PDA / PtCuO CDs-Gel, preparation method thereof and application of PDA / PtCuO CDs-Gel in preparation of antibacterial therapeutic agent

Abstract

The invention discloses PDA / PtCuO CDs-Gel nano-enzyme composite gel and a preparation method and application thereof in preparation of an antibacterial therapeutic agent.The nano-enzyme composite gel is of a gel-wrapped nano-enzyme structure, PtCuO serves as a core of the nano-enzyme, a polydopamine layer is arranged outside the core, the polydopamine layer is modified with carbon quantum dots, and the carbon quantum dots are modified with PtCuO. The PtCuO is CuO nano particles of which the surfaces are modified with Pt nano particles. According to the nano-enzyme composite gel, H2O2 does not need to be added as an enzyme reaction substrate, the catalytic efficiency is high, the antibacterial performance is high, the cell penetrability and the photothermal conversion efficiency are high, and multi-mode antibacterial treatment can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial nanomaterials, and particularly relates to PDA / PtCuO@CDs-Gel, a preparation method thereof, and an application thereof in the preparation of antibacterial therapeutic agents. Background Art

[0002] Bacterial infections pose a major threat to human health. According to the data statistics released by the World Health Organization in 2022, approximately 5 million people die globally each year due to drug-resistant bacteria. It is precisely due to the overuse of antibiotics and the inherent potential of bacteria to resist traditional antibiotics that the emergence of antibiotic-resistant strains has made the infection and spread of drug-resistant bacteria a major challenge faced clinically. In addition, the continuous increase in multi-drug resistant strains will lead to an increase in antibiotic doses and an extension of the treatment time, which further causes problems such as bacterial mutations and the prevalence of wound infections. To solve these problems, it is urgent to develop the next generation of novel antibacterial agents to reduce bacterial drug resistance and make up for the deficiencies of traditional antibiotics.

[0003] With the progress of nanotechnology, antibacterial nanomaterials are expected to become substitutes for traditional antibiotics. Antibacterial nanomaterials are small in size, large in specific surface area, high in biocompatibility, have a broad antibacterial spectrum, can be adjusted in terms of size, shape, surface chemistry, and elemental composition, have no drug resistance, and can kill bacteria and inhibit the growth and reproduction of bacteria. Antibacterial nanomaterials can achieve multi-mode synergistic antibacterial therapy. Among them, the production of reactive oxygen species (ROS) by nanozymes is the main reason for their antibacterial effect. Nanozymes act on the cell membrane of bacteria, stimulating oxygen to produce singlet oxygen and hydroxyl radicals, damaging the basic structure of bacteria, and causing bacteria to die.

[0004] However, a single nanozyme with peroxidase-like activity needs to add H2O2 as the substrate for the enzyme reaction, which may damage healthy cells at the infection site. Therefore, a multi-nanozyme-mediated cascade enzyme catalytic system has emerged. Combining the ability of oxidase (OXD) to reduce O2 to H2O2 with the ability of peroxidase (POD) to consume H2O2 to produce hydroxyl radicals to form a cascade enzyme catalytic system can self-circulate and synergistically enhance the antibacterial effect of nanozymes.

[0005] Photothermal therapy (PTT) of nanomaterials is a non-invasive treatment method that has also attracted much attention due to its advantages such as a short treatment cycle, low cost, low toxicity, strong controllability, local application, and high drug delivery efficiency. PTT utilizes a photothermal conversion agent (PTA) under the irradiation of a near-infrared light (NIR) light source. The surface plasmon band of the PTA converts electronic radiation into heat, converting light energy into heat energy. By rapidly increasing the local area temperature, it destroys the cell membrane of bacteria, denatures and inactivates proteins or causes protein leakage, causing irreversible damage, and achieving the purpose of antibacterial therapy through the thermal ablation of bacteria.

[0006] However, reports on the synergistic antibacterial effect of nanomaterials also have drawbacks such as low catalytic efficiency, poor biosafety, and unsatisfactory bactericidal effect. Single photothermal therapy is also insufficient in the application of antibacterial therapy.

[0007] To effectively address multi-bacterial infections in wounds, it is highly necessary to design a multimodal therapeutic nanoplatform that can coordinate multiple treatment methods and minimize unnecessary damage to surrounding healthy tissues. Summary of the Invention

[0008] Based on the deficiencies of the above-mentioned existing technologies, the present invention provides a nanozyme composite gel for multimodal antibacterial therapy that simultaneously has good biosafety, biocompatibility, and strong antibacterial activity. The nanozyme composite gel uses gel (Gel) as the carrier of the overall nanoantibacterial material, loads Pt nanoparticles with OXD and POD activities on CuO with POD activity to form PtCuO with higher catalytic activity, and wraps polydopamine (PDA) around it and then loads carbon dots (CDs) to form a multimodal antibacterial therapeutic nanomaterial. The catechol group of PDA with redox activity can convert O2 into H2O2, and H2O2 is decomposed by Pt to generate more hydroxyl radicals, while the oxidation product quinone can be reduced back to catechol by glutathione in bacteria. The self-circulation of PDA synergistically enhances the antibacterial performance of the cascade enzyme PtCuO, and jointly constructs a PDA / PtCuO cascade enzyme system. CDs are connected to PDA through dehydration condensation reactions of amino and carboxyl groups, and under NIR-II (808 nm) light irradiation, CDs generate high heat to kill bacteria, showing high cell penetration and photothermal conversion efficiency, and jointly constituting the nanozyme composite gel for multimodal antibacterial therapy.

[0009] To achieve the above object, the present invention adopts the following technical solutions: A PDA / PtCuO@CDs-Gel nanozyme composite gel, the nanozyme composite gel is a nanozyme structure wrapped by gel, the nanozyme has PtCuO as the core, the core is surrounded by a polydopamine (PDA) layer, and carbon quantum dots (CDs) are modified on the polydopamine layer, and the PtCuO is a CuO nanoparticle with Pt nanoparticles modified on its surface.

[0010] Preferably, the particle size of the CuO nanoparticles is 2.5 - 4.5 nm.

[0011] Preferably, the polydopamine layer is in-situ polymerized on the surface of the core.

[0012] Preferably, the gel is a copolymer gel of poly(N-isopropylacrylamide) (PNIPAM) and acrylic acid (AAc).

[0013] Preferably, the gel further contains through breathable pores with a pore diameter of more than 1 mm.

[0014] The second object of the present invention is to provide a method for preparing the above-mentioned PDA / PtCuO@CDs-Gel nanozyme composite gel, which includes the following steps: (1) Disperse CuO nanoparticles in a precursor solution of Pt, and add a reducing agent solution under stirring to obtain PtCuO; (2) Disperse PtCuO in a buffer solution, and then add dopamine hydrochloride for in-situ polymerization reaction to obtain PDA / PtCuO; (3) Ultrasonically disperse PDA / PtCuO and carbon quantum dots (CDs) in water and stir in the dark to obtain PDA / PtCuO@CDs; (4) Add PDA / PtCuO@CDs to a solution containing N-isopropylacrylamide, acrylic acid, a cross-linking agent and a photoinitiator, and under the induction of ultraviolet light, the gel polymerizes outside PDA / PtCuO@CDs to obtain the nanozyme composite gel.

[0015] Preferably, steps (1), (2), and (3) further include a step of washing and then drying, and step (4) further includes a step of soaking with a buffer solution to remove unreacted monomers.

[0016] Preferably, in step (1), the precursor solution of Pt is a solution of H2PtCl6, and the reducing agent is NaBH4.

[0017] Preferably, in step (2), the buffer solution is PBS buffer solution. More preferably, the concentration of the PBS buffer solution is 0.01 M and the pH is 7.0.

[0018] Preferably, in step (4), the photoinitiator is 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).

[0019] Preferably, in step (4), the cross-linking agent is N,N'-methylenebisacrylamide (BIS).

[0020] Preferably, step (4) further includes a step of providing through air holes in the gel. More preferably, the polymerization in step (4) is carried out in a mold, and the mold is provided with several columns penetrating the gel. After polymerization, the space occupied by the columns in the gel forms breathable pores.

[0021] The third object of the present invention is to provide the application of the above-mentioned PDA / PtCuO@CDs-Gel nanozyme composite gel in the preparation of antibacterial therapeutic agents.

[0022] The beneficial effects of the present invention are as follows: In the present invention, Pt nanoparticles with OXD and POD activities are loaded on CuO with OXD activity, and a PDA coating is applied on the outside of PtCuO to construct a cascade enzyme catalytic system that does not require the addition of H2O2 as an enzyme reaction substrate and has higher catalytic efficiency. Connecting PDA with water-soluble CDs improves the photothermal conversion rate and biocompatibility. Constructing a nano material for multi-modal antibacterial therapy of oxidative damage and PTT enhances the antibacterial therapy ability and controllability of the nano material, which is of positive significance for resisting antibiotic-resistant strains.

[0023] In the present invention, CuO nanoparticles are used as a template, and Pt nanoparticles are reduced and loaded on the surface of CuO by the NaBH4 reduction method. PDA is coated on the outside of PtCuO by the oxidative self-polymerization of dopamine, and water-soluble CDs are modified on the PDA coating by the dehydration condensation reaction of amino and carboxyl groups. Finally, P(NIPAM-AAc) copolymer gel is used as the carrier of the overall antibacterial nano material, and PDA / PtCuO@CDs is loaded by copolymerization. PDA / PtCuO@CDs has a high absorbance. Under the irradiation of an 808 nm laser, the surface plasmon band converts the electron radiation into heat, converting light energy into heat energy to form PTT, which destroys the cell membrane of bacteria, denatures or inactivates proteins, or causes protein leakage, resulting in the thermal ablation of bacteria to achieve the purpose of antibacterial therapy. PTT in the NIR spectral region has been proven to achieve higher photothermal conversion efficiency and greater target penetration, thereby improving the antibacterial effect. The bactericidal effect mediated by PTT is affected by the surface physicochemical properties of CDs. Good hydrophobicity can increase the bactericidal efficacy of PTT and promote the decomposition of bacterial biofilms. In addition, the PDA / PtCuO cascade enzyme catalytic system exhibits high peroxidase-like activity and peroxidase-like activity in the environment of bacterial infection. It can catalyze the generation of ·OH by H2O2 to attack the bacterial cell membrane and synergistically enhance the antibacterial therapy through self-circulation. Description of the Drawings

[0024] Figure 1 Are transmission electron microscope photos of each particle, where I is the CuO particle, II is PtCuO, III is PDA / PtCuO, IV is PDA / PtCuO@CDs, and the circled area in the figure is the CDs loaded on the outer layer of PDA.

[0025] Figure 2 Are particle size distribution diagrams of CuO, PtCuO, PDA / PtCuO, and PDA / PtCuO@CDs.

[0026] Figure 3 Are scanning electron microscope photos of PDA / PtCuO@CDs-Gel at different magnifications.

[0027] Figure 4XRD patterns of CuO, PtCuO, PDA, PDA / PtCuO, and PDA / PtCuO@CDs.

[0028] Figure 5 Zeta potential diagrams of CuO, PtCuO, PDA / PtCuO, CDs, and PDA / PtCuO@CDs.

[0029] Figure 6 Infrared thermal images at different times after laser irradiation of gels containing different concentrations of PDA / PtCuO@CDs.

[0030] Figure 7 Temperature-time curves of gels containing different concentrations of PDA / PtCuO@CDs after laser irradiation.

[0031] Figure 8 Temperature change-time curves of PDA / PtCuO@CDs-Gel under laser irradiation with different power densities.

[0032] Figure 9 Temperature-time curves of PDA / PtCuO@CDs-Gel during single laser irradiation and after irradiation stop.

[0033] Figure 10 Temperature-time curves of PDA / PtCuO@CDs-Gel during multiple laser irradiations and after irradiation stop.

[0034] Figure 11 Negative natural logarithm curve of the driving force temperature based on the cooling cycle and calculation of the photothermal conversion rate of PDA / PtCuO@CDs-Gel photothermal.

[0035] Figure 12 UV spectra of the TMB system in Example 1 (4-1) in the presence of different concentrations of nanozymes (in the presence of H2O2), and spectral comparison of different nanozymes at the same concentration. Among them, a is the corresponding spectrum of different concentrations of CuO, b is the corresponding spectrum of different concentrations of PtCiO, c is the corresponding spectrum of different concentrations of PDA / PtCuO, and d is the spectral comparison of CuO, PtCuO, and PDA / PtCuO at the same concentration. The photos in a, b, and c are the appearance diagrams of the corresponding samples. In the appearance diagrams, the nanozyme concentration increases from left to right in sequence.

[0036] Figure 13 UV spectra of the TMB system in Example 1 (4-2) in the presence of PtCuO and PDA / PtCuO nanozymes (in the absence of H2O2).

[0037] Figure 14 SEM photos of the antibacterial effects of each group of nanomaterials against different bacteria.

[0038] Figure 15 Colony counting charts of the antibacterial effects of each group of nanomaterials against different bacteria.

[0039] Figure 16 In Example 2(2), in vivo infrared thermal imaging photos of the infected wounds of mice with Escherichia coli ( E. coli ), and Staphylococcus epidermidis ( S. sepider ) on the wounds of PDA / PtCuO@CDs, PDA / PtCuO@CDs gel, and PDA / PtCuO@CDs b-gel before and after 405 s of laser irradiation. In each group of pictures, the left picture is before irradiation and the right picture is after irradiation.

[0040] Figure 17 In Example 2(2), fluorescence imaging pictures of PDA / PtCuO@CDs gel and PDA / PtCuO@CDs b-gel and wound photos of mice after irradiation on two consecutive treatment days after the first laser irradiation (0 h, 3 h, 9 h, 45 h, 48 h, 54 h).

[0041] Figure 18 In Example 2(2), calculation of the change curve of fluorescence intensity and the release rate of PDA / PtCuO@CDs from PDA / PtCuO@CDs gel and PDA / PtCuO@CDs b-gel.

[0042] Figure 19 In Example 2(3), representative photos of the wound recovery of mice within 15 days.

[0043] Figure 20 In Example 2(3), the change curve of the body weight of infected mice during the treatment process with different reaction systems.

[0044] Figure 21 In Example 2(3), on the 1st day and the 15th day after treatment with different dressings, E. coli and S. sepider H&E staining pictures (scale bar, 50 µm) and local magnified views of the infected wound tissues.

[0045] Figure 22 In Example 2(3), the change curves of the wound areas of infected mice within 15 days after treatment with PDA / PtCuO@CDs, PDA / PtCuO@CDs Gel, and PDA / PtCuO@CDs b-gel. E. coli and S. sepider Detailed implementation manners Example 1

[0046] (1) Preparation of PDA / PtCuO@CDs-Gel: Take 0.18 mL of glacial acetic acid and 50 mL of 0.02 M copper acetate aqueous solution, and mix them in a round-bottom flask. Heat the mixture to boiling with stirring at 800 r / min. Then, quickly add 3.5 mL of 0.04 g / mL NaOH aqueous solution to the above boiling solution. Centrifuge the precipitate and wash it three times with ethanol, and then dry it to obtain CuO nanoparticles (NPs).

[0047] Disperse 660 μL of CuO solution (170 mg·L -1 ) in 2.34 mL of water, sonicate for 10 min, and add 23.5 μL of H2PtCl6·6H2O aqueous solution (19.3 mM). Stir the mixture in an ice-water bath for 15 min. Then stir at 1000 r / min and dropwise add an aqueous solution of NaBH4 (4.8 mM, 1 mL). After stirring for 2 h, centrifuge the resulting solution and wash it 3 times, and then lyophilize to obtain PtCuO.

[0048] Add 10 mg of PtCuO NPs to 5 mL of PBS buffer (0.01 M, pH 7.0), and sonicate for 5 minutes. Then add dopamine hydrochloride (0.5 mg / mL) to the mixed solution, and stir at 800 r / min at room temperature for 1 h. Finally, centrifuge (10 min, 10000 rpm), wash 3 times with ultrapure water, and dry to obtain PDA / PtCuO.

[0049] Add 10 mg of PDA / PtCuO and 2 mg of CDs to 20 mL of ultrapure water, sonicate and break for 15 min, and then stir in the dark for 12 h. Centrifuge the resulting solution at 10000 rpm for 15 min, and wash 3 times with ethanol. After lyophilization in the dark, PDA / PtCuO@CDs is obtained.

[0050] Add 2 g of N-isopropylacrylamide monomer, 200 μL of acrylic acid, and 60 mg of N,N-methylenebisacrylamide (BIS) to 3 mL of ethanol / ethylene glycol (1:1) solution, and sonicate until completely dissolved. Then add 40 μL of 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP), and sonicate for 10 min. Subsequently, add 136 μL of PDA / PtCuO@CDs solution (5 mg / mL), and induce gel polymerization by ultraviolet light irradiation (30 W, 10 min). Finally, soak in PBS buffer (10 mM) to remove the unreacted monomers on the gel surface to obtain PDA / PtCuO@CDs-Gel.

[0051] (2) Characterization of PDA / PtCuO@CDs-Gel: First, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and hydrodynamic particle size analyzer were used to characterize the structural morphology and size of CuO, PtCuO, PDA / PtCuO, PDA / PtCuO@CDs, and PDA / PtCuO@CDs-Gel, respectively.

[0052] CuO NPs have a spherical morphology with a narrow size distribution ( Figure 1 I), and the measured average particle size is about 3 - 4 nm ( Figure 2 ). TEM images show that uniformly sized Pt NPs are formed on the surface of CuO NPs after loading Pt NPs ( Figure 1 II), and the average size of PtCuO is about 35 - 40 nm ( Figure 2 ). Figure 1 The TEM image of III shows that PtCuO is completely encapsulated in PDA, the thickness of the PDA coating is about 10.1 ± 2 nm, and the overall size is about 105 - 115 nm ( Figure 2 ). The successful loading of CDs was determined by TEM ( Figure 1 IV). Due to the small size of CDs, the particle size of the composite does not change significantly before and after loading ( Figure 2 ).

[0053] Scanning electron microscopy imaging was performed on PDA / PtCuO@CDs-Gel to analyze its internal structure. As Figure 3 shown, the interior of the freeze-dried hydrogel has a network structure, and some magnified images show that PDA / PtCuO@CDs nanoparticles are irregularly loaded in the hydrogel matrix.

[0054] X-ray diffractometer (XRD) was used to characterize the phase analysis of the composite ( Figure 4 ). Among them, the X-ray diffraction pattern of CuO NPs shows diffraction peaks at 2θ = 32.56, 35.62, 38.78, 48.88, 53.62, 58.18, 61.5, 66.14, and 67.96. These peaks correspond to the (110), (002), (111), (111), (200), (202), (020), (202), (113), (311), and (220) reflection planes of CuO NPs, which correspond to the crystal planes of CuO NPs. The crystal structure of PtCuO was determined by XRD ( Figure 4), the main diffraction peaks at 46.95, 40.55, and 68.85 point to the (20 - 2) of CuO and the (111), (220) of Pt respectively, proving the successful encapsulation of Pt. In addition, PDA itself has no obvious diffraction peaks and is amorphous. The XRD after loading CDs is mainly composed of broad peaks, and the smaller diffraction angle (27.1°) indicates that the CDs have low crystallinity and the ordered layer stacking on the basal plane is disrupted. In summary, the XRD test results can preliminarily prove the successful preparation of the composite material.

[0055] The Zeta potentials of CuO, PtCuO, PDA / PtCuO, CDs, and PDA / PtCuO@CDs were analyzed using a Zeta potential analyzer ( Figure 5 ). The zeta potentials of CuO, PtCuO, PDA / PtCuO, CDs, and PDA / PtCuO@CDs are 46.25 eV, -4.6 eV, -38.4 eV, 1.49 eV, and -4.76 eV respectively. The reduced Pt can be anchored on the surface of CuO NPs through electrostatic force. The change in Zeta potential indicates that Pt was successfully loaded on the surface of CuO. The Zeta potential of PtCuO decreased with the encapsulation of PDA, from -4.6 to -38.4 eV. The Zeta potential further increased after loading CDs, confirming the interaction between the positively charged carbon dots and PDA, effectively improving the physiological stability of the composite material.

[0056] (3)Photothermal performance of PDA / PtCuO@CDs - Gel: The photothermal performance of PDA / PtCuO@CDs - Gel was detected using a laser with λ = 808 nm. First, the photothermal conversion ability of different concentrations of PDA / PtCuO@CDs in the gel was explored ( Figure 6 、 7 ). Gels (1 mL) with PDA / PtCuO@CDs concentrations of 200, 400, 600, and 800 μg / mL were prepared respectively, and the surface of the gels was irradiated with a laser with a power density of 0.8 W / cm -2 . The real - time temperature was recorded using an infrared thermal imager, and the temperature change of the solution within 405 s was monitored. It can be seen that the increase in the temperature of PDA / PtCuO@CDs - Gel has a significant concentration - dependence. The gel with a concentration of 400 μg / mL of PDA / PtCuO@CDs increased its temperature to 60.1℃ within 405 s ( Figure 6 ), which is sufficient to cause thermal ablation of bacteria and destroy the basic structure of bacteria, thus killing bacteria.

[0057] The influence of different powers on the photothermal conversion ability is as Figure 8As shown, the results indicate that the photothermal effect of PDA / PtCuO@CDs-Gel also exhibits power dependence. As the power density increases from 0.3 W / cm -2 to 1.0 W / cm -2 , the temperature change also increases from 33.4 °C to 44 °C.

[0058] These two results provide a basis for the controllable antibacterial treatment of this nanozyme composite gel, and the treatment can be controlled by adjusting the laser power, material concentration, and illumination time.

[0059] In addition, the single heating-cooling recovery performance of PDA / PtCuO@CDs-Gel ( Figure 9 , red data points under laser irradiation conditions and blue data points under cooling conditions after stopping irradiation) and the 5-cycle heating-cooling curves ( Figure 10 ) indicate the recyclable treatment of PDA / PtCuO@CDs-Gel.

[0060] After calculation, as Figure 11 shown, the photothermal conversion efficiency (PCE) value of PDA / PtCuO@CDs-Gel is 57.13% (808 nm, 0.8 W / cm -2 ), indicating that this nanozyme composite gel has good photothermal performance.

[0061] (4) Enzyme activity: (4-1) The peroxidase-like activities of CuO, PtCuO, and PDA / PtCuO were verified using the 3,3'-5,5'-tetramethylbenzidine (TMB) system in the presence of H2O2 ( Figure 12 ).

[0062] First, 800 μL of acetate buffer (0.2 M NaAC, 0.2 M HAC), 50 μL of TMB solution (10 mM), 50 μL of H2O2 solution (10 mM), and CuO, PtCuO, PDA / PtCuO (concentrations of 2.5, 5.0, 7.5 μg / mL) were mixed to a total volume of 1 mL and allowed to stand for 10 min. The absorbance values of each group at λ = 653 nm were measured using a UV-visible spectrophotometer ( Figure 12 a-c).

[0063] It can be observed that among samples with the same concentration, the enzyme activity of the PDA / PtCuO group is the highest, followed by the PtCuO group, and the CuO group is the lowest ( Figure 12d). This is because the active ingredient of PDA / PtCuO with the same mass is the highest. After loading Pt, both Pt and CuO have peroxidase activity, synergistically enhancing the activity of the PtCuO active component. And because PDA contains benzene rings and hydroxyl groups in its molecular structure, these functional groups can participate in redox reactions, thus endowing it with peroxidase activity. Therefore, the encapsulation of PDA further enhances the enzymatic activity of PtCuO, which fully demonstrates the strong peroxidase-like activity of PDA / PtCuO.

[0064] (4-2) The peroxidase-like activity of PtCuO and PDA / PtCuO was verified using the TMB system in the absence of H2O2 ( Figure 13 ).

[0065] Take 800 μL of acetate buffer (0.2M NaAC, 0.2 M HAC), 50 μL of TMB solution (10 mM), PtCuO, PDA / PtCuO (7.5 μg / mL), mix a total of 1 mL and let it stand for 10 min, and measure the absorbance value at λ = 653 nm using a UV-visible spectrophotometer ( Figure 13 ). It can be observed that the enzymatic activity of the PDA / PtCuO group is greater than that of PtCuO. This is because the PDA molecular structure contains phenolic and ketone groups. These functional groups have oxidizing properties under certain conditions, which can promote the oxidation reaction of polydopamine, thus showing peroxidase-like activity. In addition, certain parts of the PDA molecule can also form complexes with metal ions, further enhancing its oxidizing property. These characteristics make PDA / PtCuO have excellent peroxidase-like activity. Therefore, PDA self-circulation synergistically enhances the antibacterial performance of the cascade enzyme PtCuO, fully demonstrating the good application prospects of PDA / PtCuO in the construction of multi-mode antibacterial therapy nanomaterials.

[0066] (5) Antibacterial therapy of PDA / PtCuO@CDs-Gel: To prove that the PDA / PtCuO@CDs-Gel nanoplatform has broad-spectrum antibacterial activity against bacteria, 2 Gram-positive bacteria (Bacillus subtilis B. subt, Staphylococcus epidermidis S. epider) and 2 Gram-negative bacteria (Escherichia coli E. coli, Enterobacter aerogenes E. aero) were selected for antibacterial experiments, and their morphological changes were monitored by SEM.

[0067] Specifically, configure concentrations of 200 μg / mL of PtCuO, PDA / PtCuO, PDA / PtCuO@CDs, and PDA / PtCuO@CDs-Gel and add them to the LB medium of B. subt, S. epider, E. coli, and E. aero (10 8in CFU / mL) (the system is 1 mL, incubated at 37 °C and 100 rpm for 12 h). Among them, the PDA / PtCuO, PDA / PtCuO@CDs, and PDA / PtCuO@CDs-Gel systems need to be irradiated with an 808 nm laser for 7 min every 3 h. The group without the addition of nanomaterials is the blank group (control).

[0068] As Figure 14 shown, the multi-mode synergistic sterilization groups using PDA / PtCuO, PDA / PtCuO@CDs, and PDA / PtCuO@CDs-Gel have better sterilization effects. The cell walls of the 4 bacteria without treatment with antibacterial nanomaterials are intact and smooth. The addition of PtCuO causes the bacteria to lose cell integrity, and with the loading of the material PDA, the bacteria are further damaged. After loading CDs, PTT completely destroys the basic structure of the bacteria, denatures and inactivates or leaks proteins, causing irreversible damage and leading to thermal ablation of the bacteria, showing excellent antibacterial treatment effects( Figure 14 ).

[0069] In addition, the plate coating method was used to assist in proving the antibacterial performance of the composite nanomaterials: the plate counting chart shows( Figure 15 ), the antibacterial treatment effects of the PDA / PtCuO@CDs and PDA / PtCuO@CDs-Gel systems are the best, indicating that this multi-mode antibacterial treatment nanoplatform can effectively combat and treat multiple target bacterial infections. Example 2

[0070] (1) Preparation of the gel with air holes: Similar to Example 1(1), the difference is only that the ultraviolet light-induced gel polymerization is carried out in a mold, and several columns penetrating the solution at different heights are set in the mold. The cross-sectional size of the columns is 1 mm×1 mm. After the gel polymerization, the mold is demolded, and the space occupied by the columns forms air holes penetrating the gel, and the obtained material is named PDA / PtCuO@CDs b-gel.

[0071] (2) Antibacterial treatment of PDA / PtCuO@CDs-Gel and PDA / PtCuO@CDs b-gel: To evaluate the potential performance of multi-mode synergistic treatment in bacterial-infected wounds, taking Escherichia coli( E. coli ) and Staphylococcus epidermidis( S. sepider ) as examples, the photothermal performance of PDA / PtCuO@CDs, PDA / PtCuO@CDs-Gel, and PDA / PtCuO@CDs b-gel (gel with air holes) under NIR irradiation (808 nm) was evaluated.

[0072] To reduce the light damage of NIR laser to healthy tissues, a lower power density of 0.8 W cm −2 was used to irradiate the infected drug administration site. The treatment was carried out every other day. On the treatment day, two light irradiations with a single treatment duration of 405 s and an interval of 6 h were performed. That is, the first irradiation on day 1 was recorded as 0 h, the second irradiation was at 6 h, the third irradiation was at 48 h (day 3), and the fourth irradiation was at 54 h (day 3), and so on. An infrared thermal imager was used to record the infrared thermal images and temperature changes of PDA / PtCuO@CDs, PDA / PtCuO@CDs-Gel, and PDA / PtCuO@CDs b-gel under 808 nm laser irradiation.

[0073] The infrared thermal imaging signals of each experimental group gradually increased with time, all showing an increase in the temperature of the infected site. After 405 s of 808 nm irradiation (0.8 W cm −2 ), the temperature of the E. coli-infected wound surface in the PDA / PtCuO@CDs b-gel group increased significantly from 16 °C to 56.6 °C ( Figure 16 ).

[0074] In addition, due to factors such as surface defects, functional groups, quantum confinement effects, and conjugated structures, Π-Π transitions are induced under the action of the excitation light, and PDA / PtCuO@CDs will generate fluorescence signals, providing the ability for the localization and quantitative analysis of drugs. PDA / PtCuO@CDs-Gel and PDA / PtCuO@CDs b-gel were applied to the wounds of mice with bacterial infections, the fluorescence changes of the gels were traced, and the wound surface photos of the mice were taken and recorded on two consecutive treatment days (natural days were day 1 and day 3) after the two irradiations on the same day, as shown in Figure 17 Figure. Gel fluorescence imaging was performed at specific times (before and after laser irradiation of the gel for wound treatment, 0 s and 405 s). By monitoring the changes in the mouse wounds and the fluorescence changes of the gels, the release of PDA / PtCuO@CDs was judged and calculated. The release rates were 47% (PDA / PtCuO@CDs-Gel) and 44.7% (PDA / PtCuO@CDs b-gel) ( Figure 18 ).

[0075] (3) Pure gauze wrapping, PDA / PtCuO@CDs drop coating, PDA / PtCuO@CDs-Gel dressing, and PDA / PtCuO@CDs b-gel dressing were used as treatment methods respectively. Taking E. coli and S. sepider as examples, light treatment was carried out at an interval of 1 day, and two laser irradiations were performed on the treatment day. As shown in Figure 19As shown, the size of the treated wound was photographed and recorded every other day. During all treatments, there was no significant change in the body weight of the mice ( Figure 20 ), which means that all modes had little adverse effect on the health of the mice. Hematoxylin-eosin (H&E) staining was used to observe the histological changes of wound regeneration after treatment with different dressings. As Figure 21 shown, H&E staining of the tissue around the infected wound on the first day of treatment showed that a large area of inflammatory cell infiltration was visible in both the treatment group and the untreated group. On the 15th day, the dermis tissues of the PDA / PtCuO@CDs, PDA / PtCuO@CDs-Gel, and PDA / PtCuO@CDs b-gel treatment groups were arranged neatly and there were fewer inflammatory cells. In the group of model mice (Control) infected but untreated, H&E staining showed skin cell necrosis, and the wound was still mainly characterized by acute inflammatory reaction with many inflammatory cells, indicating that neither of the two bacterial infections was inhibited.

[0076] After comparison and statistical processing with the control group, the results showed that regardless of the type of bacterial infection, the PDA / PtCuO@CDs b-gel group had the best wound healing condition and the smallest wound size after 15 days of treatment ( Figure 22 ).

[0077] There are several reasons for the optimal synergistic bactericidal effect of PDA / PtCuO@CDs b-gel: 1) The respiratory pores of PDA / PtCuO@CDs b-gel allow the entry of O2, which can promote the proliferation of fibroblasts. Fibroblasts are important cells for wound repair, and their functions of proliferation and collagen synthesis require sufficient O2. And O2 is necessary for neutrophils and macrophages to eliminate bacteria through oxidation, and sufficient O2 enhances the control of infection by immune cells; 2) Due to the air permeability of b-gel, the increase in local O2 concentration helps to inhibit the release of excessive inflammatory factors and promote healing. 3) Dressings with good air permeability are more likely to regulate the wet environment of the wound. In a moist environment, the nanozyme is in full contact with O2 and the catalytic efficiency is improved, and it helps to remove necrotic tissue and promote tissue reconstruction. Although the airtight PDA / PtCuO@CDs b-gel can also maintain humidity, it may lead to excessive accumulation of wound exudate, which is not conducive to wound healing.

Claims

1. A PDA / PtCuO@CDs-Gel nanozyme composite gel, characterized in that: The nanozyme composite gel is a gel-wrapped nanozyme structure, wherein the nanozyme has PtCuO as a core, a polydopamine layer outside the core, the polydopamine layer is modified with carbon quantum dots, and the PtCuO is a CuO nanoparticle whose surface is modified with Pt nanoparticles.

2. The nanozyme composite gel according to claim 1, characterized in that The particle size of the CuO nanoparticles is 2.5-4.5 nm.

3. The nanozyme composite gel according to claim 1, characterized in that The polydopamine layer is obtained by in-situ polymerization on the surface of the core.

4. The nanozyme composite gel according to claim 1, characterized in that The gel is a copolymer gel of poly(N-isopropylacrylamide) and acrylic acid.

5. The nanozyme composite gel according to claim 1, characterized in that The gel also contains penetrating air pores, and the diameter of the air pores is greater than 1 mm.

6. The method for preparing the PDA / PtCuO@CDs-Gel nanozyme composite gel according to any one of claims 1 to 5, characterized in that: The steps include: (1) Dispersing CuO nanoparticles in a Pt precursor solution, adding a reducing agent solution under stirring, and obtaining PtCuO; (2) PtCuO is dispersed in a buffer solution, and then dopamine hydrochloride is added to perform an in-situ polymerization reaction to obtain PDA / PtCuO; (3) PDA / PtCuO and carbon quantum dots were ultrasonically stirred in water in the dark to obtain PDA / PtCuO@CDs; (4) PDA / PtCuO@CDs is added to a solution containing N-isopropylacrylamide, acrylic acid, a crosslinker and a photoinitiator. Under the induction of ultraviolet light, the gel is polymerized outside the PDA / PtCuO@CDs to obtain the nanozyme composite gel.

7. The preparation method according to claim 6, characterized in that: The Pt precursor solution described in step (1) is a solution of H2PtCl6, and the reducing agent is NaBH4.

8. The preparation method according to claim 6, characterized in that: The buffer solution described in step (2) is PBS buffer.

9. The preparation method according to claim 6, characterized in that: Step (4) also includes the step of providing penetrating pores in the gel. Preferably, the polymerization in step (4) is carried out in a mold, and a plurality of columns penetrating the gel are provided in the mold. After polymerization, the spaces occupied by the columns in the gel form pores.

10. Use of the PDA / PtCuO@CDs-Gel nanozyme composite gel according to any one of claims 1 to 5 in the preparation of an antibacterial therapeutic agent.

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