Calcium copper phosphate nanosheet and preparation method thereof
By preparing calcium copper phosphate nanosheets through high-temperature hydrothermal reaction in oleic acid, ethanol and surfactant solutions, the problems of uneven morphology and insufficient biocompatibility in the existing technology are solved, and the uniform dispersion of nanosheets and excellent biocompatibility and antibacterial properties are achieved, which has broad prospects for biomedical applications.
Patent Information
- Application Number
- CN202510752975.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-09-05
AI Technical Summary
There are few reports on the synthesis of copper calcium phosphate nanosheets in the existing technology, and their uneven morphology, poor dispersion, insufficient biocompatibility and antibacterial properties limit their application in the biomedical field.
Calcium salt, copper salt and phosphate aqueous solution are added to a mixed solution of oleic acid, ethanol and surfactant, and copper calcium phosphate nanosheets are prepared through high-temperature hydrothermal reaction. The reaction conditions such as temperature and time are controlled, and a polytetrafluoroethylene reactor is used to seal the reaction. The nanosheets are then centrifuged and washed.
The prepared calcium copper phosphate nanosheets have uniform morphology and good dispersion, and have high hydrophilicity, good biocompatibility and antibacterial properties, and are suitable for the biomedical field.
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Figure CN120589712A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a copper calcium phosphate nanosheet and a preparation method thereof. Background Art
[0002] In the field of biomedical materials, two-dimensional nanostructures offer unique advantages over traditional spherical, granular, or rod-shaped nanomaterials. They possess a larger specific surface area and provide a smoother surface, which facilitates cell adhesion, proliferation, and differentiation. They may be superior to other nanomaterial forms in bone repair and tissue engineering. This gives nanosheets unique advantages in drug delivery, tissue engineering, bone repair, and antibacterial therapy. Calcium copper phosphate (Ca3Cu3(PO4)4) is composed of calcium, copper, and phosphate ions, but reports on its synthesis are limited. Summary of the Invention
[0003] The present invention aims to address the shortcomings of the prior art by providing a method for preparing calcium copper phosphate nanosheets. The calcium copper phosphate nanosheets prepared by this method exhibit uniform morphology, good dispersion, high hydrophilicity, good biocompatibility, hemocompatibility, and excellent antibacterial properties, and have promising application prospects in the biomedical field.
[0004] To solve the above technical problems, the present invention adopts a technical solution: a method for preparing calcium copper phosphate nanosheets, characterized by comprising: adding a calcium salt aqueous solution, a copper salt aqueous solution and a phosphate aqueous solution to a mixed solution of oleic acid, ethanol and a surfactant at a (Ca+Cu) / P molar ratio of 1.5 and a Cu / Ca molar ratio of 1:1 to 7:2; stirring evenly, transferring the mixture to a polytetrafluoroethylene reactor, sealing the mixture, and placing the mixture in an oven for a high-temperature hydrothermal reaction; and naturally cooling the mixture to room temperature after the reaction is completed, centrifuging the mixture to obtain a precipitate, and washing the mixture with anhydrous ethanol and water to obtain calcium copper phosphate nanosheets.
[0005] The above-mentioned method for preparing copper calcium phosphate nanosheets is characterized in that the surfactant is polyethylene glycol, β-cyclodextrin or polyvinyl pyrrolidone, and the concentration of the surfactant in the reaction system is 2.5 mg / mL to 20 mg / mL.
[0006] The above-mentioned method for preparing calcium copper phosphate nanosheets is characterized in that the calcium salt is Ca(NO3)2, the copper salt is Cu(NO3)2, and the phosphate is Na3PO4, Na2HPO4 or NaH2PO4.
[0007] The above-mentioned method for preparing calcium copper phosphate nanosheets is characterized in that the volume ratio of oleic acid, ethanol and salt solution is (1-4):(5-10):(5-20), wherein the salt solution refers to calcium salt aqueous solution, copper salt aqueous solution and phosphate aqueous solution.
[0008] The above-mentioned method for preparing calcium copper phosphate nanosheets is characterized in that the concentrations of the calcium salt aqueous solution, the copper salt aqueous solution and the phosphate aqueous solution are all 0.05 mol / L to 0.4 mol / L.
[0009] The above-mentioned method for preparing calcium copper phosphate nanosheets is characterized in that the temperature of the high-temperature hydrothermal reaction is 110° C. to 200° C., and the reaction time is 1 hour to 12 hours.
[0010] Furthermore, the present invention provides calcium copper phosphate nanosheets prepared by the above method.
[0011] The above-mentioned copper calcium phosphate nanosheets are characterized in that they have uniform morphology and good dispersion, a length of 100nm to 300nm, a width of 100nm to 200nm, and good biocompatibility, blood compatibility and antibacterial properties.
[0012] Compared with the prior art, the present invention has the following advantages:
[0013] 1. The raw materials used in this invention are readily available and abundant, resulting in low cost. The synthesis process is simple and easy to implement, resulting in stable product quality and good process reproducibility. The calcium copper phosphate nanosheets prepared in this invention exhibit uniform morphology, good dispersion, high hydrophilicity, good biocompatibility, hemocompatibility, and excellent antibacterial properties, and have promising application prospects in the biomedical field.
[0014] 2. The present invention can control the preparation of copper calcium phosphate nanosheets. The preparation method is significantly innovative and practical. The preparation method is simple. The prepared copper calcium phosphate nanosheets have good biocompatibility and antibacterial properties. They can be used as degradable tissue repair materials to promote the healing of defective tissues. They can be added to medical dressings as drug-loaded core materials to inhibit the growth of wound bacteria in the long term and promote the repair of infectious wounds.
[0015] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the XRD pattern of the calcium copper phosphate nanosheets of Example 1 of the present invention.
[0017] Figure 2 This is a TEM image of the calcium copper phosphate nanosheets of Example 1 of the present invention.
[0018] Figure 3This is the EDS image of the copper calcium phosphate nanosheets of Example 1 of the present invention.
[0019] Figure 4 This is a Fourier infrared spectrum of the calcium copper phosphate nanosheets of Example 1 of the present invention.
[0020] Figure 5 This is a graph showing the biocompatibility of L929 cells cultured with the extract of copper calcium phosphate nanosheets according to Example 1 of the present invention.
[0021] Figure 6 This is a diagram showing the co-culture results of the copper calcium phosphate nanosheets according to Example 1 of the present invention with Escherichia coli and Staphylococcus aureus.
[0022] Figure 7 This is a blood compatibility diagram of the copper calcium phosphate nanosheets according to Example 1 of the present invention. DETAILED DESCRIPTION
[0023] The following describes the specific embodiments of the present invention in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.
[0024] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.
[0025] Example 1 Preparation method of calcium copper phosphate nanosheets
[0026] To a mixture of 4 mL of oleic acid, 16 mL of ethanol, and 0.6 g of PEG10000, 2.0 mL of a 0.3 mol / L aqueous solution of Ca(NO3)2, 3.0 mL of a 0.3 mol / L aqueous solution of Cu(NO3)2, and 5.0 mL of a 0.2 mol / L aqueous solution of Na3PO4 were added, sequentially, while stirring. After stirring for 10 minutes, the mixture was sealed in a Teflon reactor and hydrothermally reacted in a 150°C oven for 10 hours. After the reaction, the mixture was cooled to room temperature and centrifuged to obtain the precipitate, which was then washed three times with anhydrous ethanol and then water to obtain calcium copper phosphate nanosheets.
[0027] The product was identified by X-ray powder diffraction as calcium copper phosphate. Figure 1 As shown; the results of transmission electron microscopy showed that the product was a monodisperse nanosheet with a lateral size of 100 to 200 nm and a longitudinal size of 100 to 300 nm. The morphology was uniform and well dispersed. Figure 2 As shown; the EDS test results of the product are as shown Figure 3As shown in the figure, the results show that the nanosheets are composed of calcium, copper, phosphorus and oxygen elements. The Fourier infrared spectrum of the product is shown in the figure Figure 4 As shown, it is at 3450 and 1656 cm -1 The peak at is due to the stretching vibration peak of hydroxyl group, indicating that it has good hydrophilicity.
[0028] Example 2: Preparation method of calcium copper phosphate nanosheets
[0029] To a mixture of 4 mL of oleic acid, 20 mL of ethanol, and 0.18 g of β-CD, 6 mL of a 0.4 mol / L aqueous solution of Ca(NO3)2, 21 mL of a 0.4 mol / L aqueous solution of Cu(NO3)2, and 18 mL of a 0.4 mol / L aqueous solution of Na2HPO4 were added, sequentially, while stirring. After stirring for 10 minutes, the mixture was sealed in a Teflon reactor and hydrothermally reacted in a 110°C oven for 12 hours. After the reaction, the mixture was cooled to room temperature and centrifuged to obtain the precipitate, which was then washed three times with anhydrous ethanol and then water to obtain calcium copper phosphate nanosheets.
[0030] The copper calcium phosphate nanosheets obtained in this example have similar physical and chemical properties to the copper calcium phosphate nanosheets obtained in Example 1.
[0031] Example 3: Preparation method of calcium copper phosphate nanosheets
[0032] To a mixture of 4 mL of oleic acid, 10 mL of ethanol, and 0.34 g of PVP(K30), 6 mL of a 0.05 mol / L aqueous solution of Ca(NO3)2, 6 mL of a 0.05 mol / L aqueous solution of Cu(NO3)2, and 8 mL of a 0.05 mol / L aqueous solution of NaH2PO4 were added, sequentially, while stirring. After stirring for 10 minutes, the mixture was sealed in a Teflon reactor and placed in a 200°C oven for one hour. After the reaction, the mixture was cooled to room temperature and centrifuged to obtain the precipitate, which was then washed three times with anhydrous ethanol and then water to obtain calcium copper phosphate nanosheets.
[0033] The copper calcium phosphate nanosheets obtained in this example have similar physical and chemical properties to the copper calcium phosphate nanosheets obtained in Example 1.
[0034] Example 4: Biocompatibility Experiment of Calcium Copper Phosphate Nanosheets
[0035] The copper calcium phosphate nanosheets prepared in Example 1 were sterilized in Co60, dispersed in 1640 complete medium (containing 10% serum and 1% double antibody), and extracted at 37°C for 72h to obtain sample extracts with concentration gradients of 4, 8, 16, 32, 64, 128, 256, and 512 μg / mL. The extracts were sealed and stored in a refrigerator at 4°C for later use. Mouse fibroblasts (L929 cells) were cultured in 1640 complete medium in a 96-well plate until the cells adhered to the wall. The experimental group and the control group were divided into the following groups: Replace the original culture medium with the sample extract and 1640 complete culture medium, continue to culture the cells for 24 hours, add 50 μL of MTT to each well, incubate for 3 hours, discard the liquid in the wells, add 150 μL of DMSO to each well, measure the absorbance at 490 nm, and calculate the cell viability by the following formula: Cell viability (%) = [(A sample - A blank) / (A pair - A blank)] х 100%, where A sample is the absorbance of the sample to be tested, A blank is the absorbance of the culture medium, and A pair is the absorbance of the control group.
[0036] Figure 5 This is a biocompatibility diagram of L929 cells cultured with the extract of copper calcium phosphate nanosheets (sample 1). The results show that after 24 hours of culture of L929 cells with the extract, the cell survival rate decreased with the increase of the concentration of sample 1. When the extract concentration was less than 64 μg / mL, the cell survival rate was greater than 80%, showing good cell compatibility.
[0037] Example 5: Antibacterial activity of calcium copper phosphate nanosheets
[0038] An appropriate amount of the copper calcium phosphate nanosheets prepared in Example 1 was sterilized in Co60. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were selected as model bacteria.
[0039] After activating the model bacteria with LB liquid medium, the activated bacteria were diluted to 1×10 5 The mixture was incubated in a 37°C incubator for 24 hours, and the bacterial solution was diluted 10 6 100 μL of the diluted bacterial solution was evenly spread on LB solid medium and cultured in a 37°C constant temperature incubator for 24 hours. The antibacterial activity of the sample against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was calculated by colony counting. No sample was added to the control group.
[0040] Figure 6The antibacterial test results of the copper calcium phosphate nanosheets prepared in Example 1 show that the inhibition rate of copper calcium phosphate nanosheets against Escherichia coli and Staphylococcus aureus gradually increases with the increase of sample concentration. When the concentration is 128 μg / mL, the inhibition rate can reach 90%, showing a good antibacterial effect.
[0041] Example 6: Blood compatibility test of calcium copper phosphate nanosheets
[0042] An appropriate amount of the copper calcium phosphate nanosheets prepared in Example 1 was placed in Co60 for sterilization.
[0043] Sterilized copper calcium phosphate nanosheet samples were extracted in normal saline at a mass-to-volume ratio of 0.1 g / mL for 72 hours and then filtered through a 0.22 μm filter membrane. Fresh whole blood was collected from rabbits (ear vein) and centrifuged at 2000 rpm for 5 minutes to obtain red blood cells (RBCs), which were then diluted to 2% (v / v) with normal saline. Equal volumes of the RBC suspension and sample extract were added to centrifuge tubes. Positive controls (deionized water) and negative controls (normal saline) were set up and incubated in a 37°C incubator for 1 hour. After centrifugation, the supernatant was collected, and the absorbance at 540 nm was measured and photographed.
[0044] Figure 7 The blood compatibility results of the copper calcium phosphate nanosheets (Sample 1) prepared in Example 1 are shown. The quantitative analysis results in the figure show that the hemolysis rate of the nanomaterial is less than 5% when the hemolysis rate in the saline group is 0%. The inset in the figure shows that the supernatants of both the saline and nanomaterial (Sample 1) groups are clear, with red blood cells remaining intact at the bottom. In contrast, the hemolytic activity in the deionized water group is poor, with the liquid appearing red. These results demonstrate that the copper calcium phosphate nanosheets prepared in Example 1 of the present invention have excellent blood compatibility.
[0045] The above results show that the copper calcium phosphate nanosheets prepared by the present invention have a good crystal structure, uniform morphology and good dispersibility, and have good cell and blood compatibility and antibacterial properties.
[0046] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.
Claims
1. A method for preparing calcium copper phosphate nanosheets, characterized in that: include: A calcium salt aqueous solution, a copper salt aqueous solution and a phosphate aqueous solution are added to a mixed solution of oleic acid, ethanol and a surfactant at a (Ca+Cu) / P molar ratio of 1.5 and a Cu / Ca molar ratio of 1:1 to 7:
2. The mixture is stirred evenly and then transferred to a sealed polytetrafluoroethylene reactor and placed in an oven for a high-temperature hydrothermal reaction. After the reaction is completed, the mixture is naturally cooled to room temperature, centrifuged to obtain a precipitate, and washed with anhydrous ethanol and water to obtain copper calcium phosphate nanosheets.
2. The method for preparing calcium copper phosphate nanosheets according to claim 1, wherein The surfactant is polyethylene glycol, beta-cyclodextrin or polyvinyl pyrrolidone, and the concentration of the surfactant in the reaction system is 2.5 mg / mL to 20 mg / mL.
3. The method for preparing calcium copper phosphate nanosheets according to claim 1, wherein The calcium salt is Ca(NO3)2, the copper salt is Cu(NO3)2, and the phosphate is Na3PO4, Na2HPO4 or NaH2PO4.
4. The method for preparing calcium copper phosphate nanosheets according to claim 1, wherein The volume ratio of oleic acid, ethanol and salt solution is (1-4):(5-10):(5-20), wherein the salt solution refers to calcium salt aqueous solution, copper salt aqueous solution and phosphate aqueous solution.
5. The method for preparing calcium copper phosphate nanosheets according to claim 1, wherein: The concentrations of the calcium salt aqueous solution, copper salt aqueous solution and phosphate aqueous solution are all 0.05 mol / L to 0.4 mol / L.
6. The method for preparing calcium copper phosphate nanosheets according to claim 1, wherein: The temperature of the high-temperature hydrothermal reaction is 110° C. to 200° C., and the reaction time is 1 hour to 12 hours.
7. Calcium copper phosphate nanosheets prepared by the method according to any one of claims 1 to 6.
8. The calcium copper phosphate nanosheet according to claim 7, characterized in that The calcium copper phosphate nanosheets have uniform morphology and good dispersion, a length of 100nm to 300nm, a width of 100nm to 200nm, and good biocompatibility, blood compatibility and antibacterial properties.