Injectable responsive hydrogel with synergistic antibacterial and anti-inflammatory properties for enhanced periodontitis treatment

Abstract

Periodontitis is a chronic inflammatory disease driven by dysbiotic microbial biofilms and localized reactive oxygen species (ROS) accumulation, with inflammation management made challenging by recurrent infections from residual pathogenic bacteria in the periodontal pockets. To address this, we engineered an injectable pH-responsive hydrogel (MH@ZIF-8/CS/β-GP) through the integration of minocycline hydrochloride (MH)-encapsulated zeolitic imidazolate framework-8 (ZIF-8) nanoparticles into a chitosan (CS) and β-glycerophosphate (β-GP) crosslinked matrix. The MH@ZIF-8 displayed broad-spectrum antimicrobial efficacy against key periodontal pathogens including Porphyromonas gingivalis (Pg), and Aggregatibacter actinomycetemcomitans (Aa), primarily attributed to the synergistic antimicrobial effects of Zn ions and MH. Additionally, MH@ZIF-8 effectively eliminated ROS by inhibiting the NLRP3/Caspase-1/IL-1β signaling pathway, demonstrating potent anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The MH@ZIF-8/CS/β-GP hydrogel, which exhibited favored cytocompatibility with human gingival fibroblasts (HGFs), undergoed a rapid sol-gel transition and pH-responsive sustained-release drug delivery under the acidic conditions of periodontal pockets, effectively responding to periodontitis microenvironment. Meanwhile, this hydrogel effectively alleviated alveolar bone loss in vivo. Overall, the developed MH@ZIF-8/CS/β-GP hydrogel presents a novel strategy for chronic periodontitis treatment and demonstrates promising clinical application potential.

Keywords: Anti-bacteria; Anti-inflammation; Chronic periodontitis; Responsive hydrogel.

Fig. 1

Schematic diagram of the responsive hydrogels for periodontitis treatment. (a) Schematic representation of the synthesis protocol for MH@ZIF-8/CS/β-GP hydrogel. (b) Biological performance profiling of the MH@ZIF-8/CS/β-GP hydrogel. (c) Mechanistic cartography of the anti-bacteria and anti-inflammatory dual actions in MH@ZIF-8/CS/β-GP hydrogel-treated periodontic dentition

Fig. 2

Preparation and characterization of MH@ZIF-8. (a) Schematic illustration of MH@ZIF-8. (b) Photograph of MH-1@ZIF-8. (c) SEM image of MH-1@ZIF-8. (d-f) EDS elemental mapping of Cl, N, Zn, in MH-1@ZIF-8. (g) FTIR spectra of ZIF-8 and MH@ZIF-8. (h) XRD pattern of MH@ZIF-8. (i) Drug loading capacity of MH-0.25@ZIF-8, MH-0.5@ZIF-8, and MH-1@ZIF-8. (j) Release profiles of MH-0.25@ZIF-8, MH-0.5@ZIF-8, and MH-1@ZIF-8 at 37 °C (pH = 7.4). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Biocompatibility evaluation of MH@ZIF-8 Nanocomposite. (a) Live/Dead staining for cells co-culture with MH@ZIF-8 (Live: green, Dead: red). (b) Quantitative analysis of Live/Dead cell staining images. (c) CCK-8 cytotoxicity assessment of cells after co-culture with MH@ZIF-8. The data are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

In vitro antimicrobial performance of MH@ZIF-8. (a) Colony counting assay of ZIF-8 and MH@ZIF-8 co-cultured with S.aureus, E.coli, Pg, and Aa. (b) Live/dead staining for S.aureus, E.coli, Pg, and Aa after co-culture with MH@ZIF-8. (c) SEM images of bacterial membrane integrity after co-culture with MH@ZIF-8. (d) Biofilm morphology of ZIF-8 and MH@ZIF-8. (e) OD₆₀₀ value of bacterial biofilm after co-culture with ZIF-8 and MH@ZIF-8. (f) Quantitative analysis of antibacterial rate. The data are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

In vitro anti-inflammatory assessment of MH@ZIF-8. (a) Antioxidant activity assay images. (b) Quantitative analysis of antioxidant activity. (c-e) RT-qPCR analysis of inflammatory-related gene expression in RAW264.7 macrophages (IL-1β, Caspase-1, NLRP3). (f) TNF-α expression under ZIF-8 and MH@ZIF-8 treatment. (g) Mechanistic diagram of the anti-inflammatory process. The data are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Characterization of MH@ZIF-8/CS/β-GP Hydrogel. (a) SEM images of freeze-dried CS/β-GP hydrogel. (b) SEM images of freeze-dried MH@ZIF-8/CS/β-GP hydrogel. (c) pH-responsive release kinetics of minocycline hydrochloride from MH@ZIF-8/CS/β-GP hydrogel at 37 °C (pH = 7.4 vs. pH = 6.0). (d) XRD analysis of MH@ZIF-8/CS/β-GP hydrogel. (e) FTIR analysis of MH@ZIF-8/CS/β-GP hydrogel. (f) In vitro degradation behavior of MH@ZIF-8/CS/β-GP hydrogel (pH = 7.4 vs. pH = 6.0). (g) Inversion testing of MH@ZIF-8/CS/β-GP hydrogels. (h) Rheological analysis of MH@ZIF-8/CS/β-GP hydrogel. (i) Step-strain (0.1% and 100% strain) test of MH@ZIF-8/CS/β-GP hydrogel. (j) Injectable performance evaluation of MH@ZIF-8/CS/β-GP hydrogel. (k) The tensile and shear adhesive strength of CS/β-GP and MH@ZIF-8/CS/β-GP hydrogels. (l) CCK-8 cytotoxicity assessment of cells after co-culture with MH@ZIF-8/CS/β-GP

Fig. 7

In vivo evaluation of MH@ZIF-8/CS/β-GP for periodontitis treatment. (a) Schematic diagram of establishment and therapeutic intervention in periodontitis model of rats. (b) Crystal violet staining of biofilm from gingival crevicular fluid after 2- and 4-week treatment. (c) Comparative micro-CT 3D reconstruction of maxillae in SD rats from Control, PBS, CS/β-GP, and MH@ZIF-8/CS/β-GP groups. (d) HE stained maxillary sections. (e) Masson’s trichrome stained maxillary sections. (f-g) Optical density Quantification of biofilm from gingival crevicular fluid after 2- and 4-week treatment. (h-i) Quantitative assessment of BMD and bone volume BV/TV around the second maxillary molar. (j-k) CEJ-ABC distance at mesial/distal sites of the second maxillary molar. The data are the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (AB: alveolar bone; PDL: periodontal ligament)

Fig. 8

Immunohistochemical analysis in maxillary sections. (a-d) Immunohistochemical staining of NLRP3, Caspase-1, IL-1β, and TNF-α. (e-h) Quantitative analysis of IHC staining