Liposomes are used as a sophisticated delivery system for drugs and other active biomolecules. A variety of molecules can be conjugated to the liposome surface to facilitate a range of functionalities, including targeted binding of liposomes to receptor molecules on specific cells, endocytosis, intracellular delivery, triggered release of cargo, reduced opsonisation and removal by reticuloendothelial system. Currently there are 25 clinical liposome products approved by FDA and EMA, none of which are yet to have reached clinical/commercial success. Possible explanations include difficulties with scale up and ensuring uniform production, poor correlation between animal models and humans, variation between patients and cancers, and the expense of clinical trials and regulatory requirements. In addition, limitations associated with conjugation strategies may also contribute to this lack of clinical success.
Kode technology is a rapid surface labelling technology that has been successfully used to modify and functionalise a range of biological and non-biological surfaces, including cells, viruses, and artificial membranes. Due to similarities between natural cell membranes and liposome membranes it was expected that Kode technology could be applied as a novel approach to modify liposomes without the need for complex chemical reactions.
To achieve this outcome and to understand the capabilities and limitations the aim of this research was to evaluate the modification of unwashed liposomes prepared by thin film hydration followed by extrusion and modification with Kode constructs (FSL). This included method development and characterisation (size, charge, morphology) of the resultant Kode modified liposomes. Insertion and retention dynamics of Kode modified liposomes were evaluated to determine the effects of synthesis method, concentration, temperature, time and liposome lipid composition.
Three FSL constructs, FSL-A2, FSL-biotin and FSL-FLRO4, and three methods to add them to liposomes, including lipid mix (LM), hydration (H) and post synthesis (PS) were investigated.
It was observed that all FSL constructs were very effective at labelling liposomes and did not significantly alter liposome size, morphology or stability.
Incorporation of FSL into liposomes (by H and PS methods) was temperature and time dependent, maximum insertion occurred within 2 hours 37°C and 72 hours at 4°C. The FSL spacer and head group did not appear to significantly affect this.
Transfer of FSL constructs from unwashed liposomes to other membranes (liposomes or red blood cells) was evaluated. Although no transfer to red blood cells was seen from LM liposomes transfer of FSL-A2 and FSL-biotin from liposomes prepared by H/PS methods, was in the range of 5-10%, while FSL-FLRO4 liposomes exhibited only 2% transfer. After extensive investigation it was concluded that transfer was likely due to FSL remaining in the liposome supernatant or adsorbed to the liposome surface, rather than dissociation of FSL from the liposome membrane.
The conclusions from this research are that Kode Technology can be successfully used to enable simple, rapid functionalisation of the surface of liposomes. The potential now exists to synthesise new FSL constructs with functional head groups selected to enable binding of liposomes to specific receptors on target cells/tissues.
