|dc.description.abstract||Medicine has met a revolution in the expansion of possibilities for therapy based upon synthetic gene delivery. Imagine the ability to correct problems of a genetic origin with a simple drug -- such science fiction fantasies are becoming future's reality. Still in relative infancy, synthetic gene therapy has such potential for so many medical issues that it is a very high priority area of research on a global level. Equally revolutionary is the growth in the use of nanotechnology, now that instruments have been developed to probe most any physical system on the nanometre scale. The relatively new appearance of these technologies leaves research rife with fruit for the picking, and forming new interdisciplinary connections only multiplies the possibilities.
Nature has developed excellent nanoscale machines -- viruses -- for gene delivery. Unfortunately for human beings, the end result is often detrimental rather than beneficial. Despite this, in typical human fashion we seek to adapt nature's solutions for our own purposes. Such endeavours are extremely difficult undertakings, but we persist for the benefit of all. So far, researchers have figured out how we can pack DNA with nanoscale carriers rather well using surfactants and lipids of various structures, and that these systems do an `okay' job of transfecting genes. However, we do not really know at all, let alone for certain, why some lipid or surfactant structures are better transfecting agents than others, or really how these carriers enter living cells and become expressed. The answers to these questions can never, ever be solved by observing these systems from the perspective of a single field of study, for in order to understand why, and thus to predict a better how, we must use the entire spectrum of science from the most fundamental to the most clinical.
Over the decades of fairly clinical and in vitro studies that have characterised gene delivery research, precious little literature exists at the extreme end of fundamental physics. And yet, so much depends on the physical interactions of the gene delivery systems and their targets that an understanding of the physics of gene delivery could lead to more focussed and efficient clinical research. For this reason, the present work aims to bring together clinical research into gene delivery with state-of-the-art nanotechnology, observed through the lens of physics. Our primary instrument in this work is the atomic force microscope, which is a type of scanning probe microscope capable of imaging surfaces on nanometre scales using a micromachined cantilever tip. Our particular instrument is one of the most advanced that is presently available to implement recent developments in Kelvin Probe Force Microscopy (KPFM), a variant of atomic force microscopy (AFM) that is designed to image electrical surface potentials on the nanoscale. In the present work, we utilise an advanced method of frequency modulation KPFM, which allows surface potential imaging with nanometre resolution.
This thesis begins with some of the most promising building blocks of surfactant gene delivery, gemini surfactants, and explores their nanoscale behaviour and interactions with other critical ingredients: lipids and DNA. Gemini surfactants have shown to achieve superior transfection efficiency, while maintaining a high level of versatility and flexibility yet requiring less material. These surfactants are also fairly inexpensive to manufacture. Such benefits make gemini surfactants an attractive candidate for synthetic gene therapy solutions. Further enhancements to transfection efficiency are made with the addition of `helper' lipids, an issue which we also explore.
A fundamental aspect of the physics of gene delivery is how the systems interact with their targets: a living cell. By constructing a model monolayer of a cell using lipids commonly found in most cell membranes, we compared the structure of a `plain' model cell monolayer with one which has been infused with gemini surfactant. We found that the gemini surfactant exhibited strong interactions with the gel-phase lipid present in the model monolayer, and that the resulting domains had a more positive surface potential. Using the unique capabilities of KPFM, we were able to show the presence of cationic surfactant in the monolayer from its electrical signal.
As an extension of the above, we added DNA into our monolayers to explore the effects of DNA binding. This binding behaviour is important to understand for the purposes of gene therapy. Our mixture of two cell membrane phospholipids (DOPC and DPPC), with gemini surfactant, showed three distinct domains, which we deduce to be DOPC, DPPC+gemini surfactant, and gemini surfactant+DNA. The latter region was the highest, exhibiting a network of thread-like domains. Most intriguingly, only the `middle' region exhibited a positive surface potential signal, a fact which can only be determined with KPFM imaging.
We studied mixtures of gemini surfactants, helper lipid and DNA in monolayer form so that we could explore the nanoscale structures that these molecules create. In this way, we were able to create controlled environments in which to study the interactions of components of gene tranfection complexes. In addition, we used a Langmuir trough to gather pressure-area curves for our monolayers to draw further conclusions on the roles of the various components. We found that gemini surfactants play a significant role in compacting DNA, and that this compaction is enhanced by the presence of the helper lipid DOPE. Furthermore, the nanoscale structure of these monolayers was affected by factors such as acidity and the ratio of helper lipid to gemini surfactant. Finally, we used AFM and KPFM to probe gemini surfactant gene transfection complexes (nanoparticles), which were directly deposited onto an atomically flat substrate. We found that their size distributions are broad, ranging from a few tens of nanometres to a few hundred nanometres.
This research demonstrates the unique capabilities of AFM and KPFM to probe systems of relevance to gene therapy, and that the nanoscale structure of transfection components is affected by a number of key factors such as the particular surfactant, amount of helper lipid, the presence of DNA, and environmental factors such as acidity. Given that the nature of these interactions is typically electrostatic in origin, it is clear that KPFM has a significant role to play. This thesis provides an introduction to novel methodologies for this purpose, illustrated by applications to gemini surfactant systems.||en