abstract: Environmentally responsive microgels have drawn significant attention due to their intrinsic ability to change volume in response to various external stimuli such as pH, temperature, osmotic pressure, or electric and magnetic fields. The extent of particle swelling is controlled by the nature of the polymer-solvent interaction. This thesis focuses on design and synthesis of environmentally responsive microgels and their composites, and encompasses methods of utilizing microgel systems in applications as vehicles for the adsorption, retention, and targeted delivery of chemical species. Furthermore, self-assembled microgel particles at ionic liquid (IL)-water interfaces demonstrate responsive colloidal lattice morphology. The thesis first reports on the fundamental aspects of synthesis, functionalization, and characteristic properties of multifunctional environmentally responsive microgels derived from poly(N-isopropylacrylamide) (PNIPAm) and other functional co-monomers. In particular, the uptake and release of active chemical species such as rheology modifiers into and from these ionic microgels is demonstrated. Moreover, a facile tunable method for the formation of organic-inorganic composites with Fe3O4 nanoparticles adsorbed and embedded within ionic microgel particles is explored. Additionally, the development of zwitterionic microgels (ZI-MG) is presented. These aqueous ZI-MG dispersions exhibit reversible parabolic swelling as a function of pH and display a minimum hydrodynamic diameter at a tunable isoelectric point (IEP). This study also elucidates the controlled uptake and release of surfactants from these particle systems. The extent of surfactant loading and the ensuing relative swelling/deswelling behaviors within the polymer networks are explained in terms of their binding interactions. The latter part of this thesis highlights the versatility of fluorescently labeled microgel particles as stabilizers for IL-water droplets. When the prepared particles form monolayers and equilibrate at the liquid-liquid interface, the colloidal lattice organization may re-order itself depending on the surface charge of these particles. Finally, it is shown that the spontaneously formed and densely packed layers of microgel particles can be employed for extraction applications, as the interface remains permeable to small active species. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2015

Tissue response to PNIPAM and HPC nanoparticles has been studied by implantation method. The results suggest that both PNIAPM and HPC nanoparticles possess good biocompatibility and they may serve as a good carrier for the applications of controlled delivery. Rheological properties of dispersions of IPN microgels composed of PNIPAM and PAAc have been studied. It is found that the IPN microgel dispersion can undergo a sol-gel transition at temperature above 33°C. In vivo drug release experiments suggest that the gelation procedure creates a diffusion barrier and thus leads to slow release. An emulsion method has been used to grow columnar crystals by mixing PNIPAM microgel dispersions with organic solvents. Effect of both temperature and microgel concentration on formation of columnar crystals has been studied. PNIPAM-co-NMA microgels have been used for the fabrication of crystalline hydrogel films by self-crosslinking microgels. The hydrogel film exhibits an iridescent. The thermally responsive properties and mechanical properties of this film have been studied. Melting temperature (Tm) of colloidal crystals self-assembled with PNIPAM-co-AAc microgels has been investigated as a function of pH, salt concentration and microgel concentration. It is revealed that Tm increases as pH value increases; Tm decreases with increase of salt concentration; Tm increases as microgel concentration increases. Phase behavior of PNIPAM-co-HEAc microgel dispersions has been investigated. It is observed that these microgel dispersions exhibit liquid, crystal, and glass phase. As microgel size increases, crystal phase shifts to low concentration range. As temperature increases, crystal phase shifts to high concentration ranges. These colloidal crystals can be stabilized by NaOH-induced gelation. Effect of NaOH concentration on formation of physical gelation has been investigated.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21811号 / 農博第2324号 / 新制||農||1066(附属図書館) / 学位論文||H31||N5183(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 松村 康生, 教授 白岩 立彦, 教授 丸山 伸之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Gewebe sind nicht nur durch ihre biochemische Zusammensetzung definiert, sondern auch durch ihre individuellen mechanischen Eigenschaften. Inzwischen ist es weithin akzeptiert, dass Zellen ihre mechanische Umgebung spüren und darauf reagieren. Zum Beispiel werden Zellmigration und die Differenzierung von Stammzellen durch die Umgebungssteifigkeit beeinflusst. Um diese Effekte in vitro zu untersuchen, wurden viele Zellkulturstudien auf 2D Hydrogelsubstraten durchgeführt. Zusätzlich dazu steigt die Anzahl von Studien an, die hydrogelbasierte 3D-Scaffolds nutzen, um 2D Studien zu validieren und die experimentellen Bedingungen der Situation in vivo anzunähern. Jedoch erweist es sich weiterhin als schwierig den Effekt von Mechanik in 3D in vitro zu untersuchen, da in den gemeinhin genutzten 3D Hydrogelsystemen immer eine Kopplung zwischen Gelporosität und Steifigkeit besteht. Zusätzlich hängt die Konzentration der biologisch aktiven Bindungsstellen für Zellen oft ebenfalls von der Steifigkeit ab. Diese Arbeit präsentiert die Entwicklung und Optimierung neuer 3D Hydrogelkugel-Scaffolds, in denen die Steifigkeit von der Porosität schließlich entkoppelt wird. Mit Hydrogelkugeln als Scaffold-Bausteine ist es nun möglich 3D Scaffolds mit definierten mechanischen Eigenschaften und konstanter Porengröße zu generieren. Während der Methodenentwicklung wurden verschiedene Prinzipien und Kultivierungskammern konstruiert und überarbeitet, gefolgt von der theoretischen Betrachtung der Sauerstoffdiffusion, um die Eignung der gewählten Kammer hinsichtlich Zellvitalität und Zellwachstum zu überprüfen. Eine Kombination aus mehreren getesteten Filtern wurde ausgewählt um HydrogelkugelScaffolds erfolgreich in der ausgewählten Kammer zu generieren. Im Weiteren wurden verschiedene Hydrogelmaterialien untersucht hinsichtlich der erfolgreichen Produktion monodisperser Hydrogelkugeln und der Erzeugung stabiler Scaffolds. Hydrogelkugeln aus Polyacrylamid (PAAm) wurden als Scaffold-Bausteine ausgewählt um damit die Eignung des entwickelten Systems zu demonstrieren lebende Zellen zu mikroskopieren. Außerdem wurde das Überleben von Fibroblasten über vier Tage in unterschiedlich steifen HydrogelkugelScaffolds erfolgreich gezeigt. Weiterhin war es möglich erste Zellmigrationsexperimente durchzuführen. Dafür wurden sowohl einfache PAAm-Hydrogelkugeln als auch mit Adhäsionsmolekülen funktionalisierte Hydrogelkugeln genutzt, um unterschiedlich steife Schichten in einem Scaffold zu erzeugen. Dadurch war es möglich nicht nur Zellmigration anhand von Zelladhäsion in 3D Scaffolds mit Steifigkeitsgradienten zu beobachten, sondern auch Zellmigration ohne Zelladhäsion.:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis / Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. For example, cell migration and stem cell differentiation is affected by stiffness. To study these effects in vitro, many cell culture studies have been performed on 2D hydrogel substrates. Additionally, the amount of 3D studies based on hydrogels as 3D scaffold is increasing to validate 2D in vitro studies and adjust experimental conditions closer to the situation in vivo. However, studying the effects of mechanics in vitro in 3D is still challenging as commonly used 3D hydrogel assays always link gel porosity with stiffness. Additionally, the concentration of biologically active adhesion sides often also depends on the stiffness. This work presents the development and optimization of novel 3D hydrogel bead scaffolds where the stiffness is finally decoupled from porosity. With hydrogel beads as scaffold building blocks it was possible to generate 3D scaffolds with defined mechanical properties and a constant pore size. During the method development, different culture devices were constructed and revised, followed by oxygen diffusion simulations to proof the suitability of the chosen device for cell survival and growth. A combination of different filter approaches was selected to generate hydrogel bead scaffolds in the culture device. Furthermore, different hydrogel materials were investigated regarding successful production of monodisperse beads and stable scaffold generation. Polyacrylamide (PAAm) hydrogel beads were chosen as scaffold building blocks to demonstrate live-cell imaging and successful cell survival over four days in differently compliant hydrogel bead scaffolds. Moreover, first cell migration experiments were performed by using plain PAAm hydrogel beads as well as PAAm hydrogel beads functionalized with adhesion molecules with differently stiff layers in one scaffold. Thereby fibroblast migration was observed not only in adhesion-dependent migration manner, but also in an adhesion-independent mode .:1 Introduction 1.1 Mechanics play a role in biology 1.2 3D cultures and scaffolds 1.3 3D hydrogel systems to study effects of mechanics 1.4 Decoupling stiffness and porosity in 3D scaffolds 2 Materials 3 Methods 3.1 Laser scanning microscopy and microscopy data processing 3.2 Atomic force microscopy (AFM) 3.3 Refractive index matching of PMMA beads 3.4 Regular PMMA bead scaffolds for developing analysis algorithm 3.5 Cell culture standards 3.6 Fluorescent labelling of ULGP agarose 3.7 Production of polydisperse ULGP agarose beads 3.8 Hydrogel bead production via microfluidics 3.9 PAAm bead functionalization 3.10 Real-time fluorescence and deformability cytometry (RT-fDC) 3.11 3D scaffolds made from hydrogel beads 3.12 Statistics 4 Results 4.1 Design of a suitable scaffold device 4.2 Theoretical oxygen supply in 3D culture system is sufficient for cell survival and proliferation 4.3 Further optimization of 3D scaffold device 4.3.1 PMMA beads can be arranged in stable scaffolds 4.3.2 Regular PMMA bead scaffolds can be achieved and analysed 4.3.3 PMMA bead scaffolds and agarose bead scaffolds act as combined filter to stack up hydrogel beads 4.4 PAAm hydrogel beads produced by microfluidics are suitable to create compliant 3D scaffolds 4.5 Reproducible, regular and stable 3D scaffolds made of hydrogel beads 4.6 NIH-3T3/GFP cell migration within 3D hydrogel bead scaffolds 5 Discussion and Concluding Remarks 6 Bibliography List of Figures List of Tables Eigenständigkeitserklärung Appendix A Appendix B FIJI macro for FFT analysis maxima Python script to determine regularity of PMMA bead scaffolds Excel macro to determine number of peaks for regularity analysis

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<p>The mechanical and chemical properties of hydrogels make them excellent vehicles to deliver drugs. However, current systems encounter difficulties with loading hydrophobic molecules into the aqueous gel network and the subsequent release of the drug from the gel matrix. Cyclodextrins (CDs) offer a potential solution to this drug delivery challenge. CDs have the unique property of possessing a hydrophilic exterior and a hydrophobic interior pocket which is capable of hydrophobic drug binding. CD molecules complexed with hydrophobic drugs have been demonstrated to significantly increase the bioavailability of those drugs in free solution. Thus, if these nanodomains are introduced into microgels or hydrogels, we anticipate that significantly higher hydrophobic drug loadings may be achieved together with improved controlled release of these drugs based on the properties of the hydrogel or microgel phase. We have fabricated <em>in situ</em> gellable and degradable hydrogels and microgels based on combinations of CDs and either functionalized carbohydrates (dextran) or thermosensitive synthetic polymers (poly(N-isopropylacrylamide), PNIPAM). To achieve this goal, we designed a series of microgels with grafted or immobilized CD groups and used multi-functional CD as a reactive crosslinker for making injectable bulk hydrogels.</p> / Master of Applied Science (MASc)

Although medical devices and biomaterial implants are used clinically in a variety of applications, the process of implanting them damages local tissue and initiates a localized non-specific inflammatory response that is detrimental to device performance. Extensive research efforts have focused on developing material surface treatments and systems to deliver anti-inflammatory agents to abrogate such biomaterial-mediated inflammation, yet long-term use of these traditional materials in vivo is limited due to continued inflammation and fibrous encapsulation. This work aims to address these limitations by developing a versatile implant coating with non-fouling properties using a system based on hydrogel microparticles (i.e. microgels). The overall objective of this project was to evaluate host responses to these microgel coatings. Microgel particles were synthesized from poly(N-isopropyl acrylamide) cross-linked with poly(ethylene glycol)-diacrylate and were successfully deposited onto polymeric substrates using a simple and reproducible spin coating technique. We determined that microgel-coated samples adsorbed significantly lower levels of human fibrinogen than controls. Further characterization using an in vitro culture system demonstrated that microgel coatings significantly reduced the adhesion and spreading of murine macrophages and primary human blood-derived monocytes compared to controls. Materials were then evaluated for early cellular responses following implantation in the intraperitoneal cavity of mice to model acute inflammation. Analyses of explanted biomaterials using immunofluorescence staining techniques revealed that microgel-coated samples significantly reduced the density of surface-adherent cells. Additional analysis using flow cytometry revealed that microgel-coated samples exhibited significantly lower levels of pro-inflammatory cytokines in adherent leukocytes compared to controls, indicating that these coatings modulate cellular pro-inflammatory activities. Finally, we implanted samples subcutaneously in rats to determine the efficacy of microgel coatings at longer time points using an established model of chronic inflammation. Explants were processed histologically and stained for various markers. Importantly, staining demonstrated that the microgel coatings significantly reduced fibrous capsule thickness, the capsules appeared less compact and structurally ordered than controls, and also contained significantly fewer cells. Collectively, these results demonstrate that microgel particles can be applied as polymeric coatings to modulate inflammation and achieve more desirable host responses in vivo, with the potential to extend implant lifetime.

Hydrogel micro- and nanoparticles (microgels and nanogels) are a promising class of drug delivery vehicles. Composed of hydrophilic polymers arranged into a cross-linked network structure, nanogels show several attractive features for the delivery of macromolecule therapeutics. For instance, the hydrated, porous internal cavity of the nanogel may serve as a high capacity compartment for loading macromolecules, whereas the periphery of the nanogel may be used as a scaffold for conjugating cell-specific targeting moieties. This dissertation presents recent investigations of nanogels as targeted delivery vehicles for oligonucleotides to cancer cells, while exploring new nanogel chemistries that enable future in vivo applications. For instance, synthetic efforts have produced particles capable of erosion into low molar mass constituents, providing a possible mechanism of particle clearance after repeated administration in vivo. In another example, the microgel network chemistry was tuned to promote the encapsulation of charged proteins. In parallel with those synthetic efforts, new light scattering methodologies were developed to accurately quantify the particle behaviors (e.g. loading, erosion). Using multiangle light scattering (MALS), changes in particle molar mass and radius were measured, providing a quantitative and direct approach for monitoring nanogel erosion and macromolecule encapsulation. The new particle chemistries demonstrated, together with enabling light scattering methods, will catalyze the development of improved delivery vehicles in the near future.

Poly(3,4-ethylenedioxythiophene), PEDOT, belongs to the group of conducting polymers and is characterized by its high stability, a moderate band gap and its optical transparency in the conductive state. A large disadvantage of conducting polymers, and also PEDOT, is their poor solubility. One way to achieve processible materials is the synthesis of colloidal particles. Thus, this work focuses on the development of conductive particles by preparing composite structures. Polymeric colloids like latex particles and microgels were used as templates for the oxidative polymerization of EDOT. Depending on template structure completely different composite morphologies with variable properties were obtained. It was found that modification with PEDOT did not only cause conductive particles for application as humidity sensor materials, but also candidates for further functionalization with gold nanoparticles (Au-NPs). Due to a multi-stage synthesis route it was possible to achieve polystyrene(core)-PEDOT(shell)-particles decored with Au-NPs. Microgels acting as “micro reactors” for the incorporation of PEDOT and Au-NPs were also used for preparing multifunctional composites for catalytic applications. / Poly(3,4-ethylendioxythiophen), PEDOT, gehört zur Gruppe der leitfähigen Polymere und zeichnet sich durch seine hohe Stabilität, eine moderate Bandlücke und seine optische Transparenz im dotierten Zustand aus. Ein Nachteil leitfähiger Polymere, wie auch von PEDOT, ist deren schlechte Löslichkeit. Die Synthese kolloidaler Partikel bietet jedoch eine Möglichkeit dieses Problem zu umgehen. In diesem Zusammenhang richtete sich der Fokus dieser Arbeit auf die Darstellung leitfähiger Partikel in Form von Kompositstrukturen. Polymerkolloide, wie Latex- und Mikrogelpartikel, sind als Template eingesetzt worden, in deren Gegenwart PEDOT durch eine oxidative Polymerisation synthetisiert wurde. In Abhängigkeit von der Struktur des Templats sind unterschiedliche Kompositmorphologien mit steuerbaren Eigenschaften erhalten worden. Auf diese Weise wurden neben Materialien für die Feuchtigkeitssensorik leitfähige Kompositpartikel hergestellt, die zusätzlich mit Gold-Nanopartikeln (Au-NP) funktionalisiert werden konnten. Durch ein mehrstufiges Syntheseverfahren sind somit Polystyrol(Kern)-PEDOT(Schale)-Partikel mit Au-NP-funktionalisierter Oberfläche synthetisiert worden. Mikrogelpartikel, die als „Mikroreaktoren“ für die Inkorporation von PEDOT- und Au-NP dienten, wurden ebenfalls eingesetzt, um multifunktionale Komposite mit katalytischen Eigenschaften herzustellen.

Gegenstand dieser Arbeit ist die Herstellung funktioneller Polymerpartikel und deren Nutzung für biomedizinische Applikationen. Die Anwendungsgebiete der resultierenden Hybridmaterialien reichen vom Einsatz als Kontrastmittel in bildgebenden Verfahren der medizinischen Diagnostik über die Verwendung als Antimikrobium bis hin zum Einsatz als „Werkzeug“ zur Zellisolierung und aktivierung. Dazu wurden kompakte Latexpartikel und sensitive, poröse Mikrogelpartikel mittels emulgatorfreier Heterophasenpolymerisation synthetisiert. Als funktionelles Monomer wurde Glycidylmethacrylat verwendet, über dessen reaktive Epoxygruppen anschließend weitere Moleküle angebunden werden können. Die Funktionalisierung der Polymerpartikel erfolgte einerseits mit anorganischen Nanopartikeln (dotierte Lanthanfluorid-Nanopartikel, Gold- und Silbernanopartikel) und andererseits mit Biomolekülen wie Nukleotiden und Antikörpern. Einige Verwendungsgebiete, wie die Stimulierung von Memory-T-Zellen mit Antikörper-Polymer-Konjugaten oder der Einsatz der Silberkomposite aufgrund ihrer antimikrobiellen Wirkung, wurden näher untersucht. / The aim of this work was the synthesis of functional polymeric particles and their use for biomedical purposes. The application areas of the resulting hybrid materials range from contrast agents in medical diagnostics and usage due to antimicrobial properties to “tools” for cell isolation and activation. Compact core-shell particles and porous microgel particles were prepared by surfactant-free heterophase polymerization in water. All particles contain glycidyl methacrylate whose epoxy groups are capable to bind other molecules covalently. On the one hand, polymeric particles were functionalized with inorganic nanoparticles (doped lanthanum fluoride nanoparticles, gold and silver nanopariticles) and on the other hand with biomolecules such as nucleotides and antibodies. Selected application fields like the stimulation of memory T-cells with polymer-antibody-conjugates or the use of the silver composites due to their antimicrobial activity were investigated in detail.