Metal polyphenol-hydroxyapatite nano delivery system as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a metal polyphenol-hydroxyapatite nano delivery system as well as a preparation method and application thereof. The nano delivery system comprises metal polyphenol and hydroxyapatite; wherein a nucleic acid molecule is loaded on the hydroxyapatite, and the hydroxyapatite is coated with the metal polyphenol; the metal polyphenol is formed by reaction of polyphenol molecules and metal ions. The metal polyphenol-hydroxyapatite nano delivery system can safely, stably and efficiently deliver a target gene; according to the present invention, the metal polyphenol-hydroxyapatite nano-delivery system cooperatively delivers the plasmid expressing the LL37 gene, such that the good treatment effect on the SD rat periodontitis is provided, the tissue regeneration promoting effect is provided, and the good prospect in the gene treatment field is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a metal polyphenol-hydroxyapatite nano-delivery system and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Gene therapy shows great promise in disease treatment. By delivering nucleic acids encoding functional proteins into patient cells to direct the expression of endogenous antimicrobial proteins, gene therapy offers a potential alternative to traditional protein therapy for genetic diseases and chronic inflammation. Compared with direct administration of recombinant antimicrobial proteins, this strategy can circumvent issues such as frequent dosing and high costs, while achieving more sustained therapeutic effects. However, effective nucleic acid delivery still faces huge challenges, as delivery vectors have poor cell compatibility, are easily degraded by enzymes, have limited cell membrane permeability, and tend to be trapped in lysosomes without being released. This requires the design of precise vectors to overcome these delivery barriers.

[0004] Currently, gene delivery vectors are divided into viral and non-viral vectors. While viral vectors (such as adeno-associated virus (AAV) and lentivirus (LV)) have high transfection efficiency, their clinical application is hampered by issues such as immunogenicity, carcinogenicity from insertional mutagenesis, and limited loading capacity. Among non-viral vectors, cationic liposomes and polymers rely on electrostatic encapsulation of nucleic acids. While this has improved safety, they still face challenges such as insufficient lysosomal escape efficiency, acute inflammation induced by cationic components, and poor physiological stability.

[0005] In the field of periodontitis treatment, existing delivery systems are unable to simultaneously meet the dual needs of targeted delivery of antibacterial genes and tissue regeneration and repair. Local injection of free plasmids has problems such as poor stability and low cellular uptake rate, and lacks a synergistic mechanism to promote the regeneration of alveolar bone and periodontal tissue. Although hydroxyapatite (HAp) is used as a bone regeneration material due to its bone induction, it cannot achieve efficient gene loading and controlled release when used alone.

[0006] Therefore, there is an urgent need to develop a multifunctional nanodelivery system that is stable in an enzyme environment, has high biocompatibility, stable loading and release capacity, and repair and regeneration functions, so as to break through the technical bottleneck of gene therapy in inflammatory tissue repair. Summary of the Invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a metal polyphenol-hydroxyapatite nano-delivery system and its preparation method and application. The present invention utilizes the coordination self-assembly characteristics of polyphenols and metal ions to successfully construct an MPN-DNA-HAp nano-delivery system. It solves the problems of poor biosafety, poor stability, and low loading efficiency of existing delivery vectors. At the same time, the present invention evaluated the application of apatite composite metal polyphenols in the ligation-induced SD rat periodontitis model through in vivo experiments. The results showed that the nano-delivery system showed good therapeutic effects on SD rats with periodontitis, and the bone tissue regeneration recovery effect was better than that of the control group. At the same time, it also has the effect of promoting tissue regeneration, which has important guiding significance for the relevant treatment of periodontitis.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a metal polyphenol-hydroxyapatite nano-delivery system, which comprises a metal polyphenol and hydroxyapatite; wherein the hydroxyapatite is loaded with nucleic acid molecules, and the hydroxyapatite is coated with the metal polyphenol; the metal polyphenol is composed of polyphenol molecules and metal ions through reaction.

[0010] Furthermore, the polyphenol molecules are selected from one or more combinations of tannic acid, gallic acid, propyl gallate, epigallocatechin gallate, catechin, and their derivatives; the metal ions are selected from Fe 3+ 、Ag + 、Zn 2+ Mg 2+ 、Cu 2+ 、Sr 3+ One of the soluble metal salt solutions.

[0011] Furthermore, the polyphenol molecules are selected from tannic acid, and the metal ions are selected from Zn 2+ or Fe 3+ .

[0012] Furthermore, the molar ratio of the polyphenol molecules to the metal ions is 1:2.5.

[0013] Furthermore, the nucleic acid molecule is selected from one or more of mRNA, siRNA, circleRNA, sgRNA, DNA, ecDNA, plasmid, and artificial nucleic acid.

[0014] Furthermore, the nucleic acid molecule is selected from a plasmid containing a gene encoding LL37.

[0015] Furthermore, the plasmid is LL37 in pcDNA3.1(+), hereinafter referred to as pLL37, and its nucleotide sequence is shown in SEQ ID NO.1.

[0016] The second aspect of the present invention provides a method for preparing the above-mentioned nanodelivery system, comprising the following steps: mixing nucleic acid and hydroxyapatite in a mass ratio of 1:(50-200), adding polyphenol molecule solution and metal ion solution in sequence, vortex mixing for 5 minutes, synthesizing MPN-DNA-HAp nanocarrier solution, and centrifuging at 8000 rpm to obtain the metal polyphenol-hydroxyapatite nanodelivery system, i.e., MPN-DNA-HAp nanocarrier particles.

[0017] Furthermore, the mass ratio of nucleic acid to hydroxyapatite is 1:100.

[0018] Furthermore, the concentration of the polyphenol molecule solution is 20-30 mM, preferably 24 mM, and the concentration of the metal ion solution is 50-70 mM, preferably 60 mM.

[0019] Furthermore, the volume ratio of the added polyphenol molecule solution to the metal ion solution is 1:1, and the volume ratio of the added polyphenol molecule solution to the MPN-DNA-HAp nanocarrier solution is (1-7):1000, preferably 3:1000.

[0020] The third aspect of the present invention provides the use of the above-mentioned nanodelivery system in the preparation of nucleic acid delivery or gene therapy drugs, wherein the nucleic acid delivery refers to the introduction of nucleic acids into cells; preferably, the drug is used to treat a disease or condition in a mammal; further preferably, the disease or condition includes tissue inflammatory infection.

[0021] Furthermore, the drug has the following uses:

[0022] (1) Used to reduce inflammation and infection of periodontal tissues;

[0023] (2) Used to promote the regeneration and repair of periodontal tissues;

[0024] (3) Used for bone tissue regeneration and recovery.

[0025] One or more of the above technical solutions have the following beneficial effects:

[0026] (1) The metal polyphenol-hydroxyapatite nanodelivery system of the present invention can deliver target genes safely, stably, and efficiently, and has great prospects in the field of gene therapy;

[0027] (2) The metal polyphenol-hydroxyapatite nano-delivery system of the present invention is simple to synthesize, and the raw material molecules are all natural compounds, which are easily metabolized in the body, and the metabolites still have positive effects on the body (calcium ions promote bone regeneration, tannic acid is anti-inflammatory and antioxidant, and zinc ions are essential trace elements for the human body).

[0028] (3) By co-delivering the plasmid expressing the LL37 gene with the metal polyphenol-hydroxyapatite nano-delivery system of the present invention, it has a good therapeutic effect on periodontitis in SD rats. At the same time, it was found that it also has the effect of promoting tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 Schematic diagram of the synthesis process of MPN-DNA-HAp nanocarrier particles in an embodiment of the present invention.

[0031] Figure 2 1 is the effect of the mass ratio of metal polyphenol to HAp on the hydrodynamic size in the examples of the present invention.

[0032] Figure 3 3. The effect of the mass ratio of metal polyphenols to HAp on the dispersibility in the examples of the present invention, wherein group 0 represents DNA-HAp nanoparticles without metal polyphenol coating; groups 1-7 represent different mass ratios of metal polyphenols to HAp.

[0033] Figure 4 Schematic diagram of the stability of MPN-DNA-HAp nanocarrier particles based on iron ions and zinc ions in DNase I in an embodiment of the present invention.

[0034] Figure 5 The stability of the MPN-DNA-HAp nanocarrier particles in 10% fetal bovine serum (FBS) culture medium in the examples of the present invention is shown.

[0035] Figure 6 The DNA loading capacity and loading efficiency of the MPN-DNA-HAp nanocarrier particles in the examples of the present invention.

[0036] Figure 7 Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) images of the MPN-DNA-HAp nanocarrier particles in the examples of the present invention.

[0037] Figure 8 The transfection efficiency of MPN-DNA-HAp nanocarrier particles was evaluated by qRT-PCR in the examples of the present invention.

[0038] Figure 9 1 is the DNA release curve of the MPN-DNA-HAp nanocarrier particles in different pH environments in the embodiment of the present invention.

[0039] Figure 10 Schematic diagram of micro-CT scanning for evaluating alveolar bone loss and regeneration in an embodiment of the present invention.

[0040] Figure 11 Schematic diagram of H&E staining and Masson staining for evaluating periodontal tissue regeneration in an embodiment of the present invention. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0042] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0043] The empty vector plasmid DNA used in the following examples is pEGFP-N1 (purchased from Genetically Modified Gene). LL37 in pcDNA3.1(+), hereinafter referred to as pLL37, has a nucleotide sequence shown in SEQ ID NO.1.

[0044] Hydroxyapatite (HAp) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS NO.: 12167-74-7.

[0045] TA (Tannic Acid) was purchased from Sigma-Aldrich Shanghai Company.

[0046] Zinc chloride and ferric chloride (FeCl3.6H2O) were purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0047] Preparation of TA solution: Accurately weigh 4.083 g of tannic acid using an analytical balance, add 80 mL of sterile deionized water, and stir using a magnetic stirrer until completely dissolved. Then, dilute to 100 mL with sterile deionized water to obtain a 24 mM TA solution. Aliquot and transfer to brown reagent bottles and store at -20°C in the dark.

[0048] Preparation of FeCl3 solution: Accurately weigh 1.622 g of FeCl3·6H2O using an analytical balance, add approximately 80 mL of sterile deionized water, and adjust the pH by adding 1-2 drops of dilute hydrochloric acid (1 M). Stir until completely dissolved, and then dilute to 100 mL with sterile deionized water to obtain a 60 mM FeCl3 solution.

[0049] Preparation of ZnCl2 solution: Accurately weigh 0.818 g ZnCl2 using an analytical balance, add about 80 mL of sterile deionized water, add 1-2 drops of dilute hydrochloric acid (1 M) to adjust the pH, stir until completely dissolved, and then dilute to 100 mL with sterile deionized water to obtain a 60 mM ZnCl2 solution.

[0050] Example 1 Synthesis of MPN-DNA-HAp and the Effect of the Mass Ratio of Metal Polyphenols to HAp on the Particle Size Stability and Dispersibility of Nanoparticles

[0051] Synthesis of MPN-DNA-HAp nanocarrier particles: First, 5 μg of plasmid DNA (pEGFP-N1) was loaded onto the surface of 1 mg of HAp nanoparticles using a simple on-ice ultrasonic treatment technique to form DNA-HAp nanoparticles. Next, 3 μL of 60 mM TA solution and 3 μL of 24 mM FeCl3 solution (the molar ratio of TA:FeCl3 is 1:2.5) were added in sequence, and the above solutions were mixed and made up to a final volume of 1 mL with ultrapure water. The mixture was vortexed for 5 minutes, and the polyphenol ligands were combined with metal ions through chemical coordination-driven self-assembly to successfully synthesize MPN-DNA-HAp nanocarrier particles (NPs). After centrifugation at 8000 rpm, the desired gene nanocarrier was finally obtained. The entire synthesis process was carried out in an ultrapure aqueous solution environment without DNase I (DNAseI), which effectively reduced the risk of nucleic acid degradation during the synthesis process ( Figure 1 ).

[0052] MPN-DNA-HAp nanocarriers were prepared by mixing according to the addition ratios shown in Table 1 and diluting to a final volume of 1 mL with ultrapure water. The effect of the mass ratio of metal polyphenols to HAP on the particle size stability and dispersibility of the nanocarrier particles was investigated.

[0053] Table 1

[0054]

[0055] Note: The concentration of TA solution in the table is 60mM, and the concentration of FeCl3 solution is 24mM.

[0056] like Figure 2 As shown in Figure 2, with the increase of the mass ratio of metal polyphenol to HAp, the particle size of the nanoparticles increased significantly. Figure 3As shown in the figure, the nanoparticles in groups 1 to 3 still maintained good dispersibility and stability after 1 week, while the dispersibility of the nanoparticles in groups 4 to 7 gradually decreased. Therefore, the addition ratio of group 3 was selected as the optimal ratio for subsequent experiments.

[0057] Example 2 Verification of the stability and loading efficiency of MPN-DNA-HAp nanocarrier particles with different metal ions

[0058] Plasmid DNA is susceptible to degradation by deoxyribonuclease I (DNase I), which is abundant in serum and cytoplasm. The stability of MPN-DNA-HAp nanocarriers in a DNase I-rich environment was analyzed by agarose gel electrophoresis.

[0059] The synthesis method of MPN-DNA-HAp nanocarriers refers to Example 1, and the addition ratio and concentration of each component refer to the concentration of Group 3 in Example 1, and MPN-DNA-HAp nanocarrier particles containing different metal ions (FeCl3, ZnCl2) are prepared respectively.

[0060] The MPN-DNA-HAp nanocarrier particles were placed in 1 mL of 2 U / mL DNase I solution and incubated for 0, 0.5, and 1 hour, respectively. The precipitate was collected by centrifugation, and 100 μL of 17% EDTA solution was added to terminate the DNase I enzyme activity and dissolve the nanocarrier particles. The content of the intact plasmid was detected by gel electrophoresis. The gel electrophoresis results are shown in Figure 2. Figure 4 As shown, compared with FeCl3-based MPN-DNA-HAp, ZnCl2-based MPN-DNA-HAp had a higher proportion of circular plasmids after DNase I treatment and had better stability.

[0061] Example 3 Stability of ZnCl2-based MPN-DNA-HAp nanocarrier particles in 10% fetal bovine serum (FBS) medium

[0062] Preparation of MPN-DNA-HAp nanocarrier particles: 10 μg of plasmid DNA (pEGFP-N1) was loaded onto the surface of 1 mg of HAp nanoparticles to form DNA-HAp nanoparticles. Subsequently, 3 μL of 24 mM TA solution and 3 μL of 60 mM ZnCl2 solution were added in sequence. The above solutions were mixed and diluted to a final volume of 1 mL with ultrapure water. The solution was vortexed for 5 minutes to obtain an MPN-DNA-HAp nanocarrier solution. After centrifugation at 8000 rpm, MPN-DNA-HAp nanocarrier particles were obtained.

[0063] The MPN-DNA-HAp nanocarrier particles were placed in 1 mL of culture medium containing 10% FBS. After 0-48 hours, the nanoparticles were collected by centrifugation at 8000 rpm. 100 μL of 17% EDTA was added to dissolve the nanoparticles. The plasmid content in the nanoparticles was detected by gel electrophoresis. Figure 5 It was shown that the MPN-DNA-HAp nanocarriers remained stable in 10% fetal bovine serum (FBS), and the MPN coating effectively protected plasmid DNA (pDNA) from degradation by enzymatic attack.

[0064] Example 4 DNA loading capacity and loading efficiency of MPN-DNA-HAp nanocarrier particles

[0065] To further explore the optimal loading ratio of plasmid DNA to hydroxyapatite, 5 μg, 10 μg, and 20 μg of plasmid DNA were loaded on the surface of 1 mg of HAp nanoparticles to form DNA-HAp nanoparticles. Then, 3 μL of 24 mM TA solution and 3 μL of 60 mM ZnCl2 solution were added in sequence. The above solutions were mixed and made up to a final volume of 1 mL with ultrapure water and vortexed for 5 minutes. The phenol ligands were combined with metal ions through chemical coordination-driven self-assembly, thereby successfully synthesizing MPN-DNA-HAp nanocarrier solution. After centrifugation at 8000 rpm, the supernatant (containing unloaded plasmid) and MPN-DNA-HAp nanocarrier particles were collected, and the nanocarrier particles were dissolved in 100 μL of 17% After EDTA solution, the supernatant and MPN-DNA-HAp nanocarrier particle solution were subjected to gel electrophoresis experiments to quantify the plasmid content in the supernatant and nanocarrier particles, and then calculate the loading capacity and loading efficiency of the nanocarrier particles loaded with DNA; loading capacity = DNA content in nanoparticles / 1mg HAp; loading efficiency = DNA content in nanoparticles / (DNA content in nanoparticles + DNA content in supernatant). Figure 6 As shown in the figure, when the mass ratio of DNA to HAp is 1:100, the MPN-DNA-HAp nanocarrier particles have both high loading capacity and loading efficiency.

[0066] Transmission electron microscopy (TEM) was used to characterize the morphology of the MPN-DNA-HAp nanocarrier particles and their particle size of approximately 200 nm. Energy dispersive X-ray spectroscopy (EDX) mapping confirmed that the MPN-DNA-HAp nanocarrier particles were successfully constructed. Figure 7 The main element composition of MPN-DNA-HAp is shown, including nitrogen element N representing plasmid DNA, oxygen element O, phosphorus element P, calcium element Ca representing HAp, and Zn element involved in the construction of metal polyphenol network.

[0067] Example 5 Verification of transfection efficiency of MPN-DNA-HAp nanocarrier particles

[0068] Preparation of MPN-DNA-HAp nanocarrier particles: 10 μg of plasmid DNA (pEGFP-N1) was loaded onto the surface of 1 mg of HAp nanoparticles to form DNA-HAp nanoparticles. Subsequently, 3 μL of 24 mM TA solution and 3 μL of 60 mM ZnCl2 solution were added in sequence. The above solutions were mixed and diluted to a final volume of 1 mL with ultrapure water. The mixture was vortexed for 5 minutes to obtain an MPN-DNA-HAp nanocarrier solution. After centrifugation at 8000 rpm, MPN-DNA-HAp nanocarrier particles were obtained. Free pEGFP-N1 and DNA-HAp nanoparticles without MPN coating were used as controls.

[0069] RAW264.7 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into 6-well plates (growth medium: DMEM + 10% FBS + 1% penicillin-streptomycin solution; culture conditions: 5% CO2, 37°C). After 24 hours, the cells were divided into four groups: blank group (cell growth medium without antibiotics); DNA group (pEGFP-N1 plasmid dissolved in cell growth medium without antibiotics at a concentration of 1 μg / mL); DNA-HAP group (DNA-HAP nanoparticles dissolved in cell growth medium without antibiotics at a concentration of 0.1 mg / mL); and DNA-HAP-MPN group (MPN-DNA-HAp nanocarrier particles dissolved in cell growth medium without antibiotics at a concentration of 0.1 mg / mL). After 12 hours of culture, 2 mL of treatment was added to each group. After 72 hours, cellular RNA was extracted using Trizol reagent. Gene expression in each group was analyzed using qRT-PCR. The results showed that the transfection efficiency of MPN-DNA-HAp nanocarrier particles was significantly higher than that of free pEGFP-N1 and DNA-HAp nanoparticles without MPN coating. Figure 8 It was shown that the EGFP gene expression was most significant in RAW264.7 cells transfected with MPN-DNA-HAp.

[0070] Example 6 DNA release curves of MPN-DNA-HAp nanocarriers under different pH environments

[0071] The preparation of MPN-DNA-HAp nanocarrier particles in this example is the same as that in Example 5.

[0072] The MPN-DNA-HAp nanocarrier particles were placed in 1 mL of PBS buffer at different pH conditions (7.4, 6.0, 5.5, and 4.7) at 37°C. The DNA release kinetics were quantitatively analyzed by agarose gel electrophoresis at preset time points to verify the acid-responsive disassembly properties of the MPN coating.

[0073] The results are as follows Figure 9 As shown, the MPN-DNA-HAp nanocarrier particles are highly pH-sensitive, with a release rate of 88.9% over 24 hours at pH 4.7, while the release rate at pH 7.4 is only 10% over 24 hours, indicating that the MPN-DNA-HAp nanocarriers are acid-responsive.

[0074] Example 7 Application of DNA-HAP-MPN Nanoparticles in Ligature-Induced SD Rat Periodontitis Model

[0075] Preparation of MPN-DNA-HAp: 10 μg of plasmid DNA (LL37 in pcDNA3.1(+)) was loaded onto the surface of 1 mg of HAp nanoparticles to form DNA-HAp nanoparticles. 3 μL of 24 mM TA solution and 3 μL of 60 mM ZnCl2 solution were added sequentially. The above solutions were mixed and diluted to a final volume of 1 mL with ultrapure water. The mixture was vortexed for 5 minutes to obtain MPN-pLL37-HAp nanocarriers. After centrifugation at 8000 rpm, MPN-pLL37-HAp nanocarrier particles were obtained.

[0076] The concentrations of components in the MPN-HAp group and pLL37-HAp group were the same as those in the MPN-pLL37-HAp group.

[0077] Each group was dissolved in PBS buffer to a concentration of 1 mg / mL for later use.

[0078] The plasmid DNA in MPN-pLL37-HAp of this example is LL37 in pcDNA3.1(+), hereinafter referred to as pLL37, and its nucleotide sequence is shown in SEQ ID NO.1.

[0079] LL37 is a human antimicrobial peptide (AMP) that has a significant ability to bind to bacterial membranes and inhibit bacterial activity, playing an important role in combating periodontal infections.

[0080] The present invention evaluated the application of apatite composite metal phenol co-delivery of plasmid (pLL37) expressing LL37 gene in ligature-induced periodontitis model of SD rats through in vivo experiments. In addition to the healthy control group, SD rats with ligature-induced periodontitis were further divided into 4 groups and received different treatments: the periodontitis group received PBS treatment after exposure to Porphyromonas gingivalis (P.gingivalis), and the MPN-HAp group, pLL37-HAp group and MPN-pLL37-HAp group received intragingival sulcus injection of corresponding nanocarrier particles, with an injection volume of 100 μL, once every 3 days. After 4 weeks of treatment, the hard tissues such as maxillary bones and teeth of the rats were collected for detailed analysis. Alveolar bone loss and root exposure were evaluated by micro-CT scanning. Figure 10 3D reconstruction images of hard tissue were displayed, showing that compared with the PBS-treated, MPN-HAp, and pLL37-HAp groups, the MPN-pLL37-HAp group had significantly less exposed tooth roots (orange-yellow area) and the best bone tissue regeneration and recovery. The MPN-pLL37-HAp-treated group demonstrated a favorable therapeutic effect.

[0081] To evaluate the regeneration of periodontal tissue, H&E staining and Masson staining were used. Figure 11 The H&E results showed that compared with the periodontitis group (PBS-treated group), MPN-HAp group and pLL37-HAp group, the MPN-pLL37-HAp-treated group had a smaller bone loss height and was not much different from the healthy group. In addition, Masson's results showed that in the healthy group, normal structural integrity of the periodontal tissue was observed, and no obvious pathological changes were observed. The alveolar ridge remained intact, with no signs of bone resorption or inflammatory cell infiltration. In contrast, the periodontitis group showed extensive inflammatory cell infiltration, accompanied by obvious destruction or breakage of periodontal fibers. Compared with the MPN-HAp group and the pLL37-HAp group, the blue collagen fibers in the MPN-pLL37-HAp group were neatly arranged, and periodontal tissue inflammation was significantly improved. Therefore, apatite-composite metal phenol synergistically delivered gene pLL37 nanoparticles have the effect of promoting tissue regeneration.

[0082] In summary, the apatite-composite metal phenol synergistic gene delivery system can deliver target genes safely, stably and efficiently, has good prospects in the field of gene therapy, can be well applied to periodontitis tissue repair, and provides new ideas for the treatment of periodontitis-related diseases.

[0083] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A metal polyphenol-hydroxyapatite nano-delivery system, characterized in that: The nano-delivery system comprises metal polyphenol and hydroxyapatite; wherein, nucleic acid molecules are loaded on the hydroxyapatite, and the hydroxyapatite is coated by the metal polyphenol; and the metal polyphenol is formed by the reaction of polyphenol molecules and metal ions.

2. The metal polyphenol-hydroxyapatite nano-delivery system according to claim 1, wherein: The polyphenol molecules are selected from tannic acid, gallic acid, propyl gallate, epigallocatechin gallate, catechin, and one or more combinations thereof; the metal ions are selected from Fe 3+ 、Ag + 、Zn 2+ Mg 2+ 、Cu 2+ Sr 3+ One of the soluble metal salt solutions.

3. The metal polyphenol-hydroxyapatite nano-delivery system according to claim 2, wherein: The polyphenol molecules are selected from tannic acid, and the metal ions are selected from Zn 2+ or Fe 3+ .

4. The metal polyphenol-hydroxyapatite nano-delivery system according to claim 1, wherein: The molar ratio of the polyphenol molecules to the metal ions is 1:2.

5.

5. The metal polyphenol-hydroxyapatite nano-delivery system according to claim 2, wherein: The nucleic acid molecule is selected from one or more of mRNA, siRNA, circleRNA, sgRNA, DNA, ecDNA, plasmid, and artificial nucleic acid.

6. The metal polyphenol-hydroxyapatite nano-delivery system according to claim 1, wherein: The nucleic acid molecule is selected from a plasmid encoding the LL37 gene whose nucleotide sequence is shown in SEQ ID NO.

1.

7. A method for preparing the metal polyphenol-hydroxyapatite nano-delivery system according to any one of claims 1 to 6, characterized in that: The following steps are involved: After nucleic acid and hydroxyapatite are mixed at a mass ratio of 1:(50-200), polyphenol molecule solution and metal ion solution are added in sequence.

8. The preparation method according to claim 7, wherein The concentration of the polyphenol molecule solution is 20-30 mM, and the concentration of the metal ion solution is 50-70 mM.

9. Use of the metal polyphenol-hydroxyapatite nanodelivery system according to any one of claims 1 to 6 in the preparation of nucleic acid delivery or gene therapy drugs, wherein the nucleic acid delivery refers to the introduction of nucleic acids into cells; preferably, the drug is used to treat a disease or condition in a mammal; further preferably, the disease or condition includes tissue inflammation infection.

10. The use according to claim 9, characterized in that The medicine has the following uses: (1) Used to reduce inflammation and infection of periodontal tissues; (2) Used to promote the regeneration and repair of periodontal tissues; (3) Used for bone tissue regeneration and recovery.

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