Bionic nano-enzyme hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses bionic nano-enzyme hydrogel and a preparation method thereof. The hydrogel is formed by compounding mesoporous silica nanoparticles loaded with glucose oxidase and copper-doped carbon dots with a carboxymethyl chitosan matrix. According to the hydrogel, glucose consumption, active oxygen regulation and control and oxygen generation are realized through cascade catalytic reaction; the broad-spectrum antibacterial and anti-inflammatory effects are realized; the biocompatibility is good. Animal experiments show that the hydrogel can remarkably promote healing of diabetic wounds and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a kind of biomimetic nano-enzyme hydrogel and its manufacturing method. BACKGROUND

[0002] Diabetes is a global chronic metabolic disease, and the number of patients is increasing year by year. Diabetic chronic wound is one of the common and serious complications of diabetes, which seriously affects the quality of life of patients, and even can lead to amputation and other serious consequences, bringing great challenges to global public health system.

[0003] The healing process of diabetic chronic wound is hindered by many factors. At the molecular level, the accumulation of advanced glycation end products (AGEs) and oxidative stress induced by persistent high glucose environment can destroy the skin barrier function, inhibit the migration of fibroblasts, and lead to the decomposition of extracellular matrix (ECM) components and the degradation of growth factors. At the cellular microenvironment level, the polarization advantage of M1 macrophages inhibits the migration ability of keratinocytes through the TNF-α / IL-6 / STAT3 signaling axis, while the imbalance of Th17 / Treg immune homeostasis exacerbates the local inflammatory cascade reaction. At the tissue and organ level, the abnormal degradation of hypoxia-inducible factor-1α (HIF-1α) hinders angiogenesis, and multiple drug-resistant bacteria form a biofilm barrier through quorum sensing mechanism, escape from host immune clearance, further inhibit neutrophil function, promote the evolution of bacterial drug resistance, and form a vicious cycle.

[0004] Currently, the methods for clinical treatment of diabetic chronic wound include surgical debridement, negative pressure wound therapy and local antibiotic application, but these methods have limited effect on the regulation of deep pathological mechanisms, are difficult to achieve multi-target synergistic regulation, and cannot effectively break the obstacles of wound healing. At the same time, traditional wound dressings such as gauze and sponge have problems such as inability to provide significant anti-infective effect, lack of moisturizing effect, easy to cause tearing and pain when removed, and unsatisfactory treatment effect for diabetic chronic infected wounds.

[0005] Nanomaterials have shown certain potential in the field of diabetic wound treatment due to their unique physicochemical properties such as good biocompatibility, good antibacterial and anti-inflammatory effect, and large specific surface area. However, when used alone, nanomaterials have problems such as difficulty in absorbing wound exudate and inability to achieve sustained drug release. Although hydrogel dressings have good hydrophilicity, biocompatibility and shape adaptability, single hydrogel still has insufficient functions in dealing with the complex pathological environment of diabetic wounds.

[0006] Therefore, a kind of biomimetic nano-enzyme hydrogel and its manufacturing method become a problem that people urgently need to solve. SUMMARY

[0007] The technical problem to be solved by the present invention is to provide a biomimetic nanozyme hydrogel and a method for preparing the same. The biomimetic nanozyme hydrogel with cascade catalytic activity can simultaneously achieve multiple functions such as glucose consumption, reactive oxygen regulation, antibacterial and anti-inflammatory, and promotion of tissue regeneration, effectively promoting the healing of diabetic wounds.

[0008] To solve the above technical problems, the present invention provides a technical solution: a method for preparing a biomimetic nanoenzyme hydrogel, comprising the following steps:

[0009] Step 1: Synthesis of Cu-CDs: Chlorhexidine gluconate and copper nitrate were mixed, reacted by a one-step hydrothermal method, dialyzed and purified, and then freeze-dried for use;

[0010] Step 2: Synthesis of MSN: Synthesize MSN using a template method and modify the surface by amino group to obtain MSN-NH2;

[0011] Step 3: Preparation of GCM composites: Cu-CDs and GOx were loaded into MSN-NH2 to form nanomaterials with cascade catalytic activity;

[0012] Step 4: Preparation of biomimetic nanozyme hydrogel: GCM was mixed with carboxymethyl chitosan solution to form a hydrogel through physical cross-linking.

[0013] Furthermore, the preparation method of the Cu-CDs is as follows:

[0014] 5 mL of chlorhexidine gluconate solution was mixed with 30 mg of copper nitrate solution, and the mixture was hydrothermally reacted at 180°C for 6 hours. After purification by dialysis, the mixture was freeze-dried to obtain light yellow Cu-CDs powder.

[0015] Furthermore, the preparation method of the MSN-NH2 is as follows:

[0016] MSN was prepared by sol-gel method with hexadecyltrimethylammonium bromide as template and ethyl orthosilicate as silicon source, and then modified with 3-aminopropyltriethoxysilane to obtain MSN-NH2.

[0017] Furthermore, the preparation method of the GCM composite material is as follows:

[0018] 10 mg of MSN-NH2 was dispersed in 5 mL of deionized water, 5 mg of Cu-CDs was added, and the mixture was stirred for 2 h. Then 0.24 mg of GOx was added and the mixture was stirred for another 2 h. The GCM complex was obtained after centrifugation and washing.

[0019] Furthermore, the preparation method of the hydrogel is as follows:

[0020] 90mg carboxymethyl chitosan was dissolved in 10mL deionized water, and the prepared GCM complex was added, stirred uniformly and then gelled to obtain a composite hydrogel.

[0021] Further, the particle size of the Cu-CDs is 5±2nm, and the surface Zeta potential is +15.2±0.35mV.

[0022] The application also provides a bionic nano-enzyme hydrogel for promoting healing of diabetic wounds, which is prepared based on the preparation method of the bionic nano-enzyme hydrogel.

[0023] Compared with the prior art, the application has the following advantages:

[0024] 1. Multifunctional synergistic treatment:

[0025] 1) Glucose oxidase catalyzes local glucose in the wound to generate H2O2 and gluconic acid, thereby reducing the pH value;

[0026] 2) Cu-CDs have peroxidase-like activity and can catalyze H2O2 to generate hydroxyl radicals (·OH), thereby playing an antibacterial role;

[0027] 3) At a low H2O2 concentration, Cu-CDs exhibit catalase-like activity and decompose H2O2 to generate oxygen, thereby improving tissue hypoxia.

[0028] 2. Excellent biocompatibility:

[0029] 1) Neither the hydrogel matrix nor the nanomaterials are cytotoxic;

[0030] 2) They can promote migration of vascular endothelial cells and fibroblasts;

[0031] 3) They can regulate polarization of macrophages from M1 type to M2 type, thereby reducing inflammatory reactions.

[0032] 3. Good clinical application performance:

[0033] 1) They can adhere to various tissue surfaces and adapt to irregular wound shapes;

[0034] 2) They can absorb wound exudates and maintain a suitable moist environment.

[0035] 4. The preparation process is simple and easy to scale up. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a solution before and after synthesis and an ultraviolet characterization graph.

[0037] Figure 2 is a Zeta potential graph of different materials. Different materials are dispersed in deionized water, and a Zeta potential instrument is used to detect the potential.

[0038] Figure 3 are transmission electron microscope (TEM) observation and mapping element analysis diagram of surface morphology of different materials. Among them, figures A-B are CuCDs and MSN transmission electron microscope morphology diagram; figures C-D are CM transmission electron microscope morphology diagram and mapping element analysis (figure E); figures F-G are GCM transmission electron microscope morphology diagram and mapping element analysis (figure H).

[0039] Figure 4 are CuCDs fluorescence performance diagram. (A) CuCDs, CM, GCM ultraviolet visible spectrum diagram; (B) CuCDs fluorescence spectrum diagram under different excitation wavelengths.

[0040] Figure 5 are Fourier transform infrared spectroscopy (FITR) diagrams of different materials.

[0041] Figure 6 are GCM glucose oxidase activity diagrams. (A) UV-visible absorption spectrum of ABTS under different treatment conditions in the presence of HRP; (B) pH value of different reaction solutions over time.

[0042] Figure 7 are GCM peroxidase activity diagrams.

[0043] Figure 8 are agar diffusion method detection of CuCDs and CM antibacterial activity against E. coli and S. aureus diagrams.

[0044] Figure 9 are spread plate method detection of CuCDs and CM antibacterial activity against E. coli, S. aureus under different concentrations diagrams.

[0045] Figure 10 are GCM antibacterial rate diagrams after 24 hours of co-culture with E. coli and S. aureus under different concentrations. Wherein * represents p<0.05; ** represents p<0.01; *** represents p<0.001; **** represents p<0.0001.

[0046] Figure 11 are E. coli, S. aureus and GCM co-culture treatment ultrastructure change and protein microleakage determination results diagrams. (A) SEM morphology diagram of bacteria after treatment; (B) Coomassie brilliant blue protein quantitative analysis after treatment. * represents p<0.05; ** represents p<0.01.

[0047] Figure 12This is the evaluation diagram of the inhibitory effect of GCM on bacterial biofilm. (A) Crystal violet staining results of E. coli and S. aureus biofilms under different treatments; (BC) After ethanol decolorization, at OD 590 The absorbance values ​​were measured and quantitatively analyzed, * indicates p < 0.05.

[0048] Figure 13 Figures show the results of a cytocompatibility assessment. (A) L929 cell viability after treatment with different materials (assessed after 24 hours of co-culture with raw materials, CuCDs, and CM); (B) L929 cell viability after 24 hours of co-culture with GCM; (C) Quantitative analysis of live / dead staining of L929 cells treated with different materials using ImageJ; green represents live cells, and red represents dead cells. Scale bar, 100 μm.

[0049] Figure 14 Figures show the results of an in vitro wound healing assay. (A-B) Inverted microscopic images of MUVEC cells treated with GCM and a control group at 0 and 24 hours, respectively, and quantitative analysis of wound healing rates using ImageJ. (B-D) Inverted microscopic images of L929 cells treated with GCM and a control group at 0, 24, and 48 hours, respectively, and quantitative analysis of wound healing rates using ImageJ. Scale bar, 100 μm. ** indicates p < 0.01.

[0050] Figure 15 Figure 2 shows the expression of in vitro inflammatory factors. (A, E) represent Arg-1, IL-10, IL-22, INOS, and TNF-a, respectively. ns indicates no statistical difference, ** indicates p < 0.01, and *** indicates p < 0.001.

[0051] Figure 16 Scanning electron micrographs and EDS elemental analyses of the synthesized CMCS, CM@CMCS, and GCM@CMCS. (AC) SEM images of CMCS, CM@CMCS, and GCM@CMCS, scale: 100 μm; (DF) Pore analysis images of CMCS, CM@CMCS, and GCM@CMCS; (GL) EDS elemental analysis of GCM@CMCS, scale: 100 μm.

[0052] Figure 17 3D representations of CMCS, CM@CMCS, and GCM@CMCS. CT images of (A) CMCS, (B) CM@CMCS, and (C) GCM@CMCS.

[0053] Figure 18 This is a picture of GCM@CMCS adhesion test. It includes mouse heart, liver, spleen, lung, kidney, and pig skin.

[0054] Figure 19 Figure 2 shows the relative activity of S. mutans, E. coli, and S. aureus after treatment with CMCS, CM@CMCS, and GCM@CMCS. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0055] Figure 20 This is the live-dead staining of MUVEC cells treated with different materials. Green represents live cells, and red represents dead cells.

[0056] Figure 21 The expression of inflammatory factors in vitro. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0057] Figure 22 Diagram of the preparation of a diabetic model in C57BL / 6 mice. (A) Diabetes model preparation flow chart; (B) Changes in mouse body weight before and after modeling; (C) Comparison of blood glucose levels between the model and control groups. ns indicates no statistically significant difference; **** indicates p < 0.0001.

[0058] Figure 23 These are the healing status diagrams of the mouse wound sites on days 1, 3, 7, 12, and 14, respectively.

[0059] Figure 24 This is a quantitative analysis of wound healing rates under different treatments using ImageJ. ** indicates p < 0.01.

[0060] Figure 25 H&E and Masson's trichrome staining of a skin section from a mouse wound. H&E and Masson's trichrome staining of a skin section from a mouse wound. Scale bar, 100 μm.

[0061] Figure 26 Quantitative analysis of collagen in mouse wounds using Masson's trichrome staining. * indicates p < 0.05, *** indicates p < 0.0001.

[0062] Figure 27 This is a picture of the plate coating after bacterial culture of local wound tissue of mice under different treatment time periods and different treatments.

[0063] Figure 28 This is an H&E staining of major organs. Hearts, livers, spleens, lungs, and kidneys from different groups of mice were fixed, embedded, and stained with H&E 14 days after the experiment. Scale bar, 100 μm.

[0064] Figure 29Figure 2 shows the expression of inflammatory factors in vivo. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. DETAILED DESCRIPTION

[0065] Various exemplary embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention.

[0066] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

[0067] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered part of the specification.

[0068] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0069] The biomimetic nanozyme hydrogel and its preparation method of the present invention are further described in detail below with reference to the accompanying drawings.

[0070] Combined with attachment Figure 1-29 , the present invention is introduced in detail.

[0071] A method for preparing a biomimetic nanozyme hydrogel comprises the following steps:

[0072] 1. Preparation of copper-doped carbon dots (Cu-CDs):

[0073] 5 mL of chlorhexidine gluconate solution was mixed with 30 mg of copper nitrate solution and hydrothermally reacted at 180 °C for 6 h. After purification by dialysis, the mixture was freeze-dried to obtain light yellow Cu-CDs powder.

[0074] 2. Preparation of amino-modified mesoporous silica (MSN-NH2):

[0075] MSN was prepared by sol-gel method using hexadecyltrimethylammonium bromide as template and ethyl orthosilicate as silicon source, and then modified with 3-aminopropyltriethoxysilane to obtain MSN-NH2.

[0076] 3. Preparation of GOx@CuCDs@MSN (GCM) composite nanomaterials:

[0077] 10 mg of MSN-NH2 was dispersed in 5 mL of deionized water, 5 mg of Cu-CDs was added, and the mixture was stirred for 2 h. Then, 0.24 mg of glucose oxidase (GOx) was added and stirred for another 2 h. The GCM complex was obtained after centrifugation and washing.

[0078] 4. Preparation of carboxymethyl chitosan (CMCS) hydrogel:

[0079] 90 mg of carboxymethyl chitosan was dissolved in 10 mL of deionized water, and the GCM complex prepared in step 3 was added. The mixture was stirred evenly and allowed to stand to form a gel to obtain a GCM@CMCS composite hydrogel.

[0080] Preferably, in step 1, the particle size of the Cu-CDs is 5±2 nm, and the surface zeta potential is +15.2±0.35 mV.

[0081] Preferably, in step 3, the mass ratio of GOx to Cu-CDs is 1:20.

[0082] The present invention also provides a bionic nanozyme hydrogel for promoting diabetic wound healing, which is prepared by the above-mentioned method for preparing the bionic nanozyme hydrogel.

[0083] The present invention further provides an application of a bionic nanoenzyme hydrogel for promoting diabetic wound healing in the preparation of a diabetic wound dressing.

[0084] The specific implementation process of the biomimetic nanoenzyme hydrogel and its preparation method of the present invention is as follows:

[0085] Example 1: Preparation and characterization of Cu-CDs

[0086] Dissolve 5 mL of a 20 wt% chlorhexidine gluconate solution and 30 mg of copper nitrate in 20 mL of deionized water and stir for 30 minutes. The mixture was transferred to a 50 mL polytetrafluoroethylene reactor and reacted at 180°C for 6 hours. After cooling, the reaction solution was centrifuged at 5000 rpm for 10 minutes. The supernatant was dialyzed using a 1000 Da dialysis bag for 48 hours. The dialyzate was freeze-dried to obtain a pale yellow Cu-CDs powder.

[0087] Transmission electron microscopy revealed that the Cu-CDs were spherical, with a particle size distribution of 1-10 nm and an average size of 5 nm. Zeta potential measurements revealed a surface charge of +15.2 mV. Fluorescence spectroscopy revealed that the Cu-CDs emitted the strongest fluorescence at an excitation wavelength of 330 nm.

[0088] Example 2: Preparation of GCM@CMCS hydrogel

[0089] 1) Dissolve 0.6 g CTAB and 0.15 g triethanolamine in 40 mL deionized water and stir at 80°C for 30 minutes;

[0090] 2) Add 4.0 g of TEOS dropwise and continue stirring for 4 hours;

[0091] 3) Collect the precipitate by centrifugation and wash it three times with ethanol;

[0092] 4) Disperse the precipitate in 50 mL of 1% ammonium nitrate ethanol solution and reflux at 60°C for 12 hours;

[0093] 5) Collect the MSNs by centrifugation and react them with toluene and APTES at 110°C for 6 hours to obtain MSN-NH2;

[0094] 6) Mix 10 mg of MSN-NH2 and 5 mg of Cu-CDs in 5 mL of water and stir for 2 hours;

[0095] 7) Add 0.24 mg of GOx, continue stirring for 2 hours, and centrifuge to obtain the GCM complex;

[0096] 8) Dissolve 90 mg of carboxymethyl chitosan in 10 mL of water, add the GCM complex, stir evenly, and let stand to form a gel.

[0097] Example 3: In vitro antibacterial experiment

[0098] GCM@CMCS hydrogel was co-cultured with Escherichia coli and Staphylococcus aureus for 24 hours. The results showed:

[0099] 1) The antibacterial rate against Escherichia coli is 93.7%;

[0100] 2) The antibacterial rate against Staphylococcus aureus was 97.7%;

[0101] 3) It can effectively inhibit the formation of bacterial biofilm, with an inhibition rate of 25%.

[0102] Example 4: Diabetes wound healing experiment

[0103] 1) Animal model: STZ-induced type 1 diabetic mice were induced with a 9 mm full-thickness skin incision on the back;

[0104] 2) Experimental groups:

[0105] Control group: covered with sterile gauze;

[0106] Treatment group: GCM@CMCS hydrogel covered and replaced every 3 days;

[0107] 3) Experimental results:

[0108] The healing rate on day 14 was 73.3% in the control group and 98.7% in the treatment group;

[0109] The collagen deposition in the treatment group was denser and the inflammatory cell infiltration was reduced;

[0110] The expressions of proinflammatory factors TNF-α and IL-1β were significantly decreased in the treatment group.

[0111] Example 5: Biocompatibility Evaluation

[0112] 1) Cytotoxicity assay: Co-cultured with L929 cells for 24 hours, cell viability >95%;

[0113] 2) H&E staining of major organs: No obvious pathological changes were found in the heart, liver, spleen, lung, and kidney.

[0114] The above examples show that the biomimetic nanozyme hydrogel provided by the present invention has excellent antibacterial, anti-inflammatory and healing-promoting properties, can significantly promote the healing of diabetic wounds, and has good biocompatibility.

[0115] The present invention and its embodiments are described above. This description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs structures and embodiments similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic nanozyme hydrogel, characterized in that: The following steps are involved: Step 1: Synthesis of Cu-CDs: Chlorhexidine gluconate and copper nitrate were mixed, reacted by a one-step hydrothermal method, dialyzed and purified, and then freeze-dried for use; Step 2: Synthesis of MSN: Synthesize MSN using a template method and modify the surface by amino group to obtain MSN-NH2; Step 3: Preparation of GCM composites: Cu-CDs and GOx were loaded into MSN-NH2 to form nanomaterials with cascade catalytic activity; Step 4: Preparation of biomimetic nanozyme hydrogel: GCM was mixed with carboxymethyl chitosan solution to form a hydrogel through physical cross-linking.

2. The method for preparing a biomimetic nanozyme hydrogel according to claim 1, characterized in that: The preparation method of the Cu-CDs is as follows: 5 mL of chlorhexidine gluconate solution was mixed with 30 mg of copper nitrate solution, and the mixture was hydrothermally reacted at 180°C for 6 hours. After purification by dialysis, the mixture was freeze-dried to obtain light yellow Cu-CDs powder.

3. The method for preparing a biomimetic nanozyme hydrogel according to claim 2, characterized in that: The preparation method of the MSN-NH2 is as follows: MSN was prepared by sol-gel method with hexadecyltrimethylammonium bromide as template and ethyl orthosilicate as silicon source, and then modified with 3-aminopropyltriethoxysilane to obtain MSN-NH2.

4. The method for preparing a biomimetic nanozyme hydrogel according to claim 3, characterized in that: The preparation method of the GCM composite material is as follows: 10 mg of MSN-NH2 was dispersed in 5 mL of deionized water, 5 mg of Cu-CDs was added, and the mixture was stirred for 2 h. Then 0.24 mg of GOx was added and the mixture was stirred for another 2 h. The GCM complex was obtained after centrifugation and washing.

5. The method for preparing a biomimetic nanozyme hydrogel according to claim 4, characterized in that: The preparation method of the hydrogel is specifically as follows: 90 mg of carboxymethyl chitosan was dissolved in 10 mL of deionized water, and the prepared GCM complex was added thereto. The mixture was stirred evenly and allowed to stand to form a gel, thereby obtaining a composite hydrogel.

6. The method for preparing a biomimetic nanozyme hydrogel according to claim 5, characterized in that: The particle size of the Cu-CDs is 5±2 nm, and the surface Zeta potential is +15.2±0.35 mV.

7. A biomimetic nanozyme hydrogel for promoting diabetic wound healing, characterized by: The biomimetic nanoenzyme hydrogel is prepared by the preparation method of any one of claims 1 to 6.

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