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Des nanoparticules d’or fonctionnelles ont été synthétisées avec pour objectifs l’élaboration de nanomatériaux pour applications biomédicales et propriétés physiques originales. L’étude de la fonctionnalisation a conduit à utiliser le chimie “click” entre des nanoparticules d’or portant des ligands thiolates azoturés et des alcynes terminaux en utilisant un catalyseur au cuivre (I) renforcé par un ligand polyazoté, ce qui a permis d’introduire des fonctions très variées. En particulier le récepteur folate a été greffé de cette façon pour la synthèse de nouveaux vecteurs du docétaxel, un puissant agent anti-cancéreux dont l’étude est menée en collaboration. Des nanoparticules d’or fonctionnelles ont aussi servi de support pour le greffage de complexes du fer (II) à transition de spin, ce qui a permis de réaliser l’étude en 2D par différentes techniques physiques en collaboration. Enfin un nouveau ligand azoté a été mis au point pour la synthèse de nanoparticules d’or originales. / The thesis concerns functionalizations and applications of gold nanoparticles (AuNPs). In the aspects of functionalization of AuNPs, we concentrated on efficiently functionalized AuNPs by “Click” chemistry. In the aspects of applications, the PEG capped AuNPs was prepared to encapsulate vitamins, which has a potential use in hydrophobic part of human body; the folate functionalized AuNPs was used for docetaxel delivery for cancer therapy; the novel synthesis of triazole stabilized AuNPs used for biosensors; and the citrate capped AuNPs introduced into the silica thin films to check the SERS effect and spin crossover of iron complexes.

This study describes the use of human serum albumin (HSA)-modified gold nanoparticles (HSA-AuNPs) for the selective extraction and enrichment of high-pI protein, lysozyme (Lyz) prior to analysis by capillary electrophoresis (CE) with UV detection. HSA-AuNPs are capable of extracting Lyz from a complicated matrix because a HSA capping layer not only stabilizes gold nanoparticles in a high-salt environment but also exhibits strong electrostatic attraction with Lyz under neutral pH condition. Efficient separation of Lyz and other high-pI proteins has been successfully achieved by the filling of cationic polyelectrolyte, poly(diallydimethylammonium chloride) (PDDAC), to the background electrolyte. After capturing Lyz with HSA-AuNPs, PDDAC-filled CE can be directly used for the analysis of the extracted Lyz without the addition of the releasing agent into the extractor. The extraction efficiency relied on the pH of the solution and the concentration of HSA-AuNPs. Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for Lyz were down to 8 nM. The combination of HSA-AuNP extraction and PDDAC-filled CE has been applied the analyses of lysozyme in chicken egg white, white wine and human tear. Also, we reveal that this NP-based extraction can be coupled to matrix-assisted desorption/ionization time-of-flight mass spectrometry.

Gold Nanoparticle-Aided Radiation Therapy (GNRT) is a new paradigm in radiation therapy which seeks to make a tumor more susceptible to radiation damage by modifying its photon interaction properties with an infusion of a high-atomic-number substance. The purpose of this study was to quantify the energy deposition due to secondary electrons from gold nanoparticles on a micrometer scale and to calculate the corresponding microscopic dose enhancement factor during GNRT. The Monte Carlo code EGSnrc was modified to obtain the spectra of secondary electrons from atoms of gold and molecules of water under photon irradiation of a tumor infused with 0.7 wt. % gold. Six different photon sources were used: 125I, 103Pd, 169Yb, 192Ir, 50kVp, and 6MV x-rays. Treating the scored electron spectra as point sources within an infinite medium of water, the event-by-event Monte Carlo code NOREC was used to quantify the radial dose distribution, giving rise to gold and water electron dose point kernels. These kernels were applied to a scanning electron microscope (SEM) image of a gold nanoparticle distribution in tissue. The dose at each point was then calculated, enabling the determination of the microscopic dose enhancement at each point. For the lower energy sources 125I, 103Pd, 169Yb, and 50 kVp, the secondary electron fluence was increased by as much as two orders of magnitude, leading to a one-to-two order of magnitude increase in the electron dose point kernel over radial distances up to 50 um. The dose was enhanced by 100% within 5 um of the nanoparticles, and by 5% as far away as 30 um. This study demonstrates a remarkable microscopic dose enhancement due to gold nanoparticles and low energy photon sources. Given that the dose enhancement exceeds 100% within very short distances from the nanoparticles, the maximum radiobiological benefit may be derived from active targeting strategies that concentrate nanoparticles in close proximity to the cancer cell and/or its nucleus.

<p>Cancer is currently the second leading cause of death in the United States. Although many treatment options exist, some of the most common, including radiotherapy and chemotherapy, are restricted by dose-limiting toxicities. In addition, the largest hurdle for translating novel biological therapies such as siRNA into the clinic is lack of an efficient delivery mechanism to get the therapeutic into malignant cells. This work aims to improve this situation by engineering a minimally invasive controlled release system that specifically delivers therapeutics to the site of malignant tissue. This platform consists of two novel material components: a thermally responsive poly[N-isopropylacrylamide-co-acrylamide] (NIPAAm-co-AAm) hydrogel and gold-silica nanoshells. Therapeutic molecules are encapsulated within a poly(NIPAAm-co-AAm) hydrogel carrier, leading to increased serum stability, circulation time, and decreased exposure to off-site tissues. Additionally, gold-silica nanoshells embedded within this hydrogel will be used to optically trigger therapeutic release from the carrier. This hydrogel-nanoshell composite material was designed to be swollen under physiologic conditions (37 oC), and expel large amounts of water and absorbed molecules at higher temperatures (40-45 oC). This phase transition can be optically triggered by embedded gold-silica nanoshells, which rapidly transfer near-infrared (NIR) light energy into heat due to the surface plasmon resonance phenomena. NIR light can deeply penetrate biological tissue with little attenuation or damage to tissue, and upon exposure to such light a rapid temperature increase, hydrogel collapse, and drug expulsion will occur. Ultimately, these drug-loaded hydrogel-nanoshell composite particles would be injected intravenously, passively accumulate in tumor tissue due to the enhanced permeability and retention (EPR) effect, and then can be externally triggered to release their therapeutic payload by exposure to an external NIR laser. This dissertation describes the synthesis, characterization, and validation of such a controlled therapeutic delivery platform.</p><p>Initial validation of poly(NIPAAm-co-AAm)-gold nanoshell composites to act as a material in site-specific cancer therapeutic delivery was accomplished using bulk hydrogel-nanoparticle composite disks. The composite material underwent a phase transition from a hydrated to a collapsed state following exposure to NIR light, indicating the ability of the NIR absorption by the nanoshells to sufficiently drive this transition. The composite material was loaded with either doxorubicin or a DNA duplex (a model nucleic acid therapeutic), two cancer therapeutics with differing physical and chemical properties. Release of both therapeutics was dramatically enhanced by NIR light exposure, causing 2-5 fold increase in drug release. Drug delivery profiles were influenced by both the molecular size of the drug as well as its chemical properties. </p><p>Towards translation of this material into in vivo applications, the hydrogel-nanoshell composite material was synthesized as injectable-sized particles. Such particles retained the same thermal properties as the bulk material, collapsing in size from ~330 nm to ~270 nm upon NIR exposure. Furthermore, these particles were loaded with the chemotherapeutic doxorubicin and NIR exposure triggered a burst release of the drug payload over only 3 min. In vitro, this platform provided increased delivery of doxorubicin to colon carcinoma cells compared to free-drug controls, indicating the irradiated nanoshells may increase cell membrane permeability and increase cellular uptake of the drug. This phenomena was further explored to enhance cellular uptake of siRNA, a large anionic therapeutic which cannot diffuse into cells easily. </p><p>This work advances the development of an injectable, optically-triggered delivery platform. With continued optimization and in vivo validation, this approach may offer an novel treatment option for cancer management.</p> / Dissertation

Accurate determination of the surface enhanced Raman scattering (SERS) enhancement factor (EF) is critically important for a fundamental understanding of the SERS phenomenon. Experimental quantification of SERS EFs is challenging. A series of instrument-, analyte-, and SERS-substrate related issues can affect the SERS intensity and thus compromise the reliability of the measured SERS EFs. This dissertation presents a series of computational and experimental studies that enhance the quantitative understanding of the SERS signal variation and identify ways to enhance the reliability of the SERS EF determination. Chapter I presents an overview of works described in this dissertation. The gold nanoparticle (AuNP) inner filter effect on SERS measurements is demonstrated in Chapter II. Using dithiopurine and ethanol as model analytes, we demonstrate that the nanoparticle will modify the analytes’ Raman signal through two competitive mechanisms: enhancing the Raman signal of the analyte on the nanoparticle surface through electromagnetic enhancement, and attenuating the analyte Raman signal through photon extinction. The significance of the AuNP inner filter effect is quantitatively evaluated using ethanol as the internal reference. A solvent internal reference method is presented in Chapter III for quantifying the SERS EFs of analytes adsorbed onto AuNPs and AgNPs. One of the key findings is that while an analyte’s SERS EF varies significantly as a function of nanoparticle aggregation, its peak SERS EF depends only on the types and sizes of nanoparticles, but not on experimental conditions including concentrations of analyte, nanoparticle, and aggregation reagent. Chapter IV presents a SERS internal reference method for the determination of the resonance Raman EFs in the SERS study of rhodamine 6G (R6G) adsorbed onto AuNPs and AgNPs. The most striking finding is that the AgNP binding reduces, instead of enhancing, the R6G resonance enhancement. Finally, the wavelength-dependent correlation between UV-vis intensities and SERS EFs of aggregated AuNPs and AgNPs were investigated under three fixed excitation wavelengths (532, 632, and 785 nm). The nanoparticle UV-vis intensity is an excellent indicator for identifying the optimal aggregation state for AgNP-based SERS acquisitions under each of the three excitation wavelengths and for the AuNP-based SERS under a 632 nm excitation.

Plasmonic nanoparticles, specifically gold and silver nanoparticles, exhibit unique optical, physical, and chemical properties that are exploited in many biomedical applications. Due to their nanometer size, facile surface functionalization and enhanced optical performance, gold and silver nanoparticles can be used to investigate cellular biology. The work herein highlights a new methodology that has exploited these remarkable properties in order to probe various aspect of cancer cell biology, such as cell cycle progression, drug delivery, and cell death. Cell death mechanisms due to localized gold and silver nanoparticle exposure were also elucidated in this work. Chapter 1 introduces the reader to the synthesis and functionalization of gold and silver nanoparticles as well as reviews their implementation in biodiagnostic and therapeutic applications to provide a foundation for Chapters 3 and 4, where their use in spectroscopic and cytotoxic studies are presented. Chapter 2 provides the reader with detailed explanations of experimental protocols for nanoparticle synthesis and functionalization, in vitro cellular biology experiments, and live-cell Raman spectroscopy experiments that were utilized throughout Chapters 3 and 4. Chapter 3 presents the use of nuclear-targeted gold nanoparticles in conjunction with a Raman microscope modified to contain a live-cell imaging chamber to probe cancer cell cycle progression (Chapter 3.1), examine drug efficacy (Chapter 3.2), monitor drug delivery (Chapter 3.3), and detect apoptotic molecular events in real-time (Chapter 3.4). In Chapter 4, the intracellular effects of gold and silver nanoparticles are explored through live-cell Rayleigh imaging, cell cycle analysis and DNA damage (Chapter 4.1), as well as through the elucidation of cytotoxic cell death mechanisms after nanoparticle exposure (Chapter 4.2) and live cell imaging of silver nanoparticle treated cancer cell communities (Chapter 4.3).

The enzyme spermidine/spermine N1-acetyltransferase (SSAT) is a candidate biomarker for various cancers as its activity in cancerous tissues is significantly increased. An artificial molecule, amantadine, is exclusively acetylated by SSAT to acetylamantadine (AcAm), levels of which in urine can serve as a proxy biomarker for malignancy. Current method of AcAm detection is laborious, time-consuming, and lacks the possibility of transforming to a point-of-care device. In this thesis, two different approaches were applied to detect AcAm in deionized water and in human urine using optical methods. The first one was fluorescence-based indicator displacement assay using cucurbit[7]uril as the receptor molecule. The second was programmed gold nanoparticle disaggregation with cucurbit[7]uril as a molecular linker. / Graduate

La nanochimie et l’auto-assemblage sont des voies prometteuses de fabrication de matériaux nanostructurés aux propriétés optiques innovantes dans le domaine visible. Dans cette étude, des nanocomposites plasmoniques anisotropes sont formulés en introduisant sélectivement des nanoparticules métalliques dans des phases ordonnées de copolymères diblocs symétriques selon différentes stratégies d’incorporation. Pour la stratégie de post-incorporation, des nanoparticules d’or présynthétisées en milieu aqueux sont introduites sélectivement dans des phases pré-ordonnées d’un copolymère dibloc amphiphile. L’incorporation directe consiste àmélanger des nanoparticules d’or présynthétisées et un copolymère dibloc dans un solvant commun.La synthèse in situ de nanoparticules consiste à réduire des précurseurs métalliques préalablement introduits dans un des deux blocs d’un copolymère via une étape de réduction. Nous étudions, en particulier, comment la taille des nanoparticules d’or et leur fraction volumique influencent la nanostructure et les propriétés optiques de ces films nanocomposites. La morphologie des films macroscopiques est étudiée par microscopie électronique à transmission et diffusion des rayons Xaux petits angles. Les films minces de nanocomposites sont caractérisés structurellement parmicroscopie à force atomique, microscopie électronique à transmission et réflectivité des rayons X. Les indices optiques déterminés par ellipsométrie spectroscopique peuvent être décrits par un modèle de Maxwell-Garnett, prenant éventuellement en compte de façon phénoménologique les effets de couplage entre nanoparticules d’or. / Nanochemistry and self-assembly are promising ways to fabricate nanostructuredmaterials with innovative optical properties for visible light. In this work, anisotropic plasmonicnanocomposites are formulated by selectively introducing metallic nanoparticles in ordered phasesof symmetric dibloc copolymers with different strategies. For the strategy of post-incorporation, presynthesizedgold nanoparticles in aqueous medium are selectively introduced in pre-ordered phasesof an amphiphilic dibloc copolymer. Direct incorporation consists in mixing pre-synthesized goldnanoparticles and dibloc copolymer in a common solvent. In situ synthesis of nanoparticles consistsin reducing metallic precursors previously introduced in one of two blocks of a copolymer via areduction step. The influence of the size and the volume fraction of gold nanoparticles on thenanostructure and the optical properties of the nanocomposite films have been particularly studied.Morphology of macroscopic films is studied by transmission electron microscopy and small angle Xrayscattering. The nanocomposite thin films are structurally characterized by force atomicmicroscopy, transmission electron microscopy and X-ray reflectivity. The optical indices obtained byspectroscopic ellipsometry can be described with Maxwell-Garnett models, which can take intoaccount phenomenologically the effects of coupling between gold nanoparticles.

Este trabalho apresenta estudos sobre a síntese química do PEDOT com o objetivo de desenvolver diferentes arquiteturas e propriedades para melhorar a taxa de degradabilidade deste polímero. As estratégias foram as preparações de uma blenda, um copolímero e um nanocompósito. O estudo foi iniciado pela síntese química oxidativa do PEDOT (poli (3,4- etilenodioxitiofeno)) em microestruturas utilizando condições brandas e ambientalmente amigáveis, porém o material obtido não apresentou solubilidade e boas condições de se produzir um filme. Então foi sintetizado quimicamente o PEDOT dopado com PSS (poli estireno sulfonado) (PEDOT:PSS), o mesmo foi usado para preparar blendas com o PLGA (poli (ácido láctico-co-glicólico), para melhorar sua degradabilidade. Foi possível produzir um filme fino e nanoestruturado através de deposição eletrostática camada por camada (LBL) que pode ser utilizado para modificação de eletrodos ou de suportes tridimensionais para engenharia celular. Para garantir a degradabilidade do material, foi realizada a síntese de copolímeros de PEDOT e PLLA (poli(lactídeo)) em que foi variada a proporção de PEDOT na cadeia polimérica. Os copolímeros foram caracterizados por IV, RMN, UV, análises térmicas e submetidos a testes de degradabilidade e de viabilidade celular, apresentando excelentes resultados. Foi possível a obtenção de microfibras deste material. A outra alternativa estudada foi a síntese de um nanocompósito, preparado através da síntese química do PEDOT, partindo do monômero EDOT (3,4- etilenodioxitiofeno)usando HAuCl4 como oxidante e NaPSS como dopante e dispersante. O nanocompósito obtido foi caracterizado apresentando diâmetros médio próximos de 4 nm e com uma estrutura caroço-casca, apresentando nanopartícula de ouro como caroço e o polímero PEDOT:PSS como casca. Foram obtidos filmes deste material por deposição por evaporação de solvente, LBL, utilizando como policátion o PDDA (cloreto de poli (dialil dimetil amônio)) e quitosana, e por deposição eletroforética, que apresentou excelentes propriedades eletrocrômicas como rápidos tempos de respostas com bons contrastes ópticos / This work presents studies about the chemical synthesis of PEDOT (Poly(3,4-ethylenedioxythiophene)) with the aim of preparing different architectures and properties to improvement the degradability rate of this polymer. The strategies used to achieve this pupose were the preparation of polymer blends, copolymers and nanocomposites. The study was started by the chemical synthesis of microstructures PEDOT in mild and environment friendly conditions, but the material did not show solubility which enable film formation. Then, PEDOT was synthetized by chemical synthesis doped with PSS (poly(styrene sulfonic acid)), (PEDOT:PSS) to prepare blends with PLGA (poly(lactic-co-glycolic acid)) to improve its degradability. It was possible to prepare a thin and nanostructured film, by electrostatic layer-by-layer deposition (LBL), which could be used for electrodes or scaffold surface modification. In order to ensure the material\'s degradability, PEDOT and PLLA (poly(lactide)) copolymers were prepared, changing PEDOT proportion in the polymeric structure. The copolymers were characterized by, IR, NMR, UV, thermal analysis and then degradability and cell viability tests, which shown important results. Fibers were able to be obtained with these materials. The next strategy was the preparation of a nanocomposite by one-spot chemical synthesis, initiated by the monomer EDOT (3,4- ethylenedioxythiophene) using HAuCl4 as oxidant and NaPSS as both dopant and dispersant. The nanocomposite obtained was characterized showing diameter of around 4nm and a core-shell structure, with gold nanoparticle as core and PEDOT:PSS as the shell. Films were obtained by this material by casting, by LBL, using PDDA (Poly(diallyldimethylammonium) chloride) and chitosan as polycations, and by electrophoretic deposition. The latter method shows excellent characteristics as fast response time with a good optical contrast