Bilirubin-like oxidase, enzyme-loaded gel microspheres, and preparation method and application of bilirubin-like oxidase and enzyme-loaded gel microspheres

Abstract

The invention discloses bilirubin-like oxidase, enzyme-loaded gel microspheres and a preparation method and application of the bilirubin-like oxidase and the enzyme-loaded gel microspheres, and belongs to the technical field of medicines and enzymes. The preparation method of the enzyme-loaded gel microspheres comprises the following steps: preparing a chitosan-polyacrylic acid solution into carrier microspheres by a solvent replacement method, and combining bilirubin-like oxidase to the surfaces of the carrier microspheres by an enzyme immobilization method to obtain enzyme-loaded chitosan-polyacrylic acid microspheres, namely the enzyme-loaded gel microspheres. The enzyme-loaded gel microspheres provided by the invention can improve the adsorption capacity of a bilirubin adsorbent in the form of simultaneous decomposition and adsorption, and the enzyme-loaded gel microspheres provided by the invention have the advantages of selective adsorption, low cost, high stability, high adsorption efficiency and the like when being used for decomposition and adsorption of bilirubin.

Description

Technical Field

[0001] The present invention relates to the field of medicine and enzyme technology, and in particular to a bilirubin oxidase, enzyme-loaded gel microspheres, and a preparation method and application thereof. Background Art

[0002] Liver failure is a group of clinical syndromes characterized by coagulation disorder, jaundice, hepatic encephalopathy and respiratory failure. Bilirubin is an important toxin in liver metabolism, which can cause irreversible damage to the brain and nervous system. It is also one of the indicators of liver function test. Bilirubin is a decomposition metabolite of heme metabolism, mainly from aging red blood cells. Heme is oxidized by heme oxygenase to form biliverdin. Biliverdin is further reduced to bilirubin by biliverdin reductase. Once formed, bilirubin forms a reversible complex with serum albumin in the circulation and is transported to the liver, where it is converted into water-soluble glucuronic acid-conjugated bilirubin by a specific glucuronyl transferase UGT1A1. Within the physiological range, bilirubin has anti-inflammatory, antioxidant and beneficial metabolic effects, but at high concentrations, it is potentially toxic. In general, once the source of bilirubin in the blood increases (such as hemolytic anemia and thalassemia) or the excretion pathway is blocked (such as biliary obstruction and liver damage, etc.), hyperbilirubinemia will occur.

[0003] Removing excess bilirubin is the key to treating hyperbilirubinemia. In clinical practice, several methods for removing bilirubin have been developed, such as plasma exchange, hemoperfusion, and albumin dialysis. Among them, hemoperfusion is widely used because of its mature technology and relatively low price. The effect of hemoperfusion depends on the adsorbent.

[0004] In the past few decades, many new adsorbents have been developed to remove bilirubin, but they are rarely used clinically. The main reason is that the preparation methods of these existing adsorbents are complicated, their blood compatibility is poor, and their high cost may be the key issues hindering their clinical transformation. Improving the adsorption capacity and selectivity of bilirubin adsorbents is the research goal of many blood purification scholars. Summary of the invention

[0005] One of the purposes of the present invention is to provide a bilirubin oxidase to solve the above problems.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a bilirubin oxidase, wherein the bilirubin oxidase is prepared by the following method: (a) mixing a 5-30 mM copper sulfate aqueous solution with a 10-50 mg / mL albumin phosphate buffer solution, stirring for 1-10 min to obtain a mixed solution; (b) The mixed solution prepared in step (a) is adjusted to a pH of 10 to 14 using tetrabutylammonium hydroxide or sodium hydroxide solution; after reacting for 2 to 8 hours at a temperature below 15° C., the mixed solution is dialyzed and freeze-dried (the dialysis temperature must be below 15° C.) to obtain a bilirubin oxidase.

[0007] As a preferred technical solution, in step (a), the volume ratio of the copper sulfate aqueous solution to the albumin phosphate buffer is 1:4 to 1:10.

[0008] As a preferred technical solution, in step (a), the albumin is human serum albumin or bovine serum albumin.

[0009] The albumin can be human serum albumin or bovine serum albumin, and the concentration cannot exceed 50 mg / L. The concentration of the copper sulfate solution cannot be lower than 5 mM. The inventors have proved through a large number of experiments that too high an albumin concentration or too low a copper sulfate concentration will significantly reduce the binding rate of the binding sites of copper ions in the albumin, thereby weakening the catalytic effect.

[0010] The second object of the present invention is to provide an enzyme-loaded gel microsphere prepared by using the above-mentioned bilirubin oxidase, wherein the enzyme-loaded gel microsphere is prepared by using chitosan and polyacrylic acid as carriers and loading the bilirubin oxidase.

[0011] The third object of the present invention is to provide a method for preparing the enzyme-loaded gel microspheres, comprising the following steps: (1) dissolving a chitosan aqueous solution with a concentration of 2 to 4 wt% in an acetic acid solution, and then adding a polyacrylic acid aqueous solution with a concentration of 0.1 to 3 wt% to obtain a white polymer gel, wherein the mass ratio of chitosan (dry weight) to polyacrylic acid (dry weight) is 1 to 15; then dropping an acid solution to convert the white polymer gel into a clear and uniform solution; (2) the solution obtained in step (1) is dripped into a mixed solution of 0.8-2 M sodium hydroxide and 20-40 vol% ethanol through a microinfusion needle connected to an electrostatic generator under the action of an electrostatic voltage of 5 kv-8 kv to obtain microspheres, and the microspheres are stirred at room temperature to solidify in the solidification solution for 1-5 h. After solidification, the microspheres are rinsed with deionized water to obtain chitosan-polyacrylic acid carrier microspheres; (3) The carrier microspheres obtained in step (2) are placed in a 0.1-1 wt% EDC (i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution) solution and stirred; then, after washing with phosphate buffer, the bilirubin oxidase is dissolved in phosphate buffer, and the chitosan-polyacrylic acid carrier microspheres are mixed into the solution to immobilize the enzyme, and the reaction is carried out for 1-5 hours to obtain enzyme-loaded gel microspheres.

[0012] As a preferred technical solution, in step (1), the acid solution is a hydrochloric acid solution.

[0013] As a preferred technical solution, in step (2), the diameter of the chitosan-polyacrylic acid carrier microspheres is 500-800 μm.

[0014] If the size of the microspheres is too small (less than 500 μm), the microspheres will pass through the filter and enter the blood circulation, causing safety problems such as embolism; if the size is too large (such as more than 800 μm), the specific surface area will be reduced and the adsorption efficiency will be reduced. The present invention preferably controls the voltage during the ball dropping process. The larger the voltage, the smaller the microsphere particle size, and the smaller the voltage, the larger the microsphere particle size. Therefore, by controlling the voltage at 5 kv to 8 kv, the size of the microspheres is controlled within the range of 500 to 800 μm, so that it can balance the fluidity and pressure drop, ensuring smooth blood flow during blood perfusion.

[0015] As a preferred technical solution, in step (3), the mass ratio of EDC (dry weight) to chitosan-polyacrylic acid carrier microspheres is 1:40 to 1:400.

[0016] Too much EDC will remain on the surface of the microspheres and occupy the enzyme loading sites; too little EDC will not fully activate the chemical groups on the surface of the microspheres, reducing the enzyme loading rate. Therefore, the amount of EDC should be within an appropriate range.

[0017] The fourth object of the present invention is to provide an application of enzyme-loaded gel microspheres prepared by the above-mentioned preparation method in the preparation of medical equipment for treating or improving hyperbilirubinemia.

[0018] As a preferred technical solution, the medical device is a hemoperfusion device.

[0019] Bilirubin oxidase (BOD) can effectively decompose bilirubin. The principle is that BOD, with the assistance of oxygen, will catalyze bilirubin into biliverdin, and further decompose it into derivatives of weak diazo reaction and highly polar diazo byproducts. These small molecule products are easily soluble in water and can be excreted from the body with urine, thereby achieving the removal of bilirubin. BOD is currently generally prepared by biological methods, which is difficult to prepare and expensive. Based on this principle, the inventor of this application has attempted to prepare the above-mentioned artificial enzyme that simulates bilirubin oxidase through theoretical analysis and a large number of experiments, and loaded it on microspheres that can adsorb bilirubin and its decomposition products for blood perfusion.

[0020] The principle of the bilirubin oxidase of the present invention is that in a metal ion solution, metal ions interact with biological molecules, which can significantly improve the stability of proteins; the present invention uses a copper ion solution as a reaction medium, the structure of albumin in the copper ion solution changes, a large number of amino acid residues are released, and the albumin self-assembles with the copper ions, thereby generating a soluble bilirubin oxidase.

[0021] Compared with the prior art, the advantages of the present invention are: (1) Saving production costs: Compared with the current biosynthesis of bilirubin oxidase, the preparation method of the artificial enzyme of the present invention is simple and low in cost. Specifically: In the present invention, the natural enzyme (BOD) simulated by the bilirubin oxidase is used in an amount of 100 times that of BOD to achieve the same catalytic effect as BOD. However, considering factors such as yield, preparation difficulty and preparation cost, for example, the production cost of 1g BOD is about 50,000 to 100,000 yuan, and the cost of producing 1g bilirubin oxidase is about 200 yuan, so the cost of BOD is 2.5 to 5 times that of bilirubin oxidase. Therefore, considering only the cost aspect, bilirubin oxidase is a better choice.

[0022] (2) High stability: The artificial enzyme of the present invention is a nanomaterial that can simulate the catalytic activity of natural enzymes. Compared with natural enzymes, artificial enzymes have higher stability and the possibility of large-scale preparation. The stability comparison is shown in the comparative example below. In order to retain its catalytic activity, BOD needs to be stored in an environment of -20°C, and bilirubin oxidase can be stored in a refrigerator at 4°C. The strict storage requirements of BOD make it not have the advantage of large-scale production.

[0023] (3) Selective adsorption: The enzyme-loaded gel microspheres of the present invention can greatly improve the adsorption capacity of the bilirubin adsorbent by catalyzing and adsorbing at the same time. At the same time, this strategy reduces the influence of the material on albumin. Bilirubin oxidase is used to first catalyze bilirubin into small molecular polar compounds such as biliverdin. After the decomposition products are dissociated from albumin, the decomposition products are adsorbed, thereby achieving the goal of only removing toxins without adsorbing albumin, thereby improving the removal rate and specificity of the adsorbent for bilirubin. (4) High adsorption efficiency: The base material of the gel microspheres is chitosan, which is a natural polysaccharide obtained by deacetylation of chitin and has good biocompatibility, degradability and adsorption properties. There is a certain electrostatic interaction between the amino group (-NH2) on the surface of chitosan and the carboxyl group (-COOH) of bilirubin, thereby achieving the adsorption of bilirubin and its decomposition products. At the same time, the surface of the chitosan microspheres is a porous structure, which increases the specific surface area of ​​the microspheres in contact with blood, is conducive to liquid-mass transmission, and further increases the adsorption capacity of the adsorbent. However, the mechanical strength of pure chitosan-based microspheres is poor. Adding an appropriate amount of polyacrylic acid can improve the mechanical properties of the chitosan base material. The bilirubin oxidase in the present invention is loaded onto the gel microspheres by chemical grafting, which can not only prevent the bilirubin oxidase from falling off, but also continue to exert a catalytic effect. The catalyzed bilirubin is decomposed into small molecules that are easily soluble in water and can be excreted from the body with urine. At the same time, the gel microspheres can also adsorb bilirubin and its decomposition products. Thereby achieving the removal of bilirubin. The method of the present invention can reduce the treatment time or treatment frequency, and save the medical expenses of the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the SEM structure of enzyme-loaded gel microspheres prepared in Example 8; Figure 2 Schematic diagram of the SEM structure of enzyme-loaded gel microspheres prepared in Example 10; Figure 3 Comparative photos of the enzyme-loaded gel microspheres in Example 10 before and after bilirubin adsorption in a plasma environment; Figure 4 The UV absorption spectra of the bilirubin solution after contacting with the enzyme-loaded microspheres of Example 10 for different time periods. DETAILED DESCRIPTION

[0025] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0026] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0027] The following Examples 1-7 are examples of bilirubin oxidase, and Examples 8-13 are examples of enzyme-loaded microspheres.

[0028] Example 1 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 5.0 mL of bovine serum albumin solution (15 mg / mL) in a volume ratio of 1:5 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M sodium hydroxide solution; after reacting at 10°C for 5 h, the mixed solution was dialyzed at 10°C and freeze-dried to obtain bilirubin oxidase.

[0029] Embodiment 2 This embodiment is a preferred embodiment of the present invention: A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 5.0 mL of bovine serum albumin solution (15 mg / mL) in a volume ratio of 1:5 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 10°C for 5 h, the mixed solution was dialyzed at 10°C and freeze-dried to obtain bilirubin oxidase.

[0030] Example 3 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 5.0 mL of bovine serum albumin solution (15 mg / mL) in a volume ratio of 1:5 were mixed and stirred for 5 min, and the pH was adjusted to 14 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15°C for 5 h, the mixed solution was dialyzed at 15°C and freeze-dried to obtain bilirubin oxidase.

[0031] Example 4 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 5.0 mL of human serum albumin solution (15 mg / mL) in a volume ratio of 1:5 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15°C for 5 h, the mixed solution was dialyzed at 15°C and freeze-dried to obtain bilirubin oxidase.

[0032] Example 5 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 5.0 mL of bovine serum albumin solution (15 mg / mL) in a volume ratio of 1:5 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 25°C for 5 h, the mixed solution was dialyzed at 25°C and freeze-dried to obtain bilirubin oxidase.

[0033] Example 6 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (20 mM) and 12.0 mL of bovine serum albumin solution (60 mg / mL) in a volume ratio of 1:12 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15°C for 5 h, the mixed solution was dialyzed at 15°C and freeze-dried to obtain bilirubin oxidase.

[0034] Example 7 A bilirubin oxidase capable of decomposing bilirubin, the preparation method of which comprises the following steps: 1.0 mL of copper sulfate solution (1 mM) and 12.0 mL of bovine serum albumin solution (15 mg / mL) in a volume ratio of 1:12 were mixed and stirred for 5 min, and the pH was adjusted to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15°C for 5 h, the mixed solution was dialyzed at 15°C and freeze-dried to obtain bilirubin oxidase.

[0035] Performance Test Example 1: Measuring the ability of bilirubin oxidase to decompose bilirubin in phosphate buffer solution The catalytic decomposition ability of Example 1-7 on bilirubin (dissolved in phosphate buffer) was determined. The specific method was as follows: 1 mg of bilirubin oxidase was weighed and placed in 3 mL of bilirubin phosphate buffer (200 mg / L). At 180 min, 100 μL of supernatant was taken to a 96-well plate, and the absorbance at a wavelength of 438 nm was scanned with an ELISA reader to calculate its bilirubin clearance rate.

[0036] Table 1. Preparation conditions and bilirubin clearance of Examples 1-7 sample Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 pH 12 12 14 12 12 12 12 Base type NaOH TBAOH TBAOH TBAOH TBAOH TBAOH TBAOH Temperature (℃) 10 10 15 15 25 15 15 Albumin type BSA BSA BSA HSA BSA BSA BSA Albumin concentration (mg / mL) 15 15 15 15 15 60 15 Copper sulfate concentration (mM) 20 20 20 20 20 20 1 <![CDATA[BSA: Volume ratio of CuSO4 solution]]> 1:5 1:5 1:5 1:5 1:5 1:12 1:12 Bilirubin clearance (%) 74.7 80.2 72.1 70.5 48.1 81.6 28.7 Note: TBAOH is tetrabutylammonium hydroxide, NaOH is sodium hydroxide, BSA is bovine serum albumin, and HSA is human serum albumin.

[0037] As can be seen from Table 1, although the bilirubin clearance rate of Example 2 is lower than that of Example 6, its albumin dosage is 0.25 times that of Example 6, so Example 2 is the optimal embodiment. By comparing Example 1 and Example 2, it can be seen that compared with sodium hydroxide, the use of tetrabutylammonium hydroxide to adjust the pH can improve the activity of the enzyme; the reason is that the carbon chain length of tetrabutylammonium hydroxide has a buffering effect on the combination of albumin and copper ions, which helps to disperse the copper ions on albumin. The better the dispersibility, the more metal active centers of the enzyme with the same amount of substance, and the better the catalytic effect. By comparing Example 3 and Example 2, it is found that when the pH is adjusted to 12, the activity of the enzyme is better, because the high pH value will accelerate the formation of copper ion clusters, thereby affecting the dispersibility of copper ions. By comparing Example 4 and Example 2, it is found that compared with human serum albumin, the enzyme catalytic effect obtained by using bovine serum albumin as the skeleton is better. By comparing Example 5 and Example 2, it is found that the activity of the enzyme obtained by controlling the temperature at a low temperature (below 15°C) during reaction and dialysis is higher. At the same time, by comparing the bilirubin clearance rates of each embodiment, it was found that the influence of temperature was significant, so in the preparation process, the temperature during the reaction and dialysis needs to be controlled. By comparing Example 2, Example 6 and Example 7, too high an albumin concentration will not increase the activity of the enzyme, and too low a copper sulfate concentration will significantly affect the activity of the enzyme. The amount of albumin in Example 6 is 4 times that of Example 2, but the difference in catalytic effect is not obvious, so increasing the amount of albumin (exceeding 50 mg / mL) is an unnecessary waste. In Example 7, the amount of copper sulfate is too low, resulting in a significant decrease in the catalytic effect. Copper ions are the key factor in enabling active centers to function, so the amount of copper ions must meet the needs of forming active centers.

[0038] Stability test examples of different enzymes In order to compare the stability of bilirubin oxidase and BOD, the bilirubin oxidase prepared in Example 2 and natural BOD were placed at -20°C, 4°C, and 25°C for 24 hours and then tested for their performance in catalyzing the decomposition of bilirubin in a PBS environment. The initial bilirubin clearance rates of the two enzymes were 79.69% (bilirubin oxidase) and 80.25% (BOD). The test results are shown in Table 2 below.

[0039] Table 2. Bilirubin clearance after 24 hours at different temperatures Temperature (℃) -20 4 25 Bilirubin oxidase bilirubin clearance rate (%) 79.43 79.57 76.38 BOD bilirubin clearance rate (%) 79.28 68.82 37.86 Note: Initially, the bilirubin clearance rate of bilirubin oxidase was 79.69%, and the clearance rate of BOD was 80.25%.

[0040] As shown in Table 2, the stability of the two enzymes can be reflected by comparing their clearance rates at different temperatures for 24 hours. After being placed at -20℃ for 24 hours, the effect on the two enzymes was not obvious. After being placed at 4℃ for 24 hours, the bilirubin clearance rate of bilirubin oxidase did not change much, and that of BOD decreased significantly. After being placed at 25℃ for 24 hours, the bilirubin clearance rate of bilirubin oxidase decreased from 79.69% to 76.38%, and the bilirubin clearance rate of BOD decreased from 80.25% to 37.86%. It can be seen that the stability of bilirubin oxidase is significantly better than that of BOD.

[0041] Long-term stability test of bilirubin oxidase To verify the long-term stability of the bilirubin oxidase prepared in Example 2, it was stored at 4°C and 25°C for 1, 7, 14, 28, and 90 days, and then its bilirubin clearance rate in a PBS environment was tested. The results are shown in Table 3 below.

[0042] Table 3. Bilirubin clearance by bilirubin oxidase on different days Time (days) 1 7 14 28 90 4℃ bilirubin clearance (%) 79.27 79.03 77.50 76.22 73.70 Bilirubin clearance at 25℃ (%) 76.38 73.93 68.27 63.27 53.60 As shown in Table 3, when the storage environment is 4°C, the stability of the enzyme decreases over time, but the difference is not obvious. When the storage environment is 25°C, the stability change is not obvious within 7 days, but by 90 days, the bilirubin clearance rate has decreased by 29.8%, which is a significant change. Therefore, this type of bilirubin oxidase can be exposed to room temperature in the short term, but it needs to be placed in a 4°C environment for long-term storage.

[0043] By comparing the above examples, the enzyme obtained in the preparation scheme of Example 2 was determined as the enzyme subsequently loaded on the surface of the microspheres, and used as the raw material of the following Examples 8-13.

[0044] Example 8 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, the preparation method of which comprises the following steps: Chitosan was dissolved in 3wt% acetic acid to obtain a chitosan solution with a concentration of 3wt%. In this example, polyacrylic acid was not added. Three drops of 37% hydrochloric acid solution were added with a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL NaOH (1 M) and ethanol (26vol%) mixed coagulation bath through a microinfusion needle (model 23 G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.1 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer (0.1M, pH = 7.4). Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1 mg / mL) solution for 1 h. Washed with phosphate buffer again to obtain enzyme-loaded gel microspheres, the SEM structure diagram of which is shown in the figure. Figure 1 shown.

[0045] Example 9 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, the preparation method of which comprises the following steps: Chitosan was dissolved in 3wt% acetic acid to obtain a chitosan solution with a concentration of 3wt%, which was then mixed with a 3wt% polyacrylic acid aqueous solution (the mass ratio of chitosan to polyacrylic acid was 1:1 based on dry weight). Three drops of 37% hydrochloric acid solution were added with a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL NaOH (1 M) and ethanol (26vol%) mixed coagulation bath through a microinfusion needle (model 23 G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.1 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer. Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1 mg / mL) solution for 1 h. They were then washed with phosphate buffer to obtain enzyme-loaded gel microspheres.

[0046] Embodiment 10 This embodiment is a preferred embodiment of the present invention: An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, wherein the preparation method comprises the following steps: Chitosan was dissolved in 3wt% acetic acid to obtain a chitosan solution with a concentration of 3wt%, which was then mixed with a 3wt% polyacrylic acid aqueous solution (the mass ratio of chitosan to polyacrylic acid was 15:1 on a dry weight basis). Three drops of 37% hydrochloric acid solution were added with a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL NaOH (1 M) and ethanol (26vol%) mixed coagulation bath through a microinfusion needle (model 23 G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.1 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer. Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1mg / mL) solution for 1 h. They were then washed with phosphate buffer to obtain enzyme-loaded gel microspheres, the SEM structure of which is shown in the figure. Figure 2 Compare Figure 1 and Figure 2 The surface structure of the microspheres shows that Figure 1 The pore structure of the microspheres is unevenly distributed and the size is inconsistent. Figure 2 The pore size is moderate and evenly distributed. The uniform porous structure and appropriate pore size can increase the specific surface area, increase the enzyme loading rate, optimize the mass transfer efficiency, and improve the stability of the microspheres.

[0047] Example 11 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, wherein the preparation method comprises the following steps: Chitosan was dissolved in 3wt% acetic acid to obtain a chitosan solution with a concentration of 3wt%, which was then mixed with a 3wt% polyacrylic acid aqueous solution (the mass ratio of chitosan to polyacrylic acid was 15:1 on a dry weight basis). Five drops of 37% hydrochloric acid solution were added with a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL NaOH (2 M) and ethanol (40vol%) mixed coagulation bath through a microinfusion needle (model 23 G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.1 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer. Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1 mg / mL) solution for 1 h. They were then washed with phosphate buffer to obtain enzyme-loaded gel microspheres.

[0048] Example 12 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, the preparation method of which comprises the following steps: Chitosan was dissolved in 3% acetic acid to obtain a chitosan solution with a concentration of 3 wt%. The obtained chitosan solution was mixed with a 3 wt% polyacrylic acid aqueous solution (the mass ratio of chitosan to polyacrylic acid was 15:1 on a dry weight basis). Five drops of 37% hydrochloric acid solution were added with a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL L NaOH (1 M) and ethanol (26 vol%) mixed coagulation bath through a microinfusion needle (model 23 G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.025 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer. Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1 mg / mL) solution for 1 h. They were then washed with phosphate buffer to obtain enzyme-loaded gel microspheres.

[0049] Example 13 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, the preparation method of which comprises the following steps: Chitosan was dissolved in 3% acetic acid to obtain a solution with a concentration of 3 wt%, which was then mixed with a 3 wt% polyacrylic acid aqueous solution (the mass ratio of chitosan to polyacrylic acid was 15:1 on a dry weight basis). Five drops of 37% hydrochloric acid solution were added using a 1 mL rubber-tipped dropper to convert the polymer mixture into a clear and uniform solution as a drop ball solution. The drop ball solution was dripped into a 500 mL NaOH (1 M) and ethanol (26 vol%) mixed coagulation bath through a microinfusion needle (model 23G) to prepare microspheres. After curing in the coagulation bath for 2 h, the carrier microspheres were rinsed with RO water. 10 g of wet carrier microspheres and 0.1 g of EDC were weighed and mixed in 20 mL of aqueous solution and stirred for 15 min. The activated microspheres were washed with phosphate buffer. Then, the microspheres were mixed with 20 mL of BOD (1 mg / mL) solution for 1 h. They were then washed with phosphate buffer to obtain enzyme-loaded gel microspheres.

[0050] Performance Test Example 2: Decomposition Ability of Microspheres of Example 10 on Bilirubin in Phosphate Buffer Solution The catalytic decomposition ability of Example 10 for bilirubin (dissolved in phosphate buffer) was determined by weighing 0.2 g of the wet gel microspheres prepared in Example 10 and placing them in 3 mL of bilirubin phosphate buffer (200 mg / L), and scanning the bilirubin solution spectrum in the wavelength range of 250-700 nm with an ultraviolet spectrophotometer at 0, 30, 60, 120, and 180 min, respectively. The change in the peak intensity of the characteristic peak of bilirubin and the appearance of a new characteristic peak proved that bilirubin was catalytically decomposed.

[0051] Depend on Figure 4 It can be seen that at 0 min, the solution contained pure bilirubin, with an obvious peak at 438 nm. As time went on, the peak at 438 nm weakened significantly, and new peaks were generated at 600 nm and 655 nm. This peak is the characteristic peak of biliverdin. This proves that bilirubin is converted into biliverdin under catalysis, and the peak intensity is significantly weakened, which proves that the concentration of bilirubin and biliverdin in the solution is weakened and adsorbed by the microspheres. Therefore, the enzyme-loaded gel microspheres can decompose and adsorb bilirubin and its decomposition products.

[0052] Performance Test Example 3: Decomposition and adsorption capacity of microspheres on bilirubin in plasma The decomposition and adsorption capacity of each embodiment for bilirubin in plasma was measured respectively. The specific method was as follows: 0.2 g of the wet gel microspheres prepared in each embodiment was weighed and placed in 3 mL of plasma of a hyperbilirubinemia rabbit. The plasma without gel microspheres was used as a blank control. After incubation at 37°C for 3 h, the concentration of bilirubin in plasma was detected using a total bilirubin kit and a direct bilirubin kit and the clearance rate was calculated.

[0053] Table 4. Bilirubin clearance of microspheres in various examples sample Example 8 Example 9 Example 10 Embodiment 11 Example 12 Example 13 Cs:PAA 1:0 1:1 15:1 15:1 15:1 15:1 NaOH concentration (M) 1 1 1 2 1 1 Ethanol concentration (vol%) 26 26 26 40 26 26 EDC: Microspheres (mass ratio) 1:100 1:100 1:100 1:100 1:400 1:100 Enzyme type Bilirubin oxidase Bilirubin oxidase Bilirubin oxidase Bilirubin oxidase Bilirubin oxidase BOD Direct bile clearance rate (%) 32.5 26.6 47.2 30.1 27.5 14.8 Total bile clearance rate (%) 44.7 38.9 56.6 42.8 38.1 22.3 Note: The total bilirubin concentration in the blank control group decreased from 108.2 μmol / L to 101.7 μmol / L; the direct bilirubin concentration decreased from 98.4 μmol / L to 92.2 μmol / L.

[0054] As shown in Table 4, the maximum clearance rate of the microspheres in each embodiment for total bilirubin in plasma is 56.6% (Example 10), and the maximum adsorption amount of direct bilirubin in plasma is 47.2%. In the enzyme fixation link, the enzyme dosage of each embodiment is consistent, so it can be inferred from the clearance rate that the enzyme fixation amount in Example 10 is the largest, and the synergistic effect of catalysis and adsorption is the best. These results also show that the enzyme-loaded gel microspheres can effectively remove bilirubin.

[0055] In addition, 0.2 g of the enzyme-loaded gel microspheres prepared in Example 10 were weighed and incubated with 3 mL of high bilirubin plasma for 3 h. Figure 3 As shown, Figure 3 The left picture shows that before the experiment, the total plasma bilirubin concentration was 108.2 μmol / L; the right picture shows that after the experiment, the total plasma bilirubin concentration was 52.4 μmol / L.

[0056] In Example 8 and Example 9, the ratio of chitosan to polyacrylic acid in the microspheres was changed. Compared with Example 10, the bilirubin removal effect was reduced. It can be seen that the amount of polyacrylic acid mixed in will affect the enzyme loading rate. No polyacrylic acid or too much polyacrylic acid mixed in is not the best choice. In Example 11, the concentration of the coagulation bath was increased, resulting in an accelerated solvent replacement rate. Compared with Example 10, the surface of the microspheres was denser, the specific surface area was reduced, and the enzyme grafting rate was reduced. In Example 12, the amount of EDC was 0.25 times that of Example 10, and the catalytic-adsorption effect was reduced. It can be seen that in the process of activating the surface of the microspheres, the amount of EDC cannot be too low, otherwise the enzyme grafting rate will be reduced. Example 13 has the lowest bilirubin removal efficiency among all the examples. It can be seen that this enzyme loading scheme is not suitable for natural enzyme BOD.

[0057] Performance Test Example 4: Determination of Mechanical Properties of Microspheres A universal testing machine is used. Based on the basic principle of mechanical property testing, the relationship between stress and strain is obtained by applying external force and measuring the deformation behavior of the material. The specific method is: measure the diameter of the microsphere, place the microsphere sample on the lower pressure plate of the testing machine, and ensure that it is directly below the pressure head. Start the test, apply compression force to the microsphere at a constant rate, and stop the test when the microsphere breaks or reaches a preset deformation amount. (During the blood perfusion process, the maximum pressure on the microsphere is 66.7 kPa, so the following Table 5 shows the strain corresponding to the stress of the enzyme-loaded gel microspheres prepared in different embodiments at 66.7 kPa).

[0058] Table 5. Strain of microspheres in various embodiments (stress is 66.7 kPa) sample Example 8 Example 9 Example 10 Embodiment 11 Example 12 Example 13 Cs:PAA 1:0 1:1 15:1 15:1 15:1 15:1 NaOH concentration (M) 1 1 1 2 1 1 Ethanol concentration (vol%) 26 26 26 40 26 26 strain(%) 49.3 24.8 21.6 45.7 22.1 22.5 Note: In the table, Cs is chitosan and PAA is polyacrylic acid.

[0059] As shown in Table 5, the mechanical properties of pure chitosan microspheres are relatively weak (such as Example 8), and their mechanical properties can be significantly improved by blending polyacrylic acid. The hydroxyl (-OH) and amino (-NH2) of chitosan can form hydrogen bonds with the carboxylic acid group (-COOH) of polyacrylic acid, enhance the binding force between molecular chains, and improve the toughness of the material. When the blending amount is too much, such as Example 9, the deformation amount is larger than that of Example 10. It can be seen from Example 11 that when the concentration of sodium hydroxide and ethanol in the coagulation bath is too high, the mechanical properties of the microspheres are significantly weakened. The reason is that when the concentration is too high, the solvent replacement speed is too fast, the molecular chains are unevenly entangled (dense outside and sparse inside), and the mechanical strength is reduced. The results show that blending polyacrylic acid in chitosan can improve the compressive properties of the microspheres, but attention should be paid to its dosage when mixing polyacrylic acid. During the solvent replacement process, the concentrations of sodium hydroxide and ethanol also need to be controlled within a certain range.

[0060] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A bilirubin oxidase, characterized in that The bilirubin oxidase is prepared by the following method: (a) mixing a 5-30 mM copper sulfate aqueous solution with a 10-50 mg / mL albumin phosphate buffer solution, stirring for 1-10 min to obtain a mixed solution; (b) adjusting the pH of the mixed solution obtained in step (a) to 10-14 with tetrabutylammonium hydroxide or sodium hydroxide solution; reacting at a temperature below 15° C. for 2-8 hours, dialyzing the mixed solution and freeze-drying it, wherein the dialysis temperature is below 15° C.; and obtaining bilirubin oxidase.

2. The bilirubin oxidase according to claim 1, characterized in that In step (a), the volume ratio of the copper sulfate aqueous solution to the albumin phosphate buffer is 1:4 to 1:

10.

3. The bilirubin oxidase according to claim 1, characterized in that In step (a), the albumin is human serum albumin or bovine serum albumin.

4. An enzyme-loaded gel microsphere prepared using the bilirubin oxidase according to claim 1, 2 or 3, characterized in that: The enzyme-loaded gel microspheres are prepared by using chitosan and polyacrylic acid as carriers and loading the bilirubin-like oxidase.

5. The method for preparing enzyme-loaded gel microspheres according to claim 4, characterized in that: The steps include: (1) dissolving chitosan in an acetic acid solution to obtain a chitosan solution with a concentration of 2 to 4 wt%, and then adding a polyacrylic acid aqueous solution with a concentration of 0.1 to 3 wt% to obtain a white polymer gel, wherein the mass ratio of chitosan by dry weight to polyacrylic acid by dry weight is 1 to 15; then dropping an acid solution to convert the white polymer gel into a clear and uniform solution; (2) the solution obtained in step (1) is dripped into a mixed solution of 0.8-2 M sodium hydroxide and 20-40 vol% ethanol through a microperfusion needle connected to an electrostatic generator under the action of an electrostatic voltage of 5 kv-8 kv to obtain microspheres, the microspheres are stirred at room temperature to solidify in the solidification solution for 1-5 h, and after solidification, they are rinsed with deionized water to obtain chitosan-polyacrylic acid carrier microspheres; (3) The carrier microspheres obtained in step (2) are placed in a 0.1-1 wt% EDC solution and stirred; then, after being washed with a phosphate buffer solution, the bilirubin oxidase is dissolved in the phosphate buffer solution, and the chitosan-polyacrylic acid carrier microspheres are mixed into the solution to fix the enzyme, and the reaction is performed for 1-5 hours to obtain enzyme-loaded gel microspheres.

6. The preparation method according to claim 5, characterized in that: In step (1), the acid solution is a hydrochloric acid solution.

7. The preparation method according to claim 5, characterized in that: In step (2), the diameter of the chitosan-polyacrylic acid carrier microspheres is 500-800 μm.

8. The preparation method according to claim 5, characterized in that: In step (3), the mass ratio of EDC to chitosan-polyacrylic acid carrier microspheres is 1:40 to 1:400 on a dry weight basis.

9. Use of the enzyme-loaded gel microspheres prepared by the preparation method of claim 5 in preparing medical devices for treating or improving hyperbilirubinemia.

10. The use according to claim 9, characterized in that: The medical device is a hemoperfusion device.

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