Nanopartículas de ouro recobertas com sílica (AuSHINs) como agentes fototérmicos na terapia de células derivadas de carcinoma mamário
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Data
2022-02-03
Autores
Kobal, Mirella Boaro
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Universidade Estadual Paulista (Unesp)
Resumo
A nova geração de agentes fototérmicos nanoestruturados tem relançado a terapia fototérmica (TFT) como tratamento clínico menos invasivo no combate ao câncer. Em particular, nanopartículas metálicas demonstram elevada eficiência na conversão de luz em calor, desencadeando o aumento de temperatura local nos tecidos, e consequente morte celular. Apesar dos efeitos de agregação em meios com elevada força iônica, nanopartículas esféricas de ouro (AuNPs) são pioneiras em aplicações in vitro e in vivo da TFT. Neste trabalho, AuNPs foram recobertas com uma camada ultrafina (2 – 5 nm) de sílica, obtendo nanopartículas de ouro isoladas (AuSHINs, do inglês gold shell-isolated nanoparticles). As AuSHINs foram aplicadas como agentes fototérmicos no cultivo in vitro de células derivadas de carcinoma mamário comparativamente a células não tumorais. O objetivo do recobrimento foi reduzir os efeitos de agregação, aumentando assim a eficiência fototérmica na redução da viabilidade celular. Para tanto, células derivadas de carcinoma mamário glandular (MCF7) e ductal (BT-474), e células mono e polimorfonucleares extraídas do sangue periférico, foram avaliadas considerando diferentes concentrações de AuSHINs (2,2x1012, 5,5x1012 e 2,2x1013 AuSHINs/mL) e períodos de incubação (30 min, 2 e 6 h), irradiadas (em 525 nm com 32,8 mW/cm2 de irradiância) e não-irradiadas. O aquecimento fotoinduzido das AuSHINs reduziu a viabilidade celular das células MCF7 e BT-474 para aproximadamente 11% com 5,5x1012 AuSHINs/mL e 2 h de incubação, o qual é significantemente maior que resultados prévios encontrados na literatura para AuNPs, enquanto nas células não tumorais essa condição não apresentou redução na viabilidade celular abaixo de 50%. Experimentos subsidiários revelaram a geração de espécies reativas de oxigênio (EROs) durante a fotoativação das AuSHINs. Além disso, imagens de microscopia de fluorescência confocal revelaram fragmentação da membrana e diminuição nuclear para ambas as linhagens celulares tumorais, enquanto nas células não tumorais as alterações morfológicas foram mínimas, mesmo após fotoativação. Este resultado corrobora com os dados de extravasamento de LDH, indicando deturpações significativas na integridade da membrana celular das linhagens tumorais (MCF7 e BT-474) à medida em que a concentração de AuSHINs fotoativadas aumenta, enquanto efeitos mínimos foram observados para as não tumorais, confirmando a resistência das células saudáveis ao aumento de temperatura local. Tais dados sugerem o comprometimento da membrana das células tumorais por desnaturação de proteínas e abertura de poros, que são provavelmente a origem da fototoxicidade observada, desencadeando maior porcentagem de morte celular via necrose, quando comparada às células não tumorais.
The new generation of nanostructured photothermal agents has relaunched the photothermal therapy (PTT) as a less invasive clinical treatment to fight cancer. Particularly, metallic nanoparticles demonstrate high efficiency in converting light into heat, triggering an increase in the local temperature and consequent cell death. Despite the effects of aggregation in media with high ionic strength, spherical gold nanoparticles (AuNPs) are pioneers in in vitro and in vivo PTT applications. Herein, AuNPs were coated with an ultrathin silica shell (2 - 5 nm), obtaining gold shell-isolated nanoparticles (AuSHINs). AuSHINs were applied as photothermal agents in the in vitro culture of cells derived from mammary carcinoma compared to non-tumor cells. The goal of the silica shell was to reduce the aggregation effects, increasing the photothermal efficiency in reducing cell viability. Cells derived from glandular (MCF7) and ductal (BT-474) mammary carcinoma, and mono- and polymorphic nuclear cells extracted from peripheral blood, were evaluated in different concentrations of AuSHINs (2.2x1012, 5.5x1012 and 2.2x1013 AuSHINs / mL) and incubation periods (30 min, 2 and 6 h), irradiated (at 525 nm with 32,8 mW/cm2 of irradiance) and non-irradiated. The photoinduced heating of AuSHINs reduced cell viability of MCF7 and BT-474 cells to approximately 11% with 5.5x1012 AuSHINs/mL and 2 h of incubation, which is significantly higher than previous results found in literature for AuNPs, while in non-tumor cells, the viability was not reduced below 50%. Subsidiary experiments revealed the generation of reactive oxygen species (ROS) during the AuSHINs photoactivation. Additionally, confocal fluorescence microscopies revealed membrane fragmentation and nuclear decrease for both tumor cell lines, but in non-tumor cells the morphological changes were minimal, even after photoactivation. This result corroborates with the LDH data, indicating significant disruptions in the cell membrane integrity of the tumor cell lines (MCF7 and BT-474) as the concentration of photoactivated AuSHINs increases, while minimal effects were observed for the non-tumor cells, confirming the resistance of healthy cells to the local temperature increase. Such data suggest that the membrane of tumor cells is compromised by protein denaturation and pore opening, which are probably the origin of the observed phototoxicity, triggering a higher percentage of cell death via necrosis, when compared to non-tumor cells.
The new generation of nanostructured photothermal agents has relaunched the photothermal therapy (PTT) as a less invasive clinical treatment to fight cancer. Particularly, metallic nanoparticles demonstrate high efficiency in converting light into heat, triggering an increase in the local temperature and consequent cell death. Despite the effects of aggregation in media with high ionic strength, spherical gold nanoparticles (AuNPs) are pioneers in in vitro and in vivo PTT applications. Herein, AuNPs were coated with an ultrathin silica shell (2 - 5 nm), obtaining gold shell-isolated nanoparticles (AuSHINs). AuSHINs were applied as photothermal agents in the in vitro culture of cells derived from mammary carcinoma compared to non-tumor cells. The goal of the silica shell was to reduce the aggregation effects, increasing the photothermal efficiency in reducing cell viability. Cells derived from glandular (MCF7) and ductal (BT-474) mammary carcinoma, and mono- and polymorphic nuclear cells extracted from peripheral blood, were evaluated in different concentrations of AuSHINs (2.2x1012, 5.5x1012 and 2.2x1013 AuSHINs / mL) and incubation periods (30 min, 2 and 6 h), irradiated (at 525 nm with 32,8 mW/cm2 of irradiance) and non-irradiated. The photoinduced heating of AuSHINs reduced cell viability of MCF7 and BT-474 cells to approximately 11% with 5.5x1012 AuSHINs/mL and 2 h of incubation, which is significantly higher than previous results found in literature for AuNPs, while in non-tumor cells, the viability was not reduced below 50%. Subsidiary experiments revealed the generation of reactive oxygen species (ROS) during the AuSHINs photoactivation. Additionally, confocal fluorescence microscopies revealed membrane fragmentation and nuclear decrease for both tumor cell lines, but in non-tumor cells the morphological changes were minimal, even after photoactivation. This result corroborates with the LDH data, indicating significant disruptions in the cell membrane integrity of the tumor cell lines (MCF7 and BT-474) as the concentration of photoactivated AuSHINs increases, while minimal effects were observed for the non-tumor cells, confirming the resistance of healthy cells to the local temperature increase. Such data suggest that the membrane of tumor cells is compromised by protein denaturation and pore opening, which are probably the origin of the observed phototoxicity, triggering a higher percentage of cell death via necrosis, when compared to non-tumor cells.
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Palavras-chave
Terapia fototérmica, AuSHINs, Carcinoma mamário, Photothermal therapy, Mammary carcinoma