Method for preparing calcium oxalate crystals of different particle sizes

Abstract

The present application provides a method for preparing stable calcium oxalate crystals with different particle sizes. The calcium oxalate crystals prepared by the method of the present application are regular in shape and stable in properties, and can be used to simulate different stages of kidney stone formation, and can be used to explore the mechanism of kidney stone formation and development and preventive measures.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical research, and particularly relates to a method for preparing calcium oxalate crystals with different particle sizes. BACKGROUND

[0002] Common symptoms of renal calculi include waist and abdominal cramps, nausea, vomiting, restlessness, abdominal distension, hematuria, etc. Renal calculi are classified into calcium oxalate calculi, urate calculi and ammonium magnesium phosphate calculi, among which calcium oxalate calculi are the most common, accounting for 71% to 84%. One of the main crystal components of calcium oxalate calculi is calcium oxalate monohydrate (COM), which is the most thermodynamically stable phase in renal calculi.

[0003] In the current scientific research related to renal calculi, researchers explore the formation and development of calcium oxalate calculi and its influence on the human body through the characterization of calcium oxalate crystals at the cellular level. However, the preparation methods of calcium oxalate crystals are not uniform, and the prepared crystals are irregular in size, which is not conducive to medical research of renal calculi. Therefore, it is very important to develop calcium oxalate crystals with regular shape and stable properties of different sizes in order to explore the different stages of formation and development of renal calculi, the mechanism of development of renal calculi and preventive measures. SUMMARY

[0004] The application provides a method for preparing stable calcium oxalate crystals with different particle sizes.

[0005] The application provides a method for preparing calcium oxalate crystals with a particle size of 5.8±0.4 μm, which comprises the following steps:

[0006] 1) preparing 1 mM sodium oxalate solution and 2 mM calcium chloride solution;

[0007] 2) adding 20 mL of calcium chloride solution and 20 mL of sodium oxalate solution into a centrifuge tube in sequence;

[0008] 3) mixing uniformly by inverting up and down;

[0009] 4) taking the center of the centrifuge tube as the center, mixing and incubating uniformly by inverting vertically at room temperature for 2 h, and the rotation speed of vertical inversion is 10 rpm;

[0010] 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0011] 6) adding 5 mL of organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0012] 7) repeating step 6;

[0013] 8) air-drying under ventilation, and calcium oxalate crystals with a particle size of 5.8±0.4 μm are obtained.

[0014] Further, the solvent used in step 1) is deionized water, and the organic solvent in step 5) is methanol.

[0015] The present application also provides a method for preparing calcium oxalate crystals with a particle size of 10±0.8 μm, which comprises the following steps:

[0016] 1) preparing a 1 mM sodium oxalate solution and a 2 mM calcium chloride solution;

[0017] 2) first adding 20 mL of the calcium chloride solution into a centrifuge tube, and then adding 20 mL of the sodium oxalate solution at a speed of 500 mL / h;

[0018] 3) placing in a rotating mixer, and incubating at room temperature at 20 rpm for 1 h;

[0019] 4) centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0020] 5) adding 5 mL of an organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0021] 6) repeating step 5;

[0022] 7) air-drying, to obtain calcium oxalate crystals with a particle size of 10±0.8 μm.

[0023] Further, the solvent used in step 1) is deionized water, and the organic solvent in step 5) is methanol.

[0024] The present application also provides a method for preparing calcium oxalate crystals with a particle size of 14±1.2 μm, which comprises the following steps:

[0025] 1) preparing a 1 mM sodium oxalate solution and a 2 mM calcium chloride solution;

[0026] 2) preheating the sodium oxalate solution and the calcium chloride solution to 70°C respectively;

[0027] 3) first adding 200 mL of the 70°C calcium chloride solution into a container, and then adding 200 mL of the 70°C sodium oxalate solution at a speed of 2500 mL / h;

[0028] 4) keeping the temperature at 70°C, and continuously stirring at 10 rpm for 1 h;

[0029] 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0030] 6) adding 5 mL of an organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0031] 7) repeating step 6;

[0032] 8) Ventilation air drying, the calcium oxalate crystal with a particle size of 14±1.2 μm is obtained;

[0033] Preferably, the solvent used in step 1) is deionized water, and the organic solvent used in step 5) is methanol; and the stirring in step 4) is magnetic stirring.

[0034] The present application also provides a method for preparing calcium oxalate crystals with a particle size of 18±2.0 μm, which comprises the following steps:

[0035] 1) preparing a 1 mM sodium oxalate solution and a 2 mM calcium chloride solution;

[0036] 2) preheating the sodium oxalate solution and the calcium chloride solution to 70°C;

[0037] 3) adding 200 mL of the 70°C calcium chloride solution into a container, and then adding 200 mL of the 70°C sodium oxalate solution at a speed of 10000 mL / h;

[0038] 4) keeping the temperature at 70°C, and stirring at 10 rpm for 1 h;

[0039] 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0040] 6) adding 5 mL of an organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant;

[0041] 7) repeating step 6;

[0042] 8) ventilation air drying, the calcium oxalate crystal with a particle size of 18±2.0 μm is obtained;

[0043] Preferably, the solvent used in step 1) is deionized water, and the organic solvent used in step 5) is methanol; and the stirring in step 4) is magnetic stirring.

[0044] The present application also provides the calcium oxalate crystals prepared by the above method.

[0045] The present application also provides a method for preparing a group of calcium oxalate crystals with different particle sizes and good stability, wherein the particle sizes of the calcium oxalate crystals are 5.8±0.4 μm, 10±0.8 μm, 14±1.2 μm and 18±2.0 μm, and the method comprises the above preparation method.

[0046] The present application also provides the use of the above calcium oxalate crystals in the research of the effects of the calcium oxalate crystals on kidney cells.

[0047] The present application also provides a method for researching the effects of the calcium oxalate crystals on kidney cells, which comprises the following steps:

[0048] 1) taking kidney cells for in-vitro culture;

[0049] 2) The different particle size calcium oxalate crystals prepared by the above method are added into the aforementioned culture system, and a control group is set, wherein the control group is not added with the crystals;

[0050] 3) The effect of the crystals on kidney cells is observed.

[0051] The temperature, centrifugation time and rotation speed for preparing the calcium oxalate crystals are strictly required, and the crystal type or particle size will change if the parameters are changed arbitrarily, and the stable calcium oxalate crystals with uniform particle size cannot be obtained.

[0052] The application provides a method for preparing calcium oxalate crystals with regular shape and stable properties, and the crystal size is 5.8±0.4 μm, 10±0.8 μm, 14±1.2 μm or 18±2.0 μm. The main component of the calcium oxalate crystals is calcium oxalate monohydrate. The different particle size calcium oxalate crystals prepared by the application can simulate different stages of kidney stone formation, and can be used for exploring the mechanism of kidney stone formation and development and preventive measures.

[0053] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the application.

[0054] The above content of the application is further illustrated in detail by the following embodiment. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology achieved based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 The SEM images of the calcium oxalate crystals with extremely small particle size are (a) 10 μm and (b) 3 μm.

[0056] Figure 2 The SEM images of the calcium oxalate crystals with small particle size are (a) 10 μm and (b) 3 μm.

[0057] Figure 3 The SEM images of the calcium oxalate crystals with medium particle size are (a) 10 μm and (b) 3 μm.

[0058] Figure 4 The SEM images of the calcium oxalate crystals with large particle size are (a) 10 μm and (b) 3 μm.

[0059] Figure 5 The infrared spectrograms of the calcium oxalate crystals with different particle sizes are shown. DETAILED DESCRIPTION

[0060] The raw materials and equipment used in the application are known products, which are obtained by purchasing commercial products.

[0061] The relevant instruments used in the experiment are as follows:

[0062] Vortex mixer: MX-RD-Pro type digital disc rotary mixing instrument.

[0063] Vertical inverting instrument: Tube Revolver, model: TR-02U (HYQ-2231).

[0064] Preparation of stable calcium oxalate crystals of different particle sizes in Example 1

[0065] I. Preparation method (I) Preparation of calcium oxalate crystals with extremely small particle size (min-COM, 5.8±0.4 μm)

[0066] 1. Prepare 1 mM sodium oxalate solution and 2 mM calcium chloride solution with ddH2O.

[0067] 2. In a 50 mL centrifuge tube, first add 20 mL of 2 mM calcium chloride solution.

[0068] 3. Immediately add 20 mL of 1 mM sodium oxalate solution.

[0069] 4. Mix well by inverting up and down.

[0070] 5. Take the center of the centrifuge tube as the center, and incubate at room temperature by vertical inverting mixing at a speed of 10 rpm for 2 h.

[0071] 6. Centrifuge at 2500 rpm for 5 min, discard the supernatant to about 5 mL.

[0072] 7. Add 5 mL of methanol, centrifuge at 2500 rpm for 5 min, discard the supernatant.

[0073] 8. Repeat step 7.

[0074] 9. Air dry for 30 min.

[0075] 10. Ultraviolet sterilization for 30 min.

[0076] (II) Preparation of calcium oxalate crystals with sub-small particle size (submid-COM, 10±0.8 μm)

[0077] 1. Prepare 1 mM sodium oxalate solution and 2 mM calcium chloride solution with ddH2O.

[0078] 2. In a 50 mL centrifuge tube, first add 20 mL of 2 mM calcium chloride solution.

[0079] 3. Add 20 mL of 1 mM sodium oxalate solution at a rate of 500 mL / h using a cross-flow pump.

[0080] 4. Incubate at room temperature for 1 h at 20 rpm on a vortex mixer.

[0081] 5. Centrifuge at 2500 rpm for 5 min, discard supernatant to about 5 mL.

[0082] 6. Add 5 mL of methanol, centrifuge at 2500 rpm for 5 min, discard supernatant.

[0083] 7. Repeat step 6.

[0084] 8. Air dry for 30 min.

[0085] 9. UV sterilization for 30 min.

[0086] (iii) Preparation of mid-sized calcium oxalate crystals (mid-COM, 14 ± 1.2 pm)

[0087] 1. Prepare 1 mM sodium oxalate solution, 2 mM calcium chloride solution with ddH2O.

[0088] 2. Preheat 1 mM sodium oxalate solution and 2 mM calcium chloride solution to 70 °C separately.

[0089] 3. Place a 500 mL beaker on a constant temperature magnetic stirrer at 100 rpm, 70 °C, first add 200 mL

[0090] 70 °C 2 mM calcium chloride solution.

[0091] 4. Add 1 mM sodium oxalate solution with a temperature of 70 °C at a rate of 2500 mL / h using a crossflow pump.

[0092] 5. Keep constant temperature at 70 °C, 10 rpm continuous stirring for 1 h.

[0093] 6. Centrifuge at 2500 rpm for 5 min, discard supernatant to about 5 mL.

[0094] 7. Add 5 mL of methanol, centrifuge at 2500 rpm for 5 min, discard supernatant.

[0095] 8. Repeat step 7.

[0096] 9. Air dry for 30 min.

[0097] 10. UV sterilization for 30 min.

[0098] (iv) Preparation of large-sized calcium oxalate crystals (max-COM, 18 ± 2.0 pm)

[0099] 1. Prepare 1 mM sodium oxalate solution, 2 mM calcium chloride solution with ddH2O.

[0100] 2. Preheat 1 mM sodium oxalate solution and 2 mM calcium chloride solution to 70°C.

[0101] 3. Put 500 mL beaker on constant temperature magnetic stirrer at 100 rpm, 70°C, first add 200 mL 70°C 2 mM calcium chloride solution.

[0102]

[0103] 4. Add 1 mM sodium oxalate solution at 70°C with cross-flow pump at a speed of 10,000 mL / h

[0104] 200 mL.

[0105] 5. Keep constant temperature at 70°C, 10 rpm, and stir for 1 h.

[0106] 6. Centrifuge at 2500 rpm for 5 min, discard supernatant to about 5 mL.

[0107] 7. Add 5 mL of methanol, centrifuge at 2500 rpm for 5 min, discard supernatant.

[0108] 8. Repeat step 7.

[0109] 9. Air dry for 30 min.

[0110] 10. Ultraviolet sterilization for 30 min.

[0111] Second, the crystal stability and component detection of the application

[0112] (I) Detection method

[0113] 1. Scanning electron microscope detection

[0114] Take the calcium oxalate crystals of various particle sizes prepared by the foregoing method, and place them for 4 months, respectively, during which the particle size change is observed by scanning electron microscope.

[0115] 2. Fourier infrared spectrum detection

[0116] Take the calcium oxalate crystals of various particle sizes prepared by the foregoing method, respectively, and determine the main components by Fourier infrared spectrum (FTIR).

[0117] (II) Detection results

[0118] 1. Scanning electron microscope detection results

[0119] After observation, the calcium oxalate crystals of various particle sizes prepared by the method of the application are very stable, and the particle size hardly changes. For example, the particle size of the extremely small calcium oxalate crystal remains at 5.8 ± 0.4 μm. And through Figures 1-4 It can be seen that, in addition to the extremely small crystal, the calcium oxalate crystal of other particle sizes is uniform, and all are punctate twins.​

[0120] 2. Fourier infrared spectrum detection result

[0121] The formed calcium oxalate crystal is calcium oxalate monohydrate crystal, which is proved by Fourier infrared spectrum (FTIR), and the FTIR spectrum is as follows: there is a characteristic peak of metal carboxylate stretching at 1319 cm -1 -1, and the water molecule coordinated with calcium oxalate molecule produces a specific peak matched with bending mode at 1629 cm -1 -1. The asymmetric and symmetric stretching peaks of the coordinated water molecule are shown by the above wide spectrum peak of 3000 cm -1 -1. In addition, two peaks near 800 cm -1 -1 are characteristic peaks of the calcium oxalate crystal.

[0122] Test Example 1: Study on kidney stone mechanism by using different particle size crystals of the application

[0123] 1. Kidney cells are taken for in-vitro culture;

[0124] 2. Different particle size crystals prepared in Example 1 are added into the above culture system, and a control group is set, which does not add crystals;

[0125] 3. The effect of the crystals on the kidney cells is observed.

[0126] In summary, the application provides a method for preparing calcium oxalate crystals with regular shape and stable properties, and the particle sizes of the crystals are 5.8±0.4 μm, 10±0.8 μm, 14±1.2 μm and 18±2.0 μm, respectively. The main component of the calcium oxalate crystal is calcium oxalate monohydrate. The different particle size calcium oxalate crystals prepared by the application can simulate different stages of kidney stone formation, and can be used for exploring the mechanism of kidney stone formation and development and preventive measures.

Claims

1. A method for preparing calcium oxalate crystals having a particle size of 5.8 ± 0.4 μm, characterized in that, The method comprises the following steps: 1) preparing 1 mM sodium oxalate solution and 2 mM calcium chloride solution; 2) adding 20 mL of calcium chloride solution and 20 mL of sodium oxalate solution into a centrifuge tube in sequence; 3) mixing uniformly by inverting up and down; 4) incubating by inverting vertically at room temperature for 2 h, and the rotating speed of vertical inversion is 10 rpm; 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 6) adding 5 mL of organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 7) repeating step 6; 8) air-drying to obtain calcium oxalate crystals with a particle size of 5.8 ± 0.4 μm. The solvent used in step 1) is deionized water, and the organic solvent in step 6) is methanol.

2. A method for preparing calcium oxalate crystals having a particle size of 10 ± 0.8 μm, characterized in that, The method comprises the following steps: 1) preparing 1 mM sodium oxalate solution and 2 mM calcium chloride solution; 2) adding 20 mL of calcium chloride solution into a centrifuge tube, and then adding 20 mL of sodium oxalate solution at a speed of 500 mL / h; 3) incubating in a rotating mixing instrument at 20 rpm for 1 h at room temperature; 4) centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 5) adding 5 mL of organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 6) repeating step 5; air-drying to obtain calcium oxalate crystals with a particle size of 10 ± 0.8 μm; the solvent used in step 1) is deionized water, and the organic solvent in step 5) is methanol.

3. A process for preparing calcium oxalate crystals having a particle size of 14 ± 1.2 μm, characterized in that, The method comprises the following steps: 1) preparing 1 mM sodium oxalate solution and 2 mM calcium chloride solution; 2) preheating the sodium oxalate solution and the calcium chloride solution to 70 ℃ respectively; 3) adding 200 mL of 70 ℃ calcium chloride solution into a container, and then adding 200 mL of 70 ℃ sodium oxalate solution at a speed of 2500 mL / h; 4) keeping the temperature at 70 ℃, and continuously stirring at 10 rpm for 1 h; 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 6) adding 5 mL of organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 7) repeating step 6; 8) air-drying to obtain calcium oxalate crystals with a particle size of 14 ± 1.2 μm; the solvent used in step 1) is deionized water, the organic solvent in step 6) is methanol, and the stirring in step 4) is magnetic stirring.

4. A process for preparing calcium oxalate crystals having a particle size of 18 ± 2.0 μm, characterized in that, The method comprises the following steps: 1) preparing 1 mM sodium oxalate solution and 2 mM calcium chloride solution; 2) preheating the sodium oxalate solution and the calcium chloride solution to 70 ℃; 3) adding 200 mL of 70 ℃ calcium chloride solution into a container, and then adding 200 mL of 70 ℃ sodium oxalate solution at a speed of 10000 mL / h; 4) keeping the temperature at 70 ℃, and continuously stirring at 10 rpm for 1 h; 5) centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 6) adding 5 mL of organic solvent, centrifuging at 2500 rpm for 5 min, and discarding the supernatant; 7) repeating step 6; 8) Air dried, the calcium oxalate crystals obtained have a particle size of 18 ± 2.0 μm; The solvent used in step 1) is deionized water and the organic solvent used in step 6) is methanol; the stirring in step 4) is magnetic stirring.

5. A method for preparing a group of calcium oxalate crystals having different particle sizes and good stability, characterized in that, The calcium oxalate crystals have a particle size of 5.8 ± 0.4 μm, 10 ± 0.8 μm, 14 ± 1.2 μm, 18 ± 2.0 μm, including the preparation methods of claims 1, 2, 3 and 4.

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