PhD thesis defence: Andreas Lauge Christensen

Quantitative Fluorescence Microscopy Applied to the Investigation of Encapsulation and Permeability of Single Nanoscale Liposomes

Abstract:

The ability of lipid membranes to capture and contain biomaterial in small confined volumes has inspired the use of liposomes as biocompatible drug delivery systems and as scaffolds for nanofluidics. Common to these two applications is the requirement for understanding how water-soluble entities can be trapped and exchanged within the liposomal lumen. In this work we used a highly parallel biochip format, containing millions to billions of liposomes immobilized on a glass surface, to investigate by quantitative fluorescence microscopy the encapsulation and permeability of single nanoscale liposome.

We found trapping of small water-soluble dyes and DNA oligonucleotides to be a non-stochastic and heterogeneous process with empty liposomes and liposomes exceeding the bulk solute concentration co-existing within the same liposome preparation. Single liposome analysis revealed that the encapsulation efficiency (EE) was enhanced more than 10-fold in small liposomes (~ 30 nm) compared to large liposomes (~ 300 nm) and that repeated freeze-thawing cycles both improved the EE and shifted the liposome size optimum, at which EE was maximized, to smaller sizes.

Furthermore we used our single liposome platform to study the passive permeability of sodium ions through lipid membranes and found that immobilized liposomes preserved their integrity and long-term stability for an extended period of four days. Finally, while actively permeabilizing liposomes with transmembrane transporters (VMAT2, TAPL, and AHA2), we demonstrated that we could measure transport across the membrane of individual liposomes and in some cases record kinetic traces of substrate translocation into the liposomal lumen.

Supervisor:

Prof. Dimitrios Stamou, Department of Chemistry, University of Copenhagen, Denmark

Assessment committee:

Prof. Jakob B. Sørensen, Department of Neuroscience and Pharmacology, University of Copenhagen, Denmark

Prof. Thomas L. Andresen, Department of Micro- and Nanotechnology, Technical University of Denmark, Denmark

Prof. Daniel T. Chiu, Department of Chemistry, University of Washington, USA