A infuência do tamanho das nanopartículas na atividade antimicrobiana do ZnO
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Data
2017-07-11
Autores
Silva, Bruna Lallo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os óxidos metálicos, como óxido de zinco (ZnO) são utilizados como agentes antimicrobianos inorgânicos frente à grande diversidade de microrganismos. Devido ao tamanho reduzido, as nanopartículas (NP) de ZnO são promissoras para combater infecções, uma vez que a diminuição do tamanho da partícula pode resultar em melhor atividade antimicrobiana, pois por terem tamanho menor, as NP podem com maior facilidade penetrarem na membrana ou induzir a maiores produções de espécies reativas de oxigênio (ERO). O objetivo desse trabalho foi desenvolver suspensões e pós de nanopartículas de ZnO com tamanho e superfície controladas, com a finalidade de avaliar a influência do tamanho e da modificação de superfície na atividade antimicrobiana do ZnO frente às bactérias Staphylococcus aureus e Escherichia coli e levedura Cândida albicans. As NP de ZnO foram preparadas pelo processo sol-gel de acordo com a metodologia proposta por Spanhel e Anderson (1991) com modificação da razão de hidrólise para controle do tamanho das NP seguida da adição de (3-Glycidyloxypropyl)trimethoxysilane (GPTMS), como modificador de superfície. Os nanomateriais obtidos foram caracterizados por diferentes técnicas: difração de raios X (DRX); espectroscopia na região do ultravioleta-visível (UV-vis), espalhamento de raios X à baixo ângulo (SAXS); espalhamento de raios X a alto ângulo (WAXS); análise termogravimétrica (TG); microscopia eletrônica de transmissão (MET); Espectroscopia de raios X por dispersão de energia (EDS) e potencial zeta. Com a técnica de DRX comprovamos a obtenção de NP de ZnO na fase wurtizita sendo possível o cálculo do tamanho do cristalito para as diferentes amostras utilizadas no estudo. Com a técnica de UV-vis identificamos o comprimento de onda limite, dessa forma foi possível calcular o raio médio das NP que foi de 2,9 nm e 5,4 nm para as NP submetidas a 3 e 24 horas de reação, respectivamente. Ainda através desta técnica foi possível provar alta estabilidade das NP. A temperatura de 600 °C foi selecionada por TG com finalidade de tratar termicamente as NP de ZnO na ordem de 5 nm resultando no aumento do seu tamanho, dessa forma, DRX foi utilizado para calcular o tamanho das NP, que passou a ser na ordem de 30 nm. De acordo com SAXS foi possível obter informações de tamanho, estrutura e crescimento das NP de ZnO, sendo que apresentaram em geral tamanho na ordem de 5 nm. O potencial zeta mostrou estabilidade das NP de ZnO dispersas em água a pH alcalino. A MET mostrou partículas relativamente esféricas com tamanho predominante de 5 nm. Assim, as diferentes técnicas utilizadas para avaliar o tamanho das NP apresentaram grande concordância entre si. Os dados de EDS provaram que as NP de ZnO modificadas apresentam alto grau de pureza. A avaliação da atividade antimicrobiana das NP de ZnO foi realizada pelos métodos de Concentração Inibitória Mínima (CIM), Concentração Bactericida Mínima (CBM) e Concentração Fungicida mínima (CFM), frente à Staplylococcus aureus, Escherichia coli e Candida albicans e Microscopia eletrônica de Varredura (MEV) em Staplylococcus aureus. As NP de ZnO mostraram melhor atividade frente a Staphylococcus aureus. Foram comparados os diferentes tamanhos das NP de ZnO em relação ao seu efeito antibacteriano sendo que as NP de ZnO de 5 nm foram mais eficazes para combater o crescimento microbiano do que as NP de 30 nm. A formação de buracos na estrutura de contorno de S.aureus foi evidenciada com a técnica de MEV após contato das bactérias com NP de ZnO. Os testes de citotoxicidade mostraram que as NP de ZnO não ocasionaram perda da viabilidade celular em linhagem celular de queratinócitos humanos (HaCat), dessa forma o material desenvolvido apresentou grande potencial para uso como antimicrobiano.
Metal oxides such as zinc oxide (ZnO) are used as inorganic antimicrobial agents against great diversity of microorganisms. Due the reduced size, ZnO nanoparticles (NP) are promising to combat infections, since the decrease in particle size may result in better antimicrobial activity, because of their smaller sizes, NP can more easily penetrate the membrane or induce a greater production of reactive oxygen species (ROS). The aim of this work was to develop suspensions and powders of ZnO nanoparticles with controlled size and surface, in order to evaluate the influence of size and surface modification on ZnO antimicrobial activity against bacteria Staphylococcus aureus and Escherichia coli and yeast Candida albicans. The ZnO NP were prepared by the sol-gel process according to the methodology proposed by Spanhel and Anderson (1991) with hydrolysis ratio modification for NP size control followed by the addition of (3-Glycidyloxypropyl) trimethoxysilane (GPTMS) as surface modifier. The nanomaterials obtained were characterized by different techniques: X-ray diffraction (XRD); Ultraviolet-visible spectroscopy (UV-vis); small angle light scattering (SAXS); high angle light scattering (WAXS); thermogravimetric analysis (TG); Transmission electron microscopy (TEM); energy dispersive spectroscopy (EDS) and zeta potential. With the XRD technique, we verified the obtaining of ZnO NP in the wurtizite phase and it was possible to calculate the crystallite size for the different samples used in the study. With the UV-vis technique we identified the limit wavelength, so it was possible to calculate the mean radius of the NP that was of 2,9 nm and 5,4 nm for the NP submitted to 3 and 24 hours of reaction, respectively and through this technique it was possible to prove high NP stability. The temperature of 600 ° C was selected by TG with the purpose of thermally treating the ZnO NP size in order about of 5 nm resulting in the increase of its size, thus, XRD was used to calculate the size of NP, which became in the order of 30 nm. According to SAXS, it was possible to obtain information on size, structure and growth of ZnO NP, with size of 5 nm in general. The zeta potential showed stability of the ZnO NP dispersed in water at alkaline pH. The MET showed spherical particles with a predominant size of 5 nm. Thus, the different techniques used to evaluate the NP size presented great agreement among themselves. The EDS data proved that the modified ZnO NP are high purity. The antimicrobial activity was performed by the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (CBM) and Minimum Fungicide Concentration (CFM) methods against Staplylococcus aureus, Escherichia coli and Candida albicans and Scanning Electron Microscopy (SEM) in Staplylococcus aureus. The ZnO NP showed better activity against Staphylococcus aureus. The different sizes of ZnO NP were compared to their antibacterial effect, and the 5nm ZnO NP 30nm ZnO NP. The formation of holes in the contour structure of S. aureus was evidenced with SEM technique after contact of the bacteria with ZnO NP. Cytotoxicity tests showed that ZnO NP did not cause loss of cell viability in in human keratinocyte cell line (HaCat), so the developed material presented great potential for use as an antimicrobial.
Metal oxides such as zinc oxide (ZnO) are used as inorganic antimicrobial agents against great diversity of microorganisms. Due the reduced size, ZnO nanoparticles (NP) are promising to combat infections, since the decrease in particle size may result in better antimicrobial activity, because of their smaller sizes, NP can more easily penetrate the membrane or induce a greater production of reactive oxygen species (ROS). The aim of this work was to develop suspensions and powders of ZnO nanoparticles with controlled size and surface, in order to evaluate the influence of size and surface modification on ZnO antimicrobial activity against bacteria Staphylococcus aureus and Escherichia coli and yeast Candida albicans. The ZnO NP were prepared by the sol-gel process according to the methodology proposed by Spanhel and Anderson (1991) with hydrolysis ratio modification for NP size control followed by the addition of (3-Glycidyloxypropyl) trimethoxysilane (GPTMS) as surface modifier. The nanomaterials obtained were characterized by different techniques: X-ray diffraction (XRD); Ultraviolet-visible spectroscopy (UV-vis); small angle light scattering (SAXS); high angle light scattering (WAXS); thermogravimetric analysis (TG); Transmission electron microscopy (TEM); energy dispersive spectroscopy (EDS) and zeta potential. With the XRD technique, we verified the obtaining of ZnO NP in the wurtizite phase and it was possible to calculate the crystallite size for the different samples used in the study. With the UV-vis technique we identified the limit wavelength, so it was possible to calculate the mean radius of the NP that was of 2,9 nm and 5,4 nm for the NP submitted to 3 and 24 hours of reaction, respectively and through this technique it was possible to prove high NP stability. The temperature of 600 ° C was selected by TG with the purpose of thermally treating the ZnO NP size in order about of 5 nm resulting in the increase of its size, thus, XRD was used to calculate the size of NP, which became in the order of 30 nm. According to SAXS, it was possible to obtain information on size, structure and growth of ZnO NP, with size of 5 nm in general. The zeta potential showed stability of the ZnO NP dispersed in water at alkaline pH. The MET showed spherical particles with a predominant size of 5 nm. Thus, the different techniques used to evaluate the NP size presented great agreement among themselves. The EDS data proved that the modified ZnO NP are high purity. The antimicrobial activity was performed by the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (CBM) and Minimum Fungicide Concentration (CFM) methods against Staplylococcus aureus, Escherichia coli and Candida albicans and Scanning Electron Microscopy (SEM) in Staplylococcus aureus. The ZnO NP showed better activity against Staphylococcus aureus. The different sizes of ZnO NP were compared to their antibacterial effect, and the 5nm ZnO NP 30nm ZnO NP. The formation of holes in the contour structure of S. aureus was evidenced with SEM technique after contact of the bacteria with ZnO NP. Cytotoxicity tests showed that ZnO NP did not cause loss of cell viability in in human keratinocyte cell line (HaCat), so the developed material presented great potential for use as an antimicrobial.
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Palavras-chave
Nanopartículas, ZnO, Staphylococcus aureus, Escherichia coli, Concentração inibitória mínima, Caracterização físico-química, Nanoparticles, Minimal inhibitory concentration, Physico-chemical characterization