Multifunctional nano-antibacterial material and preparation method thereof

Abstract

This invention discloses a multifunctional antibacterial nanomaterial and its preparation method, relating to the field of antibacterial materials technology. It includes polydopamine nanoparticles for photothermal conversion and lactoferrin for competitively binding iron ions with bacteria. The polydopamine nanoparticles and lactoferrin are connected by molecular arms. This invention couples lactoferrin to polydopamine nanoparticles to obtain a multifunctional antibacterial nanomaterial. In this multifunctional antibacterial nanomaterial, the polydopamine nanoparticles exhibit a photothermal effect, generating high temperatures under near-infrared light irradiation. Lactoferrin, on the other hand, can deprive bacteria of the iron element required for growth, forming a synergistic antibacterial effect with photothermal stimulation. This achieves the goal of reducing the temperature required for photothermal therapy and mitigating the side effects of photothermal antibacterial treatment, helping to achieve efficient antibacterial activity under lower-intensity NIR irradiation and avoiding damage to surrounding healthy tissues.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, specifically a multifunctional nano-antibacterial material and its preparation method. Background Technology

[0002] Bacterial infectious diseases are among the most common diseases. The widespread use of antibiotics has significantly improved the treatment of bacterial infectious diseases and reduced mortality, but it has also led to antibiotic resistance in bacteria.

[0003] To combat drug-resistant bacteria, researchers have developed various novel antibacterial therapies. Among them, photothermal therapy (PTT) is considered the most promising new antibacterial strategy due to its high antibacterial efficacy, controllability, and non-invasiveness. PTT utilizes a photothermal converter to convert near-infrared radiation (NIR) light energy into heat energy, raising the local temperature at the infection site. The high temperature then disrupts the integrity of the bacterial cell membrane, causing the cellular contents to leak out, and simultaneously inducing protein and enzyme denaturation and even DNA damage, thereby killing the bacteria. Studies have found that using PTT alone requires maintaining a high temperature at the infection site for an extended period to completely eliminate bacteria. Under such conditions, surrounding healthy tissue is easily burned, resulting in significant adverse reactions. To overcome the technical bottlenecks in the antibacterial application of PTT, multimodal synergistic antibacterial therapy based on photothermal therapy has become a research hotspot in recent years.

[0004] Iron is an essential nutrient for bacterial growth and metabolism, playing an irreplaceable role in biochemical reactions such as energy metabolism, gene expression, and protein synthesis. Iron starvation therapy refers to a method of inhibiting bacterial proliferation by interfering with or restricting the uptake of iron from the surrounding environment by pathogenic bacteria. Studies have found that the overall metabolic level of bacteria in an iron-starved state decreases significantly, while their sensitivity to various adverse physical and chemical factors (such as high temperature) increases markedly. Therefore, depriving bacteria of the Fe3+ required for growth, thus putting them in an iron-starved state, combined with photothermal antibacterial therapy, holds promise for achieving synergistic antibacterial effects through iron starvation and photothermal therapy.

[0005] Lactoferrin (Lf) is an iron-binding protein widely distributed in human and mammalian milk and other tissues and secretions, possessing a broad-spectrum antibacterial effect. Studies have found that Lf has a strong chelating ability for Fe3+, competitively depriving bacteria of the iron they need for growth, thus preventing bacterial proliferation due to iron deficiency.

[0006] Polydopamine (PDA) nanoparticles are nanoscale particles polymerized from dopamine molecules. These nanoparticles exhibit excellent photothermal conversion performance, with a conversion efficiency of up to 40%. More importantly, PDA nanoparticles not only have good biocompatibility, but their surface is also rich in amino, carboxyl, and catechol groups, making them easy to modify.

[0007] To address the aforementioned issues, we provide a multifunctional nano-antibacterial material and its preparation method to solve the problems mentioned above. Summary of the Invention

[0008] The purpose of this invention is to provide a multifunctional nano-antibacterial material and its preparation method to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A multifunctional nano-antibacterial material comprising polydopamine nanoparticles for photothermal conversion and lactoferrin for competitively binding iron ions with bacteria, wherein the polydopamine nanoparticles and the lactoferrin are connected by molecular arms.

[0010] As a further aspect of the present invention: the lactoferrin is iron-unsaturated lactoferrin with an iron saturation rate of less than 50%.

[0011] As a further embodiment of the present invention: the molecular arm is any one or a combination of at least two of malondialdehyde, succinal, glutaraldehyde, glyoxal, and terminal aldehyde-functionalized polyethylene glycol.

[0012] Another objective of this invention is to provide a method for preparing a multifunctional nano-antibacterial material, specifically comprising the following steps: Step 1: Preparation of polydopamine nanoparticles: Dissolve dopamine hydrochloride in 2 mL of water, then inject this solution into a mixture of 20 mL ethanol, 45 mL water, and 0.5-2.5 mL ammonia (25-28%). Stir the mixture at room temperature for 12-24 hours, then add 3 times the volume of ethanol and centrifuge. Wash the product 3 times with water. Step 2: Coupling lactoferrin to polydopamine nanoparticles: Add molecular arms to a 1-15 mg / mL polydopamine nanoparticle solution and stir at room temperature for 24 h. After the reaction is complete, centrifuge at 8000 rpm for 10 min, and wash the precipitate with water 3-5 times. Then redisperse the polydopamine nanoparticles in water, add lactoferrin, and continue the reaction for 24 h. Step 3: Purification of the product: After the reaction is complete, centrifuge at 8000 rpm for 10 min, wash the precipitate with water 3-5 times to remove uncoupled lactoferrin. After lyophilization, store the product at 4°C protected from light.

[0013] As a further embodiment of the present invention: the amount of dopamine added in step one is 50-300 mg.

[0014] As a further embodiment of the present invention: the concentration of the molecular arm in step two is 0.5%-5% (W / V), and the concentration range of the lactoferrin is 0.5-4 mg / mL.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention couples lactoferrin to polydopamine nanoparticles to obtain a multifunctional antibacterial nanomaterial. In this multifunctional antibacterial nanomaterial, the polydopamine nanoparticles have a photothermal effect and can generate high temperatures under near-infrared light irradiation; while lactoferrin can deprive bacteria of the iron element required for growth, forming a synergistic antibacterial effect with photothermal irradiation. This achieves the purpose of reducing the temperature required for photothermal therapy and reducing the side effects of photothermal antibacterial treatment, which helps to achieve efficient antibacterial effect under lower intensity NIR irradiation and avoids damage to surrounding healthy tissues. Attached Figure Description

[0016] Figure 1 This invention discloses a preparation process of a multifunctional antibacterial nanomaterial and a schematic diagram of its iron starvation-photothermal synergistic antibacterial action. Figure 2 Scanning electron microscope images of multifunctional antibacterial nanomaterials; Figure 3 Evaluation of the photothermal conversion effect of multifunctional antibacterial nanomaterials; Figure 4 Evaluation of the iron ion adsorption effect of multifunctional antibacterial nanomaterials; Figure 5 Evaluation of the bactericidal effect of multifunctional antibacterial nanomaterials on Staphylococcus aureus (A) and Escherichia coli (B) airborne bacteria; Figure 6 Evaluation of blood compatibility of multifunctional antibacterial nanomaterials. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0018] Preparation of Lf-PDA: Step 1: Preparation of PDA nanoparticles: Dissolve 200 mg of dopamine hydrochloride in 2 mL of water, then inject this solution into a mixture of 20 mL of ethanol, 45 mL of water, and 2 mL of ammonia (25-28%). Stir the mixture at room temperature for 24 hours. After the reaction is complete, add 3 times the volume of ethanol, centrifuge at 8000 rpm for 10 min, and wash the precipitate three times with pure water.

[0019] Step 2: Coupling Lf to PDA nanoparticles: Glutaraldehyde solution was added to a 3 mg / mL polydopamine nanoparticle solution until the final concentration reached 2%, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 min, and the precipitate was washed with water 3-5 times. The product was then redispersed in water, and lactoferrin was added until the final concentration reached approximately 1.0 mg / mL. The reaction was continued with stirring for 24 h.

[0020] Step 3: Purification of the product: After the reaction is complete, centrifuge at 8000 rpm for 10 min, wash the precipitate with water 3-5 times to remove uncoupled lactoferrin. After lyophilization, store the product at 4°C protected from light.

[0021] like Figures 1-2 As shown, the prepared Lf-PDA nanoparticles exhibit a relatively uniform spherical shape with an average particle size of approximately 93.4 nm. Example 2

[0022] Evaluation of Lf-PDA photothermal conversion performance: Lf-PDA was prepared into a 200 μg / mL solution using PBS, and then... 2 The dispersion was irradiated with NIR, and the system temperature was monitored using a thermocouple thermometer. The temperature value was recorded every 20 seconds until the system temperature stopped increasing. The results are as follows: Figure 3 As shown, under NIR irradiation, the temperature of the PBS solution at a concentration of 0 Lf-PDA showed no significant change, increasing by only 1.1°C after 500 seconds of irradiation. In contrast, Lf-PDA nanoparticles increased the solution temperature by approximately 33°C within 500 seconds. This demonstrates that Lf-PDA exhibits a photothermal conversion effect, capable of converting NIR light energy into heat, and can be used as a photothermal conversion agent for photothermal antibacterial purposes. Example 3

[0023] Evaluation of Lf-PDA iron ion adsorption effect: Lf-PDA or PDA was mixed with iron ion solutions of different concentrations and stirred for adsorption at room temperature for 4-6 h. After adsorption was complete, the mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was collected to determine the concentration of remaining iron ions using a graphite furnace atomic absorption spectrophotometer. The amount of iron ions adsorbed by the material was then calculated.

[0024] The results are as follows Figure 4 As shown, Lf-PDA exhibits a significantly higher adsorption capacity for iron ions than PDA. This is due to the high affinity of Lf for iron ions, enabling Lf-PDA to adsorb more iron ions. The iron ion adsorption experiment results indicate that Lf-PDA can competitively bind iron ions with bacteria, suggesting its potential to synergistic antibacterial effect with photothermal therapy. Example 4

[0025] Evaluation of the bactericidal effect of Lf-PDA on Staphylococcus aureus: Set OD=1.0 (approximately 1×10⁻⁶) 9 Staphylococcus aureus (cfu / mL) was diluted to 2×10⁻⁶ CFU / mL with TSB medium. 5 CFU / mL, take 0.5 mL of bacterial culture and mix it with 0.5 mL of Lf, PDA, or Lf-PDA. Incubate the mixture at 4°C for 4 h. After 4 h, use 0.5 W / cm² water. 2 The bacteria were irradiated with NIR for 5 min. After irradiation, the number of surviving bacteria was determined by plate counting, and the survival rate of Staphylococcus aureus in each group was calculated.

[0026] The result is Figure 5 It was found that, without NIR irradiation, the bacterial survival rates of the Lf, PDA, and Lf-PDA groups were not significantly different from those of the PBS group, indicating that Lf, PDA, and Lf-PDA had no significant killing effect on Staphylococcus aureus without NIR irradiation. However, with NIR irradiation, the bacterial survival rates of the PDA and Lf-PDA groups were significantly different from those of the PBS group (P<0.05). This is because PDA has a photothermal conversion effect, and the high temperature generated under NIR irradiation leads to bacterial death. Comparing the bacterial survival rates of the PDA and Lf-PDA groups, it was found that the bacterial survival rate of the Lf-PDA group was significantly lower than that of the PDA group. This is because in the PDA group, the bacteria's iron uptake was not affected, and the bacterial nutrient supply was relatively sufficient, resulting in a strong resistance to high temperature; while in the Lf-PDA group, Lf can compete with bacteria for iron ions, leading to iron deficiency and malnutrition. This nutrient deficiency reduces the bacteria's resistance to high temperature, thus killing more bacteria under the same NIR irradiation intensity. Example 5

[0027] Evaluation of the bactericidal effect of Lf-PDA on Escherichia coli: Set OD=1.0 (approximately 1×10⁻⁶) 9 E. coli (cfu / mL) was diluted to 2×10⁻⁶ in TSB medium. 5 For cfu / mL, 0.5 mL of bacterial suspension was mixed with 0.5 mL of Lf, PDA, or Lf-PDA, and the mixture was incubated at 4℃ for 4 h. After 4 h, the mixture was irradiated with NIR at 0.5 W / cm² for 5 min. After irradiation, the number of surviving bacteria was determined by plate counting, and the survival rate of E. coli in each group was calculated.

[0028] The result is Figure 5 It was found that without NIR irradiation, the bacterial survival rates of the Lf, PDA, and Lf-PDA groups were not significantly different from those of the PBS group, indicating that Lf, PDA, and Lf-PDA had no significant killing effect on E. coli without NIR irradiation. However, with NIR irradiation, the bacterial survival rates of the PDA and Lf-PDA groups were significantly different from those of the PBS group (P<0.05). This is because PDA has a photothermal conversion effect, and the high temperature generated under NIR irradiation leads to bacterial death. Comparing the bacterial survival rates of the PDA and Lf-PDA groups, it was found that the bacterial survival rate of the Lf-PDA group was significantly lower than that of the PDA group. This is because in the PDA group, the bacteria's iron uptake was not affected, and the bacterial nutrient supply was relatively sufficient, resulting in strong resistance to high temperature; while in the Lf-PDA group, Lf can compete with bacteria for iron ions, leading to iron deficiency and malnutrition. This nutrient deficiency reduces the bacteria's resistance to high temperature, thus killing more bacteria under the same NIR irradiation intensity. Example 6

[0029] Lf-PDA blood compatibility evaluation: Red blood cells were diluted with PBS and mixed with different concentrations of Lf-PDA, and the mixtures were incubated at 37 °C for 4 h. The mixtures were then centrifuged at 12000 rpm for 5 min at 4 °C. 300 μL of the supernatant was added to a 96-well plate, and the absorbance of the supernatant at 492 nm was measured using a microplate reader to calculate the hemolysis rate. Sterile deionized water was used as a positive control, and PBS as a negative control.

[0030] The results are as follows Figure 6 It can be seen that Lf-PDA has good blood compatibility; even at an immediate concentration of 400 μg / mL, the efficiency of hemolysis of erythrocytes remains below 5%. Comparison of the hemolysis rates of Lf-PDA and PDA reveals no significant difference in their efficiency of inducing hemolysis at the same concentration (P>0.05), indicating that modifying PDA with Lf to form a multifunctional photothermal antibacterial material does not lead to an increase in the hemolytic effect.

[0031] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Although this specification describes embodiments, not every embodiment contains only one technical solution. This method of description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A multifunctional nano-antibacterial material, characterized in that: The antibacterial material is composed of polydopamine nanoparticles for photothermal conversion and lactoferrin for competitive binding of iron ions with bacteria, wherein the polydopamine nanoparticles and the lactoferrin are connected by molecular arms. The lactoferrin in question is iron-unsaturated lactoferrin with an iron saturation rate of less than 50%. The molecular arm is any one or a combination of at least two of malondialdehyde, succinal, glutaraldehyde, glyoxal, and terminal aldehyde-functionalized polyethylene glycol.

2. The method according to claim 1, wherein, Specifically, the steps include the following: Step 1: Preparation of polydopamine nanoparticles: Dissolve dopamine hydrochloride in 2 mL of water, then inject the solution into a mixture of 20 mL of ethanol, 45 mL of water and 0.5-2.5 mL of ammonia (25-28%). Stir the mixture at room temperature for 12-24 hours, then add 3 times the volume of ethanol and centrifuge. Wash the product with water 3 times. Step 2: Couple lactoferrin to polydopamine nanoparticles: Add molecular arms to a 1-15 mg / mL polydopamine nanoparticle solution and stir at room temperature for 24 h; after the reaction is complete, centrifuge at 8000 rpm for 10 min, wash the precipitate with water 3-5 times, then redisperse the polydopamine nanoparticles in water, add lactoferrin, and continue the reaction for 24 h. Step 3: Purify the product: After the reaction is complete, centrifuge at 8000 rpm for 10 min, wash the precipitate with water 3-5 times to remove uncoupled lactoferrin, and store the product at 4℃ protected from light after lyophilization.

3. The method for preparing a multifunctional nano-antibacterial material according to claim 2, characterized in that, The amount of dopamine added in step one is 50-300 mg.

4. The method for preparing a multifunctional nano-antibacterial material according to claim 3, characterized in that, In step two, the concentration of the molecular arm is 0.5%-5% W / V, and the concentration range of lactoferrin is 0.5-4 mg / mL.

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