The design and discovery of new surface-active polymers that self-assemble on solid substrates to form brush layers will have a major impact on numerous applications. Through recombinant DNA technology, there exists the potential to harness a cell's protein synthesis machinery to produce a brush-forming poly(amino acid) (or PAA) with an exactly specified amino acid sequence, thus controlling the polymer's composition at a level unequaled by conventional organic polymer synthesis. The presented work demonstrates the cloning, expression, purification and characterization of de novo-designed PAA's designed to form brush layers on alumina surfaces. Using conventional recombinant DNA methods, the feasibility of producing a PAA consisting of a poly-glutamate block and a poly-proline block was demonstrated. However, the PAA design was limited by the inherent limitations of conventional cloning techniques. We introduce here the development of a simple and versatile strategy for producing de novo-designed, high molecular weight PAA's using recombinant DNA technology. The basis of this strategy is that small DNA modules encoding for short PAA blocks can be easily inserted directly into a commercially available and unmodified expression vector. The insertions can be made repeatedly until the gene encodes for a polymer of desired molecular weight and composition. Thus, sequential modifications can be made to the PAA without having to re-start the gene assembly process from the beginning, thereby allowing for quick determination of how these changes affect polymer structure and function. The feasibility and simplicity of this method was shown during the production of a PAA, consisting of a long zwitterionic tail block and a short acidic anchor block, designed to form optimal brush layers on alumina surfaces. The success and flexibility of this method indicates that it can be applied for production of de novo-designed polypeptides in general. It is hoped that this method will contribute towards the rapid development of bio-inspired protein-based polymers for a variety of applications. This dissertation also contains research that aimed to use phage display technology to develop a new liposome-based immunoassay against biological toxins. This work was part of a collaboration effort with the U.S. Department of Defense and Luna Innovations. / Ph. D.

This work investigates processing-microstructure-property relationships of a model cryogenically mechanically alloyed polymer-polymer system: polycarbonate (PC) and poly (ether ether ketone) (PEEK). Mechanically milled and alloyed powders were characterized using a variety of techniques including microscopy and thermal analysis. Cryogenically mechanically alloyed powders processed for 10 hours were shown to have a sub-micron level two-phase microstructure. These powders were processed into testable coupons using a mini ram-injection molder; microstructure and bulk mechanical properties of the coupons were investigated as a function of mechanical alloying and injection molding parameters. Atomic force microscopy, transmission electron microscopy, and scanning transmission X-ray microscopy revealed that the intimate blending achieved during the mechanical alloying process is not retained upon post-processing using a conventional polymer processing technique. Injection molded coupons were tested in 3-point bend mode via dynamic mechanical and quasi-static mechanical testing. Results demonstrated that no improvement in energy to break, strain at failure, or failure strength was achieved in coupons made from cryogenically mechanically alloyed powders compared to those of coupons made from non-mechanically alloyed samples. / Ph. D.

Understanding the biological mechanisms of polymeric DNA delivery is essential to develop vehicles that perform optimally. In this work, the cellular internalization mechanisms of poly(glycoamidoamine) (PGAA) DNA delivery polymers were investigated. Polymer:DNA complexes interact with cell-surface glycosaminoglycans (GAGs) in a manner independent of electrostatic interactions. Desulfation and GAG removal leads to decreased uptake. Individual polyplexes appear to have differing affinities for specific GAGs, as polyplex dissociation occurs in a charge-independent manner, and may influence binding. Internalization occurs through close interactions with GAGs, as GAGs accumulate on polyplex surfaces, resulting in negatively-charged polyplexes and decompaction of intact polyplexes is observed upon interaction with GAG. PGAA polyplexes enter cells via a complex, multifaceted internalization route. Pharmacological inhibition of endocytosis and visualization by confocal microscopy reveal that internalization occurs primarily through an actin and dynamin-dependent mechanism. Caveolae/raft-mediated endocytosis appears to be the predominant internalization mechanism, with clathrin-mediated endocytosis also significantly involved. Internalization occurs to a smaller degree via macropinocytosis and direct membrane penetration. Caveolae-mediated, but not clathrin-mediated, internalization leads to transgene expression, suggesting a targeting opportunity based on uptake mechanisms. PEGylation of PGAA polyplexes was achieved to minimize polyplex aggregation in serum. Polyplex size increased in serum, but PEGylation prevented further polyplex growth over time compared to non-PEGylated polymers. Specific targeting of hepatocytes through end-modification of PEG with galactose was unsuccessful, likely due to inaccessibility of targeting groups. Further hepatocyte targeting efforts focused on malonate-based polymers with clickable linkages for facile linkage of targeting groups. Despite favorable surface presentation of galactose, receptor-specific internalization of polyplexes was unsuccessful, as competitive inhibition in HepG2 cells resulted in significant polyplex internalization derived from nonspecific membrane interactions. Chemical modification of vehicles allows systematic study of structure-function properties leading to efficient intracellular delivery. Increasing G4 molecular weight generally increases toxicity and decreases transgene expression in HeLa cells. Incorporating galactose into a lanthanide-chelating polymer facilitated efficient cellular internalization that was visualized by two-photon microscopy. Increased gene expression was observed that correlated to increasing galactose, suggesting that polymer degradation increases gene expression. Also studied were branched peptides targeted to HIV-1 TAR, which displayed high biocompatibility and favorable internalization profiles in mammalian cells. / Ph. D.

Superparamagnetic Magnetite (Fe3O4) nanoparticles were synthesized and complexed with carboxylate-functionalized block copolymers, and aqueous dispersions of the complexes were investigated as functions of their chemical and morphological structures. The block copolymer dispersants possessed either poly(ethylene oxide), poly(ethylene oxide-co-propylene oxide), or poly(ethylene oxide-b-propylene oxide) outer blocks, and all contained a polyurethane center block with pendant carboxylate functional groups. The complexes were formed through interactions of the carboxylates with the surfaces of the magnetite nanoparticles. Initial efforts utilized an aqueous coprecipitation method for the synthesis of magnetite nanoparticles, which yielded polydisperse magnetite nanoparticles. The nanoparticle complexes were characterized with a range of solution- and solid-state techniques including TGA, XPS, TEM, VSM, DLS and zeta potential measurements. DLVO calculation methods, which sum the contributions from van der Waals, steric, electrostatic and magnetic forces were utilized to examine the interparticle potentials in the presence and absence of external magnetic fields. Compositions were identified wherein a shallow, attractive interparticle potential minimum appears once the magnetic term is applied. This suggested the possibility of tuning the structures of superparamagnetic nanoparticle shells to allow discrete dispersions without a field, yet permit weak flocculation upon exposure to a field. This property has important implications for biomedical applications where movement of particles with an external magnetic field is desirable. In a second study, well-defined, narrow size dispersity magnetite nanoparticles were synthesized via the thermolysis of an iron (III) acetylacetonate (Fe(acac)3) precursor in the presence of benzyl alcohol. The magnetite nanoparticles were coated with triblock and pentablock copolymers possessing poly(ethylene oxide) and poly(propylene oxide-b-ethylene oxide) tailblocks and the carboxylate-functional anchor block. DLVO calculations were applied to the new magnetite particles and diagrams of potential energy versus interparticle distance indicated the predominant effect of steric and magnetic interactions on the particle stability. Exposure of the pentablock copolymer-magnetite complexes in phosphate buffered saline to a 1500 Oe magnetic field with concomitant DLS measurements indicated flocculation of the magnetic nanoparticles. DLS measurements showed increased hydrodynamic radii and scattering intensities with time. / Ph. D.

Nanoparticle usage continues to increase in everyday products, from cosmetics to food preservation coatings, drug delivery to polymer fillers. Their characterization and synthesis is of utmost importance to ensure safety and improved product quality. Nanoparticles can be sourced naturally or synthetically fabricated. Cellulose nanocrystals (CNCs) are rod-like nanoparticles that can be isolated from nature. Reliable methods of characterization are necessary to ensure quality control. However, their physical characteristics cause challenges for imaging under transmission electron microscopy (TEM) with a high enough resolution for dimensional analysis. Heavy metal staining such as radioactive uranyl acetate is often used to increase contrast and TEM sample substrate preparation techniques often use expensive equipment such as glow discharge in order to prevent CNC agglomeration. A method to reliably produce TEM images of CNCs without using radioactive stains or expensive glow discharge equipment was developed, using a vanadium-based stain branded NanoVan® and bovine serum albumin to keep CNCs dispersed while drying on the TEM substrate. Due to their aspect ratio, there is also concern of toxicity to the lungs. The concentration of CNCs in air in production facilities must be monitored, but there is currently no method tailored to CNCs. A method using UV-vis spectroscopy, dynamic light scattering, TEM, and scanning mobility particle sizer in conjunction with impinger collectors was developed for monitoring aerosolized CNC concentration. Synthetic nanoparticles are often used for controlled drug delivery systems. A new peptide drug termed αCT1 has been shown to interact with cell communication in a way that promotes wound healing, reduces inflammation and scarring, and aids in cancer therapy. However, the peptide's half-life in the body is estimated to be less than a day, which is not conducive to long-term treatments. Controlling its release into the body over several weeks can decrease the number of doses required, which is especially useful for glioblastoma treatment. Poly(lactic-co-glycolic acid) (PLGA) is often used for drug encapsulation since it hydrolyzes in the body and is biocompatible. Two methods of αCT1 encapsulation in PLGA were explored. It was found that flash nanoprecipitation increased loading of αCT1 in the particles by 1-2 orders of magnitude compared with the double emulsion method. Particles released αCT1 over three weeks and were non-cytotoxic. / PHD / Nanoparticle usage continues to increase in everyday products, from cosmetics to food preservation coatings, drug delivery to polymer fillers. Understanding the nature of nanoparticles is important to ensure safety and quality of commercial products, and production of particles allows for tailoring for specific applications. In this work, a technique to more easily create samples of cellulose nanocrystals (CNCs) for electron microscopy is developed. Electron microscopy can then be used to measure the size of these rod-like particles. Then, the technique is used to help develop a method to measure the concentration of CNCs in air. CNCs may irritate the lungs, so development of a way to measure their concentration in air is important to ensure safety of plant workers and consumers of CNCs. Characterization techniques of CNCs were used for synthesized particles used for brain cancer treatment. Synthesized particles contain the drug αCT1, which has been shown to reduce glioblastoma, or brain cancer, from becoming resistant to chemotherapy. These particles were made using poly(lactic-co-glycolic acid) (PLGA), a polymer that degrades in the body into lactic acid and glycolic acid. PLGA particles released αCT1 over three weeks and are of a size that is compatible with the brain. However, loading of the drug was low when using the first synthesis method. By switching particle synthesis methods, drug loading in the particles was increased by 1-2 orders of magnitude.

Wheat gluten, wheat gliadin, and corn zein agricultural proteins were evaluated as multifunctional additives that: (1) provided reinforcement, (2) improved thermal stability, and (3) lowered the cost of polymer composites. Wheat proteins were utilized in two polymer matrices: poly(vinyl alcohol) (PVA) and synthetic cis-1,4-polyisoprene rubber (IR). The proteins were hydrolyzed and dispersed in the polymer matrix, where they cooperatively self-assembled into nanostructures called amyloids. Amyloids have the potential for high rigidity and stability due to high β-sheet content. In Chapter II, trypsin hydrolyzed wheat gluten (THWG) proteins were incubated in aqueous PVA solutions, then the composite solutions were air dried and compression molded into films. Anisotropic protein aggregates formed through a typical mechanism of β-sheet self-assembly, where a greater molding time and pressure and/or a lower PVA molecular weight allowed for more protein aggregation. The larger protein structures provided less reinforcement. In Chapters III and IV, THWG and trypsin hydrolyzed gliadin (THGd), a component protein in wheat gluten, were compounded in synthetic polyisoprene rubber to form nanocomposites. The reinforcement correlated to the protein β-sheet content and varied with protein concentration, protein batch preparation, processing temperature, and compounding time. The isotropic β-sheet containing structures were very thermally stable, even under harsh rubber compounding conditions. By optimizing the processing parameters uniform protein dispersion and optimal IR reinforcement were achieved, although the protein and IR phases had poor compatibility. In Chapter V, the THGd-IR composites were cured using a typical cure package and molding process. Protein aggregation into nanostructured β-sheets was observed during the curing process. Rubber reinforcement increased as a function of protein concentration and curing time. In Chapter VI, a hydrophobic protein (zein) was substituted for the hydrophilic protein (gliadin) used previously to improve protein-IR compatibility. The zein protein was better at reinforcing IR, while gliadin improved mechanical stability. Both zein and gliadin improved the thermal stability of IR. The results from Chapters II-VI showed an interesting concept: in situ filler formation in polymer matrices where the choice of protein, polymer, and processing conditions influenced the final morphology and composite properties. / Doctor of Philosophy / We use plastics every day for a wide range of applications, from food packaging to automobile tires. Many of these plastics are composite materials, called “polymer composites,” meaning they are made of two or more chemically distinct materials where one material is a polymer. For reference, a polymer is a long chain molecule made of many (“poly-”) units (“- mer”). Polymer composites often contain additives which modify the properties of the polymer. For example, many soft polymers, such as tire rubber, need to be made stiffer and so a “reinforcing additive” is used to improve the stiffness of the rubber. Many composite materials are made stiffer so less material can be used. This process is called “lightweighting.” The automotive industry and food packaging industry use this process to reduce weight and fuel costs. In this research, plant proteins are tested as reinforcing additives in polymer composites. Plant proteins, such as wheat gluten, are abundant, non-toxic, sustainable, and can self-assemble into extremely small, stiff structures. For these reasons, plant proteins offer an environmentally friendly alternative to typical reinforcing additives. This dissertation shows that plant proteins can reinforce two polymers with very different properties. The first polymer is poly(vinyl alcohol) (PVA), which is biodegradable, hydrophilic (i.e., “water loving”), and is commonly used in flexible food packaging. The second polymer is synthetic cis-1,4-polyisoprene rubber (IR), which is non-biodegradable, hydrophobic (i.e., “water fearing”), and is commonly used in automotive tires. In Chapters II-V, the wheat gluten protein is hydrolyzed, i.e., chemically “chopped” into short chain peptides, to encourage the self-assembly of the plant protein into small, stiff structures. The self-assembled protein structures survive typical industrial processing techniques, such harsh rubber compounding conditions which involve high heat, pressure, and shear forces (i.e., the material is pushed in opposing directions). In Chapter VI, full corn and wheat proteins are incorporated into IR using standard industrial mixing and curing processes. The corn and wheat proteins reinforce the synthetic rubber and inhibit the degradation of the chemical structure of cured rubber under high heat. At certain protein concentrations, the proteins improve the elasticity and lessen the permanent deformation in the polymer composite. Together, Chapters II-VI show that proteins from diverse plant sources can be used to improve the performance of polymers with dissimilar properties.

Poly(arylene ether)s are a well-established class of thermoplastics that are known for their mechanical toughness, thermal stability, and fabrication into membranes. These materials can undergo a myriad of modifications including backbone structure variability, sulfonation, and crosslinking. In this dissertation, structure-property relationships are considered for poly(arylene ether)s with regard to membrane applications for proton exchange and gas separation membranes. All of the proton exchange membranes in this dissertation focus on a disulfonated poly(arylene ether sulfone) based hydrophilic structure to produce hydrophilic-hydrophobic multiblock copolymers. The hydrophobic segments were based upon poly(arylene ether benzonitrile) polymers and copolymers. The oligomers were synthesized and isolated separately, then reacted under mild conditions to form the alternating multiblock copolymers. Structure-property relationships were considered for two different proton exchange membrane applications. One multiblock copolymer system was for H2/air fuel cells, and the other for direct methanol fuel cells (DMFCs). The H2/air fuel cells operate under harsh conditions and varying levels of relative humidity, while the DMFCs operate in an aqueous environment with a methanol-water mixture (typically 0.5-1 M MeOH). Thus two different approaches were taken for the multiblock copolymers. All of the multiblock copolymers were cast into membranes and after annealing resulted in drastically reduced water uptake as compared to random and non-annealed systems. The membranes were characterized with regard to composition, mechanical properties, morphology, water uptake, proton conductivity, and molecular weight. Membranes were also sent to collaborators to elicit the fuel cell performance of the proton exchange membranes. In H2/air fuel cells the approach was to increase charge density by bisphenol choice in the hydrophilic phase. This was performed by switching to a lower molecular weight monomer, hydroquinone, and a monosulfonated hydroquinone. This produced higher charge density in the hydrophilic phase, and the corresponding multiblock copolymer. With increased hydrophilicity the multiblock copolymers showed increased phase separation, proton conductivity, and better performance under relative humidity testing. In the second system for DMFCs, the primary goal was to reduce methanol permeability by bisphenol selection in the hydrophobic phase. This was done with by replacing fifty mole percent of the fluorinated monomer with a series of increasing hydrophobicity bisphenols. Addition of benzylic methyl groups on the bisphenols, was the method undertaken to increase the hydrophobicity. The combination of reduced fluorine content along with the addition of methyl groups resulted in multiblock copolymers with extremely low water uptake and methanol permeability. This allowed for a PEM with better performance than Nafion® in 1M MeOH in DMFC testing. The gas separation membranes presented in this dissertation are based upon poly(arylene ether ketone)s. Two systems were presented: one with a polymer directly synthesized with a bisphenol containing benzylic methyl groups and 4,4'-difluorobenzophenone, and the other a difunctional poly(phenylene oxide) oligomer polymerized with 4,4'-difluorobenzophenone. These systems were crosslinked via UV light through excitation of the ketone group to the triplet state and then hydrogen abstraction from the benzylic methyl. Confirmation of crosslinking was performed via differential scanning calorimetry and infrared spectroscopy. Changes in the glass transitions between crosslinked and non-crosslinked materials were characterized with respect to the concentration of ketones to elicit the effects of crosslink density on the polymers and copolymers. Gas transport properties showed a strong dependence on the ketone percentage as the selectivity was much higher for the homopolymer, while the permeability was higher for the PPO copolymer in the CO2/CH4 and O2/N2 gas pairs. / Ph. D.

Dibenzo-30-crown-10 (DB30C10) is one of the first-generation macrocyclic hosts discovered by Pedersen. Crown ethers originally attracted attention due to their ability to encapsulate metal cations and render them soluble in organic solvents. These studies helped to launch host-guest chemistry as a discipline within supramolecular chemistry. Crown ethers form complex molecules containing organic cations and neutral organic molecules. Additionally, they form components in supramolecular architectures such as catenanes, rotaxanes, and supramolecular polymers. They have been used as selective hosts in diverse applications such as wastewater treatment, switchable catalysis, therapeutic agents, sensors, molecular machines, and stimuli responsive materials "smart polymers". Despite the vigorous research activity in the field, DB30C10 has received surprisingly little attention. DB30C10 was reported in 1967 and has been commercially available since 1992; however, it has been mostly overlooked as a host in favour of smaller crown ethers such as DB24C8, B15C5, 18C6 and 15C5. Herein we present an improved synthetic route that improves the yield of the cyclization step in the synthesis of DB30C10 from 25% to 88% enabling us to prepare multiple grams of the material without the use of pseudo high-dilution techniques. The same methodology was applied to three other crown ethers with similar improvement in yield. Four new rotaxanes based on the DB30C10-paraquat binding motif were used to investigate the binding conformation of DB30C10 and paraquat. The new rotaxanes were characterized by 1H, 13C and 2D-NOESY NMR, mp, and HRMS. A single crystal X-ray structure of one of the [2]rotaxanes was obtained. To our knowledge, this is the first crystal structure of a rotaxane based on this particular binding motif. This result illustrated that DB30C10 was a suitable host for the construction of supramolecular systems and polymers. Our eventual goal is to use DB30C10 in the construction of supramolecular polymers with novel topologies. Therefore, the relative threading efficiency of DB30C10 in solution had to be determined. A series of segmented polyurethane poly(pseudorotaxanes) with paraquats in the backbone were synthesized with different crown ether or cyptand hosts. The threading efficiency was determined by 1H NMR. / Ph. D.

Yes / Condensation of a pyridyl-2-carbaldehyde derivative with 2-(bromoethyl)amine hydrobromide gave tetracyclic pyrido[1,2-a]pyrido[1’,2’:3,4]imidazo-[2,1-c]-6,7-dihydropyrazinium dications in excellent yields. Crystal structures and NOE data demonstrated the helical character of the dications, the dihedral angles between the two pyrido groups ranging from 28–458. An intermediate in the synthesis was also characterized. A much brighter emission compared to literature helicenes has been found, with quantum yields as high as 60% in the range of l=460– 600 nm. Preliminary cytotoxicity studies against HT-29 cancer cells demonstrated moderate-to-good activity, with IC50 values 12–30x that of cisplatin.

Yes / Interpolymer complexes of homopolymer macromolecules are often described as ‘laddered’ or ‘ribbon’ type structures. The proposition of the existence of these ladder structures seems to us not reasonable and here we examine this hypothesis. To address this we have used polymers enabled for Förster Energy Transfer (FRET). Chromophores bound to a macromolecular backbone can transfer energy across short distances via FRET. The close binding of poly(acrylamide) and poly(acrylic acid) interpolymer complex formation at low pH forms a structure compact enough for significant energy transfer to occur between different chains containing naphthalene and anthracene labels. In the context of the proposition that ladder polymers can form it was surprising that the distance between labels on the same polymer back-bone was equivalent regardless of whether the polymer was complexed or not. The data indicated that the bicomponent structure may be more compact than previously supposed: I.e. the complexes are not ladders composed of extended chains. This evidence suggests formation not of ordered ‘ladder’ systems but colloidal ‘co-globules’. / This work was carried out in part thanks to an EPSRC CASE funded PhD studentship at the University of Sheffield, sponsored by SNF (UK) Ltd.