Ethanol may change the liposome structure over a few microseconds to tens or hundreds of microseconds and induce a reduction in d-spacing. This assumption is crucial in terms of liposome formation and drug encapsulation. First, the d-spacing should be approximately 4-5 nm from immediately after liposome formation until prior to dialysis. The d-spacing then increases to 6.1 nm due to the removal of ethanol. Second, the change in d-spacing due to ethanol affects drug encapsulation efficiency and leakage during liposome formation. A shorter d-spacing may lead to hydrophilic or -phobic drug leakage from the inner aqueous phase or lipid bilayer, respectively.