The hydrophilic substances (ascorbic acid and kanamycin) and the mildly soluble lipophilic substances (hydrochlorothiazide and norfloxacin) were added to the hydration medium (solution CF in PBS) during lipid film hydration (aqueous addition route). Lipophilic substances (apixaban, candesartan cilexetil, hydrochlorothiazide and norfloxacin) were added during the first step of liposome preparation, i.e., they were mixed with the phospholipids and dissolved in a mixture of chloroform and methanol (lipid addition route). All samples were prepared in triplicates.