Vesosomes, a multi-compartment structure consisting of drug-loaded liposomes encapsulated within another bilayer, is a promising drug carrier with improved drug retention and stability compared with unilamellar liposomes. The vesosome preparation takes advantage of the interdigitated phase of saturated lipids, which causes lipid bilayers to form flat, open sheets at low temperature, that close to form large unilamellar vesicles at higher temperatures. During this closure, the interdigitated sheets encapsulate other lipid vesicles or colloidal particles to form and thereby become the outer membrane of the vesosome. However, vesosomes exterior shells made from a single lipid component, such as dipalmitoylphosphatidylcholine (DPPC), has an average diameter of 1.3 µm, too large for optimal intravenous use. The addition of poloxamer 188 (P188) at low concentrations preserves the interdigitated phase transition and can effectively decrease the vesosome diameter. Freeze-fracture TEM and light scattering were used to evaluate the size distribution and polydispersity of the vesosomes formed; poloxamer led to a decrease in the average diameter to about 0.6 µm. The polymer may also provide an extra steric layer protection of vesosomes against flocculation or recognition by the immune system. Applying osmotic pressure by diluting the vesosomes with buffer of increased salt concentration can further decrease the vesosome diameter.