Damage and/or loss of functional neurons can lead to detrimental cognitive and paralyzing effects in humans. Prime examples of such negative situations are well documented in patients with Parkinson's and Alzheimer's disease. In recent years, the utilization of neural stem cells and their derivation into neurons have been the focus of many research endeavors. The main reason for this is because neural stem cells are multi-potent and can differentiate into neurons, astrocytes, and oligodendrocytes. The research that will be detailed in this thesis involves the potential use of inorganic nanostructured materials to efficiently deliver bioactive molecules (i.e., retinoic acid, kinase inhibitors) to cells that can modulate the differentiation potential of certain cells into neurons. Specifically, PC12 (derived from rat pheochromocytoma) cells, as a neural model, was treated with select nanostructured materials with and without neuron inducers (molecules and ions) and the results were analyzed via biochemical assays and live-cell fluorescence microscopy. This thesis will include an in depth look into the cytocompatibility of the tested nanostructured materials that include silica nanoparticles, titanate nanotube microspheres, and carbon microparticles. / Bioengineering / Accompanied by two .avi files.

京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第23533号 / 人博第1012号 / 新制||人||239(附属図書館) / 2021||人博||1012(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 小松 直樹, 教授 津江 広人, 准教授 土屋 徹 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Copolymer-cobalt complexes have been formed by thermolysis of dicobalt octacarbonyl in solutions of copolysiloxanes. The copolysiloxane-cobalt complexes formed from toluene solutions of PDMS-b-[PMVS-co-PMTMS] block copolymers were annealed at 600-700 °C under nitrogen to form protective siliceous shells around the nanoparticles. Magnetic measurements after aging for several months in both air and in water suggest that the ceramic coatings do protect the cobalt against oxidation. However, after mechanical grinding, oxidation occurs. The specific saturation magnetization of the siliceous-cobalt nanoparticles increased substantially as a function of annealing temperature, and they have high magnetic moments for particles of this size of 60 emu g⁻¹ Co after heat-treatment at temperatures above 600 °C. The siliceous-cobalt nanoparticles can be re-functionalized with aminopropyltrimethoxysilane by condensing the coupling agent onto the nanoparticle surfaces in anhydrous, refluxing toluene. The concentration of primary amine obtained on the surfaces is in reasonable agreement with the charged concentrations. The surface amine groups can initiate L-lactide and the biodegradable polymer, poly(L-lactide), can be polymerized directly from the surface. The protected cobalt surface can also be re-functionalized with poly(dimethylsiloxane) and poly(ethylene oxide-co-propylene oxide) providing increased versatility for reacting polymers and functional groups onto the siliceous-cobalt nanoparticles.Phthalonitrile containing graft copolysiloxanes were synthesized and investigated as enhanced oxygen impermeable shell precursors for cobalt nanoparticles. The siloxane provided a silica precursor whereas the phthalonitrile provided a graphitic precursor. After pyrolysis, the surfaces were silicon rich and the complexes exhibited a substantial increase in Ms. Early aging data suggests that these complexes are oxidatively stable in air after mechanical grinding. Aqueous dispersions of macromolecule-magnetite complexes are desirable for biomedical applications. A series of vinylsilylpropanol initiators, where the vinyl groups vary from one to three, were prepared and utilized for the synthesis of heterobifunctional poly(ethylene oxide) oligomers with a free hydroxy group on one end and one to three vinylsilyl groups on the other end. The oligomers were further modified with carboxylic acids via ene-thiol addition reactions while preserving the hydroxyl functionality at the opposite terminus. The resulting carboxylic acid heterobifunctional PEO are currently being investigated as possible dispersion stabilizers for magnetite in aqueous media. / Ph. D.

Stabilization of highly sensitive noble metal nanoparticles is essential for their practical application. Bionanocomposites in which various types of noble metal nanoparticles, especially anisotropic noble metal nanoparticles, are immobilized into a macroscopic biomaterial membrane show promising applications in biomedical, catalytic, and environmental fields. This research focuses on developing two fabrication methods to generate novel bionanocomposite materials by immobilizing gold (Au) or silver (Ag) nanoparticles onto a "green" biomaterial, namely an eggshell membrane (ESM). Furthermore, the applications of the resulting bionanocomposite materials were demonstrated by studying their use as catalysts for environmental pollutant conversion and for the detection of two pollutant chemicals. The first fabrication method immobilizes ex situ synthesized nanoparticles onto a chemically modified ESM. Disulfide originating from the ESM was reduced by dithiothreitol into free thiol groups for binding to Au nanoparticles with different morphologies. The immobilization of Au nanoparticles greatly enhances their stability, making it possible to apply the resulting bionanocomposites for catalyzing the reduction reaction to convert pollutant p-nitrophenol (PNP) to p-aminophenol (PAP), with a great increase in their lifetime use from 2 to 10 reaction cycles. The second fabrication method utilizes the zwitterionic property of the protein based ESM for binding with Ag nanoparticles to form bionanocomposites. A seed mediated nanoparticle synthesis method originally performed in suspension was modified and adapted for the in situ synthesis of Ag nanodisks in this research. Ag nanoseeds were first immobilized onto an eggshell membrane using the static interaction between the nanoseeds and the membrane. Subsequently, Ag nanodisks were further grown directly on the Ag nanoseeds on the ESM. The final distribution density of Ag nanodisks can be adjusted by tuning the distribution density of Ag nanoseeds immobilized on the ESM. The performance of the resulting bionanocomposites were evaluated for both catalysis, and their application as substrates for surface enhanced Raman spectroscopy (SERS). The material performance was found to depend on the final distribution density of the Ag nanodisks on the ESM, offering the possibility to optimize bionanocomposite material performance by adjusting this density. A SERS based technique was further developed for detecting pollutant chemical species using the Ag nanodisks/ESM bionanocomposite material as a SERS substrate. Direct detection of thiram, a commonly used pesticide, was achieved at a concentration that is lower than that regulated by the US EPA. By using crystal violet as a SERS probe molecule, mercury, a heavy metal without an intrinsic Raman fingerprint, was indirectly detected not only at a limit of detection lower than most reported in the scientific literature, but also with a selectivity against a group of metal ions including Ba, Cu, Ca, Co, Mg, Mn, Ni, and Zn. It was also found that the detection sensitivity can be optimized by adjusting the Ag nanodisk distribution density on the ESM. The development of the fabrication approach and the use of ESM as a matrix material for immobilizing noble metal nanoparticles to form bionanocomposite materials demonstrates a novel strategy for meeting the needs of a variety of applications. The development of bionanocomposites for detecting pollutant species with different SERS activities by simply tuning the nanoparticle distribution density on the surface of the substrate, is a novel discovery, as it does not appear to have been previously reported in the literature. / Doctor of Philosophy / Noble metal nanoparticles exhibit special physical and chemical properties, which are totally different from the bulk material, making them promising candidates for use as novel materials in broad applications, such as catalysis, pollutant detection, antibacterial materials, etc. However, due to their high activity and poor colloidal stability (having high tendency to aggregate and lose activity), the nanoparticles require stabilization when being exploited for practical applications. A promising method to achieve this goal is to immobilize highly active noble metal nanoparticles onto a macroscopic membrane to form a nanocomposite. In this research, a "green" biomaterial, eggshell membrane (ESM), was utilized to immobilize noble metal nanoparticles. The resulting bionanocomposite materials were applied for catalyzing a reduction reaction to convert an environmental pollutant p-nitrophenol (PNP) to p-aminophenol (PAP) for environmental cleaning purposes, as well as detecting pollutant chemicals such as pesticide thiram and heavy metal mercury. General physical and chemical properties of the proteins in the ESM include rich chemical functional groups on the amino acid residue, and a zwitterionic property that allows the surface charge of the ESM to be changed under different pH levels. These properties, which have not been unleashed to immobilize noble metal nanoparticles in this field as of yet, were exploited in this research to create strong interactions between the noble metal nanoparticles and the ESM. This resulted in the formation of a bionanocomposite where the ESM served as a matrix for stably immobilizing the nanoparticles. Different bionanocomposites were fabricated using gold (Au) or silver (Ag) nanoparticles. The resulting bionanocomposite materials with gold nanoparticles were applied for catalyzing a reduction reaction for the conversion of p-nitrophenol, a commonly used chemical in the pharmaceutical photographic industries. The immobilized nanoparticles exhibited catalytic activity for ten reaction cycles and one hundred days after they were fabricated, while the colloidal nanoparticles (not immobilized nanoparticles) have catalytic activity for only two reaction cycles. For the chemical detection application, bionanocomposites with immobilized silver nanodisks were used as substrates for surface enhanced Raman spectroscopy. Different detection strategies were developed for detecting thiram with intrinsic Raman fingerprints and mercury without intrinsic Raman fingerprints. Outstanding detection sensitivities were achieved compared to those reported in the literature. For detection of mercury, a good selectivity was also obtained against a group of metal ions including Ba, Cu, Ca, Co, Mg, Mn, Ni, and Zn. The development of the fabrication approach and the use of ESM as a matrix material for immobilizing noble metal nanoparticles to form bionanocomposite materials demonstrates a good strategy for meeting the needs of a variety of applications

The delivery of nucleic acids remains the major obstacle for nucleic acid-based therapies such as gene therapy and gene silencing therapies based on RNA interference. In this dissertation we have developed and studied nucleic acid delivery vehicles based on cationic diblock glycopolymers that contain glucosamine and trehalosamine. Practical procedures were developed to synthesize 2-methacrylamido-2-deoxy glucose and 6-methacrylamido-6-deoxy trehalose starting with commercially available carbohydrates and utilizing trimethylsilyl protecting group chemistry. These monomers were polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield glycopolymers with the desired lengths and low polydispersity indexes. Glycopolymers were chain-extended with aminoethylmethacrylamide to produce cationic diblock copolymers. The ability of cationic diblock copolymers to bind nucleic acids was demonstrated with gel electrophoresis and heparin exclusion assays. Complexes of the synthesized polymers with nucleic acids were studied with dynamic light scattering to reveal nanoparticles of 100-250 nm that were stable in the presence of serum proteins. Quartz crystal microbalance experiments showed that serum proteins adsorb on polytrehalose coated gold surfaces and it was suggested that these interactions may help mask the polytrehalose coated nanoparticles from potential actions of the immune system. Polytrehalose was also shown to suppress water crystallization similarly to trehalose by lowering the energies associated with the water/ice phase transition. The property was utilized to freeze-dry siRNA containing polyplexes which could be re-dissolved in water after lyophilization to yield nanoparticles. The polyplexes formulated with cationic diblock copolymers were shown to efficiently enter cervical cancer cells (HeLa cell line) and glioblastoma cells (U-87 cell line) and to deliver their nucleic acid cargo. Polyglucose-containing polymers were efficient mediators of exogenous gene expression in HeLa cells, and polytrehalose- containing polymers were effective in promoting the target gene down-regulation via RNA interference by delivered siRNA. / Ph. D.

Polylactide-siloxane triblock copolymers with pendent carboxylic acid functional groups have been designed and synthesized for study as magnetite nanoparticle dispersion stabilizers. Magnetic nanoparticles are of interest in a variety of biomedical applications, including magnetic field-directed drug delivery and magnetic cell separations. Small magnetite nanoparticles are desirable due to their established biocompatibility and superparamagnetic (lack of magnetic hysteresis) behavior. For in-vivo applications it is important that the magnetic material be coated with biocompatible organic materials to afford dispersion characteristics or to further modify the surfaces of the complexes with biospecific moieties. The synthesis of the triblock copolymers is comprised of three reactions. Difunctional, controlled molecular weight polymethylvinylsiloxane oligomers with either aminopropyl or hydroxybutyl endgroups were prepared in ring-opening redistribution reactions. These oligomers were utilized as macroinitiators for ring-opening L-lactide to provide triblock materials with polymethylvinylsiloxane central blocks and poly(L-lactide) endblocks. The molecular weights of the poly(L-lactide) endblocks were controlled by the mass of L-lactide relative to the moles of macroinitiator. The vinyl groups on the polysiloxane center block were further functionalized with carboxylic acid groups by adding mercaptoacetic acid across the pendent double bonds in an ene-thiol free radical reaction. The carboxylic acid functional siloxane central block was designed to bind to the surfaces of magnetite nanoparticles, while the poly(L-lactide)s served as tailblocks to provide dispersion stabilization in solvents for the poly(L-lactide). The copolymers were complexed with magnetite nanoparticles by electrostatic adsorption of the carboxylates onto the iron oxide surfaces and these complexes were dispersible in dichloromethane. The poly(L-lactide) tailblocks extended into the dichloromethane and provided steric repulsion between the magnetite-polymer complexes. / Master of Science

Poly(dimethylsiloxane-b-methylvinylsiloxane) (PDMS-b-PMVS) diblock copolymers were synthesized via anionic living polymerization with controlled molecular weights and narrow molecular weight distributions. Targeted molecular weights agreed well with experimental values determined by 1H NMR, 29Si NMR, and GPC. Morphologies were investigated by DSC to analyze glass transition temperatures. Only one Tg was observed for each PDMS-b-PMVS block copolymer suggesting that the blocks were miscible in bulk. Tg's ranged from approximately -126 to -128 °C and were between the Tg's of the PDMS (-123 °C) and PMVS (-137 °C) homopolymers. The PMVS blocks were functionalized with trimethoxysilethyl or triethoxysilethyl pendent groups via hydrosilations to yield poly(dimethylsiloxane-b-[poly(methylvinyl)-co-(methyl-(2-trimethoxysilethyl)siloxane)] (PDMS-b-[PMVS-co-PMTMS]) or poly(dimethylsiloxane-b-[poly(methylvinyl)-co-(methyl-(2-triethoxysilethyl)siloxane)] (PDMS-b-[PMVS-co-PMTES]) copolymers, respectively. The PMVS blocks were either derivatized with the functional groups or half of the repeat units were functionalized. The fully hydrosilated materials were diblock copolymers, and the materials that were 50% hydrosilated had a random sequence of methylvinylsiloxy units and methyl-(trialkoxysilethyl)siloxy units. The PDMS-b-[PMVS-co-PMTES] block copolymers had Tg's ranging from -124 to -126 °C and only one Tg was observed. Surface tension measurements suggested that PDMS-b-[PMVS-co-PMTES] copolymers formed aggregates in toluene. Stable suspensions of superparamagnetic cobalt nanoparticles were prepared in toluene in the presence of PDMS-b-[PMVS-co-PMTMS] or PDMS-b-[PMVS-co-PMTES] copolymers via thermolysis of Co2(CO)8. It is hypothesized that the block copolymers functioned as micellar templates for the cobalt nanoparticles. TEM micrographs showed non-aggregated cobalt nanoparticles coated with copolymers that had mean particle diameters ranging from ≥10-15 nm. Specific saturation magnetizations of these cobalt-copolymer complexes ranged from 90-110 emu g-1 Co, comparable to literature values for this particle size. / Master of Science

Supramolecular, or non-covalent, interactions remain a hallmark of biological systems, dictating biologic activity from the structure of DNA to protein folding and cell-substrate interactions. Harnessing the power of supramolecular interactions commonly experienced in biological systems provides numerous functionalities for modifying synthetic materials. Hydrogen bonding, ionic interactions, and metal-ligand interactions highlight the supramolecular interactions examined in this work. Their broad utility in the fields of nanoparticle formulations, polymer chemistry, and additive manufacturing facilitated the generation of numerous biological materials. Metal-ligand interactions facilitated carbon nanohorn functionalization with quantum dots through the zinc-sulfur interaction. The incorporation of platinum-based chemotherapeutic cisplatin generated a theranostic nanohorn capable of real-time imaging and drug delivery concurrent with photothermal therapies. These nanoparticles remain non-toxic without chemotherapy, providing patient-specific. Furthermore, metal-ligand interactions proved vital to retaining quantum dots on nanoparticle surfaces for up to three days, both limiting their toxicity and enhancing their imaging potential. Controlled release of biologics remain highly sought-after, as they remain widely regarded as next-generation therapeutics for a number of diseases. Geometry-controlled release afforded by additive manufacturing advances next-generation drug delivery solutions. Poly(ether ester) ionomers composed of sulfonated isophthalate and poly(ethylene glycol) provided polymers well suited for low-temperature material extrusion additive manufacturing. Ionic interactions featured in the development of these ionomers and proved vital to their ultimate success to print from filament. Contrary to ionic interactions, hydrogen bonding ureas coupled poly(ethylene glycol) segments and provided superior mechanical properties compared to ionic interactions. Furthermore, the urea bond linking together poly(ethylene glycol) chains proved fully degradable over the course of one month in solution with urease. The strength of these supramolecular interactions demanded further examination in the photopolymerization of monofunctional monomers to create free-standing films. Furthermore, the incorporation of both hydrogen bonding acrylamides and ionic groups provided faster polymerization times and higher moduli films upon light irradiation. Vat photopolymerization additive manufacturing generated 3-dimensional parts from monofunctional monomers. These soluble parts created from additive manufacturing provide future scaffolds for controlled release applications. Controlled release, whether a biologic or chemotherapeutic, remains a vital portion of the biomedical sciences and supramolecular interactions provides the future of materials for these applications. / Ph. D. / Biology remains the unprecedented expert in the manipulation of non-covalent (or supramolecular) interactions to maintain structure and function. As an example, the structure of DNA maintains many hydrogen bonding units which allow for dynamic reading of genetic material but retain its characteristic structure. Proteins, made from linear chains of amino acids, utilize these interactions to fold into conformations necessary for their function. Harnessing these interactions in the creation of next-generation materials lies at the center of this work. Metal-sulfur bonds highlight initial work to encapsulate both drug and imaging agent onto a carbon nanoparticle. This complex revealed favorable biocompatibility and the ability to deliver drug in the elimination of bladder cancer cells in vitro. Furthermore, the complex revealed the maintenance of imaging capabilities over many days and continued to release low levels of chemotherapeutic during this time, potentially eradicating cancer cells long after initial treatment. Utilizing this nanoparticle, clinicians can monitor the location of nanoparticles in real-time and tailor doses specific to each patient. Ionic interactions provided enhanced mechanical properties of both water-soluble and water-insoluble polymers. The water-soluble polymers experienced significantly increased melt viscosity upon the addition of divalent cations, potentially creating non-covalent crosslinks in the molten state. Water-insoluble polymers acted as effective biological adhesives, likely arising from the interaction of ionic groups with its surrounding environment. Hydrogen bonding functioned to increase the mechanical integrity of water-soluble polymers for enhanced processing. The incorporation of urea groups into water-soluble polymers provided a readily available nitrogen source for plant growth while eliminating potential downstream environmental toxicity. Urethane functionality, generated with biologically-friendly byproducts, also provided hydrogen bonding to improve mechanical integrity of water-soluble polymers. Traditionally, stereolithography 3D printing demanded the use of covalent (or permanent) crosslinking to generate 3D shapes. Hydrogen bonding and ionic interactions coupled together to provide rapidly-formed free-standing films held together only through non-covalent interactions. Comparison of hydrogen bonding, ionic bonding, and both together provided insights onto the kinetics and strength of these films. These interactions proved strong enough to generate well-defined 3D structures through 3D printing. Furthermore, these parts proved water-soluble after fully forming, proving the reversibility of these bonds. Biologically-inspired interactions drive the future of materials research, and harnessing these interactions provides a better-performing material. Probing new materials for controlled release applications utilizing reversible interactions provided new families of ionic and hydrogen-bonding polymers. Whether soluble or insoluble, biological or not, these interactions pave the way to increase mechanical integrity of commonplace materials with the added reversibility hallmark of supramolecular interactions.

Plasmonic nanoparticles, specifically gold and silver nanoparticles, exhibit unique optical, physical, and chemical properties that are exploited in many biomedical applications. Due to their nanometer size, facile surface functionalization and enhanced optical performance, gold and silver nanoparticles can be used to investigate cellular biology. The work herein highlights a new methodology that has exploited these remarkable properties in order to probe various aspect of cancer cell biology, such as cell cycle progression, drug delivery, and cell death. Cell death mechanisms due to localized gold and silver nanoparticle exposure were also elucidated in this work. Chapter 1 introduces the reader to the synthesis and functionalization of gold and silver nanoparticles as well as reviews their implementation in biodiagnostic and therapeutic applications to provide a foundation for Chapters 3 and 4, where their use in spectroscopic and cytotoxic studies are presented. Chapter 2 provides the reader with detailed explanations of experimental protocols for nanoparticle synthesis and functionalization, in vitro cellular biology experiments, and live-cell Raman spectroscopy experiments that were utilized throughout Chapters 3 and 4. Chapter 3 presents the use of nuclear-targeted gold nanoparticles in conjunction with a Raman microscope modified to contain a live-cell imaging chamber to probe cancer cell cycle progression (Chapter 3.1), examine drug efficacy (Chapter 3.2), monitor drug delivery (Chapter 3.3), and detect apoptotic molecular events in real-time (Chapter 3.4). In Chapter 4, the intracellular effects of gold and silver nanoparticles are explored through live-cell Rayleigh imaging, cell cycle analysis and DNA damage (Chapter 4.1), as well as through the elucidation of cytotoxic cell death mechanisms after nanoparticle exposure (Chapter 4.2) and live cell imaging of silver nanoparticle treated cancer cell communities (Chapter 4.3).

Optical trapping is a method which uses focused laser light to manipulate small objects. This optical manipulation can be scaled below the diffraction limit by using interactions between light and apertures in a metal film to localize electric fields. This method can trap objects as small as several nanometers. The ability to determine the properties of a trapped nanoparticle is among the most pressing issues to the utilization of this method to a broader range of research and industrial applications. Presented here are two methods which demonstrate the ability to determine the properties of a trapped nanoparticle. The first method incorporates Raman spectroscopy into a trapping setup to obtain single particle identification. Raman spectroscopy provides a way to uniquely identify an object based on the light it scatters. Because Raman scattering is an intrinsically weak process, it has been difficult to obtain single particle sensitivity. Using localized electric fields at the trapping aperture, the Raman integrated trapping setup greatly enhances the optical interaction with the trapped particle enabling the required sensitivity. In this work, the trapping and identification of 20 nm titania and polystyrene nanoparticles is demonstrated. The second method uses an aperture assisted optical trap to detect the response of a magnetite nanoparticle to a varying applied magnetic field. This information is then used to determine the magnetic susceptibility, remanence, refractive index, and size distribution of the trapped particle. / Graduate / 0544 / 0752 / stevenjones3.14@gmail.com