Modulus-tunable ros-sensitive hydrogels encapsulating active ingredients and their preparation and use

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a modulus-adjustable ROS-sensitive hydrogel loaded with active ingredients and preparation and application thereof. The present application loads macrophages and C5a receptor antagonists into ROS-sensitive PVA-TSPBA hydrogel, which can release exogenous macrophages to achieve good antibacterial effect when placed in a high-ROS periodontitis microenvironment, wherein the modulus of the hydrogel is regulated to mechanically activate macrophages to improve phagocytosis activity of the macrophages; meanwhile, the released complement C5a receptor antagonists can eliminate the inhibitory effect of Porphyromonas gingivalis Pg on the bactericidal activity of macrophages, further amplify the bactericidal effect of macrophages, and jointly achieve significant Pg clearance, treatment effect of periodontitis, and effects of anti-inflammation and periodontal bone protection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a modulus-tunable ROS-sensitive hydrogel encapsulating active ingredients, its preparation, and its application. Background Technology

[0002] Periodontitis is the sixth most common chronic inflammatory disease, affecting more than 10% of adults worldwide and imposing a significant socioeconomic burden. Research has demonstrated a close relationship between periodontitis and periodontal microbial dysbiosis, which refers to changes in the composition of the microbial community or an imbalance between the host and the community. *Porphyromonas gingivalis* (Pg) plays a crucial role in this process. Pg initially disrupts the host's immune surveillance function, thereby promoting adaptation throughout the community and facilitating the selective expansion of periodontitis-associated bacteria. In this context, the homeostatic periodontal microbial community shifts towards disease-inducing microbial communities, thus triggering or exacerbating periodontitis.

[0003] Currently, various methods have been developed to prevent or treat periodontitis by inhibiting Pg. For example, a Chinese patent application discloses the application of nicotinamide in the preparation of drugs against Porphyromonas gingivalis. Experiments have shown that nicotinamide can inhibit the growth of Porphyromonas gingivalis, reduce its periodontal virulence, and has a certain therapeutic effect on periodontitis. However, further research has found that in order to evade immune clearance, Pg actively manipulates the natural immune cells in the periodontal niche—macrophages—inactivating their antibacterial activity. Specifically, Pg can disrupt the phagocytic capacity of macrophages through various pathways, thereby preventing internalization by macrophages. For example, it can selectively downregulate the pattern recognition receptor CD14 on the surface of macrophages (Wilensky, A., Tzach-Nahman, R., Potempa, J., Shapira, L. & Nussbaum, G. Porphyromonas gingivalis gingipains selectively reduce CD14 expression, leading to macrophage hyporesponsiveness to bacterial infection. J Innate Immun 7, 127-135, doi:10.1159 / 000365970(2015)), thus resisting macrophage-mediated phagocytosis. Worse still, even if Pg is phagocytosed, the internalized Pg can maintain high activity within the cell for a long time by weakening the bactericidal activity of macrophages (Wang, M. et al. Fimbrial proteins of porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages. J Immunol 179, 2349-2358, doi:10.4049 / jimmunol.179.4.2349(2007)). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing drugs for treating periodontitis that inhibit Pg growth and cause macrophage immune escape, and to provide a ROS-sensitive hydrogel with adjustable modulus that encapsulates active ingredients.

[0005] The purpose of this invention is to provide a method for preparing a ROS-sensitive hydrogel with adjustable modulus containing active ingredients.

[0006] Another object of the present invention is to provide the application of the modulus-tunable ROS-sensitive hydrogel containing the active ingredient.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A modulus-tunable ROS-sensitive hydrogel encapsulating an active ingredient, wherein the active ingredient is a macrophage and C5a receptor antagonist, and the modulus-tunable ROS-sensitive hydrogel is polyvinyl alcohol-N... 1 N 1 N 3 N 3- Tetramethylpropane-1,3-diammonium (PVA-TSPBA) hydrogel.

[0009] Studies have found that the secretion of virulence factors (HRgpA, RgpB) by macrophages induces macrophages to uncontrollably produce large amounts of complement C5a fragments. This pathologically activates the complement C5a receptor (C5aR) in macrophages. This Pg-triggered C5aR activation not only produces large amounts of cyclic adenosine monophosphate through co-activation with Toll-like receptor 2, but also inhibits Toll-like receptor 4-induced IL-12p70 production. Both ultimately lead to a reduction in nitric oxide, thereby increasing the intracellular survival rate of Pg and facilitating its long-term survival.

[0010] To address the issue of Pg inactivating the in situ antibacterial activity of macrophages in the periodontal niche, this invention encapsulates exogenous macrophages and a C5a receptor antagonist within a ROS-sensitive PVA-TSPBA hydrogel. When placed in a high-ROS periodontal microenvironment, the ROS-sensitive hydrogel degrades in response to ROS, releasing exogenous macrophages with enhanced phagocytic capacity and undisturbed bactericidal function. These exogenous macrophages act as a potent antibacterial tool against *Porphyromonas gingivalis*, achieving excellent antibacterial effects while avoiding the limitations of traditional direct antibacterial therapies such as antibiotic resistance. Adjusting the elastic modulus of the hydrogel significantly enhances the phagocytic activity of the encapsulated macrophages. Simultaneously, the complement C5a receptor antagonist pre-binds to the macrophage's C5aR, preventing Pg from utilizing C5aR to inhibit macrophage bactericidal activity, thus eliminating the inhibitory effect of Pg on macrophage bactericidal activity and further amplifying the bactericidal effect of macrophages. Furthermore, experiments have shown that this ROS-sensitive hydrogel with adjustable modulus and containing active ingredients also has anti-inflammatory and periodontal bone protection effects, and has a significant therapeutic effect on periodontitis.

[0011] Preferably, the macrophages are RAW264.7 macrophages or macrophages derived from the bone marrow of SD rats.

[0012] Preferably, the C5a receptor antagonist is PMX-53.

[0013] Furthermore, the degree of crosslinking of the PVA-TSPBA hydrogel is 3-9%.

[0014] Furthermore, the present invention also provides a method for preparing the ROS-sensitive hydrogel with adjustable modulus encapsulated active ingredients, specifically including the following steps:

[0015] The cross-linking agent TSPBA was dissolved in serum-free DMEM high-glucose medium at room temperature to prepare a TSPBA solution. PVA was dissolved in serum-free DMEM high-glucose medium to prepare a PVA solution. Macrophages and C5a receptor antagonists were suspended in the PVA solution and mixed evenly with the TSPBA solution. The reaction was allowed to proceed until complete.

[0016] Furthermore, the PVA is dissolved at a temperature of 80–90°C.

[0017] Furthermore, the reaction temperature is 30–40°C. Preferably, the reaction temperature is 37°C.

[0018] Furthermore, the mass ratio of TSPBA to PVA is 1:(1-3).

[0019] Furthermore, the preparation method of the crosslinking agent TSPBA includes the following steps: adding 4-(bromomethyl)phenylboronic acid and N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPA) to a polar organic solvent, reacting completely at 20-50°C, precipitating in tetrahydrofuran, filtering, and treating the precipitate to obtain the crosslinking agent TSPBA.

[0020] Preferably, the polar organic solvent is N,N-dimethylformamide.

[0021] Preferably, the reaction time is 24 to 36 hours.

[0022] Additionally, the present invention also claims the use of the modulus-tunable ROS-sensitive hydrogel containing the active ingredient in the preparation of anti-periodontalgia drugs.

[0023] In addition, the present invention also provides the application of the modulus-tunable ROS-sensitive hydrogel containing the active ingredient in the preparation of Porphyromonas gingivalis inhibitors.

[0024] The present invention has the following beneficial effects:

[0025] This invention relates to a modulus-adjustable ROS-sensitive hydrogel containing active ingredients. Macrophages and C5a receptor antagonists are encapsulated within a ROS-sensitive PVA-TSPBA hydrogel. When placed in a high-ROS periodontal microenvironment, it releases exogenous macrophages, achieving a good antibacterial effect. The hydrogel modulus is adjusted to mechanically activate macrophages, thereby enhancing their phagocytic activity. Simultaneously, the released complement C5a receptor antagonist eliminates the inhibitory effect of *Porphyromonas gingivalis* (Pg) on ​​the bactericidal activity of macrophages, further amplifying the bactericidal effect of macrophages. Together, they achieve significant Pg clearance and therapeutic effects for periodontitis, while also possessing anti-inflammatory and periodontal bone-protective effects. Attached Figure Description

[0026] Figure 1 The pure TSPBA prepared in Example 1 of this invention 1 H-NMR spectrum.

[0027] Figure 2 The mechanical properties characterization diagrams for the hydrogels in Experimental Example 1 are as follows: A is a photograph of PVA-TSPBA hydrogels with different degrees of crosslinking in Examples 1-3 of the present invention; B is a rheological experimental result diagram of the storage modulus (G′) and loss modulus (G″) of hydrogels with different degrees of crosslinking in Examples 1-3 of the present invention; C is a stress-strain curve diagram of hydrogels with different degrees of crosslinking in Examples 1-3 of the present invention under compression; D is a statistical diagram of the elastic modulus data of hydrogels with different degrees of crosslinking in Examples 1-3 of the present invention (n=3, mean±SD; ***p<0.001).

[0028] Figure 3 Figure 2 shows the test results of ROS-sensitive hydrogels with different degrees of crosslinking regulating the phagocytic ability of macrophages in Examples 1-3 of the present invention in Experiment 2: A and B are statistical graphs of the proportion of positive cells that phagocytosed CFSE-Pg after RAW264.7 macrophages wrapped in hydrogels of different moduli were co-incubated with CFSE-labeled Pg for 1 h (A) and 4 h (B) by flow cytometry; C is a confocal microscopy image of RAW264.7 macrophages wrapped in hydrogels of different moduli phagocytosing CFSE-labeled Pg.

[0029] Figure 4 The following is an example of the in vitro bactericidal effect of ROS-sensitive hydrogel encapsulating macrophages and C5A in Experiment Example 2: The left image shows the growth of remaining viable bacteria on blood agar plates after co-incubation of RAW264.7 macrophages treated with different methods and Pg; the right image shows the quantitative analysis statistics of colony forming units (CFU) of the remaining viable bacteria (n=8, mean±SD; *p<0.05; **p<0.01; ***p<0.001).

[0030] Figure 5This is a statistical graph showing the content of TNF-α, IL-1β and IL-6 in the supernatant of RAW264.7 macrophages after co-incubation with Pg for 4 h using ELISA, in the in vitro anti-inflammatory experiment of ROS-sensitive hydrogel-encapsulated macrophages and C5A.

[0031] Figure 6 The following is an example of the in vivo periodontitis treatment effect of ROS-sensitive hydrogel-encapsulated macrophages and C5A in Experiment 3: A is a flowchart of the in vivo periodontitis treatment study; B is a three-dimensional reconstructed micro-CT image of each experimental group; C is a quantitative statistical image of the micro-CT of the first molar of each experimental group, including the distance from the cementoenamel junction to the alveolar bone crest (CEJ-ABC) and the bone volume / total volume (BV / TV) (n=3,mean±SD;*p<0.05;**p<0.01;***p<0.001); D is an immunohistochemical staining image of TNF-α, IL-1β and IL-6 in sections of the right first molar of SD rats in each experimental group. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] Example 1: A ROS-sensitive hydrogel with adjustable modulus

[0035] The preparation method of the modulus-tunable ROS-sensitive hydrogel specifically includes the following steps:

[0036] S1. Preparation of crosslinking agent TSPBA (synthesized by quaternization reaction between N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPA) and 4-(bromomethyl)phenylboronic acid):

[0037] 4-(bromomethyl)phenylboronic acid and TMPA (3:1, mmol / mmol) were mixed with N,N-dimethylformamide (DMF) and stirred at room temperature for 24 h. The reaction solution was precipitated in tetrahydrofuran (THF), filtered, and further washed three times with THF. The mixture was then incubated under vacuum overnight to obtain pure TSPBA. 1 H-NMR characterization, results as follows Figure 1 As shown, this demonstrates that the corresponding compound was prepared.

[0038] S2. Preparation of PVA-TSPBA hydrogels with different degrees of crosslinking:

[0039] Add 0.9g of PVA powder to 9.1ml of deionized water and stir in an 85°C water bath until the PVA is completely dissolved to prepare a 9% PVA solution; add 0.3g of TSPBA obtained in step S1 to 9.7ml of deionized water and stir at room temperature until completely dissolved to prepare a 3% TSPBA solution; mix the 3% TSPBA solution and the 9% PVA solution at room temperature until homogeneous and fully reacted to prepare a PVA-TSPBA hydrogel, as shown below. Figure 2 As shown in Figure A.

[0040] Example 2: A ROS-sensitive hydrogel with adjustable modulus

[0041] The preparation method of the modulus-tunable ROS-sensitive hydrogel specifically includes the following steps:

[0042] S1. Preparation of crosslinking agent TSPBA (synthesized by quaternization reaction between N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPA) and 4-(bromomethyl)phenylboronic acid):

[0043] 4-(bromomethyl)phenylboronic acid and TMPA (3:1, mmol / mmol) were mixed with N,N-dimethylformamide (DMF) and stirred at room temperature for 24 h. The reaction solution was precipitated in tetrahydrofuran (THF), filtered, and washed three times with THF. The mixture was then placed under vacuum overnight to obtain pure TSPBA.

[0044] S2. Preparation of PVA-TSPBA hydrogels with different degrees of crosslinking:

[0045] Add 0.9g of PVA powder to 9.1ml of deionized water and stir in an 85°C water bath until the PVA is completely dissolved to prepare a 9% PVA solution; add 0.6g of TSPBA obtained in step S1 to 9.4ml of deionized water and stir at room temperature until completely dissolved to prepare a 6% TSPBA solution; mix the 6% TSPBA solution and the 9% PVA solution at room temperature until homogeneous and fully reacted to prepare a PVA-TSPBA hydrogel, as shown below. Figure 2 As shown in Figure A.

[0046] Example 3: A ROS-sensitive hydrogel with adjustable modulus

[0047] The preparation method of the modulus-tunable ROS-sensitive hydrogel specifically includes the following steps:

[0048] S1. Preparation of crosslinking agent TSPBA (synthesized by quaternization reaction between N,N,N′,N′-tetramethyl-1,3-propanediamine (TMPA) and 4-(bromomethyl)phenylboronic acid):

[0049] 4-(bromomethyl)phenylboronic acid and TMPA (3:1, mmol / mmol) were mixed with N,N-dimethylformamide (DMF) and stirred at room temperature for 24 h. The reaction solution was precipitated in tetrahydrofuran (THF), filtered, and washed three times with THF. The mixture was then placed under vacuum overnight to obtain pure TSPBA.

[0050] S2. Preparation of PVA-TSPBA hydrogels with different degrees of crosslinking:

[0051] Add 0.9g of PVA powder to 9.1ml of deionized water and stir in an 85℃ water bath until the PVA is completely dissolved to prepare a 9% PVA solution; add 0.9g of TSPBA obtained in step S1 to 9.1ml of deionized water and stir at room temperature until completely dissolved to prepare a 9% TSPBA solution; mix the 9% TSPBA solution and the 9% PVA solution at room temperature until homogeneous and fully reacted to prepare a PVA-TSPBA hydrogel, as shown below. Figure 2 As shown in Figure A.

[0052] Example 4: A modulus-tunable ROS-sensitive hydrogel encapsulating active ingredients

[0053] Crosslinking agent TSPBA was dissolved in serum-free DMEM high-glucose medium at room temperature to prepare TSPBA solutions with different mass fractions (3%, 6%, 9%). PVA was dissolved in serum-free DMEM high-glucose medium under water bath conditions of 80-90℃ to prepare a 9% PVA solution. After the 9% PVA solution was fully cooled, macrophages and C5a receptor antagonists were suspended in the 9% PVA solution and mixed evenly with 3%, 6%, and 9% TSPBA solutions, respectively. The reaction was completed at 37℃ to obtain PVA-TSPBA hydrogels with different degrees of crosslinking and encapsulating active ingredients.

[0054] Experimental Example 1: Mechanical Properties of Hydrogels with Different Degrees of Crosslinking

[0055] Experimental methods:

[0056] The rheological properties of the hydrogels obtained in Examples 1-3 were determined using a rheometer (Haake MARS III; USA) with a frequency scanning mode of 1 Hz to 10 Hz, 1% strain, and 25 °C.

[0057] The hydrogel was made into a cylinder (base diameter = 6 mm) and placed on a compression plate. At room temperature, the cylindrical hydrogel was compressed to 40% of its total height using an Instron 3343 (Instron Co, USA) at a compression strain rate of 1 mm / min. The elastic modulus was calculated from the linear portion of the stress-strain curve.

[0058] Experimental results (see details) Figure 2 ):

[0059] As can be seen from the figure, in rheological testing (see details...), Figure 2 B) The storage modulus (G′) of the hydrogels in all embodiments was higher than the loss modulus (G″), indicating that a flexible gel was formed rather than a viscous liquid; in addition, the storage modulus (G′) and loss modulus (G″) increased significantly with the increase of crosslinking agent (TSPBA) content.

[0060] In the elastic modulus test of hydrogels (see details...), Figure 2 C) The compressive stress of the hydrogel prepared in the example also showed an increase with the increase of TSPBA addition, and similarly, the elastic modulus at 40% compressive strain also showed this.

[0061] Specifically (see Figure 2D) The hydrogel of Example 1 containing 3% TSPBA crosslinking agent had the lowest elastic modulus, only 1.74±1.89 kPa, followed by the hydrogel of Example 2 containing 6% TSPBA, with an elastic modulus of 27.08±0.14 kPa. When the concentration of TSPBA increased to 9%, the elastic modulus of the gel rapidly increased to 106.53±15.83 kPa.

[0062] Experiment Example 2: Determination of the in vitro antibacterial and anti-inflammatory effects of hydrogels

[0063] (1) Engulfment of hydrogels

[0064] Experimental methods:

[0065] Preparation of hydrogels containing active ingredients in RAW264.7 cells and release of active substances: The hydrogels containing 3%, 6%, and 9% TSPBA crosslinking agent in Examples 1-3 were named soft hydrogel, medium hydrogel, and stiff hydrogel, respectively; RAW264.7 cells (1×10⁻⁶) were loaded with active ingredients. 6 The cells were encapsulated in the above-mentioned hydrogel (see Example 4 for specific operation), and a conventional cell culture plate was used as a control group. The cells were cultured in basal medium or basal medium with 100 μM H2O2. The addition of 100 μM H2O2 was to simulate the high ROS environment in the periodontitis microenvironment. Under these culture conditions, the hydrogel responded to the H2O2 in the culture medium and underwent colloidal degradation. The cells were completely released in about 3 days to obtain the hydrogel RAW264.7 cell release solution for later use.

[0066] Pg was stained with 10 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma, USA) for 1 hour (37°C, anaerobic conditions). Then, CFSE-labeled Pg (37°C, normoxic conditions) was co-incubated with the above hydrogel RAW264.7 cell release medium at a ratio of 10:1 (Pg to RAW264.7 cells). The proportion of CFSE-Pg positive RAW264.7 cells after 1 hour and 4 hours was quantified by flow cytometry (CytoFLEX, Beckman Coulter). Typical images were recorded and captured by an Olympus confocal microscope (Olympus FV3000, Japan).

[0067] Analysis of experimental results (see details) Figure 3 ):

[0068] like Figure 3As shown in A and 3B, the proportion of Pg-positive cells was highest in the stiff hydrogel group (65.63% at 1 h and 99.86% at 4 h), followed by the medium hydrogel group (32.75% at 1 h and 78.87% at 4 h). However, most Pg cells evaded phagocytosis by RAW264.7 cells in the control and H2O2 groups, as less than 20% of Pg-positive cells were observed in both groups after 4 hours of co-incubation. A consistent trend was observed in confocal microscopy images. Figure 3 C).

[0069] (2) Determination of the bactericidal activity of the hydrogel

[0070] Experimental methods: RAW264.7 cells cultured in well plates were named Free MΦ, and RAW264.7 cells cultured in well plates with 1 μM C5A added to the culture medium were named C5A+MΦ. Groups co-encapsulating C5A with RAW264.7 cells (encapsulation method as described in Example 4) in soft or stiff hydrogels were named Lgel@C5A / MΦ and Hgel@C5A / MΦ, respectively. 2 μg of C5A was added per 1×10⁻⁶ cells. 6 RAW264.7 cells were cultured in a basal medium supplemented with 100 μM H2O2 to simulate the high ROS environment of the periodontitis microenvironment and to facilitate the release of cells by colloidal degradation of H2O2 in the ROS-sensitive hydrogel in response to ROS-sensitive hydrogels. Pg was mixed with PBS as a control group.

[0071] Pg(1×10) 7 RAW264.7 cells treated differently from those described above were co-incubated under normoxic conditions for 2 hours, then centrifuged at 300g for 5 minutes, retaining the precipitate and supernatant; the cells were lysed to release endocytosed Pg, and the cell lysate was mixed with the corresponding supernatant (containing Pg that had not yet been phagocytosed), serially diluted, and the diluted solutions were cultured on blood agar plates in an anaerobic incubator at 37°C to count the remaining viable Pg.

[0072] Analysis of experimental results (see details) Figure 4 ):

[0073] like Figure 4 As shown, after co-incubating RAW264.7 cells with different treatments for 2 hours, nearly 80% of the Pg cells in the Free MF group remained viable, while only 50% of the Pg cells survived when co-incubated with C5A-pretreated cells. More importantly, the Pg survival rate was the lowest in the Hgel@C5A / MF group, below 10%, indicating that the method of combining RAW264.7 cells with C5A using the hydrogel prepared in this invention has a significant effect on clearing pathogenic Pg cells.

[0074] (3) Determination of the anti-inflammatory activity of the hydrogel

[0075] In the assay of the bactericidal activity of the hydrogel (2), RAW264.7 macrophages of different groups and treatments were co-incubated with Pg for 4 h and the supernatant was extracted. The amounts of TNF-α, IL-1β and IL-6 in the supernatant were detected by ELISA kit.

[0076] Analysis of experimental results (see details) Figure 5 ):

[0077] As shown in the figure, Pg induces RAW264.7 cells to secrete large amounts of TNF-α, IL-1β and IL-6. This pro-inflammatory response can be attenuated by the complement receptor antagonist C5A. When the ROS-responsive hydrogel of the present invention is used, the production of these cytokines is further reduced, which means that ROS removal helps to further alleviate the ROS-induced inflammatory response.

[0078] Experiment Example 3: Determination of the in vitro antibacterial and anti-inflammatory effects of hydrogels containing active ingredients.

[0079] Experimental methods: Four-week-old male SD rats (100-150g) were randomly divided into five groups. Four groups of rats were injected subgingivally with LPS (1mg / ml, 20μL) and Pg (5×10⁻⁶ mg / ml, 20μL). 8 A mixture of LPS and Pg (20 μL) was injected every two days for a total of 5 times to establish periodontitis. Another group of healthy rats served as the natural control group without treatment. Two days after the last LPS and Pg injection, the four groups of rats received 40 μL of PBS (Sham), a mixture of C5A and rBMDM (C5A+MФ), a soft hydrogel loaded with C5A and rBMDM (Lgel@C5A / MФ), and a stiff hydrogel loaded with C5A and rBMDM (Hgel@C5A / MФ), respectively. The number of cells injected at each site was 1 × 10-1. 6 The dosage of C5A was 5 μg; treatment was administered every 4 days for a total of 5 injections (see detailed experimental procedures). Figure 6 A) Four days after the last treatment, the rats were sacrificed, and the maxillary tissue of one side of the rat was immersed in 10% neutral formalin and fixed for 48 hours before analysis.

[0080] Among them, rBMDM is a macrophage derived from the bone marrow of SD rats.

[0081] Analysis of experimental results (see details) Figure 6 ):

[0082] like Figure 6As shown in the micro-CT 3D reconstruction image, the periodontitis rats (Sham) treated with PBS alone showed obvious alveolar bone features, which contrasted sharply with the Hgel@C5A / MФ treated rats; the Hgel@C5A / MФ treated rats showed better resistance to Pg-induced bone resorption.

[0083] In quantitative statistics ( Figure 6 C) In the Sham group, the CEJ-ABC distance was doubled compared to the natural control (1550.0±125.30μm vs 700.0±52.9μm), and all treatment groups showed significant protection against Pg-induced bone loss; the Hgel@C5A / MФ group had the shortest CEJ-ABC distance, only 950.0±45.83μm; in addition, in the BV / TV quantification results, the Hgel@C5A / MФ treatment group also showed significantly higher bone quality compared to all other treatment groups.

[0084] Immunohistochemical staining of rat first molar sections further supported the osteoprotective effect of the Hgel@C5A / MФ group, such as... Figure 6 As shown in Figure D, rats receiving only PBS (Sham) had a large number of TNF-α, IL-1β, and IL-6 positive cells around the alveolar bone; however, the expression of these three pro-inflammatory effectors was greatly reduced in the alveolar bone of rats treated with Hgel@C5A / MФ.

[0085] The above in vivo results all indicate that the Hgel@C5A / MФ strategy has good in vivo anti-inflammatory and osteoprotective effects.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A modulus-tunable ROS-sensitive hydrogel encapsulating an active ingredient, characterized in that, The active ingredient is a macrophage and a C5a receptor antagonist; the modulus-tunable ROS-sensitive hydrogel is a PVA-TSPBA hydrogel; and the C5a receptor antagonist is PMX-53.

2. The modulus-tunable ROS-sensitive hydrogel encapsulating active ingredients according to claim 1, characterized in that, The macrophages are RAW264.7 macrophages or macrophages derived from the bone marrow of SD rats.

3. The modulus-tunable ROS-sensitive hydrogel encapsulating active ingredients according to claim 1, characterized in that, The degree of crosslinking of the PVA-TSPBA hydrogel is 3-9%.

4. A method for preparing the modulus-tunable ROS-sensitive hydrogel containing active ingredients as described in any one of claims 1 to 3, characterized in that, Specifically, the following steps are included: The cross-linking agent TSPBA was dissolved in serum-free DMEM high-glucose medium at room temperature to prepare a TSPBA solution. PVA was dissolved in serum-free DMEM high-glucose medium to prepare a PVA solution. Macrophages and C5a receptor antagonists were suspended in the PVA solution and mixed evenly with the TSPBA solution. The reaction was allowed to proceed until complete.

5. The preparation method according to claim 4, characterized in that, The PVA is dissolved at a temperature of 80–90°C.

6. The preparation method according to claim 4, characterized in that, The reaction temperature is 30–40°C.

7. The preparation method according to claim 4, characterized in that, The mass ratio of TSPBA to PVA is 1:(1-3).

8. The use of the modulus-tunable ROS-sensitive hydrogel containing the active ingredient as described in any one of claims 1 to 3 in the preparation of anti-periodontalgia drugs.

9. The use of the modulus-tunable ROS-sensitive hydrogel containing the active ingredient as described in any one of claims 1 to 3 in the preparation of Porphyromonas gingivalis inhibitors.

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