Purpose : Corneal blindness is the fourth leading cause of blindness. Currently, corneal transplantation is the only remaining therapeutic option as pharmacotherapy remains limited. As the cornea has several physiological and anatomical barriers, direct delivery of therapeutics into the eye remains highly challenging. This work will study corneal penetration of carbon quantum dots (CQDs) as a function of size and charge. Highly positively charged spermidine-derived CQDs, have shown to allow the opening of the tight junctions of the corneal epithelium, the major barrier for drug delivery into the anterior segment of the eye. Methods : Synthesis of CQDs was achieved in a microwave reactor from solutions of glucosamine hydrochloride and capping agent at different temperatures. CQDs with hydrodynamic diameter varying between 1-389 nm were obtained. Zeta potential varied between -24 and +32 mV. The influence of size, surface properties and composition of CQDs on corneal permeation and the ability to reach deeper layers in the eye was investigated. CQDs were topically instilled on a bovine ex vivo eye model. Confocal microscopy imaging of the cornea was performed to determine the presence of CQDs in the corneal epithelium and endothelium. Results : Negatively charged CQDs synthesized from glucosamine hydrochloride and b-alanine show poor penetration into the corneal epithelium. Positively charged CQDs prepared from glucosamine hydrochloride and ethylenediamine as a passivating agent penetrated through the tight junctions of the corneal epithelium. The same was observed for positively charged formed using spermidine as capping agent at 200°C. Decreasing the reaction temperature results in positively charged CQDs of very small size(1.5±0.55nm) shown to reach even the corneal endothelium. Conclusions : We have shown that CQDs could cross the corneal epithelium and the corneal endothelium. We found that size is the most critical factor for permeation into the cornea. The corneal epithelium is a major barrier for the penetration of nanostructures > 20 nm, while particles < 10 nm could cross the endothelium. Also, the charge influences penetration efficacy. It was observed that positively charged particles could more easily penetrate the epithelium and reach the subsequent layers of the cornea. In contrast, negatively charged particles can barely cross the epithelium. This can be explained by the negative charge of the corneal epithelium.