PEGylated Azide-functionalized Liposomes Containing Drug Nanocrystals for Targeted Delivery

Liposomal formulations have important therapeutic applications in anti-cancer treatments but current formulations suffer from serious side effects, high dosage requirements and prolonged treatment. Research funded by Australian Research Council (ARC) Centre of Excellence in Bio-Nano Science and Technology (CBNS) show that PEGylated azide-functionalized liposomes containing drug nanocrystals were investigated with the aim of increasing the drug payload and achieving functionalization for targeted delivery. Liposomes were characterized using cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small and ultra-small angle neutron scattering (SANS/USANS) and small and wide-angle X-ray scattering (SAXS/WAXS). Cryo-TEM experiments revealed the dimensions of the nanocrystal-loaded liposomes and the change of shape from spherical to elongated after the formation of nanocrystals. Results from SANS/USANS experiments confirmed the asymmetric particle shape. SAXS/WAXS experiments confirmed that the crystalline drug only occurred in freeze-thawed samples and correlated with a new unidentified polymorphic form of ciprofloxacin. Using a small molecule dye, dibenzocyclooctyne (DBCO)-cy5, specific conjugation between DBCO groups and surface azide groups on the liposomes was confirmed; this indicates the promise of this system for tumour-targeted delivery.

PEGylated azide-functionalized liposomes were successfully prepared to contain ciprofloxacin drug nanocrystals as a model nanocrystallized cargo, analogous to the elongated non-PEGylated liposomes with the same drug nanocrystals. This is the first time that induced nanocrystallization using the freeze-thaw process has been demonstrated for a PEGylated liposome system. Cryo-TEM micrographs also showed that the PEGylated liposomes were spherical in shape before drug crystallization and stretched by the drug nanocrystal after freeze-thawing. The SANS and USANS data were consistent with the cryo-TEM observations. X-ray scattering profiles of the liposome samples showed diffraction of drug nanocrystals only in the freeze-thawed samples, confirming that drug nanocrystals formed in the thawing step of the freeze-thaw process, giving an ensemble picture of the novel drug delivery system, complementary to the cryo-TEM micrographs. The new unidentified ciprofloxacin polymorph formed was able to accommodate inside the liposomes and formed elongated liposomes opening great opportunities for using drug nanocrystals for drug delivery. Surface “click”-functionalization of the liposomes containing the nanocrystals was also confirmed. Overall, a drug delivery system of azide-functionalized PEGylated liposomes with encapsulated drug nanocrystals was successfully established presenting a new opportunity for future anti-cancer targeted delivery.