Preparation method and application of manganese-based amino acid coordination polymer

Abstract

The application discloses a preparation method and application of a manganese-based amino acid coordination polymer, and belongs to the technical field of nano-enzymes and nano-drugs. A manganese-based amino acid coordination polymer is prepared by adopting a divalent manganese salt and histidine and other amino acids to perform a coprecipitation reaction. The polymer has a nanoparticle structure, is small in size, high in stability, has an enzyme-like activity, catalyzes hydrogen peroxide to generate highly toxic hydroxyl radicals, and is used for specifically killing tumor cells. The manganese-based amino acid coordination polymer has the ability to catalyze the generation of singlet oxygen in the presence of hydrogen peroxide. Meanwhile, the manganese-based amino acid coordination polymer has glutathione oxidase-like activity, consumes glutathione to overcome the resistance of tumors to oxidative stress, improves the chemical kinetic effect, and has an anti-tumor application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and anti-tumor therapeutic drugs, and particularly relates to a preparation method and application of a manganese-based amino acid coordination polymer. Background Art

[0002] Metal complexes have demonstrated significant anticancer effects and have emerged as promising candidates for cancer therapy. Metals and amino acids (AAs), as two entities, have been widely used in the fields of biomaterials and nanomedicine. Recently, amino acid-metal coordination nanomaterials (AMCNs), newly developed nanoformulations, have shown great potential for biomedical applications in cancer therapy, antimicrobial applications, and biomedical imaging. Due to the rich biological and chemical properties of metals and AA, metalloamino acids can not only serve as drug carriers with specific tumor-targeting capabilities, but also enable synergistic and image-guided therapies.

[0003] The tumor microenvironment is characterized by acidity and overexpression of hydrogen peroxide (H2O2) and glutathione. This acidic environment not only favors tumor cell growth and invasion but also affects the catalytic activity of nanozymes. Certain nanozymes may exhibit enhanced catalytic activity under acidic conditions and, in weakly acidic conditions, exhibit peroxidase-like activity, catalyzing H2O2 to produce cytotoxic reactive oxygen species (ROS) through the Fenton reaction, thereby inhibiting tumor cell growth. Glutathione, an important intracellular antioxidant, is often overexpressed in tumor cells to protect against oxidative damage. Overexpression of glutathione can impair the effectiveness of oxidative stress therapies, such as those produced by nanozymes, because glutathione can scavenge ROS, reducing their cytotoxic effects. Nanozymes can modulate the pH and redox state of the tumor microenvironment through catalytic reactions, thereby improving tumor cell growth conditions and enhancing therapeutic efficacy. For example, nanozymes can catalyze the decomposition of hydrogen peroxide to produce oxygen, alleviating tumor hypoxia. Furthermore, they can catalyze the generation of ROS and scavenge overexpressed glutathione, weakening the tumor's antioxidant capacity. Breast cancer is one of the most common malignancies in women, with symptoms including breast lumps, nipple discharge, and breast skin changes. Nanozyme therapy can kill breast cancer cells by regulating the tumor microenvironment, generating ROS, and through immune regulation.

[0004] In the human body, amino acids (AA), organic ligands containing both carboxyl (-COOH) and amino (-NH2) groups, serve as building blocks for proteins and enzymes and as precursors for the synthesis of other important biomolecules, such as hormones and neurotransmitters. Amino acids and their derivatives have long been of great interest in pharmaceuticals. For example, as early as 1980, Gosalvez et al. developed an amino acid derivative, thiazolidine-4-carboxylic acid (thioproline), which has been used as an antitumor drug to transform tumor cells into normal cells. Amino acids have been widely used as modified ligands to improve the solubility of poorly water-soluble drugs. Amino acids possess low toxicity, low immunogenicity, and high affinity for specific receptors in the body. Their structural diversity, chirality, and diverse functional groups make them ideal building blocks for the fabrication of nanomaterials for biomedical applications.

[0005] Metal ions play crucial roles in living organisms, with the majority existing in the body as metal complexes with proteins, lipids, and carbohydrates, such as the ferrous ions in hemoglobin and myoglobin and the cupric ions in ceruloplasmin. Metal coordination chemistry offers attractive advantages for the design of bioactive molecules and nanomaterials. By precisely selecting the metal center and adjusting the coordination number, labile groups, and bioactive or auxiliary ligands, nanomaterials with diverse biological mechanisms of action can be prepared for applications in disease diagnosis, sensing, and therapy. Because transition metal centers can have higher coordination numbers than four-bonded carbon, the coordination "chemical space" of the metal is increased compared to pure organic compounds.

[0006] In natural proteases, the coordination site of metals and amino acids is usually the catalytic active site, where metal ions stabilize the structure of the protease, act as agonists or controllers in excitation and selectivity, and participate in the redox reaction process as Lewis acids. The types of surrounding amino acids with specific side chain groups determine the tertiary protein structure and substrate binding specificity.

[0007] In recent years, many metal amino acid nanomaterials have been prepared by metal amino acid coordination and have been used in drug delivery, cancer imaging and treatment, and bacterial killing. For these biomedical applications, they show unique advantages, including good biocompatibility, simple and mild manufacturing, multifunctional structure and multifunctionality, but manganese-based amino acid coordination polymers have not yet been used in the anti-tumor field. Unique advantages of manganese-based materials in biomedicine, especially in the field of nanozymes. Manganese is an essential trace element for the human body and has better biocompatibility than other transition metals (such as iron, cobalt, nickel, copper, etc.). Manganese has relatively low toxicity, high metabolism and clearance efficiency in the body, and is not easy to accumulate in the body for a long time, reducing the potential risk of long-term toxicity. More importantly, manganese has unique redox properties and has multiple valence states (Mn 2+ / Mn 3+ / Mn 4+ Manganese can also be used as a contrast agent for magnetic resonance imaging (MRI), enabling integrated diagnosis and treatment. Therefore, the development of manganese-based amino acid coordination polymers for efficient and safe tumor treatment has important theoretical and practical significance. Summary of the Invention

[0008] To achieve the above technical objectives, the present invention provides a method for preparing and applying a manganese-based amino acid coordination polymer. During the synthesis reaction, the composition, structure, morphology, and size of the complex are regulated by adjusting NaOH, the material ratio, the reaction temperature, the reaction time, and the type of amino acid. The present invention further optimizes the structure and surface functionalization by regulating the composition of the amino acids in the manganese-based amino acid coordination polymer to enhance its catalytic and anti-tumor activity. The manganese-based amino acid coordination polymer provided by the present invention has enzyme-like activity, particularly peroxidase-like activity, and can generate a large amount of reactive oxygen species while rapidly scavenging glutathione in tumor cells, overcoming the oxidative stress resistance of tumor cells.

[0009] The present invention provides a method for preparing a manganese-based amino acid coordination polymer, comprising the following steps:

[0010] (1) Dissolve sodium hydroxide and amino acid in ethanol and stir for 10-30 min to obtain a mixed solution. Then dissolve divalent manganese salt in 1 mL of deionized water and add the divalent manganese salt solution dropwise to the mixed solution of sodium hydroxide and amino acid.

[0011] Wherein, the molar ratio of divalent manganese salt to amino acid is 1:1-1:10.

[0012] The divalent manganese salt is any one of Mn(NO3)2•4H2O, MnCl2•4H2O or MnCl2•2H2O.

[0013] The amino acid is any one or more of histidine, arginine, tyrosine and lysine.

[0014] Preferably, the amino acid consists of two amino acids, one of which is histidine and the other is any one of arginine, tyrosine or lysine.

[0015] More preferably, the amino acid consists of two amino acids, one of which is histidine and the other is any one of arginine, tyrosine or lysine.

[0016] The concentration of NaOH solution is 0.1-10 M.

[0017] (2) Place the solution treated in step (1) into a beaker, place it at 25°C for 3 h, and stir it continuously with a magnetic stirrer;

[0018] (3) The mixture obtained in step (2) is washed and centrifuged, the supernatant is removed, and the mixture is dried to obtain the desired product.

[0019] The specific operations of washing and centrifugation are: washing with ethanol three times, and then centrifuging at a speed of 6000 rpm.

[0020] The manganese-based amino acid coordination polymer prepared by the above method has a uniform nanoparticle structure, a small size and a stable structure.

[0021] Experiments have confirmed that the complex has excellent singlet oxygen production and peroxidase-like activity, catalyzing H₂O₂ to generate ·OH, which kills tumor cells. Furthermore, the nanozyme has a highly efficient GSH consumption capacity, overcoming tumor cells' resistance to oxidative stress and thereby enhancing the efficacy of chemodynamic therapy. This manganese-based amino acid coordination polymer can be used to prepare anti-tumor drugs.

[0022] The nanozyme coordination polymer of the present invention mainly kills tumor cells by catalyzing hydrogen peroxide to produce hydroxyl radicals and singlet oxygen. At the same time, the nanozyme coordination polymer of the present invention can consume the overexpressed glutathione in the tumor microenvironment, overcome the oxidative stress in tumor cells, and inhibit the growth of tumor cells.

[0023] Furthermore, the manganese-based amino acid coordination polymer prepared by the present invention can be used to prepare drugs for treating breast cancer.

[0024] Furthermore, the present invention also provides a pharmaceutical composition comprising the above-mentioned manganese-based amino acid coordination polymer as one of the main active ingredients, and a pharmaceutically acceptable carrier.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention selects amino acids for coordination polymerization on the basis of manganese-based metals to synthesize nanomaterials with nanoparticle structures, which have good biocompatibility. The metallic manganese contained in the complex is an essential element for the human body, participating in the human body's metabolic process and maintaining normal physiological activities of the body, and has good biosafety. The amino acids have low toxicity, low immunogenicity and high affinity for specific receptors in the body.

[0027] 2. The manganese-based amino acid coordination polymer synthesized by the present invention can undergo a Fenton-like reaction to generate hydroxyl radicals, which have a killing effect on tumor cells and can consume glutathione, thereby producing a chemodynamic therapeutic effect on tumor cells in the tumor microenvironment.

[0028] 3. The manganese-based amino acid coordination polymer of the present invention is prepared by a coprecipitation method. The raw materials used are easily available, green and non-toxic, the synthesis method is simple, the yield is high, and it is easy to carry out large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Scanning electron micrographs of the manganese-based amino acid coordination polymers of Examples 1-4; (a) histidine, (b) histidine arginine, (c) histidine tyrosine, (d) histidine lysine;

[0030] Figure 2 This is a graph showing the particle size of the manganese-based amino acid coordination polymer prepared in Examples 1-4;

[0031] Figure 3 The X-ray powder diffraction pattern of the manganese-based amino acid coordination polymer prepared in Example 1-4;

[0032] Figure 4 This is the infrared contrast spectrum of manganese-based amino acid coordination polymer;

[0033] Figure 5 This is a diagram for evaluating the peroxidase-like activity of manganese-based amino acid coordination polymers;

[0034] Figure 6 This is a graph showing changes in glutathione consumption by manganese-based amino acid coordination polymers;

[0035] Figure 7 A comparison chart of the singlet oxygen produced by manganese-based amino acid coordination polymers over time;

[0036] Figure 8 This is the effect of manganese-based amino acid coordination polymer on the viability of breast cancer cell line 4T1 cells. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments using examples. Example 1

[0038] Preparation of manganese-histidine complexes:

[0039] First, 40 mg of NaOH (1 mmol) and 155 mg of histidine (1 mmol) were dissolved in 60 mL of 85% ethanol and stirred for 10 minutes. Then, 131.94 mg of MnCl2·4H2O (2 / 3 mmol) was dissolved in 1 mL of deionized water. The MnCl2·4H2O solution was added dropwise to the NaOH and histidine mixture. The mixture was stirred at 25°C for 3 hours. After the reaction, the precipitate was collected by centrifugation (6000 rpm, 6 minutes) and washed three times with ethanol. Finally, the manganese-histidine complex was obtained by drying. Figure 1 a is a scanning electron micrograph of the manganese-histidine complex of this embodiment; it can be seen from the figure that the manganese-histidine complex has a uniform nanoparticle structure with a size of about 100 nm. Figure 2 The particle size diagram of a also confirms the size and stability of the manganese-histidine complex. Figure 3 XRD also confirmed that the synthesized manganese-histidine was an amorphous complex. Figure 4 The infrared spectrum also shows that in the complex of manganese and histidine, the nitrogen characteristic peaks of the imidazole ring at 1588 and 1457 cm-1 are significantly shifted, proving that manganese is coordinated with the nitrogen on the imidazole ring. Although the stretching vibration of the carboxyl group is shifted, it may be affected by the intermolecular hydrogen bond effect. Due to the steric hindrance effect, the carboxyl group should be difficult to coordinate with manganese, so it is speculated that the complex is a di-coordinated complex, and each imidazole ring in the histidine provides a nitrogen atom to coordinate with manganese. Example 2

[0040] Preparation of manganese-histidine-arginine complex:

[0041] First, 40 mg of NaOH (1 mmol), 100 mg of histidine (2 / 3 mmol), and 55 mg of arginine were dissolved in 60 mL of 85% ethanol and stirred for 10 minutes. Next, 131.94 mg of MnCl2·4H2O (2 / 3 mmol) was dissolved in 1 mL of deionized water. The MnCl2·4H2O solution was added dropwise to the NaOH and histidine mixture. The reaction was stirred at 25°C for 3 hours. After the reaction, the precipitate was collected by centrifugation (6000 rpm, 6 minutes) and washed three times with ethanol. Finally, the manganese-histidine-arginine complex was obtained by drying. Figure 1 b is a scanning electron micrograph of the manganese-histidine-arginine complex of this embodiment; it can be seen from the figure that the manganese-histidine-arginine complex has a uniform nanoparticle structure with a size of about 100 nm. Figure 2 The particle size diagram in b also confirms the size and stability of the manganese-histidine-arginine complex. Figure 3XRD also confirmed that the synthesized manganese-histidine-arginine complex was an amorphous complex. Example 3

[0042] Preparation of manganese-histidine-tyrosine complexes:

[0043] First, 40 mg of NaOH (1 mmol), 100 mg of histidine (2 / 3 mmol), and 55 mg of tyrosine were dissolved in 60 mL of 85% ethanol and stirred for 10 minutes. Next, 131.94 mg of MnCl2·4H2O (2 / 3 mmol) was dissolved in 1 mL of deionized water. The MnCl2·4H2O solution was added dropwise to the NaOH and histidine mixture. The reaction was stirred at 25°C for 3 hours. After the reaction, the precipitate was collected by centrifugation (6000 rpm, 6 minutes) and washed three times with ethanol. Finally, the mixture was dried to obtain the manganese-histidine-tyrosine complex. Figure 1 c is a scanning electron micrograph of the manganese-histidine-tyrosine complex of this embodiment; it can be seen from the figure that the manganese-histidine-tyrosine complex has a uniform nanoparticle structure with a size of about 100 nm. Figure 2 The particle size diagram of c also confirms the size and stability of the manganese-histidine-tyrosine complex. Figure 3 XRD also confirmed that the synthesized manganese-histidine-tyrosine complex was an amorphous complex. Example 4

[0044] Preparation of manganese-histidine-tyrosine complexes:

[0045] First, 40 mg of NaOH (1 mmol), 100 mg of histidine (2 / 3 mmol), and 55 mg of lysine were dissolved in 60 mL of 85% ethanol and stirred for 10 minutes. Next, 131.94 mg of MnCl2·4H2O (2 / 3 mmol) was dissolved in 1 mL of deionized water. The MnCl2·4H2O solution was added dropwise to the NaOH and histidine mixture. The reaction was stirred at 25°C for 3 hours. After the reaction, the precipitate was collected by centrifugation (6000 rpm, 6 minutes) and washed three times with ethanol. Finally, the mixture was dried to obtain the manganese-histidine-lysine complex. Figure 1 d is a scanning electron micrograph of the manganese-histidine-lysine complex of this embodiment; it can be seen from the figure that the manganese-histidine-lysine complex has a uniform nanoparticle structure with a size of about 100 nm. Figure 2 The particle size diagram of d also confirms the size and stability of the manganese-histidine-lysine complex. Figure 3 XRD also confirmed that the synthesized manganese-histidine-lysine complex was an amorphous complex.Figure 4 The infrared spectrum also shows the group structure of manganese-histidine-lysine. Example 5

[0046] Study on the peroxidase-like activity of manganese-based amino acid coordination polymers:

[0047] The peroxidase-like activity was characterized by the ultraviolet absorption spectrum after the nanozyme reacted with 3,3',5,5'-tetramethylbenzidine (TMB). In order to ensure the stability of the sample, the sample was added to deionized water under ultrasonic conditions to prepare a 1 mg / mL sample solution, and an appropriate amount was mixed with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in a pH 4.0 PBS buffer solution so that the final sample, TMB, and H2O2 concentrations were 20 μg / mL, 6 mM, and 0.66 mM, respectively. The mixed solution was placed at a constant temperature of 37°C and allowed to react for 5 minutes in the dark. After the reaction, the solution turned blue and was placed in an ultraviolet spectrophotometer. The detection was performed in the wavelength range of 300 nm-1000 nm, and an obvious characteristic peak of oxidized TMB appeared. The intensity of the absorption peak at 652 nm represents the yield of hydroxyl radicals, that is, the activity of catalase. Figure 5 As shown in the figure, the absorption peak of the manganese-histidine-lysine complex at 652 nm is the highest, which confirms that the manganese-histidine-lysine complex can produce more •OH and has good peroxidase-like activity. Example 6

[0048] Study on glutathione oxidase-like activity of manganese-based amino acid coordination polymers:

[0049] The glutathione peroxidase-like activity of the samples was assessed using the DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) method. At pH 4, different samples were mixed with glutathione (0.16 mM) and stirred continuously in the dark. After the samples reacted for 10 minutes, DTNB (0.08 mM) was added. The samples were then incubated under alkaline conditions for 5 minutes. The absorption curve was measured using a UV spectrophotometer in the wavelength range of 300 nm to 600 nm. The decrease in the absorption peak at 412 nm indicated the glutathione depletion capacity, as shown in Figure 5. Figure 6 As shown in the figure, as the reaction of the manganese-based amino acid coordination polymer proceeds, glutathione is gradually consumed, indicating that the sample has good glutathione oxidase-like activity and can efficiently and quickly remove glutathione. When the reaction time is 10 minutes, the glutathione oxidase-like activity of the manganese-histidine-tyrosine complex is the best. Example 7

[0050] Study on the singlet oxygen generating activity of manganese-based amino acid coordination polymers:

[0051] Detection by the attenuation of the UV absorbance of 1,3-diphenylisobenzofuran (DPBF) 1 Generation of O2. A 20 μg / mL sample was mixed with 20 mM hydrogen peroxide and 0.1 mM DPBF for reaction, and then the absorption curve was measured at intervals using a UV spectrophotometer in the wavelength range of 300 nm-550 nm. The decrease in the absorption peak at 415 nm represents the ability of the sample to produce singlet oxygen. The principle is that the sample can catalyze hydrogen peroxide to produce singlet oxygen, and DPBF is oxidized by singlet oxygen, thereby reducing the absorption peak of DPBF at 415 nm. Figure 7 As shown in the figure, with the increase of reaction time, the absorption peak at 415 nm gradually decreases, indicating that the manganese-based amino acid coordination polymer produces singlet oxygen under the catalysis of hydrogen peroxide, among which the manganese-based histidine-lysine complex has a better ability to produce singlet oxygen. Example 8

[0052] Study on the anti-tumor activity of manganese-based amino acid coordination polymers:

[0053] Breast cancer cell line 4T1 cells were digested and counted. 4 The number of cells per well was seeded in a 96-well plate and cultured overnight in a 37°C carbon dioxide (5%) incubator. The samples were prepared into different concentrations using DMEM medium and then added to the well plate, with three replicate wells for each concentration, and incubated in the incubator for 24 hours. Then, 10 μL of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (CCK-8) solution was added to each well. After incubation for 2 hours, the absorption of each well was measured on a microplate reader, and the absorption at 450 nm was used to indicate the size of the cell viability. Figure 8 It can be seen that manganese-based amino acid coordination polymers have good tumor cell inhibition effects, among which the coordination polymer nanozymes of two amino acids show more significant anti-tumor activity.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a manganese-based amino acid coordination polymer, characterized in that: The following steps are involved: (1) Dissolve sodium hydroxide and amino acid in ethanol and stir for 10-30 min to obtain a mixed solution. Then dissolve divalent manganese salt in 1 mL of deionized water and add the divalent manganese salt solution dropwise to the mixed solution of sodium hydroxide and amino acid. The molar ratio of the divalent manganese salt to the amino acid is 1:1-1:10; the amino acid is histidine, or a combination of histidine and one or more of arginine, tyrosine and lysine; (2) Place the solution obtained in step (1) into a beaker and stir the mixture continuously with a magnetic stirrer for 2-4 hours; (3) The mixed solution obtained in step (2) is washed and centrifuged, the supernatant is removed, and the mixture is dried to obtain a manganese-based amino acid coordination polymer.

2. The method for preparing a manganese-based amino acid coordination polymer according to claim 1, wherein The divalent manganese salt in step (1) is any one of Mn(NO3)2•4H2O, MnCl2•4H2O or MnCl2•2H2O.

3. The method for preparing the manganese-based amino acid coordination polymer according to claim 1, wherein The amount of NaOH used in step (1) is 0.1-10 mmol.

4. The method for preparing a manganese-based amino acid coordination polymer according to claim 1, wherein In step (2), the reaction temperature is 20-30°C and the reaction time is 2-4 h.

5. The method for preparing a manganese-based amino acid coordination polymer according to claim 1, wherein: The specific operations of washing and centrifugation in step (3) are: washing three times with ethanol, and then centrifuging at a speed of 5000-12000 rpm.

6. A manganese-based amino acid coordination polymer, characterized in that The method according to any one of claims 1 to 5 is used to prepare the present invention.

7. Use of the manganese-based amino acid coordination polymer according to claim 6 in the preparation of anti-tumor drugs.

8. A pharmaceutical composition, characterized in that The invention comprises the manganese-based amino acid coordination polymer according to claim 6 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

Images