A polyaspartic acid manganese macromolecular complex, a preparation method and application thereof

Abstract

The application discloses a kind of polyaspartic acid manganese polymer complex, it is characterized in that, its molecular structure formula is as shown in formula I: (I) Wherein, m is 1-33, n is 0-100.The polyaspartic acid manganese polymer complex provided in the application is prepared by one-step method chelation of polyaspartic acid ligand and manganese ion, process is simple and low in cost, as contrast agent, it has high relaxation efficiency and good biological safety, can realize kidney target enrichment, greatly improve the signal-to-noise ratio of MRI scanning, has excellent application prospect in kidney function evaluation and related disease diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic materials technology, specifically relating to a polyaspartic manganese polymer complex, its preparation method, and its application. Background Technology

[0002] Kidney disease, especially acute kidney injury (AKI), is a major health problem with high morbidity and mortality worldwide. Early, accurate, and non-invasive assessment of kidney function is crucial for clinical diagnosis, treatment decisions, and prognosis improvement. Magnetic resonance imaging (MRI), with its non-ionizing radiation, excellent soft tissue resolution, and unlimited depth of penetration, has become an ideal tool for assessing kidney structure and function. Contrast-enhanced MRI (CE-MRI) with the injection of exogenous contrast agents allows for dynamic monitoring of renal blood perfusion, glomerular filtration, and renal tubular function, enabling a comprehensive assessment from anatomical structure to physiological function.

[0003] Currently, the most widely used MRI contrast agents in clinical practice are gadolinium-based contrast agents (GBCAs), such as gadopentetate dimeglumine (Gd-DTPA). However, the clinical application of GBCAs poses significant safety risks. Studies have shown that Gd... 3+ After ions dissociate from chelates, they can accumulate in patients with renal insufficiency, leading to the serious complication of systemic fibrosis (NSF), and there is evidence that they may cause gadolinium deposition in the brain. Therefore, the use of GBCAs in patients with impaired renal function, such as those with acute kidney injury (AKI), is strictly limited or even contraindicated. This contradiction highlights the urgent clinical need for novel MRI contrast agents that combine high imaging efficacy with excellent biocompatibility, particularly suitable for patients with kidney disease.

[0004] In search of alternatives to GBCAs, researchers have turned their attention to other paramagnetic metal ions. Among them, manganese ions (Mn) are particularly noteworthy. 2+ Mn exhibits unique advantages: as an essential trace element for the human body, its metabolic pathway is well-defined, and its theoretical biocompatibility is better; simultaneously, Mn... 2+With 5 unpaired electrons, it can efficiently shorten the longitudinal relaxation time (T1) of water protons, generating a strong T1-weighted positive contrast enhancement signal. In recent years, a variety of manganese-based contrast agents, including small molecule manganese chelates, manganese dioxide nanoparticles, and manganese-doped nanomaterials, have been reported. However, the existing manganese-based contrast agents still have the following limitations when applied to renal functional imaging: 1) Insufficient renal targeting and clearance: Most manganese-based preparations (especially nanoparticles) are large in size or captured by the reticuloendothelial system, making it difficult to effectively filter through the glomerulus, resulting in weak renal aggregation signal, high background noise, and the risk of long-term in vivo retention. They cannot achieve clear renal corticomedullary boundary and dynamic functional assessment, such as Nanoscale. 2011 Nov 7;3(12):4943-4945. doi: 10.1039 / c1nr11242b A polyaspartic acid manganese oxide contrast agent was disclosed, but it is difficult to achieve the purpose of kidney disease diagnosis of the present invention; 2) Relaxation efficiency and stability are difficult to balance: Although small molecule manganese chelates are easily excreted by the kidneys, their relaxation efficiency (r1 value) is relatively low, and some chelates have poor stability in the physiological environment in vivo, with potential toxic risks of premature release of manganese ions; 3) The preparation process is complicated: The synthesis steps of many high-performance manganese-based nanomaterials are cumbersome, the conditions are harsh, the reproducibility is poor, or complex raw materials with unknown biocompatibility are used, which is not conducive to large-scale preparation and clinical translation.

[0005] Polyaspartic acid (PAsp) is a water-soluble, biodegradable, and biocompatible polymer material synthesized from aspartic acid monomers. Its side chains are rich in carboxyl groups, making it resistant to various metal ions (such as Ca). 2+ Mn 2+ Gd 3+ PAsp exhibits excellent chelating ability. While existing technologies have explored its use as a drug carrier or for other ion chelation, its specific design for efficient and stable chelation of Mn is unique. 2+ Furthermore, the molecular weight, grafting modification, and chelation process of this agent have been systematically optimized to prepare a dedicated renal function MRI contrast agent that combines high relaxation efficiency, rapid renal clearance, excellent stability, and a simple preparation method. No such agent has been publicly reported yet. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a polyaspartic manganese polymeric complex and its preparation method, aiming to address the safety hazards of GBCAs and the shortcomings of other manganese-based contrast agents in renal applications.

[0007] This invention also discloses the application of polyaspartic manganese polymer complex in renal functional magnetic resonance contrast agents.

[0008] The objective of this invention is achieved through the following technical solution: A polyaspartic acid manganese polymeric complex, the molecular structure of which is shown in Formula I: (I) Where m is 1 to 33 and n is 0 to 100.

[0009] As part of the same inventive concept, the present invention also provides a method for preparing the aforementioned polyaspartic manganese polymeric complex, comprising the following steps: S1) Add manganese chloride tetrahydrate solution to sodium polyaspartate solution, stir and mix evenly to carry out coordination reaction, and form Mn-PAsp solution; After the S2 reaction is completed, the resulting Mn-PAsp solution is purified and dried to obtain a solid product, namely the polyaspartic acid manganese polymer complex.

[0010] In some specific embodiments, the concentration of the sodium polyaspartate solution in step S1) is 5-15 mg / mL.

[0011] In some specific embodiments, the concentration of the manganese chloride tetrahydrate solution in step S1) is 9.9-79 mg / mL.

[0012] In some specific embodiments, the volume ratio of sodium polyaspartate to manganese chloride tetrahydrate in step S1) is 5-15:1.

[0013] In some specific embodiments, the process conditions for the coordination reaction in step S1) are: the pH value of the reaction system is 7.0-12.0, and the reaction is carried out at room temperature for 1.5-3 hours.

[0014] In some specific embodiments, the purification described in step S2) is specifically dialysis; preferably, the Mn-PAsp solution in step S1) is transferred to a dialysis bag and dialyzed in ultrapure water for 2-5 days to remove unreacted small molecules and salt impurities.

[0015] In some specific embodiments, the drying described in step S2) is freeze drying.

[0016] As part of the same inventive concept, the present invention also provides the application of the aforementioned polyaspartic manganese polymer complex in renal function magnetic resonance contrast agents.

[0017] Compared with the prior art, the present invention has at least the following advantages: 1) The polyaspartic manganese polymer complex provided by this invention is used for MRI imaging and has a good renal accumulation effect, selectively accumulating in the kidneys and improving renal imaging capabilities; it also exhibits extremely high transverse and longitudinal relaxation efficiency (r1: 27.65 mM) under a clinical 3.0 T magnetic field. -1 s -1 r2: 52.34 mM -1 s -1 Furthermore, with an r2 / r1 ratio of 1.89, it can serve as an excellent T1 contrast agent for magnetic resonance imaging. 2) The preparation method provided by this invention uses readily available raw materials, has simple process steps, high repeatability, and is suitable for widespread application. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 A schematic diagram of the molecular structure of the polyaspartic manganese polymer complex provided by the present invention; Figure 2 This is a schematic diagram of the stability test of the polyaspartic manganese polymer complex provided by the present invention. Figure 3 The relaxation efficiency of the polyaspartic manganese polymer complex and Gd-DTPA provided by this invention was measured under clinical 3.0 T magnetic resonance imaging, and magnetic resonance scan images of solutions at different concentrations were also obtained. Figure 4 The biosafety test results of the polyaspartic manganese polymer complex provided by the present invention were verified from multiple aspects, including in vitro cytotoxicity, hemolysis experiments, and in vivo serum biochemical indicators. Figure 5 The imaging effect of the polyaspartic manganese polymer complex provided by this invention as a magnetic resonance imaging (MRI) contrast agent in normal rats was verified. Figure 6 The present invention provides a high molecular weight polyaspartic acid manganese complex for imaging diagnostic verification in rats with acute kidney injury using magnetic resonance imaging, and compares the imaging results with those of normal rats. Figure 7 The present invention provides a comparison of urine formation in normal rats and rats with acute kidney injury after tail vein injection of polyaspartic manganese; Figure 8 This is the Fourier transform infrared spectrum of the polyaspartic manganese polymer complex described in this invention. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are merely descriptive and not limiting, and should not be construed as limiting the scope of protection of the present invention.

[0021] When a quantity, concentration, or other value or parameter is described as a range, preferred range, or preferred upper and lower limits, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred values, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical range values ​​listed herein include the endpoints of the range and all integers and fractions within that range.

[0022] Unless otherwise stated, all percentages, parts, ratios, etc. in this document are by weight.

[0023] The materials, methods, and embodiments described herein are exemplary and should not be construed as limiting unless otherwise stated.

[0024] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0025] Example 1: Preparation of polyaspartic manganese polymer complex This embodiment provides a method for preparing a polyaspartic manganese polymeric complex, comprising the following steps: S1) Weigh 110 mg of sodium polyaspartate (PAsp-Na), dissolve it in 10 mL of ultrapure water, and stir thoroughly to ensure complete dissolution, thereby obtaining a sodium polyaspartate solution with a mass concentration of 110 mg / 10 mL. S2) Slowly add a 20 mg / mL, 1 mL solution of manganese chloride tetrahydrate (MnCl2·4H2O) to the sodium polyaspartate solution in step S1), and mix thoroughly under continuous magnetic stirring. Then adjust the pH of the reaction system to 10.0 with sodium hydroxide (NaOH) solution, and keep stirring for 2 hours to allow polyaspartic acid and manganese ions to fully coordinate and form a polymer complex Mn-PAsp solution. After the reaction is complete (S3), the resulting Mn-PAsp solution is transferred to a dialysis bag and dialyzed in ultrapure water for three days to remove unreacted small molecules and salt impurities. S4) The purified Mn-PAsp solution from step S3) is freeze-dried to obtain a solid product, namely the polyaspartic acid manganese polymer complex.

[0026] This application performs Fourier transform infrared spectroscopy analysis on the structure of the solid product prepared in Example 1, and the results are as follows: Figure 8 As shown in the figure, 1600 cm -1 The peak shifted to a higher wavenumber of 1614 cm⁻¹ -1 This is attributed to the stretching vibration absorption of C=O and the in-plane deformation vibration of NH; the stretching vibration peaks of OH in the hydroxyl group and NH in the amide group also increased from 3406 cm⁻¹. -1 Moved to 3425 cm -1 These results indicate that Mn 2+ The chelation affected the FT-IR spectral peaks of Mn-PAsp, and the amino and carboxyl groups of PAsp participated in the interaction with Mn. 2+ The coordination of the complex indicates that this application successfully prepared a polyaspartic manganese polymeric complex, and its molecular structural formula is as follows: Where m is 1 to 33 and n is 0 to 100.

[0027] Test example: Polyaspartic manganese polymer complex This application uses the polyaspartic manganese polymer complex prepared in Example 1 as an example to characterize, verify the safety and efficacy of the prepared polyaspartic manganese polymer complex, specifically: 1) Stability test A 0.5 mM manganese polyaspartic acid manganese polymer complex (prepared in Example 1) was mixed with different zinc trifluoroacetate solutions (0 (blank control group), 0.5, 1.0, 1.5 and 2.5 mM Zn). 2+） Mix them and observe their 1 / T1 values ​​at different time points.

[0028] The results are as follows Figure 2 As shown in the figure, at different time points, the 1 / T1 values ​​in the four zinc trifluoroacetate solutions were almost the same as those in the solution without zinc trifluoroacetate (blank control group). This indicates that the manganese ions hardly dissociated, suggesting that the polyaspartic acid manganese polymer complex exhibits good stability.

[0029] 2) Relaxation performance test Test method: The manganese concentration in the solution was determined by atomic absorption spectrometry. The polyaspartic acid manganese polymer complex was prepared into solutions with different manganese concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM. The T1 of the samples was measured using a 3.0T magnetic resonance scanner. The T1 relaxation efficiency (r1) was calculated and compared with that of clinical Gd-DTPA. The results are as follows Figure 3As shown in the figure, the relaxation efficiency of the polyaspartic manganese polymer complex at 3.0 T is r1 = 27.65 mM. -1 s -1 ( Figure 3 As shown in a), its relaxation efficiency is far higher than that of Gd-DTPA (r1=4.84 mM). -1 s -1 Furthermore, 3.0T in vitro imaging results showed that the polyaspartic manganese polymer complex exhibited excellent T1-weighted imaging performance, with significant enhancement of the magnetic resonance signal, and under the same metal concentration conditions, it showed more significant contrast enhancement than Gd-DTPA. Figure 3 b).

[0030] 3) Biosafety performance testing Test Method: In this application, a polyaspartic manganese polymeric complex was co-incubated with human renal cortical proximal tubular epithelial cells (HK-2) and compared with clinical Magendie studies. Specifically: Well-grown HK-2 cells were seeded in 96-well plates and incubated for 24 hours; then, different concentrations of manganese or gadolinium (0 μg / mL, 15 μg / mL, 30 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) were added and co-incubated for 24 hours; after washing away the cells with PBS, basal medium containing CCK-8 reagent was added and incubation continued for 2 hours; finally, the absorbance (OD) value of each well was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0031] The results are as follows Figure 4 As shown, where Figure 4 a represents the cytotoxicity results. As shown in the figure, the cell viability of cells co-incubated with Mn-PAsp remained above 90%, while the cell viability of cells co-incubated with magnus ultimately dropped to 80%, indicating that the cytotoxicity of this polyaspartic manganese polymer complex is much lower than that of Gd-DTPA. Simultaneously, this application also conducted a cell hemolysis experiment on the polyaspartic manganese polymer complex. Specifically, 1 mL of whole blood was collected from healthy rats, and erythrocytes were separated and purified. The erythrocytes were incubated with Mn-PAsp at different manganese concentrations (0 μg / mL, 15 μg / mL, 30 μg / mL, 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and Con (pure water, positive control group, i.e., the positive control group where 100% hemolysis occurred) at 37 ℃ for 4 hours, and then centrifuged to precipitate intact erythrocytes. PBS was used as a negative control, and ultrapure water was used as a positive control. After photographing, the optical density (OD) of the supernatant was measured at 545 nm using a multi-functional microplate reader, and the hemolysis rate was calculated.

[0032] The results are as follows Figure 4 As shown in Figure b, the hemolysis rate of the polyaspartic manganese polymer complex is consistently below 2%, demonstrating that the polyaspartic manganese polymer complex of the present invention has excellent cell safety and does not cause hemolysis.

[0033] Furthermore, in this application, a high dose (0.5 mmol Mn / kg) of polyaspartic manganese polymeric complex was injected into rats via the tail vein. Blood samples were collected on days 1, 7, and 15 to detect serum renal biochemical indicators creatinine and urea. The results are as follows: Figure 4 As shown in c and 4d, it can be seen from the figure that after rats were injected with polyaspartic manganese polymeric complex, blood samples were taken on days 1, 7, and 15. The serum renal biochemical indicators creatinine and urea did not show any abnormal changes, which proves that it has high biosafety.

[0034] 4) Magnetic resonance imaging This application describes the use of a polyaspartic manganese polymer complex injected via the tail vein into normal rats, followed by magnetic resonance imaging (MRI). Specifically, after tail vein injection of Mn-PAsp (0.05 mmol Mn / kg), kidney images were acquired via MRI at different time points (0 min, 5 min, 10 min, 30 min, and 1 hour). T1-weighted MRI scans were used with the following parameters: field of view (FOV) = 12 × 8.4 cm, repetition time / echo time (TR / TE) = 300 / 11 ms, slice thickness = 1 mm, and number of excitations = 4. The rats were kept in a prone position during all image acquisition.

[0035] The results are as follows Figure 5As shown in the figure, the signals in the liver and kidneys rapidly increased 5 minutes after injection, followed by slow metabolism, and the signals significantly weakened after 2 hours compared to 5 minutes. The polyaspartic acid manganese polymer complex exhibits excellent liver and kidney enhancement effects, especially in the kidneys, where accumulation is extremely significant, revealing detailed anatomical structures of the kidneys (renal cortex, renal medulla, and renal pelvis), demonstrating its potential for diagnosing kidney diseases.

[0036] In addition, magnetic resonance imaging (MRI) was performed on rats with acute kidney injury after intravenous injection of a polyaspartic manganese polymeric complex via tail vein. Specifically, after tail vein injection of Mn-PAsp (0.05 mmol Mn / kg), MRI images of the kidneys were acquired at different time points (0 min, 5 min, 10 min, 30 min, and 1 hour). T1-weighted MRI scans were used with the following parameters: field of view (FOV) = 12 × 8.4 cm, repetition time / echo time (TR / TE) = 300 / 11 ms, slice thickness = 1 mm, and number of excitations = 4. The rats were kept in a prone position during all image acquisition. Simultaneously, images were compared with those of normal rats, and the imaging diagnostic results are as follows: Figure 6 As shown in the figure, it can be clearly seen that the damaged kidney exhibits metabolic disorders and slower metabolism, with ring-like enhancement in the renal medulla, indicating that the damage site is mainly in the renal tubules of the renal medulla. This successfully verifies that the polyaspartic manganese of the present invention has the effect of diagnosing kidney diseases.

[0037] Meanwhile, the urine formation of normal rats and rats with acute kidney injury after intravenous injection of polyaspartic manganese (0.05 mmol Mn / kg) was compared with that of other rats. Figure 7 As shown in the figure, bladder accumulation in the AKI group was significantly slower, and the signal enhancement was significantly lower than that in the normal group. This result further confirms kidney damage and metabolic dysfunction.

[0038] In summary, the polyaspartic manganese polymer complex provided by this invention can be used as a T1 contrast agent for magnetic resonance imaging (MRI). Compared with currently used clinical T1 contrast agents (gadolinium), it: ① does not cause renal systemic fibrosis or carries the risk of gadolinium residue in the body; ② uses manganese-based materials, which are endogenous metal elements required by the human body, resulting in high biocompatibility; ③ has high T1 relaxation efficiency, enabling high-efficiency imaging with small doses. Furthermore, the polyaspartic manganese polymer complex of this invention exhibits excellent renal accumulation effects, can display detailed renal anatomical structures, and can achieve non-invasive diagnosis of kidney diseases.

[0039] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A polyaspartic manganese polymeric complex, characterized in that, Its molecular structure is shown in Formula I: （I） Where m is 1 to 33 and n is 0 to 100.

2. A method for preparing the polyaspartic manganese polymer complex according to claim 1, characterized in that, Includes the following steps: S1) Add manganese chloride tetrahydrate solution to sodium polyaspartate solution, stir and mix evenly to carry out coordination reaction, and form Mn-PAsp solution; After the S2 reaction is completed, the resulting Mn-PAsp solution is purified and dried to obtain a solid product, namely a polyaspartic acid manganese polymer complex.

3. The preparation method according to claim 2, characterized in that, The concentration of the sodium polyaspartate solution mentioned in step S1) is 5-15 mg / mL.

4. The preparation method according to claim 3, characterized in that, The concentration of the manganese chloride tetrahydrate solution in step S1) is 9.9-79 mg / mL.

5. The preparation method according to claim 4, characterized in that, The volume ratio of the sodium polyaspartate solution to the manganese chloride tetrahydrate solution in step S1) is (5-15):

1.

6. The preparation method according to claim 1, characterized in that, The process conditions for the coordination reaction described in step S1) are: the pH value of the reaction system is 7.0-12.0, and the reaction is carried out at room temperature for 1.5-3 hours.

7. The preparation method according to claim 6, characterized in that, The purification described in step S2) is specifically dialysis.

8. The preparation method according to claim 7, characterized in that, The drying process described in step S2) is freeze drying.

9. The application of the polyaspartic manganese polymer complex according to claim 1 in renal functional magnetic resonance contrast agents.

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