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. 2021 Dec 8;7(2):e10276.
doi: 10.1002/btm2.10276. eCollection 2022 May.

Ferrostatin-1-loaded liposome for treatment of corneal alkali burn via targeting ferroptosis

Kai Wang  1   2 , Li Jiang  3 , Yueyang Zhong  1   2 , Yin Zhang  1   2 , Qichuan Yin  1   2 , Su Li  1   2 , Xiaobo Zhang  1   2 , Haijie Han  1   2 , Ke Yao  1   2
Affiliations

Ferrostatin-1-loaded liposome for treatment of corneal alkali burn via targeting ferroptosis

Kai Wang et al. Bioeng Transl Med. .

Abstract

Alkali burn is a potentially blinding corneal injury. During the progression of alkali burn-induced injury, overwhelmed oxidative stress in the cornea triggers cell damage, including oxidative changes in cellular macromolecules and lipid peroxidation in membranes, leading to impaired corneal transparency, decreased vision, or even blindness. In this study, we identified that ferroptosis, a type of lipid peroxidation-dependent cell death, mediated alkali burn-induced corneal injury. Ferroptosis-targeting therapy protected the cornea from cell damage and neovascularization. However, the specific ferroptosis inhibitor ferrostatin-1 (Fer-1) is hydrophobic and cannot be directly applied in the clinic. Therefore, we developed Fer-1-loaded liposomes (Fer-1-NPs) to improve the bioavailability of Fer-1. Our study demonstrated that Fer-1-NPs exerted remarkable curative effects regarding corneal opacity and neovascularization in vivo. The efficacy was comparable to that of dexamethasone, but without appreciable side effects. The significant suppression of ferroptosis (induced by lipid peroxidation and mitochondria disruption), inflammation, and neovascularization might be the mechanisms underlying the therapeutic effect of Fer-1-NPs. Moreover, the Fer-1-NPs treatment showed no signs of cytotoxicity, hematologic toxicity, or visceral organ damage, which further confirmed the biocompatibility. Overall, Fer-1-NPs provide a new prospect for safe and effective therapy for corneal alkali burn.

Keywords: corneal alkali burn; corneal neovascularization; ferroptosis; ferrostatin‐1; liposome.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

FIGURE 1
FIGURE 1
The ferroptosis inhibitor Fer‐1 ameliorates alkali burn‐induced corneal injury. (a) Schematic diagram of modeling, administration, and photograph in mice. Anterior (b) and side (c) view of the anterior ocular segment in saline‐treated and 200 μM Fer‐1‐treated groups; scale bars, 1 mm; the yellow dashed line indicates the area of CNV. CNV length, CNV area (d), and clinical assessment score (e) were measured in mice treated with saline or Fer‐1 (n = 6) on day 14. (f) Cornea images after sacrificing and dissecting on day 14 (left, scale bars, 500 μm) and hematoxylin–eosin staining images (right, scale bars, 50 μm). (g) Quantitative analyses of the central corneal thickness in the healthy (without alkali burn), saline‐treated, and 200 μM Fer‐1‐treated groups (n = 3). Results were presented as the mean ± SEM. *p < 0.05 and ***p < 0.001. Significance was calculated by Student's t‐test (d, e) or one‐way analysis of variance (g). CNV, corneal neovascularization; Fer‐1, ferrostatin‐1
FIGURE 2
FIGURE 2
Fer‐1 protects cornea against ferroptosis in alkali burn injury. (a) Expression of the indicated reactive oxygen species‐related genes was measured using real‐time polymerase chain reaction in corneal tissue (n = 3). (b) Normalized Ptgs2 mRNA were measured in the indicated groups (n = 3). Representative immunohistochemistry images (c) and quantitative summary (d) of corneal sections stained with 4‐HNE (top) or Gpx4 (bottom); scale bars, 50 μm; n = 3. (e) Transmission electron microscope images showing the morphology of mitochondria in corneal tissue obtained from the healthy, saline‐treated, and 200 μM Fer‐1‐treated groups; scale bars represent 2 μm for left images and 1 μm for right images. Results were presented as the mean ± SEM. *p < 0.05 and **p < 0.01. Significance was calculated by one‐way analysis of variance. AOD, average optical density; Fer‐1, ferrostatin‐1
FIGURE 3
FIGURE 3
Self‐assembly and characterization of Fer‐1‐loaded liposomes and their predicted effects on corneal cells after alkali burn injury. (a) Liposomes were prepared using a thin‐film hydration method with Fer‐1 encapsulated into the hydrophobic layer. When administrated to the alkali‐burned cornea by eye drops, nanocarriers‐encapsulated Fer‐1 exhibit enhanced drug bioavailability than free Fer‐1. After the internalization of Fer‐1‐NPs, the drugs are released inside the cell, where they exert their inhibitory effects of ferroptosis, inflammation, and neovascularization. (b) The dynamic light scattering‐determined hydrodynamic diameter of the Fer‐1‐NPs (n = 3). (c) The cryo‐transmission electron microscope image of the Fer‐1‐NPs; scale bar, 100 nm. (d) In vitro release profiles of the Fer‐1‐NPs (n = 3). (e) Cellular uptake of free Nile Red and Nile Red‐NPs by human corneal epithelial cells. Scale bar, 50 μm. Representative fluorescence images of mice eyes (f) and quantification (n = 3) of the fluorescence signal (g) at different time points after topical administration of free Nile Red or Nile Red‐NPs. Results were presented as the mean ± SD. CNV, corneal neovascularization; DSPE‐PEG, 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy (polyethylene glycol)‐2000]; Fer‐1‐NPs, ferrostatin‐1‐loaded liposomes; PDI, polydispersity index; ROS, reactive oxygen species
FIGURE 4
FIGURE 4
Therapeutic effects of the various treatments for the alkali‐burned cornea. Anterior (a) and side (b) view of the anterior ocular segment in saline‐treated, 200 μM Fer‐1‐treated, 200 μM Fer‐1‐NPs‐treated, 200 μM Dex‐treated and 200 μM Dex‐NPs‐treated groups; scale bars, 1 mm; the yellow dashed line indicates the area of CNV. CNV length, CNV area (c), and clinical assessment score (d) were measured in the indicated groups (n = 10) on day 14. (e) Cornea images after sacrificing and dissecting on day 14 (top, scale bars, 500 μm) and hematoxylin–eosin (H&E) staining images (bottom, scale bars, 50 μm). (f) Quantitative analyses of central corneal thickness in the indicated groups (n = 3). #The Dex‐NPs‐treated group is not included in the CNV measurements, H&E staining images, and central corneal thickness analyses since most corneas in this group presented perforation and thus were not applicable for analyses. Results were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. stands for not statistically significant. Significance was calculated by one‐way analysis of variance. CNV, corneal neovascularization; Fer‐1‐NPs, ferrostatin‐1‐loaded liposomes
FIGURE 5
FIGURE 5
Fer‐1‐NPs exhibited more potent anti‐ferroptosis effects than free Fer‐1. (a) Normalized Ptgs2 and Acsl4 mRNA were measured in the indicated groups (n = 3). Representative immunohistochemistry images (b) and quantitative summary (c) of corneal sections stained with 4‐HNE (top), Acsl4 (middle), or Gpx4 (bottom); scale bars, 50 μm; n = 3. (d) Transmission electron microscope images showing the morphology of mitochondria in corneal tissue obtained from the healthy, saline‐treated, 200 μM Fer‐1‐treated, and 200 μM Fer‐1‐NPs‐treated groups; scale bars represent 2 μm for top images and 1 μm for bottom images. Results were presented as the mean ± SEM. *p < 0.05, **p < 0.01, and n.s. stands for not statistically significant. Significance was calculated by one‐way analysis of variance. AOD, average optical density; Fer‐1‐NPs, ferrostatin‐1‐loaded liposomes; 4‐HNE, 4‐hydroxynonenal
FIGURE 6
FIGURE 6
Fer‐1‐NPs exhibited more potent anti‐inflammatory and anti‐angiogenic effects than free Fer‐1. Expression of the indicated inflammatory‐related (a) and angiogenic‐related (b) genes was measured using real‐time polymerase chain reaction in corneal tissue (n = 3). Representative immunofluorescence images (c) and quantitative summary (d) of corneal sections stained with IL‐1β (top) or IL‐6 (bottom); scale bars, 50 μm; n = 3. Representative immunohistochemistry images (e) and quantitative summary (f) of corneal sections stained with α‐Sma (top) or CD31 (bottom); scale bars, 50 μm; n = 3. Results were presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. stands for not statistically significant. Significance was calculated by one‐way analysis of variance. AOD, average optical density; Fer‐1‐NPs, ferrostatin‐1‐loaded liposomes
FIGURE 7
FIGURE 7
Biocompatibility evaluation of the Fer‐1‐NPs. (a) The live/dead (calcein‐AM/PI) assay and the CCK‐8 assay (n = 3) of the human corneal epithelial cells treated with different concentrations of Fer‐1‐NPs were used to measure cell viability in vitro; scale bars, 250 μm. (b) For physiological conditions (without alkali burn), in vivo biocompatibility of no administration (healthy), saline‐treated, 200 μM Fer‐1‐treated, and 200 μM Fer‐1‐NPs‐treated groups were evaluated by slit‐lamp examination, fluorescein sodium staining, and hematoxylin–eosin (H&E) staining on day 14; scale bars represent 1 mm for slit‐lamp images (white) and 100 μm for H&E staining images (black). For pathological conditions (with alkali burn), in vivo biocompatibility of the indicated groups was evaluated by blood routine examination analyses and blood biochemistry (c) and H&E staining of main visceral organs (d); scale bars, 100 μm; n = 3. Results were presented as the mean ± SEM. ALT, alanine transferase; AST, aspartate transferase; BUN, blood urea nitrogen; CREA, creatinine; Fer‐1‐NPs, ferrostatin‐1‐loaded liposomes; HGB, hemoglobin; PLT, blood platelet; RBC, red blood cells; WBC, white blood cells
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