ADRIANA KUCHINSKI CAVALCANTE

ADRIANA KUCHINSKI CAVALCANTE

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Toxicologia in vitro e in vivo das nanopartículas de ouro sintetizadas e estabilizadas com fitoquímicos
2022 - CAVALCANTE, ADRIANA K.
As nanopartículas de ouro (AuNPs) estão entre as nanopartículas metálicas mais amplamente estudadas para aplicações biomédicas. Metabólitos presentes em diversos extratos de plantas têm sido explorados para a preparação de diferentes AuNPs. No presente estudo, os fitoquímicos escolhidos como agentes redutores e estabilizadores para síntese das AuNPs, foram a mangiferina (MGF) e o resveratrol (RESV). Devido ao aumento da produção e da utilização das AuNPs, sérias preocupações são levantadas sobre os efeitos perigosos desses nanomateriais sobre a segurança ambiental e humana. O presente estudo investigou o nível de toxicidade das AuNPs reduzidas e estabilizadas com MGF (MGF-AuNPs) e RESV (RESV-AuNPs) in vitro e in vivo, comparando-as com o método clássico de síntese de AuNPs, no qual o citrato de sódio é o agente redutor e estabilizante (CITR-AuNPs). Foi realizado o ensaio de citotoxicidade, de acordo com a norma ISO 10993-5, pelo método de incorporação do corante vermelho neutro; o ensaio de toxicidade aguda com Daphnia similis, de acordo com a norma ABNT NBR 12713; o ensaio de toxicidade aguda com embriões de zebrafish, com a presença e ausência do córion do animal (FET Test), de acordo com o protocolo da OECD 236; o ensaio de microinjeção das amostras em embriões de zebrafish e o ensaio comportamental com as larvas de zebrafish. A nanotecnologia verde provou ser uma ferramenta valiosa na síntese AuNPs, não exigindo o uso de solventes e outras substâncias, pois os elétrons disponíveis dos fitoquímicos RESV e MGF forneceram energia suficiente para reduzir o sal de ouro. A toxicidade das AuNPs variou de acordo com o ensaio e com o modelo in vitro e in vivo. No ensaio de citotoxicidade em células de roedores, o índice de citotoxicidade (IC50) foi obtido das CITR-AuNPs, cujo IC50 foi 72%, que corresponde à concentração de ouro de 74,16 μg mL-1. No ensaio de toxicidade aguda com Daphnia similis, as amostras que causaram efeito tóxico agudo foram os redutores RESV e MGF, as CITR-AuNPs e as RESV-AuNPs, sendo os redutores mais tóxicos que as nanopartículas. Não foi possível estimar a concentração da amostra que causa imobilidade a 50% dos organismos (CE50;48h) para RESV, assim a CE50;48h considerada para o RESV foi menor que 3,12%, que corresponde a concentração de 10,29 μg mL-1. Para MGF a CE50;48h foi 21,52%, que corresponde a concentração de 150,64 μg mL-1. Para as CITR-AuNPs, a CE50;48h foi 29,71%, que correponde a concentração de ouro de 30,60 μg mL-1, e para as RESV-AuNPs a CE50;48h foi 25,88%, que correponde a concentração de ouro de 38,56 μg mL-1. No FET Test com embriões na presença do córion, as RESV-AuNPs causaram atraso no desenvolvimento embrionário, no processo de eclosão, sendo obtida a concentração da amostra que causa efeito letal em 50% dos organismos (CL50;96h) no valor de 34,97%, correspondente a concentração de ouro de 52,10 μg mL-1. No FET Test com embriões na ausência do córion, assim como no ensaio de microinjeção, as amostras não causaram efeito tóxico agudo nos animais. No ensaio comportamental a alteração da atividade locomotora foi observada em todas as amostras, e a toxicidade neurocomportamental foi atribuída aos agentes redutores CITR, MGF e RESV. O presente estudo verificou importância de avaliar a toxicidade das amostras em organismos diferentes, pois Daphnia similis e embriões de zebrafish apresentaram maior sensibilidade comparada as células de mamíferos. Além disso, dados inéditos da avaliação da toxicidade de AuNPs sintetizadas e estabilizadas com fitoquímicos MGF e RESV foram apresentados neste trabalho, contribuindo com a nanotoxicologia/ nano-ecotoxicologia.
Desenvolvimento de nanopartículas de ouro (AuNPs) para teranóstica
2021 - RODRIGUES, ADRIANA de S.; LUGAO, ADEMAR B.; BATISTA, JORGE G. dos S.; KUCHINSKI, ADRIANA; FREITAS, LUCAS F. de; CRUZ, CASSIA P.C. da
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.
Species-specific in vitro and in vivo evaluation of toxicity of silver nanoparticles stabilized with gum arabic protein
2020 - MAZIERO, JOANA S.; THIPE, VELAPHI C.; ROGERO, SIZUE O.; CAVALCANTE, ADRIANA K.; DAMASCENO, KELME C.; ORMENIO, MATHEUS B.; MARTINI, GISELA A.; BATISTA, JORGE G.S.; VIVEIROS, WILLIAM; KATTI, KAVITA K.; KARIKACHERY, ALICE R.; MOHANDOSS, DARSHAKUMAR D.; DHURVAS, RASHMI D.; NAPPINNAI, MOHANAVELU; ROGERO, JOSE R.; LUGAO, ADEMAR B.; KATTI, KATTESH V.
Introduction: We report, herein, in vitro, and in vivo toxicity evaluation of silver nanoparticles stabilized with gum arabic protein (AgNP-GP) in Daphnia similis, Danio rerio embryos and in Sprague Dawley rats. Purpose: The objective of this investigation was to evaluate in vitro and in vivo toxicity of silver nanoparticles stabilized with gum arabic protein (AgNP-GP), in multispecies due to the recognition that toxicity evaluations beyond a single species reflect the environmental realism. In the present study, AgNP-GP was synthesized through the reduction of silver salt using the tri-alanine-phosphine peptide (commonly referred to as “Katti Peptide”) and stabilized using gum arabic protein. Methods: In vitro cytotoxicity tests were performed according to ISO 10993– 5 protocols to assess cytotoxicity index (IC50) values. Acute ecotoxicity (EC50) studies were performed using Daphnia similis, according to the ABNT NBR 15088 protocols. In vivo toxicity also included evaluation of acute embryotoxicity using Danio rerio (zebrafish) embryos following the OECD No. 236 guidelines. We also used Sprague Dawley rats to assess the toxicity of AgNP-GP in doses from 2.5 to 10.0 mg kg− 1 body weight. Results: AgNP-GP nanoparticles were characterized through UV (405 nm), core size (20± 5 nm through TEM), hydrodynamic size (70– 80 nm), Zeta (ζ) potential (- 26 mV) using DLS and Powder X ray diffraction (PXRD) and EDS. PXRD showed pattern consistent with the Ag (1 1 1) peak. EC50 in Daphnia similis was 4.40 (3.59– 5.40) μg L− 1. In the zebrafish species, LC50 was 177 μg L− 1. Oral administration of AgNP-GP in Sprague Dawley rats for a period of 28 days revealed no adverse effects in doses of up to 10.0 mg kg− 1 b.w. in both male and female animals. Conclusion: The non-toxicity of AgNP-GP in rats offers a myriad of applications of AgNP-GP in health and hygiene for use as antibiotics, antimicrobial and antifungal agents.
Avaliação da toxicidade de nanopartículas de prata estabilizadas com goma arábica
2019 - MAZIERO, J.S.; ROGERO, S.O.; DAMASCENO, K.C.; ORMENIO, M.B.; CAVALCANTE, A.K.; MARTINI, G.A.; BATISTA, J.G.S.; KATTI, K.V.; LUGAO, A.B.; ROGERO, J.R.
As nanopartículas de prata (NPAg), devido a seus diversos atributos (formato variado, elevada área superficial e alto poder bactericida), vem sendo amplamente utilizadas em diversos setores da indústria. Esta utilização abrangente, tem provocado grande preocupação, quanto aos impactos e riscos potenciais que as NPAg podem causar ao meio ambiente e à saúde humana. Este trabalho teve como objetivo verificar a toxicidade de uma amostra de NPAg estabilizada com Goma Arábica e reduzida com Tri-Alanina, utilizando ensaios in vitro e in vivo. O teste in vitro de citotoxicidade, foi realizado seguindo a norma ISO 10993 - 5, em células da linhagem NCTC-L929; os ensaios in vivo de ecotoxicidade aguda, de acordo com a norma brasileira ABNT NBR 12713, utilizando como organismo teste a Daphnia similis; e embriotoxicidade aguda de acordo com o protocolo da OECD 236, utilizando como organismo teste o Danio rerio. Os resultados obtidos foram: IC50 de 2,61 mg L-1, CE50 de 6,55 μg L-1 e CL50 de 673 μg L-1. Os organismos aquáticos apresentaram maior sensibilidade às NPAg do que as células em cultura, elevando a importância de se realizar mais estudos em várias espécies de interesse biológico. Além disso, mostra-se necessário verificar o descarte apropriado dessas nanopartículas, visto que no Brasil ainda não há legislações que quantifiquem os limites permissíveis para esse descarte.
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.
Comparison between gold nanoparticles synthesized by radiolysis and by EGCG-driven gold reduction
2020 - FREITAS, LUCAS F. de; CRUZ, CASSIA P.C. da; CAVALCANTE, ADRIANA K.; BATISTA, JORGE G. dos S.; VARCA, GUSTAVO H.C.; MATHOR, MONICA B.; LUGAO, ADEMAR B.
Radiolytic synthesis and phytochemical-driven gold reduction for the generation of nanoparticles are successful examples of Green Chemistry applied for nanomaterials. The present work compares these two green approaches focusing on hydrodynamic size, stability over time, optical properties and toxicity in NIH 3T3 (ATCC® CRL- 1658™) cells and Danio rerio (Zebra Fish). The radiolytic synthesis was performed by mixing 1 mM NaAuCl4; polyvinyl pyrrolidone 0.5%, AgNO3 6×10−5 M, propan-2-ol 0.2 M and acetone 0.06 M, followed by irradiation at 15 kGy (5 kGy h−1, 60Co source). The EGCG-functionalized nanoparticles were synthesized by mixing 1.6 mM of Au with 0.8 mM of EGCG in phosphate buffer (10 mM) for 2 h. Both methods yield the formation of gold nanoparticles featuring plasmon resonance bands at 520–530 nm, polydispersity above 0.3 was relevant only for the radiolytic protocol. Regarding stability over time, after 30 days, the nanoparticles synthesized radiolytically presented no relevant size changes, while some aggregation was observed for the EGCG-particles. The same nanoparticles demonstrated a lack of stability in high ionic strength medium. Slight toxicity was observed for the EGCG-nanoparticles in Danio rerio, with an IC50 calculated as 40.49%, while no IC50 was established within the concentration range of radiolysis-AuNPs used in this study. In conclusion, both green methods generated nanoparticles with good control of size and optical properties, especially via reduction by EGCG. However, the stability and toxicity results were found to be more promising for the radiolytically synthesized gold nanoparticles.
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.
In vitro and in vivo toxicity of coal fly Ash Lechatee
2018 - DAMASCENO, K.C.; CAVALCANTE, A.K.; MAZIERO, J.S.; MARTINI, G.A.; ORMENIO, M.B.; MAMEDE, F.C.; MIRANDA, C.S.; CAMPELLO, F.A.; IZIDORO, J.C.; ROGERO, S.O.; FUNGARO, D.A.; LOPES-FERREIRA, M.; ROGERO, J.R.
Coal Fly ash is a major solid waste from coal-fired power stations. In Brazil, more than 4 million tons per year of fly ash are generated and only 30% is applied as raw material for cement and concrete production. The remaining is disposed in on-site ponds, nearby abandoned or active mine sites and landfills. The inadequate disposal of fly ash may pose a significant risk to the environment due to the possible leaching of hazardous pollutants into the surrounding soil and groundwater. A combination of leaching tests, cytotoxicity and ecotoxicological assays were used in this studyin order toevaluate the possible adverse effects of coal fl y ash in non-target organisms. The sample was collected from coal-fi red power plant located in Southern Brazil and the coal fly ash was submitted to a leaching procedure using USEPA SW 864 Method 1311. The leachate was prepared in six dilutions: 1.56%, 3.12%, 6.25%, 12.5%, 25% and 50%. Acute toxicity tests were performed on NCTC clone 929 (CCIAL-020) culture cells by neutral red uptake cytotoxicity method; acute ecotoxicity usingDaphnia similisand Danio rerio embryos according to ABNT NBR 12713 and OECD 236, respectively were employed. The cytotoxicity index (CI50) obtained was 33%; the EC50of D. similis after 48 h of exposure to the leachate was 7.25% and the LC50of D. rerio after 96 h of exposure was 4.39%. The results of these bioassays indicated toxicity of the coal fly ash leachate toward exposed organisms.