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M. R. Patel, J. Gupta, S. H. S. Boddu, D. Paturi, D. Pal, A. K. Mitra; Identification and Functional Characterization of Folate Receptors on SIRC Cell Line and Rabbit Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1968.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this study is to characterize folate expression on corneal cells (SIRC) and rabbit cornea which can be potentially utilized for the targeted delivery of folate conjugated surface modified nanoparticles of various drugs to eye.
In Vitro Uptake study of [3H] folic acid was performed according to previously established methods. Radioactivity associated with the cells was quantified using a scintillation counter. Effect of time, pH, temperature and various concentrations of cold folic acid was studied on the uptake of [3H] folic acid (10 nM) across SIRC cells. To delineate the uptake mechanism, cells were incubated with an endocytotic inhibitor, colchicine (100 uM) and various pathways inhibitors. Transport of folic acid was carried out across freshly excised rabbit corneas using diffusion apparatus (side-by-side). To further confirm the presence of folate receptor on SIRC cells, RT-PCR analysis and fluorescence imaging using confocal microscope was carried out.
Folic acid uptake was found to be linear up to 30 min and was optimal at physiological temperature 370C. The uptake was found to be pH dependent. Inhibition of endocytosis process was seen at 40C and with colchicine, which proves the presence of folate receptors. The rate of uptake followed saturation kinetics with apparent Km of 14.22nM, and Vmax of 15 pico mol/(min mg) protein for folic acid. Calmodulin/PKA/PTK pathways played a significant role in the regulation of folic acid uptake. Permeability of [3H] Folic acid across rabbit corneas decreased significantly in the presence of cold folic acid due to competitive inhibition, indicating the presence of a specific carrier system for folic acid. Molecular evidence for the presence of folate receptor (FR) precursor was identified by RT-PCR analysis. Futher, the presence of folate receptors was confirmed by confocal imaging.
This work for the first time demonstrated the expression of specialized carrier-mediated system for folic acid on SIRC cell line and rabbit cornea. Folic acid carrier on rabbit cornea can thus be utilized for the targeted delivery of folate conjugated drug/nanoparticles across the cornea.
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