Antibacterial slow-release microcapsules for the treatment of periodontitis and a method for their preparation

Abstract

The application belongs to the field of periodontal tissue damage repair materials, and provides an antibacterial sustained-release microcapsule for treating periodontitis and a preparation method thereof. The antibacterial sustained-release microcapsule for treating periodontitis is composed of a capsule wall and a core material wrapped inside the capsule wall, the capsule wall is composed of a calcium alginate matrix, nanocellulose microcrystals distributed in the calcium alginate matrix, and platinum ions combined with the calcium alginate matrix and the nanocellulose microcrystals, and the core material is an aqueous solution of biocompatible polyelectrolyte containing human periodontal ligament stem cell exosomes. The microcapsule of the application releases the antibacterial component platinum ions and the repair component human periodontal ligament stem cell exosomes in an orderly manner, realizes the sustained release of the repair component on the basis of improving the local inflammatory microenvironment, and is beneficial to promoting the repair and reconstruction of periodontal supporting tissue damage.

Description

Technical Field

[0001] This invention belongs to the field of periodontal tissue damage repair materials, and relates to antibacterial sustained-release microcapsules for the treatment of periodontitis and their preparation method. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by bacteria such as *Porphyromonas gingivalis* in dental plaque invading periodontal tissues. It is characterized by the loss of periodontal supporting tissues and is a leading cause of tooth loosening and loss in adults. Conventional clinical treatments for periodontitis include supragingival scaling, subgingival scaling, and root planing; however, these methods cannot completely cure periodontitis. Their core goal is to control inflammation and halt disease progression. The focus and challenge of periodontal regenerative tissue engineering lies in the repair and reconstruction of damaged periodontal supporting tissues. Current technologies primarily use biomaterials to fill the defective areas to replace periodontal supporting tissues, but they cannot achieve true tissue regeneration.

[0003] To achieve tissue regeneration, researchers have proposed stem cell-based tissue engineering. For example, in recent years, researchers have successfully isolated oral mesenchymal stem cells from the pulp tissue of discarded teeth. Due to their low invasiveness and minimal ethical concerns during collection, they have become one of the ideal cell donors for stem cell clinical therapy. For periodontal tissue regeneration and repair, since bone regeneration is the foundation of periodontal tissue regeneration, the prerequisite for periodontal tissue regeneration is stimulating periodontal ligament stem cells to differentiate into osteogenic cells. Although periodontal ligament stem cells are derived from periodontal ligament tissue and are easy to obtain with excellent osteogenic differentiation characteristics, ethical and immune rejection issues still need to be addressed before they can be applied to clinical treatment.

[0004] Exosomes, as paracrine mediators between mesenchymal stem cells and target cells, possess similar biological activities to mesenchymal stem cells, but exhibit higher safety and lower immunogenicity. Compared to stem cells, exosomes hold greater potential for practical applications. Using exosomes from periodontal ligament stem cells for the regeneration and repair of periodontal tissues holds promise for the repair and reconstruction of damaged periodontal supporting tissues. Although there are existing reports on loading exosomes onto hydrogels, collagen scaffolds, and liposomes, for example, Yang et al. reported a hydrogel wound dressing loaded with exosomes / antimicrobial peptides, which was obtained by loading mesenchymal stem cell-derived exosomes and antimicrobial peptides onto a hydrogel via adsorption and then freeze-drying. This wound dressing can promote scarless wound healing [see Yang Y, Zhang J, Wu S, et al. Biomaterials, 2024, 308: 122558.]; Aliakbarian et al. reported a human umbilical cord mesenchymal stem cell-derived exosome-nanoliposome complex, which can cross the blood-brain barrier to reach damaged neurons, reduce the inflammatory response of the nervous system, reduce α-synuclein fiber tremors and cell death, and exhibit antioxidant and neuroprotective activities [see Aliakbari F, Marzookian K, Parsafar S, et al. Science Advance, 2024, 10(14):]. [eadl3406.]; Liao et al. reported a scaffold loaded with exosomes derived from mesenchymal stem cells, which promoted endometrial regeneration and fertility recovery by inducing M2 polarization of macrophages and reshaping the immune microenvironment at sites of endometrial injury [see Liao BX, LinXN, Feng Z, et al. Acta Biomaterialia, 2020, 113:252-266.]. However, there are currently no reports on loading exosomes from periodontal ligament stem cells into appropriate carriers for periodontal tissue regeneration and repair.

[0005] The main challenges in using periodontal ligament stem cell exosomes for the repair of periodontal supporting tissue damage are: (1) The local inflammatory microenvironment caused by periodontitis inhibits the function of exosomes. For example, there are a large number of pathogenic bacteria (such as Porphyromonas gingivalis) and inflammatory factors at the lesion site of periodontitis. This local inflammatory microenvironment will lead to the degradation of exosomes on the one hand, and inhibit the exosome-mediated anti-inflammatory-pro-regeneration pathway on the other hand; (2) For the regeneration and repair of periodontal supporting tissues, not only is it necessary to have an appropriate carrier to protect the activity of exosomes, but it is also necessary for exosomes to be continuously released from the carrier to promote the regeneration of periodontal supporting tissues in a long-term manner. Therefore, the development of periodontal tissue damage repair materials based on periodontal ligament stem cell exosomes still faces great challenges. If periodontal tissue repair materials that can effectively resist inflammation in the periodontal inflammatory microenvironment and can release periodontal ligament stem cell exosomes in a sustained manner can be developed, it will have a positive impact on the repair and reconstruction of periodontal supporting tissue damage. Summary of the Invention

[0006] To address the challenges faced by existing technologies in developing periodontal tissue regeneration and repair materials based on exosomes, such as the local inflammatory microenvironment of periodontitis easily inhibiting the function of exosomes and the difficulty in achieving long-term sustained release of exosomes while protecting their activity using carriers, this invention provides antibacterial sustained-release microcapsules for treating periodontitis and their preparation method. These microcapsules release antibacterial and repair components in an orderly manner, achieving sustained release of repair components while improving the local inflammatory microenvironment, thereby promoting the repair and reconstruction of damaged periodontal supporting tissues.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0008] An antibacterial sustained-release microcapsule for treating periodontitis, comprising a capsule wall and a core material encapsulated within the capsule wall, wherein the capsule wall comprises a calcium alginate matrix, nanocellulose microcrystals distributed in the calcium alginate matrix, and platinum ions bound to the calcium alginate matrix and nanocellulose microcrystals, and the core material is an aqueous solution of a biocompatible polyelectrolyte containing human periodontal ligament stem cell exosomes.

[0009] In the aforementioned antibacterial sustained-release microcapsule technology, the platinum ion loading process involves immersing microcapsules containing a core material and a capsule wall material of calcium alginate containing nanofibrillated cellulose microcrystals in a chloroplatinic acid aqueous solution. After sufficient reaction and solid-liquid separation, the unstable platinum ions are removed by washing with water. During the platinum ion loading process, the hydroxyl groups of calcium alginate and nanofibrillated cellulose microcrystals can act as nucleophilic ligands to replace chloroplatinate ions ([PtCl6)). 2- Platinum ions form Pt-O coordination bonds with some chloride ions in the calcium alginate matrix and nanocellulose microcrystalline hydroxyl groups, thus platinum ions mainly bind to the calcium alginate matrix and nanocellulose microcrystalline hydroxyl groups.

[0010] In the above-mentioned antibacterial sustained-release microcapsule technical solution, the mass ratio of calcium alginate matrix to nanocellulose microcrystals in the capsule wall is (1~5):1.

[0011] In the above-mentioned antibacterial sustained-release microcapsule technical solution, the mass of platinum in the sustained-release microcapsule accounts for 0.5% to 5% of the sum of the mass of the calcium alginate matrix and the nanocellulose microcrystals.

[0012] In the above-mentioned antibacterial sustained-release microcapsule technical solution, the biocompatible polyelectrolyte is carboxymethyl cellulose, polyethyleneimine, or chitosan.

[0013] In the above-mentioned antibacterial sustained-release microcapsule technical solution, the concentration of the aqueous solution of biocompatible polyelectrolyte in the core material is 5~40 mg / mL, and the concentration of human periodontal ligament stem cell exosomes is 5~100 μg / mL.

[0014] In the above-mentioned antibacterial sustained-release microcapsule technical solution, the diameter of the microcapsule is 300~800 μm.

[0015] The present invention also provides a method for preparing the above-mentioned antibacterial sustained-release microcapsules for treating periodontitis, comprising the following steps:

[0016] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0017] Human periodontal ligament stem cell exosomes were added to an aqueous solution of a biocompatible polyelectrolyte at room temperature and mixed thoroughly to obtain an inner phase fluid; nanocellulose microcrystals were fully dispersed in an aqueous solution of sodium alginate to obtain an outer phase fluid; and water-soluble calcium salts were dissolved in water to obtain a receiving solution.

[0018] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0019] The internal and external phase fluids were continuously pumped into the injection tube and collection tube of the microfluidic electro-spray device, respectively, by an injection pump. Under the action of an electric field, the fluid flowing out from the first-stage microfluidic device was induced to form Taylor cones and atomize into monodisperse droplets that entered the receiving liquid. The monodisperse droplets underwent a cross-linking reaction in the receiving liquid. The cross-linking and curing products were collected and washed with water to obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0020] (3) Platinum-loaded ions

[0021] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in an aqueous solution of chloroplatinic acid, allowed to react fully, and then separated into solid and liquid phases. The resulting solid phase was washed with water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0022] In the above preparation method, the concentration of the biocompatible polyelectrolyte in the inner phase fluid is 5-40 mg / mL, and the concentration of human periodontal ligament stem cell exosomes is 5-100 μg / mL; in the outer phase fluid, the mass ratio of sodium alginate to nanocellulose microcrystals is (1-5):1, and the concentration of sodium alginate is 10-17 mg / mL; the concentration of water-soluble calcium salt in the receiving solution is 5-40 mg / mL.

[0023] In step (2) of the above preparation method, the microfluidic electro-spray device is controlled at a voltage of 5~10kV, a receiving distance of 3~8 cm, and a flow rate ratio of (0.15~1):1 between the inner phase fluid and the outer phase fluid.

[0024] In step (3) of the above preparation method, the concentration of the chloroplatinic acid aqueous solution is 5~20 mg / mL, the reaction time of step (3) is controlled to be 0.5~3 h, and the reaction temperature is 20~40℃.

[0025] The antibacterial sustained-release microcapsules for treating periodontitis described in this invention exert their antibacterial effect and promote periodontal tissue regeneration as follows:

[0026] The antibacterial sustained-release microcapsules for treating periodontitis provided by this invention are administered via injection. They are directly injected into the periodontal pocket using a syringe and adhere to the periodontal tissue, releasing platinum ions and human periodontal ligament stem cell exosomes in an orderly manner. Platinum ions loaded on the capsule wall coordinate with the hydroxyl groups of the calcium alginate matrix and nanocellulose microcrystals. Because the binding stability of platinum ions to the hydroxyl groups of calcium alginate and cellulose microcrystals is less than the stability of the calcium alginate polymer network itself, the platinum ions loaded on the capsule wall are preferentially released after administration to exert their antibacterial effect. As the platinum ions are released, the porous network structure of the capsule wall itself is gradually exposed. Once the platinum ions have been largely released, the human periodontal ligament stem cell exosomes loaded inside the microcapsule begin to be gradually released. The initially released platinum ions exert their antibacterial effect in the inflammatory environment of periodontitis, improving the inflammatory microenvironment and creating a suitable microenvironment for the human periodontal ligament stem cell exosomes to exert their periodontal tissue repair properties (promoting osteoblast proliferation, migration, and osteogenic differentiation, etc.). This is beneficial for improving the repair of periodontal supporting tissues and the therapeutic effect of periodontitis.

[0027] The design and preparation concept of the antibacterial sustained-release microcapsules for treating periodontitis described in this invention are as follows:

[0028] An aqueous solution of a biocompatible polyelectrolyte containing human periodontal ligament stem cell exosomes was used as the inner phase fluid, an aqueous solution of sodium alginate containing nanocrystalline cellulose was used as the outer phase fluid, and an aqueous solution of water-soluble calcium salt was used as the receiving fluid. The inner and outer phase fluids were fed into a microfluidic electrospray device, allowing the more viscous inner and outer phase fluids to be successfully sheared. Under the action of an electric field, the fluid flowing out of the microchannel outlet was induced to form Taylor cones and atomize into monodisperse droplets, which then entered the receiving fluid. The monodisperse droplets underwent a cross-linking reaction and solidified in the receiving fluid, yielding microcapsules loaded with human periodontal ligament stem cell exosomes. These microcapsules were then subjected to platinum ion loading to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0029] Sodium alginate is composed of α-l-guluronic acid units, which interact with Ca through the residues of the α-l-guluronic acid units. 2+ The cross-linking forms a calcium alginate hydrogel, and the nanocellulose microcrystals in the external phase fluid can induce the residues of α-l-gurusonic acid units to bind with Ca. 2+ A higher degree of coordination occurs, increasing the density of the capsule wall. After loading platinum ions, the coordination binding between platinum ions and the hydroxyl groups of calcium alginate and nanocellulose microcrystals in the capsule wall further increases the density of the capsule wall. Increased capsule wall density not only slows down the release of exosomes from the capsule wall, improving the sustained-release performance of the microcapsule, but also better protects the exosomes loaded therein, reducing the loss of exosomes during washing, freeze-drying, and subsequent storage after platinum ion loading, thus increasing the effective loading capacity of exosomes inside the microcapsule. The entire preparation process of the method described in this invention is carried out in an aqueous system without introducing toxic or organic reagents, and the preparation conditions are mild, which helps to protect the activity of exosomes and increase the biocompatibility of the microcapsule. Simultaneously, the biocompatible polyelectrolyte, calcium alginate, and nanocellulose microcrystals have good biodegradability and biocompatibility, allowing the microcapsules of this invention to degrade naturally after exerting their effects in vivo. Furthermore, the appropriate platinum ion loading helps to avoid long-term residues of the microcapsules in vivo and prevent biotoxicity to the body.

[0030] Compared with the prior art, the technical solution of the present invention can produce the following beneficial technical effects:

[0031] 1. This invention provides an antibacterial sustained-release microcapsule for treating periodontitis, comprising a capsule wall and a core material encapsulated within the capsule wall. The capsule wall consists of a calcium alginate matrix, nanofiber cellulose microcrystals distributed within the calcium alginate matrix, and platinum ions bound to the hydroxyl groups of the calcium alginate matrix and the nanofiber cellulose microcrystals. The core material is an aqueous solution of a biocompatible polyelectrolyte containing human periodontal ligament stem cell exosomes. After administration, platinum ions loaded on the capsule wall are preferentially released to exert an antibacterial effect. As the platinum ions are released, the porous network structure of the capsule wall itself is gradually exposed. Once the platinum ions have been largely released, the human periodontal ligament stem cell exosomes loaded inside the microcapsule begin to be gradually released. The initially released platinum ions exert an antibacterial effect in the inflammatory environment of periodontitis, improving the inflammatory microenvironment, thereby creating a better microenvironment for the human periodontal ligament stem cell exosomes to exert their periodontal tissue repair properties. This is beneficial for improving the repair of periodontal supporting tissues and the therapeutic effect of periodontitis. This invention provides a feasible biomaterial for the treatment of periodontitis, which can solve the problems faced by existing technologies in developing periodontal tissue regeneration and repair materials based on exosomes. These problems include the local inflammatory microenvironment of periodontitis easily inhibiting the function of exosomes, and the difficulty in achieving long-term sustained release of exosomes while protecting their activity using a carrier.

[0032] 2. The antibacterial sustained-release microcapsules for treating periodontitis described in this invention use human periodontal ligament stem cell exosomes as bioactive components, which promote the regeneration and repair of periodontal supporting tissues. Compared with stem cells, human periodontal ligament stem cell exosomes have the advantages of being easy to obtain, having low invasiveness in obtaining them, and having fewer ethical issues. Therefore, the microcapsules described in this invention have good potential for clinical application.

[0033] 3. This invention increases the density of the capsule wall by combining the introduction of nano-cellulose microcrystals and the loading of platinum ions. Increased capsule wall density not only slows down the release of exosomes from the capsule wall and improves the sustained-release performance of the microcapsules, but also better protects the exosomes loaded within, reducing loss during washing, freeze-drying, and subsequent storage after platinum ion loading, thus increasing the effective loading capacity of exosomes within the microcapsules.

[0034] 4. This invention also provides a method for preparing antibacterial sustained-release microcapsules for treating periodontitis. This method is based on microfluidic electrospray technology, which, compared to ordinary microfluidic technology, has higher preparation efficiency and is easier to achieve continuous batch preparation. Furthermore, the entire preparation process is carried out in an aqueous system without introducing toxic or organic reagents, and the preparation conditions are mild. This helps to protect the activity of exosomes and increase the biocompatibility of the microcapsules.

[0035] 5. This invention experimentally demonstrates that, for the microcapsules PDLSC-exos@Pt prepared in Example 1 of this application, the release rate of platinum ions is relatively fast, reaching a basic equilibrium state by 48 hours. The exosomes loaded within them begin to be released from day 2, reaching a basic equilibrium state by day 15. This confirms that the release of platinum ions and exosomes in PDLSC-exos@Pt is sequential, and that the microcapsules can release exosomes sustainably. Furthermore, compared to the unloaded PDLSC-exos prepared in Comparative Example 1, the cumulative release rate of exosomes in PDLSC-exos@Pt is higher. This confirms that platinum ion loading can reduce the loss of exosomes loaded in the microcapsules during the washing and freeze-drying processes after platinum ion loading, thereby increasing the effective loading capacity of exosomes within the microcapsules.

[0036] 6. Experiments have confirmed that the microcapsules PDLSC-exos@Pt prepared in Example 1 of this application have a good antibacterial effect against Porphyromonas gingivalis. Simultaneously, PDLSC-exos@Pt has virtually no impact on the viability of normal cells, exhibiting good biocompatibility. This is mainly due to the selection of the solvent system and various raw materials in the preparation method. The biocompatible polyelectrolyte, calcium alginate, and nanocellulose microcrystals in the microcapsules have good biodegradability and biocompatibility, allowing the microcapsules to degrade naturally after exerting their effects in vivo. Furthermore, the appropriate platinum ion loading helps avoid long-term residues of the microcapsules in vivo and prevents biotoxicity. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the microfluidic electro-injection device and a schematic diagram of the preparation of microcapsules using this device.

[0038] Figure 2 This is an optical microscope image of PDLSC-exos@Pt.

[0039] Figure 3 This is the particle size distribution diagram of PDLSC-exos@Pt.

[0040] Figure 4 This is a scanning electron microscope image of the surface of PDLSC-exos@Pt after freeze-drying.

[0041] Figure 5 This is a scanning electron microscope image of a cross-section of lyophilized PDLSC-exos@Pt.

[0042] Figure 6 yes Figure 4 EDX image of platinum shown in the scanning electron microscope image.

[0043] Figure 7This is the Pt ion release curve of PDLSC-exos@Pt.

[0044] Figure 8 These are the drug release curves for PDLSC-exos and PDLSC-exos@Pt.

[0045] Figure 9 These are images showing the antibacterial effects of PDLSC-exos and PDLSC-exos@Pt.

[0046] Figure 10 Figure a shows the cell viability of PDLSC-exos and L929 cells co-cultured for different time periods. Figure 10 Figure b shows the cell viability of PDLSC-exos@Pt and L929 cells co-cultured for different time periods.

[0047] Explanation of reference numerals in the attached figures. Figure 1 In the diagram, 1 is a primary microfluidic device, 2 is a collection container, and 3 is a high-voltage DC power supply. Detailed Implementation

[0048] The following examples further illustrate the antibacterial sustained-release microcapsules for treating periodontitis and their preparation method according to the present invention. It should be noted that the following examples are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made to the present invention by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0049] In the following embodiments, Figure 1 This is a schematic diagram of the structure of a microfluidic electro-injection device and a schematic diagram of the preparation of microcapsules using this device. The microfluidic electro-injection device consists of a primary microfluidic device 1, an electric field generation system, and a collection container 2.

[0050] The primary microfluidic device 1 includes an injection tube, a connecting tube, and a collection tube, and is used in conjunction with an injection pump. The injection tube is made of cylindrical glass capillary tube, with the tail end drawn into a conical shape using a needle puller, and then polished on sandpaper until the inner diameter of the conical opening is approximately 100 μm. The outer diameter of the cylindrical section is 960 μm, and the inner diameter is 550 μm. The collection tube is also made of cylindrical glass capillary tube, with both ends polished smooth. The outer diameter of the collection tube is 960 μm, and the inner diameter is 300 μm. The connecting tube is a square glass tube, with both ends polished smooth. A square through-hole with dimensions of 1.0 × 1.0 mm is located in the center. The tail end of the injection tube is inserted into the head of the collection tube and connected via the connecting tube. The injection tube, connecting tube, and collection tube are coaxially arranged and fixed to a glass slide using AB glue. A flat-mouthed needle is fitted onto the inlet end of the syringe tubing, and the non-inlet end of the needle is secured with AB glue. Another flat-mouthed needle is then secured to the inlet end of the connecting tubing with AB glue. The outlet end of the connecting tubing is sealed with AB glue. The flat-mouthed needles are made of stainless steel, and each needle is connected to the syringe pump via a fitting.

[0051] The primary microfluidic device is arranged with the axes of the injection tube, connecting tube, and collection tube perpendicular to the water surface. The collection container 2 is located directly below the collection tube, and a metal plate is placed at the bottom of the collection container. The electric field generating system includes a high-voltage DC power supply 3, an emitter and a receiver connected to the positive and negative terminals of the high-voltage DC power supply, respectively. The emitter is connected to a flat-mouthed needle located at the inlet end of the injection tube, and the receiver is a metal plate located below the collection container.

[0052] In the following examples and comparative examples, the exosomes of human periodontal ligament stem cells were purchased from Cyagen (Guangzhou) Biotechnology Co., Ltd.; cellulose nanocrystals (CNC) were purchased from Suzhou Beike Nanotechnology Co., Ltd. (diameter 3~30 nm, length 300 nm~micron, elastic modulus 135~150 GPa); carboxymethyl cellulose (CMC) was purchased from Shanghai Aladdin Reagent Co., Ltd. (viscosity 800~1000 mPa.s); sodium alginate and calcium chloride were purchased from Shanghai Aladdin Reagent Co., Ltd.

[0053] Example 1

[0054] In this embodiment, antibacterial sustained-release microcapsules for the treatment of periodontitis are prepared by the following steps:

[0055] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0056] Preparation of the internal phase fluid: Carboxymethyl cellulose was added to pure water and stirred at 60 °C until it was completely dissolved. The resulting carboxymethyl cellulose aqueous solution was cooled to room temperature, and human periodontal ligament stem cell exosomes were added and mixed thoroughly to obtain the internal phase fluid. The concentration of carboxymethyl cellulose in the internal phase fluid was 20 mg / mL, and the concentration of human periodontal ligament stem cell exosomes was 20 μg / mL.

[0057] Preparation of external phase fluid: Sodium alginate was added to pure water and stirred at 60 °C until completely dissolved. Then, cellulose nanocrystals (CNC) were added and stirred to fully disperse the CNC to obtain the external phase fluid. In the external phase fluid, the mass ratio of sodium alginate to CNC was 3:1 and the concentration of sodium alginate was 15 mg / mL.

[0058] Preparation of receiving solution: Dissolve calcium chloride in pure water to obtain receiving solution; the concentration of calcium chloride in the receiving solution is 20 mg / mL.

[0059] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0060] Turn on the high-voltage DC power supply of the microfluidic electroinjection device, and continuously pump the internal phase fluid and external phase fluid into the injection tube and collection tube of the first-stage microfluidic device through the injection pump, respectively. Under the conditions of 8 kV voltage and 5 cm receiving distance, the fluid flowing out of the collection tube of the first-stage microfluidic device is induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid in the collection container. The monodisperse droplets are kept in the receiving liquid for 30 s to complete cross-linking and curing. Collect the cross-linking and curing product, wash it three times with pure water, and obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0061] In this step, the inner diameter of the injection tube outlet of the primary microfluidic device is 100 μm, and the inner diameter of the collection tube is 300 μm. The flow rate of the inner phase fluid is controlled at 100 μL / min, and the flow rate of the outer phase fluid is controlled at 300 μL / min. The receiving distance refers to the vertical distance between the liquid surface of the receiving liquid and the end of the outlet of the collection tube.

[0062] (3) Platinum-loaded ions

[0063] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in a chloroplatinic acid aqueous solution with a concentration of 10 mg / mL and a temperature of 25 °C for 2 h. After solid-liquid separation, the obtained solid phase was washed three times with pure water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis, denoted as PDLSC-exos@Pt.

[0064] The optical microscope image of the PDLSC-exos@Pt prepared in this embodiment is shown below. Figure 2 As shown, the particle size distribution of the PDLSC-exos@Pt prepared in this embodiment is as follows. Figure 3As shown. Combined with Figures 2-3 It can be seen that the PDLSC-exos@Pt prepared in this embodiment is spherical with uniform particle size, and the particle size distribution ranges from 420 to 530 μm, with the particle size concentrated between 470 and 500 μm.

[0065] The PDLSC-exos@Pt prepared in this embodiment was freeze-dried, and scanning electron microscope (SEM) images of the surface and cross-section of the freeze-dried PDLSC-exos@Pt were obtained. The results are as follows: Figures 4-5 As shown. Figure 6 yes Figure 4 The image shown is an EDX image of platinum in a scanning electron microscope. (Source: [Insert image here]) Figure 5 It can be seen that the PDLSC-exos@Pt prepared in this embodiment has a microcapsule structure. Figure 6 It can be seen that platinum is uniformly distributed on the surface of PDLSC-exos@Pt, which should be due to the formation of Pt-O coordination bonds between platinum and calcium alginate and the hydroxyl groups of CNC.

[0066] Comparative Example 1

[0067] In this comparative example, following the steps (1) and (2) of Example 1, microcapsules loaded with human periodontal ligament stem cell exosomes were prepared and denoted as PDLSC-exos microcapsules.

[0068] Example 2

[0069] In this embodiment, the release behavior of platinum ions in the PDLSC-exos@Pt prepared in Example 1 was examined.

[0070] (1) The PDLSC-exos@Pt prepared in Example 1 was freeze-dried to obtain freeze-dried PDLSC-exos@Pt.

[0071] (2) Accurately weigh 1.5 mg of lyophilized PDLSC-exos@Pt and add it to 2 mL of phosphate buffer (pH=7.4). Incubate in an air bath shaker at 37℃ and 100 rpm. During incubation, take out an appropriate amount of incubation solution every 2-24 h and add an equal amount of phosphate buffer to maintain a constant total volume. Measure the concentration of platinum ions in the taken incubation solution using inductively coupled plasma atomic emission spectrometry (ICP-AES), calculate the platinum ion release, and plot the platinum ion release curve.

[0072] The platinum ion release curve in PDLSC-exos@Pt plotted in this embodiment is as follows: Figure 7As shown in the figure, the release rate of platinum ions is relatively fast in the first 10 hours, reaching a cumulative release rate of approximately 53% by the 6th hour. The release of platinum ions essentially reaches equilibrium by the 48th hour, with a cumulative release rate of approximately 80%. Furthermore, this application tested the platinum ion loading in lyophilized PDLSC-exos@Pt using inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that the mass of platinum in lyophilized PDLSC-exos@Pt accounts for approximately 1.5% of the sum of the mass of the calcium alginate matrix and the nanocellulose crystals.

[0073] Example 3

[0074] In this embodiment, the release behavior of human periodontal ligament stem cell exosomes in PDLSC-exos prepared in Comparative Example 1 and PDLSC-exos@Pt prepared in Example 1 was investigated.

[0075] (1) The PDLSC-exos prepared in Comparative Example 1 and the PDLSC-exos@Pt prepared in Example 1 were freeze-dried to obtain freeze-dried PDLSC-exos and freeze-dried PDLSC-exos@Pt, which were then stored in a refrigerator.

[0076] (2) Accurately weigh 1.5 mg of lyophilized PDLSC-exos and add it to 2 mL of phosphate buffer (pH=7.4). Incubate in an air bath shaker at 37℃ and 100 rpm. During incubation, take out an appropriate amount of incubation solution every 1-2 days and add an equal amount of phosphate buffer to maintain the total volume. Use the BCA protein kit and microplate reader to detect the protein content in the taken incubation solution at a wavelength of 562 nm and plot the protein release curve.

[0077] (3) Accurately weigh 1.5 mg of lyophilized PDLSC-exos@Pt and add it to 2 mL of phosphate buffer (pH=7.4). Incubate in an air bath shaker at 37℃ and 100 rpm. During incubation, take out an appropriate amount of incubation solution every 1-2 days and add an equal amount of phosphate buffer to maintain the total volume. Use the BCA protein kit and microplate reader to detect the protein content in the taken incubation solution at a wavelength of 562 nm and plot the exosome release curve of human periodontal ligament stem cells.

[0078] In steps (2) and (3), the BCA protein kit was used to detect the total protein content carried by human periodontal ligament stem cell exosomes. The detection results were used to reflect the release of human periodontal ligament stem cell exosomes. The release curves of human periodontal ligament stem cell exosomes in PDLSC-exos and PDLSC-exos@Pt in this embodiment are shown below. Figure 8 As shown.

[0079] As shown in Figure 8, protein release was detected in PDLSC-exos from day 1, with a cumulative protein release rate of approximately 49% by day 5. By day 11, protein release had essentially reached equilibrium, with a cumulative release of nearly 60%. In contrast, protein release was only detected in PDLSC-exos@Pt from day 2, with a cumulative release rate of approximately 60% by day 9. By day 15, protein release had essentially reached equilibrium, with a cumulative release rate of approximately 76%. Once protein release reached equilibrium, the cumulative release of PDLSC-exos was close to 60%, while the cumulative release rate of PDLSC-exos@Pt was approximately 76%, representing an increase of about 16% in the cumulative protein release rate of PDLSC-exos@Pt compared to PDLSC-exos. This is mainly because platinum ion loading can appropriately increase the density of the capsule wall. Increased capsule wall density allows exosomes to be better stored inside the microcapsules, reducing the loss of exosomes during washing, freeze-drying, and refrigeration, thus increasing the effective load of exosomes inside the microcapsules. In PDLSC-exos, protein release was detected within day 1, while in PDLSC-exos@Pt, protein release was only detected from day 2 onwards. This is mainly because platinum ion loading increases capsule wall density, initially hindering the release of exosomes loaded inside the microcapsules. With the rapid release of platinum ions from the surface of PDLSC-exos@Pt on day 1 (see...), the release of exosomes is significantly reduced. Figure 7 The exosomes inside PDLSC-exos@Pt are gradually released, which achieves the purpose of first releasing platinum ions for antibacterial purposes and then releasing exosomes for periodontal tissue repair. The released platinum ions are used to inhibit the inflammatory microenvironment of periodontitis, creating a better microenvironment for exosomes to exert their periodontal tissue repair performance. On this basis, the exosomes can better exert their repair performance on periodontal tissues.

[0080] Example 4

[0081] In this embodiment, the antibacterial effects of PDLSC-exos prepared in Comparative Example 1 and PDLSC-exos@Pt prepared in Example 1 on Porphyromonas gingivalis were investigated.

[0082] (1) The PDLSC-exos prepared in Comparative Example 1 and the PDLSC-exos@Pt prepared in Example 1 were freeze-dried to obtain freeze-dried PDLSC-exos and freeze-dried PDLSC-exos@Pt, which were then stored in a refrigerator.

[0083] (2) Porphyromonas gingivalis was cultured in suspension using TSB medium, and the bacterial concentration was adjusted to approximately 10. 6 CFU / mL was used to obtain Porphyromonas gingivalis bacterial suspension.

[0084] (3) Set up a positive control group and a negative control group, wherein the positive control group is supplemented with TSB medium with a standard concentration (1×10⁻⁶). 6 The positive and negative control groups were cultured in TSB medium containing *Porphyromonas gingivalis* (CFU / mL). Both groups were incubated in an anaerobic environment at 37 °C for 72 h, and the OD values ​​at 600 nm were measured using a spectrophotometer.

[0085] (4) Accurately weigh 1.5 mg of lyophilized PDLSC-exos and add it to 2 mL of phosphate buffer (pH=7.4). Incubate at 37 °C for 96 h and collect the supernatant as the PDLSC-exos extract. Add a standard concentration (1×10⁻⁶) to TSB medium. 6 Porphyromonas gingivalis bacterial suspension (CFU / mL) was used as the PDLSC-exos experimental group. PDLSC-exos extract was added to a standard concentration (1×10⁻⁶ CFU / mL). 6 The bacterial suspension of *Porphyromonas gingivalis* (CFU / mL) was cultured in TSB medium at 37 °C for 72 h in an anaerobic environment, and the OD value at a wavelength of 600 nm was measured using a spectrophotometer.

[0086] (5) Accurately weigh 1.5 mg of lyophilized PDLSC-exos@Pt and add it to 2 mL of phosphate buffer (pH=7.4). Incubate at 37℃ for 96 h and collect the supernatant as the PDLSC-exos@Pt extraction solution. Add a standard concentration (1×10⁻⁶) to TSB medium. 6 Porphyromonas gingivalis bacterial suspension (CFU / mL) was used as the PDLSC-exos@Pt experimental group. The PDLSC-exos@Pt extract was added to a standard concentration (1×10⁻⁶ CFU / mL). 6 The bacterial suspension of *Porphyromonas gingivalis* (CFU / mL) was cultured in TSB medium at 37 °C for 72 h in an anaerobic environment, and the OD value at a wavelength of 600 nm was measured using a spectrophotometer.

[0087] The OD value at 600 nm in steps (3) to (5) can reflect the activity of *Porphyromonas gingivalis*, and thus the antibacterial effect of PDLSC-exos and PDLSC-exos@Pt extracts on *Porphyromonas gingivalis*. The antibacterial effect diagram drawn in this embodiment is shown below. Figure 9 As shown, "-" represents the negative control group, "+" represents the positive control group, PDLSC-exos represents the PDLSC-exos experimental group, and PDLSC-exos@Pt represents the PDLSC-exos@Pt experimental group. Figure 9The results showed that the OD value of the positive control group was close to 0.8, and the OD value of the negative control group was close to 0.05. The OD values ​​of the PDLSC-exos experimental group and the PDLSC-exos@Pt experimental group were significantly lower than those of the positive control group. Specifically, the OD value of the PDLSC-exos experimental group was close to 0.20, and the OD value of the PDLSC-exos@Pt experimental group was close to 0.42. The OD value of the PDLSC-exos@Pt experimental group was significantly lower than that of the PDLSC-exos experimental group. This indicates that PDLSC-exos@Pt effectively releases platinum ions and human periodontal ligament stem cell exosomes, and possesses certain antibacterial activity, effectively inhibiting the proliferation of Porphyromonas gingivalis.

[0088] Example 5

[0089] In this embodiment, the biocompatibility of PDLSC-exos prepared in Comparative Example 1 and PDLSC-exos@Pt prepared in Example 1 was examined.

[0090] (1) The PDLSC-exos prepared in Comparative Example 1 and the PDLSC-exos@Pt prepared in Example 1 were freeze-dried to obtain freeze-dried PDLSC-exos and freeze-dried PDLSC-exos@Pt, which were then stored in a refrigerator.

[0091] (2) Frozen mouse fibroblasts (L929) were thawed and then prepared into a cell suspension using complete culture medium. The suspension was incubated at 37 ℃ in a 5% CO2 incubator for 48–72 h, with cell growth observed every 24 h. Cells were passaged during the logarithmic growth phase. The passaged cells were then cultured at 37 ℃ in a 5% CO2 incubator for another 48–72 h, with daily cell proliferation dynamics recorded. The concentration of the cell suspension was measured using a cell counter, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL using complete culture medium. 4 Cells / mL were seeded into 96-well cell culture plates (100 μL / well), and cultured for 24 h at 37 ℃ in a 5% CO2 incubator before being aspirated to obtain L929 cell suspension.

[0092] (3) Set up a positive control group and a negative control group. The positive control group was prepared by adding a standard concentration (1×10⁻⁶) to the complete culture medium. 8 L929 cell suspension (CFU / mL) was used, while the negative control group received no added substances to the complete culture medium. The positive control and negative control groups were incubated at 37 ℃ and 5% CO2 for 24 h, 48 h, and 72 h, respectively. The supernatant was then aspirated, and CCK8 reagent (10% of the aspirated supernatant volume) was added. The cells were then incubated at 37 ℃ and 5% CO2 for another 1–3 h, and the absorbance was measured at 450 nm using a microplate reader.

[0093] (4) Accurately weigh 2 mg, 1 mg, 0.4 mg, 0.2 mg, and 0.1 mg of lyophilized PDLSC-exos, sterilize them under UV light, and prepare PDLSC-exos dispersions with concentrations of 1000, 500, 200, 100, and 50 μg / ml using complete culture medium. Add standard concentrations (1×10⁻⁶) to the complete culture medium. 8 L929 cell suspension (CFU / mL) was added to 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. The culture medium was then aspirated and an equal volume (100 μL) of PDLSC-exos dispersion was added. After further incubation at 37 ℃ in a 5% CO2 incubator for 24 h, 48 h, and 72 h, the supernatant was aspirated, and CCK8 reagent (10% of the aspirated supernatant volume) was added. The cells were then incubated at 37 ℃ in a 5% CO2 incubator for 1–3 h, and the absorbance was measured at 450 nm using a microplate reader.

[0094] (5) Accurately weigh 2 mg, 1 mg, 0.4 mg, 0.2 mg, and 0.1 mg of lyophilized PDLSC-exos@Pt, sterilize under UV light, and prepare PDLSC-exos@Pt dispersions with concentrations of 1000, 500, 200, 100, and 50 μg / ml using complete culture medium. Add standard concentrations (1×10⁻⁶) to the complete culture medium. 8 L929 cell suspension (CFU / mL) was added to 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The culture medium was then aspirated and an equal volume (100 μL) of PDLSC-exos@Pt dispersion was added. After further incubation at 37°C with 5% CO2 for 24 h, 48 h, and 72 h, the supernatant was aspirated, and CCK8 reagent (10% of the aspirated supernatant volume) was added. The plates were then incubated at 37°C with 5% CO2 for 1–3 h, and the absorbance was measured at 450 nm using a microplate reader.

[0095] In steps (3) to (5), the viability of L929 cells was measured using the CCK8 kit, and the absorbance at a wavelength of 450 nm was monitored using an ELISA reader. The results were used to reflect the effects of PDLSC-exos and PDLSC-exos@Pt on cell viability. The effect of PDLSC-exos on cell viability is as follows: Figure 10 As shown in Figure a, the effect of PDLSC-exos@Pt on cell viability is as follows: Figure 10 As shown in Figure b. (By...) Figure 10It was found that the cell viability of L929 cells was greater than 85% after co-culturing PDLSC-exos and PDLSC-exos@Pt with L929 cells for 24 h, 48 h, and 72 h. This experimental result indicates that PDLSC-exos and PDLSC-exos@Pt did not significantly inhibit the viability of L929 cells and had good cell compatibility.

[0096] Example 6

[0097] In this embodiment, antibacterial sustained-release microcapsules for the treatment of periodontitis are prepared by the following steps:

[0098] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0099] Preparation of the inner phase fluid: Carboxymethyl cellulose was added to pure water and stirred at 60 °C until it was completely dissolved. The resulting carboxymethyl cellulose aqueous solution was cooled to room temperature, and human periodontal ligament stem cell exosomes were added and mixed thoroughly to obtain the inner phase fluid. The concentration of carboxymethyl cellulose in the inner phase fluid was 40 mg / mL, and the concentration of human periodontal ligament stem cell exosomes was 100 μg / mL.

[0100] Preparation of external phase fluid: Sodium alginate was added to pure water and stirred at 60 °C until completely dissolved. Then CNC was added and stirred to fully disperse the CNC to obtain the external phase fluid. In the external phase fluid, the mass ratio of sodium alginate to CNC was 5:1 and the concentration of sodium alginate was 17 mg / mL.

[0101] Preparation of receiving solution: Dissolve calcium chloride in pure water to obtain receiving solution; the concentration of calcium chloride in the receiving solution is 40 mg / mL.

[0102] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0103] Turn on the high-voltage DC power supply of the microfluidic electroinjection device, and continuously pump the internal phase fluid and external phase fluid into the injection tube and collection tube of the first-stage microfluidic device through the injection pump, respectively. Under the conditions of voltage of 5~10 kV and receiving distance of 5 cm, the fluid flowing out of the collection tube of the first-stage microfluidic device is induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid in the collection container. The monodisperse droplets are kept in the receiving liquid for 30 s to complete cross-linking and curing. Collect the cross-linking and curing product, wash it three times with pure water, and obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0104] In this step, the inner diameter of the injection tube outlet of the primary microfluidic device is 100 μm, the inner diameter of the collection tube is 300 μm, and the flow rate of the inner phase fluid is controlled at 100 μL / min and the flow rate of the outer phase fluid is 300 μL / min.

[0105] (3) Platinum-loaded ions

[0106] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in a chloroplatinic acid aqueous solution with a concentration of 20 mg / mL and a temperature of 40 °C for 0.5 h. After solid-liquid separation, the obtained solid phase was washed three times with pure water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0107] In this embodiment, the effect of voltage in step (2) on the size of the antibacterial sustained-release microcapsules was investigated. It was found that under the same conditions, the diameter of the antibacterial sustained-release microcapsules decreased with the increase of voltage. When the voltage was 5 kV, the average particle size of the prepared antibacterial sustained-release microcapsules was 765.7 ± 2.3 μm, and when the voltage was 10 kV, the average particle size of the prepared antibacterial sustained-release microcapsules was 360.7 ± 1.6 μm.

[0108] Example 7

[0109] In this embodiment, antibacterial sustained-release microcapsules for the treatment of periodontitis are prepared by the following steps:

[0110] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0111] Preparation of the inner phase fluid: Carboxymethyl cellulose was added to pure water and stirred at 60 °C until it was completely dissolved. The resulting carboxymethyl cellulose aqueous solution was cooled to room temperature, and human periodontal ligament stem cell exosomes were added and mixed thoroughly to obtain the inner phase fluid. The concentration of carboxymethyl cellulose in the inner phase fluid was 15 mg / mL, and the concentration of human periodontal ligament stem cell exosomes was 15 μg / mL.

[0112] Preparation of external phase fluid: Sodium alginate was added to pure water and stirred at 60 °C until completely dissolved. Then CNC was added and stirred to fully disperse the CNC to obtain the external phase fluid. In the external phase fluid, the mass ratio of sodium alginate to CNC was 2:1 and the concentration of sodium alginate was 15 mg / mL.

[0113] Preparation of receiving solution: Dissolve calcium chloride in pure water to obtain receiving solution; the concentration of calcium chloride in the receiving solution is 15 mg / mL.

[0114] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0115] Turn on the high-voltage DC power supply of the microfluidic electroinjection device, and continuously pump the internal phase fluid and external phase fluid into the injection tube and collection tube of the first-stage microfluidic device through the injection pump, respectively. Under the conditions of 8 kV voltage and 5 cm receiving distance, the fluid flowing out of the collection tube of the first-stage microfluidic device is induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid in the collection container. The monodisperse droplets are kept in the receiving liquid for 30 s to complete cross-linking and curing. Collect the cross-linking and curing product, wash it three times with pure water, and obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0116] In this step, the inner diameter of the injection tube outlet of the primary microfluidic device is 100 μm, the inner diameter of the collection tube is 300 μm, and the flow rate of the inner phase fluid is controlled to be 50~250 μL / min and the flow rate of the outer phase fluid is 300 μL / min.

[0117] (3) Platinum-loaded ions

[0118] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in a chloroplatinic acid aqueous solution with a concentration of 10 mg / mL and a temperature of 25 °C for 2 h. After solid-liquid separation, the obtained solid phase was washed three times with pure water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0119] In this embodiment, the effect of the flow rate of the internal phase fluid in step (2) on the size of the antibacterial sustained-release microcapsules was investigated. It was found that under the same conditions, the diameter of the antibacterial sustained-release microcapsules increases with the increase of the internal phase fluid flow rate. When the internal phase fluid flow rate is 50 μL / min, the average particle size of the prepared antibacterial sustained-release microcapsules is 436.2±1.3 μm. When the internal phase fluid flow rate is 250 μL / min, the average particle size of the prepared antibacterial sustained-release microcapsules is 451.3±1.5 μm.

[0120] Example 8

[0121] In this embodiment, antibacterial sustained-release microcapsules for the treatment of periodontitis are prepared by the following steps:

[0122] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0123] Preparation of the inner phase fluid: Carboxymethyl cellulose was added to pure water and stirred at 60 °C until it was completely dissolved. The resulting carboxymethyl cellulose aqueous solution was cooled to room temperature, and human periodontal ligament stem cell exosomes were added and mixed thoroughly to obtain the inner phase fluid. The concentration of carboxymethyl cellulose in the inner phase fluid was 30 mg / mL, and the concentration of human periodontal ligament stem cell exosomes was 50 μg / mL.

[0124] Preparation of external phase fluid: Sodium alginate was added to pure water and stirred at 60 °C until completely dissolved. Then CNC was added and stirred to fully disperse the CNC to obtain the external phase fluid. In the external phase fluid, the mass ratio of sodium alginate to CNC was 3:1 and the concentration of sodium alginate was 16 mg / mL.

[0125] Preparation of receiving solution: Dissolve calcium chloride in pure water to obtain receiving solution; the concentration of calcium chloride in the receiving solution is 25 mg / mL.

[0126] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0127] Turn on the high-voltage DC power supply of the microfluidic electroinjection device, and continuously pump the internal phase fluid and external phase fluid into the injection tube and collection tube of the first-stage microfluidic device through the injection pump, respectively. Under the conditions of 8 kV voltage and 5 cm receiving distance, the fluid flowing out of the collection tube of the first-stage microfluidic device is induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid in the collection container. The monodisperse droplets are kept in the receiving liquid for 30 s to complete cross-linking and curing. Collect the cross-linking and curing product, wash it three times with pure water, and obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0128] In this step, the inner diameter of the injection tube outlet of the primary microfluidic device is 100 μm, the inner diameter of the collection tube is 300 μm, and the flow rate of the inner phase fluid is controlled to be 100 μL / min and the flow rate of the outer phase fluid is 100~500 μL / min.

[0129] (3) Platinum-loaded ions

[0130] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in a chloroplatinic acid aqueous solution with a concentration of 10 mg / mL and a temperature of 25 °C for 2 h. After solid-liquid separation, the obtained solid phase was washed three times with pure water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0131] In this embodiment, the effect of the flow rate of the external phase fluid in step (2) on the size of the antibacterial sustained-release microcapsules was investigated. It was found that under the same conditions, the diameter of the antibacterial sustained-release microcapsules increases with the increase of the external phase fluid flow rate. When the external phase fluid flow rate is 100 μL / min, the average particle size of the prepared antibacterial sustained-release microcapsules is 398.8±1.9 μm. When the external phase fluid flow rate is 500 μL / min, the average particle size of the prepared antibacterial sustained-release microcapsules is 535.8±2.7 μm.

[0132] Example 9

[0133] In this embodiment, antibacterial sustained-release microcapsules for the treatment of periodontitis are prepared by the following steps:

[0134] (1) Preparation of internal phase fluid, external phase fluid and receiving liquid

[0135] Preparation of the inner phase fluid: Carboxymethyl cellulose was added to pure water and stirred at 60 °C until it was completely dissolved. The resulting carboxymethyl cellulose aqueous solution was cooled to room temperature, and human periodontal ligament stem cell exosomes were added and mixed thoroughly to obtain the inner phase fluid. The concentration of carboxymethyl cellulose in the inner phase fluid was 5 mg / mL, and the concentration of human periodontal ligament stem cell exosomes was 5 μg / mL.

[0136] Preparation of external phase fluid: Sodium alginate was added to pure water and stirred at 60 °C until completely dissolved. Then CNC was added and stirred to fully disperse the CNC to obtain the external phase fluid. In the external phase fluid, the mass ratio of sodium alginate to CNC was 1:1 and the concentration of sodium alginate was 10 mg / mL.

[0137] Preparation of receiving solution: Dissolve calcium chloride in pure water to obtain receiving solution; the concentration of calcium chloride in the receiving solution is 5 mg / mL.

[0138] (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes

[0139] Turn on the high-voltage DC power supply of the microfluidic electroinjection device, and continuously pump the internal phase fluid and external phase fluid into the injection tube and collection tube of the first-stage microfluidic device through the injection pump, respectively. Under the conditions of 8 kV voltage and receiving distance of 3~8 cm, the fluid flowing out of the collection tube of the first-stage microfluidic device is induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid in the collection container. The monodisperse droplets are kept in the receiving liquid for 30 s to complete cross-linking and curing. Collect the cross-linking and curing product, wash it three times with pure water, and obtain microcapsules loaded with human periodontal ligament stem cell exosomes.

[0140] In this step, the inner diameter of the injection tube outlet of the primary microfluidic device is 100 μm, the inner diameter of the collection tube is 300 μm, and the flow rate of the inner phase fluid is controlled to be 100 μL / min and the flow rate of the outer phase fluid is 100~500 μL / min.

[0141] (3) Platinum-loaded ions

[0142] Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in a chloroplatinic acid aqueous solution with a concentration of 5 mg / mL and a temperature of 20 °C for 3 h. After solid-liquid separation, the obtained solid phase was washed three times with pure water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

[0143] In this embodiment, the effect of the receiving distance in step (2) on the size of the antibacterial sustained-release microcapsule was investigated. It was found that under the same conditions, when the receiving distance varied within the range of 3 to 8 cm, it had virtually no effect on the diameter of the antibacterial sustained-release microcapsule.

Claims

1. An antibacterial sustained-release microcapsule for treating periodontitis, characterized in that, The microcapsule consists of a capsule wall and a core material encapsulated within the capsule wall. The capsule wall is composed of a calcium alginate matrix, nanofiber cellulose crystals distributed within the calcium alginate matrix, and platinum ions bound to the calcium alginate matrix and nanofiber cellulose crystals. The core material is an aqueous solution of a biocompatible polyelectrolyte containing human periodontal ligament stem cell exosomes. The biocompatible polyelectrolyte is carboxymethyl cellulose or chitosan.

2. The antibacterial sustained-release microcapsule for treating periodontitis according to claim 1, characterized in that, The mass ratio of calcium alginate matrix to nanocellulose microcrystals in the capsule wall is (1~5):

1.

3. The antibacterial sustained-release microcapsule for treating periodontitis according to claim 1, characterized in that, In this microcapsule, the mass of platinum accounts for 0.5% to 5% of the sum of the mass of the calcium alginate matrix and the nanocellulose microcrystals.

4. The antibacterial sustained-release microcapsule for treating periodontitis according to any one of claims 1 to 3, characterized in that, In the core material, the concentration of the aqueous solution of the biocompatible polyelectrolyte is 5~40 mg / mL, and the concentration of human periodontal ligament stem cell exosomes is 5~100 μg / mL.

5. The antibacterial sustained-release microcapsule for treating periodontitis according to any one of claims 1 to 3, characterized in that, The diameter of the microcapsules is 300~800 μm.

6. The method for preparing the antibacterial sustained-release microcapsules for treating periodontitis according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of internal phase fluid, external phase fluid and receiving liquid Human periodontal ligament stem cell exosomes were added to an aqueous solution of a biocompatible polyelectrolyte at room temperature and mixed thoroughly to obtain an inner phase fluid; nanocellulose microcrystals were fully dispersed in an aqueous solution of sodium alginate to obtain an outer phase fluid; and water-soluble calcium salts were dissolved in water to obtain a receiving solution. (2) Preparation of microcapsules loaded with human periodontal ligament stem cell exosomes The inner phase fluid and the outer phase fluid were continuously pumped into the injection tube and collection tube of the microfluidic electrospray device by injection pumps, respectively. Under the action of electric field, the fluid flowing out of the microchannel outlet was induced to form Taylor cones and atomize into monodisperse droplets into the receiving liquid. The monodisperse droplets underwent a cross-linking reaction in the receiving liquid. The cross-linking and curing products were collected and washed with water to obtain microcapsules loaded with human periodontal ligament stem cell exosomes. (3) Platinum-loaded ions Microcapsules loaded with human periodontal ligament stem cell exosomes were immersed in an aqueous solution of chloroplatinic acid, allowed to react fully, and then separated into solid and liquid phases. The resulting solid phase was washed with water to obtain antibacterial sustained-release microcapsules for the treatment of periodontitis.

7. The method for preparing the antibacterial sustained-release microcapsules for treating periodontitis according to claim 6, characterized in that, In the inner phase fluid, the concentration of the biocompatible polyelectrolyte is 5-40 mg / mL, and the concentration of human periodontal ligament stem cell exosomes is 5-100 μg / mL; in the outer phase fluid, the mass ratio of sodium alginate to nanocellulose microcrystals is (1-5):1, and the concentration of sodium alginate is 10-17 mg / mL; in the receiving solution, the concentration of water-soluble calcium salt is 5-40 mg / mL.

8. The method for preparing the antibacterial sustained-release microcapsules for treating periodontitis according to claim 6, characterized in that, In step (2), the voltage of the microfluidic electro-injection device is controlled to be 5~10 kV, the receiving distance is controlled to be 3~8 cm, and the flow rate ratio of the inner phase fluid to the outer phase fluid is controlled to be (0.15~1):

1.

9. A method for preparing the antibacterial sustained-release microcapsules for treating periodontitis as described in any one of claims 6 to 8, characterized in that, The concentration of the chloroplatinic acid aqueous solution in step (3) is 5~20 mg / mL, and the reaction time in step (3) is controlled to be 0.5~3 h and the reaction temperature is 20~40 ℃.

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