Thermo-Responsive Hydrogel Containing Microfluidic Chitosan Nanoparticles Loaded with Opuntia ficus-indica Extract for Periodontitis Treatment

Abstract

Periodontitis is a chronic inflammatory disease resulting from the dysbiosis of periodontal bacteria and the host's immune response, leading to tissue degradation and sustained inflammation. Traditional treatments, such as mechanical debridement and antimicrobial agents, often fail to fully eradicate pathogenic bacteria, especially in deep periodontal pockets. Consequently, the need for novel therapeutic approaches has increased the interest in bioactive natural extracts, such as that of Opuntia ficus-indica, known for its anti-inflammatory, antioxidant, and antimicrobial properties. This study investigates the encapsulation of Opuntia ficus-indica extract in OFI-loaded chitosan nanoparticles (OFI-NPs) via ionotropic gelation using a microfluidic system, allowing precise control over nanoparticle characteristics and enhancing protection against enzymatic degradation. To achieve localized and sustained release in periodontal pockets, a thermo-responsive hydrogel comprising hyaluronic acid and Pluronic F127 (OFI@tgels) was developed. The transition of OFI@tgels from a solution at low temperatures to a solid at body temperature enables prolonged drug release at inflammation sites. The in vitro application of the optimized formulation eradicated biofilms of S. mutans, P. aeruginosa (PAO1), and P. gingivalis over 36 h and disrupted extracellular polymeric substance formation. Additionally, OFI@tgel modulated immune responses by inhibiting M1 macrophage polarization and promoting a shift to the M2 phenotype. These findings suggest that OFI@tgel is a promising alternative treatment for periodontitis, effectively reducing biofilm formation and modulating the immune response.

Keywords: Opuntia; anti-biofilm; macrophage modulation; microfluidic particle synthesis; periodontitis; thermo-responsive hydrogel.

Figure 1

Physicochemical characterization of optimized OFI-loaded nanoparticles (OFI-NP_s). (A) Average particle size, and (B) NTA measurement of OFI-loaded nanoparticles in suspension. The frame is a representative screenshot of the NTA video. (C) Morphology of OFI-loaded nanoparticles using TEM microscopy. (D) FTIR spectra of OFI-NPs. (E) Size, PDI, and (F) ζ-potential of OFI-NPs during three months of storage at 25 °C.

Figure 2

OFI@tsol and OFI@tgel properties. Gelling time at 33 °C (A), and temperature (B) of OFI@tsol. Appearance of OFI@tsol and OFI@tgel (C,D). OFI@tgel swelling ratio (E); water retention ratio of OFI@tgel at 35 °C (F). Adhesion properties of OFI@tgel (G).

Figure 3

OFI extract release and antibacterial activity. Cumulative OFI release from OFI@tgel simulated salivary fluid (SSF) at pH 6.5 and 35 °C after 24 h (A) and 1 week (B). Antibacterial activity of OFI@tgel relative to P. gingivalis (C), PAO1 (D), and S. mutans (E) as survival rate (%) at 6, 12, and 24 h. The results are expressed as the means of the values obtained (mean ± SD) after six measurements for each sample. Statistically significant differences: *** p < 0.001 versus @tgel.

Figure 4

Antibiofilm activity of OFI@tgel. (A) P. gingivalis, PAO1, and S. mutans biofilm inhibition was evaluated by crystal violet after 8, 24, and 36 h of incubation at 35 °C in the presence of OFI@tgel. (B) EPS production in the presence of OFI@tgel in P. gingivalis, PAO1, and S. mutans compared to hydrogel alone. (C) Fluorescence microscopy images of live/dead staining (scale bar 200 µm, 40× magnification). Live bacteria appeared green, while dead bacteria were stained red. When live and dead bacteria were in close proximity, they produced a yellow/orange color. (D) Quantification of biofilm viability with or without OFI@tgel. mRNA expression level of QS-related genes after incubation with OFI@tgel in (E) P. gingivalis, (F) PAO1, and (G) S. mutans. The expression levels of the tested genes were normalized to the mean critical threshold (CT) values of the housekeeping gene 16s rRNA using the 2^−ΔΔCt method. Hydrogel without OFI extract was used as a control (@tgel). For each sample, six independent experiments were performed, and the results were expressed as mean ± SD. *** p < 0.001 compared to @tgel.

Figure 5

Biocompatibility and antioxidant effect of OFI@tgel on human cells. (A) HGF and THP-1 proliferation after 24, 48, and 72 h in the presence of OFI@tgel. (B) Fluorescence images of HGF cells treated with OFI@tgel with coumarin-6 for 3 h: (a,c) 10× magnification; 50 μm scale bar and 40× magnification; 10 μm scale bar (b,d) (C) ROS production was determined by the DCF fluorescence intensity using a microplate reader. (D) Malondialdehyde was used as a marker of lipid peroxidation. (E) Superoxide dismutase (SOD2), (F) catalase (CAT), and (G) glutathione peroxidase (GPx) activity was measured using an assay kit. Treatment with LPS of Porphyromonas gingivalis (1 μg/mL) is a positive control. Results are expressed as the means of three independent experiments S.D. (n = 3). Statistically significant variations: ## p < 0.01 and ### p < 0.00 versus LPS.

Figure 6

OFI@tgel induction of macrophage M2 polarization. (A) mRNA expression of M1 macrophage markers (CTL. CCR7, TNF-α, ARG1, and MSR1) after LPS stimulation (1 μg/mL) for 24 h analyzed using RT-qPCR. Stimulated macrophages were compared with unstimulated cells used as control (@tgel). Data are represented as fold change over actin (2^−ΔΔCt). Inhibitory effects of OFI on the secretion (B) and gene expression (D) of inflammatory mediators TNF-α, IL-1 and IL-6 in LPS-stimulated macrophages measured using ELISA assay and RT-qPCR. Cytokine secretion (C) and gene expression (E) of IL-4, IL-10, ARG1, and MSR1 in LPS-stimulated macrophages measured using ELISA assay and RT-qPCR. THP-1 cells were pre-treated with OFI@tgel for 24 h and then stimulated with LPS for 24 h. PMA-differentiated THP-1 macrophages seeded on the culture plate without LPS stimulation were used as control (@tgel). Results are expressed as the mean of three independent experiments ± S.D. (n = 3). Statistically significant variations ### p < 0.001 versus gene expression after LPS stimulation.