An optimal drug delivery vehicle possesses a large drug loading capacity and retains its contents over the course of treatment. In addition, it circulates in the body long enough to reach the site of illness or disease and also localizes itself at the desired site to consequently deliver its contents at a rate appropriate for maximum therapeutic benefit. While liposomal systems have experienced success with extending circulation, content retention and controlled release remain problematic. The vesosome – a large lipid bilayer enclosing many smaller liposomes – is the most suitable candidate for addressing these issues. The external lipid bilayer offers a second barrier of protection for interior components and can also serve as the anchor for active targeting components. Also, internal compartmentalization permits customization of separate environments for multiple therapeutics and release triggers, highlighting the vesosome's potential as a single site, single dose, multiple component drug treatment.

The processes of vesosome formation and functionalization are based on the phase behavior exhibited by homogeneous lipid mixtures. Slight changes in lipid composition, such as the addition of PEG-lipids or fluorescently-labeled lipids, may have a large impact on the structures that form. Here we examine the effects of modifying the composition of the external bilayer on the formation process of vesosomes using a variety of microscopy techniques, including freeze-fracture and cryo transmission electron microscopy as well as fluorescence and confocal microscopy. Our results indicate that vesosome formation can still be achieved using heterogeneous lipid mixtures, a success vital to moving the vesosome toward in vivo studies.