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Master's Dissertation
DOI
https://doi.org/10.11606/D.82.2012.tde-15052012-105851
Document
Author
Full name
Lucas Freitas de Freitas
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2012
Supervisor
Committee
Plepis, Ana Maria de Guzzi (President)
Kurachi, Cristina
Perussi, Janice Rodrigues
Title in Portuguese
Ablação tumoral fototérmica in vivo utilizando nanobarras de ouro
Keywords in Portuguese
Apoptose
Câncer
CTAB
Nanobarras
Necrose
Ouro
Abstract in Portuguese
Ultimamente têm-se buscado tratamentos menos invasivos para o câncer, como os que utilizam campos magnéticos ou luz, e dentre esses últimos, aqueles que fazem uso de materiais, geralmente metálicos, com propriedade de ressonância plasmônica de superfície. O tratamento hipertérmico encaixa-se neste perfil e já apresenta resultados promissores com nanoshells de sílica recoberta por ouro e com nanobarras de ouro maciço, apesar de pouco se saber sobre o mecanismo de ação e sobre como as vias de morte celular são ativadas em tal tratamento. A síntese das nanobarras envolve o uso do composto brometo de cetiltrimetilamônio (CTAB), o qual permanece aderido à superfície das mesmas, porém é caracterizado por apresentar extrema citotoxicidade, fato que incita a modificação do recobrimento das nanopartículas por um biopolímero mais compatível. Estudos recentes indicam que o CTAB aderido à membrana não apresenta citotoxicidade considerável, porém há poucos dados que confirmem tal hipótese na literatura. Este trabalho se propôs a investigar a via de ativação da morte celular, bem como confirmar a hipótese de que as partículas recobertas por CTAB podem ser utilizadas para tratamento antitumoral fototérmico in vivo de forma segura. Para isso, nanobarras de ouro foram sintetizadas pelo método de seeding, sendo parte delas centrifugadas e lavadas com água deionizada por três vezes para retirar o CTAB e a outra parte deixada com CTAB no meio. As partículas foram testadas in vitro pelo teste de citotoxicidade pelo [brometo de 3-(4, 5-dimetiltiazol-2-yl)-2,5-difeniltetrazólio] (MTT) nas linhagens celulares HTC, HepG2, HT-29 e 786-O, e também foram testadas quanto à sua viabilidade com o tempo decorrido desde sua síntese. Após confirmar que as nanobarras centrifugadas e lavadas podem ser utilizadas no tratamento hipertérmico sem riscos à saúde e após verificar que as seeds e as nanobarras devem ser utilizadas até 48 horas depois de sua síntese, as nanopartículas foram utilizadas para tratamento de tumor de Ehrlich (induzido no dorso de camundongos). Para isso, foram organizados quatro grupos experimentais: L (camundongos não receberam nanopartículas, irradiados com laser em 808 nm), N (camundongos receberam nanopartículas, não irradiados com laser), H (camundongos receberam nanopartículas e irradiados com laser em 808 nm) e Controles (camundongos não receberam nanopartículas nem irradiação por laser). O material tumoral foi coletado após a irradiação e submetido à análise histológica, ao teste de quimiluminescência para avaliar a lipoperoxidação de membrana e ao teste de TRAP (do inglês, Total Radical-Trapping Antioxidant Parameter) para avaliar a capacidade antioxidante total. Após a irradiação com 2 W/'CM POT.2' ou 720 mW/'CM POT.2' de intensidade, houve evidente redução do volume tumoral nos animais do grupo H tratados com laser na maior potência utilizada, com um aumento de 47ºC (temperatura final de 79ºC) observado localmente. Nos animais do grupo H tratados com laser na menor potência utilizada, os danos foram menores. Os animais dos grupos L e H apresentaram semelhante lipoperoxidação, maior que no grupo N (estatisticamente significante somente nos animais tratados com laser em intensidade de 2 W/'CM POT.2'), e a capacidade antioxidante dos tumores dos animais do grupo H foi elevada no protocolo com laser em 2 W/'CM POT.2'. Os resultados indicam que a necrose é a via de morte ativada prioritariamente neste caso e que o tratamento com as nanobarras se mostrou eficaz.
Title in English
Photothermal tumor ablation in vivo using gold nanorods
Keywords in English
Apoptosis
Cancer
CTAB
Gold
Nanorods
Necrosis
Abstract in English
Less invasive cancer treatments, likewise those based on magnetic fields or light, are in the most common aims of researchers nowadays. Regarding light based treatments, those in which metallic, plasmonic materials are highlighted in research field. Hiperthermic treatment fits this profile, once it already presents promising results with gold-coated silica nanoshells and with gold nanorods, although little is known about its action mechanism or about how cell death pathways are activated. The compound cetyltrimethylammonium bromide (CTAB) is necessary for the nanorods synthesis, but is known to be extremely cytotoxic, fact that instigates the modification of nanorods surface coating by a compatible biopolymer. Recent studies indicate that surface-adhered CTAB does not present significant cytotoxicity, but there are few evidences to confirm this hypothesis in the literature. This study aims to investigate the cell death pathway that can be activated, as well as to confirm the possibility of safe CTAB-coated nanoparticles use in antitumor in vivo treatments. For that, gold nanorods were synthesized by the seeding method and part of them were centrifuged and washed with deionized water to eliminate CTAB of the solution and the rest remained with CTAB. The particles were tested in vitro by [3-(4, 5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cytotoxicity test, in HTC, HepG2, HT-29 and 786-O cancer cell lines, and investigated regarding their viability through time after their synthesis. After confirming that centrifuged and washed nanorods can be used in hiperthermic therapy without health risks, and after find out that seeds and nanorods must be used within 48 hours after their synthesis, those nanoparticles were used for in vivo hyperthermic Ehrlich tumor (induced on the back of mices) treatment. Four experimental groups were organized: L (mice did not receive nanoparticles, treated with laser at 808 nm), N (mice received nanoparticles, not treated with laser), and H (mice received nanoparticles and treated with laser at 808 nm) and Controls (mice did not receive nanoparticles and were not treated with laser). A tumor biopsy was taken after laser irradiation and was subjected to histological analysis, by a chemiluminescence assay to evaluate membrane lipoperoxidation, and by Total Radical-Trapping Antioxidant Parameter (TRAP) assay as well, to evaluate total antioxidant capacity. After irradiation with laser (intensities of 2 W/'CM POT.2' or 720 mW/'CM POT.2'), there was an evident tumor volume reduction in animals of H group treated with higher power laser, with a 47ºC rise in temperature (final temperature was 79ºC) observed locally. The damages in the tumors irradiated with lower power laser were less intense. The animals of L and H groups showed similar membrane lipoperoxidation, which was more intense than in N animals (statistically significant just in the animals treated with higher intensity of radiation). The antioxidant capacity of H animals tumor was elevated also in the animals treated with higher energy. Our results indicate that necrosis is the main activated cell death pathway in this case, and that nanorods treatment is worth it.
 
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Publishing Date
2012-05-16
 
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