Titanium-modified zingiber officinale exosome-like nanoparticles for periodontitis treatment and applications

Abstract

The application discloses titanium-modified ginger exosome-like nanoparticles for periodontitis treatment and application, and belongs to the field of oral drug preparations. The titanium-modified ginger exosome-like nanoparticles are prepared by using an atomic layer deposition technology to modify titanium atoms on the surface of ginger exosome-like nanoparticles, so as to form titanium-modified ginger exosome-like nanoparticles with controllable titanium atom layers on the surface of the ginger exosome-like nanoparticles. The application finds through experiments that the titanium-modified ginger exosome-like nanoparticles are mainly taken into periodontal ligament fibroblasts in a caveolin-dependent manner, greatly improving the exosome uptake efficiency of the periodontal ligament fibroblasts, and compared with unmodified ginger exosome-like nanoparticles, the titanium-modified ginger exosome-like nanoparticles have lower usage and better treatment effect under the condition of the same concentration.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of oral drug preparations, in particular to a titanium-modified ginger exosome-like nanoparticle for periodontitis treatment and application. BACKGROUND

[0002] Periodontitis is a chronic inflammation caused by the antagonism between pathogenic microorganisms in subgingival biofilm and various immune cells in tissues. In the pathogenesis of periodontitis, the host immune response produces a large amount of reactive oxygen species (ROS). High levels of ROS not only promote immune cells to continuously release pro-inflammatory cytokines to aggravate inflammation, but also cause damage to periodontal membrane fibroblasts and tissues, resulting in periodontal tissue defects. Ginger is a natural medicinal material with anti-inflammatory and antioxidant properties and other medicinal values. Its components have been extracted as an antioxidant to treat periodontitis and promote the regenerative repair ability of periodontal tissue. However, the raw material of ginger has a pungent and irritating taste, and its crude extract has poor stability in the body and low bioavailability, with an average terminal half-life ranging from 7.23 to 8.5 minutes. Therefore, how to efficiently utilize the antioxidant efficacy of ginger to promote the repair of periodontal tissue defects has become a problem to be solved.

[0003] Ginger exosome-like nanoparticles (GELNs) have been confirmed to be a natural nanocarrier rich in lipids, proteins, RNA and other active molecules, with a size ranging from 50 to 500 nm. GELNs have good biocompatibility, high solubility and permeability, and low immunogenicity. A patent has disclosed that the miRNA in ginger exosome-like nanoparticles can adjust inflammatory factors in cells and has anti-inflammatory function. However, there is no related report on improving the biological activity of ginger exosome-like nanoparticles through surface modification and using them for the treatment of periodontitis.

[0004] Surface modification of exosomes is a common method to change their activity. At present, there are mainly the following methods for surface modification of exosomes. 1. Modify the parent cells through genetic engineering, etc. to indirectly change the function of exosomes, which requires complicated procedures and expensive equipment, the types of surface proteins that can be loaded on exosomes are limited, and the phenotype of secreted exosomes is uncontrollable. 2. Click chemistry and covalent bond combination can only connect the modifier with azido group to the surface of exosomes through catalytic reaction, and it is difficult to control the loading amount on the surface of exosomes. Excessive loading will reduce the structural stability of exosomes. 3. Non-covalent binding includes multivalent electrostatic interaction, hydrophobic interaction, anchor peptide modification and aptamer modification, which also has the shortcomings of limited types of modifiers and difficult to control the amount of exosome surface modification, and cannot accurately regulate the uptake efficiency of exosomes. Therefore, there is an urgent need for an exosome surface modification method that can accurately regulate, has a wide range of modifiers, and is easy to operate, to improve the function of ginger exosomes. SUMMARY

[0005] The purpose of the present application is to provide a titanium-modified ginger exosome-like nanoparticle for periodontitis treatment and application to solve the problems existing in the prior art. Titanium atoms are modified on the surface of ginger exosome-like nanoparticles by atomic layer deposition technology, and the number of layers of titanium atoms on the surface of the ginger exosome-like nanoparticles can be accurately controlled by adjusting the number of cycles, thereby improving the uptake efficiency of periodontal membrane fibroblasts.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a titanium-modified ginger exosome-like nanoparticle, which is prepared by modifying titanium atoms on the surface of ginger exosome-like nanoparticles using atomic layer deposition technology to form a titanium-modified ginger exosome-like nanoparticle with controllable number of titanium atom layers on the surface of the ginger exosome-like nanoparticle.

[0008] Preferably, the preparation method of the ginger exosome nanoparticle comprises the following steps:

[0009] Exosomes in ginger are extracted and separated to obtain ginger exosomes.

[0010] The ginger exosomes are placed in a discontinuously dispersed sucrose gradient solution, centrifuged, and the precipitate is collected to obtain ginger exosome-like nanoparticles.

[0011] Preferably, the extraction and separation method of the ginger exosomes comprises the following steps:

[0012] The ginger is squeezed to remove the ginger residue, and the ginger juice is collected. The ginger juice is centrifuged to remove impurities, and the supernatant is collected.

[0013] The supernatant is subjected to 150,000-200,000g ultracentrifugation for 60-90min, the supernatant is discarded, and the precipitate is collected and resuspended to obtain ginger exosome.

[0014] Preferably, the ginger exosome is placed in a sucrose gradient solution with a mass concentration of 8%, 30%, 45% and 60% discontinuous dispersion, and subjected to 150,000-200,000g ultracentrifugation for 1-2h, and the precipitate at the critical interface of the 30% and 45% sucrose gradient solution is collected to obtain ginger exosome-like nanoparticles.

[0015] Preferably, the preparation method of the titanium atom layer controllable titanium modified ginger exosome-like nanoparticles comprises the following steps:

[0016] The freeze-dried powder of the ginger exosome-like nanoparticles is placed in a reactor, and titanium tetraisopropoxide and deionized water are alternately introduced into the vacuum reaction cavity of the reactor with nitrogen as the carrier gas to react, so that titanium atoms are deposited on the surface of the ginger exosome-like nanoparticles to prepare titanium atom layer controllable titanium modified ginger exosome-like nanoparticles.

[0017] Preferably, after the titanium tetraisopropoxide is introduced into the vacuum reaction cavity for 0.2s, it is waited for 3s, the vacuum reaction cavity is purged with nitrogen for 15s, then deionized water is introduced for 0.04s, it is waited for 3s, the vacuum reaction cavity is purged with nitrogen for 15s, and the cavity pressure of the vacuum reaction cavity is 10-200Pa; the above operation is repeated n times to obtain titanium modified ginger exosome-like nanoparticles with n layers of titanium atom layers; n is a natural number greater than or equal to 1.

[0018] Preferably, the temperature of the reaction is 35-40℃.

[0019] Preferably, the freeze-dried powder of the ginger exosome-like nanoparticles is formed by mixing the ginger exosome-like nanoparticles with a freeze-drying protective agent and freeze-drying; wherein the freeze-drying protective agent is a mixture of sucrose and mannitol in a mass ratio of 1:1.

[0020] The application also provides the use of the titanium modified ginger exosome-like nanoparticles in the preparation of a drug for treating periodontitis.

[0021] The application also provides the use of the titanium modified ginger exosome-like nanoparticles in the preparation of a drug for improving the uptake efficiency of periodontal ligament fibroblasts.

[0022] The application discloses the following technical effects:

[0023] The application utilizes atomic layer deposition technology to prepare Ti element of atomic level controllable on the surface of ginger exosomes, so that the ginger-derived exosomes are mainly taken into the periodontal ligament fibroblasts in a caveolin-dependent manner, wherein the 30Ti@GLENs greatly improve the uptake efficiency of the periodontal ligament fibroblasts to the exosomes, and compared with the unmodified exosomes, the use amount is lower and the treatment effect is better under the same concentration. The titanium-modified ginger exosome-like nanoparticles prepared by the application can be used in the treatment of periodontitis.

[0024] The Ti element modification on the surface of the ginger exosome-like nanoparticles in the application is realized by the atomic layer deposition method. The scheme is a self-limiting reaction, which forms a chemical connection with the hydroxyl group on the surface protein of the exosome. Compared with the existing scheme, the number of cycles can be adjusted to accurately control the number of surface modification layers and improve the uptake efficiency. The application provides a more optimal nanoparticle for improving the efficiency of periodontal ligament fibroblasts to uptake exosomes, and provides a theoretical basis for guiding the clinical treatment of periodontitis. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0026] Figure 1 TEM characterization results of GELNs of the application;

[0027] Figure 2 Zeta potential diagram of GELNs of the application;

[0028] Figure 3 SEM characterization results and Ti element content detection results of Ti@GELNs of the application; a: SEM diagram of ginger exosome nanoparticles GELNs without surface titanium atom modification; b: SEM diagram of Ti@GELNs; c: Ti element content diagram of EDS detection of Ti@GELNs;

[0029] Figure 4 Fluorescence density diagram of flow cytometry detection of different ginger exosome nanoparticles taken by periodontal ligament fibroblasts for 12h;

[0030] Figure 5 Fluorescence picture of laser confocal microscope detection of different ginger exosome nanoparticles taken by periodontal ligament fibroblasts for 12h; a: GELNs; b: 10Ti@GELNs; c: 30Ti@GELNs; d: 50Ti@GELNs;

[0031] Figure 6 Figure 7 is a fluorescence image of ROS content in periodontal ligament fibroblasts after treatment with different ginger exosome nanoparticles under periodontitis environment according to the present application; a: GELNs; b: 10Ti@GELNs; c: 30Ti@GELNs; d: 50Ti@GELNs;

[0032] Figure 7 Figure 8 is a fluorescence image of ROS content in periodontal ligament fibroblasts after treatment with different ginger exosome nanoparticles under periodontitis environment according to the present application, detected by flow cytometry;

[0033] Figure 8 Figure 9 is a schematic diagram of the preparation and application mechanism of titanium-modified ginger exosome-like nanoparticles according to the present application. DETAILED DESCRIPTION

[0034] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is made with reference to the accompanying drawings, wherein like reference numerals refer to like elements throughout the various figures. The present application is described with reference to the following examples.

[0035] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values of the parameter are combined with a stated value of the parameter in range form. Furthermore, each individual value of the parameter in the range and any other stated or intervening value of the parameter is incorporated into the scope of the present application. The same applies to ranges of values of other parameters.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated by reference, the content of the specification prevails.

[0037] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application in any way.

[0038] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or the like are open-ended terms that are intended to mean including, but not limited to.

[0039] Example 1

[0040] 1. Obtaining ginger exosomes

[0041] 1.1 Removing impurities: Wash the fresh ginger and obtain ginger juice using a juicer. Filter the ginger residue with a mesh strainer and place it on ice. Collect the ginger juice and place it in a pre-cooled centrifuge. Centrifuge at 4°C and 4000g for 30 min to remove fibers and large particles. Centrifuge the supernatant at 4°C and 10000g for 60 min to remove cell debris and other impurities.

[0042] 1.2 Preliminary separation of exosomes: Take the supernatant after centrifugation and place it in a centrifuge tube for ultracentrifugation. Tighten the ultracentrifuge tube cover and weigh it to balance the mass. The mass difference of each tube should not exceed 0.01g. After balancing, centrifuge at 4°C and 150000g for 90 min in an ultracentrifuge. Discard the supernatant and resuspend the precipitate with an appropriate amount of PBS to obtain ginger exosomes.

[0043] 1.3 Purification of ginger exosomes using density gradient centrifugation: Transfer the above-mentioned suspension to a discontinuous sucrose gradient (8%, 30%, 45%, and 60%) solution and centrifuge at 150,000g for 1.5h. Collect the layer between 30% / 45%. Add an equal volume of PBS to the 45% sucrose solution and centrifuge at 150000g for 1h. Collect the precipitate and wash away the sucrose to obtain ginger exosome-like nanoparticles (GELNs). Resuspend the GELNs precipitate with 200μL sterile PBS, measure the protein concentration using a BCA protein detection kit, and store at -80°C for further use.

[0044] 2. Freeze-drying of exosomes

[0045] 2.1 Select sucrose and mannitol as freeze-drying protectants. Add sucrose and mannitol (mass ratio 1:1, sucrose and mannitol added amount 50mg, 100mg and 150mg respectively) to 5mL PBS, dissolve at 55°C, resuspend the exosome precipitate in the above-mentioned 1.3 using the freeze-drying protectant solution, transfer to a 20mL vial, seal, and place in a -80°C freezer for rapid pre-freezing for 2h.

[0046] 2.2 The cold trap of the freeze-dryer is lowered to below -35°C in advance. Transfer the pre-frozen ginger exosomes to the freeze-dryer and freeze-dry for 30h.

[0047] The results show that when the added amounts of sucrose and mannose are 150mg, good freeze-drying morphology can be obtained.

[0048] 3. Preparation of GELNs with surface titanium atom modification

[0049] Take GELNs freeze-dried powder 5 g, placed in a vibration fluidized bed reactor. Using N2 as carrier gas, titanium tetraisopropoxide and deionized water are introduced into the reaction chamber alternately, the reaction temperature is 37℃. After 0.2s of titanium tetraisopropoxide is introduced, wait for 3s, purify the reaction cavity by N2 with a purity of 99.999% for 15s, then introduce deionized water for 0.04s, wait for 3s, purify the reaction cavity by N2 with a purity of 99.999% for 15s, the cavity pressure of the vacuum reaction cavity is 10-200Pa; the process is repeated for 10, 30, 50 times respectively, and 10Ti@GELNs, 30Ti@GELNs, 50Ti@GELNs are obtained respectively. After the deposition is completed, they are respectively filled into a Westlin bottle and sealed for use.

[0050] 4. Characterization of ginger exosome-like nanoparticles

[0051] 4.1 Identification of ginger exosome-like nanoparticles

[0052] Take 10μL of the PBS resuspension of ginger exosome-like nanoparticles in 1.3 above, drop it on a copper mesh and precipitate for 1min, absorb the supernatant with filter paper, drop 10μL of uranyl acetate on the copper mesh and precipitate for 1min, absorb the supernatant with filter paper, dry for several minutes at room temperature, and perform transmission electron microscopy imaging at 100kV. The results are shown in Figure 1 , which are concave structures with disc or hemispherical shapes, with a particle size of 150nm.

[0053] Take 1μL of the PBS resuspension of ginger exosome-like nanoparticles in 1.3 above, dilute it 30000 times with PBS, take 1mL of the sample, and use a Zeta potential instrument to detect it. The results are shown in Figure 2 , with a potential of -38.0mV.

[0054] 4.2 SEM characterization

[0055] Use a scanning electron microscope to take pictures of the surface morphology of GELNs and 30Ti@GELNs after freeze-drying. The results are shown in Figure 3 A and B, which are 150nm disc-shaped;

[0056] Use an energy dispersive X-ray spectrometer (EDS) to detect the Ti element content of 30Ti@GELNs. The results are shown in Figure 3 C, which shows that Ti is successfully modified on GELNs.

[0057] 5. Flow cytometry detection of the uptake efficiency of Ti@GELNs by periodontal membrane fibroblasts

[0058] Take 21 mg of 10Ti@GELNs, 30Ti@GELNs and 50Ti@GELNs respectively, add 1 mL of Dil dye to dissolve the exosomes, dye for 0.5 h, add 9 mL of PBS to wash off the dye, centrifuge at 10,000 g to remove excess dye, resuspend with 1 mL of PBS, add the exosome suspension to the cell culture medium at a ratio of 1:20 to replace the cell culture medium, incubate at 37°C for 12 h, discard the cell culture medium, trypsinize the periodontal ligament fibroblasts, resuspend the cells with 500 μL of PBS, and use a flow cytometer to detect the fluorescence density value of each group. The periodontal ligament fibroblasts cultured without exosome suspension are used as a control.

[0059] The results are shown in Figure 4 Compared with the control group, the uptake of 30Ti@GELNs was significantly increased.

[0060] 6. Laser confocal microscope detection of Ti@GELNs uptake by periodontal ligament fibroblasts

[0061] Take 21 mg of 10Ti@GELNs, 30Ti@GELNs and 50Ti@GELNs respectively, add 1 mL of Dil dye to dissolve the exosomes, dye for 0.5 h, add 9 mL of PBS to wash off the dye, centrifuge at 10,000 g to remove excess dye, resuspend with 1 mL of PBS, add the exosome suspension to the cell culture medium at a ratio of 1:20 to replace the cell culture medium, incubate at 37°C for 12 h, discard the cell culture medium, PBS rinse twice, methanol fixation of cells, FITC labeling of cytoskeleton, DAPI labeling of nucleus, then resuspend the cells with 500 μL of PBS, and observe the uptake of exosomes by laser confocal microscope.

[0062] The results are shown in Figure 5 Compared with the GELNs, 10Ti@GELNs and 50Ti@GELNs groups, more 30Ti@GELNs were taken up by fibroblasts.

[0063] 7. Ti@GELNs reduce ROS of periodontal ligament fibroblasts in periodontitis environment

[0064] Periodontal ligament fibroblasts were seeded in 6-well plates at a density of 9.5 x 10 5DCFH-DA detection results are shown in FIG. 6. Compared with the GELNs, 10Ti@GELNs and 50Ti@GELNs groups, the ROS content of periodontal ligament fibroblasts after 30Ti@GELNs treatment decreased significantly. Flow cytometry detection results are shown in FIG. 7. After 30Ti@GELNs treatment, the ROS fluorescence density decreased significantly.

[0065] DCFH-DA detection results are shown in FIG. 6. Compared with the GELNs, 10Ti@GELNs and 50Ti@GELNs groups, the ROS content of periodontal ligament fibroblasts after 30Ti@GELNs treatment decreased significantly. Flow cytometry detection results are shown in FIG. 7. After 30Ti@GELNs treatment, the ROS fluorescence density decreased significantly. Figure 6 Figure 7 DCFH-DA detection results are shown in FIG. 6. Compared with the GELNs, 10Ti@GELNs and 50Ti@GELNs groups, the ROS content of periodontal ligament fibroblasts after 30Ti@GELNs treatment decreased significantly. Flow cytometry detection results are shown in FIG. 7. After 30Ti@GELNs treatment, the ROS fluorescence density decreased significantly.

[0066] As can be seen from the above examples, the present application provides a titanium-modified ginger exosome-like nanoparticle, which uses atomic layer deposition technology to prepare atomically controllable Ti elements on the surface of ginger exosomes, so that ginger-derived exosomes are mainly taken into PDLSCs cells in a caveolin-dependent manner. Among them, 30Ti@GLENs greatly improves the uptake efficiency of periodontal fibroblasts to exosomes. Compared with unmodified exosomes, the use amount is lower and the treatment effect is better under the same use amount. The surface Ti element of the titanium-modified ginger exosome-like nanoparticle is modified by the atomic layer deposition method. This scheme is a self-limiting reaction, which forms a chemical connection with the hydroxyl group on the surface protein of the exosome. Compared with the existing scheme, the number of surface modification layers can be accurately controlled by adjusting the number of cycles, thereby improving the uptake efficiency. The schematic diagram of the above technical scheme is shown in FIG. 8. Figure 8 .

[0067] The above-described embodiments are only descriptions of preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.​

Claims

1. A titanium-modified ginger exosome-like nanoparticle, characterized in that, The titanium-modified ginger exosome-like nanoparticles are titanium atoms with controllable layer numbers on the surface of the ginger exosome-like nanoparticles, which are prepared by atomic layer deposition technology. The preparation method of the ginger exosome-like nanoparticles comprises the following steps: extracting and separating exosomes from ginger to obtain ginger exosomes; The ginger exosomes are placed in a sucrose gradient solution with a mass concentration of 8%, 30%, 45% and 60% discontinuous dispersion, and 150,000-200,000 g ultracentrifugation is performed for 1-2 h. The precipitate at the critical interface of the 30% and 45% sucrose gradient solution is collected to obtain ginger exosome-like nanoparticles. The preparation method of the titanium-modified ginger exosome-like nanoparticles with controllable titanium atom layers comprises the following steps: The freeze-dried powder of the ginger exosome-like nanoparticles is mixed with a freeze-drying protective agent to form a freeze-dried product; wherein the freeze-drying protective agent is a mixture of sucrose and mannitol with a mass ratio of 1:

1. The extraction and separation method of the ginger exosomes comprises the following steps:

2. The titanium-modified ginger exosome-like nanoparticle of claim 1, wherein, The ginger juice is filtered to remove ginger residues, and the ginger juice is collected. The ginger juice is centrifuged to remove impurities, and the supernatant is collected. The supernatant is subjected to 150,000-200,000 g ultracentrifugation for 60-90 min, the supernatant is discarded, the precipitate is resuspended, and ginger exosomes are obtained. After the titanium tetraisopropoxide is introduced into the vacuum reaction cavity for 0.2 s, it is waited for 3 s, the vacuum reaction cavity is purged with nitrogen for 15 s, then deionized water is introduced for 0.04 s, it is waited for 3 s, and the vacuum reaction cavity is purged with nitrogen for 15 s. The cavity pressure of the vacuum reaction cavity is 10-200 Pa; repeat the above operation n times to obtain titanium-modified ginger exosome-like nanoparticles with n layers of titanium atoms; n is a natural number greater than or equal to 1.

3. The titanium-modified ginger exosome-like nanoparticle of claim 1, wherein, The temperature of the reaction is 35-40℃.

4. The titanium-modified ginger exosome-like nanoparticle of claim 1, wherein, 5. Use of the titanium-modified ginger exosome-like nanoparticles according to any one of claims 1-4 in the preparation of a drug for treating periodontitis.

6. Use of the titanium-modified ginger exosome-like nanoparticles according to any one of claims 1-4 in the preparation of a drug for improving the uptake efficiency of periodontal membrane fibroblasts. ​

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