Functional characterization of folate transport proteins in Staten's Seruminstitut rabbit corneal epithelial cell line.

PURPOSE: The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line. METHODS: [(3)H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [(3)H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [(3)H]folic acid in the presence and absence of 1 mM cold folic acid. RESULTS: Linear increase in [(3)H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent K(m) of 14.2 ± 0.2 nM, V(max) of (1.5 ± 0.1)*10(-5) micro.moles/min/mg protein, and K(d) of (2.1 ± 0.2)*10(-6) min(-1). The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [(3)H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca(2+)/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [(3)H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10(-05) cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10(-05) cm/sec. CONCLUSIONS: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.

Recent perspectives in ocular drug delivery.

Anatomy and physiology of the eye makes it a highly protected organ. Designing an effective therapy for ocular diseases, especially for the posterior segment, has been considered as a formidable task. Limitations of topical and intravitreal route of administration have challenged scientists to find alternative mode of administration like periocular routes. Transporter targeted drug delivery has generated a great deal of interest in the field because of its potential to overcome many barriers associated with current therapy. Application of nanotechnology has been very promising in the treatment of a gamut of diseases. In this review, we have briefly discussed several ocular drug delivery systems such as microemulsions, nanosuspensions, nanoparticles, liposomes, niosomes, dendrimers, implants, and hydrogels. Potential for ocular gene therapy has also been described in this article. In near future, a great deal of attention will be paid to develop non-invasive sustained drug release for both anterior and posterior segment eye disorders. A better understanding of nature of ocular diseases, barriers and factors affecting in vivo performance, would greatly drive the development of new delivery systems. Current momentum in the invention of new drug delivery systems hold a promise towards much improved therapies for the treatment of vision threatening disorders.