Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: Linker effects on the relaxivity

The relaxivity displayed by Gd3+ chelates immobilized onto gold nanoparticles is the result of complex interplay between nanoparticle size, water exchange rate and chelate structure. In this work we study the effect of the length of -thioalkyl linkers, anchoring fast water exchanging Gd3+ chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd3+ chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM-1s-1 (30 MHz, 25 ºC) were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles is determined mainly by size. Small nanoparticles (HD= 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD= 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggests that functionalized gold nanoparticles hold great potential for further investigation as MRI Contrast Agents. This study contributes to understand the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd3+ complexes. It is a relevant contribution towards “design rules” for nanostructures functionalized with Gd3+ chelates as Contrast Agents for MRI and multimodal imaging.

Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper.

Selective oxidation of cyclohexene through gold functionalized silica monolith microreactors

Two simple, reproducible methods of preparing evenly distributed Au nanoparticle containing mesoporous silica monoliths are investigated. These Au nanoparticle containing monoliths are subsequently investigated as flow reactors for the selective oxidation of cyclohexene. In the first strategy, the silica monolith was directly impregnated with Au nanoparticles during the formation of the monolith. The second approach was to pre-functionalize the monolith with thiol groups tethered within the silica mesostructure. These can act as evenly distributed anchors for the Au nanoparticles to be incorporated by flowing a Au nanoparticle solution through the thiol functionalized monolith. Both methods led to successfully achieving even distribution of Au nanoparticles along the length of the monolith as demonstrated by ICP-OES. However, the impregnation method led to strong agglomeration of the Au nanoparticles during subsequent heating steps while the thiol anchoring procedure maintained the nanoparticles in the range of 6.8 ± 1.4 nm. Both Au nanoparticle containing monoliths as well as samples with no Au incorporated were tested for the selective oxidation of cyclohexene under constant flow at 30 °C. The Au free materials were found to be catalytically inactive with Au being the minimum necessary requirement for the reaction to proceed. The impregnated Au-containing monolith was found to be less active than the thiol functionalized Au-containing material, attributable to the low metal surface area of the Au nanoparticles. The reaction on the thiol functionalized Au-containing monolith was found to depend strongly on the type of oxidant used: tert-butyl hydroperoxide (TBHP) was more active than H2O2, likely due to the thiol induced hydrophobicity in the monolith.

Synthesis of thiolated and acrylated nanoparticles using thiol-ene click chemistry: towards novel mucoadhesive materials for drug delivery

Thiol- and acrylate-functionalized nanoparticles have been synthesized from pentaerythritol tetrakis(3-mercapto-propionate) and pentaerythritol tetraacrylate using thiol-ene click chemistry. Using Raman and 1H NMR spectroscopy as well as Ellman's assay, it was demonstrated that excess pentaerythritol tetraacrylate in the feed mixture led to nanoparticles with free acrylate groups on their surface, whereas nanoparticles with thiolated surfaces could be synthesized using feed mixtures with excess pentaerythritol tetrakis(3-mercapto-propionate). The possibility of fluorescent labelling of thiolated nanoparticles has been demonstrated through their reaction with fluorescein-5-maleimide. The thiolated nanoparticles were found to be mucoadhesive and exhibited retention on mucosal surface of porcine urinary bladder.

A General Approach Towards the Synthesis of Amido-Functionalized Biodegradable Polyesters

The thesis presented here describes methodologies to produce pendant group functionalized polyesters from amido-functionalized α-hydroxy acids. The synthetic methods used to produce the functionalized α-hydroxy acids are compatible with a wide array of functional groups, making this technique highly versatile. The synthesis of functionalized polyesters was investigated to develop polymers with properties that may improve the capabilities of existing biodegradable polyesters for applications in controlled release pharmaceuticals. Chemically modified a-hydroxy acids were synthesized by reacting glyoxylic acid with a primary or secondary amide. To demonstrate the utility of this reaction, fourstructurally dissimilar amide substituents were examined including 2-pyrrolidione, benzamide, acetamide and acrylamide. The reaction is synthetically simple, provides high yields and is uniquely flexible, functionalized monomer. The compatibility of this procedure with the collection of functional groups mentioned circumvents the need for syntheses. The amido-functionalized monomers were polymerized by two different techniques: melt polycondensation and solution polymerization. Melt polycondensation was conducted by heating the monomer past its melting temperature under reduced pressure. Oligomeric functionalized polyesters (= 800 g/mol) with low PDIs (= 1.05) were obtained by melt polycondensation. Melt polycondensation was not compatible with all of the synthesized monomers. Two of the monomers (containing benzamide and acrylamide functionalities) degraded before the polycondensation reaction occurred. Thermal gravimetric analysis confirmed that a process other than polyesterification was occurring, indicating that some amido-functionalized α-hydroxy acids cannot be synthesized in the melt.Solution polymerization was conducted to polymerize functionalized α-hydroxy acids that were incompatible with melt polycondensation. Several modified Steglich polyesterifications were tested including p-toluenesulfonic acid mediated and scandium (III) triflate catalyzed. Only oligomeric functionalized polyesters were formed bythis method. A number of possible side reactions including the formation of an N-acylurea and a cyclic polymer ring were possible. The utility of this procedure appears to be limited due to the complexity of the reaction and its inability to produce high molecular weight polymer.

The Use Of Amido- And Imido-Functionalized Alpha Hydroxy Acids In The Creation Of Biodegradable Polyesters: An Exploration Of Va

The purpose of this thesis was to synthesize biodegradable polyesters from a wide array of functionalized ¿-hydroxy acids. The initial strategy was to use amido-functionalized ¿-hydroxy acids and 2-bromopropanoyl bromide to form amido-functionalized cyclic diesters. Then, the resulting cyclic diesters would be used in ring opening polymerization to create biodegradable polyesters. However, the spontaneous rapid degradation of the secondary amido-functionalized cyclic diester structure, as seen with 2-benzamido-hydroxyacetic acid, limited ring formation to tertiary amido-functionalized ¿-hydroxy acids. Also, the hydrophilic nature of most ¿-hydroxy acids allowed water into the crystal structure of the ¿-hydroxy acid. Then, when the ¿-hydroxy acid was used in ring forming reactions, the associated water deactivated reactive reagents and limited cyclic diester synthesis. These issues led to the synthesis of hydrophobic and tertiary amido- and imido-functionalized ¿-hydroxy acids, 2-phthalimido-2-hydroxyacetic acid and 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid. The new ¿-hydroxy acids were used in two new polymerization techniques, melt polycondensation and solution polymerization, instead of ring open polymerization. Melt polycondensation and solution polymerization had shown previous success in forming oligomers of amido-functionalized ¿-hydroxy acids. Melt polycondensation was conducted by heating the monomer past its melting temperature under reduced pressure. The uncatalyzed melt polycondensation of 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid created polyesters (¿ 960 g/mol). The scandium(III) trifluoromethanesulfonate enhanced melt polycondensation polymerization created slightly larger oligomers (¿ 1340 g/mol). However, 2-phthalimido-2-hydroxyacetic acid was not compatible with melt polycondensation because thermal degradation occurred. Thus, solution polymerization was conducted via Steglich esterification. Only oligomeric functionalized polyesters were formed (¿ 1060 g/mol). Future work should focus on optimization of the catalyst and the reaction conditions to obtain higher molecular weight polyesters. Also, 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid should be utilized in the cyclic diester synthesis technique.

Silver coordination polymers with tri- and hexacyanoethyl-functionalized macrocyclic ligands

Tri-and hexa-cyanoethyl functionalized 17-(L-1) and 42-membered (L-2) macrocyclic compounds were obtained by [1 + 1] (for L-1) or [2 + 2] (for L-2) cyclocondensation of the corresponding dialdehyde and diethylenetriamine, followed by hydrogenation by KBH4 and subsequent cyano-functionalization with acrylonitrile. They react with silver nitrate, leading to the formation of [AgL1](NO3) (1) and of the metalorganic coordination polymers [Ag-2(NO3)(2)L-1](n) (2) and {[Ag2L2](NO3)(2)}(n) (3). The complexes were characterized by elemental analysis, H-1 NMR, C-13 NMR, IR spectroscopies, and ESI-MS; moreover, L-2, 1, 2 and 3 were also characterized by single crystal X-ray diffraction. The metal cation in 1 is pentacoordinated with a N3O2 coordination environment; in 2, the metal cations display N4O2 octahedral and N2O3 square-pyramid coordination and in 3 they are in square-planar N-4 sites. In 1, the ligand acts as a pentadentate chelator, and in the other two cases, the ligands behave as octadentate chelators in a 1 kappa N-3:kappa O-2,2 kappa N,3 kappa N,4 kappa N (in 2) or 1 kappa N-3,2 kappa N-3,3 kappa N,4 kappa N fashion (in 3). The cyanoethyl strands of the ligands are directly involved in the formation of the 2D frameworks of 2 and 3, which in the former polymer can be viewed as a net composed of hexametallic 36-membered macrocyclic rings and in the latter generates extra hexametallic 58-membered cyclic sets that form zig-zag layers. The thermal analytical and electrochemical properties of these silver complexes were also studied.

Caracterização do grânulo de amido de bananas (Musa AAA-Nanicão e Musa AAB-Terra)

O amido de bananas tem sido pesquisado na área de nutrição a partir da introdução do conceito de Amido Resistente. O amido de Musa AAA-Nanicão e Musa AAB-Terra foram caracterizados quanto as suas respostas fisiológicas [12]. Em continuidade, o presente trabalho estudou características físicas e morfológicas dos grânulos de amido de ambas as espécies de banana comparando-as com amido nativo de milho comercial. O amido de bananas foi extraído segundo CHIANG, CHU & CHU [3]. A morfologia dos grânulos foi realizada após tratamento enzimático in vitro a 37°C/24h com alfa-amilase pancreática. Foram efetuados os respectivos amilogramas e difractogramas de raios-X. Os grânulos de amido da Musa Tipo AAA-Nanicão apresentaram comprimento entre 30-40µm. Em Musa AAB-Terra, os grânulos, também ovais e alongados, eram um pouco menores, 20-30µm. A corrosão enzimática in vitro iniciava-se sobre a superfície anteriormente lisa e formavam estrias superficiais e apicais. A Microscopia Eletrônica de Varredura (MEV) mostrou que a hidrólise in vitro por 24 horas foi pequena e ocorria sobretudo nas camadas amorfas dos grânulos de ambas as espécies. O padrão de corrosão demonstrou-se distinto daquele ocorrido no amido de milho. As suspensões de amido de bananas ao viscosímetro demonstraram forte capacidade de hidratação e menor capacidade de retrogradação em relação ao milho, sobretudo do amido de Musa Tipo AAA-Nanicão; o amido de Musa Tipo AAB-Terra apresentou maior estabilidade de pasta. Na análise de difração de raios-X, os grânulos de bananas apresentaram padrão tipo B e C para Musa Tipo AAA-Nanicão e Musa Tipo AAB-Terra, respectivamente. Conclui-se que os amidos de Musa AAA-Nanicão e Musa AAB-Terra são estruturalmente distintos, justificando as respostas fisiológicas distintas encontradas posteriormente pelos mesmos autores. As distinções das propriedades físicas e bioquímicas obtidas para os grânulos, embora moderadas entre si e bastante distintas em relação ao amido de milho, não explicaram a alta resistência dos amidos das bananas à digestão enzimática. Os parâmetros obtidos nas análises permitiram, pela primeira vez avaliar as características e diferenças entre grânulos de amido de espécies distintas de banana e suas relações ao amido de milho.

Efeito do teor de amido danificado na produção de biscoitos tipo semi-duros

O trabalho objetivou estudar a danificação do amido de farinha de trigo por moagem e o seu efeito na produção de biscoitos tipo semi-duros. Amostras de trigo dos cultivares BR 23, BRS Angico e Rubi, com características diferentes quanto à dureza foram moídas em moinho de rolos através de uma passagem pelo conjunto de quebra; quebra mais três reduções na umidade 16% e quebra mais três reduções na umidade 12%. Neste tratamento, a moagem foi complementada pelo emprego de moinho de bolas. Nas farinhas, foram realizadas análises físicas, químicas, reológicas e funcionais, utilizando delineamento casualizado em planejamento fatorial completo 3 x 3, totalizando nove tratamentos. Os resultados foram analisados estatisticamente e, nos modelos significativos, as médias comparadas entre si pelo teste de Tukey a 5% de probabilidade de erro. Os resultados rendimento de quebra, proteína bruta, amido danificado, força geral do glúten (W x 10-4J) e relação tenacidade/extensibilidade (P/L) indicam que a farinha do cultivar BRS Angico apresentou melhores características para a produção de biscoitos seguido de BR 23 e Rubi. O teor de amido danificado produzido durante a moagem dos trigos influencia nas propriedades funcionais dos biscoitos apresentando melhor resultado aqueles obtidos a partir de uma passagem pelo conjunto de quebra.

Antioxidant effect of functionalized alkyl-organotellurides: a study in vitro

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 mu M) <= B (0.40 mu M) < D (0.68 mu M) < A (2.90 mu M), for Fe(2+)/EDTA, and B (0.21 mu M) <= C (0.33 mu M) < D (0.43 mu M) < A (1.21 mu M) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

Produção de alfa-amilase e glucoamilase termoestável pelo fungo termofílico Thermomyces lanuginosus TO-03 por fermentação submersa e em estado sólido e caracterização das enzimas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Alfa-amilase e maltase nos simbiontes Leucoagaricus gongylophorus Singer (Möller) (Leucocoprineae: agaricaceae) e Atta sexdens Linnaeus (Attini: formicidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito de métodos de preservação dos grãos úmidos de milho e de sorgo sobre a estrutura do endosperma, dos grânulos de amido e desempenho de leitões

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Uso do amido resistente na promoção da saúde intestinal de cães idosos: formação de produtos de fermentação e características histológicas da mucosa intestinal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Fermentação alcoólica com Saccharomyces cerevisiae em mosto aerado de hidrolisados de amido de mandioca

O presente trabalho foi desenvolvido no Centro de Raízes e Amidos Tropicais – CERAT, Na UNESP em Botucatu, estado de São Paulo onde foram realizados ensaios de fermentação alcoólica com hidrolisado de amido de mandioca. A fécula de mandioca foi utilizada como fonte de carboidrato para obtenção dos açúcares redutores consumidos no processo. Em um reator agitado foi produzido 12Kg de hidrolisado a partir de suspensão de fécula a 30% (p/p) utilizando enzimas alfa amilase na primeira etapa, seguida de amiloglucosidase na etapa posterior. As dosagens em unidades enzimáticas foram 2KNU.g-1 de amido e 2AGU.g-1 de amido respectivamente. O planejamento experimental considerou a realização de três ensaios de hidrolisados e três ensaios de fermentação a partir do mosto produzido; a) mosto aerado; b) com microaeração; c) em meio anaeróbio. Os ensaios foram realizados em erlenmeyers com 2,5 Kg de hidrolisado, ajustado a concentração de glicose a 100g.L-1 sendo inoculada a levedura do gênero Saccharomyces cerevisiae à taxa de 1,5% (p/p). Todos os erlenmeyers foram colocados sob agitação orbital e temperatura controlada de 30ºC sendo acompanhado o processo de fermentação através de coleta de amostras do mosto a cada hora. A aeração nos frascos erlenmeyers foi realizada através de mangueira coletora de válvula que regulava a vazão de ar. De acordo com os dados obtidos pode se concluir que o sistema anaeróbio em 32h foi o mais eficiente para a produção de etanol. Também foi possível observar que enquanto ocorre aeração no meio não se observa alteração significativa na concentração de etanol e quando cessa a aeração o meio torna se anaeróbio e tem início a produção de etanol. Quando aumenta a concentração de etanol no meio, o crescimento celular do sistema anaeróbio cai e etanol inibe a levedura, parando o crescimento celular.