Browsing by Author "Vilar, Madalena"
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- Are cellulose nanofibrils safe for biomedical applications?Publication . Vilar, Madalena; Lourenço, Ana Filipa; Ventura, Célia; Louro, Henriqueta; Ferreira, Paulo; Félix, Sérgio; Silva, Maria JoãoNanomedicine is based on the application of nanotechnologies into the medical field to advance and improve diagnostics, prevention and treatment of human disease. While its expansion has been enormous in the last years, its progress must go hand in hand with nanosafety, i.e., with the safety evaluation of nanomaterials in an early phase of its development or application into a product. Cellulose appears as a natural and readily available material, which fits within the supply-demand chain: ecological, abundant and low cost. Particularly, cellulose nanofibrils (CNF) show great mechanical strength and high water-uptake capability and have the ability to form translucent structures with high elasticity and selective permeability, which make them attractive e.g., as constituents of surgical dressings and membranes for bone regeneration. Bacterial nanocellulose is already being used, but CNF produced from plants are also finding potential to be applied in tissue engineering and regenerative medicine. However, CNF may bring more toxicological concerns than the bacterial type, due to impurities associated with the chemical and mechanical processes used to produce them or due to their different physicochemical properties that may underlie unforeseen biological effects. The main objective of this work was to evaluate the safety of two different CNFs, through the analysis of their cytotoxic, genotoxic and epigenetic effects in human osteoblasts. The CNFs were obtained from the same raw material – industrial bleached Eucalyptus globulus kraft pulp - by two different methods: TEMPO-mediated oxidation and enzymatic hydrolisis. The physicochemical properties of the CNF gels obtained, including fibrillation yield, degree of polymerization and size were evaluated. The CNF cytotoxicity was assessed by the MTT assay and the genotoxicity by the cytokinesis-block micronucleus assay; their epigenetic effects were evaluated through gene expression analysis of the DNA methytransferases genes, DNMT1 and DMNT3b, responsible for the cellular methylation pattern, using qRT-PCR. The results obtained for the several endpoints were integrated in order to contribute to the characterization of the potential toxic effects of these new CNF in an early phase of their lifecycle. This knowledge will be relevant to decide whether these CNF may be further developed for applications in the nanomedicine field, or shall be modified to give rise to safer CNF.
- Cyto-genotoxic effects of distinct micro/nanocelluloses in human osteoblastic and mice fibroblastic cellsPublication . Ventura, Célia; Marques, Catarina; Cadete, João; Vilar, Madalena; Pedrosa, J.F.S.; Pinto, P.; Rosa, R.R.; Fernandes, S.N.; Godinho, M.H.; Ferreira, P.J.T.; Louro, Henriqueta; Silva, Maria JoãoThe unique characteristics of micro/nanocelluloses (MNCs) have been boosting their use in a wide variety of industrial and biomedical applications. With the expected decrease of their production cost, it is likely to observe an even faster growing adoption of these materials in the coming years. With the increasing use of MNCs comes an increased likelihood of human exposure to these materials, raising concerns about potential human health effects. The number of studies on the potential adverse effects of MNCs is still scarce and sometimes inconclusive. The aim of the present study is to shed some light on the impact of human exposure to cellulose-based nanomaterials. For that, three MNCs were produced from bleached Eucalyptus kraft pulp using different pretreatments. A microfibrillated (CMF) and a nanofibrillated cellulose (CNF) were respectively obtained by enzymatic and TEMPO-mediated oxidation pretreatments followed by high-pressure homogenization. In addition, cellulose nanocrystals (CNC) were obtained by acid hydrolysis with diluted sulfuric acid. The potential cytotoxicity of the MNCs was evaluated in vitro in two mammalian cell lines (human MG-63 osteoblasts and mice V79 lung fibroblasts) by the MTT and the clonogenic assays, and the genotoxicity was assessed by the micronucleus assay. Cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNC, but not by the MTT assay. CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells. CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells. From the results, it was concluded that under the tested conditions all MNCs present cytotoxicity and genotoxicity, although at different concentration levels, which may be related to their physicochemical differences, to the availability for cell uptake, and to differences in the DNA damage response of the cell model.
- Genotoxicity of Three Micro/Nanocelluloses with Different Physicochemical Characteristics in MG-63 and V79 CellsPublication . Ventura, Célia; Marques, Catarina; Cadete, João; Vilar, Madalena; Pedrosa, Jorge F.S.; Pinto, Fátima; Fernandes, Susete Nogueira; da Rosa, Rafaela Raupp; Godinho, Maria Helena; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoBackground: Nanocellulose is an innovative engineered nanomaterial with an enormous potential for use in a wide array of industrial and biomedical applications and with fast growing economic value. The expanding production of nanocellulose is leading to an increased human exposure, raising concerns about their potential health effects. This study was aimed at assessing the potential toxic and genotoxic effects of different nanocelluloses in two mammalian cell lines; Methods: Two micro/nanocelluloses, produced with a TEMPO oxidation pre-treatment (CNFs) and an enzymatic pre-treatment (CMFs), and cellulose nanocrystals (CNCs) were tested in osteoblastic-like human cells (MG-63) and Chinese hamster lung fibroblasts (V79) using the MTT and clonogenic assays to analyse cytotoxicity, and the micronucleus assay to test genotoxicity; Results: cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNCs, but not by the MTT assay; CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells; CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells; Conclusions: All nanocelluloses revealed cytotoxicity and genotoxicity, although at different concentrations, that may be related to their physicochemical differences and availability for cell uptake, and to differences in the DNA damage response of the cell model.
- Safety assessment of nanofibrillated cellulose to human health using cellular modelsPublication . Ventura, Célia; Lourenço, Ana Filipa; Vilar, Madalena; Teixeira, Sara; Ferreira, Paulo J.T.; Silva, Maria JoãoNanomaterial - A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%. (2011/696/EU)
- Study on cellulose nanofibrils biological effects in different cellular modelsPublication . Pinto, Fátima; Ventura, Célia; Teixeira, Sara; Vilar, Madalena; Lourenço, Ana Filipa; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoCellulose nanofibrils (CNFs), is an innovative environmental friendly material that has been incorporated into several types of materials, in both pure and composite forms and holds great promise in different fields of application. This leads to a rising level of human exposure and draws considerable concerns regarding its potential toxicity in humans, which are specially motivated by the physicochemical resemblance of cellulose nanofibrils with multi-walled carbon nanotubes and asbestos that have shown deleterious health effects. Currently, several in vitro toxicological studies have been performed with the aim of predicting health effects caused by exposure to CNFs. A literature review was performed to gather and analyse recent data regarding the potential cytotoxic, genotoxic, immunotoxic and epigenetic effects, in different in vitro cell models, triggered by the exposure to CNFs produced from vegetal biomass and consequently differing in critical physicochemical characteristics. The main findings of this review work demonstrate that different functionalization affects CNFs hydrophobicity, surface charge and chemistry, which may either facilitate or difficult the uptake and interaction of its functional groups with the cell membrane, affecting the biological responses. Generally, CNFs are not phagocytized and do not cause inflammatory response, but revealed in vitro genotoxicity. In fact, our data have shown that CNFs may induce chromosome damage in some cell models, e.g., a co-culture of epithelial lung alveolar cells and macrophages. However, more studies are required to assess CNFs toxicity and to understand the relationship between their physicochemical properties, their behaviour in biological media and mechanism of action, which are of outmost importance to predict their nanosafety.
- The use of the in vitro micronucleus assay on the genotoxicity assessment of cellulose nanofibrils in mammalian cell linesPublication . Ventura, Célia; Teixeira, Sara; Marques, Catarina; Vilar, Madalena; Pinto, Fátima; Lourenço, A.F.; Sousa Mendes, A.; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoWith the expansion of innovative cellulose nanofibrils (CNF) applications, either for industrial (e.g., paper industry) or biomedical purposes (e.g., tissue regeneration and dentistry), human exposure has been increasing, raising concerns about their potential health outcomes . Being persistent high aspect-ratio nano-objects, CNFs effects may resemble those of some multi-walled carbon nanotubes, e.g., the MWCNT-7, which has been reported to induce chromosome instability, cytoskeleton alterations and dysfunction of several cancer-related cell signaling pathways . The in vitro cytokinesis-block micronucleus (CBMN) assay is a sensitive and reliable assay for analysis of chromosome damage in mammalian cells, with the potential of providing mechanistic information and predicting carcinogenic effects. In this work we evaluated the use of the CBMN assay in different mammalian cell systems to characterize the genotoxicity of a CNF produced from an industrial bleached Eucalyptus globulus kraft pulp through TEMPO-mediated oxidation followed by a high-pressure homogenization process . The CNF sample was fully characterized in order to assess the most relevant physicochemical properties. It presented a wide distribution of diameters, with the mode in the 20–25 nm range and a length of several micrometers. Preliminary doserange finding through assessment of the cytotoxic effects (MTT and clonogenic assays) in cell lines representative of the respiratory tract (A549, A549 co-cultured with macrophage-like THP-1 cells and V79 cells) and bone (MG63 osteoblasts) showed that the CNF was not cytotoxic, regardless of the exposure period. The results of the CBMN assay (following OECD guideline 487) showed that exposure to low CNF concentrations induced a significant increase of micronuclei in co-cultured alveolar (A549) cells and in osteoblasts, while no significant effect was observed in A549 cells in monoculture. These results support the suitability of the in vitro micronucleus assay to assess the genotoxicity of nanofibers like CNF, highlighting the relevance of the cell system selected. This and other factors related to the experimental design are discussed, pointing to the need of nano-specific guidelines for this assay application in nanogenotoxicology. References Ventura et al. 2020. Cellulose, DOI 10.1007/s10570-020-03176-9 Ventura et al. 2020. Nanotoxicology, 14:479 Ventura et al.2020. Toxicol Lett, 328: 7 Fenech, M. 2007. Nature Protocols 2: 1084. Lourenço et al. 2017. Cellulose 24: 349.
- Toxicidade da nanocelulose em modelos in vitro: um contributo para a avaliação da sua segurança para a saúde humanaPublication . Ventura, Célia; Pinto, Fátima; Marques, Catarina; Cadete, João; Vilar, Madalena; Lourenço, Ana Filipa; Pedrosa, Jorge F. S.; Fernandes, Susete N.; da Rosa, Rafaela R.; Godinho, Maria Helena; Ferreira, Paulo J.T.; Louro, Henriqueta; Silva, Maria JoãoAs nanoceluloses são nanomateriais inovadores com potencial para uma vasta gama de aplicações industriais e biomédicas. No entanto, a expansão da sua produção tem vindo a suscitar preocupações quanto aos possíveis efeitos, a longo prazo, na saúde humana. Este estudo teve como objetivo avaliar a segurança de algumas nanoceluloses produzidas a partir de matéria-prima nacional, através da caracterização da sua potencial toxicidade em células de mamífero. Para tal, testaram-se duas celuloses nano /microfibrilares (CNF e CMF ) e uma celulose nanocristalina (CNC). Analisou-se a citotoxicidade usando ensaios colorimétricos e o ensaio clonogénico, e a genotoxicidade pelo ensaio do micronúcleo in vitro em células pulmonares de mamífero (A549 e V79 ) e em osteoblastos humanos ( MG-63 ). A indução de espécies reativas de oxigénio (ROS) e a internalização celular foram também estudadas nas células A549. Observou- -se citotoxicidade no ensaio clonogénico, principalmente no caso da CNC, mas não nos restantes ensaios, não havendo também indução de ROS. Todas as nanoceluloses revelaram efeitos genotóxicos nalgumas concentrações, uma vez que induziram micronúcleos e /ou pontes nucleoplásmicas num dos modelos celulares. Para além disso, visualizou-se a internalização da CNF e CMF, mas não da CNC, em células A549. Esta primeira avaliação toxicológica veio contribuir para prevenir a exposição a materiais celulósicos potencialmente perigosos, procurando impulsionar o desenvolvimento de materiais inovadores e mais seguros.