Novel nano-enzyme composite material as well as preparation method and application thereof
By loading Pt nanoparticles onto the surface of APDA nanoparticles to form a nanoenzyme composite material, the problem of balancing the catalytic activity and biocompatibility of photothermal materials in diabetic foot infected wounds was solved, achieving efficient antibacterial and antioxidant effects and promoting wound healing.
Patent Information
- Application Number
- CN202511049764.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-11-04
AI Technical Summary
Existing photothermal materials struggle to balance catalytic activity and biocompatibility when treating infectious wounds of diabetic foot ulcers, and lack the ability to scavenge ROS, thus failing to effectively alleviate oxidative stress and hindering wound healing.
A novel nanoenzyme composite material was prepared by loading Pt nanoparticles onto the surface of APDA nanoparticles to form APDA@Pt, which combines photothermal properties with catalase-like activity to achieve antioxidant and antibacterial effects.
It achieves high antibacterial rates against E. coli and S. aureus, exhibits good photothermal stability and antioxidant function, and simultaneously addresses bacterial infection and oxidative stress, promoting wound healing.
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Figure CN120884699A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of nanobiomaterials, and particularly relates to a novel nanocatalyst composite material, a preparation method and application thereof. BACKGROUND
[0002] Diabetic patients often suffer from the persistent infection of diabetic foot wounds, and this clinical problem is caused by the interaction of multiple pathological mechanisms. The pathological characteristics are that the multiple bacterial infections under the high-sugar microenvironment cause the wound to be in a chronic inflammation period for a long time, and the respiratory burst of neutrophils produces excessive reactive oxygen species (ROS) in the inflammation period. When the accumulation of ROS exceeds the clearance capacity of the antioxidant system, it will cause significant oxidative stress damage. This excessive oxidative stress not only directly damages tissue cells, but also forms a positive feedback cycle by activating the pro-inflammatory signaling pathway, and further forms a vicious cycle that hinders wound healing.
[0003] At present, photothermal materials are concerned due to their excellent photothermal conversion efficiency and controllable antibacterial mechanism. However, the catalytic activity and biocompatibility of photothermal materials are often difficult to balance, and the antibacterial mechanism mainly treats bacterial infections by efficiently inhibiting or killing bacteria. The mechanism is often relatively single, and there are obvious limitations. On the one hand, there is a risk of drug resistance in long-term use. On the other hand, such materials generally lack ROS scavenging capacity and antioxidant function, and cannot effectively alleviate the excessive oxidative stress reaction at the infection site, so it is difficult to fundamentally break the vicious cycle that hinders wound healing.
[0004] Therefore, it is of great significance to develop photothermal materials that can simultaneously solve bacterial infection and oxidative stress for the treatment of bacterial infection. SUMMARY
[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a novel nanocatalyst composite material.
[0006] Another purpose of the present application is to provide a preparation method of the novel nanocatalyst composite material.
[0007] Another purpose of the present application is to provide the application of the novel nanocatalyst composite material in improving the activity of the catalase-like enzyme.
[0008] The purpose of the present application is achieved by the following technical solutions.
[0009] A novel nanocatalyst composite material, wherein the novel nanocatalyst composite material is APDA nanoparticles and Pt nanoparticles loaded on the surface of the APDA nanoparticles.
[0010] A preparation method of a novel nanocatalyst composite material, comprising the following steps:
[0011] Mixing APDA nanoparticles, chloroplatinic acid and water uniformly to obtain a mixed solution, dropping 0-4 DEG C sodium borohydride aqueous solution into the mixed solution under 0-4 DEG C, and continuing to stir for 4-6 h under 0-4 DEG C, then sequentially performing centrifugation, washing and freeze-drying to obtain a novel nano-enzyme composite material, wherein the mass fraction of APDA nanoparticles and the amount of substance of chloroplatinic acid are (100-101) :(0.3-0.34), the unit of the mass fraction is mg, and the unit of the amount of substance is μmol.
[0012] In the above technical solution, the APDA nanoparticles are dispersed in water to obtain an APDA solution, and the chloroplatinic acid aqueous solution is dropped into the APDA solution, and stirred uniformly at room temperature of 20-25 DEG C for 1-1.5 h to obtain a mixed solution.
[0013] In the above technical solution, the concentration of APDA nanoparticles in the APDA solution is 12.5-12.6 mg / mL, and the concentration of chloroplatinic acid in the chloroplatinic acid aqueous solution is 1-1.05 mM.
[0014] In the above technical solution, the mass fraction of APDA nanoparticles and the mass fraction of sodium borohydride in the sodium borohydride aqueous solution are (100-101) :(5-6).
[0015] In the above technical solution, the concentration of sodium borohydride in the sodium borohydride aqueous solution is 2.5-3 mg / mL.
[0016] In the above technical solution, the method for obtaining APDA nanoparticles comprises: dissolving dopamine hydrochloride in water to obtain a dopamine hydrochloride solution; dissolving arginine in water to obtain an arginine solution; dropping the arginine solution into the dopamine hydrochloride solution under stirring, and stirring at room temperature of 20-25 DEG C for 4-6 h, and then sequentially performing centrifugation, washing and vacuum drying to obtain APDA nanoparticles, wherein the ratio of dopamine hydrochloride and arginine is (1-1.1) :(1-1.1) in terms of mass fraction.
[0017] In the above technical solution, the mass fraction of arginine and the volume fraction of water in the arginine solution are (100-101) :(10-10.5), the unit of the mass fraction is mg, and the unit of the volume fraction is mL.
[0018] In the above technical solution, the mass fraction of dopamine hydrochloride and the volume fraction of water in the dopamine hydrochloride solution are (100-101) :(110-110.5), the unit of the mass fraction is mg, and the unit of the volume fraction is mL.
[0019] In the above technical solution, the temperature of vacuum drying is 60-63 DEG C, and the time of vacuum drying is 12-14h.
[0020] In the above technical solution, the operation of washing is rinsing 3-4 times with ultrapure water or anhydrous ethanol.
[0021] In the above technical solution, the rate of centrifugation is at least 12000 rpm, and the time of centrifugation is at least 20 min.
[0022] In the above technical solution, the temperature of freeze-drying is -80 to -79 DEG C, and the time of freeze-drying is at least 24h.
[0023] The above novel nanoscale enzyme composite material is applied to improving the catalase-like activity.
[0024] In the above technical solution, the clearance rate of the novel nanoscale enzyme composite material to ABTS is higher than 80%, and the hemolysis rate of the novel nanoscale enzyme composite material is lower than 3%.
[0025] Compared with the prior art, the present application has the following beneficial effects:
[0026] (1) The novel nanoscale enzyme composite material has both antioxidant function and antibacterial performance, and can simultaneously solve the problems of bacterial infection and oxidative stress.
[0027] (2) The novel nanoscale enzyme composite material has good photothermal stability as a photothermal agent. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 It is a reaction schematic diagram of the novel nanoscale enzyme composite material;
[0029] Figure 2 It is (a) TEM image and (b) HRTEM image of the novel nanoscale enzyme composite material prepared in Example 1;
[0030] Figure 3 It is SEM image of the novel nanoscale enzyme composite material prepared in Example 1;
[0031] Figure 4 It is (a) HAADF-STEM image and (b-e) EDS mapping image of the novel nanoscale enzyme composite material prepared in Example 1;
[0032] Figure 5 It is Zeta potential of the novel nanoscale enzyme composite material prepared in Example 1 and the nanoscale enzyme material prepared in Comparative Example 1;
[0033] Figure 6UV-Vis spectra of the new nanoscale enzyme composite material prepared in Example 1 and the nanoscale enzyme material prepared in Comparative Example 1;
[0034] Figure 7 XPS spectra of the new nanoscale enzyme composite material prepared in Example 1, wherein (a) is a full spectrum, (b) is a Pt4f spectrum, (c) is a N1s spectrum, (d) is a C1s spectrum and (e) is an O1s spectrum
[0035] Figure 8 (a) is the photothermal response curve of the new nanoscale enzyme composite material prepared in Example 1 and water, Figure 8 (b) is the photothermal response cycle curve of the new nanoscale enzyme composite material prepared in Example 1;
[0036] Figure 9 (a) is the infrared thermal imaging map of the new nanoscale enzyme composite material prepared in Example 1, Figure 9 (b) is the photothermal response curve of the new nanoscale enzyme composite material prepared in Example 1 under different concentrations;
[0037] Figure 10 The photothermal response curve of the new nanoscale enzyme composite material prepared in Example 1 under different power densities;
[0038] Figure 11 (a-b) of the (a-b) are the colony conditions of the photothermal agent light group, the control light group, the photothermal agent non-light group and the control non-light group, Figure 11 (c-d) of the (c-d) are the colony numbers of the photothermal agent light group, the control light group, the photothermal agent non-light group and the control non-light group, wherein the first column (a, c) is Escherichia coli, and the second column (b, d) is Staphylococcus aureus;
[0039] Figure 12 The bacterial morphology changes of the photothermal agent light group, the control light group, the photothermal agent non-light group and the control non-light group, wherein a is Escherichia coli, and b is Staphylococcus aureus;
[0040] Figure 13 The (first column) OD 600 value and the (second column) turbidity of the light group and the non-light group, wherein (a-b) is Escherichia coli, and (c-d) is Staphylococcus aureus;
[0041] Figure 14 The cell survival rate of L-929 cells treated with the new nanoscale enzyme composite material prepared in Example 1 under different concentrations;
[0042] Figure 15 The staining map of L-929 cells treated with the new nanoscale enzyme composite material prepared in Example 1 under different concentrations;
[0043] Figure 16Hemolysis rate of red blood cells treated by the new nanoscale enzyme composite material prepared in Example 1 at different concentrations;
[0044] Figure 17 Oxygen production of the new nanoscale enzyme composite material prepared in Example 1 at different concentrations;
[0045] Figure 18 UV absorption spectrum of H2O2 aqueous solution treated by the new nanoscale enzyme composite material prepared in Example 1 and the nanoscale enzyme material prepared in Comparative Example 1;
[0046] Figure 19 ABTS scavenging rate of the nanoscale enzyme material of the new nanoscale enzyme composite material prepared in Example 1 and water. DETAILED DESCRIPTION
[0047] The technical solutions of the present application will be further described in detail below in combination with the drawings and examples.
[0048] In the following examples, the method for obtaining APDA nanoparticles comprises: dissolving dopamine hydrochloride in ultrapure water to obtain a dopamine hydrochloride solution; dissolving arginine in ultrapure water to obtain an arginine solution; under stirring, adding the arginine solution dropwise into the dopamine hydrochloride solution, and stirring at room temperature of 20-25℃ for 4h to obtain a mixed solution; centrifuging the mixed solution at a speed of 12000rpm for 20min to remove the supernatant to obtain a precipitate; rinsing the precipitate with anhydrous ethanol for 3 times, and drying in a vacuum drying oven at 60℃ for 12h to obtain APDA nanoparticles, wherein the ratio of dopamine hydrochloride and arginine is 1:1 by mass fraction; the ratio of the mass fraction of arginine and the volume fraction of ultrapure water in the arginine solution is 100:10; and the ratio of the mass fraction of dopamine hydrochloride and the volume fraction of ultrapure water in the dopamine hydrochloride solution is 100:110, the unit of mass fraction is mg, and the unit of volume fraction is mL.
[0049] Example 1
[0050] As Figure 1As shown, a novel nanoenzyme composite material (designated: APDA@Pt) comprises the following steps: APDA nanoparticles are dispersed in ultrapure water to obtain an APDA solution; an aqueous solution of chloroplatinic acid is added dropwise to the APDA solution; the mixture is stirred at room temperature (20–25°C) for 1 hour until homogeneous, resulting in a mixed solution. At 0°C, an aqueous solution of sodium borohydride at 0°C is added dropwise to the mixed solution, and the mixture is stirred in an ice-water bath at 0°C for 4 hours. The mixture is then centrifuged at 12000 rpm for 20 minutes to obtain a precipitate. The precipitate is washed three times with ultrapure water and then freeze-dried at -80°C for 24 hours to obtain the novel nanoenzyme composite material (designated: APDA@Pt). The mass fraction of APDA nanoparticles to the molar fraction of chloroplatinic acid is 100:0.3, where mass fractions are in mg and molar fractions are in μmol. The concentration of APDA nanoparticles in the APDA solution is 0.0125 g / mL; the concentration of chloroplatinic acid in the aqueous solution is 1 mM. The mass ratio of APDA nanoparticles to sodium borohydride in the aqueous solution is 100:5, and the concentration of sodium borohydride in the aqueous solution is 2.5 mg / mL.
[0051] Comparative Example 1
[0052] A nanoenzyme material, which is APDA nanoparticles.
[0053] The morphology of the novel nanozyme composite material prepared in Example 1 was observed using transmission electron microscopy (TEM). TEM images are shown below. Figure 2 As shown in (a), by Figure 2 As shown in (a), the novel nanozyme composite material prepared in Example 1 exhibits uniformly sized spherical nanoparticles with a particle size distribution between 200-250 nm, and its surface is loaded with many amorphous small particles. Further high-resolution transmission electron microscopy (HRTEM) was performed on the novel nanozyme composite material prepared in Example 1; the HRTEM image is shown below. Figure 2 As shown in (b), by Figure 2 As shown in (b), the lattice spacing between adjacent stripes on the surface of the novel nanoenzyme composite material prepared in Example 1 was measured to be 0.23 nm, which corresponds to the (1,1,1) lattice of Pt. Therefore, the small particles are Pt nanoparticles.
[0054] The novel nanozyme composite material prepared in Example 1 was characterized by scanning electron microscopy (SEM). The SEM images are shown below. Figure 3 As shown, by Figure 3 As can be seen, the SEM images also show the same particle size, and the surface of the novel nanoenzyme composite material prepared in Example 1 exhibits a rougher morphology.
[0055] The new nano-enzyme composite material prepared in Example 1 was observed by high-angle annular dark field scanning transmission (HAADF-STEM) and energy dispersive X-ray spectroscopy (EDS), the HAADF-STEM image is shown in (a) of Figure 4 , the EDS image is shown in (b-e) of Figure 4 , it can be seen from Figure 4 that C, O, N and Pt are uniformly distributed on the surface of the new nano-enzyme composite material prepared in Example 1, and this result also proves the successful loading of Pt nanoparticles.
[0056] The new nano-enzyme composite material prepared in Example 1 and the nano-enzyme material prepared in Comparative Example 1 were analyzed by Zeta potential, the Zeta potential is shown in Figure 5 , it can be seen from Figure 5 that the potential of the new nano-enzyme composite material prepared in Example 1 is lower than that of Comparative Example 1, which further proves the successful loading of Pt nanoparticles.
[0057] The new nano-enzyme composite material prepared in Example 1 and the nano-enzyme material prepared in Comparative Example 1 were analyzed by UV-Vis, the UV-Vis spectrum is shown in Figure 6 , it can be seen from Figure 6 that the new nano-enzyme composite material prepared in Example 1 and the nano-enzyme material prepared in Comparative Example 1 both exhibit broadband absorption in the ultraviolet-visible region.
[0058] The surface element composition and valence state of the new nano-enzyme composite material prepared in Example 1 were characterized by X-ray photoelectron spectroscopy (XPS). As shown in Figure 7 , it can be seen from Figure 7As shown in (a), there are three distinct fitting peaks at 285 eV, 400 eV, and 532 eV, indicating the presence of C, N, and O. In addition, a fitting peak at 75 eV corresponds to Pt 4f, further confirming the successful loading of Pt nanoparticles onto the surface of polydopamine nanoparticles. In Pt 4f, the fitting peaks at 71.6 eV and 74.8 eV belong to zero-valent platinum (Pt(0)), while the fitting peaks at 72.8 eV and 76.4 eV belong to +2 valent Pt. In the C1s spectrum, the fitting peak at 284.8 eV belongs to the C-C bond, the fitting peak at 286.3 eV belongs to the CO bond, the fitting peak at 288.1 eV belongs to the CN bond, the fitting peak at 289.6 eV belongs to the C=O bond, and the fitting peak at 291.8 eV belongs to the π-π* satellite peak. In the N1s spectrum, the fitted peak at 397.7 eV belongs to -NC, the fitted peak at 399.9 eV belongs to -NH-, and the fitted peak at 402.2 eV belongs to -NO. In the O1s spectrum, the fitted peaks at 531.3 eV and 533 eV belong to C=O and CO bonds, respectively. In summary, Pt was successfully loaded onto the APDA surface via in-situ reduction, thus synthesizing the novel nanozyme composite material prepared in Example 1.
[0059] Example 2
[0060] Suitable photothermal temperature and stable photothermal properties are crucial for the antibacterial effect of photothermal agents and their potential application in wound healing. To evaluate the photothermal properties of the novel nanoenzyme composite material prepared in Example 1, the novel nanoenzyme composite material prepared in Example 1 was dispersed in water as a photothermal agent to obtain a photothermal agent aqueous solution. The concentration of the photothermal agent in the aqueous solution was 200 μg / mL, and the concentration was 0.75 W / cm². 2 At a power density of [value missing], an aqueous photothermal agent solution was irradiated with an 808nm near-infrared laser for 10 minutes, and the photothermal response curve was obtained. Additionally, the photothermal response curve of water was used as a control. Figure 8 As shown in (a). By Figure 8 As shown in (a), the novel nanoenzyme composite material prepared in Example 1 has a temperature of 53.5°C after 10 min of light irradiation, indicating that it has excellent photothermal properties.
[0061] Subsequently, this invention further investigated the photothermal stability of the novel nanoenzyme composite material prepared in Example 1, repeating the following steps four times: at 0.75 W / cm 2 At a power density of [value missing], the photothermal agent aqueous solution was irradiated with an 808nm near-infrared laser for 10 min, followed by 10 min in the dark to allow the solution to cool to room temperature. This cycle was repeated four times, and the resulting photothermal response cycle curve is shown below. Figure 8 As shown in (b), by Figure 8It can be known from (B) of FIG. 8 that, after four cycles of irradiation, the new nano-enzyme composite material prepared in Example 1 still shows excellent photothermal performance, and the final temperature has little change, indicating that the new nano-enzyme composite material prepared in Example 1 has good photothermal stability.
[0062] Example 3
[0063] In order to further optimize the photothermal conditions of the new nano-enzyme composite material (APDA@Pt) prepared in Example 1, the present application investigates the photothermal effect of the new nano-enzyme composite material under different power densities and different concentrations.
[0064] The new nano-enzyme composite material prepared in Example 1 is dispersed in water as a photothermal agent to obtain a photothermal agent aqueous solution, and the concentration of the photothermal agent in the photothermal agent aqueous solution is set to 50 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL respectively. Under a power density of 0.75 W / cm 2 , the photothermal agent aqueous solution is irradiated with 808 nm near-infrared laser for 10 min to obtain a photothermal response curve, as shown in FIG. 9, and the infrared thermal imaging of the photothermal agent aqueous solution at Tmin is obtained, as shown in FIG. 10(A), and the temperature of the photothermal agent in the photothermal agent aqueous solution at T=0 min, 2 min, 4 min, 6 min, 8 min and 10 min is obtained, as shown in FIG. 10(B). Figure 9 Figure 9 It can be known from FIG. 10(A) and (B) that, under the same power density, the higher the concentration of the photothermal agent in the photothermal agent aqueous solution, the higher the temperature of the photothermal agent in the photothermal agent aqueous solution. Figure 9
[0065] Table 1
[0066] Concentration 50 μg / mL 100 μg / mL 150 μg / mL 200 μg / mL Temperature 33.7℃ 43.1℃ 47.6℃ 53℃
[0067] The power density is set to 0.25 W / cm 2 , 0.5 W / cm 2 , 0.75 W / cm 2 and 1 W / cm 2 respectively, and the photothermal agent aqueous solution (the concentration of the photothermal agent in the photothermal agent aqueous solution is 200 μg / mL) is irradiated with 808 nm near-infrared laser for 10 min to obtain a photothermal response curve, as shown in FIG. 11, and the temperature of the photothermal agent in the photothermal agent aqueous solution under different power densities at 10 min is shown in Table 2. Figure 10 Figure 10 It can be known from FIG. 11 and Table 2 that, with the increase of the power density of irradiation, the temperature of the new nano-enzyme composite material prepared in Example 1 gradually increases.
[0068] Table 2
[0069] Power Density 0.25 W / cm 2 ]] 0.5 W / cm 2 ]] 0.75 W / cm 2 ]]> 1 W / cm 2 ]] Temperature 23.1℃ 40.2℃ 52.9℃ 63.6℃
[0070] Based on the above results, it is found that a higher concentration of photothermal agent is beneficial to the improvement of photothermal performance, and in consideration of the concept of green chemistry, the concentration of the photothermal agent is finally selected as 200 pg / mL, and the power density is 0.75 W / cm 2 For the following experiments.
[0071] Example 4
[0072] E. coli (ATCC8739) and S. aureus (ATCC29213) (source: China General Microbiological Culture Collection Center) were used as "bacteria" in the following experimental group steps:
[0073] Photothermal agent light group (APDA@Pt & NIR(+)) :
[0074] S1, the bacteria were inoculated in LB medium and cultured at 37℃, 260rpm for 12h to obtain a bacterial suspension with a density of 10 6 CFU / mL, 1mL photothermal agent solution was mixed with the bacterial suspension to obtain a mixed bacterial solution. The photothermal agent solution was a mixture of the photothermal agent and PBS buffer (pH = 7.4), the concentration of the photothermal agent in the photothermal agent solution was 1mg / mL, the photothermal agent was a new nanozyme composite material prepared in Example 1, and the concentration of the photothermal agent in the mixed bacterial solution was 200 pg / mL.
[0075] S2, the mixed bacterial solution was irradiated with 808nm near-infrared laser at a power density of 0.75W / cm 2 for 10min, and then incubated at 37℃ for 10min. After incubation, the mixed bacterial solution was diluted with PBS buffer to 10 4 CFU / mL, 20ul of the diluted mixed bacterial solution was evenly coated on the plate agar, and incubated at 37℃ for 12h. After incubation, the colony situation and colony number on the plate agar were observed.
[0076] Control light group (PBS & NIR(+)) : basically the same as the photothermal agent light group, except that the mixed bacterial solution in the control light group was obtained by mixing 1mL PBS buffer with the bacterial suspension (5mL).
[0077] Photothermal agent non-light group (APDA@Pt & NIR(-)) : basically the same as the photothermal agent light group, except that the mixed bacterial solution was not irradiated with 808nm laser but was directly incubated at 37℃ for 10min.
[0078] Control non-irradiation group (PBS & NIR(-)): substantially the same as the control irradiation group, except that the mixed bacterial solution was not irradiated with 808 nm laser, but was directly incubated in a 37 °C incubator for 10 min.
[0079] The irradiation group in the above experiment is marked with NIR(+). The non-irradiation group is marked with NIR(-).
[0080] The plate colony situation of the photothermal agent irradiation group, the control irradiation group, the photothermal agent non-irradiation group and the control non-irradiation group is shown in (A-B) of Figure 11 , and the number of colonies is shown in (C-D) of Figure 11 , wherein, Figure 11 A and C in represent E. coli; Figure 11 B and D in represent S. aureus; it can be seen from Figure 11 that E. coli and S. aureus in the photothermal agent non-irradiation group without irradiation basically maintain the original activity. According to The bacteriostatic rate of the new nano-enzyme composite material prepared in Example 1 on E. coli and S. aureus is obtained, the bacteriostatic rate of the new nano-enzyme composite material prepared in Example 1 on E. coli is 99.4%, the bacteriostatic rate of the new nano-enzyme composite material prepared in Example 1 on S. aureus is 99.5%, and the antibacterial effect of the new nano-enzyme composite material prepared in Example 1 on E. coli and S. aureus is consistent.
[0081] Further, the morphology change of bacteria and the integrity of the film in the photothermal agent irradiation group, the control irradiation group, the photothermal agent non-irradiation group and the control non-irradiation group were observed by SEM, as shown in Figure 12 , it can be seen from Figure 12 that, Figure 12 A of is E. coli, Figure 12 B of is S. aureus, in the photothermal agent irradiation group, after being treated with the photothermal agent, it is observed that the bacteria are obviously shrunk, and the contents are outflowed due to the perforation or even rupture of the cell wall. This is because the local high temperature caused by the photothermal performance of APDA@Pt can destroy the lipid bilayer structure of the bacterial membrane, affect the ion channels on the surface of the cell membrane, change the permeability of the cell membrane, and then lead to the overflow of the cell contents. In summary, APDA@Pt has strong antibacterial ability.
[0082] Example 5
[0083] The OD 600 value can also be used to evaluate the concentration of the bacterial solution and the growth of the bacteria. E. coli and S. aureus are respectively taken as "bacteria" to perform the following experimental group steps:
[0084] Light group: the bacteria were inoculated in LB medium and cultured at 37℃, 260 rpm for 12 h to obtain a bacterial suspension with a density of 10 6 CFU / mL; 1 mL of photothermal agent solution (the same as the photothermal agent solution in Example 4 above) or PBS buffer (as a control) was mixed with the bacterial suspension (5 mL) respectively to obtain a mixed bacterial solution. The mixed bacterial solution was irradiated with 808 nm near-infrared laser for 10 min at a light intensity of 0.75 W / cm 2 2. After irradiation, 100 μL of the mixed bacterial solution was added to LB medium (5 mL) and incubated at 37℃, 160 rpm for 12 h. After incubation, OD 600 value test and turbidity were performed. The results of the photothermal agent solution in the light group are shown as Figure 13 “APDA@Pt+NIR” in FIG. 6, and the results of the PBS buffer in the light group are shown as Figure 13 “PBS+NIR” in FIG. 6.
[0085] Non-light group: basically the same as the light group, except that the near-infrared laser irradiation was not performed, and the mixed bacterial solution was directly incubated at 37℃, 160 rpm for 12 h. After incubation, OD 600 value test and turbidity were performed on the mixed bacterial solution. The results of the photothermal agent solution in the non-light group are shown as Figure 13 “APDA@Pt” in FIG. 7, and the results of the PBS buffer in the light group are shown as Figure 13 “PBS” in FIG. 7,
[0086] Figure 13 The first row (a and b) of FIG. 6 is E. coli; Figure 13 The second row (c and d) of FIG. 6 is S. aureus, and it can be seen from Figure 13 that compared with the non-light group, after the light treatment of the new nanoscale enzyme composite material prepared in Example 1 and incubation for 12 h, the OD 600 values of E. coli and S. aureus were not more than 0.2, and the bacterial solution was clear, which indicated that the photothermal effect of the new nanoscale enzyme composite material prepared in Example 1 could effectively inhibit the proliferation of gram-positive bacteria (S. aureus) and gram-negative bacteria (E. coli).
[0087] Example 6
[0088] Cell survival experiment: L-929 cells (mouse fibroblasts, source: Chinese Academy of Sciences Cell Bank) were inoculated in 5 mL of the first culture medium (the first culture medium is RPMI 1640 medium containing 10% (v / v) fetal bovine serum) and cultured at 37°C in a cell incubator with 5% CO2 for 24 hours. The first culture medium was replaced every 48 hours after 24 hours of culture. After culture, the cells were collected with a scraper and centrifuged at 1130 rpm for 5 minutes. The supernatant was removed, 1 mL of the first culture medium was added for resuspension, and a cell suspension with a density of 1 x 10 4 cells / mL was obtained.
[0089] 10 μL of the photothermal agent solution (the photothermal agent solution is a mixture of the photothermal agent and the PBS buffer) or 10 μL of the PBS buffer (as Figure 14 “PBS”) were mixed with the cell suspension (1 mL) respectively, and incubated at 37°C in a cell incubator with 5% CO2 for 24 hours. The photothermal agent was the novel nanozyme composite material prepared in Example 1. The concentration of the photothermal agent in the photothermal agent solution was set so that the concentration of the photothermal agent in the cell suspension after mixing the photothermal agent solution with the cell suspension was 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL respectively.
[0090] After incubation, MTT was used to evaluate the cell activity, and the cell survival rate was obtained, as shown in Figure 14 As shown in Figure 14 , with the increase of the concentration, the novel nanozyme composite material prepared in Example 1 did not show obvious cytotoxicity. When the concentration was 200 μg / mL, the cell activity was still above 98%, indicating that the novel nanozyme composite material prepared in Example 1 had good biocompatibility in the range of 0-200 μg / mL.
[0091] Cell viability / dyeing staining can also be used to evaluate cell viability. Referring to the above cell survival experiment, the cells after 24 hours of incubation were washed with the PBS buffer (purchased from Shengong Bioengineering, the pH of the PBS buffer is 7.4). Under the condition of avoiding light, the cells were dyed with the Calcein / PI mixed solution (purchased from Biotium Kit, the dyeing operation is specifically referred to the instruction manual of Biotium Kit) for 30 minutes, and then the dyed cells were observed under a fluorescence microscope. As shown in Figure 15 As shown in Figure 15 “0 μg / mL” represents the mixture of the PBS buffer and the cell suspension), and Figure 15It can be seen that with the increase of the concentration of the new nanoscale enzyme composite prepared in Example 1, each group shows a large number of cell survival (green fluorescence), and the number of dead cells is extremely small (red fluorescence), and the cell growth morphology remains unchanged. Further shows that APDA@Pt has excellent biocompatibility, and has no obvious cytotoxicity in the concentration range used.
[0092] Example 7
[0093] Hemocompatibility is of great significance to ensure the safety and effectiveness of photothermal agents in the wound healing process. The present application uses red blood cell diluent (red blood cell diluent purchased from Puxitang (Tianjin) Biotechnology Co., Ltd., which includes red blood cells, and the volume percentage of red blood cells in the red blood cell diluent is 4%) to evaluate the hemolysis rate of the new nanoscale enzyme composite.
[0094] The same volume (100 μL) of aqueous solution of photothermal agent, H2O (as a positive control) and PBS buffer (as a negative control) were mixed with 1 mL of red blood cell diluent, and incubated at 37℃ in a cell incubator with 5v / v% CO2 for 4h. After incubation, the absorbance at 450nm was detected by a microplate reader, wherein the photothermal agent was the new nanoscale enzyme composite prepared in Example 1, and the concentration of the aqueous solution of the photothermal agent was set so that the concentration of the photothermal agent in the red blood cell diluent was 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL and 200 μg / mL.
[0095] The hemolysis rate after 4h of incubation was calculated, as shown in Figure 16 , the hemolysis condition is shown in the Figure 16 , as Figure 16 can be seen, the red blood cell diluent added with H2O presents bright red after 4h of incubation, indicating that it has a serious hemolysis reaction, while the red blood cell diluent added with different concentrations of photothermal agent has a slight hemolysis after 4h of incubation, and the supernatant is light yellow, the hemolysis rate is less than 3%, which is the same as the addition of PBS, indicating that APDA@Pt will not damage the components in the blood, nor will it cause the formation of blood clotting or thrombosis, and has high blood safety.
[0096] Example 8
[0097] The process of hydrogen peroxide decomposition to generate oxygen is often used as an evaluation of whether a material has peroxidase-like activity. The present application first investigates the oxygen production of APDA@Pt catalytic hydrogen peroxide decomposition at different concentrations. The photothermal agent is added to the H2O2 aqueous solution (the content of H2O2 in the H2O2 aqueous solution is 10 mM), mixed uniformly, and the oxygen production is observed after 5 min. The photothermal agent is the novel nanoscale enzyme composite material prepared in Example 1. The concentration of the photothermal agent in the H2O2 aqueous solution is 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively.
[0098] The oxygen production after 5 min is shown in Figure 17 It can be seen from Figure 17 that the higher the concentration of the novel nanoscale enzyme composite material prepared in Example 1, the faster the hydrogen peroxide decomposition speed, and the more visual oxygen bubbles. This indicates that APDA@Pt has excellent oxygen production capacity.
[0099] Example 9
[0100] Titanium sulfate reacts with hydrogen peroxide to change color, so titanium sulfate can also be used to determine whether it has peroxidase-like activity. The photothermal agent or no photothermal agent is added to the H2O2 aqueous solution, mixed uniformly to obtain a first solution. After 20 min, 1 mL of the first solution is taken and 500 μL of titanium sulfate (40 mM) aqueous solution is added to obtain a detection solution. The photothermal agent is one of the nanoscale enzyme material prepared in Comparative Example 1 and the novel nanoscale enzyme composite material prepared in Example 1. The concentration of the photothermal agent in the first solution is 100 μg / mL.
[0101] It is found that after adding titanium sulfate, the detection solution containing the novel nanoscale enzyme composite material prepared in Example 1 is colorless Figure 10 , while the detection solution containing the nanoscale enzyme material prepared in Comparative Example 1 still presents a distinct yellow color Figure 10 , indicating that the novel nanoscale enzyme composite material prepared in Example 1 consumes H2O2, so that it cannot react with titanium sulfate to generate yellow titanium peroxide. The absorption of the detection solution at 405 nm is determined by a UV absorption spectrometer, as shown in Figure 18 It can be seen from Figure 18 that the detection solution containing the nanoscale enzyme material prepared in Comparative Example 1 has the same characteristic absorption as the detection solution without the photothermal agent, while the characteristic absorption of the detection solution containing the novel nanoscale enzyme composite material prepared in Example 1 is significantly reduced, which also indicates that the novel nanoscale enzyme composite material effectively decomposes hydrogen peroxide to produce oxygen and reduces the characteristic absorption of titanium peroxide. The above results further show that the loading of Pt nanoparticles significantly improves the antioxidant performance of APDA nanoparticles and has excellent peroxidase-like activity.
[0102] Example 10
[0103] Efficiently scavenging active nitrogen is also of great significance for effectively promoting wound healing, reducing inflammation and improving vascular function. In order to further verify the antioxidant performance of the novel nanoscale enzyme composite material prepared in Example 1, the present application evaluates the scavenging ability of 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS).
[0104] ABTS, potassium persulfate and water were mixed and stirred in the dark for 12 hours to obtain an ABTS solution, the concentration of ABTS in the ABTS solution was 7 mM, and the concentration of potassium persulfate in the ABTS solution was 2.45 mM. The ABTS solution was diluted with PBS buffer to an absorbance of about 0.7 to obtain an ABTS diluent, 1 mL of APDA@Pt aqueous solution was mixed with the ABTS diluent in equal volume, and incubated in the dark for 20 min to obtain a monitoring solution (as an experimental group), the concentration of APDA@Pt in the APDA@Pt aqueous solution was 100 μg / mL. The absorption value of the monitoring solution at 734 nm was measured; in addition, the absorption value at 734 nm of a system in which ultrapure water and ABTS· solution were mixed in equal volume was determined as a control group.
[0105] The ABTS scavenging rate was calculated according to the ABTS· scavenging rate formula, as shown in Figure 19 The ABTS scavenging rate formula is as follows:
[0106]
[0107] As can be seen from Figure 19 , compared with ultrapure water, the ABTS scavenging rate of the novel nanoscale enzyme composite material (APDA@Pt) prepared in Example 1 is higher than 80%, which indicates that APDA@Pt has excellent active nitrogen scavenging ability.
[0108] The above has exemplarily described the present application, it should be explained that, in the case of not departing from the core of the present application, any simple modification, modification or other equivalent replacement which can not cost creative labor of the person skilled in the art falls into the protection scope of the present application.
Claims
1. A novel nanoenzyme composite material, characterized in that, include: APDA nanoparticles and Pt nanoparticles loaded on the surface of APDA nanoparticles.
2. The preparation method of the novel nanoenzyme composite material as described in claim 1, characterized in that, Includes the following steps: APDA nanoparticles, chloroplatinic acid, and water were mixed evenly to obtain a mixed solution. A sodium borohydride aqueous solution at 0–4°C was added dropwise to the mixed solution, and the mixture was stirred at 0–4°C for 4–6 hours. The mixture was then centrifuged, washed, and freeze-dried sequentially to obtain a novel nanoenzyme composite material. The mass fraction of APDA nanoparticles to the molar fraction of chloroplatinic acid was (100–101):(0.3–0.34), where the mass fraction was in mg and the molar fraction was in μmol.
3. The preparation method according to claim 2, characterized in that, APDA nanoparticles were dispersed in water to obtain an APDA solution. An aqueous solution of chloroplatinic acid was added dropwise to the APDA solution, and the mixture was stirred at room temperature (20-25°C) for 1-1.5 hours until homogeneous to obtain a mixed solution.
4. The preparation method according to claim 3, characterized in that, The concentration of APDA nanoparticles in the APDA solution is 12.5–12.6 mg / mL; the concentration of chloroplatinic acid in the chloroplatinic acid aqueous solution is 1–1.05 mM.
5. The preparation method according to claim 2, characterized in that, The mass ratio of APDA nanoparticles to sodium borohydride in the aqueous solution is (100-101):(5-6).
6. The preparation method according to claim 2, characterized in that, The concentration of sodium borohydride in the aqueous solution is 2.5–3 mg / mL.
7. The preparation method according to claim 2, characterized in that, The method for obtaining APDA nanoparticles includes: dissolving dopamine hydrochloride in water to obtain a dopamine hydrochloride solution; dissolving arginine in water to obtain an arginine solution; adding the arginine solution dropwise to the dopamine hydrochloride solution under stirring, stirring at room temperature of 20-25°C for 4-6 hours, and then centrifuging, washing and vacuum drying in sequence to obtain APDA nanoparticles, wherein the ratio of dopamine hydrochloride to arginine by mass is (1-1.1):(1-1.1).
8. The preparation method according to claim 7, characterized in that, The mass fraction of arginine and the volume fraction of water in the arginine solution are in the ratio of (100-101):(10-10.5), where the mass fraction is in mg and the volume fraction is in mL; the mass fraction of dopamine hydrochloride and the volume fraction of water in the dopamine hydrochloride solution are in the ratio of (100-101):(110-110.5), where the mass fraction is in mg and the volume fraction is in mL.
9. The application of the novel nanoenzyme composite material obtained by the preparation method described in claim 2 in improving catalase-like activity.
10. The application according to claim 9, characterized in that, The novel nanoenzyme composite material exhibits a clearance rate of over 80% for ABTS and a hemolysis rate of less than 3%.