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Three dimensional perfused cell culture for in vitro toxicity testingYang, Jie January 2011 (has links)
This study describes the development of a novel method of three dimensional perfused cell culture for in vitro toxicity testing. Multiple parallel perfused microbioreactors (TissueFlex<sup>TM</sup>) were adopted to provide a well-controlled cell culture environment. Alginate and collagen type I, commonly used as hydrogel scaffolds to support cell culture, were tested as the scaffolding materials for this application. Alginate supports cell proliferation, but does not support cell attachment. Collagen gel (type I), good for cell attachment but with poor mechanical strength, could be used at the high concentration of 5mg/ml to prevent the degradation of the gel. Improvement of collagen biomechanical property by a purpose-designed compressor to physically induce cross-linking showed promising results and merits further study. The suitability of alamarBlue® assay, a common non-toxic non-destructive viability assay method, was confirmed for this study and the protocol was optimised. To demonstrate the effectiveness of three dimensional perfused cell culture, human mesenchymal stem cells (MSC) seeded in collagen type I were employed to test the cell inhibition of two antibiotics, trimethoprim and pyrimethamine. The results displayed the perfusion system has greater advantage and sensitivity than the static system, as does these of 3D scaffolds, compared with 2D. Such differences are related to the continuous supply of fresh culture medium to keep cells at a stable pH, temperature, oxygen, and a more physiological like environment. The cytotoxicity of two stereoisomer compounds, obtained confidentially from Pfizer. Ltd., was assessed using the developed method and compared to conventional 2D static and perfused culture by using rat adipose mesenchymal stem cells. The results successfully distinguished toxic and non-toxic compounds and also demonstrated that the 3D perfused system improved the prediction of drug toxicity over 2D culture. 3D perfused bioreactors were applied to hepatotoxicity study using freshly isolated rat hepatocytes. Only algimatrix<sup>TM</sup> supported hepatocyte spheroid formation among those tested including collagen type I, alginate beads, poly lactic acid fibres, and Algimatrix<sup>TM</sup>. A new variation of TissueFlex<sup>TM</sup> bioreactor with micro-patterned surface, designed specifically for hepatocyte self-assembly culture without use of any scaffold, was tested. The results demonstrated that, compared with the standard sandwich culture, the self-assembly culture in the micro-patterned bioreactors showed high cell viability, biomarkers expression, as well as more physiological immunocytochemistry. Moreover, the differential gene expression indicated that self-assembly culture could provide more relevant information regarding metabolising processes than the 2D sandwich culture, which would potentially improve hepatotoxicity prediction. In conclusion, 3D perfused cell culture for in vitro toxicity testing improved the predictivity, reliability and physiological relevance of drug toxicity compared to traditional 2D culture.
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Hodnocení jaterní toxicity in vitro / Evaluation of liver toxicity in vitroKafuňková, Kateřina January 2020 (has links)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kafuňková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Evaluation of liver toxicity in vitro The subject of the diploma thesis is toxicity evaluation of newly synthesised substances on a cellular model representing a hepatocyte. The tested substances have been provided by the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University, as potential antifungal pharmaceuticals and medicine effective against Methicillin-resistant Staphylococcus aureus (MK-NO2-1, MK-NO2-2, DAB-5-K, PABA-Me-5, PABA-Et-5, MK-F-1, PABAN- 3, PABAN-5, MK-F-2, MK-CF3-1, MK-CF3- 2). In order to determine the toxicity we have implemented two methods. The first method is based upon measuring metabolic activity of cells by means of reducing tetrazolium to a colored product. The second method detects the amount of LDH released as a marker of toxicity. The human hepatoma cell line HepG2was used as a model. . The IC50 and EC50 parameters were used to assess the degree of viability and cytotoxicity. The final values obtained from the first method indicated all tested substances showed a certain level of toxicity to the hepatic tissue. MK-CF3-2 is the most...
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Studium cytotoxicity látek in vitro / Study on cytotoxicity of compounds in vitro.Vašková, Lucie January 2021 (has links)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...
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Estudo químico, biológico e toxicológico de drimys angustifolia Miers e drymis brasiliensis miersGomes, Madson Ralide Fonseca January 2012 (has links)
O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.
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Estudo químico, biológico e toxicológico de drimys angustifolia Miers e drymis brasiliensis miersGomes, Madson Ralide Fonseca January 2012 (has links)
O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.
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Estudo químico, biológico e toxicológico de drimys angustifolia Miers e drymis brasiliensis miersGomes, Madson Ralide Fonseca January 2012 (has links)
O gênero Drimys é o de maior área de distribuição geográfica da família (Winteraceae), que compreende sete gêneros e cerca de 120 espécies. No Brasil, encontra-se desde a Bahia até o Rio Grande do Sul e ocorre em duas espécies, Drimys angustifolia Miers. e Drimys brasiliensis Miers, conhecidas popularmente como “casca-de-anta”. As folhas e cascas são usadas na medicina popular como antiescorbútico, estimulante, antiespasmódica, antidiarreica, antifebril, contra hemorragia uterina, antibacteriana, no tratamento de asma, bronquite, certas afecções do trato digestivo e, algumas vezes no tratamento do câncer e inseticidas. Ambas são caracterizadas pela presença de flavonóides e sesquiterpenoides. O objetivo deste trabalho foi o de avaliar a composição química, o potencial antioxidante, antiviral, inseticida, antitumoral, antifúngico, avaliação tóxica aguda e, também, formular e caracterizar duas nanoemulsões dos óleos voláteis das duas espécies do estudo. Por meio da hidrodestilação, foi possível realizar a extração dos óleos essenciais e determinar seus rendimentos que variaram entre 0,3 e 0,5% de folhas. A identificação dos componentes foi realizada utilizando a Cromatrografia Gasosa acoplada ao espectrofotômetro de massas (CG-EM), as quais apresentaram como componentes majoritários em todas as análises, o biciclogermacreno para a espécie Drimys angustifolia e a ciclocolorenona para a espécie Drimys brasiliensis. A formulação e caracterização da nanoemulsão foi realizada dentro dos parâmetros estabelecidos (tamanho de partícula, índice de polidispersão, pH) e através da microscopia eletrônica de transmissão foi confirmado o tamanho na escala nano. No que tange a atividade inseticida, ambas as espécies mostraram-se promissoras repelindo o cupim da espécie Cryptotermes brevis com índices de repelência negativos. Para avaliação inicial do potencial larvicida de Aedes aegypti, os óleos das duas espécies não foram eficientes. No entanto, a nanoemulsão de Drimys brasiliensis levou a 69% e 89% de mortalidade, correspondendo às concentrações de 0,5 μg/mL e 0,83 μg/mL, respectivamente. Com relação a atividade antioxidante, os óleos das duas espécies não foram capazes de reduzir o radical (2,2-difenil-1-picril-hidrazil) DPPH . Já na atividade antiviral frente ao VHS-1, as duas espécies mostraram-se promissoras inibindo a replicação viral. Na atividade antitumoral nas linhagens de glioma (U-138 MG) e de bexiga (T24), somente o óleo de Drimys brasiliensis reduziu a viabilidade celular das duas linhagens pelo ensaio do MTT, com resultados similares na contagem celular. Na análise da citometria pela incorporação da Anexina-V/Iodeto de propídio caracterizou-se indução de apoptose tardia. Na atividade antifúngica, as duas espécies foram eficazes contra o fungo oportunista Acremonium, presente em diversas infecções em transplantados e imunocomprometidos. Quanto à avaliação da toxicidade aguda, as duas espécies mostraram importantes sinais de toxicidade em ratos Wistar como ptose, tremor, redução da atividade motora, exolftamia, aumento da freqüência respiratória. Os animais tratados com espécie D. brasiliensis ainda apresentaram aumento na micção e diarréia. Desta maneira, é importante ressaltar que todos os ensaios biológicos, o estudo toxicológico, e a elaboração e caracterização das nanoemulsões dos óleos essenciais foram realizados pela primeira vez para as duas espécies vegetais, destacando a importância e ineditismo do presente trabalho. / The genus Drimys presents the largest geographical distribution of the family (Winteraceae), which comprises seven genera and about 120 species. In Brazil, is found from Bahia to Rio Grande do Sul and occurs in two species, Drimys angustifolia L. and Drimys brasiliensis Miers, popularly known as "Casca-de-anta". The leaves and barks are used in folk medicine as antiscorbutic, stimulant, antispasmodic, anti-diarrheal, antipyretic, against uterine bleeding, antibacterial, to treat asthma, bronchitis, certain disorders of the digestive tract and sometimes in the treatment of cancer and insecticides. Both are characterized by the presence of flavonoids and sesquiterpenoids. The objective of this study was to evaluate the chemical composition, the antioxidant, antiviral, insecticidal, antitumor, antifungal, acute toxicological evaluation and also to formulate and characterize two nanoemulsions of the volatile oils of two species of the study. By steam distillation was possible to perform the extraction of essential oils and to determine their yields ranging between 0.3 and 0.5% of leaves. The identification of components was performed using the Gas Cromatography coupled to mass spectrometer (GC-MS), which presented as major components in all analyzis bicyclogermacrene for the Drimys angustifolia species and ciclocolorenona for the Drimys brasiliensis. The formulation and characterization of nanoemulsion was performed within the established parameters (particle size, polydispersity, pH) and by transmission electron microscopy confirming the size at the nanoscale. Regarding the insecticidal activity, both species were promising repelling termites Cryptotermes brevis with negative index of repellency. To evaluate initial potential larvicidal Aedes aegypti, the oils of the two species were not efficient, however, of the Drimys brasiliensis nanoemulsion the larvae mortality of the 69% and 89%, corresponding to concentrations of 0.5 μg/mL and 0.83 μg/mL, respectively. With respect to antioxidant activity, the oils of the two species have not been able to reduce the (2,2-diphenyl-1-picrylhydrazyl) DPPH radical. In the antiviral activity against the HSV-1, the two species were promising inhibiting viral replication. In antitumor activity in glioma cell lines (U-138 MG) and bladder (T24), only the oil Drimys brasiliensis reduced cell viability of two strains by the MTT assay, with similar results in cell counts and flow cytometry analysis of the incorporation of Anexinn-V/Propidium Iodide characterized as late apoptosis. In the antifungal activity, the two species were effective against the opportunistic fungus Acremonium, present in various infections in transplant and immunocompromised. Regarding the evaluation of acute toxicity, the two species showed significant signs of toxicity in rats as ptosis, tremor, decreased motor activity, exolftamia, shortness of breath. Animals treated with species Drimys brasiliensis also showed an increase in urination and diarrhea. Thus, it is important that all biological assays, the toxicity study, and preparation and characterization of nanoemulsions of essential oils were first performed for the two species, highlighting the importance and originality of this work.
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Etude in vitro de la toxicité de nanoparticules de boehmite. / In vitro study of boehmite nanoparticles toxicity.Pailleux, Mélanie 02 March 2012 (has links)
Cette étude a pour objectif d'apporter des éléments de réponse à la compréhension de la nature et de l'origine des effets biologiques des nanoparticules (NP) de boehmite en réalisant des tests de contacts in vitro sur des macrophages de culture (RAW 264.7), les macrophages étant la cible cellulaire privilégiée au niveau du tractus respiratoire. Différents domaines de la réponse cellulaire en relation avec les propriétés physico-chimiques des particules ont été étudiés. Dans une première partie l’activité biologique a été mesurée sur des NP de boehmite puis après broyage ou dispersion permettant d’observer en particulier l’influence de la taille des particules dans la réponse cellulaire.Les résultats soulignent que les particules industrielles de boehmite sont principalement caractérisées par une activité modérée au niveau de la réponse inflammatoire, mais sans effet cytotoxique ou sans stress oxydant significatif. Des différences en fonction de la taille des particules ont été observées sur les paramètres inflammatoire et cytotoxique.Toutefois, les mesures des biomolécules libérées dans les surnageants de culture cellulaire peuvent être biaisées par l’adsorption de ces biomolécules sur les NP en présence. Ainsi, ce mécanisme d'adsorption doit être pleinement compris pour éviter une interprétation erronée des données obtenues. Dans un second temps l’objectif a donc été d’élaborer une méthodologie afin d’évaluer les interactions organo-minérales en terme d’affinité et de quantité de biomolécules adsorbées sur les nanoparticules à l’équilibre thermodynamique et de déterminer une loi de correction. Dans ce contexte nous nous sommes intéressés en particulier au TNF-α. / This study aimed at a better understanding of the biological effects of boehmite nanoparticles by carrying out in vitro tests using a macrophage-like cell line (RAW 264.7), as macrophages represent the first target cells in the respiratory tract. Different cellular responses: inflammation (evaluated by the release of TNF-α), cell death (assessed by the release of LDH), and oxidative stress (production of ROS) were studied in relation to physico-chemical properties of the particles. In the first part of this work, the biological activity was measured using boehmite nanoparticles obtained either from industrial environment or by hydrothermal synthesis, and after grinding or dispersion that enabled us to observe the influence of particle size on the cellular response.The results point out that industrial boehmite particles are mainly characterized by a moderate pro-inflammatory activity, no cytotoxic effect and no significant oxidative stress. Differences depending on the size of the particles were observed on the inflammatory and cytotoxic parameters.However, the assessment of biomolecules released into cell culture supernatants may be biased by the adsorption of biomolecules on the nanoparticles. Thus, the adsorption mechanism must be fully understood to avoid misinterpretation of the results. Therefore, the second aims of this work were to develop a methodology to assess the organo-mineral interactions in terms of affinity and quantity of biomolecules adsorbed on the nanoparticles at the thermodynamic equilibrium and to determine a correction law. We focused more specifically our study on TNF-α.
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