JOANA DA SILVA MAZIERO

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In vitro and In vivo toxicity of Gold nanoparticles synthesized and stabilized with phytochemicals
2020 - CAVALCANTE, A.K.; BATISTA, J.G.; MAZIERO, J.d.; FERNANDES, B.V.; VIVEIROS, W.; ROGERO, S.O.; ROGERO, J.R.; LUGAO, A.B.
Gold nanoparticles (AuNPs) are among the most widely studied metal nanoparticles for biomedical applications. AuNPs can be synthesized by chemical reduction. Several methods for this type of synthesis are described in the literature, one of them is the Turkevich method, which uses sodium citrate (CITR) as a reducing agent. Other methods were developed based on the use of solvent systems during nanoparticle production. Although they are efficient production methods, they are environmentally unviable. In order to address this issue, metabolites present in various plant extracts have been explored for the preparation of different AuNPs. Green nanotechnology is the nanotechnology aspect that aims to develop protocols to generate sustainable products and production processes, in order to minimize the use of toxic compounds. In this study, the phytochemicals chosen as reducing agents and stabilizers for AuNPs synthesis were mangiferin (MGF) and resveratrol (RESV). Due to their ability to interact with biological systems, along with various applications of AuNPs, their toxicity has become one of the most important concerns. Due to the increased production and use of AuNPs, their risk of reaching different environmental compartments and becoming available increases the importance of determining toxicity in various species of biological interest, such as microcrustaceans and fish. Zebrafish is an important animal model used in the areas of developmental biology, genetics, biomedicine, nanotoxicology and is also used in ecotoxicological assays. The aim of the study is to evaluate the toxicity level of MGF (MGF-AuNPs) and RESV (RESV-AuNPs) reduced and stabilized AuNPs in vitro and in vivo by comparing them with the classical AuNPs synthesis method described by Turkvich (CITR-AuNPs). The study has two strands, one of which is to evaluate the level of nanomaterial toxicity using the zebrafish animal model as a preclinical study, and the other to evaluate the level of nanomaterial toxicity using the zebrafish animal model as a study of environmental toxicity. Cytotoxicity assay according to ISO 10993-5, Zebrafish FET TEST according to OECD 236 and Microinjection Assay in Zebrafish embryos. Green nanotechnology has proven to be a valuable tool in the synthesis of AuNPs for toxicity, not requiring the use of solvents and potentially toxic substances. The toxicity of AuNPs varied according to the assay. In the cytotoxicity assay, IC50 was obtained from CITR-AuNPs, whose IC50 was about 72%, which corresponds to the Au concentration of 74.16 µg.mL-1. In FET, RESV-AuNPs caused delays in the hatching process of zebrafish embryos, and it was the only sample that could obtain the LC50 (4.41%, corresponding to the Au concentration of 6,57 µg.mL -1). In the assay in which AuNPs were microinjected into embryos, the concentration causing toxic effect was not found in 50% of the organisms.
Avaliação da toxicidade in vitro e in vivo das nanopartículas de ouro reduzidas e estabilizadas com mangiferina e resveratrol
2019 - CAVALCANTE, A.K.; BATISTA, J.G.S.; MAZIERO, J.S.; ROGERO, S.; ROGERO, J.R.; VIVEIROS, W.; KATTI, K.; LUGAO, A.B.; ALAVARSE, R.D.
As nanopartículas de ouro (AuNPs) com diferentes tamanhos e formas têm sido amplamente estudadas em todo o mundo. A síntese de AuNPs geralmente envolve agentes de redução e solventes que apresentam problemas relacionados à toxicidade. A fim de resolver esta questão, metabólitos presentes em diversos extratos de plantas tem sido explorados para a preparação de diferentes nanopartículas. Pesquisadores mostraram que alguns fitoquímicos, como a mangiferina (MGF) e resveratrol (RESV), além de reduzirem e estabilizarem as AuNPs, são capazes de funcionalizá-las. O aumento na produção e utilização das nanopartículas tem provocado grande preocupação quanto aos impactos e riscos potenciais que estas podem causar ao meio ambiente e à saúde humana. Desta forma, o estudo teve como objetivo avaliar o nível de toxicidade das AuNPs, reduzidas e estabilizadas com MGF e RESV in vitro e in vivo, comparando-as com o método clássico de síntese de AuNPs, descrito por Turkvich, no qual o citrato de sódio é o agente redutor e estabilizante (CITR-AuNPs). Foi realizado o ensaio de citotoxicidade de acordo com a International Standard Organization (ISO 10993-5, 2009) e o ensaio de toxicidade aguda em embriões de Zebrafish (FET TEST), de acordo com o protocolo da OECD nº 236. Zebrafish é um importante modelo animal utilizado nas áreas de biologia do desenvolvimento, genética e biomedicina e é utilizado em ensaios ecotoxicológicos. Como o zebrafish apresenta 70% de similaridade genética com humanos, assim como similaridades fisiológicas e anatômicas, este modelo pode ser usado para prever efeitos de toxicidade em humanos. Além de alta taxa reprodutiva e rápido desenvolvimento embrionário, a espécie apresenta grande sensibilidade quando exposta a produtos químicos, sendo capaz de absorver rapidamente os compostos que são diretamente adicionados à água e acumulá-los em vários tecidos. O organismo é de fácil obtenção, gerenciamento e de baixo custo. Tanto as nanopartículas reduzidas e estabilizadas com MGF e RESV, quanto os redutores, não apresentaram citotoxicidade, porém as CITR-AuNPs apresentaram IC50 de 180 μg.mL-1. Em relação ao FET TEST, a taxa de letalidade dos organismos expostos a MGF e MGF-AuNPs, ambas na concentração de 350 μg.mL-1, foi de 12% e 5% respectivamente. Já a taxa de letalidade dos organismos expostos ao RESV na concentração de 165 μg.mL-1, foi de 17.5%. A CL50 obtida das RESV-AuNPs foi de 14.55 μg.mL-1, nas concentrações de 82.5 e 165 μg.mL- 1, observou-se atraso estatísticamente significativo da eclosão dos organismos, no período de 72 horas pós fecundação. A taxa de letalidade dos organismos expostos ao citrato de sódio e CITR-AuNPs, ambas na concentração de 250 μg.mL-1, foi de 3.75% e 16.25% respectivamente. Não foram encontrados valores de CL50 nas concentrações testadas das nanopartículas, exceto RESV-AuNPS. Nanopartículas com maiores concentrações do que as testadas não apresentam estabilidade, tendendo a aglomeração e precipitação.
In vitro and in vivo toxicity evaluation of silver nanoparticles stabilized with gum arabic
2018 - MAZIERO, J.S.; CAVALCANTE, A.K.; MARTINI, G.A.; DAMASCENO, K.C.; ORMENIO, M.B.; CAVALCANTE, B.K.; ROGERO, S.O.; ROGERO, J.R.; LUGAO, A.B.
Due to its properties such as size, varied shape, high surface area and high bactericidal properties, silver nanoparticles (AgNP) have been widely used in several sectors of the industry: bandages; inside food coolers, to retard spoilage; in antimicrobial insoles, to prevent odors; in air purifiers; in surgical instruments and etc [1] [2]. Considering the range of applications a great concern in the academic field, mainly ecotoxicological, as the potential impacts and risks that AgNP can cause to the environment and human health has increased. During the synthesis of AgNP it is necessary to use stabilizing agents such as gum arabic (GA), which is an exudate of dried gum, edible, from the stems and branches of Acacia senegal and A. seyal, rich in soluble fiber not viscous. GA has broad industrial use as stabilizer, thickening agent and emulsifier [3]. Based on these considerations, this work aimed to verify and compare the toxicity level of two GA-stabilized AgNP samples (Sample 1: AgNP at 147 ppm concentration, approximately 25 nm in size; and Sample 2: AgNP at concentration of 174 ppm with approximate size of 75 nm) using in vitro and in vivo assays. The in vitro cytotoxicity test was performed according to ISO 10993-5 by the neutral red uptake method in cells of the NCTC-L929 line, to obtain the IC50 (cytotoxicity index, which is the concentration of the substance causing it 50% mortality of exposed cells); and the in vivo acute ecotoxicity assay, according to the Brazilian standard ABNT NBR 12713, using Daphnia similis as the test organism to obtain EC50 (effective concentration that causes immobility in 50% of exposed organisms). The results obtained for Sample 1 were IC50 of 2.57 mg L-1 and EC50 of 4.40 μg L-1; and Sample 2: IC50 of 2.61 mg L-1 and EC50 of 6.55 μg L-1. These results demonstrated that aquatic organisms are much more sensitive to AgNP than cells in culture, raising the importance of conducting further studies related to the adversities that these nanoparticles can cause to the environment and human health. In addition, it is necessary to verify the disposal of the same in the environment, since in Brazil there are still no legislation that quantifies the permissible limits for this disposal.
Evaluation of the toxicity of gold nanoparticles produced by green nanotechnology in Zebrafish (Danio rerio)
2018 - CAVALCANTE, A.K.; BATISTA, J.G.S.; MAZIERO, J.S.; DAMASCENO, K.C.; ORMENIO, M.B.; CAVALCANTE, B.K.; ROGERO, S.O.; ROGERO, J.R.; LUGAO, A.B.
Gold nanoparticles (AuNPs) of different sizes and shapes have been extensively studied by researchers and laboratories around the world. Several studies have demonstrated the applicability of gold nanoparticles in the treatment and diagnosis of cancer, in the treatment of chronic inflammation, infections, degenerative diseases and autoimmune diseases [1]. The synthesis of AuNPs generally involves reducing agents which present problems related to toxicity. In order to address this issue, metabolites present in various plant extracts have been exploited for the preparation of different nanoparticles. The methods that use phytochemicals to reduce metal ions provide a green approach to nanotechnology, known as green nanotechnology [2]. Researchers have shown that some phytochemicals, such as mangiferin (MGF) and epigallocatechin-gallate (EGCG), in addition to reducing and stabilizing the gold nanoparticles, are able to functionalize them. These molecules have chemical groups that allow binding to overexpressed receptors on some types of tumor cells [3]. The objective of this study was to evaluate the level of toxicity of the gold nanoparticles, reduced and stabilized with epigallocatechin-gallate (EGCG-AuNPs) in Zebrafish embryos (Danio rerio), as an indication of a possible environmental effect. To assess the developmental impact of embryos, organisms were exposed to different dilutions of the EGCG-AuNPs suspension for a 96-hour period according to OECD Protocol 236 (Fish Embryo Acute Toxicity Test-FET). Zebrafish is an established vertebrate model for the study of development, disease and is being increasingly used for both pre-clinical studies and toxicological applications due to a range of favorable traits [4]. EGCG-AuNPs demonstrated toxicity, with organ lethality being less than 33% at all concentrations used. The work provided a contribution on the toxicity of AuNPs synthesized and stabilized with the epigallocatechin-gallate reducing agent and using Zebrafish embryos as an animal.
Nanopartículas de ouro com potencial teranóstico de câncer sintetizadas por meio de nanotecnologia verde
2018 - BATISTA, J.G.S.; LUGAO, A.B.; ROGERO, S.; CAVALCANTE, A.K.; MAZIERO, J.S.
Grupos de pesquisa ao redor do mundo voltaram a atenção para as nanopartículas de ouro pelo fato destas atenderem as necessidades de sistemas nanocarreadores na terapia e diagnóstico de câncer. Elas podem ser usadas na orientação e liberação de fármacos a sítios ou grupos celulares específicos e em terapias fototérmicas como agente gerador de calor. Um número significativo de estudos demonstrou suas possíveis aplicações, tais como biosensores, contraste na imagiologia biológica, em sistemas de liberação de fármacos e como teranóstico. São capazes de gerar imagens e aniquilar células cancerosas, simultaneamente. Assim, as nanopartículas de ouro são consideradas promissoras no desenvolvimento de novos compostos com potencial aplicação na medicina oncológica, no tratamento de inflamações crônicas, infecções, doenças degenerativas e autoimunes. No entanto, apesar das formas nanométricas de ouro apresentarem menor toxicidade comparada aos muitos outros nanomateriais, a toxidade dessas partículas deve ser minuciosamente avaliada. O maior desafio é propor modificações moleculares, como a funcionalização de superfície, que promova a melhora da farmacocinética desses compostos, diminua a toxicidade e possibilite o direcionamento a alvos específicos. Esse estudo visa o desenvolvimento de uma nova nanopartícula de ouro (AuNP), utilizando albumina humana (ASH) e epigalocatequinagalato (EGCG), na tentativa de diminuir a captação hepática e melhorar a biodisponibilidade dessas nanopartículas. A metodologia de síntese foi adaptada e estabelecida. A sua reprodutibilidade foi avaliada com base nos ensaios de caracterização físico-química, que foram realizados pelas técnicas de espectrofotometria UV-Vis e fluorescência, espalhamento de luz dinâmico (DLS), potencial Zeta, microscopia eletrônica de transmissão (MET). A estabilidade foi avaliada em relação à temperatura, pH e concentração de cloreto de sódio (NaCl). As nanopartículas não apesentaram citotoxicidade in vitro utilizando o método do vermelho neutro, nas concentrações testadas e se mostraram estáveis na faixa de pH entre 5 e 9, e também em concentrações de NaCL até 3%.
Avaliação da toxicidade in vitro e in vivo das Nanopartículas de Prata
2018 - MAZIERO, J.S.; ROGERO, S.O.; DAMASCENO, K.C.; ORMENIO, M.B.; CAVALCANTE, A.K.; LUGAO, A.B.; ROGERO, J.R.
Devido às suas propriedades como tamanho, formato variado, elevada área superficial e alto poder bactericida, as nanopartículas de prata (NPAg) vem sendo amplamente utilizadas em diversos setores da indústria: curativos, devido a sua capacidade bactericida; no interior de refrigeradores de alimentos, para retardar a deterioração; em palmilhas antimicrobianas, para evitar odores; em purificadores de ar; em instrumentos cirúrgicos e etc. A sua ampla utilização tem provocado grande preocupação na área acadêmica, principalmente ecotoxicológica, quanto aos impactos e riscos potenciais que estas podem causar ao meio ambiente e à saúde humana. Baseado nessas considerações, este trabalho teve como objetivo verificar o nível de toxicidade destas NPAg utilizando ensaios in vitro e in vivo. O teste in vitro de citotoxicidade, foi realizado seguindo a norma ISO 10993 - 5 pelo método de incorporação do corante vermelho neutro, em células da linhagem NCTC-L929 e os ensaios in vivo de ecotoxicidade aguda, de acordo com a norma brasileira ABNT NBR 12713, utilizando como organismo teste a Daphnia similis. Os resultados obtidos foram o IC50 (índice de citotoxicidade, que é a concentração das NPAg que causa mortalidade a 50% das células expostas), de 2,57 mg. L-1, e a CE50 (concentração efetiva das NPAg que causa imobilidade em 50% dos organismos expostos) de 4,40 μg. L-1. Estes resultados mostram que os organismos aquáticos são muito mais sensíveis às NPAg do que as células em cultura, elevando a importância de se realizar mais estudos relacionados às adversidades que essas nanopartículas podem causar. Além disso, mostra-se necessário verificar o descarte das mesmas no meio ambiente, visto que no Brasil ainda não há legislações que quantifiquem os limites permissíveis para esse descarte.
Ecotoxicology as a tool in nanotechnology
2017 - CAVALCANTE, A.K.; LUGAO, A.B.; ROGERO, J.R.; ROGERO, S.; MAMEDE, F.C.S.; MAZIERO, J.S.
The commercial applications of nanoparticles are diverse, such as use in the food industry, textile industry, electronics, water treatment and products used in medicine and health. The increase in the production and use of nanoparticles has caused great concern about the potential impacts and risks that these can cause to the environment and to human health. Nanoparticles can be released to the environment in a variety of ways and can reach the aquatic ecosystem and pose biota risks. Ecotoxicology is the study of the behavior and transformations of chemical agents and abiotic factors in the environment, as well as their effects on biota. The evaluation of the toxicity of chemical agents in the aquatic environment occurs by means of ecotoxicological tests. The purpose of this review was to summarize some ecotoxicological assays by addressing some concepts and data from nanoparticle ecotoxicity assays in order to demonstrate that ecotoxicological evaluation is an important tool for nanotechnology and that it has efficient methodologies for analyzing the Environmental health of aquatic ecosystems.
Evaluation of the in vitro and in vivo toxicity of gold nanoparticles synthesized by green nanotechnology
2017 - BATISTA, JORGE G.S.; BARROS, JANAINA A.G.; VARCA, GUSTAVO H.C.; ROGERO, SIZUE O.; CAVALCANTE, ADRIANA K.; MAZIERO, JOANA S.; ROGERO, JOSE R.; LUGAO, ADEMAR B.
Researchers and laboratories around the world have studied gold nanoparticles. In medicine, several studies demonstrate the applicability of gold nanoparticles (AuNPs) in the treatment and diagnosis of cancer. Green nanotechnology uses phytochemical agents to synthesize and stabilize nanoparticles. The phytochemical epigallocatechin-gallate (EGCG) reduces and stabilize gold nanoparticles by functionalizing the surface of the molecule. Such chemical groups allow binding to overexpressed receptors on some types of tumors as demonstrated in studies performed with PC3 prostate cancer cells. With the advancement of nanotechnology, a large number of nanoparticles are produced on a daily basis. However apart from their possible applications it is necessary to evaluate the environmental impact of these molecules as well as find ways for proper disposal. The embryonic zebrafish (Danio rerio) trial has recently emerged as an interesting method for evaluating in vivo nanotoxicity providing a more complex system analysis than in typical cell cultures and less expensive if compared to large-scale biocompatibility studies performed in rats and mice. The objective of this study was to evaluate the in vitro and in vivo toxicity of EGCG-AuNPs by means of the cytotoxicity by neutral red uptake methodology according to the International Standard Organization [ISO 10993-5, 2009] and in vivo test based on the OECD guideline on Fish Embryo Toxicity Test (FET) (OECD, 2013). The spectrophotometric band at 535 nm observed is characteristic of the formation of AuNPs. Nanoparticles synthesized with EGCG presented a size of 32 ± 4 nm as determined by transmission electron microscopy and the hydrodynamic diameter of these particles was about 60 ± 18 nm obtained by dynamic light scattering. The EGCG-AuNPs showed no cytotoxicity up to 4.2 μg.L-1. In the FET test regarding the acute ecotoxicity assay the LC50/96 hours revealed no toxicity at concentrations up to 1.8 mM.