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pH-responsive polymer nanoparticles synthesized using ARGET ATRPForbes, Diane Christine 24 February 2015 (has links)
Polycationic nanoparticles were synthesized with an activators regenerated by electron transfer for atom transfer radical polymerization-based (ARGET ATRP-based) emulsion in water method and investigated for their utility as biomaterials for drug delivery. The polycationic nanoparticles were composed of 2-(diethylamino)ethyl methacrylate (DEAEMA) for pH-responsiveness, poly(ethylene glycol) methyl ether methacrylate (PEGMA) for improved biocompatibility, tert-butyl methacrylate (tBMA) to impart hydrophobicity, and a tetraethylene glycol dimethacrylate (TEGDMA) cross-linking agent for enhanced colloidal stability. Dynamic light scattering demonstrated pH-responsive swelling, and cell-based assays demonstrated pH-dependent membrane disruption. The polycationic nanoparticles demonstrated low toxicity to cells. The polycationic nanoparticles were evaluated for use as drug delivery biomaterials by investigating the interactions with the drug and cells. Delivery remains a major challenge for translating small interfering RNA (siRNA) to the clinic, and overcoming the delivery challenge requires effective siRNA delivery vehicles. The polycationic nanoparticles demonstrated efficient siRNA loading. Evidence of siRNA-induced knockdown in cells was observed following transfection with the polycationic nanoparticle/siRNA complexes. Imaging techniques confirmed enhanced siRNA internalization using the polycationic nanoparticle/siRNA complexes compared to naked siRNA. An array of polycationic nanoparticles synthesized using ARGET ATRP or UV-initiated polymerization methods was characterized to examine the effect of polymerization method on material properties and the connection to molecular structure. An improved understanding of molecular structure, and its connection to polymerization method and material characteristics, may aid the design of advanced materials. The ARGET ATRP polycationic nanoparticles demonstrated increased nanoscale homogeneity compared to the UV-initiated polymerization polycationic nanoparticles; increased nanoscale heterogeneity in the UV-initiated polymerization polycationic nanoparticles was associated with broader transitions. The polycationic nanoparticles promoted cellular uptake of siRNA and induced knockdown, thus demonstrating potential as siRNA delivery vehicles. The ARGET ATRP method provides an alternative route to creating polycationic nanoparticles with improved nanoscale homogeneity. / text
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Natural biomaterials for enhanced oligodendrocyte differentiation and spinal cord injury repairGeissler, Sydney Amelia 30 March 2015 (has links)
Spinal cord injury is a devastating source of suffering in the spectrum of human pathophysiology; advancement for clinical therapy in this area has been stagnant in comparison to modern medical development. Current treatments are palliative, and functional recovery is minimal. During the first two weeks after injury, dense glial scar forms that is impenetrable by regenerating axons. Intervention is imperative to minimize scar formation and provide a supportive environment for axonal regeneration. Oligodendrocytes are critical to maintain the health of growing axons during development and after injury. Obtaining these cells through differentiation of neural progenitor cells (NPCs) is a viable option, but current clinical trials involving stem cells are plagued by poor cell survival and undirected differentiation. Research indicates that local extracellular matrix (ECM) is vital to progenitor differentiation and tissue regeneration. During development, spinal cord ECM is comprised of high concentrations of laminin and hyaluronic acid (HA), which provide essential cues to direct NPC migration and differentiation. The purpose of this research is to create a biomaterial optimized to direct NPC differentiation to oligodendrocytes. Natural biomaterials were optimized from distinct combinations of collagen I, HA, and laminin I to model the native ECM signals found during oligodendrocyte maturation. Four material combinations (collagen, collagen-HA-laminin, collagen-HA, and collagen-laminin) were fabricated into injectable hydrogels to mimic the range of compressive and shear mechanical properties present in neonatal central nervous system (CNS) tissue. Differentiation was assessed by culturing rodent fetal NPCs in these materials without specific soluble factors to direct cellular behavior. The three-component hydrogel performed optimally and achieved a 66% oligodendrocyte differentiation rate compared to approximately 15% in the collagen alone hydrogel. An in vivo study was then conducted using a rat contusion model of spinal cord injury with intervention using the injectable, three-component hydrogel seeded with rat NPCs. Functional recovery was assessed using six behavioral tests. Significant recovery was observed using two behavioral tests six weeks post-treatment. Lesion size was measured and correlated well with behavioral outcomes. The data obtained in this research indicate that a multi-component hydrogel mimicking native, developmental CNS tissue may address problems associated with current clinical practice. / text
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Effect of shape on cell internalization of polymeric hydrogel nanoparticlesAgarwal, Rachit, Ph. D. 11 August 2015 (has links)
Recent progress in drug discovery has enabled us to target specific intracellular molecules to achieve therapeutic effects. These next generation therapeutics are often biologics which cannot enter cells by mere diffusion. Therefore it is imperative that drug carriers are efficiently internalized by cells before releasing their cargo. Nanoscale polymeric carriers are particularly suitable for such intra-cellular delivery. Although size and surface-charge has been the most studied parameters for nanocarriers, it is now well appreciated that particle shape also plays a critical role in their transport across physiological barriers. Hence there is increasing interest in fabricating shape-specific polymeric nano and microparticles for efficient delivery of drugs and imaging agents. Nanoimprint lithography methods, such as Jet-and-flash imprint lithography (J-FIL), provide versatile top-down processes to fabricate shape-specific, biocompatible nanoscale hydrogels that can deliver therapeutic and diagnostic molecules in response to disease-specific cues. However, the key challenges in top-down fabrication of such nanocarriers are scalable imprinting with biological and biocompatible materials, ease of particle-surface modification using both aqueous and organic chemistry as well as simple yet biocompatible harvesting. Here we report that a biopolymer-based sacrificial release layer in combination with improved nanocarrier-material formulation can address these challenges. The sacrificial layer improves scalability and ease of imprint-surface modification due to its switchable solubility through simple ion exchange between monovalent and divalent cations. This process enables large-scale bio-nanoimprinting and efficient, one-step harvesting of hydrogel nanoparticles in both water- and organic-based imprint solutions. We also show that when shape is decoupled from volume, charge and composition, mammalian cells preferentially internalize disc-shaped nanohydrogels of higher aspect ratios over nanorods. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently. Uptake kinetics, efficiency and internalization mechanisms are all shape-dependent and cell-type specific. Although macropinocytosis is used by all cells, epithelial cells uniquely internalize nanodiscs using caveolae pathway. On the other hand, endothelial cells use clathrin-mediated uptake along with macropinocytosis for all shapes and show significantly higher uptake efficiency compared to epithelial cells. We also study the effect of shape and surface properties for their tissue uptake and penetration using spheroids as a 3D tumor model and show that hydrophobic particles show no difference in penetration inside such models even after 125 fold reduction in volume. These results provide a fundamental understanding of how cell and tissue behavior is influenced by nanoscale shape and surface properties and are critical for designing improved nanocarriers and predicting nanomaterial toxicity. / text
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A Composite Polymeric Drug Delivery System for Treatment of Spinal Cord InjuryBaumann, Matthew Douglas John 04 August 2010 (has links)
There are no clinically approved drug delivery strategies designed for localized and sustained release to the injured spinal cord, two features which are heavily exploited in pre-clinical demonstrations of efficacy. We have previously shown that injection of drug loaded hydrogels into the intrathecal space is safe, minimally invasive, and drug release localized to the site of injection for up to one day. In the present work we developed a platform for sustained release from 1 to 28 days based on a physical gel of methylcellulose with hyaluronan and poly(lactic-co-glycolic acid) (PLGA) nanoparticles added as gelation agents. These composite hydrogels met the design criteria of injectability, fast gelation, minimal swelling, and 28 day stability. Sustained release of 6 therapeutic molecules from the composite was achieved by encapsulation in the particles or dissolution in the hydrogel. Release of PLGA encapsulated drugs from the composite was linear for 28 days. Drugs dissolved in the hydrogel were released by Fickian diffusion. The HAMC hydrogel/PLGA nanoparticle composite was delivered to uninjured and spinal cord injured rats and the animals monitored for 14 and 28 days respectively. The composite was well tolerated in the intrathecal space with no impact on motor function as determined by the BBB scale and minimal inflammation in both studies. No increase in reactive astrocytes or cavity volume was found in clip compression spinal cord injured rats, indicating that the composite did not affect these aspects of the secondary injury cascade. We then turned to sustained release of anti-NogoA, a promising neuroregenerative molecule typically delivered for 2 - 4 weeks. Formulations of anti-NogoA or a model IgG were prepared and release was demonstrated over 28 days in vitro. Bioactivity was assessed using a novel ELISA which utilized anti-NogoA / NogoA binding to detect only active antibody, advantageous because anti-NogoA release can now be easily optimized prior to in vivo studies of efficacy. The key features of current work are the development of an intrathecal drug delivery platform, demonstration of safety in a rat model, and formulation for use with anti-NogoA.
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A Composite Polymeric Drug Delivery System for Treatment of Spinal Cord InjuryBaumann, Matthew Douglas John 04 August 2010 (has links)
There are no clinically approved drug delivery strategies designed for localized and sustained release to the injured spinal cord, two features which are heavily exploited in pre-clinical demonstrations of efficacy. We have previously shown that injection of drug loaded hydrogels into the intrathecal space is safe, minimally invasive, and drug release localized to the site of injection for up to one day. In the present work we developed a platform for sustained release from 1 to 28 days based on a physical gel of methylcellulose with hyaluronan and poly(lactic-co-glycolic acid) (PLGA) nanoparticles added as gelation agents. These composite hydrogels met the design criteria of injectability, fast gelation, minimal swelling, and 28 day stability. Sustained release of 6 therapeutic molecules from the composite was achieved by encapsulation in the particles or dissolution in the hydrogel. Release of PLGA encapsulated drugs from the composite was linear for 28 days. Drugs dissolved in the hydrogel were released by Fickian diffusion. The HAMC hydrogel/PLGA nanoparticle composite was delivered to uninjured and spinal cord injured rats and the animals monitored for 14 and 28 days respectively. The composite was well tolerated in the intrathecal space with no impact on motor function as determined by the BBB scale and minimal inflammation in both studies. No increase in reactive astrocytes or cavity volume was found in clip compression spinal cord injured rats, indicating that the composite did not affect these aspects of the secondary injury cascade. We then turned to sustained release of anti-NogoA, a promising neuroregenerative molecule typically delivered for 2 - 4 weeks. Formulations of anti-NogoA or a model IgG were prepared and release was demonstrated over 28 days in vitro. Bioactivity was assessed using a novel ELISA which utilized anti-NogoA / NogoA binding to detect only active antibody, advantageous because anti-NogoA release can now be easily optimized prior to in vivo studies of efficacy. The key features of current work are the development of an intrathecal drug delivery platform, demonstration of safety in a rat model, and formulation for use with anti-NogoA.
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Advanced polymeric scaffolds for functional materials in biomedical applicationsÖberg Hed, Kim January 2014 (has links)
Advancements in the biomedical field are driven by the design of novel materials with controlled physical and bio-interactive properties. To develop such materials, researchers rely on the use of highly efficient reactions for the assembly of advanced polymeric scaffolds that meet the demands of a functional biomaterial. In this thesis two main strategies for such materials have been explored; these include the use of off-stoichiometric thiol-ene networks and dendritic polymer scaffolds. In the first case, the highly efficient UV-induced thiol-ene coupling (TEC) reaction was used to create crosslinked polymeric networks with a predetermined and tunable excess of thiol or ene functionality. These materials rely on the use of readily available commercial monomers. By adopting standard molding techniques and simple TEC surface modifications, patterned surfaces with tunable hydrophobicity could be obtained. Moreover, these materials are shown to have great potential for rapid prototyping of microfluidic devices. In the second case, dendritic polymer scaffolds were evaluated for their ability to increase surface interactions and produce functional 3D networks. More specifically, a self-assembled dendritic monolayer approach was explored for producing highly functional dendronized surfaces with specific interactions towards pathogenic E. coli bacteria. Furthermore, a library of heterofunctional dendritic scaffolds, with a controllable and exact number of dual-purpose azide and ene functional groups, has been synthesized. These scaffolds were explored for the production of cell interactive hydrogels and primers for bone adhesive implants. Dendritic hydrogels decorated with a selection of bio-relevant moieties and with Young’s moduli in the same range as several body tissues could be produced by facile UV-induced TEC crosslinking. These gels showed low cytotoxic response and relatively rapid rates of degradation when cultured with normal human dermal fibroblast cells. When used as primers for bone adhesive patches, heterofunctional dendrimers with high azide-group content led to a significant increase in the adhesion between a UV-cured hydrophobic matrix and the wet bone surface (compared to patches without primers). / <p>QC 20140116</p>
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Inorganic-Organic Hydrogel Scaffolds for Tissue EngineeringBailey, Brennan 16 December 2013 (has links)
Analogous to the extracellular matrix (ECM) of natural tissues, properties of a tissue engineering scaffold direct cell behavior and thus regenerated tissue properties. These include both physical properties (e.g. morphology and modulus) and chemical properties (e.g. hydrophobicity, hydration and bioactivity). Notably, recent studies suggest that scaffold properties (e.g. modulus) may be as potent as growth factors in terms of directing stem cell fate. Thus, 3D scaffolds possessing specific properties modified for optimal cell regeneration have the potential to regenerate native-like tissues. Photopolymerizable poly(ethylene glycol) diacrylate (PEG-DA)-based hydrogels are frequently used as scaffolds for tissue engineering. They are ideal for controlled studies of cell-material interactions due to their poor protein adsorption in the absence of adhesive ligands thereby making them “biological blank slates”. However, their range of physical and chemical properties is limited. Thus, hydrogel scaffolds which maintain the benefits of PEG-DA but possess a broader set of tunable properties would allow the establishment of predictive relationships between scaffold properties, cell behavior and regenerated tissue properties.
Towards this goal, this work describes a series of unique hybrid inorganic-organic hydrogel scaffolds prepared using different solvents and also in the form of continuous gradients. Properties relevant to tissue regeneration were investigated including: swelling, morphology, modulus, degradation rates, bioactivity, cytocompatibility, and protein adhesion. These scaffolds were based on the incorporation of hydrophobic, bioactive and osteoinductive methacrylated star polydimethylsiloxane (PDMSstar-MA) [“inorganic component”] into hydrophilic PEG-DA [“organic component”]. The following parameters were varied: molecular weight (Mn) of PEG-DA (Mn = 3k & 6k g/mol) and PDMSstar-MA (Mn = 1.8k, 7k, 14k), ratio of PDMSstar-MA to PEG-DA (0:100 to 20:80), total macromer concentration (5 to 20 wt%) and utilizing either water or dichloromethane (DCM) fabrication solvent. The use of DCM produced solvent induced phase separation (SIPS) resulting in scaffolds with macroporous morphologies, enhanced modulus and a more homogenous distribution of the PDMSstar-MA component throughout. These hybrid hydrogel scaffolds were prepared in the form of continuous gradients such that a single scaffold contains spatially varied chemical and physical properties. Thus, cell-material interaction studies may be conducted more rapidly at different “zones” defined along the gradient. These gradients are also expected to benefit the regeneration of the osteochondral interface, an interfacial tissue that gradually transitions in tissue type. The final aspect of this work was focused on enhancing the osteogenic potential of PDMS via functionalization with amine and phosphonate. Both amine and phosphonate moieties have demonstrated bioactivity. Thus, it was expected that these properties will be enhanced for amine and phosphonate functionalized PDMS. The subsequent incorporation of these PDMS-based macromers into the previously described PEG-DA scaffold system is expected to be valuable for osteochondral tissue regeneration.
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A Comparative Analysis of the Neurochemical Properties of Olfactory Ensheathing Cells and their Biocompatibility in Various BiomatricesRawji, Khalil S 31 July 2012 (has links)
Olfactory ensheathing cells (OECs) are the chief glial population of the mammalian olfactory nervous system and are thought to be responsible for the successful directional growth of new olfactory axons throughout the life of adult mammals. Due to this unique property, OECs have been targeted as a potential cellular transplantation therapy for spinal cord injury. In order to effectively isolate OECs for intraspinal transplantation, more knowledge must be gained on their phenotypic properties. We investigated the neurochemical features of OECs in a variety of mammalian species (including hamsters, rabbits, monkeys, mice, and pigs) using three biomarkers: glial fibrillary acidic protein (GFAP), S100β, and α-smooth muscle actin (αSMA). In addition, we tested the ability of a few biomatrices to sustain and promote OEC growth and survival in vitro. The rationale for using biomatrices is to provide a supportive environment for glial and axonal growth in the spinal lesion. Here, we found that mucosal and bulbar OECs from all five of the aforementioned mammalian species express S100β. Expression of GFAP, however, was not consistent across the five species. Both mucosal and bulbar OECs of monkeys express αSMA; only bulbar OECs of hamsters and only mucosal OECs of rabbits express αSMA as well. Though αSMA immunostaining was not detected in the OECs of adult mice, in adult mutant mice lacking αSMA expression, OECs displayed perturbed ultrastructural morphology. None of the biomatrices used (methacrylated glycol chitosan, arginine-glycine-aspartic acid – grafted methacrylated glycol chitosan, and agarose) were able to promote OEC proliferation. Isolated strips of rodent olfactory lamina propria (the deep connective tissue layer in the olfactory mucosa containing primary sensory axons and OECs) showed sustained growth when cultured for 10 days. In sum, these findings highlight the following points: the efficacy of S100β and αSMA as biomarkers for mammalian OECs in vivo; the potential for isolated strips of lamina propria to provide a natural, supportive environment for OECs during intraspinal transplantation; the failure of methacrylated glycol chitosan and its derivatives, as well as agarose, to promote OEC proliferation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-07-27 15:29:47.642
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New inverse hydogel opals as protein responsive sensorsSütterlin, Martin January 2013 (has links)
In this work, the development of temperature- and protein-responsive sensor materials based on biocompatible, inverse hydrogel opals (IHOs) is presented. With these materials, large biomolecules can be specifically recognised and the binding event visualised. The preparation of the IHOs was performed with a template process, for which monodisperse silica particles were vertically deposited onto glass slides as the first step. The obtained colloidal crystals with a thickness of 5 μm displayed opalescent reflections because of the uniform alignment of the colloids.
As a second step, the template was embedded in a matrix consisting of biocompatible, thermoresponsive hydrogels. The comonomers were selected from the family of oligo(ethylene glycol)methacrylates. The monomer solution was injected into a polymerisation mould, which contained the colloidal crystals as a template. The space in-between the template particles was filled with the monomer solution and the hydrogel was cured via UV-polymerisation. The particles were chemically etched, which resulted in a porous inner structure. The uniform alignment of the pores and therefore the opalescent reflection were maintained, so these system were denoted as inverse hydrogel opals. A pore diameter of several hundred nanometres as well as interconnections between the pores should facilitate a diffusion of bigger (bio)molecules, which was always a challenge in the presented systems until now. The copolymer composition was chosen to result in a hydrogel collapse over 35 °C. All hydrogels showed pronounced swelling in water below the critical temperature. The incorporation of a reactive monomer with hydroxyl groups ensured a potential coupling group for the introduction of recognition units for analytes, e.g. proteins.
As a test system, biotin as a recognition unit for avidin was coupled to the IHO via polymer-analogous Steglich esterification. The amount of accessible biotin was quantified with a colorimetric binding assay. When avidin was added to the biotinylated IHO, the wavelength of the opalescent reflection was significantly shifted and therefore the binding event was visualised. This effect is based on the change in swelling behaviour of the hydrogel after binding of the hydrophilic avidin, which is amplified by the thermoresponsive nature of the hydrogel. A swelling or shrinking of the pores induces a change in distance of the crystal planes, which are responsible for the colour of the reflection. With these findings, the possibility of creating sensor materials or additional biomolecules in the size range of avidin is given. / In dieser Arbeit wird die Entwicklung von temperatur- und proteinresponsiven Sensormaterialien auf Basis von biokompatiblen, inversen Hydrogelopalen (IHO) vorgestellt, mit welchen die spezifische Erkennung größerer Biomoleküle visuell ausgelesen werden kann. Die Darstellung der IHOs erfolgte mittels Templatverfahren, bei dem im ersten Schritt monodisperse Silicapartikel vertikal auf Objektträger abgeschieden wurden. Die so erhaltenen Kolloidkristalle mit einer Dicke von 5 μm zeigten opaleszente Reflexionen aufgrund der gleichförmigen Anordnung der Partikel.
Im zweiten Schritt wurde das Templat in eine Matrix aus biokompatiblen, thermoresponsiven Hydrogelen eingebettet. Die Comonomere wurden aus der Familie der Oligo(ethylenglykol)methacrylate ausgewählt. Zur Synthese des Hydrogels wurde die Monomerlösung in eine Polymerisationsform injiziert, welche die Kolloidkristalle als Templat beinhaltete. Die Zwischenräume der Templatpartikel wurden mit der Monomerlösung gefüllt und das Hydrogelnetzwerk per UV-Polymerisation erhalten. Die Templatpartikel wurden anschließend nasschemisch heraus gelöst, so dass eine poröse innere Struktur erhalten wurde. Die regelmäßige Anordnung der Poren und damit die opaleszenten Reflexionen wurden dabei beibehalten, so dass diese Systeme als inverse Hydorgelopale bezeichnet werden. Ein Porendurchmesser von mehreren hundert Nanometer, sowie durchgängige Verbindungskanäle zwischen den einzelnen Poren sollten eine Diffusion von großen (Bio)molekülen erleichtern, was bei bisherigen Systemen ein Problem darstellte. Die Copolymerzusammensetzung wurde dabei so gewählt, dass ein Kollaps des Hydrogels über 35 °C stattfand. Alle Hydrogele zeigten ausgeprägte Quellung in Wasser unterhalb der kritischen Temperatur. Der Einbau von reaktiven Comonomeren mit Hydroxylgruppen gewährleistete dabei die Funktionalisierbarkeit des Hydrogels mit Erkennungsgruppen für entsprechende Analytmoleküle, wie z.B. Proteine.
Als Testsystem wurde Biotin als Erkennungseinheit für Avidin in das Hydrogel mittels polymeranaloger Steglich Veresterung eingebaut. Die Menge an zugänglichem Biotin wurde dabei per colorimetrischem Bindungsassay quantifiziert. Dabei zeigte sich, dass sich die Wellenlänge der Reflexion nach Zugabe von Avidin zum biotinylierten inversen Hydrogelopal signifikant verschob und damit das Bindungsereignis visuell auslesbar ist. Dieser Effekt beruht auf dem veränderten Quellungsverhalten des Hydrogels nach Bindung des hydrophilen Proteins Avidin in Wasser, welches durch den thermosresponsiven Charakter des Hydrogels verstärkt ist. Ein Aufweiten oder Schrumpfen der Poren ändert die Abstände der gleichmäßig angeordneten Poren, welche für die Farbe des inversen Opals verantwortlich sind. Auf Basis dieser Erkenntnisse lassen sich möglicherweise Sensormaterialen für die Erkennung weiterer Biomoleküle in der Größenordnung von Avidin erstellen.
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Harnessing microgel softness for biointerfacingHendrickson, Grant R. 13 January 2014 (has links)
Hydrogel materials have become a heavily studied as materials for interfacing with biology both for laboratory investigations and the development of devices for biomedical applications. These polymers are water swellable and can be made responsive to many different stimuli by choice of monomers, co-monomers, and cross-linkers or functionalization with pendent ligands, substrates, or charged groups. The high water content, low moduli and potential responsively of these polymers make good candidates for biomaterials. A specific type of hydrogel called a microgel or a hydrogel micro/nanoparticle has similar properties to bulk hydrogel materials. Many of the interesting results and utility of the microgels in bioapplications are due to their inherent softness of the material. Here, the softness, flexibility, and conformability of these water swollen particles is used to create an interesting sensor platform, studied in the context of a microgel passing through a pore, and used as an emulsifier to create a drug delivery platform. The unifying theme of this dissertation is the softness of microgels which is critical for all of these experiments. However, the study of individual microgel softness is challenging and complex, since the softness is composed of two different components. The first is that the microgel is a swollen polymer which can be deswollen by an external stimuli or force. The second is that the microgel is a volume conserving elastic colloid which can deform without deswelling under the certain conditions. Throughout, this dissertation will discuss the ramifications of the complex softness of microgels in each experimental result and potential application.
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