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Mahesh Shivanna, Wanachat Chaiyasan, Yueren Wang, Waree Tiyaboonchai, Uday Kompella, Sangly P Srinivas; Penetration of Fluorescent Silica Nanoparticles into the Cornea. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5152.
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Nanoparticles-based drug/gene delivery has been reported for therapeutic management of ocular surface and corneal disorders. In this study, we have examined the penetration dynamics of mono-dispersed silica nanoparticles stained with Rhodamine B (RhB) at the microscopic level.
A custom-built confocal scanning microfluorometer (CSMF) equipped with a water-immersion objective (40x; 0.75 NA and wd = 1.2 mm) was employed for depth-scanning across the thickness of pig corneas exposed to topical RhB-stained mono-dispersed silica (RhB-Si). These nanoparticles were prepared by overnight exposure of Silica nanoparticles (~7 nm; Sigma) to ~0.1 mg/mL of RhB followed by washing. The output of a blue LED, which was modulated at 10 kHz, was filtered through an interference filter (485 + 10 nm) and led to the excitation port. The RhB-Si fluorescence ( >530 nm) and scattered light through the exit slit were detected by two photomultiplier tubes (PMTs) coupled to two independent lock-in amplifiers. All of experiments were performed with freshly excised cornea mounted underneath the objective. RhB-Si nanoparticles in PBS were instilled (100 μL) on the corneal surface with (n = 6) and w/o epithelium (n = 10). The tissues were then held in a moist chamber at room temperature and scanned at 1, 3, and 6 hrs.
With the 40x objective, the depth resolution of CSMF was ~ 7 μm. Instillation of RhB-Si nanoparticles on cornea with intact epithelium led to a peak in fluorescence close to the scatter peak corresponding to superficial epithelium. Administration of RhB alone, in contrast, led to significant fluorescence from deeper layers of the epithelium and from anterior stroma. Taken together, these observations indicate that whereas RhB-Si, although of 7 nm, do not permeate the epithelium. The peak fluorescence with intact epithelium may indicate either uptake by the superficial cells and/or mere accumulation of the nanoparticles at the surface. When RhB-Si nanoparticles were instilled on the bare stroma, much higher fluorescence was observed in the stroma compared to the fluorescence obtained with instillation of RhB alone across the depth of the stroma.
Silica nanoparticles of ~ 7 nm did not permeate corneal epithelium 6-8 hrs after topical instillation. The same particles, however, showed significant penetration when the epithelium was removed. Penetration of RhB across bare stroma is enhanced when instilled as RhB-Si.
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