The work in this dissertation has two main focuses: (1) to develop sensors based on a quadruplex-forming oligonucleotide scaffold for the sensing of specific sequences, (2) to develop an indicator displacement assay for the high-throughput determination of host-guest binding capable of easy discrimination between strong and weak-binding species. Chapter 1 serves to provide a brief introduction to some shared background for both projects, through introducing basic tenants of aqueous supramolecular chemistry and nucleic acid chemistry, in addition to some general lessons in system design that can be learned from the study of biology. Chapter 2 describes the design of a sensor based on a naphthalene mono-imide (NMI) scaffold, which due to an intramolecular charge-transfer from the naphthalene to a conjugated bipyridine has a low native fluorescence emission. When exposed to curcubit[7]uril (CB[7]) a macromolecular host able to encapsulate the bipyridine unit, the intramolecular charge-transfer is interrupted, resulting in a significant increase in fluorescence quantum yield (by over an order of magnitude). This enhancement is reversible, with competing binders for CB[7] causing a return to the unbound state with quenched emission. Importantly, this sensor exhibits robust activity with no significant variance in properties throughout the range of 5-10 pH, and is amenable to secondary functionalization for surface attachment without loss of activity. A facile microplate assay was developed on the surface-bound sensor, and a proof of concept study was shown by testing for binding against a library of therapeutically relevant drug classes, resulting in the discovery of three novel guests for CB[7] possessing strong to moderate binding affinities. Chapter 3 discusses the development of an oligonucleotide sensor called a quadruplex molecular beacon (QMB) that is able to transition between a closed, intramolecular quadruplex state and an open, intermolecular duplex state on the sequential application of two stimuli in the form of specific oligonucleotide sequences. The chapter initially focuses on optimizing strand exchange mechanisms that allow for the sequential stimuli to open (and close) the system, finding that the combined use of a short toehold sequence with a targeted base-pair mismatch leads to efficient hybridization and displacement with sequential stimuli. The remaining part of the chapter examines the use of this strand exchange mechanism to drive a fully constituted QMB through sequential opening and closing in response to sequential stimuli, with a corresponding fluorescence signal. / Cooper Battle

A series of well-defined stimuli responsive water soluble [60]fullerene (C₆₀) containing polymers such as polyelectrolytes (polyacids and polybases), polyampholyte and polyzwitterionic polymers were synthesized using atom transfer radical polymerization. The aqueous solution properties of these polymers with respective external stimuli such as pH, temperature and salt were studied using potentiometric and conductivity titration, light transmittance, laser light scattering and transmission electron microscopic techniques. The influence of polymer concentration, temperature, pH and electrolyte on the hydrodynamic radius (Rh), radius of gyration (Rg) and aggregation number (Nagg) of the particles were investigated in detail to elucidate the morphology of the particles. The morphology of the aggregates was further confirmed by the TEM micrographs. The cytotoxicity of the pH responsive C₆₀ containing well-defined polymers (PAA-b-C₆₀, C₆₀-b-PAA-b-C₆₀ and PEO-b-PAA-b-C₆₀) was studied to confirm the suitability of these particles as potential drug delivery vehicles. The binding interaction between the anti-cancer drug (doxorubicin) and C₆₀ containing pH responsive polymers was studied using isothermal titration calorimetry, and the implication of the results will be discussed. / Singapore-MIT Alliance (SMA)

Energy harvesting using ferrofluid in OHP. Characterization of as-synthesized (bare) and surface-modified ferrofluid samples was performed using Fourier transform infrared spectroscopy, dynamic light scattering, X-ray powder diffraction, transmission electron microscopy, and atomic force microscopy. These ferrofluids were tested in a novel oscillating heat pipe set-up was utilized to harvest electricity, demonstrating the concept of ferrofluidic induction. Cobalterrite nanoparticles surface-modified with citric acid demonstrated good magnetic strengths and generated voltages close to those of the as-synthesized ferrofluids while maintaining dispersion. Surface modification of ferrous nanoparticles with SRP. Thermo responsive polymer poly(N-isopropylacrylamide) was successfully grown from the surface of cobalt-zinc ferrite nanoparticles. A dual responsive block copolymer, pH and thermo responsive comprised of poly(itaconic) acid and poly(N-isopropylacrylamide) was successfully polymerized from the surface of ferrous oxide nanoparticles. These composite having magnetic properties along with stimulus can be used in applications such as controlled drug delivery and similar biomedical applications.

The advent of highly sophisticated drugs designed to interfere with specific cellular functions has created the demand for “intelligent” carriers that can efficiently deliver therapeutic agents in response to a pathophysiogical condition. Nanoscale intelligent systems can maximize the efficacy of therapeutic treatments in numerous ways because they have the ability to rapidly detect and response to disease states directly at the site and sparing physiologically healthy cells and tissues, thereby improving a patient’s quality of life. Nanoparticle fabrication has primarily relied on emulsions, self-assembly and micelles based methods which inherently generate polydisperse spherical particles with little control over particle geometry. Despite significant progress in such drug delivery systems, critical limitations remain in synthesizing nanocarriers with highly controllable architecture (size, shape or aspect ratio) that can, at the same time, impart response-sensitive release mechanisms. These parameters are essential for controlling the in-vivo transport, bio-distribution, and drug release mechanisms. The objective of my dissertation is to employ the nanofabrication technique Step and Flash Imprint Lithography (S-FIL) to synthesize stimuli-responsive nanocarriers of precise architectures and composition. Applying S-FIL technology, fabrication of nanocarriers of a variety of shapes and sizes (down to 36nm length scale) that are also environmentally responsive by incorporating enzymatically-degradable peptides into the nanocarrier hydrogel matrix, to provide triggered release of encapsulated therapeutic agents in response to specific pathophysiological conditions, has been accomplished. Besides disease-responsive release, the two key properties of an effective nanocarrier are (a) efficient targeting to specific tissues and cells and (b) avoiding rapid clearance and remaining in circulation in the blood stream for a significant amount of time to increase particle uptake in target tissues. These two properties are expected to be dependent on the shape and size of the carriers. Using various shape and size S-FIL fabricated nanoparticles, the effects of particle geometry on intracellular uptake has also been evaluated. In this dissertation, I will present the extensive work that has been done in the fabrication and optimization of the S-FIL nanocarriers, evaluation of the nanocarrier’s in vitro properties, and evaluation of the effects of nanocarrier geometry on intracellular uptake. / text

The objective of this thesis is to investigate well-defined cross-linked particles synthesized via the reversible addition fragmentation chain transfer (RAFT) process that can be used for drug delivery. To achieve this aim, a wide range of cross-linked micelle systems have been synthesized and intensively investigated. Various biocompatible monomers were employed, including poly (ethylene glycol) methyl ether methacrylate, 2-hydroxyl ethyl acrylate, functionalized glucosamine and nucleotides containing monomers. Different cross-linked structures were used, for example, core-cross-linked, nexus-cross-linked and shell-cross-linked micelles. Diverse stimuli-responsive particles were used, such as pH-sensitive, thermo-sensitive and thiol-sensitive cross-linked systems. Evidences of the successful synthesis of all the resulting cross-linked products are given. They displayed better properties, as drug carriers, than non-cross-linked micelles. A thermo-responsive seven-arm star glycopolymer, synthesized via the RAFT process, was also investigated.

This dissertation presents the synthesis of stimuli-sensitive hydrophilic polymers, particularly doubly responsive hydrophilic block copolymers, by controlled radical polymerizations and the study of their solution behavior in water. By incorporating a small amount of stimuli-responsive groups into the thermosensitive block of a hydrophilic block copolymer, the lower critical solution temperature (LCST) of the thermosensitive block can be tuned by a stimulus and multiple micellization/dissociation transitions can be achieved by combining two external triggers. Chapter 1 describes the synthesis and thermosensitive properties of two new watersoluble polystyrenics with a short oligo(ethyl glycol) pendant from each repeat unit and the study of hydrophobic end group effects on cloud points of thermosensitive polystyrenics. Well-defined polymers were prepared from monomer-based initiators via nitroxide-mediated polymerization and the alkoxyamine end groups were removed by tri(n-butyl)tin hydride, yielding thermoresponsive polystyrenics with essentially no end groups. The results showed that hydrophobic end groups could significantly change the cloud points and the molecular weight dependences of cloud points of polystyrenics. Chapter 2 presents the synthesis of thermo- and light-sensitive hydrophilic block copolymers, poly(ethylene oxide)-b-poly(ethoxytri(ethylene glycol) acrylate-co-onitrobenzyl acrylate), and their responsive behavior in dilute aqueous solutions. Dynamic light scattering and fluorescence spectroscopy studies showed that these copolymers were molecularly dissolved in water at lower temperatures and self-assembled into micelles at temperatures above the LCST of the thermosensitive block. Upon UV irradiation, the oiv nitrobenzyl group was cleaved and the LCST of the thermosensitive block was increased, causing the dissociation of micelles into unimers. The resultant copolymers underwent thermo-induced reversible micellization at higher temperatures. Chapter 3 describes multiple micellization/dissociation transitions of thermo- and pH-sensitive hydrophilic block copolymers, poly(ethylene oxide)-b-poly(methoxydi(ethylene glycol) methacrylate-co-methacrylic acid), in response to temperature and pH changes. The LCST of the thermosensitive block can be reversibly tuned and precisely controlled by solution pH. Chapter 4 presents the study on multiple sol-gel-sol transitions of a 20.0 wt % aqueous solution of poly(ethylene oxide)-b-poly(ethoxytri(ethylene glycol) acrylate-co-o-nitrobenzyl acrylate) induced by temperature changes and UV irradiation. The solution underwent thermo-induced sol-gel-sol transitions. Upon UV irradiation to dissociate micelles, the gel was transformed into a free-flowing liquid, which upon heating underwent sol-gel-sol transitions again.

The objective of this thesis is to investigate well-defined cross-linked particles synthesized via the reversible addition fragmentation chain transfer (RAFT) process that can be used for drug delivery. To achieve this aim, a wide range of cross-linked micelle systems have been synthesized and intensively investigated. Various biocompatible monomers were employed, including poly (ethylene glycol) methyl ether methacrylate, 2-hydroxyl ethyl acrylate, functionalized glucosamine and nucleotides containing monomers. Different cross-linked structures were used, for example, core-cross-linked, nexus-cross-linked and shell-cross-linked micelles. Diverse stimuli-responsive particles were used, such as pH-sensitive, thermo-sensitive and thiol-sensitive cross-linked systems. Evidences of the successful synthesis of all the resulting cross-linked products are given. They displayed better properties, as drug carriers, than non-cross-linked micelles. A thermo-responsive seven-arm star glycopolymer, synthesized via the RAFT process, was also investigated.

Liposomes are promising drug delivery vesicles due to their biodegradibility, large volume and biocompatibility towards both hydrophilic and hydrophobic drugs. They suffer, however, from poor stability which limits their use in controlled delivery applications. Herein, a novel method was devised for modification of liposomes with small molecules, polymers or nanoparticles to afford stimuli responsive systems that release on demand and stay relatively stable in the absence of the trigger.. This dissertation discusses thermosensitive, pH sensitive, light sensitive and magnetically triggered liposomes that have been prepared for controlled drug delivery application. RAFT polymerization was utilized for the preparation of thermosensitive liposomes (Cholesterol-PNIPAm) and acid-labile liposomes (DOPE-PAA). With low Mw Cholesterol-PNIPAm, the thermosensitive liposomes proved to be effective for controlled release and decreased the cytotoxicity of PNIPAm by eliciting the polymer doses. By crosslinking the DOPE-PAA on liposome surface with acid-labile diamine linkers, DOPE-PAA liposomes were verified to be sensitive at low pH. The effects of polymer structures (linear or hyperbranched) have also been studied for the stability and release properties of liposomes. Finally, a dual-responsive Au@SPIO embedded liposome hybrid (ALHs) was prepared with light-induced “on-and-off” function by photo-thermal process (visible light) and instant release properties triggered by alternating magnetic field, respectively. The ALH system would be further applied into the cellular imaging field as MRI contrast agent.

Das corneale Endothel bildet die innere, einschichtige Zelllage der Cornea und ist für die Aufrechterhaltung der cornealen Transparenz zuständig. Krankheiten oder Verletzungen des cornealen Endothels können zu schweren Beeinträchtigungen des Sehvermögens führen und eine corneale Transplantation erforderlich machen. Der während und nach der Operation auftretende endotheliale Zellverlust erschwert das Überleben des Transplantates. Darum besteht ein Hauptziel des cornealen Tissue Engineerings in der Bereitstellung von transplantierbaren humanen cornealen Endothelzellsheets (HCEC-Sheets) mit einer adäquaten Zelldichte. Thermo-responsive Zellkulturträger fanden für die schonende, enzymfreie Gewinnung von Zellsheets für verschiedene Gewebetypen bereits Verwendung. HCEC stellen in diesem Kontext einen besonderen Fall dar, da sie eine starke Adhäsion zu ihrem Kultursubstrat ausbilden, was deren schonende, thermisch induzierte Ablösung als funktionelles Zellsheet erschwert. Im Rahmen dieser Arbeit wurde ein neuartiger thermo-responsiver Zellkulturträger entwickelt. Dieser basiert auf dem durch Elektronenbestrahlung immobilisierten und vernetzten thermo-responsiven Polymer Poly(vinylmethylether) (PVME) sowie dem alternierenden Co-Polymer Poly(vinylmethylehter-alt-maleinsäureanhydrid) (PVMEMA) als biofunktionalisierbare Komponente. Die Kombination dieser Polymere führte zur Etablierung eines thermo-responsiven Zellkulturträgers, dessen physikochemische und biomolekulare Eigenschaften in weiten Grenzen einstellbar und dadurch an die spezifischen Anforderungen von HCEC anpassbar waren. Das PVME-PVMEMA-Blend ermöglichte die Bildung konfluenter HCEC-Monolayer mit den morphologischen Grundlagen für ein funktionelles corneales Endothelgewebe. Durch Inkorporation von Poly(N-isopropylacrylamid) (PNiPAAm) als weitere thermo-responsive Polymerkomponente konnte das Ablösungsverhalten funktioneller HCEC-Sheets weiter verbessert werden. In einem weiteren Schritt erfolgte der Transfer abgelöster HCEC-Sheets auf ein planares, biofunktionalisiertes Kultursubstrat sowie auf endothelfreie porcine Corneae. Die HCEC-Sheets wurden auch nach dem Transfer umfassend biologisch analysiert. Diese Arbeit legt einen Grundstein für die Bereitstellung klinisch anwendbarer Alternativen für das Tissue Engineering von cornealem Gewebe.

Le travail de recherche de cette thèse se concentre sur le développement de nanoparticules de silice organo-hybrides pour des applications en nanomédecine et agroalimentaire. Ces nanoconteneurs de silice, comportant des liens disulfures, sont capables de se briser en petits fragments en présence du milieu réductif intracellulaire. Des nanoparticules présentant de larges pores ont été synthétisées pour la livraison d’un siRNA PLK1 pour le traitement du carcinome hépatocellulaire et ont démontré des résultats prometteurs in vitro et in vivo. Ces particules ont été également utilisées pour charger un peptide cytotoxique, souvent utilisé comme pesticide dans l’industrie agroalimentaire. Les nanoparticules cassables ont ensuite été miniaturisées pour le relargage d’agents thérapeutiques dans des glioblastomes humains. Le système présentait un relargage plus rapide comparé à la forme liposomale actuellement sur le marché. Enfin, des nanoparticules contenant des liens répondant aux réactifs dérivés de l’oxygène ont été développées et ont démontré une fragmentation importante en présence d’oxygène singulet. / The research work presented throughout this thesis focuses on the development of organo-hybrid mesoporous silica nanoparticles for their applications in nanomedicine and crop industry. Disulfide doped silica nanocarriers, able to break down in small pieces in presence of the intracellular reductive environment have been tailored. A large pore stimuli-responsive system was developed to deliver a PLK1 siRNA within hepatocellular carcinoma cells demonstrating promising results both in vitro and in vivo. The particles were further used to deliver a venom peptide, often utilized as esticide in the crop industry. The breakable nanocarriers were further miniaturized for the delivery of chemotherapeutic agents within human glioblastoma cells. The system presented a faster delivery compared to the commercially available liposomal form. Finally, Reactive-Oxygen-Species-responsive mesoporous silica nanoparticles were developed and demonstrated fast breakability upon incubation with singlet oxygen.