Copper phytate nano-enzyme as well as preparation method and application thereof
By constructing copper phytate nanoenzymes, using its high affinity coordination ability and enzyme catalytic activity, the complex and safety problems of existing copper-dependent nanomaterial preparation are solved, and the effect of effectively inducing copper death and inhibiting tumor growth is achieved.
Patent Information
- Application Number
- CN202411919188.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-01
AI Technical Summary
The existing copper-dependent nanomaterials are difficult to effectively induce copper death when treating tumors due to complex preparation, low safety and utilization, and thus inhibit tumor growth.
By combining phytic acid with sodium hyaluronate, copper nanoenzymes with high affinity coordination ability are constructed, and the amount of copper ions, phytic acid and sodium hyaluronate is optimized to prepare copper phytic nanoenzymes with small size and good dispersion.
It has achieved safe and effective induction of copper death, significantly inhibited tumor growth, and has a simple process and low cost, which is conducive to clinical transformation.
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Figure CN119732974A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of nanotechnology, and specifically relates to a copper phytate nanozyme and a preparation method and application thereof. Background Art
[0002] Copper death is a new type of copper-dependent programmed cell death discovered in 2022. Because the death mechanism is different from apoptosis, copper death can effectively overcome the resistance to chemotherapy drugs based mainly on apoptosis, and can also improve the efficacy of tumor immune checkpoint inhibitors and chemoradiotherapy. It is a promising anti-tumor treatment method. The induction of copper death is inseparable from the accumulation of copper ions in cells. However, some copper ion carrier drugs, such as ilisimol and disulfiram, have not been significantly effective in clinical treatment. The reason is that this type of small molecule drug has a short circulation cycle in the body, is easily cleared by the blood, and has poor drug accumulation at the tumor site. In order to overcome this challenge, copper-based nanomaterials prepared using nanotechnology have shown advantages over small molecule drugs in terms of treatment methods and drug utilization.
[0003] At present, nanomaterials used to induce copper death are mainly divided into two categories. One category is to construct copper-based nanomaterials to promote the accumulation of copper in tumor cells; the other category is to prepare nanomaterials loaded with copper ion carriers to improve the retention of copper ion carriers in tumor sites. Nanozymes are a type of nanomaterials with enzyme catalytic activity. Compared with other copper-based nanomaterials, copper-based nanozymes not only increase the accumulation of copper ions in cells, but also have enzyme catalytic activity, which can improve tumor hypoxia and reduce intracellular glutathione. These properties promote the occurrence of copper death in tumor cells. However, the preparation of these materials requires strict reaction conditions and complex components, which seriously hinders their clinical transformation. Therefore, the preparation of safe, effective and simple-to-synthesize copper-based nanozymes, which inhibit tumor growth by inducing copper death, has a bright future as nanomedicines for the adjuvant treatment of tumors. Summary of the invention
[0004] To solve the above problems, the present invention provides a phytate copper nanozyme and its preparation method and application. Phytic acid is a type of natural polyphosphate carbohydrate with non-toxic and anti-tumor properties. The phytate copper nanozyme is constructed by the high affinity coordination ability of the abundant phosphate groups on the surface of phytic acid and copper ions. A natural polysaccharide sodium hyaluronate is added in the synthesis, which can not only regulate the size of the nanozyme and improve the dispersibility, but also target CD44 highly expressed on the surface of tumor cells. The resulting new type of copper-based nanozyme with safe composition and simple synthesis can inhibit tumor growth by inducing copper death.
[0005] To achieve the above purpose, the technical solution provided by the present invention is as follows:
[0006] A copper phytate nanozyme is synthesized from copper chloride, sodium phytate and sodium hyaluronate; the P=O in the sodium phytate molecule and the oxygen atom in the PO bond can coordinate with copper ions, and sodium hyaluronate plays a role in regulating the material size, dispersibility and targeting CD44 on the tumor surface; by regulating the molar ratio between copper chloride and sodium phytate, the concentration of sodium hyaluronate, and the order of addition of the three, the interaction forms a copper phytate nanozyme with a smaller size and good dispersibility.
[0007] A method for preparing copper phytate nanozyme comprises the following steps:
[0008] (1) Preparation of precursor solution: copper chloride (CuCl2) solution and sodium hyaluronate ((C 14 H 21 NaNO 11 )n) solution as a raw material, and a precursor solution is obtained by shaking reaction;
[0009] (2) Preparation of copper phytate nanozyme solution: Add sodium phytate (C6H 18 O 24 P6·xNa + ·yH2O) solution, after shaking reaction, a phytate copper nanozyme solution is obtained;
[0010] (3) The copper phytate nanozyme solution prepared in step (2) is separated, cleaned, and dried to obtain the copper phytate nanozyme.
[0011] Preferably, the step (1) of preparing the precursor solution is more specifically as follows: the concentration of the copper chloride solution is 100 mM, and the reaction volume is 20 μL. The concentration of the sodium hyaluronate solution is 2 mg / mL, and the reaction volume is 100 μL. The two solutions are added to 1 mL of deionized water, and shaken on a mixer at room temperature for 10 min to prepare the precursor solution.
[0012] Preferably, the concentration of the sodium phytate solution in step (2) is 50 mM, and the added volume is 10 μL. The molar ratio between copper chloride and sodium phytate is 4: 1. The mixed solution is shaken on a mixer at room temperature for 30 min to obtain a copper phytate nanozyme solution.
[0013] Preferably, in step (3), the copper phytate nanozyme solution is separated in a centrifuge at a speed of 6000 rpm for 5 min. The supernatant is discarded and the copper phytate nanozyme is collected. The copper phytate nanozyme is washed three times by centrifugation with anhydrous ethanol. The drying method is in a forced air drying oven at a temperature of 70° C. for 1 hour.
[0014] The copper phytate nanozyme is used as an anti-tumor drug. The copper phytate nanozyme inhibits tumor growth by inducing copper death. The copper phytate nanozyme is used as an injectable agent, and the injection is intratumoral injection or intravenous injection.
[0015] The present invention uses lung cancer as a model, and demonstrates that copper phytate nanozymes significantly inhibit tumor growth by inducing copper death through in vitro cell and subcutaneous transplant tumor models. By detecting markers related to copper death, the application of copper phytate nanozymes as copper death nanomedicines for anti-tumor is established.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The present invention utilizes the coordination between the empty orbit of copper ions and the phosphate group in phytic acid to construct phytate copper nanozyme. Phytic acid is a natural polyphosphate carbohydrate that has been shown to have a broad spectrum of anti-tumor effects and has no effect on normal cells. Sodium hyaluronate belongs to natural polysaccharides and has the effect of regulating the size of nanomaterials and targeting tumors. Therefore, the optimization of the amount between copper ions, phytic acid and sodium hyaluronate is the key to preparing nanozymes. By optimizing the molar ratio between copper chloride and sodium phytate, the content of sodium hyaluronate, and the order of adding materials, a phytate copper nanozyme with small size and good dispersibility is obtained. Further, the present invention obtains phytate copper nanozyme for anti-tumor, showing the mechanism of copper death, and showing a significant effect of inhibiting tumor growth at the cellular level and in animal models. Compared with the previous copper-based nanomaterials that induce copper death to fight tumors, the process is simple, the material is safe, the cost is low, and it is conducive to clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 This is a transmission electron microscope image of the copper phytate nanozyme prepared in the present invention, Bar: 50 nm.
[0019] Figure 2 This is an XPS picture of the copper phytate nanozyme prepared in the present invention.
[0020] Figure 3 This is a picture of the copper phytate nanozyme prepared in comparative example 1, Bar: 50 nm.
[0021] Figure 4 This is a picture of the copper phytate nanozyme prepared in comparative example 2, Bar: 50 nm.
[0022] Figure 5 This is a picture of the copper phytate nanozyme prepared in comparative example 3, Bar: 100 nm.
[0023] Figure 6 The CCK-8 method was used to detect the cell activity experimental results of the copper phytate nanozyme prepared by the present invention on normal cells LO2 and lung cancer cells A549. Figure 6 The horizontal axis is the concentration of copper phytate nanozyme (μg / mL), and the vertical axis is the cell activity (%).
[0024] Figure 7 The experimental results of the copper phytate nanozyme induced copper death of lung cancer A549 cells prepared by the present invention. The control group was not treated with copper phytate nanozyme.
[0025] Figure 8 The results of Western blot experiments show that the copper phytate nanozyme prepared in the present invention causes oligomerization of the key copper death protein DLAT and down-regulation of FDX1 in lung cancer cells A549; the Control group is not treated with copper phytate nanozyme.
[0026] Fig. 9 The copper phytate nanozyme prepared by the present invention inhibits tumor growth in a nude mouse subcutaneous inoculation of A549 cells into a tumor model. The control group was injected with PBS solution, and the experimental group was injected with 20 mg / kg copper phytate nanozyme solution. DETAILED DESCRIPTION
[0027] The specific implementation is described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation. The raw materials and reagents used in the examples are all commercially available unless otherwise specified.
[0028] Example 1
[0029] 1 Preparation of copper phytate nanozymes
[0030] (1) Preparation of precursor solution: In this example, a 100 mM copper chloride solution and a 2 mg / mL sodium hyaluronate aqueous solution were prepared. 20 μL of the copper chloride solution and 100 μL of the sodium hyaluronate solution were added to 1 mL of deionized water. The mixed solution was shaken on a mixer at room temperature for 10 min to prepare a precursor solution.
[0031] (2) Preparation of copper phytate nanozyme solution: A sodium phytate solution was added to the precursor solution prepared in step (1) at a concentration of 50 mM and a volume of 10 μL. At this time, the molar ratio between copper chloride and sodium phytate was 4:1. The mixed solution was shaken on a mixer at room temperature for 30 min to obtain a copper phytate nanozyme solution.
[0032] (3) The copper phytate nanozyme solution prepared in step (2) was separated by a centrifuge at a speed of 6000 rpm for 5 minutes. The supernatant was discarded to obtain the copper phytate nanozyme. The precipitate was washed three times by centrifugation with anhydrous ethanol at a speed of 6000 rpm for 5 minutes. The copper phytate nanozyme was placed in a forced air drying oven at 70°C and dried for 1 hour for use. The prepared copper phytate nanozyme was labeled as CP. The results showed that the material had a spherical morphology and a small size, most of which were concentrated in the range of about 5-7nm. The dispersion and uniformity were good ( Figure 1 ). XPS graph shows the characteristic peaks of Cu, P, C, O and N elements in copper phytate nanozyme ( Figure 2 ).
[0033] Comparative Example 1
[0034] According to step (1) of Example 1, 100 mM copper chloride solution and 1 mg / mL sodium hyaluronate aqueous solution were prepared. 20 μL of copper chloride solution and 100 μL of sodium hyaluronate solution were added to 1 mL of deionized water. The mixed solution was shaken on a mixer at room temperature for 10 min to prepare a precursor solution for later use. The remaining operations were the same as steps (2) and (3) of Example 1 to prepare copper phytate nanozyme. The transmission electron microscopy image showed that the material was not fully formed ( Figure 3 ).
[0035] Comparative Example 2
[0036] According to step (2) of Example 1, the concentration of sodium phytate solution added to the precursor liquid prepared in step (1) is 100mM, and the added volume is 10μL. At this time, the molar ratio between copper chloride and sodium phytate is 2:1. The mixed solution is shaken on a mixer at room temperature for 30 minutes to obtain a copper phytate nanozyme solution for later use. The remaining operations are the same as steps (1) (3) of Example 1 to prepare copper phytate nanozyme. Using transmission electron microscopy, the material adhesion is relatively serious and the dispersion is poor ( Figure 4 ).
[0037] Comparative Example 3
[0038] Prepare 100mM copper chloride solution and 2mg / mL sodium hyaluronate aqueous solution, 50mM sodium phytate solution. Take 20μL copper chloride solution, 100μL sodium hyaluronate solution, and 10μL sodium phytate solution and add them to 1mL deionized water. Oscillate the mixed solution on a mixer at room temperature for 40min to obtain a copper phytate nanozyme solution. The remaining operations are the same as step (3) of Example 1 to prepare copper phytate nanozyme. Using transmission electron microscopy, the material was severely aggregated and had poor dispersibility ( Figure 5 ).
[0039] Based on the above results, the copper phytate nanozyme prepared in Example 1 was selected to explore its applicability.
[0040] The following application examples illustrate the present invention through the inhibitory effects of copper phytate nanozymes on lung cancer in vivo and in vitro, as well as the anti-tumor mechanism described at the molecular level, but do not mean that the present invention is limited to this application example.
[0041] Application Example 1
[0042] The effect of the copper phytate nanozyme prepared in Example 1 on the activity of lung cancer cells and normal cells.
[0043] Experimental materials: cell lines, culture medium, penicillin and streptomycin, trypsin and copper phytate nanozyme. Human lung cancer cell A549 and human normal liver cell LO2 were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. High-glucose Dulbecco's modified eagle (DMEM) medium, trypsin, penicillin and streptomycin were purchased from Merck. Fetal bovine serum (FBS) was purchased from Gibco. CCK-8 kit was purchased from Bio-Tech.
[0044] Both cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, and cultured in an incubator at 37°C and 5% CO2.
[0045] Experimental method: A549 cells were incubated with phytate copper nanozyme solutions at concentrations of 20, 50, 80 and 100 μg / mL. LO2 cells were incubated with phytate copper nanozyme solutions at concentrations of 10, 20, 50 and 100 μg / mL. After 24 hours, the activity of cells in each group was determined by CCK-8 method. The cells in the control group were not treated with phytate copper nanozyme. The specific method is as follows:
[0046] (1) Take cells in the logarithmic growth phase, digest them with 0.25% trypsin, add culture medium to terminate the digestion, and prepare a cell suspension. A549 and LO2 cells were inoculated into 96-well culture plates (100 μL per well, 6×10 3 (2) Weigh the dried copper phytate nanozyme, add the culture medium for ultrasonic dispersion, and prepare it into the concentration required for the experiment. After the cells were incubated for 24 hours, the cell culture medium was aspirated, 100 μL of culture medium without copper phytate nanozyme was added to the control group, and the same volume of culture medium with different concentrations of copper phytate nanozyme was added to the experimental group. Three replicate wells were added in each group, and the incubation was continued for 24 hours.
[0047] (3) The activity of the two cells in the control group and the experimental group was determined by the CCK-8 method using an ELISA reader. 10 μL of CCK-8 solution was added to each well and the cells were cultured for another hour. The absorbance (OD) value at 450 nm was read on the ELISA reader. The relative activity of the cells after treatment with copper phytate nanozyme was calculated based on the light absorption value.
[0048] like Figure 6 The experimental results show that within the same dosage range, the activity of liver cells treated with copper phytate nanozymes is basically above 80%. This indicates that copper phytate nanozymes have no obvious toxicity to normal cells. Copper phytate nanozymes have obvious cytotoxicity to tumor cells A549 and are concentration-dependent. When the concentration of copper phytate nanozymes is 100μg / mL, the cell activity is less than 20%. The above results show that copper phytate nanozymes significantly inhibit the activity of lung cancer cells A549.
[0049] Application Example 2
[0050] An experiment was conducted to investigate the copper phytate nanozyme prepared in Example 1 to induce copper death of tumor cells.
[0051] Experimental materials: Copper death inhibitor Tetrathiomolybdate (TTM, purchased from Merck).
[0052] The conditions for A549 cell culture, digestion and 96-well plate inoculation were as described in Application Example 1. The experiment was divided into three groups: the cells in the Control group were not treated with copper phytate nanozymes. The second group was incubated with 80 μg / mL of copper phytate nanozymes (labeled as CP) and A549 cells, and the third group was pretreated with 100 μM TTM for 1 hour, and 80 μg / mL of copper phytate nanozymes were added and incubated with A549 cells after 1 hour. After 24 hours, the cell activity of each group was detected by CCK-8 method using an ELISA reader.
[0053] like Figure 7 The experimental results showed that copper phytate nanozymes significantly reduced the activity of tumor cells A549, while copper death inhibitors could restore the activity of cells treated with copper phytate nanozymes, indicating that copper phytate nanozymes caused copper death of tumor cells.
[0054] Application Example 3
[0055] A Western blot experiment was conducted to investigate the changes in the copper death key proteins DLAT and FDX1 caused by the phytate copper nanozyme prepared in Example 1.
[0056] Materials: BCA protein assay kit, Protein Ladder, PVDF membrane, loading buffer, antibody diluent, TBST buffer, RIPA lysis buffer and blocking buffer were purchased from Bio-Tech. The required antibodies were purchased from Abcam. Electrophoresis buffer and 4-20% Tris-glycine precast gel were purchased from Shanghai Wansheng Haotian Biotechnology Co., Ltd. The specific method is as follows:
[0057] A549 cell culture and digestion conditions were as described in Application Example 1.
[0058] (1) A549 cells were grown at 6×10 5 Cells were seeded in 6-well plates at a density of 1.5 × 10 cells / well and incubated for 24 h.
[0059] (2) The cells in the control group were not treated with copper phytate nanozymes, while the cells in the experimental group were treated with different concentrations of copper phytate nanozymes. After 24 h, the cells in each group were collected. The cells were treated with RIPA lysis buffer on ice, centrifuged at 12,000 rpm, 4°C for 10 min, and the supernatant was collected for later use.
[0060] (3) The BCA protein assay kit was used to quantify the protein in the sample.
[0061] (4) A total of 30 μg of protein was added to each well, separated by SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with a rapid blocking solution for 15 min.
[0062] (5) Incubate with primary antibodies at the following dilution ratios: DLAT (1:1000), FDX1 (1:1000), and β-action protein (1:5000) as an internal control, and then incubate at 4°C overnight.
[0063] (6) The membrane was incubated with the secondary antibody (1:10000 dilution) for 1 hour. After washing three times with TBST buffer, the expression of each histone band was observed using a membrane scanner.
[0064] like Figure 8 The experimental results showed that compared with the Control group, after the cells were treated with 80 and 100 μg / mL copper phytate nanozymes, DLAT showed obvious oligomeric bands between 130kD-200kD. The expression of FDX1 in cells treated with 80, 100, 150, and 200 μg / mL copper phytate nanozymes was significantly reduced, showing a concentration-dependent manner. This proved that copper phytate nanozymes caused the oligomerization of DLAT, a key protein of copper death, and the reduction of the expression of FDX1, indicating that copper phytate nanozymes induced copper death in tumor cells.
[0065] Application Example 4
[0066] The copper phytate nanozyme prepared in Example 1 had an inhibitory effect on tumor growth in a nude mouse subcutaneous tumor model.
[0067] Ten BALB / c nude mice, 5-6 weeks old, female, weighing 16-18 g, were selected and divided into two groups, with 5 mice in each group. Each nude mouse was subcutaneously injected with 1×10 6 A549 single cell suspension. Copper phytate nanozyme was dispersed in PBS solution, the dosage was 20mg / kg, and the tumor was injected once every other day, for a total of 7 injections. The group injected with PBS solution was the Control group. The results showed that compared with the Control group, the size of the tumor after copper phytate nanozyme injection was significantly lower than that of the control group, indicating that copper phytate nanozyme inhibited tumor growth and had a significant effect ( Fig. 9 ).
[0068] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.
Claims
1. A copper phytate nanozyme, characterized in that: Copper phytate nanozyme is synthesized from copper chloride, sodium phytate and sodium hyaluronate; the P=O in the sodium phytate molecule and the oxygen atom in the PO bond can coordinate with copper ions, and sodium hyaluronate plays a role in regulating the material size, dispersibility and targeting of CD44 on the tumor surface; by regulating the molar ratio between copper chloride and sodium phytate, the concentration of sodium hyaluronate, and the order of addition of the three, the interaction forms a smaller and more dispersible copper phytate nanozyme.
2. The copper phytate nanozyme according to claim 1 is characterized in that: The copper chloride is one of anhydrous copper chloride and hydrated copper chloride; the sodium phytate is C6H 18 O 24 P6·xNa + ·yH2O, molecular weight is 660.04; the molecular weight of sodium hyaluronate is between 1.0-1.8MDa, and the molecular formula is (C 14 H 21 NaNO 11 )n; All solutions are aqueous solutions and are prepared with water.
3. A method for preparing the copper phytate nanozyme according to claim 1 or 2, characterized in that: The following steps are involved: (1) Preparing a precursor solution: using a copper chloride solution and a sodium hyaluronate solution as raw materials, and obtaining a precursor solution by shaking reaction; (2) preparing a copper phytate nanozyme solution: adding a sodium phytate solution to the precursor solution prepared in step (1), and obtaining a copper phytate nanozyme solution through a shaking reaction; (3) The copper phytate nanozyme solution prepared in step (2) is separated, cleaned, and dried to obtain a solid copper phytate nanozyme.
4. The method for preparing a precursor liquid according to claim 3, characterized in that: The specific operation of step (1) preparing the precursor solution is as follows: the concentration of the copper chloride solution is 100 mM, and the reaction volume is 20 μL; the concentration of the sodium hyaluronate solution is 2 mg / mL, and the reaction volume is 100 μL; the two solutions are added to 1 mL of water, and shaken on a mixer at room temperature for 10 min to prepare the precursor solution.
5. The method for preparing the copper phytate nanozyme solution according to claim 3, characterized in that: In step (2), the concentration of the sodium phytate solution is 50 mM, the added volume is 10 μL, and the molar ratio between copper chloride and sodium phytate is 4:1; the mixed solution is shaken on a mixer at room temperature for 30 minutes to obtain a copper phytate nanozyme solution.
6. The method for preparing the copper phytate nanozyme solution according to claim 3, characterized in that: In step (3), the copper phytate nanozyme solution is separated in a centrifuge at a speed of 6000 rpm for 5 minutes, the supernatant is discarded, the copper phytate nanozyme is collected, and the copper phytate nanozyme is washed three times by centrifugation with anhydrous ethanol, and the drying method is in a forced air drying oven at a temperature of 70° C. for 1 hour.
7. The use of the copper phytate nanozyme as an anti-tumor drug according to any one of claims 1 to 6, characterized in that: The anti-tumor drug is a drug that inhibits tumor growth, and copper phytate nanozymes inhibit tumor growth by inducing copper death.
8. The use of the copper phytate nanozyme as an anti-tumor drug according to claim 7, characterized in that: The copper phytate nanozyme is used as an injectable agent, and the injection is intratumoral injection or intravenous injection.