Amorphous zinc phosphate nanohybrid material, preparation method thereof, and application in promoting angiogenesis
Amorphous zinc phosphate nanohybrid materials were synthesized by wet chemical methods, and zinc and phosphate were stabilized using anionic polymers, which solved the problem of biosafety of zinc compounds and the difficulty of degradation of zinc phosphate crystals, and achieved the long-term stability of the material and the pro-angiogenesis effect.
Patent Information
- Application Number
- CN202310534919.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-12
- Publication Date
- 2025-08-29
- Estimated Expiration
- 2043-05-12
AI Technical Summary
Existing zinc compounds are not biosafety, zinc phosphate crystal materials are difficult to degrade and metabolize, and traditional nanoscale zinc oxide has biotoxicity and tissue residue problems.
Using zinc and phosphate in anionic polymer stable solution, amorphous zinc phosphate nanohybrid materials are synthesized by wet chemical method to form a nanonetwork structure, inhibit crystallization, and achieve long-term stability and rapid degradation.
The amorphous zinc phosphate nanohybrid material remains amorphous at room temperature for more than 6 months, and is still absent after heating at 150°C for 6 hours, which has good biosafety and pro-angiogenesis effects.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to an amorphous zinc phosphate nano-hybrid material, a preparation method thereof, and an application thereof in promoting angiogenesis. Background Art
[0002] Zinc (Zn) is an essential element in the human body. It is present in many nucleic acids and proteins, and many enzymes (such as acetyl dehydrogenase and RNA polymerase) require the participation of zinc to function. Abnormal zinc homeostasis can promote inflammation and oxidative stress in the body and interfere with pathophysiological processes such as apoptosis. [Reference 1: Physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis. Journal of Physiological Sciences, 2017, 67(2): 283-301.] Zinc ions have the ability to promote angiogenesis, cell proliferation and migration, and can regulate the process of wound healing. However, when zinc ions are excessive, they can produce significant cytotoxicity [Reference 2: ROS-responsive capsules engineered from EGCG-Zinc networks improve therapeutic angiogenesis in mouse limb ischemia. Bioactive Materials, 2021. 6(1): 1-11.]. A typical traditional zinc compound is zinc oxide, but the excessive release of zinc ions is only part of the reason for the excessive use of nano-zinc oxide. The biological toxicity of nano-zinc oxide is much greater than that of zinc ions at the same concentration. This may be because zinc oxide can cause a significant increase in reactive oxygen species (ROS), which in turn leads to oxidative damage [Reference 3: Acute ZnOnanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish. Aquatic Toxicology, 2013, 136: 49-59.]. For example, nano-zinc oxide has a significant effect on the LD50 activity of cells. 50The concentration of zinc oxide in the raw materials is about 50 μg / ml [Reference 4: Characterization and evaluation of cytotoxic effect, antioxidant and antimicrobial activities of zinc oxide nanoparticles derived from Justicia adhatoda. Applied Nanoscience, 2022.]. In addition, there is some evidence that nano-sized zinc oxide may remain in tissues and organs, especially the lungs, and may even cause serious damage to liver and kidney tissues [Reference 5: Acute toxicity of ferric oxide and zinc oxide nanoparticles inrats, Journal of Nanoscience and Nanotechnology, 2010, 10(12): 8617-8624.]. Therefore, new zinc ion compounds with higher biosafety are urgently needed.
[0003] Zinc phosphate nanoparticles are biocompatible and may be a promising material for research in the medical and biological fields [Reference 6: Recent progress in synthesis and applications of zinc phosphate nanoparticles: A review. Journal of Nano Research 2021, 73: 59-88.]. However, zinc phosphate crystals have a small specific surface area, low solubility, and poor hydrolysis, making it difficult to achieve efficient functionalization for biomedical applications [Reference 7: A review on crystal structure and properties of 3D transition metal(II)orthophosphates M3(PO4)2. Journal of Alloys and Compounds 2020: 835.]. Amorphous zinc phosphate can be regarded as the precursor and transition state of zinc phosphate crystals, consisting of a 2nm primary cluster structure, but it is often difficult to synthesize and unstable, forming a crystalline product [Reference 8: Thünemann A. Zinc phosphate nanoparticles produced in saliva. European Journal of Inorganic Chemistry, 2020: 3654. Reference 9: Understanding the Stability and Recrystallization Behavior of Amorphous Zinc Phosphate. Journal of Physical Chemistry C, 2021, 125(4): 2636-2647.]; Amorphous zinc phosphate precipitated by solution will quickly form a flaky crystal structure even after 4 minutes of reaction in a dilute solution [Reference 10: Thermally highly stable amorphous zinc phosphate intermediates during the formation of zinc phosphate hydrate. Journal of the American Chemical Society, 2015, 137(6): 2285-2294.]. Summary of the Invention
[0004] To address the bottlenecks in the field of low biosafety of existing zinc compounds and the difficulty in degradation and metabolism of crystalline materials such as zinc phosphate, the present invention provides an amorphous zinc phosphate nanohybrid material, its preparation method, and its application in promoting angiogenesis. The present invention utilizes an anionic polymer as a stabilizer to stabilize the zinc and phosphate in solution, retaining the amorphous precursor formed in the early stages of crystallization to form an amorphous zinc phosphate nanohybrid material. This material has improved biosafety and degradation and metabolism performance, and is stable over a long period of time (remaining amorphous at room temperature for more than 6 months and remaining amorphous after heating at 150°C for 6 hours), thereby achieving the angiogenesis-promoting effect of amorphous zinc phosphate.
[0005] The present invention provides a method for preparing an amorphous zinc phosphate nano-hybrid material, comprising the following steps:
[0006] (1) Solution preparation: Dissolve the inorganic zinc salt and phosphate in an aqueous solution of a polyanionic polymer to obtain solutions A and B, respectively;
[0007] (2) Wet chemical precipitation: Solutions A and B are mixed and subjected to a wet chemical synthesis reaction to obtain an emulsion;
[0008] (3) Cleaning and drying: The emulsion in step (2) is centrifuged to obtain a precipitate, and the precipitate is then dialyzed, cleaned, and dried to obtain an amorphous zinc phosphate nanohybrid material.
[0009] Preferably, in step (1), the polyanionic polymer comprises polyacrylic acid and its salts, polyaspartic acid and its salts; preferably, the molecular weight of the polyanionic polymer is above 1000; and / or
[0010] The zinc inorganic salts include zinc chloride, zinc nitrate and zinc sulfate; and / or
[0011] The phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate.
[0012] Preferably, in step (1), in solution A, the concentration of the zinc inorganic salt is 0.01-0.3 mol / L; in solution B, the concentration of the phosphate is 0.0067-0.2 mol / L; and / or
[0013] When the zinc inorganic salt is dissolved in the aqueous solution of the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-50 g / L; when the phosphate is dissolved in the aqueous solution of the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-200 g / L.
[0014] Preferably, in step (1), the molar ratio of zinc atoms to phosphorus atoms is 3:2.
[0015] Preferably, in step (1), the pH is adjusted to 3.0-5.0 while ensuring that no hydrolysis occurs to obtain solution A; and / or
[0016] The pH of the solution B is 8.0-10.0.
[0017] Preferably, in step (2), after mixing equal volumes of solution A and solution B, the pH is adjusted to 6.0-8.0 to carry out wet chemical synthesis reaction; and / or
[0018] The wet chemical synthesis reaction is to stir the mixture at room temperature; preferably, the stirring rate is 100-500 rpm, and the stirring time is 10 minutes to 24 hours.
[0019] Preferably, in step (3), the centrifugation is performed at 1000-10000 rpm for 2-10 min; and / or
[0020] The molecular weight cut-off of the dialysis is 2000-18000; preferably, the dialysis is performed by placing the dialysis bag in deionized water and stirring and cleaning; and / or
[0021] The drying is freeze drying; preferably, the freeze drying is to first freeze the cleaned product at -20°C to -80°C for 6-12 hours, and then place it in a freeze dryer for freeze drying. Preferably, the freeze drying parameters in the freeze dryer are: cold trap temperature -60°C, and ultimate vacuum degree less than 20Pa.
[0022] The present invention also provides an amorphous zinc phosphate nano-hybrid material, which is prepared by applying the above method.
[0023] The present invention also provides the use of the amorphous zinc phosphate nano-hybrid material in preparing products that promote cell proliferation and cell metastasis.
[0024] The present invention also provides the use of the amorphous zinc phosphate nano-hybrid material in preparing angiogenesis-promoting products.
[0025] Compared with the prior art, the beneficial effects of the present invention are embodied in:
[0026] 1. Polyanionic polymers are used to stabilize amorphous zinc phosphate nanohybrid materials. The composite anions in the material can combine with the zinc ions of the amorphous zinc phosphate to form a nano-network structure, inhibiting the crystallization of the amorphous zinc phosphate and ensuring that the material remains in an amorphous state for a long time.
[0027] 2. The amorphous zinc phosphate nano-hybrid material of the present invention has a smaller particle size, which is conducive to its rapid degradation and metabolism in the body and helps it to be phagocytosed and internalized by cells, thereby greatly improving the biosafety of the material.
[0028] 3. The amorphous zinc phosphate nano-hybrid material of the present invention promotes angiogenesis by releasing zinc ions, thereby achieving the angiogenesis-promoting effect of the amorphous zinc phosphate nano-hybrid material. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:
[0030] Figure 1 These are the XRD results of the amorphous zinc phosphate nanohybrid material prepared in Example 1, as well as the XRD results of the amorphous zinc phosphate nanohybrid material after being stored at room temperature for 6 months and treated at 150° C. for 6 hours.
[0031] Figure 2 The transmission electron microscope (TEM) morphology and electron diffraction results of the amorphous zinc phosphate nanohybrid material of Example 1 are shown.
[0032] Figure 3 These are the transmission electron microscope (TEM) morphology and electron diffraction results of the amorphous zinc phosphate nanohybrid material of Example 2.
[0033] Figure 4 The results show that the amorphous zinc phosphate nanohybrid material of Example 1 promotes the proliferation of human umbilical vein endothelial cells (HUVEC) after co-culture for 1 day and 3 days.
[0034] Figure 5 This is the staining result of the amorphous zinc phosphate nanohybrid material in Example 1 promoting cell migration in human umbilical vein endothelial cells (HUVEC) after a 10-hour cell migration experiment. DETAILED DESCRIPTION
[0035] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.
[0036] In view of the fact that amorphous zinc phosphate is very easy to crystallize, the present invention uses a polyanionic polymer to synthesize an amorphous zinc phosphate nanohybrid material through a wet chemical synthesis method, which includes the following steps:
[0037] (1) Solution preparation: Dissolve a zinc inorganic salt in an aqueous solution of a polyanionic polymer and adjust the pH to obtain solution A; dissolve a phosphate in an aqueous solution of a polyanionic polymer and adjust the pH to obtain solution B;
[0038] (2) Wet chemical precipitation: Mix solutions A and B and generate an emulsion under stirring at room temperature;
[0039] (3) Cleaning and freeze-drying: The emulsion is centrifuged to separate the precipitate, and then the precipitate is dialyzed, cleaned, and freeze-dried to obtain the amorphous zinc phosphate nanohybrid material.
[0040] In some embodiments, in step (1), the polyanionic polymer includes polyacrylic acid and its salts, polyaspartic acid and its salts; for example, polyacrylic acid, sodium polyacrylate, ammonium polyacrylate, polyaspartic acid, sodium polyacrylate.
[0041] According to some embodiments of the present application, in solution A and solution B, the same polyanionic polymer may be selected, or different polyanionic polymers may be selected.
[0042] According to some embodiments of the present application, the number average molecular weight of the polyanionic polymer is greater than 1000. When the number average molecular weight is less than 1000, a stable amorphous zinc phosphate nanohybrid material cannot be prepared.
[0043] In some embodiments, in step (1), the zinc inorganic salt includes zinc chloride, zinc nitrate and zinc sulfate, and the phosphate includes sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate.
[0044] In some embodiments, in step (1), the concentration of the inorganic zinc salt in solution A is 0.01-0.3 mol / L; and the concentration of the phosphate in solution B is 0.0067-0.2 mol / L. If the concentration of the inorganic zinc salt and / or phosphate is too high, the product will be uneven, while if the concentration of the inorganic zinc salt and / or phosphate is too low, it will be difficult to obtain a sufficient solid product.
[0045] According to some embodiments of the present application, the molar ratio of zinc atoms to phosphorus atoms is 3:2.
[0046] In some embodiments, in step (1), when the zinc inorganic salt is dissolved in the aqueous solution of the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-50 g / L; when the phosphate is dissolved in the aqueous solution containing the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-200 g / L. If the concentration of the polyanionic polymer in the water is too high, it will not dissolve easily, while if it is too low, a crystalline product will be produced.
[0047] In some embodiments, in step (1), the pH is adjusted to 3.0-5.0 while ensuring that no hydrolysis occurs (the solution remains clear) to obtain solution A. When the pH is lower than 3.0, the pH values of solutions A and B differ greatly. When the pH is higher than 5.0, the solution is easily hydrolyzed and quickly turns white.
[0048] In some embodiments, in step (1), the pH is adjusted to 8.0-10.0 to obtain solution B. If the pH is lower than 8.0, the mixed solution will be too acidic, and additional pH adjustment will be difficult. If the pH is higher than 10, it will easily become too alkaline to produce other phases.
[0049] In some embodiments, in step (2), solutions A and B are mixed in equal volumes.
[0050] In some embodiments, in step (2), after mixing solutions A and B, the pH is adjusted to 6.0-8.0 for wet chemical reaction. A pH below 6.0 tends to produce zinc hydrogen phosphate crystals, while a pH above 8.0 may also yield a crystalline product.
[0051] In some embodiments, in step (2), the wet chemical reaction needs to be carried out at room temperature. If the temperature is too high, a crystalline product may be produced and an amorphous state cannot be obtained.
[0052] In some embodiments, in step (2), the stirring rate is 100-500 rpm, and the stirring time is 10 minutes to 24 hours.
[0053] In some embodiments, in step (3), the centrifugation is performed at 1000-10000 rpm for 2-10 min.
[0054] In some embodiments, in step (3), the molecular weight cut-off of the dialysis is 2000-18000.
[0055] According to some embodiments of the present application, in step (3), the dialysis is performed by placing the dialysis bag in 50-200 times the volume of deionized water and stirring and cleaning it; for example, deionized water can be used for stirring and cleaning for 24 hours, during which the liquid is changed three times.
[0056] In some embodiments, in step (3), the freeze drying is to first freeze the cleaned product at -20°C to -80°C for 6-12 hours, and then place it in a freeze dryer for freeze drying. When freeze drying in the freeze dryer, conventional parameters can be used as long as the purpose of dehydration is achieved. For example, the parameters can be: cold trap temperature -60°C, ultimate vacuum degree less than 20Pa. The present invention adopts a two-step freeze drying method because the product needs to be frozen first to prevent boiling contamination of the solution during freeze drying, and to ensure that water is lost in the form of sublimation during freeze drying in the freeze dryer.
[0057] The zinc phosphate nano hybrid material obtained by the method of the invention is amorphous and has a particle size range of 20-60 nm.
[0058] The present invention is further described below by further specific embodiments:
[0059] Example 1
[0060] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0061] (1) Solution Preparation: A 0.05 wt.% solution of polyanionic polymer polyacrylic acid (number average molecular weight 2000) was prepared in 100 mL of water, 1.02 g of zinc chloride was added, and the pH was adjusted to 3.0 with aqueous ammonia to obtain Solution A. A 0.6 wt.% solution of polyacrylic acid (number average molecular weight 2000) was prepared in 100 mL of water, 0.66 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0062] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 7.0 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 1 hour to obtain an emulsion.
[0063] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 2000 rpm for 10 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 8000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0064] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 6 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0065] Figure 1 These are the XRD results of the amorphous zinc phosphate nanohybrid material prepared in Example 1, as well as the XRD results of the amorphous zinc phosphate nanohybrid material after being stored at room temperature for 6 months and treated at 150° C. for 6 hours.
[0066] Depend on Figure 1 It can be seen that the amorphous zinc phosphate nano-hybrid material of the present invention does not crystallize after being stored at room temperature for 6 months, and does not crystallize after being treated at a high temperature of 150° C. for 6 hours.
[0067] Figure 2 The transmission electron microscope (TEM) morphology and electron diffraction results of the amorphous zinc phosphate nanohybrid material of Example 1 are shown in Figure 1. A: transmission electron microscope bright field image, B: electron diffraction.
[0068] Example 2
[0069] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0070] (1) Solution Preparation: 100 mL of a 1 wt.% solution of polyanionic polymer polyacrylic acid (number average molecular weight 2000) was prepared with water, 2.04 g of zinc chloride was added, and the pH was adjusted to 3.0 with aqueous ammonia to obtain Solution A. 100 mL of a 0.5 wt.% solution of polyacrylic acid (number average molecular weight 2000) was prepared with water, 1.32 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0071] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 6.0 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 1 hour to obtain an emulsion.
[0072] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 5000 rpm for 5 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 8000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0073] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 6 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0074] Figure 3 The transmission electron microscope (TEM) morphology and electron diffraction results of the amorphous zinc phosphate nanohybrid material of Example 2 are shown in Figure 1. A: transmission electron microscope bright field image, B: electron diffraction.
[0075] Example 3
[0076] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0077] (1) Solution Preparation: 100 mL of a 0.5 wt.% solution of polyanionic polymer ammonium polyacrylate (number average molecular weight 5000) was prepared with water, 1.04 g of zinc chloride was added, and the pH was adjusted to 4.0 with aqueous ammonia to obtain Solution A. 100 mL of a 0.2 wt.% solution of ammonium polyacrylate (number average molecular weight 5000) was prepared with water, 0.66 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0078] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 7.4 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 12 hours to obtain an emulsion.
[0079] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 5000 rpm for 3 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 8000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0080] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 6 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0081] Example 4
[0082] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0083] (1) Solution Preparation: Sodium polyaspartate (molecular weight 2000-11000) was mixed with water to prepare a 0.25 wt.% solution (100 mL), 1.363 g of zinc chloride was added, and the pH was adjusted to 5.0 with sodium hydroxide solution to obtain Solution A. Sodium polyaspartate (molecular weight 2000-11000) was mixed with water to prepare a 0.5 wt.% solution (100 mL), 1.16 g of dipotassium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0084] (2) Wet chemical precipitation: Solution A was added to solution B and reacted at room temperature at 250 rpm for 12 hours to obtain an emulsion.
[0085] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 8000 rpm for 5 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 18000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0086] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 12 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0087] Example 5
[0088] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0089] (1) Solution Preparation: 100 mL of a 0.2 wt.% solution of polyacrylic acid (number average molecular weight 2000) and water was prepared, 4.08 g of zinc chloride was added, and the pH was adjusted to 3.0 with aqueous ammonia to obtain Solution A. 100 mL of a 0.5 wt.% solution of polyaspartic acid (number average molecular weight 2000) and water was prepared, 2.64 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.5 with aqueous ammonia to obtain Solution B.
[0090] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 6.5 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 2 hours to obtain an emulsion.
[0091] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 5000 rpm for 6 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 14000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0092] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 6 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0093] Example 6
[0094] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0095] (1) Solution Preparation: 100 mL of a 0.05 wt.% solution of polyacrylic acid (number average molecular weight 2000) and water was prepared, 1.02 g of zinc chloride was added, and the pH was adjusted to 3.0 with sodium hydroxide solution to obtain Solution A. 100 mL of a 0.5 wt.% solution of polyacrylic acid (number average molecular weight 2000) and water was prepared, 0.66 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0096] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 6.5 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 2 hours to obtain an emulsion.
[0097] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 5000 rpm for 10 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 14000 and washed with 5 L of deionized water for 24 hours, during which the liquid was changed three times.
[0098] (4) Freeze drying: The cleaned product was placed in a -20°C refrigerator for 6 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0099] Example 7
[0100] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0101] (1) Solution Preparation: 100 mL of a 3 wt.% solution of polyacrylic acid (number average molecular weight 5000) and water was prepared, 1.363 g of zinc chloride was added, and the pH was adjusted to 4.0 with sodium hydroxide solution to obtain Solution A. 100 mL of a 20 wt.% solution of polyacrylic acid (number average molecular weight 2000) and water was prepared, 0.880 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 9.0 with aqueous ammonia to obtain Solution B.
[0102] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 7.0 with aqueous ammonia, and the mixture was reacted at 400 rpm at room temperature for 12 hours to obtain an emulsion.
[0103] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 5000 rpm for 10 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 14000 and washed with 10 L of deionized water for 24 hours, during which the liquid was changed three times.
[0104] (4) Freeze drying: The cleaned product was placed in a -80°C refrigerator for 8 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0105] Example 8
[0106] The preparation method of an amorphous zinc phosphate nano-hybrid material of this embodiment is as follows:
[0107] (1) Solution Preparation: 1000 mL of a 5 wt.% solution of polyacrylic acid (number average molecular weight 8000) and water was prepared, 40.895 g of zinc chloride was added, and the pH was adjusted to 3.0 with sodium hydroxide solution to obtain Solution A. 1000 mL of a 1.5 wt.% solution of polyacrylic acid (number average molecular weight 2000) and water was prepared, 26.411 g of diammonium hydrogen phosphate was added, and the pH was adjusted to 8.0 with aqueous ammonia to obtain Solution B.
[0108] (2) Wet chemical precipitation: Solution A was added to solution B, the pH was adjusted to 8.0 with aqueous ammonia, and the mixture was reacted at room temperature at 500 rpm for 24 hours to obtain an emulsion.
[0109] (3) Cleaning: After the reaction, the obtained emulsion was centrifuged at 10,000 rpm for 8 min to collect the product. The centrifuged product was added to a dialysis bag with a molecular weight cutoff of 18,000 and washed with 15 L of deionized water for 24 hours, during which the liquid was changed three times.
[0110] (4) Freeze drying: The cleaned product was placed in a -60°C refrigerator and frozen for 12 hours, and then placed in a freeze dryer for freeze drying to obtain an amorphous zinc phosphate nanohybrid material; wherein the freeze dryer parameters are: cold trap temperature -60°C, ultimate vacuum degree 18Pa.
[0111] Comparative Example 1
[0112] The difference between this comparative example and Example 1 is that no polyelectrolyte is added, and all other aspects are the same. In this case, the product material obtained is a crystalline phase of zinc phosphate, and no amorphous nanomaterial can be obtained.
[0113] Comparative Example 2
[0114] The difference between this comparative example and Example 1 is that the polyelectrolyte is polyethyleneimine (PEI), and the rest are the same. In this case, the product obtained tends to form a crystalline phase of zinc phosphate, making it difficult to obtain an amorphous nanomaterial.
[0115] Comparative Example 3
[0116] This comparative example differs from Example 1 in that the molecular weight cut-off for dialysis is 500, with all other conditions being the same. In this case, because the molecular weight cut-off for dialysis is too low, the polymer is not easily dialyzed out and is mixed in the final product. This results in excess polymer in the product, leading to an inhomogeneous nanomaterial.
[0117] Application Example 1
[0118] 1. The amorphous zinc phosphate nanohybrid material prepared in Example 1 was co-cultured with human umbilical vein endothelial cells (HUVEC cells) to detect the effect of the amorphous zinc phosphate nanohybrid material on promoting HUVEC cell proliferation. The results are shown in FIG. Figure 4.
[0119] The specific method of co-culture is: adding a certain amount (25 μg / mL) of amorphous zinc phosphate nanohybrid material to the culture medium, using blank culture medium as the control group, conducting relevant cell experiments, and performing CCK8 assessment.
[0120] Figure 4 The results show that the amorphous zinc phosphate nanohybrid material of Example 1 promotes the proliferation of human umbilical vein endothelial cells (HUVEC) after co-culture for 1 day and 3 days.
[0121] 2. The amorphous zinc phosphate nanohybrid material prepared in Example 1 was subjected to a migration experiment with human umbilical vein endothelial cells (HUVEC). After 10 hours, the results of cell migration were detected. Figure 5 .
[0122] The specific method of the migration experiment is as follows: obtain the extract of amorphous zinc phosphate nanohybrid material (1 g in 20 ml culture medium) and dilute it 50 times, and use HUVEC cells to obtain a migration model. That is, after the cells are fully spread, use a 200-ul pipette tip to make cell scratches perpendicular to the well plate and line to ensure that the width of each scratch is consistent. After aspirating the old culture medium, add the culture medium of the experimental group and the control group (the control group is blank culture medium), and perform cell staining after 10 hours.
[0123] Figure 5 The results of the staining of the amorphous zinc phosphate nanohybrid material in Example 1 for promoting cell migration in human umbilical vein endothelial cells (HUVEC) after 10 hours of cell migration experiment. A is the control group, and B is the material group.
[0124] Since the amorphous zinc phosphate nanohybrid material of the present application can promote the migration of human umbilical vein endothelial cells (HUVEC), it can be expected that the amorphous zinc phosphate nanohybrid material of the present application can also be used for angiogenesis. According to the above experimental data, the effective concentration of the amorphous zinc phosphate nanohybrid material in promoting angiogenesis is estimated to be 1-25 μg / mL based on zinc ions.
[0125] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for preparing an amorphous zinc phosphate nanohybrid material, characterized in that: The following steps are involved: (1) Solution preparation: dissolving a zinc inorganic salt in an aqueous solution of a polyanionic polymer, and adjusting the pH to 3.0-5.0 while ensuring that hydrolysis does not occur, to obtain solution A; dissolving a phosphate in an aqueous solution of a polyanionic polymer, and adjusting the pH to 8.0-10.0, to obtain solution B; the polyanionic polymer includes polyacrylic acid and its salts, polyaspartic acid and its salts; the polyanionic polymer has a number average molecular weight of more than 1000; in solution A, the concentration of the zinc inorganic salt is 0.01-0.3 mol / L; in solution B, the concentration of the phosphate is 0.0067-0.2 mol / L; When the zinc inorganic salt is dissolved in the aqueous solution of the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-50 g / L; when the phosphate is dissolved in the aqueous solution of the polyanionic polymer, the concentration of the polyanionic polymer in the water is 0.5-200 g / L; (2) Wet chemical precipitation: Solution A and Solution B were mixed in equal volumes, and the pH was adjusted to 6.0-8.0 to perform a wet chemical synthesis reaction to obtain an emulsion; the wet chemical synthesis reaction was performed by stirring the mixture at room temperature; (3) Cleaning and drying: The emulsion in step (2) is centrifuged to obtain a precipitate, and the precipitate is then dialyzed, cleaned, and dried to obtain an amorphous zinc phosphate nanohybrid material.
2. The preparation method according to claim 1, wherein: In step (1), the zinc inorganic salt includes zinc chloride, zinc nitrate and zinc sulfate; and / or The phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate.
3. The preparation method according to claim 1, wherein: In step (1), the molar ratio of zinc atoms to phosphorus atoms is 3:
2.
4. The preparation method according to claim 1, wherein: The stirring rate is 100-500 rpm, and the stirring time is 10 minutes to 24 hours.
5. The preparation method according to claim 1, wherein: In step (3), the centrifugation is performed at 1000-10000 rpm for 2-10 min; and / or The molecular weight cut-off of the dialysis is 2000-18000; and / or The drying is freeze-drying.
6. The preparation method according to claim 5, characterized in that: The dialysis is performed by placing the dialysis bag in deionized water and stirring and cleaning it; and / or The freeze drying process is to freeze the cleaned product at -20°C to -80°C for 6-12 hours, and then place the product in a freeze dryer for freeze drying.
7. The preparation method according to claim 6, characterized in that: The freeze drying parameters in the freeze dryer are: cold trap temperature -60℃, ultimate vacuum degree less than 20Pa.
8. An amorphous zinc phosphate nano-hybrid material prepared by the method according to any one of claims 1 to 7.
9. Use of the amorphous zinc phosphate nanohybrid material according to claim 8 in the preparation of a product for promoting cell proliferation and cell metastasis.
10. Use of the amorphous zinc phosphate nanohybrid material according to claim 8 in the preparation of angiogenesis-promoting products.