一种纳米多层级天然酶水凝胶及其制备方法

Abstract

本发明提供了一种纳米多层级天然酶水凝胶及其制备方法。本发明的纳米多层级天然酶水凝胶，是由有机单体、聚乙烯大分子链转移剂、天然酶通过可逆加成‑断裂链转移自由基聚合以及聚合诱导自组装的方法制备得到，在聚合反应过程中通过原位自组装形成包裹天然酶的微纳米组装体，该微纳米组装体通过缠结和相互作用而形成多层级物理凝胶，实现了高浓度、稳定的天然酶负载组装体在水凝胶内部的形成，提供了一种简单、原位、温和的原位天然酶保护方式，在保护天然酶稳定性的同时，可以保证多种酶之间的相互串联反应。此外，本发明的纳米多层级天然酶水凝胶，从微观到宏观一步制备实现，成分简单，条件温和，在生物医学领域将有较好的应用前景。

Description

Technical Field

[0001] This invention relates to the fields of polymer materials and biomedical materials, and more specifically, to a nano-multilayer natural enzyme hydrogel and its preparation method. Background Technology

[0002] Hydrogels are generally three-dimensional cross-linked network materials formed by hydrophilic polymers under covalent or non-covalent interactions. They possess excellent properties, including biocompatible mechanical properties and outstanding water retention capacity, keeping wounds moist and continuously absorbing exudate. Besides sealing wounds to prevent fluid loss and purulent accumulation, they also prevent scab formation, which promotes bacterial growth and infection. Furthermore, their timely biodegradation avoids secondary damage during dressing changes, making them ideal wound dressing materials. More importantly, through structural and physicochemical design and functional integration, hydrogels can be endowed with a wide range of biological functions, such as anti-inflammatory, antioxidant, antibacterial, and cell behavior regulation, while also improving the microenvironment of chronic wounds and promoting tissue regeneration. Currently, a large number of hydrophilic polymers (including natural ones) can be used to construct multifunctional hydrogel materials through physical or chemical cross-linking.

[0003] Enzymes are large protein molecules with biocatalytic activity and excellent biocompatibility, resulting in far fewer rejection reactions and side effects than conventional drugs. Currently, various enzyme macromolecules, such as glucose oxidase and catalase, have shown good efficacy in promoting wound healing. For example, lysozyme can break the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharides of the cell wall into soluble glycopeptides, leading to cell wall rupture and the release of contents, thus lysing bacteria. Glucose oxidase can utilize glucose to produce reactive oxygen species (ROS), achieving antibacterial effects against bacterial-infected wounds. It can also catalyze a cascade with catalase to further decompose excess ROS into water and oxygen, reducing the risk of wound inflammation.

[0004] However, the application of natural enzymes in the biomedical field also faces certain challenges. For example, natural enzymes are easily decomposed, have poor stability, and are not conducive to long-term solution storage and efficacy. Furthermore, synergistic enhancement and cascade catalysis between multiple enzymes are difficult to achieve at low concentrations, which limits the conversion of natural enzyme-based materials. Currently, a series of methods, such as polyvinyl alcohol modification, have been used to modify natural enzymes to overcome their stability limitations. However, chemical coupling has a potential impact on enzyme function, and the modification process is complex, with spatial limitations imposed by the cascade reaction. Therefore, simplifying enzyme modification and protection strategies while ensuring the functionality of natural enzymes could provide a better approach for realizing the application of natural enzyme cascade reactions in various biomedical fields.

[0005] Biological organisms possess natural nanoscale multi-layered structures. For example, the skin structure mainly consists of three layers: epidermis, dermis, and subcutaneous tissue. Based on this, developing nanoscale multi-layered hydrogels can achieve better biocompatibility and leverage the effects of materials with different dimensions. The strategy for constructing polymer hydrogels with nanostructures commonly employs a two-step approach: constructing the loaded nanoparticles and constructing the gel carrier. However, this method is limited by the concentration of nanoparticles, and it is relatively time-consuming and complex. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing nanoscale multilayered natural enzyme hydrogels. Polymerization-induced self-assembly (PISA) is a cutting-edge polymer self-assembly technology that can construct various polymer micro / nano-assembled materials in one step through the spontaneous assembly behavior of monomers during the polymerization process. This method has the advantages of high efficiency and convenience, and is particularly easy to achieve high-concentration nano-assemblies. The present invention utilizes a polymerization-induced assembly system to protect and further assemble unstable bio-enzyme catalysts through in-situ self-assembly, achieving a step-by-step assembly process from nanometer to micrometer to macroscopic levels, and synthesizing therapeutic enzyme-loaded micro / nano-assembled gels with size-selective permeability in one step.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a nano-multilayer natural enzyme hydrogel, which is prepared by organic monomers, polyethylene macro-CTA, and natural enzymes through a reversible addition-fragmentation chain transfer polymerization (RAFT) method and a polymerization-induced self-assembly (PISA) method.

[0009] In the preparation process of the nano-multilayer natural enzyme hydrogel, micro-nano assemblies encapsulating natural enzymes are formed. These micro-nano assemblies encapsulating natural enzymes form a multilayer physical gel through entanglement and interaction, thus obtaining a nano-multilayer natural enzyme hydrogel with a high concentration of micro-nano assemblies encapsulating natural enzymes inside.

[0010] The polyethylene macromolecular chain transfer agent is a polyethylene glycol trithioester derivative RAFT reagent, which is obtained by esterification reaction of polyethylene glycol monomethyl ether and a carboxyl-containing RAFT reagent.

[0011] Specifically, the carboxyl-containing RAFT reagent is 4-cyano-4-[[(ethylthio)thioketonemethyl]thio]valeric acid (CEPA); the organic monomer is hydroxypropyl methacrylate (HPMA).

[0012] Specifically, the natural enzymes include, but are not limited to, glucose oxidase (Gox), catalase (CAT), horseradish peroxidase (HRP), superoxide dismutase, and peroxidase.

[0013] Preferably, the natural enzyme is a combination of two or more enzymes that have a cascade effect, and the nano-multilayer natural enzyme hydrogel has the property of protecting the cascade effect of the enzyme, which can ensure the cascade effect of the enzyme in the system and play a protective role for the enzyme; in a preferred embodiment, the natural enzyme is a combination of glucose oxidase and catalase.

[0014] Secondly, the present invention also provides a method for preparing the above-mentioned nano-multilayer natural enzyme hydrogel, comprising the following steps: dissolving polyethylene macro-CTA, hydroxypropyl methacrylate (HPMA), and natural enzyme in water to obtain a polymer mixture, degassing the polymer mixture with an inert gas, and reacting it in a photoreactor under light irradiation at a wavelength of 405 nm to obtain a nano-multilayer natural enzyme hydrogel.

[0015] Specifically, the preparation process of the polyethylene macromolecular chain transfer agent is as follows: polyethylene glycol monomethyl ether, 4-dimethylaminopyridine (DMAP), CEPA, and N,N-dicyclohexylcarbodiimide (DCC) are dissolved in a good solvent, stirred and reacted, filtered and rotary evaporated, and the initiator is obtained by precipitation with a poor solvent.

[0016] Specifically, in the preparation process of the polyethylene macromolecular chain transfer agent, the good solvent used is anhydrous dichloromethane (DCM), and the poor solvent used is anhydrous diethyl ether.

[0017] Further, the preparation process of the polyethylene macromolecular chain transfer agent is as follows: polyethylene glycol monomethyl ether, CEPA, and DMAP are placed in a sealed flask, and DCC dissolved in DCM is added under nitrogen protection. The mixture is stirred for 48-60 hours, during which DCC is added again. The mixture is filtered, concentrated by rotary evaporation, and filtered again. The product is placed in diethyl ether to precipitate, and then the precipitate is dried in a fume hood. Finally, it is vacuum dried for 12-24 hours to obtain the product polyethylene macromolecular chain transfer agent PEG-CEPA macro-CTA.

[0018] To optimize the catalytic and gelling properties of the system, a bioorthogonal experimental design was used. In the polymer mixture, the equivalent ratio of hydroxypropyl methacrylate (HPMA) to polyethylene macrochain transfer agent (PEG-CEPA macro-CTA) was 300:1 to 900:1, the mass ratio of each natural enzyme to polyethylene macrochain transfer agent was 1:3000 to 1:15, and the mass concentration of hydroxypropyl methacrylate was 10%-70%.

[0019] Specifically, the inert gas used in the preparation method of the enzyme nano-multilayer natural enzyme hydrogel is nitrogen, argon, etc.

[0020] In a preferred embodiment, the natural enzyme is a combination of glucose oxidase and catalase. In the polymer mixture, the equivalent ratio of hydroxypropyl methacrylate to polyethylene macromolecular chain transfer agent is 400:1, the mass ratio of glucose oxidase, catalase to polyethylene macromolecular chain transfer agent is 1:60, and the mass concentration ratio of hydroxypropyl methacrylate is 60%.

[0021] Furthermore, the preparation method of the enzyme nano-multilayer natural enzyme hydrogel is as follows: PEG-CEPA macro-CTA, CAT, and GOx are completely dissolved and mixed in water. HPMA is added, and the mixture is vortexed and mixed. The system is sealed, and inert gas is introduced to purge air from the system for approximately 20-30 minutes. The system is then placed in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction is complete, the sealing rubber stopper is opened, allowing the system to come into contact with air, and the reaction is terminated by vortexing.

[0022] The present invention has the following beneficial effects:

[0023] The nanoscale multilevel natural enzyme hydrogel provided by this invention is formed by the in-situ reaction of polymers and natural enzymes through a polymerization-induced assembly system, resulting in an enzyme-loaded nanoscale multilevel hydrogel. During the polymerization process, in-situ self-assembly forms micro- and nano-assemblies, which then form a nanoscale multilevel physical gel through entanglement and interaction. This physical gel possesses a high concentration of micro- and nano-assemblies, and the addition of natural enzymes allows for the achievement of high-concentration and stable natural enzyme-loaded assemblies, further forming a hydrogel. This simple, in-situ, and mild method of protecting natural enzymes ensures the tandem reactions between multiple enzymes while preserving the stability of the natural enzymes. Furthermore, this nanoscale multilevel hydrogel is prepared in one step from micro to macro scale, with simple composition and mild conditions, showing promising application prospects in the biomedical field. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the preparation of the nano-multilayer natural enzyme hydrogel of the present invention;

[0025] Figure 2 The molecular weight was characterized by gel chromatography in Preparation Example 1;

[0026] Figure 3 The UV response peak results (309 nm) for Preparation Example 1 are shown.

[0027] Figure 4 The physical morphology of the nano-multilayer natural enzyme hydrogel PHCG@Gel prepared in Example 1 is shown in the photograph.

[0028] Figure 5 Physical morphology photograph of the fluorescently modified nano-multilayer natural enzyme hydrogel PHCG@Gel prepared in Example 2;

[0029] Figure 6 Transmission electron microscopy characterization image of the PHCG assembly prepared in Example 4;

[0030] Figure 7 Transmission electron microscopy characterization of the PHC assembly prepared in Example 7;

[0031] Figure 8 Transmission electron microscopy (TEM) image of the PHCG assembly prepared in Example 1;

[0032] Figure 9 UV absorption peak diagrams of CAT and GOx with fluorescence modification prepared for Example 2;

[0033] Figure 10 Confocal microscopy characterization of the fluorescently modified PHCG assembly prepared for Example 5;

[0034] Figure 11 Fluorescence spectra of CAT and GOx with fluorescence modification prepared for Preparation Example 2;

[0035] Figure 12 Fluorescence spectrum of the fluorescently modified PHCG assembly prepared in Example 5;

[0036] Figure 13 Three-dimensional image of the fluorescently modified PHCG@Gel prepared in Example 2 under a confocal microscope;

[0037] Figure 14 A schematic diagram illustrating the principle of evaluating glucose oxidase GOx and cascade enzyme activity;

[0038] Figure 15 Figure showing the GOx enzyme activity verification results of the PHG assembly prepared for Example 6;

[0039] Figure 16 Figure showing the CAT enzyme activity verification results of the PHC assembly prepared in Example 7;

[0040] Figure 17 Graph showing the effect of GOx and CAT cascade enzymes on the PHCG assembly prepared in Example 4;

[0041] Figure 18 The evaluation results of the protective effect of the PHCG assembly prepared for Example 4 on enzyme activity are shown in the figure.

[0042] Figure 19 The physical morphology of PHCG@Gel prepared in Example 1 is shown under a scanning electron microscope. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0044] The embodiments of the present invention are described below to further illustrate the invention in detail. It should be noted that the following examples are merely examples within a suitable scope, and the specific process parameters, etc., are for reference only and are not intended to limit the specific values ​​in the examples below. Those skilled in the art can select parameters within a suitable range according to actual needs based on the description herein.

[0045] Please see Figure 1 The nanoscale multilayer natural enzyme hydrogel of this invention is formed by the in-situ reaction of a polymer and a natural enzyme through a polymerization-induced assembly system, resulting in a nanoscale multilayer hydrogel loaded with the natural enzyme. During the polymerization reaction, the natural enzyme is self-assembled in situ to form micro-nano assemblies that encapsulate the natural enzyme. These micro-nano assemblies then form a nanoscale multilayer physical gel through entanglement and interaction. This simple, in-situ, and mild method of protecting natural enzymes ensures the tandem reactions between multiple enzymes and protects enzyme stability. The formation of the physical gel network allows for controllable and specific targeting of the enzyme at its functional sites, reducing potential side effects in application.

[0046] Preparation Example 1: Synthesis of PEG-CEPA macro-CTA

[0047] Polyethylene glycol monomethyl ether, CEPA, and DMAP were placed in a glass bottle, sealed, and DCC and DCM were added. The mixture was stirred for 48-60 hours, with DCC added as needed. The mixture was filtered, concentrated by rotary evaporation, and then precipitated three times in diethyl ether. Finally, the precipitate was dried under ventilation and vacuum to obtain PEG-CEPA macro-CTA. Figure 2-3As shown, the molecular weight test results of gel chromatography and the UV test results demonstrate the successful synthesis of the polyethylene macrochain transfer agent PEG-CEPA macro-CTA in this preparation example.

[0048] Preparation Example 2: Fluorescently Modified CAT and GOx Enzyme Solutions

[0049] Mix 50 μL of succinimide ester (BODIPY-FL NHS) dye solution (20 mM) with 5 mL of CAT enzyme solution (8 mg / mL), and separately mix 60 μL of boron dipyrrole methylene dye (BODIPY-R6G NHS) dye solution (20 mM) with 3 mL of GOx enzyme solution (7 mg / mL). Stir for 12 hours, then dialyze to obtain fluorescently modified CAT enzyme solution and fluorescently modified GOx enzyme solution.

[0050] Preparation Example 3: Preparation of PEG-HPMA Assembly Solution System

[0051] 0.03 g of the PEG-CEPA macro-CTA prepared in Example 1 was dissolved in 0.61 mL of water, and 0.326 mL of HPMA was added. The mixture was thoroughly mixed, and nitrogen gas was introduced to purge the air from the system for approximately 20 minutes. The system was then sealed and placed in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction was completed, the sealing rubber stopper was opened to allow the system to come into contact with air, and the reaction was terminated by vortexing, yielding a polymer assembly solution system formed by PEG-HPMA. The Tyndall effect was observed under laser irradiation, indicating that the system was successfully synthesized.

[0052] Preparation Example 4: Preparation of PHCG Assembly

[0053] 0.03 g of the PEG-CEPA macro-CTA prepared in Preparation Example 1 was thoroughly mixed with 0.2 mL of GOx enzyme solution (8 mg / mL) and 0.05 mL of CAT enzyme solution (200,000 U / mL), dissolved in 0.36 mL of water, and 0.326 mL of HPMA was added. The mixture was thoroughly mixed, and nitrogen gas was introduced to purge the air from the system for approximately 20 minutes. The system was then sealed and placed in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction was complete, the sealing rubber stopper was opened, allowing the system to come into contact with air. The reaction was then terminated by vortexing, yielding a solution of the enzymatic polymer assembly, which was designated as the PHCG nanoassembly.

[0054] Preparation Example 5: Preparation of PHCG assemblies with fluorescence modification

[0055] 0.03 g of PEG-CEPA macro-CTA prepared in Preparation Example 1, 0.143 mL of GOx enzyme solution (7 mg / mL) and 0.125 mL of CAT enzyme solution (8 mg / mL) prepared in Preparation Example 2 were dissolved in 0.342 mL of water. 0.326 mL of HPMA was added, and the mixture was thoroughly mixed. Nitrogen gas was purged into the system for approximately 20 minutes. The system was then sealed and placed in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction, the sealing rubber stopper was opened, allowing the system to come into contact with air. The reaction was terminated by vortexing, yielding a solution of fluorescently modified enzyme-encapsulated polymer assemblies. These assemblies are designated as fluorescently modified PHCG nanoassemblies.

[0056] Preparation Example 6: Preparation of PHG Assembly

[0057] Take 0.03 g of the PEG-CEPA macro-CTA prepared in Preparation Example 1, add 2 mg of GOx and 0.61 mL of water. After the system dissolves, add 0.326 mL of HPMA, mix thoroughly, and purge the air from the system with nitrogen gas for about 20 minutes. Seal the system and then place it in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction is complete, open the sealing rubber stopper, expose the system to air, vortex, and terminate the reaction to obtain an enzyme-enzyme polymer assembly solution, which is denoted as PHG nanoassemblies.

[0058] Preparation Example 7: Preparation of PHC Assemblies

[0059] Take 0.03 g of the PEG-CEPA macro-CTA prepared in Preparation Example 1, add 0.05 mL of CAT enzyme solution (200,000 U / mL) and 0.56 mL of water. After the system dissolves, add 0.326 mL of HPMA, mix thoroughly, and purge the air from the system with nitrogen gas for about 20 minutes. Seal the system and then place it in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction is complete, open the sealing rubber stopper, expose the system to air, vortex, and terminate the reaction to obtain a solution of enzyme-encapsulated polymer assemblies, which are denoted as PHC nanoassemblies.

[0060] Preparation Example 8: Preparation of PHG assemblies with fluorescence modification

[0061] Take 0.03 g of PEG-CEPA macro-CTA prepared in Preparation Example 1, add 0.143 mL of the fluorescently modified enzyme GOx (7 mg / mL) prepared in Preparation Example 2, and 0.467 mL of water. After the system dissolves, add 0.326 mL of HPMA, mix thoroughly, and purge the air from the system with nitrogen gas for about 20 minutes. Seal the system and then place it in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction is complete, open the sealing rubber stopper, expose the system to air, vortex, and terminate the reaction to obtain a solution of fluorescently modified enzyme-encapsulated polymer assemblies. The assemblies are denoted as fluorescently modified PHG nanoassemblies.

[0062] Preparation Example 9: Preparation of a fluorescently modified PHC assembly solution

[0063] Take 0.03 g of PEG-CEPA macro-CTA prepared in Preparation Example 1, add 0.125 mL of the fluorescently modified CAT enzyme solution (8 mg / mL) prepared in Preparation Example 2 and 0.485 mL of water. After the system dissolves, add 0.326 mL of HPMA, mix thoroughly, and purge the air from the system with nitrogen gas for about 20 minutes. Seal the system and then place it in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction is complete, open the sealing rubber stopper, expose the system to air, vortex, and terminate the reaction to obtain a solution of fluorescently modified enzyme-encapsulated polymer assemblies. The assemblies are denoted as fluorescently modified PHC nanoassemblies.

[0064] Example 1: Preparation of PHCG@Gel Enzyme-Enzyme Gel

[0065] 0.03 g of the PEG-CEPA macro-CTA prepared in Preparation Example 1 was thoroughly mixed with 0.1 mL of GOx enzyme solution (5 mg / mL) and 0.1 mL of CAT enzyme solution (5 mg / mL), dissolved in 0.41 mL of water, and 0.326 mL of HPMA was added. The mixture was thoroughly mixed, and nitrogen gas was introduced to purge the air from the system for approximately 20 minutes. The system was then sealed and placed in a photoreactor for reaction. The wavelength was set to 405 nm, the rotation speed to 800 rpm, and the reaction was allowed to proceed for 3 hours. After the reaction was complete, the sealed rubber stopper was opened, allowing the system to come into contact with air. The reaction was then terminated by vortexing, yielding a nano-multilayer natural enzyme hydrogel, denoted as PHCG@Gel, with the following physical morphology: Figure 4 As shown.

[0066] Example 2: Preparation of fluorescently modified PHCG@Gel enzyme-containing gel

[0067] 0.03 g of PEG-CEPA macro-CTA prepared in Preparation Example 1, 0.071 mL (7 mg / mL) of GOx enzyme solution with fluorescence modification prepared in Preparation Example 2, and 0.063 mL (8 mg / mL) of CAT fluorescent enzyme solution were dissolved in 0.476 mL of water. 0.326 mL of HPMA was added, and the mixture was thoroughly mixed. Nitrogen gas was purged to remove air from the system for approximately 20 minutes. The system was then sealed and placed in a photoreactor with a wavelength of 405 nm and a rotation speed of 800 rpm for 3 hours. After the reaction, the sealing rubber stopper was opened, allowing the system to come into contact with air. The reaction was terminated by vortexing, yielding a fluorescently modified nano-multilayer natural enzyme hydrogel, denoted as PHCG@Gel. Its physical morphology is shown in the figure. Figure 5 As shown.

[0068] Performance Testing

[0069] (1) Physical morphology of assemblies with different enzyme amounts observed by transmission electron microscopy

[0070] Ten µL of each of the enzyme-enzyme nanoassembly solutions prepared in Preparation Examples 4 and 7, and the enzyme-enzyme gel prepared in Example 1, were dispersed on the carbon film surface of a copper mesh. After drying, they were characterized using transmission electron microscopy. The results are as follows: Figure 6-8 As shown.

[0071] Depend on Figure 6-8 It was observed that polymers and natural enzymes formed enzymatic nanoassemblies through polymerization-induced self-assembly, which facilitated the formation of more three-dimensional gel structures and exhibited a morphological transformation from microspheres and vesicles to worm-like structures. Furthermore, it was observed that when the system formed a gel in situ in one step, the number of worm-like assemblies inside increased significantly, indicating that worm-like assemblies are beneficial for the formation of physical gels during in-situ assembly.

[0072] (2) UV spectrophotometer was used to verify the fluorescence modification of GOx and CAT enzymes.

[0073] Take 2 mL of the CAT and GOx enzyme solutions prepared in Preparation Example 2, and detect their corresponding response peaks under a UV spectrophotometer. Water was used as the standard baseline solution. The results are as follows: Figure 9 show.

[0074] Depend on Figure 9 As can be seen, the CAT and GOx prepared in Preparation Example 2 have response peaks at approximately 503 nm and 522 nm, respectively, indicating that the fluorescent groups were successfully modified onto the corresponding CAT and GOx enzyme protein structures.

[0075] (3) Confocal microscopy analysis was used to observe the distribution of enzymes in the PHCG assembly.

[0076] Take 10 μL of the fluorescently modified PHCG assembly solution prepared in Example 5, add it dropwise onto a glass slide, cover with a coverslip, allow to evaporate overnight at room temperature, seal the slide, and store the sample for confocal imaging. The results are as follows. Figure 10 As shown.

[0077] Depend on Figure 10 It can be observed that compared with the control group (the CAT and GOx enzyme solution mixture prepared in Example 2), CAT and GOx in the PHCG assembly are co-localized, indicating that CAT and GOx are encapsulated in the nano-assemblies formed by the system.

[0078] (4) Detection of enzyme localization in PHCG enzyme vesicles by fluorescence spectroscopy

[0079] Take 1 mL of the fluorescently modified CAT and GOx enzyme solutions obtained in Preparation Example 2 into four-sided glass cuvettes, and scan their fluorescence spectra under a fluorescence spectrophotometer. The results are as follows. Figure 11 As shown.

[0080] Depend on Figure 11 The results show that the fluorescence spectra of the fluorescently modified CAT and GOx overlap, indicating that fluorescence resonance energy transfer can occur.

[0081] Subsequently, the excitation wavelength was fixed at 495 nm, the excitation wavelength for CAT carrying fluorescence. The CAT-GOx natural enzyme mixture and the fluorescently modified PHCG assembly solution prepared in Example 5 were scanned in the 505-650 nm range to obtain the corresponding spectra. All the above measurements were performed under the set instrument conditions: Slit 2.5, Voltage 700 V, Speed ​​240. The data were then processed to obtain... Figure 12 The results are shown.

[0082] Depend on Figure 12 The results show that, compared with the natural enzyme solution, there is a fluorescence resonance energy transfer phenomenon between CAT and GOx in the PHCG nanoassembly solution prepared in Preparation Example 5, indicating that CAT and GOx were successfully encapsulated in the polymer-formed nanoassembly in Preparation Example 5.

[0083] (5) The morphology of PHCG@Gel gel was characterized by confocal microscopy.

[0084] Using tweezers, take an appropriate amount of the PHCG@Gel prepared in Example 2 after dialysis and place it on a glass slide. Cover with a coverslip, flatten, and leave at room temperature overnight. Seal the slide to preserve the sample for confocal imaging. The imaging results are as follows: Figure 13 As shown.

[0085] Depend on Figure 13The enzyme with fluorescent modification was observed to be uniformly distributed in the three-dimensional system, indicating that the enzyme nanoassemblies were uniformly dispersed in the formed physical gel.

[0086] (6) Validation of GOx enzyme activity of the enzyme-enzyme nanoassembly PHG

[0087] Glucose oxidase (GOx) oxidizes glucose into gluconic acid and hydrogen peroxide (H₂O₂). For example... Figure 14 As shown, when H2O2 is present in the system, horseradish peroxidase (HRP) catalyzes the conversion of Apple Red dye into a fluorescent halogenated reagent. The activity of GOx can be verified by detecting the amount of H2O2. The H2O2 probe Apple Red was prepared into a 20 mM system. HRP solid powder (300 U / mg) was prepared into a 0.4 U / mL enzyme solution using phosphate-buffered saline (PBS). The probe was then diluted 250-fold with the HRP enzyme solution. 50 μL of 30 mg / mL glucose solution was mixed with 100 μL of the PHG assembly solution prepared in Preparation Example 6 (diluted 200-fold). The mixture was incubated at 37 °C for 30 minutes, followed by centrifugation at 4 °C and 8000 rpm for 5 minutes. 150 μL of the supernatant was transferred to a 96-well plate. 50 μL of the probe-enzyme mixture was added, and the plate was incubated for 10 minutes. Two replicates were prepared. Fluorescence was detected using a multifunctional microplate reader (excitation wavelength set at 530 nm, scanning wavelength range of 550 nm-620 nm). Following the same procedure, the PHG assembly solution from Example 6 was replaced with PBS as a negative control, and a 200-fold diluted natural GOx enzyme solution was used as a positive control. The data analysis is as follows: Figure 15 As shown.

[0088] Depend on Figure 15 It can be observed that both the PHG group and the natural GOx enzyme solution group have significant fluorescence values ​​compared with the PBS positive control group, and the fluorescence intensity of the PHG group and the natural GOx enzyme solution group is similar, indicating that the PHG group has good GOx enzyme activity, which means that GOx can still exert its catalytic activity well when encapsulated in nano-assemblies.

[0089] (7) Verification of CAT enzyme activity of enzyme-enzyme-encapsulated nanoassemblies PHC

[0090] Prepare a 20 mL glass bottle, add a stir bar, and add 10 mL of 1 mM H2O2. Submerge the dissolved oxygen meter probe below the liquid surface and fix it in place. Once the reading stabilizes, add the corresponding test system and observe the change in oxygen production over 5 minutes. The test system consisted of 25 μL, 50 μL, and 75 μL of a 100-fold diluted PHC assembly solution prepared in Example 7, and 50 μL of light-exposed natural CAT enzyme solution. The change in oxygen production over 5 minutes was observed, and the results are as follows: Figure 16 show.

[0091] Depend on Figure 16 The results show that the PHC assembly exhibits good CAT enzyme activity in a concentration-dependent manner. This indicates that the CAT-encapsulated PHC assembly can effectively exert CAT enzyme activity.

[0092] (8) Verification of the cascade effect of the enzyme assembly

[0093] GOx oxidizes glucose to gluconic acid and H2O2, while CAT catalyzes the decomposition of H2O2. The cascade effect of GOx and CAT can be determined by detecting the intermediate product H2O2. Figure 14 As shown, the cascade effect of GOx and CAT was verified by detecting the amount of H2O2. Following the performance test (6), the Applex Red-HRP probe-enzyme mixture was prepared. 100 μL of the PHCG assembly solution prepared in Preparation Example 4 (diluted 200 times) and the PHG assembly solution prepared in Preparation Example 6 (diluted 200 times) were respectively mixed with 50 μL of glucose solution (30 mg / mL) and incubated at 37 °C for 30 minutes. Then, the mixture was centrifuged at 4 °C and 8000 rpm for 5 minutes. 150 μL of the supernatant from each group was added to a well plate. 50 μL of the probe-enzyme mixture was added, and the mixture was incubated for 10 minutes. Two replicates were set for each group. Fluorescence was detected using a microplate reader (excitation wavelength set to 530 nm, scanning wavelength range 550 nm-620 nm). The PBS group was used as a negative control in the same manner. Results analysis is as follows. Figure 17 As shown.

[0094] Depend on Figure 17 The higher fluorescence intensity of the PHG group indicates the generation of more H2O2, while the fluorescence intensity of the PHCG group is significantly lower than that of the PHG group, indicating that some of the H2O2 generated by GOx is catalytically decomposed by CAT, and CAT and GOx in the PHCG assembly are successfully cascaded.

[0095] (9) Verification of the protective effect of the enzyme assembly on enzyme activity

[0096] The GOx natural enzyme solution, the GOx and CAT natural enzyme mixture, the PHCG assembly solution prepared in Preparation Example 4, and the PHG assembly solution prepared in Preparation Example 6 were placed at room temperature, and their enzyme activity was verified at 0, 1, 2, and 4 weeks after placement. The enzyme activity was tested using the methods described in "Performance Testing (6) and (8)" above, and the product H2O2 catalyzed by GOx was measured. The fluorescence ratio was plotted, and the results are as follows. Figure 18 show.

[0097] As the storage time increased, the fluorescence intensity difference between the natural GOx enzyme solution group and the PHG group became increasingly larger, indicating a difference in their ability to catalyze the production of H2O2. The PHG group showed higher fluorescence intensity than the natural GOx enzyme solution group, while the PHCG group showed lower fluorescence intensity than the mixed GOx and CAT enzyme solution group. This suggests that the PHCG group has a better ability to cascade and scavenge H2O2 than the mixed enzyme solution. These results indicate that encapsulating the enzyme in a polymer assembly helps to protect enzyme activity to a certain extent and improves its cascade effect.

[0098] (10) Observation of the physical morphology of PHCG@Gel using scanning electron microscopy

[0099] Take an appropriate amount of PHCG@Gel prepared in Example 1, freeze it at -80 ℃ for 2 hours, then freeze-dry it in a vacuum freeze dryer for 24 hours, then take it out, immerse it in liquid nitrogen for a few seconds, freeze-break it to obtain the gel cross section, place it on a stage with conductive adhesive with the cross section facing up, coat it with a carbon film, and observe the physical morphology of the gel under a scanning electron microscope.

[0100] Figure 19 The physical cross-section of the PHCG@Gel prepared in Example 1 is shown under a scanning electron microscope, indicating that the gel has a good network porous structure.

[0101] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the above examples, and various exploratory changes can be made according to the inventive purpose of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made based on the methods and techniques disclosed in the present invention should be considered equivalent substitutions, and all those within the spirit and principles of the present invention should be covered by the present invention.

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