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Controlled Release of Antioxidants via Biodegradable Polymer Films into Milk and Dry Milk Productsvan Aardt, Marleen 08 December 2003 (has links)
Residual value is defined as the price for which a used piece of equipment can be sold in the market at a particular time. It is an important element of the owning costs of equipment and needs to be estimated by equipment managers for making investment decisions.
The purpose of this study is to gain insights into the residual value of selected groups of heavy construction equipment and to develop a mathematical model for its prediction. Auction sales data were collected from two online databases. Manufacturer publications and an online source provided size parameters and manufacturers suggested retail prices matching the auction records. Macroeconomic indicator values were collected from a variety of sources, including government agencies. The data were brought into the same electronic format and were matched by model name and calendar date, respectively.
Data from auctions in the U.S. and in Canada were considered for this study. Equipment from four principal manufacturers of up to 15 years of age at the time of sale was included. A total of 35,542 entries were grouped into 11 different equipment types and 28 categories by size as measured by horse power, standard operating weight, or bucket volume. Equipment types considered were track and wheel excavators, wheel and track loaders, backhoe loaders, integrated toolcarriers, rigid frame and articulated trucks, track dozers, motor graders, and wheel tractor scrapers.
Multiple linear regression analyses of the 28 datasets were carried out after outliers had been deleted. Explanatory variables for the regression model were age in years, the indicator variables manufacturer, condition rating, and geographic region, and selected macroeconomic indicators. The response variable was residual value percent, defined as auction price divided by manufacturers suggested retail price. Different first, second, and third-order polynomial models and exponential and logarithmic models of age were examined. A second-order polynomial was selected from these functional forms based on the adjusted coefficient of determination. Coefficients for the 28 models and related statistics were tabulated. A spreadsheet tool incorporating the final regression model and its coefficients was developed. It allows performing the residual value prediction in an interactive and intuitive manner. / Ph. D.
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Influência da adição in vivo de vitamina E e de métodos de abate nos atributos de qualidade de filés de tilápia /Otani, Fabrizia Sayuri. January 2009 (has links)
Resumo: Este trabalho objetivou avaliar a influência da vitamina E pela suplementação por meio da dieta, em peixes submetidos a dois métodos de abate (imersão em água gelada e sangria), nos atributos de qualidade de filés congelados de tilápia (Oreochromis spp.). Peixes, com peso médio inicial de 340 g, foram alimentados por um período de nove semanas com três dietas isocalóricas (3264,09 kcal ED/kg) e isoprotéicas (24,8% PD), diferindo na adição de 100 mg/kg de ração de α-tocoferol (grupo TO), 100 mg de princípio ativo/kg de ração de acetato de α- tocoferil (grupo AC), e sem adição de vitamina E (grupo controle - CO). Ao final do período de alimentação, os peixes foram abatidos pelos métodos citados anteriormente. Utilizou-se delineamento inteiramente casualizado, em esquema fatorial 3x2x5, caracterizado por três dietas, dois métodos de abate e cinco tempos de análises dos filés. Foram realizadas análises de desempenho e corporais, e de composição centesimal, oxidação lipídica pela formação de substâncias reativas ao ácido tiobarbitúrico (SRATB), e sensorial pelo método do Índice de Qualidade (MIQ) nos filés, nos tempos zero (antes do congelamento), 45, 90, 120 e 150 dias de estocagem. A suplementação da vitamina E não afetou os parâmetros de desempenho, entretanto influenciou na composição centesimal e na oxidação lipídica, protegendo os filés ao longo do período de estocagem. Foi elaborada tabela de avaliação dos filés congelados, pelo MIQ, instrumento útil para a análise de atributos de qualidade. / Abstract: The aim of this investigation was to evaluate the influence of vitamin E dietary supplementation, and fish slaughtering methods (immersion in ice-water and exsanguination), on the fillets quality of tilapia, during frozen storage. Fish of 340 g mean initial body weight were fed for nine weeks with three isoenergetic (containing 3264,09 kcal DE/kg) and isonitrogenous (24,8% DP) diets. Two diets were supplemented with 100 mg/kg diet of α-tocopherol (TO group) and 100 mg of active source/kg diet of α-tocopheryl acetate (AC group), plus a nonsupplemented diet (control group - CO). At the termination of the 9-week feeding trial, fish were slaughtered by the summoned methods.A completely randomized design was used, in a 3x2x5 factorial scheme, characterized by the 3 vitamin E supplementation diets, 2 slaughter methods and 5 fillets analysis. The growth performance parameters, hepatosomatic and fat viscerosomatic index, centesimal composition, lipid oxidation determined by the thiobarbituric acid reactive substances (TBARS) and sensorial by Quality Index Method (QIM) analysis were analyzed in five times: before frozen storage, 45, 90, 120 and 150 days storage. The vitamin E supplementation did not influence the growth performance parameters, but centesimal composition were influenced, and vitamin E protected fillets from lipid oxidation in frozen storage. Fillets quality availability table were organized, by QIM, for to help in sensorial analysis. / Orientadora: Léa Silvia Sant'Ana / Coorientador: João Batista Kochenborger Fernandes / Banca: Cristiane Rodrigues Pinheiro Neiva / Banca: Cláudio Angelo Agostinho / Mestre
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Avaliação laboratorial e produtiva de bovinos confinados, alimentados com feno de Brachiaria sp. e suplementados com antioxidantes / Laboratory and productive evaluation of confined cattle feeded with feno of Brachiaria sp. and supplemented with antioxidantsCosta, Gustavo Lage 22 December 2014 (has links)
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Previous issue date: 2014-12-22 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The grass Brachiaria sp. brought to Brazil in the green revolution, came with the goal of
increasing the Brazilian livestock production. The introducing of the grass, starts emerging
problems such as leaf hopper pastures and hepatogenous photosensitization. The etiology of
photosensitization caused by Brachiaria sp. is still controversial. But not even knowing for
sure rightly if the cause is the steroidal saponins or the fungus Phytomices chartarium, it is
known that they occur and cause damage to livestock. The described laboratory
abnormalities are increased activity of enzymes aspartate aminotransferase (AST) and
gamma glutamyl transferase (GGT), bilerrubinemia and jaundice, and evidence of
insufficient functionof.liver. Histopathological examination can demonstrate a
cholangiohepatitis and presence of foamy macrophages. The presence of these cells is
important because its quantification was negatively correlated with the gain in cattle weight.
Thus this study evaluated 40 Nelore young bulls, with the Brachiaria sp hay consumption
adjusted to the maximum amount of forage (84%), with the goal of reproducing the hepatic
abnormalities described in cases of photosensitization. Therefore, the aim of this study was
to evaluate the possible liver changes, by the metabolic and histological profile of the liver
and lymph nodes, and also check if the offered antioxidants would be able to minimize these
changes, promoting greater weight gain and improving carcass characteristics of feedlot
cattle. As a result, the braquiaria hay with low amounts of saponin and fungal spores did not
induce clinical signs of liver damage in young bulls, but laboratory assessments indicate evidence of chronic inflammatory liver change with the presence of foamy macrophages.
The antioxidant supplementation did not alter the metabolic profile of the animals, but
reduced the amount of foamy macrophages in the liver. However this decrease did not
improve weight gain of cattle, carcass characteristics and the final quality of the meat. / O capim Brachiaria sp. trazido para o Brasil na revolução verde, veio com o
objetivo de aumentar a produção pecuária brasileira. Juntamete com o capim,
começararam a surgir problemas, como a cigarrinha das pastagens e a
fotossenssibilização hepatógena. A etiologia da fotossenssibilização causada pelo
capim Brachiaria sp. ainda é controversa. Mas mesmo não sabendo ao certo se
o causador é as saponinas esteroidais ou o fungo Phytomices chartarium, sabese
que elas ocorrem, e causam prejuízos à pecuária. As alterações laboratoriais
descritas são o aumento de atividade das enzimas aspartato aminotransferase
(AST) e gama glutamiltransferase (GGT), bilerrubinemia e icterícia, além de
indícios de insuficiência da função hepática. Os exames histopatológicos podem
demonstrar uma colangiohepatite e presença de macrófagos espumosos. A
presença destas células é importante, pois a sua quantificação foi correlacionada
negativamente com o ganho em peso de bovinos. Desta forma, o objetivo deste
estudo foi avaliar 40 tourinhos nelore, com o consumo de feno de Brachiaria sp.
ajustado para a maior quantidade possível de volumoso (84%), no intuto de
reproduzir as alterações hepáticas descritas em casos de fotossensibilização
hepatógena, que foram monitoradas por meio de testes como o perfil
metabólico e histológico do fígado e linfonodos, e ainda verificar se os
antioxidantes ofertados seriam capazes de minimizar estas alterações,
promovendo maior desenvolvimento ponderal e melhorando as características
da carcaça de bovinos confinados. Como resultados, o feno de braquiaria com
baixas quantidades de saponina e de esporos do fungo, não induziram sinais
clínicos de lesão hepática nos tourinhos. Porém as avaliações laboratoriais
indicam evidencias de alteração hepática inflamatória crônica com presença de
macrófagos espumosos. A suplementação com antioxidantes não alterou o perfil
metabólico dos animais, mas reduziu a quantidade dos macrófagos espumosos
no fígado. Essa diminuição não melhorou o desenvolvimento ponderal dos
bovinos, as características de carcaça e a qualidade final da carne.
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MECHANISTIC STUDIES OF PROTON-COUPLED ELECTRON TRANSFER REACTIONS INVOLVING ANTIOXIDANTSMeng, Kejie 01 January 2018 (has links)
The objective of the research was to investigate proton-coupled electron transfer (PCET) reactions involving antioxidants to gain insight into the detailed mechanisms of glutathione (GSH), Trolox, and α-tocopherol (α-TOH). PCET reactions are complex redox reactions that transfer electrons and protons sequentially or in concert. These reactions are ubiquitous in natural or artificial processes that produce electrochemical energy that is extractable as electricity or as chemical fuels of high energy content. Examples of processes based on PCET are photosynthesis, respiration, nitrogen fixation, carbon dioxide reduction, redox fuel cells, and artificial photosynthesis. Antioxidants were selected as a PCET model to understand the coupling between proton transfer (PT) and electron transfer (ET) in order to elucidate structure-reactivity relationships under different experimental conditions. PCET reactions were studied with a set of electrochemical techniques to propose a preliminary mechanism that could be validated with digital simulations matching the electrochemical response. In some cases, other analytical techniques were used to aid in the system characterization. This thesis presents the results and discussion of the effects of oxidant-base pairs on the mediated oxidation of GSH, the -2e-/-H+ process of Trolox in aqueous and nonaqueous solvents with various pH values, and the particle collision electrolysis of α-tocopherol in oil-in-water emulsion droplets on an ultramicroelectrode. Ultimately our goal was to determine the kinetic and thermodynamic factors that control PCET reactions so that they can be applied in designing artificial systems for the production of energy using more abundant reagents with lower cost and better yields.
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Embryogenesis is dependent upon 12-lipoxygenase, 5-lipoxygenase, and α-tocopherol to modulate polyunsaturated fatty acid status and the production of oxidized fatty acids in zebrafish / Embryogenesis is dependent upon 12-lipoxygenase, 5-lipoxygenase, and alpha-tocopherol to modulate polyunsaturated fatty acid status and the production of oxidized fatty acids in zebrafishLebold, Katherine M. 25 May 2012 (has links)
Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids required for proper embryonic development, specifically neurodevelopment. However, little is known regarding their conversion to other metabolites during embryogenesis. The oxidation of ARA gives rise to the biologically active eicosanoids and the oxidation of DHA gives rise to the biologically active docosanoids. The oxidation of ARA and DHA occurs through enzymatic processes, via lipoxygenase (LOX), or non-enzymatic processes, via radical-mediated lipid peroxidation. We hypothesize that oxidation of ARA and DHA via LOX is required for proper embryonic development. Additionally, we hypothesize that α-tocopherol, a potent lipid soluble antioxidant, mediates the conversion of ARA and DHA to their respective oxidized metabolites. Using zebrafish as a model of vertebrate embryogenesis, we found that the selective knockdown of either 12-LOX or 5-LOX decreased the production of docosanoids, altered fatty acid homeostasis, and increased the incidence of malformations and mortality in embryos by 24 hours post fertilization. α-Tocopherol deficiency also increased the incidence of malformations and mortality during embryogenesis, and in its absence, increased oxidized metabolites of ARA and DHA and decreased fatty acids concentrations. Therefore, oxidized metabolites of ARA and DHA perform crucial functions during embryonic development, but the production of oxidized fatty acids must be balanced with antioxidant bioavailability for proper embryogenesis. / Graduation date: 2012
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Atténuation des oxydations phosphorylantes et induction d'une réponse cellulaire hypoxique : effêt de l'[alpha]-tocophérol-acétate et de miR-210 sur les cellules stromales mesenchymateuses / Attenuation of oxidative phosporylation and induction of hypoxic celle response : [alpha]-tocopherolacetate and miR-210 effects on mesenchymal stromal cellsLoncaric, Darija 15 November 2019 (has links)
Dans cette thèse, nous avons combiné les approches des cultures single-cell, de cytométrie en flux,des analyses de métabolisme énergétique et de génétique moléculaire afin d’explorer les effets del’Acétate d’α-Tocopherol (α-TOA) sur les cellules stromales mésenchymateuses (MStroC) et leurs souspopulations fonctionnelles (cellules souches et progénitrices mésenchymateuses). L’autre but était de tester une molécule de miR-210 par rapport à son utilisation potentielle comme « hypoxia mimicking molecule ». Après avoir démontré l’hétérogénéité de la population MStroC et conclu que la population de premier passage est appropriée pour des expérimentations ultérieures, nous avons trouvé que l’α-TOA présentait un effet positif sur le maintien de la capacité proliférative élevée des cellules souches mésenchymateuses. Cet effet est accompagné d’une atténuation de l’activité de la chaîne de transport d’électrons (ETC) qui pourrait d’autre part expliquer l’accroissement modéré du niveau des Reactive Oxygen Species mitochondriales (mtROS) que nous avons détectées. L’augmentation du niveau de mtROS pourrait être associée à une dégradation de protéine HIF-1 dans la population MStroC exposée à l’α-TOA. Bien que nous n’ayons pas détecté d’augmentation compensatoire de la glycolyse, les phénomènes observés représentent en partie la réponse cellulaire complexe au faible niveau d’O2. Il a été établi que ce phénomène était relié au maintien de primitivité des cellules souches. Le mécanisme exact reste à clarifier ainsi que son potentiel translationnel. En outre, nous avons apporté la preuve que miR-210 fait partie intégrante de la réponse des MStroC à la faible concentration en O2. Dans cette étude, nous avons montré que l’augmentation de l’expression de miR-210 sur une période courte (jusqu’à 24 heures) et après une période étendue (jusqu’à 72 heures) d’exposition des MStroC à une faible concentration en O2. De plus, nous avons prouvé que ce micro ARN pouvait être régulé par les deux facteurs transcriptionnels HIF-1 et HIF-2, nous laissant penser que ceci faisait partie intégrante de la réponse des MStroC à une faible concentration en O2. Jusqu’à présent, nos données suggèrent que miR-210 est digne d’intérêt en tant que bonne molécule « hypoxia mimicking ». / In this thesis, we combined approaches of single-cell cultures, flow-cytometry, energetic metabolismanalysis and molecular genetics in order to get insight in the effects of α-Tocopherol-Acetate (α-TOA)on Mesenchymal Stromal Cells (MStroC) and their functional subpopulations (mesenchymal stem and progenitor cells). The other aim was to test a miR-210 molecule with respect to its potential use as hypoxia mimicking molecule. After defining MStroC population heterogeneity and concluding that the first passage population is convenient for further experiments, we demonstrated that α-TOA exhibits a positive effect on the maintenance of high proliferative capacity of mesenchymal stem cells. This effect could be associated with an attenuation of electron transport chain (ETC) activity, which, on the other hand could explain moderate increase in the level of mitochondrial Reactive Oxygen Species (mtROS) we detected. The increase in mtROS level could be associated with a decreased HIF-1 alpha protein degradation in MStroC exposed to α-TOA. Although we did not detect a compensatory increase in glycolysis, the observed phenomena depict part of a complex cellular response to the low O2 that is demonstrated to be related with maintenance of stem cell primitiveness. The exact mechanism remains to be elucidated as well as its translational potential. In addition, we provided new evidences that miR-210 is integral part in MStroC response to low O2. In the study, we showed increased in miR-210 expression in a short-term (up to 24 hours) and after extended (up to 72 hours) MStroC exposed to low O2. Moreover, we demonstrated that this micro- RNA could be regulated by both HIF-1 and HIF-2 transcriptional factors, suggesting it as integral part of MStroC response to low O2. So far, our data suggest that miR-210 is worthy to be considered as good hypoxia mimicking molecule.
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Исследование антиоксидантных свойств витаминных препаратов различной гидрофильности : магистерская диссертация / Investigation of the antioxidant properties of vitamin preparations of various hydrophilicityТимина, Д. С., Timina, D. S. January 2021 (has links)
Объектами исследования являются витамины разной гидрофильности и витаминосодержащие препараты в таблетированной форме. Цель работы: исследование антиоксидантных свойств витаминных препаратов различной гидрофильности. Исследована антиоксидантная емкость модельных растворов и смесей антиоксидантов разной гидрофильности потенциометрическим методом; подобраны оптимальные условия для проведения анализа липофильного и гидрофильного соединений в совместном присутствии; исследованы фармацевтические препараты в таблетированной форме, содержащие витамины С и Е; проведено сравнение результатов с методом Фолина-Чокальтеу. Рассмотрена возможность применения методики определения АОЕ в условиях микрофлюидного устройства. / The objects of research are vitamins of different hydrophilicity and vitamin-containing preparations in tablet form. Purpose of the work: study of the antioxidant properties of vitamin preparations of various hydrophilicity. The antioxidant capacity of model solutions and mixtures of antioxidants of different hydrophilicity was investigated by potentiometric method; the optimal conditions were selected for the analysis of lipophilic and hydrophilic compounds in their joint presence; investigated pharmaceutical preparations in tablet form containing vitamins C and E; the results are compared with the Folin-Chocalteu method. The possibility of using the method for determining AOE in a microfluidic device is considered.
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Etude et fonctionnalisation de protéines végétales en vue de leur application en microencapsulation / Study and functionalization of vegetable proteins and their application in microencapsulationNesterenko, Alla 05 December 2012 (has links)
Les protéines extraites des végétaux sont des matériaux relativement peu coûteux, non toxiques, biocompatibles et biodégradables. Elles représentent une bonne alternative aux protéines d’origine animale et aux polymères dérivés du pétrole. Dans le cadre de cette étude, les protéines extraites de graines de soja et de tournesol ont été utilisées en tant que matériaux enrobants pour la microencapsulation de la matière active hydrophobe (α-tocophérol) ou hydrophile (acide ascorbique) par le procédé d’atomisation. Les protéines de soja sont largement utilisées dans les applications alimentaires et non-alimentaires, notamment en microencapsulation. Elles sont donc étudiées dans ce travail comme matériau enrobant de référence. Les protéines de tournesol n’ont quant à elles pas d’application industrielle concrète, si ce n’est sous la forme de tourteaux dans l’alimentation animale. C’est pourquoi il nous semble pertinent de trouver des nouvelles voies de valorisation pour ce coproduit d’origine agricole. Plusieurs modifications des protéines, telles que l’hydrolyse enzymatique, l’acylation, la réticulation enzymatique et la cationisation ont été étudiées dans le but d’améliorer les propriétés encapsulantes du matériau enrobant. Dans le contexte de la chimie verte, toutes les modifications ont été effectuées sans utilisation de solvants organiques ni de catalyseurs chimiques. L’influence des modifications chimiques et enzymatiques des protéines, et des paramètres du procédé (pression d’homogénéisation, ratio matériau enrobant/matière active et concentration en protéines) sur les différentes caractéristiques des préparations liquides et des microparticules (viscosité, taille des gouttelettes dans le cas des émulsions, morphologie et taille des microparticules), ainsi que sur les paramètres liés au procédé d’atomisation (rendement et efficacité de microencapsulation) a été particulièrement étudiée au cours de ce travail. Les résultats obtenus confirment que l’extrait protéique de tournesol est tout à fait pertinent comme matériau enrobant et permet d’obtenir des efficacités de microencapsulation significativement plus élevées par rapport à celles obtenues avec l’extrait protéique de soja. / Proteins extracted from vegetables are relatively low-cost, non-toxic, biocompatible and biodegradable raw materials. They represent a good alternative to animal-based proteins and petroleum-extracted polymers. In this study, proteins derived from soybean and sunflower seeds were used as wall materials for microencapsulation of hydrophobic (-tocopherol) or hydrophilic (ascorbic acid) active material by spray-drying technique. Soybean proteins are widely used in food and non-food applications, especially in microencapsulation. They were studied in this work as wall material of reference. Sunflower proteins are not actually used in industrial application, but only in the form of oil-cake for animal feeding. That’s why new ways of valorization of this agricultural by-product should be investigated. Several proteins’ modifications such as enzymatic hydrolysis, acylation, cross-linking and cationization were studied in order to improve encapsulating properties of wall material. In the context of green chemistry, all the modifications and preparations were performed without use of organic solvents and chemical catalysts. The effect of protein chemical and enzymatic modifications, and process parameters (homogenization pressure, wall/core ratio and protein concentration) on different characteristics of liquid preparations and microparticles (viscosity, emulsion droplet size, microparticle size and morphology) and on parameters related to the spray-drying process (yield and efficiency of microencapsulation) was particularly investigated in this study. The obtained results confirmed that sunflower proteins are quite suitable as encapsulating agent and provide the microencapsulation efficiencies significantly higher compared to those obtained with soy proteins.
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The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer NanomedicineKumar, Krishan January 2015 (has links) (PDF)
The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters.
Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity
In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA
(pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay.
Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response.
Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids
Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines.
Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response.
Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids
To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario.
Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE.
Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors
The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic
studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels.
Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels.
Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells
This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol
conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine.
Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions.
Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo
Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments.
Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections.
Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide
Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA
and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications.
Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.
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