Conjugated Polymer Nanoparticles for Biological Labeling and Delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.

Phase and partitioning behavior of phenols and polycyclic aromatic hydrocarbons in offshore oil and gas produced water

Produced water constitutes the largest volume of waste from offshore oil and gas operations and is composed of a wide range of organic and inorganic compounds. Although treatment processes have to meet strict oil in water regulations, the definition of “oil” is a function of the analysis process and may include aliphatic hydrocarbons which have limited environmental impact due to degradability whilst ignoring problematic dissolved petroleum species. This thesis presents the partitioning behavior of oil in produced water as a function of temperature and salinity to identify compounds of environmental concern. Phenol, p-cresol, and 4-tert-butylphenol were studied because of their xenoestrogenic power; other compounds studied are polycyclic aromatic hydrocarbon PAHs which include naphthalene, fluorene, phenanthrene, and pyrene. Partitioning experiments were carried out in an Innova incubator for 48 hours, temperature was varied from 4ﾟC to 70ﾟC, and two salinity levels of 46.8‰ and 66.8‰ were studied. Results obtained showed that the dispersed oil concentration in the water reduces with settling time and equilibrium was attained at 48 h settling time. Polycyclic aromatic hydrocarbons (PAHs) partitions based on dispersed oil concentration whereas phenols are not significantly affected by dispersed oil concentration. Higher temperature favors partitioning of PAHs into the water phase. Salinity has negligible effect on partitioning pattern of phenols and PAHs studied. Simulation results obtained from the Aspen HYSYS model shows that temperature and oil droplet distribution greatly influences the efficiency of produced water treatment system.

Using Vibrational Infrared Biomolecular Spectroscopy to Detect Heat-Induced Changes of Molecular Structure in Relation to Nutrient Availability of Prairie Whole Oat Grains on a Molecular Basis

Background To our knowledge, there is little study on the interaction between nutrient availability and molecular structure changes induced by different processing methods in dairy cattle. The objective of this study was to investigate the effect of heat processing methods on interaction between nutrient availability and molecular structure in terms of functional groups that are related to protein and starch inherent structure of oat grains with two continued years and three replication of each year. Method The oat grains were kept as raw (control) or heated in an air-draft oven (dry roasting: DO) at 120 °C for 60 min and under microwave irradiation (MIO) for 6 min. The molecular structure features were revealed by vibrational infrared molecular spectroscopy. Results The results showed that rumen degradability of dry matter, protein and starch was significantly lower (P <0.05) for MIO compared to control and DO treatments. A higher protein α-helix to β-sheet and a lower amide I to starch area ratio were observed for MIO compared to DO and/or raw treatment. A negative correlation (−0.99, P < 0.01) was observed between α-helix or amide I to starch area ratio and dry matter. A positive correlation (0.99, P < 0.01) was found between protein β-sheet and crude protein. Conclusion The results reveal that oat grains are more sensitive to microwave irradiation than dry heating in terms of protein and starch molecular profile and nutrient availability in ruminants.

Responses to various protein and energy supplements by steers fed low-quality tropical hay. 1. Comparison of response surfaces for young steers

Response curves were established for different supplements, offered at intakes ranging from 0 to 20 g/kg liveweight (W).day to young Bos indicus crossbred steers fed low-quality Rhodes grass (Chloris gayana) hay ad libitum in two pen experiments. Supplements included protein meals of varying rumen-degradability (cottonseed meal (CSM) or fishmeal), as well as ‘energy sources’ comprising grains of high and low ruminal starch degradability (barley and sorghum) and a highly fermentable sugar source (molasses), with all diets adjusted for rumen-degradable nitrogen and mineral content. Unsupplemented steers gained 0.08 and 0.15 kg/day, in Experiments 1 and 2, respectively. Growth of steers increased linearly with intake of ‘energy source’ supplements in increasing order of molasses, sorghum and barley (all differences P < 0.05). Steer growth rate also increased linearly with fishmeal, albeit over a narrow intake range (0–4.1 g/kg W.day), whereas the response with CSM was asymptotic, showing a steep response at low intake before levelling at ~1.2 kg/day. All supplement types were associated with a linear reduction in hay intake by the steers (energy substitution) where the reduction was greater (P < 0.05) for barley and molasses (not different) than for sorghum (P < 0.05), and for fishmeal compared with CSM (P < 0.05). In concurrent metabolism studies with the same rations, organic matter digestibility of the total ration (561–578 g/kg DM, unsupplemented) was increased linearly by barley and molasses (both P < 0.05) but was unaffected by CSM and sorghum supplements. The efficiency of microbial protein synthesis in steers increased linearly, from 91 g microbial crude protein/kg digestible organic matter (unsupplemented), in both molasses and CSM-supplemented steers, with the trend for a higher response to molasses (P = 0.05), and appeared most closely related to digestible organic matter intake. The response curves from these studies provide the practical framework upon which to formulate rations for cattle grazing low-quality forages.

Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain

A series of 3 experiments were conducted to evaluate the use of microalgae as supplements for ruminants consuming low-CP tropical grasses. In Exp. 1, the chemical composition and in vitro protein degradability of 9 algae species and 4 protein supplements were determined. In Exp. 2, rumen function and microbial protein (MCP) production were determined in Bos indicus steers fed speargrass hay alone or supplemented with Spirulina platensis, Chlorella pyrenoidosa, Dunaliella salina, or cottonseed meal (CSM). In Exp. 3, DMI and ADG were determined in B. indicus steers fed speargrass hay alone or supplemented with increasing amounts of NPN (urea combined with ammonia sulfate), CSM, or S. platensis. In Exp. 1, the CP content of S. platensis and C. pyrenoidosa (675 and 580 g/kg DM) was highest among the algae species and higher than the other protein supplements evaluated, and Schizochytrium sp. had the highest crude lipid (CL) content (198 g/kg DM). In Exp. 2, S. platensis supplementation increased speargrass hay intake, the efficiency of MCP production, the fractional outflow rate of digesta from the rumen, the concentration of NH3N, and the molar proportion of branched-chain fatty acids in the rumen fluid of steers above all other treatments. Dunaliella salina acceptance by steers was low and this resulted in no significant difference to unsupplemented steers for all parameters measured for this algae supplement. In Exp. 3, ADG linearly increased with increasing supplementary N intake from both S. platensis and NPN, with no difference between the 2 supplements. In contrast, ADG quadratically increased with increasing supplementary N intake from CSM. It was concluded that S. platensis and C. pyrenoidosa may potentially be used as protein sources for cattle grazing low-CP pastures.

Aditivos químicos ou biológicos na ensilagem de cana-de-açúcar: 2. parâmetros ruminais e degradabilidade da matéria seca e das frações fibrosas

Objetivou-se avaliar o efeito da inclusão de aditivos na ensilagem de cana-de-açúcar (Saccharum officinarum L.) sobre a degradação de MS e de componentes da parede celular e sobre os parâmetros de fermentação ruminal em bovinos alimentados com dietas contendo essas silagens. Utilizaram-se cinco novilhos da raça Nelore providos de cânula ruminal, alocados em delineamento quadrado latino 5 ´ 5 e alimentados com dietas com 65% de volumoso (%) MS. Foram avaliadas cinco silagens (base úmida): controle - cana-de-açúcar, sem aditivos; uréia - cana-de-açúcar + 0,5% ureia; benzoato - cana-de-açúcar + 0,1% de benzoato de sódio; LP - cana-de-açúcar inoculada com Lactobacillus plantarum (1 ´ 10(6) ufc/g MV); LB - cana-de-açúcar inoculada com L. buchneri (3,6 ´ 10(5) ufc/g forragem). A forragem foi armazenada em silos do tipo poço por 90 dias antes do fornecimento aos animais. Os parâmetros ruminais foram afetados de forma moderada pelas silagens e tiveram forte efeito do horário de coleta de amostras. As concentrações molares médias dos ácidos acético, propiônico e butírico foram de 60,9; 19,3 e 10,2 mM, respectivamente. O ambiente ruminal proporcionado por dietas formuladas com silagens de cana-de-açúcar foi satisfatório e similar ao tradicionalmente observado em dietas contendo cana. O uso de aditivos na ensilagem influenciou, de forma não-significativa, a degradabilidade ruminal da MS e da MO, mas não alterou a degradabilidade ruminal da fração fibrosa. Os aditivos aplicados à cana-de-açúcar resultaram em pequenas alterações na maior parte das variáveis avaliadas. Apesar de a degradabilidade ruminal das silagens ter sido pouco afetada pelo uso de aditivos, os valores observados foram próximos aos observados para a cana-de-açúcar in natura.

Conjugated polymer nanoparticles for biological labeling and delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. ^ In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. ^ This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. ^ The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.^

Composição botânica e qualidade do pasto selecionado por ovelhas em caatinga raleada e enriquecida.

Resumo: O presente estudo foi conduzido com o objetivo de determinar a composição botânica e a qualidade da dieta selecionada por ovelhas, através da técnica de micro-histologia fecal, em caatinga raleada e enriquecida com capim massai (Panicum maximum cv. Massai), recebendo diferentes quantidades de concentrado (0; 200; 350 e 500 g de concentrado por dia), e em diferentes períodos do ano (águas, transição água-seca e seca). Foram estimados também o consumo e digestibilidade dos nutrientes, bem como a degradabilidade de espécies forrageiras ingeridas pelas ovelhas. Os experimentos foram realizados na Fazenda Crioula do Meio, pertencente a Embrapa Caprinos e Ovinos em Sobral, CE no período de março a novembro de 2013. No Experimento 1, para a determinação da composição botânica e qualidade da dieta selecionada, foram utilizadas dezesseis ovelhas Somalis brasileira, gestantes, multíparas e peso médio de 30,58+2,48 kg. O acompanhamento da ingestão do pasto pelas ovelhas foi feito em três períodos (águas, transição água-seca e seca), referentes aos meses de abril, junho e agosto de 2013, respectivamente. Amostras das plantas foram coletadas para o preparo das lâminas de referência, e posterior identificação e caracterização dos descritores epidérmicos. O mesmo foi feito para as fezes coletadas nas ovelhas. Com base na proporção de cada espécie identificada nas lâminas fecais que compuseram a dieta, e na composição química das forrageiras identificadas, foi possível determinar a qualidade da dieta ingerida. De 76 espécies observadas no pasto, 33 foram identificadas na dieta das ovelhas, destacando as espécies sabiá (Mimosa caesalpiniaefolia), centrosema (Centrosema sp.), ervanço (Alternanthera brasiliana), massai (Panicum maximum cv Massai) e paco-paco (Wissadula rostrata) como as mais selecionadas pelos animais ao longo dos períodos, chegando a compor mais de 50% da dieta selecionada. Com a chegada do período seco, espécies indesejáveis como o marmeleiro (Croton sonderianus) e o mofumbo (Combretum lepreosum), também fizeram parte das plantas selecionadas. Quanto ao valor nutritivo da dieta selecionada, os animais selecionaram uma dieta com valor nutritivo superior ao amostrado no pasto. No Experimento 2, na mesma condição do experimento anterior, trinta e duas ovelhas Somalis brasileira foram utilizadas para determinação do consumo e digestibilidade dos nutrientes, realizado em três ensaios (abril - terço final de gestação; junho - lactação e agosto - desmame). Para predição do consumo, o indicador LIPE® foi utilizado. Pesagens quinzenais foram realizadas para avaliação do desempenho das ovelhas e dos cordeiros nascidos. O concentrado oferecido favoreceu a maior ingestão e digestibilidade da MS e PB, com efeito substitutivo em relação ao consumo de pasto (P<0,05). Para o período seco, menores consumos foram observados em relação aos períodos das águas e de transição água-seca (P<0,05). Maiores consumos e digestibilidades dos constituintes fibrosos foram observados para as ovelhas não suplementados (P<0,05). Na avaliação do desempenho, a suplementação oferecida determinou os maiores pesos verificados durante a lactação, ao desmame e para os pesos ao nascer e ao desmame dos cordeiros (P<0,05). No Experimento 3, dois ovinos Morada Nova foram utilizados para determinação da degradabilidade da matéria seca (MS), proteína bruta (PB) e fibra em detergente neutro (FDN) de cinco das forrageiras selecionadas pelas ovelhas no Experimento 1: M. caesalpiniaefolia, A. brasiliana, P. maximum cv. Massai, jurema-preta (Mimosa tenuiflora), C. leprosum, nos tempos 0, 6, 24, 48, 72 e 96 horas de incubação. Para cada forrageira, foram determinadas equações para o desaparecimento da MS, PB e FDN. Também foi feito o fracionamento da proteína em suas porções degradáveis e não degradáveis no rúmen. Foi observado maior desaparecimento da MS, PB e FDN, além dos melhores níveis de proteína efetivamente degradada no rúmen para A. brasiliana, seguido pelo P. maximum cv. Massai e M. caesalpiniaefolia. Com as informações obtidas, conclui-se que a micro-histologia fecal apresenta-se como uma técnica viável para avaliações da composição botânica da dieta selecionada por ovinos na caatinga. Ovelhas na caatinga possuem uma grande habilidade de selecionar a dieta, modificando-a ao longo das fases fenológicas, sempre na tentativa de estabelecer uma dieta com melhor valor nutritivo. Forrageiras como A. brasiliana, M. caesalpiniaefolia e o P. maximum cv. Massai, podem ser consideradas um interessante recurso alimentar, em virtude de seu valor nutricional e aproveitamento por ovelhas criadas na caatinga. Abstract: This study was conducted in order to determine the botanical composition and diet quality selected by sheep through fecal micro-histological technique, in thinned and enriched caatinga with Massai grass (Panicum maximum cv Massai.), receiving different amounts of concentrate (0; 200; 350 and 500 g of concentrate per day) at different periods (wet, transition wet-dry and dry). Were also estimated the intake, digestibility, as well as the degradability of forage species eaten by sheep. The experiments were performed in the "Fazenda Crioula do Meio", owned by Embrapa Goats and Sheep, in Sobral, Ceará State, Brazil, from march to november 2013. In Trial 1, sixteen female, pregnant, multiparous, with average body weight of 30,58+2,48 kg Somalis brasileira breed sheep were used to determine the botanical composition and the quality of the selected diet. The monitoring of pasture intake of sheep were conducted in three phenological periods of the caatinga's pasture (wet season, transition wet-dry and dry season). Plant samples were collected for the preparation of the reference slides, with subsequent identification and characterization of epidermal descriptors. The same was done for the feces collected in sheep. Considering the proportion of each species identified in fecal slides which composed the diet, and the chemical composition of forage identified, it was possible to determine the quality of the selected diet. From 76 species observed in the pasture, 33 species was identified in the sheep selected diet, emphasizing the Sabiá (Mimosa caesalpiniaefolia), centrosema (Centrosema sp.), ervanço (Alternanthera brasiliana), massai (Panicum maximum cv Massai) e paco-paco (Wissadula rostrata) as the most selected species by sheep during the study, composing more than 50% of the selected diet. In the Dry Season, undesirable species like marmeleiro (Croton sonderianus) and mofumbo (Combretum leprosum), were also constituent of the diet. Regarding to the nutritional value of selected diet, the sheep selected a diet with more protein than the sampled in the pasture. In the final late gestation, the sheep without supplementation ate a diet above 16% of CP, higher than the selected diet by treatments 350 and 500 g of concentrate per day (P<0.05). Were also observed to the non supplemented sheep, less fiber content intake (P<0.05). In the Trial 2, in the same condition of the first Trial, thirty two Somalis brasileira female sheep were used to determine the intake and digestibility, conducted in three assays (April - third late pregnancy; June - lactation and August - weaning). To predict the intake, the marker LIPE was used. Sheep and lambs were weighted every two weeks to performance evaluation. The concentrate offered to sheep favored to higher intake and digestibility of DM and CP than non supplemented sheep, with inverse relationship to the pasture intake (P<0.05). For the Dry Season, lower intake were observed than for Wet Season and Transition Wet-Dry (P<0.05). Higher intakes and digestibility of the fiber constituents were verified to non supplemented sheep (P<0.05). To performance evaluation, the offered supplementation determined the higher weights observed during lactation and weaning of sheep, and to birth and weaning weights of lambs (P<0.05). In Trial 3, two male sheep were used to determine the degradability of dry matter (DM), crude protein (CP) and neutral detergent fiber (NDF) of five forages selected by sheep in Trial 1: M. caesalpiniaefolia, A. brasiliana, P. maximum cv. Massai, jurema-preta (Mimosa tenuiflora) and C. lepreosum, at zero, 6, 24, 48, 72 and 96 hours of incubation. For each forage, were determined equations for the disappearance of DM, CP and NDF. It was also realized the protein fractions in their degradable and non-degradable in the rumen parts. Was detected a higher disappearance of DM, CP and NDF, as also better proportion of the rumen degradable protein to A. brasiliana, followed by P. maximum cv. Massai and M. caesalpiniaefolia. With the information obtained, it is concluded that the fecal micro histological technique presents as a viable technique to evaluate the selected diet by sheep in caatinga's pasture. On this pasture, the sheep are skilled to select the diet, changing during the phenological phases, trying to form a diet of better nutritive value. Forages as A. brasiliana, P. maximum cv. Massai and M. caesalpiniaefolia, can be considered an interesting food source to ewes kept in the caatinga.