title: Interactions of phosphodiester and phosphorothioate oligonucleotides with intestinal epithelial Caco-2 cells.
abstract: OBJECTIVE
  Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells.
  
  
  METHODS
  We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides.
  
  
  RESULTS
  The cellular association of both an internally [3H]-labelled and a 5'end [32P]-labelled PS oligonucleotide (3.0% at 0.4 microM extracellular concentration) was similar and was an order of magnitude greater than that of the 5'end [32P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 microM whereas that of PO was less susceptible to competition (0.2% at 5 microM, 0.1% at 200 microM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37 degrees C to 17 degrees C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase, removed approximately 50% of the cell-associated oligonucleotide for both backbone types.
  
  
  CONCLUSIONS
  Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.
authors.affiliation: Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham, United Kingdom.
authors.forename: G F
authors.initials: GF
authors.lastname: Beck
authors.forename: W J
authors.initials: WJ
authors.lastname: Irwin
authors.forename: P L
authors.initials: PL
authors.lastname: Nicklin
authors.forename: S
authors.initials: S
authors.lastname: Akhtar
mesh_terms: D001386:Azides / Q000494:pharmacology; D018938:Caco-2 Cells / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011402:Pronase / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D019810:Sodium Azide; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013696:Temperature; D014316:Tritium
chemical_list: D001386:Azides; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D010761:Phosphorus Radioisotopes; D014316:Tritium; D012965:Sodium Chloride; D019810:Sodium Azide; D011402:Pronase