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Assessment of the biological and toxicological effects of silica-based nanoparticles: contribution to grouping of nanomaterials
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Os nanomateriais de sílica amorfa (aSiO2 NM) são cada vez mais utilizados numa vasta gama de aplicações, como biossensores, tintas, cosméticos, aditivos alimentares, sistemas de transporte e entrega de fármacos e material genético, produtos dentários e agentes de bioimagem. Com esta utilização crescente, a exposição humana a estes materiais, particularmente por inalação em ambientes profissionais, também aumentou. O presente trabalho teve como objetivo preencher lacunas de conhecimento existentes ao avaliar sistematicamente a toxicidade de aSiO2 NM em modelos in vitro e in vivo com foco na via inalatória e em como variações de tamanho e química de superfície influenciam a sua citotoxicidade e genotoxicidade ao nível pulmonar.
Assim, foram testadas sete variantes de aSiO2 NM, com diferentes tamanhos (7, 15 e 40 nm) e química de superfície (não modificadas ou revestidas com grupos fosfonato, amino e trimetilsilil). Estudos in vitro em células epiteliais alveolares de rato (RLE-6TN) avaliaram os efeitos citotóxicos e genotóxicos de todas as variantes de aSiO2 NM, incluindo alterações na integridade da membrana, atividade metabólica e danos no DNA (quebras de cadeia simples (SSB) e dupla (DSB)) através do ensaio do cometa. As variantes com maior potencial citotóxico e genotóxico (danos no DNA) (SiO2_7, SiO2_15_Unmod (não modificado), SiO2_15_Amino e SiO2_40) foram ainda analisadas quanto aos seus efeitos na progressão do ciclo celular e nos níveis de histona H2AX fosforilada (γ-H2AX), um biomarcador de resposta ao dano no DNA, por citometria de fluxo.
Os resultados revelaram que as variantes não revestidas, particularmente as SiO2_7 e SiO2_15_Unmod, induziram efeitos citotóxicos significativos, enquanto as variantes com revestimento demonstraram um menor potencial citotóxico. É interessante notar que, apesar do elevado potencial citotóxico, as variantes SiO2_7, SiO2_15_Unmod, e a variante SiO2_15_Amino não induziram danos significativos no DNA em concentrações não citotóxicas, ao passo que exposição à variante SiO2_40 aumentou significativamente o dano no DNA das células epiteliais alveolares, sem, no entanto, afetar a sua atividade metabólica. Deste modo, a modificação da superfície com grupos hidrofóbicos de organosilanos e, em menor grau, com grupos fosfonatos, parece ser uma estratégia eficaz para atenuar a toxicidade dos aSiO2 NM nas células RLE-6TN. Por outro lado, todas as variantes testadas (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino e SiO2_40) aumentaram significativamente os níveis totais de γ-H2AX. Na concentração mais elevada (28 μg/cm2), a exposição a todos os materiais testados levou a uma diminuição da subpopulação G0/G1, acompanhada de um aumento significativo das subpopulações S e G2/M, exceto no caso da variante SiO2_40, que não afetou a progressão do ciclo celular. Com base nos dados obtidos, as variantes de aSiO2 NM podem ser classificadas quanto ao seu potencial genotóxico de dano no DNA da seguinte forma: SiO2_7 = SiO2_15_Unmod = SiO2_40 > SiO2_15_Amino.
Foi realizado um estudo in vivo onde as variantes com maior toxicidade in vitro (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino e SiO2_40), foram administradas por instilação intratraqueal no rato, para avaliação dos danos primários e oxidativos no DNA e das alterações da expressão génica no tecido pulmonar. Além disso, foi efetuado um estudo de inalação a curto prazo (STIS) no rato, com a variante de menor tamanho (SiO2_7) e com o TiO2-NM105 como NM de referência. Nos estudos de instilação, o SiO2_7 ou o SiO2_40 causaram lesões oxidativas significativas no DNA, embora estas não tenham sido observadas nos grupos de recuperação, o que sugere que estas lesões tenham sido reparadas. No STIS, detetámos um aumento significativo das quebras de cadeia de DNA no tecido pulmonar de animais expostos a 0,5 mg/m³ de SiO2_7 ou 50 mg/m3 de TiO2_NM105, em ambos os grupos (grupo exposto e grupo de recuperação). As alterações observadas na expressão génica sugerem que as vias do stress oxidativo e/ou inflamatórias estão provavelmente envolvidas na indução dos danos oxidativos no DNA observados. De um modo geral, in vivo não se detetou uma toxicidade significativa dos aSiO2 NM testados quer no estudo de instilação intratraqueal, quer no STIS. Os efeitos genotóxicos da variante SiO2_7, foram concordantes em ambos os estudos in vivo realizados, no entanto, as alterações observadas nos animais do STIS foram mais permanentes, mais difíceis de reverter.
Os resultados dos estudos in vivo alinharam-se com os resultados dos estudos in vitro, indicando que a via de síntese desempenha um papel na determinação do modo de ação (MoA) do SiO2 NM, com as variantes pirogénicas (SiO2_7 e SiO2_40) a causarem maior toxicidade do que as variantes sintetizadas por precipitação (SiO2_15_Unmod e SiO2 _15_Amino). Consequentemente, as células RLE-6TN parecem ser um modelo preditivo da toxicidade pulmonar in vivo dos SiO2 NM.
Os nossos resultados contribuíram para o desenvolvimento de uma estratégia de agrupamento de nanomateriais de engenharia (ENM) no âmbito do projeto NanoToxClass, baseada na abordagem DF4nanoGrouping, com objetivo de agilizar o processo de avaliação de risco dos nanomateriais. Esta classificação sistemática revelou que o revestimento da superfície e a via de síntese influenciam significativamente o MoA dos aSiO2 NM de, revelando que as variantes pirogénicas são mais ativas do que as variantes sintetizadas por precipitação, enquanto as variantes revestidas apresentam uma menor toxicidade comparativamente às não revestidas. Além disso, os nossos resultados apoiam a utilidade de abordagens multiparamétricas para melhorar a compreensão do MoA dos ENM e a previsão do seu perigo. Eles salientam também o potencial destas abordagens e estratégias de agrupamento para se alinharem com os 3R (Substituição, Redução e Refinamento), reduzindo a necessidade de testes in vivo e promovendo um desenvolvimento de ENM seguros por conceção (safe-by-design).
Por último, o presente trabalho sublinha a necessidade de protocolos rápidos, robustos e reprodutíveis para a avaliação dos perigos dos ENM, defendendo a adaptação dos ensaios clássicos de genotoxicidade e a utilização de técnicas de elevado rendimento. Estes conhecimentos são cruciais para o avanço da nanotoxicologia, promovendo a conceção segura de ENM e facilitando o desenvolvimento eficiente de produtos com recurso à nanotecnologia novos e sustentáveis.
Amorphous silica nanomaterials (aSiO2 NM) are increasingly utilized in a wide range of products, such as biosensors, paints, cosmetics, food additives, carriers for gene and drug delivery, dentistry and bioimaging agents. Consequently, human exposure to aSiO2 NM, particularly through inhalation in occupational settings, has also increased. The present study aimed to fill in knowledge gaps regarding the hazards of aSiO2 NM by systematically assessing their pulmonary toxicity in vitro and in vivo, focusing on the influence of particle size and surface chemistry on cyto- and genotoxicity. Variants of aSiO2 NM differing in size (7, 15, and 40 nm) and surface capping (unmodified, phosphonate-, amino-, and trimethylsilyl-modified) were tested. In vitro studies in assessed the cytotoxic and genotoxic effects of all aSiO2 NM variants rat alveolar epithelial cells (RLE-6TN), including changes in membrane integrity, metabolic activity, and DNA damage (single- (SSB) and double-strand breaks (DSB)) by the comet assay. The variants with higher cytotoxic and DNA-damaging potential (SiO2_7, SiO2_15_Unmod (unmodified), SiO2_15_Amino and SiO2_40) were further analysed for effects on cell cycle progression and levels of phosphorylated histone H2AX (γ-H2AX), a biomarker of DNA damage response, by flow cytometry. The results revealed that unmodified variants, particularly SiO2_7 and SiO2_15_Unmod, exhibited significant cytotoxicity, while surface-modified variants demonstrated lower cytotoxicity. Interestingly, despite high cytotoxic potential, the variants SiO2_7, SiO2_15_Unmod, and SiO2_15_Amino did not induce significant DNA damage at noncytotoxic concentrations, whereas SiO2_40 caused notable DNA damage without affecting metabolic activity of RLE-6TN cells. Thus, surface modification with hydrophobic organosilanes, and in a lesser extent with phosphonate groups, seems to be an effective estrategy to mitigate the toxicity of aSiO2 NM in RLE-6TN cells. On the other hand, all variants tested (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 mg/cm2), exposure to all tested materials led to a decrease in the G0/G1 subpopulation, accompanied by a significant increase in the S and G2/M subpopulations, except for SiO2_40, which did not affect cell cycle progression. Based on the obtained data, aSiO2 NM variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_15_Unmod = SiO2_40 > SiO2_15_Amino. In vivo studies included intratracheal instillation in rats of the SiO2 NM variants with higher in vitro toxicity (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino and SiO2_40), evaluating primary and oxidative DNA damage and gene expression changes in the lung tissue. Additionally, a short-term inhalation study (STIS) with the smallest size variant of SiO2 NM (7 nm), and using TiO2-NM105 as a reference NM, was conducted. In the instillation studies, SiO2_7 or SiO2_40 caused significant oxidative DNA lesions in the lung tissue of the exposed animals, although those seem to be repaired as no significant changes have been detected in recovery groups. In the STIS, a significant increase in DNA strand breaks in the lung tissue of rats exposed to 0.5 mg/m³ of SiO2_7 or 50 mg/m³ of TiO2_NM105 was detected in both groups (i.e. in the exposure and recovery groups). The observed changes in gene expression suggest that oxidative stress and/or inflammatory pathways are likely implicated in the induction of oxidative DNA damage. Overall, the aSiO2 NM tested were not associated with significant in vivo toxicity following either instillation or STIS. The genotoxicity findings for SiO2_7 were concordant in both studies, however, the changes observed in the STIS animals were more permanent, more difficult to revert. The in vivo results are aligned with the in vitro findings, indicating that the synthesis route plays a role in determining the mode of action (MoA) of aSiO2 NM, with pyrogenic aSiO2 NM variants (SiO2_7 and SiO2_40) exhibiting greater toxicity than precipitated aSiO2 NM variants (SiO2_15_Unmod and SiO2_15_Amino). Consequently, RLE-6TN cells seem to be a predictive model of aSiO2 NM pulmonary toxicity in vivo. Our findings contributed to the development of a grouping strategy for engineered nanomaterials (ENM) within the NanoToxClass project, based on the DF4nanoGrouping approach, to support ENM risk assessment. This systematic classification revealed that surface coating and synthesis route significantly influence the MoA of aSiO2 NM, supporting that pyrogenic aSiO2 variants are more active than the variants synthesized by precipitation, while the surface-capped variants exhibit reduced toxicity compared to the uncoated ones. Additionally, our findings support the usefulness of multiparametric approaches to improve understanding of NM MoA and hazard prediction. They also emphasize the potential of these approaches and grouping strategies to align with the 3R (Replacement, Reduction, and Refinement) by reducing dependence on in vivo testing and promoting safer-by-design of ENM. Finally, the present work underscores the need for rapid, robust, and reproducible protocols for ENM hazard assessment, advocating for the adaptation of the classical genotoxicity assays and the use of high-throughput techniques. These insights are crucial for advancing nanotoxicology, promoting the safe design of ENM, and facilitating the efficient development of new and sustainable nano-based products.
Amorphous silica nanomaterials (aSiO2 NM) are increasingly utilized in a wide range of products, such as biosensors, paints, cosmetics, food additives, carriers for gene and drug delivery, dentistry and bioimaging agents. Consequently, human exposure to aSiO2 NM, particularly through inhalation in occupational settings, has also increased. The present study aimed to fill in knowledge gaps regarding the hazards of aSiO2 NM by systematically assessing their pulmonary toxicity in vitro and in vivo, focusing on the influence of particle size and surface chemistry on cyto- and genotoxicity. Variants of aSiO2 NM differing in size (7, 15, and 40 nm) and surface capping (unmodified, phosphonate-, amino-, and trimethylsilyl-modified) were tested. In vitro studies in assessed the cytotoxic and genotoxic effects of all aSiO2 NM variants rat alveolar epithelial cells (RLE-6TN), including changes in membrane integrity, metabolic activity, and DNA damage (single- (SSB) and double-strand breaks (DSB)) by the comet assay. The variants with higher cytotoxic and DNA-damaging potential (SiO2_7, SiO2_15_Unmod (unmodified), SiO2_15_Amino and SiO2_40) were further analysed for effects on cell cycle progression and levels of phosphorylated histone H2AX (γ-H2AX), a biomarker of DNA damage response, by flow cytometry. The results revealed that unmodified variants, particularly SiO2_7 and SiO2_15_Unmod, exhibited significant cytotoxicity, while surface-modified variants demonstrated lower cytotoxicity. Interestingly, despite high cytotoxic potential, the variants SiO2_7, SiO2_15_Unmod, and SiO2_15_Amino did not induce significant DNA damage at noncytotoxic concentrations, whereas SiO2_40 caused notable DNA damage without affecting metabolic activity of RLE-6TN cells. Thus, surface modification with hydrophobic organosilanes, and in a lesser extent with phosphonate groups, seems to be an effective estrategy to mitigate the toxicity of aSiO2 NM in RLE-6TN cells. On the other hand, all variants tested (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 mg/cm2), exposure to all tested materials led to a decrease in the G0/G1 subpopulation, accompanied by a significant increase in the S and G2/M subpopulations, except for SiO2_40, which did not affect cell cycle progression. Based on the obtained data, aSiO2 NM variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_15_Unmod = SiO2_40 > SiO2_15_Amino. In vivo studies included intratracheal instillation in rats of the SiO2 NM variants with higher in vitro toxicity (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino and SiO2_40), evaluating primary and oxidative DNA damage and gene expression changes in the lung tissue. Additionally, a short-term inhalation study (STIS) with the smallest size variant of SiO2 NM (7 nm), and using TiO2-NM105 as a reference NM, was conducted. In the instillation studies, SiO2_7 or SiO2_40 caused significant oxidative DNA lesions in the lung tissue of the exposed animals, although those seem to be repaired as no significant changes have been detected in recovery groups. In the STIS, a significant increase in DNA strand breaks in the lung tissue of rats exposed to 0.5 mg/m³ of SiO2_7 or 50 mg/m³ of TiO2_NM105 was detected in both groups (i.e. in the exposure and recovery groups). The observed changes in gene expression suggest that oxidative stress and/or inflammatory pathways are likely implicated in the induction of oxidative DNA damage. Overall, the aSiO2 NM tested were not associated with significant in vivo toxicity following either instillation or STIS. The genotoxicity findings for SiO2_7 were concordant in both studies, however, the changes observed in the STIS animals were more permanent, more difficult to revert. The in vivo results are aligned with the in vitro findings, indicating that the synthesis route plays a role in determining the mode of action (MoA) of aSiO2 NM, with pyrogenic aSiO2 NM variants (SiO2_7 and SiO2_40) exhibiting greater toxicity than precipitated aSiO2 NM variants (SiO2_15_Unmod and SiO2_15_Amino). Consequently, RLE-6TN cells seem to be a predictive model of aSiO2 NM pulmonary toxicity in vivo. Our findings contributed to the development of a grouping strategy for engineered nanomaterials (ENM) within the NanoToxClass project, based on the DF4nanoGrouping approach, to support ENM risk assessment. This systematic classification revealed that surface coating and synthesis route significantly influence the MoA of aSiO2 NM, supporting that pyrogenic aSiO2 variants are more active than the variants synthesized by precipitation, while the surface-capped variants exhibit reduced toxicity compared to the uncoated ones. Additionally, our findings support the usefulness of multiparametric approaches to improve understanding of NM MoA and hazard prediction. They also emphasize the potential of these approaches and grouping strategies to align with the 3R (Replacement, Reduction, and Refinement) by reducing dependence on in vivo testing and promoting safer-by-design of ENM. Finally, the present work underscores the need for rapid, robust, and reproducible protocols for ENM hazard assessment, advocating for the adaptation of the classical genotoxicity assays and the use of high-throughput techniques. These insights are crucial for advancing nanotoxicology, promoting the safe design of ENM, and facilitating the efficient development of new and sustainable nano-based products.
Descrição
Tese de Doutoramento em Ciência Biomédicas, apresentada ao Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 2025. https://hdl.handle.net/10216/167701
Palavras-chave
Toxicidade Pulmonar Ensaios de Genotoxicidade Classificação de Perigo Toxicologia Nanomateriais de Sílica Amorfa Toxicity Manufactured Nanomaterials Pulmonary Toxicity Amorphous Silica Nanomaterials Genotoxicity Testing Hazard Ranking