Mechanism of liposomes in the delivery of the P-glycoprotein substrate model calcein AM into Caco-2 cells

Abstract

The aim of this present study was to investigate the probable mechanisms by which liposomes enhance delivery of P-glycoprotein (P-gp) substrates into Caco-2 cells. The effect of liposomal formulation factors (inclusion of charged lipids, cholesterol content, and type of phosphatidylcholine) was also determined. Calcein AM was used as a model molecule for P-gp substrates. Calcein AM-loaded liposomes were prepared by the film-hydration method followed by extrusion through 100 nm polycarbonate membranes. The possible mechanisms of calcein AM uptake from liposomes were investigated by several techniques. To identify the effect of liposomal formulation factors, the extents of calcein AM uptake into Caco-2 cells were compared among liposomes with different compositions. The result demonstrated that neutral liposomes (phosphatidylcholine:cholesterol at 7:3 molar ratio) could enhance the delivery of calcein AM into Caco-2 cells in a time- and concentration-dependent manner. The results from confocal laser scanning microscopy and fluorescence dequenching technique studies substantiated endocytosis of calcein AM-loaded liposomes into Caco-2 cells. This mechanism was also supported by the inhibitory effect of endocytosis/metabolic inhibitors and the effect of temperature. On the contrary, the enhancement in calcein AM uptake via either the changing in cell membrane permeability or the modulation of P-gp function by liposomes was not evident. Charged lipids and type of phosphatidylcholine showed a strong influence on calcein AM uptake from liposomes. Calcein AM from both neutral and negatively charged (phosphatidylcholine: cholesterol:dicetyl phosphate at 6:3:1 molar ratio) liposomes was taken up by Caco-2 cells similarly and more efficiently than that from calcein AM solution. Positively charged (phosphatidylcholine: cholesterol:stearylamine at 6:3:1 molar ratio) liposomes failed to enhance calcein AM uptake into Caco-2 cells. Soybean phosphatidylcholine liposomes, either with or without cholesterol, could increase cellular uptake of calcein AM better than dipalmitoyl phosphatidylcholine liposomes did. On the other hand, cholesterol content inserted only a minimal effect on calcein AM uptake. The presence of 20-40 mole% of cholesterol in liposomes significantly reduced calcein AM uptake into Caco-2 cells, but with much less magnitude than the effects seen with the inclusion of charged lipids and the type of phosphatidylcholine. Overall results indicate that endocytosis was the main mechanism by which liposomes enhanced the uptake of calcein AM into Caco-2 cells. The liposomal composition also played a significant role in the delivery of the P-gp substrate calcein AM into Caco-2 cells.