Rozprawy doktorskie na temat „Nanoparticles - Biological Applications”

Les nanoparticules fonctionnalisées continuent de susciter beaucoup d’interêt dans les applications biomédicales et les essais biologiques. Elles sont devenues un élément clé dans la recherche en nanobiotechnologie. Un des axes primordiaux des travaux de recherche est le développement de stratégies polyvalentes de fonctionnalisation de surface pour différentes nanoparticules allant de nanostructures de diamants à des nanoparticules d'oxyde de fer, des particules de silice et des nanocapsules lipidiques. Un des objectifs en particulier a été l’introduction de diverses fonctionnalisations sur les mêmes nanoparticules en utilisant soit des ligands dérivés de la dopamine ou soit par chimie « click » de Cu(I) catalysé. Il en résulte des nanostructures bien dispersées fonctionnalisées avec différents ligands à leurs surfaces. Les applications de ces nanostructures pour l'inhibition des infections virales et pour la délivrance de gènes ont été étudiées. En effet, l'inhibition de l'entrée du VHC a été identifiée comme étant une stratégie thérapeutique potentielle. Il a pu être démontré que différentes nanoparticules peuvent être efficacement conçues pour afficher les propriétés de lectine et se comporter donc comme des inhibiteurs efficaces d'entrée du virus in vitro. Les pseudo-lectines étudiées ici comprennent les nanoparticules dérivées du fer, de silice, du diamant et des nanocapsules lipidiques comportant toutes des fragments d’acide boronique attachés à leurs surfaces.Par ailleurs, le potentiel des nanoparticules de diamant pour la délivrance de gènes a été étudié
Functionalized nanoparticles continue to attract interest in biomedical applications and bioassays and have become a key focus in nanobiotechnology research. One of the primal focuses of the research work was the development of versatile surface functionalization strategies for different nanoparticles ranging from diamond nanostructures to iron oxide nanoparticles, silica particles and lipid nanocapsules. One particular aim was the introduction of various functionalities onto the same nanoparticles using either dopamine-derived ligands or Cu(I) catalyzed “click” chemistry strategies. This resulted in well-dispersed nanostructures with different ligands present on the surface of the nanostructures. The possibilities to use such nanostructures for the inhibition of viral infections and for gene delivery were investigated. Indeed, inhibiting the entry of HCV has been identified as a potential therapeutic strategy. It could be demonstrated that various nanoparticles can be efficiently engineered to display “lectin-like” properties and indeed behave as effective viral entry inhibitors, in vitro. The pseudo-lectins investigated here include iron-, silica-, diamond-, (lipid nanocapsule)-derived nanoparticles all featuring surface-attached boronic acid moieties. In parallel to work on HCV entry inhibition, the potential of diamond nanoparticles as gene delivery system was investigated. Water dispersible and biocompatible polypegylated diamond particles were prepared using different dopamine ligands and their effect on gene delivery has been studied

Thesis (Ph. D.) -- University of Maryland, College Park, 2005.
Thesis research directed by: Dept. of Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Gold nanoparticles have attracted much attention in the field of biological research, especially in biological imaging and sensing due to their unique physical properties. Fluorescence is a highly-sensitive, non-invasive biological study method and has been widely used in a variety of research topics. The aim of this thesis is to study the unique optical properties of gold nanoparticles and demonstrate their application in biological imaging and sensing through fluorescence microscopic and spectroscopic techniques. An introduction of gold nanoparticles and fluorescence techniques used in this project is given in Chapter 1. In Chapter 2, the synthesis method of gold nanoparticles, dependence of optical properties on particle size and shape, the unique spectroscopic characterization and microscopic application of gold nanorods are discussed. Fluorescence lifetime imaging microscopy (FLIM) based on two-photon luminescence lifetime from gold nanorods in cell culture, and the advantag es of this method in biological imaging are demonstrated in Chapter 3. In Chapter 4, the energy transfer between a DNA dye, 4'-6-Diamidino-2-phenylindole (DAPI), and different types of gold nanoparticles in solution is demonstrated using FLIM. Biological imaging application based on energy transfer between gold particles and DAPI in cell culture is discussed as well in this chapter. A study on energy transfer process concerning different excitation conditions is reviewed in Chapter 5. Furthermore, application of fluorescence resonant energy transfer (FRET) based FLIM method in the research of intracellular pathway of gold nanoparticles in cells is demonstrated. Chapter 6 presents a systematic study on the cytotoxicity of gold nanorods in cell culture using MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The effects of particle shape, surface conditions, dosage, incubation time on the cytotoxicity and the mechanism of cytotoxicity are discussed. In Chapte 7, a brief summary and outlook to future work are presented.

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

The aim of this PhD thesis has been the study of metal nanoparticles and their applications in biological systems. Biological studies have been accomplished in collaboration with Dr Giorgio Scarì from the Department of Biology of Milan University. The research has been mailnly focused on the following arguments: - Specific design and use of 15-mer peptides as stabilisers in the gold nanoparticles preparation - Bioconjugation of peptide stabilised gold nanoparticles - A TEM study of the cellular uptake mechanism of peptide-coated GNPs into HeLa cells - Preparation of gold nanoparticles stabilised with different small organic bicompatible molecules for the selective cellular uptake into cancer cells - Use of Aloin A and Aloesin, two active components of Cape Aloe, in the preparation of gold and silver nanoparticles and their biological applications - NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles - Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use as biolabels Peptide Design for the Stabilisation of Gold Nanoparticles Gold nanoparticles can be easily functionalised with biomolecules or organic ligands, and they can be attractive tools for various applications. Stabilisation of gold nanoparticles by peptide molecules is well reported in literature [1-6]. In this PhD dissertation, 15-mer peptides were designed and used as stabilisers for gold nanoparticles. Peptides designed, having periodical sequences, were planned to allow a parallel binding to gold surface [7]. One, H2N-GC(GGC)4-G-COOH (GC15), composed of 10 glycines and 5 cysteines, the other H2N-GK(GGK)4-G-COOH (GK15) composed of 10 glycines and 5 lysines. The sequences of the peptides were planned to allow the peptide to bind gold particles along its length, as observed for leucine and lysine-containing peptide bound to carboxylate-terminated thiol capped gold nanoparticles [8]. GC15 bears many potential anchor groups (SH or NH2) that can covalently bind gold particle, although the superiority of the thiol groups in covalent bonding with gold has already been stated [9]. GK15 peptide contains only primary amines that can bind gold particles in different ways depending on the pH of the sol and the pI of the peptide [10]. In particular, an electrostatic binding of GK15 can be assumed, if the NH2 groups are protonated, as observed in the case of gold-poly-lysine systems [11]. Peptides were synthesised by a standard Fmoc solid-phase procedure, purified by preparative HPLC and characterised by mass spectrometry (ESI-MS) by the professor Giovanna Speranza’s research group of Milan University. Gold nanoparticles stabilised by GC15 and GK15 were prepared via the borohydride reduction method in water at pH 3, as well as via ligand exchange method. In the borohydride reduction method, gold precursor, AuCl4-, is reduced by NaBH4, in the presence of the peptide ligand obtaining a cherry red coloured gold sol. In the ligand exchange preparation method gold nanoparticles of 15 nm diameter were obtained via the Turkevich/Frens method [12-13], subsequently protected by addition of the peptide and purified by repeated centrifugation and redispersion. By different preparation methods gold particles of different diameters were obtained. Particles were purified by dialysis or centrifugation, depending on the particles size. The particles were characterised by UV-visible, ATR-FTIR, 1H NMR spectroscopies, while the particles dimensional and morphological characterisation was performed by TEM. NMR spectroscopy has revealed to be very useful tool for the characterisation in aqueous media after the lyophilisation and redispersion of the particles. This is an important result since very few nanoparticle systems can be stored in dry state and then redispersed in water [14] and studied by the NMR. These peptides containing several regularly spaced amine (lysine) or thiol (cysteine) functions have been introduced as very strongly binding “multidentate” ligands to stabilise gold nanoparticles [15]. Spectroscopic investigations suggest an electrostatic multiple interactions of protonated NH2 groups of GK15 with anions present on negative gold surface (AuCl4-, AuCl2-), observing breaking and formation of H-bonding. While for GC15 peptide, the coordination to gold particles was observed via the thiol functionality, as expected. A multidentate peptide for stabilisation and facile bioconjugation of gold nanoparticles There is an increasing interest in the preparation of nanoparticles that are stable in aqueous media and can be readily functionalised with bio-molecules by established bioconjugation procedures. A number of different approaches to conjugating metal nanoparticles to biomolecules have also been reported. These include click chemistry [16], biotin-avidin coupling [17-19], ligand exchange [20, 21] and a range of standard bioconjugation procedures [22, 23]. After the GC15 and GK15 peptides showed novel and successful characteristics in the gold particle stabilisation, a new peptide of this family was specifically designed for the stabilisation and subsequent bioconjugation of gold nanoparticles. This ligand (H2N-GCGGCGGKGGCGGCG-COOH)can bind to the nanoparticle via the thiol groups of four cysteine moieties and contains a central lysine that provides an amine function to which biomolecular functionality can be readily attached. This protection method is very robust and can be used either for the one-step synthesis of relatively small (2-4 nm) particles or for the stabilisation of pre-prepared, larger (10-20 nm) colloids. Water-soluble GCK15 peptide was purchased from Aldrich (purity>95%). The resulting GCK15 coated gold particles have been characterised by TEM, UV-vis, ATR-FTIR and 1H NMR spectroscopy. Gold nanoparticles of 2.4 nm diameters were prepared in a one-step reaction by borohydride reduction of AuCl4- in the presence of the stabilising GCK15 peptide. A clear brown solution was obtained indicating the formation of gold particles in the size range below 3 nm. This conclusion was confirmed by the absence of a plasmon absorption band in the UV-vis spectrum. Gold nanoparticles of 15 nm diameters were obtained via the Turkevich/Frens method [12, 13] subsequently protected by addition of our peptide and purified by repeated centrifugation and redispersion. The UV-vis spectrum shows a plasmon absorption band at 520 nm typical for gold particles of this size range, and red gold colloidal solution. The particles are extremely stable and can be centrifuged and redispersed in pure water many times without detectable loss of material, whereas the as prepared citrate-stabilised particles cannot be redispersed in pure water after the first centrifugation. The analysis of the ligand shell in the case of 15 nm GCK15 stabilised gold particles obtained via the ligand exchange method is more difficult due to the very small proportion of peptide present in the total amount of material, which is predominantly gold. However, using high resolution magic angle spinning (HR-MAS) well resolved 1H NMR spectra of 15 nm Au@GCK15 nanoparticles were obtained. The absence of citrate peaks (quartet centred at 2.5-2.7 ppm) suggests complete ligand exchange by exposure to the peptide. The sharp doublet centred at 2.90 ppm in the spectrum of the free peptide ligand is due to the 8 cysteine β-methylene groups vicinal to the thiol groups and disappears completely upon binding to the particles. This indicates that all cysteine thiol groups are involved in the surface binding process. As an example of facile bioconjugation, a biotin moiety has been introduced via a standard coupling procedure. Biotinylation of peptide-stabilised gold nanoparticles was achieved using the standard sulfo-NHS-biotin labelling agent. Binding of the biotinylated particles to streptavidin-modified agarose beads has been demonstrated leading to an intense red colouration of the beads as evidence for successful biotinylation. Particles that have not been biotinylated do not attach to the beads. In adittion, dot blot experiments also clearly indicate efficient biotinylation of the particles. The attachment of biomolecular functionality of choice, e.g. biotin, is possible due to the presence of a central lysine residue that is not involved in the binding of the ligand to the surface of the particles. Biological application of peptide stabilised gold nanoparticles. A study of the cellular uptake mechanism. Current studies in this research area have been focused on coating biorecognition molecules on the surface of NPs to mediate cellular accumulation in different cell compartments. In bionanotechnology it is very important to have stabilisers, which could be easily functionalised with other biologically important ligands. Understanding and controlling the interactions between nanoscale objects and living cells is of great importance for arising diagnostic and therapeutic applications of nanoparticles and for nanotoxicology studies [24]. In this PhD thesis, the intracellular uptake of differently sized spherical water-soluble peptide-coated gold nanoparticles into HeLa cells has been investigated [25]. HeLa cells are human epithelial cells from a fatal cervical carcinoma transformed by human papillomavirus 18 (HPV18), classic example of an immortalized cell line widely used in medical research. For this study, gold particles stabilised with GC15, GK15 and GCK15 peptides were successfully uptaken into HeLa cells, as well as biotinylated GCK15 peptide stabilised gold particles. A comparison has been made between gold particles prepared by two different preparation methods: borohydride direct reduction and the ligand exchange preparation method. It was found that the particles prepared by using the citrate displacement method enters HeLa cells in different fashion as compared with the particles prepared by borohydride reduction method. Intracellular uptake of gold particles was investigated using TEM microscopy. Samples for TEM observations were prepared by incubation of gold particles with HeLa cells at 37°C and 5% CO2 flow for 1h and the samples were then processed by a number of necessary steps (fixation, post fixation, staining, dexydration, embedding in epoxy resin, polymerisation, ultra thin cutting and mounting on TEM grids) in order to obtain 70 nm thick sections cutted with the ultra microtrome suitable for the TEM observations. Accumulation of gold particles into membrane-bound compartments inside cells, known as endosomes, is generally observed. It is shown that smaller particles (<4nm) entered cells in agglomerated form, this phenomenon were also described elsewhere [26, 27]. However, all the particles were found in endosomes, whether early or late endosomes. No particles were found in HeLa cells nuclei. In a similar fashion, biotinylated GCK15 stabilised gold particles were also found in HeLa cells endosomes. Microscopy observations have demonstrated that the mechanism of the cellular uptake of gold particles into HeLa cells is mediated via the receptor-mediated endocytosys, as evidenced by TEM micrographs of ultra thin cellular sections. It was possible to observe almost all the steps of this mechanism: 1.Specific adsorption of gold nanoparticles on the cell membrane 2.Specific recognition of gold nanoparticles by receptors present in the cell’s membrane 3.Invagination of the cell membrane with formation of a membrane-bound compartments known as endosomes 4.Observation of the endosomes formed carrying gold nanoparticles present in cell’s cytoplasm If the uptake mechanism of gold particles is endocytosis it is expected their exit via the exocytosis. This phenomenon would restrain their leftover time in cells, and consequently the toxicity for the organism. Selective cellular uptake of gold nanoparticles into cancer cells Current clinical X-ray contrast agents impose serious limitations on medical imaging: short imaging times, the need for catheterisation in many cases, occasional renal toxicity, and poor contrast in large patients [28]. Gold nanoparticles may overcome these limitations, as demonstrated by Hainfeld and co workers. Gold has higher absorption than iodine agents, usually used for these purposes, with less bone and tissue interference achieving better contrast with lower X-ray dose. Moreover, nanoparticles clear the blood more slowly than iodine agents, permitting longer imaging times. In this study, gold nanoparticles of 1.9 nm in diameter were injected intravenously into mice and images recorded over time with a standard mammography unit. Retention in liver and spleen resulted very low with elimination by the kidneys. These concepts were extended by using different gold nanoparticles to deliver a very large quantity of gold to tumours via intravenous injection. Combination with X-rays resulted in eradication of most tumours [29]. This PhD work was stimulated by the Hainfield’s study [30-33] where a synergistic effect was observed between gold nanoparticles and the X-ray treatment resulting in tumour reduction or eradication. The survival after one year of the combined therapy was of 70%. The success of this technique is related to the high ability of gold to accumulate within tumours and absorb X-rays. Instead of the intravenous injection in a tumour tissue, different cancer cells with a range of small sized gold nanoparticles were incubated. We have studied with confocal microscopy the intracellular uptake of small sized gold nanoparticles stabilised by different organic biocompatible ligands (5-aminovaleric acid, adipic acid, L-DOPA, glucose, glycolic acid, dopamine) and their use as nanogold bioconiugates with different cancer cells (K562-leucemia myelogenous cronica caucasica humana, PC12-pheochromocytoma). Selective entrance of these particles into cancer cells was found [34]. Negative control has been performed on human epithelial cells where no entrance of gold particles was found even after 8 h of incubation. A preliminary toxicity experiment in vivo has been performed on sane CD1 mice type. Aminovaleric acid coated gold nanoparticles were chosen as model particles and injected intraperitoneally in two mice. Survival after 2 years post injection was verified, as an exceptional result. This preliminary result leads us to conclude very low or total absence of toxicity effects on living tissue and inner organs. Aloin A and Aloesin stabilised gold and silver nanoparticles and their biological applications. The inner gel of Aloe vera (Aloe barbadensis Miller) leaf is widely used in various medical, cosmetic and nutraceutical applications [35]. Many beneficial effects and biological activities of this plant as anti-viral, anti-bacterical, laxative, anti-inflammation and immunostimulation have been attributed to the polysaccharides present in the leaf pulp. Different chemical compounds, responsible for its healing properties, have been isolated so far from this specie as alkaloids, anthraquinones, anthrones, chromones, flavonoids, coumarins and pyrones, and their chemistry was thoroughly studied and reported by Dagne and coworkers [36] and professor Speranza and professor Manitto research groups [37-40]. In the anticancer drugs research, the studies on Aloe vera components have been videly undertaken. It was found by Pecere and co-workers that a hydroxyanthraquinone, naturally present in Aloe vera leaves, has a specific in vitro and in vivo antineuroectodermal tumour activity [41]. Nanoparticles synthesis using biological entities is already reported in literature, including bacteria, yeast, funghi and plants [42, 43] as clean, non-toxic and environmetally acceptable routes. Many studies on the plant use in nanobiotechnology have appeared in literature in a size-controlled formation of gold nanoparticles. Different plants are involved in the both intra and extracellular formation of silver and gold nanoparticles, reporting the use of oat (Avena sativa) [44], lemongrass extract (Cymbopogon flexuosus) [45-47], leguminous shrub Sesbania drummondii [48], Brassica juncea [49], neem leaf broth (Azadirachta indica) [50], pine (Pinus desiflora), persimmon (Diopyros kaki), ginkgo (Ginko biloba), magnolia (Magnolia kobus) and platanus (Platanus orientalis) [51]. In the listed examples, nanoparticles formation is a consequence of the Au(III) to Au(0) reduction inside plant cells or tissues. On the other hand, use of various leaf extracts utilised both as reducing agents and stabilisers in the nanoparticles preparation has been reported, as for example Emblica Officinalis fruit extract [52], Aloe vera leaf extract [53] and Cinnamon camphora leaf extract [54]. Using Aloe vera leaf extract, the formation of gold nanotriangles has been achieved as a result of the slow reduction of aqueous tetrachloroaurate anions, AuCl4-, along with the shape-directing effects of carbonyl compounds present as constituents in the plant extract. With the aim to prepare novel water soluble and biocompatible nanoparticles for biological applications, in this PhD project two active components of Aloe vera, Aloin A and Aloesin have been utilised, as stabilisers for gold and silver nanoparticles. By using different reducing agents (sodium borohydride, citric and ascorbic acid) and varying the reaction conditions (temperature and reaction time) we were able to prepare extremely stabile, water soluble Aloin A and Aloesin stabilised gold particles sized from 4 to 50 nm diameter range and approximately 5 nm sized silver nanoparticles. Prior to characterisation, particles were purified by dialysis or centrifugation, depending on the particle’s size. Silver particles were characterised by UV-visible spectroscopy and the morphology and size of both silver and gold particles were investigated by TEM. Gold particles were characterised using UV-visible, ATR-FTIR and 1H NMR spectroscopies, which highlighted the interaction between gold and Aloin A and Aloesin ligand molecules. Although NMR studies of the particles ligand shell might be an issue, due to the very small content of the organic material present on the particles surface, HR-MAS 1H NMR technique has been used. Due to the very small amount of the sample needed for the analysis, this technique resulted very advantageous and promising in the studies of the particle ligand shell, appearing more functional and effective than usual NMR analysis in solution. By ligand exchange preparation method, involving citrate coated 15 nm gold particles, it was possible to exchange the citrate ligand with Aloin A and Aloesin molecules, stabilising the particles also in this way. The amount of Aloin A and Aloesin was finely tuned as well as pH of the colloidal solution allowing particle’s agglomeration studies. Agglomeration of the particles was followed by TEM microscopy. More agglomerated particles were found on lower pH values (5-7) in less protected colloidal samples (<5000 ligand molecules per particle). When the pH of the colloidal solution was adjusted to higher values (8-10) and approximately 10000 ligand molecules were set up for each gold particle good stabilisation of the particles was achieved. 50 nm Aloin A and Aloesin stabilised gold nanoparticles, prepared by two different methods were applied to the vehicle study into macrophage cells. For the biological experiments by a peritoneal washing procedure, macrophage cells were extracted from a CD1 mouse, pre-emptively sacrificed by CO2 asphyxia. Macrophages were then collected in the physiological solution and seeded in the cell culture medium (MEM) at 37°C in the CO2 atmosphere at 5% at sterile conditions. Subsequently, macrophages were treated with gold colloidal solution (50 nm Au-Aloin A and 40 nm Au-Aloesin particles obtained by citric acid reduction method). For the treatment, 50 μl of gold colloidal solution was added to 1 ml of the cell culture medium containing marcophage cells. Macrophages were then incubated with gold nanoparticles for 5, 15, 30 and 60 min in the same conditions (37°C, at 5% CO2). After the incubation, the samples were prepared for the confocal and fluorescence microscopy observations by a number of necessary steps (centrifugation, adittion of DAPI, fixation) obtaining incubated cells on a microscopy glass slides. DAPI (4',6-diamidino-2-phenylindole) fluorescent stain was used in order to stain the cells’ nuclei. Confocal microscopy observations have revealed the presence of Aloin A and Aloesin stabilised gold particles in macrophage cells cytoplasm, while the fluorescence microscopy has revealed, in some cases, the presence of these particles also in macrophages nuclei. It is an important result, since there is an emergent need for carriers that can carry bioactive agents into the cell nucleus for drug as well as gene delivery. However, in general, nanoparticles mainly localise in the cytosol (or extranucleus). Therefore, nanoparticles capable of localising into the nucleus are particularly inportant for cancer therapy. Because cancer cells have many intracellular mechanisms to limit drug molecules' access to the nucleus the direct delivery of the drug into the nucleus would circumvent these drug-resistance mechanisms [55]. NMR and IR studies of simple aminoalcohol stabilised gold nanoparticles Special properties of gold nanoparticles [56] led to their use in important applications in the areas of catalysis, optoelectronics, electron microscopy, and biology. However, the nature of the gold capping ligand bond usually remains unknown, especially for ligands bearing multiple anchor groups that can bind gold particles in different ways (carboxy, amino, hydroxy, thiol etc.). Several studies using variety of characterisation techniques have been carried out [57]. Some of the capping agents investigated include amines or amino derivatives [58]. Heath and co-workers proposed the formation of a partially covalent bond between the gold surface and the primary amines, pointing out that the stability of the Au-amine nanoparticle depends mainly on kinetic effects [59]. Further experiments were performed using amino derivatives and exploiting them as stabilisers and/or reducing agents [60-70] in the particles preparation. Various amino functionalities have been studied, including amino acids and amino-containing polymers, with the amino groups being particularly interesting due to their presence in biological and environmental systems. However, there is a lack of reports on aminoalcohols, which are an attractive class of compounds for their potential oxidation to amino acids. This PhD project research was stimulated by the novelty of gold-aminoalcohol systems, the discovery of aminoalcohol binding site(s) (NH2 vs OH functionality), and the nature of the Au-NH bond. Reproducible gold hydrosols stabilised by aminoalcohols have been prepared. Aminoalcohol stabilised gold and silver particles were characterised by spectroscopic and microscopic means giving a deep insight into the gold-nitrogen interaction. NMR spectroscopy was applied to investigate gold nanoparticles in organic solvents; [71, 72] however, many experimental difficulties (organic molecule quantity in the ligand shell, particles purity, redispersion of gold colloids) had to be overcome before the NMR technique could be applied as a valid tool in the investigation of small-sized water-soluble gold nanoparticles [73]. Aminoalcohol-stabilised gold nanoparticles in aqueous solution were prepared by the borohydride reduction of HAuCl4 in the presence of aminoalcohol. Different aminoalcohols were used as stabilisers for gold particles: ethanolamine, 2-(propylamino)ethanol, 2-amino-1,3-propanediol, DL-2-amino-1-pentanol, 3-amino-1-propanol, ()-3-amino-1,2-propanediol, 4-amino-1-butanol, 5-amino-1-pentanol. Red gold sols with 4 nm mean diameter particles were obtained. The samples were characterised by ATR-FTIR spectroscopy in solid state, and by 1H NMR, UV-vis, and TEM analyses in solution. The ATR-FTIR spectra of the solid materials suggested the gold-aminoalcohol interaction. Considering NH2 and OH stretching vibration modes in the free and coordinated ligands, storng evidence of the NH3+ groups involved in an electrostatic bond is shown in the case of 4-amino-1-butanol stabilised gold particles. To test this, 4-amino-1-butanol hydrochloride was prepared (by bubbling gaseous HCl in a 1,2-dimethoxy-ethane solution of 4-amino-1-butanol) and the IR spectrum was collected. Successful NMR measurements were obtained only after accurate purification of the particles (dialysis). 1H NMR experiments indicated that the binding site of aminoalcohols studied is the protonated amino group ligated with an ionic bond to the gold surface, as strongly supported by general chemical shift trend. A difference of ca. 0.40-0.50 ppm, from free aminoalcohols to coordinated aminoalcohols on gold particles has been observed [74]. Thus, NMR technique is proving to be a powerful tool for the characterisation of these colloidal systems, but is still remains not fully utilised, because of the inability of current preparative methods to supply enough purified nanoparticles. Fluorescence spectroscopy and microscopy studies of dye stabilised gold nanoparticles and their potential use in biological labelling The introduction of labelling agents in biological systems is required for a facile microscopy detection of biological systems. Fluorescent labels nowadays represent widely developed tools in biology and medicine [75]. Fluorescence parameters are usually used to obtain informations on living cells. In this context, modified gold nanoparticles can be used as nano reporters. Substantial progress in the ability to fabricate nanoparticles and the discovery of their novel size dependent physical and chemical features has drawn the attention of researchers in the area of biomedical imaging. The development of targeted contrast agents such as fluorescent probes has made it possible to selectively view specific biological events and processes in both living and nonviable systems with improved detection limits, imaging modalities and engineered biomarker functionality. The fabrication of luminescent-engineered nanoparticles is expected to be integral to the development of next generation therapeutic, diagnosis and imaging technologies [76]. The aim of this project was the fabrication of novel dye stabilised gold nanoparticles with favourable luminescent properties as bio-labels and the in vitro studies of their cellular uptake. Cellular uptake of luminescent gold nanoparticles into macrophages and Human Neuroblastoma IMR-32 cells was studied. Dye-stabilised gold particles have been prepared by a particular preparation method, designed in order to obtain 30 to 50 nm sized particles. For the preparation, different reducing agents (NaBH4, Ascorbic and Citric acid) and stabilisers having photoluminescence (PL) signals in different Visible spectrum region have been applied. As stabilisers for gold particles fluorescent molecules: 2’,7’-Dichlorofluorescein, 4-Methylumelliferyl phosphate, Eosin Y (2′,4′,5′,7′-Tetrabromofluorescein disodium salt), HPTS (8-Hydroxy-1,3,6-pyrenetrisulfonic acid) and Thionin acetate (3,7-Diaminophenothiazin-5-ium acetate) have been utilised. These novel gold colloids were characterised by UV-vis spectroscopy, TEM and HR-MAS NMR spectroscopy while the luminescent properties of these particles were studied by fluorescence spectroscopy. Absorption and emission spectra showed no peaks due to free ligands, and were substancially different. PL spectra of gold nanoparticles, obtained by excitation at the absorption wavelength value (523-547 nm range), showed values in a 596-661 nm range, depending on the ligand kind and particles size. We have also found that the type of reducing agent influences gold nanoparticles PL emission. Moreover, when excited at 488 nm, gold sols showed two PL peaks, centered at 525 and 581 nm, in accordance with Fluorescence and Confocal microscopy observations. Indeed, when FITC (Fluorescein Isothiocyanate) fluorescence filter is used (λex = 488 nm) the particles internalised in cells, showed green fluorescence signals, while Texas Red fluorescence filter is used (λex = 568 nm) the particles showed red fluorescence signals. Luminescent gold nanoparticles were tested as labelling agents in two different cellular systems (macrophages and neuroblastoma IMR-32 cells). Dye stabilised gold particles were incubated with macrophage and IMR-32 cells for 1 h (37°C, 5% CO2), and the cellular uptake of the particles was confirmed by Confocal microscopy and TEM. Besides these techniques, Fluorescence microscopy observations have showed interesting results. These novel systems, coupling gold with the dye, have an advantage of being visualised by all three microscopy techniques, (TEM, Confocal and Fluorescence microscopy) as they satisfy their detection requirements (gold electronic density, gold fluorescence and ligand fluorescence) as they exibit the long fluorescence lifetime. For the dye-stabilised particles uptaken into macrophages and IMR-32 cells, an almost general correlation between the fluorescence signal colour and the particles size has been found. In the case of particles smaller than 10 nm in size (3 nm Au@Citrate and 10 nm Au@4-aminovaleric acid were taken as examples) only green fluorescence signals are found applying FITC filter. On the contrary, if the particle diameters are 15 nm or larger, red fluorescence signals are found when Texas Red fluorescence filter is applied. However, in some cases, for particles with broader size distribution, green and red fluorescence signals were both observed. This allowed us to distinguish types of particles capable of internalisation into different cell compartments (eg. Thionin acetate stabilised gold particles; larger particles were found in macrophage nucleus and the smaller ones in the cell’s cytoplasm, i.e. endosomes). To confirm the efficient cellular uptake, and suggest gold nanoparticles internalisation mechanism, gold nanoparticle-cell systems have been studied by TEM. For gold nanoparticles found in cell endosomes, endocitosys as cellular uptake mechanism is suggested [26], and for those found in cell cytoplasm, an uptake via the diffusion mechanism is suggested. All these findings propose these particles as labelling agents in cell systems, and could be eventually applied in experiments in vivo. References: 1. Levy, R., Thanh, N.T.K., Doty, R.C., Hussain, I., Nichols, R.J., Schiffrin, D.J., Brust, M., and Fernig, D.G. J. Am. Chem. Soc. 126, 2004, 10076-10084 2. Levy, R. ChemBioChem. 7, 2006, 1141-1145 3. Liu, Y., Franzen, S., and Feldheim, D.L. Abstracts of Papers, 229th ACS National Meeting, San Diego, CA, United States, March 13-17, 2005 4. Fillon, Y., Verma, A., Ghosh, P., Ernenwein, D., Rotello, V.M., Chmielewski, J. J. Am. Chem. Soc. 129, 2007, 6676-6677 5. Pengo, P., Baltzer, L., Pasquato, L. and Scrimin, P. Angew. Chem. Int. Ed. 46, 2007, 400-404 6. Higuchi, M., Ushiba, K. and Kawaguchi, M. J. Colloid Int. Sci. 308, 2007, 356-363 7. G. P. Drobny et al., Langmuir, 21, 2005, 3002 8. P.V. Bower, E.A. Louie, J.R. Long, P.S. Stayton, G.P. Drobny. Langmuir, 21, 2005, 3002–3007 9. R. Levy, N.T.K. Thanh, R.C. Doty, I. Hussain, R.J. Nichols, D.J. Schiffrin, M. Brust, D.G. Fernig. J. Am. Chem. Soc. 126, 2004, 10076–10084 10. (a) Z. Zhong, A.S. Subramanian, J. Highfield, K. Carpenter, A. Gedanken, Chem. Eur. J. 11, 2005, 1473– 1478; (b) Z. Zhong, S. Patskovskyy, P. Bouvrette, J.H.T. Luong, A. Gedanken, J. Phys. Chem. B 108, 2004, 4046–4052; c) M. Larsson, J. Lindgren, J. Raman Spectrosc. 36, 2005, 394– 399; d) Z. Zhong, J. Luo, T.P. Ang, J. Highfield, J. Lin, A. Gedanken, J. Phys. Chem. B 108, 2004, 18119–18123 11. Jordan, C.E., Frey, B., Kornguth, L., Corn, R.C. Langmuir 10, 1994, 3642–3648 12. Turkevich, J., Stevenson, P. C., and Hillier J. J. Discuss. Faraday Soc. 11, 1951, 55-75 13. Frens, G. Nature: Phys. Sci. 241, 1973, 20-22 14. Feldheim D.L., Foss, C.A., in Metal Nanoparticles: Synthesis, Characterization, and Application, Marcel Dekker, New York. 2002 15. Porta, F., Speranza, G., Krpetić, Ž., Dal Santo, V., Francescato, P., and Scari, G. Mater. Sci. Eng., B. 140, 2007, 187-194 16. Brennan, J.L., Hatzakis, N.S., Tshikhudo, T.R., Dirvianskyte, N., Razumas, V., Patkar, S., Vind, J., Svendsen, A., Nolte, R.J.M., Rowan, A.E., Brust, M. Bioconjugate Chem. 17, 2006, 1373-1375 17. Park, J-A., Lee, J-J., Kim, I-S., Park, B-H., Lee, G-H., Kim, T-J., Ri, H-C., Kim, H-J., Chang, Y. Colloids Surf., A. 31, 2008, 288-291 18. Prosperi, D., Morasso, C., Tortora, P., Monti, D., and Bellini, T. ChemBioChem. 8, 2007, 1021-1028 19. Weiss, B., Schneider, M., Muys, L., Taetz, S., Neumann, D., Schaefer, U.F., Lehr, C-M. Bioconjugate Chem. 18, 2007, 1087-1094 20. Ackerson, C.J., Jadzinsky, P.D., Jensen, G-J.,Kornberg, R.D. J. Am. Chem. Soc. 128, 2006, 2635-2640 21. You, C-C., Verma, A., and Rotello, V.M. Soft Mater. 2, 2006, 190-204 22. Shang, L., Wang, Y., Jiang, J., Dong, S. Langmuir 23, 2007, 2714-2721 23. Schroedter, A., Weller, H. Angew. Chem. Int. Ed. 41, 2002, 3218-3221 24. Nativo, P., Prior, I.A., Brust M. ACS Nano 2, 2008, 1639–1644 25. (a) Boatman E. et. al., Cell Tiss. Res. 170, 1976, 1; (b) http://www.microbiologybytes.com; (c) http://en.wikipedia.org/wiki/HeLa 26. Chitrani, B.D., Ghazani, A.A., Chan, C.W., Nano Letters, 6, 2006, 662 27. Chitrani, B.D., Chan, C.W., Nano Letters, 7, 2007, 1542 28. Hainfeld, J.F., Slatkin, D.N., Focella, T.M., Smilowitz, H.M. Br. J. Radiol. 79, 2006, 248–2531 29. Hainfeld, J.F., Slatkin, D.N., Smilowitz, H.M. Phys. Med. Biol. 49, 2004, N309-N315 30. Dilmanian F.A., Qu Y., Liu S., Cool C.D., Gilbert J., Hainfeld J.F., Kruse C.A., Laterra J.S., Lenihan D., Nawrocky M.M., Pappas G., Sze C.-I., Yuasa T., Zhong N., Zhong Z., and McDonald J.W. Nuc. Inst. Meth. Phys. Res. Section A548, 2005, 30-37 31. Herold, D.M., Das, I.J., Strobbe, C.C., Iyer, R.V., Chapman, J.D., Int. J. Radiat. Biol. 76, 2000, 1357 32. Matsudaira, H., Ueno, A.M., Furuno, I. Radiat. Res. 84, 1980, 144 33. Rose, J.H., Norman, A., Ingram, M., Aoki, C., Solberg, T., Mesa, A. Int. J. Radiat. Oncol. Biol. Phys. 45, 1999, 1127 34. Krpetic, Z., Porta, F., Scarì, G. Gold Bull. 39, 2006, 66-68 35. Ni, Y., NTurner, D., Yates, K.M., Tizard, I. Int. Immunopharm. 4, 2004, 1745-1755 36. Dagne, E., Bisrat, D., Viljoen, A., Van Wyk B-E. Curr. Org. Chem. 4, 2000, 1055-1078 37. Manitto, P., Monti, D., Speranza, G. J. Chem. Soc. Perkin. Trans. 1, 1990, 1297-1300 38. Speranza, G., Fontana, G., Zangola, S., Di Meo, A. J. Nat. Prod. 60, 1997, 692-694 39. Speranza, G., Morelli, C. F., Tubaro, A., Altinier, G., Duri, L., Manitto, P. Planta Medica 71, 2005, 79-81 40. Duri, L., Morelli, C. F., Crippa, S., Speranza, G. Fitoterapia 75, 2004, 520-522 41. Pecere, T., Gazzola, M.V., Mucignat, C., Parolin, C., Vecchia, F.D., Cavaggioni, A., Basso, G., Diaspro, A., Salvato, B., Carli, M., Palù, G. Cancer Res. 60, 2000, 2800-2804 42. Mohanpuria, P., Rana, N.K., Yadav, S.K.J. Nanopart. Res. 10, 2008, 507–517 43. Sastry, M., Ahmad, A., Khan, M.I., Kumar, R. Curr. Sci. 85, 2003, 162-170 44. Armendariz, V., Herrera, I., Peralta-Videa, J.R. Jose-Yacaman, M. Troiani, H. Santiago, P. Gardea- Torresdey J. L. J. Nanopart. Res. 6, 2004, 377–382 45. Shankar, S.S., Rai, A., Ahmad, A., Sastry M. Chem. Mater. 17, 2005, 566-572 46. Rai, A., Singh, A., Ahmad, A., Sastry, M. Langmuir 22, 2006, 736-741 47. Singh, A., Chaudhari, M., Sastry, M. Nanotechnol. 17, 2006, 2399–2405 48. Sharma, N.C., Sahi, S.V., Nath, S., Parsons, J.G., Gardea-Torresdey, J.L., Pal, T. Environ. Sci. Technol. 41, 2007, 5137-5142 49. Haverkamp, R.G., Marshall, A.T., Van Agterveld, D. J. Nanoparticle Res. 9, 2007, 697–700 50. Shankar, S.S., Rai, A., Ahmad, A., Sastry, M. J. Colloid Interf. Sci. 275, 2004, 496-502 51. Song, J.Y., Kim, B.S. Bioprocess Biosyst. Eng. 2008 DOI:10.1007/s00449-008-0224-6 52. Ankamwar, B., Damle, C., Absar, A., Mural, S. J. Nanosci. Nanotechnol. 10, 2005,1665–1671 53. Chandran, S.P., Chaudhary, M., Pasricha, R., Ahmad, A., Sastry, M. Biotechnol. Prog. 22, 2006, 577– 583 54. Huang, J., Li, Q., Sun, D., Lu, Y., Su, Y., Yang, X., Wang, H., Wang, Y., Shao, W., He, N., Hong, J., Chen, C. Nanotechnology 18, 2007, 105104-105115 55. Xu, P., Van Kirk, E.A., Zhan, Y., Murdoch, W.J., Radosz, M., Shen, Y. Angew. Chem. Int. Ed 46, 2007, 4999–5002 56. Daniel, M. C., Astruc, D. Chem. Rev. 104, 2004, 293–346 57. Hasan, M., Bethell, D., Brust, M. J. Am. Chem. Soc. 124, 2002, 1132–1133 58. Xu, C., Sun, L., Kepley, L.J., Crooks, R.M. Anal. Chem. 65, 1993, 2102–2107 59. Leff, D.V., Brandt, L., Heath, J.R. Langmuir 12, 1996, 4723–4730 60. Selvakannan, P.R., Mandal, S., Phadtare, S., Gole, A., Pasricha, R., Adyanthaya, S. D., Sastry, M. J. Colloid Interface Sci. 269, 2004, 97–102 61. Newman, J.D.S., Blanchard, G.J. Langmuir 22, 2006, 5882–5887 62. Bhargava, S.K., Booth, J.M., Agrawal, S., Coloe, P., Kar, G. Langmuir 21, 2005, 5949–5956 63. Subramaniam, C., Tom, R.T., Pradeep, T. J. Nanopart. Res. 7, 2005, 209–217 64. Iwamoto, M., Kuroda, K., Zaporojtchenko, V., Hayashi, S., Faupel, F. Eur. Phys. J. D: At., Mol., Opt. Phys. 24, 2003, 365–367 65. Aslam, M., Fu, L., Su, M., Vijayamohanan, K., Dravid, V.P. J. Mater. Chem. 14, 2004, 1795–1797 66. Green, M., O’Brien, P. Chem. Commun. 2000, 183–184 67. Chen, X.Y., Li, J.R., Jiang, L. Nanotechnology 11, 2000, 108–111 68. Brown, L.O., Hutchinson, J.E. J. Am. Chem. Soc. 121, 1999, 882–88 69. Brown, L.O., Hutchinson, J. E. J. Phys. Chem. B 105, 2001, 8911–8916 70. Sastry, M., Kumar, A., Mukhrjee, P. Colloids Surf. A 181, 2001, 181, 255–259 71. Fabris, L., Antonello, S., Armelao, L., Donkers, R.L., Polo, F., Toniolo, C., Maran, F. J. Am. Chem. Soc. 128, 2006, 326–336 72. Bradley, J.S. The Chemistry of Transition Metal Colloids In Clusters and Colloids: From Theory to Applications; Schmid, G., Ed.; VCH Publishers, Inc.: New York, 1994, Chapter 6, p 517. 73. (a) Kohlmann, O., Steinmetz, W.E., Mao, X., Wuelfing, W.P., Templeton, A.C., Murray, R.W J. Phys. Chem. B 105, 2001, 8801–8809 (b) Hostetler, M.J., Wingate, J.E., Zhong, C-J., Harris, J.E., Vachet, R.W., Clark, M.R., Londono, J.D., Green, S.J., Stokes, J.J., Wignall, G.D., Glish, G.L., Porter, M.D., Evans, N.D., Murray, R.W. Langmuir 14, 1998,17–30 74. Porta, F., Krpetic, Z., Prati, L., Gaiassi, L., Scarì, G. Langmuir 24, 2008, 7061-7064 75. Wang, F., Tan, W.B., Zhang, Y., Fan, X., Wang, M. Nanotechnology, 17, 2006, R1-R13 76. Sharma P. et al., Adv. Colloid Interface Sci. 123-126, 2006, 471-485 The work in this PhD thesis has appeared in the following publications: 1. Selective entrance of gold nanoparticles into cancer cells. Krpetić, Željka; Porta, Francesca; Scari, Giorgio; Gold Bulletin 39, 2006, 66-68. 2. Gold nanoparticles capped by peptides. Porta, Francesca; Speranza, Giovanna; Krpetić, Željka; Dal Santo,Vladimiro; Francescato, Pierangelo; Scarì, Giorgio. Mater. Sci. Eng. B, 140, 2007, 187-194. 3 Gold-Ligand interaction studies of water soluble aminoalcohol capped gold nanoparticles by NMR. Porta, Francesca; Krpetić, Željka; Prati, Laura; Gaiassi, Aureliano; Scarì, Giorgio. Langmuir, 24, 2008, 7061-7064. 4. A multidentate peptide for stabilisation and facile bioconjugation of gold nanoparticles. Krpetić, Željka; Nativo, Paola; Porta, Francesca; Brust, Mathias. Submitted to Bioconjugate Chemistry.

Transition metals have been used to mediate bioorthogonal reactions within a biological environment. In particular, applications of biocompatible palladium catalysis currently range from biomolecules modification to the in cellulo synthesis or activation of drugs. Here, the scope of palladium-mediated chemistry in living systems has been further extended with the development of a new homogenous palladium catalyst. This water-soluble, biocompatible, and traceable catalysts is based on a palladium-carbene complex coupled to a fluorescent labelled homing peptide for targeted delivery inside cells. This “SMART” catalyst is designed to activate both caged fluorophores and drugs through the cleavage of protecting groups or cross-coupling reactions. A second strategy for targeted delivery of a biocompatible palladium catalysis involves metal nanoparticles loaded onto a heterogeneous solid support. This “modular” catalyst can be implanted in vivo at the desired site of action, e.g. a tumour, and locally activate biomolecules. These two catalytic systems will allow us to selectively activate pro-drugs in vivo, with spatial control, thus minimising the side effects of the treatment on the whole body.

El trabajo realizado en esta tesis se ha centrado en dos sistemas de nanopartículas moleculares que tienen un uso potencial en el campo de la nanomedicina: i) vesículas lipídicas – entidades supramoleculares que se proponen como sistemas de liberación de fármacos y ii) cuerpos de inclusión (Inclusion Bodies, IBs) – nanopartículas formadas por agregados proteicos. La primiera parte del trabajo se ha centrado en el estudio comparativo de sistemas vesiculares preparados por i) diferentes metodologías así como ii) diferentes composiciones. Los métodos comparados son DELOS-susp, un método de una sola etapa basado en fluidos comprimidos y procesos convencionales que constan de varias etapas, como hidratación de película delgada (thin film hydration, TFH) y tratamiento con ultrasonidos seguido de extrusión (ultrasound, US). La influencia de la estructura interna se ha investigado con vesículas con diferentes composiciones mixtas: CTAB-Colesterol y DOPC-Colesterol. Para este estudio hemos utilizado métodos que monitorizan la interacción de nanomaterials con superficies como la resonancia de plasmones superficiales (surface plasmon resonance, SPR) y la microbalanza de cristal de cuarzo con disipación (quartz crystal microbalance with dissipation, QCM-D). A partir del análisis de datos hemos demostrado que todos los sistemas investigados forman monocapas de vesículas enteras tras la interacción con superficies de oro. Ha sido posible calcular la absorción de masa, espesor, densidad y propiedades mecánicas de los diferentes sistemas vesiculares estudiados. Hemos concluido, que la influencia del método de preparación es muy. Es decir, en este tipo de sistemas, la arquitectura interna, una vez lograda, es la que determina las propiedades mecánicas de las entidades supramoleculares. Por otro lado, se ha encontrado una influencia de la composición interna de las vesículas en sus propiedades mecánicas. Así, las vesículas formadas por colesterol y CTAB han demostrado ser más rígidas que las formadas por colesterol y DOPC. Por otro lado, esta tesis se ha centrado en el uso de IBs para la funcionalización de superficies con diferentes patrones utilizando técnicas de litografía blanda. Posteriormente, se han caracterizado y evaluado las propiedades de estas superficies decoradas con IBs como soportes para el cultivo y guiaje de células. En el marco de esta tesis se han conseguido preparar con éxito patrones geométricos de IBs de alta resolución. Un análisis estadístico de los datos obtenidos a partir de imágenes de microscopía óptica y confocal ha permitido sacar demostrar, que la orientación, la morfología, y el posicionamiento de las células dependen de la geometría de los patrones. La siguiente etapa de la tesis estuvo centrada en la desarrollo de un dispositivo que permitiera la deposición de gradientes de IBs en superficies a partir de suspensiones coloidales. La técnica está basada en el fenómeno ampliamente conocido del “efecto de la gota de café”. Así, un dispositivo ha sido diseñado, construido, calibrado y utilizado con éxito para preparar sustratos funcionalizados con patrones, muy fáciles de modificar y con bajo coste. Con este dispositivo se ha propuesto e implementado un protocolo de la deposición de gradientes de nanopartículas. Los gradientes obtenidos se han caracterizado en cada caso confirmando la presencia de cambios lineales de concentración de IB sobre grandes áreas (ca 500 micras) tal y como se necesita para realizar estudios de motilidad celular. En la última parte de la tesis hemos utilizado los gradientes de IBs para estudiar la motilidad celular. El método de deposición permitió preparación de sustrato complejo con más de 80 diferentes entornos para el cultivo celular. Como conclusión general de esta parte podemos confirmar que existe un control claro de la motilidad celular provocada por los patrones de gradientes de IBs ingeniados. En general, con el trabajo realizado hemos demostrado que los IBs pueden considerarse una nueva herramienta muy interesante y útil para el guiaje celular, lo cual puede tener implicaciones muy interesantes en el campo de la medicina regenerativa y la ingeniería de tejidos.
This Thesis is focused on two systems of molecular nanoparticles that have a prospective use in nanomedicine. These systems are: (i) lipidic vesicles – supramolecular entities that are already used as drug delivery systems and (ii) Inclusion Bodies (IBs) - proteic nanoaggregates, that are emerging as a new tool in the light of tissue engineering The first part of this work is focused on the comparison of a vesicular system prepared by DELOS ‐ susp, a compressed fluid‐based single-step method previously developed in our group, and conventional multi‐step processes that are usually employed for vesicle production, like thin film hydratation or sonication followed by extrusion. We have compared also two different vesicle compositions: one system is based on DOPC phospholipid and cholesterol (liposome) and the other contain a quaternary ammonia amphiphile, CTAB, and cholesterol (quatsomes). To study the structural characteristics of both systems we have used the combination of two non-labelling methods: Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance with Dissipation (QCM-D) in order to obtain complementary data. We conclude that both investigated vesicular systems form layers of vesicles when interacting with gold surfaces. We have calculated the mass uptake, thickness, density and mechanical properties of the studied vesicular systems. We conclude, that the influence of the preparation method is negligible in the case of quatsomes. That is, the internal architecture, once achieved, determines the mechanical properties of these supramolecular entities. On the other hand, vesicles formed by quaternary salts and cholesterol have demonstrated to be more rigid than the liposomes based on phospholipid and cholesterol. The work developed in following three Chapters has been focused on the use IBs for surface engineering. In this part we have characterized and evaluated IBs decorated surfaces as supports for cell cultivation and guidance. The second Chapter is centred on the formation of two-dimensional microscale patterns of IBs using a soft lithography technique and evaluation of cell behaviour when cultivated on them. We have successfully prepared high resolution geometrical IBs patterns and cultivated cell on them. Basing on a deep, statistical analysis of data derived from optical and confocal microscopy images we derived conclusions about the influence of the IBs pattern geometry on cell´s behaviour. The orientation, morphology and positioning of cells clearly depends on the geometry of IBs patterns proving the usefulness of IBs for cell guidance. Moreover, the synergy between biological activity and topographical stimuli of cells by IBs has been confirmed. In the third Chapter, in order to study cell motility induced by IBs, we have focused on the design and engineering of a device allowing deposition of surface-bound IBs gradients from colloidal suspensions. The developed technique is based on the widely known coffee drop effect. A deposition device was constructed, calibrated and successfully used to prepare substrates with the desired patterns, which can be obtained in a fast (up to 1 mm per 1 min) and cost-effective manner. Also, a robust protocol for gradient deposition was proposed and implemented. The obtained gradients were characterized, confirming the presence of linear changes of IBs concentration over broad areas (c.a. 500 μm) as needed to perform cell motility studies. The last Chapter describes the use of the IBs gradients to study cell motility. A complex substrate with 80 different zones for high-throughput cell culture study was successfully produced. Individual cell movement assay were carried out using confocal microscopy time-lapse acquisition. Cell movement descriptors such as average travelled distance, directionality, etc. were quantified, enabling us to investigate in detail how factors such as the gradient slope or the concentration of IBs influence cell motility. As an overall conclusion of this part we can confirm the control over cell motility by the IBs gradient patterns. In general we have proved that IBs as well as the different two-dimensional engineering methods used are interesting and useful approaches with a prospective use in the control of cell guidance as well as a promising tools in regenerative medicine and tissue engineering.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.
Page 192 blank. Vita. Cataloged from PDF version of thesis, 2011.
Includes bibliographical references.
Iron oxides magnetic nanoparticles (MPs) of high crystallinity, high magnetization, and size-monodispersity were synthesized with oleic acid as their native ligands. These hydrophobic and non-functionalized MPs have magnetic properties that are suitable for various biological applications. Surface modifications were studied for transferring these MPs into biological environments as well as transforming them into functional nanoparticles. Certain surface modifications of MPs, such as attaching silane groups and silica coating, lead to formation of more complex structures of superparamagnetic and fluorescent silica microspheres and nanostructures. These microspheres and nanostructures comprising MPs and semiconductor quantum dots (QDs) are useful tools for biological applications such as for magnetically controlling with fluorescent tracking of particles and for bimodal imaging. Surface modifications of MPs with hydrophobically-modified polyacrylic acid (mPAA) amphiphilic polymer and catechol-derivative surfactants resulted in hydrophilic MPs that are stable in physiological environment and small in their hydrodynamic size. These MPs are also designed to possess active functional groups that are necessary for further conjugations with proteins and molecules of interest. These hydrophilic and functional MPs are useful in biological applications such as magnetic resonance imaging and sensing applications.
by Numpon Insin.
Ph.D.

2009/2010
Water-soluble gold nanoparticles represent an appealing scaffold for the preparation of robust and biocompatible bioconjugates. Indeed, many examples of gold nanoparticles-bioconjugates as new materials in several fields as material science, biology and medicine have been reported in the literature. The organic monolayer protecting the metallic core plays a key role in determining the properties of the system as stability, solubility, and specific interactions with biological environment. The present thesis is focused on the functionalization of water-soluble gold nanoparticles in order to develop new tools in diagnostics, drug-delivery and enhanced immuno-sensing. Gold nanoparticles protected by mixtures of ligands of different nature have been taken into consideration in the development of the three main projects of this thesis. The first project is about the synthesis of gold nanoparticles with a gold core of 1.7 nm suited for crystallization, in order to perform diffractometric analysis aimed to solve the structure of larger systems than that already reported and to find other geometries of the gold core. To this aim, gold nanoparticles protected by a monolayer of p-mercaptobenzoic acid have been synthesized, purified and characterized. The choice of an aromatic ligand with a carboxylic group imparts stability to the clusters and plays a strategic role in crystals formation. Crystallization trials under a variety of different conditions and preliminary observations about the stability of the nanoparticles are reported. Up to now suitable crystals for X-ray analysis could not be obtained. The second project is part of an ongoing investigation of the morphological organization of the monolayer protecting gold nanoparticles in order to complete previous studies carried out in our research group. Recent results from our laboratories, obtained by ESR measurements, support the formation of “patches” domains in the mixed-monolayer of water-soluble gold nanoparticles when mixtures of perfluoroalkyl- and alkylthiolates are used to form the monolayer. The complexity of these systems may also be increased introducing functional thiolates in the monolayer in a controlled topology. The preliminary results obtained so far should be completed with other investigations using different methodologies and supported by studies also on flat surfaces. Moreover, to understand the ability of the amphiphilic thiols to phase-segregate, we thought to study also micellar aggregates. The final goal is to use this phase-segregated monolayers to create clusters of functional thiols for multivalent recognition. Water-soluble gold nanoparticles coated by amphiphilic thiols of different lipophobicity have been prepared and characterized, and new ligands suited for the studies on micelles and on 2D self-assembled monolayers have been designed and synthesized. The results of Electron Spin Resonance (ESR), Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM) studies on these systems are reported and discussed. The third project is aimed to find new synthetic strategies to obtain biocompatible gold nanoparticles presenting multiple bioactive residues for multivalent recognition processes. In particular, a mimetic of the antigen GM3 Ganglioside Lactone with demonstrated antimelanoma reactivity was introduced in the monolayer of water-soluble gold nanoparticles for the development of a biological therapy against cancer. The preparation of nanoparticles of different size and loading of the antigen-mimetic is reported, together with their characterization and the preliminary biological investigations.
Nanoparticelle di oro solubili in mezzi acquosi rappresentano una piattaforma ideale per la sintesi di bioconiugati stabili e compatibili con le cellule. Infatti la letteratura scientifica riporta molti esempi di nanoparticelle di oro coniugate con biomolecole come prototipo di nuovi materiali applicabili in diversi ambiti tra cui la scienza dei materiali, la biologia e la medicina. Il monostrato organico che protegge il nocciolo metallico riveste un ruolo fondamentale nel determinare le proprietà dell’intero sistema quali la stabilità, la solubilità e le eventuali interazioni specifiche con i sistemi biologici. La presente tesi si focalizza sulla funzionalizzazione del monostrato di nanoparticelle di oro idrosolubili finalizzata allo sviluppo di nuovi strumenti ad uso diagnostico, terapeutico, e con applicazione nel sensing immunologico. Nello sviluppo dei tre progetti principali in cui la tesi si articola, sono state studiate nanoparticelle di oro protette da miscele di ligandi di natura diversa. Il primo progetto riguarda la sintesi di nanoparticelle di oro aventi diametro del gold core di 1.7 nm adatte alla cristallizzazione, al fine di effettuare un’analisi diffrattometrica che consentisse di risolvere la struttura di nanoparticelle di dimensioni maggiori rispetto a quelle riportate in letteratura e di individuare caratteristiche strutturali quali ad esempio la geometria del nocciolo di oro. A questo scopo sono state preparate nanoparticelle di oro protette da un monostrato composto da molecole di acido p-mercaptobenzoico, che sono state purificate e caratterizzate. La scelta di un ligando aromatico avente gruppi funzionali carbossilici conferisce particolare stabilità ai clusters e riveste un ruolo strategico nella formazione dei cristalli. Le prove di cristallizzazione in diverse condizioni sperimentali e alcune osservazioni preliminari riguardo la stabilità di queste nanoparticelle sono di seguito riportate. Finora non è stato ancora possibile ottenere cristalli adatti per le analisi diffrattometriche. Il secondo progetto è parte di un’indagine rispetto alla morfologia del monostrato protettivo delle nanoparticelle di oro, finalizzata a completare studi già avviati nel nostro gruppo di ricerca. Recenti risultati ottenuti nei nostri laboratori mediante misure ESR sono fortemente indicativi relativamente alla formazione di domini a “macchie” nel monostrato di nanoparticelle idrosolubili composto da miscele di tiolati alchilici e perfluoro-alchilici. Questi sistemi possono raggiungere un elevato livello di complessità mediante l’introduzione con controllo topologico di tiolati funzionalizzati. Il completamento dei risultati preliminari mediante l’impiego di ulteriori tecniche e il supporto mediante studi su superfici piane è un obiettivo di primaria importanza. Inoltre, la comprensione del fenomeno di segregazione tra tioli anfifilici potrebbe essere agevolata da studi su sistemi di tipo micellare. L’obiettivo finale è l’applicazione di suddetta segregazione di fase nella realizzazione di clusters con monostrati recanti tioli funzionalizzati per il riconoscimento multivalente. Sono state preparate e caratterizzate nanoparticelle di oro idrosolubili protette da tioli anfifilici aventi diversa lipofobicità, e sono stati progettati e sintetizzati nuovi ligandi adatti allo studio su aggregati di tipo micellare e su monostrati bi-dimensionali. I risultati ottenuti mediante Risonanza di Spin Elettronico (ESR), Microscopia a Scansione per effetto Tunnel (STM) e Microscopia a Forza Atomica (AFM) su questi sistemi sono di seguito riportati e discussi. Il terzo progetto è finalizzato alla realizzazione di nanoparticelle biocompatibili coniugate a molteplici unità di composti farmacologicamente attivi per il riconoscimento multivalente. In particolare, un mimetico dell’antigene GM3 Ganglioside Lattone con testata attività antitumorale nei confronti di cellule di melanoma è stato introdotto nel monostrato di nanoparticelle di oro idrosolubili nello sviluppo di una terapia antitumorale di tipo biologico. La sintesi di nanoparticelle di varie dimensioni e con diversa composizione del monostrato organico recanti il mimetico di antigene, ed i risultati ottenuti dai primi test biologici sono qui di seguito riportati.
XXIII Ciclo
1983

Les recherches décrites dans ce travail appartiennent à une branche de la science relativement jeune et interdisciplinaire, la nanophotonique. Les projets réalisés avaient pour objectif de décrire les phénomènes qui apparaissent lors de l’irradiation par un faisceau lumineux d’un matériau restreint à la dimension de quelques nanomètres à quelques centaines de nanomètres. Les phénomènes qui ont été examinés sont la génération d’absorption, de dispersion et d’émission fluorescente ainsi que le renforcement d’émission fluorescente et le renforcement du champ électromagnétique à une échelle plus petite que la limite de diffraction restreignant l’optique classique. Dans cette thèse, j’ai profité de nouvelles propriétés de la matière générées quand les dimensions sont réduites à l’échelle nanométrique (10-9 m). Elles se distinguent significativement des propriétés classiques qui caractérisent un matériau de plus grandes dimensions. Le changement de propriétés résulte de la limitation spatiale de la structure du nuage d'électrons et de l’augmentation du rapport entre la surface du matériau et son épaisseur. 23 Les particules plasmoniques, largement décrites dans ce travail, en sont un excellent exemple puisque leurs colloïdes possèdent une section efficace d'absorption très importante dans le domaine visible. Un colloïde peut présenter des couleurs différentes en fonction des formes, des dimensions et de la composition des particules qui le constituent, contrairement à une surface métallique qui ne doit son aspect qu'à la réflexion presque totale de la lumière visible et au lustre métallique. À l’échelle nanométrique, nous avons affaire à la résonance plasmonique de surface, un phénomène qui ouvre la porte à la manipulation, à la modification et au renforcement du champ électromagnétique autour de la nanostructure métallique. La possibilité de concentrer la lumière autour d’une nanoparticule au-dessous de la limite de diffraction a trouvé un bon nombre d’applications, dont la microscopie en champ proche, la spectroscopie Raman exaltée de surface (ang. Surface-enhanced Raman spectroscopy, SERS), la théranostique , la production de lecteurs de carte mémoire ou de cellules photovoltaïques. Les recherches décrites dans ce travail ont un caractère interdisciplinaire, elles améliorent nos connaissances dans le domaine de la synthèse de nanostructures plasmoniques, et des méthodes de séparation permettant d'obtenir des colloïdes qui contiennent des nanoparticules presque monodispersives. La méthode de synthèse d'un nouveau métamatériau, produit lors du transfert des nanobâtonnets d’or de l’eau à l’isopropanol, a aussi été présentée dans cette thèse. Par ailleurs, ces recherches ont montré une forte exaltation du champ électromagnétique parmi les nanoparticules. J’ai aussi dénoté une application potentielle de ce matériau en tant que substrat pour la détection de biomolécules. En outre, j’ai préparé des nanocoques d’or largement stables et dont l’épaisseur de dorure est contrôlée. À l’aide de la technique Z-scan, j’ai fait la mesure des propriétés non-linéaires des nanocoques d’or et je les ai comparées avec celles des nanobâtonnets d’or et de colorants organiques en indiquant une application possible. J’ai discuté aussi d'une nouvelle méthode de biofonctionnalisation des nanobâtonnets d’or qui m’a permis de créer un marqueur afin de visualiser des cellules vivantes. Il est aussi possible de convertir l’énergie lumineuse en énergie thermique par le biais des nanostructures plasmoniques, ce qui pourrait trouver d’autres applications intéressantes dans les recherches en théranostique
This dissertation shows the experimental results, which I strongly believe prove the possibility of application the proposed bioprobe in theranostics treatment. The advantages and disadvantages of the probe were discussed on the basis of imaging of cancer cells, toxicity and fluorescent efficiency. It is important to mention that the process of synthesis of the biomarker was controlled on each step, starting from the selection of appropriate size and shape of the core, through optical characterization, effective way of biofunctionalization and finally application in cell visualization.At first, I presented an improved method of separation of distinct shapes of gold nanoparticles from a heterogeneous mixture. The method of centrifugation in a glucose density gradient was applied in order to get homogenous fractions. The procedure of sample preparation, centrifugation and collection of the separated nanoparticles is described. Moreover, I discussed the synthesis with and without Ag+ ions added to the growth solution.Then, I had a closer look on transferring procedure of the NRs from water into IPA solvent, which induce self-organization of the nanoparticles. Optical characterization as well as recorded ATR spectra gave the foundations to understanding of the assembly process taking place. Additionally the work is enriched with the theoretical calculations indicating that individual self-assembled nanostructures show strong light polarization dependent properties. The electric field localized in the gap between NRs is estimated to be enhanced over 350 fold.In the next part of my thesis I have performed a systematic and quantitative description of the interactions of NRs with light (femtosecond laser pulses, 130 fs, 800 nm) in order to characterize the optical properties and design NRs with specific functionalities. In this work I focused on the investigation of structural changes of the NRs and the parameters influencing the reshaping, like surface modification using sodium sulfide, laser power and the position of the longitudinal surface plasmon resonance band (l-SPR) with respect to the laser wavelength.In the next part of the thesis I have quantified the probability of simultaneous absorption of two photons by plasmonic nanoparticles: gold nanorods and gold nanoshells, and by several dye molecules, by using the open-aperture Z-scan technique available in the laboratory at WUT in Poland. At first, I started from fabrication of stable and highly monodisperse NSs suspensions in water, with a varying degree of gold coverage. Then, the NLO properties of the nanoshells were quantified in terms of the two-photon absorption coefficient (α2), the nonlinear refractive index (n2), and the saturation intensity for one-photon absorption (Isat), which are extensive quantities. Then I calculated the two-photon absorption cross-section (σ2) taken per nanoparticle, which was also interpreted in terms of the merit factor σ2/M (where M is the molar mass of the nanoparticle), the quantity suitable for comparisons with other types of nonlinear absorbers.Finally, in the last chapter I have combined the results and knowledge from all previously described experiments in order to propose a new bioprobe. The probe is based on NR functionalized by DNA strand with attached fluorophore. The distance between gold surface and dye is selected in a such way as to maximize the fluorescent emission. The viability tests show low toxicity for cells and high compatibility. I showed that biofunctionalized NRs can provide fluorescent labeling of cancer cells and enable effective photothermal therapy. This is one of the first demonstrations of coupling a bioimaging application to a cancer therapy application using NRs targeted against a clinical relevant biomarker. I hope that the future studies will extend the in vitro concept demonstrated here to in vivo animal experiments

The overall goal of this project was the biological characterization by in vivo and in vitro testing of magnetic nanoparticles that can be used for various applications. The aim of this work was to understand which of the synthesized magnetic nanoparticles could be more suitable to be used as a carrier or platform for various applications in different scientific fields. Two different kinds of magnetic nanoparticles were developed: naked iron-oxide nanoparticles and silica or silica-based coated nanoparticles (core shell-type nanoparticles). Magnetic nanoparticles were prepared using a coprecipitation method. The structure, phase composition, physicochemical and surface properties, magnetic susceptibility, and release in vitro of MNPs were characterized by transmission electron microscopy, x-ray diffraction, scanning electron microscopy-energy dispersive x-ray spectroscopy, and a vibrating sample magnetometer. In vivo toxicity, in vitro toxicity, ROS production and genotoxicity were investigated. Therapeutic effects were evaluated by cell viability assays and flow cytometry assays. The tools developed in this thesis spanned a range of physical-chemical, biological and magnetic aspects and incorporate innovations on a nanometric range of scales. MNP-based technologies appear to hold a significant potential for a myriad of biomedical applications and the toxic potential of MNPs cannot be overlooked. For this reason we carried out different physicochemical and biological characterization of MNPs to identify a safe dose and formulation of MNPs. Understanding the relationship between the physicochemical properties of MNP constructs and their behavior will induce full translational potential of these nanoparticles. The magnetic nanoparticles prepared in this study have good biocompatibility and are suitable for further application in tumor hyperthermia.

Thesis (Ph. D.) -- University of Maryland, College Park, 2004.
Thesis research directed by: Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The transition metal ferrites of composition MFe2O4 where M is Fe, Co, or Ni are well established materials for various biological applications due to their interesting magnetic properties. Their elemental and stochiometric composition can be easily manipulated which allows further tuning of their ferrimagnetic properties. By changing the identity of M and by changing the crystallite size of the ferrites, nanocrystals with diverse magnetic properties can be systematically produced. Furthermore, ferrites are more stable in diverse chemical environments, as compared to metallic nanoparticles, which make ferrites particularly useful for a broad range of biomedical applications, especially in the field of magnetic resonance imaging and cell labeling. In this work, spinel ferrites of composition CoFe2O4, NiFe2O4, and Ni.5Co.5 Fe2O4 were synthesized by a polyol method utilizing ethylene glycol as the solvent, reducing agent, and surfactant. The nanoparticles produced were surface coated with 3-aminopropyltriethoxy silane to increase solubility as well as to serve as an anchor for further conjugation with targeting substrates such as peptides and antibodies. The first part of this dissertation was focused on using the polyol method to produce nanoparticles of various metallic compositions. In each case, the polyol method provided an easy one-pot method to produce metallic as well as metal oxide nanocrystals. Utilizing the polyol method, ferrites of CoFe2O4, NiFe2O4, and Ni.5Co.5 Fe2O4 were produced with size ranges between 20 nm and 50 nm depending on the reaction time in the polyol. The second part of this dissertation was concerned with the functionalization of the nanoparticles to serve as an anchor for further conjugation with targeting substrates in the immunoaffinity separation of food borne pathogens. These nanoparticles were functionalized using an anti-E. coli O157:H7 antibody, mixed with a food matrix, and then subsequently removed from the food matrix by an external magnet in order to be analyzed by Matrix Assisted Laser Desorption Ionization/Time of Flight (MALDI/TOF) Mass Spectrometry as a rapid identification method of bacterial pathogens. Furthermore, magnetic resonance imaging (MRI) was carried out on the polyol produced ferrites in order to measure the transverse relaxation time (T2) of the nanoparticles in order to investigate the size dependence and crystallite composition of the particles ability to affect the transverse relaxivity rarte (r2). Further understanding of how ferrite composition and crystallite size affect their magnetic properties and resulting MRI contrast abilities will provide insight into the best materials for the next generation of contrast agents. Lastly, the ability of nanoparticles to serve as a stationary phase material for reversed phase ultrahigh pressure liquid chromatography will be discussed as a novel separation technique.

This PhD thesis is dedicated to the preparation, characterisation and biological application of gold nanoconjugates. Gold nanoparticles are prepared by various modern preparation methods, and subsequently characterised by spectroscopic (UV-vis, ATR-FTIR, NMR), microscopic (TEM) techniques. The stability of the conjugates is evaluated both by using Zeta-potential studies and UV-vis. The nanoparticles are used in cellular uptake experiments using human glioblastoma cancer cells, and are found to possess a low cytotoxicity. The nanoparticles are found taken up by the cells and distributed in different cellular compartments.

Nanotechnology has made it possible to create materials with unique properties. This development offers new opportunities and overcomes challenges for many thermal transport applications. Yet, it requires a more fundamental scientific understanding of nanoscale transport. This thesis emphasizes how simulation, mathematical, and numerical methods can lead to more grounded studies of nanoscale thermal transport for biological and physical applications. For instance, magnetic fluid hyperthermia (MFH), an emerging cancer treatment, is a noninvasive method to selectively destroy a tumor by heating a ferrofluid-impregnated malignant tissue with minimal damage to the surrounding healthy tissue. We model the problem by considering an idealized spherical tumor that is surrounded by healthy tissue. The dispersed magnetic nanoparticles in the tumor are excited by an AC magnetic field to generate heat. The temperature distribution during MFH is investigated through a bioheat transfer relation which indicates that the P\'eclet, Joule, and Fourier numbers are the more influential parameters that determine the heating during such a thermotherapy. Thus, we show that a fundamental parametric investigation of the heating of soft materials can provide pathways for optimal MFH design. Since ferrofluid materials themselves play a key role in heating, we examine six materials that are being considered as candidates for MFH use. These are simulated to investigate the heating of ferrofluid-loaded tumors. We show that iron-platinum, magnetite, and maghemite are viable MFH candidates since they are able to provide the desired heating of a tumor which will destroy it while keeping the surrounding healthy tissues at a relatively safe temperature. Recent advances in the synthesis and nanofabrication of electron devices have lead to diminishing feature sizes. This has in turn increased the power dissipation per unit area that is required to cool the devices, leading to a serious thermal management challenge. The phonon thermal conductivity is an important material property because of its role in thermal energy transport in semiconductors. A higher thermal conductivity material is capable of removing more heat since higher frequency phonons are able to travel through it. In this thesis, the effects of surface stress on the lattice thermal conductivity are presented for a silicon nanowire. Based on a continuum approach, a phonon dispersion relation is derived for a nanowire that is under surface stress and the phonon relaxation time is employed to subsequently determine its thermal conductivity. The surface stress is found to significantly influence the phonon dispersion and thus the Debye temperature. Consequently, the phonon thermal conductivity decreases with increasing surface stress. Different magnitudes of surface stress could arise from various material coatings and through different nanofabrication processes, effects of which are generally unclear and not considered. Our results show how such variations in surface stress can be gainfully used in phonon engineering and to manipulate the thermal conductivity of a nanomaterial. The thermal transport during thermoelectric cooling is also an important property since thermoelectric devices are compact, reliable, easy to control, use no refrigerants and require lower maintenance than do more traditional refrigeration devices. We focus on the Thomson effect that occurs when there is a current flow in the presence of a temperature gradient in the material, and investigate its influence on an intrinsic silicon nanowire cooler. The temperature dependence of the Thomson effect has a significant influence on the cooling temperature. We also consider thermal nonequilibrium between electrons and phonons over the carrier cooling length in the nanowire. The results show that a strong energy exchange between electrons and phonons lowers the cooling performance, suggesting useful strategies for thermoelectric device design.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 70-82).
Materials that can interact with and transit membranes without toxic bilayer disruption or poration are of great interest in the drug delivery field. These materials can presumably bypass endocytosis to directly enter the cell cytosol through the plasma membrane, which is often the desired site of action for therapeutics, thereby avoiding potential and likely cargo degradation if trapped in endosomes. Alternatively, if these materials are endocytosed, they are often able to escape the endosome by interacting with and transiting the endosomal membrane, and moving into the cytosol. Here, we present a unique membrane-interacting, amphiphilic gold nanoparticle (amph-AuNP) system, which we show can interact with, embed within, and even penetrate through multiple adjacent lipid bilayers without evidence of membrane disruption or poration. By virtue of these key properties, we also present these amph-AuNPs as effective carriers for therapeutic molecules. Using a one-step reaction, we synthesized small -2-4 nm core size amph- AuNPs with an amphiphilic ligand shell comprised of one or two alkanethiols. Each amph-AuNP is coated by a mixture of long-chain mercaptoundecanesulfonate (MUS) terminated by a water-soluble sulfonate group, and in some cases, short-chain hydrophobic octanethiol (OT). First, we describe our efforts to adapt and develop a method to synthesize giant multilamellar model membranes to study amph-NP-membrane interaction in a well-defined setting. We show that giant membranes can be synthesized and fine-tuned by varying lipid composition and buffer salt concentration, can be fluorescently labeled with lipid tracers, and can be analyzed robustly with confocal microscopy and flow cytometry. Second, we describe our systematic analysis of amph- AuNP and membrane characteristics that influence mechanisms of NP association with bilayers. We study effects of general membrane properties such as electrostatics and phase that govern NP-membrane interactions, and found that NP penetration of bilayers was blocked under conditions where strong electrostatic repulsion or gel-phase lipids were employed. We further studied effects of AuNP core diameter, surface charge, and surface hydrophobicity on NP-membrane interactions at the nanoscale. We found that MUS particles with an optimal gold core size -2- 3nm in diameter and MUS:OT particles of a broader size range were capable of inducing hemifusion between liposomal membranes, while MUS:OT 2:1 particles of intermediate hydrophobicity were capable of spontaneously aggregating within the bilayer of vesicles to form Janus egg-like morphologies. Third, we built on these NP membrane-embedding properties to explore and characterize NP-embedding in erythrocyte membranes, with particular attention to the glycocalyx and membrane fluidity, for the future application of constructing therapeutic erythrocyte 'pharmacytes' in situ. Finally, we describe work in engineering amph-AuNPs to carry short antigenic peptide cargoes for in vivo vaccine applications, where immunization experiments have shown much promise for antigen-ferrying amph-AuNPs in eliciting robust and long-lasting CD8+ T cell responses.
by Prabhani U. Atukorale.
Ph. D.

Les Quantum Dots colloidaux (QDs) sont des nanocristaux colloidaux de semiconducteurs aux propriétés optiques uniques : finesse spectrale d’émission, large gamme spectrale d’excitation, brillance élevée. Cependant, leurs applications sont encore limitées par le clignotement de leur émission de fluorescence à l’échelle de la particule unique.Ce travail se concentre sur l’amélioration des propriétés optiques des QDs de CdSe/CdS, ainsi que sur leurs applications biologiques. Le développement d’une synthèse de nanocristaux de CdSe/CdS à coque épaisse a permis d’obtenir facilement des QDs non-clignotants à partir de cœurs de CdSe de cristallinité différente. Cependant, c’est QDs oscillent entre un état brillant et un état gris. La synthèse de QDs de CdSe/CdS à coque épaisse avec un gradient de composition entre le cœur et la coque produit des nanocristaux dont l’émission de fluorescence est parfaitement stable au cours du temps, et donc les rendements quantiques du mono- et du biexciton sont à 100% à l’air, à température ambiante. Les recombinaisons multiexcitoniques sont également efficaces permettant à un QD unique d’émettre de la lumière blanche à forte excitation. La croissance d’une coque d’or autour d’un QD (QDs-dorés) favorise le couplage entre l’exciton du semiconducteur et les plasmons du métal. Cet effet Purcell a pour conséquence d’accélérer les phénomènes radiatifs, diminuant le temps de vie et supprimant le clignotement du QD. De plus, la couche d’or agit comme une barrière contre la photooxydation et les QDs-dorés présentent une résistance plus élevée aux fortes puissantes d’excitation. Le contrôle de la forme des nanocristaux a permis la synthèse de nanoplaquettes, structures bidimensionnelles dont l’épaisseur est contrôlée à la monocouche atomique près. Une nouvelle synthèse de nanoplaquettes cœur/coque conduit à des propriétés intéressantes tant par la pureté de l’émission des nanocristaux que par leur résistance en température. Enfin, les QDs de CdSe/CdS, de par leur brillance et faible photoblanchiment, sont d’excellentes sondes fluorescentes pour l’imagerie biologique. Leur fluorescence et leur structure inorganique ont permis de réaliser de l’imagerie bimodale optique/électronique pour déterminer le nombre et la localisation précise de récepteurs synaptiques dans C. elegans. La monofonctionnalisation des QDs, nécessaire pour sonder certaines voies d’endocytose dans les cellules, a été réalisée grâce à l’encapsulation des QDs dans une nanocage d’ADN dont la formation est parfaitement contrôlée, à la base près. Ce complexe cage d’ADN – QDs a permis de suivre la dynamique d’endocytose des toxines Shiga dans la voie d’endocytose rétrograde jusqu’à l’appareil de Golgi
Colloidal Quantum Dots (QDs) are colloidal semiconductor nanocrystals with unique optical properties: narrow emission spectrum, large spectral range of excitation, high brightness. However, their applications are still limited by the blinking of their fluorescence emission at the single particle scale. This work focuses on the improvement of optical properties of CdSe/CdS QDs, as well as on the biological applications. The development of a synthesis of thick-shell CdSe/CdS nanocristals allowed easy obtaining of non-blinking QDs from CdSe cores of different crystallinity. However, these QDs flicker between an on and a grey state. The synthesis of thick-shell CdSe/CdS QDs with a composition gradient between the core and the shell produces nanocrystals whose fluorescence emission is perfectly stable with time. The quantum yields of the mono- and biexciton are 100% in air, at room temperature. Multiexcitonic recombinations are also efficient making a single QD emit white light under strong excitation. The growth of a gold nanoshell around a QD (golden-QDs) allows the coupling of the exciton of the semiconductor and the metal plasmons. This Purcell effect speeds up all the radiative processes, decreasing the lifetime and eliminating the blinking. Besides, the gold shell acts as a barrier against photooxidation and the golden-QDs show increased resistance to high excitation powers. The control of the shape of nanocrystals allowed the synthesis of nanoplatelets, bidimensionnal structures whose thickness is controlled to the atomic monolayer. A new synthesis of core/shell nanoplatelets leads to interesting properties due to the purity of the emission of the nanocrystals and to their resistance with temperature. Finally, Cdse/CdS QDs, because of the low photobleaching and high brightness, are excellent fluorescent probes for biological imaging. Their fluorescence and their inorganic structure were taken advantage of to perform bimodal optical/electron imaging to precisely localize and count synaptic receptors in C. elegans. Monofunctionalization of QDs, required to probe some endocytosis pathways in cells, was performed thanks to encapsulation of QDs in a DNA nanocage whose formation is perfectly controlled. This DNA cage – QD complex was used to study the dynamics of endocytosis of Shiga toxin in the retrograde endocytosis pathway, up to the Golgi apparatus

Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process.

Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory.

Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.

There is an increased interest in understanding the toxicity and rational design of gold nanoparticles (GNPs) for biomedical applications in recent years. Such efforts warrant reliable, viable, and biofriendly synthetic methodology for GNPs with homogeneous sizes and shapes, particularly sizes above 30 nm, which is currently challenging. In the present study, an environmentally benign, biofriendly, singlestep/ single-phase synthetic method using dextrose as a reducing and capping agent in a buffered aqueous solution at moderate temperature is introduced. The resulting GNPs are near-spherical, stable, catalytically active, place exchangeable, and water-soluble within the size range of 10-120 nm. The added advantage of the biologically friendly reaction medium employed in this new synthetic approach provides a method for the direct embedment/integration of GNPs into biological systems such as the E. coli bacterium without additional capping ligand or surface modification processes.

(Metallo-)Dendrimers in Catalysis, Nanoparticle Stabilization and Biological Application Technische Universität Chemnitz, Fakultät für Naturwissenschaften Dissertation 2011, 165 Seiten Die vorliegende Dissertationsschrift befasst sich mit der Darstellung, Charakterisierung und Anwendung neuartiger (Metallo-)Dendrimere. Den Schwerpunkt der Arbeit bildet dabei die terminale Funktionalisierung (Poly)amidoamin-basierender Dendrimere kleiner Generationen. Durch Standardpeptid-Knüpfungsreaktionen von 1,1´-(Diphenylphosphino)ferrocen-carbonsäure an dendritische (Poly)amidoamine ist eine Serie entsprechend funktionalisierter Metallodendrimere zugänglich. Die metallorganischen, Dendrimer-immobilisierten Engruppen können durch Zugabe von [Pd(3-C3H5)Cl]2 in heterobimetallische Übergangsmetallkomplexe umgewandelt werden und finden Einsatz als katalytisch aktive Systeme in C,C-Kreuzkupplungsreaktionen nach Heck. Ein weiterer Gegenstand der Arbeit ist die terminale Modifikation von (dendritischen) Ami-nen mit (Sp)-2-(Diphenylphosphino)ferrocen-1-carbonsäure. Nach erfolgter Umsetzung mit [Pd(3-C3H5)Cl]2 werden die erhaltenen planar-chiralen Verbindungen als Katalysatoren in asymmetrischen allylischen Substitutionsreaktionen eingesetzt. Ferner ist die Darstellung (Oligo)ethylenglykolether-terminierter (Poly)amidoamin-Dendrimere beschrieben. Diese werden als Stabilisatoren zur in-situ Generierung von Gold- sowie Magnetit-Nanopartikeln eingesetzt. Der Einfluss der dendritischen Template auf die Kolloidgrößen und Morphologien sowie die Eigenschaften der gebildeten Hybridmaterialien werden aufgezeigt. Darüber hinaus befasst sich die Arbeit mit der Verwendung biokompatibler (Oligo)ethylenglykolether-Dendrimere als Wirkstoffträger für Zytostatika bei der Krebsthera-pie. Die im Rahmen von in vitro Untersuchungen erhaltenen Ergebnisse werden präsentiert.

Ce travail de thèse a été réalisé au laboratoire de Physique de la Matière Condensée, au sein du groupe de Chimie du Solide. L'une des thématiques de cette équipe concerne l'élaboration de nanomatériaux luminescents, utilisés dans de nombreuses applications comme les systèmes d'éclairage ou de visualisation transparents. L'une des récentes applications de certains nanomatériaux luminescents a été dans le domaine de la biologie, tant pour le développement de biopuces, d'agents de contraste, de révélateurs de tissus. Parmi elles, l'utilisation comme sonde biologique fluorescente apparaît comme des plus prometteuses. Une sonde biologique est un objet attaché à la biomolécule ciblée qui peut facilement être détecté. Elle se divise en deux parties, la partie portant la « fonction » biologique, et la partie détectable. La détection peut se faire grâce à différentes propriétés physiques, et dans le cas présent, nous avons choisi de développer des sondes fluorescentes.

The idea of utilizing nanomaterials in bio-related applications has been extensively practiced during the recent decades. Magnetic nanoparticles (MPs), especially superparamagnetic iron oxide nanoparticles have been demonstrated as promising candidates for biomedicine. A protective coating process with biocompatible materials is commonly performed on MPs to further enhance their colloidal and chemical stability in the physiological environment. Mesoporous hollow silica is another class of important nanomaterials that are extensively studied in drug delivery area for their ability to carry significant amount of guest molecules and release in a controlled manner. In this study, different synthetic approaches that are able to produce hybrid nanomaterials, constituting both mesoporous hollow silica and magnetite nanoparticles, are described. In a two-step approach, pre-synthesized magnetite nanoparticles are either covalently conjugated to the surface of polystyrene beads and coated with silica or embedded/enclosed in the porous shell during a nanosized CaCO3 templated condensation of silica precursors, followed by acid dissolution to generate the hollow structure. It was demonstrated that the hollow interior is able to load large amount of hydrophobic drugs such as ibuprofen while the mesoporous shell is capable of prolonged drug. In order to simplify the fabrication procedure, a novel in-situ method is developed to coat silica surface with magnetite nanoparticles. By refluxing the iron precursor with mesoporous hollow silica nanospheres in polyamine/polyalcohol mixed media, one is able to directly form a high density layer of magnetite nanoparticles on silica surface during the synthesis, leaving reactive amine groups for further surface functionalization such as fluorescence conjugation. This approach provides a convenient synthesis for silica nanostructures with promising potential for drug delivery and multimodal imaging. In addition to nanoparticles, nanowires also benefit the research and development of instruments in clinical diagnosis. Semiconductive nanowires have demonstrated their advantage in the fabrication of lab-on-a-chip devices to detect many charge carrying molecules such as antibody and DNA. In our study, In2O3 and silicon nanowire based field effect transistors were fabricated through bottom-up and top-down approaches, respectively, for ultrasensitive bio- detection of toxins such as ricin. The specific binding and non-specific interaction of nanowires with antibodies were also investigated.

The work presented in this thesis follows two main branches. The first aims to develop instrumentation for 3D-STED microscopy and to apply it to the study of bulk diamond, nanoparticles and biological samples. The second aims to evaluate the application of fluorescence imaging and spectroscopy techniques to the study of luminescent defects in diamond. Building on previous work in the Photonics Group at Imperial College London, spatial light modulator (SLM) technology was incorporated into a STED system in a novel configuration to provide a robust and convenient solution for 3D-STED microscopy. This system was applied to the first reported super-resolution imaging of the interaction between two cells in their natural state. The system was further applied to STED imaging of nitrogen vacancy centres in bulk diamond and to a proof of principle experiment for novel plasmon-assisted labels for STED microscopy. The effects of wavefront aberration on STED microscopy were investigated and a predictive correction philosophy was developed based on spherical aberration induced by a refractive index mismatch. The flexibility offered by the SLM technology was taken advantage of to demonstrate recovery of STED imaging quality in glycerol and bulk diamond by active correction of spherical aberration experienced by the depletion point spread function. Confocal intensity imaging, confocal fluorescence lifetime imaging (FLIM) and multispectral fluorescence lifetime measurement were applied to the imaging of fluorescent defects in bulk diamond. It was demonstrated that FLIM can provide information that is complimentary to intensity imaging in diamond and that it is possible to spectrally distinguish defects in diamond while simultaneously measuring their lifetime using multispectral lifetime measurement methods. This thesis also presents the ongoing development of a system for STED of live samples that express green fluorescent protein (GFP).

Aquesta tesi està dedicada a la síntesi, caracterització i aplicacions de diferents nanomaterials que presenten la propietat de ser semiconductors. Aquesta dividida en tres blocs, en els quals, en el primer d'ells es parla sobre quantum dots (QDs), que són nanoparticulas fluorescents la longitud d'ona d'emissió varia amb la mida. Aquests materials s'estan utilitzant últimament com a substituts dels colorants orgànics ja que presenten avantatges, la principal és que no perden la seva emissió amb el temps. Aquests QDs han estat usats per estudiar la seva interacció amb l'or (que augmenta la seva intensitat de fluorescència), han estat encapsulats usant polímers per usar-los com a controls en citometria de flux i per silica per usar-los (un cop units a un peptido i un colorant orgànic adequat) com a detectors de fibrosi quística. Finalment també han estat usats en aquesta tesi per intentar seguir el moviment d'un receptor en plaquetes. En el segon bloc de la tesi es parla de up conversió nanoparticles, la diferència enfront dels QDs és que s'exciten a major longitud d'ona a la que emeten, pel que són capaços d'absorbir en el infraroig i emetre en el visible, fent-ideals per a aplicacions en biologia. En aquesta tesi es van usar per a reconèixer un receptor en neutrofilos i per introduir-lo dins de hidrotalcites (material que no és reconegut pel cos com estrany) per així poder alliberar-ho en l'organisme. Finalment, en el tercer bloc s'han sintetitzat materials per catalisis (sulfur de bismut) i per cel·les solars (òxid de titani)
Esta tesis esta dedicada a la sintesis, caracterizacion y aplicaciones de diferentes nanomateriales que presentan la propiedad de ser semiconductores. Esta dividida en tres bloques, en los cuales, en el primer de ellos se habla sobre quantum dots (QDs), que son nanoparticulas fluorescentes cuya longitud de onda de emision varia con el tamaño. Dichos materiales se estan usando ultimamente como sustitutos de los colorantes organicos ya que presentan ventajas, la principal es que no pierden su emision con el tiempo. Estos QDs han sido usados para estudiar su interaccion con el oro (que aumenta su intensidad de fluorescencia), han sido encapsulados usando polimeros para usarlos como controles en citometria de flujo y por silica para usarlos (una vez unidos a un peptido y un colorante organico adecuado) como detectores de fibrosis quistica. Finalmente tambien han sido usados en esta tesis para intentar seguir el movimiento de un receptor en plaquetas. En el segundo bloque de la tesis se habla de up conversion nanoparticles, cuya diferencia frente a los QDs es que se excitan a mayor longitud de onda a la que emiten, por lo que son capaces de absorber en el infrarojo y emitir en el visible, haciendolos ideales para aplicaciones en biologia. En esta tesis se usaron para reconocer un receptor en neutrofilos y para introducirlo dentro de hidrotalcitas (material que no es reconocido por el cuerpo como extraño) para asi poder liberarlo en el organismo. Finalmente, en el tercer bloque se han sintetizado materiales para catalisis (sulfuro de bismuto) y para celdas solares (oxido de titanio).
This thesis is dedicated to the synthesis, characterization and application of different nanomaterials that are semiconductors. It is divided in three blocks, in the first one we talk about quantum dots (QDs), that are fluorescent nanoparticles whose wavelength of emission changes with size. Such materials are being used as substitutes of organic dyes, due to the many advantages they present, the main one is that the fluorescence is not lost with time. These QDs have been used to study their interaction with gold ( that increases the fluorescence intensity), they have been encapsulated with polimers to be used as controls in flow cytometry or by silica to use them as sensors for cystic fibrosis (once they have been attatched to the right polymer and dye). Finally, in this thesis, they have been also used to track the movement of a platelet receptor. In the second block we talk about up conversion nanoparticles, which only difference regarding QDs is that they are excited using a longer wavelength than the emission, so they are able to absorb in the infrared and emit in the visible range of light, making them ideal for biological applications. We have use this materials to recognice an specific receptor in neutrophils as well as to be surrounded by hydrotalcite (body friendly material) so it can be released in the organism. Finally, in the third block we have syntesized materials for catalysis (bismuth sulfide) and for solar cells (titanium oxide for perovskite solar cells).

La presente Tesis Doctoral es un fruto de colaboración entre el Instituto Catalán de Nanociencia y Nanotecnología (ICN2) y el Hospital Clínic de Barcelona, implicados en el proyecto “Marató TV3 2012”, con el objetivo de utilizar las nanopartículas de óxido de Cerio (CeO2 Nps o “nanoceria”) como una nueva herramienta terapéutica en la regeneración del tejido hepático en las enfermedades del hígado. CeO2NPs son un material inorgánico fascinante, con una gran variedad de aplicaciones y muchas más por llegar. Lo que las hace tan interesantes es su alta capacidad de hacer de buffer de electrones en un entorno oxidante/reductor, gracias a su estructura electrónica incompleta en la capa 4f. Ésto hace posible que presente una habilidad de ser oxidada o reducida, seguida de captura o liberación de oxígeno o especies reactivas de oxígeno (ROS y los radicales libres, tales como OH·). Como consecuencia, nanoceria se comporta como una esponga natural de electrones libres. El desequilibrio de ROS tiene lugar en un gran número de enfermedades humanas. También, la sobreproducción de ROS es crítica en la neurodegeneración. A pesar de la atractiva capacidad antioxidante de CeO2 NPs, una controversia importante, en cuanto a su función biológica, fue descrita en la literatura actual. A lo largo de ésta Tesis, los métodos existentes de síntesis de nanoceria han sido analizados en detalle, cómo también la calidad de los productos obtenidos y los aspectos toxicológicos de ámbos (los procesos y los productos). Éste trabajo de investigación fue enfocado en sobrepasar las problemáticas existentes de la toxicidad de nanoceria (debida a la agregación de nanopartículas, la toxicidad del surfactante o el solvente, o a la contaminación de las muestras con endtoxina) y en diseño de soluciones útiles, con el objetivo de sacar el provecho máximo de las propiedades antioxidantes de CeO2NPs en recerca y aplicaciones biomédicos. Asimismo, el presente trabajo fue centrado en el estudio de las propiedades físico-químicas y bio-quimicas de nanoceria, para optimizar su preparación y tamaño (Capitulo 2), evaluar su reactividad (Capitulo 3), biodistribución (Capitulo 4) y no-toxicidad (Capitulo 2, Anexo 2). Finalmente, la biodistribución de nanoceria y sus efectos sobre los mediadores fibrogénicos e inflamatorios fueron evaluados a nivel molecular y celular, demostrando que la administración de CeO2NPs podría ser de interés terapéutico en enfermedades del hígado (Anexo 3).
The current Doctoral Thesis is the fruit of collaboration between the Catalan Institute of Nanoscience and Nanotechnology (ICN2) and the Hospital Clinic of Barcelona, involved in the project “Marató TV3 2012”, with the objective to apply Cerium oxide nanoparticles (CeO2 NPs) as a new therapeutic tool for tissue regeneration in liver diseases. CeO2NPs is a fascinating inorganic material with many applications and more to come. What makes nanoceria very appealing is its high capacity to buffer electrons from an oxidant/reducing environment due to the unfilled 4f electronic structure. This is due to its easy ability of being oxidized and reduced, followed by the capture or release of oxygen or reactive oxygen species (ROS and free radicals as OH·). As a result, nanoceria behaves as a natural electron sponge. Note that ROS disbalance takes place in an enormous number of human diseases. Also, the overproduction of ROS is critical in neurodegeneration. Despite the appealing redox catalytic capacity of CeO2 NPs, an important controversy upon biological effects of CeO2 has been numerously reported. During this Thesis, the existent methods of nanoceria preparation have been analyzed in detail, as well as the quality of the obtained products and the toxicological aspects of both (the processes and the products). This Doctoral research has been focused in overtaking the existent problematics of the nanoceria toxicity (due to aggregation of NPs, toxic surfactant or solvent, or contamination with endotoxin) and offering suitable solutions, in order to take full advantage of the antioxidant CeO2NPs properties in biomedical research and applications. Thus, the current work has been focused on the study of physicochemical and biochemical properties of CeO2 NPs, to optimize the preparation methods and the obtained product, in an environmentally-friendly way (Chapter 2, Annex 2). The optimization of the NPs size and monodispersity (Chapter 2); as well as the evaluation of the correspondent antioxidant activity (Chapter 3) were also performed. Finally, the in-vivo biodistribution study of CeO2 NPs, as well as their effects on inflammatory and fibrogenic mediators were evaluated at molecular and cellular level, demonstrating that administration of CeO2 NPs could be of therapeutic value in liver diseases (Annex 3).

Ces travaux de thèse sont dédiés à l’élaboration, la caractérisation et l’application des nanoparticules de silice mésoporeuse (MSNs) et d’organosilice (PMOs). Ces nanomatériaux qui font l’objet d’un grand intérêt dans la communauté scientifique, présentent des propriétés intéressantes telles que: une surface spécifique élevée, le contrôle de la morphologie, la porosité ajustable et enfin la facilité de fonctionnalisation de leur surface par des groupes fonctionnels. Dans cette thèse, deux familles de nanoparticules ont été étudiées : les nanoparticles de silice mésoporeuse (MSNs) et les nanoparticules d’organosilice.Premièrement, les MSNs synthétisées avec une taille de 200 nm et une surface spécifique de 810 m2/g ont été fonctionnalisées d’une manière covalente avec des antioxydants, un polyphénol l’acide caféique (CAF) qui est lié par sa fonction acide (-COOH) à une fonction amine (préalablement greffée sur la silice), ou un flavonoïde la ruine (RUT) qui est liée par l’isocianatopropylthrietoxysilane comme intermédiaire silicique. Les antioxydants ont été greffés afin de diminuer le stress oxydant. Les effets cellulaires sont étudiés sur deux lignées, une lignée de cellules issues d’un carcinome colo-rectal (lignée Caco-2) et une lignée tumorale de la peau (lignée HaCaT).Dans une deuxième partie, nous avons étudié l’influence de l’ajout d’un co-solvant «éthanol» au cours de la synthèse sur la morphologie des nanoparticules de silice mésoporeuse qui entraine la formation de nanoparticules de forme bâtonnet (MSNR) ainsi que le changement de structure de la poosité. Par la suite, les NPs de forme sphérique MSNA et les NPs de forme bâtonnet MSNR ont été chargées par la doxorubicine (DOX) et testées in vitro sur des cellules MCF-7.De plus, les synthèses de nanoparticules d’organosilice ont été réalisées. Ces nanomatériaux sont exclusivement synthétisés à partir de bis (3-méthoxysilyl propyl) -N-méthylamine et bis (triéthoxysilylpropyl) amine. Tout d'abord, la synthèse des NPs d’organosilice à cavité (HPONP) est decrite. Les HPONP ont été utilisés alors pour la délivrance de méthotrexate dans des cellules MCF-7. Deuxièmement, la synthèse d'autres types de NP d’organosilice (HMONP) obtenus par condensation du précurseur de bis (3-méthoxysilyl propyl) -N-méthylaminea été étudiée. Afin d'élargir la cavité des NP, nous avons signalé l'utilisation de TEB comme agent gonflement conduisant à la synthèse des HMLONP à large cavité. La morphologie et les compositions des NP ont été complètement caractérisées par diverses techniques et la délivrance de pepstatine à partir de HMLONP est envisagée. Pour ajouter une biodégradabilité aux nanocarriers, des nanoparticules mixtes ont été synthétisées par condensation de la bis (3-méthoxysilyl) propylméthylamine et du bis [3-(triéthoxysilyl) propyl] disulfure. Différentes nanoplatformes ont été conçues et entièrement caractérisées. La biodégradabilité a été évaluée dans des conditions quasi physiologiques. En outre, la voie de synthèse a été modifiée pour concevoir des nanoparticules d'organosilice à base d'éthylène ou de porphyrine. Ces nanoparticules ont été testées in vitro avec des cellules de cancer du sein et utilisées pour la délivrance de méthotrexate et de gemcitabine monophosphate.Enfin, on a décrit les nanoparticules organosilice de type cœur coquille. La coquille de ces nanoparticules obtenue par condensation du bis- (triéthoxysilyl) éthane et du bis (3- (triéthoxysilyl) propyl) tétrasulfure. Ces nanoparticules biodégradables du fait des groupements tetrasulfure ont été testées in vitro avec des cellules de cancer du sein pour l'imagerie et la délivrance d’un anticancéreux
This work is dedicated to the development, characterization and application of nanoparticles of mesoporous silica (MSNs) and organosilica (PMOs) nanoparicles. Silica nanoparticles became the subject of intense research worldwide for many reasons: their unique chemical and physical characteristics, high biocompatibility, various shapes ranging from spheres to rods with tunable diameter, easily functionalizable surface, and the ability to be used as a shell on different type of inorganic nanoparticles such as gold, iron oxide, lanthanide nanoparticles. In this dissertation mesoporous silica NPs and organosilica NPs have been designed, optimized and fully characterized. These two types of silica NPs have been applied for biological applications (drug delivery and bioimaging).First, mesoporous silica nanoparticles (MSNs) were designed and were covalently coated with antioxidant molecules, namely, caffeic acid (MSN-CAF) or rutin (MSN-RUT), in order to diminish the impact of oxidative stress induced after transfection into cells. Two cellular models involved in the entry of nanoparticles in the body were used for this purpose: the intestinal Caco-2 and the epidermal HaCaT cell lines. Rutin gave the best results in terms of antioxidant capacities preservation during coupling procedures, cellular toxicity alleviation, and decrease of ROS level after 24 h incubation of cells with grafted nanoparticles.Secondly, we studied the control of the shape of MSNs by the addition of ethanol (EtOH) as cosolvent. Spherical (MSNA) or Rod MSNs (MSNR) were obtained, and then loaded loaded with doxorubicin and incubated with MCF-7 breast cancer cells. MSNA and MSNR particles were efficient in killing cancer cells but their behaviour in drug delivery was altered on account of the difference in their morphology.Then, the syntheses of new organosilica nanoparticles are reported. These nanomaterials are exclusively synthesized from bis (triethoxysilylpropyl) amine (BTSPA), bis (3-methoxysilyl propyl) -N-methylamine (BMSPMA) and bis- (triethoxysilyl) ethane precursors. First, it is reported the synthesis of hollow organosilica NPs (HPONPs) obtained through the condensation of bis (triethoxysilylpropyl) amine precursor by sol-gel process. HPONPs were used then for methotrexate delivery in MCF-7 cells. Secondly, it is reported the synthesis of other types of hollow organosilica NPs (HMONPs) obtained through the condensation of bis (3-methoxysilyl propyl) -N-methylamine precursor. In order to enlarge the cavity of NPs, we reported the use of TEB as swelling agent leading to the synthesis of HMLONPs. The morphology and the compositions of the NPs were fully characterized by various techniques and the pepstatin delivery from HMLONPs are under considaration. To add biodegradability to the nanocarriers, mixed nanoparticles were synthesized through the condensation of bis (3-methoxysilyl) propyl methylamine and the bis [3-(triethoxysilyl) propyl] disulfide. Different nanoplatforms were designed and fully characterized. The biodegradability was assessed in near-physiological conditions. Furthermore, the synthesis pathway was modified to design ethylene-porphyrin based organosilica nanoparticles. These nanoparticles were tested in vitro with breast cancer cells and used for methotrexate and gemcitabine monophosphate delivery.Finally, gold core shell mixed organosilica nanoparticles were described. The mixed shell of these nanoparticles was obtained by the co-condensation of bis- (triethoxysilyl) ethane and the bis (3-(triethoxysilyl) propyl)tetrasulfide. These biodegradable nanoparticles were tested in vitro with breast cancer cells for photon fluorescence imaging and core shell NPs were studied for drug delivery

[ES] La presente tesis doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el diseño, síntesis, caracterización y evaluación de distintos nanodispositivos híbridos orgánico-inorgánicos. En concreto, se utilizan como soporte nanopartículas mesoporosas de sílice y nanopartículas de oro para su aplicación biomédica, en concreto en el campo del cáncer de mama. En el primer capítulo se introduce el marco general en el que se engloban los estudios realizados. Se presentan los conceptos relacionados con nanotecnología y nanomedicina, así como la interacción de las nanopartículas a nivel biológico con el organismo y las células. Finalmente, se introducen conceptos básicos del cáncer de mama y la aplicación de nanomateriales como terapia. A continuación, en el segundo capítulo, se exponen los objetivos de la presente tesis doctoral que son abordados en los siguientes capítulos experimentales. En el tercer capítulo se describe el primer nanomaterial para la liberación controlada de dos inhibidores (navitoclax y S63845) de las proteínas anti- apoptóticas de la familia Bcl-2. Este sistema se ha diseñado con el objetivo de superar la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. En concreto, se han preparado nanopartículas mesoporosas de sílice cargadas con navitoclax y S63845, y funcionalizadas con un aptámero dirigido a la proteína de superficie MUC1, que actúa como puerta molecular. En este trabajo hemos demostrado que las nanopartículas diseñadas son internalizadas preferentemente por células tumorales de cáncer de mama. También hemos demostrado la capacidad de las nanopartículas de revertir la resistencia a navitoclax en un modelo celular de cáncer de mama triple negativo. Además, ponemos de manifiesto la disminución del principal efecto adverso (trombocitopenia) asociado a la administración del navitoclax en su formulación libre, gracias a la encapsulación en las nanopartículas. En el capítulo cuatro se presenta un sistema sensible a pH para la liberación controlada de un cargo fluorescente y la maquinaria de edición génica basada en el sistema CRISPR/Cas9, dirigido a la edición del gen codificante de la proteína fluorescente verde (GFP, del inglés gren fluorescent protein). El nanodispositivo está constituido por nanopartículas mesoporosas de sílice cargadas con rodamina B, funcionalizadas con polietilenimina y revestidas con el plásmido codificante del sistema CRISPR/Cas9. En este trabajo se ha demostrado el escape lisosomal de las nanopartículas, mediado por el efecto esponja de protones de la PEI. Asimismo, mostramos un nanodispositivo pionero en su campo, basado en nanopartículas mesoporosas de sílice, capaz de realizar la doble función de llevar a cabo la edición del gen codificante de GFP y la liberación exitosa del cargo fluorescente. En el quinto, y último, capítulo experimental se propone una nueva aproximación para realizar una terapia enzimática prodroga empleando nanopartículas de oro como transportadores enzimáticos. En este caso, se aborda la funcionalización de nanopartículas de oro con la enzima peroxidasa de rábano (HRP, del inglés horseradish peroxidase), capaz de transformar la prodroga inocua ácido indol-3-acético en especies radicales que resultan tóxicas para las células tumorales. En este capítulo se ha demostrado el efecto terapéutico del nanodispositivo en combinación con la prodroga en modelos celulares de cáncer de mama de los subtipos luminal A y triple negativo. Además, se ha confirmado la eficacia terapéutica del sistema en esferoides tumorales formados por células de cáncer de mama triple negativo. Por último, se presentan en el capítulo seis las conclusiones extraídas del desarrollo de esta tesis doctoral. Los resultados obtenidos en este trabajo contribuirán al desarrollo de nuevos nanomateriales inteligentes con aplicación en diversas áreas de la nanomedicina.
[CA] La present tesi doctoral titulada "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" se centra en el disseny, síntesi, caracterització i avaluació de diferents nanodispositius híbrids orgànic-inorgànics. En concret, s'utilitzen com a suport nanopartícules mesoporoses de sílice i nanopartícules d'or per a la seua aplicació biomèdica, en concret en el camp del càncer de mama. En el primer capítol s'introdueix el marc general en el qual s'engloben els estudis realitzats. Es presenten els conceptes relacionats amb la nanotecnologia i nanomedicina, així com la interacció de les nanopartícules a nivell biològic amb l'organisme i les cèl·lules. Finalment, s'introdueixen conceptes bàsics del càncer de mama i l'aplicació de nanomaterials com a teràpia. A continuació, en el segon capítol, s'exposen els objectius de la present tesi doctoral que són abordats en els següents capítols experimentals. En el tercer capítol es descriu el primer nanomaterial utilitzat per a l'alliberament controlat de dos inhibidors (navitoclax i S63845) de les proteïnes anti-apoptòtiques de la família Bcl-2. Aquest sistema s'ha dissenyat amb l'objectiu de superar la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. En concret, s'han preparat nanopartícules mesoporoses de sílice carregades amb navitoclax i S63845, i funcionalitzades amb un aptàmer dirigit a la proteïna de superfície MUC1, que actua com a porta molecular. En aquest treball hem demostrat que les nanopartícules dissenyades són internalitzades preferentment per cèl·lules tumorals de càncer de mama. També hem demostrat la capacitat de les nanopartícules de revertir la resistència a navitoclax en un model cel·lular de càncer de mama triple negatiu. A més, posem de manifest la disminució del principal efecte advers (trombocitopènia) associat a l'administració del navitoclax en la seua formulació lliure, gràcies a l'encapsulació en les nanopartícules. En el capítol quatre es presenta un sistema sensible a pH per a l'alliberament controlat d'una càrrega fluorescent i la maquinària d'edició gènica basada en el sistema CRISPR/Cas9, dirigit a l'edició gènica del gen codificant de la proteïna fluorescent verda (GFP, del anglés gren fluorescent protein). El nanodispositiu està constituït per nanopartícules mesoporoses de sílice carregades amb rodamina B, funcionalitzades amb polietilenimina i revestides amb el plàsmid codificant del sistema CRISPR/Cas9. En aquest treball s'ha demostrat la fuga lisosomal de les nanopartícules, mediat per l'efecte esponja de protons de la PEI. Així mateix, vam mostrar un nanodispositiu pioner en el seu camp, basat en nanopartícules mesoporoses de sílice, capaç de realitzar la doble funció de dur a terme l'edició del gen codificant de la GFP i l'alliberament exitós de la càrrega fluorescent. En el cinqué i últim capítol experimental es proposa una nova aproximació per a realitzar una teràpia enzimàtica prodroga emprant nanopartícules d'or com a transportadors enzimàtics. En aquest cas, s'aborda la funcionalització de nanopartícules d'or amb l'enzim peroxidasa de rave (HRP, del anglés horseradish peroxidase), capaç de transformar la prodroga innòcua àcid indol-3-acètic en espècies radicals que resulten tòxiques per a les cèl·lules tumorals. En aquest capítol s'ha demostrat l'efecte terapèutic del nanodispositiu en combinació amb la prodroga en models cel·lulars de càncer de mama dels subtipus luminal A i triple negatiu. A més, s'ha confirmat l'eficàcia terapèutica del sistema en esferoides tumorals formats per cèl·lules de càncer de mama triple negatiu. Finalment, en el capítol sis es presenten les conclusions extretes del desenvolupament d'aquesta tesi doctoral. Els resultats obtinguts en aquesta tesi contriburan al desenvolupament de nous nanomaterials intel·ligents amb aplicació en diverses àrees de la nanomedicina.
[EN] This Ph.D. thesis entitled "Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications" is focused on the design, synthesis, characterisation, and evaluation of several hybrid organic-inorganic nanomaterials. We have developed mesoporous silica nanoparticles and gold nanoparticles for biomedical applications, specifically in the breast cancer area. The first chapter includes an overview of the concepts related to the research performed. Introductory notions about nanotechnology and biomedicine are presented, as well as the basis of the interactions of nanoparticles with biological systems. Finally, breast cancer disease and the application of nanomaterials as therapy are described. Next, in the second chapter, the objectives addressed in the following experimental chapters are displayed. In the third chapter, we present the first nanomaterial for the controlled delivery of two inhibitors (navitoclax and S63845) of the Bcl-2 anti-apoptotic proteins. This nanosystem has been designed to overcome navitoclax resistance in a triple-negative breast cancer cellular model. We have prepared mesoporous silica nanoparticles loaded with navitoclax and S63845 and functionalised with an aptamer targeting MUC1 surface protein as a molecular gate. In this work, the specific targeting of the nanodevice to breast cancer cells has been demonstrated. The ability to overcome navitoclax resistance has been shown in navitoclax-resistant triple-negative breast cancer cells. Furthermore, navitoclax encapsulation in the nanoparticles has proved to reduce the main adverse effect (thrombocytopenia) associated with free formulated drug administration. In the fourth chapter, we describe a pH-responsive nanosystem for the controlled co-delivery of a fluorescent cargo and the genome-editing machinery based on CRISPR/Cas9, which targets the green fluorescent protein (GFP) coding gene. The nanodevice consists of mesoporous silica nanoparticles loaded with rhodamine B, functionalised with polyethyleneimine, and capped with the CRISPR/Cas9 plasmid. In the present work, we have shown the lysosomal scape capacity of the nanodevice enhanced by the proton sponge effect of PEI. We have also demonstrated a pioneering mesoporous silica-based nanodevice efficient in the simultaneous genome editing of the GFP gene (as a model gene) and the successful release of a fluorescent cargo (as a model drug). In the fifth and last experimental chapter, we propose a new approximation to develop enzyme prodrug therapy using gold nanoparticles as enzyme carriers. In this case, we use gold nanoparticles functionalised with the enzyme horseradish peroxidase (HRP), which transforms the non-toxic prodrug indol-3-acetic acid into radical species toxic to tumour cells. In this chapter, the therapeutic effect of the nanodevice in combination with the prodrug has been demonstrated in two breast cancer cell subtypes (luminal A and triple-negative breast cancers). Also, the therapeutic effect of the material has been corroborated in multicellular tumour spheroid-like cultures formed by triple-negative breast cancer cells. Finally, in the sixth chapter, the conclusions derived from the presented studies and the general conclusions of this Ph.D. thesis are released. The obtained results will promote the development of new smart nanomaterials with diverse biomedical applications.
Gema Vivo-Llorca thanks the Generalitat Valenciana for her fellowship ACIF/2017/072. Vicente Candela-Noguera thanks the Spanish Government for his fellowship FPU15/02753. We would like to thank Servier for the workart used in the figures of this manuscript (Servier Medical Art https://smart.servier.com/). We thank the Spanish Government (project RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE); SAF2017-84689-R-B (MCUI/AEI/FEDER, UE)) and the Generalitat Valenciana (project PROMETEO/2018/024 and PROMETEO/2019/065) for support.
Vivo Llorca, G. (2021). Mesoporous silica and gold-based nanodevices: new controlled delivery platforms for biomedical applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172713
TESIS

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-01-23T17:09:40Z No. of bitstreams: 1 Thaynara_Schafer_2017.pdf: 1932282 bytes, checksum: 89dd37a8b10b1470458f461465755dfc (MD5)
Made available in DSpace on 2018-01-23T17:09:40Z (GMT). No. of bitstreams: 1 Thaynara_Schafer_2017.pdf: 1932282 bytes, checksum: 89dd37a8b10b1470458f461465755dfc (MD5) Previous issue date: 2017-11-20
The textile industries present a high polluting potential, due to the generation of large volumes of liquid waste, containing high organic load and strong coloration derived from the dyes. These effluents can be treated by physical, chemical and biological processes. Among these processes, the adsorption has been widely studied for the removal of water dyes, due to the lower costs, simplicity of operation and high efficiency. Methylene blue (AM), a dye widely used in the textile industry is responsible for the strong staining in the effluents. Even in small amounts (<5 mg Pt Co / L), just as other textile dyes are very visible and affect the appearance, transparency and solubility of the gases, damaging the environment. A material that has been applied as adsorbent for dye removal in wastewater treatment is the magnetic nanoparticle, because it has high adsorption capacity, low cost and magnetic character. The nanoparticles can be prepared by the electrochemical method, thermal decomposition, hydrothermal synthesis, microemulsion, decomposition-precipitation, coprecipitation, chemical vapor deposition and impregnation. Of the most well-known methods of preparation, the coprecipitation method is the oldest, the simplest, the most efficient, and the one that allows greater production on a large scale. In this work, the coprecipitation method was used to synthesize the iron oxide (Fe3O4) nanoparticle obtained by the stoichiometric mixture of Fe2+ and Fe3+ salts in aqueous medium. This material was characterized and applied in solutions with different concentrations of the methylene blue dye in order to study its adsorption capacity. The same procedure was performed with the nanoparticulate compound (Sigma-Aldrich), in order to compare the adsorption capacity. The nanoparticulate material was characterized by magnetization, X-ray diffraction (XRD), thermogravimetric analysis (TGA), infrared spectroscopy (FTIR), ultraviolet/ visible (UV/VIS) spectroscopy, scanning electron microscopy) and optical microscopy (MO). By magnetization it was possible to observe the movement of the dispersed particles in aqueous medium toward the magnetic field of the neodymium magnet, which is a property of the ferromagnetic materials. With the solid samples, the supported weight capacity was tested, whereas for the synthesized compound it was 85, 3 g and for the nanoparticulate compound was 105,3 g, it being possible to explain this difference by packaging the particles. The greater the packing of the particles, the greater the magnetic force exerted. By the study of the effect of the pH and temperature of the medium, it was confirmed that at pH 7 and ambient temperature, the interference of the medium does not occur by the surface charges of the adsorbents and thus favoring the dye adsorption process. By reading the absorbances of the AM solutions, it was possible to calculate the equilibrium concentrations and to determine the amount of dye adsorbed by the nanoparticles. At low concentrations (5,0 x 10-6 mol L-1 to 1,0 x 10-5 mol L-1) a linear behavior is observed due to the presence of monomers already in concentrations higher than 1,0x10-5 mol. L-1 has the alteration of the linear behavior of the adjusted line, characteristic of the formation of aggregate and alteration of the coefficient of molar absorption. Scanning and optical electron microscopy showed that the images presented different forms for the nanoparticulate and nanoparticulate nanoparticles (Sigma-Aldrich), and the nanoparticulate presented reduced size and rounded shape when compared to the synthesized compound. By the diffractograms it was inferred that the synthesized material presented in its composition the magnetite (Fe3O4) and hematite (Fe2O3), the nanoparticulate only the presence of magnetite. Through the FTIR spectra for the nanoparticle samples, the major bands corresponding to the Fe-O and O-H bonds were investigated. By means of the TGA analysis, a loss of 2,31% of mass was observed in the variation of 50 to 150ºC, attributed to the presence of water in the sample. Above 150°C, there was another loss of 1,20% corresponding to the mass of gaseous substances, possibly carbon dioxide (CO2) or oxygen gas (O2).
As indústrias têxteis apresentam um elevado potencial poluente, devido à geração de grandes volumes de resíduos líquidos, contendo alta carga orgânica e forte coloração derivada dos corantes. Esses efluentes podem ser tratados por processos físicos, químicos e biológicos. Dentre estes processos, a adsorção vem sendo amplamente estudada para a remoção de corantes de águas, devido os menores custos, simplicidade de operação e alta eficiência. O azul de metileno (AM), um corante amplamente utilizado na indústria têxtil é responsável pela forte coloração nos efluentes. Mesmo em pequena quantidade (< 5 mg Pt Co/L), assim como os demais corantes têxteis são bastante visíveis e afetam a aparência, a transparência e a solubilidade dos gases, prejudicando o meio ambiente. Um material que vem sendo aplicado como adsorvente para a remoção de corante no tratamento de águas residuarias é a nanopartícula magnética, pois possui alta capacidade de adsorção, baixo custo e caráter magnético. As nanopartículas podem ser preparadas pelo método eletroquímico, decomposição térmica, síntese hidrotérmica, microemulsão, decomposição-precipitação, coprecipitação, deposição química a vapor e a impregnação. Dos métodos de preparo mais conhecidos, o método da coprecipitação é o mais antigo, simples, eficiente e o que permite maior produção em larga escala. Neste trabalho, o método da coprecipitação foi utilizado para sintetizar a nanopartícula de óxido de ferro (Fe3O4), obtida pela mistura estequiométrica de sais de Fe2+ e Fe3+ em meio aquoso. Este material foi caracterizado e aplicado em soluções com diferentes concentrações do corante azul de metileno, a fim de, estudar a sua capacidade de adsorção. O mesmo procedimento foi realizado com o composto nanoparticulado (Sigma–Aldrich), com o propósito de comparar a capacidade de adsorção. O material sintetizado e o composto nanoparticulado foram caracterizados por magnetização, difração de raios-X (DRX), análise termogravimétrica (TGA), espectroscopia de infravermelho (FTIR), espectroscopia ultravioleta/visível (UV/VIS), microscopia eletrônica de varredura (MEV) e microscopia óptica (MO). Por magnetização foi possível observar o movimento das partículas dispersas em meio aquoso em direção ao campo magnético do ímã de neodímio, sendo esta uma propriedade dos materiais ferromagnéticos. Com as amostras sólidas, testou-se a capacidade de peso suportado, sendo que para o composto sintetizado foi de 85,3 g e para o composto nanoparticulado foi de 105,3 g, sendo possível explicar esta diferença pelo empacotamento das partículas. Quanto maior o empacotamento das partículas, maior a força magnética exercida. Pelo estudo do efeito do pH e temperatura do meio, confirmou-se que em pH 7 e temperatura ambiente, não ocorre a interferência do meio pelas cargas da superfície dos adsorventes e assim tem-se o favorecimento no processo de adsorção do corante. Por meio da leitura das absorbâncias das soluções de AM, foi possível calcular as concentrações no equilíbrio e determinar a quantidade de corante adsorvido pelas nanopartículas. Em baixas concentrações (5,0x10-6 mol. L-1 a 1,0x10-5 mol. L-1) tem-se um comportamento linear, devido à presença de monômeros, já em concentrações superiores a 1,0x10-5 mol. L-1 tem-se a alteração do comportamento linear da reta ajustada, característica da formação de agregado e alteração do coeficiente de absorção molar. Por microscopia eletrônica de varredura e óptica foi observado que as imagens apresentaram formas diferenciadas para a nanopartícula sintetizada e nanoparticulado (Sigma– Aldrich), sendo que o nanoparticulado apresentou tamanho reduzido e formato arredondado quando comparado ao composto sintetizado. Pelos difratogramas inferiu-se que o material sintetizado apresentou em sua composição a magnetita (Fe3O4) e hematita (Fe2O3), já o nanoparticulado somente a presença de magnetita. Através dos espectros de FTIR para as amostras das nanopartículas, averiguaram-se as principais bandas correspondentes as ligações Fe-O e O-H. Por meio da análise TGA, observou-se uma perda de 2,31% de massa na variação de 50 a 150ºC, atribuído a presença de água na amostra. Acima de 150ºC, houve outra perda de 1,20% correspondente a massa de substâncias gasosas, possivelmente dióxido de carbono (CO2) ou O2 (gás oxigênio).

Les nanoparticules d’or (Au NPs) ont montré des résultats prometteurs en nanomédecine appliquée à la cancérologie. Elles sont capables de s’accumuler dans les zones tumorales, d’induire un effet thérapeutique en délivrant des principes actifs ou un effet photo/radiothérapeutique grâce à leurs propriétés d’absorption d’énergie. Elles permettent aussi le diagnostic par différentes techniques d’imagerie. Cette double activité les définit comme des agents théranostics. Les nanoclusters d’or (Au NCs) forment une sous-famille intéressante de Au NPs. Ils sont composés d’une dizaine à une centaine d’atomes d’or stabilisés par des molécules organiques. Leur taille inférieure à ~8 nm leur permet d’être éliminé par les reins et d’avoir des propriétés de photoluminescence (PL) jusque dans l’infrarouge, une fenêtre spectrale adaptée à l’imagerie optique in vivo. Ils peuvent aussi induire la mort cellulaire sous irradiation en raison des propriétés intrinsèques de l’or. Leurs propriétés optiques, de circulation sanguine et d’accumulation tumorale sont sensibles à de faibles modifications de la taille des Au NCs et de leur chimie de surface. Actuellement, les résultats précliniques sont encore insuffisants pour espérer un transfert en clinique et il est nécessaire d’améliorer la caractérisation des Au NCs et d’étudier leur comportement in vitro et in vivo.Dans ce contexte, mon projet de thèse a consisté à fonctionnaliser ces Au NCs pour améliorer leur accumulation tumorale. La première stratégie repose sur l’auto-agrégation des Au NCs dans le microenvironnement tumoral. Pour cela la surface des Au NCs a été soit i) fonctionnalisée avec des molécules chimiques favorisant des réactions de chimie click bioorthogonale, soit ii) fonctionnalisée avec des monobrins d’oligonucléotides complémentaires pouvant s’hybrider. L’auto-agrégation des Au NCs en solution a confirmée l’augmentation de la PL par transfert d’énergie inter-particules. Cette propriété pourrait éventuellement améliorer l’effet thérapeutique, mais ils doivent encore être caractérisés in vivo. La seconde stratégie a consisté à augmenter l’affinité des Au NCs pour les cellules en ajoutant de l’arginine à la surface des Au NCs de façon contrôlée. En effet, l’arginine est connue pour favoriser l’interaction électrostatique avec les membranes plasmiques et l’internalisation cellulaire. Nous avons déterminé le seuil maximum d’arginine par Au NCs permettant d’augmenter la PL tout en conservant leur petite taille. Les meilleurs candidats ont une forte capacité d’interaction électrostatique avec des membranes artificielles même en présence de sérum, suggérant que l’opsonisation des Au NCs est faible. Leurs capacités d’interaction (< 5min) et d’internalisation (<30 min) sont rapides et ont été confirmées sur des cellules humaines de mélanome in vitro, sans toxicité notable. Cependant d’après une étude sur des sphéroïdes irradiés, l’ajout d’arginines aurait un effet « de pirégeage » sur la production d’espèces réactives oxygénées diminuant le pouvoir radiosensibilisant des Au NCs. La présence de charges positives sur les Au NCs contenant des arginines et leur capacité d’internalisation permettent aussi de les utiliser in vitro pour vectoriser des polymères anioniques tels que des siRNA. En revanche, ces Au NCs administrés par voie intraveineuse chez des souris porteuses de tumeurs sont tous éliminés extrêmement rapidement par voie rénale ce qui ne leur permet pas de s’accumuler suffisamment dans les tumeurs. Ces travaux démontrent donc que la fonctionnalisation des Au NCs influence fortement leurs propriétés optiques et physico-chimiques, leurs interactions avec les cellules et leurs effets théranostics. Il serait intéressant d’appliquer ces stratégies sur des Au NCs circulants plus longtemps dans le sang pour démontrer l’effet de ces fonctionnalisations sur l’accumulation tumorale
Gold nanoparticles (Au NPs) have shown promising results in nanomedicine applied to oncology. They are capable of accumulating in tumor areas, inducing a therapeutic effect by delivering drugs or a photo-/radiotherapeutic effect thanks to their energy absorption properties. They also allow diagnosis by different imaging techniques. This dual activity defines them as theranostic agents. Gold nanoclusters (Au NCs) define an interesting sub-family of Au NPs. They are composed of about ten to hundred gold atoms stabilized by organic molecules. Their size smaller than ~8 nm allows them to be eliminated by the kidneys and to exhibit photoluminescence (PL) properties until infrared wavelengths, which are suitable for in vivo optical imaging. They can also induce cell death under irradiation due to the intrinsic properties of gold. Their optical features, pharmaco-kinetic and tumor accumulation are highly sensitive to size and surface chemistry modification. Currently, preclinical results are not sufficient for clinical transfer and it is necessary to improve the characterization of Au NCs and to study their behaviour in vitro and in vivo.In this context, my thesis project focused on the functionalization of Au NCs in order to improve their accumulation in tumors. The first strategy is based on the self-aggregation of Au NCs in the tumor microenvironment. For this purpose, the surface of the Au NCs was either functionalized with i) molecules promoting bioorthogonal click chemistry reactions, or ii) complementary oligonucleotides that can hybridize. The self-aggregation of Au NCs in solution confirmed the increase in PL by inter-particle energy transfer. The self-agregation of Au NCs could potentially improve the therapeutic effect, but the Au NCs still need to be characterized in vivo. The second strategy consisted in increasing the affinity of Au NCs for cells by adding controlled amounts of arginine on their surface. Indeed, arginine is known to promote electrostatic interaction with plasma membranes and cellular internalization. We have determined the maximum arginine threshold per Au NCs, allowing to increase the PL while keeping their small size with high colloidal stability. The best candidates have a high capacity for electrostatic interaction with artificial membranes even in the presence of serum, suggesting that the opsonization of Au NCs is low. Their interaction (< 5min) and internalization (<30 min) capacities are rapid, and have been confirmed on human melanoma cells in vitro, without significant toxicity. However, according to a study on irradiated spheroids performed in our team, the addition of arginines would have a "trapping" effect on the production of reactive oxygen species, reducing the radiosensitizing power of Au NCs. The presence of positive charges on Au NCs containing arginines and their internalization capacity also can serve in vitro to deliver anionic polymers and biomolecules such as siRNA. However, these Au NCs administered intravenously to tumor-bearing mice are eliminated extremely rapidly by the kidneys, thus reducing their ability to accumulate in tumors. This work showed that the functionalization of Au NCs strongly influences their optical and physicochemical properties, their interactions with cells and their theranostic effects. It would be interesting to apply these strategies to Au NCs circulating longer in the blood to demonstrate the effect of these functionalizations on tumor diagnostics and therapy