Abstract
Abstract: :
Purpose: The uptake and binding of several plant lectins in rabbit primary conjunctival epithelial cell culture (RCEC) was previously investigated and shown to be dependent on time, concentration, and temperature. Of these lectins, potato lectin (Solanum. tuberosum, STL) has demonstrated optimal affinity and maximal uptake potential. We sought to evaluate the safety and drug delivery potential of STL in RCEC layers by examining cytokine release and the uptake of a model marker, horseradish peroxidase (HRP) conjugated to STL. Methods: Fluorescein labeled Solanum Tuberosum (Potato) Lectin (STL, Vector Labs) was used and quantified using a fluorescence spectrophotometer (F-2000, Hitachi). Confocal fluorescent microscopy was performed to examine the staining pattern of STL in RCECs. The effect of 2 µM STL for 24 hrs on cytokine release was evaluated using SearchLight Rat Cytokine Array kit (Pierce Co.) for the quantitative measurement of 9 different cytokines: IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-6, IL-10, GMCSF, IFN gamma, and TNF alpha. Histamine at 30 µM was applied as a positive control for cytokine induction in these cells. The optical density was calculated using an Array VisionTM software (Pierce Co.). Conjugation of STL to HRP was achieved using an acid-sensitive linker (cis-aconitic anhydride) and the uptake of the HRP-conjugate was compared with that of free HRP in RCECs. The concentration of HRP was quantified using a commercial kit (TMB Substrate Kit, Pierce Co.) and measuring the maximum absorbance at 450 nm. Results: STL staining was found at the plasma membrane and intracellularly (cytoplasmic and vesicular accumulation) confirming internalization. STL staining was absent in the presence of chitin (inhibitory sugar) confirming specific uptake of lectin rather than FITC in these cells. STL did not elicit any cytokine release for up to 24 hr after treatment, whereas histamine treatment caused release of several cytokines. STL did not cause any perturbation of tight junctions and ion transport properties of RCECs, as indicated by lack of changes in the transepithelial resistance (TEER) and potential difference values during 4-hr transport experiment. STL enhanced the uptake of HRP by 2-folds upon conjugation compared to HRP alone during a 4 hr study. Conclusions: Potato lectin has shown both permeability enhancing capability and lack of irritancy in RCECs, as judged by lack of cytokine release and any changes in TEER values. Therefore, potato lectin can be considered as a safe carrier for ocular drug delivery.
Keywords: conjunctiva • cytokines/chemokines • pharmacology