Glucose-responsive hydrogen sulfide release cascade nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a glucose-responsive hydrogen sulfide release cascade nano-enzyme as well as a preparation method and application thereof. The cascaded nano-enzyme takes BSA as a growth template and a carrier, and FeS and Au nano-particles are loaded. The FeS-coated Au is synthesized by adopting a biomineralization method, so that the co-loading of FeS and Au is realized, and good biocompatibility is achieved. Meanwhile, by combining the peroxidase-like activity of FeS and the glucose oxidase-like activity of Au, glucose can be catalyzed in a cascade manner to generate toxic hydroxyl free radicals, and drug-resistant bacteria are efficiently killed. In addition, the FeS (at) Au cascade nano-enzyme has a hydrogen sulfide gas release function, and can promote vascular regeneration by up-regulating a hypoxia inducible factor-1 pathway. The cascade nano-enzyme responding to the hyperglycemia microenvironment is combined with a gas treatment mechanism, blood glucose can be effectively consumed, drug-resistant bacterium infection can be eliminated, revascularization can be promoted, and a new strategy is provided for treatment of diabetes wounds infected by drug-resistant bacteria and regeneration of skin tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of cascade nanozymes, and in particular to a glucose-responsive hydrogen sulfide-releasing cascade nanozyme and a preparation method and application thereof. Background Art

[0002] Due to repeated bacterial infections and damaged neovascularization, wound healing in diabetic patients remains a huge challenge, seriously affecting patients' daily lives. The hyperglycemic microenvironment caused by diabetes is the main cause of delayed wound healing. Although glucose can provide the necessary energy for endothelial cell proliferation and vascularization, the excessive glucose concentration in the hyperglycemic microenvironment not only provides nutrients for bacterial proliferation, but also causes endothelial dysfunction, leading to an increased risk of bacterial infection and impaired angiogenesis. At the same time, bacterial infection causes the wound to stagnate in the inflammatory phase, further hindering wound healing.

[0003] With the continuous progress of materials science, new integrated multifunctional therapeutic platforms based on a variety of designed materials have been developed, which can serve as functional components or drug delivery platforms to meet the urgent needs of multi-step interventions in the diabetic wound healing process. For example, some studies have used hydrogels and nanomaterials as carriers to load antimicrobial agents (such as antimicrobial peptides and antibiotics) and pro-angiogenic growth factors or vascular endothelial growth factor (VEGF) to achieve the dual effects of treating bacterial infections and promoting angiogenesis. In addition, some integrated therapeutic delivery platforms have introduced functional components such as glucose oxidase, specific drugs and functional materials, which can reduce local blood glucose levels and synergize with other antimicrobial or pro-angiogenic components to jointly regulate the microenvironment, thereby accelerating the healing of diabetic wounds. Although these treatments have made significant progress, they usually require precise control of the timely and sequential release of each component to ensure the best effect at different stages of the treatment process. However, synchronizing the release kinetics of different components is technically challenging and often difficult to achieve. In addition, the interactions between different therapeutic components may interfere with each other, thereby reducing the overall therapeutic effect. At the same time, the stability of drugs and growth factors is also a key issue, which may affect their effectiveness and increase the risk of adverse side effects. Therefore, in order to more effectively promote diabetic wound healing, we urgently need to develop simpler and more effective multi-stage intervention strategies.

[0004] Nanozymes have shown great potential as natural enzyme substitutes in a variety of application scenarios due to their enzyme-mimicking catalytic activity. In recent years, they have been widely used in the field of diabetic wound treatment, including blood sugar regulating nanozyme therapy, antibacterial nanozyme therapy, and antioxidant nanozyme therapy. Although nanozyme-based therapies have achieved remarkable therapeutic effects, the functional diversity and synergistic effects of nanozymes have become a major problem that hinders their widespread application in diabetic wound healing. However, the emergence of nanozymes with multiple types of enzyme activities provides a solution to this problem, and they can meet the diverse needs of the diabetic wound healing process. In particular, nanozymes with glucose oxidase (GOD)-like activity catalyze glucose into gluconic acid and hydrogen peroxide (H 2 O 2 ), effectively reducing the local blood sugar level. At the same time, with the decrease of the local pH value of the wound and the addition of hydrogen peroxide (H 2 O 2 ) concentration, we can further integrate nanozymes with peroxidase-like (POD) activity. These nanozymes can catalyze hydrogen peroxide to generate highly toxic hydroxyl radicals (·OH), thereby inducing ferroptosis in drug-resistant bacteria and achieving efficient bactericidal effects. Therefore, multi-type enzyme-active nanozymes are expected to achieve multi-step intervention in the process of diabetic wound healing by constructing an efficient cascade catalytic system.

[0005] However, although a variety of nanozymes with enzyme-like activities, such as oxidase (OXD), superoxide dismutase (SOD), glucose oxidase, peroxidase, catalase (CAT), and glutathione peroxidase (GPx), have been widely reported, few studies have focused on nanozymes with pro-angiogenic activity. 2 As a gaseous transmitter, H (S) has attracted much attention due to its intrinsic physiological regulatory properties and plays an important role in a variety of key biological processes. 2 S can regulate the hypoxia-inducible factor HIF-1α (one of the upstream genes of vascular endothelial growth factor VEGF), thereby increasing the level and activity of VEGF, bringing broad development prospects to the field of angiogenesis. This is very different from directly delivering angiogenic growth factors or the widely used angiogenic agent VEGF, because growth factors are easily inactivated during use and the action process is difficult to control. In view of these significant advantages, the development of H 2 The cascade nanozymes with S-releasing ability undoubtedly provide a therapeutic strategy that is both highly efficient and multifunctional. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a glucose-responsive hydrogen sulfide-releasing cascade nanozyme. The cascade nanozyme is prepared by a biomineralization method, and the reaction conditions are mild and simple; the prepared cascade nanozyme FeS@Au can catalyze glucose to produce hydroxyl radicals (·OH) in multiple steps to kill bacteria and eliminate infection. At the same time, the H released by FeS@Au 2 S can accelerate the healing of diabetic infected wounds by upregulating the expression of angiogenic genes in the hypoxia-inducible factor-1 (HIF-1) signaling pathway, accelerating the proliferation, migration and vascularization of endothelial cells.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a method for preparing a glucose-responsive hydrogen sulfide-releasing cascade nanozyme, comprising the following steps:

[0009] (1) Preparation of FeSNPs:

[0010] BSA and FeCl 2 Add to water and stir at 330-350r / min for 5-10min to make solution A. 2 S is dissolved in water, added to the above solution A, and stirred at 330-350 r / min for 5-10 min; the mixture is heated in a water bath, and nitrogen is kept flowing into the reaction for 5-6 h; after the reaction is completed, it is placed in a dialysis bag and dialyzed at 4-5°C to obtain FeSNP;

[0011] (2) Preparation of FeS@Au:

[0012] Under stirring conditions, HAuCl 4 The aqueous solution of was added to the FeSNP prepared in step (1), stirred for 5-10 min, and then NaOH aqueous solution was added to adjust the pH of the reaction system to 10-12, stirred at room temperature for 1-6 h, and NaBH was added. 4 The aqueous solution continued to react for 10-30 minutes. After the reaction was completed, it was dialyzed in a dialysis bag at 4-5°C for 12-24 hours. After the dialysis was completed, the glucose-responsive hydrogen sulfide-releasing cascade nanozyme FeS@Au was obtained.

[0013] Preferably, the FeCl 2 and Na 2 The molar ratio of S is 1:(3.8~4.2).

[0014] Preferably, in step (2), the FeSNP and HAuCl 4 The molar ratio is (15.5~16.5):1.

[0015] Preferably, the water bath heating in step (1) is specifically: heating in a water bath to 30°C to 45°C.

[0016] Preferably, the concentration of the NaOH aqueous solution in step (2) is 0.1-5 mol / L.

[0017] The present invention also provides a FeS@Au nanoparticle composition with a core-shell structure on a growth template; the FeS@Au nanoparticle uses FeSNP as a core, and AuNC nanoparticles are loaded on the surface of the FeSNP.

[0018] Preferably, the particle size of the FeS@Au nanoparticles is 50-150 nm, and the particle size of the AuNC nanoparticles is 1-2 nm; the average Zeta potential of the FeS@Au nanoparticles is -20 to -40 mV.

[0019] The present invention also provides the use of the glucose-responsive hydrogen sulfide-releasing cascade nanozyme in the preparation of a drug for treating diabetic-infected chronic wounds.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) In the glucose-responsive hydrogen sulfide-releasing cascade nanozyme of the present invention, ultra-small (about 2 nm) Au nanoparticles (AuNC) are immobilized on FeS nanoparticles (FeSNP), and the Au nanoparticles catalyze glucose to generate gluconic acid and H 2 O 2 , not only consumes glucose, but also acidifies the wound environment; FeS continuously decomposes H in a weakly acidic environment 2 O 2 Generate ·OH and continuously release H 2 S. At the same time, the released H 2 S can significantly upregulate the expression of genes related to the HIF-1 signaling pathway, and can simultaneously achieve the bactericidal activity of OH and H 2 Angiogenesis effect of S release.

[0022] (2) The glucose-responsive hydrogen sulfide-releasing cascade nanozyme of the present invention has stimulus-responsive drug release behavior, strong and broad-spectrum antibacterial ability, significant ability to promote angiogenesis and good biocompatibility, and shows good healing effect and clinical transformation potential in the treatment of diabetic wound infections.

[0023] (3) The preparation method of the glucose-responsive hydrogen sulfide-releasing cascade nanozyme of the present invention uses BSA with good hydrophilicity and high stability as a growth template and carrier to in situ generate AuNC on FeSNP to form a uniformly distributed FeS@Au cascade nanozyme.

[0024] (4) The preparation method of the glucose-responsive hydrogen sulfide-releasing cascade nanozyme of the present invention is prepared by a biomineralization method, and the reaction conditions are mild and simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The morphology observation of the FeS@Au cascade nanozyme of Example 1 of the present invention, wherein a to d are transmission electron microscopy images of FeS@Au; Figure e is a mapping image of the corresponding elements of FeS@Au

[0026] Figure 2 This is the characterization of the FeS@Au cascade nanozyme of Example 1 of the present invention, wherein a~d are XPS spectra of FeS@Au, wherein a is the full scan spectrum, b is the Au 4f spectrum, c is the Fe 2p spectrum, and d is the S2p spectrum; e is the emission spectrum of FeS@Au under 480nm fluorescence excitation; f is the Zeta potential diagram of each nanoparticle.

[0027] Figure 3 Graph showing the cascade enzyme activity of the FeS@Au cascade nanozyme of Example 1 of the present invention and the cascade enzyme activity of the comparative example.

[0028] Figure 4 This is the paramagnetic resonance spectrum (hydroxyl radical detection) of the FeS@Au cascade nanozyme of Example 1 of the present invention.

[0029] Figure 5 The FeS@Au cascade nanozyme of Example 1 of the present invention releases H at different pH values. 2 The result of S.

[0030] Figure 6 The antibacterial properties of the FeS@Au cascade nanozyme of Example 1 of the present invention and the nanozymes of Comparative Examples 1 and 2, wherein a is the antibacterial activity of FeS@Au (FeS@Au) at different concentrations in PBS solution and glucose solution (PBS with a glucose concentration of 10 mM). 2+ b is the growth of MRSA on agar plates after incubation with FeS@Au (FeS@Au) at different concentrations (0, 0.4, 0.8, 1.6, 3.125 and 6.25 μg / mL) for 2 h; c is the growth of MRSA on agar plates after treatment; and c is the growth of FeS@Au (FeS@Au) at different concentrations in PBS solution and glucose solution (PBS with a glucose concentration of 10 mM). 2+ The survival of each group of bacteria after incubation with Pseudomonas aeruginosa (P. aeruginosa) at concentrations of 0, 0.4, 0.8, 1.6, 3.125 and 6.25 μg / mL for 2 h; d is a photo of the growth of P. aeruginosa on an agar plate after the corresponding treatment; e, f are photos of the FeS@Au cascade nanozyme (FeS@Au) in glucose solution. 2+CLSM (live / dead staining) images of MRSA and P. aeruginosa after treatment with the above-mentioned sample (concentration: 6.25 μg / mL) and comparative examples 1 and 2, respectively.

[0031] Figure 7 This is the biocompatibility test of the FeS@Au cascade nanozyme of Example 1 of the present invention and the nanozymes of Comparative Examples 1 and 2; wherein, a and b are CCK-8 activity tests on L929 and HUVECs cells, and c and d are live / dead staining of L929 and HUVECs.

[0032] Figure 8 It is the tubulation image of HUVECs on Matrigel after being treated with the FeS@Au cascade nanozyme of Example 1 of the present invention and the nanozymes of Comparative Examples 1 and 2, and the quantitative analysis of the tubular structure.

[0033] Fig. 9 It is the therapeutic effect of the FeS@Au cascade nanozyme of Example 1 of the present invention and the nanozymes of Comparative Examples 1 and 2 in the rat diabetic infection model; wherein, a is a schematic diagram of the treatment process of rats; b is a wound healing photo; c is a wound residual bacterial smear photo; d is a quantitative analysis of wound area; e is a quantitative analysis of residual bacterial activity; f is H&E staining of wound tissue; g is Masson staining of wound tissue.

[0034] Fig.10 These are H&E staining images of the heart, liver, spleen, lung, and kidney of rats treated with the FeS@Au cascade nanozyme according to Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0036] Example 1

[0037] 5 mL of BSA (6.25 mg / mL) and 1 mL of FeCl 2 The aqueous solution (40 mM) was placed in water and stirred at a low speed of 350 r / min for 5 min to prepare solution A. 2 S was dissolved in water to make 1 mL of 160 mM Na 2 S aqueous solution was added to the above solution A and stirred at a low speed of 350 r / min for 5 min, wherein FeCl 2 and Na 2 The molar ratio of S was 1:4; the mixture was placed in a water bath and heated to 37°C, and nitrogen was kept flowing for 6 hours; after the reaction, it was placed in a dialysis bag and dialyzed at 4°C for 12 hours to obtain FeSNP. The FeSNP solution was placed in a reaction bottle, and 1 mL of HAuCl was added under stirring. 4aqueous solution, in which FeSNP and HAuCl 4 The molar ratio of 16:1 was 1. After stirring for 5 min, 100 μL of NaOH aqueous solution was added to adjust the pH of the reaction system to 12. The reaction was stirred at room temperature for 1-2 h. 100 μL of NaBH was added. 4 The aqueous solution continued to react for 30 minutes. After the reaction, it was dialyzed at low temperature for 12 hours using a 20 kD dialysis bag. After 12 hours, glucose-responsive H 2 S release cascade nanozyme FeS@Au.

[0038] Comparative Example 1

[0039] FeSNP nanozymes were synthesized according to the FeSNP synthesis steps in Example 1.

[0040] Comparative Example 2

[0041] Dissolve 50 mg BSA in 5 mL deionized water and stir for 5 min. Add 1 mL HAuCl 4 After the two are mixed evenly, an appropriate amount of NaOH aqueous solution is added to adjust the pH to 12, and then the reaction is carried out at room temperature for 12 hours. After the reaction is completed, the solution system is transferred to a dialysis bag and dialyzed for 12 hours to obtain AuNC nanoparticles.

[0042] The morphology of FeS@Au prepared in this example was observed by TEM, HRTEM, EDS and other tests. Figure 1 As shown, Figure 1 a and b are TEM images of FeS@Au prepared in Example 1. It can be seen that the prepared FeS@Au has a core-shell nanostructure with a particle size of about 75 nm, and ultra-small gold nanoparticles with a particle size of about 1-2 nm are embedded on the FeS surface. Figure 1 Figure c is the HRTEM image of FeS@Au prepared in Example 1. It can be observed that the interplanar spacing corresponding to the (200) plane of Au is 0.203 nm, and the interplanar spacing corresponding to the (101) plane of FeS is 0.297 nm, proving the coexistence of Au and FeS. Figure 1 d in FIG. 1 is the HAADF image of Example 1, and it is observed that the image substantially overlaps with the TEM image. Figure 1 e in FIG. 1 is the energy dispersive spectrum analysis (EDS) of Example 1, which confirms the presence of Au, Fe, S and N elements in the spherical NPs.

[0043] FeS@Au was characterized by XPS, FL, and Zeta potential tests. Figure 2 As shown: Figure 2a in the figure is the elemental composition and chemical valence state of FeS@Au studied by X-ray photoelectron spectroscopy (XPS). The XPS full spectrum scan shows that Fe, S and Au elements coexist. Figure 2 (b) is the fine spectrum of Au in FeS@Au. The peaks at 87.6 eV and 83.9 eV can be attributed to Au 4f 5 / 2 and Au 4f 7 / 2, respectively, proving the existence of zero-valent Au. Figure 1 c is the fine spectrum of Fe in FeS@Au, and the peaks at 711.2 eV and 724.5 eV can be attributed to Fe 2p 1 / 2 and Fe 2p 3 / 2 , proving that the valence state of Fe in FeS@Au is Fe 2+ . Figure 2 The d in the figure is the fine spectrum of the s element in FeS@Au. The 2p3 / 2 peaks of the s element are located at 163.9 eV, 161.9 eV, and 167.9 eV, representing FeS, S 2- and organic sulfur, and the presence of FeS was also confirmed. Figure 1 Figure e is the emission spectrum of FeS@Au under 480nm fluorescence excitation. It can be seen from the figure that the ultra-small AuNC has an emission peak at about 640nm under 480nm fluorescence excitation wavelength; FeS@Au maintains this characteristic fluorescence emission peak, indicating that AuNC is loaded on the FeS surface. Figure f is the Zeta potential diagram of Example 1 and the comparative example. It can be seen that the surface potential of FeS@Au is lower than that of FeSNP. The decrease in the surface potential of FeS@Au verifies that AuNC is successfully loaded on the FeSNP surface.

[0044] The ability of nanozymes to catalyze glucose to generate hydroxyl radicals was tested using 3,3',5,5'-tetramethylbenzidine: The cascade catalytic glucose ability of nanozymes prepared in Example 1, Comparative Example 1, and Comparative Example 2 was characterized. The results are as follows Figure 3 shown. Figure 3 It can be seen that the characteristic absorption at 650 nm proves that FeS@Au has excellent cascade activity and can effectively catalyze the conversion of glucose into H 2 O 2 and gluconic acid, thereby continuously generating ·OH. However, no characteristic absorption appeared in the comparative AuNC and FeSNP groups, indicating that these two nanoparticles do not have cascade catalytic activity.

[0045] Using ESR to characterize the free radicals generated by cascade nanozymes:

[0046] FeS@Au was mixed with 10 mM glucose and incubated for 6 h, and the ESR spectrum of the reaction product was measured. Figure 4As shown, the ESR spectrum shows a peak ratio of 1:2:2:1, which corresponds to the characteristic peak of ·OH, further proving that FeS@Au nanoparticles catalyze glucose to produce ·OH through a cascade reaction.

[0047] Detection of H release from FeS@Au 2 S's abilities:

[0048] Due to the high concentration of H 2 S is likely to show high cytotoxicity by inducing cell apoptosis, so H 2 The controlled release of S is very important. 2 S detection kit detects H 2 The release behavior of S. FeS@Au was incubated in buffers with different pH values ​​(5.5, 6.5, and 7.4), and the supernatants were collected at different time points and quenched with H 2 S detection kit detects H 2 The concentration of S. The results are as follows Figure 5 As shown, when the pH value is 7.4 and 6.5, H 2 When the pH value dropped to 5.5, H 2 S can be stably released for 48 h, with a peak concentration of 32 μM, proving that FeS@Au can effectively serve as a H 2 S release carrier, achieving H 2 Controlled sustained release of S.

[0049] Antibacterial performance test of FeS@Au:

[0050] MRSA and P. aeruginosa were inoculated into LB medium and incubated overnight at 37°C and 220 r / min. The above two bacterial suspensions were diluted to a concentration of 10 7 CFU / mL, the bacterial suspension was incubated with gradient concentrations of Example 1 in PBS and PBS containing 10 mM glucose for 2 h, and then the bacterial suspension was diluted to 10 4 CFU / mL, take 20μL and evenly spread it on the LB plate, place it in a constant temperature incubator at 37℃ for 18h, observe and record, and count the colonies on the plate. The results showed that compared with PBS, the activity of bacteria in glucose solution was significantly inhibited ( Figure 6 a,c), the number of colonies on the LB plate was significantly reduced ( Figure 6 (b, d), this is because FeS@Au can cascade catalyze glucose to produce highly toxic ·OH, thus showing more excellent antibacterial performance.

[0051] At the same time, the live / dead staining kit was used to stain the bacteria after co-culture ( Figure 6(e, f) After MRSA and P. aeruginosa were incubated with Example 1 and the comparative example for 2 hours, the bacteria were collected by centrifugation (5000rpm / min, 5min). After staining with SYTO9 and PI for 15 minutes, the samples were rinsed with PBS 3 times. Finally, the staining was observed under a confocal laser scanning microscope (CLSM) and the experimental results were photographed. After staining, live bacteria with intact membrane structures showed green fluorescence, while dead bacteria with destroyed membrane structures showed red fluorescence. As can be seen from the results, PBS and AuNP had no toxic effects on bacteria, FeSNP had a certain antibacterial effect, and the bacteria treated with FeS@Au showed the most red fluorescence, indicating that FeS@Au killed almost all bacteria and had the best antibacterial effect.

[0052] Cascade nanozyme biocompatibility test:

[0053] Example 1 and Comparative Examples 1 and 2 were co-cultured with L929 cells and human umbilical vein endothelial cells (HUVECs) for 24 hours and then the cell activity was tested using a CCK-8 kit. Figure 7 As shown, the cell survival rate of each group of samples was basically not reduced, indicating that the cell compatibility of each group of samples was good.

[0054] Cascade nanozymes promote angiogenesis test:

[0055] By seeding HUVECs on Matrigel to simulate angiogenesis, the vascularization effect of FeS@Au on HUVECs was studied in a serum- and growth factor-free medium (neutral pH environment). Figure 8 As shown in Figure a, cells treated with PBS and AuNC mainly formed stacking and budding, with a small number of branched structures and a small number of complete tubules. In contrast, cells treated with FeSNP and FeS@Au showed more closed tubular structures. In particular, the various indicators of tubular bundle formation were quantified using image J software, and it was found that the network structure formed by cells treated with FeS@Au was the richest ( Figure 8 b) It is beneficial to the transport of oxygen and nutrients, thus accelerating wound healing.

[0056] The diabetic rat model was established by intraperitoneal injection of streptozotocin. Four circular wounds with a diameter of 10 mm were made on both sides of the rat's back. 50 μL MRSA (10 7 CFU / mL) to construct an infection model, and use Example 1 and the comparative example to treat the wound respectively. Fig. 9 A in the middle is a schematic diagram of the treatment process. Record the wound pictures on the 3rd, 6th and 10th days ( Fig. 9 b, d) and collect exudate from the wound for bacterial culture ( Fig. 9The wound skin tissue on day 10 was stained with hematoxylin and eosin (H&E) and Masson staining. Fig. 9 From Figures f and g, it can be seen that FeS@Au nanozymes can effectively reduce inflammation and promote collagen deposition.

[0057] In vivo biosafety assessment:

[0058] The main organs of the rats were taken for H&E staining. Fig.10 As shown, there were no abnormalities in the heart, liver, spleen, lung, and kidney of the rats, indicating that FeS@Au has good in vivo biosafety.

[0059] The AuNC in the dosed nanozyme of the present invention simulates the function of glucose oxidase (GOD), which not only significantly reduces the local blood glucose concentration in the wound, but also activates the peroxidase-like (POD) activity of FeSNP, catalyzing the conversion of glucose into hydrogen peroxide (H 2 O 2 ) and gluconic acid, which in turn generate hydroxyl radicals (·OH), which can effectively kill drug-resistant bacteria. In addition, glucose can also trigger H 2 The release of S significantly upregulates the expression level of hypoxia-inducible factor (HIF-1), further promotes the expression of angiogenesis-related genes, accelerates the proliferation, migration and lumen formation of human umbilical vein endothelial cells (HUVECs), and achieves efficient angiogenesis. In vivo experimental data fully prove that the cascade activity activated by glucose is related to H 2 The S-releasing ability enables FeS@Au to show excellent therapeutic effects in the treatment of full-thickness diabetic wounds infected with MRSA, not only with high antibacterial activity, but also with significant promotion of wound healing. In summary, the present invention provides a cascade nanozyme responsive to a hyperglycemic microenvironment, which combines gas therapy, blood sugar regulation, efficient antibacterial activity, significant angiogenesis-promoting ability and excellent biocompatibility.

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a glucose-responsive hydrogen sulfide-releasing cascade nanozyme, characterized in that: The following steps are involved: (1) Preparation of FeSNPs: BSA and FeCl2 were added into water, and stirred at 330-350 r / min for 5-10 min to prepare solution A; Na2S was dissolved in water, and added into the above solution A, and stirred at 330-350 r / min for 5-10 min; the mixture was heated in a water bath, and nitrogen was kept flowing into the reaction for 5-6 h; after the reaction, the mixture was placed in a 20 kD dialysis bag, and dialyzed at 4-5 °C to obtain FeSNP; (2) Preparation of FeS@Au: Under stirring conditions, an aqueous solution of HAuCl4 is added to the FeSNP prepared in step (1), and after stirring for 5 to 10 minutes, an aqueous solution of NaOH is added to adjust the pH of the reaction system to 10 to 12, and the reaction is stirred at room temperature for 1 to 6 hours. An aqueous solution of NaBH4 is added to continue the reaction for 10 to 30 minutes. After the reaction is completed, a 20 kD dialysis bag is used for dialysis at 4 to 5 ° C for 12 to 24 hours. After the dialysis is completed, the glucose-responsive hydrogen sulfide-releasing cascade nanozyme FeS@Au is obtained.

2. The method for preparing the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 1, characterized in that: In step (1), the molar ratio of FeCl2 to Na2S is 1:(3.8-4.2).

3. The method for preparing the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 1, characterized in that: In step (2), the molar ratio of FeSNP and HAuCl4 is (15.5-16.5):

1.

4. The method for preparing the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 1, characterized in that: The water bath heating in step (1) is specifically: heating the water bath to 30°C to 45°C.

5. The method for preparing the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 1, characterized in that: The concentration of the NaOH aqueous solution in step (2) is 0.1-5 mol / L.

6. A glucose-responsive hydrogen sulfide-releasing cascade nanozyme, characterized in that: The method comprises a growth template and FeS@Au nanoparticles with a core-shell structure loaded on the growth template; the FeS@Au nanoparticles use FeSNP as the core and AuNC nanoparticles are loaded on the surface of the FeSNP.

7. The glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 6, characterized in that: The particle size of the FeS@Au nanoparticles is 50-150nm, and the particle size of the AuNC nanoparticles loaded on the surface thereof is 1-2nm; the average Zeta potential of the FeS@Au nanoparticles is -20 to -40mV.

8. Use of the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to any one of claims 6 to 7 in the preparation of a drug for treating diabetic-infected chronic wounds.

Images