Tesi sul tema "Smart nanoparticles"

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The use of various ‘smart materials’ (briefly meaning materials that respond to a change in their environment) is currently of interest to both academics and industry. The primary aim of the current study was to entrap photochromic (PC) dyes in miniemulsions, as a means to improve their fatigue resistance, thus synthesizing smart nanoparticles. In the coatings industry the use of aqueous systems is becoming a common requirement for health and environmental reasons. Miniemulsion entrapment allows the direct dispersion of PC dyes into aqueous systems while allowing for the opportunity to tailor-make the host matrix in order to obtain a suitable PC response and improved fatigue resistance. The optimal instrument set-up required to establish the PC response of films of the so-called smart nanoparticles (i.e. PC miniemulsions) was determined. A UV-Vis instrument with a chip-type UV LED mounted inside for activation of the samples provided PC response results. A tungsten lamp with filter provided deactivation of the samples. A stable butyl methacrylate (BMA) miniemulsion formulation was established by conducting a design of experiments. A chromene and spironapthoxazine (SNO) PC dye were entrapped in the BMA miniemulsion. A hindered amine light stabiliser (HALS) was also entrapped with the SNO dye in the BMA miniemulsion to further improve the fatigue resistance. The following PC properties of the smart nanoparticles films were evaluated: colourability, thermal decay rate, half-life and fatigue resistance. To compare results with conventional systems, a BMA solution polymer was prepared. The SNO dye and different concentrations of the HALS were mixed with the BMA solution polymer. In comparison to the SNO smart nanoparticles the chromene smart nanoparticles films had lower colourability, but better fatigue resistance. Incorporating HALS at levels of 0.5–2% in the BMA miniemulsion with PC dye did not lead to any significant improvement in fatigue resistance, yet films of the BMA solution polymer showed some improvement. SNO dye incorporated at 1% gave similar colourability in both miniemulsion and in solution polymer, yet the fatigue resistance of the films of the PC miniemulsions was much better.
AFRIKAANSE OPSOMMING: Die gebruik van verskeie “slim materiale’ (kortliks beskryf as materiale wat reageer op `n verandering in hul omgewing) is tans van belang vir beide akademici en die industrie. Die hoofdoel van hierdie studie was om miniemulsietegnologie te gebruik om fotochromiese (FC) kleurstowwe vas te vang, vir die sintese van slim nanopartikels, om sodoende die weerstand teen afgematheid te verbeter. In die verfindustrie word die gebruik van waterbasissisteme meer algemeen weens gesondheids- en omgewingsredes. Die gebruik van miniemulsie sisteme om materiale vas te vang maak dit moontlik om FC kleurstowwe direk in waterbasissisteme te meng. Die sintese van `n unieke gasheer matriks word benodig om die optimum FC verandering te toon en weerstand teen afgematheid te verbeter. Om die FC verandering van die sogenaamde slim nanopartikel films (d.w.s. FC miniemulsies) te ondersoek was `n gepaste instrumentele opstelling nodig. Dit is vasgestel dat `n UV-Vis instrument waarin `n skyfie-tipe UV LED gemonteer is vir aktivering van die monsters, reproduseerbare resultate gegee het. Die monsters is gedeaktiveer deur gebruik te maak van `n tungsten lig met ‘n filter. `n Eksperimentele ontwerp is toegepas om `n stabiele butielmetakrielaat (BMA) miniemulsie formulasie te verkry. `n ‘Chromene’ en ‘spironapthoxazine’ (SNO) FC kleurstof is in die BMA miniemulsie vasgevang tesame met `n verhinderde amien ligstabiliseerder (VALS) om die weerstand teen afgematheid verder te verbeter. Die volgende FC eienskappe van die slim nanopartikels is gemeet: kleurintensiteit, tempo van termiese verwering, half-lewe en weerstand teen afgematheid. `n BMA polimeeroplossing is berei om resultate mee te vergelyk. Die SNO kleurstof en verskillende konsentrasies van die VALS is met die BMA polimeeroplossing gemeng. In vergelyking met die slim SNO nanopartikels het die intelligente chromene nanopartikelfilms `n swakker kleurintensiteit gehad, maar `n hoër weerstand teen afgematheid. Die gebruik van 0.5–2% VALS in die BMA miniemulsie met FC kleurstof het minimale verbetering in weerstand teen afgematheid getoon, maar daar was wel `n beduidende verbetering in die geval van films met FC kleurstof in `n BMA polimeeroplossing. Byvoeging van 1% SNO kleurstof in `n BMA miniemulsie of polimeeroplossing het dieselfde kleurintensiteit gelewer, maar die weerstand teen afgematheid van die FC miniemulsie was baie beter.

Ce travail de thèse propose une nouvelle approche pour la préparation de membranes à matrice mixte basée sur l’utilisation de copolymères à blocs et de nanoparticules inorganiques disposant de propriétés magnétiques. Des aggrégats de copolymères ont été préparés avec une morphologie variée (sphères, cylindres et vésicules) à partir du copolymère poly(acide méthacrylique)-b-poly(méthacrylate de méthyle). Ce dernier a été synthétisé par polymérisation radicalaire contrôlée par transfert de chaîne réversible par addition-fragmentation (RAFT) dans l’éthanol à 70°C. Des particules d’oxyde de fer ont, quant à elles, été préparées en présence de différents stabilisants à température variée pour permettre d’atteindre la charge de surface et les propriétés magnétiques recherchées. La structure des copolymères à bloc a permis d’obtenir à la fois des membranes hydrophobes via le procédé de séparation de phase induite par un non-solvant, ainsi que des membranes hydrophiles lorsque que la technique de spin-coating était appliquée aux aggrégats formés par auto-assemblage induit lors de la polymérisation. Grâce à l’étude détaillée des propriétés de filtration des membranes obtenues, la relation structure-propriété a été discutée sous l’action d’un champ magnétique externe. Enfin, la sensibilité au colmatage a été vérifiée via la filtration de solutions de protéines. Il a ainsi été démontré une diminution notable du colmatage sous champ magnétique, ouvrant de belles perspectives pour ces nouvelles membranes
This thesis presents a new approach to produce mix matrix membranes using block copolymers and inorganic nanoparticles having magnetic properties. The polymeric nanoparticle with different morphologies (linear, Spheres, worms, and vesicles), from poly (methacrylic acid)-b-(methyl methacrylate) diblock copolymer, were synthesized using Reversible addition−fragmentation chain transfer polymerization (RAFT) in ethanol at 70 ֠C. The inorganic counterpart, iron oxide nanoparticles were prepared using different stabilizers at various temperatures to acquire the necessary surface charge and magnetic properties. The chemistry of the particles leads to form both hydrophobic membranes using non-solvent induced phase separation as well as a hydrophilic membrane by using the simple spin coating technique with the particles from polymerization induced self-assembly. By a detailed experimental study of the membrane filtration, the influence of different parameters on the process performance has been investigated with and without magnetic field. Finally, membrane fouling has been studied using protein solution. Also, the membrane performance was examined under magnetic field revealing the successful reduction in the fouling phenomenon making them new performant membranes in the area of membrane technology

Smart nanomaterials can selectively respond to a stimulus and consequently be activated in specific conditions, as a result of their interaction with electromagnetic radiation, biomolecules, pH change, etc. These nanomaterials can be produced through distinct routes and be used in artificial skin, drug delivery, and other biomedical applications. In this thesis, two smart nanosystems were developed, viz., i) nanocapsules formed by aniline (A) and chitosan (CS) (A-CS) containing silver nanoparticles (AgNPs), with an average size of 78 ± 19 nm, and ii) polycaprolactone (PCL) nanofibers, fabricated by the electrospinning technique containing AgNP into their bulk, with a diameter of 417 ± 14 nm. A novel system, based on the incorporation of the as-prepared nanocapsules onto the surface of PCL nanofibers containing AgNps (antibacterial mats), was also developed. The methodology employed avoids the direct contact of silver nanoparticles with the host and optimizes its release to the surrounding environment. The AgNPs release was triggered by exposing the nanocapsules to light at 405 nm. Consequently, the electronic energy vibration resulting from the interaction of the irradiation with the surface plasmon band (SPR) of AgNps, breaking the hydrogen bonds of the nanocapsules and releasing of AgNPs at a time of 150 s. To understand the perturbation of AgNps-Nanocapsules against bacteria, membrane models using Langmuir technique with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phospho-(1\'-rac-glycerol) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) were employed, which are the main components of cell membrane of Escherichia coli (E. coli). The results suggest that DPPG has more influence on the incorporation of the nanoparticles on the cell membrane. The antibacterial properties of the nanofibers/nanomaterials mats towards E. coli and Staphylococcus aureus (S. aureus) were investigated using the Agar diffusion test for 8 samples. The experiments revealed that the samples based on nanofibers/nanocapsules and irradiation presented a radius of inhibition of 2.58 ± 0.28 mm for S. aureus and 1.78 ± 0.49 mm for E. coli. This nanosystem showed to be highly interesting for biomedical applications.
Nanomateriais inteligentes podem responder seletivamente a um estímulo e consequentemente ser ativados em condições específicas, como resultado da sua interação com a radiação eletromagnética, mudança do pH, campo magnético, etc. Esses materiais podem ser produzidos através de distintas rotas e utilizados em aplicações como pele artificial, liberação de fármacos, e outras aplicações biomédicas. Nessa tese, dois nanossistemas inteligentes foram desenvolvidos, a saber: i) nanocápsulas formadas por anilina (A) e quitosana (CS) (A-CS) contendo nanopartículas de prata (AgNps), com um tamanho médio de 78 ± 19 nm, e ii) nanofibras de policaprolactona (PCL), fabricadas pela técnica de eletrofiação contendo AgNps em seu interior, com diâmetro de 417 ± 14 nm. Um terceiro sistema foi desenvolvido, baseado na incorporação das nanocápsulas na superfície das nanofibras de PCL contendo AgNps (manta antibacteriana). A metodologia utilizada evita o contato direto das nanopartículas de prata com o hospedeiro e otimiza sua liberação no meio ambiente. As AgNps liberadas foram acionadas pela exposição das nanocápsulas à um fonte de luz em 405 nm. Consequentemente, a vibração da energia eletrônica resultante da interação da irradiação com a banda plasmônica de superfície (SPR) das AgNps, quebra as ligações de hidrogênio da nanocápsula e libera as AgNps no meio em um tempo de 150 s. Para entender a perturbação das AgNps-nanocapsulas contra as bactérias, modelos de membrana foram usados através da técnica de Langmuir com os fosfolipídios 1,2-dipalmitoil-sn-glicero-3- fosfo-(1\'-rac-glicerol) (DPPG) and 1,2-dimiristoil-sn-glicero-3-fosfoetanolamina (DMPE), que são os principais componentes da membrana celular de Escherichia coli (E. coli). Os resultados sugerem que DPPG tem mais influência na incorporação das nanopartículas na membrana celular. As propriedades antibacterianas das mantas de nanofibras/nanomateriais contra E. coli e Staphylococus aureus (S. aureus) foram investigadas usando o teste de difusão Agar em 8 grupos, o qual revelou que o grupo contendo a nanofibra/nanocapsula e irradiação apresentou um raio de inibição de 2.58 ± 0.28 mm para S. aureus e 1.78 ± 0.49 mm para E. coli. Este nanossistema mostrou ser altamente interessante para aplicações biomédicas.

Stimulus-responsive colloidosomes which completely dissolve upon a mild pH change are developed. pH-Responsive nanoparticles that dissolve upon a mild pH increase are synthesized by a nanoprecipitation method and are used as stabilizers for a double water-in-oil-in-water Pickering emulsion. These emulsions serve as templates for the production of pH-responsive colloidosomes. Removal of the middle oil phase produces water-core colloidosomes that have a shell made of pH-responsive nanoparticles, which rapidly dissolve above pH 7. The permeability of these capsules is assessed by FRAP, whereby the diffusion of a fluorescent tracer through the capsule shell is monitored. Three methods for tuning the permeability of the pH-responsive colloidosomes were developed: ethanol consolidation, layer-by-layer assembly and the generation of PLGA-pH-responsive nanoparticle hybrid colloidosomes. The resulting colloidosomes have different responses to the pH stimulus, as well as different pre-release permeability values. Additionally, fundamental studies regarding the role of particle surface roughness on Pickering emulsification are also shown. The pH-responsive nanoparticles were used as a coating for larger silica particles, producing rough raspberry-like particles. Partial dissolution of the nanoparticle coating allows tuning of the substrate surface roughness while retaining the same surface chemistry. The results obtained show that surface roughness increases the emulsion stability of decane-water systems (to almost twice), but only up to a certain point, where extremely rough particles produced less stable emulsions presumably due to a Cassie-Baxter wetting regime. Additionally, in an octanol-water system, surface roughness was shown to affect the type of emulsion generated. These results are of exceptional importance since they are the first controlled experimental evidence regarding the role of particle surface roughness on Pickering emulsification, thus clarifying some conflicting ideas that exist regarding this issue.

Dans de nombreux domaines, et particulièrement dans le secteur aérospatial, la corrosion est un problème majeur qui engendre d’importants coûts de maintenance et de réparation. Il est donc crucial de protéger les pièces soumises à des environnements agressifs. Dans ce but, une solution originale et prometteuse consiste à détecter et ralentir la corrosion dès ses prémices, en utilisant des revêtements capables de réagir dès son initiation. Récemment, de nombreuses études ont été réalisées en ce sens, développant des revêtements sensibles aux processus électrochimiques liés à la corrosion, faisant partie de la catégorie des revêtements dits « intelligents ». Les travaux présentés, financés par ArianeGroup, visent à développer des nanocapsules contenant un inhibiteur de corrosion. Ces capsules, formées suivant un procédé de mini-émulsion, sont ensuite intégrées à un revêtement organique, appliqué sur un substrat d’acier ou d’alliage d’aluminium. Lors d’une élévation locale de pH, observée lors de la corrosion des substrats métalliques, ces capsules sont capables de relarguer leur contenu. Les travaux présentés décrivent la synthèse et la caractérisation des nanocapsules, ainsi que leur incorporation dans une matrice organique à base aqueuse. Des capsules à cœur hydrophile et hydrophobe ont été envisagées avant de concentrer les recherches sur des capsules de silice à cœur hydrophobe dont le diamètre moyen est d’environ 180 nm. L’encapsulation et le relargage d’un inhibiteur de corrosion, le 2-mercaptobenzothiazole, sont décrits et les propriétés inhibitrices mises en évidence. Dans un deuxième temps, l‘impact de l’addition de capsules sur les propriétés du polymère est étudié, principalement par spectroscopie d’impédance électrochimique. Le système présenté peut potentiellement être adapté à la détection de la corrosion en remplaçant l’agent inhibiteur par un capteur de corrosion
In various domains, but especially in the space industry, protection of metallic structures against corrosion is a foremost objective because of maintenance and fixing costs in harsh environments. In order to preserve their integrity, a promising and original solution consists in detecting and slowing down corrosion as soon as it appears by developing a corrosion-sensitive and reactive coating. Recently, numerous studies have searched for the development of functional surfaces able to locally react to corrosion-related stimuli, belonging to the category of smart coatings. This PhD project, granted by ArianeGroup, is focused on this objective and deals with the development of nanocapsules containing corrosion inhibitors. These nanocapsules, formed through a miniemulsion process, are incorporated within an organic coating, applied on top of either steel of aluminum alloy substrates. The formed nanocapsules are then able to release their payload using a pH increase as a trigger. A pH change is indeed expected during cathodic activity, appearing at the early stages of the substrate degradation. This work describes the synthesis and characterization of nanocapsules, as well as their incorporation within an organic waterborne matrix. Both hydrophilic and hydrophobic-core capsules have been investigated before focusing on oil-core silica nanocapsules with diameter ca. 180 nm. In the first part, the release of a model inhibitor, namely 2-mercaptobenzothiazole (MBT) has been investigated and the inhibiting properties demonstrated. Then, the impact of capsules addition on coating integrity and properties has been studied using mainly electrochemical impedance spectroscopy. Potential enhancements of the proposed system are discussed. This system could be adapted and used for early detection of corrosion, using a corrosion sensor instead of the encapsulated inhibitor

Nanoparticle (NP)-based therapies have proven to offer potential solutions for conventional medicine while minimizing its side effects. In particular, glycosylated gold NPs (AuGNPs) represent attractive imaging and/or delivery platforms combining the biological activity of carbohydrates with the biocompatibility and physicochemical versatility of the metallic core. However and despite their rapid growth, the full clinical potential of these glyconanosystems will only be reached when their underlying safety concerns are understood and accurately addressed. When placed in biological fluids, AuGNPs, interact with a high number of biomolecules forming a so-called protein corona (PC), a coating that covers them and has severe consequences for their fate, efficacy and toxicity. As a consequence, AuGNPs are often recognized by the immune system and cleared from the blood stream before they have had enough circulation time to reach their therapeutic target. In order to address the first issue, the initial chapter of the present thesis deals with the preparation of a library of six different AuGNPs functionalized with three monosaccharides (α-mannose, α-galactose and α-fucose) and two polyethylene glycol alternatives (PEG5000 and alkyl-PEG600) as an anchor to the Au nanomaterials (NMs). In collaboration with CICBiomaGUNE (Spain), the interaction of these AuGNPs with different lectins is investigated using Fluorescence Correlation Spectroscopy (FCS) to evaluate the effect of the PEG length on the interaction with proteins and the carbohydrates binding specificity. The second part of the chapter is dedicated to the preparation of an additional set of AuGNPs functionalized with negatively charged monosaccharides such as mannose-6-phosphate, mannose-6-sulfate or N-acetyl neuraminic acid. The final goal is to compare the results with neutral mannosylated AuGNPs in terms of colloidal stability and protein binding. These measurements will be performed at RCSI (Ireland) by means of Differential Centrifugal Sedimentation (DCS) and Dynamic Light Scattering (DLS). The final aim of the work described in Chapter I is to provide a better understanding of the PC formation and a correlation to the main NMs’ surface characteristics influencing it. The immunosuppressive properties of AuGNPs functionalized with N-acetyl neuraminic acid will also be tested in vivo in collaboration with Mario Negri Institute (Italy). The second chapter focuses on the synthesis of ABO blood sugar epitopes, terminal di- (Fucα1-2Galβ1-O-R) and tri-saccharides (GalNAcα1-3(Fucα1-2)Galβ1-O-R and Galα1-3(Fucα1-2)Galβ1-O-R) covering the surface of human red blood cells and other body tissues. Our synthetic derivatives have been prepared adapting previously reported procedures and include a linear aglycone with a free amino group for NM conjugation. Functionalization of NMs with these self-antigens may improve their immunotolerance and biocompatibility in comparison to PEGylated counterparts, prolonging their blood circulation time, improving their delivery efficiency and granting them new therapeutic possibilities. These assumptions are to be tested in the near future in collaboration with RCSI (Ireland). Chapter III describes the functionalization of mannose (Manα1), dimannose (Manα1-2Manα1) and trimannose (Manα1-3(Manα1-6)Manα1) derivatives with an amine-ending linker at their reducing-end for Au nanorods functionalization at VITO (Belgium). The end goal of the project is to develop a simple and label-free biosensor to detect mannose binding lectin (MBL) from human plasma samples based on optical measurements. The near IR shift due to the effective lectin binding can be correlated to potential cardiovascular disease for prevention and early onset stroke diagnosis. In summary, this thesis aims to provide a better understanding of AuGNPs’ behavior in biological fluids, to face the main concerns impairing nanomedicine translation into clinics and to pave the way towards novel glyco-nanosystems with enhanced therapeutic performance for healthcare applications.

Thesis (Ph. D.)--University of Nevada, Reno, 2008.
"December 2008." Includes bibliographical references. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2009]. 1 microfilm reel ; 35 mm. Online version available on the World Wide Web.

We present a fabrication process to create bifunctional microparticles displaying two different proteins have been spatially segregated onto hemispheres. Silica and polystyrene microparticles with 2.0 m, 4.08 m, and 4.74 m diameters are processed with metal deposition to form two chemically distinct and segregated hemispheres. The surface of each hemisphere is then separately derivatized with proteins using different chemical conjugation strategies. These bifunctional Janus particles possess biologically relevant, native conformation proteins attached to a biologically-unreactive and safe substrate. They also display high densities of two types of spatially segregated proteins which may enable a range of capabilities that monofunctional particles cannot, such as improved targeting of drug carriers and bioimaging agents.

The Thesis is based on the development of a potentially-scalable methodology to prepare polyurethane/polyurea polymers to nanoencapsulate bioactive molecules. In the first part of the project, the design, optimization and characterization of the polymers were presented, and it showed that the most promising polymer was composed of hydrophilic and hydrophobic pendant tails, disulfide bonds and an amphoteric pair. The polymers synthesized were basically characterized by IR, NMR, MALDI-TOF, which indicated that the polyaddition reactions between the isocyanates and the difunctional nucleophiles proceeded as expected and the polymer grew to the desired molecular weight. The most promising polymer was used to nanoencapsulate four hydrophobic drugs in a straightforward manner. The nanocapsules were subsequently characterized by different biophysical techniques and showed that the nanosystems were monodisperse and that the average particle size was around 20 nm. In addition, surface charge measurements demonstrated a synchronized shell cationization under acidic conditions, while the nanosystem remained neutral to anionic under physiological pH. Fluorescence and morphological studies revealed that the nanocapsules were stable under physiological conditions and in protein-rich environments, while they degraded under a glutathione- based medium. Finally, UV/Vis spectroscopy demonstrated that the encapsulation efficiency was high for the four cases studied. The second part of the thesis aimed the extension of this methodology to amphiphilic molecules, such as anionophores (tambjamines). The application of the process without modifications led to nanosystems with highly cationic surfaces and low entrapment efficiencies, therefore, different changes were proposed. Firstly, certain modifications of the shell were considered, such as the use of aliphatic isocyanates with aromatic moieties, but they did not lead no any improvement. Secondly, the insertion of hydrophobizing agents was also tested and the results revealed a more homogeneous particle size distribution, a pH-responsive surface and a medium to high entrapment efficiency. The most promising nanocapsules were characterized in vitro in terms of cytotoxicity, cell uptake and cell internalization. The first parameter was evaluated in three different lung cancer cell lines, a normal cell line (fibroblasts) and two different neuroblastoma cell lines. The assays showed that unloaded nanocapsules did not induce any cytotoxicity in any of the cell lines tested, and the nanocapsules loaded with the drug led to a controlled cytotoxic effect that was maximum after 72 h of incubation. Moreover, the nanocapsules internalized the lung cancer cells by endosomal mechanisms and they were transported as late endosomes to the lysosomes, where they are hydrolyzed. The last in vitro assays demonstrated that the nanocapsules were uptaken after only 1 h of treatment and after 48 h the cells did not show signs of saturation. Finally, in vivo biodistribution studies were performed with amphoteric nanocapsules and antiGD2- functionalized nanocapsules in a neuroblastoma mice model. The ex-vivo results revealed that the amphoteric nanocapsules promoted an improved tumor accumulation compared to the antibody-targeted nanosystems. However, both types of nanocapsules were highly accumulated to detoxification organs, such as the liver and spleen. These undesired effects could be explained by the fact that an excessive dose of product was administered taking into account the small size and low weight of the tumors. Finally, the last part of the project involved the functionalization of the nanocapsules with peptides (A5 and IPEP) to target different types of breast cancer cells. To this aim, different hydrophilic linkers were synthesized to conjugate the peptides and provide a physical space between them and the nanocapsules. The nanosystems were prepared and evaluated by the typical techniques and by flow cytometry in order to determine the effect of targeting molecules on the surface. The results showed that only IPEP provided an improved cell penetration, since A5 did not induce any significant effect. This could be explained by the fact that A5 had lost its biological activity after being functionalized, since different data supported the presence of these peptides on the surface.
Aquesta Tesi es basa en el desenvolupament de metodologies potencialment escalables per la preparació de polímers de poliuretà/poliurea amb les característiques idònies per poder nanoencapsular molècules bioactives. La primera part del projecte es centra en el disseny, optimització i caracterització dels polímers, mentre que la resta de seccions mostren el procés i resultats de la nanoencapsulació. Les primeres reaccions de nanoencapsulació es van fer utilitzant molècules hidròfobes i les tècniques de caracterització van mostrar mides de partícula d’aproximadament 20 nm, morfologia gairebé esfèrica, alts rendiments d’encapsulació, degradació en condicions redox i amb càrrega superficial modulable segons el pH del medi. Aquesta última propietat es va considerar clau per aconseguir l’acumulació selectiva d’aquests nanosistemes en tumor. En els passos següents es va considerar l’aplicació d’aquesta metodologia en l’encapsulació d’altres tipus de molècules, com són les amfifíliques (tambjamines). En aquest cas, els procediments es van haver d’adaptar i van incloure l’ús d’agents hidrofobitzants per tal de millorar-ne l’encapsulació. Després de certes modificacions, els productes resultants van mostrar característiques molt similars respecte a les obtingudes amb fàrmacs hidrofòbics en quant a mida i característiques superficials. Les proves in vitro es van dur a terme en línies de càncer de pulmó i neuroblastoma i els assajos van confirmar que les nanocàpsules que contenien fàrmac eren citotòxiques i les que estaven buides no induïen cap efecte nociu. A més, altres estudis van demostrar que el mecanisme d’internalització era a través d’endosomes i que les nanocàpsules, una vegada dins la cèl·lula, co-localitzaven en lisosomes i altres orgànuls àcids. També es va observar que les nanocàpsules penetraven les cèl·lules ràpidament i que després de tractaments llargs (48 h) no hi havia saturació. Finalment, estudis de biodistribució van revelar que els nanosistemes amfòters tenien una millor acumulació a tumor comparat amb nanocàpsules funcionalitzades amb un anticòs, però que el fetge i la melsa també contenien alt contingut d’aquests nanosistemes. Es va concloure que una possible explicació era l’administració d’una dosi massa elevada de nanocàpsules, tenint en compte que la mida i pes dels tumors era molt petita. En l’últim capítol es va explorar la funcionalització de les nanocàpsules amb pèptids direccionadors (A5 i IPEP) a cèl·lules tumorals de càncer de mama. Es van sintetitzar diverses molècules espaiadores per tal de conjugar els pèptids a les nanocàpsules i es van repetir les tècniques de caracterització biofísica i biològica. Aquestes van mostrar que les nanocàpsules tenien una mida de uns 20 nm, càrrega superficial d’acord amb les característiques del pèptid i que IPEP millorava la internalització de les nanocàpsules, però només a temps curts, ja que a incubacions més prolongades les nanocàpsules amfòteres tenien també una molt bona entrada a les cèl·lules estudiades.

This thesis, entitled "Multifunctional Self-stratified Polyurethane-Polyurea Nanosystems for Smart Drug Delivery" is structured as a compendium of publications, being organized around a methodological synthetic patent and four internationally peer reviewed publications on the chemical and bio-applicability of the US and EU patented invention. Thus, this manuscript is divided into a General Introduction to drug delivery with polyurethane-polyurea nanosystems and three chapters, which include the publications as Results and Discussion and their introductions as Introduction of each chapter. Chapter 1 is focused on the synthetic methodology to produce a novel kind of self-stratified multifunctional polyurethane-polyurea nanoparticles for cancer nanotherapy. The Introduction of this chapter deals with the key characteristics of drug delivery nanosystems and presents the basis of self-stratification by hydrophobic effects. This chapter contains a methodological patent that breaks down multiple examples of nanoparticles formed by easy-tunable polyurethane-polyurea biocompatible and biodegradable polymers bearing multifunctionalities that are applied to cancer therapy and imaging. In addition, here we include a publication containing the in vitro proof-of-principle of the stratified nature, high encapsulation stability and selective targeting to cancer cells of the nanosystem. Finally, this chapter also contains a publication with the in vivo proof-of-concept for avf33 integrin targeted cancer therapy and imaging of polyurethane-polyurea nanoparticles encapsulating plitidepsin as antiangiogenic drug. This research is the consequence of a fruitful collaboration in the framework of a INNPACTO national project (Polysfera, IPT-090000-2010-1) with Dr. P. Calvo, Dr. P. Aviles and Dr. M. J. Guinan in PharmaMar SA; Dr. I. Abasolo, Dr. Y. Fernandez and Dr. S. Schwartz in Vail d'Hebron Institut de Recerca and Dr. J. Rocas in Ecopol Tech SL. Chapter 2 arises from the encouraging results obtained from Chapter 1 in novel synthetic methods of polymer nanoparticles with shell stratification capacity. The Introduction of this chapter presents the current concerns with titanium implants, the outcome of nanoparticle-coated biomaterials and the perspectives in multifunctional nanomaterials. In this regard, a fantastic collaboration with Dr. C. Mas-Moruno of the BiBiTE group in the UPC lead taking profit of the cell targeting high specificity and great encapsulation capacity of PUUa NPs to develop new generation nanobiomaterials for the enhancement of titanium implants osseointegration and bacterial infection prevention. Titanium implants were innovatively coated by interfacial functionalization with RGD-decorated and roxithromycin-loaded PUUa NPs. This methodology resulted in an outstanding improvement of osteoblastic cells adhesion as well as a dramatic reduction of S. Sanguinis bacteria adhesion onto titanium, which is of great interest to improve the outcome of metallic implants for regenerative medicine. In Chapter 3, we explore another segment of application of PUUa NPs, this is immunotherapy. In collaboration with G. Florez-Canals, Dr. D. Benitez-Ribas and Dr. J. Panes in IDIBAPS and Hospital Clínic of Barcelona we applied PUUa NPs encapsulating budesonide (BDS) corticosteroid for the improvement of BDS efficacy to induce tolerogenicity to dendritic cells (DCs). The Introduction deals with the current therapies used to treat autoimmune diseases as well as recent strategies to target dendritic cells in vivo using smart nanoparticles encapsulating immunosuppressive drugs. Herein we found that when PUUa NPs loaded with BDS were incubated with mature DCs, those differentiated into tolerogenic dendritic cells in a much more efficient manner than DCs incubated with free BDS. As shown in the included publication, levels of costimulatory molecules were enhanced and IL-10 immunosuppressive cytokine was largely secreted. Even more interestingly, fluorescently labeled PUUa NPs proved their DCs targeted behavior in a multi-cellular environment.
Esta tesis, titulada "Multifunctional Self-stratified Polyurethane-polyurea Nanosystems for Smart Drug Delivery" está estructurada como un compendio de cuatro publicaciones en revistas internacionales sobre la química y bio-aplicabilidad de nuestra técnica organizadas alrededor de una patente sintética y metodológica de la invención. El capítulo 1 se centra en la metodología sintética para producir un nuevo tipo de nanopartículas multifuncionales autoestratificadas de Poliuretano-poliurea para la terapia del cáncer. Este capítulo contiene una patente metodológica que analiza múltiples ejemplos de nanopartículas versátiles de Poliuretano-poliurea biocompatibles y biodegradables para el tratamiento del cáncer. Además, aquí se incluye una publicación que contiene la demostración de la estratificación de las nanopartículas, la alta estabilidad de encapsulación y la internalización selectiva in vitro de las nanopartículas en células cancerosas U87-MG. Por último, este capítulo también contiene una publicación con la prueba de concepto in vivo de la menor toxicidad, mejor biodistribución y farmacocinética en plasma sanguíneo de las nanopartículas de Poliuretano-poliurea encapsulando plitidepsina como fármaco antiangiogénico comparado con el fármaco libre. El capítulo 2 surge de los buenos resultados obtenidos del capítulo 1 en nuevos métodos de síntesis de nanopartículas poliméricas con capacidad de autoestratificación. Aquí se investigan nanobiomateriales de nueva generación recubiertos de nanopartículas de poliuretano y poliurea para mejorar la osteointegración y prevenir la infección bacteriana de implantes de titanio. Así pues, implantes de titanio fueron recubiertos superficialmente con PUUa NPs con péptido RGD en la membrana y cargadas de roxitromicina en su interior. Esta metodología dio lugar a una mejora excepcional en la adhesión de células osteoblásticas así como una reducción dramática de la adhesión bacterias S. Sanguinis en titanio, que resulta de gran interés para mejorar la vida útil de los implantes metálicos para medicina regenerativa. En el capítulo 3, exploramos otro segmento de aplicación de las nanopartículas de poliuretano y poliurea, esta es la inmunoterapia. Se aplicaron nanopartículas cargadas con el corticosteroide budesonida para su mejora como fármaco inmunosupresor para la terapia con células dendríticas. Aqui demostramos que la budesonida encapsulada es mucho más efectiva que la libre en células dendríticas humanas.

The fascinating optical properties of metallic nanostructures, dominated by collective oscillations of free electrons known as plasmons, open new opportunities for the development of devices fabrication based on noble metal nanoparticle composite materials. This thesis demonstrates a low-cost and versatile technique to produce stimuli-responsive ultrafast plasmonic nanostructures with reversible tunable optical properties. Albeit challenging, further control using thermal external stimuli to tune the local environment of gold nanoparticles embedded in VO2 host matrix would be ideal for the design of responsive functional nanocomposites. We prepared Au-VO2 nanocomposite thin films by the inverted cylindrical reactive magnetron sputtering (ICMS) known as hollow cathode magnetron sputtering for the first time and report the reversible tuning of surface plasmon resonance of Au nanoparticles by only adjusting the external temperature stimuli. The structural, morphological, interfacial analysis and optical properties of the optimized nanostructures have been studied. ICMS has been attracting much attention for its enclosed geometry and its ability to deposit on large area, uniform coating of smart nanocomposites at high deposition rate. Before achieving the aforementioned goals, a systematic study and optimization process of VO2 host matrix has been done by studying the influence of deposition parameters on the structural, morphological and optical switching properties of VO2 thin films. A reversible thermal tunability of the optical/dielectric constants of VO2 thin films by spectroscopic ellipsometry has been intensively also studied in order to bring more insights about the shift of the plasmon of gold nanoparticles imbedded in VO2 host matrix.

The present research project was aimed at developing smart nano-systems able to selectively respond with morphological alterations to external physio-pathologic stimuli. These systems are intended for diagnostic or therapeutic applications in tumor treatment. The responsiveness of these systems is intended to improve the site-selective targeting efficiency and reduce uncontrolled disposition in healthy tissues. Smart nano-systems have been obtained using gold nanoparticles (AuNPs) as platform. AuNPs have been surface coated with responsive polymeric components to endow stealth properties in physiologic conditions after injection in the bloodstream. The tumor interstitium is characterized by altered pH and temperature with respect to normal tissues. Thus, when the nano-system reaches the tumour, the responsive coating can undergo morphological modifications which modulate the interface properties of the nanoparticles. This will promote their interaction with the biological surfaces (i.e. cells, tissues). In the project here discussed, different functionalization strategies of AuNPs have been investigated in order to develop nano-carriers provided with multimodal targeting to the tumor. Gold nanoparticles were surface decorated with targeting agents and thermosensitive or pH-sensitive polymers. This allowed producing nano-carriers in which bio-recognition can be controlled by temperature or pH. In physiological conditions, the polymer on particles surface can hide the targeting moiety while in the tumor tissue the thermosensitive or pH sensitive polymer chains collapse provoking the targeting agent exposition and thus promoting cellular internalization. As a result, the multimodal targeting will decrease aspecific biorecognition and increase site-specificity. Gold nanoparticles were produced by Laser ablation technique in aqueous solution without addition of surfactants or stabilizing agents. This finely controlled method allows for obtaining AuNPs with a size of 18 nm. The components used for gold nanoparticles surface functionalization have been thiolated since sulphidril groups promptly react with metal gold with high yield. AuNPs have been surface decorated with biotin-SH and the 8 kDa thermosensitive polymer N-isopropylacrylamide-co-acrylamide-SH (pNIPAm-co-Am-SH), characterized by a low critical solution temperature (LCST) of 37 °C. The nanosystem obtained was tested by ELISA enzymatic assay to evaluate its ability to bind selectively avidin, chosen as model target, as a function of temperature. The results showed that the system behavior is thermally controlled since functionalized gold nanoparticles can only bind avidin when temperature is increased above the polymer LCST at which it collapses. Furthermore, the investigation highlighted the role of the biotin amount and of the biotin/polymer molar ratio on gold nanoparticles surface in affecting the system performance. The system has been further investigated, according to the information acquired, with the biotinylated thermosensitive AuNPs. Gold nanoparticles were surface functionalized with folic acid-SH and the thermosensitive polymer pNIPAm-co-Am-SH to achieve temperature controlled targeting towards cancer cells overexpressing folate receptor (HiFR). Stability studies performed in PBS showed that thermosensitive polymer is paramount to avoid nanoparticles aggregation in the presence of salts. At temperature below polymer LCST, gold nanoparticles are very stable while above the LCST, only a mild aggregation was detected. Folated thermosensitive AuNPs have been tested in vitro with tumor cells overexpressing and non-expressing the folate receptor. The results showed that folated thermosensitive nanoparticles incubated with HiFR cell lines are internalized by cells and dispose in the citosol only at temperature above the polymer LCST. The concept of multimodal targeting was extended to the development of pH-sensitive gold nanoparticles, using polymers able to respond with morphological alterations to environmental pH changes. Ideally, acid-sensitive polymers adequate for this purpose should be soluble and in coil form at pH 7.4 and convert into insoluble globular state, as a consequence of their protonation, in acidic tumor environment. To this aim, different pH-sensitive polymers were produced in order to have an array of materials with satisfactory pKa (in the physiopathological range) to chose from for gold nanoparticles functionalization. The materials were designed in order to endow acid sensitiveness to the decorated nanoparticles in the physic-pathological range. A 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate monomer (MOECHB) was chosen and pH responsive materials were obtained by living radical polymerization technique, namely reversible addition fragmentation chain transfer (RAFT). One homopolymer and various random and block copolymers using the 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate and hydrophilic monomers, namelly metoxy(polyetilenglycole methacrylate (mPEGMA475) or glycerol methacrylate (GMA), were synthesized. All the polymers obtained were characterised by potentiometric titration to determine the pKa value, and turbidimetric analysis to measure the cloud point. Furthermore, their pH-dependant behavior was investigated by dynamic light scattering at various pH conditions. One random and two block copolymers, composed by MOECHB and GMA, were selected for further investigation. Decreasing pH from 7.4 to 6.5, as in physiopathologic condition, they showed a conversion from the soluble to the aggregated state. One additional block copolymer, made by MOECHB and mPEGMA475, will be also evaluated: in the same physiopathologic condition, it outlined morphological modification, albeit without aggregation. The results displayed that pH responsiveness is modulated by the 2-(methacryloyloxy)ethyl 3-chloro-4-hydroxybenzoate/hydrophilic monomer molar ratio in the polymer backbone and by the polymer molecular weight.
Il presente progetto di ricerca ha riguardato lo sviluppo di un nano-sistema intelligente, capace di rispondere selettivamente a stimoli esterni di tipo fisiopatologico con alterazioni morfologiche. Tali sistemi sono stati disegnati per un impiego in diagnostica o nella terapia antitumorale. La responsività di questi sistemi ha lo scopo di migliorare l’ efficienza del direzionamento selettivo e di ridurre la distribuzione aspecifica nei tessuti sani. Sistemi intelligenti sono stati ottenuti a partire da nanoparticelle d’ oro (AuNPs), a cui sono stati associati polimeri responsivi, che conferiscono loro caratteristiche stealth in condizioni fisiologiche in seguito ad iniezione nel torrente circolatorio. E’ noto che l’ interstizio tumorale è aratterizzato da valori di pH e temperatura alterati rispetto al tessuto sano. Di conseguenza, quando il nano-sistema raggiunge il tumore, il rivestimento polimerico può² subire modifiche morfologiche che modulano le proprietà delle nanoparticelle. Questo promuoverà la loro interazione con le superfici biologiche, quali cellule e tessuti. Nel progetto qui discusso, sono state valutate diverse strategie di funzionalizzazione di AuNPs, allo scopo di sviluppare nano-carriers caratterizzati da un direzionamento multi-modale al tumore. Nanoparticelle d’ oro sono state derivatizzate superficialmente con agenti di direzionamento e polimeri termosensibili o pH sensibili, affinchè il riconoscimento selettivo del tessuto tumorale da parte dei nano-carriers sia controllato dalle condizioni di temperatura o di pH circostanti. In condizioni fisiologiche i polimeri presenti sulla superficie delle AuNPs schermano l’ agente di targeting, mentre nel tessuto tumorale grazie alla temperatura o pH alterati, essi collassano determinando l’ esposizione del direzionante e promuovendo l’ endocitosi cellulare. Come conseguenza, il direzionamento multi-modale diminuirà il bio-riconoscimento aspecifico a favore invece della sito-specificità. Le nanoparticelle d’ oro utilizzate in questo progetto sono state prodotte mediante laser ablation in soluzione acquosa, senza utilizzo di sostanze surfattanti e stabilizzanti. Attraverso questo processo controllato e riproducibile si sono ottenute dispersioni di particelle diluite, con diametro medio di 18 nm. Per la funzionalizzazione superficiale di nanoparticelle d’ oro i materiali utilizzati sono stati tiolati, nota l’ elevata capacità di coniugazione di gruppi sulfidrilici su superfici d’ oro metallico. Il primo sistema sviluppato in questo progetto di tesi ha riguardo l’ impiego di nanoparticelle d’ oro in grado di rispondere a stimoli termici. AuNPs sono state modificate superficialmente con biotina-SH e con un polimero termosensibile di 8 kDa, N-isopropylacrylamide-co-acrylamide-SH (pNIPAm-co-Am-SH), caratterizzato da una low critical solution temperature (LCST) di 37 °C. Il sistema ottenuto è stato testato mediante saggio enzimatico di tipo ELISA per valutarne la capacità di binding selettivo ad avidina, scelta come modello, in funzione della temperatura. I risultati hanno mostrato che il comportamento del sistema è controllato dalla temperatura, in quanto le nanoparticelle sono in grado di legare l’ avidina solo quando la temperatura è superiore all’ LCST del polimero, ovvero quando il polimero stesso collassa. Lo studio ha inoltre evidenziato che la quantità assoluta di biotina e i rapporti molari biotina/polimero sulla superficie delle nanoparticelle condizionano in modo rilevante le performance del sistema Sulla base delle ottimizzazioni realizzate con le nanoparticelle direzionate con biotina, il sistema è stato modificato superficialmente con acido folico e lo stesso polimero termosensibile (pNIPAm-co-Am-SH), allo scopo di ottenere un direzionamento verso cellule tumorali sovraesprimenti il recettore per il folato (HiFR). Gli studi di stabilità in PBS hanno mostrato come il polimero termosensibile sia indispensabile per impedire l’ aggregazione delle particelle in presenza di sali. A temperatura inferiore all’ LCST del polimero le nanoparticelle sono stabili, ed aggregano solo marginalmente a temperatura superiore. AuNPs termosensibili e funzionalizzate con acido folico sono state testate in vitro su due diverse linee celluari, sovraesprimenti e non il recettore per l’ acido folico. Gli studi hanno mostrato che solo le particelle termosensibili modificate con acido folico e incubate con linee cellulari HiFR, a temperatura superiore all’ LCST del polimero, vengono internalizzate dalle cellule e si distribuiscono nel citosol. Il concetto di targeting multi-modale è stato successivamente ampliato per lo sviluppo di nanoparticelle d’ oro pH-sensibili, utilizzando polimeri in grado di rispondere con cambiamenti morfologici ad alterazioni del pH ambientale. Idealmente, polimeri acido sensibili adeguati allo scopo devono essere solubili ed in conformazione estesa a pH 7.4, ma subire una conversione allo stato globulare insolubile in seguito a protonazione nell’ ambiente acido tumorale. Per raggiungere questo obiettivo sono stati sintetizzati diversi polimeri pH-sensibili, in modo tale da disporre di una gamma di materiali caratterizzati da valori di pKa e cloud point adeguati allo scopo, tra cui poter selezionare il più¹ adatto per la funzionalizzazione superficiale di nanoparticelle d’ oro. I materali sono stati disegnati per conferire alle AuNPs proprietà di acido-sensibilità nel range fisiopatologico. E’ stato selezionato e sintetizzato un monomero acido-sensibile, 2-(metacriloilossi)etil 3-cloro-4-idrossibenzoato (MOECHB), a partire dal quale sono stati poi prodotti polimeri pH responsivi. La metodica adottata per la reazione di polimerizzazione fa parte delle cosiddette tecniche di polimerizzazioni viventi, ovvero la reversible addition fragmentation chain transfer (RAFT). Sono stati sintetizzati un omopolimero e diversi copolimeri sia random che a blocchi utilizzando MOECHB e monomeri idrofilici, nello specifico metossi(polietilenglicole metacrilato) (mPEGMA475), e glicerolo metacrilato (GMA). Tutti i polimeri ottenuti sono stati caratterizzati mediante titolazione potenziometrica per determinarne il pKa, e sottoposti ad analisi turbidimetrica per misurare il punto di intorbidimento (cloud point). Il loro comportamento pH-dipendente è stato inoltre investigato mediante analisi di dynamic light scattering (DLS) effettuate a diversi valori di pH. Sono stati selezionati un copolimero random e due copolimeri a blocchi, composti da MOECHB e GMA, per ulteriori studi di funzionalizzazione di nanoparticelle d’ oro. Infatti questi materiali hanno mostrato una conversione da una forma solubile ad una aggregata insolubile per diminuzione del pH della soluzione da 7.4 a 6.5, che mima le condizioni fisiopatologiche. Un ulteriore copolimero a blocchi di MOECHB con mPEGMA475 sarà valutato, in quanto nelle medesime condizioni ha mostrato modifiche morfologiche, sebbene non si sia osservata la formazione di prodotti insolubili. I risultati delle analisi hanno evidenziato infine che la responsività al pH è modulata dal rapporto molare 2-(metacriloilossi)etil 3-cloro-4-idrossibenzoato/monomero idrofilico nella composizione polimerica, così come dal peso molecolare del polimero.

Strain engineering is used as a means of manufacturing micro- and nano- scale particles with the ability to reversibly alter their geometry from three dimensional tubes to two dimensional flat layers. These particles are formed from a bi-layer of two dissimilar materials, one of which is the elastomeric material polydimethylsiloxane (PDMS), deposited under stress on a sacrificial substrate. Upon the release of the bi-layer structure from the substrate, interfacial residual stress is released resulting in the formation of tubes or coils. These particles possess the ability to dramatically alter their geometry and, consequently, change some properties that are reversible and can be triggered by a stimulus. This work focuses on the material selection and manufacturing of the bi-layer structures used to create the responsive particles and methods for characterizing and controlling the responsive nature of the particles. Furthermore, the potential of using these particles for a capture/release application is explored, and a systematic approach to scale up the manufacturing process for such particles is provided. This includes addressing many of the problems associated with fabricating ultra-thin layers, tuning the size of the particles, understanding how the stress accumulated at the interface of a bi-layer structure can be used as a tool for triggering a response as well as developing methods (i.e. experiments and applications) that allow the demonstration of this response.

[ES] La presente tesis doctoral titulada "Nanodispositivos mesoporosos híbridos funcionalizados con enzimas para detección, liberación controlada y comunicación molecular" se centra en el diseño, preparación, caracterización y evaluación de distintos nanodispositivos híbridos orgánico-inorgánicos utilizando como soporte nanopartículas tipo Janus de oro y sílice mesoporosa, que se equipan con enzimas, especies fluorescentes y puertas moleculares. Como conclusión general, los estudios realizados muestran que la incorporación de enzimas sobre nanopartículas permite introducir funciones de reconocimiento con alta especificidad y diseñar nanodispositivos avanzados para distintas finalidades. La combinación de nanopartículas híbridas con grupos orgánicos como puertas moleculares, efectores enzimáticos y especies cromo- fluorogénicas o fármacos puede resultar muy versátil; y se espera que los resultados obtenidos puedan inspirar el desarrollo de nuevos materiales inteligentes con aplicación en distintas áreas como la nanomedicina y la detección de moléculas de interés.
[CAT] La present tesi doctoral titulada "Nanodispositius mesoporosos híbrids funcionalitzats amb enzims per a detecció, alliberació controlada i comunicació molecular" es centra en el disseny, preparació, caracterització i avaluació de distints nanodispositius híbrids orgànic-inorgànics utilitzant com a suport nanopartícules tipus Janus d'or i sílice mesoporosa, que s'equipen amb enzims, espècies fluorescents i portes moleculars. Com a conclusió general, els estudis realitzats mostren que la incorporació d'enzims sobre nanopartícules permeten introduir funcions de reconeixement amb alta especificitat i dissenyar nanodispositius avançats per a distintes finalitats. La combinació de nanopartícules híbrides amb grups orgànics com portes moleculars, efectors enzimàtics i espècies cromo-fluorogèniques o fàrmacs pot resultar molt versàtil; i s'espera que els resultats obsessos inspiren el desenvolupament de nous materials intel·ligents amb aplicació en distintes àrees com la nanomedicina i la detecció de molècules d'interés.
[EN] This PhD thesis entitled "Enzyme-functionalized hybrid mesoporous nanodevices for sensing, controlled release and molecular communication" is focused on the design, synthesis, characterization and evaluation of several hybrid organic-inorganic nanodevices using Janus gold-mesoporous silica nanoparticles as scaffolds, equipped with enzymes, fluorescent species and molecular gates. In conclusion, these studies show that the incorporation of enzymes on nanoparticles allows to introduce recognition capabilities with high specificity and to design advanced nanodevices for different purposes. The combination of hybrid nanoparticles with organic groups such as molecular gates, enzymatic effectors and chromo-fluorogenic species or drugs can be very versatile; and we hope that the obtained results inspire the development of new smart materials with application in different areas such as nanomedice and sensing.
Llopis Lorente, A. (2019). Enzyme-functionalized hybrid mesoporous nanodevices for sensing, controlled release and molecular communication [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/117612
TESIS

Biodegradable polymeric nanoparticles (nps) have shown to be promising forms for the delivery of a wide array of drug formulations, because of their ability to: (i) increase the drug half-life in the blood stream, (ii) enhance the solubility of poorly-water soluble drugs, which represents the main obstacle to their efficient administration (iii) improve drug bioavailability and (iv) reduce systemic toxicity. These properties are of particular interest when dealing with deseases like cancer, since they offer the possibility to overcome toxicity and administration problems associated with traditional chemotherapy. Moreover, polymeric nanoparticles are able to passively target tumour cells through the Enhanced Permeation and Retention (EPR) Effect. Tumours are composed of fastly- growing cells which need an extensive supply of oxygen and other nutrients. Their vessels are therefore highly fenestrated (200-300nm) and allow small-size particles to accumulate inside cancer cells. Moreover tumours lack of an efficient lymphatic system and are not able to eliminate these particles once they entered the cells. In this thesis work, biodegradable polymeric nanoparticles, prepared with different materials and techniques were proposed for the active and passive targeting of cancer cells and for the functionalization of biomimetic constructs for tissue engineering. The first section of the present work was mainly focused on the preparation of nanoparticles starting from preformed polyesterurethanes (PU) through two widely used and highly reproducible techniques. Chapter two describes the preparation of nanoparticles by the Solvent Displacement method with two widely used commercial polyesters, poly(ε-caprolactone) and poly(D,L lactide) and the newly synthesized polyesterurethane C-BC2000, based on poly(ε-caprolactone) blocks. The solvent displacement method was selected, since it leads to the formation of monodisperse, small particles by simple precipitation of the polymer in the non solvent (water) without the use of toxic solvents or the application of shear stresses. Paclitaxel, one of the most potent antineoplastic drugs, was encapsulated inside the carriers, which were analyzed in terms of: Morphology, by means of scanning electron microscopy (SEM) and atomic force microscopy (AFM) Size, size distribution, zeta potential xix Encapsulation efficiency and release properties Hydrolytic degradation pathways Uptake by A431 human epidermal carcinoma cells Results obtained for PU nanoparticles, regarding morphology, size, size distribution and surface charge are comparable to those observed with commercial polyesters. All particles possess a small size (<250nm), a low PDI, indicating a narrow size distribution and a regular spherical shape, with little coalescence. In addition, polyurethane nanoparticles exhibited superior properties, such as longer and more controlled release kinetics of the encapsulated drug and a higher cellular internalization. In fact, C-BC2000 nanoparticles had the highest encapsulation efficiency (89±2%) compared to only 24±7% and 18±8% for PLA and PCL, respectively. Moreover the release profile for particles prepared with commercial polyesters, showed that almost all the encapsulated drug was released within the first 48 hours and no further release was observed after this time-point. C-BC2000 nanoparticles were able to sustain the release of the drug for longer time (up to 5 days), when 30% of the active principle was released, indicating that the therapeutic effect of these carriers could last even longer. In chapter 3 two different polyesterurethanes, synthesized with different chain extenders (named C-BC2000 and NS-BC2000) and three commercial polyesters (PCL, PLA and poly(D,L lactide-co-glycolide) PLGA) were used to prepare Paclitaxel-loaded nanoparticles for the active targeting of breast cancer. A modified Single Emulsion Solvent Evaporation method was used, by selecting ethyl acetate as internal phase, Poly(vinyl alcohol) (PVA) as emulsifier and water as external phase. Particles were characterized in terms of morphology, size distribution and zeta potential. Carriers with good spherical shape, small size (<200nm) and negative surface charge were obtained, regardless of the polymer used as matrix-forming material. To augment the targeting ability of nanoparticles towards breast cancer cells, Herceptin, a monoclonal antibody able to selectively recognize a specific receptor (HER2) over- expressed on 25-30% of invasive breast cancer, was attached to the surface of the carriers by hydrophilic/hydrophobic interactions. Surface coating was successfully achieved, as shown by the changes in zeta potential detected for all carriers. xx Moreover the ability of coated particles to selectively recognize their target was tested, by using two different breast cancer cell lines: MCF7, which show a normal expression of the HER2 receptor, and SKBR3 which are known to highly over-express the same receptor. Quantitative uptake studies showed good cellular internalization, by both cell lines and an augmented uptake by SKBR3 cells, when the surface was coated with the monoclonal antibody, confirming that a targeting effect was achieved. Encapsulation efficiency and release profiles from both coated and uncoated particles were also analyzed, and the ability of drug-loaded particles (at three different doses) to effectively kill SKBR3 tumor cells was evaluated. PU showed interesting properties, being superior to commercial polyester in terms of drug release profiles, cellular internalization and cytotoxic activity. In fact, for C-BC2000 and NS-BC2000 nanoparticles, the amount of Herceptin detected on the surface was higher than for the other polymers, except for PCL. However, PCL nanoparticles showed no release of the encapsulated drug and poor cytotoxicity on SKBR3 cells. Moreover, both coated and uncoated PU nanoparticles exhibited the highest cellular uptake and good activity in killing cancer cells, as demonstrated by cytotoxicity tests. The second section of the present work was dedicated to the optimization and characterization of a new, bioactive and biomimetic bone cement containing nanoparticles for the controlled release of an anti-inflammatory agent (Indomethacin). This material is composed of two phases which are naturally present in bones: an organic phase made of chitosan (CH) and collagen as well as of β tricalcium phosphate as inorganic phase, which is expected to increase bioactivity (i.e. the ability to stimulate new bone deposition) of the material. Chitosan and collagen were selected because of their ability to improve injectability and mechanical strength, respectively. Moreover collagen is expected to confer high biomimetic ability to the material. Genipin (GP) was selected as cross-linker for chitosan, due to its natural origin and biocompatibility. The cement composition was optimized to obtain the desired hardening time and dissolution rate and then reinforced with collagen. Moreover, anti-inflammatory properties were given by including Indometacin-loaded biodegradable nanoparticles based on Poly(ε-caprolactone) (PCL) in the cement composition. Nanoparticles were prepared by the Solvent Displacement Method and their size, encapsulation efficiency as well as release profiles were characterized. xxi Injectability, compressive strength and bioactivity of the final cement were also investigated. The material showed good bioactivity, promoting the formation of apatite after 14 days of incubation in simulated body fluids (SBF), especially when high concentrations of inorganic phase were used. The material also possessed a low setting time (around 20min) and good injectability (95%), making it an optimal candidate as injectable bone substitute. Mechanical strength under compression was also analyzed, resulting in poor properties when collagen is not part of the composition. After the addition of collagen, the Young modulus for compression increased from 0,15Mpa to 1Mpa, which meets the requirements for a good vertebral substitute. Moreover the addition of nanoparticles confers anti-inflammatory properties, as well as additional mechanical strength, since the release rate of the drug is compatible with the infection raising kinetics.

Des glycopolymères greffés et dibloc, amphiphiles et photosensibles, à base de poly(acrylate d'o-nitrobenzyle) (PNBA) hydrophobe et photoclivable et de dextrane hydrophile ont été préparés avec succès en utilisant notammennt une réaction d'Huisgen (Cycloaddition Azoture-Alcyne catalysée par le Cuivre (I) - CuAAC chimie click). Dans un premier temps, la polymérisation de l'acrylate d'o-nitrobenzyle a été contrôlée avec succès grâce aux développements récents de la polymérisation radicalaire vivante par transfert d'un seul électron (SET-LRP). Nous avons alors obtenu un PNBA fonctionnalisé à son extrémitié par un brome. Ce brome a ensuite été substitué par un groupe azido. En parallèle, le dextrane a été modifié pour y introduire plusieurs fonctions alcyne (dextrane alcyne) ou une seule sur son extrémité réductrice (dextrane α-alcyne). Nous avons ensuite fait réagir ces dérivés de dextrane avec le PNBA-N3 pour obtenir respectivement les glycopolymères greffés et dibloc. Tous les glycopolymères ont été caractérisés par chromatographie d'exclusion stérique (SEC), Résonnance Magnétique Nucléaire 1H, 13C, 2D DOSY 1H et par spectrométrie FT-IR. Dans un deuxième temps, nous avons optimisé les conditions pour obtenir des nanoparticules peu disperses à partir des précédents glycopolymères. Dans certains cas, des nanoparticules ont également été obtenues en utilisant le dextrane alcyne et le PNBA-N3 dans un procédé d'émulsion/évaporation de solvant organique. La stabilité de toutes les nanoparticules vis-à-vis de solutions aqueuses de diverses forces ioniques ou d'un tensioactif compétitif a été étudiée. Enfin, l'effet de la lumière sur ces nanoparticules photosensibles a été mis en évidence à l'aide de la lampe UV. Plus précisément, nous avons pu suivre la destruction des nanoparticules par spectroscopie de fluorescence et diffusion de lumière dynamique en encapsulant le Rouge du Nil (sonde fluorescente) au sein de ces particules
Photosensitive grafted and diblock amphiphilic glycopolymers based on hydrophobic photosensitive poly(o-nitrobenzyl acrylate) (PNBA) and hydrophilic dextran were successfully prepared via grafting onto techniques through a Huisgen-type Copper(I) catalyzed Azide-Alkyne Cycloaddition (CuAAC click chemistry). Firstly, recent developments in the single-electron transfer–living radical polymerization (SET–LRP) provided us an access to control the o-nitrobenzyl acrylate polymerization and we obtained PNBA with bromide end function. Then, this bromide end function was replaced by azido (N3) group. In a parallel way, we modified dextran by introducing several alkyne groups all long the polysaccharide chain (alkynated dextran) or only one group at the reducing end-chain (α-alkyne dextran). In the second step, alkynated dextran and α-alkyne dextran were reacted with PNBA-N3 by CuAAC to obtain grafted or diblock glycopolymers. All glycopolymers were characterized by Size Exclusion Chromatography, 1H, 13C, 2D DOSY 1H NMR and FT-IR spectroscopy. Secondly, conditions to formulate nanoparticles from the previous glycopolymers were optimized. In some case, we also carried out an emulsion/evaporation process using dextran alkynated and PNBA-N3 to produce nanoparticles. Then, stability of nanoparticles were studied over rang of ionic strengths as well as stability in presence of a competitive surfactant. Finally, the effect of light on these photosensitive nanoparticles was studied using UV-lamp. More precisely, we loaded these nanoparticules by Nile Red fluorescent dye and followed thier destruction by using fluorescence spectroscopy and Dynamic Light Scattering

Cette thèse porte sur des matériaux luminescents sulfurés, notamment des astérisques moléculaires persulfurés phosphorescents, basés sur l'hexakis(p-tolylthio)benzène (HTTB), et d'un thiosphérulène (balle moléculaire soufrée). Non émissif en solution, HTTB est un des émetteurs phosphorescents les plus puissants à l'état solide, avec une émission induite par agrégation (AIE). Un des objectifs est de synthétiser des dérivés portant des acides carboxyliques en périphérie pour augmenter la solubilité aqueuse et mettre à profit la coordination sélective d'ions Pb(II) et Cd(II) afin de former des édifices supramoléculaires rigides luminescents et des capteurs intelligents. Ces fonctions permettent d’ancrer les astérisques à l'intérieur de nanoparticules de silice (NPs) pour activer la phosphorescence. Ces nouvelles NPs, dont l’émission est sensible à l’oxygène, sont un colorant pour l'imagerie in vivo par microscopie à deux photons d'excitation
This thesis focuses on sulfurated luminescent materials, including phosphorescent persulfurated molecular asterisks, based on hexakis(p-tolylthio) benzene (HTTB), and a thiospherulene (a sulfurated molecular ball). Non-emissive in solution, HTTB is one of the most powerful phosphorescent emitters in the solid state, from an aggregation-induced emission (AIE). One of the objectives is to synthesize some asterisk derivatives bearing carboxylic acids at their periphery to increase their aqueous solubility, and to take advantage of the selective coordination of Pb (II) and Cd (II) ions in order to form luminescent rigid supramolecular edifices and intelligent sensors. These functions allow to anchor the asterisks inside some silica nanoparticles (NPs) to activate phosphorescence. These new NPs, whose emission is sensitive to oxygen, are dyes for in vivo imaging by two-photon excitation microscopy

Zwei Modellreaktionen wurden mit thermoresponsiven Nanoreaktoren untersucht. Die Reduktion von 4-Nitrophenol und von Kaliumhexacyanidoferrat(III) mit Natriumborhydrid. Die Nanoreaktoren bestehen aus einem Polystyrol Kern, umgeben von einer Hydrogel Schale aus Poly-(N-Isopropylacrylamid). Die Reaktionen werden auf der Oberfläche von Metall Nanopartikeln in der Hydrogel Schale katalysiert. In einer auf Gold- und Silberkatalysatoren fokussierten Literaturstudie zeigte sich, dass der geschwindigkeitsbestimmende Reaktionsschritt zwischen beiden Metallen variieren könnte. Kinetische Studien mit Silber haben gezeigt, dass ein erfolgreich auf Gold angewandtes Modell modifiziert werden muss um auf Silber anwendbar zu sein und haben gezeigt, dass sich die Kinetik der Reaktion auf beiden Metallen unterscheidet. Die weitere Analyse ergab die typische, nicht der Arrhenius Abhängigkeit folgende, Abhängigkeit der Reaktionsrate von der Temperatur und hat gezeigt, dass die Partitionierung der Reaktanden im Hydrogel für das kinetische Modell relevant ist. Die Reduktion von Kaliumhexacyanidoferrat(III) auf Gold hat gezeigt, dass elektrostatische Effekte hier eine maßgebliche Rolle spielen. Ein kinetisches Modell wurde erarbeitet, dass die relevanten Einflussfaktoren durch Hydrogel, Geometrie der Nanoreaktoren, diffusions- und elektrostatische Effekte miteinbezieht. Die gewonnenen Daten konnten mittels eines auf der Auswertung des stationären Zustands basierenden Modells erfolgreich gefittet werden. Hierbei wurde das komplexe Zusammenspiel von elektrostatischen Effekten, deren Abschirmung und Einfluss auf die Diffusion sowie die Reaktionsrate gezeigt. Mit wenigen physikalisch aussagekräftigen Fitparametern konnten alle beobachteten Effekte erfolgreich erklärt werden. Der Vergleich der Reduktion von 4-Nitrophenol und von Hexacyanidoferrat(III) zeigt hierbei die entscheidenden Faktoren sowohl für reaktions- als auch für diffusionskontrollierte Reaktionen in thermoresponsiven Hydrogelen.
Two model reactions were investigated with thermoresponsive core-shell nanoreactors, the reduction of 4-nitrophenol and of potassium hexacyanoferrate(III), both reduced with sodium borohydride. The nanoreactors comprise of a polystyrene core surrounded by a hydrogel shell of poly-N-isopropylacrylamide (PNIPAM) crosslinked with N,N’-methylenebisacrylamide. Metal nanoparticles are immobilized inside the hydrogel shell on the surface of which the model reactions are catalyzed. In the reduction of 4-nitrophenol, special emphasis is laid on the reduction on gold and silver catalysts. A literature review of mechanistic as well as kinetic studies reveals that the rate determining step may differ between the two catalyst metals. Kinetic investigations with a silver catalyst reveal that the kinetic model derived previously for gold catalysts needs to be modified for the kinetic analysis in this study, confirming a difference in the kinetics for both catalyst metals. The temperature dependent analysis reveals the typical non-Arrhenius dependency of the reaction rate and shows that the partition ratio of the reactants is relevant for the kinetics. The reduction of potassium hexacyanoferrate(III) on gold reveals that electrostatic effects play a major role in this reaction. A new kinetic model is derived, accounting the relevant influence factors of the hydrogel, the nanoreactor geometry, diffusional and electrostatic effects. With a stationary state approach the experimental data are fitted successfully, revealing the complex interplay of electrostatic effects, the screening thereof and the influence on diffusion and reaction rate. With only a few physically meaningful fit parameters all observed effects can be explained successfully. The comparison of the reduction of 4-nitrophenol and potassium hexacyanoferrate(III) highlights the decisive factors in both, reaction and diffusion controlled reactions inside thermoresponsive hydrogels.

Les architectures des dispositifs Silicium-Sur-Isolant (SOI) représentent des alternatives attractives par rapport à celles en Si massif grâce à l’amélioration des performances des transistors et des circuits. Dans ce contexte, les plaquettes SOI doivent être d’excellente qualité.Dans cette thèse nous développons des nouveaux outils de caractérisation électrique et des modèles pour des substrats SOI avancés. La caractérisation classique pseudo-MOSFET (-MOSFET) pour le SOI a été revisitée et étendue pour des mesures à basses températures. Les variantes enrichies de -MOSFET, proposées et validées sur des nombreuses géométries, concernent des mesures split C-V et des mesures bruit basse fréquence. A partir des courbes split C-V, une méthode d'extraction de la mobilité effective a été validée. Un modèle expliquant les variations de la capacité avec la fréquence s’accorde bien avec les résultats expérimentaux. Le -MOSFET a été aussi étendu pour les films SOI fortement dopés et un modèle pour l'extraction des paramètres a été élaboré. En outre, nous avons prouvé la possibilité de caractériser des nanofils de SiGe empilés dans des architectures 3D, en utilisant le concept -MOSFET. Finalement, le SOI ultra-mince dans la configuration -MOSFET s'est avéré intéressant pour la détection des nanoparticules d'or
Silicon-On-Insulator (SOI) device architectures represent attractive alternatives to bulk ones thanks to the improvement of transistors and circuits performances. In this context, the SOI starting material should be of prime quality.In this thesis, we develop novel electrical characterization tools and models for advanced SOI substrates. The classical pseudo-MOSFET (-MOSFET) characterization for SOI was revisited and extended to low temperatures. Enriched variants of -MOSFET, proposed and demonstrated on numerous geometries, concern split C-V and low-frequency noise measurements. Based on split C-V, an extraction method for the effective mobility was validated. A model explaining the capacitance variations with the frequency shows good agreement with the experimental results. The -MOSFET was also extended to highly doped SOI films and a model for parameter extraction was derived. Furthermore, we proved the possibility to characterize SiGe nanowire 3D stacks using the -MOSFET concept. Finally thin film -MOSFET proved to be an interesting, technology-light detector for gold nanoparticles

Cette thèse porte sur des matériaux luminescents sulfurés, notamment des astérisques moléculaires persulfurés phosphorescents, basés sur l'hexakis(p-tolylthio)benzène (HTTB), et d'un thiosphérulène (balle moléculaire soufrée). Non émissif en solution, HTTB est un des émetteurs phosphorescents les plus puissants à l'état solide, avec une émission induite par agrégation (AIE). Un des objectifs est de synthétiser des dérivés portant des acides carboxyliques en périphérie pour augmenter la solubilité aqueuse et mettre à profit la coordination sélective d'ions Pb(II) et Cd(II) afin de former des édifices supramoléculaires rigides luminescents et des capteurs intelligents. Ces fonctions permettent d’ancrer les astérisques à l'intérieur de nanoparticules de silice (NPs) pour activer la phosphorescence. Ces nouvelles NPs, dont l’émission est sensible à l’oxygène, sont un colorant pour l'imagerie in vivo par microscopie à deux photons d'excitation
This thesis focuses on sulfurated luminescent materials, including phosphorescent persulfurated molecular asterisks, based on hexakis(p-tolylthio) benzene (HTTB), and a thiospherulene (a sulfurated molecular ball). Non-emissive in solution, HTTB is one of the most powerful phosphorescent emitters in the solid state, from an aggregation-induced emission (AIE). One of the objectives is to synthesize some asterisk derivatives bearing carboxylic acids at their periphery to increase their aqueous solubility, and to take advantage of the selective coordination of Pb (II) and Cd (II) ions in order to form luminescent rigid supramolecular edifices and intelligent sensors. These functions allow to anchor the asterisks inside some silica nanoparticles (NPs) to activate phosphorescence. These new NPs, whose emission is sensitive to oxygen, are dyes for in vivo imaging by two-photon excitation microscopy

Les NP han sorgit com prometedors sistemes d'administració de fàrmacs per les seves possibles aplicacions terapèutiques. El procediment comú per a aquest propòsit és dissoldre, encapsular i, finalment, adsorbir o adherir un medicament a la superfície de la NP, evitant el dany no desitjat a les cèl·lules i òrgans sans. De la mateixa manera, les nanopartícules de sílice mesoporoses (MSN) recentment han atret molta atenció en el camp de la nanomedicina per les seves característiques singulars. No obstant això, l'eficàcia terapèutica de les MSN quan s'utilitzen com a sistemes d'administració de fàrmacs sovint es veuen compromeses per l'alliberament previ del fàrmac carregat durant la circulació sanguínia i la manca de capacitat per arribar al destí desitjat. Tenint en compte aquests dos problemes, proposem en aquesta Tesi doctoral la utilització de la "protein corona" per evitar aquest alliberament prematura essent aquesta encaixada al porus. Per aconseguir això, s'han construït HSA-NP carregades amb un fàrmac antitumoral per a la direccionalitat selectiva del fetge utilitzant MSN. L'objectiu principal d'aquest treball és el desenvolupament d'un complex de nanopartícules i proteïnes, capaç de subministrar eficientment un fàrmac terapèutic. Aquest complex ha d'evitar simultàniament l'alliberament prematur i ser capaç de fer targeting al fetge per efecte de la PC. Per assolir això, en primer lloc, s'ha optimitzat la síntesi de MSN, per tal d'obtenir MSN reproduïbles i amb propietats definides. Després, la caracterització corresponent dels seus trets fisicoquímics va mostrar que, mitjançant el disseny d'un DOE, és possible dilucidar quins paràmetres tenen una major influència en la síntesi de MSN. A més, en el present treball s'ha demostrat que és possible una determinació qualitativa i quantitativa de la "protein corona" mitjançant un ús innovador d'equips analítics; el nanoDSC i l'ITC. Això permet aprofundir en el coneixement i control de PC. També s'ha avaluat l'efecte de la PC en el perfil d'alliberament d'un fàrmac antitumoral. Finalment, s'ha demostrat que la "protein corona" proporciona un alliberament més sostingut del fàrmac a una línia cel·lular de tumor hepàtic, sense afectar la viabilitat cel·lular. En conclusió, els resultats han demostrat que la relació entre la mida de porus i la mida de la proteïna en els sistemes HSA-MSN determina l'aplicació del sistema. En aquest treball s'ha proposat que, mitjançant l'enginyeria de PC, segons la relació de mida entre el porus i la proteïna es poden fer servir les mateixes proteïnes que formen la "protein corona" per bloquejar la sortida prematura del fàrmac mentre es dirigeix a l'òrgan desitjat.
Las NP han surgido como prometedores sistemas de administración de fármacos por sus posibles aplicaciones terapéuticas. El procedimiento común para este propósito es disolver, encapsular y, finalmente, adsorber o adherir un medicamento a la superficie de la NP, evitando el daño no deseado a las células y órganos sanos. De la misma manera, las MSN recientemente han atraído mucha atención en el campo de la nanomedicina debido a sus características singulares. Sin embargo, la eficacia terapéutica de las nanopartículas de sílice mesoporosas (MSN) cuando se utilizan como sistemas de administración de fármacos a menudo se ven comprometidas por la liberación previa del fármaco cargado durante la circulación sanguínea y la falta de capacidad para llegar al destino deseado. Teniendo en cuenta estos dos problemas, proponemos en esta Tesis doctoral la utilización de la “protein corona” para evitar esta liberación prematura siendo ésta encajada en el poro. Para lograr esto, se han construido HSA-NP cargadas con un fármaco antitumoral para la dirección selectiva al hígado utilizando MSN. El objetivo principal de este trabajo es el desarrollo de un complejo de nanopartículas y proteínas, capaz de un suministrar eficientemente un fármaco terapéutico. Este complejo debe evitar simultáneamente la liberación prematura y ser capaz de atacar al hígado por efecto de la PC. Para alcanzar esto, en primer lugar, se ha optimizado la síntesis de MSN, con el fin de obtener MSN reproducibles y con propiedades definidas. Luego, la caracterización correspondiente de sus rasgos fisicoquímicos mostró que, mediante el diseño de un DOE, es posible dilucidar qué parámetros tienen una mayor influencia en la síntesis de MSN. Además, en el presente trabajo se ha demostrado que es posible una determinación cualitativa y cuantitativa de la “protein corona” mediante un uso innovador de equipos analíticos; el nanoDSC y el ITC. Esto permite profundizar en el conocimiento y control de la PC. También se ha evaluado el efecto de la PC en el perfil de liberación de un fármaco antitumoral. Finalmente, se ha demostrado que la “protein corona” proporciona una liberación más sostenida del fármaco a una línea celular de tumor hepático, sin afectar la viabilidad celular. En conclusión, los resultados han demostrado que la relación entre el tamaño de poro y el tamaño de la proteína en los sistemas HSA-MSN determina la aplicación del sistema. En este trabajo se ha propuesto que, mediante la ingeniería de la PC, según la relación de tamaño entre el poro y la proteína se pueden usar las mismas proteínas que forman la “protein corona” para bloquear la salida prematura del fármaco mientras se dirige al órgano deseado.
NPs have gained promise for its potential therapeutic applications as drug delivery systems. The common procedure for this purpose is dissolving, encapsulating, and finally adsorbing or adhering a drug on the NPs surface, avoiding the undesired damage to healthy cells and organs. In the same way, mesoporous silica nanoparticles (MSN) have recently attracted a lot of attention in the nanomedicine field due to their singular characteristics. However, the therapeutic efficiency of MSNs when used as drug delivery systems is often compromised by the pre-release of loaded drug molecules during the blood circulation and the lack of ability to do targeting. Considering these two problems, we propose in this doctoral Thesis the utilization of the widely known protein corona to avoid this premature release by fitting it into the pore. To achieve this, Human Serum Albumin-NP loaded with an antitumor drug for selective liver targeting MSN has been constructed. The main objective of this work is the development of a nanoparticle-protein complex capable of an efficient delivery of a therapeutic drug. This complex should simultaneously avoid premature release and be able to do liver targeting both by effect of the PC. To reach this, first of all the synthesis of MSNs has been optimized, in order to obtain MSNs reproducible and with defined properties. Then, the corresponding characterization of their physicochemical traits showed that through the design of a DOE it is possible to elucidate which parameters have a higher influence on MSN synthesis. Furthermore, it has been here demonstrated that a qualitative and quantitative determination of the protein corona through an innovative use of analytical equipment is possible, the nanoDSC and ITC. This allows the deepening in the PC knowledge and control. It has also been assessed the effect of the protein corona in the release profile of an anti-tumor drug. Finally, the protein corona has been shown to provide a more sustained release of the drug to a liver tumor cell line, without affecting cell viability. In conclusion, results have shown that the relationship between the pore size and the protein size in HSA-MSN systems determine the system’s application. In this work it has been proposed the PC engineering according to the size ratio between the pore and the protein to use the same proteins that form the protein corona as gatekeepers while targeting the desired organ.

Pour étendre l'utilisation des composites dans des applications plus variées (applications intelligentes et multifonctionnelles), l'une des barrières est leur faible conductivité électrique et thermique. Dans le cas de composites renforcés par des fibres de carbone, la matrice organique est responsable des propriétés isolantes du composite résultant. L'une des solutions pour améliorer les conductivités des matériaux est l'utilisation des nanocharges conductrices. L'amélioration des propriétés électriques et thermiques des polymères nanochargés est une problématique récurrente dans la littérature. Cependant, étudier les propriétés des composites à fibre de carbone continue et nanochargés est moins abordée. Ce travail porte sur la fabrication et la caractérisation des composites nanochargés par du noir de carbone et des nanotubes de carbone. Tout d'abord, un intérêt particulier a été accordé à la phase délicate de la fabrication. Comme mentionné ci-dessus, la mise en œuvre des composites à renfort continu et matrice nanochargée implique des problèmes liés à l'agglomération et à la dispersion inhomogène des nanocharges dans le composite final. Pour résoudre ces problèmes, le choix de la matrice thermoplastique (Polyamide 6) était judicieux. En fait, la dispersion des nanocharges a été faite par extrusion bi-vis qui est connue comme l'une des voies les plus efficaces de séparation d'agglomérats. De plus, la méthode de fabrication à base de films de Polyamide 6, appelée film stacking, assure une partition homogène dès le début du processus. Des observations MEB ont été effectuées pour localiser les nanoparticules. Ceux-là ont montré que les particules pénétraient dans la zone des fibres. En effet, en atteignant le cœur des torons, les nano-charges ont créé un réseau de connectivité entre les fibres pour le passage de courant. Ceci explique l'amélioration constatée de la conductivité électrique des composites en présence de noir de carbone et des nanotubes de carbone. Ces essais ont été réalisés avec la méthode à 4 points. La conductivité électrique du composite à matrice « pure » est passée de 20S / cm à 80S / cm en ajoutant 8% en poids de noir de carbone et à 15S / cm en ajoutant 18% en poids de la même charge nanométrique. Pour les nanotubes de carbone, avec 2,5% en poids, la conductivité était d'environ 150S / cm. Pour les propriétés thermiques, des tests basés sur l'effet Joule ont été réalisés. L'augmentation de la température a été enregistrée en utilisant une caméra IR. Les résultats obtenus sont en accord avec ceux de la conductivité électrique, montrant une amélioration du comportement thermique en présence de nanocharges. Grâce à ces résultats, l'utilisation de ces composites comme outil de suivi d’endommagement était possible. Par ailleurs, la méthode de variation de la résistance électrique a été effectuée. Les matériaux nanochargés ont montré une meilleure sensibilité aux endommagements. Les résultats ont été comparés aux outils classiques de suivi d’endommagement. A la fin, plusieurs applications « intelligentes » ont été testées telles que : le composite à gradients de propriétés et des matériaux nanochargés cousus
To extend the use of composites in more varied application (smart applications, multifunctional issues), one of the actual barrier is their poor electrical and thermal conductivities. In the case of carbon fiber reinforced composites, organic matrix are in charge of the insulating properties of the resulting composite. One of the solutions to enhance conductivities of materials is the use of conductive nanofillers. Improving the electrical and thermal properties of nanofilled polymers has been investigated in several studies. However, studiing the properties of continuous carbon fiber nano-filled composites is less approached. This work tends to fabricate and characterize carbon black and carbon nanotubes nano-filled composites. First of all, special interest was given to the delicate phase of manufacturing. As mentioned before, processing continuous fiber reinforced nanofilled polymers implies issues related to nanofillers agglomeration and inhomogeneous dispersion in the final composite. To resolve these problems, the choice of the thermoplastic (Polyamide6) matrix seemed preferable. In fact, the dispersion of nanofillers was made by twin screw extrusion which is known as one of the most effective agglomeration separation ways. Adding to this, the fabrication method based on Polyamide 6 shects called film stacking, ensure a homogeneous partition at the beginning of the process. SEM observations were performed to localize the nano-particles. It showed that particles penetrated on the fiber zone. In fact, by reaching the fiber zone, the nano-fillers created network connectivity between fibers which means an easy pathway for the current. It explains the noticed improvement of the electrical conductivity of the composites by adding carbon black and carbon nanotube. This test was performed with the 4 points electrical circuit. It shows that electrical conductivity of 'neat' matrix composite passed from 20S/cm to 80S/cm by adding 8wt% of carbon black and to 15S/cm by adding 18wt% of the same nano-filler. For carbon nanotubes, with '2.5wt% the conductivity was around 150S/cm. For the thermal properties, tests based on Joule's effect were performed. The rise of temperature was recorded using IR camera. Results obtained are in agreement with the electrical conductivity ones, showing enhancement of the thermal behavior in presence of nanofillers. Thanks to these results, the use of these composites as a damage-monitoring tool was possible. By the way, the electrical resistance change method was performed. Nanofilled materials showed better sensitivity to damage. Results were compared with classical damage monitoring tools. At the end, several 'smart' applications were tested such as graded functionalities composite and stitched nanofilled materials

A low cost compact diagnostic has many implications in today’s society. Smart phone technology has exponentially grown and with it the imaging capabilities associated with smart phones. The goals of this research are i) to determine the feasibility of combining in the field smart phone images with color dependent assay results, ii) to develop a MatLab® image analysis code to analyze these results, and iii) compare limits of detection between the un-aided eye and MatLab® image analysis software. Orange G dye is used to create a stock solution and subsequent titers for analysis. Autocad is used to design an assay platform of 10x10 wells that are printed via a Xerox® Phaser printer with wax ink onto nitrocellulose paper. Dilutions are performed and pipetted into the wells. The image analysis code is used to determine hue, saturation, and value (HSV) values of wells. A limit of detection study using the dye is performed. HSV values are used to form calibration curves. The resulting curve fit equations are then integrated into the image analysis code to determine dye concentration. Finally, the complete capability is demonstrated by using an analogous 10x10 well experimental nitrocellulose sheet, which included a follow-up experiment via a spot check analysis. This study illustrates the feasibility of a low cost image analysis as a tool for lateral flow assay diagnostic versus the unaided eye. Future work includes using this protocol in conjunction with a lateral flow immunoassay and developing an application for the analysis.

Thermophysiological comfort is one of the most important factors for people to choose desirable gar-ments, which can be evaluated via measuring permeability of body heat and sweat. In this paper the water vapor permeability of nanocomposite nylon 6 fabrics produced from melt spun nanocomposite yarns with different TiO2 nanoparticle concentrations have been investigated. Results from measuring water vapor permeability at different environment temperatures for 4 h. indicated that sample with 0.4 wt% of TiO2 nanoparticle can provide fabric with maximum comfort properties. At low temperature 27.5% decline of permeability as compared to pure fabric causes this sample to protect body from cool weather through pre-venting loss of body heat. By increasing temperature from 12 to 35 oC water vapor permeability enhance-ment of nanocomposite improved about 99% as compared to pure one. Consequently nanocomposite with suitable nanoparticle content can provide more comfortable fabrics in different temperatures and applica-tions. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/35603

Tesis por compendio
[ES] La presente tesis doctoral se centra en el diseño, síntesis y caracterización de varios nanodispositivos híbridos orgánico-inorgánicos, utilizando como soporte nanopartículas de sílice mesoporosa equipadas con enzimas y puertas moleculares, los cuales muestran capacidades comunicativas además de la evaluación de diferentes estrategias de comunicación. El primer capítulo incluye un resumen de diferentes conceptos sobre los que se fundamentan los estudios realizados tales como nanotecnología, materiales de sílice mesoporosa, materiales con puertas moleculares que reaccionan a estímulos específicos, partículas Janus y biocomputación. Finalmente, se incluyen conceptos básicos acerca de la comunicación química, materiales y estrategias empleados hasta ahora y ejemplos representativos. A continuación, en el segundo capítulo, se presentan los objetivos generales de esta tesis doctoral que son abordados en los siguientes capítulos experimentales. El tercer capítulo muestra un sistema de biocomputación para liberación basado en nanopartículas Janus de oro-sílice mesoporosa capaces de comunicarse con el entorno procesando la información e imitando la función lógica booleana propia de un demultiplexer y que resulta en la liberación controlada de la carga. Se muestra que dicho nanodispositivo puede llevar a cabo sus funciones en medios complejos como en células cancerígenas. En el cuarto capítulo, se presenta un modelo circular de comunicación dentro de una red de tres nanopartículas diferentes basado en el intercambio jerárquicamente programado de mensajes químicos. La parte mesoporosa del nanodispositivo 1 (S1βgal) es cargada con la especie fluorescente [Ru(bpy)3]Cl2 y tapada con cadenas de oligo(etilenglicol) que contienen puentes disulfuro y que funcionan como puertas moleculares, mientras que la enzima β-galactosidasa es unida a la parte del oro. En la nanopartícula 2 (S2galox), la enzima galactosa oxidasa es inmovilizada en la cara del oro mientras que la sílice mesoporosa es cargada con 4-(bromometil)benzoato de metilo y los poros tapados con un derivado de arilboronato autoinmolante sensible a H2O2 que forma un complejo huéspedanfitrión con β-ciclodextrina. Finalmente, el nanodispositivo 3 (S3est) es funcionalizado con la enzima esterasa en la parte del oro, cargada con la especie reductora hidroclururo de tris(2-carboxietil)fosfina (TCEP) en la parte mesoporosa y tapada con una nanoválvula supramolecular que responde a pH (βciclodextrina:benzimidazol). En el quinto capítulo, se muestra un modelo interactivo de comunicación química entre una nanopartícula Janus abiótica y un organismo vivo (Saccharomyces cerevisiae). En particular, el nanodispositivo está basado en nanopartículas funcionalizadas con glucosa oxidasa en la parte del oro, cargadas con el genotóxico fleomicina y tapadas con la puerta molecular sensible a pH (βciclodextrina:benzimidazol). El microorganismo usado en el estudio es una levadura modificada que expresa GFP bajo el control del promotor del gen RNR3; la transcripción de dicho gen es inducida con la exposición a agentes que dañan el ADN. La ruta de comunicación interactiva empieza con la adición de sacarosa (estímulo de entrada) la cual es hidrolizada en glucosa por la invertasa localizada en el espacio periplásmico de las levaduras y que difunde al nanodispositivo donde es trasformada en el correspondiente ácido por la glucosa oxidasa de la parte del oro. La bajada local de pH da lugar a la apertura de la nanoválvula sensible a pH del nanovehículo y con ello a la liberación de fleomicina (mensaje de vuelta) que induce la expresión de GFP (señal de salida) en las levaduras. En el sexto capítulo, proponemos una estrategia para establecer una comunicación lineal entre dos microorganismos diferentes que no interactúan entre ellos mediada por un nanodispositivo que actúa como traductor químico. Finalmente, las conclusiones generales de la presente tesis doctoral son expuestas en el capítulo siete. El estudio de las capacidades comunicativas de los nanodispositivos mesoporosos funcionalizados con enzimas permite la construcción de estrategias de cooperación entre diferentes entidades que permiten funcionalidades que van más allá que aquellas llevadas a cabo por agentes individuales.
[CA] La present tesi doctoral es centra en el disseny, síntesi i caracterització de diversos nanodispositius híbrids orgànic-inorgànics, utilitzant com a suport nanopartícules de sílice mesoporosa equipades amb enzims i portes moleculars, i que mostren capacitats comunicatives a més de l’avaluació de diferents estratègies de comunicació. El primer capítol inclou un resum de diferents conceptes sobre els quals es fonamenten els estudis realitzats com ara nanotecnologia, materials de sílice mesoporosa, materials amb portes moleculars que reaccionen a estímuls específics, partícules Janus i biocomputació. Finalment, s’inclouen conceptes bàsics sobre la comunicació química, materials i estratègies utilitzades fins ara i exemples representatius. A continuació, en el segon capítol, es presenten els objectius generals d’aquesta tesi doctoral que són abordats en els següents capítols experimentals. El tercer capítol mostra un sistema de biocomputació per alliberament basat en nanopartícules Janus d’or-sílice mesoporosa capaços de comunicar-se amb l’entorn processant la informació i imitant la funció lògica booleana pròpia d’un demultiplexer i que resulta en l’alliberament controlat de la càrrega. Es mostra que aquest nanodispositiu pot dur a terme les seves funcions en mitjans complexos com en cèl·lules canceroses. En el quart capítol, es presenta un model circular de comunicació dins d’una xarxa de tres nanopartícules diferents basat en l’intercanvi jeràrquicament programat de missatges químics. La part mesoporosa del nanodispositiu 1 (S1βgal) es carrega amb l’espècie fluorescent [Ru(bpy)3]Cl2 i es tapa amb cadenes d’oligo(etilenglicol) que contenen ponts disulfur i que funcionen com portes moleculars, mentre que l’enzim β-galactosidasa s’immobilitza a la part de l’or. A la nanopartícula 2 (S2galox), l’enzim galactosa oxidasa s’immobilitza a la cara de l’or mentre que la sílice mesoporosa es carrega amb 4-(bromometil)benzoat de metil i els porus són tapats amb un derivat d’arilboronat autoimmolant sensible a H2O2 que forma un complex hoste-amfitrió amb β-ciclodextrina. Finalment, el nanodispositu 3 (S3est) es funcionalitza amb l’enzim esterasa en la part de l’or, es carrega amb l’espècie reductora hidroclurur de tris (2-carboxietil) fosfina (TCEP) a la part mesoporosa i es tapa amb una nanoválvula supramolecular que respon a pH (β-ciclodextrina:benzimidazol). En el cinqué capítol, es mostra un model interactiu de comunicació química entre una nanopartícula Janus abiòtica i un organisme viu (Saccharomyces cerevisiae). En particular, el nanodispositiu està basat en nanopartícules funcionalitzades amb glucosa oxidasa en la part de l’or, carregades amb el genotòxic fleomicina i tapades amb la porta molecular sensible a pH (βciclodextrina:benzimidazol). El microorganisme utilitzat en l’estudi és un rent modificat que expressa GFP sota el control del promotor del gen RNR3; la transcripció d’aquest gen és induïda amb l’exposició a agents que danyen l’ADN. La ruta de comunicació interactiva comença amb l’addició de sacarosa (estímul d’entrada) la qual és hidrolitzada en glucosa per la invertasa localitzada en l’espai periplasmàtic dels rents i que difon al nanodispositiu on és transformada en el corresponent àcid per la glucosa oxidasa de la part de l’or. La baixada local de pH dona lloc a l’obertura de la nanoválvula sensible a pH del nanovehicle i amb això l’alliberament de fleomicina (missatge de tornada) que indueix l’expressió de GFP (senyal de sortida) en el rent. En el sisé capítol, proposem una estratègia per establir una comunicació lineal entre dos microorganismes diferents que no interactuen entre ells facilitada per un nanodispositiu que actua com a traductor químic. Finalment, les conclusions generals de la present tesi doctoral són exposades en el capítol set. L’estudi de les capacitats comunicatives dels nanodispositius mesoporosos funcionalitzats amb enzims permet la construcció d’estratègies de cooperació entre diferents entitats que permeten funcionalitats que van més enllà que aquelles dutes a terme per agents individuals. Esperem que els resultats obtinguts inspiren aplicacions futures en diferents àrees com ara biomedicina, nanorobots, materials que imiten la naturalesa i tecnologies de la informació.
[EN] This PhD Thesis is focused on the design, synthesis and characterization of several hybrid organic-inorganic nanodevices using mesoporous silica nanoparticles equipped with enzymes and molecular gates which display communication capabilities as well as the design and evaluation of different communication strategies. The first chapter includes an overview of the different concepts which lay the foundations of the presented studies such as nanotechnology, mesoporous silica materials, stimuli-responsive gated materials, Janus particles and biocomputing. Basic concepts of chemical communication, materials and enabling technologies employed so far and representative examples in this field are also included. Next, in the second chapter, the general objectives of this PhD Thesis that are addressed in the following experimental chapters are presented. The third chapter shows a biocomputing delivery system based on Janus gold-mesoporous silica nanoparticles capable of chemically communicating with the environment and processing the information mimicking a demultiplexer Boolean logic function which results in a programmed cargo release. Finally, it is shown that such nanodevice is operative in complex media such as cancer cells. In the fourth chapter, it is presented a circular model of communication within a network of three different nanoparticles based on the hierarchically programmed exchange of chemical messages. The mesoporous face of nanodevice 1 (S1βgal) is loaded with the fluorescent dye [Ru(bpy)3]Cl2 and capped with disulfidecontaining oligo(ethylene glycol) chains acting as gatekeepers, whereas the enzyme β-galactosidase is attached to the gold face. In nanoparticle 2 (S2galox), the enzyme galactose oxidase is immobilized on the Au face, while the mesoporous silica is loaded with methyl 4-(bromomethyl)benzoate and the mesopores capped with a H2O2-sensitive self-immolative arylboronate derivative which forms a host-guest complex with β-cyclodextrin. Finally, the nanodevice 3 (S3est) is functionalized with the enzyme esterase on the Au face, loaded with the reductive species tris(2- carboxyethyl)phosphine hydrochloride (TCEP) in the mesoporous face and capped with a pH-responsive supramolecular nanovalve (β-cyclodextrin:benzimidazole). In the fifth chapter, it is showed an interactive model of chemical communication between an abiotic Janus nanoparticle and a living organism (Saccharomyces cerevisiae). In particular, the nanodevice is based on Janus goldmesoporous silica nanoparticles functionalized with glucose oxidase on the Au face, loaded with the genotoxin phleomycin and capped with a pH-responsive (βcyclodextrin:benzimidazole) gatekeeper. The microorganism used in the studies is an engineered budding yeast that expresses GFP under the control of the RNR3 promoter; RNR3 gene transcription is induced upon exposure to DNA-damaging agents. The interactive communication pathway starts with the addition of sucrose (input) which is hydrolyzed into glucose by invertase located in periplasmic space of yeasts and diffuses to the nanodevice where it is transformed into the corresponding acid by glucose oxidase on the Au face. The local drop in pH leads to uncapping of the pH-sensitive nanovalve in the nanocarrier and the release of phleomycin (feedback messenger) that induces GFP expression (output) in yeasts. In the sixth chapter, we propose a strategy to establish linear communication between two different non-interacting microorganisms mediated by a nanodevice which acts as a chemical “nanotranslator”. Finally, the general conclusions from this PhD Thesis are presented in chapter seven. The study of communication capabilities of enzyme-functionalized mesoporous nanodevices enables the construction of strategies of cooperation between different entities allowing sophisticated functionalities that go beyond those carried out by individual agents. We hope that the obtained results inspire future applications in different areas such as biomedicine, nanorobots, life-like materials and information technologies.
The authors wish to thank the Spanish Government (projects RTI2018-100910-B-C41 and RTI2018-101599-B-C22 (MCUI/AEI/FEDER, UE), CTQ2017-87954-P), the Generalitat Valenciana (PROMETEO 2018/024), the Comunidad de Madrid (IND2017/BMD7642) and CIBER-BBN (NANOCOMMUNITY project) for support.
De Luis Fernández, B. (2021). Development of enzyme-functionalized hybrid mesoporous nanodevices for advanced chemical communication [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171506
TESIS
Compendio

Les multicouches de polyélectrolytes, systèmes auto-assemblés par adsorptions successives de polycations et polyanions, peinent à trouver des applications concrètes en raison de leur fragilité mécanique et du temps nécessaire à leur assemblage. Pour améliorer leur tenue mécanique, nous avons développé une méthode d'assemblage couche-par-couche par liaisons covalentes de films polymères. Des films formés de polymères portant des groupements alcynes et azides ont ainsi été réticulés par une réaction click catalysée par les ions Cu+ obtenus par voie électrochimique. Pour améliorer le mode d'assemblage, l'auto-construction en une seule étape de films par approche morphogénique, a été développée. Cette approche, confinée à la surface et caractérisée par la présence en solution de l'ensemble des constituants, marque une rupture. Elle permet ainsi un contrôle spatial de l'assemblage des films et la combinaison de plusieurs modes d'interactions pendant leur assemblage. Des films dont la cohésion repose sur des interactions covalentes, hôtes-invités et supramoléculaires, ont ainsi été assemblés. L'introduction de nanoparticules métalliques dans les films (multicouches et auto-construits) a également été effectuée dans le but de développer des électrodes de grande surface spécifique.

L’oxyde de zinc dopé de type n est un excellent candidat pour la réalisation de films transparents et isolants thermiques grâce à ses propriétés d’absorption et de réflexion limitées aux domaines UV et IR. La synthèse en milieu polyol de particules nanostructurées d’oxyde de zinc dopé par du gallium a été utilisée afin de maîtriser la morphologie des cristallites. Il a été démontré expérimentalement et théoriquement que le maximum d’efficacité d’absorption IR est atteint pour un taux de dopant de 2,6 % molaire. Des suspensions de haute transmission dans le visible et absorption infrarouge significative ont été obtenues par l’encapsulation des particules avec un matériau fluoré d’indice de réfraction intermédiaire entre l’oxyde et le milieu dispersant, ainsi que par l’optimisation de l’état de dispersion de suspensions colloïdales grâce à l’adsorption de thioalcanes en surface des cristallites de ZnO dopés
Thanks to its absorption / reflexion properties limited to the UV and the IR range, n-doped zinc oxide is a promising candidate for the elaboration of transparent and insulating films in smart windows. Nanostructured particles of Ga-doped zinc oxide were elaborated by polyol process. Polyol process was used in order to control the size and the morphology of the particles. Both experimental and theoretical data show that a maximum of IR absorption efficiency is obtained for a doping rate of 2.6 molar percent. Colloidal suspensions with high transmission in the visible range combined with significant absorption of the near infrared range were obtained using two strategies. The first one is the encapsulation of the Ga-doped ZnO particles by a fluoride shells with an intermediate refractive index between ZnO and the dispersion medium. The second one is the optimization of the dispersion state of nano-colloidal suspensions thanks to the adsorption of thioalkanes on the Ga-doped ZnO crystallite surfaces

Vanadium oxides (VOx) have been extensively investigated due to their many phases and range of properties, such as eletrochromic, thermochromic and electrochemical. Therefore, this promising materials may be implemented as energy efficiency materials in many sectors like military, industrial, domestic and transport. In the present dissertation VOx phases and nanostructures have been synthesized by hydrothermal synthesis using microwave irradiation as heating source. The synthesis parameters, such as reducing agent, dissolution method and the hydrothermal parameters temperature, reducing agent concentration and ratios were vary and studied to observe their impact in morpholy and the resultant VOx phase. Several pure and combination of VOx phases were achieved, such as VO2 (B), V6O13, V3O7, VO2 (M2) and V2O5. The structural composition and morphology of the as-obtained powders were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. Additionally, to observe a possible transition temperature of the samples a thermogravimetry and differential scanning calorimetry (TG-DSC) analysis were performed. Furthermore, nanosheets were layered in fluorine doped tin oxide (FTO) and Indium tin oxide (ITO) and applied as cathode, with a maximum current of 1.75 mA for ITO and 1.46 mA for FTO.

Mesoporous silica nanoparticles provide a versatile drug-delivery platform due to their exceptional properties, such as, high payload uptake, tunable pore width and particle size and protection over the guest molecule. The external surface can be functionalize with bioactive molecules and stimuli responsive polymers, improving the biocompatibility and drug bioavailability, increasing the local dose delivered and decreasing the side effects. The objective of this work was to synthesize fluorescent hybrid MSNs, coated with a co-polymeric shell containing pH-responsive and carbohydrate blocks obtained through RAFT polymerization. Such combination opens the possibility of diagnosis, through carbohydrates recognition ability towards cancer cells, and therapy by a pH mediated controlled release. The fluorescent silica nanoparticles were prepared by the Stöber method, incorporating a high quantum yield perylenediimide (PDI) in the silica structure. The nanoparticles were characterized by transmission electronic microscopy, with diameters of 30 nm and 65 nm with low size dispersity. The homo and co-polymers were synthesized by RAFT polymerization and characterized by UV-Vis and size exclusion chromatography (SEC), with a polydispersity below 1.2. The external surface of the MSNs was functionalized to allow the immobilization of the smart polymer. Surface modifications were assessed and quantified using 1H-NMR, UV-Vis and ζ-Potential. Polymer immobilization on the MSNs external surface were performed using four different methods: “transfer to”, “graft to”, “graft from” and a hybrid method. The particles modified with the homo carbohydrate polymer were obtained with polymer weight percentage between 0.5% and 4.3%. The immobilization of the final pH-responsive/carbohydrate co-polymer was achieved with a 2% polymer weight via “graft to” method. The immobilization by the hybrid method was also achieved, with promising results, indicating that our strategy is correct.

With the remarkable advancement in the nanoparticles (NPs)-based drug delivery systems (DDS) over the past several decades, the pharmacological properties associated with conventional free drugs delivery are improved. In this thesis, we report potential candidates for the next-generation NP-based DDS. While natural DDS are promising as they possess exceptional delivery mechanisms and selective targeting, synthetic DDS are more favorable for their low immunogenicity. Our developed natural DDS called magnetotactic bacterial cages (MBC), which is based on magnetotactic bacteria (MTB) as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). Loading DNA functionalized AuNPs in MTB aided in increasing the maximum-tolerated dose of DNA functionalized AuNPs and tackled issues related to DNA functionalized AuNPs stability and systemic delivery. Natural DDS hold great advantages; however, it is difficult to make complete prediction about their immunogenicity and toxicity on the basis of preclinical trials. Thus, we assessed the efficacy of synthetic NP-based DDS. Using inorganic platforms, we were able to develop the first visual monitoring system of bacteria-NPs interaction. The system offers simultaneous sensing and inhibition of bacteria in infected cells. The system is comprised of Au nanoclusters @lysozyme (AuNC@lys) colloids MSN loaded with antibacterial agents. The applicability of the inorganic DDS in the biomedical field has been limited by the high bioaccumulation risks. Hybrid materials combine the advantages of organic, inorganic and natural carriers, offering opportunities for enhanced stability, manipulating release behavior and combine two or more functions in a single platform. To further enhance the properties our inorganic DDS, we incorporated light-responsive organic ligands to silicabased NPs. Plasmid DNA was loaded on the light-responsive bridged silsesquioxane nanocomposites (BS NPs). Light irradiation was performed to reverse the surface charge of NPs via a photoreaction of the organic fragments (silsesquioxane) within the NPs, that resulted in the release of plasmid DNA in HeLa cancer cells. Finally, we assessed a new class of organic-inorganic DDS composed of inorganic metal ions and organic linkers, zeolite imidazolate frameworks-8 (ZIF-8). These NPs showed exceptional ability to entrap large cargo due to their tunable porosity and structural flexibility.

This thesis presents a new approach to produce mix matrix membranes using block copolymers and inorganic nanoparticles having magnetic properties. The polymeric nanoparticle with different morphologies (linear, Spheres, worms, and vesicles), from poly (methacrylic acid)-b-(methyl methacrylate) diblock copolymer, were synthesized using Reversible addition−fragmentation chain transfer polymerization (RAFT) in ethanol at 70 ֠C. The inorganic counterpart, iron oxide nanoparticles were prepared using different stabilizers at various temperatures to acquire the necessary surface charge and magnetic properties. The chemistry of the particles leads to form both hydrophobic membranes using non-solvent induced phase separation as well as a hydrophilic membrane by using the simple spin coating technique with the particles from polymerization induced self assembly. By a detailed experimental study of the membrane filtration, the influence of different parameters on the process performance has been investigated with and without magnetic field. Finally, membrane fouling has been studied using protein solution. Also, the membrane performance was examined under magnetic field revealing the successful reduction in the fouling phenomenon making them new performant membranes in the area of membrane technology.
Ce travail de thèse propose une nouvelle approche pour la préparation de membranes à matrice mixte basée sur l’utilisation de copolymères à blocs et de nanoparticules inorganiques disposant de propriétés magnétiques. Des aggrégats de copolymères ont été préparés avec une morphologie variée (sphères, cylindres et vésicules) à partir du copolymère poly(acide méthacrylique)-b-poly(méthacrylate de méthyle). Ce dernier a été synthétisé par polymérisation radicalaire contrôlée par transfert de chaîne réversible par addition-fragmentation (RAFT) dans l’éthanol à 70°C. Des particules d’oxyde de fer ont, quant à elles, été préparées en présence de différents stabilisants à température variée pour permettre d’atteindre la charge de surface et les propriétés magnétiques recherchées. La structure des copolymères à bloc a permis d’obtenir à la fois des membranes hydrophobes via le procédé de séparation de phase induite par un non-solvant, ainsi que des membranes hydrophiles lorsque que la technique de spin-coating était appliquée aux aggrégats formés par auto-assemblage induit lors de la polymérisation. Grâce à l’étude détaillée des propriétés de filtration des membranes obtenues, la relation structure-propriété a été discutée sous l’action d’un champ magnétique externe. Enfin, la sensibilité au colmatage a été vérifiée via la filtration de solutions de protéines. Il a ainsi été démontré une diminution notable du colmatage sous champ magnétique, ouvrant de belles perspectives pour ces nouvelles membranes
Esta tesis presenta una nueva aproximación a la producción de membranas de matrices mixtas, mediante copolímeros bloque y partículas inorgánicas con propiedades magnéticas. Las nanopartículas poliméricas con diferentes morfologías (lineal, esferas, gusanos, y vesículas) a partir del copolímero di-bloque: ácido polimetacrílico-b-metilmetacrilato han sido sintetizadas utilizando una polimerización por adición, fragmentación y transferencia de cadena reversible (RAFT) en etanol a 70ºC. La contraparte inorgánica, nanopartículas de óxido de hierro, ha sido preparada utilizando diferentes estabilizadores a varias temperaturas para adquirir la carga en la superficie y las propiedades magnéticas necesarias.La propiedades químicas de las partículas conducen a la formación de membranas hidrofóbicas mediante separación de fases inducida por no disolventes (NIPS), así como a la formación de mebranas hidrofílicas utilizando la técnica de recubrimiento por rotación simple de las partículas mediante autoensamblaje inducido por polimerización. Mediante un estudio experimental detallado de la filtración de la membrana, la influencia de diferentes parámetros en el rendimiento del proceso ha sido analizado en presencia y ausencia de campo magnético. Finalmente, el ensuciamiento de la membrana han sido estudiadas utilizando una disolución de proteínas. Asimismo, el rendimiento de la membrana ha sido examinado en presencia de campo magnético, dando como resultado una disminución en el ensuciamientode la membrana. Estos resultados confirman que estas nuevas membranas poseen altas prestaciones en el área de la tecnología de membranas

Dissertação de mest., Ciências Biomédicas, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010
The aim of this thesis is to develop smart and targeted nanoparticle contrast agents for Magnetic Resonance Imaging. These nanoparticles were designed to improve the sensitivity of this high resolution imaging technique and thereby improve early cancer detection, which is a major factor for the reduction of cancer mortality. The designed nanoparticles are expected to accumulate in the tumor through passive and active targeting. In addition, when exposed to the characteristic low pH of the cancer microenvironment, the nanoparticles will release the contrast agent, which will turn on its imaging capability. This contrast agent consists of pH-sensitive polymeric micelles formed by self-assembly, loaded with Gadolinium (III) complexes and bioconjugated with the C595 monoclonal antibody against the human MUC1 protein, which is overexpressed during tumorigenesis since its early stages. The capability of micelles to disintegrate and release the encapsulated contrast in acidic conditions was proved by 1.5T MRI experiments. The MRI study showed no image signal from the sample with intact micelles whereas a signal enhancement was observed from the sample at low pH. After one mouse has been intramuscularly injected with Gadolinium(III)-loaded micelles in both hind legs, the MR image demonstrated a stronger signal from the right hind leg, which was previously injected with an acid solution. To assess in vitro cytotoxicity of free and encapsulated Gadolinium (III) complexes in polymeric micelles, MTT assays were performed on different cancer cell lines. Encapsulated Gadolinium (III) complexes showed significantly lower cytotoxicity than free Gadolinium (III) complexes, even at the highest concentration. To verify the target capability of nanoparticles, fluorescent dye-loaded polymeric micelles were incubated with breast cancer cells expressing MUC1 (verified by semi-quantitative RT-PCR, Western blotting and flow cytometry) and mouse bone marrow stroma cells. Antibody-conjugated micelles had superior affinity for MUC1-expressing human breast cancer cells than for mouse bone marrow cells.

In the field of nanotechnology, one of the most operative research areas is nanomedicine, which applies nanotechnology to highly specific medical interventions for the prevention, diagnosis and treatment of diseases. Currently, the major issue that nanomedicine needs to face is the smart design and production of nanoparticles (NPs) based drug delivery systems for cancer therapy. Highly efficient drug delivery based on nanoparticles could potentially reduce the drug dose needed to achieve therapeutic benefit, thus reducing the side effects associated with the systemic delivery of drugs, whit great benefit to the patient. Indeed, a site-specific delivery of the active compound can be obtained manipulating NP surface by attaching ligands, such as peptides, antibodies or aptamers. Moreover, both passive and active targeting of the drug can be easily obtained by manipulating NP size and surface characteristics. NPs can also control and sustain the release of a drug during transport to, or at, the site of localization, altering drug distribution and subsequent clearance. At present, a new family of nanovectors, defined as stimuli-responsive nanocarriers (SRNs), is emerging. The key point in their mechanism of action lay in the fact that a specific cellular or extracellular endogenous stimulus of chemical, biochemical, or physical origin can modify NP conformation thus promoting the release of the active agent in a specific biological environment [1] [2]. In particular, a large variety of enzymes, such as proteases, glucuronidase, or carboxylesterases can be used as biochemical triggers. Generally the proteases, that are extracellularly expressed, such as the matrix metalloproteases (MMPs), are up-regulated in tumour microenvironment and are responsible for the proteolysis of the extracellular matrix (ECM) and of the basement membranes along with tissue remodelling and metastasis invasion. Since that, they are commonly identified as biomarkers of malignant tissues [3]. In the light of these considerations, Chapter.1 points out a smart approach in NPs design that takes benefits from the MMPs over-expression at tumour site, in order to produce a stimuli-responsive nanocarrier that allows a site specific drug release. To this aim, we proposed the use of a novel nanoparticle able to carry safely doxorubicin (Dox) at tumour tissues, and to respond to MMP-2 enzyme. The produced NPs are made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) – block – PEG copolymer (namely PELGA), blended with a TAP (Tumour Activated Pro-drug) composed by a MMP-2-sensitive peptide bound to Dox at the C-terminus and to PLGA molecule at the N-terminus. These NPs are named PELGA-TAP NPs. The presence of the MMP-2 enzyme in situ, leads to the destruction of the bond between the peptide and the Dox, with the consequent diffusion and accumulation of the drug in the extracellular environment. This mechanism allows the drug delivery only in presence of an endogenous stimulus that comes from the very nature of the tumour tissue itself. Furthermore, the same NPs were prepared without the presence of the peptide sequence, as negative control, and were named PELGA-Dox. Spheroids of U87 (Human Glioma cells) and HDF (Human Dermal Fibroblast) cells were used as in vitro models of tumour and healthy tissue, respectively, to demonstrate NPs ability to “sense” the differences in the expression levels of endogenous MMP-2 enzymes [4]. Since the production process and effectiveness of PELGA-TAP and PELGA-Dox NPs was well established and consolidate, in Chapter.2 we tested them in a new three-dimensional microtissue (3D µTP) model, which is an in vitro tissue equivalent proposed by Brancato et al. [5]. They fabricate µTPs with the aim to replicate in vitro the composition and the functionalities of the tumour microenvironment. In this work they clearly show that µTPs better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the tumour microenvironment. In particular, the 3D model was developed using normal fibroblasts (NF) and human epithelial cell lines (MCF10), or cancer-activated fibroblasts (CAF) and human breast adenocarcinoma cells (MCF7), to produce healthy and cancer microtissues, respectively. In this scenario, PELGA-TAP and PELGA-Dox NPs were tested in terms of Dox release on these µTPs in order to further validate their efficacy and selective drug release in a more realistic in vitro model, which better resemble tumour microenvironment, closer to the in vivo conditions [6]. Moreover, Chapter.3 shows an upgrade of the PELGA-TAP NP presented above. The approach used for the production of the nanocarrier takes advantages from the layer by layer polymer deposition technique developed and optimized by Vecchione et al. [7]. This technique allows the production of a very stable nanocarrier able to load large amounts of hydrophobic drugs and prevents their systemic leakage. The delivery system we proposed is a crosslinked polyelectrolytes nanocapsule (NC) based on an oil-core and a matrix metalloproteases-2-sensitive shell. MMP-2 enzymes catalyse the disassembly of the NC, which is stabilized by a MMP-2-cleavable peptide sequence as cross-linker. Also in this case, the drug release occurs in a spatially-controlled fashion upon an endogenous stimulus coming from the very nature of the tumour itself. The same NC was also produced with a scrambled peptide sequence as negative control. These NCs were tested on a spheroidal in vitro model, in order to proof their selective shell destabilization and consequent stimuli-responsive drug release in tumour microenvironment. Spheroids of U87 and HDF were used as models of tumour and healthy tissue, respectively. Cell viability was evaluated by means of Alamar Blue Assay. Moreover, the selective disassembly of the NC shell was followed using confocal microscopy and colocalization analyses were also performed. Finally, in Chapter.4 preliminary studies aimed to point out the advantages of an extracellular drug delivery are presented.

碩士
國立清華大學
生醫工程與環境科學系
102
To improve the therapeutic efficacy of solid brain tumors by promoting tumor-targeted chemotherapy delivery and triggering drug release upon external alternating magnetic field, the tumor-tropic adipose-derived stem cells were exploited as a vehicle to carry the superparamagnetic iron oxide nanoparticles (SPION)/ paclitaxel (PTX) -loaded nanoparticles. The PTX and oleic acid-coated SPIONs were hydrophobically entrapped in the poly(lactic-co-glycolic acid)-based cores stabilized by amphiphilic lipid-containing copolymer, poly(γ-glutamic acid-co-distearin glutamate).While the particle size and polydispersity index were evaluated to be ca 106 nm and 0.11, respectively, the SPION / PTX -loaded nanoparticles were featured with a high drug loading efficiency (91.9%), corresponding to a loading capacity of 8.4 wt%. The in vitro results demonstrate that the SPION / PTX -loaded nanoparticles after being engulfed by stem cells are benign to the cellular host, thereby allowing the host to retain their innate tumor tropism. The in vivo fluorescence images reveal that the Cy5.5-labeled nanoparticles transported by tumor-homing stem cells display the considerably enhanced accumulation in the brain tumor of the ALTS1C1 intracranial tumor-bearing mice. Notably, the survival rate of the ALTS1C1 intracranial tumor-bearing mice subjected to the payload-containing stem cells via tail vein injection and high frequency magnetic field (HFMF) was significantly enhanced as compared to that of tumor-bearing mice receiving SPION / PTX -loaded nanoparticles alone. Furthermore, the treatment combining payload-containing stem cells and HFMF stimulus exhibited the superior capability of inhibiting tumor growth of the ALTS1C1 subcutaneous tumor-bearing mice. Based on the above results, the use of tumor-tropic stem cells to deliver therapeutic nanoparticles combined with the external remotely-controlled drug release shows the great potential for brain tumor treatment.

碩士
國立臺灣科技大學
材料科學與工程系
106
We compare the stability of silver nano particles by using three types of PIB-imide-PIB polymer dispersant. The results come out that PIB-ED2003-PIB has great stability to silver nano particles. Then we add organic/inorganic hybrid dispersant contain zero-dimensional and one-dimensional material inside the dispersant, and study the stability of silver nano particles. We use carbon black and carbon nano tube as dispersant, because they have aromatic group, carbon black and carbon nano tube are hydrophobic and have great conductivity. In the experiment, silver nano particles can be nano dispersion and stability because of the non-covalent force of carbon material and organic polymer dispersant. We then coat the dispersions onto polymide with thermoplastic elastomer to make flexible electrode and stretchable electrode which can be applied to Sensing element of smart textile. We sucessfully prepare high conductivity nano composite film by coating organic/inorganic nano silver blending material. The silver nano particles we use was prepare by adding polymer dispersant as organic materials, and carbon black and carbon nano tube as inorganic materials to make silver nano particles dispersed and stable. Then we coat the dispersions onto polymer substrate and process heating. Finally we make high conductivity film and sucessfully measured ECG.

Lipid bilayers are the structural building blocks of cell membranes and represent fundamental motifs in engineered soft matter. Understanding their interaction with nanomaterials, and in particular with nanoparticles (NPs), is one of the central challenges of nanomedicine and materials science, pursuing a twofold goal: i) to respond to the urgent quest for the mechanistic understanding of nano–bio interactions, which determine NPs cytotoxicity in living organisms; ii) to enable the rational design of smart NPs/lipid nanodevices of biocompatible nature for multiple technological purposes. In this work, we combine inorganic NPs with synthetic lipid bilayers, either in lamellar or non-lamellar arrangement, with two main purposes. Lipid bilayers are primarily employed as simplified and highly controllable models of cell membranes, enabling the identification of key determinants at stake in the interaction of engineered NPs with biological interfaces. Secondly, we exploit the conjugation of NPs with lipid bilayered-systems to develop hybrid nanostructures with technological relevance. The work presented here is organized in three parts. Part I focuses on the physicochemical investigation of the interaction of Turkevich-Frens citrated gold NPs (AuNPs@CT) with synthetic model bilayers of different physicochemical features: our results shed light on the peculiar clustering process of AuNPs@CT observed onto natural membranes, which, although well-known and biologically relevant, has remained largely unaddressed. In addition, we show that these results find application in the development of a new plasmon-based assay, for the determination of the mechanical properties of natural membranes. In Part II, we extend the investigation to curved-bilayered structures, ubiquitous in cells under certain conditions. Employing a library of gold nanoparticles (AuNPs) with different physicochemical features, we directly compare the interaction of AuNPs with model membranes of different symmetry, i.e. from lamellar to cubic architectures, encountered in diseased cells: these results constitute the first attempt to systematically investigate the impact of membrane curvature in the interaction with nanomaterials. Finally, in Part III, we address the interaction of cubic lipid assemblies with Superparamagnetic Iron Oxide NPs (SPIONs), showing that, beside its fundamental interest, it can be exploited for the development of smart nanostructured hybrids with potential application in the biomedical field. In summary, the results presented in this work advance our current understanding of the events occurring at Nano-bio Interfaces and pave the way for the development of new technological devices, exploiting the conjugation of NPs with lipid bilayers.

Yes
Many drug molecules can be directly used as nanomedicine without the requirement of any inorganic or organic carriers such as silica and liposome nanostructures. This new type of carrier-free drug nanoparticles (NPs) has great potential in clinical treatment because of its ultra-high drug loading capacity and biodegradability. For practical applications, it is essential for such nanomedicine to possess robust stability and minimal premature release of therapeutic molecules during circulation in the blood stream. To meet this requirement, herein, we develop GSH-responsive and crosslinkable amphiphilic polyethylene glycol (PEG) molecules to modify carrier-free drug NPs. These PEG molecules can be cross-linked on the surface of the NPs to endow them with greater stability and the cross-link is sensitive to intracellular environment for bio-responsive drug release. With this elegant design, our experimental results show that the liberation of DOX from DOX-cross-linked PEG NPs is dramatically slower than that from DOX-non-cross-linked PEG NPs, and the DOX release profile can be controlled by tuning the concentration of the reducing agent to break the cross-link between PEG molecules. More importantly, in vivo studies reveal that the DOX-cross-linked PEG NPs exhibit favorable blood circulation half-life (>4 h) and intense accumulation in tumor areas, enabling effective anti-cancer therapy. We expect this work will provide a powerful strategy for stabilizing carrier-free nanomedicines and pave the way to their successful clinical applications in the future.
The National Basic Research Program of China (2013CB933500, 2012CB932400), National Natural Science Foundation of China (61422403), Natural Science Foundation of Jiangsu Province (BK20131162), QingLan Project, Collaborative Innovation Center of Suzhou Nano Science and a Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).