2.8. In vitro corneal permeation studies

Corneas excised from whole eyes, obtained from Pel-Freez Biologicals, were used for the determination of in vitro transcorneal permeability. The whole eyes were shipped overnight in Hanks’ balanced salt solution over wet ice and were used immediately upon receipt. The corneas were excised with some scleral portion adhering to help secure the membrane between the diffusion half-cells during the course of the transport study. After excision, the corneas were washed with the IPBS (pH 7.4) and mounted on side-by-side diffusion half-cells (Perme Gear, Inc^®) with the epithelial side facing the donor chamber. The temperature of the half-cells was maintained at 34°C via a circulating water bath. The IN contents in the IN-SLN, IN-CS-SLN and IN-NLC formulations were 0.1% w/v, 0.1% w/v and 0.8% w/v, respectively. Three milliliters of the optimized IN-SLNs, IN-CS-SLNs or IN-NLCs was added to the donor chamber after adjusting the pH to 6.8. The donor IN concentration was maintained at 0.1% w/v in the SLN formulation and 0.8% w/v in the NLC formulation. The receiver chamber medium consisted of 3.2 mL RMβCD (2.5% w/v) in IPBS solution for all the transport studies. The difference in the volume of liquid between the two half-cells (3 mL on one side and 3.2 mL on the other) creates a pressure difference across the corneal membrane. Due to this difference in hydrostatic pressure, the cornea bulges out, the circumference of the corneal/scleral limbus is clamped together by the two half-cells toward the half-cell with less volume, thus mimicking the natural curvature of the cornea [25–28]. The contents of both chambers were stirred continuously with a magnetic stirrer. Aliquots (600 µL) were withdrawn from the receiver chamber at predetermined time points up to 3 hrs and replaced with an equal volume of 2.5% w/v RMβCD in IPBS. The samples were stored at −80°C until further analysis of IN using the chromatography system described in Section 2.1. Additionally, the transcorneal permeabilities of IN from the IN-SOL formulation (control) and IN-HPβCD in the presence of CS as a penetration enhancer were also determined.