| Popis: |
The research surrounding delivery carriers of hydrophobic drugs has evolved with the help of efficient tools brought together by nature’s most important process, self-assembly. This enabled scientists to create and tailor materials with multiple functionalities in order to suite specific needs. One such nanomaterial is the disc-shaped cargo carrier, named nanodiscs. Nanodiscs are noncovalent assemblies consisting of a phospholipid bilayer,whose hydrophobic edge is stabilized by two or more copies of membrane scaffold protein. Despite an abundance of research directed towards the development of an effective nanosystem for delivery into mammalian cells, superficial focus is given to characterizing the incorporation of hydrophobic drugs. This thesis describes the development of a simple approach to systematically study the relation between physical parameters related to loading and release of poorly water soluble drugs from nanodisc systems. For the nanodiscs reconstitution, two strategies have been studied, the first comprised of adding the protein to a mixture of lipid and detergent micelles, and second strategy involved adding protein stabilised in detergent micelles to the lipid. Two more strategies were employed for studying the loading of drugs into nanodiscs -the drugs were added either to a solution of nanodiscs, or loaded during the reconstitution of nanodiscs. The latter strategy showed an increase in drug incorporated per nanodiscs and a slower release rate. In the case of amiodarone and chlorambucil incorporation, a decrease in membrane thickness was seen to be correlated with the efficiency of drug loading within the POPC bilayer. In addition, the lipid composition within nanodiscs was varied in order to investigate its effect on nanodisc formation, drug loading and release kinetics. Finally, the protein component of empty and drug loaded nanodiscs was site-specifically modified via Sortase A mediated ligation,for traceable delivery in in vitro cell studies. Good internalisation of MSP by HeLa cells was observed when cells were treated with empty and chlorambucil loaded nanodiscs. |
