En esta Tesis Doctoral se propuso la síntesis de hidrogeles termo-sensibles para aplicaciones biomédicas a partir de copolímeros de alginato y N-isopropilacrilamida (NIPAAm). Los copolímeros de injerto se obtuvieron empleando tres métodos de síntesis: uno basado en la generación de los sitios reactivos por medio de un iniciador redox convencional (nitrato de amonio y cerio(IV), CAN), el otro mediante radiaciones ionizantes de alta energía, mientras que el tercer método se realizó en dos etapas consecutivas, obteniendo primero polímeros telequélicos amino-terminados los cuales se unieron luego a la cadena del alginato por medio de una reacción de condensación. Los copolímeros obtenidos fueron caracterizados química y térmicamente empleando distintas técnicas analíticas tales como Resonancia Magnética Nuclear de protón (1H-NMR), Espectroscopía Infrarroja con Transformada de Fourier (FTIR), análisis termogravimétrico (TGA), reología y mediciones de punto de nube (CP). Los copolímeros de injerto obtenidos mediante irradiación con rayos gamma de soluciones acuosas de alginato y NIPAAm presentaron buenas propiedades físicas, tal como solubilidad y termosensibilidad. La composición de los copolímeros resultó ser función de la dosis de radiación y la composición de alginato y NIPAAm en la mezcla inicial de reacción. De las otras dos vías de síntesis química utilizadas, resultó mejor aquella basada en la obtención de PNIPAAm amino terminado (PNIPAAm-NH2), y su posterior injerto en la cadena de alginato mediante la reacción de condensación entre los grupos amino del PNIPAAm y los grupos carboxílicos del alginato, ya que se obtuvieron copolímeros con estructura química controlada, con cadenas laterales de masas molares conocidas y una composición definida por la mezcla de reacción. Los hidrogeles empleados en los ensayos de hinchamiento se prepararon en forma de perlas, por medio del goteo de soluciones de copolímeros sobre una solución de cloruro de calcio. El grado de hinchamiento de los hidrogeles obtenidos a partir de los copolímeros sintetizados por irradiación presentó una dependencia con la temperatura y el contenido de PNIPAAm. Por otra parte, si bien los copolímeros obtenidos a partir del injerto de PNIPAAm-NH2 sobre el alginato presentaron termosensibilidad en solución, tal como se pudo observar en los ensayos de reología, los hidrogeles obtenidos a partir de estos copolímeros no mostraron luego este comportamiento, debido probablemente al menor contenido de PNIPAAm una vez obtenido el hidrogel hinchado. iii Para llevar a cabo ensayos de liberación se seleccionaron como matrices portadoras del fármaco modelo a aquellos hidrogeles más promisorios de acuerdo a los resultados de hinchamiento obtenidos previamente. Estudios preliminares de liberación de atenolol empleando hidrogeles termosensibles, preparados a partir de los copolímeros de injerto obtenidos por irradiación, mostraron una liberación de aproximadamente un 70 % del contenido inicial del fármaco en el hidrogel en la primer hora de ensayo a 37 °C. / In this Doctoral Thesis the synthesis of thermo-responsive hydrogels for biomedical application obtained from alginate and N-isopropylacrylamide (NIPAAm) graft copolymers has been proposed. In order to obtain the copolymers three methods of graft copolymerization have been studied: gamma-ray irradiation and two different methods of conventional grafting reactions using chemical initiator agents. Obtained graft copolymers were chemically and thermally characterized by H-Nuclear Magnetic Resonance (1H-NMR), Fourier Transform Infrared Spectroscopy (FTIR), Thermal Gravimetric Analysis (TGA), rheology, and cloud point measurements (CP). Graft copolymers obtained by gamma-ray irradiation of aqueous solutions of alginate and NIPAAm presented good physical properties such as solubility and thermosensibility. Copolymers composition is a function of radiation dose and alginate and NIPAAm composition in the initial aqueous solution. Only graft copolymers obtained from amino-terminated PNIPAAm, PNIPAAm-NH2 prepared by a redox initiator, and grafted to alginate by a condensation reaction between amino groups of PNIPAAm-NH2 and carboxylic groups of alginate, gave copolymers with a know chemical structure with side chains of defined molar mass defined and a composition given by the reaction mixture. Swelling degree studies were performed on hydrogels obtained by dropping an aqueous solution of the copolymer onto a solution of calcium chloride. Swelling degree of the hydrogels, prepared by using the copolymers synthesized by gamma irradiation, was a function of both temperature and NIPAAm content. On the other hand, even when copolymers obtained by grafting PNIPAAm-NH2 onto alginate showed an observable thermosensibility in solution, hydrogels prepared with those graft copolymers did not behave in the same way, probably due to the low PNIPAAm content in the swollen hydrogel. Controlled release studies were carried out on those hydrogels that presented thermosensitivity in the swelling study. Preliminary results on athenolol release, using the thermosensitivity hydrogels obtained from the graft copolymers synthesized by gamma radiation, gave a 70 % recovery of initial drug load after one hour at 37 ºC.

There are two primary objectives of this dissertation research. The first objective was to prepare a library of inorganic-organic hydrogels from methacrylated star polydimethylsiloxane (PDMSstar-MA) and diacrylated poly(ethylene oxide) (PEO-DA) with tunable chemical and physical properties for use as tissue engineering scaffolds. These inorganic-organic hydrogels provide a useful platform to study the effect of scaffold properties on cell behavior in tissue culture. Twenty compositionally unique hydrogels were prepared by photo-crosslinking varing molecular weights (Mn) of PEO-DA (Mn = 3.4k and 6k g/mol) and PDMSstar-MA (Mn = 1.8k, 5k and 7k g/mol) at varying weight ratios (up to 20 wt% PDMSstar-MA). Introduction of PDMSstar-MA caused formation of discrete PDMS-enriched "microparticles" dispersed within the PEO hydrogel matrix. The swelling ratio, mechanical properties in tension and compression, non-specific protein adhesion and cytotoxicity of hydrogels were studied. The second objective was to prepare thermoresponsive nanocomposite hydrogels, which are mechanically robust and can remove adhered cells via thermal modulation. Such hydrogels may be useful as "self-cleaning" membranes for implanted biosensors to extend their lifetime and efficiency. These hydrogels are comprised of a poly(Nisopropylacrylamide) (PNIPAAm) hydrogel matrix and polysiloxane colloidal nanoparticles (~220 nm and 50 nm ave. diameter). Due to the low preparation temperature, the nanocomposite hydrogels exhibited a homogeneous morphology by SEM analysis. The volume phase transition temperature (VPTT, ~33 degrees C) of the nanocomposite hydrogels was not altered versus the pure PNIPAAm hydrogel, which is near body temperature. Generally, nanoparticles led to improve mechanical properties versus pure PNIPAAm hydrogels. When these nanocomposite hydrogels are heated above the VPTT, they become more hydrophobic. When they are reversibly switched from a water-swollen to a deswollen state, the change in surface properties, as well as swelling-deswelling, was effective upon the removal of adhered cells.

The overall thrust of this dissertation is to gain a fundamental understanding of the synergistic effects between surface topography and chemical functionality of poorly adhesive materials on enhancing the adhesion of mouse embryonic fibroblasts. Cellular response to surface topography and chemical functionality have been extensively studied on their own providing valuable information that helps in the design of new and improved biomaterials for tissue engineering applications. However, there is a lack of understanding of the synergistic effect of microscale and nanoscale topography with chemical functionality and the relative impact and contribution of each in modulating cellular behavior. By understanding the relationship between these cues, in particular using materials that are poorly adhesive, this study will provide new clues as to how cells adapt to their environment and also suggest new dimensions of biomaterial design for fine-tuning cellular control. A microstructure that combined non adhesive materials with defined surface topography and surface chemistry is presented, to assess and correlate the enhancement of mouse embryonic fibroblasts cell adhesion and spreading. Poly (N-isopropylacrylamide) or PNIPAAm electrospun fibers were overlaid on PNIPAAm thin films (100 nm) at various time points to investigate the role of topography on such coatings by keeping the chemical functionality the same. After doing this, several topographical patterns were developed, spanning from sparse to dense fiber mats, and cell adhesion strongly depended on the relative available areas for attachment on either the fibers or the supporting surface. To gain a better understanding of this finding, two surface chemistries, non-adhesive (self-assembled monolayer of polyethylene glycol (PEGSAM) alkanethiol on gold) or an adhesive coating (3-aminopropyltriethoxysilane (APTES) on glass) with well characterized adhesive properties were included in this study to assess the effect of topographical cues provided by the PNIPAAm electrospun fibers on cellular responses. With the deposition of the PNIPAAm fibers onto a PEGSAM surface, cell adhesion increased to almost 100%, and unlike the PNIPAAm surface, cell spreading was significantly enhanced. With the deposition of PNIPAAm fibers onto APTES, both cell adhesion and spreading were unaffected up to 60% fiber coverage. For both surfaces, PNIPAAm fiber densities above 60% coverage lead to adhesion and spreading independent of the underlying surface. These findings indicate the presence of a sparse topographical feature can stimulate cell adhesion on a typically non-adhesive material, and that a chemical dissimilarity between the topographic features and the background enhances this effect through greater cell-surface interaction. In addition to the aforementioned studies, cell response was also assessed on PNIPAAm thin films coatings with thicknesses ranging from 100 nm to 7 nm. Cell adhesion and spreading was enhanced as the thickness of the thin film decreased. This change was more noticeable below 30 nm, wherein 7 nm shows the highest cell adhesion and spreading enhancement. The results reported are preliminary results and further experiments will be conducted, to support the data. It is believed that cellular response was enhanced due to a change in surface topography at the nanoscale level.

Poly(N-isopropylacrylamide) (PNIPAM), a temperature responsive polymer, undergoes a phase change at a lower critical solution temperature (LCST) in aqueous solutions. For PNIPAM this temperature is 32 °C in water. Below the LCST, the polymer is readily solvated by water. As the temperature of the solution increases, the polymer undergoes a phase transition so that above the LCST it is no longer water soluble. The LCST of PNIPAM may be changed by the addition of salt solutions from the Hofmeister series which will follow the Hofmeister effect for salting-in and salting-out the polymer. Temperature responsive polymers may be grafted to a surface in a variety of methods to create responsive thin films that exhibit a change in wettability. The surface wettability is directly related to the polymers ability to be solvated in its coil conformation. When PNIPAM is grafted to a surface, the surface becomes alternatively hydrophobic and hydrophilic in response to both temperature and the anions in the Hofmeister series which take the surface either above or below the LCST of PNIPAM. The synthesis of responsive nanocomposite grafts was successfully applied to glass slides and three-dimensional surfaces, porous glass frits which were capable of controlling the passive flow rate. The nanocomposite graft was assembled in a covalent layer-by-layer approach to create more chemically robust surfaces, and also to incorporate nanoparticles into the graft for increased surface roughness and therefore improve wettability response. Because of a much greater inherent roughness to a glass frit, characterization of the polymers and nanoparticles was performed before they were covalently bound to the surface. The final product, a functionalized frit with a PNIPAM/SiO2 nanocomposite graft, was analyzed by observing changes in the passive permeation rate of the frit between water and salt solutions. These changes in flow were indicative of the surface bound PNIPAM changing between its hydrophilic and hydrophobic conformation in response to water and concentrations of kosmotropic salts such as sodium sulfate and sodium citrate. In addition to the solute response, the frit was also determined to be responsive to temperature and concentration. Water exhibited a passive flow rate 1000 times faster than a kosmotropic salt but had a similar flow rate to that a chaotropic salt. By measuring the flow rate of 0.5 M Na2SO4 at ~7 °C in a cold room and at room temperature it was observed that sodium sulfate in the cold room passed through the frit at a rate 100 times faster than at room temperature. Because of the hysteresis of PNIPAM documented in literature, washing procedures were kept consistent between experiments to achieve more reproducible results. It was concluded that the frits were temperature responsive and had relative standard deviations below 25 percent for flow rates on a single frit. However, standard deviations of flow rates between frits were higher. This was likely due to a combination of factors, such as the frits’ pore size range of 10 μm resulting in the possibility of varied degrees of functionalization of each frit.

abstract: Solar energy is leading in renewable energy sources and the aspects surrounding the efforts to harvest light are gaining importance. One such aspect is increasing the light absorption, where heliotropism comes into play. Heliotropism, the ability to track the sun across the sky, can be integrated with solar cells for more efficient photon collection and other optoelectronic systems. Inspired by plants, which optimize incident sunlight in nature, several researchers have made artificial heliotropic and phototropic systems. This project aims to design, synthesize and characterize a material system and evaluate its application in a phototropic system. A gold nanoparticle (Au NP) incorporated poly(N-isopropylacrylamide) (PNIPAAm) hydrogel was synthesized as a photo-thermo-responsive material in our phototropic system. The Au NPs generate heat from the incident via plasmonic resonance to induce a volume phase change of the thermo-responsive hydrogel PNIPAAm. PNIPAAm shrinks or swells at temperature above or below 32°C. Upon irradiation, the Au NP-PNIPAAm micropillar actuates, specifically bending toward the incident light and precisely following the varying incident angle. Swelling ratio tests, bending angle tests with a static incident light and bending tests with varying angles were carried out on hydrogel samples with varying Au NP concentrations. Swelling ratios ranging from 1.45 to 2.9 were recorded for pure hydrogel samples and samples with very low Au NP concentrations. Swelling ratios of 2.41 and 3.37 were calculated for samples with low and high concentrations of Au NPs, respectively. A bending of up to 88° was observed in Au NP-hydrogel pillars with a low Au NP concentration with a 90° incident angle. The light tracking performance was assessed by the slope of the pillar Bending angle (response angle) vs. Incident light angle plot. A slope of 1 indicates ideal tracking with top of the pillar being normal to the incident light, maximizing the photon absorption. Slopes of 0.82 and 0.56 were observed for the low and high Au NP concentration samples. The rapid and precise incident light tracking of our system has shown the promise in phototropic applications. / Dissertation/Thesis / Masters Thesis Materials Science and Engineering 2016

Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.

No description available.

Ocular drug delivery to the posterior segment of the eye is extremely challenging. The delivery of the pharmaceuticals is made difficult by the numerous barriers that are present in the eye, as well as the isolated nature of the eye. The eye also consists of efficient drainage routes that eliminate the drug that has entered the eye successfully. Because of these reasons, drug delivery to the posterior segment of the eye is challenging and complicated. As a result, conventional eye drops are an inefficient way to deliver the pharmaceuticals to the eye as <5% of the administered dose is delivered to the anterior segment of the eye, and a negligible amount is delivered to the posterior tissues. The work presented in this thesis focuses on the design, synthesis, and characterization of the PLGA nanoparticles as a drug delivery vehicle to treat diseases associated with the posterior segment of the eye. The slow-release formulation was developed using PLGA nanoparticles and synthesized by the Double Emulsion Method (W1-O-W2). The PLGA nanoparticles were optimized by following various protocols and formulations to obtain the highest encapsulation efficacy and desired particle size range by changing the intensity of sonication, speed of ultracentrifugation, composition, and amount of the stabilizer and PLGA nanoparticles. The nanoparticles showed a 97% encapsulation efficiency with Bovine Serum Albumin (BSA) and a particle size of 201 nm. The slow-release formulation was further developed by immobilization of the particles in a thermogelling PNIPAAM scaffold. In vitro drug release results suggest that PNIPAAM containing PLGA nanoparticles produced in this work has the potential to be further developed and used as a drug delivery vehicle for the posterior segment of the eye. / Thesis / Master of Applied Science (MASc)

Im Rahmen dieser Arbeit wurden neuartige, temperatursensitive Polymerbürsten mit terminaler Click-Funktionalität hergestellt, die nicht nur im Detail untersucht bzw. charakterisiert wurden, sondern deren Eigenschaften zusätzlich durch die polymeranaloge Umsetzung mit einem entsprechend modifizierten Fluoreszenzfarbstoff, Polymer oder mit superparamagnetischen Nanopartikeln erweitert werden konnten. Mittels kontrolliert radikalischer Polymerisation wurde mono- und bi-funktionalisiertes Poly(N-isopropylacrylamid) mit unterschiedlichen Endgruppen und Molekulargewichten synthetisiert und über das "grafting to"-Verfahren an eine Modeloberfläche angebunden. Die kovalente Anbindung der entsprechenden Komponenten erfolgte entweder durch die Kupfer-katalysierte oder thermisch induzierte Alkin-Azid-Cycloaddition. Neben den physiko-chemischen Eigenschaften wurde im Besonderen das temperaturabhängige Schaltverhalten dieser funktionellen Polymerbürsten in situ untersucht, wobei neben der spektroskopischen Ellipsometrie als Hauptmethode, zusätzlich Rasterkraftmikroskopie, UV/VIS- und Fluoreszenzspektroskopie, sowie Elektronenmikroskopie verwendet wurden. Im Fall der Farbstoff-modifizierten Polymerbürsten zeigte sich, dass nicht nur die Eigenschaften des Farbstoffs durch das Polymer beeinflussbar sind, sondern auch das Quellverhalten der Polymerbürsten durch den Farbstoff. Die mit Nanopartikeln modifizierten Polymerbürsten zeigten eine homogene Verteilung der Partikel auf der Oberfläche, sowie eine veränderte Schaltcharakteristik, die durch das Auftreten eine dynamischen Wechselwirkungszone zwischen den Polymerbürsten und den Nanopartikeln erklärt werden kann. Mit Hilfe der thermisch induzierten Azid-Alkin-Cycloaddition konnten erstmalig definierte Kettenverlängerungen an Polymerbürsten durchgeführt und gleichzeitig der Nachteil der limitierten Pfropfungsdichte der „grafting to“-Methode überwunden werden. Darüber hinaus konnte der temperaturinduzierte Schalteffekt nicht nur erhalten bleiben, sondern in Bezug auf die Schaltamplitude auch signifikant vergrößert werden und ergab ein sensitiveres, verbessertes Polymerbürstensystem.

Im Rahmen dieser Arbeit wurden neuartige, temperatursensitive Polymerbürsten mit terminaler Click-Funktionalität hergestellt, die nicht nur im Detail untersucht bzw. charakterisiert wurden, sondern deren Eigenschaften zusätzlich durch die polymeranaloge Umsetzung mit einem entsprechend modifizierten Fluoreszenzfarbstoff, Polymer oder mit superparamagnetischen Nanopartikeln erweitert werden konnten. Mittels kontrolliert radikalischer Polymerisation wurde mono- und bi-funktionalisiertes Poly(N-isopropylacrylamid) mit unterschiedlichen Endgruppen und Molekulargewichten synthetisiert und über das "grafting to"-Verfahren an eine Modeloberfläche angebunden. Die kovalente Anbindung der entsprechenden Komponenten erfolgte entweder durch die Kupfer-katalysierte oder thermisch induzierte Alkin-Azid-Cycloaddition. Neben den physiko-chemischen Eigenschaften wurde im Besonderen das temperaturabhängige Schaltverhalten dieser funktionellen Polymerbürsten in situ untersucht, wobei neben der spektroskopischen Ellipsometrie als Hauptmethode, zusätzlich Rasterkraftmikroskopie, UV/VIS- und Fluoreszenzspektroskopie, sowie Elektronenmikroskopie verwendet wurden. Im Fall der Farbstoff-modifizierten Polymerbürsten zeigte sich, dass nicht nur die Eigenschaften des Farbstoffs durch das Polymer beeinflussbar sind, sondern auch das Quellverhalten der Polymerbürsten durch den Farbstoff. Die mit Nanopartikeln modifizierten Polymerbürsten zeigten eine homogene Verteilung der Partikel auf der Oberfläche, sowie eine veränderte Schaltcharakteristik, die durch das Auftreten eine dynamischen Wechselwirkungszone zwischen den Polymerbürsten und den Nanopartikeln erklärt werden kann. Mit Hilfe der thermisch induzierten Azid-Alkin-Cycloaddition konnten erstmalig definierte Kettenverlängerungen an Polymerbürsten durchgeführt und gleichzeitig der Nachteil der limitierten Pfropfungsdichte der „grafting to“-Methode überwunden werden. Darüber hinaus konnte der temperaturinduzierte Schalteffekt nicht nur erhalten bleiben, sondern in Bezug auf die Schaltamplitude auch signifikant vergrößert werden und ergab ein sensitiveres, verbessertes Polymerbürstensystem.