A renally metabolizable polysaccharide contrast agent and a method for its preparation

Abstract

The application belongs to the technical field of medical detection, and particularly relates to a polysaccharide contrast agent capable of renal metabolism and a preparation method thereof. The polysaccharide contrast agent has a structure as shown in formula (I), a particle size less than or equal to 8 nm, and can be rapidly metabolized through the kidney. The preparation method is to graft a metal macrocyclic ligand to a nano-crosslinked polysaccharide particle, and to prepare through a deprotection and metal ion chelation step. Since the contrast agent has the characteristic of renal metabolism, phagocytosis of the reticuloendothelial system is reduced, in vivo deposition of the contrast agent is reduced, and thus the biological safety is improved. Since a macrocyclic chelating ligand is used, good kinetic stability is achieved, the risk of in vivo metal ion deposition is reduced, and when applied to in vivo imaging, clear imaging of cardiovascular, lymph node, liver and other parts can be achieved. The preparation method is simple and easy to implement, reaction conditions are mild, is conducive to large-scale production and clinical transformation, and has a wide biomedical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically relating to a renally metabolizable polysaccharide contrast agent and its preparation method. Background Technology

[0002] With the advent of technologies such as magnetic resonance imaging (MRI), nuclear medicine imaging, and optical imaging, medical imaging has gradually become an indispensable tool in clinical disease diagnosis. In clinical applications, appropriate contrast agents are often selected to improve imaging sensitivity. For example, highly effective contrast agents can be used to accurately detect early-stage diseases, monitor responses to drug treatment, or track the migration of functional cells.

[0003] In contrast agents, nanoparticles are emerging as candidates for high-performance next-generation contrast agents due to their advantages such as small size, high specific surface area, and ease of modification. Among these, natural polysaccharides, with their multiple hydroxyl groups, good water solubility, good biocompatibility, low immunogenicity, low toxicity, and biodegradability, have been widely used in the fields of nanobiomaterials and contrast agent coatings or carriers. Examples include dextran-encapsulated iron oxide nanoparticles and hyaluronic acid-based nanomicelle contrast agents.

[0004] Unlike traditional small-molecule contrast agents, nanoparticle contrast agents are easily phagocytosed by the mononuclear phagocyte system and endothelial reticulum system in vivo, thus prolonging the retention time of the contrast agent and increasing the risk of metal ion deposition. Metal ion deposition can significantly increase the toxicity of contrast agents to the human body. For example, there are reports of end-stage renal disease patients developing nephrotic fibrosis after using gadolinium-based contrast agents (Nephrology Dialysis Transplantation, 2006, 21(4), 1104–1108). This may be because gadolinium dissociates from the chelate, and the free gadolinium ions deposit in tissues as phosphate, carbonate, hydroxide, or citrate complexes. Therefore, by reducing the in vivo retention time of contrast agents, manufacturing nanoparticles with optimal clearance properties will minimize the toxic risks and disease problems they pose.

[0005] After entering the body, nanocontrast agents are ideally eliminated through renal excretion. This is because renal metabolism reduces the likelihood of retention and cytotoxicity compared to intracellular catabolism via the hepatobiliary system. Studies have shown that molecules with a hydrodynamic diameter less than 8 nm can typically be directly filtered by the glomerulus (ACSNano, 2015, 9(7), 6655–6674). Therefore, the renal clearance rate of nanoparticles can be improved by reducing their particle size.

[0006] Therefore, there is an urgent need to design and develop novel nanocontrast agents that can be metabolized by the kidneys and have good imaging effects. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a renally metabolizable polysaccharide contrast agent and its preparation method.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first aspect of this invention provides a renally metabolizable polysaccharide contrast agent having a structure as shown in formula (I):

[0010]

[0011] Where 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 1-200 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] Metal chelates are grafted onto cross-linked polysaccharides via epoxy groups, as shown in formula (III), where 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 for 100 hours under the competition of other metal ions.

[0017] A second aspect of this invention provides a method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0018] S1. Crosslinking: Add polysaccharide stock solution and crosslinking agent to alkaline solution, and crosslink at 20-30℃ for 5-10 hours. The product obtained is crosslinked polysaccharide.

[0019] Wherein, the polysaccharide in the polysaccharide stock solution is a sugar chain and its derivative formed by monosaccharides linked by glycosidic bonds, preferably any one of dextran, carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomaltose or gel polysaccharide, and the molecular weight of the polysaccharide is 1 to 200 kDa.

[0020] S2. Grafting: The cross-linked polysaccharide is dissolved 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 a metal chelate ligand protected by a carboxyl protecting group is added, and the reaction is carried out at 75-85°C for 48-72 h;

[0021] S3. Deprotection: Remove the carboxyl protecting group from the product obtained in S2 to prepare a polysaccharide-metal chelate ligand;

[0022] S4. Chelation: Metal ions are chelated onto polysaccharide-metal chelate ligands to prepare a polysaccharide contrast agent that can be metabolized by the kidneys.

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

[0024]

[0025] Where k is a positive integer, and X is an element of fluorine, chlorine, bromine, or iodine.

[0026] More preferably, the crosslinking agent is any one of epichlorohydrin, epibromopropane, epichlorohydrin, or epibromobutane.

[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 crosslinking 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 to 10:1, and the mass-volume ratio of the cross-linked polysaccharide to the organic solvent is 0.08 to 0.3 g / ml.

[0029] As a preferred technical solution, 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. The carboxyl protecting group can be removed by hydrolysis under acid or base catalysis. The reagent for removing the carboxyl protecting group is trifluoroacetic acid, water, lithium hydroxide, sodium hydroxide, potassium hydroxide, methanol, or ethanol, or combinations thereof. Preferably, it is a combination of trifluoroacetic acid and water, lithium hydroxide and methanol, or lithium hydroxide and ethanol. To optimize the deprotection effect, phenol, triisopropylsilane, etc., can be added to the deprotection reagent. In this 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 as follows: dissolve the polysaccharide-metal chelating ligand and the metal ion in deionized water at a molar ratio of 1:10-200, adjust the pH of the solution to 6-7, heat to 60-80℃ for chelation for 12-24 hours, dialyze to remove the unchelated metal ions, and the resulting product is a renally metabolizable polysaccharide contrast agent.

[0031] In this invention, the metal chelating ligand is preferably 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), whose carboxyl group is protected by tert-butyl to form 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tritert-butyl ester (DO3Atbu), and DO3A whose carboxyl group is protected by ethyl to form 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid ethyl ester (DO3AOEt).

[0032] This invention offers the following advantages: It discloses a renally metabolizable polysaccharide contrast agent and its preparation method. The polysaccharide contrast agent has a particle size of less than or equal to 8 nm, enabling rapid renal metabolism. The preparation method involves grafting a macrocyclic metal ligand onto cross-linked nanoparticles, followed by deprotection and chelation of metal ions. Because the macrocyclic metal ligand reacts with the epoxy groups on the cross-linked polysaccharide via a secondary amine, the reaction site is controllable, resulting in a clear product structure. Due to its renally metabolizable nature, this contrast agent reduces phagocytosis by the reticuloendothelial system and in vivo deposition, thereby improving biosafety. The use of a macrocyclic chelating ligand provides good kinetic stability, reducing the risk of in vivo metal ion deposition, and enabling clear imaging of cardiovascular systems, lymph nodes, and the liver when applied to in vivo imaging. The preparation method disclosed in this invention is simple and easy to implement, with mild reaction conditions, facilitating large-scale production and clinical translation, and possesses broad prospects for biomedical applications. Attached Figure Description

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

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

[0035] Figure 3 The hydrated particle size diagram of pullulanose nanoparticle magnetic resonance contrast agent in Example 2 is shown.

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

[0037] Figure 5 The relaxation efficiency diagram is shown for the magnetic resonance contrast agent of dextran nanoparticles in Example 1.

[0038] Figure 6 The figure shows the kinetic stability results of the magnetic resonance contrast agent of dextran nanoparticles in Example 1; where A represents the dissociation of metal ions in the presence of high concentration of hydrogen ions; and B represents the dissociation of metal ions in the presence of high concentration of zinc ions.

[0039] Figure 7 The figure shows the cytotoxicity results of the magnetic resonance contrast agent for dextran nanoparticles on macrophages Raw264.7 in Example 1.

[0040] Figure 8 This is an MRI image of the blood vessels of SD rats after injection of the magnetic resonance contrast agent containing dextran nanoparticles, as described in Example 1. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following: Embodiment 1: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0042] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution and crosslinked at 20°C for 5 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the polysaccharide stock solution is mannan, and the crosslinking 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 crosslinking agent to the polysaccharide stock solution is 0.1 g / ml.

[0043] S2. Grafting: The cross-linked polysaccharide was dissolved in dimethyl sulfoxide, and then DO3Atbu was added. The reaction was carried out at 75°C for 48 hours to obtain the polysaccharide-DO3Atbu. The weight ratio of the cross-linked polysaccharide to DO3Atbu was 0.5:1, and the mass-volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide was 0.08 g / ml.

[0044] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is divided into two parts. Cyclocarpine and sodium acetate are dissolved in one part of N,N-dimethylacetamide, and the resulting mixed solution is mixture A. Tert-butyl bromoacetate is dissolved in the other part of N,N-dimethylacetamide, and the resulting mixed solution is mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 110 hours. The product obtained is DO3Atbu. The concentration of cyclocarpine in mixture A is 0.05 g / ml, and the concentration of sodium acetate is 0.08 g / ml. The concentration of tert-butyl bromoacetate in mixture B is 0.15 g / ml. The molar ratio of tert-butyl bromoacetate in mixture B to cyclocarpine in mixture A is 2.5:1.

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

[0046] S4. Chelation: This involves combining polysaccharide-DO3A nanoparticles with metal ions (Mn). 2+ Dissolve the contrast agent in deionized water at a molar ratio of 1:10, adjust the pH of the solution to 6, heat to 60°C for chelation for 12 hours, and dialyze to remove unchelated metal ions. The resulting product is a renally metabolizable polysaccharide contrast agent.

[0047] Example 2: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0048] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution and crosslinked at 30°C for 10 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the stock solution is isomaltose, and the crosslinking agent is epichlorohydrin. 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 crosslinking agent to the polysaccharide stock solution is 2 g / ml.

[0049] S2. Grafting: The cross-linked polysaccharide was dissolved in methanol, and then DO3Atbu was added. The reaction was carried out at 85°C for 72 h to obtain the product polysaccharide-DO3Atbu. The weight ratio of the cross-linked polysaccharide to DO3Atbu was 10:1, and the mass-volume ratio of the cross-linked polysaccharide to methanol was 0.3 g / ml.

[0050] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is divided into two parts. Cyclocarpine and sodium acetate are dissolved in one part of N,N-dimethylacetamide, and the resulting mixed solution is mixture A. Tert-butyl bromoacetate is dissolved in the other part of N,N-dimethylacetamide, and the resulting mixed solution is mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 130 hours. The product obtained is DO3Atbu. The concentration of cyclocarpine in mixture A is 0.12 g / ml, and the concentration of sodium acetate is 0.2 g / ml. The concentration of tert-butyl bromoacetate in mixture B is 1.5 g / ml. The molar ratio of tert-butyl bromoacetate in mixture B to cyclocarpine in mixture A is 3.5:1.

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

[0052] S4. Chelation: Combining polysaccharide-DO3A nanoparticles with metal ions 89 Zr was dissolved in deionized water at a molar ratio of 1:200, the pH of the solution was adjusted to 7, and the solution was heated to 80°C for chelation for 24 hours. Unchelated metal ions were removed by dialysis, and the resulting product was a renally metabolizable polysaccharide contrast agent.

[0053] Example 3: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0054] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution and crosslinked at 23°C for 6 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the stock solution is fucoidan, and the crosslinking 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 crosslinking agent to the polysaccharide stock solution is 0.6 g / ml.

[0055] S2. Grafting: The cross-linked polysaccharide was dissolved in dimethyl sulfoxide, and then DO3Atbu was added. The reaction was carried out at 78°C for 52 h to obtain the product polysaccharide-DO3Atbu. The weight ratio of the cross-linked polysaccharide to DO3Atbu was 1:1, and the mass-volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide was 0.15 g / ml.

[0056] The preparation method of DO3Atbu is as follows: N,N-dimethylacetamide is divided into two parts. Cyclocarpine and sodium acetate are dissolved in one part of N,N-dimethylacetamide, and the resulting mixed solution is mixture A. Tert-butyl bromoacetate is dissolved in the other part of N,N-dimethylacetamide, and the resulting mixed solution is mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 118 hours. The product obtained is DO3Atbu. The concentration of cyclocarpine in mixture A is 0.08 g / ml, and the concentration of sodium acetate is 0.12 g / ml. The concentration of tert-butyl bromoacetate in mixture B is 0.6 g / ml. The molar ratio of tert-butyl bromoacetate in mixture B to cyclocarpine in mixture A is 2.8:1.

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

[0058] S4. Chelation: Combining polysaccharide-DO3A nanoparticles with metal ions 99 Te was dissolved in deionized water at a molar ratio of 1:80. The pH of the solution was adjusted to 6.5, and the solution was heated to 65°C for chelation for 16 hours. Unchelated metal ions were removed by dialysis, and the resulting product was the contrast agent.

[0059] Example 4: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0060] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution and crosslinked at 25°C for 7 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the polysaccharide stock solution is chitosan, and the crosslinking agent is epoxybutane. 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 crosslinking agent to the polysaccharide stock solution is 1 g / ml.

[0061] S2. Grafting: The cross-linked polysaccharide was dissolved in dimethyl sulfoxide, and then 1,4,7,10-tetraazacyclododecane-1,4,7-triethyl acetate (DO3AOEt) was added. The reaction was carried out at 82°C for 65 h to obtain the polysaccharide-DO3AOEt. The weight ratio of the cross-linked polysaccharide to DO3AOEt was 4.5:1, and the mass-to-volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide was 0.25 g / 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 the acetonitrile, resulting in mixture A, which is then stirred and cooled in an ice bath at -10°C. Ethyl bromoacetate is dissolved in the other part of the acetonitrile, resulting in mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 48 hours. The product obtained is DO3AOEt. The concentration of 1,4,7,10-tetraazacyclododecane in mixture A is 0.1 g / ml, and the concentration of sodium acetate is 0.2 g / ml. The concentration of ethyl bromoacetate in mixture B is 0.16 g / ml. The molar ratio of ethyl bromoacetate in mixture B to 1,4,7,10-tetraazacyclododecane in mixture A is 2.8:1.

[0063] S3. Deprotection: The polysaccharide-DO3AOEt was deprotected to remove the ethyl group, yielding polysaccharide-DO3A; the reagent for removing the ethyl group was a combination of water and ethanol in a volume ratio of 1:1.

[0064] S4. Chelation: Polysaccharide-DO3A nanoparticles and metal ions Nd are dissolved in deionized water at a molar ratio of 1:150. The pH of the solution is adjusted to 7, and the solution is heated to 75°C for chelation for 22 hours. Unchelated metal ions are removed by dialysis. The resulting product is the contrast agent.

[0065] Example 5: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0066] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution, and crosslinking is carried out at 28°C for 8 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the polysaccharide stock solution is a gel polysaccharide, and the crosslinking 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 crosslinking agent to the polysaccharide stock solution is 1.2 g / ml.

[0067] S2. Grafting: The cross-linked polysaccharide was dissolved in dimethyl sulfoxide, and then DO3AOEt was added. The mixture was reacted at 80°C for 60 h to obtain the polysaccharide-DO3AOEt. The weight ratio of the cross-linked polysaccharide to DO3AOEt was 6:1, and the mass-volume ratio of the cross-linked polysaccharide to dimethyl sulfoxide was 0.15 g / 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 the acetonitrile, resulting in mixture A, which is then stirred and cooled in an ice bath at -10°C. Ethyl bromoacetate is dissolved in the other part of the acetonitrile, resulting in mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 48 hours. The product obtained is DO3AOEt. The concentration of 1,4,7,10-tetraazacyclododecane in mixture A is 0.05 g / ml, and the concentration of sodium acetate is 0.09 g / ml. The concentration of ethyl bromoacetate in mixture B is 0.08 g / ml. The molar ratio of ethyl bromoacetate in mixture B to 1,4,7,10-tetraazacyclododecane in mixture A is 3:1.

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

[0070] S4. Chelation: Polysaccharide-DO3A nanoparticles and metal ions Eu are dissolved in deionized water at a molar ratio of 1:60. The pH of the solution is adjusted to 7, and the solution is heated to 63°C for chelation for 13 hours. Unchelated metal ions are removed by dialysis. The resulting product is the contrast agent.

[0071] Example 6: A method for preparing a renally metabolizable polysaccharide contrast agent, comprising the following steps:

[0072] S1. Crosslinking: Polysaccharide stock solution and crosslinking agent are added to an alkaline solution and crosslinked at 25°C for 9 hours. The resulting product is crosslinked polysaccharide. The polysaccharide in the stock solution is heparin, and the crosslinking agent is epichlorohydrin. 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 crosslinking agent to the polysaccharide stock solution is 1.5 g / ml.

[0073] S2. Grafting: The cross-linked polysaccharide was dissolved in N,N-dimethylformamide (DMF), and then DO3AOEt was added. The reaction was carried out at 78°C for 70 h to obtain the product polysaccharide-DO3AOEt. The weight ratio of the cross-linked polysaccharide to DO3AOEt was 8:1, and the mass-volume ratio of the cross-linked polysaccharide to DMF was 0.28 g / 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 the acetonitrile, resulting in mixture A, which is then stirred and cooled in an ice bath at -10°C. Ethyl bromoacetate is dissolved in the other part of the acetonitrile, resulting in mixture B. Mixture B is added dropwise to mixture A at 0°C, and the mixture is magnetically stirred at room temperature for 48 hours. The product obtained is DO3AOEt. The concentration of 1,4,7,10-tetraazacyclododecane in mixture A is 0.02 g / ml, and the concentration of sodium acetate is 0.05 g / ml. The concentration of ethyl bromoacetate in mixture B is 0.05 g / ml. The molar ratio of ethyl bromoacetate in mixture B to 1,4,7,10-tetraazacyclododecane in mixture A is 3.2:1.

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

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

[0077] The following experiments illustrate the beneficial effects of this invention:

[0078] The polysaccharide in the polysaccharide stock solution of this invention is any one of dextran, carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomaltose, or gel polysaccharide. Since carboxymethyl dextran, pullulan, mannan, chitosan, fucoidan, seaweed polysaccharide, hyaluronic acid, heparin, isomaltose, or gel polysaccharide, like dextran, are derived from the condensation of multiple monosaccharide molecules or their derivatives, and all possess a large number of hydroxyl groups, crosslinking agents capable of crosslinking with hydroxyl groups can be used to crosslink the polysaccharides to form nano-polysaccharide particles. When using polysaccharides with smaller molecular weights, even smaller nano-polysaccharide particles can be produced, thus meeting the basic requirements for renal clearance. Therefore, this invention uses dextran as an example for experimental illustration.

[0079] Example 1:

[0080] (1) Crosslinking: 1.8 g of dextran T10 was weighed and dissolved in 9 ml of deionized water. 15 ml of 1.6 M sodium hydroxide solution was added to the solution. Then, 6.939 g of epichlorohydrin was slowly added dropwise to the above solution. The reaction was carried out at room temperature for 6 hours with mechanical stirring. The resulting mixture was dialyzed and precipitated twice with ethanol to obtain a white precipitate. The white precipitate was lyophilized to obtain the product, crosslinked dextran. The amount of epoxy on the nanoparticles was determined by sodium thiosulfate titration.

[0081] (2) Grafting:

[0082] Synthesis of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid tritert-butyl ester (DO3Atbu): 2 g of cyclohexane and 2.85 g of sodium acetate were dissolved in 25 ml of N,N-dimethylacetamide. 6.79 g of tert-butyl bromoacetate was dissolved in N,N-dimethylacetamide and added dropwise to the above solution at 0 °C. The mixture was stirred at 25 °C for 5 days. After post-treatment, a white powder, DO3Atbu, was obtained.

[0083] Dissolve 1.5 g of cross-linked dextran in 10 ml of dimethyl sulfoxide, and add 463.23 mg of DO3Atbu. Stir the mixture at 80 °C for 3 days. Dialyze, concentrate, and lyophilize the reaction product to obtain a yellow powder, dextran-DO3Atbu.

[0084] (3) Deprotection: Deprotection was performed using trifluoroacetic acid to obtain the product dextran-DO3A. The deprotection reagent was formulated as a mixture of trifluoroacetic acid, phenol, water and triisopropylsilane in 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 it dropwise to the above solution, adjusting the pH to 6. React the mixed solution at 60 °C for 24 hours. Then dialyze the solution and freeze-dry to obtain the product dextran-DO3A-Gd.

[0086] Example 2:

[0087] (1) Crosslinking: 1.8 g pullulanose was dissolved in 9 ml of deionized water, and 15 ml of 1.6 M sodium hydroxide solution was added to the solution. Then, 6.939 g of epichlorohydrin was slowly added dropwise to the above solution. The reaction was carried out at room temperature for 6 hours with mechanical stirring. The resulting mixture was dialyzed and precipitated twice with ethanol to obtain a white precipitate. The white precipitate was lyophilized to obtain the product crosslinked pullulanose. The amount of epoxy on the nanoparticles was determined by sodium thiosulfate titration.

[0088] (2) Grafting:

[0089] A white powder, DO3Atbu, was obtained by the method in Example 1. 1.5 g of cross-linked pullulanose 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 lyophilized.

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

[0091] (4) Chelation: Dissolve 750 mg pullulanose-DO3A in 15 ml of deionized water and adjust the pH to 5-6. Dissolve 750 mg gadolinium chloride hexahydrate in deionized water and add it dropwise to the above solution, adjusting the pH to 6. React the mixed solution at 60 °C for 24 hours. Then dialyze the solution and freeze-dry to obtain the product pullulanose-DO3A-Gd.

[0092] The contrast agents synthesized in Examples 1 and 2 were subjected to the following physicochemical property tests:

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

[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 proton nuclear magnetic resonance spectrometer. The results are as follows: Figure 1 As shown, peaks a and b indicate the grafted epoxy groups, peak c indicates the presence of a tert-butyl group on DO3Atbu, and peak d shows the presence of a methylene group on DO3A. The successful synthesis of the cross-linked polysaccharide and chelated metal ion pre-dextran nanoparticle magnetic resonance contrast agent can be confirmed by the 1H NMR spectrum.

[0095] In the cross-linking of polysaccharides, the molecular weight of the polysaccharide in the original solution is 1-200 kDa, and the molecular weight after cross-linking can be a times 1-200 kDa, where a is a positive integer. The molecular weight of polymers is usually measured using gel permeation chromatography (GPC), but the size exclusion effect changes after polysaccharide cross-linking, making accurate molecular weight determination impossible. However, by using appropriate alkali concentration and cross-linking agent concentration, cross-linked polysaccharides with particle sizes smaller than the polysaccharide raw material can be obtained. This is because intramolecular cross-linking occurs, transforming the polysaccharide from a linear state to a spherical state. Therefore, preferably, this invention uses cross-linked polysaccharides with a=1 and applies them to the preparation of contrast agents, which have the characteristic of small molecular particle size.

[0096] 2. Detection of the hydrated particle size of polysaccharide nanoparticle magnetic resonance contrast agent

[0097] (1) Dextran with a molecular weight of 10000 (T10) was used to prepare cross-linked dextran using the cross-linking method described in Example 1, resulting in cross-linked dextran T10. 10 mg of cross-linked dextran T10 and the dextran-DO3A-Gd prepared in Example 1 were dissolved in 1 ml of water, and the hydrated particle size of the nano-contrast agent was measured using a Malvern particle size analyzer. The experimental results are as follows: Figure 2 As shown, by Figure 2 It can be seen that the particle size of cross-linked dextran remained unchanged after grafting with the chelate DO3A and chelating metal ions, remaining at 4nm ± 2nm. This is because the grafted chelate DO3A is a small molecule, and grafting does not alter 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 the cross-linked dextran.

[0098] (2) 10 mg of dextran-DO3A-Gd prepared in Example 1 and 10 mg of pullulanose-DO3A-Gd prepared in Example 2 were dissolved in 1 ml of water, and the hydrated particle size of the nanocontrast agents 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 pullulanose nanoparticle magnetic resonance contrast agent are 4nm±2nm and 5nm±2nm, respectively, which are smaller than the size threshold for renal metabolism, and therefore can be metabolized by the kidneys.

[0099] (3) Take dextran with different molecular weights (T40, T100, T200) and prepare cross-linked dextran using 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 of different molecular weights (T40, T100, T200) were dissolved in 1 ml of water, and the hydrated particle size of the nano-contrast agent was measured using a Malvern particle size analyzer. The experimental results are as follows: Figure 4 As shown, after cross-linking with dextran T40, T100 and T200, the particle size is still below 8 nm. As can be seen from the experimental results of (1), 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 efficiency 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 1.5T and 3T. 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 number of 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 = 70ms; slice thickness = 2mm; echo time = 6.3ms; average number of times = 3; echo column length = 1; acquisition matrix = 256×152; flip angle = 90°. All detections were performed at room temperature.

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

[0106] 4. Evaluate the kinetic stability of dextran nanoparticle magnetic resonance contrast agents.

[0107] First, a 1 mol / L concentrated hydrochloric acid solution was prepared, and the gadolinium ion concentrations of the dextran-DO3A-Gd, Gd-DTPA, Gd-DOTA, Gd-DO3A, and GdCl3 solutions prepared in Example 1 were added to it to achieve a final gadolinium ion concentration of 1 mmol / L. The T2 relaxation time was then measured at different time points in a 3T magnetic field. Next, a 10 mM dextran-DO3A-Gd, Gd-DTPA, Gd-DOTA, Gd-DO3A, and GdCl3 solution was prepared using PBS, and a ZnCl2 solution was added to achieve a final zinc ion concentration of 10 mmol / L. The T2 relaxation time was then measured 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 strongly acidic conditions, the dissociation rate of Gd ions in dextran-DO3A-Gd is slower than that in commercially available Gd-DTPA. From... Figure 6 As can be seen from B, in the presence of high concentrations of zinc ions, the Gd ions in dextran-DO3A-Gd are stably chelated and do not gradually dissociate over time like Gd-DTPA, and can maintain stable chelation for up to 100 hours.

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

[0110] The cytotoxicity of dextran-DO3A-Gd prepared in Material Example 1 against Raw264.7 macrophages was evaluated using the CCK8 assay. Raw264.7 was tested at a concentration of 1 × 10⁻⁶ g / L. 4 Cells were seeded in 96-well plates at the specified concentrations and incubated for 24 hours. After cell attachment, diluted dextran-DO3A-Gd and commercial contrast agent gadobutrol were added to final concentrations of 0, 25, 50, 100, 200, and 500 μM, respectively, and cultured for another 24 hours. After incubation, the supernatant was discarded, and the cells were washed twice with PBS. 100 μl of serum-free 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 using a microplate reader, and cell viability was calculated.

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

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

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

[0114] The results are as follows Figure 8 As shown, compared to small molecule DO3A gadolinium chelates, dextran-DO3A-Gd exhibits clearer vascular imaging results.

[0115] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A renally metabolizable polysaccharide contrast agent, characterized in that, It has a structure as shown in equation (Ⅰ): ， Equation (I) Wherein, m and k are positive integers; n = 1 or 2; M is a metal ion; the molecular weight of the polysaccharide contrast agent is 1 to 200 kDa; and the particle size of the polysaccharide contrast agent is less than or equal to 8 nm.

2. The renally metabolizable polysaccharide contrast agent 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 Any one of Zr, Tb, Nd, or Eu.

3. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 1, characterized in that, Includes the following steps: S1. Crosslinking: Add polysaccharide stock solution and crosslinking agent to alkaline solution, and crosslink at 20-30 ℃ for 5-10 h. The product obtained is crosslinked polysaccharide. The polysaccharide in the polysaccharide stock solution is any one of dextran, pullulan, or mannan, and the molecular weight of the polysaccharide is 1–200 kDa; the crosslinking agent has a structure as shown in formula (II): ， Formula (II) Where k is a positive integer, and X is a fluorine, chlorine, bromine, or iodine element; S2. Grafting: The cross-linked polysaccharide is dissolved in an organic solvent, wherein the organic solvent is any one of dimethyl sulfoxide, N,N-dimethylformamide, cyclohexane, acetone, ethyl acetate or toluene, and a metal chelate ligand protected by a carboxyl protecting group is added, and the reaction is carried out at 75-85 °C for 48-72 h. S3. Deprotection: Remove the carboxyl protecting group from the product obtained in S2 to prepare the polysaccharide-metal chelate ligand; S4. Chelation: Metal ions are chelated onto polysaccharide-metal chelate ligands to prepare a renally metabolizable polysaccharide contrast agent.

4. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 3, characterized in that, The crosslinking agent is any one of epichlorohydrin, epibromopropane, epichlorohydrin, or epibromobutane.

5. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 3, characterized in that, The metal chelating ligand is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

6. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 3, 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 crosslinking 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 to 10:1, and the mass-volume ratio of the cross-linked polysaccharide to the organic solvent is 0.08 to 0.3 g / ml.

7. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 3, characterized in that, The carboxyl protecting group mentioned in steps S2 and S3 is any one of methyl, ethyl, propyl, butyl, allyl, benzyl, 4-methoxybenzyl, or 4-methoxyphenyl.

8. The method for preparing a renally metabolizable polysaccharide contrast agent according to claim 3, characterized in that, The specific operation of chelation in step S4 is as follows: dissolve the polysaccharide-metal chelating ligand and the metal ion in deionized water at a molar ratio of 1:10-200, adjust the pH of the solution to 6-7, heat to 60-80 ℃ for chelation for 12-24 h, dialyze to remove the unchelated metal ions, and the resulting product is a renally metabolizable polysaccharide contrast agent.

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