碳酸氢钠溶液调pH值至6。搅拌下向溶液中加入GdCl3·6H2O(5.58g,15mmol)，并将反应液pH用饱和碳酸氢钠溶液调至7后装入截留量3500的透析袋，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，浓缩溶液至50ml，加入乙醇50ml浓缩至干带走水分，重复两次得蓝色固体粉末GA-EB-GdDTPA2.18g。
健康的新西兰大白兔1只，体重为3.1kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例2中合成的GA-EB-GdDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂GA-EB-GdDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例3：
多聚糖醛酸载体为聚甘露糖醛酸(PM)；n1＝8n2＝14n3＝59n4＝19；X为氮原子(N)；linker1为二亚甲基氨基、linker2为苄基，无linker3；配体A为甘露糖(M)，摩尔含量为8％；配体B为金属螯合剂1-(p-氨基苄基)-DTPA，摩尔含量为14％；配体C为活体染剂甲苯胺蓝，摩尔含量为19％；顺磁性金属为钆(Gd)；得到造影剂PM-M-TB-GdNDTPA。包括以下步骤:
Figure PCTCN2016095825-appb-000023
将聚甘露糖醛酸PM(3.52g,20mmol)与酰化试剂EDC(3.83g,20mmol)溶解于去离子水50ml中，室温搅拌下分批缓慢加入1-O-氨基乙基甘露糖(1.42g,5mmol)，室温搅拌反应12h。反应结束，向溶液中加入K2CO3(8.3g,60mmol)后搅拌0.5h；将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到白色固体化合物PM-M 3.3g。
将上一步反应制得的PM-M 3.0g和酰化试剂EDC(1.92g,10mmol)溶解于去离子水100ml中，搅拌下向其加入1-(p-氨基苄基)-DTPA的盐酸盐(1.93g,3mmol)，室温搅拌反应12h。反应结束后向溶液中加入K2CO3(8.3g,60mmol)，搅拌0.5h。将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到白色固体化合物PM-M-NDTPA 3.1g。
将上一步反应制得的PM-M-NDTPA 3g和酰化试剂EDC(1.92g,10mmol)溶于100ml蒸馏水中，搅拌下缓缓加入甲苯胺蓝(0.306g,1mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到PM-M-TB-NDTPA溶液。将反应制得的PM-M-TB-NDTPA溶液，搅拌下向其分批加入GdCl3·6H2O(0.93g,2.5mmol)，5％的NaOH溶液调pH为5～6，室温搅拌1h后将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到蓝色 固体化合物PM-M-TB-GdNDTPA 2.66g。
健康的新西兰大白兔1只，体重为3.2kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例3中合成的PM-M-TB-GdNDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂PM-M-TB-GdNDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例4：
多聚糖醛酸载体为聚古罗糖醛酸(PG)；n1＝0n2＝21n3＝78n4＝1；X为氮原子(N)；linker1、linker2为二亚甲基氨基，无linker3；甘露糖受体(MBP)识别基团(配体A)，摩尔含量为0；配体B为金属螯合剂DTPA，摩尔含量为21％；配体C为活体染剂甲苯胺蓝，摩尔含量为1％；顺磁性金属为钆(Gd)；得到造影剂PG-TB-GdDTPA。包括以下步骤:
将聚古罗糖醛酸(PG)3.2g和酰化试剂DMT-MM(2.76g,10mmol)溶于100ml蒸馏水中，60℃搅拌5h后降温至室温，搅拌下缓缓加入烷基二胺类化合物乙二胺和活体染剂甲苯胺蓝(0.0306g,0.1mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到PG-TB-N溶液。
向以上的PG-TB-N溶液中加入酰化试剂DMT-MM(2.76g,10mmol)，搅拌至溶解后缓慢加入DTPA(5.91g,15mmol)，室温搅拌过夜后饱和碳酸氢钠溶液调pH值至6。搅拌下向溶液中加入GdCl3·6H2O(5.58g,15mmol)，并将反应液pH用饱和碳酸氢钠溶液调至7后装入截留量3500的透析袋，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，浓缩溶液至50ml，加入乙醇50ml浓缩至干带走水分，重复两次得蓝色固体粉末PG-TB-GdDTPA2.7g。
健康的新西兰大白兔1只，体重为3.2kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例4中合成的PG-TB-GdDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂PG-TB-GdDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例5：
多聚糖醛酸载体为聚甘露糖醛酸(PM)；n1＝30n2＝16n3＝40n4＝14；X为氮原子(N)；linker1为二亚甲基氨基、linker2为苄基，无linker3；配体A为甘露糖(M)，摩尔含量为30％；配体B为金属螯合剂1-(p-氨基苄基)-DTPA，摩尔含量为16％；配体C为活体染剂甲苯胺蓝，摩尔含量为14％；顺磁性金属为钆(Gd)；得到造影剂PM-M-TB-GdNDTPA。包括以下步骤:
将聚甘露糖醛酸PM(3.52g,20mmol)与酰化试剂DMT-Pip(3.62g,20mmol)溶解于去离子水50ml中，室温搅拌下分批缓慢加入1-O-氨基乙基甘露糖(2.84g,10mmol)，室温搅拌反应12h。反应结束，向溶液中加入K2CO3(8.3g,60mmol)后搅拌0.5h；将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到白色固体化合物PM-M 3.8g。
将上一步反应制得的PM-M 3.0g和酰化试剂DMT-Pip(1.81g,10mmol)溶解于去离子水100ml中，搅拌下向其加入1-(p-氨基苄基)-DTPA的盐酸盐(0.64g,1mmol)，室温搅拌反应12h。反应结束后向溶液中加入K2CO3(8.3g,60mmol)，搅拌0.5h。将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到白色固体化合物PM-M-NDTPA 3.2g。
将上一步反应制得的PM-M-NDTPA 3g和酰化试剂DMT-Pip(1.81g,10mmol)溶于100ml蒸馏水中，搅拌下缓缓加入甲苯胺蓝(0.306g,1mmol)，室 温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到PM-M-TB-NDTPA溶液。将反应制得的PM-M-TB-NDTPA溶液，搅拌下向其分批加入GdCl3·6H2O(0.93g,2.5mmol)，5％的NaOH溶液调pH为5～6，室温搅拌1h后将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到蓝色固体化合物PM-M-TB-GdNDTPA 2.87g。
健康的新西兰大白兔1只，体重为3.1kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例5中合成的PM-M-TB-GdNDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂PM-M-TB-GdNDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例6：
多聚糖醛酸载体为聚半乳糖糖醛酸(GA)；n1＝10，n2＝40，n3＝32，n4＝18；X为氮原子(N)；linker1、linker2为二亚甲基氨基，无linker3；甘露糖受体(MBP)识别基团(配体A)，摩尔含量为0；配体B为金属螯合剂DTPA，摩尔含量为40％；配体C为活体染剂伊文思蓝，摩尔含量为18％；顺磁性金属为钆(Gd)；得到造影剂GA-EB-GdDTPA。包括以下步骤:
将多聚半乳糖醛酸GA 3.32g和酰化试剂DMT-EMM(1.42g,10mmol)溶于100ml蒸馏水中，60℃搅拌5h后降温至室温，搅拌下缓缓加入烷基二胺类化合物乙二胺和活体染剂甲苯胺蓝(0.612g,2mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到GA-EB-N溶液。
向以上的GA-EB-N溶液中加入酰化试剂DMT-EMM(1.42g,10mmol)，搅 拌至溶解后缓慢加入DTPA(11.82g,30mmol)，室温搅拌过夜后用饱和碳酸氢钠溶液调pH值至6。搅拌下向溶液中加入GdCl3·6H2O(5.58g,15mmol)，并将反应液pH用饱和碳酸氢钠溶液调至7后装入截留量3500的透析袋，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，浓缩溶液至50ml，加入乙醇50ml浓缩至干带走水分，重复两次得蓝色固体粉末GA-EB-GdDTPA3.68g。
健康的新西兰大白兔1只，体重为3.1kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例6中合成的GA-EB-GdDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂GA-EB-GdDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例7：
多聚糖醛酸载体为聚半乳糖糖醛酸(GA)；n1＝15，n2＝18，n3＝37，n4＝30；X为氮原子(N)；linker1、linker2为二亚甲基氨基，无linker3；甘露糖受体(MBP)识别基团(配体A)，摩尔含量为0；配体B为金属螯合剂DTPA，摩尔含量为18％；配体C为活体染剂伊文思蓝，摩尔含量为30％；顺磁性金属为钆(Gd)；得到造影剂GA-EB-GdDTPA。包括以下步骤:
将多聚半乳糖醛酸GA 3.21g和酰化试剂CDMT(1.76g,10mmol)溶于100ml蒸馏水中，60℃搅拌5h后降温至室温，搅拌下缓缓加入烷基二胺类化合物乙二胺和活体染剂甲苯胺蓝(6.12g,20mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到GA-EB-N溶液。
向以上的GA-EB-N溶液中加入酰化试剂CDMT(1.76g,10mmol)，搅拌至溶解后缓慢加入DTPA(1.18g,3mmol)，室温搅拌过夜后用饱和碳酸氢钠溶液 调pH值至6。搅拌下向溶液中加入GdCl3·6H2O(5.58g,15mmol)，并将反应液pH用饱和碳酸氢钠溶液调至7后装入截留量3500的透析袋，使用3L蒸馏水透析，每隔4h换一次水，重复换水5次。透析完毕，浓缩溶液至50ml，加入乙醇50ml浓缩至干带走水分，重复两次得蓝色固体粉末GA-EB-GdDTPA3.48g。
健康的新西兰大白兔1只，体重为3.1kg。用氯胺酮(80mg/kg、1.6ml)及地西泮(5mg/kg、1ml)肌肉注射麻醉新西兰大白兔。将其固定在兔手术台上，进行MRI平扫。相应参数：3D Fast TOF-SPGRCE-MRA序列扫描,Flip Angle 30°,TE 1.6ms,TR4.5ms,视野280×280mm,矩阵360×224,层厚1.0mm,slah70,NEX 2。然后使用实施例7中合成的GA-EB-GdDTPA生理盐水溶液，于新西兰兔双后肢一、二、三趾蹼皮下注射，每次0.1ml，钆浓度为0.03mmol/ml。注射显影剂后，5.5h内每隔15-60min扫描一次，共扫描10次。
注射造影剂GA-EB-GdDTPA15min后双侧淋巴管和淋巴结信号迅速强化；三根一级淋巴管显影清晰，交汇于腘窝淋巴结；二级淋巴管同样显影清晰，由淋巴结向腹腔延伸。注射造影剂6h后，淋巴结和一、二级淋巴管仍保持清晰显影。根据MR图像进行动物解剖，获取染色的淋巴结。
实施例8：
多聚糖醛酸载体为聚葡萄糖醛酸(PGlu)；n1＝23n2＝0n3＝45n4＝32；X为氮原子(N)；linker1为二亚甲基氨基，无linker2以及linker3；配体A为甘露糖(M)，摩尔含量为23％；配体B为金属螯合剂1-(p-氨基苄基)-DTPA，摩尔含量为0％；配体C为活体染剂甲苯胺蓝，摩尔含量为32％；得到造影剂PGlu-M-TB。包括以下步骤:
将聚甘露糖醛酸聚葡萄糖醛酸(PGlu)(3.12g,20mmol)与酰化试剂DMT-TMM(2.32g,20mmol)溶解于去离子水50ml中，室温搅拌下分批缓慢加入1-O-氨基乙基甘露糖(2.84g,10mmol)，室温搅拌反应12h。反应结束，向溶液中加入K2CO3(8.3g,60mmol)后搅拌0.5h；将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到白色固体化合物PGlu-M 3.34g。
将上一步反应制得的PGlu-M 3g和酰化试剂DMT-TMM(2.32g,10mmol)溶于100ml蒸馏水中，搅拌下缓缓加入甲苯胺蓝(0.306g,1mmol)，室温搅拌过夜。反应完毕将反应液装入透析袋(截留量3500)，使用3L蒸馏水透析， 每隔4h换一次水，重复换水5次。透析完毕，将透析袋内溶液浓缩至100ml，得到PGlu-M-TB溶液。将反应制得的PM-M-TB溶液，5％的NaOH溶液调pH为5～6，室温搅拌1h后将溶液置于截留量为3500的透析袋中，使用去离子水5L透析，每隔6h换一次水，总共换水5次。透析结束后将溶液浓缩，冻干后得到蓝色固体化合物PGlu-M-TB 2.42g。
接下来，对由实施例1-8所得到的多聚糖醛酸为载体的活体染色造影剂的造影能力进行了比较，具体如下：
在将实施例1-8所述的多聚糖醛酸为载体的活体染色造影剂经皮下注射后，双侧一级淋巴管和淋巴结信号迅速强化。60min后双侧淋巴结仍显影清晰，动物解剖过程中发现的染色淋巴结位置形状与MR造影实验一致。表明实施例1-8所述的多聚糖醛酸为载体的活体染色造影剂具有生物活体染色和核磁共振双功能淋巴显影的能力，达到了淋巴结和淋巴管的清晰绘图和精确定位(表1)。
表1 本发明实施例中造影剂的造影能力
Figure PCTCN2016095825-appb-000024

Claims (14)

  1. 一种多聚糖醛酸为载体的活体染色造影剂，其具有以下结构：以带有羧基的多聚糖醛酸为载体，所述载体上的6位羧基通过烷基、芳基或杂环基团为连接臂，分别与甘露糖受体MBP识别基团配体A、顺磁性金属螯合物配体B或活体染剂配体C结合，具有以下通式：
    Figure PCTCN2016095825-appb-100001
    其中：n1是整数、n2、n3和n4是正整数；X为O、N或S；linker为烷基、芳香基或杂环基；配体A为甘露糖受体MBP识别基团；配体B为顺磁性金属螯合物；配体C为活体染剂。
  2. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，所述的配体A的摩尔含量占所述的多聚糖醛酸原有羧基的0-30％，配体B的摩尔含量占所述的多聚糖醛酸原有羧基的0-40％，配体C的摩尔含量占所述的多聚糖醛酸原有羧基的1-30％，其中配体A的摩尔含量、配体B的摩尔含量、配体C的摩尔含量以及多聚糖醛酸原有羧基中未反应羧基的摩尔含量总和为100％。
  3. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，所述的多聚糖醛酸载体为聚甘露糖醛酸PM、聚古罗糖醛酸PG、聚葡萄糖醛酸、聚半乳糖醛酸及它们的嵌段共聚物并包括其相应的羧酸盐形式，分子量为100-108Da。
  4. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，与多糖6位羧基通过酰胺键结合的Linker为原子数目1到20的烷基、芳香基或杂环基结构，再与甘露糖受体MBP识别基团配体A、顺磁性金属螯合物配体B或者活体染剂配体C结合。
  5. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，所述多聚糖醛酸的糖醛酸环羧基还以酰胺键连接有甘露糖受体MBP识别基团配体A，为α或β构型的甘露糖及其衍生物，具体结构如下：
    Figure PCTCN2016095825-appb-100002
    其中，R为烃基、羧基、氨基、羟基、卤素。
  6. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，所述顺磁性金属螯合物配体B由金属螯合剂与顺磁性金属离子螯合而成，金属螯合剂为含氨基的金属螯合剂，结构如下：
    Figure PCTCN2016095825-appb-100003
    或金属螯合剂为不含氨基的金属螯合剂，结构如下：
    Figure PCTCN2016095825-appb-100004
    所用顺磁性金属离子为Gd、Mn、Cr、Fe、Co、Ni、La、Tc、Dy或Cu的二价或三价离子。
  7. 根据权利要求1所述的多聚糖醛酸为载体的活体染色造影剂，其特征在于，所述的活体染剂配体C为甲苯胺蓝、伊文思蓝或对氨基偶氮苯。
  8. 一种制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，它包括以下步骤：
    第一步：将多聚糖醛酸和酰化试剂溶解于溶剂中，随后依次加入烷基二胺类化合物、活体染剂搅拌反应1-24h，反应完毕，淬灭反应，溶液经透析袋透析，除去小分子杂质，得到中间体，活体染剂、烷基二胺类化合物、酰化试剂、多聚糖醛酸所有羧基的投料摩尔比值为0-0.999：0.001-100：0.001-100：1；
    第二步：将上步所述中间体和酰化试剂溶解于去离子水中，加入金属螯合剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，向其加入金属离子的水溶液，经透析、冷冻干燥处理后得到最终产品，所述金属螯合剂、酰化试剂、金属离子与多聚糖醛酸所有羧基的投料摩尔比值为0.001-100：0.001-100：0.001-100：1。
  9. 根据权利要求8所述的制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，两步酰化反应的溶剂为水或DMSO、DMF极性非质子溶剂，反应温度在0℃至150℃之间。
  10. 根据权利要求8所述的制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，酰化反应所用到的酰化试剂选自DMT-MM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、CDMT(2-氯-4,6-二甲氧基-1,3,5-三嗪)、DMT-Pip(4-(4,6-二甲氧基三嗪-2-基)-4-甲基哌啶盐酸盐)、DMT-EMM(4-(4,6-二甲氧基三嗪-2-基)-N-乙基-N,N-二甲基盐酸盐)或DMT-TMM(4-(4,6-二甲氧基三嗪-2-基)-N,N,N-三甲基盐酸盐)中的任意一种，其中，DMT-MM的结构如式(Ⅰ)所示，EDC的结构如式(Ⅱ)所示，CDMT的结构如式(Ⅲ)所示，DMT-Pip的结构如式(Ⅳ)所示，DMT-EMM的结构如式(Ⅴ)所示，DMT-TMM的结构如式(Ⅵ)所示：
    Figure PCTCN2016095825-appb-100005
    Figure PCTCN2016095825-appb-100006
  11. 一种制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，以采取以下步骤：
    第一步：将多聚糖醛酸和酰化试剂溶解于溶剂中，随后加入甘露糖或甘露糖衍生物搅拌反应1-24h，反应完毕，淬灭反应，溶液经透析袋透析，除去小分子杂质，使用冷冻干燥法处理得到中间体1，甘露糖或甘露糖衍生物、酰化试剂、多聚糖醛酸所有羧基三者的投料摩尔比值为0-0.999：0.001-100：1；
    第二步：将上步所述中间体1和酰化试剂溶解于去离子水中，加入带氨基基团的金属螯合剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，溶液经透析袋透析，除去小分子杂质，使用冷冻干燥法处理得到中间体2；
    第三步：将上步所述中间体2和酰化试剂溶解于去离子水中，缓慢加入活体染剂搅拌反应1-24h，反应完毕淬灭反应，淬灭反应，溶液经透析袋透析，除去小分子杂质，向其加入金属离子的水溶液，经透析、冷冻干燥处理后得到最终产品，所述金属螯合剂、酰化试剂、金属离子与多聚糖醛酸所有羧基的投料摩尔比值为0.001-100：0.001-100：0.001-100：1。
  12. 根据权利要求11所述的制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，两步酰化反应的溶剂为水或DMSO、DMF极性非质子溶剂，反应温度在0至150℃之间。
  13. 根据权利要求11所述的制备多聚糖醛酸为载体的活体染色造影剂的方法，其特征在于，酰化反应所用到的酰化试剂选自DMT-MM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)、EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)、CDMT(2-氯-4,6-二甲氧基-1,3,5-三嗪)、DMT-Pip(4-(4,6-二甲氧基三嗪-2-基)-4-甲基哌啶盐酸盐)、DMT-EMM(4-(4,6-二甲氧基三嗪-2-基)-N-乙基-N,N-二甲基盐酸盐)、DMT-TMM(4-(4,6-二甲氧基三嗪-2-基)-N,N,N-三甲基盐酸盐)中的任意一种，其中，DMT-MM的结构如式(Ⅰ)所示，EDC的结构如式(Ⅱ)所示，CDMT的结构如式(Ⅲ)所示，DMT-Pip的结构如式(Ⅳ)所示，DMT-EMM的结构如式(Ⅴ)所示，DMT-TMM的结构如式(Ⅵ)所示：
    Figure PCTCN2016095825-appb-100007
  14. 一种如权利要求1中所述的多聚糖醛酸为载体的活体染色造影剂在制备诊断淋巴系统疾病的试剂中的应用。
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