Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Gold nanoparticle to antibody conjugates for diagnosis applications: molecular interactions and immunoassay development

Thesis for the Master degree in Structural and Functional Biochemistry

Detection of Arah1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter=32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody–antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3 h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut.

Study of gold nanoparticle assisted neuron stimulation

The unique proprieties exhibited by nanoscale particles compared to their macro size counterparts allow for the creation of novel neural activity manipula-tion procedures. In this sense, gold nanoparticles (AuNPs) can be used to stimu-late the electrical activity of neuron by converting light into heat. During this dissertation, AuNPs are synthesized by the citrate reduction method, resulting in a hydrodynamic diameter of approximately 16 nm and an absorbance peak of 530 nm. A system to control a 532 nm laser and measure the temperature variation was custom built from scratch specifically for this project. Temperature is then measured with recourse to a thermocouple and through changes in impedance. The built system had in consideration the necessities pre-sented by in vivo tests. Trials were performed by measuring the temperature rise of colloidal AuNP solutions, having the temperature variation reached a maximum of ap-proximately 18 ºC relative to control trials; successfully showing that light is ef-fectively transduced into heat when AuNPs are present. This novel approach enables an alternative to optogenetics, which require the animal to be genetically modified in order to allow neuron stimulation.

Preparation and applications of periodical gold nanoparticle arrays

Diese Arbeit beschäftigt sich mit der Herstellung und Anwendungen von periodischen Goldnanopartikel-Arrays (PPAs), die mit Hilfe von Nanosphären-Lithografie hergestellt wurden. In Abhängigkeit der verwendeten Nanosphären-Größe wurden dabei entweder kleine dreieckige Nanopartikel (NP) (bei Verwendung von Nanosphären mit einem Durchmesser von 330 nm) oder große dreieckige NPD sowie leicht gestreckte NP (bei Verwendung von Nanosphären mit einem Durchmesser von 1390 nm) hergestellt. Die Charakterisierung der PPAs erfolgte mit Hilfe von Rasterkraftmikroskopie, Rasterelektronenmikroskopie und optischer Spektroskopie. Die kleinen NP besitzen ein Achsverhältnis (AV) von 2,47 (Kantenlänge des NPs: (74+/-6) nm, Höhe: (30+/-4) nm. Die großen dreieckigen NP haben ein AV von 3 (Kantenlänge des NPs:(465+/-27) nm, Höhe: (1530+/-10) nm) und die leicht gestreckten NP (die aufgrund der Ausbildung von Doppelschichten ebenfalls auf der gleichen Probe erzeugt wurden) haben eine Länge von (364+/-16)nm, eine Breite von (150+/-20) nm und eine Höhe von (150+/-10)nm. Die optischen Eigenschaften dieser NP werden durch lokalisierte Oberflächenplasmon-Polariton Resonanzen (LPPRs) dominiert, d.h. von einem eingestrahlten elektromagnetischen Feld angeregte kollektive Schwingungen der Leitungsbandelektronen. In dieser Arbeit wurden drei signifikante Herausforderungen für Plasmonik-Anwendungen bearbeitet, welche die einzigartigen optischen Eigenschaften dieser NP ausnutzen. Erstens wurden Ergebnisse der selektiven und präzisen Größenmanipulation und damit einer Kontrolle der interpartikulären Abstände von den dreieckigen Goldnanopartikel mit Hilfe von ns-gepulstem Laserlicht präsentiert. Die verwendete Methode basiert hierbei auf der Größen- und Formabhängigkeit der LPPRs der NP. Zweitens wurde die sensorischen Fähigkeiten von Gold-NP ausgenutzt, um die Bildung von molekularen Drähten auf den PPAs durch schrittweise Zugabe von unterschiedlichen molekularen Spezies zu untersuchen. Hierbei wurde die Verschiebung der LSPPR in den optischen Spektren dazu ausgenutzt, die Bildung der Nanodrähte zu überwachen. Drittens wurden Experimente vorgestellt, die sich die lokale Feldverstärkung von NP zu nutze machen, um eine hochgeordnete Nanostrukturierung von Oberflächen mittels fs-gepulstem Laserlicht zu bewerkstelligen. Dabei zeigt sich, dass neben der verwendeten Fluenz die Polarisationsrichtung des eingestrahlten Laserlichts in Bezug zu der NP-Orientierung sowie die Größe der NP äußerst wichtige Parameter für die Nanostrukturierung darstellen. So konnten z.B. Nanolöcher erzeugt werden, die bei höheren Fluenzen zu Nanogräben und Nanokanälen zusammen wuchsen. Zusammengefasst lässt sich sagen, dass die in dieser Arbeit gewonnen Ergebnisse von enormer Wichtigkeit für weitere Anwendungen sind.

Supramolecular Approach to Gold Nanoparticle/Triruthenium Cluster Hybrid Materials and Interfaces

A systematic and comprehensive study of the interaction of citrate-stabilized gold nanoparticles with triruthenium cluster complexes of general formula [Ru(3)(CH(3)COO)(6)(L)](+) [L = 4-cyanopyridine (4-CNpy), 4,4`-bipyridine (4,4`-bpy) or 4,4`-bis(pyridyl)ethylene (bpe)] has been carried out. The cluster-nanoparticle interaction in solution and the construction of thin films of the hybrid materials were investigated in detail by electronic and surface plasmon resonance (SPR) spectroscopy, Raman scattering spectroscopy and scanning electron microscopy (SEM). Citrate-stabilized gold nanoparticles readily interacted with [Ru(3)O(CH(3)COO)(6)(L)(3)](+) complexes to generate functionalized nanoparticles that tend to aggregate according to rates and extents that depend on the bond strength defined by the characteristics of the cluster L ligands following the sequence bpe > 4,4`-bpy >> 4-CNpy. The formation of compact thin films of hybrid AuNP/[Ru(3)O(CH(3)COO)(6)(L)(3)](+) derivatives with L = bpe and 4,4`-bpy indicated that the stability/lability of AuNP-cluster bonds as well as their solubility are important parameters that influence the film contruction process. Fluorine-doped tin oxide electrodes modified with thin films of these nanomaterials exhibited similar electrocatalytic activity but much higher sensitivity than a conventional gold electrode in the oxidation of nitrite ion to nitrate depending on the bridging cluster complex, demonstrating the high potential for the development of amperometric sensors.

Gold nanoparticle formation in diamond-like carbon using two different methods: Gold ion implantation and co-deposition of gold and carbon

We describe work in which gold nanoparticles were formed in diamond-like carbon (DLC), thereby generating a Au-DLC nanocomposite. A high-quality, hydrogen-free DLC thin film was formed by filtered vacuum arc plasma deposition, into which gold nanoparticles were introduced using two different methods. The first method was gold ion implantation into the DLC film at a number of decreasing ion energies, distributing the gold over a controllable depth range within the DLC. The second method was co-deposition of gold and carbon, using two separate vacuum arc plasma guns with suitably interleaved repetitive pulsing. Transmission electron microscope images show that the size of the gold nanoparticles obtained by ion implantation is 3-5 nm. For the Au-DLC composite obtained by co-deposition, there were two different nanoparticle sizes, most about 2 nm with some 6-7 nm. Raman spectroscopy indicates that the implanted sample contains a smaller fraction of sp(3) bonding for the DLC, demonstrating that some sp(3) bonds are destroyed by the gold implantation. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4757029]

Gold nanoparticle aerosols for rodent inhalation and translocation studies

The intensive use of nano-sized particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of nanoparticles (NP) with biological systems after various routes of exposure needs to be investigated using well-characterized NP. We report here on the generation of gold-NP (Au-NP) aerosols for inhalation studies with the spark ignition technique, and their characterization in terms of chemical composition, physical structure, morphology, and specific surface area, and on interaction with lung tissues and lung cells after 1 h inhalation by mice. The originally generated agglomerated Au-NP were converted into compact spherical Au-NP by thermal annealing at 600 °C, providing particles of similar mass, but different size and specific surface area. Since there are currently no translocation data available on inhaled Au-NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation in rodents. For anticipated in vivo systemic translocation and dosimetry analyses, radiolabeled Au-NP were created by proton irradiating the gold electrodes of the spark generator, thus forming gamma ray emitting 195Au with 186 days half-life, allowing long-term biokinetic studies. The dissolution rate of 195Au from the NP was below detection limits. The highly concentrated, polydisperse Au-NP aerosol (1–2 × 107 NP/cm3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation and number concentration. After collection on filters particles can be re-suspended and used for instillation or ingestion studies.

Quantification of gold nanoparticle cell uptake under controlled biological conditions and adequate resolution

Aim: We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology. Original submitted 10 July 2012; Revised submitted 23 January 2013.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Gold nanoparticle induced vasculature damage in radiotherapy: Comparing protons, megavoltage photons, and kilovoltage photons

Purpose: The purpose of this work is to investigate the radiosensitizing effect of gold nanoparticle (GNP) induced vasculature damage for proton, megavoltage (MV) photon, and kilovoltage (kV) photon irradiation. Methods: Monte Carlo simulations were carried out using tool for particle simulation (TOPAS) to obtain the spatial dose distribution in close proximity up to 20 µm from the GNPs. The spatial dose distribution from GNPs was used as an input to calculate the dose deposited to the blood vessels. GNP induced vasculature damage was evaluated for three particle sources (a clinical spread out Bragg peak proton beam, a 6 MV photon beam, and two kV photon beams). For each particle source, various depths in tissue, GNP sizes (2, 10, and 20 nm diameter), and vessel diameters (8, 14, and 20 µm) were investigated. Two GNP distributions in lumen were considered, either homogeneously distributed in the vessel or attached to the inner wall of the vessel. Doses of 30 Gy and 2 Gy were considered, representing typical in vivo enhancement studies and conventional clinical fractionation, respectively. Results: These simulations showed that for 20 Au-mg/g GNP blood concentration homogeneously distributed in the vessel, the additional dose at the inner vascular wall encircling the lumen was 43% of the prescribed dose at the depth of treatment for the 250 kVp photon source, 1% for the 6 MV photon source, and 0.1% for the proton beam. For kV photons, GNPs caused 15% more dose in the vascular wall for 150 kVp source than for 250 kVp. For 6 MV photons, GNPs caused 0.2% more dose in the vascular wall at 20 cm depth in water as compared to at depth of maximum dose (Dmax). For proton therapy, GNPs caused the same dose in the vascular wall for all depths across the spread out Bragg peak with 12.7 cm range and 7 cm modulation. For the same weight of GNPs in the vessel, 2 nm diameter GNPs caused three times more damage to the vessel than 20 nm diameter GNPs. When the GNPs were attached to the inner vascular wall, the damage to the inner vascular wall can be up to 207% of the prescribed dose for the 250 kVp photon source, 4% for the 6 MV photon source, and 2% for the proton beam. Even though the average dose increase from the proton beam and MV photon beam was not large, there were high dose spikes that elevate the local dose of the parts of the blood vessel to be higher than 15 Gy even for 2 Gy prescribed dose, especially when the GNPs can be actively targeted to the endothelial cells. Conclusions: GNPs can potentially be used to enhance radiation therapy by causing vasculature damage through high dose spikes caused by the addition of GNPs especially for hypofractionated treatment. If GNPs are designed to actively accumulate at the tumor vasculature walls, vasculature damage can be increased significantly. The largest enhancement is seen using kilovoltage photons due to the photoelectric effect. Although no significant average dose enhancement was observed for the whole vasculature structure for both MV photons and protons, they can cause high local dose escalation (>15 Gy) to areas of the blood vessel that can potentially contribute to the disruption of the functionality of the blood vessels in the tumor.

High performance magnetic separation of gold nanoparticles for catalytic oxidation of alcohols

We present the magnetic separation approach to facilitate the recovery of gold nanoparticle (AuNP) catalysts. The use of magnetically recoverable supports for the immobilization of AuNPs instead of traditional oxides, polymers or carbon based solids guarantees facile, clean, fast and efficient separation of the catalyst at the end of the reaction cycle. Magnetic separation can be considered an environmentally benign separation approach, since it minimizes the use of auxiliary substances and energy for achieving catalyst recovery. The catalyst preparation is based on the immobilization of Au(3+) on the surface of core-shell silica-coated magnetite nanoparticles, followed by metal reduction using two different methods. AuNPs were prepared by thermal reduction in air and by hydrogen reduction at mild temperature. Interestingly, the mean particle size of the supported AuNPs was similar (ca. 5.9 nm), but the polydispersity of the samples is quite different. The catalytic activity of both catalysts in the aerobic oxidation of alcohols was investigated and a distinct selectivity for benzyl alcohol oxidation was observed.

Clean preparation of methyl esters in one-step oxidative esterification of primary alcohols catalyzed by supported gold nanoparticles

Methyl esters were prepared by the clean, one-step catalytic esterification of primary alcohols using molecular oxygen as a green oxidant and a newly developed SiO(2)-supported gold nanoparticle catalyst. The catalyst was highly active and selective in a broad range of pressure and temperature. At 3 atm O(2) and 130 degrees C benzyl alcohol was converted to methyl benzoate with 100% conversion and 100% selectivity in 4 h of reaction. This catalytic process is much ""greener"" than the conventional reaction routes because it avoids the use of stoichiometric environmentally unfriendly oxidants, usually required for alcohol oxidation, and the use of strong acids or excess of reactants or constant removal of products required to shift the equilibrium to the desired esterification product.

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in Biotechnology