Abstract
Purpose: :
Considering the fact that most drug targets are indeed contained within specific intracellular compartments like mitochondria or nucleus, ability of a drug to accumulate into these sites is a critical determinant in the observed drug response or lack thereof. Hence purpose of this study is to identify localization and functionality of efflux (P-glycoprotein, Pgp) and influx (peptide) transporters on mitochondria and nuclear membrane of rabbit primary corneal epithelium cells (rPCECs) for intracellular drug targeting.
Methods: :
Isolation and purification of mitochondria and nucleus from corneal cells was performed by optimized cell fractionation method. Mitochondrial and nuclear membrane integrity was measured by JC-1 uptake and high salt induced release of nucleotide respectively. Functional activity of peptide and Pgp transporters was assessed by performing in vitro radioactive uptake studies and flow cytometry analysis on isolated intracellular organelles. Confocal and western blot studies were also performed as a molecular evidence for intracellular localization of both influx and efflux transporters.
Results: :
An isolated fraction of mitochondria and nucleus was found to be pure and intact by western blot analysis and integrity assays. Uptake processes worked optimally at physiological temperature (370C) on isolated organelles showing presence of peptide and Pgp transporters on both mitochondria and nuclear membrane. Confocal and western blot images further confirmed the presence of both transporters on mitochondrial and nuclear membrane.
Conclusions: :
Peptide and Pgp transporters are generally considered to be cell surface localized. However, several cellular compartments (mitochondria or nucleus) may also be potential site for these transporters. This work demonstrates for the first time intracellular localization and functional characterization of influx (peptide) and efflux (Pgp) transporter on mitochondria and nuclear membrane of rPCECs. Mitochondria and nucleus is an attractive target for drug-delivery because there is growing confirmation to support an association between mitochondrial and/or nuclear dysfunction and a number of ocular diseases (glaucoma, diabetic retinopathy, retinoblastoma and AMD). Based on these findings, we hypothesized that localization of peptide transporter on these organelles can be utilized for the intracellular delivery of peptide prodrug across cornea to enhanced drug absorption at target site by evading Pgp mediated drug efflux.
Keywords: cornea: epithelium