Preparation method and application of an antibacterial nano-hydrogel

Abstract

The present invention discloses a preparation method and application of an antibacterial nano-hydrogel. By using free radical precipitation polymerization in combination with dialysis method to prepare the nano-hydrogel material, it effectively avoids the problem of too fast reaction rate between metal salt ions and monomers, the use of additional cross-linking agents, and reduces the operation process of reloading metal salt ions after obtaining the nano-gel. It can prepare nano-hydrogels with uniform particle size, good dispersibility, stability, antibacterial property and biocompatibility in one step. The physical and chemical properties of the nano-hydrogel are easy to regulate, and it has potential antibacterial and anti-inflammatory functions. It can inhibit the crystallization of pathological crystals through mechanisms such as chelation, dispersion and adsorption, and avoid inflammation or infection at the disease site through the controlled release of metal salt ions. It can even promote bone repair. The preparation method is green, environmentally friendly, simple and easy to implement. The obtained nano-hydrogel is of great significance for the treatment of pathological inorganic salt crystallization diseases and has broad application prospects in the biomedical field.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of an antibacterial nano-hydrogel. Background Art

[0002] Traditional drug delivery methods have problems such as large side effects, low bioavailability, and poor therapeutic effects. In recent years, new nano-drug delivery systems have shown great potential in the field of disease treatment and are regarded as effective strategies to improve the pharmacological properties and therapeutic effects of drugs. Among many nano-drugs, nano-hydrogels have the characteristics of both nano-materials and hydrogels, and have advantages such as high water content, good biocompatibility, easy functional modification, targeting, and slow release, playing an irreplaceable role in the field of biomedicine.

[0003] Pathological crystal diseases are caused by the deposition of inorganic salts in specific parts of the human body. At present, the treatment of pathological crystal diseases at home and abroad mainly focuses on oral administration or surgical removal, but it will cause many adverse reactions, long treatment cycles, and poor effects. Therefore, the research and development of new drugs that can inhibit the crystallization of pathological crystals has become the main way to solve the problem. Chinese patents CN202010491732.0, CN202010941932.1, and CN202110246193.9 all mention methods for preparing nano-gels loaded with metal salt ions, but they all first obtain blank nano-gels and then load metal salt ions. The metal salt ion loading amount is limited and easy to burst release, and the operation process and conditions are cumbersome and poorly controlled, which restricts large-scale production and its clinical use.

[0004] In view of the above problems, the present invention in-situ prepares a nano-hydrogel loaded with metal salt ions by a free radical precipitation polymerization synergistic dialysis method. By controlling the release rate of metal salt ions, it avoids problems such as uneven particle size, poor dispersion, and poor stability of the nano-hydrogel caused by too fast reaction rate between metal salt ions and monomers, and at the same time reduces the use of additional cross-linking agents. In addition, the crystallization behavior of crystals can be effectively regulated by using the easy functionalization characteristics of nano-hydrogels, and the controllable release rate of metal salt ions can be achieved by regulating experimental conditions. After literature research, no relevant literature and patents have been found reporting the preparation method of such antibacterial nano-hydrogel materials and their application in the field of regulating pathological inorganic salt crystallization. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an antibacterial nano-hydrogel that can regulate the crystallization behavior of pathological inorganic salts, which is of great significance for the prevention and treatment of pathological crystal diseases.

[0006] The technical solution of the present invention is as follows:

[0007] A preparation method of an antibacterial nano - hydrogel, characterized by comprising the following steps:

[0008] (1) Prepare a monomer dispersion;

[0009] (2) Prepare an aqueous metal salt solution and load it into a dialysis device for controlling the release rate of metal salt ions;

[0010] (3) Load the monomer dispersion in step (1) and the dialysis device containing the metal salt solution in step (2) into a customized container, place it in an oil bath at a certain temperature, and remove the oxygen in the container;

[0011] (4) Add an initiator to the system in step (3) and stir in an anaerobic environment for cross - linking reaction;

[0012] (5) Separate, wash, and dry the product obtained in step (4) to obtain an antibacterial nano - gel with uniform particle size;

[0013] The monomers mainly include structural monomers, functional monomers, and hydrophobic monomers; the structural monomers are thermosensitive monomers such as chitosan, gelatin, cellulose, N - isopropylacrylamide, methyl methacrylate, N - isopropylmethacrylamide, N - vinylcaprolactam, etc.; the functional monomers are monomers with groups such as carboxyl, amino, hydroxyl, sulfonic acid group, aldehyde group, nitro group, etc.; the hydrophobic monomers are N - tert - butylacrylamide, N - alkylacrylamide, ethyl acrylate, ethyl methacrylate, tert - butyl acrylate, tert - butyl methacrylate, 2 - fluoroacrylate, etc.; the total monomer concentration is 20 - 500 mM; the solvent for dispersing the monomers is water or an organic solvent.

[0014] The metal salts have antibacterial functions, such as silver salts, copper salts, zinc salts, gallium salts, iron salts, calcium salts, etc.; the concentration is 0.1 - 50 mM, and the dosage is 0.5 - 30 mL.

[0015] The dialysis device can be a micro - dialysis device, a suspension dialysis tube, a dialysis bag, etc., and the cut - off molecular weight of the dialysis device is 100 - 1000 Da.

[0016] The volume of the customized container is 50 - 2000 mL, and the reaction temperature is 40 - 100 °C.

[0017] The oxygen removal method is a vacuum pumping method or a gas replacement method, and the gas can be nitrogen, argon, with a flow rate of 5 - 100 mL / min -1 , and the oxygen removal time is 10 - 90 min.

[0018] The initiator is a peroxide and an azo initiator, specifically potassium persulfate, ammonium persulfate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, azobisisobutyramidine hydrochloride, azodicyanovaleric acid; the concentration of the initiator is 0.2 - 50 mM, the stirring speed is 0 - 1000 rpm, and the cross-linking reaction time is 3 - 24 h.

[0019] According to requirements, an appropriate amount of surfactant can also be added in step (1). The surfactant can be sodium linear alkylbenzene sulfonate, sodium dodecyl sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, α-olefin sulfonate, etc., and the concentration is 0 - 1 g / L. -1 .

[0020] The obtained antibacterial nano-hydrogel has a narrow particle size distribution, uniform size, and the particle size range is 50 - 1000 nm.

[0021] Provide the above preparation method of the antibacterial nano-hydrogel for the regulation of pathological inorganic salt crystal diseases.

[0022] The beneficial effects of the present invention: The present invention in-situ prepares a nano-hydrogel loaded with metal salt ions by free radical precipitation polymerization synergistic dialysis method. Through the slow release of metal salt ions, the reaction rate between metal salt ions and monomers can be effectively controlled, avoiding problems such as poor dispersibility and stability of the nano-hydrogel caused by too fast reaction rate. At the same time, without using additional cross-linking agents and further experiments to load metal salt ions, a nano-hydrogel with potential antibacterial, anti-inflammatory, and even bone repair-promoting properties can be obtained. In addition, the obtained nano-gel can inhibit pathological crystal crystallization through mechanisms such as chelation, dispersion, and adsorption. At the same time, the synchronous introduction of metal salt ions can achieve local controlled release at the diseased part, thereby avoiding inflammation or infection at the diseased part and even promoting bone repair. The preparation method is simple, controllable, green, environmentally friendly, and easy to scale up production, and has broad application prospects in the biomedical field. Description of the Drawings

[0023] Figure 1 It is a flow chart of the preparation method and application of the antibacterial nano-hydrogel.

[0024] Figure 2 It is a particle size distribution diagram of the antibacterial nano-hydrogel prepared in Example 1.

[0025] Figure 3 It is a scanning electron microscope image of the antibacterial nano-hydrogel.

[0026] In the figure: (A) The antibacterial nano-hydrogel prepared in Example 1, (B) The antibacterial nano-hydrogel prepared by directly mixing metal salt ions with monomers.

[0027] Figure 4Digital photos of sodium urate crystals prepared without adding antibacterial nanohydrogels (control group) and adding antibacterial nanohydrogels (Example 1) under the same experimental conditions.

[0028] In the figure: (A) Sodium urate crystals obtained from the control group, (B) Sodium urate crystals obtained from Example 1.

[0029] Figure 5 Ga in the antibacterial nanohydrogel prepared in Example 1 3+ Controlled release curve in simulated tissue fluid. Detailed implementation manners

[0030] The following further illustrates the detailed implementation manners of the present invention in combination with the accompanying drawings and technical solutions.

[0031] Example 1

[0032] (1) Disperse 30 mol% N-isopropylmethacrylamide, 40 mol% methacrylic acid and 20 mg sodium dodecyl sulfate in 49 mL of pure water, and disperse 30 mol% N-tert-butylacrylamide in 1 mL of ethanol;

[0033] (2) Prepare 10 mL of 30 mM gallium nitrate aqueous solution and load it into a dialysis device with a molecular weight cut-off of 100 - 500 Da;

[0034] (3) Load the monomer dispersion liquid in step (1) and the dialysis device containing the metal salt solution in step (2) into a customized container, place it in an oil bath at 60 °C, and introduce argon for 30 min with a gas flow rate of 20 mL min -1 ;

[0035] (4) Add 2 mL of 50 mM potassium persulfate aqueous solution to the system in step (3), with a stirring rate of 300 rpm, and continuously introduce argon and stir for 9 h;

[0036] (5) Separate, wash and dry the product obtained in step (4) to obtain the antibacterial nanohydrogel.

[0037] Use the obtained antibacterial nanohydrogel to regulate sodium urate crystallization on an in vitro dynamic bionic platform. The crystallization system: an aqueous solution with a sodium ion concentration of 140 mM, a pH of 7.4, a uric acid concentration of 3.5 mM, and a temperature of 20 °C. After running for one week, the nanohydrogel shows an obvious inhibitory effect on sodium urate crystallization. In addition, it can achieve the controlled release of Ga 3+ in simulated tissue fluid.

[0038] Example 2

[0039] (1) 48 mol% of N-vinylcaprolactam, 10 mol% of acrylic acid and 5 mg of sodium dodecyl sulfate were dispersed in 49 mL of pure water, and 40 mol% of N-alkylacrylamide was dispersed in 1 mL of ethanol;

[0040] (2) Prepare 5 mL of 50 mM calcium chloride aqueous solution and load it into a dialysis device with a molecular weight cut-off of 500 - 1000 Da;

[0041] (3) The monomer dispersion in step (1) and the dialysis device containing the metal salt solution in step (2) were simultaneously loaded into a customized container, placed in an oil bath at 75 °C, and nitrogen was passed through for 30 min with a gas flow rate of 20 mL min -1 ;

[0042] (4) Add 2 mL of 50 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride aqueous solution to the system in step (3), with a stirring rate of 300 rpm, and continuously pass nitrogen and stir for 6 h;

[0043] (5) Separate, wash, and dry the product obtained in step (4) to obtain an antibacterial nano-hydrogel;

[0044] The obtained antibacterial nano-hydrogel was used to regulate sodium urate crystallization on an in vitro dynamic bionic platform. The crystallization system: an aqueous solution with a sodium ion concentration of 140 mM, a pH of 7.4, a uric acid concentration of 3.5 mM, and a temperature of 20 °C. After running for one week, the nano-hydrogel showed a significant inhibitory effect on sodium urate crystallization.

[0045] Example 3

[0046] (1) 63 mol% of N-isopropylacrylamide, 15 mol% of 2-acrylamido-2-methylpropanesulfonic acid sodium salt and 10 mg of sodium dodecyl sulfate were dispersed in 49 mL of pure water, and 20 mol% of N-tert-butylacrylamide was dispersed in 1 mL of ethanol;

[0047] (2) Prepare 2 mL of 50 mM gallium nitrate aqueous solution and load it into a dialysis device with a molecular weight cut-off of 500 - 1000 Da;

[0048] (3) The monomer dispersion in step (1) and the dialysis device containing the metal salt solution in step (2) were simultaneously loaded into a customized container, placed in an oil bath at 60 °C, and nitrogen was passed through for 30 min with a gas flow rate of 10 mL min -1 ;

[0049] (4) Add 0.5 mL of 50 mM ammonium persulfate aqueous solution to the system in step (3), with a stirring rate of 300 rpm, and continuously pass argon and stir for 6 h;

[0050] (5) Separate, wash, and dry the product obtained in step (4) to obtain the antibacterial nano-hydrogel;

[0051] Use the obtained antibacterial nano-hydrogel to regulate sodium urate crystallization on an in vitro dynamic bionic platform. The crystallization system is an aqueous solution with a sodium ion concentration of 140 mM, a pH of 7.4, a uric acid concentration of 3.5 mM, and a temperature of 20 °C. After running for one week, the nano-hydrogel showed an obvious inhibitory effect on sodium urate crystallization.

[0052] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial nano-hydrogel, characterized in that, The steps are as follows: (1) Prepare a monomer dispersion; (2) Prepare an aqueous metal salt solution and load it into a dialysis device for controlling the release rate of metal salt ions; (3) Load the monomer dispersion in step (1) and the dialysis device containing the aqueous metal salt solution in step (2) into a sealed container, then place the sealed container in an oil bath at a temperature of 40-100 °C, and remove the oxygen in the sealed container; (4) Add an initiator to the system in step (3) and stir in an anaerobic environment for cross-linking reaction; (5) Separate, wash, and dry the product obtained in step (4) to obtain an antibacterial nano-hydrogel with uniform particle size; The monomer is a structural monomer, a functional monomer, and a hydrophobic monomer. The total concentration of the monomer is 20-500 mM, and the solvent for the dispersed monomer is water or an organic solvent; The structural monomer is a temperature-sensitive monomer, including N-isopropylacrylamide, N-isopropylmethacrylamide, or N-vinylcaprolactam; the functional monomer is a monomer with a carboxyl group, an amino group, a hydroxyl group, a sulfonic acid group, an aldehyde group, or a nitro group; the hydrophobic monomer is N-tert-butylacrylamide, ethyl acrylate, ethyl methacrylate, tert-butyl acrylate, tert-butyl methacrylate, or 2-fluoroacrylate.

2. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, The dialysis device is a microdialysis device, a suspension dialysis tube, or a dialysis bag, and the cut-off molecular weight of the dialysis device is 100-1000 Da.

3. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, The method for excluding oxygen in the closed container is the vacuum pumping method or the gas replacement method; the gas used in the gas replacement method is nitrogen or argon, and the flow rate is 5 - 100 mL min -1 , and the oxygen exclusion time is 10 - 90 min.

4. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, The initiator is a peroxide or an azo initiator. The concentration of the initiator is 0.2-50 mM, the stirring speed is 0-1000 rpm, and the cross-linking reaction time is 3-24 h.

5. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, The initiator is potassium persulfate, ammonium persulfate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, azobis(2-methylimidazoline) dihydrochloride, or azodicyanovaleric acid.

6. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, In step (1), a surfactant is added, and the surfactant is sodium linear alkyl benzene sulfonate, sodium dodecyl sulfate, sodium alcohol polyoxyethylene ether sulfate or sodium α-olefin sulfonate, and the concentration is not more than 1 g / L -1 and the concentration is not equal to 0.

7. The method for preparing an antibacterial nano-hydrogel according to claim 1, characterized in that, The metal salt is a copper salt, a zinc salt, a gallium salt, an iron salt, or a calcium salt; the concentration is 0.1-50 mM.

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