In the next step, 10 freeze-thaw cycles are performed to disrupt the multilamellar vesicles. The vacuum adapter on the flask is closed and the lipid suspension is placed in liquid nitrogen (or dry ice) until the contents of flask are completely frozen (Figure ). After several seconds of silence during the initial cooling of the flask and its contents in liquid nitrogen, a bubbling/sissing sound of liquid nitrogen evaporation indicates the phase transition of the lipid suspension. This sound ceases when the content of the flask is completely frozen. To thaw the liposomes, the flask is then placed in a water bath heated to 30-40°C on a magnetic stirrer, until a homogenous suspension is obtained. This freeze-thaw process is repeated 9 additional times to produce unilamellar vesicles.