Preparation method of modified antibacterial nano-hydroxyapatite

Abstract

The invention relates to a preparation method of modified antibacterial nano-hydroxyapatite, and belongs to the technical field of hydroxyapatite preparation. The solid waste eggshell is used as a calcium source of hydroxyapatite, so that resource utilization of the solid waste is realized, and the biocompatibility is further improved. Zinc is loaded on hydroxyapatite, so that the antibacterial property of hydroxyapatite is effectively improved. The zinc-loaded hydroxyapatite can effectively adsorb the ocimum gratissimum oil, the stability and durability of the antibacterial effect are enhanced through the multi-target antibacterial mechanism of the ocimum gratissimum oil, and after the zinc-loaded hydroxyapatite is combined with the zinc ion-loaded hydroxyapatite, the antibacterial performance is further improved. The carboxymethyl chitosan is coated on the surface of the hydroxyapatite, so that particle aggregation is prevented, and the stability is improved; carboxymethyl chitosan has a film-forming property, is beneficial to stabilizing antibacterial active substances, and realizes long-acting antibiosis; and the carboxymethyl chitosan also has certain antibacterial performance, so that the antibacterial effect of the zinc ions and the ocimum gratissimum oil can be further promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of hydroxyapatite preparation, and relates to a method for preparing modified antibacterial nano hydroxyapatite. Background Art

[0002] As an inorganic material with good biocompatibility and adsorption properties, hydroxyapatite has broad application prospects in the biomedical field. However, pure hydroxyapatite does not have antibacterial properties, which limits its application in some fields. Therefore, improving the antibacterial properties of hydroxyapatite has become one of the current research hotspots.

[0003] In recent years, with the continuous development of nanotechnology, nano-hydroxyapatite has attracted more and more attention due to its unique physical and chemical properties and wide application prospects. At the same time, zinc, as a metal element with antibacterial activity, is widely used in the preparation of various antibacterial materials. Therefore, introducing zinc into hydroxyapatite to prepare modified nano-hydroxyapatite with antibacterial properties has important research significance and application value. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing modified antibacterial nano-hydroxyapatite, which has the characteristics of good antibacterial performance.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for preparing modified antibacterial nano-hydroxyapatite, the specific steps of the preparation method are as follows:

[0007] S1-1: The eggshell was placed in a 75% ethanol solution and ultrasonicated for 30 minutes, then dried in a 60°C vacuum oven for 6 hours, then calcined in a muffle furnace for 2 hours at a calcination temperature of 900°C, ball-milled for 2 hours, and passed through a 200-mesh sieve to obtain powder A;

[0008] S1-2: 5 to 10 parts by weight of powder A and 1.5 to 2.5 parts by weight of zinc acetate are added to 100 parts by weight of deionized water, followed by dropwise addition of 0.3 to 0.5 parts by weight of citric acid solution, and ultrasonically stirred at 50 to 60° C. for 30 min to obtain solution B;

[0009] S1-3: 100 parts by weight of 0.06-0.12M disodium hydrogen phosphate solution was added dropwise to solution B, and the pH of the solution was adjusted to 9.5-10.5 with aqueous ammonia, and ultrasonically stirred at 70-80° C. for 2-4 hours, and then microwaved for 20-40 minutes, washed with deionized water and ethanol, and then dried in a vacuum drying oven at 60° C. for 12 hours to obtain powder C;

[0010] S1-4: Add 2 to 3 parts by weight of clove basil oil and 3 to 5 parts by weight of carboxymethyl chitosan to a beaker containing 50 parts by weight of ethanol solution, ultrasonicate for 30 minutes, then add powder C, seal the beaker with a polytetrafluoroethylene film, stir in the dark for 6 to 8 hours, and then freeze-dry in a freeze drying oven to obtain the modified antibacterial nano-hydroxyapatite.

[0011] As a preferred technical solution of the present invention, the eggshell in S1-1 is one or more of chicken eggshell, duck eggshell and goose eggshell.

[0012] As a preferred technical solution of the present invention, the heating rate of calcination in S1-1 is 5°C / min.

[0013] As a preferred technical solution of the present invention, the rotation speed of the ball mill in S1-1 is 250-350 r / min.

[0014] As a preferred technical solution of the present invention, the concentration of the citric acid solution in S1-2 is 0.1M.

[0015] As a preferred technical solution of the present invention, the concentration of the ammonia water in S1-3 is 0.5M.

[0016] As a preferred technical solution of the present invention, the power of the microwave irradiation in S1-3 is 700W, and the microwave temperature is 100-120°C.

[0017] As a preferred technical solution of the present invention, the mass fraction of the ethanol solution in S1-4 is 20%.

[0018] As a preferred technical solution of the present invention, the S1-4 is freeze-dried in a -35°C freeze drying box for 12 hours.

[0019] As a preferred technical solution of the present invention, the rotation speed of the light-shielded stirring in S1-4 is 600-800 r / min.

[0020] The main component of eggshell is calcium carbonate, and it also contains trace elements such as zinc, copper, manganese, and iron. The chemical composition and structure of eggshell-based hydroxyapatite are closer to those of living tissues, so they are more compatible with organisms. In addition, eggshells are common solid waste in daily life, with huge quantities and easy to obtain. Using eggshells as a calcium source not only realizes the resource utilization of solid waste, but also reduces the emission of solid waste and environmental pollution.

[0021] Zinc ions are a broad-spectrum antibacterial agent. Zinc ions have a positive charge and can be adsorbed on the negatively charged bacterial cell membrane, destroying its electrolyte balance, causing damage to the cell membrane, and then leaking the internal substances of the bacteria, inhibiting the growth and reproduction of the bacteria; after entering the bacteria, zinc ions can bind to the bacterial cell enzymes and inhibit their activity, thereby interfering with the normal metabolic process of the bacteria; zinc ions can also bind to the DNA of bacteria, affecting their replication and transcription processes, further inhibiting the growth and reproduction of bacteria. Loading zinc ions on hydroxyapatite can significantly improve the antibacterial properties. Citric acid helps to stabilize zinc ions, prevent them from precipitating prematurely in subsequent reactions, and ensure that zinc ions can be evenly embedded in the lattice of hydroxyapatite to form zinc-loaded hydroxyapatite. Microwave irradiation can generate heat inside the molecule, greatly increasing the generation rate of zinc-loaded hydroxyapatite, and at the same time helping to generate zinc-loaded hydroxyapatite with better crystallinity; during microwave irradiation, citric acid may absorb microwave energy through the dipole rotation inside its molecule and convert the energy into heat energy, promoting the synthesis process of zinc-loaded hydroxyapatite. In addition, zinc is one of the essential trace elements for the human body, widely present in nature, and harmless to the human body. Hydroxyapatite, as one of the main components of human bones and teeth, has good biocompatibility and stability. Therefore, the antibacterial material prepared by loading zinc ions on hydroxyapatite not only has excellent antibacterial properties, but also meets the requirements of environmental protection and non-toxicity.

[0022] Basil oil is a natural antibacterial agent containing eugenol, camphene, caryophyllene oxide and other substances, which have inhibitory effects on a variety of bacteria and fungi, and also have antioxidant and anti-inflammatory effects. When basil oil is added to nano-hydroxyapatite, zinc ion-loaded hydroxyapatite has good adsorption and can be used as a carrier of basil oil to further enhance the antibacterial properties of the material. In addition to inhibiting bacterial growth, basil oil can also interfere with the synthesis of bacterial cell walls and weaken the structural integrity of bacteria, thereby preventing their reproduction; at the same time, basil oil can also affect the synthesis process of bacterial proteins and interfere with the basic life activities of bacteria. This multi-target antibacterial mechanism enhances the stability and durability of its antibacterial effect.

[0023] Carboxymethyl chitosan is derived from chitin, which is widely present in the natural environment, and has excellent biocompatibility and non-toxicity. Carboxymethyl chitosan also has good stability and dispersibility. The carboxyl groups on its molecules can form hydrogen bonds with the hydroxyl groups on the surface of hydroxyapatite, and are coated on the surface of nano-hydroxyapatite to prevent particle agglomeration and improve the stability of the material. At the same time, carboxymethyl chitosan also has film-forming properties, which helps to stabilize antibacterial active substances such as zinc ions and clove basil oil, slow down the release rate of antibacterial active substances, and enable the antibacterial effect to be exerted for a long time. In addition, carboxymethyl chitosan itself has certain antibacterial properties. The carboxyl groups on its molecules can combine with the negative charges on the bacterial cell membrane, change the permeability of the cell membrane, and thus achieve the antibacterial effect; it can also fix bacteria on the surface of modified antibacterial hydroxyapatite through adsorption and encapsulation, further promoting the antibacterial effect of zinc ions and clove basil oil.

[0024] Beneficial effects of the present invention:

[0025] (1) Zinc ions, as a broad-spectrum antibacterial agent, inhibit the growth and reproduction of bacteria through multiple mechanisms. Loading zinc ions on hydroxyapatite can significantly improve the antibacterial properties, while microwave irradiation can increase the generation rate and crystallinity of the material; in addition, clove basil oil, as a natural antibacterial agent, combined with zinc ion-loaded hydroxyapatite, further enhances the antibacterial properties of the material, and its multi-target antibacterial mechanism enhances the stability and durability of the antibacterial effect.

[0026] (2) Carboxymethyl chitosan forms hydrogen bonds and is coated on the surface of nano-hydroxyapatite, preventing particle agglomeration and improving the stability of hydroxyapatite. Carboxymethyl chitosan also has film-forming properties, which can stabilize antibacterial active substances and slow down their release rate, thereby achieving long-term antibacterial effects. Carboxymethyl chitosan itself also has certain antibacterial properties and can fix bacteria on the surface of modified antibacterial hydroxyapatite, promoting the antibacterial effects of zinc ions and clove basil oil.

[0027] (3) Eggshell-based hydroxyapatite has higher biocompatibility due to its similarity to the chemical composition and structure of living tissues; using eggshell as the calcium source of hydroxyapatite not only realizes the resource utilization of solid waste, but also reduces environmental pollution. In the preparation process of modified antibacterial nano-hydroxyapatite, most of the raw materials come from the natural environment. The preparation and use process will not cause harm to the human body and the ecological environment, reflecting the development concept of green environmental protection. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0029] In the embodiments of the present invention and the comparative examples:

[0030] Ethanol: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0031] Zinc acetate: purchased from Lianyungang Kexin Chemical Co., Ltd.

[0032] Citric acid: purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.

[0033] Disodium hydrogen phosphate: purchased from Langfang Kangpu Huiwei Technology Co., Ltd.;

[0034] Ammonia: purchased from Zhuzhou Dayuan Industrial Co., Ltd.

[0035] Clove basil oil: purchased from Jiangxi Xinsen Natural Vegetable Oil Co., Ltd.;

[0036] Carboxymethyl chitosan: purchased from Xi'an Rongbai Biotechnology Co., Ltd.

[0037] Example 1

[0038] A method for preparing modified antibacterial nano-hydroxyapatite, the specific steps of the preparation method are as follows:

[0039] S1-1: The eggshell was placed in a 75% ethanol solution and ultrasonicated for 30 minutes, then placed in a 60°C vacuum drying oven for 6 hours, and then placed in a muffle furnace for calcination for 2 hours at a heating rate of 5°C / min, a calcination temperature of 900°C, and ball milled for 2 hours at a ball milling speed of 300 r / min. The powder was passed through a 200-mesh sieve to obtain powder A;

[0040] S1-2: 8 parts by weight of powder A and 2 parts by weight of zinc acetate were added to 100 parts by weight of deionized water, followed by dropwise addition of 0.4 parts by weight of 0.1 M citric acid solution, and ultrasonic stirring was performed at 55° C. for 30 min to obtain solution B;

[0041] S1-3: 100 parts by weight of 0.09M disodium hydrogen phosphate solution was added dropwise to solution B, and the pH of the solution was adjusted to 10 with 0.5M ammonia water, and ultrasonically stirred at 75°C for 3 hours, and then microwaved for 30 minutes, with a microwave irradiation power of 700 W and a microwave temperature of 110°C, and washed with deionized water and ethanol, and then dried in a vacuum drying oven at 60°C for 12 hours to obtain powder C;

[0042] S1-4: Add 2.5 parts by weight of clove basil oil and 4 parts by weight of carboxymethyl chitosan to a beaker containing 50 parts by weight of 20% ethanol solution, ultrasonicate for 30 minutes, then add powder C, seal the beaker with a polytetrafluoroethylene film, stir in the dark at a speed of 700 r / min for 7 hours, and then freeze-dry in a -35°C freeze drying oven for 12 hours to obtain the modified antibacterial nano-hydroxyapatite.

[0043] Example 2

[0044] A method for preparing modified antibacterial nano-hydroxyapatite, the specific steps of the preparation method are as follows:

[0045] S1-1: The duck egg shell was placed in a 75% ethanol solution and ultrasonicated for 30 minutes, then placed in a 60°C vacuum drying oven for 6 hours, and then placed in a muffle furnace for calcination for 2 hours at a heating rate of 5°C / min, a calcination temperature of 900°C, and ball milled for 2 hours at a ball milling speed of 250 r / min. The powder was passed through a 200-mesh sieve to obtain powder A;

[0046] S1-2: 5 parts by weight of powder A and 1.5 parts by weight of zinc acetate were added to 100 parts by weight of deionized water, followed by dropwise addition of 0.3 parts by weight of 0.1 M citric acid solution, and ultrasonic stirring was performed at 50° C. for 30 min to obtain solution B;

[0047] S1-3: 100 parts by weight of 0.06M disodium hydrogen phosphate solution was added dropwise to solution B, and the pH of the solution was adjusted to 9.5 with 0.5M ammonia water, and ultrasonically stirred at 70°C for 2 hours, and then microwaved for 20 minutes, with a microwave irradiation power of 700 W and a microwave temperature of 100°C, and washed with deionized water and ethanol, and then dried in a vacuum drying oven at 60°C for 12 hours to obtain powder C;

[0048] S1-4: Add 2 parts by weight of clove basil oil and 3 parts by weight of carboxymethyl chitosan to a beaker containing 50 parts by weight of 20% ethanol solution, ultrasonicate for 30 minutes, then add powder C, seal the beaker with a polytetrafluoroethylene film, stir in the dark at a speed of 600 r / min for 6 hours, and then place it in a -35°C freeze drying oven for lyophilization for 12 hours to obtain the modified antibacterial nano-hydroxyapatite.

[0049] Example 3

[0050] A method for preparing modified antibacterial nano-hydroxyapatite, the specific steps of the preparation method are as follows:

[0051] S1-1: Place the goose egg shell in a 75% ethanol solution and ultrasonicate for 30 minutes, then place it in a 60°C vacuum drying oven for 6 hours, then place it in a muffle furnace and calcine it for 2 hours, the heating rate is 5°C / min, the calcination temperature is 900°C, ball milling is performed for 2 hours, the ball milling speed is 350r / min, and the powder is passed through a 200-mesh sieve to obtain powder A;

[0052] S1-2: 10 parts by weight of powder A and 2.5 parts by weight of zinc acetate were added to 100 parts by weight of deionized water, followed by dropwise addition of 0.5 parts by weight of 0.1 M citric acid solution, and ultrasonic stirring was performed at 60° C. for 30 min to obtain solution B;

[0053] S1-3: 100 parts by weight of 0.12M disodium hydrogen phosphate solution was added dropwise to solution B, and the pH of the solution was adjusted to 10.5 with 0.5M ammonia water, ultrasonically stirred at 80°C for 4 hours, and then microwaved for 40 minutes at a power of 700 W and a microwave temperature of 120°C, washed with deionized water and ethanol, and then dried in a vacuum drying oven at 60°C for 12 hours to obtain powder C;

[0054] S1-4: Add 3 parts by weight of clove basil oil and 5 parts by weight of carboxymethyl chitosan to a beaker containing 50 parts by weight of 20% ethanol solution, ultrasonicate for 30 minutes, then add powder C, seal the beaker with a polytetrafluoroethylene film, stir in the dark at a speed of 800 r / min for 8 hours, and then place it in a -35°C freeze drying oven for lyophilization for 12 hours to obtain the modified antibacterial nano-hydroxyapatite.

[0055] Comparative Example 1

[0056] No zinc acetate was added, and the remaining steps were consistent with those in Example 1.

[0057] Comparative Example 2

[0058] Without adding clove basil oil, the remaining steps were consistent with those in Example 1.

[0059] Comparative Example 3

[0060] Without adding carboxymethyl chitosan, the remaining steps were the same as those in Example 1.

[0061] The antibacterial properties of the hydroxyapatite prepared in the examples and comparative examples were tested according to GB / T 21510-2008 standard. The indicator bacteria were Escherichia coli and Staphylococcus aureus, and the initial bacterial solution concentration was 1×10 6 cfu / mL.

[0062] The experimental data are summarized in the following table:

[0063]

[0064] It can be seen from the data of the embodiments and comparative examples that the hydroxyapatite prepared by the present invention has a significant inhibitory effect on both the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli.

[0065] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing modified antibacterial nano-hydroxyapatite, characterized in that: The specific steps of the preparation method are as follows: S1-1: The eggshell was placed in a 75% ethanol solution and ultrasonicated for 30 minutes, then dried in a 60°C vacuum oven for 6 hours, then calcined in a muffle furnace for 2 hours at a calcination temperature of 900°C, ball-milled for 2 hours, and passed through a 200-mesh sieve to obtain powder A; S1-2: 5 to 10 parts by weight of powder A and 1.5 to 2.5 parts by weight of zinc acetate are added to 100 parts by weight of deionized water, followed by dropwise addition of 0.3 to 0.5 parts by weight of citric acid solution, and ultrasonically stirred at 50 to 60° C. for 30 min to obtain solution B; S1-3: 100 parts by weight of 0.06-0.12M disodium hydrogen phosphate solution was added dropwise to solution B, and the pH of the solution was adjusted to 9.5-10.5 with aqueous ammonia, and ultrasonically stirred at 70-80° C. for 2-4 hours, and then microwaved for 20-40 minutes, washed with deionized water and ethanol, and then dried in a vacuum drying oven at 60° C. for 12 hours to obtain powder C; S1-4: Add 2 to 3 parts by weight of clove basil oil and 3 to 5 parts by weight of carboxymethyl chitosan to a beaker containing 50 parts by weight of ethanol solution, ultrasonicate for 30 minutes, then add powder C, seal the beaker with a polytetrafluoroethylene film, stir in the dark for 6 to 8 hours, and then freeze-dry in a freeze drying oven to obtain the modified antibacterial nano-hydroxyapatite.

2. The method for preparing a modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The eggshell in S1-1 is one or more of chicken eggshell, duck eggshell and goose eggshell.

3. The method for preparing a modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The heating rate of calcination in S1-1 is 5°C / min.

4. The method for preparing a modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The rotation speed of the ball mill in S1-1 is 250-350 r / min.

5. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The concentration of the citric acid solution in S1-2 is 0.1M.

6. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The concentration of the ammonia water in S1-3 is 0.5M.

7. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The power of the microwave irradiation in S1-3 is 700W, and the microwave temperature is 100-120°C.

8. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The mass fraction of the ethanol solution in S1-4 is 20%.

9. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The S1-4 was lyophilized in a -35°C freeze drying oven for 12 h.

10. The method for preparing modified antibacterial nano-hydroxyapatite according to claim 1, characterized in that: The rotation speed of the light-shielded stirring in S1-4 is 600-800 r / min.

Images