A kind of bilirubin oxidase, enzyme-loaded gel microspheres, a preparation method thereof and an application thereof

Abstract

The present invention discloses a bilirubin oxidase-like enzyme, an enzyme-loaded gel microsphere, a preparation method thereof and an application thereof, belonging to the technical fields of medicine and enzyme technology. The bilirubin oxidase-like enzyme is obtained by coordinating copper ions with albumin. The preparation method of the enzyme-loaded gel microsphere is to first prepare carrier microspheres from a chitosan-polyacrylic acid solution through a solvent replacement method, and then bind the bilirubin oxidase-like enzyme to the surface of the carrier microspheres through an enzyme immobilization method to obtain chitosan-polyacrylic acid microspheres loaded with the enzyme, namely the enzyme-loaded gel microspheres. The enzyme-loaded gel microspheres of the present invention can improve the adsorption capacity of a bilirubin adsorbent in a form of decomposing while adsorbing. When the enzyme-loaded gel microspheres of the present invention are used for bilirubin decomposition and adsorption, they have the advantages of selective adsorption, low cost, high stability, high adsorption efficiency, etc.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and enzyme technology, and particularly to a bilirubin oxidase-like enzyme, an enzyme-loaded gel microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Liver failure is a group of clinical syndromes characterized by coagulation mechanism disorders, 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 and is also one of the indicators for liver function detection. Bilirubin is a catabolic product of heme metabolism, mainly from senescent red blood cells. Heme is oxidized by heme oxygenase to form biliverdin. Biliverdin is further reduced by biliverdin reductase to bilirubin. 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 glucuronide-conjugated bilirubin by a specific glucuronosyltransferase UGT1A1. Within the physiological range, bilirubin has anti-inflammatory, antioxidant, and beneficial metabolic effects, but at high concentrations, it has potential toxicity. Generally, 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 injury, etc.), it will cause hyperbilirubinemia.

[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 has been 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 emerging adsorbents have been developed to remove bilirubin, but few have been used clinically. The main reasons are that the preparation methods of these existing adsorbents are complex, the blood compatibility is poor, and the high cost may be the key problems hindering their clinical transformation. Improving the adsorption capacity and selectivity of bilirubin adsorption materials is the research goal of the majority of hemofiltration scholars. Summary of the Invention

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

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A bilirubin oxidase-like enzyme, which is prepared by the following method:

[0007] (a) Mix an aqueous copper sulfate solution with a concentration of 5-30 mM and an albumin phosphate buffer solution with a concentration of 10-50 mg / mL, and stir for 1-10 min to obtain a mixed solution;

[0008] (b) Adjust the pH of the mixed solution obtained in step (a) to 10 - 14 with tetrabutylammonium hydroxide or sodium hydroxide solution; after reacting for 2 - 8 h under the condition of lower than 15 °C, dialyze and freeze-dry the mixed solution (the temperature during the dialysis process needs to be lower than 15 °C) to obtain bilirubin oxidase-like enzyme.

[0009] As a preferred technical solution, in step (a), the volume ratio of the aqueous copper sulfate solution to the albumin phosphate buffer solution added is 1:4 - 1:10.

[0010] As a preferred technical solution, in step (a), the albumin is human serum albumin or bovine serum albumin.

[0011] 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. Because the inventor has proved through a large number of experiments that: too high albumin concentration or too low copper sulfate concentration will lead to a significant decrease in the binding rate of copper ions in albumin, thus weakening the catalytic effect.

[0012] The second object of the present invention is to provide an enzyme-loaded gel microsphere prepared by using the above-mentioned bilirubin oxidase-like enzyme. The enzyme-loaded gel microsphere is prepared by using chitosan and polyacrylic acid as carriers and loading the bilirubin oxidase-like enzyme.

[0013] The third object of the present invention is to provide a preparation method of the above-mentioned enzyme-loaded gel microsphere, including the following steps:

[0014] (1) Dissolve an aqueous chitosan solution with a concentration of 2 - 4 wt% in an acetic acid solution, and then add an aqueous polyacrylic acid solution with a concentration of 0.1 - 3 wt% to obtain a white polymer gel, where the mass ratio of chitosan (dry weight) to polyacrylic acid (dry weight) is 1 - 15; then dropwise add an acid solution to convert the white polymer gel into a clear and uniform solution;

[0015] (2) Pass the solution obtained in step (1) through a micro-infusion needle connected to an electrostatic generator, and drop it into a mixed solution of 0.8 - 2 M sodium hydroxide and 20 - 40 vol% ethanol under the action of an electrostatic voltage of 5 kv - 8 kv to obtain microspheres. Stir at room temperature to make the microspheres solidify in the coagulating liquid for 1 - 5 h, and rinse with deionized water after solidification to obtain chitosan-polyacrylic acid carrier microspheres;

[0016] (3) Put the carrier microspheres obtained in step (2) into a 0.1 - 1 wt% EDC (i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution) solution and stir; then wash with phosphate buffer. After that, dissolve the bilirubin oxidase-like enzyme in phosphate buffer, and then mix the chitosan-polyacrylic acid carrier microspheres into the solution for enzyme immobilization, and react for 1 - 5 h to obtain enzyme-loaded gel microspheres.

[0017] As a preferred technical solution, in step (1), the acid solution is hydrochloric acid solution.

[0018] As a preferred technical solution, in step (2), the diameter of the chitosan-polyacrylic acid carrier microspheres is 500 - 800 μm.

[0019] Too small microsphere size (less than 500 μm) will cause the microspheres to pass through the filter and enter the blood circulation, leading to safety problems such as embolism; while too large size (such as exceeding 800 μm) will reduce the specific surface area and lower the adsorption efficiency. In the present invention, preferably, by controlling the voltage during the droplet formation process, the greater the voltage, the smaller the microsphere particle size, and the smaller the voltage, the larger the microsphere particle size. Therefore, by controlling the voltage within 5 kv - 8 kv, the microsphere size is controlled within the range of 500 - 800 μm, so as to balance the fluidity and pressure drop and ensure smooth blood flow during blood perfusion.

[0020] As a preferred technical solution, in step (3), the mass ratio of the dosage of EDC (dry weight) to the mass of the chitosan-polyacrylic acid carrier microspheres is 1:40 - 1:400.

[0021] Too much EDC dosage will cause it to remain on the surface of the microspheres and occupy the enzyme loading sites; too low dosage will cause the chemical groups on the surface of the microspheres not to be fully activated, reducing the enzyme loading rate. Therefore, the dosage of EDC should be within a suitable range.

[0022] The fourth object of the present invention is to provide an application of the enzyme-loaded gel microspheres prepared by the above preparation method in the preparation of medical devices for treating or improving hyperbilirubinemia.

[0023] As a preferred technical solution, the medical device is a hemoperfusion cartridge.

[0024] Bilirubin oxidase (BOD) can effectively decompose bilirubin. The principle is that BOD, with the assistance of oxygen, catalyzes bilirubin into biliverdin, and further decomposes it into derivatives of weak diazo reaction and highly polar diazo by-products. These small molecular products are soluble in water and can be excreted from the body with urine, thus achieving the clearance of bilirubin. Currently, BOD is generally prepared by biological methods, which is difficult and expensive. Based on this principle, the inventors of this application, through theoretical analysis and a large number of experiments, attempted to prepare the above-mentioned artificial enzyme that mimics bilirubin oxidase, and loaded it on microspheres that can adsorb bilirubin and its decomposition products for hemoperfusion.

[0025] The principle of the bilirubin oxidase-like enzyme of the present invention is as follows: in a metal ion solution, the metal ions interact with biomolecules, which can significantly improve the stability of proteins; the present invention uses a copper ion solution as the reaction medium, and the structure of albumin changes in the copper ion solution, releasing a large number of amino acid residues and self-assembling with copper ions, thereby generating a soluble bilirubin oxidase-like enzyme.

[0026] Compared with the prior art, the advantages of the present invention are as follows:

[0027] (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 has low cost. Specifically: in the present invention, the natural enzyme (BOD) mimicked by the bilirubin oxidase-like enzyme. To achieve the same catalytic effect as BOD, the dosage of the bilirubin oxidase-like enzyme of the present invention is 100 times that of BOD. However, considering factors such as yield, preparation difficulty, and preparation cost, for example, the production cost of 1 g of BOD is about 50,000 - 100,000 yuan, and the cost required to produce 1 g of bilirubin oxidase-like enzyme is about 200 yuan. Therefore, the cost of BOD is 2.5 - 5 times that of the bilirubin oxidase-like enzyme. Therefore, only considering from the cost aspect, the bilirubin oxidase-like enzyme is a better choice.

[0028] (2) High stability: The artificial enzyme of the present invention is a nanomaterial that can mimic the catalytic activity of natural enzymes. Compared with natural enzymes, the artificial enzyme has higher stability and the possibility of large-scale preparation. The comparison of its stability is shown in the following comparative examples. To retain its catalytic activity, BOD needs to be stored in an environment of -20 °C, while the bilirubin oxidase-like enzyme can be stored in a 4 °C refrigerator. The strict storage requirements of BOD make it not have the advantage of large-scale production.

[0029] (3)Selective adsorption: The enzyme-loaded gel microspheres of the present invention can greatly improve the adsorption capacity of bilirubin adsorbents in the form of catalysis and adsorption simultaneously. This strategy reduces the impact of the material on albumin. Using bilirubin oxidase-like enzyme to first catalyze bilirubin into small molecule polar compounds such as biliverdin, and after the decomposition products are dissociated from albumin, then adsorb the decomposition products, so as to achieve the goal of only removing toxins and not adsorbing albumin, and improve the clearance rate and specificity of the adsorbent for bilirubin.

[0030] (4)High adsorption efficiency: The gel microsphere substrate 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, thus realizing the adsorption of bilirubin and its decomposition products. At the same time, the surface of the chitosan microsphere is a porous structure, which increases the specific surface area of the microsphere in contact with blood, is conducive to liquid-mass transfer, and further improves the adsorption capacity of the adsorbent. However, the mechanical strength of pure chitosan-based microspheres is poor, and adding an appropriate amount of polyacrylic acid can improve the mechanical properties of the chitosan substrate. Loading the bilirubin oxidase-like enzyme in the present invention onto the gel microspheres by chemical grafting method can not only avoid the shedding of the bilirubin oxidase-like enzyme, but also continuously play a catalytic effect. The catalyzed bilirubin is decomposed into small molecules 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, thus realizing the clearance of bilirubin. The method of the present invention can reduce the treatment time or frequency and save the medical expenses of patients. Description of the Drawings

[0031] Figure 1 SEM structural schematic diagram of the enzyme-loaded gel microspheres prepared in Example 8;

[0032] Figure 2 SEM structural schematic diagram of the enzyme-loaded gel microspheres prepared in Example 10;

[0033] Figure 3 Comparison photos of the enzyme-loaded gel microspheres in Example 10 before and after bilirubin adsorption in a plasma environment;

[0034] Figure 4 Ultraviolet absorption spectra of bilirubin solution after contacting the enzyme-loaded microspheres in Example 10 for different times. Detailed Embodiments

[0035] The following embodiments are provided to better further understand the present invention, which is not limited to the best embodiment, and does not 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 art features falls within the protection scope of the present invention.

[0036] For those examples where specific experimental procedures or conditions are not specified, the operations or conditions of the conventional experimental procedures described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not specified, they are all conventional reagent products that can be obtained through commercial purchase.

[0037] The following Examples 1-7 are examples of bilirubin oxidase, and Examples 8-13 are examples of enzyme-loaded microspheres.

[0038] Example 1 A bilirubin oxidase capable of decomposing bilirubin, and its preparation method includes the following steps:

[0039] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:5 with 5.0 mL of bovine serum albumin solution (15 mg / mL), stir for 5 min, and adjust the pH to 12 with 1.0 M sodium hydroxide solution; after reacting at 10 °C for 5 h, dialyze the mixed solution at 10 °C and freeze-dry to obtain bilirubin oxidase.

[0040] Example 2 This example is a relatively preferred example of the present invention:

[0041] A bilirubin oxidase capable of decomposing bilirubin, and its preparation method includes the following steps:

[0042] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:5 with 5.0 mL of bovine serum albumin solution (15 mg / mL), stir for 5 min, and adjust the pH to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 10 °C for 5 h, dialyze the mixed solution at 10 °C and freeze-dry to obtain bilirubin oxidase.

[0043] Example 3 A bilirubin oxidase capable of decomposing bilirubin, and its preparation method includes the following steps:

[0044] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:5 with 5.0 mL of bovine serum albumin solution (15 mg / mL), stir for 5 min, and adjust the pH to 14 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15 °C for 5 h, dialyze the mixed solution at 15 °C and freeze-dry to obtain bilirubin oxidase.

[0045] Example 4 A bilirubin oxidase capable of decomposing bilirubin, and its preparation method includes the following steps:

[0046] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:5 and 5.0 mL of human serum albumin solution (15 mg / mL) and stir for 5 min. Adjust the pH to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15 °C for 5 h, dialyze the mixed solution at 15 °C and freeze-dry to obtain biliverdin oxidase-like enzyme.

[0047] Example 5 A biliverdin oxidase-like enzyme capable of decomposing bilirubin, and its preparation method includes the following steps:

[0048] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:5 and 5.0 mL of bovine serum albumin solution (15 mg / mL) and stir for 5 min. Adjust the pH to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 25 °C for 5 h, dialyze the mixed solution at 25 °C and freeze-dry to obtain biliverdin oxidase-like enzyme.

[0049] Example 6 A biliverdin oxidase-like enzyme capable of decomposing bilirubin, and its preparation method includes the following steps:

[0050] Mix 1.0 mL of copper sulfate solution (20 mM) with a volume ratio of 1:12 and 12.0 mL of bovine serum albumin solution (60 mg / mL) and stir for 5 min. Adjust the pH to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15 °C for 5 h, dialyze the mixed solution at 15 °C and freeze-dry to obtain biliverdin oxidase-like enzyme.

[0051] Example 7 A biliverdin oxidase-like enzyme capable of decomposing bilirubin, and its preparation method includes the following steps:

[0052] Mix 1.0 mL of copper sulfate solution (1 mM) with a volume ratio of 1:12 and 12.0 mL of bovine serum albumin solution (15 mg / mL) and stir for 5 min. Adjust the pH to 12 with 1.0 M tetrabutylammonium hydroxide solution; after reacting at 15 °C for 5 h, dialyze the mixed solution at 15 °C and freeze-dry to obtain biliverdin oxidase-like enzyme.

[0053] Performance test example 1: Measure the ability of biliverdin oxidase-like enzyme to decompose bilirubin in phosphate buffer solution

[0054] Determine the catalytic decomposition ability of Examples 1-7 on bilirubin (dissolved in phosphate buffer solution). The specific method is as follows: Weigh 1 mg of biliverdin oxidase-like enzyme and place it in 3 mL of bilirubin phosphate buffer solution (200 mg / L). At 180 min, take 100 μL of the supernatant and place it in a 96-well plate, and scan the absorbance at a wavelength of 438 nm with an enzyme-labeled instrument to calculate its bilirubin clearance rate.

[0055] Table 1. Preparation conditions and bilirubin clearance rates of Examples 1 - 7

[0056] 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 (°C) 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 rate (%) 74.7 80.2 72.1 70.5 48.1 81.6 28.7

[0057] Note: Among them, TBAOH is tetrabutylammonium hydroxide, NaOH is sodium hydroxide, BSA is bovine serum albumin, and HAS is human serum albumin.

[0058] As can be seen from Table 1, although the bilirubin clearance rate of Example 2 is lower than that of Example 6, the albumin dosage of Example 2 is 0.25 times that of Example 6. Therefore, Example 2 is the optimal example. By comparing Example 1 and Example 2, it can be seen that compared with sodium hydroxide, using tetrabutylammonium hydroxide to adjust the pH can improve the enzyme activity. The reason is that the carbon chain of tetrabutylammonium hydroxide is long, which has a buffering effect on the binding of albumin and copper ions. This effect helps the dispersion of copper ions on albumin. The better the dispersion, 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 enzyme activity is better. The reason is that a high pH value will accelerate the formation of copper ion clusters, thus affecting the dispersion of copper ions. By comparing Example 4 and Example 2, it is found that compared with human serum albumin, using bovine serum albumin as the backbone gives better enzyme catalytic effect. By comparing Example 5 and Example 2, it is found that the enzyme obtained by controlling the temperature at a low temperature (below 15 °C) during the reaction and dialysis has higher activity. At the same time, by comparing the bilirubin clearance rates of each example, it is found that the influence of temperature is significant. Therefore, during the preparation process, the temperature during the reaction and dialysis needs to be well controlled. By comparing Example 2, Example 6, and Example 7, it is found that too high albumin concentration will not improve the enzyme activity, and too low copper sulfate concentration will significantly affect the enzyme activity. The albumin dosage in Example 6 is 4 times that of Example 2, but the catalytic effect difference is not obvious. Therefore, increasing the albumin dosage (exceeding 50 mg / mL) is an unnecessary waste. The copper sulfate dosage in Example 7 is too low, resulting in a significant decrease in the catalytic effect. Copper ions are the key factors for the active center to play a role. Therefore, the dosage of copper ions needs to meet the requirements for the formation of the active center.

[0059] Examples of stability tests of different enzymes

[0060] To compare the stabilities of bilirubin oxidase-like and BOD, the bilirubin oxidase-like prepared in Example 2 and natural BOD were placed at -20 °C, 4 °C, and 25 °C for 24 h, and then their performances in catalyzing the decomposition of bilirubin in a PBS environment were tested. The initial bilirubin clearance rates of the two enzymes were 79.69% (bilirubin oxidase-like) and 80.25% (BOD). The test results are shown in Table 2 below.

[0061] Table 2. Bilirubin clearance rates after being placed at each temperature for 24 h

[0062] Temperature (°C) -20 4 25 Bilirubin oxidase-like bilirubin clearance rate (%) 79.43 79.57 76.38 BOD bilirubin clearance rate (%) 79.28 68.82 37.86

[0063] Note: Initially, the bilirubin clearance rate of bilirubin oxidase was 79.69%, and the clearance rate of BOD was 80.25%.

[0064] As can be seen from Table 2, the stability of the two enzymes was reflected by comparing their clearance rates after being placed at different temperatures for 24 h. After being placed at -20°C for 24 h, the effect on the two enzymes was not obvious. After being placed at 4°C for 24 h, the bilirubin clearance rate of bilirubin oxidase changed little, while that of BOD decreased significantly. After being placed at 25°C for 24 h, 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.

[0065] Long-term stability test of bilirubin oxidase

[0066] 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 respectively, and then its bilirubin clearance rate in PBS environment was tested. The results are shown in Table 3 below.

[0067] Table 3. Bilirubin clearance rate of bilirubin oxidase at different days

[0068] Time (days) 1 7 14 28 90 Bilirubin clearance rate at 4°C (%) 79.27 79.03 77.50 76.22 73.70 Bilirubin clearance rate at 25°C (%) 76.38 73.93 68.27 63.27 53.60

[0069] As can be seen from Table 3, when the storage environment was 4°C, the stability of the enzyme decreased with time, but the difference was not obvious. When the storage environment was 25°C, the change in stability was not obvious within 7 days, but by 90 days, the bilirubin clearance rate decreased by 29.8%, and the change was obvious. Therefore, this bilirubin oxidase can be exposed to room temperature in the short term, but it needs to be stored at 4°C for long-term storage.

[0070] By comparing the above-mentioned examples, the enzyme obtained by the preparation scheme of Example 2 was determined as the enzyme to be subsequently loaded on the surface of the microspheres and used as the raw material for the following Examples 8-13.

[0071] Example 8 A carrier enzyme gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method includes the following steps:

[0072] Chitosan was dissolved in 3 wt% acetic acid to obtain a chitosan solution with a concentration of 3 wt%. In this example, polyacrylic acid was not added. 3 drops of 37% hydrochloric acid solution were added dropwise using a 1 mL pipette to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a 500 mL NaOH (1 M) and ethanol (26 vol%) mixed coagulation bath through a micro-infusion 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 (0.1 M, pH = 7.4). Then, the microspheres were mixed with 20 mL of bilirubin oxidase (1 mg / mL) solution for 1 h. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained. The SEM structural schematic diagram thereof is as shown in Figure 1 shown.

[0073] Example 9. An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method comprises the following steps:

[0074] Chitosan was dissolved in 3 wt% acetic acid to obtain a chitosan solution with a concentration of 3 wt%, and then mixed with 3 wt% aqueous polyacrylic acid solution (by dry weight, the mass ratio of chitosan to polyacrylic acid was 1:1). 3 drops of 37% hydrochloric acid solution were added dropwise using a 1 mL pipette to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a 500 mL NaOH (1 M) and ethanol (26 vol%) mixed coagulation bath through a micro-infusion 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. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained.

[0075] Example 10. This example is a preferred example of the present invention:

[0076] An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method comprises the following steps:

[0077] Chitosan was dissolved in 3 wt% acetic acid to obtain a chitosan solution with a concentration of 3 wt%. Then it was mixed with an aqueous solution of 3 wt% polyacrylic acid (by dry weight, the mass ratio of chitosan: polyacrylic acid was 15:1). 3 drops of 37% hydrochloric acid solution were added dropwise with a 1 mL dropper to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a coagulation bath of 500 mL NaOH (1 M) and ethanol (26 vol%) through a micro-infusion 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. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained, and the SEM structural schematic diagram is as Figure 2 shown. Comparing Figure 1 with Figure 2 the surface structures of the microspheres, it can be seen that Figure 1 in [reference] the pore structure distribution on the surface of the microspheres is uneven and the sizes are inconsistent, while Figure 2 the pore size of [reference] is moderate and the distribution is uniform. 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.

[0078] Example 11 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method includes the following steps:

[0079] Chitosan was dissolved in 3 wt% acetic acid to obtain a chitosan solution with a concentration of 3 wt%. Then it was mixed with an aqueous solution of 3 wt% polyacrylic acid (by dry weight, the mass ratio of chitosan: polyacrylic acid was 15:1). 5 drops of 37% hydrochloric acid solution were added dropwise with a 1 mL dropper to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a coagulation bath of 500 mL NaOH (2 M) and ethanol (40 vol%) through a micro-infusion 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. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained.

[0080] Example 12 An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method includes the following steps:

[0081] 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 an aqueous solution of 3 wt% polyacrylic acid (by dry weight, the mass ratio of chitosan: polyacrylic acid was 15:1). 5 drops of 37% hydrochloric acid solution were added dropwise with a 1 mL dropper to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a coagulation bath of 500 mL NaOH (1 M) and ethanol (26 vol%) through a microperfusion 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. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained.

[0082] Example 13. An enzyme-loaded gel microsphere capable of decomposing and adsorbing bilirubin and its decomposition products, and its preparation method includes the following steps:

[0083] Chitosan was dissolved in 3% acetic acid to obtain a solution with a concentration of 3 wt%, and then mixed with an aqueous solution of 3 wt% polyacrylic acid (by dry weight, the mass ratio of chitosan: polyacrylic acid was 15:1). 5 drops of 37% hydrochloric acid solution were added dropwise with a 1 mL dropper to convert the polymer mixture into a clear and uniform solution as the droplet solution. The droplet solution was dropped into a coagulation bath of 500 mL NaOH (1 M) and ethanol (26 vol%) through a microperfusion 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. After washing with phosphate buffer again, enzyme-loaded gel microspheres were obtained.

[0084] Performance test example 2: Decomposition ability of the microspheres of Example 10 for bilirubin in phosphate buffer solution

[0085] The catalytic decomposition ability of Example 10 for bilirubin (dissolved in phosphate buffer solution) was measured. The specific method was as follows: 0.2 g of the wet gel microspheres prepared in Example 10 was weighed and placed in 3 mL of bilirubin phosphate buffer solution (200 mg / L). The spectrum of the bilirubin solution in the wavelength range of 250 - 700 nm was scanned with a UV spectrophotometer at 0, 30, 60, 120, and 180 min respectively. The catalytic decomposition of bilirubin was proved by the change in the peak intensity of the bilirubin characteristic peak and the appearance of a new characteristic peak.

[0086] It can be seen from Figure 4 that at 0 min, the solution contains pure bilirubin, with an obvious peak at 438 nm. As time progresses, the peak at 438 nm significantly weakens, and new peaks appear at 600 nm and 655 nm. This peak is the characteristic peak of biliverdin. It is proved that bilirubin is transformed into biliverdin under the catalytic action. At the same time, the peak intensity significantly weakens, indicating that the concentrations of bilirubin and biliverdin contained in the solution decrease and are adsorbed by the microspheres. Therefore, the enzyme-loaded gel microspheres can decompose and adsorb bilirubin and its decomposition products.

[0087] Performance Test Example 3: Decomposition and Adsorption Ability of Microspheres for Bilirubin in Plasma

[0088] The decomposition and adsorption abilities of each example for bilirubin in plasma were measured respectively. The specific method was as follows: Weigh 0.2 g of the wet gel microspheres prepared in each example and place them in 3 mL of plasma from rabbits with hyperbilirubinemia. Use the plasma without adding gel microspheres as a blank control. After incubating at 37 °C for 3 h, use a total bilirubin kit and a direct bilirubin kit to detect the concentration of bilirubin in the plasma and calculate the clearance rate.

[0089] Table 4. Clearance Rate of Bilirubin by Microspheres in Each Example

[0090] Sample Example 8 Example 9 Example 10 Example 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:Microsphere (mass ratio) 1:100 1:100 1:100 1:100 1:400 1:100 Enzyme type Bilirubin oxidase-like Bilirubin oxidase-like Bilirubin oxidase-like Bilirubin oxidase-like Bilirubin oxidase-like BOD Direct bilirubin clearance rate (%) 32.5 26.6 47.2 30.1 27.5 14.8 Total bilirubin clearance rate (%) 44.7 38.9 56.6 42.8 38.1 22.3

[0091] Note: In the blank control group, the total bilirubin concentration 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.

[0092] It can be seen from Table 4 that the maximum clearance rate of the microspheres in each example for total bilirubin in plasma is 56.6% (Example 10), and the maximum adsorption amount for direct bilirubin in plasma is 47.2%. In the enzyme immobilization step, the enzyme dosage in each example is the same. Therefore, it can be speculated from the clearance rate that the enzyme immobilization amount in Example 10 is the largest, and the synergistic effect of catalysis and adsorption is the best. These results also indicate that the enzyme-loaded gel microspheres can effectively remove bilirubin.

[0093] In addition, weigh 0.2 g of the enzyme-loaded gel microspheres prepared in Example 10 and incubate them with 3 mL of hyperbilirubin plasma for 3 h. The results are as Figure 3 shown Figure 3 The left figure is before the experiment, and the plasma total bilirubin concentration is 108.2 μmol / L; the right figure is after the experiment, and the plasma total bilirubin concentration is 52.4 μmol / L.

[0094] Example 8. In Example 9, the ratio of chitosan to polyacrylic acid in the microspheres was changed. Compared with Example 10, the bilirubin clearance effect decreased. It can be seen that the amount of polyacrylic acid incorporated affects the enzyme loading rate. Neither the absence of polyacrylic acid nor an excessive amount of polyacrylic acid is the optimal 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 decreased, and the enzyme grafting rate decreased. In Example 12, the amount of EDC used was 0.25 times that of Example 10, and the catalytic-adsorption effect decreased. It can be seen that during the process of activating the surface of the microspheres, the amount of EDC used cannot be too low, otherwise the enzyme grafting rate will decrease. Example 13 had the lowest bilirubin clearance efficiency among all the examples, indicating that this enzyme-loaded scheme is not suitable for natural enzyme BOD.

[0095] Performance Test Example 4: Determination of the Mechanical Properties of Microspheres

[0096] Using a universal testing machine, based on the basic principle of mechanical property testing, the relationship between stress and strain was obtained by applying an external force and measuring the deformation behavior of the material. The specific method was as follows: Measure the diameter of the microspheres. Place the microsphere sample on the lower platen of the testing machine to ensure that it is directly below the indenter. Start the test and apply a compressive force to the microspheres at a constant rate. Stop the test when the microspheres rupture or reach a preset deformation amount. (During the hemoperfusion process, the maximum pressure borne by the microspheres is 66.7 kPa. Therefore, Table 5 below shows the strain corresponding to the stress of 66.7 kPa for the enzyme-loaded gel microspheres prepared in different examples).

[0097] Table 5.

[0098] Strain of Microspheres in Each Example (Stress is 66.7 kPa)

[0099] Sample Example 8 Example 9 Example 10 Example 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

[0100] Note: In the table, Cs is chitosan and PAA is polyacrylic acid.

[0101] As can be seen from Table 5, the mechanical properties of pure chitosan microspheres are weak (such as in Example 8), and the mechanical properties can be significantly improved by co-mixing with polyacrylic acid. The hydroxyl (-OH) and amino (-NH2) groups of chitosan can form hydrogen bonds with the carboxyl groups (-COOH) of polyacrylic acid, enhancing the binding force between molecular chains and improving the toughness of the material. When the co-mixing amount is excessive, such as in Example 9, the deformation amount is larger than that in Example 10. As can be seen from Example 11, when the concentrations of sodium hydroxide and ethanol in the coagulation bath are 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, and the molecular chain entanglement is uneven (dense outside and sparse inside), resulting in a decrease in mechanical strength. The results show that co-mixing polyacrylic acid into chitosan can improve the compressive properties of 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.

[0102] Obviously, the above examples are only for clear illustration and not limitations on the implementation modes. For those of ordinary skill 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 implementation modes here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An enzyme-loaded gel microsphere, characterized in that: The enzyme-loaded gel microspheres are prepared by using chitosan and polyacrylic acid as carriers to load 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) 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 enzyme-loaded gel microspheres 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; and the albumin is human serum albumin or bovine serum albumin.

3. The method for preparing enzyme-loaded gel microspheres according to claim 1, 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, 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 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.

4. The preparation method according to claim 3, characterized in that: In step (1), the acid solution is a hydrochloric acid solution.

5. The preparation method according to claim 3, characterized in that: In step (2), the diameter of the chitosan-polyacrylic acid carrier microspheres is 500-800 μm.

6. The preparation method according to claim 3, 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.

7. Use of the enzyme-loaded gel microspheres prepared by the preparation method of claim 3 in preparing medical devices for treating or improving hyperbilirubinemia.

8. The use according to claim 7, characterized in that: The medical device is a hemoperfusion device.

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