A glucose-responsive hydrogen sulfide release cascade nanozyme, its preparation method and application

Abstract

This invention discloses a glucose-responsive hydrogen sulfide-releasing cascade nanozyme, its preparation method, and its applications. The cascade nanozyme uses BSA as a growth template and carrier, loading FeS and Au nanoparticles. This invention synthesizes FeS@Au using a biomineralization method, achieving co-loading of FeS and Au, exhibiting good biocompatibility. Simultaneously, by combining the peroxidase-like activity of FeS and the glucose oxidase-like activity of Au, it can cascade and catalyze the generation of toxic hydroxyl radicals from glucose, efficiently eliminating drug-resistant bacteria. Furthermore, the FeS@Au cascade nanozyme possesses hydrogen sulfide gas release functionality, which can promote angiogenesis by upregulating the hypoxia-inducible factor-1 pathway. This invention provides a hyperglycemic microenvironment-responsive cascade nanozyme, combined with a gas therapy mechanism, which can effectively consume blood glucose, eliminate drug-resistant bacterial infections, and promote angiogenesis, providing a new strategy for the treatment of diabetic wounds infected with drug-resistant bacteria and for skin tissue regeneration.

Description

Technical Field

[0001] This invention relates to the technical field of cascade nanozymes, and particularly to a glucose-responsive hydrogen sulfide-releasing cascade nanozyme, its preparation method, and its applications. Background Technology

[0002] Wound healing remains a significant challenge for diabetic patients due to recurrent bacterial infections and damage to new blood vessels, severely impacting their daily lives. The hyperglycemic microenvironment caused by diabetes is a major cause of delayed wound healing. While glucose provides the necessary energy for endothelial cell proliferation and angiogenesis, excessively high glucose concentrations in a hyperglycemic microenvironment not only provide nutrients for bacterial growth but also cause endothelial dysfunction, leading to an increased risk of bacterial infection and impaired angiogenesis. Simultaneously, bacterial infection causes the wound to remain in the inflammatory phase, further hindering wound healing.

[0003] With the continuous advancement of materials science, novel integrated multifunctional therapeutic platforms based on a variety of designed materials have been developed. These materials can serve as functional components or drug delivery platforms to meet the urgent need for multi-step interventions in the healing process of diabetic wounds. For example, some studies have utilized hydrogels and nanomaterials as carriers to load antibacterial 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. Furthermore, some integrated therapeutic delivery platforms have introduced functional components such as glucose oxidase, specific drugs, and functional materials, which can lower local blood glucose levels and synergize with other antibacterial or pro-angiogenic components to jointly regulate the microenvironment, thereby accelerating the healing of diabetic wounds. Although these therapeutic methods have made significant progress, they typically require precise control over the timely and sequential release of each component to ensure optimal efficacy at different stages of the treatment process. However, synchronizing the release kinetics of different components is technically challenging and often difficult to achieve. Moreover, interactions between different therapeutic components can interfere with each other, thereby reducing the overall therapeutic effect. Meanwhile, the stability of drugs and growth factors is also a key issue, as it may affect their effectiveness and increase the risk of adverse side effects. Therefore, in order to more effectively promote wound healing in diabetic patients, we urgently need to develop simpler and more effective multi-stage intervention strategies.

[0004] Nanozymes, with their enzyme-mimicking catalytic activity, have demonstrated great potential as natural enzyme alternatives in various applications. In recent years, they have been widely used in the treatment of diabetic wounds, including glycemic regulation nanozyme therapy, antibacterial nanozyme therapy, and antioxidant nanozyme therapy. Although nanozyme-based therapies have achieved significant efficacy, the functional diversity and synergistic effects of nanozymes have become a major obstacle to their widespread application in diabetic wound healing. However, the emergence of nanozymes with multi-enzyme activities has provided a solution to this problem, meeting the diverse needs of the diabetic wound healing process. In particular, nanozymes with glucose oxidase-like (GOD) activity effectively reduce local blood glucose levels by catalyzing glucose into gluconic acid and hydrogen peroxide (H2O2). Simultaneously, with the decrease in local wound pH and the increase in hydrogen peroxide (H2O2) concentration, we can further integrate nanozymes with peroxidase-like (POD) activity. These nanozymes can catalyze the generation of highly toxic hydroxyl radicals (·OH) from hydrogen peroxide, thereby inducing ferroptosis in drug-resistant bacteria and achieving highly efficient bactericidal effects. Therefore, multi-enzyme-active nanozymes hold promise for enabling multi-step intervention in the wound healing process of diabetes by constructing a highly efficient cascade catalytic system.

[0005] However, while various 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. Hydrogen sulfide (H2S), as a gaseous neurotransmitter, plays a crucial role in many key biological processes due to its inherent physiological regulatory properties, and has therefore attracted considerable attention. Importantly, H2S can regulate hypoxia-inducible factor HIF-1α (one of the upstream genes of vascular endothelial growth factor VEGF), thereby enhancing the level and activity of VEGF, bringing broad prospects to the field of angiogenesis. This is significantly different from the direct delivery of pro-angiogenic factors or the widely used pro-angiogenic agent VEGF, because growth factors are easily inactivated during use, and the process of action is difficult to control. Given these significant advantages, developing cascaded nanozymes with H2S release capabilities undoubtedly provides a therapeutic strategy that is both highly efficient and multifunctional. Summary of the Invention

[0006] To overcome the aforementioned shortcomings and deficiencies of existing technologies, the present invention aims to provide a method for preparing a glucose-responsive hydrogen sulfide-releasing cascade nanozyme. The nanozyme is prepared via a biomineralization method under mild and simple reaction conditions. The prepared cascade nanozyme FeS@Au can catalyze the generation of hydroxyl radicals (·OH) from glucose in multiple steps to kill bacteria and eliminate infection. Simultaneously, the H2S released by FeS@Au can upregulate the expression of angiogenesis genes in the hypoxia-inducible factor-1 (HIF-1) signaling pathway, accelerating endothelial cell proliferation, migration, and vascularization, thereby accelerating the healing of diabetic infection wounds.

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

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

[0009] (1) Preparation of FeSNP:

[0010] Add BSA and FeCl2 to water and stir at 330-350 r / min for 5-10 min to prepare solution A; dissolve Na2S in water and add it to solution A, stirring at 330-350 r / min for 5-10 min; heat the mixture in a water bath and keep nitrogen gas flowing through the reaction for 5-6 h; after the reaction is complete, place it in a dialysis bag and dialyze at 4-5 °C to obtain FeSNP;

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

[0012] Under stirring conditions, an aqueous solution of HAuCl4 was added to the FeSNP prepared in step (1). After stirring for 5-10 min, an aqueous solution of NaOH was added to adjust the pH of the reaction system to 10-12. The reaction was stirred at room temperature for 1-6 h. An aqueous solution of NaBH4 was added to continue the reaction for 10-30 min. After the reaction was completed, the mixture was dialyzed at 4-5℃ for 12-24 h using a dialysis bag. After the dialysis was completed, the glucose-responsive hydrogen sulfide release cascade nanozyme FeS@Au was obtained.

[0013] Preferably, the molar ratio of FeCl2 to Na2S is 1:(3.8 to 4.2).

[0014] Preferably, in step (2), the molar ratio of FeSNP and HAuCl4 is (15.5~16.5):1.

[0015] Preferably, the water bath heating in step (1) specifically involves heating the 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 nanoparticles use FeSNP as the core and AuNC nanoparticles are loaded on the surface of FeSNP.

[0018] Preferably, the FeS@Au nanoparticles have a particle size of 50-150 nm, and the AuNC nanoparticles have a particle size of 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 medicament for treating diabetic infected chronic wounds.

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

[0021] (1) The glucose-responsive hydrogen sulfide-releasing cascade nanozyme of the present invention comprises ultra-small (approximately 2 nm) Au nanoparticles (AuNC) immobilized on FeS nanoparticles (FeSNP). The Au nanoparticles catalyze the production of gluconic acid and H2O2 from glucose, not only consuming glucose but also acidifying the wound environment. In the weakly acidic environment, FeS continuously decomposes H2O2 to generate ·OH and continuously releases H2S. Simultaneously, the released H2S can significantly upregulate the expression of genes related to the HIF-1 signaling pathway, thus simultaneously achieving the bactericidal activity of ·OH and the angiogenesis effect of H2S release.

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

[0023] (3) The method for preparing glucose-responsive hydrogen sulfide release cascade nanozymes of the present invention utilizes BSA, which has good hydrophilicity and high stability, as a growth template and carrier to generate AuNC in situ on FeSNP, thereby forming a uniformly distributed FeS@Au cascade nanozyme.

[0024] (4) The method for preparing glucose-responsive hydrogen sulfide release cascade nanozymes of the present invention is prepared by biomineralization, and the reaction conditions are mild and simple. Attached Figure Description

[0025] Figure 1 The morphology of the FeS@Au cascade nanozyme of Example 1 of the present invention is observed, where a to d are transmission electron micrographs of FeS@Au; and e is a mapping image of the corresponding elements in FeS@Au.

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

[0027] Figure 3 The images show the activity diagrams of the FeS@Au cascade nanozyme of Example 1 and the cascade enzyme of the comparative example of the present invention.

[0028] Figure 4 Paramagnetic resonance spectroscopy (hydroxyl radical detection) of the FeS@Au cascade nanozyme of Example 1 of the present invention.

[0029] Figure 5 The results of H2S release from the FeS@Au cascade nanozyme of Example 1 of the present invention at different pH values ​​are shown.

[0030] Figure 6 To demonstrate the antibacterial properties of the FeS@Au cascade nanozyme of Example 1 and the nanozymes of Comparative Examples 1 and 2 of the present invention, wherein a is the antibacterial performance of FeS@Au (Fe 2+ The survival of bacteria in each group after incubation with methicillin-resistant Staphylococcus aureus (MRSA) at concentrations of 0, 0.4, 0.8, 1.6, 3.125, and 6.25 μg / mL for 2 h was shown in Figure 1. b shows the growth of the corresponding treated MRSA on agar plates. c shows the growth of FeS@Au (Fe...) in PBS and glucose solution (PBS with a glucose concentration of 10 mM) at different concentrations. 2+ The survival of Pseudomonas aeruginosa was observed after incubation with FeS@Au cascade nanozymes (at concentrations of 0, 0.4, 0.8, 1.6, 3.125, and 6.25 μg / mL) for 2 h. Photograph d shows the growth of the corresponding treated P. aeruginosa on agar plates. Photographs e and f show the growth of FeS@Au cascade nanozymes (FeS@Au) in glucose solution. 2+ Images of CLSM (live / dead staining) of MRSA and P. aeruginosa after treatment with a concentration of 6.25 μg / mL and comparative examples 1 and 2, respectively.

[0031] Figure 7 This is a 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 the CCK-8 activity tests of L929 and HUVECs cells, and c and d are the live / dead staining of L929 and HUVECs.

[0032] Figure 8 This is a tube formation image and quantitative analysis of the tubular structure of the FeS@Au cascade nanozyme of Example 1 and HUVECs treated with nanozymes of Comparative Examples 1 and 2 on a matrix gel.

[0033] Figure 9 This invention describes the therapeutic effects of the FeS@Au cascade nanozyme of Example 1 and the nanozymes of Comparative Examples 1 and 2 in a rat diabetic infection model; wherein, a is a schematic diagram of the rat treatment process; b is a photograph of wound healing; c is a photograph of residual bacteria smeared on the wound; 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] Figure 10 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of rats treated with the FeS@Au cascade nanozyme of Example 1 of this invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0036] Example 1

[0037] 5 mL of BSA (6.25 mg / mL) and 1 mL of FeCl2 aqueous solution (40 mM) were placed in water and stirred at a low speed of 350 r / min for 5 min to prepare solution A. Na2S was dissolved in water to prepare 1 mL of 160 mM Na2S aqueous solution, which was added to solution A and stirred at a low speed of 350 r / min for 5 min. The molar ratio of FeCl2 to Na2S was 1:4. The mixture was heated in a water bath to 37 °C and nitrogen gas was introduced for 6 h. After the reaction was completed, the mixture was placed in a dialysis bag and dialyzed at 4 °C for 12 h to obtain FeSNP. The FeSNP solution was placed in a reaction flask, and 1 mL of HAuCl4 aqueous solution was added while stirring. The molar ratio of FeSNP to HAuCl4 was 16:1. 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, and then 100 μL of NaBH4 aqueous solution was added to continue the reaction for 30 min. After the reaction was completed, the solution was dialyzed at low temperature for 12 h using a 20 kD dialysis bag. After 12 h, the glucose-responsive H2S release cascade nanozyme FeS@Au was obtained.

[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 of BSA in 5 mL of deionized water and stir for 5 min. While stirring, add 1 mL of HAuCl4 aqueous solution. After mixing thoroughly, add an appropriate amount of NaOH aqueous solution to adjust the pH to 12, and then react at room temperature for 12 h. After the reaction is complete, transfer the solution to a dialysis bag and dialyze for 12 h to obtain AuNC nanoparticles.

[0042] The morphology of the FeS@Au prepared in this embodiment was observed by TEM, HRTEM, and EDS tests, and the results are as follows: Figure 1 As shown, Figure 1 a and b in the figure 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. Ultra-small gold nanoparticles with a particle size of about 1-2 nm are embedded on the FeS surface. Figure 1 c in the image is the HRTEM image of FeS@Au prepared in Example 1. It can be observed that the interplanar spacing of Au (200) plane is 0.203 nm and the interplanar spacing of FeS (101) plane is 0.297 nm, which proves the coexistence of Au and FeS. Figure 1 In the image, d is the HAADF image of Example 1, which is observed to largely overlap with the TEM image. Figure 1 In this context, 'e' refers to the energy dispersive spectroscopy (EDS) analysis of Example 1, which confirmed the presence of Au, Fe, S, and N elements in the spherical NPs.

[0043] FeS@Au was characterized by XPS, FL, and Zeta potential measurements, and the results are as follows: Figure 2 As shown: Figure 2 In Figure 'a', the elemental composition and chemical valence state of FeS@Au were studied using X-ray photoelectron spectroscopy (XPS). The full-spectrum XPS scan showed that Fe, S, and Au elements coexisted. Figure 2 b in the figure 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 In the figure, c represents 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, respectively. 1 / 2 and Fe 2p 3 / 2 This proves that the valence state of Fe in FeS@Au is Fe. 2+ . Figure 2 The 'd' in the image represents 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 and S in BSA, respectively. 2-The presence of FeS was also confirmed by organic sulfur and organic sulfur. Figure 1 Figure e shows the emission spectrum of FeS@Au under 480 nm fluorescence excitation. As can be seen from the figure, the ultra-small AuNC has an emission peak at approximately 640 nm under 480 nm fluorescence excitation. FeS@Au retains this characteristic fluorescence emission peak, indicating that AuNC is loaded onto 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 the successful loading of AuNC onto the FeSNP surface.

[0044] The ability of nanozymes to catalyze the formation of hydroxyl radicals from glucose was tested using 3,3',5,5'-tetramethylbenzidine: The cascade catalytic ability of the nanozymes prepared in Example 1, Comparative Example 1, and Comparative Example 2 to catalyze glucose was characterized, and the results are as follows: Figure 3 As shown. Figure 3 The characteristic absorption at 650 nm demonstrates that FeS@Au possesses excellent cascade activity, effectively catalyzing the conversion of glucose into H2O2 and gluconic acid, thereby continuously generating ·OH. In contrast, the characteristic absorption was not observed in the comparative AuNC and FeSNP groups, indicating that these two nanoparticles do not possess cascade catalytic activity.

[0045] Qualitative analysis of free radicals generated by cascade nanozymes using ESR:

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

[0047] Detecting the ability of FeS@Au to release H2S:

[0048] High concentrations of H2S readily induce apoptosis and exhibit high cytotoxicity, therefore, controlled and sustained release of H2S is crucial. An H2S detection kit was used to detect H2S release behavior under different pH conditions. FeS@Au was incubated in buffer solutions at different pH values ​​(5.5, 6.5, 7.4), and the supernatant was collected at different time points. The concentration of H2S in the solution was then detected using the H2S detection kit. The results are as follows: Figure 5 As shown, the amount of H2S released was relatively small when the pH value was 7.4 and 6.5, but when the pH value was reduced to 5.5, H2S could be released stably for 48 hours with a peak concentration of 32 μM. This proves that FeS@Au can be effectively used as a release carrier for H2S to achieve controlled and sustained release of H2S.

[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 rpm. The bacterial suspensions were then 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, 20 μL was evenly spread onto LB agar plates and incubated at 37℃ for 18 h. Observation and recording were performed, and colony counts were conducted. Results showed that bacterial activity was significantly inhibited in glucose solution compared to PBS. Figure 6 In cases a and c), the number of colonies on the LB plate was significantly reduced. Figure 6 (b, d) This is because FeS@Au can cascade catalyze the formation of highly toxic ·OH from glucose, thus exhibiting superior antibacterial properties.

[0051] Simultaneously, a live / dead staining kit was used to stain the co-cultured bacteria. Figure 6 In steps e and f), MRSA and P. aeruginosa were incubated with the samples from Example 1 and the comparative example, respectively, for 2 hours. Bacteria were then collected by centrifugation (5000 rpm / min, 5 min). After staining with SYTO9 and PI for 15 min, the bacteria were washed three times with PBS. Finally, the staining was observed and the results were photographed using a laser scanning confocal microscope (CLSM). After staining, viable bacteria with intact membrane structures showed green fluorescence, while dead bacteria with damaged membrane structures showed red fluorescence. The results showed that PBS and AuNP had no toxic effect on the bacteria, FeSNP had a certain antibacterial effect, and bacteria treated with FeS@Au exhibited the most red fluorescence, indicating that FeS@Au killed almost all bacteria and had the best antibacterial effect.

[0052] Biocompatibility test of cascaded nanozymes:

[0053] After co-culturing L929 cells and human umbilical vein endothelial cells (HUVECs) in Examples 1 and 2 for 24 h, cell viability was tested using a CCK-8 assay kit. Figure 7 As shown, the cell survival rate of each group of samples did not decrease significantly, indicating that the cell compatibility of each group of samples was good.

[0054] Cascaded nanozymes promote angiogenesis assay:

[0055] By seeding HUVECs on a matrix gel to mimic angiogenesis, the vascularization-promoting effect of FeS@Au on HUVECs was investigated in a serum-free and growth factor-free medium (neutral pH environment). Figure 8 As shown in Figure a, cells treated with PBS and AuNC exhibited predominantly stacking and budding, forming a small number of branched structures and fewer intact tubules. In contrast, cells treated with FeSNP and FeS@Au showed more closed tubular structures. Specifically, quantifying various indicators of tubular formation using ImageJ software revealed that FeS@Au-treated cells formed the most abundant network structure. Figure 8 (b) This facilitates the transport of oxygen and nutrients, thereby accelerating wound healing.

[0056] A diabetic rat model was established using intraperitoneal injection of streptozotocin. Four circular incisions, each 10 mm in diameter, were created on both sides of the rat's back, and 50 μL of LMRSA (10) was instilled into each incision. 7 An infection model was constructed using CFU / mL, and wounds were treated with Example 1 and the comparative example, respectively. Figure 9 Image 'a' in the diagram illustrates the treatment process. Wound images were recorded on days 3, 6, and 10. Figure 9 (b, d) and collect exudate from the wound site for bacterial culture. Figure 9 (c, e). The wound skin tissue on day 10 was stained with hematoxylin and eosin (H&E) and Masson's stain. The results are as follows: Figure 9 The f and g values ​​in the figure show that FeS@Au nanozyme can effectively reduce inflammation while promoting collagen deposition.

[0057] In vivo biosafety assessment:

[0058] The major organs of rats were collected and stained with H&E. Figure 10 As shown, no abnormalities were found in the heart, liver, spleen, lungs, and kidneys of the rats, indicating that FeS@Au has good in vivo biocompatibility.

[0059] The AuNC in the dosage nanozyme of this invention mimics the function of glucose oxidase (GOD), significantly reducing local blood glucose concentration at the wound site and activating the peroxidase-like (POD) activity of FeSNP. This catalyzes the conversion of glucose into hydrogen peroxide (H2O2) and gluconic acid, generating hydroxyl radicals (·OH), thereby powerfully killing drug-resistant bacteria. Furthermore, glucose triggers the release of H2S, significantly upregulating the expression level of hypoxia-inducible factor-1 (HIF-1), further promoting the expression of angiogenesis-related genes, accelerating the proliferation, migration, and lumen formation of human umbilical vein endothelial cells (HUVECs), and achieving highly efficient angiogenesis. In vivo experimental data fully demonstrate that the glucose-activated cascade activity and H2S release capacity enable FeS@Au to exhibit excellent therapeutic effects in treating full-thickness diabetic wounds infected with MRSA, demonstrating not only high antibacterial activity but also significantly promoting wound healing. In summary, this invention provides a cascaded nanozyme that responds to a hyperglycemic microenvironment, which integrates gas therapy, blood glucose regulation, highly efficient antibacterial activity, significant pro-angiogenic capacity, and excellent biocompatibility.

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a glucose-responsive hydrogen sulfide-releasing cascade nanozyme, characterized in that, Includes the following steps: (1) Preparation of FeSNP: BSA and FeCl2 were added to water and stirred at 330-350 r / min for 5-10 min to prepare solution A. Na2S was dissolved in water and added to solution A, and stirred at 330-350 r / min for 5-10 min. The mixture was heated in a water bath while nitrogen was introduced into the reaction for 5-6 h. After the reaction was completed, the mixture was placed in a 20 kD dialysis bag and dialyzed at 4-5 °C to obtain FeSNP. The molar ratio of FeCl2 to Na2S was 1:(3.8-4.2). (2) Preparation of FeS@Au: Under stirring conditions, an aqueous solution of HAuCl4 was added to the FeSNP prepared in step (1). After stirring for 5-10 min, an aqueous solution of NaOH was added to adjust the pH of the reaction system to 10-12. The reaction was stirred at room temperature for 1-6 h, and an aqueous solution of NaBH4 was added to continue the reaction for 10-30 min. After the reaction was completed, the reaction was dialyzed for 12-24 h at 4-5 °C using a 20 kD dialysis bag. After the dialysis was completed, the glucose-responsive hydrogen sulfide release cascade nanozyme FeS@Au was obtained. The molar ratio of FeSNP to HAuCl4 was (15.5-16.5):

1. The particle size of the FeS@Au nanoparticles was 50-150 nm, and the particle size of the AuNC nanoparticles loaded on its surface was 1-2 nm.

2. The method for preparing the glucose-responsive hydrogen sulfide release cascade nanozyme according to claim 1, characterized in that, The water bath heating mentioned in step (1) specifically refers to heating the water bath to 30℃~45℃.

3. The method for preparing the glucose-responsive hydrogen sulfide release 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.

4. A glucose-responsive hydrogen sulfide-releasing cascade nanozyme, prepared by the method for preparing the glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to any one of claims 1 to 3, characterized in that, It consists of 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 FeSNP.

5. The glucose-responsive hydrogen sulfide-releasing cascade nanozyme according to claim 4, characterized in that, The average Zeta potential of the FeS@Au nanoparticles is -20 to -40 mV.

6. The use of the glucose-responsive hydrogen sulfide-releasing cascade nanozyme of claim 5 in the preparation of a medicament for treating diabetic infected chronic wounds.

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