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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.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.bc.ufg.br:tde/2874 |
| Date | January 2010 |
| Creators | Silva, Anderson Costa da |
| Contributors | Bakuzis, Andris Figueiroa |
| Publisher | Universidade Federal de Goiás, Programa DE Pós-graduação em Física (IF), UFG, Brasil, Instituto de Física - IF (RG) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFG, instname:Universidade Federal de Goiás, instacron:UFG |
| Rights | http://creativecommons.org/licenses/by-nc-nd/4.0/, info:eu-repo/semantics/openAccess |
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