A method for preparing an antibacterial hybrid nano apatite with high dispersity and high osteogenic activity

Abstract

The application discloses a preparation method of a novel antibacterial nano apatite with high dispersity and high osteogenic activity. The novel antibacterial nano apatite with high dispersity and high osteogenic activity is characterized in that: alkali lignin is reacted with alendronate to prepare aminated lignin, and then the aminated lignin is ring-opening synthesized with epichlorohydrin to prepare phosphorylated lignin, and then sodium phosphate solution is added to set A liquid; calcium nitrate solution is taken out, and then eugenol is added to react for 2 hours to set B liquid, the B liquid is slowly added to the A liquid, the molar ratio of Ca / P is kept as 1.67, finally, 5 wt% sodium hydroxide is used to adjust the pH value to about 12, heating and stirring are carried out at 70 DEG C for 5 hours, aging is carried out for 2 days, water washing is carried out until neutral, and drying and grinding are carried out to form powder. The hybrid nano apatite prepared by the application has good dispersity, high osteogenic activity and good antibacterial property, and therefore, the novel nano apatite can be directly used for preparing bone filling particles, and can be used as a high polymer filler to prepare high-performance composite materials, and has a good application prospect in orthopedic materials.

Description

Technical Field

[0001] This invention relates to a highly dispersible and osteogenic antibacterial hybrid nanoapatite, belonging to the field of biomedical materials. Background Technology

[0002] Nano hydroxyapatite [Ca 10 [(PO4)6(OH)2, n-HA] contains mineral components and structures similar to human bone, exhibiting good biocompatibility, osteoconductivity, and high wear resistance. It is also slightly alkaline and is considered a bioactive inorganic nanomaterial in orthopedic materials. However, when used alone as an orthopedic material, it is brittle and usually requires the use of polymers to improve its processability. Studies have shown that n-HA inorganic nanoparticles, due to their inherent tendency to aggregate, especially their hydrophilic surface, are difficult to disperse in hydrophobic solvents, making it difficult to achieve good interfacial bonding with polymers to prepare high-strength composite materials. Furthermore, the osteogenic activity of n-HA alone is insufficient to meet the requirements for vascularized bone formation and high osseointegration. In addition, n-HA itself lacks antibacterial properties, making it difficult to prevent recurrent and delayed bacterial infections in orthopedic surgical areas. Therefore, effective modification of n-HA has become a hot topic and a challenge in orthopedic material research.

[0003] To address the issue of combining inorganic n-HA particles with polymers, researchers both domestically and internationally have conducted extensive studies on surface grafting modification of n-HA. However, before grafting, most nanoparticles may have already agglomerated, resulting in minimal modification effects and a failure to significantly improve dispersibility. Introducing some macromolecular organic compounds during the n-HA preparation process can effectively improve its dispersibility. A domestic invention patent (publication number CN106924819A) proposes using phosphorylated cyclodextrin as an auxiliary phosphorus source, obtaining a novel nano-apatite with better dispersibility. However, phosphorylated cyclodextrin has low solubility in water, and the osteogenic activity and antibacterial properties of n-HA have not been improved. Therefore, further research is needed on hybrid nano-apatite with other phosphorylated macromolecules. Lignin is the second most abundant natural biomass resource after cellulose. Lignin has a complex chemical structure, composed of phenylpropane units, and is a three-dimensional network phenolic polymer formed in a nonlinear random manner based on three main monomers. It is the only non-toxic, biodegradable, and biocompatible plant resource in nature rich in benzene ring structures. In particular, its phenolic hydroxyl groups have antibacterial, antioxidant, and ultraviolet absorption functions, making it an ideal raw material for preparing functional materials. It has already been used in the fields of biomedicine and packaging materials. Therefore, if lignin can be phosphorylated and used to prepare hybrid nano-apatite, it is expected that the steric hindrance of lignin macromolecules can be utilized to improve its dispersibility.

[0004] In addition, alendronate is a bisphosphate with an amino group at the end of its side chain and is a commonly used drug for treating osteoporosis. [References] ACS Appl. Mater.&Inter. [2018, 10: 25547-25560] confirmed that alendronate and Fe3O4 can self-assemble on the surface of nano-apatite, significantly promoting osteoblast development. Therefore, if amino-containing diphosphates are first prepared into amination lignin through the chemical bonding of amino groups, and then ring-opened with epichlorohydrin to synthesize phosphorylated lignin, and then used in the preparation of n-HA, it is expected to be used to prepare hybrid nano-apatite with better osteogenic activity.

[0005] To improve the antibacterial properties of n-HA, scholars both domestically and internationally have conducted studies on n-HA using Ag. + Zn 2+ Studies on plasma doping or adsorption of bioactive substances such as vancomycin and antimicrobial peptides have been conducted, but these methods suffer from drawbacks such as difficulty in controlling the doping amount, easy burst release, and high cost. Syringic acid, the active ingredient of the traditional Chinese medicine *Malan*, is a natural phenolic compound that exhibits good antibacterial activity against multidrug-resistant *Staphylococcus aureus*. Literature [J. Mol. Liq. 2021, 322: 114872] confirms that syringic acid can react with Fe... 2+ or Fe 3+ It forms a complex, resulting in stronger antibacterial properties. Therefore, if syringic acid is introduced into phosphorylated lignin containing an alendronate structure, it can react with Ca in n-HA. 2+ By combining the preparation of hybrid nano-apatite, it is expected to endow n-HA with antibacterial properties, so that the obtained hybrid nano-apatite not only has better dispersibility, but also better osteogenic activity and antibacterial properties, so as to be better applied in orthopedic materials. Summary of the Invention

[0006] This invention provides a method for preparing a novel hybrid nanoapatite with high dispersibility and high osteogenic activity. The novel hybrid nanoapatite prepared by this method not only has good dispersibility and high osteogenic activity, but also has good antibacterial properties. Therefore, this novel hybrid nanoapatite can be directly used to prepare bone filler particles, or it can be used as a polymer filler to prepare high-performance composite materials, and it has good application prospects in orthopedic materials.

[0007] A method for preparing highly dispersible and osteogenic antibacterial hybrid nanoapatite includes the following steps:

[0008] First, alkali lignin was reacted with alendronate to prepare aminated lignin, which was then ring-opened with epichlorohydrin to synthesize phosphorylated lignin. Subsequently, sodium phosphate solution was added, wherein the introduced alkali lignin accounted for 30-40% of the mass of the novel nano-apatite, and alendronate was 1.47 times the mass of alkali lignin, designated as solution A. Separately, syringic acid was added to calcium nitrate solution and reacted for 2 hours, wherein the syringic acid content accounted for 5-15% of the total mass of the hybrid nano-apatite, designated as solution B. Solution B was slowly added dropwise to solution A, maintaining a Ca / P molar ratio of 1.67. Finally, the pH was adjusted to approximately 12 with 5 wt% sodium hydroxide solution, heated and stirred at 70 ℃ for 5 hours, aged for 2 days, washed with water until neutral, dried, and ground into powder.

[0009] The advantages of the preparation method of highly dispersible and osteogenic antibacterial hybrid nano-apatite provided by this invention are as follows:

[0010] (1) In terms of material properties, compared with existing n-HA, the nano-apatite structure prepared in this invention contains phosphorylated lignin macromolecules, which endow it with steric hindrance and achieve high dispersibility. At the same time, the phosphorylated lignin structure contains alendronate, which promotes osteogenic activity, thus improving its osteogenic activity. In addition, syringic acid, which has antibacterial properties, is also introduced into the hybrid lignin, thus endowing the hybrid nano-apatite with antibacterial properties. It can be seen that this novel hybrid lignin is a highly dispersible and highly osteogenic antibacterial hybrid nano-apatite.

[0011] (2) The raw materials used in this invention are readily available, the preparation steps are simple, the reaction conditions are mild, the reagents used are environmentally friendly, and the reaction time is short, making it suitable for large-scale production.

[0012] (3) The hybrid nano-apatite prepared by the present invention contains lignin macromolecules in its structure, so it can not only be directly processed into bone defect filling particles, but also be used as a polymer filler to prepare high-performance composite materials. It has great application prospects in various orthopedic fields such as bone tissue engineering scaffold materials, bone tissue regeneration membranes, and drug-loaded materials. Attached Figure Description

[0013] Figure 1 The images show the dispersion of different powders in dichloromethane in the examples. Implementation

[0014] Example 1: 10.00 g of alkali lignin was dissolved in 100 ml of 2 mol / L NaOH solution, followed by 14.67 g of alendronate solution and 4 mL of 37% formaldehyde. The mixture was stirred at 40°C for 3 h, then heated to 85°C and stirred for another 3 h to obtain aminated lignin. Next, 3.12 g of sodium dihydrogen phosphate was introduced, dissolved in water, and 2.19 g of epichlorohydrin was added. 3 ml of 10% dilute hydrochloric acid was added to adjust the environment to acidity, and the mixture was stirred at 85°C for another 5 h to obtain phosphorylated lignin. Then, 45.62 g of trisodium dodecahydrate solution was added, and the reaction was carried out for 2 h, designated as solution A. Separately, 47.23 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water, and 1.5 g of syringic acid was added, reacting for 2 h, designated as solution B. Solution B was slowly added dropwise to solution A. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide, and the mixture was heated and stirred at 70°C for 5 h. The mixture was aged for 48 hours, washed with deionized water until neutral, dried, and ground into powder. It dispersed well in dichloromethane after standing for 2 hours, and after soaking in simulated body fluids for 2 weeks, it showed deposits of bone-like apatite with an antibacterial rate of 70%.

[0015] Example 2: 15.00 g of alkali lignin was dissolved in 200 ml of 2 mol / L NaOH solution, followed by 22.00 g of alendronate phosphate solution and 6 mL of 37% formaldehyde. The mixture was stirred at 40°C for 3 h, then heated to 85°C and stirred for another 3 h to obtain aminated lignin. Next, 4.68 g of sodium dihydrogen phosphate was introduced, dissolved in water, and 3.29 g of epichlorohydrin was added. 3 ml of 10% dilute hydrochloric acid was added to adjust the environment to acidity, and the mixture was stirred at 85°C for another 5 h to obtain phosphorylated lignin. Then, 79.83 g of trisodium dodecahydrate solution was added, and the reaction was carried out for 2 h, designated as solution A. Separately, 82.65 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water, and 3.0 g of syringic acid was added, reacting for 2 h, designated as solution B. Solution B was slowly added dropwise to solution A. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide, and the mixture was heated and stirred at 70°C for 5 h. The mixture was aged for 48 hours, washed with deionized water until neutral, dried, and ground into powder. It dispersed well in dichloromethane after standing for 2 hours, and after soaking in simulated body fluids for 2 weeks, it showed deposits of bone-like apatite with an antibacterial rate of 75%.

[0016] Example 3: 10.00 g of alkali lignin was dissolved in 200 ml of 2 mol / L NaOH solution, followed by 22.00 g of alendronate phosphate solution and 4 mL of 37% formaldehyde. The mixture was stirred at 40°C for 3 h, then heated to 85°C and stirred for another 3 h to obtain aminated lignin. Next, 3.12 g of sodium dihydrogen phosphate was introduced, dissolved in water, and 2.19 g of epichlorohydrin was added. 3 ml of 10% dilute hydrochloric acid was added to adjust the environment to acidity, and the mixture was stirred at 85°C for another 5 h to obtain phosphorylated lignin. Then, 34.21 g of trisodium dodecahydrate solution was added, and the reaction was carried out for 2 h, designated as solution A. Separately, 35.42 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water, and 2.5 g of syringic acid was added, reacting for 2 h, designated as solution B. Solution B was slowly added dropwise to solution A. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide, and the mixture was heated and stirred at 70°C for 5 h. After aging for 48 hours, the product was washed with deionized water until neutral, dried, and ground into powder. It dispersed well in dichloromethane after standing for 2 hours, and after soaking in simulated body fluids for 1 week, it showed deposits of bone-like apatite with an antibacterial rate of 82%.

[0017] Example 4: 10.00 g of alkali lignin was dissolved in 200 ml of 2 mol / L NaOH solution, followed by 22.00 g of alendronate phosphate solution and 4 mL of 37% formaldehyde. The mixture was stirred at 40°C for 3 h, then heated to 85°C and stirred for another 3 h to obtain aminated lignin. Next, 3.12 g of sodium dihydrogen phosphate was introduced, dissolved in water, and 2.19 g of epichlorohydrin was added. 3 ml of 10% dilute hydrochloric acid was added to adjust the environment to acidity, and the mixture was stirred at 85°C for another 5 h to obtain phosphorylated lignin. Then, 34.21 g of trisodium dodecahydrate solution was added, and the mixture was reacted for 2 h, designated as solution A. Separately, 35.42 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water, and 4.0 g of syringic acid was added, reacting for 2 h, designated as solution B. Solution B was slowly added dropwise to solution A. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide, and the mixture was heated and stirred at 70°C for 5 h. After aging for 48 hours, the powder was washed with deionized water until neutral, dried, and ground into powder. It dispersed well in dichloromethane after standing for 2 hours and showed an antibacterial rate of 95% when the bone-like apatite deposited after soaking in simulated body fluids for 1 week.

[0018] Comparative Example 1: 11.81 g of calcium nitrate tetrahydrate was dissolved in 50 ml of water and stirred for 2 hours. Then, 11.38 g of sodium phosphate dodecahydrate dissolved in 50 ml of water was slowly added dropwise to the above solution. The pH was adjusted to approximately 10 with 10 wt% sodium hydroxide. The mixture was heated and stirred at 70 °C for 4 hours. After standing for 48 hours, it was washed 5 times with deionized water and then 3 times with ethanol. After drying, it was ready for use. It began to separate into layers after standing in dichloromethane for 5 minutes. Immersion in simulated body fluids for 4 weeks resulted in the deposition of bone-like apatite, but showed no antibacterial effect.

[0019] Comparative Example 2: 10.00 g of alkali lignin was dissolved in 200 ml of 2 mol / L NaOH solution, followed by the addition of 34.21 g of trisodium phosphate dodecahydrate solution. The reaction was allowed to proceed for 2 hours. Separately, 35.42 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water and slowly added dropwise. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide. The mixture was heated and stirred at 70 °C for 5 hours, aged for 48 hours, washed with deionized water until neutral, dried, and ground into powder. The powder dispersed well in dichloromethane after standing for 1 hour. Immersion in simulated body fluids for 4 weeks resulted in the deposition of bone-like apatite, but showed no antibacterial effect.

[0020] Comparative Example 3: 10.00 g of alkali lignin was dissolved in 200 ml of 2 mol / L NaOH solution, followed by 22.00 g of alendronate phosphate solution and 4 mL of 37% formaldehyde. The mixture was stirred at 40°C for 3 h, then heated to 85°C and stirred for another 3 h to obtain aminated lignin. 3.12 g of sodium dihydrogen phosphate was then introduced, dissolved in water, and 2.19 g of epichlorohydrin was added. 3 ml of 10% dilute hydrochloric acid was added to adjust the environment to acidity, and the mixture was stirred at 85°C for another 5 h to obtain phosphorylated lignin. Subsequently, 34.21 g of trisodium dodecahydrate solution was added and reacted for 2 h. 35.42 g of calcium nitrate tetrahydrate was dissolved in 200 ml of water and slowly added dropwise. Finally, the pH was adjusted to above 10 with 1 mol / L sodium hydroxide, heated and stirred at 70°C for 5 h, aged for 48 h, washed with deionized water until neutral, dried, and ground into powder. It did not separate into layers when left to stand in dichloromethane for 2 hours, and showed no antibacterial effect when soaked in simulated body fluid for 1 week, with deposits of bone-like apatite.

Claims

1. A method for preparing a highly dispersible and osteogenic antibacterial hybrid nanoapatite, characterized in that: First, alkali lignin is reacted with alendronate to prepare aminated lignin, and then ring-opened with epichlorohydrin to synthesize phosphorylated lignin. Subsequently, sodium phosphate solution is added, wherein the introduced alkali lignin accounts for 30-40% of the mass of hybrid nano-apatite, and alendronate is 1.47 times the mass of alkali lignin; this is designated as solution A. Separately, syringic acid is added to calcium nitrate solution and reacted for 2 hours, wherein the syringic acid content is 5-10 wt% of the hybrid nano-apatite content; this is designated as solution B. Solution B is slowly added dropwise to solution A, maintaining a Ca / P molar ratio of 1.

67. Finally, the pH is adjusted to above 10 with 1 mol / L sodium hydroxide, heated and stirred at 70 ℃ for 5 hours, aged for 48 hours, washed with deionized water until neutral, dried, and ground into powder for later use.

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