As of 2010, cancer remains the leading cause of death in Canada, and second in the United States of America. This is despite decades of research into development of chemotherapeutics and diagnostics. A number of major challenges have prevented new discoveries from translating into a reduction in mortality rates. One challenge is the poor efficiency with which anti-cancer agents (diagnostic contrast agents and therapeutics) reach deregulated cells in the body. Therefore, development of new methods and technologies for improving efficiency of delivery has been a focus of research. Nanoparticles are leading candidates for improving the efficiency of delivery because they can act as payload vehicles for anti-cancer agents, because it is possible to mediate their interaction with biological systems and thus their pharmaockinetics, and because they can exploit inherent vulnerabilities of tumours. This thesis describes the results from a series of research projects designed to progress our understanding of how nanoparticles behave in vivo, and how their design can be optimized to improve tumour targeting.

1,3-β-glucans are a class of natural polysaccharides with unique pharmacological properties and the ability to form triple helical structures and resilient gels. Curdlan and other 1,3-β-glucans have found application pharmacologically in the treatment of cancers and acceleration of wound healing in humans and in the impartation of infection resistance in animal husbandry. Structurally, these polysaccharides have found application in food science as thermal gels, in nanostructure formation as helical scaffolds, and in drug delivery as nanocarriers for drugs and as inclusion complexes with polynucleotides. A literature review of the important work on Curdlan research reveals two streams of research: investigation of the pharmacological significance of these polymers and their application in increasing host immunocompetency and investigation of the nature of the triple helix and its application in a variety of fields from food gels to drug delivery. Two significant contributions to the field of Curdlan research have been completed including 1) The development of a Curdlan nanoparticle drug delivery platform and 2) A new multi-component liquid crystalline hydrogel providing a new route to form polynucleotide inclusion complexes with Curdlan for gene delivery. The developed nanoparticle platform exhibited high encapsulation of chemotherapeutic drugs and a 24-hour controlled release with a particle size of 109.9 nm. The liquid crystalline hydrogel exhibited homogeneous inclusion of DNA into amorphous and crystalline phases of Curdlan and delayed and triggered release of polynucleotide content. This work has been a significant demonstration of the potential of Curdlan as a new polymer for multi-functional drug delivery.

Plasmonic properties of self-assembled silver nanoparticles/nanowires array on periodically patterned Si (100) substrate are reported with special attention on the mechanism of nanoparticles self-assembly. The advantage of this bottom up approach over other self-assembling and lithographic methods is the flexibility to tune array periodicity down to 20 nm with interparticle gaps as low as 5 nm along the ripple. Ripple pattern have shallow modulation (~2 nm) still particles self-assembly was observed in non-shadow deposition. Therefore adatoms diffusion and kinetics is important on ripple surface for the self-assembly. PVD e-beam evaporation method used for deposition has proven to be superior to sputter deposition due to lower incident flux and lower atom energy. It was found that particles self-assembly largely dependent on angle of incidence, substrate temperature, and deposition direction due to ripple asymmetric tilt. Ostwald ripening observed during annealing on ripples substrate has striking dependency on ripple periodicity and was found to be different compared to Ostwald ripening on flat Si surface. In-situ RBS measurements of deposited silver on flat and rippled substrate confirmed different sticking of atoms on the two surfaces. The difference between maximum and minimum of the calculated local flux show a peak at an incidence angle of 70o with respect to surface normal. This explains the best alignment of particles at this angle of incidence compare to others. Self-assembled nanoparticles are optically anisotropic, i.e. they exhibit a direction dependent shift in LSPR. The reason of the observed anisotropy is a direction dependent plasmonic coupling. Different in plane and out of the plane dielectric coefficients calculated by modelling Jones matrix elements, confirms that nanoparticle/nanowire array are biaxial anisotropic (ex ¹ ey ¹ ez). The nanoparticles are predominantly insulating while nanowires are both metallic and insulating depending on the dimension. Silver nanoparticles/nanowires self-aligned on pre-patterned rippled substrate are presented for the first time as an active SERS substrate. Anisotropic SERS response in such arrays is attributed to different field enhancement along and across the ripples. Strong plasmonic coupling in elongated nanoparticles chain results in significantly higher SERS intensity then spherical nanoparticles/nanowires and non-ordered nanoparticles. Higher SERS intensity across the nanowires array in comparison to along the array (bulk silver) confirms electromagnetic field enhancement (hot-junction) is responsible for SERS phenomenon. Self-assembly of cobalt nanoparticle on ripple pattern substrate is also reported. Due to less adatom mobility and higher sticking cobalt self-assembly is possible only at much higher temperature. A strong uniaxial magnetic anisotropy was observed not observed for non ordered cobalt particles.

京都大学 / 新制・論文博士 / 博士(理学) / 乙第13478号 / 論理博第1578号 / 新制||理||1692(附属図書館) / (主査)教授 北川 宏, 教授 吉村 一良, 教授 竹腰 清乃理 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DGAM

abstract: Nanotechnology is a scientific field that has recently expanded due to its applications in pharmaceutical and personal care products, industry and agriculture. As result of this unprecedented growth, nanoparticles (NPs) have become a significant environmental contaminant, with potential to impact various forms of life in environment. Metal nanoparticles (mNPs) exhibit unique properties such as increased chemical reactivity due to high specific surface area to volume ratios. Bacteria play a major role in many natural and engineered biogeochemical reactions in wastewater treatment plants and other environmental compartments. I have evaluated the laboratory isolates of E. coli, Bacillus, Alcaligenes, Pseudomonas; wastewater isolates of E. coli and Bacillus; and pathogenic isolate of E. coli for their response to 50 & 100 nm sized Cu nanoparticles (CuNPs). Bactericidal tests, scanning electron microscopy (SEM) analyses, and probable toxicity pathways assays were performed. The results indicate that under continuous mixing conditions, CuNPs are effective in inactivation of the selected bacterial isolates. In general, exposure to CuNPs resulted in 4 to >6 log reduction in bacterial population within 2 hours. Based on the GR, LDH and MTT assays, bacterial cells showed different toxicity elicitation pathways after exposure to CuNPs. Therefore, it can be concluded that the laboratory isolates are good candidates for predicting the behavior of environmental isolates exposed to CuNPs. Also, high inactivation values recorded in this study suggest that the presence of CuNPs in different environmental compartments may have an impact on pollutants attenuation and wastewater biological treatment processes. These results point towards the need for an in depth investigation of the impact of NPs on the biological processes; and long-term effect of high load of NPs on the stability of aquatic and terrestrial ecologies. / Dissertation/Thesis / Ph.D. Engineering 2012

Passenger vehicle disk brakes produce large amounts of wear debris that needs to be analyzed to verify its environmental and health impact because its effects are amplified by the massive extent of vehicular transportation around the world. An efficient and thorough method for wear debris analysis should be derived from this research to assist brake manufacturers and researchers in understanding the characteristics of wear particulates being released from current brake materials. In addition, a test method allowing inspection of brake formulations in development phase should be provided and several model "environmentally friendly" brake lining materials should be developed. The most hazardous constituents in a known brake lining formula should be replaced with non-hazardous materials that will obtain performance specifications comparable to the original formula. The brake manufacturing industry and general public should be notified of important findings from this research. Model brake linings fabricated on-site with known constituents were run through an AO4D performance dynamometer test to collect airborne and non-airborne wear debris that is comparable to wear debris produced by an average passenger vehicle. Based on previous experience with analysis of brake linings, this wear debris will be analyzed with scanning electron microscopy equipped with energy dispersive X-ray microanalysis (SEM-EDX), transmission electron microscopy with EDX (TEM-EDX), X-ray diffraction (XRD), polarized light microscopy (PLM), X-ray fluorescence (XRF), and laser scattering particle size distribution analyzer (LSPSDA). Research presented in this report indicates clearly that the previous work published by several groups is lacking sufficient information and the findings should be corrected. For instance the particle size distribution detected by particle size analyzers and impactors (recommended by EPA) is not accurate and TEM as well as SEM studies allowing direct observation of wear particles revealed considerable differences between sizes detected by these analyzers and realistic particle size. EDX in combination with XRF and XRD allows for reliable identification of chemical species and crystalline phases present in wear debris. True airborne wear debris should be sampled from brake exhaust from an appropriate distance. Degummed hemp shows promising results as a copper replacement in low-metallic friction material.

The bursa of Fabricius is a central lymphoid organ essential for B cell development and generation of humoral immune responses in birds. The bursa is connected dorsally to the cloaca and continually “samples” the intestinal fluid phase for the presence of antigen. It is comprised of folds or plicae, which are seeded with individual follicles, where antigen processing and presentation occurs for B cell development as well as generation of antibody responses. The plicae are separated from the bursal lumen by interfollicular epithelium (IFE) and specific regions of follicle-associated epithelium (FAE). The FAE is comprised of M cell-like cells, which are specialized for transcytosis of antigen from the bursal lumen into underlying follicles. The uptake of particulate and soluble antigen in the bursa of Fabricius has been previously demonstrated, but how particle size affects their internalization within bursal FAE and the transport of particulate antigen into deeper follicles has not been explored. It has been shown that nanoparticles (NPs) ≤40 nm are most efficiently internalized by the epithelium of the murine intestine and vaginal tract. Therefore, we examined the uptake of 0.04 - 2 μm fluorescent polystyrene NPs in bursa at 1 hour or 6 hours after bursal administration to determine whether bursal epithelium is similarly constrained. Using immunofluorescence microscopy (IFM) and spectrofluorophotometry we found that NP uptake is inversely correlated with NP size. NPs ≤40 nm are most efficiently internalized by the bursal epithelium and bursal follicles, while NPs ≥500 nm are not effectively taken up by the bursal epithelium within 6 hours of administration. Moreover, once the size-limited capabilities of the bursal epithelium were established, we also found that bursal priming of chickens with 20 nm NPs conjugated to IMP-1, a protective antigen of an important avian pathogen Eimeria maxima, induces IMP-1-specific serum IgG following sub-cutandous boosting. We induced similar IMP-1-specific serum antibody responses in chickens primed bursally and sub-cutaneously boosted as those primed and boosted sub-cutaneously. Whether this route of immunization is able to elicit long-term protection must be investigated. A number of infectious diseases, including Infectious Bursal Disease Virus (IBDV), which directly targets the bursa of young birds and prevents the development of the immune system and causes mortality, are prevalent in the poultry industry. While vaccines exist for many of these diseases they confer only partial or incomplete protection; therefore, alternative vaccine strategies must be investigated. In addition, the bursa is an ideal in vivo model for the investigation of endocytic mechanisms for uptake of particulate antigen. Therefore, further characterizing the mechanisms of NP uptake at mucosal surfaces and their immunogenicity will be important for the development of NP-based mucosal vaccines for agriculturally relevant species such as poultry.

The depletion of natural resources has long been a concern since the rapid increase in energy consumption in recent years. The gradual increase of pollution worldwide accompanied by energy generation process also started to post threats to the environment. With the evolution of technology and materials, power generation and energy storage with significant improved efficiency can be made possible, and further benefits the reduction of degree of pollution generated. In this research, synthesis, processing, characterization and application of nano materials towards energy generation and energy storage devices are studied. In chapter 2, superior corrosion resistance properties of HVOF thermal spray of TiC and TiB2 coatings on 304H stainless steel, 430, and P91 steels were reported. The coatings successfully served at a protection layer by limiting oxygen penetration, sulfur attack, and decreased the formation of pits and cracks on the substrates at 750°C for up to 800 hours. In chapter 3, continuous smooth TiC nanofibers were successfully synthesized by carbothermal reduction of electrospun titanium based nanofibers. XRD and HR-TEM analysis results indicated the synthesized nanofibers were composed of high purity TiC. Electric conductivity of a single fiber was in the 2.00×10^5 range. Symmetrical cyclic voltammetry curve further indicated good electrochemical properties of the fibers. In addition, the TiC nanofibers also exhibited excellent sintering properties over TiC or TiB2 nanoparticles. Studies on morphology and electrochemical properties of MnOx nanofiber and nanoparticles is reported in chapter 4. MnOx, MnOx/SnO2, and MnOx/CNT nanofibers synthesized using electrospinning method showed specific capacitance of 166.12 F/g, 182 F/g for, and 472 F/g at scan rate of 10mV/s. Analysis results also showed positive impact of conductivity and fiber morphology on the electrochemical properties of the fibers. morphology and electrochemical properties of the MnOx nanoparticles synthesized using solvents with different polarity with gelation pH of 8.5, 9.0 and 10.0 were also studied. Analysis results show the impact of particle sized and morphology on the electrochemical properties. Highest specific capacitance measured for the synthesized nanoparticles was 231.38F/g@10mV/s and 165.13F/g@10mV/s for methanol and mixture of methanol and propanol based MnOx respectively. Effect of solvent polarity of the manganese sol on MnOx formation and phase transformation temperature is also shown in the chapter.

Doctor of Philosophy / Department of Agronomy / Masaaki Tamura / Over the years proteins/peptides have evolved as promising therapeutic agents in the treatment of cancer. Considering the advantages of peptides such as their small size, ease of synthesis, tumor-penetrating ability and bio-compatibility, present report discusses proof of concept for 1. C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine combination treatment for peritoneally disseminated pancreatic cancer and 2. dTAT peptide nanoparticles mediated gene (angiotensin II type 2 receptor gene) therapy for lung cancer. 1. A significant reduction in intraperitoneally (IP) transplanted pancreatic carcinoma growth was demonstrated with C1B5 peptide and gemcitabine co-treatment in an immunocompetent mouse model. Increased number of Granzyme B positive cells was observed in treated mice ascites, suggesting the involvement of immune response in tumor attenuation. The strong effect observed in combination treatment might be because of increase in lymphocyte recruitment by gemcitabine followed by C1B5 peptide mediated CD8+ T-cells or NK cells activation apart from direct cancer cell apoptosis. 2. To test dTAT peptide nanoparticles (dTAT NPs) mediated therapeutic gene delivery, luciferase reporter gene containing dTAT nanoparticles were synthesized (dTAT/pLUC/Ca2+). Synthesis conditions for nanoparticles were optimized based on dTAT/pLUC/Ca2+ nanoparticles transfection efficiency. With the optimized conditions, dTAT NPs containing AT2R, TRAIL or miR-34a pDNA (dTAT/pAT2R, dTAT/TRAIL or dTAT/miR- 34a) were synthesized. Therapeutic potential of these NPs was analyzed in lung adenocarcinoma containing mice by administering them intravenously (IV) or/and intratracheally (IV). Combination treatment with the IV injection of the new dTAT/pAT2R/Ca2+ formulation and the IT injection of the original dTAT/pAT2R/Ca2+ formulation is effective in attenuation of developed human bronchioloalveolar carcinoma in the SCID mouse lungs. Findings from the above mentioned studies have vital clinical relevance as it implies that peptides alone or when used as gene delivery systems may prove to be beneficial in the treatment of various stages of cancer.

Neste trabalho, desenvolvemos um gerador de nanopartículas (NPs) como uma adaptação para um sistema de magnetron sputtering. Com ele, somos capazes de produzir NPs de materiais diversos e codepositá-las em matrizes dielétricas ou metálicas. A adaptação consiste em incluir uma região de alta pressão relativa de Ar no caminho do vapor atômico removido do alvo. A aglomeração ocorre termodinamicamente devido a diminuição da energia cinética após colisões com o gás. Desenvolvemos também, uma metodologia para colimar o fluxo de NPs dentro da região de alta pressão. A deposição é feita no substrato na forma de uma mancha com alguns milímetros de diâmetro e o tempo de preparação da amostra é significativamente curto. Desenvolvemos um modelo fenomenológico para explicar a condensação e a colimação do nosso sistema. Este, apesar de não sofisticado, explica bem ambos os fenômenos e consegue prever o diâmetro das nanopartículas para certas condições. Em paralelo ao desenvolvimento, produzimos e caracterizamos nanopartículas de cobalto. Da caracterização morfológica, através de microscopia eletrônica, concluímos que as NPs produzidas tem diâmetros médios de 10 nm com uma dispersão de 13 %. Através de análises de retro espalhamento Rutherford estudamos a distribuição do material sobre o substrato e observamos que este segue uma distribuição Gaussiana de espessuras. Além disso, devido a colimação, observamos que as taxas de deposição são da ordem de 50 vezes maiores que as taxas usuais de um sistema de sputtering. Estudos estruturais através de difração de raios X mostraram que as nanopartículas são nanocristalinas e imagens em alta magnificação de microscopia eletrônica de transmissão comprovaram esta hipótese. Finalmente, estudos magnéticos mostraram que as NPs não possuem eixos preferenciais de magnetização. Desenvolvemos condições padrões de operação e estabilizamos o sistema que atualmente produz amostras confiáveis e reprodutíveis. Além do Co, nanopartículas de Cu e SmCo foram produzidas em condições parecidas. A morfologia destas partículas foi investigadas por microscopia eletrônica e seus tamanhos se mostraram próximos dos das NPs de Co. Estes resultados ilustraram a universalidade do nosso sistema de deposição de nanopartículas. / We have developed a nanoparticle (NP) generator by adapting one of the sputtering guns on a magnetron sputtering system. With it, we are able to produce nanoparticles with different types of material. The adaptation consists of including a high-pressure region in the path of the atomic vapor removed from the sputtering target. The condensation happens thermodynamically through the loss of kinetic energy that the atomic vapor suffers after collisions with the gas. We have also developed a methodology to collimate the flow of nanoparticles inside the high pressure region. The deposition on the substrate is in the form of a stain with a few millimeters in diameter. The sample preparation time is also relatively short. We created a phenomenological model to explain both the condensation and collimation phenomena in our system. Despite being relatively simple, this model explain both quite well. In parallel to the development of the system, we produced and characterized cobalt nanoparticles. From a morphological analysis, carried out using electron microscopy, we determined that the nanoparticles mean diameter is of about 10 nm with a dispersion of 13 %. Through Rutherford back-scattering analysis, we studied the thickness distribution of the sample along the substrate. We observed that it follows a Gaussian distribution. Also, because of the collimation of the material, the deposition rates are about 50 times higher than in a regular sputtering system. Using X ray diffraction we were able to determine that the NPs are nano-crystalline which is corroborated with high resolution transmission electron microscopy images. Finally, magnetic measurements showed that the nanoparticles do not have any preferential magnetization axis. We developed standards of operations and stabilized the system. The samples we produce are trustworthy and reproducible. Besides Co, Cu and SmCo NPs were produced using this system with conditions similar to the ones used on the Co NPs. Through morphological analysis, we determined that their sizes are also similar. These results illustrate the universality of our system.