Development of self organising size-limited liposomal clusters using asymmetric Janus-textured liposomes and DNA-amphiphiles

Current technologies focus on the use of single drug delivery carriers delivering a single drug species, or multiple species, in a compromisingly loaded or damaged state. Here we aim to develop controlled liposome (vesicle) clusters as potential multi-drug carriers for applications in nanomedicine. Specifically; applications include but are not limited to, combinational therapeutics and for the simultaneous delivery of prodrug and complementary activating enzyme. To this end, we are utilising a platform where different liposomes can be connected through DNA linkers, in hope to deliver multiple species in close proximity whilst keeping the individual cargos separate. Liposome clusters can be formed using complementary lipid-DNA conjugates integrated into a vesicle s surface. To ensure strong directional liposome bridging, a patch of localised DNA allows the assembly of size-limited clusters through the directionality of the adhesive interactions. Studies have localised DNA through phase coexistence, mixing saturated (DPPC) and unsaturated (DOPC) lipids with cholesterol, forming lo patches of saturated lipid surrounded by a ld phase of unsaturated lipids. However, these studies displayed poor saturated lipid-DNA partitioning to the lo ordered patches, allowing a lack of specificity in directional interactions between functionalised domains. To enhance saturated lipid-DNA lo partitioning, 10 mole percent (mol%) cardiolipin (CL) was added. CL increases the free energy required for a saturated lipid to insert into the ld phase, increasing DNA partition coefficient to the lo phase by an order of magnitude. Here, a new four component phase diagram incorporating 10 mol% CL was plotted using liposomes with diameters of ~100 nm, an appropriate particle magnitude required for cellular uptake. Förster Resonance Energy Transfer (FRET) was implemented to map out the four component phase diagram as vesicle size was below optical microscopy limits. Results showed similar phase topology to the analogous three component diagram with the addition of potential lo-HII phase coexistence. Using the new phase diagram in conjunction with lipid-DNA conjugates, dynamic light scattering (DLS) displayed size limited tethered clusters < 200 nm in diameter could be achieved. This Indicated 10 mol% CL and phase diagram position limits the average number of tethered vesicles to single figures. Moreover, DNA-lipid bridging stab off analysis, where aggregates were shown to be stable at homeostatic temperature, pH and salt concentration. Furthermore, through the implementation of DNA i-motifs, small clusters were shown to be able to disassemble at pH 3, forming ~single vesicles, and to reassemble when the pH was raised to pH 7.4.