Chitosan-Rapamycin Carbon Dots Alleviate Glaucomatous Retinal Injury by Inducing Autophagy to Promote M2 Microglial Polarization

Abstract

Introduction

Glaucoma is a prevalent cause of irreversible vision impairment, characterized by progressive retinal ganglion cells (RGCs) loss, with no currently available effective treatment. Rapamycin (RAPA), an autophagy inducer, has been reported to treat glaucoma in rodent models by promoting RGC survival, but its limited water solubility, systemic toxicity, and pre-treatment requirements hinder its potential clinical applications.

Methods

Chitosan (CS)-RAPA carbon dot (CRCD) was synthesized via hydrothermal carbonization of CS and RAPA and characterized by transmission electron microscopy, Fourier transform infrared spectra, and proton nuclear magnetic resonance. In vitro assays on human umbilical cord vein endothelial and rat retinal cell line examined its biocompatibility and anti-oxidative capabilities, while lipopolysaccharide-stimulated murine microglia (BV2) assays measured its effects on microglial polarization. In vivo, using a mouse retinal ischemia/reperfusion (I/R) model by acute intraocular pressure elevation, the effects of CRCD on visual function, RGC apoptosis, oxidative stress, and M2 microglial polarization were examined.

Results

CRCD exhibited good water solubility and anti-oxidative capabilities, in the form of free radical scavenging. In vitro, CRCD was bio-compatible and lowered oxidative stress, which was also found in vivo in the retinal I/R model. Additionally, both in vitro with lipopolysaccharide-stimulated BV2 cells and in vivo with the I/R model, CRCD was able to promote M2 microglial polarization by activating autophagy, which, in turn, down-regulated pro-inflammatory cytokines, such as IL-1β and TNF-α, as well as up-regulated anti-inflammatory cytokines, such as IL-4 and TGF-β. All these anti-oxidative and anti-inflammatory effects ultimately aided in preserving RGCs, and subsequently, improved visual function.

Discussion

CRCD could serve as a potential novel treatment strategy for glaucoma, via incorporating RAPA into CDs, in turn not only mitigating its toxic side effects but also enhancing its therapeutic efficacy.

Graphical Abstract

Keywords:

• retina
• ischemia/reperfusion injury
• rapamycin
• carbon dots
• microglial polarization
• autophagy

Acknowledgments

We thank Prof. Zhengbo Shao for her valuable suggestions on this manuscript, Drs. Weijie Wang and Fengyi Guo for their timely assistance in data acquisition, Ms. Alina Yao for her assistance with manuscript preparation and editing, as well as Mr. Jia for his support in schematic diagram drawing and UPLC data analysis.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This work was supported by grants from the National Natural Science Foundation of China (82070956, 52061135204, 51972086, 81970799, and 82000889), the Applied Technology Research and Development Program of Heilongjiang Provincial Science and Technology Department (GA20C008), Interdisciplinary Research Foundation of HIT (IR2021105), State Key Laboratory of Urban Water Resource and Environment of Harbin Institute of Technology (2022TS21), Ministry of Science and Higher Education of the Russian Federation (075-15-2022-1114), China Postdoctoral Science Foundation (2021M701022), Heilongjiang Postdoctoral Fund (LBH-Z21184), and Heilongjiang Provincial Key R&D project (No. 2022ZX02C24).