Preparation method and application of protein crown modified zinc coordination polymer multilevel structure composite microspheres

Abstract

The invention belongs to the field of medical instruments, and particularly relates to a preparation method and application of protein crown modified zinc coordination polymer multilevel structure composite microspheres. The preparation method of the zinc coordination polymer multilevel structure composite microsphere comprises the following steps: by taking a natural polymer microsphere as a template, carrying out reaction on a zinc ammonium ethanol solution and dopamine, thereby obtaining the zinc coordination polymer multilevel structure composite microsphere. According to the invention, natural polymer microspheres are used as a template, and the zinc coordination polymer skeleton submicron microspheres with a nanopore structure are prepared by a one-step method. The zinc coordination polymer multilevel structure composite microspheres modified by the protein crown are obtained by modifying the surfaces of the microspheres to form the protein crown, and the zinc coordination polymer multilevel structure composite microspheres modified by the protein crown have the functions of efficient toxin removal, antibacterial protection and anticoagulation, and provide an innovative solution for breaking through the clinical bottleneck of a blood perfusion device.

Description

Technical Field

[0001] The present invention belongs to the field of medical devices, and particularly relates to a preparation method and application of a protein corona-modified zinc coordination polymer multi-level structure composite microsphere. Background Art

[0002] With the increasing incidence of clinical diseases such as end-stage renal disease, severe infections, and poisoning, hemodialysis, as a core blood purification technology, has been widely used. However, its dependence on the characteristics of the semi-permeable membrane results in significant clearance of small molecule metabolic wastes, while the ability to remove macromolecular or middle molecule toxins (such as bilirubin, inflammatory mediators, endotoxins, etc.) is limited. Therefore, perfusion technology has been introduced into the blood purification system to rapidly remove toxic substances in the blood through an extracorporeal circulation system equipped with an adsorption device, improve systemic and visceral circulation, maintain organ function, and promote cell regeneration.

[0003] The core function of the perfusion device is to target and remove specific macromolecular toxins, inflammatory factors, or endotoxins by using physical and chemical adsorption mechanisms (such as van der Waals forces, electrostatic forces, hydrophobic forces, hydrogen bonds, ion exchange, and coordination bonds), especially suitable for scenarios such as septic shock and severe sepsis that require regulating the immune response and reducing the risk of organ damage. Multi-modal combined purification (such as hemodialysis combined with perfusion) can comprehensively remove toxins and improve the survival rate and quality of life of patients, and has now become an important measure for the treatment of liver and kidney failure. The core of perfusion technology lies in the performance of the adsorbent, which needs to meet the following criteria: having strong adsorption ability and high selectivity, having a large specific surface area, suitable pore structure to optimize the adsorption efficiency, ensuring good biocompatibility, avoiding damage to blood components, and having stable physical and chemical properties, sufficient mechanical strength, and convenient disinfection and storage conditions, and the preparation process needs to be simple and efficient to reduce production costs.

[0004] The currently used perfusion device adsorption materials are mainly activated carbon and synthetic resin adsorption materials. Activated carbon has become the most widely used adsorption material in the early stage due to its high specific surface area and rich microporous structure. It can capture a variety of small molecules and some middle molecule toxins through physical adsorption mechanisms. Synthetic resin adsorption materials can achieve high-selectivity adsorption of toxins within a specific molecular weight range by controlling the pore size distribution and surface chemical properties. It has good mechanical strength and is convenient for long-term use in perfusion devices, and has been applied in various blood purification systems. However, the above two types of adsorption materials have low selectivity for macromolecules in practical applications, are prone to adsorbing blood proteins, leading to coagulation, and at the same time, they will also cause problems of bacterial infection, which may ultimately endanger the life and health of patients.

[0005] An increasing number of novel nanomaterials and composite materials have been applied to the adsorption field of hemoperfusion devices and achieved very important results. However, the serious problems of hemoperfusion devices in clinical practice caused by premature coagulation and bacterial infection have still not been solved. Therefore, it is very important to design an adsorbent material for hemoperfusion devices with a high specific surface area, strong adsorption ability, high selectivity, good biocompatibility, and the ability to effectively solve coagulation and bacterial infection. Summary of the Invention

[0006] The purpose of the present invention is to provide a zinc coordination polymer multi-level structure composite microsphere.

[0007] Another purpose of the present invention is to provide a preparation method for the above-mentioned zinc coordination polymer multi-level structure composite microsphere.

[0008] Another purpose of the present invention is to provide the application of the above-mentioned zinc coordination polymer multi-level structure composite microsphere.

[0009] Another purpose of the present invention is to provide a protein corona-modified zinc coordination polymer multi-level structure composite microsphere.

[0010] According to the preparation method of the zinc coordination polymer multi-level structure composite microsphere of the specific embodiment of the present invention, the preparation method includes the following steps:

[0011] Using a natural polymer microsphere as a template, it is prepared by reacting with a zinc ammonium ethanol solution and dopamine.

[0012] According to the preparation method of the zinc coordination polymer multi-level structure composite microsphere of the specific embodiment of the present invention, in the zinc ammonium ethanol, the concentration of zinc ions is 0.01 - 0.09 mol / L, and / or, in the zinc ammonium ethanol, the volume ratio of ethanol is 25 - 50 vol.%.

[0013] According to the preparation method of the zinc coordination polymer multi-level structure composite microsphere of the specific embodiment of the present invention, the volume ratio of the packed volume of the natural polymer microsphere to the volume of the zinc ammonium ethanol solution is 1:0.5 - 3.

[0014] According to the preparation method of the zinc coordination polymer multi-level structure composite microsphere of the specific embodiment of the present invention, the final concentration of dopamine is 1 - 5 mg / mL.

[0015] According to the preparation method of the zinc coordination polymer multi-level structure composite microsphere of the specific embodiment of the present invention, the natural polymer microsphere is selected from any one or more of agarose microspheres, chitosan microspheres, albumin microspheres, gelatin microspheres, starch microspheres, or sodium alginate microspheres.

[0016] Natural polymer microspheres are mainly prepared from natural polymer materials derived from animals, plants or microorganisms, and have biocompatibility and degradability. Moreover, the interior of the natural polymer microspheres has a certain porosity, providing the necessary conditions for the formation of a stacked structure of nanoflower microspheres with a nanoporous structure inside.

[0017] Agarose microspheres are linear polysaccharides extracted from seaweeds, with a three-dimensional network porous structure. The pore size is usually in the micron to submicron range, and the porosity is high.

[0018] Chitosan microspheres are natural polycationic polysaccharides obtained by deacetylating chitin, and can be prepared by chemical cross-linking or ion curing methods, with a porous or sponge-like structure.

[0019] Albumin microspheres use bovine serum albumin or human serum albumin as carriers and are made by emulsification or cross-linking techniques. Their interior shows a porous or honeycomb-like structure.

[0020] Gelatin microspheres are protein materials prepared by hydrolyzing collagen, and they present a sponge-like porous structure after swelling.

[0021] Starch microspheres use natural starch as raw materials and are prepared by cross-linking or emulsification-solvent evaporation methods, with a microporous network structure.

[0022] Sodium alginate microspheres are polyanionic polysaccharides extracted from brown algae. Gel microspheres are formed by ionic cross-linking (such as Ca 2+ ), and a cross-linked network is formed by the reaction of sodium alginate solution with Ca 2+ . After solvent replacement, micropores are retained.

[0023] According to the preparation method of the zinc coordination polymer multi-level structure composite microspheres of the specific embodiment of the present invention, the preparation method includes the following steps:

[0024] Using natural polymer microspheres as templates, adding zinc ammonium ethanol solution and dopamine, and reacting under constant temperature and shaking.

[0025] The present invention also provides zinc coordination polymer multi-level structure composite microspheres prepared by the above preparation method.

[0026] The present invention also provides the application of the above zinc coordination polymer multi-level structure composite microspheres in the adsorption material of a hemoperfusion device.

[0027] According to the preparation method of the protein corona-modified zinc coordination polymer multi-level structure composite microspheres of the specific embodiment of the present invention, the preparation method includes the step of reacting the above zinc coordination polymer multi-level structure composite microspheres with proteins.

[0028] According to the preparation method of the protein corona-modified zinc coordination polymer multi-level structure composite microspheres of the specific embodiment of the present invention, the preparation method includes the following steps:

[0029] The zinc coordination polymer multi - level structure composite microspheres are added to the protein solution, and a constant - temperature oscillation reaction is carried out to obtain the product. Among them, the mass ratio of the zinc coordination polymer multi - level structure composite microspheres to the protein is 1:0.1 - 1.

[0030] The present invention also provides protein - corona - modified zinc coordination polymer multi - level structure composite microspheres prepared by the above - mentioned scheme.

[0031] The beneficial effects of the present invention:

[0032] In the present invention, natural polymer microspheres are used as templates, and zinc coordination polymers are obtained by reacting with soluble zinc ions and dopamine. Preferably, agarose microspheres are used as templates in the present invention, and Aga - PDA / Zn microspheres are prepared by a one - step method with zinc ammonium ethanol solution and dopamine. It is a large microsphere structure formed by the accumulation of sub - micron microspheres with a zinc coordination polymer skeleton having a nanoporous structure. The surface of the agarose microspheres has nanoflower microspheres with a nanoporous structure. The interior of Aga - PDA / Zn is entirely composed of nanoflower microspheres with a nanoporous structure stacked together. The total pore volume of the Aga - PDA / Zn microspheres is increased to 4.1 mL / g, and the total pore area of the Aga - PDA / Zn microspheres is increased to 139.1 m 2 / g. The close - packed growth between the nanoflower microspheres does not change the original framework and properties of the Aga microspheres. The Young's modulus of the Aga - PDA / Zn microspheres can reach 6375 kPa and can maintain long - term stability.

[0033] In the present invention, a protein corona is modified on the surface of the Aga - PDA / Zn microspheres to form HAS - Aga - PDA / Zn microspheres (abbreviated as HAP / Zn microspheres). The HAP / Zn microspheres have good anticoagulant effects, and their anticoagulant functions are achieved through dual mechanisms: on the one hand, it inhibits the adsorption of pro - coagulant proteins such as fibrinogen and blocks the coagulation cascade reaction; on the other hand, the HAP / Zn microspheres synergistically anticoagulate through multi - dimensional effects such as regulating surface properties, preventing the activation of coagulation factors, reducing platelet adhesion and activation, and regulating the complement system. The Aga - PDA / Zn microspheres integrate high - efficiency toxin clearance, antibacterial protection, and anticoagulant functions, providing an innovative solution for breaking through the clinical bottleneck of hemoperfusion devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0035] Figure 1 Scanning electron microscopy characterization images of the surfaces and cross-sections of different microspheres; among them,

[0036] (a) Surface and morphology of Aga microspheres;

[0037] (b) Surface and morphology of Aga-PDA microspheres;

[0038] (c) Surface and morphology of Aga-PDA / Zn microspheres;

[0039] (d) Cross-sectional morphology of Aga microspheres;

[0040] (e) Cross-sectional morphology of Aga-PDA microspheres;

[0041] (f) Cross-sectional morphology of Aga-PDA / Zn microspheres.

[0042] Figure 2 Show the surface morphology of Aga-PDA / Zn microspheres at different concentrations of zinc ammonium solution. Among them,

[0043] (a) 0.8, (b) 0.7, (c) 0.6, (d) 0.5, (e) 0.4, (f) 0.3, (g) 0.2, (h) 0.1 mol / L.

[0044] Figure 3 Show the effect of different reaction times of zinc ammonium dopamine solution modified Aga microspheres on Aga-PDA / Zn microspheres. Among them,

[0045] (a) Growth of nanoflower microspheres on the surface of Aga-PDA / Zn microspheres when the reaction durations are (a1) 1 h, (a2) 2 h, (a3) 6 h, and (a4) 12 h respectively;

[0046] (b) Zinc ion content of Aga-PDA / Zn microspheres after different reaction durations.

[0047] Figure 4 Investigation of the mechanical properties of microspheres; among them,

[0048] (a) Microsphere strength test process;

[0049] (b) Young's modulus values of microspheres before and after storage for 30 days;

[0050] (c) Microsphere strength force vs. displacement curve;

[0051] (d) Young's modulus values of microspheres after 3 repeated compressions;

[0052] (e) Change in Young's modulus of microspheres after 3 repeated compressions.

[0053] Figure 5(a) Porosity, (b) total pore volume, and (c) total pore area of Aga, Aga-PDA, and Aga-PDA / Zn microspheres; (d) Curve of the adsorption amount of Aga-PDA / Zn microspheres for FITC-HSA versus time; (e) Adsorption amount of Aga, Aga-PDA, and Aga-PDA / Zn microspheres after reacting with FITC-HAS for 24 h; (f) Curve of the surface fluorescence intensity of Aga, Aga-PDA, and Aga-PDA / Zn microspheres changing with time.

[0054] Figure 6 (a) EDX spectrum of HAP / Zn microspheres; (b) Element mass percentage content of Aga-PDA / Zn and HAP / Zn microspheres; (c) Surface Zeta potential of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres.

[0055] Figure 7 . (a) Hemolysis rate of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres interacting with whole blood, using deionized water as the positive control and normal saline as the negative control; The inset is the digital photo of each group. (b) White blood cell count, (c) red blood cell count, and (d) platelet count after Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres contacting with blood.

[0056] Figure 8 . (a) Function relationship between clot formation and time of the blank group, Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres; (b) Quantitative clotting time.

[0057] Figure 9 . (a) Colony growth photos of Escherichia coli and Staphylococcus aureus after being treated with Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres, and the blank group is without any treatment; (b) Comparison of the antibacterial efficiency of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres against Escherichia coli and Staphylococcus aureus (p < 0.0001); (c) Growth curves of Escherichia coli and Staphylococcus aureus co-cultured with Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres for 0 - 24 h (p < 0.0001).

[0058] Figure 10 . (a) Curve of the adsorption amount of Aga-PDA / Zn microspheres for bilirubin versus adsorption time; (b) Adsorption amount of Aga, Aga-PDA, and Aga-PDA / Zn microspheres after adsorption equilibrium for bilirubin; (c) Adsorption amount of Hag, HAP, and HAP / Zn microspheres after adsorption equilibrium for bilirubin (p < 0.0001). DETAILED DESCRIPTION

[0059] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0060] Experimental reagents:

[0061] Dopamine hydrochloride was purchased from SigmaAldrich (USA).

[0062] LIVE / DEAD BacLight Bacterial Viability Kit (L7012) was purchased from Invitrogen (USA).

[0063] Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were purchased from Solarbio (China).

[0064] Ultrapure water was obtained from the Milli-Q system (Millipore, USA).

[0065] Human serum albumin and FITC-HSA were purchased from Solarbio (China).

[0066] Phosphate buffer solution (pH, 7.4) was purchased from Gibco (China).

[0067] Unless otherwise stated, all other chemical reagents were purchased from Aladdin (China).

[0068] The zinc ion content and microsphere stability were characterized by ICP-MS. The bilirubin adsorption experiment was performed using an ultraviolet spectrophotometer UV-2700 (SHIMADZU, Japan). The protein adsorption experiment was characterized by a fluorescence spectrophotometer (RF-5301PC, Shimadzu Corporation, Japan).

[0069] In the present invention, the packed volume refers to the total volume of the granular materials after being tightly packed in a specific container, including the volume of the granules themselves and the volume of the voids between the granules.

[0070] The present invention provides a method for preparing zinc coordination polymer multi-level structure composite microspheres, comprising the following steps:

[0071] The microspheres are prepared by taking natural polymer microspheres as templates and reacting with zinc ammonium dopamine solution.

[0072] Preferably, in the zinc ammonium dopamine solution, the concentration of zinc ions is 0.01 - 0.09 mol / L, and / or the zinc ammonium dopamine solution contains ethanol with a volume ratio of 25 - 50 vol.%, and / or the final concentration of dopamine is 1 - 5 mg / mL.

[0073] When there is no ethanol in the reaction system, this kind of submicron microsphere with a zinc coordination polymer framework having a nanoporous structure cannot be formed on the surface and inside of the agarose microspheres. Zinc ions can form metal coordination bonds with -O and -N in the polydopamine formed during the oxidation process of dopamine, and this metal coordination effect is the main reason for the microspheres to have a nanoflower network structure.

[0074] Preferably, the volume ratio of ethanol is 25 - 50 vol.%, more preferably, the volume ratio of ethanol is 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 36%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or the volume of ethanol is any value within the above range, which will not be elaborated here one by one.

[0075] Preferably, the concentration of zinc ions is 0.01 - 0.09 mol / L, more preferably, the concentration of zinc ions is 0.04 - 0.06 mol / L, or the concentration of zinc ions is 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or the concentration of zinc ions is any value within the above range, which will not be elaborated here one by one.

[0076] Preferably, the final concentration of dopamine is 1 - 5 mg / mL, more preferably, the final concentration of dopamine is 2 - 4 mg / mL, or the final concentration of dopamine is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or the final concentration of dopamine is any value within the above range, which will not be elaborated here one by one.

[0077] Among them, as long as the zinc ions, dopamine, and ethanol in the reaction solution meet the above concentration requirements, the microspheres of the present invention can be prepared. Among them, the zinc ions can be derived from zinc chloride, zinc nitrate, zinc sulfate, etc. Dopamine can be derived from dopamine hydrochloride, dopamine sulfate, etc. Of course, the present invention provides an optional preparation method for the zinc ammonium dopamine solution. For example,

[0078] (1) Add the zinc ion solution to the ammonia water solution to produce a zinc ammonium complex and form a zinc ammonium solution;

[0079] (2) Ethanol is added to the zinc ammonium solution to obtain a zinc ammonium ethanol solution;

[0080] (3) Dopamine is added to the zinc ammonium ethanol solution to obtain a zinc ammonium dopamine solution.

[0081] In the zinc ammonium dopamine solution provided by the above preparation method, the concentration of zinc ions is 0.01 - 0.09 mol / L, and / or the zinc ammonium dopamine solution contains ethanol with a volume ratio of 25 - 50 vol.%, and / or the final concentration of dopamine is 1 - 5 mg / mL.

[0082] Preferably, the volume ratio of the packed volume of the natural polymer microspheres to the volume of the zinc ammonium dopamine solution is 1:0.5 - 3. More preferably, the volume ratio of the two is 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0. At the same time, the volume ratio of the two can also be any value within the range of 1:0.5 - 3, which will not be elaborated here one by one.

[0083] Preferably, the natural polymer microspheres are selected from any one or more of agarose microspheres, chitosan microspheres, albumin microspheres, gelatin microspheres, starch microspheres or sodium alginate microspheres. More preferably, the natural polymer microspheres are selected from agarose microspheres or gel microspheres.

[0084] The present invention provides a preparation method of zinc coordination polymer multi - level structure composite microspheres, including the following steps:

[0085] Using agarose microspheres as a template, adding a zinc ammonium ethanol solution and dopamine, and carrying out a repeated cyclic oscillation reaction in a constant temperature water bath.

[0086] Among them, in the zinc ammonium ethanol solution, the concentration of zinc ions is 0.02 - 0.08 mol / L, the final concentration of dopamine is 2 - 4 mg / ml, and the volume ratio of the packed volume of agarose microspheres to the volume of the solution is 1:8 - 12.

[0087] Preferably, the reaction is carried out by reciprocating cyclic oscillation in a 25 °C constant temperature water bath, and / or the reaction time is 1 - 24 h.

[0088] The preparation method of the protein - corona - modified zinc coordination polymer multi - level structure composite microspheres of the present invention, the preparation method includes the step of reacting the above - mentioned zinc coordination polymer multi - level structure composite microspheres with proteins.

[0089] Specifically, the preparation method includes the following steps:

[0090] Add the zinc coordination polymer multi - level structure composite microspheres to the protein solution and carry out a constant - temperature oscillation reaction to obtain the product. Among them, the mass ratio of the zinc coordination polymer multi - level structure composite microspheres to the protein is 1:0.1 - 1.

[0091] Preferably, the protein is selected from human serum albumin or bovine serum albumin.

[0092] Preferably, the mass ratio of the zinc coordination polymer multi - level structure composite microspheres to the protein is 1:0.1 - 1. Or, the mass ratio of the two is 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.7, 1:0.9, 1:1. Or, the mass ratio of the zinc coordination polymer multi - level structure composite microspheres to the protein can be any value within the above range, which will not be elaborated here.

[0093] Preferably, the constant - temperature oscillation reaction is carried out at 37 °C for 12 - 36 h under constant - temperature oscillation.

[0094] Example 1 Preparation of Aga - PDA / Zn Microspheres

[0095] Add 13.63 g of ZnCl2 to 50 mL of secondary pure water to obtain a 2 mol / L Zn 2+ solution. Take 39 mL of 25% ammonia water and gradually add it to 50 mL of the Zn 2+ solution until the white floccules just disappear to obtain a zinc - ammonium complex. According to the conservation of the amount of zinc ions, the zinc - ammonium concentration at this time is 1.124 mol / L.

[0096] Add 6.55 mL of secondary pure water to 3 mL of absolute ethanol, and then add 0.45 mL of 1.124 mol / L zinc - ammonium solution to finally obtain a 0.05 mol / L zinc - ammonium ethanol solution (30%).

[0097] Take 5 mL of the above zinc - ammonium ethanol solution, add it to a six - well cell culture plate containing agarose template microspheres, and add 3 mg / mL of dopamine to it. Then, carry out reciprocating cyclic oscillation coating in a constant - temperature shaking water bath for 12 h (25 °C, 120 rpm). After the reaction, wash the microspheres with secondary pure water multiple times to obtain Aga - PDA / Zn microspheres. The microspheres are dispersed in pure water or PBS for standby.

[0098] Example 2

[0099] Use the Aga - PDA / Zn microspheres prepared with zinc - ammonium solutions of different concentrations as the control experimental group, and their reaction conditions are the same. The solution ratios are shown in Table 1.

[0100] Table 1. Preparation ratio of zinc - ammonium ethanol solutions with different concentrations

[0101]

[0102] To investigate the influence of ethanol on the reaction system, an aqueous solution with a zinc ammonium concentration of 0.05 mol / L was used as a control test group, and the other experimental conditions remained unchanged. To investigate the influence of reaction time on the reaction system, the reaction time was changed to 1 h, 2 h, 6 h, and 12 h for the preparation of Aga-PDA / Zn microspheres, while the other experimental conditions remained unchanged.

[0103] Scanning electron microscope (SEM) images of blank agarose template microspheres (Aga), Aga-PDA, and Aga-PDA / Zn microspheres in the dry state were taken using a Phenom G2 Pro (Phenom World, Netherlands).

[0104] As Figure 1 shown, the surface of the blank Aga microspheres is smooth without protrusions, and there are also small pore structures. Its cross-section is relatively smooth, and it can be seen that there are tiny channels inside. After modification with PDA, there are many dopamine particles on the surface of the Aga microspheres, making the overall surface of the microspheres relatively rough. There is no obvious change inside the Aga microspheres of the Aga-PDA microspheres.

[0105] After modification with zinc ammonium dopamine solution, nanosized flower microspheres with a surface nanometer porous structure and a diameter of about 1 micrometer grew uniformly on the surface of the Aga microspheres (Aga-PDA / Zn), and the microspheres were closely packed together. At the same time, the inside of the Aga-PDA / Zn was entirely composed of nanosized flower microspheres with a nanometer porous structure that were closely packed. The close packing growth of the nanosized flower microspheres does not change the original framework and properties of the Aga microspheres. This large microsphere structure formed by the packing of nanosized flower microspheres is called a hierarchical structure composite. The special nature of its structure makes it theoretically have a high porosity and a high specific surface area, which can provide sufficient adsorption sites for the adsorption of various toxins in the blood.

[0106] As Figure 2 shown, the influence of different zinc ammonium solution concentrations on the above hierarchical structure composite was investigated. When the concentration of the zinc ammonium ethanol solution was 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mol / L, the above nanosized flower microspheres with a size of about 1 micrometer did not appear. Therefore, in subsequent experiments, the Aga-PDA / Zn hierarchical structure composite microspheres prepared when the ethanol concentration of the zinc ammonium solution was 0.05 mol / L were used as the research object.

[0107] As Figure 3As shown in the figure, the present invention studied the influence of the reaction duration (the reaction time of zinc ammonium dopamine solution modified Aga microspheres) on the growth of nanoflower microspheres on Aga. When the reaction duration was only 1 h, only a part of the nanoflower microspheres were formed on the surface of Aga microspheres. After the reaction duration was extended to 2 h, the growth quantity of nanoflower microspheres increased significantly and gradually accumulated closely, but it was found that there were still many relatively large voids between the nanoflower microspheres. When the reaction duration was extended to 6 h, the quantity of nanoflower microspheres on the surface of Aga microspheres increased significantly and accumulated closely. When the reaction duration was further extended to 12 h, the packing density of the nanoflower microspheres on its surface did not change significantly.

[0108] The zinc ion content of Aga-PDA / Zn with different reaction durations was tested by ICP-OES ( Figure 3 b). With the increase of the reaction duration, the zinc ion content of Aga-PDA / Zn microspheres gradually increased. The zinc ion content was 51.2 mg / g when the reaction duration was 1 h, increased to 88.1 mg / g when the reaction duration was 2 h, and increased to 114.8 mg / g when the reaction duration was 6 h. It is worth noting that the zinc ion content was 116.8 mg / g when the reaction duration was extended to 12 h, which was close to that at 6 h.

[0109] The above results show that nanoflower microspheres with closely packed surfaces can be obtained after the Aga microspheres are modified with zinc ammonium dopamine solution for 6 h. Therefore, in the subsequent experiments, the Aga-PDA / Zn microspheres prepared with a reaction duration of 6 h were selected for the next step of research.

[0110] Example 3 investigated the mechanical properties of Aga-PDA / Zn microspheres

[0111] Microspheres need to have certain mechanical strength and long-term stability to be applied in hemoperfusion devices. Therefore, the present invention tested the strength of Aga, Aga-PDA, and Aga-PDA / Zn microspheres by a particle strength tester.

[0112] As Figure 4 shown in a, both the Aga-PDA microspheres modified by dopamine and the Aga-PDA / Zn microspheres modified by zinc ammonium dopamine can be compressed and quickly restored to a spherical shape within 2 s like the Aga blank microspheres. The Young's modulus of Aga microspheres is 4625 kPa. The Young's modulus of Aga-PDA microspheres increased to 5400 kPa compared with Aga microspheres because dopamine forms a dense network structure through intermolecular hydrogen bonds, π-π stacking, and covalent cross-linking during the formation of polydopamine, significantly improving the rigidity of the microspheres.

[0113] The Young's modulus of Aga-PDA / Zn microspheres increased to 6375 kPa ( Figure 4b), which is due to the formation of the zinc coordination-poly dopamine network. The coordination of Zn-O and Zn-N has a high bond energy and can serve as additional crosslinking points, significantly increasing the crosslinking density and stiffness of the material. At the same time, the closely packed structure of the nanoflower microspheres in the Aga-PDA / Zn microspheres can reduce interfacial slippage and enhance the stress transfer efficiency, thereby suppressing plastic deformation, and thus the Aga-PDA / Zn microspheres have a high Young's modulus.

[0114] Even after storing different microspheres in PBS solution with pH = 7.4 for 30 days, the Young's modulus of the microspheres hardly changed, indicating that the Aga-PDA / Zn microspheres can maintain long-term stability under the condition of having a higher Young's modulus.

[0115] To investigate the repeated compression resistance of the same microsphere, the present invention performed repeated compression and rebound tests on the same microsphere three times, and the force-distance curve is as Figure 4 shown in c. It can be found that the force-displacement curves of the second and third times of the Aga, Aga-PDA, and Aga-PDA / Zn microspheres during repeated compression basically coincide with the first compression curve of the microspheres themselves. The Young's modulus of the Aga, Aga-PDA, and Aga-PDA / Zn microspheres hardly changed during the three repeated compression processes ( Figure 4 d), and the percentage change in their Young's modulus is less than 5%. The above results indicate that the Aga-PDA / Zn hierarchical structure composite microspheres have a higher Young's modulus and better rigidity compared to the Aga and Aga-PDA microspheres, and at the same time do not change the anti-extrusion ability and long-term stability of the Aga microspheres themselves.

[0116] The Aga, Aga-PDA, and Aga-PDA / Zn were tested by a mercury intrusion porosimeter. The porosity of the Aga microspheres is 73.3%, the Aga-PDA microspheres is 79.4%, and the Aga-PDA / Zn microspheres is 84.4% ( Figure 5 a). The total pore volume of the Aga microspheres is 2.1 mL / g, the total pore volume of the Aga-PDA microspheres after dopamine modification is 2.9 mL / g, and the total pore volume of the Aga-PDA / Zn microspheres after reaction with zinc ammonium dopamine solution increases to 4.1 mL / g ( Figure 5 b).

[0117] The total pore area of the Aga microspheres is only 75.5 m 2 / g, the Aga-PDA microspheres is 103.2 m 2 / g, and the total pore area of the Aga-PDA / Zn microspheres increases to 139.1 m 2 / g ( Figure 5 c).

[0118] The Aga microspheres themselves have a relatively high porosity and total pore area, which can provide sufficient space and sites for the zinc ammonium dopamine solution to enter the interior for reaction. Therefore, the Aga-PDA / Zn microspheres have closely packed nano-flower microspheres from the inside out, thus forming a unique multi-level structure composite.

[0119] The higher porosity, total pore volume and total pore area of the Aga-PDA / Zn microspheres can provide sufficient storage and reaction space for the modification of human serum albumin molecules (HSA), enhance the permeability of HSA inside the microspheres, improve the reaction activity of the microspheres, and thus increase the modification efficiency of HSA.

[0120] Example 4 Preparation of HAP / Zn Microspheres

[0121] Adsorption Kinetics:

[0122] Weigh 1 mg of Aga, Aga-PDA and Aga-PDA / Zn microsphere samples into 5 mL centrifuge tubes, and add FITC-HSA solution with a concentration of 100 μg / mL to them. Seal the centrifuge tubes and place them in a constant temperature shaking water bath at 37 °C and 170 rpm for adsorption. Take 1 mL of the supernatant at fixed times and use a fluorescence spectrophotometer to measure the remaining absorbance of the solution until the adsorption reaches equilibrium (set 3 parallel groups for each group).

[0123] The standard curve is determined as follows: Prepare FITC-HAS solution with a concentration of 100 μg / mL using a volumetric flask, and then dilute it stepwise to different concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625 μg / mL. Measure the fluorescence intensity using a fluorescence spectrophotometer and plot the standard curve.

[0124] Figure 5 d is the curve showing the relationship between the adsorption amount and time of the Aga-PDA / Zn microspheres for the adsorption of FITC-labeled HAS (FITC-HSA). After 24 h of reaction, the adsorption amount basically reaches equilibrium. Therefore, the reaction time between different microspheres and HSA is determined to be 24 h. The results show that after 24 h of reaction, the adsorption amount of the Aga microspheres is only 18.1 mg / g, and the adsorption amount of the Aga-PDA microspheres for HSA is 49.4 mg / g. This is because polydopamine can bind to HSA through hydrophobic interaction and hydrogen bond, thus increasing the modification of human serum albumin on Aga-PDA to a certain extent.

[0125] The adsorption amount of the Aga-PDA / Zn microspheres for HSA is as high as 95.3 mg / g ( Figure 5 e), almost 5 times that of the Aga microspheres. This is because in addition to binding to HSA through hydrophobic interaction and hydrogen bond with PDA, Zn 2+It can bind to the cysteine and histidine residues (such as His-242, His-288) of HSA through coordination, and Zn 2+ It can also bind to human serum albumin (isoelectric point ~ 4.7, negatively charged at physiological pH) through electrostatic interaction. In addition, the unique multi-level structure composite of Aga-PDA / Zn microspheres can also provide enough sites for the modification of HSA.

[0126] Figure 5 Figure f shows the fluorescence intensity characterization of the surfaces of Aga, Aga-PDA, and Aga-PDA / Zn microspheres with FITC-HSA at different reaction times by confocal laser scanning microscopy. The results also show that with the extension of the reaction time, the fluorescence intensity on the surface of Aga-PDA / Zn microspheres gradually increases, and at the same moment, the fluorescence intensity on the surface of Aga-PDA / Zn microspheres is much greater than that of Aga and Aga-PDA microspheres.

[0127] Figure 5 Figure f inset shows the fluorescence images of the surfaces of Aga, Aga-PDA, and Aga-PDA / Zn microspheres at 24 h. It can be found that there is almost no fluorescence on the surface of Aga, a little fluorescence on the surface of Aga-PDA microspheres, and a large amount of fluorescence on the surface of Aga-PDA / Zn microspheres.

[0128] The above results indicate that multiple interactions and the unique multi-level structure composite of Aga-PDA / Zn microspheres promote the modification of a large amount of HSA on the surface and inside of the microspheres, which is beneficial to the formation of a protein corona on the surface of the nanoflower microspheres, and the formation of the protein corona will be beneficial to prolonging the blood coagulation time during hemoperfusion.

[0129] Preparation of HAP / Zn microspheres:

[0130] Take 10 mg of Aga-PDA / Zn microsphere sample and add it to 20 mL of HSA solution with a concentration of 200 μg / mL, and react in a constant temperature shaking water bath at 37 °C and 170 rpm for 24 h (the optimal adsorption time obtained from the protein adsorption experiment). After the reaction, wash the sample with PBS buffer solution multiple times to obtain HAP / Zn microspheres.

[0131] Aga and Aga-PDA microspheres were used as control groups and reacted under the same conditions to obtain HAa microspheres and HAP microspheres.

[0132] To determine that HSA was successfully loaded on the surface of Aga-PDA / Zn microspheres, the present invention performed elemental analysis on HAP / Zn microspheres by EDX, and its spectrum is as Figure 6As shown in Figure 6 b), the results show that the Aga-PDA / Zn microspheres contain C (25 wt%), N (3 wt%), O (25 wt%) and Zn (47 wt%) elements, while the mass percentage of each element in the HAP / Zn microspheres is C (20 wt%), N (3 wt%), O (26 wt%), Zn (45 wt%) and S (6 wt%). The above results further illustrate the successful loading of HSA on the Aga-PDA / Zn microspheres.

[0133] The potential results of different microspheres are shown in Figure 6 c. The surface potential of the Aga microspheres is -2.38 mV, and that of the Aga-PDA microspheres is -3.92 mV. The increase in the negative potential is due to the fact that the pKa of the catechol group in PDA is approximately 9.5, and partial deprotonation occurs at physiological pH (7.4) to form phenolate anions (-O-), resulting in an increase in the negative potential. In addition, due to the nano-scale rough surface of the Aga-PDA microspheres, the specific surface area is increased, allowing more phenolate anions to be exposed, thus enhancing the accumulation of negative charges. The negative potential on the surface of the Aga-PDA / Zn microspheres further increases to -5.39 mV. This is because the unique nano-flower stacked multi-level structure composite of the Aga-PDA / Zn microspheres has a larger specific surface area, exposing more deprotonated phenolate anions (-O-) at the interface, strengthening the accumulation of negative charges. At the same time, zinc ions bind to the phenolic hydroxyl groups in PDA through coordination to form stable Zn-O coordination bonds, reducing the pKa of the hydroxyl groups and promoting the release of protons more easily at physiological pH, increasing the surface negative charge density. The surface potential of HAP / Zn is -9.02 mV. This is because the isoelectric point (pI) of HSA is approximately 4.7. Under physiological pH (7.4) conditions, the surface carboxylic acid groups (-COO-) are completely deprotonated, while the amino groups (-NH3 + ) are partially protonated. Therefore, it has a net negative charge as a whole, resulting in a larger surface negative potential compared to the Aga-PDA / Zn microspheres, which also indicates the successful loading of HSA on the surface of the Aga-PDA / Zn microspheres.

[0134] Example 5 Blood compatibility and anticoagulant properties of HAP / Zn microspheres

[0135] The microspheres also need to have excellent blood compatibility when applied to hemoperfusion. The hemolysis rate is an index to measure the degree of damage of a certain substance or material to red blood cells, mainly used to evaluate its biocompatibility and blood safety, and is the golden standard index of blood compatibility. The standard requires that the hemolysis rate of direct contact materials should be < 5% (ISO 10993-4).

[0136] 5.1 Hemolysis rate

[0137] Direct method: Add 10 mL of normal saline into a centrifuge tube, and add 5 mg of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microspheres into the normal saline; the group with only 10 mL of normal saline added is set as the negative control group, and the group with 10 mL of deionized water added is set as the positive control group (three parallel groups are set for each group). Place all test tubes in a constant temperature water bath at 37 °C for incubation for 30 min. Dilute 4 mL of sodium citrate rabbit whole blood (New Zealand white rabbit) in 5 mL of normal saline, and take 0.2 mL of the diluted sodium citrate rabbit whole blood and add it to the incubated test tubes respectively. After mixing, place them in a constant temperature water bath at 37 °C for continued incubation for 1 h; pour the original solution into a centrifuge tube, centrifuge at a speed of 3000 r / min for 5 min, carefully aspirate 150 μL of the supernatant after centrifugation, add it to a 96-well plate, and measure the absorbance value at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0138] The hemolysis rate calculation formula is as follows: Hemolysis rate = (sample absorbance value - negative absorbance value) / (positive absorbance value - negative absorbance value).

[0139] The experimental results show that the hemolysis rate of the Aga group is 0.89%, the hemolysis rate of the Aga-PDA group is 0.97%, the hemolysis rate of the Aga-PDA / Zn group is 0.72%, while the hemolysis rate of the HAP / Zn group is only 0.16%, far lower than 5%. This indicates that the microspheres modified with HAS have better biocompatibility and hardly cause the rupture of red blood cells, suggesting that the HAP / Zn microspheres are materials with biological safety( Figure 7 a).

[0140] 5.2 Blood routine test

[0141] The adhesion performance of microspheres to blood cells was evaluated by hemocytometry. Weighed 5 mg of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microsphere samples and pre-soaked them in PBS solution for more than 2 h. Added 4 U / mL of sodium heparin solution to fresh rabbit whole blood, and then added 10 mmol / L of CaCl2, gently shaken to make it evenly distributed in the whole blood. Took 1 mL of the above whole blood and added it to the Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microsphere samples soaked in PBS. The glass bead group was used as the positive control group, and the blank control group was only added with rabbit whole blood (3 parallel groups were set for each group). Then gently inverted the PP tube to fully mix the sample with rabbit whole blood, and placed it in a water bath at 37 °C with a rotation speed of 60 rpm for incubation for 60 min. After the reaction, added 5 mmol / L of EDTA, and then used the blood cell analyzer BC-2800Vet (mindray, China) for detection.

[0142] After different microspheres were contacted with blood, the routine blood indices of the blood after contact were tested. White blood cell count is usually used to reflect the immune and inflammatory status, and red blood cell count is usually used to evaluate the oxygen-carrying capacity. The experimental results showed ( Figure 7 b, c), except that the white blood cell count in the glass bead positive group was significantly lower than that in the normal saline negative group, the white blood cell count and red blood cell count in the other groups basically did not change. This indicates that neither the Aga-PDA microspheres modified with dopamine, the Aga-PDA / Zn microspheres modified with dopamine zinc ammonium solution, nor the HAP / Zn microspheres modified with HAS will cause immune reactions and inflammation, and will not reduce the oxygen-carrying capacity of red blood cells.

[0143] Platelet count is usually used to evaluate the coagulation function. When the platelet count decreases or the morphology is abnormal, it may indicate that the material activates platelets and causes consumption. The platelet count in the glass bead positive control group was significantly lower than that in the normal saline negative group. The platelet counts in the Aga, Aga-PDA, and Aga-PDA / Zn microsphere groups also decreased compared with the negative group, from 85.7×10 9 / L decreased to 74×10 9 / L, 76×10 9 / L, and 72.3×10 9 / L, respectively. The platelet count in the HAP / Zn microsphere group modified with HAS protein was 83.3×10 9 / L, which was almost unchanged compared with the negative group. This indicates that the HAS protein corona on the surface of the HAP / Zn microspheres can change the interaction between the nanoflower microspheres and platelets, reduce platelet adhesion and activation, and thus further inhibit the occurrence of coagulation ( Figure 7 d).

[0144] 5.3 Whole blood clotting time experiment

[0145] Weigh 1 mg of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microsphere samples, incubate them in PBS buffer solution for at least 2 h, and then place the samples in a 96-well plate. Add 0.2 mol / L CaCl2 solution to sodium citrate rabbit whole blood (10 μL to 100 μL of whole blood), gently mix, and then pipette 50 μL of blood into the 96-well plate containing the material. Immediately add normal saline to rinse three times at regular intervals, take pictures to compare the coagulation effects of each group, and record the coagulation time in detail. Set three parallel groups for each group, and the group without adding the material is the blank control group.

[0146] Evaluate the anticoagulant ability of HAP / Zn microspheres by monitoring the coagulation time after direct contact between different microspheres and blood in the 96-well plate ( Figure 8 a). Under normal conditions, without any intervention, the blood in the blank group coagulates within 4.48 ± 0.05 min. The coagulation times of the Aga, Aga-PDA, and Aga-PDA / Zn groups did not change significantly, being 4.43 ± 0.07 min, 4.44 ± 0.05 min, and 4.45 ± 0.05 min, respectively. However, in the HAP / Zn group, due to the presence of HAS crown on the microsphere surface, it can effectively reduce platelet adhesion and activation, thus inhibiting coagulation, and the coagulation time of the HAP / Zn group increased to 5.40 ± 0.08 min ( Figure 8 b). The above results indicate that HAP / Zn microspheres have excellent anticoagulant performance and can be applied to hemoperfusion cartridges.

[0147] Example 6 Antibacterial properties of HAP / Zn microspheres

[0148] Relative bactericidal rate:

[0149] Weigh 5 mg of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microsphere samples and place them in a 48-well plate (the control group is without any sample), then add 500 μL of a bacterial (Escherichia coli or Staphylococcus aureus) suspension diluted to 1.0×10 3 cfu / mL. Place the plate in a 37 °C constant temperature shaker overnight (12 h), take 100 μL of the bacterial suspension from the plate and spread it on an agar plate (set three parallel groups for each sample). Invert the agar plate and incubate it in a 37 °C oven for 24 h, and finally calculate the bacterial mortality rate based on the number of colonies:

[0150] Relative bactericidal rate=(n c -n s ) / n c ×100% (2)

[0151] where n c is the number of bacteria in the control group, n s The number of bacteria in the experimental group

[0152] The bacterial growth curve method is mainly used to characterize the inhibitory effect of bactericidal materials on the dynamic proliferation process of bacterial populations under specific environmental conditions. Weigh 5 mg of Aga, Aga-PDA, Aga-PDA / Zn and HAP / Zn microspheres and place them in a 48-well plate (the control group does not place any sample), then add 500 μL of diluted to 1.0×10 3 cfu / mL of bacterial (Escherichia coli or Staphylococcus aureus) suspension, the well plate was placed in a 37°C constant temperature shaker for incubation, and the well plate was taken out at 0, 1, 2, 6, 12 and 24 h, respectively, and a microplate reader (Varioskan LUX, Thermo Fisher) was used to read the OD 600 Measure the absorbance at different times.

[0153] Aga, Aga-PDA, Aga-PDA / Zn and HAP / Zn microspheres were co-cultured with Escherichia coli and Staphylococcus aureus at 37°C (100 rpm) and then the OD values were measured at 0, 1, 2, 6, 12 and 24 h. 600nm ) Test the absorbance value of bacterial solution ( Figure 9 c). From the experimental results, it can be seen that the absorbance values of the Bare group, Aga group and Aga-PDA group increased significantly during the bacterial growth cycle, regardless of Escherichia coli and Staphylococcus aureus, which indicates that both bacteria have proliferated a lot during the growth cycle (24h). Figure 9 a). However, the growth curves of the Aga-PDA / Zn and HAP / Zn treatment groups did not change significantly during the bacterial growth cycle, indicating that PET-PDA / Zn and HAP / Zn microspheres have excellent inhibitory effects on the dynamic proliferation of Escherichia coli and Staphylococcus aureus ( Figure 9 b). At the same time, the PET-PDA / Zn and HAP / Zn microspheres treatment groups showed significant statistical differences from the other groups, p<0.0001. The above results show that PET-PDA / Zn and HAP / Zn microspheres not only have excellent spectral bactericidal properties against Gram-negative and Gram-positive bacteria, but also have excellent growth inhibition effects on the dynamic proliferation process of the two types of bacteria. Based on the excellent biocompatibility and anticoagulant properties of HAP / Zn microspheres, combined with the high porosity and high specific surface area characteristics given by its multi-level pore multi-level structure composite, it is expected that this material can provide abundant adsorption sites and efficient mass transfer channels for toxins (such as bilirubin) in the blood; it can be used in blood perfusion devices to achieve effective adsorption of toxin molecules in the body, while effectively reducing the risk of coagulation and bacterial infection.

[0154] Example 7 HAP / Zn Microsphere Bilirubin Clearance Performance

[0155] Bilirubin is the main product of human hemoglobin metabolism, which is transported by albumin to the liver for processing and excreted through bile. The normal total serum bilirubin level is 0.1 - 1.0 mg / dL -1 . However, people with liver failure cannot eliminate bilirubin in time. When the bilirubin concentration in the blood abnormally increases (hyperbilirubinemia), it may cause multi-system damage and even lead to death. Therefore, removing excessive bilirubin from the blood is of great significance to patients with liver failure and is also a major challenge in clinical practice.

[0156] To obtain a uniform bilirubin (BLB) solution, a certain amount of BLB was first dissolved in a small volume of 0.1 mol / L NaOH, and then diluted to 25, 50, 100, 200, 300, 400 μg / mL with PBS for standby.

[0157] Adsorption Kinetics: Weigh 1 mg of Aga, Aga-PDA, Aga-PDA / Zn, and HAP / Zn microsphere samples and add them to 2 mL of 200 μg / mL BLB solution. Conduct the adsorption experiment in the dark in a constant-temperature shaking water bath at 37 °C and 170 rpm. Take 1 mL of the supernatant at fixed times and measure the absorbance of the solution at a wavelength of 438 nm using a UV spectrophotometer until adsorption equilibrium is reached, and calculate the adsorption capacity through the standard curve (set 3 parallel groups for each group). The standard curve was determined as follows: Prepare a 200 μg / mL BLB standard solution using a volumetric flask and dilute it stepwise to 100, 50, 25, 12.5, 6.25, 3.125 μg / mL. Measure the absorption values at a wavelength of 438 nm using a UV spectrophotometer and plot the standard curve. The adsorption capacity can be calculated by Equation (1).

[0158] The adsorption equilibrium time of Aga-PDA / Zn microspheres for bilirubin ( Figure 10 a), it can be found that it reaches equilibrium after 120 min of adsorption. Therefore, 120 min of adsorption is used as the adsorption saturation time in subsequent adsorption experiments. The adsorption capacity of Aga microspheres is only 23.87 mg / g after 120 min of adsorption. After dopamine modification, the adsorption capacity of Aga-PDA microspheres increases to 112.21 mg / g ( Figure 10b) The increase in adsorption is mainly due to the following aspects: (1) the nanoscale rough structure formed by PDA on the surface of Aga microspheres through self-polymerization, and the higher total pore area and total pore volume of Aga-PDA microspheres can further increase the contact area of bilirubin, thereby improving the adsorption kinetics; (2) the aromatic ring in bilirubin and the π electron cloud of PDA form strong adsorption through π-π stacking, which is also the main driving force for bilirubin adsorption; (3) the hydroxyl (-OH) and amino (-NH2) in PDA can form a hydrogen bond network with the keto group (C=O) and imino group (-NH) of bilirubin.

[0159] The adsorption capacity of bilirubin by Aga-PDA / Zn microspheres was 140.00 mg / g, which was further improved compared with Aga-PDA. This is because in addition to the interaction between PDA and bilirubin, Aga-PDA / Zn microspheres also have the following advantages: (1) they can be adsorbed through the metal polyphenol network (Zn 2+ -PDA) structure 2+ It coordinates with the propionic acid carboxyl group (-COOH) of bilirubin to form a stable ternary complex, thereby further improving the adsorption efficiency of bilirubin; (2) The multi-level structure complex formed by the unique nanoflower microsphere stacking of Aga-PDA / Zn microspheres can provide more adsorption sites for bilirubin.

[0160] Figure 10 c is the adsorption of bilirubin by Aga, Aga-PDA and Aga-PDA / Zn microspheres after HSA was modified on the surface. The adsorption of bilirubin by Hag microspheres after Aga surface was modified with HSA was 21.79 mg / g, which was almost unchanged compared with blank Aga microspheres. This is because the adsorption capacity of Aga microspheres themselves is limited, resulting in insignificant differences. The adsorption of bilirubin by HAP microspheres decreased from 112.21 mg / g of Aga-PDA microspheres to 77.13 mg / g. This is because (1) after HSA was modified on the surface of Aga-PDA microspheres, it would occupy some active sites or block the pores of microspheres, resulting in a decrease in nonspecific adsorption originally dominated by PDA; (2) HSA (molecular weight of about 66 kDa, diameter of about 7 nm) is large in size, which may hinder the diffusion of bilirubin into the interior of Aga-PDA microspheres, thereby reducing mass transfer efficiency; (3) although free HSA can specifically bind to bilirubin through Sudlow site I / II (Ka≈10 7 -10 8 M -1 ), but PDA immobilization may lead to changes in the three-dimensional structure of HSA, reduced exposure of the hydrophobic cavity, and reduced specific binding ability.

[0161] It should be noted that the adsorption capacity of HAP / Zn microspheres for bilirubin is 149.32 mg / g, which is increased compared with Aga-PDA / Zn microspheres. This is mainly due to (1) Zn 2+ can coordinate with the histidine residues (such as His67 and His247) of HSA, fix its three-dimensional structure, prevent denaturation during the immobilization process, maintain the activity of the Sudlow site, and thus increase the specific recognition ability of HAP / Zn microspheres for bilirubin; (2) Zn that forms a ternary stable structure with PDA and HSA 2+ can further form coordination bonds with the carboxylic acid group (-COOH) of bilirubin or the nitrogen atom in the pyrrole ring and directly participate in the adsorption, thereby forming a stable multi-component structure; (3) The special nano-flower porous multi-level structure composite of PDA / Zn can effectively avoid pore blockage caused by HSA modification, thus ensuring the efficient mass transfer of bilirubin on the surface and inside of the microspheres.

[0162] Based on the in-situ construction of Aga-PDA / Zn multi-level structure composite microspheres with nano-flower microspheres, after its surface is modified with HSA, the active conformation of HSA can be stabilized through metal-ligand interactions, and at the same time, the multi-level pore structure of the microspheres can be maintained. This system realizes the efficient adsorption of bilirubin through the synergistic mechanism of specific binding of HSA and bilirubin, metal complexation of PDA / Zn, and non-specific adsorption such as π-π stacking and hydrogen bonding. Combining the anticoagulant performance of HAP / Zn microspheres and excellent antibacterial performance, the metal coordination-biological molecule synergistic modification strategy proposed in the present invention provides a new idea for developing highly efficient hemoperfusion materials with both anticoagulant and antibacterial dual functions by dual-regulating the specific / non-specific adsorption path.

[0163] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all of them should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Preparation method of zinc coordination polymer multi-level structure composite microspheres, characterized in that, The preparation method comprises the following steps: It is prepared by using natural polymer microspheres as a template and reacting with a zinc ammonium ethanol solution and dopamine.

2. The preparation method of the zinc coordination polymer multi - level structure composite microspheres according to claim 1, characterized in that, In the zinc ammonium ethanol, the concentration of zinc ions is 0.01 - 0.09 mol / L, and / or, in the zinc ammonium ethanol, the volume ratio of ethanol is 25 - 50 vol.%.

3. The preparation method of the zinc coordination polymer multi-level structure composite microspheres according to claim 1, characterized in that, The volume ratio of the packing volume of natural polymer microspheres to the volume of the zinc ammonium ethanol solution is 1:0.5 - 3.

4. The preparation method of the zinc coordination polymer multi-level structure composite microsphere according to claim 1, characterized in that The final concentration of dopamine is 1 - 5 mg / mL.

5. The preparation method of the zinc coordination polymer multi-level structure composite microsphere according to claim 1, characterized in that, The natural polymer microspheres are selected from any one or more of agarose microspheres, chitosan microspheres, albumin microspheres, gelatin microspheres, starch microspheres, or sodium alginate microspheres.

6. The preparation method of the zinc coordination polymer multi-level structure composite microsphere according to any one of claims 1-5, characterized in that, The preparation method comprises the following steps: Using natural polymer microspheres as a template, adding a zinc ammonium ethanol solution and dopamine, and performing a repeated cyclic oscillation reaction under constant temperature conditions.

7. A zinc coordination polymer multi - level structure composite microsphere prepared by the preparation method according to any one of claims 1 - 6.

8. The application of the zinc coordination polymer multi - level structure composite microsphere according to claim 7 in the aspect of an adsorbent material for a hemoperfusion device.

9. Preparation method of protein corona modified zinc coordination polymer multi-level structure composite microspheres, characterized in that, The preparation method comprises the step of reacting the zinc coordination polymer multi - level structure composite microsphere according to claim 7 with a protein.

10. The preparation method of the protein corona-modified zinc coordination polymer multi-level structure composite microspheres according to claim 9, characterized in that, The preparation method comprises the following steps: Adding the zinc coordination polymer multi - level structure composite microsphere into a protein solution, and performing a constant temperature oscillation reaction to obtain the product, wherein the mass ratio of the zinc coordination polymer multi - level structure composite microsphere to the protein is 1:0.1 - 1.

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