Copper-loaded photoresponse nano-material, preparation method and application of copper-loaded photoresponse nano-material in removal of periodontal biofilm

Abstract

The invention provides a method for removing a periodontal biofilm by using a copper-loaded photoresponse nano material. The method breaks through the single function limitation that a PDA-based material only sterilizes or only promotes repair in periodontal treatment, and the PDA-coated Cu NPs photoresponse nano material is prepared by adopting a reduction-deposition method. According to the method, the remarkable advantages of excellent biocompatibility and antioxidant activity of PDA and broad-spectrum antibacterial and bone regeneration promotion of copper are perfectly fused, and the problem that an existing PDA-based material lacks targeted removal of a biological membrane is solved. Experiments prove that the charge property of the PDA is reversed due to introduction of Cu, and negative charges are converted into positive charges. The PDA-coated Cu can be specifically combined with bacterial cells with negative charges on the surfaces through electrostatic interaction, so that efficient removal of bacteria is realized. The bottleneck of traditional periodontal treatment is broken through, and certain clinical transformation value is achieved.

Description

Technical Field

[0001] This invention focuses on the field of dental biomedical materials, specifically relating to the application of a copper-loaded photoresponsive nanomaterial for the efficient removal of periodontal biofilm under light irradiation. Technical Background

[0002] Periodontitis is the sixth most common disease worldwide, a non-infectious, chronic inflammatory disease. According to the 2022 World Health Organization (WHO) Global Oral Health Status Report, the global prevalence of severe periodontitis is approximately 19%. Its core pathogenic mechanism is the continuous stimulation of periodontal tissues by dental plaque biofilm, triggering an inflammatory response and leading to alveolar bone resorption. Clinically, periodontitis often causes tooth loosening and loss, and it is closely linked to the occurrence and development of systemic diseases such as diabetes, cardiovascular disease, rheumatoid arthritis, and chronic nephritis. Currently, clinical treatment mainly employs mechanical scaling and antibiotics, but due to the strong resistance of biofilms, the effectiveness of traditional treatments is very limited. Therefore, there is an urgent need to explore innovative antibacterial and anti-inflammatory strategies.

[0003] Copper, with its broad-spectrum antibacterial properties and significant advantages in promoting bone regeneration, has become an emerging strategy for biofilm removal. It exhibits broad-spectrum antibacterial activity against periodontal pathogens, such as *Porphyromonas gingivalis*, and can also promote bone regeneration by activating osteoblast differentiation and collagen synthesis, making it a promising alternative to photodynamic therapy (PDT) and polydopamine (PDA) for periodontal disease treatment. CN118593719A discloses a biomimetic fusion membrane encapsulating polydopamine nanomedicine for treating periodontitis. The core is polydopamine, and the outer shell is a fusion membrane composed of *Porphyromonas gingivalis* outer membrane vesicles and ginger exosome-like nanoparticles. This membrane can be used for ROS removal and helper T cell regulation in the periodontal microenvironment, thereby achieving periodontitis treatment. However, existing PDA-based materials have limitations in periodontal treatment due to their single-function limitations, only killing bacteria or only promoting repair, and lack the synergistic effect of highly efficient targeted removal of biofilms and bone regeneration. Summary of the Invention

[0004] This invention proposes the construction of Cu-loaded PDA-based photoresponsive nanomaterials (Cu-PDA NPs), opening up new avenues for more efficient periodontal disease treatment. Cu is an important component of biological proteins and enzymes. Compared with other antibacterial metals such as Ag / Au, Cu²⁺ achieves an optimal balance between antibacterial efficacy and photothermal synergy. It also exhibits low-cost cytotoxicity. However, Cu²⁺ has a relatively long bactericidal process and exhibits certain toxicity at high concentrations. By binding with PDA, the release of Cu²⁺ can be controlled, achieving a mild and long-lasting antibacterial effect. Simultaneously, the release of Cu²⁺ synergistically promotes periodontal bone regeneration. This research not only breaks through the bottlenecks of traditional periodontal treatment but also provides new material science insights for the repair of infected bone defects, possessing significant clinical translational value.

[0005] The development of Cu-loaded PDA-based photoresponsive nanomaterials (Cu-PDA NPs) encompasses three key stages: the design and synthesis of Cu-PDA NPs; the characterization and exploration of the properties of Cu-PDA NPs; and the testing of the periodontal bacterial inhibition rate of Cu-PDA NPs. The aim is to improve the efficiency of periodontal biofilm removal through innovative technological means.

[0006] This invention aims to overcome two inherent defects of current PDA nanoparticles: easy aggregation and insufficient non-radiative conversion and light absorption in the NIR region. The nanomaterial designed in this invention can efficiently remove stubborn biofilms, and this material possesses a wide range of visible light absorption and good antibacterial properties. Cu-PDA NPs can specifically bind to bacterial cells through electrostatic interactions, thereby achieving efficient bacterial removal. This invention is applied to the treatment of periodontal disease, and revolves around three core steps: the design and synthesis of Cu-PDA NPs; the characterization of Cu-PDA NPs; and the testing of the inhibition rate of Cu-PDA NPs against periodontal bacteria.

[0007] This invention provides a method for removing periodontal biofilm using copper-loaded photoresponsive nanomaterials, comprising the following steps:

[0008] To contain Cu 2+ A suspension of polydopamine nanoparticles (PDA NPs) was added dropwise to an ion dispersion, and ascorbic acid solution was added dropwise to prepare copper nanoparticles by reduction. After sufficient reaction, Cu-loaded PDA-based photoresponsive nanomaterials (Cu-PDA NPs) were obtained.

[0009] Specifically, it includes the following steps:

[0010] Step 1: Mix the ammonia solution (1 mL) with anhydrous ethanol (20 mL) and deionized water (45 mL).

[0011] Step 2: Add dopamine hydrochloride (250 mg) dissolved in deionized water (5 mL) to the above mixed solution and stir gently at 37 °C for 24 hours.

[0012] Step 3: Separate PDA NPs by high-speed centrifugation (12,000 rpm, 8 min) and wash four times with deionized water.

[0013] Step 4: Dilute the synthesized PDA NPs to 5 mg / mL for subsequent use.

[0014] Step 5: Add 0.25 g of copper chloride dihydrate to a 250 ml round-bottom flask containing 20 ml of deionized water, and heat the copper chloride solution to 85 ℃.

[0015] Step 6: While stirring continuously (300 rpm), simultaneously add ascorbic acid solution (0.625 M) and PDA NPs suspension (10 mg / mL) and react for 60 minutes.

[0016] Step 7: Centrifuge at 12,000 rpm for 8 minutes to obtain PDA@Cu NPs.

[0017] Step 8: Wash the obtained PDA @Cu NPs four times with deionized water, dilute to 5 mg / mL, and store in a refrigerator at 4 ℃ for later use.

[0018] The present invention also provides a Cu-loaded PDA-based photoresponsive nanomaterial obtained by the above method.

[0019] The PDA-based photoresponsive nanomaterials with positively charged Cu on their surface have wide applications in targeted bacterial removal and bone regeneration, especially in the treatment, rehabilitation, and rehabilitation of periodontitis.

[0020] The beneficial effects achieved by this invention are as follows:

[0021] This invention utilizes a reduction-deposition method to prepare PDA@Cu NPs photoresponsive nanomaterials. These materials not only possess strong visible light absorption capabilities but also the ability to target and eliminate bacteria. Attached Figure Description

[0022] Figure 1 It is a TEM image of the PDA;

[0023] Figure 2 This is a TEM image of PDA@Cu;

[0024] Figure 3 These are the Zeta potentials of PDA and PDA@Cu;

[0025] Figure 4 This is the UV-Vis absorption spectrum of PDA@Cu NPs;

[0026] Figure 5 The inhibition rate of F. nucleatum after treatment with 200 μg / ml PDA@Cu and irradiation for different durations. Detailed Implementation

[0027] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the detailed content of the present invention will be described below in conjunction with specific embodiments. It should be noted that the following embodiments are only for explaining the present invention and not for limiting its scope of protection; any equivalent modifications or substitutions based on the concept of the present invention should be covered by the claims of the present invention.

[0028] Example 1

[0029] PDA@Cu photoresponsive nanomaterials were prepared using a reduction-deposition method. 0.25 g of copper chloride dihydrate was added to a 250 mL round-bottom flask containing 20 mL of deionized water. While continuously stirring (300 rpm), ascorbic acid solution and a PDA NPs suspension were added dropwise, and the reaction proceeded for 60 minutes. The mixture was then centrifuged at 12,000 rpm for 8 minutes to obtain PDA@Cu NPs. The morphology of the prepared PDA and PDA@Cu was characterized using transmission electron microscopy.

[0030] The following experiments were used to verify the effectiveness of the invention:

[0031] Reference Figure 1 , Figure 1 This is a TEM image of the PDA. The image shows that the PDA exhibits a uniform spherical structure, and its elemental mapping image shows that C, O, and N elements are evenly distributed.

[0032] Figure 2 The images show TEM images of PDA@Cu. Transmission electron microscopy reveals copper nanoparticles (CuNPs) dispersed on the PDA@Cu surface. The Cu-modified PDA nanoparticles maintain a relatively uniform spherical structure with an average diameter of approximately 220 nm. Furthermore, elemental mapping images confirm the uniform distribution of Cu, O, N, and C elements, indicating the successful fabrication of PDA@Cu photoresponsive nanomaterials.

[0033] Figure 3 The surface charge properties of PDA and PDA@Cu were characterized and evaluated based on the Zeta potential of PDA and PDA@Cu, and the results are as follows: Figure 3As shown, the Zeta potential of PDA is -29.7 mV, while that of PDA@Cu is +10.57 mV. This phenomenon originates from the consumption of some negatively charged groups in the PDA molecule during the reduction of Cu²⁺ to Cu, leading to a significant increase in the potential of PDA@Cu. Simultaneously, the introduction of Cu reverses the charge property of PDA, changing it from negative to positive. According to basic principles of physics, like charges repel each other while unlike charges attract each other. Therefore, PDA@Cu can specifically bind to negatively charged bacterial cells through electrostatic interactions, thereby achieving highly efficient bacterial removal.

[0034] Figure 4 The UV-Vis absorption spectrum of PDA@Cu NPs is shown. The absorption intensity of visible light by a material is a crucial factor affecting the efficacy of photodynamic therapy for periodontitis. Stronger light absorption means the material can capture more photons, thereby exciting the generation of more reactive oxygen species and other substances. These substances can effectively disrupt the periodontal biofilm structure, kill pathogenic bacteria, and enhance the inhibitory effect on periodontal inflammation, thus improving the overall efficacy of photodynamic therapy. The light absorption properties of PDA and PDA@Cu were studied using UV-Vis absorption spectroscopy. Both PDA and PDA@Cu have a wide visible light absorption range, but PDA@Cu has a stronger visible light absorption capacity than PDA.

[0035] Figure 5 To investigate the inhibition rate of *F. nucleatum* after treatment with 200 μg / ml PDA@Cu for different light exposure times, we conducted related experiments. The results showed that the inhibition rate of *F. nucleatum* by PDA@Cu increased continuously with prolonged light exposure.

[0036] In the initial stage of illumination, the inhibition rate steadily increased over time, indicating that illumination can enhance the inhibitory effect of PDA@Cu on *F. nucleatum* to some extent. When the illumination time reached 4 minutes, the inhibition rate significantly increased to 91%, demonstrating a strong inhibitory effect. However, when the illumination time was further extended to 5 minutes, the inhibition rate did not change and remained at the 91% level. This indicates that under the experimental conditions, when the illumination time reached 4 minutes, the inhibitory effect of PDA@Cu on *F. nucleatum* reached saturation, and further increasing the illumination time could not improve its inhibition rate.

[0037] The Cu-loaded PDA-based photoresponsive nanomaterials constructed in this invention perfectly combine the excellent biocompatibility and antioxidant activity of polydopamine (PDA) with the significant advantages of copper's broad-spectrum antibacterial properties and bone regeneration promotion. PDA doped with Cu nanoparticles exhibits the strongest visible light absorption capacity, making it an excellent photosensitizer material. This result lays a solid foundation for subsequent research on PDA@Cu nanomaterials targeting and eliminating bacteria to promote bone regeneration through photodynamic therapy.

Claims

1. A method for preparing copper-loaded photoresponsive nanomaterials, characterized in that, Includes the following steps: To contain Cu 2+ A suspension of polydopamine nanoparticles was added dropwise to an ion dispersion, followed by the addition of ascorbic acid solution. Copper nanoparticles were prepared by liquid-phase reduction and fully reacted to obtain copper-loaded photoresponsive nanomaterials PDA@Cu NPs.

2. The method according to claim 1, characterized in that, Includes the following steps: 20 ml of a copper chloride solution containing 12.5 g / L copper chloride dihydrate was heated to 85 °C. While stirring continuously at 300 rpm, 5 ml of 0.625 M ascorbic acid solution and 5 ml of 10 mg / mL PDA NPs suspension were added dropwise and reacted for 60 minutes. Copper-loaded photoresponsive nanomaterials PDA@Cu NPs were prepared by reduction-deposition method.

3. The method according to claim 1, characterized in that, Includes the following steps: 1) Mix 1 mL of ammonia solution with 20 mL of anhydrous ethanol and 45 mL of deionized water; 2) Dissolve 250 mg of dopamine hydrochloride in 5 mL of deionized water, add it to the mixed solution in step 1), and stir gently at 37 °C for 24 hours; 3) PDA NPs were separated by high-speed centrifugation at 12000 rpm for 8 min and washed four times with deionized water; 4) Dilute the PDA NPs synthesized in step 3) to 5 mg / mL for subsequent use; 5) Add 0.25g of copper chloride dihydrate to 20ml of deionized water, place the solution in a 250ml round-bottom flask, and heat the copper chloride solution to 85℃; 6) While stirring continuously at 300 rpm, simultaneously add 5 ml of 0.625 M ascorbic acid solution and 5 ml of 10 mg / mL PDA NPs suspension to the copper chloride solution in step 5), and react for 60 minutes; 7) Centrifuge at 12000 rpm for 8 minutes to obtain PDA@Cu NPs; 8) Wash the obtained PDA @Cu NPs four times with deionized water, dilute to 5 mg / mL, and store in a refrigerator at 4°C for later use.

4. A copper-loaded photoresponsive nanomaterial obtained by the method according to any one of claims 1-3.

5. The material according to claim 4, characterized in that, The material has a spherical structure with a diameter of 220 nm, and the Cu, O, N and C elements are uniformly distributed on the surface of the material.

6. The use of a copper-loaded photoresponsive nanomaterial as described in any one of claims 4 or 5 in the preparation of antibacterial and / or antibacterial materials.

7. The application according to claim 6, characterized in that, Compared to polydopamine, the material exhibits a significantly higher Zeta potential. This is manifested in the process of Cu²⁺ being reduced to Cu, during which some negatively charged groups in the PDA molecule are consumed, leading to a significant increase in the potential of PDA@Cu NPs. The charge properties are reversed, changing from negative to positive, and through electrostatic interaction, it specifically binds to bacterial cells, achieving highly efficient removal of bacteria.

8. The application according to claim 6, characterized in that, Compared to polydopamine, the material exhibits significantly increased absorption intensity of visible light, demonstrating stronger visible light absorption; the material also has a stronger photon-trapping ability, which can stimulate the generation of more reactive oxygen species, thereby enhancing the overall effect of photodynamic therapy.

9. The application according to claim 6, characterized in that, Under illumination, it exhibits a high inhibition rate against F. nucleatum for a short period of time, with an inhibition rate of up to 91% when the illumination time reaches 4 minutes.

10. The application according to claim 6, characterized in that, Includes the following steps: The material is uniformly coated onto the area to be sterilized and / or inhibited, and the coated surface is exposed to light for 5 minutes to complete the sterilization and / or inhibition treatment of the area to be treated.

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