Abstract

Liposomes have long been explored as versatile drug delivery systems. Recently, achieving and maintaining asymmetry of liposomes has been the focus of liposomal research. The maintenance of asymmetry remains a challenge due to the flip-flop of lipids within the vesicle’s bilayer (inner and outer leaflets).
This research aims to provide a novel method for formulating asymmetric liposomes, focusing on the development and optimisation of asymmetric liposome formulation using a novel cyclodextrin (CD)-lipid exchange method. In the literature, asymmetric liposomes are generally formulated using complex methods that are time-consuming and require expensive equipment. Moreover, the simpler methods tend to have short stability (48 hours). The method used in the research enhances the stability of the liposomes while ensuring simpler formulation techniques. The method involves dissolving cyclodextrin in buffer while heating. The phospholipid is then dissolved in methanol and added to the cyclodextrin solution. The mixture is gently mixed to allow for complexation and methanol evaporation. Complexation of the lipid and cyclodextrin is confirmed by several methods. Then, large unilamellar vesicles are formulated using thin-film hydration. Finally, the cyclodextrin-lipid complex is added to the acceptor vesicle suspension and mixed to allow lipid exchange.
The novel method was tested on several liposomal formulations using bromocresol green as a model drug, and it was found that the most optimised formulation was the one with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the outer leaflet and POPC (30%), Dioleoyl-3-trimethylammonium propane (DOTAP) (30%), and cholesterol (40%) in the inner leaflet. Further optimisation was trialled on this method, and it was found that increasing the DOTAP to 45% has increased the entrapment efficiency from 41.11% to 44.22%, a small increase but expected considering that DOTAP increased by only 15%. The liposomes were analysed using size and zetapotential analysis, microscopy and UV/VIS spectrophotometry plus pH gradient method to calculate entrapment efficiency. The novel cyclodextrin-lipid method offers a major improvement over existing asymmetric liposome formation techniques. It simplifies the process by eliminating the need to form multilamellar vesicles (explained further in chapter 3), making it much easier to create the donor complex and separate the asymmetric liposomes from the rest of the suspension.
The final formulation containing POPC in the outer leaflet and POPC (15%), DOTAP (45%), and cholesterol (40%) was used to entrap salmon sperm DNA. Three different encapsulation techniques were trialled, and the most optimised technique was entrapping the DNA during symmetric liposomes formation then performing the lipid exchange. The entrapment efficiency was determined using nanodrop lite machine. When 6µl of DNA is added to asymmetric liposomes (final concentration 2mM) at a ratio of 19.4:1 liposome to DNA, the entrapment efficiency was 92.5%. The liposomes were then stored at 4°C and remained stable for 1 week. In conclusion, the novel method was successful in formulating asymmetric liposomes. These liposomes were able to efficiently entrap DNA and were stable for 1 week at 4 °C. With this method, liposomes were able to stay stable much longer than those made using the conventional cyclodextrin-exchange method, which lasted only 48 hours (explained in chapter 1).