Amorphous copper phosphate nanocluster material and its preparation method and application
Amorphous copper phosphate nanocluster materials were prepared by polyelectrolyte stabilizers and wet chemical synthesis, which solved the problem of large particle size and poor hydrophilicity of copper phosphate crystals, and achieved rapid degradation and efficient treatment of tumor cells in the tumor microenvironment.
Patent Information
- Application Number
- CN202310534906.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-12
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2043-05-12
AI Technical Summary
The existing copper phosphate crystal materials have large particle size and poor hydrophilicity, making it difficult to achieve efficient tumor treatment effects.
Polyelectrolyte is used as a stabilizer to prepare amorphous copper phosphate nanocluster materials by wet chemical synthesis, and the re-aggregation of clusters is reduced through concentration purification technology to form nanoclusters with excellent water solubility.
The material rapidly degrades in the tumor microenvironment, increases ROS accumulation of tumor cells, and has efficient tumor treatment capabilities, especially in the treatment of colon cancer.
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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 copper phosphate nanocluster material and a preparation method and application thereof. Background Art
[0002] Copper (Cu) is an essential element in the human body. Many key enzymes and transcription factors require copper for their activity [Reference 1: Copper in Medicine: Perspectives and Toxicity. Biomedical Applications of Metals, 2018: 95-112.]. In recent years, research has focused on the catalytic properties of copper ions and the development of copper-based nanomaterials for therapeutic applications. These copper-based nanomaterials often work by utilizing endogenous or self-supplied H2O2 in the tumor microenvironment [Reference 2: Review of Iron-free Fenton-Like Systems for Activating H2O2 in Advanced Oxidation Processes. Journal of Hazardous Materials, 2014, 275: 121-135.]. Furthermore, under the precise regulation of copper, cells can produce a novel cell death mechanism that differs from traditional known cell death mechanisms [Reference 3: Copper induces cell death by targeting lipoylated TCA cycle proteins. Science, 2022, 375(6586): 1254.]. This has led to an urgent need for copper-based nanomaterials with tumor therapeutic capabilities.
[0003] Copper phosphate is biocompatible and may be a promising material for treating tumors in the field of medicine and biology [Reference 4: Amplified oxidative stress therapy by a degradable copperphosphate nanozyme coated by the in situ polymerization of PEGDA. Journal of Materials Chemistry B, 2021, 9(38): 8094-8108. Reference 5: Preparation of Fetal Bovine Serum-Copper Phosphate Hybrid Particles under Cell Culture Conditions for Cancer Cell Treatment. ACS Omega 2022, 7(33): 29495-29501.]. However, the particle size of copper phosphate crystal materials is often large and their hydrophilicity is very poor, making it difficult to achieve efficient functionalized tumor treatment effects [Reference 6: A review on crystal structure and properties of 3d transition metal (II)orthophosphates M3(PO4)2; Journal of Alloys and Compounds 2020,835:155028. Document 7: Synthesis, characterization of imidazole-based copper complex mixtures and study of their thermal behavior. Special Issue: Progress in Alternative Fuels and Energies 2021,45(6):9179-9192.]. Summary of the Invention
[0004] To address the bottleneck problem of copper-based nanocrystalline compounds often having large particle sizes and poor hydrophilicity, which hinder their full effectiveness, the present invention provides an amorphous copper phosphate nanocluster material, as well as a preparation method and application thereof. This method utilizes a polyelectrolyte as a stabilizer to stabilize the copper and phosphate in solution, preserving the amorphous precursor clusters formed in the early stages of crystallization while simultaneously enhancing the aqueous dispersion of the nanocluster material, thereby forming an amorphous copper phosphate nanocluster material. Subsequently, concentration and purification techniques are used to reduce cluster reagglomeration, resulting in the material having excellent water solubility.
[0005] The material of the present invention can maintain a stable amorphous state for a long time, effectively treating tumors. It has excellent dispersion ability, is easily degraded in the tumor microenvironment, and accelerates the accumulation of ROS in tumor cells, demonstrating its high tumor therapeutic potential. It is a highly promising copper-based anti-tumor nanomaterial.
[0006] The technical solutions of the present invention are as follows:
[0007] The present invention provides a method for preparing an amorphous copper phosphate nanocluster material, comprising the following steps:
[0008] (1) Solution preparation: Dissolve a copper inorganic salt in an aqueous solution of a polyelectrolyte to obtain solution A, and dissolve a phosphate in an aqueous solution of a polyelectrolyte to obtain solution B;
[0009] (2) Wet chemical synthesis: Solution A and solution B are mixed and subjected to a wet chemical synthesis reaction to obtain a solution containing copper phosphate clusters;
[0010] (3) Post-treatment: The cluster solution obtained in step (2) is first concentrated and then freeze-dried to obtain an amorphous copper phosphate nanocluster material.
[0011] Preferably, in step (1), the polyelectrolyte comprises polyacrylic acid and its salts, polyaspartic acid and its salts; preferably, the molecular weight of the polyelectrolyte is above 1000; and / or
[0012] The copper inorganic salts include copper chloride, copper nitrate and copper sulfate; and / or
[0013] The phosphates include sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate.
[0014] Preferably, in step (1), in solution A, the concentration of the copper inorganic salt is 18-180 mmol / L; in solution B, the concentration of the phosphate is 12-120 mmol / L; in solution A and solution B, the concentration of the polyelectrolyte aqueous solution is 0.5-50 g / L;
[0015] As a further preference, the molar ratio of the copper atoms to the phosphorus atoms is 1.3-1.7:1; most preferably, the molar ratio of the copper atoms to the phosphorus atoms is 1.5:1;
[0016] As a further preferred method, after dissolving phosphate in an aqueous solution of a polyelectrolyte, the pH is adjusted to 8.0-10.0 to obtain solution B.
[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 concentration is performed by dialysis concentration of the cluster solution obtained in step (2), and the molecular weight cut-off of the dialysis is 2000-18000.
[0020] Preferably, in step (3), the dialysis concentration is to place the cluster solution obtained in step (2) into a dialysis bag, and then place the dialysis bag in an aqueous solution containing 5-20% of a hydrophilic polymer for concentration; the molecular weight of the hydrophilic polymer is greater than the molecular weight cut-off of the dialysis bag, and the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10-1:50;
[0021] As further preferred, the hydrophilic polymer is polyethylene glycol;
[0022] As a further preference, the dialysis bag after dialysis concentration is washed with deionized water.
[0023] The present invention provides an amorphous copper phosphate nanocluster material, which is prepared by applying the above method.
[0024] The present invention also provides the use of the amorphous copper phosphate nanocluster material in preparing drugs for treating tumors.
[0025] Preferably, the tumor comprises colorectal cancer.
[0026] The present invention also provides a medicine, the active ingredient of which is the amorphous copper phosphate nanocluster material; preferably, the dosage form of the medicine is an injection.
[0027] Compared with the prior art, the beneficial effects of the present invention are embodied in:
[0028] 1. Metastable amorphous precursor cluster structures exist in natural phosphates, but the cluster structures are extremely unstable. The present invention uses a polyelectrolyte stabilization strategy to stabilize amorphous copper phosphate in nanomaterials for a long time.
[0029] 2. The amorphous copper phosphate nanocluster material obtained by the method of the present invention has a small particle size and good dispersibility in water, 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 biological efficacy of the material.
[0030] 3. The amorphous copper phosphate nanocluster material of the present invention can be rapidly degraded in the tumor microenvironment and increase the ROS accumulation of tumor cells through the Fenton reaction. It has extremely high efficacy in tumor treatment, especially in the treatment of colon cancer. This cluster nanomaterial has significant application value prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] 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:
[0032] Figure 1 This is the XRD test result of the amorphous copper phosphate nanocluster material in Example 1.
[0033] Figure 2 This is the XRD test result of the amorphous copper phosphate nanocluster material in Example 1 after being stored at room temperature for 3 months.
[0034] Figure 3 This is the Malvern laser particle size analyzer test result of the amorphous copper phosphate nanocluster material in Example 1.
[0035] Figure 4 This is the TEM morphology of the amorphous copper phosphate nanocluster material in Example 1 and the morphology image under a high-resolution TEM electron microscope.
[0036] Figure 5 This is the transmission electron microscope electron diffraction pattern of the amorphous copper phosphate nanocluster material in Example 1.
[0037] Figure 6 This is the XPS spectrum of the amorphous copper phosphate nanocluster material in Example 1.
[0038] Figure 7 This is the infrared spectrum of the amorphous copper phosphate nanocluster material of Example 1.
[0039] Figure 8 These are the results of toxicity experiments on amorphous copper phosphate nanocluster materials and HT29 colon cancer cells.
[0040] Figure 9 These are the experimental results of the effect of amorphous copper phosphate nanocluster materials on ROS reactive oxygen species generation in cells.
[0041] Figure 10 The therapeutic effect of amorphous copper phosphate nanocluster materials on subcutaneous colon cancer in mice.
[0042] Figure 11 These are the experimental results of the degradation of the amorphous copper phosphate nanocluster material of the present invention in cells. DETAILED DESCRIPTION
[0043] 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.
[0044] The present invention utilizes polyelectrolytes as stabilizers to stabilize copper ions and phosphates in a solution, synthesizes long-term stable amorphous copper phosphate nanocluster materials by wet chemical synthesis, and reduces the reagglomeration of the nanomaterials by a set concentration, purification, and drying technology. In the present invention, the polyelectrolyte is preferably an anionic high molecular polymer.
[0045] The preparation method of the amorphous copper phosphate nanocluster material of the present invention is as follows:
[0046] (1) Solution preparation: Dissolve a copper inorganic salt in an aqueous solution of a polyelectrolyte to obtain solution A, and dissolve a phosphate in an aqueous solution of a polyelectrolyte to obtain solution B;
[0047] (2) Wet chemical synthesis: Solutions A and B were mixed and the pH was adjusted to generate a solution containing copper phosphate clusters under stirring at room temperature;
[0048] (3) Post-treatment: The cluster solution obtained in step (2) is first concentrated, and then freeze-dried and dehydrated to obtain an amorphous copper phosphate nanocluster material.
[0049] In some embodiments, in step (1), the polyelectrolyte includes polyacrylic acid and its salts, polyaspartic acid and its salts; for example, polyacrylic acid, sodium polyacrylate, ammonium polyacrylate, polyaspartic acid, sodium polyacrylate.
[0050] According to some embodiments of the present application, in solution A and solution B, the same polyelectrolyte may be selected, or different polyelectrolytes may be selected.
[0051] According to some embodiments of the present application, the concentrations of the polyelectrolyte in solution A and solution B may be the same or different.
[0052] According to some embodiments of the present application, the number average molecular weight of the polyelectrolyte is greater than 1000. When the number average molecular weight is less than 1000, a stable amorphous copper phosphate nanocluster material cannot be obtained.
[0053] In some embodiments, in step (1), the copper inorganic salt includes copper chloride, copper nitrate and copper sulfate; and the phosphate includes sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium dihydrogen phosphate, dipotassium hydrogen phosphate and diammonium hydrogen phosphate.
[0054] In some embodiments, in step (1), the concentration of the copper inorganic salt in solution A is 18-180 mmol / L; the concentration of the phosphate in solution B is 12-120 mmol / L; and the concentration of the polyelectrolyte aqueous solution in both solution A and solution B is 0.5-50 g / L. If the concentration of the copper inorganic salt and / or phosphate is too low, the yield is insufficient and centrifugation is difficult to produce a product; if the concentration is too high, the product is uneven, agglomerates significantly, and crystalline products are easily obtained. If the concentration of the polyelectrolyte in water is too high, it is difficult to dissolve; if the concentration is too low, a crystalline product will be produced.
[0055] Because the wet chemical reaction product is subsequently concentrated by dialysis in the present invention, the polyelectrolyte concentration in solution B can be lower than, equal to, or higher than that in solution A. In each case, a uniform wet chemical reaction product can be produced, thereby obtaining an amorphous product. In other words, the polyelectrolyte concentrations in solutions A and B are not required to be within the ranges specified in the present invention.
[0056] According to some embodiments of the present application, the molar ratio of copper atoms to phosphorus atoms is 1.3-1.7: 1, preferably 1.5: 1. At this ratio, the formed amorphous material is closer to copper phosphate in terms of atomic composition.
[0057] In some embodiments, in step (1), after the copper inorganic salt is dissolved in the aqueous solution of the polyelectrolyte, the pH can be adjusted to ensure that no hydrolysis occurs (the solution remains clear) so that the pH of the AB solutions in the reaction system are similar to each other, thereby obtaining solution A.
[0058] In some embodiments, in step (1), after dissolving the phosphate in the aqueous solution of the polyelectrolyte, the pH is adjusted to 8.0-10.0 to obtain solution B. The pH is not more than 10 to prevent the over-alkalinity product from becoming hydroxyapatite, and the pH is not less than 8 to allow the AB solutions to quickly form cluster nuclei after contact, thereby reducing local over-alkalinity when subsequently adding alkali to adjust the pH.
[0059] In some embodiments, in step (2), solutions A and B are mixed in equal volumes.
[0060] In some embodiments, in step (2), after mixing solutions A and B, the pH is adjusted to 6.0-8.0 for wet chemical synthesis. If the pH is lower than 6, calcium hydrogen phosphate crystals may be produced, and if the pH is higher than 8, the over-alkaline product may be hydroxyapatite.
[0061] 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.
[0062] In some embodiments, in step (2), the stirring rate is 100-500 rpm, and the stirring time is 10 minutes to 24 hours.
[0063] In some embodiments, in step (3), the concentration is performed by dialysis concentration of the cluster solution obtained in step (2), and the molecular weight cut-off of the dialysis is 2000-18000.
[0064] According to some embodiments of the present application, the dialysis is to put the cluster solution obtained in step (2) into a dialysis bag, place the dialysis bag in an aqueous solution containing 5-20% by weight of a hydrophilic polymer, wherein the molecular weight of the hydrophilic polymer is greater than the molecular weight cut-off of the dialysis bag, and utilize the concentration difference to concentrate the liquid in the dialysis bag, for example, concentrating once for 6-24 hours; the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10-1:50.
[0065] In the present application, an aqueous solution containing 5-20% by weight of a hydrophilic polymer is used. If the hydrophilic polymer content is less than 5%, it may result in insufficient efficiency, and if it is greater than 20%, the hydrophilic polymer is not easily dissolved.
[0066] According to some embodiments of the present application, the hydrophilic polymer is polyethylene glycol.
[0067] According to some embodiments of the present application, the concentrated dialysis bag is dialyzed and cleaned with deionized water, and the dialysis can be performed three times, each time for 12-24 hours.
[0068] In the present application, there is no specific requirement for the freeze-drying method, as long as the purpose of dehydration can be achieved.
[0069] The copper phosphate nanocluster material obtained by the above method is in an amorphous state, and its particle size ranges from 5 to 300 nm (here is the data obtained under a transmission electron microscope).
[0070] The present invention will be further described below in conjunction with the embodiments:
[0071] Example 1
[0072] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0073] (1) 1.8 mmol of copper chloride dihydrate and 0.6 g (0.3 mmol) of polyacrylic acid (PAA) with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water to prepare solution A; 1.2 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.5) and 0.06 g (0.03 mmol) of PAA with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water, and the pH was adjusted to 9.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B;
[0074] (2) Solution A was slowly added dropwise to solution B at 300 rpm and ammonia was added with a dropper to keep the pH at 7.4. The mixture was stirred at room temperature for 1 h.
[0075] (3) Prepare a 20% by mass polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 14,000-18,000), and place it in a 20% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:20), and use the concentration difference to concentrate the liquid in the dialysis bag once (12 hours); wash and dialyze with deionized water 3 times (12 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0076] Figure 1 This is the XRD test result of the amorphous copper phosphate nanocluster material in Example 1.
[0077] Figure 2 This is the XRD test result of the amorphous copper phosphate nanocluster material in Example 1 after being stored at room temperature for 3 months.
[0078] After the amorphous copper phosphate nanocluster material was dispersed in water, it was tested using a Malvern laser particle size analyzer. The test results are shown in Figure 3 , Figure 3 This is the hydrated particle size, which will be larger due to agglomeration.
[0079] Figure 3 This is the Malvern laser particle size analyzer test result of the amorphous copper phosphate nanocluster material in Example 1.
[0080] Figure 4 The TEM morphology and high-resolution TEM electron microscope morphology of the amorphous copper phosphate nanocluster material of Example 1 are shown. Among them, (a) is a bright field transmission electron microscope image, and (b) is a high-resolution transmission electron microscope image.
[0081] Figure 5 This is the transmission electron microscope electron diffraction pattern of the amorphous copper phosphate nanocluster material in Example 1.
[0082] Figure 6This is the XPS spectrum of the amorphous copper phosphate nanocluster material in Example 1.
[0083] Figure 7 This is the infrared spectrum of the amorphous copper phosphate nanocluster material of Example 1.
[0084] Example 2
[0085] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0086] (1) 3 mmol of copper chloride dihydrate and 1 g (0.5 mmol) of polyacrylic acid (PAA) with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water to prepare solution A; 2 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.5) and 0.05 g (0.025 mmol) of PAA with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water, and the pH was adjusted to 10.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B;
[0087] (2) Solution A was slowly added dropwise to solution B at 300 rpm and ammonia was added with a dropper to maintain the pH at 6.5; the mixture was stirred thoroughly at room temperature for 1 h.
[0088] (3) Prepare a 20% mass fraction polyethylene glycol (PEG, molecular weight: 8000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 2000), and place it in a 20% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10). Use the concentration difference to concentrate the liquid in the dialysis bag once (24 hours); wash and dialyze with deionized water three times (12 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0089] Example 3
[0090] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0091] (1) 12 mmol of copper chloride dihydrate and 10 g (2% by mass) of polyaspartic acid (PASP) with a molecular weight of 2000-11000 were dissolved in 500 mL of ultrapure water to prepare solution A. 8 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.5) and 0.5 g (0.1% by mass) of PAA with a molecular weight of 2000 were dissolved in 500 mL of ultrapure water, and the pH was adjusted to 10.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B.
[0092] (2) Solution A was slowly added dropwise to solution B at 300 rpm and ammonia was added with a dropper to maintain the pH at 7.4; the mixture was stirred at room temperature for 2 h.
[0093] (3) Prepare a 10% mass fraction of polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 14,000-18,000), and place it in a 10% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:20). Use the concentration difference to concentrate the liquid in the dialysis bag once (6 hours); wash and dialyze with deionized water 3 times (24 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0094] Example 4
[0095] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0096] (1) 6 mmol of copper chloride dihydrate and 2.5 g (0.5 mmol) of polyacrylic acid (PAA) with a number average molecular weight of 5000 were dissolved in 100 mL of ultrapure water to prepare solution A; 4 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.5) and 0.05 g (0.025 mmol) of PAA with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water, and the pH was adjusted to 9.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B;
[0097] (2) Solution A was slowly added dropwise to solution B at 300 rpm and ammonia was added with a dropper to maintain the pH at 7.8; the mixture was stirred at room temperature for 1 h.
[0098] (3) Prepare a 5% polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 14,000-18,000), and place it in a 5% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10). Use the concentration difference to concentrate the liquid in the dialysis bag once (12 hours); wash and dialyze with deionized water three times (12 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0099] Example 5
[0100] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0101] (1) 18 mmol of copper chloride dihydrate and 10 g (5% by mass) of polyaspartic acid (PASP) with a molecular weight of 2000-11000 were dissolved in 200 mL of ultrapure water to prepare solution A. 12 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.5) and 1 g (0.5% by mass) of polyaspartic acid with a molecular weight of 2000-11000 were dissolved in 200 mL of ultrapure water and the pH was adjusted to 10.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B.
[0102] (2) Solution A was slowly added dropwise to solution B at 500 rpm and ammonia was added with a dropper to maintain the pH at 6.0; the mixture was stirred thoroughly at room temperature for 1 h.
[0103] (3) Prepare a 15% mass fraction polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 8,000), and place it in a 15% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10). Use the concentration difference to concentrate the liquid in the dialysis bag once (6 hours); wash and dialyze with deionized water 3 times (24 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0104] Example 6
[0105] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0106] (1) 18 mmol of copper chloride dihydrate and 5 g (1 mmol) of polyacrylic acid (PAA) with a number average molecular weight of 5000 were dissolved in 100 mL of ultrapure water to prepare solution A; 13.8 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.3) and 5 g (2.5 mmol) of PAA with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water, and the pH was adjusted to 10.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B;
[0107] (2) Solution A was slowly added dropwise to solution B at 500 rpm and ammonia was added with a dropper to maintain the pH at 8.0; the mixture was stirred at room temperature for 24 h.
[0108] (3) Prepare a 20% mass fraction of polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 14,000-18,000), and place it in a 20% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:50). Use the concentration difference to concentrate the liquid in the dialysis bag once (24 hours); wash and dialyze with deionized water three times (each time for 24 hours); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0109] Example 7
[0110] The preparation method of the amorphous copper phosphate nanocluster material of this embodiment is as follows:
[0111] (1) 1.8 mmol of copper chloride dihydrate and 0.05 g (0.025 mmol) of polyacrylic acid (PAA) with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water to prepare solution A; 1.1 mmol of diammonium hydrogen phosphate (Cu / P molar ratio of 1.7) and 0.5 g (0.25 mmol) of PAA with a number average molecular weight of 2000 were dissolved in 100 mL of ultrapure water, and the pH was adjusted to 8.0 with concentrated aqueous ammonia (25-28 wt.% ammonium hydroxide solution) to prepare solution B;
[0112] (2) Slowly add solution A to solution B at 100 rpm and add ammonia water with a dropper to keep the pH at 8.0; stir thoroughly at room temperature for 10 minutes;
[0113] (3) Prepare a 10% mass fraction polyethylene glycol (PEG, molecular weight: 20,000) solution with deionized water, put the solution after the reaction in step (2) into a dialysis bag (molecular weight cutoff of 14,000-18,000), and place it in a 10% PEG solution (the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:20). Use the concentration difference to concentrate the liquid in the dialysis bag once (6 hours); wash and dialyze with deionized water 3 times (12 hours each time); freeze-dry and dehydrate to obtain an amorphous copper phosphate nanocluster material.
[0114] Comparative Example 1
[0115] The difference between this comparative example and Example 1 is that no polyelectrolyte is added, and all other conditions are the same. In this case, the product obtained is a crystalline phase, and an amorphous material cannot be obtained.
[0116] Comparative Example 2
[0117] The difference between this comparative example and Example 1 is that the polyelectrolyte is PEI, and the rest are the same. In this case, the product obtained easily transforms into a crystalline phase, and it is difficult to obtain a stable amorphous material.
[0118] Comparative Example 3
[0119] This comparative example differs from Example 1 in that the molecular weight cut-off for dialysis is 500, with all other conditions remaining the same. In this case, because the molecular weight cut-off for dialysis is too low, the polymer is difficult to dialyze out and is mixed with the final product. This results in a large amount of polyelectrolyte in the obtained product, resulting in a large amount of unnecessary impurities and an uneven product.
[0120] Application Examples
[0121] 1. Toxicity test of the amorphous copper phosphate nanocluster material of the present invention
[0122] Different concentrations of amorphous copper phosphate nanocluster materials prepared in Example 1 of the present invention were dissolved in culture medium and seeded into HT29 cells in a 96-well plate at a density of 8000 cells / well. After culturing for 24 h, the original culture medium was aspirated and the nanomaterial culture medium was added. After incubation for another 24 h, CCK8 staining was performed. The number of viable cells was determined by the OD value, and the median lethal dose (IC50) of the amorphous copper phosphate nanocluster material was obtained by the probability unit regression method. 50 ) is 40 μg / mL (the concentration here is the equivalent concentration of copper ions in amorphous copper phosphate nanocluster materials), and begins to level off at a concentration of 115 μg / mL, indicating that this concentration has reached the concentration of complete cell death. The experimental results are shown in Figure 8 .
[0123] Figure 8 The results of the toxicity test of amorphous copper phosphate nanoclusters on HT29 colon cancer cells are shown in Figure 1. (a) is the total lethal dose, and (b) is the median lethal dose.
[0124] 2. ROS generation experiment of the amorphous copper phosphate nanocluster material of the present invention
[0125] The fluorescent probe DCFH-DA was used to detect ROS in HT29 cells incubated with amorphous copper phosphate nanocluster materials in step 1. The control group was a culture medium group without nanomaterials. The green fluorescence signal of ROS was detected by a fluorescence microplate reader. The detection results are shown in Figure 9 .
[0126] Figure 9 These are the experimental results of the effect of amorphous copper phosphate nanocluster materials on ROS reactive oxygen species generation in cells.
[0127] Figure 9 Here, “nanomaterials” refer to “amorphous copper phosphate nanocluster materials”.
[0128] Depend on Figure 9It can be seen that the fluorescence signal of the amorphous copper phosphate nanocluster material prepared in Example 1 of the present invention is significantly enhanced after incubating cells, and a high dose of ROS is generated.
[0129] 3. The therapeutic effect of the amorphous copper phosphate nanocluster material of the present invention on colon cancer
[0130] 8-week-old male mice weighing 20-25 g were injected subcutaneously in the right axilla with 200 μL of cells at a density of 8 × 10 6 / mL HT29 cell suspension was used to construct the HT29 nude xenograft model. When the average tumor volume reached 100mm 3 Around 10 days later, the patients were randomly divided into two groups. The amorphous copper phosphate nanocluster material prepared in Example 1 of the present invention was dissolved in physiological saline to prepare a solution with a concentration of 10 mg / mL (the concentration here is the equivalent concentration of copper ions in the amorphous copper phosphate nanocluster material). The solution was injected intratumorally, once every two days, for a total of two injections. The control group was injected with physiological saline. The tumor size was observed on the 10th day after the injection of the nanomaterial. The experimental results are shown in Figure 10 .
[0131] Figure 10 The therapeutic effect of amorphous copper phosphate nanocluster materials on subcutaneous colon cancer in mice.
[0132] Figure 10 In the figure, the “material group” is “amorphous copper phosphate nanocluster material”.
[0133] Depend on Figure 10 It can be seen that there was no significant fluctuation in the weight of mice in the control group and the nanomaterial group. The tumor in the control group continued to grow, and the tumor weight was 0.27g when the sample was finally taken, while the tumor in the nanomaterial injection group had completely disappeared.
[0134] Depend on Figures 8-10 It can be seen that the amorphous copper phosphate nanocluster material of the present invention can be used for tumor treatment. The half-lethal effective concentration of the amorphous copper phosphate nanocluster material for colon cancer cells is 40 μg / mL, calculated and evaluated with copper ions. When the concentration is above 20 μg / mL, the cancer cells can be effectively killed. In addition, 0.1-20 mg / mL of the amorphous copper phosphate nanocluster material can be injected intratumorally in a mouse tumor model to achieve the purpose of treating tumors.
[0135] 4. Degradation experiment of the amorphous copper phosphate nanocluster material of the present invention in cells
[0136] The amorphous copper phosphate nanocluster material of the present invention is encapsulated with FITC green fluorescence to obtain FITC-ACuP nanoparticles.
[0137] HT29 cells were incubated with FITC-ACuP nanoparticles for 4 hours. After absorbing the nanoparticles for 4 hours, the HT29 cells were cultured in normal culture medium for 12 hours. Figure 11 .
[0138] Figure 11 Figure 3 shows the experimental results of the degradation of the amorphous copper phosphate nanocluster material of the present invention in cells. Figure a shows HT29 cells incubated with FITC-ACuP nanoparticles for 0 hours, Figure b shows HT29 cells incubated with FITC-ACuP nanoparticles for 1 hour, Figure c shows HT29 cells incubated with FITC-ACuP nanoparticles for 4 hours, and Figure d shows HT29 cells incubated with normal culture medium for 12 hours after absorbing FITC-ACuP nanoparticles for 4 hours.
[0139] Depend on Figure 11 As can be seen, Figure (a) shows the blank control group, where no green fluorescence is observed in the cells. As the incubation time increases, as can be seen in Figures (b) and (c), the green fluorescence intensity is significantly higher after 4 hours of incubation than after 1 hour, while the green fluorescence disappears after 12 hours. This phenomenon indicates that the nanoparticles continue to enter the cells. Furthermore, the overlay of green, blue, and red staining reveals the colocalization of FITC-encapsulated green fluorescent nanoparticles with lysosomes (yellow). This demonstrates that the nanoparticles are internalized by HT29 cells, enter the lysosomes, and are rapidly degraded.
[0140] 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 copper phosphate nanocluster material, characterized in that: The following steps are involved: (1) Solution preparation: dissolving a copper inorganic salt in an aqueous solution of a polyelectrolyte, adjusting the pH while ensuring that hydrolysis does not occur so that the pH of the A and B solutions in the reaction system are similar, thereby obtaining solution A; dissolving a phosphate in an aqueous solution of a polyelectrolyte, and adjusting the pH to 8.0-10.0, thereby obtaining solution B; the polyelectrolyte comprises polyacrylic acid and its salts, polyaspartic acid and its salts; and the molecular weight of the polyelectrolyte is greater than 1000; In solution A, the concentration of the copper inorganic salt is 18-180 mmol / L; in solution B, the concentration of the phosphate is 12-120 mmol / L; in solution A and solution B, the concentration of the polyelectrolyte aqueous solution is 0.5-50 g / L (2) Wet chemical synthesis: equal volumes of solution A and solution B are mixed, the pH is adjusted to 6.0-8.0, and a wet chemical synthesis reaction is performed to obtain a solution containing copper phosphate clusters; the wet chemical synthesis reaction is performed by stirring the mixture at room temperature; (3) post-processing: concentrating the cluster solution obtained in step (2) and then freeze-drying it to obtain an amorphous copper phosphate nanocluster material; The concentration is to perform dialysis concentration on the cluster solution obtained in step (2), wherein the molecular weight cutoff of the dialysis is 2000-18000; the dialysis concentration is to place the cluster solution obtained in step (2) into a dialysis bag, and then place the dialysis bag in an aqueous solution containing 5-20% of a hydrophilic polymer for concentration; the molecular weight of the hydrophilic polymer is greater than the molecular weight cutoff of the dialysis bag, and the volume ratio of the solution in the dialysis bag to the solution outside the dialysis bag is 1:10-1:50; The hydrophilic polymer is polyethylene glycol.
2. The preparation method according to claim 1, wherein: In step (1), the copper inorganic salt includes copper chloride, copper nitrate and copper 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: The molar ratio of copper atoms to phosphorus atoms is 1.3-1.7:
1.
4. The preparation method according to claim 3, wherein: The molar ratio of copper atoms to phosphorus atoms is 1.5:
1.
5. 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.
6. The preparation method according to claim 1, wherein: The dialysis bag after dialysis concentration was rinsed with deionized water.
7. An amorphous copper phosphate nanocluster material prepared by the method according to any one of claims 1 to 6.
8. Use of the amorphous copper phosphate nanocluster material according to claim 7 in the preparation of a drug for treating tumors; the tumor is colorectal cancer.
9. A drug, characterized in that: The active ingredient of the medicine is the amorphous copper phosphate nanocluster material according to claim 7.
10. A medicine according to claim 9, characterized in that: The dosage form of the medicine is injection.