Nano-particles with antibacterial and osteogenesis promoting effects as well as preparation method and application of nano-particles

Abstract

The invention discloses nano-particles with antibacterial and osteogenesis promoting effects as well as a preparation method and application of the nano-particles, and belongs to the technical field of antibacterial nano-materials. On one hand, the invention provides the nano-particles with antibacterial and osteogenesis promoting effects and the preparation method thereof, and on the other hand, the invention provides the application of the nano-particles. The nanoparticles obtained by the method disclosed by the invention have good stability under physiological conditions (PH = 7.4); polyphenol, metal ions and AMP (adenosine monophosphate) can be slowly released in an inflammatory (weakly acidic) environment, and the functions including but not limited to bacterium capturing, bacterium killing, inflammation and oxidation resisting, osteogenesis promoting and the like are realized; the nano-particles can be used for preparing a bone infection treatment drug, and the bone infection treatment drug has a broad-spectrum antibacterial effect and cannot cause antibiotic drug resistance; the treatment medicine can efficiently prevent and treat bone infection and improve bone repair efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial nanomaterials, and particularly relates to a nanoparticle with both antibacterial and osteogenic functions, a preparation method thereof, and an application thereof. Background Art

[0002] Bone infection is a serious disease, usually caused by a variety of pathogenic microorganisms invading the bone marrow through different routes, mainly cocci and bacilli. During the treatment of bone infection, bactericidal and osteogenic processes after bacteria capture play crucial roles in the repair of infected bone. However, currently, the treatment of bone infection mainly relies on open debridement, systemic and local use of antibiotics. This method only considers the elimination of bacteria and does not consider osteogenesis; moreover, the antibiotic administration method is inefficient, often leading to problems such as drug resistance and difficult bone healing.

[0003] The construction strategy of metal polyphenol network (MPN) and the polyphenol-polypeptide assembly strategy based on supramolecular interactions provide ideas for the design of bioactive materials with customized functions. MPN is a simple, mild, and effective method that can assemble into stable MPN nanoparticles while retaining the structures and bioactivities of different components; supramolecular interactions can assemble polypeptides and MPN nanoparticles through non-covalent interactions to achieve the construction of nanoparticles and retain the structures and activities of polypeptides. Therefore, using this construction strategy to provide a biomaterial with both good antibacterial and osteogenic functions is worthy of further exploration. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a technical solution for a nanoparticle with both antibacterial and osteogenic functions, a preparation method thereof, and an application thereof. This nanoparticle has good biocompatibility, the ability to capture and kill bacteria, and excellent osteogenic activity, and can be used to promote the bone infection repair process.

[0005] The present invention is specifically implemented by the following technical solutions:

[0006] The first aspect of the present invention provides a preparation method of a nanoparticle with both antibacterial and osteogenic functions, which includes the following steps:

[0007] 1) Dissolve natural polyphenols in a NaOH solution to prepare a polyphenol solution;

[0008] 2) Dissolve metal ions with osteogenic functions in pure water to prepare a metal ion aqueous solution;

[0009] 3) Synthesize an antibacterial peptide AMP with the following sequence:

[0010] H2N-GWKDWAKKAGGWLKKKGPGMAKAALKAAMQ-OH;

[0011] Dissolve the antimicrobial peptide AMP in a buffer to prepare an AMP solution;

[0012] 4) Under ultrasonic oscillation treatment, mix the above-mentioned polyphenol solution and the above-mentioned metal ion solution to obtain a metal polyphenol complex MPN; subject the metal polyphenol complex MPN to centrifugation treatment, and resuspend the precipitate under ultrasonic waves to obtain MPN nanoparticles;

[0013] 5) Under ultrasonic oscillation treatment, mix the MPN nanoparticles with the AMP solution, centrifuge, resuspend the precipitate under ultrasonic waves, and remove the free AMP to obtain MPN@AMP nanoparticles.

[0014] Furthermore, the natural polyphenols in step 1) include but are not limited to at least one of curcumin, quercetin, anthocyanin, and catechin.

[0015] Furthermore, the molar concentration of the polyphenol solution in step 1) is 10 - 1000 mM.

[0016] Furthermore, the metal ions with osteogenic promoting effects in step 2) include but are not limited to at least one of magnesium, calcium, and strontium ions.

[0017] Furthermore, the molar concentration of the metal ion solution in step 2) is 30 - 3000 mM.

[0018] Furthermore, the buffer in step 3) includes at least one of tris(hydroxymethyl)aminomethane - hydrochloric acid buffer solution and phosphate buffer solution; the concentration of the buffer is 1 mM - 1 M; the pH of the buffer is 7 - 9.

[0019] Furthermore, the mass concentration of the AMP solution in step 3) is 0.1 - 10 mg / mL.

[0020] The second aspect of the present invention provides a kind of nanoparticle with both antibacterial and osteogenic promoting effects prepared by any of the above preparation methods.

[0021] The third aspect of the present invention provides the application of a nanoparticle with both antibacterial and osteogenic promoting effects in the preparation of a bone infection treatment drug, and this bone infection treatment drug has the effects of antibacterial, anti - inflammatory, antioxidant stress, and osteogenic promoting.

[0022] The beneficial effects of the present invention are as follows: The nanoparticles obtained by the method of the present invention have good stability under physiological conditions (pH = 7.4); they can slowly release polyphenols, metal ions, and AMP in an inflammatory (weakly acidic) environment, achieving functions including but not limited to capturing bacteria, killing bacteria, anti-inflammatory and antioxidant effects, and promoting osteogenesis; the nanoparticles of the present invention can be used to prepare bone infection treatment drugs, which have a broad-spectrum antibacterial effect and do not cause antibiotic resistance; the treatment drug can effectively prevent and treat bone infections and improve the bone repair efficiency. Description of the Drawings

[0023] Figure 1 Typical transmission electron microscope images of the nanoparticles of Comparative Example 1, Example 1, and Example 2.

[0024] Figure 2 Particle size distributions of the nanoparticles of Comparative Example 1, Example 1, and Example 2.

[0025] Figure 3 (a) XPS spectra and (b) elemental distributions of the nanoparticles of Comparative Example 1, Example 1, and Example 2.

[0026] Figure 4 Relative cell viability of bone marrow mesenchymal stem cells co-incubated with Comparative Example 2, Example 1, and Example 2.

[0027] Figure 5 Inhibitory effects of the nanoparticles of Comparative Example 1, Example 1, and Example 2 on the growth of Staphylococcus aureus and Escherichia coli.

[0028] Figure 6 Typical pictures of ALP staining of osteoblasts after co-culture with the nanoparticles of Comparative Example 1, Example 1, and Example 2 for 14 days.

[0029] Figure 7 Typical HE pictures of the femur tissues of rats with infectious bone defects after treatment with Example 1 and Comparative Example 1. Detailed Description of the Invention

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It should be clear that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the claims of the present invention.

[0031] Example 1

[0032] In this example, a nanoparticle material with both antibacterial and osteogenic promoting effects was prepared, and the specific steps are as follows:

[0033] 1) Dissolve natural polyphenol curcumin (Curcumin, Cur, C 21 H 20 O6, 368.38 MW) in 0.2 M NaOH solution to prepare a Cur solution with a molar concentration of 100 mM;

[0034] 2) Dissolve strontium chloride (SrCl2) in pure aqueous solution to prepare an aqueous solution of Sr ions with a molar concentration of 300 mM;

[0035] 3) Dissolve the antibacterial peptide (AMP) of H2N - GWKDWAKKAGGWLKKKGPGMAKAALKAAMQ - OH with antibacterial effect in 10 mM tris - hydroxymethylaminomethane - hydrochloric acid buffer solution at pH = 7 to prepare an AMP solution with a mass concentration of 1 mg / mL;

[0036] 4) Add the Cur solution and Sr ion solution successively under ultrasonic oscillation to form Cur / Sr - MPN, centrifuge at 15000 rpm for 10 minutes, discard the supernatant, resuspend the precipitate with pure water by ultrasonic treatment, and repeat the centrifugation - ultrasonic washing 3 times to obtain Cur / Sr - MPN nanoparticles;

[0037] 5) Add the AMP solution to the Cur / Sr - MPN suspension under ultrasonic oscillation, mix well and continue ultrasonic treatment for 60 min, remove the free AMP by centrifugation - ultrasonic washing 3 times to obtain Cur / Sr - MPN@AMP nanoparticles; resuspend with an appropriate amount of pure water to 1 mg / ml and store at room temperature.

[0038] Example 2

[0039] This example prepared a nanoparticle material with both antibacterial and osteogenic promotion effects, and the specific steps are as follows:

[0040] 1) Dissolve natural polyphenol proanthocyanidins (Proanthocyanidins, PC, C 30 H 26 O 13 , 594.52 MW) in 0.2 M NaOH solution to prepare a PC solution with a molar concentration of 100 mM;

[0041] 2) Dissolve magnesium chloride (MgCl2) in pure aqueous solution to prepare an aqueous solution of Mg ions with a molar concentration of 300 mM;

[0042] 3) Dissolve the antibacterial peptide (AMP) of H2N - GWKDWAKKAGGWLKKKGPGMAKAALKAAMQ - OH with antibacterial effect in 10 mM tris - hydroxymethylaminomethane - hydrochloric acid buffer solution at pH = 7 to prepare an AMP solution with a mass concentration of 1 mg / mL;

[0043] 4) Under ultrasonic oscillation, PC solution and Mg ion solution were added successively to form PC / Mg-MPN. It was centrifuged at 15,000 revolutions per minute for 10 minutes. After discarding the supernatant, the precipitate was resuspended ultrasonically with pure water, and the centrifugation-ultrasonic washing was repeated 3 times to obtain PC / Mg-MPN nanoparticles;

[0044] 5) AMP solution was added to the PC / Mg-MPN suspension under ultrasonic oscillation. After mixing, it was ultrasonically treated for another 60 min. Free AMP was removed by centrifugation-ultrasonic washing 3 times to obtain PC / Mg-MPN@AMP nanoparticles; It was resuspended with an appropriate amount of pure water to 1 mg / ml and stored at room temperature.

[0045] Example 3

[0046] Based on Example 1, a Cur solution with a molar concentration of 1000 mM was prepared; an aqueous Sr ion solution with a molar concentration of 3000 mM was prepared, and other conditions remained unchanged.

[0047] Example 4

[0048] Based on Example 1, a Cur solution with a molar concentration of 10 mM was prepared; an aqueous Sr ion solution with a molar concentration of 30 mM was prepared, and other conditions remained unchanged.

[0049] Comparative Example 1

[0050] A kind of MPN nanoparticle material was prepared in this comparative example. The specific steps are as follows:

[0051] 1) Natural polyphenol curcumin (Curcumin, Cur, C 21 H 20 O6, 368.38 MW) was dissolved in 0.2 M NaOH solution to prepare a Cur solution with a molar concentration of 100 mM;

[0052] 2) Strontium chloride (SrCl2) was dissolved in pure aqueous solution to prepare an aqueous Sr ion solution with a molar concentration of 300 mM;

[0053] 3) Under ultrasonic oscillation, Cur solution and Sr ion solution were added successively to form PC / Sr-MPN. It was centrifuged at 15,000 revolutions per minute for 10 minutes. After discarding the supernatant, the precipitate was resuspended ultrasonically with pure water, and the centrifugation-ultrasonic washing was repeated 3 times to obtain Cur / Sr-MPN nanoparticles; It was resuspended with an appropriate amount of pure water to 1 mg / ml and stored at room temperature.

[0054] Comparative Example 2

[0055] A control group of simply physically mixing 1 mg / ml Cur solution and 1 mg / ml SrCl2 solution.

[0056] Experimental Example 1: Morphology Observation Experiment

[0057] The morphology of the nanoparticles prepared in Comparative Example 1, Example 1 and Example 2 was characterized by transmission electron microscopy (TEM). Figure 1 ) The typical microscopic images showed that Cur / Sr-MPN, PC / Mg-MPN@AMP and Cur / Sr-MPN@AMP all presented a quasi-circular structure.

[0058] Experimental Example 2: Dynamic Light Scattering Experiment

[0059] The hydrodynamic diameter of the nanoparticles prepared in Comparative Example 1, Example 1 and Example 2 was analyzed by dynamic light scattering (DLS) technology. Figure 2 ) The test data showed that the hydrodynamic diameter of Cur / Sr-MPN was about 50.7 nm. After functional modification, the particle size of Cur / Sr-MPN@AMP increased significantly to about 106.0 nm, with an increase of 109%, and this doubling of the particle size confirmed the effective coating of AMP; compared with Cur / Sr-MPN@AMP, the particle size of PC / Mg-MPN@AMP was smaller, about 28.8 nm.

[0060] Experimental Example 3: Surface Element Analysis Experiment

[0061] The elements of the nanoparticles prepared in Comparative Example 1, Example 1 and Example 2 were characterized by X-ray photoelectron spectroscopy (XPS). Figure 3 ) The wide-scan spectra showed that Cur / Sr-MPN and Cur / Sr-MPN@AMP both presented characteristic Sr 3d orbital peaks at 133.9 eV, with atomic percentages of 2.69% and 1.57% respectively, and PC / Mg-MPN@AMP presented a characteristic Mg orbital peak at eV, with an atomic percentage of 4.2%; while Cur / Sr-MPN@AMP and PC / Mg-MPN@AMP showed significant N1s characteristic peaks at 400.06 eV (atomic percentages were 3.67% and 5.98% respectively). This demonstrated the successful construction of Cur / Sr-MPN, Cur / Sr-MPN@AMP and PC / Mg-MPN@AMP nanoparticles and their element distributions. Experimental Example 4: Cell Compatibility Evaluation Experiment

[0062] Bone marrow mesenchymal stem cells (BMSCs) were seeded at 2×10 4 cells / cm 2For density culture, add the nanoparticle suspensions of Example 1 and Example 2 and the mixed solution of Comparative Example 2 with a final concentration of 0.125 mg / ml. After culturing for 24 hours, incubate with 10% of the culture medium volume of CCK-8 solution for 2 hours, then transfer 100 μL of the solution to a 96-well plate and measure the absorbance value at a wavelength of 450 nm at room temperature to reflect cell viability. As Figure 4 shown, Example 1 and Example 2 showed better cell viability compared with Comparative Example 2 and exceeded the control group (100%) without adding any materials, indicating that the nanoparticles obtained in Example 2 have good cell compatibility.

[0063] Test Example 5: Particle antibacterial test

[0064] Take 10 μL of the nanoparticle mixed solutions of Example 1, Comparative Example 1, and Example 2 respectively, and mix them with 1 mL of PBS suspension containing Staphylococcus aureus or Escherichia coli (10 7 CFU / mL), and incubate in a constant temperature shaker at 37 °C (200 rpm) for 4 h. After fully dispersing the bacteria by ultrasonic fragmentation method (40 kHz, 5 min), take 100 μL of the bacterial solution and evenly coat it on an LB agar plate with Φ = 10 cm, and observe the number of surviving colonies after culturing at 37 °C for 24 h ( Figure 5 ). The results show that the nanoparticles obtained in Example 1 and Example 2 exhibit excellent antibacterial properties.

[0065] Test Example 6: Particle osteogenic promotion test

[0066] Seed 1×10 4 BMSCs on the surface of a 2 cm 2 glass slide. In the experimental groups, add the nanoparticles of Example 1, Comparative Example 1, and Example 2 with a final concentration of 0.125 mg / mL respectively. After culturing all samples in osteogenic induction medium for 14 days, fix them with 4% paraformaldehyde. Detect the alkaline phosphatase (ALP) activity by BCIP / NBT staining method (ALP is used as an early osteogenic differentiation marker). As Figure 6 shown, on the 7th day, the groups of Comparative Example 1, Example 1, and Example 2 all showed blue-stained positive regions. Among them, compared with Comparative Example 1, both Example 1 and Example 2 showed a more significant increase in blue staining, indicating that the multifunctional nanoparticles can significantly promote the differentiation of BMSCs into osteoblasts.

[0067] Test Example 7: Infectious bone defect treatment test

[0068] First, establish an infectious bone defect model in SD rats. Prepare a non-penetrating bone defect with a diameter of 1 mm in the middle of the femur, and then inject 10 μL of PBS bacterial suspension containing Staphylococcus aureus into the defect cavity (concentration 1×10 7CFU / mL). After successful modeling, equal volumes (10 μL) of the corresponding suspensions were injected into the bone defect sites of the animals in Example 1 and Comparative Example 1. The experimental animals were sacrificed 1 week after postoperative treatment, and femoral specimens were taken for pathological analysis. Observation through tissue section preparation and hematoxylin-eosin (HE) staining showed that ( Figure 7 ), compared with the Comparative Example 1 group, the treatment group of Example 1 presented the following characteristic changes: (1) significantly reduced infiltration of inflammatory cells, and the density of neutrophils decreased by about 65%; (2) active formation of new bone callus, and osteoblasts could be seen arranged in a palisade; (3) increased local blood vessel density and reduced degree of fibrous tissue hyperplasia. These pathological features suggest that the nanoparticles of Example 1 have significant anti-infective and bone regeneration-promoting effects in vivo.

Claims

1. A preparation method of nanoparticles with both antibacterial and osteogenic effects, characterized in that, Comprising the following steps: 1) Dissolve natural polyphenols in an NaOH solution to prepare a polyphenol solution; 2) Dissolve osteogenic metal ions in pure water to prepare an aqueous metal ion solution; 3) Synthesize an antimicrobial peptide AMP with the following sequence: H2N-GWKDWAKKAGGWLKKKGPGMAKAALKAAMQ-OH; And dissolve the antimicrobial peptide AMP in a buffer to prepare an AMP solution; 4) Under ultrasonic oscillation, mix the above polyphenol solution and the above metal ion solution to obtain a metal polyphenol complex MPN; Centrifuge the metal polyphenol complex MPN, and resuspend the precipitate under ultrasound to obtain MPN nanoparticles; 5) Under ultrasonic oscillation, mix the MPN nanoparticles with the AMP solution, centrifuge, resuspend the precipitate under ultrasound, and remove the free AMP to obtain MPN@AMP nanoparticles.

2. The preparation method of a nanoparticle with both antibacterial and osteogenic effects according to claim 1, characterized in that, The natural polyphenols in step 1) include but are not limited to at least one of curcumin, quercetin, anthocyanin, and catechin.

3. The preparation method of a nanoparticle with both antibacterial and osteogenic effects as described in claim 1, characterized in that, The molar concentration of the polyphenol solution in step 1) is 10 - 1000 mM.

4. The preparation method of a nanoparticle with both antibacterial and osteogenic effects as described in claim 1, characterized in that, The osteogenic metal ions in step 2) include but are not limited to at least one of magnesium, calcium, and strontium ions.

5. The preparation method of a nanoparticle with both antibacterial and osteogenic effects as described in claim 1, characterized in that, The molar concentration of the metal ion solution in step 2) is 30 - 3000 mM.

6. The preparation method of a nanoparticle with both antibacterial and osteogenic effects as described in claim 1, characterized in that, The buffer in step 3) includes at least one of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution and phosphate buffer solution; The concentration of the buffer is 1 mM - 1 M; The pH of the buffer is 7 - 9.

7. The preparation method of a nanoparticle with both antibacterial and osteogenic functions according to claim 1, characterized in that, The mass concentration of the AMP solution in step 3) is 0.1 - 10 mg / mL.

8. A nanoparticle with both antibacterial and osteogenic effects prepared by any of the preparation methods of claims 1 - 7.

9. Use of a nanoparticle with both antibacterial and osteogenic effects as claimed in claim 8 in the preparation of a bone infection treatment drug, the bone infection treatment drug having antibacterial, anti-inflammatory, antioxidant stress, and osteogenic effects.

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