Prof. Dr Leila Aparecida Chiavaccifavorin

Dr Renata C K Kaminski

Bruna Brunalallo

Chiari

João, Mariluci, Flavia Gabriela Neima

Mariana Sato

Any, André, Júnior, Thais, Benedict Cristina Leila

Boris, Frank, Elodie, Sarah Chau

Dr Vincent Faivre

Dr Jean-Jacques David

Angélina Angelova

Dr Gillian Fany

Barratt

Alice Claire

Dr. Didier Desmaele Christine Vauthier

Sara Bachner

Rodrigo ( Dido

Kellen Dani

Paris Má

Sarah

Cidinha

Marina, Roberto Milena Patty

Carol, André (Presidente Tia Rô

Mrs Sonia

Mr Antônio

Gisele Cavalim

Tia Eliana

Tia Eloísa

Gledson The Brazilian

Gustave Rue

Sarah Geffroy

Dr Celso V Santilli

Dr Sandra Helena Pulcineli

the colleagues Nalva Peter Hammer

Title : Zinc oxide (ZnO) based quantum dots for bioimaging applications of lipid nanocarriers Keywords : ZnO, Quantum Dots, bioimaing, and lipid nanocarriers Abstract:Theranostic systems consist of a single device containing therapeutics and diagnosis agents and have increased attention in the actual researches because these devices can improve the disease therapy such as cancer, decrease the side effects and the toxicity in nocancer cells and permit monitoring the treatment. The aim of this work was to develop theranostic systems consisted of lipid based nanocarriers containing ZnO based quantum dots (QDs) as cancer cell luminescent guides, and a model drug for cancer therapy. Firstly, it was study the synthesis of ZnO/ZnS QDs aiming to achieve improved luminescent properties. In this step, X-Rays Absorption Spectroscopy, together with other usual characterization techniques, could identify the synthesis condition in which core-shell structures were formed. Nevertheless, the emission of ZnO/ZnS QDs in the visible range was not promising. Therefore, Mg-doped ZnO QDs were synthesized and their luminescence went through a maximum for a 20 mol% nominal concentration of Mg 2+ ions in the reaction medium. Zn 0.8 Mg 0.2 O QDs presented quantum yield (QY) six times higher (QY = 64%) than undoped ones (QY = 10%). ZnO and Zn 0.8 Mg 0.2 O QDs capped by oleic acid (OA) were synthesized and formed stable colloidal dispersions in chloroform and toluene. The QY of OA-Zn 0.8 Mg 0.2 O was about 4 times (around 40%) higher than that of the OA-ZnO QDs. Zn 0.8 Mg 0.2 O QDs and OA-Zn 0.8 Mg 0.2 O QDs could be incorporated into lipid based nanocarriers of average hydrodynamic diameter around 100 -220 nm. The luminescent solid lipid nanoparticles (SLN) were stable in different media at 37°C during 3 hours. The fluorescence association study showed enhanced emission of the J774 macrophage-like cells treated with 2 mg/mL of luminescent SLN during 50 min, suggesting partial internalization of the nanoparticles into the macrophages. The internalization study using the videomicroscope and fluorescence microscope were not successfully, once the equipment condition used could not overcome the cells autofluorescence phenomena.

Titre : Conception de Quantum dots à base d'oxyde de zinc (ZnO) pour des applications en bioimagerie de nanosystèmes lipidiques Mots clés : ZnO, Quantum Dots, bioimagerie etnanosystèmes lipidiques Résumé :Les systèmes théranostiques sont constitués d'un dispositif unique contenant des agents thérapeutiques et diagnostiques et attirent actuellement l'attention pour améliorer le traitement de maladies telles que le cancer ; ils pourraient réduire les effets secondaires (e.g. la toxicité pour les cellules non-cancéreuses) et permettre le suivi du traitement. Les quantum dots (QDs) sont utilisés comme agents d'imagerie in vitro et in vivo. Parmi ceux-ci, ZnO est moins cher, plus biocompatible et moins toxique que ceux utilisés couramment (SeCd, PbS, etc.). Le but de notre étude était de développer des systèmes théranostiques constitués de nanoparticules lipidiques contenant des QDs à base de ZnO comme agent luminescent, l'objectif final étant d'incorporer un principe actif pour le traitement du cancer. Nous avons d'abord étudié la synthèse de QDs de ZnO/ZnS (structure coeur/coquille) par le procédé sol-gel, afin d'améliorer les propriétés de luminescence de ZnO grâce à la passivation de la surface par le ZnS. Pour cela, deux voies de synthèse ont été explorées: le thioacétamide (TAA), utilisé comme source d'ions soufre, a été ajouté à (i) la solution de précurseur de l'acétate de zinc, et (ii) la suspension colloïdale de ZnO. Différentes concentrations de thioacétamide (TAA) ont été utilisées (1,5, 5 et 50 mM). Pour distinguer la formation de la structure coeur/coquille (ZnO / ZnS) d'un simple melange de particules de ZnO et ZnS, les échantillons ont été caractérisés par plusieurs techniques (DRX, XAS, SAXS, UV-vis, pH, HRTEM et PL). Parmi celles-ci, l'absorption des rayons X (XAS) a été essentielle pour montrer que seule la réaction effectuée à partir de la suspension colloïdale de ZnO contenant 5 mm de TAA donnait lieu à la structure ZnO/ZnS. Cependant, les QDs de ZnO/ZnS n'ont pas montré une intensité de l'émission dans le visible plus grande que celle des QDs de ZnO. Les QDs de ZnO ont donc été dopés pour générer des électrons ou des trous supplémentaires dans leur structure, entraînant des changements de leurs propriétés de luminescence. Différentes quantités de Mg (2,5, 5, 10 et 20% en (SeCd, PbS, etc). O objetivo deste trabalho foi desenvolver sistemas teranósticos constituídos de nanocarreadores à base de lipídeos contendo pontos quânticos a base de ZnO, como guias luminescentes de células cancerígenas, e um fármaco modelo para tratamento do câncer. Primeiramente, foi estudada a síntese de pontos quânticos de ZnO/ZnS (estrutura de casca/caroço) pelo processo sol-gel, a fim de melhorar as propriedades luminescentes do ZnO através da passivação da sua superfície provocada pelo ZnS. Para isto, as reações de síntese tiveram como base (i) a solução precursora de acetato de Zinco e (ii) a suspensão coloidal de ZnO. Além disso, foram variadas as concentrações de tioacetamida (TAA) (1.5, 5 e 50mM), usada como fonte de íons de enxofre. Para diferenciar a formação de estruturas casca/caroço (ZnO/ZnS) e não uma mistura de partículas de ZnO e ZnS, várias técnicas foram utilizadas para caracterizar as amostras (DRX, XAS, SAXS, UV-vis, pH, HRTEM e PL). Dentre elas, XAS foi crucial para identificar que somente a reação realizada a partir da suspensão coloidal de ZnO com TAA 5mM deu origem à estrutura de ZnO/ZnS do tipo casca/caroço. Entretanto, os PQs de ZnO/ZnS não apresentaram maior intensidade da emissão na região do visível quando comparado ao ZnO. Portanto, PQs de ZnO foram dopados com Mg para gerar elétrons ou buracos extras na sua estrutura levando a mudanças em suas propriedades luminescentes. Diferentes quantidades de Mg (2.5, 5, 10 e 20 % em mol) foram usadas durante a síntese de ZnO pelo processo sol-gel para poder obter, assim, partículas com maior luminescência na região do visível. DRX, HRTEM, ICP-MS, SAXS, UV-Vis e PL foram usadas a fim de avaliar as características físico-químicas dos diferentes To all those who directly or indirectly contributed to the realization of this thesis, thank you very much. (TANDON et al., 2000;KOLEVA e STOILOVA, 2002;CUSCÓ et al., 2007;DE GELDER et al., 2007) Because of ZnO/ZnS structures did not demonstrate desirable optical properties, the optimization of ZnO QDs for imaging was performed differently. Chapter IV described ZnO QDs doped with Mg 2+ ions with enhanced luminescence emission. Specifically, we have shown that the maximum quantum yield (QY = 64%) for Mg precursor concentration of 20 mol% was six times higher than for undoped ZnO QDs (QY = 10%). Mg-doped ZnO QDs were characterized in order to establish a relationship between the composition and structure of the QDs and their luminescent properties.

List of Figures

List of tables

List of abbreviations

The surface modification of the QDs was then performed using oleic acid (OA) to Cell internalization studies using luminescent SLN were performed using a macrophage-like cell line. However, cell autofluorescence prevented to unambiguously evidence the internalization of SLN into the cells.

  mole) ont été ajoutées lors de la synthèse du ZnO par la voie sol-gel, afin d'obtenir des particules ayant une luminescenceplus forte dans le visible. Plusieurs techniques (XRD, HRTEM, ICP-MS, SAXS, UV-Vis et PL) ont été utilisées pour déterminer les caractéristiques physicochimiques des différents Zn 1-x Mg x O QDs obtenus. Il a été observé une diminution de la taille des particules avec l'augmentation de la concentration de Mg. Lors de l'ajout d'une concentration nominale de Mg 2+ dans le milieu réactionnel de 20% molaire (Zn 0.8 Mg 0.2 O), le rendement quantique (QY) était six fois plus élevé (QY= 64%) que celui de la suspension de ZnO non dopé (QY= 10%). Pour utiliser les QDs dans un environnement biologique tout en conservant des substances hydrophiles pour les rendre stables dans l'eau, ou des substances hydrophobes pour permettre leur intégration dans des systèmes/nanoparticules à base de lipides ou de polymères. La surface des QDs de ZnO et Zn 0.8 Mg 0.2 O a été recouverte par de l'acide oléique (AO) ; les QDs ainsi modifiés forment une dispersion colloïdale stable dans le chloroforme et le toluène. La spectroscopie Raman a confirmé la présence d'acide oleique à la surface des QDs. Le rendement quantique des QDs de Zn 0.8 Mg 0.2 O recouverts d'AO était environ 4 fois plus élevé (QR = 40%) que celui des QDs de ZnO recouverts d'AO. Enfin, les QDs de Zn 0.8 Mg 0.2 O, recouverts ou non d'AO, ont été incorporés dans des nanoparticules lipidiques pour une utilisation ultérieure dans des études d'internalisation cellulaire. Des formulation à base de DPPC, DSPE-Peg et des nanoparticules lipidiques solides (SLN) contenant des QDs ont été préparées avec une taille hydrodynamique variant entre 100 et 220 nm. Les formulations à base de DSPE-Peg ont montré une intensité d'émission plus élevée dans le visible, comparé aux SLNs. L'étude de stabilité réalisée avec des SLNs luminescentes dans différents milieux (eau, PBS, PBS avec du Mg et du Ca, RPMI avec du sérum à 10%) à 37°C pendant 3 heures a montré que la taille ne changeait pas fortement au cours du temps. Dans cette étude, la taille maximale était de l'ordre de 300 nm, montrant que même en présence de protéines, il ne se forme pas de gros agrégats. Trois méthodes ont été utilisées pour l'étude de l'internalisation cellulaire dans des macrophages J774. Lorsque la longueur d'onde d'excitation correspond à celle des QDs, les mesures d'intensité de fluorescence sur les suspensions de cellules ont montré une faible augmentation de l'intensité de l'émission visible pour les cellules incubées avec 2 mg/mL de SLNs luminescentes pendant 50 minutes, en comparaison avec les cellules témoins. Ce résultat suggère une internalisation partielle des SLNs dans les macrophages. Malheureusement, ces résultats n'ont pas pu être confirmés par vidéo-microscopie et microscopie de fluorescence sur les cellules parce que les conditions expérimentales ( longueurs d'onde d'excitation et d'émission possibles) ne permettaient pas d'observer un signal supérieur à celui de l'auto-fluorescence des cellules. En conclusion, cette étude a permis d'optimiser la composition et la synthèse par voie sol-gel de QDs à base de ZnO pour les rendre aptes à être utilisés comme sondes luminescentes dans des nanoparticules lipidiques. La relation entre leur structure et leurs propriétés de luminescence a été étudiée; les QDs dopés Zn 0.8 Mg 0.2 O, qui ont le rendement quantique le plus élevé, ont pu être incorporés dans des nanoparticules lipidiques pouvant contenir des agents thérapeutiques. Título :Pontos Quânticos à base de óxido de zinco (ZnO) para aplicações em bioimagem de nanocarreadores lipídicos Palavras-chaves : ZnO, Pontos Quânticos, bioimageme nanocarreadores lipídicos Resumo :Sistemas teranósticos consistem em um único dispositivo contendo agentes terapêuticos e de diagnóstico e tem ganhado atenção nas pesquisas atuais por melhorar o tratamento de doenças como câncer, podendo diminuir efeitos colaterais, diminuir toxicidade em células não cancerígenas e permitir o monitoramento do tratamento. Os pontos quânticos (PQs) vem sendo usado como agentes de imagem para realizar o monitoramento ótico em investigações in vivo e in vitro. Dentre eles, o ZnO tem se destacado por ser mais barato, mais biocompatível e menos tóxico do que os utilizados comumente

  Zn 1-x Mg x O obtidos. Observou-se a diminuição do tamanho das partículas com o aumento da concentração de Mg. Quando se utilizou 20 % em mols em concentração nominal de íons Mg 2+ no meio reacional (Zn 0.8 Mg 0.2 O), o rendimento quântico (RQ) foi seis vezes maior (RQ = 64%) do que a suspensão de ZnO não dopado (RQ = 10%). Para utilizar os PQs em meio biológico mantendo sua propriedade fotoluminescente, sua superfície normalmente é modificada com substâncias hidrofílicas para torná-los estáveis em água, ou com substâncias hidrofóbicas para permitir sua incorporação em sistemas/carreadores à base de lipídeos ou polímeros. Os PQs de ZnO e de Zn 0.8 Mg 0.2 O foram revestidos com ácido oleico (AO) formando uma dispersão coloidal estável em clorofórmio e tolueno. A espectroscopia RAMAN permitiu confirmar a modificação da superfície dos PQs pela presença dos grupamentos característicos do AO nessas amostras. O RQ do Zn 0.8 Mg 0.2 O revestido com AO foi em torno de 4 vezes maior (RQ = 40%) que o RQ de ZnO revestido com AO. Por fim, os PQs de Zn 0.8 Mg 0.2 O revestidos e nãorevestidos com AO foram incorporados em nanocarreadores lipídicos para serem utilizados posteriormente nos estudos de internalização celular. Formulações à base de DPPC, DSPE-Peg e nanopartículas lipídicas sólidas (NLS) contendo PQs foram obtidos com diâmetro hidrodinâmico entre 100-220 nm. A formulação preparada com DSPE-Peg apresentou maior intensidade de emissão no visível quando comparadas com as NLS. O estudo de estabilidade realizado com as NLS luminescentes em diferentes meios (água, PBS, PBS com Mg e Ca, RPMI com soro à 10%) à 37°C durante 3 horas, mostrou que o tamanho delas não variou bruscamente no tempo analisado. Nesse estudo o tamanho máximo encontrado foi de 300 nm, mostrando que na presença de proteínas, elas não formam grandesagregados que podem favorecer a fagocitose pelo sistema mononuclear. Foram utilizados três métodos para realizar o estudo de internalização celular. O estudo da associação de fluorescência mostrou aumento da intensidade de emissão no visível de células de macrófagos J774 tratadas com 2 mg/mL de NLS luminescente durante 50 min, comparado com as células sem tratamento. Este resultado sugere internalização parcial das NLS nos macrófagos. A vídeo-microscopia e a microscopia de fluorescência não tiveram su-cesso para realizar este estudo, uma vez queas condições experimentais utilizadas não puderam superar o efeito de auto-fluorescência das células. Para concluir, a síntese de PQs à base de ZnO pelo processo sol-gel possibilitou estudar as estruturas e propriedades luminescentes destes, permitindo otimizar sua síntese para adequá-los para uso como guias luminescentes. Desta forma, Zn 0.8 Mg 0.2 O com alto rendimento quântico foi incorporado em nanocarreadores lipídicos e sua aplicação como guia luminescente foi evidenciada através do teste de associação de fluorescência. The synchrotron SOLEIL for providing beamtime at the SAMBA beamline (project number: 20131299),Dr. Valérie Briois for her support during EXAFS experiments and all her knowledge, discussions, and help during my thesis; SWING beamline (project number: 20131299) J. Perez, Y. Liatimi and Pierre Roblin for their support during SAXS experiments; Stéphanie Blanchandin and Karine Chaouchi for WAXS measurements and their kindly support at the Chemistry Laboratory of SOLEIL. The LNLS (project number: 14321) for SAXS measurements, Dr. Florian Meneau for his support during SAXS understanding, motivation and friendship, LNNano for HRTEM measurements. The Service of electronic microscopy of the University Pierre et Marie Curie for HRTEM measurements. The secretaries of Post-GraduationTechnique Section of Facultyof PharmaceuticalSciences, Claudia, Daniela, Joyce, Flávia and the secretary of Doctoral School"Innovation thérapeutique du fondamental à l'appliqué" Mme Lucie Landry. FAPESP(Process number: 2012/07570-4 and BEPE process number: 2015/01198-4) and PADC/FCF-UNESP for the financial support. My thesis was conducted at the Institut Galien of University Paris -Sud during one year supported by the International Cooperation Program CAPES/COFECUB (ME 767-13), financed by CAPES -Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil.

Figure I.

  Figure I.1. Schematic structures of the nanocarriers: (a) Polymeric nanoparticles, (b)

1 .

 1 Figure I.1. Schematic structures of the nanocarriers: (a) Polymeric nanoparticles, (b) Liposomes, (c) Lipid nanoparticles and nanoemulsions, (d) Micelles, (e) Cyclodextrins, (f) Dendrimers, (g) Carbon nanotubes, (h) Gold nanoparticles.Erro! Indicador não definido.

Figure I. 2 .

 2 Figure I.2. Illustration of the EPR effect of drug-loaded nanocarriers (macromolecule) in a malignant tissue. ................................................................. Erro! Indicador não definido.

Figure I. 3 .

 3 Figure I.3. Absorption coefficient of oxyhemoglobin, hemoglobin and water from 400 to 1000 nm. The inset shows the lowest absorption coefficient in the NIR region around 650-900 nm. ...................................................................................... Erro! Indicador não definido.

Figure I. 4 .

 4 Figure I.4. Schematic representation of lipid-based nanocarriers.Erro! Indicador não definido.

Figure I. 5 .

 5 Figure I.5. Schematic structures of the three generations of liposomes.Erro! Indicador não definido.

Figure I. 6 .

 6 Figure I.6. Schematic size-dependent luminescence properties of QDs: correlation between the electronic structure of QDS and the QD radius which leads to the blue shift of the emitted ration due to quantum confinement as the QD size decreases.Erro! Indicador não definido.

Figure I. 7 .

 7 Figure I.7. Solutions of CdSe/CdS core-shell QDs with different size and shell thickness under normal indoor light (no UV radiation). .................... Erro! Indicador não definido.

Figure I. 8 .

 8 Figure I.8. Schematic structure of the core-shell quantum dot capped by amphiphilic molecules. ........................................................................... Erro! Indicador não definido.

Figure

  Figure I.9. A mouse under UV light (λexc =330 nm) (A) after 5 min after intradermal injection of green QDS, and (B) 60 min after intradermal injection of green and yellow QDS. ................................................................................... Erro! Indicador não definido.

Figure I. 10 .

 10 Figure I. 10. Schematical structure of wurtzite ZnO, where Zn is the grey ball and O is the red ball, both Zn and O ions fourfold coordinated. .................. Erro! Indicador não definido.

Figure I. 11 .

 11 Figure I.11. Suggested mechanisms involved in the ZnO photoluminescent processes: (a) typical exciton emission (UV emission), (b) recombination of a shallowly trapped electron with a deeply trapped hole, and (c) recombination of a shallowly trapped hole with a deeply trapped electron (visible emission). In the maps VB and CB are the valence band and the conductance band, respectively. ................... Erro! Indicador não definido.

Figure

  Figure I.12. PL spectra of the ZNO nanoparticles synthesized at (a) 50, (b) 100, (c) 150 and (d) 200 °C, measured at room temperature with excitation wavelength 280 nm. ....... Erro! Indicador não definido.

Figure I. 13 .

 13 Figure I.13. Schematic diagram of Co-doped ZnO QDs structure.Erro! Indicador não definido.

Figure II. 1 .

 1 Figure II.1. Molecule of (a) DPPC, (b) DSPE-Peg 2000, and (c) oleic acid.Erro! Indicador não definido.

Figure II. 2 .

 2 Figure II.2. Experimental setup for ZnAc precursor preparation.Erro! Indicador não definido.

Figure II. 3 .

 3 Figure II.3. The Bragg description of diffraction in terms of the reflection of a plane wave (wavelength λ) incident at an angle θ to atomic planes of spacing d.Erro! Indicador não definido.

Figure II. 4 .

 4 Figure II.4. (a) Schematic SAXS setup and (b) X-ray beam paths from the source to the detector, both elements located far away from the sample. Erro! Indicador não definido.

Figure II. 5 .

 5 Figure II.5. Schematical setup used at SWING beamline. ........ Erro! Indicador não definido.

Figure II. 6 .

 6 Figure II.6. Illustration of a typical X-ray absorption, µx versus E, for PD and a brief explanation of the principle of X-ray absorption spectroscopy both in terms of XANES and EXAFS spectroscopy................................................... Erro! Indicador não definido.

Figure

  Figure II.7. ZnO QDs UV absorption spectra appointing λ determined by Nedelijkovic. . Erro! Indicador não definido.

Figure

  Figure II.8. Schematic figure of the plate used to perform the determination of the fluorescence association by fluorescence spectroscopy. .... Erro! Indicador não definido.

Figure II. 9 .

 9 Figure II. 9. Schematic figure of the plate used to perform the cell treatment with luminescent nanoparticles for further observation by fluorescence microscope.Erro! Indicador não definido.

Figure III. 1 .

 1 Figure III.1. (a)XRD profiles of ZnO and ZnS standards, ZnO and ZnS QDs, and ZnO/ZnS core-shell QDs with different concentration of the sulphur source. (b)Marked lines indicating the hexagonal wurtzite phase (black) and cubic zinc blende phase (red) are shown. Zoom of the peaks (111), (100), (002) and (101) in the 2θ ranging from 23 to 40 ° of the ZnO QDs, ZnS QDs, SUSP_TAA5mM and PREC_TAA5mM.Erro! Indicador não definido.

Figure III. 2 .

 2 Figure III.2. (a) XANES spectra and (b) Fourier Transforms of EXAFS spectra recorded for the different samples and ZnO and ZnS standard references.Erro! Indicador não definido. Figure III.3. HRTEM image of (a) SUSP_TAA5mM and (b) a zoom of the sample SUSP_TAA5mM which shows the interplanar spacing of ZNO and ZNS phases. .... Erro! Indicador não definido.

Figure III. 4 .

 4 Figure III.4. UV-vis spectra of (a) ZnO QDs, ZnS QDs and the mixture of ZnO and ZnS QDs and (b) ethanolic solution of TAA. .................................... Erro! Indicador não definido.

Figure III. 5 .

 5 Figure III.5. Selected UV-vis spectra measured at the indicated reaction time (min) of the reactions using the Route 1 on the left ((a) SUSP_TAA1.5mM, (b) SUSP_TAA5mM and (c) SUSP_TAA50mM) and the Route 2 on the right ((d) PREC_TAA1.5mM, (e) PREC_TAA5mM and (f) PREC_TAA50mM). ................. Erro! Indicador não definido.

Figure III. 6 .

 6 Figure III.6. (a) Zoom of selected UV-vis spectra measured at the indicated reaction time (min) in the region of the excitonic peak of ZnO QDs (330 to 360 nm) of the SUSP_TAA50mM reaction, and (b) UV-vis spectrum of SUSP_TAA50mM reaction product washed and re-suspended in etanol. ...................... Erro! Indicador não definido.

Figure III. 7 .

 7 Figure III.7. Time evolution of the pH during the reactions using Route 1 (a) and Route 2 (b). ............................................................................................ Erro! Indicador não definido.

Figure III. 8 .

 8 Figure III.8. Selected in situ SAXS profiles measured at the indicated reaction time (min) of ZnO QDs (a) and the reactions using the Route 1 (b, c and d) and the Route 2 (e, f and g). ............................................................................................ Erro! Indicador não definido.

Figure III. 9 .

 9 Figure III.9. Time evolution of the Rg (nm) during 40 min of the reactions (except for the reaction SUSP_TAA5mM which presented the plateau in the Guinier region up to 20 min). ................................................................................... Erro! Indicador não definido.

Figure

  Figure III.10. PL spectra of the ZnO QDs, samples prepared using the Route 1 (SUSP_TAA1.5mM, SUSP_TAA5mM and SUSP_TAA50mM) and the Route 2 (PREC_TAA1.5mM, PREC_TAA5mM and PREC_TAA50mM) excited at 353 nm. ............................................................................................ Erro! Indicador não definido.

Figure III. 11 .

 11 Figure III.11. Time evolution of (a) the pH, ZnO and TAA UV-vis absorbance intensity of the SUSP_TAA50mM and (b) the pH and TAA UV-vis absorbance intensity of the PREC_TAA50mM. ............................................................ Erro! Indicador não definido.

Figure IV. 1 .

 1 Figure IV.1. (a) XRD profiles of the ZnO standard and nanocrystals from the reactions with 0,

Figure IV. 2 .

 2 Figure IV.2. HRTEM images of undoped ZnO QDs (a) and Zn 0.8 Mg 0.2 O QDs (b). ........ Erro! Indicador não definido.

Figure IV. 3 .

 3 Figure IV.3. Three-dimensional stacked log-log plots of the SAXS curves as a function of time recorded in situ during the formation of ZnO QDs (a) and selected in situ SAXS profiles measured at the indicated reaction time (min) (b). Erro! Indicador não definido.

Figure IV. 4 .

 4 Figure IV.4. Three-dimensional stacked log-log plots of the SAXS curves as a function of time recorded in situ during the formation of Zn 0.8 Mg 0.2 O QDs (a) and selected in situ SAXS profiles measured at the indicated reaction time (min) (b).Erro! Indicador não definido.

Figure IV. 5 .

 5 Figure IV. 5. Comparison of the time evolution of the radius of gyration (Rg) determined by the Guinier region and the radius of Sphere (R) determined by the sphere model of (a) ZnO QDS and (b) Zn 0.8 Mg 0.2 O QDs. ................................. Erro! Indicador não definido.

Figure IV. 6 .

 6 Figure IV.6. Raman spectra of ZnO standard, ZnO QDs, Zn 0.8 Mg 0.2 O QDs, OA, OA-ZnO QDs and OA-Zn 0.8 Mg 0.2 O QDs with indications of the principal vibrations (a) from 200 to 800 cm -1 and (b) from 800 to 1800 cm -1 . ....................... Erro! Indicador não definido.

Figure IV. 7 .

 7 Figure IV.7. Absorption spectra of the undoped and 2.5, 5, 10 and 20% Mg doped ZnO colloidal suspensions. ......................................................... Erro! Indicador não definido.

Figure

  Figure IV. 8. PLE and PL spectra of the undoped and 2.5, 5, 10 and 20 mol% Mg doped ZnO colloidal suspensions. The inset shows the photography of the undoped (right) and Zn 0.8 Mg 0.2 O (left) colloidal suspensions under UV lamp (λexc = 365 nm). ............... Erro! Indicador não definido.

Figure IV. 9 .

 9 Figure IV.9. PL spectra of the OA-ZnO QDs and OA-Zn 0.8 Mg 0.2 O QDs dispersed in chloroform. The insets show the photographs of each sample under UV lamp (λexc = 365 NM). ................................................................................... Erro! Indicador não definido.

Figure V. 1 .

 1 Figure V.1. Principal nanocarriers endocytic pathways in mammalian cells: (a) Phagocytosis, an actin-based mechanism, closely associated with opsonization; (b) Clathrin-mediated endocytosis, associated with the formation of a clathrin lattice; (c) Caveolae-mediate endocytosis, typical flask-shaped invaginations of the membrane coated with caveolin dimers; (d) Macropinocytosis, also an actin-based pathway, less selective than

Figure V. 2 .

 2 Figure V.2. SAXS patterns of DPPC based liposomes formulations collected at 30°C. ... Erro! Indicador não definido.

Figure V. 3 .

 3 Figure V.3. SAXS patterns of DPPC based liposomes formulations collected at 50°C. ... Erro! Indicador não definido.

Figure V. 4 .

 4 Figure V.4. Cryo-TEM images of formulation similar to D5 in a large excess of water, after extrusion.. ........................................................................... Erro! Indicador não definido.

Figure V. 5 .

 5 Figure V.5. SAXS patterns of DSPE-Peg based formulations collected at 30 °C. ............ Erro! Indicador não definido.

Figure V. 6 .

 6 Figure V.6. SAXS patterns of DSPE-Peg/DPPC based formulations collected at (a) 30°C (continuous lines) and (b) 50°C (dotted lines). .................. Erro! Indicador não definido.

Figure V. 7 .

 7 Figure V. 7. Cryo-TEM images of formulations DPeg6. .......... Erro! Indicador não definido.

Figure V. 8 .

 8 Figure V.8. Photos of (a)the dried film, (b)the hydrated film under visible light and (c) under UV lamp (λexc = 356nm) of the DPeg6 formulation. ........ Erro! Indicador não definido.

Figure V. 9 .

 9 Figure V.9. Cryo-TEM images of formulation (a) G12, and (b) THF.Erro! Indicador não definido.

Figure V. 10 .

 10 Figure V.10. Photos of the formulations G1-G9 (from left to right) under UV lamp (λexc = 356nm). .............................................................................. Erro! Indicador não definido.

Figure V. 11 .

 11 Figure V.11. Absorption spectra of the diluted formulations G4, G8, and DPeg6. ........... Erro! Indicador não definido.

Figure

  Figure V.12. (a) PLE and (b) PL spectra of the formulations G4, G8 and DPeg6. ............ Erro! Indicador não definido.

Figure V. 13 .

 13 Figure V.13. Absorption spectra of the water and cells suspension.Erro! Indicador não definido.

Figure V. 14 .

 14 Figure V.14. PL spectra of (a) cells suspension excited at 270, 340, 350, and 365 nm and (b) a zoom in the PL spectra excited at 340, 350, and 365 nm. .. Erro! Indicador não definido.

Figure V. 15 .

 15 Figure V.15. Evolution of the mean size of the formulation G4 in different media during the time. .................................................................................... Erro! Indicador não definido.

Figure V. 16 .

 16 Figure V.16. PL spectra of lysed cells without treatment, cells treated with 1 and 2 mg/mL of G4 nanopaticles excited at (a) 340 nm and (b) 365 nm. .... Erro! Indicador não definido.

Figure VI. 1 .

 1 Figure VI.1. Schematical final products obtained in the reactions starting from ZnO colloidal

  QDs: ZnO quantum dots with oleic acid as surface modifier OA-Zn 1-x Mg x O QDs: Mg-doped ZnO quantum dots with oleic acid as surface modifier PBSemission spectroscopy XPS: X-ray Photoelectron Spectroscopy ZnO Susp: ZnO colloidal suspensions Zn 1-x Mg x O QDs: Mg-doped ZnO quantum dots with x amount of Mg CONTENT INTRODUCTION ................................................................................................................. CHAPTER I -Bibliography I.1. Nanomedicine as a theranostic system ............................................................................ I.2. Lipid-based nanocarriers ................................................................................................. I.2.1. Liposomes .................................................................................................................... I.2.2. Micelles ........................................................................................................................ I.2.3. Solid lipid nanoparticles (SLN) ................................................................................... I.2.4. Lipid nanocapsules (LNC) ........................................................................................... I.3. Quantum Dots for optical imaging .................................................................................. I.3.1. Core-shell QDs ............................................................................................................. I.3.2. Surface-modification of QDs with organic molecules ................................................. I.3.3. In vitro imaging using QDs .......................................................................................... I.3.4. In vivo imaging using QDs .......................................................................................... I.3.5. ZnO QDs ...................................................................................................................... I.3.5.1. Sol-gel synthesis ........................................................................................................ I.3.5.2. Photoluminescence properties ................................................................................

  photoblinking effect, and QDs surface modification with organic molecules can provide colloidal stability in aqueous media or non-polar solvents. Nevertheless, nontoxic QDs, such as ZnO or ZnS QDs, began to be used due to their biocompatility and promising photoluminescent properties.The photoluminescence of ZnO QDs is directly related to their size and surface defects. Because of these important characteristics, the synthesis route strongly interferes with the final properties of the material. Since 1980, researchers have studied the quantum confinement and luminescent properties of ZnO QDs synthesized using the sol-gel route,

  hinder their aggregation and to provide them colloidal stability in non-polar environment. Still in the Chapter IV, we could show that Mg-doped ZnO QDs capped by OA formed stable colloidal suspensions in toluene and chloroform, while preserving their photoluminescence with QY around 40 %, promising for bioimaging. In the Chapter V, the OA surface modified Mg-doped QDs were incorporated in lipid based nanocarriers in order to analyze the nanoparticles structure, stability and luminescence. At low QD concentrations, QDs were embedded within 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) bilayers, without perturbing their stacking. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] ammonium salt(DSPE-Peg) /QDs formulations formed aggregates displaying a close-packed, layered structure, of QDs likely stabilized by a layer of DSPE-Peg. Solid lipid nanoparticles (SLN) loaded with QDs demonstrated good stability in biological media as shownby the absence of large aggregates.

Figure VI. 1 .

 1 Figure VI.1.Schematical final products obtained in the reactions starting from ZnO colloidal suspensions (Route 1) and ZnAc precursor (Route 2) using three TAA concentrations (1.5, 5 and 50mM).

Figure VI. 2 .

 2 Figure VI.2.Schematical structures of ZnO and Zn 0.8 Mg 0.2 O QDs with and without OA capping, their photographs and respective QY.

Finally

  , we have tried to incorporate highly luminescent Zn 0.8 Mg 0.2 O and OA-Zn 0.8 Mg 0.2 O QDs into lipid-based nanocarriers.

Figure VI. 3

 3 Figure VI.3 shows examples of photographs of liposomes, DSPE-Peg based formulations and SLNs, loaded with QDs. As observed in this figure, liposomes presented lower luminescence compared to the DSPE-Peg formulation and SLN. SLNs were fairly stable in biological media, as shown by the absence of large aggregates. However, further investigations are needed to optimize the preparation and photoluminescence properties of the different nanocarriersfor bioimaging applications.

Figure VI. 3 .

 3 Figure VI.3.Photographs of QDs loaded liposomes, a DSPE-Peg based formulation and an example of SLN (from left to right, respectively) under UV lamp.

Table III .

 III 1. EXAFS structural parameters for the first Zinc coordination sphere of ZnO, for the first Zinc coordination sphere of ZnS, and the percentages of ZnO and ZnS deduced from the Linear Combination Fittings (LCF) of XANES spectra.Erro! Indicador não definido. TableIII.2. Initial (time = 1 min) and Final (time = 40 min) ZnO QDs Radius (nm) deduced from UV-vis spectra using Brus Equation, and Initial (time = 1 min) and Final (time = 40 min) Rg (nm) deduced from SAXS curves using Guinier Equation.Erro! Indicador não definido.

Table IV .

 IV 1. Amount of Mg incorporated into ZnO QDs obtained by ICP, Crystallite size deduced from X-ray diffraction peaks using the Debye-Scherrer relation, Bandgap measured using UV-vis absorption, Wavelength of the PLE peak maximum, Wavelength of the PL emissionpeak maximum, Quantum Yield of each sample.Erro! Indicador não definido. Table IV.2. Variables N (particle number density), s (polidispersitiy), and X0 (mean radius) used to fit the ZnO and Zn 0.8 Mg 0.2 O SAXS curvesand the values of I(0) obtained by Guinier equation ………................................................................…………………Erro! Indicador não definido. Table IV. 3. Raman shift (cm-1) and assignment of the main vibrations observed in the following samples: ZnO standard, ZnO QDS, Zn 0.8 Mg 0.2 O QDS, OA, OA-ZnO QDS and OA-Zn 0.8 Mg 0.2 O QDs. Raman shifts are compared with values from References

  Table IV.4. Quantum Yield (QY) of the ZnO and Zn 0.8 Mg 0.2 O QDs capped with oleic acid Table V.2. OA-Zn 0.8 Mg 0.2 O QDs, DSPE-Peg and water quantities used in the formulations, Table V.3. OA-Zn 0.8 Mg 0.2 O QDs, DPPC, DSPE-Peg and water quantities used in the formulations, QD/lipids ratio (mg/mg) and d-spacing at 30 and 50 °C.Erro! Indicador não definido. Table V.4. Zn 0.8 Mg 0.2 O QDs and Gelcuire concentrations, ratio of Zn 0.8 Mg 0.2 O QDs:Gelucire 50/13® and ethanol:water used. ......................................... Erro! Indicador não definido.

. ....................................................... Erro! Indicador não definido. (OA). .................................................................................. Erro! Indicador não definido.

Table V.1. OA-Zn 0.8 Mg 0.2 O QDs, DPPC and water quantities used in the liposomes formulations, QD/DPPC ratio (mg/mg) and d-spacing at 30 and 50 °C.Erro! Indicador não definido. QD/DSPE-Peg ratio (mg/mg) and d-spacing at 10, 30 and 50 °C.Erro! Indicador não definido.

  ... I.3.6. Doped ZnO QDs .......................................................................................................... I.3.7. Toxicity of quantum dots ............................................................................................. I.4. References ....................................................................................................................... CHAPTER II -Materials and methods II.1. Materials ....................................................................................................................... II.2. Synthesis ........................................................................................................................ II.2.1. ZnO QDs ..................................................................................................................... II.2.2. ZnS QDs ..................................................................................................................... II.2.3.ZnO/ZnS core-shell QDs and ZnO and ZnS QDs mixtures ........................................ II.2.4.Zn 1-x Mg x O QDs ........................................................................................................... II.2.5.Powder obtainment ......................................................................................................

Acknowledgements

Firstly, I thank God for always guide me.

CHAPTER VI

Final conclusions and perspectives

Chapter VI -Final conclusions and perspectives

VI.1. Final conclusions

The aim of this thesis was to explore ZnO based QDs as bioimaging agents due to their promising photoluminescent properties, when synthesized via sol-gel route, and low toxicity compared to the usual QDs, which contain heavy metals such as Cd, Pb, etc.

In a first step, attempts were made to improve the photoluminescence properties of ZnO QDs by synthesizing ZnO/ZnS core-shell QDs and doping ZnO QDs with Mg 2+ ions.

Hereafter, QD surface modification was achieved for later incorporation in lipid based nanocarriers. Finally, the study of the structure, stability, photoluminescence properties and cell internalization of lipid nanocarriers loaded with QDs was carried out.

We could monitor the reactions tentatively used for obtaining ZnO/ZnS core-shell QDs and characterize the final products. We could observe that a small amount of TAA in the reaction medium was not sufficient to produce ZnS; an intermediate amount of TAA led to the formation of both species (ZnO and ZnS); and higher quantities of TAA gave mainly rise to ZnS nanoparticles. The samples obtained in each reaction could be characterized by UV-vis, XRD and XAS techniques; however, it is noteworthy that only XAS allowed identifying core-shell structure when the reaction proceeded in ZnO colloidal suspension mixed with TAA 5 mM (Route1).

For cell internalization studies, the limitations due to the cell auto-fluorescence phenomena might be overcome if ZnO QDs were excited at the optimal excitation wavelength with high intensity beam and short exposure time.Appropriate setups are available at the DISCO beamline at SOLEIL. Besides, the luminescent nanoparticle formulation, concentration and time of incubation with cells should be optimized.

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