A head-to-head multi-parametric high content analysis of a series of medium chain fatty acid intestinal permeation enhancers in Caco-2 cells

Abstract

There is debate over the narrow safety margin of many oral permeation enhancers. The sodium salts of medium chain fatty acids (MCFAs) are components of selected oral peptide formulations being assessed in advanced clinical studies. The aim of the study was to examine the effects of the C8–C12 series on filter-grown Caco-2 monolayers and cells grown on 96 well plates in order to dissect the relationship between paracellular permeability enhancement (Papp of [14C]mannitol, reduction in TEER), critical micellar concentration (CMC), and sub-lethal cytotoxicity (high content analysis (HCA). There was a high degree of correlation between the EC50 for increasing the Papp with reduction in transepithelial electrical resistance (TEER), and with CMC values. C8 had the highest EC50 and highest CMC value and was the least cytotoxic, while C12 had the reverse, suggesting a close association between increases in chain length with increases in permeability, hydrophobicity and cytotoxicity. HCA revealed further association between MCFA-induced intracellular calcium increases and plasma membrane permeability reductions in Caco-2 cells with the EC50 to increase the Papp across monolayers. HCA identified sub-lethal cytotoxicity in a series of MCFA and related cell parameters to physicochemical properties and efficacy as intestinal permeation enhancers. These mild surfactants therefore non-specifically partition into the plasma membrane causing membrane fluidization, which is associated with concentration-dependent increases in permeability.