Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-08-01T13:01:53Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao_Anderson Costa da Silva.pdf: 1776105 bytes, checksum: e9ddfbafe65f7344ec61ee2426b1f505 (MD5) / Made available in DSpace on 2014-08-01T13:01:53Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao_Anderson Costa da Silva.pdf: 1776105 bytes, checksum: e9ddfbafe65f7344ec61ee2426b1f505 (MD5) Previous issue date: 2010 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this work we investigated the internalization process of magnetite nanoparticles, surface coated with dextran, by mice tumour cells of Sarcoma 180 (S180) through the tech- niques of vibrating sample magnetometer (VSM) and static magnetic birefringence (SMB). The magnetic fluid sample, stable in physiological conditions, was prepared by the coprecip- itation method. The growth of nanoparticles occurred in conjunction with the nanoparticle surface coating process by dextran. The crystal structure was confirmed by X-ray diffraction. The nanoparticles were characterized by high resolution transmission electronic microscopy. The Sturges method was used to obtain the polydispersity in diameter, which was fitted by a lognormal size distribution obtaining a modal diameter of 5.5 ± 0.1 nm and dispersity of 0.18 ± 0.02.The mice tumour cell sarcoma 180 was obtained using protocol established by the American Type Culture Collection (ATCC, Rockville, MD, USA). Studies of cytotoxicity, using the MTT method, were obtained for a nanoparticle volumetric fraction of φ = 0.00065 after one and five hours of exposure of cells S180 to the nanoparticles. In particular, we found a cellular viability of 87 ± 11 % after one hour of exposure proving that there was no appreciable cell death in the time interval in which the measurements of MAV and BME were performed. Magnetization measurements were performed to obtain the volume fraction of nanoparticles. Tests regarding the effect of centrifugation of nanoparticles suspended in cell culture medium RPMI 1640 showed a extremely low sedimentation of magnetic nanoparticles. A procedure, using a acceleration of 260×g for 10 minutes, was used to separate cells containing internalized nanoparticles from nanoparticles suspended in RPMI 1640. Measurements of magnetization of S180 cells containing nanoparticles were performed in a wide range of exposure time (100 iv minutes). Between 10 and 70 minutes the amount of nanoparticles in mass unit increased from 52 ± 20 pg/cell to 110 ± 15 pg/cell. Indeed magnetometry data indicate that the process of internalization had achieved saturation between 30 to 40 minutes. Magneto-optical technique of SMB was also used to investigate the process of inter- nalization of nanoparticles. Firstly, SMB measurements were performed in control samples consisting of magnetic nanoparticles suspended in RPMI 1640. We investigated the effects of nanoparticle concentration and aging time (related to the dynamics of nanoparticle agglom- eration). In particular, the average size of the agglomerate (Q), associated with the number of nanoparticles forming a linear chain, remained basically constant, Q = 4.8 ± 0.2 for a full- time of 70 minutes. Magnetic birefringence saturation data also remained stable in this time interval. Additionally, analysis of the measurements of SMB were also used to estimate the thickness of the coating layer (dextran), from which we found 1.70 ± 0.02 nm. Unlike VSM data, SMB measurements were obtained on samples containing both S180 cells and magnetic nanoparticles inside the RPMI medium 1640. Data were obtained in a wide range of time (120 min.). Initially it was observed that the SMB signal decreases in a time range and then increases again (between 30-40 min.). The fit of the experimental data indicate that the mag- netic birefringence saturation (∆ns) decreases in the first 30 minutes and then increases again smoothly, while the average size of the cluster has the opposite behavior, i.e. increases in the first 30 minutes and then decreases. In particular, for a exposure time, t(exp), of 10 min. the average size of the agglomerate (magnetic birefringence saturation) changed from 4.18 ± 0.04 (∆n(s) = 3.41 ± 0.02 ×1018 cm−3 min. As the birefringence saturation is proportional to the number of nanoparticles contribut- ing to the magneto-optical signal one can conclude that the decrease in the magneto-optical signal was due to the process of internalization of magnetic nanoparticles by cells S180. On the other hand, the analysis of the aging time dependence of the mean size of the agglomerate also suggests that the process of internalization occurs primarily with anisometric nanoparticles or nanostructures forming small agglomerates. Finally, after reaching saturation of the process ) to 5.22 ± 0.08 (∆ns = 2.75 ± 0.02 ×1018 cm−3 ) at texp = 30 v of nanoparticle internalization we found a formation of small agglomerates in the RPMI 1640 medium, which is responsible for the increased intensity of the magneto-optical signal, as well as the decrease of the mean size of the agglomerate for times longer than 30 minutes. / Neste trabalho investigamos o processo de internaliza ̧c ̃ao de nanopart ́ıculas magn ́eticas de magnetita, recobertas com dextrana, por c ́elulas neopl ́asicas de Sarcoma 180 (S180), por meio das t ́ecnicas de magnetometria de amostra vibrante (MAV) e birrefringˆencia magn ́etica est ́atica (BME). A amostra de fluido magn ́etico, est ́avel em pH fisiol ́ogico, foi preparada pelo m ́etodo de coprecipita ̧c ̃ao. O crescimento das nanopart ́ıculas ocorreu conjuntamente com o recobrimento molecular por dextrana. A estrutura cristalina foi confirmada por difra ̧c ̃ao de raios-X. As nanopart ́ıculas foram caracterizadas por microscopia eletrˆonica de transmiss ̃ao de alta resolu ̧c ̃ao. O m ́etodo de Sturges foi utilizado para obter a polidispers ̃ao de diˆametros, que foi ajustada por uma distribui ̧c ̃ao do tipo lognormal com diˆametro modal de 5, 5 ± 0, 1 nm e dispersidade 0, 18 ± 0, 02. A linhagem tumoral de camundongo Sarcoma 180 foi obtida segundo protocolo estabelecido pela American Type Culture Collection (ATCC, Rockville, MD, USA). Estudos de citotoxicidade, utilizando o m ́etodo MTT, foram feitos para uma fra ̧c ̃ao volum ́etrica de nanopart ́ıculas de φ = 0, 00065 ap ́os uma e cinco horas de exposi ̧c ̃ao das c ́elulas S180 as nanopart ́ıculas. Em particular, foi encontrada uma viabilidade celular de 87 ± 11% ap ́os uma hora de exposi ̧c ̃ao provando que n ̃ao houve morte celular significativa no intervalo de tempo em que as medidas de MAV e BME foram realizadas. Medidas de magnetiza ̧c ̃ao foram feitas para obter a fra ̧c ̃ao volum ́etrica de nanopart ́ıculas. Testes do efeito de centrifuga ̧c ̃ao das nanopart ́ıculas suspensas em meio de cultura celular RPMI 1640 revelaram uma sedimenta ̧c ̃ao de nanopart ́ıculas magn ́eticas extremamente baixa. Um procedimento, utilizando acelera ̧c ̃ao de 260×g por 10 minutos, foi adotado para separar c ́elulas contendo nanoparticulas internalizadas daquelas suspensas no meio RPMI 1640. Medidas de magnetiza ̧c ̃ao das c ́elulas S180 contendo nanopart ́ıculas foram realizadas numa larga faixa de tempo de exposi ̧c ̃ao (100 minutos). Entre 10 e 70 minutos a quantidade de nanopart ́ıculas em unidade de massa passou de 52 ± 20 pg/c ́elula para 110 ± 15 pg/c ́elula. De fato os dados de magnetometria indicam que o processo de internaliza ̧c ̃ao atingiu a satura ̧c ̃ao entre 30 a 40 minutos. A t ́ecnica de magneto ́optica de BME tamb ́em foi utilizada para investigar o processo de internaliza ̧c ̃ao das nanopart ́ıculas. Primeiramente, medidas de BME foram feitas em amostra controle consistindo de nanopart ́ıculas magn ́eticas suspensas em meio RPMI 1640. Foram investigados efeitos de concentra ̧c ̃ao de nanopart ́ıculas e de tempo de envelhecimento (associado a dinˆamica de forma ̧c ̃ao de aglomerados). Em particular, o tamanho m ́edio do aglomerado (Q), associado ao n ́umero de nanopart ́ıculas formando uma cadeia linear, manteve-se basicamente constante, Q=4, 8 ± 0, 2, para uma faixa de tempo de 70 min. Dados de birrefringˆencia de satura ̧c ̃ao tamb ́em permaneceram est ́aveis neste intervalo. Adicionalmente, medidas de BME foram utilizadas para estimar a espessura da camada de cobertura (dextrana) sendo encontrado 1, 70 ± 0, 02 nm. Diferentemente dos dados de MAV, as medidas de BME foram feitas em amostras contendo tanto c ́elulas S180 quanto nanopart ́ıculas no meio RPMI 1640. Dados foram obtidos numa larga faixa de tempo (120 min.). Inicialmente observou-se que o sinal de BME decresce num intervalo de tempo e depois volta a crescer (entre 30-40 min.). O ajuste dos dados de BME indicam que a birrefringˆencia de satura ̧c ̃ao (∆ns) decresce nos primeiros 30 minutos e depois volta a crescer de forma suave, enquanto o tamanho m ́edio do aglomerado possui um comportamento oposto, ou seja cresce nos primeiros 30 minutos e depois volta a decrescer. Em particular, no tempo de exposi ̧c ̃ao, texp, de 10 min. o tamanho m ́edio do aglomerado (birrefringˆencia de satura ̧c ̃ao) variou de 4, 18±0, 04 (∆ns = 3, 41±0, 02 ×1018cm−3 0, 08 (∆ns = 2, 75 ± 0, 02 ×1018cm−3 ́e proporcional ao n ́umero de nanopart ́ıculas contribuindo para o sinal magneto ́optico conclui- se que o decr ́escimo do sinal magneto- ́optico foi decorrente do processo de internaliza ̧c ̃ao de nanopart ́ıculas magn ́eticas pelas c ́elulas S180. Por sua vez, a an ́alise da dependˆencia temporal do tamanho m ́edio do aglomerado tamb ́em sugere que o processo de internaliza ̧c ̃ao ocorre ) em texp=30 min. Como a birrefringˆencia de satura ̧c ̃ao primeiramente com nanopart ́ıculas anisom ́etricas ou com nanoestruturas formando pequenos aglomerados. Finalmente, ap ́os atingir a satura ̧c ̃ao no processo de internaliza ̧c ̃ao, observa-se a forma ̧c ̃ao de pequenos aglomerados no meio RPMI 1640, que ́e o respons ́avel pelo aumento da intensidade do sinal magneto- ́optico e diminui ̧c ̃ao do tamanho m ́edio do aglomerado para tempos maiores que 30 minutos.