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. 2024 Nov 1:19:11163-11179.
doi: 10.2147/IJN.S480800. eCollection 2024.

Zeolitic Imidazolate Framework-8 Offers an Anti-Inflammatory and Antifungal Method in the Treatment of Aspergillus Fungal Keratitis in vitro and in vivo

Xueyun Fu #  1 , Xue Tian #  1 , Jing Lin  1 , Qian Wang  1 , Lingwen Gu  1 , Ziyi Wang  1 , Menghui Chi  1 , Bing Yu  1 , Zhuhui Feng  1 , Wenyao Liu  1 , Lina Zhang  1 , Cui Li  1 , Guiqiu Zhao  1
Affiliations

Zeolitic Imidazolate Framework-8 Offers an Anti-Inflammatory and Antifungal Method in the Treatment of Aspergillus Fungal Keratitis in vitro and in vivo

Xueyun Fu et al. Int J Nanomedicine. .

Abstract

Background: Fungal keratitis is a serious blinding eye disease. Traditional drugs used to treat fungal keratitis commonly have the disadvantages of low bioavailability, poor dispersion, and limited permeability.

Purpose: To develop a new method for the treatment of fungal keratitis with improved bioavailability, dispersion, and permeability.

Methods: Zeolitic Imidazolate Framework-8 (ZIF-8) was formed by zinc ions and 2-methylimidazole linked by coordination bonds and characterized by Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Zeta potential. The safety of ZIF-8 on HCECs and RAW 264.7 cells was detected by Cell Counting Kit-8 (CCK-8). Safety evaluation of ZIF-8 on mice corneal epithelium was conducted using the Draize corneal toxicity test. The effects of ZIF-8 on fungal growth, biofilm formation, and hyphae structure were detected by Minimal inhibit concentration (MIC), crystal violet staining, Propidium Iodide (PI) testing, and calcofluor white staining. The anti-inflammatory effects of ZIF-8 on RAW 246.7 cells were evaluated by Quantitative Real-Time PCR Experiments (qPCR) and Enzyme-linked immunosorbent assay (ELISA). Clinical score, Colony-Forming Units (CFU), Hematoxylin-eosin (HE) staining, and immunofluorescence were conducted to verify the therapeutic effect of ZIF-8 on C57BL/6 female mice with fungal keratitis.

Results: In vitro, ZIF-8 showed outstanding antifungal effects, including inhibiting the growth of Aspergillus fumigatus over 90% at 64 μg/mL, restraining the formation of biofilm, and destroying cell membranes. In vivo, treatment with ZIF-8 reduced corneal fungal load and mitigated neutrophil infiltration in fungal keratitis, which effectively reduced the severity of keratitis in mice and alleviated the infiltration of inflammatory factors in the mouse cornea. In addition, ZIF-8 reduces the inflammatory response by downregulating the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β after Aspergillus fumigatus infection in vivo and in vitro.

Conclusion: ZIF-8 has a significant anti-inflammatory and antifungal effect, which provides a new solution for the treatment of fungal keratitis.

Keywords: MOFs; ZIF-8; anti-inflammatory; antifungal; fungal keratitis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1
Scheme 1
Schematic diagram of the synthesis of ZIF-8 and its effects in vitro and in vivo.
Figure 1
Figure 1
Characteristic of ZIF-8. (A) SEM image with 200 nm scale of ZIF-8. (B) SEM image with 1μm scale of ZIF-8. (C–F) EDS elemental mapping of ZIF-8. (G) Distribution histogram of 92 particles. (H) Zeta Potential of ZIF-8. (I) Powder X-ray diffraction (PXRD) picture of ZIF-8.
Figure 2
Figure 2
The biological toxicity of ZIF-8 in vitro and in vivo. (A and B) Cell viability of RAW 264.7 cells and HCECs in the presence of different concentrations of ZIF-8. (C and D) Photographs of Corneal fluorescein staining (CFS) of mice corneas and scores. (ns, significance, **** p<0.0001).
Figure 3
Figure 3
Anti-inflammatory effect of ZIF-8 in RAW 264.7 cells. ZIF-8 decreases the expression of pro-inflammatory factors (A) IL-6, (B) IL-1β, (C) TNF-α, (D) TLR-4, and Inflammasome (E) NLRP3 mRNA and increases the expression of anti-inflammatory factor (F) HO-1 in RAW 264.7 cells. (*** p<0.001, **** p<0.0001).
Figure 4
Figure 4
Anti-fungal activity of ZIF-8 against Aspergillus fumigatus. (A) MIC of ZIF-8 for A. fumigatus. (B) Inhibitory effect of ZIF-8 on biofilm. Hyphae treated with (C) PBS and (D) ZIF-8. (ns, no significance, **** p<0.0001).
Figure 5
Figure 5
(A) PI staining reflects the disruptive effects of ZIF-8 and natamycin on hyphae cell membranes. (B) Calcofluor White Staining reveals the impact of ZIF-8 and natamycin on the hyphal cell wall.
Figure 6
Figure 6
ZIF-8 therapeutic approach ameliorates the prognosis of FK in mice. (A) Photographs of corneas of FK-infected mice after the ZIF-8 regimen and (B) scoring and corneas of Aspergillus fumigatus infected mice after natamycin management. (C) Plates of viable fungus in the cornea 3 days after infection. (D) Quantitative analysis of fungal load. (ns, no significance; * p<0.05, *** p<0.001, **** p<0.0001).
Figure 7
Figure 7
Pathological changes of corneas after ZIF-8 medical intervention. (A) HE staining of corneas of different groups of Aspergillus fumigatus keratitis mice 3 days after infection (magnification: 400×). (B) Immunofluorescence staining of PBS and ZIF-8-treated neutrophils (magnification: 400×).
Figure 8
Figure 8
The effect of ZlF-8 on reducing inflammatory proteins in RAW 264.7 cells and mice corneas. ZIF-8 decreases the expression of pro-inflammatory proteins (A) TNF-α, (B) IL-1β, and (C) lL-6 in cells after hyphae stimulation. ZlF-8 reduces the expression of proinflammatory proteins (D) TNF-α, (E) lL-1β, and (F) lL-6 in the cornea of mice infected with Aspergillus fumigatus 3 days after infection (ns, no significance, **** p<0.0001).
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