In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction. / Singapore-MIT Alliance (SMA)

INTRODUÇÃO: A doença de Chagas e a leishmaniose são doenças tropicais supernegligenciadas, que afetam regiões de extrema pobreza. Os fármacos disponíveis para estas duas doenças são escassos, de eficácia limitada, de alta toxicidade e suscitam casos de resistência. OBJETIVO: Considerando-se a necessidade de desenvolvimento de novos agentes antichagásicos e leishmanicidas, a importância da latenciação no aprimoramento de fármacos/compostos bioativos e a versatilidade de transportadores dendriméricos, o objetivo deste trabalho foi a síntese de pró-fármacos dendriméricos de primeira geração de 3-hidroxiflavona, composto que apresenta potencial atividade tripanomicida e leishmanicida. Desta forma, pretendeu-se obter liberação controlada, melhora da permeabilidade, toxicidade reduzida e aumento da eficácia deste agente bioativo. MATERIAL E MÉTODOS: Para a obtenção desses dendrímeros empregaram-se as abordagens divergente e convergente, compostas por várias etapas de síntese com reações de proteção, desproteção e acoplamentos. RESULTADOS E DISCUSSÃO: A abordagem convergente apresentou problemas sintéticos, devido à instabilidade dos derivados contendo 3-hidroxiflavona nas diferentes condições reacionais e de purificação testadas. No entanto, há indícios da síntese dos pró-fármacos dendriméricos de 3-hidroxiflavona, mas esses compostos apresentam-se impuros. Devido a essa instabilidade e a dificuldade de purificação na abordagem convergente, optou-se pela síntese divergente, no qual o composto bioativo é acoplado na etapa final. Os estudos sintéticos mostraram a obtenção dos intermediários puros formados pelos focos centrais propano- e hexano-diamina acoplados ao ácido málico protegido. CONCLUSÃO: Há indicativos da obtenção de pró-fármacos dendriméricos de 3-hidroxiflavona, ainda que impuros. As maiores dificuldades encontradas foram a purificação e a estabilidade dos compostos obtidos. / INTRODUCTION: Chagas\' disease and leishmaniasis are super neglected tropical diseases that affect primarily areas of extreme poverty. The drugs available for these diseases are scarce and of limited effectiveness, toxic and rouse resistance. OBJECTIVE: Considering that the development of new antichagasic and leishmanicide agents are urgently needed, the importance of prodrug design to the improvement of drugs and bioactive compounds and the versatility of dendrimers as drug carriers, the objective of this work was the synthesis of dendrimer prodrug of 3-hydroxyflavone, which shows potential antichagasic and leishmanicide activities. Thus, we intended to obtain controlled release, improvement of the permeability, reduction of the toxicity and increase of efficacy of this bioactive agent. MATERIAL AND METHODS: Convergent and divergent approaches have been used to synthesize those compounds. Synthetic steps consist of protection, deprotection and coupling reactions. RESULTS AND DISCUSSION: The convergent approach presented problems due to the instability of the 3-hydroxyflavone derivatives, in different reaction and purification conditions. However, there is evidence of the synthesis of dendrimer prodrugs, though still impure. Due to instability and purification difficulty of intermediate, we performed the divergent synthesis. We obtained the pure intermediates composed by cores propanediamine and hexanediamine coupled to the protected malic acid as spacer group. CONCLUSION: Synthetic studies suggested the synthesis of dendrimer prodrugs, although impure. The greatest difficulties were the purification and the instability of compounds.

O ancoramento do complexo trans-[RuIII(NH3)4(SO4)ina]Cl em dendrímeros PAMAM de geração 0 e 2 (G0 e G2) foi realizada por meio de uma ligação peptídica, e esses produtos foram submetidos à reação com NO(g) gerando os respectivos nitrosilo complexos G0/RuNO e G2/RuNO. A caracterização desses compostos por infravermelho, UV-vis, voltametria cíclica, RMN de 1H e 13C, e análise elementar indica que os nitrosilo complexos foram imobilizados na superfície dos PAMAM G0 e G2. Os espectros de infravermelho para G0/RuNO e G2/RuNO apresentaram apenas um estiramento &nu;NO+, respectivamente em 1933 e 1937 cm-1, e para o produto RuNO (não ligado ao dendrímero) em 1933 cm-1. O espectro eletrônico para esses três compostos apresentou bandas nas regiões de 230, 270 e 330 nm, e por meio de voltametria cíclica observou-se o processo eletroquímico relativo a NO+/NO0 com ENO+/NO0 vs ECS igual a -0,173 V para G0/RuNO, -0,178 V G2/RuNO e -0,175 V para RuNO. O espectro de 1H RMN do complexo RuNO apresentou dois dubletos com deslocamentos químicos centrados em 8,73 e 8,35 ppm, referentes aos hidrogênios aromáticos respectivamente nas posições orto e meta do ligante ina coordenado ao metal. Para G0/RuNO e G2/RuNO esses sinais foram observados em 8,73 e 8,36 ppm, e os sinais referentes aos dendrímeros nesses produtos foram verificados entre 2,7 e 4,0 ppm. O espectro de RMN 13C para o complexo RuNO apresentou quatro sinais, e para G0/RuNO e G2/RuNO, respectivamente, dez e doze sinais, conforme esperado para esses compostos. Apesar dos resultados supracitados indicarem que o ancoramento ocorreu de forma satisfatória, os dados de análise elementar apresentaram desvios significativos entre o valor teórico e o experimental, principalmente para G2/RuNO. Em adição, foram realizados ensaios em células do baço de camundongos para verificar a toxicidade dos nitrosilo complexos às células saudáveis, e os resultados indicaram baixa citotoxicidade (<15%) para RuNO, G0/RuNO e G2/RuNO. Também foram realizados experimentos sobre a atividade in vitro desses compostos contra os parasitos Trypanosoma cruzi e Leishmania major. Os melhores resultados, ainda que preliminares, foram obtidos com a maior concentração, 200&micro;M (em relação à Ru), em que observou-se atividade tripanocida (média) em torno de 88% para G2/RuNO, 82% para G0/RuNO e 72% para RuNO, enquanto que para o Bz (referência) esse valor foi de 96%. Já a atividade leishmanicida (concentração de 200&micro;M) desses compostos ficou entre 60 a 70% (65% para G2RuNO, 69% para G0/RuNO e 60% para RuNO). / The anchoring of the complex trans-[RuIII(NH3)4(SO4)ina]Cl on PAMAM dendrimers of generation 0 and 2 (G0 and G2) was performed by a peptide bond, and the products were submitted to reaction with NO (g) generating the related nitrosyl complexes G0/RuNO and G2/RuNO. The characterization of these compounds by IR, UV-vis, cyclic voltammetry, 1H and 13C NMR, and elemental analysis indicated that the nitrosyl complexes were immobilized on the surface of PAMAM G0 and G2. Infrared spectra for G0/RuNO and G2/RuNO showed only one &nu;NO+ band in 1933 and 1937 cm-1 respectively, and for RuNO (complex not bounded to the dendrimer) at 1933 cm-1. Electronic spectra for these three compounds showed bands in the regions of 230, 270 and 330 nm, and by cyclic voltammetry it was possible to observe the electrochemical process relative to NO+/NO0 with ENO+/NO0 equal to -0.173 V vs SCE for G0/RuNO , -0.178 V for G2/RuNO and -0.175 V for RuNO. The 1H NMR spectra for RuNO complex showed two doublets with chemical shifts centered at 8.73 and 8.35 ppm, respectively referring to the aromatic hydrogens in the ortho and meta positions of the ina ligand coordinated to the metal. The same signals obtained for G0/RuNO and G2/RuNO were observed in 8.73 and 8.36 ppm, and signals related to dendrimers between 2.7 and 4.0 ppm. The 13C NMR spectrum for RuNO exhibited four signals, and for G0/RuNO and G2/RuNO, respectively, ten and twelve signals, as expected for these compounds. Despite the results above, which indicate that anchoring occurred satisfactorily, the elemental analysis showed significant deviations between the theoretical and experimental values, especially for G2/RuNO. In adition, in vitro assays were performed on mice spleen cells to determine the toxicity of the nitrosyl complex to healthy cells, and the results showed low cytotoxicity (<15%) for RuNO, G0/RuNO and G2/RuNO. In vitro experiments were also carried out to determine the activity of these compounds against the parasite Trypanosoma cruzi and Leishmania major. The best results (preliminary) were obtained with the highest concentration 200&micro;M (relative to Ru), which was observed trypanocidal activity (average) around 88% for G2/RuNO, 82% for G0/RuNO and 72% for RuNO, while for Bz (reference) it was around 96%. The leishmanicidal activity (concentration of 200&micro;M) of these compounds was in the range of 60 to 70% (65% for G2RuNO, 69% for G0/RuNO and 60% for RuNO).

Two specific drug-delivery applications were sought in this work using polyamidoamine (PAMAM) dendrimers. One drug-delivery system used a novel dendrimer hydrogel (DH) for sustained delivery of anti-glaucoma drugs. In this work, PAMAM G3.0 dendrimers were covalently bonded with poly(ethylene glycol) (PEG­12000) molecules which were subsequently acrylated, resulting in photocurable DH conjugates. For pharmacological studies, DH were loaded with a solution of intraocular pressure lowering drugs, brimonidine and timolol maleate, and were characterized for in vitro release and ex vivo transport and uptake. DH formulations were shown to increase the loading of drug molecules, increase transcorneal drug delivery, and exhibit sustained-delivery of drug molecules. A second drug-delivery system, utilizing cell-surface engineering, intended to increase the targeting ability of highly toxic anti-cancer drugs to curtail systemic effects. In particular, Qdots and 5-(aminoacetamido) fluorescein-labeled polyamidoamine dendrimer G4.5, both of which were coated with amine-derivatized polyethylene glycol, were immobilized to the sodium periodate-treated surface of RAW264.7 macrophages through a transient Schiff base linkage. Further, a reducing agent sodium cyanoborohydride was applied to reduce Schiff bases to stable secondary amine linkages. The distribution of nanoparticles on the cell surface was observed by fluorescence microscopy and was found to be dependent on the stability of the linkages tethering nanoparticles to the cell surface.

NANOMEDECINE DRUG DELIVERY ACROSS MUCOUS MEMBRANES By Michael G. Lancina III A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth Univeristy, 2017. Major Director: Dr. Hu Yang, Associate Professor, Chemical and Life Science Engineering Control over the distribution of therapeutic compounds is a complex and somewhat overlooked field of pharmaceutical research. When swallowing a pill or receiving an injection, it is commonly assumed that drug will spread throughout the body in a more or less uniform concentration and find its way to wherever it is needed. In truth, drug biodistribuition is highly non-uniform and dependent on a large number of factors. The development of advanced drug delivery systems to control biodistribution can produce significant advances in clinical treatments without the need to discover new therapeutic compounds. This work focuses on a number of nanostructured materials designed to improve drug delivery by direct and efficient transfer of drugs across one of the body’s external mucous membranes. Chapter 1 outlines the central concept that unites these studies: nanomaterials and cationic particles can be used to delivery therapeutic compounds across mucous membranes. Special attention is given to dendritic nanoparticles. In chapter 2, uses for dendrimers in ocular drug delivery are presented. The studies are divided into two main groups: topical and injectable formulations. Chapter 3 does not involve dendrimers but instead another cationic particle used in transmembrane drug delivery, chitosan. Next, a dendrimer based nanofiber mat was used to deliver anti-glaucoma drugs in chapter 4. A three week in vivo efficacy trial showed dendrimer nanofiber mats outperformed traditional eye drops in terms of intra-ocular pressure decrease in a normotensive rat model. Finally, we have developed a new dendrimer based anti-glaucoma drug in chapter 5. Collectively, these studies demonstrate some of the potential applications for nanotechnology to improve transmembrane drug delivery. These particles and fibers are able to readily adhere and penetrate across epithelial cell lays. Utilizing these materials to improve drug absorption through these portals has the potential to improve the clinical treatment of wide variety of diseases.

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Flat dendrimers, consisting of a hexavalent aromatic core and rigid ethynyl units locked in place by ether connections were developed based upon the divergent synthetic method. Alternating functional groups were adopted on each site of the hexa-substituted benzene, in order to avoid undesired cyclization pathways. The flat structures of conjugated dendrimers would allow investigation on the discotic liquid crystal properties. In addition, these ethylnyl dendrimers are expected to show directed energy and electron transfer with a highly conjugated system, and thus are effective in the preparation of photoreactive materials such as electronic sensors or light harvesting materials. Conjugated polycyclic aromatic hydrocarbons, consisting of naphthalene, anthracene, pyrene, and phenanthrene groups connected via 1,3,5-triethynylbenzene cores, were synthesized. These molecules exhibited luminescence properties and the π-complexation with a mercury trifunctional lewis acid are expected to enhance the phosphorescence in the presence of the heavy metal due to the spin-orbit coupling. Besides, owing to the presence of heavy metal atom in the Au (I) complexes linked by s-bonded triethynyltriphenylene luminophore, the phosphorescence occurs from a metal-centered emission. The conjugated organic luminophores have been developed to produce excellent quantum efficiencies, brightness, and long lifetimes.

Cancer is the second leading cause of death in the world. It is therefore critically important to detect cancer in its early stage to significantly increase the survival rate of cancer patients. Circulating tumour cells (CTCs) are cancer cells that peel off from primary tumour and enter bloodstream in early stage of a cancer, and thus it has been established that these CTCs are reliable targets for early cancer diagnosis. However, background signal reduction and optimization of CTC capturing mechanisms are still significant challenges in CTC detections with high sensitivities and accuracies. To this end, we have developed an aptamers and dendrimers based ultra non-fouling microfluidic detection system for sensitive detections of circulating tumour cells. More specifically, we demonstrate a simple strategy to bind PDMS and SU-8 surfaces in order to prepare a hybrid microfluidic device and subsequently modify both surfaces simultaneously using poly(amidoamine) (PAMAM), a highly hydrophilic dendrimer to improve non-fouling properties of the hybrid microfluidic channel. The resulting hybrid microfluidic system shows a remarkable non-specific adsorption suppression of 99.7% when tested with hydrophobic microbead suspension, an ultra non-fouling performance that has not been reported before. This is significantly important for detections with high sensitivities. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and water contact angle are used to characterize and confirm surface modifications. In addition, we investigate the combined effects of surface properties on surface non-fouling performance to both live and dead cells. (3-aminopropyl)-trimethoxysilane (APTMS), carboxyl functionalized PAMAM dendrimer (PAMAM-COOH) and amino functionalized PAMAM dendrimer (PAMAM-NH2) are used to provide different surfaces with various surface hydrophilicity, electric charge and roughness. We show that electric charge of a surface is the most important factor influencing non- specific adsorption of live cells to the surface while hydrophilicity/hydrophobicity of a surface is the most important factor for dead cells. Atomic force microscopy, water contact angle and microscopy are used to characterize and confirm surface modifications. To further exploit and improve capturing efficiency of target cancer cells, we investigate the effect of the length of spacers that tether capturing aptamer to the microfluidic surfaces on capturing performance of CCRF-CEM circulating tumour cells. Aptamers with different lengths of thymine base spacers are immobilized onto PAMAM dendrimer modified surfaces in microfluidic channels. We demonstrate that ten thymine bases spacer has the best length for sgc8 aptamer to form its secondary structure for CCRF-CEM cell capture. Water contact angle, and microscopy are used to characterize and confirm surface modifications. Taken together, the results of this study significantly highlight the importance of different considerations on surface modification and its optimizations when designing a microfluidic system for high sensitivity detection and biosensing applications.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22660号 / 工博第4744号 / 新制||工||1741(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 大内 誠, 准教授 佐々木 善浩 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19738号 / 工博第4193号 / 新制||工||1647(附属図書館) / 32774 / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 近藤 輝幸, 教授 辻 康之, 教授 大江 浩一 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM