Liposomes based approach for biosensing, protein-membrane interaction studies and targeted drug delivery.

摘要

The objective of this study is to utilize the versatile nature of liposomes for application in various research fields. Liposomes are artificial vesicles with the ability to entrap a myriad of molecules such as proteins, small molecules, nanoparticles and drugs. Further, by manipulation of their membrane properties, they can be converted to mini-reactors, enabling us to monitor reactions in real time. In this work, we created nanoporous proteoliposomes with the assistance of a-Hemolysin (a-HL) toxin and carried out detection of glucose and ethanol. The limit of detection achieved for glucose and ethanol were 0.39 mM and 0.54 mM, respectively. We also studied the effect of liposome functionalization on the pore-forming ability of a-HL. The magnetic nanoparticlemediated immobilization proved effective whereas the functionalization with biotin compromised a-HL's pore forming ability.;Another interesting aspect of liposomes is the potential to emulate a cellular environment. Using liposomes as a membrane model, we investigated the protein-membrane interactions underlying the pathogenesis of botulism. The causative agent botulinum neurotoxin (BoNT) causes blockade of acetylcholine neurotransmitter release at the neuromuscular junctions. It has been proposed that the N-terminal region of the heavy chain (HC) subunit of toxin forms an endosomal membrane channel at low pH for translocation of the light chain (LC) subunit in to the cytosol. Structural analysis of BoNT/HC in the presence of liposomes showed conformational changes at low pH suggesting membrane interactions of the protein that is relevant to low-pH induced channel formation. Also, the effect of low pH on purified BoNT as well as BoNT complex, which consists of BoNT and a group of neurotoxin associated proteins (NAPs) suggested dramatically different structural responses.;Finally, we exploited the drug delivery potential of Iiposomes for developing a therapeutic strategy for botulism. Some of current therapeutic measures include the administration of antitoxins, delivery of peptide and small molecule inhibitors to BoNT intoxicated cells. Nevertheless, ineffective neuronal delivery of therapeutic molecules has remained a bottleneck in the successful treatment of botulism. Using liposomes as a drug carrier and with BoNT/HC as a neuronal cell recognition ligand, we demonstrated the delivery of FITC-dextran reporter molecule to neuronal cells. This strategy could thus be applied for effective delivery of BoNT antidotes.;In summary, liposomes are at the mainstay in pharmaceutical, biosensor, and biochemical studies because of their tunable membrane properties and their ability to entrap a range of molecules. Based on this rationale, the overall goal of this project was to harness the immense potential of liposomes for biosensor applications and in the field of botulinum research for exploring the toxin-membrane interactions and in the delivery of therapeutics against BoNT.