A composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions, its preparation method and applications

Abstract

The present invention discloses a composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions, its preparation method and application, which relates to the technical field of biomedicine, and specifically includes the following steps: S1: Hypoxic engineering treatment of human periodontal ligament stem cells; S2: Preparation of a functionalized membrane coating; S3: Preparation of the composite biomimetic nanomaterial. The composite biomimetic nanomaterial of the present invention can be used to target the periodontal inflammatory area, neutralize the over-accumulated virulence products and cytokines, improve the over-inflammatory state, and promote the control of periodontal tissue inflammation and disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a composite biomimetic nanomaterial having oral periodontal inflammation targeting and inflammatory neutralizing functions, a preparation method thereof, and an application thereof. Background Art

[0002] Periodontitis is the oral disease with the highest incidence rate at the present stage, mainly manifested as gingival inflammation, periodontal attachment loss, alveolar bone resorption, etc. In the late stage, tooth loosening, displacement and even loss occur, which is the main cause of tooth loss in adults. Its prevalence is high, the onset is hidden, and the consequences are serious, which have a great impact on the oral health, function, aesthetics and quality of life of patients. The latest research shows that periodontitis is closely related to systemic diseases such as cardiovascular diseases, diabetes, Alzheimer's disease, etc. Therefore, the prevention and treatment of periodontitis and the repair and regeneration of defective tissues are crucial and are difficult problems concerned all over the world.

[0003] From the perspective of etiology, periodontitis is a chronic infectious disease mainly caused by dental plaque biofilm. The accumulation of bacterial virulence products and the induced excessive immune response of the body are the key pathogenic mechanisms for the progressive destruction of periodontal tissues. During the occurrence of periodontitis, when the balance of host-biofilm changes, periodontal pathogens such as Porphyromonas gingivalis ( P. gingivalis, Pg ), Treponema denticola ( Treponema denticola ), and Tannerella forsythia ( Tannerella forsythia ), etc. colonize and grow in large numbers. The plaque biofilm formed by long-term accumulation, as the initiating factor, contains a large number of pathogenic microorganisms and their secreted virulence products. Lipopolysaccharide (LPS) is the most important pathogen-associated pattern molecule and virulence factor of the main periodontal pathogen Pg, which activates the toll-like receptor (TLR) of immune cells and then leads to a series of inflammatory reactions, and is closely related to periodontal tissue destruction and alveolar bone loss.

[0004] With the in-depth exploration of the immune microenvironment of periodontal tissues, it has now been proven that cytokine-mediated host immune responses play a key role in the destruction, development, and outcome of periodontal tissues. A large number of pathogenic microorganisms and their toxin products activate pattern recognition receptors and downstream signal transduction, stimulating the activation of immune cells such as neutrophils and macrophages in tissues and the secretion of pro-inflammatory cytokines. These excessive inflammatory factors are further amplified in a cascade reaction, recruiting and activating specific immune cells and forming an inflammatory cytokine storm in tissues, affecting osteoblast bone formation and osteoclast bone resorption. Among them, the TNF family, IL-1 family, IL-6 family, etc., as the most important inflammatory mediators, are secreted in large amounts under inflammatory conditions and participate in pathological processes such as immune responses, alveolar bone resorption, and connective tissue destruction, and are most closely related to the occurrence and development of chronic periodontitis. The long-term infiltration of a large number of pro-inflammatory cytokines leads to a decline in the regenerative potential of periodontal ligament stem cells (PDLSCs) and the long-term inflammatory polarization of immune cells, ultimately resulting in the persistence of the periodontal over-inflammatory state and difficulty in healing.

[0005] The basic treatment plan in the current clinical diagnosis and treatment plan for periodontitis is to mechanically remove local dental calculus, plaque, and bacteria by ultrasonic or manual scaling and root planing. Then, after several years of clinical practice, it has been proven that the tissue regeneration effect of basic periodontal treatment is very limited. On the one hand, the periodontitis environment is complex, with areas such as the bottom of the periodontal pocket, the root bifurcation area, and many areas that are difficult to reach with instruments. On the other hand, basic treatment can only eliminate dental plaque, but the bacterial virulence products accumulated in the microenvironment and the excessive inflammatory cytokines in the tissue reaction will not be cleared. Most of the existing periodontitis drugs in clinical practice are auxiliary treatment methods, and due to disadvantages such as short action time, low effective concentration, and large systemic side effects, effective and safe treatment drugs are still scarce.

[0006] Based on the fact that basic periodontal treatment and new therapies can only eliminate dental plaque, but the bacterial toxin products and inflammatory cytokines accumulated in the tissue reaction will not be cleared, it is necessary to suspend the immune system's response to bacteria in the gingival tissue by regulating the level of cytokines. However, at present, targeted strategies for inflammatory cytokines such as blocking antibodies and targeted inhibitors have the risks of high cost, single effect, and uncontrollable side effects, and there is an urgent need to develop a safe, efficient, and widely sourced targeted treatment strategy for periodontitis. Therefore, developing a targeted strategy that synergistically targets the key pathologies of periodontitis occurrence (virulence products) and development (a large number of cytokines), that is, a plan based on the etiology and progression of periodontal inflammation, and simultaneously neutralizing the virulence product LPS and pro-inflammatory cytokines, has great potential for periodontitis treatment and tissue regeneration. At the same time, aiming at the key problem that the physical environment of periodontitis is complex and traditional materials and treatment methods cannot accurately reach the lesion area, it is necessary to enhance the inflammatory targeting function of the treatment plan and accurately exert an anti-inflammatory effect. Summary of the Invention

[0007] In view of the above problems, the present invention provides a composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions, as well as a preparation method and application thereof, which solves the problem that in the prior art, periodontal basic treatment regimens can only eliminate dental plaque, while the bacterial virulence products accumulated in the inflammatory microenvironment and the excessive inflammatory cytokines in tissue reactions are not cleared.

[0008] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0009] The present invention provides a preparation method of a composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions, specifically including the following steps:

[0010] S1: Hypoxic engineering treatment of human periodontal ligament stem cells:

[0011] S11: Obtain periodontal ligament tissue from the surface of discarded human third molars or orthodontic tooth roots collected clinically, and isolate and culture periodontal ligament stem cells (PDLSCs);

[0012] S12: Place the P3-P5 generation of periodontal ligament stem cells in a three-gas incubator for hypoxic culture;

[0013] S2: Preparation of a functionalized membrane coating:

[0014] S21: Prepare a cell membrane protein extraction kit in advance, take membrane protein extraction reagent A, and add a protease inhibitor before use;

[0015] S22: Wash the periodontal ligament stem cells after hypoxic culture twice with ice-cold PBS, scrape the cells with a cell scraper, and directly centrifuge to collect the cell pellet;

[0016] S23: On ice, add 1 mL of membrane protein extraction reagent A containing a protease inhibitor to every 20 million cells, and gently blow to suspend the cells to obtain a cell suspension;

[0017] S24: Quick-freeze the cell suspension in S23 in liquid nitrogen, then thaw at 37 °C, take the cell suspension for trypan blue staining to detect the cell fragmentation degree, and stop freeze-thawing when the cell fragmentation degree is greater than 80%;

[0018] S25: Centrifuge the cell suspension after stopping freeze-thawing in S24 at 4 °C and 700 g for 10 min to collect the supernatant to obtain a solution containing cell membrane fragments, then centrifuge at 4 °C and 16,000 g for 30 min, discard the supernatant and take the precipitate to obtain cell membrane proteins;

[0019] S26: Resuspend the cell membrane proteins with an EDTA solution, disperse them by ultrasonic treatment at 4 °C, and store them at -80 °C;

[0020] S3: Preparation of the composite biomimetic nanomaterial:

[0021] S31: Mix the cell membrane protein obtained in step S26 evenly with the aqueous solution of hollow mesoporous manganese dioxide nanoparticles to obtain a cell membrane manganese dioxide nanoparticle mixed solution, and load it into a liposome extruder.

[0022] S32: Use the liposome extruder to extrude the cell membrane manganese dioxide nanoparticle mixed solution obtained in step S31, successively pass through polycarbonate membranes with pore sizes of 400 nm and 200 nm, and extrude repeatedly for 20 - 25 cycles to fully coat the nanoparticles with cell membrane proteins, obtaining a cell membrane-coated manganese dioxide nanoparticle mixed solution.

[0023] S33: Treat the cell membrane-coated manganese dioxide nanoparticle mixed solution obtained in step S32 under ultrasonic conditions, centrifuge, and wash to obtain the composite biomimetic nanomaterial (hPMNPs).

[0024] Further, in step S12, the O2 content in the triple-gas incubator is 2%, the CO2 content is 5%; the hypoxic culture time is 24 h.

[0025] Further, in step S21, the concentration of the membrane protein extraction reagent A with protease inhibitor added is 1 mM.

[0026] Further, in step S24, the quick-freezing time is 5 min, the thawing time is 3 min; the volume of the cell suspension for trypan blue staining is 5 - 10 μL.

[0027] Further, in step S26, the concentration of the EDTA solution is 0.2 mM.

[0028] Further, in step S31, the concentration of the aqueous solution of hollow mesoporous manganese dioxide nanoparticles is 1 mg / mL, the particle size of the nanoparticles is 150 - 200 nm; the mass ratio of the cell membrane protein to the aqueous solution of hollow mesoporous manganese dioxide nanoparticles is 2:1.

[0029] Further, in step S33, the ultrasonic time is 10 min, the ultrasonic frequency is 40 KHZ, the ultrasonic power is 100 W; the centrifugation speed is 5000 rpm, and the centrifugation time is 10 min.

[0030] The present invention also provides a composite biomimetic nanomaterial prepared by the preparation method of a composite biomimetic nanomaterial having oral periodontal inflammation targeting and inflammatory neutralization functions.

[0031] The present invention also provides an application of a composite biomimetic nanomaterial having oral periodontal inflammation targeting and inflammatory neutralization functions.

[0032] Compared with the prior art, the beneficial effects of the present invention:

[0033] In the present invention, human periodontal ligament stem cells are cultured under hypoxic conditions to obtain cell membranes, and a functionalized cell membrane coating is obtained. Then, it is compounded with hollow mesoporous manganese dioxide nanoparticles to prepare a composite biomimetic nanosystem in which human periodontal ligament stem cell membranes encapsulate manganese dioxide nanoparticles, which can be used to target the periodontal inflammatory region and neutralize the over-accumulated virulence products and cytokines. The composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions of the present invention can specifically target the periodontal inflammatory microenvironment, neutralize excessive pro-inflammatory cytokines and bacterial virulence product LPS in the environment, reverse the problem of decreased stem cell regeneration potential in the inflammatory microenvironment, and at the same time has good biocompatibility in vitro and in vivo, and is expected to be used as a new strategy for the treatment of periodontitis. Brief Description of the Drawings

[0034] Figure 1 It is a statistical chart of the transcriptional expression of surface characteristic molecules of PDLSCs after hypoxic culture detected by qRT-PCR in the present invention.

[0035] Figure 2 It is a TEM image of hPMNPs and MnO2 nanoparticles in the examples of the present invention, where: Figure 2 (A) is the TEM image of hPMNPs, Figure 2 (B) is the TEM image of MnO2 nanoparticles;

[0036] Figure 3 It is a particle size and potential analysis chart of hPMNPs and MnO2 nanoparticles obtained by a dynamic light scattering instrument (DLS) in the present invention, where: Figure 3 (A) is the hydrated particle size analysis chart, Figure 3 (B) is the Zeta potential analysis chart;

[0037] Figure 4 It is a fluorescence three-dimensional reconstruction image of hPMNPs and MnO2 nanoparticles obtained by Transwell experiments in the examples of the present invention, where: Figure 4 (A) is the fluorescence three-dimensional reconstruction image of MnO2 nanoparticles, Figure 4 (B) is the fluorescence three-dimensional reconstruction image of hPMNPs;

[0038] Figure 5 It is a neutralization curve chart of hPMNPs and MnO2 nanoparticles in the examples of the present invention neutralizing pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and LPS at gradient concentrations, where: Figure 5 (A) is the neutralization curve chart of TNF-α; Figure 5 (B) is the neutralization curve chart of IL-6; Figure 5 (C) is the neutralization curve chart of IL-1β; Figure 5(D) is the neutralization curve of TNF-α. Detailed implementation mode

[0039] In order to make the purpose and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0041] In this embodiment, the membrane protein extraction kit (containing membrane protein extraction reagent A, protease inhibitor, 0.4% trypan blue solution, etc.) was purchased from Wuhan Sanying Biotechnology Co., Ltd., brand Proteintech, product number PK10015; the aqueous solution of hollow mesoporous manganese dioxide (MnO2) nanoparticles was purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., product number XF158, with a concentration of 1 mg / mL and a particle size of 150-200 nm.

[0042] Example 1

[0043] This embodiment provides a preparation method of a composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions, specifically including the following steps:

[0044] S1: Hypoxic engineering treatment of human periodontal ligament stem cells:

[0045] S11: Obtain periodontal ligament tissue from the surface of discarded human third molars or orthodontic tooth roots collected clinically, and isolate and culture periodontal ligament stem cells (PDLSCs). Cells of passages P3-P5 are used in the subsequent process;

[0046] S12: Place P3-generation PDLSCs in a three-gas incubator containing 2% O2 and 5% CO2 for hypoxic culture; after hypoxic culture for 24 hours, collect the cells and detect the mRNA expression of characteristic surface molecules (CXCR4, IL-1R1, IL-6R, TNFR1, TLR2, TLR4) on PDLSCs by qRT-PCR. Among them, PDLSCs cultured under standard oxygen concentration of 20% O2 are used as the control group (see Experimental Example 1 for details).

[0047] S2: Preparation of functionalized membrane coating:

[0048] S21: Prepare a cell membrane protein extraction kit in advance. Take reagent A for membrane protein extraction and add a protease inhibitor before use to a final concentration of 1 mM.

[0049] S22: Wash the periodontal ligament stem cells that have completed hypoxic culture twice with ice-cold PBS. Scrape the cells with a cell scraper and directly centrifuge to collect the cell pellet.

[0050] S23: On ice, add 1 mL of reagent A for membrane protein extraction containing a protease inhibitor per 20 million cells, and gently blow to suspend the cells to obtain a cell suspension.

[0051] S24: Place the cell suspension after freeze-thawing in step S23 into liquid nitrogen for rapid freezing for 5 min, then thaw at 37 °C for 3 min. Take 5 - 10 μL of the cell suspension for trypan blue staining (0.4% trypan blue solution) to detect the degree of cell lysis. Stop freeze-thawing when the degree of cell lysis is greater than 80%.

[0052] S25: Centrifuge the cell suspension sample in step S24 at 4 °C and 700 g for 10 min to collect the supernatant, obtaining a solution containing cell membrane fragments. Then centrifuge at 4 °C and 16,000 g for 30 min, discard the supernatant, and take the precipitate to obtain the cell membrane protein.

[0053] S26: Resuspend the cell membrane protein with 0.2 mM EDTA solution, disperse it by ultrasonic treatment at 4 °C, and store it at -80 °C.

[0054] S3: Preparation of composite biomimetic nanomaterials:

[0055] S31: Mix the cell membrane protein obtained in step S26 evenly with an aqueous solution of hollow mesoporous manganese dioxide nanoparticles (concentration: 1 mg / mL, particle size: 150 nm) (the mass ratio of cell membrane protein to MnO₂ nanoparticles is 2:1) to obtain a cell membrane - manganese dioxide nanoparticle mixed solution, and load it into a liposome extruder.

[0056] S32: Use the liposome extruder to extrude the cell membrane - manganese dioxide nanoparticle mixed solution obtained in step S31 successively through polycarbonate membranes with pore sizes of 400 nm and 200 nm, and repeat extrusion 20 - 25 cycles to ensure that the nanoparticles are fully coated with the cell membrane protein, obtaining a cell membrane - coated manganese dioxide nanoparticle mixed solution.

[0057] S33: Treat the cell membrane - coated manganese dioxide nanoparticle mixed solution obtained in step S32 under ultrasonic conditions for 10 min (ultrasonic frequency: 40 KHZ, ultrasonic power: 100 W), then centrifuge at 5000 rpm for 10 min, and wash to obtain the composite biomimetic nanomaterials (hPMNPs).

[0058] This example also provides a composite biomimetic nanomaterial (hPMNPs) with oral periodontal inflammation targeting and inflammatory neutralization functions prepared by the above method.

[0059] This example also provides an application of a composite biomimetic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions.

[0060] Example 2

[0061] The difference from Example 1 is that the PDLSCs of passage 4 are used for hypoxic culture in step S12; in step S31, the particle size of the hollow mesoporous manganese dioxide nanoparticles is 175 nm.

[0062] Example 3

[0063] The difference from Example 1 is that the PDLSCs of passage 5 are used for hypoxic culture in step S12; in step S31, the particle size of the hollow mesoporous manganese dioxide nanoparticles is 200 nm.

[0064] Experimental Example 1

[0065] qRT-PCR was used to detect the transcriptional expression of surface characteristic molecules of PDLSCs after hypoxic culture:

[0066] (1) Experimental conditions: After hypoxic culture for 24 hours, cells were collected and the mRNA expression of characteristic surface molecules (CXCR4, IL-1R1, IL-6R, TNFR1, TLR2, TLR4) of PDLSCs was detected by qRT-PCR. Among them, PDLSCs cultured under the standard oxygen concentration of 20% O2 were used as the control group. The specific steps were as follows: After the cell culture was completed, cells were lysed with Trizol reagent and RNA was extracted. 1 μg of RNA was reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Kit reagent (reverse transcription conditions: 37 °C, 15 minutes; 85 °C, 5 seconds). The expression levels of related genes were detected using the SYBR ® Green Premix Pro Taq HS qPCR Kit and the ABI 7500 Real-time PCR system. The relative expression of mRNA was quantified using the 2 −ΔΔCt -ΔΔCt method, and endogenous GAPDH was used as an internal reference.

[0067] (2) Results: As Figure 1The results showed that compared with PDLSCs cultured under standard oxygen concentration conditions, the mRNA expression of characteristic surface molecules (CXCR4, IL-1R1, IL-6R, TNFR1, TLR2, TLR4) of PDLSCs cultured under hypoxia was significantly increased, and the results were statistically significant. The above results indicate that hypoxia treatment of PDLSCs provides a molecular basis for the targeting and neutralization functions required for functional membrane coatings.

[0068] Experimental Example 2

[0069] Transmission electron microscopy characterization of the morphological characteristics of hPMNPs:

[0070] (1) Experimental conditions: The above-mentioned MnO2 nanoparticles and hPMNPs prepared in Example 1 were diluted with deionized water to adjust the concentration to 20 μg / mL, 10 μL was added dropwise to the copper mesh, and then 10 μL of 2% phosphotungstic acid dye solution was added dropwise. After standing for 5 min, the excess dye solution was aspirated and dried at room temperature. The copper mesh loaded with the material sample was placed in the sample chamber, vacuumed, and then its morphology and structure were observed using a transmission electron microscope.

[0071] (2) Results: Figure 2 (A) shows that the shadow-like structure of the cell membrane coating is clearly observed on the surface of the MnO2 nanoparticles, with a thickness of about 10 nm; Figure 2 (B) shows that the MnO2 nanoparticles present a characteristic hollow mesoporous structure with uniform particle size and regular morphology; thus proving that the MnO2 nanoparticles are successfully encapsulated by the cell membrane, proving that the synthesis method used in this example has good feasibility.

[0072] Experimental Example 3

[0073] Dynamic light scattering detection of hPMNPs particle size potential distribution:

[0074] (1) Experimental conditions: The above-mentioned MnO2 nanoparticles and hPMNPs prepared in Example 1 were prepared into aqueous solutions with deionized water at a concentration of 1 mg / mL. 1 mL of each solution was added to a sample cuvette and loaded into a potential cell. The zeta potential and hydrated particle size of the MnO2 nanoparticles and hPMNPs were measured by dynamic light scattering (DLS).

[0075] (2) Results: Figure 3 (A) Dynamic light scattering analysis results show that MnO2 nanoparticles and hPMNPs carry negative potentials, with potentials of -29.8±0.36 mV and -27.96±0.89 mV, respectively. Figure 3 (B) Particle size analysis showed that the hydrated particle size of MnO2 nanoparticles was 267.6±5.8 nm, while after encapsulating the cell membrane, the hydrated particle size of hPMNPs increased to 297.9±9.32 nm.

[0076] Experimental Example 4

[0077] Evaluating the inflammatory targeting function of hPMNPs by Transwell assay:

[0078] (1) Experimental conditions: The Transwell assay was used to evaluate the ability of hPMNPs to target inflammation in an in vitro inflammatory environment of periodontitis. The specific steps were as follows: (A) Construction of the in vitro inflammatory environment of periodontitis: PDLSCs were cultured in an inflammatory medium (normal complete medium supplemented with 10 ng / mL TNF-α and 5 ng / mL IL-1β) for 3 days, and then the cells were washed 2 - 3 times with PBS and changed to normal complete medium without pro-inflammatory cytokines TNF-α and IL-1β for 24 hours. After 24 hours, the medium was collected, centrifuged at 4000 rpm for 10 min, and the supernatant was retained, which was defined as the in vitro inflammatory environment of periodontitis. (B) Detection of the targeting ability of hPMNPs: First, MnO2 nanoparticles were labeled with FITC dye (green fluorescence), and then the nanoparticles were coated with cell membranes to synthesize hPMNPs loaded with FITC dye. 500 μL of the medium collected in experimental condition (A) was placed in the lower chamber of the Transwell, and hPMNPs dispersed in 100 μL of type I rat tail collagen were inoculated in the upper chamber of the Transwell and co-incubated at 37°C for 2 hours. After 2 hours, the medium in the lower chamber of the Transwell was collected and transferred to a 96-well glass-bottom culture plate, and the content of green fluorescence was observed and photographed using a confocal microscope.

[0079] (2) Results: As Figure 4 (A) showed, no obvious green fluorescence was observed in the MnO2 nanoparticle group. In contrast, Figure 4 (B) more and stronger green fluorescence was observed in the hPMNPs group. The results indicate that the hPMNPs system wrapped with cell membranes, carrying CXCR4 protein on the surface, can respond to the periodontal inflammatory microenvironment and target to the inflammatory area, proving that the hPMNPs of the present invention have good inflammatory targeting ability.

[0080] Experimental Example 5

[0081] Evaluating the inflammatory neutralization function of hPMNPs by ELISA assay:

[0082] (1)Experimental conditions: ELISA was used to detect the neutralizing ability of MnO2 nanoparticles and hPMNPs at gradient concentrations against pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and LPS in an in vitro environment (serum-free medium containing 10% fetal bovine serum). The specific steps are as follows: TNF-α (10 ng / mL), IL-6 (5 ng / mL), IL-1β (5 ng / mL), and LPS (10 ng / mL) were respectively mixed with MnO2 nanoparticles and hPMNPs at gradient concentrations (final concentrations were 4, 16, 64, 128, and 256 μg / mL), and co-incubated at 37 °C for 2 hours. The precipitated nanoparticles bound were removed by centrifugation at 20000 rpm for 10 min, and the supernatant was retained. The cytokine content in it was detected according to the steps of the ELISA detection kit respectively.

[0083] (2)Results: The ELISA results are as Figure 5 (A)- Figure 5 (D)showed that compared with pure MnO2 nanoparticles, the content of pro-inflammatory cytokines and LPS in the culture medium supplemented with hPMNPs decreased significantly, and with the increase of hPMNPs concentration, the neutralizing ability increased significantly. The above results indicate that hPMNPs have a significantly enhanced neutralizing ability against pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and LPS in the in vitro environment, demonstrating that the present invention has good inflammatory neutralizing ability.

[0084] Furthermore, the hPMNPs prepared in Example 2 and Example 3 were respectively subjected to the experiments of Experimental Examples 1-5, and the same experimental results as those in Example 1 were obtained.

[0085] In summary, this embodiment is based on the key technical problem of the accumulation of bacterial virulence products and the excessive immune response of the body induced by them in the periodontal inflammatory microenvironment, generating a large number of pro-inflammatory cytokines. Based on the basic principle of natural cell membrane-coated biomimetic nanoparticles, a biomimetic system of engineered human periodontal ligament stem cell membrane-coated manganese dioxide nanoparticles was constructed. Through hypoxia pretreatment, the cell membrane coating highly expresses chemokine receptor CXCR4, LPS-binding receptor TLR4, TLR2, and inflammatory cytokine receptors TNFR1, IL-1R1, IL-6R, which can be used to target the periodontal inflammatory microenvironment and neutralize the virulence products and cytokines that have accumulated excessively in the periodontal inflammatory environment, improve the excessive inflammatory state, and promote the control of periodontal tissue inflammation and disease treatment.

[0086] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite bionic nanomaterial with oral periodontal inflammation targeting and inflammation neutralization functions, characterized in that: The specific steps include: S1: Hypoxia-engineered treatment of human periodontal ligament stem cells: S11: Periodontal ligament tissue was obtained from the root surface of discarded human third molars or orthodontic teeth collected clinically, and periodontal ligament stem cells were isolated and cultured; S12: placing the P3-P5 generation periodontal ligament stem cells in a three-gas incubator for hypoxic culture; the three-gas incubator has an O2 content of 2% and a CO2 content of 5%; and the hypoxic culture time is 24 hours; S2: Preparation of functionalized membrane coating: S21: Prepare a cell membrane protein extraction kit in advance, take a membrane protein extraction reagent A, and add a protease inhibitor before use; the cell membrane protein extraction kit was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., brand Proteintech, item number PK10015; the concentration of the membrane protein extraction reagent A with the protease inhibitor added is 1 mM; S22: After the hypoxic culture, the periodontal ligament stem cells were washed twice with ice-cold PBS, the cells were scraped off with a cell scraper, and the cell pellets were directly collected by centrifugation; S23: On ice, add 1 mL of membrane protein extraction reagent A containing protease inhibitors for every 20 million cells, and gently blow to suspend the cells to obtain a cell suspension; S24: The cell suspension in S23 was quickly frozen in liquid nitrogen, and then thawed at 37°C. The cell suspension was taken for trypan blue staining to detect the degree of cell disruption. When the degree of cell disruption was greater than 80%, the freezing and thawing was stopped. S25: The cell suspension after freeze-thawing in S24 was centrifuged at 4°C and 700g for 10 min to collect the supernatant to obtain a solution containing cell membrane fragments, and then centrifuged at 4°C and 16000g for 30 min, the supernatant was discarded and the precipitate was obtained to obtain cell membrane protein; S26: Resuspend the cell membrane proteins with EDTA solution, disperse them by ultrasonic at 4°C, and store them at -80°C; S3: Preparation of composite bionic nanomaterials: S31: uniformly mixing the cell membrane protein obtained in step S26 with the aqueous solution of hollow mesoporous manganese dioxide nanoparticles to obtain a cell membrane manganese dioxide nanoparticle mixed solution, and loading the solution into a liposome extruder; S32: using a liposome extruder to extrude the cell membrane manganese dioxide nanoparticle mixed solution obtained in step S31, and sequentially passing through a 400 nm and a 200 nm polycarbonate membrane, and repeatedly extruding for 20 to 25 cycles, so that the nanoparticles are fully coated with cell membrane proteins, to obtain a cell membrane coated manganese dioxide nanoparticle mixed solution; S33: The mixed solution of cell membrane-coated manganese dioxide nanoparticles obtained in step S32 is treated under ultrasonic conditions, centrifuged, and washed to obtain a composite bionic nanomaterial.

2. The method for preparing a composite bionic nanomaterial having oral periodontal inflammation targeting and inflammation neutralization functions according to claim 1, characterized in that: In step S24, the quick freezing time is 5 minutes, and the thawing time is 3 minutes; the volume of the cell suspension for trypan blue staining is 5-10 μL.

3. The method for preparing a composite bionic nanomaterial having oral periodontal inflammation targeting and inflammation neutralization functions according to claim 1, characterized in that: In step S26, the concentration of the EDTA solution is 0.2 mM.

4. The method for preparing a composite bionic nanomaterial with oral periodontal inflammation targeting and inflammation neutralization functions according to claim 1, characterized in that: In the step S31, the concentration of the hollow mesoporous manganese dioxide nanoparticle aqueous solution is 1 mg / mL, the particle size of the nanoparticles is 150-200 nm, and the mass ratio of the cell membrane protein to the hollow mesoporous manganese dioxide nanoparticle aqueous solution is 2:

1.

5. The method for preparing a composite bionic nanomaterial with oral periodontal inflammation targeting and inflammation neutralization functions according to claim 1, characterized in that: In step S33, the ultrasonic time is 10 minutes, the ultrasonic frequency is 40 kHz, the ultrasonic power is 100 W, the centrifugal speed is 5000 rpm, and the centrifugal time is 10 minutes.

6. A composite bionic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions prepared by the preparation method of the composite bionic nanomaterial with oral periodontal inflammation targeting and inflammatory neutralization functions as described in any one of claims 1 to 5.

7. Use of the composite bionic nanomaterial as claimed in claim 6 in the preparation of a composite bionic nanomaterial with oral periodontal inflammation targeting and inflammation neutralization functions, characterized in that: The composite bionic nanomaterial is used to target the periodontal inflammatory microenvironment and neutralize the excessively accumulated toxic products and cytokines in the periodontal inflammatory environment, thereby improving the excessive inflammatory state.

Images