An optimal drug delivery vehicle should circulate long enough to reach the site of illness or disease, possess a large drug loading capacity, retain its contents over the course of treatment, and be able deliver its contents at a rate appropriate for maximum therapeutic benefit at the site of interest. The vesosome, a large lipid bilayer enclosing multiple, smaller liposomes, is our solution to addressing these needs. The external lipid bilayer offers a second barrier of protection for interior components and can also serve as the anchor for active targeting components. Furthermore, internal compartmentalization permits customization of separate environments for multiple therapeutics and release triggers. Previous work established the ability of the vesosome to retain its contents in vitro an order of magnitude longer than liposomes. To be viable in vivo, the vesosome must be functionalized for biocompatibility and tracking, and its synthetic procedure must be repeatable, reliable and result in a purified product.

The vesosome was functionalized by introducing biocompatible polymers, such as poly(ethylene glycol) (PEG), and fluorescent dyes in their lipid-bound forms into the external membrane of the vesosome. The external vesosomal membrane is formed from large, flat lipid sheets in the interdigitated (L _βI) phase which, when heated, are used to encapsulate smaller drug-containing vesicles. Through X-Ray diffraction (XRD) and freeze-fracture transmission electron microscopy (FF-TEM), we established that the molar amounts of functionalized lipid required to label the vesosome for tracking and biocompatibility (∼5–7mol% total) did not prevent the formation of the interdigitated phase. Thus, functionalization of the external vesosome membrane can be achieved through functionalization of interdigitated sheets. For in vivo testing, functionalized vesosomes must be separated from unencapsulated vesicles and purification was performed using size exclusion chromatography (SEC) and centrifugation.

Having functionalized vesosomes for biocompatibility, PEGylated vesosomes were examined in vitro and in vivo. The presence of surface-grafted PEG was shown to reduce vesosome-vesosome aggregation when exposed to human blood and the circulation half-life was determined to be approximately 2 hours. The evolution of biodistribution was examined by functionalizing the vesosome with a near-infrared dye for in vivo fluorescence imaging and preliminary active targeting experiments show increased vesosome presence at the targeted sites. Ex vivo organ analysis showed the ability of the vesosome to maintain structural integrity for at least 24 hours post-injection.

By functionalizing the vesosome for biocompatibility and tracking through a repeatable and reliable synthesis, we have obtained a biocompatible vesosome. Through proof-of-concept live animal testing, we have demonstrated the feasibility of the vesosome as a single site, single dose, multi-therapeutic drug delivery vehicle.