Modified dextran, t1-weighted iron-based contrast agent, and preparation method and application thereof

Abstract

The application provides a modified dextran, a T1 weighted iron-based contrast agent and a preparation method and application thereof. The modified dextran template has a general structure (I), and the preparation method comprises the following steps: esterification reaction of hydroxyl in dextran and a dicarboxylic acid, a diacyl halide or a dicarboxylic anhydride containing an olefin double bond in a polar solvent to generate a carboxyl dextran derivative containing an olefin double bond; the carboxyl dextran derivative containing the olefin double bond is subjected to a thiol-alkene click reaction with a monovalent carboxylic acid containing a thiol group to generate the modified dextran with a dicarboxylic acid functional group. The T1 weighted iron-based contrast agent prepared by using the modified dextran as a template comprises iron oxide nanoparticles and the modified dextran; wherein the iron oxide nanoparticles are wrapped in the modified dextran, and the iron oxide nanoparticles and the modified dextran are connected through a chemical chelate bond. The T1 weighted iron-based contrast agent has small toxic and side effects and excellent magnetic resonance imaging performance.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a modified dextran, a T1-weighted iron-based contrast agent, its preparation method, and its application. Background Technology

[0002] Magnetic Resonance Imaging (MRI), compared to currently used clinical techniques such as Computed Tomography (CT), Positron Emission Tomography / Computed Tomography (PET-CT), and Ultrasound (US), offers advantages such as no ionizing radiation, no radioactive radiation, and high imaging resolution. It can provide non-invasive and high-contrast clear imaging of various organs in the human body, making it one of the most powerful diagnostic tools in modern clinical practice. To improve the contrast between tissues in MRI, contrast agents are frequently used for clinical enhancement. Based on their mechanism of action, contrast agents can be divided into longitudinal relaxation contrast agents (T1-weighted contrast agents) and transverse relaxation contrast agents (T2-weighted contrast agents). T1-weighted contrast agents primarily accelerate longitudinal relaxation and produce a "bright" contrast effect in T1-weighted images, while T2-weighted contrast agents primarily increase the transverse relaxation rate and produce a "dark" contrast effect.

[0003] Currently, commonly used T1-weighted contrast agents for magnetic resonance imaging (MRI) are mainly gadolinium-based small-molecule chelates, such as gadoteric acid (Gd-DOTA), gadopentetate dimeglumine (Gd-DTPA), gadoxetate disodium (Gd-EOB-DTPA), and gadodiamide (Gd-DTPA-BMA). As a lanthanide rare-earth heavy metal, gadolinium ions exhibit high biotoxicity; therefore, clinical gadolinium-based chelates, based on highly polar molecules, also carry significant risks during use. Studies have shown that gadolinium-based contrast agents may leak small amounts of gadolinium ions during use, resulting in a high rate of adverse reactions, and large-scale use carries the risk of metabolic tissue fibrosis. Clinically, gadolinium-based contrast agents should be used with caution in patients with severe renal impairment, epilepsy, hypotension, asthma, other allergic respiratory diseases, and those with allergic tendencies.

[0004] To overcome this limitation, research reports have described the preparation of novel non-gadolinium-based T1-weighted contrast agents using various methods, such as iron-based, manganese-based, and copper-based contrast agents. Currently, iron-based MRI contrast agents have been widely reported; however, the imaging performance of iron-based T1-weighted contrast agents is difficult to surpass that of gadolinium-based agents, and their preparation methods are complex, making mass production and clinical translation challenging. To date, no pure T1-weighted iron-based contrast agent has been applied in clinical T1-weighted MRI. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a T1-weighted iron-based contrast agent using modified dextran as a template. The T1-weighted iron-based contrast agent contains ultra-small iron oxide nanoparticles internally, and its exterior is modified with a layer of modified dextran template material. This prepared T1-weighted iron-based contrast agent exhibits the clinical efficacy of gadolinium-based T1-weighted contrast agents, and is superior to clinical gadolinium-based T1-weighted contrast agents in angiography.

[0006] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0007] In a first aspect, the present invention provides a modified dextran, the structure of which is as shown in general formula (I):

[0008]

[0009] In the general formula (I), -OH represents the glucan backbone; -OH represents the hydroxyl group in the glucan molecule; m represents 0 to 10; n represents 0 to 10.

[0010] Furthermore, the molecular weight of the dextran backbone is 1000–100000 Da, and 1%–100% of the hydroxyl groups in the dextran backbone are modified by chemical reaction into a polymer containing a dicarboxyl group structure. Because the modified dextran molecule in general formula (I) contains a dicarboxyl group structure, its dicarboxyl group structure has a high function of stabilizing iron oxide nanoparticles.

[0011] In a second aspect, the present invention provides a method for preparing modified dextran, the method comprising: (1) reacting the hydroxyl groups in the dextran with a dicarboxylic acid, a diacyl halide or a dicarboxylic anhydride containing an olefin double bond in a polar solvent to generate a carboxylated dextran derivative containing an olefin double bond; (2) reacting the carboxylated dextran derivative containing an olefin double bond with a monocarboxylic acid containing a thiol group via a thiol-olefin click reaction to generate the modified dextran having a dicarboxylic acid functional group.

[0012] Further, in step (1), the molar ratio of the dicarboxylic acid, diacyl halide, or dicarboxylic anhydride to the hydroxyl group in the dextran is 0.01–2; the polar solvent is one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or water; the reaction temperature is 60°C–80°C, and the reaction time is 6h–48h; in step (2), the reaction is protected by nitrogen, the reaction temperature is 25°C–60°C, and the reaction time is 6h–24h.

[0013] Further, the preparation method specifically includes: dissolving maleic anhydride and dextran in dimethyl sulfoxide at a molar ratio of 1:1 (maleic anhydride to hydroxyl groups in dextran), stirring and reacting at 60°C for 12 h to obtain a reaction solution; adding 10 times the volume of ethyl acetate solution to the reaction solution to precipitate the precipitate, filtering and drying to obtain a carboxylated dextran derivative containing olefin double bonds; placing the carboxylated dextran derivative containing olefin double bonds and mercaptopropionic acid in a water bath at 25°C with stirring, nitrogen protection, and reacting for 12 h to obtain a reaction product; removing the small molecule mercaptopropionic acid from the reaction product by dialysis, freeze-drying, and obtaining the modified dextran in white powder form.

[0014] Secondly, the present invention provides a T1-weighted iron-based contrast agent prepared using modified dextran as a template, the T1-weighted iron-based contrast agent comprising iron oxide nanoparticles and the modified dextran as described above; wherein the iron oxide nanoparticles are encapsulated within the modified dextran, and the iron oxide nanoparticles and the modified dextran are connected by chemical chelate bonds.

[0015] Thirdly, the present invention provides a method for preparing a T1-weighted iron-based contrast agent, the method comprising: dissolving the modified dextran in an aqueous solution and stirring; then adding an iron ion solution to the modified dextran solution at a ratio of 0% to 100% of ferrous ions to total iron to obtain a first reaction solution; placing the first reaction solution in a constant temperature water bath at 25°C to 90°C and stirring for 10 min to 60 min to obtain a second reaction solution; then adjusting the pH of the second reaction solution to 8 to 12 with an inorganic base; after the stirring reaction is completed, removing the excess inorganic base from the second reaction solution to obtain the T1-weighted iron-based contrast agent; wherein, the total iron element is the sum of ferrous ions and ferric ions.

[0016] Further, the preparation method specifically includes: dissolving the modified dextran in an aqueous solution and stirring in a constant temperature water bath at 37°C; then adding an iron ion solution to the modified dextran solution at a molar ratio of 50% for ferrous ions to ferric ions; rapidly stirring the solution in a constant temperature water bath at 37°C at 500 rpm; then adjusting the pH of the reaction solution to 9 with a 1M sodium hydroxide solution at a flow rate of 2.5 mL / s; after stirring the reaction for 30 min, removing excess sodium hydroxide, and finally preparing an iron-based magnetic resonance imaging agent prepared using modified dextran as a template.

[0017] Furthermore, the particle size range of the iron oxide nanoparticles is 2nm to 10nm; the inorganic base used to adjust the pH is preferably an aqueous solution of sodium hydroxide or potassium hydroxide; the method for removing excess inorganic base from the solution is dialysis or acid-base neutralization with dilute hydrochloric acid solution.

[0018] Fourthly, the present invention provides the T1-weighted iron-based contrast agent as described above for blood pool contrast imaging in magnetic resonance imaging and T1-weighted enhanced contrast imaging in clinical magnetic resonance imaging, as well as its application as a clinical iron supplement.

[0019] The effects of this invention are as follows:

[0020] 1. The T1-weighted iron-based contrast agent prepared in this invention consists of ultra-small iron oxide nanoparticles internally and an externally modified layer of dextran molecules. Compared to clinically commonly used T1-weighted gadolinium-based magnetic resonance imaging (MRI) contrast agents, the prepared T1-weighted iron-based contrast agent exhibits higher biocompatibility. Furthermore, the raw materials used in its preparation are iron and dextran, which, compared to gadolinium chelated with macrocyclic complexes used clinically, make the preparation process green, convenient, pollution-free, and inexpensive.

[0021] 2. The T1-weighted iron-based contrast agent prepared using modified dextran as a template in this invention can be used for T1-weighted tissue enhancement and T1-weighted angiography in magnetic resonance imaging (MRI). The T1-weighted iron-based contrast agent prepared using modified dextran as a template in Example 1 demonstrates performance comparable to clinically used Gd-DTPA in T1-weighted MRI, and its effectiveness is superior, especially in angiography of Wistar rats, New Zealand rabbits, and beagle dogs.

[0022] 3. The T1-weighted iron-based contrast agent prepared using modified dextran as a template in this invention exhibits stronger T1-weighted aperture brightness than Gd-DTPA under magnetic fields of 3.0T and below. Currently, most MRI machines used in domestic hospitals are 3.0T and below, therefore this contrast agent has broad clinical applicability.

[0023] 4. The T1-weighted iron-based contrast agent prepared by the present invention using modified dextran as a template has a particle size that can be controlled below 10 nm. Therefore, the nano-contrast agent prepared by this method can be metabolized and excreted from the body through the kidneys and liver and gallbladder, thus having lower biological toxicity side effects.

[0024] 5. Compared with T1-weighted iron-based contrast agents prepared by commonly used high-temperature pyrolysis methods, the T1-weighted iron-based contrast agent prepared by this invention using modified dextran as a template does not require the complex step of oil-phase to aqueous-phase conversion. Furthermore, the prepared T1-weighted iron-based contrast agent has higher nanoparticle water solubility, solution stability, and resistance to non-specific adsorption of biomacromolecules. Therefore, compared with the traditional high-temperature pyrolysis preparation method, the T1-weighted iron-based contrast agent prepared by this invention is more beneficial for clinical application.

[0025] 6. Compared with iron-based magnetic resonance contrast agents prepared by the commonly used high-temperature pyrolysis method, the T1-weighted iron-based contrast agent prepared by this invention using modified dextran as a template has weaker internal crystallinity of iron oxide nanoparticles than that prepared by the high-temperature pyrolysis method. The lower crystallinity of the iron oxide nanoparticles is more conducive to T1-weighted magnetic resonance imaging of iron oxide nanoparticles.

[0026] 7. Currently, reported T1-weighted iron oxide-based magnetic resonance nanoparticles rarely outperform clinical gadolinium-based contrast agents in magnetic resonance imaging (MRI), and no iron oxide-based MRI is currently used clinically for T1-weighted contrast-enhanced imaging. However, the T1-weighted iron-based contrast agent prepared using modified dextran as a template in this invention exhibits superior MRI contrast-enhanced effects compared to clinical gadolinium-based contrast agents. Therefore, this invention, which prepares a T1-weighted iron-based contrast agent using modified dextran as a template, possesses significant innovative and clinical translational advantages.

[0027] 8. The T1-weighted iron-based contrast agent prepared by the present invention using modified dextran as a template has a large number of dicarboxyl groups modified on its surface. The abundant carboxyl functional groups are beneficial to endowing the magnetic resonance contrast agent with corresponding targeting and responsive functions through coupling reaction with amino groups. These are difficult to achieve with other iron oxide-based magnetic resonance nanoparticles. Attached Figure Description

[0028] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments, and unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0029] Figure 1 This is a schematic diagram of the general structural formula of the T1-weighted iron-based contrast agent in this invention;

[0030] Figure 2 This is a flowchart illustrating the general reaction steps in the preparation process of the modified dextran in this invention.

[0031] Figure 3 This is a general formula diagram of the preparation process of the T1-weighted iron-based contrast agent in this invention;

[0032] Figure 4 This describes the synthesis steps of the T1-weighted iron-based contrast agent (iDMMA) prepared using modified dextran as a template in Example 1 of this invention;

[0033] Figure 5 The above are the proton nuclear magnetic resonance spectra of the raw materials dextran (Dextran), carboxylated dextran derivative containing olefin double bonds (D-MAH), and modified dextran (DMMA) in Example 1 of this invention.

[0034] Figure 6This is a high-resolution transmission electron microscope image of the iDMMA in Embodiment 1 of the present invention;

[0035] Figure 7 This is a high-resolution transmission electron microscope (TEM) histogram of the particle size distribution of iDMMA in Example 1 of this invention.

[0036] Figure 8 This is an image of iDMMA in a high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) in Embodiment 1 of the present invention.

[0037] Figure 9 This is a histogram of the hydrated particle size distribution of iDMMA in Embodiment 1 of the present invention;

[0038] Figure 10 The infrared spectra of Dextran, DMMA, and iDMMA in Embodiment 1 of the present invention are shown below.

[0039] Figure 11 This is a comparison of the longitudinal and transverse relaxation rates of iDMMA and Gd-DTPA prepared in Example 1 of this invention under 1.0T magnetic resonance.

[0040] Figure 12 This is a comparison of the longitudinal and transverse relaxation rates of iDMMA and Gd-DTPA prepared in Example 1 of this invention under 1.47T magnetic resonance.

[0041] Figure 13 This is a comparison of the longitudinal and transverse relaxation rates of iDMMA and Gd-DTPA prepared in Example 1 of this invention under clinical 3.0T magnetic resonance imaging.

[0042] Figure 14 This is a comparison chart of UV data for 5-hydroxytryptamine (5-HT), 5-hydroxytryptamine-modified T1-weighted iron contrast agent (iDMMA-HT), and iDMMA in Example 2 of this invention.

[0043] Figure 15 The image shows a comparison of the T1-weighted aperture brightness of the T1-weighted iron-based contrast agent (iDMMA-c(RGDfK)) modified with c(RGDfK) peptide, iDMMA, and Gd-DTPA prepared in Example 2 of this invention under 1.0T magnetic resonance.

[0044] Figure 16 Comparison of T1-weighted aperture brightness of iDMMA-c(RGDfK), iDMMA and Gd-DTPA prepared in Example 2 of this invention under clinical 3.0T magnetic resonance T1-weighted sequence;

[0045] Figure 17The image shows a comparison of T1-weighted aperture brightness of iDMMA-c(RGDfK), iDMMA, and Gd-DTPA prepared in Example 2 of this invention, as prepared in Example 3 of this invention, under a clinical 3.0T magnetic resonance angiography 3D Trick sequence.

[0046] Figure 18 The metabolism of iDMMA prepared in Example 1 of this invention after injection of contrast agent into the bladder of mice under clinical 3.0T magnetic resonance imaging at a concentration of 0.1 mmol / kg;

[0047] Figure 19 Comparison of angiography images of iDMMA and Gd-DTPA prepared in Example 1 of this invention under clinical 3.0T magnetic resonance imaging in Wister rats with an injection concentration of 0.1 mmol / kg;

[0048] Figure 20 Comparison of angiography images of iDMMA and Gd-DTPA prepared in Example 1 of this invention under clinical 3.0T magnetic resonance imaging in New Zealand rabbits at an injection concentration of 0.1 mmol / kg;

[0049] Figure 21 This is a comparison of angiography images of iDMMA and Gd-DTPA prepared in Example 1 of the present invention under clinical 3.0T magnetic resonance imaging at an injection concentration of 0.1 mmol / kg in beagle dogs. Detailed Implementation

[0050] As is known from the background art, no pure iron-based T1-weighted contrast agent has yet been applied in clinical magnetic resonance imaging. Therefore, this invention utilizes modified dextran as a template and ultra-small iron oxide nanoparticles to prepare a T1-weighted iron-based contrast agent. This contrast agent exhibits the clinical T1-weighted contrast effect of gadolinium-based agents and is superior to clinical gadolinium-based agents in angiography.

[0051] Specifically, in a first aspect, the present invention provides a modified dextran, the structure of which is as shown in general formula (I):

[0052]

[0053] General Formula (I)

[0054] In the general formula (I), The denoted represents the dextran backbone; -OH represents the hydroxyl group in the dextran molecule; m represents 0–10; n represents 0–10. It should be noted that the dextran backbone in the formula is a complete chain. m and n can be the same number or different numbers.

[0055] Secondly, the present invention provides a T1-weighted iron-based contrast agent prepared using modified dextran as a template, such as... Figure 1As shown, the T1-weighted iron-based contrast agent comprises iron oxide nanoparticles and modified dextran as described above; wherein the iron oxide nanoparticles are encapsulated within the modified dextran, and the iron oxide nanoparticles and the modified dextran are connected by chemical chelate bonds.

[0056] Thirdly, the present invention provides a method for preparing a T1-weighted iron-based contrast agent, such as... Figure 3 As shown, the preparation method includes: dissolving the modified dextran in an aqueous solution and stirring; then adding the modified dextran solution in a ratio of 0% to 100% of ferrous ions to total iron to obtain a first reaction solution; placing the first reaction solution in a constant temperature water bath at 25℃ to 90℃ and stirring for 10 min to 60 min to obtain a second reaction solution, as shown. Figure 2 Step (I) is performed; then the pH of the second reaction solution is adjusted to 8-12 with an inorganic base. After the reaction is completed by stirring, excess inorganic base in the second reaction solution is removed to obtain the T1-weighted iron-based contrast agent, as shown in the figure. Figure 2 Step (II) in the process; where the total iron element is the sum of ferrous ions and ferric ions.

[0057] Furthermore, the iron oxide nanoparticles prepared in this invention (e.g., Example 1) have template materials (e.g., dextran—a plasma volume expander (also known as plasma substitute); maleic anhydride—hydrolyzed to maleic acid (also known as dehydrated malic acid, an acidulant in food and beverages); mercaptopropionic acid—a pharmaceutical intermediate for phenalool) covering their surfaces that are all green, safe, and certified by the Food and Drug Administration (FDA) as safe materials for human use. Therefore, the high-performance T1-weighted iron-based contrast agent prepared using modified dextran as a template in this invention has the potential to replace the commonly used gadolinium-based magnetic resonance contrast agents in clinical practice, exhibiting high safety and promising clinical translation prospects.

[0058] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0059] Example 1

[0060] This embodiment provides a method for preparing modified dextran, and a T1-weighted iron-based contrast agent prepared using modified dextran as a template. The preparation steps are as follows: Figure 4 As shown, it specifically includes:

[0061] (1) Using dextran with a molecular weight of 20000 Da and maleic anhydride as raw materials, 5 g of dextran and 9.08 g of maleic anhydride were dissolved in 50 mL of DMSO solution at a molar ratio of 1:1 (hydroxyl group in dextran to maleic anhydride). The mixture was stirred at 60 °C for 12 h. After the reaction, the reaction solution containing DMSO was added dropwise to 10 times the volume of rapidly stirred ethyl acetate solvent to obtain a white viscous crude product. Subsequently, the obtained white viscous crude product was placed in rapidly mechanically stirred ethyl acetate solvent for slurry purification to remove DMSO solvent and unreacted maleic anhydride from the crude product. After filtration and vacuum drying, a pure white powder containing olefin double bonds of carboxylated dextran derivative, namely D-MAH, was obtained.

[0062] (2) Using the D-MAH prepared in the previous step and mercaptopropionic acid as raw materials, according to the molar ratio of double bonds in D-MAH to mercaptopropionic acid of 1:1.5, 4g of D-MAH and 8mL of mercaptopropionic acid were mixed and stirred in a nitrogen-protected environment. Then, the pH of the reaction solution was adjusted to neutral with 6M NaOH (about 12mL of 6M NaOH was added), and the reaction was carried out at room temperature (25℃) for 12h under nitrogen protection. After the reaction was completed, the excess unreacted mercaptopropionic acid raw material was removed by dialysis, and the dialysate was freeze-dried to obtain modified dextran, i.e., DMMA.

[0063] The 1H NMR spectra of DMMA, D-MAH, and dextran raw materials prepared by this method are shown below. Figure 5 As shown, Figure 5 The appearance of two chemical shift peaks at the 6.0-7.0 ppm chemical shift of maleic acid, corresponding to the hydrogen atoms on the double bond, indicates that maleic anhydride successfully underwent esterification with the hydroxyl groups on the dextran, proving the successful synthesis of D-MAH. Furthermore, the olefin double bond in D-MAH underwent a mercapto-olefin click reaction with mercaptopropionic acid. Figure 5 The disappearance of the two hydrogen chemical shift peaks in the "olefin double bond" at 6.0-7.0 ppm in the DMMA product indicates that the double bond in D-MAH was completely reacted by excess mercaptopropionic acid, proving the successful synthesis of DMMA.

[0064] (3) Using the DMMA synthesized in the previous step as raw material, dissolve 8g of DMMA in 800mL of deionized water and stir in a water bath at 37℃ for 10min. Then proceed according to Fe... 2+ with Fe 3+In a 1:1 molar ratio, 100 mL of 9.9 mg / mL ferrous chloride tetrahydrate solution and 100 mL of 13.46 mg / mL ferric chloride hexahydrate solution were added sequentially to the stirred solution. Then, 50 mL of 1M NaOH was added to the reaction solution at a flow rate of 2 mL / s to adjust the pH to alkaline, and the reaction was rapidly stirred for 30 min. The reaction solution was then dialyzed in a dialysis bag with a molecular weight cutoff of 14000 Da, ultimately yielding a T1-weighted iron-based contrast agent, iDMMA, prepared using modified dextran as a template.

[0065] The prepared iDMMA was characterized, such as... Figure 3-7 As shown:

[0066] Figure 6 This is a high-resolution transmission electron microscope image of the iDMMA in Embodiment 1 of the present invention. Figure 7 for Figure 6 The particle size distribution histogram. Based on... Figure 6 and Figure 7 It can be seen that the iron oxide nanoparticles prepared using this invention patent have a particle size of 2.52±0.06nm. The prepared nanoparticles are extremely small and have a uniform particle size distribution, with good particle dispersibility.

[0067] Figure 8 This is a high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of iDMMA in spherical aberration electron microscopy in Embodiment 1 of the present invention. According to... Figure 8 It can be seen that the prepared iDMMA nanoparticles have many lattice defects and low crystallinity. Therefore, with the interruption of the crystal structure, the spins on the nanoparticle surface become disordered. Since these surface spins are not perfectly aligned with the overall spin of the nanoparticle, they form a magnetic dead region on the particle surface. As the core size decreases, the proportion of the magnetic dead region in the particle volume increases, which is more conducive to T1-weighted enhanced magnetic resonance imaging.

[0068] Figure 9 This is a histogram of the hydrated particle size distribution of iDMMA in Embodiment 1 of the present invention. According to... Figure 9 It can be seen that the hydrated particle size of the prepared iDMMA nanoparticles is between 5-20 nm.

[0069] Figure 10 The infrared spectra of dextran, DMMA, and iDMMA in Example 1 of this invention are shown. The infrared spectrum is located at 1730 cm⁻¹. -1 1582cm -1 and 1400cm -1 The characteristic peaks of the carboxyl functional group further confirm the successful synthesis of the modified dextran. Additionally, the peak at 1640 cm⁻¹ further confirms this. -1 and 1346cm -1The characteristic peaks indicating the binding of the carboxyl group to the iron element suggest that the modified dextran and iron oxide nanoparticles are stably bound together by chelate bonds.

[0070] Figure 11-13 This is a comparison of the longitudinal relaxation rate (r1) and transverse relaxation rate (r2) of iDMMA and Gd-DTPA prepared in Example 1 of this invention under magnetic resonance at 1.0T, 1.47T, and 3.0T. The longitudinal relaxation rate (r1) of iDMMA at 1.0T, 1.47T, and 3.0T is 10.22 mM. -1 ·S -1 8.22mM -1 ·S -1 and 6.41mM -1 ·S -1 All of these values ​​are higher than the longitudinal relaxation rate (r1) of 4.09 mM of Gd-DTPA at 1.0T, 1.47T, and 3.0T. -1 ·S -1 3.30mM -1 ·S -1 and 3.32mM -1 ·S -1 .

[0071] Example 2

[0072] This embodiment provides a process for surface functionalization of nanoparticles using carboxyl groups on the surface of a T1-weighted iron-based contrast agent prepared with modified dextran as a template and amino-containing 5-hydroxytryptamine, specifically including:

[0073] Take 60 mL of iDMMA (5 mM concentration) prepared in Example 1, add 4 mL of N-hydroxysuccinimide (NHS) at a concentration of 8.5 mg / mL, and activate by stirring at room temperature for 2 h. Then add 4 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) at a concentration of 14.2 mg / mL and 4 mL of 5-HT at a concentration of 11.19 mg / mL, and react at room temperature for 24 h. Finally, remove impurities by dialysis to obtain a 5-hydroxytryptamine-modified T1-weighted iron-based contrast agent, iDMMA-HT.

[0074] Figure 14 This is a comparison chart of UV data for iDMMA-HT, 5-HT, and iDMMA in Embodiment 2 of the present invention. From... Figure 11 The characteristic absorption peak of 5-HT in the 250-300nm UV absorption of iDMMA-HT proves that amino-containing 5-hydroxytryptamine was successfully modified onto the surface of the T1-weighted iron-based contrast agent prepared with modified dextran as a template and containing carboxyl functional groups.

[0075] Example 3

[0076] This embodiment provides a process for surface functionalization of nanoparticles using carboxyl groups on the surface of a T1-weighted iron-based contrast agent prepared with modified dextran as a template and an amino-containing tumor-targeting cyclic peptide c (RGDfK). Specifically, the process includes:

[0077] Take 60 mL of iDMMA (5 mM concentration) prepared in Example 1, add 4 mL of N-hydroxysuccinimide (NHS) at a concentration of 8.5 mg / mL, and activate by stirring at room temperature for 2 h. Then add 4 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) at a concentration of 14.2 mg / mL and 4 mL of amino-containing tumor-targeting cyclic peptide c(RGDfK) at a concentration of 10 mg / mL, and react at room temperature for 24 h. Finally, remove impurities by dialysis to obtain a T1-weighted iron-based contrast agent modified with c(RGDfK) peptide, i.e., iDMMA-c(RGDfK).

[0078] Figure 15-16 The aperture brightness of the materials prepared for clinical Gd-DTPA and iDMMA in Examples 1 and 3 of this invention, iDMMA-c(RGDfK), is measured at 1.0T and 3.0T, respectively. Figure 15 The aperture brightness under spin echo SE sequence at 1.0T is shown to be that iDMMA and iDMMA-c(RGDfK) are stronger than clinical Gd-DTPA in contrast imaging, and the contrast effect of iDMMA after surface modification with c(RGDfK) is not significantly reduced. Figure 16 The aperture brightness under spin echo SE sequence at 3.0T is shown to be comparable to that of Gd-DTPA in T1-weighted contrast-enhanced angiography at 3.0T in routine clinical settings. Figure 17 The aperture brightness of iDMMA and iDMMA-c(RGDfK) under 3.0T using the 3D Trick angiography sequence is shown to be significantly higher than that of clinically used Gd-DTPA, indicating that iDMMA and iDMMA-c(RGDfK) are significantly superior to clinically used Gd-DTPA in angiography.

[0079] Example 4

[0080] This embodiment provides a method for preparing modified dextran, and a T1-weighted iron-based contrast agent prepared using modified dextran as a template, specifically including:

[0081] (1) Using dextran with a molecular weight of 1000 Da and fumarate chloride as raw materials, 5 g of dextran, 0.127 g of fumarate chloride, and 0.053 g of N,N-diisopropylethylamine (DIPEA) were dissolved in 50 mL of DMF solution at a molar ratio of 99:1 and stirred at 25 °C for 6 h. After the reaction was completed, the reaction solution containing DMF was added dropwise to 10 times the volume of rapidly stirred ethyl acetate solvent to obtain a white viscous crude product. Subsequently, the obtained white viscous crude product was placed in rapidly mechanically stirred ethyl acetate solvent for slurry purification to remove DMF solvent and unreacted fumarate chloride from the crude product. After filtration and vacuum drying, a pure white powder of fumarate chloride-modified carboxylated dextran derivative containing olefin double bonds, namely D-FC, was obtained.

[0082] (2) Using the D-FC prepared in the previous step and thioglycolic acid as raw materials, 4g of D-FC and 4mL of thioglycolic acid were mixed and stirred in a nitrogen-protected environment. Then, the pH of the reaction solution was adjusted to neutral with 6M KOH, and the reaction was carried out at room temperature (25℃) for 24h under nitrogen protection. After the reaction was completed, excess unreacted thioglycolic acid raw material was removed by dialysis, and the dialysate was freeze-dried to obtain the modified dextran prepared from fumarate thioglycolic acid.

[0083] (3) Using the DMMA synthesized in the previous step as raw material, dissolve 8g of DMMA in 800mL of deionized water and stir in a water bath at 37℃ for 10min. Then proceed according to Fe... 3+ with Fe 2+ A 1:0 molar ratio of 200 mL of 13.46 mg / mL ferric chloride hexahydrate solution was added to the stirred solution. Then, 1 M KOH was added to the reaction solution at a flow rate of 2 mL / s to adjust the pH to alkaline, and the reaction was rapidly stirred for 30 min. The reaction solution was then dialyzed in a dialysis bag with a molecular weight cutoff of 14000 Da to finally prepare a T1-weighted iron-based contrast agent using modified dextran as a template.

[0084] Example 5

[0085] This embodiment provides a method for preparing modified dextran, and a T1-weighted iron-based contrast agent prepared using modified dextran as a template, specifically including:

[0086] (1) Using dextran with a molecular weight of 100,000 Da and fumaric acid as raw materials, 5 g of dextran was dissolved in 50 mL of DMSO solution with 3.22 g of fumaric acid and 5 mL of concentrated sulfuric acid at a molar ratio of 1:1. The mixture was stirred at 100 °C for 24 h. After the reaction, the sulfuric acid and fumaric acid were removed by dialyzing. The dialysate was freeze-dried to obtain a pure white powder of fumaric acid-modified carboxylated dextran derivative containing olefin double bonds (D-FMC).

[0087] (2) Using the D-FMC and 11-mercaptodecanoic acid prepared in the previous step as raw materials, 4g of D-FMC and 4.85mg of 11-mercaptodecanoic acid were mixed and stirred in a nitrogen-protected environment. Then, the pH of the reaction solution was adjusted to neutral with 6M NaOH, and the reaction was carried out at room temperature (25°C) for 24h under nitrogen protection. After the reaction was completed, excess unreacted 11-mercaptodecanoic acid raw material was removed by dialysis. The dialysate was freeze-dried to obtain the modified dextran prepared from fumaric acid and 11-mercaptodecanoic acid.

[0088] (3) Using the DMMA synthesized in the previous step as raw material, dissolve 8g of DMMA in 800mL of deionized water and stir in a water bath at 37℃ for 10min. Then proceed according to Fe... 3+ with Fe 2+ A 0:1 molar ratio of 200 mL of 9.9 mg / mL ferrous chloride tetrahydrate solution was added to the stirred solution. Then, 1 M NaOH was added to the reaction solution at a flow rate of 2 mL / s to adjust the pH to alkaline, and the reaction was rapidly stirred for 30 min. The reaction solution was then dialyzed in a dialysis bag with a molecular weight cutoff of 14000 Da, ultimately yielding a T1-weighted iron-based contrast agent prepared using modified dextran as a template.

[0089] Example 6

[0090] This embodiment provides a method for preparing modified dextran, and a T1-weighted iron-based contrast agent prepared using modified dextran as a template, specifically including:

[0091] (1) Dextran with a molecular weight of 1000 Da and fumarate chloride were selected as raw materials. 5 g of dextran, 0.127 g of fumarate chloride, and 0.053 g of DIPEA were dissolved in 50 mL of DMF solution at a molar ratio of 99:1. The mixture was stirred at 25 °C for 6 h. After the reaction, the DMF-containing reaction solution was added dropwise to 10 times its volume of rapidly stirred ethyl acetate solvent to obtain a white, viscous crude product. Subsequently, the obtained white, viscous crude product was placed in rapidly stirred ethyl acetate solvent for purification to remove the DMF solvent and unreacted fumarate chloride. After filtration and vacuum drying, a pure white powder of fumarate chloride-modified carboxylated dextran derivative containing olefin double bonds (D-FC) was obtained.

[0092] (2) Using the D-FC prepared in the previous step and thioglycolic acid as raw materials, 4g of D-FC and 4mL of thioglycolic acid were mixed and stirred in a nitrogen-protected environment. Then, the pH of the reaction solution was adjusted to neutral with 6M KOH, and the reaction was carried out at room temperature (25℃) for 24h under nitrogen protection. After the reaction was completed, excess unreacted thioglycolic acid raw material was removed by dialysis, and the dialysate was freeze-dried to obtain the modified dextran prepared from fumarate thioglycolic acid.

[0093] (3) Using the DMMA synthesized in the previous step as raw material, dissolve 8g of DMMA in 800mL of deionized water and stir in a water bath at 37℃ for 10min. Then proceed according to Fe... 3+ with Fe 2+ A 1:0 molar ratio of 200 mL of 13.46 mg / mL ferric chloride hexahydrate solution was added to the stirred solution. Then, 1 M KOH was added to the reaction solution at a flow rate of 2 mL / s to adjust the pH to alkaline, and the reaction was rapidly stirred for 30 min. The reaction solution was then dialyzed in a dialysis bag with a molecular weight cutoff of 14000 Da to finally prepare a T1-weighted iron-based contrast agent using modified dextran as a template.

[0094] Example 7

[0095] This embodiment provides a method for preparing modified dextran, and a T1-weighted iron-based contrast agent prepared using modified dextran as a template, specifically including:

[0096] (1) Using dextran with a molecular weight of 10000 Da and (E)-tetradecyl-2-enediol chloride as raw materials, 5 g of dextran, 7.12 g of (E)-tetradecyl-2-enediol chloride, and 2.65 g of DIPEA were dissolved in 50 mL of DMF solution and stirred at 25 °C for 12 h. After the reaction was completed, the reaction solution containing DMF was added dropwise to 10 times the volume of rapidly stirred ethyl acetate solvent to obtain a white viscous crude product. Subsequently, the obtained white viscous crude product was placed in rapidly mechanically stirred ethyl acetate solvent for slurry purification to remove the solvent and unreacted (E)-tetradecyl-2-enediol chloride from the crude product. After filtration and vacuum drying, a pure white powder of (E)-tetradecyl-2-enediol chloride modified carboxylated dextran derivative containing olefin double bonds (D-TEC) was obtained.

[0097] (2) Using the D-TEC prepared in the previous step and mercaptopropionic acid as raw materials, 4g of D-TEC and 2.36g of mercaptopropionic acid were mixed and stirred in a nitrogen-protected environment. Then, the pH of the reaction solution was adjusted to neutral with 6M NaOH, and the reaction was carried out at room temperature (25°C) for 12 hours under nitrogen protection. After the reaction was completed, excess unreacted mercaptopropionic acid raw material was removed by dialysis. The dialysate was freeze-dried to obtain the modified dextran prepared from (E)-tetradecyl-2-enediol chloride and mercaptopropionic acid.

[0098] (3) Using the DMMA synthesized in the previous step as raw material, dissolve 8g of DMMA in 800mL of deionized water and stir in a water bath at 37℃ for 10min. Then proceed according to Fe... 3+ with Fe 2+ In a 1:1 molar ratio, 100 mL of 9.9 mg / mL ferrous chloride tetrahydrate solution and 100 mL of 13.46 mg / mL ferric chloride hexahydrate solution were added to the stirred solution. Then, 1 M NaOH was added to the reaction solution at a flow rate of 0.5 mL / s to adjust the pH to alkaline, and the reaction was rapidly stirred for 30 min. The reaction solution was then dialyzed in a dialysis bag with a molecular weight cutoff of 14000 Da, ultimately yielding a T1-weighted iron-based contrast agent prepared using modified dextran as a template.

[0099] Fourthly, the present invention provides the T1-weighted iron-based contrast agent as described above for blood pool contrast imaging in magnetic resonance imaging and T1-weighted enhanced contrast imaging in clinical magnetic resonance imaging, as well as its application as a clinical iron supplement.

[0100] The following experiments were conducted using the T1-weighted iron-based contrast agent prepared in Example 1 with modified dextran as a template to further illustrate the performance of the contrast agent.

[0101] Experimental Example 1: iDMMA Magnetic Resonance Cystography

[0102] I. Experimental Methods

[0103] Adult female Balb / c mice, weighing approximately 20g, were anesthetized with 1% sodium pentobarbital. The mice were then placed under a clinical Philips 3.0T MRI scanner for bladder imaging. The iDMMA dosage was 0.1 mmol / kg (iron). Imaging parameters: TR = 600 ms; TE = 20 ms; slice thickness 2 mm. The bladders were scanned before iDMMA injection and at 4, 12, 16, and 50 minutes after iDMMA injection.

[0104] II. Experimental Results

[0105] Figure 18 The metabolism of iDMMA prepared in Example 1 of this invention after injection of contrast agent into the bladder of mice under clinical 3.0T magnetic resonance imaging at a concentration of 0.1 mmol / kg. Figure 18 It can be seen that the bladder began to light up 4 minutes after iDMMA injection, the bladder was half full after 12 minutes, and the bladder was fully lit up after 50 minutes. The experimental results indicate that iDMMA can be metabolized by the kidneys and excreted through urine.

[0106] Experimental Example 2: Wister Rat Angiography Imaging

[0107] I. Experimental Methods

[0108] Adult Wister rats, weighing approximately 250g, were anesthetized with 1% sodium pentobarbital. The rats were then placed in a clinical GE 3.0T MRI head coil, and vascular angiography was performed using the 3D Trick sequence. iDMMA and Gd-DTPA were administered at a dose of 0.1 mmol / kg (iron or gadolinium). Imaging parameters: 3D Trick sequence; TR = 3.3 ms; TE = 1.2 ms; slice thickness 1 mm; 30 phases.

[0109] II. Experimental Results

[0110] Figure 19This image shows a comparison of angiography results of iDMMA and Gd-DTPA prepared in Example 1 of this invention under clinical 3.0T MRI in Wister rats at an injection concentration of 0.1 mmol / kg. The image shows that iDMMA has a longer imaging window in rat arteries, and its imaging effect on head and neck vessels is superior to that of clinical Gd-DTPA. After 110 seconds of contrast agent injection, the imaging effect of Gd-DTPA on rat head and neck vessels significantly decreased, while iDMMA maintained good vascular imaging performance. In conclusion, iDMMA exhibits superior T1-weighted angiography performance compared to Gd-DTPA in small animal vessels.

[0111] Experimental Example 3: Angiography of New Zealand Rabbits

[0112] I. Experimental Methods

[0113] Adult New Zealand rabbits, weighing approximately 2.5 kg, were anesthetized with Serta-50 and then placed in a clinical GE 3.0T MRI head coil. 3D Trick sequence imaging was performed on the blood vessels of the rabbit's head and neck. iDMMA and Gd-DTPA (iron or gadolinium) were administered at a dose of 0.1 mmol / kg via a high-pressure injector. Imaging parameters: 3D Trick sequence; TR = 3.0 ms; TE = 1.1 ms; slice thickness 1 mm; phase 48.

[0114] II. Experimental Results

[0115] Figure 20 This image shows a comparison of angiography results of iDMMA and Gd-DTPA prepared in Example 1 of this invention under clinical 3.0T MRI in New Zealand rabbits at an injection concentration of 0.1 mmol / kg. The image shows that iDMMA has a longer imaging window in the arteries of New Zealand rabbits, and the imaging effect on the upper body vessels of New Zealand rabbits is superior to that of clinical Gd-DTPA. After 217 seconds of contrast agent injection, the imaging performance of Gd-DTPA in New Zealand rabbits significantly decreased, while iDMMA maintained good vascular imaging performance. In summary, iDMMA has better T1-weighted angiography performance than Gd-DTPA in medium-sized New Zealand rabbits.

[0116] Experimental Example 4: Angiography of a Beagle Dog

[0117] I. Experimental Methods

[0118] Adult beagle dogs, weighing approximately 20 kg, were sedated with atropine and then anesthetized with a combination of salbutamol and xylazine hydrochloride. The dogs were then placed in a clinical GE 3.0T MRI head coil for 3D Trick sequence imaging of the canine head vessels. iDMMA and Gd-DTPA (iron or gadolinium) were administered at a dose of 0.1 mmol / kg via a high-pressure injector. Imaging parameters: 3D Trick sequence; TR = 3.5 ms; TE = 1.3 ms; slice thickness 1 mm; phase 48.

[0119] II. Experimental Results

[0120] Figure 21 This image shows a comparison of angiography results of iDMMA and Gd-DTPA prepared in Example 1 of this invention under a clinical 3.0T MRI scan at an injection concentration of 0.1 mmol / kg in beagle dogs. The image shows that iDMMA has a longer imaging window in the cephalic arteries of beagle dogs, and its imaging effect on the head and neck vessels is superior to that of clinical Gd-DTPA. After 254 seconds of contrast agent injection, the imaging effect of Gd-DTPA in beagle dogs significantly decreased, while iDMMA maintained good vascular imaging performance. In summary, iDMMA maintains good imaging performance in large animals like beagle dogs, and its T1-weighted angiography performance is superior to that of clinical Gd-DTPA.

[0121] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A modified dextran, characterized in that, The structure of the modified dextran is as general formula (I): General Formula (I) In the general formula (I), represents a glucan backbone; -OH represents a hydroxyl group in the glucan molecule; m represents 0 to 10; n represents 0 to 10; More than or equal to 1% and less than 100% of the hydroxyl groups in the dextran main chain are modified into units containing dicarboxyl structures through chemical reactions.

2. The modified dextrans according to claim 1, characterized in that, The molecular weight of the dextran main chain is 1000-100000 Da.

3. The method for producing a modified dextran according to claim 1 or 2, characterized in that, The preparation method comprises: (1) esterification reaction of hydroxyl groups in dextran with dicarboxylic acid, dicarboxylic acid halide or dicarboxylic anhydride containing olefin double bond in a polar solvent to generate carboxyl dextran derivative containing olefin double bond; (2) thiol-alkene click reaction of the carboxyl dextran derivative containing olefin double bond with monobasic carboxylic acid containing thiol to generate the modified dextran with dicarboxylic acid functional group.

4. The method for producing a modified dextran according to claim 3, characterized by, In the step (1), the molar ratio of the dicarboxylic acid, dicarboxylic acid halide or dicarboxylic anhydride to the hydroxyl groups in dextran is 0.01-2; The polar solvent is one of dimethyl sulfoxide, N,N-dimethylformamide and water; The reaction temperature is 60-80℃, and the reaction time is 6-48 h; In the step (2), the reaction is protected by nitrogen, the reaction temperature is 25-60℃, and the reaction time is 6-24 h.

5. The method for preparing a modified dextran according to claim 3, characterized in that, The preparation method specifically comprises: according to the molar ratio of maleic anhydride to the hydroxyl groups in dextran being 1:1, maleic anhydride and dextran are dissolved in dimethyl sulfoxide and stirred at 60℃ for 12 h to obtain a reaction solution; the reaction solution is dropped into a solution of 10 times volume of ethyl acetate, a precipitate is separated out, filtered and dried to obtain the carboxyl dextran derivative containing olefin double bond; The carboxyl dextran derivative containing olefin double bond and mercaptopropionic acid are placed in a 25℃ water bath for stirring, nitrogen protection and reaction for 12 h to obtain a reaction product; the reaction product is subjected to a dialysis method to remove small molecular mercaptopropionic acid, and freeze-drying is performed to obtain the modified dextran in white powder form.

6. A Tl-weighted iron-based contrast agent, characterized in that, The T1-weighted iron-based contrast agent comprises iron oxide nanoparticles and the modified dextran as claimed in claim 1 or 2; The iron oxide nanoparticles are wrapped in the modified dextran, and the iron oxide nanoparticles and the modified dextran are connected through a chemical chelate bond.

7. The method for preparing the T1-weighted iron-based contrast agent according to claim 6, characterized in that, The preparation method comprises: stirring the modified dextran in an aqueous solution, then adding an iron ion solution into the modified dextran solution according to the ratio of divalent iron ions to total iron elements being 0%-100% to obtain a first reaction solution; stirring the first reaction solution in a constant-temperature water bath at 25-90℃ for 10-60 min to obtain a second reaction solution; then adjusting the pH of the second reaction solution to 8-12 with an inorganic base, and removing the excess inorganic base in the second reaction solution after the stirring reaction is completed to obtain the T1-weighted iron-based contrast agent; The total iron elements are the sum of divalent iron ions and trivalent iron ions.

8. The method of claim 7, wherein the T1 -weighted iron-based contrast agent is prepared by the method comprising the steps of: The preparation method specifically comprises the following steps: dissolving the modified dextran in an aqueous solution and stirring in a 37 DEG C constant-temperature water bath, then adding an iron ion solution into the modified dextran solution according to a molar ratio of divalent iron ions to trivalent iron ions of 50%, placing the solution in a 37 DEG C constant-temperature water bath and stirring at 500 r / min, then adjusting the pH of the reaction solution to 9 by using a 1 M sodium hydroxide solution at a flow rate of 2.5 mL / s, stirring for 30 min, and then removing the excess sodium hydroxide, so as to finally prepare the iron-based magnetic resonance contrast agent prepared by using the modified dextran as a template.

9. The method of claim 8, wherein the T1 -weighted iron-based contrast agent is prepared by, The particle size of the iron oxide nanoparticles ranges from 2 nm to 10 nm; The inorganic base used for adjusting the pH is an aqueous solution of sodium hydroxide or potassium hydroxide; The method for removing the excess inorganic base in the solution is dialysis or acid-base neutralization by using a dilute hydrochloric acid solution.

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