Hypo-Osmotic Stress Enhances the Uptake of Polyethylenimine/Oligonucleotide Complexes in A549 Cells via Ca 2+ Mobilization from Intracellular Stores

Abstract

To determine the mechanism of osmolarity involved in polyethylenimine (PEI)/oligonucleotide (ON) complex transfection in cells, we measured the fluorescence intensities of fluorescein isothiocyanate-labeled ONs complexed with PEI and the changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in A549 cells, and we found that uptake of PEI/ON complexes was improved in the cells along with a rise of [Ca²⁺]_c in A549 cells challenged by 50% hypotonic medium. Further experiments showed that the enhanced uptake efficiency and the rise in [Ca²⁺]_c in A549 cells were almost completely abolished from cells loaded with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)-N,N,N,N-tetraacetic acid–acetoxymethyl ester. 2-Aminoethoxydiphenyl borate or 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate, two potent antagonists of inositol 1,4,5-trisphosphate-mediated Ca²⁺ release that blunt [Ca²⁺]_c elevation via Ca²⁺ release from endoplasmic reticulum, inhibited the enhanced uptake of PEI/ON complexes induced by Ca²⁺-free hypo-osmotic stress. In summary, the results strongly suggest that calcium-dependent transfection is responsible for the uptake of PEI/ON complexes into A549 cells under hypotonic conditions.