Dissertations / Theses on the topic 'Biological and Biomedical Applications'

Computer models are used extensively to investigate biological systems. Many of these systems can be described in terms of fields???spatially- and temporally- varying scalar, vector and tensor properties defined over domains. For example, the spatial variation of muscle fibers is a vector field, the spatial and temporal variation in temperature of an organ is a scalar field, and the distribution of stress across muscle tissue is a tensor field. In this thesis I present my research on how to represent fields in a format that allows researchers to store and distribute them independently of models and to investigate and manipulate them intuitively. I also demonstrate how the work can be applied to solving and analysing biomedical models. To represent fields I created a two-layer system. One layer, called the Field Representation Language (FRL), represents fields by storing numeric, analytic and meta data for storage and distribution. The focus of this layer is efficiency rather than usability. The second layer, called the Abstract Field Layer (AFL), provides an abstraction of fields so that they are easier for researchers to work with. This layer also provides common operations for manipulating fields as well as transparent conversion to and from FRL representations. The applications that I used to demonstrate the use of AFL and FRL are (a) a fields visualisation toolkit, (b) integration of models from different scales and solvers, and (c) a solver that uses AFL internally. The layered architecture facilitated the development of tools that use fields. A similar architecture may also prove useful for representations of other modelled entities.

In this project we, for the first time, integrated microfluidics with thermo-plasmonics. While microfluidics is a popular platform allowing experiments with small volumes of fluid, thermo-plasmonics can be used for powerful particle manipulation including capturing, mixing, filtering and projection. Combined, these two techniques give us an opportunity to work with numerous complex fluids containing particles, cells, and micro-beads. Here we designed, developed and tested several devices demonstrating various aspects of this exciting hybrid technology. This required use of soft lithography, metal deposition, 3D printing, oxygen plasma treatment and several other surface modification techniques. Additional challenges were in the fabrication of a multi-layer chip with several types of surfaces binding at several interfaces. The detailed design optimization was conducted, and many characteristics of the microfluidic channel were varied. After that, optimal flow patterns were determined using high-quality syringe pumps. An experiment with the simultaneous flow of two colored solutions through the same microfluidic chip demonstrated controlled laminar flow with minimal mixing. Next, thermo-plasmonic experiments were conducted in optimized micro-fluidic channels. Efficient capturing of microbeads were demonstrated using low power green laser with a wavelength 532 nm. In future, these experiments have many important applications including separation of bacteria from blood on a microfluidic chip. This might help with treatment of sepsis, analysis of blood pathogens and better prescription of antibiotics.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Silicon carbide (SiC) has been used for centuries as an industrial abrasive and has been actively researched since the 1960's as a robust material for power electronic applications. Despite being the first semiconductor to emit blue light in 1907, it has only recently been discovered that the material has crucial properties ideal for long-term, implantable biomedical devices. This is due to the fact that the material offers superior biocompatibility and hemocompatibility while providing rigid mechanical and chemical stability. In addition, the material is a wide-bandgap semiconductor that can be used for optoelectronics, light delivery, and optical sensors, which is the focus of this dissertation research. In this work, we build on past accomplishments of the USF-SiC Group to develop active SiC-based Brain Machine Interfaces (BMIs) and develop techniques for coating other biomaterials with amorphous SiC (a-SiC) to improve device longevity. The work is undertaken to move the state of the art in in vivo biomedical devices towards long term functionality. In this document we also explore the use of SiC in other bio photonics work, as demonstrated by the creation of the first reported photosensitive capacitor in semi-insulating 4H-SiC, thus providing the mechanism for a simple, biocompatible, UV sensor that may be used for biomedical applications. Amorphous silicon carbide coatings are extremely useful in developing agile biomaterial strategies. We show that by improving current a-SiC technology we provide a way that SiC biomaterials can coexist with other materials as a biocompatible encapsulation strategy. We present the development of a plasma enhanced chemical vapor deposition (PECVD) a-SIC process and include material characterization analysis. The process has shown good adhesion to a wide variety of substrates and cell viability tests confirm that it is a highly biocompatible coating whereby it passed the strict ISO 10993 standard tests for biomaterials and biodevices. In related work, we present a 64-channel microelectrode array (MEA) fabricated on a cubic 3C-SiC polytype substrate as a preliminary step in making more complex neurological devices. The electrode-electrolyte system electrical impedance is studied, and the device is tested against the model. The system is wire-bonded and packaged to provide a full neural test bed that will be used in future work to compare substrate materials during long-term testing. Expanding on this new MEA technology, we then use 3C-SiC to develop an active, implantable, BMI interface. New processes were developed for the dry etching of SiC neural probes. The developed 7 mm long implantable devices were designed to offer four channels of single-unit electrical recording with concurrent optical stimulation, a combination of device properties that is indeed at the state-of-the-art in neural probes at this time. Finally, work in SiC photocapacitance is presented as it relates to radio-frequency tuning circuits as well as bio photonics. A planar geometry UV tunable photocapacitor is fabricated to demonstrate the effect of below-bandgap optical tuning. The device can be used in a number of applications ranging from fluorescence sensing to the tuning of antennas for low-power communications. While technology exists for a wide variety of in vivo interfaces and sensors, few active devices last in the implantable environment for more than a few months. If these devices are going to reach a long-term implant capability, use of better materials and processing strategies will need to be developed. Potential devices and strategies for harnessing the SiC materials family for this very important application are reviewed and presented in this dissertation to serve as a possible roadmap to the development of advanced SiC-based biomedical devices.

Commercial organic dyes are widely used for cellular staining due to their small size, high brightness, and chemical functionality. However, their blinking and photobleaching are not ideal for studying dynamics inside live cells. An improvement over organics and much larger quantum dots, silver nanodots (Ag NDs) exhibit low cytotoxicity and excellent brightness and photostability, while retaining small size. We have utilized ssDNA hairpin structures to encapsulate Ag NDs with excellent spectral purity, high concentration, and good chemical and photophysical stability in a variety of biological media. Multi-color staining of fixed and live cells has been achieved, suggesting the promise of Ag NDs as good fluorophores for intracellular imaging. The great brightness and photostability of Ag nanodots indicate that they might be outstanding imaging agents for in vivo studies when encapsulated in delivery vehicles. In addition, Ag NDs can be optically modulated, resulting in increased sensitivity within high backgrounds. These good characteristics are combined with delivery vehicles such as PLGA and nanogels. After encapsulation, Ag nanodots still retain their good photophysical properties and modulation. It might be useful for in vivo applications in the near future

La tribocorrosion peut être définie comme l’ étude de l’influence des facteurs environnementaux (chimiques et/ou électrochimiques) et mécanique (frottement) sur le comportement tribologique de surfaces en mouvement relatifs. En raison de leurs caractéristiques particulières: performances mécaniques, associées à une faible densité, bonne tenue à la corrosion, biocompatibilité, le titane et ses alliages sont souvent utilisés dans le domaine médical comme implants dentaires et orthopédiques. Cependant, leur faible résistance vis-À-Vis du frottement en milieu agressif et plus spécifiquement biologique reste un frein à leur usage courant dans le domaine prothétique. Pour améliorer la résistance à la corrosion et à l'usure du titane et de ses alliages, différentes méthodes de modification de la surface ont été proposées durant ces dernières décennies. Dans ce cadre, le but de ce travail est de comparer les comportements en corrosion et tribocorrosion du titane commercialement pur (cp Ti), avec ce même matériau ayant subi au préalable les traitements suivants :- soit une étape d'oxydation thermique à 650 °C à l’air durant 48 h (formation d’un film d'oxyde de titane (TiO2) en surface),- soit un dépôt électrochimique de calcium phosphate (CaP) en surface,- soit un dépôt électrochimique de calcium phosphate (CaP) suivi d’une tape d’oxydation thermique à 650 °C à l’air durant 6 h (formation d’un dépôt de type CaP/TiO2 en surface). Les phases cristallines des films modifiés ont été identifiées par diffraction des rayons X (XRD). La microscopie électronique à balayage (MEB) en combinaison avec la spectroscopie à dispersion d'énergie (EDS) a été utilisée pour caractériser la morphologie et la composition de ces films.Le comportement en corrosion pure des échantillons cp Ti avec ou sans modifications de surface à été étudié in situ à partir des mesures électrochimiques de suivi du potentiel en circuit ouvert (OCP), de la spectroscopie d'impédance électrochimique (EIS) et du tracé de courbes de polarisation potentio-Dynamiques.Le comportement en tribocorrosion à été étudié quant à lui à l'aide d'un tribomètre de type pion-Disque apte à travailler en milieu aqueux et permettant outre l’enregistrement des paramètres tribologiques classiques, la mise en œuvre in situ des techniques électrochimiques utilisées lors de l’étude en corrosion pure. Caractérisation et analyses de la surface(composition, morphologie, rugosité …) sont effectuées avant et après chaque étude de comportement (corrosion et tribocorrosion). Un protocole pour la culture des cellules sur la surface de titane a été validé, en se basant sur les résultats expérimentaux préliminaires
Tribocorrosion is defined as the study of the interplay between chemical, electrochemical and mechanical processes that leads to a degradation of passivating materials in a corrosive environment. Due to the low density, excellent mechanical properties, high corrosion resistance and good biocompatibility, titanium and its alloys are widely used as dental and orthopedic implants. However, the poor wear resistant and bio-Inert properties limit their further development as more efficient and economic biomedical implants. To improve the corrosion-Wear resistance and even bioactivity of metallic implants, different surface modification methods are imposed in the past decades.The aim of this work is to provide a deep insight in the area of corrosion and tribocorrosion behavior of commercially pure titanium (cp Ti) under the guidance of a tribocorrosion protocol for passivating materials. And then three different surface modification treatments, as:- one-Step thermal oxidation at 650 °C for 48 h in air atmosphere to form a titania (TiO2) film on the surface of cp Ti.- one-Step electrochemical deposition of calcium phosphate (CaP) bioactive film on the surface of cp Ti.- electrochemical deposition of CaP bioactive film followed by thermal oxidation at 650 °C for 6 h in air atmosphere to form a CaP/TiO2 bioceramic film on cp Ti surface.The crystalline phases of the modified films were identified by X-Ray diffraction (XRD). Scanning electron microscopy (SEM) combined with energy dispersive spectroscopy (EDS) was used to characterize the morphology and composition of these films on cp Ti surface. In situ electrochemical measurements, like open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization are used to characterize the corrosion behavior of cp Ti samples without or with surface modification. The tribocorrosion behavior was investigated in an aqueous environment by combining a pin-On-Disc tribometer with the in situ electrochemical equipment. The classical tribological parameters could be also recorded under mechanical loaded condition. Surface characterization and analysis (like chemical composition, morphology, roughness...) are carried out before and after each corrosion and tribocorrosion test. A protocol for the culture of cells on the surface of titanium was validated, basing on the preliminary experimental results

Doctor Scientiae - DSc
Semiconductor nanocrystals or quantum dots (QDs) are fluorescent nanometer-sized particles which have physical dimensions that are smaller than the excitonic Bohr radius, large surface area-to-volume ratios, broad absorption spectra and very large molar extinction coefficients. Biomedical applications of QDs are mainly based on II-VI QDs containing cadmium, such as CdSe/ZnS. These cadmium-based systems are associated with high toxicity due to cadmium. As a result, potential replacements of cadmium-based QDs in biological applications are needed. In this study, InP/ZnSe QDs were synthesized for the first time using a one-pot hot injection method. Furthermore, a growth-doping method was used for silver, cobalt and iron incorporation into the InP core. Water compatibility was achieved through ligand exchange with 3- mercaptopropionic acid. In vitro cytotoxicity and imaging/internalization of the as-prepared MP A-InP/ZnSe and MP A-capped CdTe/ZnS QDs were evaluated. InP/ZnSe QDs were successfully synthesized with ZnSe shell causing a 1.4 times reduction in trap-related emission.

Photoacoustic tomography (PAT), as well as thermoacoustic tomography (TAT), utilize electromagnetic radiation in its visible, near infrared, microwave, and radiofrequency forms, respectively, to induce acoustic waves in biological tissues for imaging purposes. Combining the advantages of both the high image contrast that results from electromagnetic absorption and the high resolution of ultrasound imaging, these new imaging modalities could be the next successful imaging techniques in biomedical applications. Basic research on PAT and TAT, and the relevant physics, is presented in Chapter I. In Chapter II, we investigate the imaging mechanisms of TAT in terms of signal generation, propagation and detection. We present a theoretical analysis as well as simulations of such imaging characteristics as contrast and resolution, accompanied by experimental results from phantom and tissue samples. In Chapter III, we discuss the further application of TAT to the imaging of biological tissues. The microwave absorption difference in normal and cancerous breast tissues, as well as its influence on thermoacoustic wave generation and the resulting transducer response, is investigated over a wide range of electromagnetic frequencies and depths of tumor locations. In Chapter IV, we describe the mechanism of PAT and the algorithm used for image reconstruction. Because of the broad bandwidth of the laser-induced ultrasonic waves and the limited bandwidth of the single transducer, multiple ultrasonic transducers, each with a different central frequency, are employed for simultaneous detection. Chapter V further demonstrates PAT’s ability to image vascular structures in biological tissue based on blood’s strong light absorption capability. The photoacoustic images of rat brain tumors in this study clearly reveal the angiogenesis that is associated with tumors. In Chapter VI, we report on further developing PAT to image deeply embedded optical heterogeneity in biological tissues. The improved imaging ability is attributed to better penetration by NIR light, the use of the optical contrast agent ICG (indocyanine green) and a new detection scheme of a circular scanning configuration. Deep penetrating PAT, which is based on a tissue’s intrinsic contrast using laser light of 532 nm green light and 1.06 µm near infrared light, is also presented.

The last fifty years has seen an increase in the production of synthetic or artificial enzyme substrates used to identify and quantify enzymes. These substrates have found applications in a range of biomedical science disciplines. Used in biochemistry and clinical chemistry to identify and measure enzymes, some of these substrates have been adapted for use in microbiology, particularly bacterial diagnosis and, in more recent years, molecular biology. The use of artificial chromogenic and fluorogenic enzyme substrates to identify certain bacteria is now common place in medical laboratories worldwide. Not all bacteria can be identified with existing and commercially available artificial substrates. Some of these can be slow to yield results, imprecise, expensive or require a technical method too complicated to provide a viable laboratory test. Therefore, the search for new, more efficient, biochemical tests has progressed, with novel substrates and inventive applications being developed continually. In this study, core compounds were synthesised by various condensation reactions and their characteristics evaluated with respect to colouration/fluorescence and possible enhancement of these properties by metal chelation. Promising candidates were selected for glycosidation, via modified Koenigs-Knorr reactions, in an attempt to synthesise artificial substrates. Several commercially available core molecules were also subjected to glycosidation. The more successful substrates included glycosides of alizarin, nitrosalicylaldehyde and 3- hydroxyflavone. The galactoside of nitrosalicylaldehyde was evaluated in solid agar media and found to be selective for certain Gram-negative bacteria. When similarly investigated, the 3- hydroxyflavone-β-D-glucoside showed the possibility of being used in a procedure for the isolation of the clinically significant pathogens including Listeria monocytogenes. The enzyme kinetics of β-glucosidase with this substrate were also determined in a novel fluorescence assay and compared favourably to the well documented 4-methylumbelliferyl-β-D-glucopyranoside. Alizarin-2-yl-β-D-galactoside and p-naphtholbenzein-β-D-galactoside were successfully utilized for the screening of recombinant and non-recombinant Escherichia coli transformants produced routinely in molecular biology. Aminopeptidase substrates have been shown to be useful for the detection of enzymes which hydrolyse peptides that are specific to certain bacteria. To allow the evaluation of novel aminopeptidase substrates, that were to be subsequently synthesised, a cost effective, large scale source of recombinant leucyl aminopeptidase enzyme was developed via gene cloning techniques. Consequently, the products of this study may serve a beneficial purpose in future enzymatic investigations, medical diagnosis and molecular biology.

Heparin binding (HB) proteins mediates a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins could bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to the currently available methods. One of the most important classes of heparin binding protein is the fibroblast growth factors (FGFs) and its receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak amberlite cation (IRC) exchanger. This approach is an alternative to conventional affinity column chromatography, which exhibit several disadvantages, including time-consuming experimental procedures and regeneration and results in high cost for production of recombinant proteins. Authenticity of the purified proteins was verified by SDS-PAGE and MALDI mass spectrum analysis. Results of the heparin binding chromatography and steady state fluorescence experiments showed that the FGF-1 and the D2 are in a native biologically active conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules.

Novel surgical strategies for spinal disc repair are currently being developed that require materials that (1) possess the appropriate mechanical properties to mimic the tissue the material is replacing or repairing and (2) maintain their mechanical function for long durations without negatively affecting the tissue response of adjacent tissue (i.e. bone). Polymers formed through photopolymerization have emerged as candidate biomaterials for many biomedical applications, but these materials possess limited toughness in vivo due to the presence of water inherent in most tissues. Therefore, the overall objective of this research was to develop photopolymerizable (meth)acrylate networks that are both mechanically and biologically compatible under physiological conditions to be implemented in spinal repair procedures. The fundamental approach was to determine structure-property relationships between toughness and network structure in the presence of phosphate buffered saline (PBS) using several model copolymer networks in order to facilitate the design of photopolymerizable networks that are tough in physiological solution. It was demonstrated that networks toughness could be optimized in PBS by tailoring the Tg of the copolymer network close to body temperature and incorporating the appropriate "tough" chemical structures. The ability to maintain toughness up to 9 months in PBS was dependent upon the viscoelastic state and overall hydrophobicity of the network. In tandem, the effect of network chemistry and stiffness on the response of MG63 pre-osteoblast cells was assessed in vitro. The ability of MG63 cells to differentiate on (meth)acrylate network surfaces was found to be primarily dependent on surface chemistry with PEG-based materials promoting a more mature osteoblast phenotype than 2HEMA surfaces. Amongst each copolymer group, copolymer stiffness was found to regulate osteoblast differentiation in a manner dependent upon the surface chemistry. In general, photopolymerizable (meth)acrylate networks that were deemed "tough" were able to promote osteoblast differentiation in a manner comparable if not exceeding that on tissue culture polystyrene (TCPS). This research will impact the field of biomaterials by elucidating the interrelationships between materials science, mechanics, and biology.

Proteins are large macromolecules constituted by amino acids that are responsible for most of the biological processes within a cell. Proteins showing high complementary affinity may bind forming protein-protein complexes. In this context, antibodies are proteins that recognize abnormal particles in the body (known as epitopes), and are elicited by means of random recombinatory events followed by strict screening selection processes. Along their production, antibodies can be modified by mutation events leading to potent antibody variants. In this sense, there is an industrial and biomedical interest for the artificial optimization of antibodies. The rise of the computational era together with the deeper understanding of structural biology allowed the design and implementation of predictive algorithms for simulating the effects of mutations in protein-protein complexes. This process usually involves, among others, the prediction of changes in Gibbs free energy upon mutation and the use of other computational simulations for unveiling motions and binding patterns, such as Molecular Dynamics and Monte Carlo techniques. During this thesis, we have developed and implemented predictive algorithms focused on the design of potent antibody variants. We developed UEP, an open-source code for predicting the effects of mutations in protein-protein complexes. UEP differs from the state-of-the-art and employs other sources of knowledge rather than experimental binding affinity determinations upon mutation. Moreover, we designed a PELE protocol to simulate the binding affinity of antibodies against hypermutated HIV-1 viral isolates. Finally, we describe three different computational workflows for antibody optimization. We particularly focused on the challenge of increasing the binding potency of the N6 antibody, one of the best antibodies against HIV-1. Each computational workflow has been evaluated experimentally by our collaborators from Irsicaixa, and such combined computational and experimental effort resulted in the design of an improved variant of the N6 antibody against HIV-1.

Carbon dots (CD) have emerged as the new eye-catching theranostic nanomaterials due to their distinctive features, including tunable emission, facile surface modification, high biocompatibility and low cytotoxicity. These distinguishing features allow full customizations of CD according to the needs of various studies. Of note, they have been widely employed as nano-vehicles with live-tracking systems in many biological applications to deliver medicine with low bioavailability to targeted sites. Candida albicans, a commonly seen commensal fungus accounts for life-threatening infections in humans, is the leading cause of oral candidiasis. Yet, the efficacy of the "gold standard" Amphotericin B (AmB) has been limited due to poor water solubility and dose-dependent cytotoxicity. In addition, the interactions of CD with Candida cells/biofilms and human epithelial tissues have not been fully investigated, and very limited studies have been done on CD-based antifungal drugs delivery for topical administration. Herein, AmB-conjugated guanylated CD (CD-Gu + -AmB) tackling oral fungal infections were synthesized and possessed potent antifungal/anti-biofilm effects against C. albicans. Moreover, CD-Gu + -AmB exhibit low cytotoxicity to primary human oral keratinocytes and can selectively accumulate in the cell nuclei. Above all, the first evidence of studying the penetration and exfoliation profiles of CD in a three- dimensional organotypic human oral epithelial tissue model was provided, and the accumulation of CD-Gu + -AmB in the epithelial tissue can form a 'shielding' layer on oral epithelia against C. albicans. This study demonstrates that CD-Gu + -AmB may serve as a promising antifungal agent for tackling C. albicans and Candida-induced oral candidiasis through fast epithelial penetration, extra-/intra-cellular embedding and gradual exfoliation

L'amélioration de la visualisation in vivo des lésions précancéreuse (dysplasies) du col utérin est essentielle pour mieux identifier les zones à biopsier et pour optimiser la définition des limites d'exérèse chirurgicale. Dans ce but nous étudions une nouvelle technique d'imagerie polarimétrique en rétrodiffusion, que nous avons mise en oeuvre sur des échantillons ex vivo dans des configurations expérimentales variées afin d'optimiser le diagnostic in vivo. Comme cette optimisation passe par la compréhension des contrastes polarimétriques observés, nous avons réalisé de nombreuses simulations de la propagation de lumière polarisée dans des structures multicouche représentatives des tissus. Ces structures comprennent typiquement une couche comportant des diffuseurs dans une matrice homogène et représentant l'épithélium ou le tissu conjonctif superficiel, et un substrat lambertien totalement dépolarisant pour les couches plus profondes. Ces simulations ont été effectuées au moyen d'un code Monte Carlo que nous avons adapté à notre problématique. Nous avons ainsi montré que la contribution des noyaux cellulaires est très faible en rétrodiffusion. Pour le tissu conjonctif, les fibres de collagène, modélisées par des diffuseurs sphériques de 200 nm de rayon, donnent une contribution plus importante que les noyaux, mais ne reproduisent pas la réponse polarimétrique de type Rayleigh observée dans tous les tissus étudiés, qu'ils soient sains ou pathologiques. En revanche, l'inclusion de diffuseurs de taille nettement inférieure à la longueur d'onde, modélisés par des sphères de 50 nm, permet de reproduire cette réponse de manière très stable. Ces diffuseurs correspondent a priori aux protéines intracellulaires. Dans le cadre de ce modèle, les contrastes observés entre tissus sains et cancéreux s'expliquent essentiellement par une variation de la concentration de ces petits diffuseurs. Ce résultat, encore préliminaire, suggère que l'imagerie polarimétrique en rétrodiffusion peut être sensible non seulement à la morphologie, mais également à l'état physiologique du tissu, ce qui peut s'avérer important pour la détection sélective des dysplasies.

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

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

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

DEVELOPMENT AND CHARACTERIZATION OF PHOSPHOLIPID ENCAPSULATED QUANTUM DOT CONSTRUCTS FOR BIOLOGIC APPLICATIONS The American Cancer Society predicts that 577,190 cancer-related deaths and 1,638,910 newly diagnosed cases of cancer will occur in 2012. As these statistics show, cancer is a prevalent and devastating health issue; determined by the Mayo Clinic to be the second leading cause of death in the United States. Skin cancer is the most common form of cancer in the United States. In 2012 more than 68,000 Americans will be diagnosed with melanoma, 48,000 will be diagnosed with an early form of the disease that has not yet reached the lower levels of the epidermis, and more than 2 million people will be treated for basal cell or squamous cell skin cancer. Early and accurate detection is the most reliable way to ensure a positive outcome and the ultimate survival of the patient. As the most aggressive form of skin cancer, survival of melanoma is especially connected to early detection. Current methods for the initial detection of potential cancerous masses and lesions rely on visual examination, palpitation, and biopsy. Accurate determination of the presence of cancerous cells in a biopsy is especially difficult at the early stages when only a small percentage of cells in the biopsied mass show the morphological traits associated with being cancerous. This circumstance often results in a false negative (FN), delaying the necessary treatment until the cancer has reached a more developed stage. Developing more accurate methods for the detection of cancerous cells within a biopsy would aid in alleviating this problem. An improvement to the conventional method of visually examining biopsied tissues for the presence of cells with abnormal morphologies can be offered by utilizing the model of functionalized quantum dot (QD) constructs. Quantum dots are nano-particles composed of semi-conducting materials that fluoresce at discrete wavelengths when irradiated by a high energy UV source. QD constructs are cadmium-selenium/zinc-sulfide (CdSe/ZnS) quantum dots encapsulated within a bovine derived milk phospholipid micelle. QD constructs provide a potential mechanism for the identification of cancerous cells within a biopsy. Appreciating the scope of the clinical problem and understanding the potential of QDs, the objective of this thesis is to develop a primary model for the solubilization, encapsulation, and primary phospholipid functionalization of two distinct sizes of CdSe/ZnS QDs. The first stage of this thesis optimized the currently utilized protocol for synthesizing cadmium-selenium (CdSe) quantum dots to develop a set of parameters for consistently producing white fluorescing CdSe cores (WFCs) and CdSe/ZnS QDs of 505nm and 555nm (+/- 10nm). The application of synthesis times, temperatures, and quenching methods were employed to achieve this. The second stage developed a phospholipid encapsulation method for the initial functionalization and suspension of the hydrophobic QDs in aqueous media via encapsulation within phospholipid micelles. The final stage of this thesis focused on the successful introduction of the QD constructs into keratinocyte cells. Calcein and Ethidium homodimer-1 stains were applied to determine cell viability, Histochoice was applied as a fixative, and Hoechst staining was employed for cell nuclei identification. Analysis using confocal microscopy suggests successful attachment of QD constructs, in 0.1% w/v keratinocyte media, to the exterior of keratinocyte cell membranes with a 30% average cell survival rate at 24 hours after sample introduction. Future research investigating the interaction of QD constructs with biologic mediums of greater physiological complexity, as well as application of a secondary functionalization, are the next steps on the path toward achieving a viable mechanism for targeting and identifying cancerous cells within a biopsy.

Cette thèse a pour objectif l’étude d’un lien effectif potentiel entre la motilité cellulaire, la mécanique cellulaire, et l’activité biochimique de ces mêmes cellules. Ce couplage a été étudié dans divers systèmes biologiques, et aussi bien dans des cultures de cellules qu’à l’intérieur de tissus plus complexes. Notamment, nous avons particulièrement cherché à détecter un couplage électromécanique dans des neurones qui pourrait être impliqué dans la propagation du message nerveux.Pour ce faire, nous avons dû développer deux microscopes optiques à la sensibilité extrême. Ces microscopes se composent de deux parties principales. La première sert à détecter des mouvements axiaux plus petits que la longueur d’onde optique, soit en dessous de 100 nanomètres. La deuxième partie permet la détection d’un signal de fluorescence, offrant la possibilité de suivre l’évolution biochimique de la cellule. Avec ces deux microscopes multimodaux, il est donc possible de suivre de manière simultanée un contraste de motilité, un contraste mécanique, un contraste structurel et un contraste biochimique. Si l’un de ces systèmes est basé sur la tomographie de cohérence optique plein champ et permet de faire de telles mesures en 3-D et en profondeur dans les tissus biologiques, le second ne permet que des mesures dans des cultures de cellules, mais est bien plus robuste au bruit mécanique. Dans ce manuscrit, nous allons essentiellement décrire le développement de ces deux appareils, et préciser les contrastes auxquels ils sont sensibles spécifiquement.Nous développerons également deux des applications principales de ces microscopes que nous avons étudié dans le détail au cours de cette thèse. La première application développe l’intérêt d’un de nos microscopes pour la détection sans marquage des principaux composants cellulaires et structuraux de la cornée et de la rétine. La seconde application tend à détecter et à suivre des ondes électromécaniques dans des neurones de mammifères
This PhD project aims to explore the relationship that might exist between the dynamic motility and mechanical behavior of different biological systems and their biochemical activity. In particular,we were interested in detecting the electromechanical coupling that may happen in active neurons, and may assist in the propagation of the action potential. With this goal in mind, we have developed two highly sensitive optical microscopes that combine one modality that detects sub-wavelength axial displacements using optical phase imaging and another modality that uses a fluorescence path. Therefore, these multimodal microscopes can combine a motility, a mechanical,a structural and a biochemical contrast at the same time. One of this system is based ona multimodal combination of full-field optical coherence tomography (FF-OCT) and allows the observation of such contrast inside thick and scattering biological tissues. The other setup provides a higher displacement sensitivity, but is limited to measurements in cell cultures. In this manuscript, we mainly discuss the development of both systems and describe the various contrastst hey can reveal. Finally, we have largely used our systems to investigate diverse functions of the eye and to look for electromechanical waves in cell cultures. The thorough description of both biological applications is also provided in the manuscript

L’évolution et l’utilisation croissante des systèmes de communications mobiles sont associées à des études en laboratoire pour s’assurer de l’innocuité d’une exposition aux ondes électromagnétiques radiofréquences. Dans ce contexte, ce mémoire se concentre sur la caractérisation de systèmes d'exposition permettant des études en laboratoire sur des modèles cellulaires in vitro. Une double approche, numérique et expérimentale, est mise en œuvre pour réaliser la dosimétrie de ces dispositifs, afin de déterminer et maîtriser les niveaux d’exposition. Une des problématiques liées à cette dosimétrie est due aux dimensions micrométriques mises en jeu. Aussi, une technique de microscopie basée sur un marqueur fluorescent dépendant de la température, nommé Rhodamine B, a été mise en place et évaluée. Il ressort de cette évaluation une recommandation sur la concentration du marqueur de l’ordre de 50 μM. Après calibration, il est alors possible d’estimer le débit d’absorption spécifique (DAS) à partir de la variation de température, et ce même pour de faibles niveaux de DAS (< W/kg) avec une résolution spatiale inférieure à la dizaine de micron. On parle alors de microdosimétrie. Les deux principaux systèmes d’exposition étudiés, basés sur des réseaux de microélectrodes (MEA), permettent l’enregistrement d’une activité électrophysiologie de neurones. L’exposition aux ondes électromagnétiques est réalisée simultanément en insérant ces MEA dans des cellules TEM. De la dosimétrie effectuée à 1.8GHz, il ressort une plus forte sensibilité du premier MEA à son environnement. On a montré que les modifications apportées au second MEA, taille de l’ouverture et plan de masse, ont permis de limiter de façon significative l’influence de l’environnement. La microdosimétrie a mis en évidence une bonne homogénéité du DAS entre les électrodes avec une valeur estimée à 7±1W/kg pour1W incident. Enfin, un dispositif d'exposition microfluidique basé sur un guide d'ondes coplanaire a été caractérisé en conditions statiques
The evolution and increasing use of mobile communications systems was associated with laboratory investigations to study radiofrequency electromagnetic waves exposure safety. In this context, this thesis focuses on the characterization of exposure systems allowinglaboratory in vitro studies on cell models. A dual numerical and experimental approach is implemented to perform the devices dosimetry allowing to determine and control the exposure levels. One of the limitations associated with this dosimetry is due to the micrometric dimensions involved. Therefore, a microscopy technique based on a temperature-dependent fluorescent dye named Rhodamine B was set up and evaluated. This assessment recommends an optimal concentration of the dye at around 50 μM. After calibration, it is possible to estimate the specific absorption rate (SAR) from the temperature variation, even for low levels of SAR (< W / kg) with a spatial resolution of less than ten micrometers i.e. microdosimetry. The two main exposure systems studied, based on microelectrode arrays (MEA), allow the recording of neurons electrophysiological activity. Exposure to electromagnetic waves is achieved simultaneously by inserting these MEAs into TEM cells exposure systems. Dosimetry carried out at 1.8 GHz shows a higher sensitivity of one MEA to its environment. It was shown that the modifications made to the second MEA such as its aperture size and ground planes, have reduced the proximity environment influence. The microdosimetry demonstrated good homogeneity of the SAR between the electrodes with an estimated value of 7 ± 1 W / kgfor 1 W incident power. Finally, a microfluidic exposure device based on a coplanar waveguide was characterized under static conditions

Learning is central to infusing intelligence to any biologically inspired system. This study introduces a novel Cross-Correlated Delay Shift (CCDS) learning method for spiking neurons with the ability to learn and reproduce arbitrary spike patterns in a supervised fashion with applicability tospatiotemporalinformation encoded at the precise timing of spikes. By integrating the cross-correlated term,axonaland synapse delays, the CCDS rule is proven to be both biologically plausible and computationally efficient. The proposed learning algorithm is evaluated in terms of reliability, adaptive learning performance, generality to different neuron models, learning in the presence of noise, effects of its learning parameters and classification performance. The results indicate that the proposed CCDS learning rule greatly improves classification accuracy when compared to the standards reached with the Spike Pattern Association Neuron (SPAN) learning rule and the Tempotron learning rule. Network structureis the crucial partforany application domain of Artificial Spiking Neural Network (ASNN). Thus, temporal learning rules in multilayer spiking neural networks are investigated. As extensions of single-layer learning rules, the multilayer CCDS (MutCCDS) is also developed. Correlated neurons are connected through fine-tuned weights and delays. In contrast to the multilayer Remote Supervised Method (MutReSuMe) and multilayertempotronrule (MutTmptr), the newly developed MutCCDS shows better generalization ability and faster convergence. The proposed multilayer rules provide an efficient and biologically plausible mechanism, describing how delays and synapses in the multilayer networks are adjusted to facilitate learning. Interictalspikes (IS) aremorphologicallydefined brief events observed in electroencephalography (EEG) records from patients with epilepsy. The detection of IS remains an essential task for 3D source localization as well as in developing algorithms for seizure prediction and guided therapy. In this work, we present a new IS detection method using the Wavelet Encoding Device (WED) method together with CCDS learning rule and a specially designed Spiking Neural Network (SNN) structure. The results confirm the ability of such SNN to achieve good performance for automatically detecting such events from multichannel EEG records.

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

Dans le cadre du traitement chirurgical du cancer, des sondes agissant comme des compteurs de radioactivité ont été introduites en bloc opératoire pour assister en temps réel le chirurgien lors de l'ablation des tumeurs radio-marquées. Cette technique de radio-guidage permet d'accéder à la localisation précise et à l'ablation complète des tissus tumoraux. Pour renforcer cette pratique chirurgicale nous avons développé une mini gamma-caméra appelée POCI (Per-Operative Compact Imager). L'objectif de cette thèse était de déterminer l'apport de cette nouvelle génération de détecteur pour assister le chirurgien dans l'exérèse des lésions tumorales et d'aborder également la cancérologie à travers les études in vivo sur petits animaux. Du point de vue instrumental, le principe de détection reposant sur une photodiode à localisation intensifiée validé par un premier prototype a été étendu à l'imagerie à grande surface d'analyse avec la double contrainte de ne pas dégrader les performances spatiales et de réduire l'encombrement de l'imageur. La caméra ainsi réalisée offre un champ de vue de 40mm de diamètre et une masse de 1.2kg. A 140 keV, la résolution spatiale est de 2.1mm pour une efficacité de détection de 2.8 10^-4%. POCI a été évalué à travers le protocole du ganglion sentinelle dans le cadre du cancer du sein selon deux approches : l'une basée sur une étude comparative des performances de détection d'une sonde et de POCI et l'autre reposant sur une évaluation clinique menée en collaboration avec l'Institut Gustave Roussy. Cette étude a permis d'établir la complémentarité entre POCI et la sonde en fonction des différentes configurations cliniques. Les performances de détection de POCI ont également été évaluées chez la souris pour étudier la biodistribution de l'iode dans la thyroïde et les glandes mammaires. L'ensemble de ces résultats prometteurs permet d'envisager l'utilisation du détecteur dans un plus large cadre d'investigations tant cliniques que biologiques.

Modern day medicine is on the brink of a new age of therapy, which aims to harness the natural power of molecular biology for disease treatment. This therapy could include replacement of dysfunctional genes that cause disorders such as cystic fibrosis (Lommatzsch and Aris, 2009), or silencing the overexpression of genes that cause disorders such as cancer (Pelengaris and Khan, 2003). In both examples, the treatment of these genetic diseases lies in the delivery of synthetic nucleic acids into diseased cells, the former being called gene replacement therapy (Dobson, 2006a), and the latter being called RNA interference (RNAi) therapy (Whitehead et al., 2009). While these techniques have long been in use as genetic research tools for gene transfection or silencing in vitro, their translation for use in clinical disease treatment has yet to be achieved. The main problem facing the development of these novel therapies is the specific delivery of nucleic acids into diseased cells within the body. It is hoped that nanoparticles (NPs) can be used to overcome this problem, by acting as vehicles to transport nucleic acids through the body for specific delivery into diseased cells. This feat can be aided by the attachment of additional functional molecules such as cell penetrating peptides (CPPs), targeting peptides, additional drug types and molecules for imaging during treatment. Many different NP design strategies are currently under development. It is essential for new designs to be extensively tested for toxicity and efficiency in human cells before they can be successfully released into the clinic. As part of this effort, this PhD project has investigated two different NP design strategies for drug delivery: 1) the use of a magnetic field (MF) and a CPP to increase the delivery of iron oxide magnetic NPs (mNPs) to cells grown in tissueequivalent 3D collagen gels, and 2) gold NPs (AuNPs) for the delivery of siRNA to silence the c-myc oncogene for cancer treatment. In the first investigation, a MF and the CPP penetratin were found to increase mNP delivery to cells grown in 3D. In the second investigation, AuNPs were assessed in a range of different cell types (grown in 2D) for their performance in 4 main areas; cellular toxicity, cellular uptake, c-myc knockdown and effect on the cell cycle.

Surface topography is known to be important in biomedical applications such as scaffolds for tissue regeneration and has been shown to affect wettability and cell behaviour. Traditionally, topographical effects such as surface texturing have been generated using methods such as photolithography, soft lithography, thermal embossing, and laser/electron beam techniques. This thesis introduces a relatively new technique known as photoembossing to create surface texturing for biomedical applications. Photoembossing is used to produce surface texturing on polymer surfaces by patterned ultraviolet (UV) exposure of a photopolymer blend without an etching step or an expensive mould. After a short general introduction and a literature review, the first experimental chapters describe surface patterning of poly(methyl methacrylate) (PMMA) photopolymer substrates by photoembossing. PMMA is blended with an acrylate monomer and photoinitiator by dissolution in a volatile solvent and processed into films by wire bar coating, and fibres are produced by electrospinning. Surface texture is achieved on both films and fibres by photoembossing. Endothelial cell culture shows that the substrates are biocompatible and cells readily adhere to the surface. In tissue regeneration applications, scaffold degradation is often important to allow tissue in-growth. Thus, in subsequent studies polylactide-co-glycolide (PLGA) is used as a polymer binder. PLGA blended with a triacrylate monomer showed partial degradation after 10 weeks, with a cross-linked acrylate network remaining. Endothelial cell adhesion was even better on the PLGA photopolymer substrates compared to PMMA. Furthermore, surface texture improved cell adhesion and proliferation on the PLGA photopolymer. To obtain completely degradable substrates, thiol monomer was used in addition to the acrylate to produce ester bonds after the thiol-ene reaction, which is cleavable by hydrolysis. Accelerated degradation in sodium hydroxide (NaOH) showed complete degradation of this photopolymer system. The degradation rate of the photopolymer could be tuned by the molecular weight of the acrylate monomer, with low molecular weight monomers degrading more slowly than high molecular weight species. Furthermore, the height of the surface relief structures could be enhanced by using low-molecular-weight acrylate monomers. Endothelial cell culture revealed biocompatibility of the blend and cells were able to adhere after 24 hours of seeding. This thesis demonstrates that photoembossing is a viable technique in producing surface texture for tissue engineering applications. This surface texture can be achieved on both biocompatible and biodegradable photopolymer films and fibres.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

This thesis presents the analysis and design of biological interfacing technologies in light of a need for radical improvements in scalability. It focuses primarily on structural and functional neural data acquisition, but also extends to other problems including genomic editing and nanoscale spatial control. Its main contributions include analysis of the physical limits of large-scale neural recording, experimental development of a screening platform for ion-dependent molecular recording devices, characterization of the design space for molecularly-annotated neural connectomics, and new designs for high-speed genome engineering and bio-nano-fabrication. Articulating governing principles and roadmaps for these domains has contributed to the initiation of multi-institutional projects that are strategically targeted towards scalability.

Methods are presented for improving the accuracy of the value of the diffusion coefficient used in continuum theory based simulation of ion channel permeation. A mathematical framework is derived for computing the diffusion coefficient of a spherical species undergoing diffusion confined within an arbitrarily defined container. Methods for standardizing the coordinates of the atoms constituting biological ion channels are presented. From its standardized coordinates, a method is described for rendering the channel's pore lining surface. This surface representation is used as the definition of the container in application of the mathematical framework for diffusion coefficient estimation within the channel's pore. These methods are applied to Gramicidin A (GA). Results for the value of the diffusion coefficient computed in pore of GA via these methods are comparable to those produced from much more complex and costly molecular dynamics simulations. Predictions about the nature of diffusion in highly confined geometries based on the mathematical framework for computing the diffusion coefficient are presented. Their implications for design with ion channels are discussed.
Cette thèse présente des méthodes pour évaluer plus précisément le coefficient de diffusion utilisé dans les simulations fondées sur la théorie du continuum de la perméation des canaux ioniques. On établit tout d'abord un cadre mathématique pour le calcul des coefficients de diffusion d'espèces sphériques lors d'une diffusion restreinte à un contenant arbitrairement délimité. On présente ensuite des méthodes pour standardiser les coordonnées d'atomes formant des canaux ioniques biologiques. En utilisant ces coordonnées standardisées, on démontre comment on peut représenter la surface d'un pore intérieur d'un canal ionique. On utilise alors cette représentation comme contenant ainsi que le cadre mathématique présenté précédemment afin d'évaluer le coefficient de diffusion dans le pore du canal. On applique ensuite ces méthodes à la gramicidine A (GA) pour calculer le coefficient de diffusion du pore de la GA, avec des résultats comparables à ceux obtenus par des simulations de dynamique moléculaire beaucoup plus complexes et coûteuses. Finalement, à l'aide du cadre mathématique servant à calculer le coefficient de diffusion, on formule des prévisions à propos de la diffusion dans des espaces très restreints, et on considère leur incidence sur la conception appliquée aux canaux ioniques.

Often in biology, rare individuals within a population dominate the population’s overall behavior, and we wish to extract those individuals for further analysis. We design a sorting instrument as a flexible platform for the development of novel microfluidic sorting techniques. We demonstrate a microfluidic cell sorter, which screens cells at rates approaching those of commercially-available fluorescence-activated cell sorters. This device incorporates a three-dimensional flow-focusing nozzle with a slanted ceiling groove to enhance the capabilities of a surface acoustic wave (SAW) transducer by harnessing the component of the SAW oriented normal to the plane of the substrate. The device achieves sorting at a rate of 9000 events/s with 54% purity and yields 89% purity, while operating at 1000 events/s; this level of performance approaches that of a FACS operating in its high-purity mode. We also present a rare event sorting technique, which can successfully extract desired droplets from a sample containing nearly a billion droplets. The technique yields pure samples after two rounds. The preliminary round is fast, capable of screening 10 ml of droplets at 100 ml/h, but each sort also captures many other droplets together with the droplet of interest. The second round of sorting enriches the sample to nearly 100% purity, using known designs for high purity drop-by-drop sorting. Thus, we devise a method, which can sort droplets rapidly and achieve high purity of few droplets from samples containing large numbers of droplets. The two disparate approaches to microfluidic sorting use a common platform to create new methods for sorting with biological applications.
Engineering and Applied Sciences - Engineering Sciences

[Truncated abstract] In the past decade, the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) has received considerable attention due to their potential applications in biomedical fields. However, success in size and shape control of the SPIONs has been mostly achieved through organic routes using large quantities of toxic or/and expensive precursors in organic reaction medium at high reaction temperature. This has limited the biomedical applications of SPIONs and therefore, development of a synthetic method under aqueous condition that is reproducible, scalable, environmentally benign, and economically feasible for industrial production is of paramount importance in order to fully realise their practical applications. Spinning Disc Processing (SDP) has been used to synthesise superparamagnetic magnetite (Fe3O4) nanoparticles at room temperature via a modified chemical precipitation method under continuous flow condition and offer a potential alternative to be applied to SPIONs production. SDP has extremely rapid mixing under plug flow conditions, effective heat and mass transfer, allowing high throughput with low wastage solvent efficiency. The synthesis process involves passing ammonia gas over a thin aqueous film of Fe2+/3+ which is introduced through a jet feed close to the centre of a rapidly rotating disc (500-2500 rpm). Synthetic parameters such as precursor concentrations, temperature, flow rate, disc speed, and surface texture influence the particle sizes. ... Magnetic silica microspheres are receiving great attention for possible applications in magnetic targeting drug delivery, bioseparation and enzyme isolation. However, the current available methods for preparation suffer from the setback of low loading of Fe3O4 nanoparticles in the silica microsphere, which result in low magnetic moment, thereby limiting their practical applications. Therefore it is of considerable importance to develop new alternative synthetic methods for fabricating magnetic silica microspheres with high magnetic nanoparticles loading. Superparamagentic Fe3O4 nanoparticles (8-10 nm diameter) and curcumin have been encapsulated in mesoporous silica in a simple multiplestep self assembly approach process with high Fe3O4 nanoparticles loading (37%). The synthesis involves loading of curcumin in the Cetyltrimethylammonium bromide (CTAB) micellar rod in the presence of superparamagnetic Fe3O4 nanoparticles via a parallel synergistic approach. The synthesised magnetic mesoporous silica composite material is stable, superparamagnetic with high saturation magnetisation before and after curcumin leaching experiment. Under physiological pH in phosphate buffer, the curcumin is slowly released over several days. These magnetic mesoporous silica are expected to have great potential as targeted drug delivery systems.

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Switchable oligopeptides, able to expose of conceal biomolecules on a surface, upon the application of an electrical potential, represent a versatile tool for the development of novel devices, presenting potential biomedical applications. Recently, several studies have demonstrated the applicability of smart devices for the control of protein binding and cellular response. In this work; a detailed analysis of the steric requirements necessary to develop a mixed oligopeptide Self-Assembled Monolayer (SAM) presenting an optimum switching ability will be described. The influence of both the SAM components surface ratio and the switching unit length on the mixed SAMs switching performance will be investigated. The findings of this investigation will be used to develop, for the first time, a platform, based on electrically switchable oligopeptides, able to control the interaction between an antigen and its relative antibody. The influence of the biological medium on the oligopeptide switching ability will also be investigated. Finally, an orthogonal functionalisation strategy, will be investigated in detail, together with a new platform able to promote human sperm cells adhesion. The results of this research thesis will also represent the first building blocks towards the development of glass-gold rnicropattemed surfaces able to control the calcium signalling in human sperm cells, presenting potential applications in the improvement of in-vitro fertilisation (NF) treatments success rates.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2017.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 170-176).
Research on biomedical applications of magnetic nanoparticles (MNPs) has increasingly sought to demonstrate noninvasive actuation of cellular processes and material responses using heat dissipated in the presence of an alternating magnetic field (AMF). By modeling the dependence of hysteresis losses on AMF amplitude and constraining AMF conditions to be physiologically suitable, it can be shown that MNPs exhibit uniquely optimal driving conditions that depend on controllable material properties such as magnetic anisotropy, magnetization, and particle volume. "Magnetothermal multiplexing," which relies on selecting materials with substantially distinct optimal AMF conditions, enables the selective heating of different kinds of collocated MNPs by applying different AMF parameters. This effect has the potential to extend the functionality of a variety of emerging techniques with mechanisms that rely on bulk or nanoscale heating of MNPs. Experimental investigations on methods for actuating deep brain stimulation, drug release, and shape memory polymer response are summarized, with discussion of the feasibility and utility of applying magnetothermal multiplexing to similar systems. The possibility of selective heating is motivated by a discussion of various models for heat dissipation by MNPs in AMFs, and then corroborated with experimental calorimetry measurements. A heuristic method for identifying materials and AMF conditions suitable for multiplexing is demonstrated on a set of iron oxide nanoparticles doped with various concentrations of cobalt. Design principles for producing AMFs with high amplitude and ranging in frequency from 15kHz to 2.5MHz are explained in detail, accompanied by a discussion of the outlook for scalability to clinically relevant dimensions. The thesis concludes with a discussion of the state of the field and the broader lessons that can be drawn from the work it describes.
by Michael G. Christiansen.
Ph. D.