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

Abstract

The invention 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. Divalent manganese salt, histidine and other amino acids are subjected to a co-precipitation reaction to prepare the manganese-based amino acid coordination polymer, and the polymer is of a nanoparticle structure, small in size and high in stability, has enzyme-like activity, catalyzes hydrogen peroxide to generate high-toxicity hydroxyl free radicals and is used for specifically killing tumor cells. The catalyst has the capability of catalytically generating singlet oxygen in the presence of hydrogen peroxide. Meanwhile, the manganese-based amino acid complex has glutathione oxidase-like activity, consumes glutathione to overcome the resistance of tumors to oxidative stress, improves the chemical kinetic curative effect, and has anti-tumor application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of nano materials 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 shown significant anticancer effects and have emerged as candidates with great potential for cancer therapy. Metals and amino acids (AA), as two entities, have been widely used in the fields of biomaterials and nanomedicine. Recently, amino acid-metal coordination nanomaterials (AMCNs), as newly developed nanoformulations, have shown great potential for biomedical applications in cancer therapy, antibacterial applications, biomedical imaging, etc. Since metals and AA each have rich biological and chemical properties, metallo-amino acids can not only serve as drug carriers with specific tumor targeting capabilities, but also achieve synergistic therapy and image-guided therapy, etc.

[0003] The tumor microenvironment has the characteristics of acidity and overexpression of hydrogen peroxide (H2O2) / glutathione. The acidic environment is not only conducive to the growth and invasion of tumor cells, but also affects the catalytic activity of nanozymes. Some nanozymes may show higher catalytic activity under acidic conditions and peroxidase-like activity under weak acid conditions, catalyzing H2O2 to produce cytotoxic reactive oxygen species (ROS) through the Fenton reaction, thereby inhibiting tumor cell growth. Glutathione is an important intracellular antioxidant that is often overexpressed in tumor cells to protect cells from oxidative damage. Overexpression of glutathione weakens the effect of oxidative stress therapy (such as ROS produced by nanozymes) because glutathione can scavenge ROS, thereby reducing its killing effect. Nanozymes can regulate the pH value and redox state of the tumor microenvironment through catalytic reactions, thereby improving the growth conditions of tumor cells and improving the therapeutic effect. For example, nanozymes can catalyze the decomposition of hydrogen peroxide to produce oxygen, alleviating the hypoxic state of tumors; at the same time, they can also catalyze the production of ROS and scavenge overexpressed glutathione, weakening the antioxidant capacity of tumors. Breast cancer is one of the most common malignant tumors in women, and its symptoms include breast lumps, nipple discharge, breast skin changes, etc. Nanozyme therapy can kill breast cancer cells by regulating the tumor microenvironment, producing ROS and immune regulation.

[0004] In the human body, amino acids (AA), as organic ligands containing both carboxyl (-COOH) and amino (-NH2) groups, are the basic building blocks of proteins and enzymes, and are also precursors for the synthesis of other important biomolecules, such as hormones and neurotransmitters. Amino acids and their derivatives have long been molecules of great interest in pharmacy. 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 have low toxicity, low immunogenicity, and high affinity for specific receptors in the body. Their structural diversity, chirality, and multiple functional groups make them ideal building blocks for the manufacture of nanomaterials for biomedical applications.

[0005] Metal ions play a vital role in living organisms, and most of them exist in the body as metal complexes with proteins, lipids, and carbohydrates, such as ferrous ions in hemoglobin and myoglobin, and cupric ions in ceruloplasmin. Metal coordination chemistry provides attractive features for the design of bioactive molecules and nanomaterials. By precisely selecting metal centers, adjusting coordination numbers, unstable groups, and bioactive or auxiliary ligands, nanomaterials with different biological mechanisms of action can be prepared and applied to disease diagnosis, sensing, and treatment. Since transition metal centers may have higher coordination numbers than four-bonded carbons, the coordination "chemical space" of the metal is increased compared to pure organic matter.

[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 applied to 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 its multiple valence states (Mn 2+ / Mn 3+ / Mn 4+ Manganese can also be used as a contrast agent for magnetic resonance imaging to achieve integrated diagnosis and treatment. Therefore, it is necessary to develop manganese-based amino acid coordination polymers to achieve efficient and safe treatment of tumors, which has important theoretical basis 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. In the synthesis reaction, the components, structure, morphology and size of the complex are regulated by adjusting NaOH, material ratio, reaction temperature, reaction time and amino acid type. The present invention further optimizes the structure and surface functionalization by regulating the components of amino acids in the manganese-based amino acid coordination polymer to improve its catalytic activity and anti-tumor activity. The manganese-based amino acid coordination polymer provided by the present invention has enzyme-like activity, among which the peroxidase-like activity is particularly prominent, and can generate a large amount of reactive oxygen, and can quickly remove 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: (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.

[0010] Wherein, the molar ratio of divalent manganese salt to amino acid is 1:1-1:10.

[0011] The divalent manganese salt is any one of Mn(NO3)2•4H2O, MnCl2•4H2O or MnCl2•2H2O.

[0012] The amino acid is any one or more of histidine, arginine, tyrosine and lysine.

[0013] Preferably, the amino acids consist of two amino acids, one of which is histidine, and the other is any one of arginine, tyrosine or lysine.

[0014] More preferably, the amino acids consist of two amino acids, one of which is histidine, and the other is any one of arginine, tyrosine or lysine.

[0015] The concentration of NaOH solution is 0.1-10 M.

[0016] (2) Place the solution treated in step (1) into a beaker, place at 25 °C for 3 h, and stir continuously with a magnetic stirrer; (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.

[0017] The specific operations of washing and centrifugation are: washing with ethanol three times, and then centrifuging at a speed of 6000 rpm.

[0018] The manganese-based amino acid coordination polymer prepared by the above method has a uniform nanoparticle structure, a small size and a stable structure.

[0019] Experiments have confirmed that the complex has good singlet oxygen production activity and peroxidase-like activity, and can catalyze H2O2 to generate OH, which has a killing effect on tumor cells. At the same time, the nanozyme has a high efficiency of GSH consumption, which overcomes the resistance of tumor cells to oxidative stress and thus improves the effect of chemodynamic therapy. The manganese-based amino acid coordination polymer can be used to prepare anti-tumor drugs.

[0020] The nanozyme coordination polymer of the present invention mainly achieves the effect of killing tumor cells by catalyzing hydrogen peroxide to produce hydroxyl free 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.

[0021] Furthermore, the manganese-based amino acid coordination polymer prepared by the present invention can be used to prepare drugs for treating breast cancer.

[0022] 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.

[0023] The beneficial effects of the present invention are: 1. The present invention selects amino acids for coordination polymerization on the basis of manganese-based metals to synthesize nanomaterials with nanoparticle structure, which has good biocompatibility; and the metallic manganese contained in the complex is an element necessary for the human body, participates in the human body's metabolic process and maintains the body's normal physiological activities, and has good biosafety. The amino acids are completely low in toxicity, low in immunogenicity and have high affinity for specific receptors in the body.

[0024] 2. The manganese-based amino acid coordination polymer synthesized by the present invention can undergo a Fenton-like reaction to generate hydroxyl free 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.

[0025] 3. The manganese-based amino acid coordination polymer of the present invention is prepared by a coprecipitation method. The raw materials used are easy to obtain, 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

[0026] Figure 1 The scanning electron micrographs of the manganese-based amino acid coordination polymers of Examples 1-4 are: (a) histidine, (b) histidine arginine, (c) histidine tyrosine, (d) histidine lysine; Figure 2 The particle size diagram of the manganese-based amino acid coordination polymer prepared in Examples 1-4; Figure 3 The X-ray powder diffraction pattern of the manganese-based amino acid coordination polymer prepared in Example 1-4; Figure 4 This is the infrared contrast spectrum of manganese-based amino acid coordination polymer; Figure 5 This is a diagram for evaluating the peroxidase-like activity of manganese-based amino acid coordination polymers; Figure 6 This is a graph showing changes in glutathione consumption by manganese-based amino acid coordination polymers; Figure 7 A comparison chart of the singlet oxygen produced by manganese-based amino acid coordination polymers over time; Figure 8 The effect of manganese-based amino acid coordination polymer on the viability of breast cancer cell line 4T1 cells. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific implementation examples. Example 1

[0028] Preparation of manganese-histidine complex: First, 40 mg NaOH (1mmol) and 155 mg histidine (1mmol) were dissolved in 60 mL ethanol (85%) and stirred for 10 min. Then, 131.94 mg MnCl2·4H2O (2 / 3mmol) was dissolved in 1 mL deionized water, and the MnCl2·4H2O solution was added dropwise to the mixed solution of NaOH and histidine. The reaction was stirred at 25 °C for 3 h. After the reaction, the precipitate was collected by centrifugation (6000rpm, 6 min), and the precipitate was washed with ethanol 3 times. Finally, the manganese-histidine complex was obtained by drying. Figure 1 a is a scanning electron microscope image 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 2The 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 the influence of the intermolecular hydrogen bond effect. Due to the steric hindrance effect, the carboxyl group should be difficult to coordinate with manganese. Therefore, 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

[0029] Preparation of manganese-histidine-arginine complex: First, 40 mg NaOH (1mmol), 100 mg histidine (2 / 3 mmol) and 55 mg arginine were dissolved in 60 mL ethanol (85%) and stirred for 10 min. Then, 131.94 mg MnCl2·4H2O (2 / 3mmol) was dissolved in 1 mL deionized water, and the MnCl2·4H2O solution was added dropwise to the mixed solution of NaOH and histidine. The reaction was stirred at 25 °C for 3 h. After the reaction, the precipitate was collected by centrifugation (6000rpm, 6 min), and the precipitate was washed with ethanol 3 times. 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 3 XRD also confirmed that the synthesized manganese-histidine-arginine complex was an amorphous complex. Example 3

[0030] Preparation of manganese-histidine-tyrosine complex: First, 40 mg NaOH (1mmol), 100 mg histidine (2 / 3 mmol) and 55 mg tyrosine were dissolved in 60 mL ethanol (85%) and stirred for 10 min. Then, 131.94 mg MnCl2·4H2O (2 / 3mmol) was dissolved in 1 mL deionized water, and the MnCl2·4H2O solution was added dropwise to the mixed solution of NaOH and histidine. The reaction was stirred at 25 °C for 3 h. After the reaction, the precipitate was collected by centrifugation (6000rpm, 6 min), and the precipitate was washed with ethanol 3 times. Finally, the manganese-histidine-tyrosine complex was obtained by drying. Figure 1 c is a scanning electron microscope image 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

[0031] Preparation of manganese-histidine-tyrosine complex: First, 40 mg NaOH (1mmol), 100 mg histidine (2 / 3 mmol) and 55 mg lysine were dissolved in 60 mL ethanol (85%) and stirred for 10 min. Then, 131.94 mg MnCl2·4H2O (2 / 3mmol) was dissolved in 1 mL deionized water, and the MnCl2·4H2O solution was added dropwise to the mixed solution of NaOH and histidine. The reaction was stirred at 25 °C for 3 h. After the reaction, the precipitate was collected by centrifugation (6000rpm, 6 min), and the precipitate was washed with ethanol 3 times. Finally, the manganese-histidine-lysine complex was obtained by drying. Figure 1 d is a scanning electron microscope image 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

[0032] Study on the peroxidase-like activity of manganese-based amino acid coordination polymers: 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 make the sample stable, 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, so that the final sample, TMB, and H2O2 concentrations were 20 μg / mL, 6 mM, and 0.66 mM, respectively. The mixed solution was kept at a constant temperature of 37°C and allowed to react for 5 minutes in the dark. When the solution after the reaction turned blue, it was placed in an ultraviolet spectrophotometer and detected in the wavelength range of 300 nm-1000 nm. 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, 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

[0033] Study on glutathione oxidase-like activity of manganese-based amino acid coordination polymers: The glutathione peroxidase activity of the samples was investigated according to the DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) method. Under pH=4, different samples were mixed with glutathione (0.16mM) for reaction, and the mixture was stirred continuously under light-proof conditions. After the sample reacted for 10 min, DTNB (0.08mM) was added, and then incubated under alkaline conditions for 5 min. The absorption curve was measured by a UV spectrophotometer with a wavelength range of 300 nm-600 nm. The decrease in the absorption peak at 412 nm represents the ability to consume glutathione, such as Figure 6 As shown, 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

[0034] Study on the singlet oxygen generating activity of manganese-based amino acid coordination polymers: Detection by the attenuation of the ultraviolet absorbance of 1,3-diphenylisobenzofuran (DPBF) 1O2 generation. 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 with a UV spectrophotometer at regular intervals, with a wavelength range of 300 nm-550 nm. The reduction value of 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

[0035] Study on the anti-tumor activity of manganese-based amino acid coordination polymers: Breast cancer cell line 4T1 cells were digested and counted. 4 The number of cells per well was inoculated in a 96-well plate and cultured overnight in a carbon dioxide (5%) incubator at 37°C. The samples were prepared with DMEM medium at different concentrations 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, and the absorption of each well was measured on an ELISA reader after 2 hours of incubation, 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.

[0036] 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 principle of the present invention. These improvements and modifications should also be regarded as 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. (2) Place the solution obtained in step (1) into a beaker and stir the mixture with a magnetic stirrer for 2-4 h; (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 the manganese-based amino acid coordination polymer according to claim 1, characterized in that: The molar ratio of the divalent manganese salt to the amino acid is 1:1-1:

10.

3. The method for preparing the manganese-based amino acid coordination polymer according to claim 1, characterized in that: The divalent manganese salt described in step (1) is any one of Mn(NO3)2•4H2O, MnCl2•4H2O or MnCl2•2H2O; the amino acid is one or more of histidine, arginine, tyrosine and lysine.

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

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

6. The method for preparing the manganese-based amino acid coordination polymer according to claim 1, characterized in that: The specific operation of washing and centrifugation in step (3) is: washing three times with ethanol respectively, and then centrifuging at a speed of 5000-12000 rpm.

7. A manganese-based amino acid coordination polymer, characterized in that: The manganese-based amino acid coordination polymer is prepared by the method described in any one of claims 1 to 6; the manganese-based amino acid coordination polymer is a nanoparticle structure with a small size and high stability.

8. The manganese-based amino acid coordination polymer according to claim 7, characterized in that: The manganese-based amino acid coordination polymer has peroxidase-like activity, glutathione oxidase-like activity and singlet oxygen-producing activity.

9. The use of the manganese-based amino acid coordination polymer according to claim 7, characterized in that: The manganese-based amino acid coordination polymer is used in the preparation of anti-tumor drugs.

10. A pharmaceutical composition, characterized in that The invention comprises the manganese-based amino acid coordination polymer as claimed in claim 7 as one of the main active ingredients, and a pharmaceutically acceptable carrier.

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