Application of InNNi3 in preparation of antibacterial product

Abstract

The invention discloses an application of InNNi3 in preparation of an antibacterial product. It is found that InNNi3 can effectively destroy biological membranes of bacteria or fungi, so that the bacteria or fungi are killed, and the antibacterial effect is achieved.

Description

Technical Field

[0001] This invention relates to the use of InNNi3 in the preparation of antibacterial products. Background Technology

[0002] The skin is a vital barrier against pathogens, but it is also susceptible to damage, leading to wounds. Wound healing involves complex physiological processes such as inflammatory responses, tissue regeneration, and repair. However, bacterial infection often becomes a major obstacle to wound healing. Persistent bacterial infection of open wounds can exacerbate the inflammatory response, prolong healing time, and may even cause serious complications. Clinically, antibiotics are widely used to treat bacterial infections, but their overuse has led to the emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Klebsiella pneumoniae (MDR-Klebsiella pneumoniae), which exhibit high resistance to common antibiotics. More seriously, the development of new antibiotics lags far behind the evolution and mutation of bacterial resistance. Therefore, developing novel antibacterial agents beyond antibiotics is particularly important for anti-infective therapy.

[0003] Photoresponsive therapies, such as photothermal antibacterial therapy and photocatalytic antibacterial therapy, have attracted widespread attention due to their advantages of spatiotemporal selectivity, minimal invasiveness, relative clinical safety, and low likelihood of inducing drug resistance. Although many photothermal agents and photocatalysts have been studied to date, the development of novel phototherapy antibacterial agents that combine high extinction coefficient, broad spectral absorption, high photothermal conversion efficiency, good photothermal stability, controllable large-scale preparation, good biocompatibility, and low biotoxicity is of great significance for developing phototherapy strategies for practical clinical applications.

[0004] ANX3-type anti-perovskite nitride materials (where A and X are metallic components, and N represents nitrogen; typically, A / X represents transition metals such as Co, Cu, and Ni, and A sites include In and Ga) possess a unique inverted electronic coordination structure. In the octahedral coordination structure of the crystal, the N element occupies the central position of the octahedron, carrying a partial negative charge, exhibiting a nitrogen anion state; while the transition metal X occupies the vertex position of the octahedron, carrying a partial positive charge, exhibiting a mixed valence state with both low and high valences, acting as a transition metal cation. Therefore, the surface of this type of material is usually also positively charged. This type of material exhibits conductor-like electronic properties, possessing a quasi-continuous distribution of energy levels near the Fermi level, resulting in absorption across the entire solar spectrum. Therefore, the strong and broad solar spectrum absorption characteristics of ANX3 (e.g., InNNi3) give it excellent potential for phototherapy and antibacterial applications. However, current technologies do not focus on the antibacterial applications of anti-perovskite nitrides; most research focuses on their chemical applications.

[0005] CN117753980A discloses an electrocatalytic reduction catalyst for carbon dioxide from an anti-perovskite substrate. The molecular formula of this catalyst is InNNi. 3-x V x Where x = 0.2, 0.4, 0.6, or 0.8. The working electrode prepared using this anti-perovskite catalyst can be used for the electrocatalytic reduction of carbon dioxide to formic acid, but its antibacterial properties are not involved.

[0006] CN118198255A discloses a negative electrode sheet. This negative electrode sheet includes a negative electrode material layer, which comprises a lithiophilic material and a three-dimensional framework loaded with the lithiophilic material. The lithiophilic material is InNNi3 with an anti-perovskite crystal structure. The material of the three-dimensional framework includes at least one of carbon, copper, titanium, iron, or nickel. The InNNi3 in this negative electrode sheet is mainly used in secondary batteries and does not involve antibacterial properties.

[0007] CN119679940A discloses the application of a perovskite-porous carrier composite material in the preparation of antibacterial and antiviral products, antitumor drugs, bioimaging agents, or photothermal agents for thermotherapy. The composite material comprises: a porous carrier, and a perovskite material loaded on the porous carrier. The perovskite material includes one or more of oxide perovskite materials, halide perovskite materials, bis-perovskite materials, and anti-perovskite materials. The perovskite material is selected from one or more of ABX3, A2B6, A3B2X5, and A2BX4, wherein A is selected from one or more of La, Ce, Cs, FA, and MA, B is selected from one or more of Fe, Mn, Bi, Ag, In, and Ti, and X is selected from one or more of O, Br, Cl, and I. Although this composite material may include anti-perovskite materials, it does not involve ANX3-type anti-perovskite materials. Summary of the Invention

[0008] The purpose of this invention is to provide a use of InNNi3 in the preparation of antibacterial products. This invention discovers that InNNi3 can effectively disrupt the biofilm of bacteria or fungi, thereby eliminating bacteria or fungi and achieving an antibacterial effect.

[0009] The present invention achieves the above objectives through the following technical solutions.

[0010] This invention provides a use of InNNi3 in the preparation of antibacterial products.

[0011] According to the intended use described in this invention, preferably, the antibacterial product is a topical product; the antibacterial product is selected from at least one of pharmaceuticals and health products.

[0012] According to the intended use described in this invention, preferably, the antibacterial product is a drug.

[0013] According to the intended use of the present invention, preferably, the drug forms an antibacterial pharmaceutical preparation; the pharmaceutical preparation comprises InNNi3 and also comprises pharmaceutically acceptable excipients.

[0014] According to the use described in this invention, preferably, the drug is a pharmaceutical formulation that forms an anti-biofilm.

[0015] According to the intended use described in this invention, preferably, the content of InNNi3 in a unit pharmaceutical preparation is 0.5 mg or more.

[0016] According to the intended use of the present invention, preferably, in the cytotoxicity experiment, when the concentration of InNNi3 in the cell culture medium is 0.5-2 mg / mL, the survival rate of human embryonic kidney cells and mouse embryonic fibroblasts after 24 h of culture is both above 90%; in the hemolytic activity experiment, when the concentration of InNNi3 in 2% erythrocyte suspension is 0.5-2 mg / mL, the hemolysis rate of erythrocytes is below 5%.

[0017] According to the application described in this invention, preferably, in the in vitro antibacterial experiment, when the concentration of InNNi3 in the bacterial solution is 0.5 mg / mL, and the optical power density is 1.0 W / cm², 2 Under the condition of irradiation with a near-infrared laser with a wavelength of 808nm for 10 minutes, the antibacterial efficiency against bacteria or fungi after 18 hours of culture is over 99.9%.

[0018] According to the intended use of the present invention, preferably, in an in vivo antibacterial experiment, the infected wound is treated with an aqueous solution of InNNi3 at a concentration of 0.5 mg / mL, followed by treatment with an optical power density of 1.0 W / cm². 2 The infected wound was irradiated with a near-infrared laser with a wavelength of 808nm for 10 minutes, and the wound healing rate of the infected wound after 14 days was more than 93.71%; wherein the infected wound is a circular wound with a diameter of 9cm and full-thickness skin defect that has been infected with bacteria or fungi for 30 minutes.

[0019] According to the intended use of the present invention, preferably, the bacteria are selected from at least one of Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans.

[0020] InNNi3 is commonly used as a material for preparing catalysts or electrodes. This invention unexpectedly discovered that InNNi3 possesses low cytotoxicity and good biocompatibility, effectively eliminating bacteria or fungi and significantly improving the healing rate of infected wounds. Attached Figure Description

[0021] Figure 1 The cell survival rates of 293T cells and NIH 3T3 cells treated with different concentrations of InNNi3 solution are shown.

[0022] Figure 2 The hemolysis of erythrocytes treated with different concentrations of InNNi3 solution is shown; (a) shows the hemolytic activity of erythrocytes treated with different concentrations of InNNi3 solution; (b) shows the hemolysis rate of erythrocytes treated with different concentrations of InNNi3 solution.

[0023] Figure 3 To determine whether to use 1 W / cm with or without InNNi3 solution of different concentrations. 2 Photographs and quantitative results of bacteria treated with 808nm NIR laser irradiation for 10 min; where (a) is a colony photograph of E. coli, (b) is the quantitative result of E. coli, (c) is a colony photograph of MRSA, (d) is the quantitative result of MRSA, (e) is a colony photograph of C. albicans, and (f) is the quantitative result of C. albicans; the pentagrams in the figure indicate that less than 1% of the colonies survive; data are expressed as mean ± standard deviation, n = 3. * indicates statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

[0024] Figure 4 For 1W / cm 2 Colony images and quantitative results of bacteria treated with InNNi3 under 808nm NIR laser irradiation after 10 irradiation exposure cycles.

[0025] Figure 5 Images show the live / dead staining of biofilms formed after different treatments; (a) is the live / dead staining image of E. coli biofilm, and (b) is the live / dead staining image of MRSA biofilm; the scale bar in the figures is 30 μm.

[0026] Figure 6 This image shows a schematic diagram and results of an experiment using InNNi3 solution combined with NIR laser treatment of rat wounds; (a) is a schematic diagram of the experiment, (b) is a photothermal image of the rat wound, and (c) shows the wound treated with 0.5 mg / mL InNNi3 solution followed by a 1 W / cm² laser. 2 Photothermal heating curve after 10 minutes of irradiation with an 808nm NIR laser.

[0027] Figure 7This study examines the healing progress of wounds from day 0 to day 14 after treatment. (a) shows images of rat skin wounds on days 0, 3, 7, 1, and 14; (b) shows a schematic diagram of the wound healing process from day 0 to day 14 in different treatment groups; (c) shows a quantitative graph of the functional relationship between wound area and treatment time (n=3); (d) shows MRSA colony diagrams of wounds on LB agar plates on days 3 and 7; and (e) shows a statistical graph of colony counts. Data in the figures are expressed as mean ± standard deviation, n=3, and * indicates statistical significance (*p<0.05, **p<0.01, ***p<0.001).

[0028] Figure 8 The results of H&E staining of wound tissue are shown below; (a) shows the H&E staining results on days 3 and 10 (red arrows indicate inflammatory infiltrating cells, blue arrows indicate fibroblasts, green arrows indicate neovascularization, orange double arrows indicate granulation tissue, and the spacing between black dashed lines indicates scar width), (b) shows the quantitative analysis results of regenerated granulation tissue on day 10, and (c) shows the quantitative analysis results of wound scar width on day 10; data are expressed as mean ± standard deviation, n = 3, * indicates statistical significance (*p < 0.05, **p < 0.01).

[0029] Figure 9 Masson staining and CD31 staining results of wound tissue; (a) Masson staining on days 3 and 10, (b) quantitative analysis of collagen deposition on day 10 in (a), (c) CD31 staining of newly formed blood in the wound area on days 3 and 10, (d) quantitative analysis of microvessels on day 3 in (c), and (e) quantitative analysis of microvessels on day 10 in (c); data are expressed as mean ± standard deviation, n = 3, * indicates statistical significance (*p < 0.05, **p < 0.01).

[0030] Figure 10 The images show the H&E staining results of the major organs of rats on day 3 and day 10 after treatment; (a) shows the H&E staining images of the major organs (heart, liver, spleen, lung and kidney) of rats in different treatment groups on day 3, and (b) shows the H&E staining images of the major organs (heart, liver, spleen, lung and kidney) of rats in different treatment groups on day 10; the scale bar in the images is 100 μm.

[0031] Figure 11 The graph shows the change in body weight of rats in different treatment groups over time. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0033] <Purpose>

[0034] This invention relates to a novel use of InNNi3. InNNi3 is commonly used as a material in the preparation of catalysts or electrodes. This invention unexpectedly discovered that InNNi3 possesses low cytotoxicity and good biocompatibility, effectively disrupting bacterial or fungal biofilms and eliminating bacteria or fungi, particularly against Escherichia coli, methicillin-resistant Staphylococcus aureus, and Candida albicans. InNNi3 can also significantly improve the healing rate of infected wounds. Therefore, this invention provides a use of InNNi3 in the preparation of antibacterial products.

[0035] According to one embodiment of the present invention, the antibacterial product can be a topical product, preferably at least one of a drug or health product, and more preferably a drug. The drug can be an active pharmaceutical ingredient (API) or a formulation.

[0036] In this invention, InNNi3 can be a commercially available product or prepared using existing methods as needed.

[0037] In this invention, the drug can form an antibacterial pharmaceutical formulation, preferably an antibiofilm-forming pharmaceutical formulation. The pharmaceutical formulation may contain InNNi3 and may also contain pharmaceutically acceptable excipients.

[0038] In this invention, the dosage form of the pharmaceutical preparation can be any dosage form of topical medicine known in the art, and is not particularly limited herein. For example, it can be a liquid, ointment, powder, aerosol, etc.

[0039] In this invention, the excipients can be any type of excipient known in the art for topical medications, and are not particularly limited herein. For example, they can be selected from matrix materials, emulsifiers, stabilizers, transdermal penetration enhancers, preservatives, antioxidants, lubricants, etc. Matrix materials can be selected from water, oils, waxes, powders, etc. Water can be ultrapure water, deionized water, or antibacterial water, etc. Oils can be selected from petrolatum, glycerin, sesame oil, peanut oil, rapeseed oil, etc. Waxes can be selected from beeswax, paraffin wax, etc. Powders can be selected from talc, starch, zinc oxide, calamine, etc. Emulsifiers can be selected from sodium lauryl sulfate, polysorbate, etc. Stabilizers can be selected from sodium carboxymethyl cellulose (CMC-Na), methyl cellulose (MC), hydroxypropyl methylcellulose (HPMC), etc. Transdermal penetration enhancers can be selected from azone, propylene glycol, etc. Preservatives can be selected from sodium benzoate, parabens, etc. Antioxidants can be selected from vitamin E, tert-butylhydroxyanisole (BHA), etc.

[0040] According to one embodiment of the present invention, InNNi3 may be the sole active ingredient in an antibacterial drug. According to another embodiment of the present invention, in addition to InNNi3, the antibacterial drug may also contain other active ingredients with antibacterial activity, or active ingredients that do not have antibacterial activity themselves but can assist the antibacterial activity of InNNi3.

[0041] According to one embodiment of the present invention, the content of InNNi3 in a unit pharmaceutical preparation can be 0.5 mg or more, preferably 0.5 to 2 mg, and more preferably 0.5 to 2 mg per mL of pharmaceutical preparation.

[0042] According to one embodiment of the present invention, in a cytotoxicity experiment, when the concentration of InNNi3 in the cell culture medium is 0.5–2 mg / mL, the survival rate of human embryonic kidney cells and mouse embryonic fibroblasts after 24 hours of culture is both above 90%. Preferably, when the concentration of InNNi3 in the cell culture medium is 0.5–0.75 mg / mL, the survival rate of human embryonic kidney cells and mouse embryonic fibroblasts after 24 hours of culture is both above 95%. More preferably, when the concentration of InNNi3 in the cell culture medium is 0.5 mg / mL, the survival rate of human embryonic kidney cells 293T after 24 hours of culture reaches 100%, and the survival rate of mouse embryonic fibroblasts NIH 3T3 after 24 hours of culture is above 95%.

[0043] According to one embodiment of the present invention, in the hemolytic activity test, when the concentration of InNNi3 in a 2% erythrocyte suspension is 0.5–2 mg / mL, the hemolysis rate of erythrocytes is 5% or less. Preferably, when the concentration of InNNi3 in a 2% erythrocyte suspension is 0.5–0.75 mg / mL, the hemolysis rate of erythrocytes is 3% or less. More preferably, when the concentration of InNNi3 in a 2% erythrocyte suspension is 0.5 mg / mL, the hemolysis rate of erythrocytes is 1% or less.

[0044] According to one embodiment of the present invention, in an in vitro antibacterial experiment, when the concentration of InNNi3 in the bacterial solution is 0.5 mg / mL, and the optical power density is 1.0 W / cm², 2 Under near-infrared (NIR) laser irradiation at a wavelength of 808 nm for 10 min, the antibacterial efficiency against bacteria or fungi after 18 h of culture is above 99.9%. Preferably, the bacteria or fungi are selected from at least one of Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. More preferably, the bacteria or fungi are selected from at least one of Escherichia coli BL 21, MRSA ATCC 43300, and Candida albicans ATCC 10231.

[0045] According to one embodiment of the present invention, in an in vivo antibacterial experiment, the infected wound was treated with an InNNi3 aqueous solution at a concentration of 0.5 mg / mL, followed by an optical power density of 1.0 W / cm². 2 The infected wound was irradiated with a near-infrared laser with a wavelength of 808 nm for 10 minutes, and the wound healing rate after 14 days was over 93.71%. Preferably, the infected wound is a circular wound with a full-thickness skin defect of 9 cm in diameter from a rat, which has been infected for 30 minutes with at least one of Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. More preferably, the rat is an SD (Sprague-Dawley) rat, and the bacteria or fungus is selected from at least one of Escherichia coli BL 21, MRSA ATCC 43300, and Candida albicans ATCC10231.

[0046] This invention reveals that InNNi3 exhibits significant antibacterial effects and the ability to accelerate wound healing under near-infrared laser irradiation with appropriate optical power density and wavelength; simultaneously, InNNi3 also possesses low cytotoxicity and good biocompatibility, making it suitable as an antibacterial product.

[0047] <Ingredient Description>

[0048] Unless otherwise specified, all raw materials used in the following examples are commercially available products.

[0049] The water used was ultrapure water. InNNi3 was prepared in-house using existing typical synthesis methods. The InNNi3 solution was prepared by placing InNNi3 particles with a particle size of 200 nm in ultrapure water.

[0050] Example 1 - In-NNi3 In Vitro Biocompatibility Experiment

[0051] 1.1 Cytotoxicity Detection

[0052] 1.1.1 Experimental Materials

[0053] Mouse embryonic fibroblasts (NIH 3T3): cultured in-house. Human kidney epithelial cells (293T): cultured in-house. Fetal bovine serum (FBS) was purchased from Adamas Life, catalog number: P1692553. Penicillin-PS antibodies were purchased from Adamas Life, catalog number: P2127484. DMEM was purchased from Adamas Life. Trypsin digestion solution (0.25% purity) was purchased from Adamas Life. The CCK-8 cell counting kit was purchased from Shanghai Titan Technology Co., Ltd. The cell culture medium was DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-PS antibodies.

[0054] 1.1.2 Experimental Methods

[0055] Mouse embryonic fibroblasts 293T (denoted as 293T cells) and human renal epithelial cells NIH 3T3 (denoted as NIH3T3 cells) were removed from a -80°C freezer and thawed in a 45°C water bath. After thawing, the cells were aspirated into DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin antibiotics (PS), centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. The cell pellet was resuspended in 3 mL of cell culture medium, and then the cell culture medium containing the cell pellet was evenly dispersed in a culture dish containing 10 mL of DMEM medium. The cells were cultured and observed until they adhered to the culture dish. The adhered cell culture dish was placed in a sterile laminar flow hood, the DMEM medium was removed, and the cells were digested with 5 mL of trypsin digest for 5 min. The digested cell solution was then centrifuged at 1500 rpm for 3 min, the supernatant was discarded, and the cell pellet was resuspended in 2 mL of cell culture medium to form a cell suspension. 10 μL of a well-mixed cell suspension was placed on a cell counting plate for cell counting and dilution calculation. Then, 100 μL of the diluted cell suspension was added to each well of a 96-well plate. 200 μL of PBS (phosphate-buffered saline) was added around the perimeter of each well. The 96-well plate was then incubated at 37°C and 5% CO2 for 10 hours until cell attachment. After cell attachment, the supernatant was aspirated. For the experimental group, 100 μL of different concentrations of InNNi3 solution was added to each well; for the control group, 100 μL of cell culture medium was added to each well. The 96-well plate was then incubated at 37°C and 5% CO2 for 24 hours. After incubation, 10 μL of CCK-8 cell counting reagent was added to each well. Cell viability was assessed by measuring absorbance at 450 nm and calculated based on the absorbance values. The formula for calculating cell viability is as follows:

[0056]

[0057] Where B represents the absorbance value of the group without added materials (control group), and B0 represents the absorbance value of the blank well group (zeroing group) containing only culture medium. t This indicates the absorbance value of the InNNi3 treatment group (experimental group).

[0058] 1.1.3 Experimental Results

[0059] The experimental results are as follows Figure 1As shown, at a concentration of 0.5 mg / mL, the survival rate of 293T cells reached 100% after 24 hours of culture, and the survival rate of NIH 3T3 cells was over 95% after 24 hours of culture, indicating good biocompatibility of InNNi3. Furthermore, although cell survival rate decreased slightly with increasing InNNi3 concentration, it remained at 90% at the highest tested concentration (2 mg / mL). This indicates that the cytotoxicity of InNNi3 is low-concentration dependent, further demonstrating its potential as a safe antibacterial agent.

[0060] 1.2 Hemolytic Activity Detection

[0061] 1.2.1 Experimental Materials and Instruments

[0062] The bispecific antibody (PS) was sourced from the same source as above. Triton X-100 was purchased from Shanghai Titan Technology Co., Ltd. Defibrinated sheep blood was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: J10IM2019710. The multi-functional microplate reader was a Spectra Max Plus 384 manufactured by Molecular Devices.

[0063] 1.2.2 Experimental Methods

[0064] Take 1 mL of defibrinated sheep blood and place it in a 10 mL centrifuge tube. Centrifuge at 4℃ and 3000 rpm for 5 min. Discard the supernatant (which contains plasma, white blood cells, and platelets). Collect the red blood cell pellet and wash it 5 times with PBS under the same centrifugation conditions. Dilute the washed red blood cell pellet to a 4% red blood cell suspension. The experimental groups and treatments are as follows: Experimental group: 4% red blood cell suspension was mixed with equal volumes of different concentrations of InNNi3 solution. Positive control group: 4% red blood cell suspension was mixed with equal volumes of 0.1% Triton X-100. Negative control group: 4% red blood cell suspension was mixed with equal volumes of PBS. After 4 h of treatment, each group was centrifuged at 3000 rpm for 5 min. Then, 200 μL of the supernatant was collected, and the absorbance (OD) at 540 nm was measured using a multi-mode microplate reader. 540 The hemolysis rate is calculated based on the absorbance value. The formula for calculating the hemolysis rate is as follows:

[0065]

[0066] Where A represents the absorbance value of the red blood cell supernatant in the experimental group; A0 represents the absorbance value of the red blood cell supernatant in the negative control group (PBS-treated group); A t The absorbance value represents the red blood cell supernatant of the positive control group (0.1% Triton X-100 treatment group).

[0067] 1.1.3 Experimental Results

[0068] The experimental results are as follows Figure 2 As shown. The InNNi3-treated red blood cell solution was similar in color to the negative control group (PBS), with no significant redness. Figure 2 (a) At concentrations of 0.25–2 mg / mL, the hemolysis rate showed a significant difference compared to the positive control group (0.1% Triton X-100). The hemolysis rate of InNNi3 at concentrations of 0.25–2 mg / mL was below the critical value of 5%. In particular, at InNNi3 concentrations of 0.5–0.75 mg / mL, the hemolysis rate of erythrocytes was below 3%. At an InNNi3 concentration of 0.5 mg / mL, the hemolysis rate of erythrocytes was below 1%. Figure 2 (b) This indicates that InNNi3 has low hemolytic activity, which meets the requirements of clinical trials.

[0069] Example 2 - In vitro antibacterial and anti-biofilm experiments of InNNi3

[0070] 2.1 In vitro antibacterial activity test

[0071] 2.1.1 Experimental Materials

[0072] Escherichia coli BL21 (E. coli): cultured in-house. Methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA): cultured in-house. Candida albicans ATCC 10231 (C. albicans): cultured in-house. LB medium was prepared as follows: 2g sodium chloride, 2g peptone, and 1g yeast extract were weighed and dissolved in 200mL of ultrapure water. YPD medium was prepared as follows: 2g glucose, 2g peptone, and 1g yeast extract were weighed and dissolved in 200mL of ultrapure water. A multi-functional microplate reader was used as described above. The 808nm laser (NIR) was a LE-LS-808-3000TFCIL model manufactured by Leo Optoelectronics Technology Co., Ltd.

[0073] 2.1.2 Experimental Methods

[0074] 2.1.2.1 Bacterial culture:

[0075] *Escherichia coli* BL 21 (E. coli), methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), and *Candida albicans* ATCC 10231 (C. albicans) were selected as experimental subjects. *E. coli* and MRSA were inoculated into 20 mL of LB (Lysogeny Broth) medium and cultured with shaking at 37℃ and 165 rpm for 12 hours. *C. albicans* was inoculated into 20 mL of YPD (Yeast Extract Peptone Dextrose Medium) medium and cultured with shaking at 28℃ and 165 rpm for 12 hours. After culture, 1 mL of the bacterial culture was transferred to a 1.5 mL centrifuge tube (PE tube), centrifuged at 7100 rpm for 1 min, the supernatant was discarded, the precipitate was washed twice with ultrapure water, and finally resuspended in ultrapure water. Next, 200 μL of the washed bacterial culture was taken and its OD was measured using a multi-functional microplate reader. 670 The bacterial culture was diluted to OD value. 670 =0.03 for backup.

[0076] 2.1.2.2 In vitro antibacterial activity experiment of InNNi3

[0077] The bacterial cultures (OD) of E. coli, MRSA and C. albicans were respectively... 670 =0.03) was divided into four groups for different treatments, as follows: Control group I (no NIR laser irradiation, denoted as NIR(-)), Control group II (with NIR laser irradiation, denoted as NIR(+)), InNNi3 treatment group I (no NIR laser irradiation, denoted as InNNi3-NIR(-)), and InNNi3 treatment group II (with NIR laser irradiation, denoted as InNNi3-NIR(+)). In the experiment, the concentration of InNNi3 in the bacterial culture was 0.5 mg / mL. The light power density was 1.0 W / cm². 2 Near-infrared laser with a wavelength of 808nm (denoted as 1.0W / cm²) 2 After irradiation with an 808nm NIR laser for 10 min, the mixture was diluted. The mixture of E. coli and MRSA was diluted 500-fold, and the mixture of C. albicans was diluted 25-fold. After dilution, 100 μL of the bacterial culture was spread onto agar plates and then incubated at 37°C for 18 h in a thermostatic incubator (C. albicans was incubated at 28°C). Colony counts were calculated using the plate count method (each experiment was repeated three times).

[0078] From the InNNi3-NIR(+) group following the aforementioned antibacterial experiment, a mixture of InNNi3 and bacteria (InNNi3 concentration 4 mg / mL) was taken, sonicated for 1 min, and then centrifuged at 4100 rpm for 2 min. After discarding the supernatant, the precipitate was resuspended in 100 μL of ultrapure water and reacted with 100 μL of bacterial culture (OD200). 670 =0.03) Mix, then place in 1.0W / cm 2 Irradiated with an 808nm NIR laser for 10 minutes. The control group consisted of bacterial culture (OD200...). 670 =0.03) was mixed with an equal volume of ultrapure water and left to stand for 10 min. After the reaction was complete, the mixture was diluted: the mixture for E. coli and MRSA was diluted 500 times, and the mixture for C. albicans was diluted 25 times. After dilution, 100 μL of each diluted solution was spread onto agar plates and then incubated in a thermostatic incubator at 37 °C for 18 h (C. albicans was incubated at 28 °C). The colony count was calculated by plate counting. This process was repeated 10 times to evaluate the antibacterial effect of InNNi3 under NIR laser irradiation.

[0079] 2.1.3 Experimental Results

[0080] The results of the first antibacterial test are as follows Figure 3 As shown, the survival rates of *E. coli*, *MRSA*, and *C. albicans* in the experimental groups irradiated with InNNi3 and 808 nm NIR lasers were significantly reduced, which was significantly better than the experimental groups without NIR laser irradiation and the control group. At 1.0 W / cm² 2 Under 808 nm NIR laser irradiation, the antibacterial efficiency of InNNi3 solution increased with increasing InNNi3 concentration, reaching 99.9% inhibition efficiency against bacteria / fungi at 0.5 mg / mL, demonstrating highly efficient and broad-spectrum antibacterial properties. This result indicates that InNNi3 has significant potential for antibacterial applications.

[0081] The cyclic antibacterial results of InNNi3 are as follows: Figure 4 As shown, a 2 mg / mL InNNi3 solution, combined with near-infrared laser treatment, showed no significant decrease in antibacterial efficiency for E. coli, MRSA, and C. albicans in 10 cycles of antibacterial experiments, demonstrating the stability of InNNi3 in photothermal antibacterial applications. Under NIR laser irradiation, InNNi3 can continuously generate heat, disrupting the structure of microorganisms and thus achieving a highly efficient antibacterial effect.

[0082] 2.2 In vitro anti-biofilm experiment of InNNi3

[0083] 2.2.1 Experimental Materials and Instruments

[0084] Escherichia coli BL 21 was sourced as above. Methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA) was sourced as above. The multi-functional microplate reader was used as above. The 808nm laser (NIR) was used as above. The laser scanning confocal microscope was a Leica SP8 manufactured by Leica GmbH, Germany.

[0085] 2.2.2 Experimental Methods

[0086] After resuscitation, 1 mL of E. coli and MRSA bacterial culture was transferred to 1.5 mL centrifuge tubes (PE tubes), centrifuged at 7100 rpm for 1 min, and the supernatant was discarded. The precipitate was then washed twice with LB medium (pH 8.0), and finally resuspended in LB medium to allow for final OD500. 670 The value was 0.03. Subsequently, 1 mL of bacterial dilution was placed in each well of a 12-well plate and incubated for 48 h to form a biofilm. After biofilm formation, the biofilm was washed three times with physiological saline to remove suspended bacteria. The subsequent grouping and treatment were as follows: Control group: 1 mL of LB medium solution was added to each well for 4 h; InNNi3 treatment group: 1 mL of InNNi3 sample solution was added; NIR(+) was performed at 1.0 W / cm². 2 Irradiation with 808nm NIR laser for 10 minutes, NIR(-) without using 1.0W / cm 2 Irradiation with an 808 nm NIR laser. Finally, after staining with Calcein-AM and propidium iodide (PI), the activity of the biofilm was evaluated by laser scanning confocal microscopy.

[0087] 2.2.3 Experimental Results

[0088] The results of observations on the biofilm status of E. coli and MRSA using laser scanning confocal microscopy are as follows: Figure 5 As shown in the figure, Calcein-AM emits green fluorescence to label live bacteria, while PI emits red fluorescence to label dead bacteria. In the control group, the biofilm remained intact and exhibited bright green fluorescence regardless of whether it was irradiated by NIR laser, indicating that the integrity of the bacterial biofilm did not change significantly. In contrast, the biofilm treated with InNNi3 showed a small amount of red fluorescence in some areas even without NIR laser irradiation, which may be due to the electrostatic interaction between InNNi3 and the biofilm, causing some degree of damage to the biofilm. Under NIR laser irradiation, the InNNi3-treated biofilm showed strong red fluorescence, indicating that it had a significant bactericidal effect on the bacteria within the biofilm. The results show that InNNi3 not only has highly efficient antibacterial properties but also has the potential to disrupt biofilms.

[0089] Example 3 - Experiment on InNNi3 on wound healing

[0090] Animal experiments were conducted in accordance with the National Research Council of the United States’ Guidelines for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Ethics Committee of Fujian Medical University (No.: IACUC FJMU 2023-Y-0552).

[0091] 3.1 Experimental Materials

[0092] SD (Sprague-Dawley) rats were purchased from Beijing Huafukang Biotechnology Co., Ltd. 4% PFA fixative was purchased from Biosharp (catalog number: BL539A). An 808nm NIR laser was used as described above. An infrared thermal imager was a FLIR E53 model. An inverted fluorescence microscope was a Leica DMi8 model from Leica GmbH, Germany.

[0093] 3.2 Experimental Methods

[0094] The wound healing ability of InNNi3 in SD rats was evaluated using a full-thickness skin defect model. Thirty-six male SD rats weighing 165g were selected as experimental subjects. Before the formal experiment, all rats underwent a one-week acclimatization period: they were provided with free access to water and a fixed amount of food (by the time of the formal experiment, their weight had increased to 200g). Rats were randomly divided into four groups according to weight: control + NIR(-) group, InNNi3 + NIR(-) group, control + NIR(+) group, and InNNi3 + NIR(+) group, with nine rats in each group. InNNi3 indicates treatment with a 0.5mg / mL InNNi3 solution, NIR(+) indicates NIR laser irradiation treatment, and NIR(-) indicates no NIR laser irradiation treatment. Rats were anesthetized with 0.3% sodium pentobarbital, and their backs were shaved. Then, a circular wound with a diameter of 9 mm was created on the back of the rat using a biopsy needle, and 10 μL (7.5 × 10⁻⁶) of full-thickness skin was instilled into the wound. 8 MRSA bacterial suspension (CFU / mL). After 30 minutes of infection, different treatments were administered according to the groups. The NIR(+) group was placed in a 1W / cm² solution. 2 Rats were irradiated with an 808nm NIR laser for 10 minutes, and the temperature change of the wound area was recorded every 30 seconds using an infrared imager. Subsequently, wound photographs were taken on days 0, 3, 7, 11, and 14 (day 0 being the modeling day), and changes in wound size were measured using ImageJ. The wound residue rate was calculated using the following formula:

[0095]

[0096] Where A0 is the initial wound area on day 0, An It is the wound area on day n of treatment (n = 3, 7, 11, 14).

[0097] On days 3 and 10, exudate was collected from the wound using swabs and further diluted with physiological saline. 100 μL of each dilution was then plated onto LB agar plates and incubated at 37°C for 18 h. Colony counts were determined using the plate count method.

[0098] On days 3 and 10 of the experiment, three rats from each group were sacrificed. Wounds and surrounding tissues were surgically removed, and all excised tissues were fixed in 10% PFA (paraformaldehyde) solution. After fixation, the tissues were dehydrated, embedded, and sectioned. The sections were then stained sequentially with hematoxylin and eosin (H&E), Masson's stain, and immunohistochemically (CD31) staining. The newly formed blood vessels stained with CD31 were quantitatively analyzed by measuring integrated optical density (IOD) to analyze the pathological morphological changes in the tissues surrounding the wound. Heart, liver, spleen, lung, and kidney organs were collected from rats, sectioned, and stained with H&E to observe the histological reactions of these organs and assess the in vivo biosafety of InNNi3 under NIR laser irradiation. The weight of rats was monitored from day 0 to 14 during the experiment to observe their growth and health status.

[0099] 3.3 Experimental Results

[0100] Figure 6 This invention illustrates and presents the results of an experiment using InNNi3 solution combined with NIR laser treatment of rat wounds. InNNi3 solution and 1 W / cm² were used in this invention. 2 A schematic diagram illustrating the synergistic effect of 808nm NIR laser irradiation on rats and its promotion of wound healing is shown below. Figure 6 As shown in (a). The photothermal imaging results are as follows. Figure 6 As shown in (b), compared with the control-NIR(+) group, the InNNi3-NIR(+) group showed a higher temperature at the wound site on the back of the rats, indicating that InNNi3 has a highly efficient photothermal conversion capacity in rats. After 10 min of 808 nm NIR irradiation, the local temperature of the wound in the InNNi3-treated group increased by 13.25 ± 0.45 °C, while the local temperature of the wound in the control group only increased by 2.83 ± 0.66 °C. Figure 6 (c)). In the InNNi3-NIR(+) group, this significant temperature increase disrupted the integrity of the MRSA cell membrane, leading to leakage of MRSA contents and thus rapid inactivation. This process not only effectively kills MRSA but also shortens the duration of the inflammatory response, thereby promoting wound healing.

[0101] The monitoring of wound healing progress between 0 and 14 days is as follows: Figure 7 As shown. Figure 7 (a) As shown, on day 3, suppuration and biofilm formation appeared in the wounds of rats in the control group, indicating that the MRSA infection model had been successfully established. In contrast, the suppuration in the wounds of rats in the InNNi3-NIR(-) group was significantly reduced compared to the control group, suggesting that treatment with InNNi3 solution alone can produce a mild antibacterial effect against MRSA. On day 3, no biofilm formation was observed in the wounds of rats in the InNNi3-NIR(+) group, and the wounds had already scabbed over, indicating a faster wound healing rate. Figure 7 (b) and Figure 7 (c) shows that on day 7, the wound healing rate of rats in the InNNi3-NIR(+) group reached 55.43±3.96%, while the wound healing rates of rats in the control-NIR(-), control-NIR(+), and InNNi3-NIR(+) groups were only 36.17±5.78%, 35.55±3.01%, and 43.56±5.74%, respectively. On day 14, the wound healing rate of rats in the InNNi3-NIR(+) group was as high as 93.71±2.63%, while the wound healing rates of rats in the control-NIR(-), control-NIR(+), and InNNi3-NIR(-) groups were only 86.29±2.36%, 84.79±2.38%, and 87.86±3.54%, respectively. Compared with the other groups, the InNNi3-NIR(+) group showed a significantly improved degree of wound healing in rats with infection. The results of plate smears and colony counts of wound exudate collected on days 3 and 7 are as follows: Figure 7 (d) and Figure 7 As shown in (e), the InNNi3-NIR(+) group exhibited an inhibition rate of approximately 72% on day 3, which increased to 99.9% on day 7. This indicates that InNNi3, after NIR laser irradiation, maintains stable and sustained antibacterial activity throughout the wound healing process. These results demonstrate the potential and reliability of InNNi3 as an antibacterial agent and show its significant efficacy in both anti-infection and accelerating wound healing.

[0102] H&E staining results of wound tissue as follows Figure 8 As shown. From Figure 8(a) As shown, by day 3, the wound tissues of rats in the control-NIR(-) group, control-NIR(+) group, and InNNi3-NIR(-) group all exhibited significant inflammatory cell infiltration. In contrast, the number of inflammatory cells in the wound tissue of rats in the InNNi3-NIR(+) group was relatively moderate. This suggests that MRSA infection may exacerbate the inflammatory response in the wound area, while InNNi3 synergistically with NIR laser irradiation in the InNNi3-NIR(+) group killed most of the bacteria at the wound site, helping to control infection and alleviate the inflammatory response. By day 10, the number of inflammatory cells in the wound tissue of rats in the InNNi3-NIR(+) group further decreased, indicating that InNNi3 treatment and NIR irradiation effectively reduced inflammation. The reduction in the inflammatory response created a more suitable microenvironment for wound healing, reduced tissue damage, and thus promoted wound healing. Fibroblasts play an important role in the proliferative phase of wound healing. In the wound tissue of rats in the InNNi3-NIR(+) group, fibroblast migration was significantly enhanced on day 10, promoting scar formation. Figure 8 As shown in (b), on day 10, the InNNi3-NIR(+) group exhibited thicker granulation tissue in its wound tissue compared to the control-NIR(-), control-NIR(+), and InNNi3-NIR(-) groups, indicating the best wound healing effect. Figure 8 As shown in (c), on day 10, the wound tissue of rats in the InNNi3-NIR(+) group had a narrower scar width, approximately 2.58 ± 0.05 mm, while the scar widths of the wound tissue of rats in the control-NIR(-), control-NIR(+), and InNNi3-NIR(-) groups were 4.88 ± 0.21 mm, 4.52 ± 0.17 mm, and 4.08 ± 0.03 mm, respectively. This further demonstrates that InNNi3 has a significant ability to fight infection and accelerate wound healing.

[0103] Masson staining and CD31 staining results of wound tissue are as follows: Figure 9 As shown. Figure 9 (a) and Figure 9 (b) It was found that there were significant differences in collagen deposition in the wound tissue among the groups. The InNNi3-NIR(+) group had the highest collagen deposition rate, at 60.52±5.54%. This rate was higher than that of the control-NIR(-) group, the control-NIR(+) group, and the InNNi3-NIR(-) group, which were 21.68±1.05%, 21.19±0.18%, and 32.54±2.33%, respectively. This again demonstrates that InNNi3 can enhance the organized deposition of collagen by effectively inactivating bacteria and reducing the inflammatory response, thus contributing to the formation of narrower and more mature scars. Figure 9 As shown in (d), the integrated optical density (IOD) of the wound tissue in the InNNi3-NIR(+) group on day 3 was 57471±1764, significantly higher than that in the control-NIR(-), control-NIR(+), and InNNi3-NIR(-) groups. The integrated optical densities of the control-NIR(-), control-NIR(+), and InNNi3-NIR(-) groups were 22829±705, 21547±119, and 37398±1959, respectively. By day 10, the angiogenic activity of the wound tissue in the InNNi3-NIR(+) group was significantly reduced. Figure 9 (c) and Figure 9 (e) This indicates that once a wound reaches a sufficient level of vascularization, intrinsic regulatory mechanisms inhibit angiogenesis, thereby preventing unnecessary angiogenesis. Masson staining and CD31 staining results of wound tissue show that InNNi3 can significantly accelerate wound healing by reducing the inflammatory response, stimulating granulation tissue formation, and promoting collagen deposition and early angiogenesis.

[0104] The H&E staining results of major organs (heart, liver, spleen, lung, and kidney) of rats in different treatment groups on day 3 and day 10 after treatment are shown below. Figure 10 As shown. From Figure 10 It can be seen that during the experiment, no abnormalities or pathological changes were observed in the organs and tissues of rats in all experimental groups on days 3 and 10, which fully demonstrates that InNNi3 has good safety in vivo.

[0105] The body weight of rats during the experimental period from 0 to 14 days is as follows: Figure 11 As shown. Figure 11 It was observed that, regardless of whether it was the experimental group or the control group, the weight of rats in each group showed a steady increase without any irregular weight fluctuations. This consistent weight gain trend further demonstrates the safety of InNNi3, indicating that InNNi3 has good biocompatibility with various organs, has the potential for clinical antibacterial and wound healing promotion applications, and will not have any negative impact on the health of the organism.

[0106] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. An application of InNNi3 in the preparation of antibacterial products.

2. The use according to claim 1, characterized in that, The antibacterial product is for external use; the antibacterial product is selected from at least one of drugs and health products.

3. The use according to claim 2, characterized in that, The antibacterial product is a drug.

4. The use according to claim 3, characterized in that, The drug forms an antibacterial pharmaceutical preparation; the pharmaceutical preparation contains InNNi3 and also contains pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that, The drug is a pharmaceutical preparation that forms an anti-biofilm.

6. The use according to claim 4, characterized in that, The content of InNNi3 in a unit of drug preparation is 0.5 mg or more.

7. The use according to claim 6, characterized in that, In the cytotoxicity experiment, when the concentration of InNNi3 in the cell culture medium was 0.5–2 mg / mL, the survival rate of human embryonic kidney cells and mouse embryonic fibroblasts was over 90% after 24 h of culture; in the hemolytic activity experiment, when the concentration of InNNi3 in 2% erythrocyte suspension was 0.5–2 mg / mL, the hemolysis rate of erythrocytes was less than 5%.

8. The use according to claim 6, characterized in that, In in vitro antibacterial experiments, when the concentration of InNNi3 in the bacterial solution was 0.5 mg / mL, and the optical power density was 1.0 W / cm², the antibacterial effect was achieved. 2 Under the condition of irradiation with a near-infrared laser with a wavelength of 808nm for 10 minutes, the antibacterial efficiency against bacteria or fungi after 18 hours of culture is over 99.9%.

9. The use according to claim 6, characterized in that, In in vivo antibacterial experiments, infected wounds were treated with an aqueous solution of InNNi3 at a concentration of 0.5 mg / mL, followed by an optical power density of 1.0 W / cm². 2 The infected wound was irradiated with a near-infrared laser with a wavelength of 808nm for 10 minutes, and the wound healing rate of the infected wound after 14 days was more than 93.71%; wherein the infected wound is a circular wound with a diameter of 9cm and full-thickness skin defect that has been infected with bacteria or fungi for 30 minutes.

10. The use according to any one of claims 1 to 9, characterized in that, The bacteria are selected from at least one of Escherichia coli, methicillin-resistant Staphylococcus aureus, and Candida albicans.

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