Highly efficient antibacterial zinc oxide nanoparticles with bone targeting property, preparation method and application thereof

Abstract

This invention belongs to the field of biomedical materials technology and discloses a highly efficient antibacterial zinc oxide nanoparticle with bone targeting capability. The nanoparticle comprises zinc oxide nanoparticles with active sites and a bone-targeting material grafted onto the surface of the zinc oxide nanoparticles via the active sites. The active site is an amino group; the bone-targeting material is one of aspartic oligopeptide and glutamic acid oligopeptide. This invention also discloses the preparation method and application of the aforementioned highly efficient antibacterial zinc oxide nanoparticle with bone targeting capability. The highly efficient antibacterial zinc oxide nanoparticle with bone targeting capability provided by this invention can be directed to the site of bone infection through the targeting ability of the bone-targeting material for hydroxyapatite in bone tissue, and then exert the broad-spectrum antibacterial ability of the zinc oxide nanoparticles to kill bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a highly efficient antibacterial zinc oxide nanoparticle with bone-targeting properties, its preparation method, and its application. Background Technology

[0002] Bacterial infections in bone tissue are acute or chronic inflammations caused by microbial infection. Osteomyelitis is a typical example of orthopedic infection. It is an inflammatory process often accompanied by bone destruction caused by purulent microbial infection. It is mostly caused by the local spread of infection after trauma, orthopedic surgery or joint replacement surgery. It can be limited to a certain part of the bone, or it can involve multiple parts such as bone marrow, cortex, periosteum and surrounding soft tissues. It can lead to severe pain in bones and joints and degenerative changes in bone.

[0003] The main pathogens causing osteomyelitis include Gram-negative bacteria such as *Pseudomonas aeruginosa* and *Escherichia coli*, and Gram-positive bacteria such as *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Enterococcus*, and *Streptococcus*. *Staphylococcus aureus* is the most common pathogen in osteomyelitis; it can destroy bone tissue by inducing osteoblast apoptosis, activating osteoclast formation, and secreting toxins. The routine clinical treatment of osteomyelitis involves long-term, high-dose antibiotic therapy combined with multiple surgical debridements. Since the discovery of penicillin in 1928, antibiotics have been widely used in the field of antibacterial treatment, effectively treating various infectious diseases. However, the overuse of antibiotics has led to severe antibiotic resistance in bacteria. Therefore, the development of effective and safe antibacterial drugs for the treatment of osteomyelitis has become an urgent need.

[0004] Zinc oxide (ZnO) nanoparticles (NPs) have been shown to possess potent antibacterial activity. Composed of metal ions, they can be activated in biocides. While exhibiting relatively low stability under high temperature / pressure, they demonstrate good robustness compared to organic antibacterial agents. ZnO nanoparticles alter the composition of bacterial cell membranes, causing distortion and resulting in the loss of intracellular substances, ultimately leading to bacterial cell death. Zinc oxide nanoparticles possess broad-spectrum antibacterial activity, including against both Gram-negative and Gram-positive bacterial strains. Their significant ability to inhibit bacterial growth and proliferation has attracted considerable interest.

[0005] In recent years, significant progress has been made in the development and application of bone-targeting biomaterials. Nanocarriers, through targeted drug delivery strategies, offer a promising approach, providing more effective treatments for bone-related diseases, improving therapeutic outcomes, and reducing side effects. In bone, active targeted drugs utilize specific interactions with osteoids to enhance selective accumulation at the target site, thereby improving disease treatment and reducing side effects on healthy tissue. Developed bone-targeting groups are a class of small molecules with high affinity for bone tissue, such as bisphosphonate systems, oligopeptide systems, tetracycline drugs, and aptamer systems.

[0006] Therefore, the main technical problem to be solved by this invention is how to combine ZnO nanoparticles with bone-targeting biomaterials to develop zinc oxide nanoparticles with bone targeting. Summary of the Invention

[0007] The purpose of this invention is to address the technical problems existing in the prior art by providing a highly efficient antibacterial zinc oxide nanoparticle with bone targeting and its preparation method. By modifying the surface of the zinc oxide nanoparticle and grafting bone-targeting materials, the targeted antibacterial ability of the nano zinc oxide is improved.

[0008] The present invention also provides the use of the above-mentioned bone-targeting, highly efficient antibacterial zinc oxide nanoparticles for the preparation of bone tissue therapeutic drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] This invention provides a highly efficient antibacterial zinc oxide nanoparticle with bone targeting, comprising zinc oxide nanoparticles with active sites and bone targeting materials grafted onto the surface of the zinc oxide nanoparticles via the active sites.

[0011] The aforementioned bone-targeting, highly efficient antibacterial zinc oxide nanoparticles have an active site of amino groups; the bone-targeting material is one of aspartic oligopeptides, glutamic acid oligopeptides, etc. Preferably, the bone-targeting material is aspartic hexapeptide; the aspartic hexapeptide group has multiple carboxyl groups; the zinc oxide nanoparticles with active sites bind to the terminal carboxyl groups of the aspartic hexapeptide (Asp6) via amide bonds, which can ensure the stability of the binding between the two materials and retain the bone-targeting performance of the material.

[0012] This invention also provides a method for preparing the above-mentioned bone-targeting, highly efficient antibacterial zinc oxide nanoparticles, which includes the following steps:

[0013] (1) Zinc oxide nanoparticles were modified by surface amination using silane coupling agent to obtain surface-modified zinc oxide nanoparticles.

[0014] (2) Preparation of bone-targeting zinc oxide nanoparticles, specifically:

[0015] Surface-modified zinc oxide nanoparticles were added to a bone-targeting material buffer solution with a pH of 5-5.5, and the pH of the resulting reaction system was adjusted to 5.5-6.5. The mixture was then stirred for 4-5 hours, and after filtration, washing, and drying, highly efficient antibacterial zinc oxide nanoparticles with bone-targeting properties were obtained. The mass ratio of the surface-modified zinc oxide nanoparticles to the bone-targeting material was 10:1.

[0016] The purpose of step (1) above is to modify the surface of zinc oxide nanoparticles by grafting highly reactive amino groups. The silane coupling agent is one of γ-aminopropyltriethoxysilane (KH550) or γ-methacryloyloxypropyltrimethoxysilane (KH570). Silane coupling agent surface modification of zinc oxide nanoparticles consumes the hydroxyl groups on the surface of the nanoparticles, effectively improving the tendency of the nanoparticles to aggregate. Simultaneously, the surface of the silane-modified zinc oxide nanoparticles is rich in highly reactive amino groups, which is beneficial for multifunctional modification of the nanoparticles.

[0017] In the specific implementation method, step (1) includes the following sub-steps:

[0018] (11) Add the silane coupling agent to the uniformly dispersed zinc oxide nanoparticle dispersion and sonicate for 20-40 min; the silane coupling agent is 20%-22% of the mass of the zinc oxide nanoparticles.

[0019] (12) The ultrasonically treated dispersion was centrifuged, washed and dried multiple times to obtain surface-modified zinc oxide nanoparticles.

[0020] In step (11) above, zinc oxide nanoparticles are added to anhydrous ethanol and ultrasonically dispersed to obtain a zinc oxide nanoparticle dispersion with a concentration of 20 mg / mL. The ultrasonic dispersion time here is 15-20 min.

[0021] In step (12) above, the dispersion after ultrasonic treatment is first centrifuged to obtain a precipitate, and the precipitate is then washed with anhydrous ethanol. This centrifugation and washing operation is repeated at least three times. The product from the last washing is then centrifuged, and the product obtained by centrifugation is dried to obtain surface-modified zinc oxide nanoparticles. The centrifugation conditions are: 5000-8000 rpm, 5-10 min. The drying conditions are: 50-80℃, 12-24 h.

[0022] In step (2) above, bone-targeting materials are grafted onto the surface of surface-modified zinc oxide nanoparticles to obtain bone-targeting zinc oxide nanoparticles; this is mainly achieved through surface grafting of bone-targeting materials. After grafting with bone-targeting materials (preferably aspartic hexapeptide (Asp6)), the targeting ability of the bone-targeting materials to hydroxyapatite in bone tissue is used to direct the particles to the site of bone infection, thereby exerting the broad-spectrum antibacterial ability of the zinc oxide nanoparticles to kill bacteria.

[0023] First, the bone-targeting material was dissolved in a first buffer solution to obtain a bone-targeting material buffer solution with a concentration of 0.5 g / L. The pH of the bone-targeting material buffer solution was then adjusted to 5-5.5 using acid. The first buffer solution was a MES buffer solution containing EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (NHS is N-hydroxysuccinimide); the mass ratio of EDC to NHS was 1:1; the total mass of EDC and NHS to the volume ratio of the MES buffer solution was 10 mg-20 mg:1 mL. The acid was hydrochloric acid with a concentration of 0.1 M. After adding the acid, the bone-targeting material buffer solution was stirred at room temperature for 30-40 min to ensure uniform mixing and to fully activate the carboxyl groups of the bone-targeting material.

[0024] The surface-modified zinc oxide nanoparticles were then added to a bone-targeting material buffer solution, and the pH of the resulting reaction system was adjusted to 5.5-6.5 using a second buffer solution. The second buffer solution was commercially available PBS buffer with a pH range of 7.2-7.4. After the reaction system was stirred at room temperature, the precipitate was collected by centrifugation and filtration. The precipitate was washed with ultrapure water and dried to obtain highly efficient antibacterial zinc oxide nanoparticles with bone-targeting properties. The drying conditions were: 50-60℃, 12-24h.

[0025] Unless otherwise specified, all operations in steps (1)-(2) above shall be performed at room temperature.

[0026] This invention also provides the application of the above-mentioned bone-targeting, highly efficient antibacterial zinc oxide nanoparticles in the preparation of bone tissue therapeutic drugs, mainly in the preparation of osteomyelitis therapeutic drugs.

[0027] Osteomyelitis caused by pyogenic microbial infection is an inflammatory response accompanied by bone destruction. It is often caused by the local spread of infection after trauma, orthopedic surgery, or joint replacement surgery. It can be localized to a specific area of ​​bone or involve multiple sites such as the bone marrow, cortex, periosteum, and surrounding soft tissues. After pathogens invade the bone area, they release proteolytic enzymes and free radicals, inducing inflammation, destroying surrounding tissues, and causing blood flow to the infected area, increasing the chance of bone destruction. Hydroxyapatite (HA) is an important component of bone. Compared with traditional osteomyelitis treatments, the aspartic hexapeptide (Asp6) modified zinc oxide nanoparticles provided in this invention can target hydroxyapatite in the bone tissue at the site of bacterial infection. The zinc oxide nanoparticles with aspartic hexapeptide (Asp6) bound to their surface can exert a broad-spectrum antibacterial effect at the site of infection, thereby killing bacteria. Compared with traditional systemic antibiotic administration, the bone-targeting zinc oxide nanoparticles provided in this application can achieve efficient bacterial clearance at the bone infection site while reducing the dosage, showing promising clinical application prospects for the treatment of osteomyelitis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The bone-targeting high-efficiency antibacterial zinc oxide nanoparticles provided by the present invention graft bone-targeting materials onto the surface of zinc oxide nanoparticles, which can be directed to the bone infection site through the targeting ability of bone-targeting materials to hydroxyapatite in bone tissue, and then exert the broad-spectrum antibacterial ability of zinc oxide nanoparticles to kill bacteria.

[0030] 2) This invention utilizes activated and modified zinc oxide nanoparticles with highly reactive sites on their surface to bind to the terminal carboxyl group of aspartic acid hexapeptide (Asp6) via amide bonds. Simultaneously, aspartic acid hexapeptide (Asp6) can also bind to Ca in mineralized tissues. 2+ The interaction generates a strong affinity, thereby forming a stable structure between zinc oxide nanoparticles and aspartic acid hexapeptide (Asp6), which in turn enables the prepared material to have both bone-targeting properties and broad-spectrum antibacterial capabilities. Attached Figure Description

[0031] Figure 1 This describes the preparation process of the bone-targeting, highly efficient antibacterial zinc oxide nanoparticles ZnO-Asp6 in Example 1;

[0032] Figure 2 This is a schematic diagram illustrating the preparation principle of ZnO-Asp6, a highly efficient antibacterial zinc oxide nanoparticle with bone-targeting properties, in Example 1.

[0033] Figure 3The surface-modified ZnO nanoparticles prepared for the example were observed by scanning electron microscopy; where (a) is the zinc oxide nanoparticles before modification (10000x magnification), (b) is the zinc oxide nanoparticles after modification (10000x magnification), and (c) is the zinc oxide nanoparticles after modification (4000x magnification).

[0034] Figure 4 FTIR curves for successfully prepared nano-zinc oxide powder with highly reactive sites on its surface;

[0035] Figure 5 The in vitro binding capacity of ZnO-Asp6 bone-targeting nanoparticles prepared in Example 1 to hydroxyapatite is shown in (a) and (b) is a schematic diagram of the principle.

[0036] Figure 6 The antibacterial activity of the ZnO-Asp6 bone-targeting nanoparticles prepared in Example 1 is shown in (a) and (b) is parallel experimental group 2. Detailed Implementation

[0037] The technical solutions of various 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.

[0038] Example 1

[0039] The zinc oxide nanoparticles used in this embodiment have a particle size of 20-200 nm; the silane coupling agent used is γ-aminopropyltriethoxysilane (KH550); and the bone-targeting material used is aspartic acid hexapeptide (Asp6).

[0040] The preparation process of ZnO-Asp6, a highly efficient antibacterial zinc oxide nanoparticle with bone-targeting properties, provided in this embodiment is as follows: Figure 1 As shown, the zinc oxide surface was first activated using KH550; the specific reaction formula is shown in [reference needed]. Figure 2 As shown, KH550 is grafted onto the surface of zinc oxide nanoparticles. On the one hand, it consumes the hydroxyl groups on the surface of zinc oxide nanoparticles, effectively improving the tendency of nanoparticles to aggregate. On the other hand, it modifies the surface of zinc oxide nanoparticles by amylation. Then, based on the amidation reaction, aspartic hexapeptide Asp6 is grafted onto the modified zinc oxide nanoparticle surface through its carboxyl group to obtain the ZnO-Asp6 surface.

[0041] Specifically, in this embodiment, bone-targeting, highly efficient antibacterial zinc oxide nanoparticles ZnO-Asp6 are prepared according to the following steps:

[0042] (1) Surface modification of zinc oxide nanoparticles by silane coupling agent to obtain surface-modified zinc oxide nanoparticles; specifically including the following steps:

[0043] (11) Add 0.2g of zinc oxide nanoparticles to 10mL of anhydrous ethanol and sonicate for 15min to disperse evenly; then add 38μL of KH550 to the evenly dispersed zinc oxide nanoparticle dispersion (KH550 mass is about 20% of zinc oxide nanoparticles) and continue sonication for 30min.

[0044] (12) Transfer the ultrasonically treated dispersion to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 5 min, retaining the precipitate; then add 40 mL of anhydrous ethanol for washing; repeat the above centrifugation and washing operation three times, and then centrifuge the product after the last washing. Place the product obtained by centrifugation in a vacuum oven and dry at 60 °C for 24 h to obtain surface-modified zinc oxide nanoparticles.

[0045] (2) Preparation of bone-targeting zinc oxide nanoparticles, specifically:

[0046] 100 mg EDC and 100 mg NHS were dissolved in 10 mL of MES buffer and mixed thoroughly to obtain the first buffer (EDC / NHS-MES buffer). 5 mg of aspartic hexapeptide (Asp6) was dissolved in 10 mL of the first buffer to obtain an aspartic hexapeptide (Asp6) buffer with a concentration of 0.5 g / L. The pH of the aspartic hexapeptide (Asp6) buffer was adjusted to 5.5 using 0.1 M hydrochloric acid, and then stirred at room temperature for 30 min. 50 mg of surface-modified zinc oxide nanoparticles were added to the aspartic hexapeptide (Asp6) buffer, and the pH of the resulting reaction system was adjusted to 6.0 using PBS buffer. The mixture was then stirred at room temperature for 4 h. The resulting product was collected by centrifugation and filtration, and the precipitate was washed with ultrapure water and then dried in a vacuum oven at 60 °C for 24 h to obtain bone-targeting, highly effective antibacterial zinc oxide nanoparticles ZnO-Asp6.

[0047] Example 2

[0048] The zinc oxide nanoparticles used in this embodiment have a particle size of 20-200 nm; the silane coupling agent used is γ-methacryloyloxypropyltrimethoxysilane (KH570); and the bone-targeting material used is aspartic acid hexapeptide (Asp6).

[0049] This embodiment prepares bone-targeting, highly efficient antibacterial zinc oxide nanoparticles ZnO-Asp6 according to the following steps:

[0050] (1) Surface modification of zinc oxide nanoparticles by silane coupling agent to obtain surface-modified zinc oxide nanoparticles; specifically including the following steps:

[0051] (11) Add 0.2g of zinc oxide nanoparticles to 10mL of anhydrous ethanol and sonicate for 15min to disperse evenly; then add 43μL of KH570 to the evenly dispersed zinc oxide nanoparticle dispersion (KH570 mass is about 22% of zinc oxide nanoparticles) and continue sonication for 20min.

[0052] (12) Transfer the ultrasonically treated dispersion to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min, retaining the precipitate; then add 40 mL of anhydrous ethanol for washing; repeat the above centrifugation and washing operation three times, and then centrifuge the product after the last washing. Place the product obtained by centrifugation in a vacuum oven and dry at 80 °C for 12 h to obtain surface-modified zinc oxide nanoparticles.

[0053] (2) Preparation of bone-targeting zinc oxide nanoparticles, specifically:

[0054] 50 mg EDC and 50 mg NHS were dissolved in 10 mL of MES buffer and mixed thoroughly to obtain the first buffer (EDC / NHS-MES buffer). 5 mg of aspartic hexapeptide (Asp6) was dissolved in 10 mL of the first buffer to obtain an aspartic hexapeptide (Asp6) buffer with a concentration of 0.5 g / L. The pH of the aspartic hexapeptide (Asp6) buffer was adjusted to 5 using 0.1 M hydrochloric acid, and then stirred at room temperature for 30 min. 50 mg of surface-modified zinc oxide nanoparticles were added to the aspartic hexapeptide (Asp6) buffer, and the pH of the resulting reaction system was adjusted to 5.5 using PBS buffer. The mixture was then stirred at room temperature for 4 h. The resulting product was collected by centrifugation and filtration, and the precipitate was washed with ultrapure water and then dried in a vacuum oven at 50 °C for 24 h to obtain bone-targeting, highly efficient antibacterial zinc oxide nanoparticles ZnO-Asp6.

[0055] Example 3

[0056] The zinc oxide nanoparticles used in this embodiment have a particle size of 20-200 nm; the silane coupling agent used is γ-aminopropyltriethoxysilane (KH550); and the bone-targeting material used is glutamic acid hexapeptide (Glu6).

[0057] This embodiment prepares bone-targeting, highly efficient antibacterial zinc oxide nanoparticles ZnO-Glu6 according to the following steps:

[0058] (1) Surface modification of zinc oxide nanoparticles by silane coupling agent to obtain surface-modified zinc oxide nanoparticles; specifically including the following steps:

[0059] (11) Add 0.2g of zinc oxide nanoparticles to 10mL of anhydrous ethanol and sonicate for 20min to disperse evenly; then add 38μL of KH550 to the evenly dispersed zinc oxide nanoparticle dispersion (KH550 mass is about 20% of zinc oxide nanoparticles) and continue sonication for 30min.

[0060] (12) Transfer the ultrasonically treated dispersion to a 50 mL centrifuge tube and centrifuge at 8000 rpm for 5 min, retaining the precipitate; then add 35 mL of anhydrous ethanol for washing; repeat the above centrifugation and washing operation three times, and then centrifuge the product after the last washing. Place the product obtained by centrifugation in a vacuum oven at 50 °C for 24 h to obtain surface-modified zinc oxide nanoparticles.

[0061] (2) Preparation of bone-targeting zinc oxide nanoparticles, specifically:

[0062] 100 mg EDC and 100 mg NHS were dissolved in 10 mL of MES buffer and mixed thoroughly to obtain the first buffer (EDC / NHS-MES buffer). 5 mg of glutamate hexapeptide (Glu6) was dissolved in 10 mL of the first buffer to obtain a 0.5 g / L glutamate hexapeptide (Glu6) buffer. The pH of the glutamate hexapeptide (Glu6) buffer was adjusted to 5.5 with 0.1 M hydrochloric acid, and then stirred at room temperature for 40 min. 50 mg of surface-modified zinc oxide nanoparticles were added to the glutamate hexapeptide (Glu6) buffer, and the pH of the resulting reaction system was adjusted to 6.5 with PBS buffer. The mixture was then stirred at room temperature for 5 h. The resulting product was collected by centrifugation and filtration, and the precipitate was washed with ultrapure water and then dried in a vacuum oven at 60 °C for 24 h to obtain bone-targeting, highly effective antibacterial zinc oxide nanoparticles ZnO-Glu6.

[0063] (I) Structural Characterization

[0064] The morphology of the surface-modified zinc oxide nanoparticles prepared in Example 1 was examined using scanning electron microscopy, and the results are as follows: Figure 3 As shown in Figure (a), the zinc oxide nanoparticles before modification are granular with a size between 20-200 nm and obvious agglomeration. Figures (b) and (c) show the zinc oxide nanoparticles after modification with KH550 silane coupling agent. It can be seen that the degree of agglomeration has been improved and the particle size has not changed significantly.

[0065] Infrared spectroscopy was performed on the surface-modified zinc oxide nanoparticles prepared in Example 1, and the results are as follows: Figure 4 As shown in the figure, the infrared spectrum of the unmodified zinc oxide nanoparticles shows a peak density of 500-600 cm⁻¹. -1 A strong absorption peak is observed at 3675-3131 cm⁻¹, corresponding to the bending vibration of the Zn-O bond in zinc oxide. -1 Peak within the range (≈3411cm) -1 The vibration of Si-O is a tensile vibration of the -OH group. After modification with KH550 coupling agent, the vibrational peak of Si-O is approximately 546 cm⁻¹. 1 The location was originally at 3411cm. -1 The stretching vibration peak of the -OH group at the position showed a shift to lower wavenumbers and a broadening of the peak. This is due to the successful grafting of -NH2, indicating the successful modification of zinc oxide nanoparticles by KH550.

[0066] (II) Characterization of the specific binding ability of ZnO-Asp6 to hydroxyapatite powder

[0067] 1. Material preparation:

[0068] 1) Weigh 20 mg of hydroxyapatite powder that has passed through a 400-mesh sieve and disperse it in 200 mL of PBS buffer (commercially available, pH approximately 7.2-7.4). Set the stirring speed to 500 rpm and stir overnight.

[0069] 2) Prepare PBS dispersions of ZnO-Asp6 (prepared in Example 1) with different concentration gradients (0.056M, 0.112M, 0.224M, 0.336M) for later use.

[0070] 2. Experimental methods:

[0071] 900 μL of hydroxyapatite powder dispersion was mixed with 100 μL of ZnO-Asp6 PBS dispersion of different concentrations and placed in a shaker at 37°C for 24 h.

[0072] After shaking for 24 hours, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected and retained. 500 μL of the supernatant was then diluted to 3000 μL with 2500 μL of PBS buffer and added to a cuvette. The absorbance of the diluted solution at 220 nm was measured using a UV spectrophotometer. The results are as follows. Figure 5 As shown in (a).

[0073] 3. Experimental Results:

[0074] Generally, the higher the Asp6 content in the dispersion, the higher the absorbance at 220 nm should be. Comparing the absorbance at 220 nm of four groups of ZnO-Asp6 PBS dispersions with different concentrations after mixing with hydroxyapatite dispersion for 24 hours reveals that in the experimental group of this invention, as the initial peptide concentration increases, the absorbance of the supernatant after centrifugation for 24 hours after mixing with hydroxyapatite dispersion shows an opposite trend; that is, the higher the initial peptide concentration, the lower the absorbance. This indicates that during the mixing process, the ZnO-Asp6 nanoparticles, due to the bone-targeting ability of Asp6, attract the entire nanoparticle to bind with the hydroxyapatite nanoparticles (e.g., ...). Figure 5 (b) As shown, after centrifugation of the supernatant, Asp6, which has a certain solubility in water, is separated as a precipitate during centrifugation due to its binding with hydroxyapatite. The higher the concentration of ZnO-Asp6 nanoparticles in the group, the more extensive the binding with hydroxyapatite, resulting in the separation of a large amount of Asp6 and a decrease in the absorbance of the supernatant. These results confirm that the carboxyl groups on Asp6 grafted onto ZnO nanoparticles bind to calcium ions on HA crystals, exerting a strong anchoring effect on calcium ions on the surface of hydroxyapatite.

[0075] (III) Antibacterial effect of ZnO-Asp6 against Staphylococcus aureus

[0076] 1. Experimental strain:

[0077] The test strain, Gram-positive Staphylococcus aureus (S. aureus, CMCC26003), was purchased from the Shanghai Center for Preservation of Biotechnology.

[0078] 2. Experimental methods:

[0079] To further verify the bactericidal effect of ZnO-Asp6, Staphylococcus aureus, the most common pathogen of osteomyelitis, was selected for antibacterial experiments.

[0080] Bacterial culture medium: a nutrient broth medium containing Staphylococcus aureus, with an inoculation density of 1×10⁻⁶. 8 CFU / mL. The nutritional broth consists of 10 g / L peptone, 3 g / L beef extract powder, and 5 g / L sodium chloride. The specific preparation process is as follows: Weigh 1.8 g of the nutritional broth ingredients, add 100 mL of deionized water, heat to boiling to dissolve, and autoclave at 121°C for 30 min.

[0081] Solid culture medium: Nutrient agar medium. The nutrient agar composition is 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, and 15 g / L agar. The specific preparation process is as follows: Weigh 3.3 g of nutrient agar, add it to 100 mL of deionized water, heat to boiling to dissolve, and autoclave at 121°C for 30 min.

[0082] The specific steps are as follows: Take 6 sterilized filter paper pieces... Soak in 1 mL of different concentration dispersions (control group pure PBS buffer, PBS dispersion of 1 mg / mL Asp6, PBS dispersion of 2 mg / mL ZnO-Asp6 (prepared in Example 1), PBS dispersion of 4 mg / mL ZnO-Asp6 (prepared in Example 1), PBS dispersion of 2 mg / mL ZnO, and PBS dispersion of 4 mg / mL ZnO) for 1 h before use.

[0083] Bacterial Culture Medium: Take 100 μL of bacterial culture and add it dropwise to sterilized solid culture medium (the solid culture medium should be in a non-clumping state, at approximately 50-60℃). Prepare the bacterial culture medium by pouring the medium onto a plate. After standing for 8 hours, use a dispenser or sterile forceps to firmly attach the filter paper discs (two parallel experimental groups) to the culture dish. The center-to-center distance between each disc should be ≥24 mm. The discs should not be moved after attachment. Incubate at 37℃ for 24 hours, observe, and measure the diameter of the inhibition zone using Image-J. The results are shown in Table 1 and... Figure 6 As shown.

[0084] Table 1. Measurement results of inhibition zone diameter

[0085] Material concentration Diameter of inhibition zone (parallel experimental group 1) Diameter of inhibition zone (parallel experimental group 2) Pure PBS - 9.4mm 9.74mm Asp6 1 mg / mL 11.35mm 6.86mm ZnO-Asp6 4mg / mL 17.29mm 12.43mm ZnO-Asp6 2mg / mL 16.79mm 12.55mm ZnO 4mg / mL 17.14mm 16.07mm ZnO 2mg / mL 16.98mm 12.69mm

[0086] 3. Experimental Results:

[0087] from Figure 6 As shown in Table 1, inhibition zones of a certain size appeared in all the culture dishes after the filter paper was soaked in the material dispersion. The reason why the filter paper co-cultured with PBS buffer and ASP6 dispersion showed smaller inhibition zones may be that after the filter paper was soaked and placed on the culture dish, there was still a trace amount of liquid infiltration, which diluted the local bacterial concentration and showed a certain size of inhibition zone.

[0088] Filter paper immersed in ZnO-Asp6 and ZnO dispersions showed more obvious and larger inhibition zones. The filter paper immersed in 4 mg / mL ZnO-Asp6 dispersion had an inhibition zone diameter of 17.29 mm, which was much larger than that of the control group (pure PBS and ASP6) and close to that of the 4 mg / mL ZnO dispersion. This indicates that the surface modification of ZnO nanoparticles in this invention did not affect their ability to kill bacteria and that they have a good antibacterial effect.

[0089] Those skilled in the art will recognize that the embodiments described herein are intended to help the reader understand the principles of the invention, and should be understood that the scope of protection of the invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical teachings disclosed in this invention without departing from the spirit of the invention, and these modifications and combinations are still within the scope of protection of this invention.

Claims

1. Application of a bone-targeting, highly efficient antibacterial zinc oxide nanoparticle in the preparation of a drug for treating osteomyelitis; The preparation method of the bone-targeting, highly efficient antibacterial zinc oxide nanoparticles includes the following steps: (1) Surface modification of zinc oxide nanoparticles by silane coupling agent to obtain surface-modified zinc oxide nanoparticles; step (1) includes the following sub-steps: (11) Add zinc oxide nanoparticles to anhydrous ethanol and disperse them evenly by ultrasonication to obtain a zinc oxide nanoparticle dispersion with a concentration of 20 mg / mL; add silane coupling agent to the evenly dispersed zinc oxide nanoparticle dispersion and ultrasonically treat for 20-40 min; the silane coupling agent is 20%-22% of the mass of zinc oxide nanoparticles; the silane coupling agent is γ-aminopropyltriethoxysilane or γ-methacryloyloxypropyltrimethoxysilane; (12) The ultrasonically treated dispersion was centrifuged, washed and dried multiple times to obtain surface-modified zinc oxide nanoparticles. (2) Preparation of bone-targeting zinc oxide nanoparticles, specifically: Surface-modified zinc oxide nanoparticles were added to a bone-targeting material buffer solution with a pH of 5-5.5, and the pH of the resulting reaction system was adjusted to 5.5-6.

5. The mixture was then stirred for 4-5 hours, and after filtration, washing, and drying, highly efficient antibacterial zinc oxide nanoparticles with bone-targeting properties were obtained. The mass ratio of the surface-modified zinc oxide nanoparticles to the bone-targeting material was 10:

1. The bone-targeting material was aspartic acid hexapeptide.

2. The application according to claim 1, characterized in that, In step (2), the bone-targeting material is dissolved in the first buffer solution to obtain a bone-targeting material buffer solution with a concentration of 0.5 g / L, and the pH value of the bone-targeting material buffer solution is adjusted to 5-5.5 by acid.

3. The application according to claim 2, characterized in that, The first buffer is a MES buffer containing EDC and NHS; the mass ratio of EDC to NHS is 1:1; the total mass of EDC and NHS to the volume ratio of MES buffer is 10-20 mg: 1 mL.

4. The application according to claim 1, characterized in that, Surface-modified zinc oxide nanoparticles were added to a bone-targeting material buffer, and the pH of the resulting reaction system was adjusted to 5.5-6.5 using a second buffer; the second buffer was PBS buffer with a pH range of 7.2-7.4.

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