Penetration of fluorescent silica nanoparticles into the cornea

Abstract

Nanoparticles-based drug/gene delivery is being investigated for therapeutic management of various ocular disorders. In this study, we have examined the penetration of monodispersed silica nanoparticles (NPs), which were stained with Rhodamine B (RhB) or labelled with fluorescein isothiocyanate (FITC), across freshly isolated porcine cornea at a microscopic level using a custom-built confocal scanning microfluorometer (CSMF).CSMF had been developed previously for quantitative measurements of trans-corneal fluorescence. With a 40x objective (water immersion, 1.2 mm working distance; Zeiss), CSMF enables a depth resolution of ∼ 7 µm in trans-corneal fluorescence measurements with a signal/noise ratio > 50. The excitation in the CSMF is in the form of a slit beam, which is obtained by filtering the output of a white LED (10 W; modulated as a sine wave at 10 kHz) through an interference filter (470 ± 10 nm). The emission, collected through exit slit held in the eyepiece (referred to as the collection slit), is passed through a barrier filter with a cutoff at 510 nm. The collection and excitation slits are held confocal to each other so that depth-resolved measurements are obtained during depth scanning. The emission, as well as scattered light are detected by photomultiplier tubes and amplified using a lock-in amplifier. The sync signal of the function generator employed for modulation of the LED was coupled to the reference input of the lock-in amplifier.Penetration of the RhB-stained or FITC-labelled NPs were characterized with excised porcine eyes mounted underneath the objective of the microscope on a nanostage. Topical administration of RhB in solution partitioned readily into the corneal epithelium and distributed into the stroma as observed by an increase in RhB fluorescence over time. Administration of RhB-stained silica NPs also led to an increase in fluorescence emission, but it was largely from the epithelium and weakly from the anterior stroma. On the other hand, topical FITC-labelled NPs produced an increase in fluorescence only from the epithelium but not from the stroma. When RhB-stained or FITC-labelled silica NPs were administered on the bare stroma, a significant fluorescence was observed even from deeper layers of the tissue.Based on our observations, we conclude that while silica NPs are transported across the epithelium possibly by iterative endocytosis and exocytosis, their penetration into the corneal stroma is negligible at 24 hrs. The RhB fluorescence from the anterior stroma following topical RhB-stained NPs suggests a slow release of the dye from the NPs and its subsequent penetration into the stroma. Furthermore, a pronounced penetration of NPs into the stroma following their administration on the bare stroma is possibly accelerated by a convective mechanism secondary to imbibition pressure that exists in the corneal stroma