Rozprawy doktorskie na temat „Nanotechnology - Biomedical Application”

Two batches of CdSe QDs with different sizes were synthesized for immobilizing in polyisoprene (PI), polymethylmethacrylate (PMMA), and low-density polyethylene (LDPE). The combinations of QDs and polymer substrates were evaluated for their analytical fit-for-use in applicable immunoassays. Hydrogen peroxide standards were injected into the flow injection analyzer (FIA) constructed to simulate enzyme-generated hydrogen peroxide reacting with bis-(2,4,6-trichlorophenyl) oxalate. Linear correlations between hydrogen peroxide and chemilumenscent intensities yielded regression values greater than 0.9750 for hydrogen peroxide concentrations between 1.0 x 10-4 M and 1.0 x 10-1 M. The developed technique’s LOD was approximately 10 ppm. Variability of the prepared QD-polymer products was as low as 3.2% throughout all preparations.Stability of the preparations was tested during a 30-day period that displayed up to a four-fold increase in the first 10 days. The preparations were decently robust to the FIA system demonstrating up to a 15.20% intensity loss after twenty repetitive injections.

The overall aim of this thesis is to investigate the combination of supramolecular cylinders with DNA nanotechnology and assess any effects that can occur through binding and any applications this could have in biomedical therapy applications. From this base it is hoped that insight can be gained as to whether supramolecular chemistry can be used to create DNA nano-machines, capable of triggered release of cargo. The thesis begins with a review of DNA discovery, structure and binding by small molecules, followed by a review of the field of DNA nanotechnology. By expanding on the field of DNA nanotechnology recognition, chapters 2 and 3 will highlight the advantages of supramolecular chemistry when combined with DNA nanotechnology in both nano-machines and inside cell systems with a focus on DNA tetrahedral nanostructures. Chapter 4 researches the photocleavage capabilities of a ruthenium cylinder and the possibilities of selective release and photodynamic therapy using a DNA tetrahedron. Chapter 5 illustrates a new class of anti-viral agents capable of structure recognition regardless of RNA sequence. The chapter focuses on the inhibition of binding between the TAR RNA and ADP-1 peptide found in the HIV-1 virus.

Le travail de recherche de cette thèse consiste en le développement de nouveaux matériaux hybrides organiques-inorganiques pour des applications en nanotechnologie, nanomédicine et diagnostic. Dans ce contexte, des cristaux poreux de zéolite-L ont été utilisé comme nano-vecteur pour faire de la transfection d’ADN et d’ANP, en combinaison avec le relargage de molécules hôtes placées dans les pores. Des nanoparticules de silice mesoporeuses multifonctionnelles ont été utilisées pour traiter le glioblastome, en combinant la thérapie génique avec l’administration durable d’un principe actif. Des nano-coquilles hybrides biodégradables ont été encore développés pour encapsuler des protéines et les relâcher dans les cellules vivantes. Dans le domaine de la détection d’acides nucléiques, des fibres optiques à cristal photonique, fonctionnalisées avec des sondes d’ANP, ont été exploitées comme plateformes optiques pour faire de la détection ultra-sensible d’oligonucléotides ou d’ADN génomique. Enfin, la squelette de l’ANP a été modifié à créer des sondes fluorescentes pour reconnaître et détecter la présence des séquences cibles spécifiques
The research work presented throughout this thesis focuses on the development of novel organic-inorganichybrid materials for applications in nanotechnology, nanomedicine and diagnostics. In such a context, porous zeolite-L crystals have been used as nanocarriers to deliver either DNA or PNA in live cells, in combination with the release of guest molecules placed into the pores. Multifunctional mesoporous silica nanoparticles have been designed to treat glioblastoma, combining gene therapy with the sustained delivery of a chemotherapy agent. Biodegradable hybrid nano-shells have been furthermore created to encapsulate proteins and release them in living cells upon degradation of the outer structure in reductive environment. In the field of nucleic acid detection, photonic crystal fibers, functionalized with specific PNA probes, have been exploited as optical sensing devices to perform ultra-sensitive detection of DNA oligonucleotides or genomic DNA. Eventually, the PNA backbone has served as scaffold to synthesize fluorescent switching probes able to recognize and to detect the presence of specific target sequences

Lanthanide-doped upconversion nanoparticles (UCNPs) are perceived as promising novel near-infrared (NIR) bioimaging agents characterised by high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied and the corresponding intracellular uptake pathways remain unclear. Herein, my research work involved the use of hydrothermal method and ligand exchange approach to prepare UCNP-PVP, UCNP-PEI, and UCNP-PAA. These polymer-coated UCNPs demonstrated good water dispersibility, the similar size distribution as well as similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as revealed by cellular uptake studies. Meanwhile, it was discovered that cationic UCNP-PEI could be effectively internalized mainly through the clathrin endocytic machanism. This study is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, it allows us to control the UCNP-cell interactions by tuning surface properties. Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors in humans. Small molecule MRI contrast agents are used for GBM diagnosis and preoperative tumor margin delineation. However, the conventional gadolinium-based contrast agents have several disadvantages, such as a relatively low T1 relaxivity, short circulation half lives and the absence of tumor targeting efficiency. Multimodality imaging probes provide a better solution to clearly delineate the localization of glioblastoma. My research work also involved the development of multimodal nanoprobes for targeted glioblastoma imaging. Two targeted paramagnetic/fluorescence nanoprobes were designed and synthesized, UCNP-Gd-RGD and AuNP-Dy680-Gd-RGD. UCNP-Gd-RGD was prepared through PEGylation, Gd3+DOTA conjugation and RGD labeling of PEI-coated UCNP-based nanoprobe core (UCNP-NH2). It adopted the cubic NaYF4 phase, had an average size of 36 nm by TEM, and possessed a relatively intense upconversion luminescence of Er3+ and Tm3+. It also exhibited improved colloidal stability and reduced cytotoxicity compared with UCNP-NH2, and a higher T1 relaxivity than Gd3+DOTA. AuNP-Dy680-Gd-RGD was synthesized through bioconjugation of amine-modified AuNP-based nanoprobe core (AuNPPEG- NH2) by a NIR dye (Dy680), Gd3+DOTA and RGD peptide. It demonstrated a size of 3–6 nm by TEM, relatively strong NIR fluorescence centered at 708 nm, longterm physiological stability, and an enhanced T1 relaxivity compared with Gd3+DOTA. Targeting abilities of both UCNP-Gd-RGD and AuNP-Dy680-Gd-RGD towards overexpressed integrin αvβ3 receptors on U87MG cell surface was confirmed by their enhanced cellular uptake visualized by confocal microscopy imaging and quantified by ICP-MS, where their corresponding control nanoprobes were used for comparison. Furthermore, targeted imaging capabilities of UCNP-Gd-RGD and AuNP-Dy680-Gd- RGD towards subcutaneous U87MG tumors were verified by in vivo and ex vivo upconversion fluorescence imaging studies and by in vivo and ex vivo NIR fluorescence imaging and in vivo MR imaging studies, respectively. These two synthesized targeted nanoprobes, with surface-bounded cyclic RGD peptide and numerous T1 contrast enhancing molecules, are applicable in targeted MR imaging glioblastoma and delineating the tumor boundary. In addition, UCNP-Gd-RGD favors the upconversion luminescence with NIR-to-visible nature, while AuNPDy680- Gd-RGD possesses NIR-to-NIR fluorescence, and both lead to their potential applications in fluorescence-guided surgical resection of gliomas.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

We present a versatile biosensing strategy that uses nucleic acids programmed to undergo an isothermal toehold mediated strand displacement in the presence of analyte. This rearrangement results in a double biotinylated duplex formation that induces the rapid aggregation of streptavidin decorated quantum dots (QDs). As biosensor reporters, QDs are advantageous to organic fluorophores and fluorescent proteins due to their enhanced spectral and fluorescence properties. Moreover, the nanoscale regime aids in an enhanced surface area that increase the number of binding of macromolecules, thus making cross-linking possible. The biosensing transduction response, in the current approach, is dictated by the analysis of the natural single particle phenomenon known as fluorescence intermittency, or blinking is the stochastic switching of fluorescence intensity ON (bright) and OFF (dark) states observed in single QD or other fluorophores. In contrast to binary blinking that is typical for single QDs, aggregated QDs exhibit quasi-continuous emission. This change is used as an output for the novel biosensing techniques developed by us. Analysis of blinking traces that can be measured by laser scanning confocal microscopy revealed improved detection of analytes in the picomolar ranges. Additionally, this unique biosensing approach does not require the analyte to cause any fluorescence intensity or color changes. Lastly, this biosensing method can be coupled with therapeutics, such as RNA interference inducers, that can be conditionally released and thus used as a theranostic probes.

La presente tesis doctoral, titulada "Nanotecnología y química supramolecular en procesos de liberación controlada y reconocimiento molecular para aplicaciones biomédicas", se centra en dos temas importantes: el reconocimiento molecular y los procesos de liberación controlada. Esta tesis doctoral está estructurada en cuatro capítulos. El primer capítulo introduce el concepto de materiales híbridos orgánicos-inorgánicos funcionalizados con puertas moleculares y sus aplicaciones biomédicas como nanomateriales para dirigir y controlar la liberación controlada de fármacos. Además se introduce una breve descripción sobre sensors colorimétricos basados en la base de la quimica supramolecular, particularmente en los procesos de reconocimiento molecular. En particular, el capítulo 2 describe la preparacion de cinco nanodispositivos que responden a enzimas. Estos materiales híbridos se componen de dos unidades principales: un soporte mesoporoso basado en sílice inorgánica, capaz de encapsular moléculas orgánicas y un compuesto orgánico anclado en la superficie externa del soporte mesoporoso inorgánico que actúa como puerta molecular. Todos los sistemas propuestos utilizan puertas moleculares peptídicas que responden a temperatura o enzimas como estímulo. La segunda parte de esta tesis doctoral se centra en el diseño y desarrollo de un nuevo compuesto químico capaz de detectar monóxido de carbono in vivo. En resumen, para todos los resultados antes mencionados podemos decir que esta tesis doctoral constituye una contribución científica original al desarrollo de la química supramolecular. Sus resultados derivados de los estudios presentados dejan rutas abiertas para continuar el estudio y el desarrollo de nuevos materiales híbridos y sensors químicos más eficientes para aplicaciones biomédicas y terapeuticas.
This PhD thesis entitled "Nanotechnology and supramolecular chemistry in controlled release and molecular recognition processes for biomedical applications", is focused on two important subjects: molecular recognition and controlled delivery processes. This PhD thesis is structured in four chapters. The first chapter introduces the concept of organic-inorganic hybrid materials containing switchable "gate-like" ensembles and their biomedical applications as nanomaterials for targeting and control drug delivery. Furthermore, is introduced a short review about chromo-fluorogenic chemosensors based on basic principles of supramolecular chemistry, particulary in molecular recognition processes. In particular, in chapter 2 is focus on the development of enzymatic-driven nanodevices. These hybrid materials are composed of two main units: an inorganic silica based mesoporous scaffold, able to store organic molecules and an organic compound anchored on the external surface of the inorganic mesoporous support than acts as molecular gate. All the systems proposed use peptidic gates that respond to temperature or enzimatic stimulis. The second part of this PhD thesis is focused on the design and development of a new chemical compound capable of detecting carbon monoxide in vivo. In summary, for all the results above mentioned we can say that this PhD thesis constitutes an original scientific contribution to the development of supramolecular chemistry. Its results derived from the studies presented leaves open routes to continue the study and development of new hybrid materials and more efficient chemical sensors with biomedical and therapeutic applications.
La present tesi doctoral, titulada "Nanotecnologia i química supramolecular en processos d'alliberament controlat i reconeixement molecular per a aplicacions biomèdiques", es centra en dos temes importants de la química: el reconeixement molecular i els processos d'alliberament controlat. Aquesta tesi doctoral està estructurada en quatre capítols. El primer capítol introdueix el concepte de materials híbrids orgànics-inorgànics funcionalitzats amb portes moleculars i les seves aplicacions biomèdiques com nanomaterials per dirigir i controlar l'alliberament controlat de fàrmacs. A més s'introdueix una breu descripció sobre sensors colorimètrics fonamentats en la base de la química supramolecular, particularment en els processos de reconeixement molecular. En particular, el capítol 2 descriu la preparació de cinc nanodispositius que responen a enzims. Aquests materials híbrids es componen de dues unitats principals: un suport mesoporos basat en sílice inorgànica, capaç d'encapsular molècules orgàniques i un compost orgànic ancorat a la superfície externa del suport mesoporós inorgànic que actua com a porta molecular. La segona part d'aquesta tesi doctoral es centra en el disseny i desenvolupaent d'un nou compost químic capaç de detectar monòxid de carboni in vivo. En resum, per a tots els resultats abans mencionats podem dir que esta tesi doctoral constituïx una contribució científica original al desenvolupament de la química supramolecular. Els seus resultats derivats dels estudis presentats deixen rutes obertes per a continuar l'estudi i el desenvolupament de nous materials hibrids i sensors químics més eficients per a aplicacions biomèdiques i terapeutiques.
De La Torre Paredes, C. (2017). Nanotechnology and supramolecular chemistry in controlled release and molecular recognition proceses for biomedical applications" [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/94043
TESIS

Chitosan is a naturally derived polymer, which represents one of the most technologically important classes of active materials with applications in a variety of industrial and biomedical fields. Polymeric materials can be regarded as promising candidates for next generation devices due to their low energy payback time. These devices can be fabricated by high-throughput processing methodologies, such as spin coating, inkjet printing, gravure and flexographic printing onto flexible substrates. However, the extensive applications of polymeric films are still limited because of disadvantages such as poor electromechanical properties, high brittleness with a low strain at break, and sensitivity to water. For certain critical applications the need for modification of physical, mechanical and electrical properties of the polymer is essential. When blends of polymer films with other materials are used, as is commonly the case, device performance directly depends on the nanoscale morphology and phase separation of the blend components. To prepare nanocomposite thin films with the desired functional properties, both the film composition and microstructure have to be thoroughly characterized and controlled.

Chitosan reinforced bio-nanocomposite films with varying concentrations of gold nanoparticles were prepared through a solution casting method. Gold nanoparticles (∼ 32 nm diameter) were synthesized via a citrate reduction method from chloroauric acid and incorporated in the prepared Chitosan solution. Uniform distribution of gold nanoparticles was achieved throughout the chitosan matrix and was confirmed by SEM images. Synthesis outcomes and prepared nanocomposites were characterized using TEM, SAED, SEM, EDX, XRD, UV-Vis, particle size analysis, zeta potential and FT-IR for their physical, morphological and structural properties. Nanoscale mechanical properties of the nanocomposite films were characterized at room temperature, human body temperatures and higher temperatures using instrumented indentation techniques. The obtained films were confirmed to be biocompatible by their ability to support the growth and proliferation of human tissue cells in vitro. Statistical analysis on mechanical properties and biocompatibility results, were conducted. Results revealed significant enhancement on both the mechanical properties and cell adherence and proliferation. The results will enhance our understanding of the effect of nanostructures reinforcement on these important functional polymeric thin films for potential biomedical applications.

Thesis (Ph.D.)--University of California, San Francisco with the University of California, Berkeley, 2010.
Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Francis C. Szoka.

The tumor microenvironment has been demonstrated to be a key determinant in the progression of cancer. Unfortunately, the mechanisms behind the different microenvironments (cytokine gradients, hypoxia, hypoglycemia, etc) have not been fully elucidated. Identifying these mechanisms can lead to targeted, individualized therapy to prevent metastasis. The Nano Intravital Device (NANIVID) is a microfabricated, implantable device designed to initiate specific microenvironments in vivo so that the time course of the effects can be observed. With both spatial and temporal control over the induced environments, the affected regions of the tumor can be compared to the rest of the tumor. The NANIVID was first used to establish cytokine gradients to monitor the migration of invasive cancer cells. The three projects that comprise this work expand the applications of the NANIVID to establish the device as a robust platform for investigating tumor microenvironment interactions. The first project released chemical mimics from the device to induce the cellular hypoxic response in tumors to determine how hypoxia affects the fate of disseminated tumor cells. The second project used the NANIVID in combination with an atomic force microscope to investigate the altered mechanics of migrating invasive cancer cells. The final project was to develop a cell counter to monitor the isolation of the invasive subpopulation of cells that were drawn into the device using a chemoattractant. These three projects demonstrate the potential of the NANIVID as a platform for investigating the tumor microenvironment.

The advancements in nanotechnology enabled the development of new diagnostic tools and drug delivery systems based on nanosystems, which offer unique features such as large surface area to volume ratio, cargo loading capabilities, increased circulation times, as well as versatility and multifunctionality. Despite this, the majority of nanomedicines do not translate into clinics, in part due to the biological barriers present in the body. Synthetic nano- and micromotors could be an alternative tool in nanomedicine, as the continuous propulsion force and potential to modulate the medium may aid tissue penetration and drug diffusion across biological barriers. Enzyme-powered motors are especially interesting for biomedical applications, owing to their biocompatibility and use of bioavailable substrates as fuel for propulsion. This thesis aims at exploring the potential applications of urease-powered nanomotors in nanomedicine. In the first work, we evaluated these motors as drug delivery systems. We found that active urease- powered nanomotors showed active motion in phosphate buffer solutions, and enhanced in vitro drug release profiles in comparison to passive nanoparticles. In addition, we observed that the motors were more efficient in delivering drug to cancer cells and caused higher toxicity levels, due to the combination of boosted drug release and local increase of pH produced by urea breakdown into ammonia and carbon dioxide. One of the major goals in nanomedicine is to achieve localized drug action, thus reducing side-effects. A commonly strategy to attain this is the use moieties to target specific diseases. In our second work, we assessed the ability of urease-powered nanomotors to improve the targeting and penetration of spheroids, using an antibody with therapeutic potential. We showed that the combination of active propulsion with targeting led to a significant increase in spheroid penetration, and that this effect caused a decrease in cell proliferation due to the antibody’s therapeutic action. Considering that high concentrations of nanomedicines are required to achieve therapeutic efficiency; in the third work we investigated the collective behavior of urease-powered nanomotors. Apart from optical microscopy, we evaluated the tracked the swarming behavior of the nanomotors using positron emission tomography, which is a technique widely used in clinics, due to its noninvasiveness and ability to provide quantitative information. We showed that the nanomotors were able to overcome hurdles while swimming in confined geometries. We observed that the nanomotors swarming behavior led to enhanced fluid convection and mixing both in vitro, and in vivo within mice’s bladders. Aiming at conferring protecting abilities to the enzyme-powered nanomotors, in the fourth work, we investigated the use of liposomes as chassis for nanomotors, encapsulating urease within their inner compartment. We demonstrated that the lipidic bilayer provides the enzymatic engines with protection from harsh acidic environments, and that the motility of liposome-based motors can be activated with bile salts. Altogether, these results demonstrate the potential of enzyme-powered nanomotors as nanomedicine tools, with versatile chassis, as well as capability to enhance drug delivery and tumor penetration. Moreover, their collective dynamics in vivo, tracked using medical imaging techniques, represent a step-forward in the journey towards clinical translation.
Recientes avances en nanotecnología han permitido el desarrollo de nuevas herramientas para el diagnóstico de enfermedades y el transporte dirigido de fármacos, ofreciendo propiedades únicas como encapsulación de fármacos, el control sobre la biodistribución de estos, versatilidad y multifuncionalidad. A pesar de estos avances, la mayoría de nanomedicinas no consiguen llegar a aplicaciones médicas reales, lo cual es en parte debido a la presencia de barreras biológicas en el organismo que limitan su transporte hacia los tejidos de interés. En este sentido, el desarrollo de nuevos micro- y nanomotores sintéticos, capaces de autopropulsarse y causar cambios locales en el ambiente, podrían ofrecer una alternativa para la nanomedicina, promoviendo una mayor penetración en tejidos de interés y un mejor transporte de fármacos a través de las barreras biológicas. En concreto, los nanomotores enzimáticos poseen un alto potencial para aplicaciones biomédicas gracias a su biocompatibilidad y a la posibilidad de usar sustancias presentes en el organismo como combustible. Los trabajos presentados en esta tesis exploran el potenical de nanomotores, autopropulsados mediante la enzima ureasa, para aplicaciones biomédicas, y investigan su uso como vehículos para transporte de fármacos, su capacidad para mejorar penetración de tejidos diana, su versatilidad y movimiento colectivo. En conjunto, los resultados presentados en esta tesis doctoral demuestran el potencial del uso de nanomotores autopropulsados mediante enzimas como herramientas biomédicas, ofreciendo versatilidad en su diseño y una alta capacidad para promover el transporte de fármacos y la penetración en tumores. Por último, su movimiento colectivo observado in vivo mediante técnicas de imagen médicas representan un significativo avance en el viaje hacia su aplicación en medicina.

Biosensors are nowadays a powerful tool to enable the detection of specific biological interactions and to evaluate the concentration dependence in the response. A biosensor usually consists of three different parts: the sample to be measured, the transducer and the electronic system that amplifies the signal, analyzes the data and brings a result to the final user. The transducer includes the bioreceptor (which specifically interacts with the sample) and the interface that transforms the recognition from the bioreceptor into a measurable signal. When the analyte interacts with the bioreceptor, the transducer sends a signal that is processed by the electronics. All this process occurs in an efficient, quick, cheap, easy, simple and specific way. Regarding the type of the transductor, the biosensors can be electrochemical, optical, acoustic, magnetic or thermometric; but overall the most powerful ones are the optical biosensors, and among them the grating coupler. As a technique for investigating processes at the solid/liquid interface, presents high mechanical stability, immunity to electromagnetic interferences and pushes the sensitivity to levels even higher than other techniques and allows for the direct monitoring of macromolecular adsorption. Taking advantage of the last advances in nanotechnology, the goal of this thesis is to study the versatility of an Optical Grating Coupler Biosensor. The design of new grating sensor chips will be investigated, a new calibration technique for the sensors will be proposed and, taking advantage of the technique, different biomedical scenarios will be tested.
Esta tesis consiste en el diseño, fabricación y test de un Biosensor Óptico basado en redes de difracción y sus aplicaciones en biomedicina. Los biosensores ópticos son dispositivos que detectan interacciones biomoleculares específicas mediante un transductor óptico. Exhiben alta sensibilidad, alta estabilidad mecánica, son inmunes a las interferencias electromagnéticas y permiten medidas no destructivas. En los Biosensores Ópticos por Onda Evanescente un modo guiado se propaga a lo largo de la guía de ondas mientras que la onda evanescente interactúa con la superficie del sensor, reconociendo cualquier interacción biomolecular que provoque una modificación en el índice de refracción efectivo de la guía óptica. En este caso, la inserción de luz láser en la guía óptica se produce con ayuda de una red de difracción grabada en la superficie del sensor. Para un ángulo muy preciso se excita un modo guiado. Como consecuencia de las reacciones en la superficie se produce un cambio en el ángulo de acoplo. La medida en tiempo real del ángulo de acoplo, en función de la actividad bioquímica en la superficie es la base de este tipo de biosensor óptico. El objetivo es fabricar sensores de bajo coste en polímero y también en distintos materiales que permitan calibrar otras técnicas. Otro objetivo de esta tesis es la calibración de los sensores y de las distintas soluciones buffer comúnmente usadas en biosensado. Como aplicación, se ha usado un equipo comercial (Optical Waveguide Lightomode Spectroscopy, OWLS, MicroVacuum) para estudiar, mediante control electroquímico, el crecimiento y la liberación de multicapas de PLL/DNA para aplicaciones en administración de fármacos. También se ha usado el OWLS para optimizar la inmovilización de receptores olfativos en un dispositivo biosensor para el desarrollo de una nariz bioelectrónica.

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Durante las últimas décadas la construcción de dispositivos basados en materiales moleculares funcionales se ha convertido en uno de los principales objetivos para la ciencia de los materiales. Aunque las propiedades fundamentales de dichos materiales (electrónicas, magnéticas, ópticas, mecánicas, etc) vienen determinadas por las propiedades de sus constituyentes moleculares, la funcionalidad final de dichos dispositivos vendrá determinada en gran medida por las diferentes técnicas de procesado y estructuración empleadas durante su construcción. En este contexto, el objetivo principal de esta tesis se centra en el uso de diferentes técnicas de procesado y estructuración de compuestos con actividad medioambiental y biomédica para el desarrollo de nuevos materiales funcionales. Los metales pesados, y especialmente el mercurio, son especies altamente tóxicas cuya presencia, debida a causas tanto naturales como antropogénicas, se ha visto incrementada de forma global generando una situación de riesgo no solo para el medio ambiente sino también para el ser humano. Así pues, el desarrollo de nuevos sensores capaces de llevar a cabo la detección sensible y selectiva de iones Hg(II) disuelto en medio acuoso supone un desafío para la ciencia actual. La primera parte de esta tesis doctoral se ha basado en el procesado y nano-estructuración de dos compuestos orgánicos (1 y 2) derivados del 2,3-diaza-1,3-butadieno capaces de llevar a cabo la detección selectiva de iones Hg(II) mediante métodos ópticos. Como resultado se han obtenido diversos sistemas heterogeneos capaces de detectar la presencia dichos iones en medios acuosos. El primero de ellos, basado en un proceso de fisisorción de los indicadores previamente descritos sobre una membrana de celulosa, dio como resultado el desarrollo de sondas sensoras de un solo uso y bajo coste para la detección de Hg(II). Dichas sondas fueron preparadas siguiendo dos técnicas de estructuración diferentes. La más intuitiva de las cuales, que denominamos "técnica de revelado", se basó en el uso de un receptor orgánico sensible a la presencia de iones Hg(II) como agente revelador de una membrana de celulosa previamente impregnada con la muestra contaminada. Aunque los resultados obtenidos usando el receptor colorimétrico 1 como agente de revelado mostraban buena selectividad y reproducibilidad, la sensibilidad de dicho sistema frente a los iones Hg(II) se pudo establecer en la decenas de ppm, lejos de las unidades de ppb establecidas por la Unión Europea (EU) y la Agencia de Protección Medioambiental Estadounidense (EPA) como máximo para la presencia de iones Hg(II) en agua potable. Sin embargo este proceso sirvió como una prueba de concepto para el desarrollo de sondas sensoras basadas en el uso de materiales baratos y renovables como la celulosa para la detección de contaminantes. El otro proceso de estructuración empleado involucra la producción y deposición de nanoparticulas de los receptores orgánicos 1 y 2 sobre la superficie de membranas nanoporosas de celulosa. La obtención de estas membranas híbridas supuso un importante incremento en la sensibilidad de las sondas obtenidas alcanzando (con aquellas basadas en el uso del receptor 2) los niveles de detección exigidos por la EU y la EPA (ppb de Hg(II)). En una segunda aproximación se llevó a cabo el anclaje químico de los receptores 3 y 4 sobre la superficie de un substrato sólido para la obtención de un sensor de Hg(II) de alto rendimiento. En este caso llevamos a cabo el desarrollo de un sensor basado en la resonancia superficial de plasmón (SPR) capaz de detectar la presencia de picomoles de iones Hg(II) en medios acuosos. El diseño racional de los receptores (3 y 4) se llevó a cabo para optimizar la sensibilidad, selectividad y fiabilidad del sensor, lo cual nos permitió mejorar los parámetros establecidos por la EPA y la EU en tres ordenes de magnitud. El creciente desarrollo de la medicina regenerativa en general y la regeneración de tejidos en particular ha traído consigo una enorme mejora de la calidad de vida para decenas de miles de personas por todo el mundo. Aunque la mayoría de los biomateriales usados hoy en día presentan la estructura y resistencia adecuada para ser usados en medicina regenerativa, la interacción de dichos materiales con el entorno biológico no se controla de forma completa aun, lo cual genera, en algunos casos, efectos secundarios indeseados. El trabajo desarrollado en esta segunda parte de la tesis se centra en el estudio, caracterización y procesado de un nuevo tipo de material proteinaceo nano-particulado conocido como cuerpos de inclusión (IBs). La primera parte de esta investigación se centró en la caracterización nanoscópica de las propiedades fisico-químicas y estructurales de esta nueva familia de agregados, conocidos como IBs. Así pues, IBs provenientes de diferente trasfondo genético fueron caracterizados mediante diferentes técnicas como la dispersión dinámica de luz (DLS) la microscopia de fuerza atómica (AFM) o el ángulo de contacto (CA). Los resultados obtenidos indicaron que los IBs producidos en ausencia de diferentes elementos de la maquinaria de control de shock térmico celular (genes de la DnaK, ClpA y ClpP) exhiben una diferente distribución de tamaños, y propiedades fisico-químicas. De esta manera es posible concluir que existe una relación directa entre la conformación de las proteínas recombinantes que forman los IBs y sus propiedades. Una distribución aleatoria de IBs con diferente trasfondo genético se usó para decorar una serie de superficies químicamente modificadas con grupos amino, las cuales fueron sometidas a diferentes ensayos de proliferación celular obteniendo diferentes resultados según el origen genético de los IBs empleados. Dicho experimento probó que es posible utilizar los IBs para modificar las superficies de los materiales con objeto de obtener diferentes comportamientos de proliferación celular, expandiendo el posible uso de dichos materiales para aplicaciones en regeneración de tejidos.
During the last decades the construction of devices based on molecular functional materials with specific properties has become one of the major objectives of materials scientists, since they can offer new and exciting functionalities to the present human activities. Although their basic properties will be guided by the fundamental -electronic, magnetic, optical, mechanical, etc- properties of their molecular constituent units, the final functionality of a device will depend, in a major way, on the processing and structuring techniques used during its construction. In this context, the main objective of this Thesis has been the use of different processing and structuring techniques for the development of new functional materials based on already tested environmentally and biologically active compounds. Among all the environmentally hazardous substances present in our environment, heavy metal ions, and specially mercury, are highly toxic elements which contamination, due to both natural and anthropogenic reasons, has become severe in some parts of the world, resulting in health damage to their inhabitants. Therefore, the developing of new sensors able to detect selectively and sensitively Hg2+ on aqueous media is still an actual challenge. In this work we present two 1,4-disubstituted-2,3-diaza-1,3-butadiene derivatives (1 and 2) able to selectively perform optical detection of Hg2+ in aqueous media, that combined with different nanostructuring and anchoring techniques allowed us to obtain highly sensitive solid-supported mercury detection systems. The first of them is based on the physisorption of the diaza butadiene indicators on porous cellulose membranes obtaining indicator coated probes that could be used as new cheap and reliable Hg2+ sensing systems. In order to do that, two different structuring techniques have been used. The most intuitive one, which we have named “developing technique”, is founded on the use of the optically active Hg2+ organic receptor 1 as a Hg2+ developing agent of a cellulose substrate, previously impregnated with the contaminated solution. Although Hg2+ detection tests performed using this colorimetric chemosensing probes, based on receptor 1, showed good selectivity and reproducibility, they presented a limited sensitivity vs. Hg2+. The detection limit of the probes was set on tens of ppm (10−2g/l), far away from the 1 ppb (μg/l) fixed by the European Union (EU) and the North American Environmental Protection Agency (EPA) as the maximum amount of Hg2+ allowed in drinkable water. Nevertheless, this procedure served as a prove of concept for the developing of probes based on the use of cheap and renewable materials to be applied on the in situ detection of contaminants. The other structuring technique used is based on a new physisorption procedure, involving the production and deposition of nanoparticles of the organic sensing molecules on nanoporous cellulose membranes for the fabrication of hybrid membranes. In this case, excellent Hg2+ detection results showing a high Hg2+ sensitivity and selectivity were obtained for the receptor 2 based cellulose probes. In contrast to the previous case, the detection limit obtained matched the EU and EPA requirements for drinkable water, reaching the level of ppb (μg/l). On a second approach the covalent bonding was used as a driving force for the receptor anchoring onto a solid substrate. In this case we developed a surface plasmon resonance (SPR) sensor able to perform picomolar detection of Hg2+ on aqueous systems. The rational design of the Hg2+ receptors (3 and 4) optimizes the sensitivity and reliability of the sensor allowing us to selectively detect, in presence of other divalent cations, Hg2+ concentrations on aqueous systems on the picomolar range, meliorating on three orders of magnitude the EU and EPA Hg2+ detection limit on drinkable water. As contamination control and pollutant removal, regenerative medicine in general and particularly in tissue engineering (TE) has the enormous potential of improving the quality of life for many thousands of people throughout the world. Although most of the more commonly used biomaterials match all the structural and mechanical resistance requirements to be applied in regenerative medicine, the interaction of such materials with the surrounding biological media is still not well controlled, leading to undesired immunological responses such as infections or uncontrolled inflammation in some cases. The work developed on the second part of this thesis has been focused on the study, characterization and processing of a new kind of proteinaceous nanoparticulate biomaterial, known as inclusion bodies (IBs), as a promising additive for cell proliferation enhancement. The first part of the research regarding the processing and structuring of biologically active materials is centered on the characterization of the nanoscale, physicochemical and structural properties of a novel family of proteinaceous aggregates known as “inclusion bodies” (IBs). Thus, IBs coming from different genetic backgrounds have been characterized by means of light dispersion and surface analysis techniques, such as dynamic light scattering (DLS), atomic force microscopy (AFM) or contact angle (CA). Results obtained indicated that IBs produced in absence of different elements of the cellular heat shock machinery (DnaK, ClpA, and ClpP genes) exhibit a range of sizes, wettability and stiffness values, that let us conclude the existence of a direct relationship between the conformation status of the recombinant proteins inside the IBs and their physicochemical and structural properties. Randomly distributed IBs, from different genetic backgrounds, were used to decorate amine terminated silicon surfaces. It was possible to observe how cultured mammalian cells respond differentially to IB variants when used as particulate materials to engineer the physicochemical surface properties, proving that the actual range of referred mechanical as well as other physicochemical properties is sensed and discriminated by biological systems. To further prove the validity of IBs as stimulator of cell proliferation, microstructuring of the IBs onto the same substrate was performed using the Microcontact Printing (μCP) technique. The obtained results confirmed again the ability of IBs to stimulate cell proliferation on surfaces initially not suitable for cell growth. Therefore, it is possible to conclude that the tuning opportunities offered through adjusting the genetic background of the cell where the IBs are produced, definitively expands the spectrum of biomedical applications of this novel bacterial nanomaterial.

En la present tesi doctoral es descriu un procediment general per a l'obtenció de nanopartícules mesoporoses de sílice (MSNs) regioselectivament bifuncionalizades de forma ortogonal amb diferents grups funcionals. L'estratègia sintètica consisteix en la preparació de MSNs mitjançant co-condensació, seguit d'una posterior funcionalització covalent, mentre el tensioactiu es troba encara present en l'estructura de les MSNs. Seguint aquesta metodologia, s'han sintetitzat les nanopartícules bifuncionalizades amina-azida (MSN-(NH2)i(N3)o), amina-isotiocianat (MSN-(NH2)i(NCS)o) i amina-aldehid (MSN-(NH2)i(CHO)o), per al seu ús en aplicacions biomèdiques. En primer lloc, s'han sintetitzat i caracteritzat de forma homogènia i reproduïble les nanopartícules de referència (MSN-NH2) que permetran les successives funcionalitzacions. Aquestes nanopartícules aminades s'han fet servir posteriorment per a la síntesi de sensors de naftalimida. S'ha aconseguit desenvolupar un procediment general per a la introducció de 4-amino-1,8 naftalimides. Aquestes naftalimides han estat provades com a sensor i portes lògiques per a la detecció de H+ i F-. D'altra banda, s'ha descrit un protocol per preparar amino-azida-MSNs de forma regioselectiva. Aquestes MSNs han estat funcionalitzades per primera vegada amb foldàmers catiònics i la seva capacitat per creuar membranes citoplasmàtiques i viabilitat ha estat estudiada, així com l'ús d'aquests sistemes per a l'alliberament intracel·lular de Doxorubicina (DOX) de forma controlada. També s'ha realitzat un nou protocol per preparar MSNs amb isotiocianat en la seva estructura. La metodologia sintètica és general i es pot aplicar, en principi, a qualsevol MSNs aminada. L'eficiència de la funcionalització és comparable a la cicloaddició de coure (CuAAC) evitant els protocols d'aïllament i d'eliminació del metall. Seguint aquesta metodologia, s'han preparat unes noves amino-isotiocianat-MSNs per al disseny d'un nano-contenidor capaç d'alliberar el fàrmac Ataluren de forma controlada, per el seu us en la distròfia muscular de Duchenne (DMD). S'han aconseguit sintetitzar amina-aldehid-MSNs. Aquestes MSNs s'han aplicat com una nanoplataforma simple i versàtil capaç d'alliberar de forma dual una barreja CPT/DOX per al tractament del càncer, mitjançant l'ús d'estímuls de pH. Mentre un fàrmac és absorbit dins de la superfície interior, l'altre està unit covalentment a la superfície externa, actuant així, a la vegada, com a fàrmac i com agent bloquejant de porus. Aquest sistema respon als estímuls de pH i tots dos fàrmacs són només alliberats en un medi àcid.
En la presente tesis doctoral se describe un procedimiento general para la obtención de nanopartículas mesoporosas de sílice (MSNs) regioselectivamente bifuncionalizadas de forma ortogonal con distintos grupos funcionales. La estrategia sintética consiste en la preparación de MSNs mediande co-condensación, seguido de una posterior funcionalización covalente, mientras el tensioactivo se encuentra todavía presente en la estructura de las MSNs. Siguiendo esta metodología, se han sintetizado las nanopartículas bifuncionalizadas amina-azida (MSN-(NH2)i(N3)o), amina-isotiocianato (MSN-(NH2)i(NCS)o) y amina-aldehído (MSN-(NH2)i(CHO)o), para su uso en aplicaciones biomédicas. En primer lugar, se han sintetizado y caracterizado de forma homogénea y reproducible las nanopartículas aminadas de referencia (MSN-NH2) que permitirán las sucesivas funcionalizaciones, con un tamaño de 50 nm y 100 nm aproximadamente. Estas nanopartículas aminadas se han usado posteriormente para la síntesis de sensores de naftalimida. Se ha conseguido desarrollar un procedimiento general para la introducción de 4-amino-1,8 naftalimidas. Estas naftalimidas han sido probadas como sensores y puertas lógicas para la detección de H + y F-. Por otra parte, se ha descrito un protocolo para preparar amino-azida-MSNs de forma regioselectiva. Estas MSNs han sido funcionalizadas por primera vez con foldámeros catiónicos y su capacidad para cruzar membranas citoplasmáticas y viabilidad ha sido estudiada, así como el uso de estos sistemas para la liberación intracelular de doxorubicina (DOX) de forma controlada. También se ha realizado un nuevo protocolo para preparar MSNs con isotiocianato en su estructura. La metodología sintética es general y puede aplicarse, en principio, a todo tipo de MSNs aminadas. La eficiencia de la funcionalización es comparable a la cicloadición de cobre (CuAAC) evitando los protocolos de aislamiento y de eliminación del metal. Siguiendo esta metodología, se han preparado unas nuevas amino-isotiocianato-MSNs para el diseño de un nano-contenedor capaz de liberar el fármaco Ataluren de forma controlada. Se ha logrado sintetizar amina-aldehído-MSN. Estas MSNs se han aplicado como una nanoplataforma simple y versátil capaz de liberar de forma dual una mezcla CPT/DOX para el tratamiento del cáncer, mediante el uso de estímulos de pH. Mientras un fármaco es absorbido dentro de la superficie interior, el otro está unido covalentemente a la superficie externa, actuando así como fármaco y como agente bloqueante de poro. Este sistema responde a los estímulos de pH y ambos fármacos son solamente liberados en un medio ácido.
In this PhD dissertation, a general procedure for the obtaining of different regioselective orthogonal bifunctionalized mesoporous silica nanoparticles (MSNs) has been carried out. The strategy consists of a covalent functionalization of co-condensed monodispersed and uniform aminated-MSNs, where tensioactive is still present in its structure. Three bifunctionalized MSNs, amine-azide (MSN-(NH2)i(N3)o), amine-isothiocyanate (MSN-(NH2)i(NCS)o) and amine-aldehyde (MSN-(NH2)i(CHO)o), with efficient “click” reactions, have been synthetized for its use in biomedical applications. First, a well characterized batch of precursor aminated-MSNs (MSN-(NH2)) has been prepared. The best conditions for the synthesis of homogenous and reproducible MSN-(NH2) with a size between 50-100 nm have been studied. These aminated-MSNs have been used for the synthesis of naphthalimide sensors where a general procedure for the introduction of 4-amine-1,8-naphthalimides has been developed. These naphthalimides have been tested as potential logic gates for the detection of H+ and F-. A straightforward protocol to prepare amine-azide MSNs has been described. These MSNs have been functionalized with quinolin cationic foldamers for the first time. The ability of these foldamer-MSNs to cross cytoplasmic membranes and its viability has been studied. The penetrating capacity of foldamer-MSNs have been used for intracellular delivery of Doxorubicin (DOX). A new protocol to prepare isothiocyanate functionalized MSNs is described. The synthetic methodology is general and can be applied, in principle, to all type of aminated MSNs. The efficiency of the functionalization is comparable to the copper cycloaddition (CuAAC) avoiding isolation and copper removal protocols. Following this methodology, new amino-isothiocyanate-MSNs have been prepared for the design of a nano-container able to release the drug Ataluren in a controlled manner, for the treatment of Duchenne muscular dystrophy (DMD). Regioselective bifunctionalized amine-aldehyde-MSNs have been synthetized. These MSNs have been applied as a versatile nanoplatform able to release dual synergistic CPT/DOX mixture for cancer treatment only by using pH stimuli. While CPT is absorbed at the inner surface, DOX is covalently linked to the external surface acting both as an active and a capping agent (pH=4).

Present PhD thesis aimed to investigate relatively unknown properties of halloysite nanoparticles, as well as to further examine HNTs as potential drug nanocarriers. NPs loading and release characteristics were studied using model active molecules: magnesium monoperoxyphthalate (MMPP), aspirin and epirubicin. The research was fulfilled with formation of complex multi-functional nanoarchitectures, which apart from ability to deliver incorporated drugs, showed the potential of controlled and sustain release of therapeutics, biocompatible and bioresorbable characteristics as well as potential targeting abilities. Great attention was dedicated to characterization of formed halloysite-based nanoarchitectures in qualitative as well as quantitative manner. Investigations performed in this thesis also faced the problem of exceeding dimensions of halloysite units, nanoparticles aggregation, poor loading capability and dose dumping effect. Subsequently, studies for trying to find a solution to these obstacles were undertaken. Fully characterized halloysite nanoconstructs were further examined in biological field, employing different cancer cell lines. Studies on pristine halloysite nanotubes: Physico-chemical and biological properties of halloysite nanoparticles were evaluated using microscopic techniques, spectroscopic analysis, surface studies regarding charge, porosity and wettability. The thermal and time-based examination of pristine halloysite was performed as well, showing stability of HNTs alumino-silicate skeletons up to ~400 ℃ and over a long period of time (2 years) at room temperature, however with a variable amount of incorporated water molecules. Biological performance of HNTs was determined in vitro in multiple cellular systems by toxicity, cellular uptake, colocalization and accumulation studies using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction (MTT) assay and set of microscopic techniques. Aiming to deeply characterize halloysite nanoparticles, the study proceeded with employment of non-standard techniques, as multiphoton microscopy that drove to discovery of novel NPs promising capabilities. It was revealed that halloysite is able to convert light to its second harmonic, at twice of the frequency (and therefore half of the wavelength) while using high intensity femtosecond pulsed laser. Halloysite Second Harmonic Generation (SHG) signal was detected over a broad wavelength range, showed stability over a long period of time, polarization properties and quadratic dependence on the intensity of incident light. The analysis also pointed out characteristic structure properties of the nanoparticle that is lack of the center of symmetry and the high crystalline structure organization. Among a wide spectrum of domains where discovered HNTs characteristics can be utilized (e.g. optoelectronics, biosensors), we have explored its application in alternative label-free bioimaging. The proposed multiphoton method of analysis showed advantages over the standard confocal microscopy, since e.g. nanoparticles did not have to be stained prior the analysis, thus no possible alterations of HNTs including size, surface chemistry and consequent cellular uptake were induced. Therefore, for the first time, halloysite nanotubes were exploited as imaging agents, taking advantage of their endogenous properties. Along the research it was revealed that the length of pristine HNTs and the strong aggregation limit their ability to pass intracellular membranes and thus minimize their effectiveness as drug nanocarriers. Therefore, efforts were devoted to the development of facile methodology to efficiently disperse and shorten HNTs units. Set of characterizations techniques, such as Scanning Electron Microscopy (SEM) analysis with size distribution profile and nitrogen adsorption Brunauer–Emmett–Teller (BET) method revealed that the applied ultrasonication procedure resulted with longest tubes breaking and favored obtaining HNTs below 300 nm in length (39.1 % to 76 % of the batch). The number of voids among the pristine nanoparticles when packing together (123–43 nm) greatly increased (total pore volume from 0.23 cm^3/g to 0.30 cm^3/g), meaning that the nanomaterial was efficiently disaggregated as well. In vitro internalization and colocalization studies by Scanning Electron and Multiphoton Microscopy demonstrated that the sonicated halloysite were preferentially internalized via macropinocytosis within 60 min and accumulated in the perinuclear region within 24 h. Halloysite application in nanomedicine: To study halloysite potential as a carrier for drugs, we set up the preparation and characterization of hybrid nanoconstructs with model molecules such as magnesium monoperoxyphthalate hexahydrate (MMPP), a negatively charged oxidizing agent, aspirin, an anti-inflammatory drug and epirubicin, a chemotherapeutic. Chosen molecules were incorporated with 3.5 %wt, 1.1 %wt and 5.1 %wt capacity, respectively for MMPP, aspirin and epirubicin. Loading efficiency (LE) improvement was achieved through the choice of the right solvent (1), enhancement of electrostatic forces between nanoparticle and the drug, via functionalization of HNT surfaces with an active linker (2), as well as NPs structure modification leading to increase of inner lumen volume (3). Specifically, the use of water: EtOH (7:3 v/v) as a solvent instead of water, increased MMPP loading capacity up to 6.1 %wt. Poor incorporation of aspirin was improved by enhancing electrostatic forces between deprotonated aspirin molecules and modified HNTs inner walls with amine-rich organosilane. It was also demonstrated that by enlarging volume of the NPs cavities, more molecules could be loaded. To do that, pristine HNTs were treated with 0.1M aqueous solution of NaOH, which resulted in an exfoliation of bilayers located inside the lumen. At the same time, outer surface of the halloysite tubules was preserved. As a consequence of the base treatment, halloysite cylinders gained more volume in the inner cavity as concluded from Transmission Electron Microscopy (TEM) and nitrogen adsorption BET analysis. The actual test on loading capacity using model MMPP molecule revealed increased MMPP incorporation from 6.1 % wt to 11.7 %wt. To evaluate if the activity of MMPP as an oxidizing agent remained unchanged upon incorporation and release from halloysite, and therefore to demonstrate the inactivity of the inorganic skeleton towards carried molecule, we tested HNT-MMPP nanoconstruct with selective fluorescent 1,3-diphenylisobenzofuran (DPBF) probe. Among available modifications of halloysite nanoparticles via covalent bond, the surface silanization is commonly recognized as one of the most efficient and widespread reaction while HNT manipulation. Up to date, the halloysite nanotubes functionalized with silanes have been used as a support for versatile applications in diverse scientific domains, including enzymes immobilization and biosensing. Willing to explore the halloysite functionalization with those active linkers, we have performed grafting reactions with representative organosilanes carrying the same backbone, while varying in the content of terminal groups, namely (3-aminopropyl)triethoxysilane (APTES), 3-(2-aminoethylamino)propyldimethoxymethylsilane (AEAPS), (3-mercaptopropyl)trimethoxysilane (MPS). Successful HNTs surfaces functionalization with organosilanes was demonstrated by means of quantitative thermogravimetric analysis (TGA) that allowed to estimate the loading capacity of organosilanes to be of 5.7 %wt for APTES, 7.4 %wt for AEAPS and 0.7 %wt for MPS. In addition, particular attention was dedicated to further quantify incorporated organosilane (APTES), since only one method has been so far reported, that is the destructive thermogravimetric analysis (TGA). For this reason, we set up and optimized a Fmoc based method by performing the following three reactions: (i) synthesis of “APTES-Fmoc” molecule; (ii) halloysite functionalization with “APTES-Fmoc”; and (iii) time-dependent Fmoc deprotection reaction in piperidine: EtOH (20 %) solution, resulting in dibenzofulvenepiperidine adduct (DBF-pip) formation. The UV-visible spectroscopic analysis of supernatant solutions demonstrated that the DBF-pip deprotection from halloysite support needs 5 h to be completed. Therefore, it was evidenced that HNT Fmoc-method showed strong coherence with already existing TGA method (± 2 % measurement error) and stood out as a valuable complementary technique for quantification of silane grafting on HNTs surface with additional low-cost and nondestructive advantages. The possibility of using halloysite nanotubes as a non-viral gene delivery nanosystem for therapeutic treatments was studied as well. Aiming to immobilize plasmid DNA (pDNA) based on the Green Fluorescent Protein (GFP) on HNTs support, the layer-by-layer (LbL) adsorption technique was applied. Obtained multi-component assembly was characterized qualitatively by monitoring variation in nanoparticle physico-chemical properties including surface charge, mass weight, presence of functional groups at each step of hybrid formation, which confirmed the successful nanoarchitecture formation. In order to additionally demonstrate the presence of GFP encoding plasmid (pGFP) on HNTs, the nanoarchitecture was treated with the bovine pancreatic deoxyribonuclease (DNase) enzyme, which induced the pGFP degradation through hydrolytic cleavage of phosphodiester linkages in DNA backbone. Thus, as expected, such nanoform with deposed genetic material varied in physico-chemical properties, expressing similar ones of the nanoconstruct without pGFP plasmid attached. The biological efficiency of HNTs-pGFP nanosystem was checked by means of Multiphoton microscopy. Successful pGFP plasmid transportation into cells was verified by detection of GFP expression, which yielded fluorescence emission. The interesting and innovative aspect of this case study was the simultaneous observation of GFP expression via fluorescence detection, and colocalization of halloysite nanoparticles by their SHG signal. This study proved that halloysite can act as an efficient carrier of genetic material, since free pGFP cannot be internalized by same cells, due to its large size and significant charge. Drug-loaded halloysite nanoconstructs (HNT-MMPP, HNT-APTES-aspirin) were also examined on the drug release kinetics, demonstrating long-term MMPP leakage taking 18 days and aspirin over 60 min. However, great drug liberation into the solvent of release was observed in the first minutes, followed by desired sustained drug release. The initial molecule liberation (dose dumping effect) is known to entail local toxicity. Herein, trying to find a solution to this problem, the coating of HNTs with the natural collagen polymer was investigated. Two strategies for the loading with this biopolymer were studied: (i) formation of a covalent bond between collagen and APTES-modified HNTs using glutaraldehyde cross-linker or (ii) noncovalent adsorption of collagen into pores of NPs. Immobilization of collagen on the surface of HNTs was estimated to be 3.7 %wt (i) and 1.8 %wt (ii). Other supplementary characterization techniques, such as water contact angle, ζ–potential analysis, Kaiser test, ultraviolet and visible (UV-vis) spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) were in accordance and proved nanoarchitectures formation. For the visualization purpose of HNTs encapsulated in collagen shell, the innovative characterization technique was implemented, namely 3D Multiphoton microscopy. It revealed that the biopolymer coating blocked the entrances of the hollow tubes thus, entrapping the drug in NPs. Mimicking tumor microenvironment (TME), the pH and/or enzyme triggered release was performed. LC-Mass analysis revealed that the collagen coating slowed down the release of aspirin from HNTs. Studies on cells showed that the collagen coating on HNTs is biocompatible and cell viability assay performed on 5637 urinary bladder and HeLa cervical cancer cell lines demonstrated the sustained release of the entrapped epirubicin chemotherapeutic agent in the biological context. Industrial application of halloysite: During a stage in BASF SE (USA), validation and properties enhancement of halloysite-based products potentially manufacturable in the company on an industrial scale were studied. In particular, the research was dedicated to aspects such as the pH-dependent dispersion behavior of halloysite nanotubes and iron coarse impurities removal from bulk samples. Applied methodologies and set of physico-chemical characterization techniques generated and revealed decreased percentage of present aggregates, maintained low shear viscosity under the threshold value and increased solids loading capacity in final halloysite-based products. Conclusions: In conclusion, PhD studies here reported contributed to the exploration of halloysite nanotubes for their application in the nanomedical and industrial fields. The investigations suggest a facile manipulation and functionalization of HNTs, useful for properties modification and improved NPs performance. Specifically, the study was directed toward formation of multi-functional nanocarriers with controlled drug delivery and release properties, together with targeting and imaging abilities. Moreover, the research was completed with halloysite-related technology transfer to the BASF SE, for the purpose of knowledge increase in the halloysite-field and bringing forward placement of halloysite-based products on the market. The systematic study on HNTs characterization and application performed in this PhD thesis will contribute to the development of HNTs as a high performance structural and functional material.

The motivation of this study is to investigate the size dependent properties of Gadolinium silicide nanoparticles and their potential applications in Biomedicine. We use two approaches in our investigation - size dependence and possible exchange interaction in a core-shell structure. Past results showed Gd5Si4 NPs exhibit significantly reduced echo time compared to superparamagnetic iron oxide nanoparticles (SPION) when measured in a 7 T magnetic resonance imaging (MRI) system. This indicates potential use of Gd5Si4 ferromagnetic nanoparticles as T2 contrast agents for MRI. Until recently most contrast agents (CA) that are used in Magnetic Resonance Imaging (MRI) studies have been paramagnetic. However, ferromagnetic CAs are potentially more sensitive as T2 CAs than T1 paramagnetic compounds due to their large magnetic moments. Furthermore, the need for better MRI images without the need of upgrading to the higher magnetic field strength can be achieved using better CA such as Gd5Si4 NP. The quality of the image contrast in MRI is improved by shortening T1 and T2 relaxation times at the site or close proximity to the CA. In this study, effect of Gd5Si4 NP of varying sizes and with different concentrations are investigated on T1, T2 and T2* (effective/observed T2) relaxations times. Further study was carried out on possible exchange interaction between Fe3O4 and Gd5Si4 to enhance the magnetic properties of the Gd5Si4 which could be later used to synthesize core-shell structures. Exchange interaction / bias is a phenomena associated with the exchange anisotropy created at the interface between the two magnetic materials. Therefore, thin films of varying thickness was deposited and studied for their magnetic properties.

Nanoparticles are often considered as efficient drug delivery vehicles for precisely dispensing the therapeutic payloads specifically to the diseased sites in the patient’s body, thereby minimizing the toxic side effects of the payloads on the healthy tissue. However, the fundamental physics that underlies the nanoparticles’ intrinsic interaction with the surrounding cells is inadequately elucidated. The ability of the nanoparticles to precisely control the release of its payloads externally (on-demand) without depending on the physiological conditions of the target sites has the potential to enable patient- and disease-specific nanomedicine, also known as Personalized NanoMedicine (PNM). In this dissertation, magneto-electric nanoparticles (MENs) were utilized for the first time to enable important functions, such as (i) field-controlled high-efficacy dissipation-free targeted drug delivery system and on-demand release at the sub-cellular level, (ii) non-invasive energy-efficient stimulation of deep brain tissue at body temperature, and (iii) a high-sensitivity contrasting agent to map the neuronal activity in the brain non-invasively. First, this dissertation specifically focuses on using MENs as energy-efficient and dissipation-free field-controlled nano-vehicle for targeted delivery and on-demand release of a anti-cancer Paclitaxel (Taxol) drug and a anti-HIV AZT 5’-triphosphate (AZTTP) drug from 30-nm MENs (CoFe2O4-BaTiO3) by applying low-energy DC and low-frequency (below 1000 Hz) AC fields to separate the functions of delivery and release, respectively. Second, this dissertation focuses on the use of MENs to non-invasively stimulate the deep brain neuronal activity via application of a low energy and low frequency external magnetic field to activate intrinsic electric dipoles at the cellular level through numerical simulations. Third, this dissertation describes the use of MENs to track the neuronal activities in the brain (non-invasively) using a magnetic resonance and a magnetic nanoparticle imaging by monitoring the changes in the magnetization of the MENs surrounding the neuronal tissue under different states. The potential therapeutic and diagnostic impact of this innovative and novel study is highly significant not only in HIV-AIDS, Cancer, Parkinson’s and Alzheimer’s disease but also in many CNS and other diseases, where the ability to remotely control targeted drug delivery/release, and diagnostics is the key.

The potential of magnetic nanoparticle-vesicle assemblies (MNP-V) as remote controlled drug delivery platforms capable of inducing cellular responses under magnetic stimuli has been previously demonstrated in the Webb group at the University of Manchester. To create these magnetoresponsive nanomaterials biotin-avidin and Cu-histidinyl multivalent recognition were employed. This thesis describes an exploration of the potential of thiol-thioester exchange reactions (leading to native chemical ligation, NCL) to create magnetoresponsive materials, which potentially have applications in biomedicine. Firstly, iron oxide magnetic nanoparticles have been synthesised using a thermal co-precipitation method followed by chemical modification with sulfhydryl motifs for use as smart biomaterials. Knowing that the behaviour and reactivity of nanoparticles is highly influenced by their physicochemical properties, a thourough characterisation of these particles has been obtained. Secondly, during this project, several thioester derivatives have been synthesised that can be incorporated into the membranes of 800 nm liposomes. Among these, the spectrophotometric properties of synthetic lipid 38 allowed the investigation of trans-thioesterification rates with cysteinyl functionalities, both in solution and at the phospholipid membrane interface of liposomes. Product identification has been achieved using mass spectrometry and 1H-NMR spectroscopy. Finally, the conditions required to induce the release of a dye (e.g. 5(6)-CF) from MNP-V upon exposure to an AMF pulse have been established. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases has been investigated as interesting payload due to its fluorescent and anti-viral properties.

En aquesta tesi hem desenvolupat materials microestructurats basats en silici macroporós, centrant-nos en la producció de microestructures per la seva aplicació en biomedicina. El silici macroporós es forma per atac electroquímic de silici en electròlits basats en àcid fluorhídric. Es fabriquen mostres de silici macroporós ordenat i aleatori. Amb un procés litogràfic, es pot crear un patró predisenyat en el silici, i així definir els punts de nucleació i aconseguir porus amb un creixement ordenat i un diàmetre uniforme. L’oxidació tèrmica del silici macroporós permet la formació de noves estructures, com micropilars de SiO2. El SiO2 es normalment acceptat com un material biocompatible. Tot i això, utilitzem l’espectroscòpia infraroja per realitzar una caracterització exhaustiva i una modificació adequada de la química de superfície orientada cap a la conjugació de biomolècules. La peculiar arquitectura d’aquests substrats va permetre la creació de partícules multifuncionals amb una doble funcionalització selectiva en les cares interior i exterior. Aquestes microestructures van ser concebudes com a materials per al transport de fàrmacs. Així doncs, aquestes micropartícules de SiO2 van ser proposades com a sistemes d’alliberament de fàrmacs per control de pH quan es combinen amb polielectròlits sensibles al pH. Finalment, la doble funcionalització va ser explotada per crear micropartícules multifuncionals per l’alliberament de fàrmacs dirigida cap a cèl•lules diana. La viabilitat del sistema va ser provada amb cèl•lules cancerígenes in vitro.
En esta tesis hemos desarrollado materiales microestructurados basados en silicio macroporoso, centrándonos en la producción de plataformas y partículas para su aplicación en biomedicina. El silicio macroporoso se forma por ataque electroquímico de silicio en electrolitos basados en ácido fluorhídrico. Se fabricaron muestras de silicio macroporoso ordenado y aleatorio. Con un proceso litográfico, se puede crear un patrón prediseñado en el silicio, y así definir los puntos de nucleación y conseguir poros con un crecimiento ordenado y un diámetro uniforme. La óxidación térmica del silicio macroporoso permite la formación de nuevas estructuras, como micropilares de SiO2. El SiO2 es normalmente aceptado como un material biocompatible. A pesar de esto, utilizamos la espectroscopía infraroja para realizar una caracterización exhaustiva y una modificación adecuada de la química de superficie orientada hacia la conjugación de biomoleculas. La peculiar arquitectura de estos sustratos permitió la creación de partículas multifuncionales con una doble functionalización selectiva en las caras interior y exterior. Estas microestructuras fueron concebidas como materiales para el transporte de fármacos. Así pues, estas micropartículas de SiO2 fueron propuestas como sistemas de liberación de fármacos por control de pH cuando se combinan con polielectrolitos sensibles al pH. Finalmente, la doble funcionalización fue explotada para crear micropartículas multifunctionales para la liberación de fármacos dirigida hacia células diana. La viabilidad del sistema fue probada con células cancerígenas in vitro.
This thesis has explored the fabrication of silicon oxide (SiO2) microstructures based on macroporous silicon (macro-pSi), with a focus on producing suitable platforms and particles for application in biomedicine. Macroporous silicon was formed by the electrochemical etching of low doped p-type silicon in hydrofluoric acid based solutions. Both random and ordered structures were fabricated. A patterning lithography prior etching led to an ordered pore nucleation and consequently tubular structures of uniform size were produced. Thermal oxidation of macro-pSi allowed the formation of novel structures such as SiO2 micropillars, with identical arrangement and dimensions of those in the preceding macro-pSi. SiO2 is generally accepted as a biocompatible material; nevertheless, a methodical study of the surface chemistry and its modification was performed by infrared (IR) spectroscopy to generate surfaces capable of interfacing with living cells. The particular architecture of these substrates allowed creating multifunctional particles with a selective dual functionality in nanometrically separated internal and external sides. We also foresaw these microstuctured materials as drug carriers. Thus, SiO2 microparticles were proposed as pH-controlled drug delivery system when they are combined with pH-responsive polyelectrolytes. Finally, a dual-functionalization of the inner/outer sides was employed for creating multifunctional microparticles, which were demonstrated to be cancer-targeted in in vitro tests.

In this presentation it is demonstrated that the unique magnetic properties of superparamagnetic cobalt-spinel ferrite nanoparticles can be employed in several novel applications. A method to selectively capture and remove pathogens from infected organisms to improve longevity is presented. Evidence is provided to show that automated methods using modified forms of hemofiltration or peritoneal dialysis could be used to eliminate the particle/pathogen or particle/infected cell conjugates from the organism postoperatively. It is shown that disparately functionalized nanoparticles can be used in concert as drug carrier and release mechanisms. Lastly, we provide preliminary evidence to support the use of magnetic nanoparticles for controlling reaction kinetics.

Plasmonics, the science of the excitation of surface plasmon polaritons (SPP) at the metal-dielectric interface under intense beam radiation, has been studied for its immense potential for developing numerous nanophotonic devices, optical circuits and lab-on-a-chip devices. The key feature, which makes the plasmonic structures promising is the ability to support strong resonances with different behaviors and tunable localized hotspots, excitable in a wide spectral range. Therefore, the fundamental understanding of light-matter interactions at subwavelength nanostructures and use of this understanding to tailor plasmonic nanostructures with the ability to sustain high-quality tunable resonant modes are essential toward the realization of highly functional devices with a wide range of applications from sensing to switching. We investigated the excitation of various plasmonic resonance modes (i.e. Fano resonances, and toroidal moments) using both optical and terahertz (THz) plasmonic metamolecules. By designing and fabricating various nanostructures, we successfully predicted, demonstrated and analyzed the excitation of plasmonic resonances, numerically and experimentally. A simple comparison between the sensitivity and lineshape quality of various optically driven resonances reveals that nonradiative toroidal moments are exotic plasmonic modes with strong sensitivity to environmental perturbations. Employing toroidal plasmonic metasurfaces, we demonstrated ultrafast plasmonic switches and highly sensitive sensors. Focusing on the biomedical applications of toroidal moments, we developed plasmonic metamaterials for fast and cost-effective infection diagnosis using the THz range of the spectrum. We used the exotic behavior of toroidal moments for the identification of Zika-virus (ZIKV) envelope proteins as the infectious nano-agents through two protocols: 1) direct biding of targeted biomarkers to the plasmonic metasurfaces, and 2) attaching gold nanoparticles to the plasmonic metasurfaces and binding the proteins to the particles to enhance the sensitivity. This led to developing ultrasensitive THz plasmonic metasensors for detection of nanoscale and low-molecular-weight biomarkers at the picomolar range of concentration. In summary, by using high-quality and pronounced toroidal moments as sensitive resonances, we have successfully designed, fabricated and characterized novel plasmonic toroidal metamaterials for the detection of infectious biomarkers using different methods. The proposed approach allowed us to compare and analyze the binding properties, sensitivity, repeatability, and limit of detection of the metasensing devices

Elastin like polypeptides (ELPs) are a class of naturally derived biomaterials that are non-immunogenic, genetically encodable, and biocompatible making them ideal for a variety of biomedical applications, ranging from drug delivery to tissue engineering. Also, ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides and functionalization of ELPs. This work utilizes ELPs for regenerative medicine and drug delivery. The goal of the first study was to synthesize a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in the treatment of chronic wounds. EGF plays a crucial role in wound healing by inducing epithelial cell proliferation and migration, and fibroblast proliferation. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not protect exogenous EGF from degradation, or prevent it from diffusing away from the application site. We created an EGF-ELP fusion protein to combat these issues. As demonstrated through the proliferation of human skin fibroblasts in vitro, the EGF-ELP may be able to aid in the treatment of chronic wounds. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration. The objective of the second study was to create an injectable hydrogel platform that does not require conjugation of functional moieties for crosslinking or biological activity. Hydrogels are three-dimensional polymer networks that are able to absorb water and biological fluids without dissolving. Their high water content gives them physical properties similar to soft tissues, making them useful as scaffolds for cell migration and drug delivery vehicles. Injectable hydrogels that crosslink in situ are particularly useful because they can form to the shape of the defect, providing a near perfect fit. However, many hydrogel platforms cannot be crosslinked in situ because cytotoxic crosslinking reagents are required. Additionally, hydrogels typically require the chemical conjugation of crosslinking domains and bioactive peptides to the polymer backbone, adding more steps and time required for hydrogel production. We devised an injectable hydrogel platform that can be synthesized in a single step using photoreactive ELPs as the polymer backbone. Leucine auxotrophic Eshcherichia coli expressed ELPs containing photoleucine, a leucine analog and photoreactive diazirine crosslinker, which is substituted for leucine periodically throughout the ELP sequence. Upon exposure to ultraviolet radiation (~370 nm), photoleucine is able to form covalent crosslinks with amino acid side chains, forming a polymer network for hydrogel formation. Additionally, recombinant growth factors and morphogens can be encoded into the ELP sequence providing a simple method of hydrogel functionalization for regenerative medicine applications. The potential for this platform was demonstrated through in vivo crosslinking of photoreactive ELPs in the expression hosts. Though the production of the photoreactive ELP was not as forthright as originally assumed. The substitution of noncanonical amino acids typically requires the auxotrophic expression hosts to be starved of the amino acid that they are auxotrophic for. A noncanonical analog of said amino acid can then be supplemented into expression media, maximizing incorporation. In this investigation, it was found the addition of photoleucine alone inhibited photoreactive ELP expression. ELP expression only occurred in the presence of photoleucine if valine or leucine was also present in the media. Furthermore, valine was found to aid the production of ELPs as much as leucine. It was postulated the bacterial translational machinery might need to be altered for optimal ELP expression.

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.

There is a growing interest among researchers on the interaction of living systems and biomaterials from the perspective of advanced biomedical engineering applications. Nanomaterials are employed in different biological applications (biosensing, molecular imaging, delivering drug particles, anticancer therapy etc.) because of their noble properties. Recently, boron nitride (BN NP) have attracted significant interest due to its superior chemical, physical and thermal properties and have found practical applications in fields like industrial tool manufacturing, electrical devices, photocatalysis and lubrication. However, studies to assess boron nitride for biomedical applications have been largely limited. This project aims at evaluating BN NP as a potential tool for advanced bioengineering applications. The study is conducted by focusing on four key aspects: nanomaterial characteristics, biocompatibility, uptake process and effect on biophysical properties. Simultaneously, Hydroxyapatite (HAP) was also assessed as a point of reference. Both BN NP and HAP were characterised based on their size, shape, surface charge and porosity to quantify the physical parameters of materials that dictate cellular response to nano-sized materials. The cytotoxicity of BN NP was extensively studied by conducting a number of biological assays. Overall, BN NP was found to be biocompatible within certain concentration range (0-50 μg/ml). Once the biocompatibility of BN NP was established, focus was placed on studying the uptake process and adopted mechanism. Cells were sectioned into thin slides (80 nm) after being cultured with nanomaterials and later imaged using transmission electron microscopy (TEM). Nanomaterials were observed inside cell cytoplasm, which confirmed successful internalisation of BN NP by human cells. The uptake process was extensively studied by analysing the microscopic images in a time dependent manner. The uptake mechanism of both BN NP and HAP was observed to be endocytosis. Finally, the effect of nanomaterial uptake on the biophysical properties of cells was investigated. While assessing nanomaterials, previous studies were largely limited to biological assay. However, in this study, it was hypothesised that, apart from biological consequences, nanomaterials uptake will also affect the physical properties of cells. Robust and accurate experimental techniques were developed to quantify the cell stiffness and adhesion property using Atomic force microscopy (AFM). The obtained results revealed increase in cell stiffness for BN NP treated cells (50 and 100μg/ml) and a significant decrease in adhesion property for HAP treated cells (100μg/ml). Together, these results demonstrated the effect of nanomaterial uptake on biophysical properties of cells and explained the underlying mechanism. This was an innovative way of studying the physical wellbeing of cells, which also contributed in the existing knowledge of nanomaterial toxicity. In summary, BN NP was evaluated in this study through an organised approach considering a number of key aspects. Collectively, this research develops a better understanding of the interaction between BN NP and human cells in in vitro condition and establishes a primary framework for nanomaterial assessment for biomedical use. The results validate BN NP's potential as a suitable biomedical engineering tool and emphasises the need for more research efforts in this field.

Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials.

However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward.

This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.

The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models.

The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.

In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.

Dissertation

Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.

Dissertation

When a material’s size is reduced to the nanoscale, it is well understood that in addition to the dramatically increased surface area to volume ratio, new properties emerge distinct from those present in the bulk material. When graphite is exfoliated down to graphene, several altered and highly attractive properties emerge which may enable new photothermal roles as well as allowing significant improvements to existing strategies. One such nanosize tuning method that is highly suitable for layered materials is surfactant assisted liquid exfoliation of the bulk material to single and few layered sheets. Successfully isolated graphene presents a range of remarkable properties such as an unsurpassed thermal conductivity of ≈ 5000 W m-1 K-1,1-5 a very high mechanical strength with a Young’s modulus of ≈ 1 TPa,6-7 a broad optical absorptivity with a transmission of 97.7 % independent of wavelength in the visible spectrum (and a large portion of the infrared),8-9 as well as an ultrahigh electron mobility of ≈ 200,000 cm2 V-1 s-1.10-12 The development of photothermal agents for a range of biomedical applications is an area of huge interest and promise with high impact outcomes possible. In this study, single and few layer graphene has been explored for use as a photothermal agent for a range of biomedical roles such as thermal ablation of cancerous cells and photothermally controllable drug release. With this focus, several biomedical photothermal applications were explored including the thermal ablation of cancerous glioma-neuroblastoma cells through photothermal conversion at the target site by the graphene microplates. By exploiting the significant absorption of graphene in the near-infrared, substantial amounts of energy can be delivered deep within biological tissue allowing a highly-localized region of dramatic heating to be achieved resulting in tissue ablation and cell death. 7 This study also explores the use of graphene as a photothermal trigger to activate the controlled release of drug payloads in three different carrier systems. These systems include a graphene loaded (and stabilised) Pickering emulsion carrier with both oil in water and water in oil types possible. A photothermal coring of the emulsions was successfully achieved demonstrating the photothermally induced collapse of the emulsion. Several graphene entrained lipid nanocarrier systems were explored with near-infrared activation inducing phase transitions. Small angle X-ray scattering was used to dynamically monitor photo-activated, reversible phase transitions. An injectable hybrid graphene-surfactant-α-cyclodextrin thermoreversible gel system with graphene as an intrinsic component was also explored within this study. Photothermal drug release switching and controllable release rates were demonstrated with this biocompatible carrier system showing a highly versatile photothermally activatable drug depot. Graphene is a material well suited for photothermal biomedical applications particularly when prepared via liquid exfoliation. This study explores the interactions of specific light energies with surfactant assisted liquid exfoliated graphene for photothermal applications and shows that graphene has a high transduction efficiency, is thermally stable and is intrinsically suitable towards stealth strategies, suggesting that graphene could be a significant addition to a range of photothermal biomedical applications.

Yes
Electrospinning is a versatile technique that has gained popularity for various biomedical applications in recent years. Electrospinning is being used for fabricating nanofibers for various biomedical and dental applications such as tooth regeneration, wound healing and prevention of dental caries. Electrospun materials have the benefits of unique properties for instance, high surface area to volume ratio, enhanced cellular interactions, protein absorption to facilitate binding sites for cell receptors. Extensive research has been conducted to explore the potential of electrospun nanofibers for repair and regeneration of various dental and oral tissues including dental pulp, dentin, periodontal tissues, oral mucosa and skeletal tissues. However, there are a few limitations of electrospinning hindering the progress of these materials to practical or clinical applications. In terms of biomaterials aspects, the better understanding of controlled fabrication, properties and functioning of electrospun materials is required to overcome the limitations. More in vivo studies are definitely required to evaluate the biocompatibility of electrospun scaffolds. Furthermore, mechanical properties of such scaffolds should be enhanced so that they resist mechanical stresses during tissue regeneration applications. The objective of this article is to review the current progress of electrospun nanofibers for biomedical and dental applications. In addition, various aspects of electrospun materials in relation to potential dental applications have been discussed.

In this dissertation, molecular simulation techniques are used for the theoretical prediction of nanoscale properties for peptide-based materials. This work is focused on two particular systems: peptide nanotubes formed by cyclic-D,L peptide units and peptide nanotubes formed by phenylalanine dipeptides [-Phe-Phe-]. Mechanical characterization of cyclic peptide nanotubes is a challenging problem due the anisotropy resulting from the nature of their molecular interactions. To address rigorously the thermo-mechanical stability of cyclic peptide nanotubes (CPNTs), a homogeneous deformation method combined with the generalized elasticity theory and molecular dynamics simulations (MD) were used for the calculation of second order anisotropic elastic constants. The results for anisotropic elastic constants, yield behavior and engineering Young’s modulus show remarkable mechanical stability for these materials supporting experiments for the development of their applications. Furthermore, the heat capacity, thermal expansion coefficient and isothermal compressibility were predicted using numerical difference methods and molecular dynamics. In order to understand the transport properties of confined water in cyclic peptide nanotubes, the influence of nanotube diameter was studied and self-diffusion coefficient, dipole correlation functions and hydrogen bond probabilities were calculated via molecular dynamics and statistical mechanics. Enhanced transport and higher diffusion rates for water were obtained in cyclic peptide nanotubes (CPNTs) compared with commonly used biomedical channels like carbon nanotubes (CNTs). The greater transport efficiency in CPNTs is attributed to the hydrophilic character and high hydrogen bonding presence along their tubular structure, versus the hydrophobic core of CNTs. One of the most important opportunities for cyclic peptide nanotubes is their utilization as artificial ion channels in antibacterial applications. Here, molecular dynamics methods were used to investigate the effect of confinement on the transport properties of Na+ and K+ ions under the influence of electric field; the ion mobility, selectivity, radial distribution function, coordination number and effect of temperature were studied and results from simulations proved their ability to transport ions. Additionally, the molecular organization of phenylalanine dipeptides into ordered peptide nanotubes was investigated, a model for the molecular structure of these nanotubes was proposed and optimized through molecular simulations; a helical pattern was found and characterized. Thermal stability results show that phenylalanine dipeptide nanotubes are stable up to about 400K; above this temperature, a significant decrease in hydrogen bonding was observed and the perfect pattern was altered. Findings from this work open new opportunities for research in the area of peptide based materials and provide tools and methods to study these systems efficiently at nanoscale.

abstract: Biogenic silica nanostructures, derived from diatoms, possess highly ordered porous hierarchical nanostructures and afford flexibility in design in large part due to the availability of a great variety of shapes, sizes, and symmetries. These advantages have been exploited for study of transport phenomena of ions and molecules towards the goal of developing ultrasensitive and selective filters and biosensors. Diatom frustules give researchers many inspiration and ideas for the design and production of novel nanostructured materials. In this doctoral research will focus on the following three aspects of biogenic silica: 1) Using diatom frustule as protein sensor. 2) Using diatom nanostructures as template to fabricate nano metal materials. 3) Using diatom nanostructures to fabricate hybrid platform. Nanoscale confinement biogenetic silica template-based electrical biosensor assay offers the user the ability to detect and quantify the biomolecules. Diatoms have been demonstrated as part of a sensor. The sensor works on the principle of electrochemical impedance spectroscopy. When specific protein biomarkers from a test sample bind to corresponding antibodies conjugated to the surface of the gold surface at the base of each nanowell, a perturbation of electrical double layer occurs resulting in a change in the impedance. Diatoms are also a new source of inspiration for the design and fabrication of nanostructured materials. Template-directed deposition within cylindrical nanopores of a porous membrane represents an attractive and reproducible approach for preparing metal nanopatterns or nanorods of a variety of aspect ratios. The nanopatterns fabricated from diatom have the potential of the metal-enhanced fluorescence to detect dye-conjugated molecules. Another approach presents a platform integrating biogenic silica nanostructures with micromachined silicon substrates in a micro/nano hybrid device. In this study, one can take advantages of the unique properties of a marine diatom that exhibits nanopores on the order of 40 nm in diameter and a hierarchical structure. This device can be used to several applications, such as nano particles separation and detection. This platform is also a good substrate to study cell growth that one can observe the reaction of cell growing on the nanostructure of frustule.
Dissertation/Thesis
Doctoral Dissertation Materials Science and Engineering 2014

伴隨著納米科技的發展，越來越多的納米顆粒系統已經應用於生物醫學领域。其中，二氧化硅納米顆粒因其簡單易操作的表面化學性質和在生理環境中的良好穩定性，已被廣泛認為是最有前途的治療和診斷載體之一。
在本論文中，我首先對二氧化硅納米顆粒與人類細胞之間的作用進行了系統研究。這些作用包括了以下主要特點: 胞吞和胞吐被分別確定為納米顆粒主要的進入和離開細胞的主要途徑; 大部份的納米顆粒被發現存在於有膜結構的細胞器里，這些細胞器相當穩定（不易破損），只有很少的一部份納米顆粒被釋放到細胞質里; 納米顆粒和細胞之間的作用是動態的，它們進入細胞內的數量由其在細胞培養液中的數量和形態（聚集的程度）所決定。正是這些特點決定了二氧化硅顆粒在低濃度時的低細胞毒性。
緊接著我比較了兩種最常見的二氧化硅納米顆粒（晶體態和無定型態）引入細胞后對細胞所帶來的影響。儘管兩種形態的納米顆粒所造成的細胞毒性都比較低，但是更細緻的分析揭示了它們對細胞及其衍化途徑的不同影響。細胞吞入晶體態的二氧化硅納米顆粒后，其內部的活性氧物質含量顯著提高，這種變化會導致細胞線粒體功能受損（表現為線粒體增生）並且最終將細胞導向死亡。不過只有在p53基因缺失的細胞中才有這種由活性氧物質水平升高導致的細胞損傷，p53正常的細胞卻能抵禦這種來自晶體態二氧化硅納米顆粒的刺激。而無定型態二氧化硅納米顆粒對生物系統無損害，因而有發展為藥物載體的巨大潛力。
基於對二氧化硅顆粒細胞毒性研究的理解，我們設計了一種新型納米載體--金核/二氧化硅殼層（Au@SiO₂）納米顆粒用於藥物輸運。在這一體系中，無定形態二氧化硅和金納米顆粒的優勢被整合在一起，同時光敏劑（PS）藥物分子被裝載在二氧化硅殼層內。對比於自由形式的PS，裝載在Au@SiO₂納米顆粒中的PS展示出增強的藥效。需要強調的是，用這種納米顆粒處理的細胞以阻梗壞死為主要的死亡途徑，代替了凋亡這種不太有效的方式。在光照下，金的等離子體效應被發現能促進PS的光響應過程，這使得細胞殺死率得到了大幅度增強。這一效應得益于我們把PS束縛在金核的表面，同時保證金表面等離子體振盪能量和PS吸收能量的配對。此外，把PS裝載在二氧化硅中會引起PS有益的光化學改變。這些作用結合在一起導致了藥效的提高。這些機理能被普遍應用於納米顆粒裝載藥物分子的設計中，為最優化設計提供指導。
With recent development of nanotechnology, various nanoparticulate systems have been proposed to serve as functional units for biomedical applications in many innovative ways. Among various possible choices, silica nanoparticles (NPs) enjoys easily modifiable surface chemical characteristics and excellent stability in physiological environment. Therefore, it is considered as one of the most promising carrier candidate for therapeutic and diagnostic applications.
A systematic study on the interaction between silica nanoparticles and human cells is first carried out in the present thesis work. Endocytosis and exocytosis are identified as major pathways for NPs entering, and exiting the cells, respectively. Most of the NPs are found to be enclosed in membrane bounded organelles, which are fairly stable (against rupture) as very few NPs are released into the cytoplasma. The nanoparticle-cell interaction is a dynamic process, and the amount of NPs inside the cells is affected by both the amount and morphology (degree of aggregation) of NPs in the medium. These interaction characteristics determine the low cytotoxicity of SiO₂ NPs at low feeding concentration.
Experiments were then designed to compare the the biological consequence of two most common form of SiO₂ nanoparticles, i.e., crystalline and amorphous NPs, when they were introduced to human cells. Although the apparent cytotoxicity of both types of NPs seems to be low, more detailed characterizations disclose the profound difference induced by the crystalline and amorphous ones, resulting in significantly different cell evolution pathways. Crystalline NPs but not amorphous ones are found to drastically increase the recative oxygen species (ROS) level in the cells, which can cause mitochondria dysfunction (being expressed as mitochondria proliferation), and eventually direct the cell into apoptosis. Nonetheless, only p53 deficient cells are subjective to such ROS induced cell damage, while p53 proficient cells can accommodate the stimulation from crystalline SiO₂ NPs. The amorphous SiO₂ NPs are found to be benign in the biological systems, and have great potential to be developed as nanomedicine.
Base on the understanding obtained from the toxicology study of the SiO₂ NPs, we have designed a special nanocarrier system for drug delivery. We have combined advantages of both SiO₂ and Au NPs by constructing Au-core/SiO₂-shell (Au@SiO₂) nanocarriers with the photosensitizer (PS) drug embedded in the SiO₂ shell layer. Compared with free PS, PS loading in the Au@SiO₂ NPs showes a enhanced drug efficacy. In particular, the cells treated with the NP drug take necrosis as a major death path instead of apoptosis, which is a much less effective route. The Au plasmonic effect is found to promote the photo-response of the PS drug under light irradiation, contributing to the largely decreased cell viability. Nevertheless, one shall note that spatial confinement of the drug moledules to the close proximity of the Au core and an energy match between the drug absorption and the Au surface plasmon resonance are critical in manifesting the plasmonic effect. At the same time, embedding the drug in the SiO₂ matrix leads to favorable change in the photochemical process. The combined effects brought by the Au@ SiO₂ NP carrier is responsible for the high drug efficacy. These mechanisms can be generally valid in engineering drug molecule incorporation into NP carriers and also give guidance for the optimum design of the NP drug carrier.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chu, Zhiqin = 二氧化硅納米顆粒與人類細胞的作用及其在生物醫學方面的應用 / 褚智勤.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 120-137).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Chu, Zhiqin = Er yang hua gui na mi ke li yu ren lei xi bao de zuo yong ji qi zai sheng wu yi xue fang mian de ying yong / Chu Zhiqin.
Table of contents --- p.VIII
List of figures --- p.XIII
List of tables --- p.XIX
Chapter Chapter 1 --- Introduction --- p.1
Chapter Chapter 2 --- Background --- p.4
Chapter 2.1 --- Overview of the silica-based nanoparticles for bio-medical applications --- p.4
Chapter 2.2 --- Health issue on the silica-base nanoparticles --- p.5
Chapter 2.3 --- Understanding the nano-bio interface --- p.6
Chapter 2.3.1 --- Nano-bio interface in vitro --- p.7
Chapter 2.3.2 --- Nano-bio interface in vivo --- p.10
Chapter 2.4 --- Bio-application of silica-based nanoparticles --- p.11
Chapter 2.4.1 --- Use of silica nanoparticle as imaging agent --- p.11
Chapter 2.4.2 --- Use of silica nanoparticle as drug carrier --- p.12
Chapter 2.4.3 --- Use of silica nanoparticle as coating media --- p.12
Chapter 2.5 --- Surface plasmon of gold nanostructures and its bio-application --- p.13
Chapter 2.5.1 --- Introduction to the SPR of gold nanostructures --- p.13
Chapter 2.5.2 --- Synthesis of gold NRs and their SPR effect --- p.13
Chapter 2.5.3 --- SPR of gold NRs in bio-application --- p.16
Chapter Chapter 3 --- Experimental --- p.18
Chapter 3.1 --- Standard methodologies for nanoparticle preparation and their feeding to the cells --- p.18
Chapter 3.2 --- Cell sampling for room temperature TEM study --- p.18
Chapter 3.3 --- Developing methods to distinguish NPs in cell sample under TEM --- p.20
Chapter 3.4 --- Confocal microscopy study --- p.21
Chapter 3.4.1 --- Study the photoluminescence of various dye molecules --- p.21
Chapter 3.4.2 --- Study the two photon luminescence (TPL) of Au NRs --- p.23
Chapter 3.5 --- UV-Vis-NIR spectrophotometer and fluorescence spectrophotometer --- p.25
Chapter 3.6 --- Flow-cytometry --- p.26
Chapter 3.7 --- Western plot --- p.28
Chapter 3.8 --- Colormetric assays and other biological labels --- p.28
Chapter 3.8.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test --- p.28
Chapter 3.8.2 --- Mitochondria, lysosome and nucleus staining --- p.30
Chapter 3.8.3 --- Detection of apoptosis --- p.31
Chapter 3.8.4 --- Detection of various reactive oxygen species (ROS) --- p.31
Chapter Chapter 4 --- Silica NPs interact with human cells --- p.34
Chapter 4.1 --- Introduction --- p.34
Chapter 4.2 --- Characterization of silica nanoparticles --- p.36
Chapter 4.3 --- General description of the NPs’ uptaking and excreting process --- p.42
Chapter 4.4 --- Tracking of NPs inside the cells --- p.53
Chapter 4.5 --- Factors influencing the NP-cell interaction and exocytosis process --- p.55
Chapter 4.5.1 --- The effect of serum (in the incubation medium) on cellular uptake --- p.55
Chapter 4.5.2 --- Crystallinity effectdistribution of amorphous and crystalline SiO₂ NPs in the cells --- p.57
Chapter 4.5.3 --- Factors affecting the exocytosis process --- p.59
Chapter 4.6 --- Cytotoxic effect of silica NPs --- p.60
Chapter 4.7 --- Conclusion --- p.63
Chapter Chapter 5 --- Genotoxic effect specifically induced by crystalline SiO₂ nanoparticles in p-53 deficient human cells --- p.65
Chapter 5.1 --- Introduction --- p.65
Chapter 5.2 --- The difference between crystalline and amorphous silica NPs --- p.66
Chapter 5.2.1 --- Mitochondria multiplication specially induced by crystalline silica NPs --- p.68
Chapter 5.2.2 --- DNA fragmentation specially observed in crystalline silica NPs treated cells --- p.71
Chapter 5.3 --- The cell line sensitive cytotoxicity of crystalline silica NPs --- p.79
Chapter 5.3.1 --- A general phenomenon of mitochondria increase in p-53 negative cell lines --- p.80
Chapter 5.3.2 --- General biological consequence of such mitochondria increase --- p.82
Chapter 5.4 --- Conclusion --- p.83
Chapter Chapter 6 --- Surface plasmon enhanced drug efficacy for PDT using core shell Au@SiO₂ nanoparticle carrier --- p.84
Chapter 6.1 --- Introduction --- p.84
Chapter 6.1.1 --- Brief introduction to the photodynamic therapy (PDT) and photosensitizer (PS) --- p.84
Chapter 6.1.2 --- Brief introduction to the SPR enhanced generation of ROS --- p.86
Chapter 6.2 --- Using Au@SiO₂ NPs as drug carrier --- p.88
Chapter 6.2.1 --- Growth of gold NRs and their controllable oxidation --- p.88
Chapter 6.2.2 --- Preparation and characterization of Au@(SiO₂-MB) NPs --- p.90
Chapter 6.2.3 --- Confirmation of MB loading into silica shell --- p.92
Chapter 6.3 --- Enhanced PDT drug (MB) efficacy when loaded in Au@SiO₂ NPs --- p.95
Chapter 6.3.1 --- Cellular uptake pathway of free MB and Au@SiO₂ NPs --- p.95
Chapter 6.3.2 --- Comparing the efficacy of free MB, SiO₂-MB NPs and Au@(SiO₂MB) NPs --- p.98
Chapter 6.4 --- Studying the behavior of free MB and Au@(SiO₂-MB) NPs as PDT agent --- p.100
Chapter 6.4.1 --- Comparing the ability of generating ROS by free MB and Au@(SiO₂MB) NPs --- p.100
Chapter 6.4.2. --- Comparing the types of ROS generated by free MB and Au@(SiO₂MB) NPs --- p.103
Chapter 6.4.3 --- Comparing the cellular death pathway triggered by free MB and Au@(SiO₂-MB) NPs --- p.105
Chapter 6.5. --- Discussion on the mechanism for the enhanced efficacy --- p.109
Chapter 6.5.1 --- Excluding the photothermal effect of Au NRs core --- p.109
Chapter 6.5.2 --- The role of SiO₂ in the Au@SiO₂ NPs carrier --- p.111
Chapter 6.5.3 --- Attributing the enhanced efficacy to plasmonic effect of Au NRs core --- p.112
Chapter 6.6 --- Exploring the potential of using Au@SiO₂ NP carrier in vivo --- p.114
Chapter 6.7 --- Conclusion --- p.116
Chapter Chapter 7 --- Conclusion --- p.118
References --- p.120

Superparamagnetic iron oxide (Fe3O4 and γ-Fe2O3) nanoparticles (IONPs) are of great in-terest as a diagnostic and/or therapeutic aid. Several IONPs with biocompatible polymer coatings have been approved for clinical use, as MRI contrast agents. IONPs conjugated to targeting ligands and therapeutic agents are being investigated for targeted drug deliv-ery applications. The superparamagnetic properties of IONPs are also helpful for magnetic field assisted localization to specific target sites and for in situ MRI applications. This thesis primarily focuses on the synthesis and surface modifications (with biocompatible polymers including dextran, poly(ethylene glycol) (PEG), dextran, poly(ethyl methacry-late) (PEMA), poly(hydroxyethyl methacrylate) (PHEMA), etc.) of IONPs. The IONPs were prepared following the classical co-precipitation method and a novel reduction-hydrolysis method. Initial studies used bovine serum albumin (BSA) to examine the ca-pabilities of polymer coated IONP to deliver a model protein therapeutic. Gel migration studies using BSA physisorbed onto polymer coated IONP under gradient magnetic field of an MRI showed that the IONPs had limited control in transporting the protein. Cova-lent linking of therapeutics to IONP core can improve the time window of formers con-trollability using magnetic field. To facilitate covalent conjugations, functional silane coated IONPs (with surface amino and carboxylic acid) were prepared as general precur-sors. The utility of silane coated IONPs for bioconjugations was demonstrated by cova-lently linking PEG diacid through surface amino groups and by linking of BSA through surface carboxylic acid groups. The biocompatibility of the IONPs synthesized following the novel reduction-hydrolysis method were assessed in vitro on cell culture models using toxicity assays. The versatile reduction-hydrolysis method was further extended, as a general method to prepare several early transition metal oxide NPs (manganese oxide (Mn3O4), cobalt oxide (Co3O4), nickel/nickel oxide (Ni/NiO), copper/copper oxide (Cu/Cu2O) and zinc oxide (ZnO) NPs), silica nanoparticles with surface IONPs, and iron/iron oxide nanosheets.

Materials science today is a multidisciplinary effort comprising an accelerated convergence of diverse fields spanning the physical, applied, and engineering sciences. This diversity promises to deliver the next generation of advanced functional materials for a wide range of specific applications. In particular, the past decade has seen a growing interest in the development of nanoscale materials for sophisticated technologies. Aqueous colloidal microgels have emerged as a promising class of soft materials for multiple biotechnology applications. The amalgamation of physical, chemical and mechanical properties of microgels with optical properties of nanostructures in hybrid composite particles further enhances the capabilities of these materials. This work covers the general areas of responsive polymer microgels and their composites, and encompasses methods of fabricating microgel-based drug delivery systems for controlled and targeted therapeutic applications. The first part of this thesis is devoted to acquainting the reader with the fundamental aspects of the synthesis, functionalization and characteristic properties of stimulus-responsive microgels constructed from poly(N-isopropylacrylamide) (poly(NIPAm)) and other functional comonomers. In particular, the role of electrostatics on the swelling-deswelling transitions of polyampholyte microgels upon exposure to a range of environmental stimuli including pH, temperature, and salt concentration are discussed. The templated synthesis of bimetallic gold and silver nanoparticles in zwitterionic microgels is also described. The latter part of this thesis focuses on the rational development of microgel-based drug delivery systems for controlled and targeted drug release. Specifically, the development of a biofunctionalized, pH-responsive drug delivery system (DDS) is illustrated, and shown to effectively suppress cancer cells when loaded with an anticancer agent. In another chapter, the design of tailored hybrid particles that combine the thermal response of microgels with the light-sensitive properties of gold nanorods to create a DDS for photothermally-induced drug release is discussed. The photothermally-triggered volume transitions of hybrid microgels under physiological conditions are reported, and their suitability for the said application evaluated. In another component of this work, it is explicitly shown that electrostatic interactions were not needed to deposit gold nanorods on poly(NIPAm)-derived particles, thereby eliminating the need for incorporation of charged functional groups in the microgels that are otherwise responsible for large, undesirable shifts and broadening of the phase transition.

Over the past few decades, inorganic nanostructured materials have elicited a lot of interest due to their high surface-to-volume ratio and many size dependent properties which stem from their nanoscale dimensions. Owing to these distinct properties, they have found applications in widespread fields like catalysis, energy storage, electronics, and biotechnology. In the field of biotechnology, nanotubes and mesoporous materials are attractive vehicles for drug delivery because of their hollow and porous structures and facile surface functionalization. Their inner void can take up large amounts of drug as well as act as gates for the controlled release of drug. These hollow structures can also be used for confining biomolecules like proteins and peptides. The study on protein conformation in biocompatible materials is very important in materials sciences for the development of new and efficient biomaterials(sensors, drug delivery systems or planted devices). Titania(TiO2)has been widely explored for applications in photovoltaic cells, batteries, desalination, sensing, and photocatalysis, to name only a few. The work presented in this thesis focuses on titania based nanostructures for drug delivery and protein confinement. First part of the work focusses on synthesis and characterization of Fe-doped TiO2 nanotubes. Fe-doped TiO2 nanotubes were demonstrated as controlled drug delivery agents. In vitro cytotoxic effects of Fe-doped titania nanotubes were assessed by MTT assay by exposing Hela cell line to the nanotubes. Second part of the work focusses on synthesis and characterization of TiO2 nanotubes by two synthesis procedures, namely hydrothermal and sol-gel template synthesis. Myoglobin, a model globin protein was encapsulated in hydrothermally synthesized TiO 2 nanotubes(diameter 5 nm) and sol-gel template synthesized TiO2 nanotubes(diameter 200 nm). Effect of encapsulating myoglobin these nanotubes was studied. The electrochemical activity and structure of myoglobin were studied by cyclic voltammetry and circular dichroism respectively. Direct electron transfer was found to be enhanced upon confinement in 200 nm diameter nanotubes. No such enhancement was observed upon encapsulation in hydrothermally synthesized nanotubes. In addition to this, the thermal stability of myoglobin was found to be enhanced upon confinement inside 200 nm diameter TiO 2 nanotubes.

Over the past few decades, inorganic nanostructured materials have elicited a lot of interest due to their high surface-to-volume ratio and many size dependent properties which stem from their nanoscale dimensions. Owing to these distinct properties, they have found applications in widespread fields like catalysis, energy storage, electronics, and biotechnology. In the field of biotechnology, nanotubes and mesoporous materials are attractive vehicles for drug delivery because of their hollow and porous structures and facile surface functionalization. Their inner void can take up large amounts of drug as well as act as gates for the controlled release of drug. These hollow structures can also be used for confining biomolecules like proteins and peptides. The study on protein conformation in biocompatible materials is very important in materials sciences for the development of new and efficient biomaterials(sensors, drug delivery systems or planted devices). Titania(TiO2)has been widely explored for applications in photovoltaic cells, batteries, desalination, sensing, and photocatalysis, to name only a few. The work presented in this thesis focuses on titania based nanostructures for drug delivery and protein confinement. First part of the work focusses on synthesis and characterization of Fe-doped TiO2 nanotubes. Fe-doped TiO2 nanotubes were demonstrated as controlled drug delivery agents. In vitro cytotoxic effects of Fe-doped titania nanotubes were assessed by MTT assay by exposing Hela cell line to the nanotubes. Second part of the work focusses on synthesis and characterization of TiO2 nanotubes by two synthesis procedures, namely hydrothermal and sol-gel template synthesis. Myoglobin, a model globin protein was encapsulated in hydrothermally synthesized TiO 2 nanotubes(diameter 5 nm) and sol-gel template synthesized TiO2 nanotubes(diameter 200 nm). Effect of encapsulating myoglobin these nanotubes was studied. The electrochemical activity and structure of myoglobin were studied by cyclic voltammetry and circular dichroism respectively. Direct electron transfer was found to be enhanced upon confinement in 200 nm diameter nanotubes. No such enhancement was observed upon encapsulation in hydrothermally synthesized nanotubes. In addition to this, the thermal stability of myoglobin was found to be enhanced upon confinement inside 200 nm diameter TiO 2 nanotubes.