Rozprawy doktorskie na temat „Fluorescent Nanoparticle”

During this work, we have addressed two aspects of the properties of the fluorescent organic nanoparticles made of Adambodipy: their spectroscopy and their production with controlled sizes. We have produced micro-crystals (100x10x1µm3) by precipitation in solutions of low supersaturation. We have measured their spectroscopy under microscope in the range 380nm to 900nm. The microcrystals are birefringent and dichroic. By adding polarizers on a microscope we have measured their refraction index along the two neutral axes according to the method of Swanepoel. We have measured the two absorption spectra along the neutral axis. We have calculated these absorption spectra using the model of the dipolar coupling for Frenkel excitons. The amplitude of this coupling has been estimated according to the classic model. But for two particular pairs of the cell, we have compared this estimation with the value that can be deduced from the quantum calculation of a dimer by TDDFT. The calculated spectra reproduce the dichroism, the spectral broadening of the absorption spectra but not the experimental peak shape probably because our micro-spectrophotometer levels up at high absorbance. The calculated fluorescence spectra predict a polarized transition along the b direction of the cell. The experiment shows two other red shifted bands. The study of their polarization, as well as their fluorescence lifetime allows us to attribute them to defects in the crystal. The spectra of the nanoparticles produced in the second part of this work are not those of crystals. We have been able to reproduce them theoretically by introducing an orientation disorder inside the periodic structure. The 3D hydrodynamic focusing enables us to produce nanoparticles with controlled size without precipitation of Adambodipy on the wall. We have used the PDMS technology and we moved to a glass tube approach, in order to avoid the diffusion of fluorescence into the PDMS. By adjusting the flow ratio between the inner organic solution of the dye and outer aqueous solution, we can control the size of the nanoparticle between 100nm and 300nm. The stability of the colloidal suspension is maintained by the surfactant CTACl below the CMC. Indeed above the CMC, the nanoparticles exist together with dyes dispersed in micelles. We have simulated using COMSOL the precipitation of the nanoparticles. We have introduced in the calculation the hydrodynamic and mutual diffusion of water and ethanol, as well as the diffusion of the Adambodipy. From our studies of the solubility of Adambodipy in water/ethanol mixtures, we have obtained the saturation curve and we have built the supersaturation maps in the micro-device. We have used Fluorescence lifetime imaging microscopy to follow in situ the precipitation process. From the decay collected in different positions can be attributed to the coexistence of three species : the monomers, the nanoparticles and an intermediate species supposed to be the nuclei. The FLIM shows a precipitation in the diffusion area of the two solvents as well as a massive precipitation after a few hundred of millisecond. The FLIM images are very close to the COMSOL predictions.

The literature on nanomaterials has been flooded with new shapes, sizes, and compositions of nanostructures. The process of developing and characterizing these particles has been broadly accomplished and many interesting and promising properties have been revealed for application in current and developing technologies. In particular, the phenomenon of surface plasmon resonance seen in metallic gold and silver nanoparticles has drawn substantial interest. It has been established that the electromagnetic fields surrounding plasmonic particle surfaces can influence the properties of nearby systems, causing them to experience effects such as enhanced absorption and emission of light or drastically increased conductivity. For this reason, plasmonic nanoparticles are being applied to an endless number of applications for new materials. This thesis investigated the effects of silver nanocube (AgNC) arrays on the photophysical properties of poly(paraphenyleneethynylene) (PPE) fluorescent polymers, a particularly relevant material to the applications of organic-electronics. AgNCs were selected because of their particularly strong plasmonic field, which is enhanced at the sharp features of the cubes. The PPE polymer is an exceptionally fluorescent conjugated polymer that often serves as a building block for polymer-based sensing applications. By monitoring the absorption and emission of the PPE polymer, a better understanding of plasmonic effects on this polymer system was obtained. Compression of the monolayer of AgNCs on the surface of a Langmuir-Blodgett trough can be used for control of interparticle distance and, thus, the plasmon field intensity felt by an adsorbed layer of PPE polymer. In the Chapter 4, PPE (n = 15) emission was monitored as a function of the AgNC plasmonic field. A two-photon process was found to explain the unusual increase then decrease of the fluorescence intensity. This observation was attributed to exciton-exciton annihilation processes within the polymer. The annihilation process is initiated by large enhancements of the polymer absorption rate when plasmonic fields are at their highest (when the AgNCs are compressed to short interparticle distances). In chapter 5, the optical properties of PPE polymers as a function of their chain length and the AgNC density were examined. A simple study was conducted to consider the conformational/geometrical effects on PPE that were caused by the deposition of PPE onto the AgNC topography. In this study, the structure of the absorption and emission profiles were evaluated and used as evidence of polymer interchain interactions, planarization, and even the potential generation of oligomeric species through breaking of conjugation. Fundamental interactions between materials must be evaluated and optimized prior to their use in devices. This thesis serves to shed a little bit of light on the interaction of a well-defined plasmonic particle with a conjugated polymer. The Langmuir-Blodgett technique serves as a critical tool in applying these colloidally produced nanoparticles to 2D arrays in practical applications. The observation of exciton-exciton annihilation at low-energy excitation is an entirely new phenomenon that was initiated by the plasmonic properties of metal nanoparticles. It is the hope of the author that the results contained herein can aide in the use of plasmonic nanoparticles in future devices.

Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Dickson, Robert; Committee Member: Brown, Ken; Committee Member: Curtis, Jennifer; Committee Member: Payne, Christine; Committee Member: Perry, Joseph.

Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)

L’objectif de ce travail est d’utiliser les nanoparticules (NPs) de nanosondes fluorescentes de température en particulier dans les films lubrifiants. Le développement de ces nanosondes nécessite la détermination de leurs sensibilités thermiques afin de pouvoir sélectionner les NPs les plus prometteuses. Pour atteindre cet objectif, nous avons présenté deux méthodes d’élaboration utilisées pour la synthèse des nanostructures à base de SiC-3C, la méthode d’anodisation électrochimique et la méthode d’attaque chimique. Dans le premier cas, les analyses FTIR,RAMAN et MET des NPs finales ont montré que la nature chimique de ces NPs est majoritairement formée de carbone graphitique. L’étude détaillée de la photoluminescence de ces NPs a montré que le processus d’émission dépend de la chimie de surface des NPs, du milieu de dispersion et de sa viscosité, de la concentration des suspensions et de la température du milieu. Pour la deuxième famille de NP de SiC, les analyses cohérentes MET, DLS et PL ont montrées une taille moyenne de 1.8 nm de diamètre avec une dispersion de ±0.5nm. Le rendement quantique externe de ces NPs est de l’ordre de 4%. Les NPs dispersées dans l’éthanol, n’ont pas montré une dépendance à la température exploitable pour notre application. Par contre, les NPs de SiC produites par cette voie, étant donné la distribution en taille resserrée et le rendement quantique « honorable » pour un matériau à gap indirect, sont prometteuses pour des applications comme luminophores en particulier pour la biologie grâce à la non toxicité du SiC. Dans le cas des NPs de Si, nous avons également étudié deux types différents de NPs. Il s’agit de : (i) NPs obtenues par anodisation électrochimique et fonctionnalisées par des groupements alkyls (décène, 1-octadécène). Nous avons mis en évidence pour la première fois une très importante variation de l’énergie d’émission dEg/dT avec la température de type red-shift entre 300 et 400K. Les mesures de(T) conduisent à une sensibilité thermique de 0.75%/°C tout à fait intéressante par rapport aux NPs II-VI. De plus il a été montré que la durée de vie mesurée n’est pas fonction de la concentration. (ii) NPs obtenue par voie humide et fonctionnalisées par le n-butyl. Pour ce type de NPs nous avons mis pour la première fois en évidence un comportement de type blue-shift pour dEg/dT de l’ordre de -0.75 meV/K dans le squalane. Pour ces NPs, la sensibilité thermique pour la durée de vie de 0.2%°C est inférieure à celle des NPs de type (i) mais largement supérieure à celle des NPs de CdSe de 4 nm (0.08%/°C). La quantification de cette la sensibilité à la température par la position du pic d’émission dEg/dT et de la durée de vie nous permet d’envisager la conception de nanosondes de température basée sur les NPs de Si avec comme recommandations l’utilisation de NPs obtenues par anodisation électrochimique et de la durée de vie comme indicateur des variations en température
The goal of this study is the use of Si and SiC nanoparticles (NPs) as fluorescent temperature nanoprobes particularly in lubricating films. The development of these nanoprobes requires the determination of their thermal sensitivity in order to select the best prospects NPs. To achieve this goal, we presented two preparation methods used for the synthesis of 3C-SiC based nanostructures : (i) anodic etching method and (ii) chemical etching method. In the first case, the FTIR, Raman and TEM analysis of final NPs showed that the chemical nature of these NPs is formed predominantly of graphitic carbon. The detailed photoluminescence study of these NPs showed that the emission process depends on the surface chemistry of the NPs, the dispersion medium and its viscosity, the suspension concentration and temperature of the environment.. In the second case, coherent TEM, DLS and PL analyzes showed an average size of 1.8 nm in diameter with a dispersion of ±0.5 nm. The external quantum efficiency of these NPs is 4%. NPs dispersed in ethanol, did not show an exploitable fluorescence dependence on temperature for our application. On the other hand, 3C-SiC NPs produced by this way, given the narrow size distribution and the reasonably high quantum yield for an indirect bandgap material, are promising for applications such as luminophores in particular in the biology field thanks to nontoxicity of SiC. In the case of Si we studied also two different types of NPs. (i) NPs obtained by anodic etching and functionalized by alkyl groups (decene, octadecene). We have demonstrated for the first time an important red-shift in the emission energy dEg/dT with temperature from 300 to 400K. The PL lifetime measurement(T) lead to a thermal sensitivity of 0.75% /°C very interesting compared to II-VI NPs. Furthermore it has been shown that t is not depending on the concentration. (ii) NPs obtained by wet-chemical process and functionalized with n-butyl. For this type of NPs we have identified for the first time a blue-shift behavior of dEg dT in the order of -0.75 meV/K in squalane. The thermal sensitivity for the PL lifetime of these NPs is 0.2%/°C, which is lower than that of NPs obtained by anodic etching method, but much greater than that of CdSe NPs with 4 nm of diameter (0.08%/°C). Quantification of the temperature sensitivity by the position of emission peak dEg/dT and the PL lifetime dτ/dT allows us to consider the realization of temperature nanoprobes based on Si NPs with recommendations to use Si NPs obtained by anodic etching method and PL lifetime as an indicator of temperature changes

Photodynamic therapy (PDT) is an alternative treatment of cancer requiring the use of chromophore molecules (photosensitizers), which can induce cell death after light excitation. Gold nanoparticles (AuNP), exhibiting localized Surface Plasmon Resonance, can enhance the photophysical response of chromophores located in their vicinity, and thus improve their therapeutic action. Moreover, the use of highly localized two-photon chromophores (photosensitizers and fluorophores), capable to undergo a localized excitation by light in the Near InfraRed region, should increase the penetration depth into tissues, thus improve the treatment efficiency (by PDT) and the imaging (by fluorescence microscopy) of cancer tissues.In this work, we describe the elaboration of water-soluble hybrid nano-objects for PDT and fluorescence bioimaging applications, composed of two-photon chromophore-polymer conjugates grafted onto gold nanoparticles. In order to obtain these nano-objects we follow a multistep strategy: i) the synthesis of a well-defined water-soluble chromophore-polymer conjugates; ii) the end-group oriented grafting of chromophore-polymer conjugates onto 20 nm AuNP. The coupling of hydrophobic two-photon chromophores on linear water-soluble copolymer chains (poly(N-acryloylmorpholine-co-N-acryloxysuccinimide)), obtained by controlled/living RAFT polymerization, resulted in well-defined water-soluble chromophore-polymer conjugates, with different polymer lengths (2 000 g.mol-1 < Mn < 37 000 g.mol-1) and architectures (random or block), and a controlled number of chromophores per chain (varying between 1 and 21). Their grafting onto 20 nm AuNP gave water-soluble hybrid nano-objects with high grafting densities (~0.5 chains/nm²). The role of the polymer chain being to tune the distance between chromophores and AuNP surface, we have evidenced the increase in the polymer corona thickness of grafted AuNP (estimated by TEM) with the increasing polymer Mn, corroborating with the corresponding distance-dependent fluorescence properties of those. Finally, the in cellulo biological properties of two-photon chromophore-polymer conjugates, before and after grafting onto AuNP, have been investigated, highlighting their potential for two-photon bioimaging and PDT applications.

Nanotechnology has recently emerged as a strong contributor toward research efforts to develop targeted systems of drug delivery in cancer therapy. Our work investigates the therapeutic potential of Targeted Charge-Reversal Nanoparticles (TCRNs), a novel nanoparticle with in vitro evidence of nuclear drug delivery. Using M12 prostate cancer cells, MDA-MB-231 breast cancer cells, and modified derivatives of these cell lines, we investigated the ability of Folic Acid-tagged TCRNs to deliver Nile Red and Dimethyl Indole Redfluorescent (DiR) fluorescent dyes to the nucleus of cells using confocal microscopy and in vivo biphontonic imaging using Xenogen® Technology. Confocal imaging with the SCP28 derivative of MDA-MB-231 cells shows nuclear association of the TCRNs over time, although specific nuclear deposition was unclear. Biophotonic imaging with M12 and SCP28 xenograft tumors in athymic nude mice shows retention of TCRNs in animals out to 7 days with minimal localization of TCRNs to tumor tissues. Our findings suggest that further characterization and manipulation of both the cells and the nanoparticle is necessary in order to make definitive claims regarding the TCRN’s ability to deliver fluorescent dyes, and eventually therapeutic compounds, to the nucleus of cells.

During the past two decades, increasing research attention has been devoted to nanomaterials (materials in the range of 10-100 nm) because of their unique optoelectronic properties. In particular, inorganic nanomaterials, such as quantum dots, metal-based nanoparticles and silica nanoparticles, have been investigated extensively. Instead, nanomaterials based on organic molecules are been subject of research only since very recent years. This thesis presents an extensive study of novel fluorescent organic nanoparticles and fluorescent organic binary and ternary nanoassemblies. In particular the attention is focused on the preparation and characterization of organic nanoparticles and new nanocomposites obtained from different types of small organic chromophores, their stabilization and the use of these materials for biological and optoelectronics applications.

Les performances des techniques de bioimagerie et de biodétection peuvent être améliorées grâce aux nanoparticules fluorescentes (NPs) permettant un transfert d’énergie résonante de type Förster (FRET) efficace. Le but de mon projet de thèse est le développement de NPs polymériques brillantes et ultrastables encapsulant des fluorophores, capables de produire un FRET au-delà du rayon de Förster. Il a été montré que les groupements encombrés sont essentiels pour minimiser l’auto-extinction et le blanchiment des fluorophores encapsulés. Par ailleurs, la matrice polymérique joue un rôle crucial dans le contrôle de l’effet collaboratif entre fluorophores du au transfert d’énergie d’excitation. Puis, en utilisant cet effet collaboratif entre fluorophores, nous avons conçu des NPs présentant une photocommutation efficace, ainsi qu'un phénomène de "light harvesting" très important. Enfin, de très petites NPs avec un FRET efficace à leur surface ont été élaborées et appliquées pour la détection ultra-sensible de protéines. Les résultats obtenus fournissent de nouvelles perspectives dans le développement des nanoparticules brillantes avec un transfert d'énergie efficace, ainsi que des nano-sondes pour la détection de molécules uniques
Performance of biosensing and bioimaging techniques can be improved by fluorescent nanoparticles (NPs) capable of efficient Förster resonance energy transfer (FRET). The aim of my PhD project is to develop bright and photostable dye-loaded polymer NPs capable to undergo efficient FRET beyond the Förster radius. We showed that bulky groups are essential for minimizing self-quenching and bleaching of encapsulated dyes. Moreover, polymer matrix plays a crucial role in controlling the inter-fluorophore communication by excitation energy transfer. Then, by exploiting communication of dyes, we designed NPs exhibiting efficient photoswitching as well as giant light-harvesting. Finally, very small NPs with efficient FRET to their surface were developed and applied for ultra-sensitive molecule detection of proteins. The obtained results provide new insights in the development of bright nanoparticles with efficient energy transfer as well as nano-probes for single-molecule detection

Ce projet de thèse consiste dans un premier temps à synthétiser des systèmes moléculaires multifonctionnels possédants un grand nombre d’entités fluorescentes et photochromes. De telles architectures nécessitent ensuite une étude photophysique poussée pour caractériser leurs propriétés d’émission photo-activée pour jouer le rôle de sondes moléculaires fluorescentes super-résolutives en imagerie de fluorescence. En effet, l’accès aux observations nanométriques par microscopie optique est actuellement un domaine de recherche extrêmement actif et prometteur. Ici, la combinaison astucieuse de molécules photochromes (de type diaryléthène) et fluorescentes (de type dicyanométhylène pyranes ou benzophénoxazines) à l’échelle nanométrique, grâce à des plateformes moléculaires telles que des oligosaccharides et des peptides fonctionnalisés par “chimie click”, doit permettre d’obtenir des structures aux géométries variées avec des distances et orientations inter-chromophores diverses, dont l’optimisation doit conduire à des interactions efficaces menant à l’émergence d’effets collectifs coopératifs. Pour ces assemblages moléculaires mixtes, il est attendu que la présence d’une unité photochrome puisse engendrer l’extinction de fluorescence de plusieurs fluorophores lorsque ces entités sont situées à une distance appropriée pour que le transfert d’énergie ait lieu de manière amplifiée. Par ailleurs, même une faible conversion des photochromes doit permettre d’atteindre un contraste de fluorescence extrêmement efficace avoisinant 100% et ainsi obtenir des super-molécules dont la photo-commutation serait rapide et économe en photons. Il s’agira enfin de démontrer que les systèmes moléculaires multichromophoriques ainsi optimisés présentent le comportement souhaité à l’échelle de la molécule unique pour l’imagerie de fluorescence super-résolution
The synthesis of photoswitchable emissive molecular system still represents a challenge, in order to develop fluorescence-based devices for nanotechnologies. In the last decade, excitation energy transfer processes (EET) have been advantageously employed to design photoswitchable fluorescent molecular systems between a photochromic dye and an appropriate fluorescent molecule. More recently, photochromic dyes of particular interest, showing no emission in the open-form (P-OF) but a strong emission signal in the closed-form (P-CF), have been developed. Such molecules allow a multi-emission switch when combined with a fluorescent moiety. In this project, we will design and synthesize fluorescent photochromic diarylethene covalently linked to different fluorophores, such as benzothiadiazole (BTD) and BODIPY derivatives. By choosing a fluorophore moiety with blue/green emission (F) and a photochromic moiety with orange/red emission in closed-form (P-CF), EET can occur between F and P-CF leading to photoswitchable multi-emission properties. Photophysical characteristics of dyads will be studied by spectroscopy with the aim of presenting their light-controllable optical properties and the intramolecular EET processes between fluorescent and photochromic moieties

Les techniques de fluorescence sont des outils de choix pour l’étude et la compréhension fine des processus biologiques. Ceci requiert toutefois l’utilisation de sondes fluorescentes parfaitement adaptées au but visé et répondant aux différentes exigences requises pour l’application visée. Dans ce cadre, nous nous sommes plus particulièrement intéressés à l’élaboration de sondes biphotoniques de pH adaptées à une mesure très sensible de faibles variations de pH autour du pH neutre. Les variations et gradients de pH sont en effet impliqués dans un certain nombre de processus biologiques importants et peuvent être associées à des dysfonctionnements liés à certaines maladies. Dans ce cadre, nous avons développé de nouvelles sondes fluorescentes de pH fluorescentes présentant à la fois un comportement ratiométrique, une forte sensibilité autour du pH neutre et facilement excitables dans le proche IR par absorption à deux photons. Ces sondes de structure quadrupolaire et bolamamphiphile permettent ainsi la détection ratiométrique du pH dans des environnements biologiques au moyen d'une excitation biphotonique dans le proche IR. En parallèle, nous nous sommes intéressés à l’élaboration de nanoparticules hyperbrillantes dédiées à l’imagerie biologique par microscopie de fluorescence induite par excitation à deux photons. Nous nous sommes plus particulièrement attachées au design de nanoparticules organiques fluorescentes constituées de molécules organiques de bas poids moléculaire (FONs). Cette approche offre en effet une grande flexibilité et la possibilité d’accéder à des nanosondes ayant des brillances comparables aux très populaires quantum dots mais moins toxiques et plus facilement dégradables. L’ingénierie moléculaire des fluorophores utilisés pour la préparation des FON est cruciale puisqu’elle influence fortement à la fois les propriétés photophysiques (brillance, couleur…) et leur propriétés physico-chimiques (stabilité chimique et structurale, stabilité colloïdale). Dans ce contexte, une librairie de nouveaux chromophores dipolaires a été synthétisée et utilisées pour la préparation de FON par la méthode de nano-précipitation. Leurs propriétés ont été étudiées afin de déterminer la relation entre la structure du chromophore et les propriétés globales des nanoparticules constituées de ces colorants. Ce travail a permis d’identifier les paramètres structuraux permettant d’accéder à des nanoparticules présentant à la fois une brillance exceptionnelle, une émission modulable du vert au rouge et proche IR et une remarquable stabilité colloïdale. Ces nanoparticules présentent des potentialités majeures pour l’imagerie in vivo par excitation et détection dans le proche IR
Fluorescence-based techniques are popular tools for the study and understanding of biological processes. This has prompted continuous research aimed at the development of a wide range of fluorescent probes specifically designed for specific applications. Among them, fluorescent pH probes are of much interest as pH variations or gradients are involved in many biological events and anomalous alterations are often related to the onset of dysfunctions and diseases. In this framework we have developed a series of promising two-photon pH fluorescent molecular probes. These quadrupolar bolaamphiphilic probes are of great interest, as they combine a steep pH dependence of their optical properties close to neutral pH, ratiometric behavior and large response to two-photon (2P) excitation in the NIR region. As such they offer much promise for ratiometric detection of the pH in biological environments and in situ monitoring of acidification. In parallel, we have been interest in the design of ultrabright nanoparticles for bioimaging purpose (in particular highly sensitive optical imaging). We chose to focus on Fluorescent Organic Nanoparticles made of organic molecules with low molecular weight (FONs) as they offer a flexible route and promising alternatives to toxic quantum dots. In this case the design of the dye used as building blocks of the FONs is of crucial importance and strongly influence the chemical and physical properties of the nanoparticles generated, such as their one and two-photon brightness and both their structural and colloidal stability. In that context a library of novel dipolar chromophores have been synthesized and used to prepare FONs using the nanoprecipitation method. Their properties were thoroughly investigated in order to determine the relationship between the molecular design of the isolated dye and the overall properties of the nanoparticles made of these dyes. As a result, Hyperbright FONs emitting in the green to NIR region and combining giant brightness and remarkable stability have been achieved. They offer major promise for bioimaging based on both excitation and detection in the NIR region

L’encapsulation dans des nanomatériaux de polymères de colorants ioniques à l’aide de contre-ions hydrophobes volumineux apparaît être une méthode très efficace pour générer des nanoparticules (NPs) fluorescentes ultra-brillantes pour la bioimagerie. Nous avons d’abord étendu cette approche par contre-ions aux colorants cyanine opérant dans la gamme du bleu au proche infra-rouge. A partir de NPs chargés en cyanines, une methode de code-barre multicolore pour le traçage cellulaire à long terme a été développé. Ensuite, le rôle des contre-ions hydrophobes volumineux dans l’auto-assemblage des colorants cationiques à l’intérieur des NPs de polymères a été étudié en testant une large collection d’anions. Nous avons montré qu’une forte hydrophobicité du contre-ion augmente l’encapsulation du colorant, régule son clustering et empêche l’agrégation de nanoparticules, alors qu’une grande taille empêche l’auto-inhibition de fluorescence. Enfin, nous avons introduit les contre-ions à base d’aluminates et de barbiturates, qui sur-performent les tetraphénylborates fluorés. Ce travail procure une base solide au concept d’émission et d’encapsulation augmentées par contre-ions pour la préparation de NPs chargés en colorants fluorescents
Encapsulation of ionic dyes with help of bulky hydrophobic counterions into polymer nanomaterials emerged as powerful method for generating ultrabright fluorescent nanoparticles (NPs) for bioimaging. Here, this counterion-based approach is extended to cyanine dyes, operating from blue to near-infrared range. Based on cyanine-loaded NPs, a multicolour cell barcoding method for long-term cell tracking is developed. Second, the role of bulky hydrophobic counterion in self-assembly of cationic dyes inside polymeric NPs is studied by testing a large library of anions. We show that high hydrophobicity of a counterion enhances dye encapsulation, prevents particle aggregation and tunes dye clustering, while large size prevents dyes from self-quenching. Third, counterions based on aluminates and barbiturates are shown to outperform fluorinated tetraphenylborates. This work provides a solid basis for counterion-enhanced encapsulation and emission concept in preparation of dye-loaded fluorescent NPs

La production et l’utilisation croissante des nanomatériaux et nanoparticules (NP) dans de nombreux secteurs d’activité conduisent inévitablement à un relargage de NP dans l'environnement et notamment dans les eaux, devenant ainsi une pollution émergente dans le schéma de la production d’eau potable. Les membranes d’ultrafiltration (UF) semblent présenter un réel potentiel de rétention envers les NP du fait de leur taille de pores proche de 20 nm. La filtration de NP fluorescentes de diamètre 100, 10 et 1,5 nm en suspensions, seules ou en mélange, a été étudiée. Les tailles des NP ainsi sélectionnées permettent de travailler avec des dimensions plus grande, plus petite et du même ordre de grandeur que la taille des pores. La considération de la concentration des flux de la filtration en nombre de NP a permis d’estimer le nombre de NP bloquées sur et/ou dans la membrane. Une méthodologie précise et fiable permettant la localisation de ces NP bloquées a été consolidée par une précision de mesure plus importante grâce à une caractérisation multi-échelle. Des profils de pénétration des NP fluorescentes dans la membrane ont été réalisés grâce à une imagerie au Microscope Confocal à Balayage Laser (MCBL). L’application des modèles de colmatage aux données expérimentales a montré une bonne adéquation avec la localisation microscopique des NP et les résultats expérimentaux obtenus. L’influence des conditions opératoires, de la présence de sel et/ou de la polydispersité de la suspension d’alimentation sur l’établissement et la localisation du colmatage a pu être déterminée, notamment grâce à la mise en place de plan d'expérience
The increasing use of nanotechnologies and nanoparticles (NPs) in many sectors of activities leads to their inevitable discharge in the environment and thus in water. The properties and the toxicity of these NP are still unclear, so that this new pollution have to be considered in drinking water treatment plant. Ultrafiltration (UF) membranes, which have nanometric pore size, present a great potential for NP retention. The mass transport through the UF membrane of fluorescent NP, with size of 100, 10 and 1.5 nm, has been studied. The influence of operating conditions (transmembrane pressure, volumetric concentration factor) have been treated by experimental design. Consideration of the NP concentration in number in each flux thanks to a Nanosight NS300, allowed to estimate the number of NPs blocked on and/or into the membrane. An accurate and reliable methodology allowed the localization of the fouling by Confocal Laser Scanning Microscopy (CLSM). Application of fouling models to experimental data showed good agreement with microscopic NP location. The influence of the operating conditions, the salinity and/or the polydispersity of the feed suspension on the fouling establishment and fouling profile has been studied

La nanomédecine est un secteur d’activité en plein essor depuis le développement des liposomes, nanovecteurs permettant d’encapsuler des molécules hydrosolubles ou amphiphiles pour la délivrance de médicament. Les nouveaux agents thérapeutiques synthétisés étant de plus en plus lipophiles, le développement de nouveaux vecteurs nanoparticulaires permettant leur transport constitue aujourd’hui un enjeu majeur. Les lipidots, nanoparticules lipidiques solides biocompatibles de 50 nm de diamètre, composées d’un mélange d’huile, de cire et de lécithine stabilisées par une couronne de surfactants pegylés, permettent de transporter des composés hautement hydrophobes en leur cœur. Des agents nucléaires pour la tomographie à émission monophotonique et des agents de fluorescence ont pu y être encapsulés pour l’évaluation de la biodistribution de ces nanoparticules chez la souris saine ainsi que dans différents modèles tumoraux. Un peptide de ciblage, le cRGD a également été greffé à la surface des lipidots afin d’adresser préférentiellement, dans les 3 premières heures après injection, les nanoparticules vers les tumeurs surexprimant les intégrines αvβ3. Une biodistribution particulière des lipidots dans les organes stéroïdiens (surrénales, ovaires) a été observée avec une localisation spécifique dans les zones synthétisant les hormones stéroïdiennes (le corps jaune pour les ovaires, la zone corticale pour les surrénales). Cette affinité unique a été mise à projet pour cibler des tumeurs hormono-dépendantes, ainsi que pour encapsuler des hormones ovariennes pour le traitement hormonal substitutif ou le contrôle de la procréation
Nanomedicine is a fast growing field since the development of liposomes, nanovectors able to encapsulate in their core or phospholipid bilayer hydrophilic or amphiphilic molecules for drug delivery purposes. Nowadays, most of the new synthetized therapeutic compounds are hydrophobic, necessitating the development of new types of nanocargos. Lipidots, 50 nm diameter biocompatible solid lipid nanoparticles, composed of a mixture of oil, wax and lecithin stabilized by a shell of pegylated surfactants, are used to encapsulate highly hydrophobic compounds in their core for vectorization purpose. Nuclear agents for simple photon emission tomography, or near infrared fluorescent dyes, have been encapsulated in the lipidot core, to assess the biodistribution of these nanoparticles in healthy mice as well as in a large range of tumor models. A targeting peptide, the cRGD motif, was also grafted on the surface of lipidots to vectorize preferentially the nanoparticles to tumors overexpressing αvβ3 integrins. An unexpected lipidot biodistribution in steroid organs (adrenal, ovary) was observed, with a specific localization in areas of steroid hormones synthesis (corpus luteum in ovaries, cortex for adrenals). This unique lipidot affinity was used to target hormono-dependent cancer cells, as well as to encapsulate ovarian hormones, like estradiol or ethynil estradiol, for hormone substitution therapy or birth control

El estudio de nuevas nanopartículas orgánicas fluorescentes (FONs) que puedan superar los límites de las comunes sondas fluorescentes como fluoróforos moleculares, proteínas fluorescentes y nanopartículas inorgánicas es un tema de gran interés para los científicos de materiales que desarrollan nuevas sondas para microscopía de fluorescencia y teranóstica. En los últimos años, se han desarrollado nuevas nanovesículas no liposómicas, basadas en el autoensamblaje de tensioactivos y esteroles, denominados Quatsomes (QSs), que constituyen unos prometedores vehículos de fármacos multifuncionales. Dentro de este escenario, el objetivo principal de esta Tesis (realizada en el marco del proyecto Marie Skłodowska-Curie ITN “Nano2Fun”) es explorar la posibilidad de utilizar los Quatsomes como vehículos para la nano-estructuración en medios acuosos de varias moléculas fluorescentes, independientemente de sus propiedades físico-químicas y ópticas, con el fin de obtener nuevas nanopartículas orgánicas fluorescentes (FONs) con elevada estabilidad coloidal y características fluorescentes optimales, especialmente en relación al brillo. El auto-ensamblaje de fluoróforos orgánicos moleculares, esteroles y tensioactivos de amonio cuaternario en Quatsomes fluorescentes se logró mediante el método DELOS-SUSP, un proceso a base de CO2 comprimido, que garantiza una composición de membrana y una disposición supramolecular altamente homogéneas y, como consecuencia, nanovesículas con elevadas propiedades ópticas. Se han explorado diferentes estrategias para la nano-estructuración en medios acuosos, por medio de QSs, de moléculas fluorescentes con diferentes propiedades fisicoquímicas, incluyendo aquellas solubles y no solubles en agua, analizando el impacto de la nano-estructuración sobre las propiedades ópticas de las FONs obtenidas. De esta manera, los fluoroforos aniónicos solubles en agua, como la fluoresceína, fueron nano-estructurados encima de los QSs. Por otra parte, los fluoróforos lipófilos y no solubles en agua, modificados con largas cadenas alquílicas pueden incorporarse de forma estable en la membrana de los QSs, como se muestra en el caso de varias familias de colorantes, por ejemplo, las cianinas, los diketopirrolopirroles (DPP) y los derivados del fluorene. Los QSs fluorescentes mostraron una estabilidad óptica coloidal excepcional (hasta varios meses), un alto grado de homogeneidad estructural y altas eficiencias de fluorescencia, mostrando mejores prestaciones en comparación con otras nanoestructuras de los mismos fluoroforos. Además, con el objetivo de obtener nanopartículas multicolores, los Quatsomes permitieron cargar simultáneamente diferentes fluoroforos dentro de sus membranas, mostrando un mecanismo de transferencia de energía de resonancia de fluorescencia (FRET) altamente eficiente, una interesante herramienta para monitorear la integridad del carrier durante la administración del fármaco y para la adquisición de imágenes multicolores. En conclusión, los Quatsomes fluorescentes se probaron como nano-sondas para la obtención de imágenes de células in vitro. Se ha demostrado que los Quatsomes que incorporan derivados del fluorene (denominados LysoQS) constituyen una sonda lisosómica altamente específica, ideal para la adquisición de imágenes en tiempos largos. Además, los Quatsomes cargados de cianinas se utilizaron como sondas para técnicas de microscopía superresolución (STORM), que permitió la visualización y resolución de Quatsomes individuales tras la internalización en las células. Los resultados de esta tesis muestran que los Quatsomes fluorescentes, gracias a las ventajas ofrecidas en comparación con otros marcadores fluorescentes comúnmente empleados, son unas nanosondas altamente prometedoras, con posibles aplicaciones futuras en bioimagen, teragnostica y, en general, nanomedicina.
Finding new fluorescent organic nanoparticles (FONs) with the potential to overcome the limits of common fluorescent probes as molecular fluorophores, fluorescent proteins and inorganic nanoparticles is a subject of strong interest for materials scientists developing new probes for fluorescence microscopy and theranostics. In the recent years, innovative non-liposomal nanovesicles, based on the self-assembly of quaternary ammonium surfactants and sterols, named Quatsomes (QSs), have been developed as promising candidates for applications as multifunctional drug carriers. Within this scenario, the main objective of this Thesis (conducted in the framework of the Marie Skłodowska-Curie ITN “Nano2Fun”) is to explore the possibility of using Quatsomes as a vehicle for nanostructuring in aqueous media several dye molecules, irrespective of their physicochemical and optical properties, in order to obtain new fluorescent organic nanoparticles (FONs) with superior colloidal stability and enhanced fluorescent features, especially with high brightness, in relation to single molecule flurofores and other type of FONS. The self-assembly of molecular organic fluorophores, sterols and quaternary ammonium surfactants into fluorescent Quatsomes was achieved by the DELOS-SUSP method, a compressed CO2 –based process which guarantees a highly homogeneous membrane composition and supramolecular arrangement, which have impact on the optical properties of the obtained FONs. Different strategies have been explored to nanostructurate in aqueous media, by mean of QSs, molecular dyes with different physicochemical properties, including those water- and non-water soluble, analyzing the impact of their nanostructuration on the optical properties of the obtained FONs. Thus, anionic water-soluble dyes, such as fluorescein, were nanostructured over QSs surface, taking advantage of anionic/cationic interaction among dye and vesicles surface. On the other hand, lipophilic and non-water soluble dyes modified with long alkyl chains can be stably incorporated into QSs membrane, as shown in the case of several dyes families, including cyanine, diketopyroolopyrrole (DPPs) and fluorene derivatives. The fluorescent QSs showed superior colloidal and optical stability (up to several months), a high degree of structural homogeneity and high fluorescence performances, overcoming those of other nanostructures of the same dyes. Furthermore, aiming to obtain multicolor nanoparticles, Quatsomes allowed the simultaneous loading within their membrane of different dyes, which showed a highly efficient fluorescence resonance energy transfer (FRET) mechanism, an interesting tool for monitoring the carrier integrity during the drug delivery and for multiplexed imaging applications. Finally, fluorescent Quatsomes were tested as nanoprobes for in vitro cells imaging. It has been demonstrated that fluorene-based Quatsomes (named LysoQS) constitute a strongly specific lysosomal probe ideal for long-term imaging. Furthermore, cyanines-loaded Quatsomes were used as probes for super-resolution microscopy technique (STORM) which allowed visualizing and resolving single Quatsomes structures upon internalization in cells. The results of this Thesis showed that fluorescent Quatsomes, thanks to the advantages offered in comparison with other commonly employed fluorescent labels, constitute a promising fluorescent nanoprobes with possible future applications in bioimaging, theranostics and, generally, nanomedicine.

En aquesta tesi s'han utilitzat diferents estratègies per obtenir control en la producció i detecció de diferents espècies reactives d'oxigen (ROS), especialment per a l'oxigen singlet (1O2). En la primera part de la tesi, l'enfoc principal consisteix en entendre la generació de ROS i intentar potenciar-ne el seu efecte. En primer lloc, demostrem que la modificació de diferents fotosensibilitzadors afegint-hi un catió de trifenilfosfoni com a element diana produeix derivats amb una excel·lent activitat fotoantimicrobiana contra bacteris Gram-positius (S. aureus i E. faecalis). En segon lloc, descobrim una sèrie de nous aspectes de la reacció de “-phenyl quenching” per derivats de 9-fenilfenalenona. La fototoxicitat d’aquests derivats ja es troba esmentada en el llibre: “L'origen de les espècies” de C. Darwin. També es suggereix una via metabòlica mediada per la reacció BPQ en la biosíntesi dels pigments vegetals derivats de fluorones. A més, si el grup fenil és substituït per altres grups arils, s'observa diferencies en la reacció de BPQ. En tercer lloc, s’ha demostrat que diferents antraquinones d’origen natural indueixen fototoxicitat en biofilms de C. tropicalis a causa de la generació de O2•, tenint l’1O2 un rol menor. En quart lloc, es demostra el fàrmac antitumoral Doxorubicina produeix quantitats significants d’1O2, però es redueix la seva generació quan es complexa amb el ADN. En cinquè lloc, s’ha estudiat l’efecte d’adsorció o unió covalent d’un fotosensibilitzador a nanopartícules mesoporoses de sílice. A més a més, s’han derivatitzat per afegir-hi elements diana. Sisè i últim, s’ha estudiat les propietats fotoquímiques d’una nova diada que conté un bromo-bodipy com a fotosensibilitzador i trampa química de ROS (que desactiva la capacitat del bromo-bodipy de generar 1O2). Un cop oxidada la trampa química, la diada recupera la capacitat de generar 1O2 i causar dany cel·lular. S’observa diferents propietats foto-antitumorals d’aquesta diada en funció de l’estrès cel·lular o de la localització cel·lular. En la segona part de la tesi, s'ha centrat en la detecció de ROS. En primer lloc, s’han dissenyat, sintetitzat i caracteritzat nanosondes fluorescents per la detecció d'1O2 en sistemes biològics. La nanovehiculització elimina algunes de les limitacions de les diferents sondes fluorescents d’1O2. En aquest sentit, diverses sondes tals com SOSG, ADPA o furil-vinil-naftooxazol s’han unit covalentment a nanopartícules utilitzant diferents cadenes espaiadores per tal d’optimitzar la seva reactivitat front 1O2. A diferència de quan es troben lliures en solució, les nanosondes són fàcilment internalitzades per cèl·lules eucariotes i procariotes i es minimitza la interacció amb proteïnes (com per exemple, l’albúmina de sèrum boví). Les diferents nanosondes responen a l’1O2 generat intracel·lular. Com a prova de concepte, també s’ha desenvolupat una nanosonda fluorescent per la detecció no selectiva de ROS, basada en 2’,7’-diclorodihidrofluoresceina. En segon lloc, s’ha caracteritzat la estructura i reactivitat de la sonda fluorescent: CellROX Deep Red. En tercer lloc, s’ha desenvolupat la primera sonda d’optoacústica per la detecció de ROS basada en l’oxidació de la tetrametilbenzidina. S’ha aconseguit detectar 1O2 produït per bacteris utilitzant tal sonda. Finalment i com a prova de concepte, s’ha dissenyat un “self-reporter” nanofotosensibilitzador. El nanosistema és capaç de produir i detectar 1O2 simultàniament. Aquest nanodispositiu s'ha utilitzat amb èxit per la fotoinactivació de S. aureus, observant-se una correlació entre el canvi de fluorescència de la sonda i la mort bacteriana.
En esta tesis se han utilizado distintas estrategias para obtener el control en la producción y detección de diferentes especies reactivas de oxígeno (ROS), especialmente para el oxígeno singlete (1O2). En la primera parte de la tesis, el enfoque principal consiste en entender la generación de ROS e intentar potenciar su efecto. En primer lugar, demostramos que la modificación de distintos fotosensibilizadores, añadiendo un catión de trifenilfosfonio como elemento diana, produce derivados con una excelente actividad fotoantimicrobiana contra bacterias Gram-positivas (S. aureus y E. faecalis). En segundo lugar, descubrimos una serie de nuevos aspectos de la reacción de "-phenyl quenching" por derivados de 9-fenilfenalenona. La fototoxicidad de estos derivados ya se encuentra mencionada en el libro: "el origen de las especies" de C. Darwin. También se sugiere una vía metabólica mediada por la reacción BPQ en la biosíntesis de los pigmentos vegetales derivados de fluorenonas. Además, si el grupo fenilo es sustituido por otros grupos arilos, se observan diferencias en la reacción de BPQ. En tercer lugar, se ha demostrado que distintas antraquinonas de origen natural inducen fototoxicidad en biofilms de C. tropicalis debido a la generación de O2•, teniendo el 1O2 un rol menor. En cuarto lugar, se demuestra que el fármaco antitumoral Doxorubicina produce cantidades significantes de 1O2, pero se reduce su generación cuando se compleja con el ADN. En quinto lugar, se ha estudiado el efecto de adsorción o unión covalente de un fotosensibilizador a nanopartículas mesoporosas de sílice. Además, se han derivatizado para añadir elementos diana. Sexto y último, se han estudiado las propiedades fotoquímicas de una nueva diada que contiene un bromo-bodipy como fotosensibilizador y trampa química de ROS (que desactiva la capacidad del bromo-bodipy para generar 1O2). Una vez oxidada la trampa química, la diada recupera la capacidad para generar 1O2 y causar daño celular. Se observan diferentes propiedades foto-antitumorales de esta diada en función del estrés celular o de la localización celular. La segunda parte de la tesis, se ha centrado en la detección de ROS. En primer lugar, se han diseñado, sintetizado y caracterizado nanosondas fluorescentes para la detección de 1O2 en sistemas biológicos. La nanovehiculización elimina algunas de las limitaciones de las distintas sondas fluorescentes de 1O2. En este sentido, varias sondas tales como SOSG, ADPA o furilo-vinilo-naftooxazol se han unido covalentemente a nanopartículas utilizando distintas cadenas espaciadoras para optimizar su reactividad frente 1O2. A diferencia de cuando se encuentran libres en solución, las nanosondas son fácilmente internalizadas por células eucariotas y procariotas y se minimiza la interacción con proteínas (como por ejemplo con la albúmina de suero bovino). Las distintas nanosondas responden al 1O2 generado intracelular. Como prueba de concepto, también se ha desarrollado una nanosonda fluorescente para la detección no selectiva de ROS, basada en 2',7'-diclorodihidrofluoresceina. En segundo lugar, se ha caracterizado la estructura y reactividad de la sonda fluorescente: CellROX Deep Red. En tercer lugar, se ha desarrollado la primera sonda de optoacústica para la detección de ROS basada en la oxidación de la tetrametilbenzidina. Se ha logrado detectar 1O2 producido por bacterias emprando tal sonda. Finalmente, y como prueba de concepto, se ha diseñado un "self-reporter" nanofotosensibilitzador. El nanosistema es capaz de producir y detectar 1O2 simultáneamente. Este nanodispositivo ha sido utilizado con éxito para la fotoinactivación de S. aureus, observándose una correlación entre el cambio de fluorescencia de la sonda y la muerte bacteriana.
In this thesis, different strategies have been used in order to gain control in reactive oxygen species (ROS) production and detection, especially for singlet oxygen (1O2). In the first part of the thesis, the main focus is towards understanding ROS generation and try to potentiate its effect. First, we demonstrate that modification of different photosensitisers with the triphenylphosphonium cation yields derivatives with an excellent photoantimicrobial activity against Gram‐positive bacteria (i.e., S. aureus and E. faecalis). Second, we uncover a number of new aspects of -phenyl quenching reaction in 9-phenylphenalenone scaffold, whose phototoxicity was already mentioned in Darwin’s Origin of Species. It is suggested an excited state-mediated metabolic pathway in the biosynthesis of fluorone plant pigments. Moreover, if phenyl moiety is substituted for other aryl groups, it is observed that the electrocyclic ring opening back to ground state ketones have lifetimes between miliseconds and picoseconds. Third, we demonstrate that the main photosensitizing mechanism, involved in the photo-induced C. tropicalis antibiofilm activity by natural anthraquinones, is via O2• production, whereas 1O2 participation seems of lesser importance. Fourth, we demonstrate that doxorubicin produces significant amounts of 1O2, however, this is largely suppressed when bound to DNA. Fifth, we studied the effect of PS adsorption or covalently bond onto the surface of mesoporous silica nanoparticles. Moreover, we further derivatitze them for attach targeting elements. Sixth and last, we studied the activation a new dyad comprising a bromo-bodipy, which acts as PS, plus a non-selective ROS chemical trap, which quenches the ability of bromo-bodipy to produce 1O2. For that aPS we observe a differential behaviour in function of the cellular stress or even in function of the organelle. In the second part of the thesis, focus has been shifted towards ROS detection. First, we designed, synthesized, and characterized biocompatible fluorescent nanoprobes for 1O2 detection in biological systems that circumvents many of the limitations of the different molecular 1O2 fluorescent probes. Under that purpose different 1O2 probes (Singlet Oxygen Sensor Green, anthracene dipropionic acid and furyl-vinyl-naphthoxazole) were covalently linked to nanoparticles core using different architectures to optimize their response to 1O2. In contrast to its molecular counterpart, the optimum nanoprobes are readily internalized by prokaryotic and eukaryotic cells and they do not interact with proteins (i.e. bovine serum albumin). Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellular generated 1O2 with the corresponding change in fluorescence. As a proof of concept, a non-selective ROS fluorescent nanoprobe, based on diacetyl 2’,7’-dichlorodihydrofluorescein, has been synthetized and successfully used for detecting intracellular ROS. Second, we have performed the chemical characterization of the CellROX Deep Red, a new commercial non-selective ROS fluorescent probe, ascertained its putative chemical structure and evaluated its reactivity towards different reactive oxygen/nitrogen species and light in solution. Third, we developed the first ROS optoacoustic probe based on the oxidation of tetramethylbenzidine and successfully used for detecting 1O2 produced by bacteria. Finally, as proof of concept we have designed a self-reporter nanophotosensitizer. The nanosystem is capable to produce and detect the 1O2 generated simultaneously. It has been successfully used for S. aureus photoinactivation in which a correlation was observed between fluorescent change of the probe and bacterial cellular death.

Dans ce travail de thèse nous avons étudié les propriétés de fluorescence de films minces d’or dopés avec des molécules organiques Nous avons montré par imagerie électroniques MEB et TEM qu’ils sont structurés en agglomérats de nanocristaux (5 à 10nm) recouverts de molécules formant un gap nanométrique. Dans les spectres d’extinction nous avons observé la présence de la résonance plasmon du métal ainsi que d’une autre résonance à 600nm que nous attribuons au plasmon de gap.Les spectres d’émission et d’excitation de fluorescence ont confirmé que ces films dopés fluorescents avec une composante venant de la fluorescence de l’or, et une autre caractéristique de la présence des molécules fluorescentes. Les décalages spectraux en excitation et en émission à la fois de l’or et des molécules sont les signes d’un couplage fort entre leurs états électroniques, ce qui serait en accord avec la très forte concentration de molécules dans le film (1/100 molaire)L’observation non intuitive de la fluorescence des molécules insérées dans la couche d’or aurait pour origine l’augmentation considérable de leur taux radiatifs qui a été récemment observé dans les nanogaps
In this thesis work we studied the fluorescence properties of gold thin films doped with organic molecules. We have shown by electronic imaging SEM and TEM that they are structured in agglomerates of nanocrystals (5 to 10 nm) covered with molecules forming a nanometric gap. In the quenching spectra we observed the presence of the plasmon resonance of the metal as well as another resonance at 600nm that we attribute to the gap plasmon.The fluorescence emission and excitation spectra confirmed that these fluorescent doped films with a component coming from the fluorescence of gold, and another characteristic of the presence of fluorescent molecules. The spectral shifts in excitation and in emission of both the gold and the molecules are the signs of a strong coupling between their electronic states, which would be in agreement with the very high concentration of molecules in the film (1/100 molar)The non-intuitive observation of the fluorescence of the molecules inserted into the gold layer is due to the considerable increase in their radiative levels that has recently been observed in nanogaps

Développer de nouveaux fluorophores ayant une forte section efficace d’absorption à deux photons (ADP) et des propriétés d'émission dans le rouge lointain est important, en particulier pour l’imagerie in-vivo profonde. Cette gamme de longueur d'onde correspond en effet à la fenêtre de transparence optique des tissus. Cette thèse étudie le potentiel de nouveaux fluorophores émettant dans rouge construits sur un noyau fluorène pour la microscopie à deux photons in-vivo, en privilégiant l'imagerie du système vasculaire, d'une part, et la mesure optique de la pression d'oxygène dissous, d'autre part.Ainsi, une famille de chromophores asymétriques a été conçue et synthétisée. La plupart des chromophores présentent une forte émission dans le proche IR, induite par l'agrégation. De plus, une stratégie de co-protection basée sur un système micellaire / silice a été utilisé pour préparer des nanoparticules avec un intérieur apolaire et conserver les propriétés optiques des chromophores dipolaires en solution aqueuse. Des mesures de fluorescence excitée à deux photons ont été menées en solvant organique et en suspension aqueuse. Les agrégats et les nanoparticules ont été utilisés avec succès en imagerie biphotonique du système vasculaire sur petit animal en utilisant un modèle de tumeur à l'intérieur de la peau de l'oreille de la souris. Les nanoparticules de silice montrent une coloration exceptionnelle du système vasculaire qui en fait de parfaits marqueurs du système vasculaire.Dans un deuxième temps, quatre nouveaux chromophores, absorbant à deux photons, ont été synthétisés et leurs propriétés photo physiques à un et à deux photons ont été étudiées. Le chromophore le plus adapté a ensuite été greffé de manière covalente par chimie click, à un complexe de palladium avec un ligand porphyrine, cœur phosphorescent dont l’émission est sensible à la présence d’oxygène. Deux composés contenant quatre ou huit absorbeur à deux photons ont été obtenus et étudiés. Les résultats démontrent que l'incorporation d'un chromophore ADP approprié peut effectivement augmenter les propriétés d’ADP du système, ce qui permet une sensibilité efficace vis-à-vis de l'oxygène
The development of fluorophores with efficient two-photon absorption (TPA) and emission properties in the far red/NIR is important, especially for in depth in-vivo optical imaging as this wavelength range corresponds to the optical transparency window of tissues. This thesis investigates the potential of new red emitting fluorophores containing a fluorene ring for in-vivo two-photon microscopy focusing on vascular imaging on one hand and on oxygen pressure measurement on the other hand.A new series of asymmetrical fluorene-based chromophores were designed and synthesized. Their structure-property relationships were systematically investigated. It was found that most of chromophores exhibit aggregation-induced emission behaviors in the NIR region. In addition, a micelle/silica coprotection strategy was proposed to prepare nanoparticles with a less polar interior, which can be used to conserve optical properties of dipole chromophores in aqueous solution. The two-photon excited fluorescence (TPEF) measurements indicate that they all display obvious TPA activities in organic solvent and aqueous suspension. Both the NIR-emissive aggregates and nanoparticles have been successfully used for TPEF imaging of blood vessels inside mouse ear skin. The silica nanoparticles show outstanding staining of the vascular system making them perfect blood pool markers.On a second part, four new fluorene-based two-photon absorbing chromophores have been synthesized and their one- and two-photon photophysical properties were investigated. The optimum chromophore was successfully attached covalently to an oxygen responsive phosphorescent Pd-porphyrin complex by click chemistry. Two new compounds contain four or eight TPA chromophores donor connected to the phosphorescent core. The result demonstrate that the incorporation of a suitable TPA chromophore can effectively enhance the TPA of the system, allowing efficient sensitivity towards oxygen

Pour une application optimale des nanovecteurs comme système de délivrance de médicaments, il est nécessaire de caractériser pleinement leur intégrité, et leurs propriétés d’encapsulation et de libération de leur contenue. Mon projet de doctorat consiste à développer des méthodes basées sur la fluorescence pour caractériser l'intégrité de ces nanovecteurs lipidiques et la libération des molécules actives in vitro et in vivo. Premièrement, en utilisant le FRET entre deux fluorochromes infrarouges spécialement conçus, l'intégrité des nanovecteurs lipidiques dans le sang et la tumeur cible a été évaluée et quantifiée par imagerie ratiométrique dans le proche infrarouge chez les souris vivantes. Deuxièmement, nous avons développé un test rapide et simple basé sur la FCS pour la quantification in situ de la libération du contenu des différents nanovecteurs. Troisièmement, en utilisant le blanchiment du Nil Rouge par le dithionite de sodium, nous avons établi une approche originale pour étudier l'état physique des nanovecteurs et le niveau d'encapsulation des fluorochromes. En conclusion, nous avons montré que les nanovecteurs lipidiques encapsulant des fluorochromes apparaissent comme un outil prospectif pour la libération contrôlée par la lumière de molécules actives in vitro et in vivo
For effective application of nanocarriers as drug delivery system, it is necessary to fully characterize their integrity, encapsulation, and release properties. The aim of my PhD project is to develop fluorescence-based methods for characterizing integrity of lipid nanocarriers and the release of active molecules in vitr o and in vivo. First, using FRET between specially designed near-infrared dyes the integrity of lipid nanocarriers in bloodstream and tumor was assessed and quantified by near-infrared ratiometric imaging in living mice. Second, we have developed fast and simple FCS-based assay for in situ quantification of release from different NCs. Third, using Nile Red bleaching by sodium dithionite, we established an original approach to study the physical state of the nanocarriers and the level of dye encapsulation. Finally, we showed that dye-loaded lipid nanocarriers appear as a prospective tool for light-controlled release of active molecules in vitro and in vivo

Le design et l’ingénierie de nanoparticules d’or (AuNPs) polyfonctionnelles suscitent un intérêt considérable en vue d’applications en nanomédecine, reconnaissance moléculaire, dans le domaine des capteurs et en catalyse dans un environnement aqueux. Cette thèse a été dédiée à une variété de fonctionnalisations, en particulier à l’aide de la méthode “click” impliquant la catalyse par le cui vre (I) de lacycloaddition des alcynes terminaux avec les azotures avec le catalyseur [Cu(hexabenzyltren)] Br pour l’introduction de polyéthylène glycol, carborane,ferrocène, coumarine, cyclodextrine, médicaments et molécules fluorescentes sur les AuNPs. Les ligands dits “click”, c’est-à-dire des 1,2,3-triazoles fonctionnalisés en positions 1,4 et formés de cette façon ont été ici largement utilisés afin de stabiliser des AuNPs pour des applications biomédicales et catalytiques en collaboration
The design and molecular engineering of multi-functional gold nanoparticles (AuNPs) is of considerable interest towards applications in nanomedicine, molecular recognition, sensing and catalysis in aqueous environments. This thesis has been devoted to a variety of functionnalizations, in particular with the copper(I)-catalyzed Alkyne Azide cycloaddition (CuAAC) using thecatalyst [Cu(I)(hexabenzyltren] Br for the introduction of polyethylene glycol,carborane, ferrocene, coumarin, cyclodextrin, drugs and fluorescent probes. The so called “clicked” ligands, 1,4 -bifunctional triazoles, that were formed in this way have been exensively used to stabilize AuNPs for biomedical and catalytic collaborative applications

L'étude de la dispersion de matériaux inorganiques dans des médias organiques et aqueux est un facteur déterminant pour leur mise en œuvre industrielle. Ces matériaux trouvent leur application dans de nombreux domaines allant de l'industrie pétrolière à celle du polissage. Nous nous sommes intéressés à la stabilité de ces matériaux dans des médias tels que : (i) les solvants aqueux utilisés pour la formulation de suspensions abrasives, (ii) les eaux d'exploitations pétrolières et (iii) le pétrole brut et ses dérivés. Ceci dans le but d'améliorer les propriétés de luminescence de ces matériaux pour l'industrie pétrolière et les propriétés abrasives pour l'industrie du polissageNous avons ainsi développé des formulations stables de complexes de lanthanides (Ln) dérivés du DOTA et de nanoparticules (NPs) de sulfure de zinc dopées au manganèse, dans le but de tracer les eaux d'injections pétrolières. Ces matériaux ont été détecté par fluorescence en temps-résolu (TRF) et ce afin de contourner les problématiques liées à l'auto-fluorescence des produits pétroliers présents dans les eaux d'exploitation. Nous avons également étudié la dispersion de composés spécifiques dans des carburants, dans le but de proposer des nouveaux marqueurs anticontrefaçon pour le pétrole brut et ses dérivés. Enfin, nous avons cherché à améliorer la dispersion de nanoparticules de diamant dans des médias aqueux et ce afin d'optimiser l'efficacité des suspensions abrasives à base diamantées, utilisées pour le polissage du saphir plan a
The dispersion of inorganic materials in organic and aqueous media is a decisive factor for their industrial implementation. These materials are used in many areas from the oil industry to polishing. Herein, we focus on studying the stability of these materials in different media such as: (i) the aqueous solvents used for the formulation of abrasive slurries, (ii) the waters of oil operations and (iii) crude oil and its derivatives. Our objective is to improve the luminescence properties of these materials for the oil industry and the abrasive properties for polishing applications.We have developed stable formulations of lanthanide complexes (Ln) derived from DOTA and nanoparticles (NPs) of zinc sulfide doped with manganese for water-management in oil fields. These materials were characterized by time-resolved fluorescence (TRF) to solve the problems associated with the auto-fluorescence of petroleum products in the operating water.Furthermore, we studied the dispersion of specific compounds in different fuels in order to develop new anti-counterfeiting markers for crude oil and its derivatives. Finally, we tried to improve the dispersion of diamond nanoparticles in aqueous media in order to enhance the efficiency of diamond abrasive suspensions based for polishing sapphire a-plan

Le but de cette thèse est la synthèse, l’optimisation et la fonctionnalisation de nanoparticules coeur-coquille organique@inorganique qui constituent une nouvelle classe de nanotraceurs pour l’imagerie profonde à deux photons de la vascularisation des tumeurs. Ces NPs cœur-coquille qui contiennent un cœur nanocristallin organique (ca 40-50 nm) enrobé d’une coquille de silice sont synthétisées en utilisant une méthode de séchage d’aérosol originale développée dans notre groupe. Le procédé est basé sur la nucléation et la croissance confinées d’un nanocristal organique ayant lieu simultanément avec la formation d’une croûte de silice par le séchage rapide de gouttelettes contenant des oligomères de silice un colorant organique et du solvant dans un flux d’air à 150-200 °C. Ce procédé en une étape est rendu possible grâce au contrôle à la fois de la chimie sol-gel (polycondensation) et du procédé de nanocristallisation qui ont lieu simultanément. Les précurseurs silicatés sont des alcoxydes de silicium : le TMOS (tetraméthoxysilane) et le TMSE (bis(triméthoxysilyl)éthane) choisis pour formés la coquille d’organosilice. De plus, l’organosilane AzPTES ((3-azidopropyl)triéthoxysilane) est utilisé pour inclure des fonctions azoture aux NPs pour une fonctionnalisation ultérieure avec des fragments organiques contenant des fragments alcyne par CuAAC (cycoaddition alcyne-azoture catalysée au cuivre). Les colorants organiques constituant le cœur organique sont non commerciaux et conçus pour fluorescer de façon très brillante à l’état solide sous excitation biphotonique dans le proche infra-rouge (fenêtre de transparence biologique). Ils ont en outre les propriétés physico-chimiques appropriées pour permettre leur nanocristallisation. Des NPs sphériques et sans défaut ont été obtenues, qui ont pu être mises en suspension colloïdale dans l’eau après dissolution basique partielle des coquilles puis neutralisation à pH physiologique.Afin de circuler de façon prolongée dans le flux sanguin pour permettre l’utilisation de ces NPs comme traceurs fluorescents, les NPs synthétisées ont été dérivatisées avec différentes fonctions pour augmenter leur stabilité colloïdale par des effets de charge ou stériques. L’influence de la fonctionnalisation a été étudiée en utilisant différentes techniques de caractérisation comme la spectroscopie de fluorescence, la diffusion dynamique de la lumière ou le potentiel zêta en conditions physiologiques. La fonctionnalisation par différents types de PEG (polyéthylène glycol) de différentes longueurs et modifiés par des fonctions alcyne a été effectuée. La spectroscopie infrarouge a permis de montrer le succès de la fonctionnalisation grâce à la diminution de l’intensité de la bande azoture et à l’apparition de vibrations CH. Les suspensions colloïdales de NPs fonctionnalisées par du PEG5000 ont été traitées dans l’eau ou dans du fluide biologique simulé, à 25 ou 37 °C. Dans tous les cas, la DLS a montré une bonne stabilité avec des diamètres moyens inférieurs à 200 nm dans tous les cas. La spectroscopie de fluorescence avant et après fonctionnalisation montre des brillances comparables ce qui suggère l’absence de blanchiment dans les conditions de fonctionnalisation. Les suspensions colloïdales une fois fonctionnalisées montrent une perte d’intensité de moins de 10% sur 8 h, ce qui suggère une stabilité colloïdale satisfaisante.L’interaction de ces NPs cœur-coquille avec différentes protéines sanguines a aussi été étudiée par DLS, et une très faible agrégation en présence de doses élevées de protéines a été montrée. Des tests d’imagerie par fluorescence à deux photons sur souris sont en cours
The aim of this work is the synthesis, optimization and functionalization of organic@inorganic core-shell nanoparticles (NPs), which constitute a novel class of nanoparticulate tracers, to be used for two-photon deep tissue imaging of tumor vascularization. These core-shell NPs, which comprise an organic dye nanocrystal core (ca 40-50 nm) surrounded by a silicate crust, are synthesized using an original spray-drying method developed in our group. This process is based on the confined nucleation and growth of an organic nanocrystal concomitantly with the formation of a silicate crust by fast drying of sprayed droplets containing silicate oligomers, organic dye and solvent under an air flux at 150-200 °C. This one-step synthesis is made possible thanks to the control of both the sol-gel chemistry (polycondensation) and the nanocrystallization process, which occur simultaneously. Alkoxide precursors, TMOS (tetramethoxysilane) and TMSE (1.2-bis(trimethoxysilyl)ethane) are chosen to form the silicate shell. Additionally, an organosilane, (3-azidopropyl) triethoxysilane (AzPTES), is used to impart an azide functionality to the NPs for further functionalization with alkyne-modified moieties using the Cu(I)-catalyzed 1,3-dipolar cycloaddition of organic azides to alkynes (CuAAC). The organic dyes for the nanocrystalline core are non-commercial and designed to exhibit high fluorescence intensity in the solid state under two-photon excitation in the near infrared (biological window) and the appropriate physico-chemical properties to enable their nanocrystallization. Spherical defect-free NPs were obtained. Colloidal NP suspensions were obtained after a basic partial dissolution of the shells of the NPs followed by acidic neutralization to pH 7.4, to match the pH of physiological media.In order to provide long circulation time of the NPs in the bloodstream to enable the use of these NPs as tracers for deep-tissue imaging, the synthesized NPs were derivatized with different moieties to improve their colloidal stability by charge/steric stabilization. The effects of the functionalization were studied using different characterization tools such as fluorescence spectroscopy, dynamic light scattering (DLS) and zeta potential under physiological conditions.Functionalization with different forms of alkyne-modified polyethylene glycol (PEG), differing in chain length and structure was done using CuAAC, to render them furtive and increase their circulation time in the bloodstream. The functionalized NPs, when compared with the initial core shell NPs (prior to functionalization) using IR spectroscopy, showed positive results, with reduction in the azide band intensity and appearance of bands corresponding to the C-H bonds of the PEG in the functionalized NPs. DLS performed on colloidal suspensions of the core-shell NPs functionalized with a long-chain (Mn :5000) PEG in two media, (a) water and (b) Simulated body fluid (SBF) solution, each tested at two different temperatures (i) 25 °C and (ii) 37 °C resulted in size distributions centered at less than 200 nm in all four cases, thereby indicating stability of the functionalized core-shell NP suspensions under physiological conditions. Fluorescence spectroscopy of the NP suspensions before and after functionalization also exhibited good results, with comparable brightness after functionalization, suggesting that no quenching occurred in the presence of Cu salts. The colloidal suspensions were found to have lost less than 10 % of the fluorescence signal, suggesting colloidal stability.The interactions of these core-shell NPs with different plasma proteins were also investigated, with minimal aggregation in the presence of high concentrations of proteins. Two-photon fluorescence imaging tests in mice are underway. In conclusion, bright, red-emitting core-shell NPs have been produced, which are promising for use in bio-imaging

Semiconductor nanoparticles, often referred to as quantum dots (QDs), have attracted considerable interest due to their unique photophysical properties such as high photostability and fluorescence quantum yields. In the biomedical arena, QDs are being studied both for their diagnostic and therapeutic applications, in particular the possibility of using QDs in photodynamic therapy, which is based on the destruction of tissue with light through photosensitised formation of reactive oxygen species (ROS). In this thesis, the ability QDs to induce the formation of ROS through Type I photoinduced electron transfer and Type II energy transfer mechanisms was investigated. Firstly, the effectiveness of quantum dot-photosensitiser complexes for photosensitised production of ROS was investigated. A sulfonated phthalocyanine was found to form stable complexes with water soluble pegylated quantum dots. Efficient Förster resonance energy transfer (FRET) between the quantum dot and phthalocyanine was demonstrated to generate singlet oxygen by the Type II mechanism with a quantum yield of up to 0.15. Secondly, the potential of QDs alone to produce ROS in aqueous solutions and cellular environments was studied. In aqueous solution, the production of superoxide radical anions by photoinduced electron transfer to molecular oxygen was demonstrated with a quantum yield of 0.005. The yield could be significantly enhanced via a Type I mechanism in the presence of the electron transfer agent, NADH, which was demonstrated by oxygen consumption measurements, electron paramagnetic resonance (EPR) spin trapping and cytochrome c reduction. Production of hydroxyl radicals was shown using a fluorescence probe. In the cellular studies, QD uptake could be significantly enhanced by conjugation with the cell penetrating Tat peptide, which enabled studies of phototoxic effects induced by QD photosensitised ROS production. QDs can also be used as fluorescent imaging probes in vivo for a variety of biological applications; however their fluorescence properties in tissue have not been widely investigated. The uptake of intravenously administered CdSeTe/ZnS QDs in a range of organs including liver, was investigated. By comparing a range of fluorescence quantitation methods, including fluorescence microscopy and chemical extraction, it was found that these QDs were highly resistant to degradation under physiological conditions, demonstrating their effectiveness as imaging probes in vivo.

Lipid liquid crystalline nanoparticles can find application as nanocarriers in several fields of the daily life but, very likely, the pharmaceutical arena is the most relevant. Indeed, several problems encountered in drugs administration (e.g. critical sideeffects from antitumor drugs) require alternative, less invasive, but simultaneously efficient therapeutic routes to be explored. Novel fields of personalized nanomedicine are developing in this direction. One of the most interesting is theranostic, which calls for the design of platforms capable of combining therapeutic and diagnostic functionalities. In this optic, we explored the potential of monoolein-based cubosomes and hexosomes as nanocarriers for theranostic purposes. Our work focussed on the design of lipid nanoparticles able to deliver antineoplastic drugs and imaging probes for fluorescent optical in vitro and in vivo imaging. We developed cubosome formulations loaded with antineoplastic drugs and useful for the fluorescence imaging of cells. Such formulations were also actively targeted to cancer cells and coupled with a NIR-emitting fluorophore, which was the promise for in vivo applications. We also investigated hexosomes with encouraging results encapsulating in their lipid matrix a BODIPY derivative with solvatochromic properties, helpful for the understanding of the dye localization. Importantly, we reported (manuscript submitted) the first proof-of-principle for in vivo fluorescence optical imaging application using monoolein-based cubosomes in a healthy mouse animal model. Finally, since relatively little is known about the interaction of cubosomes with biological systems, their effects on lipid droplets, mitochondria and lipid profile of HeLa cells were deeply studied. This thesis is divided in two main parts. The introduction section reports on the essential background of the research field, and it is followed by the publications (published or submitted) resulting from these three years of work.

Ce travail de thèse a eu pour but d'étudier la thermodynamique de petits objets (nanoparticules ou édifices biomoléculaires) dans des milieux fortement hors-équilibre. Deux milieux miroirs ont été explorés, à savoir des gouttelettes micrométriques chargées et des microbulles générées sous irradiation laser intense. Ces deux systèmes sont par nature difficiles à sonder par des méthodes traditionnelles, c'est pourquoi, une thermographie innovante multi-échelle par méthodes optiques a été développée. Ainsi, une thermographie locale (au sein des nano-objets) et globale (dans le milieu environnant) a été réalisée. Cette thermographie utilise la fluorescence induite par laser - méthode simple, non invasive et efficace pour fournir une mesure de température avec une bonne résolution spatiale, temporelle et thermique - grâce à des colorants thermochromiques directement en solution dans le milieu ou bien incorporés dans les nano-objets. Cette thermographie est également complétée avec d'autres mesures physiques, notamment la taille des gouttelettes et des bulles micrométriques pour aller vers une étude thermodynamique exhaustive de ces systèmes. Ces études thermodynamiques ont été menées autour des deux thématiques : Edifices biomoléculaires dans des gouttelettes micrométriques chargées en cours de déshydratation. Les sources électrospray, devenues un outil incontournable en spectrométrie de masse, présentent une thermodynamique riche et encore mal comprise. Des mesures de taille et de température de gouttelettes micrométriques chargées en cours d'évaporation ont pu être confrontées dans le but d'avoir une description thermodynamique complète d'une source électrospray. De plus, afin de pouvoir suivre la conformation des édifices biomoléculaires (protéines) en cours de déshydratation, le concept d'anisotropie de fluorescence comme sonde conformationnelle a été validé en solution et pourra être transposé pour une analyse in situ dans la plume de l'électrospray.Nanoparticules dans des microbulles générées sous irradiation laser intense. Il s'agit ici d'étudier l'influence de nanoparticules lors de la génération de microbulles par nano-cavitation induite par irradiation laser intense, thématique au coeur du projet « ERTIGO ». Le principal objectif scientifique de ce projet a été d'obtenir une compréhension des mécanismes d'absorption d'une solution contenant des nanoparticules irradiées. A cet effet, une source lumineuse atypique (laser aléatoire) a été employée
The aim of this thesis was to study the thermodynamics of small objects (nanoparticles or biomolecule) in out-of-equilibrium media. Two mirror media were explored, namely charged micrometric droplets and microbubbles generated under intense laser irradiation. By nature, these two systems are difficult to probe by traditional methods, which is why an innovative multi-scale thermography by optical methods has been developed. Thus, a local (within the nano-objects) and a global (in the surrounding medium) thermography were carried out. Such thermography uses Laser-Induced Fluorescence - a simple, non-invasive and efficient method for providing temperature measurements with good spatial, temporal and thermal resolution - using thermochromic dyes directly in solution or incorporated into nano- objects. This thermography is also supplemented with other physical measurements, in particular the size of microdroplets and microbubbles, towards a complete thermodynamic study of these systems. These thermodynamic studies were carried out around the two following themes: Biomolecular structures in charged microdroplets during dehydration. Electrospray sources, which have become an essential tool in mass spectrometry, present a rich and still poorly understood thermodynamics. Measurements of the size and temperature of charged micrometric droplets during evaporation have been possible in order to obtain a complete thermodynamic description of an electrospray source. Moreover, in order to be able to follow the conformation of biomolecules (proteins) during dehydration, the concept of fluorescence anisotropy as a conformational probe has been validated in solution and can be transposed for an in situ analysis into the electrospray plume. Nanoparticles in microbubbles generated by intense laser irradiation. Here, we want to study the influence of nanoparticles during the generation of microbubbles by nano-cavitation induced by intense laser irradiation. This thematic is at the heart of the project "ERTIGO". The main scientific objective of this project was to obtain an understanding of the absorption mechanisms of a solution containing irradiated nanoparticles. For this purpose, an atypical light source (random laser) has been used in order to be able to illustrate this complex out-of-equilibrium system as a function of time by optical microscopy. In parallel, a local measurement of the temperature of the nanoparticles is envisaged

Les nanoparticules inorganiques luminescentes ont suscité un intérêt croissant au cours des dernières décennies, notamment pour leur application en imagerie biologique. Un certain nombre d’entre elles présentent toutefois des limitations telles que toxicité, absence de biodégradabilité, faible brillance, clignotements…. Dans cette optique, les nanoparticules fluorescentes à base de petites molécules organiques (FONs) offrent une solution alternative prometteuse aux nanoparticules inorganiques pour l'imagerie biologique. Le principal défi réside dans l'élaboration des nanoparticules organiques possédant une brillance élevée, une bonne stabilité dans l'eau (y compris en milieu biologique), une bonne biocompatibilité ainsi qu'une émission accordable dans le visible et au-delà dans le proche infrarouge (pour une détection plus aisée en milieu diffusant). Dans cette optique, nous avons utilisé une stratégie basée sur l’utilisation de chromophores dipolaires de type "push pull" « adaptés ». Au cours du travail, la synthèse de séries de chromophores homologues bâtis sur le même système conjugué et ayant en commun un groupe donneur de type triphénylamine (destiné à préserver les propriétés de luminescence) présentant ou non des motifs encombrants positionnés a été réalisée. Les nanoparticules correspondantes ont été préparées selon un protocole classique, simple et rapide à mettre en oeuvre (précipitation). L’étude des propriétés photophysiques des nanoparticules organiques fluorescentes ainsi obtenues a été réalisée et mise en perspective avec celles des chromophores en solution dans des solvants organiques de polarité variable. Une étude systématique de l’évolution dans le temps des propriétés optiques des nanoparticules organiques a été réalisée permettant de mettre en lumière des relations entre la structure des sous-unités chromophoriques et la stabilité colloïdale et « optique » des nanoparticules. Ces études ont permis d’identifier des nanoparticules émettant dans le proche infrarouge extrêmement brillantes et présentant une stabilité colloïdale remarquable dans l’eau, une photostabilité accrue et une très bonne biocompatibilité. De ce fait, ces nanoparticules ont pu être utilisées avec succès dans l'imagerie biologique des cellules et le suivi (tracking) à l'échelle de la particule unique, démontrant l'intérêt de la démarche d'ingénierie mise en oeuvre
During the last decades, luminescent inorganic nanoparticles have attracted a large interest in different fields including biological imaging. However, a number of them have drawbacks such as toxicity and absence of biodegradability. Recently, molecular-based fluorescent organic nanoparticles (FONs) have emerged as a promising alternative to inorganic nanoparticles for bioimaging. The main challenge lies in the elaboration of organic nanoparticles that combine large brightness, good colloidal stability in biological environments) and biocompatibility as well as NIR emission (to allow improved detection in thick tissues). To achieve this objective, we have implemented a molecular engineering strategy based on dedicated polar and polarizable "push pull" chromophore built from a triphenylamine donor moiety and a specific pi-conjugated system. The corresponding nanoparticles were readily prepared by the reprecipitation method. In the present manuscript, the synthesis of the chromophores and the preparation and characterization of the organic fluorescent nanoparticles is described. A comprehensive investigation of their photophysical properties and study of their colloidal stability is presented allowing to derive structure-property relationships. The implemented study led to innovative NIR-emitting nanoparticles combining large brightness (superior to those of QDs and NIR-emitting organic dyes), remarkable colloid stability and suitable photostability. These nanoparticles have been successfully used for single particle tracking and imaging in cells, while no toxic effect was observed

This project is carried out within the context of nano medicine, which is the branch of research that tries to implement nanotechnology for therapeutic applications. The specific target of this project was the development of artificial multivalent systems for the selective recognition of liver cancer cells. Multivalency, which is very common in nature, relies on the ability of a system to develop multiple interactions with a target, resulting in a very high overall binding strength. Among various synthetic multivalent systems available (dendrimers, micelles, liposomes, …) it was chosen to use gold nanoparticles as a multivalent scaffold. These are small cluster of gold atoms with dimensions that typically vary between 1 and 20 nanometers, which can be covered with a monolayer of organic molecules. The monolayer is spontaneously formed upon the addition of molecules able to form a stable bond with the gold surface. Terminal functional groups on these molecules are exposed to the solvent and can be used for recognition or catalytic purposes. For the formation of the monolayer, it was relied on the gold-sulfur interaction, which is sufficiently strong to provide for stability under physiologically relevant conditions. The particular research target of this project was the development of ‘universal’ nanoparticles, which are intended as a class of monolayer protected Au nanoparticles that can be further functionalized after their synthesis and purification. Here, the key issue is the development of a post-functionalization reaction compatible with the stability of the nanoparticles. For this purpose the liver cancer cells form an attractive biological target as it is known that multiple copies of the preS1 peptide bind strongly to the transmembrane protein SCCA localized on the surface of the cells. A first approach was aimed at obtaining monolayer protected gold nanoparticles that could be further functionalized through covalent bond formation. For that purpose, gold nanoparticles coated with aldehyde-terminating thiols were investigated, since the aldehyde-group is perfectly suited for covalent imine-bond formation with amines. As the aldehyde group is prone to nucleophilic attack by thiols, this approach critically relies on the use of appropriate protecting groups. At first, aromatic aldehydes, protected as dimethyl acetal, were studied, since these form particularly stable imines. Regrettably, after monolayer formation it appeared impossible to remove the aldehyde protecting group without destroying the nanoparticle system. This problem found its origin in the excessive stability of the protecting group owing to the presence of a nitro-group in para-position. However, removal of the nitro-group created the opposite problem of a protecting group with too low stability unable to protect the aldehyde against nucleophilic attack. Subsequently, it was tried to synthesize nanoparticles containing aliphatic aldehydes as end groups, but in this case only insoluble products were obtained. Identical problems were obtained when it was tried to oxidize vicinal diols to aldehydes. Abandoning the aldehyde pathway, it was tried to perform post-functionalization reactions through a nucleophilic attack of amines on gold nanoparticles coated with brominated thiols. Also these attempts yielded exclusively insolubile products. Finally, also the use of carboxylic acid as a reactive group proved unsuccessful for the same reason, independent whether the carboxylic acid was used in a mixed monolayer or not. The low success of the covalent post-functionalization approach led us to consider a noncovalent alternative, relying on the use of electrostatic interactions between small oligoanions and a cationic monolayer. For this purpose, gold nanoparticles coated with a cationic monolayer composed of thiols terminating with ammonium of TACN∙Zn(II) groups were used. The interaction between oligoanionic peptide sequences was studied using fluorescence, taking advantage of the fact that gold nanoparticles quench the fluorescence of bound fluorophores. In order to study the possibility of forming a heterofunctionalized surface on top of the monolayer, two probes were prepared consisting of either coumarin 343 or coumarin 2 linked to an oligopeptide composed of three aspartic acids. These fluorophores were chosen for their ability to give fluorescence resonance energy transfer (FRET) when in close proximity. In the experiments, the fluorescence emission of the receiver (cum 343) was measured upon exciting the donor (cum2). This was done for various ratios of the probes at a constant surface saturation concentration of the monolayer surface. From these studies it emerged that, when anchored on the cationic surface, the probes are able to communicate with each other. In the absence of gold nanoparticles, or in the presence of a large excess of ATP, a strong competitor for binding, FRET was not detected. This provides unequivocal evidence for the possibility of creating a multivalent heterofunctionalized surfaces relying on a double self-assembly strategy. The final part of the research project was then dedicated to the development of a peptide-nanoparticle conjugate using the developed strategy. The peptide pre S1 (21-47) is known for its ability to recognize specifically SCCA, a transmembrane protein overexpressed in cancerous hepatocytes. The first studies were aimed at studying the possibility of ligating multiple copies of the preS1 peptide to the surface of cationic gold nanoparticles. The difference in binding affinity of preS1 and preS1 equipped with an anionic triAsp tail were studied by a series of experiments, relying on direct fluorescence titrations (λexcitation = 280, λemission = 360), fluorescence displacement studies (λeccitazione = 305, λemissione = 370), and inhibition studies of TACN∙Zn(II) catalysed hydrolysis (measuring the release of p-nitrophenate, absorbance 400 nm). Alll these studies not only confirmed the possibility of forming a multivalent peptide-nanoparticle structure through self-assembly, but also pointed to a slight stabilizing effect of the triAsp anionic tail. Next the ability of the system to interact with SCCA was investigated.From literature studies it is known that the preS1 peptide in tetrameric form has a higher affinity for SCCA compared to the single peptide. It was thus hypothesized that the presence of SCCA would cause an enhanced stabilization of the nanoparticle-peptide complex. Regrettably, from fluorescence displacement studies it was impossible to obtain evidence that this was indeed the case. No difference in stability was observed for the same system in the presence or absence of SCCA. Since the assay was not conclusive, the stability of the system was studied using a series of other analytical techniques. DLS measurements provided the increase of hydrodynamic radius of the system as consequence of complex formation, but aggregation phenomena made these data difficult to interpret. SPR analysis was used to quantify the strength of interaction using the variation of the surface plasmon resonance band of a functionalized gold chip. In this case, unspecific binding interactions of the nanoparticles and the dextran layer of the gold chip prevented an analysis of the binding event. In conclusion, the research described in this thesis has led towards the development of multivalent peptide-nanoparticle conjugates that can be obtained through a hierarchical self-assembly process. In the first step, an organic monolayer is spontaneously formed on the surface of gold nanoparticles. Subsequently, oligoanionic peptide sequences spontaneously cover the surface through electrostatic interactions with the monolayer. The formation of multivalent peptide surfaces has been demonstrated, but their effective application in multivalent recognition has proven difficult for the lack of an appropriate analytical readout system
Questo progetto si inquadra nell’ambito della nanomedicina. Sfruttando sistemi multivalenti esso si propone di ottenere nanostrutture che siano in grado di riconoscere specificatamente le cellule tumorali epatiche. La multivalenza, molto frequente in natura, si basa sulla capacità di un sistema di instaurare molteplici interazioni deboli, ottenendo un’interazione complessiva superiore rispetto al singolo legame forte. Essa si presta ad essere utilizzata in applicazioni pratiche come il riconoscimento molecolare e la catalisi. Tra i vari sistemi multivalenti artificiali utilizzabili (dendrimeri, micelle, liposomi …), la scelta è ricaduta sulle nanoparticelle d’oro: esse sono piccoli nuclei di atomi d'oro con diametri che tipicamente variano da 1 a 20 nanometri, che possono facilmente essere funzionalizzate tramite l'autoassemblaggio di un monostrato di molecole sulla superficie. Tale monostrato può portare sulla superficie esterna gruppi funzionali di interesse, dotati di capacità catalitica o di riconoscimento. Per legare le molecole del monostrato organico ai nuclei d’oro è stata utilizzata l’interazione oro-zolfo; lo zolfo, infatti, lega l’oro con un legame più forte rispetto alla maggior parte degli altri elementi, come azoto e fosforo. La ricerca descritta in questa tesi si è focalizzata sull’ottenimento di un sistema nanoparticellare “universale”, cioè un sistema che si presta ad una post-funzionalizzazione dopo la sintesi e la purificazione. Tale post-funzionalizzabile deve essere compatibile con la sopravvivenza delle nanoparticelle stesse, permettendo di assemblanrvi un monostrato misto sulla sua superficie. L’ottenimento di un metodo di riconoscimento per le cellule tumorali epatiche sarebbe stato un ottimo campo di prova per verificare in tal senso le capacità del sistema. Innanzitutto è stata verificata la possibilità di ottenere nanoparticelle funzionalizzabili tramite legami covalenti. Per farlo, sono state sintetizzate AuNPs ricoperte con tioli dotati di gruppi aldeidici all’estremità, in grado di reagire in modo quantitativo con ammine tramite la formazione di legami imminici.. I gruppi aldeidici, però, sono facilmente soggetti agli attacchi nucleofili da parte dei tioli a livello del loro carbonile elettrofilo. È stato quindi necessario trovare un sistema di protezione delle aldeidi da rimuovere dopo la sintesi. Le prime aldeidi erano aromatiche, protette in forma di acetale metilico, scelte per la loro capacità di formare immine con alta stabilità termodinamica. Purtroppo non sono stati superati gli ostacoli della deprotezione del gruppo protettore dovuto ad un’eccessivamente stabilità. Questa alta stabilità era in parte dovuta alla presenza di un gruppo nitro in posizione para rispetto all’aldeide, inserito per aumentarne la reattività. Rinunciando a tale gruppo frunzionale, invece, la protezione stessa è risultata troppo labile, incapace di impedire la polimerizzazione dei tioli prima dell’ottenimento delle NPs. Si è quindi passati all’utilizzo di aldeidi alifatiche. Si è provato a sintetizzare nanoparticelle ricoperte con tioli la cui aldeide terminale fosse protetta come dietilacetale, ma si è andati incontro a numerosi problemi di insolubilità. L’utilizzo di dioli ossidabili ad aldeidi non ha dato migliori risultati. Cambiando approccio, si è cercato di sfruttare la sostituzione nucleofila su AuNPs ricoperte con tioli bromurati. Purtroppo, la post-funzionalizzazione con ammine ha avuto come risultati la sola formazione di prodotti insolubili. Infine, la sintesi di nanoparticelle ricoperte con tioli aventi, nell’estremità esterna del monostrato, acidi carbossilici ha dato ulteriori problemi di solubilità, fossero essi in monostrati misti con tioli alchilici oppure no. Visto l’insuccesso della via covalente ci si è successivamente concentrati sull’utilizzo di interazioni elettrostatiche per la funzionalizzazione di AuNPs. Sono state sintetizzate nanoparticelle ricoperte con un monostrato cationico, composto da tioli dotati di gruppi ammonici oppure di gruppi TACN∙Zn(II). Esse sono state usate come AuNPs “universali”, per poi essere coperte con molecole oligoanioniche tramite la formazione di complessi ad alta affinitá. Il metodo scelto per la rilevazione della formazione di questi complessi è stata la fluorescenza, sfruttando il principio secondo il quale una sonda fluorescente vede smorzata la propria emissione in prossimità di una nanoparticelle d'oro. Come sonde sono state scelte cumarina 343 (λeccitazione 450, λemissione 492) e cumarina 2 (λeccitazione 353, λemissione 450), entrambe legate a un oligopeptide anionico formato da tre acidi aspartici (TriAsp-Cum343 e TriAsp-Cum2). Con esse è stata studiata la possibilità di formare un secondo monostrato eterofunzionalizzato, sfruttando il fenomeno FRET. Esso si basa sulla capacità di due fluorofori di comunicare tra loro quando si trovano vicini. Negli esperimenti effettuati, è stata registrata l’emissione della ricevente (cum343) eccitando solamente la donatrice (cum2). Questo è stato fatto in presenza di diversi rapporti percentuali tra le due molecole, alla concentrazione di saturazione del monostrato. Gli esperimenti sono stati condotti anche in presenza di un grande eccesso di ATP, oppure in assenza di nanoparticelle cationiche. Da queste prove è emerso che, quando co-localizzate sullo stesso monostrato, le due sonde fluorescenti sono in grado di generare un segnale FRET. Quando invece sono libere in soluzione, per assenza di nanoparticelle o perché in presenza di un eccesso del competitore ATP, non è stato osservato il medesimo effetto. Il risultato ottenuto ha mostrato che, tramite interazioni elettrostatiche, è possibile assemblare un monostrato eterofunzionalizzato su nanoparticelle “universali” pre-sintetizzate. A questo punto si è proceduto allo sviluppo di un sistema nanoparticellare coperto con il peptide preS1 per il riconoscimento della proteina SCCA. Il peptide preS1 è noto per la capacità di riconoscere specificatamente SCCA, una proteina di membrana sovraespressa negli epatociti cancerosi. Innanzitutto è stato verificato quale fosse la diversa affinità per le AuNPs-TACN∙Zn(II) da parte del peptide tal quale piuttosto che legato all’ancora anionica TriAsp4-. Sono state effettuate prove di fluorescenza diretta (λeccitazione = 280, λemissione = 360, smorzamento del triptofano), fluorescenza indiretta (λeccitazione = 305, λemissione = 370, spiazzamento F-ATP) e di inibizione della catalisi di HPNP operata dai gruppi TACN∙Zn(II) (con liberazione di p-nitro-fenato, assorbanza 400 nm). Tutti e tre gli esperimenti hanno dimostrato la capacità di TriAsp-preS1 di formare un complesso polivalente con le nanoparticelle. È stato osservato che la presenza della sequenza TriAsp ha causato un piccolo miglioramento dell’affinità rispetto al peptide privo dell’ancora anionica. Successivamente è stato verificato il legame del sistema nanoparticella-peptide con la proteina SCCA. Il peptide preS1, quando si trova in forma tetramerica, è in grado di legare con maggiore affinità la proteina SCCA. È stato quindi teorizzato che la presenza della proteina SCCA dovesse stabilizzare il nanosistema composto da nanoparticelle e preS1. Purtroppo non sono state rilevate grandi differenze di stabilità in presenza o in assenza di SCCA, sia per il peptide preS1 che per TriAsp-preS1. Il metodo di analisi basato su studi di spiazzamento è stato quindi accantonato, poiché non garantiva una valida valutazione dell’interazione. Sono stati usati altri metodi per verificare l’interazione del sistema nanoparticella-peptide con la proteina. Tramite analisi DLS è stato visualizzato l’aumento di volume delle nanoparticelle a seguito del legame col peptide prima e con la proteina poi, ma fenomeni di aggregazione hanno reso poco attendibili le misurazioni. Analisi SPR, invece, miravano a quantificare l’intensità dell’interazione tra il sistema nanoparticella-peptide e la proteina SCCA sfruttando la variazione della banda plasmonica superficiale. Questo non è stato possibile a causa di interazioni aspecifiche tra le nanoparticelle e il supporto di destrano del sistema di analisi. Il tempo a disposizione non ha poi permesso di mettere a punto un protocollo di analisi adeguato

Fluorescence plays a key role in a growing number of disciplines, from molecular biology, to analytical chemistry, to optoelectronics. Its high spatial and temporal resolution and excellent signal-to-noise ratio make fluorescence an ideal tool for studying the structure and dynamics of matter and living systems on a molecular and nanometric scale. The most common fluorophores, organic molecules or metal complexes, are plagued by low brightness and photostability. These limitations can be overcome by embedding in ceramic or polymeric nanoparticles. Inclusion of organic fluorophores in silica nanoparticles, in particular, offers several advantages. Silica is an ideal matrix, transparent to visible light and relatively inert to photophysical processes. The embedded fluorophores, protected from the environment, enjoy a high resistance to photobleaching, while their presence in large numbers inside each nanoparticle makes for a much higher brightness. Silica nanoparticles are also extremely versatile. The ease of synthesis allows for the creation of complex structures through core-shell architectures with multiple layers, each doped with a different species. The surface can in turn be functionalized with molecules or macromolecules, which can control the chemical interactions of the nanoparticels with the environment and their colloidal stability in various solvents, or act as multivalent scaffold for the realization of supramolecular systems. These systems find application especially in the fields of drug delivery, intracellular imaging and sensing. Their intense and stable emission allows for the trafficking of nanoparticles in the intracellular environment to be easily tracked by fluorescence microscopy. Dye-doped silica nanoparticles are therefore ideally suited as vehicles for the delivery of therapeutic payloads, acting at the same time as tracers for in vivo imaging, or as fluorescent probes for in vitro imaging applied to fundamental biological problems. This thesis, in particular, deals with the development of vehicles for the delivery of an antitumor drug and with novel synthetic strategies paving the way for the realization of more complex vehicles. Chapter 1 presents the properties, synthesis and some applications of dye-doped silica nanoparticles. Special attention is given to biological applications. Chapter 2 briefly introduces photodynamic therapy (PDT), a non-invasive modality for the treatment of various diseases. Its clinical potential has been known for more than a century, but its use in oncological therapy is relatively recent. The treatment is administered in two stages: administration of a photoactive drug (a photosensitizer) which accumulates in the target tissues, and selective irradiation of the target area with focalized light. The photoactivation of the drug triggers a cascade of events leading to the destruction of the irradiated tissues. Chapter 3 describes the synthesis and characterization of silica-based carriers for covalently-linked photosensitizers. Conjugation with molecules capable of endowing the carrier with the desired functionality is one of the key advantages of using nanoparticels in photodynamic therapy. In order to make this kind of modification easier and more versatile, a modular carrier was conceived, its surface decorated with grafting sites for molecules bearing a complementary functionality. This functionalization strategy was tested by coating the nanoparticles with a poly(ethylene glycol) derivative. Chapter 4 reports the preparation of organically modified silica nanoparticles (ORMOSIL) doped non-covalently with meta-tetra(hydroxyphenyl)chlorin (mTHPC), a second generation photosensitizer. The investigation of the fate of these nanocarriers and of the embedded molecule after exposure to biological fluids and living cells yielded unexpected results. These results suggest that the delivery of drugs embedded in nanosystems may be more complex than it seems. A fluorimetric assay is presented, based on intraparticle energy trasfer processes, which can be used to tell unambiguously whether a physically embedded drug is delivered into living cells still associated with the nanosystem or follows a different path. Chapter 5 presents some peculiar features of mesoporous silica nanoparticles and the problems associated with their synthesis. Some ingenuous applications in the fields of controlled release and sensing are also shown. Chapter 6 describes an alternative carrier, based on mesoporous silica nanoparticles. A novel synthetic route was conceived, where the templating agent is a hydrolytically unstable inorganic phase, and the template removal treatment is a simple solvent exchange with water at room temperature. The proposed synthesis is an adaptation on a nanometric scale of a process that has been known for decades: the production of silica glasses with nanometric pores starting from borosilicates melts, obtained by inducing a phase separation and hydrolytically removing the boron-rich phase. Chapter 7 collects the experimental procedures related to the research reported in this thesis.
Le applicazioni della fluorescenza rivestono un ruolo chiave in un numero crescente di discipline, dalla biologia molecolare, alla chimica analitica, all'optoelettronica. L'elevata risoluzione spaziale e temporale, insieme a un eccellente rapporto segnale-rumore, rendono la fluorescenza un metodo ideale per lo studio della struttura e della dinamica della materia e dei sistemi viventi su scala molecolare e nanometrica. I fluorofori più comunemente usati, molecole organiche o complessi metallici, presentano di frequente problemi di luminosità e fotostabilità. Questi limiti possono essere superati attraverso l’incapsulazione in nanoparticelle ceramiche o polimeriche. L'inclusione di fluorofori organici in nanoparticelle di silice, in particolare, offre numerosi vantaggi. La silice rappresenta una matrice ideale, trasparente alla luce visibile e relativamente inerte rispetto ai processi fotofisici. I fluorofori, protetti dall’ambiente esterno, godono di una elevata resistenza al photobleaching, mentre la loro presenza in numero elevato all'interno di ogni particella conferisce a queste un’elevata luminosità. Le nanoparticelle di silice sono anche sistemi estremamente versatili. La facilità della sintesi consente la realizzazione di strutture complesse attraverso architetture core-shell a strati multipli, ciascuno drogato con una specie diversa. La superficie, a sua volta, può essere funzionalizzata con molecole o macromolecole che ne controllino l'interazione chimica con l'ambiente e la stabilità colloidale in diversi solventi, o fungere da piattaforma multivalente per la realizzazione di sistemi supramolecolari. Questi sistemi trovano applicazione soprattutto nei campi del drug delivery, dell’imaging cellulare e della sensoristica. L’emissione di fluorescenza intensa e stabile consente infatti di seguire il movimento delle nanoparticelle nell'ambiente intracellulare tramite microscopia ottica. Queste si prestano quindi ad essere sfruttate, ad esempio, come vettori per il trasporto di carichi terapeutici che fungano allo stesso tempo da traccianti per l'imaging dei tessuti malati, oppure come sonde fluorescenti per l’imaging cellulare in vitro, applicato allo studio di problemi biologici di base. Questo lavoro di tesi tratta, in particolare, dello sviluppo di vettori per un farmaco antitumorale e di nuove strategie sintetiche che aprono la strada alla realizzazione di vettori più complessi. Nel capitolo 1 sono illustrate le proprietà, i metodi di sintesi e alcune applicazioni delle nanoparticelle di silice drogate con specie fluorescenti. Particolare attenzione è dedicata alle applicazioni biologiche. Nel capitolo 2 viene introdotta la terapia fotodinamica (o PDT, acronimo di photodynamic therapy), un trattamento non invasivo per la cura di una varietà di malattie tumorali e di altra natura. Le sue potenzialità cliniche sono note da più di un secolo, ma l’uso in terapia oncologica è relativamente recente. Il trattamento si articola in due fasi: la somministrazione di un farmaco fotoattivo (un fotosensibilizzatore) che si accumula nei tessuti malati, e l’irraggiamento selettivo di questi con luce focalizzata. La fotoattivazione del farmaco innesca una cascata di eventi che conduce alla distruzione dei tessuti irraggiati. Nel capitolo 3 vengono descritti la sintesi e la caratterizzazione di vettori per fotosensibilizzatori basati su nanoparticelle di silice nelle quali il farmaco è legato covalentemente alla matrice. La coniugazione con molecole che conferiscano al vettore le funzionalità desiderate è uno dei vantaggi chiave dell’uso di nanoparticelle in terapia fotodinamica. Per facilitare queste modifiche e renderle il più possibile versatili, è stato progettato un vettore modulare la cui superficie fosse decorata con siti di ancoraggio per molecole recanti una funzionalità complementare. Questa strategia di funzionalizzazione è stata messa alla prova funzionalizzando le nanoparticelle con un derivato del polietilenglicole. Nel capitolo 4 viene descritta la preparazione di nanoparticelle di silice modificate con gruppi organici (ORMOSIL) drogate con meta-tetra(idrossifenil)clorina (mTHPC), un fotosensibilizzatore di seconda generazione. Lo studio del destino di questi nanovettori e della molecola incapsulata al loro interno in seguito all’esposizione a fluidi biologici e cellule viventi ha fornito risultati inattesi. I risultati ottenuti suggeriscono che il trasporto di farmaci incapsulati in nanosistemi possa essere più complesso di come appare. Viene presentato un saggio fluorimetrico basato sul trasferimento energetico che può essere utile per determinare senza ambiguità se un farmaco intrappolato fisicamente venga trasportato nelle cellule ancora associato al nanosistema o segua una strada diversa. Nel capitolo 5 vengono illustrate le caratteristiche salienti delle nanoparticelle di silice mesoporose e i problemi legati alla loro sintesi. Vengono inoltre presentate alcune applicazioni nel campo del drug delivery e della sensoristica. Nel capitolo 6 viene presentato un vettore alternativo, basato su nanoparticelle di silice mesoporose. È stata concepita una strategia sintetica innovativa, nella quale l’agente templante è una fase inorganica idroliticamente instabile, e il trattamento di rimozione del templante consiste nel semplice scambio di solvente con acqua a temperatura ambiente. La sintesi proposta è la trasposizione su scala nanometrica di un processo noto da decenni: la produzione di vetri di silice con pori di dimensioni nanometriche a partire da vetri borosilicati, ottenuta inducendo una separazione di fase e rimuovendo selettivamente la fase ricca di boro per idrolisi. Nel capitolo 7 sono raccolte le procedure sperimentali relative all’attività di ricerca descritta in questo lavoro di tesi.

Le 20ème siècle a vu le recul des maladies infectieuses grâce aux antibiotiques. Cependant leur importante utilisation a rendu certaines bactéries, comme Staphylococcus aureus ou Pseudomonas aeruginosa (multi)résistantes. Un des moyens de lutte est de réduire la consommation d’antibiotiques ou de cibler ceux qui seront actif sur une souche identifiée. Nous souhaitons développer des surfaces et des dispositifs sensibles pour la détection précoce, rapide de bactéries pathogènes dans des fluides. Cela permettra de limiter la contamination et donc l’usage de médicaments. Ce projet regroupe 3 partenaires qui travaillent en synergie en mettant à profit leur expertise en physico-chimie, chimie de synthèse et microbiologie. Des nano-objets fluorescents, biocompatibles, et sensibles à la croissance bactérienne seront immobilisés sur des surfaces de verre. Ils seront rendus sélectifs de bactéries pathogènes par des traitements post-synthétiques. Il s’agit in fine de mettre au point un dispositif de détection miniaturisé et de tester la résistance aux antibiotiques des pathogènes détectés
Infectious diseases have recessed during the 20th century thanks to antibiotics. However, some bacterial strains like Staphylococcus aureus or Pseudomonas aeruginosa have become (multi)resistant to antibiotic treatments because of overuse. One way to combat this is to reduce consumption of drugs or to better target those that will eliminate a given strain. We wish to develop sensitive surfaces and devices for the early and rapid detection of pathogenic bacteria in fluids. They will help limit contaminations and the use of drugs. The project gathers 3 partners working in synergy because they combine expertise in physical-chemistry, synthetic chemistry and microbiology. Fluorescent nanoobjects that are biocompatible and sensitive to bacterial growth will be immobilized on glass surfaces. They will be selective for pathogenic bacteria by post-synthetic modifications. The final goal is to build miniaturized sensitive devices that can detect pathogens and further test their resistance to antibiotics

Depuis deux décennies, la modernisation des techniques d’imagerie de fluorescence a permis decomprendre et d’étudier de nombreux processus biologiques. Ce formidable essor a néanmoins étéralenti par les faibles performances des sondes fluorescentes disponibles pour ce type d’imagerie. Cemanque d’objets luminescents a motivé des recherches intenses dans le but d’optimiser les sondesmoléculaires fluorescentes déjà existantes afin de leur permettre d’être plus fluorescentes, mais aussiplus stables. En parallèle à ces optimisations, l’avancée des nanotechnologies à permis de développerde nouveaux outils pour l’imagerie biologique. Du fait de leurs propriétés optiques, de leur excellentephotostabilité, ainsi que la possibilité de fonctionnaliser leur surface, les nanoparticules semiconductrices(QDs) ont connu un succès phénoménal dans les domaines de l’imagerie. Au fur et àmesure de leur utilisation, de nombreuses questions partiellement élucidées sur la toxicité des QDs ontémergé. Dans le but de proposer une alternative aux QDs, de nombreux groupes de recherche onttravaillé sur la préparation de nanoparticules (ONPs) à base de molécules organiques, ce qui a mis finà l’hégémonie des QDs en imagerie biologique. Parmi ces ONPs, les nanoparticules fluorescentes àbase de molécules organiques (FONs) se sont imposées comme des candidats sérieux pour sonder levivant. Dans cette optique, nous développons de nouveaux chromophores qui, par un designmoléculaire adapté, peuvent former des FONs avec des brillances à un et deux photons comparables àcelle des QDs tout en étant biocompatibles
Since decades, bioimaging technics increased a lot, thus leading to a better understanding of biologicalprocesses. However, the fluorescence imaging was limited due to the low photostability and brightnessof available organic dyes. In order to improve the lack of photostability and brightness of thecommercially available dyes, lots of effort was done by organic chemists. In the mean time with theincoming of nanotechnologies, new tools such as the semi-conductors QDs raised as promisingluminescent probes for bioimaging. Due to their intrinsic properties, QDs are bright and photostablenano-objects, furthermore, the labeling of the surface of the QDs is of interest for targeting (such asthe detection of cancer cells). Despite their popularity, with time, issues about the clearance and thetoxicity of the QDs have been adressed and are still not fully solved. So the development of safer andbiocompatible tools for bioimaging emerged as evidence for scientists. In that context, the use ofnanoparticles made of organic materials gain in interest and particularly the fluorescent organicnanoparticles (FONs). We aimed to propose biocompatible and safer alternative to QDs, so wedeveloped new nanotools, based on the self aggregation of propeller-shaped fluorophores as FONs.Because of the specific design of the chromophores, these FONs have large one and two-photonbrightness combine with good photostability. These features made these FONs promising tools foreither one-photon and two-photon fluorescence imaging in cells and in vivo

L’objectif de ces travaux de thèse repose sur la conception de nanoparticules pour l’upconversion (UCNPs) visant à réaliser de l’imagerie médicale par fluorescence. L’intérêt de ces nano-objets s’appuie sur l’excitation et l’émission conjointe dans le domaine du proche infrarouge, permettant de travailler dans la fenêtre thérapeutique des tissus. Une première partie présente l’élaboration des nanoparticules sphériques de NaYF 4 , dont le taux de dopage a permis de mettre en avant les conversions possibles grâce aux phénomènes optiques d’upconversion (IR → NIR, IR → visible et IR → UV). De plus, l’étude d’hydrophilisation, primordiale pour des applications médicales, a montré un fort intérêt pour l’enrobage par échange de ligands et plus particulièrement par des molécules dendritiques. Dès lors que les nano-objets ont été validés et stabilisés dans un milieu aqueux, ces derniers ont été testés in vitro et ont permis de montrer une cytotoxicité faible. La dernière partie a permis d’adapter l’équipement IVIS SPECTRUM® à nos conditions optimales d’utilisation des UCNPs. Cette installation a validé la preuve de concept de nos UCNPs in vivo, permettant ainsi d’être imagées chez le petit animal. Ce dispositif, encore unique en Europe ouvre ainsi la voie pour des études plus approfondies de l’utilisation des UCNPs pour la détection de tissus spécifiques, tels que les tissus tumoraux
The aim of this PhD work deals with the design of upconverting nanoparticles (UCNPs) to perform medical imaging by fluorescence. The interest of these nano-objects is based on both excitation andemission in the near infrared range, allowing the work in the therapeutic window of tissue. The first part presents the development of spherical nanoparticles of NaYF 4 , which the doping concentration has highlighted the transitions permits through upconversion phenomena (IR → NIR, IR → visible and IR → UV). In addition, the hydrophilization part, necessary for medical application, has shown a strong interest in the coating by ligand exchange and particularly by dendritic molecules. Since the nanoparticles have been validated and stabilized in aqueous medium, they were tested in vitro and have shown low cytotoxicity. The last part was to adapt the IVIS SPECTRUM® equipment to our optimal conditions of use of UCNPs. This installation has validated the proof of concept of our UCNPs in vivo, which is to image in small animals. This device, also unique in Europe, opens the way for further use of UCNPs studies for the detection of specific tissues such as tumor tissues

This thesis mainly consists of two parts. The first part is concentrated on synthesizing amine/carboxylic-functionalized carbon nanoparticles (CNP) and investigating its fundamental properties in the aid of capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). In this part, CNP is synthesized from citric acid (CA) and 1,2-ethylenediamine (EDA) by a microwave-assisted pyrolysis method. The resultant CNP is characterized by absorption and photoluminescence (PL) spectroscopy, transmission electron microscopy (TEM) and infrared spectroscopy (IR) to determine its overall optical properties, morphology and composition. Followed by this, the CNP product is separated and analyzed by CZE coupled with UV absorption and laser-induced fluorescence (LIF) detections. Under optimal pH and concentration of run buffer, the effect of reaction time and mole ratio of amine (NH2) to carboxylic acid (COOH) moieties of the precursors on the CNP species present in CNP products are studied. Our results show that the synthesis of CNP could be improved by lengthening the microwave irradiation time and optimizing the initial mole ratio of NH2/COOH in the precursors. Negatively charged CNP are obtained only when the amount of CA exceeds that of EDA, i.e., the mole ratio of NH2/COOH is 0.250.80. By contrast, when the quantity (in mole) of NH2 in EDA is equal to or larger than that of COOH in CA, only positively charged and neutral CNP species are formed, inferring that the CNP species are predominantly covered by the surface-attached ammonium and amido moieties. This work highlights the merit of CZE to identify the composition of an as-prepared CNP product which is pretty much dependent on the mole ratio of NH2/COOH. In addition, we carry out reversed-phase high-performance liquid chromatography coupled with fluorescence detection (RP-HPLC-FD) methodology to study the properties of each individual CNP species. Under optimal mobile phase and elution gradient conditions, the effect of mole ratio of NH2/COOH in the initial reagents on CNP product is studied. At NH2/COOH = 0.67, the strongest fluorescence CNP sample is obtained. The separated CNP fractions are collected and further characterized by UV-visible absorption and PL spectroscopy, CE, TEM, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The absorption and PL emission bands (λem) of the fractions are bathochromatically shifted with the elution order of CNP on RP-HPLC. The TEM images prove that CNP are eluted from the smallest to the largest. The MALDI-TOF MS data show that CNP undergo fragmentations, closely relating to their surface-attached carboxylic acid and amide/amine moieties. This work highlights the merit of RP-HPLC coupled with fluorescence detection, TEM and MS for isolation and characterization of individual CNP species present in a CNP sample. By application of CE and HPLC separation for CNP product, better understandings of the hidden fundamental properties of CNP are achieved. The two separation techniques are well complementary to each. On one hand, CE is able to separate both positively and negatively charged CNP species which cannot be retained and separated by HPLC column, facilitating the investigation of different charge states of CNP species present in a CNP product. On the other hand, the preparative property of HPLC allows for multi-collection of the separated CNP fractions which is difficult in the case of CE analysis owning to its low sample injection volume. By using HPLC separation, the individual CNP fractions can be collected for more precious investigation of their unique photophysical and chemical properties by absorption and PL spectroscopy, TEM and MALDI-TOF MS. The second part is focused on preparing CNP from naturally available bioresources and investigating the effect of doped heteroatoms on nitrogen (N) and sulfur (S)-doped carbon nanoparticles (N,S-CNP) based on our proposed modern RP-HPLC-FD methodology which has been proved to be useful in separating and analyzing CNP in the first part of this work. In this part, ultrasmall N,S-CNP is prepared by microwave-assisted pyrolysis of precursors of rice as carbon source and N-acetyl-L-cysteine (NAC) as N and S dopants. The obtained N,S-CNP are fully characterized by elemental analysis, FTIR, x-ray photoelectron spectroscopy, TEM, UV-vis absorption and PL spectroscopy. Meanwhile, undoped CNP (derived from rice only) is also synthesized and characterized. The chemical compositions, sizes and spectral properties of undoped CNP (derived from rice only) and N,S-CNP are demonstrated to be different from each other. With the assistance of RP-HPLC-FD, the effect of different mass ratios of NAC to rice (NAC/rice) on N,S-CNP is investigated. When the NAC/rice increases from 0.20 to 0.80, the signals of the later eluted peaks increase progressively, indicating that higher NAC/rice benefits the generation of CNP with stronger fluorescence emissions. The HPLC-separated N,S-CNP fractions are collected and further characterized by MALDI-TOF MS, UV-vis absorption and PL spectroscopy, showing that the structural changes induced by doping with heteroatoms N and S plays a key role in regulating the PL properties of the N,S-CNP

Boîtes quantiques (QDs) et nanoparticules fluorescentes de silice (NPs) ont influencé le domaine de la bioimagerie de par leur forte luminosité et photostabilité. Par rapport aux QDs, les NPs organiques peuvent s’avérer être encore plus brillantes et entièrement biodégradables, avec une bonne biocompatibilité et sans contenir aucun élément toxique. Nous avons développé quatre types de ces NPs : en premier, des nano-gouttelettes lipidiques chargées de colorants lipophiles (flavone et Nil Rouge) pour l'imagerie in vivo chez le poisson zèbre ; en second, l’association ionique entre rhodamine B alkylée et tétraphénylborate fluoré (TPB) donne des NPs de 11-20 nm avec un rendement quantique de ~60% ; une troisième type de NPs consiste en des micelles de 7 nm obtenus par co-assemblage de cyanine amphiphiles et contre-ions TPB ; enfin, la polymérisation de micelles de calix[4]arène par agents de réticulation bi-fonctionnels à base de cyanine donne des NPs de 7 nm présentant un comportement fluorogène et une bonne stabilité en milieu intracellulaire. Ces NPs plus brillantes et de taille inférieure aux QDs apparaissent comme des outils prometteurs en bioimagerie
Quantum dots (QDs) and fluorescent silica nanoparticles (NPs) have impacted the domain of bioimaging by their high brightness and robust photostability. In comparison to QDs, organic NPs can be even brighter and fully biodegradable, as well biocompatible and not containing toxic elements inside. Herein, we developed four types of these NPs. At first, lipid nano-droplets loaded with lipophilic flavone and Nile Red dyes for in vivo imaging in zebrafish; second, ion-association of alkyl rhodamine B with fluorinated tetraphenylborate (TPB) counterions result in 11-20 nm NPs with fluorescence quantum yield up to 60%; third, 7 nm micellar NPs obtained by co-assembly of cyanine amphiphiles with TPB counterions; finally, polymerization of calix[4]arene micelles using bi-functional cyanine crosslinkers giving 7 nm NPs, that show fluorogenic behavior and high intracellular stability. These NPs, being of smaller size and brighter than QDs, have emerged as promising tools for bioimaging

Les nanoparticules polymériques fluorescentes apparaissent comme des outils importants pour l'imagerie en temps réel des processus biologiques au niveau moléculaire et cellulaire. L’objectif de mon projet de doctorat a été d’optimiser les nanoparticules polymériques fluorescentes pour l’imagerie biologique. Premièrement, nous avons pu, en faisant varier la chimie des polymères, obtenir un très bon contrôle de leur taille. Ceci a permis de mettre en évidence l’importance de la taille des NPs pour des applications intracellulaires avec une taille maximale de 23 nm pour une distribution dans tout le cytosol. Deuxièmement, nous avons pu montrer que la simple adsorption d’un amphiphile PEGylé de type Pluronic permet la stabilisation des nanoparticules dans des milieux biologiques. Le nombre de molécules incorporées et leur stabilité ont été étudiés en combinant des techniques de FRET et de FCS. Les meilleures formulations résultent en une stabilité des nanoparticules in vivo, ce qui a permis leur imagerie en tant que particules individuelles dans les vaisseaux sanguins du cerveau de souris. Troisièmement, le transfert d’énergie entre différents fluorophores encapsulés dans les NPs a été étudié et optimisé
Fluorescent polymeric nanoparticles appear as important tools for real-time imaging of biological processes at the molecular and cellular level. The objective of my PhD project was to optimize fluorescent polymeric nanoparticles for biological imaging. First, by varying the chemistry of the polymers, we have been able to obtain a very good control of their size. This made it possible to highlight the importance of NPs size for intracellular applications with a maximum size of 23 nm for optimal distribution throughout the cytosol. Secondly, we have shown that simple adsorption of a PEGylated amphiphiles pluronic family allows the stabilization of nanoparticles in biological media. The number of incorporated molecules and their stability has been studied by combining FRET and FCS techniques. The best formulations result in nanoparticle stability in vivo, which allowed their imaging as individual particles in the blood vessels of the mouse brain. Third, energy transfer among different fluorophores encapsulated in NPs has been studied and optimized

Background: Quantum dots (QDs) have emerged as one of the most exciting fluorescent nanoparticles with a potential for diagnostic and therapeutic application in the field of nanomedicine. The aim of this study was to synthesize water soluble QDs; bio-conjugating these QDs with RGD peptides prior to linking the QD-conjugated peptide to cancer cells with the aim to study cytotoxicity and assess its feasibility for in vivo studies. Methods: Water soluble Cadmium Telluride (CdTe) QDs were synthesized by the reaction of cadmium chloride with sodium tellurite in the presence of buffer solution of Mercaptosuccinic acid (MSA) as a capping ligand. Water soluble red emitting QDs thus obtained were characterized using spectrophotometric analysis. These QDs revealed a wide absorption spectrum with an excitonic absorption peak of 380nm and a narrow symmetrical emission spectrum of 630nm. The size and pattern of these QDs were studied using Transmission Electron Microscopy (TEM). These nanocrystals revealed their configuration in the form of isolated crystals or clusters measuring from 5-10nm in diameter. X-ray microanalysis combined with TEM permitted analysis of the elemental configuration of these QDs. These CdTe QDs were subsequently bound to HT 29 colon cancer cells to study the interaction of QDs in vitro. As colon cancer cells over-express integrins, QDs were conjugated with RGD (Lysine) and RGD (Cysteine) peptides for the purpose of active binding with HT29 colon cancer cells. The conjugated QDs were applied to colorectal cancer cells to assess their affinity to cellular adhesion molecules. The toxicity of naked and conjugated QDs was also assessed by analyzing cell survival and cell death after exposure to C2C12 mouse skeletal muscle cells. In vivo experiment using Sprague 5 Dawley (SD) rat established feasibility of biodistribution studies with a small dose of 10μg/ml. Results: These water soluble fluorescent CdTe nanocrystals were synthesized using relatively stable precursors. It was possible to demonstrate binding of these red emitting QDs to the HT29 colon cancer cells in vitro. Significant and stable binding was noted after QDs were conjugated with RGD peptides. Toxicity assay evaluation studies suggested that both nonconjugated and conjugated QDs were nontoxic to C2C12 mouse skeletal muscle cells at a concentration of 50 μg /ml indicating that they are less toxic to normal cells, and are safe to be applied to in vivo models. Further in vivo experimentation in SD rats established feasibility for imaging sentinel lymph nodes following interdigital web space injection of QDs. Conclusions: RGD-conjugated QDs can selectively target HT29 colorectal cancer cells with low toxicity to normal muscle cells offering a potential novel detection strategy for colorectal cancer. This property can be explored for early diagnostic and therapeutic purpose by selectively targeting cancer cells. Further studies are required in an in vivo model to analyze systemic biodistribution and toxicity studies.

Quantum dots (QDs) are semiconductor nanocrystals (<100 nm), which are emerging as a novel class of multifunctional fluorescent probes for many potential biological and medical applications. In comparison to conventional organic fluorescent probes (organic dyes), QDs have substantial advantages, such as, bright fluorescence, narrow emission, broadband excitation, photostability and extended half-life. Imaging with diagnostic assessment, plays an important part in clinical settings for determining disease (cancer) progression and therapy. However, current imaging techniques have certain limitations, and they include insufficient sensitivity to detect low numbers of cancer cells at primary or metastatic sites and appropriate probes to detect specific cancer cell surface markers. To address these limitations, studies were conducted (1) to develop an aqueous synthesis of a series of near infrared (NIR) QDs, incorporating cadmium (Cd), tellurium (Te), cobalt (Co) and mercury (Hg) in its core (2) to minimize its potential toxicities, by developing coating strategies with a novel coating nanomaterial, mercaptopolyhedral oligomeric silsequioxane (MPOSS) while maintaining strong emission, stability and biocompatibility, (3) to apply conjugated NIR QDs as probes in targeting and detecting immunogenic apoptosis in cancer cells in vitro and mapping biodistribution in vivo. The inclusion of mercury (Hg (ClO4)2) and cobalt (Co) to the QD core resulted in NIR emission at 800 nm with paramagnetic properties. Characterization by transmission electron microscopy (TEM) confirmed size of the QDs. Detoxification of QDs was demonstrated, by toxicity studies, using two different vital stains, Alamar Blue and Neutral Red on human umbilical vein endothelial cells (HUVECs), human breast cancer cells (MCF-7), colorectal cancer cells (SW620) and prostate cancer cells (PC3). A short synthetic peptide to calreticulin (CRT) was chemically synthesised and antibodies generated against the peptide (Anti-CRT) with specificity to the native CRT protein (a cancer cell immunogenic apoptosis marker). The presence of functional groups on the coatings of QDs provided an additional advantage for conjugation to Anti-CRT for targeting, and carbon nanotubes (CNT) for thermal strategy. QDs conjugated to Anti-CRT showed specificity to cancer cells in vitro undergoing apoptosis when exposed to the following: 1) Doxorubicin (an anticancer drug), 2) cadmium and 3) QD-CNT (photothermal effect). Characterization by Fourier Transform Infrared Spectrophotometry (FTIR) confirmed conjugation of QDs to Anti-CRT. Confocal microscopy images further confirmed targeted and non-targeted QDs in vitro, and NIR sensitive camera for in vivo imaging. These studies and findings demonstrate the feasibility of applying these engineered nanocrystals for clinical diagnostics, drug delivery and therapy.

The human body and many living organisms are comprised of very complex biological system with distinct metabolism. In order to understand life activities, we need to monitor the individual chemical interactions happening in vivo. Bioimaging with naked organic dye molecules always suffers from drawbacks such as photobleaching and biocompatibility issues. Silica matrix protects the fluorophores from external environment and provides hydrophilic shell, which improves the photostability and biocompatibility of dye molecules. A nanocarrier, which is highly compatible with the target metabolic system, may be beneficial for therapeutic and diagnostic applications in living organisms. Mesoporous silica nanoparticles (MSNs) are highly biocompatible and safe for biological applications and may provide the solution. Therefore, this project focused on the synthesis of dye-doped mesoporous silica nanoparticles, coupling them with various bioactive carbohydrate molecules, and investigation of these nanoparticles for their potential biological applications in microorganisms. Rhodamine B, fluorescein, and methylene blue dyes were employed for doping into amine modified mesoporous silica matrix through covalent and non-covalent approaches. The results revealed that all dyes were successfully doped into the silica matrix and showed bright fluorescence. In the next stage, methylene blue encapsulated amine grafted mesoporous silica nanoparticles (MB AMSNs) were utilized for coupling with carbohydrates- glucose, maltose, ribose, and raffinose by employing N, N'-carbonyldiimidazole as a coupling agent. The chemical and physical characterization showed the successful conjugation of carbohydrates onto amine-modified silica surface. Finally, glucose conjugated methylene blue doped mesoporous silica nanoparticles (Glu-MB MSN) were used in bioimaging and toxicity assessments. The as-synthesized nanoparticles were investigated in E.Coli and B.Subtilis bacterial samples. The characteristic results revealed bright fluorescence in bacteria like formations via confocal microscopy. Therefore, Glu-MB MSN may be useful for bioimaging purposes. SEM images showed bacterial aggregation after treatment with nanoparticles. This interaction is relatively higher in the case of B.Subtilis. Moreover, the bacterial cell structure appeared unaltered after incubation with the nanoparticles. This suggested that the nanoparticles were not toxic to these specific bacteria. However, more studies need to be performed to confirm these results.

La thérapie génique est probablement une des approches la plus ambitieuse de l'histoire de l'humanité pour éliminer des maladies résistantes à tout autre traitement. Cependant, c'est une approche qui doit encore être développée afin d'obtenir un meilleur contrôle du processus de délivrance des médicaments et aussi de réduire les coûts. À cette fin, ce projet de thèse est axé sur l’optimisation et le contrôle de la délivrance d’oligonucléotides basée sur l'utilisation de nanobâtonnets d'or (Gold NanoRods, GNRs). De telles nanoparticules (40 nm de long et 10 nm de diamètre) sont internalisées par les cellules et grâce à leurs propriétés physiques extraordinaires permettent de délivrer les médicaments dans le cytoplasme de manière contrôlée. En effet, leur absorption très élevée dans le proche infrarouge du spectre électromagnétique permet de convertir l’énergie lumineuse en chaleur à l’intérieur et autour des nanobâtonnets, sans affecter la cellule. L’avantage d’une absorption dans l’infrarouge est qu’à cette longueur d’onde la lumière pénètre profondément dans les tissus humains (3 cm). Le contrôle de la température autour des nanoparticules permet la libération d'oligonucléotides par simple dénaturation du duplex à un instant donné.L’obtention de nanoparticules pouvant être considérées comme un « cargo » implique de remplir les conditions suivantes : stabilité de la forme colloïdale en milieu complexe, conservation des propriétés physiques et chimiques une fois administrées et possibilité d’immobiliser et de libérer le médicament de manière contrôlée.La première étape de mon projet a consisté à établir un protocole de synthèse de nanobâtonnets d’or afin d’obtenir une solution colloïdale mono-disperse et dont la bande d’absorption de plasmon longitudinal soit dans le proche infrarouge. L'étape suivante était d’optimiser un protocole de fonctionnalisation de la surface des GNRs. Le défi ici est associé à l'agrégation des GNRs lorsque le surfactant (CTAB) nécessaire au maintien des GNRs en solution est remplacé par des biomolécules (oligonucléotides). Cependant, après une étude systématique et détaillée, la déstabilisation de la couche protectrice de surfactant sur la surface métallique et l’ajout d’oligonucléotides ayant une fonction thiol à une des deux extrémités dans un rapport approprié ont permis une bio-fonctionnalisation efficace des nanobâtonnets. En conséquence, les nanoparticules fonctionnalisées, après redispersion dans la solution, ont les propriétés physico-chimiques nécessaires. En outre, l’immobilisation des oligonucléotides sur la surface des nanoparticules est spécifique (via la liaison thiol-Au) et permet leur transfert dans des solutions tamponnées ou dans des milieux complexes sans affecter leur stabilité. Après hybridation entre le simple brin immobilisé sur la surface des nanobâtonnets et le brin complémentaire, j’ai démontré que les oligonucléotides étaient stables et que le nombre de doubles brins qui se forment par hybridation peut être contrôlé. L’analyse des propriétés des nanomatériaux a constitué la seconde étape clé de mon travail, car elle revêt une importance cruciale pour la délivrance contrôlée de médicaments. J'ai décidé d'appliquer des méthodes uniquement optiques couvrant l'absorption des nanobâtonnets et l'analyse de la fluorescence des oligonucléotides marqués et des images TEM.Au cours du projet, il a donc été possible d’établir une nouvelle approche de fonctionnalisation et de créer un protocole de caractérisation efficace, axé sur les oligonucléotides
Gene therapy is probably one of the most ambitious approaches in human history that aims to eliminate diseases, often those completely resistant to other treatments. However, this approach requires further development in order to obtain better control over the process of drug delivery and reduce costs. For this purpose, this project has focused on delivery of oligonucleotides using gold nanorods (GNRs). Such nanoparticles, (40 mm in length and 10 nm in diameter) can be internalized by cells and their extraordinary physical properties allow the delivery of drugs to the cytoplasm of cells in a controlled manner. Indeed, their strong absorption in the near-infrared part of the electromagnetic spectrum allows conversion of the energy of light into heat around the nanorods without affecting the cells. The advantage of absorption in the infrared is that at this wavelength the light can penetrate human tissues (3 cm). Control of the temperature around the nanoparticles allows the release of oligonucleotides by simple denaturation of the duplex at a given time.Obtaining nanoparticles that can be considered as a "cargo ship" implies fulfilling the following conditions: stability of the colloidal form in a complex medium, preservation of the physical and chemical properties once administered and the ability to immobilize and release the drug in a controlled manner.The first step of my project was to establish a nanorods synthesis protocol in order to obtain a monodisperse colloidal solution whose longitudinal absorption band is in the near infrared. The next step was to optimize the functionalisation protocol of the surface of the GNRs. The challenge here is associated with the aggregation of GNRs when the surfactant (CTAB) needed to maintain the GNRs in solution is replaced by biomolecules (oligonucleotides). However, after a systematic and detailed study, the destabilisation of the surfactant protective layer on the metal surface and the addition of oligonucleotides having a thiol function at one of the two extremities in a suitable ratio allowed an efficient bio-functionalisation of the nanoparticles. As a consequence, the functionalised nanoparticles, after redispersion in solution, possess the necessary physicochemical properties. In addition, the immobilisation of oligonucleotides on the surface of the nanoparticles is specific (via the thiol-Au bond) and allows their transfer into buffered solutions or in complex media without affecting their stability. After hybridisation between the single strand immobilized on the surface of the nanorods and the complementary strand, I demonstrated that the oligonucleotides were stable and that the number of double strands that are formed by hybridization can be controlled. The analysis of the properties of nanomaterials was the next important part of the work, as it is of crucial importance for the controlled delivery of drugs. I decided to apply only optical methods covering nanorods absorption and fluorescence analysis of labeled oligonucleotides and TEM images.In summary, during the project it was possible to establish a new functionalization approach and create a protocol for efficient characterization, focused on oligonucleotides. We expect that these observations will aid further research in the field of gene delivery based on gold nanoparticles

L'obtention de matériaux structurés sur plusieurs échelles de longueurs permet d'obtenir des propriétés physiques innovantes par rapport aux propriétés individuelles des constituants élémentaires. Dans cette thèse nous nous sommes intéressé à l'obtention de matériaux possédant des propriétés optiques nouvelles. Ainsi des bâtonnets semi-conducteurs anisotropes de type cœur-coquille ont été synthétisés. Leur forme permet de les assembler dans des phases de type cristal-liquides. Après fonctionnalisation de la surface des bâtonnets par des molécules hydrophiles possédant une charge négative, une méthode originale de séchage entre un substrat et un moule microstructuré a permis l'obtention de structures macroscopiques organisées sur plusieurs échelles. D'autres méthodes d'assemblages ont également été utilisées comme des membranes organiques forçant la structuration selon la phase cristalline désirée, mais également l'hybridation sélective de brins d'ADN complémentaire entre les bâtonnets et des nanoparticules métalliques. Les structures de ces matériaux ont alors été analysées par SAXS et microscopie électronique et les propriétés optiques par spectroscopie de fluorescence. Plusieurs types ont montré une exaltation de la fluorescence
Obtaining structured materials on multiple scales provides innovative physical properties, which differ from the individual properties of the constituent building blocks. In this thesis we focused on obtaining materials with novel optical properties. Thus semiconductor anisotropic core/shell rods have been synthesized. Their shape allows them to assemble into liquid-crystalline phases. After functionalization of the surface by hydrophilic molecules having a negative charge, an innovative method of drying the rods suspension between a substrate and a microstructured mold was applied and macroscopic structures organized on several scales were obtained. Other assembly methods have also been studied as the use of an organic mold forcing the structuration according to a particular crystalline phase, but also the selective hybridization of complementary DNA strands between rods and metal nanoparticles. The optical properties of these materials were then analyzed by fluorescence spectroscopy and several cases have shown an enhancement of the fluorescence intensity

La modification des propriétés du graphène, notamment l’ouverture d’une bande interdite par la nanostructuration, est un véritable enjeu pour la physique et pour les applications du graphène. La nanostructuration peut se faire suivant l’approche « top-down » ou « bottom-up ». Au cours de cette thèse nous nous sommes intéressés à la seconde approche. L’approche « bottom-up » permet de contrôler à l’atome près la structure des matériaux. L’objectif de cette thèse est de fabriquer par synthèse chimique des boites quantiques de graphène et des motifs graphéniques contenant un réseau périodique de trous (nanomesh) et d’en étudier les propriétés physiques. Dans une première partie, une « famille » de nanoparticules de graphène a été préparée par synthèse organique via des réactions de Diels-Alder et de Scholl et les propriétés optiques ont été étudiées sur des solutions et à l’échelle de la molécule unique. Dans une deuxième partie, un nouveau type de structures graphéniques intermédiaires entre les boites quantiques et les nanorubans, des nano-bâtonnets de graphène (nanorods) ont été synthétisés. Enfin, plusieurs précurseurs ont été synthétisés pour la réalisation de nanomeshs de graphène. Ces précurseurs permettront d’obtenir, en utilisant le dépôt chimique en phase vapeur dans la chambre d’un microscope à effet tunnel, des nanomesh de graphène présentant des structures différentes
The manipulation of the electronic properties of graphene, and in particular the bandgap opening by nano-patterning, is a crucial issue for both physics and applications. The nanostructuration can be done either through the top-down approach or the bottom-up approach. This bottom-up approach allows controlling at the atomic level the structure of the materials. The aim of this thesis is to prepare graphene quantum dots and graphene nanomeshes (regular arrays of holes in a graphene sheet) by chemical synthesis, and to study their physical properties. In the first part, a “family” of graphene quantum dots was prepared with organic chemistry via Diels-Alder and Scholl reactions and the optical properties were studied both in solution and at the single molecule scale. In the second part, a new type of graphenic structures intermediate between quantum dots and nanoribbons were synthesized and we named them “graphene nanorods”. These objects are one dimensional but have a controlled length compared to nanoribbons prepared via polymerization. Finally, various precursors were synthesized to create graphene nanomeshes. These precursors will allow the formation, using chemical vapor deposition in a scanning tunneling microscope chamber, of nanomeshes exhibiting different structures and morphology

Une ingénierie moléculaire a été menée. On a ainsi obtenu des fluorophores émettant, à l'état solide, dans le rouge et le proche infrarouge avec des rendements quantiques performants. Nous nous sommes particulièrement intéressés à des petites molécules de type push-pull, facile à synthétiser, permettant ainsi d'obtenir un grand nombre de molécules indispensable pour cette ingénierie moléculaire. Il a notamment été constaté que certaines règles établies en solution sont également valables à l'état solide. Ensuite, les chromophores répondant au cahier des charges fixé par la méthode de séchage par spray ont été sélectionnés et encapsulés, d'une part, en couche mince sol-gel afin d'observer leur comportement en milieu confinée. D'autre part, pour ceux ayant un bon comportement en matrice sol-gel (contrôlé par spectroscopie de fluorescence à un photon), ils ont été insérés dans une coquille du même type. Les conditions d'élaboration pour chaque chromophore ont été optimisées en se basant, essentiellement, sur la technique de microscopie électronique à balayage. Nous avons, en particulier, pu démontrer que l'étape d'encapsulation en couche mince sol-gel, était une très bonne technique, rapide et facile à mettre en œuvre, pour s'assurer que les composés ne souffraient ni de polymorphisme ni de protonation dans une matrice sol-gel avant la synthèse de nanoparticules cœur-coquille. Enfin, une étape de fonctionnalisation et d'ajout d'agent de ciblage permettra d'effectuer des premiers tests in vivo des nanoparticules comme agents imageant
A molecular engineering was led. We obtained fluorophores emitting, in the solid state, in the red and the near infrared with efficient quantum yields. We were particularly interested in small push-pull molecules, easy to synthesize, to obtain a large number of molecules essential for this molecular engineering. Especially, it was noticed that certain rules established in solution are also valid in the solid state. Then, chromophores which correspond to the specifications fixed by the spray drying method was selected and encapsulated, on one hand, in a sol-gel thin-layer to observe their behavior in an environment confined. On the other hand, for those having a good behavior in the sol-gel matrix (controlled by one photon fluorescence spectroscopy), they were confined in a sol-gel shell of the same type. Elaboration's conditions for every compound were optimized, essentially, with the technique of scanning electron microscopy. We demonstrated that the encapsulation step in a sol-gel thin-layer was a very good technique, fast and easy to operate, to make sure that compounds had neither polymorphism nor protonation in a sol-gel matrix before the synthesis of the nanoparticles. Finally, a functionalization step and addition of new targeting functions will allow making first in vivo tests of nanoparticles as biological labels

I materiali luminescenti aventi dimensioni nell’ordine di grandezza dei nanometri, come Nanoparticelle (NPs) che presentano proprietà di Upconversion (UC) o Quantum Dots (QDs), sono attualmente molto studiati per le loro interessanti proprietà, trovando possibili applicazioni in campo biomedico come marcatori ottici, ad esempio come bio-sensori, per il bio-imaging e nel drug delivery. In particolare UC-NPs possono essere utilizzate per ottenere un elevato contrasto nella tecnica di imaging eliminando il rilevante problema dell'autofluorescenza tissutale e permettendo, quindi, di ottenere una maggiore accuratezza nella misura. Lo studio dell'interazione tra NPs e bio-molecole ha coinvolto, nei recenti anni, molti campi di ricerca. Tali interazioni permettono di ottenere nuove ed importanti proprietà per le NPs, come un comportamento idrofilico, bio-compatibilità e bio-degradabilità, che permettono, inoltre, di acquisire una miglior conoscenza sul processo di internalizzazione del bio-nanomateriale da parte delle cellule. La specificità delle bio-molecole permette anche di ottenere differenti bio-distribuzioni del bio-nanomateriale. Queste nuove proprietà migliorano, quindi, le possibili applicazioni delle NPs in campo biomedico. I processi di Energy Transfer (ET), tra due sistemi, coinvolgono l'assorbimento di luce da parte di un partner, detto donatore, che trasferisce l'energia immagazzinata all'altro partner, detto accettore, il quale, di conseguenza, emette l'energia sotto forma luminosa. Tale ET può avvenire mediante processi di tipo radiativo o non-radiativo, dipendenti dalle caratteristiche del sistema. Un esempio di ET è quella che viene definita come FRET, Fluorescence Resonance Energy Transfer, che è un processo fisico distanza-dipendente. L’utilizzo dei processi ET permettono l’analisi di una grande varietà di fenomeni biologici. I fluorofori comunemente utilizzati come donatori e accettori appartengono ad un gruppo di proteine auto-fluorescenti chiamate genericamente GFPs (Green Fluorescent Protein). In particolare le tecniche di imaging basate sull'ET utilizzando GFPs, sono state molto importanti per la determinazione dell'organizzazione cellulare e per tracciare i movimenti di differenti proteine all'interno delle cellule stesse. In questo lavoro studiamo l'interazione tra differenti tipi di Nanoparticelle e GFP, in particolare il processo di Energy Transfer da UC-NPs/QDs a GFP utilizzando varie tecniche spettroscopiche, come spettroscopia di assorbimento ed emissione, e anche tecniche calorimetriche, come ITC (Isotermal Titration Calorimetry). ITC è una tecnica molto promettente che permette di investigare la termodinamica relativa all'interazione tra due sistemi, nel nostro caso tra UC-NPs/QDs e GFP.

It is well established that when in close proximity to gold nanoparticles the optical properties of local fluorescent molecules are dramatically altered. When the localised surface plasmon resonance (LSPR), tuned to the fluorophore absorption band is excited a strong optical enhancement is observed near the nanoparticle due to enhancement in the excitation rate. Both the radiative and non-radiative decay rates undergo significant modification, resulting in either quantum efficiency enhancement, or fluorophore quenching, and a corresponding reduction in the fluorescence lifetime. These effects depend on fluorophore and nanoparticle separation the fluorophore quantum efficiency and the alignment of fluorophore excitation and emission wavelength with the LSPR. Fluorescence lifetime imaging microscopy (FLIM) is used to create high-resolution spatial maps of molecular lifetime and intensity values of single gold nanoparticles deposited on a thin fluorescent-doped polymer film, separated by a SiO2 spacer layer. A strong enhancement in emission intensity is observed in the region of a single nanoparticle. The fluorescence lifetime images are described well using two contributions to fluorescence decay; an unmodified term, allowing for the fact that the diffraction limited focus is significantly larger than the nanoparticle, and some average modified term accounting for the reduction in fluorescence lifetime. Large numbers of nanoparticles are interrogated, giving a statistical distribution of intensity enhancement and lifetime reduction, associated with varying nanoparticle size and shape. These nanoparticle populations are measured for a variety of excitation wavelengths, LSPRs, and yes, allowing analysis of the relationship between LSPR and fluorophore excitation and emission wavelength. Additionally dark-field information is collected for individual nanoparticles, allowing a direct comparison between LSPR and modified lifetime in order to investigate a correlation between peak lifetime reduction, and optimal overlap of the fluorophore emission and the location of the LSPR.

Le travail présenté dans ce manuscrit inclut la synthèse organique des différents types de calixarènes, l'étude de leurs propriétés optiques, des simulations théoriques pour déterminer leurs conformations favorables et leurs utilisations pour stabiliser des nanoparticules. Des nanoparticules d’argent, d’or, de platine et des nanoparticules bimétalliques (Ag-Au) ont été synthétisées en utilisant par réduction radiolytique ainsi que la synthèse la photochimique. Ces nanoparticules sont stabilisées en utilisant des calixarènes et divers polymères. Les nanomatériaux ont été caractérisés par spectroscopie d’absorption UV-Visible et spectroscopie de fluorescence et par des observations en microscopie électronique en transmission. Comme les nanoparticules métalliques sont connues pour leurs applications dans divers domaines, des propriétés antibactériennes de nanoparticules d'argent et des propriétés électrocatalytiques des nanoparticules d'or ont été testées
The work presented in this manuscript includes the organic synthesis of different types of calixarenes, the study of their optical properties, computational studies for determination of their favourable conformations and their use in the stabilisation of nanoparticles. Silver, gold, platinum and bimetallic (Ag-Au) nanoparticles were synthesised using radiolytic reduction as well as photochemical method. These nanoparticles were stabilised by calixarenes and also other ligands which included several polymers. The nanomaterials were characterised using UV-Visible absorption and fluorescence spectroscopy and transmission electron microscopy (TEM) measurements. As metal nanoparticles are known for their applications in various fields, the antibacterial properties of silver nanoparticles and the electrocatalytic properties of gold nanoparticles were tested