Gold nanorods characterization for nanomedical applications

In recent years, several groups have investigated the changes of chemical and physical properties of materials with size in the nanometer scale. These studies have highlighted a number of possible applications for nanostructures, which are now employed, for example, in biology and medicine for imaging, disease detection, diagnosis, sensing and therapy. In noble metals, the coherent collective oscillation of electrons in the conduction band (Surface Plasmon Resonance, SPR), induces large surface electric fields which greatly enhance the radiative properties of gold and silver NPs when they interact with resonant electromagnetic radiation. The coupling of SPR with the electromagnetic field may lead to a huge enhancement of both the absorption and scattering cross sections. The SPR, tunable in the visible (for spherical gold NPs) and near-infrared region (for anisotropic gold NPs) of the electromagnetic spectrum, can also interact, with the fluorescence emission of dyes and substantially modify their brightness and excited-state lifetime. One of the considerations that has inspired the present project is the expect that any change in the dielectric constant of the NP surface, induced, for example, by a biorecognition process that occurs on the surface itself, can produce a change in the emission properties of the fluorophores. SPR effect becomes also important when combined with two-photon excitation (TPE), which consists in the simultaneous absorption of two photons, each carrying about half the energy necessary to excite the molecule. In fact, the luminescence (TPL) induced by TPE is enhanced (when coupled with an appropriate plasmon resonance) by many orders of magnitude in gold nanorods respect to a standard fluorophore. These properties promise to improve the usefulness of these nanoparticles for in-vivo imaging in the NIR region of the electromagnetic spectrum and have inspired the second part of the project. The aim of the first part has been studied the interaction of gold nanoparticles a few nanometers in size with fluorophores and to exploit the changes of the dye excited-state lifetime and brightness induced by our interaction in solution under physiological conditions. I have investigated the system based on 5 and 10 nm gold NPs coupled (via a biotin-streptavidin linker) to a fluorophore (FITC) and to a specific protein antibody. The binding of protein to the gold NPs through antigen-antibody recognition modifies the dye excited-state lifetime, which change can then be used to measure the protein concentration. In particular, we have tested the nanodevice measuring the change of the fluorophore excited-state lifetime after the binding of the model protein bovine serum albumine (BSA) and p53 protein, a marker for early cancer diagnosis and prognosis (both in standard solution and in total cell extracts). These studies have been published in {J. Phys. Chem. C 2009 113(7) 2722-2730} and {J. Biom. Nanotech. 2009 5 683-691}. In the second part of the project I focused on the use of anisotropic gold nanoparticles as probe in cellular imaging. I have studied the optical properties of gold nanorods synthesized with standard surfactant CTAB (cetyl trimethylammonium bromide) and asymmetric branched gold nanoparticles synthesized with zwitterionic surfactant LSB (laurylsulphobetaine). The sample have been analyzed with a number of structural techniques to obtain a complete characterization: absortpion spectra, TEM images, Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS) experiments in suspensions. From the analysis of the data, I have gained information on the nanoparticles shapes, dimension and aggregation numbers. In particular, three different populations have been found: nanospheres with diameter lower than 20 nm, nanostars characterized by large trapezoidal branches, and asymmetric branced nanoparticles with high aspect ratio (≈ 4-5) (published {Chem. Comm., 2011; 47; 1315-1317}). Moreover, TPL (two-photon luminescence) was finally exploited to study by optical microscopy the problems of internalization and toxicity of gold nanoparticles by different cell line (macrophages, HEK and A549). Presently, is in preparation a manuscript on TPL, cellular uptake and cytotoxicity of branched gold NPs. In the last year I have demonstrated the potential use of NPs, gold nanorods and magnetic nanoparticles (MNPs), as a novel contrast reagent for selective thermal therapy of cancer cells using a near-infrared low energy laser and an AC magnetic field, respectively. Photothermal therapy for cancer have been widely investigated as a minimally invasive treatment modality in comparison with other methods. The appeal of gold NRs as contrast agents is amplified by their additional capability to absorb photons and to convert into heat by nonradiative processes (phonon-phonon interaction). Magnetic nanoparticles heat inductively due to magnetic losses associated with three mechanisms: hysteresis, N\'eel relaxation and Brownian relaxation; moreover they are considered as a low toxicity material with gold and biological compatibility. These two kinds of NPs (gold and magnetic nanoparticles) are delivered to cancer cells and heated to induce apoptosis (programmed cell death). Within this last part of the project, I have first evaluated the increase in temperature of NR and MNP solutions, using a direct visualization by means of a sensitive thermocamera. Then I have developed a nano-sensor to measure the local temperature on the surface of the nanoparticles under excitation. The rationale is to bind an organic chromophore whose excited state lifetime (ESLT) is particularly sensitive to the temperature, and to refer to its lifetime which is an intensive parameter. Rhodamine B is such a fluorophore, with a temperature dependence of the excited state lifetime ≈ 0.029 ± 0.001 ns/°C, as we also measure here. Moreover this nanoconstruct can target tissues or single cells and used for imaging before the therapy. Part of these experimental results have been successfully composed to numerical simulations of light induced heating of gold nanorods, using the Two Temperature Model (TTM) in order to calculate raising in temperature due to laser irradiation. Finally, I have developed methods and knowledge in the field of the use of NPs made of gold or oxide ({Nanotoxicology, 2011; doi:10.3109/17435390}) in biological and medical research and applications. These studies have produced four publications and two additional manuscripts are under preparation on the cytotoxicity of these NPs and their use for imaging and phototherapy.