Characterizing the Structural Arrangement of Lipoplexes by Pyrene Excimer Formation

Abstract

Lipoplexes formed through the complexation of cationic surfactants and DNA were investigated by a combination of steady-state and time-resolved fluorescence with the fluorescent probe PyO-3-12, dynamic light scattering (DLS), and transmission electron microscopy (TEM). PyO-3-12 is an asymmetric gemini surfactant consisting of two dimethylammonium bromide headgroups linked by a propyl spacer, with one headgroup bearing a dodecyl chain and the other a 1-pyrenemethoxyhexyl derivative. Its sensitivity to the local pyrene concentration and polarity of the local environment were utilized to probe the hydrophobic microdomains generated inside lipoplexes. The overarching goal of this thesis is to demonstrate how pyrene excimer formation (PEF) between an excited and a ground-state pyrenyl labels can be applied to probe the interactions between surfactants and DNA and the resulting morphology of the lipoplexes. The first chapter of the thesis presents a review of the interactions between surfactants and water-soluble polymers such as DNA and the fundamentals of PEF with the perspective of their application for the characterization of lipoplexes. Other techniques used to probe lipoplexes such as DLS and TEM are briefly described as well. In Chapter 2, the ability of PyO-3-12 to behave as a complexing agent for the formation of lipoplexes was investigated. The interactions between PyO-3-12 and DNA were monitored as PyO-3-12 was held at a fixed concentration of 16 and 56 µM and the DNA concentration was varied between 1/10th and a 10-fold excess of the PyO-3-12 concentration. In terms of (-/+) ratio, representing the concentration of negative phosphates divided by the concentration of positive ammonium ions, the (-/+) ratio ranged from 0.1 to 10 in these experiments. Upon complexation onto DNA, PyO-3-12 showed increased PEF and indicated a more hydrophobic environment as would be expected. These results reflect the morphology of the PyO-3-12/DNA lipoplexes at the molecular level were supported by TEM and DLS experiments, which described the lipoplexes at the macroscopic level. In Chapter 3, the integrity of the PyO-3-12/DNA lipoplexes prepared with a (-/+) ratio of 1.5 and a PyO-3-12 concentration of 16 and 56 mM was investigated as they interacted with sodium dodecyl sulfate (SDS) for SDS concentrations ranging from 0 to 100 mM. The anionic surfactant generated a competing interaction for PyO-3-12 to induce the release of the negatively charged DNA. The existence of tertiary aggregates between all three species was demonstrated by fluorescence at the equicharge point between PyO-3-12 and SDS, followed by the loss of electrostatic interactions between PyO-3-12 and CT-DNA at higher SDS concentrations resulting in the release of DNA from the lipoplex. The evolution of the complexes formed between PyO-3-12, DNA, and SDS could be followed by TEM but DLS was less informative due to the polydispersity of the system that hampered the analysis of the DLS data. Chapter 4 represents the first example in the literature where PyO-3-12 was employed to probe the hydrophobic domains generated inside a lipoplex by two cationic surfactants, namely dodecyltrimethylammonium bromide (DTAB) and the gemini surfactant 12-3-12 constituted of two dimethylammonium headgroups held together by a propyl linker and bearing one dodecyl chain each. Analysis of the fluorescence data indicated that the 12-3-12/DNA lipoplexes were denser than the DTAB/DNA lipoplexes which was confirmed by conducting DLS and TEM experiments. In summary, this thesis demonstrated that PyO-3-12 is an interesting fluorescent probe to characterize the interactions between PyO-3-12 and anionic molecules such as SDS and DNA and study the interactions of other cationic surfactants such as 12-3-12 and DTAB with DNA. It opens the path for using PyO-3-12 in the molecular level characterization of soft matter generated through the interactions between surfactants and macromolecules.