We determined the permeability coefficient of a model hydrophilic drug, calcein, encapsulated within saturated lipid-based nano-sized liposomes of various lipid profiles. We demonstrated that the addition of cholesterol to liposomes containing saturated lipids increased the permeability of the liposomal membrane to calcein via a decrease in the membrane bending modulus, as determined by means of atomic force microscopy. We found an inverse correlation between the membrane bending modulus of saturated lipid-based nano-sized liposomes and the permeability coefficient of encapsulated calcein, demonstrating that bending modulus, as determined by means of atomic force microscopy, is a quantitative parameter describing the permeability of liposomal membranes to calcein.