Structural, dynamical and optical properties of gold nanorod/DNA complexes for gene delivery applications

Structural, dynamical and optical properties of gold nanorod/DNA complexes for gene delivery applications Luca Digiacomo Supervisor: Giulio Caracciolo Internal supervisor: Stefano Luin Co-supervisor: Stefano Coppola Enhancement of the optical response by nanometric metallic systems (e.g. gold and silver nanoparticles) has raised large fundamental and technological interest during the past decade, due to their localized surface plasmon resonances (LSPRs). Such enhancement is associated with the collective resonant electron oscillation, that is driven by an external electromagnetic field. Gold nanorods (AuNRs) have broadly tunable LSPRs, which can be adjusted in the visible and near-infrared (NIR) range by acting on their shape, size, and composition. The geometrical aspects of gold nanoparticles (AuNPs) can be modulated in the synthesis procedure. Generally for spherical objects, with the increase of the particle size the surface plasmon absorption maximum redshifts, when the shape of the particle changes from sphere-like to rod-like, a degree of anisotropy is introduced. As a consequence, the LSPR spectrum splits into two bands and the absorption spectrum strongly depends on the aspect ratio (i.e. the length-to-diameter ratio). Due to their interesting and versatile features, the high tolerability in living organisms and the ease of the functionalization, noble metal nanoparticles have several applications in different fields of nanotechnology, including surface-enhanced Raman spectroscopy, luminescence and fluorescence correlation spectroscopy, biological imaging and biomedicine. The biosensing and biomedical potential of these metallic nanoparticles depends on how they interact with the NIR light and the biological environment. The size affects cellular uptake, diffusion and cytotoxicity. The shape mainly rules the plasmon resonance wavelengths, therefore the optical response. They can both be modulated in the synthesis procedure. Hence, an intriguing perspective is to gain a substantial control over the principal properties of these nanoparticles, efficiently and systematically, in order to optimize them for biotechnological applications. Our work is focused on the structural and optical analysis of the systems under study, involved in gold-nanoparticle-mediated gene delivery applications: the NR/DNA complexes represent a non-viral vector, the nucleic acid (DNA) within them is their cargo, which has to be protected during the cellular uptake, while the complexes overcome several extra- and intra-cellular barriers. Once efficiently internalized, the complexes have to traverse the cytoplasm and only in the proximity of the nucleus the DNA should be released. One of the most interesting aspects of gold nanorods is the controlled gene-payload release. It can be mediated and driven by the optical response of NRs to a laser beam, tuned at the wavelength of the localized surface plasmon resonance. The electronic excitations and the subsequent nonradiative decays induce an increase of the kinetic energy, a local overheating and a rapid and deep alteration of the structure. The bonds between nanoparticles and nucleic acid are broken and, as a consequence, the DNA is released. The formation of the complexes is a self-assembling process, driven by the Coulomb interaction between the nucleic acid and a shell of cationic surfactant, namely cetyltrimethylammonium bromide (CTAB), which coats the gold nanorods. The nanoparticle functionalization with the surfactant is performed in the synthesis procedure. The size, charge, symmetry, geometrical and dynamical properties strongly depend on the molar ratio between their constituents, while the nanorod uniaxial anisotropy affects the optical response and defines the way that particles and DNA can be organized in three dimensions. In this work we investigate CTAB-capped gold nanorods and nanorod/DNA complexes, through the following experimental techniques. The trasmission electron microscopy represents the most direct approach to estimate size and shape of NRs. It provides high accuracy and allowed us to evaluate the CTAB-capped AuNR dimensions, as well as the slight deviation from the cylindrical geometry. The system azimuthal symmetry is peculiar in further analysis, i.e. synchrotron small angle X-ray scattering (SAXS) and dynamic light scattering (DLS) experiments. The former provides an estimation of the NR optical element structure. Indeed, because of its intrinsic features, the synchrotron SAXS technique allowed us to investigate the gold core of the larger CTAB-gold unit. Then, NR length and radius have been related to the localized surface plasmon resonance (LSPR) wavelengths, which have been measured by UV-Vis absorption spectroscopy. Further, we studied the NR dynamics through DLS, by measuring the translational and rotational brownian diffusion coefficients. The DLS approach represents also a relevant starting point for the NR/DNA complexes study. More precisely, the system symmetry affects the response to an incident light beam, thus it can be considered as a discriminating element for our analysis. We evaluated NR/DNA complexes symmetry, size and dynamics as a function of the component molar ratio and coupled them to the corresponding zeta potentials, which have been measured through electrophoretic light scattering (ELS). The most suitable complexes as drug delivery systems can be selected through this structural analysis, which allowed us to determine a specific molar ratio that corresponds to a class of potential gene delivery systems. Finally, we investigated the nucleic acid thermal stability through the UV-Vis absorption spectroscopy, both for naked DNA and within the complexes. The Coulomb interaction between DNA and CTAB is responsible for the self-assembling process, but it slightly affects the nucleic acid secondary structure. This binding effect is however negligible in a temperature range wide enough to ensure the DNA stability for the expected application perspectives. The thesis is divided into 4 chapters. Chapter 1 underlines some physical aspects of gold nanoparticles and how the optical properties of noble metal systems are related to the nanobiotechnology. After a brief historical introduction, the application in gene delivery is presented, then the physical background of the surface plasmon resonance, the approximations adopted, the theories developed and the validity ranges are discussed. In Chapter 2 a presentation of the materials involved precedes the sections about the techniques adopted, within which the theoretical and experimental aspects are explained. All the results obtained are discussed in Chapter 3 and, finally, Chapter 4 gives a work overview, focused on the conclusions related to the experimental results and the consequent perspectives of application