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. 2021 May 5;6(12):4447-4454.
doi: 10.1016/j.bioactmat.2021.04.032. eCollection 2021 Dec.

A long-term retaining molecular coating for corneal regeneration

Yi Zhang  1   2 , Chenglin Li  1   2 , Qiuwen Zhu  1   2 , Renjie Liang  1   2 , Chang Xie  1   2 , Shufang Zhang  1   2   3 , Yi Hong  1   2   3 , Hongwei Ouyang  1   2   4   3
Affiliations

A long-term retaining molecular coating for corneal regeneration

Yi Zhang et al. Bioact Mater. .

Abstract

Corneal injuries will cause corneal surface diseases that may lead to blindness in millions of people worldwide. There is a tremendous need for biomaterials that can promote corneal regeneration with practical feasibility. Here we demonstrate a strategy of a protein coating for corneal injury regeneration. We synthesize an o-nitrosobenzaldehyde group (NB)-modified gelatin (GelNB), which could adhere directly to the corneal surface with covalent bonding to form a thin molecular coating. The molecular coating could avoid rapid clearance and provide a favorable environment for cell migration, thereby effectively accelerating corneal repair and regeneration. The histological structure of the regenerated cornea is more similar to the native cornea. This molecular coating can be used conveniently as an eye drop solution, which makes it a promising strategy for corneal regeneration.

Keywords: Corneal regeneration; Eye drops; GelNB molecular coating; Long-term adhesion.

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Conflict of interest statement

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Design, synthesis, and characterization of GelNB coating for the corneal defect. (A) Schematic of the molecular coating strategy for corneal regeneration. (B) Schematic of the chemical reaction for GelNB formation. (C) Schematic diagram illustrating the photo-triggered chemical structures transformation of the GelNB hydrogel. O-nitrobenzene is converted to o-nitrosobenzaldehyde groups under UV exposure. Then the active aldehyde group could subsequently crosslink with amino groups to form Schiff bases. (D) Schematic of GelNB forming hydrogel and coating under different feed ratios. (E) Different gelling performance of GelNB with various feed ratios of NB. (a–d) represent 0.5,1,2,4 NB feed ratio of GelNB, respectively.
Fig. 2
Fig. 2
Characterization of GelNB molecular coating solution. (A) Gross view of gelatin, unactivated modified GelNB-4 molecular coating solution, and GelNB-4 solution after UV illumination. (B) The ATR-FTIR spectra of gelatin, unactivated GelNB-4, and UV-activated GelNB-4.
Fig. 3
Fig. 3
In vitro adhesion performance characterization of GelNB molecular coating. (A) SEM images of the injured corneal surface, gelatin, and GelNB-4 protein coating treated corneal surface. Scale bars: 30 μm (top panels); 40 μm (bottom panels, enlarged). (B) Fluorescence images of labeled gelatin and GelNB-4 protein coating treated corneal surface at different time points. Scale bars: 200 μm. (C) X-ray photon spectroscopy (XPS) of GelNB-4 bonding to tissue. The bond energies of the peptide -C–NH– and amino amine group C–NH2 shifting respectively with the appearance of a Cformula imageN bond peak reveals the UV-induced formation of Schiff bases.
Fig. 4
Fig. 4
In vitro biocompatibility characterization. (A) Representative fluorescence images showed human corneal epithelial cells treated with or without modified gelatin on days 1, 3, and 5. Scale bars: 200 μm. (B) Scratch assay images of corneal epithelial cells migration into the scratched area cultivated with gelatin and GelNB-4 coating treated samples. Scale bars: 200 μm. (C) The cytotoxicity of GelNB-4 coating to human corneal epithelial cells after incubation for 1, 3, and 7 days. (D) Viability quantification of the epithelial cells culture at different time points. (E) Quantification of cell migration area after incubation of 12 and 24 h (*p < 0.05 and **p < 0.01; n ≥ 3).
Fig. 5
Fig. 5
In vivo function of the GelNB molecular coating on the corneal defect. (A) Representative photographs taken by slit lamp demonstrated a recovery of smooth and clear slit beam of the eyes. (B) Fluorescein staining imaged with cobalt blue showed the recovery of the corneal epithelial by the progressive reduction staining size after the application of GelNB-4. (C) H&E stained images of rabbit cornea section for the native rabbit cornea, untreated cornea following the surgery, and corneal defect after application of UV-activated GelNB eyedrop on the defect. Scale bars: 100 μm (top panels); 100 μm (bottom panels, enlarged). (D) Immunofiuorescence staining of wounded cornea treated with and without GelNB (DAPI and α-SMA marker). Scale bars: 100 μm (top panels); 100 μm (bottom panels, enlarged). (E) Corneal epithelial migration was quantified by comparing the size of green staining in the corneal center, with respect to day 1. (F) The measurements of epithelial layer thickness in different groups obtained from histological images. (****p < 0.0001; n = 3.).
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