Polysaccharide contrast agent capable of realizing kidney metabolism and preparation method thereof

Abstract

The invention belongs to the technical field of medical detection, and particularly relates to a polysaccharide contrast agent capable of realizing kidney metabolism and a preparation method thereof. The polysaccharide contrast agent has a structure as shown in a formula (I), the particle size is smaller than or equal to 8 nm, and the polysaccharide contrast agent can be rapidly metabolized through the kidney. The preparation method comprises the following steps: grafting a metal macrocyclic ligand to nano cross-linked polysaccharide particles, and carrying out deprotection and metal ion chelation to prepare the nano cross-linked polysaccharide. The contrast agent has the characteristic of kidney metabolism, so that the phagocytosis of a reticuloendothelial system is reduced, the in-vivo deposition of the contrast agent is reduced, and the biological safety is improved. Due to the use of the macrocyclic chelating ligand, good dynamic stability is achieved, the risk of in-vivo metal ion deposition is reduced, and clear imaging can be conducted on cardiovascular, lymph nodes, livers and other parts when the compound is applied to in-vivo imaging. The preparation method disclosed by the invention is simple and easy to implement, mild in reaction condition and beneficial to large-scale production and clinical conversion, and has a wide biomedical application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a polysaccharide contrast agent capable of being metabolized by kidney and a preparation method thereof. Background Art

[0002] With the emergence of technologies such as Magnetic Resonance Imaging (MRI), Nuclear Medicine Imaging, and Optical Imaging, medical imaging technology has gradually become an indispensable tool in clinical disease diagnosis. In clinical applications, it is often necessary to select corresponding contrast agents to improve the sensitivity of imaging. For example, with the help of efficient contrast agents, early-stage diseases can be accurately detected, responses to drug treatments can be monitored, or functional cell migration can be tracked.

[0003] Among contrast agents, nanoparticles are becoming candidates for high-performance next-generation contrast agents due to their small size, high specific surface area, and easy modification. Among them, natural polysaccharides have been widely used in the field of nanobiomaterials and contrast agent coatings or carrier materials due to their polyhydroxylation, good water solubility, good biosafety, low immunogenicity, low toxicity, and degradability. For example, dextran-coated iron oxide nanoparticles, hyaluronic acid-based nanomicelle contrast agents, etc.

[0004] Unlike traditional small molecule contrast agents, nanoparticle contrast agents are easily engulfed by the mononuclear phagocyte system and the endothelial reticular system in the body, thereby prolonging the retention time of the contrast agent in the body and increasing the risk of metal ion deposition in the body. The deposition of metal ions will greatly increase the toxicity of contrast agents to the human body. For example, it has been reported that patients with end-stage renal disease will induce nephrogenic fibrotic skin disease after using gadolinium-based contrast agents (Nephrology Dialysis Transplantation, 2006, 21(4), 1104–1108.). The reason may be that gadolinium dissociates from the chelate, and the free gadolinium ions will be deposited in the tissue in the form of phosphate, carbonate, hydroxide or citrate complexes. Therefore, by reducing the retention time of contrast agents in the body, manufacturing nanoparticles with optimal clearance characteristics will minimize the toxicity risks and disease problems caused by them.

[0005] After entering the body, nanocontrast agents are excreted through the kidneys, which is an ideal route for their elimination. Compared with intracellular catabolism in the hepatobiliary system, renal metabolism reduces the possibility of retention and cytotoxicity. Studies have shown that molecules with a hydrodynamic diameter of less than 8 nm can usually be directly filtered by the glomerulus (ACS Nano, 2015, 9(7), 6655–6674.). Therefore, the renal clearance rate of nanoparticles can be improved by reducing the particle size of nanoparticles.

[0006] Therefore, there is an urgent need to design and develop new nanocontrast agents that can be metabolized by the kidney and have good contrast effects. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a polysaccharide contrast agent that can be metabolized by the kidney and a preparation method thereof.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention provides a polysaccharide contrast agent that can be metabolized by the kidney, having a structure as shown in formula (I):

[0010]

[0011] Wherein, m and k are positive integers; n=1, 2, 3, 4; M is a metal ion; and the molecular weight of the polysaccharide contrast agent is 1 to 200 kDa.

[0012] As a preferred technical solution, the metal ion M is Gd 3+ , Mn 2+ , Fe 3+ , Fe 2+ , 64 Cu, 68 Ga, 89 Zr, 99 Any one of Te, Tb, Nd or Eu.

[0013] The metal chelate is grafted onto the cross-linked polysaccharide via epoxy groups, as shown in formula (III), wherein n=1, 2, 3, 4. Preferably, n=2.

[0014]

[0015] As a preferred technical solution, the particle size of the polysaccharide contrast agent is less than or equal to 8 nm.

[0016] The polysaccharide contrast agent of the present invention has good metal chelation kinetic stability, and the polysaccharide contrast agent can maintain chelation stability within 100 hours under the competition of other metal ions.

[0017] The second aspect of the present invention provides a method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0018] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent into alkaline solution, cross-link at 20-30°C for 5-10h, and the obtained product is cross-linked polysaccharide;

[0019] The polysaccharide in the polysaccharide stock solution is a sugar chain formed by glycosidic bonds of monosaccharides and its derivatives, preferably any one of dextran, carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomaltoside or curdlan, and the molecular weight of the polysaccharide is 1 to 200 kDa;

[0020] S2. Grafting: dissolving the cross-linked polysaccharide in an organic solvent, wherein the organic solvent is any one of dimethyl sulfoxide, methanol, N,N-dimethylformamide, cyclohexane, acetone, ethyl acetate or toluene, preferably dimethyl sulfoxide; then adding a metal chelate ligand protected by a carboxyl protecting group, and reacting at 75-85°C for 48-72h;

[0021] S3. Deprotection: removing the carboxyl protecting group of the product obtained in S2 to obtain a polysaccharide-metal chelate ligand;

[0022] S4. Chelation: Chelate metal ions on polysaccharide-metal chelate ligands to produce polysaccharide contrast agents that can be metabolized by the kidneys.

[0023] As a preferred technical solution, the cross-linking agent in step S1 has a structure as shown in formula (II):

[0024]

[0025] Wherein, k is a positive integer, and X is fluorine, chlorine, bromine or iodine.

[0026] More preferably, the cross-linking agent is any one of epichlorohydrin, epibromohydrin, epichlorohydrin or epibromohydrin.

[0027] As a preferred technical solution, the concentration of the alkaline solution in step S1 is 0.3-5 mol / L, the concentration of the polysaccharide stock solution is 0.02-0.5 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.2-2:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 0.1-2 g / ml;

[0028] In step S2, the weight ratio of the cross-linked polysaccharide to the metal chelate ligand is 0.5-10:1, and the mass volume ratio of the cross-linked polysaccharide to the organic solvent is 0.08-0.3 g / ml.

[0029] As a preferred technical solution, the carboxyl protecting group described in steps S2 and S3 is any one of methyl, ethyl, propyl, butyl, tert-butyl, allyl, benzyl, 4-methoxybenzyl or 4-methoxyphenyl. The carboxyl protecting group can be hydrolyzed and removed under the catalysis of acid or base. The reagent for removing the carboxyl protecting group is trifluoroacetic acid, water, lithium hydroxide, sodium hydroxide, potassium hydroxide, methanol or ethanol and a combination thereof. Preferably, it is a combination of trifluoroacetic acid and water, a combination of lithium hydroxide and methanol, or a combination of lithium hydroxide and ethanol. In order to optimize the deprotection effect, phenol, triisopropylsilane, etc. can be added to the deprotection reagent. In the present invention, the deprotection reagent is more preferably a combination of trifluoroacetic acid, phenol, water and triisopropylsilane.

[0030] As a preferred technical solution, the specific operation of chelation in step S4 is: dissolving the polysaccharide-metal chelate ligand and the metal ion in deionized water at a molar ratio of 1:10-200, adjusting the pH value of the solution to 6-7, heating to 60-80°C for chelation for 12-24 hours, and dialyzing to remove the unchelated metal ions. The resulting product is a polysaccharide contrast agent that can be metabolized by the kidney.

[0031] In the present invention, the metal chelate ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), the carboxyl group of which is protected by a tert-butyl group to become 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester (DO3Atbu), and the carboxyl group of DO3A is protected by an ethyl group to become 1,4,7,10-tetraazacyclododecane-1,4,7-triethyl ester (DO3AOEt).

[0032] The present invention has the following advantages: the present invention discloses a polysaccharide contrast agent that can be metabolized by the kidney and a preparation method thereof. The particle size of the polysaccharide contrast agent is less than or equal to 8nm and can be rapidly metabolized by the kidney. The preparation method is to graft a metal macrocyclic ligand onto nano cross-linked particles and prepare the contrast agent through the steps of deprotection and chelation of metal ions. Since the metal macrocyclic ligand reacts with the epoxy group on the cross-linked polysaccharide through a secondary amine, the reaction site is controllable and the product structure is clear. Since the contrast agent has the characteristic of being metabolizable by the kidney, the phagocytosis of the reticuloendothelial system is reduced, the in vivo deposition of the contrast agent is reduced, and thus the biosafety is improved. Since a macrocyclic chelating ligand is used, the contrast agent has good kinetic stability, reduces the risk of metal ion deposition in the body, and can clearly image the cardiovascular, lymph nodes, liver and other parts when applied to in vivo imaging. The preparation method disclosed by the present invention is simple and easy to implement, the reaction conditions are mild, it is conducive to large-scale production and clinical transformation, and has broad prospects for biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1This is the H NMR spectrum of the intermediate product of the synthesis process of the dextran nanoparticle magnetic resonance contrast agent in Example 1. a and b in the figure are characteristic peaks of the epoxy group, c is the characteristic peak of the tert-butyl group, and d is the characteristic peak of DO3A.

[0034] Figure 2 The hydrated particle size diagram of cross-linked dextran T10 and dextran-DO3A-Gd.

[0035] Figure 3 This is a diagram of the hydrated particle size of the pullulan nanoparticle magnetic resonance contrast agent of Example 2.

[0036] Figure 4 The hydrated particle size diagram of cross-linked dextran with different molecular weights.

[0037] Figure 5 This is a relaxation efficiency diagram of the dextran nanoparticle magnetic resonance contrast agent of Example 1.

[0038] Figure 6 This is a diagram showing the kinetic stability results of the dextran nanoparticle magnetic resonance contrast agent of Example 1; wherein A is the dissociation of metal ions in the presence of high concentrations of hydrogen ions; and B is the dissociation of metal ions in the presence of high concentrations of zinc ions.

[0039] Figure 7 This is a graph showing the experimental results of the cytotoxicity of dextran nanoparticle magnetic resonance contrast agent on macrophage Raw264.7 in Example 1.

[0040] Figure 8 This is the vascular MRI signal image of SD rats after injection of the dextran nanoparticle magnetic resonance contrast agent of Example 1. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and examples, and the protection scope of the present invention is not limited to the following: Example 1: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0042] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 20°C for 5 hours, and the resulting product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is mannan, and the cross-linking agent is epichlorohydrin. The concentration of the alkaline solution is 0.3 mol / L, the concentration of the polysaccharide stock solution is 0.02 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.2:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 0.1 g / ml;

[0043] S2. Grafting: dissolving a cross-linked polysaccharide in dimethyl sulfoxide, adding DO3Atbu, and reacting at 75°C for 48 hours to obtain a product of polysaccharide-DO3Atbu; the weight ratio of the cross-linked polysaccharide to DO3Atbu is 0.5:1, and the mass volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide is 0.08 g / ml;

[0044] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is taken and divided into two parts, and cyclopentane and sodium acetate are dissolved in one part of N,N-dimethylacetamide to obtain a mixed solution as mixed solution A; tert-butyl bromoacetate is dissolved in another part of N,N-dimethylacetamide to obtain a mixed solution as mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 110 hours, and the product obtained by the reaction is DO3Atbu; wherein the concentration of cyclopentane in the mixed solution A is 0.05 g / ml, and the concentration of sodium acetate is 0.08 g / ml; the concentration of tert-butyl bromoacetate in the mixed solution B is 0.15 g / ml; the molar ratio of tert-butyl bromoacetate in the mixed solution B to cyclopentane in the mixed solution A is 2.5:1;

[0045] S3. Deprotection: Deprotection of polysaccharide-DO3Atbu to remove the tert-butyl group to obtain polysaccharide-DO3A; the reagent for removing the tert-butyl group is a combination of trifluoroacetic acid and dichloromethane, with a volume ratio of 2:1;

[0046] S4. Chelation: Chelating polysaccharide-DO3A nanoparticles and metal ions Mn 2+ The solution is dissolved in deionized water at a molar ratio of 1:10, the pH value of the solution is adjusted to 6, heated to 60°C for chelation for 12 hours, and dialyzed to remove unchelated metal ions. The resulting product is a polysaccharide contrast agent that can be metabolized by the kidney.

[0047] Example 2: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0048] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 30°C for 10 hours, and the resulting product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is isomaltose, and the cross-linking agent is epibromopropane. The concentration of the alkaline solution is 5 mol / L, the concentration of the polysaccharide stock solution is 0.5 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 2:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 2 g / ml;

[0049] S2. Grafting: dissolving the cross-linked polysaccharide in methanol, adding DO3Atbu, and reacting at 85°C for 72h to obtain a product of polysaccharide-DO3Atbu; the weight ratio of the cross-linked polysaccharide to DO3Atbu is 10:1, and the mass volume ratio of the cross-linked polysaccharide to methanol is 0.3g / ml;

[0050] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is taken and divided into two parts, and cyclopentane and sodium acetate are dissolved in one part of N,N-dimethylacetamide to obtain a mixed solution as mixed solution A; tert-butyl bromoacetate is dissolved in another part of N,N-dimethylacetamide to obtain a mixed solution as mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 130 hours, and the product obtained by the reaction is DO3Atbu; wherein the concentration of cyclopentane in the mixed solution A is 0.12 g / ml, and the concentration of sodium acetate is 0.2 g / ml; the concentration of tert-butyl bromoacetate in the mixed solution B is 1.5 g / ml; the molar ratio of tert-butyl bromoacetate in the mixed solution B to cyclopentane in the mixed solution A is 3.5:1;

[0051] S3. Deprotection: The polysaccharide-DO3Atbu is deprotected to remove the tert-butyl group to obtain the polysaccharide-DO3A; the reagent for removing the tert-butyl group is a combination of trifluoroacetic acid and water, with a volume ratio of 19:1;

[0052] S4. Chelation: Chelating polysaccharide-DO3A nanoparticles and metal ions 89 Zr is dissolved in deionized water at a molar ratio of 1:200, the pH value of the solution is adjusted to 7, heated to 80°C for chelation for 24 hours, and dialyzed to remove unchelated metal ions. The resulting product is a polysaccharide contrast agent that can be metabolized by the kidney.

[0053] Example 3: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0054] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 23°C for 6 hours, and the resulting product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is fucoidan, and the cross-linking agent is epichlorohydrin. The concentration of the alkaline solution is 0.5 mol / L, the concentration of the polysaccharide stock solution is 0.1 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.5:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 0.6 g / ml;

[0055] S2. Grafting: dissolving a cross-linked polysaccharide in dimethyl sulfoxide, adding DO3Atbu, and reacting at 78°C for 52 hours to obtain a product of polysaccharide-DO3Atbu; the weight ratio of the cross-linked polysaccharide to DO3Atbu is 1:1, and the mass volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide is 0.15 g / ml;

[0056] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is taken and divided into two parts, and cyclopentane and sodium acetate are dissolved in one part of N,N-dimethylacetamide, and the obtained mixed solution is mixed solution A; tert-butyl bromoacetate is dissolved in another part of N,N-dimethylacetamide, and the obtained mixed solution is mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 118 hours, and the reaction product is DO3Atbu; wherein the concentration of cyclopentane in the mixed solution A is 0.08 g / ml, and the concentration of sodium acetate is 0.12 g / ml; the concentration of tert-butyl bromoacetate in the mixed solution B is 0.6 g / ml; the molar ratio of tert-butyl bromoacetate in the mixed solution B to cyclopentane in the mixed solution A is 2.8:1;

[0057] S3. Deprotection: Deprotection of polysaccharide-DO3Atbu to remove the tert-butyl group to obtain polysaccharide-DO3A; the reagent for removing the tert-butyl group is a combination of trifluoroacetic acid, water and phenol in a volume ratio of 19:5:1;

[0058] S4. Chelation: Chelating polysaccharide-DO3A nanoparticles and metal ions 99 Te is dissolved in deionized water at a molar ratio of 1:80, the pH value of the solution is adjusted to 6.5, heated to 65°C for chelation for 16 hours, and dialyzed to remove unchelated metal ions. The resulting product is a contrast agent.

[0059] Example 4: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0060] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 25°C for 7 hours, and the obtained product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is chitosan, and the cross-linking agent is butyl bromide. The concentration of the alkaline solution is 1.6 mol / L, the concentration of the polysaccharide stock solution is 0.2 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.7:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 1 g / ml;

[0061] S2. Grafting: dissolving a cross-linked polysaccharide in dimethyl sulfoxide, adding 1,4,7,10-tetraazacyclododecane-1,4,7-triethyl acetate (DO3AOEt), and reacting at 82°C for 65h to obtain a product of polysaccharide-DO3AOEt; the weight ratio of the cross-linked polysaccharide to DO3AOEt is 4.5:1, and the mass volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide is 0.25g / ml;

[0062] The preparation method of DO3AOEt is as follows: acetonitrile is divided into two parts, 1,4,7,10-tetraazacyclododecane and sodium bicarbonate are dissolved in one part of acetonitrile, the obtained mixed solution is mixed solution A, and the mixture is stirred and cooled in an ice bath at -10°C; ethyl bromoacetate is dissolved in another part of acetonitrile, the obtained mixed solution is mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 48 hours, and the reaction product is DO3AOEt; wherein the concentration of 1,4,7,10-tetraazacyclododecane in the mixed solution A is 0.1 g / ml, and the concentration of sodium acetate is 0.2 g / ml; the concentration of ethyl bromoacetate in the mixed solution B is 0.16 g / ml; the molar ratio of ethyl bromoacetate in the mixed solution B to 1,4,7,10-tetraazacyclododecane in the mixed solution A is 2.8:1;

[0063] S3. Deprotection: deprotecting the polysaccharide-DO3AOEt to remove the ethyl group, thereby obtaining the polysaccharide-DO3A; the reagent for removing the ethyl group is a combination of water and ethanol in a volume ratio of 1:1;

[0064] S4. Chelation: Dissolve polysaccharide-DO3A nanoparticles and metal ions Nd in deionized water at a molar ratio of 1:150, adjust the pH value of the solution to 7, heat to 75°C for chelation for 22 hours, and dialyze to remove unchelated metal ions. The resulting product is a contrast agent.

[0065] Example 5: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0066] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 28°C for 8 hours, and the resulting product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is gellan, and the cross-linking agent is epichlorohydrin. The concentration of the alkaline solution is 2.5 mol / L, the concentration of the polysaccharide stock solution is 0.3 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.8:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 1.2 g / ml;

[0067] S2. Grafting: dissolving a cross-linked polysaccharide in dimethyl sulfoxide, adding DO3AOEt, and reacting at 80°C for 60h to obtain a product of polysaccharide-DO3AOEt; the weight ratio of the cross-linked polysaccharide to DO3AOEt is 6:1, and the mass volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide is 0.15g / ml;

[0068] The preparation method of DO3AOEt is as follows: acetonitrile is divided into two parts, 1,4,7,10-tetraazacyclododecane and sodium bicarbonate are dissolved in one part of acetonitrile, the obtained mixed solution is mixed solution A, and stirred and cooled in an ice bath at -10°C; ethyl bromoacetate is dissolved in another part of acetonitrile, the obtained mixed solution is mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 48 hours, and the reaction product is DO3AOEt; wherein the concentration of 1,4,7,10-tetraazacyclododecane in the mixed solution A is 0.05 g / ml, and the concentration of sodium acetate is 0.09 g / ml; the concentration of ethyl bromoacetate in the mixed solution B is 0.08 g / ml; the molar ratio of ethyl bromoacetate in the mixed solution B to 1,4,7,10-tetraazacyclododecane in the mixed solution A is 3:1;

[0069] S3. Deprotection: The polysaccharide-DO3Atbu is deprotected to remove the ethyl group to obtain the polysaccharide-DO3A; the reagent for removing the ethyl group is a combination of sodium hydroxide, ethanol and water in a volume ratio of 1:15:15;

[0070] S4. Chelation: Dissolve polysaccharide-DO3A nanoparticles and metal ions Eu in deionized water at a molar ratio of 1:60, adjust the pH value of the solution to 7, heat to 63°C for chelation for 13 hours, and dialyze to remove unchelated metal ions. The resulting product is a contrast agent.

[0071] Example 6: A method for preparing a polysaccharide contrast agent that can be metabolized by the kidney, comprising the following steps:

[0072] S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent to alkaline solution, cross-link at 25°C for 9 hours, and the resulting product is cross-linked polysaccharide; wherein the polysaccharide in the polysaccharide stock solution is heparin, and the cross-linking agent is epibromopropane. The concentration of the alkaline solution is 4 mol / L, the concentration of the polysaccharide stock solution is 0.4 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.5:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 1.5 g / ml;

[0073] S2. Grafting: dissolving the cross-linked polysaccharide in N,N-dimethylformamide (DMF), adding DO3AOEt, and reacting at 78°C for 70h to obtain a product of polysaccharide-DO3AOEt; the weight ratio of the cross-linked polysaccharide to DO3AOEt is 8:1, and the mass volume ratio of the cross-linked polysaccharide to DMF is 0.28g / ml;

[0074] The preparation method of DO3AOEt is as follows: acetonitrile is divided into two parts, 1,4,7,10-tetraazacyclododecane and sodium bicarbonate are dissolved in one part of acetonitrile, the obtained mixed solution is mixed solution A, and the mixture is stirred and cooled in an ice bath at -10°C; ethyl bromoacetate is dissolved in another part of acetonitrile, the obtained mixed solution is mixed solution B; mixed solution B is added dropwise to mixed solution A at 0°C, and magnetic stirring is performed at room temperature for 48 hours, and the product obtained by the reaction is DO3AOEt; wherein the concentration of 1,4,7,10-tetraazacyclododecane in the mixed solution A is 0.02 g / ml, and the concentration of sodium acetate is 0.05 g / ml; the concentration of ethyl bromoacetate in the mixed solution B is 0.05 g / ml; the molar ratio of ethyl bromoacetate in the mixed solution B to 1,4,7,10-tetraazacyclododecane in the mixed solution A is 3.2:1;

[0075] S3. Deprotection: deprotect the polysaccharide-DO3AOEt to remove the ethyl group, thereby obtaining the polysaccharide-DO3A; the reagent for removing the ethyl group is a combination of methanol, ethanol and water in a volume ratio of 1:2:2;

[0076] S4. Chelation: Chelating polysaccharide-DO3A nanoparticles and metal ions 89 Zr is dissolved in deionized water at a molar ratio of 1:190, the pH value of the solution is adjusted to 7.5, and the solution is heated to 60°C for chelation for 23 hours. Unchelated metal ions are removed by dialysis, and the resulting product is a contrast agent.

[0077] The beneficial effects of the present invention are described below by experiments:

[0078] The polysaccharide in the polysaccharide stock solution of the present invention is any one of dextran, carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomalt or curdlan, because carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomalt or curdlan are all condensed by multiple monosaccharide molecules or their derivatives like dextran, and all have a large number of hydroxyl groups. Therefore, the use of a cross-linking agent that can cross-link with hydroxyl groups can make the polysaccharide cross-linked to form nano polysaccharide particles. When a polysaccharide with a smaller molecular weight is used, nano polysaccharide particles with a smaller particle size can be produced to meet the basic requirement of kidney clearance. Therefore, the present invention uses dextran as an example to carry out experimental description.

[0079] Example 1:

[0080] (1) Cross-linking: Weigh 1.8 g of dextran T10 and dissolve it in 9 ml of deionized water. Add 15 ml of 1.6 M sodium hydroxide solution to the solution. Then slowly drop 6.939 g of epichlorohydrin into the solution. React at room temperature for 6 hours under mechanical stirring. Dialyze the mixture after the reaction and precipitate it with ethanol twice to obtain a white precipitate. Freeze-dry the white precipitate to obtain the product cross-linked dextran. Use sodium thiosulfate titration to determine the amount of epoxy on the nanoparticles.

[0081] (2) Grafting:

[0082] Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tri-tert-butyl ester (DO3Atbu): Dissolve 2 g of cyclopentane and 2.85 g of sodium acetate in 25 ml of N,N-dimethylacetamide. Dissolve 6.79 g of tert-butyl bromoacetate in N,N-dimethylacetamide and add dropwise into the above solution at 0°C. Stir at 25°C for 5 days. After post-treatment, white powder DO3Atbu is obtained.

[0083] 1.5 g of cross-linked dextran was dissolved in 10 ml of dimethyl sulfoxide, and 463.23 mg of DO3Atbu was added. The mixture was stirred at 80°C for 3 days. The reaction product was dialyzed, concentrated, and freeze-dried to obtain yellow powder dextran-DO3Atbu.

[0084] (3) Deprotection: Deprotection with trifluoroacetic acid to obtain the product dextran-DO3A. The deprotection reagent formula is: a mixture of trifluoroacetic acid, phenol, water and triisopropylsilane, with a volume ratio of 88:5:5:2.

[0085] (4) Chelation: Dissolve 750 mg of dextran-DO3A in 15 ml of deionized water and adjust the pH to 5-6. Dissolve 750 mg of gadolinium chloride hexahydrate in deionized water and add dropwise to the above solution to adjust the pH to 6. The mixed solution is reacted at 60°C for 24 hours. The solution is then dialyzed and freeze-dried to obtain the product dextran-DO3A-Gd.

[0086] Example 2:

[0087] (1) Cross-linking: Weigh 1.8 g of pullulan and dissolve it in 9 ml of deionized water. Add 15 ml of 1.6 M sodium hydroxide solution to the solution. Then slowly add 6.939 g of epichlorohydrin dropwise to the solution. React at room temperature for 6 hours under mechanical stirring. Dialyze the reaction mixture and precipitate it twice with ethanol to obtain a white precipitate. Freeze-dry the white precipitate to obtain the product cross-linked pullulan. Use sodium thiosulfate titration to determine the amount of epoxy on the nanoparticles.

[0088] (2) Grafting:

[0089] White powder DO3Atbu was obtained by the method of Example 1. 1.5 g of cross-linked pullulan was dissolved in 10 ml of dimethyl sulfoxide, and 463.23 mg of DO3Atbu was added. The mixture was stirred at 80° C. for 3 days. The reaction product was dialyzed, concentrated, and freeze-dried.

[0090] (3) Deprotection: The yellow powder obtained was deprotected using trifluoroacetic acid to obtain the product pullulan-DO3A. A mixture of trifluoroacetic acid, phenol, water and triisopropylsilane was prepared in a volume ratio of 88:5:5:2.

[0091] (4) Chelation: Dissolve 750 mg of pullulan-DO3A in 15 ml of deionized water and adjust the pH to 5-6. Dissolve 750 mg of gadolinium chloride hexahydrate in deionized water and add dropwise into the above solution to adjust the pH to 6. The mixed solution is reacted at 60°C for 24 hours. The solution is then dialyzed and freeze-dried to obtain the product pullulan-DO3A-Gd.

[0092] The contrast agents synthesized in Example 1 and Example 2 were tested for the following physicochemical properties:

[0093] 1. Detection of the synthesis process of dextran nanoparticle magnetic resonance contrast agent by H NMR spectrum

[0094] 10 mg of each of dextran T10, cross-linked dextran, dextran-DO3Atbu, and dextran-DO3A were dissolved in deuterated water or deuterated dimethyl sulfoxide and detected on a 400 MHz nuclear magnetic resonance hydrogen spectrometer. Figure 1 As shown, peaks a and b indicate grafted epoxy groups, peak c indicates the presence of tert-butyl groups on DO3Atbu, and peak d shows the presence of methylene groups on DO3A. The successful synthesis of cross-linked polysaccharides and pre-chelated metal ion dextran nanoparticle magnetic resonance contrast agents can be demonstrated by hydrogen nuclear magnetic resonance spectra.

[0095] In the cross-linking of polysaccharides, the molecular weight of the polysaccharide in the polysaccharide stock solution is 1 to 200 kDa, and the molecular weight after cross-linking can be a times of 1 to 200 kDa, where a is a positive integer. The molecular weight of polymers is usually measured by gel chromatography (GPC), but the volume exclusion effect changes after the polysaccharide is cross-linked, and its molecular weight cannot be accurately determined. However, if a suitable alkali solution concentration and cross-linking agent concentration are used, a cross-linked polysaccharide with a particle size smaller than that of the polysaccharide raw material can be obtained. The reason is that due to the occurrence of intramolecular cross-linking, the polysaccharide is transformed from a linear state to a spherical state. Therefore, preferably, the present invention uses a cross-linked polysaccharide with a=1 and applies it to the preparation of a contrast agent, which has the characteristic of a small molecular particle size.

[0096] 2. Detection of the Hydration Particle Size of Polysaccharide Nanoparticle Magnetic Resonance Contrast Agents

[0097] (1) Take dextran (T10) with a molecular weight of 10,000, and prepare cross-linked dextran using the cross-linking method in Example 1 to obtain cross-linked dextran T10. Take 10 mg of cross-linked dextran T10 and dextran-DO3A-Gd prepared in Example 1 and dissolve them in 1 ml of water respectively, and measure the hydrated particle size of the nanocontrast agent under a Malvern particle size analyzer. The experimental results are shown in FIG. Figure 2 As shown by Figure 2 It can be seen that the particle size of cross-linked dextran did not change after grafting chelate DO3A and chelating metal ions, and was 4nm±2nm. The reason is that the grafted chelate DO3A is a small molecule, and whether it is grafted or not will not change the hydrated particle size of the nanoparticles. Therefore, we believe that the particle size of the synthesized polysaccharide nanoparticle contrast agent is consistent with that of cross-linked dextran.

[0098] (2) 10 mg of dextran-DO3A-Gd prepared in Example 1 and 10 mg of pullulan-DO3A-Gd prepared in Example 2 were dissolved in 1 ml of water respectively, and the hydrated particle size of the nanocontrast agent was measured using a Malvern particle size analyzer. The results are as follows: Figure 2 and Figure 3 As shown, the hydrated particle sizes of the synthesized dextran nanoparticle magnetic resonance contrast agent and pullulan nanoparticle magnetic resonance contrast agent are 4nm±2nm and 5nm±2nm, respectively, which are smaller than the size threshold of kidney metabolism and can therefore be metabolized by the kidney.

[0099] (3) Dextran with different molecular weights (T40, T100, T200) was taken separately, and cross-linked dextran was prepared by the cross-linking method in Example 1 to obtain cross-linked dextran with different molecular weights (T40, T100, T200).

[0100] 10 mg of cross-linked dextran with different molecular weights (T40, T100, T200) were dissolved in 1 ml of water, and the hydrated particle size of the nanocontrast agent was measured using a Malvern particle size analyzer. Figure 4 As shown, after cross-linking with dextran T40, T100, and T200, the particle size is still below 8 nm. From the experimental results of (1), it can be seen that the particle size of the corresponding contrast agent is also below 8 nm, which meets the basic particle size requirements for glomerular filtration.

[0101] 3. Evaluation of the Relaxation Efficacy of Dextran Nanoparticle Magnetic Resonance Contrast Agents

[0102] Dextran-DO3A-Gd prepared in Example 1 was prepared with final Gd concentrations of 0.4, 0.3, 0.25, 0.1, and 0.03 mmol / L, and its T1 relaxation efficiency was tested at 1.5 T and 3 T. The measurement parameters are as follows:

[0103] 1.5T: Scan sequence: spin echo; repetition time = 150.0ms; slice thickness = 3mm; echo time = 13.6ms; average times = 5; echo sequence length = 1; slice spacing = 3mm; acquisition matrix = 192\0\0\224; flip angle = 90°; field of view = 140mm*120mm.

[0104] 3.0T: Scan sequence: spin echo; repetition time = 70 ms; slice thickness = 2 mm; echo time = 6.3 ms; average times = 3; echo train length = 1; acquisition matrix = 256 × 152; flip angle = 90°. All tests were performed at room temperature.

[0105] The results are as follows Figure 5 As shown, the T1 relaxation efficiency at 1.5T is 18.5Gd mM -1 s -1 ， The T1 relaxation efficiency at 3T is 16.7Gd mM -1 s -1 .

[0106] 4. Evaluation of the Kinetic Stability of Dextran Nanoparticle Magnetic Resonance Contrast Agents

[0107] First, prepare concentrated hydrochloric acid with a concentration of 1 mol / L, and add the dextran-DO3A-Gd, Gd-DTPA, Gd-DOTA, Gd-DO3A, and GdCl3 solutions prepared in Example 1 to make the final concentration of gadolinium ions 1mmol / L, and detect the T2 relaxation time at different time points in a 3T magnetic field. Secondly, prepare dextran-DO3A-Gd, Gd-DTPA, Gd-DOTA, Gd-DO3A, and GdCl3 solutions with a concentration of 10mM using PBS, and add ZnCl2 solution to make the final concentration of zinc ions 10mmol / L. Detect the T2 relaxation time at different time points in a 3T magnetic field.

[0108] The results are as follows Figure 6 As shown, Figure 6 A shows that under strong acid conditions, the dissociation rate of Gd ions in dextran-DO3A-Gd is slower than that of commercial Gd-DTPA. Figure 6 B shows that in the presence of high concentration of zinc ions, the Gd ion chelation in dextran-DO3A-Gd is stable and does not gradually dissociate over time like Gd-DTPA, and can maintain chelation stability within 100 hours.

[0109] 5. Evaluation of the biosafety of dextran nanoparticle magnetic resonance contrast agents

[0110] The CCK8 method was used to evaluate the cytotoxicity of dextran-DO3A-Gd prepared in Material Example 1 on macrophage Raw264.7. Raw264.7 was incubated at 1×10 4 The cells were seeded in a 96-well plate at a concentration of 100 μM per well and incubated for 24 hours. After the cells adhered, dextran-DO3A-Gd and commercial contrast agent gadobutrol diluted in culture medium were added to a final concentration of 0, 25, 50, 100, 200, and 500 μM, and cultured for another 24 hours. After the incubation was completed, the supernatant was discarded and washed twice with PBS, and 100 μl of serum-free culture medium containing 10% CCK-8 was added to each well. After incubation in the dark for 1.5 hours, the absorbance at 450 nm was measured in a microplate reader and the cell survival rate was calculated.

[0111] The results are as follows Figure 7 As shown in the results, neither dextran-DO3A-Gd nor the commercial contrast agent gadobutrol produced significant toxicity to macrophages at low or high concentrations.

[0112] 6. Evaluation of the in vivo vascular imaging effect of dextran nanoparticle magnetic resonance contrast agent

[0113] The in vivo MRI effect was evaluated by tail vein injection of dextran-DO3A-Gd (dose: 0.05 mmol Gd / kg body weight) prepared in Example 1 into SD rats (180-200 g), and the control group was injected with the same concentration of gadobutrol. The specific steps were as follows: first, a tail vein indwelling needle was placed in the SD rat, and the vascular MRI signal image of the SD rat before injection was obtained (3.0T magnetic resonance scanning system, Siemens), and then the vascular MRI signal image of the SD rat was obtained again 1 minute after the contrast agent was injected through the indwelling needle.

[0114] The results are as follows Figure 8 As shown, compared with the small molecule DO3A gadolinium chelate, dextran-DO3A-Gd has a clearer vascular imaging effect.

[0115] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A polysaccharide contrast agent metabolizable by the kidney, characterized in that: It has a structure as shown in formula (I): Wherein, m and k are positive integers; n=1, 2, 3, 4; M is a metal ion; and the molecular weight of the polysaccharide contrast agent is 1 to 200 kDa.

2. A polysaccharide contrast agent metabolizable by the kidney according to claim 1, characterized in that: The metal ion M is Gd 3+ , Mn 2+ , Fe 3+ , Fe 2+ , 64 Cu, 68 Ga, 89 Zr, 99 Any one of Te, Tb, Nd or Eu.

3. A polysaccharide contrast agent metabolizable by the kidney according to claim 1, characterized in that: The particle size of the polysaccharide contrast agent is less than or equal to 8 nm.

4. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 1, characterized in that: The following steps are involved: S1. Cross-linking: Add polysaccharide stock solution and cross-linking agent into alkaline solution, cross-link at 20-30°C for 5-10h, and the obtained product is cross-linked polysaccharide; The polysaccharide in the polysaccharide stock solution is any one of dextran, carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomaltoside or curdlan, and the molecular weight of the polysaccharide is 1 to 200 kDa; S2. Grafting: dissolving the cross-linked polysaccharide in an organic solvent, wherein the organic solvent is any one of dimethyl sulfoxide, methanol, N,N-dimethylformamide, cyclohexane, acetone, ethyl acetate or toluene, and then adding a metal chelate ligand protected by a carboxyl protecting group, and reacting at 75-85°C for 48-72h; S3. Deprotection: removing the carboxyl protecting group of the product obtained in S2 to obtain a polysaccharide-metal chelate ligand; S4. Chelation: Chelate metal ions on polysaccharide-metal chelate ligands to produce polysaccharide contrast agents that can be metabolized by the kidneys.

5. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 4, characterized in that: The cross-linking agent in step S1 has a structure as shown in formula (II): Wherein, k is a positive integer, and X is fluorine, chlorine, bromine or iodine.

6. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 5, characterized in that: The cross-linking agent is any one of epichlorohydrin, epibromohydrin, epichlorohydrin or epichlorohydrin.

7. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 4, characterized in that: The metal chelate ligand is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

8. The method for preparing a polysaccharide contrast agent that can be metabolized by the kidney according to claim 4, characterized in that: In step S1, the concentration of the alkaline solution is 0.3-5 mol / L, the concentration of the polysaccharide stock solution is 0.02-0.5 g / ml, the volume ratio of the alkaline solution to the polysaccharide stock solution is 1.2-2:1, and the mass volume ratio of the cross-linking agent to the polysaccharide stock solution is 0.1-2 g / ml; In step S2, the weight ratio of the cross-linked polysaccharide to the metal chelate ligand is 0.5-10:1, and the mass volume ratio of the cross-linked polysaccharide to the organic solvent is 0.08-0.3 g / ml.

9. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 4, characterized in that: The carboxyl protecting group in steps S2 and S3 is any one of methyl, ethyl, propyl, butyl, tert-butyl, allyl, benzyl, 4-methoxybenzyl or 4-methoxyphenyl.

10. The method for preparing a polysaccharide contrast agent metabolizable by the kidney according to claim 4, characterized in that: The specific operation of chelation in step S4 is: dissolving the polysaccharide-metal chelate ligand and the metal ion in deionized water at a molar ratio of 1:10-200, adjusting the pH value of the solution to 6-7, heating to 60-80°C for chelation for 12-24 hours, and dialyzing to remove the unchelated metal ions. The resulting product is a polysaccharide contrast agent that can be metabolized by the kidney.

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