Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis

PLoS One. 2014 Jan 8;9(1):e84856. doi: 10.1371/journal.pone.0084856. eCollection 2014.

Abstract

Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB) on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8) cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetophenones / pharmacology
  • Benzopyrans / pharmacology
  • Biomarkers / metabolism
  • Caco-2 Cells
  • Cell Differentiation / drug effects
  • Endocytosis* / drug effects
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Epithelial Cells / cytology*
  • Epithelial Cells / drug effects
  • Fluorescent Dyes / metabolism*
  • Humans
  • Intestines / cytology*
  • Permeability / drug effects
  • Protein Transport / drug effects
  • beta-Cyclodextrins / metabolism*
  • rab5 GTP-Binding Proteins / metabolism

Substances

  • Acetophenones
  • Benzopyrans
  • Biomarkers
  • Fluorescent Dyes
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • rottlerin
  • rab5 GTP-Binding Proteins

Grants and funding

This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 National Excellence Program. This work was supported by the following grants: TÁMOP-4.2.2.A- 11/1/KONV-2012-0025, OTKA 72762 and OTKA-101337. The preparation of FITC-RAMEB was supported by Marie Curie Program # 237962 CYCLON (FP7-PEOPLEITN-2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.