Síntese de de nanopartículas de prata através de açúcares redutores e seu potencial

UNIVERSIDADE FEDERAL DO CEARÁ CENTRO DE CIÊNCIAS

PROGRAMA DE PÓS-GRADUAÇÃO EM QUÍMICA TESE DE DOUTORADO

EDUARDO JOSÉ JUCÁ MALLMANN

GREEN SYNTHESIS OF SILVER NANOPARTICLES TROUGH REDUCING SUGARS AND THEIR ANTIMICROBIAL POTENTIAL

EDUARDO JOSÉ JUCÁ MALLMANN

GREEN SYNTHESIS OF SILVER NANOPARTICLES TROUGH REDUCING SUGARS AND THEIR ANTIMICROBIAL POTENTIAL

Tese apresentada ao curso de Doutorado em Química do Pós-Graduação em Química do Centro de Ciências da Universidade Federal do Ceará, como parte dos requisitos para obtenção do Título de Doutor em Química. Área de Concentração: Físico-Química.

Orientador: Prof. Dr. Pierre Basílio Almeida Fechine

Ao meu amigo Afrânio, autor da inesquecível frase “daqui a dez anos

seremos todos doutores”.

AGRADECIMENTOS

Um doutorado não é construído sozinho, de modo que várias pessoas contribuem para a realização deste projeto. Cabe então destacar aqueles que, de modo relevante, ajudaram na execução da pesquisa.

Primeiramente, agradeço a Deus, a grande fonte de força e inspiração para continuar seguindo em frente.

Agradeço a meus pais pelo incentivo contínuo aos estudos, e por todo o apoio dado a mim, o que é um alicerce tremendo que me permite trilhar caminhos mais ousados.

À minha noiva, Auriana, por sempre me apoiar, dar forças, estar presente, compreender, estudar comigo, me ouvir e ser também uma grande amiga nas horas em que mais preciso.

Ao professor Pierre, como orientador, sempre buscando mostrar o caminho certo, aconselhando e repreendendo nos momentos propícios. Incontáveis foram os e-mails com artigos para se estudar, prazos a cumprir, sugestões de atividades... É um espelho de inspiração e alguém de admirável conhecimento e caráter. Desde que fui seu aluno de físico-química para farmácia, percebi sua grandeza intelectual e como ser humano, e tive a sorte de tê-lo como orientador no mestrado e no doutorado. Como amigo, sei que posso confiar e esperar lealdade, e deixo a recíproca registrada como verdadeira.

Ao meu amigo e colega de doutorado Afrânio Cunha, alguém por quem tenho uma admiração enorme e como modelo de professor desde quando fui seu aluno na graduação. Sua contribuição para a execução deste trabalho de doutorado foi imensurável e certamente sem seu apoio as dificuldades pareceriam fatalmente intransponíveis. Com seu espírito solidário e sua imensa energia e potência de trabalho, suas incansáveis lições me inspiram e fazem continuar e dar o melhor no magistério. Seus intermináveis compartilhamentos de ideias, suas ligações telefônicas nos mais variados horários com propostas de trabalho e de pesquisa sensacionais, suas eternas cobranças o transformam em um verdadeiro modelo de pesquisador a ser seguido.

tardes de estudo, dividindo toda a evolução no caminhar desta disciplina sensacional.

Ao professor da referida disciplina, Dr. Luiz Constantino (“Luizão”) por todo o conhecimento compartilhado não somente técnico e acadêmico, mas verdadeiramente filosófico e como farol orientador para a vida.

À professora Selma Mazzetto por sempre manter abertas as portas de seu laboratório (LPT) para o que quer que precisemos. Além disso, sua ajuda foi importantíssima para uma conquista pessoal minha ao longo desta jornada doutoral. Ainda, ao seu orientando e colega de doutorado Eufrázio Júnior, que sempre se demonstra solícito e ágil para contribuir com análises termográficas.

Da mesma forma, agradeço aos professores Dr. Luiz Gonzaga e Drª Idalina Carvalho por, em um momento inicial e outro projeto de pesquisa do doutorado, ter disponibilizado seu laboratório, material e pessoal para que a pesquisa fosse desenvolvida, além de manterem sempre abertas as portas para as incontáveis análises de espectroscopia de infravermelho. É também por este espírito colaborador que faz com que nosso programa de pós-graduação em Química seja tão bem conceituado.

À professora Drª Nágila Ricardo, pois assim como os professores supracitados, abriu as portas de seu laboratório para que uma pesquisa inicial envolvendo polímeros fosse realizada sob sua supervisão e auxílio. Sou eternamente grato por todo seu suporte e carinho. Da mesma forma, à professora Drª Cristiane Pinto.

Agradeço também ao professor Dr. Antoninho Valentini, pessoa extremamente solícita, que sempre deixou abertas as portas de seu laboratório para que as análises espectroscópicas fossem lá realizadas. Também manifesto minha gratidão aos colegas de doutorado Moacir Júnior e Bárbara Sales, que sempre estiveram disponíveis para ajudar nas análises.

Minha gratidão à colega de doutorado Clara, que muito contribuiu para minha pesquisa realizando as análises de DLS, assim como meu muito obrigado à professora Drª Judith, por ceder espaço para as mesmas.

Ao professor Dr. Ayala e sua aluna Silmara Alves pela ajuda e contribuição nas análises de raios-x e infravermelho.

Às professoras Drª Wladiana Matos e Drª Gisele Lopes, assim como a seu aluno Luan, pelas amostras de análise de espectroscopia de absorção atômica – ICP-OES.

Toda minha gratidão à minha colega de bancada e aluna de iniciação científica, Valdirene Oliveira. As sínteses preliminares do doutorado não seriam as mesmas sem sua companhia, ajuda e otimismo, repetindo sempre o mantra “vai dar certo” a cada tentativa e etapa realizada. Muito obrigado Val!

Agradeço à minha amiga Jéssica Miranda pelo auxílio durante as mesmas sínteses preliminares citadas anteriormente, colaborando no que foi necessário com instruções técnicas e fornecimento de material, além do mais importante: seu tempo.

Meus agradecimentos ao meu amigo Davino, companheiro de caracterização de estabilidade e tamanho de amostras. Sempre solícito e atencioso, devo bastante à sua contribuição.

Minha profunda gratidão ao meu amigo Rafael que sempre ouviu e aconselhou em momentos chave da pesquisa, além de ter sido meu companheiro de estudo e preparação para o concurso de docente da UECE. Sua força e contribuição foram fundamentais e essenciais para que lograsse êxito nesse certame.

Ao meu amigo Victor, companheiro de bancada da primeira etapa de projeto de doutorado, dividindo experiências, expectativas, frustrações, alegrias, conhecimento... Sou grato por toda sua contribuição para este trabalho e desenvolvimento no doutorado.

A todos os integrantes do GQMAT, que formam uma grande família em que todos se apoiam, se ajudam, compartilham momentos diversos e estão sempre contribuindo uns com os outros para o crescimento como pesquisador e como ser humano. Vocês são sensacionais!

com novas possibilidades e novas resoluções de impasses e contratempos, além de novas parcerias firmadas para a caracterização de nossos materiais. Muito obrigado, craques da química!

À Central Analítica do Departamento de Física, onde as análises de microscopia foram cuidadosamente realizadas.

Ao professor Dr. Everardo por, desde minha graduação em Química, permitir o uso de seu Laboratório de Microbiologia da Faculdade de Farmácia da UFC. Sem dúvidas, um grande diferencial nesse doutorado foi seu companheirismo com o desenvolvimento da pesquisa.

A toda equipe de professores do curso de Química da Universidade Estadual do Ceará – Campus Itapipoca pela compreensão e apoio no desenvolvimento deste trabalho de pesquisa doutoral. Em especial, à minha amiga professora Doutora Edinilza Maria Anastácio Feitosa, que deu todo o suporte necessário na coordenação do PIBID, compreendendo a divisão de tempo e de tarefas entre o programa e o desenvolvimento deste trabalho doutoral. Sem este suporte, conciliar o trabalho com o doutorado seria ainda mais árduo, esgotante e beiraria a barreira do intransponível. Ainda ao meu amigo Walber, alguém de uma inteligência ímpar e companheiro de viagem, de ideias e de projetos.

Agradeço ainda a todos os meus alunos que sempre demonstraram entusiasmo em aprender sobre minha pesquisa e desta forma me incentivavam sempre a continuar crescendo acadêmica e intelectualmente para melhor servi-los. O corpo discente do curso de Química da UECE Itapipoca tem minha eterna gratidão e admiração.

Agradeço aos meus amigos externos às Instituições, pois sempre se demonstraram compreensivos quanto ao andamento do meu trabalho e minhas constantes ausências em momentos de confraternização, além de serem sempre interessados no objeto de minha pesquisa. Cabe citar nomes de grande relevância como Helder, Cássio, Roberto, Paulo André, Marcelo, Renar e Airton.

Agradeço à CAPES pela bolsa concedida nos anos iniciais de doutorado e à Universidade Federal do Ceará pela oportunidade de realizar este crescimento profissional, retornando à casa agora como aluno de doutorado.

A todos que, embora não tenham sido citados nestes agradecimentos, contribuíram direta ou indiretamente para a confecção deste trabalho. Sem a ajuda e o apoio de cada um, teria sido bem mais difícil!

RESUMO

Nanopartículas de prata (AgNPs) são materiais promissores em diversas áreas da ciência e tecnologia. Métodos tradicionais de síntese geram resíduos tóxicos e indesejáveis. A síntese verde de AgNPs a partir de açúcares redutores surge como uma opção viável de obtenção deste material. Neste trabalho foram utilizados os açúcares arabinose, glicose, ribose e xilose para obtenção de AgNPs, além dos sais de sódio citrato e dodecil sulfato como agentes estabilizantes. A caracterização destes materiais foi realizada por meio das técnicas de espectroscopia de absorção na região do UV-Vis _– identificando a banda plasmônica – difração de raios X, espalhamento dinâmico de luz, potencial zeta, microscopia eletrônica de varredura, microscopia de força atômica e espectroscopia de absorção na região do infravermelho. As soluções apresentaram boa estabilidade (potencial zeta médio de -46,4mV), partículas com tamanhos que variaram de ±10nm a ±100nm e diferentes morfologias de grãos. A partir das AgNPs sintetizadas, materiais – filmes de ágar e gel de carbômero – foram obtidos para serem caracterizados e aplicados. A aplicação das soluções de AgNPs e destes materiais obtidos foi majoritariamente na verificação de atividade microbiológica contra fungos de diferentes espécies de

Candida spp e Aspergillus spp, além das bactérias patogênicas Escherichia coli, Staphylococcus aurus e Pseudomonas aeruginosa. Buscou-se ainda investigar os efeitos de associação entre as AgNPs e o Itraconazol, um medicamento disponível para tratamento de infecções fúngicas. Todas as nanopartículas que foram aplicadas tiveram atividade antibiótica eficaz contra os microrganismos, bem como os materiais desenvolvidos também apresentaram resultados satisfatórios. As AgNPs apresentaram ainda resultados que apontam para a potencialização do antifúngico Itraconazol, quando utilizado combinado com as nanopartículas. A relação custo x benefício da síntese de AgNPs, suas características e suas aplicações tornam o material desenvolvido nesta pesquisa um potencial coadjuvante no combate aos microrganismos multirresistentes.

ABSTRACT

Silver nanoparticles (AgNPs) are promising materials in many areas of Science and technology. Traditional methods of synthesis generate undesirable and toxic residues. The green synthesis of AgNPs starting from reducing sugars emerges as a viable option of obtaining this material. At this present work, there were used the sugars arabinose, glucose, ribose and xylose to obtain AgNPs, as well as sodium citrate and sodium dodecyl sulfate as capping agents. The characterization of these materials were done trough the UV-Vis absorption – identifying the plasmon band _– X-ray diffraction, dynamic light scattering, zeta potential, scanning electron microscopy, atomic force microscopy and infrared spectroscopy techniques. The solutions showed a good stability (mean zeta potential -46.4mV), particles size ranging from ±10nm to ±100nm and different grains morphology. Starting from synthesized AgNPs, materials – agar films and carbomer gel – were obtained to be characterized and applied. The application of AgNPs solutions and derivate materials was predominantly at verifying of microbiological activity against fungi of different species of Candida spp and

Aspergillus spp, as well as pathogenic bacteria such Escherichia coli,

Staphylococcus aureus and Pseudomonas aeruginosa. It was also investigated the effects of association between AgNPs and Itraconazole, a medicine to be used against fungal infections. All the nanoparticles applied showed antibiotic activity against the microorganisms, as well as the developed materials. The AgNPs showed also results that aim to the potentiation of Itraconazole, when combined with nanoparticles. The relation cost x benefits of the synthesis of AgNPs, its characteristics and applications turn the developed material on this research a potential adjunctive in the battle against multi-resistant microorganisms.

LIST OF ILLUSTRATIONS

Chapter 1

Figure 1.1 _– Different scales of materials and organisms 20 Figure 1.2 –Evolution of “nanoresearch” along 15 years 21 Figure 1.3 – Silver nanoparticles solutions: different sizes show different

colors 24

Figure 1.4 – Chemical structure of sugars monomers: gliceraldehyde (a),

glycogen (b), starch (c) e cellulose (d) 27

Figure 1.5 – L-arabinose (a), D-ribose (b) and D-xylose (c) 28 Figure 1.6 – Last fifteen years of publishing works involving the term silver

nanoparticles 30

Figure 1.7 – Physical and chemical methods of nanoparticles synthesis 32 Figure 1.8 – Stabilization: electrostatic (a), steric (b) and electrosteric (c) 34 Figure 1.9 – Stabilization of AgNP trough polymer (a), surfactant (b) and

ligand (c) 35

Figure 1.10 – Sodium citrate (a) and sodium dodecyl sulfate (b) 35 Figure 1.11 – Silver nanoparticles with capping agent (a) and without it

(b) 36

Figure 1.12 – Plasmon band 37

Chapter 2

Figure 2.1 – AgNP synthesis: illustrated steps (citrate series). 54 Figure 2.2 – Solution before (left) and after (right) centrifugation. 55 Figure 2.3 –Aspergillus fumigatus (a), A. terreus (b) and A. niger (c). 55 Figure 2.4 – UV-Vis absorption of AgAC-01, AgAC-02 and AgAC-03. 57 Figure 2.5 – UV-Vis absorption of AgRC-01, AgRC-02 and AgRC-03. 58 Figure 2.6 – UV-Vis absorption of 01, 02, 03,

AgXC-04, AgXC-05 and AgXC-06. 59

Figure 2.13 – Zeta potential of AgXC-06 sample. 67 Figure 2.14a – SEM images from AgRC-01 at 78,022x. 69 Figure 2.14b – SEM images from AgRC-01 at 424,973x. 70 Figure 2.15a – SEM images for AgRC-02 at 100,000x. 71 Figure 2.15b – SEM images for AgRC-02 at 187,802x. 71 Figure 2.16a – SEM images for AgRC-03 at 100,000x. 72 Figure 2.16b - SEM images for AgRC-03 at 200,000x. 73 Figure 2.17 – Histogram of AgRC-01 (a), AgRC-02 (b) and AgRC-03 (c)

samples. 74

Figure 2.18 –Aspergillus fumigatus: control (a), with AgRC-01 (b),

AgRC-02 (c) and AgRC-03 (d). 75

Figure 2.19 –Aspergillus terreus: control (a), with AgRC-01 (b), AgRC-02

(c) e AgRC-03 (d). 76

Figure 2.20 – Aspergillus niger: control (a), with AgRC-01 (b), AgRC-02

(c) e AgRC-03 (d). 77

Figure 2.21 – Growing of A. niger along six days. 79 Figure 2.22 – Growing of A. terreus along six days. 80 Figure 2.23 – Growing of A. fumigatus along six days. 81 Chapter 3

Figure 3.1 – Identification of C. tropicalis (a) and C. albicans (b) on

chromogenic medium. 87

Figure 3.2 _– AgNP solution. 88

Figure 3.3 – UV-Vis specter of AgNP. 89

Figure 3.4 – Size distribution by volume (DLS). 89 Figure 3.5 – Fungicidal activity of AgNP: marked as R (AgNP synthesized by ribose), marked as G (AgNP synthesized by glucose – for comparative purposes, only) and marked as A (amphotericin B). 91 Chapter 4

Figure 4.1 – AgFilm. 102

Figure 4.2 – UV-Vis spectroscopy of AgNP and AgFilm. 103

Figure 4.3 _– DLS. 104

Figure 4.4 – FTIR spectroscopy. 105

Figure 4.6 – SEM of AgFilm (a), EDX of AgFilm (b) and histogram for

AgNP base on SEM (c). 108

Figure 4.7 – AgFilm AFM (a), AgFilm AFM on topographic mode (b) and

histogram for AgNP based on AFM. 109

Figure 4.8 – Halos around AgFilm (a) and (b) and halo measurement

average (c). 111

Chapter 5

Figure 5.1 – Obtaining the gel functionalized with silver nanoparticles. 120 Figure 5.2 – UV–VIS absorption spectra of AgNPs. 123 Figure 5.3 – XRPD patterns of the AgNPs and diffraction peaks from JCPDS (04-0783) used for identification and comparison. 124 Figure 5.4 – SEM image of the AgRC-03 at an amplification of

100,000X(a) and 238,000X (b) and size distribution histogram (c). 125 Figure 5.5 – Shear stress (a), viscosity x deformation (b) and viscosity x

time (c). 126

Figure 5.6 – Chemical stability of AgNP during the stability testing of gel

(1 and 2%) at 25°C (a) and 40°C (b). 130

Figure 5.7– (a) Antibacterial activity against Staphylococcus aureus for 0.5% AgNP gel (marked as 1), 1.0% AgNP gel (marked as 2), 2.0% AgNP gel (marked as 3) and 1.0% silver sulfadiazine (marked as 4). (b) Antibacterial activity against Escherichia coli for gel without AgNPs (marked as 5), 1.0% silver sulfadiazine (marked as 6), 2.0% AgNP gel (marked as 7) and 1.0% AgNP gel (marked as 8). (c) Antibacterial activity against Pseudomonas aeruginosa for 0.5% AgNP gel (marked as 9), 1.0% AgNP gel (marked as 10), 2.0% AgNP gel (marked as 11)

and 1.0% silver sulfadiazine (marked as 12). 132 Chapter 6

Figure 6.1 – UV-Vis spectrum 144

Figure 6.2 – DLS of nanoparticles showing (a) centrifuged AgNPs, (b) AgNPs + Itraconazole 3.2 µg mL-1 _{and (c) AgNPs + Itraconazole 4.8 µg}

mL-1_{. 146}

Figure 6.4 – SEM of (a) centrifuged AgNPs only, (b) AgNPs + ITR 3.2 µg

mL-1_{, and (c) AgNPs + ITR 4.8 µg mL}-1_{. 149}

Figure 6.5 – EDX of (a) centrifuged AgNPs only, (b) AgNPs + ITR 3.2 µg

mL-1_{, and (c) AgNPs + ITR 4.8 µg mL}-1_{. 150}

Figure 6.6 – Histogram of (a) centrifuged AgNPs only, (b) AgNPs + ITR 3.2 µg mL-1_{, and (c) AgNPs + ITR 4.8 µg mL}-1 ₁₅₁ Figure 6.7 – SEM images of samples of centrifuged AgNPs (a) and its compositional map shown at (b). SEM of AgNPs + ITR 3.2 µg mL-1 _(c) and its compositional map (d). Finally, SEM of AgNPs + ITR 4.8 µg mL-1

(e) and its compositional map (f). 153

Figure 6.8 – Chromogenic medium to identify C. parapsilosis. 154 Figure 6.9 – Sensibility test of C. parapsilosis against six different combinations. From twelve o’clock and clockwise: ITR 4.8 mg mL-1_, centrifuged AgNPs, ITR 3.2 mg mL-1_{, ITR 80 mg mL}-1_{, AgNPs + ITR 4.8} mg mL-1 _{and ending, AgNPs + ITR 3.2 mg mL}-1_{. 155} Figure 6.10 – Itraconazole as control drug and its associations with silver

nanoparticles. 156

TABLES LIST

Chapter 2

Table 2.1 _– AgNPs samples and temperature of synthesis. 54 Table 2.2 – Particle medium size according FWHM. 63 Table 2.3 – Size distribution by intensity of light scattering. 66

Table 2.4 – Zeta potential values 68

Table 2.5 – Growing of A. niger versus time 79 Table 2.6 _– Growing A. terreus versus time. 80 Table 2.7 _– Growing of A. fumigatus versus time. 81 Chapter 3

Table 3.1 – Effect of AgNPs produced by green synthesis and Amphotericin B against C. albicans and C. tropicalis. 90 Chapter 5

Table 5.1 – Physicochemical characteristics of 1.0% and 2.0% Gel-AgNP

stored at 25ºC /60%RH. ₁₂₈

Table 5.2 –Physicochemical characteristics of 1.0% and 2.0% AgNP gel

stored at 40ºC /75%RH. ₁₂₈

Table 5.3 – Chemical stability of AgNP gel (1.0 and 2.0%) during

SUMMARY

Chapter 1

1 INTRODUCTION 20

1.1 Nanoscience and nanotechnology 20

1.2 Silver nanoparticles 23

1.3 Green synthesis and reducing sugars 24

1.4 Multi-resistant microorganisms 29

2 LITERATURE REVIEW 30

2.1 Silver nanoparticles 30

2.1.1 Synthesis of silver nanoparticles 32

2.1.2 Capping agents used to stabilize silver nanoparticles 33

2.1.3 Plasmon band 37

3 OBJECTIVES 38

3.1 General objective 38

3.2 Specifics objectives 38

3.2.1 Characterization of AgNP trough 38

3.2.2 Obtaining and characterization of composite materials with

AgNPs 38

3.2.3 Perform tests as 38

REFERENCES 39

Chapter 2

1 INTRODUCTION 53

2 EXPERIMENTAL 53

2.1 Materials 53

2.2 Methods 53

2.2.1 Concentrating AgNPs 55

2.2.2 Preliminary antimicrobial assays 55

3 CHARACTERIZATION 56

3.1 UV-Vis spectroscopy 56

3.2 X-ray diffraction (XRD) 56

3.3 Dynamic light scattering (DLS) and zeta potential 56

4 RESULTS AND DISCUSSION 57

4.1 UV-Vis spectroscopy 57

4.2 XRD 64

4.3 DLS and zeta potential 65

4.4 SEM 69

4.5 Antimicrobial activity 75

5 CONCLUSIONS 82

REFERENCES 83

Chapter 3

1 INTRODUCTION 86

2 EXPERIMENTAL 87

3 CHARACTERIZATION 88

4 RESULTS AND DISCUSSION 88

5 CONCLUSION 92

REFERENCES 93

Chapter 4

1 INTRODUCTION 98

2 EXPERIMENTAL 99

2.1 Materials 99

2.2 2.2 AgNPs and AgFilm fabrication 99

3 CHARACTERIZATION 99

3.1 3.1 Ultraviolet-visible (UV-Vis) spectroscopy 99

3.2 Size 99

3.3 Fourier transform infrared (FT-IR) spectroscopy 100

3.4 Powder X-ray diffraction (PXRD) 100

3.5 AgFilm thickness 100

3.6 Microscopic features 100

3.7 Antifungal activity of AgFilm 101

4 RESULTS AND DISCUSSION 102

5 CONCLUSION 111

REFERENCES 112

Chapter 5

2 EXPERIMENTAL 119

2.1 Materials 119

2.2 Methods 120

2.2.1 Synthesis and functionalization of AgNPs 120

2.2.2 AgNPs characterization 121

2.2.3 Formulation of AgNPs based gel. 121

2.2.4 Antibacterial activity 121

2.2.5 Stability evaluation 122

3 RESULTS AND DISCUSSION 123

3.1 AgNPs characterization 123

3.2 AgNPs based gel and properties 126

4 CONCLUSION 133

REFERENCES 134

Chapter 6

1 INTRODUCTION 140

2 EXPERIMENTAL 141

2.1 Materials 141

2.2 Methods 141

2.2.1 Synthesis of AgNPs 141

2.2.2 Association of AgNPs with Itraconazole 141

2.2.3 Antifungal activity of AgNPs + ITR 142

2.2.4 Statistic data 142

2.3 Characterization 142

2.3.1 UV-Vis spectroscopy 142

2.3.2 Infrared spectroscopy 143

2.3.3 X-ray diffraction spectroscopy 143

2.3.4 Dynamic light scattering 143

2.3.5 Scanning electron microscopy 143

3 RESULTS AND DISCUSSION 144

4 CONCLUSIONS 158

CHAPTER 1

CHAPTER 1 _– NANOTECHNOLOGY, SILVERNANOPARTICLES AND GREEN SYNTHESIS: AN OVERVIEW

1 INTRODUCTION

1.1 Nanoscience and nanotechnology

Richard Feynman, who is considered the father of nanoscience, made the following question into a lecture at Annual Meeting of American Physical Society (1959): - “Why can't we write the entire 24 volumes of the Encyclopedia Britannica on the head of a pin?” [1]. It is true: it is not possible yet. However, a lot of progress in nanoscience had been obtained, on a way that this science reveals a vast area of researches and applications to be done. Once it is possible to control the characteristics of nanosized particles, the properties of these materials and their functions in different devices may be considerably improved. The term Nanotechnology was first pronounced by the Japanese professor and researcher Norio Taniguchi, in 1974, to describe the technological and scientific progress of these reduced materials [2].

Nanometer (nm = 10-9_{m) may be realized as the billion part of the meter.} A material is considered nanosized when its dimensions area among 0.1 and 100 nm. Figure 1.1 shows a comparison of different materials that exist in various scales.

Figure 1.1 – Different scales of materials and organisms.

To verify the state of the art, a research on the scientific database

ScienceDirect (http://www.sciencedirect.com) reveals the arising of researches envolving the term “nano”, in a projection of the earlier 2000’s to 2015, shown at Figure 1.2

Figure 1.2 –Evolution of “nanoresearch” along 15 years, till 2015.

Source: author

The data shown help us to understand, in a certain way, the importance of nanotechnology along the last years. The performed search reveals that, back at 2000, there were 2,546 published works about the subject. Five years later, this number arises to 10,143 and at the end of 2015, there were 33,055 works on the referred site! This represents a considerable arising of ten times over the beginning of researches.

Then, after searching for the term nanotechnology, it were found 1,043 Brazilian works – at Scopus database (http://www.scopus.com) – from a total of 104,394 published papers. This discrete participation of Brazil represents 0.99%, against 32.16% (USA) and 12.98% (China). Despite having few publications, Brazil leads in the South America. The second country that publishes in a

considerable way is Argentina, with 238 works, which traduces a 0.22%. Brazil occupies the 20th _{position when the searched term is nanotechnology. Still on this} database, when the terms nanoparticles and nanomedicine are searched, it is found 33,387 published works from the first and 8,371 from the last one, which Brazil contributes with 482 and 161 publications, respectively.

These numbers show the running for the technological development on this area of science, and Brazil, despite occupies 20th _{position among all} countries, is working to contribute continuously. Somehow, it is a reasonable overview, when we consider that only in 2004 our country began massive investments on this area [4]. Recently [5] Brazil had firmed a partnership with The US to the Nanotechnology, Energy and Materials Innovation Consortium. This partnership consolidates an investment made by Federal Government, trough Ministry of Science and Technology, of R$ 100 million to the three years 2012-2015 [6].

The research in nanotechnology produced considerable progresses on treatment of several diseases, which originated a new area: nanomedicine. Autoimmune diseases such as rheumatics, conjunctive tissue disorders, neurological, endocrine and dermatologic disorders are now subjects of researches in nanomedicine [7], such as cardiovascular diseases, [8], infections [9] and neoplastic ones, promoting also progress in veterinary medicine.

Zhu and co-workers [10] cataloged several activities and applications of nanoparticles in microbiological infections, especially at diagnoses, treatment, medical devices and vaccines.

The size of nanoparticles or nanodevices applied in nanomedicine is decisive, once they act in cell scale, and many times entering in cells to perform effective activity.

So, today it is understood that nanotechnology is a very promising area of Science, in a way that it expands possibilities to obtain new materials with different properties when nanoscaled [26]. It is very important that researches be performed and constantly encouraged, aiming the production of new materials that supply the demand of industry, academy, health and a lot of areas that may be beneficed.

1.2 Silver nanoparticles

Silver - atomic number 47 and molecular weight 107.86 g mol-1 _{- is a} noble metal found in the earth's crust in the form of minerals such as argentita (Ag2S), chloride (AgCl) or metallic silver [27]. An efficient way to enhance and

improve the biocompatibility of materials is to reduce its size. Nanomaterials once obtained can be modified to improve its efficiency and applicability in different areas such as bioscience, biomedicine and biomaterials.

Among different possibilities of obtaining and using nanomaterials, there are silver nanoparticles. Like other nanostructured particles of other metals, their physico-chemical properties differ from silver metal [28].

Figure 1.3 – Silver nanoparticles solutions: different sizes show different colors.

Source: http://nanocomposix.com/collections/silver (access may 13th ₂₀₁₅₎

1.3 Green synthesis and reducing sugars

With the advances in research for obtaining new materials and modernization of industrial processes, there is an increasing demand for raw matter to accompany this development. Furthermore, there is a constant concern for the type of material used in these processes, since it seeks to reduce the toxic and polluting them. In this way, a so-called Green Synthesis technique had been developed over the years to minimize such effects [29-33].

Green Synthesis involves processes that should be in accordance with the Twelve Principles of Green Chemistry [34]. They are the prevention, saving atoms, synthesis of less dangerous products, product design safe, safer solvents and auxiliary, search for energy efficiency, use of renewable raw material, avoiding the formation of derivatives, catalysis, drawing for degradation, real-time analysis for the prevention of intrinsically safe and chemical pollution to prevent accidents . In this way, some methods of green synthesis of silver nanoparticles are cited in the study by Sharma and colleagues [35], and are also highlighted:

cases, also as reducing agents. An example is the use of _-D-glucose as reducing sugar and starch as a stabilizing agent, used simultaneously under slight heating [33]. The use of starch prevent the use of toxic organic solvents [36]. These produced nanoparticles can be easily integrated into biological and pharmaceutical use systems.

b) Tollens method: the Tollens reaction basically is to reduce the silver from the diaminesilver complex - [Ag(NH3)2]+ _{- by an aldehyde. The} reaction can be envisaged as:

[Ag(NH3)2]+(aq) + RCHO(aq) → Ag(s) + RCOOH(aq)

It is possible to considerably reduce the concentration in the modified procedure of the Tollens reaction and the silver ions are then reduced by the saccharide in the presence of ammonia, yielding silver nanoparticle films with sizes ranging from 50 to 200 nanometers.

c) Irradiation method: a series of irradiation methods - laser, microwave, radiolysis, photolysis - can be used to reduce silver ions and to produce nanoparticles. It is common, for example, using laser in an aqueous solution containing silver salt and a surfactant. This technique can produce nanoparticles with well-defined shapes and uniform size distribution [37]. This technique does not need reducing agents. The replacement of the laser source with a mercury lamp it is also possible to produce nanoparticles [38], and the visible light radiation employment [39].

e) Polyoxometalates method: this method has the potential to synthesize silver nanoparticles due to the fact that the polyoxometalates (POM) are water soluble and have the processor capacity to polielectronics reduction-oxidation reactions (more electrons en-ware) without a disorder in their [42, 43]. structures. In this synthesis, the POM act as photocatalytic agents, reducing and stabilizing agents, which confers a high efficiency of the method. Zhang and colleagues [44] conducted a study of synthesis and satisfactory stabilization of gold nanoparticles from polioxometalates.

Figure 1.4 – Chemical structure of sugars monomers: gliceraldehyde (a), glycogen (b), starch (c) and cellulose (d).

H

O

H

OH

O

H

O

H

O

H

H

O

H

O

H

O

H

H OH

O

H

OH

(d)

Source: author

The monosaccharaides can be further classified as aldoses or ketoses. Both can be classified according to the number of carbons in its chain. Aldoses with four, five or six carbon atoms are termed tetroses, pentoses and hexoses, and successively. The ketoses are classified as the ketotetroses, ketopentoses and ketoexoses for the same number of carbon atoms above-mentioned mind. It is a common occurrence among sugars that are isomers, once asymmetric carbons (stereogenic centers) can be found in these saccharides. Among these possibilities of isomers, we highlight the epimers, which are diastereoisomers: structures that differ in the configuration of the chiral carbon only.

Figure 1.5 – L-arabinose (a), D-ribose (b) and D-xylose (c).

O

H

O

H

O

H

H

H

H

O

H

O

_H

O

H

O

H

H

H

H

O

H

O

H

O

_H

O

H

H

H

O

H

H

O

H

O

H

O

_H

In this context, the use of reducing sugars for the production of the silver nanoparticles is within the expected of green chemistry. These sugars may be aldoses or ketoses. The aldehydes undergo oxidation to form carboxyl groups characteristic of acids. This reaction, also, is the basis for the verification of aldoses. The ketoses, when isomerize as aldoses also assume the character of reducing sugars [45]. In redox reactions, in so far as the sugars are oxidized, another participant of reaction is reduced. Thus, the silver ions present in solution are reduced by these saccharides, and reduction half-reaction may be satisfactory written by Equation 1.1:

1.4 Multi-resistant microorganisms

According to the World Health Organization (WHO), resistance to antimicrobials occurs when a microorganism does not respond to the originally drug developed for the treatment of infections caused by the same. These microorganisms (bacteria, fungi, viruses and parasites) are resistant to the attack of antimicrobial agents, so the standard treatments become ineffective and the infection continues, increasing the risk of spreading to others [46].

Also according to the WHO, the evolution of resistant strains is a natural phenomenon that occurs with microorganisms which erroneously replicate or when resistance traits are exchanged between them. Indiscriminate use of antibiotics accelerates the higher incidence of multidrug-resistant strains. In addition, poor practices of infection control, inadequate and poor sanitary quality conditions in the treatment of foods contribute to the propagation of antimicrobial resistance.

Bacteria are simple, unicellular organisms and prokaryotes, what implicates that their genetic material is spread in the cytoplasm. Since fungi are eukaryotes and more complex bacteria, they may present as uni or multicellular beings. The first are the yeasts: simpler forms of fungi and larger than bacteria. The second are molds: visible and are due to the formation of a mass called mycelium and comprises several filaments called hyphae [47].

The antimicrobial agents used against bacteria and fungi can be classified as antibiotic and antifungal agents, respectively. Both pathogens may develop resistance to antimicrobials. This condition represents a health risk, since the infection by these pathogens can be fatal.

2 LITERATURE REVIEW 2.1 Silver nanoparticles

It is possible to understand that silver nanoparticles – AgNP – had been widely studied along last years, according to data obtained at Science Direct database, showed at Figure 1.6.

Figure 1.6 – Last fifteen years of publishing works involving the term silver nanoparticles.

Source: author

The silver nanoparticles are highlighted for showing uncommon physical-chemical and biological properties. This leads to an extensive application in health care. A lot of products are available, such as materials for surgery and prosthesis [48]. Still according to Heilman, the studies involving silver nanoparticles in Brazil are not significant yet, and need a massive investment to grow according to the demands.

When investigating the diverse possibilities of applications of silver, it can be highlighted its employment on electronic devices, which aims to obtain best responses in conduction [49-51], catalyst [52, 53], increase the efficiency of paints and prototypes in printers [54-56], obtaining of paint with bactericide properties [57], confection of wound healing dressings with anti-inflammatory and antimicrobial properties [58-60], functionalization of tissues on textile industries [40-42], cosmetic industries [44], as sensor to identify, qualitative and quantitative, other molecules [45-48], despite its large use as controlling of microbial growth [49-54].

number of surface atoms will lead to an antimicrobial particle compared with the massive metal [75].

The silver nanoparticles are commonly firstly characterized by UV-Vis spectroscopy, through the appearing of plasmon band, which _max will depend on the shape and size of particles [80-82], as well as the composition of system [83, 84].

2.1.1 Synthesis of silver nanoparticles

The nanoparticles may be obtained by physical (top down) or chemical (bottom up) processes. Simplifying, on physical methods, the nanoparticles are obtained through maceration of a macro-material. By the other way, chemical processes involve the manipulation of material at atomic scale, which offers a higher size and properties control [85]. Figure 1.7 shows a short scheme differencing both processes.

Figure 1.7 – Physical and chemical methods of nanoparticles synthesis

Source: adapted from Santos [86].

One way to provide the synthesis is by using plants. In a work performed in 2011, Vidhu and co-workers [87] synthesized AgNPs trough an aqueous extract of Macrotiloma uniflorum. A similar study was made by Rastogi and Arunachalam [88], when they synthesized silver nanoparticles by using garlic –

Allium sativum – aqueous extract and sunlight, and evaluated the antibacterial potential of the obtained nanoparticles. Moldovan and co-workers [89] synthesized AgNPs trough Sambucus nigra and studied its antioxidant activity. Other works had shown a tendency to use aqueous extract of plants to synthesize silver nanoparticles [90-93].

Another way is trough photo induction. El-Sherbiny and co-workers [94] synthesized silver nanoparticles trough a photo-induced process obtaining functionalized silver nanoparticles. Patra [95] worked on a photo-mediated synthesis of silver nanoparticles. Also, antioxidant, antibacterial and anticandidal activities were investigated. A similar work was performed by Verma [96]. In this last work, the system containing AgNPs was synthesized by exposing the mixture to sunlight over 45 min, and the yield increased each minute of synthesis.

It is possible to synthesize silver nanoparticles also trough a micro-wave assisted process, as performed Pal [97]. On his work, he used ethanol as reducing agent. Hydrotermal synthesis is possible as well [98] as sonochemical technique [99, 100] to obtain silver nanoparticles. However, the traditional method to synthesize silver nanoparticles is trough sodium borohydride [101-106]. This method is largely employed, but generates toxic products and does not attend the green synthesis purpose.

2.1.2 Capping agents used to stabilize silver nanoparticles

Figures 1.8a, 1.8b and 1.8c show different types of stabilization of nanoparticles, such as electrostatic, steric and electrosteric.

Figure 1.8 – Stabilization: electrostatic (a), steric (b) and electrosteric (c).

(a) (b) (c)

Source: author, inspired on Santos [86].

It is important to consider the kind of stabilizer, once there are some possibilities of stabilize the nanoparticles. The capping agent must be able to avoid the van der Waals forces that exist between the particles, and this mechanism may be done by three ways: electrostatic, showed at Figure 1.8a, which there are electric forces surrounding the nanoparticle and the repulsion among them produces the stability in solution. The electrostatic stabilization occurs when there are ions adsorbed at the surface of metallic nanoparticles. This adsorption is multilayer, and originates a coulombic repulsive force among the AgNPs. Another way is the steric stabilization, as shown in Figure 1.8b, which the stabilization does not occur because of electrostatic forces, but it is intrinsically about the steric occupation of spaces, and this steric hindrance avoids the contact among nanoparticles, which also confers to the system stability at solution. This kind of stabilization is as better as higher voluminous is the ligand. And as last, the electrosteric stabilization, as shown at Figure 1.8c, which works with both principles: electrostatic and steric. This kind of stabilization may be observed at polioxoanions [109, 110].

Figure 1.9 – Stabilization of AgNP trough polymer (a), surfactant (b) and ligand (c).

(a) (b) (c)

Source: author, inspired on Santos [86].

Therefore, the capping agents play a very important role on the synthesis of nanoparticles as a whole. There are a lot of capping agents. Two of these capping agents are objects of this work: sodium citrate and sodium dodecyl sulfate, illustrated in Figure 1.10.

Figure 1.10 – Sodium citrate (a) and sodium dodecyl sulfate (b).

O

_O

O

H

O

-O

O

O

-Na

Na

Na

C

H

O

S

O

O

O

-10

Na

Figure 1.11 – Silver nanoparticles with capping agent (a) and without it (b).

Source: author (inspired in the work of Shabbeer and co-workers [113])

2.1.3 Plasmon band

Metallic nanoparticles show different colors when in solution. It is common to observe colors ranging from blue to red of silver nanoparticles, depending on the shape. This changing of colors is due to a phenomenon known as surface plasmon resonance. This occurs because of the collective oscillation of electrons from the surface of a material, which interacts with the light that focuses on it. This collective oscillation leads the electrons, even for a short time, to be cumulated over a certain place on the material. The phenomena is thermodynamically unstable, because reduces the entropy of system, which responds tending to restore its initial condition. This restoring force originates an oscillation frequency inside the particle, then it is possible to observe the plasmon band [118].

Figure 1.12 – Plasmon band

Source: author

3 OBJECTIVES

3.1 General objective

To perform a green synthesis to obtain silver nanoparticles (AgNPs), through reducing sugars and silver nitrate, and promote the stabilization of the system by using sodium citrate and sodium dodecyl sulfate.

3.2 Specifics objectives

Once the AgNPs are obtained, several steps are taken, as described below:

3.2.1 Characterization of AgNP trough:

- UV-vis spectroscopy; - X-ray diffraction;

- Dynamic scattering of light; - Zeta potential;

- Scanning electron microscopy - Atomic force microscopy.

3.2.2 Obtaining and characterization of composite materials with AgNPs:

- Agar films; - Carbomer gel.

3.2.3 Perform tests as:

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CHAPTER 2