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.

L’exaltation de fluorescence par un métal est de plus en plus utilisée pour augmenter la sensibilité de détection dans les systèmes utilisant la fluorescence. Au cours de ce travail de thèse, nous avons étudié ce phénomène dans des nanoparticules hybrides Métal@SiO2 possédant des émetteurs de fluorescence immobilisés sur la silice. Dans un premier temps, nous avons élaboré les nanoparticules cœur-coquille Métal@SiO2 (Métal = Au ou Ag) en utilisant différentes méthodes et en les comparant pour choisir la plus adaptée selon le diamètre du cœur métallique. Dans un deuxième temps, nous avons étudié les propriétés de fluorescence exaltée des nanoparticules hybrides. Deux types d’émetteurs de fluorescence ont été sélectionnés : des nanoparticules semi-conductrices (SiC) et des fluorophores organiques (cyanine 3 et fluorescéine). Après fonctionnalisation de la silice, les émetteurs de fluorescence ont été greffés à la surface des nanoparticules Métal@SiO2. L’exaltation de leur fluorescence a été analysée en fonction de leur densité surfacique, de leur distance par rapport au cœur métallique (fixée par l’épaisseur de silice), du diamètre du cœur métallique et de la longueur d’onde d’excitation. Le facteur d’exaltation le plus important (de l’ordre de 103) a été obtenu avec une faible épaisseur de silice (10 nm) pour les nanoparticules de SiC dont le rendement quantique intrinsèque est très faible (inférieur à 1%). Enfin, la surface de nanoparticules hybrides a été fonctionnalisée avec des nanoparticules d’oxyde de fer de manière à obtenir une combinaison de propriétés optiques (fluorescentes et plasmoniques) et magnétiques à l’intérieur d’une même nanoparticule hybride. / For the past decade, metal-enhanced fluorescence (MEF) has attracted much attention as it improved the sensitivity of fluorescence detection. In this work, MEF was investigated in hybrid Metal@SiO2 nanoparticles with fluorescent emitters immobilized onto silica. In the first part, core-shell Metal@SiO2 (Metal = Au or Ag) nanoparticles were elaborated and several elaboration methods were compared. A comparison was given in order to choose the most suitable method depending on metal core diameter. The second part was dedicated to MEF properties of hybrid nanoparticles. Two kinds of fluorescent emitters were selected: quantum dots (SiC) and organic dyes (cyanine 3 and fluorescein). After silica surface modification, fluorescent emitters were linked to Metal@SiO2 nanoparticles. MEF phenomenon was investigated by tuning the distance between metal and fluorescent emitter (fixed by silica thickness), metal diameter, the fluorescent emitter surface density, and the excitation wavelength. The highest enhancement factor (almost 103) was obtained for a low silica thickness (10 nm) with SiC nanoparticles whose intrinsic quantum yield is very low (lower than 1%). Finally, we functionalized the surface of hybrid nanoparticles with iron oxide nanoparticles to obtain a combination of optical (fluorescence and plasmonics) and magnetic properties inside one hybrid nanoparticle.

This study focuses on preparing surface enhanced fluorescence (SEF) substrates for use in the enhancement of the emission signal of rhodamine B and fluorescein dyes. Fluorescence spectroscopy has been widely utilized owing to its high sensitivity. SEF is a process where the interactions of fluorophores with the localized surface plasmons of metal nanoparticles results in fluorescence enhancement, increased photostability and rates of system radiative decay which leads to a decreased lifetime. One of the most important factors of SEF studies is to provide a uniform distance between fluorophore and metal nanoparticle in a controlled manner / otherwise, F&ouml / rster resonance energy transfer takes place from fluorophore to metal nanoparticle and emission intensity of fluorophore is quenched. The spherical gold and silver nanoparticles were prepared using the well known and straightforward chemical reduction method, in which sodium citrate acted both as a reducing agent and a stabilizer around the formed nanoparticles. Silver and gold were chosen because of their high plasmon field enhancement. Since plasmon field strongly depends on the shape and size of the nanoparticles, the prepared nanoparticles were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Prior to deposition of silver or gold nanoparticles on glass slides, the slides were derivatized by immersing them into an aqueous solution of 3-Aminopropylethoxysilane (APTES). Following derivatization, silver or gold nanoparticles were deposited by immersing the slides into the colloid mixture. Metal nanoparticle coated slides were characterized using absorption spectroscopy and field emission scanning electron microscopy (FE-SEM). Surface enhanced Raman scattering (SERS) measurements were carried out to observe the plasmon efficiency of the deposited nanoparticles. The SERS measurements were repeated for the duration of two weeks in order to check the stability of the plasmon efficiency. In this study, different types of materials (silica, zinc oxide, gold, stearic acid.) were employed as spacers to observe their effects on fluorescence enhancement. Physical vapor deposition (PVD) and Langmuir-Blodgett (LB) film deposition techniques were used for the formation of the spacer within the substrate. Fluorescence enhancement of rhodamine B and fluorescein was observed on the prepared SEF substrates. Obtained enhancement factors indicate that SEF substrates have the potential for sensitivity improvements of fluorescence sensing in many fields.

La photothérapie dynamique (PTD) est un traitement alternatif du cancer qui nécessite l’utilisation de chromophores (photosensibilisateurs) capables d’induire la mort cellulaire suite à une irradiation lumineuse. Les nanoparticles d’or (AuNP), grâce à leur phénomène de résonance plasmon localisée, peuvent exalter les propriétés photophysiques des chromophores localisés à leur surface. L’utilisation d’une excitation biphotonique, dans le proche infrarouge, peut être utilisée pour améliorer l’action thérapeutique (PTD) ou diagnostique (imagerie de fluorescence) des chromophores en augmentant la profondeur de pénétration dans les tissus et la résolution tridimensionnelle de la microscopie biphotonique.Lors de ce travail, l’élaboration de nouvelles nanoparticules hybrides est proposée, présentant des applications potentielles en bioimagerie (sondes brillantes) et comme photosensibilisateurs pour la PTD. Ces nanoparticules sont composées d’un cœur d’or sur lequel sont greffés des conjugués polymère-chromophores biphotoniques. La stratégie de synthèse a consisté à : i) synthétiser des conjugués polymère-chromophores biphotoniques solubles dans l’eau ; ii) les greffer de manière orientée à la surface des AuNP. La synthèse des conjugués polymère-chromophores hydrosolubles on été synthétisés via le couplage efficace de chromophores hydrophobes en position latérale des copolymères P(NAM-co-NAS) bien-définis, obtenus par la techniques de polymérisation radicalaire contrôlée RAFT. Cette stratégie permet le contrôle à la fois de la longueur des chaînes polymère formées (2 000 g.mol-1 < Mn plus < 37 000 g.mol-1) et du nombre de chromophores couplés par chaîne (de 1 à 21). Le greffage orienté de ces conjugués à la surface des AuNP a été mis en évidence (TEM, ATG) et les densités de greffage se sont avérées élevées (~0.5chaîne/nm²). Un des rôles de la chaîne polymère étant de contrôler la distance entre les chromophores et la surface des AuNP. Il a été mis en évidence que l’augmentation de la longueur de chaine des conjugués induisait, outre une augmentation de la couronne greffée (par MET) sur les AuNP, une augmentation de l’émission de fluorescence de ces conjugués polymère-chromophores. Enfin, les propriétés biologiques des conjugués avant et après greffage sur les AuNP ont été évaluées in cellulo, mettant en valeur leur potentiel pour des applications thérapeutiques et diagnostiques. / 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.