Antibacterial nano-drug with photoresponse nano-enzyme activity as well as preparation method and application of antibacterial nano-drug

Abstract

The invention belongs to the technical field of nano-material antibiosis, and particularly relates to an antibacterial nano-drug with photoresponse nano-enzyme activity as well as a preparation method and application of the antibacterial nano-drug. The Zn-TCPP-Pt nano-enzyme antibacterial agent is prepared from metalloporphyrin and Zn metal ions through a method from bottom to top, and the preparation method specifically comprises the following steps: dissolving Zn (NO3) 2.3 H2O, PVP and pyrazine in a solvent to obtain a mixture A; dissolving the PtTCPP in a solvent to obtain a mixture B; dropwise adding and uniformly mixing the mixture A and the mixture B, and reacting to obtain the Zn-TCPP-Pt nano-enzyme. The enzyme utilizes the light absorption capacity of porphyrin and the electron transmission capacity of metalloporphyrin to realize photoresponse OXD activity, ROS can be generated in the absence of H2O2, ROS is generated in a light-controllable manner, bacteria are killed, wound healing is promoted, and meanwhile, excessive ROS is effectively prevented from damaging normal cells and tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial nanomaterials, and specifically relates to an antibacterial nanomedicine with light-responsive nanoenzyme activity, and a preparation method and application thereof. Background Art

[0002] As the main drug for treating bacterial infections, antibiotics play a vital role in the treatment of human diseases. However, with the abuse and irregular use of antibiotics, more and more drug-resistant infections have occurred, seriously threatening human health. According to the authoritative British medical journal The Lancet, 1.27 million people died directly from antibiotic resistance in 2019, and 4.95 million deaths were related to antibiotic-resistant infections. Therefore, it has become a top priority to find new antibiotics and develop highly effective antimicrobial agents to avoid drug resistance.

[0003] Nanozymes have become a promising antibacterial nanoagent due to their high catalytic activity, high stability and mild catalytic conditions. Among them, peroxidase (POD)-like and oxidase (OXD)-like enzymes can efficiently produce ROS to oxidatively destroy bacterial cell membranes and biomolecules, thereby effectively inactivating bacteria, and therefore dominate in antibacterial activities. However, although POD-like and OXD-like enzymes have great potential in the field of antibacterial activities, there are still some challenges and limitations. POD-like enzymes mainly kill bacteria by catalyzing H2O2 to produce ROS, and cannot play an antibacterial role in the absence of H2O2. The necessity of hydrogen peroxide and uncontrollable activity are the main disadvantages of POD-like nanozymes. OXD-like enzymes can catalyze H2O or O2 to produce ROS without the participation of H2O2. From the perspective of whether H2O2 is needed, OXD-like enzymes are superior to POD-like enzymes. However, uncontrollable excess ROS may be produced, damaging normal cells, tissues or biomolecules, causing adverse effects, or even reversing the therapeutic effect. Therefore, it is of great significance to develop an antibacterial nanoagent with controllable activity for antibacterial therapy that is activated only when needed.

[0004] In the prior art, the CN117959428A patent reports a photoresponsive multifunctional nanozyme and its preparation method and application, which is prepared by hydrothermal synthesis using Pt and Te precursors. The nanozyme has multiple enzyme activities such as oxidase, peroxidase, catalase, and glutathione peroxidase, and has good photothermal effect and photodynamic performance. The CN118527647A patent discloses a near-infrared light-responsive Au / Pt / RGDC nanomaterial, which is based on gold nanobipyramids with near-infrared light absorption and peroxidase-like catalytic activity, and platinum nanoparticles are modified to the tip of the gold nanobipyramids by chemical reduction. The nanomaterial can be catalytically treated by photothermal therapy in coordination with nanozyme under NIR irradiation, thereby achieving a rapid and efficient bactericidal effect. Summary of the invention

[0005] In view of this, the present invention proposes an antibacterial nanomedicine with light-responsive nanoenzyme activity and its preparation method and application. The present invention uses metalloporphyrin and Zn metal ions as raw materials to synthesize antibacterial nanomedicine, which has the ability of light-responsive OXD-like activity, can use light to controllably produce toxic ROS, play a bactericidal role, and then promote wound healing.

[0006] One of the purposes of the present invention is to provide an antibacterial nanomedicine with light-responsive nanozyme activity.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention discloses an antibacterial nanomedicine with light-responsive nanoenzyme activity, wherein the antibacterial nanomedicine is prepared by coordinating metal porphyrin with Zn metal ions.

[0009] Furthermore, the nanozyme is an oxidase-like enzyme.

[0010] A second object of the present invention is to provide a method for preparing the aforementioned antibacterial nanomedicine.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] The preparation method of the aforementioned antibacterial nanomedicine uses metal porphyrin and Zn metal ions to prepare Zn-TCPP-Pt nanozyme through a bottom-up method.

[0013] The antibacterial nano drug proposed by the present invention is a metal organic framework material (MOF), which is formed by metal ions or metal clusters and organic ligands interacting at the molecular level through coordination bonds to form an integral material with a regular structure. More specifically, the present invention uses metal porphyrin (PtTCPP) as a ligand and zinc nitrate as a metal source to prepare Zn-TCPP-Pt nanozyme. The obtained nanozyme is a layered nanosheet structure. The enzyme utilizes the light absorption ability of porphyrin and the electron transmission ability of metal porphyrin to achieve light-responsive OXD activity, avoids the generation of ROS in the absence of H2O2, and light-controllable generation of ROS, avoids excessive ROS from damaging normal cells and tissues. At the same time, the generated ROS is sufficient to produce a bactericidal effect on drug-resistant pathogens, and further can promote the healing of infected wounds, showing excellent antibacterial effects.

[0014] Further, the method comprises the following steps:

[0015] (1) dissolving Zn(NO3)2·3H2O, PVP and pyrazine in a solvent to obtain a mixture A;

[0016] (2) dissolving PtTCPP in a solvent to obtain a mixture B;

[0017] (3) Add the mixture A obtained in step (1) and the mixture B obtained in step (2) dropwise and mix well, and react to obtain Zn-TCPP-Pt nanozyme.

[0018] Furthermore, the molar ratio of the Zn(NO3)2·3H2O, the PVP, the pyrazine and the PtTCPP is 8-12:8-12:8-12:4-6.

[0019] Preferably, the molar ratio of the Zn(NO3)2·3H2O, the PVP, the pyrazine and the PtTCPP is 1:1:1:0.5.

[0020] Most preferably, in step (1), the ratio of the mixed solution of Zn(NO3)2·3H2O, PVP, pyrazine, DMF and ethanol is 3 mg:20 mg:0.4 mg:12 mL.

[0021] Most preferably, in step (2), the ratio of the mixed solution of PtTCPP, DMF and ethanol is 4.4 mg:4 mL.

[0022] Furthermore, in step (1) and step (2), the solvent is a mixed solution of DMF and ethanol, and the volume ratio of DMF to ethanol in the solvent is 2.5 to 5:1, preferably 3:1.

[0023] Furthermore, in step (3), the reaction temperature is 70 to 90° C., and the reaction time is 18 to 24 hours.

[0024] Preferably, the reaction temperature is 80°C and the reaction time is 24h.

[0025] As a preferred solution, step (3) further includes the following steps: after the reaction is completed, cooling to room temperature, collecting the flocculent precipitate by centrifugation, and washing with ethanol to obtain Zn-TCPP-Pt nanozyme.

[0026] As a preferred solution, the PtTCPP is prepared by the following method:

[0027] 1) dissolving methyl p-formylbenzoate and pyrrole in propionic acid respectively, adding the two dropwise and mixing them, and refluxing at 120-180° C. for 8-14 hours to obtain TMCPP;

[0028] 2) The TMCPP obtained in step 1) and PtCl2 are dissolved in benzonitrile, and reacted under N2 at 180-220°C for 12-48 hours; the reaction product is purified by column chromatography and hydrolyzed with KOH to obtain PtTCPP.

[0029] More preferably, in step 1), after the reflux reaction is completed, the mixture is cooled to room temperature, the precipitate is collected, and washed with ethanol, ethyl acetate and tetrahydrofuran, respectively, and dried to obtain TMCPP.

[0030] More preferably, the molar ratio of the methyl p-formylbenzoate to the pyrrole is 1:1;

[0031] More preferably, the molar ratio of the TMCPP to the PtCl2 is 3:1.

[0032] The third object of the present invention is to provide an application of the aforementioned antibacterial nanomedicine in antibacterial infection.

[0033] To achieve the above object, the present invention adopts the following technical solutions:

[0034] The application of the aforementioned antibacterial nanomedicine in combating bacterial infection.

[0035] Furthermore, the bacteria include methicillin-resistant Staphylococcus aureus.

[0036] Furthermore, light is used to control the activity of oxidases and thus the generation of ROS.

[0037] The beneficial effects of the present invention are:

[0038] 1. The present invention uses photosensitive porphyrin molecules with easy coordination in the central cavity to prepare light-responsive nanozymes, and combines the unique light-responsiveness of porphyrin to achieve controllable enzyme-like activity. At the same time, metal-anchored porphyrin is more conducive to the separation of photogenerated carriers during the light response process, thereby controllably generating more 1 O2、·OH、·O 2- , so that it can achieve good antibacterial effect.

[0039] 2. The Zn-TCPP-Pt nanozyme antibacterial agent prepared by the present invention has a good antibacterial effect on drug-resistant Staphylococcus aureus, and the bactericidal rate is increased by 90-99% compared with the positive control drug.

[0040] 3. The Zn-TCPP-Pt nanozyme antibacterial agent of the present invention is applied to wound infection. Under light therapy, ROS can be generated in a controllable manner, which can kill bacteria and promote wound healing while effectively preventing excessive ROS from damaging normal cells and tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the spectrum of the nanozyme oxidizing the substrate TMB;

[0042] Figure 2 This is the effect diagram of light-controlled nanozyme;

[0043] Figure 3 This is a graph showing the antibacterial effect of nanozymes on methicillin-resistant Staphylococcus aureus;

[0044] Figure 4 Schematic diagram of wound healing of mice in the Control group and Zn-TCPP-Pt+L group from day 0 to day 10;

[0045] Figure 5 The figure shows the wound healing effect of mice in the Control group and the Zn-TCPP-Pt+L group;

[0046] Figure 6 It is a graph showing the wound area of ​​mice in the Control group and the Zn-TCPP-Pt+L group from day 0 to day 10;

[0047] Figure 7 This is a scanning electron microscope image of the sample Zn-TCPP-Pt in Example 1. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0049] In the embodiment of the present invention, the structural formula of the porphyrin carboxylic acid ligand (PtTCPP) is shown in Formula I below.

[0050]

[0051] In an embodiment of the present invention, the porphyrin carboxylic acid ligand (PtTCPP) is prepared by the following method: a certain amount of methyl paraformylbenzoate and pyrrole are weighed, dissolved in propionic acid respectively, the two are added dropwise and mixed, the mixture is refluxed at 120-180°C for 8-14h, cooled to room temperature, the precipitate is collected, and washed with ethanol, ethyl acetate and tetrahydrofuran (THF) respectively, and dried to obtain TMCPP. The obtained TMCPP and PtCl2 are dissolved in benzonitrile, and the mixture is reacted at 180-220°C under N2 for 12-48h. The solvent is removed, and the obtained residue is purified by column chromatography and hydrolyzed in the presence of KOH to obtain PtTCPP. Among them, the molar ratio of methyl paraformylbenzoate to pyrrole is 1:1; the molar ratio of TMCPP to PtCl2 is 3:1. In addition, PtTCPP can also be purchased as a commercial product, and its CAS number is 94288-45-6.

[0052] In the embodiment of the present invention, the mixed solution of DMF and ethanol is prepared by mixing DMF and ethanol in a volume ratio of 3:1.

[0053] Example 1. Preparation method of antibacterial nanomedicine with light-responsive nanozyme activity

[0054] The layered nanosheet structure is synthesized by a bottom-up method using metalloporphyrin and Zn metal. The specific steps are as follows:

[0055] (1) 3 mg Zn(NO3)2·3H2O, 20 mg PVP and 0.4 mg pyrazine were fully dissolved in a mixed solution of 12 mL DMF and ethanol to obtain a mixture A;

[0056] (2) dissolving 4.4 mg of PtTCPP in a mixed solution of 4 mL of DMF and ethanol to obtain a mixture B;

[0057] (3) The mixture A obtained in step (1) and the mixture B obtained in step (2) were added dropwise and mixed, and reacted in an oven at 80° C. for 24 h; after cooling to room temperature, the flocculent precipitate was collected by centrifugation and washed with ethanol several times to obtain Zn-TCPP-Pt nanozyme, whose scanning electron microscopy image is shown in FIG. Figure 1 shown.

[0058] Example 2. Preparation of Zn-TCPP nanozyme

[0059] 3 mg Zn(NO3)2·3H2O, 20 mg PVP and 0.4 mg pyrazine were fully dissolved in a mixed solution of 12 mL DMF and ethanol (3:1) to obtain mixture C; 4.4 mg TCPP was dissolved in a mixed solution of 4 mL DMF and ethanol to obtain mixture D; mixture C and mixture D were added dropwise and mixed, and reacted at 80°C for 24 h; after cooling to room temperature, the flocculent precipitate was collected by centrifugation and washed several times with ethanol to obtain Zn-TCPP nanozyme.

[0060] Example 3. Light-responsive enzyme activity

[0061] The enzyme-like activity of Zn-TCPP-Pt prepared in Example 1 and Zn-TCPP prepared in Example 2 was compared using a typical TMB reaction. Usually, the prepared nanozyme (10 μL, 1 μmol / mL) was placed in 500 μL acetate buffer solution (NaAc-HAc, pH 3.5) in a 1 mL total system, TMB (100 μL, 10 mM) was used as a substrate, and the mixture was illuminated under an LED lamp for 10 min, the color change was observed by the naked eye, and a wavelength scan of 550-800 nm was performed using a UV-visible spectrophotometer to evaluate its light-responsive oxidase-like activity, wherein the oxidation of the substrate TMB under illumination (L) / no illumination was used as a control group.

[0062] Results: Figure 1 As shown in the figure, Zn-TCPP, Zn-TCPP-Pt and TMB cannot be oxidized without light irradiation, while Zn-TCPP and Zn-TCPP-Pt nanozyme materials quickly oxidize TMB after 10 minutes of light irradiation. This is because the nanozyme produces ROS under light irradiation to oxidize TMB; at the same time, by continuously turning off and on light irradiation, the OXD-like activity of Zn-TCPP-Pt shows a step-like behavior, see for details. Figure 2 The results show that Zn-TCPP-Pt nanozyme has good light-responsive enzyme activity.

[0063] Example 3. Antibacterial effect of nanozymes on drug-resistant Staphylococcus aureus

[0064] Methicillin-resistant Staphylococcus aureus was selected as the model bacteria. The bacteria were inoculated in TSB medium and cultured at 37°C overnight. The next day, they were re-inoculated in fresh TSB medium to grow to the logarithmic growth phase, and then diluted to about 2×10 7 Colony forming unit (CFU). This dilution was mixed with 2.5 μL, 5 μL, and 10 μL of nanozyme (1 μmol / mL) respectively, and exposed to LED light for 10 min. 100 μL of the treated bacterial solution was diluted / undiluted and spread, incubated at 37°C for 18 h, and the plate was counted.

[0065] The results are as follows Figure 3 As shown in the figure, under illumination, the antibacterial effects of Zn-TCPP-Pt and Zn-TCPP are positively correlated with the material concentration, and the dilution factor required for the plate count of Zn-TCPP is higher than that of Zn-TCPP-Pt, indicating that Zn-TCPP-Pt has better bactericidal toxicity than Zn-TCPP, which may be due to the fact that metalloporphyrin shortens the transmission distance of electrons, resulting in stronger light response ability and more ROS production.

[0066] Example 4. Nanozymes promote wound healing

[0067] A circular skin wound with a diameter of ≈9 mm was made on the back of female Kunming mice (6-8 weeks old). 30 μL of Staphylococcus aureus (≈2×10 8 CFU) and incubated overnight to establish an infected wound model. Then, the mice were randomly divided into two groups (I) and (II) and instilled with PBS (40 μL) / Zn-TCPP-Pt (1 μmol mL -1 , 40 μL), and then light treatment, namely: I) infection untreated group and II) material light treatment group. The wound healing of mice was recorded.

[0068] The results are as follows Figures 4 to 6 As shown, the wound area of ​​the material light treatment group (Zn-TCPP-Pt+L) was significantly smaller than that of the infected untreated group (Control) on the third day of treatment, indicating that the material light treatment produced sufficient ROS, which could kill bacteria in time and promote wound healing.

Claims

1. Antibacterial nanomedicine with light-responsive nanozyme activity, characterized in that: The antibacterial nano drug is prepared by coordinating metal porphyrin with Zn metal ions.

2. The antibacterial nanomedicine according to claim 1, characterized in that: The nanozyme is an oxidase-like enzyme.

3. The method for preparing the antibacterial nanomedicine according to any one of claims 1 to 2, characterized in that: Zn-TCPP-Pt nanozyme was prepared by a bottom-up approach using metalloporphyrin and Zn metal ions.

4. The preparation method according to claim 3, characterized in that: The method comprises the following steps: (1) dissolving Zn(NO3)2·3H2O, PVP and pyrazine in a solvent to obtain a mixture A; (2) dissolving PtTCPP in a solvent to obtain a mixture B; (3) Add the mixture A obtained in step (1) and the mixture B obtained in step (2) dropwise and mix well, and react to obtain Zn-TCPP-Pt nanozyme.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the Zn(NO3)2·3H2O, the PVP, the pyrazine and the PtTCPP is 8-12:8-12:8-12:4-6.

6. The preparation method according to claim 4, characterized in that: In step (1) and step (2), the solvent is a mixed solution of DMF and ethanol, and the volume ratio of DMF to ethanol in the solvent is 2.5 to 5:

1.

7. The preparation method according to claim 4, characterized in that: In step (3), the reaction temperature is 70 to 90° C. and the reaction time is 18 to 24 hours.

8. The preparation method according to claim 4, characterized in that: The PtTCPP is prepared by the following method: 1) dissolving methyl p-formylbenzoate and pyrrole in propionic acid respectively, adding the two dropwise and mixing them, and refluxing at 120-180° C. for 8-14 hours to obtain TMCPP; 2) The TMCPP obtained in step 1) and PtCl2 are dissolved in benzonitrile, and reacted under N2 at 180-220°C for 12-48 hours; the reaction product is purified by column chromatography and hydrolyzed with KOH to obtain PtTCPP.

9. Use of the antibacterial nanomedicine according to any one of claims 1 to 2 in preventing bacterial infection.

10. The use according to claim 9, characterized in that: Such bacteria include methicillin-resistant Staphylococcus aureus.

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