Cation-mediated interaction between dna and anionic lipid surfacesexperimental and simulation study

  1. Germán Luque Caballero
Supervised by:
  1. Alberto Martín Molina Director
  2. Victor Manuel Quesada Perez Director
  3. Julia Maldonado Valderrama Director

Defence university: Universidad de Granada

Fecha de defensa: 24 July 2014

Committee:
  1. Miguel Ángel Cabrerizo Vílchez Chair
  2. Ana Belén Jódar Reyes Secretary
  3. M. Carmen Morán Badenas Committee member
  4. Alberto Antonio Caria Canelas Pais Committee member
  5. Elena Junquera González Committee member

Type: Thesis

Abstract

ABSTRACT The object of this survey is the interaction between polyanions and negatively charged membranes as mediated by multivalent cations. This phenomenon appears in the formation of anionic lipoplexes, which are mesoscopic assemblies formed from negatively charged liposomes and polynucleotides in the presence of multivalent cations. The interest in this sort of systems is due to its capability to transport genetic material inside the cell, allowing access to a therapeutic target at a molecular level. Their main advantage over other kinds of vectors used in gene therapy is that, since they are formed by lipids occurring in the cell membrane, their toxicity is much lower. The basic research we address in this project about the nature of the interactions involved in anionic lipoplexes is essential to understand the mechanisms underlying their formation, stability and function. Thus, in the present work we carry out a characterization of the membrane-cation-polyanion system through a combination of experimental and theoretical approaches. The manuscript begins with an introduction where we summarize the state of the art of the biophysics of anionic lipoplexes. Firstly, we list the results about the structure and function of a variety of systems prepared in solution. Next, we introduce the study of complexation at the air-water interface as an experimental model to look into the influence of other factors such as lateral packing. Finally, we describe the application of theoretical models to predict the factors governing the formation and structure of this kind of complexes. Later on, we formulate the aims of this research project in agreement with the approaches mentioned in the introduction. In the methodology section, we detail the experimental and simulation protocols used. In particular, we have set up the Langmuir-Blodgett technique for film deposition and atomic force microscopy imaging of the structures at the interface, in our laboratory. We have performed infrared reflexion-absorption spectroscopy measurements during a short training stay in the Max Planck Institute for Colloids and Interfaces in Potsdam (Germany). We have also programmed the source code used in coarse grain simulations. We have carried out fluorescence spectroscopy measurements through a collaboration in the Physical Chemistry Department in the University of Granada. Afterward, we thoroughly discuss the results obtained and the publications to which they have given rise are listed in the annex. We have organized result presentation from the strategy employed to study the complexation. First, we show the results obtained from the characterization of the interaction at the air-solution interface, visualizing the resulting structures and identifying the functional groups involved; then, we evaluate DNA encapsulation capability in solution and, in the end, we show the results concerning the theoretical study of the role of electrostatic interactions in complex formation and stability. Finally, we enunciate the conclusions based on the results of this work. In this way, we have learned that interactions of different nature combine to form anionic lipoplexes. Especially, surface effects derived from the lateral packing modulate the morphology of the structures formed at the interface, while electrostatic interactions affect in a decisive way to complex stability. After assessment of the work done, we consider that, based on the knowledge gained in this research we begin to understand the mechanisms involved in DNA/anionic lipid complexation. However,it is necessary to improve the approaches chosen here in combination with other strategies, in order to elucidate the group of interactions that take place in these complex systems. Only by understanding the fundamentals of formation, stability and function of anionic lipoplexes, we may be able to rationally design optimized formulations for a biomedical performance. Bibliografía / References (1) Martín-Molina A.; Luque-Caballero G.; Faraudo J.; Quesada-Pérez M.; Maldonado-Valderrama J. Adsorption of DNA onto Anionic Lipid Surfaces. Adv Colloid Interface Sci. 2014, 206, 172-175. (2) Luque-Caballero G.; Martin-Molina A.; Sanchez-Trevino A.Y.; Rodriguez-Valverde M.A.; Cabrerizo-Vilchez M.A.; Maldonado-Valderrama J. Using AFM to Probe the Complexation of DNA with Anionic Lipids Mediated by Ca2+: the Role of Surface Pressure. Soft Matter. 2014, 10, 2805-2815. (3) Luque-Caballero G.; Martin-Molina A.; Quesada-Perez M. Polyelectrolyte Adsorption onto Like-Charged Surfaces Mediated by Trivalent Counterions: a Monte Carlo Simulation Study. J Chem Phys. 2014, 140, 174701.