Utilization of Nitrogen-Doped Graphene Quantum Dots to Neutralize ROS and Modulate Intracellular Antioxidant Pathways to Improve Dry Eye Disease Therapy

Abstract

Purpose

Patients afflicted with dry eye disease (DED) experience significant discomfort. The underlying cause of DED is the excessive accumulation of ROS on the ocular surface. Here, we investigated the nitrogen doped-graphene quantum dots (NGQDs), known for their ROS-scavenging capabilities, as a treatment for DED.

Methods

NGQDs were prepared by using citric acid and urea as precursors through hydrothermal method. The antioxidant abilities of NGQDs were evaluated through: scavenging the ROS both extracellular and intracellular, regulating the nuclear factor-erythroid 2-related factor (Nrf2) antioxidant pathway of human corneal epithelial cells (HCECs) and their transcription of inflammation related genes. Furthermore, NGQDs were modified by Arg-Gly-Asp-Ser (RGDS) peptides to obtain RGDS@NGQDs. In vivo, both the NGQDs and RGDS@NGQDs were suspended in 0.1% Pluronic F127 (w/v) and delivered as eye drops in the scopolamine hydrobromide-induced DED mouse model. Preclinical efficacy was compared to the healthy and DPBS treated DED mice.

Results

These NGQDs demonstrated pronounced antioxidant properties, efficiently neutralizing free radicals and activating the intracellular Nrf2 pathway. In vitro studies revealed that treatment of H₂O₂-exposed HCECs with NGQDs induced a preservation in cell viability. Additionally, there was a reduction in the transcription of inflammation-associated genes. To prolong the corneal residence time of NGQDs, they were further modified with RGDS peptides and suspended in 0.1% Pluronic F127 (w/v) to create RGDS@NGQDs F127 eye drops. RGDS@NGQDs exhibited superior intracellular antioxidant activity even at low concentrations (10 μg/mL). Subsequent in vivo studies revealed that RGDS@NGQDs F127 eye drops notably mitigated the symptoms of DED mouse model, primarily by reducing ocular ROS levels.

Conclusion

Our findings underscore the enhanced antioxidant benefits achieved by modifying GQDs through nitrogen doping and RGDS peptide tethering. Importantly, in a mouse model, our novel eye drops formulation effectively ameliorated DED symptoms, thereby representing a novel therapeutic pathway for DED management.

Keywords:

• dry eye disease
• antioxidant
• nitrogen doped graphene quantum dots
• Nrf2 antioxidant pathway

Abbreviations

DED, dry eye disease; ROS, reactive oxygen species; NGQDs, nitrogen-doped graphene quantum dots; HCECs, human corneal epithelial cells; (), superoxide anion; H₂O₂, hydrogen peroxide; ·OH, hydroxyl radicals; GQDs, Graphene quantum dots; Nrf2, Nuclear factor E2-related factor 2; RGDS, Arg-Gly-Asp-Ser; HRTEM, high-resolution transmission electron microscopy; AFM, atomic force microscopy; XPS, X-ray photoelectron spectroscopy; UV‒Vis, ultraviolet‒visible; FT-IR, Fourier transform infrared; TMB, 3.3’,5,5’-Tetramethylbenzidine; HRP, horseradish peroxidase; FRAP, ferric reducing ability of plasma; Fe³⁺-TPTZ, Ferric-tripyridyltriazine; FBS, fetal bovine serum; NC, negative control group; DCFH-DA, 2’-7’-dichlorofluorescin diacetate; RT‒PCR, reverse transcription polymerase chain reaction; H&E, hematoxylin eosin; PAS, periodic acid-Schiff; DHE, dihydroethidium.

Acknowledgments

Financial supports for this work from the Zhejiang National Nature Science Foundation (LY20C100002, LQ21H120008), the Wenzhou Bureau of Science and Technology (Y20220150), the Medical and Health Technology Project in Zhejiang Province (2023RC088), the Agricultural and Social Development Science and Technology Plan Project in Yinzhou, Ningbo City (2023AS017), the Ningbo science and technology plan project (2023J211), the project of Ningbo Eye Hospital (2022QN002), the Zhejiang Medical and Health Science and Technology Project (No. 2023RC088), and the Yinzhou Agricultural and Social Development Scientific Research Project (2023AS017) are acknowledged.

Disclosure

The authors report no conflicts of interest in this work.