A single-atom nanoscale enzyme based on a red blood cell template, a preparation method and application thereof

Abstract

The application belongs to the technical field of single-atom nanoscale enzyme synthesis, and particularly relates to a single-atom nanoscale enzyme based on a red blood cell template, a preparation method and application. The single-atom nanoscale enzyme based on the red blood cell template comprises red blood cells, a fixing solution and a salt, wherein the red blood cells serve as a template, the fixing solution is used for fixing the red blood cells, and the salt is incorporated into the red blood cells. The single-atom nanoscale enzyme derived from the red blood cells prepared by the application has high peroxidase-like activity, and simultaneously has excellent photothermal performance, and the combination of the two promotes the generation of a large amount of ROS, so as to kill bacteria, and has a significant antibacterial and wound healing promoting effect in vivo and in vitro. Meanwhile, the loose and porous structure can promote the combination of the nanoscale enzyme with thrombin and fibrinogen, and has a significant hemostatic effect in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of single-atom nanoscale enzyme synthesis, and particularly relates to a single-atom nanoscale enzyme based on a red blood cell template, a preparation method and application. BACKGROUND

[0002] A single-atom nanoscale enzyme refers to a nanoscale enzyme with atomically dispersed metal active sites, and its chemical nature is a single-atom catalyst. In theory, in a single-atom nanoscale enzyme, the utilization efficiency, catalytic activity and stability of metal atoms are maximized. The preparation method is usually to introduce exogenous metal or non-metal elements into a carbon carrier and then prepare it by high-temperature calcination. However, in actual preparation, this method often leads to the agglomeration of nanoparticles, so that most of them exist in the form of atomic clusters rather than single atoms, which limits the catalytic performance and application of single-atom nanoscale enzymes. To solve this problem, we propose to use red blood cells as a template to synthesize single-atom nanoscale enzymes. Red blood cells contain a large amount of hemoglobin, about 260 million hemoglobin molecules in one red blood cell, and each hemoglobin structure contains 4 Fe atoms coordinated with the heme plane. Coincidentally, the catalytic active center structure of natural horseradish peroxidase also contains an iron porphyrin structure, which makes red blood cells an ideal iron source for preparing single-atom nanoscale enzymes to simulate the catalytic activity of horseradish peroxidase. This provides a new idea for the preparation of single-atom nanoscale enzymes. SUMMARY

[0003] In view of the existing problems in the prior art, the application provides a single-atom nanoscale enzyme based on a red blood cell template, a preparation method and application.

[0004] In a first aspect, the application provides a single-atom nanoscale enzyme based on a red blood cell template, which comprises red blood cells, a fixing solution and a salt, wherein the red blood cells act as a template, the fixing solution is used to fix the red blood cells, and the salt is incorporated into the red blood cells.

[0005] Further, the red blood cells are derived from red blood cells in animal blood, including chicken, duck, goose, pig, cow, sheep, rat, mouse, rabbit, etc., and preferably red blood cells in mouse blood.

[0006] Further, the salt is a common inorganic salt, and preferably NaCl. The ratio of the red blood cells to the salt is 1 mL of a 0.5M-6M, preferably 1M, salt solution per 400mg of red blood cells.

[0007] Further, the fixing solution is a common cell fixing solution, and preferably 4% paraformaldehyde. The volume ratio of the red blood cells to the fixing solution is 1:1-1:10.

[0008] In a second aspect, the application provides a method for preparing a red blood cell-derived single-atom nanoscale enzyme, comprising the following steps: collecting red blood cells, adding a fixing solution for fixation, adding a salt solution and then drying, high-temperature calcination in an inert gas atmosphere, washing off excess salt and drying to obtain the single-atom nanoscale enzyme.

[0009] Further, the collecting step of the red blood cells is: low-speed centrifugation of anticoagulated whole blood, discarding the supernatant and obtaining a red blood cell precipitate; preferably, the centrifugation speed is 500-4000 rpm and the centrifugation time is 2-10 min.

[0010] Further, the drying method after adding the salt solution is vacuum drying, preferably vacuum freeze-drying.

[0011] Further, the gas atmosphere during high-temperature calcination is an inert gas such as nitrogen or argon, preferably a nitrogen atmosphere; the high-temperature calcination temperature is 200-900℃, preferably 800℃, and the pyrolysis time is 0.5-4 h, preferably 2 h.

[0012] In a third aspect, the application provides a use of a red blood cell-derived single-atom nanoscale enzyme in antibiosis and wound healing promotion.

[0013] Further, the antibiosis includes gram-positive bacteria and gram-negative bacteria, and the wound is a scratch or bleeding.

[0014] The application has the following beneficial effects:

[0015] The red blood cell-derived single-atom nanoscale enzyme prepared by the application has high peroxidase-like activity and excellent photothermal performance, and the combination of the two promotes the generation of a large amount of ROS, thereby killing bacteria and having a significant antibacterial effect and wound healing promotion effect in vivo and in vitro. At the same time, the loose and porous structure can promote the combination of the nanoscale enzyme with thrombin and fibrinogen, and has a significant hemostatic effect in vivo and in vitro. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a preparation process of a red blood cell-derived single-atom nanoscale enzyme (ETN).

[0017] Figure 2 Figure 2 is a scanning electron microscope characterization of the ETN preparation process. (1) Fixed red blood cells (scale bar: 1 μm); (2) Salted red blood cells after freeze-drying (scale bar: 1 μm); (3) The final prepared ETN (scale bar: 5 μm); (4) Local magnification of the prepared ETN (scale bar: 1 μm).

[0018] Figure 3Transmission electron microscopy characterization of ETN. a, Transmission electron microscopy characterization of ETN (scale bar: 5 nm); b, spherical aberration electron microscopy characterization (scale bar: 5 nm); c, elemental energy dispersive spectroscopy characterization of ETN (scale bar: 1 μm).

[0019] Figure 4 ETN from different species. a, ETN from chicken; b, ETN from duck; c, ETN from goose; d, ETN from pig; e, ETN from cow; f, ETN from sheep; g, ETN from rat; h, ETN from rabbit (scale bar: 5 μm (left) and 1 μm (right)).

[0020] Figure 5 Scanning electron microscopy characterization of unsalted ETN (scale bar: 5 μm).

[0021] Figure 6 Peroxidase-like activity assay of ETN.

[0022] Figure 7 Oxidase-like activity assay of ETN.

[0023] Figure 8 Superoxide dismutase enzyme activity assay of ETN.

[0024] Figure 9 Catalase-like activity assay of ETN.

[0025] Figure 10 Coagulation-promoting effect of ETN at different concentrations (***P < 0.01).

[0026] Figure 11 Coagulation-promoting effect of ETN at different times (***P < 0.01, ****P < 0.001).

[0027] Figure 12 Western Blot results of prothrombin and fibrinogen after ETN-treated plasma.

[0028] Figure 13 In vivo coagulation-promoting effect of ETN (***P < 0.01).

[0029] Figure 14 Temperature change under different conditions of laser irradiation (***P < 0.01).

[0030] Figure 15 Survival of MRSA under different treatment conditions (*P < 0.5, ***P < 0.01, ****P < 0.001).

[0031] Figure 16Survival of E. coli under different treatment conditions (*P<0.5, **P<0.1, ****P<0.001).

[0032] Figure 17 Survival of S. aureus under different treatment conditions (*P<0.5, **P<0.1, ***P<0.01).

[0033] Figure 18 Photographic results of wound sites in different treatment groups.

[0034] Figure 19 Wound area statistics in different treatment groups (*P<0.5).

[0035] Figure 20 Colony count results statistics of wound sites in different treatment groups (*P<0.5).

[0036] Figure 21 H&E staining results of wound sites in different treatment groups. DETAILED DESCRIPTION

[0037] The present application will be further illustrated below with examples. It should be understood that the examples are only used to further illustrate and explain the present application, and are not intended to limit the present application.

[0038] Example 1 Preparation of single-atom nanoszyme based on red blood cell template

[0039] The preparation process of single-atom nanoszyme (ETN) based on red blood cell template is shown in Figure 1 , centrifuge mouse anticoagulant whole blood at 1500 rpm for 10 min, collect the red blood cells at the bottom, add 4% paraformaldehyde at a volume ratio of 1:1, mix well, and then fix at 4°C. Centrifuge again to discard the fixing solution, then add 1M NaCl, mix well, and then vacuum freeze-dry. Take the dried powder, heat it to 800°C at a rate of 4°C / min in a nitrogen atmosphere, and calcine it for 2h. After washing to remove excess salt, dry it to obtain red blood cell-derived single-atom nanoszyme. The morphology characterization during the preparation process is shown in Figure 2 , as shown in Figure 3 , the same method is used to prepare red blood cell-derived single-atom nanoszyme from different species, and the salt-free red blood cell-derived single-atom nanoszyme is prepared after fixing without adding salt. Figure 4 , Figure 5 .

[0040] Test results: as shown in Figure 2 , the morphology of the fixed red blood cells remains the concave round cake shape of the two sides of the red blood cells. After salt freeze-drying, a layer of salt particles is uniformly attached to the surface of the red blood cells. After high-temperature carbonization, the morphology of the red blood cells changes greatly, forming a honeycomb structure.

[0041] The results are shown in Figure 3 The results are shown in

[0042] The results are shown in Figure 4 and Figure 5 The results are shown in

[0043] Example 2 Activity determination of single-atom nanoszyme based on red blood cell template

[0044] The peroxidase-like activity of ETN was determined in NaAc buffer (1 mL, 0.1 M, pH 4.5) with TMB (20 μL, 10 mg / mL in DMSO) and H2O2 (15 μL, 10 M) as substrates. The absorbance of oxidized TMB at 652 nm was recorded by a microplate reader at a specific time, indicating the peroxidase-like activity of ETN, and the results are shown in Figure 6

[0045] The oxidase-like activity of ETN and ETN without NaCl treatment was determined in NaAc buffer (1 mL, 0.1 M, pH 4.5) with TMB (20 μL, 10 mg / mL in DMSO) as substrate. The absorbance of oxidized TMB at 652 nm was recorded by a microplate reader at a specific time, and the results are shown in Figure 7

[0046] The SOD-like activity of ETN was determined using a superoxide dismutase assay kit. First, different concentrations (0-1 mg / mL) of ETN were mixed with 200 μL WST-1 working solution, and then 20 μL xanthine oxidase solution was added to start the reaction. After incubation at 37 °C for 20 min, the decrease in absorbance was quantitatively determined at 450 nm, indicating the SOD-like activity of ETN, and the results are shown in Figure 8 The catalase-like activity of ETN was determined using a specific oxygen electrode on a multi-parameter analyzer to monitor the increase in dissolved oxygen concentration. ETN (2 μg / mL) was mixed with H2O2 (500 mM). The reaction was carried out in a total system of 5 mL, and the results are shown in Figure 9

[0047] The results are shown in Figures 6-9 ​​​As shown, ETN exhibited multiple types of enzyme catalytic activities, in which, under acidic conditions, oxidase and peroxidase activities were dominant, while under neutral and basic conditions, superoxide dismutase and catalase activities were dominant.

[0048] Example 3 Coagulation-promoting effect of single-atom nanoszyme based on red blood cell template

[0049] Take 90 μL of blood and mix with 5 μL of calcium chloride (0.2 M), then add 5 μL of ETN (dispersed in water at different concentrations), and store at 37°C for 5 min, and centrifuge at 500 rpm for 3 min. Then slowly add 1 mL of ddH2O, take a photo, and measure the absorbance of the supernatant at 575 nm, and the results are shown in Figure 10 、 Figure 11 .

[0050] Test results: As shown in Figures 10-11 , the coagulation-promoting effect of ETN is concentration-dependent, and the coagulation time is shortened to 2-3 min.

[0051] In order to identify whether ETN can bind to thrombin and fibrinogen, rabbit anticoagulant whole blood was centrifuged at 4000 rpm for 10 min at 4°C. After centrifugation, the supernatant, i.e. rabbit plasma, was collected. In 90 μL of rabbit plasma, 5 μL of ETN at different concentrations (0, 62.5, 125, 500 μg / mL) was added, respectively, and incubated at 37°C for 5 min. Finally, centrifugation was performed at 12000 rpm for 5 min at 4°C, and the precipitate was collected after centrifugation to obtain the pretreated sample. The obtained sample was subjected to Western Blot test with anti-fibrinogen antibody and anti-thrombin antibody, respectively. The results are shown in Figure 12 .

[0052] Test results: As shown in Figure 12 , the contents of fibrinogen and thrombin in the plasma after ETN treatment gradually increased with the increase of ETN concentration, indicating that fibrinogen and thrombin were combined with ETN.

[0053] In order to determine the in vivo coagulation-promoting effect of ETN, before the experiment, the experimental group cotton balls were soaked in 0.5 mg / mL ETN dispersed in sodium acetate solution (ETN group), the buffer group cotton balls were soaked in sodium acetate solution, and the control group cotton balls were soaked in water. Eight-week-old wild-type balb / c mice were divided into three groups: control group, buffer group and ETN group. After the mice were anesthetized, part of the liver tissue was taken out and placed on the above-mentioned three kinds of treated cotton balls to press the wound surface until the bleeding stopped. The bleeding time and the weight of the excised liver were recorded, and the weight of the cotton ball before and after treatment was also recorded. After the wound of the mouse was hemostatic, it was sutured, and then the bleeding amount per unit mass of liver was calculated, as shown in Figure 13 . Test results: As shown in Figure 13As shown, compared with the control group, the amount of liver bleeding of the mice in the sodium acetate buffer group alone did not significantly decrease, indicating that it had no obvious hemostatic effect, while the amount of bleeding in the ETN soaking group significantly decreased, indicating that ETN had a significant hemostatic effect.

[0054] Example 4 Photothermal performance of single-atom nanoszyme based on red blood cell template

[0055] The temperatures of NaAC, H2O2(100 μM), ETN(500 μg / mL) and ETN+H2O2 under 808 nm laser irradiation (2 W / cm 2 , 6 min) were recorded with a thermometer, respectively, and the results are shown in Figure 14 .

[0056] Test results: as shown in Figure 14 , after laser irradiation, the temperature of the ETN group and the ETN+H2O2 group increased significantly to 47.5℃ within 1 min, indicating that ETN has good photothermal performance.

[0057] Example 5 Antibacterial effect of single-atom nanoszyme based on red blood cell template

[0058] Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300), Staphylococcus aureus (ATCC 29213) and gram-negative bacteria Escherichia coli (CMCC(B)44102) were used for antibacterial test. MRSA was cultured in standard LB (Luria-Bertani) medium. ETN was placed in an ep tube, and MRSA (1 mL, 1×10 6 CFU / mL) was incubated with NaAc buffer, H2O2(100 μM), NaAc+NIR, ETN, H2O2+ETN(500 μg / mL), ETN+H2O2+NIR, ETN+NIR, H2O2+NIR, respectively. The (NIR+) sample was irradiated with 808 nm NIR laser (2 W / cm 2 ) for 6 min, and the results are shown in Figures 15-17 .

[0059] Test results: after treatment with ETN+H2O2+NIR, the number of colonies of methicillin-resistant Staphylococcus aureus, Escherichia coli and Staphylococcus aureus decreased by 2.32-log, 2.26-log and 1.79-log, respectively, which was significantly better than ETN+NIR and ETN+H2O2 alone, indicating that the bactericidal effect was carried out by peroxidase-like activity combined with photothermal effect.

[0060] Example 6 Wound healing promoting effect of single-atom nanoszyme based on red blood cell template

[0061] Mice were depilated on the back and anesthetized with isoflurane. A 10 mm diameter full-thickness skin wound was made on the back of the mice by a puncher. 30 μL of MRSA mixed suspension (5 x 10 7 CFU) was injected in the center of each wound. The mice were randomly divided into 8 groups (n = 6) as follows: (1) control group; (2) NaAC + NIR; (3) H2O2 + NIR; (4) H2O2; (5) ETN + NIR; (6) NIR; (7) ETN + H2O2 + NIR; (8) ETN + H2O2. After 24 h, 20 μL (ETN 500 μg / mL, H2O2 100 μM, both dispersed in NaAC) was dropped on the wound of ETN + H2O2 + NIR group, and irradiated with 808 nm laser (2 W / cm 2 ) for 6 min. The same method was used for the other 7 groups. NaAC was added during laser irradiation to avoid wound burning. The wound area of each group was photographed and measured every day (as shown in Figure 18 , 19 ). On the 7th day, the skin tissue of the wound was ground, diluted with LB medium, and 100 μL of the diluted solution was added to the agar culture plate, which was incubated at 37 °C for 18 h. The in vivo antibacterial performance was detected by CFU. The results are shown in Figure 20 . In addition, the wound part of all mice was taken for H&E staining. The results are shown in Figure 21 .

[0062] Experimental results: As shown in Figures 18-21 , compared with the ETN + NIR group and the ETN + H2O2 group, the treatment effect of the ETN + H2O2 + NIR group was the most significant, indicating that the peroxidase-like activity of ETN combined with photothermal effect jointly resisted bacteria.

[0063] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The present disclosure is further defined by the following Examples.

Claims

1. A method for the preparation of red blood cell-derived single atom nanoszymes for antimicrobial use, characterized by: The method comprises the following steps: Collecting red blood cells and adding a fixing solution for fixation; After adding a salt solution, drying, high-temperature calcination in a nitrogen atmosphere; Washing off excess salt and drying to obtain the single-atom nanoscale enzyme.

2. The method of claim 1, wherein: The red blood cells are derived from red blood cells in animal blood, and the animals include chicken, duck, goose, pig, cow, sheep, rat, mouse, and rabbit.

3. The method of claim 2, wherein: The red blood cells are red blood cells in mouse blood.

4. The production method according to claim 1, characterized by: The salt is NaCl; the ratio of red blood cells to salt is 1 mL of a 0.5 M-6 M salt solution added to every 400 mg of red blood cells.

5. The method of claim 4, wherein: The ratio of red blood cells to salt is 1 mL of a 1 M salt solution added to every 400 mg of red blood cells.

6. The method of claim 1, wherein: The fixing solution is 4% paraformaldehyde, and the volume ratio of red blood cells to fixing solution is 1:1-1:

10.

7. The method of claim 1, wherein: The step of collecting red blood cells is to centrifuge anticoagulated whole blood at a low speed, discard the supernatant, and obtain a red blood cell precipitate.

8. The method of claim 7, wherein: In the low-speed centrifugation, the centrifugal speed is 500-4000 rpm, and the centrifugation time is 2-10 min.

9. The method of claim 1, wherein: The drying method after adding the salt solution is vacuum drying.

10. The method of claim 1, wherein: The high-temperature calcination temperature is 200-900°C, and the pyrolysis time is 0.5-4 h.

11. The method of claim 10, wherein: The high-temperature calcination temperature is 800°C, and the pyrolysis time is 2 h.

12. The use of red blood cell-derived single-atom nanoscale enzymes prepared by the preparation method of any one of claims 1-11 in antibiosis.

13. Use according to claim 12, characterized in that: The bacteria for antibiosis include gram-positive bacteria and gram-negative bacteria.

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