Fabrication of nanozyme thixotropic anionic hydrogel for treating fungal keratitis by Dectin-1/p38 pathway

Abstract

Fungal keratitis (FK) is a major cause of corneal blindness, particularly in China, where treatment is often limited by systemic side effects and antifungal drug resistance. We propose a nanozyme (carbon nanotube (CNT))-based anionic hydrogel coating (NHC) loaded with itraconazole (IZ) (NTH@CNT/IZ) as a treatment for FK to overcome these challenges. This formulation was designed to enhance ocular drug delivery, improve antifungal efficacy, and reduce inflammation. In vitro assays against Aspergillus fumigatus demonstrated potent antifungal activity, including significant reductions in colony-forming units and biofilm formation at 50 µg/mL. Cell viability tests using ARPE-19 cells revealed high biocompatibility, with no observed morphological alterations or apoptosis, despite increased ROS and DNA proliferative activity. Importantly, NTH@CNT/IZ markedly downregulated inflammatory mediators-Dectin-1, IL-1β, and TNF-α-and inhibited phosphorylation of p38 MAPK, indicating suppression of the Dectin-1/p38 MAPK signaling pathway. In vivo results further confirmed its therapeutic potential, showing reduced corneal fungal burden and inflammation, along with effective penetration through the corneal epithelium, overcoming mucosal and fungal barriers. Together, these findings highlight NTH@CNT/IZ as a promising localized treatment strategy for fungal keratitis, offering targeted antifungal action and immune modulation to improve clinical outcomes and preserve ocular integrity. KEY POINTS: Nanozyme hydrogel for ocular health In vitro antifungal efficacy.

Keywords: Anionic hydrogel; Anti-inflammation; Fungal keratitis; Itraconazole.

Scheme 1

Preparation and application of NTH@CNT/IZ

Fig. 1

Physical characterization of CNT and NHC. SEM images of NHC@CNT (A, B). XPS spectra of tested samples (C). Binding energy with XPS of CNT and NHC@CNT (D, E)

Fig. 2

Characterization of CNT and NHC. The fluctuations in storage moduli G′ and loss moduli G″ (A–C). Viscosity-shear rate curves (D). Thixotropy test (E). Zeta potential of NHC (F). Water content (G). pH of tested samples (H). O₂ generating performance of tested samples with different time intervals (I)

Fig. 3

Evaluation of enzyme mimic activity. The absorption spectra of the reaction systems with different compositions with different materials (A, B). The accumulative drug release at different pH of NHC@CNT (C). Absorption spectra of prepared material with different pH (D). Magnetic field of the NHC@CNT (E). Absorption spectra of autoxidation with CNT (F). *p < 0.01 statistical significance was observed

Fig. 4

Antifungal activity on the zone of inhibition on A. fumigatus (A). CFU counting done with different types of sample materials (B). Growth of inhibition % (C). OD value absorbance at 590 nm (D). Fungal viability CFU counting with log CFU/mL (E). Fluorescence of dead fungal cells expressed at different materials (F). *p < 0.01 significant difference between control

Fig. 5

Anti-biofilm activity done through A. fumigatus at different samples at 100 µm with × 10 magnification (A). Absorbance of anti-biofilm activity with 570 nm (B). Statistical differences among groups were observed

Fig. 6

Cell viability of ARPE-19 cell line morphological observation done through phase contrast microscope treated with different samples (A). Absorbance through 570 nm (B)

Fig. 7

ARPE-19 cell lines were subjected to oxidative stress when exposed to different samples. ROS production in ARPE-19 cells following a 24-h exposure to maximum 100 µg/mL, 100 µm, and × 10 magnification (A). ROS percentage with significant asterisk exhibits statistical significance among groups, **p < 0.001 (B)

Fig. 8

Blue fluorescent dye (DAPI-negative) was used to label the nuclear DNA of proliferating cells. The presence of high levels of blue fluorescence of chromatin condensation at 100 µm and the magnification of × 10 (A). Fluorescence density of ARPE-19 cells measured by flow cytometry (B). Cell proliferation for ARPE-19 cell lines with various periods of 12 h was examined with significant variations, ***p < 0.0001 (C). Fluorescent intensity was measured with sample (D)

Fig. 9

The severity of fungal keratitis infection (A). Clinical scores of before and after treatment (B). HE staining of the cornea in normal and treated groups corneal infection with A. fumigatus in mice after treatment with 1, 3, and 5 days (C). Histological scores were observed through statistical analysis (D). The asterisk indicates statistical significance among groups: **p < 0.001, ***p < 0.0001